prr.hec.gov.pkprr.hec.gov.pk/jspui/bitstream/123456789/9362/1/nadia afsheen... · web viewin the...
TRANSCRIPT
In the name of ALLAH, the Gracious, The Merciful, All prays belong to ALLAH, Lord of All words,
The Gracious, The Merciful. Master of the Day of Judgment. Thee alone do we worship and
Thee alone do we Implore for help. Guide us in the right path….
The path of those on whom Thou hast Bestowed Thy blessings, Those who have not incurred. Thy
Displeasure, and Those Who have not gone astray.
1
Biochemical Profiling and Cardioprotective Potential of Various Combinations of Medicinal Plants
By NADIA AFSHEEN
(M. Phil. UAF)
A thesis submitted in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHYIN
BIOCHEMISTRY
DEPARTMENT OF BIOCHEMISTRY
FACULTY OF SCIENCES,UNIVERSITY OF AGRICULTURE,
FAISALABAD2016
2
DECLARATION
I hereby declare that the content of the thesis “Biochemical profiling and cardioprotective
potential of various combinations of medicinal plants” are product of my own research and no
part has been copied from any published source (except the references, standard mathematical
models/equation/protocols). I further declare that this work has not been submitted for the award
of any other diploma/degree. The university may take action if the information provided is found
inaccurate at any stage.
SIGNATURE OF THE STUDENT
3
The Controller of Examinations,
University of Agriculture,
Faisalabad.
“We, the supervisory Committee, Certify that the contents and form of thesis submitted by Miss
Nadia Afsheen, Reg. no. 2007-ag-526, have been found satisfactory and recommend that it
should be processed for evaluation, by the External Examiner(s) for the award of degree”.
Supervisory committee:
1. Chairman ------------------------------------ (Prof. Dr. Khalil-ur-Rehman)
2. Member ----------------------------------- (Prof. Dr. Khalid Mahmood Khan)
3. Member ------------------------------------ (Dr. Muhammad Anjum Zia)
4. Member ------------------------------------ (Dr. Nazish Jahan)
4
DEDICATED To
MY ADORABLE AND AFFECTIONATE
“PARENTS”“Mr. & Mrs. Rana Irshad
Ahmed”WHO
Taught me the First Word to SpeakThe First Alphabet to Write
The First Step to TakeAnd
Who those burnt themselves to make me a candle&To
My BelovedHusband
“Rana Shafaqat Ali”
5
6
AcknowledgementsFirst and the foremost, I would like to give my humble thanks and praise to the Almighty Allah for His
grace and blessings throughout my entire life and in the completion of this dissertation particularly.
Without the Blessings of Almighty Allah and His teachings taught by Prophet Muhammad (PBUH), my
life is nothing.
During the completion of this thesis, there were many kinds of supports I have got. I would like to express
my deepest thanks and gratitude to my supervisor Prof. Dr. Khalil-ur-Rehman, (Department of
Biochemistry University of Agriculture, Faisalabad), for the valuable guidance, assistance and
constructive advice throughout the entire project. I would like to make my sincere appreciation and
pleasure and sincerest thanks to my committee members Dr. Khalid Mehmood Khan, Dr. Muhammad
Anjum Zia (Department of Biochemistry University of Agriculture Faisalabad) and Dr. Nazish Jahan
(Department of Chemistry, University of Agriculture, Faisalabad), for valuable assistance and guidance.
I also pay homage to all teachers goal of my academic carrier with light of knowledge and enable me to
touch a section in my life.
Very special thanks to Higher Education Commission (HEC) of Pakistan for its moral & financial
support, without its assistance this dissertation was merely a dream.
No acknowledgement would ever adequately express my delegation to my beloved parents Mr. & Mrs.
Rana Irshad Ahmed and my parents in law Mr. & Mrs. Rana Sadiq for their day and night prayers
which boost my moral to fly high to accomplish my goal. The names of my parents will always be in front
of my eyes, as I will look on the cover of my life. I also express my gratitude to my beloved Husband,
Rana Shafaqat Ali for his sincere help and inspiring assistance.
I feel my immense pleasure to express my deepest gratitude and sincere thanks to my brother Rana Asif
and brother-in-law Rana Rafaqat Ali, Dr. Shahid Hafeez and Rana Moeen and for their encouraging
and inspiring cooperation all the time. I have no words to express my sweet sensation to my beloved
Sisters, my cousins and my all family for their moral boost, encouragements and countless prayers for
me to achieve higher ideal of life.
I have no words to express my sweet sensations to my loving friends Saman Hina and Sofia Parveen,
Zynab Ahmed who are near and dear to me. Finally, May Allah’s blessings be upon all these people with
His countless favors and I would like to pray for their happy and peaceful lives (Ameen)!
NADIA AFSHEEN
7
CONTENTSChapter
No. Title Page No.
1. INTRODUCTION 1
2. REVIEW OF LITERATURE 6
3. MATERIALS AND METHODS 16
4. RESULTS AND DISCUSSIONS 35
5. SUMMARY 130
LITERATURE CITED 132
8
LIST OF TABLES
Table No. Title Page No.
3.1Selected parts of plants for evaluation of cardioprotective
potential 17
3.2 Protocol of the mutgenicity assay 22
3.3Experimental design suggested by Response Surface
Methodology to optimize the dose of Salbutamol23
3.4The Central Composite Design for the treatment of selected
medicinal plants25
3.5 Formation of different herbal combinations 26
3.6 Dehydration treatment procedure of histopathology 33
3.7 Clearing treatment procedure of histopathology 33
3.8 Infiltration treatment procedure of histopathology 33
3.9Detailed protocol of Hematoxylin and Eosin (H&E) staining for
histopathology34
4.1Angiotensin converting enzyme inhibitory activity (%) of studied
medicinal plants 37
4.2 DPPH radical scavenging activity of selected medicinal plants 53
4.3 Hemolytic activity (%) of extracts of selected medicinal plants 58
4.4The mutagenicity of standard, Background and extracts of
selected medicinal plants63
4.5Analysis of variance (ANOVA) for the fitted model of CK-MB,
LDH and SGOT activity as a function of independent variables68
4.6Effects of optimized dose of salbutamol on different cardiac
markers suggested by Response Surface Methodology69
4.7
Analysis of variance (ANOVA) for response surface
methodology of CK-MB (IU/L) as a function of independent
variables
73
9
4.8Optimized concentrations of medicinal plants for CK-MB (IU/L)
against salbutamol induced Myocardial infarction74
4.9
Analysis of variance (ANOVA) for response surface
methodology of SGOT (IU/L) as a function of independent
variables
77
4.10Optimized concentrations of medicinal plants for SGOT (IU/L)
against salbutamol induced Myocardial infarction78
4.11
Analysis of variance (ANOVA) for response surface
methodology of LDH (IU/L) as a function of independent
variables
81
4.12Optimized concentrations of medicinal plants for LDH (IU/L)
against salbutamol induced Myocardial infarction82
4.13
Analysis of variance (ANOVA) for Response Surface
Methodology of HDL (mg/dL) as a function of independent
variables
86
4.14Optimized concentrations of medicinal plants for HDL (mg/dL)
against salbutamol induced myocardial infarction87
4.15
Analysis of variance (ANOVA) for response surface
methodology of LDL (mg/dL) as a function of independent
variables
91
4.16Optimized concentrations of medicinal plants for LDL (mg/dL)
against salbutamol induced myocardial infarction92
4.17
Analysis of variance (ANOVA) for response surface
methodology of TGs (mg/dL) as a function of independent
variables
95
4.18Optimized concentrations of medicinal plants for triglycerides
(mg/dL) against salbutamol induced myocardial infarction96
4.19
Analysis of variance (ANOVA) for response surface
methodology of TC (mg/dL) as a function of independent
variables
99
10
4.20
Optimized concentrations of medicinal plants for Total
Cholesterol (mg/dL) against salbutamol induced myocardial
infarction
100
4.21
Analysis of variance (ANOVA) for response surface
methodology of SOD (IU/mg) as a function of independent
variables
104
4.22
Analysis of variance (ANOVA) for response surface
methodology of GPX (IU/mg) as a function of independent
variables
106
4.23
Analysis of variance (ANOVA) for response surface
methodology of Catalase (IU/mg) as a function of independent
variables
108
4.24Hematological analysis of different groups of rats treated with
various concentrations of selected medicinal plants109
4.25Formulation of different herbal combinations of selected
medicinal plants 112
4.26Hemodynamic analysis of herbal combination against surgically
induced myocardial infarction120
11
LIST OF FIGURES
Figure No. Title Page No.
2.1 Schematic representation of the progression of myocardial necrosis after coronary artery occlusion 7
2.2Role of reactive oxygen species and its prevention by Natural
antioxidants 9
4.1Graphical presentation of angiotensin converting enzyme
inhibition (%) of studied medicinal plants 37
4.2 Full mass spectrum of Terminalia arjuna 40
4.3MS-MS of 685.58 with CID showing Termiarjunoside I at
667.50 m/z 40
4.4 Mass spectrum of T. arjuna showing Quercetin at 301.08 m/z 41
4.5 Mass spectrum of T. arjuna showing Gallic acid at 169.08 m/z 41
4.6 MS-MS CID (30.00) of peak 169 m/z 41
4.7 Mass spectrum indicating the presence of Myricetin 42
4.8MS/MS of T. arjuna of peak 317 at CID (21.00) showing
Ferulic acid at 193 m/z and Catechin at 289 m/z 42
4.9Mass spectrum of C. oxyacantha showing proanthocynidine at
593.17 m/z 43
4.10MS2 CID (20.00) of peak 591.42 showing Ursolic acid at
457.25 m/z 44
4.11Mass spectrum of C. oxyacantha showing Crateagolic acid at
471.08 m/z 44
4.12 MS2 of 381 of C. oxyacantha with CID (20.00) showing Quercetin at 301.17 m/z 45
4.13Mass spectrum of R. serpentina showing Yohimbine at 355.33
and Ajmaline at 327.25 m/z 46
4.14 R. serpentina showing MS-MS at peak 327 with CID (25.00) 46
12
4.15MS2 of peak 327 with CID (25.00) showing Ajmailicine at
353.25 m/z 47
4.16Mass spectrum of R. serpentina showing serpentine at 349.25
m/z 47
4.17 Mass spectrum of A. sativum 48
4.18 MS2 CID 25.00 of 896 of A. sativum showing Myricetin at 319.25 m/z 48
4.19 Mass spectrum of A. sativum showing Apigenin at 327.25 m/z 49
4.20 Full Mass spectrum of C. sativum 49
4.21 Mass Spectrum of C. sativum showing Caffeic acid at 179.08 m/z and isorhamnetin-3-O-glucoside at 477.17 m/z 50
4.22 Mass spectrum of Coriandrum sativum showing apigenin-6-C-glucoside at 593.25 m/z 50
4.23 Mass Spectrum of E. cardamom showing Terpinylacetate at 195.17 m/z 51
4.24 Mass Spectrum of E. cardamom showing Sabinene at 137.08 m/z 51
4.25 Mass Spectrum of P.nigrum showing pipercide at 219.08 m/z 52
4.26Graphical presentation of DPPH radical scavenging activity of
selected medicinal plants 54
4.27 DNA plasmid pBR322 55
4.28
Agarose gel electrophoresis pattern of pBR322 plasmid DNA treated with 30 mM H2O2 in the presence and absence of different plants extracts [Lane 1: pBR322 DNA + 30mM H2O2+ P1 (100 µg/mL), Lane 2: pBR322 DNA + 30mM H2O2+ P1 (500 µg/mL), Lane 3: pBR322 DNA + 30mM H2O2+ P1 (1000 µg/mL), Lane 4: pBR322 DNA + 30mM H2O2+ P2 (100 µg/mL), Lane 5: pBR322 DNA + 30mM H2O2+ P2 (500 µg/mL), Lane 6: pBR322 DNA + 30mM H2O2+ P2 (1000 µg/mL), Lane 7: pBR322 DNA + 30mM H2O2+ P3 (100 µg/mL), Lane 8: pBR322 DNA + 30mM H2O2+ P3 (500 µg/mL), Lane 9: pBR322 DNA + 30mM H2O2+ P3 (1000 µg/mL), Lane 10: pBR322 DNA + 30mM H2O2+ P4 (100 µg/mL), Lane 11: pBR322 DNA + 30mM H2O2+ P4 (500µg/mL), Lane 12: pBR322 DNA + 30mM H2O2+ P4 (1000 µg/mL)
56
13
4.29
Agarose gel electrophoresis pattern of pBR322 plasmid DNA treated with 30 mM H2O2 in the presence and absence of different plants extracts: [Lane13: pBR322 DNA + 30mM H2O2+ P5 (100 µg/mL), Lane 14: pBR322 DNA + 30mM H2O2+ P5 (500 µg/mL), Lane 15: pBR322 DNA + 30mM H2O2+ P5 (1000 µg/mL), Lane 16: pBR322 DNA + 30mM H2O2+ P6 (100 µg/mL), Lane 17: pBR322 DNA + 30mM H2O2+ P6 (500 µg/mL), Lane 18: pBR322 DNA +30mM H2O2+ P6 (1000 µg/mL), Lane 19: pBR322 DNA + 30mM H2O2+ P7 (100 µg/mL), Lane 20: pBR322 DNA + 30mM H2O2+ P7 (500 µg/mL), Lane 21: pBR322 DNA + 30mM H2O2+ P7 (1000 µg/mL)]
57
4.30 Graphical presentation of % Hemolysis of extracts of selected medicinal plants at different concentrations 60
4.31 Standard S. typhimurium TA 98 62
4.32 Background plate 62
4.33 Mutagenicity of plants extracts 63
4.34 Response surface plots of CK-MB vs. time and concentration 65
4.35 Response surface plot of SGOT vs time and concentration 66
4.36 Response surface plot of LDH vs. time and concentration 67
4.37Graphical representation of optimized concentration of medicinal plants for CK-MB (IU/L) against salbutamol induced Myocardial infarction
72
4.38Graphical representation of optimized concentration of medicinal plants for SGOT (IU/L) against salbutamol induced Myocardial infarction
76
4.39Graphical presentation of optimized concentration of medicinal plants for LDH (IU/L) against salbutamol induced Myocardial infarction
81
4.40Graphical presentation of optimized concentration of medicinal plants for HDL (mg/dL) against salbutamol induced Myocardial infarction
85
4.41Graphical presentation of optimized concentration of medicinal plants for LDL (mg/dL) against salbutamol induced Myocardial infarction
90
4.42Graphical presentation of optimized concentration of medicinal plants for Triglycerides (mg/dL) against salbutamol induced Myocardial infarction
94
4.43Graphical presentation of optimized concentration of medicinal plants for T. cholesterol (mg/dL) against salbutamol induced Myocardial infarction
99
14
4.44 Graphical representation of doses optimization of medicinal plants for SOD 104
4.45 Graphical representation of doses optimization of medicinal plants for GPX 106
4.46 Graphical representation of doses optimization of medicinal plants for CAT 108
4.47
Graphical representation of Cardioprotective effect of herbal combinations of plant extracts on CK-MB level (IU/L) in the serum of experimental groups through the preventive mode of treatment
115
4.48
Graphical representation of Cardioprotective effect of herbal combinations of plant extracts on SGOT level (IU/L) in the serum of experimental groups through the preventive mode of treatment
117
4.49
Graphical representation of Cardioprotective effect of herbal combinations of plant extracts on LDH level (IU/L) in the serum of experimental groups through the preventive mode of treatment
118
4.50 Graphical representation of hemodynamic parameters of various groups treated with different herbal combinations 122
4.51 The histopathological representation of cardiac tissue of normal control group 123
4.52 The histopathological representation of cardiac tissue surgically induced MI control group 124
4.53 The histopathological representation of cardiac tissue of HC1 treated group 125
4.54 The histopathological representation of cardiac tissue of HC2 treated group 125
4.55 The histopathological representation of cardiac tissue of HC3 treated group 126
4.56 The histopathological representation of cardiac tissue of HC4 treated group 126
15
ABSTRACTMyocardial infarction (MI) is the most dreaded menace and its incidences are increasing
gradually. Although many of the major and minor risk factors impart a crucial role in the onset of
MI, however the hypertension and hyperlipidemia are its major risk factors. In spite of
significant pharmacological advancements regarding drug development has been made, but most
of the available drugs have a long list of side effects which limit their use in clinical medicine.
Hence there is a dire need to integrate complementary and alternative medications into the
practice of conventional medicines, for the treatment of MI. The research was planned to be
carried out into two sections including in vitro and in vivo analysis. In vitro analysis involved the
screening of medicinal plants by Angiotensin Converting Enzyme inhibition assay. Among all
the selected medicinal plants, methanolic extracts of Terminalia arjuna, Piper nigrum,
Coriandrum sativum, Allium sativum, Rauvolfia serpentina, Eletaria cardamom and Crataegus
oxyacantha showed maximum ACE inhibition potential. These medicinal plants were further
subjected to LC-MS analysis which proved the existence of vital phytoconstituents and phenolic
acids in extracts. The antioxidant execution of selected medicinal plants has performed by DPPH
and DNA protection assay. The dose dependant response for antioxidative potential i.e, the
activity of all the medicinal plants in term of % age inhibition increased with increase in
concentration. The toxicity assay of selected medicinal plants exhibited no hemolytic effect and
considered to be safe herbal product for effective fighting against various diseases. Section- II
comprised of In vivo analysis was conducted in three phases. The phase-I included the
preliminary trial, in which the RSM optimized the dose of salbutamol (80 mg/kg b. wt.) to
induce myocardial infarction. In phase-II, the optimal concentrations of selected medicinal plants
were evaluated against salbutamol induced myocardial infarction by using Response Surface
Methodology. In case of Phase-III, the optimized doses of selected medicinal plants were used
to formulate four different herbal combinations with appropriate ratio. The herbal combination
(HC4) showed maximum restoration of cardiac markers (CK-MB, AST and LDH) and
haemodynamic parameters (MAP, HR, LVEDP). The histopathological examination also
confirmed the cardioprotective potential of HC4. Thus the HC4 being safe, inexpensive and
cardioprotective herbal combination, could be considered an alternate of synthetic drug.
Keywords: Myocardial Infarction, Angiotensin converting enzyme, LCMS, Herbal
combinations.
16
CHAPTER #1 INTRODUCTIONCardiovascular diseases (CVDs) which usually stem from vascular dysfunctions are the
major factors causing morbidity and mortality in developing countries (Lee and Kim, 2014).
Cardiovascular diseases cause 17.1 million deaths every year and this number will raise up to 20
million in 2020 (Velavan et al., 2008; Gunjal et al., 2010; Upaganlawar et al., 2011). Although it
is considered as a disease of developed countries, but its incidence is increasing in the
developing world as well (Torabian et al., 2009; El-Sayed et al., 2011). In Pakistan, the
condition has become very critical as CVDs are responsible for 25% of deaths (Maruthappan and
Shree, 2010; Radhika et al., 2011; Manimegalai and Venkatalakshmi, 2012).
A variety of synthetic drugs for the treatment and management of CVD are now
available. Significant improvements regarding synthetic drug development have been made.
Contrary to this advancement these available drugs are taking their toll in the form of side effects
(Thippeswamy et al., 2009; Gielen and Landmesser, 2014). Hence there is a need to integrate
conventional drugs into the practice of synthetic drugs, for the treatment of CVD (Ittagi et al.,
2014).
Among CVD, the Myocardial infarction (MI) is the most dreaded menace. MI is defined
as extended myocardial ischemia with necrosis of myocytes due to disruption of blood supply to
a part of heart resulting in death of cardiac tissue (Kumar and Gurusamy 2014; Subhashini et al.,
2011; Ittagi et al., 2014). Multiple biochemical alterations, lipid peroxidation, free radical
damage, hyperglycemia and hyperlipidemia occur during MI (Siddiq et al., 2012; Krushna et al.,
2009; Alamgeer et al., 2015). The patients of MI mostly presents as chest pain, palpitations,
sweating and anxiety. These symptoms may be gradual or instantaneous (Thygesen et al., 2007;
Radhika et al., 2011). MI causes loss of some of the functioning of myocardium, hence
increasing the work load of the healthy myocardial tissue causing its hypertrophy. The cardiac
hypertrophy leads to compensation of perfusion pressure of ventricles. If work load on the heart
increases for a long time, it will result in compromised cardiac functioning and heart failure
(Cohen and Shah 2004).
The prevalence of major risk factors of cardiovascular diseases including hypertension,
diabetes and impaired glucose tolerance is increasing in Pakistan (Jafary et al., 2007; Jaffery et
al., 2014). This increase in the prevalence of risk factors may be responsible for increase in the
morbidity and mortality rate in Pakistan (Pham et al., 2007; Jaffery et al., 2014). Although a
17
variety of major and minor risk factors are involved in the onset and progression of CVD
(Zahidullah et al., 2012) but the hypertension and hyperlipidemia are considerd as most
remarkable risk factors. The hypertension doubles the rate of coronary artery disease and triples
the rate of congestive heart failure (Mendis et al., 2011).
The blood pressure in body is monitored mainly by Renin Angiotensin Aldosterone
System (RAAS) and to some extent by Kallikrein Kinin system (Hammoud et al., 2007). The
Angiotensin converting enzyme (ACE) (EC 3.4.15.1) is one of the members of a series of
enzymatic reactions in RAAS which converts angiotensin I to angiotensin II. Angiotensin II acts
as a potent vasoconstrictor for vascular smooth muscle cells (Chen et al., 2009; Hernan-
dezLedesma et al., 2011). It also acts on proximal convulated tubules of kidney and causes Na+
and water retention, leading to increase in blood pressure. Thus inhibition of the ACE lowers the
blood pressure by opposing its effects (Ansor et al., 2013; Sharifi et al., 2013). The drugs used in
the management of hypertension include diuretics, β-blockers, calcium channel blockers,
angiotensin II receptor blockers and ACE inhibitors. Among these ACE inhibitors are commonly
used to manage hypertension (Sharifi et al., 2013). A number of synthetic ACE inhibitors
including Captopril, Lisinopril, Enalpril and Rampril have been used as first line of treatment for
hypertension. These ACE inhibitors have many common side effects like cough, dizziness,
headach, weakness, renal failure and angioneuretic edema. Hence there is a need to explore the
herbal alternatives having same mechanism of action as that of ACE inhibitor but with no side
effects (Balasuriya and Rupasinghe 2011). Mostly the natural ACE inhibitors are protein
hydrolysates and peptides attained from animal and plant sources (Belovic et al., 2011; Belovic
et al., 2013). A number of crude and purified herbal extracts have been used to assess ACE
inhibition potential (Chen et al., 2009; Hernan-dezLedesma et al., 2011). Active substances
present in medicinal plants may act as ACE inhibitors and reduce the blood pressure to normal
(Sharifi et al., 2013).
Major biochemical changes during MI due to hyperlipidemia and peroxidation lead to
loss of plasma membrane integrity. The hyperlipidemia is highly responsible for the
development and complcations of atherosclerosis and coronary heart diseases (Gosain et al.,
2010; Joshi and Jain 2014). Proatherogenic cholesterols are risk factors for thrombotic
cardiovascular diseases including MI (Rudenko et al., 2010; Mohanty et al., 2009; Radhika et
al., 2011) but the antiatherogenic cholesterol HDL possess protective effect (Hajizadeh et al.,
18
2011; Joshi and Jain 2014). There are several synthetic drugs such as HMG-Co-A reductase
inhibitors, Fibrates, Niacin, Bile acid binding resins and Probucol that may help to lower plasma
lipid. Owing to high costs and more side effects, associated with synthetic drugs, the scientists
and researchers are trying to substitute the natural components as an alternate of synthetic drugs
(Mahmood et al., 2010; Joshi and Jain 2014; Khursheed et al., 2010).
Free radicals are required during many biochemical processes in living organisms
(Velavan et al., 2007) but the amplified production of toxic Reactive Oxygen Species exerts
severe oxidative stress on myocardium leading to ischemic heart disease, cardiomyopathy,
cardiac failure and arrhythmia (Ittagi et al., 2014). ROS also formed in ischemic tissues cause
lipid peroxidation of membrane and DNA damage. These alterations lead to anatomical and
physiological damages of cardiac cells as well. In this process, mitochondrial, endoplasmic
reticular and extraceluular Ca2+ is released in cytosole through damaged cell membrane hence
increasing cytosolic Ca2+. This cytosolic Ca2+ activates: 1) ATPase, exhausting ATP and
increasing cyclic AMP, 2) Phospholipase, decreasing phospholipids, 3) protease, disrupting
membrane and cytoskeleton proteins, 4) endonucleases, causing nuclear chromatin damage
(Prabha et al., 2014). ROS increases angiotensin II which causes hypertension and also activate
NADPH oxidase. This increase in NADPH oxidase further rise the ROS (Sharifi et al., 2013)
leading to progression of ischemic injury (Manjunatha et al., 2011).
The antioxidant potential of herbal medicines has shielding effect against cardiovascular
diseases (Ayesha et al., 2013). This will be natural protective strategy and would be freely
available with low cost as compared to synthetic drugs (Dianat et al., 2014). The use of
indigenous medicinal plants against variety of diseases is successfully in practice by
complementary and alternative medical practitioner but without knowing their pharmacokinetics
and therapeutic details. However it is also predicted that about one quarter of accepted medicines
have been orignated from medicinal plants (Adaramoye, 2009).
Pakistan is bestowed with a wide range of plants species with unique biodiversity in
different climatic zones. There are about 6000 wild plants available; out of these 600 are used as
medicinal plants (Khan et al., 2012). These medicinal plants have been used in scientific
research for the therapeutical intentions in human beings (Vasu et al., 2009; Shreya et al., 2013).
The bioactive substances like tannins, alkaloids, carbohydrates, terpenoids, steroids and
flavonoids found in medicinal plants impart a crucial role in physiological and biochemical
19
pathways during different ailments (Edoga et al., 2005, Slusarczyk et al., 2009; Soni and Sosa,
2013). Various phytoconstituents present in medicinal plants like aloin, mangiferin, bromelain,
curcuminoids, glycosides, ginkgolides, crocin, neriine, emblicanin, piperine etc are well
documented for their cardioprotective potential (Ramesh et al., 2008; Ragavendran et al., 2011;
Lakshmy et al., 2014). These phytoconstituents may lessen the rate of heart diseases and other
degenerative tissue injuries (Palasuwan and Soogarun 2014). Epidemiological studies also
claimed that there is a considerable synergism between intake of fruits, vegetables and herbs.
Thus the medicinal plants and other natural food constituents are now the focus of attention for
decreasing the risk factors of MI (Goyal et al., 2010; Ojha et al., 2010). The number of drugs and
chemotherapeutics extracted from plants are now used as medicine with reliability. That’s why
the use of traditional medicinal plants in most developing countries is increasing (Alsarhan et al.,
2014).
Currently available synthetic cardioprotective drugs exhibit a number of side effects and
are out of reach for poor community. Many researches have studied the cardioprotective
potential of some medicinal plants which are safe and inexpensive (John 2014; Beaulah et al.,
2014; Susila et al., 2013; Ramadoss et al., 2012; Ittagi et al., 2014). Therefore the natural
cardioprotective drugs divert the attention of entire world population towards green medicines.
Its therapeutic efficacy is because of the primary and secondary metabolites (Beaulah et al.,
2014). According to World Health Organization (WHO) herbal medicines are used as primary
health care in 80% of total world’s population and recommended for therapeutic uses as safe
alternative medicines all over the world (Menaka et al., 2011).
Keeping in view the above facts the study was planned to assess biochemical profiling
and cardioprotective potential of medicinal plants by inducing myocardial infarction, chemically
as well as surgically in experimental animals. The research work was divided into two sections
comprised of in vitro and in vivo studies. The in vitro studies included the screening of medicinal
plants for ACE inhibition potential. After screening, the said medicinal plants were subjected to
biochemical profiling including, LC-MS analysis, antioxidant and toxicological assays. The
animal models impart crucial role in drug discovery for management of various diseases. The
chemically provoked myocardial infarction in animals is a well known model to study the
important outcomes of various medicinal plants against cardiac disorder (Ahsan et al., 2014;
Sahreen et al., 2011). Hence after in vitro characterization of medicinal plants, the In vivo
20
analysis was planned comprising of three phases. In phase-I, the dose of salbutamol was
optimized to induce myocardial infarction. The Phase-II involved the dose optimization of
cardioprotective medicinal plants by using Response Surface Methodology (RSM). In
combination bioactive phytoconstituents of medicinal plants imparts synergic therapeutic
potential (Li et al., 2009). In case of Phase-III, herbal combinations were made by using the
optimized concentrations of selected medicinal plants to evaluate their cardioprotective potential
through surgically induced MI. The hemodynamic parameters, activities of cardiac enzymes in
serum and antioxidants enzymes in cardiac tissue of different groups of experimental animals
were analyzed to evaluate the best herbal combination which showed the maximum potential
against MI and also suppress the risk factors associated to MI.
Aims and Objectives:
The objective of this research was to get an alternative, innocuous and effective herbal
formulation that enable to ameliorate the MI and its risk factors.
21
CHAPTER # 2 REVIEW OF LITERATURE
Anatomically the heart comprises of arterial, ventricular and conductive muscle fibers
(Harika et al., 2014). The cardiac muscle fibers, which are anatomically as well as
physiologically similar to that of skeletal muscle, are formed by typical myofibrils containing
actin and myosin filaments. During cardiotoxicity any damage to the heart muscle can lead to
small changes in blood pressure, arrhythmias and cardiomyopathy (Koti et al., 2009).
Cardiomyopathy and the subsequent loss of heart function is continuation of a variety of
cardiovascular pathologies. Generally in cardiomyopathy the heart muscle becomes enlarged,
thick, or rigid and in unusual cases the muscle tissue itself is substituted with scar tissue thus
consequently heart would be unable to pump blood throughout the body.
2.1 Cardiovascular Diseases:
Cardiovascular diseases (CVD) are complex multifactorial disease (Rahman and Lowe
2006). Not only under developing countries like Pakistan, but the developed countries also could
not able to give the successful solution for the management and treatment of CVD (Aslam et al.,
2015). According to the World Health Organization, CVD are one of the major reasons of
fetality and account for 30% of all deaths in 2005 (Lee and Kim 2014). The Global Burden of
Disease estimated that 29.6% of all worldwide deaths were caused by CVD in 2010 (Nichols et
al., 2014). It is estimated that by year 2020, it will account for one third of the deaths all over the
world (Rajalakshmy et al., 2011). Many risk factors such as elevated low density lipoprotein,
low levels of high density lipoprotein, diabetes mellitus and hypertension increase the prevalence
of CVD (Toth, 2007). Among these the hypertension is a major risk factor contributes to the
development of CVD by 13%. WHO estimation showed that 45% of CVD deaths are associated
with hypertension as about 15-37% of the adult population suffered from high blood pressure,
while at 60 years of age this prevalence increased to 50% of population (Banjari et al., 2013).
The oxidized form of low density lipoprotein-cholesterol (LDL-c) and high concentration
of cholesterol is also one of the major risk factors of heart diseases that lead to progressive
atherosclerosis (Attar, 2006; Mansour et al., 2009; Maruthappan and Shree 2010). This is
responsible for blockage of coronary arteries and infarction of cardiac muscles (Maruthappan
and Shree 2010; Radhika et al., 2011; Manimegalai and Venkatalakshmi, 2012).
22
2.2: Myocardial infarction:
It is well established that among the CVD, the Myocardial infarction (MI) is necrosis of
myocardium and perpetually followed by several biochemical alterations like hyperglycemia,
hyperlipidaemia and lipid peroxidation (bhandari et al., 2008). Moreover, during pathological
progression of MI, there is a large scale alteration in normal biochemical processes because of
metabolic shift (Taegtmeyer et al., 2004). This “metabolic shift” leads to dysfunctioning of
energy metabolism, oxidative stress and inflammation in plasma membrane (Jiang et al., 2011).
The thrombotic occlusion in coronary arteries may also lead to myocardial cell death (Jiang et
al., 2014; Graham et al., 2007; Devlin and Henry 2008). As the plaque increases in size, the
coronary arteries get narrower and cause the hindrance in blood flow to cardiac musculature.
This reduced blood flow and oxygen supply effects heart structurally leading to MI, ischemia,
unstable angina and sudden death (Dhevi et al., 2014).
Fig. 2.1 Schematic presentation of the development of myocardial infarction after blockage of coronary artery
The process of necrosis starts from a small zone of myocardium beneath the endocardium
surface. This necrosed region is oxygenated by diffusion of blood, circulating into the ventricle.
This region of myocardium (shaded) depends on the occluded vessel for its perfusion (Fig. 2.1).
23
The necrotic region of cardiac musculature losses its integrity and viability, this myocardiam is
called infracted and the process is called myocardial infarction.
In developing countries, over 80% of deaths take place due to CVD in underdeveloped
countries (Susila et al., 2013). The classical symptoms of myocardial infarction included nausea,
vomiting, chest pain, palpitations, acute coronary syndrome, anxiety, sweating or feelings of
impending doom. The onset of these symptoms is usually gradual and rarely instantaneous
(Thygesen et al., 2007; Radhika et al., 2011).
2.3: Salbutamol induced myocardial infarction:
The cardioprotective potential of medicinal plants against chemically induced MI in
animal models has been executed in many studies (Siddiq et al., 2012; Beaulah et al., 2014).
This method is simple for executing the biochemical and histological alteration during acute
myocardial infarction (Gomathi et al., 2014; Ittagi et al., 2014; Prabha et al., 2014; Ramadoss et
al., 2012). Salbutamol is synthetic beta adrenergic receptor agonist that causes severe oxidative
stress in myocardium (Shiny et al., 2005; Hina et al., 2010; Jahan et al., 2012; Kumar and
Gurusamy 2014; Zafar et al., 2015) and alterations in membrane permeability which is
responsible for loss of anatomical and physiological integrity of myocardium (Ramadoss 2012;
Beaulah et al., 2014).
The mechanism of action of salbutamol, to induce MI, is to create hyperlipidemia by
enhancing adenylate cyclase activity. This results in increased cyclic AMP formation and
accumulation of lipid. The cytotoxic action of salbutamol causes lipolysis and peroxidation of
endogenous lipids. These biochemical and pathological changes including hyperlipidemia,
peroxidation and loss of plasma membrane integrity lead to MI (Khursheed et al., 2010).
Considerable clinical and experimental evidences also suggested that the generation of free
radicals and ROS are involved in the pathogenesis of salbutamol induced MI. Therefore,
salbutamol induced MI would be a well established model to study the protective potential of
medicinal plants against MI (Kumar and Gurusamy 2014). Several studies have demonstrated
that nutrients, antioxidant and/or complementary medicine strengthen the LDL oxidation
susceptibility and increase the antiatherosclerotic impact of high density lipoproteins which is
performing a key role in prevention of cardiovascular diseases (Rivas-Arreola, 2010).
24
2.3: Oxidative stress and Natural antioxidants:
Free radicals are essential for chemical signaling, detoxification, energy supply and
immune functions during normal physiological function. However excess amount of free radicals
initiate the oxidation of biomolecules that may lead to numerous diseases along with cell injury.
The production of reactive oxygen species (ROS) is balanced by the endogenous antioxidative
defense system. The deleterious oxidative stress is generated as a result of imbalance between
the production and elimination of ROS (Alam et al., 2013). This oxidative stress on myocardium
may lead to the development of MI (Yun et al., 2013). The free radicals and ROS damage the
cell membrane and consequently attributed to the structural and biochemical alterations which
ultimately lead to cell death (Tappia et al., 2001; Prabha et al., 2014).
Free radical scavenging activity
Fig. 2.2 Role of reactive oxygen species and its prevention by Natural antioxidants
The ROS can adversely damage tissues by reacting with polyunsaturated fatty acids,
nucleotides and critical sulfhydryl bonds (Kaja et al., 2014). Antioxidants delay the oxidation
process by inhibiting the initiation of series of oxidizing reactions (Ayesha et al., 2013). Owing
to the presence of antioxidants, medicinal plants have emerged as substantial therapeutic agents
to cure various diseases like, cardiovascular diseases, cancer and diabetes (Biapa et al., 2007).
Most phenolic compounds are usually present in medicinal plants have been reported as free
radical scavengers and good therapeutic agent (Zhang and Zuo 2006; Mradu et al., 2012). Thus
25
Hypercholestrolemia, Diabetes Mellitus, Hypertension
Free radicals
Altered vasomotionVascular smooth muscle growth
Adhesion molecule expression
Lipid oxidation Apoptosis
Cardiovascular diseases
Natural antioxidants (Curcuma longa, Crataegus oxyacantha, Terminalia arjuna, Coriandrum sativum, Trigonella foenum)
to understand the therapeutic potential of medicinal plants, it is necessary to study the existence
of phytoconstituents of herbal medicines (Wang and Zuo 2011).
The medicinal plants are nature’s gift to have disease free healthy life (Begum 2009;
Gomathi et al., 2014). Therefore, the scintists and researchers are taking interest in the
assessment of prophylactic and therapeutic effects of medicinal plants in order to decrease CVD
as they are economical, effective and harmless (Ramadoss et al., 2012; Berman, 2000). More
than 100,000 of the active compounds have been found in medicinal plants (Souravi and
Rajasekharan 2014). The Phytoconstituents being part of medicinal plants are the natural
bioactive compounds, and found to be the integral part of defense system against various
diseases and stress conditions (Mathangi and Prabhakaran 2013; Nonita and Mylene 2010).
Experimental and epidemiological studies also proved that there is an inverse relation between
intake of phytoconstituents and progression of diseases (Devasagayam et al., 2004). Human
health could be maintained by consumption of medicinal plants (Lichtenstein et al., 2006;
Halliwell, 2012; Othman et al., 2014) because a number of plant-derived phenolic compounds
having antioxidant potential are able to challenge oxidative stress in hman body (Halliwell, 2012;
Othman et al., 2014).
Nature designs the metabolism of our body to oppose the excess of free radicals by the
formation of endogenous antioxidants. Their effectiveness is increased by endogenous free
radical scavenging enzymes and vitamins. The redox stress triggers the activation of immune
cells which release proinflammatory cytokines, reactive oxygen and nitrogen species that lead to
damage of biological molecules and inducing imbalance in physiological and pathological
pathways (Lonkar and Dedon, 2011; Babu et al., 2013). Any alteration in the balance of
oxidative metabolites and antioxidant scavangers, may lead to many disorders (Rashed, 2014).
For these reasons, information on the antioxidant properties of natural product is becoming
relavant to the nutrition and nutraceutical fields. Owing to its complexity, the single method is
unable to provide comprehensive picture of the antioxidant profile therefore, Multimethod
approach is required to assess the antioxidant activity (Rivas-Arreola et al., 2010).
In 1985 Farnsworth et al., identified 119 secondary metabolites which were used as
therapeutic agents. The World Health Organization recommended that out of 225 basic and
essential drugs 11% are obtained from natural precursors. These Phytochemicals are known to
possess antibacterial (Nair et al., 2005), antifungal (Khan and Wassilew, 1987), antioxidant
26
(Wong et al., 2009), antidiabetic (Singh and Gupta, 2007; Kumar et al., 2008), anti-inflammatory
(Kumar et al., 2008) and radio-protective activity (Jagetia et al., 2005). Owing to these
properties the medicinal plants are commonly used for therapeutic purpose.
2.4: Medicinal plants:
According to the WHO the medicinal plants could be used for various therapeutic
purposes due to presence of active phytoconstituents (Brussels, 2001; Kumari et al., 2011).
Pakistan is rich in medicinal herbs because of its varied climate and wide distribution over a
large area. About 600 plant species are identified as medicinal plants having potent therapeutics
(Husain et al., 2008). In Pakistan medicinal plants are mostly used in crude form by hakims.
Thus it should be processed for the extraction of various active constituents by pharmaceutical
industries and researchers (Mahmood et al., 2003; Husain et al., 2008).
More than 2000 plants being a part of Traditional systems of medicine are used to treat
the people suffering from cardiovascular diseases (Arya and Gupta 2011; Rajalakshmy et al.,
2011). About 300 species of medicinal plants are used worldwide in the pharmaceutical
industries (Deshmukh et al., 2012; Harisaranraj et al., 2009) because of the presence of many
bioactive substances which are used for treatment and management of various diseases (Hu et
al., 2008; Harisaranraj et al., 2009; Mitta et al., 2012).
2.5: A promising approach for natural therapy:
A variety of compounds in traditionally used medicinal plants having therapeutic
properties are now the focous of attention for many researchers (Reddy et al., 2010). Many plant
products are rich in polyphenolics which are different in chemical structure, characteristics and
widely recognized as naturally occurring antioxidants (Rajadurai and Prince 2007). The active
compounds in their natural formulations are more potant, as they contain both dotes and
antidotes (Khopde et al., 2001; Krushna et al., 2012). Moreover the side effects of synthetic
medicines motivated the researchers to explore the therapeutic potentials of medicinal plants to
treat myocardial infarction. Being a natural source the plant extracts have been used for
medicinal purposes without any side effects. The prophylactic and therapeutic effects of many
plants in reducing isoprenaline induced cardiotoxicity have been discussed (Prashee et al., 2008;
Panda and Naik 2008). It also revealed that the antioxidants present in medicinal plants not only
suppress the formation of ROS but also have a modulatory effect (Merzenich et al., 2009). That’s
why herbal medicine is increasingly attaining appreciation from medical professionals due to
27
advancement in understanding of the mechanisms by which herbs positively influence health and
quality of life (Panda and Naik 2008).
2.6: Medicinal Plants with Cardioprotective Properties:
Many medicinal plants have been found to possess beneficial cardioprotective effects
(Siddiq et al., 2012; Beaulah et al., 2014). Ayurveda has identified many plants which possess
cardiotonic and cardioprotective effects. Some of them are Allium sativum, Allium cepa,
Asparagus racemosus, Caesalpinia bonducella, Cassia fistula, Curcuma longa, Emblica
officinalis, Garcinia indica, Hemidesmus indicus, Ocimum sanctum, Phyllanthus amarus,
Terminalia arjuna, Trigonella foenum-graecum, Vitis vinifera, Withania somnifera and Zingiber
officinalis (Tilak-Jain and Devasagayam 2006; Niero et al., 2010; Silva and Fernandes 2010;
Rajalakshmy et al., 2011; Hassan, 2012, Alaribe, 2008). Thus, it is usually referred that the
“extracts of plants” not the plants themselves or their parts are used for medicinal effects. These
substances have therapeutical potential; hence could be used for different ailments with respect
to human physiology (Nwachukwu et al., 2010).
In this study the sixteen medicinal plants were selected on the basis of folk literature data
showing the effectiveness and therapeutical potential against cardiovascular diseases and its
related risk factors. All these plants were screened and out of these seven medicinal plants were
chosen for further biochemical and cardioprotective potential against myocardial infarction.
Crataegus oxyacantha:
Crataegus oxyacantha, commonly known as Hawthorn, a member of Rosaceae family
(Amy 2006; Verma et al., 2007). Hawthorn berries supported for its beneficial cardiovascular
properties due to the high content of bioflavonoids (oligomeric procyanidins, vitexin, quercetin,
and hyperoside) (Mohanty et al., 2013). Hawthorn preparations have been used to treat the early
stages of congestive heart failure, hypertension and total plasma cholesterol. The in vitro analysis
demonstrated that Hawthorn is more active antioxidant than vitamins E and C (Saadatian et al.,
2014). Previous investigations have confirmed the efficacy, non toxicity and reliability of this
plant and its active ingredients (Verma et al., 2007; Ebrahimzadeh and Bahramian, 2009).
Eletaria cardamom:
Eletaria cardamom is commonly known as ‘chhoti elaichi’ and is also rightly known as
the ‘Queen of Spices’. Fruits and seeds of the Cardamom are economically important parts of the
plant and also act as antiseptic, carminative, anti-spasmodic and diuretic (Kumari and Nirmala,
28
2015). Cardamom is effective as an antioxidant and could increase the levels of glutathione, a
natural antioxidant in body (Amma et al., 2010). It also acts as remedy in case of digestive
problems, urinary complaints, asthma, bronchitis and several other human ailments (Kaushik et
al., 2010; Ali and Shahnaz 2014; Abbas and Maliki 2011). Its valuable part, the seeds are used to
cure tumors of the uterus (Krishnamurthy, 2010) dyspepsia, nausea and even during pregnancy
(Jazila et al., 2007; Savan and Zehra 2013).
Coriandrum sativum:
Coriandrum sativum is herbaceous plant well known for its diuretic, carminative,
digestive, anthelmintic, antioxidative, hepatoprotective and antibacterial activities (Pandey et al.,
2011). C. sativum has been used as an antifungal, antioxidant, hypolipidemic, antimicrobial,
hypocholesterolemic and anticonvulsant substance (Joshi et al., 2014). The major compounds
present in essential oil are 𝛼-pinene, 𝛾-terpinene, geranyl acetate and geraniol (Nadeem et al.,
2013). C. sativum showed protection against the deleterious effects in lipid metabolism (Chithra
and Leelamma, 1999). C. sativum have strong antioxidant potential which might be responsible
for its therapeutic potential (Kousar et al., 2011).
Terminalia arjuna:
Terminalia arjuna belongs to family Combretaceae, is a large and evergreen tree
commonly known as Arjuna (Rameshkumar et al., 2014). It is traditionally claimed that the bark
of T. arjuna is mostly used for medicinal purposes. The cardioprotective potential of T. arjuna
bark against isoproterenol induced myocardial injured supported this traditional claim (Jahan et
al., 2012; Sivakumar and Rajeshkumar, 2014). Phytochemicals including tannins, triterpenoids,
saponins, arjunic acid, arjunolic acid and arjungenin in the bark of T. arjuna, are responsible for
its medicinal properties (Manna et al., 2007). The bark also helps in maintaining the cholesterol
level near to normal due to its antioxidant potential. It also showed significant antidiabetic,
cardioprotective and antimicrobial activities (Jahan et al., 2011a; Ramya et al., 2008; Jahan et
al., 2011b; Jahan et al., 2012). Two new cardenolide cardiac glycosides were extracted from the
root and seed of T. arjuna. The mechanism of action of cardenolides is to increase the
intracellular sodium and calcium and increasing force of myocardial contractility (Amol et al.,
2014).
29
Rauvolfia serpentina:
Rauvolfia serpentina belongs to the family Apocynaceae is an evergreen, woody and
perennial shrub having tuberous roots (Deshmukh et al., 2012; Singh et al., 2009). The common
name of plant is Sarpagandha (Mallick et al., 2012). The metabolites present mainly in the
leaves, roots and rhizomes of R. serpentina are taking attention of practitioners due to its
medicinal importance (Poonam et al., 2013; Mittal et al., 2012). R. serpentina is considered an
important medicinal herb in the pharmaceutical world because of numerous alkaloids in its roots.
The roots have been reported to treat cardiovascular diseases, hypertension, arrhythmia
(Kirillova et al., 2001; Kumari et al., 2013) and human leukemia (Dey et al., 2010). Reserpine is
the main alkaloid that shows complex pattern of activity in brain and act as anti-diuretic (Rani et
al., 2014; Mittal et al., 2012; Kumari et al., 2013). Reserpine, the active ingredient of R.
serpentina is responsible for antihypertensive potential due to its action on central nervous
system (Kumari et al., 2013). Researches also showed that the root of R. serpentina has anti
hypercholesterolemic effects with no side effects (Qureshi and Shamsa 2009).
Piper nigrum:
Piper nigrum (Black pepper) belongs to the family Piperaceae, known as ‘King of
Spices’ (Srinivasan, 2007). The Piperine, an alkaloid of P. nigrum improves the bioavailability
of a variety of structurally and functionally diverse drugs (Khajuria et al., 2002; Duangjai et al.,
2013). It also has small amounts of various bioactive compounds (Hussain et al., 2011) which
may be responsible for its high antioxidant and free radical scavenging property (Nahak and
Sahu 2011). The methanolic extract of P. nigrum showed a considerable protection against
chemically induced cardiotoxicity due to presence of various antioxidantspresent in it (Aruna et
al., 2014; Wakade et al., 2008). During an experimental high fat diet along with black pepper
were given to rats which showed the elevated level of HDL-c, reduced the LDL-c and VLDL-c
levels in the plasma as compared to rats treated merely with high fat feed (Vijayakumar et al.,
2002). Black pepper, being rich in vanadium, is responsible for its cardioprotective potential
during myocardial infarction and hypertrophy (Shenuarin and Fukunaga, 2009).
Allium sativum:
Allium sativum is present extensively in all over the world and used as a popular
medication for treatment of a various diseases like dermatitis, rheumatism, abdominal disorders
and diabetes mellitus. In many of the experimental studies Garlic has been most encouraging as a
30
complementary therapy for hypertension and cardiovascular diseases (Capraz et al., 2006). The
vasoactive ability of garlic was also confirmed by in vitro study, whereby red blood cells convert
garlic organic polysulfides into endogenous cardioprotective signaling molecule (Papu et al.,
2012). The two major risk factors including high blood pressure and high blood serum
cholesterol levels that may lead to heart disease and the therapeutic action of garlic directly
reduced the impact of these risks. In India, 432 patients suffering from coronary artery disease
were grouped and half of them were supplied with garlic juice in milk depicted curative
therapeutic potential as compared to other groups that were not supplied with garlic juice (Papu
et al., 2012). Hence the literature presented the garlic a bioactive agent for prevention and
treatment of cardiovascular and other metabolic diseases (Khan et al., 2008). Moreover, garlic
also responsible for antioxidant potential against MI in rats (Asdaq and Inamdar, 2010; Anoush
et al., 2009).
31
CHAPTER # 3 MATERIALS AND METHODSThe research was planned to explore the Biochemical profiling and cardioprotective
potential of medicinal plants in various combinations. The major part of the research was
conducted in the Clinico-Medical Biochemistry Lab (CMBL), Department of Biochemistry,
University of Agriculture, Faisalabad (U.A.F). The surgical procedure of the experimental
animal was conducted in the Department of Clinical Medicine and Surgery, U.A.F. A part of
research was also accomplished in National Institute of Biotechnology and Genetic Engineering
(NIBGE) Faisalabad. The sixteen medicinal plants were selected on the basis of information
available in the literature showing the cardioprotective potential and also able to minimize the
risk factors of myocardial infarction. This research was an effort to develop an herbal
combination possessing good cardioprotective potential but with less or no side effect. For this
purpose the research work was divided in two sections, in vitro and in vivo analysis. The in vitro
analysis involved the screening of medicinal plants for their Angiotensin Converting Enzyme
(ACE) inhibition potential. The plants which exhibited the good ACE inhibition potential were
selected for further biochemical profiling including, LC-MS analysis, antioxidant and
toxicological evaluation. Second part of the research was comprised of in vivo studies and
subdivided into three phases. In phase-I, the dose of salbutamol was optimized that can induce
myocardial infarction. Phase-II consisted of the series of experiments in which the dose of
cardioprotective medicinal plants were optimized. In phase-III, different herbal combinations of
studied medicinal plants were formulated against surgically induced MI to get the best
formulation having cardioprotective potential and also able to restrain the risk factors related to
MI.
3.1 Collection of Medicinal plants:
Different parts of medicinal plants were collected from Botanical Garden of University of
Agriculture, Faisalabad and from the local herbal market. All the plants’ parts were identified by
the Plant Taxonomist in the Department of Botany, University of Agriculture, Faisalabad,
Pakistan. The parts of the plants, selected for research work are listed in Table. 3.1. These parts
of the plants were washed and allowed to dry under shadow at room temperature and ground and
sieved to get fine powder.
32
Table. 3.1 Selected parts of plants for evaluation of cardioprotective potential
Parts used Plants
Seeds Trachyspermum ammi, Coriandrum sativum, Foeniculum vulgarea, Bunium
bulbocastanum, Eletaria cardamom and Ocimum sanctum
Roots Rauvolfia serpentina, Cichorium intybus and Curcuma longa
Leaves Piper nigrum, Aloe vera and Lepidium sativum
Fruits Allium sativum, Crataegus oxyacantha and Terminalia chebula
Bark Terminalia arjuna
3.1.1 Preparation of Herbal Extract:
The powdered plants (5 g of each) were macerated in methanol (50 mL). The macerate
was kept in orbital shaker for four days. The supernatant was poured and the residue was
remacerated with methanol. The pooled supernatants were combined and filtered with
Whatman’s filter paper No. 1. The filtrate was concentrated by rotary evaporator under reduced
pressure and dried by using lyophilizer (Jahan et al., 2012).
Section-I (In vitro analysis)3.2 Screening of medicinal plants:
The methanolic extracts of selected medicinal plants were screened by using Angiotensin
Converting Enzyme inhibition assay. Although various in vitro methods are available for
evaluation of ACE inhibition activity but the most elaborated method established by Cushman
and Cheung, 1971 (Gao et al., 2010; Rinayanti et al., 2013) was followed.
3.2.1 ACE inhibition assay:3.2.1.1 Principle:
The Hippuryl L-Histidyl Leucine (HHL) is hydrolysed by Angiotensin Converting
Enzyme. The Hippuric acid formed in the reaction is estimated by measuring the absorbance at
228 nm. The difference between absorbance in the absence and presence of inhibitor is
proportional to the inhibitory activity of tested sample.
3.2.1.2 Reagents: The main reagents used in the ACE inhibition assay included, 1) Angiotensin converting
enzyme (EC 3.4.15.1) that was extracted from rabbit’s lungs, 2) Hippuryl Histidyl Leucine (Hip
His Leu) and Captopril purchased from Sigma Aladrich (U.S.A).
3.2.1.3 Angiotensin converting enzyme extraction:
33
Angiotensin converting enzyme (ACE) was extracted from rabbit’s lungs powder. For the
formation of rabbit’s lungs acetone powder, the lungs were obtained from freshly slaughtered
rabbits and washed with 0.8% saline solution. The lungs were homogenized with phosphate
saline buffer by tissue homogenizer and filtered through cheese cloth. More volume of buffer
was added and centrifuged at 4000 rpm for 10 minutes. The residues were washed several times
with acetone to complete dehydration. The acetone dip residues were placed overnight for
evaporation. The dry rabbit’s lungs acetone powder was ground to get fine powder and preserved
in polythene bag at 4oC (Nemerson, 1969; Luna et al., 2009). The dry lung acetone powder was
used for crude extraction of ACE. One gram of this lung powder was mixed in 10 mL of 100
mM phosphate buffer (pH 8.3). It was stirred on magnetic stirrer overnight and centrifuged at
4000 rpm for 45 minutes. The supernatant was dialyzed against 100 mM phosphate buffer of pH
8.3 by using dialyzing membrane of 12 KD cutoffs and lyophilized to get ACE extract.
3.2.1.4 Preparation of Captopril solution:
Captopril (25 mg) was grounded and extracted with distilled water (25 mL) in ultrasonic bath
for 10 minutes. The obtained extract was filtered by using filter paper (0.45 μm pore size).
Captopril solution (1 mg/mL) was used as the positive control (Duncan et al., 1999; Donath-
Nagy et al., 2011).
3.2.1.5 Procedure:
ACE solution (50 μL) and 50 μL of borate buffer were incubated at 37°C for 10 min. The
reaction mixture was incubated for 80 min at 37°C after addition of 8.3 mM Hip His Leu (150
μL). After that 250 μL of 1 M HCl was added to terminate the reaction. The hippuric acid
formed in this reaction was extracted by 1500 μL of ethyl acetate and centrifuged at 800 rpm for
15 min. The upper layer (750 μL) was poured into test tube and dried under laminar air flow at
37°C. At the end 1 mL of distilled water was used to dissolve hippuric acid and the absorbance
was measured at 228 nm by UV/Vis spectrophotometer (Cintra 303, GBC Scientific Equipment,
Australia). The reaction blank was also prepared by following the same process except the HCl
was added before the enzyme (Belovic et al., 2013). Extracts of plants and captoprill standards
were prepared by replacing the same quantity of buffer with samples. The sample blank was
prepared as the reaction blank, by replacing buffer to the tested sample. The ACE inhibition was
calculated by given formula.
% IACE = 100[ (A-B) – (C-D)]
34
(A-B)Where “A” depicts the absorbance in the presence of ACE, “B” represents the absorbance
of the reaction blank, “C” is the absorbance in the presence of ACE and inhibitor and “D”
indicates the absorbance of the sample blank. (HernandezLedesma et al., 2011; Cushman and
Cheung, 1971).
3.3 Biochemical profiling:
Biochemical profiling through LC-MS analysis, antioxidant assay and toxicological
evaluation of screened medicinal plants, possessing high ACE % age inhibition, was performed.
3.3.1 Liquid chromatography mass spectrometry (LC-MS):
These medicinal plants were analyzed by using Liquid Chromatography combined with
Electron Spray Ionization Mass Spectrometry (LC-ESI-MS) from National Institute of
Biotechnology and Genetic Engineering (NIBGE) Faisalabad, Pakistan. The plant extracts
prepared in section 3.1.1 were filtered by using a 0.45 µm syringe filter before analysis. Surveyor
plus HPLC System equipped with Surveyor auto (Thermo Scientific, San Jose, CA, USA) were
used for separation. A Luna Reverse Phase C-18 analytical column (4.6×150 mm, 3.0 µm
particle size) (Phenomenex, USA) was attached with pump. LC-MS grade methanol and
acidified water (0.5 % formic acid v/v) were used as the mobile phase A and B respectively.
Solvent elution consisted of gradient system run at a flow rate of 0.3 mL/min. The gradient
elution was programmed as follows. A 20 minutes re-equilibration time was used after each
analysis. The column was maintained at 25ºC and the injection volume was 5.0 µL. The effluent
from the HPLC column was directed to electron spray ionization mass spectrometer (LTQ XLTM
linear ion trap Thermo Scientific River Oaks Parkway, USA). Negative ion mode with spectra
posessing mass range from m/z 260 to 2000 was used for analysis of parameters (Adom and Liu,
2002). The accurate mass spectra data of the molecular ions was processed through X-caliber
software (Thermo Fisher Scientific Inc, Waltham, MA, USA) (Jiao and Zuo, 2009; Zuo et al.,
2002).
3.3.2 Antioxidant assay:
In order to evaluate the antioxidant potential of methanolic extracts of plants, the “1, 1-
Diphenyl-2-Picrylhydrazyl (DPPH) free radical scavenging assay" and “DNA protection assay”
were used.
3.3.2.1 1, 1, Diphenyl-2-Picrylhydrazyl (DPPH) free radical scavenging assay:
35
DPPH free radical scavenging assay was used to explore the antioxidant potential of
extracts of selected plants (Sahu et al., 2013). The extracts of secreened plants, prepared in
section 3.1.1 were further used to perform the DPPH free radical scavenging assay.
3.3.2.1.1 Procedure:
The antioxidant activity was resoluted by using DPPH as a free radical. Stock solutions
(10 mg/mL) of extracts of plants were prepared in methanol. Different concentrations (20, 40,
60, 80 and 100 µg/mL) of extracts of selected plants and methanolic solution of DPPH (0.1 mM)
were mixed in equal volume. The mixture was kept for 30 minutes in dark and the absorbance
was noted at 517 nm against a blank solution. Ascorbic acid acts as a standard. The DPPH
inhibition (% ) was measured by using given formula.
DPPH Inhibition (%) = [1 - A1/A0] x 100
Where “A1” is the absorbance of sample and “A0” is the absorbance of control (Yadav et al.,
2014; Ayesha et al., 2013).
3.3.2.2 DNA protection assay:
The antioxidant activity was also confirmed by DNA protection assay following the
method given by Riaz et al, (2012). pBR 322 DNA plasmid (0.5 µL) was diluted by using
sodium phosphate buffer (50 mM, pH 7.4). The diluted pBR 322 DNA (3 µL) was treated with 5
µL of different concentrations (100, 500 and 1000 µL) of extracts of all the plants. After that 4
µL of 30% H2O2 was added to make the volume up to 15 µL with sodium phosphate buffer (pH
7.4). The comparative variation in the migration between the native and oxidized DNA was
determined on 1% agarose by horizontal DNA gel electrophoresis using a wide mini system
(Techview, Singapore). 1% agarose was ready by mixing 1g agarose in 100 mL of 1X×TAE
buffer and placed it in microwave oven for two minutes. It was cooled and poured in casting
plate. After solidification, gel was kept in the sodium phosphate buffer and samples were loaded
in the wells one by one. The gel was stained with ethidium bromide and documented by Syngene
model Gene Genius unit (Syngene, Cambridge, UK).
3.3.3 Toxicity assay:
The toxicological evaluation of some of the screened medicinal plants was performed
through “Hemolytic activity” and “Mutagenicity assay”.
3.3.3.1 Hemolytic activity:
36
The haemolytic activity, a useful starting point for toxicological evaluation, provides the
primary information about the interaction between bioactive compounds of medicinal plants and
biological entities (Da Silva et al., 2004).
3.3.3.1.1 Preparation of erythrocytes suspension:Five milliliter of blood was withdrawn from individuals having good health and
centrifuged at 1500 rpm for three minutes. Plasma (supernatant) was discarded and the washing
of pellet was performed thrice with PBS (pH 7.2±0.2) by centrifugation at 1500 rpm for 5 min.
The cells were resuspended in normal saline and maintained the cell count at 108 cell/mL (Kumar
et al., 2011).
3.3.3.1.2 Procedure:Cell suspension (0.5 mL) was mixed with 0.5 mL various concentrations of extracts of
said plants (100, 500 and 1000 μg/mL in PBS). The incubation of mixture was done for 30 min
at 37°C in an incubator (Sanyo, MIR-254, Japan) and centrifuged at 1500 rpm for 10 min in a
laboratory centrifuge (22331 Hamburg). The free haemoglobin in the supernatant was measured
in UV-Vis spectrophotometer (Dynamica, Halo BD-20, UV-Vis spectrophotometer, Australia) at
540 nm. Phosphate buffer saline and Triton-X were used as negative and positive hemolytic
control respectively. The hemolysis in term of percentage by the plants extracts was quantified
by using the given formula,
% Hemolysis = At - An
Ac
Where “At” is the absorbance of test sample, “An” showed the absorbance of the saline
control and “Ac” is the absorbance of the water control (Kumar et al., 2011).
3.3.3.2 Mutagenicity assay:
The mutagenicity test was performed by using “Ames bacterial reverse mutation assay”.
3.3.3.2.1 Test bacterial strain:
The mutant strain S. typhimurium TA98 was maintained on nutrient agar and incubated at
37˚C for 18-24 hr prior to the test (Razak et al., 2007).
3.3.3.2.2 Preparation of reagent mixture:
Reagent mixture was prepared by mixing Devis Mingoili salt (21.62 mL), D-Glucose
(4.75 mL), Bromocresol purple (2.38 mL), D-Biotin (1.19 mL) and L-Histidine (0.06 mL)
37
aseptically in a sterile bottle. The mutagen sodium azide (0.5 µg/100mL) was used for S.
typhimurium TA 98.
3.3.3.2.3 Herbal extraction:
The extracts of all the selected plants prepared in section 3.1.1 were reconstituted in
Dimethyl Sulfoxide (DMSO) to form 10, 000 µg/mL stock solution.
3.3.3.2.4 Procedure:
Herbal extracts, reagent mixture, sterile distilled water and standard mutagen were mixed
in bottles with the quantity indicated in Table. 3.2 and inoculated with homogenous culture broth
of S. typhimurium. The contents of each bottle were dispensed into each well of a 96 well micro
titration plate and plates were incubated at 37˚C for 4 days.
3.3.3.2.5 Interpretation of result:
The yellow wells were considered as positive and the purple wells were considered as
negative. The herbal extract was said to be mutagenic if the positive well are prominently higher
in number as compared to the positive well in the background plate.
Table. 3.2 The protocol for mutagenicity assay
Treatment Volume (mL)Mutagen standard
Extract
Reagent mixture
Deionized water
S. typhimurium
Blank - - 2.5 17.5 -Background
- - 2.5 17.5 0.005
Standard mutagen
0.1 - 2.5 17.4 0.005
Test samples
- 0.005 2.5 17.5 0.005
3.4 Section-II (In vivo study)
3.4.1 Selection of animals:
The experimental animals including rats and dogs were housed in cages and acclimatized
for one week under laboratory conditions (27°C in 12 hr dark/light cycle). They were fed with
standard feed and water. The husk in the cages of rats was changed thrice a week to ensure
hygienist. All the animals were kept in Animal House, Department of Clinical Medicine and
Surgery, University of Agriculture, Faisalabad.
38
3.4.2 Phase-I: Dose optimization of salbutamol:The rats were orally administered with varying concentrations of salbutamol as suggested
by “Central Composite Design” of Response Surface Methodology (RSM-CCD) for two
consecutive days. Furthermore different time intervals from 0 to 116 hr, for blood sampling were
also proposed by RSM-CCD (Table. 3.3). All the blood samples were centrifuged to separate the
serum for the analysis of cardiac markers (CK-MB, SGOT and LDH). The procedures for the
estimation of cardiac markers have been given in section 3.4.6. The response of cardiac markers
was analyzed by the same statistical tool “RSM” to get the optimal dose of salbutamol at which
it could induce MI.
Table. 3.3 Experimental design suggested by Response Surface Methodology to optimize the dose of Salbutamol
Sr. No. Time of blood sampling (hr)X1
Conc. of salbutamol (mg/kg)
X2
1 0 502 96 503 0 1254 96 1255 0 886 116 887 48 348 48 1419 48 8810 48 8811 48 8812 48 8813 48 88
3.4.3 Phase-II: Dose response experiment: A pilot study:
In the dose response experiment the rats were randomly divided into following groups.
The rats in control group were fed with normal diet throughout the experimental period of 23
days. In positive control group the rats were treated with normal diet for 21 days and after that
the optimized dose of salbutamol in phase-I was given orally twice at an interval of 24 hr.
Moreover seven treatment groups were run and each treatment group was pretreated with
different concentrations of its respective plant as suggested by “Central Composite Design”
39
(Table. 3.4), once daily for three weeks. After that the optimized dose of salbutamol was
administered for two consecutive days. The blood sampling was performed after 24 hr of
administration of salbutamol. The cardiac biomarkers (CK-MB, SGOT and LDH) as well as lipid
profile (HDL-c, LDL-c, TC and TG) were analysed to assess the cardioprotective worth of
selected medicinal plants at various concentrations. At the end of experiment the rats were
sacrificed and their hearts were excised for the estimation of antioxidants (SOD, GPX and CAT).
The protocols of all these biomarkers have been given under the section 3.4.6.
40
Table. 3.4 The Central Composite Design for the treatment of selected medicinal plants
Grps. Plants Conc. (mg/kg)
Grps. Plants Conc. (mg/kg)
Grps. Plants Conc. (mg/kg)
G1 T. arjuna 80 P. nigrum 110 A. sativum 200 T. arjuna 80 P. nigrum 170 A. sativum 200 T. arjuna 110 P. nigrum 200 A. sativum 140 T. arjuna 170 P. nigrum 200 G6 R. serpentina 80 T. arjuna 200 P. nigrum 140 R. serpentina 80 T. arjuna 200 G4 C. sativum 80 R. serpentina 110 T. arjuna 140 C. sativum 80 R. serpentina 170
G2 C. oxyacantha 80 C. sativum 110 R. serpentina 200
C. oxyacantha 80 C. sativum 170 R. serpentina 200
C. oxyacantha 110 C. sativum 200 R. serpentina 140 C. oxyacantha 170
C. sativum 200 G7 E. cardamom 80 C. oxyacantha 200 C. sativum 140
E. cardamom 80 C. oxyacantha 200 G5 A. sativum 80 E. cardamom 110
C. oxyacantha 140 A. sativum 80 E. cardamom 170 G3 P. nigrum 80 A. sativum 110 E. cardamom 200
P. nigrum 80 A. sativum 170 E. cardamom 200 E. cardamom 140
3.4.4 Phase-III (Herbal combination therapy):In phase-III the synergestic cardioprotective potential of selected medicinal plants was
observed. For this purpose the dogs were selected as experimental animals and myocardial
infarction was induced surgically. The dogs were divided into three groups. The first group of
dogs was the control group, to which normal diet was fed for 23 days. The second group was
surgically induced MI control group, in which the dogs were treated with normal diet for 22 days
and after that the ligation of left anterior descending coronary artery was performed on 23rd day.
The third group was treatment group which was further divided into four subgroups. Each
subgroup was pretreated with its respective herbal combination (Table. 3.5) for 22 days. On day
23 all the dogs of treatment group underwent left anterior descending coronary artery ligation.
After completion of surgical procedure the blood samples were taken at various time intervals (0
41
to 48 hr) to analyze the biomarkers (CK-MB, SGOT and LDH). At the end of the experiment the
dogs were sacrificed by an overdose of anesthesia and hearts were excised for histopathological
studies. The procedures and principles of the said biomarkers are given in section 3.4.6.
Table. 3.5 Formation of different herbal combinations
Groups R.serpentin
a
E.cardamo
m
P.nigru
m
A.sativu
m
T.arjun
a
C.oxyacanth
a
C.
sativum
Herbal ratio
HC1 1 0.5 1 0.5 - - 0.5
HC2 1 0.5 1 0.25 - 1 0.5
HC3 1 1 0.5 - 1 - 0.5
HC4 0.5 - - 0.5 1 0.5 1
3.4.5 Surgical induction of myocardial infarction:The dogs were sedated with pentabarbitone sodium (60 mg/kg) and additional 4mg/kg
were given when required. To keep the heart rate elevated during surgical procedure and to
reduce the bronchotracheal secretions atropine was given subcutaneously at a dose of 0.1 mg/kg
once before surgery. The animals were ventilated with room air from a positive pressure by using
compressed air at the rate of 90 stroke/min and tidal volume of 10 mL/kg. Neck was opened and
left tracheotomy was performed to open the thoracic cavity. Anatomy of Left Anterior
Descending (LAD) coronary artery was ligated 4-5 mm and end of this ligature was passed
through polyethylene tube to form snare. At the end of surgery the heart was returned to its
normal position in thoracic cavity (Ojha et al., 2010; Ojha et al., 2012).
3.4.5.1 Estimation of hemodynamic variables:
The mean arterial pressure (MAP) and heart rate of dogs in all the groups was calculated.
The left thoracic cavity was opened by an incision at the fifth intercostal space and the heart was
exposed. A metal cannula was introduced in cavity of left ventrical from the posterior epical
region of heart for measuring left ventricular dynamics throughout the surgical procedure
(Nandave et al., 2013; Ojha et al., 2010; Ojha et al., 2012).
3.4.6 Biochemical analysis:
The principles and protocols of all the studied biochemical parameters have given under
the following headings.
3.4.6.1 Creatine Kinase MB (CK-MB):
42
3. 4.6.1.1 Reagent preparation:
The reaction mixture was organized by adding four parts (400 µL) of reagent 1 (R1) and
one part (100 µL) of reagent 2 (R2). In the reaction mixture 50 µL serum samples were added
and the absorbance was measured at 340 nm after 5 minutes.
3.4.6.2 Serum Glutamate Oxaloacetate Transaminase (SGOT):
Serum Glutamate Oxaloacetate Transaminase (SGOT) was determined through kinetic
method.
3.4.6.2.1 Principle:
The kinetic method was optimized for determination of aspartate amino transferase
without perodoxal phosphate in accordance with International Federation of Clinical Chemistry
(I.F.C.C) recommendation. Aspartate aminotransferase (AST/GOT) catalyzed α- Ketoglutarate
and L-Aspartat by the coupled reaction of malate dehydrogenase and the relative coenzyme
(NADH), oxaloacetate is reduced to malate with the co-enzyme oxidation.
3.4.6.2.2 Reagent preparation:
Reaction mixture was prepared by mixing 9 parts (450 µL) of reagent 1 (R1) and 1 part
(50 µL) of reagent 2 (R2). In the reaction mixture 50 µL serum samples were added and the
absorbance was measured at 340 nm after 120 seconds.
3.4.6.3 Lactate Dehydrogenase (LDH):
3. 4.6.3.1 Principle:
Lactate Dehydrogenase was measured in accordance with Deutsche Gesell schaft for
Klinische Chemie (DGKC) recommendation. The oxidation rate of NADH is proportional to the
LDH catalytic activity. The LDH activity of the sample was calculated by taking the absorbance
at 340 nm.
3. 4.6.3.2 Reagent preparation:
Reaction mixture was prepared by mixing 4 parts (400 µL) of reagent 1 (R1) and 1 part
(100 µL) of reagent 2 (R2). In the reaction mixture 50 µL serum samples were added and the
absorbance was taken at 340 nm after 30 seconds.
3. 4.6.4 Cholesterol:3. 4.6.4.1 Principle:
43
Cholesterol esterase oxidized the cholesterol ester releasing hydrogen peroxide.
Hydrogen peroxide reacted with a phenol substitute and 4-aminopyrine. The intensity of red
compound formed is directly proportional to the total cholesterol in the sample.
3. 4.6.4.2 Reagent preparation:
The reaction mixture contained reagent 2 (1000 µL), standard solution (1000 µL) and
sample (1000 µL). In the reaction mixture 50 µL serum samples were added and the absorbance
was measured.
3. 4.6.4.3 Calculation:
Cholesterol (mg/dL) = (A) Sample×200 (A) Standard
3.4.6.5 Triglycerides:
3.4.6.5.1 Principle:
The quantitative determination of Triglycerides was done by measuring the intensity of
red color produced. It is directly proportional to triglycerides in the sample.
3.4.6.5.2 Reagent preparation: The reagent R1 and R2 were mixed with 10:1 ratio respectively and 10 µL samples was mixed and incubated for 5 min at 37˚C and the absorbance was taken.
3.4.6.5.3 Calculation:
Triglycerides (mg/dL) = (A) Sample×200 Standard
3.4.6.6 High density lipoprotein (HDL):
3.4.6.6.1 Principle:
HDL-Cholesterol is obtained during the preparation of LDL and VLDL, where the HDL
remained in solution. At the end the color formation was observed and absorbance was measured
at 546 nm that was proportional to HDL cholesterol concentration in sample.
3.4.6.6.2 Procedure:
Reaction mixture was made by mixing 1.0 mL BioMed Cholesterol reagent and sample.
The mixture was then incubated for 5 min at 37˚C and absorbance was measured.
3.4.6.7 Low density lipoprotein (LDL):
3.4.6.7.1 Principle:
44
High density lipoprotein and very low density lipoprotein remained in the supernatant,
the LDL is precipitated by addition of Heparine and after centrifugation it is measured
enzymatically. The concentration of LDL cholesterol is calculated by the difference of total
cholesterol and cholesterol in the supernatant.
3.4.6.7.2 Procedure:
Sample (50 µL) and precipitating reagent (500 µL) were mixed at room temperature and
centrifuged for 20 minutes.
3.4.6.7.3 Calculation:Cholesterol in supernatant (mg/dL) = Sample×conc. Std. (mg/dL)
StandardThe standard concentration is the concentration of the total cholesterol in the cholesterol
standard solution.
LDL cholesterol (mg/dL) = Total cholesterol (mg/dL) - cholesterol in the supernatant
3.4.6.8 Antioxidant assay:
Antioxidant enzymes were evaluated from cardiac tissue by using following protocol.
3.4.6.8.1 Enzyme assay of superoxide dismutase (SOD):
The potential of superoxide dismutase was estimated by its ability to inhibit the photo
reduction of Nitro Blue Tetrazol (NBT) following the method of Gianopolitis and Ries (1977).
3.4.6.8.2 Required reagents for enzyme assay:
1 M EDTA solution containing 0.3 mM NaCN.
0.067 mM potassium phosphate buffer, pH 7.8.
1.5 mM NBT
0.12 mM Riboflavin.
3.4.6.8.3 Preparation of 0.067 mM Phosphate buffer, pH 7.8:
0.14 g of KH2PO4 was taken in a flask and then 0.74 g of K2HPO4 was added in it. The
volume was made up to 80 mL in flask by adding distilled water.
3.4.6.8.4 Preparation of EDTA and NaCN solution:
0.16 g of EDTA was taken in flask and then 0.08 mg NaCN was added in it. The distilled
water was added to make the volume 5.4 mL.
3.4.6.8.5 Preparation of riboflavin solution:
45
0.06 mg riboflavin was taken in flask and distilled water was added to make its volume
up to 1.3 mL and stored in cold dark bottle.
3.4.6.8.6 Preparation of Nitro Blue Tetrazol (NBT) solution:
3.23 mg NBT was taken in flask and its volume was made up to 2.64 mL with the
addition of distilled water and stored in cold dark bottle.
3.4.6.8.7 Procedure:
The buffer (1 mL) was taken in cuvette as blank and absorbance was measured at 560 nm
in spectrophotometer. After that 5-6 cuvettes were set in a light box having light bulb of 30 watt.
1 mL of buffer, 0.05 mL of enzyme extract and 0.016 mL of ribolflavin was added to each
cuvette and incubated in light box for 12 minutes. The cuvettes were moved to
spectrophotometer where EDTA/NaCN solution (0.067 mL) and NBT (0.033 mL) were added to
illuminate reaction mixture. The absorbance was measured after 20 seconds of the reaction. The
specific activity was measured in IU/mL/min/mg of protein by multiplying enzyme activity with
the protein contents (mg) of the extract.
3.4.6.8.8 Calculation:
% age inhibition = Blank-sample (Abs)×100 Blank (Abs)
Specific Activity= Enzyme Activity ×mg of protein
3.4.6.9 Glutathion peroxidase:
3.4.6.9.1 Preparation of enzyme extract:
To remove the RBCs, the disected organs were washed with 0.2 M phosphate buffer of
pH 6.5 and homogenized in blender. After that the samples were centrifuged for 15 minutes at
10,000 rpm and 4ºC. The supernatants were stored at 4ºC for enzyme assay (Civello 1995).
3.4.6.9.2 Reagents for enzyme assay:
0.2M phosphate buffer, pH 6.5
Hydrogen peroxide
Guaiacol
3.4.6.9.3 Preparation of 0.2 M phosphate buffer (pH 6.5)
4g NaH2PO4 and 1g Na2HPO4 was dissolve in distilled water. Then volume was maid up
to 200 mL.
3.4.6.9.4 Preparation of buffer substrate solution:
46
Guaiacol (750 µL) and phosphate buffer (47 mL) were mixed well. After agitation, H2O2
(0.3 mL) was added to buffer solution.
3.4.6.9.5 Procedure:
A cuvette having 3 mL of blank solution was put into spectrophotometer at wavelength of
470 nm. The 0.06 mL of enzyme extract was added to it and the absorbance was noted after 3
min. Enzyme activity was calculated and this activity was multiplied with the protein contents
(mg) of the extract to measure the specific activity (U/mL/min/mg of Protein).
3.4.6.9.6 Calculation:
Activity U/mL = ΔA/3____26.6×60/300
Specific activity= enzyme activity× mg of protein
3.4.6.10 Catalase:3.4.6.10.1 Isolation of catalase enzyme:3.4.6.10.1 Procedure:
The organs were weighed and the phosphate buffer was addedwith ratio of 1:4.
Homogenized it by using pestle and mortar.
To remove the biomass homogenized tissue was passed through muslin cloth.
The obtained liquid was filtered with Whatman’s filter paper No. 1 and centrifuged at
10,000 rpm for 15 minutes.
Both supernatants and sediments were separated and stored at 4˚C.
3.4.6.10.3 Enzyme activity:
The activity of catalase was measured by its ability of to decompose H2O2 at 240 nm
(Chance and Mehlay, 1977).
3.4.6.10.4 Reagents:
60 mM phosphate buffer (pH 7.0)
10 mM hydrogen peroxide
3.4.6.10.5 Preparation of 60 mM phosphate buffer (pH 7.0):
0.224 g of NaH2PO4 and 0.1632 g of Na2HPO4 were added in distilled water to make the
total volume up to 50 mL.
3.4.6.10.6 Preparation of buffer substrate solution:
47
Buffer substrate solution of 10mM of H2O2 was prepared in 60 mM phosphate buffer by
dissolving 0.442 H2O2 in 60 mM phosphate buffer.
3.4.6.10.7 Procedure: A cuvette having 2 mL of blank solution was kept into spectrophotometer at the
wavelength of 240 nm.
Another cuvette having buffered substrate solution was put in the spectrophotometer.
0.05 mL enzyme extract was added into buffered substrate solution.
The second cuvette was incubated for three minutes to complete the reaction.
The absorbance was measured at 240 nm.
3.4.6.10.8 Calculation:Enzyme activity was calculated then this activity was multiplied with the protein content
(mg) of the extract and specific activity was measured in U/mL/min/mg of protein.
Activity (U/mL) = ΔA/min×dilution×2mL 0.04mM-1cm-1×0.05mL
Where, ΔA Absorbance at 240 nmMin: Reaction time buffer0.04 mm-1cm-1 Standard factor0.05 mL Enzyme concentration used2 mL Amount of enzyme and substrateSpecific activity: Enzyme activity× mg of protein
3.4.6.11 Histopathology:
Reserved organs were processed for histopathaological examination (Bancfort and
Gamble) by following steps as described below.
3.4.6.11.1 Washing and dehydration:
After fixation in 10% buffered formaline 5 mm chunky pieces of tissue were washed by
tap water overnight to remove all debrisis. Slices of tissues were then dehydrated as follows.
Table. 3.6 Dehydration treatment procedure of histopathology
48
Dehydrating agents Time (hour)
Alcohol 70% 8
Absolute alcohol-1 2
Absolute alcohol-2 2
Alcohol 85% 4
Alcohol 95% 4
3.4.6.11.2 Cleaning:
The elimination of dehydrating agent and substitution with some fluid was done during
cleaning process.
Table. 3.7 Clearing treatment procedure of histopathology
Clearing agent Time (minutes)
Xylene+Alcohol (50+50) 30
Xylene-1 15
Xylene-2 15
3.4.6.11.3 Infiltration:
Infiltration was carried out in liquid paraffin at 59-60 ºC as shown below:
Table. 3.8 Infiltration treatment procedure of histopathologyInfiltrating agent Time (hrs)
Paraffin-1 2
Paraffin-2 2
Paraffin-3 2
3.4.6.11.4 Embedding:
Tissues in Steel mold were poured with wax and let it solidify at -1 to 5 ˚C in the plastic
moults. On solidification of paraffin, the steel moult was removed from the block.
3.4.6.11.5 Sectioning and Mounting:
Microtome was used for tissue sectioning.
The thickness of section was about 4-5 µm.
Tissue blocks were faced by wheel of microtome to form smooth ribbon of sections.
These sections were kept in the warm water (~50˚C).
49
Thin smeared of egg albumin was made onto glass slides and the slides were swelled by
dipping the slides under the section floating in water bath.
Slides were dried in incubator at 45-55˚C for 30 minutes to remove fragments of the
paraffin.
3.4.6.11.6 Staining:
Staining was performed by using Hematoxylin and Eosin stain (H&E stain) by adopting
protocol as given in Table. 3.9.
Table. 3.9 Detailed protocol of Hematoxylin and Eosin (H&E) staining for histopathology
Staining agents Time (minutes)Xylene-1 3Xylene-2 3Absolute Alcohol-1 3Absolute Alcohol-2 3Alcohol 70% 3Water 5Hematoxylin 5-8Acid Alcohol 1-2 dropsWater 3Ammonia Alcohol 3Water 3Alcohol 70% 3Eosin y 1-2Alcohol 70% 3Absolute Alcohol-1 3Absolute Alcohol-2 3Xylene-1 3Xylene-1 3
Afterward a drop of mixture of distyrene, a plasticizer and xylene was added on the cover slip
and placed on the stained section.
3.4.7 Statistical analysis:
The statistical analysis was done by ANOVA and Response Surface Methodlogy (Myers
et al., 2002).
CHAPTER # 4 RESULTS AND DISCUSSION
50
The research work was conducted in the Clinico-Medical Biochemistry Laboratory,
Department of Biochemistry, University of Agriculture, Faisalabad. The research work was
divided into two sections. The first included in vitro and the second was comprised of in vivo
studies. In the in vitro section, screening of some medicinal plants through ACE inhibition assay
was made. After that, the medicinal plants those showed high % age of ACE inhibition were
further subjected to biochemical profiling including LC-MS analysis, antioxidant and
toxicological evaluation. The in vivo study (section-II) was comprised of three phases. In phase-
I, the concentration of salbutamol required to induce cardiotoxicity in terms of MI, was
confirmed. The phase-II involved the evaluation of different concentrations of selected medicinal
plants for their therapeutic potential against MI. The phase-III included the assessment of
synergistic cardioprotective potential of different herbal combinations in surgically induced MI.
The findings of all the said experiments have been described under the following headings.
Section-I (In vitro study):
4.1 Screening of medicinal plants:
Untreated hypertension may leads to various complications including coronary and
peripheral heart diseases and even stroke (Chen et al., 2009; HernandezLedesma et al., 2011).
The Angiotensin Converting Enzyme (ACE) inhibitors are one of the most valuable medications
for the management of hypertension (Sharifi et al., 2013). The medicinal plants possessing ACE
inhibition potential would be the better alternative of synthetic drugs for the treatment of
hypertension, which is one of the major risk factors of MI.
4.1.1 Angiotensin converting enzyme inhibition assay:
The ACE inhibition of both methanolic and ethanolic extracts of various parts of some
selected medicinal plants has been demonstrated in Table. 4.1. The comparative ACE inhibition
(%) of methanolic and ethanolic extract of each selected medicinal plant has been presented
graphically in Fig. 4.1. The methanolic extracts of the selected medicinal plants showed
relatively higher ACE inhibition potential relative to the corresponding ethanolic extracts (Fig.
4.1). The difference in ACE inhibition potential of both types of extracts might be due to the
reason, that the ethanol masked the inhibitory activity of the examined samples (Belovic et al.,
2013). The methanolic extracts of Trachyspermum ammi (50.76±1.342), Terminalia arjuna
(56.23±3.427), Piper nigrum (63.03±0.153), Coriandrum sativum (63.033±0.153), Foeniculum
vulgarea (50.26±0.75), Allium sativum (52.9±2.621), Rauvolfia serpentina (63.467±3.198),
51
Eletaria cardamom (53.467±0.55) and Crataegus oxyacantha (64.267±0.2) showed more than
50% ACE inhibition potential (Table. 4.1). All these medicinal plants with good ACE inhibition
activity have already been reported to possess cardiotonic, diuretics and antilipidemic potential
(Tassell et al., 2010; Kousar et al., 2012; Jahan et al., 2012). Although the captoprill showed
maximum inhibition of ACE (Fig. 4.1), but the rising cost and side effects of this and other
contemporaneous antihypertensive drugs stimulate the assessment of medicinal plants as cheaper
and safer natural alternatives (Filho, 2006).
The high ACE inhibition of medicinal plants might be due to the presence of secondary
metabolites like terpenoids and polyphenolic compounds (Balasuriya and Rupasinghe 2011).
Most of the flavonoids were reported to be competitive inhibitors that can compete with the
substrate in binding to the active site of the enzyme (Balasuriya et al., 2011). The presence of
polyphenolics like procyanidin, catchein and epicatechin in T. arjuna (Goretta et al., 2006) is
responsible for its antihypertensive potential (Dwivedi and Chopra 2014). The dimers and
hexamers of the epicatechins present in T. arjuna were found to be competitive inhibitors of
angiotensin converting enzyme (Goretta et al., 2003).
Procyanidin, the active bioflavonoid of Crataegus oxyacantha was claimed to have the
vasorelaxant effect (Verma et al., 2007) due to its ACE inhibition potential (Sharifi et al., 2013).
The dimers and tetramers of procyanidins are responsible for its competitive ACE inhibition
(Ottaviani et al., 2006). The extract of C. oxyacantha can normalize the blood pressure, both
systolic (from 160 to 150 mm Hg) and diastolic (from 89 to 84 mm Hg) (Murray, 1995; Mills
and Bone 2000) in hypertensive patients in a similar fashion to synthetic ACE inhibitors
(Meschino 2014).
The apigenin, a flavon, present in A. sativum is responsible for its antihypertensive
potential (Loizzo et al., 2009; Balasuriya and Rupasinghe 2011). Daily use of Allium sativum
may keep the blood pressure normal due to presence of the Phe-Tyr (dipeptide) at its N-terminal
sequence. The peptides are responsible for ACE inhibition by chelating zinc, which being a part
of ACE is mandatory for ACE activity (Kumar et al., 2011).
Table. 4.1 Angiotensin Converting Enzyme inhibitory activity (%) of studied medicinal
plants
52
Botanical names Family name Parts used
% age ACE inhibition of
Methanolic extract
% age ACE inhibition of
Ethanolic extractCaptoprill 85.87±1.503 81.08±2.98
Trachyspermum ammi
Apiaceae Seed 50.76±1.342 44.03±1.95
Terminalia arjuna Combretaceae Bark 56.23±3.427 23.9±1.082
Piper nigrum Piperaceae Leaves 63.03±0.153 31.67±0.907
Curcuma longa Zingiberaceae Powder 31.1667±3.427 22.7±0.754
Coriandrum sativum Umbelliferae Seed 63.033±0.153 49.67±0.305
Foeniculum vulgarea Umbelliferae Seed 50.267±0.75 30±0.36
Bunium bulbocastanum
Apiaceae Seed 44.2±1.136 29.4±0.721
Allium sativum Amaryllidaceae Fruit 52.9±2.621 38.633±0.551
Rauvolfia serpentine Apocynaceae Root 63.467±3.198 56.033±0.636Eletaria cardamom Zingiberaceae Seed 53.467±0.55 39.66±0.666
Lepidium sativum Curciferae Leaves 36.767±2.914 18.3±0.794
Ocimum sanctum Lamiaceae Seed 49.167±0.838 12.933±1.001
Cichorium intybus Compositae Root 18.367±2.35 16.76±0.152
Crataegus oxyacantha Rosaceae Fruit 64.267±0.2 21.9±0.567
Terminalia chebula Combretaceae Fruit 16.466±1.32 16.43±0.61
Aloe vera Liliaceae Leaves and gel
38.1±0.954 26±2
53
Captoprill
T. ammi
T. arju
n
P. nigrum
C. longa
C. sativu
m
F. vulgarea
B. bulbocasta
num
A. sativu
m
R. serpen
tine
E. cardamom
L. sativu
m
O. sanctu
m
C. intyb
us
C. oxyc
antha.
T. cheb
ula
A. vera
0
10
20
30
40
50
60
70
80
90
100
Methanolic extractEthanolic extract
% a
ge A
CE in
hibi
tion
Fig. 4.1 Graphical presentation of angiotensin converting enzyme inhibition (%) of studied
medicinal plants
The antihypertensive property of Rauvolfia roots are attributed to reserpine due to its
inhibitory action on central and peripheral nervous system (Kumari et al., 2013). The binding of
reserpine stops the normal accumulation of catecholamine and serotonin (Nammi et al., 2005)
which is involved in maintaining the blood pressure, heart rate, cardiac contraction and
peripheral resistance (Kumari et al., 2013).
Generally ACE inhibition of extracts of medicinal plants is due to chelation of metal ion
co-factor with secondary metabolites. Other possible mechanism is the formation of hydrogen
bridges between the inhibitor and amino acids near the active site of ACE (Balasuriya et al.,
2011).
54
4.2 Biochemical profiling:
Biochemical profiling of the methanolic extracts of seven plants including T. arjuna, C.
oxyacantha, R. serpentina, C. sativum, A. sativum, E. cardamom and P. nigrum which showed
higher ACE inhibition potential, was performed through LC-MS, antioxidants and toxicological
evaluation.
4.2.1 Liquid chromatography mass spectrometry (LC-MS):
The HPLC analysis generated extremely narrow peaks thus the high speed data handling
performance was required in the MS segment of the analysis. This LC/MS greatly contributes for
the identification of some of the phenolics and flavonoids compounds (Zhang and Zuo 2004;
Ogura and Sakamoto 2012). Moreover, the purpose was to have detailed analysis of some of the
peak fragmentation to discriminate between different compounds by Collision Induced
Dissociation (CID) using MS technique. Thus the structural elucidation of specific bioactive
compounds responsible for different therapeutic potential was made through LC-MS by the
courtesy of National Institute of Biotechnology and Genetic Engineering (NIBGE), Pakistan.
4.2.1.1 Biochemical profiling of Terminalia arjuna:
LC-MS analysis of methanolic extract of Terminalia arjuna was performed to evaluate
the phytoconstituents including phenolics, flavonoids and alkaloids. The full mass spectrum
obtained by LC-MS analysis was presented in Fig. 4.2. The mass spectrum depicted the high
peaks at 413.42, 511.50, 321.33, 589.33 and 685.58. The CIDMS-MS-ESI fragment ion of
685.58 peak resulted in three abundant peaks at 667.50, 523.33 and 457.25. The peak of 667.50
indicated the presence of Termiarjunoside 1,3,9,22-tetraol-12-en-28-oicacid-3-D-
glucopyranoside (Fig. 4.3). The presence of Termiarjunoside I from the bark of T. arjuna was
also reported in study by Ali et al. (2006). The fast atom bombardment mass spectroscopy
(FABMS) of T. arjuna also displayed a molecular ion peak at m/z = 666 [M]+ indicating the
presence of Termiarjunoside I, with a molecular formula of C36H58O11, which was also supported
by 13C and Distortionless enhancement by polarization transfer (DEPT) NMR spectra.
55
Fig. 4.2 Full mass spectrum of Terminalia arjuna
Fig. 4.3 MS-MS of 685.58 with CID showing Termiarjunoside I at 667.50 m/z
The mass spectrum obtained from LC-MS analysis in Fig. 4.4 revealed the highest peak
at 301.08 which may indicate the presence of quercetin in T. arjuna. The quercetin prominantly
decreased the impairment of cardiac functions following ischemia by improving mitochondrial
function (Kumar and Agnihotri 2008).
The Fig. 4.5 depicted the high peaks at 301.08, 523.42 and 169.08 (m/z). The peak at
169.08 indicated the presence of gallic acid which was further confirmed by MS-MS using CID
(30.00). The peak at 125.08 is the concequence of the removal of COO - from gallic acid (Fig.
4.6). Gallic acid (GA) is also found to be strong antioxidant which impart a vital role in number
of biological and pharmacological activities (Kim, 2007; Nikolic et al., 2011).
56
Fig. 4.4 Mass spectrum of T. arjuna showing Quercetin at 301.08 m/z
Fig. 4.5 Mass spectrum of T. arjuna showing Gallic acid at 169.08 m/z
Fig. 4.6 MS-MS CID (30.00) of peak 169 m/z
57
Fig. 4.7 Mass spectrum indicating the presence of Myricetin
The mass spectrum of T. arjuna depicted the presence of myricetin at peak 317.25 (Fig.
4.7). The MS-MS of the peak 317.25 by CID (21.00) showed the highest peaks at 302.08,
241.08 and 179.06. However the peaks at 193 and 289 may indicate the presence of ferulic acid
and catechin respectively. The mass spectrum and structures of these compounds were presented
in Fig. 4.8. Ferulic acid is responsible for wide range of therapeutic effects against cancer,
diabetes, lungs and cardiovascular diseases (Paiva et al., 2013).
Fig. 4.8 MS/MS of T. arjuna peak 317 at CID (21.00) showing Ferulic acid at 193 m/z and
Catechin at 289 m/z
The ferulic acid present in T. arjuna is not only a good antioxidant against protein
oxidation but also have some potential against lipid peroxidation in various biological systems
(Kanski et al., 2002). The HPLC analysis of T. arjuna bark by Jahan et al. (2012) exhibited
excellent concentration of polyphenols and phenolic acids including ferulic acid followed by
gallic acid, caffeic acid and catechin (Jahan et al., 2012).
58
The cardioprotective potential of flavonoids is because of its various actions as the intake
of flavonoids stop the endothelial dysfunction hence responsible to reduce arterial pressure
(Narayana et al., 2001; Kurosawa et al., 2005). Flavonoids may also help in uptake of oxdatively
modify LDL-c due to its free radicals scavenging property. Various studies showed that the
quercetin suppress the LDL oxidation and exert considerable vasorelaxation (Burns et al., 2000).
The presence of such potent bioactive compounds in T. arjuna is responsible for its
cardioprotective and antioxidant activity (Jahan et al., 2012).
4.2.1.2 Crataegus oxyacantha:The LC-MS analysis of C. oxyacantha was executed to assess the phytoconstituents
which help to cure the myocardial infarction. The mass spectrum of C. oxyacantha showed the
peaks at 591.33, 553.42 and 593.17. The peak at 593.17 indicated the presence of bioactive
compounds proanthocynidine with positive mode of ESI (Fig. 4.9). The MS-MS of peak 593 in
Fig. 4.10 gave the highest peaks 429.25, 457.17, 411.25 and 401.17, where the peak at 457.17
might indicate the presence of ursolic acid.
Fig. 4.9 Mass spectrum of C. oxyacantha showing proanthocynidine at 593.17 m/z
59
Fig. 4.10 MS2 CID (20.00) of peak 591.42 showing Ursolic acid at 457.25 m/z
The ursolic acid has also been reported to act as competitive inhibitor of Na+/K+ ATPase
at the digitaloid binding site (Verma et al., 2007). The presence of ursolic acid in C. oxyacantha
is responsible for its ACE inhibiting and cardioprotective potential (Lacaille et al., 2001). The
LC-MS-ESI also revealed the spectrum and structure of cratagolic acid at peak of 417 (m/z) as
given in Fig. 4.11. The ESI is commonly used for ionization of molecules where the sample was
ionized by application of high electric charge. Moreover ESI covers a broad range of
metabolites, since it operates ionization in negative and positive modes (Mallick et al., 2012).
Fig. 4.11 Mass spectrum of C. oxyacantha showing Crateagolic acid at 471.08 m/z
The CID MS-MS of the peak 381 of C. oxyacantha showed the highest peak of 191,
207.08 and 249.17. The peak at 301.17 may give the idea of the presence of quercetine (Fig.
4.12).
60
Fig. 4.12 MS2 of 381 of C. oxyacantha with CID (20.00) showing Quercetin at 301.17 m/zThe most important active constituents identified in C. oxyacantha are the phenols and
the flavonoids (Verma et al., 2007; Meschino et al., 2014). These bioactive phytoconstituents
showed cardioprotective potential by inhibiting phosphodiesterase which increases the
intracellular cyclic nucleotides (Verma et al., 2007).
4.2.1.3 Rauwolfia serpentina:
R. serpentina is a versatile medicinal plant and several researchers have investigated it
due presence of various phytoconstituents (Mittal et al., 2012; Singh et al., 2009; Mallick et al.,
2012; poonam et al., 2013). Various secondary metabolites such as alkaloids, phenols, tannins
and flavonoids present in the roots of R. serpentina are responsible for its cardioprotective
potential (Morales, 2000b). The LC-MS analysis of roots extract of R. serpentina was performed.
The full mass spectrums along with the highest peaks at 327.25 and 355.33 indicated the
presence of ajmaline and yohimbine respectively (Fig. 4.13).
61
Fig. 4.13 Mass spectrum of R. serpentina showing yohimbine at 355.33 and ajmaline at
327.25 m/z
Fig. 4.14 R. serpentina showing MS-MS of peak 327 with CID (25.00)
Ajmaline being a sodium channel blocker when given intravenously illustrated the
therapeutic potential (Brugada et al., 2000; Kostin et al., 1986). It has also been claimed to
stimulate respiration and intestinal movements. The action of ajmaline on systemic and
pulmonary blood pressure is similar as that of serpentine (Gawade et al., 2012). The MS-MS
with CID of 25.00 at peak 327 produced different fragments ion peaks (Fig. 4.15). Among these
peaks the peak at 353.25 m/z may indicate the presence of ajmailacine. The ajmalicine is derived
from tryptophan. It prevents strokes and also helps in maintaining blood pressure (Srivastava et
al., 2006).
62
Fig. 4.15 MS2 of peak 327 with CID (25.00) showing Ajmailicine at 353.25 m/z
The Fig. 4.16 showed the mass spectrum and structure of serpentine at the peak 349.52.
Serpentine is relatively weak tertiary base and is useful to cure hypertension, cardiovascular and
neurological diseases (Gawade et al., 2012; Weiss et al., 2000). R. serpentina is a hopeful
medication in the world of pharmacy due to the existance of considerable bioactive constituents
in roots (Rolf et al., 2003).
Fig. 4.16 Mass spectrum of R. serpentina showing Serpentine at 349.25 m/z
4.2.1.4 Allium sativum:
A. sativum was subjected to LC-MS analysis to evaluate the presence of
phytoconstituents that might be responsible for cardiovascular diseases, dyslipidemia and
hypertension. The LC-MS analysis of A. sativum depicted the highest peaks at 896.92, 917.75
and 782.58 (Fig. 4.17). The MS-MS of 896.92 with CID (25.00) gave the peak at 319.25,
indicated the presence of myricetin. The mass spectrum along with structure of myricetin is
presented in Fig. 4.18. The myricetin due to its specific chemical structure counteracts oxidative
63
stress generated as a result of Reactive Oxygen Species (ROS) (Mariee et al., 2012; Larson et al.,
2012).
Fig. 4.17 Mass spectrum of A. sativum
Fig. 4.18 MS2 CID 25.00 of 896 of A. sativum showing Myricetin at 319.25 m/zThe mass spectrum in Fig. 4.19 showed the presence of apigenin at peak 269.08. The
apigenin showed its peak at negative mode of ESI. Naturally occurring apigenin is found mostly
in hydroxylated form and has been demonstrated to inhibit tumor cell proliferation, angiogenesis
and induce apoptosis (Fang et al., 2007).
64
Fig. 4.19 Mass spectrum of A. sativum showing Apigenin at 327.25 m/z4.2.1.5 Coriandrum sativum:
LC-MS analysis of seeds extract of C. sativum was performed to evaluate the active
phytoconstituents including phenolics, flavonoids and alkaloids. The full mass spectrum is given
in Fig. 4.20. The Fig. 4.21 was the presentation of mass spectrum and structure of Caffeic acid at
peak 179.08 and Isorhamnetin-3-O-glucoside at 478.17 m/z. The Caffeic acid is the main
representative of the phenolic acids showing peak at negative mode of electron spray ionization.
The mass spectrum of C. sativum also showed apigenin-6-C-glucoside at peak 593.25 m/z with
negative mode of electron spray ionization (Fig. 4.22).
Fig. 4.20 Full Mass spectrum of C. sativum
65
Fig. 4.21 Mass Spectrum of C. sativum showing Caffeic acid at 179.08 m/z and Isorhamnetin-3-O-glucoside at 477.17 m/z
Fig. 4.22 Mass spectrum of Coriandrum sativum showing Apigenin-6-C-glucoside at 593.25 m/z
Caffeic acid is a potent antioxidant and has several therapeutic properties including
antiviral, antioxidants, anti-inflammatory and anticarcinogenic. It has been reported that caffeic
acid inhibits both lipoxygenase activity and suppresses lipid peroxidation thus completely blocks
the production of Reactive Oxygen Species (Jayanthi and Subash 2010).
4.2.1.6 Eletaria cardamom:
Cardamom fruit is used against cardiac disorders, renal and vesicular calculi, dyspepsia,
debility, halitosis and gastrointestinal disorders (Husain and Ali 2014). Phytochemical
66
investigation of cardamom has revealed highly bioactive components. Therefore, phytochemical,
biological and chemical compositional studies of this genus should be intensified (Savan and
Kucukbay 2013). In this research the mass spectrum obtained by LC-MS analysis of E.
cardamom represented the high peaks at 195.17 133.06 and 333.33. The peak at 195.17 indicated
the presence of terpinyl acetate (Fig. 4.23).
Fig. 4.23 Mass Spectrum of E. cardamom showing Terpinylacetate at 195.17 m/z The mass spectrum also depicted the presence of sebinen at 137.08 peak (Fig. 4.24). The
literature also reported the presence of terpinyl acetate in E. cardamom extract (Menon et al.,
1999 and Singh et al., 2008).
Fig. 4.24 Mass Spectrum of E. cardamom showing Sabinene at 137.08 m/z
67
4.2.1.7 Piper nigrum:The methanolic extract of P. nigrum is subjected to LC-MS analysis to determine its
bioactive compounds that impart crucial role in cardioprotection. The pippercide, an active
ingredient of P. nigrum showed its peak at 219.08. The mass spectrum and structural
representation of pipercide is shown in Fig. 4.25. Pipercide, important secondary metabolite,
play a vital role in disease resistance (Sruthi et al., 2013).
Fig. 4.25 Mass Spectrum of P.nigrum showing Pipercide at 219.08 m/z
4.2.2 Antioxidant assay:
Antioxidant potential of extracts of selected medicinal plants (Rauvolfia serpentina,
Terminalia arjuna, Coriandrum sativum, Piper nigrum, Eletaria cardamom, Allium sativum and
Crataegus oxyacantha) was determined through “DPPH free radical scavenging activity” and
“DNA protection assay”.
4.2.2.1 DPPH free radical scavenging activity:
The DPPH free radical scavenging activity (in term of % age inhibition) of R. serpentina,
T. arjuna, C. sativum, P. nigrum, E. cardamom, A. sativum and C. oxyacantha at various
concentrations (20, 40, 60, 80 and 100 µg/mL) was recorded (Table. 4.2). The T. arjuna
(62.5±1.624) and A. sativum (60.7±2.83) showed high antioxidant potential at the least
concentration of 20 µg/mL as compared to the same concentrations of other selected plants
(Table. 4.2). The C. sativum and P. nigrum showed higher than 50% inhibition at the
concentration of 40 µg/mL. However the R. serpentina depicted maximum antioxidant potential
(56.2±0.871) at the concentration of 100 µg/mL as compared to other concentrations of this
plant. While the E. cardamom presented relatively low antioxidant potential (47.7±2.52) even at
68
its higher concentration of 100 µg/mL. In case of C. oxyacantha, the increase in antioxidant
potential was very low with increase in concentration from 20 to 40 µg/mL, but it rapidly
increased with further increase in concentration from 60 to 100 µg/mL. The antioxidant potential
of C. oxyacantha is mainly due to the presence of procynidine and quercetine in it (Sokol-
Letowska et al., 2007; Kashyap et al., 2012; Guo et al., 2003; Mary et al., 2010).
Table. 4.2 DPPH free radical scavenging activity of selected medicinal plants
Plants extract Concentration (µg/mL)
20 µg/mL 40 µg/mL 60 µg/mL 80 µg/mL 100 µg/mL
R. serpentina 29.307±1.279
36.1±1.605 42.5±1.473 48.4±1.64 56.24±0.871
T. arjuna 62.5±1.624 65.78±2.678 71.57±3.25 89.607±1.14 91.093±0.349
C. sativum 31.1±2.505 58.067±0.95 64.267±1.419 75.53±1.45 77.83±0.21
P nigrum 37.97±1.285 53.73±2.618 58.03±1.88 67.13±1.25 76.77±4.12
E. cardamom 13.20±1.923 16.54±1.47 24.48±2.54 39.87±2.44 47.7±2.52
A. sativum 60.7±2.835 56.13±3.362 70.4±4.53 75.03±2.16 80.33±2.80
C. oxyacantha 33.8±2.212 44.73±1.124 65.033±1.32 77.23±1.42 92.31±1.06
The high antioxidant potential of T. arjuna is because of the presence of its flavonoids
(Nema et al., 2012). The LC-MS analysis also revealed the abundance of Termiarjunoside I,
catchein, gallic acids, quercetin and myricetin in the methanolic extract of T. arjuna bark
(4.2.1.1) that strongly endorsed its antioxidant potential (Mandal et al., 2013). The high contents
of antioxidant in the bark of T. arjuna contribute significantly to its therapeutic profile and also
used in various degenerative diseases (Nema et al., 2012).
The graphical presentation depicted the dose dependant response for free radical
scavenging potential i.e, the activity of all the extracts of plants in term of % age inhibition
increased with increase in concentration (Fig. 4.26). The dose dependant response of extracts of
various plants for their antioxidant potential has also been supported by other studies (Shukla et
al., 2013; Soni and Sosa et al., 2013; Yadav et al., 2014; Ayesha et al., 2013). This dose
dependant manner might be due to gradual increase in phenolic components, phenolic acids and
phenolic diterpenes which have very strong antioxidant power (Soni and Sosa, 2013).
69
R. serpentine T. arjun C. sativum P. nigrum E. cardamom A.sativum C. oxycantha0
10
20
30
40
50
60
70
80
90
100
20 µg/ml40 µg/ml60 µg/ml80 µg/ml100 µg/ml
% a
ge in
hibi
tion
Fig. 4.26 Graphical presentation of DPPH radical scavenging activity of selected medicinal
plants
Free radical production in the biological system is a routine and continuous phenomenon
and the healthy individuals balance it by the antioxidative defense system (Gulcin et al., 2005;
Soni and Sosa 2013). The oxidative stress is produced when there is high free radical generation
as a result of reduction in antioxidant levels (Kratchanova et al., 2010). The antioxidant level
could be maintained by phytoconstituents present in medicinal plants, which claimed to act as
powerful chain breaking antioxidants (Padmanabhan and Jangle 2012) hence have a positive
correlation with antioxidant activity (Sahu et al., 2013).
Different kind of mechanisms including inhibition of enzymes, chelation of metal ions
and scavenging of free radicals are involved in antioxidative properties of flavonoids (Mandal et
al., 2013; Soumia et al., 2014). The selected medicinal plants could be beneficial to mankind by
virtue of their effective antioxidant activity which may impart cardioprotective potential against
various cardiovascular diseases.
70
4.2.2.2 DNA protection assay:
The DNA protection assay of extracts of various selected plants (Terminalia arjuna,
Crataegus oxyacantha, Piper nigrum, Rauvolfia serpentina, Allium sativum, Coriandrum
sativum and Eletaria cardamom) was performed by using pBR322 plasmid DNA (Fig. 4.27).
The pBR322 plasmid has been used in number of researches to investigate the DNA protection
of various plants against H2O2 damage (Bhawya and Anilakumar 2011; Guha et al., 2011; Kalita
et al., 2012; Kutlu et al., 2014).
Fig. 4.27 DNA plasmid pBR322
The protective effect of different concentrations (100, 500 and 1000 µg/mL) of extracts
of said plants was evaluated against pBR322 plasmid DNA damaged by oxidative stress. The
response of seven selected methanolic extracts of medicinal plants with varying concentrations
on DNA damage along with positive control (H2O2 and FeSO4) has been presented in Fig. 4.28
and 4.29. The free radicals produced in response of H2O2 and FeSO4 caused the strand cleavage
of pBR322 plasmid DNA and resulted in DNA band streaking (Fig. 4.28). All the plants
exhibited protection of plasmid DNA against H2O2 damage as compared to the plasmid DNA
merely treated with H2O2. High phenolic compounds, in extracts of all plants could be considered
as the key reason behind the antioxidant potential of the plants (Kalita et al., 2012; Golla et al.,
71
2014). The strength of DNA protection increased in concentration dependant manner of extracts
of all the plants, which showed the protective effect of extracts against H2O2 induced damage.
The least concentration (100 µg/mL) of P. nigrum showed the band streaking in lane: 7 of Fig.
4.28, while the lane: 8 and 9 exhibited the good protection of P. nigrum against pBR322 plasmid
DNA damage at the corresponding concentration of 500 and 1000 µg/mL. In case of E.
cardamom, the concentrations of 100 µg/mL and 500 µg/mL in lane: 19 and 20 of Fig. 4.29 did
not show the protection, while the concentration of 1000 µg/mL showed the marked DNA
protection against H2O2 damage. Shyur et al. (2005) has also investigated the dose dependent
DNA protection of some selected medicinal plants and the results showed significant inhibition
of free radical and superoxide anion with increase in concentration of medicinal plants.
Fig. 4.28 Agarose gel electrophoresis pattern of pBR322 plasmid DNA treated with 30 mM H2O2 in the presence and absence of different plants extract. [Lane 1: pBR322 DNA + 30mM H2O2+ P1 (100 µg/mL), Lane 2: pBR322 DNA + 30mM H2O2+ P1 (500 µg/mL), Lane 3: pBR322 DNA + 30mM H2O2+ P1 (1000 µg/mL), Lane 4: pBR322 DNA + 30mM H2O2+ P2 (100 µg/mL), Lane 5: pBR322 DNA + 30mM H2O2+ P2 (500 µg/mL), Lane 6: pBR322 DNA + 30mM H2O2+ P2 (1000 µg/mL), Lane 7: pBR322 DNA + 30mM H2O2+ P3 (100 µg/mL), Lane 8: pBR322 DNA + 30mM H2O2+ P3 (500 µg/mL), Lane 9: pBR322 DNA + 30mM H2O2+ P3 (1000 µg/mL), Lane 10: pBR322 DNA + 30mM H2O2+ P4 (100 µg/mL), Lane 11: pBR322 DNA + 30mM H2O2+ P4 (500µg/mL), Lane 12: pBR322 DNA + 30mM H2O2+ P4 (1000 µg/mL)
72
Fig. 4.29 Lane13: pBR322 DNA + 30mM H2O2+ P5 (100 µg/mL), Lane 14: pBR322 DNA + 30mM H2O2+ P5 (500 µg/mL), Lane 15: pBR322 DNA + 30mM H2O2+ P5 (1000 µg/mL), Lane 16: pBR322 DNA + 30mM H2O2+ P6 (100 µg/mL), Lane 17: pBR322 DNA + 30mM H2O2+ P6 (500 µg/mL), Lane 18: pBR322 DNA +30mM H2O2+ P6 (1000 µg/mL), Lane 19: pBR322 DNA + 30mM H2O2+ P7 (100 µg/mL), Lane 20: pBR322 DNA + 30mM H2O2+ P7 (500 µg/mL), Lane 21: pBR322 DNA + 30mM H2O2+ P7 (1000 µg/mL)
P1= T. arjuna, P2= C. oxyacantha, P3= P. nigrum, P4= R. serpentina, P5= A. sativum, P6 =C. sativum and P7= E. cardamom.
H2O2 generates ●OH radicals, which cause massive oxidative damage. The ●OH radicals
bound to DNA strand may leads to base modification, deoxy sugar fragmentation and strand
breakage. Moreover, ●OH and other ROS causes oxidation of lipids which is responsible to
generate end products, like malondialdehyde and unsaturated aldehydes, that can attach to DNA
and produce mutagenic adducts (Guha et al., 2011). These oxidative modifications of DNA have
been suggested to contribute in the development of aging and various diseases (Bhawya and
Anilakumar, 2011).
The treatment of C. oxyacantha against hydroxyl radical induced DNA damage suggested
the DNA protection against oxidative damage. As the C. oxyacantha fruit is a traditional
medicine, hence used to cure the cardiovascular diseases because of its antioxidant potential
(Park et al., 2010). The mechanism by which the plants protect the DNA against H2O2 damage is
ambiguous because there are several potential inhibition pathways. However the antioxidants
may control the oxidative stress by reacting directly with H2O2 or reacting with intermediates
formed from enzymes and H2O2 (Kanwal et al., 2011).
73
A large number of plants have been examined to protect DNA against oxidative stress
due to UV induced photolysis of H2O2 (Rajkumar et al., 2010; Guha et al., 2011). The aqueous
extract of Curcuma amada showed the concentration dependant increase in protective potential
against oxidative stress induced in pBR322 plasmid DNA (Vishnupriya et al., 2012). However,
the established view is that the antioxidant potential of medicinal plants are related to DNA
protection activity by various mechanisms including chelation of transition metal (Asker et al.,
1996) inhibition of enzymes required for the initiation reaction of DNA cleveage (Bibi et al.,
2011).
4.2.3 Toxicity assay:
Toxicological testing is an essential prerequisite for the evaluation of medicinal plants as
therapeutics agent (Kumar et al., 2011). The cytotoxicity extracts of selected medicinal plants
(R. serpentina, T. arjuna, C. sativum, P. nigrum, E. cardamom, A. sativum and C. oxyacantha)
was evaluated by conducting “Hemolytic assay” and “Mutagenic assay”.
4.2.3.1 Hemolytic assay:
Hemolytic assay of the selected methanolic extracts was performed against human
erythrocytes using triton X-100 as positive control. The hemolytic activity of extracts of various
plants is shown in term of percentage hemolysis and given as mean ± SD of three replicates. The
percentage hemolysis of individual plants at different concentrations (100, 500 and 1000 μg/mL)
was expressed in Table. 4.3. None of the plant extract showed any hemolytic effect against
erythrocytes thus all the results showed no hemolysis. So pharmacologically these plants are safe
to use for human beings as a source of therapeutic drug. The T. arjuna and C. oxyacantha
showed very low % age hemolysis as compared to other selected medicinal plants. The P.
nigrum showed 8.10±0.232 and 9.21 ±0.106 % age hemolysis at the corresponding concentration
of 500 and 1000 μg/mL. However, extracts of all the plants showed dose dependent increase in
hemolytic activity, which showed that further increase in concentration, may cause hemolysis.
Thus further investigations are needed to evaluate hemolysis at higher concentrations.
74
Table. 4.3 Hemolytic activity (%) of extracts of selected medicinal plants
SamplesHemolysis (%)
100µg/mL 500 µg/mL 1000 µg/mL
C. sativum 5.47±0.281 6.03±0.131 7.30±0.012 -
C. oxyacantha 2.86±0.237 4.45±0.281 5.71±0.0321 -
T. arjuna 2.49±0.164 3.18±0.071 5.15±0.046 -
R. serpentina 4.20±0.082 5.26±0.051 5.39±0.102 -
A. sativum 3.46±0.067 5.18±0.201 7.26±0.341 -
E. cardamom 4.78±0.201 5.92±0.291 7.92±0.210 -
P. nigrum 6.30±0.014 8.10±0.232 9.21±0.106 -
Positive control Triton X 100
97.38±0.11
Negative control PBS
1.02±0.82
Hemolysis is due to obliteration of red blood cells which resulted from lysis of membrane
lipid bilayer. The hemolytic activity is related to chemical composition, concentration and
potency of each extract (Zohra and Fawzia, 2014). Several reports indicated that the membranes
of human erythrocytes have varying stability as determined from the mean corpuscular fragility.
The extracts of plants may affect the membrane of red blood cells in a positive way (Freitas et
al., 2008) but many plants may also have serious adverse effects, which include initiation of
hemolytic anemia (Zohra and Fawzia, 2014). Hemolytic activity of the aqueous extracts of
different Acacia species were accessed against human erythrocytes and showed low to mild
hemolytic effect. The hemolytic percentage of extracts was found to be increased with increase
in concentration of extracts of plants (Sulaiman and Gopalakrishnan, 2013).
75
C. sativu
m
C. oxya
cantha
T. arju
na
R. serpen
tina
A. sativu
m
E. card
amom
P. nigr
um
Positive
control Tr
iton X 100
Negative
control P
BS0
20
40
60
80
100
120
100µg/mL500 µg/mL1000 µg/mL
Samples
Hem
olys
is (%
)
Fig. 4.30 Graphical presentation of % Hemolysis of extracts of selected medicinal plants at
different concentrations
The aqueous extracts of Aerva lanata, Calotropis gigantea and Elaeocarpus ganitrus
with various concentrations of 125, 250, 500, 1000 μg/mL were analyzed for the hemolytic
activity against human erythrocytes. All the samples exhibited very low hemolytic effect as
compared to positive control and also showed the dose dependent increase in hemolytic activity
(Kumar et al., 2011).
Solvent extracts of Syzigum cuminii, Cratevan urvula and Achyranthes aspera also have
been studied for their hemolytic activity and reported to possess no haemolytic effect (Mathur et
al., 2011; Priya et al., 2010). The phytochemicals constituents present in extracts of various
medicinal plants might be responsible for their antihemolytic activity (Kumar et al., 2011;
76
Lakshmi et al., 2014). Actually, investigation of the toxicity of certain medicinal plants is
considered to be important because of their uses for medicinal purpose (Das et al., 2010).
4.2.3.2 Mutagenicity assay (Ames test):
The Ames test was applied to confirm whether the selected medicinal plants were
mutagenic or not (Zeiger et al., 2001; Kim et al., 2010; Abudayyak et al., 2015), so that these
may be used as safe therapeutics for various CVD.
The mutagenic probability of selected medicinal plants including R. serpentina, T.
arjuna, C. sativum, P. nigrum, E. cardamom, A. sativum and C. oxyacantha was evaluated by
using test strain S. typhimurium TA 98. The standard S. typhimurium TA 98 showed considerable
mutagenicity with high number (94/96) of positive wells (yellow) (Fig. 4.31). The mutagenic
property of all the selected medicinal plants was counted and compared with background plate
(11/96 positive wells) (Fig. 4.32). The medicinal plants are assumed to be mutagenic if the
number of positive wells is two folds higher as compared to the background plate (Razak and
Aidoo, 2011). The C. sativum, T. arjuna and R. serpentina showed 8/96 while the E. cardamom,
A. sativum and P. nigrum presented 16/96 yellow wells in microplate (Table. 4.4). Among these
the C. oxyacantha showed good results with no positive well in microplate. Therefore none of
the selected medicinal plants exhibited the mutagenic property. The variation in response of
these examined medicinal plants might be due to the differences in their active constituents (Aqil
et al., 2008).
77
Fig. 4.31 Standard S. typhimurium TA 98
Fig. 4.32 Mutagenicity in Background plate
Fig. 4.33 Mutagenicity of extracts of selected medicinal plants. A= C. sativum, B= C.
oxyacantha, C= E. cardamom, D= P. nigrum, E= A. sativum, F= T. arjuna, G= R. serpentina
Table. 4.4 The mutagenicity of standard, Background and extracts of selected medicinal
plants
Sr. No. Plant extracts +ve/total Results Interpretation
78
1 Standards 94/96 + Mutagenic2 Background 11/963 C. sativum 08/96 - Non mutagenic
4 C. oxyacantha 0/96 - Non mutagenic5 E. cardamom 16/96 - Non mutagenic6 P. nigrum 16/96 - Non mutagenic
7 A. sativum 16/96 - Non mutagenic
8 T. arjuna 8/96 - Non mutagenic9 R. serpentina 8/96 - Non mutagenic
The Ames test is based on the number of Histidine revertants, produced by crude plant
extracts in S. typhimurium strain (Taylor et al., 2003; Luseba et al., 2007). Hence the absence of
mutagenic response of these selected medicinal plants against S. typhimurium bacterial strains is
an astounding approach in determining the safe use of these medicinal plants (Ghazali et al.,
2011).
Luseba et al. (2007) also observed the non mutagenicity of some medicinal plants
through Ames in S. typhimurium (TA98) test strain. This non-mutagenic property of medicinal
plants might be owing to the existance of active phytoconstituents. Lack of mutagenicity
suggested that the medicinal plants are safe tool for effective fighting against various diseases
(Luseba et al., 2007).
4.5 Section- II (In vivo analysis):
Section-II is related to in vivo analysis and it has been completed in three Phases. In
phase-I, the preliminary trial was run to optimize the dose of salbutamol at which it induced
myocardial infarction. The phase-II involved the optimization of various concentrations of
selected medicinal plants (R. serpentina, T. arjuna, C. sativum, P. nigrum, E. cardamom, A.
sativum and C. oxyacantha) to get more suitable cardiopotant dose against salbutamol induced
myocardial infarction. In Phase-III of in vivo analysis, the cardioprotective potential of herbal
combinations was evaluated against surgically induced myocardial infarction to get the more
cardiopotent herbal product.
4.5.1 Phase-I:
4.5.1.1 Preliminary trial (Dose optimization of Salbutamol):
79
In preliminary trial the rats were administered with different concentrations of salbutamol
(mg/kg) for two consecutive days and the blood samples were taken at different time intervals
(hr). The varying concentrations and time intervals, both were suggested by “Central Composite
Design” of RSM. The cardiac markers including CK-MB, SGOT and LDH, being diagnostic
features were analysed to estimate the severity of myocardial infarction (Dianita et al., 2015).
The relationship between dependant (cardiac markers) and independent variables (concentrations
of salbutamol and time intervals) was graphically presented by 3D Response surface plots in
Figures. 4.34-36.
The interaction of different concentrations of salbutamol and time intervals of blood
sampling on the level of CK-MB has been given in Fig. 4.34. The increased in concentration of
salbutamol from 34 to 88 mg/kg.b.wt. after 48 hr of salbutamol administration resulted in
elevation in CK-MB level from 152 to 297 IU/L. Further increase in concentration from 88 to
141 mg/kg of salbutamol showed comparatively low elevation in the CK-MB level. However
after 116 hr of salbutamol administration (88 mg/kg.b.wt), there was considerable decline in
enzyme level from 293 to 184 IU/L. The possible explaination could be that the CK-MB level
fall to normal with passage of time intervals after 48 to 72 hr of MI development (Dianita et al.,
2015). In case of rats, administered the salbutamol at concentration of 50 and 125 mg/kg did not
show the considerable elevation (148 and 198 IU/L) in the level of CK-MB after intervals of 96
hr. The variation in quantity of enzymes released depends upon the degree of cellular damage
(Azmat et al., 2012; Khan et al., 2014).
80
Design-Expert® Sof tware
CK-MB299
144
X1 = A: TimeX2 = B: Conc
0.00
24.00
48.00
72.00
96.00
50.00
68.75
87.50
106.25
125.00
140
180
220
260
300
CK
-MB
A: Time B: Conc
Fig. 4.34 Response surface plot of CK-MB vs. time and concentration
The Fig. 4.35 described the relation between SGOT (dependent variable) conc. and time
intervals (independent variables). The salbutamol at the concentrations of 34, 88 and 141 mg/kg
b.wt depicted the inclined in the level of SGOT with corresponding values of 51, 128 and 132
IU/L after 48 hr of salbutamol administration. This might be due to the reason that salbutamol
causes leakage of SGOT from cardiomyocytes into the blood. It occurs due to collapse of cellular
and subcellular compartments that reflect pathological alterations in myocardium (Jaffe et al.,
2006). After 96 hr of salbutamol administration at the concentration of 50 and 125 mg/kg, the
SGOT level was comparatively low. This decline in SGOT level might be due to the increase in
time intervals as the SGOT level shifted towards normal with passage of time (Dianita et al.,
2015).
81
Design-Expert® Sof tware
SGOT264
36
X1 = A: TimeX2 = B: Con.
0.00
41.00
82.00
123.00
164.00
50.00
68.75
87.50
106.25
125.00
50
110
170
230
290
SG
OT
A: Time B: Con.
Fig. 4.35 Response surface plot of SGOT vs time and concentration
In case of LDH, the increase in concentrations of salbutamol from 34 to 88 mg/kg b.wt.
resulted in increase in level of LDH from 271 to 512 IU/L respectively after 48 hr interval of
salbutamol administration. The LDH is detectable from 8 to12 hours of post MI and reached at
its peak at 24-72 hours (Rosenblat et al., 2012). Further increase in time interval of 116 hr after
salbutamol administration the rats treated with salbutamol at the concentration of 88 mg/kg
depicted the considerable fall in the level of LDH from 512 to 298 IU/L (Fig.4.36). This decline
in LDH level supported the fact that serum levels of LDH approaches to normal value after 96
hours after MI (Wang et al., 2006). The level of LDH was 278 and 357 IU/L in response to the
corresponding concentrations of 50 and 125 mg/kg b.wt. after 96 hr intervals of salbutamol
administration. This increase in the level of cardiac markers in serum indicated the altered
membrane permeability and leakage of these enzymes into blood stream (Abirami and
Kanagavalli, 2013).
The increase in enzyme level is due to the oxidative stress and myocardial cell necrosis
caused by salbutamol (Dianita et al., 2015; Zafar et al., 2015). The extent of leakage of cardiac
markers (CK-MB, LDH and SGOT) indicated the onset of myocardial infarction and act as
sensitive markers of myocyte injury (Nandave et al., 2007).
82
Design-Expert® Sof tware
LDH518
265
X1 = A: TimeX2 = B: Conc
0.00
24.00
48.00
72.00
96.00
50.00
68.75
87.50
106.25
125.00
260
325
390
455
520
LD
H
A: Time B: Conc
Figure: 4.36 Response surface plot of LDH vs. time and concentration
The cardiotoxicity due to administration of salbutamol is reflected by the increased
cardiac markers, lipid profile and alleviated antioxidant enzymes as compared to normal control
group (Zafar et al., 2015). The histopathological examination of heart after salbutamol
administration also endorsed the development of myocardial infarction in another animal trial
(Kousar et al., 2012; Aslam et al., 2015).
The analysis of variance (ANOVA) presented the statistical significance of the fitted
quadratic polynomial model (Table. 4.5). The F values of model were, 48.18, 27.27 and 122.30
for CK-MB, SGOT and LDH respectively which suggested the significance of quadratic model
for cardiac markers. The model is adequate to present the relationship between the response of
dependent (cardiac markers) and independent variables (concentrations of salbutamol and time
intervals). The value of determination coefficient (R2) for CK-MB, SGOT and LDH was 0.9718,
0.9512 and 0.9887 respectively with non significant lack of fit at P>0.05, which means that the
statistical model could explain more than 97.18 of the result. Meanwhile, a relatively lower value
83
of coefficient of variation, 6.86, 12.9 and 4.20 for CK-MB, SGOT and LDH respectively showed
a better precision and reliability of the experiment.
Table. 4.5 Analysis of variance (ANOVA) for the fitted model of CK-MB, LDH and SGOT activity as a function of independent variables
Parameter Source SS Df MS F Value P>F
CK-MB R2=0.9718 CV=6.86
Model 54546.23 5 10909.25 48.18 <0.0001A-Time 1188.22 1 1188.22 5.25 0.0557B-Conc 4585.08 1 4585.08 20.25 0.0028
AB 400.00 1 400.00 1.77 <0.0001
A2 34111.31 1 34111.31 150.65 <0.0001
B2 20304.00 1 20304.00 89.67 <0.0001Residual 1585.00 7 226.43
Lack of Fit 1564.20 3 521.40 100.27 <0.0521Pure Error 20.80 4 5.20
L
SGOT R2=0.9512CV=12.96
Model 17167.89 5 3433.58 27.27 0.0002A-Time 969.95 1 969.95 7.7 0.0275B-Con 3459.58 1 3459.58 27.48 0.0012
AB 387.04 1 387.04 3.07 0.123A2 11359.67 1 11359.67 90.22 < 0.0001B2 2036.11 1 2036.11 16.17 0.0051
Lack of Fit 731.36 3 243.79 6.5 0.0541Residual 881.36 7 125.91
Pure Error 150 4 37.5
LDHR2=0.9887
CV=4.20
Model 1.571E+005 5 31423.93 122.30 < 0.0001A-Time 2680.09 1 2680.09 10.43 <0.0145B-Conc 7444.98 1 7444.98 28.97 <0.0010
AB 1521.00 1 1521.00 5.92A2 99216.21 1 99216.21 386.13
B2 64680.21 1 64680.21 251.72Residual 1798.64 7 256.95
Lack of Fit 1777.44 3 592.48 111.79 0.0013Pure Error 21.20 4 5.30
The purpose of dose optimization of salbutamol was to find out the optimal concentration
which indicated the onset of myocardial infarction in experimental animals. The optimal time of
blood sampling (hr) and concentration of salbutamol (mg/kg) was suggested by RSM (Table.
4.6). Hence the RSM predicted the 80 mg/kg as optimum dose of salbutamol at which it may
elevate the SGOT level up to 99 IU/L after 20 hr of salbutamol administration while in case of
84
our experiment the concentration of 141 mg/kg.b.wt. elevated the SGOT level at its maximum
extant (132 IU/L). In case of our experimental study the concentration of 88 mg/kg elevated the
level of CK-MB (293 IU/L) and LDH (515 IU/L). The RSM suggested the concentration of 80
mg/kg.b.wt which raised the level of CK-MB and LDH up to 265 and 467 IU/L after 23 hr of
two concecutive doses (Table. 4.6) which was near to our experimental approach. The
cardioprotective potential of Coriandrum sativum against salbutamol induced cardiac injury also
favored the increased in CK-MB level up to 203 IU/L after administration of two doses of
salbutamol (Kousar et al., 2012).
Table. 4.6 Effects of optimized dose of salbutamol on different cardiac markers suggested by Response Surface Methodology
Parameter Time (hr) Concentration(mg/Kg) Optimized dose (IU/L) Desirability
CK-MB 23 80 265 0.907
SGOT 20 80 99 0.786
LDH 23 80 467 0.912
Exhaustion in levels of cardiac LDH and CK-MB isoenzymes during myocardial
infarction indicated the alteration in membrane integrity and causes the leakage of these
biomarkers into blood stream (Nandave et al., 2013). The formation of free redicals due to
oxidative stress resulted in Increase in cardiac enzymes which consequently responsible for
variations in membrane permeability thus ultimately lead to the failure of myocardial membranes
functions (Barman et al., 2013). The optimum dose (80 mg/kg) of salbutamol suggested by RSM
was further used to establish the experimental model of chemically induced MI.
4.6. Phase-II
4.6.1 Dose response experiment (A pilot study):
The dose response evaluation of selected parts of the medicinal plants like roots of R.
serpentina, bark of T. arjuna, seeds of C. sativum and E. cardamom, leaves of P. nigrum and
fruit of A. sativum and C. oxyacantha, was carried out followed by in vitro characterization. In
this phase different groups of rats were pretreated with said medicinal plants with varying
concentrations as suggested by RSM. After administration of two optimized doses of salbutamol
(Phase-I) the blood samples of rats were drawn to evaluate different biomarkers including
85
cardiac markers (CK-MB, SGOT and LDH), lipid profile (LDL, HDL, T. Cholesterol and
Triglycerides) and antioxidant enzymes (SOD, CAT and GSH). The results of all these
biomarkers are described accordingly under the following headings.
4.6.1.1.1 Cardiac markers:
The elevated levels of CK-MB, SGOT and LDH during myocardial infarction act as
highly sensitive diagnostic markers (Sabeena et al., 2004; Gurgun et al., 2008; Mastan et al.,
2009; Siddiq et al., 2012). The effect of different concentrations of medicinal plants against MI
was evaluated by estimation of cardiac markers (Fig. 4.37-39). These cardiac markers were
analysed by RSM to have the optimum concentration of each medicinal plant (Table. 4.8, 4.10,
4.12).
4.6.1.1.1.1 CK-MB:
CK-MB is considered as standard for diagnosis of MI and hence preferred in selective
situations (Jagannadha et al., 2010). The CK-MB level reaches at its peak after 24 hr of MI
development and fall to normal with passage of time intervals after 48 to 72 hr (Dianita et al.,
2015).
The CK-MB level in normal control group was found 150 IU/L. The positive control
group, to which only salbutamol (80 mg/kg) was given, showed considerable elevation (296
IU/L) in the CK-MB level. This might be due to the reason that salbutamol causes leakage of
CK-MB from cardiomyocytes into the blood stream. It occurs due to collapse of cellular and
subcellular compartments that reflect pathological alterations in myocardium (Jaffe et al., 2006).
Moreover, the rats in different treatment groups were treated with various concentrations (80,
110, 140, 170 and 200 mg/kg) of its respective medicinal plants. The effect of these medicinal
plants on the level of CK-MB has been given graphically (Fig. 4.37).
The first group was pretreated with different concentrations of T. arjuna. The increased
in concentration from 80-140 mg/kg b.wt. of T. arjuna maintained the level of CK-MB from
188-151 IU/L which display a decline of 12.38%. While the increase in concentration of this
plant up to 170 mg/kg showed very nominal effect i.e 153 IU/L level of CK-MB. However, with
further increase in concentration from 170 to 200 mg/kg, the level of CK-MB was maintained up
to 162 IU/L. Therefore the findings of the study gave the impression that 140 mg/kg b.wt is a
good dose to maintain the level of CK-MB. In another study the T. arjuna bark at the
concentration of 200 mg/kg b.wt, could maintained the CK-MB level with value of 146 IU/L
86
near to normal (Jahan et al., 2012). This cardioprotective potential of T. arjuna is credited to the
potent antioxidants and their free radical scavenging activity (Aslam et al., 2015).
The second group of rats was pretreated with different concentrations of C. oxyacantha.
The dose dependent response was observed when treated with concentration of 80 to 170 mg/kg
of C. oxyacantha with level of CK-MB from 223-162 mg/dL. The rats pretreated with
concentration of 200 mg/kg depicted slight elevation in CK-MB level (170 IU/L) as compared to
the concentration of 170 mg/kg. Thus the concentration of 170 mg/kg would be considered as the
dose of choice against MI. The C. oxyacantha berry is probably deemed as best known
cardiotonic. Hence it helps to improve the blood supply to heart by dilating peripheral and
coronary blood vessels and attenuating symptoms in early period of heart failure (Zafar et al.,
2015).
The third group of rats was pretreated with different concentrations of P. nigrum to get its
optimal concentration against myocardial infarction. With increase in concentrations the level of
CK-MB was also maintained gradually. The dose of 80 mg/kg b.wt. of P. nigrum could not
maintain the level of CK-MB (262 IU/L) with in limits in contrast to the same dose of other
selected medicinal plants. The treatment with concentrations of 170 and 200 mg/kg depicted
almost same effect on the levels of CK-MB with values of 180 and 188 IU/L respectively.
The fourth group was treated with different concentrations of C. sativum. The dose
dependent response was observed to maintaine the level of CK-MB appreciably. The
concentration of 200 mg/kg b.wt showed the substantial maintenance in the level of CK-MB
(171 IU/L) as compared to the positive control (296 IU/L) presented in Fig. 4.37. This reduction
in enzyme level could be due to its protective action on membrane integrity and restricting the
leakage of this enzyme (Aslam et al., 2015).
The fifth group pretreated with different concentrations of A. sativum also showed the
increase in the maintenance of CK-MB level with increase in its concentrations. There was not
much difference between the response of 140 and 170 mg/kg which showed almost equal
protection with CK-MB level of 162 and 168 IU/L. The concentration of 200 mg/kg appreciably
restored the level of CK-MB (157 IU/L) near to normal value. A. sativum has therapeutic
potential for hypertension and hypercholesterolemia. German Commission and the World Health
Organization have approved the A. sativum for its hyperlipidemia and atherosclerotic vascular
changes (Blumenthal et al., 2000).
87
Design-Expert® Sof tware
CK-MB
B1 T. arjunB2 C. oxy canthaB3 P. nigrumB4 C. sativ umB5 A. sativ umB6 R. serpentineB7 E. cardamom
X1 = A: Conc.X2 = B: Plant
Plant species
80.00 110.00 140.00 170.00 200.00
Conc.
CK
-MB
140
172.5
205
237.5
270
Fig. 4.37 Graphical presentation of optimized concentration of medicinal plants for CK-
MB against salbutamol induced Myocardial infarction
The pretreatment with R. serpentina showed the prominent effect in keeping the level of
CK-MB from 214 IU/L to 163 IU/L with increased in corresponding concentrations from 80 to
170 mg/kg. There was abrupt elevation in the level of CK-MB (175 IU/L) at the concentration of
200 mg/kg. This increase in CK-MB level might be due to the intake of high concentration of R.
serpentina which also cause a depletion of nor-epinephrine resulting in tranquilizing effect.
Moreover very high dose can also cause a loss of nerve coordination (Huang et al., 1999). The
seventh group was treated with E. cardamom to get the optimized dose at which it showed
preventive potential against salbutamol induced MI. The E. cardamom showed the dose
dependent response as the increase in concentrations considerably maintained the level of CK-
MB. In case of the dose of 80 mg/kg the CK-MB level was maintained at 261 IU/L while there
88
was better maintenance of CK-MB (219 IU/L) when the rats were treated with concentration of
110 mg/kg. However the concentration of 200 mg/kg b.wt depicted the maximum preventive
potential as it maintained the level of CK-MB up to 179 IU/L.
Overall the T. arjuna showed better maintenance even at lower concentration of 80
mg/kg as compared to the same dose of other selected medicinal plant. Cardioprotective potential
of T. arjuna extract can be correlated with presence of polyphenolic fraction and its antioxidant
activity (Jahan et al., 2012). The bark of T. arjuna reported to possess cardioprotective
(Gauthaman et al., 2001; Singh et al., 2008; Sivakumar and Rajeshkumar 2014; Aslam et al.,
2015; Zafar et al., 2015), hypocholesterolemic and antioxidant effect (Jahan et al., 2011a).
The experimental data was statistically analyzed using the Design-expert 7.0 for ANOVA
(Table. 4.7) which explained the significance of quadratic model. The F-value (437.43) depicted
the model is significant and also indicated the interaction strength of each parameter. The closer
the R2 (0.9945) value to unity, better the model fits to the actual data (Wani et al., 2013). The
value of Predicted R-Squared (0.9898) was close to the Adjusted R-Squared value (0.9922) as
expected. This may indicate a small block effect or a possible problem.
Table. 4.7 Analysis of variance (ANOVA) for response surface methodology of CK-MB
(IU/L) as a function of independent variables
Source SS Df MS F Value Prob > FModel 45186.46 14 3227.60 437.43 < 0.0001 Significant
A-Conc. 25348.74 1 25348.74 3435.46 < 0.0001 .B-Plant 11898.25 6 1983.04 268.76 < 0.0001
AB 2697.00 6 449.50 60.92A2 5242.48 1 5242.48 710.50
Residual 250.87 34 7.38Lack of Fit 250.37 20 12.52 350.52 < 0.0691 Non
SignificantPure Error 0.50 14 0.036Cor Total 45437.33 48R-Squared 0.9945 Standard Deviation 2.72
Adj R-Squared 0.9922 Mean 190.84Pred R-Squared 0.9898 Coefficient of Variation (CV) % 1.42Adeq Precision 72.594 Prediction Error Sum of Squares (PRESS) 463.20
The optimum concentration of selected medicinal plants predicted by Response Surface
Methodology is given in Table. 4.8. The RSM suggested 165, 172 and 164 mg/kg b. wt. as
89
optimum concentration for T. arjuna, C.oxyacantha and R. serpentina respectively that may
sustain CK-MB level near to normal which was related to experimental findings (140-170
mg/kg.b.wt). According to experimental approach the concentration of 170 mg/kg.b.wt. of P.
nigrum maintained the enzyme level against salbutamol induced MI while the response surface
methodology suggested the concentration of 186 mg/kg that may able to maintain the level of
CK-MB near to normal. The RSM predicted the optimum concentration of 170 mg/kg for A.
sativum while the experimental value depicted the concentration of 170-200 mg/kg to keep the
level of CK-MB within range. The RSM endorsed the concentration of 183 and 190 mg/kg for
C.sativum and E. cardamom which was near to our experimental approach (200 mg/kg). The
ability of medicinal plants to maintain the CK-MB level might be due to the presence of
flavanoids and antioxidants (Abirami and Kanagavalli 2013).
Table. 4.8 Optimized concentrations of medicinal plants for CK-MB (IU/L) against
salbutamol induced Myocardial infarction
Sr.
No.
Plants Optimum concentrations of selected medicinal plants (mg/kg)
CK-MB (IU/L)
Based upon biochemical evaluation
Suggested by RSM
1 T. arjuna 140-170 165 150 (165)*
2 C.oxyacantha 140-170 172 163
3 P.nigrum 170 186 182
4 C.sativum 200 183 170
5 A. sativum 170-200 170 155
6 R. serpentina 140-170 164 164
7 E. cardamom 200 190 178
Among all the studied medicinal plants, T. arjuna showed maximum restoration of CK-
MB at the optimum concentration. Cardioprotective potential of T. arjuna extracts can be
correlated with polyphenolic fraction and antioxidant activity (Jahan et al., 2012).
4.6.1.1.1.2 SGOT:
The SGOT is an index of cardiac damage and is also a clinical biomarker during
myocardial infarction (Rafatullah et al., 2008; Mahaswari et al., 2008). The normal control group
90
illustrated 36 IU/L of SGOT while the positive control group, merely treated with salbutamol,
showed 96 IU/L level of SGOT. The considerable elevation in the levels of SGOT in salbutamol
provoked group indicated the onset of myocardial injury. Amount of SGOT released depends
upon the degree of cellular damage (Alla et al., 2007; Azmat et al., 2012; Khan et al., 2014). A
number of medicinal plants have been reported to maintain the elevated level of SGOT during
cardiac damage (Orhue and Nawanze, 2004). The graphical presentation (Fig. 4.38) showed the
response of different treatments on SGOT against salbutamol induced MI. The first treatment
group of rats was administered with different concentrations of T. arjuna. A decreasing trend of
SGOT from 75 to 45 IU/L was found, with increase in concentration of T. arjuna from 80 to 170
mg/kg. The rats pretreated with the concentration of 140 and 170 mg/kg showed parallel effect
on the enzymatic level (45 IU/L). The effective maintenance of SGOT is due to the presence of
active phytoconstituents in T. arjuna bark (Gauthaman et al., 2001). The high concentration (200
mg/kg) of T. arjuna could not maintained the level of SGOT (55 IU/L) as compared to the
concentrations of 140 and 170 mg/kg b.wt. This depicted the slight deviation from dose
dependant response that might be due to the side effects of high dose of T. arjuna.
The second group of rats was pretreated with different concentrations of C. oxyacantha
(Fig. 4.38). All the concentrations of C. oxyacantha showed effective maintenance of SGOT in
rats against salbutamol even at its least concentration of 80 mg/kg.b.wt, as compared to the same
dose of other selected medicinal plants. High cardioprotective effect with 49 IU/L of SGOT was
observed with the concentration of 200 mg/kg. There was not any report related to adverse
effects of low doses of C. oxyacantha but its higher doses may increase the risk of hypotension
and sedation (Verma et al., 2007). The German Commission has also approved the use of C.
oxyacantha as a heart remedy and has become a part of many prescriptions for the common
cardiovascular disorders (Verma et al., 2007).
The third group of rats treated with P. nigrum maintained the SGOT level at 53 IU/L with
the corresponding concentration of 170 mg/kg.bwt. Further increase in concentration from 170 to
200 mg/kg did not show any considerable difference in order to maintain the level of SGOT (60
IU/L). None of the concentrations of P. nigrum from 80 to 140 mg/kg b.wt. could maintain the
level of SGOT as compared to normal control group. Therefore 170 mg/kg of P. nigrum might
be considered as the effective concentration that may cope with complications related to MI. The
fourth group of rats treated with C. sativum showed the dose dependent behaviour on the level of
91
SGOT as the increase in concentrations resulted in gradual decline in the enzyme level. The
outcomes of results gave an idea that the concentration of 200 mg/kg may exert a remarkable
protection from the toxic effect of salbutamol.
The A. sativum given to fifth group of rats, showed the better maintenance of SGOT level
with increase in its concentrations from 180 to 200 mg/kg b.wt. (Fig. 4.38). The concentration of
200 mg/kg maintained the level of SGOT at 46 IU/L that depicted very close effectiveness to the
response of concentration of 170 mg/kg.b.wt. The sixth group pretreated with R. serpentina
considerably sustained the level of SGOT almost within normal range (35 to 55 IU/L) at the
corresponding concentrations of 140-200 mg/kg b.wt. Hence this range could be considered the
effective range to maintain the enzyme level near to normal as compared to positive control
group (96 IU/L).
Design-Expert® Sof tware
SGOT
B1 T. arjunB2 C. oxy canthaB3 P. nigrumB4 C. sativ umB5 A. sativ umB6 R. serpentineB7 E. cardamom
X1 = A: Conc.X2 = B: Plant
Plant species
80 110 140 170 200
Conc.
SG
OT
40
53
65
78
91
Fig. 4.38 Graphical representation of optimized concentration of medicinal plants for
SGOT against salbutamol induced Myocardial infarction
The seventh group of rats pretreated with E. cardamom showed the decrease in SGOT
level from 88 IU/L to 54 IU/L with corresponding concentration of 80 to 170 mg/kg. Further
increase in concentration up to 200 mg/kg b.wt. could not sustain the level of SGOT (64 IU/L)
92
near to control group. Previously considerable maintenance of enzymes level has been observed
in the rats pretreated with 100 and 200 mg/kg.b.wt. of E. cardamom with concomitant
administration of isoproterenol. Moreover, the cardioprotective effect was supported by better
histopathological changes, which specify the salvage of cardiomyocytes from the harmful effects
of isoproterenol (Goyal et al., 2015).
The response of above said medicinal plants at varying concentration gave an idea that
the C. oxyacantha and T. arjuna are relatively more effective for maintaining the SGOT level.
This maintenance in enzyme level could be due to the presence of antioxidant and polyphenols in
these medicinal plants, thereby preventing the secretion of enzymes from myocardium (Jahan et
al., 2012). The bark of T. arjuna enhances the generation of endogenous antioxidant compounds
of rats’ hearts and prevent oxidative stress associated with ischemic reperfusion injury of the
heart (Singh et al., 2008).
The Analysis of Variance (ANOVA) indicated the model F-value of 114.98, which
implies the significance of quadratic model (Table 4.9). The value of R2 (0.9793) for SGOT was
closer to unity, indicating better the empirical model fits the actual data (Fan et al., 2008).
Moreover, the accuracy and reliability of the experiment was also confirmed by the coefficient of
variation (3.71) for SGOT. This suggested that the predicted quadratic model defined well the
behavior of the studied markers.
Table. 4.9 Analysis of variance (ANOVA) for response surface methodology of SGOT (IU/L) as a function of independent variables
Source SS Df MS F Value Prob > F Model 8591.59 14 613.69 114.98 < 0.0001 Significant
A-Conc. 4741.56 1 4741.56 888.41 < 0.0001 .B-Plant 1909.99 6 318.33 59.64 < 0.0001
AB 399.52 6 66.59 12.48A2 1540.52 1 1540.52 288.64
Residual 181.46 34 5.34Lack of Fit 174.95 20 8.75 18.80 < 0.0864 Non
Significant Pure Error 6.52 14 0.47Cor Total 8773.05 48 R-Squared 0.9793 Standard Deviation 2.31
Adj R-Squared 0.9708 Mean 62.34Pred R-Squared 0.9633 Coefficient of Variation (CV) % 3.71Adeq Precision 36.813 Prediction Error Sum of Squares (PRESS) 321.95
93
The RSM depicted optimized concentration of 165, 157, 173 and 170 mg/kg b.wt for
maximum protective potential of T. arjuna, C. oxyacantha, P.nigrum and E. cardamom
respectively and the biochemical analysis showed the effective concentration ranged from 140-
170 mg/kg. The RSM suggested optimal concentration of 178 mg/kg of C. sativum, which fall in
our experimental range of 170-200 mg/kg which noticeably maintained the level of SGOT. In
case of A. sativum and R. serpentina the range of 140-200 mg/kg b.wt. gave the best respons
while the RSM suggested 174 and 156 mg/kg b.wt.
Table. 4.10 Optimized concentrations of medicinal plants for SGOT (IU/L) against
salbutamol induced Myocardial infarction
Sr. No. Plants extract Optimum concentrations of selected medicinal
plants (mg/kg)
SGOT (IU/L)
Based upon
biochemical evaluation
Suggested by RSM
1 T. arjuna 140-170 165 502 C.oxyacantha 140-170 157 41
3 P.nigrum 140-170 173 56
4 C.sativum 170-200 178 52
5 A. sativum 140-200 174 46
6 R. serpentine 140-200 156 52
7 E. cardamom 140-170 170 59
4.6.1.1.1.3 LDH:
Lactate dehydrogenase (LDH) is a cardiospecific enzyme, existed in myocardium and
released into the blood stream following myocytes injury and disintegration of the subcellular
and cellular compartments (Mnafgui et al., 2015).
94
The normal and positive control groups presented 250 and 519 IU/L level of LDH
respectively. The high level of LDH in positive control group is due to the reason that salbutamol
causes leakage of LDH from cardiomyocytes into the blood stream. Different treatment groups
were administered with varying concentrations of selected medicinal plants and their effects on
the level of myocardium specific LDH (H4) have been presented graphically in Fig. 4.39. The
first treatment group was administered with different concentrations of T. arjuna prior to the
salbutamol intoxication. The increase in concentration from 80 to 170 mg/kg resulted in decrease
in the level of LDH (437 to 299 IU/L). The concentrations of 140 and 200 mg/kg b.wt. of T.
arjuna did not show much variation in maintaining the level of LDH against salbutamol induced
MI. However 170 mg/kg.b.wt of T. arjuna would be considered as the dose of choice that
depicted the effective response to uphold the LDH level (299 IU/L) near to normal. The
maintenance of LDH by T. arjuna has been widely explored in the scientific researches all over
the world owing to its powerful antioxidant activities (Aslam et al., 2015). Traditional remedial
system also documented the cardioprotective properties of T. arjuna (Nema et al., 2012).
The second group treated with different concentrations of C. oxyacantha illustrated the
dose dependent response to maintain the level of LDH against salbutamol intoxication. The
increase in concentration from 80 to 170 mg/kg showed the gradual decline in the level of LDH
from 399 to 283 IU/L. The concentration of 200 mg/kg of b.wt. depicted the closs effectiveness
to the concentration of 170 mg/kg.b.wt. in order to maintain the level of LDH. Thus the
concentration of 170 mg/kg b.wt. could be recommended as therapeutic dose.
In third treatment group, increased in concentrations of P. nigrum from 80 to 170 mg/kg
b.wt resulted in increase in protective potential against salbutamol induced MI with respect to
LDH level. The concentration of 200 mg/kg did not show any protective effect as it kept the
level of LDH at 316 IU/L. Therefore the concentration of 170 mg/kg b.wt. was the only dose of
choice that could keep the level of LDH near to normal.
The C. sativum given to forth group of rats showed the dose dependant response in order
to sustain the level of LDH near to normal control group. The increased in concentration from 80
to 200 mg/kg decreased the level of LDH from 421 to 305 IU/L. Several phytochemicals and
pharmacological studies of the various parts of C. sativum also supported its cardioprotective
potential against MI (Momin et al., 2012; Iqbal et al., 2012).
95
In case of treatment group five, different concentrations of A. sativum was given to rats
and found that the increase in concentration of A. sativum resulted in increase in protective
potential to maintain the level of LDH against salbutamol induced damage. The rats pretreated
with concentration of 200 mg/kg b.wt exhibited substantial maintenance of LDH at 287 IU/L,
which could be considered as therapeutic dose to maintain the level of LDH.
The sixth group pretreated with R. serpentina also showed the dose dependant response
with respect to decrease in the LDH level (442 to 306 IU/L) when treated with concentration of
80 to 170 mg/kg b.wt. The concentration of 200 mg/kg.b.wt showed an inconsequential
maintenance in the level of LDH (334 IU/L). The findings suggested that none of the
concentration of R. serpentina could maintain the level of LDH against salbutamol induced MI.
Thus comparatively the concentration of 170 mg/kg could be considered as better dose to
maintain the level of LDH.
Design-Expert® Sof tware
LDH
B1 T. arjunB2 C. oxy canthaB3 P. nigrumB4 C. sativ umB5 A. sativ umB6 R. serpentineB7 E. cardamom
X1 = A: Conc.X2 = B: Plant
Plant species
80.00 110.00 140.00 170.00 200.00
Conc.
LDH
270
335
400
465
530
96
Fig. 4.39 Graphical presentation of optimized concentration of medicinal plants for LDH
against salbutamol induced Myocardial infarction
The seventh treatment group of E. cardamom also presented the dose dependant decline
in enzyme level against MI. The least concentration of 80 mg/kg of E. cardamom did not show
any protective effect on the LDH level (519 IU/L) while further increase in concentrations from
110 to 200 mg/kg effectively decreased the level of LDH gradually from 420 to 292 IU/L. Goyal
et al. (2015) also demonstrated the cardioprotective potential of E. cardamom with concentration
of 200 mg/kg b.wt. against MI by bring back the level of endogenous antioxidants, maintaining
histopathology of myocardium, thus enhancing cardiac function.
The overall response of all the treatment groups gave a picture that C. oxyacantha and T.
ajuna restored the LDH level better even at their lower concentrations as compared to other
groups pretreated with various concentrations of selected medicinal plants. The reduction in
LDH leakage, by C. oxyacantha pretreatment endorsed the protection of the cell membrane from
myocardial injury (Kashyap et al., 2012; Panda and Naik 2009).
The ANOVA for the fitted quadratic model was presented in Table. 4.11. The value of
determination coefficient (R2) was 0.9832 which means that the calculated model was able to
explain 98.32% of the results. The significance of the model was also judged by F-test, which
showed that model had a high F-value (142.17).
Table. 4.11 Analysis of variance (ANOVA) for response surface methodology of LDH
(IU/L) as a function of independent variables
Source SS Df MS F Value Prob > FModel 2.076E+005 14 14828.25 142.17 < 0.0001 Significant A-Conc. 1.655E+005 1 1.655E+005 1587.14 < 0.0001 . B-Plant 15205.97 6 2534.33 24.30 < 0.0001 AB 11629.36 6 1938.23 18.58 A2 15226.65 1 15226.65 145.99Residual 3546.09 34 104.30Lack of Fit 3545.59 20 177.28 4963.82 < 0.0781 Non
significant Pure Error 0.50 14 0.036Cor Total 2.111E+005 48R-Squared 0.9832 Standard Deviation 10.21Adj R-Squared 0.9763 Mean 360.26Pred R- 41.125 Coefficient of Variation (CV) % 2.83
97
SquaredAdeq Precision 10.823 Prediction Error Sum of Squares (PRESS) 6086.98
The RSM suggested the optimized concentrations of selected medicinal plants which may
able to cure the MI (Table. 4.12). The RSM suggested that T. arjuna and C. oxyacantha may
restore the level of LDH near to normal at the corresponding optimum concentration of 186 and
179 mg/kg which was in accordance to our experimental range from 140 to 200 mg/kg. The
RSM recommended the concentration of 193 mg/kg b.wt. for P.nigrum which showed slight
deviation from our experimental findings (140-170 mg/kg b.wt). The RSM endorsed the
optimum concentration of 181 mg/kg as effective dose for C. sativum that was under our
experimental range of 170-200 mg/kg. The optimize dose suggested by RSM for A.sativum and
E. cardamom was 200 mg/kg which is in accordance to our experimental findings (200 mg/kg).
Table. 4.12 Optimized concentrations of medicinal plants for LDH (IU/L) against
salbutamol induced Myocardial infarction
The T. arjuna and C.oxyacantha presented the effective response to maintain the level of
cardiac enzymes (CK-MB, SGOT and LDH) during myocardial infarction. Jahan et al. (2012)
treated different groups of rabbits with T. arjuna extracts (200 mg/kg b.wt.) which significantly
stopped the isoproterenol induced leakage of all cardiac diagnostic markers (CK-MB, LDH and
AST), sensitive index to assess the degree of myocardial necrosis. The amount of these cardiac
98
Sr. No. Plant extract Optimum Concentration (mg/kg) of selected
medicinal plants
LDH
(IU/L)
Based upon
experimental evaluation
Suggested by RSM
1 T. arjuna 140-200 186 298
2 C.oxyacantha 140-200 179 284
3 P.nigrum 140-170 193 302
4 C.sativum 170-200 181 310
5 A. sativum 200 200 296
6 R. serpentina 200 180 317
7 E. cardamom 200 200 296
biomarkers in heart shows the changes in plasma membrane permeability. Due to therapeutic
potential of medicinal plants, these are considered valuable remedies. Plants having flavanoids
have been reported to possess strong antioxidant properties which is responsible for its
cardioprotective potential (Abirami and Kanagavalli 2013).
4.6.1.1.2 Antilipidemic profile:The lipid profile is a group of clinical tests comprising of total cholesterol, triglycerides,
HDL-c and LDL-c. The hypertriglyceridemia and low level of HDL-c are associated with the
development of cardiovascular diseases (Spalding et al., 2009). Hence the lipid profile along
with other risk factors is used to assess the risk level of development of cardiovascular disease
(Beaulah et al., 2014). The antilipidemic potential of varying concentrations (80, 110, 140, 170
and 200 mg/kg b.wt.) of the selected medicinal plants was evaluated and its findings were
presented graphically in Figures 4.40-43.
4.6.1.1.2.1 HDL-c:The HDL-c is inversely associated with risks of CHD and is a key component for
predicting the chances of development of cardiovascular diseases. The HDL-c shuttles the
cholesterol back to the liver for recycling hence preventing the cholesterol deposition in
bloodstream. Problem arises when there is no enough HDL to carry the cholesterol back to liver
hence creating an imbalance that may lead to heart diseases (Rader and Hovingh 2014).
The normal control group showed the HDL-c level (53 mg/dL) more than the positive
control group (23 mg/dL), indicating the reduction of good cholesterol due to intoxication of
salbutamol. The salbutamol might be responsible for accumulation of cholesterol in Cardiac
membranedue to rapid mobility of LDL-cholesterol from blood into the myocardial membranes
(Sangeetha and Quine 2006).
The response of varying concentrations of selected medicinal plants towards HDL-c
against salbutamol induced MI has been indicated graphically (Fig. 4.40). The HDL-c level was
28 and 24 mg/dL in rats pretreated with T. arjuna at the concentration of 80 and 110 mg/kg
respectively. This means that the said doses could not maintain the level of HDL-c relative to
normal control group. The HDL-c level was found well maintained at all the high doses of 140,
170 and 200 mg/kg.b.wt of T. arjuna with corresponding HDL values of 33, 48 and 37 mg/dL.
The best response regarding the maintenance of HDL-c was observed in rats pretreated with
concentration of 170 mg/kg. The excellent hypolipidemic potential of T. arjuna bark is due to its
99
ability to increase in hepatic clearance of cholesterol, down regulation of lipogenic enzymes and
inhibition of HMG- CoA reductase (Patil et al., 2011).
The second group was treated with different concentrations of C. oxyacantha through
preventive mode. The rats to which the dose of 80 mg/kg was given did not show any
considerable maintenance of HDL level (27 mg/dL). Increase in concentration of C. oxyacantha
from 110 to 200 mg/kg revealed the effective maintenance of HDL level from 37 to 53 mg/dL
respectively. The concentration of 200 mg/kg b.wt. of C. oxyacantha was considered as the dose
of choice to sustain the level of HDL near to normal control. The C. oxyacantha acts as lipid
regulating agent (Verma et al., 2007) and the cardioprotective potential of it is due to its
antioxidative strength and presence of oligomeric procyanidins (Chatterjee et al., 1996).
Design-Expert® Sof tware
HDL
B1 T. arjunB2 C. oxy canthaB3 P. nigrumB4 C. sativ umB5 A. sativ umB6 R. serpentineB7 E. cardamom
X1 = A: Conc.X2 = B: Plant
Plant species
80.00 110.00 140.00 170.00 200.00
Conc.
HD
L
21
30
39
48
57
Fig. 4.40 Graphical presentation of optimized concentration of medicinal plants for HDL
(mg/dL) against salbutamol induced Myocardial infarction
100
The rats treated with P. nigrum at the concentrations of 80, 170 and 200 mg/kg depicted
very close effectiveness in order to maintain the level of HDL with corresponding values of 34,
36 and 33 mg/dL. However comparatively, the concentrations of 110 and 140 mg/kg showed
better impact on the HDL level (40 and 46 mg/dL).
The fourth group of rats was pretreated with different concentrations of C. sativum. The
dose dependant response of C. sativum was observed to maintain the level of HDL-c near to
normal value (54 mg/dL). The concentration of 200 mg/kg of C. sativum showed maximum
potential to maintaine the level of HDL-c (54 mg/dL). The preventive treatment of C. sativum
against salbutamol induced MI is due to its natural antioxidant property, which reduces the
complications related to cardiac diseases (Kousar et al., 2011).
The response of A.sativum at varying concentrations towards HDL-c against salbutamol
induced toxicity has been presented graphically. The concentration of 110 and 140 mg/kg did not
show the effective response to control the level of HDL (24 and 25 mg/dL) against MI while
further increase in concentration (200 mg/kg) substantially sustained the HDL level up to 49
mg/dL. The A. sativum is reported to maintain the cholesterol level, blood pressure and also
delayed the progression of atherosclerosis thus preventing the heart diseases (Rottblatt et al.,
2002).
The sixth treatment group treated with R. serpentina sustained the HDL-c at the level of
27, 29, 36, 39 and 54 mg/dL at corresponding concentrations of 80, 110, 140, 170 and 200 mg/kg
b.wt. This showed the graduall maintenance in the level of HDL-c during MI. Therefor, the 200
mg/kg b.wt. of R. serpentina roots might be considered as therapeutic dose. R. serpentina has
been reported to enrich with almost 50 indole alkaloids (Deshmukh et al., 2012) that may be
responsible for maintenance of HDL-c level.
The E. cardamom given to seventh group of rats also showed the dose dependent
response. The increase in concentration resulted in considerable maintenance of HDL-c level.
However the concentrations of 110, 140 and 170 mg/kg depicted very close effectiveness to
maintain the level of HDL-c with values of 35, 38 and 40 mg/dL respectively. The E. cardamom
at its concentration of 200 mg/kg maintained the HDL level (46 mg/dL) near to normal control
group (53 mg/dL). Hence the concentration of 170 and 200 mg/kg b.wt. would be recommended
as the potant dose to maintain the level of HDL-c during MI. This suggested the cardamom as a
beneficial mediator in delaying the progression and development of MI.
101
It is assumed that HDL can eliminate the cholesterol within arteries and transfer it back to
the liver for reutilization, that’s why HDL-c is considered as “good cholesterol.” The effective
maintenance in the level of HDL-c during MI by selected medicinal plants might be due to the
reasons that these plants enhance the production of HDL-c or increase the activity of the protein
lipase (Prince et al., 2008; Hamid et al., 2013; Gomathi et al., 2014; Shatoor et al., 2014). Many
of the medicinal plants are reported to maintain the lipid profile during MI (Jahan et al., 2011;
Murugesan et al., 2012; Adi et al., 2013).
The statistical analysis was carried out through ANOVA and the findings have been
presented in Table. 4.13. The ANOVA showed the Pred R2 of 0.9235 is closed to the Adjusted R-
Squared 0.8639 as expected which indicated a small block effect (Loong et al., 2014). The
adequate precision measures the signal to noise ratio (Liu et al., 2010). Here the adequate
precision was 12.515 which indicated the importance of model.
Table. 4.13 Analysis of variance (ANOVA) for Response Surface Methodology of HDL
(mg/dL) as a function of independent variables
Source SS Df MS F Value Prob > F
Model 3756.24 21 178.87 15.51 < 0.0001 Significant A-Conc. 2237.79 1 2237.79 194.08 < 0.0001B-Plant 354.98 6 59.16 5.13 0.0012 AB 694.16 6 115.69 10.03 < 0.0001 A2 2.24 1 2.24 0.19 0.6631
Residual 311.31 27 11.53Lack of Fit 278.31 13 21.41 9.08 0.0501 Non
significantPure Error 33.00 14 2.36Core Total 4067.55 48
R-Squared 0.9235 Standard Deviation 3.40Adj R-Squared 0.8639 Mean 37.27Pred R-Squared 0.7738 Coefficient of Variation (CV) % 9.11Adeq Precision 12.515 Prediction Error Sum of Squares (PRESS) 920.01
The RSM was applied to get the optimal concentrations of selected medicinal plants
(Table. 4.14). The experimental values of T. arjuna ranged from the concentration of 170-200
102
mg/kg depicted the maximum cardioprotective potential and the RSM supported this range as it
suggested the concentration of 200 mg/kg as optimal dose. The concentration of 110 to 200
mg/kg of C.oxyacantha tried to maintain the HDL level while the RSM recommended the
optimal concentration of 200 mg/kg which was in accordance to given finding. The experimental
value of P. nigrum depicted that the concentration of 140 mg/kg prominently maintained the
HDL level near to normal but the RSM suggested the concentration of 110 mg/kg showing the
deviation from our experimental approach. The RSM presented the concentration of 200 mg/kg
for C.sativum and A. sativum to keep the level of HDL in normal range against salbutamol that
was also related to our findings. The recommended dose of R. serpentina by RSM was 180
mg/kg while the experimental finding suggested the maximum potential in rats pretreated with
concentration of 170-200 mg/kg. In case of E. cardamom the RSM proposed the concentration of
170 mg/kg while in our experimental analysis concentration of 140 to 200 mg/kg showed
effectiveness in order to maintain the level of HDL near to normal. Thus these suggested
concentrations could be used to maintain the HDL level during MI.
Table. 4.14 Optimized concentrations of medicinal plants for HDL (mg/dL) against salbutamol induced myocardial infarctionSr. No. Plants extract Optimum Conc. (mg/dL) of selected medicinal
plants
HDL (mg/dL)
Based upon biochemical
evaluation
Suggested by RSM
1 T. arjuna 170-200 200 39.11842 C.oxyacantha 110-200 200 53.80093 P.nigrum 140 110 38.2934 C.sativum 200 200 53.2935 A. sativum 200 200 42.78516 R. serpentine 170-200 180 50.92797 E. cardamom 140-200 170 45.1343
4.6.1.1.2.2 Low density lipoprotein cholesterol LDL:
103
A high level of LDL-c in serum is considered as a main risk factor for development of
coronary heart disease (Lamarche et al., 1997; Austin et al., 1988). It is also involved in
activating the inflammatory state which ultimately causes the heart diseases (Colpo 2005).
The positive control groups revealed high LDL-c level with the value of 154 mg/dL as
compared to normal control group (33 mg/dL). The elevated level of LDL-c in positive control
group indicated the salbutamol induced hyperlipidemia. Highly oxidative metabolites of
salbutamol accelerate the rate of peroxidation in membrane phospholipids. Salbutamol is
responsible for release of free fatty acids into plasma by the action of phospholipase A2 (Panda
and Naik 2009).
The effects of various concentrations of selected medicinal plants in different treatment
groups on LDL level against MI have been shown in Fig. 4.41. The first group of rats was treated
with various concentrations of T. arjuna. The rats treated with concentration of 80 and 110
mg/kg b.wt. showed LDL-c level 80 and 63 mg/dL respectively. Both of these concentrations
could not maintain the level of LDL considerably as compared to normal control group (33
mg/dL). However the rats treated with concentration of 140, 170 and 200 mg/kg b.wt. of T.
arjuna revealed considerable maintenance in the level of LDL-c (53, 51 and 55 mg/dL).
Although there is no remarkable difference in response of these concentrations therefore the
concentration of 140 mg/kg, being the lower concentration, may be considered as the dose of
choice to maintain the LDL level.
The second group was treated with various concentrations of C. oxyacantha showed the
dose dependant response as the increase in concentration from 80 to 170 mg/kg b.wt. tend to
decrease the LDL-c level from 62 to 46 mg/dL. The concentration of 170 mg/kg showed
maximum potential against salbutamol intoxication and could be considered as a potant dose to
maintain the level of LDL-c (46 mg/dL) near to normal control group. The antilipidemic
potential of C. oxyacantha may be due to stabilization of heart membrane and scavenging of
highly oxidized metabolites produced by salbutamol (Zafar et al., 2015).
The third group of rats was treated with various concentrations (80, 110, 170 and 200
mg/kg b.wt.) of P. nigrum. The increase in concentrations from 80 to 200 mg/kg depicted the
considerable decline in LDL level from 120 to 41 mg/dL. Therefore 200 mg/kg b.wt. would be
considered the effective concentration to maintain the LDL level near to normal control group.
104
The protective effect of P. nigrum is because of its antioxidant property that defence against
oxidation of LDL (Vijver 1997).
The treatment of rats with different concentrations of C. sativum showed that the
concentration of 80 mg/kg did not maintain the LDL level (105 mg/dL) against salbutamol
intoxication. However the rats pretreated with concentrations of 110, 140 and 170 mg/kg showed
the LDL-c level at 52, 56 and 48 mg/dL respectively. This showed that the concentrations of 170
mg/kg b.wt. is the dose of choice to maintain the level of LDL-c. The oral administration of
aqueous extracts of coriander seeds was reported to decrease the metabolic syndrome,
atherosclerotic indices and increased the cardioprotective potential (Aissaoui et al., 2011).
Design-Expert® Sof tware
LDL
B1 T. arjunB2 C. oxy canthaB3 P. nigrumB4 C. sativ umB5 A. sativ umB6 R. serpentineB7 E. cardamom
X1 = A: Conc.X2 = B: Plant
Plant species
80.00 110.00 140.00 170.00 200.00
Conc.
LDL
36
58
80
102
124
Fig. 4.41 Graphical presentation of optimized concentration of medicinal plants for LDL
(mg/dL) against salbutamol induced Myocardial infarction
The group of rats treated with A. sativum with various concentrations from 80 to 170
mg/kg depicted the decline in the level of LDL from 98 to 55 mg/dL. The rats pretreated with
concentration of 200 mg/kg b.wt. was less effective to maintain the LDL level (62 mg/dL) in
105
comparision to the 170 mg/kg b.wt. Thus the concentration of 170 mg/kg sustain the level of
LDL near to normal against salbutamol induced MI and could be referred as dose of choice. The
observed cardioprotective potential might be due to presence of flavanoid and its antioxidant
activity (Balasuriya and Rupasinghe 2011).
The group treated with various concentrations of 80, 110 170 and 200 mg/kg b.wt. of R.
serpentina showed the level of LDL-c 79, 66, 61 and 64 respectively. However the rats treated
with concentration of 140 mg/kg of R. serpentina effectively maintained the LDL-c at 58 mg/dL
near to normal.
The group of rats pretreated with different concentrations of E. cardamom showed the
decrease in the LDL-c level (116 to 61 mg/dL) with increase in concentrations from 80 to 140
mg/kg b.wt. However further increase in concentration up to 200 mg/kg b. wt. did not show any
considerable effect against salbutamol intoxication as it showed the LDL-c level 73 mg/dL. The
concentration of 140 mg/kg maintained the LDL level (61 mg/dL) up to some extant. Therefore
the concentration of 140 mg/kg of E. cardamom would be considered as therapeutic dose.
The results suggested that the C. sativum, T. arjuna and C. oxyacantha showed good
maintenance of LDL level as compared to other selected medicinal plants against salbutamol
induced MI. The lipid lowering effect of these medicinal plants may be due to stabilization of
heart membrane and scavenging of highly oxidized metabolites produced by salbutamol (Zafar
et al., 2015). The prior administration of extracts of various plants also showed significant
reduction in elevated serum lipid profile during myocardial infarction and responsible for normal
structural and architectural integrity of cardiac myocytes (Siddiq et al., 2012).
The experimental data were statistically analyzed using the Design-expert 7.0 (Table
4.15) The ANOVA depicted the significance of quadratic model with F value of 53.22. The
linear terms of concentrations (A) and plants (B) are also significant which showed that both
have important impact on the LDL profile. In addition, the coupling term AB is also significant
which indicated a positive interaction between the two variables (Conc. and Plants) to maintain
the tendency of LDL-c towards normalization (Noshadi et al., 2012). Moreover the values of the
determination coefficient R2 (0.9564) and the adjusted determination coefficient (0.9384)
showed the significance of model. Comparatively low value of the CV 7.52 % indicated a
improved accuracy and consistency of experimental study.
106
Table. 4.15 Analysis of variance (ANOVA) for response surface methodology of LDL
(mg/dL) as a function of independent variables
Source SS Df MS F Value Prob > F
Model 19560.68 14 1397.19 53.22 < 0.0001 Significant
A-Conc. 9533.46 1 9533.46 363.14 < 0.0001
B-Plant 3280.41 6 546.73 20.83 < 0.0001
AB 3717.43 6 619.57 23.60 < 0.0001
A2 3029.38 1 3029.38 115.39 < 0.0001
Residual 892.59 34 26.25
Lack of Fit 678.59 20 33.93 2.22 0.0654 Non significan
Pure Error 214.00 14 15.29
Cor Total 20453.27 48
R-Squared 0.9564 Standard Deviation 5.12
Adj R-Squared 0.9384 Mean 68.12
Pred R-Squared 0.9112 Coefficient of Variation (CV) % 7.52
Adeq Precision 27.358 Prediction Error Sum of Squares (PRESS) 1815.72
The optimal concentrations of selected medicinal palnts suggested by response surface
mehdology heva been presented in Table. 4.16. The RSM suggested 160, 144 and 170 mg/kg for
T. arjuna, C. oxyacantha and P. nigrum respectively that was related to our experimental
therapeutic range 140-170 mg/kg b.wt. The concentrations proposed by RSM for C. sativum, A.
sativum and E. cardamom was 173, 168 and 175 mg/kg respectively that were close to the
107
experimental finding (170 mg/kg b. wt). The RSM proposed concentration of 152 mg/kg for R.
serpentina to maintain the LDL level with in range that was near to experimental finding (140
mg/kg).
Table. 4.16 Optimized concentrations of medicinal plants for LDL (mg/dL) against
salbutamol induced myocardial infarction
Sr. No. Plants extract Optimum Conc.(mg/kg) of selected
medicinal plants
LDL(mg/dL)
Based upon biochemical
evaluaton
Suggested by RSM
1 T. arjuna 140-170 160 482 C.oxyacantha 140-170 144 433 P.nigrum 140-170 200 414 C.sativum 170 173 465 A. sativum 170 168 566 R. serpentina 140 152 547 E. cardamom 170 175 64
4.6.1.1.2.3 Triglycerides:
Triglycerides are rich in apo C-III that delays the lipolysis of VLDL and inhibits its
uptake and clearance from plasma (Aminoff 2004). Elevated level of triglycerides increases 1.9
and 1.8 folds risk of coronary heart diseases in men and women respectively (Nadeem et al.,
2013).
The normal and positive control group showed the level of triglyceride with
corresponding values of 46 and 131 mg/dL. The effect of different concentrations of selected
medicinal plants on the level of triglycerides has been presented in Fig. 4.42. The group of rats
treated with bark extract of T. arjuna at varying concentrations against salbutamol induced MI
showed that the concentration of 170 mg/kg b.wt. considerably maintained the level of TGs (57
108
mg/dL) near to normal. The excellent hypolipidemic effect of T. arjuna bark seems to be
mediated through increased hepatic clearance of cholesterol and inhibition of HMG- CoA
reductase (Oben et al., 2006; Kanakavalli et al., 2014).
The C. oxyacantha showed the dose dependent response, as the increase in concentrations
from 80 to 170 mg/kg resulted in the decline in level of TGs from 89 to 57 mg/dL. However the
concentration of 200 mg/kg showed the slight deviation from the dose dependent response by
keeping the level of TGs at 66 mg/dL. Thus the concentration of 170 mg/kg of C. oxyacantha
would be recommended as therapeutic dose to maintain the level of TGs. This finding was also
supported by Weikl et al. (1996) who studied the effect of C. oxyacantha at the concentration of
160 mg/kg during human clinical trial. The findings depicted that there was no changes in
electrolytes, liver enzymes and ESR and therefore the concentration of 160 mg/kg was
considered as safe dose. The higher concentrations may increase the chances of hypertension but
there are no reports of side effects with low doses. (Verma et al., 2007).
Design-Expert® Sof tware
TG
B1 T. arjunB2 C. oxy canthaB3 P. nigrumB4 C. sativ umB5 A. sativ umB6 R. serpentineB7 E. cardamom
X1 = A: Conc.X2 = B: Plant
Plant species
80.00 110.00 140.00 170.00 200.00
Conc.
TG
46
62.75
79.5
96.25
113
Fig. 4.42 Graphical presentation of optimized concentration of medicinal plants for
Triglycerides (mg/dL) against salbutamol induced Myocardial infarction
109
The rats treated with concentration of 80 mg/kg of P. nigrum did not show the effective
response to maintain the TGs level (81 mg/dL) against salbutamol induced MI as compared to
normal control group (43 mg/dL). However the concentration of 200 mg/kg considerably
maintained the level of TGs (48 mg/dL) near to normal and could be recommended as
therapeutic dose to maintain the level of TGs.
The group treated with C. sativum also showed the dose dependant decline in the level of
TGs from 86 to 53 mg/dL with corresponding increase in concentration from 80 to 170 mg/kg.
The concentrations of 200 mg/kg did not show any considerable effect to maintain the TGs level
(66 mg/dL) in comparision to the concentration of 170 mg/kg b.wt. and could not be used as
therapeutic dose. The concentration of 170 mg/kg depicted the maximum therapeutic potential
and could be used to maintain the level of TGs near to normal against salbutamol intoxication.
The ability of C. sativum to maintain the level of TGs might be due to presence of
phytoconstituents in it (Momin et al., 2012). These phytoconstituents also play a crucial role in
ethnomedicine, pharmaceutical and food industries (Burdock and Carabin, 2009). The dried
seeds of C. sativum have been used as herb in ethnomedicine for the treatment of a variety of
diseases (Chithra and Leelamma, 1999; Momin et al., 2012).
The group of rats treated with various concentrations of A. sativum prior to salbutamol
intoxication showed that the concentration of 80 mg/kg was unable to sustain the level of TG (99
mg/dL) near to normal control group (43 mg/dL). All other concentrations of A. sativum from
110 to 200 mg/kg b.wt. decreased the triglycerides level from 77 to 55 mg/dL. Therefore, the
concentration of 200 mg/kg b.wt. would be recommended as the effectiv dose of A. sativum.
The rats treated with various concentrations (80-200 mg/kg b.wt.) of R. serpentina
resulted in decrease in TGs level (104 to 73 mg/dL). This showed that there was no considerable
maintenance in TGs level as compared to the concentrations of other selected medicinal plants.
Similarly the rat treated with various concentrations (80-200 mg/kg b.wt.) of E. cardamom
demonstrated that none of the concentration was able to maintain the level of TGs within normal
range. Even the rats pretreated with concentration of 200 mg/kg depicted poor effect in order to
maintain the level of TGs (67 mg/dL).
The good lipid lowering effect of T. arjuna, C. sativum and C. oxyacantha is because of
constraint of liver cholesterol synthesis, higher bile acid discharge and enhancement of receptor
110
mediated catabolism of LDL cholesterol (Hamid et al., 2013; Gomathi et al., 2014). Moreover,
plant extracts also reported to activate the generation of HDL which enhance the activity of the
protein lipase (Prince et al., 2008; Shatoor et al., 2014).
The Analysis of Variance (ANOVA) indicated the model F-value of 114.98, which
implies the significance of quadratic model (Table 4.17). Closer the R2 value (0.9243) to unity,
better the empirical model fits the actual data (Fan et al., 2008). The Adequate Precision
measures the signal to noise ratio, should above 4 so that it can be considered desirable (Loong
et al., 2014). In this case, the Adequate Precision obtained was 20.970 and this indicated this
model is significant. Moreover, the accuracy and reliability of the experiment was confirmed by
the coefficient of variance (6.82 %) for TGs. This suggested that the predicted quadratic model
defined well the effect of the different concentrations on level of TGs.
Table. 4.17 Analysis of variance (ANOVA) for response surface methodology of TGs
(mg/dL) as a function of independent variables
Source SS Df MS F Value
Prob > F
Model 10094.08
14 721.01 29.64 < 0.0001 Significant
A-Conc. 6160.01 1 6160.01 253.25 < 0.0001 B-Plant 2478.53 6 413.09 16.98 < 0.0001 AB 716.83 6 119.47 4.91 <0.0010 A2 738.72 1 738.72 30.37 < 0.0001Residual 827.02 34 24.32Lack of Fit 697.02 20 34.85 3.75 <0.0773 Non significant Pure Error 130.00 14 9.29R-Squared 0.9243 Standard Deviation 4.93Adj R-Squared 0.8931 Mean 72.35Pred R-Squared 0.8549 Coefficient of Variation (CV) % 6.82Adeq Precision 20.970 Prediction Error Sum of Squares (PRESS) 1585.06
The optimized doses of selected medicinal plants recommended by RSM have been given
in Table. 4.18. The RSM suggested the concentration of 176 mg/kg of T. arjuna to maintain the
TGs level in normal range that is near to our experimental value (170 mg/kg). RSM predicted the
possible concentration of 160 mg/kg of C.oxyacantha, to keep the level of TGs near to normal
against salbutamol induced MI that lies in our experimental therapeutic range from 110 to 170
mg/kg.b.wt. For P. nigrum and C.sativum, the RSM suggested the concentration of 193 and 175
111
mg/kg respectively that approaches to our experimental range (170-200 mg/kg). The RSM
recommended 200 and 195 mg/kg for A. sativum and R. serpentine respectively which is in
accordance to our experimental value 200 mg/kg. In case of E. cardamom, the dose suggested by
RSM was 160 mg/kg while in our experimental analysis the concentration of 200 mg/kg was
able to control the level of TG.
Table. 4.18 Optimized concentrations of medicinal plants for triglycerides (mg/dL) against
salbutamol induced myocardial infarction
Sr.No. Plants name Optimum concentration of selected
medicinal plants
Triglycerides
(mg/dL)
Based upon
biochemical evaluation
Suggested by
RSM
1 T. arjuna 170 176 62
2 C.oxyacanth
a
110-170 160 60
3 P.nigrum 170-200 193 50
4 C.sativum 170-200 175 60
5 A. sativum 200 200 54
6 R. serpentine 200 195 74
7 E. cardamom 200 160 64
Polyphenols are potent antioxidant that not only neutralize the lipid free radicals but also
prevent the decomposition of hydroperoxides into free radicals (Li et al., 2009; Rohman et al.,
2010). Medicinal plants significantly ameliorated the elevated lipid profile in rats to which MI
was induced by salbutamol due to presence of polyphenols. The cardioprotective potential of
medicinal plants may also be due to scavenging of highly oxidized metabolites produced by
salbutamol and stabilization of integrity of heart membrane with a consequent decrease in the
leakage of these markers (Beaulah et al., 2014; Zafar et al., 2015).4.6.1.1.2.4 Total Cholesterol:
High levels of total blood cholesterol are associated with the onset of coronary heart
disease (Carol and Merrily, 1984). Different herbs and natural products are highly effective in
lowering the cholesterol levels (Chand et al., 2007).
112
In normal control group Total Cholesterol (TC) level was 43 mg/dL and the positive
control group that was treated with salbutamol only, depicted the elevated TC level (196 mg/dL).
Different groups of rats were treated with various concentrations of selected medicinal plants
prior to salbutamol administration and their effects against MI have been given in Fig. 4.43. The
first group treated with T. arjuna illustrated that the increase in concentrations from 80 to 170
mg/kg b.wt. consequently decreased the level of TC from 109 to 56 mg/dL. The rats pretreated
with the concentration of 200 mg/kg of this plant sustained the level of T. cholesterol at the value
of 67 mg/dL. Thus the concentration of 170 mg/kg was considered as the concentration which
may use to maintain the level of T. cholesterol within range during MI.
The second group was pretreated with C. oxyacantha showed the considerable
maintenance of TC at 60 and 59 mg/dL when treated with corresponding concentrations of 170
and 140 mg/kg b. wt. Therefore the concentration of 140 mg/kg b.wt being the least
concentration would be rcommended to maintain the level of TC near to normal.
The concentration of 80 mg/kg of P. nigrum did not maintain the level of TC (119
mg/dL) but further increase in concentration from 110 to 170 mg/kg b.wt. resulted in decrease
the level of TC from 81 to 41 mg/dL. The concentration of 170 mg/kg b. wt. being most effective
dose and would be considered as therapeutic dose.
C. sativum considerably illustrated the dose dependant response as the level of TC was 92
to 59 mg/dL with increase in concentrations from 80 to 170 mg/kg b.wt. The concentration of
200 mg/kg showed the slight deviation from the dose dependant response as the level of TC was
68 mg/dL. Hence the concentration of 170 mg/kg would be considered as therapeutic dose.
The fifth group of rats pretreated with concentration of 80 to 170 mg/kg of A. sativum
depicted decline in TC level (101 to 65 mg/dL) against salbutamol induced MI. The
concentrations of 170 and 140 mg/kg presented the same effect on the level of T. cholesterol
therefore, the concentration of 140 mg/kg b.wt. being the least concentration might be considered
as appropriate dose to overcome the elevated level of TC during salbutamol intoxication.
113
Design-Expert® Sof tware
Chol.
B1 T. arjunB2 C. oxy canthaB3 P. nigrumB4 C. sativ umB5 A. sativ umB6 R. serpentineB7 E. cardamom
X1 = A: Conc.X2 = B: Plant
Plant species
80.00 110.00 140.00 170.00 200.00
Conc.
Cho
l.
41
60.5
80
99.5
119
Fig. 4.43 Graphical presentation of optimized concentration of medicinal plants for T.
cholesterol (mg/dL) against salbutamol induced Myocardial infarction
The preventive treatment of R. serpentina showed the dose dependent response in order
to decrease the level of TC from 102 to 58 mg/dL with corresponding concentrations of 80 to
200 mg/kg b.wt. The concentration of 200 mg/kg of R. serpentina proved as effective dose to
keep the TC near to normal range. The group of rats treated with various concentrations of E.
cardamom prior to salbutamol intoxication did not maintain the level of T. cholesterol near to
normal control group. However the concentration of 170 mg/kg b. wt. revealed the maximum
potential to control the level of TC with value of 72 mg/dL.
The Analysis of Variance (ANOVA) indicated the model F-value of 59.98, which
implies the significance of quadratic model (Table 4.19). The value of R2 (0.9451) for TC was
closer to unity, indicating better the empirical model fits the actual data (Fan et al., 2008).
Moreover, the accuracy and reliability of the experiment was also confirmed by the coefficient of
114
variation (5.66) for TC. This suggested that the predicted quadratic model defined well the
behavior of the studied markers.
Table. 4.19 Analysis of variance (ANOVA) for response surface methodology of TC (mg/dL) as a function of independent variablesSource SS Df MS F Value Prob > F
Model 16582.63 14 1184.47 59.98 < 0.0001 Significant
A-Conc. 10496.03 1 10496.03 531.54 < 0.0001
B-Plant 1347.14 6 224.52 11.37 < 0.0001
AB 1677.86 6 279.64 14.16 < 0.0001
A2 3061.59 1 3061.59 155.05 < 0.0001
Residual 671.37 34 19.75
Lack of Fit 577.87 20 28.89 4.33 0.0937 Non signifcant
Pure Error 93.50 14 6.68
Cor Total 17254.00 48
R-Squared 0.9611 Standard Deviation 4.44
Adj R-Squared 0.9451 Mean 78.57
Pred R-Squared
0.9259 Coefficient of Variation (CV) % 5.66
Adeq Precision 27.116 Prediction Error Sum of Squares (PRESS) 1278.23
The Response Surface Methodology suggested the optimal concentrations of selected
medicinal plants given in Table. 4.20. The RSM suggested the concentration of 175, 163, 159
and 161 mg/kg of T. arjuna, C.oxyacantha, C. sativum and E. cardamom to maintain the TC
level that was related to our experimental ranged from 140-170 mg/kg. RSM predicted the
possible concentration of 193 and 174 mg/kg for P. nigrum and R. serpentine respectively which
is in accordance to our experimental range 170-200 mg/kg.
Table. 4.20 Optimized concentrations of medicinal plants for Total Cholesterol (mg/dL) against salbutamol induced myocardial infarction
115
Sr. No. Plants
extract
Optimum Conc. (mg/kg) of selected medicinal
plants
T.Cholesterol
(mg/dL)
Based upon biochemical
evaluation
Suggested by RSM
1 T. arjuna 140-170 175 632 C.oxyacantha 140-170 163 613 P.nigrum 170-200 193 464 C.sativum 140-170 159 585 A. sativum 140-170 159 676 R. serpentina 170-200 174 607 E. cardamom 140-170 161 74
The observed elevated levels of TC, TGs and LDL in salbutamol induced control group
indicated the presence of hyperlipidemia. The oxidative stress produced by salbutamol is mainly
responsible for damage to myocardial membrane, lipids leakage and prominent decrease in HDL
cholesterol levels (Fravin et al., 2004). The highly oxidative metabolites of salbutamol lead lipid
peroxidation responsible for the destructive reactions in cellular mechanism of myocardial
infarction (Sivakumar et al., 2007). These oxidative metabolites also also accelerate the rate of
peroxidation in membrane phospholipids that lead to hyperlipidemia (Panda and Naik 2009). The
mechanism involved in increase in lipids level by salbutamol was due to increase in adenylate
cyclase action causes to enhance cAMP production, which lead to increase of lipid accumulation
in myocardium (Adi et al., 2013).
The treatment of experimental animals with selected medicinal plants at different doses
decreased salbutamol induced hyperlipidemia. With preventive mode of treatment the levels of
lipid profile reduced closer to the normal level because of the remedial action of medicinal
plants. The group which was treated with C. oxyacantha, T. ajuna and R. serpentina showed the
maximum potential against MI and minimized the elevated enzymatic level considerably as
compared to other selected plants. The presence of terminoarjunoside I and other antioxidants are
responsible for the efficacy of T. arjuna. In our findings the LC-MS analysis of these selected
plants showed the presences of many phytochemicals responsible for its cardioprotective
potential. Quercetin present in C. oxyacantha, T. arjuna and A. sativum may be responsible to
116
cure the impairment of cardiac functions, possibly via a mechanism involving the improvment in
mitochondrial function during ischemia (Agnihotri et al., 2008). In case of lipid profile the
preventive treatment of coriander decreased the LDL-c and triglycerides level but increase the
HDL-c level considerably.
4.6.1.1.2 Antioxidant profiling:
Endogenous antioxidative defense is a very important source to neutralize the free radical
mediated tissue injuries. Superoxide dismutase and Catalase, the primary free radical scavenging
enzymes, are the first line of cellular defense against oxidative injury, decomposing O2 and H2O2
before their interaction to form more reactive hydroxyl radical (Kumar and Gurusamy, 2014).
The antioxidant profiling of the entire treatment groups has been discussed as follows.
4.6.1.1.2.1 Superoxide dismutase:
SOD, a free radical scavenging enzyme, is the first line of cellular defense against
oxidative injury decomposing superoxide and hydrogen peroxide before interacting to form the
more reactive hydroxyl radical. The equilibrium of enzyme is an important process for the
effective removal of oxygen free radicals (Dormandy, 1978; Bhattacharya et al., 2000).
The antioxidative strength of selected medicinal plants at various concentrations against
salbutamol oxidative stress has presented graphically in Fig. 4.44. The normal control group
showed the SOD with value of 12 IU/mg while the positive control group that was treated with
salbutamol resulted in decrease in SOD level (5 IU/mg). Reductions in myocardial SOD activity
strongly suggested the overwhelming superoxide radical generation and hydrogen peroxide
formation following catecholamine administration (Rathore et al., 2000).
The first group of rats treated with T. arjuna prior to salbutamol intoxication showed the
increase in maintenance of SOD with increase in its concentrations. However the concentration
of 200 mg/kg b. wt. of T. arjuna showed good maintenance of SOD level (10 IU/mg). The rats
treated with concentration of 170 mg/kg also showed maintenance in the level of SOD (9 IU/mg)
almost similar to the concentration of 200 mg/kg b.wt. Hence the concentration of 170 mg/kg
b.wt. of T. arjuna could be recommended as effective dose to kept the level of SOD within
normal range.
In second group, the rats treated with concentration of 110 and 140 mg/kg b. wt. of C.
oxyacantha did not show the maintenance of SOD level (5 and 6 IU/mg) and these
concentrations were unable to cope the complications related to salbutamol intoxication. Further
117
increased in concentration up to 200 mg/kg b.wt. showed the effective maintenance of SOD (11
IU/mg). The increase in endogenous antioxidants contributes to hawthorn’s cardioprotective
effect during MI and this property is primarily correlated with the oligomeric procyanidins
(Verma et al., 2007).
The third group pretreated with different concentrations of P. nigrum demonstrated the
dose dependant response as the antioxidative power increased with increase in concentration.
The concentration of 200 mg/kg showed the good maintenance of SOD level (10 IU/mg) and
could be considered as the dose of choice. Ahmad et al. (2012) reported the prevention of
oxidative stress by arresting free radicals and maintaining the level of SOD. The ability of P.
nigrum to maintain the level of antioxidant may be due to presence of flavonoids and phenolic
contents present in it.
The group of rats treated with C. sativum also showed dose dependant response against
oxidative stress induced by salbutamol. The rats treated with concentration of 200 mg/kg showed
effective maintenance of SOD with value of 10 IU/mg. In case of the group of rats treated with
different concentrations of A. sativum, the concentration of 80 and 110 mg/kg showed the equal
maintenance of SOD level (8 IU/mg) against salbutamol induced oxidative stress. However the
concentration of 200 mg/kg b. wt. depicted the considerable maintenance of SOD level (11
IU/mg). The group of rats treated with varying concentrations of R. serpentina also showed dose
dependant response. The rats pretreated with concentration of 110 and 140 mg/kg sustained the
SOD at the level of 7 IU/mg. While the further increase in concentration up to 200 mg/kg
b.wt.resulted in increase in the maintenance of SOD. This may be due to the presence of
therapeutic phytochemicals.
118
Design-Expert® Sof tware
SOD
B1 T. arjunB2 C. oxy canthaB3 P. nigrumB4 C. sativ umB5 A. sativ umB6 R. serpentineB7 E. cardamom
X1 = A: Conc.X2 = B: Plant
Plant species
80.00 110.00 140.00 170.00 200.00
Conc.
SO
D
4.2
6.15
8.1
10.05
12
Fig. 4.44 Graphical representation of dose optimization of medicinal plants for superoxide
dismutase
The rats pretreated with different concentrations of E. cardamom depicted the dose
dependant response. The maintenance of SOD level increased with increase in concentration
from 80 to 110 mg/kg but the level of SOD decreased with further increase in concentration up
to 140 mg/kg. The rats pretreated with concentration of 200 mg/kg prominently maintained the
level of SOD (12 IU/mg).
The ANOVA depicted the F-value 12.97 which implied the significance of cubic model
(Table. 4.21). The linear terms of concentration (A) and plant (B) are also significant which
showed that both have important impact on the level of SOD. In addition, the coupling term AB
is significant which indicated a positive interaction between the two variables including
concentrations and plants. This suggested that the predicted cubic equation defined well the real
behavior of the interaction. In addition, the closer the adjusted R2 value (0.8397) to the R2
(0.9098) depicted the significance of model (Hismath et al., 2011). It has also proved that the
cubic model is the best model as it showed the characteristic of a good model.
119
Table. 4.21 Analysis of variance (ANOVA) for response surface methodology of SOD (IU/mg) as a function of independent variablesSource SS Df MS F Value Prob > FModel 161.02 21 7.67 12.97 < 0.0001 Significant A-Conc. 120.07 1 120.07 203.13 < 0.0001 B-Plant 13.27 6 2.21 3.74 0.0077 AB 6.76 6 1.13 1.91 0.1162A2 9.62 1 9.62 16.27 0.0004Residual 15.96 27 0.59Lack of Fit 12.46 13 0.96 3.83 0.0090Pure Error 3.50 14 0.25Cor Total 176.98 48R-Squared 0.9098 Standard Deviation 0.77Adj R-Squared
0.8397 Mean 8.02
Pred R-Squared
0.7124 Coefficient of Variation (CV) % 9.59
Adeq Precision
11.951 Prediction Error Sum of Squares (PRESS) 50.90
4.6.1.1.2.2 Glutathione peroxidase:
GPX is implicated in cellular defence against xenobiotics and not only protects cell
membrane from oxidative damage, but also help to maintain the sulphydryl groups of many
proteins in reduced form, required for their normal function (Saiprasanna et al., 2012).
The normal control group of rats showed the 1.44 IU/mg of GPX while the group of rats
merely treated with salbutamol depicted the decrease in GPX level (0.531 IU/mg). Reduction in
myocardial GPx activities strongly suggested the overwhelming superoxide radical generation
and hydrogen peroxide formation following catecholamine administration (Rathore et al., 2000).
The rats pretreated with various concentrations of selected medicinal plants were divided into
seven groups and their response against oxidative stress was presented graphically (Fig. 4.45).
Although all the concentrations of selected medicinal plants showed dose dependant response as
the increase in concentration from 80 to 200 mg/kg of all the plants depicted increase in the
maintenance of the level of glutathione peroxidase however the concentration of 200 mg/kg
showed the prominent protection of GPX. The increase in antioxidant potential may be due to
increase in phenolic components such as flavonoids, phenolic acids and phenolic diterpenes.
120
These phenolic components possess many hydroxyl groups including O-dihydroxy group which
have very strong antioxidant power (Soni and Sosa, 2013). Among all the selected medicinal
plants, T. arjuna, P. nigrum and C. sativum showed good maintenance of GPX even at the
concentration of 80 mg/kg. T. arjuna has significant antioxidant properties and proved as a good
heart tonic (Jahan et al., 2012).
Design-Expert® Sof tware
GSH
B1 T. arjunB2 C. oxy canthaB3 P. nigrumB4 C. sativ umB5 A. sativ umB6 R. serpentineB7 E. cardamom
X1 = A: Conc.X2 = B: Plant
Plant species
80.00 110.00 140.00 170.00 200.00
Conc.
GS
H
0.4
0.7
1
1.3
1.6
Fig. 4.45 Graphical representation of dose optimization of medicinal plants for glutathione
peroxidase
The endogenous antioxidant enzyme level increased the synthesis of glutathione
peroxidase and attributed to free radical scavenging capacity of antioxidant polyphenols in
medicinal plants (Karthikeyan et al., 2007). Piper nigrum being a very good antioxidant is used
to treat cardiac diseases (Jahan et al., 2011). C. sativum has also been reported to increase in
catalase and Glutathione (GSH) content considerably in rabbits. This revealed the protective
antioxidant action of C. sativum on cells suffering from oxidative stress induced by free radicals
(Joshi et al., 2012).
121
The F value of 63.57 in ANOVA suggested the significance of model (Table. 4.22). The
value of R2 (0.9832) for GPX gave an idea that closer the R2 value to unity, better the empirical
model fits the actual data (Fan et al., 2007). Additionally, the accuracy and reliability of the
experiment was confirmed by the coefficient of variance (4.87 %) for GPX. The model was
deemed appropriate in this study based on the significance of model F-value, good agreement
between adjusted and predicted R2. Adequate Precision measures the signal to noise ratio should
above 4 so that it can be considered desirable. In this case, the Adequate Precision obtained was
31.061 and this indicated the significance of model.
Table. 4.22 Analysis of variance (ANOVA) for response surface methodology of GPX (IU/mg) as a function of independent variablesSource SS Df MS F Value Prob > F Model 3.23 21 0.15 63.57 < 0.0001 Significant A-Conc. 2.38 1 2.38 981.90 < 0.0001 B-Plant 0.43 6 0.072 29.72 < 0.0001 AB 0.057 6 9.542E-003 3.94 0.0059 A2 0.16 1 0.16 67.73 < 0.0001Residual 0.065 27 2.420E-003Lack of Fit 0.065 13 5.021E-003 1124.63 < 0.0001Pure Error 6.250E-005 14 4.464E-006Cor Total 3.30 48R-Squared 0.9802 Standard Deviation 0.049Adj R-Squared
0.9648 Mean 1.01
Pred R-Squared
0.9399 Coefficient of Variation (CV) % 4.87
Adeq Precision
31.061 Prediction Error Sum of Squares (PRESS) 0.20
4.6.1.1.2.3 Catalase:
Catalase, the primary free radical scavenging enzyme, is the first line of cellular defense
against oxidative injury, decomposing O2 and H2O2 before their interaction to form the more
reactive hydroxyl radical (Kumar and Gurusamy, 2014). Catalase is also responsible for
breakdown of H2O2, formed during the reaction catalyzed by SOD (Lobo et al., 2010).
The normal control group showed 34 IU/mg of catalase and the positive control group to
which the optimized dose of salbutamol was given depicted the 12 IU/mg of catalase.
Presumably, a decrease in CAT activity could be attributed to cross-linking and inactivation of
122
the enzyme protein in the lipid peroxides (Prabha et al., 2014). The preventive effect of selected
medicinal plants has been presented graphically in Fig. 4.46. Although all the plants showed
maximum protection against salbutamol induced oxidative stress but the C. sativum, R.
serpentina and C. oxyacantha showed good maintenance of catalase level even at the lower
concentration of 80 mg/kg b. wt.
The groups treated with various concentrations (80, 110, 140, 170 and 200 mg/kg b.wt.)
of T. arjuna, C. sativum and E. cardamom illustrated the dose dependent response. The increased
in concentrations resulted in increase in antioxidant activity of these selected medicinal plants.
Joshi et al. (2012) examined the antioxidant potential of C. sativum and reported the considerable
increased in catalase level in rabbits against oxidative stress induced by free radicals. The
cardioprotective potential of T. arjuna extracts in terms of preventive mode can be correlated
with polyphenolic fraction and antioxidant activity (Karthikeyan et al., 2007).
Although the group of rats pretreated with various concentrations of P. nigrum showed
the increase in the antioxidative strength with increase in concentration but the rats pretreated
with concentration of 170 and 200 mg/kg of P. nigrum showed almost equal effect on catalase
level 27 and 26 IU/mg respectively.
Design-Expert® Sof tware
CAT
B1 T. arjunB2 C. oxy canthaB3 P. nigrumB4 C. sativ umB5 A. sativ umB6 R. serpentineB7 E. cardamom
X1 = A: Conc.X2 = B: Plant
Plant species
80.00 110.00 140.00 170.00 200.00
Conc.
CA
T
14
20.75
27.5
34.25
41
Fig. 4.46 Graphical representation of dose optimization of medicinal plants for catalase
123
The maintenance of antioxidants level near to normal can be correlated to the free radical
scavenging potential of the medicinal plants during oxidative stress generated by myocardial
infarction (Mohanty et al., 2013; Goyal et al., 2010). Medicinal Plants are noticed to generate a
prominent effect on functional revival and improvement in the tissue defense antioxidant
network (Aslam et al., 2015).
The ANOVA indicated the model F value 29.95 for CAT which suggested the
significance of model (Table. 4.23). The accuracy and reliability of the experiment was
confirmed by the coefficient of variance (6.11) for catalase. The model was considered
appropriate in this study based on the good agreement between adjusted and predicted R2. In
addition, the closer the adjusted R2 value (0.9268) to the R2 (0.9588) showed the significance of
the model. It has also proved that the cubic model is the best model as it showed the
characteristic of a good model.
Table. 4.23 Analysis of variance (ANOVA) for response surface methodology of Catalase as a function of independent variablesSource SS Df MS F Value Prob > F Model 1669.13 21 79.48 29.95 < 0.0001 Significant A-Conc. 1104.20 1 1104.20 416.14 < 0.0001 B-Plant 417.63 6 69.61 26.23 < 0.0001 AB 66.86 6 11.14 4.20 0.0041 A2 12.87 1 12.87 4.85 0.0364Residual 71.64 27 2.65Lack of Fit 68.64 13 5.28 24.64 < 0.0001Pure Error 3.00 14 0.21Cor Total 1740.78 48R-Squared 0.9588 Standard Deviation 1.63Adj R-Squared
0.9268 Mean 26.67
Pred R-Squared
0.8762 Coefficient of Variation (CV) % 6.11
Adeq Precision
21.186 Prediction Error Sum of Squares (PRESS) 215.50
The presence of polyphenols in medicinal plants is responsible for their antioxidant
potential, neutralization of lipid free radicals and also prevents decomposition of hydroperoxides
into free radicals. The cardioprotective potential may also be due to scavenging of highly
124
oxidized metabolites produced by salbutamol and stabilization of heart membrane by herbal
medicines with a consequent decrease in the leakage of cardiac markers.
4.6.1.1.3 Hematological analysis:
The hematological analysis of various groups of rats treated with different concentrations
of T. arjuna, C. oxyacantha, C. sativum, P. nigrum, E. cardamom, A. sativum and R. serpentina
against salbutamol induced MI was executed (Table. 4.24). The higher concentration 200 mg/kg
b.wt. of T. arjuna, P. nigrum and R. serpentina depicted the significant decreased in the count of
RBC along with Hb contents. However, no effect could be observed on PCV, MCH, MCHC and
platelet count also remained unaffected. All other concentrations did not showed any negative
effect on hematological parameters. While the group that merely treated with salbutamol showed
significant changes in hematological parameters.
Table. 4.24 Hematological analysis of different groups of rats treated with various
concentrations of selected medicinal plants
Runs
Plplant ant Cco WBCs (×10^3/uL)
LYM(×10^3/uL)
GRA(×10^3/uL)
RBC×10^12/L
HGB g/dl
MCHC g/dl
MCV fl
PLT×10^3
Control 9.84 5.35 1.74 6.72 13.7 32.04 68.78 1144
Positive control
2.92 3.54 1.84 3.21 6.54 22.98 79.83 845
1 T. arjuna 80 10.47 7.70 0.42 5.22 11.0 33.27 63.36 1038 2 T. arjuna 80 9.63 7.38 0.37 6.49 13.5 31.16 66.71 1219
3 T. arjuna 110 8.43 6.79 0.20 4.55 10.22 35.17 63.78 1249 4 T. arjuna 170 6.86 4.54 0.69 8.34 17.1 30.67 66.83 1222
5 T. arjuna 200 9.75 5.40 0.20 3.79 7.5 31.82 62.22 988 6 T. arjuna 200 9.9 8.86 0.61 3.88 7.3 31.39 66.21 997 7 T. arjuna 140 10.86 6.68 1.32 6.20 12.4 31.68 64.23 14428 C. oxyacantha 80 11.84 7.35 1.04 8.72 15.7 34.04 65.85 1244
9 C. oxyacantha 80 10.45 5.45 1.25 7.45 12.4 32.28 63.43 1398
10 C. oxyacantha 110 10.43 8.35 1.81 7.92 15.3 33.57 64.64 1352
11 C. oxyacantha 170 11.04 6.87 1.76 7.21 13.5 32.09 63.81 1265
12 C. oxyacantha 200 8.90 7.37 1.86 7.59 14.4 31.07 67.44 899
13 C. oxyacantha 200 8.99 7.65 1.67 6.1 10.3 32.04 63.12 863
14 C. oxyacantha 140 9.26 8.23 1.56 8.21 13.9 32.76 66.35 1344
125
15 P. nigrum 80 9.88 5.35 1.73 6.62 13.1 30.04 65.85 1122
16 P. nigrum 80 10.09 6.65 0.49 5.91 9.8 32.84 60.81 869
17 P. nigrum 110 10.43 6.15 1.81 7.35 14.3 30.57 63.64 1242
18 P. nigrum 170 15.68 6.23 0.93 5.16 10.6 34.01 60.46 997
19 P. nigrum 200 10.80 3.27 1.86 4.19 10.4 31.07 64.44 898
20 P. nigrum 200 12.17 3.16 0.61 4.68 9.6 32.88 62.41 895
21 P. nigrum 140 15.25 6.32 0.78 5.69 10.12 33.47 61.42 1029
22 C. Sativum 80 10.41 4.03 2.78 5.31 12.9 32.42 74.94 1142
23 C. Sativum 80 13.57 3.92 0.68 4.46 10.01 32.64 70.73 1145
24 C. Sativum 110 10.47 6.42 129 7.17 14.3 30.62 65.13 1229
25 C. Sativum 170 13.42 7.29 1.85 5.64 12.0 34.47 61.71 1035
26 C. Sativum 200 11.57 3.87 1.15 7.65 10.9 31.32 66.32 900
27 C. Sativum 200 12.13 2.41 1.06 7.71 9.4 31.13 64.10 911
28 C. Sativum 140 7.21 5.23 0.98 6.67 11.23 32.65 62.81 1327
29 A. sativum 80 10.84 6.35 1.14 7.72 14.7 33.04 64.85 1244
30 A. sativum 80 11.04 7.12 1.29 7.87 10.1 32.84 62.81 1201
31 A. sativum 110 11.43 7.35 1.21 7.42 14.3 31.57 62.64 1352
32 A. sativum 170 12.08 8.02 1.09 7.51 15.12 32.81 63.29 1269 33 A. sativum 200 11.80 6.77 1.36 5.59 5.4 32.07 64.44 799
34 A. sativum 200 12.88 7.88 1.28 5.34 5.98 31.07 66.23 798
35 A. sativum 140 10.26 8.21 0.95 7.98 15.21 32.90 66.12 1193
36 R. serpentina 80 12.92 7.15 1.5 7.70 15 30.84 63.17 1256
37 R. serpentina 80 7.8 7.33 6.37 5.51 12.5 31.83 61.66 1119
38 R. serpentina 110 13.50 7.04 2.65 7.18 14.1 30.7o 64.40 1128
39 R. serpentina 170 13.62 6.22 2.37 6.39 12.4 31.78 61.11 1103
40 R. serpentina 200 3.27 3.22 1.16 1.42 6.7 21.58 66.43 879
41 R. serpentina 200 4.70 4.78 1.63 1.52 6.4 33.90 61.35 836 42 R. serpentina 140 10.29 4.58 1.63 4.52 10.28 32..81 62.19 1328 43 E. cardamom 80 9.84 5.35 1.74 6.72 13.7 32.04 65.85 1144 44 E. cardamom 80 10.69 7.31 0.93 3.12 7.8 30.82 68.74 1504
45 E. cardamom 110 10.43 6.35 1.01 7.12 13.3 31.57` 64.64 1252
126
46 E. cardamom 170 10.06 7.94 0.34 5.20 10.5 32.54 62.01 1325 47 E. cardamom 200 8.80 6.27 1.86 8.19 15.4 31.07 62.44 999
48 E. cardamom 200 9.37 8.15 0.36 7.18 10.6 34.40 61.67 962
49 E. cardamom 140 11.32 8.39 1.98 6.78 10.34 32.67 62.63 1224
The assessments of haematological parameters are used to determine the extent of
decreased count of RBC that showed the suppression of erythrocytes. Reduction in RBC, Hb and
PCV is an indication of either the destruction of RBC or their decreased production, which may
lead to anemia (Adedapo et al., 2007; Onyeyilli et al., 1998). Excessive intake of variety of
medicinal plants or their active ingredients have been reported to generate hypo proliferative or
non regenerative anemia, which is a stem cell disorder distinguished by reduced bone marrow
production of all blood components in the absence of a primary disease process infiltrating the
bone marrow or suppressing haematopoiesis (Olson et al., 1984). It showed that regular intake of
extract of medicinal plants may also cause these disorders in experimental animals. It may also
compromise the principal function of white blood cells (Adedapo et al., 2007).
4.7 Phase-III:
The optimal concentration of each herbal extract obtained in Phase-II, was further used to
formulate four herbal combinations (HCs) against the extent of jeopardized myocardium. To
achieve better results for the development of therapeutic approaches, the synergestic
cardioprotective potential of herbal combinations was evaluated against surgically induced
myocardial infarction. The surgical model to induce myocardial infarction facilitates a precise
timing and location of coronary events consequently leading to more reproducible results
(Klocke et al., 2007). The MI was induced by ligating the left anterior descending coronary
artery (LADCA) in dogs. The dogs were selected as experimental animal due to the relevance of
large animal models to human physiological and pathophysiological processes which is very
important (Lukacs et al., 2012).
4.7.1 Herbal combination therapy:
It is usually expected that the medicinal plants when used in combination, impart
additional therapeutic benefits due to the interaction among various active ingredients (Che et
al., 2013). Although the therapeutic effects of various herbal combinations have been
documented but still there is need to explore the synergestic potential of medicinal plants.
127
Different herbal combination of Rauvolfia serpentina, Terminalia arjuna, Coriandrum sativum,
Piper nigrum, Eletaria cardamom, Allium sativum and Crataegus oxyacantha were formulated
by using their optimized concentrations and with consultation of Complementry and alternative
medical practitionars with varying ratios (Table. 4.25).
Table. 4.25 Formulation of different herbal combinations of selected medicinal plants
HC R.serpenti
na
E.cardamo
m
P.nigru
m
A.sativum T.arjun
a
C.oxyacant
ha
C.
sativum
Herbal ratio
HC
1
1 0.5 1 0.5 - - 0.5
HC
2
1 0.5 1 0.25 - 1 0.5
HC
3
1 1 0.5 - 1 - 0.5
HC
4
0.5 - - 0.5 1 0.5 1
4.7.2 Effect of herbal combinations on cardiac markers:
The dogs were administered with said herbal combinations for three weeks before
ligating left anterior descending coronory artery. To investigate whether the combinations of
herbal extracts under investigation would offer any added advantage over individual herbal
treatment, the effects of HCs were compared with normal and the surgically induced MI group.
The potential of herbal combinations was evaluated by analysing the cardiac markers (CK-MB,
SGOT and LDH) as described under following headings.
4.7.2.1 CK-MB:
CK-MB is restricted primarily in the heart and considered a valuable diagnostic tool for
myocardial infarction. The damage to myocardium during MI would result in increase in the CK-
MB level in serum (Loh et al., 2007). The effect of different herbal combinations on CK-MB
level against surgically induced MI has been presented graphically in Fig. 4.47. The normal
control group showed the CK-MB level 173±3.51 IU/L throughout the experimental period.
There was considerabrle increase in CK-MB level (274±2.08 IU/L) in surgically induced MI
128
group after 12 hr of left anterior descending coronary artery ligation while the level of enzyme
was further raised up to 294.3±1.53 IU/L after 24 hr. This prominent increase in level of CK-MB
in surgically induced MI group might be due to enhanced susceptibility of damage to myocardial
cell membrane because of ligation (Beaulah et al., 2014). Alterations in integrity, fluidity and
permeability of myocardial membrane have been believed to be a reason for the leakage of CK-
MB (Ojha et al., 2012).
The first herbal combination (HC1) did not show any considerable maintenance in CK-
MB level (253±1 and 224 ±3 IU/L) after 12 and 24 hr of ligation as compared to normal control
group. In comparision of HC1, the group pretreated with HC2 showed better maintenance of CK-
MB level, 215±2 and 201±2.65 IU/L after 12 and 24 hr of ligation respectively. This
improvement in efficacy of HC2 might be due to the addition of C. oxyacantha that possessed
more cardioprotective polyphenols (Verma et al., 2007). The antioxidant and lipid neutralizing
potential of polyphenols in combination may synergise the therapeutic effect against myocardial
infarction (Li et al., 2009; Rohman et al., 2010). The group treated with HC3 showed the level of
CK-MB 241±2 IU/L after 12 hr and 203±1 IU/L after 24 hr of ligation. A decrease in CK-MB
level (213±1.73 IU/L) was observed in group pretreated with HC4 after 12 hr of ligating left
anterior descending coronary artery. After 24 hr of ligation, this group showed considerable
decline in the level of CK-MB with value of 192±1.53 IU/L that was very close to the control
group. The prior administration of HC4 depicted the better maintenance of the serum CK-MB as
compared to other herbal combinations. The phytoconstituents and antioxidants present in this
herbal combination might be responsible for the cardioprotection (Aslam et al., 2015). The HC4
may render the myocytes less leaky by preventing myocardial membrane destruction and
disorganization (Aslam et al., 2015).
129
Treatment
Time_hr
350
300
250
200
150
100
50
0
CK-M
B le
vel (
IU/L
)
HC1HC2HC3HC4Normal controlSurg
Treatment
24
82
96
27
61
792
00
22
42
53
174
.667
18
32
03
24
11
66
169
20
121
51
71
17
21
922
13
168
17
31
73
173
174
Fig. 4.47 Graphical representation of Cardioprotective effect of herbal combinations of plant extracts on CK-MB level (IU/L) in the serum of experimental groups through the preventive mode of treatment4.7.2.2 SGOT:
The increase in level of SGOT usually results in hemolytic anemia, myocardial infarction
and cholestatic disease of the liver and may be a useful tool in assessing the extent of damage
(Gupta, 2004; Javanmardi et al., 2003).
The effect of different herbal combinations on the level of SGOT has been presented in
Fig. 4.48. In normal control group the SGOT level was 43±2 and 46±1.05 IU/L with time
intervals of 12 and 24 hr respectively. The SGOT level raised up to 115±1.527 IU/L and
130
123±1.154 IU/L after the corresponding time intervals of 12 and 24 hr of LADCA ligation in
surgically control group. The ligation of coronary artery imparts an additional workload on the
remaining viable myocytes that may be unbearable, resulting in pathological stimuli and death
signals. The treatment with HCs might salvage these viable myocytes, which are at risk of injury,
thus prevent cell loss induced by necrosis (Mohanty et al., 2013). The HC1 showed the SGOT
level with value of 94±1.53 IU/L after 12 hr and 74±1 IU/L after 24 hr of ligation. The
pretreatment of HC2 showed the SGOT level 75±1 IU/L after 12 hr of surgery and considerably
maintained at level of 62±0.57 IU/L after 24 hr of ligation in LADCA as compared to surgical
control group (123±1.154 IU/L). The preventive treatment of HC3 after 12 and 24 hr of LADCA
ligation indicated the corresponding 84±1.53 and 73±1.53 IU/L level of SGOT (Fig. 4.48). There
was no considerable variation in the outcomes of HC1 and HC3. However the pretreatment of
HC4 showed maximum potential against myocardial infarction as it uphold the SGOT level 73±1
IU/L after 12 hr and 53±1.53 IU/L after 24 hr of LADCA ligation. The HC4 gave better results
and modulated the SGOT level near to normal control, suggesting the beneficial action of HC4
as compared to other groups. Pretreatment of HC4 improved the status of antioxidants that
further contributes to its cardioprotective property (Orhue and Nawanze, 2004; Rafatullah et al.,
2008; Mahaswari et al., 2008). The cardioprotective potential of herbal combinations is because
of the correlation of the total contents of phytoconstituents and biological activities (Sapna et al.,
2007).
The depletion of cardiomyocytes specific enzymes CK-MB, SGOT and LDH in ischemic
reperfusion control group revealed the injury of heart. However treatment with I. racemosa
significantly restored the myocardial antioxidant status and prevented the leakage of
cardiomyocytes specific enzymes (Ojha et al., 2010).
131
Tre atme nt
Time_hr
140
120
100
80
60
40
20
0
SG
OT lev
el (IU
/L)
HC1HC2HC3HC4Normal controlSurg
Treatment
13
91
23
11
545
687
494
45
677
384
44.3
33
3
566
275
444653
73
4546
44
44
43
Fig. 4.48 Graphical representation of Cardioprotective effect of herbal combinations of plant extracts on SGOT level (IU/L) in the serum of experimental groups through the preventive mode of treatment
4.7.2.3 LDH:
LDH has been used as traditional diagnostic tool for myocardial infarction and usually
raised within 6-12 hours of MI (Prabha et al., 2014). The preventive treatment of herbal
combinations against surgically induced MI on the level of LDH has been presented graphically
in Fig. 4.49. The serum analysis of normal control group revealed 223±1.15 to 235 IU/L of LDH
132
from 0 to 48 hr respectively. The LDH level in surgically induced MI group was considerably
higher (565.3±2.31 IU/L) as compared to normal control group. The group of dogs pretreated
with HC1 showed 382.33±1.53 IU/L of LDH after 12 hr and 283±1.15 IU/L after 24 hr of
ligation. In dogs treated with HC2, the LDH level was 291.67±1.15 IU/L and 264±2.08 IU/L at
corresponding time intervals of 12 and 24 hr after LADCA ligation. While the pretreatment of
HC3 showed 343±1.53 IU/L level of LDH after 12 hr and maintained at the level of 250±1 IU/L
after 24 hr of ligation. The preventive treatment of HC4 revealed considerable maintenance of
LDH level (285±2 IU/L) after 12 hr of ligation. The HC4 sustained the level of LDH up to
264±1.52 IU/L after 24 hr of ligating LADCA (Fig. 4.49).
Treatment
Time_hr
600
500
400
300
200
100
0
LD
H le
vel (
IU/L
)
HC1HC2HC3HC4Normal controlSurg
Treatment
328
405
561
23427
728
338
222
7245
250
343
226.
667
24226
4.66
729
122
6
234.
333
26428
522
6
235
227
225
223
Fig. 4.49 Graphical representation of Cardioprotective effect of herbal combinations of plant extracts on LDH level (IU/L) in the serum of experimental groups through the preventive mode of treatment
133
The HC2 and HC4 showed the prominent cardioprotective potential by maintaining the
cardiospecific markers (CK-MB, SGOT and LDH) near the normal against surgically induced
myocardial infarction. The HC3 also showed relatively better maintenance of enzyme level after
24 hr of LADCA ligation. Although the precise mechanism of the cardioprotective effects of
HCs in surgically induced myocardial injury is not fully understood but the cardioprotection by
HC4 treatment may be attributed to its favorable myocardial adaptogenic properties.
Furthermore, these herbal extracts might have the potential for the management of patients at
risk of myocardial infarction.
The increased in CK-MB, LDH and SGOT levels in surgically induced MI group as
compared to normal control group consistent with idea that development of degenerative changes
in myocardial cell membrane resulted in the leakage of myocardial enzymes from the tissue to
the plasma (Gokkusu et al., 2003). The therapeutic properties of plants have been widely
explored in the scientific researches owing to their powerful biological activities. Traditional
remedial system also documented several plants having cardioprotective properties. The
presence of flavonoids, glycosides and tannins are responsible for antioxidant, antihypertensive
and cardioprotective properties of Terminalia arjuna (Nema et al., 2012). The roots of Rauvolfia
serpentina are reported to enclose almost fifty indole alkaloids that attributed to its
cardioprotective and antihypertensive potential (Deshmukh et al., 2012). Seeds of Elettaria
cardamom are documented to use for the treatment of cardiac disorders (Verma et al., 2009). The
flowers, leaves and berries of Crataegus oxyacantha are reported to possess a diversity of
flavonoid compounds that minimize the risks of CVDs (Rasmussen, 2011). All these plants
showed sufficient therapeutic effects because of their synergetic potential and thus exhibited no
side effects (Tende et al., 2015). The results gave an idea that it is preferable to use herbal
combination instead of depending on single herb (Prince et al., 2008; Baber et al., 2012;
Rajalakshmy et al., 2011).
The herbal combinations (HC2 and HC4) considerably ameliorated cardiotoxicity by
keeping the levels of biochemical parameters towards the normal. Phytoconstituents and
antioxidants present in these herbal combinations might be responsible for their cardioprotection
against surgically induced myocardial necrosis, thus it can be optimally used in herbal
preparations to form novel herbal drugs for the treatment of cardiovascular diseases. In case of
HC4 the bioactive compounds present in different plants exert synergistic biofunctionalities in
134
combination rather than acting alone. This membrane stabilizing effect of plant mixture on the
myocardium secured the cardiac injury, and thus limiting the escape of these enzymes (Aslam et
al., 2015). Thus HC4 can be used as an alternative effective drug for the treatment of myocardial
infarction.
4.7.3 Effect of herbal combinations on heamodynamic variable:
4.7.3.1 The Mean arterial pressure:
Measurement of the hemodynamic variables was also incorporated into the experimental
design for better understanding and more precise information of the corelation between
biochemical and functional changes in the myocardium subjected to surgically induced damage.
The normal control group depicted the 85±6.81 mean arterial pressure (MAP) while the positive
control group showed the decline in MAP (33±4.35) after occlusion in LADCA (Fig. 4.50). The
pretreatment of HC1 tried to sustain the level of MAP up to 52±5.13. However the group treated
with HC2 and HC4 substantialy maintained the MAP 76 ±4.04 and 77± 5.13 respectively as
compared to other groups.
Similarly in the positive control group there was an abrupt increase in heart rate (HR)
(277±8.02) as compared to normal control group (186±4.04). It is well documented that a
considerable fall in MAP and increased HR indicated hemodynamic impairment and ventricular
dysfunction due to increased generation of ROS (Ojha et al., 2012). While the pretreatment of
herbal combinations to surgically induced MI group revealed considerable maintenance of HR
especially in HC4 (213±4.36) and HC2 group (224±3.61). A fall in MAP due to coronary
occlusion is expected to increase HR and myocardial contractility by activating the baroreceptor
reflex, which may subsequently result in reflex vasoconstriction and thus worsening the
imbalance, between myocardial oxygen demand and supply (Gupta et al., 2004).
Cardioprotective potential of inula racemosa in experimental model of ischemic reperfusion
injury also depicted the clear restoration of MAP and HR (Ojha et al., 2010).
4.7.3.2 Effect of herbal combinations on ventricular function:
A significant decline in Left Ventricular End Diastolic Pressure (LVEDP) (9±3.05)
marked the onset of myocardial infarction in surgically induced MI group which remained
decreased throughout the experimental period in comparison of normal control group (32±5.51)
(Fig. 4.50). The pretreatment with HC4 and HC2 effectively maintained the LVEDP level
25±2.52 and 18±1.53 respectively as compared to surgically induced ischemic group. The HC1
135
and HC3 also tried to sustain the LVEDP with corresponding values 12±4.04 and 08±1.53. The
increase in blood flow through the subendocardial region of the left ventricular muscle is the
major consequence of the reduction in LVEDP in surgically induced infarction group. Under
ischemic conditions, the disproportionate reduction in microcirculation and blood flow to the
subendocardial regions of the heart occour, which is subjected to the greatest extravascular
compression during systole. In addition, the therapeutic efficacy of HC4 against surgically
induced MI might be due to the improvement in both inotropic and lusitropic function of the
heart and considerable maintainenance of antioxidant defense capacity of the myocardium
(Mohanty et al., 2013).
Table. 4.26 Hemodynamic analysis of herbal combination against surgically induced myocardial infarction
Hemodynamic parameters
Control Surg HC1 HC2 HC3 HC4
MAP 85±6.81 33±4.35 52±5.13 76±4.04 57±4.16 77±5.13
HR 186±4.04 277±8.02 252±6.51 224±3.61 231±9.29 213±4.36
LVEDP 32±3.05 9±5.51 12±4.04 18±1.53 08±1.53 25±2.52
LVSP 120±4.58 79±5.56 91±8.33 107±3.21 102±4.04 112±2.08
The surgically induced myocardial infarction group showed the significant decrease in
left ventricular systolic pressure (LVSP) (79±5.56) as compared to normal control group
(120±4.58). The pretreatment of HC1 maintained the LVSP up to 91±8.33 while the HC2 tried to
sustain the level up to 107±3.21 (Table. 4.26). However the HC3 maintained the level of systolic
pressure 102±4.04 while the pretreatment of HC4 showed the marked restoration as compared to
other groups as it elevated the the level of LVSP (112±2.08) near to normal control group. It is
materialized that the HC4 are more potent in preventing the hemodynamic deteriorations
observed in the surgically induced MI group. The hydroalcoholic extract of Andrographis
paniculata was evaluated against MI induced by LADCA ligation. The LADCA ligation resulted
in significant cardiac dysfunction evidenced by reduced mean arterial pressure (MAP) and
increased heart rate (HR). The left ventricular contractile function was also altered (Ojha et al.,
2012).
136
Mohanty et al. (2009) also studied the efficacy of herbal combinations to limit the
myocardial injury in left anterior descending coronary artery ligation model. The animals treated
with herbal combination prior to left anterior descending coronary artery ligation maintained the
heamodynamic level near to normal as compared to positive control group. Many experimental
and clinical studies have observed that natural products could be used prophylatically in the
treatment of various disorders because of their antioxidants and adaptogenic properties (Nandave
et al., 2013).
Control Surgery HC1 HC2 HC3 HC40
50
100
150
200
250
300
MAPHRLVEDPLVSP
Fig. 4.50 Graphical representation of hemodynamic parameters of various groups treated
with different herbal combinations
137
The combination of herbal extracts significantly ameliorated the MI compromised
enzymatic status and hemodynamic alterations as compared to control ischemic group. The HCs
decreased the severity of pathological changes and significantly preserved the myocardial
markers confirming its salvaging effects (Tende et al., 2015).
4.7.4 Histopathological examination:
The histopathological findings of myocardial tissue in normal control group illustrated
clear integrity of the myocardial cell membrane. The myofibrillar structure was normal with no
inflammatory cell infiltration. The nuclei were also normal without any pyknotic changes (Fig.
4.51). The histopathological examination of surgically induced MI group showed extensive
myofibrillar degeneration related to infiltration and disruption of cardiac myofibers. There was
marked necrosis in the ventricular region. Pyknotic changes in nuclei were also observed (Fig.
4.52).
Fig. 4.51 The histopathological representation of cardiac tissue of normal control group
138
Fig. 4.52 The histopathological representation of cardiac tissue surgically induced MI control group
The treatment of HC1 prior to ligation showed myofibrilation (Fig. 4.53) while the
pretreatment with HC2 demonstrated marked improvement in surgically induced alterations but
there was cellular infilteration at few places. The nuclei were also normal (Fig. 4.54). The group
treated with HC3 did not protect the cardiac dysfunctions as compared to other groups.
Myocardial fibrillation as well as some pyknotic changes in nuclei were also seen in group
treated with HC3 (Fig. 4.55). The histopathological examination of group treated with HC4
showed that there was no myofibrilation and the cardiac parenchyma was also normal. This
confirmed the potential of herbal combination (HC4) over oxidative stress related to cardiac
ailment (Fig. 4.56).
139
Fig. 4.53 The histopathological representation of cardiac tissue of HC1 treated group
Fig. 4.54 The histopathological representation of cardiac tissue of HC2 treated group
140
Fig. 4.55 The histopathological representation of cardiac tissue of HC3 treated group
Fig. 4.56 The histopathological representation of cardiac tissue of HC4 treated group
141
The cardioprotective and histopathological examination of animals treated with herbal
mixture (Terminalia arjuna, Rauvolfia serpentina, Elettaria cardamom and Crataegus
oxyacantha) considerably ameliorated the cardiotoxicity by bringing back the cardiospecific
enzymes towards the normal (Aslam et al., 2015). Therefore it can be optimally used in herbal
preparations for the treatment of cardiovascular diseases with no side effects. Histopathological
examination of heart proved the cardioprotective potential of herbal combination that was in line
with many previous studies (Hina et al., 2010; Fathiazad et al., 2012; Mohanty et al., 2013;
Sahreen et al., 2011; Yousefi et al., 2014).
Conclusively the HC4 prepared by optimized medicinal plants showed better synergistic
cardioprotective potential. The bioactive compounds present in medicinal plants exert synergistic
biofunctionalities in combination by interacting with one another, rather than acting alone. HC4
can be used as an alternative of synthetic drugs for the treatment and management of
cardiovascular diseases as it considerably maintained the cardiac markers as well as
hemodynamic parameters against surgically induced MI because of its synergistic
cardioprotective potential. In view of the safety, efficacy and traditional acceptability of these
plants extract, well controlled clinical trials should be contemplated to establish the efficacy of
herbal combination in the treatment of myocardial infarction.
142
Chapter # 5 SUMMARY
Myocardial infarction (MI) and the resulting complications in cardiac functions are
leading cause of morbidity and mortality in developed countries. MI is a complex phenomenon
affecting the mechanical, electrical, structural and biochemical properties of the cardiac system
(Ramadoss et al., 2012). The research was planned in two parts including in vitro and in vivo
analysis. In vitro analysis involved the screening of selected medicinal plants by Angiotensin
Converting Enzyme inhibition assay. Out of all selected medicinal plants, the methanolic extracts
of Terminalia arjuna (56.23±3.427), Piper nigrum (63.03±0.153), Coriandrum sativum
(63.033±0.153), Allium sativum (52.9±2.621), Rauvolfia serpentina (63.467±3.198), Eletaria
cardamom (53.467±0.55) and Crataegus oxyacantha (64.267±0.2) showed maximum ACE
inhibition. These selected medicinal plants were further subjected to LC-MS analysis which
confirmed the presence of important phytoconstituents and phenolic acids responsible for
antioxidative and cardioprotective potential of these plants. The antioxidant execution of these
selected medicinal plants at different concentrations was performed by DPPH and DNA
protection assay. The plants showed the antioxidant potential with descending order Terminalia
arjun> Crataegus oxycantha> Allium sativum> Coriandrum sativum > Piper nigrum>
Rauvolfia serpentine> Eletaria cardamom. Moreover all the said plants showed the dose
dependant response for free radical scavenging potential. This may be due to increase in phenolic
components such as flavonoids, phenolic acids and phenolic diterpenes. These phenolic
components possess many hydroxyl groups including o-dihydroxy group which have very strong
antioxidant power (Soni and Sosa, 2013). The toxicity assay was also performed to get relatively
safe product. The cytotoxicity findings showed that all the selected medicinal plants are safe and
may be used as herbal medicine for various therapeutic purposes. In vivo analysis was conducted
in three phases. The phase-I included the preliminary trial, where the different concentrations of
salbutamol were optimized by using the Response Surface Methodology. The analysis revealed
that the concentration of 80 mg/kg b.wt of salbutamol considerably elevated the level of cardiac
markers including CK-MB, SGOT and LDH. This elevated cardiac markers indicated the onset
of myocardial infarction. In phase-II, various concentrations (80, 110, 140, 170 and 200 mg/kg
b.wt) of selected medicinal plants suggested by RSM were optimized against salbutamol induced
myocardial infarction. The cardiac markers and lipid profile were analyzed to get the optimal
143
concentration of each medicinal plant that could provide maximum protection against MI.
Among all the studied medicinal plants T. arjuna, C. oxyacantha and C. sativum showed good
cardioprotective potential even at their least concentration. In Phase-III, the optimized doses of
selected medicinal plants were used to formulate four different herbal combinations with
appropriate ratio. These herbal combinations were further evaluated for their cardioprotective
potential against MI induced by ligating left anterior descending coronary artery in dogs. The
pretreatment of HC4 including R.serpentina, A.sativum, T.arjuna, C.oxyacantha, C. sativum with
corresponding ratio of 0.5:0.5:1:0.5:1 sustained the cardiac marker enzymes (CK-MB, SGOT
and LDH) near to normal. The HC4 also maintained the hemodynamic parameters (MAP, HR,
LVEDP) against surgically induced MI. The medicinal plants in herbal combination (HC4) with
appropriate ratio showed better synergistic cardioprotective potential by interacting with one
another, rather than acting alone. The combination of herbal extracts significantly ameliorated
the MI compromised cardiac markers because of its bioactive contents.
144
LITERATURE CITED
Abbas, D.R. and D.M.A. Maliki. 2011. Isolation and identification of phenolic compounds from Elettaria cardamom seeds and study of their medicinal activity against pathogenic bacteria of prostate gland. J. of Missan Res. 8: 25-39.
Abirami, M. and U. Kanagavalli. 2013. Cardioprotective effect of grapeseed proanthocyanidin on doxorubicin induced myocardial injury in rats. Int. J. of Pharmacy and Life Sciences. 4(1).
Abudayyak, M., E.O. Nath and G. Ozhan. 2015. Toxic potentials of ten herbs commonly used for aphrodisiac effect in Turkey. Turk. J. Med. Sci. 45: 496-506.
Adaramoye, O.A. 2009. Comparative effects of vitamin E and kolaviron (a biflavonoid from arcinia kola) on carbon tetrachloride-induced renal oxidative damage in mice. Pakistan. J. Biol. Sci. 12: 1146–1151.
Adedapo, A.A., M.O. Abatan and O.O. Olorunsogo. 2007. Effects of some plants of the spurge family on haematological and biochemical parameters in rats. Vet. Arhiv. 77(1): 29-38.
Adi, K., K. Metowogo, A. Mouzou, P. Lawson, K. Gadegbeku, A. Agbonon, C. Lamboni, K. Essien, K. Aklikokou and M. Gbeassor. 2013. Evaluation of cardioprotective effects of Parkia biglobosa (Jacq. Benth) Mimosaceae stem bark. J Appl Pharm Sci. 3: 60-64.
Agnihotri, A.K., P. Lakhanpal and D.K. Rai. 2008. Role of cardiovascular diseases. Int. J. of Medical. 4: 31-49.
Ahmed, M. 2015. Some medicinal plant resources and traditional uses in Pakistan. Journal of Plant Breeding and Crop Science. 7(5): 157-161.
Ahsan, F., H.H Siddiqui, T. Mahmood, R.K Srivastav and A. Nayeem. 2014. Evaluation of cardioprotective effect of Coleus forskohlii against isoprenaline induced myocardial infarction in rats. Ind J Pharm Biol Res. 2: 17-25.
Aissaoui, A., S. Zizi, Z.H. Israili and B. Lyoussi. 2011. Hypoglycemic and hypolipidemic effects of Coriandrum sativum L. in Meriones shawi rats. J. of Ethnopharmacology. 137(1): 652-661.
Alam, M.N., J.B. Nusrat and M.D. Rafiquzzaman. 2013. Review on in vivo and in vitro methods evaluation of antioxidant activity. Saudi Pharm. J. 21(2): 143−152.
Alamgeer, M.N., H. Malik, S. Bashir, I.U. Khan, S. Karim, M.N. Mushtaq, H.U. Khan, M. Rashid, H. Naz and S. Samreen. 2015. Cardiotonic and vasoconstriction effects of aqueous methanolic extract of Paspalidium flavidum. Pakistan. J. Pharm. Sci. 28(2): 437-441.
Alaribe, S.I. 2008. A Survey of the Importance and Problems of Traditional Health care medicine, A case study of Ezinihitte Mbaise L.G.A. Imo State. Unpublished B.Sc. project, A.I.F.C.E. Owerri, Imo State.
Ali, A., M. Ali and M. Alam. 2006. Two new oleanane triterpene glycosides from the bark of Terminalia arjuna. Zeitschrift für Naturforschung, 61(10): 1282-1286.
Ali, M., S. Shahnaz and Husain. 2014. Analysis of volatile oil of the fruits of Elettaria cardamom (L.) Maton and its antimicrobial activity. World J. of Pharmacy and Pharma. Sci. 3: 45-54.
Alla, F., F. Zannad and G. Filippatos. 2007. Epidemiology of acute heart failure syndromes. Heart Fail Rev, 12: 91-95.
145
Alsarhan, A., N. Sultana, A. Al-Khatib and M.R.A. Kadir. 2014. Review on some malaysian traditional medicinal plants with therapeutic properties. J. of basic and appli. sci. 10: 149-159.
American Heart Association (AHA). Understand your risk of heart attack. 2012.Aminoff, M.J. 2004. Nervous System. In: Current medical diagnosis and Treatment. The
McGraw- Hill Company, 43rd ed. 956-963.Amma, K.P.A.P., M.P. Rani, I. Sasidharan and V.N.P. Nisha. 2010. Chemical composition,
flavonoid phenolic contents and radical scavenging activity of four major varieties of cardamom. Int. J. of Biol. and Medi. Res. 1(3): 29-76.
Amol, A., A. Dambal, A.D. Bhagwat and D.S.S. Khadabadi. 2014. Pharmacognostic Studies of the Terminalia arjuna. J. of Chem. and Pharma. Sci. Res. 2(5): 837-840.
Amy, B., M. Deanna and W. Christy. 2006. Hawthorn (online). Available from URL. 3: 34-65.Anouch, M., M.A. Eghbal, F. Fathiazad, H. Hamzery and N.S. Kouzehkonani. 2009. The
protective effects of garlic extracts against acetaminophen induced oxidative stress and glutathione depletion. Park .J. Biol. Sci. 12(10): 765- 771.
Ansor, N. M., N. Abdullah and N. Aminudin. 2013. Anti-angiotensin converting enzyme (ACE) proteins from mycelia of Ganoderma lucidum (Curtis) P. Karst. BMC Compl. and Alternative Med. 13: 256-289.
Aqil, F., M. Zahin and I. Ahma. 2008. Antimutagenic activity of methanolic extract of four ayurvedic medicinal plants. Indian journal of experimental biology, 46(9), 668.
Aruna, R., P.V. Sathiyarajeswaran, K. Gopakumar and R.S. Ramaswamy. 2014. Cardioprotective effects of kitchen culinaries mentioned in Siddha literature. J. of Pharmacog. and Phytochem. 3(3): 71-79.
Arya, V. and V.K. Gupt. 2011. Chemistry and pharmacology of plant cardioproectives. A review. IJPSR. 2: 1156-1166.
Asdaq, S. M. and M.N. Inamdar. 2010. Pharmacodynamic interaction of captopril with garlic in isoproterenol induced myocardial damage in rat. Phytother. Res. 24: 720-725.
Asker V., V.D.D.J. Berg and M.N.J.L. Tromp. 1996. Structural aspects of antioxidant activity of flavonoids. Free Radic. Biol. Med. 20: 331-42.
Aslam, S., N. Jahan and K.M. Khan. 2015. Efficacy of Herbal Mixture for the Treatment of Salbutamol Induced Myocardial Necrosis in Rabbits. Pakistan Vet. J. 35(3).
Attar, A.M. 2006. Comparative physiological study on the effect of Rosemary, Tarragon and Bay leaves extract on serum lipid profile of quail, Coturnix coturnix. Saudi J. Biol. Sci. 13: 91‒98.
Austin, M.A., J.L. Breslow, C.H. Hennekens, J.E. Buring, W.C. Willett and R.M. Krauss. 1988. Low-density lipoprotein subclass patterns and risk of myocardial infarction. JAMA. 260: 1917–1921.
Ayesha, N. Jahan., K.U. Rahman and S. Nosheen. 2013. Gemmomodification: An emerging source of natural antioxidants from Silybum marianum. Pakistan. J. Pharm. Sci. 26(3): 585-591.
Azmat, H., M. Javed and G. Jabeen. 2012. Acute toxicity of aluminium to the fish (Catla catla, Labeo rohita and Cirrhina mrigala). Pak Vet J, 32: 85-87.
Azmat, H., M. Javed and G. Jabeen. 2012. Acute toxicity of aluminium to the fish (Catla catla, Labeo rohita and Cirrhina mrigala). Pakistan Vet. J. 32: 85-87.
Babar, W., Z. Iqbal, M.N. Khan and G. Muhammad. 2012. An inventory of the plants used for parasitic ailments of animals. Pakistan. Vet. J. 32: 183-187.
146
Babu, D., P. Gurumurthy, S.K. Borra and K.M. Cherian. 2013. Antioxidant and free radical scavenging activity of triphala determined by using different in vitro models. J. of Med. Plant Res. 7(39): 2898-2905.
Balasuriya, B.W.N. and H.P.V. Rupasinghe. 2011. Plant flavonoids as angiotensin converting enzyme inhibitors in regulation of hypertension. Functional Foods in Health and Disease. 5: 172-188.
Banjari, I., S.B. Labovic, A. Misir and B. Huzjak. 2013. Mediterranean diet and cardiovascular diseases. J. of Regional Section of Serbian Medical Association in Zajecar. 38: 46-49.
Baqar, S.R. 2001. Text book of Economic Botany. Ferozsons (PVT) Ltd, Rawalpindi, Lahore, Karachi. 23-100.
Barman, N.R., P.K. Kar, P.K. Hazam and H.S. Pal. 2013. Cardioprotective effect of Urtica parviflora leaf extract against doxorubicin-induced cardiotoxicity in rats. Chin. J. Nat. Med. 11: 38-42.
Beaulah, A.G., M. Sadiq, V. Sivakumar and J. Santhi. 2014. Cardioprotective activity of methanolic extract of Croton sparciflorus on isoproterenol induced myocardial infarcted wistar albino rats. J. of Medicinal Plants Studies. 2(6): 01-08.
Begum, M.S.F. and V. Vimalnath. 2009. Bioassay directed screening of selected medicinal plants for antibacterial activity. Asian J. of Microbiol. Biotechnol. Env. Sci. 11(1): 965-1000.
Belovic, M., J. Mastilovic, A. Torbica, J. Tomic, D. Stanic and N. Dzinic. 2011. Potential of bioactive proteins and peptides for prevention and treatment of mass non communicable diseases. Food and Feed Res. 38(2): 51-61.
Belovic, M.M., N.M. Ilic, A.N. Tepic and Z.M. Sumic. 2013. Selection of conditions for angiotensin-converting enzyme inhibition assay: influence of sample preparation and buffer. Food and Feed Research. 40(1): 11-15.
Berman, F.A. 2000. Herbs and dietary supplements in the prevention and treatment of cardiovascular disease. Prev Cardiol. 3: 24-32.
Bhandari, U., M.N. Ansari, F. Islam. 2008. Cardioprotective effect of aqueous extract of Embelia ribes Burm fruits against isoproterenol induced myocardial infarction in albino rats. Indian J Exp Biol. 46: 35-40.
Bhattacharya, S.K., K.S. Satyan and S. Ghosal. 2000. Antioxidant activity of Bacopa monniera in rat frontal cortex, straitum and hippocampus. Phytotherapy. 14: 174-179.
Bhawya, D. and K.R. Anilakumar. 2011. Antioxidant DNA damage protection and antibacterial effect of Psoralea corylifolia. Asian J. Pharm. Clin. Res. 4(2): 149-155.
Biapa, N.P.C., G.A. Agbor, J.E. Oben and J.Y. Ngogan. 2007. Phytochemical studies and antioxidant properties of four medicinal plants used in Cameroon. Afr. J. Tradit. Complement. Altern. Med. 4(4): 495-500.
Bibi, G., I. Haq, N. Ullah, A. Mannan and B. Mirza. 2011. Antitumor, cytotoxic and antioxidant potential of Aster thomsonii extracts. Afr. J. of Pharm. and Pharmacol. 5(2): 252-258.
Blumenthal, M., A. Goldberg and J. Brinckmann. (Eds). 2000. Herbal medicine expanded Commission E monographs. Austin, TX: American Botanical Council; Newton, MA: Integrative Medicine Communications.
Brugada, R., J. Brugada, C. Antzelevitch, G.E. Kirsch, D. Potenza, J.A. Towbin and P. Brugada. 2000. Sodium channel blockers identify risk for sudden death in patients with ST-segment elevation and right bundle branch block but structurally normal hearts. Circulation. 101(5): 510-515.
147
Brussels. 2001. Third United Nations Conference on the least developed countries Profile. Medicinal Plants. 2: 21-31.
Burdock, G.A. and I.G. Carabin. 2009. Safety assessment of coriander (Coriandrum sativum L.) essential oil as a food ingredient. Food Chem. Toxicol. 47: 22–34.
Burns, J., P.T. Gardner, J.O. Neil, S. Crawford, I. Morecroft, D.B. McPhail, C. Lister, D. Matthews, M.R. MacLean, M.E. Lean, G.C. Duthie and A. Crozier. 2000. Relationship among antioxidant activity, vasodilatation capacity and phenolic content of red wines. J. Agri. Food Chem. 48: 220-230.
Capraz, M., M. Dilek and T. Akpolat. 2006. Hypertension and patient education. Int. J. Cardiol. 3: 15-19.
Carol, J.W.S. and F.C. Merrily. 1984. Nutrition principles and application in health promotion. 2nd. (Ed. J. B. Lippincott)
Chance, M. and A.C. Mehaly. 1977. Assay of catalase and peroxidase. Method Enzymol. 2: 764-817.
Chand, N., F.R. Durrani, M.S. Qureshi and Z. Durrani. 2007. Role of Berberis lycium in reducing serum cholesterol in broilers. Asian Aust. J. of Animal Sci. 20(4): 563.
Chatterjee, S. 1996. Antioxidant and human neutrophil elastase inhibitory activity of Crataegus extract (WS- 1442) Standardized or Oligomeric procyanidins (OPC). Proceedings of the 2nd International Congress on Phytomedicine. Sep. 11-14.
Che, C. T., Z.J. Wang, M.S.S. Chow and C.W.K. Lam. 2013. Herb-herb combination for therapeutic enhancement and advancement: theory, practice and future perspectives. Molecules. 18(5): 5125-5141.
Chen, H. and Y. Zuo. 2007. Identification of flavonol glycosides in American cranberry fruit. Food Chemistry. 101(4): 1357-1364.
Chen, H. Y. Zuo and Y. Deng. 2001. Separation and determination of flavonoids and other phenolic compounds in cranberry juice by high-performance liquid chromatography. Journal of Chromatography A. 913(1): 387-395.
Chen, Z.Y., C. Peng, R. Jiao, Y.M. Wong, N. Yang and Y. Huang. 2009. Antihypertensive nutraceuticals and functional foods. J. Agric. Food Chem. 57: 4485-4499.
Chithra, V. and S. Leelamma. 1999. Coriandrum sativum-mechanism of hypoglycaemic action. Food Chem. 67: 229–231.
Cohen J. and P.M. Shah. 2004. Cardiac hypertrophy and cardiomyopathy. Circ Res. 35: 1-2.Colpo, A. 2005. LDL Cholesterol:" Bad" Cholesterol or Bad Science?.Journal of American
Physicians and Surgeons. 10(3): 83.Cushman, D.W. and H.S. Cheung. 1971. Spectrometric assay and properties of the Angiotensin-
Converting Enzyme of rabbit lung. Biochem. Pharmacol. 20:1637-1648. Da Silva, E., P. Shahgaldian and A.W. Coleman. 2004. Haemolytic properties of some water-
soluble para-sulphonato-calix-[n]-arenes. Int. J. Pharm. 273: 57-62.Das, A., J.H. Wang and E.J. Lien. 1994. Carcinogenicity, mutagenicity and cancer preventing
activities of flavonoids: A structure-system activity relationship (SSAR) analysis. Prog. Drug Res. 42: 133–167.
Deshmukh, S.R., S.A. Dhanashree and B.A. Patil. 2012. Extraction and evaluation of indole alkaloids from Rauvolfia serpentina for their antimicrobial and antiproliferative activities. Int. J. of Pharmacy and Pharmaceutical Sci. 4(5): 329-334.
148
Devasagayam, P.A., J.C. Tilak, K.K. Boloor, K.S. Sane, S.S. Ghaskadbi and R.D. Lele. 2004. Free radicals and antioxidants in human health: Current status and Future Prospects. J. Associ. Phys. Ind. 52: 794−804.
Devlin, R.J. and J.A. Henry. 2008. Clinical review: major consequences of illicit drug consumption. Crit. Care. 12: 202.
Dey, A. and J.N. De. 2010. Rauvolfia serpentina (L). Benth. Ex Kurz. - A Review, Asian Journal of Plant Sciences. 9(6): 285-298.
Dhande, S.R., P.P. Dongare, A.A. Kaikini, K.A. Patil and V.J. Kadam. 2014. In vitro antioxidant and thrombolytic activities of medicinal plants. IJUPBS. 3: 78–87.
Dhevi, R., V. Elango and K. Gayathri. 2014. Cardioprotective and antioxidant effects of seeds of spermacoce hispida on isoproterenol induced myocardial infarction in rats. World J. of Pharmacy and Pharmaceutical Sci. 3(9): 1150-1158.
Dianat, M., M. Radan, M. Badavi and A. Sarkaki. 2014.The evaluation of inotropic properties and antidysrhythmic effect of vanillic acid and exercise on CaCl2 induced arrhythmia in young and aged rats. Res. J. of Pharmaceutical Biol. and Chemical Sci. RJPBCS. 5(3): 1545.
Dianita, R., I. Jantan, A.Z. Amran and J. Jalil. 2015. Protective effects of Labisia pumila var. alata on biochemical and histopathological alterations of cardiac muscle cells in isoproterenol-induced myocardial infarction rats. Molecules. 20(3): 4746-4763.
DonathNagy, G., S. Vancea and S. Imre. 2011. Comparative study of captopril derivatization reaction by LCUV, LCMS and CEUV methods. Croat. Chem. Acta. 84(3): 423-427.
Dormandy, T.L. 1978. Free radical oxidation and antioxidants. Lancet. 1: 647-650. Duangjai, A., K. Ingkaninan, S. Praputbut and N. Limpeanchob. 2013. Black pepper and piperine
reduce cholesterol uptake and enhance translocation of cholesterol transporter proteins.Journal of natural medicines. 67(2): 303-310.
Duncan, A.C., A.K. Jager and J.V. Staden. 1999. Screening of Zulu medicinal plants for angiotensin converting enzyme (ACE) inhibitors. J. of Ethnopharmacol. 68: 63-70.
Dwivedi, S. and D. Chopra. 2014. Revisiting Terminalia arjuna – An Ancient Cardiovascular Drug. J. Tradit. Complement. Med. 4(4): 224–231.
Ebrahimzadeh, M.A. and F. Bahramian. 2009. Antioxidant activity of Crataegus pentaegyna fruit extract used as traditional medicine in Iran. Pakistan J. Biol. Sci. 12: 413-419.
Edoga, H.O., D.E. Okwu and B.O. Mbaebie. 2005. Phytochemicals constituents of some Nigerian medicinal plants. Afr J. Biotechnol. 4(7): 685-688.
El-Sayed, E.M., A.S.A. El-azeem, A.A. Afify, M.H. Shabana and H.H. Ahmed. 2011. Cardioprotective effects of Curcuma longa L. extracts against doxorubicin-induced cardiotoxicity in rats. J Medicinal Plant Res. 5: 4049-4058.
Fan, J., X. Ding and W. Gu. 2008. Radical-scavenging proanthocyanidins from sea buckthorn seed. Food Chemistry. 102: 168–177.
Farvin, K.H.S., R. Anandan, S.H.S. Kumar, K.S. Shiny, T.V. Sankar and T.K. Thankappan. 2004. Effect of squalene on tissue defense system in isoproterenol induced myocardial infarction in rats. Pharmacol. Res. 50: 231-236.
Fathiazad, F., A. Matlobi and A. Khorrami. 2012. Phytochemical screening and evaluation of cardioprotective activity of ethanolic extract of Ocimum basilicum L. (basil) against isoproterenol induced myocardial infarction in rats. DARU J. of Pharmaceutical.
Filho, B., M. Jose, V.K.M. Martins, L.A. Rabelo, M.D. Moura, M.S. Silva, E.V.L. Cunha, M.F.V. Souza, R.N. Almeida and A.I. Medeiros. 2006. Natural Products Inhibitors of the
149
Angiotensin Converting Enzyme (ACE). A review between 1980 – 2000. Brazilian J. of Pharmacognosy. 16(3): 421-446.
Freitas, M.V.D., R.C. Netto, J.C.D.C. Huss, T.M.D. Souza, J.O. Costa, C.B. Firmino and N. Penha-Silva. 2008. Influence of aqueous crude extracts of medicinal plants on the osmotic stability of human erythrocytes. Toxicol. 22: 219-24.
Gao, D., T. Chang, H. Li and Y. Cao. 2010. Angiotensin I-converting enzyme inhibitor derived from cottonseed protein hydrolysate. African J. of Biotechnol. 9(53): 8977–8982.
Gauthaman, K., M. Maulik, R. Kumari, S.C. Manchanda, A.K. Dinda and S.K. Maulik. 2001. Effect of chronic treatment with bark of Terminalia arjuna: a study on the isolated ischemic reperfused rat heart. J. Ethnopharmacol. 75: 197‒201.
Gawade, B.V. and S.A. Fegade. 2012. Rouvolfia (reserpine) as a potential antihypertensive agent – a review. Int. J. of Pharmaceutical and Phytopharmacological Res. 2(1): 46-49.
Ghazali, A.R., R. Abdullah, N. Ramli, N.F. Rajab, M.S. Ahmad-Kamal and N.A. Yahya. 2011. Mutagenic and antimutagenic activities of Mitragyna speciosa Korth extract using Ames test. Journal of Medicinal Plants Research. 5(8): 1345-1348.
Giannopolitis, C.N. and S.K. Ries. 1997. Superoxide dismutase occurrence in higher plants. Plant Physiol. 59: 309-314.
Gielen, S. and U. Landmesser. 2014. The Year in Cardiology 2013: cardiovascular disease prevention. Eur. Heart J. 551.
Gokkusu, C. and T. Mostafazadeh. 2003. Changes of oxidative stress in various tissues by longterm administration of vitamin E in hypercholesterolemic rats. Clin. Chm. Acta. 328: 155-61.
Golla, U., and S.S.R. Bhimathati. 2014. Evaluation of antioxidant and DNA damage protection activity of the hydroalcoholic extract of Desmostachya bipinnata L. Stapf. The Scientific World Journal. 2014.
Gomathi, R., M. Vijipriya and A. Kush. 2014. Cardioprotective effect of ethanolic extract of Medicago sativa stem on isoproterenol induced myocardial infarction in wistar albino rats. Int. J. of Pharmacy and Pharmaceutical Sci. 6(2): 56-87.
Goretta, A.L., J. Ottaviani and C.G. Fraga. 2006. Inhibition of angiotensin converting enzyme activity by flavanol rich foods. J. Agri. Food Chem. 54(1): 229-235.
Gosain, S., R. Ircchiaya, C.P. Sharma, S. Tharejad, A. Kalra, A. Deep and Y. Bhardwaj. 2010. Hypolipidemic effect of ethanolic extract from the leaves of Hibiscus sabdariffa L. in hyperlipidemic rats. Acta Pol Pharm-Drug Res. 67(2): 179-184.
Goyal, S.N., C. Sharma, U.B. Mahajan, C.R. Patil, Y.O. Agrawal, S. Kumari, D.S. Arya, and S. Ojha. 2015. Protective Effects of Cardamom in isoproterenol induced myocardial infarction in rats. International journal of molecular sciences. 16(11): 27457-27469.
Goyal, S.N., S. Arora, A.K. Sharma, S. Joshi, R. Ray, J. Bhatia, S. Kumari and D.S. Arya. 2010. Preventive effect of crocin of Crocus sativuson hemodynamic, biochemical, histopathological and ultrastuctural alterations in isoproterenol-induced cardiotoxicity in rats. Phytomedicine. 17: 227-232.
Graham, I., D. Atar and K. B. Johnsen. 2007. European guidelines on cardiovascular disease prevention in clinical practice: executive summary. Eur. Heart J. 28: 2375–2414.
Guha, G., V. Rajkumar, L. Mathew and R.A. Kumar. 2011. The antioxidant and DNA protection potential of Indian tribal medicinal plants. Turk J. Biol. 35: 233-242.
Gulcin, I. 2005. The antioxidant and radical scavenging activities of black pepper seeds. Int. J. Food Sci. Nutr. 56: 491-499.
150
Gunjal, M.A., A.S. Shah, A.S. Wakade and A.R. Juvenkar. 2010. Protective effect of aqueous extract of Moringa oleifera Lam. Stem bark on serum, lipids, marker enzymes and heart antioxidants parameters in isoproterenol induced cardiotoxicity. Indian J. Nat. Prod. Res.1: 485‒492.
Guo, C. 2003. Antioxidant activities of peel, pulp and seed fractions of common fruits as determined by FRAP assay. Nut. Res. 23: 1719–1726.
Gupta, A., A. Mittal, K.K. Jha and A. Kumar. 2011. Nature’s treasurer: plants acting on colon cancer. J of Stress Physio and Biochem. 7 (4): 217–231.
Gupta, S.K., I. Mohanty, K.K. Talwar, A. Dinda, S. Joshi and P. Bansal. 2004. Cardioprotection from ischemia and reperfusion injury by Withania somnifera: a hemodynamic, biochemical and histopathological assessment. Mol. Cell Biochem. 260: 39-47.
Gurgun, C., M. Ildizli, O. Yavuzgil, A. Sin, A. Apaydin and C. Cinar. 2008. The effects of short term statin treatment on left ventricular function and inflammatory markers in patients with chronic heart failure. Int. J. Cardiol. 123(2): 102-107.
Guyton, A., C. Hall and E. John. 2006. Text book of medical physiology 11 Edition. Philadelphia, Saunders Publisher.
Hajizadeh, R.M. and E. Mirzajani. 2011. The effects of consumption of raw garlic on serum lipid level, blood sugar and a number of effective hormones on lipid and sugar metabolism in hyperglycemic and/or hyperlipidemic individuals. Adv. Biol. Chem. 1(2): 29-33.
Halliwell, B. 2012. Free radicals and antioxidants: updating a personal view. Nutrition Reviews. 70(5): 257-265.
Hamid, M., and N. Jahan. 2013. Cardioprotective and antilipidemic effect of gemmotherapeutically treated Glycyrrhiza glabra against isoproterenol induced myocardial injury. Eur. J. of Medicinal Plants. 3(3): 405.
Hammoud, R.A., C.S. Vaccari, S.H. Nagamia and B.V. Khan. 2007. Regulation of the renin-angiotensin system in coronary atherosclerosis: a review of the literature. Vasc Health Risk Manag. 3(6): 937-987.
Harika, K., S. Mondi, D.J.L. Bai, V. Chidravar and U.M. Rao. 2014. A comprehensive review on cardioprotective medicinal plants. Int. J. Inv. Pharm. Sci. 2(4): 793-799.
Harisaranraj, R., K. Suresh and S. Saravanabab. 2009. Evaluation of the chemical composition Rauvolfia serpentina and Ephedra vulgeris. Adv. in Biol. Res. 3(6): 174-178.
Hassan, B.A.R. 2012. Medicinal Plants (Importance and Uses). Pharmaceutica Anal Acta. 3: 10.HernandezLedesma, B., D.M. Contreras and M. Recio. 2011. Antihypertensive peptides:
Production, bioavailability and incorporation into foods. Adv. in Colloid and Interface Sci. 165: 23-35.
Hina, S., K. Rehman and Z.H. Dogar. 2010. Cardioprotective effect of gemmotherapeutically treated Withania somnifera against chemically induced myocardial injury. Pakistan J. of Bot. 42(3): 1487–1499.
Hismath, I., W.M. Wan Aida and C.W. Ho. 2011. Optimization of extraction conditions for phenolic compounds from neem (Azadirachta indica) leaves. Int Food Res J. 18(3): 931-939.
Hu, X.J., H.P. He, H. Zhou, Y.T. Di, X.W. Yang, X.J. Hao and L.Y. Kong. 2008. New indole alkaloids from Rauvolfia verticillatae. Helvetica Chimica Acta. 89: 1344-1350.
Huang, X., C.S. Atwood, M.A. Hartshorn, G. Multhaup, L.E. Goldstein, R.C. Scarpa and R.E. Tanzi. 1999. The Aβ peptide of Alzheimer's disease directly produces hydrogen peroxide through metal ion reduction.Biochemistry. 38(24): 7609-7616.
151
Husain, S.S., and M. Ali. 2014. Analysis of volatile oil of the fruits of Elettaria cardamomum (L.) maton and its antimicrobial activity. World Journal of Pharmacy and Pharmaceutical Sciences. 3: 1798-1808.
Husain, S.Z., R.N. Malik, M. Javaid and S. Bibi. 2008. Ethonobotanical properties and uses of medicinal plants of Morgah biodiversity park, Rawalpind. Pakistan. J. Bot. 40(5): 1897-1911.
Hussain, A., S. Naz, H. Nazir and Z.K. Shinwari. 2011. Tissue culture of black pepper (Piper nigrum L.) in Pakistan. Pakistan. J. Bot. 43(2): 1069-1078.
Iqbal, Z., W. Babar, Z.U.D. Sindhu, R.Z. Abbas and M.S. Sajid. 2012. Evaluation of anthelmintic activity of different fractions of Azadirachta indica A. Juss seed extract. Pak. Vet. J. 32: 579‒583.
Ittagi, S., V.K. Merugumolu and R.S. Siddamsetty. 2014. Cardioprotective effect of hydroalcoholic extract of Tecoma stans flowers against isoproterenol induced myocardial infarction in rats. Asian Pac. J. Trop. Dis. 4(1): 378-384.
Jafary, M.H., A. Samad, M. Ishaq, S.A. Jawaid, M. Ahmad and E.A. Vohra. 2007. Profile of Acute Myocardial Infarction (AMI) In Pakistan. Pakistan J. Med. Sci. 23(4): 485-589.
Jaffe, A.S., L. Babuin and F.S. Apple. 2006. Biomarkers in acute cardiac disease: the present and the future. J. Amer. Coll. Cardiol. 48: 1-11.
Jaffery, M.H., K. Shaikh, G.H. Baloch and S.Z.A. Shah. 2014. Acute myocardial infarction; hypomagnesemia in patients. Professional Med. J. 21(2): 258-263.
Jagannadha, R.P., A.M. Jarari, A. Hai, A.K. Rawal, S.D. Kolla, S. Sreekumar, L. Khurana and N.R. Sidhanathi. 2010. Cardiac Biomarkers: The Troponins and CK- MB. J. Med. BS. 2(5): 190-197.
Jagetia, G.C., M.S. Baliga and P. Venkatesh. 2005. Influence of seed extract of Syzygium Cumini (Jamun) on mice exposed to different doses of γ-radiation. J. Radiat Res. 46(1): 59-65.
Jahan, N., K. Rehman, S. Ali and I.A. Bhatti. 2011b. Antioxidant activity of gemmotherapeutically treated indigenous medicinal plants. Asian J. Chem. 23: 3461-3470.
Jahan, N., K.U. Rahman, S. Ali, M.R. Asi and A. Akhtar. 2012. Cardioprotective potential of gemmomodified extract of Terminalia arjuna against chemically induced myocardial injury in rabbits. Pakistan Vet. J. 32(2): 255-259.
Jahan, N., K.U. Rehman and S. Ali. 2012. Cardioprotective and Antilipidemic potential of Cyprus rotundas in chemically induced cardio toxicity. Int. J. of Agric. and Biol. 14: 989-992.
Jahan, N., K.U. Rehman, S. Ali and I.A. Bhatti. 2011a. Antimicrobial potential of gemmo-maodified extracts of Terminalia arjuna and Euophorbia tirucalli. Int. J. Agri. Biol. 13: 1001-1005.
Javanmardi, J., C. Stushnoff, E. Locke and J.M. Vivanco. 2003. Antioxidant activity and total phenolic content of Iranian Ocimum accessions. Food chemistry. 83(4): 547-550.
Jayanthi, R., and P. Subash. 2010. Antioxidant effect of caffeic Acid on oxytetracycline induced lipid peroxidation in albino rats. Indian Journal of Clinical Biochemistry. 25(4): 371-375.
Jazila, E.M., D.A. Mount and H. Amarouch. 2007. Antimicrobial activity of Elettaria cardamomum: Toxicity, biochemical and histological studies. Int. J. of Drug Formulation and Res. Food Chem. 104: 32-39.
Jiang, M., L. Kang, Y. Wang, X. Zhao, X. Liu, L. Xu and Z. Li. 2014. A metabonomic study of cardioprotection of ginsenosides, schizandrin and ophiopogonin D against acute
152
myocardial infarction in rats. BMC Complementary and Alternative Medicine. 14: 350-357.
Jiang, P., W. Dai, S. Yan, Z. Chen, R. Xu, J. Ding, L. Xiang, S. Wang, R. Liu and W. Zhang. 2011. Biomarkers in the early period of acute myocardial infarction in rat serum and protective effects of Shexiang baoxin Pill using a metabolomic method. J. Ethnopharmacol. 138: 530–536.
Jiao, Y. and Y. Zuo. 2009. Ultrasonic extraction and HPLC determination of anthraquinones, aloe‐emodine, emodine, rheine, chrysophanol and physcione, in roots of Polygoni multiflori. Phytochemical Analysis. 20(4): 272-278.
John, J. 2014. Therapeutic potential of Withania somnifera: a report on phyto-pharmacological properties. IJPSR. 5(6): 2131-2148.
Joshi, S.C. and P.K. Jain. 2014. A review on hypolipidemic and antioxidant potential of some medicinal plants. World J. of Pharmacy and Pharmaceutical Sci. 3(11): 357-380.
Kaja, L.K., P.V.A. Rao, A.D.N. Kumar, B.G. Babu and R.R. Malla. 2014. Evaluation of antioxidant potentials of selected indian medicinal plants. Int. J. of Pharmaceutical Sci. and Res. IJPSR. 5(5): 1819-1829.
Kalita, S., G. Kumar, L. Karthik and K.V.B. Rao. 2012. In vitro antioxidant and DNA damage inhibition activity of aqueous extract of Lantana camara L.(Verbenaceae) leaves. Asian Pacific J. of Tropical Biomedicine. 2(3): S1675-S1679.
Kamel, A.M.F., M.R. Dina and H.A. Amin. 2010. Protective effect of Ginkgo biloba against experimental cardiotoxicity induced by isoproterenol in adult male albino rats, A Histological and Biochemical Study. Egypt. J. Histol. 33(4): 735 – 744.
Kanakavalli, K., S. Thillaivanan, P. Parthiban, G. Vijayalakshmi, M. Sudha and J. Sutha. 2014. Antihyperlipidemic herbs in siddha system of medicine. Int. J. of Pharma. Sci. 4(3): 541-545.
Kanski, J., M. Aksenova, A. Stoyanova and D.A. Butterfield. 2002. Ferulic acid antioxidant protection against hydroxyl and peroxyl radical oxidation in synaptosomal and neuronal cell culture systems in vitro: structure–activity studies. J. of Nutri. Biochem. 13: 273–281.
Kanwal, S., N. Ullah, I.U. Haq, I. afzal and B. Mirza. 2011. Antioxidant, antitumor activities and phytochemical investigation of Hedera Nepalensis, an important medicinal plant from Pakistan. Pak. J. Bot. 43: 85-89
Karthikeyan, K., B.R.S. Bai and S.N. Devaraj. 2007. Efficacy of grape seed poranthocyanidins on serum and heart tissue lipids in rats subjected to isoproterenol induced myocardial injury. Vascul Pharmacol. 40: 566-595.
Kashyap, C.P., V. Arya and N. Thakur. 2012. Ethnomedicinal and phytopharmacological potential of Crataegus oxyacantha - A review. Asian Pac. J. of Trop. Biomed. S1194-S1199.
Kaushik, P., P. Goyal, A. Chauhan and G. Chauhan. 2010. In Vitro evaluation of antibacterial potential of dry fruit extracts of Elettaria cardamomum Maton (Chhoti Elaichi). Iranian J. of Pharmaceutical Res. 9(3): 287.
Khajuria, A., N. Thusu and U. Zutshi. 2002. Piperine modulates permeability characteristics of intestine by inducing alterations in membrane dynamics: influence on brush border membrane fluidity, ultrastructure and enzyme kinetics. Phytomedicine. 9(3): 224-231.
153
Khan, G., S.E. Haque, T. Anwer, M.N. Ahsan, M.M. Safhi and M.F. Alam. 2014. Cardioprotective effect of green tea extract on doxorubicin induced cardiotoxicity in rats. Acta Pol Pharm Drug Res. 5: 861- 868.
Khan, M. and S.W. Wassilew. 1987. Natural pesticides from the Neem tree and other tropical plants. (Eds) Schmutterer H and Asher KRS, Germany: Digitalverlag GmbH. 645-650.
Khan, M.A., M.A. Khan, G. Mujtaba and M. Hussain. 2012. Ethnobotanical study about medicinal plants of poonch valley Azad Kashmir. The J. of Animal & Plant Sci. 22(2): 493-500.
Khan, S.H., S. Hassan, R. Sarder and M.A. Anjum. 2008. Effects of dietary garlic powder on cholesterol concentration in native desi laying hens. Am. J. Food. Technol. 3: 207-213.
Khopde, S.M., K.I. Priyadarsini and H. Mohan. 2001. Characterizing the antioxidant activity of amla (Phyllanthus emblica) extract. Current Sci. 81: 185–190.
Khursheed, F., K. Rehman, M.S. Akhtar, M.Z. Dogar and B. Khalil. 2010. Comparative antilipidemic effects of native and gemmotreated Withania somnifera (asghand) extracts. J. Appl. Pharm. 1(2): 47-59.
Kim, S.J., K.T. Rim, H.Y. Kim and J.S. Yang. 2010. Mutagenicity of octane and tetrasodium pyrophosphate in bacterial reverse mutation (Ames) test. J. Toxicol. Sci. 35: 555-562.
Kim, Y.J. 2007. Antimelanogenic and antioxidant properties of gallic acid. Biol Pharm Bull. 30: 1052-1059.
Kirillova, N.V., M.G. Smirnova and V.P. Komov. 2001. Sequential isolation of superoxide dismutase and ajmaline from tissue culture of Rauvolfia serpentina Benth. Appl. Biochem. and Microbiol. 37: 181-185.
Klocke, R., W. Tian, M.T. Kuhlmann and S. Nikol. 2007. Surgical animal models of heart failure related to coronary heart disease. Cardio. res. 74(1): 29-38.
Kostin, Y.V., E.I. Melokhova, E.I. Gendenshtein, N.D. Volkova, T.V. Astakhova and E.K. Saveleva. 1986. Antiarrhythmic activity of the total alkaloids from a Rauvolfia serpentina tissue culture. Pharma. Chem. J. 20(3): 214-217.
Koti, B.C., A.H. Vishwanathswamy, J. Wagwade and A.H. Thippeswamy. 2009. Cardioprotective effect of lipistat against doxorubicin induced cardiotoxicity in albino rats. Ind. J. Exp. Biol. 47: 227-32.
Kousar, S., N. Jahan, K.U. Rehman and S. Nosheen. 2011. Antilipidemic activity of Coriandrum Sativum.Bioscience Research. 8(1): 08-14.
Kratchanova, M., P. Denev, M. Ciz, A. Lojek and A. Mihailov. 2010. Evaluation of antioxidant activity of medicinal plants containing polyphenol compounds. Comparison of two extraction systems. Acta Biochim Pol. 57(2): 229-234.
Krishnamurthy, K.H. 2010. Elaaichi or cardamom (Elettaria cardamomum); Journal of New Approaches to Medicine and Health. 18: 32-41.
Krushna, G., M.A. Kareem and K.L. Devi. 2009. Antidyslipidemic effect of Aegle marmelos Linn. fruit on isoproterenol induced myocardial injury in rats. Int. J. Pharmacol. 6: 89-101.
Krushna, G.S.S., M.A. Kareem, V.D. Reddy, P. Padmavathi, S.A. Hussain and L.D. Kodidhela. 2012. Aegle marmelos fruit extract attenuates isoproterenol-induced oxidative stress in rats. J. Clin. Biochem. Nutr. 50(3): 199–204.
Kumar, A., R. Ilavarasan, T. Jayachandran, M. Deecaraman, M.R. Kumar, P. Aravindan, N. Padmanabhan and M.R.V. Krishan. 2008. Anti inflammatory activity of Syzigiumcumini seed. African J. of Biotechnol. 7(8): 941-943.
154
Kumar, G., L. Karthik and K.V.B. Rao. 2011. Hemolytic activity of Indian medicinal plants towards human erythrocytes: an in vitro study. Elixir Appl. Botany. 40: 5534-5537.
Kumar, S., J. Kamboj and S. Sharma. 2011. Overview for various aspects of the health benefits of Piper longum linn. fruit. J. of acupuncture and meridian studies. 4(2): 134-140.
Kumar, V.D.R. and K. Gurusamy. 2014. Antioxidant effect of Garcinia indica linn fruit extract against isoprenaline hydrochloride induced myocardial necrosis in rats. Int. J. Pharm. Sci. Drug Res. 6(3): 220-223.
Kumari, O.S. and N.B. Rao. 2015. Phytochemical analysis of Elettaria cardamom leaf extract. World J. of Pharmaceutical Res. 4(1): 30-37.
Kumari, R., B. Rathi, A. Rani and S. Bhatnagar. 2013. Rauvolfia serpentina L. Benth. ex Kurz. Phytochemical, Pharmacological and Therapeutic Aspects. Int. J. Pharm. Sci. Rev. Res. 23(2): 348-355.
Kumari, S., G. Shukla and A.S. Rao. 2011. The present status of medicinal plants aspects and prospects. Int. J. of Res. in Pharmaceutical and Biomed. Sci. 2(1): 19-23.
Kurosawa, T., F. Itoh, A. Nozaki, Y. Nakano, S. Katsuda, N. Osakabe, H. Tsubone, K. Kondo and H. Itakura. 2005. Suppressive effects of cacao liquor polyphenols (CLP) on LDL oxidation and the development of atherosclerosis in Kurosawa and Kusanagi hypercholesterolemic rabbits. Atherosclerosis. 179: 237-246.
Kutlu, T., K. Takim, B. Çeken and M. Kizil. 2014. DNA damage protecting activity and in vitro antioxidant potential of the methanol extract of Cherry (Prunus avium L). J. of Medicinal Plants Res. 8(19): 715-726.
Lacaille, D.V., U. Franck and H. Wagner. 2001. Search for potential angiotensin converting enzyme (ACE) inhibitors from plants. Phytomedicine. 8: 47-52.
Lakshmi, G., N. Smitha, S.V. Ammu, C.L. Priya and R.K.V. Bhaskara. 2014. Phytochemical Profile, In vitro Antioxidant and Hemolytic Activities of various leaf extract of Nymphaea nouchali linn: an in vitro study. Int. J. of Pharmacy and Pharmaceutical Sci. 6(6): 548-552.
Lamarche, B., A. Tchernof and S. Moorjani. 1977. Small dense low-density lipoprotein particles as a predictor of the risk of ischemic heart disease in men. Prospective results from the Quebec cardiovascular study. Circulation. 95: 69–75.
Larson, A.J., J.D. Symons and T. Jalili. 2012. Therapeutic potential of quercetin to decrease blood pressure: review of efficacy and mechanisms. Advances in Nutrition: An Int. Rev. J. 3(1): 39-46.
Lee, C.H. and J.H. Kim. 2014. A review on the medicinal potentials of ginseng and ginsenosides on cardiovascular diseases. J. Ginseng Res. 38: 161-166.
Li, H.Y., Z.B. Hao, X.L.Wang, L. Huang and J.P. Li. 2009. Antioxidant activities of extracts and fractions from Lysimachia foenum-graecum Hance. Bioresource Technology. 100(2): 970–974.
Lichtenstein, A.H., L.J. Appel, M. Brands, M. Carnethon, S. Daniels, H.A. Franch, B. Franklin, P. Kris-Etherton, W.S. Harris, B. Howard, N. Karanja, M. Lefevre, L. Rudel, F. Sacks, H. L. Van, M. Winston and J. Wylie-Rosett. 2006. Diet and lifestyle recommendations revision: a scientific statement from the American Heart Association Nutrition Committee. Circulation. 114: 82–96.
Lobo, V., A. Patil, Phatale and N. Chandra. 2010. Free radicals antioxidants and functional foods impact on human health. Pharmacognosy Reviews. 4: 118-126.
155
Loizzo, M.R., R. Tundis, F. Conforti, G.A.S. Menichini. 2009. Inhibition of angiotensin converting enzyme activity by Senecio Species. Pharm. Biol. 47(6): 516-530.
Lonkar, P. and P.C. Dedon. 2011. Reactive species and DNA damage in chronic inflammation: reconciling chemical mechanisms and biological fates. Int. J. Cancer. 128(9): 1999-2009.
Loong, N.K., P.F. Wahida and C.H. Chong. 2014. Optimisation of extraction of thymol from Plectranthus amboinicus leaves using response surface methodology. Journal of Engineering Science and Technology. 79 –88.
Lukacs, E., B. Magyari, L. Toth, Z. Petrasi, I. Repa, A. Koller, and I. Horvath. 2012. Overview of large animal myocardial infarction models (review). Acta Physiological Hungarica. 99(4): 365-381.
Luna, H., A. Pacheco, A. Solis, H.I. Perez, N. Manjarrez and J. Cassani. 2009. Study towards the improvement of the enantioselective hydrolysis of Naproxen esters by sheep liver acetone powder. J. of Biotech. Res. 1: 21-27.
Luseba, D., E.E. Elgorashi, D.T. Ntloedibe and J.V. Staden. 2007. Antibacterial, anti-inflammatory and mutagenic effects of some medicinal plants used in South Africa for the treatment of wounds and retained placenta in livestock. South African J. of Bot. 73: 378–383.
Mahaswari, C., R. Maryammal and R. Venkatamarayanan. 2008. Hepatoprotective activity of Orthosiphon stamineus on liver damage caused by paracitamol in rats. Jord. J. Biol. Sci. 1: 105-108.
Mahmood, A., M. Ahmad, A. Jabeen, M. Zafar and S. Nadeem. 2003. Pharmacognostic studies of some indigenous medicinal plants of Pakistan. Journal of ethnobotanical leaflets SIUC, USA. 31-35.
Mahmood, Z.A., M. Sualeh, S.B.Z. Mahmood and M.A. Karim. 2010. Herbal treatment for cardiovascular disease the evidence based therapy. Pakistan J. Pharm. Sci. 23(1): 119–124.
Mallick, S.R., R.C. Jena and K.C. Samal. 2012. Rapid in vitro multiplication of an endangered medicinal plant sarpgandha (Rauvolfia serpentina). Amer J. of Plant Sci. 3: 437-442.
Mandal, S., A. Patra, A. Samanta, S. Roy, A. Mandal, T.D. Mahapatra, S. Pradhan, K. Das and D.K. Nandi. 2013. Analysis of phytochemical profile of Terminalia arjuna bark extract with antioxidative and antimicrobial properties. Asian Pac. J. Trop. Biomed. 3(12): 960-966.
Manimegalai, S. and P. Venkatalakshmi. 2012. Cardioprotective effect of Cassia auriculata Linn., petal extract on isoproterenol induced myocardial infarction in male albino rats. Int. J. Pharm. Sci. Res. 3: 848‒852.
Manjunatha, P.M., S. Karia and D. Goli. 2011. Preventive effect of Rhododendron arboreum on cardiac markers, lipid peroxides and antioxidants in normal and isoproterenol induced myocardial necrosis in rats. African J. of Pharmacy and Pharmacol. 5(6): 755-763.
Manna, P., M. Sinha and P.C. Sil. 2007. Phytomedicinal activity of Terminalia arjuna against carbon tetrachloride induced cardiac oxidative stress. Pathophysiol. 1(2): 71-78.
Mansour, S.Z., K.S.K. Hassan and A.S.A. Hegazi. 2009. Evaluation of the antilipidemic effect of polyoxyethylenated cholesterol on rats fed high fat diet. J. Appl. Sci. Res. 5: 613‒621.
Mariee, A.D., G.M. Abd-Allah and H.A. El-Beshbishy. 2012. Protective effect of dietary flavonoid quercetin against lipemic-oxidative hepatic injury in hypercholesterolemic rats. Pharmaceutical biology. 50(8): 1019-1025.
156
Maruthappan, V. and K.S. Shree. 2010. Effects of Phyllanthus reticulates on lipid profile and oxidative stress in hypercholesterolemic albinorats. Ind. J. Pharmacol. 42: 388–391.
Mary, C.T., R. Kingston, D. Gilroy, M. Lehane and A. Furey. 2010. Hawthorn (Crataegus spp.) in the treatment of cardiovascular disease. Pharmacogn Rev. 4(7): 32–41.
Mastan, S.K., G. Chaitanya, L.T. Bhavya, A. Srikanth, G. Sumalatha and G.E. Kumar. 2009. Cardioprotective effect of methanolic extract of Syzygium cuminiseeds on isoproterenolinduced myocardial infarction in rats. Der Pharmacia Lettre. 1(1): 143-149.
Mathangi, T. and P. Prabhakaran. 2013. DPPH free radical scavenging activity of the extracts of the aquatic fern Marsilea quadrifolia Linn. Int. J. Curr. Microbiol. App. Sci. 2(10): 534-536.
Mathur, A., R. Purohit, D. Mathur, G.B.K.S. Prasad and V.K. Dua. 2011. Pharmacological investigation of methanol extract of Syzigumcuminii seeds and Crateva nurvula bark on the basis of antimicrobial, antioxidant and anti-inflammatory properties. Der. Chemica Sinica. 2(1): 174-181.
Menaka, C.T., R.N. Jadeja, S.S. Ansarullah, J.D. Karn, D.K. Shah, S.P. Patel, G.S. Salunke, R.V. Padate, A.V. Devkar and Ramachandran. 2011. Cardioprotective effect of Sida rhomboidea. Roxb extract against isoproterenol induced myocardial necrosis in rats. Experimental and Toxicol. pathology. 63: 351-356.
Mendis, S., P. Puska and B. Norrving. 2011. Global Atlas on Cardiovascular Disease Prevention and Control. World Health Organization (in collaboration with the World Heart Federation and World Stroke Organization), Geneva. 204-670.
Merzenich, U.G., H. Zeitler, H. Vetter and K. Kraf. 2009. Synergy research: vitamins and secondary plant components in the maintenance of the redox-homeostasis and in cell signaling. Phytomedicine. 16(1): 2-16.
Meschino, D.C.J. et al. 2014. Hawthorn (Crataegus oxyacantha). Natural Health Products. Compendium Herbals. 7: 1-5.
Mills, M., K. Bone and K. Churchill. 2000. Principles and Practice of Phytotherapy. Livingstone. 444-445
Mittal, B., A. Meenakshi, A. Sharma and V.K. Gothecha. 2012. Phytochemical and pharmacological activity of Rauvolfia serpentina - a review. Int. J. of Ayurvedic and Herbal Medicine. 2(3): 427-434.
Mnafgui, K., R. Hajji, F. Derbali, I. Khlif, F. Kraiem, H. Ellefi, A. Elfeki, N. Allouche, N. Gharsallah. 2015. Protective effect of hydroxytyrosol against cardiac remodeling after isoproterenol-induced myocardial infarction in rat. Cardiovasc. Toxicol.
Mohanty, I.R., D.S. Arya and S.K. Gupta. 2009. Dietary Curcuma longa protects myocardium against isoproterenol induced hemodynamic, biochemical and histopathological alternations in rats. Int. J. Appl. Res. Nat. Prod. 1: 19‒28.
Mohanty, I.R., S.K. Gupta, N. Mohanty , D. Joseph and Y. Deshmukh. 2012. The beneficial effects of herbs in cardiovascular diseases. Global Journal of Medical research. 12 (4): 38-58.
Momin, A.H., S.S. Acharya and V. Gajjar. 2012. Coriandrum sativum - review of advances in phytopharmacology. Int. J. Pharm. Sci. Res., 3: 1233–1239.
Montalescot, G., J. Dallongeville and E.V. Belle. 2007. STEMI and NSTEMI: are they so different? 1 year outcomes in acute myocardial. Eur. Heart J. 12: 1409-1417.
Morales, A. 2000b. Yohimbine in erectile dysfunction: the facts, Int. J. of Impotence Res. 12(1): S70-74.
157
Mradu, G., S. Saumyakanti, M. Sohini and M. Arup. 2012. HPLC profiles of standard phenolic compounds present in medicinal plants. Int. J. Pharmacog. Phytochem. Res. 4: 162-167.
Murray, M.T., 1995. The Healing Power of Herbs (2nd edition), Prima Publishing.Murugesan, M., M. Ragunath, S. Nadanasabapathy, R. Revathi and V. Manju. 2012. Protective
role of fenugreek on isoproterenol induced myocardial infarction in rats. Int. Res. J. of Pharmacy. 3(2): 211–216
Myers, R.H. and D.C. Montgomery. 2002. Response surface methodology process and product optimization using designed experiments. 2nd ed., John Wiley & Sons, Inc., New-York, USA.
Nadeem, M., F.M. Anjum, M.I. Khan, S. Tehseen, A. El-Ghorab and J.I. Sultan. 2013. Nutritional and medicinal aspects of coriander (Coriandrum sativum L.): a review. British Food J. 115(5): 743–755.
Nahak, G. and R. K. Sahu. 2011. Phytochemical evaluation and antioxidant activity of Piper cubeba and Piper nigrum. J. of Appl. Pharma. Sci. 01(8): 153-157.
Nair, R., T. Kalariya and C. Sumitra. 2005. Antibacterial activity of some selected Indian medicinal flora. Turkey J. of Bio. 29: 41-47.
Nammi, S., K. M. Boini, S. Koppula and S. Sreemantula. 2005. Reserpine-induced central effects: pharmacological evidence for the lack of central effects of reserpine methiodide, Canadian. J. of Physio. and Pharma. 83(6): 509-515.
Nandave, M., S.K. Ojha, S. Kumari, T.C. Nag, R. Mehra, R. Narang and D.S. Arya. 2013. Cardioprotective effect of root extract of Picrorhiza kurroa (Royle Ex Benth) against isoproterenol-induced cardiotoxicity in rats. Ind. J. of Experimental Bio. 51: 694-701.
Narayana, K.R., M.S. Reddy, M.R. Chaluvadi and D.R. Krishna. 2001. Bioflavonoids classification, pharmacological, biochemical effects and therapeutic potential. Ind. J. Pharmcol. 33: 2-16.
Natural Medicines Comprehensive Database. http://www.naturaldatabase.com/. Date accessed: June 11, 2006.
Nema, R., P. jain, S. Khare, A. Pradhan, A. Gupta and D. Singh. 2012. Antibacterial and antifungal activity of Terminalia arjuna leaves extract with special reference to flavanoids. Basic Res. J. Med. Clin. Sci. 1(5): 63-65.
Nemerson, Y. 1969. Characteristics and lipid requirements of coagulant proteins extracted from lung and brain: the Specificity of the protein component of tissue factor. J. of Clinic. Investigation. 48: 322-331.
Nichols, M., N. Townsend, P. Scarborough and M. Rayner. 2014. Cardiovascular disease in Europe epidemiological update. Euro. Heart J. 23-26.
Niero, R. 2010. Farmacos, fitofarmacos e fitoterapicos: abordagem economica e de mercado. In: Bresolin TMB, Cechinel Filho V, editors. Farmacos e medicamentos. Uma abordagem multidisciplinar. Sao Paulo: Editora Santos. 1-15.
Nikolic, G., A. Veselinovic, Z. Mitic and S. Zivanovic. 2011. HPLC‐DAD Study of Gallic acid autoxidation in alkaline aqueous solutions and the Influence of Mg(II) Ion. Sci. J. of the Faculty of Medicine in Nis. 28(4): 219-224.
Nonita, P.P. and M.U. Mylene. 2010. Antioxidant and cytotoxic activities and phytochemical screening of four Philippine medicinal plants. J. Med. Plant Res. 4: 407 414.
Noshadi, I., N.A. Amin and R.S. Parnas. 2012. Continuous production of biodiesel from waste cooking oil in a reactive distillation column catalyzed by solid heteropolyacid: Optimization using response surface methodology (RSM). Fuel. 94: 156.
158
Nwachukwu, C.U., C.N. Umeh and I.G. Kalu. 2010. Identification and traditional uses of some common medicinal plants In Ezinihitte mbaise L.G.A., of Imo State, Nigeria. Report and Opinion. 2(6): 32-34.
Oben, J., D. Kuate and G. Agbor. 2006. The use of Cissus quadrangularis formulation in the management of weight loss and metabolic syndrome. Lipids in Health and Disease. 5: 24.
Ogura, T., and Y. Sakamoto. 2012. Application of metabolomics techniques using LC/MS and GC/MS profiling analysis of green tea leaves. SHIMADZU Corporation (10). application note.
Ojha, S., M. Nandave, S. Kumari and D.S. Arya. 2010. Cardioprotection by Inula racemosa Hook in experimental model of myocardial ischemic reperfusion injury. Indian J of experimental Biology. 48: 918-924.
Ojha, S.K., S. Bharti, S. Joshi, S. Kumari and D.S. Arya. 2012. Protective effect of hydroalcoholic extract of Andrographis paniculata on ischaemia reperfusion induced myocardial injury in rats. Indian J. Med. Res. 135: 414-421.
Olson, C.T., W.C. Keller, D.F. Gerken and S.M. Reed. 1984. Suspected tremetol poisoning in horses. J. Am. Vet. Med. Assoc. 185: 1001-1003.
Onyeyilli, P.A., C.L. Iwuoha and J.A. Akinniyi. 1988. Chronic toxicity study of Fiscus platyphylla blume in rats. West. Afr. J. Pharmacol. Drug. Res. 14: 27-30.
Orhue, N.E.L., and E.A.C. Nwanze. 2004. Effect of Scoparia dulcis on Trypanosome brucee induced alterations in serum Transaminase, Alkaline phosphatase and Bilirubin in the rabbit. J. Med. Sci. 4: 194-197.
Othman, A., N.J. Mukhtar, N.S. Ismail and S.K. Chang. 2014. Phenolics, flavonoids content and antioxidant activities of 4 Malaysian herbal plants. Int. Food Res. J. 21(2): 759-766.
Ottaviani, J.I., L. Actis-Goretta, J.J. Villordo and C.G. Fraga. 2006. Procyanidin structure defines the extent and specificity of angiotensin I converting enzyme inhibition. Biochimie. 88: 359-65.
Padmanabhan, P. and S.N. Jangle. 2012. Evaluation of DPPH Radical Scavenging Activity and Reducing Power of Four Selected Medicinal Plants and Their Combinations. Int. J. of Pharmaceutical Sci. and Drug Res. 4(2): 143-146.
Paiva, B.D., R. Goldbeck, W.D.D. Santos and F.M. Squina. 2013. Ferulic acid and derivatives: molecules with potential application in the pharmaceutical field Livia Brazilian. J. of Pharmaceutical Sci. 3: 395-411.
Palasuwan, A. and S. Soogarun. 2014. Total antioxidant activity of Thai medicinal plants associated with the treatment of cardiovascular diseases, diabetes and cancers. J. Chem. Pharm. Res. 6(10): 27-31.
Panda, V.S. and S.R. Naik. 2008. Cardioprotective activity of Ginkgo biloba phytosomes in isoproterenol induced myocardial necrosis in rats: a biochemical and histoarchitectural evaluation. Exp Toxicol Pathol. 60: 397-404.
Panda, V.S. and S.R. Naik. 2009. Evaluation of cardioprotective activity of Ginkgo biloba and Ocimum sanctum in rodents, Alternative Medicine Review. 14(2): 161–171.
Pandey, A., P. Bigoniya, V. Raj and K. K. Patel. 2011. Pharmacological screening of Coriandrum sativum Linn for hepatoprotective activity. J. of pharmacy bioallied sciences. 3: 435-441.
Papu, S., S. Jaivir, S. Sweta and B.R. Singh. 2012. Medicinal values of Garlic (Allium sativum L.) in human life: An Overview. Greener J. of Agricultural Sci. 3: 6-9.
Park, J.H., C. Li, W. Hu and M.H. Wang. 2010. Antioxidant and free radical scavenging activity of different fractions from hawthorn fruit. J. of Food Sci. and Nut. 15(1): 44-50.
159
Patil, R.H., K. Prakash and V.L. Maheshwari. 2011. Hypolipidemic effect of Terminalia arjuna in experimentally induced hypercholesteremic rats. Acta Biologica Szegediensis. 55(2): 289-293.
Pham, P.C., P.M. Pham, S.V. Pham, J.M. Miller and P.T. Pham. 2007. Hypomagnesemia in patients with type 2 diabetes. Clin. J. Am Soc. Nephrol. 2(2): 366-737.
Poonam, S.A. and S. Mishra. 2013. Physiological, biochemical and modern biotechnological approach to improvement of Rauvolfia serpentina. Journal of Pharmacy and Biological Science. 6(2): 73-78.
Prabha, S.P., A. Nitha, P.N. Ansil and M.S. Latha. 2014. Cardioprotective effect of methanolic extract of Gardenia gummifera linn. On isoproterenol induced myocardial infarction in rats. International Journal of Pharmaceutical Sciences and Research. BIJPSR. 5(9): 3817-3828.
Prashee, A.P., P.C. Trivedi, P.B. Nigade, M.M. Ghaisas and A.D. Deshpande. 2008. Cardioprotective effect of Azadirachta indica A. Juss. on isoprenaline induced myocardial infarction in rats. Int. J. Cardiol. 126: 123–134.
Prince, P.S.M., S. Suman, P.T. Devika and M. Vaithianathan. 2008. Cardioprotective effect of ‘marutham’ a polyherbal formulation on isoproterenol induced myocardial infarction in wistar rats. Fitoterapia. 79: 433-438.
Qureshi, S.A. and S.K. Udani. 2009. Hypolipidaemic activity of Rauwolfia serpentina benth. Pakistan Journal of Nutrition. 8(7): 1103-1106.
Rader, D.J. and G.K. Hovingh. 2014. HDL and cardiovascular disease. The Lancet. 384: 618-625.
Radhika, S., K.H.I. Smila and R. Muthezhilan. 2011. Cardioprotective activity of Hybanthus enneaspermus (Linn.) on isoproterenol induced rats. Indian Journal of Fundamental and Applied Life Sciences. 1(3): 90-97.
Rafatullah, S., A. Al-Sheikh, S. Alqsoumi, M. Al-Yahya, K. El-Tahir and A. Galal. 2008. Protective effect of fresh radish juice (Raphanus satnus L.) against carbon tetrachloride induced hepatotoxicity. Int. J. Pharm. 40: 1811-7775.
Ragavendran, P., D. Sophia, C. Arulraj and V.K. Gopalakrishnan. 2011. Cardioprotective effect of aqueous, ethanol and aqueous ethanol extract of Aerva lanata againt doxorubicin induced cardiomyopathy in rats. Asian pacific J. of tropical biomedicine.7: 1-7.
Rahman, K., and G.M. Lowe. 2006. Garlic and cardiovascular disease: a critical review. J Nutr. 136(3): 736S-740S.
Rajadurai, P. and P.S. Prince. 2007. Preventive effect of naringin on isoproterenol induced cardiotoxicity in wistar rats an in vivo and in vitro study. Toxicology. 232: 216–225.
Rajalakshmy, I., R. Pydi and S. Kavimani. 2011. Cardioprotective medicinal plants. Int J Pharm Inven. 1: 24-41.
Rajkumar, V., G. Gha and A. Kumar. 2010. Evaluation of antioxidant activities of Bergenia ciliata rhizome. Rec. Nat. Prod. 4(1): 38-48.
Ramadoss, S., K. Kannan, K. Balamurugan, N.S. Jeganathan and R. Manavalan. 2012. Cardioprotective effect of Cyathula prostrata Linn on isoproterenol induced myocardial infarction in rat. International Journal of Research in Pharmaceutical and Biomedical Sci. 3(2): 24-29.
Ramesh, T.M., R. Sureka, C. Begum and V. Hazeena. 2008. Cardioprotective effects of Sesbania grandiflora in cigarette smoke-exposed Rats. J. of Cardio. Pharmacology. 52: 338-343.
160
Rameshkumar, M.R., M.G. Jagadeesan, P. Rajendran and N.J. Arunagirinathan. 2014. Antibacterial activity of bark extracts of Terminalia arjuna (Roxb.) against extednded spectrum β-Lactamase producing multi drug resistant bacteria from urinary tract infections. Res. J. of Recent Sci. 3(7): 28-33.
Ramya, S., T. Kalaivani, C. Rajasekaran, P. Jepachanderamohan, N. Alaguchamy, M. Kalayansundaram and R. Jayakumararaj. 2008. Antimicrobial activity of aqueous extracts of bark, root, leaves and fruits of Terminalia arjuna Wight & Arn. Ethnobot Leafs. 12: 1192- 1197.
Rani, B.A., M. Kumar and S. Kumar. 2014. Effect of growth regulators on micropropagation of Rauvolfia serpentina. Journal of Applied and Natural Science. 6(2): 507-511.
Rashed, K. 2014. Medicinal Plants with antioxidant Potential. A review. Hygeia. J. D.Med. 6 (1): 106-110.
Rathore, N., M. Kale, S. John and D. Bhatnagar. 2000. Lipid peroxidation and antioxidant enzymes in isoproterenol induced oxidative stress in rat erythrocyte. Indian Journal of Physiology and Pharmacology. 44: 161–166.
Razak, M. F. A., and K.E. Aidoo. 2011. Toxicity studies of Eurycoma longifolia (Jack)-Based remedial products. Asian J. Pharm. Clin. Res. 4: 23-27.
Razak, M.K., E. Abd, K.E. Aidoo and A.G.G. Candlish. 2007. Mutagenic and cytotoxic properties of three herbal plants from Southeast Asia. Tropical Biomedicine. 24(2): 49-59.
Reddy, V.D., P. Padmavathi, M. Paramahamsa and N.C. Varadacharyulu. 2010. Amelioration of alcohol induced oxidative stress by Emblica officinalis (Amla) in rats. Ind. J. Biochem. Biophys. 47: 20–25.
Rinayanti, A., M. Radji, M.I. Abdul and F.D. Suyatna. 2013. Screening of Angiotensin Converting Enzyme (ACE) Inhibitor Activity of Antihypertensive Medicinal Plants from Indonesia. International J. of Pharmacy Teaching and Practices. 4(1): 527-532.
Rivas-Arreola, M.J., N.E.R. Guzman, J.A.G. Infante, R.F.G. laredo, M.R. Castro, J.R. Bacon, R. (Tsao) Cao, A. Proulex and P. Ortega. 2010. Antioxidant activity of Oak (Quercus) leaves infusions against free radicals and their cardioprotective potential. 2010. Pakistan. J. Biol. Sci. 13(11): 537-545.
Rohman, A., S. Riyanto, N. Yuniarti, W.R. Saputra, R. Utami and W. Mulatsih. 2010. Antioxidant activity, total phenolic, and total flavaonoid of extracts and fractions of red fruit (Pandanusconoideus Lam). Int Food Res J. 17(1): 97–106.
Rolf, S., H.J. Bruns, T. Wichter, P. Kirchhof, M. Ribbing, K. Wasmer, M. Paul, G. Breithardt, W. Haverkamp and L. Eckardt. 2003. The ajmaline challenge in Brugada syndrome: diagnostic impact, safety, and recommended protocol. Eur. Heart J. 24(12): 1104-1112.
Rosenblat, J., A. Zhang and T. Fear. 2012. Biomarkers of myocardial infarction: Past, present
and future. UWOMJ. 81(1): 23-25.
Rottblatt, M. and I. Ziment. 2002. eds. Garlic (Allium Sativum). In: Evidence Based Herbal
Medicine. Philadelphia: Hanley and Belfus, Inc. 193–200.
Rudenko, G., S. Huang, L. Henery, H.J. Pownall and Y.K. Ho. 2010. Mechanism of LDL binding and release probed by structure-based mutagensis of the LDL receptor. J Lipid Res. 51(2): 297-308.
161
Saadatian, M., F. Peshawa, K. Asiaban, A. Karzan and H. Muhammad. 2014. Determination of biochemical content and some pomological characteristics of 4 Hawthorn species (Crataegus spp.) Grown in erbil Province, Kregion, Iraq. Advances in Environmental Biology. 8(7): 2465-2468.
Sabeena, F.K.H., R. Anandan, S.H. Kumar, K.S. Shiny, T.V. Sankar and T.K. Thankappan. 2004. Effect of squalene on tissue defense system in isoproterenol induced myocardial infarction in rats. Pharmacol. Res. 50(3): 231-236.
Sahreen, S., M.R. Khan and R.A. Khan. 2011. Hepatoprotective effects of methanol extract of Carissa opaca leaves on CCl4 induced damage in rat. BMC Complementary and Alternative Medicine. 11: 48.
Sahu, R.K., M. Kar and R. Routray. 2013. DPPH Free Radical Scavenging Activity of Some Leafy Vegetables used by Tribals of Odisha. Indian Journal of Medicinal Plants Studies. 1(4): 21-27.
Saiprasanna, B., M.S. Babu, R.Y. Ramani, P.C. Kumar and P. Rajeshree. 2012. Cardioprotective effect of Pongamia pinnata hydro-alcoholic leaf extract against Isoproterenol induced myocardial infarction in wistar rats. Int. J. Med. Pharmaceut. Sci. 2(3): 1-15.
Sangeetha, T. and S.D. Quine. 2006. Preventive effect of S-allyl cysteine sulphoxide (Alliin) on mitochondrial dysfunction in normal and isoproterenol induced cardiotoxicity in male Wistar rats: a histopathological study. J. Pharm. Pharmacol. 58: 617-623.
Savan, E.K.F. and Z. Kuçukbay. 2013. Essential oil composition of Elettaria cardamom Maton. J. of Appl. Bio. Sci. 78-79.
Shanmukha, S.A., T.M. Jyoti and G. Kamlesh. 2012. Cardioprotective effect of Spathodea campanulata bark on isoproterenol induced myocardial infarction in rats. Asian Pacific Journal of Tropical Disease. 1-5.
Sharifi, N., E. Souri, S.A. Ziai, G. Amin and M. Amanlou. 2013. Discovery of new angiotensin converting enzyme (ACE) inhibitors from medicinal plants to treat hypertension using an in vitro assay. J. of Pharmaceutical Sci. 21: 74-89.
Shatoor, A.S., M. Atif and S. Ahmed. 2014. Cardioprotective effect of Crataegus aronia syn. Azarolus (l) aqueous extract against doxorubicin induced cardiotoxicity and heart failure in Wistar rats. J. Basic Appl. Sci. Res. 4: 102-114.
Shenuarin, B. and K. Fukunag. 2009. Cardioprotection by vanadium compounds targeting Akt-mediated signaling. J. Pharmacol Sci. 110: 1-13.
Shiny, K.S., S.H. Kumar, K.H. Farvin, K.H. Anandanx and K. Devadasan. 2005. Protective effect of taurine on myocardial antioxidant status in isoprenaline induced myocardial infarction in rats. J. Pharm. Pharmacol. 57: 1313-1317.
Shreya, M., A. Patra, A. Samanta, S. Roy, A. Mandal, T.D. Mahapatra, S. Pradhan, K. Das and D.K. Nandi. 2013. Analysis of phytochemical profile of Terminalia arjuna bark extract with antioxidative and antimicrobial properties. Asian Pac. J. Trop Biomed. 3(12): 960-966.
Shukla, S.K., A. Kumar, M. Terrence, J. Yusuf, V.P. Singh and M. Mishra. 2013. The probable medicinal usage of Cassia tora : An overview. J. of bio. sci. 13(1): 13-17.
Shyura, L.F., J.H. Tsunga, J.H. Chenb, C.Y. Chiua and C.P. Lo. 2005. Antioxidant properties of extracts from medicinal plants popularly used in Taiwan. Int. J. of Appl. Sci. and Engineering. 3: 195-202.
162
Siddiq, A., T.M. Shanmukha and G. Kamlesh. 2012. Cardioprotective effect of Spathodea campanulata bark on isoproterenol induced myocardial infarction in rats. Asian Pacific Journal of Tropical Disease. 1-5.
Silva, N.C.C. and F. Júnior. 2010. A. Biological properties of medicinal plants: a review of their antimicrobial activity. The Journal of Venomous Animals and Toxins including Tropical Diseases. 16(3): 402-413.
Singh, G., A.T. Sing, A. Abraham, B. Bhat, A. Mumherjee, R. Verma, S.K. Agarwal, S. Jha, Mukherjee and A.C. Burman. 2008. Protective effects of Terminalia arjuna against doxorubicin induced cardiotoxicity. J. of ethnopharmacology. 117: 123-129.
Singh, N. and M. Gupta. 2007. Effect of ethanolic extract of Syzygium cumini seed powder on pancreatic islets of alloxen diabetic rats. Indian Journal of Experimental Biology. 45: 861-867.
Singh, P., A. Singh, A.K. Shukla, L. Singh, V. Pande and T.K. Nailwal. 2009. Somatic embryogenesis and in vitro regeneration of an endangered medicinal plant sarpgandha (Rauvolfia serpentina. L). Life Science Journal. 6(3): 74-79.
Sivakumar, R., R. Rajesh, S. Buddhan, R. Jeyakumar, D. Rajaprabhu, B. Ganesan and R. Anandan. 2007. Antilipidemic effect of chitosan against experimentally induced myocardial infarction in rats. J. of Cell and Animal Biology. 1:071-077.
Sivakumar, V. and S. Rajeshkumar. 2014. International Journal of Pharma Sciences and Research (IJPSR). 5: 11-13.
Slusarczyk, S., M. Hajones, K. S. Wozniak and A. Mathowski. 2009. Antioxidant activity of polyphenols from Lycopuslucidus. Turcz., 113: 134-138.
Sokol-Letowska, A., J. Oszmianski and A. Wojdylo. 2007. Antioxidant activity of phenolic compounds of Hawthorn, pine, skullcap. Food Chem. 103(3): 853-859.
Soni, A. and S. Sosa. 2013. Phytochemical analysis and free radical scavenging potential of herbal and medicinal plant extracts. J. of Pharma. and Phytochemist. 2(4): 22-29.
Soumia, K., D. Tahar, L. Lynda, B. Saida, C. Chabane and M. Hafidha. 2014. Antioxidant and antimicrobial activities of selected medicinal plants from Algeria. J. of Coastal Life Medi. 2(6): 478-483.
Souravi, K. and P.E. Rajasekharan. 2014. A review on the pharmacology of Embelia ribes burm. Threatened medicinal plant. Journal of Pharma and Bio Sciences. Bio Sci. 5(2): 443–456.
Spalding, A., J. Kernan and W. Lockette. 2009. The metabolic syndrome: a modern plague spread by modern technology. J. Clin. Hypertens. 11: 755–760.
Srinivasan, K. 2007. Black pepper and its pungent principle-piperine: A review of diverse physiological effects Critical Rev. Food Nut. 47: 735-748.
Srivastava, A., A.K. Tripathi, R. Pandey, R.K. Verma and M.M. Gupta. 2006. Quantitative determination of reserpine, ajmaline and ajmalicine in Rauvolfia serpentina by reversed-phase high-performance liquid chromatography. Journal of Chromatographic Science. 44: 557-560.
Sruthi, D., J.T. Zachariah, N.K. Leela and K. Jayarajan. 2013. Correlation between chemical profiles of black pepper (Piper nigrum L.) var. Panniyur-1 collected from different locations. Journal of Medicinal Plants.
Subhashini, N., G. Nagarajan, O. Shahinsha, K. Leelanvitha and S. Kavimani. 2011. Cardioprotective effect of Garcinia combogia on isoproterenol induced myocardial infarction in rats. Pharma Prof. 1: 91-100.
163
Sulaiman, C.T. and V.K. Gopalakrishnan. 2013. Radical scavenging and In-Vitro Hemolytic Activity of Aqueous Extracts of Selected Acacia Species. J. of Applied Pharmaceutical Sci. 3(3): 109-111.
Susila, R., J. Gladys, R. Balagurusamy and K. Mubarak. 2013. A Review of Siddha Cardiology and Cardioprotective Herbs. Int. J. of Herbal Medicine. 1(4): 71-75.
Szollosi, R. and I.S. Varga. 2002. Total antioxidant power in some species of Labiatae (Adaptation of FRAP method). Acta Biol Szeged. 46 (4): 125 - 159.
Taegtmeyer, H., L. Golfman, S. Sharma, P. Razeghi and M.V. Arsdall. 2004. Linking gene expression to function: metabolic flexibility in the normal and diseased heart. Ann N Y Acad Sci. 1015: 202–213.
Tappia, P.S., T. Hets, L. Hozaima, M.S. Sandhu, V. Panagia and N.S. Dhalla. 2001. Role of oxidative stress in catecholamine induced changes in cardiac sarcolemmal Ca2+ transport. Archives of Biochemistry and Biophysics. 377: 85 – 92.
Tassell, M.C., R. Kingston, D. Gilroy, M. Lehane and A. Furey. 2010. Hawthorn (Crataegus spp.) in the treatment of cardiovascular disease. Pharmacogn Rev. 4(7): 32–41.
Taylor, J.L.S., E.E. Elgorashi, A.V. Maes, U.D. Gorp, N.V. Kimpe, N.V. Staden and J.L. Verschaeve. 2003. Investigating the safety of plants used in South African traditional medicine: testing for genotoxicity in the micronucleus and alkaline comet assays. Environmental and Molecular Mutagenesis. 42: 144–154.
Tende, J.A., J.O. Ayo, A. Mohammed and A.U. Zezi. 2015. Blood pressure lowering and cardioprotective effects of garlic (Allium sativum) and ginger (Zingiber officinale) extracts in some laboratory animals. Int. J. Med. Med Sci. 7: 8-13.
Thippeswamy, B.S., S.P. Thakker, S. Tubachi, G.A. Kalyani, M.K. Netra and U. Patil. 2009. Cardioprotective effect of Cucumis trigonus Roxb on isoproterenol-induced myocardial infarction in rat. Am. J. Pharmacol Toxicol. 4(2): 29-37.
Thygesen, K., J.S. Alpert and H.D. White. 2007. Universal definition of myocardial infarction. Eur. Heart J. 28(20): 2525–38.
Tilak-Jain, J.A. and T.P.A. Devasagayam. 2006. Cardioprotective and other beneficial effects of some Indian medicinal plants. J. Clin.Biochem. Nutr. 38: 9-18.
Torabian, S., E. Haddad, S. Rajaram, J. Banta and J. Sabate. 2009. Acute effect of nut consumption on plasma total polyphenols, antioxidantcapacity and lipid peroxidation. J. Hum. Nut. and Diet. 22: 64-71.
Toth, P.P. 2007. Making a case for quantitative assessment of cardiovascular risk. J Clin Lipidol. 1: 234-241.
Upaganlawar, A., H. Gandhi and R. Balaram. 2011. Isoproternol induced myocardial infarction; protective role of natural products. J. Pharm. Toxicol. 6: 1‒17.
Vasu, K., J.V. Goud, A. Suryam and M.A.S. Chary. 2009. Biomolecular and phytochemical analyses of three aquatic angiosperms. Afr. J.Microbiol. Res. 3(8): 418-421.
Velavan, M.T., I. Aegil and K. Gokulakrishnan. 2008. Protective effect of Vitis vinifera against myocardial ischemia induced by isoproterenol in rats. Pharmacology. 3: 958‒967.
Velavan, S., K. Nagulendran and R. Mahesh. 2007. In vitro antioxidant activity of Asparagus racemosus root. Pharmacog Mag. 3: 26-33.
Verma, S.K., V. Jain and S.S. Katewas. 2009. Blood pressure lowering, fibrinolysis enhancing and antioxidant activities of cardamom (Elettaria cardamomum). Ind. J. Biochem. Biophy. 46: 503-506.
164
Verma, S.K., V. Jain, D. Verma and R. Khamesra. 2007. Crataegus oxyacantha - A cardioprotective herb. J. of Herbal Medicine and Toxicol. 1(1): 65-71.
Vijayakumar, R.S., D. Surya, R. Senthilkumar and N. Nalini. 2002. Hypolipidemic effect of black pepper (Piper nigrum Linn.) in rats fed high fat diet. J. Clin Biochem Nutr. 32: 31-42.
Vijver, D.V.L., 1997. Low-density lipoprotein oxidation, antioxidants and risk of atherosclerosis.Vishnupriya, M., S. Nishaa, J.M. Sasikumar and V.K. Gopalakrishnan. 2012. Antioxidant
activity and hydroxyl radical induced DNA damage protection effect of aqueous extract of Curcuma amada. Res. J. of Pharm, Bio. and Chemical Sci. 3(4): 89-96.
Wakade, S.A., S.A. Shah, P.M. Kulkarni and R.A. Juvekar. 2008. Protective effect of Piper longum L. on oxidative stress induced injury and cellular abnormality in adriamycin induced cardiotoxicity in rats. Indian J. Exp. Biol. 46: 528-533.
Wang, C. and Y. Zuo. 2011. Ultrasound-assisted hydrolysis and gas chromatography–mass spectrometric determination of phenolic compounds in cranberry products. Food chemistry. 128(2): 562-568.
Wang, J., H. Bo, X. Meng, Y. Wu, Y. Bao and Y. Li. 2006. A Simple and Fast Experimental Model. Texas Heart Institute Journal. 33(3).
Wang, Y., Z. Zhang, Y. Wu, J. Zhan, X.H. He and Y. Wang. 2013. Honokiol protects rat hearts against myocardial ischemia reperfusion injury by reducing oxidative stress and inflammation. Experimental and Therapeutical Medicine. 5: 315–319.
Wani, T. A., S.M. Wani, A.G. Shah and F.A. Masood. 2013. Optimizing conditions for antioxidant extraction from sea buckthorn leaf (Hippophae rhamnoides L.) as herbal tea using Response Surface Methodology (RSM). Int Food Res J. 20(4): 1677-1681.
Weikl, A., K.D. Assmus, A.N. Schmidt. Crategeus special extract WS 1442: Assessment of objective effectiveness in patients with heart failure. Fortschr Med. 114: 291-96.
Weiss, R.F., and V. Fintelmann. 2000. Herbal medicine, 2nd ed. Thieme, Stuttgart. 229-230, 387-416.
Wong, S.K., Y.Y. Lim and E.W.C. Chan. 2009. Antioxidant properties of Hibiscus species variation, altitudinal change costal influence and floral colour change. J. of Tropical Forest Sci. 21: 307-315.
World Health Organization. 2011. Global Atlas on cardiovascular disease prevention and control. Geneva: World Health Organization.
Yadav, A., R. Bhardwaj and R.A. Sharma. 2014. Free radical scavenging potential of the Solanum surattense burm f. an important medicinal plant. Int. J. of Pharmacy and Pharmaceutical Sci. 6(3): 39-42.
Yang, Z.G., H.X. Sun and W.H. Fang. 2005. Haemolytic activities and adjuvant effect of Astragalus membranaceus saponins (AMS) on the immune responses to ovalbumin in mice. Vaccine. 23(44): 5196-5203.
Yousefi, K., F. Fathiazad, H. Soraya, M. Rameshrad, N. Maleki- Dizaji and A. Garjani. 2014. Marrubium vulgare L. methanolic extract inhibits inflammatory response and prevents cardiomyocyte fibrosis in isoproterenol induced acute myocardial infarction in rats. BioImpacts. 4(1): 21–27.
Yun, W., Z.Z. Zhang, Y. Wu, J. Zhan, Xiang, X.H. He and Y.L. Wang. 2013. Honokiol protects rat hearts against myocardial ischemia reperfusiol injury by reducing oxidative stress and inflammation. Experimental and Therapeutical Medicine. 5: 315–319.
165
Zafar, F., N. Jahan, K.U. Rahman, A. Khan and W. Akram. 2015. Cardioprotective potential of polyphenolic rich green combination in catecholamine induced myocardial necrosis in rabbits. Evidence Based Complementary and Alternative Medicine. 2015: 1-9.
Zahidullah, M., M. Aasim, I. Khan, H. Muhammadzai, M.A. Shah, N. Ali, A. Mohammad, A. Muzahir and M. Rehman. 2012. Evaluation of patients with coronary artery disease for major modifiable risk factors for Ischemic heart disease. J Ayub Med Coll Abbottabad. 24(2): 315-318.
Zeiger, E. 2001. Mutagens that are not carcinogenic: faulty theory or faulty tests? Mutation Res. 492: 29–38.
Zhang, K. and Y. Zuo. 2006. Natural phenolic antioxidants in human fluids: analytical approaches and antioxidant capacity studies. Pakistan Journal of Analytical Chemistry. 7(1): 39-47.
Zhang, K. and Y. Zuo. 2004. GC-MS determination of flavonoids and phenolic and benzoic acids in human plasma after consumption of cranberry juice. Journal of agricultural and food chemistry. 52(2): 222-227.
Zohra, M. and A. Fawzia. 2014. Hemolytic activity of different herbal extracts used in Algeria. Int. J. of Pharma Sci and Res. 5(8): 495-500.
Zuo, Y. H. Chen and Y. Deng. 2002. Simultaneous determination of catechins, caffeine and gallic acids in green, Oolong, black and pu-erh teas using HPLC with a photodiode array detector. Talanta. 57(2): 307-316.
166