effect of methanolic root extract of rauwolfia...

EFFECT OF METHANOLIC ROOT EXTRACT OF

RAUWOLFIA SERPENTINA (BENTH) ON

ANTIOXIDANT ENZYMES & DIABETIC

DYSLIPIDEMIA

MUHAMMAD BILAL AZMI

Department of Biochemistry

University of Karachi

Karachi-75270, Pakistan

2014

EFFECT OF METHANOLIC ROOT EXTRACT OF

RAUWOLFIA SERPENTINA (BENTH) ON

ANTIOXIDANT ENZYMES & DIABETIC

DYSLIPIDEMIA

Thesis submitted for the fulfillment of the degree of

DOCTOR OF PHILOSOPHY

By

MUHAMMAD BILAL AZMI



Karachi-75270, Pakistan

2014

DECLARATION

I hereby undertake that this research entitled ―Effect of Methanolic Root

Extract of Rauwolfia Serpentina (Benth) on Antioxidant Enzymes &

Diabetic Dyslipidemia‖ is an original research work of my approved

synopsis from the Board of Advance Studies and Research, University of

Karachi and no part of said thesis falls under plagiarism. None of the

context of this work was previously submitted by another person which has

been accepted for the award of any degree or diploma of the university or

the institute of higher education, except where due acknowledgment has

been made in text.

MUHAMMAD BILAL AZMI

Enrolment No. SCI/BCH/KU-33455/2012

Department of Biochemistry,

University of Karachi.

Dated: 29th Dec, 2014

CERTIFICATE

I hereby certified that Mr. Muhammad Bilal Azmi, (Enrolment No.

SCI/BCH/KU-33455/2012) PhD student of Department of Biochemistry,

University of Karachi, has successfully completed his research study

entitled, ―Effect of Methanolic Root Extract of Rauwolfia Serpentina

(Benth) on Antioxidant Enzymes & Diabetic Dyslipidemia‖ under my

supervision. The entire content is his own work and that, to the best of my

knowledge and belief, it contains no material previously published or

written by anyone else nor material which has been accepted for the award

of any other degree or diploma of the university of higher learning, except

due acknowledgment made in the text.

Dr. SHAMIM A. QURESHI

Assistant Professor &

Research Supervisor



Dated: 29th Dec, 2014

DEDICATION

This effort is dedicated to my beloved grandfather, who his-self, the example

of inspiration for me. There are not enough words I can say to describe just

how important he was to me, and what a powerful influence he continues to

be. He is no more with me but I still remember his words as

“How hard the tasks are for you, just put your best and honest efforts without

demanding your own share in its”

(May Allah rest his soul with eternal peace).

To my father, for me his uncompromising principles, that were the key secret

of this success,

&

To my Mother, who consumed her-self, just to nurtured and candle my

career. Especially her prayers are fuel that enables me to do the impossible.

Now at this moment, I am proudly quoting what I am today is due to her

existence.

May Allah bless all of us

Acknowledgments

My all thanks are sincerely due to the ALMIGHTY ALLAH and the last

Prophet Hazrat Mohammad (S.A.W) of ISLAM by blessing me with this great

opportunity. I believe that I was not been able to timely complete this research work

without HIS blessing which ALLAH showered upon me and able me to enjoy ecstasy

moments.

My sincere thanks and gratitude are always available for my kind supervisor

Dr. Shamim A. Qureshi, Assistant Professor, Department of Biochemistry, University

of Karachi. I very gratefully express that her supervision turned out to be a blessing for

me. She knew that I was not perfect in research but her continuous support waking me up

to perform quality work. I believe her constant attention with kind reminders are the key

ingredients that perfectly utilize my abilities and time in constructive manner. I still

remember many glorious moments with wonderful memories that made my stay at the

best which I passed with her during the completion of this research work. Especially,

when I presented a talk in BioCon 2012 Conference organized by Department of

Biochemistry. It was never a forgettable day of my life. I received the Young Scientist

Award and my talk which stood best among all presenters. Another, brilliant day for me

was when I presented a research talk in 2nd

National Conference of Pharmacy, organized

by Dow University of Health Sciences, where I was only non-pharmacist among all

presenters. My talk was declared as 2nd

Best Research Presentation which belongs to this

work. I feel extremely fortunate to have had Dr. Shamim Qureshi as my supervisor.

I very respectfully convey thanks to my respected Chairperson and all teachers of

Department of Biochemistry, University of Karachi (UoK).

I render my whole hearted thanks to all my lab colleagues Dr. Rabia Badar, Ms.

Tooba Lateef, Mr. Ali Zia, Ms. Shamsa Kanwal, Ms. Saleha Sultana and all others. They

all helped me with their continuous, creative, and valuable assistance.

I also convey my gratitude to Prof. Dr. Iqbal Azhar, Chairman, Department of

Pharmacognosy, Faculty of Pharmacy, UoK, for providing me facility of rotary vaccuum

evaporator. Here I forward my special thanks to Prof. Dr. Iqbal Choudhry, Dr. Shakeel

Ahmed and Dr. Shamsa Kanwal of H.E.J. Research Institute of Chemistry, UoK for their

unconditional support. I am also grateful to Prof. Dr. Nazrul Hasnain, Head of

Biochemistry Department, Dow International Medical College, Dow University of

Health Sciences (DUHS) and Dr. Jameel Ahmed, Former Chief Scientist, Pakistan

Atomic Energy Comission (PAEC) for the critical appraisal of my dissertation. I am also

thankful to Dr. Zameer Khnazada, Incharge Laboratory Animal Health Sciences, DUHS,

for his valuable coordination. I am also especially thankful to Mr. Fahim, Mr. Zaid

Kundan and all other technical staff for their value added support and co-operation

extended towards me.

I am indebted to my dearest friend Dr. Waqasuddin Khan who encouraged me

during the entire span of this journey. His support and advices were always much

constructive to me.

I have no words for the unconditional support and selfless devotion of my parents

and sisters (Sarah and Maria). I am also thankful to my dearest wife Ms. Warda Jabeen

for her love and motivation, which always strengthened my shoulders to complete this

big task smoothly.

My words are finally inadequate to express my gratitude to Ms. Shehla Naz,

Assistant Director of Quality Enhancement Cell, DUHS for her extended supports.

Thank you all,

Muhammad Bilal Azmi

2014

Table of Contents

S. No. Title of Contents Page No.

Summary i

Summary (Urdu) vii

Chapter 1 Introduction

1.1 Diabetes 01

1.2 Prevalence 02

1.3 History 03

1.4 Types 04

1.5 Complications 08

1.6 Treatments 12

1.6.1 Pharmaceutical Medicines as Conventional

Treatment 12

1.6.2 Medicinal plants as an Alternative Treatment 14

1.7 Plant for the Present study 21

1.7.1 Classification of Rauwolfia serpnetina 22

1.7.2 Morphology and Distribution 22

1.8 Literature Review of R. serpentina 22

1.8.1 Chemical Aspects of R.serpentina 22

1.8.2 Pharmacological Aspects of R.serpentina 22

1.9 Objective of the Present Research Work 31

Flow Chart Showing Scheme of Present Research work 33

Chapter 2 Extraction, Phytochemistry, Acute Toxicity and Oral

Glucose Tolerance Test (OGTT) of Root Extracts of

R.serpentina

2.1 Materials and Methods 34

2.1.1 Animals 34

2.2 Plant and Experimental Material 34

2.2.1 Identification and Authentication of Experimental Material 35

2.2.2 Preparation of Root Extracts 35

2.2.3 Percent Yield of Root Extracts 35

2.3 Analysis of Phyto-constituents in Root Extracts 36

2.3.1 Qualitative Analysis 36

2.3.2 Quantitative Analysis 39

2.4 In Vivo Evaluation of Root Extracts 43

2.4.1 Determination of the Acute Toxicity of Root Extracts 43

2.4.2 Determination of the Median Lethal Dose (LD50) of

Root Extracts 44

2.4.3 Determination of the Effect of Root Extracts on

Glucose Tolerance in Mice 44

2.5 Results 49

2.5.1 Percent Yield of Root Extracts 49

2.6 Analysis of Phyto-constituents in Root Powder

and Extracts 49

2.6.1 Qualitative Analysis 49

2.6.2 Quantitative Analysis 49

2.7 In Vivo Evaluation of Root Extracts 56

2.7.1 Acute Toxicity and Median lethal Dose (LD50) of

Root Extracts 56

2.7.2 Effect of Root Extracts on Glucose Tolerance in Mice 59

2.8 Discussion 62

Chapter 3 Antidiabetic Effect of Root Extracts of R.serpentina in Alloxan-induced

Type 1 Diabetic Mice Model


3.1.1 Animals 66

3.1.2 Chemicals, Medicines and Enzymatic Kits

3.1.3 Apparatus /Instruments 67

3.2 Determination of the Effect of Root Extracts in

Type 1 Diabetic Mice Model 67

3.2.1 Induction of Type 1 Diabetes in Mice 67

3.2.2 Determination of Physical Parameters 71

3.2.3 Collection of Samples for Biochemical

and Hematological Parameters 71

3.2.4 Determination of Hematological Parameters 72

3.3 Statistical Analysis 88

3.4 Results 89

3.4.1 Effect of Root Extracts in Alloxan-Induced

(Type 1) Diabetic Animal Model 89

3.5 Discussion 118

Chapter 4 Antidiabetic Effect of Root Extracts of R.serpentina in Fructose-induced



4.1.1 Determination of the Effect of Root Extracts

in Type 2 Diabetic Mice Model 125

4.1.3 Determination of Activity of Rate-Regulatory

Enzyme of Cholesterol Biosynthesis 129

4.2 Results 131

4.2.1 Effect of Root Extracts on Fructose-Induced

(Type 2) Diabetic Animal Model 131

4.3 Discussion 162

Chapter 5 Fourier Transform Infrared Spectroscopy (FTIR) of MREt and

AqMREt of R.serpentina

5.1 Material and Method 171

5.1.1 Fourier Transform Infrared Spectroscopy (FTIR)

of Root Extracts 171

5.2 Results 172

5.2.1 FTIR Analysis of Root Extracts of R. Serpentina 172

5.3 Discussion 175

5.4 Conclusion 177

5.5 Future Prospects of Present Research 178

Chapter 6 References 179

Publiation from thesis 208

Regeant Glossary 214

LIST OF FIGURES

S. No. Figures Page No.

Figure 1: Generation of free radicals, their action

and elimination in cellular environment 19

Figure 2: Flow chart showing scheme of present research work 33

Figure 3: Standard curve of gallic acid 42

Figure 4: Log-dose verses probit for calculation of LD50

of MREt of R.serpentina 45

Figure 5: Log-dose verses probit for calculation of LD50

of AqMREt of R.serpentina 46

Figure 6: Effect of MREt on glucose tolerance in mice 47

Figure 7: Effect of AqMREt on glucose tolerance in mice 48

Figure 8: Qualitative analysis of phyto-constituents of

Root extracts of R.serpentina 51

Figure 9: Effect of MREt in Alloxan-Induced type 1

Diabetic Mice 69

Figure 10: Effect of AqMREt in Alloxan-Induced type 1

Diabetic Mice 70

Figure 11: Standard curve of dextrin 80

Figure 12: Standard curve of ALT 84

Figure 13: Effect of Root Extracts on Percent (%) Body

Weight Change of Alloxan-Induced Diabetic Mice 91

Figure 14: Effect of Root Extracts on Percent (%) Glycemic

Change of Alloxan-Induced Diabetic Mice 97

Figure 15: Effect of Root Extracts on Serum Insulin Levels of

Alloxan-Induced Diabetic Mice 100

Figure 16: Effect of Root Extracts on Glycogen in Liver

Tissues of Alloxan-Induced Diabetic Mice 110

Figure 17: Effect of Root Extracts on Total Lipids in Liver

Tissues of Alloxan-Induced Diabetic Mice 111

Figure 18: Effect of MREt in Fructose-Induced Type 2 Diabetic Mice 127

Figure 19: Effect of AqMREt in Fructose-Induced Type 2

Diabetic Mice 128

Figure 20: Effect of Root Extracts on Percent (%) Body

Weight Change of Fructose-Induced Diabetic Mice 134

Figure 21: Effect of Root Extracts on Percent (%) Glycemic

Change of Fructose-Induced Diabetic Mice 140

Figure 22: Effect of Root Extracts on Glycogen in Liver Tissues

of Fructose-Induced Diabetic Mice 154

Figure 23: Effect of Root Extracts on Total Lipids in Liver

Tissues of Fructose-Induced Diabetic Mice 155

Figure 24: FTIR of Root Extracts of R. serpentina 174

List of Tables

S. No. Tables Page No.

Table 1: Compounds isolated from the roots of R.serpentina 24

Table 2: Absorbance of gallic acid 41

Table 3: Qualitative analysis of phyto-constituents of root

extracts of R.serpentina 50

Table 4: Quantitative analysis of phyto-constituents of root

extracts of R.serpentina 55

Table 5: Acute toxicity of MREt 57

Table 6: Acute toxicity of AqMREt 58

Table 7: Effect of MREt on glucose tolerance in mice 60

Table 8: Effect of AqMREt on glucose tolerance in mice 61

Table 9: Absorbance of dextrin 79

Table 10: Absorbance of ALT 83

Table 11: Effect of MREt on body weights of Alloxan-

Induced diabetic mice 90

Table 12: Effect of AqMREt on body weights of Alloxan-

Induced diabetic mice 92

Table 13: Effect of MREt on wet and relative organs body

weights of Alloxan-Induced diabetic mice 94

Table 14: Effect of AqMREt on wet and relative organs

body weights of Alloxan-Induced diabetic mice 95

Table 15: Effect of MREt on blood glucose levels of Alloxan-

Induced Diabetic Mice 96

Table 16: Effect of AqMREt on blood glucose levels of Alloxan-


Table 17: Effect of MREt on Hb, HbA1c and HbA1c / Hb Ratio in

Alloxan-Induced Diabetic Mice 101

Table 18: Effect of AqMREt on Hb, HbA1c and HbA1c / Hb Ratio

in Alloxan-Induced Diabetic Mice 102

Table 19: Effect of MREt on Lipid Profile of Alloxan-Induced

Diabetic Mice 104

Table 20: Effect of AqMREt on Lipid Profile of Alloxan-


Table 21: Effect of MREt on Cardioprotective and Antiatherogenic

Indices of Alloxan-Induced Diabetic Mice 107

Table 22: Effect of AqMREt on Cardioprotective and Antiatherogenic

Indices of Alloxan-Induced Diabetic Mice 108

Table 23: Effect of MREt on Liver Specific and Antioxidant

Enzymes of Alloxan-Induced Diabetic Mice 112

Table 24: Effect of AqMREt on Liver Specific and Antioxidant

Enzymes of Alloxan- Induced Diabetic Mice 113

Table 25: Effect of MREt on Blood Profile of Alloxan-


Table 26: Effect of AqMREt on Blood Profile of Alloxan-


Table 27: Effect of MREt on Body Weights of Fructose-


Table 28: Effect of AqMREt on Body Weights of Fructose-


Table 29: Effect of MREt on Wet and Relative Organs Body

Weights of Fructose-Induced Diabetic Mice 136

Table 30: Effect of AqMREt on Wet and Relative Organs Body

Weights of Fructose-Induced Diabetic Mice 137

Table 31: Effect of MREt on Blood Glucose Levels of Fructose-


Table 32: Effect of AqMREt on Blood Glucose Levels of Fructose-


Table 33: Effect of MREt on Serum Insulin and FIRI of Fructose-


Table 34: Effect of AqMREt on Serum Insulin and FIRI of Fructose-


Table 35: Effect of MREt on Hb, HbA1c and HbA1c / Hb ratio of

Fructose-Induced Diabetic Mice 147

Table 36: Effect of AqMREt on Hb, HbA1c and HbA1c / Hb Ratio

of Fructose-Induced Diabetic Mice 148

Table 37: Effect of MREt on Lipid Profile of Fructose-


Table 38: Effect of AqMREt on Lipid Profile of Fructose-


Table 39: Effect of MREt on Cardioprotective and Antiatherogenic

Indices of Fructose-Induced Diabetic Mice 151

Table 40: Effect of AqMREt on Cardioprotective and Antiatherogenic

Indices of Fructose-Induced Diabetic Mice 152

Table 41: Effect of MREt on Liver Specific and Antioxidant

Enzymes of Fructose-Induced Diabetic Mice 157

Table 42: Effect of AqMREt on Antioxidant Enzymes of

Fructose-Induced Diabetic Mice 158

Table 43: Effect of MREt on Blood Profile of Fructose-


Table 44: Effect of AqMREt on Blood Profile of Fructose-


Table 45: FTIR profile of Root Extracts of R. serpentina 173

Summary The increase in prevalence of diabetes worldwide characterizes its identity as one

of the silent threat for future generation of both genders. Pakistan is also affected from

this health hazard and ranked 6th

largest country with diabetic statistics. The

characteristic feature of diabetes is hyperglycemia which may be due to the absolute

(type 1 diabetes) or relative insulin deficiency and/or insulin resistance (type 2 diabetes)

that also alters lipid and protein metabolism thereby increases the risk of chronic

complications like hypertension, atherosclerosis, and other cardiovascular diseases. In

spite of the presence of commercially available pharmaceutical medicines, herbal

medicines are also found effective in the treatment of diabetes by observing a good count

of population dependent on plant derived medicines all over the world.

Rauwolfia serpentina (Apocynaceae) is a well-known antihypertensive medicinal

plant. Past literature also described its therapeutic spectrum in the treatment of variety of

other ailments like gastrointestinal tract disorders, skin cancer, eczema, burns, breast

cancer, etc. More than fifty (50) indole alkaloids were isolated and reported with

antihypertensive activity from this valuable medicinal plant. The prime aim of the

present study was to evaluate the effect of methanolic (MREt) and 80% aqueous

methanolic root (AqMREt) extracts of R. serpentina on antioxidant enzymes and diabetic

dyslipidemia in alloxan-induced (type 1) and fructose-induced insulin resistance (type 2)

diabetic model. The present study is the first elaborative approach to enlighten the

antidiabetic activity of root extracts of R.serpentina.

Preliminary qualitative and quantitative analysis of phyto-constituents was

performed in MREt and AqMREt. Qualitative analysis of extracts has revealed the

presence of alkaloids, carbohydrates, flavonoids, glycosides, cardiac glycosides,

phlobatannins, resins, saponins, steroids, tannins, triterpenoids in MREt. Same

constituents were also observed in AqMREt except saponins and carbohydrates.

Alkaloids, flavonoids and saponin were also quantitatively determined in ground root

powder and found as 7, 97 and 20 mg/g of starting material respectively. However, a

good quantity of total phenols was estimated in MREt and AqMREt as 233.33 and 321.6

i

mg/g of extract respectively. The presence of alkaloids and phenols was also confirmed

by fourier transform infrared spectroscopic (FTIR) analysis of both root extracts. Acute

toxicity of MREt and AqMREt was determined in terms of hypoglycemic activity,

behavior pattern (sedative or not) and mortality rate in normal Wister mice. In which, test

doses of MREt (10-250 mg/kg) and AqMREt (5-2500 mg/kg) were orally administered

in overnight fasted mice. Of which, MREt in doses 10, 30 and 60 mg/kg was found non-

sedative and decreased (p< 0.0001) mean blood glucose level from 80 to 62 mg/dl after

30 minutes of its administration. Similarly, AqMREt in doses of 5-250 mg/kg was found

non-sedative and non-toxic after 4 hours of its oral administration. However, doses of

MREt from 100-250 mg/dl showed sedation and mortality rate from 17 to 100% within 4

hours of their administration while doses of AqMREt from 500-2500 mg/kg were found

sedative and showed mortality rate as same as MREt. Hence the median lethal doses

(LD50) of MREt and AqMREt were found as 141.25 mg/kg (log LD50 = 2.15 mg/kg) and

1412.54 mg/kg (log LD50 3.15 mg/kg) from graph plotted between log-dose verses

probit.

After determining the acute toxicity and LD50, MREt (10, 30, 60 & 100 mg/kg)

and AqMREt (10, 50, 100 & 150 mg/kg) were used to perform oral glucose tolerance test

(OGTT) to justify the hypoglycemic potential of selected doses. Orally administrated

doses of MREt 10, 30 and 60 mg/kg showed significant (p< 0.01) percent reduction in

blood glucose level ranging from 31- 65%, 39 - 49% and 25 - 51% respectively in their

respective test groups after oral glucose load (2g/kg) from 0 to 120 minutes as compared

to control and negative control groups which were treated with 1 ml of distilled water

and 0.05% dimethylsulfoxide (DMSO) respectively after glucose load on same time

interval. However, MREt in dose of 100 mg/kg induced slight sedation along with

hypoglycemic activity (0 to 120 min) in its test group. Through these findings, the doses

of 10, 30 and 60 mg/kg of MREt were found non-sedative and hypoglycemic in mice

while dose ≥100 mg/kg was excluded. Similarly, AqMREt in doses of 50, 100 and 150

mg/kg showed significant (p< 0.01) hypoglycemic activity ranging from 6 - 31%, 16 -

34 % and 14 -33 % from 0 to 120 min after glucose load (2g/kg) as compared to control

ii

and negative control groups, whereas 10 mg/kg of same extract was not found effective

and thus excluded.

In alloxan-induced type 1 diabetic mice model, mice were made diabetic by

single intra-peritoneal injection of alloxan monohydrate (150 mg/kg), after 72 hours of

this injection fasting blood glucose (FBG) level of each mouse was estimated. Mice

which showed FBG ≥ 190 mg/dl were divided into diabetic control group (distilled water

1ml/kg), negative control (0.05% DMSO 1ml/kg), positive control (glibenclamide

5mg/kg), and six test groups. Three of which treated with MREt 10, 30 & 60 mg/kg and

other three treated with AqMREt 50, 100 & 150 mg/kg. Six mice were run as normal

control (treated with distilled water 1ml/kg). Each treatment was given orally for 14 days

consecutively. Test doses of MREt and AqMREt significantly (p< 0.01) improved the

percent body weights as compared to diabetic control groups. In addition, both root

extracts did not produce any alteration in wet and relative organs weights in their

respective test groups that proves non-toxic effect of these extracts. All three doses of

MREt and AqMREt produced a significant (p< 0.0001) percent glycemic reduction from

- 47 to -51 % and -39 to -53 % in FBG levels of their test groups. Serum insulin levels

were markedly lowered in diabetic and negative control groups mainly due to the

destruction of pancreatic β-cells induced by alloxan injection. However, MREt and

AqMREt doses significantly (p< 0.01) upgrade the serum insulin levels in their

respective test groups. Similarly, total hemoglobin (Hb) levels were upgraded and

glycosylated hemoglobin (HbA1c) levels were fairly controlled (p<0.0001) in all test

groups especially AqMREt decreased HbA1c dose-dependently from 4.5 -3.77%. Thus,

HbA1c / Hb ratio was significantly (p< 0.0001) improved in all test groups.

Lipid profile in terms of total cholesterol (TC), triglycerides (TG), high-density

lipoprotein (HDL-c), low-density lipoprotein (LDL-c) and very low-density lipoprotein

(VLDL-c) cholesterols was also estimated in all groups of alloxan-induced diabetic type

1 model. Increased TC, TG, LDL-c and VLDL-c levels were observed in diabetic control

groups while improvement (p< 0.05 & p< 0.01, p< 0.0001) in lipid profile was noticed

in MREt and AqMREt treated test groups, by showing significant (p< 0.05 & p< 0.0001)

iii

increase in HDL-c level and decrease in TC, TG, LDL-c and VLDL-c levels. Hence a

prominent (p< 0.05 & p< 0.01, p< 0.0001) decrease in TG/HDL-c and TC/HDL-c ratio

and increase in HDL-c/LDL-c ratio were observed in all MREt and AqMREt treated test

groups. In addition, antiatherogenic index was improved in MREt and AqMREt

treatments. These findings validate the cardioprotective and antiatherogenic potential of

root extracts of R.serpentina which could be beneficial in the treatment of diabetic

dyslipidemia.

Decrease in glycogen and increase in total lipids in hepatic tissues of diabetic

control groups were observed while an entirely opposite scenario related to both these

parameters was found in MREt and AqMREt treatments. Positive control

(glibenclamide) also showed the same picture. Hepatoprotective effect of MREt and

AqMREt was also confirmed by observing normal levels of liver-specific enzyme

alanine transaminase (ALT) in all test groups. Antioxidant potential of MREt and

AqMREt test doses was strongly supported by observing a significant (p < 0.05 & p <

0.01, p < 0.0001) decrease in percent inhibition of catalase (CAT) and superoxide

dismutase (SOD) enzymes in all root extracts treated test groups. Similarly,

hematopoietic (hematinic) effect of MREt and AqMREt was established by observing a

gradual and significant (p< 0.01) improvement in red blood cells (RBC) count, packed

cell volume (PCV), mean corpuscular volume (MCV), mean corpuscular hemoglobin

(MCH) and mean corpuscular hemoglobin concentration (MCHC) with reduction in

white blood cells (WBC) count as compared to diabetic control groups that showed

entirely opposite picture of all these hematological parameters.

The effect of MREt (10, 30 & 60 mg/kg) and AqMREt (50, 100 & 150 mg/kg) of

R.serpentina was also investigated in fructose-induced insulin resistance type 2 diabetic

mice model. Insulin resistance was induced in overnight fasted mice by giving 10%

fructose solution 1 ml/kg orally for 14 days consecutively, side by side these mice were

divided in different groups including controls and test groups as same as in alloxan-

induced type 1 diabetic model except pioglitazone (15mg/kg) was used in positive

control group. The mode of administration of each treatment was oral. At the end of trial,

iv

significant increase in percent body weight (7-9%) was observed in diabetic control and

negative control groups while significant (p < 0.05 & p < 0.01, p < 0.0001) dose-

dependent decrease in percent body weight (-2.49 to -3.41%) was noticed in test groups

treated doses of MREt and AqMREt. Similarly, no alteration in wet and relative body

organs weights was observed in same test groups. Most importantly, a good (p < 0.0001)

reduction (p < 0.01 & p < 0.0001) in percent glycemic change was observed in MREt

treated test (25 - 17%) and AqMREt treated test (48 - 3.95%) groups as compared to

fructose-induced diabetic control groups that showed 77 to 114% increase in FBG levels.

Positive control (pioglitazone) also found effective in reducing (24%) the FBG level.

Likewise hyperinsulinemia was observed in diabetic control groups whereas serum

insulin level was significantly (p < 0.01 & p < 0.0001) decreased (0.87- 0.43µU/ml) by

test doses of MREt and almost came to the insulin level that was found in normal control

group. However, increment in serum insulin level was observed in AqMREt test groups

suggested its insulin secretary effect. Decrease in fasting insulin resistance index (FIRI)

also validated the dose-dependent (p < 0.01 & p < 0.0001) increase in insulin sensitivity

in MREt treated test groups. MREt and AqMREt treatments significantly decreased (p <

0.0001) the HbA1c (4.80 - 4.25%) and increased (p < 0.01) total Hb (12.7-12.9%) levels

as compared to diabetic control groups that showed increased HbA1c (6.8-7.3%) and

decreased total Hb (7.15-7.55%) levels. Therefore, both extracts found to improve (p <

0.01 & p < 0.0001) glycation index (HbA1c / Hb ratio) in all test groups.

Hypertriglyceridemia and hypercholesterolemia are the major characteristics of

fructose-induced type 2 diabetes which were clearly observed in diabetic control groups.

On contrary, test doses of MREt and AqMREt significantly reduced (p < 0.05 & p <

0.01, p < 0.0001) the levels of TC, TG, LDL-c and VLDL-c and uplift the level of HDL-

c in their test groups. Improvement in 3-hydroxy-3-methylglutryl coenzyme A (HMG

Co-A) / mevalonate ratio showed a good and significant (p < 0.05 & p < 0.01, p <

0.0001) decrease in cholesterol biosynthesis in test groups which confirmed the

inhibitory effect of MREt and AqMREt on HMG Co-A reductase, the rate-limiting of

enzyme of cholesterol biosynthesis. Similarly, cardioprotective indices including

v

TG/HDL-c, HDL-c/ LDL-c, TC/HDL-c ratios and antiatherogenic index were also

improved (p < 0.05 & p < 0.01, p < 0.0001) in test groups of both root extracts. Increase

in glycogen and decrease in total lipid contents were also observed in hepatic tissues of

mice of all test groups. Pioglitazone also produced same effect in positive control group.

Normal levels of ALT were observed in MREt and AqMREt treated groups.

Significantly (p < 0.05 & p < 0.01, p < 0.0001) less percent inhibition of antioxidant

enzymes including CAT and SOD was observed in test groups while elevated percent

inhibition of these enzymes were noticed in fructose-induced diabetic control groups.

Administration of MREt test doses effectively (p < 0.01 & p < 0.0001) improved the

RBC count moreover it was improved (p < 0.05 & p < 0.01) dose-dependently in

AqMREt treated test groups. WBC count was gradually and significantly (p < 0.05 & p <

0.01, p < 0.0001) reduced in MREt and AqMREt treated groups. In addition, PCV,

MCV, MCH and MCHC levels are significantly improved all test groups.

Therefore the results conclude that oral administration of test doses of MERt and

AqMREt improves hyperglycemia and diabetic dyslipidemia by showing antiglycation,

antioxidant and hematinic effects in alloxan-induced type 1 and fructose-induced insulin

resistance type 2 diabetic mice models. The antidiabetic effect of root extracts of

R.serpentina in alloxan-induced type 1 diabetes may be due to extra-pancreatic or

pancreatic action either by inhibiting glucose absorption in gastro intestinal tract, or

inducing glucose tolerance in mice by accelerating the glucose uptake in target tissues of

insulin thereby stimulating glucose oxidation and storage processes or enhancing the

release of insulin (pancreatic action) from β-cells of pancreas which in turn induce

glucose homeostasis. However, in fructose-induced insulin resistance type 2 diabetes, the

hypoglycemic potential of MREt and AqMREt is possibly due to extra-pancreatic action

by reducing hypertriglyceridemia and hypercholesterolemia thereby enhancing insulin

sensitivity and maintaining glucose homeostasis or active ingredient (alkaloids/ phenols)

of root extracts may mimic the action of insulin by acting as insulin receptor sensitizer.

Therefore, the present work is successful in strengthening the new antidiabetic aspect of

well-famous antihypertensive plant R.serpentina.

vi

Chapter 1

Introduction

Effect of Methanolic Root Extract of Rauwolfia Serpentina (Benth)

on Antioxidant Enzymes & Diabetic Dyslipidemia

1. Introduction

1.1 Diabetes

Diabetes is the most prevalent endocrine disorder in the world which affects both

genders in developing and well-developed countries (Zimmet et al., 2014). It is

characterized by elevated blood glucose level (hyperglycemia) and its excessive

excretion in urine (glucosuria or glycosuria). The primary cause of diabetes is the

absolute or relative insulin deficiency or insulin resistance (Clark et al., 2007; El Khoury

et al., 2014). Insulin is a hormone (hetero-dimeric peptide in structure) release from beta

(β)-cells of Islets of Langerhans of pancreas and involves in maintaining the blood

glucose level (70-110 mg/dl) by increasing the glucose uptake in target tissues viz., liver,

adipose tissues, muscles, etc, by up-regulating the glucose transporters where glucose is

not only utilized for energy (adenosine triphosphate, ATP) production through processes

like glycolysis and tricarboxylic acid cycle (TCA) but also for reserving energy in the

form of glycogen and lipid (triglycerides, TG) through processes of glycogenesis and

lipogenesis (Barrett, 2003; Kaur, 2014). In order to produce all anabolic effects, insulin

binds with its receptor present on outer surface of plasma membrane of target cells or

tissues and mediates its action through tyrosine kinase cascade mechanism (Guo, 2014).

Therefore, due to complete deficiency of insulin or availability of low units of insulin

(relative insulin deficiency) or decrease insulin sensitivity (insulin resistance) for its

receptors, glucose influx in target tissues stops and remains elevated in blood to creates a

hyperglycemic condition, the prominent feature of diabetes. This hyperglycemia, slowly

and gradually, disturbs various biological mechanisms and contributes irreversible life-

threatening complications of this disorder.

1

1.2 Prevalence

According to the International Diabetes Federation‘s (IDF) statistics, 366

million people were suffering from diabetes in 2011 and this figure may rise to 552

million by 2030 (Whiting et al., 2011). The federation also claimed that more than

80% of diabetic population is contributed by low and middle-income countries, the most

affected age ranging from 40-59 years (Whiting et al., 2011). This increased prevalence

of diabetes in developing countries is actually the result of adopting sedentary life-style,

westernized diet with high intake of wheat and rice, fizzy drinks, heavy smoking and

alcohol consumption (Abate and Chandalia, 2003). In addition, inappropriate nutrition

during pregnancy and in old age also enhanced the risk of diabetes (Adeghate et al.,

2006). The prevalence of type 2 diabetes is also increasing day by day in children

because of high intake of junk food or fizzy drinks plus long-sitting in front of computers

for playing video games, watching television for entertainment and lack of play grounds

because of increasing urbanization, all these factors are gradually increasing obesity and

insulin resistance in children (Kaufman, 2002; Quan et al., 2013). The same situation is

also seen in case of type 1 diabetes as, a study reported that almost 78,000

children developed type 1 diabetes annually (Stanescu et al., 2012). Furthermore,

diabetic patients are at high risk of associated complications which ultimately result in

increased hospitalization, disability and mortality count (Young et al., 2008). Another

study reported that diabetes is one of the top-ten causes of death in both developed and

developing countries (Alberti and Zimmet, 2014).

Pakistan ranks sixth among the highly populated countries in the world with 191

million residents according to the world population data sheet issued by US Population

Reference Bureau in 2013 (Chhutto et al., 2009; Ramachandran et al., 2014). Of these,

6.9 million people are affected with diabetes and this could double by the end of 2025

according to IDF. On the basis of these data, Pakistan ranked seventh among the

countries with high diabetic prevalence (Qidwai and Ashfaq, 2010). A national survey

describes the prevalence of diagnosed diabetes in urban areas as 5.1% in men and 6.8%

in women whereas in rural areas it constitutes 5.0% in men and 4.8% in women (Aziz et

al., 2009). No doubt, this is an alarming situation for our country but it can be prevented

2

http://www.sciencedirect.com/science/article/pii/S0889852912001004

http://www.sciencedirect.com/science/article/pii/S016882271300394X

by educating people about diet control and providing medical facilities at reasonable cost

in all over the Pakistan.

1.3 History

The history of diabetes started from long time back when Ancient Egyptians

(1500 B.C.E.) recognized an uncommon condition of frequent urination in persons with

apparently low body weight. A Greek physician Aretaeus (80-138 C.E) provided a

complete description about the same condition. Another Greek Apollonius of Memphis

(230 B.C.E) introduced the word ―diabetes‖ for the condition characterized by excessive

urine with sweet taste. An Arabic physician Avicenna (960-1037) described the diabetic

complications include peripheral neuropathy, gangrene and erectile dysfunction in his

well-known book ―Al Qanoon‖ which remained a classic and standardized medical

literature in numerous European Universities while Al-Razi, a Muslim scientist,

mentioned the treatment of polyuria and obesity (Savona-Ventura, 2009). Interestingly,

the types of diabetes were identified by two Indian physicians Sushrant and Charaka

(400-500 C.E) who described that type 1 is found in young people and type 2 is

associated with obesity (Shaikh, 2010).

The second word ―mellitus‖ which generally stands for honey, was added to

diabetes by Thomas Willis (1675), though, Matthew Dobson (1776) measured surplus

glucose in urine of diabetic patients (Dobson, 1776). In 1812, diabetes was recognized as

a separate clinical entity and F. C. Bernard (1857) gave a significant concept that glucose

level not only elevated by dietary intake but also by synthesizing from non-carbohydrate

precursors in liver (Robin and Bernard, 1979). Later, the role of pancreas in the

pathogenesis of diabetes and maintaining the glucose homeostasis was recognized by J.

von Mering and O. Minkowski (1889) whereas this concept was extended by E. A.

Sharpey-Schafer (1910) who theorized that diabetes is due to the deficiency of chemical

named ―insulin‖ produced by pancreas (von Mering J, Minkowski, 1890; Brogard et al.,

1992).

Many noble prizes have been awarded to different scientists in the fields of

Medicine and Chemistry who were involved in discovering universal aspects of diabetes

3

like F. Banting and C. Best (1921) discovered insulin and won Nobel prize (1923) with J.

Collip and J. Macleod for the discovery of insulin from bovine pancreas and used it for

the treatment of diabetes (Banting et al., 1991). Similarly, C. F. Cori and G. T. Cori

(1947) won the same prize for the discovery of catalytic conversion (synthesis and

breakdown) of glycogen by describing the key enzymes of glycogen metabolism (Cori

and Cori, 1946). In the same year, B. A. Houssay received this prestigious Prize for

discovering the role of anterior pituitary hormones in glucose metabolism (Houssay,

1942). The Noble prize was also awarded to F. Sanger (1958) for developing method

which helped to study amino acid sequence in insulin while the same prize was given to

E. W. Sutherland (1971) for describing the role of cyclic AMP (cyclic 3‘5‘-adenosine

monophosphate) in the mechanism of action of hormones epinephrine and glucagon

which are involved in increasing the blood glucose level and constituted a diabetic

hyperglycemia (Sanger, 1945; Sutherland, 1972). R. Yalow and S. Berson received that

honor (1977) for the development of method named radioimmunoassay and made the

quantitative analysis of insulin and other metabolites possible in animals and human

(Yalow and Berson, 1959). In 1992 the efforts of E. H. Fischer and E. G. Krebs made

dramatic advancement in the field of diabetes by discovering the reversible protein

phosphorylation as a biological regulatory mechanism (Fischer, 2010). The journey of

discoveries concerning to treatment pattern of diabetes, patients‘ awareness and

education is still going on, however, steadily rise in diabetes prevalence declared it is a

worldwide epidemic and most serious medical disorder of mankind.

1.4 Types

Diabetes is classified as,

Type 1 (Insulin-dependent diabetes mellitus, IDDM)

Type 2 (Non insulin-dependent diabetes mellitus, NIDDM)

Gestational diabetes

Experimentally-induced diabetes

4

http://en.wikipedia.org/wiki/Diabetes_mellitus_type_1


1.4.1 Type 1 Diabetes (Insulin-Dependent Diabetes Mellitus, IDDM)

Type 1 diabetes is due to the inability of pancreas to synthesize insulin. This

failure is due to either congenital absence or destruction of pancreatic β-cells (insulin-

producing cells of the islets of Langerhans in pancreas) that leads to absolute insulin

deficiency. The destruction of β-cells may be either due to toxic chemicals or drugs or

viruses or immune-mediated mechanism in which autoimmune antibodies involved in

CD8+ T-cell mediated destruction of β-cells thereby inhibiting the release of insulin

(Rother, 2007). This type of autoimmune diabetes is often linked with other endocrine

disorders especially those associated with adrenal and thyroid glands such as Addison‘s

disease, Grave‘s disease and Hashimoto‘s thyroiditis (Barker, 2006). The autoimmune

type 1 diabetes has also been reported in people with age above 30 years termed as latent

autoimmune diabetes of adulthood (LADA) (Landin-Olsson, 2002). The onset of this

type 1 is due to the involvement of multiple genes, the strongest gene IDDM 1 is located

in the major histocompatibility complex (MHC) class II region of chromosome 6 (Pociot

and McDermott, 2002). Other genetic causes of type 1 diabetes include mutation in gene

that encodes for insulin or defects in post-translational modification of insulin especially

in pro-insulin conversion (Pociot and McDermott, 2002).

The prevalence of type 1 diabetes is about 10% of total diabetic population

(Daneman, 2006). The cases of this type are reported in children (juvenile diabetes) and

in adults up to 25 years of age while its onset is acute and may or may not be associated

with family history (Aanstoot et al., 2007). The symptoms of type 1 is imbalanced and

irregular hyperglycemia accompanied with diabetic ketoacidosis, polyuria, polydipsia,

polyphagia, xerostomia (dry mouth), fatigue and weight loss (Clark et al., 2007). The

patients suffering from this type are completely dependent on exogenous insulin

injections for its treatment (Barrett, 2003).

1.4.2 Type 2 Diabetes (Non insulin-Dependent Diabetes Mellitus, NIDDM)

Type 2 diabetes is most commonly reported and constitutes about 90% cases in

all diabetic population. It is reported in adults having age 35 years and above thus

referred as adult-onset diabetes or maturity onset diabetes of the young (MODY)

5


http://en.wikipedia.org/wiki/Beta_cell

http://en.wikipedia.org/wiki/Islets_of_Langerhans

http://en.wikipedia.org/wiki/Hyperglycemia

http://en.wikipedia.org/wiki/Ketosis

(Schellenberg et al., 2013). Type 2 primarily results from relative insulin deficiency or

insulin resistance which may be due to mutation in insulin receptor or post-receptor

deformities (Quan et al., 2013). The onset of type 2 is gradual and often associated with

family history (Valdez, 2009). Its symptoms include hyperglycemia which may or may

not be associated with ketoacidosis, however, three Ps viz., polyuria, polydipsia,

polyphagia with fatigue and weight gain is observed (Vijan, 2010). The treatments

include diet, exercise, oral hypoglycemic medicines, insulin injections (in severe cases)

or combination of these (Ripsin et al., 2009).

The secondary causes of type 2 diabetes include life-style and dietary pattern,

obesity, hypertension, endocrine disorders like Cushing‘s syndrome, gigantism,

acromegaly and other genetic conditions like cystic fibrosis, fibrocalculous

pancreatopathy (Schellenberg et al., 2013; Risérus et al., 2009; Resmini et al., 2009;

Mohan et al., 2008; Sharer et al., 1998). Medicines are also reported to induce insulin

resistance like protease inhibitor, anti-inflammatory glucocorticoids, interferon, etc

(Ripsin et al., 2009; Vijan, 2010).

1.4.3 Gestational Diabetes

Gestational diabetes is a condition which some women experience during

pregnancy especially in the third trimester of gestation in which high blood glucose level

is observed though not previously diagnosed (Buchanan and Xiang, 2005). In pregnancy,

the malfunctioning of insulin receptor occurs due to the presence of human placental

lactogen which interferes with the sensitivity of insulin receptors that result in elevated

blood glucose level (Kapoor et al., 2007). Studies mentioned that gestational diabetes

affects 3-10% of pregnancies and untreated gestational diabetes may leads to certain

complications of delivery, hypoglycemia, seizures or stillbirth and jaundice (Haram

and Gjelland, 2007; Nold and Georgieff, 2004). Studies reported that women who

experienced gestational diabetes have higher chances of preeclampsia and caesarean

section plus are at high risk of developing type 2 (or type 1) diabetes after few years of

pregnancy (Metzger et al., 2008). This type of diabetes if remains unmanaged by mother

could produce some consequences in offspring like being prone to developing childhood

6

http://en.wikipedia.org/wiki/Insulin_resistance

http://www.ncbi.nlm.nih.gov/pubmed?term=Buchanan%20TA%5BAuthor%5D&cauthor=true&cauthor_uid=15765129

http://www.ncbi.nlm.nih.gov/pubmed?term=Xiang%20AH%5BAuthor%5D&cauthor=true&cauthor_uid=15765129

http://www.ncbi.nlm.nih.gov/pubmed?term=Haram%20K%5BAuthor%5D&cauthor=true&cauthor_uid=17952149


http://www.ncbi.nlm.nih.gov/pubmed?term=Gjelland%20K%5BAuthor%5D&cauthor=true&cauthor_uid=17952149

http://en.wikipedia.org/wiki/Pre-eclampsia

http://en.wikipedia.org/wiki/Caesarean_section

http://en.wikipedia.org/wiki/Caesarean_section

http://en.wikipedia.org/wiki/Type_2_diabetes_mellitus

obesity while in later age of life it could convert into type 2 diabetes (Boney et al., 2005;

Metzger et al., 2008).

1.4.4 Experimentally-Induced Diabetes

Diabetes has been experimentally induced in animals through certain well-

reported methods including administration of various toxicants (chemicals), drugs

(steroids or glucocorticoids), genetic and surgical manipulations (Etuk and Muhammed,

2010). Of which, two experimental animal models or methods are alloxan-induced type 1

diabetic and fructose-induced type 2 diabetic animal models. These models are also

adopted in the present study to investigate the antidiabetic effect of experimental plant.

Alloxan-Induced Diabetes: Alloxan is a widely used pyrimidine derivative to

induce type 1 diabetes in laboratory animals. Alloxan has been found toxic to pancreatic

β-cells specifically as it can enter in these cells as glucose analogues. Beside this, alloxan

mediates its cytotoxic effect chiefly by generating reactive oxygen species (ROS).

Alloxan and its reduced product (dialuric acid) are involve in establishing a redox cycle

by forming superoxide radicals that may undergo dismutation to hydrogen peroxide

(H2O2) and highly reactive hydroxyl radicals through Fenton reaction. In addition, the

massive increase in cytosolic calcium concentration ultimately leads a rapid degeneration

of β-cells of pancreatic islets by causing destruction of DNA of these cells thereby

inhibiting insulin release (Federiuk et al., 2004; Rohilla and Ali, 2012).

Fructose/Sucrose-Induced Diabetes: High (10% w/v) and continuous intake of

fructose or sucrose (a disaccharide consists of fructose and glucose) was reported to

induce obesity which in turn induce insulin resistance, the characteristic feature of type 2

diabetes in rats and mice models by producing hypercholesterolemia,

hypertriglyceridemia and hyperinsulinemia (Wilson and Islam, 2012; Adeneye, 2012;

Johnson et al., 2009). Fructose is a lipogenic agent which after intestinal absorption

enters into liver cells via GLUT-5 (insulin-independent glucose transporter) where it

produces dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (GA3P)

without passing through the step catalyzed by phosphofructokinase, one of the rate-

7

http://en.wikipedia.org/wiki/Type_2_diabetes

regulatory enzymes of glycolysis, thereby accelerates the synthesis of glucose via

gluconeogenesis and triglycerides (TG) that leads to hyperglycemia and

hypertriglyceridemia (Feinman and Fine, 2013; Bray, 2007). This increased amount of

TG not only decrease the insulin sensitivity (insulin resistance) to its receptor by

masking them on target tissues which results in hyperinsulinemia but also act as an

alternate source of energy for the body results in increased production of acetyl

coenzyme A that cannot be easily handled by tricarboxylic acid cycle (TCA) thus induce

cholesterol biosynthesis and leads to hypercholesterolemia (Feinman and Fine, 2013;

Bray, 2007; Johnson et al., 2009). In spite of these reported and well-known harmful

effects, high fructose corn syrup (HFCS) is one of the widely used sweeteners in

commercial food stuffs where it acts as a slow poison for increasing the risk of metabolic

disorders especially type 2 diabetes globally (Bray et al., 2004; Gorana et al., 2012).

1.5 Complications

1.5.1 Acute Complications

Diabetic ketoacidosis: In diabetic condition, the body starts utilizing fatty acids

as an alternate source of energy through beta-oxidation, this results in the production

and accumulation of acetyl-CoA which cannot be easily handled by tricarboxylic acid

(TCA) cycle. This increased amount of acetyl-CoA is then involved in the formation of

ketone bodies (acetone, acetoacetic acid and beta-hydroxybutyric acid). Accumulation

of these ketone bodies in blood creates a condition called ketonemia and because of

acidic nature, these are responsible to shift the pH of blood towards acidic side and

create ketoacidosis. Ketonemia is also accompanied with ketonuria (excretion of ketone

bodies in urine). This ketoacidosis may also lead to many deleterious chronic

complications of diabetes (Sheikj-Ali et al., 2008).

Hyperosmolar hyperglycemic condition: Polyuria is one the symptoms of

diabetes induced by hyperglycemia, as a result of which large amount of water along

with sugar excrete out in urine that leads to dehydration and hyperosmolar state in body.

8

This condition brings weakness, sweating, agitation and other physiological problems

like coma and death in extreme cases (Kitabchi et al., 2009).

Others: Persistent hyperglycemia may also results in decreased immunity in

body that may enhance the susceptibility of various bacterial and fungal infections in

body like pneumonia, influenza, foot infections, etc (Lavery et al., 2006).

1.5.2 Chronic Complications

Uncontrolled hyperglycemia gradually and severely affects small and large blood

vessels that may leads to number of complications which can be categorized as follows:

1.5.2.1 Micro-vascular Problems

Diabetic retinopathy: Chronic hyperglycemia impairs visual clarity and leads to

the development of cataract, if this condition remains untreated, it induces blindness. The

key enzyme aldose reductase (AR) of polyol pathway is involved in diabetic

complication by converting glucose into sorbitol (glucose alcohol) upon reduction and

accumulates it in cells including retinal cells as the endothelial cells of retinal vessels are

rich in AR (Pathania et al., 2013; Gabbay, 1973). The sorbitol formation in the presence

of AR requires NADPH, this depletes NADPH pool which is essentially needed by

glutathione reductase, the enzyme involved in maintaining the intracellular pool of

reduced glutathione (GSH), an important antioxidant protein in cells (Barnett et al.,

1986). The second reaction of same pathway, catalyzed by sorbitol dehydrogenase

(SDH) converts sorbital into fructose which after phosphorylation forms fructose-3-

phosphate that converts into 3-deoxyglucosone. These two compounds are powerful

glycosylating agents which non-enzymatically glycate cell proteins and formed advanced

glycation end (AGEs) products that produced osmotic stress. Hence in diabetes, because

of the excess glucose, polyol pathway is actively operational in retinal vicinity and

generates osmotic and oxidative stresses that ultimately disturb the fluid homeostasis in

retina therefore, leads to cataract, macular edema, micro-aneurysms, apoptosis in

vascular cells and retinal ischemia (Engerman, 1989; Lorenzi, 2007). The diabetic

retinopathy categorized into background retinopathy characterized by dot hemorrhages

9

http://care.diabetesjournals.org/search?author1=Abbas+E.+Kitabchi&sortspec=date&submit=Submit

http://care.diabetesjournals.org/search?author1=Lawrence+A.+Lavery&sortspec=date&submit=Submit

and proliferative retinopathy characterized by white areas on retina. If both remain

untreated, blindness can occur due to damage of vitreous chamber of an eye (Lorenzi,

2007).

Diabetic nephropathy: In diabetes, excess glucose induces osmotic pressure

which results an increase excretion of urine (polyuria) that alters the re-absorbing

capacity of kidney for glucose. This frequent urination also bring sodium chloride out of

body as a result of which juxta-glomerular apparatus starts synthesizing a local hormone

rennin to induce vasoconstriction in order to slow down the entry of blood in glomerulus.

This process affects glomerular capillaries hence called as intercapillary

glomerulonephritis (Cooper, 1998). With the passage of time, it induces structural

deterioration of glomeruli results in proteinuria (microalbuminuria), decreased

glomerular filtration rate (GFR) by showing nodular glomerulosclerosis and finally end-

stage renal failure (Chang, 2008). Beside this, other pathways including polyol, protein

kinase C (PK-C) and hexosamine pathways are involved in producing advance glycation

end (AGE) products, reactive oxygen species (ROS), pro-inflammatory cytokines,

growth factors and cell adhesion molecules, all in turn induce renal structural change and

contribute to diabetic nephropathy (Tan et al., 2007).

Diabetic neuropathy: Neuropathy refers to ―nerve damage‖. In diabetes,

NADPH becomes depleted by accelerating polyol pathway which affects the synthesis of

reduced glutathione (GSH) and nitric oxide (NO) that act as antioxidant protein and

vasodilator respectively. In addition, the same pathway disturbs myo-inositol production,

required for nerve conduction. Due to the deficiency of NO, vasoconstriction can occurs

that decreases the blood flow and induces the oxygen deficiency that results in neural

ischemia. Secondly, elevated AGEs and ROS induce damage to the walls of blood

vessels by affecting membrane proteins. Thirdly, decreased production of myo-inositol

and increased production of diacylglycerol (DAG) through PK-C pathway affects neural

conductivity. All these mechanisms together induce neural dysfunction (Wu et al., 2013;

Edwards et al., 2008). This neuropathy affects almost all peripheral nerves (pain fibers),

motor neurons and the autonomic nervous system. The general symptoms of diabetic

10

neuropathy include numbness, tingling, dysesthesia, diarrhea, erectile dysfunction,

urinary incontinence, and eyelid drooping, vision changes, dizziness, muscular

weakness, speech impairment, fasciculation, anorgasmia, etc (Boyko, 1998). It was also

stated that more than 50% population with diabetes have diabetic neuropathy and

persistence of this situation leads to many other complications (Aring et al., 2005).

1.5.2.2 Macro-vascular Problems

In diabetes, AGE products and oxidative stress play an important role in the

development of heart problems especially atherosclerosis which is the beginning of other

cardiovascular diseases like angina, myocardial infarction, stroke, etc (Jay et al., 2006).

AGE product- induced cross-linked formation results in arterial stiffening and loss of

elasticity of large blood vessels. In addition, ROS modify low-density lipoprotein

cholesterol (LDL-c) into oxidized LDL-c (ox-LDL) that can easily engulfed by

macrophages to form foam cell and deposited on walls of coronary arteries to form

atherosclerotic plaque or atheroma (Qureshi et al., 2010). A muscle wasting (diabetic

myonecrosis) problem has occasionally been seen in diabetic patients especially in

females while irregular fatigue-related pain in leg and foot (claudication) has been

observed in both genders (Sran et al., 2014). Stroke (ischemic type) is also reported in

diabetic patients (Sacco, 2002). Diabetes also impairs cognitive capabilities

(encephalopathy) that enhanced the risk of dementia in patients (Mijnhout et al., 2006;

Sima, 2010). Neuropathy in combination with peripheral vascular problems, diabetic

patients feel numbness in feet and in severe cases, skin infections, foot ulcers and

gangrene have also been reported (Yasuda, 2013). Literature also established that

diabetes in women may also induce reproductive complications like increase risk

of infertility, delayed puberty, menarche, menstrual irregularities

(especially oligomenorrhoea), mild hyperandrogenism, polycystic ovarian syndrome and

possibly earlier menopause (McCance, 2011; Jonasson et al., 2007).

11

http://en.wikipedia.org/wiki/Macrovascular_disease

http://en.wikipedia.org/wiki/Atherosclerosis

http://en.wikipedia.org/wiki/Diabetic_myonecrosis

http://en.wikipedia.org/wiki/Diabetic_myonecrosis

http://en.wikipedia.org/wiki/Intermittent_claudication

http://en.wikipedia.org/wiki/Stroke

http://en.wikipedia.org/wiki/Dementia

http://en.wikipedia.org/wiki/Oligomenorrhoea

http://en.wikipedia.org/wiki/Hyperandrogenism

http://en.wikipedia.org/wiki/Polycystic_ovarian_syndrome

http://en.wikipedia.org/wiki/Menopause

http://care.diabetesjournals.org/search?author1=Junmei+Miao+Jonasson&sortspec=date&submit=Submit

1.6 Treatments

The main objective of the treatment of diabetes is to keep the glucose

homeostasis in body. In this regard, numbers of strategies have been in practice including

controlled diet plan, exercise, antidiabetic medicines (ADM) and insulin injections.

Beside these, herbal medicines are also gaining fame in both developed and developing

countries (Tiwari, 2008; Aggarwal and Shishu, 2011). These preventive measures lower

the risk of chronic complications of this disorder.

1.6.1 Pharmaceutical Medicines as Conventional Treatment

Type 1

The treatment of type 1 diabetes becomes the part of daily routine of patients

which include restricted diet, proper exercise and insulin injections or commercially

manufactured insulin analogs or their combinations (Atkinson and Eisenbarth, 2001;

Shaikh et al., 2011). All these strategies required monitoring of glucose more than once a

day regularly. The route of insulin is usually subcutaneous while in severe condition it

may also be given intravenously for prompt action. Commercially there are three types

of insulin preparations available which are categorized on the basis of their duration of

action, as

i. Insulin for rapid action

ii. Insulin for intermediate action

iii. Insulin for long action

Type 2

The patients of type 2 diabetes also require its treatment throughout the life-time in

the form of antidiabetic medicines (their selection strictly depends on the exact cause of

type 2 diabetes) in combination with controlled diet and exercise. However, 30-40 % of

these patients also need insulin injections, if medicines are unable to control

12



hyperglycemia (DeFronzo, 1999; Ripsin et al., 2009). A vast range of ADM are

commercially available with different antidiabetic or hypoglycemic potential, as

i. ADM that enhances the insulin release from β-cells of pancreas are

referred as insulin secretagogues such as sulfonylureas do the task by suppressing

the KATP channel. This class includes toulbutamide, chloropropamide (first generation)

and glyburide, glipizide, glimepiride (second generation). First generation has long

duration of action while the second generation is more efficacious with fewer side effects

like weight gain (Rang et al., 2003).

Meglitinide is a famous short-acting secretagogues which activates both pancreas

and insulin production. Its action is as same as sulfonylureas but the binding site is

distinct and it closes the potassium channels of β-cells of pancreas and facilitates the

opening of calcium channels, ultimately enhances the insulin secretion. Its side effects

include weight gain and hypoglycemia (Blicklé, 2006).

Another group of insulin secretagogues named incretins have been introduced

recently. It includes exenatide and liraglutide are two long-lasting analogues of incretins

and administrated subcutaneously. They mimic the action of glucagon like peptide-

1 (GLP-1), a gastrointestinal peptide hormone which stimulates insulin secretion while

inhibits glucagon secretion and hepatic glucose production. In addition, it slows the

nutrient absorption rate in blood stream by reducing the gastric emptying and may

directly suppresses intake of food through central nervous system pathways though

incretins have side effects like weight loss, nausea and gastro-intestinal problems

(Drucker and Nauck, 2006).

ii. ADM that promotes the sensitivity of target tissues to insulin are referred as

insulin sensitizers such as metformin (a biguanides). It increases the uptake of glucose by

peripheral tissues like skeletal muscle and reduces output of hepatic glucose by

inhibiting the process of gluconeogenesis. Metformin is the most commonly used

hypoglycemic agent and widely prescribed medicine for newly diagnosed type 2 diabetic

patients. However, precautions must be taken in those patients that have hepatic and

13

http://en.wikipedia.org/wiki/Secretagogue

http://en.wikipedia.org/wiki/ATP-sensitive_potassium_channel

http://en.wikipedia.org/wiki/Meglitinide


http://www.ncbi.nlm.nih.gov/pubmed?term=Drucker%20DJ%5BAuthor%5D&cauthor=true&cauthor_uid=17098089

http://www.ncbi.nlm.nih.gov/pubmed?term=Nauck%20MA%5BAuthor%5D&cauthor=true&cauthor_uid=17098089

renal impairments. It does not induce gain in body weight and found effective in

reducing the plasma triglyceride (TG) and low-density lipoprotein cholesterol (LDL-c)

levels (Dunn and Peters, 1995; Hundal and Inzucchi, 2003).

Another group of sensitizer is thiazolidinediones (TZDs) or glitazones such as

rosiglitazone and pioglitazone. These TZDs bind with gamma peroxysome proliferator

activated receptor (PPARγ) that induces the transcription of genes involved in the

regulation of glucose and lipid metabolism thereby improving the utilization of glucose

by target tissues. This group also found effective in decreasing plasma TG and LDL-c

levels. However, weight gain, peripheral edema, anemia and hepatoxicity were reported

as side effects of TZDs (Gale, 2001; Loke et al., 2011).

iii. ADM that effectively lowers the absorption of glucose from the GIT like α-

glucosidase inhibitors including acarbose and miglitol responsible to inhibit the activity

of enzyme α- glucosidase that involved in the conversion of complex carbohydrate into

glucose thereby delaying its absorption in gastro-intestinal tract (Karla, 2014).

A new amylin analogue, pramlintide, that can be given subcutaneously as adjunct

to insulin in both type 1 and 2 diabetic patients. It is also responsible to slow down the

gastric emptying thereby slowing the glucose absorption in blood stream (Edelman et al.,

2008).

1.6.2 Medicinal plants as an Alternative Treatment

Tradition to utilize medicinal plants as natural remedy for different ailments is as

old as the civilization of mankind. Interestingly, this science has had religious as well as

spiritual background that supports its therapeutic effectiveness since long (Badoni and

Badoni, 2001). A study described that even in this modern era 80% population of

African and Asian countries depends on phytomedicines (Tiwari, 2008). The safe and

cost-effective nature of these medicines holds a strong competitive place among

allopathic medicines (Meena et al., 2009). It was estimated that the entire market of

plant-based products was nearly 10 billion US$ in late nineties (1997) and forecast with

approximately 7% growth in this figure annually (Rates, 2001).

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http://en.wikipedia.org/wiki/Peroxisome_proliferator-activated_receptor_gamma

Many basic and life-saving medicines are derived from plant sources, for

example, ajmalicine (antihypertensive) from Rauwolfia serpentina, atropine

(anticholinergic) from Atropa belladonna., digitalin, digitoxin & digoxin (cardiotonic)

from Digitalis purpurea, morphine & codeine (analgesic) from Papaver somniferum.,

quinine (antimalarial) & quinidine (antiarrhythmic) from Cinchona ledgeriana, and

vincristrine & vinblastine from Catharanthus roseus, etc (Siddiqui and Siddiqui, 1931;

Shu, 1998; Rates, 2001). Similarly, nearly 12000 compounds from plant origin with

different pharmacological activities have been isolated and investigated (Habeck, 2003;

Marles and N. R. Farnsworth, 1995) but there is more left for investigation since a

variety of active compounds are present in a single plant. Therefore, in these days of

modern technology there is a need for discovering the new drug molecules that are still

hidden in natural sources by applying extensive experimental measures and

technological advancement.

1.6.2.1 Role of Medicinal Plants in the Treatment of Diabetes and Diabetic

Dyslipidemia

The role of medicinal plants in the management of diabetes is well-reported in

scientific literature, especially the literature derived from developing regions of the world

where modern facilities and healthcare resources are not easily accessible to common

man (Patel et al., 2012). Many ethnobotanical and ethnopharmacological studies have

revealed the use of more than 1200 plant species with prominent hypoglycemic activity

(Habeck, 2003; Yang, 2014). Amazingly, metformin (one of the commonly used

medicines in type 2) has been originally derived from plant Galega officinalis (Bailey

and Day, 2004). In spite of this, ethanopharmacologists are still working and looking for

a big breakthrough to develop a safest drug for the management of diabetes without any

side effect. Some of the well-reported medicinal plants that have been used

conventionally all across the world for the treatment of diabetes, are

Allium cepa: Commonly known as ―onion‖ (family: Liliaceae). Its various

fractions and dried powder exhibited significant hypoglycemic potential in alloxan-

induced diabetic rats where it improved diabetic dyslipidemia (Modak et al., 2007). In

15

addition, a clinical trial on diabetic patients justified its postprandial blood glucose

lowering activity (Taj Elhadin et al., 2010).

Allium sativum: Commonly known as ―garlic‖ (family: Liliaceae). Its

antidiabetic potential in both animal and human subjects is well-reported by describing

its ameliorative effect on fasting blood glucose (FBG), TG, total cholesterol (TC), lipid

peroxidation, liver glycogen and enzymes viz., hexokinase & glucose-6-phosphatase

(Koscielny et al., 1999; Eidi et al., 2006; Londhe et al., 2011). In addition, it was

reported to enhance the insulin secretion from β-cells (Albajali et al., 2011).

Aloe vera (Syn: Aloe barbadensis): It is considered as home grown plant

(family: Xanthorrhoeaceae) with multiple uses in folk medicine (Pal et al., 2013).

Mucilaginous gel from the leaves of this plant is reported in producing prominent fall in

blood glucose and TG levels while other studies also supported its antidiabetic effects

with the release of insulin from β-pancreatic cells (Ajabnoor, 1990; Lanjhiyana et al.,

2011).

Azadirachta indica: Commonly called as ―neem‖ (family: Maliaceae). Vast

researches established its antihyperglycemic potential by enhancing insulin release from

β-cells of pancreas and showing positive effect on diabetic dyslipidemia. Thus A.indica

characterized as a significant phytomedicinal remedy for the treatment of diabetes

(Biswas et al., 2002; Bhat et al., 2011).

Coccinia indica: Its administration in diabetic patients was reported to accelerate

the lipase activity with improvement in diabetic status while other studies also supported

its hypoglycemic potential by inducing glucose tolerance in animal models (Kamble et

al., 1998; Modak et al., 2007).

Eugenia jambolana: Commonly known as ―jamun‖ (family: Myrtaceae). The

hypoglycemic and antidiabetic activities of fruit pulp, seeds powder and extract of

E.jambolana were reported in different animal models and in type 2 diabetic patients

(Waheed et al., 2007). Studies also reported its insulin secretary potential from β-cells

(Chaturvedi et al., 2009).

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Ginseng species: For a long time in Far East, the roots of Ginseng species

(family: Araliaceae) have been used as health-promoting agent (Cefalu et al., 2008). Two

species of ginseng including Panax ginseng (Asian ginseng) and P. quinquefolius

(American ginseng) have prominent hypoglycemic potential especially in type 1 diabetes

with improving effect on glycosylated (glycated) hemoglobin (HbA1c) and body weights

while it was found to lowers postprandial blood glucose level in type 2 diabetic patients

(Uzayisenga et al., 2014; Sen et al., 2013). The antidiabetic activity of Ginseng species is

due to inhibiting glucose absorption in intestine, reducing destruction of β-cells and

insulin resistance (Mucalo et al., 2012; Yoo et al., 2012).

Gymnema sylvestre: Commonly termed as ―gurmar‖ (family: Asclepiadaceae)

and in US market it was also characterized as ―sugar blocker‖ (Anjum and Hasan, 2013).

A clinical trial on type 2 diabetic patients demonstrated its effectiveness on blood

glucose and HbA1c levels (Kumar et al., 2010). The antidiabetic potential of same plant

was claimed due to the regeneration of β-cells thus elevates insulin level and glucose

absorption in intestinal mucosa (Al-Romaiyan et al., 2013).

Momordica charantia: Commonly known as ―bitter melon‖ or ―karela‖ (family:

Cucurbitaceae) and it is easily available in Asia, Africa and America. In both pre-clinical

and clinical trials, its seed powder and fresh juice have been well-reported for significant

antidiabetic effect by showing insulin secretagogue or insulinomimmetic action (Patel et

al., 2012). Its subcutaneous administration in type 1 diabetic subjects showed prominent

fall in FBG level due to the presence of insulin-like peptide, known as polypeptide-P

(almost similar to bovine insulin in action) in this plant that showed significant result by

inhibiting gluconeogenesis (Efird et al., 2014). Other pharmacological studies on this

plant described its effect in type 2 patients by improving glucose tolerance, postprandial

blood glucose and HbA1c levels (Habicht et al., 2014).

Trigonella foenum-graecum: Commonly termed as ―fenugreek” or ―meethi‖

(family: Fabaceae) and is used all over Asia for the management of diabetes (Rizvi and

Mishra, 2013). The oral administration of defatted seeds extract of T. foenum-graecum

in type 2 diabetic patients was found to decrease fasting and postprandial blood glucose

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levels while improves lipid profile and insulin level (Saxena and Vikram, 2004; Kassaian

et al., 2009). However, its effectiveness in type 1 diabetic patients was contributed to its

effect on reversing the activities of glucose and lipid metabolizing enzymes thus

established glucose homeostasis (Modak et al., 2007; Sauvaire et al., 1998).

Some other antidiabetic plants: Acacia Arabica (Babhul) showed promising

antidiabetic activity and attributed with insulin secretagogues potential from pancreatic

region (Wadood et al., 1989; Hegazy et al., 2013). Aegle marmelos (Bengal quince)

showed prominent reduction in glucose, cholesterol and urea levels. The decrease in

blood glucose level was found by delaying the carbohydrate absorption (Narender et al.,

2007; Kumar et al., 2013). Capparis decidua proved its hypoglycemic and

hypolipidemic effects with improvement in lipid peroxidation (Zia-Ul-Haq et al., 2011;

Chahlia, 2009; Yadav et al., 1997). Phyllanthus amaranthus was also reported in the

treatment of diabetes with antioxidative potential (Kim et al., 2006). Pterocarpus

marsupium has antidiabetic activity by showing prominent insulinogenic effect (Ahmad

et al., 1989). Ocimum sanctum leaves extract showed hypoglycemic activity with

improvement in diabetic dyslipidemia in pre-clinical models (Vats et al., 2004). Oral

administration of Tinospora cordifolia resulted in lowering of blood and urine glucose

levels with improvement in body weights and also induced glucose tolerance in rodents

(Saha and Ghosh, 2012). To date, many plants were screened for their potential

antidiabetic activities and bulk of literature represents the active involvement of

phytotheraphy in diabetes and diabetic dyslipidemia.

1.6.2.2 Role of Medicinal Plants as Antioxidants

An antioxidant represents an enzyme or any organic substance which prevents the

body from harmful impacts of oxidants (Tiwari, 2001). The oxidation represents a

biochemical reaction that involves the transfer of electrons or hydrogen atoms from a

substance to an oxidizing agent which ultimately generates free radicals in the cellular

environment (Davies, 2001). The term free radical describes the molecular entity with

one or more unpaired electrons in its outer shell (Blois, 1958). These radicals can

generally be classified into reactive oxygen species (ROS) and reactive nitrogen species

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http://en.wikipedia.org/wiki/Oxidizing_agent

(RNS) like hydroxyl (OH•), superoxide (O2•ˉ), nitric oxide (NO•), nitrogen dioxide

(NO2•), peroxyl (ROO•) and lipid peroxyl (LOO•) radicals while other non-reactive

species like hydrogen peroxide (H2O2), ozone (O3), singlet oxygen (1O2), hypochlorous

acid (HOCl), nitrous acid (HNO2), peroxynitrite (ONOOˉ), dinitrogen trioxide (N2O3),

lipid peroxide (LOOH) are also categorized as oxidants (Pham-Huy et al., 2008). The

sources of these reactive species include electron transport chain in mitochondria,

NADPH oxidase and myeloperoxidase of neutrophils and xanthine oxidase of

endothelial cells (Seifu et al., 2012). The ROS or RNS have harmful impacts on body by

reacting with or oxidizing all macromolecules including carbohydrates, lipids, nucleic

acids and proteins (Davies, 2001). These ROS-linked damages reported to induce many

problems like aging, atherosclerosis, cancer, cardiovascular diseases, diabetes,

inflammatory consequences, due to the imbalance between radical generating and

scavenging systems in body that constitute oxidative stress (Seifu et al., 2012).

Interestingly, nature equipped living machinery with important defense systems to

detoxify radicals which include enzymes like catalase (CAT), superoxide dismutase

(SOD), glutathione peroxidase (GP), glutathione reductase (GR) and non-enzymes like

albumin, ceruloplasmin, ferritin (high molecular weight) while ascorbic acid, α-

tocopherol, β-carotene, glutathione (GSH) and uric acid (low molecular weight)

antioxidants (Pham-Huy et al., 2008; Seifu et al., 2012). These antioxidants prevent the

radical-induced oxidative process either by direct scavenging the free radicals or

preventing their formation by inhibiting the activity of particular oxidase enzyme which

thereafter improves the defense mechanisms of body (Figure 1).

Figure 1: Generation of free radicals, their action and elimination in cellular environment

(adopted from Becker, 2004).

19

Antioxidant can be grouped into two, as

Synthetic Antioxidants: These are the synthetic analogues of antioxidants

commonly used in commercial foods and beverages examples include butylated

hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and propyl gallate (PG) (Seifu

et al., 2012; Kahl and Kappus, 1993).

Natural Antioxidants: Natural (plant) sources have significant antioxidant

activity due to the presence of versatile secondary metabolites which can be sub-grouped

into three major domains, as

i. Phenolic compounds: These compounds possess one or more aromatic ring and

hydroxyl group(s). They are further sub-categorized into phenolic acid and flavonoids.

Flavonoids are potential antioxidant compounds either by showing their free radical

scavenging activities or by exhibiting metal-chelating properties (Seifu et al., 2012).

They are also considered as strong inhibitors of radical generating enzymes including

aldose reductase, xanthine oxidase and NADPH oxidase, thereby inhibiting the

formation of ROS (Katalinic et al., 2006; Pereira et al., 2009).

ii. Terpenoids: These belong to most diverse class of secondary plant metabolites

and have multiple roles which act as plant hormones, electron carriers and membrane

structural components (phytosterols). Besides being antioxidant, terpenoids also possess

other activities like antibacterial, antiviral, anti-inflammatory, antidiabetic, cholesterol

lowering, immunomodulatory and anticancer (Bohlmann and Keeling, 2008; Seifu et al.,

2012). The antioxidant role of terpenoids (caretonoids) is due to its capacity of either

quenching singlet-oxygen which considered as mutagenic agent or scavenging the

peroxyl radicals specifically at low oxygen tension (Zwenger and Basu, 2008).

iii. Alkaloids: These belong to a group of low-molecular weight nitrogen containing

compounds with significant effects in treating Alzheimer disease, myasthenia gravis,

myopathy, gout (Crozier et al., 2006; Tener et al., 2003). These can also act as muscle

relaxant, antiviral and anticholinergic agents (Facchini, 2001). The alkaloids showed

strong antioxidant activities like berberine has free radical scavenging potential (Hsu et

al., 2012; Seifu et al., 2012). These not only found to inhibit the lipid peroxidation but

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also inhibit lipoxygenase and xanthine oxidase activities thereby prevent the generation

of ROS (Rackova et al., 2007; Ahmad et al., 2010). In addition, they also uplift the

antioxidant enzyme (SOD) status in vivo (Seifu et al., 2012; Facchini, 2001).

The most popular antidiabetic plants also contain different antioxidant chemical

constituents like phenolic acids (protocatechuic, p-hydroxybenzoic, vanillic, and ferulic)

are reported in A.cepa and A. sativum (Gorinstein et al., 2008). Five (05) phytosterol

(lophenol, 24-methyl-lophenol, 24-ethyl-lophenol, cycloartanol, and 24-methylene-

cycloartanol) were isolated from Aloe vera (Aloe barbadensis) gel (Tanaka et al., 2006).

Presence of prenylated flavanones (5,7,4‗-trihydroxy-8-prenylflavanone, 5,4‗-dihydroxy-

7-methoxy-8-prenylflavanone, 5,7,4‗-trihydroxy-3‗,8-diprenylflavanone, and 5,7,4‗-

trihydroxy-3‗,5‗-diprenylflavanone) were identified in A. indica (Nakahara et al., 2003).

Few acylated flavonol glycosides [myricetin-3-methylether-5-O-(6‖-trans-caffeoyl)-β-D-

glucoside and querctin-3-methoxy-5-O-(6‖ trans-caffeoyl)-β-D-gluocside] were isolated

from E. jambolana that potentially exhibit antioxidant activity (Yadav and Verma,

2011). Beside this, pharmacologically active sterols have been identified in ginseng

species (Kim et al., 2013). Similarly, a number of cucurbitane triterprnoids have been

isolated from Momordica charantia (Liu et al., 2009). Another well-known antidiabetic

plant Trigonella foenum-graecum contains some antioxidant compounds include

protodioscin, Diosgenin (a steroidal saponin) and 4-hydroxyisoleucine (Hibasami et al.,

2003; Raju et al., 2004). Researchers claimed that the presence of antioxidant

compounds enhanced the antidiabetic potential of medicinal plants (Rahimi et al., 2005).

1.7 Plant for the Present study

Rauwolfia serpentina (L.) Benth. ex Kurz was selected for the present study.

21

1.7.1 Classification of Rauwolfia serpnetina (Kritikar and Basu, 1993)

Kingdom: Plantae

Division: Magnoliophyta

Class: Magnoliopsida

Order: Gentianales

Family: Apocynaceae

Genus: Rauwolfia

Species: serpentina

1.7.2 Morphology and Distribution

It is a perennial shrub (erect) with a long, irregular, nodular and yellowish root

stock. Leaves are whorls of three, lanceolate and bright green in color. Flowers are in

irregular corymbose cymes, white, often tinged with violet. Fruit are drupe and shining

black. The flowering time of this shrub is from March to May in Asian countries. Its

roots and leaves are famous for medicinal purposes. More than 80 species of this snake-

bite antidote genus are widely distributed in areas like tropical part of the Himalayas,

Indian peninsula, Sri Lanka, Burma, and Indonesia (Dey and De, 2010).

1.8 Literature Review of R. serpentina

Many aspects of R. serpentina are well-reported, as

1.8.1 Chemical Aspects of R.serpentina

More than 50 alkaloids chiefly indole alkaloids and other compounds have been

isolated and reported from roots of R.serpentina (Table 1).

1.8.2 Pharmacological Aspects of R. serpentina

The pharmacological aspect of R. serpentina can be categorized according to its

traditional and current therapeutic uses, as

Animal, Insect and Snake Bite: Literature from ancient time mentioned the use

of R. serpentina as natural medicine to cure snake and insect bites (Singh, 2008; Dey

22

and De, 2011). Many current surveys also describes the traditional use of roots and

leaves of same plant in different areas of India and Bangladesh where they serve as an

antidote to suppress the toxic effects of snake venom and scorpion sting (Srivastava

and Pandey, 2006). A study also reported the importance of root paste of R.serpentina

as an effective remedy for dog bites (Dey and De, 2011).

Hypertension: In literature R. serpetina was famously reported as an

antihypertensive medicine like its clinical significance was reported in patients with

―essential‖ and ―renal‖ hypertension (Bhatia, 1942). The same plant was found effective

in epileptic patients by decreasing the systolic blood pressure up to 80, diastolic 56, and

the pulse rate to 54 (Gupta et al., 1983). A researcher reported his ten years experience of

working with same plant and confirmed the medical importance of R.serpentina in

hypertensive patients (Vakil, 1955). Similarly, other studies also supported the

antihypertensive aspect of R.serpentina by describing it as a hypotensive drug of

moderate potency with sedative effect and found effective in providing symptomatic

relief in manic patients by slowing down the pulse rate markedly (Roy, 1950;

Chakravarty et al., 1943; Wilkins and Judson, 1953). Interestingly, the various pre-

clinical trial of same plant in hypertensive rats also described its effectiveness in

lowering the blood pressure (de Jongh and Proosdij-Hartzema, 1955) and found that it

could be due to the presence of various alkaloids (Glynn, 1955; Ibanez and Gijon, 1955).

23

Table 1: Compounds isolated from the roots of R. serpentina

S.

No. Identified Compound Chemical (IUPAC) Name Reference Source

1. Reserpine (1R,15S,17R,18R,19S,20S)-6,18-dimethoxy-17-(3,4,5-trimethoxybenzoyl)oxy-1,3,11,12,14,15,16,17,18,

19,20,21-dodecahydroyohimban-19-carboxylate Madhusundanan et al., 2008

2. Ajmaline (1R,9R,10S,13R,14R,16S,18S)-13-ethyl-8-methyl-8,15-diazahexacyclo [14.2.1.0^1,9.0^2,7.0^10,

15.0^12,17] nonadeca-2(7),3,5-triene-14,18-diol Srivastava et al., 2006

3. Isoajmaline 13-ethyl-8-methyl-8,15 diazahexacyclo [14.2.1.0^{1,9}.0^{2,7} .0^{10,15}.0^{12,17}]nonadeca-

2(7),3,5-triene-14,18-diol Yarnell, 2007

4. Neoajmaline 3-Ethyl-4,8-dihydroxy-13-methyl-5-propyl-1,3,4,7,8,13,13a,13b-octahydro-2H,6H-2,7-cyclo-6,8a-

methano-pyrido[1',2':1,2]azepino[3,4-b]indol-5-ium Yarnell, 2007

5.

Ajmalicine (19α)-16,17-didehydro- 19-methyloxayohimban- 16-carboxylic acid methyl ester Srivastava et al., 2006;

6. Raubasine Methyl (1S,15R,16S,20S)-16-methyl-17-oxa-3,13 diazapentacyclo [11.8.0.0^{2,10} .0^{4,9}.0^{15,20}]

henicosa-2(10),4,6,8,18-pentaene-19-carboxylate Yarnell, 2007

7. Yohimbine (1S,15R,18S,19R,20S)-18-hydroxy-1,3,11,12,14,15,16,17,18,19,20,21-dodecahydroyohimban-19-

carboxylate Gerasimenko et al., 2001.

8. Deserpidine Methyl (1R,15S,17R,18R,19S,20S)-18-methoxy-17-(3,4,5-trimethoxybenzoyloxy) -3,13-diazapentacyclo

[11.8.0.0^{2,10}.0^{4,9}.0^{15,20}]henicosa-2(10),4,6,8-tetraene-19-carboxylate Varchi et al., 2005

9. Rescinnamine

Methyl(1R,15S,17R,18R,19S,20S)-6,18-dimethoxy-17-{[(2E)-3-(3,4,5-trimethoxyphenyl)prop-2-

enoyl]oxy}-3,13diazapentacyclo [11.8.0.0^{2,10}.0^{4,9}.0^{15,20}]henicosa-2(10),4(9),5,7-tetraene-

19-carboxylate

Gerasimenko et al., 2001.

10. Serpentinine

11-{2-[(3Z)-3-ethylidene-1H,2H,3H,4H,6H,7H,12H,12bH-indolo[2,3-a]quinolizin-2-yl]-3-methoxy-3-

oxopropyl}-19-(methoxycarbonyl)-16-methyl-17-oxa-3,13$l^{5}-diazapentacyclo [11.8.0.0^ {2,10}

.0^{4,9} .0^{15,20}] henicosa-1(13),2(10),4,6,8,11,18-heptaen-13-ylium Yarnell, 2007

11. Corynanthine

Methyl (1S,15R,18S,19S,20S)-18-hydroxy-3,13-diazapentacyclo [11.8.0.0^{2,10}.0^{4,9}. 0^{15,20}]

henicosa-2(10),4,6,8-tetraene-19-carboxylate Zaidi, 1986.

12. Papaverine 1-[(3,4-dimethoxyphenyl)methyl]-6,7-dimethoxyisoquinoline Zaidi, 1986.

13. Sarpagine (1S,12S,13R,14S,15E)-15-ethylidene-13-(hydroxymethyl)-3,17-diazapentacyclo[12.3.1.0^{2,10}

.0^{4,9}.0^{12,17}]octadeca-2(10),4(9),5,7-tetraen-7-ol Sheludko et al., 2002.

14. Serpentine (15R,16S,20S)-19-(methoxycarbonyl)-16-methyl-17-oxa-3,13$l^{5}-diazapentacyclo

[11.8.0.0^{2,10}.0^{4,9} .0^{15,20}]henicosa-1(13),2(10),4,6,8,11,18-heptaen-13-ylium Wachsmuth & Matusch, 2002

15. Serpinine (1R,9R,10S,12R,13E,16S,17R,18R)-13-ethylidene-8-methyl-8,15-diazahexacyclo[14.2.1.0^

{1,9}.0^{2,7}.0^{10,15}.0^{12,17}]nonadeca-2,4,6-trien-18-ol Zaidi, 1986

16. Alstonine (15S,16S,20S)-19-(methoxycarbonyl)-16-methyl-17-oxa-3,13$l^{5}-diazapentacyclo

[11.8.0.0^{2,10}.0^{4,9}.0^ {15,20}]henicosa-1(13),2(10),4(9),5,7,11,18-heptaen-13-ylium-3-ide Yarnell, 2007

17. Renoxidine Methyl 6,18-dimethoxy-13-oxo-17-(3,4,5-trimethoxybenzoyloxy)-3,13$l^{5}-diazapentacyclo

[11.8.0.0^{2,10}.0^{4,9}.0^{15,20}]henicosa-2(10),4,6,8-tetraene-19-carboxylate Mittal et al., 2012.

18. Reserpiline Methyl (1R,15S,16S,20S)-6,7-dimethoxy-16-methyl-17-oxa-3,13-diazapentacyclo [11.8.0.0^{2,10 Mittal et al., 2012.

24

}.0^{4,9}.0^{15,20}]henicosa-2(10),4,6,8,18-pentaene-19-carboxylate

19. Reserpinine Methyl (1R,15S,17R,18R,19S,20S)-6,18-dimethoxy-17-[(E)-3-(3,4,5-trimethoxyphenyl)prop-2-

enoyl]oxy-1,3,11,12,14,15,16,17,18,19,20,21-dodecahydroyohimban-19-carboxylate Itoh et al., 2005.

20. Ophioxylin 5-hydroxy-2-methyl-1,4-dihydronaphthalene-1,4-dione Mittal et al., 2012

21. Rauwolscine Methyl (1S,15S,18S,19S,20S)-18-hydroxy-3,13-diazapentacyclo [11.8.0.0^{2,10} .0^{4,9}.0^{15,20}]

henicosa-2(10),4,6,8-tetraene-19-carboxylate Dey & De, 2011

22. Thebaine (1S,5R,13R)-10,14-dimethoxy-4,5-dimethyl-12-oxa-4-azapentacyclo [9.6.1.0^{1,13} .0^{5,17}.0^

{7,18}]octadeca-7(18),8,10,14,16-pentaene Zaidi, 1986

23. 7-Dehydrositosterol (1S,2R,5S,11R,15R)-14-[(2R,5S)-5-ethyl-6-methylheptan-2-yl]-2,15-dimethyl- tetracyclo [8.7.0.0^

{2,7}.0^{11,15}]heptadeca-7,9-dien-5-ol Phillips et al., 2012.

24. 2,6-Dimethoxybenzoquinone 2,6-dimethoxycyclohexa-2,5-diene-1,4-dione Itoh et al., 2005

25. Tetraphyllicine (1R,9R,10S,12R,13E,16S,17R,18R)-13-ethylidene-8-methyl-8,15-diazahexacyclo [14.2.1.0^{1,9}

.0^{2,7}.0^{10,15}.0^{12,17}]nonadeca-2,4,6-trien-18-ol Zaidi, 1986

26. Raucaffricine 13-ethylidene-14-{[3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy}-8,15-diazahexacyclo

[14.2.1.0^{1,9}.0^{2,7}.0^{10,15}.0^{12,17}] nonadeca-2,4,6,8-tetraen-18-yl acetate Ruppert et al., 2006;

27. Vomilenine (1R,10S,12R,13E,14R,16S)-13-ethylidene-14-hydroxy-8,15-diazahexacyclo

[14.2.1.0^{1,9}.0^{2,7}.0^{10,15}.0^{12,17}]nonadeca-2,4,6,8-tetraen-18-yl acetate Madhusudanan et al., 2008

28. Raumacline (1S,12S,13S,18S)-1,11,12,13,18-pentahydrogenio-17-(1-hydrogenioethyl)-3-methyl-15-oxa-3,20-

diazapentacyclo[10.7.1.0^{2,10}.0^{4,9}.0^{13,18}]icosa-2(10),4(9),5,7-tetraen-14-ol Bailey et al., 2008

29. Rauwolfine (9R,14R)-13-ethyl-8-methyl-8,15-diazahexacyclo[14.2.1.0^{1,9}.0^{2,7}.0^{10,15}.0^{12,17}]

nonadeca-2,4,6-triene-14,18-diol Ganugapati et al., 2012.

30. Rauwolfinine 13-ethyl-8-methyl-19-oxa-8,15-diazahexacyclo[14.3.1.0^{1,9}.0^{2,7}.0^{10,15}.0^{12,17}]icosa-

2(7),3,5-trien-18-ol Yarnell, 2007

31. Rescinnamidine

Methyl(1R,15S,17R,18R,19S,20S)-6,18-dimethoxy-17-{[3-(3,4,5-trimethoxyphenyl)propanoyl]oxy}-

3,13-diazapentacyclo[11.8.0.0^{2,10}.0^{4,9}.0^{15,20}]henicosa-2(10),4(9),5,7-tetraene-19-

carboxylate

Haider, 1986.

32. Rescinnaminol 1-({7-methoxy-1,10-diazapentacyclo[11.8.0.0^{3,11}.0^{4,9}.0^{15,20}]henicosa-3(11),4(9),5,7-

tetraen-18-yl}oxy)-3-(3,4,5-trimethoxyphenyl)propan-1-ol Haider, 1986.

33. Tetraphylline Methyl(1S,15R,16S,20S)-6-methoxy-16-methyl-17-oxa-3,13-diazapentacyclo-[11.8.0.0^

{2,10}.0^{4,9}.0^{15,20}]henicosa-2(10),4(9),5,7,18-pentaene-19-carboxylate Yarnell, 2007

34. Indobine Benzyl 3-(1H-indol-3-yl)propanoate Haider, 1986.

35. Indobinine Cyclohexyl 3-(1H-indol-3-yl)propanoate Zaidi, 1986.

36. Isorauhimbine Methyl(1R,15S,18S,19S,20S)-18-hydroxy-3,13-diazapentacyclo[11.8.0.0^{2,10}.0^{4,9}.0^

{15,20}]henicosa-2(10),4,6,8-tetraene-19-carboxylate Itoh et al., 2005.

37. Rauhimbine Methyl(1S,15R,18S,19S,20S)-18-hydroxy-3,13-diazapentacyclo[11.8.0.0^{2,10}.0^{4,9}

.0^{15,20}]henicosa-2(10),4,6,8-tetraene-19-carboxylate Itoh et al., 2005.

38. Sandwicolidine 14-ethyl-13-methoxy-8-methyl-8,15-diazahexacyclo[14.2.1.0^{1,9}.0^{2,7}.0^{10,15}.0^{12,17}]

nonadeca-2(7),3,5-trien-18-ol

Zaidi, 1986.

39. Sandwicoline 14-(1-hydroxybutan-2-yl)-3,16-dimethyl-3,16-diazapentacyclo[10.3.1.1^{10,13}.0^{2,10}.0^{4,9}] Zaidi, 1986.

25

heptadeca-4(9),5,7-trien-17-ol

40. Yohambinine 12-methyl-3,13-diazapentacyclo [11.8.0.0^{2,10}.0^{4,9}.0^{15,20}]henicosa-2(10),4(9),5,7-tetraene Zaidi, 1986.

41. Diisobutyl phthalate 1,2-bis(2-methylpropyl) benzene-1,2-dicarboxylate Reddy et al.

42. Deserpidic acid lactone (1S,2S,4R,18S,20R,23R)-23-methoxy-21-oxa-6,16-diazahexacyclo

[18.2.1.0^{2,18}.0^{4,16}.0^{5,13}.0^{7,12}]tricosa-5(13),7(12),8,10-tetraen-22-one Varchi et al., 2005

43. Vallesiachotamine Methyl(2S,12bS)-2-[(E)-1-oxobut-2-en-2-yl]-1,2,6,7,12,12b-hexahydroindolo[2,3-a]quinolizine-3-

carboxylate Sheludko et al., 2002.

44. N(b)-Methylajmaline (1R,9R,12S,16S)-13-ethyl-14,18-dihydroxy-8,15-dimethyl-8,15-diazahexacyclo[14.2.1.0^{1,9

}.0^{2,7}.0^{10,15}.0^{12,17}]nonadeca-2(7),3,5-trien-15-ium Itoh et al., 2005.

45. N(b)-Methylisoajmaline (1R,9R,12S,13R,14S,16S)-13-ethyl-14,18-dihydroxy-8,15-dimethyl-8,15-diazahexacyclo[14.2.1

.0^{1,9}.0^{2,7}.0^{10,15}.0^{12,17}]nonadeca-2(7),3,5-trien-15-ium

Itoh et al., 2005.

46. 3-Hydroxysarpagine (1R,12S,14S)-15-ethylidene-13-(hydroxymethyl)-3,17-Diazapentacyclo[12.3.1.0^{2,10}.0^{4,9

}.0^{12,17}]octadeca-2(10),4(9),5,7-tetraene-1,7-diol Itoh et al., 2005.

47. Yohimbinic acid (1S,15R,18S,19R,20S)-18-hydroxy-3,13-diazapentacyclo[11.8.0.0^{2,10}.0^{4,9} .0^{15,20}] henicosa-

2(10),4,6,8-tetraene-19-carboperoxoic acid Itoh et al., 2005.

48. Isorauhimbinic acid (1S,15R,18S,19R,20S)-18-hydroxy-3,13-diazapentacyclo-[11.8.0.0^{2,10}.0^{4,9}.0^{15,20}] henicosa-

2(10),4,6,8-tetraene-19-carboperoxoic acid Itoh et al., 2005.

49. 7-Epiloganin Methyl (1R,2R,3aS,7aS)-2-hydroxy-1-methyl-7-{[3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy}-

2,3,3a,6,7,7a-hexahydro-1H-indene-4-carboxylate Itoh et al., 2005.

50. 6′-O-(3,4,5-trimethoxy-

benzoyl) glomeratose A

(3,4,5-trihydroxy-6-{[4-hydroxy-2,5-bis(hydroxymethyl)-3-{[(2E)-3-(3,4,5-trimethoxyphenyl)prop-2-

enoyl]oxy}oxolan-2-yl]oxy}oxan-2-yl)methyl-3,4,5-trimethoxybenzoate Itoh et al., 2005.

51. Normacusine B [(15Z)-15-ethylidene-3,17-diazapentacyclo[12.3.1.0^{2,10}.0^{4,9}.0^{12,17}]octadeca-2(10),4,6,8-

tetraen-13-yl]methanol Itoh et al., 2005.

52. Geissoschizol 2-[(2S,4R,5E)-5-ethylidene-7,17-diazatetracyclo[8.7.0.0^{2,7}.0^{11,16}]heptadeca-1(10),11(16),12,14-

tetraen-4-yl]ethan-1-ol Itoh et al., 2005.

53. Rhazimanine Methyl(11S,12E,18S)-12-ethylidene-17-oxo-14-azapentacyclo[9.5.3.0^{1,10}.0^{2,7}.0^{14,18}]

nonadeca-2,4,6,8-tetraene-10-carboxylate Itoh et al., 2005.

54. 18Hydroxyepiallo-yohimbine Methyl17,18-dihydroxy-3,13-diazapentacyclo [11.8.0.0^{2,10}.0^{4,9}.0^{15,20}] henicosa-2(10),4,6,8-

tetraene-19-carboxylate Itoh et al., 2005.

55. Methyl reserpate Methyl 17-hydroxy-6,18-dimethoxy-3,13-diazapentacyclo[11.8.0.0^{2,10}.0^{4,9}.0^{15,20}] henicosa-

2(10),4(9),5,7-tetraene-19-carboxylate Varchi et al., 2005

56. Loganic acid (1S,4aS,6S,7R,7aS)-6-hydroxy-7-methyl-1-{[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan

-2-yl]oxy}-1H,4aH,5H,6H,7H,7aH-cyclopenta[c]pyran-4-carboxylic acid Itoh et al., 2005.

57. 7-Deoxyloganic acid 7-methyl-1-{[3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy}-1H,4aH,5H,6H,7H,7aH-cyclopenta[c]

pyran-4-carboxylic acid Itoh et al., 2005.

58. Secoxyloganin 2-[(2S,3R,4S)-3-ethenyl-5-(methoxycarbonyl)-2-{[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-

(hydroxymethyl)oxan-2-yl]oxy}-3,4-dihydro-2H-pyran-4-yl]acetic acid Itoh et al., 2005.

59. Glomeratose A 4-hydroxy-2,5-bis(hydroxymethyl)-2-{[3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy}oxolan-3-

yl(2E)-3-(3,4,5-trimethoxyphenyl)prop-2-enoate

Itoh et al., 2005.

60. 16-Epinormacusine B [(15E)-15-ethylidene-3,17-diazapentacyclo[12.3.1.0^{2,10}.0^{4,9}.0^{12,17}]octadeca-2(10),4(9),5,7-

tetraen-13-yl]methanol Itoh et al., 2005.

26

61. Swertiaside 6-(3-hydroxybenzoyloxy)-7-methyl-1-{[3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy}-

1H,4aH,5H,6H,7H,7aH-cyclopenta[c]pyran-4-carboxylic acid

Itoh et al., 2005.

62. 3,4,5,6-

Tetradehydroyohimbine

(15R,18S,19R,20S)-18-hydroxy-19-(methoxycarbonyl)-3,13$l^{5}-diazapentacyclo

[11.8.0.0^{2,10}.0^{4,9}.0^{15,20}]henicosa-1(13),2(10),4,6,8,11-hexaen-13-ylium-3-ide Wachsmuth & Matusch, 2002.

63. 3,4,5,6-Tetradehydro-(Z)-

geissoschizol

(2R,3Z)-3-ethylidene-2-(2-hydroxyethyl)-1H,2H,3H,4H,12H-5$l^{5},12-indolo[2,3-a]quinolizin-5-

ylium-12-ide Wachsmuth & Matusch, 2002.

64. 3,4,5,6-

Tetradehydrogeissoschizol

(2R,3E)-3-ethylidene-2-(2-hydroxyethyl)-1H,2H,3H,4H,12H-5$l^{5},12-indolo[2,3-a]quinolizin-5-

ylium-12-ide Wachsmuth & Matusch, 2002.

65.

3,4,5,6-

Tetradehydrogeissoschizine-

17-O-b-d-glucopyranoside

(2S,3E)-3-ethylidene-2-[(1Z)-3-methoxy-3-oxo-1-{[3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-

yl]oxy}prop-1-en-2-yl]-1H,2H,3H,4H,12H-5$l^{5},12-indolo[2,3-a]quinolizin-5-ylium-12-ide Wachsmuth & Matusch, 2002

66. Ajmalimine 13-ethyl-14-hydroxy-8-methyl-8,15-diazahexacyclo [14.2.1.0^{1,9}.0^{2,7}.0^{10,15}.0^{12,17}]

nonadeca-2,4,6-trien-18-yl 3,4,5-trimethoxybenzoate Hanhinen & Lounasmaa,

2001.

67. Tetrahydroalstonine (19α)-16,17-didehydro- 19-methyloxayohimban- 16-carboxylic acid methyl ester Sheludko et al., 2002

68. 19(S),20(R)-

dihydroperaksine

[(1S,12S,13R,15R,16S)-15-(hydroxymethyl)-16-methyl-3,17-diazapentacyclo [12.3.1.0^{2,10}.0^{4,9}

.0^{12,17}]octadeca-2(10),4(9),5,7-tetraen-13-yl]methanol

Sheludko et al., 2002

Edwankar et al., 2011.

69. 19(S),20(R)-

dihydroperaksine-17-al

(1S,12S,13R,15R,16S)-15-(hydroxymethyl)-16-methyl-3,17-diazapentacyclo[12.3.1.0^{2,10}.0^{4,9}.0^

{12,17}]octadeca-2(10),4(9),5,7-tetraene-13-carbaldehyde Sun et al., 2008

70. 10-Hydroxy-19(S),20(R)-

dihydroperaksine

(1S,12S,13R,15R,16S)-13,15-bis(hydroxymethyl)-16-methyl-3,17-diazapentacyclo[12.3.1.0^{2,10}.0^

{4,9}.0^{12,17}]octadeca-2(10),4(9),5,7-tetraen-7-ol Edwankar et al., 2011.

71. 19(S),20(R)-(O)-

Acetylpreperakine

[(1S,12S,13R,15R,16S)-15-formyl-16-methyl-3,17-diazapentacyclo[12.3.1.0^{2,10}.0^{4,9}.0^{12,17}]

octadeca-2(10),4(9),5,7-tetraen-13-yl]methyl acetate Edwankar et al., 2011.

72. Reserpoxidine Methyl(1R,15S,17R,18R,19S,20S)-6,18-dimethoxy-13-oxo-17-(3,4,5-trimethoxybenzoyloxy)-3,13$l^{5}

-diazapentacyclo[11.8.0.0^{2,10}.0^{4,9}.0^{15,20}]henicosa-2(10),4,6,8-tetraene-19-carboxylate Mittal et al., 2012.

73. 12-Hydroxyajmaline 13-ethyl-8-methyl-8,15-diazahexacyclo[14.2.1.0^{1,9}.0^{2,7}.0^{10,15}.0^{12,17}]nonadeca-2(7),3,5-

triene-6,14,18-triol Sheludko et al., 2002.

74. 3-Epi-alpha-yohimbine Methyl(1R,15S,18S,19S,20S)-18-hydroxy-3,13 diazapentacyclo [11.8.0.0^{2,10}.0^{4,9}.0^{15,20}]

henicosa-2(10),4,6,8-tetraene-19-carboxylate Sheludko et al., 2002

75. 18-beta-hydroxy-3-epi-alpha-

yohimbine

Methyl(1S,15R,17S,18S,19R,20R)-17,18-dihydroxy-3,13-diazapentacyclo [11.8.0.0^{2,10}.0^{4,9}.0^

{15,20}]henicosa-2(10),4,6,8-tetraene-19-carboxylate Sheludko et al., 2002.

76. 17-O-acetyl-ajmaline (1R,9R,10S,12R,13S,14R,16S)-18-(acetyloxy)-13-ethyl-14-hydroxy-8-methyl-8,15-diazahexacyclo

[14.2.1.0^{1,9}.0^{2,7}.0^{10,15} .0^{12,17}]nonadeca-2,4,6-trien-15-ium

Hanhinen & Lounasmaa,

2001.

77. Strictosidine Methyl(2S,3R,4S)-3-ethenyl-4-[[(1S)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl]methyl]-2-[(2S,3R,

4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-3,4-dihydro-2H-pyran-5-carboxylate Sheludko et al., 2002;

78. Strictosidine lactam 19-ethenyl-18-{[3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy}-17-oxa-3,13-diazapentacyclo

[11.8.0.0^{2,10}.0^{4,9}.0^{15,20}]henicosa-2(10),4(9),5,7,15-pentaen-14-one Sheludko et al., 2002.

79. Isosandwicine 13-ethyl-8-methyl-8,15-diazahexacyclo[14.2.1.0^{1,9}.0^{2,7}.0^{10,15}.0^{12,17}]nonadeca-2(7),3,5-

triene-14,18-diol Gerasimenko et al., 2001.

80. Sandwicine (9R,14S)-13-ethyl-8-methyl-8,15-diazahexacyclo[14.2.1.0^{1,9}.0^{2,7}

.0^{10,15}.0^{12,17}]nonadeca-2(7),3,5-triene-14,18-diol Gerasimenko et al., 2001.

27

81. Acetylcorynanthine Methyl(1S,15R,18S,19S,20S)-18-(acetyloxy)-3,13-diazapentacyclo

[11.8.0.0^{2,10}.0^{4,9}.0^{15,20}]henicosa-2(10),4(9),5,7-tetraene-19-carboxylate Stöckigt et al., 2002.

82. Raunescine Methyl(1R,15S,17R,18R,19S,20S)-18-hydroxy-17-(3,4,5-trimethoxybenzoyloxy)-3,13-

diazapentacyclo[11.8.0.0^{2,10} .0^{4,9}.0^{15,20}]henicosa-2(10),4,6,8-tetraene-19-carboxylate Stöckigt et al., 2002.

83. Isosandwicimine (1R,9R,10S,12R,13R,14S,16S,18S)-13-ethyl-18-hydroxy-8-methyl-8,15-diazahexacyclo

[14.2.1.0^{1,9}.0^{2,7}.0^{10,15}.0^{12,17}]nonadeca-2(7),3,5-trien-14-yl acetate Haider, 1986.

84. Seredine Methyl18-hydroxy-6,7-dimethoxy-3,13-diazapentacyclo[11.8.0.0^{2,10}.0^{4,9}.0^{15,20}]henicosa-

2(10),4(9),5,7-tetraene-19-carboxylate Haider, 1986.

85. Ajmalicidine Methyl(1S,15R,16S,18S,20R)-18-hydroxy-16-methyl-17-oxa-3,13-diazapentacyclo

[11.8.0.0^{2,10}.0^{4,9}.0^{15,20}]henicosa-2(10),4(9),5,7-tetraene-3-carboxylate Zaidi, 1986.

86. Ajmalinimine 4-acetyl-13-ethyl-14-hydroxy-8-methyl-8,15-diazahexacyclo[14.2.1.0^{1,9}.0^{2,7}.0^{10,15} .0^

{12,17}]nonadeca-2,4,6-trien-18-yl acetate Haider, 1986.

87. Raugalline (9R,14R)-13-ethyl-8-methyl-8,15-diazahexacyclo[14.2.1.0^{1,9}.0^{2,7}.0^{10,15}.0^{12,17}]

nonadeca-2,4,6-triene-14,18-diol Zaidi, 1986

88. Perakenine 13-ethyl-8-methyl-19-oxa-8,15-diazahexacyclo[14.3.1.0^{1,9}.0^{2,7}.0^{10,15}.0^{12,17}]icosa-

2(7),3,5-trien-18-ol Zaidi, 1986

89. Serpine Methyl 18-hydroxy-3,13-diazapentacyclo[11.8.0.0^{2,10 }.0^{4,9} .0^{15,20}]henicosa-2(10),4(9),5,7-

tetraene-19-carboxylate Zaidi, 1986

90. Acetylajmalicidine Methyl (1S,15R,16S,18R,20R)-18-(acetyloxy)-16-methyl-17-oxa-3,13-diazapentacyclo[11.8.0.0^

{2,10}.0^{4,9}.0^{15,20}]henicosa-2(10),4(9),5,7-tetraene-3-carboxylate Zaidi, 1986

91. Acetylsandwicoline (2S)-2-[(1S,2R,10R,12S,14R,17S)-17-(acetyloxy)-3,16-dimethyl-3,16-diazapentacyclo

[10.3.1.1^{10,13}.0^{2,10}.0^{4,9}]heptadeca-4(9),5,7-trien-14-yl]-2-methylbutyl acetate Zaidi, 1986

92. (+)-17R-O-(3',4',5'-trime-

thoxybenzoyl)ajmaline

(13S,14R)-13-ethyl-14-hydroxy-8-methyl-8,15-diazahexacyclo [14.2.1.0^{1,9}

.0^{2,7}.0^{10,15}.0^{12,17}]nonadeca-2(7),3,5-trien-18-yl 3,4,5-trimethoxybenzoate Hanhinen &Lounasmaa, 2001.

93. Ajmalicimine Methyl(4S,8S,9R)-8-methyl-7-oxa-11,21-diazatetracyclo[12.7.0.0^{4,9}.0^{15,20}]henicosa-

1(14),5,15(20),16,18-pentaene-5-carboxylate Haider, 1986.

94. Acetylajmalicimine Methyl (4S,8S,9R)-11-acetyl-8-methyl-7-oxa-11,21-diazatetracyclo [12.7.0.0^{4,9}.0^{15,20}]henicosa-

1(14),5,15(20),16,18-pentaene-5-carboxylate Haider, 1986

28

The effect of reserpine and rescinnamine (alkaloids of Rauwolfia) on arterial pressure,

blood sugar and behavior was also reported and concluded that the sedative effect of

rescinnamine is approximately 3 times more than that of reserpine (Gijon and Ibanez,

1955) while the hyperglycemic effect of reserpine is 3 times than that of rescinnamine

(Gijon and Ibanez, 1955). However, both were found effective in decreasing the blood

pressure of rats and rabbits (de Jongh and van Proosdij-Hartzema, 1955; Yada, 1957).

Later, new alkaloids including ajmaline, serpentine, serpentinine, and two unknown

substances from the roots of R. serpentina were isolated and found more potent

hypotensive agents without being sedative and depressant (Siddiqui, 1957; Siddiqui and

Alauddin, 1958; Zipf, 1957). The chemistry, mechanism of action, hemodynamic

changes, toxicity, pharmacokinetic, clinical and sedative or tranquilizing actions of

Rauwolfia‘s alkaloids were reported by many researchers (Dey and De, 2010). The effect

of R.serpentina in the treatment of arrhythmia was also briefly described (Bazika, 1969).

Diabetes: A preliminary study described the effect of total alkaloids of R.

serpentina and reserpine on blood sugar level in anesthetized cats (Chatterjee et al.,

1957). Similar action of R.serpentina was determined in 30 diabetic patients and

observed that glucose was decreased in blood and urine of those patients (Cazzaroli and

Dall‘Oglio, 1958). Another study was conducted for evaluating the same aspect of

Rauwolfia alkaloid in patients having diabetes and hypertension both (Cohen et al.,

1959).

Central Nervous System (CNS) and Mental Disorders: The tranquilizing and

antipyschotic effects of reserpine is related to depleting the amount of catecholeamines

(dopamine, norepinephrine and epinephrine) and serotonins in CNS while its

antihypertensive effect is related to the same action in peripheral nerves (Sreemanthula et

al., 2005). The differential effect of reserpine analogues on monoamines of brain was

also reported (Trcka and Carlsson, 1967). When reserpine and morphine were applied

alone or in combination to determine their effect on CNS of mouse, it was found that

reserpine increased ATP and decreased coenzyme-A levels thus counteracts the

stimulation of CNS caused by morphine (Leuschner et al., 1958). Reserpine methonitrate

29

is the quaternary analogue of reserpine, it does not produce any behavior change by not

interfering the bran bioamines level but it enhances the urinary excretion of

vanillylmandelic acid (VMA) and 5-hydroxyindoleacetic acid (5-HIAA), the end

products of catecholamines and serotonin metabolism by showing its effect on peripheral

nerves (Sreemantula et al., 2004). It was reported that a polyherbal formulation, Anximin

(contains R. serpentina as one of the ingredients) significantly up-regulated the

benzodiazepine receptors and showed anxiolytic activity in rats (Msihra et al., 2008).

Therefore, R. serpentina (especially its roots) is well- reported for the treatment of

anxiety, excitement, epilepsy, insanity, insomnia, mental agitation, schizophrenia, and

other mental disorders (Dey and De, 2011).

Gastrointestinal Disorders: Intravenous injection of reserpine-free extract in dogs

has no effect on the gastric acid secretion (Barre and Lieber, 1957) while number of

studies showed that reserpine induced hypermotality of stomach and marked increase in

the secretion of same acid. Beside this, the roots and leaves of R.serpentina were

used to treat abdomen & liver pains, intestinal disorders, intestinal worms, colic,

diarrhea, dysentery (Barre and Gillo, 1957; Schneider and Clark, 1957; Dey and De,

2011). A study also described its use in treating spleen diseases (Mia et al., 2009).

Skin diseases: Roots paste of R. serpentina was reported to treat scabies

(Mohanta et al., 2006) while other studies also supported its use in the

management of cut, wound, boils, itches, eczema and urticaria (Dey and De,

2011).

Other Diseases: The ethanolic extract of R. serpentina was reported to inhibit

the growth of some bacterial strains (Jigna et al., 2005). The anti-inflammatory effect

of pectic polysaccharide named rauvolfin isolated from same plant was also reported

(Popov et al., 2007). R.serpentina was also reported to possess remarkable activity

against breast cancer (Stanford et al., 1986) and human promyelocytic leukemia (HL-

60) cell lines (Itoh et al., 2005). Traditionally, R. serpentina has been used in

treating headache, fever, edema, eye inflammation, rheumatism, malaria,

pneumonia, asthama and respiratory diseases (Dey and De, 2011).

30

1.9. Objective of the Present Research Work

The objective of present research work was to investigate the antidiabetic and

antioxidant effects of root extracts of R. serpentina in alloxan-induced diabetic (type 1)

and fructose-induced insulin resistance (type 2) models. Following are the steps decided

to conduct in order to achieve the basic objective of the present study.

1. To prepare the methanolic (MERt) and 80% aqueous methanolic root (AqMREt)

extracts of R.serpentina.

2. To determine the qualitative and quantitative phytochemical analyses of both root

extracts (MREt and AqMREt) of R. serpentina.

3. To determine the acute toxicity and lethal median doses (LD50) of both root

extracts (MREt and AqMREt) of R. serpentina.

4. To study the effect of both root extracts (MREt and AqMREt) of R. serpentina on

glucose tolerance in overnight fasted male mice by conducting the oral glucose tolerance

test (OGTT).

5. To study the antidiabetic effect of both root extracts (MREt and AqMREt) of R.

serpentina in alloxan-induced (type 1) diabetic mice by observing their effect on percent

body weight & glycemic change, serum insulin, total cholesterol (TC), triglycerides

(TG), high density (HDL-c), low-density (LDL-c) & very low-density lipoprotein

(VLDL-c) cholesterols, total hepatic glycogen & lipid contents, glycosylated (HbA1c) &

total (Hb) hemoglobins, hematocrit (HCT), red (RBC) & white blood (WBC) cells

counts, mean cell volume (MCV), mean cell hemoglobin (MCH), mean cell hemoglobin

concentration (MCHC) and on antioxidant enzymes including catalase (CAT) &

superoxide dismutase (SOD).

6. To study the antidiabetic effect of both root extracts (MREt and AqMREt) of R.

serpentina in fructose-induced insulin resistance (type 2) diabetic mice by observing

their effect on same parameters which are mentioned in point 5.

7. To analyze results obtained from OGTT, type 1 and type 2 diabetic mice models

through statistical software.

31

8. To detect the presence of possible compounds in both root extracts (MREt and

AqMREt) of R. serpentina through spectroscopic technique.

32

Flow Chart Showing Scheme of

Present Research Work

Figure 2: Flow Chart Showing Scheme of Present Research Work

CHAPTER 5

CHAPTER 4

CHAPTER 2

Ground Roots of Rauwolfia serpentina

Methanolic Roots extract

(MREt)

Aqueous (80%) Methanolic

Roots extract (AqMREt) EXTRACTION

Phytochemical Analyses

(Qualitative and quantitative)

Determination of Acute toxicity

and lethal median doses (LD50)

Oral glucose tolerance test

(OGTT) to determine three

effective doses

Antidiabetic and

antioxidant effects of

both roots extracts

(MREt and AqMREt)

of R. serpentina in

fructose-induced

(type 2) diabetic mice

Antidiabetic and

antioxidant effects of

both roots extracts

(MREt and AqMREt)

of R. serpentina in

alloxan-induced

(type 1) diabetic mice

Fourier Transform Infrared

(FTIR) spectroscopy of both

extracts

Long term activities

after dose selection

CHAPTER 3

33

Chapter 2

Extraction, Phytochemistry, Acute

Toxicity and Oral Glucose Tolerance

Test (OGTT) of Root Extracts of

R.serpentina

Chapter 2 Extraction, Phytochemistry, Acute Toxicity

and Oral Glucose Tolerance Test (OGTT) of Root Extracts

of R.serpentina

Background

The current chapter deals with the extraction and phytocehmical analyses (both

qualitative and quantitative) of the methanolic root extract (MREt) and aqueous

methanolic root extract (AqMREt) of R.serpentina. The acute toxicity and OGTT of both

extracts was also performed. This chapter describes the materials and methods, results

and discussion of the above content.

2.1 Materials and Methods

2.1.1 Animals

Healthy male albino Wister mice (25-35 grams) were purchased from breeding

house of Dow University of Health Sciences (DUHS), Karachi, Pakistan. The mice were

acclimatized and maintained individually in cages in an air conditioned room with

temperature 23 ± 2oC (Relative humidity 55%) for one week prior to the experiment in

the conventional animal house of the same university. They were given standard

laboratory diet with free access to water ad libitum and no physical stress was provided.

During the entire experimental period, the care and handling of these mice were in

accordance with internationally accepted standard guidelines. The present study was

conducted in collaboration with DUHS, therefore, its experimental protocol was

approved by Board of Advance Study and Research (BASR), University of Karachi and

the Institutional Ethical Review Board (IERB) of DUHS.

2.2 Plant and Experimental Material

Medicinally important plant Rauwolfia serpentina Benth of family Apocynacea

was selected for this study. Roots of this plant were used as an experimental material and

purchased from the Hamdard Dawakhana, Saddar, Karachi, Pakistan. Chemicals used in

34

qualitative and quantitative photochemical analyses were purchased from Merck (Pvt.)

Ltd.

2.2.1 Identification and Authentication of Experimental Material

Roots of R. serpentina were identified and authenticated by taxonomist of

Department of Botany, University of Karachi, Pakistan and kept in Department of

Biochemistry of the same university as a specimen No. SAQ/BCH/KU-02. These were

stored in clean airtight bottle at room temperature until used.

2.2.2 Preparation of Root Extracts

Finely ground roots powder (40 g) of R. serpentina was soaked overnight in

methanol (1L; 95%). It was filtered through Whatman No. 1 filter paper (twice) and

concentrated till dryness at 40 oC in a rotary vacuum evaporator (Eylea-NE) to obtain a

brown residue that was referred as methanolic root extract abbreviated by MREt

(Qureshi et al, 2009). For preparing 80% aqueous methanolic root extract (AqMREt),

method for determining total flavonoids was adopted (Boham and Kocipai, 1974), in

which, 10 gm of ground root powder of R.serpentina was extracted with 100 ml of 80%

aqueous methanol repeatedly at room temperature and filtered through Whatman filter

paper No 42 (125 mm). Filtrate was transferred into a crucible which was placed on

water bath for evaporation. This process continues till dryness and weighed. Both MREt

and AqMREt were kept in an airtight container separately and stored in refrigerator

below 10oC until used.

2.2.3 Percent Yield of Root Extracts

The percent yield of MREt and AqMREt was determined by using the following

formula

( )

Note: Y = grams (g) of MREt or AqMREt obtained.

A = grams (g) of total root powder (40 or 10 gm) used for extraction.

35

2.3 Analysis of Phyto-constituents in Root Extracts

2.3.1 Qualitative Analysis

The root extracts (MREt and AqMREt) of R. serpentina were tested for different

phyto-constituents like alkaloids, anthraquinones, carbohydrates, flavonoids, glycosides,

resins, saponins, steroids, tannins, triterpenoids, etc, by standard methods

(Chandrashekar et al., 2010; Edeoga et al., 2005; Shanmugam et al., 2010).

2.3.1.1 Test for Alkaloids

1. Dragendroff’s Test

5 ml of distilled water was added to 2 ml of extract, followed by the addition of

2M hydrocholric acid (HCl) till an acid reaction occurred. Then 1 ml of Dragendroff‘s

reagent was added, resulted in the formation of orange or orange-red precipitate that

indicated the presence of alkaloids.

2. Hager’s Test

Few drops of Hager‘s reagent were added to 1ml of extract which produced

yellow precipitates and confirmed the presence of alkaloids.

3. Mayer’s Test

Few drops of Mayer‘s reagent were added to 2 ml of extract which produced

white or pale yellow precipitates and indicated the presence of alkaloids.

4. Wagners’ Test

1 ml of extract was acidified with HCl, then few drops of Wagner’s reagent was

added which resulted in the formation of yellow or brown precipitates that confirmed the

presence of alkaloids.

2.3.1.2 Test for Anthraquinones (Borutrager’s Test)

Equal volumes of extract and 10% ferric chloride (FeCl3) were mixed, followed

by the addition of 0.5 ml concentrated HCl, boiled and filtered. The filtrate was treated

36

with 1 ml of diethyl ether and concentrated ammonia which produced pink or deep red

color in test tube and confirmed the presence of anthraquinones.

2.3.1.3 Test for Carbohydrates

1. Benedict’s Test

2 mg of extract was mixed with 10 ml of distilled water and filtered. The filtrate

was concentrated and treated with 5 ml of Benedict‘s reagent, boiled for 5 minutes,

during that period brick red colored precipitates were formed and indicated the presence

of carbohydrates.

2. Fehling’s Test


was concentrated and treated with 1 ml of Fehling‘s solution (mixture of equal parts of

Fehling‘s solutions A and B), boiled for few minutes, during that brick red precipitates

were appeared which reflected the presence of reducing sugar.

3. Molisch’s Test


was concentrated and treated with 2 drops of (freshly prepared) alcoholic solution of α-

naphthol (20%) and 2 ml of concentrated sulfuric acid (H2SO4) that resulted in the

formation of red violet ring and indicated the presence of carbohydrates.

2.3.1.4 Test for Flavonoids

1. Ammonia Test

A strip of filter paper was dipped in 1 ml of extract and exposed to ammonia

vapours, resulted in the appearance of yellow spot on filter paper that indicated the

presence of flavonoids.

37

2. Shinoda Test

10 drops of diluted HCl was added to 2 ml of extract. To this, small piece of

magnesium strip was added which resulted in the formation of reddish pink color and

indicated the presence of flavonoids.

2.3.1.5 Test for Glycosides

Molisch‘s test was performed for detecting the presence of glycosides as same as

it is described for the detection of carbohydrates in section 2.3.1.3.

2.3.1.6 Test for Cardiac Glycosides (Keller-Killani Test)

5 ml of extract was treated with 2 ml of glacial acetic acid, one drop of FeCl3

solution and 1 ml of concentrated H2SO4 resulted in the formation of three rings of

brown, violet and brown colored (one after the other) at the interface which confirmed

the presence of cardiac glycosides.

2.3.1.7 Test for Resins

1 ml of extract mixed with 1 ml of acetone added in a test tube containing

2 ml of distilled water, resulted in the formation of turbidity which confirmed the

presence of resins.

2.3.1.8 Test for Saponins

A drop of 3% sodium bicarbonate solution was added in 5 ml of extract, shaken

vigorously and kept for 3-5 minutes. During that honeycomb like froth was formed at the

bottom of test tube which indicated the presence of saponins.

2.3.1.9 Test for Steroids (Liebermann-Burchard Test)

0.5 ml of extract was treated with 2 ml of acetic anhydride, boiled and cooled. To

this 1ml of concentrated H2SO4 was added along with the sides of test tube which

produced green color, indicated the presence of the steroids.

38

2.3.1.10 Test for Tannins

Few drops of 5% FeCl3 solution was added in 2 ml of extract resulted in the

appearance of green color that confirmed the presence gallotannins while appearance of

brown color indicated the presence of pseudotannins.

2.3.1.11 Test for Phlobatannins

1 ml of extract was treated with 1% HCl and boiled. Red precipitates appeared

and indicated the presence of phlobatannins.

2.3.1.12 Test for Triterpenoids (Salkowski Test)

2 ml of chloroform was added into 5ml of extract and shaken. To this, 3 ml of

H2SO4 was added slowly along the sides of the test tube which produced reddish brown

color and indicated the presence of the steroids.

2.3.2 Quantitative Analysis

2.3.2.1 Determination of Alkaloids

Five (05) gm of ground root powder of R.serpentina was dissolved in 200 ml of

10% acetic acid in ethanol, covered, kept for 4 hours and filtered. The filtrate was

concentrated on a water bath till 1/4th

of its original volume remained. Concentrated

ammonium hydroxide was added drop wise to it until the precipitation was completed.

The precipitates were collected and washed with dilute ammonium hydroxide and

filtered. The residues were alkaloids, oven-dried and weighed (Harborne, 1973).

2.3.2.2 Determination of Flavonoids

Ten (10) g of ground root powder of R.serpentina was extracted repeatedly at

room temperature with 100 ml of 80% aqueous methanol. Then filtered through

Whatman filter paper No 42 (125 mm) and filtrate was transferred into a crucible which

was placed on water bath for evaporation. It was done till dryness and weighed (Boham

and Kocipai, 1974).

39

2.3.2.3 Determination of Total Phenolic Content

Test was prepared by adding 0.1 mg of extract into 1 ml of Folin–Ciocalteu

reagent (1:10 diluted with distilled water). After 3 minutes, 3 ml of 2% sodium carbonate

was added and incubated for 2 hours at room temperature with intermittent shaking. The

absorbance was measured at 760 nm against a reagent blank. The standard curve was

prepared by using 0.01-0.1 mg of gallic acid (Table 2 and Figure 3). The total phenolic

content was expressed in mg/g of extract (Sengul et al., 2009).

40

Table 2: Absorbance of Gallic acid

S. No Standards Concentration of gallic acid (mg) Absorbance at 760nm

1 S1 0.01 0.101

2 S2 0.02 0.085

3 S3 0.03 0.180

4 S4 0.04 0.220

5 S5 0.05 0.307

6 S6 0.06 0.334

7 S7 0.07 0.380

8 S8 0.08 0.436

9 S9 0.09 0.482

10 S10 0.1 0.517

41

Figure 3: Standard Curve of Gallic acid

y = 5.4249x

R² = 0.9761

0

0.1

0.2

0.3

0.4

0.5

0.6

0 0.02 0.04 0.06 0.08 0.1 0.12

Ab

sorb

an

ce a

t 760n

m

Concentration of gallic acid (mg)

42

2.3.2.4 Determination of Saponins

Twenty (20) grams of ground root powder of R.serpentina was dissolved in 200

ml of 20% aqueous ethanol and kept in a water bath at about 55°C for 4 hours with

continuous stirring. The mixture was filtered and the residue re-extracted with another

200 ml of same aqueous ethanol. The combined extracts were concentrated at 90°C till

40 ml was left to its original volume. The concentrate was transferred into a 250 ml

separatory funnel and 20 ml of diethyl ether was added and shaken vigorously. The

aqueous layer was recovered while the diethyl ether layer was discarded. 60 ml of n-

butanol was added to recover aqueous layer. The combined n-butanol extracts were

washed twice with 10 ml of 5% aqueous sodium chloride. The remaining solution was

transferred to crucible and heated in a water bath for evaporation till a constant weight

was achieved (Obdoni and Ochuko, 2001).

2.4 In Vivo Evaluation of Root Extracts

2.4.1 Determination of the Acute Toxicity of Root Extracts

Overnight fasted mice (approximately 12-14 hrs) were randomly divided into

eight groups. Each group contained six (06) mice. A single dose of each of 10, 30, 60,

100, 150, 200 and 250 mg/kg of methanolic root extract (MREt) of R. serpentina was

separately administrated orally to mice in their respective test groups. Six mice used as

control group and treated with distilled water 1 ml/kg through the same route. The mice

in both test and control groups were then allowed free access to food and water, and their

activity was observed over a period of 12 hr for behavior change (sedative or not) and

mortality rate (Bhatia and Mishra, 2010). In addition, hypoglycemic activity was

determined after 30 min of administration of each dose in its respective group with the

help of glucometer (Azmi and Qureshi, 2012).

Similar protocol was followed for determining the acute toxicity of 80% aqueous

methanolic root extract (AqMREt) of R. serpentina but overnight fasted mice were

divided into eleven (11) groups including ten (10) test and one (01) control groups

(6/group). A single dose of each of 05, 10, 50, 100, 250, 500, 1000, 1500, 2000 & 2500

43

mg/kg of AqMREt was separately administrated orally to mice of its respective group

and six mice in control group were treated with distilled water 1 ml/kg orally. However,

behavior change was observed as same as in MREt treated model.

2.4.2 Determination of the Median Lethal Dose (LD50) of Root Extracts

Median lethal dose (LD50) of root extracts including MREt and AqMREt of R.

serpentina were determined by log-probit graphical method (Miller and Tainter, 1944)

(Figure 4 and 5).

2.4.3 Determination of the Effect of Root Extracts on Glucose Tolerance in

Mice

Overnight fasted (12-14 hrs) experimental mice were divided into different

groups (6/group) according to the treatments (Figure 6 and 7). Each group after receiving

its respective treatment orally was immediately administered with glucose load (2 g/kg)

from the same route. Blood glucose level was monitored from the tail vein of mice of

each group at 0, 30, 60, and 120 min with the help of glucometer.

44

Figure 4: Log-dose verses Probit for Calculation of LD50 of MREt of R.serpentina

45

45

Figure 5: Log-dose verses Probit for Calculation of LD50 of AqMREt of R.serpentina

46

Figure 6: Effect of MREt on Glucose Tolerance in Mice

Test Group VI

(MREt 60 mg/kg + glucose

load 2 g/kg)

Animal Grouping (6/group)

Group I: Control

(Distilled water 1 ml/kg +

glucose load 2 g/kg)

Group II: Negative control

(0.05% DMSO 1 ml/kg +


Group III: Positive control

(Glibenclamide 5 mg/kg +


Test Groups

Test Group VI


load 2 g/kg)

Test Group V


load 2 g/kg)

Test Group VII

(MREt 100 mg/kg +

glucose load 2 g /kg)

47

Figure 7: Effect of AqMREt on Glucose Tolerance in Mice


Group I: Control

(Distilled water1 ml/kg +


Group II: Negative control

(0.05% DMSO 1 ml/kg +


Group III: Positive control

(Glibenclamide 5mg/kg +


Test Groups

Test Group I

(AqMREt 10 mg/kg +


Test Group II

(AqMREt 50 mg/kg +


Test Group III

(AqMREt 100 mg/kg +


Test Group IV

(AqMREt 150 mg/kg +


48

2.5 Results

2.5.1 Percent Yield of Root Extracts

The total yields of methanol root (MREt) and 80% aqueous methanol root extract

(AqMREt) extracts of R. serpentina were 5 and 10 % g/g of starting material

respectively. The quality of extracts was maintained by keeping in air-tight containers

separately and stored in refrigerator below 10C until used.

2.6 Analysis of Phyto-constituents in Root Powder and Extracts

2.6.1 Qualitative Analysis

The qualitative analysis of MREt showed the presence of alkaloids,

carbohydrates, flavonoids, glycosides, cardiac glycosides, phlobatanins, resins, saponins,

steroids, tannins and triterpenoids (Table 3 & Figure 8). Whereas the qualitative analysis

of AqMREt showed the presence of alkaloids, flavonoids, glycosides, cardiac glycosides,

steroids, tannins, triterpenoids, resins, phlobatanins but no carbohydrates (by giving no

reaction with Fehling‘s and Benedict‘s reagents) and saponins were observed (Table 3 &

Figure 8).

2.6.2 Quantitative Analysis

The amount of alkaloids, flavonoids, saponins in root powder were found as 7, 20

and 97 mg/g respectively while total phenols in MREt and AqMREt were 233.33 41.63

and 321.67 27.54 mg / g of extract (Table 4).

49

Table 3: Qualitative Analysis of Phyto-constituents of Root Extracts of R. serpentina

Phyto-constituents Qualitative analysis

MREt AqMREt

Alkaloids Positive Positive

Anthraquinones Negative Negative

Carbohydrates Positive Negative

Flavonoids Positive Positive

Glycosides Positive Positive

Cardiac glycosides Positive Positive

Phlobatannins Positive Positive

Resins Positive Positive

Saponins Positive Negative

Steroids Positive Positive

Tannins Positive Positive

Triterpenoids Positive Positive

50

Figure 8: Qualitative Analysis of Phyto-constituents of Root Extracts of R. serpentina

D r a g e n d r o f f ’ s T e s t M a y e r ’ s T e s t

H a g e r ’ s t e s t W a g n e r ’ s T e s t

51

B e n e d i c t ’ s T e s t F e h l i n g ’ s T e s t

M o l i s c h ’ s T e s t S h i n o d a T e s t

52

C a r d i a c g l y c o s i d e s T e s t T r i t e r p e n o i d s T e s t

R e s i n s T e s t S a p o n i n s T e s t

53

T a n n i n s T e s t P h l o b a t a n n i n s T e s t

54

Table 4: Quantitative Analysis of Phyto-constituents of Root Extracts of R. serpentina

Phyto-constituents Quantitative analysis

MREt (mg/g) AqMREt (mg/g)

Alkaloids 7* -

Flavonoids 97* -

Saponins 20* -

Total Phenols 233.33 ± 41.63 321.6 ± 27.54

*mg/g of root powder

55

2.7 In Vivo Evaluation of Root Extracts

2.7.1 Acute Toxicity and Median lethal Dose (LD50) of Root Extracts

The MREt of R. serpentina from 10 to 60 mg/kg was found non-sedative and

produced percent reduction in blood glucose level from -33 to -48 % after 30 min of its

oral administration in overnight fasted normoglycemic mice as compared to the control

group (p<0.05). Whereas the same extract in a dose of 100 mg/kg though found

hypoglycemic but gave 17% mortality within 4 hr of its oral administration. On the other

hand, the doses of MREt in doses of 150, 200 and 250 mg/kg showed acute toxicity by

making mice extremely sedative and giving 50, 83 and 100% mortalities respectively

(Table 5). Therefore, the value of median lethal dose (LD50) of MREt was found as

141.25 mg/kg (log LD50 = 2.15 mg/kg) from graph plotted between log-doses verses

probits (Figure 4).

The AqMREt of R.serpentina from 5 to 250 mg/kg in normoglycemic mice of

test groups was found non-sedative as these mice showed normal behavior pattern as

same as behavior observed in mice of control group. Whereas the same extract from 500-

2500 mg/kg found mild to extreme sedative in their respective test groups and showed

17-100% mortalities within 4hr of oral administration (Table 6). Therefore, the LD50 of

AqMREt was obtained as 1412.54 mg/kg (log LD50 = 3.15mg/kg) through a graph plotted

between log doses verses probits (Figure 5).

56

Table 5: Acute Toxicity of MREt

Treatments Hypoglycemic activity* Sedative behavior Mortality rate (%)

Control (distilled water 1ml/kg) 119.33 12.56 - 0

MREt (10 mg/kg) 80 2.56*** (- 33) - 0

MREt (30 mg/kg) 65 1.23*** (- 45) - 0

MREt (60 mg/kg) 62 2.83*** (- 48) - 0

MREt (100 mg/kg) 55 3.56*** (- 54) + 17

MREt (150 mg/kg) 26 0.82*** (- 78) ++ 50

MREt (200 mg/kg) - +++ 83

MREt (250 mg/kg) - +++ 100

* Mean blood glucose level (n=6) after 30 minutes of oral administration of each treatment.

Values in parenthesis represent percent blood glucose decrease (-) compared to control group

*** = p< 0.0001 when compared with control group

- = No sedation was observed

+ = slight sedation and 17 % mortality were observed within 4 hours of oral administration of dose

++ = moderate sedation and 50 % mortality were observed within 4 hours of oral administration of dose.

+++ = Acute toxicity with extreme sedation and 83-100 % mortality was observed.

57

Table 6: Acute Toxicity of AqMREt

Treatments Behavioral change

Normal Locomotive Sedative Mortality rate (%)

Control (distilled water 1ml/kg) √ - - 0

AqMREt (5 mg/kg) √ - - 0



AqMREt (100 mg/kg) √ - - 0

AqMREt (250 mg/kg) √ - - 0

AqMREt (500 mg/kg) - - + 17

AqMREt (1000 mg/kg) - - + 33

AqMREt (1500 mg/kg) - - ++ 50

AqMREt (2000 mg/kg) - - +++ 83

AqMREt (2500 mg/kg) - - +++ 100

n = 6 in each group.

- = sedation was not observed

+ = slight sedation and 17 % mortality were observed within 4 hours of oral administration of dose

++ = moderate sedation and 50 % mortality were observed within 4 hours of oral administration of dose.

+++ = Acute toxicity with extreme sedation and 83-100 % mortality was observed.

58

2.7.2 Effect of Root Extracts on Glucose Tolerance in Mice

The MREt of R. serpentina in doses of 10, 30 and 60 mg/kg and glibenclamide

5mg/kg produced significant percent reduction in blood glucose levels ranging from 31-

64.60%, 38.95 - 48.87%, 25 -51.01% and 36-56.16% respectively at 0, 30 and 60 min

after oral glucose load (2 g/kg) in their respective test groups as compared with control

and negative groups (p <0.01, p < 0.001 & p <0.0001). However, the same extract in a

dose of 100 mg/kg was found hypoglycemic from 0 to 60 min after glucose load but

induced slight sedation in experimental mice (Table 7).

In case of AqMREt, out of its four doses tested, three doses viz., 50, 100 & 150

mg/kg induced significant percent reduction (p<0.05, p<0.001 & p<0.0001) ranging

from 6.15-30.68%, 15.73-33.70% and 13.57-33.15% in blood glucose levels of

normoglycemic mice at 0, 30, 60 and 120 minutes after oral administration of glucose

load (2 g/kg) as compared to control group which were only administrated with glucose

load. Whereas the same extract in a dose of 10 mg/kg was not found effective as same as

its other three doses. Significant percent reduction in blood glucose level was also

observed at each time interval in positive control group treated with glibenclamide

(5mg/kg) after oral glucose load (Table 8).

59

Table 7: Effect of MREt on Glucose Tolerance in Mice

Groups Treatments Blood glucose level (mg/dl)

0 min 30 min 60min 120 min

Control Distilled water (1ml/kg) +

glucose load (2g/kg) 150 26.33 252.3 25.8 185.6 17.62 92 23.30

Negative

control

0.05% DMSO (1ml/kg) +

glucose load (2g/kg) 140 25.16 250 5.57 178.6 8.02 98.6 10.60

Positive

control

Glibenclamide (5mg/kg) +

glucose load (2g/kg) 96 16.70 **

ab

(-36)

110.6 6.66*** ab

(-56.16)

85.3 18.04 *** ab

(-54.04)

81 10.4

(-11.95)

Test

MREt (10mg/kg) +

glucose load (2g/kg) 102.3 23.71*

ab

(-31.8)

89.3 19.73*** ab

(-64.60)

104 15.72*** ab

(-43.96)

94.3 17.16

(+2.5)

MREt (30mg/kg) +

glucose load (2g/kg) 84 16.09**

ab

(-44)

129 0.22*** ab

(-48.87)

113.3 11.72*** ab

(-38.95)

93.3 8.02

(+1.41)

MREt (60mg/kg) +

glucose load (2g/kg) 112 7.21*

ab

(-25)

123.6 7.77*** ab

(-51.01)

114.6 9.07*** ab

(-38.25)

100.6 8.14

(+9.34)

MREt (100mg/kg) +

glucose load (2g/kg) 86.6 14.15**

ab

(-42.2)

116.6 40.38*** ab

(-53.08)

109 36.29** ab

(-47.27)

106.3 12.01

(+15.54)

*, ** & *** = p< 0.01, p< 0.001 & p<0.0001 when compared with respective control (a) and negative control (b) groups. All values are expressed as

mean SD (n=6). Values in parenthesis represent percent blood glucose decrease (-) / increase (+) compared with respective time interval in control group

60

Table 8: Effect of AqMREt on Glucose Tolerance in Mice

Groups Treatments Blood glucose level (mg/dl)

0 min 30 min 60min 120 min

Control Distilled water(1ml/kg) +

glucose load (2g/kg) 113 6.08 198.67 44.07 179 22 135.67 5.51

Negative

control

0.05% DMSO (1ml/kg) +

glucose load (2g/kg) 141 7.94 260.33 16.92 184 18.03 128.33 20.21

Positive

control

Glibenclamide (5mg/kg) +

glucose load (2g/kg) 106 12.58

*b

(-6.19%)

117 25.63*a***b

(-41.11%)

96.67 12.06**ab

(-45.99%)

82.67 7.64***a**b

(-39.07%)

Test

AqMREt (10mg/kg) +

glucose load (2g/kg) 111.67 7.37

*b

(-1.18%)

203.33 53.72

(+2.35%)

175.67 51.54

(-1.86%)

130 24.98

(-4.18%)

AqMREt (50mg/kg) +


*a***b

(-30.68%)

170.67 30.99*b

(-14.09%)

131.67 14.57*ab

(-26.44%)

127.33 10.69

(-6.15%)

AqMREt (100mg/kg) +


*a***b

(-22.42%)

138 41.33**b

(-30.54%)

118.67 11.24*ab

(-33.70%)

114.33 9.07

(-15.73%)

AqMREt (150mg/kg) +


*b

(-13.57%)

134.33 21.55*a**b

(-32.39%)

119.67 14.47*ab

(-33.15%)

96.67 6.66*ab

(-28.75%) *, ** & *** = p< 0.05, p< 0.001 & p<0.0001 when compared with respective control (a) and negative control (b) groups. Values are expressed as mean SD (n=6).

Values in parenthesis represent percent blood glucose decrease (-) / increase (+) compared with respective time interval in control group.

61

2.8 Discussion

Plants and herbal formulations have been used as remedy from a long time for the

treatment of various ailments like hypertension, diabetes, gastrointestinal tract disorders,

inflammation, ulcers and cardiovascular problems. More than 800 plants are reported to

have versatile ethnopharmacological properties with no or few side effects and also

found cost effective (Aggarwal and Shishu, 2011). It has been reported that more than

80% population from developing and developed world use herbal medicines or natural

products in combating many diseases (Tiwari, 2008). In spite of the availability of

pharmaceutical preparations in market, medicinal plants are gradually and steadily

making their prominent position as they contain variety of secondary metabolites which

together or alone found effective in solving number of health problems (Pitchai et al.,

2010; Ye et al., 2013).

Rauwolfia serpentina has been well-known for its anti-hypertensive activity from

last few decades. However, this herb is also reported in the treatment of various

neurological disorders like schizophrenia, anxiety, insanity, etc. The sedative and

antihypertensive activities of roots of R. serpentina are due to the presence of variety of

indole alkaloids including ajamlicine, ajmaline, reserpine, etc, that have been reported in

depleting the level of catecholamines in the central and peripheral nervous system

(Pandey et al., 2010; Salma et al., 2008; Mashour et al., 1998; Von Poser et al., 1990).

The present work introduces the antidiabetic activity of methanolic (MREt) and 80%

aqueous methanolic root (AqMREt) extracts of R.serpentina in the scientific world for

the first time. The qualitative phyto-chemical analysis confirmed the presence of

alkaloids and other chemical constituents including carbohydrates, flavonoids,

glycosides, cardiac glycosides, phlobatannins, resins, saponins, steroids, tannins and

triterpenoids in MREt. Similar constituents except carbohydrates and saponins were also

found in AqMREt of R.serpentina. The quantitative assessment of ground root powder of

R.serpentina demonstrated the presence of alkaloids (7mg/g), flavonoids (97mg/g) and

saponins (20mg/g) while AqMREt and MREt demonstrated the presence of good

62

concentration of total phenolic compounds as 321.6 and 233.3 mg/g of extract

respectively. The presence of secondary metabolites including flavonoids, phenols /

polyphenols, terpenoids, alkaloids, etc, in medicinal plants uplift their worth as

antihyperglycemic, antihyperlipidemic, antiinflammatory, antiulcer, hepatoprotective,

antiviral, anticancer, antioxidant, and antimicrobial agents (Pereira et al., 2009; Seifu et

al., 2012).

In early 1960, the hypoglycemic activity of R. serpentina in hypertensive patients

and in anesthetized cats has been briefly reported (Cazzaroli and Dall‘Oglio, 1958;

Cohen et al., 1959; Chatterjee et al., 1960). After that no strong proof was available that

highlights the same worth of R.serpentina. In 2009, a short-term scientific claim based

on a single dose (30 mg/kg) of MREt of R. serpentina was published which proved that

this extract has hypoglycemic and hypolipidemic activities in alloxan-induced diabetic

rats without producing any alteration in liver-specific ALT level (Qureshi et al., 2009).

This study brought a nice breakthrough in the scientific world related to R.serpentina. To

evaluate this claim, the present study initially aimed to determine the median lethal dose

(LD50) of root extracts (MREt & AqMREt) of R.serpentina as roots of same plant were

previously reported to rich in alkaloids having tranquilizing effect (Vakil, 1955). In this

regard, different doses of MREt (10-250 mg/kg) and AqMREt (5-2500 mg/kg) were

orally administrated in overnight fasted mice for determining the acute toxicity of these

extracts in term of behavioral change (sedative or not) and mortality rate. Observation

confirmed that MREt from 10-60 mg/kg were found non-toxic and possess significant

(p< 0.0001) hypoglycemic potential by lowering the blood glucose level from 33 to 48%

after 30 min of administration as compared to hyperglycemic control group. Whereas,

doses of same extract from 100-250 mg/kg produced sedative effect with mortality rate

from 17-100% in overnight fasted mice within 4 hours of their oral administration.

Therefore, the LD50 of MREt was found as 141.25 mg/kg (log LD50 = 2.15 mg/kg) by

plotting a graph between log dose and probit. On contrary, the LD50 of AqMREt was

calculated as 1412.54 mg/kg (log LD50 = 3.15 mg/kg) from the same graph since the

extract from 5-250 mg/kg was found safe by showing no sedation and mortality in their

63

respective groups of overnight fasted mice whereas its doses from 500-2500 mg/kg

produced mild to severe sedation and 17-100% mortalities within 4 hours of oral

administration. Therefore, the concentrations of MREt from 10-100 mg/kg and AqMREt

from 10-150 mg/kg were selected to conduct oral glucose tolerance test (OGTT) as

AqMREt found safer than MREt.

The significance of OGTT is to assess the ability to tolerate orally administrated

glucose which helps to diagnose pre-diabetic and diabetic states (Islam et al., 2009).

Beside this, it also helps to diagnose inappropriate absorption of sugar in blood stream

from gastro-intestinal tract (GIT) in any malabsorption syndrome (Rang et al., 2008).

The MREt in doses of 10, 30 and 60 mg/kg was found significant in lowering blood

glucose level after 0, 30 and 60 min of their oral administration with glucose load (2 g/kg

orally) and induced reduction in same parameter by 25-65% when these groups

compared with respective hyperglycemic control and negative control mice groups (p<

0.01, p< 0.001 & p< 0.0001). No doubt, 100 mg/kg of same extract was found effective

in lowering blood glucose level from 0-60 minutes but induced slight sedation in its

respective test group. Similarly, AqMREt in doses of 50, 100 and 150 mg/kg was found

effective in reducing the blood glucose levels at 0, 30, 60 and 120 minutes immediately

after glucose load and produced percent reduction in same parameter from 6-34 % as

compared to hyperglycemic control group which was only administrated by glucose load

(p< 0.05, p< 0.001 & p< 0.0001). Interestingly, the same AqMREt in a dose of 10 mg/kg

was not found effective in this aspect. The positive control, glibenclamide (5 mg/kg),

used in the present study, produced significant (p< 0.05, p< 0.001 & p< 0.0001) percent

glucose decrease at each time interval from 0 to 120 min. The antihyperglycemic effect

of both root extracts of R.serpentina observed in OGTT possibly due to its extra-

pancreatic or pancreatic action either by adopting any of the following mechanisms,

i. Inhibiting glucose absorption in GIT (Azmi and Qureshi, 2012a; 2014)

ii. Inducing glucose tolerance in mice by accelerating the glucose uptake in target

tissues of insulin like muscles and liver thereby stimulating the processes of glycolysis

and glycogenesis (Ju et al., 2008; Azmi and Qureshi, 2012a).

64

iii. Enhancing the release of insulin (pancreatic action) from β-cells of pancreas

which in turn induce glucose homeostasis as glibenclamide, an efficacious oral

hypoglycemic agent belongs to a class of sulfonylurea, responsible to enhance the

secretion of insulin from functional β-cells of pancreas (Azmi and Qureshi, 2012b) also

found to improve glucose tolerance in experimental mice in the present study. There are

several medicinal plants reported to have insulin releasing effect from β-cells of pancreas

such as Gymnema sylvestre, Ocimum sanctum, Phyllanthus species (Al-Romaiyan et al.,

2010; Daisy et al., 2004; Hannan et al., 2006; Qureshi et al., 2009).

According to the results obtained from OGTT, three doses of each of MREt (10,

30 & 60 mg/kg) and AqMREt (50, 100, 150 mg/kg) were selected for conducting the

antidiabetic activities in alloxan-induced type 1 and fructose-induced type 2 diabetic

male Wister mice models in order to evaluate the appropriate mechanism(s) that involved

in maintaining blood glucose homeostasis.

65

Chapter 3

Antidiabetic Effect of Root Extracts of

R.serpentina in Alloxan-Induced


Chapter 3 Antidiabetic Effect of Root Extracts of

R.serpentina in Alloxan-induced Type 1 Diabetic Mice

Model

Background

The current chapter deals with the antidiabetic effect of the methanolic root extract

(MREt) and aqueous methanolic root extract (AqMREt) of R.serpentina on type 1 animal

model, including their dyslipidemia along with the additional therapeutic characteristic.

This chapter describes the materials and method, results and discussion sections of the

above contents.


3.1.1 Animals

It was as same as described in chapter 2 section 2.1.1

3.1.2 Chemicals, Medicines and Enzymatic Kits

Alloxan monohydrate (2,4,5,6-tetraoxypyrimidine,5,6-dioxyuracil) was

purchased from AppliChem GmbH Darmstadt, Germany. White crystalline powder of D

(-) Fructose with molecular weight 180.16 and formula O.CH2(CH.OH)3.C(OH)CH2OH

was purchased from AnalaR, BDH Laboratories Supplies, England. Analytical reagent

grade of dimethyl sulphoxide (DMSO) was purchased from Fisher Scientific, United

Kingdom (UK). Chemicals used for estimating percent inhibition of catalase and

superoxide dismutase were purchased from Merck (Pvt.) Ltd.

Antidiabetic medicines including glibenclamide (Doanil, 5mg/kg) and

pioglitazone (Zolid, 15mg/kg) for type 1 and type 2 diabetes were purchased from

Sanofi-aventis and Getz Pharma, Pakistan Ltd. respectively. Commercially available

enzymatic kits for estimating triglycerides (TG), total cholesterol (TC), HDL-cholesterol

66

(HDL-c) and alanine aminotransferase (ALT) in serum were purchased from Randox,

UK.

3.1.3 Apparatus /Instruments

Glucometer of Optium Xceed, Diabetes Monitoring system by Abbott

Laboratories Pakistan Ltd was used to monitor blood glucose levels of mice.

Homogenizer (Janke and Kunkel, IKA-Labortechnik Ultra-turrac T25) was used to

homogenize liver tissue. Digital weighing balance (Sartorius Secura®) was used to weigh

chemicals accurately for reagent preparation. Kitchen Scale 1800 was used to measure

body weights of mice. Micro-pipettes (10-1000µl) of Eppendorf, Germany were used to

measure small quantities of samples and reagents accurately. Spectrophotometer

(Jennway 5600) was used to read absorbance of all biochemical parameters. Sysmex

(XS-1000i) automated hematology analyzer (America Inc.) was used to analyze

complete blood count (CBC). Automatic clinical (Roche/Hitachi 902) analyzer was used

to estimate serum insulin and glycosylated hemoglobin (HbA1c).

3.2 Determination of the Effect of Root Extracts in Type 1 Diabetic Mice

Model

3.2.1 Induction of Type 1 Diabetes in Mice

Type 1 diabetes was induced in overnight fasted mice (12-14 h) by a single intra-

peritoneal injection of alloxan monohydrate (150 mg/kg). After 72 hours of injection,

fasting blood glucose levels were monitored from tail vein of mice with the help of

glucometer and mice those showed glucose level ≥ 190 mg/dl were selected for study

(Sun et al., 2008)

3.2.1.1 Protocol

Overnight fasted alloxan-induced diabetic mice (blood glucose level ≥ 190 mg/dl)

were randomly divided into seven groups (6 / group) according to the treatments (Figure

9 and 10). Each treatment was given orally once in a day for consecutively 14 days. At

the end of animal trial, mice were sacrificed to collect whole blood, serum and body

67

tissues including heart, kidneys, liver and spleen which were carefully dissected out

(Qureshi et al., 2009).

68

Figure 9: Effect of MREt in Alloxan-Induced Type 1 Diabetic Mice


Group I

Control

(Distilled water

1 ml/kg)

Alloxan monohydrate (150 mg/kg) i.p.

After 72hr, Mice having blood glucose level ≥ 190mg/dl were included in study

Test Groups

Group V

MREt 10 mg/kg

Group IV

Positive Control

(Glibenclamide

5 mg/kg)

Group III

Negative Control

(0.05% DMSO

1 ml/kg)

Group II

Diabetic Control

(Distilled water

1 ml/kg)

Group VI

MREt 30 mg/kg

Group VII

MREt 60 mg/kg 6

9

Figure 10: Effect of AqMREt in Alloxan-Induced Type 1 Diabetic Mice

Group III

Negative Control

(0.05% DMSO

1ml/kg)


Group I

Control

(Distilled water

1ml/kg)

Alloxan monohydrate (150 mg/kg) i.p.

After 72hr, Mice having blood glucose level ≥ 190mg/dl were included in study

Test Groups

Group V

AqMREt 50 mg/kg

Group IV

Positive Control

(Glibenclamide

5 mg/kg)

Group II

Diabetic Control

(Distilled Water

1ml/kg)

Group VI

AqMREt 100 mg/kg

Group VII

AqMREt 150 mg/kg

70

3.2.2 Determination of Physical Parameters

3.2.2.1 Determination of Percent Gain / Loss in Body Weights

The percent gain/loss in body weight was calculated with the help of the

following formula (Saba et al., 2010).

( ) (

)

3.2.2.2 Determination of Wet Organs Weight and their Relative Weights

(Percent Body Weight)

On final day (14th

day) of animal trial, after sacrificing mice, body organs

including heart, kidneys, liver and spleen were dissected out carefully, tabbed on filter

paper and their weights were recorded in grams (g) by using digital weighing balance

and finally their relative weights (percent body weight) were calculated with the help

of following formula (Adaramoye et al., 2012).

( ) (

)

3.2.3 Collection of Samples for Biochemical and Hematological

Parameters

At the end of trial, mice were sacrificed a portion of whole blood was

collected in a commercially available plastic gel tube (yellow-topped tube) and kept

them undisturbed at room temperature for at least 30-45 minutes then centrifuged at

3000 rpm for 15 minutes to collect serum, saved in eppendrof and stored at 2-8°C.

Whereas another portion of same whole blood was collected in ethylenediamine tetra

acetic acid (EDTA) containing tubes (purple-topped tube) for analyzing

hematological parameters.

71

3.2.4 Determination of Hematological Parameters

3.2.4.1 Glycosylated Hemoglobin (HbA1c)

The HbA1c was estimated by using turbidimetric inhibition immunoassay

(TINIA) on automatic clinical analyzer (Roche/Hithachi 902). The principle of this

immunoassay is that HbA1c in hemolyzed sample reacts with anti-HbA1c antibody to

form soluble antigen-antibody complexes. Since only one specific HbA1c antibody

site is present on HbA1c molecule so formation of insoluble complexes does not take

place. Addition of polyhaptens reacts with excess anti-HbA1c antibodies to form an

insoluble antibody-polyhapten complex which can be determined turbidimetrically.

Whereas, hemoglobin (Hb) concentration is determined in a second channel, in which

liberated Hb is converted to a derivative having characteristic absorption spectrum

and measured bichromatically. The final result is expressed as % HbA1c.

3.2.4.2 Complete Blood Profile

Complete blood profile (CBP) including total hemoglobin (Hb), red blood

cells (RBC), white blood cells (WBC), packed cell volume or hematocrit (PCV),

haemoglobin concentration (Hb), mean corpuscular volume (MCV), mean

corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration

was determined by Sysmex (XS-1000i) automated hematology analyzer.

3.2.5 Determination of Biochemical Parameters

3.2.5.1 Determination of the Blood Glucose Level and Percent Glycemic

Change

Fasting blood glucose levels were monitored by pricking the tail vein of each

mice of each group by using glucometer at initial (0) and final (14th

) day of trial and

recorded as milligram per deciliter (mg/dl). To determine percent glycemic change,

following formula was used (Perfumi and Tacconi, 1996).

( ) (

)

Where, Go = blood glucose level at 0 day and Gx = blood glucose level at 14 day.

72

3.2.5.2 Determination of the Fasting Insulin Level

The serum fasting insulin level was measured by electro-chemiluminescence

immunoassay ―ECLIA‖ on Cobas e411 automated analyzer. The principle of this

immunoassay is based on the formation of sandwich complex of insulin between

biotinylated monoclonal insulin-specific antibody and a monoclonal insulin-specific

antibody labeled with a ruthenium complex. The sandwich complex then allow to

binds with streptavidin-coated microparticles, via interaction of biotin and

streptavidin. These micro-particles are later magnetically captured onto the surface of

electrode and after applying voltage to the electrode, induced chemiluminescent

emission which is measured by a photomultiplier. The results are automatically

displayed and determined through a calibration curve which is instrument-specific

(Sapin et al., 2001).

3.2.5.3 Determination of Lipid Profile

1. Total Cholesterol (TC) by Enzymatic Endpoint Method (Trinder, 1969)

Principle

Cholesterol ester + H2O Cholesterol + Fatty acids

Cholesterol + O2 Cholestene-3-one + H2O2

2H2O2 + phenol + 4·Aminoantipyrine Quinoneimine + 4H2O

Reagents

1. Buffer reagent (R1, pH 6.8): 4-Aminoantipyrine 0.30 mmol/l,

Phenol 6 mmol/l, peroxidase ≥ 0.5 U/ml, cholesterol esterase ≥ 0.15 U/ml,

cholesterol oxidase ≥ 0.1U/ml, Pipes Buffer 80 mmol/l.

2. Standard: Cholesterol 200mg/dl.

Cholesterol esterase

Cholesterol oxidase

Peroxidase

73

Procedure

Reagent Blank Standard Sample

Distilled water 10 µl __ __

Standard __ 10 µl __

Serum __ __ 10 µl

Reagent 1000 µl 1000 µl 1000 µl

Mixed and incubated for 5 min at 37°C. Absorbance of the sample (Asample) against the

reagent blank was measured at 500 nm.

Calculation

( )

( )

2. High Density Lipoprotein-Cholesterol (HDL-c) by Precipitant

Method (Lopes-Virella et al., 1977)

Principle

Low density lipoproteins (LDL-c and VLDL-c) and chylomicron fractions are

precipitated quantitatively by the addition of phosphotungstic acid in the presence of

magnesium ions. After centrifugation, the cholesterol concentration in the HDL (high

density lipoprotein) fraction remained in the supernatant and determined.

Reagents

1. HDL-Cholesterol Precipitant (R1): Phosphotungstic acid 0.55 mmol/l.

2. Diluted Precipitant (R2): R1 was diluted in 4:1 ratio with re-distilled water

before used.

3. Magnesium chloride(MgCl2): MgCl2 25 mmol/l

74

Procedure

1. Precipitation

Semi Macro

Serum 200 µl

Diluted Precipitant (R2) 500 µl

Mixed, kept for 10 minutes at room temperature and centrifuged for 15 minutes at

3,000 rpm to separate clear supernatant. Determination of cholesterol content was

done as same as method described for TC estimation.

Calculation

( )

( )

3. Triglyceride (TG) by GPO-PAP Method (Tietz, 1990)

Principle

Triglycerides + H2O glycerol + fatty acids

Glycerol + ATP glycerol-3-phosphate + ADP

Glycerol-3-phosphate + O2 dihydroxyacetone + phosphate + H2O2

2H2O2 + 4-aminophenazone + 4 chlorophenol quinoneimine + HCI + 4H2O

Reagents

1. Buffer (R1a): Pipes Buffer 40 mmol/l, 4 chloro-phenol 5.5 mmol/l,

Magnesium-ions I7.5 mmol/l

Glycerol-3-phosphate oxidase

Lipase

Glycerol kinase

Peroxidase

75

2. Enzyme Reagent (R1b): 4-aminophenazone 0.5 mmol/l, ATP 1.0 mmol/l,

Lipases ≥ 150 U/ml, Glycerol-kinase ≥ 0.4 U/ml, Glycerol-3-phosphate

oxidase ≥ 1.5 U/ml, Peroxidase ≥ 0.5 U/ml

3. Reagent (R1): One vial of R1b was dissolved with 15 ml of R1a before used.

4. Standard: Triglycerides 194 mg/dl

Procedure

Reagent Blank Standard Sample

Standard - 10 µl -

Serum - - 10 µl

Reagent (R1) 1000 µl 1000 µl 1000 µl

Mixed and incubated for 5 minutes at 37°C. Absorbance of the sample (Asample) and

standard (Astandard) against the reagent blank was measured at 500 nm within 60

minutes.

Calculation

( )

( )

4. Low Density Lipoprotein-Cholesterol (LDL-c)

LDL-c was calculated by using Friedewald formula (Friedewald et al., 1972)

given on Randox kit, as

LDL-c (mg/dl) = TC – (TG/5) – HDL-c

5. Very Low Density Lipoprotein-Cholesterol (VLDL-c)

VLDL-c was calculated by using Friedewald formula (Friedewald et al.,

1972), given below

VLDL-c = TG /5

76

3.2.5.4 Determination of Cardioprotective Ratios and Indices from

Estimated Values of Lipid Profile and Serum Insulin

1. TG / HDL-c Ratio

This ratio was calculated by dividing the value of TG (mg/dl) with HDL-c

(mg/dl) (Kang et al., 2012).

2. TC / HDL-c Ratio

This ratio was calculated by dividing the concentration of TC (mg/dl) with

HDL-c (mg/dl). It is also called coronary risk index and abbreviated by CRI

(Adeneye and Olagunju, 2009).

3. HDL-c / LDL-c Ratio

This ratio was calculated by dividing the concentration of HDL-c (mg/dl) with

LDL-c (mg/dl) (Barter et al., 2007).

4. Anti-Atherogenic Index (AAI)

This index was calculated according to the formula (Waqar and Mahmood,

2010), given as follows

AAI (%) = 100 x [HDL-c / TC- HDL-c]

5. Fasting Insulin Resistance Index (FIRI)

This index was calculated according to the formula (Duncan et al., 1995)

mentioned below,

( ) ( )

3.2.5.5 Determination of Liver Glycogen by Phenol-Sulfuric Acid Method

Principle

Simple sugars, oligosaccharides, polysaccharides and their derivatives

(including the methyl ethers with free or potentially free reducing groups) give an

orange–yellow color when treated with phenol and concentrated sulfuric acid, its

absorbance can be measured at 490 nm (Dubois et al, 1956).

77

Reagents

1. Stock Dextrin solution (1mg/ml): 0.1 g of dextrin was dissolved in 1000 ml of

distilled water.

2. Working Dextrin solution (0.1 mg/ml): Dilute the stock solution in 1:10 ratio.

3. Sulfuric acid (concentrated).

4. Phenol reagent (5%)

Procedure

1. Preparation of Sample

0.5 g of liver tissue was homogenized in 100 ml of distilled water. Then 0.5 ml

of homogenate was diluted up to 10 ml with distilled water. Finally 0.1ml of diluted

sample was taken in a test tube marked as test and subjected to color development.

2. Preparation of Blank, Standards, Test and Color Development

S1 S2 S3 S4 S5 B T C

Diluted Homogenate _ _ _ _ _ _ 0.1 ml 0.1 ml

Dextrin Solution 0.2 ml 0.4 ml 0.6 ml 0.8 ml 1 ml _ _ _

Distilled water 0.8 ml 0.6 ml 0.4 ml 0.2 ml _ 1 ml 0.9 ml 0.9 ml

Phenol Reagent 1 ml 1 ml 1 ml 1 ml 1 ml 1 ml 1 ml 1 ml

Sulfuric acid 5 ml 5 ml 5 ml 5 ml 5 ml 5 ml 5 ml 5 ml

78

Table 9: Absorbance of Dextrin

S. No Standards Concentration of dextrin (mg) Absorbance at 490 nm

1 S1 0.02 0.1

2 S2 0.04 0.2

3 S3 0.06 0.3

4 S4 0.08 0.4

5 S5 0.1 0.5

79

Figure 11: Standard Curve of Dextrin

y = 5x

R² = 1

0

0.1

0.2

0.3

0.4

0.5

0.6

0 0.02 0.04 0.06 0.08 0.1 0.12

Ab

sorb

an

ce a

t 490 n

m

Concentartion of Dextrin (mg)

80

Mixed well and incubated all the tubes at room temperature for 30 minutes.

Absorbance was read at 490 nm.

Calculation

The concentration of liver glycogen gm /gm of liver tissue was expressed and

calculated from standard curve of dextrin from 0.02-0.12 mg (Table 3 and Figure 11).

3.2.5.6 Total Lipids by Gravimetric Method

Principle

The lipid including both free and bound as lipoproteins comprise complex

mixture of different classes of compounds. Lipids occurring in the human tissue are

hydrocarbon, alcohol, fatty acids, neutral glycerides (mono-, di-, triglycerides), sterols

(cholesterol), glycerol-phospholipids and sphingolipids. Organic solvent are used to

extract lipids. Substances other than lipids are removed by washing the extract with

aqueous salt solution (Folch et al., 1956).

Procedure

Liver tissue (0.5 gm) was homogenized with 100 ml of chloroform: methanol

(2:1) mixture, filtered and evaporated the filtrate at room temperature until the

residues were remained either on the walls or at the bottom of test tube. Finally the

residues were weighed as total lipids.

Calculation

W = W2 – W1

W = Weight of total lipids (g / 0.5 g of liver tissue).

W1 = Weight of empty test tubes.

W2 = Weight of test tubes containing residues.

Finally, results are expressed as g of total lipids / g of liver tissue.

3.2.5.7. Determination of Liver-specific Enzyme

1. Alanine Aminotarnsferase (ALT)

Principle

α-oxoglutarate + L-alanine L-glutamate + pyruvate

ALT

81

The ALT activity is measured by monitoring the concentration of pyruvate

hydrazone formed with 2, 4-dinitrophenyl- hydrazine (Reitman and Frankel, 1957).

Reagents

1. Buffer (R1): Phosphate buffer (pH 7.4) 100 mmol/l, L-alanine 200 mmol/l

α-oxoglutarate 2 mmol/l,

2. 2, 4-dinitrophenyt-hydrazine (2,4- DNP; R2): 2.0 mmol/l

3. Sodium hydroxide (R3): 4.0 mol/l

4. Standard: Pyruvate 1.97mmol/l

Procedure

Reagent Blank Sample

Sample - 0.1 ml

Buffer (R1) 0.5 ml 0.5 ml

Distilled Water 0.1 ml -

Mixed and incubated for exactly 30 min at 37oC

2.4-DNP (R2) 0.5 ml 0.5 ml

Mixed and allowed to stand for exactly 20 min at 20-25oC

Sodium hydroxide (R3) 5 ml 5 ml

Mixed and incubated for 5 min at room temperature. Absorbance of sample against

the reagent blank was read at 530nm.

82

Table 10: Absorbance of ALT

S. No. Standards Concentration (U/L) Absorbance at 560nm

1 S1 4 0.025

2 S2 8 0.05

3 S3 12 0.075

4 S4 17 0.1

5 S5 21 0.125

6 S6 25 0.15

7 S7 29 0.175

8 S8 34 0.2

9 S9 39 0.225

10 S10 43 0.25

11 S11 48 0.275

12 S12 52 0.3

13 S13 57 0.325

14 S14 62 0.35

15 S15 67 0.375

16 S16 72 0.4

83

Figure 12: Standard Curve of ALT

y = 0.0057x

R² = 0.9976

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0 10 20 30 40 50 60 70 80

Ab

sorb

an

ce a

t 56

0n

m

Concentration of ALT (U/L)

84

Calculation

The ALT activity (U/l) was calculated through calibration curve (Table 4 and

Figure 12) given in Randox kit.

3.2.5.8 Determination of Antioxidant Enzymes

1. Catalase (CAT)

Principle

2H2O2 2H2O + O2

The CAT activity is directly proportional to decomposition of H2O2 which is

reflected by decrease in absorbance measured at 620 nm for 3 min in the presence of

color developer dichromate-acetic acid reagent (Pari and Latha, 2004).

Reagents

1. Phosphate buffer 0.01 M (pH 7.0): 1.735 g disodium hydrogen phosphate and

sodium dihydrogen phosphate was dissolved in 1000 ml of distilled water.

2. Liver homogenate (0.5%): 0.50 g liver tissue was homogenized in 100 ml of

distilled water.

3. Hydrogen peroxide (H2O2) 2 M: 6.8 ml H2O2 was dissolved in 93.2 ml of distilled

water.

4. Dichromate-acetic acid reagent: 5% potassium dichromate and glacial

acetic acid (1:3) were freshly mixed before use.

CAT

85

Procedure

Reagents Control Test

Phosphate buffer 1.0 ml 1.0 ml

Distilled H2O 0.1 ml -

Homogenate - 0.1 ml

Hydrogen peroxide (H2O2) 0.4 ml 0.4 ml

Dichromate-acetic acid 2.0 ml 2.0 ml

Calculation

( ) (

)

Where, C = Absorbance of control and T = Absorbance of Test

2. Superoxide Dismutase (SOD)

Principle

SOD catalyzes the dismutation of superoxide ion (O2-) into oxygen and hydrogen

peroxide and this superoxide radical is involved in autoxidation of epinephrine at high

pH. This method is based on the ability of SOD to inhibit the autoxidation of epinephrine

at alkaline pH which can be reflected by increase in absorbance, measured at 480 nm

(Misra and Fridovich, 1972).

Reagents

1. Liver homogenate (0.5%): 0.50 g liver tissue was homogenized in 100 ml of

distilled water.

2. Ethylenediamine tetraacetic acid (EDTA) 0.6 mM: 0.175 g EDTA was dissolved

in 1000 ml of distilled water.

86

3. Carbonate-bicarbonate buffer 0.1 M: (pH 10.2)

4. Epinephrine 1.8 mM: 0.329 g of epinephrine was dissolved in 1000 ml of distilled

water.

5. Ethanol (95%)

6. Chloroform.

Procedure

The increase in absorbance was measured at 480 nm for 3 min.

Calculation

( ) ( )

( ) (

)

Where C = Absorbance of control

Reagents Control Test

Ethanol 0.75 ml 0.75 ml

Chloroform 0.15 ml 0.15 ml

Homogenate - 0.1 ml

Centrifuged for 10 min at 3000 rpm

Supernatant - 0.5 ml

EDTA (0.6mM) 0.5 ml 0.5 ml

Buffer (pH 10.2) 1 ml 1 ml

Epinephrine (1.8mM) 0.5 ml 0.5 ml

87

3.3 Statistical Analysis

Results of the present study are expressed as Mean ± SD (Standard Deviation).

The data were analyzed by using one-way ANOVA followed by LSD (least significant

difference) test at p< 0.05 available in statistical package for social science (SPSS

version 18). The differences of means of test groups were considered significant at p<

0.05, p< 0.01, p< 0.001 and p< 0.0001 when compared with means of diabetic control

groups.

88

3.4 Results

3.4.1 Effect of Root Extracts in Alloxan-Induced (Type 1) Diabetic Animal

Model

3.4.1.1 Effect of Root Extracts on Body Weights of Alloxan-Induced Diabetic

Mice

There was a marked reduction observed in percent body weight up to -11.08 and -

9.25 % respectively in diabetic and negative control groups as compared to control mice.

Whereas + 8.45 % gain in body weight was observed in mice treated with glibenclamide

as compared to diabetic and negative controls (p<0.0001). Though all three doses of

MREt of R.serpentina slightly reduced (p<0.05 & p<0.01) the body weights of test mice

in their respective groups but the reduction was not as high as it was observed in both

diabetic control groups (Table 11 & Figure 13).

Similarly, all three doses (50, 100 and 150 mg/kg) of AqMREt of R.serpentina

gradually and significantly (p<0.05 & p<0.01) improved the body weight with respect to

increase in dose magnitude like 150 mg/kg showed 3.88 percent gain in body weights of

mice in its respective test group as compared to diabetic and negative controls. However,

diabetic and negative control groups showed marked reduction in percent body weights

up to -10.12 and -10.82 % respectively as compared to control mice. Glibenclamide

showed 6.73 % gain in body weights of mice in positive control group (Table 12 &

Figure 13).

89

Table 11: Effect of MREt on Body Weights of Alloxan-Induced Diabetic Mice

Groups Treatment

Body weights (g)

Initial day Final day % Change

Group I Distilled water

(1 ml/kg) 28.25 ± 1.71 31.25 ± 1.71 10.68 ± 2.97

Group II Alloxan

(150 mg/kg) 29.25 ± 3.20 26 ± 2.83 -11.08 ± 2.96

Group III Alloxan (150 mg/kg) +

0.05% DMSO (1 ml/kg) 29.50 ± 1.73 26.75 ± 1.26 -9.25 ± 1.24

Group IV Alloxan (150 mg/kg) +

Glibenclamide (5 mg/kg) 29.50 ± 1.0 32 ± 1.41 8.45 ± 1.80

***ab

Group V Alloxan (150 mg/kg) +

MREt (10 mg/kg) 30.75 ± 2.63 29 ± 2.45 -5.69 ± 1.56

*a

Group VI Alloxan (150 mg/kg) +

MREt (30 mg/kg) 28.50 ± 3.11 26.75 ± 3.30 -6.25 ± 2.09

*a

Group VII Alloxan (150 mg/kg) +

MREt (60 mg/kg) 27.50 ± 3.0 26.75 ± 3.77 -2.94 ± 4.75

***a**b

Values are expressed as mean ± SD (n = 6). *p < 0.05, **p< 0.01, and ***p < 0.0001, when compared with respective group II (a) and III (b).

Negative (−)/positive (+) signs represent loss/gain in weights of mice on final day (14th

day).

90

Figure 13: Effect of Root Extracts on Percent (%) Body Weight Change of Alloxan-Induced Diabetic Mice

Values are expressed as mean ± SD (n = 6). *p < 0.05, *p < 0.01 and *p < 0.0001, when compared with respective group II (a) and III (b). Negative

(−)/positive (+) signs represent loss/gain in body weights of mice on final day (14th

day).

-25

-20

-15

-10

-5

0

5

10

15

20

Group I Group II Group III Group IV Group V Group VI Group VII

Bo

dy

wei

gh

ts ch

an

ge

(%)

MREt AqMREt

***ab

*a *a

***a**b

***ab *ab

**ab

91

Table 12: Effect of AqMREt on Body Weights of Alloxan-Induced Diabetic Mice

Groups Treatment

Body weights in (g)



(1 ml/kg) 28.54 ± 2.75 31.14 ± 2.42 + 9.28 ± 2.33

Group II Alloxan

(150 mg/kg) 29.81 ± 2.54 26.76 ± 2.33 -10.12 ± 5.33


0.05% DMSO (1 ml/kg) 29.48 ± 3.12 26.20 ± 1.34 -10.82 ± 8.87


Glibenclamide (5 mg/kg) 29.64 ± 1.77 31.59 ± 1.24 + 6.73 ± 4.04

***ab


AqMREt (50 mg/kg) 30.06 ± 2.40 27.99 ± 2.63 - 6.94 ± 1.62


AqMREt (100 mg/kg) 29.47 ± 1.63 29.04 ± 0.77 - 1.21 ± 6.84

*ab


AqMREt (150 mg/kg) 29.68 ± 2.14 30.74 ± 0.66 3.88 ± 5.96

**ab

Values are expressed as mean ± SD (n = 6). *p < 0.05, **p < 0.01, and ***p < 0.0001, when compared with respective group II (a) and III (b).

Negative (−) / positive (+) signs represent loss / gain in weights of mice on final day (14th

day).

92

3.4.1.2 Effect of Root Extracts on Wet Organs Weight and their Relative

Weights (Percent Body Weight)

The wet organs weights and their relative weights (percent body weights) in all

MREt and AqMREt treated mice were found as same as observed in their respective

control groups including normal, alloxan-induced diabetic, negative and positive control

groups which depict the unaltered and non-toxic effect of root extracts of R. serpentina

on body organs (Table 13-14).

3.4.1.3 Effect of Root Extracts on Blood Glucose Level in Alloxan-Induced

Diabetic Mice

Significant (p<0.0001) percent glycemic reduction was found as -51.33, - 47.17

and - 49.33 % in test groups treated with MREt of R.serpentina in doses of 10, 30 and 60

mg/kg respectively as compared to diabetic and negative control groups (p<0.0001)

which showed increased blood glucose levels. Similarly - 40.10% reduction was

observed in blood glucose level in positive control group (Table 15 & Figure 14).

Similarly, AqMREt of same plant in doses of 50, 100 and 150 mg/kg produced

significant percent glycemic reduction in test groups respectively as -39.27, - 44.63 and –

53.82 % when compared to their respective diabetic and negative controls (p<0.0001).

Moreover, - 43.09% reduction (p<0.0001) in blood glucose level was also observed in

positive control group (Table 16 & Figure 14).

93

Table 13: Effect of MREt on Wet and Relative Organs Body Weights of Alloxan-Induced Diabetic Mice

Group Wet organ weight (g) Relative Weight (% body weight)

Kidney Liver Spleen Heart Kidney Liver Spleen Heart

Group I 0.40 ± 0.04 1.44 ± .05 0.11 ± 0.02 0.10 ± 0.01 1.29 ± 0.14 4.52 ± 0.28 0.35 ± 0.07 0.32 ± 0.04

Group II 0.40 ± 0.05 1.44 ± 0.07 0.11 ± 0.03 0.11 ± 0.02 1.56 ± 0.31 5.58 ± 0.76 0.42 ± 0.13 0.40 ± 0.05

Group III 0.39 ± 0.03 1.46 ± 0.08 0.11 ± 0.04 0.10 ± 0.01 1.45 ± 0.15 5.46 ± 0.52 0.39 ± 0.13 0.35 ± 0.03

Group IV 0.40 ± 0.05 1.41 ± 0.07 0.10 ± 0.02 0.10 ± 0.02 1.25 ± 0.15*a

4.41 ± 0.12*ab

0.31 ± 0.07 0.32 ± 0.05*a

Group V 0.40 ± 0.03 1.40 ± 0.12 0.10 ± 0.02 0.11 ± 0.02 1.38 ± 0.13 4.84 ± 0.41 0.35 ± 0.10 0.37 ± 0.03

Group VI 0.38 ± 0.02 1.47 ± 0.04 0.12 ± 0.02 0.10 ± 0.03 1.44 ± 0.17 5.56 ± 0.77 0.44 ± 0.12 0.36 ± 0.09

Group VII 0.40 ± 0.03 1.43 ± 0.07 0.10 ± 0.02 0.11 ± 0.01 1.52 ± 0.27 5.42 ± 0.61 0.38 ± 0.09 0.40 ± 0.03

Values are expressed as mean ± SD (n = 6). *p < 0.05 when compared with respective group II (a) and III (b).

94

Table 14: Effect of AqMREt on Wet and Relative Organs Body Weights of Alloxan-Induced Diabetic Mice

Group

Wet organ weight (g) Relative Weight (% body weight)


Group I 0.43 ± 0.07 1.44 ± 0.13 0.08 ± 0.02 0.09 ± 0.01 1.40 ± 0.24 4.62 ± 0.23 0.27 ± 0.04 0.29 ± 0.05

Group II 0.42 ± 0.06 1.42 ± 0.09 0.10 ± 0.03 0.11 ± 0.01 1.60 ± 0.30 5.33 ± 0.58 0.36 ± 0.09 0.40 ± 0.07

Group III 0.43 ± 0.05 1.39 ± 0.07 0.09 ± 0.02 0.09 ± 0.01 1.63 ± 0.16 5.34 ± 0.51 0.36 ± 0.10 0.33 ± 0.02

Group IV 0.42 ± 0.10 1.39 ± 0.10 0.10 ± 0.02 0.10 ± 0.02 1.32 ± 0.31 4.40 ± 0.32**ab

0.31 ± 0.04 0.32 ± 0.07

Group V 0.46 ± 0.03 1.42 ± 0.06 0.10 ± 0.02 0.10 ± 0.02 1.67 ± 0.22 5.09 ± 0.32 0.34 ± 0.07 0.34 ± 0.09

Group VI 0.48 ± 0.02 1.35 ± 0.11 0.09 ± 0.01 0.09 ± 0.02 1.64 ± 0.06 4.63 ± 0.38*ab

0.31 ± 0.04 0.30 ± 0.06*a

Group VII 0.42 ± 0.08 1.39 ± 0.06 0.10 ± 0.02 0.10 ± 0.02 1.36 ± 0.24 4.52 ± 0.21**ab

0.31 ± 0.08 0.33 ± 0.06

Values are expressed as mean ± SD (n = 6). *p < 0.05 and **p < 0.01 when compared with respective group II (a) and III (b)

95

Table 15: Effect of MREt on Blood Glucose Levels of Alloxan-Induced Diabetic Mice

Groups Treatment Blood glucose level (mg/dl)

% Glycemic Change Initial day Final day


(1 ml/kg) 102.25 ± 5.43 98 ± 11.69 -3.71 ± 14.71

Group II Alloxan

(150 mg/kg) 198.25 ± 9.81 245.50 ± 41.32 24.59 ± 26.25


0.05% DMSO (1ml/kg) 199.50 ± 11.24 257.25 ± 45.36 28.47 ± 17.61


Glibenclamide (5mg/kg) 195.25 ± 6.24 116.50 ± 28.92

***ab -40.10 ± 16.07

***ab


MREt (10 mg/kg) 198.25 ± 10.01 96.25 ± 9.88

***ab -51.33 ± 5.87

***ab


MREt (30 mg/kg) 197.50 ± 12.40 105 ± 24.78

***ab -47.17 ± 9.61

***ab


MREt (60 mg/kg) 195.25 ± 11.32 98 ± 13.09

***ab -49.93 ± 4.71

***ab

Values are expressed as mean ± SD (n = 6). *p < 0.0001, when compared with respective group II (a) and III (b). Negative (−)/positive (+) signs

represent decrease / increase in blood glucose level of mice on final day (14th

day).

96

Figure 14: Effect of Root Extracts on Percent (%) Glycemic Change of Alloxan-Induced Diabetic Mice

Values are expressed as mean ± SD (n = 6). *p < 0.0001 when compared with respective group II (a) and III (b). Negative (−)/positive (+)

signs represent decrease/increase in blood glucose level of mice on final day (14th

day).

-80

-60

-40

-20

0

20

40

60


Gly

cem

ic c

ha

ng

e (%

) MREt AqMREt

***ab

***ab ***ab

***ab

***ab

***ab ***ab

***ab

97

Table 16: Effect of AqMREt on Blood Glucose Levels of Alloxan-Induced Diabetic Mice

Groups Treatment

Blood glucose level (mg/dl)

% Glycemic Change

Initial day Final day


(1 ml/kg) 108.89 ± 17.87 108.29 ± 13.76 +0.06 ± 6.55

Group II Alloxan

(150 mg/kg) 194.75 ± 9.95 224.25 ± 11.79 +15.29 ± 6.89


0.05% DMSO (1ml/kg) 199.50 ± 4.51 216.25 ± 8.99 +8.43 ± 4.97


Glibenclamide (5 mg/kg) 200.50 ± 9.57 114.25 ± 11.93

***ab -43.09 ± 4.96

***ab


AqMREt (50 mg/kg) 225.50 ± 43.38 133.50 ± 5.74

***ab -39.27 ± 11.01

***ab


AqMREt (100 mg/kg) 211.75 ± 49.12 116.75 ± 27.11

***ab -44.63 ± 8.14

***ab


AqMREt (150 mg/kg) 253.25 ± 94.73 105.25 ± 8.66

***ab -53.82 ± 16.63

***ab

Values are expressed as mean ± SD (n = 6). *p < 0.0001, when compared with respective group II (a) and III (b). Negative (−)/positive (+) signs

represent decrease / increase in blood glucose level of mice on final day (14th

day).

98

3.4.1.4 Effect of Root Extracts on Serum Insulin Level in Alloxan-Induced

Diabetic Mice

All three doses of MREt (10, 30 & 60mg/kg) of R.serpentina significantly

(p<0.0001) improved the serum insulin levels in their respective test groups as compared

to diabetic and negative control groups. Similarly, prominent increase (p<0.0001) in

serum insulin level was also induced by glibenclamide in positive control group (Figure

15). Same significant improvement (p<0.0001) in serum insulin levels was observed in

all three test groups treated with AqMREt of same plant in doses of 50, 100 & 150

mg/kg as compared to their respective diabetic and negative control groups (Figure 15).

3.4.1.5 Effect of Root Extracts on Glycosylated Haemoglobin (HbA1C)/ Total

Haemoglobin (Hb) Ratio in Alloxan-Induced Diabetic Mice

All three doses of MREt of R.serpentina and glibenclamide significantly

improved (p<0.0001) the HbA1c/Hb ratio in test and positive control groups by

recovering the total Hb level from 12.10 to 12.98 g/dl and decreasing the HbA1c level

from 6.38 -7.40 % as compared to diabetic and negative control mice that showed 10 g/dl

total Hb and 10.38-11.83% HbA1c (Table 17). On the other hand, all three doses of

AqMREt of same plant was also found effective in improving HbA1c / Hb ratio in their

respective test groups by decreasing the percentage of HbA1c and recovering the amount

of total Hb as compared to their respective diabetic and negative control groups

(p<0.0001) that showed HbA1c from 10.55-10.84 % and total Hb from 7.25-10.91 g/dl

(Table 18).

99

Figure 15: Effect of Root Extracts on Serum Insulin Levels of Alloxan-Induced Diabetic Mice

Values are expressed as mean ± SD (n = 6). **p < 0.01, and ***p < 0.0001, when compared with respective group II (a) and III (b)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9


Ser

um

in

suli

n (

µU

/ml)

MREt AqMREt

***ab

***a**b

***ab

***ab

***ab

***ab

***ab

***ab

100

Table 17: Effect of MREt on Hb, HbA1c and HbA1c /Hb Ratio in Alloxan-Induced Diabetic Mice

Groups Hb (%) HbA1c (%) HbA1c/Hb ratio

Group I 13.55 ± 0.41 6.10 ± 0.39 0.45 ± 0.03

Group II 10.22 ± 0.22 11.83 ± 1.31 1.16 ± 0.14

Group III 10.35 ± 0.38 10.78 ± 0.71 1.04 ± 0.05

Group IV 12.10 ± 0.14***ab

6.7 ± 0.80***ab

0.56 ± 0.07***ab

Group V 12.85 ± 0.54***ab

7 ± 0.47***ab

0.55 ± 0.04***ab

Group VI 12.98 ± 0.15***ab

7.40 ± 0.44***ab

0.57 ± 0.04***ab

Group VII 12.73 ± 0.43***ab

6.38 ± 0.56***ab

0.50 ± 0.05***ab

Values are expressed as mean ± SD (n = 6). ***p < 0.0001, when compared with respective group II (a) and III (b)

101

Table 18: Effect of AqMREt on Hb, HbA1c and HbA1c/Hb Ratio in Alloxan-Induced Diabetic Mice

Groups Hb (%) HbA1c (%) HbA1c / Hb ratio

Group I 13.67 ± 0.70 5.99 ± 1.16 0.44 ± 0.09

Group II 10.14 ± 0.26 10.84 ± 0.69 1.07 ± 0.07

Group III 9.93 ± 0.53 10.55 ± 0.68 1.07 ± 0.10

Group IV 12.36 ± 0.59**ab

6.12 ± 0.80***ab

0.50 ± 0.07***ab

Group V 7.25 ± 0.61***ab

4.52 ± 0.42***ab

0.62 ± 0.03***ab

Group VI 10.43 ± 0.85 3.89 ± 0.63***ab

0.37 ± 0.04***ab

Group VII 10.91 ± 1.75 3.77 ± 0.72***ab

0.36 ± 0.10***ab

Values are expressed as mean ± SD (n = 6). **p < 0.01 and ***p < 0.0001, when compared with respective group II (a) and III (b)

102

3.4.1.6 Effect of Root Extracts on Lipid Profile of Alloxan-Induced Diabetic

Mice

MREt in doses of 10, 30 and 60 mg/kg in test mice induced drastic decrease

(p<0.0001) in serum TC levels from 129.33 -146.19 mg/dl as compared to diabetic and

negative controls which showed TC levels up to 260.42 and 254.11 mg/dl respectively.

Similarly, LDL- and VDL-c levels were also decreased in three of the test groups from

63.46-83.83 mg/kg and 25.65-29.82 mg/kg as compared to the same control groups that

showed elevated levels of same two parameters (p<0.05, p<0.01& p<0.0001). However,

low levels of HDL-c were observed in test groups. Serum TG levels was also reduced up

to 128.27 and 138.75 mg/dl by same extract in doses of 10 and 60 mg/kg in test mice

(p<0.05 & p<0.01) while in dose of 30 mg/kg extract was not found statistically

significant in reducing TG level in its respective test group. Glibenclamide, the positive

control, produced the same picture on TC, TG, HDL-c, LDL-c and VLDL-c in its

respective group (Table 19).

In second animal model, three doses (50, 100 and 150 mg/kg) of AqMREt

produced a gradual decrease (p<0.0001) in a dose-dependent manner in serum TC, TG,

LDL-c, VLDL-c levels and increase in HDL-c level in test groups respectively ranging

from 169.50-128.57, 132.87-90.36, 89.85-43.12, 26.57-18.08 mg/dl and 53.08-83.33

mg/dl as compared to diabetic and negative control groups. Glibenclamide produced

almost the same significant picture of all of these parameters in positive control group

(Table 20).

103

Table 19: Effect of MREt on Lipid Profile of Alloxan-Induced Diabetic Mice

Groups TC (mg/dl) TG (mg/dl) HDL-c (mg/dl) LDL-c (mg/dl) VLDL-c (mg/dl)

Group I 141.65 ± 26.10 148.86 ± 4.16 53.08 ± 16.93 66.56 ± 19.21 29.77 ± 0.83

Group II 260.48 ± 20.36 161.33 ± 7.66 50.69 ± 15.85 175.09 ± 31.52 32.27 ± 1.53

Group III 254.11 ± 29.88 173.50 ± 38.09 45.63 ± 20.01 164.64 ± 39.39 34.70 ± 7.62

Group IV 178.40 ± 31.90***a**b

121.15 ± 6.77*a**b

31.20 ± 7.17 122.98 ± 36.69*a**b

24.23 ± 1.35*a**b

Group V 146.19 ± 20.01***ab

128.27 ± 13.19*a**b

36.71 ± 10.79 83.83 ± 14.18*a**b

25.65 ± 2.64*a**b

Group VI 129.33 ± 38.69***ab

149.12 ± 30.92 36.07 ± 14.11 63.46 ± 47.37 29.82 ± 6.18

Group VII 138.50 ± 18.58***ab

138.75 ± 21.31*b

37.45 ± 8.06 73.30 ± 17.91*b

27.75 ± 4.26*a

Values are expressed as mean ± SD (n = 6). *p < 0.05, **p < 0.01, and ***p < 0.0001, when compared with respective group II (a) and III (b)

104

Table 20: Effect of AqMREt on Lipid Profile of Alloxan-Induced Diabetic Mice

Groups TC

(mg/dl)

TG

(mg/dl)

HDL-c

(mg/dl)

LDL-c

(mg/dl)

VLDL-c

(mg/dl)

Group I 154.15 ± 21.27 147.30 ± 17.35 62.70 ± 14.22 72.47 ± 23.11 29.46 ± 3.45

Group II 255.73 ± 28.32 195.12 ± 16.71 39.02 ± 17.70 181.23 ± 27.64 39.03 ± 3.34

Group III 253.34 ± 27.52 206.70 ± 18.41 34.74 ± 10.84 173.68 ± 36.02 41.34 ± 3.68

Group IV 175.71 ± 32.11***ab

139.55 ± 23.79**a***b

44.04 ± 8.73 104.83 ± 20.85***a**b

27.91 ± 4.76**a***b

Group V 169.50 ± 22.52***ab

132.87 ± 27.23**a***b

53.08 ± 9.77 89.85 ± 18.19***ab

26.57 ± 5.45**a***b

Group VI 146.31 ± 4.26***ab

105.87 ± 29.51***ab

60.18 ± 0.65*ab

65.10 ± 2.54***ab

21.18 ± 5.89***ab

Group VII 128.57 ± 23.80***ab

90.36 ± 14.01***ab

83.33 ± 23.52***ab

43.12 ± 26.11***ab

18.07 ± 2.79***ab

Values are expressed as mean ± SD (n = 6). *p < 0.05, **p < 0.01, and ***p < 0.0001, when compared with respective group II (a) and II (b)

105

3.4.1.7 Effect of Root Extracts on Cardioprotective indices of Alloxan-

Induced Diabetic Mice

TG/ HDL-c ratio was found improved in test groups especially group V and VII

treated MREt (10 & 60 mg/kg). Similarly, improved HDL-c/LDL-c and TC/HDL-c ratios

were observed in test groups from 0.43-1.07 and 4.11-3.8 respectively compared with

diabetic and negative controls which showed low values (0.29 to 0.3) of former and high

values (6.06-6.52) of latter ratio, even glibenclamide found ineffective on all these ratios.

Likewise, the values of AAI were significantly became better from 33 to 53 % in test

groups as compared to diabetic, negative and positive controls that showed the same AAI

from 23 to 24 % (Table 21).

The TG/ HDL-c ratio was found lowered dose-dependently and significantly

(p<0.05, p<0.01 & p<0.0001) from 3.31 to 1.19 in positive control and test groups

treated with glibenclamide and AqMREt in doses of 50, 100 & 150 mg/kg respectively.

Similarly, nice improvement (p<0.05, p<0.01& p<0.0001) was also observed in HDL-

c/LDL-c and TC/HDL-c ratio in test groups treated with MREt from 0.59 -3.01 and 3.22-

1.71 respectively compared with diabetic and negative controls which showed low values

(0.23 to 0.21) of former and high values (7.28 to 7.72) of latter ratio, even glibenclamide

was not found as effective as MREt on both of these ratio. Therefore, AAI was found

much better in test groups (45.31 -69.57%) and positive control (36.73%) compared to

diabetic and negative controls that showed the same AAI from 16.01-17.88% (Table 22).

106

Table 21: Effect of MREt on Cardioprotective and Antiatherogenic Indices of Alloxan-Induced Diabetic Mice

Groups TG/HDL-c Ratio HDL-c/LDL-c Ratio TC/HDL- Ratio AAI (%)

Group I 3.02 ± 0.93 0.91 ± 0.62 2.88 ± 0.96 37.31 ± 12.87

Group II 3.47 ± 1.25 0.30 ± 0.12 5.73 ± 2.66 24.98 ± 9.24

Group III 3.95 ± 1.49 0.29 ± 0.18 6.52 ± 3.09 23.67 ± 13.36

Group IV 4.02 ± 0.84 0.29 ± 0.18 6.06 ± 2.12 23.56 ± 12.78

Group V 3.72 ± 1.13 0.43 ± 0.10 4.11 ± 0.66 33.34 ± 7.67

Group VI 4.56 ± 1.64 1.07 ± 1.16*ab

4.05 ± 1.99 53.29 ± 48.08

Group VII 3.81 ± 0.80 0.54 ± 0.20 3.8 ± 0.78 37.54 ± 9.17


107

Table 22: Effect of AqMREt on Cardioprotective and Antiatherogenic Indices of Alloxan-Induced Diabetic Mice

Groups TG/HDL-c ratio HDL-c/LDL-c ratio TC/HDL-c ratio AAI (%)

Group I 2.41 ± 0.40 0.95 ± 0.41 2.50 ± 0.26 67.99 ± 10.65

Group II 5.66 ± 1.96 0.23 ± 0.12 7.28 ± 2.22 17.88 ± 7.54

Group III 5.38 ± 1.95 0.21 ± 0.10 7.72 ± 1.96 16.01 ± 5.30

Group IV 3.31 ± 1.16*a***b

0.44 ± 0.15 4.16 ± 1.31**ab

36.73 ± 17.23*b

Group V 2.65 ± 1.00**a***b

0.59 ± 0.01 3.22 ± 0.17***ab

45.31 ± 3.43**ab

Group VI 1.76 ± 0.47***ab

0.95 ± 0.01 2.44 ± 0.03***ab

69.57 ± 1.75***ab

Group VII 1.19 ± 0.51***ab

3.01 ± 0.51***ab

1.71 ± 0.52***ab

48.71 ± 28.07**ab


108

3.4.1.8 Effect of Root Extracts on Liver Glycogen and Total Lipids in

Alloxan-Induced Diabetic Mice

Improved liver glycogen amount was found in test groups as well as in positive

control from 1.16 to 1.63 g/g of liver tissue as compared to diabetic and negative control

groups that showed its reduced levels from 0.73 to 0.77 g/g tissue (Figure 16). Whereas

total lipid content in liver tissues was increased in diabetic and negative control groups

(0.12 and 0.15 g/g tissue) as compared to MREt treated mice and positive control group

that showed significantly reduced (p<0.0001) level of total lipids in their livers from

0.05-0.06 g/g (Figure 17).

Similarly, all doses of AqMREt induced a significant increase (p<0.05, p<0.01&

p<0.0001) in glycogen (0.74 to 1.2 g/g) and decrease (p<0.01 &p<0.0001) in total lipid

(0.05 - 0.07g/g) contents were observed in livers of all mice belonging to groups V-VII

as compared to diabetic and negative control groups that showed glycogen from 0.49-

0.49 g/g and total lipids from 0.74 to 1.48 g/g in liver tissue (Figure 16-17).

3.4.1.9 Effect of Root Extracts on Liver-specific Enzyme in Alloxan-Induced

Diabetic Mice

Elevated levels of liver-specific enzyme alanine aminotransferase (ALT) was

observed in both diabetic and negative control groups while glibenclamide and doses of

MREt significantly (p<0.0001) recovered the levels of this enzyme in their respective

groups (Table 23). Likewise, all doses of AqMREt was also found effective (p<0.05 &

p<0.01) in this same aspect as compared to its respective diabetic control groups II & III

(Table 24).

109

Figure 16: Effect of Root Extracts on Glycogen in Liver Tissues of Alloxan-Induced Diabetic Mice


0

0.5

1

1.5

2

2.5


Gly

cog

en (

g/g

) MREt AqMREt

*ab *ab

***ab

*ab

**ab

110

Figure 17: Effect of Root Extracts on Total Lipids in Liver Tissues of Alloxan-Induced Diabetic Mice


0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

0.18


To

tal

lip

ids

(g/g

) MREt AqMREt

***a**b

***ab

***ab

***ab

***ab

**ab

**a***b ***ab

111

Table 23: Effect of MREt on Liver Specific and Antioxidant Enzymes of Alloxan-Induced Diabetic Mice

Groups Treatment ALT (U/L)

Percent Inhibition (%)

CAT SOD

Group I Distilled water (1 ml/kg) 15.63 ± 0.75 36.93 ± 5.99 43.70 ± 10.32

Group II Alloxan (150 mg/kg) 16.93 ± 0.69 73.65 ± 5.50 76.12 ± 12.16

Group III 0.05% DMSO (1 m/kg) 16.26 ± 0.38 70.48 ± 14.37 76.01 ± 3.92


Glibenclamide (5 mg/kg) 9.82 ± 0.21

***ab 44.52 ± 6.69

**a*b 25.26 ± 2.66

***ab


MREt (10 mg/kg) 7.68 ± 0.80

***ab 29 ± 10.68

**ab 42.81 ± 12.24

***ab


MREt (30 mg/kg) 6.75 ± 0.41

***ab 27.50 ± 8.66

**ab 34.29 ± 14.16

***ab


MREt (60 mg/kg) 7.25 ± 0.47

***ab 26.59 ± 15.63

**ab 39.16 ± 5.0

***ab


112

Table 24: Effect of AqMREt on Liver Specific and Antioxidant Enzymes of Alloxan-Induced Diabetic Mice




CAT SOD


Group II Alloxan (150 mg/kg) 61.25 ± 11.15 83.65 ± 5.47 78 ± 14.19


0.05% DMSO (1 ml/kg) 65.50 ± 5.07 82.12 ± 5.72 73.15 ± 11.42


Glibenclamide (5 mg/kg) 41.25 ± 10.73

*a

44.16 ± 15.21

**ab 29.27 ± 12.03

***ab


AqMREt (50 mg/kg) 48.67 ± 10

42.73 ± 12.85

**ab 39.33 ± 4.5

***a**b


AqMREt (100 mg/kg) 44.75 ± 10.62

*b

31.88 ± 10.47

***ab 54.26 ± 1.23

*ab


AqMREt (150 mg/kg) 40.25 ± 10.45

*a**b

44.12 ± 9.19

**ab 30.61 ± 3.26

***ab

113

3.4.1.10 Effect of Root Extracts on Antioxidant Enzymes in Alloxan-Induced

Diabetic Mice

All three doses (10-60 mg/kg) of MREt significantly (p<0.0001, p<0.01 &

p<0.05) decreased the percent inhibition of CAT and SOD in group V, VI and VII as

compared to diabetic (group II) and negative (group III) controls that showed high rate of

percent inhibition of these enzymes. Similar significant (p<0.0001, p<0.01 & p<0.05)

effect was also observed by glibenclamide in group IV on these antioxidant enzymes

(Table 23).

In second model, all three doses (50-150 mg/kg) of AqMREt also significantly

(p<0.0001, p<0.01 & p<0.05) decreased the percent inhibition of CAT and SOD in

group V, VI and VII as compared to their respective diabetic and negative control groups

that showed high percent inhibition of these enzymes. Of course significant (p<0.0001 &

p<0.01) effect was also produced by glibenclamide in group IV on these antioxidant

enzymes (Table 24).

3.4.1.11 Effect of Root Extracts on Blood Profile of Alloxan-Induced

Diabetic Mice

All three doses (10, 30 & 60mg/kg) of MREt accelerated the RBC count

significantly (p<0.0001, p<0.01 & p<0.05) so gradual improvement was observed in test

groups treated with least to high doses of same extract when compared with diabetic and

negative groups which showed reduced levels of RBC. In contrast, WBC count were

decreased significantly (p<0.0001, p<0.01 & p<0.05) while its marked elevated levels

were observed in diabetic and negative controls (Table 25). A significant positive

progress (p<0.0001, p<0.01 & p<0.05) was also observed in PCV, MCV, MCH and

MCHC levels in test groups exponentially as compared to diabetic control groups (Table

25).

In case of AqMREt, all doses (50, 100 & 150 mg/kg) of this extract significantly

improved the RBC count (p<0.0001, p<0.01 & p<0.05) in all three test groups from

4.60-6.20 106/µl when compared with diabetic and negative groups which showed

114

reduced levels of RBC from 2.78-2.99 106/µl while WBC count were gradually and

significantly decreased from 2.90-3.35x103/µl (p<0.01) in test groups. However, diabetic

and negative controls groups showed its elevated levels from 5.11-5.30 x 103/µl (Table

26). A beneficial effect (p<0.01 & p<0.05) of extract was also observed on PCV, MCV,

MCH and MCHC levels in three test groups in comparison of diabetic control groups

(Table 26).

In both animal models, glibenclamide found effective in improving the levels of

RBC, PCV, MCH, and MCHC in positive control group (Table 25-26).

115

Table 25: Effect of MREt on Blood Profile of Alloxan-Induced Diabetic Mice

Groups RBC (106/µl) WBC (103/µl) PCV (%) MCV (fl) MCH (pg) MCHC (g/dl)

Group I 4.85 ± 0.59 3.5 ± 0.94 22.93 ± 5.28 46.20 ± 5.83 17.22 ± 2.19 34.78 ± 4.48

Group II 3.15 ± 0.77 5.08 ± 0.87 18.30 ± 4.01 36.36 ± 7.26 14.49 ± 2.10 20.21 ± 5.54

Group III 3.45 ± 0.70 5.40 ± 0.89 17.93 ± 5.60 36.15 ± 6.47 13.46 ± 2.60 22.76 ± 4.53

Group IV 3.60 ± 0.72 3.08 ± 0.60**ab

23.28 ± 5.86 45.84 ± 3.14*ab

17.94 ± 2.70*b

35.41 ± 4.50***a**b

Group V 4.38 ± 0.57*a

2.88 ± 0.91**a***b

23.91 ± 5.17 52.19 ± 5.97**a***b

16.84 ± 3.77 32.15 ± 3.69**a**b

Group VI 4.68 ± 0.66**a*b

3.55 ± 1.04*a**b

28.01 ± 7.10*ab

53.36 ± 4.51***ab

18.02 ± 3.97*b

34.01 ± 5.36***a**b

Group VII 5.28 ± 0.66***a**b

2.88 ± 0.59**a***b

29.51 ± 8.62*ab

55.13 ± 4.24***ab

19.50 ± 2.78*a

36.89 ± 3.29***ab


11

6

Table 26: Effect of AqMREt on Blood Profile of Alloxan-Induced Diabetic Mice

Groups RBC (106/µl) WBC (103/µl) PCV (%) MCV (fl) MCH (Pg) MCHC (g/dl)

Group I 4.59 ± 0.54 3.67 ± 0.75 23.80 ± 8.77 51.87 ± 10.86 14.99 ± 3.80 35.43 ± 6.62

Group II 2.99 ± 0.51 5.11 ± 1.04 15.82 ± 3.93 30.96 ± 4.75 10.78 ± 0.80 22.89 ± 9.30

Group III 2.78 ± 0.68 5.30 ± 0.95 16.58 ± 9.07 34.37 ± 7.43 10.54 ± 3.08 21.54 ± 7.20

Group IV 3.80 ± 0.28*b

3.22 ± 0.85**ab

24.19 ± 6.12 44.91 ± 11.92*a

17.02 ± 4.26**ab

33.74 ± 5.11*a**b

Group V 4.60 ± 0.73**ab

3.15 ± 1.22**ab

24.90 ± 1.15 54.85 ± 6.18***a**b

16.30 ± 0.92**ab

29.90 ± 1.62*b

Group VI 6.20 ± 0.41***ab

3.35 ± 0.40**ab

32.10 ± 0.69**ab

51.90 ± 2.31**ab

15.80 ± 0.12*a**b

30.50 ± 1.15*b

Group VII 6.08 ± 1.19***ab

2.90 ± 0.35**ab

31.70 ± 8.89**ab

51.45 ± 4.56**ab

15.40 ± 1.04*ab

29.90 ± 0.58*b


117

3.5 Discussion

Alloxan is an oxygenated pyrimidine analogue and the most commonly used

type 1 diabetes inducer in experimental animals (Sun et al., 2008; Azmi and Qureshi,

2012b). This toxicant is meant for permanent destruction of β-cells thus inhibiting the

release of insulin from pancreas and induces hyperglycemia, diabetic dyslipidemia and

oxidative stress via producing reactive oxygen species (Rohilla and Ali, 2012). The most

apparent symptom of type 1 diabetes is the loss of body weight, the same was observed

in alloxan-induced diabetic control mice in the present study that showed rapid reduction

in their body weights. However, this scenario was significantly (p< 0.05) improved in

mice treated with 10 and 30 mg/kg of MREt for 14 days consecutively while dose of 60

mg/kg of same extract found more effective in improving the same physical parameter

(p< 0.01 & p< 0.0001) in its respective group. Similarly, significant improvement (p<

0.05 & p< 0.01) in body weights of test mice treated with three doses of AqMREt of

R.serpentina was also observed. In diabetes, body consume triglycerides in place of

glucose to fulfill its energy requirement this process is also accompanied with loss of

tissue proteins that results in decrease in fat and lean body mass which ultimately leads a

significant fall in total body weight (Bishop et al., 2010; Wolfsthal et al., 2006).

Therefore, both root extracts of R. serpentina may protect tissue proteins by inhibiting

catabolism and help to regain the body weight as glibenclamide did in positive control

group. In addition, no deleterious effect of MREt and AqMREt of R.serpentina was

found by observing no change in wet weights and relative weights of body organs

including kidney, liver, spleen and heart of test mice treated with extracts which

confirmed the non-toxic status of extracts.

Hyperglycemia, the characteristic feature of type 1 diabetes is associated with

the disturbance in glucose uptake and its metabolism due to the insulin deficiency

(Daneman, 2006). Intra-peritoneal injection of alloxan (150 mg/kg) has elevated the

blood glucose level (23-28%) in diabetic control groups after its 72 hours of

administration by interfering the release of insulin from β-cells of pancreas either by

completely or partially destroying these cells. However, doses (10-60 mg/kg) of MREt

118

induced a significant (p< 0.0001) percent glycemic reduction (- 47 to -51 %) in fasting

blood glucose levels of all three alloxan-induced diabetic test groups. Similarly,

AqMREt (50-150mg/kg) also induced significant (p< 0.0001) percent glycemic

reduction (-39 to - 53%) in their respective alloxan-induced diabetic test groups. These

results support previous findings obtained in OGTT that MREt of R.serpentina improves

the glucose tolerance in mice either by inhibiting glucose absorption in intestine or

enhancing glucose utilization in hepatic and peripheral tissues (Azmi and Qureshi,

2012a). In the same alloxan-induced type 1 model, glibenclamide 5 mg/kg, one of the

secretagogues (sulphonylurea) which is responsible for enhancing the insulin release

from functional β-cells of pancreas (Kaku et al., 1995) also found effective and produced

significant percent glycemic reduction in positive control group. This finding indicates

another possible mechanism of antidiabetic action of root extracts that they may has

some pancreatic action. As it may possible that few β-cells remain alive in alloxan-

induced type 1 diabetic mice from which root extracts uplift the insulin release as same

as glibenclamide possibly did in its respective group. In order to prove this claim, serum

insulin levels were estimated in alloxan-induced diabetic test groups treated with MREt

and AqMREt. Interestingly a significant (p< 0.01 & p< 0.0001) increase in serum insulin

levels was observed in test mice treated with each extract in dose-dependent manner as

compared to diabetic control groups that showed very low levels of same hypoglycemic

hormone. The same improvement in serum insulin level observed in glibenclamide

treated positive control group. These results clearly prove that both root extracts of

R.serpentina may have both pancreatic and extra-pancreatic actions as same as the

glibenclamide to normalize blood glucose level.

Hyperglycemia induced glycation plays an important role in gradual development

of diabetic complications (Azmi and Qureshi, 2013). In diabetes, excess blood glucose

non-enzymatically combines with hemoglobin (Hb) to form glycosylated (glycated)

hemoglobin (HbA1c) and elevates its level above 6 % by slightly affecting the level of

total Hb (Silverman et al., 2011). The same was observed in alloxan-induced diabetic

groups that showed elevated amount of HbA1c (10-11%) and decreased amount of total

119

Hb (9-10 %) as compared to normal control group. On the other hand, test doses of

MREt showed a fair control (p< 0.0001) on HbA1c by decreasing its levels up to 6-7%

and improving the total Hb levels from 12.7-12.9%, thus significantly improved HbA1c /

Hb ratio. Similarly, all three doses of AqMREt showed a good (p< 0.0001) control of

HbA1c levels in test groups by bringing its level equals to or less than 4% and side by

side a nice improvement was also brought in levels of total Hb and of course in HbA1c /

Hb ratio in same test groups. Literature evidenced unrecognized mild anemia in diabetic

condition (Thomas et al., 2003). Therefore, the present findings prove that MREt not

only has antidiabetic activity but also has in vivo antiglycation ability by normalizing the

amount of HbA1c which is now the most preferable diagnostic marker for diabetes used

globally.

Elevated levels of serum TC, TG, LDL-c and VLDL-c with decreased serum

HDL-c levels constitute the diabetic dyslipidemia which enhanced the risk of

athrosclerosis and other cardiovascular problems (Bishop et al., 2010). Alloxan-induced

diabetes is accompanied with hypertriglyceridemia and hypercholesterolemia (Qureshi et

al., 2009). The same was observed in alloxan-induced diabetic control groups of present

study which showed elevated levels of serum TC, TG, LDL-c, VLDL-c and low levels of

HDL-c. However, all three doses of MREt found effective (p< 0.0001) in reducing the

levels of TC, TG, LDL-c, VLDL-c and induced slight elevation in HDL-c level in test

groups. Likewise, all doses of AqMREt of R.serpentina exhibited the same

hypolipidemic activity in their respective test groups by decreasing the levels of all

atherosclerotic developing lipids (TC, TG, LDL-c, VLDL-c) while it was found more

efficient in increasing the level of good cholesterol (HDL-c) in test group as compared to

MREt. Both low-density lipoproteins (LDL-c and VLDL-c) are involved in transporting

TC and TG from liver to peripheral tissues and depositing them on the walls of coronary

arteries to form atherosclerotic plaques (Qureshi et al., 2010). Reduced serum levels of

LDL-c and VLDL-c found in test groups treated with root extracts is one of the

beneficial aspects of this present research and proved the anti-atherosclerotic potential of

root extracts while HDL-c involves in reverse transport of cholesterol from peripheral

120

tissues to liver to accelerate its metabolism and excretion (Kwiterovich, 2000). The

hypolipidemic effect of root extracts is secondary to their potent hypoglycemic effect

observed in the present study. Beside this, hypocholesterolemic effect may be due to

inhibiting the activity of 3-hydroxy-3-methyl glutaryl coenzyme A reductase (HMG-

CoA reductase), the rate-limiting enzyme of cholesterol biosynthesis or increase

excretion of cholesterol in bile (Lateef and Qureshi, 2014). On the other hand, the

hypotriglyceridemic effect of root extracts may be due to inhibiting the activity of acetyl

CoA carboxylase (one of the lipogenic enzymes) thus inhibit fatty acid biosynthesis or

enhanced lipolysis by accelerating the activity of hormone-sensitive lipase (Nelson and

Cox, 2008; Bishop et al., 2010).

The significant hypolipidemic activity of MREt and AqMREt of R.serpentina

found in present study was also strengthened by observing the improvement in all cardio-

protective ratios including TG/HDL-c, HDL-c/LDL-c and TC/HDL-c ratios in their

respective treated test groups. The value of TG/HDL-c ratio is preferable less than 4 and

ideal less than 2 whereas HDL-c/LDL-c ratio is preferable greater than 0.3 and ideal

greater than 0.4 (Gaziano et al., 1997). The MREt produced preferable results in case of

TG/HDL-c ratio by decreasing its value ≤ 4 while produced ideal results in case of HDL-

c/LDL-c ratio by increasing its value ≥ 0.4 in test groups as compared to diabetic control

groups. However, both of these ratios are ideally improved by doses of AqMREt in their

respective test groups (p< 0.05, p< 0.01 & p< 0.0001). Past researches described that

these two ratios have remarkable importance and serve as good indicators of

cardiovascular problems and insulin resistance by showing increase in TG/HDL-c and

decrease in HDL-c/LDL-c ratios (Marotta et al., 2010). A significant reduction ≤ 4 by

MREt and dose-dependent gradual reduction ≤ 3 (p< 0.0001) by AqMREt were observed

in coronary risk index (CRI) in term of TC/HDL-c ratio in their respective test groups as

compared to diabetic control groups. Studies proved that CRI showing values greater

than 5 indicates the higher coronary risk while its values less than 3.5 predict safe from

all coronary problems (Barter et al., 2007; Kang et al., 2012). Both root extracts (MREt

& AqMREt) found efficient in reducing the CRI in their respective test groups. These

121

findings are also supported by calculating anti-atherogenic index (AAI) which found

increased from 33-53% in MREt treated test groups and 45-69% (p< 0.05, p< 0.01, p<

0.0001) in AqMREt treated test groups as compared to diabetic control groups.

Therefore, in the present study, both root extracts of R.serpentina found good

hypolipidemic agents that could reduce the life-threatening consequences of diabetic

dyslipidemia induced by type 1 diabetes.

MREt and AqMREt were found efficient in increasing the glycogen content in

liver and decreasing the content of total lipids in same tissue of test groups (p< 0.05, p<

0.01 & 0.0001) as same as glibenclamide in positive control group while diabetic control

groups showed an entirely opposite picture of same parameters. These findings also

support one of the possible antidiabetic actions of root extracts that they could enhance

the uptake of glucose in liver either directly or indirectly by enhancing the insulin

secretion from β-cells of pancreas thereby accelerating the process of glycogenesis and

inhibiting the processes of lipogenesis and hepatic glucose production via inhibiting

glycogen phosphorylase (glycogenolysis) and glucose-6-phosphatase (gluconeogenesis)

activities, thus normalized blood glucose level. Orally administrated drug must pass

through hepatic metabolism therefore should not be toxic to liver as it is the chief site of

all metabolism in body (Bishop et al., 2010; Azmi and Qureshi, 2012b). In this regard,

the level of ALT, the liver-specific enzyme was estimated and found normal in MREt

(p< 0.0001) and AqMREt (p< 0.05 & p< 0.01) treated test groups as compared with

diabetic control groups. The normal ALT level stated as 0-37 U/l and its elevated level

represents hepatic damage (Moss and Henderson, 1996; Reitman and Frankel, 1957).

Therefore, all three doses of each root extract found non-toxic to liver function and safe

for consumption.

Persistent hyperglycemia and diabetic dyslipidemia also accompanied by

increased production of reactive oxygen species (ROS) which in turn involved in

oxidative damage of body tissues including heart, kidneys, eyes, nerves, liver, blood

vessels, etc and become the initiator of diabetic complications (Saba et al., 2010). Body

is God-gifted with antioxidant enzymes system to prevent the toxic effect of these ROS

122

(Seifu et al., 2012; Pham-Huy et al., 2008). In order to evaluate the in vivo antioxidant

activity of root extracts of R.serpentina, the percent inhibition of catalase (CAT) and

superoxide dismutase (SOD) was estimated and interestingly, both MREt and AqMREt

found to uplift the status of these antioxidant enzymes in their respective test groups.

SOD converts superoxide anion into hydrogen peroxide which later hydrolyzes by CAT

into water and oxygen, thereby protecting the body tissues from oxidative damage

induced by free radicals (Mao et al., 1993). Test doses of MREt showed a significant (p<

0.01 & p< 0.0001) decrease in percent inhibition of CAT and SOD when compared with

diabetic control groups which showed high percent inhibition of these enzymes. Almost

same antioxidant activity was also observed by test doses of AqMREt by showing a

significant (p< 0.05, p< 0.01, p< 0.0001) decrease in percent inhibition of CAT and

SOD. Therefore, R.serpentina significantly elevates the CAT and SOD activities thereby

reducing the oxidative stress induced by ROS in diabetes. The antioxidant activity of

root extracts of R.serpentina is supported by a current computational study which

described that some indole alkaloids present in roots of R.serpentina serve as potent

inhibitors of aldose reductase, the key enzyme involved in formation of ROS in diabetic

condition (Pathania et al., 2013).

In diabetes, hyperglycemia induced glycation and oxidative stress also disturb the

osmotic fragility of red blood cells (RBC) thus reduces the half-life (t1/2) of these cells by

subjecting them to hemolysis (Saba et al., 2010). Beside this, studies reported that

erythropoietin (EPO) production from kidneys is also affected in diabetes (Thomas,

2008). EPO is a glycoprotein hormone and controls the RBC production (McGill and

Bell, 2006). In the present study, three doses of each of MREt and AqMREt showed a

gradual and dose-dependent increase (p< 0.05, p< 0.01, p< 0.0001) in RBC count in their

respective test groups as compared to diabetic control groups and confirmed the

hematopoietic effect of R.serpentina which also supports the total Hb improving effect of

same root extracts observed earlier in the present study. On the other hand, the increased

WBC count found in diabetic control groups represents the pancreatic assault induced by

alloxan (Tanko et al., 2011). Administration of MREt and AqMREt significantly (p<

123

0.05, p< 0.01, p< 0.0001) normalized the WBC count in all test groups. Thus reduces the

pancreatic inflammation induced by alloxan. Other hematological parameters including

PCV, MCV, MCH and MCHC are also found to improve (p< 0.05, p< 0.01, p< 0.0001)

in MREt and AqMREt treated test groups as compared to diabetic controls. The decrease

in all these hematological parameters represents the anemia as they reflect the average

Hb concentration per RBC and per given volume of packed red blood cells (Hoffman et

al., 2005). Studies claimed that mild anemia has also been seen in diabetes (Thomas et

al., 2003). These findings enlighten one more potential aspect of this medicinal plant that

it may have hematinic effect in alloxan-induced diabetic mice.

Therefore, the results conclude that MREt and AqMREt of R.serpentina

are powerful antidiabetic agents by showing significant hypoglycemic, antiglycation,

hypolipidemic, antioxidant and hematopoietic effects in alloxan-induced type 1 diabetes

mice. All these effects may be due to the presence of different chemical constituents

especially alkaloids, polyphenolic compounds in same root extracts that observed in the

start of present study. AqMREt found better than MREt of R.serpentina, no doubt its

high doses have been selected from OGTT and used in present study but it was observed

that it has more phenolic content as compared to MREt. On contrary, MREt found rich in

alkaloids and as efficient as AqMREt in its small doses in alloxan-induced type 1

diabetic mice.

124

Chapter 4

Antidiabetic Effect of Root Extracts of

R.serpentina in Fructose-Induced


Chapter 4 Antidiabetic Effect of Root Extracts of

R.serpentina in Fructose-induced Type 2 Diabetic Mice

Model

Background

The current chapter deals with the antidiabetic effect of the methanolic root extract

(MREt) and aqueous methanolic root extract (AqMREt) of R.serpentina on type 2 animal

model, including their dyslipidemia along with the additional therapeutic characteristic.

This chapter describes the materials and method, results and discussion sections of the

above content.


4.1.1 Determination of the Effect of Root Extracts in Type 2 Diabetic Mice

Model

Except Induction of type 2 diabetes in animals and determination of the rate-regulatory

enzyme of cholesterol biosynthesis named 3-hydroxy-3-methyl-glutaryl coenzyme A

(HMG-CoA) reductase activity all the materials and methods was same as used and

described in section 3.1 of chapter 3 previously.

4.1.1.1. Induction of Type 2 Diabetes in Mice

Type 2 diabetes was induced in overnight fasted mice (12-14 hrs) by daily oral

administration of 10% solution of D (-) fructose (1ml/kg) 30 min before providing

standard laboratory diet (Neeharika et al., 2012).

4.1.1.2. Protocol

Overnight fasted mice (12-14 h) were randomly divided into seven groups

(6/group), according to the treatments (Figure 18 and 19). Each treatment was given

orally once in a day for consecutively 14 days. At the end of animal trial, mice were

125

sacrificed to collect whole blood, serum and body tissues including heart, kidneys, liver

and spleen (Neeharika et al., 2012).

126

Figure 18: Effect of MREt in Fructose-Induced Type 2 Diabetic Mice


Group I

Control

(Distilled water

1ml/kg)

10% Fructose solution (1ml/kg) orally for 14 consecutive days

Test Groups

Group V

(MREt 10 mg/kg)

Group IV

Positive Control

(Pioglitazone

15 mg/kg)

Group III

Negative Control

(0.05% DMSO

1ml/kg)

Group II

Diabetic Control

(Distilled water

1ml /kg)

Group VI

(MREt 30 mg/kg)

Group VII

(MREt 60 mg/kg)

Diabetes Induction

127

Figure 19: Effect of AqMREt in Fructose-Induced Type 2 Diabetic Mice

Diabetes Induction


Group I

Control

(Distilled water

1ml/kg)

10% Fructose solution (1ml/kg) orally for 14 consecutive days

Test Groups

Group V

(AqMREt 50 mg/kg)

Group IV

Positive Control

(Pioglitazone 15

mg/kg)

Group III

Negative Control

(0.05 % DMSO

1ml/kg)

Group II

Diabetic Control

(Distilled water

1ml/kg)

Group VI

(AqMREt (100 mg/kg)

Group VII

(AqMREt 150 mg/kg)

128

4.1.3 Determination of Activity of Rate-Regulatory Enzyme of Cholesterol

Biosynthesis

The rate-regulatory enzyme of cholesterol biosynthesis named 3-hydroxy-3-

methyl-glutaryl coenzyme A (HMG-CoA) reductase activity was measured in term of

HMG-CoA / mevalonate ratio in liver tissue (Rao and Ramakrishnan, 1975).

Principle

This is an indirect method used to assess variation in HMG-CoA reductase

activity in liver tissues. HMG-CoA and mevalonate concentrations in the tissue

homogenate are estimated in terms of absorbance and the ratio between the two is taken

as an index of the activity of reductase, which catalyzes the conversion of HMG-CoA to

mevalonate. The ratio increases under conditions in which activity of this enzyme in

liver is reported to decrease (e.g., fasting, cholesterols feedings) and decrease in the

same ratio under conditions in which the activity of this enzyme is reported to increase

(e.g., Triton injection, Phenobarbital treatment).

Reagents

1. Homogenate: 0.1 gm of liver tissue was homogenized with 10 ml of saline arsenate

solution.

2. Sodium Arsenate Solution: 1 gm of sodium arsenate was dissolved in 1 liter of

physiological saline.

3. Dilute Perchloric Acid (PCA): 50 ml perchloric acid was dissolved per liter of

distilled water.

4. Physiological Saline: 0.9 g of sodium chloride (NaCl) was dissolved in 100 ml of

distilled water.

5. Hydroxylamine hydrochloride reagent (2 mol/liter): 138.98 gm hydroxylamine

hydrochloride was dissolved in 1 liter of distilled water.

129

A. Hydroxylamine hydrochloride reagent for Mevalonate (pH: 2.1 or 2.2): equal

volumes of hydroxylamine hydrochloride reagent and water were mixed freshly before

use.

B. Hydroxylamine hydrochloride reagent for HMG-CoA (pH: 5.5-5.9): equal

volumes of hydroxylamine hydrochloride reagent and NaOH solution were mixed freshly

before use.

6. Sodium hydroxide (NaOH; 4.5 mol): 180 gm of NaOH was dissolved in liter of

distilled water.

7. Ferric chloride (FeCl3) reagent: 5.2 g of tricarboxylic acid (TCA) and 10 grams of

ferric chloride were dissolved in 50 ml of 0.65 M HCl and made up the volume to 100

ml with the later.

8. HCl (0.65 mol): 53.6 ml of concentrated HCl was dissolved in 946.4 liter of distilled

water.

Procedure

1. Preparation of Sample

Liver homogenate (10%) was prepared in sodium arsenate (0.1%) solution and

filtered. The filtrate was used for the estimation of HMG-CoA and mevalonate

concentrations.

2. Preparation of tests

Reagent HMG-CoA Mevalonate

Filtrate 1.0 ml 1.0 ml

PCA (diluted) 1.0 ml 1.0 ml

Incubated for 5 min and centrifuged for 15 min at 3000 rpm

Supernatant 1.0 ml 1.0 ml

Hydroxyamine reagent

(2M, pH 5.5) 0.5 ml --

130

Hydroxyamine reagent

(2M, pH 2.1) -- 0.5 ml

Incubated for 5 min at room temperature

FeCl3 reagent 1.5 ml 1.5 ml

Mixed well and incubated for 10 min at room temperature

Absorbance was read at 540 nm against simultaneously treated saline arsenic blank.

Calculation

The ratio of HMG-CoA to mevalonate was calculated (Rao and Ramakrishnan,

1975).

4.2 Results

4.2.1 Effect of Root Extracts on Fructose-Induced (Type 2) Diabetic Animal

Model

4.2.1.1 Effect of Root Extracts on Body Weights of Fructose-Induced

Diabetic Mice

There was marked increment observed in body weights of diabetic and negative

control group as 9.13 and 6.44 % as compared to positive control group (1.6 %).

Whereas, in all three MREt dose treated test group showed gradual improvement in the

body weights like doses 10 and 30 mg/kg of extract showed 4.99 and 2.09% increments

in body weights respectively in their test groups while dose 60 mg/kg of MREt showed

significant (p<0.01 & p<0.05) decrease approximately -3.41% in body weight when

compared with diabetic and negative control groups (Table 27 & Figure 20). Similarly,

AqMREt produced significant improvement (p<0.05) in body weights of test groups in

dose-dependent manner like 50 and 100 mg/kg of same extract showed 6.65 and 2.95%

increase in their test groups while its concentration 150 mg/kg induced a marked decrease

(p<0.001) in body weight approximately -2.49% in its test group as compared to its

respective diabetic control groups (Table 28 & Figure 20).

131

4.2.1.2 Effect of Root Extracts on Wet Organs Weight and their Relative

Weights (Percent Body Weight) in Fructose-Induced Diabetic Mice

The wet organs weights and their relative weights (percent body weights) in all

MREt and AqMREt treated mice were found as same as observed in their respective

control groups including normal, alloxan-induced diabetic, negative and positive control

groups which depict the unaltered and non-deleterious effects of root extracts of R.

serpentina on body organs (Table 29-30).

132

Table 27: Effect of MREt on Body Weights of Fructose-Induced Diabetic Mice

Groups Treatment

Body weights (g)


Group I Distilled water (1 ml/kg) 29.83 ± 1.75 31.84 ± 1.19 + 6.93 ± 5.49

Group II 10% Fructose (1 ml/kg) 27.90 ± 1.96 30.43 ± 2.32 + 9.13 ± 5.51

Group III 10% Fructose (1ml/kg)

+ 0.05% DMSO (1 ml/kg) 29.81 ± 1.76 31.73 ± 1.99 + 6.44 ± 2.70

Group IV 10% Fructose (1ml/kg)

+ Pioglitazone (15 mg/kg) 28.63 ± 1.71 29.10 ± 1.98 + 1.60 ± 1.88

Group V 10% Fructose (1ml/kg)

+ MREt (10 mg/kg) 28.92 ± 2.30 30.34 ± 2.02 + 4.99 ± 2.15

Group VI 10% Fructose (1ml/kg)

+ MREt (30 mg/kg) 28.27 ± 3.10 28.65 ± 1.19 + 2.09 ± 3.59

Group VII 10% Fructose (1ml/kg)

+ MREt (60 mg/kg) 29.57 ± 3.89 28.44± 2.67 -3.41 ± 4.93

**a*b

Values are expressed as mean ± SD (n = 6). *p < 0.05 and **p < 0.01, when compared with respective group II (a) and III (b).

Negative (−)/positive (+) signs represent loss/gain in weights of mice on final day (14th

day).

133

-10

-5

0

5

10

15

20


Bo

dy

wei

gh

t ch

an

ge

(%)

MREt AqMREt

**a*b

**ab **a*b

***ab

Figure 20: Effect of Root Extracts on Percent (%) Body Weight Change of Fructose-Induced Diabetic Mice

Values are expressed as mean ± SD (n = 6). *p < 0.05, **p < 0.01 and *p < 0.0001, when compared with respective group II (a) and III (b). Negative (−)/positive

(+) signs represent loss/gain in body weights of mice on final day (14th

day).

134

Table 28: Effect of AqMREt on Body Weights of Fructose-Induced Diabetic Mice

Groups Treatment

Body weights (g)


Group I Distilled water (1 ml/kg) 30.16 ± 2.63 31.10 ± 2.48 +3.17 ± 2.63

Group II 10% Fructose (1 ml/kg) 28.53 ± 2.35 30.98 ± 1.87 +8.74 ± 2.45


+ 0.05% DMSO (1 ml/kg) 28.24 ± 2.15 30.26 ± 1.89 +7.23 ± 2.25


+ Pioglitazone (15 mg/kg) 28.47 ± 0.66 28.82 ± 0.94 +2.09 ± 1.44

**ab


+ AqMREt (50 mg/kg) 29.24 ± 1.53 31.16 ± 1.16 +6.65 ± 1.75


+ AqMREt (100 mg/kg) 28.78 ± 1.57 29.62 ± 1.51 +2.95 ± 0.54

**a*b


+ AqMREt (150 mg/kg) 29.56 ± 1.72 28.77 ± 0.53 -2.49 ± 4.22

***ab

Values are expressed as mean ± SD (n = 6). *p < 0.05, **p < 0.01, and ***p < 0.0001, when compared with respective group II (a) and III (b). Negative (−)/positive (+)

signs represent loss/gain in weights of mice on final day (14th

day).

135

Table 29: Effect of MREt on Wet and Relative Organs Body Weights of Fructose-Induced Diabetic Mice

Group



Group I 0.39 ± 0.07 1.38 ± 0.14 0.10 ± 0.02 0.09 ± 0.01 1.26 ± 0.21 4.47 ± 0.63 0.31 ± 0.08 0.29 ± 0.06

Group II 0.39 ± 0.04 1.45 ± 0.10 0.11 ± 0.02 0.09 ± 0.02 1.28 ± 0.16 4.81 ± 0.60 0.35 ± 0.08 0.29 ± 0.06

Group III 0.38 ± 0.01 1.46 ± 0.04 0.10 ± 0.02 0.09 ± 0.01 1.20 ± 0.07 4.63 ± 0.30 0.33 ± 0.06 0.29 ± 0.03

Group IV 0.41 ± 0.06 1.38 ± 0.08 0.08 ± 0.03 0.10 ± 0.02 1.40 ± 0.26 4.74 ± 0.24 0.29 ± 0.09 0.35 ± 0.08

Group V 0.38 ± 0.06 1.53 ± 0.06 0.11 ± 0.01 0.10 ± 0.02 1.26 ± 0.28 5.06 ± 0.47 0.35 ± 0.03 0.32 ± 0.08

Group VI 0.37 ± 0.02 1.42 ± 0.08 0.09 ± 0.02 0.09 ± 0.01 1.29 ± 0.04 4.97 ± 0.41 0.31 ± 0.08 0.32 ± 0.02

Group VII 0.41 ± 0.04 1.47 ± 0.15 0.11 ± 0.03 0.10 ± 0.01 1.46 ± 0.16 5.18 ± 0.62 0.37 ± 0.10 0.34 ± 0.04

Values are expressed as mean ± SD (n = 6). Values in group IV, V, VI and VII were found non-significant when compared with respective group II and III.

136

Table 30: Effect of AqMREt on Wet and Relative Organs Body Weights of Fructose-Induced Diabetic Mice

Group



Group I 0.44 ± 0.08 1.39 ± 0.13 0.10 ± 0.02 0.08 ± 0.02 1.41 ± 0.30 4.48 ± 0.49 0.32 ± 0.06 0.25 ± 0.06

Group II 0.46 ± 0.05 1.43 ± 0.10 0.08 ± 0.01 0.09 ± 0.01 1.48 ± 0.23 4.61± 0.11 0.27 ± 0.03 0.28 ± 0.03

Group III 0.44 ± 0.07 1.39 ± 0.12 0.10 ± 0.03 0.09 ± 0.02 1.47 ± 0.31 4.59 ± 0.46 0.31 ± 0.09 0.29 ± 0.06

Group IV 0.43 ± 0.06 1.42 ± 0.06 0.09 ± 0.02 0.10 ± 0.03 1.47 ± 0.22 4.88 ± 0.37 0.32 ± 0.06 0.33 ± 0.10

Group V 0.43 ± 0.06 1.37 ± 0.03 0.08 ± 0.02 0.08 ± 0.02 1.37 ± 0.20 4.38 ± 0.15 0.27 ± 0.06 0.27 ± 0.06

Group VI 0.43 ± 0.07 1.45 ± 0.05 0.10 ± 0.01 0.09 ± 0.01 1.44 ± 0.21 4.92 ± 0.27 0.34 ± 0.05 0.32 ± 0.04

Group VII 0.45 ± 0.03 1.34 ± 0.12 0.09 ± 0.01 0.09 ± 0.02 1.57 ± 0.10 4.65 ± 0.50 0.30 ± 0.04 0.30 ± 0.07

Values are expressed as mean ± SD (n = 6). Values in group IV, V, VI and VII were found non-significant, when compared with respective group II and III

137

4.2.1.3 Effect of Root Extracts on Blood Glucose Level in Fructose-Induced

Diabetic Mice

At 14th

day all the three doses of MREt (10, 30 & 60 mg/kg) showed significant

((p<0.01 & p<0.0001) reduction in blood glucose level by inducing decrease percent

glycemic change in their respective group like 25.38%, 18.64% and 17.40% while similar

pattern was also observed in positive control group (pioglitazone 15 mg/kg) as compared

to diabetic and negative control groups which showed marked increase in percent

glycemic change as 80.06% and 76.88% respectively (Table 31 & Figure 21).

In case of AqMREt, a gradual significant (p<0.0001 & p<0.0001) decrement was

observed in blood glucose level of test groups in comparison with respective diabetic and

negative control groups like test groups treated with doses of extract (50, 100 and 150

mg/kg), significantly (p<0.01, 0.0001) showed percent glycemic change as +48.43%,

+3.95% and + 9.65% as compared to both diabetic and negative control groups that

showed high percent glycemic increment as +114.14 and +95.89%. Similarly,

pioglitazone 15 mg/kg also found effective (p<0.05 & p<0.0001) in controlling the

percent glycemic change by showing only +24.15% increase in blood glucose level of

mice in positive control group (Table 32 & Figure 21).

138

Table 31: Effect of MREt on Blood Glucose Levels of Fructose-Induced Diabetic Mice



Group I Distilled water (1 ml/kg) 104.92 ± 14.33 103.43 ± 8.85 -0.78 ± 7.41

Group II 10% Fructose (1ml/kg) 103.33 ± 5.44 185.83 ± 4.23 +80.06 ± 5.73

Group III

10% Fructose (1ml/kg)

+ 0.05% DMSO (1 ml/kg) 99 ± 7.26 176 ± 39.66 +76.88 ± 32.69

Group IV


+ Pioglitazone (15 mg/kg) 95.67 ± 20.01 116.33 ± 13.10

***ab +24.91 ± 25.63

***a**b

Group V


+ MREt (10 mg/kg) 97.33 ± 9.29 122 ± 15.94

***ab +25.38 ± 11.70

***a**sb

Group VI


+ MREt (30 mg/kg) 99 ± 6.68 116.75 ± 6.55

***ab +18.64 ± 14.28

***ab

Group VII


+ MREt (60 mg/kg) 95 ± 10.68 110.33 ± 3.40

***ab +17.40 ± 15.26

***ab

Values are expressed as mean ± SD (n = 6). **p < 0.01, and ***p < 0.0001, when compared with respective group II (a) and III (b). Negative

(−)/positive (+) signs represent decrease/increase in blood glucose level of mice on final day (14th

day).

139

Figure 21: Effect of Root Extracts on Percent (%) Glycemic Change of Fructose-Induced Diabetic Mice

Values are expressed as mean ± SD (n = 6). *p < 0.05, **p < 0.01 and*p < 0.0001, when compared with respective group II (a) and III (b). Negative

(−)/positive (+) signs represent decrease/increase in blood glucose level of mice on final day (14th

day).

-50

0

50

100

150

200

250


Gly

cem

ic c

ha

ng

e (%

)

MREt AqMREt

***a**b ***a**b

***ab ***ab **a*b

**ab **a*b

140

Table 32: Effect of AqMREt on Blood Glucose Levels of Fructose-Induced Diabetic Mice



Group I Distilled water (1 ml/ kg) 98.75 ± 20.84 99.75 ± 13.45 +4.94 ± 27.28

Group II 10% Fructose (1 ml/kg) 99.75 ± 25.77 197.25 ± 48.84 +114.14 ± 102.97

Group III


+ 0.05% DMSO (1 ml/kg) 99.25 ± 15.11 190.50 ± 9.15 +95.89 ± 35.01



***ab +24.15 ± 28.62

**a*b


+ AqMREt (50 g/kg) 86.25 ± 15.92 128 ± 25.15

**ab + 48.43 ± 10.58


+ AqMREt (100 g/kg) 111.33 ± 9.57 115.67 ± 10.66

***ab +3.95 ± 5.24

**ab


+ AqMREt (150 g/kg) 104.33 ± 19.14 109.50 ± 9.18

***ab +9.65 ± 23.39

**a*b


Negative (−)/positive (+) signs represent decrease/increase in blood glucose level of mice on final day (14th

day).

141

4.2.1.4 Effect of Root Extracts on Serum Insulin Level and Fasting Insulin

Resistance index (FIRI) in Fructose-Induced Diabetic Mice

In MREt dose treated mice (group V, VI & VII), a dose-dependent gradual

decrease in serum insulin level was observed from 0.87 to 0.43 µU/ml as compared to

diabetic and negative control mice belongs to group II and III respectively (p<0.01 &

p<0.0001). Even the highest dose of extract 60mg/kg showed serum insulin level as same

as showed by control group (group I). Similarly, pioglitazone found effective (p<0.0001)

in this aspect (Table 33). As a result of which FIRI also found improved

(p<0.01&p<0.0001) in all extract and pioglitazone treated mice (group VI, V, VI and

VII) mice as compared to diabetic control groups (Table 33). On contrary, increased

serum insulin level was found in all test groups treated with AqMREt as same as

observed in diabetic and negative control groups. However, positive control showed

marked and significant (p<0.01) decreased as compared with diabetic controls (Table

34). Similarly, FIRI was also not improved in test groups, however this situation

appeared opposite in positive control group (Table 34).

142

Table 33: Effect of MREt on Serum Insulin and FIRI of Fructose-Induced Diabetic Mice

Groups Treatment Serum Insulin (µU/ml) FIRI

Group I Distilled water (1 ml/ kg) 0.48 ± 0.03 1.97 ± 0.27

Group II 10% Fructose (1 ml/kg) 1.81 ± 0.75 13.48 ± 5.67

Group III


+ 0.05% DMSO (1 ml/kg) 1.86 ± 0.78 12.92 ± 5.64


+ Pioglitazone (15 mg/kg) 0.19 ± 0.02

***ab 0.89 ± 0.17

***ab


+ MREt (10 mg/kg) 0.87 ± 0.31

**ab 4.35 ± 1.92

**ab


+ MREt (30 mg/kg) 0.71 ± 0.48

**ab 3.30 ± 2.14

***ab


+ MREt (60 mg/kg) 0.43 ± 0.20

***ab 1.93 ± 0.94

***ab


143

Table 34: Effect of AqMREt on Serum Insulin and FIRI of Fructose-Induced Diabetic Mice

Groups Treatment Serum Insulin (µU/ml) FIRI

Group I Distilled water (1 ml/kg) 0.49 ± 0.16 1.89 ± 0.38

Group II 10% Fructose (1ml/kg) 1.80 ± 0.51 14.08 ± 4.51

Group III


+ 0.05% DMSO (1 ml/kg) 1.83 ± 0.43 14 ± 3.61

Group IV 10% Fructose 1ml/kg

+ Pioglitazone (15 mg/kg) 0.22 ± 0.14

**ab 0.96 ± 0.53

***ab


+ AqMREt (50 mg/kg) 1.42 ± 0.56 7.39 ± 3.5

*ab


+ AqMREt (100 mg/kg) 2.94 ± 0.01

*ab 13.60 ± 1.29


+ AqMREt (150 mg/kg) 2.41 ± 0.44 10.56 ± 2.34


144

4.2.1.5 Effect of Root Extracts on Glycosylated Hemoglobin (HbA1C)/ Total

Hemoglobin (Hb) Ratio in Fructose-Induced Diabetic Mice

There was a marked increase was found in HbA1c and decrease in total Hb levels

up to 7 % in diabetic and negative control groups. These parameters were found

improved (p<0.01 & p<0.0001) in MREt treated group V, VI and VI and pioglitazone

treated group IV like HbA1c appeared from 4.25 -5.6 % and Hb from 7-9.6%. Therefore,

HbA1c / Hb ratio was also improved in all test groups (Table 35). Similarly, all doses of

AqMREt were also found effective in controlling the HbA1c (p<0.0001) and total Hb

levels thus improved (p<0.05, p<0.01 & p<0.0001) HbA1c / Hb ratio in their test groups

as compared to their respective diabetic control groups (Table 36).

4.2.1.6 Effect of Root Extracts on Lipid Profile of Fructose-Induced Diabetic

Mice

MREt in doses of 10, 30 and 60 mg/kg in test mice induced significant (p<0.01)

decrease in serum TC levels up to 176.73, 163.50 and 165.75mg/dl respectively as

compared to diabetic and negative controls which showed same parameter up to 199.63

and 237.80 mg/dl respectively even pioglitazone treated positive group showed 193.50

mg/dl TC as compared to same diabetic controls. Significant (p<0.01 & p<0.0001)

decrease was also found in serum TG levels up to 178.14, 144.46 and 146.66 mg/dl in

test group V, VI and VII treated with extract (10, 30 and 60 mg/kg) respectively as

compared to diabetic (group II) and negative (group III) controls that showed same

parameter up to 261.51 and 219.54 mg/dl. Similarly, LDL-c levels in test mice were

significantly decreased (p<0.01 & p<0.0001) up to 90.30, 57.61 and 23.25 mg/dl and

also VLDL-c levels (p<0.01& p<0.0001) as 35.63, 28.89 and 29.33 mg/dl in MREt dose

treated mice. However, HDL-c level showed gradual increase in test groups with respect

to increase in doses of extract as 50.82, 77 and 113.17 mg/dl. Interestingly, pioglitazone

was not found as effective as MREt in improving lipid profile in positive control group

(Table 37).

145

In case of AqMREt, its doses 50, 100 and 150 mg/kg induced a significant

(p<0.05, p<0.01 & p<0.0001) and dose-dependent gradual decrease in serum TC

(183.68, 158.56 & 127.54 mg/dl) and TG (152.62, 130.02 & 107.22 mg/dl) levels in

their respective test groups V, VI and VII as compared to diabetic and negative controls

which showed TC levels from 200-239 mg/dl and TG from 221-262 mg/dl. Similarly,

AqMREt significantly reduced the levels of LDL-c (p<0.05) and VLDL-c (p<0.01 &

p<0.0001) in test groups. However, same extract significantly increased (p<0.01 &

p<0.0001) the HDL-c levels in all test group mice from 68 -90 mg/dl when compared

with diabetic and negative control groups (Table 38).

4.2.1.7 Effect of Root Extracts on Cardioprotective indices in Fructose-


The TG/HDL-c, HDL-c/LDL-c and TG/HDL-c ratios were significantly (p<0.05)

and dose-dependently improved in MREt treated groups V, VI & VII as compared to

diabetic and negative control groups. This beneficial effect of extract has also elevated

(p<0.05 & p<0.0001) AAI in all three test groups (Table 39).

As compared to MREt, AqMREt of R.serpentina found more effective in

improving (p<0.05 & p<0.0001) TG/HDL-c, HDL-c/LDL-c and TG/HDL-c ratios in test

groups treated with 50, 100 & 150mg/kg of same extract, thus significantly induced rise

in AAI of same test groups as compared to diabetic and negative control groups (Table

40). Unfortunately, pioglitazone was not found effective in improving cardioprotective

ratios which resulted in decreased AAI in positive control group (Table 39-40).

146

Table 35: Effect of MREt on Hb, HbA1c and HbA1c / Hb ratio of Fructose-Induced Diabetic Mice


Group I 12.58 ± 1.36 6.13 ± 0.72 0.49 ± 0.06

Group II 7.55 ± 0.06 7.13 ± 0.88 0.95 ± 0.12

Group III 7.15 ± 1.29 6.76 ± 1.02 0.96 ± 0.27

Group IV 5.27 ± 0.56**ab

5.68 ± 0.31**ab

1.08 ± 0.05

Group V 7.63 ± 0.26 4.35 ± 0.16***ab

0.57 ± 0.02***ab

Group VI 9.6 ± 1.25**ab

4.25 ± 0.20***ab

0.45 ± 0.05***ab

Group VII 9.13 ± 0.85*a**b

4.80 ± 0.41***ab

0.53 ± 0.02***ab


147

Table 36: Effect of AqMREt on Hb, HbA1c and HbA1c / Hb Ratio of Fructose-Induced Diabetic Mice


Group I 12.52 ± 1.27 5.99 ± 1.17 0.49 ± 0.13

Group II 7.31 ± 0.73 7.35 ± 0.72 1.02 ± 0.17

Group III 7.43 ± 1.46 6.76 ± 0.98 0.95 ± 0.26

Group IV 5.93 ± 0.76 5.84 ± 0.41**a

0.99 ± 0.08

Group V 7.77 ± 3.02 4.54 ± 0.57***ab

0.67 ± 0.35*a

Group VI 9.10 ± 0.74 4.51 ± 0.22***ab

0.50 ± 0.05**ab

Group VII 10.13 ± 1.70*ab

4.26 ± 0.40***ab

0.43 ± 0.10***a**b


148

Table 37: Effect of MREt on Lipid Profile of Fructose-Induced Diabetic Mice

Groups TC

(mg/dl)

TG

(mg/dl)

HDL-c

(mg/dl)

LDL-c

(mg/dl)

VLDL-c

(mg/dl)

Group I 139.74 ± 28.64 140.21 ± 14.63 54.52 ± 15.18 65.22 ± 19.42 28.04 ± 2.92

Group II 199.63 ± 42.71 261.51 ± 27.22 58.16 ± 28.20 94.64 ± 58.99 52.30 ± 5.44

Group III 237.80 ± 42.31 219.54 ± 62.85 61.25 ± 10.56 132.64 ± 36.59 43.91 ± 12.57

Group IV 193.50 ± 34.42 167.74 ± 15.43***a*b

43.37 ± 14.80 115.47 ± 38.77 33.55 ± 3.09***a*b

Group V 176.73 ± 16.69**b

178.14 ± 21.17**a

50.82 ± 3.07 90.30 ± 9.50 35.63 ± 4.23**a

Group VI 163.50 ± 25.48**b

144.46 ± 24.55***a**b

77 ± 11.11 57.61 ± 11.45**b

28.89 ± 4.91***a**b

Group VII 165.75 ± 7.76**b

146.66 ± 12.49***a**b

113.17 ± 6.98***ab

23.25 ± 5.61**a***b

29.33 ± 2.50***a**b


149

Table 38: Effect of AqMREt on Lipid Profile of Fructose-Induced Diabetic Mice

Groups TC

(mg/dl)

TG

(mg/dl)

HDL-c

(mg/dl)

LDL-c

(mg/dl)

VLDL-c

(mg/dl)

Group I 138.70 ± 18.82 139.58 ± 9.31 54.99 ± 18.15 64.39 ± 20.30 27.92 ± 1.86

Group II 200. 65 ± 40.16 262.12 ± 27.59 54.80 ± 24.79 93.30 ± 18.74 52.42 ± 5.52

Group III 238.65 ± 29.59 221.96 ± 48.80 48.15 ± 13.38 108.79 ± 22.99 44.39 ± 9.76

Group IV 190.99 ± 29.26*b

163.33 ± 20.42***a**b

51.67 ± 13.82 97.56 ± 28.33 32.67 ± 4.08***a**b

Group V 183.68 ± 7.22**b

152.62 ± 0.74***a**b

68.97 ± 6.97 83.89 ± 0.07 30.52 ± 0.15***a**b

Group VI 158.56 ± 18.78*a***b

130.92 ± 7.80***ab

90.07 ± 8.79**ab

42.55 ± 28.50*b

26.18 ± 1.56***ab

Group VII 127.14 ± 34.36**a***b

107.22 ± 26.93***ab

81.67 ± 18.55*a**b

66.53 ± 83.73 21.43 ± 5.37***ab


150

Table 39: Effect of MREt on Cardioprotective and Antiatherogenic Indices of Fructose-Induced Diabetic Mice

Groups HMG CoA/

Mevalonate Ratio TG/HDL-c Ratio HDL-c/LDL-c Ratio TC/HDL-c Ratio AAI (%)

Group I 1.24 ± 0.24 2.69 ± 0.58 0.93 ± 0.55 2.72 ± 0.82 84.32 ± 9.3

Group II 0.46 ± 0.18 5.92 ± 1.68 0.67 ± 0.13 4.09 ± 1.94 40.12 ± 17.05

Group III 0.65 ± 0.07 3.56 ± 0.74 0.49 ± 0.17 3.93 ± 0.73 34.97 ± 7.81

Group IV 0.81 ± 0.24 4.26 ± 1.56 0.42 ± 0.22 4.91 ± 2.13 24.06 ± 6.90

Group V 0.82 ± 0.25 3.50 ± 0.23 0.57 ± 0.09 3.50 ± 0.54 41.55 ± 8.64

Group VI 1.08 ± 0.14*a

1.89 ± 0.33**a

1.35 ± 0.16*a

2.12 ± 0.14*ab

90.08 ± 11.57*ab

Group VII 1.63 ± 0.27***a**b

1.31 ± 0.19**a

5.10 ± 1.36***ab

1.48 ± 0.05**ab

216.66 ± 25.76***ab


151

Table 40: Effect of AqMREt on Cardioprotective and Antiatherogenic Indices of Fructose-Induced Diabetic Mice

Groups HMG Co-A/

Mevalonate Ratio

TG/HDL-c Ratio HDL-c /LDL-c Ratio TC/HDL-c Ratio AAI (%)

Group I 1.23 ± 0.22

2.70 ± 0.65 0.96 ± 0.61 2.72 ± 0.83 85.23 ± 12.23

Group II 0.48 ± 0.14

5.74 ± 2.90 0.58 ± 0.20 4.25 ± 1.88 41.64 ± 26.57

Group III 0.65 ± 0.10

4.67 ± 0.42 0.47 ± 0.19 5.20 ± 1.40 25.81 ± 8.46

Group IV 0.85 ± 0.19*a

3.37 ± 1.12*a

0.55 ± 0.15 3.90 ± 1.22 39.57 ± 17.10

Group V 1.02 ± 0.10***a*b

2.23 ± 0.25**a*b

0.83 ± 0.09 2.67 ± 0.18*a**b

60.04 ± 6.19

Group VI 1.19 ± 0.22***ab

1.46 ± 0.12***a**b

5.81 ± 2.02**ab

1.80 ± 0.39**a***b

164.84 ± 8.5***ab

Group VII 1.34 ± 0.08***ab

1.41 ± 0.54***a**b

5.22 ± 1.35*ab

1.55 ± 0.17**a***b

198.94 ± 5.9***ab


152

4.2.1.8 Effect of Root Extracts on 3-Hydroxy-3-Methyl-Glutaryl Coenzyme A

(HMG-CoA) Reductase in Fructose-Induced Diabetic Mice

All three doses (10-60 mg/kg) of MREt inhibited the activity of HMG-CoA

reductase by showing significant improvement (p<0.0001, p<0.01 & p<0.05) in HMG

Co-A/Mevalonate ratio in all test groups. Similarly, all three doses (50-150 mg/kg) of

AqMREt were also found effective (p<0.0001 & p<0.05) in inhibiting the activity of

same reductase by improving HMG Co-A/Mevalonate ratio in their respective test

groups. However, pioglitazone was not found as effective as both root extracts (Table 39-

40).

4.2.1.9 Effect of Root Extracts on Liver Glycogen and Total Lipids in

Fructose-Induced Diabetic Mice

The liver glycogen amount was found improved in MREt treated mice belong to

group V, VI & VII and in pioglitazone treated mice (group IV) from 0.52-0.86 g/g of

tissue while total lipid content was found decreased (p<0.01 &p<0.0001) from 0.06-0.04

g/g of tissue in same test and positive control groups as compared to diabetic and

negative control groups that showed decreased amount of glycogen from 0.49 to 0.56 g/g

and increased amount of total lipids 0.11 g/g of tissue (Figure 22-23). However, all doses

of AqMREt found more effective in improving (p<0.01) liver glycogen from 0.60-1.20

g/g of tissue in their respective test groups and decreasing (p<0.05 & p<0.01) total lipids

from 0.07-0.05 g/g of tissues in same test groups as compared to their respective diabetic

control groups (Figure 22-23).

153

Figure: 22: Effect of Root Extracts on Glycogen in Liver Tissues of Fructose-Induced Diabetic Mice

Values are expressed as mean ± SD (n = 6). **p < 0.01, when compared with respective group II (a) and III (b)

0

0.5

1

1.5

2

2.5


Gly

cog

en (

g/g

) MREt AqMREt

**ab

**ab

154

Figure 23: Effect of Root Extracts on Total Lipids in Liver Tissues of Fructose-Induced Diabetic Mice

Values are expressed as mean ± SD (n = 6). *p < 0.05, **p < 0.01 and ***p < 0.0001, when compared with respective group II (a) and III (b)

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16


To

tal

lip

ids

(g/g

) MREt AqMREt

***ab

**ab ***ab

***ab

*a**b

*b

*b *a**b

155

4.2.1.10 Effect of Root Extracts on Liver-specific Enzyme in Fructose-


Normal levels of liver-specific enzyme ALT was found in mice belong to groups

treated with pioglitazone, doses of MREt and AqMREt while elevated levels of same

enzyme were observed in both diabetic control groups including II and III (Table 41-42).

4.2.1.11 Effect of Root Extracts on Antioxidant Enzymes in Fructose-Induced

Diabetic Mice

The decreased percent inhibition of CAT from 40-55% and SOD from 38-63%

was observed in positive control and test groups treated pioglitazone and doses (10-60

mg/kg) of MREt as compared to diabetic and negative control groups (group II & III) that

showed high percent inhibition of these enzymes up 78 and 83% respectively (Table 41).

Similarly, all three doses (50-150 mg/kg) of AqMREt significantly (p<0.0001, p<0.001

&p<0.05) decreased the percent inhibition of CAT from 42-58% and SOD from 33-67%

in their respective test groups (Table 42).

156

Table 41: Effect of MREt on Liver Specific and Antioxidant Enzymes of Fructose-Induced Diabetic Mice



CAT SOD


Group II 10% Fructose (1 ml/kg) 56.63 ± 7.27 78.77 ± 3.01 81.90 ± 4.73

Group III 10% Fructose (1 ml/kg)

+ 0.05% DMSO (1 ml/kg) 51 ± 5.35 78.32 ± 6.64 83.69 ± 4.39

Group IV 10% Fructose (1 ml/kg)


*ab 42.11 ± 18.42

**ab

Group V 10% Fructose (1 ml/kg)

+ MREt (10 mg/kg) 53 ± 8.12 55.21 ± 10.73

*ab 63.73 ± 12.34

Group VI 10% Fructose (1 ml/kg)

+ MREt (30 mg/kg) 48.75 ± 6.98 49.71 ± 10.91 35.43 ± 9.73

***ab

Group VII 10% Fructose (1 ml/kg)

+ MREt (60 mg/kg) 45.50 ± 7.77 43.70 ± 12.07

*ab 38.67 ± 4.54

***ab


157

Table 42: Effect of AqMREt on Antioxidant Enzymes of Fructose-Induced Diabetic Mice


Groups Treatment ALT (U/L) Percent Inhibition (%)

CAT SOD

Group I Distilled water (1 ml/ kg) 33.27 ± 5.13 29.79 ± 12.05 38.15 ± 8.5

Group II 10% Fructose (1 ml/kg) 61.89 ± 7.43 77.76 ± 12.26 78.65 ± 9.16


+ 0.05% DMSO (1 ml/kg) 53.87 ± 8.58 77.14 ± 12.19 74.53 ± 18.40

Group IV 10% Fructose (1 ml/kg)


***ab 37.14 ± 14.12

**a*b

Group V 10% Fructose (1 ml/kg)

+ AqMREt (50 mg/kg) 48 ± 3.25 58.18 ± 10.70

*a 67.87 ± 13.52

Group VI 10% Fructose (1 ml/kg)

+ AqMREt (100 mg/kg) 44.67± 4.98

*a 42.15 ± 10.79

**ab 33.83 ± 10.59

**ab

Group VII 10% Fructose (1 ml/kg)

+ AqMREt (150 mg/kg) 38.33 ± 4.78

**a*b 42.40 ± 11.72

**ab 51.99 ± 12.01

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4.2.1.12 Effect of Root Extracts on Blood Profile of Fructose-Induced

Diabetic Mice

Parameters of blood profile including RBC, WBC, PCV, MCV, and MCHC were

significantly improved (p<0.05) in test groups treated with 30 and 60 mg/kg of MREt

when compared with diabetic and negative control groups (Table 43). Beside this, all

three doses of AqMREt of R.serpentina have more significant impact on RBC count

(p<0.05 & p<0.001) and showed gradual improvement in same count like 3.12, 4.39 and

5.29 x 106µ/l in their respective test groups when compared with diabetic and negative

groups which showed reduced levels of same count from 2.78-3.14 x 106µ/l. In contrast,

WBC count was decreased significantly (p<0.05) with respect to increase in dose

magnitude as 3.17, 1.83 and 1.27 x 103µ/l while their elevated levels were observed in

diabetic and negative controls. Similarly, PCV (p<0.01), MCV (p<0.0001), MCH and

MCHC (p<0.05) levels were significantly and gradually improved in all AqMREt treated

test groups (Table 44).

159

Table 43: Effect of MREt on Blood Profile of Fructose-Induced Diabetic Mice


Group I 4.10 ± 0.42 3.18 ± 0.50 24.13 ± 7.50 41.41 ± 7.75 18.78 ± 2.90 37.41 ± 6.19

Group II 3.16 ± 0.18 2.08 ± 0.30 13.83 ± 2.80 37.53 ± 5.04 22.89 ± 8.48 64.70 ± 8.09

Group III 3.41 ± 0.65 3.60 ± 0.86 14.85 ± 8.07 36.30 ± 8.76 20.76 ± 7.79 50.75 ± 16.58

Group IV 3.01 ± 0.48 2.51 ± 0.31 *b

17.46 ± 1.38 36.17 ± 15.88 19.49 ± 7.80 39.94 ± 10.05

Group V 2.71 ± 0.77 1.30 ± 0.67***b

14.07 ± 3.81 52.34 ± 4.89*ab

22.50 ± 3.02 43.67 ± 9.78

Group VI 5.01 ± 0.46***a**b

1.85 ± 0.82**b

25.83 ± 3.74*ab

54.78 ± 4.62**ab

24.30 ± 0.71 46.47 ± 1.47

Group VII 3.34 ± 0.72 0.95 ± 0.53*a***b

20.86 ± 7.39 55.28 ± 4.78**ab

24.65 ± 0.86 48.70 ± 5.47


160

Table 44: Effect of AqMREt on Blood Profile of Fructose-Induced Diabetic Mice


Group I 4.19 ± 1.06 3.06 ± 0.49 23.20 ± 6.07 41.35 ± 15.01 21.65 ± 6.03 37.95 ± 4.95

Group II 2.78 ± 0.79 3.03 ± 0.81 14.78 ± 3.26 35.76 ± 9.51 26.92 ± 7.35 58.83 ± 9.67

Group III 3.14 ± 0.96 2.80 ± 1.54 15.38 ± 5.77 37.46 ± 12.46 21.64 ± 12.71 50.52 ± 16.55

Group IV 3.48 ± 0.58 2.38 ± 0.39 18.98 ± 4.09 35.24 ± 17.11 21.56 ± 3.20 36.10 ± 6.42

Group V 3.12 ± 1.60 3.17 ± 0.91 14.27 ± 7.07 46.33 ± 1.70 28.07 ± 6.68 60.60 ± 14.09

Group VI 4.39 ± 0.80*a

1.83 ± 0.93 18.40 ± 6.02 41 ± 6.16 30.30 ± 3.83 64.73 ± 17.78

Group VII 5.29 ± 1.02**ab

1.27 ± 0.63*ab

27.83 ± 5.93**ab

52.33 ± 1.70*a

32.27 ± 1.32 65.83 ± 3.76

Values are expressed as mean ± SD (n = 6). *p < 0.05 and **p < 0.01 , when compared with respective group II (a) and III (b)

161

4.3 Discussion

Type 2 diabetes constitutes more than 90% cases of total diabetic population

globally (Schellenberg et al., 2013). It is characterized by decrease insulin secretion from

β-cells of pancreas or insulin resistance in which insulin is secreted normally but cannot

bind with its receptor present on target tissues like liver, muscles and adipose tissues.

This resistance may occur due to abnormally synthesized receptor or masking of

receptors by fatty deposits that lead to hyperglycemia and hyperinsulinemia (Quan et al.,

2013; Patel et al., 2013). Genetic or acquired obesity plays an important role in the

development of insulin resistance, thus enhance the prevalence of type 2 diabetes in all

over the world. Among all, life-style and diet pattern (especially high-fat and high

carbohydrate diets) are the chief initiators of obesity and insulin resistance (Singh, 2011;

Schellenberg et al., 2013). Therefore, after having significant results in alloxan-induced

type 1 diabetic mice model, the study has been designed to investigate the effect of

MREt and AqMREt of R.serpentina in fructose-induced insulin resistance type 2 diabetic

mice models. This is again the new aspect of antidiabetic activity of R.serpentina.

Fructose is a ketohexose, abundantly present in plant sources and commonly referred as

fruit sugar (Feinman and Fine, 2013). Commercially, high fructose corn syrup (HFCS) is

used as an artificial sweetener. However, studies described that its high intake induces

hypertriglyceridemia that gradually and warmly welcome to obesity and insulin

resistance (type 2 diabetes) worldwide (Bray et al., 2004).

The most important and prominent feature of type 2 diabetes is the weight gain

(Ijaz and Ajmal, 2011). The same was observed in mice of fructose-induced diabetic

control groups that showed a drastic percent gain in body weight from 7- 9% at the end

of the trail after consuming 10% fructose solution 1 ml/kg daily in fasting state. Whereas

test doses of MREt and AqMREt of R.serpentina prevent the percent body weight gain in

their respective test groups by inducing gradual and dose-dependent decrease in this

physical parameter especially the highest dose of each of these extract found most

effective (p< 0.05, p< 0.01 & p< 0.01) in this regard. These findings are also comparable

to the results obtained by pioglitazone (the most commonly used drug in type 2 diabetes)

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on the same physical parameter in positive control group of present study. Studies

reported that pioglitazone belongs to a group of antidiabetic medicines named

thiazolidinedione, put an additional weight as its side-effect (Gale, 2001). However,

preventing the percent body weight gain in test groups by test doses of both root extracts

in dose-dependent manner established the distinguished characteristic of R.serpentina in

controlling body weights. In addition, the wet organ weight and relative weight of

visceral body organs (kidney, liver, spleen and heart) of test groups were also analyzed

and reported the non-toxic aspect of both root extracts by observing no change in their

weights.

Hyperglycemia in type 2 diabetes is the biggest threat for all the future

complications of this disorder (Vijan, 2010). Orally administrated fructose significantly

increased the percent glycemic change from 76 to 114% in diabetic control groups.

Whereas, repeated administration of the selected doses of MREt found effective in

controlling (p< 0.01 & p< 0.0001) the hyperglycemia from 25 to 17 % in dose-

dependent manner in test groups. Similarly, administration of AqMREt doses also

improved (p< 0.05 & p< 0.01) fructose-induced glycemia by inducing prominent

reduction in percent glycemic change from 48 to 9 % in their respective test groups dose-

dependently as compared to diabetic control groups that showed massive glycemic

increase. Analogous effect on same parameter was also observed in positive control

group treated with pioglitazone that satisfactorily reduced the blood glucose level. The

hypoglycemic effects of both root extracts validates our previous claims and findings

related to the possible antidiabetic mechanism(s) of R.serpentina that it may inhibit sugar

(glucose) absorption in GIT or induce glucose tolerance in mice by accelerating glucose

uptake in hepatic and peripheral tissues (Azmi and Qureshi, 2012a; Azmi and Qureshi,

2012b). An in vitro study also demonstrated that R.serpentina is a potent inhibitor of α-

amylase and α-glucosidase, enzymes involved in carbohydrate absorption (Aadil et al.,

2013). Therefore, it supports that both root extracts may help in slowing down the

process of carbohydrate absorption in GIT thus preventing the elevation in blood glucose

level even after the administration of good concentration of fructose. There are many

163

medicinal plants reported to up-regulate the expression of glucose transporters including

GLUT-2 (insulin independent; present on liver, intestine, kidney and pancreas) and

GLUT-4 (insulin dependent; present on adipocytes and skeletal muscles) thereby

accelerating the glucose uptake in liver and peripheral tissues. This also supports our

second possible mechanism of antidiabetic action of root extracts of R.serpentina.

Fructose-induced hyperglycemia is accompanied with hyperinsulinemia which is

actually the reflection of insulin resistance (Wilson and Islam, 2012; Adeneye, 2012;

Johnson et al., 2009). The same was observed in present study where diabetic control

groups showed the significant elevation in serum insulin levels and fasting insulin

resistance index (FIRI) as compared to normal control group. On contrary, consecutive

treatment with MREt significantly (p< 0.01 & p< 0.0001) decrease the serum insulin

level with increase in dose magnitude in test groups and which is to some extent

comparable with serum insulin levels observed in positive control group plus almost

equal to the level of same hormaone found in normal control group. Additionally, dose-

dependent reduction (p< 0.01 & p< 0.0001) was found in FIRI which authenticates the

decrease in insulin resistance in test groups. However, the AqMREt showed an entirely

different picture where its doses, instead of normalizing insulin level, were enhancing the

insulin release from the pancreatic β-cells of mice as normally does by sulfonylurea

(Kaku et al., 1995). This finding is compatible to our previous observation found in

alloxan-induced type 1 diabetic test mice where it improved insulin release plus it did not

improve FIRI as same as MREt of same plant. This finding suggests that MREt of

R.serpentina may act as insulinomimetic agent or both root extracts of this plant may

reduce insulin resistance via inhibiting the oxidation of membrane lipids and proteins in

cells and elevating antioxidant enzymes levels in vivo or by decreasing the blood TG

level thereby enhancing the insulin sensitivity for its receptors (Azmi and Qureshi,

2012b; Azmi and Qureshi, 2013).

Hyperglycemia induced the formation of HbA1c, the confirmatory diagnostic

marker for diabetes (Azmi and Qureshi, 2013). Researches earlier reported that HbA1c

level ≤ 4 reflects the glucose homeostasis whereas increased from this limit (upto 6%)

164

alter the scenario (Silverman et al., 2011). The results of MREt treated test groups

showed an effective (p< 0.0001) lower level of HbA1c and similar significant (p< 0.0001)

findings were also found in AqMREt treated test groups. However, fructose-induced

diabetic control groups showed an increased percentage of same parameter which is due

to the fructose-induced hyperglycemia involved in non-enzymatic glycation of Hb that

elevates the amount of HbA1c from 6% to onwards (Cederberg et al., 2010). The effect of

both root extracts in preventing glycation of Hb was also confirmed by observing gradual

improvement in total Hb levels in their respective test groups as compared to fructose-

induced diabetic control groups (p< 0.05 & p< 0.01). Therefore, the glycosylation index

(HbA1c / Hb ratio) significantly (p< 0.05 & p< 0.01, p< 0.0001) decreased in all root

extracts treated test groups and this ratio almost comes to the level which was found in

normal control group. Hence, these findings confirm the antidiabetic and antiglycation

effects of root extracts of R.serpentina in type 2 diabetes.

High intake of dietary fructose may serve as a lipogenic agent that enters in

hepatic cells through GLUT-5 (glucose transporter; insulin-independent) after absorption

where it involves in the production of glycerol-3-phopsphate, acyl coenzyme A and

acetyl coenzyme A (Johnson et al., 2009). The first two components accelerate the

formation of triglycerides (fats) and its transporting vehicle VLDL-c which enhance the

deposition of newly synthesized triglycerides on membranes of peripheral tissues,

thereby possibly involve in masking of insulin receptors and induce insulin resistance

(Rutledge and Adeli, 2007). Likewise, acetyl-CoA speeds up the synthesis of cholesterol

and cholesterol transporting protein, LDL-c thus encouraging the hypercholesterolemia

and discouraging the role of HDL-c that involves in cholesterol efflux from peripheral

tissues (Johnson et al., 2009; Rutledge and Adeli, 2007; Simonen et al., 2002). Both of

these hyperlipidemic effects of fructose provoke the risk of life-threatening heart

problems in insulin resistance type 2 diabetes (Toth et al., 2012). Interestingly, there was

a significant (p< 0.01 & p< 0.0001) decrease observed in serum TG concentration in test

groups after oral administration of their respective doses of MREt and AqMREt as

compared to diabetic control groups. Of which, test doses of AqMREt produced

165

prominent and dose-dependent decrease in same parameter (p< 0.01 & p< 0.0001) as

compared to MREt. Similarly, the significant decrease (p< 0.05 & p< 0.01, p< 0.0001)

was also observed in TC, LDL-c, VLDL-c levels and increased in HDL-c level in both

root extracts treated test groups. On contrary, pioglitazone only reduced TG, VLDL-c but

did not show fruitful effect on TC and LDL-c levels in positive control group. Decrease

content of TG was possibly due to glucose homeostasis maintained by root extracts of

R.serpentina in test groups thereby preventing hypertriglyceridemia and improving

insulin sensitivity for its receptor. There are many medicinal plants reported to decrease

TG biosynthesis in fructose-induced type 2 diabetic rats (Adeneye, 2012). The

hypocholesterolemic effect showed by MREt and AqMREt of R.serpentina was also

authenticated by estimating 3-hydroxy-3-methyl glutaryl Coenyme A (HMG-CoA) /

mevalonate ratio. The dose-dependent significant (p< 0.05 & p< 0.01, p< 0.0001)

increase in same ratio was observed in test groups treated with MREt and AqMREt. The

increase in HMG-CoA / mevalonate ratio clearly depicts that root extracts of

R.serpentina involve in the inhibition of HMG-CoA reductase activity, the key rate-

limiting enzyme in cholesterol anabolism. The inhibition of this enzyme is a well-known

mechanism of action of statins, a group of orally administrated hypolipidemic agents

(Lateef and Qureshi, 2014). However, the decreased values of HMG-CoA/mevolanate

ratio were observed in diabetic control groups.

Computed values of cardioprotective indices also strengthen the hypolipidemic

effect of root extracts of R.serpentina observed in the present study like TG / HDL-c

ratio should remain under 4 is preferable while half of this value represents an ideal

situation (Gaziano et al., 1998). The present finding on the same ratio fortified (p< 0.05

& p< 0.01, p< 0.0001) the cardioprotective effect of both root extracts of R.serpentina

except the smallest dose (10 mg/kg) of MREt, two of the higher doses of MREt and three

of the test doses of AqMREt have brought the values of TG / HDL-c ratio within an ideal

limit. Similarly, HDL-c / LDL-c ratio forecast the cardioprotective status if its range

preferably remains over 0.3 and ideal over 0.4 (Gaziano et al., 1998). Both the root

extracts of R.serpentina exhibited an ideal and significant (p< 0.05 & p< 0.01, p<

166

0.0001) status of same ratio in their respective test groups while its lower values

observed in diabetic control groups. According to the literature, high TG / HDL-c ratio

and low HDL-c / LDL-c ratio serve as a fine forecaster for cardiac disorders and insulin

resistance (Marotta et al., 2010). The cardioprotective effect of root extracts becomes

stronger after computing coronary risk index (CRI) in term of TC/HDL-c ratio, its values

above 5 especially in insulin resistance (type 2 diabetes) shows the susceptibility towards

cardiac problems while values under 3.5 predicts ideal status (Barter et al., 2007; Kang et

al., 2012). In the present study, test doses of MREt and AqMREt showed an ideal (p<

0.05 & p< 0.01, p< 0.0001) CRI below 3.5 in their respective test groups, this index

decreases with the increase in doses of each root extract while increase levels of CRI

were observed in diabetic control groups. Insulin resistance-induced

hypertriglyceridemia and hypercholesterolemia double the risk of atherogenicity in type

2 diabetes that leads to myocardial infarction (Meisinger et al., 2009). Therefore,

antiatherogenic index (AAI) is commonly used to assess the antiatherogenic potential of

any remedy and its values are inversely proportional to the risk of atherogenesis (Waqar

and Mehmood, 2010; Azmi and Qureshi, 2012b). Interestingly, a significant (p< 0.05 &

p< 0.0001) and dose-dependent increase was found in AAI in test groups of both root

extracts of R.serpentina as compared to diabetic control groups even positive control

groups did not show any improvement in this index. These findings have also verified

the efficient improvement in cardioprotective indices observed in alloxan-induced

diabetic mice (Azmi and Qureshi, 2012b).

Another important aspect of present study is the improved status of hepatic

glycogen content in MREt and AqMREt treated test groups dose-dependently (p< 0.01).

However, the entirely opposite situation was observed in all the same test groups in case

of hepatic total lipids which were gradually decreased from small to high doses of each

root extract (p< 0.05 & p< 0.01, p< 0.0001). Pioglitazone was also found effective in

increasing the content of hepatic glycogen and decreasing the content of total lipids in

same tissue in positive control group. However, deposition of total lipids in liver

normally happens in type 2 diabetes which may leads to hepatomegaly or fatty liver

167

(Roden, 2006). The significant activity of root extracts observed in present study may be

due to the reduction of deposition of lipid droplets in hepatic cells possibly by enhancing

the activity of lipases that hydrolyze excess TG and LDL-c (Ado et al., 2013). The

increase of glycogen content in test groups indicates an effective decrease in glucose

production in hepatic cells by inhibiting the processes of glycogenolysis,

gluconeogenesis and enhancing the processes of glycogenesis and glycolysis (Azmi and

Qureshi, 2012b). These findings may also support the possibility of improvement in

insulin sensitivity for its receptors on target tissues including liver thereby accelerating

its anabolic effects or it may possible that chemical constituents present in root extract

mimic the action of insulin or act as insulin receptor sensitizers. A current computational

study also confirmed that few alkaloids present in roots of this plant serve as a potent

sensitizer of insulin receptors (Ganugapati et al., 2012).

Hepatoprotective effect of MREt and AqMREt was also confirmed by observing

normal levels of ALT in their respective test groups (p< 0.05 & p< 0.01) that showed an

improvement in liver function as compared to diabetic control groups which showed

elevated levels of same liver-specific enzyme. ALT is an important liver biomarker and

its elevated levels in serum reflects the liver malfunctioning (Qureshi et al., 2010). The

liver is the most important organ of the body, the essential role of this tissue/organ is to

metabolize all orally administrated compounds plus detoxify compounds which are

injurious to health (Qureshi et al., 2009). In addition, it also act as weaker vessel as any

fluctuation taking place in metabolism also affects the liver function (Bishop et al.,

2010).

Increase intake of fructose not only induced hyperglycemia, hyperinsulinemia but

also accelerate the formation of ROS to create oxidative stress (Johnson et al., 2009;

Bray, 2007). Hyperglycemia may lead to the activation of polyol pathway, an alternate

pathway of glucose metabolism in diabetic condition that involve in reduction of sweet

substance (glucose) to sorbitol (alcohol) in the presence of aldose reductase, the key

enzyme of polyol pathway (Pathania et al., 2013). This increased sorbitol production in

turn leads to the formation of ROS (superoxide anions, hydroxyl radicals, etc) and

168

advanced glycation end products (AGE) which accelerate the development of micro-

vascular complications of diabetes by inducing lipid peroxidation and alter membrane

structure and function (Barnett, 1986). On the other hand, this oxidative stress suppresses

the activity of antioxidant enzymes system in body (Pham-Huy, 2008). The same was

observed in present investigation where high percent inhibition of CAT and SOD

activities were found in fructose-induced diabetic groups whereas the percent inhibition

of CAT and SOD was effectively (p< 0.05 & p< 0.01, p< 0.0001) decreased in both root

extracts (MREt and AqMREt) treated test groups. Interestingly, a current computational

study based on our reported antidiabetic activity of R.serpentina described that few

alkaloids present in roots of R.serpentina are potent inhibitors of aldose reductase

enzyme thereby inhibiting the formation of ROS and reduce oxidative stress (Pathania et

al., 2013).

In diabetes, it was reported that the half-life (t1/2) of RBC become shortened due

to oxidative stress and non-enzymatic glycation of RBC membrane proteins (Saba et al.,

2010). In addition, increased amount of HbA1c and decreased amount of total Hb also

affects RBC count (Azmi and Qureshi, 2013). Hb improving effect of root extracts of

R.serpentina that observed in present study also improves (p< 0.01 & p< 0.0001) the

RBC count in their respective test groups especially AqMREt found more effective in

this aspect and produced dose-dependent significant improvement (p< 0.05 & p< 0.01) in

RBC count. This finding also correlates with the hematopoietic effect of MREt and

AqMREt found in alloxan-induced type 1 diabetic mice model and of course secondary

to the antiglycation and antioxidant activities of both root extracts of R.serpentina

thereby stabilizing the osmotic fragility of RBC membrane. On the other hand, the

significantly (p< 0.05 & p< 0.01, p< 0.0001) lowered and better WBC count was found

in test groups as compared to elevated level of same cells count observed in diabetic

control groups which reflects the inflammatory response occur in these control groups

that may be either due to hyperglycemic or hyperlipidemic conditions induced by high-

fructose intake (Johnson et al., 2009; Tanko et al., 2011). Ameliorative effect of MREt

and AqMERt on total Hb level and RBC count also induced improvement in other

169

hematological parameters including PCV, MCV, MCH and MCHC in test groups (p<

0.05). Therefore, root extracts of R.serpentina may possibly minimize the chances of

mild anemia which normally observed in diabetes especially after prolong use of

thiazolidinediones (Gale, 2001).

The results conclude that MREt and AqMREt of R.serpentina may prevent

insulin resistance in fructose-induced type 2 diabetic mice by reducing

hypertriglyceridemia, hypercholesterolemia and maintaining glucose homeostasis. In

addition, the same root extracts are also found as antiglycation, antioxidant and

hepatoprotective agents in same type 2 diabetic mice.

170

Chapter 5

Fourier Transform Infrared

Spectroscopy (FTIR) of MREt and

AqMREt of R.serpentina

Chapter 5 Fourier Transform Infrared Spectroscopy

(FTIR) of MREt and AqMREt of R.serpentina

Background

The current chapter deals with the FTIR spectrcopy of of the methanolic root extract

(MREt) and aqueous methanolic root extract (AqMREt) of R.serpentina. This chapter

describes the materials and method, interpretation of results and discussion sections.

5.1 Material and Method

5.1.1 Fourier Transform Infrared Spectroscopy (FTIR) of Root Extracts

5.1.1.1 Preparation of Samples for FTIR

The preparation of MREt of R. serpentina was done by soaking the 0.4 g of

ground root powder in 10 ml of methanol (95%) for overnight, sonicated for 10-15 min

then 7-8 ml of methanol was added, vortexes for 5-10 minutes and centrifuged for 10

minutes at 4000 rpm to separate the supernatant. It was transferred to 50 ml volumetric

flask. The whole procedure was repeated till colorless extract will appear. However, 5-10

mg of AqMREt extract of R. serpentina was dissolved in 10 ml of 80% aqueous

methanol and finally made up the volume till 50 ml with the same 80% aqueous

methanol.

The root samples were kept separately on water bath at 40-50 oC until the dried or

solid state of both extracts was obtained which then subjected to FTIR spectroscopic

analysis.

5.1.1.2 FTIR Profiling of Root Extracts

FTIR analysis of root extracts (MREt and AqMREt) were performed on Nicolet

6700 Thermo Fischer Scientific Infrared spectrophotometer. In conjugation with infrared

spectroscopy Attenuated Total Reflectance (ATR) of smart orbit of Thermo Scientific

(Diamond 30,000 – 200 cm-1

) was used. The spectrum was recorded in a region of 4000

– 500 cm-1

.

171

5.2 Results

5.2.1 FTIR Analysis of Root Extracts of R. Serpentina

FTIR spectroscopic analysis was used to identify the characteristics peaks of

functional groups of possible active compounds present in both root extracts of R.

serpentina.

5.2.1.1 FTIR Analysis of Root Extracts

MREt and AqMREt samples showed almost similar pattern of spectra while to

some extent few divergence in peak order was also observed. The first peak was

observed in MREt at 3277.24 cm-1

and in AqMREt at 3264.67 cm-1

. Interestingly, both

peaks showed the presence of phenol functional group with O-H bend. Secondly, two

consecutive peaks of MREt at 2922.13 cm-1

, 2852.06 cm-1

and AqMREt peak at 2927.61

cm-1

showed alkyl C-H stretch. Third, MREt peak at 1618.34 cm-1

and AqMREt peak at

1623.75 cm-1

showed the C = C stretch of alkenes. Fourth, MREt peak at 1411.93 cm-1

and AqMREt peak at 1405.96 cm-1

showed the presence of C-C stretch in aromatic ring.

Another single peak of MREt appeared at 1253.49 cm-1

that showed the C-N stretch of

alkaloids. MREt peak at 1040.75 cm-1


showed C-O

stretch of phenols. Similarly, MREt peak of 829.67 cm-1

showed the presence of N-H

wagging of secondary amines in alkaloids while this was not observed in AqMREt

spectrum. MREt peak at 520.45 cm-1


showed the C-Cl

stretch of alkyl halide (Table 45 and Figure 24).

172

Table 45: FTIR profile of Root Extracts of R. serpentina

S. No. Functional Groups MREt wave number

(cm-1)

AqMREt wave number

(cm-1) References

1

O – H (Phenol)

3277.24 3264.67 Pavia et al., 2001a

2

Alkyl C – H stretch

2922.13

2852.06 2927.61

Ahmad et al., 2014

3

C = C stretch (alkenes)

1618.34 1623.75 Ahmad et al., 2014

4

C – C stretch

(aromatic ring)

1411.93 1405.96 Bobby et al., 2012

5

C – N stretch (alkaloid)

1253.49 ----- Ahmad et al., 2014

6

C – O stretch (Phenols)

1040.74 1045.77 Ahmad et al., 2014

7

N – H wagging of

secondary amine

(alkaloids)

829.67 ---- Bobby et al., 2012

8

C – Cl stretch

(alkyl halide)

520.45 539.71 Pavia et al., 2001b

173

Figure 24: FTIR of Root Extracts of R. serpentina

174

5.3 Discussion

FTIR is a time-saving analytical technique which evaluates the functional groups

possess by compounds present in extracts of natural sources (Jagdish and Irudayaraj,

2004; Gamal et al., 2011). In the present study, this technique is used to reveal the

functional groups present in both MREt and AqMREt that could give an idea of possible

active compounds present in both of these root extracts responsible for significant

antidiabetic activity observed in alloxan-induced type 1 and fructose-induced type 2

diabetic mice models. The spectrum of each root extract showed peaks at different wave

numbers from 4000 to 500 nm. Of which, the FTIR analysis of both MREt and AqMREt

showed the presence of phenols by displaying peaks for their characteristic functional

groups (O-H & C-O) and aromatic ring at 3277.24 cm, 3264.67 cm and 1040.74 cm,

1045.77 cm respectively. This finding also verified the initial quantitative analyses of

both root extracts of R.serpentina through manual methods conducted in the present

study and observed that these are rich in phenols especially AqMREt contained more

total phenols as compared to MREt. Literature proved that medicinal plants rich in

phenolic compounds are significant antidiabetic agents and also ameliorate

hyperglycemia-induced oxidative stress in experimental diabetic models (Yang et al.,

2012). It has also been well-reported that phenolic compounds inhibited the aldose

reductase, NADPH oxidase and xanthine oxidase enzymes, thereby diminishing the

production of ROS in cells and enhancing the antioxidant capacity of biological system

(Seifu et al., 2012).

It has been reported that the roots of R.serpentina are rich in indole alkaloids (that

contains indole ring like ajmaline, ajmalicine, yuhimbine, etc) having hypotensive and

sedative properties (Itoh et al., 2005; Azmi and Qureshi, 2012a). The possible presence

of these indole alkaloids in MREt of R.serpentina was also proved by observing

characteristic peaks of C-N and N-H (wagging of secondary amines) stretches of

alkaloids in FTIR spectrum at 1253.49 and 829.67 nm. Whereas these peaks are

completely absent in FTIR spectrum of AqMREt that indicates the presence of negligible

amount of alkaloids in AqMREt. This also supports the results obtained in determining

175

the acute toxicity of both root extracts where MREt of R.serpentina was found to induce

sedation from 100mg/kg to onward whereas AqMREt was found safe till 250mg/kg as a

result of which 10 times high LD50 of AqMREt was found as compared to MREt.

Alkaloids are nitrogen containing compounds of low molecular weights and various

studies described their therapeutic potential in the management of clinical disease like

Alzheimer, myasthenia gravis, myopathy and dyslipidemia (Seifu et al., 2012). In

addition, different biological activities of alkaloids including antiinflammatory,

analgesic, gout suppressing, antineoplastic, antiviral, anticholinergic, antioxidant, etc are

reported (Mukhopadhyay et al., 2012; Pelletier, 1991). It was also reported that

alkaloids can inhibit lipid peroxidation thereby protecting the cell against LDL-c

oxidation by inhibiting lipoxygenase and xanthine oxidase enzymes which mostly

considered as ROS generating sources and /or accelerating the SOD enzyme activity

which ultimately provide protection against oxidative stress (Rackova et al., 2007;

Ahmad et al., 2010; Seifu et al., 2012; Facchini, 2001).

Therefore, it may be possible that presence of alkaloids and phenols (especially

flavonoids) together established the antidiabetic, antihyperlipidemic, antioxidant,

antiglycation and hematinic characteristics of MREt and AqMREt in both alloxan-

induced type 1 and fructose-induced type 2 mice models in present study.

176

5.4 Conclusion

The results of qualitative analysis conclude that MREt and AqMREt of R.

serpentina contain number of secondary metabolites including alkaloids, flavonoids,

glycosides, cardiac glycosides, phlobatannins, resins, steroids, tannins and triterpenoids

except carbohydrates and saponins which are only present in MREt but not in AqMREt.

Similarly, quantitative assessment conclude that both root extracts are rich in total

phenols especially AqMREt while MREt contain more alkaloids than AqMREt. It has

been confirmed by FTIR analysis of both root extracts. These results are verified by

observing small LD50 of MREt as 141.25 mg/kg (log LD50 = 2.15 mg/kg) as compared to

high LD50 of AqMREt as 1412.54 mg/kg (log LD50 = 3.15 mg/kg) in determining acute

toxicity of both root extracts. This study clearly describes that MREt from 100 mg/kg to

onward induce mild to severe sedation and 17-100% mortalities in overnight fasted test

groups whereas test doses of AqMREt from 500-2500 mg/kg induce the same acute toxic

effects in experimental mice. Therefore, test doses of 10, 30 & 60 mg/kg of MREt and

50, 100 & 150 mg/kg of AqMREt are selected and found to ameliorate post-prandial

blood glucose levels in their respective test groups after glucose load by showing

hypoglycemic potential at each time interval from 0 to 120 min in OGTT.

Oral administration of test doses of MERt and AqMREt improves hyperglycemia

and diabetic dyslipidemia by preventing glycation, oxidative stress, elevating antioxidant

enzymes system and inducing hematopoietic effects in alloxan-induced type 1 and

fructose-induced insulin resistance type 2 diabetic mice models. The antidiabetic effect

of root extracts of R.serpentina in alloxan-induced type 1 diabetes may be due to extra-

pancreatic or pancreatic action either by inhibiting glucose absorption in GIT, or

inducing glucose tolerance in mice by accelerating the glucose uptake in target tissues of

insulin like muscles and liver thereby stimulating the processes of glycolysis and

glycogenesis or enhancing the release of insulin (pancreatic action) from β-cells of

pancreas which in turn induce glucose homeostasis. However, in fructose-induced

insulin resistance type 2 diabetes, the hypoglycemic potential of MREt and AqMREt is

177

possibly due to extra-pancreatic action by reducing hypertriglyceridemia and

hypercholesterolemia thereby enhancing insulin sensitivity and maintaining glucose

homeostasis or active ingredient (alkaloids/ phenols) of root extracts may mimic the

action of insulin by acting as insulin receptor sensitizer. Therefore, the present work is

successful in enlightening the new antidiabetic aspect of well-famous antihypertensive

plant R.serpentina. However, more work has to be done to authenticate the use of root

extract of same plant as antidiabetic agent.

5.5 Future Prospects of Present Research

The results of the present study established the importance of roots of R.

serpentina in treatment of diabetes and diabetic dyslipidemia by improving the

antioxidant enzymes status in both insulin dependent (type 1) and insulin resistance (type

2) diabetic mice. Followings are the possible suggestions for future extension of this

research work.

Fractionations of crude root extracts (MREt and AqMREt) of R.serpentina that

were used in the present study should be done by using different organic solvents

with least to high polarity.

Evaluation of antidiabetic activity should be done in each of the prepared fraction

of crude root extract of R.serpentina which may help in isolating an active principle

involve in antidiabetic aspect of this plant and which could be a base of

manufacturing new antidiabetic medicine with negligible side effect in

pharmaceutical industry.

Evaluation of possible mechanism of antidiabetic action suggested in the present

study.

Evaluate the antidiabetic activity of root extracts of R.serpentina in type 1 and type

2 diabetic human volunteers which help in authenticating its use as antidiabetic

agent in herbal medicines.

178

Chapter 6

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Publications & Presentations

LIST OF RESEARCH PUBLICATIONS

Muhammad Bilal Azmi Department of Biochemistry


Karachi-75270

RESEARCH PAPER PUBLISHED FROM THESIS

1. Qureshi SA, Nawaz A, Udani SK, Azmi MB. 2009. Hypoglycaemic and

hypolipidemic activities of Rauwolfia serpentina in alloxan-induced diabetic rats.

International Journal of Pharmacology, 5(5): 323-326.

2. Azmi MB, Qureshi SA. 2012. Methanolic Root Extract of Rauwolfia serpentina

improves the glucose tolerance in mice. Journal of Food and Drug Analysis, 20(2):

484-488.

3. Azmi MB, Qureshi SA. 2012. Methanolic root extract of Rauwolfia serpentina

Benth improves the glycemic, anti-atherogenic and cardioprotective indices in

alloxan induced diabetic mice. Advances in Pharmacological Sciences, 2012,

Article ID 376429: 11 pages.

4. Azmi MB, Qureshi SA. 2013. Rauwolfia serpentina ameliorates hyperglycemia,

haematinic and antioxidant status in alloxan-induced diabetic mice. Journal of

Applied Pharmaceutical Sciences, 3(7): 136-141.

5. Azmi MB, Qureshi SA. 2014. Glucose lowering potential of hydromethanolic

extract of Rauwolfia. World Journal of Pharmaceutical Sciences, 2(3):219-223.

208

OTHER PUBLICATIONS

1. Qureshi SA, Kamran M, Asad M, Zia A, Lateef T, Azmi MB. 2010. A Preliminary

Study of Santalum album on Serum Lipids and Enzymes. Global Journal of

Pharmacology, 4(2): 71 – 74.

2. Azmi MB, Qureshi SA, Lateef T, Arshad HM. 2010. Health Hazards of work

related stress. Journal of Dow University of Health Sciences, 4(3): 115-118.

3. Lateef T, Rukash H, Bibi F, Azmi MB, Qureshi SA. 2010. Effect of Convalaria

Majalis on Kidney function. Journal of Dow University of Health Sciences ,

4(3):94-97.

4. Azmi MB, Shamim S, Hussain M, Masood A, Mirza Z. 2011. Factors And

Demographic Characteristics Related To Nursing Workplace Satisfaction:

Perspectives of Nursing Care Providers At Tertiary Care Hospitals Of Karachi.

Journal of Dow University of Health Sciences, 5(3): 92-98.

5. Qureshi SA, Rehman MMU, Azmi MB, Hasnat S. 2011. Most Prevalent Diseases

with Relation of Basal Metabolic Index (BMI) & Waist Circumference (WC) in

Karachi, Pakistan. Journal of Dow University of Health Sciences, 5(3): 85-91.

6. Mudassir HA, Azmi MB, Qureshi SA. 2011. Phytochemical investigation of

ethanolic seed extract of Anethum Graveolens and its effect on glucose tolerance in

rabbits. FUUAST Journal of Biology, 1(2):41-45.

7. Arshad HM, Mohiuddin OA, Azmi MB. 2012. Comparative in vitro antibacterial

analysis of different brands of cefixime against clinical isolates of Staphylococcus

209

aureus and Eschericia coli. Journal of Applied Pharmaceutical Sciences, 2(1): 109-

113.

8. Khan F, Azmi MB, Hussain M, Azmi SS. 2013. Assessment of job stress;

demographic factors in doctors working at the tertiary care hospitals of Karachi.

The Professional Medical Journal, 20(1):152-159.

9. Azmi MB, Sohail A, Hussain M, Azmi SS, Qureshi SA. 2013. Work associated

stress on female medical house officers of Karachi, Pakistan. FUUAST Journal of

Biology, 3(1): 63-70.

ORAL PRSENTATIONS FROM THESIS

1. Presented Paper entitled “Rauwolfia serpentina improves the heamatological

indices in alloxan-induced diabetic mice” in Golden Jubilee Symposium of Jinnah

Postgraduate Medical Centre held from March 23-29, 2014 at Jinnah Postgraduate

Medical Centre, Karachi.

2. Presented Paper entitled “Ameliorative effect of Rauwolfia serpentina on insulin

resistance and cardioprotective indices in fructose-induced diabetic mice” in 2nd

National Conference on Research in Pharmacy and Poster Competition, at Dow

College of Pharamcy DUHS, held on Saturday 7th

September, 2013.

3. Presented Paper entitled “Antidiabetic and antiatherogenic potential of flavonoid

rich extract of Rauwolfia serpentina in alloxan-induced diabetic mice” in 3rd

Symposium on Health care from bench to bedside and beyond, April 26-27, 2013,

organized by Bahria University Medical and Dental College, Karachi, Pakistan.

4. Presented paper entitled “Methanolic root extract of Rauwolfia serpentina

ameliorates lipids and glucose homeostasis in high-fructose fed insulin resistant

210

mice” in two days conference on Climate Change and Bioresources of Pakistan,

March 21-22, 2013, organized by Federal Urdu University of Arts, Science and

Technology, Karachi, Pakistan.

5. Presented paper entitled “Glycemic improvement by methanolic root extract of

Rauwolfia serpentina in high fructose fed type 2 diabetic mice” in 1st

International

Conference on Endorsing Health Science Research, February 2-3, 2013, organized

by Advance Educational Institute and Research Center (AEIRC).

6. Presented paper entitled “Biochemical and haematological effects of methanolic

root extract of Rauwolfia serpentina Benth in alloxan-induced diabetic mice” in

“BioCon 2012, Organized by Dept. of Biochemistry University of Karachi, July 9-

11, 2012. Won 1st

prize among all oral Presenter and award of Young Scientist for

year 2012.

7. Presented paper entitled “Methanolic root extract of Rauwolfia serpentina Benth

improves the antiatherogenic and cardioprotective indices in alloxan-induced

diabetic mice” in “7th

Liaquat National Symposium, Liaquat National Hospital and

Medical College (LNHMC), Karachi, February 24-26, 2012.

8. Presented paper entitled Phytochemical investigation of methanolic root extract of

Rauwolfia serpentina Benth and its effect on glycosylated hemoglobin (HbA1C) in

alloxan-induced diabetic mice” in “3rd

International Conference/ Workshop on

Dendrochronology and Plant Ecology”, organized by Federal Urdu University of

Arts, Science & Technology, Karachi, November 25- 30, 2011.

POSTER PRSENTATIONS FROM THESIS

1. Presented Poster entitled “Rauwolfia serpentina prevents glycation in alloxan-

induced diabetic mice” in 2nd

National Conference on Research in Pharmacy and

211

Poster Competition, in 2nd

National Conference on Research in Pharmacy and

Poster Competition, at Dow College of Pharamcy, DUHS held on September 7,

2013.

2. Presented Poster entitled “Flavonoid rich extract of Rauwolfia serpentina develops

glucose tolerance in normoglycemic mice with no toxicity” in 3rd

Symposium on

Health Care from Bench to Bedside and Beyond, April 26-27, 2013, organized by

Bahria University Medical and Dental College, Karachi, Pakistan.

3. Presented Poster and Abstract entitled: “Methanolic root extract of Rauwolfia

serpentina improves lipids and glucose homeostasis in high-fructose fed insulin

resistant mice” in 4th

international Symposium-cum-training Course on Molecular

Medicine and Drug Research Januray 7-10, 2013, organized by International

Center for Chemical and Biological Sciences (ICCBS), Univesrity of Karachi,

Pakistan.

4. Presented Poster entitled: “Rauwolfia serpentina ameliorates diabetic

dyslipidemia” in 13th

International Symposium on Natural Product Chemistry

(ISNPC-13), HEJ Research Institute of Chemistry, University of Karachi,

September 22 – 25, 2012.

5. Presented Poster entitled: “Assessment of acute toxicity and glucose tolerance of

flavonoid fraction of Rauwolfia serpentina in Wister mice”, in 13th

International

Symposium on Natural Product Chemistry (ISNPC-13), HEJ Research Institute of

Chemistry, University of Karachi, September 22 – 25, 2012.

6. Presented poster entitled: “Antihyperlipidemic activity of Rauwolfia serpentina”, at

Symposium cum Workshop on Tools in Biomedical Research. Department of

Biochemistry, University of Karachi March 01-03, 2011.

212

7. Presented Poster entitled:“Oral hypoglyceamic activities of methanolic root extract

of Rauwolfia serpentina: A therapeutic dose selection study” in 10th

International

Biennial Conference of Pakistan Society of Biochemistry & Molecular Biology

“Biomolecular Sciences in Development” held at Khan institute of Biotechnology

and Genetic Engineering (KIBGE), University of Karachi, December 1 – 5, 2010.

213

Reagent Glossary

Reagent Glossary

1. Benedict's Solution (qualitative reagent for glucose): Solution A: dissolve 173 g

of sodium citrate and 100g of Na2CO3 in 800 ml of distilled water with slight

heating. Filter, if necessary. Solution B: dissolve 17.3 g of CuSO4.5H2O in 100

ml of distilled water. Mix solution A with solution B with constant stirring and

make up the volume up to 1 liter.

2. Dragendroff’s Reagent (reagent for alkaloid detection): Solution A: dissolve

1.7 g bismuth nitrate in 100 ml of distilled water: acetic acid (4:1). Solution B:

dissolve 40 g potassium iodide (KI) in 100 ml of distilled water. Mix 5 ml of

solution A + 5 ml of solution B + 20 ml of acetic acid + 70 ml of distilled water.

3. Fehling’s Solution (reagent for reducing sugar): Solution A (copper sulfate

solution): Dissolve 7g of CuSO4.5H2O in 100ml of distilled water plus two drops

of dilute sulfuric acid. Solution B (alkaline tartrate solution): Dissolve 35g of

potassium sodium tartrate (Rochelle salts, KNaC4H4O6.4H2O) and 12 g of NaOH

in 100ml of distilled water. Mix equal volumes of solution A and B before use.

4. Hager's Reagent (reagent for alkaloids detection): Dissolve 1g of picric acid in

100ml of distilled water.

5. Mayer's Reagent (reagent for alkaloids detection): Dissolve 1.358g of mercuric

chloride (HgCl2) in 60ml of distilled water and 5g of potassium iodide (KI) in 10

ml of distilled water. Mix both the solutions together and make the volume up to

100 ml with the distilled water.

6. Wagner's Reagent (reagent for alkaloids detection): Dissolve 2g of iodine and

6g of potassium iodide (KI) in 100 ml of distilled water.

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effect of methanolic root extract of rauwolfia...

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