possible protective effect of methanolic crude …
TRANSCRIPT
POSSIBLE PROTECTIVE EFFECT OF METHANOLIC CRUDE EXTRACT OF
PANSIT-PANSITAN (Peperomia pellucida (L.) HBK) AGAINST ELEVATED
BLOOD GLUCOSE LEVEL AND CARDIAC AND AORTIC HISTOLOGY OF
STZ-INDUCED DIABETIC ICR MICE (Mus musculus)
KRISTIANNE DANIELLE BUCU BARON
DANNA LORRAINE TUDOR DECENA
Submitted to the
Department of Biology
College of Arts and Sciences
University of the Philippines – Manila
Padre Faura, Manila
In partial fulfillment of the requirements
for the degree of
Bachelor of Science in Biology
June 2016
ii
Department of Biology
College of Arts and Sciences
University of the Philippines – Manila
Padre Faura, Manila
ENDORSEMENT
The thesis attached hereto, entitled Possible Protective Effect of Pansit-Pansitan
(Peperomia pellucida) Against Elevated Blood Glucose Levels and Abnormal Cardiac
and Aortic Histology in STZ-induced Diabetic Mice (Mus musculus) prepared and
submitted by Kristianne Danielle B. Baron and Danna Lorraine T. Decena, in partial
fulfillment of the requirements for the degree of Bachelor of Science in Biology was
successfully defended on May 16, 2016.
This undergraduate thesis is hereby officially accepted as partial fulfillment of the
requirements for the degree of Bachelor of Science in Biology.
ELENA M. RAGRAGIO, M.A. LEONARDO R. ESTACIO JR., Ph.D.
Chair Dean
Department of Biology College of Arts and Sciences
UP Manila UP Manila
KIMBERLY BELTRAN-BENJAMIN, M.Sc.
Thesis Adviser
ROHANI B. CENA, D.V.M., M.Sc.
Thesis Co-Adviser
ELISA L. CO, Ph.D.
Thesis Reader
iii
TABLE OF CONTENTS
PAGE
TABLE OF CONTENTS …………………………………………………………......... iii
LIST OF TABLES ……………………………………………………………………..... v
LIST OF FIGURES ……………………………………………………………….......... vi
LIST OF APPENDICES ………………………………………………………………. vii
ACKNOWLEDGMENTS …………………………………………………………...... viii
ABSTRACT …………………………………………………………………………..…. x
INTRODUCTION ………………………………………………………………………. 1
Background of the Study ………………………………………………………... 1
Statement of the Problem ………………………………………………………... 3
Research Objectives ……………………………………………………………... 3
Hypothesis ……………………………………………………………………….. 4
Significance of the Study ………………………………………………………... 4
Scope and Limitations ………………………………………………………….... 4
REVIEW OF RELATED LITERATURE ………………………………………………. 6
Medicinal properties of Peperomia pellucida ………………………………….... 6
Diabetes mellitus ………………………………………………………………… 7
Cardiovascular diseases …………………………………………………………. 8
Induced diabetes in Mus musculus ………………………………………………. 9
Protective effects of Peperomia pellucida ……………………………….…….. 11
Cardioprotective effects of family Piperaceae ……………………………...….. 12
Metformin …………………………………………………………………….... 13
iv
MATERIALS AND METHODS ……………………………………………….……… 15
Plant collection and extraction …………………………………………………. 15
Acquisition and acclimatization of mice ……………………………………….. 15
Procurement of chemical reagents …………………………………………...… 15
Induction of diabetes and treatment protocol …………………………..………. 16
Slide preparation and processing ………………………………………...…….. 17
Histopathological analysis …………………………...………………………… 18
Statistical analysis ……………………………………………………………… 19
RESULTS ……………………………………………………………………………… 20
Blood glucose levels …………………………………………...………………. 20
Blood cholesterol levels ………………………………………………………... 21
Histopathological analysis of the heart ……………………….………………... 22
Histopathological analysis of the aorta ……………………………………...…. 27
DISCUSSION ………………………………………………………………………….. 28
Blood glucose levels ………………………...…………………………………. 28
Blood cholesterol levels …………………………………………….………….. 29
Histopathological analysis of the heart …………….……………………...…… 29
Histopathological analysis of the aorta ………………………………………… 32
CONCLUSION AND RECOMMENDATIONS ……………………………………… 34
REFERENCES ………………………………………………………………………… 35
GANTT CHART / LINE-ITEM BUDGET …………………………………….……… 42
APPENDICES …………………………………………………………………………. 43
CURRICULUM VITAE ……………………………………………………………….. 51
v
LIST OF TABLES
TABLE PAGE
1 Histopathological assessment of the heart ……………………………………... 26
vi
LIST OF FIGURES
FIGURE PAGE
1 Mean ± SEM blood glucose levels at initial and final levels …………….…….. 20
2 Mean ± SEM blood cholesterol values at initial and final values …………..….. 21
3 Photomicrographs of longitudinal sections of the mouse heart at
LPO (100x) .......................................................................................................... 22
4 Photomicrographs of longitudinal sections of the heart at HPO (400x)
and OIO (1000x, inset) ………………………………………………………… 24
5 Photomicrographs of transverse sections of the aorta at HPO (400x) …………. 27
vii
LIST OF APPENDICES
APPENDIX PAGE
A Blood glucose levels of mice ……………………...…………………..……….. 43
B Blood cholesterol values of mice ………………………………………………. 45
C Plant Identification/Certification Form ………………………………………… 46
D IACUC Certificate of Approval ………………………………………………... 48
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ACKNOWLEDGMENTS
We would like to express our sincerest gratitude to:
Our beloved thesis adviser, Prof. Kimberly Beltran-Benjamin, for staying by our
side through countless ups and downs, and keeping our spirit alive in times of failure,
depression, and difficulties;
Our dearest thesis co-adviser, Dr. Rohani Cena, for sharing her expertise with us
and guiding us throughout the course of accomplishing this thesis;
Our beloved thesis reader, Prof. Elisa Co, for sharing her valuable insights on our
thesis and encouraging our best efforts;
Our thesis groupmates, Dawn S. De Loreto and Junel Carla O. Magbuhat, for
sharing their resources and being with us through the difficulties that came with taking care
of the mice;
The National Institutes of Health - UP Manila, for their generosity in sheltering our
mice at the Animal House and allowing us access to their laboratory and equipment;
Kuya Mel of the NIH Animal House, for his skill, patience, and tireless efforts in
assisting us with the handling of our mice, to which we are extremely grateful for;
Our families, for their never-ending support in all things, and for motivating us to
keep believing in our dreams no matter how difficult achieving them might seem;
Our friends, for their contributions and help in this endeavor, as well as the joy and
relief their company brought with every bump in the road;
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To all the people who, in one way or another, gave their precious time, talent and
skill in making this thesis a success;
Finally, to Almighty God, for bestowing upon us wisdom, knowledge, patience,
and dedication, whom this thesis would never have come to fruition without.
x
ABSTRACT
Peperomia pellucida is a medicinal herb that has been reported to have anti-diabetic effects.
However, its cardioprotective effects have not yet been studied. This study aimed to
determine if the methanolic crude extract of P. pellucida has a protective effect on elevated
blood glucose levels and abnormal histology of the heart and aorta in streptozotocin-
induced diabetic mice. Thirty male ICR mice were divided into 6 groups: negative control
(Con), diabetic mice treated with 150 mg/kg b.wt. Metformin (Met), diabetic mice treated
with 200 mg/kg b.wt. P. pellucida (PPE 200), diabetic mice treated with 400 mg/kg b.wt.
P. pellucida (PPE 400), diabetic mice treated with 800 mg/kg b.wt. P. pellucida (PPE 800),
and untreated diabetic mice (STZ/HFD). Streptozotocin (STZ) was used in order to induce
diabetes. Blood glucose and blood cholesterol levels were monitored throughout the
experiment. The heart and aorta were harvested after 2 weeks of treatment and evaluated
based on histopathological parameters such as intercellular fibrosis, fatty infiltration,
endocardial thickening, cardiomyocyte degeneration, and changes in cardiomyocyte nuclei.
Results showed that P. pellucida methanolic crude extract was ineffective at lowering
elevated blood glucose levels, but it displayed potential protective effects on the diabetic
heart and aorta. Histopathological analysis indicated that the P. pellucida methanolic crude
extract was cardioprotective at 800 mg/kg b.wt..
Keywords: Peperomia pellucida, diabetes mellitus, streptozotocin
1
INTRODUCTION
Background of the Study
Diabetes mellitus is a metabolic disease in which glucose, the main source of
energy in the human body, cannot reach its target cells and instead accumulates in the
bloodstream. This is due to either a lack of insulin produced by the pancreas, or the misuse
of insulin by the liver or muscles, as insulin is needed to allow glucose entry to the cells.
A high amount of glucose in the blood can damage blood vessels and nerves, and can
eventually lead to loss of vision, kidney malfunctions, and more notably, heart failure
(NIDDK, 2013).
Heart failure causes death in 70% of people with diabetes. This occurs due to the
excessive deposition of fatty materials into the bloodstream, characteristic of dyslipidemia
and hyperglycemia, which causes blockage of blood vessels, particularly the aorta, and
disruption of the normal flow of blood. Heart failure may also be brought about by
contractile dysfunction of the heart due to loss of microvessels and abnormalities in the
extracellular matrix (ECM) of cardiac muscle cells. It can be said that diabetes leads to a
greater risk of contracting cardiovascular diseases such as atherosclerosis, coronary artery
disease, or diabetic cardiomyopathy (Miki et al., 2013).
According to the Department of Health, diabetes mellitus is ranked 8th in the top 10
leading causes of mortality in the Philippines. In 2014 alone, there were 3.2 million cases
of diabetes recorded in the country, 53,549 of which resulted in death in adults. It can be
said that the number of cases of diabetes in the Philippines has greatly increased from the
2.7 million cases of diabetes recorded in 2000, and the World Health Organization predicts
that by 2030, the Philippines will have 7.8 million cases of diabetes. Moreover, diabetes is
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highly associated with the increased risk of cardiovascular diseases, which may be a reason
why heart disease remains the number one cause of mortality in the Philippines. Thus, there
exists an urgent need to find a suitable means of controlling the prevalence of diabetes in
the present time.
Due to the growing prevalence of diabetes, there are a number of anti-diabetic drugs
commercially available today, the most effective of which is Metformin, a synthetic drug
used to assist glucose uptake and decrease its levels in the bloodstream (Boyle et al., 2010).
However, most of these drugs are chemically based and have many reported side effects
such as nausea, vomiting, skin rash, abdominal pain, headaches and diarrhea. This includes
even Metformin, which has been reported to cause hepatotoxicity, lactic acidosis, and even
allergy development in certain cases (Aksay et al., 2007; Wiwanitkit, 2011). Given this,
natural alternatives to synthetic anti-diabetic drugs must be considered.
While medicinal plants have been used in traditional medicine for treating diabetes
in the past, there have been few scientific studies to prove their validity. Fortunately, these
herbal medicines, such as those derived from Peperomia pellucida, are now being
researched further for their anti-diabetic properties, as there are fewer side effects during
treatment and the costs are much cheaper (Modak et al., 2007).
Peperomia pellucida, commonly known as pansit-pansitan or ulasimang bato in the
Philippines, is one of the 10 medicinal herbs approved by the Department of Health for
public use. The plant has been used in traditional medicine to treat fatigue, gout, arthritis,
skin diseases, migraines, abdominal pains and kidney pains (Mutee et al., 2010; Beltran-
Benjamin et al., 2013).
3
Peperomia pellucida is known to have medicinal properties, most, if not all, of
which have been proven by numerous scientific studies. It has antimicrobial (Akinnibosun,
et al., 2008; Oloyede et al., 2011; Mensah et al., 2013), antipyretic (Khan et al., 2008),
anti-inflammatory (Arrigoni-Blank et al., 2002; Mutee et al., 2010), neuropharmacological
(Khan et al., 2008), and antioxidant (Hamzah et al., 2012; Beltran-Benjamin et al. 2013)
properties. It has also been suggested that P. pellucida has antidiabetic (Hamzah et al.,
2012) properties; however, this has yet to be examined in further detail, particularly in
terms of its protective effect on the heart of diabetic patients.
Statement of the Problem
Does Peperomia pellucida (L.) HBK have a protective effect on elevated blood
glucose levels and abnormal cardiac and aortic histology of STZ-induced diabetic ICR
mice?
Research Objectives
The general objective of the study is to determine the protective effect of Peperomia
pellucida (L.) HBK methanolic crude extract on the elevated blood glucose levels and
abnormal cardiac and aortic histology of STZ-induced diabetic ICR mice.
Specific objectives include: a) to measure and compare the blood glucose levels of
all 6 groups of mice; b) to compare the cardiac and aortic histology of the 6 groups of mice;
and c) to determine the most effective cardioprotective dose of Peperomia pellucida
methanolic crude extract.
4
Hypothesis
Ho: Peperomia pellucida has no protective effect in STZ-induced diabetic ICR mice.
Ha: Peperomia pellucida protects against elevated blood glucose levels and
abnormal cardiac and aortic histology in STZ-induced diabetic ICR mice.
Significance of the Study
Previous studies have shown that Peperomia pellucida has gastroprotective and
hepatoprotective effects on rats (Rosalida & Noor Aini, 2009; Beltran-Benjamin et al.,
2013; Alfonso & Riego de Dios, 2015). However, to date, there have not been any
published studies on the protective activity of this plant against hyperglycemia and
abnormal histology of the heart and aorta in diabetic mice. Therefore, this study helps to
provide new insights on P. pellucida as a potential treatment for both diabetes and
cardiovascular diseases that may be brought about by diabetes such as atherosclerosis and
diabetic cardiomyopathy.
With the growing prevalence of diabetes in the Philippines, alternative sources of
treatments are also being sought after in the pharmacological field. In line with this, the
study can provide more information on a possible substitute that is cheaper and safer than
the commercially available anti-diabetic drugs sold today.
Scope and Limitations
This study investigated the effect of Peperomia pellucida on the elevated blood
glucose levels, as well as abnormal cardiac and aortic histology of STZ-induced diabetic
ICR mice (Mus musculus). The mice were aged 4-6 weeks old, each weighing 18-24 grams.
5
The mice were induced with Type II diabetes mellitus by means of a high-fat diet and
streptozotocin (STZ) injection. To ensure the consistency of the results, P. pellucida plants
were collected from a local plantation in Tarlac.
The study did not concern itself with the treatment of Type I diabetes mellitus. The
isolation of any active anti-diabetic compounds from Peperomia pellucida were also not
involved. Furthermore, the study did not examine the effects of P. pellucida on the
physiology and behavior of the mice.
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REVIEW OF RELATED LITERATURE
Medicinal properties of Peperomia pellucida
Pansit-pansitan (Peperomia pellucida), also known as ulasimang bato, is a member
of the Piperaceae family. It is an annual herb that usually grows in clumps on damp areas
during the rainy season. This plant is commonly found in Asian and South American
countries. It can grow from 15 cm up to 45 cm in height. The leaves are cordate, around 4
cm in length, possess a shiny luster, and have an alternate phyllotaxy. The stems are erect,
glabrous, succulent, translucent pale green, and 5 mm in diameter. The inflorescence is
composed of one to several spikes. The fruits are small, round to oblong in shape, and are
green at first but eventually turn black in color when ripe (Majumder et al., 2011).
As listed by the Department of Health, pansit-pansitan is one of the 10 most
accepted herbal medicines in the Philippines, and scientific researches continue to reinforce
the effectiveness of this plant’s medicinal properties (Philippine Herbal Medicine, 2014).
Studies have shown that Peperomia pellucida exhibits anti-inflammatory properties by
inhibiting the increase of swelling in hind paw edema (Arrigoni-Blank et al., 2002; Mutee
et al., 2010). The plant’s extracts also decrease levels of fever induced in rabbits (Khan et
al., 2008); induce depression in mice (Khan et al., 2008); inhibit microbial growth
(Akinnibosun, et al., 2008; Oloyede et al., 2011; Mensah et al., 2013); and exhibit
antioxidant properties (Hamzah et al., 2012; Beltran-Benjamin et al. 2013). Some have
even isolated the active compounds present in the plant that are responsible for its
medicinal capacity (Akhila et al., 2012; Rojas-Martinez et al., 2013).
Although it is not usually described as such in folkloric medicine, recent studies
have shown that Peperomia pellucida has anti-diabetic potential. In a study by Hamzah et
7
al. (2012), the blood glucose levels of diabetic and non-diabetic rats were measured and
compared, and it was found that the groups of diabetic rats treated with 10% w/w or 20%
w/w P. pellucida had lower blood glucose levels compared to the diabetic rats that were
not subjected to treatment. There was also a slight decrease in the blood glucose levels of
non-diabetic rats treated with P. pellucida extracts, as well.
In another study by Akhila et al. (2012), the bioactive compounds responsible for
the anti-diabetic property of P. pellucida were determined in silico. Of all the components
of P. pellucida subjected to docking studies against aldose reductase, yohimbine was the
only one that showed a greater binding energy than the standard quercetin, the ligand that
binds with aldose reductase. This indicated that yohimbine was the most active anti-
diabetic component in P. pellucida.
Diabetes mellitus
Diabetes mellitus is a group of metabolic diseases that results from the body’s
inability to regulate its own blood sugar levels, thus causing persistent hyperglycemia and
dyslipidemia, among other symptoms. The disease may develop as a result of impaired
insulin secretion, defective insulin action, or both (Pittas, n.d.). It is without a known cure;
thus, it is a common cause of increasing mortality and morbidity rates worldwide, both for
its acute and chronic forms.
At present, the Philippines is considered one of the world’s emerging diabetes
hotspots. According to the International Diabetes Federation, the country is included in the
Top 15 most diabetes-prevalent countries in the world, with more than 4 million people
8
diagnosed with the disease as of 2014. It has remained the 8th leading cause of mortality in
the country since 2009, based on surveys from the Department of Health.
Diabetes is classified into two types based on their etiology. Type 1 diabetes,
normally manifesting in children aged less than 20 years old and accounting for 5-10% of
all diabetes cases, involves the auto-immune destruction of beta islet cells in the pancreas;
thus, all patients require insulin for survival. On the other hand, Type 2 diabetes occurs at
adult onset, and involves insulin resistance, insulin deficiency, or both. Cases are often
asymptomatic until chronic complications arise. Type 2 diabetes is characterized by the
body not being able to produce enough insulin or the body developing a resistance to its
produced insulin. It is considered the most common type of diabetes, as well as the most
prevalent, composing 90% of all known cases of diabetes worldwide (Pittas, n.d.).
Diabetes mellitus is associated with higher incidence rates of atherosclerotic,
cardiovascular, peripheral, arterial, and cerebrovascular diseases, according to the
American Diabetes Association. It increases the risk of contracting cardiovascular diseases
due to its effect on the cardiac muscles; because diabetes leads to higher blood glucose
levels and higher lipid profiles, it can cause systolic or diastolic heart failure (Dokken,
2008).
Cardiovascular diseases
Cardiovascular disease is considered an umbrella term for coronary artery disease,
heart failure, and stroke. The Nutritionist-Dietitians’ Association of the Philippines stresses
that it is primarily a consequence of other related diseases such as obesity, hypertension,
and most notably in this case, diabetes. According to a 2013 survey by the Department of
9
Health, heart disease has been the top leading cause of mortality in the Philippines since
2009.
As mentioned previously, cardiovascular diseases are strongly correlated with
diabetes. This is mainly due to the latter’s characteristic hyperglycemia and dyslipidemia
congesting the blood vessels, which impairs the normal passage of blood and results in a
significantly higher risk of atherosclerosis and subsequent heart failure. Diabetes also
causes contractile dysfunction of the heart due to the loss of microvessels and changes in
the extracellular matrix of cardiac muscle cells (Miki et al., 2013). Heart failure is
considered the main cause of death for 70% of patients with diabetes, whether by
atherosclerosis, coronary heart disease or diabetic cardiomyopathy (Cade, 2008).
Diabetic cardiomyopathy is one of the causes for the increasing number of diabetic
patients with heart failure. This occurs when patients without coronary artery disease and
hypertension experience ventricular dysfunction (Bugger & Abel, 2009). This condition is
characterized by diastolic dysfunction and eventually followed by systolic dysfunction
(Bayeva et al., 2013). The mechanism of diabetic cardiomyopathy is not yet fully
understood.
Induced diabetes in Mus musculus
Rodent models are considered essential tools in research concerning human
diseases. In particular, mice (Mus musculus) are widely accepted as a general multi-
purpose research model for studies involving genetics, nutrition, and drug toxicity testing.
Although experiments involving rats and mice yield similar results, the latter are commonly
preferred as animal models because of their ease of handling and their less aggressive
10
nature compared to the former. Mice also have good reproductive capabilities, such that
researchers are readily able to observe the activity of certain genes or compounds as they
carry over from one generation to the next in a practical amount of time (Johnson, 2012).
In a study by Wang et al. (2013), rodent models are said to exhibit diabetic
symptoms similar to that of diabetic humans. Specifically, they can spontaneously
reproduce the main features of type II diabetes mellitus. Therefore, mice can also be
considered an ideal animal model for diabetes, especially since they share 99% of their
DNA sequence with humans. Moreover, mice possess a 4-chambered heart consisting of
ventricles and atria, much like the human heart. With such similarities, it may be inferred
that the morphology and physiology of mouse organs is similar to that of humans (Johnson,
2012).
Diabetes can be induced in mice either orally (fat-fed or fructose-fed diets) or
through streptozotocin (STZ) injections. However, a recent study by Wilson & Islam
(2012) has suggested the effectiveness of using both fructose-feeding and STZ-injection
methods to induce the disease; the former is utilized to induce insulin resistance while the
latter serves to destroy the beta cell islets in the pancreas, effectively imitating the
conditions of type II diabetes (Skovsø, 2014).
The normal fasting blood glucose level of mice is around 100-130 mg/dl, which is
close to a human’s normal blood glucose level of 80-120 mg/dl (Mandal, 2013). Zhang
(2011) indicates that mice with blood glucose levels of more than 140 mg/dl are considered
diabetic.
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Protective effects of Peperomia pellucida
A study by Roslida & Noor Aini (2009) showed the anti-ulcerogenic activity of
Peperomia pellucida crude extracts, the first study on its gastroprotective effect. With the
exception of one group which was treated with 1% Tween 80 to serve as a negative control,
the researchers measured the total area of gastric lesions in all groups of rats, treated with
varying doses of the crude extract. The most effective dose was 300 mg/kg of 70%
ethanolic extract of P. pellucida in groups induced with 80% ethanol and 0.2M NaOH with
the total area of lesion of 10.17 ± 3.37mm2 and 2.80 ± 0.80mm2 respectively. For the groups
induced with 25% NaCl and 0.6M HCl, the most effective dose was 100 mg/kg of 70%
ethanolic extract of P. pellucida with a total area of lesion of 1.60 ± 0.40mm2 and 2.33 ±
0.84mm2 respectively. The results showed that the total area of lesions for the groups
treated with the extracts decreased significantly compared to the total area of lesions of the
group treated with 1% Tween 80. For the set-up induced with 30 mg/kg indomethacin, the
most effective dose was the 10 mg/kg of 70% ethanolic extract of P. pellucida with a total
area of lesion of 23.20 ± 4.09mm2. The extracts were not as effective as the 150 mg/kg
cimetidine, with only a total area of lesion of 1.00 ± 1.00mm2.
In another study by Rojas-Martinez et al. (2013), dillapiole was isolated from
Peperomia pellucida and screened for gastroprotective activity. In the experiment, the
researchers used absolute ethanol to induce ulcer in male Wistar rats. Extractions were
done with dichloromethane, hexane, and methanol. Results showed that dichloromethane
extract has the most gastroprotective effect, with a gastroprotection of 82.3% for the 100
mg/kg dose of the extract. The obtained fraction from the silica gel column was compared
12
using spectrophotometry with the known compounds, and the most active compound for
gastroprotection was identified as dillapiole.
Beltran-Benjamin et al. (2013) studied the hepatoprotective effects of the
methanolic crude extract of Peperomia pellucida on male Sprague-Dawley rats. They
induced oxidative stress by injecting trichloroethylene (TCE). The group of rats not treated
with TCE and injected with 400 mg/kg of the extract showed the highest superoxide
dismutase (SOD) and catalase (CAT) activity and the lowest thioltransferase (TT) and
thioredoxin reductase (TrxR) activity. The groups of rats induced with oxidative stress
showed a higher SOD and CAT activity and a lower TT and TrxR activity compared to the
negative control group. These results indicated that the methanolic crude extract of P.
pellucida had a protective effect on the liver of the rats.
Cardioprotective effects of family Piperaceae
Although gastroprotective and hepatoprotective effects have been reported for
Peperomia pellucida, there has been little to no record of the effects of P. pellucida on the
blood glucose and cardiovascular system of both normal and STZ-induced diabetic mice.
However, there are studies that seem to suggest such a possibility, as other members of the
plant family Piperaceae have also been reported to have protective effects. According to a
study by Arya et al. (2010), Piper betle has been reported to have protective effects against
isoproterenol-induced myocardial infarction in rats. Treatment with P. betle extract
revealed a dose-dependent protective effect, wherein the myofibrillar tissues of the heart
exhibited less degeneration and infiltration by leukocytes with increasing concentrations
of the extract during treatment.
13
The methanolic extract of Piper longum has also been shown to be effective against
isoproterenol-induced acute myocardial infarction in rats. Results showed a decrease in
vascular and fatty degeneration, as well as a decrease in hyaline muscle fiber necrosis
(Chauhan, 2010).
The effect of Piper sarmentsoum crude extract on the histology of the heart and
proximal aorta of STZ-induced diabetic Sprague-Dawley rats has been researched, as well.
The study displayed the profound effect of diabetes on the thickness of the proximal aorta,
with the untreated diabetic group possessing a thicker aorta than that of the treated diabetic
group. Moreover, the hearts of the diabetic mice that were treated with P. sarmentsoum
showed significantly reduced histological and oxidative damage than the untreated group,
having a lower number of connective tissue deposits and fewer changes in their
cardiomyocyte nuclei (Thent et al., 2012).
Metformin
Of the anti-diabetic drugs being sold today, Metformin remains one of the most
effective drugs for the treatment of diabetes. Metformin is a biguanide synthesized from
the French lilac plant, Gallega officinalis, in Europe, which was known in traditional
medicine to treat diabetes (Rojas & Gomes, 2013). Due to its proven anti-hyperglycemic
effects, this drug has been commonly prescribed for the treatment of diabetes. Several
studies have shown that this drug lowers blood glucose levels through several mechanisms.
Hundal et al. (2000) found that Metformin lowers endogenous glucose production in type
2 diabetes patients by decreasing the amount of glucose produced in gluconeogenesis.
Metformin activates adenosine monophosphate activated protein kinase (AMPK), which
14
in turn helps lower blood glucose levels. When AMPK is activated, the sensitivity of
insulin, amount of lipids and fats stored, and amount of glucose uptake increases (Boyle et
al., 2010).
Despite being an effective hypoglycemic drug, Metformin should be taken with
caution as this has numerous reported side effects. Although Metformin does not have any
effect on pregnancy, Bertoldo et al. (2014) found a decrease in the size of testis, Sertoli
cell number and size of seminiferous tubules in male mouse offspring exposed to the drug
while in the uterus. Lactic acidosis is another side effect of Metformin (Aksay et al., 2007;
Boyle, 2010). This side effect, together with rare hepatotoxicity, was observed in a 52-
year-old male (Aksay et al., 2007). Metformin was also known to cause allergies
(Wiwanitkit, 2011).
15
MATERIALS AND METHODS
Plant collection and extraction
The aerial parts of Peperomia pellucida were gathered from a local plantation in
Tarlac and taken to the Botany Division of the National Museum of the Philippines for
verification. The plants were air-dried for 3 weeks before they were ground into fine
powder using a homogenizer. The powder was soaked in 95% methanol for 3 days, then
filtered and extracted with a rotary evaporator to produce the crude methanolic extract. The
crude extract was stored in an airtight bottle before use.
Acquisition and acclimatization of mice
Thirty male mice (Mus musculus), ICR strain, each at 4-6 weeks old and weighing
between 18-24 grams, were obtained from the Food and Drugs Administration in Alabang,
and were housed individually in cages at the Animal House of the National Institutes of
Health (NIH), University of the Philippines Manila.
The mice were acclimatized for 1 week at room temperature (27 ± 2°C) and at
standard photoperiod (9-hour light, 15-hour dark cycle). Mineral water and standard feeds
were administered ad libitum throughout the week. All procedures were performed
following the guidelines of the Institutional Animal Care and Use Committee (IACUC)
and the Ethics Review Board of the NIH.
Procurement of chemical reagents
Streptozotocin (STZ) powder and phosphate buffered saline (PBS) were acquired
from Belman Laboratories, Philippines. Chemical reagents for citrate buffer were procured
16
from the Chemistry Laboratory Stockroom of the College of Arts and Sciences, University
of the Philippines Manila.
Induction of diabetes and treatment protocol
The initial weights were measured and recorded prior to treatment proper. 5 μl of
blood was milked from the periorbital sinus of each mouse and measured using Accu-
Chek® Active Blood Glucose Meter System and blood glucose test strips to get the initial
blood glucose levels. Measurement of the weights and blood glucose levels were done once
per week. The mice were then randomly divided into 6 groups: negative control (Con),
diabetic mice treated with 150 mg/kg b.wt. Metformin (Met), diabetic mice treated with
200 mg/kg b.wt. P. pellucida (PPE 200), diabetic mice treated with 400 mg/kg b.wt. P.
pellucida (PPE 400), diabetic mice treated with 800 mg/kg b.wt. P. pellucida (PPE 800),
and untreated diabetic mice (STZ/HFD).
The Met, PPE 200, PPE 400, PPE 800, and STZ/HFD groups were induced with
diabetes by feeding the mice a high-fat diet (HFD) composed of butter and rodent pellets,
mixed at a ratio of 5 grams of butter to 8 grams of pellets. The diet was then administered
alongside the mineral water ad libitum. This was maintained for 2 weeks, with each mouse
given 8 grams of the diet daily. The blood cholesterol levels of the mice fed with HFD
were measured using EasyTouch® Blood Cholesterol Test Kit and cholesterol strips.
After 2 weeks of HFD, the mice were injected intraperitoneally with 40 mg/kg b.wt.
STZ, prepared by mixing the STZ powder with 0.4 M citrate buffer pH 4, for 5 days. On
the third day after induction, the blood glucose levels of the mice were measured. Mice
with blood glucose levels of 140 mg/dL and above were considered diabetic (Zhang, 2011).
17
The blood cholesterol levels of the mice injected with STZ were also measured. The diet
of the diabetic mice was switched back to standard rodent pellets and mineral water. Mice
that remained non-diabetic despite being injected with STZ were not included in the study.
The treatment method was taken from Beltran-Benjamin et al. (2013) with slight
modifications regarding groupings and dosages. Group Con served as the negative control,
composed of non-diabetic mice with normal diet. Group PPE 200 was treated with 200
mg/kg of P. pellucida extract, Group PPE 400 with 400 mg/kg P. pellucida extract, and
Group PPE 800 with 800 mg/kg P. pellucida extract. Group Met served as the positive
control and was treated with 150 mg/kg Metformin. Finally, Group STZ/HFD consisted of
untreated diabetic mice. P. pellucida dosages were individually prepared and mixed in
phosphate buffered saline (PBS) before being administered through oral gavage.
Metformin was mixed in distilled water and also given via oral gavage. The negative
control group and untreated diabetic mice group were given PBS only. The mice were
treated daily according to their respective treatment patterns for 2 weeks.
Slide preparation and processing
Final blood glucose levels and blood cholesterol levels were measured before each
mouse was anesthetized with 0.1 mL of Zoletil 50 and euthanized via cervical dislocation.
The hearts and dorsal aortas of each mouse were excised after euthanasia. The weight of
each heart was measured and recorded, before being fixed in individual sterile cups
containing 10% buffered formalin.
18
The samples were taken to Hi-Precision Diagnostics, Remedios Branch for
histological processing. The prepared slides were then viewed under a compound light
microscope at LPO and HPO.
Histopathological analysis
Selected longitudinal sections of the heart and transverse sections of the aorta were
photo-documented and evaluated for changes in their histology based on each group’s
subjected treatment.
The epicardium, myocardium, and endocardium of the mouse hearts were
examined under hematoxylin and eosin (H&E) staining. A histopathological assessment
scale was derived from Kataoka et al. (2010), but slightly modified by Benjamin (2016) to
suit the histological findings of the study. The following parameters were considered: 1)
the degree of intercellular fibrosis in the subendocardium, myocardium, and
subepicardium; (2) the degree of infiltration of fatty materials into the myocardium; (3) the
thickness of the endocardium; (4) cardiomyocyte size and vascular degeneration; and (5)
size and shape of cardiomyocyte nuclei. The results were then scored based on the degree
of histopathological change. A score of “0” was given for the parameter if the myocardium
exhibited little to no damage (0-20%), “1” for mild damage (21-40%), “2” for moderate
damage (41-60%), and “3” for severe damage (61-100%).
The tunica intima, tunica media, and tunica adventitia of the aorta were also
analyzed under H&E stain. The thickness of the tunica intima and tunica media was
measured at four different angles with an ocular micrometer, and the average thickness of
each aorta was computed.
19
Statistical Analysis
Microsoft Excel 2013 was used to perform statistical tests for the current study. A
one-way Analysis of Variance (ANOVA) test was performed at <0.05 level of significance
to determine significant differences in the blood glucose levels between and within all 6
groups of mice. Tukey’s HSD post-hoc test was used to determine which specific groups
differed statistically from each other.
For the histopathological analysis, a Kruskal Wallis test was used in order to
determine significant differences between the 7 groups based on their assessment scale
results. Mann-Whitney U Test was then used as a post-hoc test to determine significant
differences between pairs of specific groups.
20
RESULTS
Blood glucose levels
Figure 1. Mean ± SEM blood glucose levels at initial and final levels. Different letters denote significance
(P < 0.05) within the groups.
Results showed that the final blood glucose levels of the diabetic mice increased
after they were given their respective treatments. There were no significant differences
between the blood glucose levels of all groups. Only PPE 800 group had a statistically
significant increase (P < 0.05) within their group. The rest of the groups showed no
significant result (P > 0.05) within their group.
All three P. pellucida dosages were not able to decrease blood glucose levels
significantly. Metformin was also not able to lower blood glucose levels, despite being a
commercially available anti-diabetic drug.
0
50
100
150
200
250
300
350
400
Con Met PPE 200 PPE 400 PPE 800 STZ/HFD
Me
an b
loo
d g
luco
se le
vels
(m
g/d
L)
Treatment Groups
Initial
Final
a b
21
Blood cholesterol levels
Figure 2. Mean ± SEM blood cholesterol values (mg/dL) of all groups showing initial values after STZ
injection (blue) and final values (green)
One-way ANOVA between all the groups showed no significant difference (P >
0.05). The decrease within the Met, PPE 400, and PPE 800 groups were not significant, as
well as the increase in the blood cholesterol values of the PPE 200 and STZ/HFD groups.
The negative control group also had no significant difference in blood cholesterol level
upon using t-Test to analyze their values.
0
20
40
60
80
100
120
140
160
180
200
Con Met PPE 200 PPE 400 PPE 800 STZ/HFD
Me
an
blo
od
ch
ole
ste
rol va
lue
s (
mg
/dL
)
Treatment Groups
After STZ
Final
22
Histopathological analysis of the heart
Figure 3. Photomicrographs of longitudinal sections of the mouse heart at LPO (100x) showing general
structure of myocardium. Negative control group (Con) showed normal arrangement of cardiomyocytes.
Groups treated with Metformin (Met), 200 mg/kg b.wt. P. pellucida (PPE 200), and 400 mg/kg b.wt. P.
pellucida (PPE 400) showed slightly disarrayed cardiomyocytes, but had less damage compared to the
untreated diabetic group (STZ/HFD). The group treated with 800 mg/kg b.wt. P. pellucida (PPE 800)
displayed normal arrangement of cardiomyocytes and had the closest appearance to the negative control.
Untreated diabetic group (STZ/HFD) exhibited greatest amount of damage with moderately disarrayed
arrangement of cardiomyocytes.
Results showed that common anomalies seen among the treatment groups include
irregular arrangement of myofibers in the myocardium. The myocardium was examined
because it is the layer of the heart in which the effects of diabetes are most clearly seen.
Type 2 diabetes is highly associated with the development of heart failure, which manifests
visibly in the myocardium as extracellular fibrosis in myocardial fibers and increased
interstitial (Marwick, 2006).
Con
PPE 400
PPE 200
PPE 800
Met
STZ/HFD
23
Out of all the treatment groups, the group treated with 800 mg/kg b.wt. P. pellucida
exhibited the least amount of damage, having a grade of 0 (0-20% damage) for
cardiomyocyte degeneration and a grade of 1 (21-40% damage) in terms of nuclear
enlargement/abnormality (Table 1). Of the three P. pellucida groups, the PPE 800 group
showed the closest appearance to the negative control group.
On the other hand, the groups treated with 200 mg/kg b.wt., 400 mg/kg b.wt., and
Metformin had the same amount of damage with a grade of 1 in the histopathological scale,
amounting to 21-40% damage for both cardiomyocyte and nuclei appearance (Table 1).
All three were shown to have disarrayed cardiomyocytes, although the damage was
considerably less than that of the diabetic group.
The untreated diabetic group suffered the greatest amount of damage, with a grade
of 2 (41-60% damage) for both cardiomyocyte and nuclei appearance (Table 1). Under
LPO, disarrayed cardiomyocytes can be seen.
24
Figure 4. Photomicrographs of longitudinal sections of the heart at HPO (400x) and OIO (1000x, inset)
showing myocardial fibers (MF), cardiomyocyte nuclei (N, black arrows), and size of cardiomyocyte nuclei
(inset) in cardiac tissue. The negative control group (Con) exhibited normal histology with single, central,
oval nuclei and linear arrangement of cardiomyocytes. Diabetic mice treated with Metformin (Met) showed
thinner MF and larger N compared to the negative control, but lesser damage compared to untreated diabetic
mice (STZ/HFD). Groups treated with 200 mg/kg b.wt. P. pellucida (PPE 200), 400 mg/kg b.wt. P. pellucida
(PPE 400), and 800 mg/kg b.wt. P. pellucida (PPE 800) also showed lesser damage and slightly larger N,
with PPE 800 being most similar in appearance to the negative control. Untreated diabetic group (STZ/HFD)
showed disarrayed cardiomyocytes, thinner MF, and displaced and larger N.
Results indicated that there were aberrations in the cardiomyocytes and nuclei of
all treatment groups except for the negative control. The negative control (Con) exhibited
normal heart histology with central, single, oval nuclei and a relatively linear arrangement
of myofibrils and branching pattern of the cardiac muscles.
On the other hand, the diabetic group (STZ/HFD) showed disarrayed
cardiomyocyte arrangement, thinner myocardial fibers, and cardiomyocyte nuclei that
Con PPE 200
STZ/HFD
MF
N
MF
PPE 800
MF
N
MF
Met
N
MF
N
PPE 400
M
F
N
N
25
were displaced from the center of the cardiomyocytes. The nuclei were also irregular in
shape, and found to be larger than those found in the negative control group.
The groups treated with P. pellucida showed less damage compared to the
STZ/HFD group. Of the 3 treatment dosages, the appearance of the cardiomyocytes in the
PPE 800 group was almost similar to the negative control group. On the other hand, the
group treated with 200 mg/kg b.wt. sustained the most damage among the 3 P. pellucida
doses.
The positive control group treated with Metformin (Met) showed cardiomyocytes
of normal size but having irregular arrangement. Slight hypertrophy of cardiomyocyte
nuclei was also observed. However, the damage was of a lesser extent than the STZ/HFD
group.
26
Table 1. Histopathological assessment of the heart. Ranking scale: “0” – 0-20% damage; “1” – 21-40%
damage; “2” – 41-60% damage; “3” – 61-100% damage. a and b denote significance between groups
with respect to cardiomyocyte degeneration. c and d denote significance between groups with respect
to nuclei enlargement/abnormality.
Groups
Histological Parameters
Intercellular
Fibrosis
Fatty
Infiltration
Endocardial
Thickening
Cardiomyocyte
Degeneration
Nuclei
Enlargement/
Abnormality
Con 0 0 0 0a 0c
Met 0 0 0 1b 1d
PPE 200 0 0 0 1b 1d
PPE 400 0 0 0 1b 1d
PPE 800 0 0 0 1a 0c
STZ/HFD 0 0 0 2b 2d
There were no observations of intercellular fibrosis, fatty infiltration, or endocardial
thickening in any of the groups. Performing a Kruskal-Wallis test showed a significant
difference between groups in terms of the cardiomyocyte degeneration and nuclei shape
parameters. Specifically, a Mann-Whitney U post-hoc test showed significant differences
between Con and Met; Con and PPE 200; Con and PPE 400; and Con and STZ/HFD groups
for both parameters.
27
Histopathological analysis of the aorta
Figure 5. Photomicrographs of transverse sections of the aorta at HPO (400x) showing tunica intima (black
arrows) and tunica media (asterisks) of the aortas. The negative control group (Con) displayed normal aortic
histology with no foam cells or lipid deposits observed in the blood vessel walls. Groups treated with
Metformin (Met), 200 mg/kg b.wt. P. pellucida (PPE 200), 800 mg/kg b.wt. P. pellucida (PPE 800), and
untreated diabetic mice (STZ/HFD) exhibited uneven thickness in tunica media but did not display any lipid
deposits or foam cells. The group treated with 400 mg/kg b.wt. P. pellucida had no foam cells or lipid deposits
and showed the closest resemblance to the negative control.
All groups exhibited normal aortic histology. There were no observances of foam
cells or lipid deposits, which are characteristic of atherosclerosis (Björkegren et al., 2014).
There were no abnormal thickenings observed in the tunica intima. The elastic fibers of the
tunica media were intact.
STZ/HFD
Con PPE 200
PPE 400 PPE 800
Met
*
*
*
*
*
*
28
DISCUSSION
Blood glucose levels
In the experiment, the blood glucose levels of the positive control (Met), PPE 400,
PPE 800, and STZ/HFD groups increased after the mice received their respective
treatments. However, PPE 800 group was the only group that had a significant increase
(Figure 1). Hamzah et al. (2012) proposed that the mechanism of action of P. pellucida is
the same with sulfonylureas, which lower blood glucose levels by stimulating insulin
secretion. P. pellucida contains metabolites (flavonoids, glycosides, alkaloids) that can
stimulate insulin secretion (Sheikh et al., 2013; Gaikwad et al., 2014). P. pellucida had
fewer β-cells to stimulate because of STZ, which selectively destroyed pancreatic β-cells
continuously. Thus, the extract was not effective at lowering blood glucose levels.
However, partial β-cell islet recovery can occur after 120 days (Yin et al., 2006). Therefore,
it is possible that P. pellucida extract may decrease blood glucose levels after this period
of time. Results also showed that Metformin did not lower blood glucose levels, despite
being the most widely used anti-diabetic drug. This is because of the lack of time for
treatment, as well as the STZ still taking effect in the pancreas of the mice.
Results were not consistent with previous studies (Hamzah et al., 2012; Sheikh et
al., 2013). These discrepancies are due to the use of a different solvent for extraction by
previous researchers, having a longer period for treatment, and most notably, using alloxan
in inducing diabetes. Alloxan and STZ both target the beta cells of the pancreas; however,
alloxan is a less stable diabetes-inducing chemical. Some alloxan-induced diabetic animals
show fluctuations in their blood glucose levels and even return within the normal range
(Jain & Arya, 2011; Kumar et al., 2012).
29
Blood cholesterol levels
The total blood cholesterol levels of the mice have no significant differences (P >
0.05) between and within groups. The lack of significant differences between and within
the groups is due to the proven anti-hypercholesterolemic effect of P. pellucida extract
(Hamzah et al., 2012; Alfonso & Riego de Dios, 2015; Mazroatul et al., 2016). Alfonso &
Riego de Dios (2015) conducted a study on the hypocholesterolemic effect of P. pellucida
methanolic crude extract on hypercholesterolemic rats. Their study showed that all P.
pellucida doses (200 mg/kg b.wt.; 400 mg/kg b.wt.; 800 mg/kg b.wt.) decreased the blood
cholesterol levels of hypercholesterolemic rats. There were also no significant differences
between the groups treated with the extract, indicating that all doses were effective in
decreasing high cholesterol. Saponins are active compounds in the plant that lower blood
cholesterol. Metformin can also lower blood cholesterol levels (Salpeter et al., 2008;
Geerling et al., 2014). Metformin decreases the LDL by 5% and increases HDL by 5%,
which reduces risk of contracting cardiovascular diseases (Salpeter et al., 2008).
Histopathological analysis of the heart
Diabetes has been shown to cause irregularities in the macrovascular structure of
the heart; histological aberrations in the cardiac tissue may often lead to complications like
diabetic cardiomyopathy and heart failure in the diabetic heart (Unachukwu & Ofori, 2012).
These abnormalities include intercellular fibrosis, fatty infiltration, endocardial thickening,
cardiomyocyte degeneration, and changes in cardiomyocyte nuclei (Kataoka et al., 2010).
In the study, aberrations were limited only to cardiomyocyte degeneration and
nuclei enlargement. Cardiomyocyte degeneration was found to be most prominent in the
30
diabetic group; while the negative control group had hearts with a relatively neat and linear
arrangement of myocardial fibers, the diabetic mouse hearts showed thinner myofibrils,
accompanied by wider interstitial spaces that contained scattered neutrophils. The thinner
myofibrils observed in the STZ/HFD group were caused by their high blood glucose levels.
A study by Dyntar et al. (2006) showed that exposing cardiac cells in vitro to high blood
glucose concentrations reduced their capability of forming myofibrils. The effect of
glucose on myofibril formation was prevented by antioxidant regimens (Dyntar et al.,
2006). The nuclei of the cardiomyocytes in diabetic hearts were found to be displaced and
larger in size than those found in the negative control group. The groups treated with
Metformin and P. pellucida also exhibited slight enlargement of nuclei, albeit less than that
of the untreated group. Thinner myofibrils and enlargement of cardiomyocyte nuclei are
early symptoms of STZ-induced diabetic cardiac dysfunction (Cosyns et al., 2007; Thent
et al., 2012). These traits also point to early stages of myocardial infarction in the diabetic
hearts (Eckhouse & Spinale, 2012).
The groups treated with P. pellucida sustained myocardial damage due to high
glucose levels. However, the level of damage was lower than that of the untreated diabetic
group. This is due to P. pellucida having proven antioxidant properties that reduce
oxidative damage to the myocardium (Hamzah et al., 2012; Beltran-Benjamin, 2013).
Comparing the three doses of P. pellucida given, the PPE 800 group had the least
damage while the PPE 200 group suffered the most damage. This suggests that P.
pellucida’s cardioprotective effect is concentration-dependent, meaning that there is less
damage at high doses of P. pellucida and more damage at low doses. This is supported by
the results of the histopathological scale modified from Kataoka et al. (2010), particularly
31
in regards to cardiomyocyte degeneration and nuclei hypertrophy. The negative control
was significantly different from all other groups except for the PPE 800 group. This implies
that only the PPE 800 group had a relatively normal histology when compared to the non-
diabetic heart. P. pellucida is cardioprotective at 800 mg/kg b.wt.
Comparing the diabetic group with the Met group, it was observed that the hearts
of mice treated with Metformin displayed less myocardial damage. Metformin is known to
be an effective anti-hyperglycemic drug, and recent studies have indicated that it also has
a cardioprotective effect (Eurich & McAlister, 2011; Eurich et al., 2013).
There were no signs of fibrosis observed in the subendocardium, myocardium, and
subepicardium in all groups. Fibrosis occurs in cardiac tissues when there is increased
collagen production by myocardial fibroblasts, promoting stiffness and loss of contractile
functions in the heart (Conrad et al., 1995). Of particular note is interstitial fibrosis, a type
of fibrosis that mainly affects the interstitial spaces between myocardial fibers and one that
may arise due to metabolic problems caused by hyperglycemia, which is characteristic of
diabetes (Mewton et al., 2011). While the blood glucose levels of the mice were elevated,
there was not enough time for fibrosis to be induced on the myocardium. Chen (2014)
states that it takes 2 ½ months for diabetic cardiomyopathy and heart failure to develop in
the STZ-induced diabetic heart. Since the treatment period of the experiment only lasted 2
weeks, fibrosis was not observed.
Similarly, there were no occurrences of endocardial thickening in any of the groups.
Thickening of the endocardium is highly associated with intercellular and endomyocardial
fibrosis, accompanied by cardiomyocyte necrosis in both the endocardium and adjacent
32
myocardial fibers, which eventually leads to heart failure (Zaidi et al., 1982; Seki et al.,
2013). Since there was no fibrosis in the mouse hearts, endocardial thickening is also not
expected to occur. Both intercellular fibrosis and endocardial thickening are signs of
cardiomyopathy (Seki et al., 2013). As previously stated, it takes 2 ½ months for the onset
of diabetic cardiomyopathy, which means that there was not enough time for both
parameters to be observed (Chen, 2014).
The infiltration of adipose tissue in the myocardium is associated with obesity and
Type 2 diabetes; it signals hypercholesterolemia and the subsequent impediment of proper
conduction within the heart (Balsaver et al., 1967; Iozzo, 2011). However, there was no
fatty infiltration observed in any of the groups. As previously stated, initial and final blood
cholesterol levels in all groups did not show any significant decrease or increase (Figure
2), suggesting that all values were more or less close to normal. Moreover, the
accumulation of excess myocardial fat content is highly unlikely due to P. pellucida having
an anti-hypercholesterolemic effect (Hamzah et al., 2012; Mazroatul et al., 2016).
Histopathological analysis of the aorta
There were little to no abnormalities observed in the histology of the aortas in all
groups. This is due to the short amount of time in the study. It takes at least 12 weeks for
hypercholesterolemic mice to develop atherosclerosis (Bjørklund et al., 2014). However,
the treatment period only lasted for 2 weeks.
33
In diabetic mice, hyperglycemia alone has no effect in progression of
atherosclerosis (Chait & Bornfeldt, 2009). However, hyperglycemia accompanied with
hyperlipidemia accelerates development of atherosclerosis (Kanter et al., 2007). No group
of mice had hypercholesterolemia. Moreover, as stated earlier, P. pellucida and Metformin
have anti-hypercholesterolemic effects, which contributed to preventing atherosclerosis
from developing in the aortas of the mice.
34
CONCLUSION AND RECOMMENDATIONS
The results of the present study showed that P. pellucida methanolic crude extract
has a cardioprotective effect on the heart of STZ-induced diabetic mice, and is most
effective at 800 mg/kg b.wt based on histopathological evidence. However, the extract was
found to be ineffective at lowering blood glucose levels.
The researchers recommend that a longer period of time should be allocated for the
study to ensure that diabetes is properly induced. The effect of the P. pellucida extract on
blood glucose levels and histology of the heart and aorta should also be evaluated. Usage
of special stains during histological processing is also recommended for better visualization
of myocardial damage (e.g. Masson’s Trichrome Stain for better visualization of fibrosis).
Further studies should be conducted on P. pellucida and its effect on diabetes in
order to rectify discrepancies on its effect as an anti-diabetic extract. The exact mechanisms
and the specific active compounds behind the supposed anti-diabetic properties of the P.
pellucida extract should be explored and discussed in further detail.
35
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42
GANTT CHART
LINE-ITEM BUDGET
Mice ............................................................................................................ Php 1, 700.00
Equipment and processing fees ...............………………………………... Php 27, 503.00
Housing fees …………………………………………………………....... Php 4, 920.00
Reagents ………………………………………………………………..... Php 18, 368.00
Histological sectioning fees ……………………………………………... Php 12, 600.00
Transportation ………………………………………………………........ Php 1,000.00
Miscellaneous …………………………………………………………….Php 2,000.00
TOTAL …………………………………………………………............. Php 68, 091.00
43
APPENDIX A
Blood glucose levels of mice
Table A-1. Blood glucose values (mg/dL) of each mouse for the whole duration of the experiment starting
from their initial values (Wk 1), 2 weeks of HFD for diabetic mice (Wk 2-3), after injection of STZ for
diabetic mice (Wk 4), and the remaining weeks for treatment (Wk 5-8)
By Week
Grou
p
Mice Wk 1 Wk 2 Wk 3 Wk 4 Wk 5 Wk 6 Wk 7 Wk
8
Con
1A 153 160 138 142 155 98
1B 137 146 133 133 163 146
1C 125 154 101 132 149 137
1D 111 150 113 84 73 86
1E 124 139 126 105 117 130
1F 146 168 131 129 160 133
Averag
e
132.7 152.8 123.7 120.8 136.2 121.7
Met
4D 149 150 199 384 129 220
5C 104 149 195 205 99 170
4A 140 81 125 107 175 220 232
2F 148 151 149 168 170 272 275
Extra 124 165 143 97 174 134 134
Averag
e
133 139.2 162.2 192.2 149.4 203.2 213.6
7
PPE
200
3F 118 121 144 264 105 194
6B 106 146 148 239 168 468
5A 128 153 176 399 188 275
5E 118 132 148 164 167 164 239
2E 91 92 127 124 139 131 224
Averag
e
112.2 128.8 148.6 238 153.4 246.4 231.5
PPE
400
2B 109 135 120 233 264 134
4B 106 146 168 416 156 411
5B 94 171 186 122 210 455 481
5F 133 151 174 124 203 229 302
3D 118 130 180 123 196 332
Averag
e
112 146.6 165.6 203.6 205.8 312.2 391.5
44
PPE
800
2D 83 111 126 157 118 160 251 366
3A 111 114 154 137 100 181 264 232
3B 113 114 153 147 127 180 423 367
3C 120 148 146 166 115 167 293 156
6C 104 64 134 106 137 148 210 408
Averag
e
106.2 110.2 142.6 142.6 119.4 167.2 288.2 305.
8
STZ/
HFD
2A 136 119 dead dead dead dead
3E 139 127 189 300 dead dead
6A 92 140 148 116 246 205 378
2C 152 150 134 173 103 211 151
4E 146 134 126 178 80 371 454
OLD
6D
150 122 181 253 194 179
OLD 6E 134 180 204 116 171 133
OLD 6F 147 145 165 212 165 162
Averag
e
137 139.6
3
163.8
6
192.5
7
159.8
3
210.1
7
45
APPENDIX B
Blood cholesterol levels of mice
Table B-1. Blood cholesterol values of representative mice of diabetic groups after 2 weeks of HFD, after
injection of STZ, and after 2 weeks of treatment
Group Mice HFD After
STZ
Treatment
Wk 1
Treatment
Wk 2
PPE
200
3F 167 138
5A 160 202
5E 120 174 138
Average 140 170.5 159.3
PPE
400
4B 131 138
5B 160 121 102
3D 129 156 118
Average 130 158 119.5 120
PPE
800
2D 128 130 148
3C 196 106
6C 152
Average 140 163 127
Met
4A 158 132
2F 121 170
Extra 170 101
Average 139.5 170 116.5
STZ/HFD
2C 98 106 110
4E 149 148 149
Average 123.5 127 129.5
46
APPENDIX C
Plant Identification/Certification Form
47
48
APPENDIX D
IACUC Certificate of Approval