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

viii

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;

ix

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

2

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.

6

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.

11

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