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EFFECTS OF XANTHONE GLYCOSIDE FROM Peperomia pellucida (L.) Kunth

AGAINST HYPERGLYCEMIA AND ALTERATIONS IN PANCREAS OF HFD/STZ-

INDUCED DIABETIC MICE Mus musculus

DIANNE MAE B. TAN

JOLAINE ASHLEY T. VALLO

An Undergraduate Thesis

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

May 2018

Department of Biology

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TABLE OF CONTENTS

TABLE OF CONTENTS………………………………..……………………………………......ii

LIST OF FIGURES………………………………..……………………………………….…......v

LIST OF TABLES………………………………..…………………………………….…….......vi

LIST OF APPENDICES………………………………..……………………………………......vii

ACKNOWLEDGEMENT………………………………..……………………………………..viii

ABSTRACT………………………………..…………………………………………………......1

INTRODUCTION………………………………..………………………………………….........2

Background of the Study..………………………………………….....….....…..…...........2

Statement of the Problem..…………………………………………...….....…....…..........4

Research Objectives..………………………………………………………….....….........4

Significance of the Study..……………………………………...….....………………......5

Scope and Limitations of the Study..………………………………………........…..........5

REVIEW OF RELATED LITERATURE………..…………………………………………........7

Morphology of Peperomia pellucida…..…………………………………………............7

Chemical Constituents of Peperomia pellucida…..………………………………............7

Xanthone Glycoside…..…………………………………………......................................8

Pharmacological Properties of Xanthone Glycoside…..……………………………..…..9

Diabetes Mellitus…..………………………………………….........................................10

Type II Diabetes Mellitus…..…………………………………………............................11

Gross Morphology and Histology of the Pancreas...………………………………...…..11

Histopathological Studies of Islets in Type II Diabetes Mellitus…..………………........13

HFD/STZ Mouse Model…..…………………………………………….....…….…........13

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MATERIALS AND METHODOLOGY…………………………………….....………..............15

Plant Extraction and Fractionation…………………………………….....…….…...........15

Purification of Xanthone Glycoside…………………………………….....…….….........15

Acclimatization of Mice…………………………………….....…….…..........................17

Induction of Diabetes…………………………………….....……….................…...........17

Experimental Set-up…………………………………….....…….…....….........................18

Obtaining Body Weight…………………………………….....…….….…......................19

Blood Glucose Analysis…………………………………….....…….…….......................19

Examination of Gross Morphology and Histopathology of Mice Pancreas…….….........19

Statistical Analysis…………………………………….....…….…...................................21

RESULTS…..…………………………………………….....…….…..........................................22

Body Weight…..…………………………………………….....…….…..........................22

Blood Glucose Levels…..…………………………………………….....…….…............24

Gross Morphology of Pancreas…..…………………………………….....…….…..........26

Islet Diameter…..…………………………………………….....……………............…..28

Histopathology of Pancreas…..……………………………………….....…….…...........29

DISCUSSION…..…………………………………………….....…….…....……….....………...33

Induction of Type 2 Diabetes…..……………………………………….....…….….........33

Effect on Body Weight…..…………………………………………….....…….…..........33

Effect on Blood Glucose Levels…..……………………………………….…….…........35

Pancreas Morphology, Histology and Islet Diameter…………………….……...............41

Group A: Negative Control (Standard Diet)……….…….…..….…..….…..........43

Group B: HFD + STZ + Metformin……….…….…....….…..….…..….……......43

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Group C: HFD + STZ + 10 mg/kg Xanthone Glycoside……….…….….............46

Group D: HFD + STZ + 20 mg/kg Xanthone Glycoside……….…….….............48

Group E: HFD + STZ + 40 mg/kg Xanthone Glycoside……….…….….............50

Group F: Positive Control (HFD + STZ)……….…….…….…..….…..…...........51

CONCLUSION……….…….…......….…….…......….…….…......….…….…............................53

RECOMMENDATION……….…….….....….…….…......….…….…......….…….....…............54

LITERATURE CITED……….…….….…….…......….…….…......….…….…..........…............55

APPENDICES……….…….…….…….…......….…….…......….…….…...................................69

BIOGRAPHICAL DATA……….……….…….…......….…….…......….…….….......…............77

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LIST OF FIGURES

FIGURE PAGE

1 Mean body weights of all groups of male ICR Mus musculus from

the week after acclimatization and after STZ/HFD induction………………….23

2 Mean body weights of groups of male ICR Mus musculus from

the initial and final week of treatment with xanthone glycoside………….……23

3 Mean blood glucose levels of all groups of male ICR Mus musculus

before and after STZ induction………………………………………………….25

4 Mean blood glucose levels (mg/dL) of all groups of male ICR

Mus musculus before and after treatment of xanthone glycoside……………….25

5 Gross Morphology of the Pancreas……………………………………………...27

6 Mean diameters of the pancreatic Islets of Langerhans per male ICR

mice group…………………………………..…………………………………...28

7 Mean histological scores of pancreatic tissue samples from HFD/STZ-induced

hyperglycemic male ICR mice……………………………..…………………....29

8 Representative Micrographs of Hematoxylin and Eosin-Stained (H&E)

Pancreatic Islets of Mus musculus…………………………………………….....30

9 Representative Micrographs of Hematoxylin and Eosin-Stained (H&E)

Pancreatic Acinar Cells of Mus musculus……………………………………......31

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LIST OF TABLES

TABLE PAGE

1 Scoring system for histological alterations as indicators of damages in

the pancreatic acinar and islets of Langerhans of diabetic male ICR mice…......20

2 Summary of the histological alterations observed in pancreatic islets

treated with xanthone glycoside obtained from P. pellucida

methanolic extract.………….……………….……………….………………….32

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LIST OF APPENDICES

APPENDIX PAGE

A Letter of Approval for Implementation from the IACUC, NIH………………...69

B Certificate of Authentication from Institute of Biology, College of Science,

University of the Philippines Diliman…………………………………………..70

C Phytochemical Analysis Results of Butanol Fraction from P. pellucida

from NIH, UP Manila…………………………………………………………..71

D Computation for Total Xanthone Glycoside Needed…………………………...73

E Computation for Streptozotocin Amount………………….…………….……...73

F1 Paired T-Test Results Between Before and After HFD/STZ Induction

Body Weights……………….…………….…………….…………….………...73

F2 Paired T-Test Results Between Body Weights of Mouse Subjects

Before and After Treatment……….…………….…………….…………….….74

G1 Paired T-Test Results Between Blood Glucose Levels of Mouse Subjects

Before and After STZ Induction……….…………….…………….…………...74

G2 Paired T-Test Results Between Blood Glucose Levels of Mouse Subjects

Before and After Treatment……….…………….…………….……...………...74

H1 Kruskal-Wallis One-Way Analysis Results for Mean Histological

Index Scores……………….…………….…………….…………….…..……...75

H2 Mann-Whitney U Test Results for Mean Histological Index Scores...………....75

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ACKNOWLEDGEMENT

The researchers would like to express our deepest and wholehearted gratitude to the following:

Our thesis adviser, Prof. Kimberly S. Beltran-Benjamin, for her unwavering guidance,

patience and support, and for simply believing that we could successfully make it until the end;

Dr. Elisa L. Co and Dr. Thucydides L. Salunga for sharing their wonderful insights and

criticisms on our study;

Kuya Edgar and Kuya Max for all the encouragements and for cheering us up when things go

bad, and as well as for having patience when we borrow laboratory equipment;

Kuya Mel of UP-NIH for teaching and assisting us in mice handling during the duration of the

experiment;

Mang Mon of UP Diliman for helping us in collecting and authenticating our plant samples;

To our home for four years, the Department of Biology, for always pushing us to always strive

for excellence and for helping us prepare for our future careers;

To our fellow blockmates and friends who have been with us for four years for all the times we

keep each other sane and happy;

To our families who have been always there and constantly supporting us all throughout our

thesis.

Most of all, we thank our Almighty God for endlessly guiding us in all of our endeavors

especially in conducting this study.

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ABSTRACT

Type II diabetes mellitus (T2DM) known to be the most prevalent form of diabetes, is

caused by defects in insulin secretion, decreased sensitivity of insulin target cells, and β-cell

dysfunction. This study tests the effects of xanthone glycoside from Peperomia pellucida on

blood glucose levels and pancreas histology of HFD/STZ- induced diabetic Mus musculus.

The experiment used 30 mice divided equally into 6 groups. Two groups served as the

negative and positive control, while the remaining 4 groups were administered with

metformin, 10 mg/kg xanthone glycoside, 20 mg/kg xanthone glycoside, and 40 mg/kg

xanthone glycoside. Body weight, blood glucose, islet diameter, and pancreas histology were

obtained and analyzed. Results indicate that 20 mg/kg xanthone glycoside from P. pellucida

significantly lowered blood glucose levels and can reduce alterations in pancreatic histology

of HFD/STZ induced diabetic Mus musculus.

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INTRODUCTION

Background of the Study

According to the International Diabetes Federation (IDF) (2015), approximately 415

million adults have diabetes and by 2040 it will rise up to 642 million adults. About 90% of

the cases belong to Type II Diabetes because of unhealthy lifestyle. IDF estimated the

occurrence of diabetes in Filipino adults ages 20-79 years of age is around 6.5% and will

increase to 7.9% by 2025; thus, there is an alarming growth of diabetes among adult Filipinos

in the Philippines (Higuchi, 2010). Diabetes mellitus is a metabolic disorder characterized by

chronic high levels of blood glucose levels resulting in abnormalities in insulin secretion,

action, or both (American Diabetes Association, 2014). Additionally, insulin is a hormone

that facilitates the absorption of glucose from blood in skeletal muscle cells and hepatocytes

(Reeven, 1988). There two types of diabetes: Type I (Insulin Dependent Diabetes Mellitus)

and Type II (Non-insulin Dependent Diabetes Mellitus). Chronic effects of diabetes mellitus

include: blood vessel and nerve damages, multiple organ failures, and death (National

Diabetes Data Group, 1979).

Currently, synthetic medicines are made available for diabetic patients- an example of

which being metformin. Metformin is an oral drug used to control blood glucose levels in the

body specifically on Type II Diabetes patients (Viollet, et al., 2012). It is also part of World

Health Organization’s Model List of Essential Medicines as of 2011. Despite being a first-

line treatment for T2DM, metformin has several side effects. Some adverse effects include

lactic acidosis, acute renal failure, and vitamin B12 deficiency (Fitzgerald, Mathieu & Ball,

2009; De Jager, et al., 2010).

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Plants have also been used as traditional folk medicine for several types of diseases.

The Philippines holds a high number of medicinal plants due to its existing plant diversity

(Balinado & Chan, 2017). Medicinal plants are easy to access, simple to prepare, and

believed to be safe and effective (Savithramma, Linga Rao, & Ankanna, 2011). Moreover,

utilizing medicinal plants in the Philippines would allow for those in the marginalized sector

to receive quality health care and services. (Balinado & Chan, 2017). In 2007, the Philippine

Department of Health (DOH) endorsed ten medicinal plants through the Philippine Institute

of Traditional and Alternative Health Care (PITAHC) under the Traditional Program. The list

includes: Cassia alata (akapulko), Momordica charantia (ampalaya), Allium sativum

(bawang), Psidium guajava (bayabas), Vitex negundo (lagundi), Quisqualis indica L. (niyog-

niyogan), Blumea balsamifera (sambong), Ehretia microphylla Lam. (tsaang gubat),

Clinopodium douglasii (yerba buena), and Peperomia pellucida L. (pansit-pansitan). These

herbs have been tested and clinically proven to have medicinal value (Department of Health,

2007).

P. pellucida, locally known as pansit-pansitan or ulasimang-bato, has several

medicinal properties such as anti-inflammatory (Arrigoni-Blank et al., 2004; Mutee, et al.,

2010), analgesic, antipyretic (Khan, Rahman, & Islam, 2008), antibacterial (Wei, et al.,

2011), anti-cancer (Xu, et al., 2006; Wei, et al., 2011), anti-arthritis (Mutee, et al., 2010) ,

antihyperuricemic (Sio, Sia, & Cortes-Maramba, 2001), anti-ulcer (Roslida & Noor Aini,

2009) and hypoglycemic (Hamzah, et al., 2012). In the Philippine ethnomedicine, P.

pellucida is traditionally used for treating arthritis and lowering of uric acid levels (Heinrich

& Jäger, 2015). The Peperomia species are known to have the presence of xanthones.

Xanthones are secondary metabolites found in lichens, fungi and higher plant families. Their

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structures are similar to that of flavonoids, but only appear in a limited number of plant

families. Xanthones are often attached with parent polyhydroxylated compounds or with

glycosides (Negi et al., 2013). According to Bo and Liu (2004), xanthones are of great

interest due to its several pharmacological properties.

In a study done by Hamzah and his colleagues (2012), it was presented that P.

pellucida had hypoglycemic properties. Furthermore, a study conducted by De Loreto and

Magbuhat (2016) showed that the plant exhibited hypoglycemic effects on HFD/STZ

induced diabetic mice. As such, this study aims to test a specific phytochemical constituent

of P. pellucida, a xanthone glycoside, and its effect on blood glucose levels and pancreas

histology of HFD/STZ induced diabetic mice. The antihyperglycemic properties of xanthone

glycoside from P. pellucida remain to be acknowledge.

Statement of the Problem

Does xanthone glycoside from P. pellucida (L.) Kunth have a significant effect on

blood glucose levels and pancreas histology of HFD/STZ-induced diabetic Mus musculus?

Research Objectives

In general, this study aims to test the potential effects of xanthone glycoside obtained

from P. pellucida (L.) Kunth on blood glucose levels and pancreas histology of HFD/STZ-

induced diabetic Mus musculus. Specifically, this research seeks (1) to compare the effects of

xanthone glycoside from P. pellucida with commercially sold drug metformin; (2) to

determine the effective dosage of xanthone glycoside for reducing elevated blood glucose in

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Mus musculus, and (3) to assess the effect of xanthone glycoside on the pancreas histology of

Mus musculus.

Significance of the Study

The use of herbal medicinal products and supplements has continued to expand

rapidly over the past three decades with not less than 80% of the world population depending

on them as primary healthcare (Ekor, 2014). Studies on P. pellucida, one of the top ten

medicinal herbs endorsed by the Department of Health (DOH) (Saguibo and Elegado, 2012)

particularly on its preventive and curative properties makes a high value research as it may

provide new and important leads for drug discovery. To date, there are multitudes of study on

P. pellucida and its various potential medicinal uses. In particular, its potential in reducing

hyperglycemic blood glucose levels has been a topic of discussion in several researches

(Beltran-Benjamin et. al., 2013; Hamzah et. al., 2012; Gbolade 2009) albeit the main

composition of P. pellucida responsible for its anti-hyperglycemic properties was not

discussed. By expanding the prospects on xanthone glycoside, a component of P. pellucida,

and its anti-hyperglycemic effects, novel drugs against Diabetes Mellitus can be discovered.

Scope and Limitations of the Study

The study was limited to the assessment of three different dosages of xanthone

glycoside (10 mg, 20 mg, and 40 mg) obtained from P. pellucida and its anti-hyperglycemic

effects in Mus musculus through measurement of blood glucose levels and analysis of

pancreas histology. The three different dosages, together with commercially sold drug

metformin (40 mg/kg), was compared based on their efficacy. In addition, positive control

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and experimental groups would be fed with High Fat Diet/Streptozotocin. Lastly, only male

mice aged 8 weeks, and weighing ~35g would be studied.

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REVIEW OF RELATED LITERATURE

Morphology of Peperomia pellucida

P. pellucida (L.) Kunth, of the Piperaceae family, is a common and annual herb,

native to tropical Asia, Africa, and Central and South America. In the Philippines, it is

commonly known as “pansit-pansitan” or “ulasimang bato”. The plant is erect, usually 15-45

cm long, with fibrous and shallow roots. It has a thread-like but angular trailing stem;

although those growing in rich habitats have succulent round stems. The internodes are

glabrous and usually measure 3-8 cm long. Its leaves are palmately veined, alternate in

phyllotaxy, cordate-shaped, and smooth in texture. The plant possesses tiny greenish-yellow

bisexual flowers which are loosely arranged in a spike inflorescence. Its fruit is a globose

drupe, green when unripe, and turns black as it matures (Theresa-Ibibia, 2012; Dy & Olotu,

2016; Guillermo, 2015).

Chemical Constituents of Peperomia pellucida

A variety of chemical constituents were found in P. pellucida. Preliminary

phytochemical screening revealed that there are secondary metabolites such as tannins,

saponins, flavonoids, phenols, phytosterols, steroids, terpenoids, sesquiterpenes, alkaloids,

and glycosides. Such compounds are known for their potential medicinal properties

(Savithramma, Linga Rao & Ankanna, 2011). The confirmation of such chemical

constituents further supports the medicinal properties of P. pellucida.

Tannins were found to have strong free radical scavenging activities, the ability to

treat bleeding, wounds and skin afflictions, and dysentery (Hu et al., 2004; Scalbert, 1991).

Flavonoids have antioxidant and free radical scavenging properties (Middleton, Kandaswami

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& Theoharides, 2000). Its mechanism in treating diseases is its ability to inhibit specific

enzymes, simulate certain hormones and neurotransmitters and scavenge free radicals

(Havsteen, 2002). Plant-derived triterpenoids and saponins exhibit anti-inflammatory and

hypocholesterolemic properties (Balandrin, 1996). Meanwhile, alkaloids are the most diverse

group of secondary metabolites. Alkaloids are known to have analgesic and anti-microbial

properties. Alkaloids and its derivatives are also used as muscle relaxants and

parasympathetic agents (Roberts & Wink, 2013; Garba & Okeniyi, 2011).

Dill apiole was another prominent chemical constituent found in P. pellucida

(Francois, et al., 2013; da Silva, et al., 1999). Different oil compositions of P. pellucida from

different countries reveals that dill apiole is a characteristic constituent. The essential oils

found in different origins only significantly different in a few major and minor chemical

constituents. This further supported by a study done by Narayanamoorthi and his constituents

(2015) where apiol is the most common constituent. Apiole is a known organic compound

that was first seen in parsley and dill. It can alleviate amenorrhea but also an abortifacient

(Ragan, 2010.) Another notable chemical constituent that is often found in P. pellucida are

the secoligans. These secoligans are named peperomins. Some examples of which are

Peperomins B and E, which are said to exhibit inhibitory effect on tumor cells. Other effects

include anti-HIV and anti-inflammatory (Xu, et al., 2006).

Xanthone Glycoside

Xanthones are a class of naturally and synthetically-occurring, oxygen-containing

heterocyclic compounds, that are prominent in the field of medicinal chemistry due to their

extensive biological activities. Xanthone compounds always occur in the families

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Gentianaceae, Guttiferae, Moraceae, Clusiaceae, and Polygalaceae (Negi et. al., 2013). Their

wide usage can be attributed to their chemical structure which allows a variety of substituents

to position on its aromatic ring. Among the compound’s many derivatives include xanthone

glycosides, which is any member of the class of xanthones having one or more glycosyl

residues attached at unspecified positions (Shagufta & Ahmad, 2016). To be more specific,

xanthones glycosides are further classified as either C-glycosides or O-glycosides. In C-

glycosides, a C-C bond links the sugar moiety to the xanthone nucleus and they are therefore

resistant to acidic enzymatic hydrolysis whereas the O-glycosides have typical glycosidic

linkage (Sultanbawa, 1979).

Pharmacological Properties of Xanthone Glycosides

Several studies have elucidated the various applications of xanthone glycosides in

medicine. In a study conducted by Teng and her colleagues (1989), xanthone glycosides were

tested for their anti-platelet activities in washed rabbit platelets and found that it inhibited

platelet aggregation and release reaction. In another study by Lin and his collaborators

(1984), results claimed xanthone glycosides to have a remarkable central stimulant effect in

mice and a depressant effect in rats. In an antimycotic study conducted by Khan and his

associates (2010), they isolated patuloside, a xanthone glycoside from P. pellucida, and

found that it had significant antibacterial activity against both Gram-positive bacteria and

Gram-negative bacteria.

In a similar investigation of Muruganandan and his fellow researchers (2005), they

studied the effect of mangiferin, a xanthone glucoside from the leaves of Mangifera indica,

and its atherogenic potential in streptozotocin-induced diabetic rats. Furthermore, their study

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showed significant anti-hyperlipidemic and anti-atherogenic activities of the compound. With

chronic administration (14 days), it significantly improved oral glucose tolerance in glucose-

loaded normal rats suggesting its potent antihyperglycemic activity. Their preliminary

investigations, however, showed that rats exhibited signs of depression when administered

with mangiferin with doses beyond 100 mg/kg through the intraperitoneal route. Thus, their

study tested for 10 and 20 mg/kg xanthone glycoside concentration administered

intraperitoneally, which showed no gross signs of toxicity. Moreover, mangiferin was

reported to have poor bioavailability in the oral route in rats (Geodakyan et. al., 1992) and

showed signs of pharmacological effects when administered intraperitoneally (Guha, Gusal,

& Chattopadhyay, 1996).

Diabetes Mellitus

Diabetes mellitus is defined as a group of metabolic diseases characterized by

abnormally high levels of blood glucose or hyperglycemia. Diabetes is commonly caused by

defects in insulin secretion, insulin action, or both (American Diabetes Association, 2014).

There are two types of diabetes mellitus: Type I and Type II. Type I diabetes or juvenile

diabetes, formerly known as insulin-dependent diabetes mellitus (IDDM), is caused by

pancreatic islet β-cell destruction. Ketoacidosis is a common feature of this kind of diabetes,

and often attributed to autoimmune disease. Type II diabetes, formerly known as non-insulin-

dependent diabetes mellitus (NIDDM), is caused by defects in insulin secretion, decreased

sensitivity of insulin target cells, and β-cell dysfunction. This type of diabetes always lead to

insulin resistance (Alberti & Zimmet, 1998). Both types of diabetes lead to hyperglycemia,

polyuria (frequent urination), polydipsia (excessive thirst), ketonuria (presence of ketones in

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urine), and sudden weight loss, together with gross and unequivocal elevation of plasma

glucose (PG) (National Diabetes Data Group, 1979).

Type II Diabetes Mellitus

Type II diabetes mellitus (T2DM) occurs when the pancreas does not produce enough

insulin. It is the most prevalent form of diabetes, accounting for about 90% of the total

diabetic patients (Stumvoll, van Haeften, & Goldstein, 2005); and can be improved, and in

some instances reversed (Peterson, 2014) by making positive lifestyle choices such as eating

a healthy diet, exercising on a regular basis, and losing excess weight.

To further understand the pathophysiology of T2DM, it is necessary to conceptualize

how glycemia is controlled. Insulin is the principal hormone responsible in mediating blood

glucose levels. For a normoglycemic individual, as blood glucose levels rise, or in the case of

after a meal (2-hour Plasma Glucose of less than 200 mg/dL), insulin levels likewise

increase. Conversely, once blood glucose levels drop, there will be decreased release of

insulin and increased release of glucagon for glycogenolysis or the conversion of glycogen to

glucose (American Diabetes Association, 2017; Lin & Sun, 2009). Specifically, insulin

regulates the uptake of glucose from the blood into the cells. Important to the regulation of

insulin is the sensitivity of pancreatic β-cells to adapt to changes in insulin action.

Gross Morphology and Histology of the Pancreas

In rodents, the pancreas is characterized as a lobular and tan gland suspended in the

mesentery, between the stomach, duodenum and ascending and transverse colons. The gland

is divided into irregular lobes and lobules, with several excretory ducts penetrating the gland

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(Longnecker, 2014). Unlike the human pancreas that consists of five parts, mouse pancreas is

divided into three parts: the duodenal, gastric, and splenic lobes. However, there are no

definite boundaries that separate each lobe (Nakagawa, 2015).

When examined microscopically, the mouse pancreas is similar to that of the human

pancreas, both containing exocrine and endocrine portions that are enclosed in a fibrous

capsule. Just like most organs, the pancreas is divided into two general parts: the storm and

the parenchyma. The stroma comprises the capsule and associated septa and reticular fibers.

On the other hand, the parenchyma comprises an exocrine and an endocrine portion

(Lehmann et al., 2007).

The pancreatic acinar cells, described as clusters of pyramidal shaped cells with basal

nuclei, represent the exocrine part of the pancreas. Characteristically, these acinar cells have

a basal part that is basophilic due to abundance of rough endoplasmic reticulum, and an

acidophilic apical part due to the presence of secretory granules.

Meanwhile, the Islets of Langerhans, responsible for the endocrine function of the

pancreas, are described as pale, spherical, scattered, and vascularised collections of cells

arranged as anastomosing plates (Kilimnik et al., 2012). The endocrine pancreas contains

five types of cells, namely: alpha cells, beta cells, delta cells, G cells and F cells. Alpha cells

are acidophilic cells responsible for producing glucagon. Antagonistic to its function are the

beta cells which are large, spherical, and the most abundant cells in the islets (>60%),

responsible for the production of insulin. Other cells of the Islets include delta cells, G cells,

and F cells, which produce somatostatin, gastrin, and pancreatic polypeptide respectively. It

is important to take note that these cells of the Islets cannot be differentiated from one

another by routine stains like H&E stain (Dolenšek, Rupnik, & Stožer, 2015).

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Histopathological Studies of Islets in Type II Diabetes Mellitus

Type II diabetes mellitus subjects manifest both defective insulin action and

abnormalities in their pancreatic islets. Whether islet abnormalities are a primary defect or

are a consequence of defective insulin action is unknown.

In a study by Deng and his colleagues (2004), human pancreatic islets were recovered

from type 2 diabetic cadaveric donors and normal donors, with matching age, BMI, and cold

ischemia time. The two samples were compared, and it was found that there was significantly

less and smaller islets, defined by Lehmann and his colleagues (2007) to be between 50 and

150 μm in humans, recovered from the diabetic donor. Histologically, islets from the diabetic

donor contained a higher proportion of glucagon-producing α-cells. In similar investigations

(Nurdiana et. al., 2017; Jelodar, Mohsen, & Shahram, 2007), the histopathology of diabetic

rat pancreas was examined. The acinar cells were swollen, with almost cells observed

containing vacuoles. Interlobular ducts were lined with flattened epithelium and Islet β-cells

are almost entirely destroyed in STZ-treated (Nurdiana et. al., 2017) and Alloxan-treated

(Jelodar, Mohsen, & Shahram, 2007) rats. Destroyed islets were characterized to be

hyperplastic and hypertrophic, and with vascular congestion or hemorrhage. The Islet

periphery was described to be intruded with skeins of fibrous tissue, while the central portion

infiltrated by lymphocytes (Nurdiana et. al., 2017).

HFD/STZ Mouse Model

The employment of High Fat Diet-Streptozotocin (HFD/STZ) protocol has been

reported by several studies (Skovsø, 2014; Nakagawa, 2015; Kim et. al., 2016; Li et. al.;

2014). Such protocol was classified by Nakagawa (2015) as a Diet in Combination with

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Chemotoxic Agent Model. Furthermore, according to Wu and Huan’s (2008) investigation,

the HFD/STZ model has been routinely applied to mice. Streptozotocin (STZ) is an antibiotic

that has a diabetogenic property which is characterized by selective destruction of pancreatic

islet β-cells, causing insulin deficiency, hyperglycemia, polydipsia, and polyuria, all of which

mimic human diabetes mellitus (Wu & Huan, 2008). Meanwhile, the high fat diet (HFD)

induces hyperinsulinemia, insulin resistance, and glucose intolerance while promoting

obesity; all these are the conditions during prediabetes (De Loreto & Magbuhat, 2016).

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MATERIALS AND METHODOLOGY

Plant Extraction and Fractionation

The collection of P. pellucida was done at the University of the Philippines Diliman,

Quezon City, and the aerial parts of the plant samples were verified and authenticated in the

Institute of Biology Jose Vera Santos Memorial Herbarium, College of Science, Diliman,

Quezon City. The aerial parts of the plants were washed and air dried for two weeks and

grounded into powder with a mortar and pestle. About 750 g of ground P. pellucida were

soaked in 7 L 95% methanol for five days. This is performed because xanthone glycosides

are usually crystallized from MeOH (Negi et. al., 2013). The crude extracts were stored in

erlenmeyer flasks at low temperatures (5-15 oC) before use.

Succeedingly, the methanolic crude extract was filtered using a Whatman Filter #1

and evaporated using a rotary evaporator to obtain a blackish-green mass.

Purification of Xanthone Glycoside

The mode of extraction of xanthone glycoside was based on the selective partitioning

of glycosides by Kuwajima and his colleagues (1992). The initial solid isolate obtained from

the rotary evaporator, and which weighed 38.04 g, was resuspended in a 3L-solution of 30%

Methanol : 70% water. This solution was subjected to solvent-solvent extraction using a

separatory funnel, with the solution as the aqueous solvent and hexane as the organic solvent.

The methanol-water solution and hexane were mixed and allowed to stand until two definite

layers was seen. The lower layer, also known as the organic layer was discarded. This step

specifically removes wax, lipids, and fatty acid constituents of the plant material. The upper

16

layer, also known as the aqueous layer, was collected and subjected to another solvent-

solvent extraction, this time, chloroform as the organic solvent.

The aqueous aliquot from the first extraction is mixed with chloroform and allowed to

stand and separate into two definite layers. From this system, the lower layer was discarded,

removing hydrophobic secondary metabolites such as terpenoids, carotenoids, and phenols.

The upper layer or aqueous layer was obtained and subjected to a final solvent-solvent

extraction system.

At this point, a solution of 90% water and 10% aqueous aliquot from the second

extraction was made. This solution served as the aqueous layer while butanol served as the

organic layer. This mixture was allowed to stand in a separatory funnel until two definite

layers were seen. This time, the organic layer or the lower layer is collected for final

purification because glycosides are butanol-soluble compounds.

The butanol fraction was evaporated using a rotary evaporator where 700 mg of a

blackish-green mass was obtained. To verify the identity of the mass obtained, 30 mL of the

butanol fraction was submitted to the Institute of Pharmaceutical Sciences, National Institute

of Health, Manila for phytochemical analysis. From the results obtained, only glycosides

tested positive, verifying that the isolate obtained was indeed purified glycoside.

From there, we obtained the percent yield of xanthone glycoside from the glycosides

of Peperomia pellucida. Based on the study of Khan and his associates (2010), the xanthone

glycoside content of P. pellucida was found to be at 1% of the total glycosides. Using this

information, we computed the amount of xanthone glycoside, through ratio and proportion,

present in the obtained 700 mg of the blackish-green mass confirmed to be pure glycosides.

17

After computation, we found out that our compound contained 0.075 mg of pure xanthone

glycoside.

Acclimatization of Mice

Thirty (30) male Mus musculus (ICR-Strain Laboratory Mouse), aged 6 to 8 weeks

old, approximately weighing 20-30 g, were obtained from the National Institutes of Health,

University of the Philippines Manila. The mice were housed individually in plastic cages,

each with 8.5 inches x 5.5 inches x 5.9 inches floor area with autoclaved wood shavings as

bedding, at the College of Public Health Animal House, University of the Philippines Manila.

The mice were acclimatized for one week following standard conditions: Temperature

ranging from 18-26 oC; Relative humidity at 50 ± 5%; Light intensity at 12 hours light : 12

hours dark cycles. The temperature was maintained by an air conditioning unit while

fluorescent lights were for the photoperiodicity of the mice. The mice were fed with high fat

diet (HFD) food pellets and given access to water. The HFD food pellets were composed of

58% fat from lard, 25.6% carbohydrates, and 16.4% protein. All experimental procedures

were checked and approved by the Institutional Animal Care and Use Committee (IACUC)

of the National Institute of Health.

Induction of Diabetes

Diabetes was induced using Streptozotocin (STZ) together with high fat diet (HFD).

After two weeks of consumption of HFD, the mice were fasted for 20 hours and were given

an intraperitoneal injection of STZ (40 mg/kg) for a span of five days. STZ was dissolved in

0.05 M citrate buffer at pH 4.5 (Byrne, et al., 2015). Each mouse was held in dorsal position

18

during the intraperitoneal injection of STZ. The injection site was swabbed with povidone-

iodine solution, and then STZ were injected in the caudal abdominal cavity of the mouse with

a sterile syringe (Abeeleh et al., 2009). Blood glucose were checked after five days of STZ

treatment for confirmation of diabetes. Diabetic mice were then randomly assigned and

distributed to different treatment groups.

Experimental Set-up

Thirty (30) male ICR Mus musculus were distributed into six groups with five

individuals each. Group A served as the negative control. This group was fed with a standard

diet of quality mice pellets obtained from local pet stores. For succeeding groups, instead of

being fed with a standard diet of quality mice pellets, high fat diet was used. The HFD food

pellets were composed of 58% fat from lard, 25.6% carbohydrates, and 16.4% protein.

Groups B, C, D, and E would serve as the treatment groups. Group B were fed with STZ,

HFD, and metformin. Meanwhile, for Group C, mice were fed with STZ, HFD, and 10 mg

xanthone glycoside. For Group D, mice were fed with STZ, HFD, and 20 mg xanthone

glycoside. As for Group E, mice were fed with STZ, HFD, and 40 mg xanthone glycoside.

The last group, Group F, served as the positive control and would consist of mice fed with

Streptozotocin (STZ) and high fat diet (HFD).

The reference drug was metformin (40 mg/kg) (De Loreto & Magbuhat, 2016) that

was purchased from Mercury Drug Store. It was administered via intraperitoneal injection,

on a daily basis, for three weeks, by dissolving it in distilled water containing 0.9%

(weight/volume) sodium chloride (Cheng, et al., 2006). Meanwhile, xanthone glycoside was

administered through intraperitoneal injection for a span of three weeks. This mode of

19

administration was based on the studies of Geodakyan and his colleagues (1992) and Guha

and his associates (1996).

Obtaining Body Weight

The weight of the mice was closely monitored during the entire course of the study.

Specifically, we measured them closely at the beginning of the experiment, before and after

treatment with metformin and xanthone glycoside of selected groups, and before the mice’s

cervical dislocation. From these data, the initial and final body weight as well as the before

and after treatment weight data was compared respectively for statistically significant

changes.

Blood Glucose Analysis

Blood samples were drawn from the periorbital sinus of the mice and closely

monitored weekly for 6 weeks. Specifically, blood glucose before and after treatment with

metformin and xanthone glycoside were compared for statistically significant changes. Blood

glucose levels were determined using Advan electronic glucometer.

Examination of Gross Morphology and Histopathology of Mice Pancreas

After treatment, the mice were subjected to overnight fasting and blood was drawn

for blood glucose analysis. They were killed via cervical dislocation, anesthetizing them first

with 30 mg/kg Zoletil injected intraperitoneally. The pancreas was excised for

histopathological analysis.

20

Table 1. Scoring system for histological alterations as indicators of damages in the

pancreatic acinar and islets of Langerhans of diabetic male ICR mice modified by Professor

Benjamin.

Parenchymal Necrosis 0 - No necrosis

0.5 - Focal occurrence of 1–4 necrotic cells

1 - Diffuse occurrence of 1–4 necrotic

cells/HPF

1.5 - Same as 1 + focal occurrence of 5–10

necrotic cells/HPF

2 - Diffuse occurrence of 5–10 necrotic

cells/HPF

2.5 - Same as 2+ focal occurrence of 11–16

necrotic cells/HPF

3 - Diffuse occurrence of 11–16 necrotic

cells/HPF

3.5 - Same as 3 focal occurrences of >16

necrotic cells/HPF

4 - ≥ 16 necrotic cells

Steatosis 0 – Absent

1 - 25% has steatosis

2 - 50% has steatosis

3 - 75-100% present

Fibrosis 0 - No fibrosis

1 - Focal, <10% of the pancreas parenchyma

2 - Mild, between 11 to 50% of the pancreas

parenchyma

3 - Diffuse, between 51 to 75% of the

pancreas parenchyma

4 - Severe, >76% of the pancreas parenchyma

Hemorrhage 0 – Absent

1 - Present

Acinar Cells 1 - No acinar cell destruction

2 - Acinar cell destruction <25% of acinar

cells

3 - Acinar cell destruction <26–50% of acinar

cells

4 - Acinar cell destruction <51–75% of acinar

cells

5 - Acinar cell destruction >75% of acinar

cells

21

The excised pancreas were examined for morphological alterations. After which, the

pancreas were fixed in 10% buffered formaldehyde and brought to Hi-Precision Diagnostics

for histological processing of the selected tissue for histological examination. The tissue

sample was sectioned, stained with hematoxylin and eosin, and analyzed under the light

microscope. The histological slides were examined for fibrosis, damage in acinar cells, fat

deposits, parenchymal necrosis, hemorrhage and lymphocyte infiltration. In addition, the

diameter of the islets of Langerhans was measured with a micrometer. The mice pancreas

tissue were analyzed by using a histological alteration scoring system modified by Prof.

Benjamin (De Loreto & Mangubat, 2016).

The index scores were subjected to non-parametric tests, Kruskal-Wallis H Test

followed by Mann-Whitney U Test, respectively, to determine if there are significant

differences in between groups, and to determine specifically what groups bring about the

significance.

Statistical Analysis

All changes in weight, and blood glucose values before and after treatments were

subjected to paired T-test. Final body weight, blood glucose, and islet of Langerhans

diameter values were analyzed by one-way ANOVA followed by Tukey’s HSD Test. While

the histological index scores were subjected to Kruskal-Wallis H test, followed by Mann-

Whitney U test.

22

RESULTS

Body Weight

The body weights of all mice in all groups were noted at the first week of the

experiment and were monitored throughout the duration of the study. To know whether Type

2 Diabetes have been successfully induced in mice, the mean body weights were measured

before and after introduction of STZ and HFD. Figure 1 shows that all groups have exhibited

an increase in weight. After the introduction of STZ to the treatment groups, the mean body

weight of the control group fed with standard diet (Group A) increased by 14.07%, while the

mean body weight of the untreated diabetic group (Group F) increased by 14.28%. The mean

body weights of the diabetic groups treated with metformin, 10 mg/kg xanthone glycoside,

20 mg/kg xanthone glycoside and 40 mg/kg xanthone glycoside increased by 27.37%,

14.50%, 16.00%, and 14.40% respectively. Eventually, the mean body weights were

subjected to paired t-test. As seen in the figure 1, all the groups exhibited a significant

increase in body weight.

After successful induction of Type 2 diabetes, the mice received various

concentration of xanthone glycoside to know whether this compound can reduce not only

body weight but even blood glucose level mean body weight of diabetic mice. Figure 2

shows the mean body weight of diabetic mice before and after introduction of xanthone

glycoside. The results showed that there is a decrease in weight by 5.17% and 1.44% in

Groups B and C respectively, while an increase in weight by 11.85% and 7.97% for Groups

D and E respectively. The mean body weights before and after treatment were also subjected

to paired-T test. The results show that only Groups A, D and F exhibited significant increases

in body weight after the treatment period.

23

Figure 1. Mean body weights (in g) of all groups of male ICR Mus musculus from the week after

acclimatization and after STZ/HFD induction. Different letters denote significant changes within each group

using Paired T-test (p<0.05). ANOVA Test (p<0.05) yielded no significant differences between groups.

Figure 2. Mean body weights (in g) of groups of male ICR Mus musculus from the initial and final week of

treatment with xanthone glycoside. Different letters denote significant changes within each group using Paired

T-test (p<0.05). ANOVA Test (p<0.05) yielded no significant differences between groups.

24

Blood Glucose Levels

Blood glucose levels of all mouse subjects were tested after acclimatization and were

closely monitored weekly throughout the study. Similar to the body weight, mean glucose

level were also determined before and after introduction of high fat diet and STZ and before

and after treatment. Figure 3 exhibited an increase of blood glucose levels across all

treatment groups. Comparison of before and after STZ induction blood glucose levels of all

mice showed 16.67% increase for Group A, 104.27% increase for Group B, 124.22%

increase for Group C, 88.64% increase for Group D, 147.95% increase for Group E, and

3.33% for Group F. Upon subjecting the results to statistical analysis, results show that only

Group D and E have significant increase in blood glucose after injecting STZ and feeding

HFD.

After induction of diabetes, the mice were given different xanthone glycoside

concentrations, 10 mg/kg, 20 mg/kg and 40 mg/kg, for three weeks to test whether or not

blood glucose level change or not. Figure 4 shows an increase blood glucose levels in all

groups except Group D. The mean blood glucose level before and after treatment shows that

Group A had 9.47% increase, Group B had 20.80% increase, Group C had 11.27% increase,

Group D had 11.74% decrease, Group E had 63.97% increase, and lastly, Group F had

17.94%. Upon application of paired-T test, only Groups D and E have significant changes in

blood glucose levels before and after treatment. The results of the one-way ANOVA show a

significant difference between Groups A and E.

25

Figure 3. Mean blood glucose levels (mg/dL) of all groups of male ICR Mus musculus before and after STZ

induction. Different letters denote significant changes within each group using Paired T-test (p<0.05). ANOVA

(p<0.05) yielded no significant difference between groups.

Figure 4. Mean blood glucose levels (mg/dL) of all groups of male ICR Mus musculus before and after

treatment of xanthone glycoside. Different letters denote significant changes within each group using Paired T-

test (p<0.05). Different numbers denote significant changes in between groups using ANOVA (p<0.05).

26

Gross Morphology of Pancreas

All mice pancreas were initially subjected to gross morphological examination using

a stereoscope. The following characteristics were noted during the gross analysis: color,

shape, size, and the presence or absence of black blotches which are gross manifestations of

necrosis and hemorrhage. Sizes were quantitatively measured with a ruler, and later on

categorized as small (less than 1 cm), medium (1 - 3 cm), or large (greater than 3 cm) based

on the scale of Bowen (2017).

Group A exhibited a normal tan color of the pancreas, lobular shape, and was

measured to be of medium size at 2.82 cm. No red or black blotches were observed in the

pancreas of group A. These features are in accordance to the descriptions of Bowen (2017) of

normal gross pancreas characteristics. Group B exhibited a pancreas that is whitish in color

across the entire pancreas, lobular shape, medium sized at 2.75 cm, and with black to red

blotches specifically observed at the head and tail of the pancreas. Particularly, blotches of

Group B were dominant at the tail region of the pancreas and minimal at the head region.

Group C exhibited a pancreas that is whitish in color across the entire pancreas, lobular

shape, medium-sized at 2.70 cm, and with black to red blotches specifically observed

minimally at both the head and body of the pancreas. Group D exhibited a pancreas that has

whitish spots, lobular shape, medium size at 2.80 cm, and with black to red blotches

observed at the head and body of the pancreas. Group E exhibited a pancreas that has whitish

spots, lobular shape, medium size at 2.50 cm, and with black to red blotches observed at the

uncinate, body, and tail region of the pancreas. Group F exhibited a pancreas that is pinkish

in color, lobular in shape, medium-sized at 2.65 cm, with presence of black to red blotches

observed specifically at the tail region of the pancreas. In terms of frequency of blotches,

27

Group F exhibited the most abundant blotches, suggesting extensive necrosis and

hemorrhage. Next to Group F is Group B, followed by groups E, C, and D, arranged with

decreasing prevalence of blotches.

Figure 5. Gross Morphology of the Pancreas. The black arrow shows the areas where black-red blotches are

seen. The letters U, H, B and T signifies uncinate, head, body and tail of the pancreas. A: Pancreas of Group A

which exhibits normal gross morphological appearance. It is characterized as tan in color and lobular in shape,

with absence of black to red blotches (10x). B: Pancreas of Group B which is whitish in color, lobular in shape,

and exhibits black to red blotches particularly dominant at the tail and minimal on the head region of the

pancreas (10x). C: Pancreas of Group C which is whitish in color, lobular in shape, and with black to red

blotches at the head and body of the pancreas (10x). D: Pancreas of Group D which is whitish in color, lobular

in shape, and with minimal black to red blotches at the head and body of the pancreas (10x). E: Pancreas of

Group E which is whitish in color, lobular in shape, and with black to red blotches at the uncinate, body, and

tail of the pancreas (10x). F: Pancreas of Group F which is characterized as pinkish in color, lobular in shape,

and with black to red blotches at the tail region of the pancreas (10x).

28

Islet Diameter

During the histological analysis, islet diameter was measured. It was found that

Group C, the group administered with HFD, STZ, and 10 mg/kg xanthone glycoside, had the

highest mean islet diameter at 191.10 μm. This group was followed by Group D (HFD + STZ

+ 20 mg/kg xanthone glycoside) at 186.04 μm Group A (Negative Control) at 180.46 μm,

Group E (HFD + STZ + 40 mg/kg xanthone glycoside) at 163.04 μm, and Group B (HFD +

STZ + 40 mg/kg metformin) at 141.68 μm. Group F, the positive control, had the lowest islet

diameter at 81.39 μm.

Statistical analysis was performed using ANOVA, where a statistically significant

difference among the groups. Post-hoc analysis specifically revealed that Groups C and F,

and Groups D and F have respectively significant differences in terms of islet diameter.

Figure 6. The mean diameters of the pancreatic Islets of Langerhans per male ICR mice group. Different

numbers denote significant changes in between groups (p<0.05).

29

Histopathology of Pancreas

After gross examination of the pancreas, microscopic examination was performed. It

was found that Group A (Negative Control) exhibited normal pancreas architecture. Both the

Islets of Langerhans and the surrounding acinar cells remained intact and free of

abnormalities. Meanwhile, histological abnormalities such as parenchymal necrosis,

steatosis, fibrosis, hemorrhage, lymphocyte infiltration, and acinar cell destruction have been

observed among Groups B to E (Treatment Groups) and Group F (Positive Control).

Figure 7. The mean histological scores of pancreatic tissue samples from HFD/STZ-induced hyperglycemic

male ICR mice with selected groups treated with xanthone glycoside obtained from P. pellucida. Different

numbers denote the similar groups (p<0.05).

30

Figure 8. Representative Micrographs of Hematoxylin and Eosin-Stained (H&E) Pancreatic Islets of Mus

musculus. The red arrow signifies hemorrhage, black arrow signifies necrosis, yellow arrow signifies fibrosis

and white arrow signifies lymphocyte infiltration. A: The islet of Group A exhibits a normal histological

appearance (400x). B: The islet of Group B (metformin) has hemorrhage and a few necrotic cells (400x). C:

The islet of Group C (XG10) exhibits several necrotic cells and fibrosis (400x). D: The islet of Group D

(XG20) shows cell necrosis (400x). E: The islet of Group E (XG40) shows fibrosis, necrotic cells and cells

undergoing hypertrophy (400x). F: The islet of Group F has shown a remarkable decrease in islet diameter as

well as presence of hemorrhage and fibrosis (400x).

31

Figure 9. Representative Micrographs of Hematoxylin and Eosin-Stained (H&E) Pancreatic Acinar Cells

of Mus musculus. The red arrow signifies hemorrhage, black arrow signifies necrosis, yellow arrow signifies

fibrosis and white arrow signifies lymphocyte infiltration. IL shows the intralobular septum A: The acinar cells

of Group A exhibits a normal histological appearance (400x). B: The acinar cells of Group B (metformin) has

exhibited necrosis and fibrosis(400x). C: The acinar cells of Group C (XG10) exhibits lymphocyte infiltration,

cell necrosis and fibrosis (400x). D: The acinar cells of Group D (XG20) shows hemorrhage, lymphocyte

infiltration, several necrotic cell, fibrosis and hypertrophic cells (400x). E: The acinar cells of Group E (XG40)

shows necrotic cells and cells undergoing hypertrophy (400x). F: The acinar cells of Group F has shown

necrotic cell, fibrosis and hemorrhage (400x).

IL

32

Table 2. Summary of the histological alterations observed in pancreatic tissues treated with

xanthone glycoside obtained from P. pellucida methanolic extract.

Histological

Alterations

A

(Negative

Control)

B

(HFD/ STZ

+

Metformin)

C

(HFD/ STZ

+ 10 mg/kg

XG)

D

(HFD/ STZ

+ 20

mg/kg)

E

(HFD/ STZ +

40 mg/kg

XG)

F

(Positive

Control)

Parenchymal

Necrosis

Absent Present

(32.5%)

Present

(62.5%)

Present

(50%)

Present

(62.5%)

Present

(50%)

Fat Deposits /

Steatosis

Absent Present

(18.95%)

Present

(8.85%)

Present

(22.50%)

Present

(55.80%)

Present

(61.25%)

Fibrosis Absent Present

(27%)

Present

(25.75%)

Present

(44.29%)

Present

(33.55%)

Present

(37.81%)

Acinar

Damage

Absent Present

(22.06%)

Present

(18.35%)

Present

(37.76%)

Present

(29.50%)

Present

(25.66%)

Hemorrhage Absent Present Present Present Present Present

Lymphocyte

Infiltration

Absent Present Present Present Present Present

Average

Histological

Scores Per

Group

0.2 1.42 1.58 1.67 1.92 1.83

33

DISCUSSION

Induction of Type 2 Diabetes

Type 2 Diabetes Mellitus (T2DM) is clinically characterized by reduced insulin

secretion, decreased sensitivity of insulin target cells and β-cell dysfunction (Alberti &

Zimmet, 1998). Other characteristics may include obesity and, at some degree, β-cell failure.

(Yu et al., 2017). Induction of T2DM through high fat diet (HFD) and multiple low doses of

Streptozotocin (STZ) mimics the pathological progression of diabetes in humans. HFD

allowed obesity, hyperinsulinemia and insulin resistance to develop in mice subjects

(Srinivasan, et al., 2005). On the other hand, STZ directly damages the β-cells which results

in hyperglycemia and hypoinsulinemia. Depending on the dose, STZ can induce diabetes in

two ways: 1) At a singly high dose, STZ acts as an alkylating agent directly to the β-cell, and

2) at multiple low doses, STZ elicits immune and inflammatory responses. Destruction of β-

cells and induction of hyperglycemia are mainly caused by lymphocyte infiltration in

pancreatic tissues, especially in the islets (Graham, et al., 2011).

In this study, two weeks of HFD were done to induce insulin resistance in mice

subjects, while the 5-day STZ treatment led to dysfunctional pancreatic islets.

Effect on Body Weight

Body weights of all groups were obtained after acclimatization and after STZ/HFD

induction of experiment. In addition to this, body weights of Groups B, C, D, and E were

noted before and after treatment with metformin and xanthone glycoside.

In this study, a significant increase of 14.07%, 27.37%, 14.50%, 16.00%, 14.40%,

and 14.28% between the initial and final body weights (g) of Groups A, B, C, D, E, and F

34

respectively, were observed. Many factors are known to influence body weight in mice, and

some of the strongest and best-known factors are age, sex, and diet. For instance, old mice, or

mice that are older than 8 weeks, are significantly heavier than young mice mostly because of

the increase in fat mass due to normal body metabolism. There are also sex-specific

differences in mice body weight such that male mice tend to have larger bodies and thus have

greater propensity to gain more weight than female mice. Diet can also have a large impact

on body weight (Yang et al., 2014; Houtkooper et al., 2011; Korou et al., 2013). In this study,

treatment groups (Groups B, C, D, and E) and the positive control (Group F) were fed with

high fat diet, specifically composed of 58% fat from lard, 25.6% carbohydrates, and 16.4%

protein, which promoted weight gain. This weight gain associated with hyperglycemic blood

glucose levels suggests successful induction of Type 2 Diabetes.

Meanwhile, no significant changes in body weight have been observed in Groups B,

C, and E before and after treatment administration. Specifically, there was an insignificant

decrease in body weights for Groups B and C; while an insignificant increase was observed

in Group E. Meanwhile, a significant increase of 11.85% in body weight was observed in

Group D.

The decrease in body weight observed in Group B can be attributed to metformin, a

prescription medication used to treat Type 2 Diabetes by increasing insulin sensitivity.

Associated side effects of this drug which include a decreased appetite and an upset stomach,

may lead to modest reduction in weight — thus accounting for the insignificant decrease in

body weight observed in Group B (Seifarth, Schehler, & Schneider, 2013).

Similarly, an insignificant decrease was observed in the body weight of Group C. On

the other hand, an insignificant increase in body weight was observed for Group E. These

35

insignificant changes may therefore imply the dose-dependent nature of xanthone glycoside

in terms of its control in body weight.

Interestingly, Group D, the group administered with 20 mg/kg xanthone glycoside,

was found to have a significant increase in body weight. This result is corroborated by the

studies of Dineshkumar and his colleagues (2010), Muruganandan and his fellow researchers

(2005), and Ratwita and his associates (2017) who all found a significant increase in body

weights of STZ-induced diabetic rats after treatment with mangiferin, a xanthone glucoside

found in the family Anacardiaceae. All three studies agree in their claim that mangiferin,

specifically of 20 mg/kg, was found to significantly improve body weight loss.

Effect on Blood Glucose Levels

Blood glucose concentration level is defined as a function of the rate of glucose

entering circulation balanced by the rate of glucose removal from circulation. Glucose comes

from three major sources: intestinal absorption, glycogenolysis and gluconeogenesis. Normal

muscle glucose metabolism involves the activation of the glucose transport system which

leads glucose to insulin target cells. An increase in blood glucose levels means that the rate of

glucose entering is greater than the rate of removal. These glucose influxes are regulated by

hormones such as insulin and glucagon (Aronoff, et al., 2004). In this experiment, the high

levels of blood glucose are attributed to β-cell destruction by the STZ and increased levels of

free fatty acids by the high fat diet.

Induction of high fat diet causes hyperlipidemia, a blood lipid disorder that shows an

abnormally high levels of blood lipid in the blood, in mice. Presence of hyperlipidemia is a

high-risk factor for obesity and T2DM (Park & Lee, 2013). In a study done by Buettner and

36

his colleagues (2006), lard-based diet led to a pronounced manifestation of obesity and

insulin resistance. Rodents with this type of high fat diet gained more weight, had high

plasma glucose levels and showed inefficient insulin-induced glucose uptake. Lard contains

high amounts of saturated fatty acids which can increase blood cholesterol levels in the body.

Overnutrition on fatty acids can eventually lead to ectopic fat accumulation which promotes

insulin resistance in organs such as the liver. Increased ectopic fat levels in the body also

affect the primary functions of pancreatic β-cells and muscles (Skovsø, 2014). As a

consequence, skeletal muscles will have impaired glycogen synthesis which can be

associated to insulin resistance. In prediabetic or early stages of T2DM, insulin resistant

muscles redirect glucose to the liver. The liver promotes lipogenesis which induces hepatic

insulin resistance. Instead of promoting glycogenesis, the liver promotes gluconeogenesis

which causes hyperglycemia to the body (Defronzo & Tripathy, 2009; Skovsø, 2014).

Moreover, streptozotocin (STZ), a potent and selective β-cell toxin which causes necrosis of

β-cells, was used in this study to decrease islet activity, lowering antioxidants such as

superoxide dismutase, catalase, and glutathione peroxidase. Destruction of pancreatic β-cells

eventually leads to decreased synthesis and secretion of insulin to the bloodstream (Qinna &

Badwan, 2015).

In the pathogenesis of diabetes, the chronic exposure to hyperglycemia results in the

generation of reactive oxygen species which in turn results in oxidative stress in β-cells, and

later as β-cell dysfunction. This phenomenon is known as glucose toxicity and results to

impaired insulin secretion by the β-cells and ultimately proceeds as diabetes. In a previous

study by Benjamin and her fellow researchers (2013), the potential antioxidant effect of P.

pellucida crude extract was confirmed. Their study showed that P. pellucida crude extract

37

elevated superoxide dismutase (SOD), and catalase (CAT) activity, which are considered as

first line defense antioxidants (Ighodaro & Akinloye, 2017). Moreover, according to Khan

and his colleagues (2010), xanthone glycosides found in P. pellucida were found to have an

antioxidative property by inhibiting lipid peroxidation and scavenging radicals. With these

information, we can deduce that the efficacy of xanthone glycosides as an antihyperglycemic

agent is based on its antioxidant properties. In particular, xanthone glycosides as

antioxidants, increase activity in the islet by increasing superoxide dismutase, catalase, and

glutathione peroxidase, thereby suppressing β-cell dysfunction and providing possible

intervention to hyperglycemia or diabetes (De Loreto & Magbuhat, 2016). This is

substantiated by the result of the study of Muruganandan and his associates (2005) which

showed the potent antihyperglycemic property of mangiferin, a xanthone glucoside, against

STZ-induced diabetic rats.

To date, the most commonly prescribed drug administered in the treatment of non-

insulin-dependent diabetes mellitus (NIDDM) is metformin. It improves glycemic control,

specifically through non-pancreatic mechanisms, thus it does not cause hypoglycemia and

hyperinsulinemia -- which are common side effects of other antidiabetic drugs (Nasri &

Kopaei, 2014; Gong et al., 2012). Metformin works mainly by decreasing hepatic glucose

production through the reduction of gluconeogenesis, glycogenolysis, and fatty acid

oxidation. According to a study of Nasri and Kopaei (2014), metformin specifically

phosphorylates and activates the enzyme adenosine monophosphate kinase (AMPK) which is

an inhibitor of vital enzymes involved in gluconeogenesis, glycogenolysis, and fatty acid

oxidation in the liver. It is important to take note that although metformin largely acts on

38

hepatic metabolism, it is not metabolized by the liver, and is excreted unchanged in the urine

and feces (Gong et al., 2012; Rice et al., 2011).

Outside the liver, metformin works by reducing intestinal absorption of glucose.

According to several studies (Gong et al., 2012; McCreight et al., 2016; Bailey, Wilcock, &

Scarpello., 2008), metformin works by increasing glucose utilization by the intestine,

particularly through anaerobic glucose metabolism, which contributes to the reduction of

glucose intestinal absorption from the luminal into the serosal side of the intestine.

Metformin also works by increasing insulin-mediated glucose uptake in the peripheral tissues

by increasing translocation of glucose transporters, notably GLUT-4, thereby increasing

cellular glucose uptake. Several side effects of metformin include modest weight reduction,

dyspepsia, nausea, and diarrhea (Gong et al., 2012).

In this study, blood glucose was monitored weekly after acclimatization until the

death of the mice. Particularly, blood glucose levels before and after STZ administration

were statistically compared, as well as blood glucose levels before and after treatment. As

seen in Figure 3, blood glucose levels before and after STZ administration, increased for all

groups; however, only Groups D and E exhibited significant increases in this aspect. This

trend after STZ administration was expected due to the nature of STZ as a potent β-cell toxin,

capable of decreasing islet activity and thereby impairing insulin secretion. In mammalian

cells, STZ uptake and metabolism involves free radical generation which causes DNA and

chromosomal damage. Particularly, STZ toxicity is related to the glucose moiety in its

chemical structure which enables it to enter the beta cell via the low affinity glucose 2

transporter in the plasma membrane. Since β-cells of the pancreas are more active than other

39

cells in taking up glucose, they are more sensitive than other cells to STZ damage (Eleazu et

al., 2013).

As for blood glucose levels before and after treatment, results show insignificant

increases in Groups A, B, C, and F. This means that, statistically, these mentioned groups

had the same blood glucose levels before and after administration of treatment. This is

expected of Group A, because being the negative control, it was not administered any

treatment. As for Group B, an unexpected increase in blood glucose levels was observed.

Although this increase was statistically insignificant, a decrease is theoretically expected

after treatment with metformin.

One crucial information which is key to understanding this result is the mechanism of

pancreatic β-cell proliferation. Prior to treatment with metformin, Group B was fed with HFD

and injected with STZ. The combined effects of HFD and STZ on the pancreas resulted in

the toxicity of the islets, particularly the β-cells, resulting to impairment of insulin

production. Normally, pancreatic β-cells have normal proliferative potential after a short

quiescence period of several days. However, this quiescence period is lengthened with age.

Further analysis suggests that replicated β-cells are less likely to divide again compared to

unreplicated β-cells, and that they enter a prolonged refractory period, estimated in months

(Salpeter et al., 2010; Teta et al., 2007). This idea corroborates the idea of a study by Desgraz

and Herrera (2009), suggesting that a single β-cell undergoes only two or three replications

over the course of its lifetime. Considering the age of the mice during the time of

administration of STZ, they were about 10 weeks old, an adult age for mice (McCutcheon &

Marinelli, 2009). From this data, we can deduce that the age of the mice affected the normal

proliferative potential of the β-cells. Thus, the toxicity-induced and impaired β-cells at the

40

time of HFD and STZ administration might have remained in the pancreas of Group B,

accounting for the insignificant increase in blood glucose rather than the expected significant

decrease in blood glucose levels.

In the experiment, Groups C, D, and E, or groups administered with 10, 20, and 40

mg/kg xanthone glycoside respectively, exhibited insignificant increase, significant decrease,

and significant increase in blood glucose levels, respectively.

The principle of drug optimal dose can be used to explain the results in these

treatment groups. Notably, only Group D, administered with 20 mg/kg xanthone glycoside,

exhibited a significant decrease in blood glucose levels. This result is corroborated by the

studies of Dineshkumar and and his colleagues (2010), Muruganandan and his fellow

researchers (2005), and Ratwita and his associates (2017) who all claim that mangiferin, a

xanthone glucoside, specifically of doses 20 mg/kg is an effective antihyperglycemic agent.

As for Groups C and E, non-optimal doses of xanthone glycoside have been

administered. Because Group C had been administered only 10 mg/kg xanthone glycoside—

a dose lower than the optimal— the effects of xanthone glycoside were not fully maximized.

This is reflected on the insignificant increase in blood glucose levels after treatment.

Meanwhile, Group E, administered the highest dose of xanthone glycoside at 40 mg/kg,

exhibited a significant increase in blood glucose levels. This group may have been provided a

toxic dose. This can be corroborated by a study of Pal, Sinha, and Sil (2013) who observed

that high doses of mangiferin, specifically 50 mg/kg promotes Pb(II)-induced hepatic

damage. From this information, we can agree with Daughton and Ruhoy (2013) that reduced

doses of drugs help prevent adverse side effects, drug diversion, and poisonings.

41

Therefore, the results obtained reflect that xanthone glycoside’s potency as an

antihyperglycemic agent is concentration or dose-dependent. This also suggests that 20

mg/kg dose of xanthone glycoside is the most effective of the three doses.

As for Group F, it exhibited an insignificant increase in blood glucose levels.

Theoretically, this should have been a significant increase since no intervention or treatment

was administered in this group. A principle that can explain the results obtained in this group

is the associated genetic variability in the strain of mice used. The mice utilized in this

experiment is ICR mice, a strain of albino mice, oftenly used in pharmacology and toxicity

studies, safety and efficacy testing, cancer research, aging studies, transgenic experiments,

and gene mapping strategies. This specific strain of mice exhibits genetic diversity and thus it

is helpful in product safety testing because its responses are reflective of the human

population (Lee et al., 2017). This genetic variability of the ICR mice strain suggests that

different individuals may respond to different administered drugs, which explains the

unprecedented insignificant increase in blood glucose levels.

It is also important to compare, that while toxicity-induced β-cells have remained in

groups administered with STZ, Group B, administered with Metformin did not have a

profound effect on blood glucose levels as compared with Group D, administered the optimal

dose of xanthone glycoside. This suggests that an optimal dose of xanthone glycoside is

better than metformin in terms of antihyperglycemic activity.

Pancreas Morphology, Histology and Islet Diameter

Pancreatic gross morphology and histology is reflective of the condition of the organ

and its capability of carrying out its functions. In this study, observed gross morphological

42

and histological alterations from the normal pancreatic architecture suggests an underlying

pathology. Observed alterations in the gross morphology of the pancreas include deviations

from the normal tan color of the organ and the presence of black to red blotches, indicative of

necrosis and hemorrhage. Meanwhile, observed alterations in the histology of the pancreas

include parenchymal necrosis, steatosis, fibrosis, hemorrhage, lymphocyte infiltration, and

acinar cell destruction.

Chronic exposure to hyperglycemia, defined by Davidson and his colleagues (2016)

as 3 weeks or more, eventually leads to a phenomenon known as glucose toxicity (Robertson

et al., 2003). This phenomenon is marked by a decrease in insulin secretion by the pancreatic

β-cells and an increase in insulin resistance. Concurrently, the excessive glucose is able to

react with amino groups specifically through glycosylation. This process leads to protein

modification, which results in functional changes in enzymatic activity, production of

reactive oxygen species, and increased vulnerability to oxidative stress. With the

administration of STZ, a potent selective β-cell toxin, β-cell compensation or the increase in

β-cell mass and insulin secretion is halted. With the presence of a β-cell toxin, and the

continuous administration of HFD leading to oxidative stress, total β-cell dysfunction ensued

leading to alterations in normal pancreas architecture. The presence of lymphocytes that have

infiltrated the pancreas are crucial mediators of β-cell destruction as this signifies chronic

inflammation of the pancreas (Cerf, 2013). According to De Loreto and Magbuhat (2016),

such alterations occur in the chronological manner of hypertrophy, necrosis, immune cell

infiltration, fibrosis, and hemorrhage. Thus, the manifestation of these abnormalities is

reflective of the individual’s state of diabetes (Robertson et al., 2003; Kawahito et al., 2009;

Wolff & Dean, 1987).

43

Likewise, reflective of an underlying pathology of the pancreas is the islet diameter.

In studies performed by Kilimnik and his associates (2012), when islet diameter of normal

and diabetic patients were compared, it was found that there was a preferential loss of large

islets (>60 µm in diameter) in diabetic patients. This is also corroborated by the study of

Rawal and her colleagues (2013), where islet diameter of diabetic patients were found to be

significantly smaller than islet diameter of nondiabetic patients. In this study, this was

substantiated with islet disruption in diabetic patients, resulting in a smaller diameter.

Group A: Negative Control (Standard Diet)

This group has been fed with a standard diet of mice pellets throughout the duration

of the study. No gross morphological abnormalities nor alterations in normal pancreas

histology have been observed. Both the Islets of Langerhans and the surrounding acinar cells

remained intact and free of abnormalities. Notably, this group exhibited the lowest

histological score of 0.2, which indicates no incidence of histological alterations or damages

in the pancreatic tissue. This group also exhibited a relatively high mean islet diameter,

ranking third among all groups, suggesting a healthy pancreas. These results are attributed to

standard conditions with which this group was subjected as well as the absence of

administration of HFD and STZ.

Group B: HFD + STZ + Metformin

This group has been administered with HFD and STZ, followed by a treatment of 40

mg/kg of Metformin. After gross morphological analysis, it was found that the pancreas was

whitish in color, lobular in shape, and exhibited black to red blotches, indicative of necrosis

and hemorrhage. These blotches were specifically observed with great abundance in the tail

region of the pancreas and minimally in the head region of the pancreas.

44

The tail region of the pancreas is the region closest to the spleen, an organ of the

lymphatic system. According to Debi and his colleagues (2013), this region is the most

susceptible to pancreatic injury. In relation to this, pancreatic injuries are rarely a solitary

event, and in majority of instances, multiple organ injuries occur. Notably, the spleen, a

secondary lymphoid organ nearest the tail of the pancreas, can contribute to pancreatic injury

(Kshirsagar et al., 2015). This contribution to pancreatic injury by the spleen can be

attributed to the vascular connections between the two organs. According to Ley and his

colleagues (2012), there is increasing evidence that the spleen harbors stem cells that act as

precursors to insulin-producing cells. In this case, β-cell precursors get delivered to the

pancreas through the splenic artery and its pancreatic branches which drain largely into the

tail region and minimally into the body of the pancreas. From this information, we can

deduce that most β-cell precursors initially reside in the tail region until they migrate in

different regions of the pancreas. Since STZ is a selective β-cell toxin, β-cell precursors

initially residing in the tail are also affected. Increase in circulation of β-cell precursors from

the spleen into the tail region of the pancreas occurs to compensate β-cell destruction in due

to STZ. This cycle of delivery and destroy of β-cells in the tail region explains the abundant

gross morphological aberrations observed (Debi et al., 2013; Kshirsagar et al., 2015).

White spots observed in the pancreas are a result of pancreatic enzymes which liquefy

fat cell membranes. These pancreatic enzymes are released in response to injury to the

pancreas, such as infection, toxins, viruses, trauma, or ischemia. The resulting fatty acids

from this enzymatic breakdown combine with calcium to form the grossly visible chalky

white nodules, which can later on develop into fat necrosis (Lack, 2003; Lombardi, Estes, &

Longnecker, 1975).

45

Histological abnormalities observed in this group include parenchymal necrosis,

steatosis, fibrosis, hemorrhage, and lymphocyte infiltration. Although metformin is used in

the treatment of hyperglycemic blood glucose levels, histological aberrations were still found

to be present. This can be attributed to the mechanism of proliferation of β-cells. Normally,

pancreatic β-cells have normal proliferative potential after a short quiescence period of

several days. However, this quiescence period is lengthened with age. Further analysis

suggests that replicated β-cells are less likely to divide again compared to unreplicated β-

cells, and that they enter a prolonged refractory period, estimated in months (Salpeter et al.,

2010; Teta et al., 2007). This idea corroborates the idea of a study by Desgraz and Herrera

(2009), suggesting that a single β-cell undergoes only two or three replications over the

course of its lifetime. Toxicity-induced and impaired β-cells at the time of HFD and STZ

administration might have remained in the pancreas of Group B, accounting for the

insignificant difference with that of Group F’s islet diameter.

Although several histological abnormalities were observed, this group was second to

Group A, in terms of the best histological score. However, in terms of islet diameter, it

ranked second to the lowest, and had an insignificant difference with that of Group F, the

group with the smallest islet diameter. Although there is a scale discrepancy, our findings

suggest that histological analysis is a more reliable parameter than islet diameter for analysis

of diabetes-induced aberrations. This is because some islets may indeed be large in size, but

this is actually due to the presence of hypertrophic cells. These instances are further

explained in the pancreatic histological analysis of Group C.

46

Group C: HFD + STZ + 10 mg/kg Xanthone Glycoside

The pancreas of Group C which was treated with 10 mg/kg of xanthone glycoside

exhibited multiple red to black blotches in their pancreas and as well as hemorrhage. These

blotches were particularly observed at the head and body of the pancreas. Occasionally, a few

fat deposits were found around and within the pancreas.

The head and uncinate regions of the pancreas are two of the most resistant regions of

pancreas when it comes superficial fat necrosis (Biffl, et al., 2013; Howard & Wagner,

1989). Within these locations, the main pancreatic duct and intrapancreatic ducts can be

found. In relation to this, a study by Butler and his colleagues (2010) indicated the

correlation between high fat diet and obesity with upregulation of pancreatic duct cell

replication. The increase in the rate of replication of pancreatic duct cells causes necrosis to

the excess cells due to the lack of oxygen coming from the blood vessels.

A portion of the body of the pancreas share the same the blood vessels of the tail

forming the corporocaudate segment (Busnardo, DiDio & Thomford, 1988). The body of the

pancreas also receives a portion of the β-cells progenitor stem cells from the splenic artery.

STZ is a toxin specific for β-cells and its precursors thus necrosis will likely occur in the area

where there are numerous β-cell stem cells. Presence of necrosis at body of the pancreas can

also be caused by a disruption of the main pancreatic ducts (Gmeinwieser, et al., 2000).

Pancreatic enzymes, when reacted with the fat deposits, causes superficial fat necrosis.

Histological abnormalities observed in this group are severe parenchymal necrosis,

fibrosis, mild steatosis, hemorrhage and lymphocyte infiltration. This group exhibited the

largest islet diameter out of the six groups. However, it was observed that the large size of

the islets was attributed to hypertrophic cells. In addition, the islets were severely damaged

47

with multiple necrotic cells and fibrosis. The histological index scores of Group C is the

lowest among the treatment groups. The acinar cells of this group are relatively normal with

minimal hemorrhage and hypertrophy of cells when compared to Group A (Negative

Control). The low scores given to the acinar cells may have contributed to the overall mean

histological score. Despite that, severe damages to the islets still suggest that 10 mg/kg of

xanthone glycoside is not the optimal dosage due to the lack of protective effects, such as

alleviating cell necrosis and atrophy, on the islet itself.

The mode of islet cell death in STZ-induced diabetes is necrosis. According to

Fehsel, Kolb-Bachofen and Kröncke (2003), in vitro treatment of pancreatic islets with high

concentrations of STZ showed evidence of necrosis. Under the electron microscope,

disrupted plasma membrane and spilled-out cellular constituents were evident in the

pancreatic islets. DNA damage in necrotic cells are caused by the activation of enzymes such

as intranuclear DNAse and poly(ADP-ribose) polymerase. In a study done by Leist and his

colleagues (1997), availability of ATP is a decisive factor on the mode of cell death. During

DNA damage in necrotic cells, the reduced frequency of base excision repair-induced strand

breaks and activation of poly(ADP-ribose) polymerase (PARP) causes the depletion of ATP.

ATP depletion leads to cell necrosis (Cardinal, et al., 2001).

The results suggest that this dose is not optimal because the given dose was not

enough or underdosed. An underdose of a drug or medication cause the treatment to be

ineffective. A study showed that a low dose of xanthone prevented the GLUT-4 expression in

diabetic mice (Ratwita, et al., 2017). As seen in the islets, several necrotic cells are still

evident on the β-cells thus this suggest that xanthone glycoside failed to induce its protective

effects on the islets.

48

Group D: HFD + STZ + 20 mg/kg Xanthone Glycoside

The pancreas found in Group D exhibited a pale white color and few black blotches

particularly observed at the head and body of the pancreas. Similar to Group C, the whitish

color of the pancreas can be attributed to the reaction of fats and pancreatic enzymes found at

the exocrine portion of the pancreas. Necrosis at the head and body portions of the pancreas

can be attributed to the damages and excessive cell proliferation at the main pancreatic ducts

(Gmeinwieser, et al., 2000; Butler, et al., 2010).

Mild parenchymal necrosis, severe fibrosis, steatosis, hemorrhage and lymphocyte

infiltration were the histological abnormalities found in this group. Its high histological score

can be attributed to the acinar damage and fibrosis found in the acinar portion of the

pancreas. However, islets of this group only show a few occurrences of necrotic cells, and its

islets are relatively bigger than Group A (Negative Control) and is significantly different

from Group F (Positive Control). This suggest the possibility that 20 mg/kg of xanthone

glycoside is the optimal dose.

Oxidative stress plays an important role in the development of diabetes. One such

metabolic complication involves the overproduction of mitochondrial superoxide in

endothelial cells of blood vessels. The increased production causes the activation of polyol

pathway flux, increased formation of advanced glycation end-products (AGEs), increased

expression of the receptor for AGEs and its activating ligands, activation of protein kinase C

(PKC) isoforms, and overactivity of the hexosamine pathway which are major complications

in diabetic patients (Giacco & Brownlee, 2010). Moreover, islets are susceptible to reactive

oxygen species (ROS)-induced damages because they exhibit low antioxidant activity. Major

antioxidant enzymes such as superoxide dismutase (SOD), catalase, and glutathione

49

peroxidase (GPx) are suppressed or inactive (Robertson, et al., 2003). Factors involving beta

cell dysfunction includes generation of ROS due to hyperglycemia and susceptibility of the

islet to oxidative stress. A study done by Benjamin and her colleagues (2013) reported that P.

pellucida increase antioxidant activities of the SOD and catalase to prevent accumulation of

ROS or free radicals. Prevention of free radical accumulation may lead to prevention of

oxidative stress of the pancreatic islets. In one study done by Hasnain and his co-workers

(2016), suppression of oxidative stress can revert a β-cell to its normal structure and function.

This suggest that P. pellucida has this property to alleviate ROS-related damages to the

pancreas.

P. pellucida also have anti-inflammatory components such as peperomin E (Xu, et al.,

2006). Low doses of STZ causes inflammation and lymphocyte infiltration in the β-cells of

the pancreatic islets which in turn decreases synthesis and secretion of insulin. P. pellucida

can combat the effects of inflammation thus preventing lymphocytes from infiltrating in.

High amounts of ROS associated with the production of inflammatory cytokines are involved

in the NF-κB pathway (Lawrence, 2009). Therefore, this suggests that P. pellucida prevents

necrosis, immune cell infiltration and inflammation by suppressing oxidative stress and

inhibition of the NF-κB pathway.

The xanthone family are also known to be powerful inhibitors of α-glucosidases. α-

Glucosidases are membrane-bound enzymes of the glycoside hydrolase family 31 which

function in hydrolyzing large carbohydrate molecules to glucose and other monosaccharides.

Inhibition of this enzymes delay the release of glucose thus delaying glucose absorption of

target cells. α-Glucosidase inhibitors are useful in treating diabetes and other prediabetic

conditions. Xanthones are also possible inhibitors of aldose reductase, an enzyme found in

50

the polyol pathway responsible for reducing glucose to sorbitol (Zheng, et al., 2014). In

diabetic patients, aldose reductase become more active and produces more sorbitol thus

activating the polyol pathway. This pathway is responsible for transforming glucose to

fructose, however it uses large amounts of NADPH and NAD. Reduced NADPH levels

prevents formation of glutathione which helps in reducing hydrogen peroxides to water.

Increased NADH levels allow high levels of oxidative stress which allows more diabetic

complications such as cardiovascular problems to happen (Tang, Martin and Hwa, 2012;

Nishimura, 1998). This suggest a possible antihyperglycemic function of the xanthone

glycoside found in P. pellucida.

Group E: HFD + STZ + 40 mg/kg Xanthone Glycoside

The gross morphology of the pancreas of Group E shows small black blotches,

observed at the body, tail, and uncinate process of the pancreas, which may indicate the

presence of necrotic cells or internal bleeding.

In particular, blotches were observed abundantly at the middle of the body and tail of

the pancreas. Based on reports by Debi et al. (2013), Kshirsagar et al. (2015), and Ley et al.

(2012), these blotches were brought about by the accumulation of β-cell precursors and their

apparent destruction by STZ in that area. Minimal blotches were observed at the uncinate

process of the pancreas. Blotches observed in this area are attributed to the conclusion of

Butler and his colleagues (2010) who indicated that following high fat diet administration,

there is upregulation of pancreatic duct cell regulation. This occurs over time and eventually,

as cells overcrowd in that region, cells at the center become deprived of their vascular

supply, causing necrosis.

51

The color which is white to pinkish also shows possible signs of

inflammation. Histological abnormalities observed in this group are parenchymal necrosis,

fibrosis, severe steatosis, severe hemorrhage and lymphocyte infiltration. From the islet

diameter, this group ranked fifth overall which signifies its relatively small diameter

compared to Group A and the other treatment groups. With high histological score, low islet

diameter, and presence of black blotches in the pancreas, the data suggest that 40 mg/kg may

not be the optimal dose of xanthone glycoside intake as it did not exhibit protective effects on

the pancreas of diabetic mice.

Many xanthone derivatives from other plants, such as 1,7-Dihydroxyxanthone and

Gartanin, have cytotoxic effects. Often times, cytotoxic drugs are used for cancer treatment.

Compounds with cytotoxic effects affect both healthy and cancerous tissues. Its mode of

action is disrupting the normal dividing cells thus effectively halting mitosis and proliferation

of cells. (Mazimba, et al., 2013). High amounts of compounds with cytotoxic effects may be

detrimental to the healthy cells of the subject. This may suggest the damage found on the

pancreatic cells are both inflicted by the xanthone compound and HFD/STZ. However,

xanthone glycosides found in P. pellucida are not fully studied yet in terms of its side effects.

Group F: Positive Control (HFD + STZ)

This group has been administered with HFD and STZ, with no treatment administered

afterwards. After gross morphological analysis, it was found that the pancreas was reddish in

color, lobular in shape, and exhibited black to red blotches observed at the tail region of the

pancreas.

The tail region of the pancreas, the region closest to the spleen is most susceptible to

pancreatic injury. This is because most β-cell precursors delivered by the spleen to the

52

pancreas initially reside in the tail region where they are destroyed by STZ, a potent β-cell

toxin. This cycle of delivery and destroy of β-cells in the tail region explains the abundant

gross morphological aberrations observed (Debi et al., 2013; Kshirsagar et al., 2015).

Histological abnormalities observed in this group include parenchymal necrosis,

steatosis, fibrosis, hemorrhage, and lymphocyte infiltration. This group had the third highest

histological score obtained. Notably, this group had the lowest islet diameter. This may be

attributed to the effect of STZ, a potent β-cell toxin, coupled with no administration of any

treatment or intervention afterwards.

53

CONCLUSION

This study demonstrated the effects of xanthone glycoside from Peperomia pellucida

on HFD/STZ induced diabetic mice through changes in body weights, blood glucose levels,

and evaluation of gross morphology, islet diameter and histopathological index scores of the

pancreas.

Treatment of xanthone glycoside for three weeks showed significant decrease in

blood glucose levels in groups administered with 20 mg/kg and significant increase in blood

glucose levels in groups administered with 40 mg/kg. This suggests that 20 mg/kg xanthone

glycoside is the optimal dose which can reduce hyperglycemia. This also suggests that at the

optimal dose, xanthone glycoside from P. pellucida can be an alternative to metformin in the

treatment of T2DM.

In terms of the histological effect of xanthone glycoside, it did not prevent

histological abnormalities from occurring. Surrounding acinar cells and islets still exhibited

abnormalities. However, when administered the optimal dose, islets were less affected and

even maintained a large diameter, like that of a normal and healthy pancreas.

54

RECOMMENDATIONS

Modification of the protocol, specifically by adjusting the treatment duration and

intervals, should allow ample time for β-cell recovery from STZ before introducing

treatments. Future diabetic studies should also look into the blood cholesterol levels of their

subjects, as these can further substantiate results obtained. It is also suggested that further

studies utilize the recommended dose of 20 mg/kg xanthone glycoside, and to test its effect

on other organs of the body, primarily the liver.

55

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APPENDIX

Appendix A. Letter of Approval for Implementation from the IACUC, NIH

70

Appendix B. Authentication from Institute of Biology, College of Science, University of

the Philippines Diliman

71

Appendix C. Phytochemical Analysis Results of Butanol Fraction from Peperomia

pellucida from NIH, UP Manila

72

73

Appendix D. Computations for Total Xanthone Glycoside Needed

*Assumed weight of mouse is 30 g

0.3 mg XG x 5 mice x 21 days = 31.5 mg XG

0.6 mg XG x 5 mice x 21 days = 63 mg XG

1.2 mg XG x 5 mice x 21 days = 126 mg XG

Total XG: 220.5 mg XG

Appendix E. Computation for Streptozotocin Amount

*Assume the weights of the mice are around 20-30 g

0.8 mg STZ x 25 mice x 5 days = 100 mg STZ

1.2 mg STZ x 25 mice x 5 days = 150 mg STZ

TOTAL: 100-150 mg of STZ

Appendix F1. Paired T-Test Results Between Before and After HFD/STZ Induction

Body Weights (g)

Treatment

Group T-critical p-value Significance

A 2.776 0.017 SIGNIFICANT

B 3.182 0.012 SIGNIFICANT

C 2.776 0.004 SIGNIFICANT

D 2.776 0.006 SIGNIFICANT

E 2.776 0.009 SIGNIFICANT

F 3.182 0.035 SIGNIFICANT

74

Appendix F2. Paired T-Test Results Between Body Weights (g) of Mouse Subjects

Before and After Treatment

Treatment

Group T-critical p-value Significance

A 2.776 0.020 SIGNIFICANT

B 3.182 0.228 NOT SIGNIFICANT

C 2.776 0.432 NOT SIGNIFICANT

D 2.776 0.039 SIGNIFICANT

E 2.776 0.141 NOT SIGNIFICANT

F 3.182 0.006 SIGNIFICANT

Appendix G1. Paired T-Test Results Between Blood Glucose Levels (mg/dL) of Mouse

Subjects Before and After STZ Induction

Treatment

Group T-critical p-value Significance

A -1.520 0.102 NOT SIGNIFICANT

B -1.025 0.191 NOT SIGNIFICANT

C -1.956 0.061 NOT SIGNIFICANT

D -3.093 0.018 SIGNIFICANT

E -3.531 0.012 SIGNIFICANT

F -0.272 0.402 NOT SIGNIFICANT

Appendix G2. Paired T-Test Results Between Blood Glucose Levels (mg/dL) of Mouse

Subjects Before and After Treatment

Treatment Group T-critical p-value Significance

A -0.701 0.261 NOT SIGNIFICANT

B -0.839 0.232 NOT SIGNIFICANT

C -1.053 0.176 NOT SIGNIFICANT

D 2.937 0.022 SIGNIFICANT

E -3.518 0.012 SIGNIFICANT

F -1.503 0.115 NOT SIGNIFICANT

75

Appendix H1. Kruskal-Wallis One-way ANOVA Results for Mean Histological Index

Scores

Appendix H2. Mann-Whitney U Test Results for Mean Histological Index Scores

Treatment

Group

Mann-

Whitney U

Wilcoxon

W Z

Asymp. Sig.

(2-tailed)

Exact

Sig. Significance

A B 0.000 21.000 -3.108 0.002 0.002 SIGNIFICANT

C 0.000 21.000 -3.108 0.002 0.002 SIGNIFICANT

D 0.000 21.000 -3.102 0.002 0.002 SIGNIFICANT

E 0.000 21.000 -3.089 0.002 0.002 SIGNIFICANT

F 0.000 21.000 -3.089 0.002 0.002 SIGNIFICANT

B

C 15.500 36.500 -0.436 0.663 0.699

NOT

SIGNIFICANT

D 16.500 37.500 -0.258 0.796 0.818

NOT

SIGNIFICANT

E 11.000 32.000 -1.185 0.236 0.310

NOT

SIGNIFICANT

F 13.000 34.000 -0.846 0.397 0.485

NOT

SIGNIFICANT

C

D

17.500 38.500 -0.086 0.931 0.937 NOT

SIGNIFICANT

76

E 14.000 35.000 -0.677 0.498 0.589

NOT

SIGNIFICANT

F 16.000 37.000 -0.339 0.735 0.818

NOT

SIGNIFICANT

D E 0.000 10.000 -2.530 0.011 0.029 SIGNIFICANT

F 0.000 10.000 -2.381 0.017 0.029 SIGNIFICANT

E F 7.500 17.500 -0.643 0.521 0.556

NOT

SIGNIFICANT