EFFECT OF THE AQUEOUS EXTRACT OF

MUNTINGIA CALABURA LINN. ON THE BLOOD

GLUCOSE LEVELS OF INDUCED DIABETIC MICE
AT FASTED STATE

Research Proposal

C-2

Lim, Phoebe
Lim, Syndel
Lipana, Kirk

Lucila, Ana Marie

Liu, Johanna

Lustre, Alexis Lorenz

Llamas, Alay

Macabagdal, Jesmarie

Llego, Nasreen

Macalma, Glenn

Lopez, Camille

Macapagal, Justin

Lopez-Dee, Bernadette

Madrid, Bianca

Lorenzo, Adrian

Malabanan, Michelle

Lorilla, Richardson

Malaca, Chester

Lu, Pei-Chei

Mallari, Romina
Malvar, Alfred
Mamauag, Mary Jo

CHAPTER I

INTRODUCTION

BACKGROUND OF THE STUDY

Diabetes mellitus is the most common metabolic disorder, its prevalence varying widely
worldwide (Takrouri, 2009). The frequency of the diabetes escalate worldwide, with a major impact on the
population of developing countries (Park's textbook of preventive and social medicine. 15th edition, 1997).
(I ERASED THE USA PART I JUST FOCUSED IN THE PHILIPPINES) In the Philippines, diabetes ranked
8th in the leading causes of mortality (Department of Health, 2005). As many as 1.4 million Filipino adults
(aged 20 and above) acquired diabetes in the last five years mainly because of unhealthy diets and
sedentary lifestyles, according to data compiled by anti-diabetes advocates. It has been proven that the
prevalence of diabetes is constantly on the rise (Takrouri, 2009). According to a study made by Wild et al,
the total number of people with diabetes is projected to rise from 171 million in 2000 to 366 million in 2030
worldwide(Diabetes Care, 2004).

Diabetes mellitus is a chronic disease that requires long-term medical attention both to limit the
development of its devastating complications and to manage them when they do occur. It is a
disproportionately expensive disease; in 2002, the per-capita cost of health care was $13,243 for people
with diabetes, while it was $2,560 for those without diabetes (Scott et. al, 2009). Correctly determining
whether a patient has type 1 or type 2 diabetes is important because patients with type 1 diabetes are
dependent on a continuous source of exogenous insulin and carbohydrate for survival and this will surely
be an additional expense for the patient.

Complications, particularly cardiovascular disease, are the major sources of expenses for
patients with diabetes mellitus. Approximately two-thirds of people with diabetes die from heart disease or
stroke (Ligaray & Isley, 2009). Adults with diabetes have two- to four-fold increased risk for death from
heart disease than adults without diabetes. The risk for stroke is likewise two to four times higher among
people with diabetes. In 2003 to 2004, 75% of adults with diabetes have hypertension. Diabetic
retinopathy causes 12,000 to 24,000 new cases of blindness each year. More than 60% of nontraumatic
lower-limb amputations are performed in people with diabetes (National Diabetes Information
Clearinghouse, 2008).

Type 1 DM in particular is associated with a high morbidity and premature

mortality due to complications. The annual financial cost from diabetes overall

exceeds $100 billion, almost $1 of every $7 dollars of US health expenditures in

terms of medical care and loss of productivity (Aneela Naureen Hussain, MD, FAAFM,
Assistant Professor, Department of Family Medicine, State University of New York
Downstate Medical Center).

Advances in treatment that permit tight glycemic

control and control of comorbidities (hyperlipidemia) can greatly reduce the

incidence of microvascular and macrovascular complications .

STATEMENT OF THE PROBLEM

This study aims to determine the effectiveness of Muntingia calabura Linn.

Specifically, the

purpose of the study is to determine the effect of the aqueous extract from the leaves of Muntingia
calbura Linn. on blood glucose levels of Alloxan-induced diabetic mice on fasted state.

STATEMENT OF HYPOTHESIS
The researchers hypothesis is that Muntingia calabura Linn. will have a hypoglycemic effect on
the diabetes-induced mice.

OBJECTIVES OF THE STUDY

The objectives of the study are as follows:
1. to determine if there is a significant decrease in blood glucose level with the use of Muntingia
calabura Linn.
2. to determine the extent of reduction in blood glucose level with the use of Muntingia calabura
Linn.

SIGNIFICANCE OF THE STUDY

The Philippines is blessed with more than 500 medicinal plants approved by the Philippine
Department of Health (DOH) as an alternative medicine in treating particular disorders . In recent years,
there is an increase in the use of traditional herbal drugs due to their increased efficacy, low cost and gain
in popularity. Herbal medicines or their extracts are widely prescribed, even before their biologically active
compounds are explored. The World Health Organization approved the use of plant-based drugs for
different ailments, including diabetes mellitus (Kumar, 2007). It is therefore useful to look into alternative
treatments for diabetes, specifically herbal medicines.

( I erased the part of Banaba.. coz accdg to Dr. Matro he thought daw n a impt un kasi nauna pa
pero wala pala daw importance.. so I decided to completely delete it nlng.. ^_^)
One such herb is Muntingia calabura Linn., locally known as saresa or aratiles, is a plant of the
family Elaeocarpaceae/ Muntingiaceae/ Tiliaceae. It is proven by the Philippine Department of Health
(DOH) that aratiles is used as a treatment for headaches, colds and decoction of flowers is used to
relieve abdominal cramps and good antiseptic. In addition to those proven uses the people in La Paz,
Tarlac, Philippines are using decoction from the leaves of Muntingia calabura Linn. as an adjunct
treatment for diabetes mellitus. However until now no scientific investigation had been carried out on to
shed light in the anti-diabetic, hypoglycemic property of Muntingia calabura Lin .
Providing conclusive evidence of the effectiveness of this herb in decreasing blood glucose levels
in diabetics may prove to be valuable in any probable future use of Muntingia calabura Linn. as an
alternative treatment for diabetes.

SCOPE AND LIMITATION

The research shall provide a scientific basis for the folkloric use of the decoction of Muntingia
calabura Linn.in the treatment of diabetes melitus. It will deal with the determination if there is a significant
reduction in blood glucose level. The results gathered from the blood glucose levels of the diabetic mice
will be analyzed using Statistical Evaluation. However, the following should not be determined in the
study: (ginawa ko ng bulleted since parang mejo sakit sa ulo kung in paragraph form eh puro will not do
etc lang.. thats my point of view lang nmn.. kasi thats what we did sa thesis nmn before ^_^)

active components present in the extract of Muntingia calabura Linn. and its mechanism of action
responsible for its hypoglycemic effect.

determination of toxic and lethal doses of the aqueous extract of Muntingia calabura Linn
including its adverse effect.

effectiveness of the extract in fed state Alloxan-induced diabetic mice. (hehehe.. ito ung di ko ma
gets why.. kasi db we did this naman..)

DEFINITION OF TERMS

Average lethal dose (LD50) median lethal dose of a toxic substance is the dose required to kill
half the members of a tested population

Blood glucose term used to refer to the amount of glucose in the blood

Decoction a method of extraction of herbal or plant material, which includes, but is not limited to
leaves, flowers, stems, roots, bark, and rhizomes

Effective dose (ED50) the amount of drug that produces a therapeutic response in 50% of the
people taking it

Fasting blood sugar - measures blood glucose after you have not eaten for at least 8 hours.

Hyperglycemia a condition in which an excessive amount of glucose circulates in the blood

plasma

Hypoglycemic agent an anti-diabetic drug used to treat diabetes mellitus. They usually work by
lowering the glucose levels in the blood

Insulin an anabolic polypeptide hormone that regulates carbohydrate metabolism. It is produced

in the Islets of Langerhans in the pancreas

Oral gavage route of administration used to deliver fluids or drugs to experimental mice directly
to the esophagus
CHAPTER II

REVIEW OF RELATED LITERATURE

MUNTINGIA CALABURA LINN.

Description
Muntingia calabura Linn. also known as Aratiles (Tag.); Seresa (Ilk.); Cereza(Sp.); Datiles (Tg.,
Bik); Latires (Tag); Zanitas (Ilk., Ibn); Ratiles (Tag.); Jamaican Cherry (Eng.) (Quisumbing, 1985).

Aratiles is a tree growing from five to ten meters in height, with spreading branches. The leaves
are hairy, sticky, alternate, distichous, oblong ovate to broadly oblong-lanceolate, and 8 to 13 centimeter
long, with toothed margins, the apex pointed and the base inequilateral, one side rounded, and the other
acute. The flowers are about two centimeters in diameter, white, extra-axillary, and solitary or in pairs. The
sepals are five and are green, reflexed, lanceolate, and about one centimeter long. The petals are white,
obovate, about one centimeter long, deciduous, and spreading. The fruit (berry) is rounded, about 1.5
centimeters in diameter, red, smooth, very fleshy, sweet, and filled with very numerous, small seeds
(Quisumbing, 1985).

Phytochemistry
Aratiles leaves are said to contain the following: (1) two new dihydrochalcones, 2,3-dihydroxy-43,4,5-tetramethoxydihydrochalcone, (2) 4,2,4-trihydroxy-3-methoxydihydrochalcone, and (3) a new

flavanone, (2R,3R)-(-)-3,5-dihydroxy-6,7-dimethoxyflavanone, together with nineteen known compounds

that have been isolated from the leaves of Muntingia calabura. Among the isolates, 2, 3-dihydroxy-4, 3,
4, 5-tetramethoxydihydrochalcone, 5,7-dihydroxy-3-methoxyflavone, 5,7-dihydroxy-6-methyl gallate are
responsible for the anti-platelet aggregation activity (Chen et al, 2007).
Su et al (2003) extracted from Muntingia calabura a flavanone with an unsubstituted B-ring, (2R,
3R)-7-methoxy-3,5,8-trihydroxyflavone

and

also

(2S)-5-hydroxy-7-methoxyflavanone,

2,4-

dihydroxychalcone, 4,2,4-trihydroxychalcone, 7-hydroxyisoflavone and 7,3,4-trimethoxyisoflavone that

were found to induce quinine reductase activity in his study.

Aratiles fruits are identified to contain Ascorbic acid, Ash, Beta- Carotene, Calcium,
Carbohydrates, Fat, Fiber, Iron, Moisture, Niacin, Phosphorus, Potassium, Protein, Riboflavin, Sodium,
Thiamin and Water (United States Department of Agriculture, 2000).

Pharmacologic Use

In a study made by Zakaria, et al (2007), Muntingia calabura was found to possess anti-tumor
properties as well as antinociceptive activity. Muntingia calabura leaves seemed to possess heat-stable
antinociceptive activity that is partly mediated by an opioid receptor. In the same study, it was again found
that Muntingia calabura possessed anti-inflammatory and additional antipyretic activities. Further studies
that have been carried out also revealed the involvement of non-opioid receptors like -adrenergic and
muscarinic receptors in Muntingia calabura antinociceptive activity. In addition, this activity was also found
to resist the effect of extreme acidic and alkaline conditions. These researchers also studied the in-vitro
antibacterial activity of Muntingia calabura extracts. Cheng-Dean et al investigated in 2006 the
hypotensive effect of the crude methanol extract from the leaf of Muntingia calabura by the activation of
nitric oxide. Further studies of have demonstrated that the aqueous extract of Muntingia calabura leaves
has peripheral antinociception and anti-inflammatory effects which involved the activation of -opioid,

-adrenergic and muscarinic receptors (Zakaria, 2007).

Methods of Extraction

In the study of Zakaria et al in 2007, leaves of Muntingia calabura were washed and rinsed with
water to remove all the dirt and unwanted particles, after which the leaves were oven-dried for 72 hours at
the temperature of 40oC. The dried leaves were then ground into small particles, weighed and mixed with

dH 2 O at the ratio of 1: 25 (w/v). This mixture was then left for 24 hours and the supernatant was
collected and filtered using Whatman No. 1 filter paper while the remaining plant residue was kept in an
oven for future use. The supernatant obtained, labelled as MCAE and considered as stock solution with
100% concentration, was diluted to the concentrations of 1, 5, 10, and 50% with distilled water for
antinociceptive study. A 30-ml volume of the obtained supernatant divided equally (10 ml each) was put
into three plastic test tubes and then subjected to a freezing-drying process to determine the amount of
crude dried MCAE present in every 10 ml of the supernatant.

Male ICR mice (2530 g; 57 weeks old) were used in this study. The animals were obtained
from the Veterinary Animal Unit, Faculty of Veterinary Medicine, University Putra Malaysia (UPM),
Malaysia and kept at room temperature (27 8 2 oC; 70 80% humidity; 12-hour light/darkness cycle) in the
Animal Holding Unit for at least 48 h before use. Food and water were supplied ad libitum up to the
beginning of the experiments. At all times, the mice were handled in accordance with current UPM
guidelines for the care of laboratory animals and the ethical guidelines for investigations of experimental
pain in conscious animals. All experiments (n = 10) were conducted between 09.30 and 18.30 hours to
minimize the effects of environmental changes.

DIABETES MELLITUS

The term diabetes mellitus describes a group of metabolic diseases characterized by high blood
glucose levels that result from defects in insulin secretion, or response, or both. Over time, diabetes can
lead to blindness, kidney failure and nerve damage. These are the result of damage to small blood
vessels. Diabetes is also an important factor in accelerating atherosclerosis, leading to stroke, coronary
heart disease and other large blood vessel diseases (Mathur, 2009).

There are two major types of diabetes, called type 1 and type 2. In type 1 diabetes (formerly
called insulin-dependent diabetes or juvenile-onset diabetes), the pancreas undergoes an autoimmune
attack by the body itself (Mathur, 2009). Insulin-producing cells of the pancreas are permanently
destroyed, and the pancreas produces little or no insulin. Most people who have type 1 diabetes develop
the disease before age 30 (Kishore, 2008).

In type 2 diabetes (formerly called non-insulin-dependent diabetes or adult-onset diabetes),the

pancreas continues to produce insulin, sometimes even at higher-than-normal levels. However, the body
develops resistance to the effects of insulin, therefore there is not enough insulin to meet the bodys

needs (Kishore, 2008). A major feature of type 2 diabetes is a lack of sensitivity to insulin by the cells of
the body (particularly fat and muscle cells) (Mathur, 2009). Type 2 diabetes was once rare in children and
adolescents but has recently become more common. However, it usually begins in people older than 30
and becomes progressively more common with age (Kishore, 2008).

THE FASTING AND POSTPRANDIAL BLOOD GLUCOSE STATES

Though blood glucose concentrations fluctuate throughout the day, they can be divided into two
basic states. The Diabetes Association of Greater Cleveland distinguishes the fasting and postprandial
blood glucose states (Blood Sugar 101).

The Fasting State

In the fasting state, the liver keeps blood glucose concentration at a normal level by continually
releasing small amounts of glucose from the glycogen it has stored after meals or by producing new
glucose from protein (Blood Sugar 101). If a person doesnt have diabetes, his or her body reacts by
producing insulin, which prevents hyperglycemia (high blood sugar). However, if ones body cannot
generate enough insulin or cannot appropriately respond to insulin, fasting blood sugar levels will stay
high (Close, 2008).

A normal, healthy liver is also sensitive to insulin levels. The less circulating insulin it senses in
the blood stream, the harder the liver will work to put more glucose into the blood. In a healthy person, the
liver keeps the fasting blood sugar concentration near 85 mg/dl (4.7 mmol/L) at all times (Blood Sugar
101). The normal, nondiabetic range for blood glucose is from 70 to 110 mg/dL (MedicineNet, 2009).

The Postprandial State

After eating, any pure glucose that was present in your food will be absorbed into your
bloodstream within fifteen minutes. During this postprandial state, the concentration of glucose in blood
will begin to rise as the glucose liberated from the food comes pouring in. In a healthy body, this rise is
brief and not very high because as soon as the concentration of glucose in the body starts to rise, it

stimulates the -cells in pancreas to produce a large burst of insulin. Insulin's function is to activate
receptors on body's cells which enable these cells to remove the circulating glucose molecules from the
bloodstream and either burn them for fuel or store them for future use (Blood Sugar 101).
The goals for blood glucose in the control of diabetes for postprandial state is <8.9 mmol/L or
<160 mg/dL (Blood Sugar 101). The magnitude and time of the peak plasma glucose concentration
depend on a variety of factors, including the timing, quantity, and composition of the meal. In nondiabetic
individuals, plasma glucose concentrations peak approximately 60 minutes after the start of a meal,
rarely exceed 140 mg/dL, and return to preprandial levels within 2-3 hours. Even though glucose
concentrations have returned to preprandial levels by 3 hours, absorption of the ingested carbohydrate
continues for at least 5-6 hours after a meal (American Diabetes Association, 2009).

ALLOXAN

Alloxan is a cyclic urea derivative reported to be a potent diabetogenic agent. It is widely used for
the induction of experimental diabetes in animal species by damaging the insulin-secreting pancreatic cells. This results in a decrease in endogenous insulin release, which, in turn, leads to decreased
utilization of glucose by the tissues. Although the exact mechanism of action of Alloxan is not fully
understood, evidences indicate that the pancreatic -cell damage is mediated through the
generation of cytotoxic oxygen free radicals (Kumar, 2007).

INDUCTION OF EXPERIMENTAL DIABETES

Gupta et al (2005) induced diabetes to rats by injecting intraperitoneally a freshly prepared

solution of Streptozotocin (50 mg/kg) in 0.1 M citrate buffer, pH 4.5, and to rabbits by intravenous injection
of Alloxan (80 mg/kg). Fasting blood glucose level was estimated at the time of induction of diabetes and
postprandial glucose was checked regularly until stable hyperglycemia, usually one week with
Streptozotocin and two weeks with Alloxan. Depending on the glucose levels of the animals, they were
divided into three groups: (1) subdiabetic, with nearly normal fasting blood glucose of 80-120 mg/dL, (2)
diabetic, with fasting blood glucose of 120-250 mg/dL, and (3) severely diabetic, with fasting blood
glucose above 250 mg/dL.

An estimation of the percent reduction in blood glucose of mice was done by Santiago-Mendoza
& Ysrael (2001) by measuring the average blood glucose concentration of fasted mice taken at 0 minute,
60 minutes and 120 minutes after oral administration of M. paradisiaca.

CHAPTER III

METHODOLOGY

COLLECTION OF THE PLANT

Fresh young leaves of Muntingia calabura Linn. were collected from La Paz, Tarlac. Time of
collection was four in the morning. The leaves were then air-dried for two weeks.

500 grams of the air-dried Muntingia calabura Linn. leaves were powderized. This sample was
boiled with 10 liters of distilled water in medium heat for eight hours. The resulting decoction was filtered
using cheesecloth and centrifuged at the speed of 10,000 revolutions per minute for four minutes. The
centrifuged decoction was kept in a freezer when not in use for the duration of the research. To promote
lyophilization, the decoction was freeze-dried for 72 hours at the Chemical Department of De La Salle
University until dried powdered extract was achieved. The lyophilized aqueous extract was then weighed
and diluted to compute for the concentration.

PREPARATION OF LABORATORY ANIMALS

Mice weighing 20-30 grams purchased from the Research Institute for Tropical Medicine were
used in the study. They were fed a normal diet consisting of small pellets for mice and an adequate
supply of purified water. The researchers acclimatized the mice 5 days prior to bioassay.

REAGENTS

The reagents used in this experiment were (1) distilled water for aqueous extraction, and (2)
Alloxan for the induction of diabetes in mice.

INDUCTION OF DIABETES USING ALLOXAN

The mice were fasted four hours prior to diabetes induction. Type 1 diabetes mellitus was then
induced by a single intraperitoneal injection of freshly prepared aqueous solution of Alloxan monohydrate
at a dose of 100mg/kg mouse body weight. On the third week after complete Alloxan injection, the basal
blood glucose levels of the mice were determined. Mice with fasting glucose levels of greater than 126
mg/dL were considered diabetic.

DETERMINATION OF EFFECTIVE DOSE

Twenty eight male mice were used in the determination of the Approximate Effective Dose (AED)
of the aqueous extract from the leaves of Muntingia calabura Linn. The dose was computed using
Logarithmic Method starting at 0.039 mg/g body and +0.6 log spacing. The mice were fasted for sixteen
hours, and then given the computed dose of the extract through oral gavage based on body weight.

Observation for 120 minutes of a decrease in blood glucose level followed. Blood sample from
the mices tails were taken at 0, 1 and 2 hours after plant extract administration. Glucose levels were
measured using a glucometer. The dose where 50% of the mice showed a decrease to normal levels of
blood glucose will be considered approximate effective dose.