A Comparative Study of the Hypoglycemic Effect of Aqueous and

Methanolic Extracts of Pansit- Pansitan (Peperoma pellucida) on

Alloxan Induced Sprague Dawley Rats

Researchers

Aguilar, Charmaine Vhea V.

Calagui, Marshiel- Vee A.
Castillo, Sheryll G.
Frias, Edralynne Joy C.
Olivas, Ma. Cristina G.
Verano, Joezer G.
 Chapter I

THE PROBLEM AND ITS BACKGROUND

Introduction

One of the serious and widespread global health problems that

are characterized by hyperglycemia is Diabetes mellitus, which
is cause by the absolute or relative deficiency of insulin
resistance at the cellular level. In fact, Excessive food
consumption regarding to high calorie, obesity, cardiovascular
disease, stress, and lack of exercise are risk factors for
diabetes mellitus. According to International Diabetes
Federation currently around 40.9 million of peoples are diabetic
and number is expected to rise to 69.9 million by 2025 unless
progressive and effective preventive measures are steps to make
and deal with.

Different types of oral hypoglycemic agents are available along

with insulin for the management of diabetes mellitus. Although
new and more efficacious diabetes medications and improved
medication delivery systems have been developed, the majority of
diabetic patients do not achieve optimal blood glucose control,
leading to poor health outcomes and needing the news alternative
drugs for treatment. However, herbal treatment is an alternative
in the treatment of this pathophysiology in as much as plants
are used to cure diabetes.

Pancit- pancitan is an herb which its availability here in the

Philippines is distributed in loose and damp soil and
conspicuous in most rocky parts of waterways which has an
incredibly property for depressing the blood pressure and
glucose values. In circumstance, it has also an analgesic and
anti- inflammatory action, anti – cancer activity, antioxidant,
anti – bacterial activity. It also use for the Uric Acid
reduction in blood as well as the depressant activity.

Furthermore, this plant would be the central creed of curing the

most common metabolic syndrome, the Diabetes mellitus that
causes disability, early death, and many other complications
like neuropathy, nephropathy, retinopathy, amputation, erectile
dysfunction and cardiovascular disease.

Conceptual Framework

In this study we will use fasting plasma glucose test and an

oral glucose tolerance test to determine the effect of Pancit
Pancitan leaves and stem on the Diebetes mellitus Alloxan
induced Sprague Dawley Rats. In this method, the sprague dawley
rats were divided into 2 groups of 3 rats in the different
concentrations and 1 rat in each of the controls. All of the
groups will be induced with Alloxan. Body mass of each diabetic
mouse in a group was measured and amount of extract for each
test group was calculated, weighed, dissolved in distilled water
and administered to each mouse PO. Group 1-3: Diabetic mice
treated with crude aqueous extract of Pansit Pansitan a dose of
500 mg/kg, 750 mg/kg, and 1000 mg/kg PO. Group 4-6: Diabetic
mice treated with methanolic extract of Pansit Pansitan at dose
of 500 mg/kg, 750 mg/kg, and 1000 mg/kg PO. Group 7: Diabetic
mice treated standard drug Glibenclamide 10 mg/kg PO [20]. Group
8: Untreated diabetic mice (Diabetic control) given equal volume
of distilled water PO. Group 9: Non-diabetic reference group
(Normal Control) given equal volume of distilled water P.O.
Blood glucose level of these mice was measured using glucometer
at interval of 1 hr after treatment for consecutive 5 hrs. by
drawing blood from tail vein. The result of troponin test will
be measured.

Republic Act No. 8423 known as the traditional and Alternative

Medicine Act (TAMA) of 1997 is a policy of the state that seek a
legally workable basis by which student will study a certain
ethno medicinal plant that will undergo a research as a
treatment for certain disease. It is a guide for the researchers
to improve and give the Filipinos a cheap alternative medicine.

Tuklas Lunas, the Essential National Health Research (ENHR)

Program of the Philippines, Department of Health (DOH) is also a
guide for the researchers to improve their study. It covers a
guideline for a quality research and a scientific based study
regarding the search for the alternative plants that could
provide a medicinal effect for certain disease.

Philippine Institute of Traditional and Alternative Health Care

(PITAHC) -Cagayan Valley Herbal Processing Plant (CVHPP)
This is our guide for the plant and extract that we utilized for
our study. It is capable of formulating efficient herbal plants
for Filipinos. It helps us perform the proper procedure for the
extract. Furthermore, It helps in providing a low cost medicine
with same effect as the commercially available drugs that are
subjected for approval as per substantiated in the Article III
Section 6 of the RA 8423.
 INPUT PROCESS OUTPUT

A. Methanolic Leaf
A. Acute toxicity A. Oral acute
Extract and crude
test in Sprague toxicity of
aqueous extract
Dawley Rats Pansit Pansita
(500 mg/kg, 750
aqueous
mg/kg, and 1000
extracts
mg/kg PO
) B. Phytochemical
screening
a. Diabetes mellitus B. Hypoglycemic
effect of
A. Positive Control: C. Fasting plasma aqueous and
Glibenclamide 10 glucose test methanolic
mg/kg PO extracts
D. Oral glucose
B. Negative Control: of Pansit
tolerance test
Distilled Water Panitan

Fi

Figure 1: Research Paradigm showing the input-process- output

schematic illustration of the study.
Objectives of the Research

The main objective of this study is to determine the

effectiveness of Pansit-pansitan on its hypoglycemic of its
methanolic and aqueous leaf extract introduced in Sprague Dawley
rats. Specifically, it aims to:

 Determine which of the concentration between 25%, 50%, 75%

and 100% of each methanolic and aqueous extract is more
eefective to Diabetes melitus.
 Determine the different effectiveness of both aqueous and
methanolic leaf extract of Pansit-pansitan when tested to
Sprague Dawley Rats through Acute Toxicity test, fasting
plasma glucose test and oral glucose tolerance test.
 Determine if there is significant difference on the
methanolic and aqueous extract in:
a. Glibenclamide
b. Distilled water

Hypothesis: suppressant property

Ho: There is no significant difference of the methanolic leaf

extract and aqueous leaf extract of Pansit-pansitan compared to
the positive and negative control.

Significance of the Study

This study is expected to benefit:

Department of Health (DOH): The DOH can promote the use of

Pansit- pansitan as an alternative medicine.
Department of Science and Technology (DOST): Through this study
DOST can fund and support further investigations on the property
of the plant.

Department of Agriculture (DA): DA can propagate the Pansit-

pansitan for future use.

Herbal Processing Plant in the Philippines: they can utilize the

plant as a raw material for the production of herbal medicines.

The Public: they can utilize the plant to prevent and cure
Cardiac markers of the The Researchers.

The study can serve as a future reference on related researches

will be conducted in the future.

Scope and Delimitation

The study is limited only to the leaves of Pansit pansitan

(Peperomia pellucida). The leaves selected with the single
species of matured leaves of Pansit pansitan gathered from a
single plant to ensure that there is no variation in the test
result. Alloxan will be induced to the 6 rat to elevate the
glucose inside of their body to give the same case regardless of
its effect to the hyperglycaemia . The plant is harvested in
Tabuk, Kalinga. The Sprague Dawley rats were purchased at
Philippine Institute of Traditional and Alternative Health Care
(PITAHC), Carig, Tuguegarao City, Cagayan. The method that will
be used in this study is Acute toxicity test in Sprague Dawley
Rats, Phytochemical screening, fasting plasma glucose test, oral
glucose tolerance test. The controls used in this study for
Diabetes Mellitus and Glibenclamide as a positive control,
distilled water is used as negative control for both tests.

Definition of Terms:

 Pansit-pansitan - utilized as an experimental plant

evaluated for its effect on the Cardiac Markers of
Ibuprofen induced Sprague Dawly Rats.

 Diabetes mellitus - commonly referred to as diabetes, is a

group of metabolic disorders characterized by high blood
sugar levels over a prolonged period. Symptoms of high
blood sugar include frequent urination, increased thirst,
and increased hunger. If left untreated, diabetes can cause
many complications.

 Alloxan - used as an inducer on Sprague Dawley Rats.

 Sprague Dawley Rats - experimental animal used in this

controlled clinical trial.

 Methanolic extract- ideal solvent because of its polarity.

It can extract both hydrophilic and lipophilic molecules
from plant parts. After extraction we can remove the
 solvent at low temperature by distillation as methanol is
highly volatile.
 Aqueous extract - an extract prepared by evaporating a
watery solution of the soluble principles of a vegetable
drug (such as licorice) to a semisolid or solid
consistency.

Chapter II
 REVIEW OF RELATED LITERATURE

A. Pansit-pansitan

Taxonomical
Classification

Pansit-pansitan came
from the kingdom
Plantae, division
Tracheophyta, class
Magnoliopsida, order
Piperales, family
Piperaceae, genus
Peperomia and species P.
pellucida.

Botanical Description

(Peperomia pellucida (Linn.) (HBK)) It belongs to the family

Piperaceae.

It is an herbaceous annual plant with succulent stems

found in many South American and Asian countries. The plant
grows to a height of 15 to 45 cm. Its leaves
are alternate, heart-shaped and turgid, as transparent and
smooth as candle wax. Tiny dot like flowers scattered along
solitary and leafopposed stalk maturing gradually from the base
to the tip and turning brown when ripe. It propagates by seeds.
Numerous tiny seeds drop off when mature and grow easily in
clumps. The species develops during rainy periods and thrives in
loose, humid soils under the shade of trees (www.drugs.com).

The decoction of leaves is used for gout and arthritis

(http://www.stuartxchange.org/Pansit.html). Also the pounded
stems are applied on the face for boils and pimples and have
said to have anti bacterial property against Bacillus subtilis
and Staphylococcus aureus. (Lans, Journal of hEthnobiology and
Ethnomedicine,2007)

Chemical Content of Botanical Extract

Pansit-pansitan (Peperomia pellucida (Linn.) (HBK)) It was found

out that the plant leaf contains ascorbic acid, beta-
carotene,calcium, carbohydrates, fat, fiber, iron, niacin,
phosphorus, potassium, protein, riboflavin, sodium, thiamine,
water (http://www.ars-grin.gov/cgibin/duke/farmacy2.pl). Other
substances are benzenoid, steroids and apiol found throughout
the plant (Manalo JB, et al. 1983). Alkaloids and tannin are
mainly abundant in leaves (De Padua LS, Pancho JV.
1983).Alkaloids are plant-derived compound that is toxic or
physiologically active. It has a complex structure and is of
limited distribution in the plant kingdom. Alkaloids have many
uses such as poisons which build up nerve impulses transmitted
by the brain which results in restlessness, convulsions, nausea,
vomiting and death (Dales MJ, 1996). Quinine which is a type of
alkaloid has anti-malarial compound. Another form of alkaloids
is atropine and is used to dilate the pupils of the eye.
Atropine is also a CNS stimulant and is used as a treatment for
nerve gas poisoning (Murray G, Frank M, 1973). Apiol (C12H14O4)
is an essential oil usually found in some succulent plant
species. It is green, non-viscous, oily liquid with a peculiar
odor and a disagreeable acrid taste (United States National
Library of Medicine). In humans extremely high doses of the
chemical may produce effects such as ringing in the ears,
headache and vertigo. In pregnant women it can cause an
accumulation of blood in the pelvic-uterus area and liver
damage. High doses of some oils, especially containing apiol can
have narcotic or paralyzing effects
(http://www.juniperandsage.com/oils/toxicity.php).

Tannins are polyphenols that are obtained from various parts of

different plants belonging to multiple species. It is found in
abundance in the tree bark, wood, fruit, leaves, and roots and
also in plant gall. Interestingly, tannins are found almost in
all plants and in all climates all over the world however its
percentage varies depending on plant species. Tannins are
usually found in large quantities in the bark of trees where
they act as a barrier for micro-organisms like bacteria and
fungi and protect the tree. It is also used in dyeing,
photography, astringent, stops infection, and refining beer and
wine. As tannins often lower the absorption of some materials
into the body,tannins are also often known as anti-nutrients.
Tannins can be classified into two broad groups - hydrolysable
tannins and condensed tannins. Hydrolysable tannins are
basically derived from simple phenolic acids like gallic acid
that when heated they give away pyrogallol. Pyrogallol is also
known as hepatotoxic and has antiseptic as well as caustic
properties. Condensed tannins do not split easily and hence it
is difficult to analyze these. When condensed tannins are
heated, catechol emerges as the final product. Unlike
hydrolysable tannins, condensed tannins do not possess any trace
of hepatotoxicity or any adverse side effects and hence are
favorable for use (Harborne Jeffery, H. Baxter. 1983).

Geographical Distribution
An annual herb, favouring shady, damp and loose soil all over
Asia and the Americas. It often grows in groups in nooks in the
garden and yard. It is conspicuous in rocky parts of canals. It
is a pantropic species of American origin.

Phytochemical and Active Metabolites

Preliminary phytochemical screening of methanol extracts of

stems yielded carbohydrates

Folkloric Uses

Ethno medicinal uses for plant vary. Infusion and decoction of

leaves and stems are used for gout and arthritis. Decoction of
k\leaves is used for urinary tract infection. Externally, as a
facial rinse for complexion problems. In Ayurveda, used to
pacify vitiated cough pitta, constipation, kidney diseases,
urinary retention dysuria, urinary tract infection, elimination
edema and general weakness. Pounded whole plant used as a warm
poultice for boils, pustules and pimples.

In Jamaica and the Caribbean used for colds and as a diuretic

for kidney problems. In South America, solution of fresh juice
of stem and the ,leaves used foe eye inflammation.

In Brazil, used for abscesses and conjunctivitis. In Bolivia,

decoction of roots used for fever; aerial parts of the wounds.In
Nigeria used for hypertension. In Bangladesh, leaves used in the
treatment of excited mental disorders.
In Africa, used in convulsions and tumors used for headaches,
rheumatic pains, impotence

In Camerron used for fracture healing. In Brazil, used to lower

cholesterol; for treatment of abscesses for arthritis and gout\
B. Diabetes mellitus

Cause and Etiologic

During digestion, your body breaks down carbohydrates from foods

— such as bread, rice and pasta — into various sugar molecules.
One of these sugar molecules is glucose, a main energy source
for your body. Glucose is absorbed directly into your
bloodstream after you eat, but it can't enter the cells of most
of your tissues without the help of insulin — a hormone secreted
by your pancreas.

When the level of glucose in your blood rises, it signals your

pancreas to release insulin. The insulin, in turn, unlocks your
cells so that glucose can enter and provide the fuel your cells
need to function properly. Any extra glucose is stored in your
liver and muscles in the form of glycogen.

This process lowers the amount of glucose in your bloodstream

and prevents it from reaching dangerously high levels. As your
blood sugar level returns to normal, so does the secretion of
insulin from your pancreas.

Diabetes drastically diminishes the effects of insulin on your

body, either because your pancreas is unable to produce enough
insulin (type 1 diabetes) or because your body is resistant to
the effects of insulin or doesn't produce enough insulin to
maintain a normal glucose level (type 2 diabetes). As a result,
glucose tends to build up in your bloodstream (hyperglycemia)
and may reach dangerously high levels if not treated properly.
Insulin or other drugs are used to lower blood sugar levels.
Pathogenesis

The conventional pathways that generate NADH by breaking down

glucose via glycolysis and the Krebs cycle. The enzymes involved
in NADH/NAD+ recycling are shown. *DLDH stands for
dihydrolipoamide dehydrogenase and is the component in each
given enzyme complex that actually makes NADH from NAD+

NADH oxidation by complex I in the electron transport chain.

Electrons from NADH are transported via CoQ and cytochrome c to
molecular oxygen. This process involves proton pumping that is
tightly linked to superoxide production. ATP synthesis by
complex V driven by the proton gradient is also shown.

The branch-off pathways that are activated to dispose excess

glucose when glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is
inactivated by ROS. These five alternative pathways in addition
to the electron transport chain shown in Figure 2, are linked to
ROS production, thus further exacerbating oxidative stress.
Inset shows the polyol pathway. Pathways in the grey area would
no longer efficiently break down glucose when GAPDH is
inactivated by posttranslational modifications.

Hyperglycemia induces overproduction of NADH and mitochondrial

ROS that inhibit GAPDH activity. This inhibition then activates
the alternative glucose metabolic pathways, which further
produce ROS involved in glucotoxicity that is responsible for
the development of diabetes and diabetic complications. ETC:
electron transport chain.
Treatment and Prevention

Dietary Management and Physical Activity

Modifying eating habits and increasing physical activity are
typically the first steps toward reducing blood sugar levels. At
UCSF Medical Center, all patients work with their doctor and
certified dietician to develop a dietary plan. Our Teaching
Center conducts workshops that provide patients with information
on food nutrient content, healthy cooking and exercise.

Insulin Therapy
People with type 1 diabetes require multiple insulin injections
each day to maintain safe insulin levels. Insulin is often
required to treat type 2 diabetes too. Using an insulin pump is
an alternative to injections. The pump is about the size of a
pager and is usually worn on your belt. Insulin is delivered
through a small tube (catheter) that is placed under the skin
(usually in the abdomen).

Oral Medications
Sometimes blood sugar levels remain high in people with type 2
diabetes even though they eat in a healthy manner and exercise.
When this happens, medications taken in pill form may be
prescribed. The medications work in several different ways.
These include improve the effectiveness of the body's natural
insulin, reduce blood sugar production, increase insulin
production and inhibit blood sugar absorption. Oral diabetes
medications are sometimes taken in combination with insulin.
Test Parameters

Fasting blood sugar level from 100 to 125 mg/dL (5.6 to

6.9mmol/L) is considered prediabetes. If it's 126 mg/dL (7
mmol/L) or higher on two separatetests, you have diabetes.

Glucose tolerance test-For this test, you fastovernight, and

the fasting blood sugar level is measured

C. Sprague Dawley Rats

The Sprague Dawley rat is an outbred multipurpose breed of

albino rat used extensively in medical research. Its main
advantage is its calmness and ease of handling. The average
litter size of the Sprague Dawley rat is 10.5. The adult body
weight is 250–300g for females, and 450–520g for males. The
typical life span is 2.5–3.5 years.

D. Controls

Glibenclamide, also known as glyburide, is an antidiabetic drug in

a class of medications known as sulfonylureas, closely related
to sulfonamide antibiotics. It was developed in 1966 in a
cooperative study between Boehringer Mannheim (now part of
Roche) and Hoechst (now part of Sanofi-Aventis).

Glibenclamide is a type of medicine called a sulphonylurea. It

is used to help control blood sugar levels in people with type 2
diabetes. People with type 2 diabetes (non-insulin
dependent diabetes) have a deficiency of a hormone called
insulin.