1

CHAPTER I
INTRODUCTION

A BACKGROUND OF THE STUDY

Diabetes mellitus is one of the common metabolic disorders that have microvascular
complications such as neuropathy, nephropathy, retinopathy and macrovascular complications
(atheroma) that results in significant morbidity and mortality worldwide ( 1, 2, 3, 4).
Diabetes is a global concern that cuts across geographical boundaries regardless of race,
sex, status and age. 347 million worldwide have diabetes. More than 80% of people with
diabetes live in low- and middle-income countries. World Health organization (WHO) projects
that deaths due to diabetes will double between 2005 and 2030, and will be the 7th leading cause
of death in 2030. (5)
The occurrence of diabetes is emergent in all parts of the world. According to Dona
Pazzibugan of the Philippine Daily Inquirer (6), a survey conducted in the Philippines on 2007
by the Philippine Cardiovascular Outcome Study on Diabetes Mellitus (PhilCOS-DM) further
shows that 3 out of 5 adults are already diabetic or on the verge of developing diabetes. Health
authorities and the medical community are alarmed by the rapid development of diabetes
especially among children.
Due to the decreasing economic stability in the Philippines, the coordination between
private and public health care providers needs improvement since the health system suffers from
inadequate and irregular insurance coverage, thus reducing access of the poor and marginalized
sector to adequate health care, adding to high prices of pharmaceutical products and medical
devices and meager penetration of generic medicine (7). Consequently, there is an increasing

demand by patients to use natural products with anti-diabetic activity due to side effects
associated with the use of insulin and oral hypoglycemic agents (8, 9, 10).
WHO (5) stated that the positive features of using natural products in traditional medicine
include its diversity, flexibility, easy accessibility, broad continuing acceptance in developing
countries and increasing popularity in developed countries, relative low cost, low levels of
technological input, relative low side effects, and its growing economic importance.
An ethno-medicinal survey conducted in the Philippines led many people in the discovery
of Calabash tree (Crescentia cujete) as being part of the traditional medicine in treating many
illnesses particularly diabetes (11). In the Philippines, it is also used as a form of herbal
medicine. The Department of Science and Technologys (DOST) Philippine Council for Health
Research and Development (PCHRD) noted that local communities have used the Calabash tree
to treat various illnesses (12). The pulp of the fruit has medicinal properties. Among which, is its
effectivity as a hypoglycemic agent. It can reduce the bodys sugar uptake that can be attributed
to its high reserves of pectin (13).
Diabetes is evidently one of the most imperative medical problems of our time (5).
Because of its chronic nature, the financial burden of diabetes approaches the development of
alternative treatments and herbal medicines (6, 7). However, many people are not aware of its
phytochemical constituents that are more likely to cause toxicity resulting to different organ
damage and health problems if not taken with the appropriate dosage. The guiding principle of
the study is to affirm the hypoglycemic effects of the calabash fruit and to determine the
appropriate dosage to which it is effective. Another is to check the direct toxic effect of the fruit.
This is to ensure complete safety, before the use of Calabash fruit will be introduced and
promoted to the public.

B OBJECTIVES OF THE STUDY

This study generally aimed to determine the acute toxicity of Calabash (C. cujete) fruit
pulp decoction in rabbits and its Approximate Effective Dose (AED) and Median Effective Dose
(ED50) in alloxan-induced hyperglycemic rats (Spague dawley).
It specifically aimed:
1 To determine the Acute Toxicity Dose of the Calabash decoction on New Zealand White rabbits;
2 To determine the Approximate Effective Dose (AED) of the Calabash decoction on the alloxan3

induced hyperglycemic rats (Sprague dawley);

To determine the Effective Dose (ED50) of the Calabash decoction on the alloxan-induced

hyperglycemic rats (Sprague dawley);

4 To determine the blood glucose level of alloxan-induced diabetic rats (Sprague dawley) and the
peak effect of Calabash (C. cujete) fruit pulp decoction at 0, 0.5, 1, 3, 5, 8, 10, 12, 14, 16, 18, 20,
22, 24 hours treated with:
a Normal Saline Solution (Negative control group)
b Calabash Decoction (Test group);
5 To determine the fasting blood glucose levels of the three alloxan-induced diabetic rat (Sprague
dawley) groups on the 0, 5th, 10th and 15th day treated with:
a Metformin (Positive control group)
b Normal Saline Solution (Negative control group)
c Calabash Decoction (Test group) ; and
6 To determine whether there is a significant difference among the fasting blood glucose levels of
the three diabetic rat (Sprague dawley) groups treated with:
a Metformin (Positive control group)
b Normal Saline Solution (Negative control group)
c Calabash Decoction (Test group)

C SIGNIFICANCE OF THE STUDY

The study was conducted significantly for the benefits of:
Medical Students. There is unfamiliarity of the chemical content of the fruit sample
Calabash (C. cujete) and its acute toxicity levels. Thus, this can be a significant endeavor in

acquiring and spreading information as additional knowledge that can be applied or used in the
present or even in their future profession.
Researchers and future researchers. This can provide basis of additional knowledge
and be a reference for current or prospective studies.
Community. As the Calabash (C. cjuete) is being grown to a lot of places here in
Philippines and is considered by many as a traditional alternative medicine, people will now be
aware of its safety and toxicity dosages.
Different Organizations/Agencies (DOST, DOH, DOA). The information this study
provides can strengthen the research and development department of such organizations. Design
of new or improved programs for health and production of the fruit can also rise.
Healthcare providers. Lastly, healthcare providers can utilize the study as basis for their
recommendations and information to patients who use Calabash (C. cuejete) as an adjunct for
different health conditions, especially as a blood glucose-lowering agent.
D SCOPE AND LIMITATION
This study focused on the determination of the Acute Toxicity dose of Calabash (C. cujete)
decoction in New Zealand White rabbits and the Approximate Effective Dose (AED) and
Effective Dose (ED50) of the fruit decoction on alloxan-induced hyperglycemic rats (Sprague
dawley). Only female adult New Zealand White rabbits weighing 2-6 kg 20% were used in the
determination of acute toxicity dose. Rats (Sprague dawley) of both genders weighing 250-350
grams were used in AED, ED50 and bioassay.

The Calabash (C. cujete) fruit was collected in Fatima Village, Circumferential Road, Davao
City. Test was conducted at Davao Medical School Foundation Inc., Davao City. This experiment
covered the months of November until January of the 2nd semester of school year 2015-2016.

E DEFINITION OF TERMS
1 Acute Hypoglycemic Effect determines the anti hyperglycemic activity of Calabash
2

(C. cujete) fruit pulp decoction in a period of 15 days.

Acute Toxicity Test establishes the concentration on the relative toxicity likely to
arise from a single exposure, observed by death, within 14 days using New Zealand

White (NZW) rabbits as test animal.

Alloxan the crystalline oxidation product of uric acid that will be used to induce
hyperglycemia on the rabbits by selective destruction of pancreatic beta cells, which

produce insulin.
Approximate Effective Dose (AED) the dose wherein the Calabash (C. cujete) fruit
pulp decoction concentration is found to lower elevated glucose levels of adult rats as

a test animal.
Bioassay a scientific procedure wherein the effectivity of Calabash (C. cujete) fruit
pulp decoction as a hypoglycemic agent will be quantitatively measured together with
the negative control (normal saline solution) and positive control (Metformin) on

alloxan-induced diabetic rats.

Fasting Blood glucose level - the concentration of glucose in the blood expressed in
mg/dL taken after an overnight fast of less than 16 hours (no food taken except

water).
Calabash fruit (Crescentia cujete) the test plant that will be utilized by the
researchers for determining its lethal and effective dose.

Fruit Pulp Decoction - the method of extraction by boiling the fruit pulp excluding the

seeds.
Effective Dose (ED50) the amount of a substance required to produce a specific

effect in half of an animal population comprising a test sample.

10 Glucometer a portable glucose-measuring device that utilizes enzymatic sticks and
uses LED monitor to quantify and display the glucose level
11 Peak Effect determines the time where Calabash (C. cujete) fruit pulp decoction is
in its highest percentage of hypoglycemic activity.
12 Rabbit a healthy, randomized, properly identified, nulliparous, nonpregnant, and 812 weeks old New Zealand white female rabbit, that weighs 2-6 kg 20% at the start
of the experiment.
13 Rat a healthy, randomized, properly identified, fasted adult Sprague dawley rat of
either gender that weighs 250-350 g at the start of the experiment.
F LIST OF ACRONYMS
1 AED Approximate Effective Dose
2 ANOVA Analysis of Variance
3 CDER - Center for Drug Evaluation and Research
4 DOST Department of Science and Technology
5 ED50 Median Effective Dose
6 FBG Fasting Blood Glucose
7 FDA Food and Drug Administration
8 NZW New Zealand White
9 OECD Organization for Economic Co-operation and Development
10 PCHRD Philippine Council for Health Research and Development
11 PhilCOS-DM Philippine Cardiovascular Outcome Study on Diabetes Mellitus
12 PITAHC Philippine Institute of Traditional and Alternative Health Care.
13 WHO - World Health Organization

CHAPTER II
REVIEW OF RELATED LITERATURE

A RELATED LITERATURE AND STUDIES

1 Diabetes Mellitus
Etiology
Diabetes mellitus is a metabolic disease characterized by chronic uncontrolled hyperglycemia
due to defects in insulin secretion or action, or both. The causes of increased blood glucose levels
could be linked to a wide range of pathologies which roots from the destruction of -cells of the
pancreas, which is the main insulin-producing cells of the body. Insulin insufficiency, amount or
in function, are associated with deregulations in the metabolism of carbohydrates, fats, and
proteins. These impairments eventually manifests as hyperglycemia, the hallmark sign of the
disease. (14)
There are two major classifications of Diabetes Mellitus, namely Type I and Type II. Type I
diabetes have been described by its cause which is associated with the destruction of insulin
producing pancreatic -cells (52). In the study, Alloxan Induced Diabetes: Mechanisms and
Effects, the experimental animals have been induced to develop Type I Diabetes. Repeated
blood glucose monitoring were performed on the animals to determine the presence of
uncontrolled hyperglycemia induced by the administered drug alloxan. Alloxan is known to
induce diabetes mellitus in animals either by its accumulation in the beta cells as glucose
analogues or increase in calcium ions resulting to damage of beta cells (53).

Diagnosing Diabetes Mellitus

Fasting Blood Glucose (FBG)

The FBG test, more commonly known as Fasting Blood Sugar, measures the blood glucose
levels after an 8-hour period of fasting. It is used to detect diabetes and pre-diabetes. The FBG
test measures blood glucose and is most reliable when given in the morning. Physiologically,
blood glucose levels decreases after a period of fasting, and thus, low levels of plasma glucose is
to be expected. However, in diabetes mellitus, persistent increase in levels of blood glucose after
fasting can be observed, thus, diagnostic of the disease. FBG testing has been commonly used in
diagnosing diabetes mellitus because of convenience and cost-effectiveness compared to other
tests such as Oral Glucose Tolerance Test (OGTT), A1C, and others.

Figure 1. Diagrammatic representation of glucose metabolic pathways in normal and

diabetic case.
Prevalence in the Philippines and Local Setting
The incidence of diabetes is rapidly rising. Diabetes is listed as one of the top ten leading
causes of morbidity worldwide and Philippines is included in the top 15 countries with high
prevalence rates of the disease (15). Philippines is home to more than 4 million people diagnosed
with the disease, exclusive of those who are undiagnosed and yet to be diagnosed. According to
Philippine Diabetes Statistics (16), the survey predicts that one out of every five Filipinos

already have or will develop diabetes. There were 3.4 million diabetes cases in the country in
2010, representing a prevalence rate of 7.7 percent according to Dr. Joey Miranda, secretary of
the American Association of Clinical Endocrinology-Philippines. In addition, he cited data from
the World Health Organization and International Diabetes Foundation that by 2030, the
prevalence rate is projected to rise to 8.9 percent or 6.16 million cases (17). A more pressing
concern is a growing trend where populations of children as young as 5 years old are being
diagnosed to have developed diabetes type II and thus contributing to the already rising
population of people with diabetes now and the future, according to Philippine Diabetes
Statistics (16).
2

Calabash Fruit (Crescentia cujete)

Plant Profile of Miracle Fruit (C. cujete)

Figure 2. Taxonomic classification (left) of the test plant,calabash tree (C. cujete) and its
fruits (right), which will be used in the study.

Common Names

10

Calabash Tree (C. cujete) is also known as Ayale or gourd tree (English), Calabacero (Spain),
Totumo (Panama and Venezuela), Cujete (Spain), and Miracle Fruit (Philippines) (18).
Description
Calabash (C. cujete) belongs to the family of Bignoniaceae. It is a tree reaching 6 to 10 m
tall with a wide crown and long branches covered with clusters of tripinnate leaves and gourdlike fruit. The branches have simple elliptical leaves clustered at the anode. The greenish flowers
arise from the main trunk and blooms at night. It is propagated either by seed or stem cuttings.
Calabash fruit is a seasonal fruit that develops after pollination by bats. It appears at the end of
dry season, and the fruit is up to 12 to 14 cm in diameter. It is globular with smooth hard green
woody shell. It takes about six to seven months to ripen and eventually falls to the ground. Small
flat seeds are embedded in the pulp. (18)
Biological Source
The Calabash tree is widely distributed in the Caribbean region, Mexico, Northern and
Southern American, and later introduced to tropical Africa from Senegal to Cameroon then to
other parts of Asia. Virtually, all parts of the tree have been found to be useful. The wood is used
for tool handles, ribs in boat building, and cattle yokes; and the gourd for cups, containers, and
musical instruments. The fruit and leaves are reported to have medicinal application. (21)
Calabash as a Hypoglycemic Agent and its Chemical Constituents
The Philippine Council for Health Research and Development of Department of Science
and Technology stated that the local communities in Philippines have been using Calabash tree to
treat various illnesses. With its many uses in folkloric healing, the Calabash tree is considered
as miracle tree of General Santos City, as the PCHRD said (12).

11

The said department confirmed that the miracle fruit Calabash has the ability to lower
blood glucose levels. They stated that the one responsible for the decrease of blood sugar level
was attributed to the effects of phytochemicals found in the Calabash fruit in which it stimulates
insulin release. (54)
Crescentia cujete has been reported to contain principally tartaric acid which improves the
lowering of glucose and A1C levels. Moreover, improved glucose tolerance by the body was also
observed (19). It has moderate fructose content that elucidates why it is being used in treating
diabetes (20). Pectin which is also an essential component of the fruit has the important function
of reducing the rate of sugar uptake. It has been noted to have a role in gastric emptying as it is a
good detoxifying agent (13).
It has been found out that the fruit contains high mean concentrations of mineral elements
such as sodium that functions as electrolytes and plays key role in ion and extracellular fluid
balance and a major factor in nerve impulse transmission (70); and phosphorus in its own
contribution functions in combination with calcium for the formation of bones, teeth and nerve
cells (70); and low mean concentrations of calcium which helps in regulating the passage of
nutrients through cell walls and the correct contraction of the muscle and also helps in the
clotting of blood and the transfer of signal by the nerves (69,70); magnesium that provides bone
and tooth strength, helps in blood clotting, aids nerve impulse transmission required for muscle
contraction (69,70,71); and potassium which is essential for keeping a normal water balance
between the cell and body fluids, that is, it plays an important role in proper heart function
(21,70). Also, several mineral constituents were obtained in the fruit such as manganese that
functions in enzyme reactions with regards to blood sugar metabolism and thyroid hormone
function (22), and zinc is said to be important in protein and carbohydrate metabolism (23).

12

Flavonoids found in C. cujete can act as anti-oxidants and protect the cells of the body from
free radical damage; free radicals are reputed to damage cell and contribute to various health
related problems (72).
Quercetin, a flavonoid with anti-inflammatory and antioxidant properties, helps defend
against diabetes-induced exaggerated vasoconstriction and reduces the elevated blood pressure
(75). In addition, it has the ability to regenerate pancreatic islets increasing insulin release as well
as inhibits diabetes associated increased collagen deposition endothelial pyknosis, and leukocyte
infiltration (76).
Ursolic Acid, also present in the fruit pulp, is capable of increasing skeletal muscle AKT
activity, which could stimulate muscle growth and convey resistance to fatty liver disease,
glucose intolerance, and obesity. It also has a protective effect on kidneys in diabetic rats, thus, a
potential treatment for diabetic nephropathy. (77, 78)
Koffi, et al. (55) conducted an ethnomedical investigation comprising of twenty eight
species of plants, including Calabash, as a means of treatment for diabetes. They assumed that
the component in Calabash that stimulates the release of insulin is cyanhidric acid and the one
involved in glycogenesis is its alkaloids. Hence, Calabash has a pharmacological potential in
decreasing the levels of blood glucose.
Toxicity of Calabash
Several studies revealed the usefulness of calabash fruit in human health and nutrition.
However, there were few studies that have reported that the fruit pulp has carcinogenic activity
and can produce severe diarrhea (44). In the study, The chemical constituents of calabash (C.
cujete) conducted by Ejenolu (21), the value recorded for lead, chromium, nickel, cadmium, and

13

arsenic in the fruit sample were found to be 0.17, 0.07, 0.10 0.01, and 0.01 ppm respectively.
Lead is known to be a very toxic element and its presence in humans and animal diet is not ideal
(24).
The relatively low concentrations of cadmium, chromium, nickel, and arsenic obtained
from the fruit extract do not exhibit significant health effects. However, the concentration of lead
remains a matter of great health concern. The WHO recommended safety value for lead in
portable water is 0.01 mg/L (45). The mean value of lead in the fruit greatly exceeds this,
suggesting that the consumption of the fruits by man or animal could be a good source of lead
toxicity.
The main values of most metals are relatively low, however, the continual consumption
of the fruit may trigger accumulation and toxicity and hence, it should be discouraged for health
reasons (24).
The mean value of the hydrogen cyanide in the C. cujete fruit sample was found to be
0.11 ppm. Higher amounts of HCN were recorded by Ogbuagu (13), 028 ppm and 0.23 ppm for
wet and dry method respectively. The hydrogen cyanide concentration of 0.11 ppm recorded in
the fruit extract was found to exceed the WHO cyanide value for drinking water (0.01 mg/L),
meaning that the continual consumption of C. cujete fruit extract may eventually lead to
hydrogen cyanide toxicity. Hydrogen cyanide, a chemical asphyxiant, prevents the tissue from
exploiting oxygen making it a potential poison (28).
As cited by Dvorkin and Whelan (29), the fruit pulp of calabash (C. cujete) has
carcinogenic activity inducing possible neoplasms related to leukemia and lymphoma. It can also
produce severe diarrhea. The juice and infusion are considered safe if taken properly.

14

Acute Toxicity Studies

A study conducted by Amilhasan et al (30), concentrated more on the blood glucose lowering

agent of the C. cujete fruit. The aim of the study is to determine the fruits Acute Toxicity Dose,
Effective Dose (ED50) and Approximate Effective Dose (AED). An oral dose of the C. cujete
decoction was given to Swiss mice in order to establish the fruits toxicity. Based on the OECD
guideline, the result of category 5 of the fruit decoction categorized as non- toxic. Alloxaninduced rabbits were used in the study to determine ED 50 and AED. ED50 of the fruit was 9.88
mg/kg while AED of the fruit ranged from 3.98 to 15.84 mg/kg.
According to the research done by Alegre et al (31), a randomized type experimental design
was used to screen and test the toxicity and blood glucose lowering capacity of calabash fruit
pulp decoction to alloxan induced rats. They calculated the set LD 50 of 8.57% through the Brine
shrimp assay. They also found out that together with metformin the calabash fruit decoction was
even more effective six hours after the single dose administration, achieving almost 75% of
blood glucose reduction. Phytochemical compounds such as alkaloids, pectin, flavonoids,
cyanhidric acid, quercetin, and ursolic acid contributed to the hypoglycemic effects of the
Calabash fruit. However, the dosage was only limited from 10 to 30% of concentration due to the
highly toxic effects of the Calabash fruit.
According to Food and Drug Administration (FDA) and Center for Drug Evaluation and
Research (CDER), two or more species are generally tested because a drug may affect one
species differently from another (56). As a potential study for clinical trial, this prompted the
investigators to utilize such animals. Results of this study may validate, support and affirm

15

previous studies done or negate, oppose and otherwise present values different from the
references cited.
4

Bioassay
Experimental pharmacologic studies conducted on animal subjects

utilizes different formats in measuring bioassay levels that would allow

investigators to achieve more with less, that is, safer and greater efficacy,
with less time, staff and resources (57).
In an animal study conducted by Amilhasan et al (30), sixty (60) rabbits were
administered with alloxan to induce an animal model for diabetes mellitus and thus,
hyperglycemia. After administration, animals who qualified for the inclusion criteria were then
randomized using fish bowl technique into Groups A, B, and C. Each group were assigned with
three specific treatments, namely plain normal saline solution, standard metformin dose and
calabash decoction, respectively. Treatments were then administered and blood glucose levels
were measured at intervals, 0, 3, and 6 hours after. The bioassay results showed constant blood
glucose over time with normal saline solution (283 mg/dL), while the metformin (261.87 mg/dL)
and Calabash (241.4 mg/dL) groups had a significant decrease over time. Mean measurements of
the three groups were not equal, thus rejecting the null hypothesis.
Published studies using calabash fruit pulp were meager, however, there were several
studies investigating in vivo hypoglycemic effect of different plant extracts on alloxan-induced
hyperglycemic rats to which this thesis were patterned.
Determination of Hypoglycemic Activity and its Peak Effect

16

In an in vivo study conducted by Nkambo et al (58), wherein hypoglycemic effect of

methanolic fruit extract of Momordica charantia L. in rats was studied, blood glucose levels
were measured at 0, 0.5, 1, 2, 3, 5, 8 and 12 hours after administration. In another study by
Kumar et al (59), blood glucose levels of Alloxan-induced rats were measured at intervals of 0,
1, 2, 4 and 8 hours after administration. Both studies divided the qualified animals into specific
groups with six rats each.
In this study, the researchers will be measuring blood glucose levels of alloxan-induced
rats on an hourly basis for 24 hours or until blood glucose levels of the hyperglycemic animal
subjects will be within baseline levels as measured prior to administration of Alloxan. With this
interval pattern, the peak effect in a 24-hour period of the calabash fruit decoction will be
determined and be subject for comparison with the positive control group and the negative
control group.
Acute Hypoglycemic Effect
In a study by Sebai et.al (60), adult male rats were divided into 4 groups (n=12) after
Alloxan induction of diabetes. Treatments of Groups I, non-diabetic rat (control) with NaCl; II,
diabetic rat (Alloxan, 220 mg/kg b.w.) with NaCl (0.9%); III, non-diabetic rat with plant extract;
and IV, diabetic rat (Alloxan, 220 mg/kg b.w. i.p.). The duration of their study was 15 days.
Twenty hours after the last injection, animals were sacrificed, blood was collected and processed
for biochemical parameter determinations.
In a study by Shah and Khan (61), Alloxan induced diabetic animals were divided into
five groups (n=5). Four groups were comprised of diabetic animals and one group of normal
animals. Animals of different groups were given treatment according to their respective group for

17

15 days. After 15 days, fasting blood glucose concentration was measured and animals were
dissected and processed for histological examination and for tissue antioxidant enzymes assays.
In the study by Kumar et al (59) mentioned previously, proceeding the series of blood
extractions on the 1st day, the treatment was continued for the next 21 days and blood samples
were collected. Body weights were measured on the 4th, 7th, 14th and 21st days after 1 h
administration.

Animal Test Subjects

Rabbits for Acute Toxicity

According to Organization for Economic Co-operation and Development (OECD)
Guideline for Testing of Chemicals (OECD Guideline No. 423), in acute toxicity tests, utilization
of smaller number of animals should be considered. Doses should be carefully selected and every
effort should be made not to exceed moderately toxic doses. In such tests, administration of
lethal doses of the test substance should be avoided. Then the substance is tested using a stepwise
procedure, each step using three animals of a single sex. Moreover, it was also indicated that
aside from the preferred species, the rats, other rodent species may be used. Normally, healthy
young adult females, between 8 and 12 weeks old, with weight not falling in an interval within
20% of the mean weight, nulliparous, and non-pregnant, are used. (47)

18

Table 1. Basic Parameters of Acute Toxicity

Studies conducted by Mir and Darzi (32), have demonstrated characteristics such as
convenient size, longer life span, strain specific, good temperaments, easily to be handled and are
relatively inexpensive which possess by rabbits suited to for a laboratory animal model.
As many as 76 different breeds of rabbit are recognized by the British Rabbit Council but the
New Zealand White (NZW), bred in the 1920s has become the one most commonly used in
research (33).
Rats for AED, ED50, and Bioassay
Along with mice and other rodents, rats make up more than 90 percent of animals used
for biomedical research, making their group an important contributory factor in advancing
human health, as emphasized by the National Institutes of Health. According to Bayne, the
importance of using rats in research studies lies in the fact that their genetics and physiology are
closely related to those of human beings. Because of those reasons, this kind of living organisms
can even be used to virtually study every system of the human body. (34)

19

Physiological data can be most probably provided by rats and most of them are known to
manifest a number of human diseases compared to other animals. Aside from the advantage of
being physiologically and genetically close to human beings, other advantages of using rats were
emphasized, such as the availability of resources and cost. Rats can also be fed readily with
almost any kind of diet because they are omnivorous and because of their sizes, they can also be
handled easily. (34)
Fasting Blood Glucose of Rats
On a study done by Nowland et al (48) demonstrating the effects of fasting in Sprague
dawley rats, rats fasted for 6 and 16 hours had considerably lower serum glucose than the nonfasted rats. Other values showed no difference from the control except for rats fasted for 24 hours
which expressed elevated corticosterone levels. Thus, to minimize negative experiences such as
stress for the test animals, an overnight fast of less than 16 hours was indicated.
Blood Chemistry of Rats
Syahida and colleagues (35) conducted a blood hematology and serum biochemistry of
Sprague dawley rats using Sour sop in vivo 28-day repeated doses, was dosed at 0 (CD, control
dose), 0.5 (LD, low dose), 1.0 (MD, medium dose), 2.0g/kg (HD, high dose) body weight. The
study revealed that blood glucose contents were increased as the dose was increased but were
still within normal laboratory range of 90 to 201.6 mg/dL and no significant difference was
noted.
Whereas according to Janvier Labs, Sprague dawley rat of 10-week old has a normal glucose
value of 1.6 0.1 g/L for male and 2.1 0.2 g/L for female or average of 160 mg/dL and 210
mg/dL for male and female respectively (62).

20

Alloxan as Inducer of Hyperglycemia

Alloxan (2, 4, 5, 6-pyrimidinetetrone), an oxygenated pyrimidine derivative, is a colorless

powder, melting at 256 C, and is easily soluble in water and alcohol. Also, it is chemical
diabetes caused by treatment with an organic compound, which is related to physiological body
substances (73).
It is present as alloxan hydrate in aqueous solution. It is an oxidation product of uric acid
that is found in the human intestine in diarrhea. Alloxan has been used to produce diabetes in
experimental animals by destroying the insulin-secreting islet cells of the pancreas (36).
Biological Effects
Alloxan is a toxic glucose analogue, which selectively destroys insulin-producing beta
cells in the pancreas when administered to rodents and many other animal species. This causes
an insulin-dependent diabetes mellitus, called "Alloxan Diabetes", in these animals with
characteristics similar to type 1 (DM I) diabetes in humans. Alloxan is selectively toxic to
insulin-producing pancreatic beta cells because it preferentially accumulates in beta cells through
uptake via the GLUT2 glucose transporter (74).
In the presence of intracellular thiols, it generates reactive oxygen species (ROS) in a
cyclic reaction with its reduction product, dialuric acid. The beta cell toxic action of alloxan is
initiated by free radicals formed in this redox reaction. The study of Lenzen (37) suggests that
alloxan does not cause diabetes in humans. Others found a significant difference in Alloxan
plasma levels in children with and without diabetes Type 1 (37).

21

Alloxan induced diabetes mellitus served as a pathological biomodel for testing a

substance with supposed antioxidant activities in vivo. One of the targets of the reactive oxygen
species is DNA of pancreatic islets. Its fragmentation takes place in beta cells exposed to alloxan
(38). The increase in oxygen free radicals in diabetic conditions is mainly because of the effect of
the diabetogenic agent Alloxan. According to Etuk (39), chemical induction with alloxan appears
to be the easiest, reliable and the most practicable method of inducing diabetes mellitus in
rodents.
Impact upon Beta-Cells
Because it selectively kills the insulin-producing beta-cells found in the pancreas,
Alloxan is used to induce diabetes in laboratory animals. This occurs most likely because of
selective uptake of the compound due to its structural similarity to glucose as well as the betacell's highly efficient uptake mechanism (36).
However, Alloxan is not toxic to the human beta-cell, probably due to differing glucose
uptake mechanisms in humans and rodents. But in very high doses, it is toxic to the liver and the
kidneys (40; 41).
Induction of Diabetes
Fasted animals were more susceptible to alloxan, whereas increased blood glucose provided
partial protection (84). In the study of Viswanathaswamy AH, rats were weighed and diabetes
were induced with a single injection of 4% alloxan freshly prepared at a dose of 150 mg/kg
(85). Intraperitoneal dose below 150mg/kg body weight may be insufficient for inducing
diabetes in the rat. In an experiment done by Gauta MK et al, rats were treated with 5% glucose
solution to prevent hypoglycemia for 24 hours. After one hour of Alloxan induction, the animals

22

were fed ad libitum. Diabetes was confirmed on the third day of Alloxan post-administration by
glucose determination on the tail vein. Their blood glucose was measured using a wellcalibrated glucometer prior to and after Alloxan induction, the former served as the baseline.
Those that will meet the expected glucose level higher than 240mg/dl after inducing Alloxan
shall be selected and numbered. (86).

Metformin HCl as Positive Control

Metformin (Glucophage), the only member of the biguanide class of oral hypoglycemic

drugs available for use today (79), is considered currently the first drug of choice for the
treatment of type 2 diabetes (80).

Figure 3. Structural formula of the Metformin (N,N-Dimethylimidodicarbonimidicdiamide)

Metformin increases the activity of the AMP-dependent protein kinase (AMPK) which
when activated stimulates fatty acid oxidation, glucose uptake, and non-oxidative metabolism,
and it reduces lipogenesis and gluconeogenesis resulting to increase glycogen storage in skeletal
muscle, lower rates of hepatic glucose production, increase insulin sensitivity and lower blood
glucose levels. (79)

23

Metformin, an insulin sensitizer, reduces blood glucose levels without causing overt
hypoglycemia (79). It could also modulate multiple components of the incretin axis and recently
reported to have acutely increases glucagon-like peptide 1 (GLP-1) levels in the plasma and
induces isclet incretin receptor gene expression dependent on peroxisome proliferator-activated
receptor (PPAR)- (maida). It is also capable of inhibiting hepatic gluconeogenesis through
changes enzyme activities, reduction in hepatic uptake of gluconeogenesis substrates and in
hepatocytes, predominant expression of organic cation transporter 1 (OCT1), which has been
shown to facilitate cellular uptake of metformin (80, 82).
Metformin is the only antidiabetic drug that could prevent complications of the
cardiovascular system by reducing LDL cholesterol and triglyceride levels without association
with weight gain (7).
Common adverse effects of metformin include gastrointestinal upset, and lactic acidosis
as a consequence of overdosing and/or prescribed to contraindicated patients. However, when
appropriately prescribed, there is no significant risk. (49)

24

B. THEORETICAL FRAMEWORK
The nutritive as well as anti-nutritive contents of calabash fruit pulp was studied by
Ogbaugu, NM (13) and found that the fruit pulp contains 4.88% of dietary fibers including
pectin. Pectin helps in reducing the rate of sugar uptake thus helping in the treatment of diabetes.
Pectin is metabolized into galacturonic acid and acetic acid and acetic acid is found to inhibit
several activity of various carbohydrate digesting enzymes like amylase and sucrase (87). In
2011,Ejenolu (19), BC and his colleagues have studied the chemical constituents of calabash
fruit in terms of mineral composition and phytochemical properties. Aside from its beneficial and
nutritive components, it was also found that the fruit contains hydrogen cyanide and lead.
According to the, Code of Practice for the Prevention and Reduction of Lead Contamination
in Foods (50), lead is a toxic heavy metal with widespread industrial uses, but no known
nutritional benefits. Chronic exposure to lead at relatively low levels can result in damage to the
kidneys and liver, and to the reproductive, cardiovascular, immune, hematopoietic, nervous, and
gastrointestinal systems. The most critical effect of low-level lead exposure is reduced cognitive
and intellectual development in children.
Hydrogen Cyanide (HCN), a source of cyanide ion is an asphyxiant due to an inhibitory
action on metabolic enzyme system and can be rapidly fatal. Cyanide exerts this effect because it
inactivates certain enzymes (51). It was further elaborated that the lead content was 0.17mg/L
and hydrogen cyanide was 0.11ppm, both of which are beyond the World Health Organization
recommendation of 0.01mg/L for lead and 0.01mg/L for cyanide. Therefore, although the other
chemical components of the fruit are beneficial to humans and animals, the presence of heavy
metals can cause toxicity.

25

Cellular respiration is coupled to the production of adenosine triphosphate (ATP) by

oxidative phosphorylation in the mitochondria of cells, thus naming them as powerhouses.
Mitochondria have a lot of enzymes, but in relevance to this research, the enzymes of great
significance are of those located in the inner membrane which is responsible for the respiratory
chain. Electrons flow through the respiratory chain passing through three large protein
complexes: NADH-Q oxidoreductase (Complex I), where electrons are transferred from NADH
to coenzyme Q; Q-cytochrome c oxidoreductase (Complex III), which passes the electrons on to
cytochrome c; and cytochrome c oxidase (Complex IV), which completes the chain, passing the
electrons to O2 and reducing it to H2O. The three complexes act as proton pumps which creates
an electrochemical potential difference or proton motive force, thereby driving the mechanism of
ATP synthesis. ATP synthase spans the membrane and acts like a rotary motor using the potential
energy of the proton motive force to synthesize ATP from ADP and P i. ATP has been called the
energy currency of the cell because it passes on the free energy to drive those cellular
processes that requires energy. However, there are many well-known poisons that can disrupt
these processes, and one of which is cyanide. Cyanide completely blocks the enzyme
cytochrome oxidase (Complex IV) resulting to disrupted or arrested respiratory chain process
and inability of the cells or tissues to use oxygen even when there is plenty available (42).

26

C. CONCEPTUAL FRAMEWORK
Independent Variable

Calabash Decoction
Concentration

Dependent Variable

Acute Toxicity Dose

AED and ED50

Figure 4. Conceptual Framework showing Acute Toxicity Dose and AED and ED50 as
dependent variables.

Independent Variable

Calabash Decoction
Positive Control
(Metformin)
Negative Control
(Normal Saline

Dependent Variable

Fasting Blood Sugar

(mg/dl)

Figure 5. Conceptual Framework showing Bioassay as dependent variable.

Figure above shows the Calabash decoction, metformin and normal saline solution as the
independent variables in the study and their corresponding effects such as acute toxicity
measured in rabbits as well as approximate effective dose and effective dose in rats.

D NULL HYPOTHESIS
Ho

There is no significant difference in the hypoglycemic effects of Metformin,

Normal Saline Solution, and Calabash.

27

CHAPTER III
METHODOLOGY

A Research Design
Experimental design was employed in this study comprising of two (2) parts: part 1,
determination of acute toxicity dose of calabash fruit pulp decoction in rabbits, and part 2,
determination of the approximate effective dose (AED), median effective dose (ED50) of calabash
fruit pulp decoction in rats, and Bioassay.

B Research Locale
The Calabash fruit was harvested in Fatima Village, Circumferential Road, Davao City. The
preparation of the drug (pulp aqueous extraction, preparing and packaging of decoction) was
done at Davao Medical School Foundation, Inc., Laboratory. The administration of the
decoction, blood extraction, and blood glucose level determination was conducted at one of the
residences of the members (GSIS, Matina) where the test animals were kept and monitored.

C Sources of Data

The primary sources of data were from the results of fasting blood glucose determination
using glucometer which was recorded and observed. Secondary sources of data was from books,
journals, and reliable sources from the internet.

D Data Gathering Instrument/Materials

28

The instruments that were used throughout the study were analytical weighing scale, and
glucometer with glucose test strips. All instruments were calibrated and double checked if they
are in good condition before each procedure.

E Sampling Technique

Randomized sampling was utilized in the various aspects of the study. Forty-Four (44)
rats (Sprague dawley) were diabetically induced (See Induction of Diabetes). Determined
diabetic rats were taken and grouped in one cage which then will serve as the pool of diabetic
rats. In determination of Approximate Effective Dose (AED), ten (10) rats (five males, and five
females) were randomly picked from the pool. Using opaque envelopes, one of each gender were
randomly assigned to a specific dose.
In determination of the Median Effective Dose (ED 50), sixteen (16) rats were randomly
selected from the pool (eight females and eight males). Again, using opaque envelopes, the rats
were randomly assigned into four groups having four (4) rats (two from each gender) each group.
For the bioassay, determination of the hypoglycemic activity and its peak effect, utilized
twelve (12) rats randomly taken from the pool then assigned to two (2) groups (Negative Control
Group and Test Group).
For the determination of acute hypoglycemic effect, eighteen (18) rats were randomly
taken from the pool and assigned into three (3) groups: the Negative, Positive and Test Groups.

F RESEARCH SUBJECTS

29

Forty-four (44) healthy Sprague Dawley rats, male and female of 4-8 weeks old adult, and
three (3) female rabbits with average weights of 2-6 kg 20% were acquired from Philippine
Institute of Traditional and Alternative Health Care (PITAHC), a government office that provides
certified animals for experimentation. The test animals were grouped accordingly (see Sampling
Technique) and each group were kept in separate and appropriate cages. Furthermore, these
animals were acclimatized first for a period of at least five (5) days and were maintained under
standard environmental conditions of temperature, relative humidity, and dark-light cycle.
The physical conditions that were attended are as follows: 1.) At day time, the cloth covering
of the cages were removed for proper ventilation; 2.) During night time, the cages were covered
with cloth to protect them from coldness and lights were turned off to stimulate the condition of
the natural environment. The rabbits were housed in compartmentalized cages and are were fed
with standard rabbit pellets and drinking water ad libitum.

G VARIABLES
Table 2. Dependent Variables of the Study and Ways of Measurement.

Variables

Measures

Fasting Blood Glucose

mg/dL

30

PROCEDURE
Approval from different Authorities:
- Pharmacology Department of
Medicine
- Animal Ethics Committee
- In-house Veterinarian
Animals Acquisition at
PITAHC:
- Rabbits
- Rats

Acquisition of Calabash from Davao

Fruit Identification, Certification

and Authentication by a licensed

Acclimatization of test

Fruit Decoction (done as

mentioned in the methodology)

Determination of Acute Toxicity

dose on Rabbits in accordance
with the OECD Guidelines 423

Determination of
Approximate Effective

Determination of
Effective Median Dose

Bioassay on

Treatment with:
- Calabash Decoction (Test
Group)
- Metformin (Positive
Control)

Data recording and

Statistical Analysis and

Results Interpretation

Conclusio
n

Figure 5. Plan of Analysis. This flow chart shows the plan of analysis of the study
prior to experimentation until interpretation and conclusion.

31

Calabash Fruit Acquisition and Certification

The harvested Calabash fruits from Fatima Village, Circumferential Road, Davao City have

undergone certification and authentication by a licensed taxonomist in University of Immaculate

Conception Department of Biology (see Appendix 2a).

2 Fruit Decoction / Preparation of Drug

Fresh mature Calabash fruit was harvested, cut open, and the pulp scooped up. Flat seeds
were carefully removed from the pulp. The pulp of specific amount depending on the test
procedure done was placed in a stainless steel cooking pot, and cooked at minimum heat of
approximately 65C for one hour. Trial and error computation was done to compute for the
estimated amount or weight of the fruit pulp used using the general formula in Appendix 3.
During the course of cooking, formed bubbles were scraped and thrown. The cooked pulp was
strained using a mesh cloth. The formed decoction was poured in tight sealed containers and
placed in the refrigerator at -12 to -20C for proper storage.

Calabash pulp decoction

concentration was then computed.

3 Animal Acquisition

Three healthy young adult female rabbits weighing between 2-6kg 20% and forty-four (40)
healthy 4-8 weeks old Sprague dawley rats, were acquired from Philippine Institute of
Traditional and Alternative Health Care (see Appendix 2b). Animals were marked for their
identification and for other future purposes (e.g. in utilizing random selection all throughout the
experiment).

32
4 Acclimatization of Test Animals

The animals were kept in well-constructed separate cages in one of the residences of the
group members and were acclimatized for a period of at least five (5) days. The animals were
placed in a spacious quiet, open-air room, and maintained in a clean, and good condition
everyday by the housekeeper. The animals were fed, taken care of, and monitored daily. The
temperature in the experimental animal room were kept at constant room temperature with
humidity at relatively at least 30% and not exceeding 70%. Lighting of the room was artificial,
with the sequence being 12 hours light, 12 hours dark. For feeding, conventional laboratory diets
were used or as prescribed by a licensed veterinarian with an unlimited supply of drinking water.
Animals were group-caged by dose.
5 Determination of Acute Toxicity Dose on Rabbits

The determination of acute toxicity on rabbits were based on the OECD Guidelines 423
through acute oral toxicity (acute toxic class method). This was a stepwise procedure with the
use of a minimum number of animals, i.e., three rabbits of a single sex (female) per step (see
Appendix 4). Calabash decoction was administered orally to the rabbits using a stepwise
procedure. Absence or presence of compound-related mortality of the animals dosed at one step
would determine the next step. The rabbits were weighed by a standard calibrated analytical
weighing scale, wherein their weights should fall in an interval within 20% of the mean weight.
The rabbits were weighed shortly before Calabash decoction administration, and at least weekly
thereafter, and lastly, at the end of the test for those surviving animals. Weight changes were
calculated and recorded. Animals were randomly selected using opaque sealed envelopes (see
Appendix 1).
6. Preparation of Doses
The maximum dose for the rabbits was tested, which must not be exceeded, is 2mL/100g
of body weight. Doses were prepared shortly prior to administration for stability. The starting

33

dose was 2000 mg/kg since based on previous studies (42), mortality was unlikely at smaller
doses. If animals were likely to survive, a limit test with a dose of 5000 mg/kg was administered.
Administration of doses
The Calabash decoction was administered in a single dose by gavage. The rabbit was
fasted for a total of twelve hours prior to dosing. Following the period of fasting, the test animals
were weighed, and Calabash decoction was administered. Time interval between treatment
groups was fourteen (14) days or were delayed until one was confident of survival of the
previously dosed animals based on observations. If the rabbits survived the first regimen, the
dosage would be increased until toxic dosage would be reached.
Observations
Animals were observed individually after dosing at least once during the first 30 minutes,
periodically during the first 24 hours, with special attention given during the first 4 hours, and
daily thereafter, for a total of 14 days. However, duration was not fixed rigidly but was
determined by the toxic reactions, time of onset and length of recovery period, and might extend
when considered necessary. All observations were systematically recorded with individual
records being maintained for each animal. Observations included changes in skin, fur, eyes, and
mucous membranes, and also respiratory, circulatory, autonomic and central nervous systems,
and somatomotor activity and behavior pattern. Attention was directed to observations of
tremors, convulsions, salivation, diarrhea, lethargy, sleep and coma. Animals that were found in a
moribund condition or showing severe pain shall be humanely killed. Including those that were
found dead, time of death were recorded as precisely as possible.
Data and Reporting

34

Individual animal data was provided, all summarized in tabular form (see Appendix 5).
7. Induction of Diabetes
Before induction, the rats were fed and blood glucose level were checked to ensure that their
glucose level was higher than the normal value of 160mg/dl and 210 mg/dl for male and female
respectively, since increased blood glucose provided partial protection (84) so as not to induce
further morbidity to test animals.
Rats were weighed and diabetes were induced with a single injection of 4% alloxan freshly
prepared at a dose of 150 mg/kg body weight. Rats were treated with 5% glucose solution to
prevent hypoglycemia for 24 hours. After one hour of alloxan induction, the animals were fed
ad libitum. Diabetes was confirmed on the third day of alloxan post-administration by glucose
determination on the tail vein. Their blood glucose was measured using a well-calibrated
glucometer prior to and after alloxan induction, the former served as the baseline. Those that
will meet the expected glucose level higher than 240mg/dl after inducing alloxan shall be
selected and numbered.

8 Determination of Approximate Effective Dose (AED) of Calabash Decoction (Modified

Guevarra Method)
The method that was utilized for AED determination was through Single Dose Method.
Random sampling using opaque sealed envelopes (see Appendix 1) were utilized to determine
the groupings of the test animals that received different dosage. A pair of diabetic rats of
different gender were randomly assigned into different dosages of the calabash fruit pulp
decoction. Each pair received a specific dosage starting from an arbitrary dose of 1 mg/kg of
calabash decoction then increase logarithmically point 6 logarithmic interval. The blood glucose

35

levels of all the groups were monitored consecutively from day 1 to day 7. Any significant
decrease of the blood glucose of the rats were noted. Approximate Effective Dose were
computed (see Appendix 6).

9 Determination of Effective Median Dose (ED50) (Modified Guevarra Method)

The adult diabetic rats received different concentrations of the decoction based on the
computed AED. They were randomly grouped into five, with 4 diabetic rats each of equal
number of gender. Each group was assigned with a specific computed dosage (see appendix 8).
The calabash fruit pulp decoction was administered according to their assigned dosages through
oral gavage. Rats were observed and monitored for a period of 7 days conferring to the Random
Blood Sugar Level before and after the treatment. Approximate effective dose was used to
determine Median Effective Dose (ED50) that will be calculated following approximate probit
method of L.C. Miller and M.L. Tainter or the more definite Litchfield and Wilcoxons method
(Guevarras Method). A significant decrease of Blood sugar was noted. Probit Algebraic method
was used for the computation.

10 Bioassay

Determination of Hypoglycemic Activity and Its Peak Effect (Modified procedure of

Nkambo et al and Kumar et al)
The diabetic rats of Group I was randomly divided into 2 subgroups of 6 animals each:
Group IA, negative control treated with normal saline solution; and Group IB, test group treated
with calabash fruit pulp decoction. After an overnight fast of 8 hours (less than 16 hours),
diabetic rats was administered with their specific treatment, using a syringe and endogastric tube.

36

Post administration of the substances, blood glucose level was measured using a glucometer at 0,
0.5th , 1st, 3rd, 5th, and 8th hour. Continued blood glucose determination was done with a 2-hour
interval (maximum at 24th hour) until either the blood glucose levels would normalize and return
to its baseline glucose level or when the calabash decoction would lose its effect evidenced by
the rise of blood sugar levels in comparison to the blood sugar level from the previous hour. This
was done in order to calculate the percentage change in blood glucose by using the formula %
Change in Glycemia = [(Ax-A0)/A0] x 100 (63).

Acute Hypoglycemic Effect (Modified Procedure of Sebai et.al, Shah and Khan, and
Kumar et al)
Diabetic rats of Group II were divided into 3 groups and each group consisted of 6 rats:
Group IIA, negative control treated with normal saline solution; Group IIB, positive control
treated with Metformin solution; and Group IIC, test group treated with Calabash fruit pulp
decoction. Specified treatments were administered to the rats every day morning at the same time
for 15 days by using a syringe and endogastric tube. Blood samples were collected from the tail
veins before the start of the treatment and on the 5th, 10th and 15th day. Blood glucose
determination were done on the hour of its peak effect post-administration.
I.

Statistical Treatment
For the group under the Short Treatment Period (Acute Period), results were analyzed

using t-test in order to reveal any significant difference between the two groups (Normal Saline
& Calabash). In addition, Percentage Change in Glycemia with the formula taken from the study
of T.R Fagbohun and K.T Odufunwa were calculated in order to determine the percentage
change in the blood glucose levels exerted by the calabash starting from the time right after
administration (zero hour) up to the time of either the blood glucose levels would normalize or

37

the times the calabash decoction would lose its effect evidenced by the rise of blood sugar levels
in comparison to the blood sugar level from the previous hour.
For the Long Treatment Period Group, results were analyzed utilizing analysis of
variance (ANOVA) with 0.01 as level of significance to find any significant difference in the
results within the three groups of rats (Metformin group, Normal Saline group, Calabash group).
The researchers required the aid of a statistician that the data would be analyzed through
SPSS program.

J. Ethical Considerations
Animals for experiments were procured from PITAHC, a recognized facility for source of
animals for research and experimentation. In accordance with Animal Protection Act, the
approach known as the 3 Rs were considered at all times; replacement, reduction, and
refinement (64).

Replacement
Replacement defined as the substitution for conscious living higher animals of insentient
material. Replacement may be relative, where animals were still required to provide cells or
tissue, but experiments were conducted in vitro. Advantages to replacement included utilizing
pre-existing knowledge for teaching, applying known principles to new systems to look for

38

similarities, and using less expensive animals or models to screen large numbers of agents for
toxicity or mutagenicity (64).
Reduction
The goal of reduction, was to reduce the numbers of animals used to obtain information of a
given amount and precision. To achieve this, the study was designed to be scientifically and
statistically valid and only the minimum numbers of animals were used and would not be
repeated unnecessarily. The principle of reduction of numbers of animals were not applied at the
expense of greater suffering to individual animals and the number of animals used would satisfy
statistical requirements -neither too few nor too many (64).
Refinement
Refinement was any decrease in the incidence or severity of 'inhumane' procedures applied to
those animals that still have to be used. The researchers assessed the impact of any procedure or
condition on the well-being of the animal and employed strategies to eliminate or minimise that
impact (64).

Housing guidelines
Experimental animals (rats and rabbits) were housed in a suitable metal cage that were of
suitable size for each species of animal and would have adequate arrangement for feeding and
watering. All animals were checked daily, including weekends and holidays, no exceptions. This
check included monitoring room conditions monitoring animals for health problems, monitoring

39

food and water levels, monitoring for proper cage/enclosure conditions. Documentation of daily
checks were provided in the form of log or check list (65).
Adequate light levels for the animal to perform normal behaviors and for the animal care
giver to perform their duties were ensured. Ventilation for rooms housing mammalian species
would be provided for oxygen and remove chemical, biological, and heat waste. There were
fresh air supply and 100% exhaust air to the outside. Ventilation ducts and filters were cleaned at
regularly. Temperature in rooms were maintained in a range suitable for the rats and rabbits and
the animals were protected from abrupt changes. Noise in animal rooms were minimized
whenever possible. Noise from mechanical equipment in adjacent areas were avoided. Room
surfaces were constructed of material that was easily sanitized. Acceptable primary enclosures
were allowed for the normal physiologic and behavioral needs of the animals, including urination
and defecation, maintenance of body temperature, normal movement and postural adjustments
which made it possible for the animals to remain clean and dry. Adequate ventilation was
ensured. Animals access to food and water and easy filling, refilling, changing, servicing, and
cleaning of food and water utensils were permitted. Secure environment that did not allow
escape of or accidental entrapment of animals or their appendages between opposing surfaces or
by structural openings were provided in such that free of sharp edges or projections that could
cause injury to the animals were prevented. Observation of the animals with minimal disturbance
of them was allowed. Primary enclosures were constructed with materials that balance the needs
of the animal with the ability to provide for sanitation. The cages were housed as far away from
human habitations as possible and not exposed to dust, smoke, noise, wild rodents, insects and
birds. Strict barriers were provided to avoid the entry of wild rodents, insects and pests (66).

40

Health Monitoring
Animal health status were monitored at least once daily. Changes in behavior, food or water
consumption, fecal or urine output, reduction in grooming behavior, aggression, muscular
rigidity, hair coat, reaction to handling were monitored because these can be nonspecific signs of
distress or disease. More specific signs or objective measurements of organ dysfunction were
monitored if indicated by the animals condition or the expected impact of the experiment.
Animals were fed commercially available complete diets appropriate for their physiologic status.
They were maintained on a balanced diet containing protein, carbohydrates, fat, minerals,
vitamins, and water in required proportions, balanced pelleted feeds available commercially were
used to feed the animals. For most purposes tap water from a potable water faucet was adequate
for research mammals. No drug, hormone or antibiotic were added in the feed to avoid abnormal
metabolism of the animals and produce biased results. For experimental purposes, animals were
kept fasting overnight but had free access to water. The test animals were given standard and
healthy feeds for the duration of the study as prescribed by a licensed veterinarian (66).

Restraint and handling of animals

Restraining of rats were handled by the tail, with emphasis on only grasping the tail base.
Holding the tail distal to the base could result in a de-gloving injury to the tail that would require
surgical repair or euthanasia. Rabbits were handled by sliding one hand under the chest and
gently lifting it with the other arm cradling the body, the head nestled in the crook of the arm.

41

Rabbits were never lifted by the ears or by the scruff of the neck (65). An experienced
veterinarian were available for health care, monitoring, diagnosis and treatment of diseases and
injuries.
Death or Moribundity
Common signs of moribundity that would closely monitored on experimental animals
included, but are not limited to: a) lack of responsiveness to manual stimulation; b) immobility;
and/or c) an inability to eat or drink. Animals involved in experiments that would lead to
moribundity or death would be monitored at least daily by personnel experienced in recognizing
signs of morbidity (illness, injury, or abnormal behavior) for at least the following: abnormal
posture, rough hair coat, head tucked into abdomen, exudates around eyes and/ or nose, skin
lesions, or abnormal breathing, difficulty with ambulation, decreased food or water intake, or
self-mutilation. The frequency of observation would be increased when animals exhibit the
above or other signs of morbidity. An assessment of the animals' condition would be made as
soon as possible and a plan of action established. Consideration would be given to moving
animals to individual cages when their condition deteriorated to the point that injury from other
animals was likely. Dead animals were promptly removed. Written records were kept of
monitoring (67).
Attempts to restore the normal health of test animals were performed at the conclusion of
the experiment. The researchers ensured that they have adequate training in handling research
animals. Reasonable efforts were realized to ensure minimal discomfort, infection and illness to
the test animals. The procedures involving the use of test animals were done in manner that
induced less or no stress to the test animals. The procedures which were considered painful were

42

conducted under appropriate anesthesia as recommended and would be given for the full
duration of experiment and at no stage the animal is conscious to perceive pain during the
experiment (65).
Euthanasia
When it was appropriate that an animals life be terminated, it proceeded rapidly, with an
effort to minimize pain and in accordance with accepted procedures. Euthanasia was resorted to
in the event an animal was required to be sacrificed on termination of an experiment or otherwise
for ethical reasons. This was done without causing anxiety, pain or distress, minimum
physiological and psychological disturbances. In rats the following can be employed; carbon
dioxide (CO2), Sodium Pentobarbital 100 or > mg/kg IV, IP, Commercial Euthanasia Solution
(Sodium pentobarbital 390 mg + sodium phenytoin 50 mg/ml) (e.g. Beuthanasia, Euthasol,
Fatal-Plus, Somlethal) 0.22 ml/kg IV, IP (~86 mg/kg pentobarbital). In rabbits, sodium
Pentobarbital 100 or > mg/kg IV, commercial Euthanasia Solution (Sodium pentobarbital 390 mg
+ sodium phenytoin 50 mg/ml) (e.g. Beuthanasia, Euthasol, Fatal-Plus, Somlethal) 0.22
ml/kg IV, IP (~86 mg/kg pentobarbital). In lieu of demonstrated technical competency, animals
were unconscious or anesthetized (68). (see Appendix 13 for additional checklists.)

43

CHAPTER IV
PRESENTATION, ANALYSIS & INTERPRETATION OF DATA

This chapter presents the data gathered and the analysis for each result given on the
hypoglycemic activity of Calabash Fruit (Crescentia cujete) decoction in alloxan-induced
hyperglycemic rats (Sprague dawley). The procedures involve the determination of acute oral
toxicity test, approximate effective dose as hypoglycemic agent, median dose (ED 50)
determination and Bioassays. Statistical analyses were conducted to determine which of the
three test drugs (Metformin, plant extract and Placebo Drug) significantly decreased the blood
glucose level of alloxan-induced hyperglycemic rats.

Determination of Acute Oral Toxicity

Table 3. Acute Oral Toxicity Test of Calabash Fruit (Crescentia cujete) decoction in
Alloxan-induced hyperglycemic rats (Sprague dawley) based on OECD Guidelines.
Type of Fruit
decoction
Ripe Calabash

Dose Level,
N
Observed Toxicity*
4
hours
7th day
14 days
mg/kg
2000
3
None
None
None
5000
3
None
None
None
*None of the test animals exhibit symptoms of toxicity.

Remarks
Survived
Survived

The three rabbits showed no signs of toxicity which include changes in skin and fur, eyes
and mucous membranes, and also respiratory, circulatory, autonomic and central nervous
systems, and somatomotor activity and behavior pattern. Likewise, special attention was directed
to observations of tremors, convulsions, salivation, diarrhea, lethargy, sleep and coma. Based
from the results of the test, none of the test animals died posttreatment which indicate that the
plant used is nontoxic. Amilhasan, S.J, et. al (2010) conducted acute toxicity test on mice with
the same starting dose of 2000mg/kg & maximum dose of 5000 mg/kg which also revealed that

44

the Calabash decoction is nontoxic and safe for further testing. Summary of the test results are
shown in table 1 while raw data are depicted in appendix 5.
Determination of Approximate Effective Dose
The hypoglycemic activity of Calabash Fruit (Crescentia cujete) decoction in alloxaninduced hyperglycemic rats (Sprague dawley) was conducted based on the computed dose which
starts at 1.00mg/kg dose. Succeeding doses were computed based on 0.60 logarithmic interval
wherein the highest dose administered was 251.20mg/kg. The test was conducted in duplicate
and results are shown in table 2 while the raw data are shown in appendix 7.
Table 4. Approximate Effective Dose of Calabash Fruit (Crescentia cujete) decoction in
alloxan-induced hyperglycemic rats (Sprague dawley).

Rat
No.

Gender

C M
H F
B M
F F
E M
G F
P value of <0.05

Dosage
(mg/kg)
15.84
mg/kg
63.1
mg/kg
251.20
mg/kg

Pretest
Blood
Glucose
(mg/dL)
221
174
195
211
194
228

Post-Alloxan
Induction
Blood
Glucose
(mg/dL)
254
258
261
251
254
256

PostTest
Blood
Glucose
231.75
208.75
237.75
220.5
223.75
211.5

P value

Remarks*

0.166595

Not
Significant

0.00061

Significant

0.010249

Significant

Results of approximate effective dose determination revealed substantial decreased in

fasting blood glucose level at 63.1 and 251.20 mg/kg. However, based on the P-value, the 63.1
mg/kg dose showed more significant result than 251.20 mg/kg dose. The findings clearly
indicated that the plant extract used is a potential hypoglycemic agent which showed capability
of lowering the blood glucose level of alloxan-induced hyperglycemic rats. As such, the
approximate effective dose for the test plant material on Sprague dawley rats is about between

45

15.84 to 63.1 mg/kg. In the study done by Amilhasan, S.J, et. al (2010), AED tested on Sprague
dawley rats was found to range between 3.98 to 15.84 mg/kg.
Determination of Effective Dose (ED50
Table 5. Effective Dose 50 (ED50) of Calabash Fruit(Crescentia cujete) decoction in
alloxan-induced hyperglycemic rats (Sprague dawley).
DOSE
(mg/kg)

PREALLOXA
N
27.655
205.75
39.47
214.25
51.285
206.25
63.1
195.5
P value of <0.05

POSTALLOXA
N
254.75
256
269
259.25

DAY 1 to
DAY 7

P Value

Remarks

256.5
255.5
270.5
262

0.27575
0.66075
0.276
0.00675

Not Significant
Not Significant
Not Significant
Significant

In ED50 determination, four rats are assigned to each dose namely 27.655, 39.47, 51.285,
and 63.1 mg/kg. Table 3 shows the mean results of the rats per dose received as well as the mean
results of their fasting blood glucose level monitoring from day 1 until day 7. Results revealed
that the dose 63.1 mg/kg caused a substantial decrease in fasting blood glucose level. Based from
the findings, the researchers proceeded with the bioassay testing using 63.1 mg/kg dose for
Calabash Group, while 7 mg/kg dose for Metformin, and 0.2 ml/kg for normal saline as placebo.
The selected test animals were randomly selected for treatment with Metformin (Positive
control), Test Drug (Experimental Group) and Placebo (Negative Control). Summarize results of
bioassay are shown in table 4 while the overall data are shown in appendix 9.

Determination of Calabash Hypoglycemic Activity and Peak Effect

46

30 m 1
0

10

12

14

16

18

20

22

24

-5

Rat G

-10

Rat K
Rat S
Rat Z
Rat CC
Rat EE

-15

-20

-25

Figure 7. Calabash Hypoglycemic Activity and Its Peak Effect.

Figure above shows the effect of single dose of calabash decoction on the blood glucose
levels of 6 rats. The blood glucose level of some rats have begun to lower down by 30 minutes
post calabash administration, however, its substantial effect can be seen at the 14 th hour and still
exerting its hypoglycemic action beyond this hour. However, it is inconclusive whether or not the

47

decoction is still capable of lowering the blood glucose level beyond the 24 th hour. Raw data are
shown in appendix D. Calabash contains high levels of pectin which has the ability to reduce the
rate of sugar uptake (13). According to studies done, pectin is decomposed chiefly in the colon of
man through bacterial enzymes. Pectin is metabolized into galacturonic acid and acetic acid and
acetic acid is found to inhibit several activity of various carbohydrate digesting enzymes like
amylase and sucrase. As a result, some sugars and starches temporarily pass through the intestine
without being digested (87). The time it takes for the intestinal flora of the rats intestine to
decompose the pectin in the calabash decoction in order to release acetic acid may be the reason
behind its substantial peak effect on lowering blood glucose level on the 14 th hour. Summarize
results of bioassay are shown in Figure 1 while the overall data are shown in appendix 10.

Table 6. Bioassay of Calabash Fruit (Crescentia cujete) decoction in versus Placebo &
Metformin alloxan-induced hyperglycemic rats (Sprague dawley).
GROUP/RAT

Weight
(kg)

Calabash
Placebo
Metformin

0.099
0.116
0.118

Volume
administere
d (mL)
0.238
0.200
0.592

DAY 1

DAY 5

DAY 10

DAY 15

258.000
270.167
243.667

207.833
282.000
232.333

208.400
281.500
221.500

195.400
280.833
187.333

Table 4 shows the mean results of bioassay conducted. The test revealed decrease in
blood glucose level of calabash and metformin group of test animals which were previously
induced hyperglycemia. Meanwhile, negative control group (treated with Placebo drug) do not
show substantial reduction in glucose level posttreatment. The findings demonstrated the fact
that Metformin a positive control test drug can substantially decreased the blood glucose of test
animals.

Moreover, experimental group treated with Calabash Fruit (Crescentia cujete)

48

decoction extract likewise showed decreased in blood glucose level thus confirming its
hypoglycemic activity.

Table 7. Multiple Mean Comparisons of Blood Glucose Level Lowered by Various

Treatments (Metformin, Negative control and Calabash Fruit Decoction) in alloxaninduced hyperglycemic rats (Sprague dawley).
(I) Types of
Treatment
Calabash group
Placebo group
Metformin group

(J) Types of
Treatment
Placebo group
Metformin group
Calabash group
Metformin group
Calabash group
Placebo group

Mean
Difference (IJ)
-59.8068*
-2.3902
59.8068*
57.4167*
2.3902

Sig.

Decision*

.000
.444
.000
.000
.444

Significant
Not significant
Significant
Significant
Not significant

-57.4167*

.000

Significant

*Calculation was performed at the 0.05 level of significance

Multiple mean comparison test using Least Significant Difference (LSD) revealed that
there is no significant difference (p>0.05) in the blood glucose level lowered by test animals
treated with Metformin and Calabash fruit (Crescentia cujete) decoction. The findings account to
the potential of both test drugs to significantly decrease the glucose level of alloxan-induced
hyperglycemic rats.

However, negative control group (treated with placebo drug) did not

significantly showed decrease in blood glucose level posttreatment. In this case, placebo drug
has no hypoglycemic activity towards the test animals. Amilhasan and colleagues study (30)
done on rabbits revealed that calabash and metformin had comparable effects.

49

Table 8. Multiple Mean Comparisons of Blood Glucose Level at various

Observation Time in alloxan-induced hyperglycemic rats (Sprague dawley).
(I) Observation
Time

Day 1

Day 5

Day 10

Day 15

(J) Observation
Time

Sig.

Decision*

.000
.000
.000
.000
.596
.000
.000
.596
.000
.000

Significant
Significant
Significant
Significant
Not significant
Significant
Significant
Not significant

Day 5

-18.0163

.000

Significant

Day 10

-16.1176*

.000

Significant

Day 5
Day 10
Day 15
Day 1
Day 10
Day 15
Day 1
Day 5
Day 15
Day 1

Mean
Difference (IJ)
16.5556*
18.4542*
34.5719*
-16.5556*
1.8987
18.0163*
-18.4542*
-1.8987
16.1176*
-34.5719*

Significant

*Calculation was performed at the 0.05 level of significance

Post hoc test was also determined at various observation times as shown above. Multiple
mean comparison test using Least Significant Difference (LSD) revealed that there is significant
difference (p<0.05) in the blood glucose level lowered by various observation time except
between Day 5 and Day 10. It can be shown that significant decrease of the glucose level of
alloxan-induced hyperglycemic rats were at Day 15.

50

CHAPTER V
SUMMARY, CONCLUSIONS AND RECOMMENDATIONS

Summary
This study aimed to determine the hypoglycemic activity of Calabash Fruit (Crescentia
cujete) decoction in alloxan-induced hyperglycemic rats (Sprague dawley).

To confirm its

activity, simultaneous testing with positive control (Metformin), and negative control were done
in the bioassay. Likewise, acute toxicity test, approximate effective dose and median effective
dose determine were done.
Based on the findings, Calabash Fruit (Crescentia cujete) decoction in alloxan-induced
hyperglycemic rats (Sprague dawley) was found to have hypoglycemic activity similar to the
activity of the Metformin. It had shown clear significant reduction of blood glucose level in the
test animals. Meanwhile, negative control group showed no significant decrease in the blood
glucose level when analyzed according to types of treatment and observation time.
With these findings, it can be inferred that Calabash Fruit (Crescentia cujete) decoction is
a potential hypoglycemic agent towards alloxan-induced hyperglycemic rats (Sprague dawley).

Conclusions
Based from the results of the tests, the researchers deduced the following conclusions:
1. The plant material is non-toxic. No signs of toxicity which include changes in skin and
fur, eyes and mucous membranes, and also respiratory, circulatory, autonomic and central
nervous systems, and somatomotor activity and behavior pattern were observed on the
test animals

51

2. Results of approximate effective dose determination revealed substantial decrease in

blood glucose level at 15.84 to 63.1 mg/kg. The findings clearly indicated that fruit
decoction used is a potential hypoglycemic agent which showed capability of lowering
the blood glucose level of alloxan-induced hyperglycemic rats.
3. Results of median effective dose determination revealed substantial decrease in blood
glucose level at 63.1 mg/kg. The outcomes evidently showed that Calabash fruit pulp
decoction utilized is a potential hypoglycemic agent which exhibited blood lowering
capability on alloxan-induced hyperglycemic rats.
4. A single dose Calabash decoction had its peak effect in lowering the blood glucose level
at the 14th hour and still exerting its hypoglycemic action beyond this hour.
5. There is significant difference (p<0.05) in the blood glucose level post-treatment of the
test animals when analyzed according to types of treatment (Metformin, Negative control
and Calabash Fruit (Crescentia cujete) decoction) and observation time (Day 1, Day 5,
Day 10, and Day 15).
6. There is no significant difference on the blood glucose lowering effect of Metformin and
Calabash Fruit (Crescentia cujete) decoction towards alloxan-induced hyperglycemic rats
(Sprague dawley).
7. There is a significant difference (p>0.05) in the blood glucose level lowered by various
observation time except between Day 5 and Day 10. It was shown that significant
decrease of the glucose level of alloxan-induced hyperglycemic rats were at Day 15.

Recommendations

52

The researchers propose the subsequent recommendation to imminent researchers in the

advancement of the study which is useful in the procedural scheme and valuable role to humans
with blood glucose level problems. These were indicated as:
1. Clinical assessment of Calabash Fruit (Crescentia cujete) decoction may be done to
evaluate its activity towards diabetic patients.
2. Increased number of subjects may be included to better evaluate the different factors that
could contribute to the potential difference in the blood glucose levels.
3. Prolonged duration of studies to assess the long-term or chronic effect of Calabash.
4. Histopathological evaluation of the subjects may be done to better assess the potential
toxicity of Calabash.
5. Other dosage formulas (e.g. capsule, tablet, and tea bags) may be prepared to determine
its therapeutic activity in glucose monitoring test.

53

APPENDIX 1
RANDOM SAMPLING: OPAQUE SEALED ENVELOPE
Test animals (female rabbits and rats of either gender) acquired from
PITAHC are placed in a separate enclosure.
As for rats, male and female are further placed in separate enclosures.

All test animals who meet the inclusion criteria are

labeled with their unique corresponding number.

Their numbers are placed in an opaque, sealed

envelope.

For bioassay, rats who qualified for testing will be

divided into 3 groups.

Each group will have their own separate enclosure

and further divided according to gender.

Figure 8. Opaque Sealed Envelope Technique.

APPENDIX 2
CERTIFICATION

54

Appendix 2a. Certification of Calabash Fruit

55

Appendix 2b. Certification of Test Animals

56

APPENDIX 3

GENERAL FORMULA USED FOR COMPUTATION IN FRUIT DECOCTION AND

DRUG PREPARATION

VolumemL be administered= ( Animal weightkg ) Dose

mg
Volume water mL
kg Weight of pulpdrugmg

)(

57

APPENDIX 4
ACUTE TOXICITY TESTING PROCEDURE

Figure 9. Test Procedure with a Starting Dose of 2000 mg/kg Body Weight.

58

APPENDIX 5
Raw Data For Acute Oral Toxicity Test
Table 9. Acute Toxicity with 2,000 mg/kg dose.

Rabbit 1

Weight of Rabbits (kg)

4 hours
Day 7
2.1
2.2

Day 14
2.5

Rabbit 2

2.1

2.0

1.9

Rabbit 3

2.0

1.9

2.0

Observations

Remarks

No significant
changes
observed.
No significant
changes
observed.
No significant
changes
observed.

Survived

Observations

Remarks

No significant
changes
observed.
No significant
changes
observed.

Survived

No significant
changes
observed.

Survived

Survived

Survived

Table 10. Acute Toxicity with 5,000 mg/kg dose.

Rabbit 1

Weight of Rabbits (kg)

4 hours
Day 7
2.6
2.4

Day 14
2.3

Rabbit 2

1.8

2.2

2.3

Rabbit 3

1.8

2.0

2.2

Survived

59

APPENDIX 6
APPROXIMATE EFFECTIVE DOSE (AED)
Starting Dose: 1 mg/kg
Dose#1: 1 mg/kg
Dose#2: log 1 mg/kg + 0.6 = 3.98 mg/kg
Dose#3: 3.98 mg/kg x antilog 0.6 = 15.84 mg/kg
Dose#4: 15.84 mg/kg x antilog 0.6 = 63.10 mg/kg
Dose#5:63.10 mg/kg x antilog 0.6 = 251.20 mg/kg
Dose#6: 251.20 mg/kg x antilog 0.6 = 1000 mg/kg
Dose in mL to be administered:
Weight of animal (kg) x desired dose in mg/kg x Stock Solution or Concentration
Computation for Concentration:
(Weight of plant material before extraction Weight if residual material after extraction)
divided by the volume extracted in Ml = Concentration in mg or g/Ml
Determination of AED Factor:
AED Factor = (upper limit lower limit) divided by 5 (arbitrary number)

60

APPENDIX 7
BLOOD GLUCOSE MONITORING FOR AED DETERMINATION

Table 11. Raw Data for Approximate Effective Dose

Rat
No.

Gender

D M
J
A
I
C
H
B
F
E
G

F
M
F
M
F
M
F
M
F

Dosage
(mg/kg)

PreWeight
(kg)

1 mg/kg

0.293

0.204

0.02

206

PostAlloxan
Induction
Blood
Glucose
(mg/dL)
296

3.98
mg/kg
15.84
mg/kg
63.1
mg/kg
251.20
mg/kg

0.163
0.245
0.151
0.210
0.145
0.156
0.110
0.115
0.088

0.116
0.140
0.100
0.177
0.121
0.156
0.131
0.127
0.115

0.01
0.08
0.05
0.26
0.18
0.76
0.49
2.23
1.55

216
186
196
221
174
195
211
194
228

259
258
266
254
258
261
251
254
256

PostWeight
(kg)

Volume of
Calabash
Decoction
(mL)

Pretest
Blood
Glucose
(mg/dL)

Table 12. Fasting blood glucose of Alloxan-induced Hyperglycemic rats for AED

PostTest
Blood
Glucose
233
238.25
238.5
228.25
231.75
208.75
237.75
220.5
223.75
211.5

61

Test Animal
(Gender/Number
)
D
M
J
F
A
M
I
F
C
M
H
F
B
M
2
F
E
M
G
F

Dose
1
1 mg/kg

276
256
3.98 mg/kg 257
240
15.84 mg/kg 249
218
63.1 mg/kg 268
261
251.20
241
215
mg/kg

Fasting Blood Glucose in Day (mg/dL)

2
3
4
5
6
229
216
246
226
246
236
239
218
251
221
201
211
249
218
219
201
231
211
218
216
APPENDIX 8

211
225
215
216
206
205
216
201
212
197

MEDIAN EFFECTIVE DOSE (ED50)

Determination of Effective Dose levels:
Dose#1: 15.84 + 11.815 = 27.655 mg/kg
Dose#2: 27.655 mg/kg + 11.815 = 39.47 mg/kg
Dose#3: 39.47 mg/kg + 11.815 = 51.285 mg/kg
Dose#4: 51.285 mg/kg + 11.815 = 63.1 mg/kg

210
223
198
212
201
198
211
196
207
188

207
221
192
208
191
194
208
190
195
180

7
206
218
186
199
179
185
194
178
183
173

62

APPENDIX 9
Table 13. BLOOD GLUCOSE MONITORING ON ED50 DETERMINATION
RAT/
GEND
ER
16/M
2/F
15/M
9/F
14/M
3/F
11/M
5/F
10/M
6/F
17/M
1/F
12/M
7/F
19/M
8/F

PREALLOX
AN
180
201
212
197
215
198
211
226
206
211
194
186
205
231
213
201

POSTALLO
XAN
250
271
255
261
254
249
258
261
250
260
254
256
251
278
257
291

DAY 1

DAY 2

DAY 3

DAY 4

DAY 5

DAY 6

DAY 7

248
271
256
258
251
251
256
261
251
260
259
268
254
279
265
290

226
268
251
254
214
241
248
259
212
241
226
241
218
258
258
268

218
254
243
249
218
238
241
241
208
238
215
238
211
212
246
217

215
254
221
238
215
218
218
226
206
221
208
219
202
211
208
215

212
249
216
235
212
208
210
216
203
207
201
216
195
189
201
211

210
244
213
217
206
205
203
204
199
201
190
205
186
187
189
199

206
241
207
212
203
198
196
193
188
189
182
189
177
180
174
183

63

APPENDIX 10
DETERMINATION OF CALABASH HYPOGLYCEMIC ACTIVITY AND PEAK
EFFECT
Day
1

0m

30 m

10

12

14

16

18

20

22

261

261

260

259

255

254

250

249

249

248

238

216

211

260

260

260

260

258

256

251

251

251

245

241

240

240

251

251

251

250

250

250

247

247

247

241

236

230

221

254

253

253

254

252

250

246

246

246

240

238

221

218

CC

258

258

257

256

256

255

255

255

255

239

231

231

226

EE

268

267

267

265

265

265

264

264

263

251

248

244

239

Table 14. GROUP A (TREATMENT/CALABASH GROUP) BLOOD GLUCOSE LEVEL

Day
1

0m

30 m

10

12

14

16

18

20

22

246

246

246

248

248

248

249

248

249

250

255

255

260

276

276

276

276

277

281

280

281

281

286

286

285

287

258

258

259

259

263

263

263

264

263

265

265

265

268

276

277

277

277

279

279

280

282

286

286

287

287

287

291

291

291

291

291

292

292

292

292

298

297

297

298

DD

269

270

272

274

274

271

272

286

286

288

288

288

289

Table 15. GROUP B (NSS/NGATIVE CONTROL GROUP) BLOOD GLUCOSE LEVEL

APPENDIX 11
BLOOD GLUCOSE MONITORING FOR BIOASSAY
Table 16. Overall Data for Bioassay
GROUP/RAT

Weight
(kg)

Volume
administere

DAY 1

DAY 5

DAY 10

DAY
15

64

GROUP A
(Treatmen
t Group)

GROUP B
(Negative
Control)

GROUP C
(Positive
Control)

0.121

d (mL)
0.29

K
S
Z
CC
EE
A
H
N
P
Y
DD
C
D
E
M
U
V

0.093
0.112
0.086
0.090
0.090
0.113
0.179
0.118
0.102
0.085
0.098
0.112
0.145
0.147
0.109
0.113
0.083

.22
.27
.21
.22
.22
.2
.2
.2
.2
.2
.2
.56
.73
.74
.51
.57
.44

260

196

260
251
253
257
267
246
276
259
277
291
272
244
238
239
241
239
261

225
208
191
207
220
271
287
268
288
290
288
239
231
228
230
226
240

X (died) X
(died)
225
200
206
196
190
185
201
195
220
201
270
269
287
287
268
268
287
292
291
288
286
281
233
201
228
196
216
185
221
164
211
186
220
192

Appendix 12
Overall Data of Statistical Test
Table 17. Descriptive Statistics
Dependent Variable: Blood Glucose Level
Types of
Observation
Mean
Treatment
Time
Day 1
258.0000
Day 5
207.8333
Treatment group Day 10
208.4000
Day 15
195.4000
Total
218.8182
Day 1
270.1667
Day 5
282.0000
Negative control Day 10
281.5000
Day 15
280.8333
Total
278.6250
Positive control
Day 1
243.6667
Day 5
232.3333

Std.
Deviation
5.72713
13.16688
14.22322
6.34823
26.92253
15.66418
9.77753
9.85393
10.18659
11.95212
8.75595
5.81951

N
6
6
5
5
22
6
6
6
6
24
6
6

65

Total

Day 10
Day 15
Total
Day 1
Day 5
Day 10
Day 15
Total

221.5000
187.3333
221.2083
257.2778
240.7222
238.8235
222.7059
240.1429

7.96869
12.92543
23.18166
15.11838
33.12153
34.43152
45.43094
35.10163

6
6
24
18
18
17
17
70

Appendix 12 (continued)
Table 18A. Tests of Between-Subjects Effects
Dependent Variable: Blood Glucose Level
Source
Type III Sum
df
Mean
F
of Squares
Square
Corrected
78600.671a
11
7145.516
64.596
Model
Intercept
3982727.291
1 3982727.291 36004.019
TT
55411.959
2
27705.980
250.463
TimeTreat
11467.740
3
3822.580
34.556
TT * TimeTreat
13146.059
6
2191.010
19.807
Error
6415.900
58
110.619
Total
4121818.000
70
Corrected Total
85016.571
69
a. R Squared = .925 (Adjusted R Squared = .910)

Table 18B. Tests of Between-Subjects Effects

Dependent Variable: Blood Glucose Level

Sig.
.000
.000
.000
.000
.000

66

Source

Type III Sum

of Squares

df

Mean Square

Sig.

Corrected
78600.671a
11
7145.516
64.596
Model
Intercept
3982727.291
1 3982727.291 36004.019
TT
55411.959
2
27705.980
250.463
TimeTreat
11467.740
3
3822.580
34.556
TT * TimeTreat
13146.059
6
2191.010
19.807
Error
6415.900
58
110.619
Total
4121818.000
70
Corrected Total
85016.571
69
a. R Squared = .925 (Adjusted R Squared = .910)

.000
.000
.000
.000
.000

Appendix 12 (continued)
Table 19. Post Hoc Tests
Multiple Comparisons
(I) Types of (J) Types of
Treatment Treatment

Mean
Std. Error Sig.
Difference (IJ)

Negative
-59.8068* 3.10439
Treatment control
group
Positive
-2.3902 3.10439
control
Treatment
59.8068* 3.10439
Negative
group
control
Positive
57.4167* 3.03616
control
Treatment
2.3902 3.10439
group
Positive
control
Negative
-57.4167* 3.03616
control
Based on observed means.
The error term is Mean Square(Error) = 110.619.

95% Confidence Interval

Lower Bound
Upper Bound

.000

-66.0209

-53.5927

.444

-8.6043

3.8240

.000

53.5927

66.0209

.000

51.3391

63.4942

.444

-3.8240

8.6043

.000

-63.4942

-51.3391

67

*. The mean difference is significant at the .05 level.

(I)
(J)
Mean
Std. Error
Observati Observatio Difference (Ion Time
n Time
J)
Day 5
16.5556* 3.50585
Day 1
Day 10
18.4542* 3.55703
Day 15
34.5719* 3.55703
Day 1
-16.5556* 3.50585
Day 5
Day 10
1.8987 3.55703
Day 15
18.0163* 3.55703
Day 1
-18.4542* 3.55703
Day 10
Day 5
-1.8987 3.55703
Day 15
16.1176* 3.60749
Day 1
-34.5719* 3.55703
Day 15
Day 5
-18.0163* 3.55703
Day 10
-16.1176* 3.60749
Based on observed means.
The error term is Mean Square(Error) = 110.619.
*. The mean difference is significant at the .05 level.

Sig.

.000
.000
.000
.000
.596
.000
.000
.596
.000
.000
.000
.000

95% Confidence Interval

Lower Bound
Upper Bound
9.5378
11.3341
27.4517
-23.5733
-5.2215
10.8962
-25.5744
-9.0189
8.8965
-41.6921
-25.1365
-23.3388

23.5733
25.5744
41.6921
-9.5378
9.0189
25.1365
-11.3341
5.2215
23.3388
-27.4517
-10.8962
-8.8965

68

APPENDIX 13
ETHICAL CONSIDERATION

Appendix 13a.
ETHICS REVIEW AND APPROVAL OF RESEARCH STUDIES INVLOVING
ANIMALS

Checklist for Review

YE
S

NO

1. Demonstrates that potential benefit outweigh harm through

literature reviews and preview trials
2. The proposal contains the following information:
A. RATIONALE OF THE STUDY
1. How the study will advance knowledge for the welfare of
humans and animals
2. The study provides benefits to humans.
3. The study provide benefits to animals.
B. ACQUISITION OF ANIMALS
1. Where the animals are purchased?
Animals are purchased from Philippine Institute of Traditional and Alternative
Health Care (PITAHC).

N/A

69

2. How the animals are purchased?

Animals are purchased directly from PITAHC office.
3. How the animals are transported to the DMSF?
Test animals are placed inside cages with appropriate food and water and loaded in a
spacious air-conditioned vehicle. Researchers responsible for handling and
transportation are knowledgeable and able to recognize signs of distress and pain of
animals. Excessive noise and vehicle movement are avoided. No unnecessary delays are
done in transferring animals to housing.
C. PERSONNEL INVOLVED IN THE STUDY
1. Who will handle the experiment?
The student researchers supervised by licensed veterinarian will handle the experiment.
2. Qualification/s of person/s who sill handle the experiment.
The student resesearchers are are trained by a licensed veterinarian in
terms of handling, managing, and other procedures done in the study.
3. Protection of researcher/s frpm bites/risks of infection
Aside from the training done, contact numbers of professional health
personnels and the licenced veterinarian are made readily available in
case of emergencies.
A.
1.
a.
b.
c.
d.
e.
2.
a.
b.
3.

CARE OF ANIMALS
Feeding procedures
What to feed
Who will feed
How to feed
Management of pain (drug/dose/route
Management of infection
Handling of animals
How to carry/transport animal/s fromone place to another
Protection of animals from hazardous reagents/chemicals
Housing
a. Type of housing
b. Temperature control
c. Protection from insects and vermins
E. ADVERSE EFFECTS
1. How to handle adverse effects

B. MORTALITY
1. Procedure on how to conduct autopsy
2. Report/Documentation

70

3. Disposition of carcass
C. POST-RESEARCH ANIMAL MANAGEMENT
1. What to do with sick animals
2. What to do with healthy animals (where to
endorse for care and ownership)

71

Appendix 13b.
ETHICAL CONSIDERATIONS CHECKLIST

Animal experiments undertaken only after due consideration of their

relevance for human or animal health and the advancement of knowledge. 72

The animals selected are of an appropriate species and quality, and

minimum number used to obtain scientifically and statistically valid results.

Investigators and other personnel treat animals with kindness and should
take proper care by avoiding or minimizing discomfort, distress or pain.
4 Use a procedure subjecting animals to pain, stress or privation only when an
alternative procedure is unavailable and the goal is justified by its
prospective scientific, educational or applied value.
5 Perform surgical procedures under appropriate anesthesia and follow
techniques to avoid infection and minimize pain during and after surgery.
6 Proceed rapidly when it is appropriate that an animal's life be terminated,
with an effort to minimize pain and in accordance with accepted procedures.
7 The best possible living condition should be provided to animals used for
research purpose.
8 Care of animals should be under the supervision of a veterinarian or a
person having adequate experience in laboratory.
9 Ensure Restraining procedures are appropriately done.
10 Researchers must know the clinical signs of pain for the species to which
they are working.
11 Analgesia (or euthanasia) must be promptly provided to all animals
demonstrating pain or suffering, unless specifically exempted as a Pain
Category E experiment.
12 Sick or injured animal(s) with weight loss of 20% or more must be noted on
the vivarium health report or euthanized.
13 Only the specific drugs, methods, and materials approved may be used on
animals, unless written documentation is provided from a veterinarian
permitting and justifying their immediate use.
14 Barrier procedures must be adhered to by everyone entering a barrier
facility/room, including wearing of protective clothing, disinfection of
equipment, and proper handling of animals/cages.
15 Species-specific physical and psychological environmental enrichment will
be provided for all animals. Exemptions for providing environmental
enrichment must be scientifically justified in the protocol.
16 Housing social animals in pairs or groups is the standard method of
housing. Single housing of any animal must be based on social
incompatibility, veterinary-related concerns, scientific justification, or end
of study animals /singly housed breeders.
17 Researchers are responsible for labeling the cage card of singly housed
rodents to signify the cause for separation: fighting/social incompatibility ,
veterinary care reason, or for scientific justification.
18 Animal feed/diet (special or standard) must be stored in a tight fitting
container and labeled with the feed type and expiration or mill date. Feed
taken to the laboratory must follow same requirements.
19 Animals in cages or on carts should always be concealed by a drape or bag
during transport outside of the vivarium or laboratory. Animals should be
placed in a clean cage with a secured lid. Public corridors and elevators
should be avoided during transport. Recommend placing animals in clean
cage before the animal leaves the vivarium if there are plans for the animal
to return to the vivarium following surgery and/or biohazard inoculation.
3

73

REFERENCES

74

1. Borries RP. Natural Product researches, Perespective from a major pharmaceutical

company. Ethnopharmacology; 1996.
2. Alison MG. Peter, R.F. Antihyperglycemic action of Eucalyptus globulus are associated
with pancreatic and extra- pancreatic effects in mice. J. Nutr; 1998.
3. Alves TMA, Silva AF, Brandao M, Grandi TSM, Smania A, Zani CL. Biological
Screening of Brazilian medicinal plants. MemInst Oswaldo Cruz; 2000.
4. Kivack BMT, Tansel H. Antimicrobial and Cytotoxic activities of Ceratoniasiliqua L.
extracts. Turk. J. Biol; 2001.
5. World Health Organization [internet]. About
http://www.who.int/diabetes/action_online/basics/en/

diabetes.

Available

from

6. Pazzibugan D. Philippine daily inquirer. 2009.

7. Batangan D. The Prices People Have to Pay for Medicines in the Philippines; 2009.
8. Holman RR, Turner RC. Oral Agents and Insulin in the Treatment of Diabetes,
Blackwell, Oxford. 1991; (467c469).
9. Rao EV, et. al. Essential oil of Ageratum conyzoides. Planta Med. 1988;54:5557
10. Kameswara R, Giri R, Kesavulu MM, Apparao C. Herbal medicine: In the management
of diabetes mellitus. ManpharVaidhyaPatrica. 1997; (3335).
11. Balangcod TD, Balangcod, AKD. Ethnomedicinal knowledge of plants and healthcare
practices among the Kalanguya tribe in Tinoc, Ifugao, Luzon, Philippines. Indian
Journal of Traditional Knowledge. 2011: 10; pp. 227-238.
12. Cancer cure seen in Miracle Tree. Manila bulletin. [2013]
13. Ogbuagu MN. The Nutritive and anti-nutritive compositions of calabash (Crescentia
cujete)
Fruit
Pulp.
2008.
[June
2015].
Available
from:
http://medwelljournals.com/abstract/?doi=jftech.2008.267.270
14. The DECODE Study Group.: Glucose tolerance and mortality: comparison of WHO and
American Diabetic Association diagnostic criteria. Lancet [1999]354 : 617621
15. International Diabetes Federation (2014). Bringing Research inn Diabetes to Global
Environments and Systems: Philippines. Retrieved September 30, 2015, from
International
Diabetes
Foundation
Website:
https://www.idf.org/BRIDGES/map/philippines

75

16. Philippine Diabetes Statistics (2012). Retrieved September 30, 2015, from All About
Diabetes Website: http://www.allaboutdiabetes.net/philippine-diabetes-statistics/
17. Crisostomo S (2013). 6.16 M Pinoy diabetics seen by 2030: Once-a-day pill to improve
medication adherence. Retrieved September 30, 2015, from The Philippine Star Website:
http://www.philstar.com/science-and-technology/2013/03/14/919260/6.16-m-pinoydiabetics-seen-2030-once-day-pill-improve
18. Gilman D, Watson D. Cresentia cujete: calabash tree. [July 2015]. Available from:
https://edis.ifas.ufl.edu/st216
19. Zhao Z, Au DT, Wu J, Jiang Z, Chen H, Lu G. Ethnobotanical study of medicinal plants
used by Hakka in Guangdong, China..Journal of Ethnopharmacology 2008. Available
from
http://www.ncbi.nlm.nih.gov/pubmed/18313871?
dopt=Abstract&holding=f1000,f1000m,isrctn

20. Morton JF. Atlas of medicinal plants of middle America: Bahamas to Yucatan.
Springfield. 1981.
21. Ejelonu BC, Lasisi AA, Olaremu AG, Ejelonu OC. The chemical constituents of
calabash
(Crescentiacujete).
2011.
[June
2015].
Available
from:
http://www.ajol.info/index.php/ajb/article/view/99140
22. Realtime (2011)

23. Popenoe D. Vertebrate endocrinology: fundamentals of biomedical implications,

volume2
24. Reis MF, Sampaio C, Brantes A, Aniceto P, et. al. Human exposure to heavy metals in
the vicinity of Portuguese solid waste incineratorsPart 2: biomonitoring of lead in
maternal and umbilical cord blood. J Hyg Environ Heal. 2007;210:447454. doi:
10.1016/j.ijheh.2007.01.020.[PubMed] [Cross Ref]
25. Bernard A. Cadmium & its adverse effects on human health. Indian J Med Res. 2008;
128: 557564.
26. Fields S. Cadmium Cause and Effect. Environmental Health Perspectives. 2002:
110(12), A764A765.
27. Faroon O, Ashizawa A, Wright S, et al. Toxicological Profile for Cadmium. Atlanta
(GA): Agency for Toxic Substances and Disease Registry (US). 2012 Sep.

76

28. Brown, et. Al. blood hematology and serum biochemistry of Sprague Dawley rats; 2001.

29.
Dvorkin-Camiel L,
Whelan J. Tropical American plants in the
treatment of infectious diseases. 2008.
30.
Amilhasan SJ, et. al. Acute toxicity dose and approximate
effective dose of calabash (Crescentia cujete) decoction as a
hypoglycemic agent in alloxan-induced rabbits; 2010
31. Alegre, et. Al. Toxicity and blood glucose lowering capacity of calabash (Cresenticujete)
fruit pulp decoction in alloxan-induced hypergysemic albino rats (Rattussp); 2009.
32. Mir SH, Darzi MM, Histopathological abnormalities of prolonged alloxan-induced
diabetes mellitus in rabbits, International Journal of Experimental Pathology. 2009: vol.
90(1): pp. 6673.
33. Suckow MA, Douglas F, The Laboratory Rabbit, a volume in the Laboratory Animal
Pocket Reference Series (Boca Raton: CRC Press, 1997)
34. Savory B..the beneficial and harmful effects of organic drugs; 1863.
35. Syahida M, Maskat, M. Y, Suri, R., et.al. Soursop (Anonamuricata L.): Blood
hematology and serum biochemistry of Sprague-Dawleyrat.sInternational; 2012.
36. Mosby, et. Al. Alloxan in inducing diabetes. 2009.
37. Lenzen S. The mechanisms of alloxan- and streptozotocin-induced diabetes.
Diabetologia 2008;51:216-226
38. Erato P, Adebola PO, Grierson DS, Afolayan AJ. An ethnobotanical study of plants used
for the treatment of diabetes in the Eastern Cape Province, South Africa. African Journal
of Biotechnology; [2005] 4: 1458-1460.
39. Etuk EU. Animals models for studying diabetes mellitus. Agric Biol J N Am
2010;1:130-4.
40. Tyberg, et al. Effects of alloxan in inducing diabetes to rodents; 2001.
41. Eizirik, et al.Plants of great uses. 3rd Edn. Richmond, 1994.
42. Murray, RK, et. al. Complete amino acid sequence and structure characterization of the
taste-modifying protein, miraculin; 2012.

77

43. Abdullah MR, et. Al. Acute Toxicity Dose in Mice, Approximate Effective Dose,
Effective Dose (ED50) and Bioassay of Calabash (Crescentiacujete) Fruit Decoction as a
Hypoglycemic Agent in Alloxan-induced Hyperglycemic Rabbit; 2013. vol. 2. pp. 13-19
44. Plants Database (2004). Crescentia cujete L. common calabash tree. Retrieved
September 30, 2015, from http://plants.usda.gov/core/profile?symbol=CRCU.
45. World Health Organization (2011). Lead in Drinking-water. Retreived September 30,
2015, from http://www.who.int/water_sanitation_health/dwq/chemicals/lead.pdf
46. Environmental Protection Agency (2014). Arsenic Rule. Retreived September 30, 2015,
from http://water.epa.gov/lawsregs/rulesregs/sdwa/arsenic/regulations.cfm
47. Organization for Economic Co-operation and Development (2015). OECD Guidelines
for the Testing of Chemicals. Retrieved September 30, 2015, from
http://www.oecd.org/chemicalsafety/testing/41761436.pdf
48. Nowland MH, Hugunin KM, Rogers KL. (2011). Effects of Short-Term Fasting in Male
SpragueDawley Rats. Comparative Medicine, 61(2), 138144.
49. Clinical Guidelines Task Force, International Diabetes Federation (2005)."Glucose
control: oral therapy" ). In: Global Guideline for Type 2 Diabetes. Brussels: International
Diabetes
Federation,
358.
Retrieved
September
30,
2015,
from
http://www.idf.org/webdata/docs/GGT2D%2009%20Oral%20therapy.pdf
50. CAC/RCP 56-2004. Code of practice for the prevention and reduction of lead
contamination in foods. CAC/RCP 56-2004. Code of practice for the prevention and
reduction of lead contamination in foods. Retrieved September 15, 2015, from
http://www.codexalimentarius.org/download/standards/10099/CXP_056e.pdf
51. Centers for Disease Control and Prevention (1978). Occupational Health Guideline for
Hydrogen
Cyanide.
Retrieved
September
30,
2015,
from
http://www.cdc.gov/niosh/docs/81-123/pdfs/0333.pdf
52. Ozougwun JC, Obimba KC, Belonwu CD, and Unakalamba CB. (2013). The
pathogenesis and pathophysiology of type 1 and type 2 diabetes mellitus. Journal of
Physiology and Pathophysiology, 4(4), 46-57.
53. Rohilla A, & Ali S. (2012). Alloxan Induced Diabetes: Mechanisms and Effects.
International Journal of Research in Pharmaceutical and Biomedical Sciences, 3 (2).
54. The Philippine Council for Health Research and Development of Department of Science
and Technology : http://www.pchrd.dost.gov.ph/index.php/news/r-d-updates/3133calabash-fruit-found-potential-in-lowering-blood-sugar

78

55. Ethnobotanical Study of Plants Used to Treat Diabetes in Traditional Medicine, by

Abbey and Krobou Populations of Agboville (Cte-dIvoire) / N'guessan Koffi et al /
American Journal of Scientific Research, ISSN 1450-223X, Issue 4 (2009), pp 45-58
56. Fda.gov [homepage on the internet]. US Food and Drug Association [updated 2015;
cited
2015].
Available
from
http://www.fda.gov/Drugs/DevelopmentApprovalProcessHowDrugsareDevelopedandAp
proved/ApprovalApplications/InvestigationalNewDrugINDApplication/ucm176522.htm
57. Chapman K, Holzgrefe H, Black L, Brown, Chellman G, Copeman C, editors.
Pharmaceutical toxicology: Designing studies to reduce animal use, while maximizing
human translation; 2013.

58. Nkambo W., Anyama N., Onegi B. (2013). In vivo hypoglycemic effect of methanolic
fruit extract of Momordica charantia L. African Health Sciences,13(4), 933939.
http://doi.org/10.4314/ahs.v13i4.11
59. Yashwant Kumar A, Nandakumar K, Handral M, Talwar S, & Dhayabaran D. (2011).
Hypoglycaemic and anti-diabetic activity of stem bark extractsErythrina indica in
normal and alloxan-induced diabetic rats. Saudi Pharmaceutical Journal: SPJ, 19(1),
3542. http://doi.org/10.1016/j.jsps.2010.10.001
60. Sebai H, Selmi S, Rtibi K, Souli A, Gharbi N, Sakly M. (2013). Lavender (Lavandula
stoechas L.) essential oils attenuate hyperglycemia and protect against oxidative stress in
alloxan-induced diabetic rats. Lipids in Health and Disease, 12, 189.
http://doi.org/10.1186/1476-511X-12-189
61. Shah NA, & Khan MR. (2014). Antidiabetic Effect of Sida cordata in Alloxan Induced
Diabetic
Rats. BioMed
Research
International, 2014,
671294.
http://doi.org/10.1155/2014/671294
62. http://www.janvierlabs.com/tl_files/_media/images/FICHE_RESEARCH_MODEL_SPRAGUE_DAWLE
Y.pdf
63. Hypoglycemic Effect of Methanolic Extract fo Anacardium occidentale leaves in
Alloxan-Induced Diabetic Rats. Fagbohun, T.R, Odufunwa, K.T. 2010.
64. NSW Department of Primary Industries and Animal Research Review Panel initiative,
Decemer, 2014 http://www.animalethics.org.au/three-rs
65. Research Animal Resources, 2003 https://www.ahc.umn.edu/rar/housing.html

79

66. Guidelines for care and use of animals in scientific research from
http://oacu.od.nih.gov/ac_cbt/guide3.htm
67. Guidelines for Endpoints in Animal Study Proposals, (March 10, 2013)
http://oacu.od.nih.gov/ARAC/documents/ASP_Endpoints.pdf
68. Guidelines on the care and use of animals for scientific purposes; 2004 from
http://www3.ntu.edu.sg/Research2/Grants%20Handbook/NACLAR-guide%20Lines.pdf
69. Suzanne NS. 2002. Introduction to Chemical Analysis of Food. pp. 137-164.
70. Gordon MW. 2000. Contemporary Nutrition: Issues and Insights. 4th Edn. McGraw Hill
Companies New York. pp. 102-256.
71. Barbara AB, Robert MR. 2001. Present knowledge in Nutrition. 8th Edn. pp157-184.
72. Harborne JB. 1973. Phytochemical Methods Chapman and Hall, London.
73. Goldner MG. 1944. Alloxan Diabetes Its Production and Mechanism, Chicago.
74. Mrozikiewicz A. et al., 1994. Blood Levels of Alloxan in Children with IDDM, Acta
Diabetologica 31 (4): 236-237.
75. Mahmoud MF, Hassan NA, El Bassossy, HM, Fahmy A. (2013). Quercetin Protects
against Diabetes-Induced Exaggerated Vasoconstriction in Rats: Effect on Low Grade
Inflammation. PLoS ONE, 8(5), e63784. http://doi.org/10.1371/journal.pone.0063784
76. Vessal M, Hemmati M, Vasei M. Antidiabetic effects of quercetin in streptozocininduced diabetic rats.Comp Biochem Physiol C Toxicol Pharmacol. 2003;135C:35764.
77. Kunkel SD, Elmore CJ, Bongers KS, Ebert SM, Fox DK, Dyle MC, Adams CM. (2012).
Ursolic Acid Increases Skeletal Muscle and Brown Fat and Decreases Diet-Induced
Obesity, Glucose Intolerance and Fatty Liver Disease. PLoS ONE, 7(6), e39332.
http://doi.org/10.1371/journal.pone.0039332
78. Ling C, Jinping L, Xia L, Renyong Y. (2013). Ursolic Acid Provides Kidney Protection
in Diabetic Rats. Current Therapeutic Research, Clinical and Experimental, 75, 5963.
http://doi.org/10.1016/j.curtheres.2013.07.001
79. Brunton L, Chabner B, Knollman B. Goodman and Gilmans Pharmacological Basis of
Therapeutics.12th ed. McGraw-Hill Education; 2011.
80. Viollet B, Guigas B, Sanz Garcia N, Leclerc J, Foretz M, Andreelli F. (2012). Cellular
and molecular mechanisms of metformin: an overview. Clinical Science (London,
England: 1979), 122(6), 253270. http://doi.org/10.1042/CS20110386

80

81. Maida A, Lamont BJ, Cao X, Drucker DJ. Metformin regulates the incretin receptor axis
via a pathway dependent on peroxisome proliferator-activated receptor-alpha in
mice. Diabetologia. 2011;54:339349.
82. Shu Y, Sheardown SA, Brown C, Owen RP, Zhang S, Castro RA, Giacomini KM.
(2007). Effect of genetic variation in the organic cation transporter 1 (OCT1) on
metformin action. Journal of Clinical Investigation,117(5), 14221431.
http://doi.org/10.1172/JCI30558
83. http://www.niddk.nih.gov/health-information/health-topics/Diabetes/diagnosis-diabetesprediabetes/Pages/index.aspx
84. Szkudleski T. The Mechanism of Alloxan and Streptozocin Action in B cells of the Rat
Pancreas; 2001.
85. Viswanathaswamy AHM, Koti BC, Gore A, Thippeswamy AHM, & Kulkarni RV
(2011). Antihyperglycemic and Antihyperlipidemic Activity ofPlectranthus
Amboinicus on Normal and Alloxan-Induced Diabetic Rats. Indian Journal of
Pharmaceutical Sciences, 73(2), 139145.
86. Guatam MK, et. al. Studies on the hypoglycemic effects of Murraya paniculata Linn.
extract on alloxan-induced oxidative stress in diabetic and non-diabetic models; 2012.
87. Huisjes EH, de Hulster E, van Dam JC, Pronk JT, van Maris AJA. (2012). Galacturonic
Acid Inhibits the Growth of Saccharomyces cerevisiae on Galactose, Xylose, and
Arabinose. Applied
and
Environmental
Microbiology, 78(15),
50525059.
http://doi.org/10.1128/AEM.07617-11
88. Lattimer JM, Haub MD. Effects of Dietary Fiber and Its Components on Metabolic
Health; 2010.