TITLE: SYNERGISTIC EFFECT OF ASHITABA (ANGELICA KEISKEI KOIDZUMI)

LEAF EXTRACT AND AMPALAYA (MOMORDICA CHARANTIA) FRUIT EXTRACT

IN LOWERING THE BLOOD SUGAR LEVEL OF ALBINO MICE

CHAPTER ONE

THE PROBLEM: RATIONALE AND BACKGROUND

Diabetes Mellitus is a group of metabolic disease characterized by increased levels

of glucose in the blood (hyperglycemia) resulting from defects in insulin secretion,

insulin action, or both. Blood, normally contains certain amount of glucose. The

major sources of this glucose are from food nutrient absorbed in the gastrointestinal

tract and glucose formation by the liver in the process called glyconeogenesis

(American Diabetes Association, 2004).

Insulin, a hormone produced in the pancreas, controls the level of glucose in

the blood by regulating the production and storage of glucose. In diabetes, the cells

may stop responding to insulin or the pancreas may stop producing insulin entirely.

This lead to hypoglycemia, which may result in acute metabolic complication such

as diabetic ketoacidosis (DKA) and hyperglycemia hyperomolar non-ketotic

syndrome (HHNS).
 Diabetes is the third most common cause of death worldwide. In 2000, the

worldwide estimate of the prevalence of diabetes was 170 million people, and by

2030, this is expected to increase to 366 million. Diabetes is especially prevalent in

the elderly ; as many as 50% of people older than 65 years of age have some

degree of glucose intolerance. People 65 years and older account for almost 40% of

people with diabetes.

From 1980 through 2002, the prevalence of diagnosed diabetes increased

in all age groups. People between 65 and 74 years of age had the highest

prevalence, followed by people younger than 45 years of age. In 2002, the

prevalence diagnosed diabetes among people 65 to 74 years of age (16.8%) was

almost 14 times of people younger than 45 years of age (1.2%). However, many

people with diabetes are undiagnosed (Wild, Roglic, Green, et al., 2004).

The main goal of diabetes treatment is to normalize insulin activity and

blood glucose levels to reduce the development vascular and neuropathic

complications. Different synthetic drugs were develop to manage diabetes, but all

offers had adverse side effect like severe hypoglycemia, coma, or seizure. Thus, a

lot of researches were conducted to find a natural substance from plants that will

aid in the Management of diabetes. Among those studies, Ampalaya (Momordica

charantia) and Ashitaba (Angelica keiskei koidzumi) gains much popularity.

Ampalaya (Momordica charantia) also known as bitter melon, is a tropical

and subtropical vine belonging to the family of cucurbitaceae (Garau et al. 2003). It

is widely used as alternative medicine in treating various diseases including

Diabetes Milletus (Yadav et al. 2005). Momordica charantia is an antidiabetic agent

has been used in India. There are 3 nonsteroidal hypoglycemic compounds isolated
from Ampalaya fruit and there are charantin, vicine, polypeptide-p or p-insulin and

momordicin( Srivastava et al. 1993).

Ashitaba (Angelica Keiskei Koidzumi) or Tomorrow's leaf, is a herbal plant

originated from Central Japan belonging to the family of Umbelliferae. Which was

found to posses metabolites chalcone known to have hypoglycemic effect.

Chalcone is a yellow sap flavanoids that enhances hypoglycemia and is classified

into two major type; the Xanthoangelol (XA) and 4-Hydroxyderricin (4HD) Ezimar et

al. 2013).

There are several studies that proves the Effectiveness of Ashitaba (Angelica keiskei

koidzumi) and Ampalaya (Momordica charantia) as a natural hypoglycemic agents.

Some remarkable studies are:

The result of the study conducted by Mahmoud 2017, "Research on the

Antidiabetic Activities of Momordica charantia fruit extract", concluded that

Momordica charantia presents antidiabetic and antioxidant activities. Thus, has

great potential as the new source for diabetes treatment whether it is used for

prophylaxis or treatment.

In the field of Pharmacy, some substances with the same effect are combined to

produce a new substance with stronger or a more desirable effect, the process

called Synergistic Reaction. And it is within this premises of synergism that the

researchers wants to investigate the possible Synergistic Effect of Ashitaba

(Angelica keiskei koidzumi) and Ampalaya (Momordica charantia) in lowering the

blood sugar level of Albino Mice.

STATEMENT OF THE PROBLEM

The Synergistic Effect of Ashitaba (Angelica keiskei koidzumi) leaf extract and

Ampalaya (Momordica charantia) fruit extract in lowering the blood sugar level of

Albino mice. The following are the specific questions:

1. Does Ashitaba (Angelica Keiskei koidzumi) leaf extract and Ampalaya (Momordica

charantia) fruit extract lowers the blood sugar level of albino mice?

2. Does Ashitaba (Angelica keiskei koidzumi) leaf extract and Ampalaya (Momordica

charantia) fruit extract has a synergistic effect in lowering the blood sugar level of

albino mice?

3. Is there a significant difference in the hypoglycemic effect among Ashitaba

(Angelica Keiskei koidzumi) leaf extract , Ampalaya ( Momordica charantia) fruit

extract and the combined Ashitaba leaf extract and Ampalaya fruit extract?

OBJECTIVES OF THE STUDY

The main purpose of this study is to determine if there is a Synergistic Effect of

Ashitaba (Angelica keiskei koidzumi) leaf extract and Ampalaya (Momordica

charantia) fruit extract in lowering the blood sugar level of Albino mice. The

following are the specific objectives:

1. To determine if the Ashitaba (Angelica keiskei koidzumi) leaf extract and

Ampalaya (Momordica charantia) fruit extract lowers the blood sugar level of albino

mice?
2. To determine if the Ashitaba (Angelica keiskei koidzumi) leaf extract and

Ampalaya (Momordica charantia) fruit extract has a synergistic effect in lowering

the blood sugar level of albino mice?

3. To determine if there is a significant difference in the hypoglycemic effect among

Ashitaba (Angelica keiskei koidzumi) leaf extract , Ampalaya (Momordica charantia)

fruit extract and the combined Ashitaba leaf extract and Ampalaya fruit extract?

NULL HYPOTHESIS

1. The Ashitaba (Angelica keiskei koidzumi) leaf extract and Ampalaya (Momordica

charantia) fruit extract doesn't lowers the blood sugar level of albino mice?

2. The Ashitaba (Angelica keiskei koidzumi) leaf extract and Ampalaya (Momordica

charantia) fruit extract has no synergistic effect in lowering the blood sugar level of

albino mice?

3. There is no significant difference in the hypoglycemic effect among Ashitaba

(Angelica keiskei koidzumi) leaf extract , Ampalaya (Momordica charantia) fruit

extract and the combined Ashitaba leaf extract and Ampalaya fruit extract?

THEORETICAL/CONCEPTUAL FRAMEWORK

The result of research conducted by Tonog (2011), "Determination of the

Primary and Secondary metabolites Present in the Crude Leaf Extracts of Ashitaba

(Angelica Keiskei Koidzumi)" , isolates the yellow sap pigment known as Chalcones.
 Chalcone is naturally found in Ashitaba, it is a metabolites in which it

induces adipocyte differentiation and enhances the activity of glucose uptake

inhibiting hyperglycemia. Chalcone as insulin-like substance is responsible for

lowering the blood glucose by allowing the glucose to enter into the body cells, or

stored in the liver as glycogen or used as energy. Otherwise, absence of insulin nor

insulin-like substances, glucose cannot transport into the cells and remains on blood

necessity of viscosity when prolonged, Diabetes Mellitus occurs. Chalcone in

Ashitaba can reduces and improve the blood glucose control (Campbell, 2011).

Another investigation conducted by Tatsuji Etnoki (2004), "Antidiabetic

activities of Ashitaba (Angelica keiskei joidzumi); Induction of Adipocyte,

Differentiation and Enhancement of Glucose Uptake in Adipocyte"; identified two

major Chalcones that possess a preventive activity against diabetes. The Chalcone

has 2 major compounds that inhibits hypoglycemia the Xantoangelol (XA) and 4-

hydroxyderricin (4HD). Strongly promoting activity of glucose uptake and

preventive effect on the progression of diabetes. The study proves the lowering of

blood sugar level when dose take orally (Etnoki et al. 2010).

Another plant found abundant of antidiabetic substance is Ampalaya (Momordica

charantia). The study published by APJTD (Asian Pacific Journal of Tropical Disease)

in 2013 entitled, "Antidiabetic effects of Momordica Charantia (Bittermelon) and its

Medicinal Potency" reveals the constituents of Ampalaya responsible for

hypoglycemic activity. Having the presence of Charantin, Polypeptide-p, alkaloid

and kakra compounds has a significant effect in lowering blood glucose level.

Charantin is a potential substance of Ampalaya (Momordica charantia) that posses

antidiabetic property, containing two compounds, sitosteryl glucoside and

stigmateryl glucoside (Chen et al.). In addition, charantin demonstrated that it

could be used as treatment for diabetes and has a potential to replace the

systematic treatment (Pitiphanpong et al.).

Polypeptide-p is found in Ampalaya to used in controlling diabetes.

Polypeptide-p or insulin-p, has an insulin-like hypoglycemic protein that can lower

blood glucose levels in species belonging to gebrils, langus and humans when

injected simultaneously. It imitates the action of human insulin in the body. Thus,

proclaims that it can be used as plant-based insulin replacement.

Wang et al. Experimented by cloning and expressed 498 Polypeptide-p

gene sequence coding of Ampalaya (Momordica charantia). Polypeptide-p gene has

a hypoglycemic effect of the recombinant polypeptide to induced diabetic mice. In

addition, the oral intake of the Ampalaya seed extract produced lowering blood

glucose action to Streptozotocin (STZ) induced type-1 diabetic rats. Hence,

indicating Ampalaya seeds can be used as antidiabetic substance. Vicine it is glycol

alkanoid isolated from Ampalaya seeds. This pyrimidine nucleoside has been shown

to have effect in hypoglycemia in non-diabetic fasting rats. Momordicin a substance

that is oftentimes called as plant insulin, responsible for it's bitter taste of plant's

leaves and fruits which attributed that Ampalaya has the blood sugar lowering

property.

SCOPE AND LIMITATION OF THE STUDY

This study scopes only (about the determining if there is a Synergistic

activity between Ashitaba (Angelica Keiskei Koidzumi) and Ampalaya (Momordica

Charantia) in lowering the blood sugar level of albino mice.

 The study will limits accordingly to the result of the experimental tool

glucometer which it will measure each test animals if there is really a hypoglycemia.

DEFINITION OF TERMS

Albino Mice - is a small mammal of the order Rodentia which is breed and used for

scientific research. It is commonly used mammalian research model and are used

for research in genetics, psychology, medicine and other scientific disciplines.

Alternative Medicine- it is the the used in this study referring to practices claimed to

have the healing effects of medicine but still not proven.

Antidiabetic- it is a drug used to alleviate diabetes such as Glibenclamide.

Ampalaya- a tropical and subtropical plant often called as bitter melon or bitter

gourd used to treat diabetes.

Ashitaba- is a large herb originated from Central Japan wherein it's roots, stem and

leaf are used to develop medicine.

Chalcone- an organic compound called flavonoids recently found in Ashitaba which

is responsible for antidiabetic effect.

Concentration- the act or process of the extracts be concentrated.

Diabetes Mellitus- commonly referred to as metabolic disorder in which there are

increase of blood glucose level.

Extract- it is the concentrated liquid extracted from the fruit and leaves.

Glucose- it refers to the sugar that is used as source of energy.

Hyperglycemia- refers to increase of blood glucose level.

Hypoglycemia- refers to decrease of blood glucose level.

Insulin- it is a hormone released by the pancreas to control blood glucose level.

Synergistic- refers to the interaction of the two components (Ashitaba leaf extract

and Ampalaya fruit extract) forming a combined effect.

CHAPTER TWO:

REVIEW OF RELATED LITERATURE, STUDY AND LEGAL BASIS

RELATED LITERATURE

Ashitaba is scientifically name Angelica keiskei koidzumi which originated

from Japan and belong to the family Umbelliferae. It is a herbaceous, perennial plant

growing to a height of 50 to 120 centimeters. Roots are stout, either conical or

cylindrical. The flowers are hermaprodite (presence of both sex). Ashitaba plant is

self-fertile and regenerative. Harvesting a leaf at the break of day often results in a

new sprout growing overnight, hence explained called as "Tomorrow Leaf" (Kumor

2011).

Ashitaba (Angelica keiskei koidzumi) has yellow sap, organic compound

flavanoid containing Chalcones a fights against diabetes. Ashitaba has two major
compound identified in Chalcone the xanthoangelol (XA) and 4 hydroxyderricin

(4HD) which induces hyperglycemia (Etnoki et al. 2010).

Ampalaya with a scientific name Momordica charantia known as bitter melon

or gourd distinguished by its bitter taste due to presence of momordicin, is a

tropical and subtropical vine belong to the family of Cucurbitaceae. Ampalaya is

also a herbaceous, perennial climbing plant that grows up to 5m, with tendrils up to

20 cm long. Leaves are heart-shaped, 5 to 10cm across, cut into 5 to 7 lobes. Each

plant bears separate yellow male and female flowers. It bears freshly green fruit,

oblong-shaped with pointed ends, ribbed and wrinkled bursting when mature

released seeds appeared flat and ruminated margins (Plant. Net 2017).

Ampalaya (Momordica charantia), has major constituents inhibits

hypoglycemia. Previous studies have isolate antidiabetic agents of Ampalaya, the

Charantin, polypeptide-p, vicine and momordicin. Charantia is a potential substance

composed of two mixture sitosteryl and stigmateryl glucoside. In which can

potentially used as alternative medicine. Polypeptide-p also known as p-insulin, is a

metabolites that has as insulin-like hypoglycemic protein. It can imitate the action of

human insulin, thus, it can replace synthetic drugs to lower blood glucose level.

Then, vicine, a glycol alkaloid found in Ampalaya seeds and momordicin the

substance responsible for it's bitter taste that used in hypoglycemia (Chen et

al.____).

Insulin, is a hormone releases by the Pancreas actively for lowering the blood

glucose by permitting the entry of ingested glucose to the cells in all organs of the

body. The cells need glucose to burn as energy it used or stored as glycogen. Insulin

also promotes the storage of fat in adipose fats and protein synthesis in the various
body tissues. Otherwise, absence of insulin glucose won't be accommodated. In

which it will stayed on blood responsible for viscosity when prolonged it will be

excreted as urine output, resulting to a condition known as Diabetes Mellitus. It is

the increase of levels of glucose in the blood. High levels of glucose in blood can

damage the tiny blood vessels in your kidneys, heart, eyes and even nervous

system that's why if diabetes left untreated it leads to further damages such as

heart disease, stroke, kidney disease, blindness and nerve damage to nerves in the

feet.

Diabetes milletus is commonly referred to as diabetes, a chronic disease

associated by abnormal high levels of sugar(glucose) in the blood, due to

inadequate production of insulin by the Pancreas and also inadequate sensitivity of

cells to the action of insulin (American Diabetes Association).

Furthermore, diabetes Mellitus is recently one of the five leading causes

of deaths in the world. A major global health alarming concerning with a projected

increase of prevalence from 171 million last 2000 to 366 million in the future 2030.

This is a syndrome of disordered metabolism, usually due to hereditary and

environmental phenomena causing of abnormality increased of blood sugar levels

forming hyperglycemia. Being a one of the degenerative disease, it has been found

all over the world, becoming the third most lethal disease to mankind. It is the

common cause of endocrine disorder, affecting 16 million people in the United

States and over 200 million individuals worldwide.

Glibenclamide is the drug choice as a positive control for the blood

sugar level. It is an antidiabetic drug in a class of medication known as sulfonylurea.

This drug is used to treat the type 2 diabetes and the major cause of drug that
generates hypoglycemia. The drug works by binding to and activating the ATP-

sensitive potassium channels (KATP) inhibitory regulatory sub unit sulfonylurea

receptor 1(SUR1) in pancreatic beta cells. This inhibition make cell membrane

depolarization opening voltage- department calcium channel, this results in an

increase in intracellular calcium in the beta cells and subsequentl stimulation of

insulin release (Smeltzer 2008).

RELATED STUDY

FOREIGN STUDIES

In 2017, Mahmoud et al. on their study about the Research on

Antidiabetic activities of Momordica Charantia fruit extract caused a significant

reduction of serum glucose, total cholesterol, triglycerides, levels, insulin resistance

index and pancreatic malon-dialdehyde content. While it induced a significant

increase of serum insulin, HOL-cholesterol, total antioxidant capacity levels, B cell

function percent and pancreatic reduced glutathione (GSH) content and improved

histo pathological changes of the pancreas.

In 2010, Etnoki on his study about the "Antidiabetic Activities of Ashitaba

(Angelica Keiskei Koidzumi); Induction of adipocyte, differentiation and

enhancement of Glucose uptake in adipocyte", evaluated that the chalcone (4-

hydroxyderricin ;4HD) has an insulin like activity in vitro and vivo, as it induces

adipocyte differentiation and glucose uptake and inhibit the hyperglycemia in

genetically diabetic mice (KKay).

CHAPTER THREE:

METHODOLOGY

LOCALE OF THE STUDY

This study will be conducted at the Science Laboratory Room of Bobon School for

Philippine Craftsmen (BSPC). A Government Secondary School located along the

Maharlika Highway of Brgy. Magsaysay, Bobon Northern Samar.

RESEARCH DESIGN

This is experimental since it seeks to investigate the Synergistic Effect of Combined

Ashitaba leaf extract and Ampalaya fruit extract on Albino mice.

Experimental Design

The experiment will be using Randomized Complete Block Design (RCBD). Five

treatments referring to have all 75% concentrations of Ashitaba (Angelica keiskei

koidzumi) leaf extract, Ampalaya (Momordica charantia) fruit extract, and the

combined Ashitaba leaf extract and Ampalaya fruit extract and the positive and

negative control will be included in the testing of the experiment. And because of

limited resources each experimental lot were contains only 1 mouse.

FIGURE 2. THE EXPERIMENTAL LAYOUT FOLLOWING RCBD SHOWING THE

DIFFERENT TREATMENT THE POSITIVE AND NEGATIVE CONTROL AND THEIR

REPLICATIONS.

Treatment Replicate I Replicate II Replicate III

For Ashitaba

T-1 (75% concentration)

T-2 (50% concentration)

T-3 (25% concentration)

For Ampalaya

T-4 (75% concentration)

T-5 (50% concentration)

T-6 (25% concentration)

For Combined Ashitaba and Ampalaya

T-7 (75% concentration)

T-8 (50% concentration)

T-9 (25% concentration)

T-10 Glibenclamide (+

control)

T-11 Distilled water (-

control)

Preparation of the Test Animal

Forty-five mice, regardless of sex will be used as experimental animals. The mice

will weighed individually on a double pan beam balance. The animals will be housed

in screen cages of 10 x 20 x 10 inches in size, with three mice in each cage. The
mice will be placed in a suitable environment where disturbances are unnecessary.

Within, it will be provided regular food and water for their daily diet

Preparation of the Test Substances

Collection of the Plant Materials

The leaves of Ashitaba (Angelica Keiskei Koidzumi) will be gathered within the

Municipality of Bobon Northern Samar. As well as the fruit of Ampalaya (Momordica

Charantia). These two be brought to the School Laboratory of Bobon School for

Philippine Craftsmen for further identification and preparation of the materials for

extraction.

Preparation of the Plant Materials Extraction

In Ashitaba (Angelica Keiskei Koidzumi), the collected leaves will be washed with

tap water to cleansed and remove dust and insect. Afterwards, it will be weighed

with approximately 1000g of Ashitaba leaves to be used in extraction.

The Researchers will be using Decoction Method in order to obtain the crude leaf

extract. The leaves will be soaked in cold water for 30 minutes before it undergo to

boiling. Through this, it enables the plant cells to expand and release their active

compound needed when boiled. One thousand ml of water will be boiled first and

afterwhich the 1000g of soaked Ashitaba leaves will be added accordingly. The heat

will be maximize from 50 degrees Celsius up to 60 degrees Celsius in order to

maintain a slow simmer for 15 minutes long. Then, it will be remove from heat and

transfer to cool and strain using a clean cheese cloth. The obtain crude leaf extract
will be placed in a transparent plastic container and stored in the refrigerator until

use for the preparation of the different concentration.

Preparation of different concentration

The preparation of each concentrations ( 75%, 50%, and 25%) of Ashitaba (Angelica

Keiskei Koidzumi) leaf extract, Ampalaya (Momordica Charantia) fruit extract and

the Combined Ashitaba and Ampalaya extract was followed: For 75%,7.5ml of leaf

crude extract, fruit crude extract and the combined crude extract will be dissolve in

10 ml of distilled water, for 50%, 5ml of leaf crude extract, fruit crude extract and

the combined crude extract will be dissolve in 10 ml of distilled water and for 25%,

2.5 ml of leaf crude extract, fruit crude extract and the combined crude extract will

be dissolve in 10 ml of distilled water.

Positive and Negative Controls

Glibenclamide, also known as glyburide, is an antidiabetic drug with an insulin-like

property will be used as the positive control for blood sugar level, while the distilled

water will be used as the negative control.

Administration of the Test substance and Positive control to the Test

Animals

Each of the test substance will be administered orally by means of their daily diet.

In which, the 0.2 ml per gram body weight (0.2 ml/gbw), will be mixed in their

drinking water three times a day for one week. While, glibenclamide as the positive

control for blood sugar level will be administered also the same process

aforementioned but with a dosage of 1mg per gram body weight (1gm/gbw).

Determination of Blood Sugar level

The concentration of glucose in the blood of Albino mice will be determined through

the use of Terumo Medisafe glucometer using Blood Glucose Test (TIP) before and

after 7 days of Ashitaba Crude leaf extract, Ampalaya Crude fruit extract and the

Combined Crude extract administration. The glucometer code together with the

strip will be inserted within with an arrow indicating that it is ready for used. A drop

of blood will be gathered from the tail of the Albino Mice. Then, it will be drop unto

the TIP of glucometer. After ten seconds, the reading will be taken and recorded on

the recording sheet.