Republic of the Philippines

Department of Education

Region 2

Division of Quirino

MADDELA COMPREHENSIVE HIGH SCHOOL

Maddela, Quirino

Hypoglycemic Properties of Robusta Coffee Leaves on

Alloxan

Capstone Project Proposal

Presented to:

Mr. Nhorvien Jay P. Libao

By:

Abraham, John Carl Hero P.

April, 2023
CHAPTER 1

INTRODUCTION

RATIONALE

Diabetes Mellitus is a condition that imparts the body’s ability to process blood glucose,

otherwise known as blood sugar. This disease can be acquired at any age without proper diet

which leads to long- term manifestation of diabetes that may contribute complications such as

cardiovascular diseases which affects the nerves and its functions.

Coffea arabica and Coffea Robusta belongs to the family of rubiaceae (Nayeem, N. et al.,

2011) Coffee is often used traditionally as anti-bacterial and anti-oxidant. A coffee contains

compounds such as phenolic acids, tannins, and flavonoids that have anti-bacterial constituents

which helps in lowering blood glucose. According to Vinayagam, R. et al., (2016), Phenolic

acids are found in fruits, vegetables, and wines and they contribute natural properties for the

health. Phenolic acids increase glucose uptake and glycogen synthesis, which improve glucose

and lipid profiles of certain diseases such as diabetes mellitus. Moreover, tannins enhance

glucose uptake and helps in aiding pathological oxidative state of a diabetic patient (Kumari &

Jain, 2012). Natural sources from flavonoids have bioactive compounds that possesses anti-

diabetic property (Jaitak, 2019).

Synthetic drugs are commonly used to treat diabetes despite the side effects that a person

may acquire. However, considering the herbal plants which have anti- diabetic properties may

lessen the amount of chemical intake and significantly contribute to the environment in favor of

natural medicine.
STATEMENT OF THE PROBLEM

The research study pursues a main objective which is to create a product (alternative anti-

diabetic medicine) as a ready-to-use medicine. Henceforth, the researcher formulated these

following objectives:

a) To extract the crude extract sample of robusta coffee leaves that can be used as a

hypoglycemic medicine.

b) To evaluate the phytochemical constituents of robusta coffee leaves.

c) Is there significant difference in the effectiveness of a commercial medicine and the

robusta coffee leaves medicine?

FORMULATION OF HYPOTHESIS (null)

There are no significant difference in the effectiveness of a commercial medicine and the robusta

coffee leaves medicine.

SIGNIFICANCE OF THE STUDY

This medicine will benefit the people under the field of medicine because they will be

able to use Robusta leaves as alternative medicine and manufacture fewer synthetic drugs which

can harm the human body. In agriculture for this will help farmers maximize the use of Robusta

coffee leaves as herbal medicine and may serve as additional income. In addition, this will serve

as an information to consumers on how Robusta leaves reduces blood sugar level among patients

with diabetes. Lastly, to the future researchers in which this study will serve as a reference in

their study and may help the future researchers to strengthen their research paper in testing
validity of other related findings. Over all, this study will benefit the society because using

alternative and natural medicine will be more effective and safer than synthetic drugs. Also, to

lessen the production of synthetic drugs.

SCOPE AND DELIMITATIONS

This study will be conducted to determine the hypoglycemic properties of Robusta coffee

leaves that can be effective in lowering blood sugar. It will be studied to extract the crude extract

sample of Robusta coffee leaves that can be used as a hypoglycemic medicine, to evaluate the

phytochemical constituents of Robusta coffee leaves, to identify the toxicity level of the

concentration of Robusta coffee leaves, and to determine the blood glucose level of the mice

using glucometer and evaluate hyperglycemic activity.

DEFINITION OF TERMS

Hypoglycemic - deficiency of glucose in the bloodstream.

Diabetes mellitus - a group of diseases that affect how the body uses blood sugar.

Rubiaceae - a family of flowering plants, commonly known as the coffee, madder, or bedstraw

family.

Phenolic acids - are bioactive molecules that impact a plant's physiology, ecology, and

development.

Tannins - a yellowish or brownish bitter-tasting organic substance present in some galls, barks,

and other plant tissues.

Flavonoids - a class of polyphenolic secondary metabolites found in plants

REVIEW OF RELATED LITERATURE/ REVIEW OF RELATED STUDIES

Plants are considered cost-effective because it can be found in any specific habitat. The

role of medicinal plants is to furnish answers to diseases and maintain health management.

Nowadays, research tools and equipment are evolving which makes researches feasible and

analytical. Synthetic drugs are made to cure diseases such as cancer, diabetes, minor pains and

many more but they are harmful to the body when ingested always and beyond the prescribed

dosage. Hence, discoveries to new compounds and alternative medicines are enormous that has

been continuously examined and tested to avert adverse reaction to the body.

Diabetes

Jimeno K. et al., (2015) stated that in year 2008, 7.2% of the entire nation are diabetic

approximately 6.377 million in a population of 88.57 million. Diabetes, as referred by

Moradabadi, L. et al., (2013), is a global health problem in the public which is considered

epidemic worldwide. The prevalence of increasing the population of diabetic patients will be

doubled between 2005-2030 in the countries of Asia, South America and Africa (Rowley et al.,

2017).

According to Shokouh.P, et al., (2019), the effects of chronic coffee exposure in models of type

2 diabetes mellitus (T2D) is scarcely studied, and the efficacy of the main coffee species has

never been compared. They also tested the hypothesis that long-term consumption of arabica and

Robusta coffee may differentially delay and affect T2D development in Zucker diabetic fatty

rats. Both coffee species reduced liver triglyceride content and area under the curve of fasting

and postprandial insulin. At study end, plasma adiponectin, total cholesterol and high-density
lipoprotein levels were higher in the robust group compared with both arabica and control

groups. Robusta coffee was seemingly superior to arabica coffee with respect to effects on lipid

profile, adiponectin level and hepatic gene expression.

Xiumin (2018) directed an examination about the phytochemicals, bioactivities, and utilizations

of espresso leaves and as of late, considerations have been paid to their medical advantages to

individuals on account of plenteous bioactive parts in espresso leaves. Espresso leaves are

promising asset in the territories of nourishment and industry, particularly, in the drink business.

The researchers about in understanding effects of the handling techniques on the phytochemicals,

catalysts, bioactivities, and kind of espresso leaves are exceptionally required.

As expressed by Herawati D. et al., (2019), the bioactive compound arrangements of Robusta

espresso mix and the cancer prevention agent, hostile to glycosidase and against glycation

exercises of these mixes are properties significant for diabetes the executives. Past investigations

detailed that bioactive mixes in Robusta espresso blend display cancer prevention agent action

that might be associated with the anticipation of non-transferable illnesses (Ludwig et al., 2012;

Vignoli et al., 2014). This property is likewise bolstered by epidemiological examinations that

show espresso blend utilization forestalls type 2 diabetes mellitus (T2DM) (Sarriá et al., 2016).

As per Lopez, N. et al., (2012), they examined the physiological and biochemical capacities of

Robusta espresso leaves for acclimation to adapt to worldly changes in light accessibility

wherein the impacts of fluctuating forces of light on plants rely upon when they happen,

regardless of whether the aggregate sum of light got is kept consistent. They planned an

investigation utilizing two clones of Robusta espresso (Coffea Canephora) intercropped with

cover trees so that permitted us to think about espresso brambles concealed in the first part of the

day (SM) with those concealed toward the evening (SA), and afterward standing up to both with
hedges accepting full daylight through the span of the day (FS). All in all, concealing may

improve the physiological presentation of espresso shrubs developing in unforgiving, tropical

situations.

A study conducted by Nayeem N. et.al. (2011), determined the contents of flavonoids, phenolic

acid and tannins of Arabica coffee leaves (L. Coffea Arabica) and Robusta coffee leaves (L.

Coffea Canephora) by testing the methanolic extracts to compare the anti-oxidant and anti-

bacterial property of both leaves. They concluded that Robusta coffee leaves (L. Coffea

Canephora) showed more anti-bacterial and anti-oxidant property than the Arabica coffee leaves

(L. Coffea Arabica) which also implies that Robusta coffee leaves contains more flavonoid,

phenolic acids and tannins than Arabica coffee leaves.

Isolated flavonoids were examined by In-vitro α-glucosidase enzyme inhibition activity

in the research of Shah, S. (2019), to determine the possible in-vitro anti-diabetic potentials of

these flavonoids. Moreover, Salvigenin, eupatilin and cirsilineol were observed in in-vivo anti-

diabetic activity to test the toxicological effects using mice. Results showed that flavonoids have

high natural products that fights against diabetes mellitus. (Ahmed et al., 2018)

The hyperglycemia test on diabetes-induced mice conducted by Riany, H. (2019)

identified the possible effects of hyperglycemia and the central focus of the researcher is on

habitual consuming of coffee which can overcome diabetes. The study made use of five

experimental treatment namely: metformin, Arabica, robust, liberica and aquades) with three

replications. Results showed that Arabica, robust and liberica coffee can reduce blood sugar in

hyperglycemia mice in 16 days.

various purposes such as therapeutic agents and raw materials that can be transformed to semi

synthetic compounds. Similarly, robusta coffee leaves contains anti-diabetic compounds such as

flavonoids, phenolic acids and tannins that inhibits hyperglycemia and can be a product for anti-

oxidant and anti-bacterial. Some of the medicines used nowadays came from plants that have

been screened and examined to treat diseases. The literature review showed the beneficial effect

of coffee primarily robusta coffee in lowering blood glucose in the body. Coffee beans are

processed to produce different types of coffee blends. However, the leaves are often left behind

which was proven that have anti-diabetic properties. Preceding studies showed that coffee in

treating diabetes mellitus was scarcely studied and has never been compared. Thus, this study

will focus in robusta coffee leaves to determine the hypoglycemic properties on hyperglycemic

mice.

The coffee leaves are a part of the coffee plant that is considered waste and has not been used

well. The various phytochemicals group in the coffee leaves such as alkaloids, flavonoids,

terpenes, tannins, xanthones, phenolic acids, phytosterol, amino acid, and carotenoids, contribute

to the antioxidant, anti-inflammatory, anti-hypertensive, antibacterial, anti-fungi activities.

Because of the presence of beneficial phytochemicals in coffee leaves, there is growing interest

in the application of coffee leaves such as tea drinks, functional dietary supplements, and

ethnomedicine. The coffee plant is a plant that contains high antioxidants. Antioxidants are

molecules that inhibit free radical reactions and inhibit cellular damage. Oxidation reactions can

produce free radicals. When the chain reaction occurs in a cell, it can cause damage or death to

the cell. These low- molecular-weight antioxidants can safely interact with free radicals and

terminate the chain reaction before vital molecules are damaged. Some antioxidants are produced
during normal metabolism in the body. The presence of antioxidants in the body is already

available in the form of enzymes known as endogenous antioxidants, but the amount is often not

balanced with the number of free radicals that enter the body so that exogenous antioxidants are

needed. One area that has coffee plants is Alor Island, NTT, with robusta coffee species. Based

on the data obtained, in addition to using coffee beans for drinks, some people in the area use

yellowed coffee leaves to be used as tea drinks.

According to Chen (2018), the antioxidant activity of coffee grown in one area and another has

different characteristics according to the age of the plant used, harvest time, the environment in

which was grown, or the ecology of the highlands. Also, the phytochemical profile of coffee

leaves varies according to plant species, cultivar, growing region, climate, stage of development,

and processing methods affect the phytochemical composition and bioactivity of coffee leaves.

Therefore, it is necessary to test the best phytochemical and antioxidant activity on Robusta

coffee leaves as seen based on differences in the ecology of the highlands on Alor Island - NTT,

which is not widely known to the public and no research has been carried out in the area.

CHAPTER 3

METHODOLOGY

Collection And Preparation

The robusta coffee leaves will be collected at Dipantan Nagtipunan, Quirino using

scissors and hands. The leaves of robusta coffee will be collected and will be placed in a clean

garbage bag that will be taken home. To prepare for air drying.
Air Drying Method

After collecting the plant samples the researchers then prepare the leaves for air

drying. The researchers will lay the leaves in a space where the temperature is at room

temperature which is approximately 35 to 40 degree Celsius. The leaves where left to dry for two

weeks without any exposure to sunlight. After two weeks of air drying the leaves where then

brought to school in a sterile container to be powdered using a blender at the Senior High School

classroom in Maddela Comprehensive High School.

Ethanolic Crude Extraction

The leaves will be air-dried under a shade, in room temperature. Once the leaves turn

crisp, the researchers will turn the dried leaves into powder. The pulverized dried leaves of the

robusta plant was placed into beaker and will be submerged into volume ethyl alcohol. The

beaker will be covered with aluminum foil and the plant material was soaked for 48 hours. After

48 hours, the soaked pulverized leaves will be filtered using a Buchner funnel. The residue will

be discarded and the filtrate collected into another container for filtering. After filtering, the

solution will be water bathed in 50ºC for 8 hours until the solution became paste-like. The paste-

like remnant in the beaker will be the ethanolic crude extract of C. canephora leaves and the

container will be labeled with Coffea canephora crude extract. Said container will be covered

with an aluminum foil and will be stored in a cold temperature to prevent any fungal growth

(Guevara, 2005).
Phytochemical Screening

After extraction, the extracted robusta coffee leaves (L. Coffea canephora) will be

applied by using fine capillary cylinders on TLC (Thin-layer chromatography) plates and will be

placed in a TLC chamber. After sometime in the TLC chamber the developed TLC plates will be

taken out of the chamber to be air dried at 254 nm and 355 nm under ultra-violet UV light.

(Sharma & Paliwal, 2013).

Animal Handling and Preparation of Alloxan-induced Mice

The researchers will wear protective gadgets such as mask, gloves, and lab gowns for

proper animal handling and safety. The laboratory mice will be obtained from the pet shop and

will be checked regularly. The mice will be contained at the Center for Natural Sciences Animal

House from the day these will be obtained until they will be acclimatized. Alloxan will be

administrated depending on the body mass of the mice in order to induce high blood glucose

levels. The blood glucose of the mice will be tested for hyperglycemia prior to the treatment of

ethanolic crude extracted robusta leaves. Five mice will be treated using a gavage with the

ethanolic crude extract; five mice will be given water as negative control; and five will be treated

with Glibenclamide as the positive control.

Induction of Diabetes

Venkatesh et al., 2003 stated that diabetes will be induced in the mice by administering alloxan

monohydrate into the 24 hours fasted rats. Blood samples will be collected after 24 hours and

blood glucose levels will be estimated. Blood glucose of experimental mice beyond 150 mg/dL
will be selected. Mice exceeding 150 mg/dL will be considered diabetic. The blood glucose

levels will be monitored for three days.

Toxicological and Pharmaco- toxicological Effects Assay

Test organisms should not exceed a maximum volume of the test drug of 2 mL. The

concentration will be identified through body mass of the mice. According to Earnest and

Ajaghaku (2014) the standard dose for extracted plant is 150 mg.

Hypoglycemic Assay

Procedures taken form Njogu et.al., (2016) indicated the methods for

Hypopolycemic testing. The mice will be injected with Alloxan to become diabetic. Blood

samples will be taken from diabetic mice prior to treatment and feeding. Mice will be tested and

treated for 10 consecutive days. The tail will be sterilized using 70% ethanol. “Milking” the tail

from the caudal part toward the tip will be applied to collect the blood samples in mice.

Glucometer will be used to determine blood glucose level. Hyperglycemic mice have 200

mg/dL.

Treatment of Data

The result will be subjected to statistical analysis. The comparison of both negative

and positive control with the Alloxan- induced mice will use One-Way Analysis of Variance

(ANOVA) and blood glucose level will be identified through Mean ± Standard deviation (SD).

Formulation of Capsule Drug

The final product will be an oral liquid solution that will be placed in a bottle and

then decorated to improve its visual appeal.

The final product will be presented to the research adviser and also to the research

department of Maddela Comprehensive High School.