San Pedro College of Davao

12 C. Guzman Street, 8000 Davao City, Philippines

HYPOGLYCEMIC ACTIVITY OF THE PULP AND PEELS OF

RED DRAGON FRUIT (Hylocerus costaricensis) EXTRACTS: AN IN VITRO STUDY

A Thesis Proposal Presented to

The Faculty of the Pharmacy Department
San Pedro College, Davao City

In Partial Fulfillment
of the Requirements for the Degree of
BACHELOR OF SCIENCE IN PHARMACY

Proponents:
SOGUILON, Selina Grace M.
ABRENICA, Frances Faye R.
ALQUIZA, Trinity Grace C.
ARANETA, Aires R.
MONTERO, Jamie M.
MULA, Karen Joy S.
RODA, Jona Dave Garyn R.
TESIORNA, Jian Kristiene H.

BS Pharmacy – 3A

August 2019
 Chapter I
INTRODUCTION

Background of the Study

According to WHO (2019), diabetes mellitus is defined as a chronic disease caused by

inherited and/or acquired deficiency in production of insulin by the pancreas, or by the
ineffectiveness of insulin produced. This deficiency results from increased concentrations of
glucose in the blood which may lead to damage of many body systems, blood vessels and nerves
in particular. Of the two principle forms of diabetes, the study shall focus on Type 2 diabetes, also
known as non-insulin dependent diabetes mellitus (NIDDM). In this form, the body becomes
unable to respond properly to the action of insulin produced by the pancreas. It commonly occurs
among adults, with a present approximate of 425 million people being affected worldwide. In the
Western Pacific region, the number of cases can rise from 159 million to 183 million by the year
2045. This can result from population growth, unhealthy diets, obesity and sedentary lifestyle. In
the Philippines, diabetes cases were over 3,721,900 in 2017 (International Diabetes Federation,
2019).

There are ways to prevent and manage the diabetes mellitus. One of which is through non-
pharmacological treatment of diet and physical activity. It is a must to follow a healthy meal plan
and being active in order to manage one’s blood glucose level. Food groups recommended for
people with high blood glucose levels include vegetables, fruits, grains, protein, and dairy.
Consequently, upon the discovery of plants and plant products in the aid of disease treatment,
dragon fruit (Hylocereus spp.) has been one of the many fruits made popular of having health
benefits. Reports have shown that dragon fruit is rich in proline, potassium, magnesium, calcium,
sodium, and also contains iron, zinc, and copper (Khalili 2006; Le Bellec et al. 2006). Red dragon
fruit (Hylocereus costaricensis) is also high in antioxidant activity compared to other fruits and
vegetables (Mahattanatawee et al. 2006). These results suggest that the fruit has a potential in
preventing risk of acquiring non-communicable diseases, such as cardiovascular diseases,
hypertension, diabetes mellitus, and anemia. In addition, dragon fruit is also rich in fibers, vitamin
C, minerals and phytoalbumins which are highly valued for their antioxidant properties. It helps

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the digestive process, prevent colon cancer and diabetes, neutralize toxic substances such as heavy
metal, reduce cholesterol levels and high blood pressure and, if consumed regularly, can help
against asthma and cough.

Having this information, our study seeks to shed light on the hypoglycemic activity of the
Red Dragon Fruit (Hylocereus costaricensis) utilizing its pulp and peel extracts, which can
contribute to the management of blood glucose levels and potential treatment of diabetes mellitus
type 2.

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Review of Related Literature

Hylocereus costaricensis, Red Dragon Fruit

Kingdom: Plantae

Subkingdom: Tracheobionta

Superdivision: Spermatophyta

Division: Magnoliophyte

Class: Magnoliopsida

Subclass: Caryophyllidae

Order: Caryophyllales

Family: Cactaceae

Genus: Hylocereus

Species: H. costaricensis

Graphical Representation of H. costaricensis

(retrieved from: http://www.hear.org/species/hylocereus_costaricensis/)

Dragon fruit (Hylocereus spp.) is a plant genus that originating from South America
(Bauer, 2003). It has a unique flower that it was introduced as an ornamental plant. It is cultivated
for the sole purpose of consumption for being known for its complex nutritional contents (Krisanto,

3
2003). The dragon fruit is a cactus fruit perhaps most common as the red dragon fruit. Sweet
pitayas or the dragon fruits have a creamy pulp and a delicate aroma. To prepare a dragon fruit for
consumption, the fruit is cut open to expose the flesh. The fruit's texture is compared to that of the
kiwi fruit due to the presence of black, crunchy seeds. The flesh, which is eaten raw, is mildly
sweet and low in calories. The seeds are eaten together with the flesh (Abdul Azis Ariffin et al.,
2008). Red dragon fruit have high antioxidant activity being compared to the other fruits
(Mahattanatawee et al., 2006). Dragon fruit is one of the medicinal plants that reported to control
blood glucose level which it has a potential in the treatment of diabetes mellitus (Nalinee Poolsup,
et al., 2017).

A meta-analysis that appeared in the journal Plos One looks at the effects of consuming
dragon fruit on blood glucose in those with prediabetes and those with type 2 diabetes. According
to the paper, previous animal studies had shown a potential link between dragon fruit consumption
and better control of diabetes. This is because dragon fruit encourages the growth of pancreatic
cells that produce insulin. (Nall, R. 2019, March 8).

Polyphenols

Polyphenols are mainly natural chemicals containing multiples of phenol structures. It is

said that these natural chemical “may influence glycemia and type 2 diabetes (T2D) through
different mechanisms, such as promoting the uptake of glucose in tissues, and therefore improving
insulin sensitivity” (Guasch-Ferré).

One study showed significant evidence from epidemiological investigations that dietary
polyphenols might manage and prevent type 2 diabetes (T2D). Polyphenols from coffee, guava
tea, whortleberry, olive oil, propolis, chocolate, red wine, grape seed, and cocoa have been reported
to show anti-diabetic effects in T2D patients through increasing glucose metabolism, improving
vascular function as well as reducing insulin resistance and HbA1c level (Hui Cao, et al., 2018).

In addition, “…Based on several in vitro animal models and some human studies,
polyphenols may play a role in many metabolic processes. They can modulate carbohydrate and
lipid metabolism, attenuate hyperglycemia, dyslipidemia and insulin resistance, improve adipose
tissue metabolism, and alleviate oxidative stress and stress-sensitive signaling pathways and

4
inflammatory processes (Testa).” (Retrived from: https://www.ncbi.nlm.nih.gov/pmc/articles/
PMC4882722/)

Flavanoids

Flavanoids are class of secondary metabolites in plants and fungi. Chemically, flavonoids
have the general structure of a 15-carbon skeleton, which consists of two phenyl rings (A and B)
and a heterocyclic ring (C). They are divided into flavonoids/bioflavonoids, isoflavanoids, and
neoflavanoids.

According to a Research Article (BioMed Research International Volume 2017, article ID

8386065) the best described pharmacological property of flavonoids is their capacity to act as
potent antioxidant that has been reported to play an important role in the alleviation of diabted
mellitus. The protective effects of flavonoids in biological systems are ascribed to their capacity
to transfer hydrogen or electrons free radical, activate antioxidant enzymes, chelate metal catalyst,
reduce α-tocopherol radicals, and inhibit oxidases. Relative Oxygen Species (ROS) are capable of
oxidizing cellular proteins, nucleic acids, and lipids. Studies and clinical evidences have shown
that the generation of ROS increases in both types of diabetes and that the onset of diabetes in
closely associated with oxidative stress mainly through oxidation, nonenzymatic protein glycation,
and oxidative degradation of glycated proteins. Elevation of ROS such as mitochondrial
superoxide in endothelial cell and endoplasmic reticulum stress followed by reduced antioxidant
defense mechanism provokes cellular and enzyme damage, and lipid peroxidation which
subsequently lead to the development and progression of insulin resistance and hyperglycemia.
Studies have also shown that antioxidants are able to improve insulin action. Since the early 1980s,
the potential effects of flavonoids in diabetes mellitus have been studied eminently for type 2
compared to that type 1. Flavonoids showing potent antioxidant activity have suggested being
beneficial in the management of diabetes mellitus. The ability of antioxidants to protect against
the deleterious effects of hyperglycemia and also to enhance glucose metabolism and uptake
should be considered as an alternative in diabetes mellitus treatment. Being a radical scavenger,
flavonoids can effectively prevent and/or manage type 2 diabetes mellitus.

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Betalains

Betalains are indole-derived pigments “characteristically red to violet (betacyanins) and

yellow to orange (betaxanthyns) present in different plant parts of members of the Caryophyllales,
including the cactus family” (Anderson, 2001). (Retrieved from: https://www.sciencedirect.com/
topics/agricultural-and-biological-sciences/betalains).

A study by Choo W.S (2018) betalains are plant derivative natural pigments that are
presently gaining popularity for uses a natural colorant in the food industry. Betalains are from
four main sources such as red beet, amaranth, prickly pear, and red pitahaya. They are found in a
much smaller group of plants. Our study focuses on the red dragon fruit or red pitahaya that
contains betalains and is well known for its aesthetically pleasing deep purple color pulp with
numerous small soft seeds. A broad definition of antioxidant is “any substrate that, when at low
concentrations compared to those of an oxidizable substrate, significantly delays or prevents
oxidation of the substrate” (Halliwell B 1990). The term “oxidizable substrate” includes almost
everything found in foods and in living tissues including proteins, lipids, carbohydrates, and DNA.
The bioavailabilty of betalains is at least as high as flavonoids, which are well-accepted natural
antioxidants. Betalains, as natural antioxidants, may provide protection against oxidative stress-
related disorders.

Diabetes Mellitus

Diabetes is one of the common diseases that characterized by too much glucose in the
blood. It is a multifactorial metabolic disorder. It occurs when the pancreas is not properly producing
insulin. When insulin is not enough there are risk in health including: heart, kidney and nerve
disease, impotence in male, and vision problems, that are some of the complications of diabetes
(William Isley et al., 2005).

Type 2 diabetes mellitus

Type 2 diabetes is a serious and common chronic disease resulting from a complex
inheritance-environment interaction along with other risk factors. Type 2 diabetes and its
complications constitute a major worldwide public health problem, affecting almost all

6
 populations with high rates of diabetes-related morbidity and mortality. Non-insulin-
dependent diabetes mellitus (type 2 diabetes mellitus, T2DM). T2DM is the most common
form of DM, which accounts for 90% to 95% of all diabetic patients (Yanling Wu et al.,
2014).

Hypoglycemic agents

For the management of type 2 diabetes mellitus, various oral hypoglycemic agents are
available, such as dipeptidyl peptidase‐4 (DPP‐4) inhibitors, α‐glucosidase inhibitors (αGI),
biguanides, sulfonylureas, thiazolidine insulin sensitizers and glinides (Hitoshi Ishii et al., 2017).
The choice of medication should depend on individual patient factors while strictly adhering to
clinical guidelines. Physicians can be expected to choose hypoglycemic medications in
consideration of factors that influence the overall health and clinical outcome of each patient, with
particular concern regarding cardiovascular diseases (Kazuya Fujihara et al.,2017).

Acarbose

It is an inhibitor of alpha glucosidase that retards the digestion and absorption of

carbohydrates in the small intestine and hence reduces the increase in blood-glucose
concentrations after a carbohydrate load. It is given orally to non-insulin dependent
diabetes mellitus patients where diet modification or oral hypoglycemic agents do not
control their condition.

A safe and effective medication for type 2 diabetes, acarbose works by slowing the
action of certain chemicals that break down food to release glucose (sugar) into your blood.
Slowing food digestion helps keep blood glucose from rising very high after meals. it
lowers the postprandial increase in blood glucose levels in NIDDM patients. Its medical
effect is based on a decreased release of glucose from starch- and sucrose-containing foods
in the human intestine, which leads to reduced levels of blood glucose and serum insulin
(Wendler S et al., 2014).

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α-Amylase

Alpha amylases catalyze the hydrolysis of α-1,4-glucosidic linkages in starch, glycogen,

and various oligosaccharides and further degraded to absorbable monosaccharides by α-
glucosidase, readily available for the intestinal absorption.

One of the therapeutic approaches is to delay the absorption of glucose by carbohydrate

hydrolyzing enzymes, α-amylases and α-glucosidase inhibition, in the digestive tract of
humans (Honda, 1990)

α-Glucosidase

Alpha glucosidase and alpha amylase are the important enzymes involved in the digestion
of carbohydrates. Alpha Amylase is involved in the breakdown of long chain carbohydrates and
alpha glucosidase breaks down starch and disaccharides to glucose. They serve as the major
digestive enzymes and help in intestinal absorption. Alpha amylase and glucosidase inhibitors are
the potential targets in the development of lead compounds for the treatment of diabetes.
(Subramanian R, 2008)

The goal of diabetic patient treatment is to maintain near normal levels of glycemic control,
in both the fasting and postprandial states. Many natural resources have been investigated with
respect to the suppression of glucose production from carbohydrates in the gut or glucose
absorption from the intestine (T. Matsui, 2007).

8
Theoretical Framework

The pitaya roja or red dragon fruit is known for containing betalains, red and yellow indole-
derived pigment antioxidants that may prevent against the oxidation of low-density lipoproteins,
commonly extracted from Beta vulgaris as a supplement as well as a variety of flavonoids and
phenolic agents. Containing these antioxidants may reduce oxidative species as well as “improve
insulin resistance through decreasing fibroblast growth factor–21 (FGF-21) expression and
increasing level of FGF-21 related genes (Klb, FGFR2, Egr1, and cFos) in the liver (Song et.al.,
2015).” Tropical regions such as the Philippines can thereby utilize the dragon fruit as an ideal
native source for extracting these powerful antioxidants.

Research pertaining to the plant of the same genus, the white dragon fruit, Hylocereus
undatus, outlined “improved insulin resistance, hepatic steatosis and adipose hypertrophy, with no
influence on body weight gain in mice”, when extracted for betacyanins in the peel and applied to
high-fat-diet-fed mice (Song et.al., 2015). In addition, “preventive effects were observed on
histopathological picture of pancreatic β cells in alloxan induced diabetic rats by reducing reactive
oxidative species” from these same antioxidant species (Poolsup, et al., 2017).

For these reasons, the researchers have chosen to study Hylocereus costaricensis pulp and
peel extracts for potential application in anti-diabetic medicine, especially in tropical regions such
as the Philippines. The researchers aim to test whether or not the betalains or another component
of the red dragon fruit pulp or peels may also exhibit alpha-glucosidase and/or alpha-amylase
inhibitory capacity, which would aid in the regulation of carbohydrate digestion and reduction of
glucose absorption rate.

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 α-GLUCOSIDASE ENZYME INHIBITION

Mechanism of action of alpha-glucosidase inhibitors

Patil, Prasad & Mandal, Surajit & Kumar Tomar, Sudhir & Anand, Santosh. (2015)

(AG: alpha glucosidase, AGI: alpha glucosidase inhibitor)

“The development of the alpha-glucosidase inhibitor acarbose provides a new approach in

the management of diabetes. By competitive and reversible inhibition of intestinal alpha-
glucosidases, acarbose delays carbohydrate digestion, prolongs the overall carbohydrate digestion
time, and thus reduces the rate of glucose absorption. After oral administration of acarbose, the
postprandial rise in blood glucose is dose-dependently decreased, and glucose-induced insulin
secretion is attenuated. Because of diminished postprandial hyperglycemia and hyper-insulinemia
by acarbose, the triglyceride uptake into adipose tissue, hepatic lipogenesis, and triglyceride
content are reduced. Therefore, acarbose treatment not only flattens postprandial glycemia, due to
the primary and secondary pharmacodynamic effects, but also ameliorates the metabolic state in
general” (Bischoff 1995).

“Glucosidase inhibitors are indicated as adjuncts to diet and exercise in type 2 diabetic
patients not reaching glycemic targets. They can also be used in combination with other oral
antidiabetic agents or insulin” (Goodman, 2011).

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 α-AMYLASE ENZYME INHIBITION

Mechanism of α-Amylase Carbohydrate Digestion

(http://biology.kenyon.edu/BMB/jsmol2015/3BLKAmylase/index4.html)

Alpha-amylase, (α-amylase) is a protein enzyme that hydrolyzes alpha bonds of large,

alpha-linked polysaccharides. It is one of the major secretory products of the pancreas (about 5-
6%) and salivary glands. It plays a role in the digestion of starch and glycogen, and also found in
microorganisms, plants, and higher organisms. Moreover, the amylase presents a three-
dimensional structure capable of binding to substrate and, by the action of highly specific catalytic
groups, promote the breakage of glycoside links (de Sales, et.al., 2011). Long-chain carbohydrates
(amylose and amylopectin strands) can be broken down by α-amylase and give maltotriose and
maltose from amylose, or maltose, glucose and limit dextrin from amylopectin.

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Conceptual Framework

Dependent Variable (Y)

Independent Variable (X)
Hypoglycemic Activity
 Pulp and Peel Extracts of
Dragon Fruit (Hylocereus  Percent inhibition of
costaricensis) α–Amylase Enzyme
 Acarbose (Positive control)  Percent inhibition of
α–Glucosidase Enzyme

By extracting the peel and pulp from Hylocereus costaricensus grown in Davao region and
comparing each to the control (acarbose) based on percent inhibition of alpha-glucosidase and
alpha-amylase, the researchers can evaluate whether or not the pulp and/or peel of Philippine
Hylocereus consaricensis can be utilized as a source for an adjunct treatment for diabetes for its
enzyme-inhibition alongside its previously exhibited benefits.

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Statement of the Problem

The study aims to determine if there is a significant difference in the hypoglycemic activity of the
red dragon fruit (Hylocerus costaricensis) pulp and peel extract, α-Amylase and α-Glucosidase
inhibitions in particular, and that of the positive control, Acarbose.

Specifically, it seeks to answer the following questions:

1. What are the physicochemical characteristics of the red dragon fruit pulp and peel extracts?
a. Moisture Content Determination
b. Organoleptic Test
c. pH Determination
2. How much is the Phenolic and Flavonoid Content of the Ethanolic Extract of Hylocerus
costaricensis pulp and peel extract using UV-Vis Spectrophotometry?
3. Is there a significant difference in the in vitro hypoglycemic activity of Red Dragon Fruit
(Hylocerus costaricensis) Pulp Extract in the following assay:
a. α-Amylase and Acarbose
b. α-Glucosidase and Acarbose
4. Is there a significant difference in the in vitro hypoglycemic activity of Red Dragon Fruit
(Hylocerus costaricensis) Peel Extract in the following assay:
a. α-Amylase and Acarbose
b. α-Glucosidase and Acarbose
5. Is there a significant relationship in the in vitro hypoglycemic activity of red dragon fruit
(Hylocerus costaricensis) pulp and peel extracts and in the following assay:
a. α-Amylase
b. α-Glucosidase

Null Hypothesis

There is no significant difference between the α-Amylase and α-Glucosidase inhibitions

of Acarbose and Red Dragon Fruit (Hylocerus costaricensis) Pulp Extract based on percent
inhibition.

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 There is no significant difference between the α-Amylase and α-Glucosidase inhibitions
of Acarbose and Red Dragon Fruit (Hylocerus costaricensis) Peel Extract based on percent
inhibition.

Scope and Limitations

This is only limited to the utilization of the pulp and peel extracts of red dragon fruit (H.
costaricensis). The study is centered to the analysis of the in vitro hypoglycemic activity of the
pulp and peel extracts of red dragon fruit (H. costaricensis) extracts. The fruits will be obtained
from Digos City, Davao del Sur. The span to accomplish the study is about 5 months.

Significance of the Study

The results of the study will be beneficial to the following:
Future Researchers. The outcome of this study can provide information and serve as a
reference to guide future researchers who will undertake similar studies.
Public Health Pharmacists. As health advocates and educators, public health pharmacists
can offer information about diseases, specific to diabetes mellitus type 2, and treatment alternatives
with red dragon fruit in mind.
Community. The outcome of this study can well benefit the society in opening
opportunities for development of new drug that is affordable, accessible, safe and effective for the
remedy of hyperglycemia and/or treatment for diabetes.

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 Chapter 2

METHODOLOGY

This chapter provides the details on how the study shall be conducted. It comprises the
research design, materials, setting, and procedures. It shall also include the method of data
collection, limitations, statistical treatment, and analysis to be applied to characterize and
determine the in vitro α–Amylase and α–Glucosidase inhibitory activities of red dragon fruit (H.
costaricensis) feel and peel extracts.

Research Design

In this study, the researchers will be conducting an In Vitro Enzyme Inhibitory Study using
α-amylase and α-glucosidase enzyme kits to evaluate the hypoglycemic activity of the pulp and
peel extracts of red dragon fruit (Hylocereus costaricensis). Acarbose is the chosen positive control
for this study. A microplate will be used to obtain statistical result for the enzyme inhibition
activity of the H. costaricensis extract. The plant extract will also undergo Physical (Organoleptic
properties, pH, Moisture content), Phytochemical: Quantitative and Qualitative (TLC for
Flavonoid Content, Phenol Content, and Quercetin Content). Toxicological (limit test for Mercury
and Lead), and Microbiological tests (limit test for Staphylococcus aureus and Escherichia coli).

Chemicals and Reagents

Acarbose (1000ppm), 4-Nitrophenyl-α-D-glucopyranoside and α-glucosidase enzyme

(Maltase from yeast), Phosphate buffer (pH 6.6) and 10% Folin-Ciocalteu solution will be
purchased outside the premises of San Pedro College. The other chemicals and reagents that will
be utilized in the experiment are already available.

Plant Material

The Red Dragon Fruit (Hylocereus costaricensis) will be obtained from a farm in Digos
City, Davao del Sur.

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Research Setting

Extraction, Physical test, Phytochemical Screening and In Vitro α-amylase will be

conducted at the Science Laboratory of San Pedro College. For the microplate reading, assay of α-
glucosidase inhibition, Microbiological Test, Toxicological limit test will be performed outside
the Science Laboratory of San Pedro College.

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Study Flow

Plant Collection Plant Authentication

Lyophilization Process

Pulverized pulp and peel Physical: Moisture

content

Plant Extraction by Maceration

95% Ethanol Extract

Characterization of the Dragon In Vitro test

Fruit pulp and peel extracts

Physical: Organoleptic pH

α -Amylase α -Glucosidase Acarbose

Inhibitory Inhibitory (Control)
Phytochemical screening: Assay Assay
(Quantitative and Qualitative
Analysis): TLC- Flavonoid,
Phenol Content, Iodoform Content

Microbiological: limit test for S.

aureus and E. coli

Toxicological: Limit test for

mercury and lead

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Data Gathering Procedure

A. Collection
The Hylocereus costaricensis fruit will be collected at Digos City, Davao del Sur. It will
undergo garbling, thereafter the pulp and peel will be lyophilized. After the lyophilizing
process, the pulp and peel will be pulverized by the use of grinder and weighed.

B. Extraction
An adequate amount of the pulverized pulp and peel samples will be macerated. The plant
samples will be soaked with 95% ethanol for 24 hours. After which, the plant samples will
be filtered using a funnel with a muslin cloth and filter paper and the plant residue will be
discarded. The volume of the filtered plant extract will be transferred into a sterile container
and stored in a cold place about 15 degrees Celsius.

Calculate the plant extract concentration using the formula:

Percentage (%) yield = Weight of the viscous extract x 100

Weight of the pulverized leaves plant sample

C. Moisture Content Determination of the Pulverized Pulp and Peel

Tare the moisture content analyzer. Place test sample on the aluminum plate over the
moisture content analyzer. Start moisture content determination and note the percentage of
moisture content.

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D. Physical tests

1. Organoleptic test
Place the plant extract in an evaporating dish. The color, odor, and taste of the extract
will also be observed.

2. pH determination
A pH meter probe will be dipped into the Hylocereus costaricensis pulp and peel
extract. The pH value displayed in the pH meter screen is recorded as its pH value.

E. Phytochemical Screening: Qualitative and Quantitative Analyses

I. Preliminary Test: Thin Layer Chromatography Analysis

1. Prepare the test solution. Add 30mL of 95% ethanol to 3g of powdered of the
ethanolic extract. Heat on a water bath for 15 minutes. Allow to cool. Filter.
2. Prepare mobile phase. Glacial Acetic Acid, Distilled water, Propanol, Butanol
(20:20:25:35 V/V/V/V).
3. Draw a thin mark at the bottom of the plate with a pencil.
4. Dipped the capillary tube into the liquid sample. The plate will spot just below
the thin mark. Air-dry the spots between applications.
5. Pour the mobile phase into the TLC Chamber to a level of few centimeters
above the chamber bottom. Place the filter paper moistened in mobile phase on
the inner wall of the chamber to maintain equal humidity in the entire chamber
and there avoids edge effect.
6. Place the prepared plate with sample spotting in the TLC chamber such that the
side of the plate with sample line is toward the mobile phase. Immerse the plate
such that the sample spots are well above the mobile phase but not immerse in
the solvent. Close the chamber with a lid.
7. Give sufficient time for the development of the spots.

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 8. Remove the plate and allow to dry.
9. Spray the sample spots with Potassium Ferricyanide-ferric chloride to enhance
the appearance of the spots and then visualize in suitable UV light chamber.

To solve the Retention Factor, use the formula below:

Retention Factor = Distance Traveled by the Solute

Distance Traveled by the Solvent

II. Determination of Quercetin Content from Red Dragon Fruit Pulp and Peel Extracts

1. To make 1mg/mL concentration of solution, weigh 100mg quercetin and mix with
100mL 95% ethanol.
2. To make 0.25mg/mL concentration of solution, get 50 mL from the 1mg/mL solution
and mix with 50 mL 95% ethanol.
3. To make 0.50mg/mL concentration of solution, get 50 mL from the 0.25mg/mL
solution and mix with 50 mL 95% ethanol.
4. To make 0.125mg/mL concentration of solution, get 50 mL from 0.50mg/mL solution
and mix with 50 mL 95% ethanol.
5. After serial dilution, use spectrophotometer to determine the absorbance of each diluted
solutions.

III. Determination of Phenolic Content from Red Dragon Fruit Pulp and Peel Extracts

1. Mix 0.2 mL of test solution (1mg/mL) with 1 mL of 10% Folin-Ciocalteu solution and
0.9 mL of 7.5% sodium carbonate.
2. Incubate the mixture for 1 hour at room temperature.
3. Measure the absorbance and convert to phenolic contents according to the calibration
curve of Gallic Acid.
4. Total phenolic content of plant material is express as mg Gallic Acid equivalents/mL
Extracts.

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 IV. Iodoform Test

1. Four drops of plant sample into a test tube.

2. Add 0.5 mL of distilled water.
3. Add 0.25 mL of 6M NaOH and 0.25 mL distilled water.
4. Add 5 drops of KI3.
5. Record the data after 10 minutes.
6. A yellow cloudy bottom precipitate indicates a positive result.

F. Toxicological Test

The Red Dragon Fruit Pulp and Peel Extracts will undergo limit test for heavy metals (Hg
and Pb) at the Science Resource Center of the University of the Immaculate Concepcion.

G. Microbiological Test

The Red Dragon Fruit Pulp and Peel Extracts will be submitted to the Science Resource
Center of the University of Immaculate Conception for the limit test of Staphylococcus aureus and
Escherichia coli.

H. In Vitro Hypoglycemic Tests

α – amylase Enzyme Inhibition Assay

The determination of α – amylase inhibition will be carried out by quantifying the reducing
sugar that will liberate during assay conditions. This assay will be carried out using a modified
procedure of McCue and Shetty (2004).

1. A total of 250 µL of extract (triplicates of 1 mg/ml, 0.5 mg/ml, 0.25 mg/ml, and 0.125
mg/ml concentrations) will be placed in a tube and 250 µL of 0.02 M sodium phosphate
buffer (pH 6.9) that contains α – amylase solution will be added.

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 2. This solution will be pre-incubated at 25°C for 10 minutes, after which 250 µL of 1%
starch solution in 0.02 M sodium phosphate buffer (pH 6.9) will be added at time
intervals and will be further incubated at 25°C for 10 minutes.
3. The reactions will be terminated after incubation by adding 500 µL of dinitrosalicylic
acid (DNS) reagent.
4. The tubes will be incubated in boiling water for 5 minutes and will be cooled at room
temperature.
5. The reaction mixtures will be diluted with 5 ml distilled water and the absorbance will
be measured at 540 nm using spectrophotometer.

A control will be prepared using the same procedure replacing the extract with distilled
water. The α – amylase inhibitory activity will be calculated as percentage inhibition.

% Inhibition = (Abscontrol – Absextracts)/Abscontrol x 100

α – glucosidase Enzyme Inhibition Assay

The procedures of this study are the study of Richemae Grace R. Lebosada and Ivy L.
Librando. Various dilutions of 1 mg/ml, 0.5 mg/ml, 0.25 mg/ml, 0.125 mg/ml and 0.0625 mg/ml
will be obtained by preparing a stock solution of the Hylocereus costaricensis (Red Dragon Fruit)
pulp and peel extracts and the positive control, Acarbose (1000ppm). The dilutions of the Red
Dragon Fruit pulp and peel extracts, Acarbose, 4-Nitrophenyl- α –D-glucopyranoside and the α –
Glucosidase enzyme will be done with phosphate buffer (Ph 6.6)

1. 60 µL of Red Dragon Fruit pulp and peel extracts and 50 µL α –Glucosidase enzyme
(0.2U/mL) will be placed in a 96 well plates. In the Red Dragon Fruit pulp and peel
extracts, all 5 concentrations (1 mg/mL, 0.5 mg/mL, 0.25 mg/mL, 0.125 mg/mL and
0.0625 mg/mL) will be used.
2. The solution will be pre-incubated for 10 minutes at 25°C.
3. 50 µL of 4-Nitrophenyl- α –D-glucopyranoside solution will be added into the solution
to start the reaction.
4. The reacting mixture will be incubated for 20 minutes at 25°C.

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 5. The extent of the enzymatic hydrolysis will be determined by measuring the absorbance
at 405 nm with BIOBASE-EL10A Elisa Reader.

Percentage inhibition of enzyme by various test compounds will be calculated with the
formula:

% inhibition = (ABS of control – ABS of sample) x 100

ABS of Control

Disposal of Wastes, Chemicals, and Reagents

Proper disposal requires information about the properties of the waste. It is recommended that all
chemicals used or generated be identified and their source must be defined clearly on the container
in order to prevent cross-contamination of chemicals which will lead to lab accidents.

Method of Data Collection

For the collection of data for this study, two methods will be used; Observation and literature
analysis. Observation allows researchers to recognize and understand much more about what goes
on in complex real-world situations. The researcher will also use already existing literatures for
references as these documents served as backbone to support our results during the study.

Data Analysis

For the statistical analysis of the results, T-test for independent samples will be use in this study.
The significant p value will be set to 0.05. Using this method, the significant difference in percent
inhibition of the α-glucosidase and α-amylase activity of the Hylocereus costaricensis pulp and
peel extract will be evaluated.

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Limitation of the Study

Hylocereus costaricensis is the herbal that will be studied on. The pulp and peel of the plant will
be extracted by maceration with 95% ethanol after 7 days air-drying and pulverization. Further
safety procedures will be implemented by the analysis of these preparations with stability,
microbiological and toxicological testing. This study will consider the In Vitro Hypoglycemic
potential of the Red Dragon Fruit pulp and peel extract by inhibition of α-amylase and α-
glucosidase enzyme. The results will be evaluated using T-test for Independent samples.

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