Duration of Project: ___________________

SAINT MARY’S UNIVERSITY

Date Completed: _____________________
SENIOR HIGH SCHOOL
Signature of Program Leader:
RESEARCH DEPARTMENT
____________________________________
CAPSULE RESEARCH PROPOSAL
Project Research Leader: Marquez Queenie Mae Lucas
Family Name First Name Middle Name

Researchers: Klyde Ferdinand Bañaga

Mark Lenard dela Cruz
John Russel Miguel
Christopher Moses Latar
Ivy Calubaquib
Jhanezza May Gadiano
Caryle Joy Numbalin
Kamylle Paula Tan
Rayah Angela Viloria

Proposed Project Title: In- Vivo Assessment of Ponkan (Citrus reticulata) Vizcaya Peel Capsule
in Alleviating Diabetes Mellitus in Alloxan- Induced Mice

Project Cost: Php 4 000

Collaborators/Research Mr. Mark Balonquita
Adviser (State Affiliation):
Brief Summary/ Rationale There are approximately 422 million reported cases of people with
(Brief problem statement): DM worldwide. WHO (2016) reported that the major impediment to
successful treatment and decrease of complications and premature death of
Diabetes milletus (DM) patients is the lack of access to affordable insulin
and oral hypoglycemic agents. Diabetes milletus (DM) is a chronic,
progressive disease characterized by abnormal high level of blood sugar
caused by either insufficient supply of insulin in the body or generated
insulin does not work efficiently (International Diabetes Federation, 2018).
In the Philippines, DM is considered as one of the leading causes of
mortality in the country (DOH, 2017). This disease can lead to acute and
chronic complications and can increase the overall risk of dying prematurely
(Piemonte, 2019). Moreover, according to WHO (2018) almost 79% of DM
patients live in low and middle-income countries because essential synthetic
medicines in gaining control of this disease are frequently unavailable in
their area so continuous exploration for natural and cheap medicine with
high effectiveness, lesser side-effects and relatively low cost arises
(Vazquez et al., 2016).
According to Food and Agriculture Organization (2015), large
amount of citrus fruit peels is tossed as waste from fruit processing industry
yearly. The by-products derived from these citrus wastes are an economic
and renewable source for valuable compounds which can be used in
pharmaceutical, nutraceutical, food and cosmetic industries. Citrus fruits are
rich source of essential vitamins, minerals, fibers and array of
phytochemicals like flavonoids, alkaloids, coumarins, limonoids,
carotenoids, phenol acids and essential oils. These constituents show several
 bioactivities that is vital to human health, including anti-oxidative, anti-
inflammatory, anti-cancer, as well as cardiovascular protective effects, and
neuroprotective effects (Ly, X., 2015).
Citrus has been identified as one of the top high-value crops in the
Philippines, with the Cagayan Valley as one of the major citrus-producing
regions, to which Kasibu, Nueva Vizcaya, belongs (Antonio, 2015). One
type of citrus produced in the province is the Citrus reticulata, a type of
Mandarin that is locally known as Ponkan. In traditional medicine, products
derived from the peel have been used to treat numerous health problems like
gastrointestinal disturbances, respiratory ailments as agent for cough,
insomnia, stress disorders, epilepsy, and anxiety (Matesz, 2010). In several
studies, the Citrus reticulata exemplified hyperglycemic effect on alloxan-
induced rats and streptozotocin-induced rats. Moreover, most of the plant
subjects are from foreign countries and the citrus pulp is usually used.
With the increasing interest in finding natural antidiabetic
compounds encouraged by the increasing awareness of health-related risks
of synthetic compounds, Ponkan (Citrus reticulata) can be a promising
replacement of synthetic antidiabetic medicine in the market. This study
aims to explore the potential of Citrus reticulata peel in treating diabetes
mellitus.
Objective/s Main Objective:
(Identify specific
To evaluate effectiveness of Ponkan (Citrus reticulata) Vizcaya peel
objectives that will help
achieve the overall goal capsule in treating diabetes mellitus.
of the project. Specific
objectives should be
simple, measurable, Specific Objectives:
achievable, realistic, and  To obtain ethanol crude extract from the C. reticulata peel
 To find out the toxicological and pharmaco-toxicological level of the
time-bound (SMART))

crude extract
 To see the presence of phytochemical properties present in C.
reticulata peel
 To examine the antioxidant property of the extract
 To evaluate the hypoglycemic activity of the C. reticulata capsule in
alloxan-induced mice.
 To make a capsule from the C. reticulata peel

Significance This project will be significant and utmost importance to people

(State the issues the research aims suffering from diabetes milletus that cannot afford commercial medicines
to address, and the significance of
this research project in relation to and can experience side effects from commercial medicines. This will also
other researches on the same help the community to lessen food waste and gain organic medicine.
topic/s consider social and
technological impact of the research
project):
Literature Review Folk Medicine: A Review on the Bioactive Components and
(Comprehensive review of related Pharmacological Properties of Citrus Peels
literature and studies):

Citrus peels are considered an environmental waste in many

developing countries but in folk medicine, it was used in managing some
progressive diseases. The medicinal properties of the citrus peels also
promote as functional food because it is generally regarded as safe and
consumed in forms of candies, wines, infusions, and additives. Studies on
the structures for the antioxidant, anti-diabetic, cardioprotective,
neuroprotective and anticancer activities of the peel have established the
interaction with some key enzymes relevant to the management of diseases
(Ademosun et al., 2018)

Analysis of Bioactive Compounds in Seven Citrus Cultivars

Citrus fruits and derived products have a helpful impact on the

human health because of its nutritional and antioxidant properties.
Moreover, phenolic compounds and antioxidant activities in citrus peel have
been widely investigated and they have been found to possess antiallergenic,
anti-atherogenic, anti-inflammatory, antimicrobial, antioxidant,
antithrombotic, cardioprotective and vasodilatory effects. Its major
constituents include flavonoids, mainly polymethoxylated flavonoids,
terpenoids and other volatile oils. In this report, flavonoids identified in over
60 types of Citrus contains flavanone, flavone and flavonol compounds. In
addition, the highest concentrations of flavonoids in citrus fruit occur in the
peel (Bermejo & Cano, 2016).

Functional benefits of citrus fruits in the management of diabetes

Citrus flavonoids Hesperidin and Naringin, Naringenin, and

Polymethoxylated flavones are tested on Type 2 diabetic mouse, LDL-
receptor null mouse and Fructose- induced insulin resistant hamster
respectively. Beneficial from improving hyperlipidemia and hyperglycemia,
corrected many metabolic disturbances linked to insulin resistance, and can
ameliorate hyperglygeridemia. Promising therapeutic approach for
metabolic syndrome (Aruoma et al., 2015).

Antidiabetic Properties of Naringenin: A Citrus Fruit Polyphenol

Polyphenols have been established to have antioxidant properties

and possess a variety of other specific biological effects. Naringenin is found
mainly in citrus fruits. Naringenin has been studied for its pharmacological
effects, including antioxidant, anti-inflammatory, immunomodulatory,
hepatoprotective, nephroprotective, neuroprotective, anti-cancer, anti-
atherosclerotic, and anti-diabetic properties. The review is focused on the
anti-diabetic effects of naringenin and all existing in vitro and in vivo animal
and human studies are presented. A PubMed search was performed using
the key words: naringenin, skeletal muscle, adipocyte, hepatocyte, β-cell,
pancreas, streptozotocin (STZ), alloxan, high-fat diet, obesity and diabetes.
These key words were searched in multiple different combinations to ensure
that all existing in vitro and in vivo animal and human studies were included.
In Streptozotocin (STZ)-Induced Diabetes models, studies indicate
that naringenin administration in STZ-induced diabetic animals resulted in
restoration of blood glucose and lipid levels, increased GLUT4 translocation
in adipose tissue, and reduced diabetic nephropathy. In addition, naringenin
administration resulted in anti-inflammatory and antioxidant properties.
Also, in Alloxan-Induced Diabetes model, treatment of alloxan-induced
diabetic mice with naringenin (75 mg/kg b.w.) for 7 days, resulted in a
significant increase in body weight and an overall improvement of cellular
blood component levels compared to control diabetic mice. Hematological
and immunological blood parameters, including macrophage, leukocyte,
erythrocyte, hemoglobin, hematocrit, and platelets levels were all increased
following treatment with naringenin. Additionally, cholesterol levels in the
naringenin treated group were significantly reduced when compared to the
control diabetic mice. Overall, the survival of diabetic mice was
significantly improved by naringenin treatment. In a study by Sirovina et al.,
(2016) oral administration of naringenin (50 mg/kg/day) for 2 days to
alloxan-induced diabetic mice reduced lipid peroxidation levels in the liver
and kidney tissue.
Overall, all available in vitro and in vivo animal studies examining
the effects of naringenin indicate that it can reduce glucose adsorption by
the intestinal brush border, reduce renal glucose reabsorption, and increase
glucose uptake and utilization by muscle and fat tissues. In hepatocytes,
naringenin treatment reduces triglyceride production and gluconeogenesis
resulting in the attenuation of hyperglycemia and hyperlipidemia. Evidence
also shows a productive effect of naringenin in the pancreas, salvaging the
remaining β-cells and increasing their glucose-sensing capacity and
response to glucose. The limited human studies indicate that naringenin
administration Biomolecules can result in significant (micromolar range)
plasma naringenin levels. Furthermore, the limited epidemiologic evidence
indicates anti-diabetic effects of naringenin. Thus, the health benefits of
naringenin are widespread, and the low toxicity of the molecule makes it a
prime candidate for medicinal use against insulin resistance and T2DM (Den
& Tsiani, 2019).

A Comparative Study of Antidiabetic Activity of Hexane-Extract of Lemon

Peel (Limon citrus) and Glimepiride in Alloxan-Induced Diabetic Rats

Diabetes mellitus is the world’s largest endocrine disease of multiple

etiologies involving metabolic disorders of carbohydrate, fat and protein. At
present the treatment of diabetes are debatable due to unwanted side effects
the effectiveness of these compounds. Thus, with a disturbing rise in the
prevalence of the metabolic disease and associate health care cost, interest
in natural alternative or complementary therapies has grown. The present
study has been carried out to evaluate the effects of the hexane extract of C.
limon fasting blood glucose on alloxan induced diabetic rats. From the
gathered results, single dose of glimepiride has produced significant
reduction of blood glucose level compared to normal control group in 96 hrs
of the experiment. Also, the hexane extract of C. limon significantly reduce
blood glucose level in 96 hrs of experiment. These results have showed that
the hexane extract of C. limon significantly possess blood glucose lowering
activity in alloxan induced diabetic rats.

Evaluation of Antihyperglycemic Activity of Citrus Reticulata Fruit Peel in

Streptozotocin-Induced Diabetic Rats

The present study shows that the methanol extract of the fruit peels
of C. reticulata potent antihyperglycemic activity against STZ-induced
diabetes as well as having hypoglycemic activity in normoglycemic rats and
in glucose overloaded rats. The Citrus plants are rich in flavonoids which
are polyphenolic compounds having potent antioxidant property. As
discussed earlier in the introduction section, the fruit peel of Citrus reticulata
contains the flavonoids hesperidin and naringin. Hesperidin and naringin
both are proven to be potent hypoglycaemic agents, and their hypoglycaemic
activity is postulated to be partly mediated by hepatic glucoseregulating
enzymes in C57BL/KsJ-db/db mice. Dietary hesperidin also exerts
hypoglycemic and hypolipidemic effects in streptozotocin-induced diabetic
rats. Naringin provided a significant amelioration of hypoglycaemic and
antioxidant activity in STZ-induced diabetic rats. Therefore, it can be
postulated that the presence of flavonoids in the extract might be the reason
of the antihyperglycemic action shown by MECL (Bala et al., 2016).

Potential Hypoglycemic Effect of Essential Oil of Citrus Reticulata in Wistar

Rats

The ancient Indian literature has prescribed various herbs for cure of
diabetes mellitus, several plants have been investigated and reported to
possess hypoglycemic activity including Citrus reticulata. In traditional
medicine the essential oil from citrus fruit rind was advised for cutaneous
complaints, snake bite, fever loss of taste, chronic rheumatism,
stomachache, menorrhagia, splenomegaly, edema and cardiac diseases.
Present study was therefore planned to confirm the hypoglycemic activity of
essential oil of Citrus reticulata and to explore effect of fruit rind oil on
glucose in euglycemic and alloxan induced hyperglycemic Wistar rats.
Findings of the present study clearly indicate that acute (single dose)
treatment with essential oil of Citrus reticulata showed significant
hypoglycemic activity. Whereas the single dose administration of the oil in
sub hypoglycemic dose with that of Glibenclamide did not show any
significant hypoglycemic activity. Though the present study was not aimed
to elicit the mechanism of hypoglycemic activity of the extract, the
phytochemical constituents of Citrus reticulata bioflavonoid, sterols, D-
limonene, linalool could be responsible for its hypoglycemic activity by
virtue of their antioxidant property. Antioxidants have been reported to exert
beneficial effects on pancreatic β-cell function by preventing or delaying β-
cell dysfunction due to glucose toxicity (Burli et al., 2017).

A Comparative Study Of In Vitro Total Antioxidant, In Vivo Antidiabetic And

Antimicrobial Activities Of Essential Oils From Leaves And Rind Of Citrus
Reticulata Blanco Cv. Murcot (Honey)

There is increasing interest in the radical scavenging activities of

some natural antioxidants, especially those found in edible plants, which
may play a significant role in preventing various diseases. The present
investigation aimed to study the composition of essential oils of Citrus
reticulata Blanco cv. Murcot (honey) and to analyze its antioxidant,
antimicrobial, and antidiabetic activities. The data presented here shows that
Citrus reticulata Blanco cv. Murcot (Honey) extracts have great antioxidant
and radical scavenging activity and thus may be used as a good source of
 natural antioxidants. Both low and high concentrations of essential oils have
exhibited hypoglycemic effect. Immediate drop in the blood glucose levels
can be seen. Effect of high dose of essential oil is about almost equal to the
low dose. All these findings suggest that essential oils may be acting through
some mechanism to improve the receptor-responsiveness to insulin causing
increased sugar uptake by the tissue (Khan et al., 2014).

Expected Output of the The expected output is a capsule from Ponkan Vizcaya peel with
Research Project 25%, 50%, and 100% concentration. The product will then be effective to
(Provide the expected relieve Diabetes Milletus in alloxan- induced mice.
results/deliverables for each of the
objective above. Please be specific
and quantify outputs as much as
possible):
Methodology
(Comprehensive procedure of the Gathering of the Plant
study):
The materials to be used for the testing of antidiabetic effect will be
peels from Ponkan that will be purchased in Nueva Vizcaya Pasalubong
Center that is acquired from Kasibu, Nueva Vizcaya.
The collected fruit rinds will be macerated and will be air dried at room
temperature. Afterwards, the dried peels will be powdered using a blender.

Test Animals
Healthy mice reared and maintained at the animal house of the
institution will be used. The mice will be feed with commercial animal fee
and water ad libitum. The mice weighing between 150-250 g will be housed
separately in groups with a distinct identity for about a week for
acclimatization. All experimental protocols will be prepared and performed
based on ethical guidance of Institutional Animal Ethical Committee.

Preparation of Ethanolic Peel Extract

Approximately 50 g of powdered Ponkan peels will be suspended in
500 mL of 95% ethanol in a round bottom flask and gently boiled on a water
bath for 2 hrs. The ethanolic extracts will be removed by vacuum filtration
and then concentrated under reduced pressure to obtain crude viscous
extracts. The yield of the green viscous masses was determined and was
stored at 4˚C until further use in experiment

Toxicological and Pharmaco- toxicological Assay

Toxicological screening will be adopted from Chunlara et al. (2004)
which uses Acute toxicity test.
According to the World Health Organization (WHO) guideline
(WHO, 2000) and the Organization of Economic Co-operation and
Development (OECD) guideline for testing of chemicals, 10 rats were
randomly divided into two groups of 5 animals per sex. The extract at a
single dose of 5,000 mg/kg body weight was given orally to the treated group
(the extract at concentration 2,500 mg/ml in distilled water), while the
control group received water vehicle. Body weight, signs of toxicity and
mortality were observed after the administration at the first, second, fourth
and sixth hour and once daily for 14 days. On the 15th day, all rats were
fasted for 16-18 hours, and then sacrificed for necropsy examination. The
internal organs were excised and weighed. The gross pathological
observations of the tissues were performed by histopathological
examination.

Phytochemical Screening
The phytochemical screening of the ethanol extract of C. reticulata
peel will be adopted from Rana et al. (2017). The crude ethanol extracts of
ponkan peel will be tested for the presence of Carbohydrates, Steroids,
Proteins, Alkaloids, Tannic Acid and Phenolic compounds, Sulphates,
Chlorides, and Flavonoids. The qualitative results will be expressed as (+)
for the presence and (-) for the absence of phytochemicals.

DPPH Free Radical Scavenging Assay

The antioxidant activity of the extracts will firstly be evaluated by
monitoring its ability in quenching the stable free radical DPPH. The radical
scavenging activity of the extracts and fractions against DPPH free radicals
will be measured using the method of Clarke et al. (2013) slightly modified
as follows: 20 μl of appropriately diluted samples or Vitamin C solutions
(10, 50, 100, 500, and 1000 μg/ml) was added to 190 μl of DPPH solution
(100 μM) in a well of 96-well plate. The mixture will be shaken vigorously
and allow to reach a steady state at room temperature for 30 min.
Discoloration of DPPH was determined by measuring the absorbance at
517 nm with a Beckman spectrophotometer. All determination was carried
out in triplicate. Ascorbic acid was used as a positive control. The DPPH
radical scavenging activity was calculated according to the following
equation:

Where 𝐴0 was the absorbance of the control (blank, without extract), 𝐴1 the
absorbance in the presence of the extract, and 𝐴2 the absorbance without
DPPH.

Antidiabetic Test
Hyperglycemia will be induced in overnight fasted adult mice
weighing 150-250 g by a single intraperitoneal injection of freshly prepared
alloxan monohydrate in normal saline (150 mg/kg body weight) in a volume
1 ml/kg body weight. Hyperglycemia will be confirmed by the elevated
glucose level in blood, will be determined at 48 h after injection. The mice
that will be found hyperglycemic will be screened for the antihyperglycemic
study.
Animals will be divided into five groups of six mice each. Test group is
administered with the extract at dose of 200 mg/kg body weight by oral
route. Standard and control animals will be treated with standard drug
glibenclamide at an oral dose of 1 mg/kg body weight and distilled water
respectively. All doses will start 48 h after alloxan injection. The above
treatments will be given once daily for 30 days and on 31st day under ether
anesthesia cardiac blood 2ml will be collected for estimation of glucose.
Glucometer will be used to estimate the blood glucose level.
 Statistical analysis
The results will be analyze using one-way ANOVA followed by
dunnets test, p<0.05 will be significant.

Encapsulation of the Plant Material

The encapsulation of the extracts will be carried out by the standard
procedure.

Research Consultant Research Adviser

Signature over Printed Name Signature over Printed Name

Important note: Please provide the source or reference of information cited in the proposal on separate page.
References

Ademosun, A., Oboh, G., Olasehinde, & T., Adeoyo, O.(2018). Folk Medicine: A Review on the
Bioactive Components and Pharmacological Properties of Citrus Peels. Retrieved from
https://doi.org/10.1007/s13596-017-0292-8.
Bermejo, M.J. Llosa & A. Cano. (2016). Food Science and Technology International: analysis of bioactive
compounds in seven citrus cultivars. DOI: 10.1177/1082013210368556.
Aruoma, O., Landas, B., Ramful, D., Bhujun, E., Wagner, K., & Bahorun, T. (2015). 1(1) pp 6-9.
International Journal of Pharmaceutical Applications: functional benefits of citrus fruits in the
management of diabetes.
Den, D., & Tsiani, E., (2019). Antidiabetic Properties of Naringenin: A Citrus Fruit Polyphenol. Retrieved
from
Sen, S., Haldar, P., Gupta, M., Mazumder, U., Saha, P., Bala, A., Bhattacharya, S., & Kar, B. (2016).
ISRN Endocrinology: evaluation of antihyperglycemic activity of citrus reticulata fruit peel in
streptozotocin-induced diabetic rats. doi:10.5402/2011/869273.
Burli, S., Gavimath, C., Jadhav, N., & Kangralkar, V. (2017). Potential Hypoglycemic Effect of Essential
Oil of Citrus Reticulata in Wistar Rats. Retrieved from
Mehmood, F., Khan, Z., Shahzadi, P., Yaseen, T., Mughal, T., Raza, S., & Qasim, M. (2014). A
Comparative Study Of In Vitro Total Antioxidant, In Vivo Antidiabetic And Antimicrobial
Activities Of Essential Oils From Leaves And Rind Of Citrus Reticulata Blanco Cv. Murcot
(Honey). Retrieved from