INTRODUCTION

Background of the Study

Diabetes mellitus is a heterogeneous group of disorders that interferes
with normal carbohydrate and lipid metabolism. Diabetes may be classified as
Type I, an auto-immune disease resulting to insufficient insulin secretion, or
Type II, the more common type, caused by a combination of insulin insensitivity
and a deficiency in pancreatic insulin secretions. Impairment in insulin secretion
and action results to high levels of blood glucose or hyperglycemia which is
associated with complications such as heart disease, stroke, kidney
dysfunction, blindness, nerve problems, gum infections and amputation (Hui et
al., 2009).
Diabetes continues to be a major health problem in the world (WHO,
2014). Last 2013, 382 million people were reported to have diabetes. By 2035,
this number is projected to increase to 592 million (International Diabetes
Federation, 2013). Valisno (2013) reports 3.4 million diabetes cases locally in
2010, representing a prevalence rate of 7.7 which is projected to rise to 8.9
percent or 6.16 million cases, making it the 7 th leading cause of death by 2030.
In 2009, Type II diabetes mellitus was ranked as the eighth leading cause of
death in the Philippines (Gatbonton et al., 2013).
While oral hypoglycemic drugs used in the management of diabetes are
effective in regulating blood glucose, synthetic drugs have certain limitations and
side effects such as severe hypoglycemia, lactic acidosis, idiosyncratic liver cell
1

injury, permanent neurological deficit, digestive discomfort, headache and

dizziness (Hui et al., 2009). To address this, the World Health Organization
Expert Committee on Diabetes (1980), encourages further study on traditional
medicinal herbs which, aside from possibly having less side effects, also cost
less and are accessible to locals.
Canarium ovatum Engl. (pili) is an indigenous tree nut abundant in the
Philippines. Tree nuts are possible sources of agents which can improve both
glycemic control and serum lipids in Type II diabetic patients (Jenkins et al.,
2011). Individual studies by Laforteza and Tripon (2000) showed that extracts of
pili pulp oil have potential hypoglycemic effects. Moreover, the kernel oil of pili
was found to contain glycerides of oleic acid. Oleic acid was reported to be
effective in reversing the inhibition of insulin production and in lowering blood
glucose levels (Vassiliou et al., 2009). In addition, pili kernel oil is also low in
saturated fat and high in polyunsaturated fatty acids and monounsaturated fatty
acids (Zarinah et al., 2014) which may improve insulin sensitivity in people with
Type II diabetes, thereby improving blood glucose control (Parillo et al., 1992).
While previous studies have shown that Canarium ovatum is a potential
source of antihyperglycemic agents, there are still no clinical and experimental
studies regarding the use of the kernel oil as an antihyperglycemic. This study
will thus evaluate the antihyperglycemic properties of C. ovatum kernel oil. In
addition, this study will determine if the effect of the kernel oil is dose dependent
and its efficacy will be compared with the oral hypoglycemic Metformin.

Statement of the Problem

Does the kernel oil extract of C. ovatum have an antihyperglycemic effect
on the blood and urine glucose levels of diet-induced hyperglycemic mice?
Objectives of the Study
This study aims to determine if the kernel oil of locally available C.
ovatum has antihyperglycemic effects on the blood and urine glucose levels of
diet-induced hyperglycemic mice. It specifically intends (1) to determine the
effect of treatment with pili kernel oil extract and Metformin by measuring the
percent change in blood glucose levels, (2) to determine if the effect of pili
kernel oil on blood and urine glucose levels is dose dependent, (3) to evaluate
the effect of C. ovatum kernel oil on blood glucose levels, urine glucose levels,
and weight of mice, (4) and to test the presence of antihyperglycemic agents in
C. ovatum kernel oil.
Significance of the Study
While commercially available hypoglycemic drugs are effective in blood
glucose level management and are frequently prescribed, side effects such as
severe hypoglycemia, lactic acidosis, idiosyncratic liver cell injury, permanent
neurological deficit, digestive discomfort, headache and dizziness (Hui et al.,
2009), warrant the search for safer alternatives. Herbal remedies can be grown
on accessible land, come at lower costs and may reduce unwanted side effects.
The results of this study will provide information about the possible
antihyperglycemic effects of C. ovatum kernel oil. This would promote its local
utilization as an herbal plant and consequently, the need for its preservation and
3

propagation as a native plant species. This could also encourage the

development of an efficient and large-scale process of extracting pili kernel oil.
Moreover, findings can serve as a basis for further study on the chemical
composition, active ingredients and mechanisms of action of C. ovatum kernel
oil. The findings in this study will be useful as reference for succeeding related
studies.

Scope and Limitations of the Study

Only the kernel oil of Canarium ovatum was used because of its potential
as an antihyperglycemic agent (Vassiliou et al., 2009; Laforteza & Tripon,
2000). Nontoxic doses of 300 mg/kg body weight and 3000 mg/kg body weight
(Mokiran et al., 2014; Martinod, 2005; Tang & Reed, 2001; Fujita et al., 2005)
were used to determine if the antihyperglycemic effect of pili kernel oil is dose
dependent.
The experimental design is a randomized block design which involved
treatment and non-treatment with C. ovatum kernel oil extract. Hyperglycemia
was induced by feeding the mice orally with a high carbohydrate (corn syrup)
diet for the entire duration of the study. Blood samples were collected after 21
days of high carbohydrate diet to confirm successful induction of hyperglycemia.
Mice having blood sugar level readings between 120 to 200 mg/dL were
considered hyperglycemic (Serreze et al., 2000; Keren et al., 2000).
Twenty-four (24) eight (8) week old male ICR mice (Mus musculus) were
distributed randomly into four groups, with six animals in each group. Male mice

were chosen because the progress of hyperglycemia is slower and less uniform
in females (Rakoczy et al., 2010). The grouping was as follows: (1) No Oil
group, (2) Low Dose group (300 mg/kg), (3) High Dose group (3000 mg/kg) and
(4) Metformin group (150 mg/kg).
Antihyperglycemic effect was evaluated through collection of blood
samples on the 1st and 14th day of treatment using tail puncture technique and
estimation of blood glucose level using a glucometer (Infopia Co., Ltd.). In
addition, urine glucose levels were evaluated through collection of urine
samples using modified metabolic cages and estimation of urine glucose levels
using colorimetric strips.
This study does not aim to offer an absolute treatment for diabetes. It will
only assess the antihyperglycemic activity of C. ovatum kernel oil. This study
does not include the isolation of the active compound in C. ovatum kernel oil as
well as the determination of its mechanism of action.

REVIEW OF RELATED LITERATURE

Diabetes mellitus is becoming a prevalent endocrine disorder. According
to Shaw et al. (2010), an estimated 6.4% of the world population or 285 million
of adults aged 20 to 79 were afflicted by the year 2010 and this percentage is
expected to rise to 7% or 439 million adults by the year 2030. An increase of
69% is predicted in the number of diabetic cases in developing countries from
2010 to 2030 (Shaw et al., 2010). Last 2014, 387 million cases were recorded
(International Diabetes Federation, 2014). According to the World Health
Organization (WHO), people from Southeast Asia and the Western Pacific are
most at risk of this disease, dubbed one of the major killers of our time (Tiwari
and Rao, 2002).
Locally, Soria et al. (2009) noted increased fasting blood glucose in
respondents from national regions from 1998 to 2007 and this trend is expected
to continue in years to come. Gatbonton et al. (2013) reported 4.2 million
diabetes cases in the Philippines as of 2011, and by 2030, 7.4 million cases are
projected. The growing prevalence of diabetes among Filipinos is accompanied
by increasing mortality caused by the disease. Gatbonton et al. (2013) reported
low high-density-lipoprotein-cholesterol levels, abdominal obesity, high blood
pressure, high triglyceride levels and elevated fasting blood sugar as risk factors
associated with diabetes.
Diabetes mellitus is a disease requiring prevention and immediate
treatment upon diagnosis as several other health problems are associated with
it.

Kaczmar

(1998)

enumerates

retinopathy,
6

neuropathy,

nephropathy,

atherosclerotic coronary artery disease and peripheral atherosclerotic vascular

disease as major complications associated with diabetes. According to the
same study, nearly 85% of diabetics develop retinopathy, 25 to 50% develop
kidney disease and 60 to 70% suffer mild to severe nerve damage. Diabetics
are also twice or four times more at risk of developing cardiovascular disease or
suffer a stroke. Since these complications can arise from hyperglycemia,
prevention lies in immediate and effective regulation of increased blood glucose
levels (Kaczmar, 1998).
There are two types of diabetes. Type I, also called juvenile onset
diabetes or insulin dependent diabetes mellitus (IDDM), is an auto-immune
disease destroying the beta cells in the Islets of Langerhans of the pancreas
resulting in inadequate insulin production. Insulin injections are used in the
treatment of this type of diabetes (Said et al., 2008). On the other hand, Type II,
also called maturity onset or non-insulin dependent diabetes mellitus (NIDDM),
is caused by impairment in insulin secretion and action. It is the more common
type, comprising 90 to 95% of all diabetes cases (Tiwari & Rao, 2002). Initially
the cell exhibits insensitivity to insulin, but this eventually leads to inadequate
insulin production by the pancreas (Hui et al., 2009). By 2025, the number
cases of Type II diabetes in Asian-Pacific countries is expected to increase by
30 to 60%, attributable to high-calorie diets and sedentary habits (Chan et al.,
2006). Treatment of Type II diabetes mellitus involves changes in diet and
supplementary oral hypoglycemic drugs (Wadkar et al., 2007).

Oral hypoglycemics are used in the treatment of Type II diabetes

7

mellitus. Metformin is a biguanide commonly used locally to treat Type II

diabetes mellitus (De Luna, 2011). While Park et al. (2009) remark that
Metformin is effective in reducing weight gain, hyperinsulinemia and
hyperglycemia in adults with Type II diabetes, it can also cause discomfort and
have minor side effects. In the same study by Park et al. (2009), gastrointestinal
problems were the most common reported side effect frequently reported from
Metformin use (Park et al., 2009). Metformin can also cause Vitamin B 12
malabsorption and deficiency as a result of drug action interference with calcium
ion absorption on which vitamin B12 absorption is dependent (Bauman et al.,
2000). Furthermore, long-term use of Metformin was found to contribute to
cobalamin malabsorption in elderly people (Andres et al, 2004). While rare,
cases of lactic acidosis resulting from Metformin use have been reported
(Chang et al., 2002). Due to reactions to the drug, Metformin cannot be used by
all diabetic patients. A number of cases of lactic acidosis occurred in the
presence of renal, hepatic or cardiovascular disease, thus it is advised not to
prescribe Metformin to diabetic patients with these ailments (Phillips et al.,
2008).
Aside from having possible side effects, anti-diabetic drugs are costly.
Healthcare expenditures for diabetes account for 11% of the health
expenditures in the country. To add, the countrys estimated 360,000 USD
expenditure from 2010 is expected to rise to around 670,000 USD by 2030
(Zhang et al., 2010). Considering this, people are driven to search for more
economical options (De Luna, 2011).

Since herbal remedies come from natural sources, they are perceived to
have less side effects (Chandira & Jaykar, 2013). While several herbal
treatments remain anecdotal, certain species used in traditional medicine have
been found to be effective hypoglycemics. A familiar plant, bitter gourd
(Momordica charantia) is used in alternative treatment for diabetes (Basch et
al., 2003). Traditionally used in Ayurvedic medicine as treatment for diabetes,
M. charantia has exhibited hypoglycemic effects similar to Glibenclamide, a
synthetic antidiabetic drug (Virdi et. al, 2003). Banaba (Lagerstroemia speciosa
L.), a tree commonly found in the Philippines and other tropical countries, is also
traditionally used as an herbal hypoglycemic. Its extract, due to its corosolic acid
content, was found to have the insulin-like action capable of speeding up
glucose uptake by cells (Deocaris et al., 2005). According to a Department of
Health report in 2012, traditional, complementary and alternative medicines are
widely used in the Philippines. It states that the WHO WPRO estimated that
70% of the population uses traditional and complementary medicines
(Department of Health, 2012). To further encourage the safe use of traditional
medicines, the Department of Health launched the Traditional Medicine
Program in 1992. In addition, the Republic Act 8423 or the Traditional and
Alternative Medicine Act of 1997, which promotes the use of traditional
medicine, was passed under the Ramos administration.
Canarium species, belonging to the family Burseraceae, were reported
to have antidiabetic properties. The methanol extract of stem bark of Canarium
schweinfurthii Engl. was reported to have antidiabetic activity. In a study by

Kamtchouing et al. (2006), it was reported that a dose of 300 mg/kg causes a
67.1% reduction in blood glucose levels after a single daily subcutaneous
injection on Streptozotocin-induced diabetic male rats over 14 days. Weight
gain was only 6.6% and there was a significant reduction in food and fluid
consumption by 68.5% and 79.7%. These results show that the extract could
reverse hyperglycemia, polyphagia and polydipsia provoked by Streptozotocin,
thus having antidiabetic activity (Kamtchouing et al., 2006). In a study by
Mokiran et al. (2014), Canarium odontophyllum fruit extract had a noticeable
plasma glucose level lowering effect at a concentration of 600 mg/kg body
weight in obese-diabetic rats. While the fruit extract did not increase the insulin
level, it was able to reduce insulin resistance (Mokiran et al., 2014). In China, a
functional food made up of Canarium album and balsam pear was invented and
patented. It contains 3 to 18 parts of C. album and 1 to 10 parts of balsam pear
by weight. The product has many health benefits including the improvement of
blood sugar level and blood pressure. It can also possibly be used to prevent
and treat Type II diabetes mellitus and cardiovascular diseases. It can further be
developed as an anti-diabetic in Chinese medicine. (Tang & Tang, 2012).
Other members of the Family Burseraceae also showed antidiabetic
potential. In a study by Goji et al. (2009), the aqueous ethanolic stem bark
extract of Commiphora africana produced a dose-dependent, significant
reduction in blood glucose levels of fasted normal rats. Three doses (100, 200,
and 400 mg/kg) of the extract were administered orally. While the 100 mg/kg
dose showed no significant decrease in the blood glucose level, a significant
decrease in the blood glucose levels after 5 and 7 days of administration were
10

observed with doses 200 mg/kg and 400 mg/kg (Goji et al., 2009). Garuga
pinnata

Roxb.

(Burseraceae)

aqueous

bark

extract

showed

potential

antidiabetic property. Two doses of the plant extract exhibited a significant

decrease in fasting blood glucose levels in Streptozotocin-induced diabetic mice
(Shirwaikar, 2006).
Canarium ovatum
Canarium ovatum Engl. belongs to the family Burseraceae consisting of
18 genera and about 400 species, of which, four genera and about 40 species
are found in the Philippines. Of the seventy-five species of Canarium, nine are
found in the Philippines, namely, C. indicum, C. luzonicum (Bl.) A. Gray, C.
odontophyllum Miq., C. asperum (pili-pili), C. vrieseamum, C. gracile (pilingokai), C. euryphyllum, C. hirsutum (hagushus) and C. ovatum (Coronel, 1983).
Canarium ovatum, locally called pili, is endemic to the Philippines. It is
found in low to medium elevation primary forests mostly in Bicol Region,
Cordillera Administrative Region, Western Visayas, Central Visayas, Eastern
Visayas and Mindanao.

Pili can easily be cultivated either by using

seedlings/seeds or by asexual methods such as marcotting, grafting, and

budding. It easily grows in areas where rainfall is almost evenly distributed
throughout the year. It is a sturdy tree which is resistant to typhoons and most
pests. An average pili tree starts to bear fruits after four or five years of planting.
On average, each tree produces 1,000 to 2,000 nuts per year. As the tree gets
older, it bears more fruits. Some pili trees seasonally produce fruits while others
produce fruits all year round. The present system of harvesting pili fruits is
laborious. The harvesters climb the tree and manually detach the fruits from the
11

shoots. For commercial pili orchards, however, ripe fruits can simply be allowed
to fall to the ground and be collected manually or using a machine. Canarium
ovatum is considered the most important nut-producing Canarium species in the
Philippines and has great potential as a major export crop in the country
(Coronel, 1996).
At present, most of the pili production in the Philippines is
concentrated in the Bicol Region. It accounts for 82% of the total volume of pili
nut production having an existing area of 7,746 hectares with 221,250 fruit
bearing trees, although most of this production is small-scale and there are few
commercial plantations of pili. With this, the government is assisting the locals
and launching projects to promote the large-scale production of this crop
(Bureau of Agricultural Research, 2009). The government encourages
businessmen to invest in pili farming and product development due to its
promising local and foreign marketability. Due to the many uses of pili, it is
often called as the second tree of life. There is an increasing domestic and
foreign demand for pili so the pili producers need to upgrade their production
and postharvest operations to a larger scale. In order to promote pili products,
most pili producers participate in local, national, and international trade fairs
sponsored by the DTI, DA, DOST, and DOT. Most pili products are also sold at
various pasalubong centers and supermarkets in the Philippines and other
countries (Bureau of Agricultural Research, 2009).
Canarium ovatum is a semi-deciduous tree which can reach a height of
35 meters and about a meter in diameter. It has large, compound alternate and
pinnate leaves which are about 40 cm long. Leaves have three pairs of opposite
12

ovate-oblong leaflets and a terminal leaflet (Lanting & Palaypayon, 2002).

Leaflets are odd-pinnate, thick, smooth, dark green, entire, rounded at the base,
pointed at the tip, and prominently veined (Coronel, 1996).
The plant is dioecious. The fruit is a drupe consisting of a pulp, a shell
and a seed. The young fruit is green and gradually turns purple black as it
ripens. The mature fruit is a smooth ovoid drupe, 65 mm long and 23 to 38 mm
in diameter, weighing 15.7 to 45.7 g. The thin exocarp of the fruit is smooth and
shiny. The yellow green pulp (mesocarp) is thick and fibrous and it exudes
green or brown resin. The pulp oil is clear, greenish yellow in color and it is
composed of 56.7% oleic glycerides, 13.5% linoleic glycerides and 29.3%
saturated fatty acids. The pili pulp makes up about 64.5% of the fruit by weight
and contains 73% moisture. Its dry weight (per 100g) contains 8% protein,
33.6% fats, 3.4% crude fiber, 9.2% ash, and 45.8% carbohydrates. It contains
35.6 to 51.4% moisture, 11.5 to 15.7% protein, 69.2 to 76.6% monounsaturated
fats, and 2.59 to 4.32% carbohydrates. It also contains the following minerals
and vitamins (per 100g): 119 mg calcium, 508 mg phosphorus, 2.6 mg iron, 489
mg potassium, 45 IU vitamin A, 0.95 mg thiamine, 0.12 mg riboflavin, 0.4 mg
niacin, and traces of vitamin C (Coronel, 1996).
The thick hard endocarp, pointed at one end, protects the seed. A fibrous
seed coat under the shell covers the dicotyledenous embryo. The kernel
weighing 0.74 to 5.14g makes up 4.4 to 16.6% of the whole fresh fruit. It is oily
and has a turpentine odor. The ripe pili fruit weighs 15.7 to 45.7 g. The pili
kernel oil is composed of glycerides of oleic (59.6%) and palmitic (38.2%) acids.
It is light yellow in color and has an agreeable odor and taste (Coronel, 1996).
13

The kernel also contains vitamin E, magnesium, copper, and manganese

(Tadayyon, 2013).
The different parts of the pili plant have many uses. Its resinous wood
can be used as firewood, while the young shoots and pulp are edible. The
young shoot can be added to salads and its pulp is usually boiled and eaten.
The kernel is the most commonly used part of the plant. It is edible and can be
roasted, fried or coated with sugar. Extracted pulp oil can be used for cooking
and lighting. It shares similarities with olive oil and can be used as cottonseed
oil substitute (Coronel,1996).
Canarium ovatum also has medicinal uses. The seed kernel is used as a
laxative. The bark is used in treatment of malaria while the leaves are used in
the treatment of vertigo (Barwick, 2004). The resin is used as an ointment for
healing wounds. Although pili has a lot of potential, not much has been studied
about the pili kernel in terms of its health benefits (Kris-Etherton, 2013).
In a study by Laforteza (2000), the hypoglycemic, cytotoxic and
antifungal activities of C. ovatum pulp extracts were compared. The plant
extracts and glucose were injected intraperitoneally. The percent reduction in
blood glucose was measured using the glucose tolerance test. The results
showed that pressed pili pulp extract exhibited an 84% decrease in blood sugar
levels of diabetic mice. Also, the pili pulp nonpolar fraction showed a 59.65%
reduction in blood glucose. This indicates that pili pulp extract is potentially
hypoglycemic (Laforteza, 2000). Another study by Tripon (2000) showed that
the pili pulp ethanol extract caused a 42% reduction in blood glucose level.

14

Percent reduction in blood glucose was also tested using the glucose tolerance
test. These studies suggest that pili extracts have potential hypoglycemic
activity.
Canarium ovatum belongs to a group referred to as tree nuts. Most tree
nuts are healthy and have lots of health benefits and research suggests that
consuming tree nuts can improve blood sugar levels in people with Type II
diabetes (Jenkins et al., 2011). Tree nuts can also improve blood cholesterol
levels. Studies also reveal that tree nuts can be a replacement for
carbohydrates in the diet of diabetic patients. Monounsaturated fatty acids in
diabetic diets preserve high density lipoprotein cholesterol and improve
glycemic control. In a study by Jenkins et al. (2011), the diet of a total of 117
Type II diabetic patients were randomly supplemented with mixed nuts
(75g/day), muffins, or half portions of both. This was conducted for three months
and results showed that two ounces of nuts daily as a replacement for
carbohydrate foods improved both glycemic control and serum lipids in Type II
diabetic patients.
Djarkasi (2011) found that tree nuts such as almond, cashew, walnut,
Brazil nut, hazelnut, pecan, macadamia, and Canarium are rich in bioactive
compounds. In general, the bioactive compounds often found in fruits or seeds
of tree nuts are phenolic compounds, carotenoids, phylosterols, and tocopherol.
These compounds are beneficial to human health and can decrease the
likelihood

of

acquiring

degenerative

diseases

like

high

cholesterol,

hypertension, diabetes, and cataract. (Djarkasi, 2011). In the case of Canarium

15

ovatum, however, not much of these compounds have been isolated. There is
still a need to isolate and identify the specific bioactive compounds present in C.
ovatum.
According to Mogana & Wiart (2011), only about 12% of the total
Canarium L. species have been studied for chemistry and pharmacological
activities. Thus, there is still a need to study Canarium L. species which have
potential as drugs. Among the secondary metabolites isolated from members of
the genus Canarium L. are terpenes (monoterpenes, triterpenes, tetraterpenes),
carboxylic acids, coumarins, furans, lipids, and phenols (flavonoids, tannins,
phenolic acids). The derived extracts were reported to have a variety of
pharmacological activities such as antioxidant, antibacterial, antifungal,
antitumor, anti-inflammatory, hepatoprotective, analgesic, and antidiabetic
(Mogana & Wiart, 2011). A phytochemical screening of the leaves of C. ovatum
showed that it contains tannins, saponin, terpenoids, flavonoid, glycoside, and
phenolic compounds (Hernandez & Paguigan, 2009). The pili kernel also
contains flavonoids and phenols (Urtal, 2008). These compounds are mostly
found in hypoglycemic plants. Substances like glycosides, alkaloids, terpenoids,
and flavonoids are usually regarded as having antidiabetic effects (Mukesh &
Namita, 2013).
Pili kernel oil also contains glycerides of oleic (59.6%) and palmitic
(38.2%) acids (Coronel, 1996). Oleic acid was found to be effective in reversing
insulin production inhibition. Vassiliou et al. (2009) reported that insulin
production was enhanced in rat pancreatic beta cell line INS-I following
treatment with oleic acid and peanut oil which is rich in oleic acid. Also, blood
16

glucose levels significantly decreased in Type II diabetic mice given a high oleic
acid diet derived from peanut oil. This shows that oleic acid can have beneficial
effect to those with Type II diabetes (Vassiliou et al., 2009). Since other
members of its family and genus were found to have hypoglycemic properties
and potential hypoglycemic agents were found in the kernel, it is possible that
C. ovatum kernel oil also has antihyperglycemic effects.

17

METHODOLOGY

Canarium ovatum Kernel Oil Extract

Canarium ovatum nuts (pili nuts) were collected from Catarman, Northern
Samar. The plant species was verified by the Botany Division at the Philippine
National Herbarium (Appendix E). A total of 550 g of pili nuts were washed
thoroughly with tap water. These were dried, ground, weighed and manually
extracted using an oil extractor and was then refrigerated at 4 C to prevent
growth of bacteria (Martinod, 2005). The total volume of oil extracted from 550 g
of pili nuts was 54.6 ml with a percentage yield of 8.84%. A sample of pili kernel
oil was sent to the Industrial Technology Development Institute Department of
Science and Technology (ITDI-DOST) for fatty lipid profile analysis.

Test Animals

Twenty-four (24) 8-week old male ICR mice (Mus musculus Linn.)
weighing 30 to 40 g were procured from the Research Institute for Tropical
Medicine and housed at the animal room of the University of the Philippines
College of Medicine throughout the experimental period. The experiment
protocol was approved by the Institutional Animal Care and Use Committee
(IACUC) of the University of the Philippines Manila National Institutes of Health
(Appendix F). The animals were kept individually in clean cages with the
temperature maintained at 25 2C and a regular 12 hour light/ 12 hour dark
cycle. Assignment to cages was done randomly and cages were marked for

18

identification.
The mice were acclimatized to the laboratory environment for seven (7)
days and were provided with standard pellets and distilled water ad libitum.
After the seven-day acclimatization period, they were randomly distributed into
four (4) groups of six (6). The grouping was as follows: (1) No Oil group, (2) Low
Dose group, (3) High Dose group and (4) Metformin group.
The body weight of each mouse was measured on Day 1, Day 22, and
Day 36 of the experimental period. Each mouse was placed in a beaker and
weighed using a digital balance (Appendix D).

Induction of Hyperglycemia
The mice were given a high carbohydrate diet containing corn syrup
(67% carbohydrates) all throughout the experiment (35 days) to render them
hyperglycemic (Ip et al., 2014). The corn syrup was added to the pellets and
water. The high carbohydrate diet was used to induce diabetes because it
strongly resembles the metabolic abnormalities of diabetes in humans. Also,
diet-induced hyperglycemic mice were observed to have a significant increase
in blood glucose levels (Noonan & Banks, 2000; Pierroz et al., 2002).
To confirm the induction of hyperglycemia, blood was drawn from the tail
of conscious mice after 21 days and glucose content was estimated using a
digital glucometer. This was recorded as the hyperglycemic blood glucose level.
Mice having blood sugar level readings between 120 to 200 mg/dL were
considered hyperglycemic (Serreze et al., 2000; Keren et al., 2000). All mice
became hyperglycemic and were included in the treatment.
19

Administration of Canarium ovatum Kernel Oil Extract

Twenty-one (21) days after the induction of hyperglycemia, the C.
ovatum kernel oil was administered through oral gavage at a volume depending
on their body weight. The following treatment was administered to the
hyperglycemic mice once daily for 14 days (Kamtchouing et al., 2006): (1) No
Oil group given mineral oil daily, (2) Low Dose group given pili kernel oil at a
dose of 300 mg/kg body weight (Mokiran et al., 2014; Martinod, 2005), (3) High
Dose group given pili kernel oil at a dose of 3000 mg/kg body weight (Mokiran
et al., 2014; Martinod, 2005), and (4) Metformin group given at a dose of 150
mg/kg body weight. Metformin was dissolved in mineral oil. These doses are
considered nontoxic and could produce possible reduction in blood glucose
levels (Mokiran et al., 2014; Martinod, 2005; Tang & Reed, 2001; Fujita et al.,
2005).

Measurement of Glucose Levels

Blood glucose levels of all mice were measured using a glucometer
(Infopia Co., Ltd.) on the 1st and 14th day of treatment at nine in the morning.
Approximately 10 l of blood was collected using the tail puncture method with
the lateral vein as the source of blood. The blood vessel was superficially and
aseptically nicked. The drop of blood was placed on the glucose strip and the
blood glucose level was displayed automatically within 5 seconds on the digital
glucose meter. Mice having blood glucose level readings between 60 to 120
mg/dL were considered normoglycemic while mice having blood glucose levels

20

of 120 to 200 mg/dL were considered hyperglycemic (Serreze et al., 2000;

Keren et al., 2000).
The urine glucose level of each mouse was measured twice, before the
beginning of the treatment (Day 22) and after the treatment period (Day 36).
Modified metabolic cages were used to collect urine samples. The modified
metabolic cages consist of two 4 L plastic mineral water bottles, one cut in the
middle, where a 250 mL beaker was laid at the center under a 3 in. x 3 in.
netted wire, and the other bottle cut 2 cm from the flat end with a 3 cm latch for
opening and closing, positioned such that the mouth resembles a V facing the
netted wire. The mouse was placed on double netted wire 3 cm below the latch.
The cage had a food container and a hole big enough for the drinking tube of
the water bottle (Appendix D). A period of 24 hours was allotted for mouse
micturition. Urine glucose was measured using urine test strips. These strips
follow a colorimetric assay of glucose, pH, specific gravity and protein isolates.
Color change is a positive indicator of glucose (Appendix D).
Oral Glucose Tolerance Test
The Oral Glucose tolerance test is a standard procedure that determines
how fast glucose is cleared from the blood and can be used to exhibit
alterations of glucose metabolism (Zhang, 2011). The OGTT was accomplished
a day after the 14-day experimental period. The mice were fasted for six hours
and their blood glucose level was measured. A glucose load of 1.5 mg/kg was
given to the mice via oral gavage. This was followed by administration of the
respective treatment Low Dose (300 mg/kg), High Dose (3000 mg/kg) and

21

Metformin (150 mg/kg) via oral gavage. The blood glucose levels were
measured at 30 minutes before the OGTT and 30, 60, 90, and 120 minutes after
OGTT using a glucometer (Ayala et al., 2010).

Data Processing and Analysis

The data on the change in blood glucose was presented as mean

standard error (SE). Using the SPSS software version 21, statistical analyses
were performed to determine the following: (1) One-way ANOVA, (2) Tukeys
test as post hoc analysis and (3) Paired t-test. The level of statistical
significance was set at p 0.05 for all tests.

22

RESULTS
Percent Yield of Oil
The fresh weight of Canarium ovatum (pili) kernels harvested is 3.35
kilograms. The kernels without the shell weighed 550 grams. These were dried
and oil was obtained by manual pressing. The kernel oil obtained was 54.6 ml,
which weighs 48.594 grams giving a percent yield of 8.84%.
Composition of Oil
The pili kernel oil was analyzed at the Industrial Technology Development
Institute (ITDI) DOST. The oil was found to contain the antihyperglycemic
agents such as oleic acid (38.3%) and linoleic acid (20.8%), as well as the fatty
acids linolenic acid (0.110%), palmitic acid (27.4%) and stearic acid (13.3%).
After the analysis, the effect of kernel oil on weight, blood glucose and urine
glucose in mice were tested.
Body Weight Changes
Mice had initial weights ranging from 34.7 to 36.85 g (Table 1). The mean
weights at Day 1 did not differ significantly from each other. After the induction of
hyperglycemia (Day 22), there was a significant increase in the mean weight of
mice in all groups except for the High Dose group (p 0.05). Mean body weights
ranged from 35.7 g to 40.3 g. After the treatment period (Day 36), the weight
increased in all subjects, however, only the increase observed in the Low Dose
group was significant (p = 0.008). The final (Day 36) mean body weights of the
No Oil and Metformin groups were significantly different from the final mean
23

body weights of the Low Dose and High Dose groups (p 0.05) (Figure 1). The
final weights of the mice ranged from 37.2 g to 41.8 g.
Blood Glucose Concentrations
Blood glucose levels ranged from 178 to 243 mg/dl after the 21 day
induction of hyperglycemia alone (Table 2) and were not statistically different
from each other. After treatment, the No Oil group had the highest mean reading
of 212 mg/dl, followed by the Low Dose group (300 mg/kg) with a reading of 203
mg/dl, the Metformin group with 188 mg/dl and the High Dose group (3000
mg/kg) with 183 mg/dl. Blood glucose levels increased significantly in the No Oil
group and decreased significantly in the Low Dose, High Dose and Metformin
groups (Figure 2). These differ by 4.50% (Low Dose), 15 % (High Dose) and
12% (Metformin) from the No Oil group. Blood glucose levels decreased by
6.24%, 9.05%, and 12.28% in the Low Dose, High Dose, and Metformin groups
respectively, however the difference among groups was not significant (Figure
2).
OGTT
Blood glucose concentration was measured thirty minutes after a 1.5
mg/kg of glucose was orally administered to the mice. Thirty minutes after the
glucose load, the blood glucose concentrations in all treatment groups
increased. At 90 minutes, the Low Dose group exhibited an increase which is
11.58% higher than the increase in the No Oil group. Finally, at 120 minutes,

24

both the No Oil and Low Dose groups showed a decrease of 2.58% and 6.84%
respectively (Table 3).
On the other hand, the blood glucose trends for the High Dose and
Metformin groups continuously decreased at 60, 90, and 120 minutes. There
was a decrease of 3.06% (High Dose) and 10.45% (Metformin) in blood glucose
levels respectively from 30 to 60 minutes which continued to decrease to 5.97%
(High Dose) and 10.67% (Metformin) at 90 minutes. Compared to the blood
glucose levels at 30 minutes, the High Dose and Metformin group exhibited a
16.94% and 19.20% decrease, respectively, at 120 minutes (Table 3).
Compared to the No Oil group, the High Dose group had a significant 18.22%
blood glucose reduction at 120 minutes. Despite the decrease, however, the
blood glucose levels did not fall within the normal range.
Similar trends can be observed in the glucose tolerance curves of the No
Oil and Low Dose groups, and with the High Dose and Metformin groups (Figure
3). This is supported by statistical analysis which revealed no significant
difference between the mean blood glucose levels of the No Oil and Low Dose
groups and the High Dose and Metformin groups (Appendix A-9). A significant
difference was observed between the No Oil group and the High Dose group and
between the Low Dose and Metformin groups (p 0.05). Also, a significant
difference between the Low Dose and High Dose groups was observed
(Appendix A-9).

25

Urine Glucose Concentration

Urine glucose levels were measured after the induction of hyperglycemia
(Day 22) and after the treatment period (Day 36). Traces of glucose ranging
from 0 to 5 mg/dl were observed in all groups during the first reading. After the
treatment, no glucose was observed in the No Oil, and High Dose and Metformin
groups, while a reading of 2000 mg/dl appeared in the Low Dose group.

26

DISCUSSION
Oil Extraction and Percent Yield
Using the manual extraction method (Martinod, 2005), the kernel oil
extract of Canarium ovatum had a percent yield of 8.84%. Other commonly used
methods of extracting seed oils are the cold press method and the solvent
extraction method (Food and Agriculture Organization, 2014). Cold press
machines use high pressure to extract oil, yielding oil of high quality. However,
the machine used is very expensive and produces lower quantity of oil compared
to the solvent extraction method (FAO, 1994). To achieve greater oil yield, most
oil manufacturing companies use the solvent extraction method, which makes
use of a solvent such as hexane to extract oil. However, the use of solvents
impairs the quality of oil produced (Anderson, 2011).
One advantage of the manual extraction method is that pure oil or virgin
oil is obtained, without chemicals added (FAO, 1994). However, this method has
low oil yield as demonstrated in this study. While a high total yield of 65.7% of pili
kernel oil was reported from using the cold press method of oil extraction
(Zarinah et al., 2014), another study by Kamtchouing et al. (2006) only obtained
a 10.9% yield from Canarium schweinfurthii extract. Despite the lower yield, the
extract was able to reduce the blood glucose by 71.7% (Kamtchouing et al.,
2006). A study by Kouambou et al. (2007) attained a low 3.83% yield from
Canarium schweinfurthii bark extract and significantly reduced blood glucose
levels by 73.7%. Also, in a study by Mokiran et al. (2014), the yield of the
Canarium odondophyllum fruit extract was only 3% using solvent extraction
27

method but still showed a 30% decrease in blood glucose levels. These studies
show that the extract yield is independent of its blood glucose lowering effect.
The yield of the Canarium plant extract is independent of its pharmacological
effectivity such as antioxidant, antimicrobial, hepatoprotective, and antidiabetic
(Mogana et al., 2011). Thus, 8.84% is a low yield but it is still an acceptable
amount of extract.
C. ovatum Lipid Profile
Analysis of the lipid profile of C. ovatum kernel oil revealed high
percentages of the unsaturated fatty acids oleic and linoleic acid. Unsaturated
fats are liquid at room temperature and are divided into two main groups:
polyunsaturated and monounsaturated. Making unsaturated fats, also known as
healthy fats, part of the diet is encouraged as they have benefits like helping
reduce the risk of heart disease, lowering cholesterol levels, and controlling type
2 diabetes (Dietitians Association of Australia, 2014). Monounsaturated fatty
acids (MUFAs) may benefit insulin levels and control blood glucose levels and
polyunsaturated fatty acids

(PUFAs) improve

blood

cholesterol levels,

consequently decreasing the risk of heart disease. The PUFAs also help
decrease the risk of type 2 diabetes (Mayo Clinic, 2014). Kotake et al. (2004)
found that a high-MUFA diet decreases blood glucose levels and improves
impaired glucose tolerance in diabetic mice, thereby improving glucose
metabolism disorders. The glucose lowering observed in this study may have
been caused by the presence of unsaturated fatty acids in the pili kernel oil.

28

The crude C. ovatum kernel oil used in this study was composed mostly
of oleic acid (38.3%) and linoleic acid (20.8%). Oleic acid is a widely distributed
monounsaturated fatty acid that is abundant in nature. It is commonly found in
animal and vegetable oils (PubChem, n.d.). It protects the beta cells and insulin
target tissues, thereby promoting insulin sensitivity. In a study by Bermudez et al.
(2014), oleic acid improved glycemic control by optimizing the insulin production
of the pancreas and causing immediate lowering of blood glucose levels after
meals. Oleic acid can also be beneficial to patients with type 2 diabetes as it
stimulates the secretion of the antidiabetic hormone called glucagon-like peptide
1 (GLP-1) (Rocca et al., 2001). Moreover, Ahmad et al. (2012) demonstrated
that the seed oil of Momordica charantia, which contained a high percentage of
oleic acid and linoleic acid, caused a large inhibition of 79% for -glucosidase
and 38% for -amylase, making it a potential antidiabetic agent. On the other
hand, linoleic acid is a polyunsaturated fatty acid that occurs widely in plant
glycosides. It is an essential fatty acid in mammals and is also used in the
biosynthesis of prostaglandins and cell membranes (PubChem, n.d.). A separate
study by Ezekwe et al. (2013) and Matravadia et al. (2014) showed that linoleic
acid inhibits hyperglycemia in Alloxan-induced diabetic rats, probably through
oxidative reaction or production of prostaglandins and can prevent insulin
resistance (Matravadia et al., 2014). Therefore, both oleic acid and linoleic acid
are potential antihyperglycemic agents and may have caused the blood glucose
lowering effects observed in this study.

29

Vassiliou et al. (2009) found that feeding Type II diabetic mice oleic acidrich peanut oil reversed high glucose levels in all mice after a 21-day treatment
and concluded that oleic acid increased insulin production in the rat beta cell line
INS-I. Aside from possibly having anti-hyperglycemic effects, oleic acid was
found to be a better diet substitute compared to other fatty acids (Reaven et al.,
1993). While oleic acid was found to reduce lipoprotein cholesterol and oxidative
stress associated with early atherosclerosis (Nicolosi et al., 2004), a study by
Sundram et al. (2003), suggests that an excess of it in the diet can lead to
atherosclerotic lesions. The advisable amount of oleic acid intake can be further
investigated.
Effect on Body Weight
Increase in body weight due to high carbohydrate intake is usually
correlated to hyperglycemia (Thomassian, 2013). In the study, there was a
significant increase in body weights of mice from the first day of hyperglycemic
induction to the first day of treatment. Along with the body weight increase, the
blood glucose levels were above 120 mg/dl, indicating that the mice became
hyperglycemic (Serreze et al., 2000; Keren et al., 2000). The weight increase of
mice in all treatment groups probably indicates an increase in fat deposition. In a
study by Ferreira et al. (2011), mice fed a high carbohydrate diet showed
increase in body weights and adipose mass tissue enhanced by 120%. Also, at 8
weeks, the physiological mode to increase body weight of a mouse is most
commonly through fat depostion (Dickerson, 1947).

30

Most diabetes medications can cause weight gain and too much
hypoglycemia, thereby reducing their clinical benefits. These conditions prompt
most drug developers to create a diabetic medication that is either weight neutral
or induces weight loss and lessens weight gain (Mitri and Hamdy, 2009). The
present study revealed a non-significant increase in the mean body weights of
the No Oil, High Dose, and Metformin groups. This suggests that a higher dose
is more effective in maintaining body weight. Controlling weight during
hyperglycemia or diabetes is important because excess weight aggravates
hyperglycemia,

increases insulin

resistance,

the

risk for hypertension,

hyperlipidemia, and other conditions that lead to cardiovascular diseases (Scnell

et al., 2005).

During hyperglycemia, weight loss or maintenance greatly

contributes to improved glucose control (Inzucchi et al., 2012). The Low Dose,
however, showed significant increase in mean body weight suggesting that it is
not as effective in maintaining the body weight of mice. However, the mean body
weight increase may have only been a consequence of the continuous high
carbohydrate diet given to the mice all throughout the study. As such, pili kernel
oil can still be considered a good alternative blood glucose lowering agent.
Effect on Blood Glucose Levels
For humans, fasting blood glucose levels are considered normal at 99
mg/dl and below, prediabetic at 100 to 125 mg/dl, and diabetic at 126 mg/dl
and above (American Diabetes Association, 2012). For mice, blood glucose
level readings are considered normoglycemic between 60 to 120 mg/dL,
hyperglycemic between 120 to 200 mg/dL, and diabetic above 200 mg/dl
31

(Serreze et al., 2000; Keren et al., 2000).

In the present study, oral administration of Canarium ovatum at a dose of
300 mg/kg (Low Dose) and 3000 mg/kg (High Dose) and Metformin at 150
mg/kg body weight caused blood glucose levels to decrease significantly. Mean
blood glucose levels in the Low Dose, High Dose and Metformin groups
decreased by 6.24%, 9.05% and 12.3% respectively. These differ by 4.50%
(Low Dose), 15 % (High Dose) and 12% (Metformin) from the No Oil group. No
significant difference in the mean blood glucose levels was observed among the
three groups after treatment, suggesting that they are similarly effective in
lowering blood glucose levels and that treatment with C. ovatum kernel oil is
comparable with that of Metformin. In a study by Tripon (2000), pressed pili pulp
oil was found to produce a greater percent decrease in blood glucose levels than
Metformin. Likewise, Laforteza (2000) observed a decrease in blood sugar levels
after treatment with pili pulp extract. In this study, however, while there was a
significant decrease in blood glucose levels, these did not reach the normal
reading of 120 mg/dl or below (Klueh, 2006) possibly because of continued
intake of a high carbohydrate diet.
Other members of the family Burseraceae have also been found to
demonstrate antihyperglycemic effects at different doses. Commiphora africana
produced a significant decrease in blood glucose levels at doses of 200 mg/kg
and 400 mg/kg (Goji et al., 2009). Treatment with methanol extracts of Canarium
schweinfurthii Engl. at 300 mg/kg reduced blood glucose levels in diabetic rats
(Kamtchouing et al., 2006). Canarium odontophyllum fruit extract had blood

32

glucose lowering effects at 600 mg/kg (Mokiran et al., 2014). In this study, since
treatment with a Low Dose caused a significant decrease in blood glucose
levels, it is an indication that pili kernel oil has a high bioavailability or that an
adequate amount of the active component reaches the systemic circulation.
Hydrophobic substances like the kernel oil are readily absorbed. Also,
substances in liquid form are more easily absorbed and have a higher
bioavailability than solids (Merck Manuals, 2015).
Oral Glucose Tolerance Test
The Oral Glucose Tolerance Test (OGTT) is a standard clinical
procedure that determines how fast glucose is cleared from the blood and can
be used to exhibit alterations in glucose metabolism (Zhang, 2011). In humans,
OGTT is commonly used to diagnose Type II diabetes. While in animal
research, it is used to assess the effect of insulin or other drugs on the bodys
ability to metabolize glucose (Stoppler, 2014).

It is a widely used test to

determine whether an individual, in this study, a mouse, is glucose intolerant

and diabetic. Glucose ingested is absorbed in the intestinal lining, enters the
splanchnic circulation and then into the systemic circulation. This causes an
increased blood glucose concentration which in turn stimulates the pancreatic
beta cells to release insulin. Insulin stimulates glucose uptake of the peripheral
tissues (Pacini et al., 2013). In this test, blood glucose levels are measured four
times over a period of two hours after glucose administration. In individuals
without diabetes, blood glucose levels rise and then fall quickly within two
hours, while in diabetics, blood glucose levels rise higher than normal and do
33

not decrease (Stoppler, 2014). Different variables such as fasting duration,

state of consciousness, route and amount of glucose administered may have a
significant effect on the blood glucose level readings in mice following glucose
administration (Andrikopoulos, 2008).
In the study, the blood glucose levels of mice in all groups significantly
increased at thirty minutes after the glucose and treatment administration.
According to Andrikopoulos et al. (2008), delivering glucose load orally
significantly increases the glucose and insulin profiles of mice on a high
carbohydrate diet which is most evident after 15 to 30 minutes. Similar trends
were observed in the glucose tolerance curves of the High Dose and Metformin
groups with a continuous reduction in blood glucose levels, suggesting that
treatment with a high dose of pili kernel oil and intake of Metformin are more
efficient methods of lowering blood glucose levels. The antihyperglycemic effect
in the High Dose group is more evident with a difference of 18.2% blood glucose
level reduction compared to the No Oil group.
Similar trends were observed in the glucose tolerance curves of the No
Oil and the Low Dose groups. Although the Low Dose group has higher mean
blood glucose levels than the No Oil group at 120 minutes, statistical analysis
showed that the two groups are not significantly different. The mean blood
glucose levels of both groups are are still high. Vijayvargia et al. (2000) found
that blood glucose lowering action may only initiate only after a longer time
interval, and since the OGTT is only done for two hours, treatment with a low
dose may not be observed to fully take effect. Extending the time of the test to 5

34

hours or more can conclusively demonstrate the effect of a low dose of the
extract (Vijayvargia, 2000).
Other studies have found lower doses of plant extracts to have blood
glucose lowering effects over a short period of time. A study by Mokiran et al.
(2014) showed that Canarium odontophyllum fruit extract (600 mg/kg) caused a
20% reduction in blood glucose levels compared to an untreated group.
Kouambou et al. (2007) found that acute treatment with Canarium schweinfurthii
bark extract at 75 mg/kg, resulted to a 33.8% glucose reduction after a two hour
treatment. Both C. odontophyllum and C. schweinfurthii showed greater blood
glucose reduction than C. ovatum at lower doses, suggesting these plant
extracts are more effective than C. ovatum in lowering blood glucose levels over
a short period of time; however, differences in parameters such as animals used,
plant part used, and route of glucose and extract administration should be taken
into consideration. Although C. ovatum was less effective in lowering blood
glucose over a short period of time, it was able to reduce the in blood glucose
levels significantly at a high dose, showing that the C. ovatum kernel oil still has
short-term blood glucose lowering activity.
Antihyperglycemic agents have different mechanisms of lowering blood
glucose. Other studies are cited since the determination of the mechanism of
action is beyond the scope of this study. Metformin manages glucose through
several mechanisms. One proposed mechanism is by increasing insulin
sensitivity through insulin receptor expression and tyrosine kinase activity and
another is through its inhibition of hepatic gluconeogenesis (Viollet et al., 2012).
35

Antihyperglycemic plants can increase insulin production and secretion (Puri,

2001), improve insulin sensitivity (Ludvik et al., 2003) and regulate the activity of
carbohydrate metabolizing enzymes (Zuo et al., 2008). In a study by Urtal
(2008), pili kernel oil was found to have flavonoids and phenols. Flavonoids are
suggested to have antidiabetic effects (Mukesh & Namita, 2013) through their
inhibition of aldose reductase and -glucosidase (Yoshikawa et al., 1998). C.
ovatum kernel oil may have a blood glucose lowering effect through one of more
of these mechanisms.
Effect on Urine
Increased glucose excretion was not observed and no glucose was
excreted after treatment. Under normoglycemic conditions, glucose from the
filtrate is almost completely reabsorbed via active transport as the filtrate goes
through the proximal tubule on its way to the loop of Henle (Whaley et al., 2012).
Glucose excreted only reached trace amounts of up to 5 mg/dl, indicating that
the mice did not become diabetic. The detection of glucose by the urine test
strips depends on a peroxide mediated reaction (Surgitech Reagent Strip For
Urinalysis, n.d.). False positives may have appeared due to the presence of
oxidizing agents or peroxidase from disinfectants used to clean the cages (Bayer
Multistix Reagent Strips, 2005). No relationship can be drawn between blood
glucose decrease and glucose excretion. Blood glucose levels were observed to
decrease without an accompanying change in glucose excretion. This suggests
that C. ovatum kernel oil reduces blood glucose levels through a mechanism

36

other than glucose excretion, possibly through increasing insulin secretion and
subsequently increasing blood glucose uptake by the cells.

37

CONCLUSION AND RECOMMENDATION

The study has shown that the Canarium ovatum kernel oil has an
antihyperglycemic effect on diet-induced hyperglycemic mice. The presence of
oleic acid and linoleic acid as possible antihyperglycemic agents was confirmed
through the lipid analysis of the C. ovatum kernel oil. In this study, intake of 300
mg/kg, 3000 mg/kg C. ovatum kernel oil and 150 mg/kg Metformin decreased
blood glucose levels significantly over a prolonged period of treatment by 6.24%,
9.05% and 12.28%, respectively. However, only the High Dose and Metformin
caused a significant decrease over a short period of time, suggesting that the
antihyperglycemic activity of the kernel oil is time dependent. The mean weights
for all treatment groups increased but only the increase in the weight mean of
the Low Dose group is significant. Ingestion of the two doses of oil had minimal
effect on the urine glucose of mice fed a high carbohydrate diet. Based on this
result, glucose may either be absorbed by body cells or glycogen synthesis
occurs in the liver and the reduction of blood glucose exhibited is not through
glucose excretion. The results of the study suggest that oral administration of C.
ovatum kernel oil significantly lowers blood glucose levels in a time dependent
manner, maintains body weight and does not affect the urine glucose excretion
of mice. Moreover, the High Dose is more effective as it was able to reduce
blood glucose levels even during a short term and maintain body weight.
The researchers recommend that the C. ovatum kernel oil be evaluated
for its antihyperglycemic activity in humans, for a longer period of treatment and
at a higher dose. Further researches should consider the different methods of
38

extraction to obtain a greater yield of the kernel oil. Researches should be done
on the bioactive compounds in C. ovatum oil and their blood glucose lowering
mechanism.

39

LITERATURE CITED
Adam, Z., Ismail, A., Khamis, S., Mohktar, M.H., & Hamid, M. (2011).
Antihyperglycemic Activity of F. deltoidea Ethanolic Extract in Normal
Rats. Sains Malaysiana, 40(5), 489-495.
Ahmad, Z., Zamhuri, K., Yaacob, A., Siong, C., Selvarajah, M., Ismail, A., &
Hakim M. (2012). In Vitro Anti-diabetic Activities and Chemical
Analysis of Polypeptide-k and Oil Isolated from Seeds of Momordica
charantia
(Bitter
Gourd).
Molecules,
17,
9631-9640.
doi:10.3390/molecules17089631
American Diabetes Association. (2012). Standards of Medical Care in
Diabetes. Diabetes Care, 35, S12.
Anderson, G.E. (2011). Edible Oil Processing Solvent Extraction. The
AOCS Lipid Library. Retrieved from http://lipidlibrary.aocs
.org/processing/solventextract/index. htm
Andres, E., Loukili, N., Noel, E., Kaltenbach, G., Abdelgheni, M., Perrin, A.,
Noblet- Dick, M., Maloisel, F., Schlienger, J., & Blickle, J. (2004).
Vitamin B 12 (cobalamin) deficiency in elderly patients. Canadian
Medical
Association
Journal,
171,
251-259.
doi:
10.1503/cmaj.1031155
Andrikopoulos S., Blair, A., Deluca, N., Fam, B., & Proietto J. (2008).
Evaluating the glucose tolerance test in mice. American Journal of
Physiology Endocrinology and Metabolism, 295(6), 1323-1332.
Ayala, J.E., Samuel, V.T., Morton, G.J., Obici, S., Croniqer, C.M., Shulman,
G.I., Wasserman, D.H., & McGuinness, O.P. (2010). Standard
Operating Procedures for Describing and Performing Metabolic Tests
of Glucose Homeostasis in Mice. Disease Models & Mechanisms,
3(9-10), 525-534.
Azlan, A., Prasad, K.N., Khoo, H.E., Abdul-Aziz, N., Mohamad, A., Ismail,
A., & Amom, Z. (2010). Comparison of Fatty Acids, Vitamin E and
Physicochemical Properties of Canarium odontophyllum Miq. (dabai),
Olive and Palm Oils. Journal of Food Composition and Analysis, 23,
772-776.
Barwick, M. (2004). Tropical and Subtropical Trees A Worldwide
Encyclopaedic Guide. London, England: Thames & Hudson.

40

Basch, W., Gabardi, S., & Ulbricht, C. (2003). Bitter Melon (Momordica
charantia): A Review of Efficacy and Safety. American Journal of
Health- System Pharmacy, 60, 356-359.
Bauman, W., Shaw, S., Jayatilleke, E., Spungen, A., & Herbert, V.
(2000). Increased Intake of Calcium Reverses Vitamin B 12
Malabsorption Induced by Metformin. Diabetes Care, 23, 1227-1231.
Bayer Multistix Reagent Strips. (2005). Chemical Principles of Procedures
and Ingredients. Elkhart, Indiana: Bayer HealthCare.
Bermudez B., Gomez A., Varela L., Villar J., Abia R., Muriana F., & Lopez
S. (2014). Clustering effects on postprandial insulin secretion and
sensitivity in response to meals with different fatty acid compositions.
Food and Function, 5, 1374. doi:10.1039/c4fo00067f.
Bureau of Agricultural Research. (2009). Pili. Retrieved from http://www.bar.
gov.ph/agfishtech-home/crops/205-fruit-crops/1266-pili-con-t
Chan, J. Deerochanawong, C., Shera, A., Yoon, K., Adam, J., Binh, T.,
Chan, S., Fernando, R., Horn, L., Khue, N., Litonjua, A., Soegondo,
S., & Zimmet, P. (2006). Role of metformin in the initiation of
pharmacotherapy for type 2 diabetes: An Asian-Pacific perspective.
Diabetes Research and Clinical Practice, 75, 255-256.
doi:10.1016/j.diabres.2006.06.023
Chandira, R. Margaret & Jaykar, B. (2013). Extraction, Pharmacological
Evaluation and Formulation of Selected Medicinal Herbs for
Antidiabetic Activity. International Journal of Pharmacy Teaching &
Practices (IJPTP),
4, 458-482.
Chang, C., Chen, Y., Fang, J., & Huang, C. (2002). Metformin-associated
lactic acidosis: case reports and literature review. Journal of
Nephrology, 15, 398-402.
Coronel, R. (1996). Pili Nut Canarium ovatum Engl.: Promoting the
conservation and Use of Underutilized and Neglected Crops 6. Italy:
Institute of Plant Genetics and Crop Plant Research.
Coronel, R. (1983) Promising Fruits of the Philippines. Los Banos,
Philippines: College of Agricutlture, University of the Philippines.
De Luna, W., Diaz, F., & Martinez, V. (2011). Interaction between
Ipomoea batatas L. (Family Convolvulaceae) Leaf Extract and
Metformin and Its Anti-Diabetic Activity in Alloxan-Induced Diabetic
41

Swiss Mice. Undergraduate thesis , Pharmaceutical Chemistry,

College of Pharmacy , University of the Philippines Manila.
Deocaris, C., Aguinaldo, R., dela Ysla, J., Asencion, A., & Mojica, E.
(2005).Hypoglycemic
Activity
of
Irradiated
Banaba
(Lagerstroemia
speciosa Linn.) Leaves. Journal of Applied
Sciencse Research, 1, 95-98.
Department of Health. (2012). Philippine Health Service Delivery Profile.
Retrieved from www.wpro.who.int/health_services/service_delivery_
profile_ philippines.pdf
Dickerson G. (1947). Hereditary obesity and efficient food utilization in mice.
Science, 105, 496.
Dietitians Association of Australia. (2014). Unsaturated Fats. Retrieved from
http://daa.asn.au/for-the-public/smart-eating-for-you/nutrition-az/unsaturated-fats/
Djarkasi, G., Numali, E., Sumual, M. & Lalujan, L. (2011). Analysis of
Bioactive Compound in Canarium Nut (Canarium indicum L.). Texas:
Tropical Plant Curriculum Project.
Ezekwe C, Nwodo O, & Ezea S. (2013). Oil extract from Gongronema
latifolium leaves exhibit anti-diabetic and anti-ulcer activities. African
Journal
of
Biotechnology,
12(45),
6419-6423.
doi:10.5897/AJB2013.13076.
Ferreira A, Mario E, Porto L, Andrade S, & Botion L. (2011). HighCarbohydrate Diet Selectively Induces Tumor Necrosis Factor-
Production in Mice Liver. Inflammation, 34 (2), 39-145.
Food and Agriculture Organization. (1994). Vegetable and Animal Oils and
Fats. Retrieved from http://www.fao.org/es/faodef/fdef14e.htm
Food and Agriculture Organization. (2014). Processing and Refining Edible
Oils.
Retrieved
from
http://www.fao.org/docrep/v4700e/
v4700e0a.htm
Fujita, H., Fujishima, H., Koshimura, J., Hosoba, M., & Yoshioka N. (2005).
Effects of Antidiabetic Treatment with Metformin and Insulin on
Serum and Adipose Tissue Adinopectin Levels in db/db Mice.
Endocrine Journal, 52(4), 427-433.
Gatbonton, P., De los Santos, G., Domingo, F., Bader, G., & Barangan, G.
(2013). Effectiveness and Tolerability of Vildagliptin Versus
42

Other
Oral Antidiabetic Drugs in Patients with Type 2 Diabetes
Mellitus in the Philippines: Results from One Year Observational
Study (EDGE). Philippines Journal of Internal Medicine, 51, 1-6.
Goji, A.D.T., Dikko, A.A.U., Bakari, A.G., Mohammed, A., Ezekiel I., &
Tanko, Y., (2009). Effect of Aqueous-Ethanolic Stem Bark Extract of
Commiphora africana on Blood Glucose Levels on Normoglycemic
Wistar Rats. International Journal of Animal and Veterinary
Advances, 1 (1), 22-24.
Handa, S.S., Khanuja, S.P.S., Longo, G., & Rakesh, D.D. (2008). Extraction
Technologies for Medicinal and Aromatic Plants. Trieste, Italy:
International Center for Science and High Technology.
Hernandez, C.L. & Paguigan, N.D. (2009). Antimutagenic Potential and
Phytochemical
Analysis
of
Selected
Philippine
Plants.
Pharmacognosy Magazine, 20(5), 388-393.
Hui, H., Tang, G., & Go, V.L. (2009). Hypoglycemic herbs and their
action mechanisms. Chinese Medicine, 2, 1-11. doi: 10.1186/17498546-4-11
International Diabetes Federation. (2013). Diabetes Atlas. Retrieved from
www.idf.org/diabetesatlas
International Diabetes Federation. (2014). Diabetes in the Philippines.
Retrieved from http://www.idf.org/membership/wp/the-philippines
Inzucchi, S.E., Bergenstal, R., Buse, J., Diamant, M., Tsapas, A., Wender,
R., & Matthews D. (2012). Management of hyperglycemia in Type 2
diabetes: A Patient-Centered Approach. Diabetes Care, 35(6), 13641379. doi: 10.2337/dc12-0413
Ip, B., Liu, C., Smith, D., Ausman, L., Wang, X. (2014) High-refinedcarbohydrate and high-fat diets induce comparable hepatic tumorigenesis
in male mice. Journal of Nutrition,144 (5), 647-653.
Jenkins, D.J., Kendall, C.W., Banach, M.S., Srichaikul, K., Vidgen, E.,
Mitchell, S., Parker, T., Nishi, S., Bashyam, B., de Souza, R., Ireland,
C., & Josse, R.G. (2011). Nuts as a Replacement for Carbohydrates
in the Diabetic Diet. Diabetes Care, 34(8), 1706-1711. doi:
10.2337/dc11-0338
Kakuda, T., Nozawa, A., & Unno, T. (2000). Inhibiting effest of theanine on
caffeine stimulation evaluated by EEF in the rat. Biosci. Biotechnol.
Biochem, 64, 287-293.

43

Kamtchouing P., Kahpui S.M., Dzeufiet P.D., Djomeni T., Asongalem L., &
Dimo T. (2006). Antidiabetic Activity of Methanol/Methylene Chloride
Stem Bark Extracts of Terminalia superba and Canarium
schweinfurthii on Streptozotocin-induced Diabetic Rats. Journal of
Ethnopharmacology, 104, 306-309.
Kaczmar, T. (1998). Herbal Support for Diabetes Management. Clinical
Nutrition Insights, 6, 1-4.
Keren, P., George, J., Shaish, A., Levkovitz, H., Janakovic, Z., Afek,
A., Goldberg, I., Kopolovic, J., Keren, G., & Harats, D.. (2000). Effect
of Hyperglycemia and Hyperlipidemia on Atherosclerosis in LDL
Receptor-deficient Mice: Establishment of a Combined Model and
Association with Heat Shock Protein 65 Immunity. Diabetes, 49(6),
1064-1069.
Kotake, J., Tanaka, Y., Umehara, N., Miyashita, A., Tsuru, T., Hikida, S., &
Mizote, H. (2004). Effects of a High-Monounsaturated Fat Diet on
Glucose and Lipid Metabolisms in Normal and Diabetic Mice. J Nutr
Sci Vitaminol (Tokyo), 50 (2), 106-113.
Kouambou, C., Dimo, T., Dzeufiet, P., Ngueguim, F., Tchamadeu, M.,
Wembe, E., & Kamtchouing, P. (2007). Antidiabetic and
Hypolipidemic Effects of Canarium schweinfurthii Hexane Bark
Extract in Streptozotocin-Diabetic Rats. Pharmacologyonline, 1, 209219.
Klueh, U., Liu, Z., Cho, B., Ouyang, T., Feldman, B., Henning, T., Kaur, M.,
& Kreutzer, D. (2006). Continuous Glucose Monitoring in Normal
Mice and Mice with Prediabates and Diabetes. Diabetes Technology
& Therapeutics, 8(3), 402-412.
Kurien, B. & Scofield, R.H. (1999). Mouse Urine Collection Using Clear
Plastic
Wrap.
Laboratory
Animals,
33,
83.
doi:
10.1258/002367799780578525
Kris-Etherton, P.M. (2013, August 5). Pili Nuts Promise Vitamins and
Minerals.
The
Wall
Street
Journal.
Retrieved
from
http://online.wsj.com/news/
articles/SB1000142412
7887323968704578650020593473051
Laforteza, J.A.M. (2000). A Comparative Study of the Hypoglycemic,
Cytotoxicity and Antifungal Activities of Extracts of Canarium ovatum
Engl. (Undergraduate Thesis). University of the Philippines, Diliman,
Quezon City.

44

Lanting, M.V., Jr., & Palaypayon, C.M. (2002). Forest Tree Species with
Medicinal Uses, 11, 19. Laguna, Philippines: Department of
Environment and Natural Resources.
Ludvik, B., Waldhausl, W., Prager, R., Kautzky-Willer, A., & Pacini, G.
(2003). Mode of action of Ipomoea batatas (Caiapo) in type 2 diabetic
patients. Metabolism, 52(7), 875-880.
Martinod, S. (2005). Canarium indicum Nut Oil. Retrieved from
www.fda.gov/OHRMS/Dockets/ dockets/95s0316/95s-0316-rpt028904-vol227.pdf)
Matravadia, S., Herbst, E., Jain, S., Mutch, D., & Holloway, G. (2014). Both
linoleic and alpha-linolenic acid prevent insulin resistance but have
divergent impacts on skeletal muscle mitochondrial bioenergetics in
obese Zucker rats. American Journal of Physiology, 307(1), 102-114.
doi: 10.1152/ajpendo.00032.2014.
Mayo Clinic. (2014). Dietary Fats: Know which Types to Choose. Mayo
Foundation for Medical Education and Research. Retrieved from
http://www.mayoclinic.org/healthy-living/nutrition-and-healthyeating/in-depth/fat/art-20045550
Merck Manuals. (2015). Drug Bioavailability. Whitehouse Station, New
Jersey: Merck Sharp & Dohme Corp.
Mitri J., & Hamdy O. (2009). Diabetes medications and Body weight. Expert
Opin Drug Saf. 8(5), 573-584. doi: 10.1517/14740330903081725
Mogana R. & Wiart C. (2011). Canarium L.: A Phytochemical and
Pharmacological Review. Journal of Pharmacy Research, 4(8), 24822489.
Mokiran N., Ismail A., Azlan A., Hamid M., & Hassan F. (2014). Effect of
Dabai (Canarium odontophyllum) Fruit Extract on Biochemical
Parameters of Induced Obese-Diabetic Rats. Journal of Functional
Foods, 8, 139-149.
Morris, L., McGee, J., & Kitabachi, A. (1981). Correlation between plasma
and urine glucose in diabetes. Annals of Internal Medicine, 94(4),
469-471.
Mukesh R., & Namita P. (2013). Medicinal Plants with Antidiabetic Potential
A Review. American-Eurasian J. Agric. & Environ. Sci., 13, 81-94.

45

Nicolosi, R., Woolfrey, B., Wilson, T., Scollin, P., Handelman, G., & Fisher,
R. (2004). Decreased aortic early atherosclerosis and associated risk
factors in hypercholesterolemic hamsters fed a high- or mid-oleic acid
oil compared to a high-linoleic acid oil. The Journal of Biochemistry,
15 (9), 540-547.
Noonan, W. & Banks, R. (2000). Renal Function and Glucose Transport in
Male and Female Mice with Diet-induced Type II Diabetes Mellitus.
Proceedings of the Society for Experimental Biology and Medicine,
225, 221-230
Pacini G., Omar B., & Ahren B. (2013). Methods and Models for Metabolic
Assessment in Mice. Journal of Diabetes Research.
Park, M., Kinra, S., Ward, K., White, B., & Viner, R. (2009). Metformin for
Obesity in Children and Adolescents: A Systematic Review.
Diabetes Care, 32, 1743-1745.
Parillo, M., Rivellese, A.A., Ciardullo, A.V., Capaldo, B., Giacco, A.,
Genovese, S, & Riccardi, G. (1992). A High-Monounsaturated-Fat/
Low-Carbohydrate Diet Improves Peripheral Insulin Sensitivity in
Non-Insulin Dependent Diabetic Patients. Metabolism, 41(12), 13731378.
Phillips, P. J., Scicchitano, R., Clarkson, A. R., & Gilmore, H. R. (1978),
Metformin Associated Lactic Acidosis. Australian and New
Zealand Journal
of
Medicine,
8, 281284.
doi:10.1111/j.14455994. 1978.tb04524.x
Pierroz, D., Zipotopoulou, M., Ungsunan, L., Moschos, S., Flier, J., &
Mantzoros, C. (2002). Effects of Acute and Chronic Administration of
the Melanocortin Agonist MTII in Mice with Diet-Induced Obesity.
Diabetes, 51, 1337-1345.
Prasad, S.K., Kulshreshtha, A., & Qureshi, T.N. (2009). Antidiabetic
Activity of Some Herbal Plants in Streptozotocin Induced Diabetic
Albino Rats. Pakistan Journal of Nutrition, 8, 551-557.
Pub Chem. (n.d.). Compound Summary for Linoleic Acid. National Center for
Biotechnology Information. Retrieved from http://pubchem.ncbi.
nlm.nih.gov/ compound /linoleic_acid#section=Top

46

Pub Chem. (n.d.). Compound Summary for Oleic Acid. National Center for
Biotechnology
Information.
Retrieved
from
http://pubchem.
ncbi.nlm.nih.gov/ compound/ oleic_acid#section=Information-Sources
Puri, D. (2001). The insulinotropic activity of a Nepalese medicinal plant
Biophytum sensitivum: preliminary experimental study.
J
Ethnopharmacol , 78(1), 89-93.
Rakoczy, E.P., Ali Rahman, I.S., Binz, N., Li, C., Vagaja, N.N., de Pinho, M.
& Lai, C. (2010). Characterization of a Mouse Model of
Hyperglycemia and Retinal Neovascularization. American Journal of
Pathology, 177(5), 2659-2670.
Ravi, K., Sathish Sekar, D., & Subramanian, S. (2003). Hypoglycemic
Activity of Inorganic Constituents in Eugenia jambolana Seed on
Streptozotocin- Induced Diabetes in Rats. Biological Trace
Element Research, 99, 145-155. doi: 10.1385/BTER:99:1-3:145
Reaven, P., Parthasarathy, S., Grasse, B.J., Miller, E., Almazan, F.,
Mattson, F.H., Khoo, J.C., Steinberg, D., & Witztum, J.L. (1993).
Feasibility of using an oleate-rich diet to reduce the susceptibility of
low-density lipoprotein to oxidative modification in humans. Am. J.
Clin. Nutr. 1991, 54, 701706.
Rocca, A., LaGreca, J., Kalitsky, J., & Brubaker P. (2001). Monounsaturated
fatty acid diets improve glycemic tolerance through increased
secretion of glucagon-like peptide-1. Endocrinology, 142(3), 11481155.
Said, O., Fulder,S., Khalil, K., Azaizeh, H., Kassis, E., & Saad, B.
(2008).Maintaining A Physiological Blood Glucose Level with
Glucolevel, A Combination of Four Anti-Diabetes Plants Used in the
Traditional Arab Herbal Medicine. eCam, 5, 421-428. doi:
10.1093/ecam/nem047
Scnell, A., Gregg, E., Bowman, B., Morris, S., Zhang, X., Avenell, A, Gregg,
E., Bowman, B., Schmid, C., & Lau, J. (2005). Long-term
effectiveness of weight-loss interventions in adults with pre-diabetes:
a review. Am J Prev Med, 28, 126139.
Serreze, D.V., Ottendorfer, E.W., Ellis, T.M., Gauntt, C.J., & Atkinson, M.A.
(2000). Acceleration of Type I Diabetes by a coxsackievirus Infection
Requires a Preexisting Critical Mass of Autoreactive T-cells in
pancreatic Islets. Diabetes, 49(5), 708-711.

47

Shaw, J., Sicree, R., & Zimmet, P. (2010). Global estimates of the
prevalence of diabetes for 2010 and 2030. Diabetes Research and
Clinical Practice, 87, 4-14. doi: 10.1016/j.diabres.2009.10.007
Shirwaikar, A., Rajendran, K., & Barik, R. (2006). Effect of Aqueous Bark
Extract of Garuga pinnata Roxb. in Streptozotocin-nicotinamide
Induced Type-II Diabetes Mellitus. Journal of Ethnopharmacology,
107(2), 285-290.
Soria, M., Sy, R., Vega, B., Ty-Willing, T., Abenir-Gallardo, A., Velandria, F.,
& Punzalan, F. (2009). The incidence of type 2 diabetes mellitus in
the Philippines: A 9-year cohort study. Diabetes Research and
Clinical Practice, 86, 130-133. doi:10.1016/j.diabres.2009.07.014
Stoppler, M. (2014). Glucose Tolerance Test. Medicine Net. Retrieved from
www.medicinenet.com/glucose_tolerance_test/page3.htm
Sundram, K., French, M.A. & Clandinin, T.A. (2003) Exchanging partially
hydrogenated fat for palmitic acid in the diet increases LDLcholesterol
and
endogenous
cholesterol
synthesis
in
normocholesterolemic women. Eur J Nutr. 42, 188-194.
Surgitech Reagent Strip For Urinalysis. (n.d.). Methodology: Glucose.
Brussels, Belgium: Surgitech.
Tadayyon, B. (2013). The Miracle of Nuts, Seeds and Grains. Gordon, New
South Wales: Xlibris Corporation.
Tang, D. & Tang, J. (2012). Canarium album Granule with Hypolipidemic
and Hypoglycemic Effects. Fuzhou, Fujian: Fuzhou Gulou District
Date Biotechnology.
Tang, T. & Reed, M. (2001). Exercise Adds to Metformin and Acarbose
Efficacy in db/db Mice. San Francisco, CA: Shaman Pharmaceuticals.
Tiwari, A. & Rao, J.M. (2002). Diabetes mellitus and multiple therapeutic
approaches of phytochemicals: Present status and future prospects.
Current Science, 83, 30-38.
Thomassian, B. (2013). Diabetes Detectives Finding Uncommon
Conditions. Diabetes Educational Services. Retrieved from
http://www.diabetesed.
net/~diabetes/page
/_files/DiabetesDetectives-Finding-Uncommon-Conditions-PDF-144-KB_259.PDF

48

Tripon, J. (2000). The Screening for Hypoglycemic, Antimicrobial, and

Molluscicidal Activity of Pili Pulp and Nut Oils and a Further Study on
th Hypoglycemic Activity of Alugbati. (Undergraduate Thesis).
University of the Philippines, Diliman, Quezon City.
Urtal, A. (2008). Antioxidant Potential, Total Phenolic Content and Total
Flavonoid Content of Pili (Canarium ovatum Engl.) Kernel Subjected
to Different Drying Temperatures. (Undergraduate Thesis). University
of the Philippines, Los Baos, Laguna.
Valisno, J.O. (2013). Diabetes and the Filipino Diet. Retrieved from
www.pchrd.dost.gov.ph
Vassillou, E.K., Gonzalez, A., Garcia, C., Tadros, J.H., Chakraborty, G., &
Toney, J.H. (2009). Oleic Acid and Peanut Oil High in Oleic Acid
Reverse the Inhibitory Effect of Insulin Production of the Inflammatory
Cytokine TNF- both in vitro and in vivo Systems. Lipids in Health
and Disease, 8, 25. doi:10.1186/1476-511X-8-25
Vijayvargia, R., Kumar, M., & Gupta S. (2000). Hypoglycemic effect of
aqueous extract of Enicostemma littorale Blume (chhota chirayata) on
alloxan induced diabetes mellitus in rats. Indian Journal of
Experimental Biology, 38, 781-784
Viollet, B., Guigas, B., Sanz Garcia, N., Leclerc, J., Foretz, M., & Andreelli,
F. (2012). Cellular and molecular mechanisms of metformin: an
overview. Clinical Science (London, England: 1979), 122(6), 253
270. doi:10.1042/CS20110386
Virdi, J., Sivakami, S., Shahani, S., Suthar, A., Banavalikar, M., &
Biyani, M. (2003). Antihyperglycemic effects of three extracts from
Momordica charantia. Journal of Ethnopharmacology, 88, 107-111.
Wadkar, K.A., Magdum, C.S., Patil, S.S., & Naikwade, N.S. (2008). Antidiabetic potential and Indian medicinal plants. Journal of Herbal
Medicine and Toxicology, 2, 45-50.
Wang, L. & Weller, C.L. (2006). Recent Advances in Extraction of
Nutraceuticals from Plants. Trends in Food Science and Technology,
17, 300-312
Wang X., Lu W., Huang G., Lu F., Tu Q., Zhang Q., Ogihara T., Sato N.
(2010). Effects of the TCM Formulation Wangs Ketsumeisei
Extract in the Treatment of Diabetes Mellitus. Integrative Therapy
Research.

49

Wanjek, C. (2010). Striking Gold in Rodent Urine Collection. NIH Catalyst.

Retrieved from http://www.nih.gov/catalyst
Whaley, J. M., Tirmenstein, M., Reilly, T. P., Poucher, S. M., Saye, J.,
Parikh, S., & List, J. F. (2012). Targeting the kidney and glucose
excretion with dapagliflozin: preclinical and clinical evidence for
SGLT2 inhibition as a new option for treatment of type 2 diabetes
mellitus. Diabetes, Metabolic Syndrome and Obesity: Targets and
Therapy, 5, 135148. doi:10.2147/DMSO.S22503
World Health Organization. (2014). Diabetes: The Cost of Diabetes.
Retrieved from http://www. who.int/mediacentre/factsheets/fs236/en/)
World Health Organization Expert Committee on Diabetes Mellitus.
(1980). Second Report, World Health Organization Technical
Report Series 646, 66. Geneva, Switzerland.
Yoshikawa, M., Shimada, H., Nishida, N., Li, Y., Toguchida, I., Yamahara,
J., & Matsuda, H. (1998). Antidiabetic principles of natural medicines.
II.Aldose reductase and alpha-glucosidase inhibitors from Brazilian
natural medicine, the leaves of Myrcia multiflora DC. (Myrtaceae):
structures of myrciacitrins I and II and myrciaphenones A and B.
Chem Pharm Bull (Tokyo), 46(1), 113-119.
Zarinah, Z., Maaruf, A.G., Nazaruddin, R., Wong, W.W.W., & Xueding, X.
(2014). Extarction and Determination of Physico-chemical
Characteristics of Pili Nut Oil. International Food Research Journal,
21(1), 297-301.
Zhang, P. (2011). Glucose Tolerance Test in Mice. Bio Protocol. Retrieved
from http://www.bio-protocol.org/e159
Zhang, P., Zhang, X. Brown J., Vistisen, D., Sicree, R., Shaw, J., & Nichols,
G. (2010). Global healthcare expenditure on diabetes for 2010 and
2030. Diabetes Research and Clinical Practice, 87, 293-301.
Zuo, Y.Q., Liu, W.P., Niu, Y.F., Tian, C.F., Xie, M.J., Chen, X.Z., & Li, L.
(2008). Bis(alphafurancarboxylato)oxovanadium(IV) prevents and
improves dexamethasone-induced insulin resistance in 3T3-L1
adipocytes. J Pharm Pharmacol, 60(10), 1335-1340.

50

TABLES
Table 1. Mean SD of the body weight (g) of male mice (n=24) given different dosages
of C. ovatum kernel oil
Experimental
Period

No Extract (0
mg/kg)

Low Dose (300

mg of kernel oil
per kg B.W.)

High Dose
(3000 mg of
kernel oil per
kg B.W.)

Metformin (150
mg of
Metformin per
kg B.W.)

Day 1 (first day

of high
carbohydrate
diet)

36.50 1.63

34.85 0.88

34.70 2.62

36.85 0.69

Day 22 (first day

of treatment)

40.32.85

36.12 1.3

35.75 3.33

40.3 2.12

Day 36 (last day

of treatment

41.83 2.57

38.27 1.41

37.2 1.67

41.62 1.66

Table 2. Mean SD of the blood glucose levels (mg/dl) of male mice (n=24) before and
after the two-week administration of kernel oil and metformin
Treatment
Group

Day 22 (start of treatment)

Day 36 (end of treatment)

No Extract (0
mg/kg)

197.17 15.22

212.17 17.52

Low Dose (300

mg of kernel oil
per kg B.W.)

216.33 16.33

202.83 17.88

High Dose (3000

mg of kernel oil
per kg B.W.)

200.67 17.93

182.5 16.43

Metformin (150
mg of metformin
per kg B.W.)

214.5 17.03

188.17 12.61

51

Table 3. Mean SD of the blood glucose levels (mg/dl) of male mice (n=24) at specific
time periods after oral administration of high glucose
Treatment
Group

Before
OGTT

No Extract
(0 mg/kg)

Minutes After Oral Glucose Tolerance Test (OGTT)

30 min

60 min

90 min

120 min

212.17
17.52

217.33
12.27

210.00
21.68

232.83
19.71

226.83
20.64

Low Dose
(300 mg of
kernel oil per
kg B.W.)

202.83
17.88

217.83
9.89

228.83
23.66

263.33
27.71

245.17
24.49

High Dose
(3000 mg of
kernel oil per
kg B.W.)

182.5
16.43

223.33
32.37

216.5
33.76

210.00
28..45

185.5
15.71

Metformin
(150 mg of
metformin
per kg B.W.)

188.17
12.61

224.83
18.80

201.33
16.61

200.83
14.29

181.67
10.33

52

Weight (g)

FIGURES
50
45
40
35
30
25
20
15
10
5
0

Day 22
Day 36

No Oil (0
mg/kg)

Low Dose (300 High Dose

mg/kg)
(3000 mg/kg)

Metformin
(150 mg/kg)

Group

Figure 1. Comparison of mean weights (g) among treatment groups during the
experiment. Means with asterisks indicate a significant change.

Blood glucose concentration (mg/dl)

250
*

200

150
100

Day 22
Day 36

50
0
No Oil (0
mg/kg)

Low Dose (300 High Dose

Metformin
mg/kg)
(3000 mg/kg) (150 mg/kg)
Group

Figure 2. Comparison of Mean blood glucose levels (mg/dl) among treatment groups
before (Day 22) and after treatment (Day 36). Means with asterisks indicate a significant
change.

53

Blood glucose concentration (mg/dl)

300
250
200

No Oil (0 mg/kg)

150

Low Dose (300 mg/kg)

100

High Dose (3000 mg/kg)

50

Metformin (150 mg/kg)

0
0

30

60

90

120

TIme (min after administration)

Figure 3. Glucose tolerance curves of hyperglycemic mice given different dosages of C.

ovatum kernel oil and Metformin

Blood glucose concentration (mg/dl)

300
250
200
No Oil (0 mg/kg)

150

Low Dose (300 mg/kg)

High Dose (3000 mg/kg)

100

Metformin (150 mg/kg)

50
0
0

30

60

90

120

Time (min after administration)

Figure 4. Blood glucose levels of the four treatment groups during the oral glucose
tolerance test

54

APPENDICES
Appendix A-1. Analysis of Variance for Initial Weights (g) of male mice at first
day of hyperglycemic induction (Day 1)
Source

SS

df

MS

P-value

Treatment

22.095

7.365

2.735

0.071

Error

53.850

20

2.693

Total

75.945

23

Appendix A-2. Analysis of Variance for weights (g) of male mice at start of
treatment (Day 22)
Source

SS

df

MS

P-value

Treatment

114.810

38.270

6.024

0.004

Error

127.063

20

6.353

Total

241.873

23

Appendix A-3. Analysis of Variance for weights (g) of male mice after two-week
treatment period (Day 36)
Source

SS

df

MS

P-value

Treatment

99.155

33.052

9.327

0.000

Error

70.875

20

3.544

Total

170.030

23

55

Appendix A-4. Paired t-tests between weights (g) at first day of hyperglycemic
induction and first day of treatment
Treatment
Group

t critical

P value

Significance

No Oil

6.884

0.001

Significant

Low Dose

-6.697

0.001

Significant

High Dose

1.364

0.231

Not significant

Metformin

5.186

0.004

Significant

Appendix A-5. Paired t-tests between weights (g) at first day and last day of
treatment period
Treatment
Group

t critical

P value

Significance

No Oil

-1.958

0.108

Not significant

Low Dose

-4.313

0.008

Significant

High Dose

-1.602

0.170

Not significant

Metformin

-1.896

0.116

Not significant

Appendix A-6. Analysis of Variance for blood glucose levels (mg/dl) of male mice
at first day of treatment (Day 22)
Source

SS

df

MS

P-value

Treatment

1680.333

560.111

2.019

0.144

Error

5549.000

20

277.450

Total

7229.333

23

56

Appendix A-7. Analysis of Variance for blood glucose levels (mg/dl) of male mice
at last day of treatment (Day 36)
Source

SS

df

MS

P-value

Treatment

3305.833

1101.944

4.176

0.019

Error

5278.000

20

263.900

Total

8583.833

23

Appendix A-8. Paired t-tests between blood glucose levels (mg/dl) of male mice
at first and last day of treatment period
Treatment Group

t critical

P value

Significance

No Oil

-2.567

0.050

Significant

Low Dose

2.723

0.042

Significant

High Dose

2.590

0.049

Significant

Metformin

3.458

0.018

Significant

57

Appendix A-9. Tukey-HSD Test for blood glucose levels in different treatment
groups

Appendix A-10. Tukey-HSD Test Homogenous Subset of Treatment Groups with

Non-statistically Significant Differences in Blood Glucose Levels

58

Appendix A-11. Multivariate Analysis of Variance of Blood glucose levels and

weights of male mice at first day of treatment (Day 22)

59

Appendix B-1. Body weights (g) of male mice

Treatment Group

Group 1
No Oil
(0 mg/kg)

Group 2
Low Dose
(300 mg/kg)

Group 3
High Dose
(3000 mg/kg)

Group 4
Metformin
(150 mg/kg)

Body Weight (grams)

Day 1
Day 22
Day 36
(first day of
(First day of
(last day of
hyperglycemic
treatment)
treatment)
induction)
35.0
39.0
37.9
35.0
37.1
40.9
34.5
41.6
42.2
39.4
45.3
45.6
36.4
39.8
43.2
36.2
39.0
41.2
30.4
36.7
37.4
33.2
35.1
37.1
34.5
35.2
39.6
34.4
35.4
37.1
34.4
38.5
40.4
30.7
35.8
38.0
32.6
39.5
38.8
34.2
36.1
35.9
30.0
34.3
36.2
30.1
32.0
35.3
34.8
39.8
39.5
33.4
32.8
37.5
36.0
39.0
40.9
36.8
39.6
43.0
35.6
38.2
38.7
37.7
44.2
42.6
37.0
41.0
43.0
37.0
39.8
41.5

60

Appendix B-2. Blood Glucose Levels (mg/dl) of individual male mice in all
treatment groups
Treatment
Group

Group 1
No extract
(0 mg/kg)

Group 2
Low Dose
(300 mg/kg)

Group 3
High Dose
(3000 mg/kg)

Group 4
Metformin
(150 mg/kg)

Blood Glucose Levels

During Two-week Treatment
Period

Day 22 (First
day of
treatment)
222
180
189
188
207
197
242
202
222
197
223
212
216
188
222
189
211
178
192
204
215
214
243
219

Day 36
(Last day of
treatment)
227
181
212
215
208
230
214
200
215
168
211
209
163
176
199
177
206
174
172
194
189
182
183
209

61

Blood Glucose Levels after OGTT

30 mins

60 mins

90
mins

120
mins

238
210
207
207
217
225
220
213
232
220
220
202
208
213
203
206
222
288
229
194
223
253
226
224

238
201
221
202
176
222
250
240
251
189
226
217
188
207
215
201
205
283
209
188
221
202
212
176

272
223
217
229
228
228
271
296
272
230
282
229
185
204
212
200
265
194
205
183
201
185
214
217

255
248
200
220
219
219
240
267
263
219
268
214
176
177
192
183
214
171
195
173
177
169
185
191

Appendix C. Urine Glucose of Male mice

Treatment Group

Group 1
No extract
(0 mg/kg)

Group 2
Low Dose
(300 mg/kg)

Group 3
High Dose
(3000 mg/kg)

Group 4
Metformin
(150 mg/kg)

Urine Glucose
Day 22 (First Day of
Day 36 (Last Day of
treatment)
treatment)
5 - trace
Negative
5 - trace
Negative
5 - trace
Negative
5 - trace
Negative
5 - trace
Negative
5 - trace
Negative
5 - trace
Negative
5 - trace
>= 2000 mg/dl
5 - trace
>= 2000 mg/dl
5 - trace
Negative
5 - trace
Negative
5 - trace
Negative
5 - trace
Negative
5 - trace
Negative
5 - trace
Negative
5 - trace
Negative
5 - trace
Negative
5 - trace
Negative
5 - trace
Negative
5 - trace
Negative
5 - trace
Negative
5 - trace
Negative
5 - trace
Negative
5 - trace
Negative

62

Appendix D

Canarium ovatum (Pili) tree

Canarium ovatum (Pili) fruit

63

Canarium ovatum (Pili) kernels

Dried Kernels

64

Oil Extractor

Modified Metabolic Cages

65

Weighing of Mouse

Mouse in Restraint Tube

66

Blood Extraction

Glucometer

67

Oral Gavage

Urine Glucose Strips

68