1

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 7th 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

2

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.

3

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

4

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

5

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.

6

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, neuropathy, nephropathy,

7

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

8

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 B12

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 country’s 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).

9

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

10

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

11

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 (piling-

okai), 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

12

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

13

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).

14

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.

15

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

16

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

17

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.

18

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 ± 2°C and a regular 12 hour light/ 12 hour dark

cycle. Assignment to cages was done randomly and cages were marked for

19

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.

20

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 µl 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

21

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

22

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) Tukey’s

test as post hoc analysis and (3) Paired t-test. The level of statistical

significance was set at p ≤ 0.05 for all tests.

23

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

24

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,

25

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).

26

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.

27

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

28

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.

29

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 α-glucosidase

and 38% for α-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.

30

Vassiliou et al. (2009) found that feeding Type II diabetic mice oleic acid-

rich 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).

31

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

32

(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

33

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 body’s

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

34

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

35

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).

36

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 α-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

37

other than glucose excretion, possibly through increasing insulin secretion and

subsequently increasing blood glucose uptake by the cells.

38

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

39

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.

40

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 B12 (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.

41

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 B12 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

42

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-a-z/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). High-

Carbohydrate Diet Selectively Induces Tumor Necrosis Factor-α 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

43

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: 10.1186/1749-8546-4-11

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), 1364-1379. doi: 10.2337/dc12-0413

Ip, B., Liu, C., Smith, D., Ausman, L., Wang, X. (2014) High-refined-carbohydrate 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.

44

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, 209-

219.

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.

45

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-rpt0289-04-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-healthy-

eating/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), 2482-2489.

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.

46

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), 1373-1378.

Phillips, P. J., Scicchitano, R., Clarkson, A. R., & Gilmore, H. R. (1978),

Metformin Associated Lactic Acidosis. Australian and New Zealand Journal of Medicine, 8, 281–284. 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

47

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, 701–706.

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), 1148-1155.

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, 126–139.

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.

48

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 LDL-cholesterol 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/Diabetes-

Detectives-Finding-Uncommon-Conditions-PDF-144-KB_259.PDF

49

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 Baños, 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-α 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). Anti-

diabetic 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 “Wang’s Ketsumeisei Extract” in the Treatment of Diabetes Mellitus. Integrative Therapy Research.

50

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, 135–148. 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.

51

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)

40.3±2.85 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

52

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

Minutes After Oral Glucose Tolerance Test (OGTT)

30 min 60 min 90 min 120 min

No Extract

(0 mg/kg)

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

53

FIGURES

Figure 1. Comparison of mean weights (g) among treatment groups during the

experiment. Means with asterisks indicate a significant change.

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.

*

0

5

10

15

20

25

30

35

40

45

50

No Oil (0mg/kg)

Low Dose (300mg/kg)

High Dose(3000 mg/kg)

Metformin(150 mg/kg)

We

igh

t (g

)

Group

Day 22

Day 36

**

**

0

50

100

150

200

250

No Oil (0mg/kg)

Low Dose (300mg/kg)

High Dose(3000 mg/kg)

Metformin(150 mg/kg)

Blo

od

glu

cose

co

nce

ntr

atio

n (

mg/

dl)

Group

Day 22

Day 36

54

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

ovatum kernel oil and Metformin

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

tolerance test

0

50

100

150

200

250

300

0 30 60 90 120

Blo

od

glu

cose

co

nce

ntr

atio

n (

mg/

dl)

TIme (min after administration)

No Oil (0 mg/kg)

Low Dose (300 mg/kg)

High Dose (3000 mg/kg)

Metformin (150 mg/kg)

0

50

100

150

200

250

300

0 30 60 90 120

Blo

od

glu

cose

co

nce

ntr

atio

n (

mg/

dl)

Time (min after administration)

No Oil (0 mg/kg)

Low Dose (300 mg/kg)

High Dose (3000 mg/kg)

Metformin (150 mg/kg)

55

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 F P-value

Treatment 22.095 3 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 F P-value

Treatment 114.810 3 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 F P-value

Treatment 99.155 3 33.052 9.327 0.000

Error 70.875 20 3.544

Total 170.030 23

56

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 F P-value

Treatment 1680.333 3 560.111 2.019 0.144

Error 5549.000 20 277.450

Total 7229.333 23

57

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 F P-value

Treatment 3305.833 3 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

58

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

59

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

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

60

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

Treatment Group

Body Weight (grams)

Day 1 (first day of

hyperglycemic induction)

Day 22 (First day of treatment)

Day 36 (last day of treatment)

Group 1 No Oil

(0 mg/kg)

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

Group 2

Low Dose (300 mg/kg)

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

Group 3

High Dose (3000 mg/kg)

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

Group 4

Metformin (150 mg/kg)

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

61

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

treatment groups

Treatment

Group

Blood Glucose Levels

During Two-week Treatment

Period

Blood Glucose Levels after OGTT

Day 22 (First

day of

treatment)

Day 36

(Last day of

treatment)

30 mins 60 mins 90

mins

120

mins

Group 1

No extract

(0 mg/kg)

222 227 238 238 272 255

180 181 210 201 223 248

189 212 207 221 217 200

188 215 207 202 229 220

207 208 217 176 228 219

197 230 225 222 228 219

Group 2

Low Dose

(300 mg/kg)

242 214 220 250 271 240

202 200 213 240 296 267

222 215 232 251 272 263

197 168 220 189 230 219

223 211 220 226 282 268

212 209 202 217 229 214

Group 3

High Dose

(3000 mg/kg)

216 163 208 188 185 176

188 176 213 207 204 177

222 199 203 215 212 192

189 177 206 201 200 183

211 206 222 205 265 214

178 174 288 283 194 171

Group 4

Metformin

(150 mg/kg)

192 172 229 209 205 195

204 194 194 188 183 173

215 189 223 221 201 177

214 182 253 202 185 169

243 183 226 212 214 185

219 209 224 176 217 191

62

Appendix C. Urine Glucose of Male mice

Treatment Group

Urine Glucose

Day 22 (First Day of

treatment)

Day 36 (Last Day of

treatment)

Group 1

No extract

(0 mg/kg)

5 - trace Negative

5 - trace Negative

5 - trace Negative

5 - trace Negative

5 - trace Negative

5 - trace Negative

Group 2

Low Dose

(300 mg/kg)

5 - trace Negative

5 - trace >= 2000 mg/dl

5 - trace >= 2000 mg/dl

5 - trace Negative

5 - trace Negative

5 - trace Negative

Group 3

High Dose

(3000 mg/kg)

5 - trace Negative

5 - trace Negative

5 - trace Negative

5 - trace Negative

5 - trace Negative

5 - trace Negative

Group 4

Metformin

(150 mg/kg)

5 - trace Negative

5 - trace Negative

5 - trace Negative

5 - trace Negative

5 - trace Negative

5 - trace Negative

63

Appendix D

Canarium ovatum (Pili) tree

Canarium ovatum (Pili) fruit

64

Canarium ovatum (Pili) kernels

Dried Kernels

65

Oil Extractor

Modified Metabolic Cages

66

Weighing of Mouse

Mouse in Restraint Tube

67

Blood Extraction

Glucometer

68

Oral Gavage

Urine Glucose Strips