The Effect of Agarwood Leaf Extracts on Blood Glucose Level of Type II Diabetes Mellitus in ICR Male Mice

 

Aws Ali Fayyadh¹, Husni Ibrahim¹*, Haniza Hanim Mohd Zain¹, Mothanna Sadiq Al-Qubaisi²

¹Department of Biology, Faculty of Science and Mathematics, University Pendidikan Sultan Idris, 35900 Tanjong Malim, Perak, Malaysia

²Institute of Bioscience, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia

 

ABSTRACT:

In order to study the potential of Aquilaria malaccensis (agarwood) leaves extracts to combat diabetes mellitus, in vivo animal models might provide a true representation of the range of the human diabetic conditions. The model used here, the streptozotocin-induced diabetic male ICR mice. A group of ICR male mice, aged 8 weeks were induced by a single intraperitoneal injection of 100 mg /kg body weight of streptozotocin (STZ). Mice with fasting blood glucose over 200 mg/dl were used in this experiment. Diabetic mice were divided into eight groups, 6 groups were force-fed with three doses of plant extract (50, 100 and 150) mg plant extract /kg body weight for 14 consecutive days each and the rest groups were the control groups. Two different types of solvents were used in this study i.e. distilled water and 95% methanol. Low doses of aqueous and methanolic extracts (50 mg/Kg b.w) exhibited valuable treatments for hyperglycemia via lowering glucose levels to within the normal range in diabetic mice. Glucose levels in blood were reduced on the most recent days of treatment and, sometimes, last days of treatments, at the lowest concentration (50 mg/Kg b.w), were more effective in glucose levels reductions.

 

 

1. INTRODUCTION:

Diabetic Mellitus (DM) is a heterogeneous disease defined by high blood glucose levels and dyslipidemia. Diabetes is usually accompanied by a major metabolic deficiency which is the failure of peripheral tissues in the body to properly exploit glucose, thereby leading to chronic hyperglycemia^1-3. Diabetes also increases the oxidative stress levels and usually leads to intricate health problems such as cardiovascular, neurological, retinal, renal diabetic problems^4,5. Moreover, DM is obviously resulting in a serious economic burden worldwide due to its increasing prevalence rates⁶.

 

There had been an increased interest in studies of other potential oral anti-diabetic drugs and renewed for attention to herbal medicines and traditional therapies^7-12.

 

This has stimulated a new wave of studies in alternative medicine and the World Health Organization (WHO) encourages the exploitation of traditional herbal remedies in national health care programs because of their safety, availability, low-cost and people have faith in them^13,14. Over 1100 plant species have been identified to be able to treat diabetic symptoms¹⁵ by improving insulin secretion¹⁶, or by imitating insulin activity¹⁷. For instance, Ocimum sanctum leaves were reported to increase glucose tolerance and insulin resistance¹⁸, also the seeds, leaves, and fruit of Momordica charantia was reported to increase glucose tolerance and insulin resistance as well¹⁹, moreover Annona squamosal leaves were found to decrease blood glucose level and cholesterol level²⁰. It is worth mentioning that the extraction methods are used pharmaceutically and they involve the separation of medicinally active portions of plant tissues from the inactive/inert components by using selective solvents. During extraction, solvents diffuse into the solid plant material and solubilize compounds with similar polarity ¹⁴. More than 200 pure compounds of the plant, origin has been shown to produce a hypoglycemic effect²¹ including glycosides, alkaloids, polysaccharides, galactomannan gum, organic ions, amino acids, carbohydrates, glycopeptides, and steroids.

 

Aquilaria malaccensis, the traditional herbal oral hypoglycaemic plant, might provide a useful source of new oral anti-diabetic constitutes for development as pharmaceutical entities, or as simple dietary adjuncts to existing therapies^22-24. The known use of Aquilaria malaccensis for diabetes dates about 1400 BC and is used throughout the world^25,26. After the introduction of sulfonylureas, metformin, and other therapies, the use of traditional treatments for non-insulin-dependent diabetes mellitus greatly declined in societies, although many plant extracts (like Aquilaria malaccensis) are still used as prophylactics and adjuncts to conventional medicine ^27-31. For this purpose, the natives of Southeast Asia use a hot water decoction made from the different parts of Aquilaria malaccensis²⁶. This plant has been shown to improve glucose tolerance activity³², suppress postprandial hyperglycemia and possess hypoglycemic activity in laboratory animals^24,33. Studies on the antidiabetic mechanisms of Aquilaria malaccensis leaves extract showed that the plant has the ability to repair the damaged pancreatic β2 cells^32,34; increases insulin levels and enhances insulin sensitivity²². Although there are many reports on agarwood which indicates that it has hypoglycemic activity on diabetic rats²², it is clear that there are not any documented reports on the antidiabetic activity of Aquilaria malaccensis on diabetic ICR. In this study, the effect of aqueous and methanolic extracts of Aquilaria malaccensis on blood glucose levels was determined on streptozotocin-induced diabetic ICR male mice.

 

2. MATERIALS AND METHODS:

The framework followed in this research is illustrated briefly in Figure 1

 

 

Figure 1: Methodology flowchart.

 

2.1    Chemicals:

All the chemicals utilized in this research were purchased from (Sigma Aldrich) and they include Methanol (95 %), Ethanol (70 %), Normal saline (0.9 %), Streptozotocin (STZ 98 %, WGK Germany) and Formalin.

2.2 Plant material:

Leaves of Aquilaria malaccensis were obtained from Al-Hilmi plantation in Slim River, Ipoh after cutting the branches of the trees. The fresh leaves of Aquilaria malaccensis (aqueous and methanol) prepared according to the method described by Najafi [35] with modification. An amount of two kilograms of leaves washed using tap water and then washed again using distilled water. To prevent the decomposition of chemical constituents, the leaves were dried in shade at room temperature (25°C) for 21 days until a constant weight achieved. After that, the dried leaves were grounded into a fine powder using an electric grinder. The fine powder was stored in an airtight container at room temperature to avoid humidity.

 

2.3 Preparation of plant extract:

For aqueous extraction, fine powder immersed in distilled water at solid to solvent ratio of (1:10) (W/V) in a sterile bottle for 24 hours at room temperature with occasional stirring. Then the samples were filtered using Whatman No 1 filter paper. The extractant liquid was stored in containers at -20°C in the refrigerator until using the scanvac freeze dryer. The lyophilized liquid put in Scanvac freeze-dryer (Alpha 1-2 LDplus Entry Freeze Dryer, Germany) for 72 hours to remove the distilled water. The samples were kept at -20°C up to utilize it. The resultant powder mixed with distilled water to give the desired dose: 50, 100 or 150mg plant extract/kg body weight of mice.

 

For methanolic extraction, the fine powder was soaked in 95% methanol solution at solid to solvent ratio of (1:10) (W/V) in a sterile bottle for 72 hours, followed by separating the drenched powder from the extractant solution and soaking it again in the same ratio of clean methanol solution. This procedure was repeated over 9 days and the collected extractant solution was then filtered using Whatman No 1 filter paper. The filtrate liquid was dried using a rotary evaporator apparatus. The methanol was removed under reduced pressure at 40°C until the appearance of brown gummy solids. The samples were stored at room temperature. The gummy solids were mixed with olive oil to give the desired dose: 50, 100 or 150mg plant extract/kg body weight.

 

2.4 Induction of diabetes in ICR mice:

In this experiment, diabetes was induced in 8-week-old male ICR mice by intraperitoneal (i.p.) injection with 100mg/kg Streptozotocin (STZ) at 9 am. All methods applied in this study was done under proper research ethics and care that was approved by Universiti Pendidikan Sultan Idris research committee. These animals were housed under standard housing animal conditions with controlled lighting (12 hours dark-light cycles) and temperature (25±2°C), During the entire experimental period, the animals were provided with food and water ad libitum, except for the short fasting period where the drinking water was still in free access but no food supply was provided.

 

Mice fasted overnight before STZ injection. STZ was dissolved in 0.9% of normal saline (pH 4.5) freshly before injection. Mice were screened to confirm hyperglycemia at one week after the first dose of STZ injection by testing the blood glucose. Blood glucose was determined by using the glucometer at 9 am in the morning. 1-2µl tail vein blood was produced by snipping the tail tip with sharp scissors.

 

2.5 Experimental design:

The rats were divided into eight groups; each group consisted of five male ICR mice to see the effect of agarwood of aqueous extract and methanol extract; as shown below:

·       Group 1: diabetic male mice were orally administered with aqueous extract at 50 mg/kg body weight of the plant.

·       Group 2: diabetic male mice were orally administered with aqueous extract at 100 mg/kg body weight.

·       Group 3: diabetic male mice were orally administered with aqueous extract at 150 mg/kg body weight.

·       Group 4: diabetic male mice were orally administered with methanol extract at 50 mg/kg body weight of the plant.

·       Group 5: diabetic male mice were orally administered with methanolic extract at 100 mg/kg body weight of the plant.

·       Group 6: diabetic male mice were orally administered with methanolic extract at 150 mg/kg body weight of the plant.

·       Group 7: diabetic male mice were with no administration of treatment.

·       Group 8: nondiabetic normal male mice were with no administration of treatment.

 

The first six groups were forced feeding with agarwood leaf aqueous extract and agarwood leaf methanolic extract at seven times per week for 14 consecutive days (figure 2), while both 7^th and 8^th groups were orally fed distilled water solely. The blood glucose level was determined by collecting venous blood for mice tail after seven days of STZ injection. On the first experimental day, food was removed from all animal cages, four hours prior to STZ injection. The water was provided as normal. Later, the food was provided with 10% sucrose water and the mice were closely monitored every 2 hr for 12hr in order to observe the hypoactivity, unresponsiveness, or convulsions. On the third day of the experiment, 10% sucrose water was replaced with regular water. On the seventh day of the experiment, all mice fasted for 6 hr (i.e. from 7 a.m. to 1 p.m.). After sevend days from the STZ injection, the treatment experimnt has only carried out for mice with fasting blood glucose levels equal to or high than 200mg/dl and these mice were defined as successfully induced by Non-insulin-dependent diabetes mellitus (NIDDM). The blood glucose level was checked directly before treatment administration and two hours after treatment and mice were fasted overnight before testing the blood glucose levels.

 

Figure 2: Experimental schedule of the feeding of agarwood extracts and test of blood glucose levels in ICR mice with STZ-induced diabetes

 

3. RESULTS AND DISCUSSION:

3.1 Study of aqueous and methanolic agarwood extract antidiabetic effect on male mice:

In this study, three different doses of Aquilaria malaccensis leaves extracts were chosen; 50mg/kg b.w., represent low dose, 100mg/kg b.w., represent moderate dose and 150mg/kg b.w., represent high dose. This range of doses was selected because it was widely used to evaluate the hypoglycemic activity of plants extracts ^36-40. Furthermore, the selected doses were found to be toxicologically safe as reported by Zulkifle et al., (2018) that the oral acute administration of 2000 mg/kg of Aquilaria malaccensis leaves methanol extract produced neither mortality nor changes in behavior or any other physiological activities in Sprague-Dawley Rats⁴¹.

 

3.1.1 Investigation of aqueous extract antidiabetic activity:

The initial blood glucose levels of normal ICR male mice were in the range of 8.0-8.5 mmol/L whereas blood glucose levels of streptozotocin-induced diabetic ICR male mice were in the range of 30-34 mmol/L. Figure 3 shows the effect of agarwood aqueous extract (at a concentration of 50mg/Kg b.w) on the blood glucose levels in streptozotocin-induced diabetic ICR male mice. A reduction of blood glucose levels in aqueous extract at 50mg/Kg b.w-treated diabetic ICR male mice group was 14.6%, 16.2%, 51.1%, 56.1%, 56.0%, 58.2% and 74.5%, respectively at days; 0, 1, 3, 6, 9, 12 and 14 when a compared to untreated at starting time (zero-day) (figure 3). In addition, a significant change (p < 0.05) of fasting blood glucose levels between pre-treatment and post-treatment was also observed at days; 3, 6, 9, 12 and 14. No significant difference of blood glucose levels was found in mice at days; 0 and 1 post-treatment when a compared to pre-treatment (figure 3). However, the mean blood glucose levels were significantly increased (about 400%) in the diabetic control group as compared to the non-diabetic group at all tested days. Meanwhile, blood glucose levels in the 100mg/Kg b.w aqueous leaf extract treated- diabetic ICR male mice (Group 2) significantly (p<0.05) decreased by 25.0%, 42.2%, 51.2%, 45.9%, 45.7 and 42.9% from day-0 (31.140± 1.036 mmol/L) to: day-1 (23.460± 4.143 mmol/L), day-3 (18.060±8.258 mmol/L), day-6 (15.240±2.849 mmol/L), day-9 (16.920±6.266 mmol/L), day-12 (16.980±5.542 mmol/L) and day-14 (17.860±4.132mmol/L), respectively (Figure 3). There was no significant (p<0.05) effects on reduction of blood glucose levels at 150µg/Kg-treated diabetic ICR male mice group (group-3) after days; 1, 3, 9, 12 and 14 when a compared to untreated at starting time (zero-day) (Figure 3). The present study demonstrated that the aqueous extract of Aquilaria malaccensis leaves reduced levels of blood glucose in a time-dependent manner, at the lowest concentration (50mg/Kg) in the streptozotocin-induced diabetic ICR male mice. The increase in the concentration of treatment did not lead to more decreasing in blood glucose levels. This observation suggested that low concentration might contain high water-soluble compounds that possess insulin secretion activity.

 

 

Figure 3: Two weeks effects of agarwood leaf aqueous extract (ALAE) at different concentrations: (a) 50 mg/ kg, (b) 100 mg/ kg, (c)150 mg/kg on blood glucose levels in mice with STZ-induced diabetes. Note: Mean ± standard deviation (n=5 mice/treatment). *P <0.05 compared with the pre-treated mice.

 

3.1.2 Investigation of methanolic extract antidiabetic activity:

The post-treatment levels of fasting blood glucose levels in 50µg/Kg methanolic leaf extract treated-streptozotocin-induced diabetic ICR male mice were significantly decreased compared to pre-treatment levels by 25.3%, 22.2%, 24.8% and 39.2% at days-0, 9, 12 and 14, respectively (figure 4). In group-8, the fasting blood glucose levels of day-1, day-6, day-9 and day-12 were decreased significantly (p < 0.05) 45.4%, 46.3%, 33.3% and 34.2%, respectively, after two hours post-treatment with 100mg/Kg b.w of agarwood leaf methanolic extract when a compared to pretreated streptozotocin-induced diabetic ICR male mice, on the other hand, slight reduction (17%) in blood glucose was seen in day-3 of the same group. The blood glucose levels in diabetic ICR male mice were not reduced the after 1, 3, 9, 12 and 14 days after treatment with 150 mg/Kg b.w methanolic leaf extract when a compared to day-0 (figure 4).

 

Figure 4: Two weeks effects of agarwood leaf methanolic extract (ALME) at different concentrations: (a) 50 mg/ kg, (b) 100 mg/ kg, (c)150 mg/kg on blood glucose levels in mice with STZ-induced diabetes. Note: Mean ± standard deviation (n=5 mice/treatment). *P <0.05 compared with the pre-treated mice.

 

The methanolic extract of Aquilaria malaccensis leaves decreased blood glucose levels elevation significantly at all concentrations evaluated (50, 100 and 150mg/Kg). However, the extract at a concentration of 50mg/Kg was shown to exhibit the highest inhibition of blood glucose levels in the treated streptozotocin-induced diabetic ICR male mice (figure 4) as this concentration reduced glucose level to less than 72%. However, the efficacy of such low concentration may be taken into account because the active compounds present in such concentrations may not cause death due to its protective role for the vital organs as approved by many deep studies^42-44.

 

Polyphenols compound have a high affinity in aqueous solution, so, it is dissolved in polar solvents like water^45-47. The aqueous extract can have much higher efficacy than methanol because it has medium polarity in nature ^48-50. The aqueous extract is effectively used to treat diabetes and inflammation which is induced due to high blood glucose^51,52. On the other hand, the key feature of using alcoholic (methanol) extract is its ability to extract more organic constituents. Alcoholic (methanol) extract can enhance the inhibition potency of diabetic disorder ⁵³ and compare to the aqueous extract, it can be more toxic to cancers^{54, 55} and microbes⁵⁶.

 

4. CONCLUSION:

Aqueous and methanolic extracts of Aquilaria malaccensis leaves exhibited a significant reduction of fasting hyperglycemia in diabetic mice at the lowest concentration. Both extracts reduced hyperglycemia earlier after the first or third day of administration and this was observed for all doses of extracts. For methanolic extract, low and moderate doses, 50 and 100 mg/kg b.w. reduced fasting hyperglycemia in diabetic mice after 2 hours of administration whereas in mice treated with the highest dose, 150mg/kg b.w., a slight reduction of hyperglycemia occurs after 2 hours of administration. This could possibly be due to that the highest dose of methanolic extract contains non-bioactive compounds which may antagonize the activity of bioactive compounds. For both extracts, the most potent dose in reducing fasting hyperglycemia of diabetic mice was 50mg/kg b.w. followed by 100 and 150mg/kg. Thus, from the present study, the aqueous and methanolic extracts were observed to exert anti-hyperglycemic property which can reduce hyperglycemia in streptozotocin-induced diabetic male mice in fasting and long term treatment. However, further efforts are needed to identify the antidiabetic effect mechanism to optimize the utilization of these extracts for diabetic treatment.

 

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Received on 20.04.2019           Modified on 26.06.2019

Accepted on 30.07.2019         © RJPT All right reserved