Comparative Study of the Effect of Ethanol Extract of Psidium Guajava Linn Leaves with Glibenclamide on Experimentally Induced Diabetes Mellitus in Rats

Background: Diabetes Mellitus is a major health problem not only in urban but also in the rural areas of Bangladesh. Regarding its treatment, a suitable drug is yet to be available which can permanently cure this disease. Over 400 traditional plants have been reported for the treatment of diabetes, but only a small number have received scientific and medical evaluation to assess their efficacy. Objective: The study was performed to compare the anti diabetic effect of Psidium guajava Linn leaves with an oral anti diabetic drug (Glibenclamide), in experimentally induced diabetic rats. Materials and method: The experiment was carried out in the department of Pharmacology & Therapeutics of Dhaka Medical College, Dhaka, Bangladesh from July 2012 to June 2013. Twenty four healthy rats of Long Evans Norwegian strain were divided into 4 groups (group A, B, C and D) comprising 6 rats in each. Group A (normal control) received standard rat food for 14 days. Diabetes was induced by administration of Alloxan 120 mg/kg/body weight in group B, C and D. Group B (diabetic control group) was given standard rat food. Group C and D was treated with ethanol extract of Psidium guajava leaves 100 mg/kg body weight and Glibenclamide 1.5 mg/kg/day orally respectively. Total duration of the experiment was 15 days. Results: Administration of ethanol extract of Psidium guajava leaves in group C and Glibenclamide in group D produced a significant reduction (p<0.001) in blood glucose level as compared to group B (diabetic control group). Conclusion: Ethanol extract of Psidium guajava leaves can be considered as a potential anti diabetic agent like Glibenclamide but it requires further investigations.


Introduction
Now a days type 2 DM is one of the major public health concerns in both developing and developed countries in Asian-Pacific region.It has become epidemic in a number of countries, particularly in newly industrialized nations.The direct and indirect, social and economic costs of treating Assistant Professor, Dept. of Pharmacology and Therapeutics, Delta Medical College, Dhaka, Bangladesh.
Assistant Professor, Dept. of Pharmacology and Therapeutics, University Dental College, Dhaka, Bangladesh.
Assistant Professor, Dept. of Pharmacology and Therapeutics, Delta Medical College, Dhaka, Bangladesh.

5.
diabetes and its complications have the potential to cripple the countries' healthcare budget.In recent times, a new dimension has been added with the increasing appearance of type 2 diabetes in adolescents and even in children. 1 Excess mortality attributable to diabetes in the year 2000 was estimated to be 2.9 million deaths, equivalent to 5.2% of all deaths.It accounted for 2-3% of deaths in poorest countries and over 8% in the U.S., Canada and Middle East.In people between the age of 35-64 years old, 6-27% of deaths were attributable to diabetes. 2 Bangladesh, the magnitude of diabetes mellitus is increasing.Findings of 10 screening studies carried out from 1996 to 2003 has shown that in mid 60s, the prevalence of diabetes mellitus was around 1.5% which has increased to more than 15% in recent years, (where blood glucose >7.8 mmol/L after 2 hours of 75 gram glucose was considered as an impaired glucose tolerance). 3aditional medicine derived from medicinal plants is used by about 60% of the world's population.The World Health Organization has listed 21000 plants, which are used for medicinal purposes around the world.Among these 2500 species are in India.Out of which 150 species are used commercially on a fairly large scale.A list of medicinal plants with proven anti diabetic and related beneficial effects and of herbal drugs in the treatment of diabetes is compiled.This includes Allium sativum, Eugenia jambolana, Momordica charantia, Ocimum sanctum, Phyllantus amarus, Pterocaprus marsupium, Tinospora cordifolia, Trigonella foenum graecum and Withania somniferum. 4 Psidium guajava is popularly known as guava and has been used traditionally as a medicinal plant throughout the world for a number of ailments.Guava tree is basically from Meso American area.It can also be found in tropical and subtropical areas.Guava tree is a member of myrtaceae family.All the parts of this tree are widely used in curing many health problems.A lot of pharmacological researches have been done to demonstrate the use of extract from guava leaves which proved that it is a very useful medicine, widely used by doctors and pharmacists.The main constituents of guava leaves are phenolic compounds, isoflavonoid, gallic acid, catechin, epicatechin, rutin, naringenin, and kaempferol. 5,6rld health organization (WHO) also says that plants would be the best source of obtaining different types of medicines and drugs.These natural products are widely used by human for its effective results.
The leaves of guava are rich in flavonoids, in particular, quercetin.Quercetin is the main flavonoid in guava leaves which contribute to its anti hyperglycemic effect.Guava leaves also have antioxidant properties which is attributed to the polyphenols found within. 7 has also been demonstrated that flavonoids of guava leaves can act as insulin secretagogues or insulin mimetics, probably by influencing the pleiotropic mechanism, attenuate the diabetic complications, and also stimulate glucose uptake in peripheral tissues, and regulate the activity and/or expression of the rate-limiting enzymes involved in carbohydrate metabolism pathway.In a study it was found that flavonoids act directly on pancreatic beta cells leading to activation of the cAMP/PKA signaling cascade to exert an insulinotropic effect. 8Glibenclamide (INN), also known as glyburide (USAN), is an anti diabetic drug in a class of medications known as sulfonylureas, closely related to sulfa drugs.It is used in the treatment of type 2 diabetes.The drug works by inhibiting ATP-sensitive potassium channels in pancreatic bets cells.This inhibition causes cell membrane depolarization opening voltage-dependent calcium channel.This results in an increase in intracellular calcium in the beta cell and subsequent stimulation of insulin release. 9 A retrospective study showed that in type 2 diabetic patients already taking glyburide, NH stroke scale scores were improved on discharge compared to diabetic patients not taking glyburide. 10th this background information, in this study, attempt has been taken to make comparison of blood glucose lowering effect of Psidium guajava leaves with an oral anti diabetic drug, Glibenclamide in experimental diabetic rats.Alloxan has been chosen to induce diabetes mellitus in rats.Blood glucose level has been estimated to the extent of pancreatic damage.

Materials and method
This experimental study has been performed in the department of Pharmacology & Therapeutics at Dhaka medical college, Dhaka, Bangladesh from July 2012 to June 2013.
A total number of 24 healthy rats of Long Evans Norwegian strain weighing between 140-150 gm and age between 8-10 weeks which were purchased from Bangladesh Centre for Scientific and Industrial Research (BCSIR) Lab were used for the present study.The rats were allowed to live at room temperature with 12 hours of light and 12 hours dark schedule.They were fed normal rat diet and given water ad libitum.
The rats were divided into 4 groups (A, B, C and D).Each group consisted of 6 rats.

Plant materials
Psidium guajava leaves were collected from local garden.Leaves were dried under shade at room temperature less than 40 degree Celsius.

Preparation of extract
Ethanol extract was made in the Drug Research Laboratory of Center for Advanced Research of Sciences (CARS) of Dhaka University.One kg of Psidium guajava (Guava) leaves were cleaned and shade dried.Then it was crushed into coarse powder and soaked in absolute ethanol (2L) with continuous shaking (40 rmp) at 25°C for three days and filtered by filter paper.The ethanol extract was evaporated under vacuum rotator evaporator at 35°C temperature to obtain final deep green semisolid extract.A total of 30 gram extract was found in this way.

Experimental design
It is comprised of 24 rats which were divided into 4 groups (Group A, B, C and D) having 6 rats in each.All the rats were fasted overnight before collection of blood.
Group A (Control group) received standard rat food for 14 days.Fasting blood glucose was estimated on day 1, 4 and day 15.
Group B (Diabetic control group) was given alloxan 120 mg/kg intraperitonially for induction of diabetes on day 1.After alloxan injection rats were given standard food.Fasting blood glucose level were estimated on day 1 (before alloxan), on day 4 on day 15 of the experiment.
Group C was given alloxan 120 mg/kg intraperitoneally on day 1.After alloxan injection rats were given standard food.Then after 3 days ethanol extract of Psidiam guajava (guava) leaves 100 mg/kg/day was given orally along with standard food for 10 days.Fasting blood glucose level was estimated on day 1 (before alloxan), on day 4 and on day 15 of the experiment.
Group D was given alloxan 120 mg/kg intraperitoneally on day 1.After alloxan injection rats were given standard food.Then after 3 days, Glibenclamide 1.5mg/kg/day was given orally along with standard food for 10 days. 9Fasting blood glucose was estimated on day 1 (before alloxan), on day 4 and on day 15 of the experiment.

Original Article
Delta Med Col J. Jan 2017;5(1) In group D blood glucose levels (mean±SD) in mmol/L were 5.80±0.48,15.01±3.39 and 7.05±0.90on day 1, day 4 and day 15 respectively.Percentage change on day 4 and day 15 was 152.45 and 28.86 respectively from day 1.

Percentage change of blood glucose level in diabetic rats after EEPG leaves and Glibenclamide treatment
In group B, the blood glucose levels (mean±SD) in mmol/L were 5.70±0.39 and 16.00±2.85on day 1 and day 15 respectively.Percentage change was 190.38%.
In group C, the blood glucose levels (mean±SD) in mmol/L were 5.40±0.65 and 8.60±2.25 on day 1 and day 15 respectively.Percentage change was 43.55%.
In group D, the blood glucose levels (mean±SD) in mmol/L were 5.80±0.48and 7.05±0.90on day 1 and day 15 respectively.Percentage change was 28.86%.Unpaired Student's 't' test was done between group B and group C and between group B and group D. Differences on day 15 were statistically significant (p<0.001)(Table-III).

Table III: Effect of EESG (ethanol extract of Psidium guajava) leaves and Glibenclamaide on blood glucose level in diabetic rats with the percentage changes
ns= not significant (p<0.05),***= highly significant (p<0.001).

Effect of Alloxan on fasting blood glucose (FBG) level of Group B in comparison to group A on day 1, 4 and 15
In group A, the blood glucose levels (mean±SD) in mmol/L were 5.80±0.51,5.80±0.49and 5.80±0.52 on day 1, day 4 and day 15 respectively.Percentage change on day 15 from day 1 was 2.30.
In group B, the blood glucose levels (mean±SD) in mmol/L were 5.70±0.39,15.00±3.35and 16.00±2.85on day 1, day 4 and day 15 respectively.Percentage change on day 4 and day 15 was 170.85 and 190.38 respectively from day 1.The results are shown in Table I.

Table I: Effect of Alloxan on blood glucose level on day 1, day 4 and day 15
ns= not significant (p<0.05)***= significant at p <0.001 Comparison of FBG level on day 1, day 4 and day 15 with control (group A) done by unpaired student's 't' test.

Effects of Alloxan, Ethanol extract of Psidium guajava (EEPG) leaves, and Glibenclamide on blood glucose level of group B, group C and Group D rats on day 1, day 4 and day 15
In group B, blood glucose levels (mean±SD) in mmol/L were 5.70±0.39,15.00±3.35and 16.00±2.85on day 1, day 4 and day 15 respectively.Percentage change on day 4 and day 15 was 170.85 and 190.38 respectively from day 1.
In group C, blood glucose levels (mean±SD) in mmol/L were 5.40±0.65,15.35±4.55 and 8.60±2.25 on day 1, day 4 and day 15 respectively.Percentage change on day 4 and day 15 was 160.74 and 43.55 respectively from day 1.

Discussion
The present study was carried out to compare the glucose lowering effect of ethanol extract of Psidium guajava (guava) leaves with Glibenclamide on experimentally induced diabetic rats.Diabetes was induced by alloxan.The dose and route of administration of alloxan monohydrate was selected from Andrade et al. and Kim et al. 11,12 The blood glucose levels in animals were measured 72 hours after administration of alloxan which was done according to experiment of Etuk et al. 13 In this study, intraperitonial (ip) administration of single dose of alloxan (120mg/kg) increased blood glucose level significantly.Similar observations were reported by number of researchers.Ghosh et al. 14 observed the condition of diabetes after 24 hours of intravenous injection of sterile, freshly prepared 1% alloxan monohydrate solution at a dose of 40mg/kg in albino rats.Jeloder et al. 15 in their experiment observed the effect of fenugreek, onion and garlic on blood glucose and histopathology of pancreas of diabetic rats, by inducing diabetes in 20 out of 25 adult male albino rats by intraperitonial injection of 185mg/kg of alloxan.In the present study, the rise of blood glucose level in experimental diabetic rats was also very high.
The dose of Psidium guajava leaves (100mg/kg body weight), and duration used in this study was selected in keeping conformity with the dose and duration used in research work by Yesmin. 7 The leaves of guava are rich in flavonoid, in particular, quercetin.Quercetin is the main flavonoid in guava leaves which contribute to its anti hyperglycemic effect. 7Flavonoid compounds act against diabetes mellitus either through their capacity to avoid glucose absorption or by improving glucose tolerance. 16he epicatechingallate, myricetin, quercetin, apigenin, epigallocatechin gallate, and epigallocatechin demonstrated a marked reduction in glucose absorption, when compared with control, by competitive inhibition of sodium dependent glucose transporter. 17In several studies it was found that flavonoids act directly on pancreatic beta cells leading to activation of the cAMP/PKA signaling cascade to exert an insulinotropic effect. 8s a result bioflavonoids are now a days regarded as promising and significantly attractive natural substance to enrich the current therapy options against diabetes.
It was observed that the ethanol extract of Psidium guajava leaves has glucose lowering effect in alloxan induced diabetic rats.It has the capability of reducing blood glucose level in diabetic rat as well as Glibenclamide.
The result suggests that the ethanol extract of Psidium guajava (EEPG) leaves may be useful anti diabetic agent in treatment of diabetes mellitus.The observation and results of the study provided a rationale for the use of Psidium guajava leaves in the development of a new drug for treatment of Diabetes mellitus.It is recommended that further studies should be carried out regarding pharmacokinetics, pharmacodynamics and toxicology of the ethanol extract of Psidium guajava leaves.It is suggested to measure plasma insulin level, HbA1C, liver glycogen level, lipid hydroperoxidation level and free radical in the tissues after treatment with ethanol extract of Psidium guajava leaves.Despite all these limitations, interpretation of the results obtained in this study was made carefully and cautiously.