https://imcjms.com Ibrahim Medical College Journal of Medical Science https://imcjms.com/registration/journal_full_text/220 Tue, 16 May 2017 14:40:35 +0000 Background and purposes: Glycosmis pentaphylla (Retz.) Correa, a medicinal plant is popularly used as herbal remedy for various ailments in Bangladesh. It was also reported that GP has both anti-hyperglycemic and analgesic effects and being widely used to reduce blood glucose and to alleviate pain for many years in this region though published literatures are scarce. The present study was designed to evaluate whether ethanolic extract of Glycosmis pentaphylla (GP) have anti-hyperglycemic and analgesic effects. A total of 60 Swiss Albino male mice of nine weeks (weight, 20-25g) were used for investigation. Of them, 40 were made diabetic by alloxan. They were investigated in two groups – a) 20 mice by oral glucose tolerance test (4 samples OGTT) – at 0, 30, 90 and 120 min; and b) 20 mice for a week-long antihyperglycemic activity on day 0, 1, 3 & 7. Both the groups were subdivided into four, each having 5 mice – i) the ‘control’ received only 0.5% methyl cellulose as vehicle; ii) ‘Standard’ received vehicle plus metformin; iii & iv) test ‘DGP250’ & ‘DGP500’ received vehicle plus GP extract with 250 & 500 mg /kg, respectively. For the analgesic activity, 20 mice were investigated in four subgroups, each having 5 mice and similar steps were adopted. Here, vehicle was used 1% Tween 80 and intra-peritoneal injection of Acetic acid for eliciting pain in all four subgroups. The ‘standard’ group got diclofenac sodium for comparison with the test groups ‘GP250’ and ‘GP500’. In OGTT, Ethanolic extract of GP250 and GP500 reduced blood glucose at 90 min. But the levels of reduction were more significant at 120 min, 50.7% by GP250 and 66% by GP500 (p<0.001). The reduction is almost comparable with that induced by metformin. Likewise, for a weeklong anti-hyperglycemic activity, the GP extracts were found as equally effective as metfomin, which was also dose dependent. In addition to antihyperglycemic effect, the ethanolic extract of GP showed significant analgesic effect that was also dose dependent. Our results indicate that GP extract has antihyperglycemic effect in both short and in weeklong duration, which is almost comparable to Metformin HCL, a known and widely used antihyperglycemic agent. The GP extract was also found to have an analgesic effect almost comparable to diclofenac sodium, a known analgesic drug. Further study is needed to confirm the anti-hyperglycemic and analgesic effect of GP including its side effects in long term use. Address for Correspondence: Dr. Kohinur Begum, Department of Pharmacy, Bangladesh University, 15/1 Iqbal Road, Mohammadpur, Dhaka-1207. E-mail: kohinur025@yahoo.com   Diabetes is a global disease with a huge adverse impact on health and mortality particularly of cardiovascular disorders. Diabetes mellitus (DM) is major clinical disorder affecting nearly 10% of the populations all over the world.1 In Bangladesh, the situation is most vulnerable; the number of people with diabetes will rise from 3.2 million in 2000 to 11.7 million by 2030.2 Patient with diabetes have an increased risk of coronary heart disease, peripheral vascular disease, strokes and may account for more than 65% death among people with diabetes mellitus.3,4 Multiple pathophysiologic mechanisms play a role in the risk of cardiovascular events in the metabolic syndrome including glucose intolerance, hyperglycemia, hypertension, dyslipidemia, atherosclerosis that are caused primarily by insulin resistance.5,6 Traditional medicines are used to reduce blood glucose level as well as have beneficial effects on complication of diabetes.7 Glycosmis pentaphylla is a small trees or shrubs belonging to family of Rutaceae, which grows to a height 5 m. It is widely distributed in tropical forest at low altitudes like India, South China, Thailand, Malaysia, Indonesia and Philippines Islands.11 Arborine [2-benzyl-1-methyl-4-quinazolone] was isolated as the major compound from ethyl acetate soluble fraction of leaf extract of Glycosmis pentaphylla.12 The root bark contains alkaloid skimmianine, g-fagarine, dictamine, arbone, 3-methoxycarbazone, glycone, glycozoline and glycozolicine and beta-sitosterol.13 A paste prepared from leaves of GP and ginger can be used for treatment of eczema and other skin infection.11 Extract of leaves of GP is used in fever, liver complains, cough and jaundice.14 But still no scientific and methodical investigation has so far been reported in literature regarding its anti-diabetic and analgesic activity. Therefore as a part of our ongoing phytochemical and pharmacological investigations on local medicinal plants of Bangladesh, the present study has been designed to examine anti-diabetic and analgesic activity of ethanolic extracts of leaf of Glycosmis pentaphylla. Methods and Materials Fresh leafs of Glycosmis pentaphylla. (Vernicular name- tooth brush plant) was collected from Savar, Dhaka in September 2009 and plant authenticity was confirmed from the Bangladesh National Herbarium, Dhaka. Preparation of ethanol extract   The active drug, Metformin hydrochloride and Diclofenac-Na were collected from Square Pharmaceuticals Ltd; Pabna Bangladesh. Alloxan was purchased from Sisco Research Laboratories Pvt. Ltd. Mumbai, India. These sixty mice were divided into three experimental groups, each group with 20 mice. Two groups (20 + 20 = 40) were studied for antihyperglycemic activity – one for short term and the other for a weeklong duration. Both of the two groups were made diabetic by alloxan (alloxan is selectively toxic to insulin-producing pancreatic beta cells because it preferentially accumulates in beta cells through uptake via the GLUT2 glucose transporter). These alloxan induced two diabetic groups were investigated for a short term – a) by oral glucose tolerance test (n=20, 4 samples OGTT) – at 0, 30, 90 and 120 min; and b) for a week-long antihyperglycemic activity (n=20) on day 0, 1, 3 & 7. Both the groups were subdivided into four, each having 5 mice – i) the ‘control’ received only 0.5% methyl cellulose as vehicle; ii) ‘Standard’ received vehicle plus metformin; iii & iv) test ‘DGP250’ & ‘DGP500’ received vehicle plus GP extract with 250 & 500 mg /kg, respectively. All blood samples were taken from tail-vein for estimation of blood glucose by Glucometer, a reflectance photometer. For the analgesic activity, 20 mice were investigated in four subgroups, each having 5 mice and similar steps were adopted as for the antihyperglycemic effect. Here, vehicle was used 1% Tween 80 and intra-peritoneal injection of Acetic acid for eliciting pain in all four subgroups. The ‘standard’ group got diclofenac sodium for comparison with the test groups ‘GP250’ and ‘GP500’. Oral glucose tolerance test (OGTT)   Short term test: One ml (50mg/ml) of glucose solution in a dose of 2 gm/kg body weight was administered to all groups by gastric tube. Simultaneously, one ml of 0.5% methyl cellulose for the control (DC) and 1 ml (2.5mg/ml of 0.5% methyl cellulose) of standard drug metformin and one ml of ethanolic extract for group 250 (6.25mg/ml) and for group 500 (12.5mg/ml) were administered orally to respective groups. The blood glucose content was measured after 30 mins, 90 mins and 120 mins.   Analgesic activity of ethanolic extract was studied by acetic acid-induced writhing test in mice. Mice were divided into four groups (each group comprises five mice). Control group mice received vehicles (1% Tween 80 in water), Standard Group received Diclofenac-Na 10 mg/kg body weight , Test Group I and Test Group II were received 250 and 500 mg/kg b. wt. of ethanolic extract of GP. Test samples and vehicle were administered orally 30 mins before intra-peritoneal administration of 0.7% acetic acid and Diclofenac-Na (0.25mg/ml ) was administered intra-peritoneally 15 mins before injection of acetic acid. After 5 mins interval, mice were observed for specific contraction of body referred to as “writhing” for next 10 mins.15 Phytochemical screening   The results were expressed as mean ± Standard Error of Mean (SEM). Statistical analysis was performed by using ANOVA followed by Tukey’s test using Graph pad Prism Software version 5.03 (Graph Pad Software, San Diego, CA, USA, www.graphpad.com). P values < 0.05 were considered as statistically significant. Results After oral administration of glucose, blood glucose levels were significantly higher in mice and results shown in Figure 2. In diabetic control peak blood glucose concentration was observed after 30 mins and remained high after next hour. Mice treated with extract in Group DGP-250 and Group DGP-500 showed a significant decrease in blood glucose concentration 50.7% and 66% respectively, at 120 mins compared with diabetic control mice. Anti hyperglycemic effect of ethanolic extract in diabetic mice hypoglycemic test was performed and compared with diabetic control (DC group). After 7 days of treatment with extract glucose level were significantly lowered 48.1% and 63.10% in Group DGP-250 and DGP-500, respectively (figure 3). Hypoglycemic effect was found dose dependent. Analgesic activity of ethanolic extract   Chemical constituents were identified by characteristic color changes. The screening results were as follows: Alkaloids + ve; Carbohydrates - ve; Proteins and amino acids + ve; Phenols + ve; Flavonoids + ve; Saponin + ve and Tannins + ve. Where + ve and – ve indicates presence and absence of compounds.   Fig 2. Glucose tolarence effect of Glycosmis pentaphylla extract in diabetic mice. Values were expressed in Mean ±SEM. Each group comprises 5 mice. Control group received 0.5% Methyl cellulose and standard group received Metformin 100 mg/kg. *p<0.05, **p<0.01, and ***p<0.001 indicate compared with diabetic control.     Fig 4. Analgesic effect of ethanolic extract of Glycosmis pentaphylla. Values were expressed in Mean ± SEM.  Control group mice received vehicles (1% Tween 80 in water), Standard Group received Diclofenac-Na 10 mg/kg body weight, Test Group I and Test Group II were received 250 and 500 mg/kg b. wt. of ethanolic extract of GP, respectively. Discussion Glycosmis pentaphylla has not been subjected to pharmacological investigations so far analgesic screening to provide scientific justification to its traditional claim in various pains. Therefore, present study has shown to establish remarkable analgesic potential of GP (figure 4). Acetic acid-induced writhing model represent pain sensation by triggering localized inflammatory response. The Prostaglandins mainly prostacyclin and prostaglandin-E have been reported to be responsible for pain sensation by exciting A-fibres. Activities in A-fibres cause a sensation of sharp well localized pain. Any agent that lowers the number of writhing will demonstrate analgesia preferably by inhibition of prostaglandin synthesis, a peripheral mechanism of pain inhibition.19 The response is thought to be mediated by peritoneal mast cells, acid sensing ion channels and prostaglandin pathway.20 Flavonoids being powerful antioxidants are reported to play a role in analgesic activity by targeting prostaglandins.21 Overall analgesic action of GP is assumed to be due to inhibition of prostaglandin synthesis. Conclusions   1.   Burke JP, Williams K, Narayan KMV. A population perspective on diabetes prevention; whom- weight gain. Diabetes Care 2003; 26: 1999-2004. 3.   Brown WV. Lipoprotein disorders in diabetes mellitus. The medicinal clinics of North America 1994; 87: 143-161. 5.   Reaven GM. Role of insulin resistance in human disease. Diabetes 1988; 37: 1595-1607. 7.   Dixit PP, Londhe JS, Ghashadi SS and Devasagayam TPA. Antidiabetic and Related beneficial properties of Indian medicinal plants in Herbal Drug Research. In Sharma, R.K. and Arora R eds. A twenty first century perspective. Jaypee brothers medical publisher Limited 2006; 377-386. 9.   Raquibul Hassan, et al. Analgesic and Antioxidant Activity of the Hydromethnolic Extract of Mikania scandens (L.) Wild. Leaves. American Journal of Pharmacology and toxicology 2009, 4(1): 1-7. 11.Samy J, Sugumaran M. and Lee KLW. Herbs of Malaysia. Federal Publications Sdn. Berhod 2005, 114-115. 13.Daniels M. Medicinal plants, Chemestry and Properties. Science Publishers Enfield, USA 2005; 43. 15.Ahmed F, Selim MST, Das AK. and Choudhuri MSK. Anti-inflammatory and antinociceptive activities of Lippa nodiflora Linn. Pharmazie 2004; 59: 329-333. 17.Gold AH. The effect of diabetes and insulin on liver glycogen synthetase activation. J Biol Chem.1970; 245: 903–5. 19.Brown JE and Evans CAR. Luteolin rich artichoke extract protects low density lipoprotein from oxidation in vitro. Free Radic. Res. 1998; 29: 24255. 21.Rajanarayana KMS, Reddy MR. Chaluvadi and Krishna DR. Biflavonoids classification, pharmacological, biochemical effects and therapeutic potential. Ind. J. Pharmacol 2001; 33: 2-16. 2026 Ibrahim Medical College. All rights reserved.