ETHIOPIAN PHARMACEUTICAL JOURNAL Ethiop. Pharm. J. 28, 12-20 (2010) http://dx.doi.org/10.4314/epj.v28i1.2 Antidiabetic Activity of the Leaf Extracts of Pentas schimperiana Subsp. schimperiana (A. Rich) Vatke on Alloxan-Induced Diabetic Mice Tadele Dinku1, Solomon Tadesse1 and Kaleab Asres1* 1 Department of Pharmaceutical Chemistry and Pharmacognosy, School of Pharmacy, Addis Ababa University, P.O. Box 1176, Addis Ababa, Ethiopia Pentas schimperiana (A. Rich) Vatke (Rubiaceae) is widely used for the treatment of diabetes mellitus and various other ailments in the traditional medical practices of Ethiopia. This study reports the antidiabetic and free radical scavenging activities of extracts and solvent fractions prepared from the leaves of P. schimperiana grown in Ethiopia on alloxan-induced diabetic mice and 2,2-diphenyl-1-picrylhydrazil (DPPH) assay, respectively. A single dose of 500 mg/kg of each of the aqueous dried leaf, hydroalcoholic fresh and dried leaf extracts of P. schimperiana did not show significant antidiabetic effect. However, at a dose of 1000 mg/kg, the extracts lowered blood glucose level by 26.97%, 21.90% and 26.70%, respectively. At a dose of 500 mg/kg, the aqueous and methanol fractions prepared from the dried plant material lowered blood glucose level by 23% and 27.2%, respectively, as compared with diabetic untreated mice. Treatment with a known antidiabetic drug glibenclamide (5 mg/kg of body weight) lowered blood glucose by 38%. The antidiabetic activity of the dried plant material was shown to be better than that of the fresh plant material. Both the hydroalcoholic extracts and the methanol fraction of the dried leaves of P. schimperiana displayed a very good DPPH scavenging activity with IC50 values of 7.83 and 8.84 µg/ml, respectively. In this study ascorbic acid showed an IC50 value of 4.42 µg/ml. Acute toxicity study of the aqueous and the hydroalcholic extracts of P. schimperiana performed on Swiss albino mice indicated that the median lethal dose (LD50) of the extract is above 4000 mg/kg. Phytochemical screening carried out on the total leaf extracts of the plant confirmed the presence of flavonoids, saponinns, steroids and tannins. The results of the current study support the traditional use of the plant in the management of diabetes. Key words: Pentas schimperiana, antidiabetic activity, alloxan, 2,2-diphenyl-1-picrylhydrazil, free radical scavenging activity INTRODUCTION Diabetes mellitus is a chronic metabolic disease characterized by elevated blood glucose level and disturbances in carbohydrate, fat and protein metabolism (Mastan and Kumar, 2009). It results from defects in insulin secretion, insulin action or both. If the glucose level in the blood remains high over a long period of time, it can result in long term damage to organs such as kidneys, liver, eyes, nerves, heart, teeth and blood vessels. Complications in some of these organs are considered as one of the five leading causes of death in the world (Moore et al., 2003; Semwal et al., 2009). The prevalence of diabetes mellitus is increasing with ageing and life style changes * Correspondence: kasres@phar.aau.edu.et associated with rapid urbanization. The disease is found in all part of the world and rapidly increasing in its coverage. It affects nearly 10% of the population all over the world and is the leading cause of deaths in humans and animals. It is also the fastest growing metabolic disease in the world (Sobngwi et al., 2003; Parsad et al., 2009). The global number of individuals with diabetes in 2000 was estimated to be 171 million (2.8% of the world population). A figure projected to increase in 2030 to 366 million (6.5% of world population) 298 million of whom will live in developing countries (Kengne et al., 2005). Approximately 80% diabetic patients live in poor and developing countries (Sadicot, 2009). In Ethiopia, community based studies are non existent at the national level, and 13 T. Dinku et al. hospitals may give figures of those diabetic patients who come for treatment and follow up. The national estimate is based on neighboring countries with similar socioeconomic situation. According to Yemane et al. (2007) the number of deaths attributed to diabetes in Ethiopia reached over 21,000. Analysis of medical admissions from Addis Ababa and a number of provincial hospitals have shown prevalence ranging from 0.5 to 8.4% (Abdulkadir and Reja, 2006). Diabetes management concentrates on keeping blood sugar levels with normal limits, through diet exercise and use of a p p r o p r i a t e me d i c a t i o n s . D i e t a r y modification, oral hypoglycaemic, and insulin, have limitations of their own (Jung et al., 2006). Traditionally plants are also used for the treatment of diabetes mellitus throughout the world. Many studies confirmed the benefits of medicinal plants with hypoglycaemic effects in the management of diabetes mellitus. The effects of these plants may delay development of diabetic complications and correct the metabolic abnormalities. During the past few years some of the new bioactive drugs isolated from hypoglycaemic plants showed antidiabetic activity with more efficacy than oral hypoglycaemic agents used in clinical therapy. So, management of diabetes without any side effect is still a challenge for the medical system, which has led to an increasing search for improved antidiabetic drugs (Bnouham et al., 2006; Koski, 2006). P. schimperiana is shrubby and semi woody herb which reaches up to 2 m high. The leaves are narrow at both ends and the petiole is ranging up to nearly half inch. It has purplish black woody stems and the corolla is tube funnel shaped and the fruits are 4-6 mm long. It occurs in altitude range between 1710-2350 m. Its habitat is thickest at stream side, marshy edges and open ground (Puff, 2003). P. schimperiana has two subspecies: P. schimperiana subsp. schimperiana (A. Rich) Vatke and P. schimperiana subsp. occidentalis (Hook). Subspecies schimperiana is found in Ethiopia while subspecies occidentalis occur in Cameroon. In Ethiopia P. schimperiana is known by its vernacular name “Woinagrefet” Its fresh dry root bark powder mixed with water is taken orally for epilepsy (Mesfin et al., 2009) and the decoctions of leaves are also used for the treatment of various diseases including diabetes (Abate, 1989). MATERIALS AND METHODS Materials: Plant materials: The leaves of P. schimperiana were collected in February 2009 near Menagesha state forest about 30 km west of Addis Ababa, Ethiopia. The plant material was authenticated by Ato Melaku Wondafrash, the National Herbarium, Department of Biology, Addis Ababa University where voucher specimen was deposited (collection number TD 001). Part of the leaves were air dried under shed at room temperature and the dried plant material was powdered and kept in polyethylene bags until use. The remaining fresh leaves were directly extracted by using water and hydoalcholic solution as a solvent separately. Animals: Swiss albino mice of either sex weighing of 20 - 33 g were obtained from Ethiopian Health and Nutrition Research Institute (EHNRI), Addis Ababa. Before and during the experiment, the mice were allowed free access to standard pellet diet and water ad libtum. After randomization into various groups and before initiation of the Ethiop Pharm J 28, 12-20 (2010) http://dx.doi.org/10.4314/epj.v28i1.2 ANTIDIABETIC ACTIVITY OF PENTAS SCHIMPERIANA experiment, the mice were acclimatized at the animal house of the School of Pharmacy, Addis Ababa University. Prior to each study the animals were made to fast for 12-14 h but had free access to water. Methods: Extraction: Dried and fresh leaves of P. schimperiana (400 g each) were extracted with 80% methanol for 72 h (solvent was changed every 24 h) by percolation. The combined percolate was filtered with a filter paper (Whatman No. 3, Whatman Ltd, England) and concentrated under reduced pressure using rota vapor (Buchi Rota Vapor R-2000) at 40 °C. The resulting semisolid extract was placed on a water bath set at 40 °C till it dried. The dried leaves (400 g) of P. schimperiana were also macerated in distilled water for 72 h (solvent was changed every 24 h), filtered and concentrated in an oven (< 40 °C). The dried extracts were transferred into vials and kept in a refrigerator for further use. Fractionation: The hydroalcoholic dried leaf extract of P. schimperiana (30 g) was fractionated successively by soxhlet extraction using different solvents of increasing polarity (chloroform, acetone and methanol). Extraction in each solvent was carried out for 72 h and before changing the solvent the marc was left in air till all the solvent was evaporated. The different fractions were concentrated under reduced pressure and dried using water bath at a temperature not exceeding 40 °C. The mark left after methanol extraction was macerated with distilled water and dried in an oven with a temperature not exceeding 40 °C. The dried fractions were then transferred into separate vials and stored for further use. 14 Phytochemical screening: Standard screening tests of the hydroalcoholic extract was carried out for various plant constituents such as alkaloids, steroidal compounds, phenolics, flavonoids, saponins, tannins and anthraquinones (Trease and Evans, 1978; Sofowara, 1993; William et al., 2006). Antidiabetic activity study: Induction of experimental diabetes. Swiss albino mice were fasted overnight (12-14 h) and their weight and fasting blood glucose levels were recorded. Mice were made diabetic by a single intraperitoneal injection of alloxan monohydrate solubilized in sodium citrate (150 mg/kg of body weight). Alloxan was first weighed for each group of animal according to their average weight prior to injection. Food and water were presented to the animals 30 min after drug administration (Decarvalho et al., 2003; Mistry, 2008). Two days after alloxan injection plasma blood glucose level of each animal was determined and animals with fasting blood glucose above 150 mg/dl were included in the study. Blood samples were collected from the tail of mice, glycemia was determined by the glucoseoxidase peroxidase method with reagent strips, and evaluation was made on Glab active glucometer (GLAB Ltd, Germany) (Etuk and Mohammed, 2009). Experimental design. Animals were divided into 9 different groups, 5 mice in each. The first group serves as a diabetic control and treated with distilled water while the seconed group served as a positive control and received glibenclamide dissolved in distilled water at a dose of 5 mg/ kg of body weight. The other groups were given the plant material orally (extracts or fractions) at 500 mg/ kg or 1000 mg/ kg doses dissolved in distilled water. Animals were fasted for 12Ethiop Pharm J 28, 12-20 (2010) http://dx.doi.org/10.4314/epj.v28i1.2 15 T. Dinku et al. 14 h prior to drug administration allowing them access to water only. Blood samples were collected from the tail tip of mice and blood glucose level was detrmined at 0, 1, 2, 3 and 4 h after extract administration. Statistical analysis: Blood glucose levels in the groups were expressed as mean ± standard error of mean (S.E.M.). The data were statistically analyzed by one way ANOVA followed by Dunnet’s test. P values less than 0.05 were considered significant. Antioxidant activity study: The hydrogen-donating ability of the extracts was examined based on the method of Blois (1958) in the presence of 2,2diphenyl-1-picrylhydrazil (DPPH) stable radical. The samples or the positive control, vitamin C, were diluted with methanol to prepare sample solution equivalent to 1000, 500, 250, and 125 μg of dried extract or fraction/ml solutions. 5 ml of 0.004% DPPH solution was pipetted into each test tube followed by the addition of 50 ml of the sample solution. The mixture was incubated at 37 °C for 30 min. Absorbance of the methanolic DPPH-tincture was measured at 517 nm (Jenway Model 6500 spectrophotometer). The inhibition percentage (%) of radical scavenging activity was calculated using the following equation: Inhibition (%) = ((Ao − As) / Ao)100 where, Ao is absorbance of the control and As is absorbance of the sample at 517 nm. IC50 values were calculated by linear regression of plots, where the abscissa represented the concentration of tested plant extracts and the ordinate represented the average percent of scavenging capacity from three replicates. Acute toxicity test: Acute toxicity test of the aqueous and the hydroalcholic extracts of P. schimperiana was carried out on Swiss albino mice. The mice were fasted overnight and the weight of each mouse was recorded just before use. Animals were divided randomly into a control and three treatment groups for each extract, each group consisting of five mice. Control group received only the vehicle (distilled water), each treatment group received orally the aqueous or the hydroalcholic extracts of P. schimperiana dried leaves at a dose of 1000, 2000 and 5000 mg/ kg. Animals were kept under close observation for 4 h after administering the extracts, and then they were observed daily for fourteen days for any change in general behaviour and other physical activities (Barik et al., 2008). RESULTS AND DISCUSSION Extraction and fractionation The fresh and dried leaves of P. schimperiana were extracted by maceration using 80% methanol to obtain the total extracts of the plant. The dried leaves were also extracted by using distilled water. The percentage yields of the hydroalcholic and aqueous extracts from the dried leaves were found to be 20.5% and 20%, respectively. Similarly, the percentage yield of the fresh leaf hydroalcoholic extract of P. schimperiana was 32%. Since the hydroalcoholic dried leaf extract showed higher antidiabetic activity, it was further fractionated successively by chloroform, acetone, methanol and distilled water. The yields were 7.0%, 6.8%, 25.0% and 11.6%, respectively. The results of the phytochemical screening of P. schimperiana hydro -alcholic extract have indicated the possible presence of saponins, flavonoids, tannins, steroidal and phenolic compounds. Ethiop Pharm J 28, 12-20 (2010) http://dx.doi.org/10.4314/epj.v28i1.2 16 ANTIDIABETIC ACTIVITY OF PENTAS SCHIMPERIANA hydroalcoholic dried leaf extract reduced blood sugar level by 19.27% at a dose of 1000 mg/kg. A maximum reduction (38%) in blood glucose level was achieved by glibenclamide 4 h after administration of the drug. The fresh leaf hydroalcoholic and dried leaf aqueous extracts showed maximum reduction of about 27% at a dose of 1000 mg/kg at the 3rd h. Antidiabetic Activity Aqueous and hydroalcoholic extracts Tables 1 and 2 show the hypoglycaemic activity of the aqueous and hydroalcoholic extracts of P. schimperiana when orally administered to alloxan-induced diabetic mice at doses of 500 mg/kg and 1000 mg/kg. When comparisons were made at 0 h versus different intervals (Table 3), blood glucose levels declined from 296.2 to 258.6 mg/dl on the 3rd h after oral feeding of 500 mg/ kg of the fresh leaf hydroalcoholic extract. Similarly, blood glucose level declined from 283.2 to 227.6 mg/dl and from 264.6 to 213mg/dl on the 3rd h after oral feeding of 500 mg/kg of each of the hydroalcoholic and the aqueous dried leaf extract, respectively. At a dose of 5 mg/kg, glibenclamide decreased blood glucose level on the 3rd h from 374.4 to 241.6 mg/dl. At a dose of 1000 mg/kg, the maximum percent reduction in the blood glucose level for fresh leaf hydroalcoholic and dried leaf aqueous extracts were 26.97 % and 26.7%, respectively. As shown in Table 2 the Solvent fractions of P. schimperiana As shown in Table 3, the methanol and aqueous fractions exerted a significant hypoglycaemic activity at a dose of 500 mg/ kg whereas the chloroform and acetone fractions were devoid of such activity. Hence, it may be deduced that the constituents of P. schimperiana, which possess hypoglycaemic activity, are polar in nature. Antioxidant activity Alloxan causes the generation of free radicals that damage beta cells of pancreas resulting in diabetes mellitus. Therefore free Table 1. Effect of oral administration of the fresh and dry leaf extracts of Pentas schimperiana on fasting blood glucose level (mg/dl) in alloxan-induced diabetic mice at a dose of 500 mg/kg. Group Blood glucose level in mg/dl 0 h 1h 2h 3h Diabetic control 290.8±60.87 308.4±55.94 332.0±54.60 315.0±61.13 (NS) 302.4±51.81 (NS) Hydroalcoholic dried leaf extract 283.2±56.40 289.6±54.60 (NS) 261.6±49.16 (NS) *** 27.6±46.50 (19.63) *** Aqueous dried leaf extract 264.6±41.11 257.6±56.08 (NS) * 228.0±49.49 (NS) *** 213.0±45.30 (19.50) *** Hydroalcoholic fresh leaf extract 296.0±36.07 279.0±25.17 (NS) 269.8±32.50 (NS) ** 258.8±38.20 (12.56) ** Glibenclamide (5 mg/kg) 374.4±28.20 353.2±32.77 305.6±37.23 *** *** 241.6±27.23 (35.00) 4h 227.2±51.29 (19.50) 213.0±45.84 (19.50) 255.2±38.70 (13.78) 231.4±25.30 (38.00) Values are given as mean ± SEM for groups of five animals; Figures in parenthesis indicate maximum percent reduction in blood glucose level; NS = non significant; Comparison is made at 0 time, 1, 2, 3 and 4 h; Values are statistically significant at *p<.05, **p<.01 and ***p<0.001. Ethiop Pharm J 28, 12-20 (2010) http://dx.doi.org/10.4314/epj.v28i1.2 17 T. Dinku et al. Table 2. Effect of oral administration of the fresh and dried leaf extracts of Pentas schimperiana on fasting blood glucose level (mg/dl) in alloxan-induced diabetic mice at a dose of 1000 mg/kg. Group Blood glucose level in mg /dl 0h 1h 2h 3h Diabetic control 290.8±60.87 308.4±55.94 332.0±54.60 315.0±61.13 (NS) 302.4±51.81 (NS) Hydroalcoholic dried leaf extract 324.8±48.15 340.4±68.90 (NS) 308.4±47.30 (NS) ** 263.0±40.8 0(19.27%) ** Aqueous dried leaf extract 298.5±55.44 285.8±45.45 (NS) 227.4±39.32 (NS) ** 208.6±47.8 3(26.97%) * 218.0±47.49 (26.96%) Hydroalcoholic fresh leaf extract 214.2±26.06 193.2±33.80 (NS) 162.4±29.70 (NS) * 150.6±26.20 (26.70%) * 156.4±28.11 (26.98%) Glibenclamide (5 mg/kg) 374.4±28.20 353.2±32.77 305.6±37.23 ** ** 241.6±27.2 3(35.00%) 4h 253.6±33.00 (21.90%) 231.4±25.30 (38.00%) Values are given as mean ± SEM for groups of five animals; Figures in parenthesis indicate maximum percent reduction in blood glucose level; NS= non significant; Values are statistically significant at *<0.01 and**<0.001. Comparison is made at 0 time, 1, 2, 3 and 4 h. radical scavenging substances can protect this damage by scavenging the free radicals generated (Oberley, 1998). In order to delineate the possible antidiabetic mechanism of the plant, DPPH free radical scavenging activity study was carried out on the hydroalcoholic dried leaf extract and the methanol fraction of P. schimperiana, which Table 3. Effect of oral administration of solvent fractions of Pentas schimperiana on fasting blood glucose level (mg/dl) in alloxan-induced diabetic mice at a dose of 500 mg/kg. Group Blood glucose level in mg/dl 0h 1h 2h 3h Diabetic control 290.8±60.87 308.4±55.94 332.0±54.60 315.0±61.13 302.4±51.81 Chloroform 334.0±43.89 375.0±57.32 367.8±58.12 (NS) 360.4±59.08 (NS) 344.2±59.12 (NS) Acetone 317.0±35.28 352.0±42.68 364.2±49.10 (NS) 327.2±38.30 (NS) 318.0±40.60 (NS) Methanol 409.0±32.40 355.6±37.50 * 29.2±41.18 * 312.4±33.02 (23.6%) * 297.8±26.90 (27.2%) Aqueous 363.0±35.27 347.6±23.92 * 299.8±26.69 * 284.6±28.50 (21.6%) * 279.2±30.80 (23.0%) Glibenclamide (5 mg/kg) 374.4±28.20 353.2±32.77 305.6±37.23 * * 241.6±27.23 (35.0%) 4h 231.4±25.30 (38.0%) Values are given as mean ± SEM for groups of five animals; Figures in parenthesis indicate maximum percent reduction in blood glucose level; NS= non significant; Comparison is made at 0 time, 1, 2, 3 and 4 h; Values are statistically significant at *<0.001. Ethiop Pharm J 28, 12-20 (2010) http://dx.doi.org/10.4314/epj.v28i1.2 ANTIDIABETIC ACTIVITY OF PENTAS SCHIMPERIANA 18 100 % Inhibition 80 60 Ascorbic acid 40 Hydroalcoholic extract 20 Methanol fraction 0 1 2.5 5 10 Concentration (µg/ml) Figure 1. Percentage inhibition of DPPH radical by the hydroalcoholic extracts and methanol fraction of Pentas schimperiana dried leaves in comparison with ascorbic acid. showed good antidiabetic activity. Extent of DPPH radical scavenged was determined by the decrease in intensity of violet colour in the form of IC50 values, a lower IC50 value representing a higher antioxidant activity. The antioxidant activity was compared with ascorbic acid (Tenpe et al., 2008). As shown in Figure 1, both the hydroalcoholic dried leaf extract and the methanol fraction of the dried leaves showed DPPH scavenging activity with IC50 values of 7.83 and 8.84 μg/ ml, respectively, while ascorbic acid showed an IC50 of 4.42 μg/ml. Acute toxicity test A preliminary acute toxicity study was carried out in order to determine the safety of the plant and to determine the safe dose to be used for antidiabetic activity study. The study revealed that up to 5000 mg/kg, the total aqueous and hydroalcholic extracts of P. schimperiana did not produce changes in behaviours such as restlessness, motor activity, breathing and diarrhoea. Also, none of the experimental animals died at the maximum dose employed i.e. 5000 mg/kg. All the animals were active during the experimental period. CONCLUSION The following conclusions may be made based on the findings of the current study. P. schimperiana leaf extract has significant antidiabetic activity at a dose of 1000 mg/kg. The methanol and aqueous fractions showed pronounced activity at 500 mg/kg as compared to the chloroform and acetone fractions. Phytochemical investigation of P. schimperiana revealed the presence of flavonoids, saponins, steroids and tannins. The hydroalcoholic extract and the methanol fraction of P. schimperiana were found to have antioxidant activity. This activity may be attributed to the presence of polar phenolic compounds such as flavonoids, tannins and saponnins. The antidiabetic activity of the plant may also be related to the presence of one or more of these compounds as compounds belonging to these groups are known to have antioxidant property thus active for the management of diabetes. The results of the current study support the traditional use of the plant in the management of diabeties. Acknowledgements Tadele Dinku would like to acknowledge the Graduate Studies and Research Office of Addis Ababa University for sponsoring this study. Ethiop Pharm J 28, 12-20 (2010) http://dx.doi.org/10.4314/epj.v28i1.2 19 T. Dinku et al. REFERENCES Abate G (1989). Etse Debdabe (Ethiopian Traditional Medicine). Biology Department, Science Faculty, Addis Ababa: Addis Ababa University Press, p 97. Abdulkadir J, Reja A (2006). 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Ethiop Pharm J 28, 12-20 (2010) http://dx.doi.org/10.4314/epj.v28i1.2 ANTIDIABETIC ACTIVITY OF PENTAS SCHIMPERIANA 20 Yemane T, Belachew T, Asaminew B, Befekadu O (2007). Type ІІ diabetes mellitus in Jimma town south west Ethiopia. Ethiop J Health Sci 17: 13-20. Ethiop Pharm J 28, 12-20 (2010) http://dx.doi.org/10.4314/epj.v28i1.2 View publication stats