- Email: [email protected]
methanol extract on streptozotocin-diabetic rats
Journal of Ethnopharmacology 110 (2007) 434–438
Effect of Sclerocarya birrea (Anacardiaceae) stem bark methylene chloride/methanol extract on streptozotocin-diabetic rats Th´eophile Dimo a,∗ , Silvere V. Rakotonirina a , Paul V. Tan a , Jacqueline Azay b , Etienne Dongo c , Pierre Kamtchouing a , G´erard Cros b a
Department of Animal Biology and Physiology, Faculty of Science, University of Yaounde I, P.O. Box 812, Yaounde, Cameroon b Laboratoire de Pharmacologie et Physiopathologie Exp´ erimentales, Universit´e Montpellier I, 34 093 Montpellier, France c Department of Organic Chemistry, Faculty of Science, University of Yaounde I, P.O. Box 812, Yaounde, Cameroon Received 7 January 2005; received in revised form 29 August 2006; accepted 12 October 2006 Available online 26 October 2006
Abstract Sclerocarya birrea (Anacardiaceae) is used as a traditional treatment of diabetes in Cameroon. In this study, we investigated the possible antidiabetic effect of the stem bark extract in diabetic rats. Diabetes was induced by intravenous injection of streptozotocin (STZ, 55 mg/kg) to male Wistar rats. Experimental animals (six per group), were treated by oral administration of plant extract (150 and 300 mg/kg body weight) and metformin (500 mg/kg; reference drug) for comparison, during 21 days. The stem bark methanol/methylene chloride extract of Sclerocarya birrea exhibited at termination, a significant reduction in blood glucose and increased plasma insulin levels in diabetic rats. The extract also prevented body weight loss in diabetic rats. The effective dose of the plant extract (300 mg/kg) tended to reduce plasma cholesterol, triglyceride and urea levels toward the normal levels. Four days after diabetes induction, an oral glucose tolerance test (OGTT) was also performed in experimental diabetic rats. The results showed a significant improvement in glucose tolerance in rats treated with Sclerocarya birrea extract. Metformin, a known antidiabetic drug (500 mg/kg), significantly decreased the integrated area under the glucose curve. These data indicate that Sclerocarya birrea treatment may improve glucose homeostasis in STZ-induced diabetes which could be associated with stimulation of insulin secretion. © 2006 Published by Elsevier Ireland Ltd. Keywords: Sclerocarya birrea; Glucose; Insulin; Streptozotocin; Diabetes
1. Introduction Sclerocarya birrea {(A. Rich.) Hochst.} is a tree which grows up to 10–15 m with grey fissured bark, stout branchlets and pale foliage. The leaves are compound, pinnate and the flowers greenish-white or reddish. The fruits are yellow, resembling a mango. In Africa, the tree is commonly found in savannah regions and its geographical distribution stretches from Gambia in the West across to Nigeria and Cameroon in Central Africa, and to Ethiopia and Sudan in the East (Berhaut, 1971). In Ghana, the leaves are used to treat snakebite, and pruritus (filarial); the stem bark, the root and the fruits are used to treat pharyngitis, splenomegaly and goitre, respectively (Mshana et al., 2000). In South Africa, the Zulu people use bark decoc-
∗
Corresponding author. E-mail address: [email protected] (T. Dimo).
0378-8741/$ – see front matter © 2006 Published by Elsevier Ireland Ltd. doi:10.1016/j.jep.2006.10.020
tions to treat diarrhoea, dysentery, fevers, stomach ailments, ulcers and for bacteria-related diseases (Eloff, 2001). Sclerocarya birrea is widely used in traditional medicine in Africa against hypertension, stomach-ache or gastro-enteritis, cough, and as antihyperglycemic (Laurens et al., 1984; Belemtougri et al., 2001). Phytochemical studies of Sclerocarya birrea have revealed the presence of alkaloids, anthocyans, flavonoids, tannins and saponosides (Watt and Breyer-Brandwijk, 1962; Belemtougri et al., 2001). A compound ((−)-epicatechin-3-galloyl ester) extracted from the stem bark of Sclerocarya birrea has secretagogue activity (Galvez et al., 1992). Pharmacological tests have shown that various leaf extracts of Sclerocarya birrea inhibit intracellular caffeine-induced Ca2+ release from skeletal muscle sarcoplasmic reticulum (Belemtougri et al., 2001). In addition, Eloff (2001), has demonstrated the antibacterial activity of the bark extract of Sclerocarya birrea against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Enterococcus faecalis.
T. Dimo et al. / Journal of Ethnopharmacology 110 (2007) 434–438
Information provided by practitioners of traditional medicine in North Cameroon suggest that Sclerocarya birrea possesses useful antidiabetic properties, although this was not cited in the national ethno-botanical survey of Cameroonian medicinal plants conducted by Adjanohoun et al. (1996). Recent studies (Ojewole, 2003) have revealed that moderate to high doses (100–800 mg/kg) of the stem bark aqueous extract possesses hypoglycaemic activity. In the present study, we have examined the antidiabetic activity of low doses of the organic (methanol/methylene chloride) extract of the stem bark of Sclerocarya birrea on streptozotocin (STZ)-induced diabetic rats. 2. Materials and methods 2.1. Animals
435
tion, orally to normal and STZ-diabetic rats fasted for 18 h. Distilled water (control groups), Sclerocarya birrea extract at a dose of 150 or 300 mg/kg body weight and a reference drug, metformin at a dose of 500 mg/kg were orally administrated to groups of six rats each. One hour later, glucose (5 g/kg) was administrated. Blood glucose levels from the tail vein were evaluated before (60 min) and at 0, 30, 60, 120, 180 and 300 min after glucose administration using Glucometer 4 Ames (Bayer Diagnostics). Calculation of the area under the curve (AUC) was made according to the following equation: AUC = basal glycaemia × 0.25 + glycaemia 0.5 h × 0.5 + glycaemia 1 h × 0.75 + glycaemia 2 h + glycaemia 3 h + glycaemia 5 h × 0.5.
The fresh stem bark of Sclerocarya birrea was harvested from Maroua in the Far North Province of Cameroon in June, and identified in Yaounde where voucher specimen No. 7770 is deposited in the National Herbarium. Two kilograms of the air-dried powdered stem bark were macerated in a mixture of methanol/methylene chloride (1:1) for 7 days (with occasional stirring) at room temperature. The mixture was filtered with Whatman No. 1 filter paper. The filtrate was concentrated under reduced pressure to obtain 120 g of a dark solid. Three grams of this extract were dissolved in 1 mL of dimethyl sulfoxide (DMSO) and the solution adjusted to 100 mL with distilled water to obtain a final extract concentration of 30 mg/mL.
2.4.2. Repeated administration of Sclerocarya birrea extract in STZ-diabetic rats Four days after STZ induction of diabetes in male rats, the non-fasted blood glucose levels were measured. Twenty-four hyperglycaemic rats were divided on Day 0 into equal groups of six animals each, two control groups were given by gastric intubations, either distilled water or 500 mg/kg metformin, and two test groups were given 150 or 300 mg/kg of extract per kilogram body weight. Six normal rats taken as negative controls were treated with vehicle. Treatment administration, food and water intakes were monitored every day between 8.00 and 9.00 am for 3 weeks. Plasma glucose and body weight were closely monitored twice a week. On the 15th day, the effect of the plant extract was studied up to 10 h after administration of the extract. Animals were fasted at 0 h, and the blood glucose levels were evaluated at 1.5, 3, 6, 8 and 10 h after administration of the extract. The treatment of rats was terminated on Day 21 at which time plasma samples were collected for biochemical determinations. Plasma glucose, cholesterol, triglycerides and urea were determined using a Hitachi 704 automatic analyzer. Plasma insulin was determined by the radio immunological method of Herbert et al. (1965) using rat insulin as standard.
2.3. Induction of experimental diabetes mellitus
2.5. Statistical analysis
The non-fasted Wistar rats were made diabetic with streptozotocin (55 mg/kg) (Sigma, St. Louis, MO, USA) by a single injection via the penis vein. The streptozotocin was freshly dissolved in physiological saline solution. The injection volume was 1 mL/kg. Control rats were injected with the same physiological solution only. Diabetes was confirmed in the STZtreated rats by measuring glucose levels 96 h post injection using glucose reagent strips with a Glucometer 4 Ames (Bayer Diagnostics). The rats with non-fasted blood glucose levels above 19.44 mmol/L were considered to be diabetic and were used in the experiment.
Statistical analysis of data was performed using a one-way analysis of variance (ANOVA) followed by Dunnett test for comparison between groups. Values for p < 0.05 were considered statistically significant. The data are shown as mean ± standard error of the mean (S.E.M).
Male Wistar rats from Iffa Credo, France, initially weighing between 180 and 200 g, were used. The rats were kept in a laboratory animal unit with a 12 h light/dark cycle. Throughout the experiment, room temperature was maintained at 25 ◦ C. The rats were maintained on a standard chow diet and water ad libitum. 2.2. Preparation of the plant extract
2.4. Experimental procedure 2.4.1. The oral glucose tolerance test (OGTT) in STZ-diabetic rats Four days after diabetes induction, the glucose tolerance test was performed by feeding glucose in the form of a solu-
3. Results 3.1. Effects of Sclerocarya birrea treatment on body weight, food and fluid intake Table 1 shows the evolution of body weight, food and water intake in control, untreated diabetic and treated diabetic rats during the 21-day treatment. At the end of the study, body weight of untreated diabetic rats were significantly lower than those of control rats. The increase in body weight of Sclerocarya birrea (300 mg/kg) groups was similar to that seen in control rats. The results shown in Table 1 also indicate that Sclero-
436
T. Dimo et al. / Journal of Ethnopharmacology 110 (2007) 434–438
Table 1 Body weight, water and food intakes in control and STZ-diabetic rats on Day 1 and Day 21 of treatment with Sclerocarya birrea extract and metformin Treatment
Body weight (g) Day 1
Control Diabetic S. b (150 mg/kg) S. b (300 mg/kg) Met (500 mg/kg)
257 256 253 258 255
± ± ± ± ±
Water (mL/rat/day) Day 21
6 7 16 7 5
308 240 243 305 279
± ± ± ± ±
8* 10 15 14* 14
Day 1 22.06 166.78 135.25 93.77 78.53
Food (g/rat/day) Day 21
± ± ± ± ±
1.60* 14.19 4.82* 11.01* 8.39*
18.78 218.55 142.46 90.45 75.90
Day 1 ± ± ± ± ±
1.65* 14.93 16.62* 9.13* 9.78*
20.40 34.56 32.21 27.03 23.86
Day 21 ± ± ± ± ±
0.96 2.39 1.89 0.49* 1.76*
20.15 48.40 35.63 30.04 28.00
± ± ± ± ±
1.05* 2.75 2.15* 1.96* 0.33*
Each value represents mean ± S.E.M., n = 6. * p < 0.05 as compared with untreated diabetic rats; S. b, Sclerocarya birrea, Met, metformin.
carya birrea treatment of diabetic rats resulted in a significant reduction of food intake as compared to untreated diabetic animals. The induction of STZ-diabetes resulted in elevated fluid intake which, like food intake, increased throughout the 3-week period in the untreated diabetic group. Polydipsia was significantly reduced with the administration of Sclerocarya birrea extract and metformin as compared to the diabetic control rats.
(in a concentration-dependent manner) to 942.58 ± 37.26 and 716.42 ± 54.85 mg/dL (p < 0.05), respectively, for the 150 and 300 mg/kg doses. Metformin treatment reduced area under the curve by 52% when compared with untreated diabetic rats.
3.2. Effects of Sclerocarya birrea extract on blood glucose in glucose-fed hyperglycemic rats
The mean blood glucose concentration of untreated diabetic and treated rats on Day 15 following the administration of Sclerocarya birrea extract at various time intervals are shown in Table 2. On Day 15 after the start of treatment, the plant extract (300 mg/kg) alone caused a significant decrease in unfasted blood glucose levels. Sclerocarya birrea extract at the dose of 300 mg/kg significantly decreased blood glucose levels throughout the entire experimental period. Metformin (500 mg/kg) also significantly reduced blood glucose concentration at the above time intervals. The plant extract at the doses of 150 and 300 mg/kg caused a maximum reduction in blood glucose concentrations of 65.8 and 67.0%, respectively, at the end of 10 h, as compared with the basal level. The most important decrease was induced by metformin (500 mg/kg), 10 h after it was administrated, with an 82.4% reduction in the basal glycaemia.
Fig. 1 shows the blood glucose levels of normal control, diabetic control, Sclerocarya birrea- and metformin-treated rats after oral administration of glucose (5 g/kg body weight). In diabetic control rats, the peak increase in blood glucose concentration was observed after 1 h, and remained high over the next hour. Sclerocarya birrea (300 mg/kg), like metformin (500 mg/kg), caused a significant decrease in blood glucose concentration 30 and 60 min after administration, respectively, when compared to the diabetic control rats. Mean glucose concentration calculated from the area under the response curves revealed a significant increase in glucose concentration of untreated diabetic rats (1240.33 ± 49.52 mg/dL) as compared to control rats (481.50 ± 18.70 mg/dL). Administration of the plant extract during glucose load markedly reduced glucose concentration
3.3. Effects of Sclerocarya birrea extract on blood glucose in diabetic rats
3.4. Effects of Sclerocarya birrea treatment on plasma parameters At the end of the experimental period, various plasma parameters were measured in all the groups. Plasma glucose, cholesterol and urea levels in untreated diabetic animals were significantly higher while the level of plasma insulin was significantly decreased (Table 3). Treatment with Sclerocarya birrea extract (300 mg/kg) significantly reduced plasma glucose levels by 70% as compared with untreated diabetic rats. Plasma insulin levels were significantly decreased in untreated diabetic animals as compared to the control rats. The administration of Sclerocarya birrea (300 mg/kg) and metformin (500 mg/kg) tended to bring insulin levels significantly towards normal values. 4. Discussion
Fig. 1. Effect of Sclerocarya birrea extract on blood glucose level in glucosefed hyperglycaemic rats. Values are means ± S.E.M., n = 6. * p < 0.05 compared with untreated diabetic rats.
In the present study, the administration of streptozotocin to rats, as expected, resulted in polyphagia, polydipsia, hyperglycemia, hypoinsulinemia, increased cholesterol, triglycerides and urea levels, and decreased body weight. The mechanism of
T. Dimo et al. / Journal of Ethnopharmacology 110 (2007) 434–438
437
Table 2 Effects of Sclerocarya birrea extract on blood glucose levels in STZ-diabetic rats on Day 15 of treatment Treatment
Blood glucose level (mM/L) 0h
Control Diabetic Sclerocarya birrea (150 mg/kg) Sclerocarya birrea (300 mg/kg) Met (500 mg/kg)
6.55 29.11 26.16 14.61 23.16
1.5 h ± ± ± ± ±
0.11* 1.33 1.22 0.94* † 1.11
6.38 28.22 24.66 12.94 14.66
3h ± ± ± ± ±
0.11*
6.22 27.72 20.77 11.44 7.88
1.38 1.44† 0.77* 1.05*
6h ± ± ± ± ±
0.16 1.72 1.44† 1.00* 0.77*
6.50 25.94 18.72 11.55 5.72
8h ± ± ± ± ±
0.22* 1.55 1.33† 0.72* 0.44*
6.11 22.88 13.77 6.00 4.66
10 h ± ± ± ± ±
0.11 1.05 1.50* † 0.61* 0.50*
6.00 21.50 8.94 4.83 4.05
± ± ± ± ±
0.05* 1.44 0.55* † 0.22* 0.33
Each value represents mean ± S.E.M. for six rat per group. * p < 0.05 as compared with untreated diabetic rats, †p < 0.05 as compared with metformin-treated rats. S. b, Sclerocarya birrea; Met, metformin.
STZ action has been fully described elsewhere (Meyerovitch et al., 1987; Lang and Dobrescu, 1989; Mongold et al., 1990; Pushparaj et al., 2001; Matsuda et al., 2002). In our study, we observed a significant reduction of food and fluid intakes and plasma glucose levels when STZ-diabetic rats were treated with Sclerocarya birrea extract at the dose of 300 mg/kg. Despite the reduction food and fluid intake by diabetic treated rats, at the end of 3 weeks of treatment, there was no loss in body weight when the effective dose of the plant extract was administrated, indicating that Sclerocarya birrea extract possibly compensated for the energy supplied from food intake, or contributed to the improved metabolism in diabetic treated animals. It has long been known that in the STZ-induced diabetes mellitus, the rise in blood glucose is accompanied by an increase in plasma cholesterol, triglycerides and urea (Cam et al., 1993; Pari and Saravanan, 2002). The Sclerocarya birrea dose (300 mg/kg body weight per day) used in this study had a tendency to reduce plasma cholesterol, triglyceride and urea levels, but the difference between treated diabetic and untreated diabetic groups did not reach statistical significance after 3 weeks. The effect of the plant extract on diabetic hypercholesterolemia and hypertriglyceridemia could be through its control of hyperglycaemia. This is in agreement with the fact that the level of glycemic control is the major determinant of total and very low density lipoprotein triglyceride concentrations (Laakso, 1995). The single high dose STZ-induced diabetic rat is one of the animal models of human insulin dependent diabetes mellitus (IDDM) or type I diabetes mellitus. In this model, diabetes arises from irreversible destruction of the -islet cells of the pancreas, causing degranulation or reduction of insulin secretion. In this type I model of diabetes, insulin is markedly depleted, but not absent (Pushparaj et al., 2001). Although insulin has become one of the most important therapeutic agents known to medicine, there is a continuing effort to find insulin substitutes,
secretagogues, or sensitizers from synthetic or plant sources for the treatment of diabetes. Hypoglycaemic effects have been reported for some plants that contain flavonoids (Pushparaj et al., 2001; Hosseinzaded et al., 2002) and we have obtained preliminary unpublished phytochemical results which show that Sclerocarya birrea contains flavonoids. In addition, Ojewole (2003) has discussed the possibility that arginine, glutamic acid, coumarins and/or terpenoids which are contained in the aqueous extract of Sclerocarya birrea could be responsible for the hypoglycaemic effect. The present study demonstrated that the methanol/methylene chloride stem bark extract of Sclerocarya birrea has marked hypoglycaemic property when given for 3 weeks to STZ-diabetic rats. Our results also revealed that Sclerocarya birrea treatment, like metformin, significantly reduced mean glucose concentration calculated from the area under the curve. Such an effect might be accounted for, in part, by a decrease in the rate of intestinal glucose absorption, as noted, e.g. when plant fibre is given orally with glucose (Mukherjee et al., 1997). Hence, it may be presumed that the glucoselowering effect of the plant extract was achieved by an extra intestinal action. Plasma insulin levels in the Sclerocarya birrea (300 mg/kg)-treated group was significantly higher on Day 21 compared to the untreated diabetic group. The elevation in plasma insulin levels in the Sclerocarya birrea-treated STZdiabetic rats could be due to substances present in the plant extract which stimulate insulin secretion or which protect the intact functional -cells from further deterioration so that they remain active and continue to produce insulin. Protection of the -cells could be at least, in part, a result of the reduction in blood glucose, eliminating gluco-toxicity to the -cells. This possibility is strengthened by the observation that insulin concentration where also increased by metformin, an agent which definitely does not stimulate insulin secretion but which lowers blood glucose through extra pancreatic mechanisms.
Table 3 Plasma blood glucose, cholesterol, triglyceride, urea and insulin levels of normal and experimental groups at the termination (21 days) Control Glucose (mM/L) Cholesterol (mM/L) Triglycerides (mM/L) Urea (mM/L) Insulin (pM/L)
6.61 1.24 0.88 4.16 296.21
Diabetic ± ± ± ± ±
0.16* 0.02 0.04 0.17 50.40*
37.28 1.76 1.41 7.66 69.89
± 1.66‡ ± 0.12‡ ± 0.16 ± 0.50‡ ± 10.12‡
Sclerocarya birrea (150 mg/kg) 38.61 1.50 1.25 8.83 60.95
± ± ± ± ±
1.83‡ 0.20 0.10 1.16‡ 9.10‡
Sclerocarya birrea (300 mg/kg) 10.50 1.45 1.06 6.83 186.97
± ± ± ± ±
3.77* 0.07 0.12 1.66 32.75*
Metformin (500 mg/kg) 30.66 1.56 1.68 6.00 186.24
± ± ± ± ±
3.27‡ 0.07‡ 0.80‡ 0.50 44.92*
Values are given as mean ± S.E.M. for six rats in each group. * p < 0.05 as compared with untreated diabetic rats; ‡p < 0.05 as compared with normal control rats.
438
T. Dimo et al. / Journal of Ethnopharmacology 110 (2007) 434–438
In conclusion, the present study shows that the antihyperglycemic action of the methanol/methylene chloride stem bark extract of Sclerocarya birrea on STZ-diabetic rats is associated with an increased plasma insulin concentration. Further comprehensive biochemical and pharmacological investigations are needed to elucidate the exact mechanism of the hypoglycemic effect of Sclerocarya birrea. Acknowledgements This project was supported by the Third World Academy of Sciences (TWAS), Trieste, Italy, through grant No. 03460/RG/BIO/AF/AC in collaboration with the “Institut pour la Recherche et le D´eveloppement (IRD)”, Paris, France for the provision to carry out a part of this study. References Adjanohoun, J.E., Aboubakar, N., Dramane, K., Ebot, M.E., Ekpere, J.A., EnowOrock, E.G., Focho, D., Gbile, Z.O., Kamanyi, A., Kamsu Kom, J., Keita, A., Mbenkum, T., Mbi, C.N., Mbiele, A.L., Mbome, L.L., Mubiri, N.K., Nancy, W.L., Nkongmeneck, B., Satabie, B., Sofowora, A., Tamze, V., Wirmum, C.K., 1996. Traditional Medicine and Pharmacopoeia: Contribution to Ethnobotanical and Floristic Studies in Cameroon. Organisation of African Unity Scientific. Technical and Research Commission. Centre National de Production de Manuels Scolaires, Porto Novo, 641 pp. Belemtougri, R.G., Constantin, B., Cognard, C., Raymond, G., Sawadogo, L., 2001. Effects of Sclerocarya birrea (A. rich) hochst (Anacardiaceae) leaf extract on calcium signalling in cultured rat skeletal muscle cells. Journal of Ethnopharmacology 76, 247–252. Berhaut, J., 1971. Flore Illustr´ee du S´en´egal. Dycotyl´edones. Tome I. Acanthac´ees a` avicenniac´ees. Minist`ere du D´eveloppement Rural. Direction des Eaux et Forˆet, Dakar, 277–279. Cam, C.M., Cros, H.G., Serrano, J.J., Lazaro, R., McNeill, H.J., 1993. In vivo antidiabetic actions of naglivan, an organic vanadyl compound in streptozotocin-induced diabetes. Diabetes Research and Clinical Pratctice 20, 111–121. Eloff, J.N., 2001. Antibacterial activity of Murula (Sclerocarya birrea (A. rich) Hochst. Subsp. Caffra (Sond) Kokwaro) (Anacardiaceae) bark and leaves. Journal of Ethnopharmacology 76, 305–308. Galvez, P.J., Zarzuelo, A., Busson, R., Cobbaert, C., De Witte, P., 1992. (−)-Epicatechin-—galloyl ester: a secretagogue compound from bark of Sclerocarya birrea. Planta Medica 58, 174–175.
Herbert, V., Lau, K.S., Gottlieb, C.W., Bleicher, S.J., 1965. Coated Charcoal immunoassay of insulin. Journal of Clinical Endocrinology and Metabolism 25, 1375–1384. Hosseinzaded, H., Ramezani, M., Danaei, A.R., 2002. Antihyperglycaemic effect of Securigera securidaca L. seed extract in mice. Phytotherapy Research 16, 745–747. Laakso, M., 1995. Epidemiology of diabetic dislipidemia. Diabetes Review 3, 408–422. Lang, H.C., Dobrescu, C., 1989. Sepsis-induced changes in in vivo insulin action in diabetic rats. American Journal of Physiology 257, E301–E308. Laurens, A., Giono-Barber, P., Mosser, J., Sylla, O., Giono-Barber, H., 1984. Activit´e antidiab´etique d’extraits de feuilles de Poupartia birrea (Hochst.) Aubr. Annal Pharmaceutiques Franc¸aise 41, 547–551. Matsuda, M., Kawasaka, F., Mikami, Y., Takeuchi, Y., Saito, M., Eto, M., Kaku, K., 2002. Rescue of beta-cell exhaustion by diazoxide after the development of diabetes mellitus in rats with steptozotocin-induced diabetes. European Journal of Pharmacology 431, 141–148. Meyerovitch, J., Farfel, Z., Sack, J., Shechter, Y., 1987. Oral administration of vanadate normalizes blood glucose levels in streptozotocin-treated rats. The Journal of Biological Chemistry 262, 6658–6662. Mongold, J.J., Cros, H.G., Vian, L., Tep, A., Ramanadham, S., Siou, G., Diaz, J., McNeill, J.H., Serrano, J.J., 1990. Toxicological aspects of vanadyl sulphate on diabetic rats: effects on vanadium levels and pancreatic ß-cell morphology. Pharmacology and Toxicology 67, 192–198. Mshana, N.R., Abbiw, D.K., Addae-Mensah, I., Adjanouhoun, E., Ahyi, M.R.A., Ekpere, J.A., Enow-Orock, E.G., Gbile, Z.O., Noamesi, G.K., Odei, M.A., Odunlami, H., Oteng-Yeboah, A.A., Sarpong, K., Sofowora, A., Tackie, A.N., 2000. Traditional medicine and pharmacopoeia. Contribution to the revision of ethnobotanical and floristic studies in Ghana. Organisation of African Unity/Scientific, Technical and Research Commission, p. 55. Mukherjee, K.P., Saha, K., Pai, M., Saha, B.P., 1997. Effect of Nelumbo nucifera rhizome extract on blood sugar level in rats. Journal of Ethnopharmacology 58, 207–213. Ojewole, J.A., 2003. Hypoglycemic effect of Sclerocarya birrea [(A. Rich)] Hochst [Anacardiaceae] stem bark aqueous extract in rats. Phytomedicine 10, 675–681. Pari, L., Saravanan, G., 2002. Antidiabetic effect of Cogent db, a herbal drug in alloxan-induced diabetes mellitus. Comparative Biochemistry and Physiology Part C 131, 19–25. Pushparaj, N.P., Tan, H.K.B., Tan, H.C., 2001. The mechanism of hypoglycemic action of the semi-purified fractions of Averrhoa bilimbi in streptozotocindiabetic rats. Life Sciences 70, 535–547. Watt, J.M., Breyer-Brandwijk, M.G., 1962. The Medicinal and Poisonous Plants of Southern and Eastern Africa. E.S. Livingstone, Edinburgh.
Effect of Sclerocarya birrea (Anacardiaceae) stem bark methylene chloride/methanol extract on streptozotocin-diabetic rats Th´eophile Dimo a,∗ , Silvere V. Rakotonirina a , Paul V. Tan a , Jacqueline Azay b , Etienne Dongo c , Pierre Kamtchouing a , G´erard Cros b a
Department of Animal Biology and Physiology, Faculty of Science, University of Yaounde I, P.O. Box 812, Yaounde, Cameroon b Laboratoire de Pharmacologie et Physiopathologie Exp´ erimentales, Universit´e Montpellier I, 34 093 Montpellier, France c Department of Organic Chemistry, Faculty of Science, University of Yaounde I, P.O. Box 812, Yaounde, Cameroon Received 7 January 2005; received in revised form 29 August 2006; accepted 12 October 2006 Available online 26 October 2006
Abstract Sclerocarya birrea (Anacardiaceae) is used as a traditional treatment of diabetes in Cameroon. In this study, we investigated the possible antidiabetic effect of the stem bark extract in diabetic rats. Diabetes was induced by intravenous injection of streptozotocin (STZ, 55 mg/kg) to male Wistar rats. Experimental animals (six per group), were treated by oral administration of plant extract (150 and 300 mg/kg body weight) and metformin (500 mg/kg; reference drug) for comparison, during 21 days. The stem bark methanol/methylene chloride extract of Sclerocarya birrea exhibited at termination, a significant reduction in blood glucose and increased plasma insulin levels in diabetic rats. The extract also prevented body weight loss in diabetic rats. The effective dose of the plant extract (300 mg/kg) tended to reduce plasma cholesterol, triglyceride and urea levels toward the normal levels. Four days after diabetes induction, an oral glucose tolerance test (OGTT) was also performed in experimental diabetic rats. The results showed a significant improvement in glucose tolerance in rats treated with Sclerocarya birrea extract. Metformin, a known antidiabetic drug (500 mg/kg), significantly decreased the integrated area under the glucose curve. These data indicate that Sclerocarya birrea treatment may improve glucose homeostasis in STZ-induced diabetes which could be associated with stimulation of insulin secretion. © 2006 Published by Elsevier Ireland Ltd. Keywords: Sclerocarya birrea; Glucose; Insulin; Streptozotocin; Diabetes
1. Introduction Sclerocarya birrea {(A. Rich.) Hochst.} is a tree which grows up to 10–15 m with grey fissured bark, stout branchlets and pale foliage. The leaves are compound, pinnate and the flowers greenish-white or reddish. The fruits are yellow, resembling a mango. In Africa, the tree is commonly found in savannah regions and its geographical distribution stretches from Gambia in the West across to Nigeria and Cameroon in Central Africa, and to Ethiopia and Sudan in the East (Berhaut, 1971). In Ghana, the leaves are used to treat snakebite, and pruritus (filarial); the stem bark, the root and the fruits are used to treat pharyngitis, splenomegaly and goitre, respectively (Mshana et al., 2000). In South Africa, the Zulu people use bark decoc-
∗
Corresponding author. E-mail address: [email protected] (T. Dimo).
0378-8741/$ – see front matter © 2006 Published by Elsevier Ireland Ltd. doi:10.1016/j.jep.2006.10.020
tions to treat diarrhoea, dysentery, fevers, stomach ailments, ulcers and for bacteria-related diseases (Eloff, 2001). Sclerocarya birrea is widely used in traditional medicine in Africa against hypertension, stomach-ache or gastro-enteritis, cough, and as antihyperglycemic (Laurens et al., 1984; Belemtougri et al., 2001). Phytochemical studies of Sclerocarya birrea have revealed the presence of alkaloids, anthocyans, flavonoids, tannins and saponosides (Watt and Breyer-Brandwijk, 1962; Belemtougri et al., 2001). A compound ((−)-epicatechin-3-galloyl ester) extracted from the stem bark of Sclerocarya birrea has secretagogue activity (Galvez et al., 1992). Pharmacological tests have shown that various leaf extracts of Sclerocarya birrea inhibit intracellular caffeine-induced Ca2+ release from skeletal muscle sarcoplasmic reticulum (Belemtougri et al., 2001). In addition, Eloff (2001), has demonstrated the antibacterial activity of the bark extract of Sclerocarya birrea against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Enterococcus faecalis.
T. Dimo et al. / Journal of Ethnopharmacology 110 (2007) 434–438
Information provided by practitioners of traditional medicine in North Cameroon suggest that Sclerocarya birrea possesses useful antidiabetic properties, although this was not cited in the national ethno-botanical survey of Cameroonian medicinal plants conducted by Adjanohoun et al. (1996). Recent studies (Ojewole, 2003) have revealed that moderate to high doses (100–800 mg/kg) of the stem bark aqueous extract possesses hypoglycaemic activity. In the present study, we have examined the antidiabetic activity of low doses of the organic (methanol/methylene chloride) extract of the stem bark of Sclerocarya birrea on streptozotocin (STZ)-induced diabetic rats. 2. Materials and methods 2.1. Animals
435
tion, orally to normal and STZ-diabetic rats fasted for 18 h. Distilled water (control groups), Sclerocarya birrea extract at a dose of 150 or 300 mg/kg body weight and a reference drug, metformin at a dose of 500 mg/kg were orally administrated to groups of six rats each. One hour later, glucose (5 g/kg) was administrated. Blood glucose levels from the tail vein were evaluated before (60 min) and at 0, 30, 60, 120, 180 and 300 min after glucose administration using Glucometer 4 Ames (Bayer Diagnostics). Calculation of the area under the curve (AUC) was made according to the following equation: AUC = basal glycaemia × 0.25 + glycaemia 0.5 h × 0.5 + glycaemia 1 h × 0.75 + glycaemia 2 h + glycaemia 3 h + glycaemia 5 h × 0.5.
The fresh stem bark of Sclerocarya birrea was harvested from Maroua in the Far North Province of Cameroon in June, and identified in Yaounde where voucher specimen No. 7770 is deposited in the National Herbarium. Two kilograms of the air-dried powdered stem bark were macerated in a mixture of methanol/methylene chloride (1:1) for 7 days (with occasional stirring) at room temperature. The mixture was filtered with Whatman No. 1 filter paper. The filtrate was concentrated under reduced pressure to obtain 120 g of a dark solid. Three grams of this extract were dissolved in 1 mL of dimethyl sulfoxide (DMSO) and the solution adjusted to 100 mL with distilled water to obtain a final extract concentration of 30 mg/mL.
2.4.2. Repeated administration of Sclerocarya birrea extract in STZ-diabetic rats Four days after STZ induction of diabetes in male rats, the non-fasted blood glucose levels were measured. Twenty-four hyperglycaemic rats were divided on Day 0 into equal groups of six animals each, two control groups were given by gastric intubations, either distilled water or 500 mg/kg metformin, and two test groups were given 150 or 300 mg/kg of extract per kilogram body weight. Six normal rats taken as negative controls were treated with vehicle. Treatment administration, food and water intakes were monitored every day between 8.00 and 9.00 am for 3 weeks. Plasma glucose and body weight were closely monitored twice a week. On the 15th day, the effect of the plant extract was studied up to 10 h after administration of the extract. Animals were fasted at 0 h, and the blood glucose levels were evaluated at 1.5, 3, 6, 8 and 10 h after administration of the extract. The treatment of rats was terminated on Day 21 at which time plasma samples were collected for biochemical determinations. Plasma glucose, cholesterol, triglycerides and urea were determined using a Hitachi 704 automatic analyzer. Plasma insulin was determined by the radio immunological method of Herbert et al. (1965) using rat insulin as standard.
2.3. Induction of experimental diabetes mellitus
2.5. Statistical analysis
The non-fasted Wistar rats were made diabetic with streptozotocin (55 mg/kg) (Sigma, St. Louis, MO, USA) by a single injection via the penis vein. The streptozotocin was freshly dissolved in physiological saline solution. The injection volume was 1 mL/kg. Control rats were injected with the same physiological solution only. Diabetes was confirmed in the STZtreated rats by measuring glucose levels 96 h post injection using glucose reagent strips with a Glucometer 4 Ames (Bayer Diagnostics). The rats with non-fasted blood glucose levels above 19.44 mmol/L were considered to be diabetic and were used in the experiment.
Statistical analysis of data was performed using a one-way analysis of variance (ANOVA) followed by Dunnett test for comparison between groups. Values for p < 0.05 were considered statistically significant. The data are shown as mean ± standard error of the mean (S.E.M).
Male Wistar rats from Iffa Credo, France, initially weighing between 180 and 200 g, were used. The rats were kept in a laboratory animal unit with a 12 h light/dark cycle. Throughout the experiment, room temperature was maintained at 25 ◦ C. The rats were maintained on a standard chow diet and water ad libitum. 2.2. Preparation of the plant extract
2.4. Experimental procedure 2.4.1. The oral glucose tolerance test (OGTT) in STZ-diabetic rats Four days after diabetes induction, the glucose tolerance test was performed by feeding glucose in the form of a solu-
3. Results 3.1. Effects of Sclerocarya birrea treatment on body weight, food and fluid intake Table 1 shows the evolution of body weight, food and water intake in control, untreated diabetic and treated diabetic rats during the 21-day treatment. At the end of the study, body weight of untreated diabetic rats were significantly lower than those of control rats. The increase in body weight of Sclerocarya birrea (300 mg/kg) groups was similar to that seen in control rats. The results shown in Table 1 also indicate that Sclero-
436
T. Dimo et al. / Journal of Ethnopharmacology 110 (2007) 434–438
Table 1 Body weight, water and food intakes in control and STZ-diabetic rats on Day 1 and Day 21 of treatment with Sclerocarya birrea extract and metformin Treatment
Body weight (g) Day 1
Control Diabetic S. b (150 mg/kg) S. b (300 mg/kg) Met (500 mg/kg)
257 256 253 258 255
± ± ± ± ±
Water (mL/rat/day) Day 21
6 7 16 7 5
308 240 243 305 279
± ± ± ± ±
8* 10 15 14* 14
Day 1 22.06 166.78 135.25 93.77 78.53
Food (g/rat/day) Day 21
± ± ± ± ±
1.60* 14.19 4.82* 11.01* 8.39*
18.78 218.55 142.46 90.45 75.90
Day 1 ± ± ± ± ±
1.65* 14.93 16.62* 9.13* 9.78*
20.40 34.56 32.21 27.03 23.86
Day 21 ± ± ± ± ±
0.96 2.39 1.89 0.49* 1.76*
20.15 48.40 35.63 30.04 28.00
± ± ± ± ±
1.05* 2.75 2.15* 1.96* 0.33*
Each value represents mean ± S.E.M., n = 6. * p < 0.05 as compared with untreated diabetic rats; S. b, Sclerocarya birrea, Met, metformin.
carya birrea treatment of diabetic rats resulted in a significant reduction of food intake as compared to untreated diabetic animals. The induction of STZ-diabetes resulted in elevated fluid intake which, like food intake, increased throughout the 3-week period in the untreated diabetic group. Polydipsia was significantly reduced with the administration of Sclerocarya birrea extract and metformin as compared to the diabetic control rats.
(in a concentration-dependent manner) to 942.58 ± 37.26 and 716.42 ± 54.85 mg/dL (p < 0.05), respectively, for the 150 and 300 mg/kg doses. Metformin treatment reduced area under the curve by 52% when compared with untreated diabetic rats.
3.2. Effects of Sclerocarya birrea extract on blood glucose in glucose-fed hyperglycemic rats
The mean blood glucose concentration of untreated diabetic and treated rats on Day 15 following the administration of Sclerocarya birrea extract at various time intervals are shown in Table 2. On Day 15 after the start of treatment, the plant extract (300 mg/kg) alone caused a significant decrease in unfasted blood glucose levels. Sclerocarya birrea extract at the dose of 300 mg/kg significantly decreased blood glucose levels throughout the entire experimental period. Metformin (500 mg/kg) also significantly reduced blood glucose concentration at the above time intervals. The plant extract at the doses of 150 and 300 mg/kg caused a maximum reduction in blood glucose concentrations of 65.8 and 67.0%, respectively, at the end of 10 h, as compared with the basal level. The most important decrease was induced by metformin (500 mg/kg), 10 h after it was administrated, with an 82.4% reduction in the basal glycaemia.
Fig. 1 shows the blood glucose levels of normal control, diabetic control, Sclerocarya birrea- and metformin-treated rats after oral administration of glucose (5 g/kg body weight). In diabetic control rats, the peak increase in blood glucose concentration was observed after 1 h, and remained high over the next hour. Sclerocarya birrea (300 mg/kg), like metformin (500 mg/kg), caused a significant decrease in blood glucose concentration 30 and 60 min after administration, respectively, when compared to the diabetic control rats. Mean glucose concentration calculated from the area under the response curves revealed a significant increase in glucose concentration of untreated diabetic rats (1240.33 ± 49.52 mg/dL) as compared to control rats (481.50 ± 18.70 mg/dL). Administration of the plant extract during glucose load markedly reduced glucose concentration
3.3. Effects of Sclerocarya birrea extract on blood glucose in diabetic rats
3.4. Effects of Sclerocarya birrea treatment on plasma parameters At the end of the experimental period, various plasma parameters were measured in all the groups. Plasma glucose, cholesterol and urea levels in untreated diabetic animals were significantly higher while the level of plasma insulin was significantly decreased (Table 3). Treatment with Sclerocarya birrea extract (300 mg/kg) significantly reduced plasma glucose levels by 70% as compared with untreated diabetic rats. Plasma insulin levels were significantly decreased in untreated diabetic animals as compared to the control rats. The administration of Sclerocarya birrea (300 mg/kg) and metformin (500 mg/kg) tended to bring insulin levels significantly towards normal values. 4. Discussion
Fig. 1. Effect of Sclerocarya birrea extract on blood glucose level in glucosefed hyperglycaemic rats. Values are means ± S.E.M., n = 6. * p < 0.05 compared with untreated diabetic rats.
In the present study, the administration of streptozotocin to rats, as expected, resulted in polyphagia, polydipsia, hyperglycemia, hypoinsulinemia, increased cholesterol, triglycerides and urea levels, and decreased body weight. The mechanism of
T. Dimo et al. / Journal of Ethnopharmacology 110 (2007) 434–438
437
Table 2 Effects of Sclerocarya birrea extract on blood glucose levels in STZ-diabetic rats on Day 15 of treatment Treatment
Blood glucose level (mM/L) 0h
Control Diabetic Sclerocarya birrea (150 mg/kg) Sclerocarya birrea (300 mg/kg) Met (500 mg/kg)
6.55 29.11 26.16 14.61 23.16
1.5 h ± ± ± ± ±
0.11* 1.33 1.22 0.94* † 1.11
6.38 28.22 24.66 12.94 14.66
3h ± ± ± ± ±
0.11*
6.22 27.72 20.77 11.44 7.88
1.38 1.44† 0.77* 1.05*
6h ± ± ± ± ±
0.16 1.72 1.44† 1.00* 0.77*
6.50 25.94 18.72 11.55 5.72
8h ± ± ± ± ±
0.22* 1.55 1.33† 0.72* 0.44*
6.11 22.88 13.77 6.00 4.66
10 h ± ± ± ± ±
0.11 1.05 1.50* † 0.61* 0.50*
6.00 21.50 8.94 4.83 4.05
± ± ± ± ±
0.05* 1.44 0.55* † 0.22* 0.33
Each value represents mean ± S.E.M. for six rat per group. * p < 0.05 as compared with untreated diabetic rats, †p < 0.05 as compared with metformin-treated rats. S. b, Sclerocarya birrea; Met, metformin.
STZ action has been fully described elsewhere (Meyerovitch et al., 1987; Lang and Dobrescu, 1989; Mongold et al., 1990; Pushparaj et al., 2001; Matsuda et al., 2002). In our study, we observed a significant reduction of food and fluid intakes and plasma glucose levels when STZ-diabetic rats were treated with Sclerocarya birrea extract at the dose of 300 mg/kg. Despite the reduction food and fluid intake by diabetic treated rats, at the end of 3 weeks of treatment, there was no loss in body weight when the effective dose of the plant extract was administrated, indicating that Sclerocarya birrea extract possibly compensated for the energy supplied from food intake, or contributed to the improved metabolism in diabetic treated animals. It has long been known that in the STZ-induced diabetes mellitus, the rise in blood glucose is accompanied by an increase in plasma cholesterol, triglycerides and urea (Cam et al., 1993; Pari and Saravanan, 2002). The Sclerocarya birrea dose (300 mg/kg body weight per day) used in this study had a tendency to reduce plasma cholesterol, triglyceride and urea levels, but the difference between treated diabetic and untreated diabetic groups did not reach statistical significance after 3 weeks. The effect of the plant extract on diabetic hypercholesterolemia and hypertriglyceridemia could be through its control of hyperglycaemia. This is in agreement with the fact that the level of glycemic control is the major determinant of total and very low density lipoprotein triglyceride concentrations (Laakso, 1995). The single high dose STZ-induced diabetic rat is one of the animal models of human insulin dependent diabetes mellitus (IDDM) or type I diabetes mellitus. In this model, diabetes arises from irreversible destruction of the -islet cells of the pancreas, causing degranulation or reduction of insulin secretion. In this type I model of diabetes, insulin is markedly depleted, but not absent (Pushparaj et al., 2001). Although insulin has become one of the most important therapeutic agents known to medicine, there is a continuing effort to find insulin substitutes,
secretagogues, or sensitizers from synthetic or plant sources for the treatment of diabetes. Hypoglycaemic effects have been reported for some plants that contain flavonoids (Pushparaj et al., 2001; Hosseinzaded et al., 2002) and we have obtained preliminary unpublished phytochemical results which show that Sclerocarya birrea contains flavonoids. In addition, Ojewole (2003) has discussed the possibility that arginine, glutamic acid, coumarins and/or terpenoids which are contained in the aqueous extract of Sclerocarya birrea could be responsible for the hypoglycaemic effect. The present study demonstrated that the methanol/methylene chloride stem bark extract of Sclerocarya birrea has marked hypoglycaemic property when given for 3 weeks to STZ-diabetic rats. Our results also revealed that Sclerocarya birrea treatment, like metformin, significantly reduced mean glucose concentration calculated from the area under the curve. Such an effect might be accounted for, in part, by a decrease in the rate of intestinal glucose absorption, as noted, e.g. when plant fibre is given orally with glucose (Mukherjee et al., 1997). Hence, it may be presumed that the glucoselowering effect of the plant extract was achieved by an extra intestinal action. Plasma insulin levels in the Sclerocarya birrea (300 mg/kg)-treated group was significantly higher on Day 21 compared to the untreated diabetic group. The elevation in plasma insulin levels in the Sclerocarya birrea-treated STZdiabetic rats could be due to substances present in the plant extract which stimulate insulin secretion or which protect the intact functional -cells from further deterioration so that they remain active and continue to produce insulin. Protection of the -cells could be at least, in part, a result of the reduction in blood glucose, eliminating gluco-toxicity to the -cells. This possibility is strengthened by the observation that insulin concentration where also increased by metformin, an agent which definitely does not stimulate insulin secretion but which lowers blood glucose through extra pancreatic mechanisms.
Table 3 Plasma blood glucose, cholesterol, triglyceride, urea and insulin levels of normal and experimental groups at the termination (21 days) Control Glucose (mM/L) Cholesterol (mM/L) Triglycerides (mM/L) Urea (mM/L) Insulin (pM/L)
6.61 1.24 0.88 4.16 296.21
Diabetic ± ± ± ± ±
0.16* 0.02 0.04 0.17 50.40*
37.28 1.76 1.41 7.66 69.89
± 1.66‡ ± 0.12‡ ± 0.16 ± 0.50‡ ± 10.12‡
Sclerocarya birrea (150 mg/kg) 38.61 1.50 1.25 8.83 60.95
± ± ± ± ±
1.83‡ 0.20 0.10 1.16‡ 9.10‡
Sclerocarya birrea (300 mg/kg) 10.50 1.45 1.06 6.83 186.97
± ± ± ± ±
3.77* 0.07 0.12 1.66 32.75*
Metformin (500 mg/kg) 30.66 1.56 1.68 6.00 186.24
± ± ± ± ±
3.27‡ 0.07‡ 0.80‡ 0.50 44.92*
Values are given as mean ± S.E.M. for six rats in each group. * p < 0.05 as compared with untreated diabetic rats; ‡p < 0.05 as compared with normal control rats.
438
T. Dimo et al. / Journal of Ethnopharmacology 110 (2007) 434–438
In conclusion, the present study shows that the antihyperglycemic action of the methanol/methylene chloride stem bark extract of Sclerocarya birrea on STZ-diabetic rats is associated with an increased plasma insulin concentration. Further comprehensive biochemical and pharmacological investigations are needed to elucidate the exact mechanism of the hypoglycemic effect of Sclerocarya birrea. Acknowledgements This project was supported by the Third World Academy of Sciences (TWAS), Trieste, Italy, through grant No. 03460/RG/BIO/AF/AC in collaboration with the “Institut pour la Recherche et le D´eveloppement (IRD)”, Paris, France for the provision to carry out a part of this study. References Adjanohoun, J.E., Aboubakar, N., Dramane, K., Ebot, M.E., Ekpere, J.A., EnowOrock, E.G., Focho, D., Gbile, Z.O., Kamanyi, A., Kamsu Kom, J., Keita, A., Mbenkum, T., Mbi, C.N., Mbiele, A.L., Mbome, L.L., Mubiri, N.K., Nancy, W.L., Nkongmeneck, B., Satabie, B., Sofowora, A., Tamze, V., Wirmum, C.K., 1996. Traditional Medicine and Pharmacopoeia: Contribution to Ethnobotanical and Floristic Studies in Cameroon. Organisation of African Unity Scientific. Technical and Research Commission. Centre National de Production de Manuels Scolaires, Porto Novo, 641 pp. Belemtougri, R.G., Constantin, B., Cognard, C., Raymond, G., Sawadogo, L., 2001. Effects of Sclerocarya birrea (A. rich) hochst (Anacardiaceae) leaf extract on calcium signalling in cultured rat skeletal muscle cells. Journal of Ethnopharmacology 76, 247–252. Berhaut, J., 1971. Flore Illustr´ee du S´en´egal. Dycotyl´edones. Tome I. Acanthac´ees a` avicenniac´ees. Minist`ere du D´eveloppement Rural. Direction des Eaux et Forˆet, Dakar, 277–279. Cam, C.M., Cros, H.G., Serrano, J.J., Lazaro, R., McNeill, H.J., 1993. In vivo antidiabetic actions of naglivan, an organic vanadyl compound in streptozotocin-induced diabetes. Diabetes Research and Clinical Pratctice 20, 111–121. Eloff, J.N., 2001. Antibacterial activity of Murula (Sclerocarya birrea (A. rich) Hochst. Subsp. Caffra (Sond) Kokwaro) (Anacardiaceae) bark and leaves. Journal of Ethnopharmacology 76, 305–308. Galvez, P.J., Zarzuelo, A., Busson, R., Cobbaert, C., De Witte, P., 1992. (−)-Epicatechin-—galloyl ester: a secretagogue compound from bark of Sclerocarya birrea. Planta Medica 58, 174–175.
Herbert, V., Lau, K.S., Gottlieb, C.W., Bleicher, S.J., 1965. Coated Charcoal immunoassay of insulin. Journal of Clinical Endocrinology and Metabolism 25, 1375–1384. Hosseinzaded, H., Ramezani, M., Danaei, A.R., 2002. Antihyperglycaemic effect of Securigera securidaca L. seed extract in mice. Phytotherapy Research 16, 745–747. Laakso, M., 1995. Epidemiology of diabetic dislipidemia. Diabetes Review 3, 408–422. Lang, H.C., Dobrescu, C., 1989. Sepsis-induced changes in in vivo insulin action in diabetic rats. American Journal of Physiology 257, E301–E308. Laurens, A., Giono-Barber, P., Mosser, J., Sylla, O., Giono-Barber, H., 1984. Activit´e antidiab´etique d’extraits de feuilles de Poupartia birrea (Hochst.) Aubr. Annal Pharmaceutiques Franc¸aise 41, 547–551. Matsuda, M., Kawasaka, F., Mikami, Y., Takeuchi, Y., Saito, M., Eto, M., Kaku, K., 2002. Rescue of beta-cell exhaustion by diazoxide after the development of diabetes mellitus in rats with steptozotocin-induced diabetes. European Journal of Pharmacology 431, 141–148. Meyerovitch, J., Farfel, Z., Sack, J., Shechter, Y., 1987. Oral administration of vanadate normalizes blood glucose levels in streptozotocin-treated rats. The Journal of Biological Chemistry 262, 6658–6662. Mongold, J.J., Cros, H.G., Vian, L., Tep, A., Ramanadham, S., Siou, G., Diaz, J., McNeill, J.H., Serrano, J.J., 1990. Toxicological aspects of vanadyl sulphate on diabetic rats: effects on vanadium levels and pancreatic ß-cell morphology. Pharmacology and Toxicology 67, 192–198. Mshana, N.R., Abbiw, D.K., Addae-Mensah, I., Adjanouhoun, E., Ahyi, M.R.A., Ekpere, J.A., Enow-Orock, E.G., Gbile, Z.O., Noamesi, G.K., Odei, M.A., Odunlami, H., Oteng-Yeboah, A.A., Sarpong, K., Sofowora, A., Tackie, A.N., 2000. Traditional medicine and pharmacopoeia. Contribution to the revision of ethnobotanical and floristic studies in Ghana. Organisation of African Unity/Scientific, Technical and Research Commission, p. 55. Mukherjee, K.P., Saha, K., Pai, M., Saha, B.P., 1997. Effect of Nelumbo nucifera rhizome extract on blood sugar level in rats. Journal of Ethnopharmacology 58, 207–213. Ojewole, J.A., 2003. Hypoglycemic effect of Sclerocarya birrea [(A. Rich)] Hochst [Anacardiaceae] stem bark aqueous extract in rats. Phytomedicine 10, 675–681. Pari, L., Saravanan, G., 2002. Antidiabetic effect of Cogent db, a herbal drug in alloxan-induced diabetes mellitus. Comparative Biochemistry and Physiology Part C 131, 19–25. Pushparaj, N.P., Tan, H.K.B., Tan, H.C., 2001. The mechanism of hypoglycemic action of the semi-purified fractions of Averrhoa bilimbi in streptozotocindiabetic rats. Life Sciences 70, 535–547. Watt, J.M., Breyer-Brandwijk, M.G., 1962. The Medicinal and Poisonous Plants of Southern and Eastern Africa. E.S. Livingstone, Edinburgh.