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Journal of Ethnopharmacology 72 (2000) 69 – 76

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Effects of A6errhoa bilimbi leaf extract on blood glucose
and lipids in streptozotocin-diabetic rats
P. Pushparaj a, C.H. Tan a, B.K.H. Tan b,*
a

Department of Biochemistry, Faculty of Medicine, National Uni6ersity of Singapore, Singapore 119260, Singapore
Department of Pharmacology, Faculty of Medicine, National Uni6ersity of Singapore, 10 Kent Ridge Crescent,
Singapore 119260, Singapore

b

Received 18 June 1999; received in revised form 5 December 1999; accepted 28 February 2000

Abstract
The present study was designed to investigate the hypoglycemic and hypolipidemic activities of an ethanolic extract
of A6errhoa bilimbi Linn. leaves (Oxalidaceae, Common name: Bilimbi) in streptozotocin (STZ)-diabetic rats. The
optimal hypoglycemic dose (125 mg kg − 1) was determined by performing the oral glucose tolerance test (OGTT) in
both normal and STZ-diabetic rats. To investigate the effect of repeated administration of an ethanolic extract of
A6errhoa bilimbi (ABe) leaves, diabetic rats were treated with vehicle (distilled water), ABe (125 mg kg − 1) or
metformin (500 mg kg − 1) twice a day for 2 weeks. Like metformin, ABe significantly lowered blood glucose by 50%
and blood triglyceride by 130% when compared with the vehicle. ABe also significantly increased the HDL-cholesterol
concentrations by 60% compared with the vehicle. ABe thus significantly increased the anti-atherogenic index and
HDL-cholesterol/total cholesterol ratio. However, like metformin, ABe did not affect total cholesterol and LDLcholesterol concentrations, but significantly reduced the kidney lipid peroxidation level. These data show that ABe has
hypoglycemic, hypotriglyceridemic, anti-lipid peroxidative and anti-atherogenic properties in STZ-diabetic rats.
© 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: A6errhoa bilimbi Linn.; Diabetes mellitus; Hypoglycemia; Hypolipidemia; Streptozotocin; Sprague – Dawley rat

1. Introduction
The use of medicinal plants for the treatment of
diabetes mellitus dates back from the Ebers papyrus of about 1550 BC. Many traditional plant
treatments for diabetes mellitus are used through* Corresponding author. Tel.: +65-8743272; fax: +657730579.
E-mail address: phctankh@nus.edu.sg (B.K.H. Tan).

out the world. After the introduction of insulin
therapy the use of the traditional treatments for
diabetes mellitus greatly declined in the occidental
societies, although some traditional practices are
continued for prophylactic purposes and as adjuncts to conventional therapy (Swanston Flatt et
al., 1990). Few of the traditional plant treatments
for diabetes have received scientific scrutiny, and
the World Health Organization has recommended
that this area warrants attention (WHO, 1980).

0378-8741/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 3 7 8 - 8 7 4 1 ( 0 0 ) 0 0 2 0 0 - 2

MO. (Oxalidaceae. Standard pelleted diet (Glen Forest. National University of Singapore. The STZ was freshly dissolved in citrate buffer (0. The SD rats with blood glucose level above 350 mg dl − 1 were considered to be diabetic and were used in the experiment (Lisa et al. bilimbi fruits and leaves (Tan et al. 1993). Induction of experimental diabetes mellitus The overnight fasted SD rats were made diabetic with streptozotocin (STZ) (Sigma. Preparation of the plant extract The fresh leaves of A. A dried specimen was deposited in the herbarium (Voucher specimen No. which has been widely used in traditional medicine as a cure for cough. Blood samples were collected from the tail vein by tail milking at − 30 (just before the ABe and metformin administration).p)..3.. 1985).2. St Louis. 1 filter paper (Whatman International. i. Materials and methods 2. 1998). 0 (just before the . glucose (3 g kg − 1) was orally administered to each rat with a feeding syringe (Al-awadi et al.70 P. and postpartum protective properties (Goh et al. Singapore Botanic Gardens. or each of three different doses of ABe (125. / Journal of Ethnopharmacology 72 (2000) 69–76 This paper describes the study of A6errhoa bilimbi Linn. The OGTT in normal and STZ-diabetic SD rats Prior to an oral glucose tolerance test (OGTT) rats were fasted for 16 h. Diabetes was confirmed in the STZ-treated rats by measuring the fasting blood glucose concentration 72 h after injecting STZ. pH 4. A. a reference drug metformin (500 mg kg − 1). Australia) and water were given ad libitum. WA. bilimbi (1 kg) were blended and extracted with 80% ethanol (10 l) until exhaustion.4. UK). Animals All experiments were performed on male Sprague–Dawley (SD) rats aged 10 weeks (200– 250 g) obtained from the Laboratory Animal Holding Unit. The filtrate was centrifuged for 10 min at 10 000 × g to remove particulate substances. itches.. 1992). bilimbi leaves on the blood glucose level and serum lipid profile in streptozotocin-diabetic Sprague–Dawley (SD) rats. anti-scorbutic. anti-bacterial. 2. syphilis. Keeper of Herbarium and Library. The clear supernatant was concentrated at 40°C by a rotavapor (Buchi Labortechnik. In Indonesia it has a considerable medicinal reputation as a potent folk remedy in the treatment of diabetes mellitus (Wee Yeow Chin. cold. whooping cough. yielding about 40 g of yellowish-green pow- der..5. The animals were acclimatized for 1–2 weeks before being used for the experiments. a common plant in Asia.1. 1996). 2. The extract was suspended in distilled water before use. Animals were maintained under a constant 12-h light and dark cycle and an environmental temperature of 21–23°C (Niyonzima and Vlietinck. Materials The plant was collected from a private garden and identified as A6errhoa bilimbi by Dr Ruth Kiew. 2. This study was thus initiated with the aim of evaluating the effects of an ethanolic extract of A. bilimbi has been widely reported for its multiple ethnopharmacological properties such as anti-inflammatory. BT 2). the injection volume was 1 ml kg − 1 (Hamilton et al.5) and maintained on ice prior to use. boils. diabetes. The concentrate was freezedried. rheumatism.1. Switzerland) to 1 l. Thirty minutes later.01 M. 1998).. and hypertension (Goh et al. 2. Experimental procedure 2. 2. 1995). astringent. 60 mg kg − 1. A preliminary study in our laboratory showed the reduction of blood glucose and food intake in diabetic rats given with extracts of A. 250 and 500 mg kg − 1) were then orally administered to groups of 4–5 rats each. 1995). The mixture was filtered with Whatman No. Animals had free access to food and water after the STZ injection. Distilled water (control). In addition. common name: Bilimbi).5. Singapore.. Pushparaj et al.

After the . The hyperglycemic rats (blood glucose \350 mg dl − 1) were divided on day zero into three groups (each with 5 – 6 rats). positive control and the treatment groups. the rats were decapitated and the blood was collected for estimation of the fasting blood glucose. 50% (v/v) and 1.5 ml of 1 M potassium phosphate buffer and the required volume of 1. Boehringer Mannheim.2 ml of distilled water were added. all the rats were decapitated and their livers and kidneys were removed quickly. The rat liver (2 g) was homogenized in ice cold 1. Sigma. Protein content in each microsomal suspension was determined by Lowry’s method (Lowry et al. and 180 min after glucose load for the assay of glucose (Trinder method. pH 7. total cholesterol. Germany).7. total cholesterol.1 ml of 25% homogenate. Briefly to 0. after 16 h fasting. The molar extinction coefficient of microsomal P450 at the lmax of 450 nm was 91 mM − 1 cm − 1.6. / Journal of Ethnopharmacology 72 (2000) 69–76 oral administration of glucose). metformin (positive control) and ABe-treated groups. 2.2 ml of 8. On day 15. The organs such as liver and kidney were isolated. Six animals were used for distilled water (control). followed by 1 ml of 0.5. respectively for 2 weeks.2.15% KCl (20 ml) using a glass Potter-Elvejhem homogeniser.6. MO). 2. 1974). The microsomal preparation was placed in two cuvettes and initially saturated with carbon monoxide. A small amount of sodium dithionite (not more than 2 mg) was added to the sample cuvette only. The microsomal pellet was resuspended in a volume of glycerol buffer [200 mM potassium phosphate buffer. The determination of the lipid peroxidation level in the liver and kidneys was performed by the thiobarbituric acid method (Uchiyama and Mihara. 1964) was adopted in this assay to eliminate the absorption peak at 420 nm due to contamination by hemoglobin in the sample.1% sodium dodecyl sulfate (SDS). LDL-cholesterol.5 ml of 1% phosphoric acid and 0.6% 2-TBA. Assay of li6er microsomal cytochrome P450 content An aliquot of microsomal preparation of 1 mg protein ml − 1 was obtained by adding 0. metformin (500 mg kg − 1) and ABe (125 mg kg − 1) were then administered orally twice a day to control.. triglycerides. triglycerides.6. and HDL-cholesterol (using diagnostic kits. weighed and stored at −70°C for the assay of hepatic cytochrome P450 and thiobarbituric acid reactive substances (TBARS) in both liver and kidney. Liver microsomes were prepared.2. Repeated administration of the ABe in STZ-diabetic SD rats One week after STZ induction of diabetes in male SD rats. food and water intakes were monitored daily for 2 weeks. The OGTTs were performed in STZ-diabetic rats using the same procedure as described for the normal rats. 120.1.15% (w/v) potassium chloride (5:8:7)] equivalent to twice the liver weight.15% KCl to make a 25% (w/v) homogenate. Body weight. 2. the fasting blood glucose levels were measured. A modified technique (Omura and Sato. The fasting blood glucose level. Li6er microsomal cytochrome P450 content 2.. The microsomal P450 content was then determined from the difference in absorbance values between the dithionite-reduced and control microsomal preparations using a Shimadzu UV-dualbeam spectrophotometer. HDL-cholesterol. Pushparaj et al. Preparation of li6er microsomes After 14 days of treatment.15% KCl.4. rinsed in ice-chilled normal saline and weighed. Distilled water. 60. using the ultra-centrifugation method (El Defrawy El Masry et al. 0. 2. and LDL-cholesterol concentrations were also measured on day zero. Sigma Diagnostics. 1951).P. 1. Assay of TBARS The liver and kidney samples were minced and homogenized by a polytron homogeniser in icecold 1. The pellet was further homogenized with seven strokes of the pestle after which aliquots of the microsomal suspension were stored at − 70°C. 1978). The homogenate obtained was centrifuged at 71 10 000× g for 10 min at 4°C and the supernatant obtained recentrifuged at 100 000× g for 45 min at 4°C. St Louis. The mixture was heated for 45 min in a boiling water bath.

p. the fasting blood glucose levels were 4–5 times higher than that of the normal SD rats. 500 mg kg−1. p. 125. food and water intakes during the 14 days of treatment period were compared by two-way ANOVA.05 were considered to be statistically significant.6** −66 91.01). * PB0. 500 mg 609 7.9 32914.7* 500 mg kg−1.8 3 91.7** Values are expressed as the mean9S. p. the lowest dose. 125 mg kg − 1 caused a significant attenuation in the blood glucose at 180 min when compared to the vehicle-treated control group (PB 0. Pushparaj et al. *** PB0.01) after the oral glucose load (Table 1B). the lowest dose (125 mg kg − 1) was found to be most effective in improving glucose tolerance (P B0.P. The data on blood glucose.o.3** −129 6.3 kg−1. Results 3. 120 and 180 min was calculated from the corresponding 0-h value (just before the oral administration of glucose) in each group.8. for 4–6 rats in each group. HDL-cholesterol. Of the three doses of ABe tested.05). P-values of B 0. Metformin −29 9. .M.M.9 11911.6** −669 5.2* 419 12. After centrifugation at 1000× g for 10 min.9 kg−1. Effects of ABe on glucose tolerance in normal and STZ-diabetic SD rats The blood glucose levels of the normal rats reached a peak 60 min after the oral administration of glucose and gradually decreased to the pre-glucose load level (Table 1A). p. Metformin (500 mg kg − 1) produced a significant decrease (P B0.9 −26912. ABe and metformin-treated normal and diabetic rats after oral glucose load (3 g kg−1) in the oral glucose tolerance test (OGTT)a Group Percentage change in blood glucose level 60 min A. i. 120 min (PB 0. 250 mg kg−1. triglycerides. p. the absorbance of the n-butanol layer was determined at 535 nm.01) when compared to the vehicle-treated group. p.001). and anti-atherogenic index were analysed by the Student’s t-test (two-tailed).5 2 9 6.0 ml of n-butanol. The changes in body weight.2 −29 6. TBARS. the cold TBA reactants were extracted with 4.7*** 180 min 39 12. even at 180 min. total cholesterol.7* 47 9 6. p.E.o.01) and 180 min (PB 0.01 compared with the corresponding control. STZ-diabetic rat Control ABe 125 mg kg−1.5 34 98. 250 and 500 mg kg − 1.o. Of the three different doses. B.o. Normal rat Control 76 916 ABe 125 mg 39 98.o.1 −2699.E.6 159 3.5 89 9. 3. Table 1 The mean percentage changes in the blood-glucose levels over the 0-h control.1. / Journal of Ethnopharmacology 72 (2000) 69–76 72 reaction.01) in blood glucose level 180 min after the administration of an oral glucose load. a 120 min 38 9 11. The ABe at a dose of 125 mg kg − 1 produced a significant attenuation in the blood glucose (PB 0.6 23 93.05 compared with the corresponding control.o.05) at 120 and 180 min (PB 0. Metformin (500 mg kg − 1) caused significant attenuation at 60 min (PB 0.6 −18 95.o. viz.o.5 1798.2 7 9 1.1 kg−1. The percentage change in blood glucose at 60.3 64 99. p.001 compared with the corresponding control. In the diabetic rats.e. However. ABe at a dose of 500 mg kg − 1 caused a significant attenuation (PB 0. ** PB0. 2. Statistical analysis The results are presented as means9 S. There was no significant attenuation in the rats administered 250 mg of ABe kg − 1. the mixture was cooled in an ice bath. Metformin 500 mg kg−1.01) in the blood glucose only at 180 min when compared to the vehicle-treated group. Hence it was selected for the 2-week study. 250 mg 679 16. LDL-cholesterol.5 369 8. The TBARS values thus obtained were expressed as nmol of malonaldehyde per 25 mg wet tissue.5* 89 6 −18 94.

Similarly. Effects of 2 -week administration of ABe and metformin on STZ-diabetic SD rats The body weights in the ABe-treated and the metformin-treated groups were increased significantly (PB0.001) when compared to the vehicle-treated group of rats.and vehicle-treated control STZ-diabetic SD rats (P\ 0. The anti-atherogenic index was significantly increased in the ABe-treated group (PB 0.01) in the blood glucose level in STZ-diabetic SD rats when compared to vehicle and day zero values.05) and a significant increase in HDL-cholesterol (P B0. there was no significant difference in the hepatic microsomal cytochrome P450 content between ABe. As shown in Table 3.05).01) when compared to the vehicle-treated control rats. ABe (125 mg kg−1) and metformin (500 mg kg−1) twice a day for 2 weeksa Treatment group Vehicle ABe Metformin Body weight (g) Water intake (ml rat−1 day−1) Food intake (g rat−1 day−1) Before After Before After Before After 222911 259918 258913 205 914 282931* 323947* 149 9 27 92.05) in the ABe-treated SD rats when compared to the vehicle-treated control SD rats. However. 4.4 95 34 94* 29 91. a 3.05).1 9 27 131 9 23 89 9 12* 77. However.05). the water intake was significantly reduced (PB0.P.2. However there was a significant reduction in the hepatic microsomal cytochrome P450 content in the metformin-treated group when compared to the vehicle-treated group (PB0. Discussion Our present studies show that ABe possesses definite hypoglycemic. However there was no significant difference in TBARS levels in the livers of both ABeand metformin-treated diabetic rats when compared to the vehicle-treated control diabetic rats.05) in the HDL-cholesterol/total cholesterol ratio in the metformin-treated group when compared to the vehicle-treated group.05). The HDLcholesterol/total cholesterol ratio was significantly increased in the ABe-treated group when compared to the vehicle-treated group (PB 0. As shown in Table 4. It also failed to increase the HDL-cholesterol. Metformin. The daily administration of metformin (500 mg kg − 1 twice a day) for 14 days to STZ-diabetic SD rats caused a significant decrease in the serum triglycerides (PB 0. The food intake was significantly lowered in the ABetreated and metformin-treated groups (P B0. Similarly. ABe did not decrease the serum cholesterol and LDL-cholesterol significantly (P\ 0. * Significantly different from vehicle at PB0.001) when compared with the vehicle-treated group.E.6 926* 37 95 28 9 7 26 9 6 44. anti- . did not de- crease serum cholesterol and LDL-cholesterol.01). (n= 5 or 6).and metformin-treated STZ-diabetic SD rats (PB 0.M.1* Values are expressed as mean 9S.001. The TBARS levels were significantly reduced in the kidneys of both ABe.6 9 30 101.001) on day 14 when compared with the vehicle-treated group (Table 2).01) rats and day zero value (P B0. / Journal of Ethnopharmacology 72 (2000) 69–76 73 Table 2 Body weight. However. there was no significant difference in the anti-atherogenic index of the metformin and the vehicle-treated groups (P\ 0. hypotriglyceridemic.05). Pushparaj et al. the daily administration of the ABe (125 mg kg − 1 twice a day) for 14 days in STZ-diabetic SD rats caused a significant reduction in the blood glucose level when compared with the vehicle-treated control (P B0.001) in both ABe-treated and metformin-treated groups (Table 2). repeated administration of metformin (500 mg kg − 1 twice a day) for 14 days caused a significant reduction (PB 0.01). water and food intakes in STZ-diabetic rats before and after oral treatment with vehicle (distilled water). however. there was no significant difference (P\ 0. There was a significant decrease in serum triglycerides (P B 0.

9 35.4b.0 21. ABe (125 mg kg−1) and metformin (500 mg kg−1)a Treatment group Vehicle Abe Metformin Liver cytochrome P450 content (nmol mg−1 protein) 1. ** Significantly different from vehicle at PB0.05c.2b.25 2.M.6 44.3690.04 2. e Significantly different from zero day at PB0.4 59 9 8.2b.3 18.7 920. a Lipid peroxidation level (nmol of malonaldehyde per 25 mg of tissue) Liver Kidney 3.897.08 90.01.3 20.6924.1 78.799.99 62. d Significantly different from vehicle at PB0.5 219 2.05.491.2 90.E.74 Table 3 Metabolic variables in STZ-diabetic rats before and after oral treatment with vehicle (distilled water).e 202.197.01 90.6 258.4 522.5 90.03 68.49 0.4931.1 394.996.797.89 37.9 24.2 3.4 917.46 90.99 0.8 9 0.5913.03* Values are expressed as mean 9S.7 28.1 21.08 1. a b c Table 4 Liver cytochrome P450 content and lipid peroxidation level in the kidney and liver of STZ-diabetic rats after 2 weeks of oral treatment twice a day with vehicle (distilled water).499.29 0.6940.f 26 9 6.3 90. / Journal of Ethnopharmacology 72 (2000) 69–76 Treatment group .1 57.792.001.2 70.8 44.390.3 9 46. Pushparaj et al.25** P.2c.2 9 0.5 194.03 After 0.05.07 1.89 4.5 72.02 0.4 173.293.593.01.01. * Significantly different from vehicle at PB0.8 19.195.f 0.89 0.49 57.1 90.79 30.17** 3.0d. Significantly different from vehicle at PB0.05.f 81.3 0.0 20.7 130.M.13 4. Significantly different from vehicle at PB0.1914. (n = 5 or 6).1 110913.e 25.5 509.11 Values are expressed as mean 9 S.E. ABe (125 mg kg−1) and metformin (500 mg kg−1) twice a day for 2 weeksa Vehicle ABe Metformin Fasting blood glucose (mg dl−1) Total cholesterol (mg dl−1) Triglycerides (mg dl−1) Before After Before After Before 498.598.4 0.3 9 0.f 0.2b.0 LDL-cholesterol (mg dl−1) HDL-cholesterol (mg dl−1) Antiatherogenic index (%) HDL-C/TC ratio After Before After Before After Before After Before 185. f Significantly different from zero day at PB0.394. (n=5 or 6).39 0.599.7 2192.39 17.4 14.193.2910.e 59.

but it increases glucose utilisation in the extrahepatic tissues. 1998). (Bailey and Flatt. The mechanism by which metformin reduces the cytochrome P450 content is not known. 1996). The accumulation of TxB2 along with thromboxane-A2 (TxA2) can cause platelet aggregation and promote thrombosis (Sushil Jain et al. 1998). resulting in the formation of hydroperoxy fatty acids and endoperoxides. anti-lipid peroxidative as well as the antiatherogenic properties of ABe might be due to different types of active principles. The cytochrome P450 content in the liver has been found to be increased in diabetic animals (Lucas et al. a biguanide. and an increase in the body weight in STZ-diabetic rats. Pushparaj et al.. a pure compound isolated from Larrea tridentata (Reed et al. 1990) does not induce the secretion of insulin from the b-islet cells of pancreas. and anti-lipid peroxidative properties in STZ-diabetic rats after 2 weeks of treatment. ABe thus has the potential to prevent the formation of atherosclerosis and coronary heart disease which are the secondary diabetic complications of severe diabetes mellitus (Fontbonne et al. However a reduction was found in the metformin-treated group. hypotriglyceridemic. Department of Pharmacology. metformin failed to increase the HDL-cholesterol level and did not increase the anti-atherogenic index and HDL-cholesterol/total cholesterol ratio. 1998). Metformin. ABe may contain a hypolipidemic principle(s). Since ABe has the ability to reduce the formation of TBARS.. 1997). National University of Singapore. The daily administration of ABe (125 mg kg − 1) and metformin (500 mg kg − 1) to STZ-diabetic rats twice a day for 2 weeks caused a statistically significant reduction in food and water intakes... In this study cytochrome P450 content of the ABe-treated group was similar to that of the vehicle-treated group. Since ABe reduced the blood glucose potently in the STZ-diabetic SD rats like metformin. Hence. it significantly increased the anti-atherogenic index and HDL-cholesterol/total cholesterol ratio. which subsequently generates free radicals such as O2 − and OH°. the hypoglycemic. 1992) and increases the expression of insulin receptors in the liver plasma membranes (Kanigur-Sultuybek et al. This increases the formation of malonaldehyde (MDA) and thromboxane-B2 (TxB2). 1995). Pushparaj. 1989). However it has been reported that metformin can reduce the blood lipid parameters in non-diabetic patients with coronary heart disease (Carlsen et al. it could potentially prevent platelet aggregation and thrombosis. The reduction in the insulin levels in the diabetic state also causes an increase in the level of cytochrome P450 enzymes (Woodcroft and Novak. reduces hepatic gluconeogenesis (Bailey. The hypoglycemic activity of ABe was observed at the lowest dose (125 mg kg − 1) in normal as well as STZ-diabetic rats and was similar to the action of metformin. .. / Journal of Ethnopharmacology 72 (2000) 69–76 atherogenic. it may have hypoglycemic principle(s) that are similar in action to metformin. 75 The cytochromes are the primary system (phase I detoxification enzymes) responsible for chemical defense in animals (Elizabeth Gillam. The ABe might reduce the triglycerides by decreasing the serum non-esterified fatty acids (NEFA) in the STZ-diabetic rats similar to masoprocol (nordihydroguaiaretic acid). In contrast. The authors also acknowledge the excellent technical assistance of Annie Hsu. Further biochemical and pharmacological investigations are now in progress to isolate and identify the active compound(s) in ABe. Acknowledgements The authors wish to thank the National University of Singapore for the research grant (RP 960329) and a research scholarship awarded to P. In conclusion. This could be the result of improved glycemic control produced by ABe and metformin. 1999). which could act in a way different from that of metformin.P. Since ABe increased HDL-cholesterol. Singapore. These reactive compounds can cause peroxidation of lipids. each with a single or a diverse range of biological activities. The hyperglycemia in the STZ-treated rats leads to the formation of hydrogen peroxide..

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