- Email: [email protected]
Traditional Indian anti-diabetic plants attenuate progression of renal damage in streptozotocin induced diabetic mice
Journal of Ethnopharmacology 76 (2001) 233– 238 www.elsevier.com/locate/jethpharm
Traditional Indian anti-diabetic plants attenuate progression of renal damage in streptozotocin induced diabetic mice J.K. Grover a,*, V. Vats a, S.S. Rathi b, R. Dawar c a
Department of Pharmacology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110049, India b St. Boniface Institute of Cardio6ascular Sciences, Winnipeg, Canada c Department of Pathology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110049, India Received 20 June 2000; received in revised form 10 March 2001; accepted 11 April 2001
Abstract The purpose of the study was to investigate the effects of daily oral feeding Momordica charantia (MC) (200 mg/kg), Eugenia jambolana (EJ) (200 mg/kg), Mucuna pruriens (MP) (200 mg/kg) and Tinospora cordifolia (TC) extracts for 40 days on blood glucose concentrations and kidney functions in streptozotocin (STZ)-diabetic rats. Plasma glucose levels, body weight, urine volume and urinary albumin levels were monitored on every 10th day over a 40-day period while plasma creatinine levels were assessed at the beginning and end of experiment. Renal hypertrophy was assessed as the ratio between the kidney weight and total body weight. Plasma glucose concentrations in STZ-diabetic mice were reduced by the administration of extracts of MC, EJ, TC and MP by 24.4, 20.84, 7.45 and 9.07%, respectively (PB 0.005 for MC, EJ, MP and P B 0.05 for TC). Urine volume was significantly higher (PB0.005) in diabetic controls and MC, EJ, MP and TC treatment prevented polyuria (P B 0.001, 0.0001, 0.01 and 0.001, respectively). After 10 days of STZ administration urinary albumin levels (UAE) were over 6 fold higher in diabetic controls as compared to normal controls. Treatment with MC, EJ, MP and TC significantly prevented the rise in UAE levels from day 0 to 40 in comparison to diabetic controls (PB 0.0001, 0.0001, 0.05, 0.05, respectively). Renal hypertrophy was significantly higher in diabetic controls as compared to non-diabetic controls. MC and EJ partially but significantly (PB0.05) prevented renal hypertrophy as compared to diabetic controls. TC and MP failed to modify renal hypertrophy. Results indicate that these plant drugs should be studied further. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Momordica charantia; Eugenia jambolana; Tinospora cordifolia; Mucuna pruriens; Experimental diabetes; Diabetic nephropathy; Streptozotocin
1. Introduction For various reasons in the recent years, the popularity of complimentary medicine has increased. Dietary measures and traditional plant therapies as prescribed by Ayurvedic and other indigenous systems of medicine have been used commonly in India. Surveys conducted in Australia and US indicate that almost 48.5 and 34% of respondents had used at least one form of unconventional therapy including herbal medicine (Eisenberg et al., 1993; Maclennan et al., 1996). Indian figures are not * Corresponding author. Tel.: + 91-11-6594897; fax: +91-116862663. E-mail address: [email protected] (J.K. Grover).
available. The World Health Organization (1980) has also recommended the evaluation of the effectiveness of plants in conditions where we lack safe modern drugs (Upadhayay and Pandey, 1984). The primary objective of this study was to assess the efficacy of selected traditional anti-diabetic plants in diabetic nephropathy. Momordica charantia (MC), commonly known as bitter gourd belongs to Cucurbitaceae family. It is a widely used vegetable in India and its fruit, leaves and roots are recommended for treating diabetes mellitus (Warier, 1995). The hypoglycemic activity of extract of MC fruit has been previously confirmed in experimental animals and reviewed elsewhere (Gupta and Seth, 1962; Leatherdale et al., 1981; Raman and Lau, 1996). Seeds of MC have been
0378-8741/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 1 ) 0 0 2 4 6 - X
234
J.K. Gro6er et al. / Journal of Ethnopharmacology 76 (2001) 233–238
shown to exert hypoglycemic activity comparable to glibenclamide (Kedar and Chakrabarti, 1982). Eugenia jambolana (EJ) belongs to the Myrtacae family and is commonly called Black Berry. Jamun seeds have been used by the Indian natives as an anti-diabetic remedy (Chopra et al., 1958). Various medicinal properties of EJ including its astringent, stomachic, astringent, diuretic and anti-diabetic properties have been described in traditional medicine (Nadkarni, 1992). Mucuna pruriens (MP), commonly known as Cowitch in English belongs to the Papillonaceae family and has been reported to be useful in diabetes (Dhawan et al., 1980). Hypoglycemic effects of seeds of MP have been demonstrated in normal rats (Pant and Joshi, 1970) but not in alloxan treated rats (Joshi and Pant, 1970). Tinospora cordifolia (TC) belongs to Menispermaceae and has been indicated in Ayurvedic treatment as a tonic, vitalizer and anti-diabetic (Nadkarni, 1954; Chopra et al., 1958). Scientific reports describing anti-diabetic (Gupta et al., 1967), immunomodulatory (Atal et al., 1986), anti-hepatotoxic (Peer and Sharma, 1989), anti-pyretic (Vedavathy and Rao, 1991) and anti-stress activity (Sarma et al., 1996) are available.
2. Materials and methods
2.1. Preparation of extracts 2.1.1. Aqueous extracts Fruit of MC and kernels of EJ were purchased from the local market in June 1997 and were authenticated by Head, Department of Botany, Miranda House, University of Delhi (India). The green outer skin (i.e. cuticle) of the MC fruit was peeled off and the rest was macerated in an electric mixer (Electro Com, New Delhi). The kernels of EJ were ground in an electric grinder and macerated as described above. This pulp (1 kg each) was then soaked separately in equal amount of water (1 l) and stirred intermittently and then left overnight. This pulp was then filtered through a coarse sieve and the filtrate was dried at reduced temperature. Respective yields MC and EJ after partial drying was 80.4 g/kg of fruit and 73 g/kg of kernel. To increase the shelf life and uniformity, the extracts were completely lyophilised by continuous freeze drying operation of 54 h (Christ freeze dryer, alpha 1-4, Germany), yielding 64 and 51 g/100 g; of MC and EJ extract. 2.1.2. Extracts of Tinospora cordifolia and Mucuna pruriens Alcoholic and aqueous extracts of TC and MP were received as gift from Brawn Pharmaceuticals Ltd., Faridabad (India). All the extracts were suspended in 1% carboxymethylcellulose (Central Drug House, New Delhi) and given orally.
2.2. Experimental design Albino mice (30–50 g) of both sexes were obtained from the experimental animal facility of the All India Institute of Medical Sciences. Before the start and during the experiment, mice were fed standard chow diet. The animals were randomised in the following groups: Mice in group I received saline plus CMC daily and served as normal control. Mice in group II–VI received a single intraperitoneal injection of 150 mg/kg STZ. Group II received saline daily and served as a diabetic control. Group III received 200 mg of lyophilised powder of MC, group IV received 200 mg of lyophilised powder of EJ, and group V received 400 mg of aqueous extract of TC and group VI received 200 mg/day of alcoholic extract of MP. All the extracts were dissolved in CMC and given PO everyday for 40 days by force-feeding using a 5-ml syringe. After randomisation into various groups, the rats were acclimatised for a period of 2–3 days before initiation of experiment. Animals described as fasting had been deprived of food for at least 16 h but had been allowed free access to drinking water.
2.3. Preparation of diabetic nephropathic animals The method of Yotsumoto et al., 1997 was used for experimental induction of diabetic nephropathy. After acclimatisation, overnight fasted animals were injected a bolus of STZ (150 mg/kg of dissolved in 3 mM citrate buffer pH 4.5) intraperitoneally. Ten days after the single dose of STZ injection, only those mice exhibiting plasma glucose levels \ 300 mg/dl were included in the study.
2.4. Sample collection Fasting blood sample was collected in fresh vials containing sodium fluoride and sodium oxalate as anticoagulant/anti-glycolytic agents, retro-orbitally every 10th day till the end of experiment (i.e. 50th day) from the inner canthus of the eye under light ether anaesthesia using capillary tubes (Micro Hematocrit Capillaries, Mucaps). Plasma was separated in a T8 electric centrifuger (Remi Udyog, New Delhi) at 2000 rpm for 2 min.
2.5. Biochemical analysis 2.5.1. Plasma glucose Glucose levels were estimated by commercially available glucose kits based on glucose oxidase method (Trinder, 1969) (Autopak®, Bayer Diagnostics, Baroda).
J.K. Gro6er et al. / Journal of Ethnopharmacology 76 (2001) 233–238 Table 1 The effect of 40 days treatment with selected doses of four different plant extracts on glucose levels (mg/%) and serum creatinine (mmol/l) in STZ (150 per mg/kg) diabetic mice Serum glucose 10th day
Serum creatinine 50th day
10th day
NC 94.98 9 4.67 92.03 9 4.32 DC 441.16 9 19.90b 439.5 9 14.25b MC 449.33 9 22.33 339.66 914.22b (24.4) EJ 456.59 22.90 361.83 9 31.192b (20.84) TC 464.66 9 15.57 430 914.97b (7.45) MP 462.83 9 14.48 420.83913.36a (9.07)
50th day
41.5 93.08 46.6 9 3.05 46.89 4.49 50.09 1.67 45.39 1.63 47.5 9 3.08 46.39 4.63 42.3 9 3.92
235
2.5.4. Renal hypertrophy On the 40th day, animals were sacrificed and the kidney weight was determined gravimetrically and the degree of renal hypertrophy was expressed as the ratio of the weight of the two kidneys to total body weight. 2.6. Statistical analysis All results are expressed as the mean9 SD. The results were analysed for statistical significance by one way ANOVA test using computerised software, Microcal Origin version 2.9, Northampton, USA.
46.0 9 6.32 47.1 97.54 42.8 9 3.90 51.0 95.36
Values are given as mean 9SD for groups of six animals each. Diabetic control was compared with the corresponding value of the non-diabetic controls. Experimental groups were compared with their corresponding values on the 0th day. NC: non-diabetic control; DC: diabetic control; MC: Momordica charantia (lyophilised powder 200 mg/kg); EJ: Eugenia jambolana (lyophilised powder 200 mg/kg); MP: Mucuna pruriens (alcoholic extract 200 mg/kg) and TC: Tinospora cordifolia (aqueous extract 400 mg/day). a Values are statistically significant at PB0.05. b Values are statistically significant at PB0.005.
2.5.2. Urinary albumin and creatinine le6els Albumin and creatinine were measured by commercially available kits (based on quantitative colorimetric assay) (Spectrum Medical India Pvt. Ltd, New Delhi). 2.5.3. Body weight and urine 6olume Weight of individual animals was measured gravimetrically on every 10th day. For urine collection, animals were placed individually in metabolic cages for 24-h urine collection and urine volume was gravimetrically measured.
3. Results
3.1. Plasma glucose The effect of STZ and the plant extracts on plasma glucose levels is shown in Table 1. The plasma glucose levels were markedly raised (up to 4.5 times) in the diabetic controls as compared with non-diabetic controls on the 10th and 40th day of the experiment (PB0.005). Treatment with MC, EJ, TC and MP for 40 days significantly reduced the plasma glucose levels (PB 0.005 for MC, EJ, MP and PB 0.05 for TC) with percentage reduction of 24.4, 20.84, 7.45 and 9.07, respectively.
3.2. Body weight The effect of STZ and different plant extracts on the body weight of mice is summarised in Table 2. The basal values in the controls and treated groups were not significantly different from each other. The percentage increase in the body weight in the non-diabetic controls was significantly higher as compared to diabetic controls (27.519 2.55 versus 14.969 11.76; PB0.05). Treatment with plant extracts did not affect the body weight through out the experimental period.
Table 2 The effect of 50 days treatment with selected doses of four different plant extracts on body weight (g) in STZ (150 per mg/kg) diabetic mice
NC DC MC EJ MP TC
0th day
10th day
20th day
30th day
40th day
% Rise
26.66 91.96 289 2.44 26.66 93.77 28 92.44 26.66 92.58 25 93.16
28.16 9 2.22 29.83 9 2.56 28.83 9 2.04 29.83 9 2.56 27.5 9 2.42 26.16 9 3.25
30.16 92.40 30.33 9 3.26 29.66 92.58 30.33 93.26 29.5 9 2.34 2793.93
32.33 92.87 32.33 94.36 30.5 9 3.01 32.33 9 4.36 31.33 9 2.50 29.83 93.43
34.0 9 2.52 32.5 9 3.50 32.16 9 4.02 33.5 9 3.50 32.83 9 2.48 30.2 9 3.30
27.51 9 2.55 14.96 9 11.76a 20.07 9 13.87 21.4 912.5 19.61 9 5.98 23.55 9 9.06
Values are given as mean 9 SD for groups of six animals each. Diabetic control was compared with the normal. Abbreviations as for Table 1. a Values are statistically significant at PB0.05.
J.K. Gro6er et al. / Journal of Ethnopharmacology 76 (2001) 233–238
236
Table 3 The effect of 40 days treatment with selected doses of four different plant extracts on mean urine volume (ml) in six STZ (150 per mg/kg) diabetic mice
NC DC MC EJ MP TC
0th day
10th day
20th day
30th day
40th day
Mean rise from days 0–40
1.56 90.28 21.95 90.28b 17.43 93.07 20.83 91.83 21.16 91.47 22.13 90.75
1.56 9 0.13 23.969 4.10b 19.41 9 1.09 20.16 9 1.72 22.019 2.72 24.12 9 1.35
1.36 9 0.20 31.019 1.93b 19.58 9 1.78 19.83 9 0.75 22.1 93.72b 28.5 9 2.12
1.47 90.07 31.96 90.39b 20.96 9 4.08 21.16 9 2.04 25.16 92.99b 33.4 9 1.1
1.53 9 0.19 32.00 90.30b 20.45 9 3.08 20.33 9 1.5 26.05 93.59b 30.23 9 0.65
Negligible 10.5 9 0.432 1.93 9 2.24b 1.5 9 1.56c 4.88 9 3.32a 8.08 9 0.94b
Values are given as mean 9 SD for groups of six animals each. Diabetic control was compared with the normal on 0, 10th, 20th, 30th, 40th day and the mean rise in urine volume from day 0–40. Abbreviations as for Table 1. a Values are statistically significant at PB0.01. b Values are statistically significant at PB0.001. c Values are statistically significant at PB0.0001. Table 4 The effect of 40 days treatment with selected doses of four different plant extracts on urinary albumin (mg/24 h) in STZ (150 per mg/kg) diabetic mice
NC DC MC EJ MP TC
0th day
10th day
20th day
30th day
40th day
Mean rise in UAE levels from days 0–40
170.83 922.01 1055.33 9 18.6 1043.5 929.69 1097.16 90.87 1064.66 9 20.39 1079.16 952.65
205.339 11.48 1552.66948.68 1396.33964.83 1465.59 59.19 1527 9 26.55 1449.839 72.41
369.66 9 17.85 1504.8 9 150.78 1159.3 9 151.85 1151.5 9 74.54 1510 9 59.294 1410.5 9 47.60
474.83 921.67 1488.6 9102.27 1078.83 9 45.66 1083.83 954.13 1420.33 9 82.62 1406.33 915.76
582.83 9 23.7 1411.33 9 72.69 1072.83 9 73.49 1041.5 9 91.492 1310 990.54a 1377.5 9 18.9
352.33 9 153.15 387 9 70.9 46.66 9 45.79b 59.33 9 57.34b 246.16 9102.64a 298.33 9 63.74a
Values are given as mean 9SD for groups of six animals each. Diabetic control was compared with the normal on 0, 10th, 20th, 30th and 40th day. Abbreviations as for Table 1. a Values are statistically significant at PB0.05. b Values are statistically significant at PB0.0001.
3.3. Urine 6olume The effect of STZ on urine volume of mice is shown in Table 3. Ten days after STZ injection, urine volume was significantly higher (P B 0.005) in diabetic controls as compared to non-diabetic controls (21.959 0.28 versus 1.5690.28 ml/day, respectively). The polyuria in diabetic animals continued till the end of experiment (31.98 90.29 versus 1.5391.96 ml/day, respectively, PB 0.005). The mean rise in urine volume from day 0 to 40 in diabetic control was 109 0.43 ml and it was significantly more than compared to MC, EJ, MP and TC treated groups (1.939 2.24, 1.59 1.56, 4.889 3.32 and 8.089 0.94, respectively; respective P values were B 0.001, B 0.0001, B0.001 and B0.001).
3.4. Urinary albumin le6els Urinary albumin levels in the STZ treated and nondiabetic control is shown in Table 4. The mean rise in diabetic controls over the period of 40 days was : 3 folds over the baseline values (170.839 22.01 versus 582.839 23.7 on 0th and 40th day, respectively; PB 0.005). The basal levels in the diabetic controls were
over 6 fold high as compared to normal controls on 0 day (1055.339 18.6 versus 170.839 22.01, respectively). The mean rise in UAE levels from day 0 to 40 in diabetic control was 387970.9 mg/24 h and it was significantly more in comparison to MC, EJ, MP and TC treated groups (46.669 45.79, 59.339 57.34, 246.169 102.64 and 298.339 63.74; respective P values were B0.0001, B 0.0001, B 0.05, B 0.05).
3.5. Renal hypertrophy Table 5 summarises the effect of STZ and treatment with different plant extracts on renal hypertrophy. The net two weight of kidneys was significantly higher in diabetic controls as compared to controls (0.50119 0.0286 versus 0.5969 0.0508; PB 0.005). Treatment with MC and EJ partially but significantly (PB0.05) prevented renal hypertrophy as compared to diabetic controls along with reduction in kidney weight. TC and MP failed to modify renal hypertrophy. Renal hypertrophy measured as the ratio of kidney weight to total body weight in control, diabetic controls, MC, EJ, MP and TC treated groups was 0. 0147, 0.0183, 0.0161, 0.0153, 0.0167 and 0.0191, respectively.
J.K. Gro6er et al. / Journal of Ethnopharmacology 76 (2001) 233–238
3.6. Serum creatinine The effect of STZ and treatment with plant extracts on the creatinine levels is shown in Table 1. No significant difference between normal controls and diabetic control animals or the treated groups was noted throughout the duration of the experiment.
4. Discussion STZ induced diabetes in rodents results in development of nephropathy similar to early stage clinical diabetic nephropathy (Rasch and Mogensen, 1980; Sassy-Prigent et al., 1995). Yotsumoto et al. (1997) showed that the use of mice was beneficial as they developed nephropathy earlier than rats (Mauer et al., 1978) and required a lower less amount of the diabetogenic agent. Therefore, mice were used in the present study. The effects of STZ induced diabetes in mice in the present case were similar to those reported earlier (Yotsumoto et al., 1997). Therefore, the present study reinforces the findings of Yotsumoto et al. (1997) and recommends the use of mice to produce experimental diabetic nephropathy. Furthermore, STZ has no longterm effects on the kidney other than those mediated by diabetes mellitus (Rasch, 1979). Urinary albumin levels are a selective marker of glomerular injury and elevated rates of albumin excretion are a harbinger of progressive nephropathy (Viberti et al., 1982), Rodents such as rats and mice exhibit albuminuria normally (Mauer et al., 1978; Yotsumoto et al., 1997) and during a period of 40 days, normal controls showed a rise in UAE by over 3 folds. UAE levels in the diabetic group, however, were many folds higher than normal controls. Treatment with MC, EJ, MP and TC prevented the rise in UAE levels with varying degree as seen in diabetic controls. The effect was more with MC and EJ and less with MP and TC. The observations of the present study indicate that the extracts of MC and EJ protect glomerulous from the injurious effects of diabetes. The histopathological examination of the kidneys from different groups sup-
237
ports this point as MC and EJ treated kidneys were within normal limits. Diabetes induction by STZ has been known to produce increase in kidney weight relative to body weight (Seyer-Hansen, 1976; Kang et al., 1982). In the present study, the average net weight of both kidneys of diabetic controls was significantly higher than non-diabetic controls. This is consistent with the previous finding of Yotsumoto et al., 1997. Treatment with MC and EJ prevented renal enlargement while MP and TC failed to bring any effect on this parameter. Previous studies on this respect have correlated the degree of renal enlargement with the degree of glycemic control (Rasch, 1979) while others have contradicted the glycemic theory (Wiseman et al., 1985), However, in the present study although MC and EJ exerted anti-hyperglycemic effect and prevented renal enlargement, the serum glucose levels remained well above 300 mg/dl. Thus, it is likely that mechanism or mechanisms independent of anti-hyperglycemic properties played a role in preventing renal enlargement. Rasch (1980), reported that the rise in body weight was far less in the poorly controlled diabetic rats as compared to well-controlled diabetic rats. Similar observations were made in this study. However, body weight was not significantly affected by any of the plant extracts, possibly due to poor glycemic control offered by the treatment. No statistical significant intra-group variation was seen in serum creatinine levels. Polyuria is a characteristic symptom of diabetes. Twenty-four hour urine volume was significantly increased in diabetic controls versus normal controls. MC, EJ, MP and TC treatment prevented polyuria in comparison with diabetic controls. Administration of plant extracts (notably MC and EJ) to diabetic mice prevented the increase in urine volume, UAE excretion, renal hypertrophy as well as caused a marginal reduction in plasma glucose levels. The results of the present study cannot be explained entirely on the basis of glycemic theory as treatment with MC and EJ was unable to achieve an euglycemic state and yet halted the progression of diabetic nephropathy (prevented renal enlargement in animals
Table 5 The effect of 50 days treatment with selected doses of four different plant extracts on renal hypertrophy i.e. kidney weight (g) in STZ (150 per mg/kg) diabetic mice
Two kidney weight (g) Ratio (kidney weight/total body weight)
NDC
DC
MC
EJ
MP
TC
0.50119 0.0286 0.0147
0.5969 0.0508b 0.0183
0.5193 90.0264a 0.0161
0.515 90.0219a 0.0153
0.549 90.0379 0.0167
0.577 9 0.307 0.0191
Values are given as mean 9SD for groups of six animals each. Diabetic control was compared with the normal. Experimental groups were compared with diabetic control. Abbreviations as for Table 1. a Values are statistically significant at PB0.05. b Values are statistically significant at PB0.005.
238
J.K. Gro6er et al. / Journal of Ethnopharmacology 76 (2001) 233–238
and attenuated the rate of increase in microalbuminuria along with reduction in renal hypertrophy). Regardless of the mechanism of action, the present study indicates that these plant drugs (MC and EJ) should be studied further.
References Atal, C.K., Sharma, M.L., Kaul, A., Khajuria, A., 1986. Immunomodulating agents of plant origin. 1. Preliminary screening. J. Ethnopharmacol. 18, 133 –141. Chopra, R.N., Chopra, I.C., Handa, K.L., Kapur, L.D., 1958. Indigenous Drugs of India, second ed. Dhar and Sons, Calcutta, pp. 686– 689. Dhawan, B.N., Dubey, M.P., Mehrotra, B.N., Rastogi, R.P., Tandon, J.S., 1980. Screening of Indian plants for biological activity. Part IX. Indian J. Exp. Biol. 18, 594 – 606. Eisenberg, D.M., Kessler, R.C., Foster, C., Norlock, F.E., Calkins, D.R., Delbanco, T.L., 1993. Unconventional al medicine in the United States. Prevalence, costs, and patterns of use. New Engl. J. Med. 328, 246 – 252. Gupta, S.S., Seth, C.B., 1962. Effect of Momordica charantia Linn. (Karela) on glucose tolerance in albino rats. J. Indian Med. Assoc. 39, 581 – 584. Gupta, S.S., Verma, S.C.L., Garg, V.P., Rai, M., 1967. Anti-diabetic effect of Tinospora cordifolia part I. Effect on fasting blood sugar level, glucose tolerance and adrenaline induced hyperglycemia. Indian J. Med. Res. 55, 733 –745. Joshi, L.D., Pant, M.C., 1970. Hypoglycemic effect of Glycine soja, Dolichos biflorus and Mucuna pruriens seed in albino rats. Annual Conference of Indian Pharmacological Society. Lucknow, India. Indian J. Pharmacol. 2, 29. Kang, S.S., Fears, R., Noirot, S., Mbanya, J.N., Yudkin, J., 1982. Changes in metabolism of rat kidney and liver caused by experimental diabetes by dietary sucrose. J. Diabetol. 22, 285 – 288. Kedar, P., Chakrabarti, C.H., 1982. Effects of bittergourd (Momordica charantia) seed and glibenclamide in streptozotocin induced diabetes mellitus. Indian J. Exp. Biol. 20, 232 –235. Leatherdale, B.A., Panesar, R.K., Singh, G., Atkins, T.W., Bailery, C.J., Bignell, A.H.C., 1981. Improvement in glucose tolerance due to Momordica charantia (Karela). BMJ 282, 1823 – 1824. Maclennan, A.H., Wilson, D.H., Taylor, A.W., 1996. Prevalence and cost of alternative medicine in Australia. Lancet 347, 569 – 573. Mauer, S.M., Brown, D.M., Matas, A.J., Steeffes, M.W., 1978. Effects of pancreatic islets transplantation on the increased urinary albumin excretion rates in intact and unephrectomized rats with diabetes mellitus. Diabetes 27, 959 – 964. Nadkarni, A.K., 1954. Indian Materia Medica. Popular Prakashan, Bombay, p. 1221. Nadkarni, A.K., 1992. Indian Materia Medica, vol. I. Popular Prakashan, Bombay, p. 157.
.
Pant, M.C., Joshi, L.D., 1970. Identification of pharmacologically active substances in the seeds of Mucuna pruriens (Abs). Proceedings of Annual Conference of Indian Pharmacological Society. Lucknow, India. Indian J. Pharmacol. 2, 24. Peer, F., Sharma, M.C., 1989. Therapeutic evaluation of Tinospora cordifolia on blood glucose and total lipid levels of normal and alloxan diabetic rats. Plant. Med. 58, 131 – 136. Raman, A., Lau, C., 1996. Antidiabetic properties and phytochemistry of Momordica charantia L. (Cucurbitaceae). Phytomedicine 2, 349 – 362. Rasch, R., 1979. Prevention of diabetic glomerulopathy in streptozotocin diabetic rats by insulin treatment. Kidney size and glomerular volume. Diabetologia 16, 124 – 128. Rasch, R., 1980. Prevention of diabetic glomerulopathy in streptozotocin diabetic rats by insulin treatment. Diabetologia 18, 413 – 416. Rasch, R., Mogensen, C.I., 1980. Urinary excretion of albumin and total protein in normal and streptozotocin diabetic rats. Acta Endocrinol. 95, 376 – 381. Sassy-Prigent, C., Heudes, D., Jouquey, S., Auberval, D., Belair, M.F., Michel, O., Hamon, G., Bariety, J., Bruneval, P., 1995. Morphometric detection of incipient glomerular lesions in diabetic nephropathy in rats. Protective effects of ACE inhibition. Lab. Invest. 73, 64 – 71. Sarma, D.N.K., Khosa, R.L., Chansouria, J.P.N., Sahai, M., 1996. Anti-stress activity of Tinospora cordifolia Miers and Centella asiatica (L.) extracts. Phytotherapy Res. 10, 181 – 183. Seyer-Hansen, K., 1976. Renal nephropathy in streptozotocin diabetic rats. Clin. Sci. Mol. Med. 51, 551 – 555. Trinder, P., 1969. Determination of glucose using glucose oxidase with an alternative oxygen acceptor. Annu. Clin. Biochem. 6, 24 – 27. Upadhayay, V.P., Pandey, K., 1984. Ayurvedic approach to diabetes mellitus and its management by indigenous resources. In: Bajaj, J.S. (Ed.), Diabetes Mellitus in Developing Countries. Interprint, New Delhi, pp. 375 – 377. Vedavathy, S., Rao, K.N., 1991. Antipyretic activity of six indigenous medicinal plants of Tirumala Hills, Andhra Pradesh, India. J. Ethnopharmacol. 33, 193 – 196. Viberti, G.C., Hill, R.D., Jarrett, R.J., Argyropoulous, A., Mahmud, U., Keen, H., 1982. Microalbuminuria as a predictor of clinical nephropathy in insulin-dependent diabetes mellitus. Lancet 1, 1430 – 1432. Warier, P.K., 1995. Momordica charantia Linn. In: Warrier, P.K., Nambiar, V.P.K., Ramankutty, C. (Eds.), Indian Medicinal Plants. Orient Longman, Chennai, pp. 48 – 51. Wiseman, M.J., Saunders, A.J., Keen, H., Viberti, G., 1985. Effect of blood glucose control on increased glomerular filtration rate and kidney size in insulin dependent diabetes. New Engl. J. Med. 312, 617 – 621. World Health Organization, 1980. Second Report of the WHO Expert Committee on Diabetes Mellitus. Technical report series. 646,66. Yotsumoto, T., Naitoh, T., Shikada, K., Tanaka, S., 1997. Effects of specific antagonists of angiotensin II receptors and captopril on diabetic nephropathy in mice. Jpn. J. Pharmacol. 75, 59 –64.
Traditional Indian anti-diabetic plants attenuate progression of renal damage in streptozotocin induced diabetic mice J.K. Grover a,*, V. Vats a, S.S. Rathi b, R. Dawar c a
Department of Pharmacology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110049, India b St. Boniface Institute of Cardio6ascular Sciences, Winnipeg, Canada c Department of Pathology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110049, India Received 20 June 2000; received in revised form 10 March 2001; accepted 11 April 2001
Abstract The purpose of the study was to investigate the effects of daily oral feeding Momordica charantia (MC) (200 mg/kg), Eugenia jambolana (EJ) (200 mg/kg), Mucuna pruriens (MP) (200 mg/kg) and Tinospora cordifolia (TC) extracts for 40 days on blood glucose concentrations and kidney functions in streptozotocin (STZ)-diabetic rats. Plasma glucose levels, body weight, urine volume and urinary albumin levels were monitored on every 10th day over a 40-day period while plasma creatinine levels were assessed at the beginning and end of experiment. Renal hypertrophy was assessed as the ratio between the kidney weight and total body weight. Plasma glucose concentrations in STZ-diabetic mice were reduced by the administration of extracts of MC, EJ, TC and MP by 24.4, 20.84, 7.45 and 9.07%, respectively (PB 0.005 for MC, EJ, MP and P B 0.05 for TC). Urine volume was significantly higher (PB0.005) in diabetic controls and MC, EJ, MP and TC treatment prevented polyuria (P B 0.001, 0.0001, 0.01 and 0.001, respectively). After 10 days of STZ administration urinary albumin levels (UAE) were over 6 fold higher in diabetic controls as compared to normal controls. Treatment with MC, EJ, MP and TC significantly prevented the rise in UAE levels from day 0 to 40 in comparison to diabetic controls (PB 0.0001, 0.0001, 0.05, 0.05, respectively). Renal hypertrophy was significantly higher in diabetic controls as compared to non-diabetic controls. MC and EJ partially but significantly (PB0.05) prevented renal hypertrophy as compared to diabetic controls. TC and MP failed to modify renal hypertrophy. Results indicate that these plant drugs should be studied further. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Momordica charantia; Eugenia jambolana; Tinospora cordifolia; Mucuna pruriens; Experimental diabetes; Diabetic nephropathy; Streptozotocin
1. Introduction For various reasons in the recent years, the popularity of complimentary medicine has increased. Dietary measures and traditional plant therapies as prescribed by Ayurvedic and other indigenous systems of medicine have been used commonly in India. Surveys conducted in Australia and US indicate that almost 48.5 and 34% of respondents had used at least one form of unconventional therapy including herbal medicine (Eisenberg et al., 1993; Maclennan et al., 1996). Indian figures are not * Corresponding author. Tel.: + 91-11-6594897; fax: +91-116862663. E-mail address: [email protected] (J.K. Grover).
available. The World Health Organization (1980) has also recommended the evaluation of the effectiveness of plants in conditions where we lack safe modern drugs (Upadhayay and Pandey, 1984). The primary objective of this study was to assess the efficacy of selected traditional anti-diabetic plants in diabetic nephropathy. Momordica charantia (MC), commonly known as bitter gourd belongs to Cucurbitaceae family. It is a widely used vegetable in India and its fruit, leaves and roots are recommended for treating diabetes mellitus (Warier, 1995). The hypoglycemic activity of extract of MC fruit has been previously confirmed in experimental animals and reviewed elsewhere (Gupta and Seth, 1962; Leatherdale et al., 1981; Raman and Lau, 1996). Seeds of MC have been
0378-8741/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 1 ) 0 0 2 4 6 - X
234
J.K. Gro6er et al. / Journal of Ethnopharmacology 76 (2001) 233–238
shown to exert hypoglycemic activity comparable to glibenclamide (Kedar and Chakrabarti, 1982). Eugenia jambolana (EJ) belongs to the Myrtacae family and is commonly called Black Berry. Jamun seeds have been used by the Indian natives as an anti-diabetic remedy (Chopra et al., 1958). Various medicinal properties of EJ including its astringent, stomachic, astringent, diuretic and anti-diabetic properties have been described in traditional medicine (Nadkarni, 1992). Mucuna pruriens (MP), commonly known as Cowitch in English belongs to the Papillonaceae family and has been reported to be useful in diabetes (Dhawan et al., 1980). Hypoglycemic effects of seeds of MP have been demonstrated in normal rats (Pant and Joshi, 1970) but not in alloxan treated rats (Joshi and Pant, 1970). Tinospora cordifolia (TC) belongs to Menispermaceae and has been indicated in Ayurvedic treatment as a tonic, vitalizer and anti-diabetic (Nadkarni, 1954; Chopra et al., 1958). Scientific reports describing anti-diabetic (Gupta et al., 1967), immunomodulatory (Atal et al., 1986), anti-hepatotoxic (Peer and Sharma, 1989), anti-pyretic (Vedavathy and Rao, 1991) and anti-stress activity (Sarma et al., 1996) are available.
2. Materials and methods
2.1. Preparation of extracts 2.1.1. Aqueous extracts Fruit of MC and kernels of EJ were purchased from the local market in June 1997 and were authenticated by Head, Department of Botany, Miranda House, University of Delhi (India). The green outer skin (i.e. cuticle) of the MC fruit was peeled off and the rest was macerated in an electric mixer (Electro Com, New Delhi). The kernels of EJ were ground in an electric grinder and macerated as described above. This pulp (1 kg each) was then soaked separately in equal amount of water (1 l) and stirred intermittently and then left overnight. This pulp was then filtered through a coarse sieve and the filtrate was dried at reduced temperature. Respective yields MC and EJ after partial drying was 80.4 g/kg of fruit and 73 g/kg of kernel. To increase the shelf life and uniformity, the extracts were completely lyophilised by continuous freeze drying operation of 54 h (Christ freeze dryer, alpha 1-4, Germany), yielding 64 and 51 g/100 g; of MC and EJ extract. 2.1.2. Extracts of Tinospora cordifolia and Mucuna pruriens Alcoholic and aqueous extracts of TC and MP were received as gift from Brawn Pharmaceuticals Ltd., Faridabad (India). All the extracts were suspended in 1% carboxymethylcellulose (Central Drug House, New Delhi) and given orally.
2.2. Experimental design Albino mice (30–50 g) of both sexes were obtained from the experimental animal facility of the All India Institute of Medical Sciences. Before the start and during the experiment, mice were fed standard chow diet. The animals were randomised in the following groups: Mice in group I received saline plus CMC daily and served as normal control. Mice in group II–VI received a single intraperitoneal injection of 150 mg/kg STZ. Group II received saline daily and served as a diabetic control. Group III received 200 mg of lyophilised powder of MC, group IV received 200 mg of lyophilised powder of EJ, and group V received 400 mg of aqueous extract of TC and group VI received 200 mg/day of alcoholic extract of MP. All the extracts were dissolved in CMC and given PO everyday for 40 days by force-feeding using a 5-ml syringe. After randomisation into various groups, the rats were acclimatised for a period of 2–3 days before initiation of experiment. Animals described as fasting had been deprived of food for at least 16 h but had been allowed free access to drinking water.
2.3. Preparation of diabetic nephropathic animals The method of Yotsumoto et al., 1997 was used for experimental induction of diabetic nephropathy. After acclimatisation, overnight fasted animals were injected a bolus of STZ (150 mg/kg of dissolved in 3 mM citrate buffer pH 4.5) intraperitoneally. Ten days after the single dose of STZ injection, only those mice exhibiting plasma glucose levels \ 300 mg/dl were included in the study.
2.4. Sample collection Fasting blood sample was collected in fresh vials containing sodium fluoride and sodium oxalate as anticoagulant/anti-glycolytic agents, retro-orbitally every 10th day till the end of experiment (i.e. 50th day) from the inner canthus of the eye under light ether anaesthesia using capillary tubes (Micro Hematocrit Capillaries, Mucaps). Plasma was separated in a T8 electric centrifuger (Remi Udyog, New Delhi) at 2000 rpm for 2 min.
2.5. Biochemical analysis 2.5.1. Plasma glucose Glucose levels were estimated by commercially available glucose kits based on glucose oxidase method (Trinder, 1969) (Autopak®, Bayer Diagnostics, Baroda).
J.K. Gro6er et al. / Journal of Ethnopharmacology 76 (2001) 233–238 Table 1 The effect of 40 days treatment with selected doses of four different plant extracts on glucose levels (mg/%) and serum creatinine (mmol/l) in STZ (150 per mg/kg) diabetic mice Serum glucose 10th day
Serum creatinine 50th day
10th day
NC 94.98 9 4.67 92.03 9 4.32 DC 441.16 9 19.90b 439.5 9 14.25b MC 449.33 9 22.33 339.66 914.22b (24.4) EJ 456.59 22.90 361.83 9 31.192b (20.84) TC 464.66 9 15.57 430 914.97b (7.45) MP 462.83 9 14.48 420.83913.36a (9.07)
50th day
41.5 93.08 46.6 9 3.05 46.89 4.49 50.09 1.67 45.39 1.63 47.5 9 3.08 46.39 4.63 42.3 9 3.92
235
2.5.4. Renal hypertrophy On the 40th day, animals were sacrificed and the kidney weight was determined gravimetrically and the degree of renal hypertrophy was expressed as the ratio of the weight of the two kidneys to total body weight. 2.6. Statistical analysis All results are expressed as the mean9 SD. The results were analysed for statistical significance by one way ANOVA test using computerised software, Microcal Origin version 2.9, Northampton, USA.
46.0 9 6.32 47.1 97.54 42.8 9 3.90 51.0 95.36
Values are given as mean 9SD for groups of six animals each. Diabetic control was compared with the corresponding value of the non-diabetic controls. Experimental groups were compared with their corresponding values on the 0th day. NC: non-diabetic control; DC: diabetic control; MC: Momordica charantia (lyophilised powder 200 mg/kg); EJ: Eugenia jambolana (lyophilised powder 200 mg/kg); MP: Mucuna pruriens (alcoholic extract 200 mg/kg) and TC: Tinospora cordifolia (aqueous extract 400 mg/day). a Values are statistically significant at PB0.05. b Values are statistically significant at PB0.005.
2.5.2. Urinary albumin and creatinine le6els Albumin and creatinine were measured by commercially available kits (based on quantitative colorimetric assay) (Spectrum Medical India Pvt. Ltd, New Delhi). 2.5.3. Body weight and urine 6olume Weight of individual animals was measured gravimetrically on every 10th day. For urine collection, animals were placed individually in metabolic cages for 24-h urine collection and urine volume was gravimetrically measured.
3. Results
3.1. Plasma glucose The effect of STZ and the plant extracts on plasma glucose levels is shown in Table 1. The plasma glucose levels were markedly raised (up to 4.5 times) in the diabetic controls as compared with non-diabetic controls on the 10th and 40th day of the experiment (PB0.005). Treatment with MC, EJ, TC and MP for 40 days significantly reduced the plasma glucose levels (PB 0.005 for MC, EJ, MP and PB 0.05 for TC) with percentage reduction of 24.4, 20.84, 7.45 and 9.07, respectively.
3.2. Body weight The effect of STZ and different plant extracts on the body weight of mice is summarised in Table 2. The basal values in the controls and treated groups were not significantly different from each other. The percentage increase in the body weight in the non-diabetic controls was significantly higher as compared to diabetic controls (27.519 2.55 versus 14.969 11.76; PB0.05). Treatment with plant extracts did not affect the body weight through out the experimental period.
Table 2 The effect of 50 days treatment with selected doses of four different plant extracts on body weight (g) in STZ (150 per mg/kg) diabetic mice
NC DC MC EJ MP TC
0th day
10th day
20th day
30th day
40th day
% Rise
26.66 91.96 289 2.44 26.66 93.77 28 92.44 26.66 92.58 25 93.16
28.16 9 2.22 29.83 9 2.56 28.83 9 2.04 29.83 9 2.56 27.5 9 2.42 26.16 9 3.25
30.16 92.40 30.33 9 3.26 29.66 92.58 30.33 93.26 29.5 9 2.34 2793.93
32.33 92.87 32.33 94.36 30.5 9 3.01 32.33 9 4.36 31.33 9 2.50 29.83 93.43
34.0 9 2.52 32.5 9 3.50 32.16 9 4.02 33.5 9 3.50 32.83 9 2.48 30.2 9 3.30
27.51 9 2.55 14.96 9 11.76a 20.07 9 13.87 21.4 912.5 19.61 9 5.98 23.55 9 9.06
Values are given as mean 9 SD for groups of six animals each. Diabetic control was compared with the normal. Abbreviations as for Table 1. a Values are statistically significant at PB0.05.
J.K. Gro6er et al. / Journal of Ethnopharmacology 76 (2001) 233–238
236
Table 3 The effect of 40 days treatment with selected doses of four different plant extracts on mean urine volume (ml) in six STZ (150 per mg/kg) diabetic mice
NC DC MC EJ MP TC
0th day
10th day
20th day
30th day
40th day
Mean rise from days 0–40
1.56 90.28 21.95 90.28b 17.43 93.07 20.83 91.83 21.16 91.47 22.13 90.75
1.56 9 0.13 23.969 4.10b 19.41 9 1.09 20.16 9 1.72 22.019 2.72 24.12 9 1.35
1.36 9 0.20 31.019 1.93b 19.58 9 1.78 19.83 9 0.75 22.1 93.72b 28.5 9 2.12
1.47 90.07 31.96 90.39b 20.96 9 4.08 21.16 9 2.04 25.16 92.99b 33.4 9 1.1
1.53 9 0.19 32.00 90.30b 20.45 9 3.08 20.33 9 1.5 26.05 93.59b 30.23 9 0.65
Negligible 10.5 9 0.432 1.93 9 2.24b 1.5 9 1.56c 4.88 9 3.32a 8.08 9 0.94b
Values are given as mean 9 SD for groups of six animals each. Diabetic control was compared with the normal on 0, 10th, 20th, 30th, 40th day and the mean rise in urine volume from day 0–40. Abbreviations as for Table 1. a Values are statistically significant at PB0.01. b Values are statistically significant at PB0.001. c Values are statistically significant at PB0.0001. Table 4 The effect of 40 days treatment with selected doses of four different plant extracts on urinary albumin (mg/24 h) in STZ (150 per mg/kg) diabetic mice
NC DC MC EJ MP TC
0th day
10th day
20th day
30th day
40th day
Mean rise in UAE levels from days 0–40
170.83 922.01 1055.33 9 18.6 1043.5 929.69 1097.16 90.87 1064.66 9 20.39 1079.16 952.65
205.339 11.48 1552.66948.68 1396.33964.83 1465.59 59.19 1527 9 26.55 1449.839 72.41
369.66 9 17.85 1504.8 9 150.78 1159.3 9 151.85 1151.5 9 74.54 1510 9 59.294 1410.5 9 47.60
474.83 921.67 1488.6 9102.27 1078.83 9 45.66 1083.83 954.13 1420.33 9 82.62 1406.33 915.76
582.83 9 23.7 1411.33 9 72.69 1072.83 9 73.49 1041.5 9 91.492 1310 990.54a 1377.5 9 18.9
352.33 9 153.15 387 9 70.9 46.66 9 45.79b 59.33 9 57.34b 246.16 9102.64a 298.33 9 63.74a
Values are given as mean 9SD for groups of six animals each. Diabetic control was compared with the normal on 0, 10th, 20th, 30th and 40th day. Abbreviations as for Table 1. a Values are statistically significant at PB0.05. b Values are statistically significant at PB0.0001.
3.3. Urine 6olume The effect of STZ on urine volume of mice is shown in Table 3. Ten days after STZ injection, urine volume was significantly higher (P B 0.005) in diabetic controls as compared to non-diabetic controls (21.959 0.28 versus 1.5690.28 ml/day, respectively). The polyuria in diabetic animals continued till the end of experiment (31.98 90.29 versus 1.5391.96 ml/day, respectively, PB 0.005). The mean rise in urine volume from day 0 to 40 in diabetic control was 109 0.43 ml and it was significantly more than compared to MC, EJ, MP and TC treated groups (1.939 2.24, 1.59 1.56, 4.889 3.32 and 8.089 0.94, respectively; respective P values were B 0.001, B 0.0001, B0.001 and B0.001).
3.4. Urinary albumin le6els Urinary albumin levels in the STZ treated and nondiabetic control is shown in Table 4. The mean rise in diabetic controls over the period of 40 days was : 3 folds over the baseline values (170.839 22.01 versus 582.839 23.7 on 0th and 40th day, respectively; PB 0.005). The basal levels in the diabetic controls were
over 6 fold high as compared to normal controls on 0 day (1055.339 18.6 versus 170.839 22.01, respectively). The mean rise in UAE levels from day 0 to 40 in diabetic control was 387970.9 mg/24 h and it was significantly more in comparison to MC, EJ, MP and TC treated groups (46.669 45.79, 59.339 57.34, 246.169 102.64 and 298.339 63.74; respective P values were B0.0001, B 0.0001, B 0.05, B 0.05).
3.5. Renal hypertrophy Table 5 summarises the effect of STZ and treatment with different plant extracts on renal hypertrophy. The net two weight of kidneys was significantly higher in diabetic controls as compared to controls (0.50119 0.0286 versus 0.5969 0.0508; PB 0.005). Treatment with MC and EJ partially but significantly (PB0.05) prevented renal hypertrophy as compared to diabetic controls along with reduction in kidney weight. TC and MP failed to modify renal hypertrophy. Renal hypertrophy measured as the ratio of kidney weight to total body weight in control, diabetic controls, MC, EJ, MP and TC treated groups was 0. 0147, 0.0183, 0.0161, 0.0153, 0.0167 and 0.0191, respectively.
J.K. Gro6er et al. / Journal of Ethnopharmacology 76 (2001) 233–238
3.6. Serum creatinine The effect of STZ and treatment with plant extracts on the creatinine levels is shown in Table 1. No significant difference between normal controls and diabetic control animals or the treated groups was noted throughout the duration of the experiment.
4. Discussion STZ induced diabetes in rodents results in development of nephropathy similar to early stage clinical diabetic nephropathy (Rasch and Mogensen, 1980; Sassy-Prigent et al., 1995). Yotsumoto et al. (1997) showed that the use of mice was beneficial as they developed nephropathy earlier than rats (Mauer et al., 1978) and required a lower less amount of the diabetogenic agent. Therefore, mice were used in the present study. The effects of STZ induced diabetes in mice in the present case were similar to those reported earlier (Yotsumoto et al., 1997). Therefore, the present study reinforces the findings of Yotsumoto et al. (1997) and recommends the use of mice to produce experimental diabetic nephropathy. Furthermore, STZ has no longterm effects on the kidney other than those mediated by diabetes mellitus (Rasch, 1979). Urinary albumin levels are a selective marker of glomerular injury and elevated rates of albumin excretion are a harbinger of progressive nephropathy (Viberti et al., 1982), Rodents such as rats and mice exhibit albuminuria normally (Mauer et al., 1978; Yotsumoto et al., 1997) and during a period of 40 days, normal controls showed a rise in UAE by over 3 folds. UAE levels in the diabetic group, however, were many folds higher than normal controls. Treatment with MC, EJ, MP and TC prevented the rise in UAE levels with varying degree as seen in diabetic controls. The effect was more with MC and EJ and less with MP and TC. The observations of the present study indicate that the extracts of MC and EJ protect glomerulous from the injurious effects of diabetes. The histopathological examination of the kidneys from different groups sup-
237
ports this point as MC and EJ treated kidneys were within normal limits. Diabetes induction by STZ has been known to produce increase in kidney weight relative to body weight (Seyer-Hansen, 1976; Kang et al., 1982). In the present study, the average net weight of both kidneys of diabetic controls was significantly higher than non-diabetic controls. This is consistent with the previous finding of Yotsumoto et al., 1997. Treatment with MC and EJ prevented renal enlargement while MP and TC failed to bring any effect on this parameter. Previous studies on this respect have correlated the degree of renal enlargement with the degree of glycemic control (Rasch, 1979) while others have contradicted the glycemic theory (Wiseman et al., 1985), However, in the present study although MC and EJ exerted anti-hyperglycemic effect and prevented renal enlargement, the serum glucose levels remained well above 300 mg/dl. Thus, it is likely that mechanism or mechanisms independent of anti-hyperglycemic properties played a role in preventing renal enlargement. Rasch (1980), reported that the rise in body weight was far less in the poorly controlled diabetic rats as compared to well-controlled diabetic rats. Similar observations were made in this study. However, body weight was not significantly affected by any of the plant extracts, possibly due to poor glycemic control offered by the treatment. No statistical significant intra-group variation was seen in serum creatinine levels. Polyuria is a characteristic symptom of diabetes. Twenty-four hour urine volume was significantly increased in diabetic controls versus normal controls. MC, EJ, MP and TC treatment prevented polyuria in comparison with diabetic controls. Administration of plant extracts (notably MC and EJ) to diabetic mice prevented the increase in urine volume, UAE excretion, renal hypertrophy as well as caused a marginal reduction in plasma glucose levels. The results of the present study cannot be explained entirely on the basis of glycemic theory as treatment with MC and EJ was unable to achieve an euglycemic state and yet halted the progression of diabetic nephropathy (prevented renal enlargement in animals
Table 5 The effect of 50 days treatment with selected doses of four different plant extracts on renal hypertrophy i.e. kidney weight (g) in STZ (150 per mg/kg) diabetic mice
Two kidney weight (g) Ratio (kidney weight/total body weight)
NDC
DC
MC
EJ
MP
TC
0.50119 0.0286 0.0147
0.5969 0.0508b 0.0183
0.5193 90.0264a 0.0161
0.515 90.0219a 0.0153
0.549 90.0379 0.0167
0.577 9 0.307 0.0191
Values are given as mean 9SD for groups of six animals each. Diabetic control was compared with the normal. Experimental groups were compared with diabetic control. Abbreviations as for Table 1. a Values are statistically significant at PB0.05. b Values are statistically significant at PB0.005.
238
J.K. Gro6er et al. / Journal of Ethnopharmacology 76 (2001) 233–238
and attenuated the rate of increase in microalbuminuria along with reduction in renal hypertrophy). Regardless of the mechanism of action, the present study indicates that these plant drugs (MC and EJ) should be studied further.
References Atal, C.K., Sharma, M.L., Kaul, A., Khajuria, A., 1986. Immunomodulating agents of plant origin. 1. Preliminary screening. J. Ethnopharmacol. 18, 133 –141. Chopra, R.N., Chopra, I.C., Handa, K.L., Kapur, L.D., 1958. Indigenous Drugs of India, second ed. Dhar and Sons, Calcutta, pp. 686– 689. Dhawan, B.N., Dubey, M.P., Mehrotra, B.N., Rastogi, R.P., Tandon, J.S., 1980. Screening of Indian plants for biological activity. Part IX. Indian J. Exp. Biol. 18, 594 – 606. Eisenberg, D.M., Kessler, R.C., Foster, C., Norlock, F.E., Calkins, D.R., Delbanco, T.L., 1993. Unconventional al medicine in the United States. Prevalence, costs, and patterns of use. New Engl. J. Med. 328, 246 – 252. Gupta, S.S., Seth, C.B., 1962. Effect of Momordica charantia Linn. (Karela) on glucose tolerance in albino rats. J. Indian Med. Assoc. 39, 581 – 584. Gupta, S.S., Verma, S.C.L., Garg, V.P., Rai, M., 1967. Anti-diabetic effect of Tinospora cordifolia part I. Effect on fasting blood sugar level, glucose tolerance and adrenaline induced hyperglycemia. Indian J. Med. Res. 55, 733 –745. Joshi, L.D., Pant, M.C., 1970. Hypoglycemic effect of Glycine soja, Dolichos biflorus and Mucuna pruriens seed in albino rats. Annual Conference of Indian Pharmacological Society. Lucknow, India. Indian J. Pharmacol. 2, 29. Kang, S.S., Fears, R., Noirot, S., Mbanya, J.N., Yudkin, J., 1982. Changes in metabolism of rat kidney and liver caused by experimental diabetes by dietary sucrose. J. Diabetol. 22, 285 – 288. Kedar, P., Chakrabarti, C.H., 1982. Effects of bittergourd (Momordica charantia) seed and glibenclamide in streptozotocin induced diabetes mellitus. Indian J. Exp. Biol. 20, 232 –235. Leatherdale, B.A., Panesar, R.K., Singh, G., Atkins, T.W., Bailery, C.J., Bignell, A.H.C., 1981. Improvement in glucose tolerance due to Momordica charantia (Karela). BMJ 282, 1823 – 1824. Maclennan, A.H., Wilson, D.H., Taylor, A.W., 1996. Prevalence and cost of alternative medicine in Australia. Lancet 347, 569 – 573. Mauer, S.M., Brown, D.M., Matas, A.J., Steeffes, M.W., 1978. Effects of pancreatic islets transplantation on the increased urinary albumin excretion rates in intact and unephrectomized rats with diabetes mellitus. Diabetes 27, 959 – 964. Nadkarni, A.K., 1954. Indian Materia Medica. Popular Prakashan, Bombay, p. 1221. Nadkarni, A.K., 1992. Indian Materia Medica, vol. I. Popular Prakashan, Bombay, p. 157.
.
Pant, M.C., Joshi, L.D., 1970. Identification of pharmacologically active substances in the seeds of Mucuna pruriens (Abs). Proceedings of Annual Conference of Indian Pharmacological Society. Lucknow, India. Indian J. Pharmacol. 2, 24. Peer, F., Sharma, M.C., 1989. Therapeutic evaluation of Tinospora cordifolia on blood glucose and total lipid levels of normal and alloxan diabetic rats. Plant. Med. 58, 131 – 136. Raman, A., Lau, C., 1996. Antidiabetic properties and phytochemistry of Momordica charantia L. (Cucurbitaceae). Phytomedicine 2, 349 – 362. Rasch, R., 1979. Prevention of diabetic glomerulopathy in streptozotocin diabetic rats by insulin treatment. Kidney size and glomerular volume. Diabetologia 16, 124 – 128. Rasch, R., 1980. Prevention of diabetic glomerulopathy in streptozotocin diabetic rats by insulin treatment. Diabetologia 18, 413 – 416. Rasch, R., Mogensen, C.I., 1980. Urinary excretion of albumin and total protein in normal and streptozotocin diabetic rats. Acta Endocrinol. 95, 376 – 381. Sassy-Prigent, C., Heudes, D., Jouquey, S., Auberval, D., Belair, M.F., Michel, O., Hamon, G., Bariety, J., Bruneval, P., 1995. Morphometric detection of incipient glomerular lesions in diabetic nephropathy in rats. Protective effects of ACE inhibition. Lab. Invest. 73, 64 – 71. Sarma, D.N.K., Khosa, R.L., Chansouria, J.P.N., Sahai, M., 1996. Anti-stress activity of Tinospora cordifolia Miers and Centella asiatica (L.) extracts. Phytotherapy Res. 10, 181 – 183. Seyer-Hansen, K., 1976. Renal nephropathy in streptozotocin diabetic rats. Clin. Sci. Mol. Med. 51, 551 – 555. Trinder, P., 1969. Determination of glucose using glucose oxidase with an alternative oxygen acceptor. Annu. Clin. Biochem. 6, 24 – 27. Upadhayay, V.P., Pandey, K., 1984. Ayurvedic approach to diabetes mellitus and its management by indigenous resources. In: Bajaj, J.S. (Ed.), Diabetes Mellitus in Developing Countries. Interprint, New Delhi, pp. 375 – 377. Vedavathy, S., Rao, K.N., 1991. Antipyretic activity of six indigenous medicinal plants of Tirumala Hills, Andhra Pradesh, India. J. Ethnopharmacol. 33, 193 – 196. Viberti, G.C., Hill, R.D., Jarrett, R.J., Argyropoulous, A., Mahmud, U., Keen, H., 1982. Microalbuminuria as a predictor of clinical nephropathy in insulin-dependent diabetes mellitus. Lancet 1, 1430 – 1432. Warier, P.K., 1995. Momordica charantia Linn. In: Warrier, P.K., Nambiar, V.P.K., Ramankutty, C. (Eds.), Indian Medicinal Plants. Orient Longman, Chennai, pp. 48 – 51. Wiseman, M.J., Saunders, A.J., Keen, H., Viberti, G., 1985. Effect of blood glucose control on increased glomerular filtration rate and kidney size in insulin dependent diabetes. New Engl. J. Med. 312, 617 – 621. World Health Organization, 1980. Second Report of the WHO Expert Committee on Diabetes Mellitus. Technical report series. 646,66. Yotsumoto, T., Naitoh, T., Shikada, K., Tanaka, S., 1997. Effects of specific antagonists of angiotensin II receptors and captopril on diabetic nephropathy in mice. Jpn. J. Pharmacol. 75, 59 –64.