Iranian Journal of Pharmaceutical Research (2008), 7 (1): 61-68 Received: June 2006 Accepted: September 2006

Copyright 2008 by School of Pharmacy Shaheed Beheshti University of Medical Sciences and Health Services

Original Article

Antidiabetic and In Vivo Antioxidant Activity of Ethanolic Extract of Bacopa monnieri Linn. Aerial Parts: A Possible Mechanism of Action
Tirtha Ghosha*, Tapan Kumar Maityb, Pinaki Sengupta b, Deepak Kumar Dash b and Anindya Bose a
Institute of Pharmacy and Technology, Salipur, Cuttack district, Orissa, India. bDepartment of Pharmaceutical Technology, Jadavpur University, Kolkata, India.
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Abstract

Keywords: Bacopa monnieria; Diabetes; Antioxidant activity; Antihyperglycemic activity.

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Diabetes mellitus is a metabolic disorder affecting carbohydrate, fat and protein metabolism that affects nearly 10% of the population every year. The treatment of diabetes mellitus has been confined to use of oral hypoglycemic agents and insulin, the former being reported to possess serious side effects. This leads to increasing demand for herbal products with antidiabetic factor with little side effects. This article describes the antihyperglycaemic activity, in vivo antioxidant potential, effect on glycosylation of hemoglobin and in-vitro peripheral utilisation of glucose of the ethanolic extract of the aerial parts of Bacopa monnieri. The extract produced significant decrease in the blood glucose level when compared with the controls in alloxan induced hyperglycemic rats both in the single dose as well as multiple dose experiment at the tested dose level and is comparable with the standard drug glibenclamide. It was observed that the ethanolic extract reversed the weight loss of the diabetic rats and they returned to near normal. The extract prevented significant elevation of glycosylated hemoglobin in vitro, with IC50 value being 11.25 g/ml that is comparable with the reference drug -tocopherol. Administration of the exract and glibenclamide significantly decreased the levels of TBARS, increased the content of GSH and increased the activity of SOD and CAT in liver of diabetic rats. The extract increased peripheral glucose utilisation in the diaphragm of diabetic rats in vitro, which is comparable with the action of insulin. Thus, the extract might have insulin like activity and the antihyperglycemic effect of the extract might be due to an increase in peripheral glucose consumption as well as protection against oxidative damage in alloxanised diabetes.

Introduction Diabetes mellitus is a metabolic disorder affecting carbohydrate, fat and protein metabolism. A worldwide survey reported that
* Corresponding author: E-mail: [email protected]

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diabetes mellitus is affecting nearly 10% of the population every year (1). The treatment of diabetes mellitus in clinical practice has been confined to use of oral hypoglycemic agents and insulin, the former being reported to be endowed with characteristic profiles of serious side effects (2). This leads to increasing demand for herbal products with antidiabetic factor with little side

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Ghosh T, Maity TK, Sengupta P, Dash DK and Bose A / IJPR (2008), 7 (1): 61-68

Experimental

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Plant Material The plant was identified by the taxonomists of the Botanical Survey of India, Govt. of India, Shibpur, Howrah, India. After authentication, fresh aerial parts of the young and matured plants were collected in bulk from the rural belt of Salipur, Orissa, India during early summer, washed, shade dried and then milled in to coarse powder by a mechanical grinder.

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Preparation of extract The powdered plant material (400 g) was defatted with petroleum ether (60-80 C) and then extracted with 1.5 litre of ethanol (95%) in a soxhlet apparatus. The solvent was removed under reduced pressure, which obtained a greenish-black sticky residue (yield: 11.6% w/w with respect to dried plant material). The dried extract was stored in a desiccator till further study. Animals Used Wistar albino rats of either sex, weighing 180-250 g supplied by M/s Ghose enterprises, Kolkata, India were used. The selected animals
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effects. A large number of plants have been recognized to be effective in the treatment of diabetes mellitus (3). Bacopa monnieri L. (Fam. Scrophulariaceae) is a creeping, glabrous, succulent herb, rooting at nodes, distributed throughout India in all plain districts, ascending to an altitude of 1,320 m. The plant is reported to show sedative, antiepileptic, vasoconstrictor and anti-inflammatory activity (4). It has been reported that the plant contains tetracyclic triterpenoid saponins, bacosides A and B, hersaponin, alkaloids viz. herpestine and brahmine and flavonoids (4, 5). In folklore practice, some of the tribes of Orissa, India use the decoction of the aerial parts of the plant for the treatment of diabetes mellitus. In the present paper we report the antihyperglycaemic activity, in vivo antioxidant potential, effect on glycosylation of hemoglobin and in vitro peripheral utilisation of glucose of the ethanolic extract of the aerial parts of B. monnieri using standard laboratory procedures.

were housed in acrylic cages in standard environmental conditions (25-30 C). They were allowed free access to standard dry pellet diet (Hindustan Lever, Kolkata, India) and water ad libitum. All experiments were carried out as per the guidelines of the Institutional Animal Ethical Committee. Drugs and chemicals used Bovine serum albumin (Sigma chemical St. Louis, MO, USA), thiobarbituric acid, nitro blue tetrazolium chloride (NBT), hemoglobin (Loba Chemie, Mumbai, India), trichloro acetic acid (Merck Ltd, Mumbai, India), 5,5-dithio bis-2nitrobenzoic acid (DTNB) were used. All the solvents were of analytical grade and purchased from local market. Screening for antidiabetic activity The method of Joy and Kuttan was followed (6). The acclimatized animals were kept fasting for 24 h with water ad libitum and injected intraperitoneally a dose of 150 mg/kg of alloxan monohydrate in normal saline. After one hour, the animals were provided feed ad libitum. The blood glucose level was checked before and 72 h after alloxan injection. The animals were considered diabetic when the blood glucose level was raised beyond 300 mg/dl of blood. This condition was observed at the end of 72 h after alloxan injection. Effect on oral glucose tolerance in rats After overnight fasting, a 0-min blood sample was taken from the tip of the tail of each rat of different groups under mild ether anesthesia. Without delay a glucose solution (2 g/kg) was administered by a gavage. Four more samples were taken at 30, 60, 90 and 120 min after glucose administration (7). All blood samples were taken for the estimation of the blood glucose. Estimation of blood glucose was carried out with the haemoglucostrips supplied by M/s Lifescan, Inc. USA with the help of a Johnson & Johnson ONE TOUCH blood glucometer. Single dose study The animals were segregated into five groups of six rats in each. Group I and II rats were randomly selected from normal rats that

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Antidiabetic and Antioxidant Activity of Bacopa monnieri Linn.

received only distilled water and the extract (300 mg/kg, p.o.) respectively. Group III to Group V animals were selected from the alloxanised rats. Group III animals served as diabetic control. Group IV animals received glibenclamide (600 g/kg) and group V was treated with the extract (300 mg/kg) in a similar manner. Blood samples were collected from the tip of tail of each rat under mild ether anesthesia at 0 h, 1 h, 2 h and 4 h after the administration of test samples and tested for glucose concentration as above.
Multidose study For multidose study, administration of test samples was continued for 10 days, once daily through oral route. Blood samples were collected from the tip of tail and the estimation of blood glucose was carried out as above on the 1, 3, 7 and 10 day of the drug administration. Body weights of all the animals were recorded just prior to and on the 10th day of the study to determine the change in the body weight, if any. Determination of in vivo antioxidant activity On the 10th day following study, the animals were deprived of food overnight and sacrificed by cervical dislocation. The livers were dissected out, washed in ice-cold saline, patted dry and weighed. A 10 % w/v of homogenate was prepared in 0.15 M TrisHCl buffer and processed for the estimation of lipid peroxidation by the method of Fraga et al. (8). A part of homogenate after precipitating proteins with trichloro acetic acid (TCA) was used for estimation of glutathione by the method of Ellman et al. (9). The rest of the homogenate was centrifuged at 15000 rpm for 15 min at 4 C. The supernatant thus obtained was used for the estimation of SOD by the method described by Kakkar et al. (10) and CAT activity was measured by the method of Maehly et al. (11).

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Determination of peripheral consumption of glucose in vitro The method of Chattopadhyay et al. was followed (13). Peripheral glucose consumption was studied in rat diaphragm preparation from animals fasted for 36 h prior to the experiment. The animals were sacrificed by cervical dislocation and the diaphragms were quickly taken out; followed by dividing each diaphragm into four pieces. The pieces of diaphragms were incubated in the nutrient solution with constant oxygenation and shaking (90 cycles / min) at 37 C for 90 min in accordance with the procedure. The nutrient solution with the diaphragms was aerated for 10 min and used immediately. Glucose was added to a final concentration of 500 mg%. Each piece of diaphragm is incubated in 2.5 ml of glucose nutrient mixture. The results were expressed as glucose consumption per 10 mg of dry diaphragm (by subtracting glucose concentration after incubation from glucose concentration before incubation). The dry weight was determined after oven drying the diaphragm at 105 C for 2 h. Statistical analysis Statistical significance was determined by one way analysis of variance (ANOVA) followed by Dunnets t-test. P<0.05 indicates significant difference between group means. Results and Discussion Table 1 shows the blood glucose level of

Determination of in vitro glycosylation of hemoglobin The degree of glycosylation of hemoglobin in vitro was measured colorimetrically as suggested by Fluckiger et al. (12). Hemoglobin,
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5gm/ml in 0.01 M phosphate buffer (pH 7.4) was incubated for 72h in presence of 2 g/100ml concentration of glucose in order to find out the best condition for hemoglobin glycosylation. The assay was performed by adding 1 ml of glucose solution, 1ml of hemoglobin solution and 1ml of gentamicin (20 mg/100ml) in 0.01 M phosphate buffer (pH 7.4). The mixture was incubated in dark at room temperature. The degree of glycosylation of hemoglobin in presence of different concentration of the extract and their absence was measured colorimetrically at 440 nm. -tocopherol was used as standard.

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Ghosh T, Maity TK, Sengupta P, Dash DK and Bose A / IJPR (2008), 7 (1): 61-68

Table 1. Effect of ethanolic extract of Bacopa monnieri aerial parts (300 mg/kg, p.o.) on oral glucose tolerance test (OGTT) in normal and alloxan induced diabetic rats. Groups I II III IV V Treatment Normal Normal + Extract Diabetic control (Alloxan only) Diabetic+ Extract Diabetic+ Glibenclamide Blood sugar level (mg/dl) Fasting 75.000.77 74.830.79 NS 250.333.10* 77.501.50* 76.50 2.02* 30 min 149.83 2.32 146.50 1.78NS 322.33 4.16* 141.83 2.91* 151.56 3.45* 60 min 176.832.09 181.17 2.39NS 374.175.16* 176.17 3.52* 185.332.53* 90 min 125.172.83 124.17 3.78NS 319.33 3.29* 127.50 2.80* 126.83 2.46* 120 min 80.16 1.83 85.67 3.18NS 317.83 2.67* 87.50 1.43* 92.50 1.50*

Values are mean SEM for n=6; *P < 0.05 = significant; NS = Not significant; Group II and III are compared with group I while Group IV and V are compared with group III.

Table 2. Effect of single dose treatment of ethanolic extract of Bacopa monnieri aerial parts (300 mg/kg, p.o.) on blood glucose level in normal and alloxan induced diabetic rats. Group I II III IV V Treatment Normal Normal + Extract Diabetic control (Alloxan only) Diabetic+ Glibenclamide Diabetic+ Extract Blood glucose level (mg/dl) Basal value 76.330.71 76.17 0.87NS 349.672.95* 343.17 5.12
NS

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normal and experimental animals after oral administration of glucose (2 g/kg). Extract as well as standard drug treated animals showed more significant decrease in peak blood glucose level after 1 h. After 2 h, the extract treated animals tended to bring the values near normal. The results of Table 2 reveals that the extract produced significant decrease in the blood glucose level when compared with the controls in alloxan induced hyperglycaemic rats in the single dose experiment at the tested dose level and is comparable with the standard drug glibenclamide. In the multi dose study (Table 3), the test extract constantly maintained significant reduction of the glucose level in diabetic rats throughout the experimental period suggesting the antihyperglycaemic property of the extract. Diabetes mellitus causes failure to use of glucose for energy that leads to increased utilization and decreased storage of protein responsible for reduction of body weight essentially by depletion

of the body proteins (14). In the present study, it was observed that the ethanolic extract reversed the weight loss of the diabetic rats and they returned to near normal. During diabetes the excess glucose present in the blood reacts with hemoglobin to form glycosylated hemoglobin. The rate of glycosylation is directly proportional to concentration of blood glucose and with improvement of glycemic control glycosylated hemoglobin also decreases (15). Hence the estimation of glycosylation of hemoglobin is a well established parameter useful in the management and prognosis of the disease (16). Our study gave a clear view that the ethanolic extract prevented significant elevation of glycosylated hemoglobin in vitro, with IC50 value being 11.25 g/ml that is comparable with the reference drug - tocopherol (Table 4). Further, since the non-enzymatic glycosylation of hemoglobin is an oxidative reaction (17), an antioxidant is expected to inhibit the

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1h 76.170.65 75.83 0.70NS 350.172.71* 319.505.35* 289.334.89*

2h 75.830.95 75.17 0.75NS 349.832.62* 298.833.91* 266.833.37*

4h 76.17 0.79 74.330.71NS 350.17 2.79* 284.83 3.65* 246.83 3.20*

338.50 3.19NS

Values are mean SEM for n=6; *P < 0.05 = significant; NS = Not significant; Group II and III are compared with group I while Group IV and V are compared with group III.

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Antidiabetic and Antioxidant Activity of Bacopa monnieri Linn.

Table 3. Effect of multiple dose treatment of ethanolic extract of Bacopa monnieri aerial parts (300 mg/kg, p.o., once daily) on blood glucose level and change in body weight after 15 days in normal and alloxan induced diabetic rats.
Group I II III IV V Treatment Normal Normal + Extract Diabetic control (Alloxan only) Diabetic + Glibenclamide Diabetic + Extract Blood glucose level (mg/dl) Basal value 76.33 0.71 76.17 0.87NS 349.67 2.95* 343.17 5.12NS 338.50 3.19NS Day 1 76.17 0.48 75.00 0.73NS 356.83 2.83* 264.33 4.07* 219.67 3.58* Day 3 75.83 0.40 75.17 0.54NS 353.83 3.39* 235.83 3.57* 209.33 3.96* Day 7 76.17 0.65 74.83 0.60NS 354.33 3.90* 219.33 4.28* 200.33 2.68* Day 10 76.50 0.56 73.33 0.67* 354.17 3.83* 205.33 3.65* 186.50 1.80* Change in body weight (g) (+) 9.83 1.47 (+) 10.00 0.97NS (-) 8.83 0.87* (+) 8.83 0.98* (+) 9.16 1.08*

Values are mean SEM for n=6; *P < 0.05 = significant; NS = Not significant; Group II and III are compared with Group I while Group IV and V are compared with Group III.

Table 4. Effect of ethanolic extract of Bacopa monnieri on percent inhibition of hemoglobin glycosylation in vitro. Group I II III IV V Treatment Normal Normal + Extract Diabetic control (Alloxan only) Diabetic + Glibenclamide Diabetic + Extract Blood glucose level (mg/dl) Basal value 76.33 0.71 76.17 0.87NS 349.67 2.95* 343.17 5.12
NS

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reaction. The extract is thus expected to possess antioxidant activity. Lipid peroixidation is one of the characteristic features of chronic diabetes. Alloxan gives rise to dialuric acid, which undergoes oxidation and leads to generation of O2, H2O2 and OH (18). Dialuric acid has been observed to stimulate lipid peroxidation in vitro. In this context, a marked increase in the concentration of TBARS was observed in liver of diabetic rats. Increased lipid peroxide concentration in the liver of diabetic animals has already been reported (19). Administration of the exract and glibenclamide significantly decreased the levels of TBARS in diabetic rats (Table 5). Glutathione (GSH), a tripeptide present in all the cells is an important antioxidant (20). Decreased glutathione levels in diabetes have been considered to be an indicator of increased

oxidative stress (21). GSH also functions as free radical scavenger in the repair of radical caused biological damage (22). A decrease was observed in GSH in liver during diabetes. Administration of the extract and glibenclamide increased the content of GSH in liver of diabetic rats (Table 5). The cellular radical scavenging systems include the enzymes such as superoxide dismutase (SOD), which scavenges the superoxide ions by catalysing its dismutation and catalase (CAT), a haeme enzyme which removes hydrogen peroxide (23). Therefore, reduction in the activity of these enzymes (SOD, CAT) results in a number of deleterious effects due to the accumulation of superoxide anion radicals and hydrogen peroxide. Administration of ethanolic extract and glibenclamide increased the activity of SOD and catalase in diabetic rats

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76.17 0.65 75.83 0.70NS

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4h 76.17 0.79 74.33 0.71NS 350.17 2.79* 284.83 3.65* 246.83 3.20*

75.83 0.95 75.17 0.75NS 349.83 2.62* 298.83 3.91* 266.83 3.37*

350.17 2.71* 319.50 5.35* 289.33 4.89*

338.50 3.19NS

Values are Mean S.D. for n=3; r = regression co-efficient.

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Ghosh T, Maity TK, Sengupta P, Dash DK and Bose A / IJPR (2008), 7 (1): 61-68

Table 5. Changes in the levels of TBARS and Reduced glutathione in liver of normal and diabetic rats. Group I II III IV V Treatment Normal Normal + Extract Diabetic Diabetic + Extract Diabetic + Glibenclamide TBARSa 2.32 0.06 2.07 0.08NS 5.27 0.10* 3.53 0.11* 4.20 0.17* Reduced glutathioneb 58.07 1.27 54.49 1.39NS 27.30 1.57* 52.28 0.88* 48.47 1.07*

a = nmole of MDA/mg of protein; b = g/ mg of protein; values are Mean S.D. for n=6; Group II and III are compared with group I while Group IV and V are compared with group III. *P < 0.05 = significant; NS = not significant.

Table 6. Changes in the activity of Catalase and Superoxide dismutase in liver of normal and diabetic rats. Groups I II III IV V Treatment Normal Normal + Extract Diabetic Diabetic + Extract Diabetic + Glibenclamide Catalasec 68.71 4.72 66.52 4.85
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(Table 6). Alloxan has been found to induce free radical generation and cause tissue injury (24). Since the extract showed in vivo antioxidative activity in normal and diabetic rats, improvement of the liver tissues and the subsequent increase in the uptake and utilisation of blood glucose might be the mechanism of action of this extract as antidiabetic agent. Alloxan causes irreversible destruction of pancreas -cells (25). Thus, the antihyperglycemic activity might be due to extra pancreatic mechanism. Hence, the effect of ethanolic extract of B. monnieri aerial parts on peripheral consumption of glucose was investigated. The result suggests that the extract produces an antidiabetic action mediated by an increase in peripheral glucose consumption in the rat diaphragm of diabetic rats, especially at a concentration of 600 g/ml (Table 7). Insulin increased the peripheral glucose consumption in normal and diabetic rats. Thus, the extract might have an insulin like activity and the antihyperglycemic effect of the extract might be due to an increase in peripheral glucose

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consumption. The ethanolic extract of B. monnieri is reported to be rich in saponins (26). Saponins are reported to possess antidiabetic (27) and antioxidant activity (28). Presence of saponins in the ethanolic extract was confirmed through our preliminary phytochemical screening also. Thus, the saponins in the extract may be suspected to possess the activity that may be attributed to their protective action on lipid peroxidation and at the same time the enhancing effects on cellular antioxidant defense contributing to the protection against oxidative damage in alloxanised diabetes.

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40.78 2.4*

The authors are thankful to the management of Institute of Pharmacy and Technology, Salipur, India and the authorities of Jadavpur University, Kolkata, India for providing necessary facilities to carry out the research work. The authors are also thankful to the taxonomists of Botanical Survey of India, Shibpur, Howrah, India for proper identification of the plant.

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52.26 3.51* 48.65 4.85*

Acknowledgement

Superoxide dismutased 5.93 0.70 6.25 0.79NS 4.22 0.55* 5.74 0.57* 5.31 0.57*

c = mole of H2O2 consumed/ min / mg of protein; d = Units/ mg of protein; One unit of activity means enzyme reaction responsible for 50% inhibition of NBT per min. Values are Mean S.D. for n=6; Group II and III are compared with group I while Group IV and V are compared with group III. *P < 0.05 = significant; NS = not significant

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Antidiabetic and Antioxidant Activity of Bacopa monnieri Linn.

Table 7. Effect of ethanolic extract of Bacopa monnieri on in vitro peripheral glucose consumption in diaphragm of normal and diabetic rats. Glucose consumption (mg/10 mg of diaphragm dry weight) Control Normal rats Diabetic rats 0.47 0.03 0.49 0.03 Ethanolic extract (g/ml) 300 (g/ml) 0.59 0.05NS 0.64 0.04* 600 (g/ml) 0.68 0.05* 0.78 0.06* Insulin 5 U/ml 0.82 0.06* 0.90 0.06*

Values were expressed as Mean SEM for n=6. *P < 0.05 = Significant; NS = not significant when compared to control

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