Oxidative Stress Plays An Important Role

Published Date: 02 Nov 2017

9 present study, the antioxidant effect of oral administration of aqueous extract of Mucuna

1211 pruriens seed on tissue antioxidant enzymes and lipid peroxidation in liver and kidney of

14 streptozotocin-induced diabetic rats was evaluated. Administration of seed extract to

16 diabetic rats significantly (p<0.05) decreased the levels of serum glucose, cholesterol,

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19 triglycerides and increased HDL-C. The diabetic rats showed significant (p<0.05) lower

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21 activities of superoxide dismutase, catalase and reduced glutathione content in liver and

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24 kidney, which were restored by treatment with the aqueous extract of seed in a dose

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26 dependant manner. The increased levels of lipid peroxidation in liver and kidney tissues

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2928 in diabetic rats was significantly (p<0.05) decreased by M. pruriens seed extract in a dose

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31 dependant manner. The present study reveals the aqueous extract of M. pruriens seed was

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33 effective in the amelioration of diabetes, which may be attributed to its hypoglycemic

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36 property along with its antioxidant potential due to presence of L-dopa in the extract.

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41 Key words:

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44 Oxidative stress, Mucuna pruriens, Diabetes, Antioxidant

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7 Oxidative stress plays an important role in chronic complications of diabetes and it is

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109 postulated to be associated with increased lipid peroxidation.1-3 Enhanced oxidative stress

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12 and changes in antioxidant capacity, observed in both clinical and experimental diabetes

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15 mellitus, are thought to be the etiology of diabetic complications.

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Mechanisms by which

17 increased oxidative stress is involved in the diabetic complications are partly known,

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19 including activation of transcription factors, advanced glycated end products (AGEs) and

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22 protein kinase C. Elevated glucose level causes slow but significant non-enzymatic

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24 glycosylation of proteins in diabetes.5 The oxidatively modified proteins may be

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2726 recognized as ‘foreign’ by the immune system, triggering the antibody formation.6 There

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29 are several potential sources of increased free radical production in diabetes including

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3231 auto-oxidation of plasma glucose, activation of leucocytes and increased transition metal

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34 bioavailability.7 Several in vivo and in vitro studies have demonstrated that reactive

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3736 oxygen metabolites including free radicals like superoxide radical, hydroxyl radical and

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39 hydrogen peroxide are important mediators of tissue injury.8 The concentration of the

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41 reactive oxygen species are modulated by antioxidant enzymes such as glutathione

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44 peroxidase (GPx), superoxide dismutase (SOD), catalase (CAT) and non-enzymatic

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46 scavengers like reduced glutathione (GSH).9 Marked reductions in antioxidant enzyme

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4948 activities and tissue GSH concentrations have been reported in diabetes.10,11

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52 Many indigenous Indian medicinal plants have been found to be useful to successfully

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5554 manage diabetes.12-14 Despite the introduction of hypoglycemic agents from natural and

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57 synthetic sources, diabetes and its secondary complications continue to be a major

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59 problem in the world population. Mucuna pruriens Linn. DC. is an annual climbing

7 especially Africa, India and the West Indies. In India, the plant is known by different

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9 local names in different languages like, Alkushi in Eastern India (Bengal), Kavatch in

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1211 Gujarati, Kavacha, Kuhili, Kanchkuri in Marathi. In Hindi, Kiwach, Kaunch, Goncha,

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14 Atamagupta in Sanskrit, Poonaipidukkan in Tamil, Dulagondi in Telugu. In Ayurvedic

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16 system of medicine, M. pruriens was used for the management of male infertility,

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19 nervous disorders and also as an aphrodisiac.16 M. pruriens seed powder contains high

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21 amount of L-DOPA, neurotransmitter precursor used in the treatment of Parkinson’s

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24 disease.17 M. pruriens seed in addition to levodopa, contains tryptamine, 5-

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26 hydroxytryptamine (5-HT), mucunine, mucunadine, prurienine and prurieninine.18 It is

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2928 also rich in fatty content.19 M. pruriens have been reported to decrease blood glucose and

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31 cholesterol levels in rats.20 Powdered seeds of M. pruriens have been reported to decrease

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33 21,22

34 blood glucose level in normal and alloxan-diabetic rabbits.

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Grover et al. reported that

36 blood glucose lowering activity of alcoholic extract upon daily oral feeding for 40 days in

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38 STZ-diabetic mice.23 In vitro Lipid peroxidation and antimicrobial activity of methanolic

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41 extract of M. pruriens has been reported by Rajeshwar et al.24 Alcoholic extract of M.

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43 pruriens inhibit iron-induced lipid peroxidation.25 M. pruriens contain L-dopa which is

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46 reported to decrease free radical generation in various in vitro models for radical

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48 scavenging activity.26 In light of above, the present study was aimed to investigate to

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5150 study the effect of M. pruriens seed extract on tissue lipid peroxides and antioxidant

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53 parameters in STZ-diabetic rats.

10 The seeds of Mucuna pruriens (L.) DC. (MP) were purchased from the United Chemicals

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13 and Allied Products, Kolkata, India. It was authenticated by Dr. B. C. Patel, Botany

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15 Department, Modasa, India. A voucher specimen was retained in our laboratory for

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1817 further reference. For the extract, the seeds were powdered in a mechanical grinder. One

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20 kg seed powder of M. pruriens was initially defatted with 750 ml of petroleum ether (60-

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22 80°C) then aqueous extract was prepared by cold maceration method in that extract was

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25 shaken intermediately and CHCl3 was added to prevent bacterial growth. After seven

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27 days, the extract was filtered using Whatman filter paper (No. 1) and then concentrated in

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30 vacuum and dried. The yield was 10.05 % w/w with respect to dry powder.

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32 2.2. Standardization of extract

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35 Standardization of extract was carried out by high performance thin layer

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3837 chromatography. The samples were spotted in the form of bands with a Camag microlitre

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40 syringe on a precoated silica gel plates 60 F254 (10 cm ×10 cm with 0.2 mm thickness, E.

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42 Merck, Darmstadt, Germany) using a Camag Linomat V Automatic Sample Spotter

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45 (Muttenz, Switzerland). The plates were prewashed by methanol and activated at 60 °C

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47 for 5 min prior to chromatography. The plate was developed in a solvent system (6.0 ml)

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50 of n-butanol-acetic acid-water (4:1:1, V/V/V) in a CAMAG glass twin–through chamber

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52 (10 x 10 cm) previously saturated with the solvent for 30 min (temperature 25 ± 2oC,

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5554 relative humidity 40%). The development distance was 8 cm. Subsequent to the scanning,

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57 TLC plates were air dried and scanning was performed on a Camag TLC scanner III in

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59 absorbance mode at 280 nm and operated by Win Cats software. Evaluation was via peak

7 found to be linear in the range of 10-120 µg/ml.

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10 2.3. Animals

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13 Male Sprague Dawley rats (weighing between 200-250 g each) were used for the study.

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15 They were maintained under standard environmental conditions and were fed a standard

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1817 pellet diet with water ad libitum. The study was approved by Institutional Animal Ethical

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20 Committee, Shri B. M. Shah College of Pharmaceutical Education and Research,

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22 Modasa, Gujarat, India (IAEC/BMCPER/04/2005-06).

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25 2.4. Induction of diabetes

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28 Diabetes was induced by single tail vein injection of streptozotocin (STZ) (45 mg/kg)

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31 [Sigma, St. Luis, MO, USA] to male Sprague Dawley rats (200-250 g). Animals showing

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33 glucosuria more than 2% (Diastix, Bayer Diagnostics, India) or blood glucose level (>140

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36 mg/dl) 48 h after STZ injection were selected for the experiment.

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3938 2.5. Experimental design

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4241 Animals were divided into five groups of 6 rats each: Group 1: non diabetic control rats.

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44 Group 2: non diabetic control rats treated with M. pruriens at the dose of 400 mg/kg, p.o.

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46 daily for six weeks. Group 3: diabetic control rats. Group 4: diabetic rats treated with M.

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49 pruriens at the dose of 200 mg/kg, p.o. daily for six weeks. Group 5: diabetic rats treated

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51 with M. pruriens at the dose of 400 mg/kg, p.o. daily for six weeks.

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54 2.6. Blood sampling and biochemical analysis

9 HDL-C (Bayer Diagnostics Kit, India).

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12 2.7. Oral glucose tolerance test

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15 Rats were subjected to an oral glucose tolerance test (OGTT). Glucose (1.5 g /kg) was

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18 administered to 12 hours fasted rats. Blood samples were collected at 0, 30, 60, 120

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20 minutes. Serum was separated immediately and analyzed for glucose and insulin. The

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2322 results of OGTT were expressed as integrated areas under the curves for glucose

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25 (AUCglucose) over a period of 0-120 minutes.

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28 2.8. Estimation of lipid peroxidation and antioxidant parameters

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31 Twenty-four h after the last dose, animals were anesthetized with sodium pentobarbitone

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33 (40 mg/kg i.p.). Liver and kidney were excised and immediately frozen in dry ice and

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36 stored at -20 0C. Frozen tissue from each rat was homogenized in ice cold 0.1 M Tris-HCl

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38 buffer (pH 7.4). The homogenate was centrifuged and supernatant was used for the assay

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41 of lipid peroxidation, superoxide dismutase, catalase, glutathione and total protein

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43 estimation.27-31

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46 2.9. Statistical analysis

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49 Results were expressed as mean  standard error of mean (S.E.M.). Result were analyzed

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52 statistically using analysis of variance (ANOVA) followed by Tukey's test. Values of p<

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54 0.05 were considered significant.

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57 3. Results

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60 3.1. Standardization of extract

7 Comparision of absorption spectrum of the band in the sample track with that of standard

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9 L-dopa at Rf 0.39 by overlaping confirmed the presence of L-dopa in the sample and it

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1211 was found to be one of the major components (Figure 1).

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14 3.2. Effect on serum glucose and AUCglucose

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17 STZ-diabetic rats exhibited a significant (p<0.05) hyperglycemia as compared to non

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19 diabetic control animals. Treatment with M. pruriens at all doses significantly (p<0.05)

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22 prevented STZ-induced hyperglycemia (Figure 2). The decrease in glucose levels by M.

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24 pruriens was dose dependent. AUCglucose was significantly (p<0.05) higher in diabetic

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27 rats as compared to non diabetic control rats during oral glucose tolerance test. Treatment

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29 with M. pruriens significantly (p<0.05) lowered AUCglucose in a dose dependant manner

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3231 in diabetic rats (Figure 2).

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3534 3.3. Effect on lipid profile

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3837 STZ-diabetic rats produced significant (p<0.05) hypercholesteremia and

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40 hypertriglycedemia as compared to non diabetic control animals. Treatment with M.

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42 pruriens showed dose dependent decrease in both cholesterol and triglyceride levels

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45 (Figure 4). Serum HDL-C levels was significantly (p<0.05) decreased in STZ diabetic

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47 rats as compared to non diabetic control. Treatment with aqueous extract significantly

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50 (p<0.05) increased HDL-C levels as compared to diabetic control rats in a dose

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52 dependent manner (Table 1).

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55 3.4. Effect on lipid peroxidation and antioxidant parameters

9 extract of M. pruriens at a dose of 200 mg/kg and 400 mg/kg caused a dose dependant

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1211 decrease in lipid peroxides which were increased in diabetic rat. There was a significant

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14 (p<0.05) decrease in superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH)

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16 levels in liver and kidney tissue as compared to non diabetic control. Treatment with

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19 aqueous extract produced dose dependant increase in superoxide dismutase, catalase and

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21 glutathione levels. Aqueous extract alone in rats did not produce any significant change

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24 in lipid peroxides and SOD, CAT and GSH in liver and kidney (Table 2 and 3).

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27 4. Discussion

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30 STZ-diabetic rats showed significant increase in serum glucose levels as compared to non

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32 diabetic control rats. Treatment with M. pruriens significantly decreased glucose level in

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3534 dose dependant manner. During OGTT when further glucose load was given in STZ-

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37 diabetic rats there was further rise in glucose levels as compared to non diabetic control

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4039 rats. M. pruriens treatment significantly decreased AUCglucose in diabetic rats. Earlier

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42 reports suggest that alcoholic extract of M. pruriens seed significantly decrease serum

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44 glucose in alloxanized as well as STZ diabetic mice.21-23 Decrease in serum glucose by

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47 M. pruriens may be due to presence of L-dopa in the extract and we also found that

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49 aqueous extract of M. pruriens contain 5.6 % of L-dopa. The effects of L-dopa were

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5251 linked to endorenal dopamine synthesis and dopamine-1 receptor stimulation.32

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54 Dopomine receptor is reported to cause improvement in insulin sensitivity and there by

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5756 cause decrease in glucose levels33-35. Dopaminergic agonists are reported to reduce the

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7 phosphoenol pyruvate carboxykinase (PEPCK).36

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10 STZ-diabetic rats produced significant increase in cholesterol, triglycerides and decrease

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12 HDL levels as compared to non-diabetic control rats. Alterations in lipid levels by STZ-

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1514 diabetics have been explained on the basis of alterations in insulin levels or insulin

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17 sensitivity.37 Treatment of diabetic rats with M. pruriens at a dose of 200 mg/kg and 400

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19 mg/kg caused a significant decrease in cholesterol, triglycerides and increase in HDL

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22 levels as compared to diabetic control rats. Dopaminergic agonist, bromocriptine and

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24 SKF 38393 are reported to reduce basal lipolysis and adipose tissue lipoprotein lipase

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2726 activity resulting in reduced serum free fatty acid concentrations.36,38

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29 Oxygen derived free radicals generated in excess in response to various stimuli can be

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3231 cytotoxic to several tissues. Most of the tissue damage is considered to be mediated by

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34 these free radicals by attacking membranes through peroxidation of unsaturated fatty

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3736 acids.39 Induction of diabetes in rats with STZ uniformly results in an increase in lipid

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39 peroxidation (TBARS), an indirect evidence of intensified free radical production.40 In

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41 the present study the concentrations of lipid peroxides was significantly increased in liver

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44 and kidney tissue of diabetic rats, indicating an increase in the generation of free radicals.

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46 Increased lipid peroxidation in diabetes can be due to increased oxidative stress in the cell

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49 as a result of depletion of antioxidant scavenger systems. The present finding indicates

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51 significantly increased lipid peroxidation of rats exposed to STZ and its attenuation by

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5453 seed extract of M. pruriens. Rajeshwar et al. reported that seed extract of M. pruriens

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56 decrease antitumor activity and in vivo antioxidant status of against ehrlich ascites

9 L-dopa present in the extract.

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12 Reduced glutathione (GSH) is known to protect the cellular system against the toxic

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14 effects of lipid peroxidation.43 GSH functions as a direct free radical scavenger, as a

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1716 cosubstrate for GPx activity and as a cofactor for many enzymes and forms conjugates in

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19 endo and xenobiotic reactions.44 A marked decrease in the level of GSH in liver and

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21 kidney during diabetes was observed. Several studies support the hypothesis that in

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24 diabetes, chronic hyperglycemia increases the polyol pathway as well as advanced

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26 glycation end products (AGEs) formation and free radical generation rates, leading to

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29 increased GSH oxidation. A relative depletion of NADPH due to aldose reductase

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31 activation and secondary to reduced production through the pentose cycle impairs GSH

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3433 generation and leads to depletion of this free radical scavenger.45 The significant recovery

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36 of GSH content by treatment with aqueous extract of M. pruriens indicates the protective

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3938 effect of extract on antioxidants.

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41 Reduced activities of SOD and CAT in liver and kidney of diabetic rats have been

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43 observed in our study. The decreased activities of SOD and CAT in liver during diabetes

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46 may be due to increased production of reactive oxygen radicals that can themselves

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48 reduce the activity of these enzymes8. SOD is an important defense enzyme, which

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5150 converts superoxide radicals to hydrogen peroxide.46 CAT is a hemeprotein, which

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53 decomposes hydrogen peroxide and protects the tissues from highly reactive hydroxyl

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56 radicals.

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The reduction in the activity of these enzymes may result in a number of

58 deleterious effects. Administration of M. pruriens extract increased the activity of

7 diabetes.

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9 In conclusion, M. pruriens seed extract was effective in the amelioration of diabetes,

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1211 which may be attributed to its hypoglycemic property along with its antioxidant potential

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14 due to presence of L-dopa in the extract.

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9 Mucuna pruriens. Key to peak identity: 1, L-dopa.

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12 Figure 2 Effect of aqueous extract of Mucuna pruriens on serum glucose and AUCglucose

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14 in STZ-diabetic rats. Each bar represents Mean  S.E.M. number of animals in each

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1716 group = 6. R1 = non diabetic control, R2 = non diabetic control treated with M. pruriens

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19 (400 mg/kg), R3 = diabetic control, R4 = diabetic treated with M. pruriens (200 mg/kg),

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2221 R5 = diabetic treated with M. pruriens (400 mg/kg) * significantly different from non-

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24 diabetic control, ** significantly different from diabetic control p<0.05.

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