Proteome and phytochemical analysis of Cynodon dactylon leaves extract and its biological activity in diabetic rats

Biomedicine & Preventive Nutrition 1 (2011) 49–56

Original article

Proteome and phytochemical analysis of Cynodon dactylon leaves extract and its biological activity in diabetic rats D. Karthik , S. Ravikumar ∗ Department of Biotechnology, PRIST University, Thanjavur, Tamil Nadu 613 403, India

a r t i c l e

i n f o

Article history: Received 17 May 2010 Accepted 16 September 2010 Keywords: Cynodon dactylon Alloxan diabetes 2D electrophoresis

a b s t r a c t In Indian traditional system of medicine, herbal remedies are prescribed for treatment of various diseases including diabetes mellitus. In recent years, plants are being effectively tried in a various pathophysiological states. Cynodon dactylon is one of them. In the present study, aqueous extract of leaves of C. dactylon was found to have potent antidiabetic, antioxidant and hypolipidemic efficacy in alloxan-induced diabetic rat. Supplementation of this aqueous extract orally at the dose of 450 mg/kg body weight per day in alloxan-induced diabetic rats. Plant extract administered for 15 days resulted in a significant diminution of fasting blood sugar level. The lipid profile shows a significant (P < 0.05) decrease in HDL and increase in (P < 0.05) cholesterol, triglyceride, LDL and VLDL in alloxan diabetic rats, which were normalized after the plant extract treatment. We also investigated the role of AST, ALT, ALP, AP, LDH and CPK (P < 0.05) activities, which were found to decrease significantly in the aqueous extract supplemented group with respect to diabetic group. C. dactylon extract were further evaluated with gas chromatography coupled to mass spectrometry (GC–MS). In this, seven chemical constituents were identified from crude leaf sample. 2D electrophoresis analysis of proteins extracted from C. dactylon was performed using pH range 3–10 revealed 95 protein spots in the gel. Our findings suggested that C. dactylon aqueous extract is effective for alleviating hyperglycemia and improving lipid profile in diabetic rats and these could be used in diabetic and coronary heart disease (CHD) management. © 2011 Elsevier Masson SAS. All rights reserved.

1. Introduction Diabetes mellitus is a disease due to abnormality of carbohydrate metabolism and it is mainly linked with low blood insulin level or insensitivity of target organs to insulin. It is the most prevalent chronic disease in the world affecting nearly 25% of the population. From literature review, it is revealed that 15–20% of diabetic patients are suffering from insulin-dependent diabetes mellitus (IDDM) or type-I [1]. The IDDM is noted both in adult and childhood [2]. In modern medicine, no satisfactory effective therapy is still available to cure the diabetes mellitus. Though insulin therapy is also used to manage diabetes mellitus there are several drawbacks like insulin resistance [3], anorexia nervosa, brain atrophy and fatty liver observed [4] even after treatment. In recent years, there has been renewed interest in plant medicine [5] as an alternative treatment against different diseases as herbal drugs are generally non-toxic [6] as reported from research work conducted on experimental animal. The toxicological effect is not studied in human so far. Pure compounds have been

∗ Corresponding author. Tel.: +91 9443724611; fax: +91 4362 265140. E-mail address: [email protected] (S. Ravikumar). 2210-5239/$ – see front matter © 2011 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.bionut.2010.09.001

screened from various plants having “folk medicine reputation” by biochemical test for its antidiabetogenic effect [7]. Cynodon dactylon (L.) Pers. (Fam: Poaceae) is commonly known as “Doob” in India. It is a weed and has been regarded to possess varied medicinal properties. It has been used to treat urinary tract infection, calculi and prostatitis. The plant possesses antimicrobial and antiviral activity [8]. The aqueous fluid extract of the rhizome is used as anti-inflammatory, diuretic, anti-emetic, purifying agent and also in dysentery [9]. The plant extract also has significant application in dropsy and secondary syphilis [10]. The present work deals with the antidiabetic, antioxidant as well as hypolipidemic activity of aqueous extract of C. dactylon leaves in alloxan-induced diabetic rats. In addition, to characterize the chemical components and proteins in the C. dactylon leaves extract. 2. Materials and methods 2.1. Plant material Fresh leaves of C. dactylon were collected from PRIST University area. The plants were taxonomically identified and authenticated by Rev Dr S John Britto SJ, Director, The Rapinat Herbarium and

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Centre for Molecular Systematics, St. Joseph’s College (Autonomous), Tiruchirappalli, Tamil Nadu, India. The voucher specimens are deposited at the Rapinat herbarium and the voucher number is RHCD02. 2.2. Preparation of aqueous extract of C. dactylon leaves Fresh leaves of C. dactylon were air dried in shade and powdered. The powdered plant up to 450 g was extracted with boiling water for 10 h. The resulting extract was filtered and concentrated in rotavapour under reduced pressure. The concentrated extract was lyophilized to get a powder (yield 13.5%, w/w). This was used as the crude leaf extract to study the glycemic and lipidemic effect in alloxan-induced diabetes.

2.5. Experimental animal Male adult Wistar rats of 130–150 g body weight were used for all experiments. They were housed in separate cages under 12 h light and dark periods. Rats have free access to standard food and water ad libitum. The animal experimental protocol has been approved by Institutional Ethics Committee. Diabetes was induced by a single intraperitoneal injection of 150 mg/kg of alloxan monohydrate in sterile saline [11]. After 72 h of alloxan injection, the diabetic rats (glucose level > 200 mg/dl) were separated and used for the study. 2.6. Experimental design

2.3. Gas chromatography coupled to mass spectrometry (GC–MS) analysis

It is already been found that the most effective dose for treatment is 450 mg/kg [12]. Therefore, this dose was taken for evaluation in the case of severe diabetic animals. The experiment was carried on three groups (I, II and III) of six rats each:

Aqueous extract of C. dactylon was analyzed by gas chromatography equipped with mass spectrometry (GC-MS-QP2010Shimadzu). The chromatographic conditions were as follows: Column: DB-% ms (length 30.0 m, diameter 0.25 mm, film thickness 0.25 ␮m). The 1 ␮l DG aqueous extract was injected into the GC–MS in split less mode at 200 ◦ C. The column oven temperature was held at 45 ◦ C for 1 min, then programmed at 10 different rates up to 280 ◦ C and held for 18 min. Helium carrier gas was maintained at a flow rate of 1.4 ml/min.

(a) group I: normal ad libitum; (b) group II: diabetic rats were made diabetic by single intraperitoneal injection of 150 mg/kg of alloxan monohydrate in sterile saline; (c) group III: diabetic rats treated with aqueous extract of C. dactylon leaves: aqueous extract of C. dactylon leaves was given orally to diabetic rats in the dosage of 450 mg/kg body weight at 24 h intervals during the entire period of the experiment.

2.4. Two-dimensional gel electrophoresis The powder was resuspended in 2 ml of plant extraction buffer containing 20 mm Tris pH 8.0, 5 mm EDTA, 0.05% SDS. Then icecold acetone-10% TCA (Sigma) added to supernatant and sonicated for 1 min. The homogenate was kept for precipitation overnight at −20 ◦ C. After centrifugation at 10,000 rpm for 15 min at 4 ◦ C, the supernatant was removed by decanting immediately and the pellet was rinsed twice in ice-cold acetone for four times. The pellet was then air-dried, resuspended in a lysis buffer containing (85 mM Tris pH 6.8, 2% SDS). Iso-electric focusing (IEF) gels were made in glass tubing (160 × 3 mm inside diameter) and contained 10.3 g urea, 7.125 ml distilled water, 2.44 ml acrylamide (28.38% acrylamide, 1.62% bis-acrylamide), 0.750 ml carrier ampholytes 3/10, 0.375 ml NP-40, 34.625 ␮l ammonium persulphate (10%) and 12.5 ␮l TEMED. After half an hour’s polymerisation, a pre-run for focusing the ampholytes was performed by loading 30 ␮l lysis solution (9.8 M urea, 2% NP-40 [10% in distilled water], 2% carrier ampholytes 8/10, 25 mM DTT) and over 30 ␮l overlay solution (8 M urea, 1% carrier ampholytes 8/10, 5% NP-40, 25 mM DTT). Upper running buffer (20 mM NaOH) was degassed for 10 min, but the lower one (10 mM H3 PO4 ) was not. The electrophoretic conditions of the rod gels during the IEF were 200 V for 15 min, 300 V for 30 min and 400 V for 1 h. The solution was removed from the upper tank. Meanwhile, the sample (350 ␮g) was prepared by adding lysis solution in a 1:2 proportion and heating for 2 min at 100 ◦ C. After loading, 30 ␮l of overlay solution was added above every sample, which was then run at 400 V for 16 h. After focusing, IEF gels were maintained in equilibrating buffer (6 M urea, 75 mM Tris–HCl pH 8.8, 29.3% glycerol, 2% SDS, 0.002% bromophenol blue) for 30 min. Then, the IEF rod gels were immediately applied to an SDS-polyacrylamide gel that contained 12% acrylamide, but the stacking gel was replaced by IEC rod gels fixed to the SDS-PAGE gel with an agarose solution (1% agarose, 0.002% bromophenol blue in the first equilibrating buffer). The analytical gels were stained with colloidal blue.

The animals were considered as diabetic, if their blood glucose values were >200 mg/dl on the third day after alloxan injection. The treatment was started on the third day after alloxan injection and continued for 15 days. All biochemical parameters were estimated after 15th day of treatment. 2.7. Biochemical parameters Blood glucose was estimated by Ashwell method [13]. Oral glucose tolerance was evaluated by feeding 10 g/kg glucose after the treatment with 450 mg/kg extract. The specific activities of different enzymes viz. lactate dehydrogenase (EC 1.1.1.27), aspartate aminotransferase (EC 2.6.1.1), alanine aminotransferase (EC 2.6.1.2) [14], acid phosphatase (EC 3.1.3.2), alkaline phosphatase (EC 3.1.3.1) [15] were estimated with standard colorimetric and photometric techniques. Protein content of enzyme samples were determined colorimetrically [16]. Plasma total cholesterol and HDL-cholesterol levels were determined by the Lieberman–Bourchard method (Naito [17]). Plasma triglyceride was determined by the methods of Soloni [18]. LDL cholesterol level and Atherogenic index were calculated by formula [19]. VLDL-cholesterol levels were calculated by formula [20]. 2.8. Biochemical assays The collected plasma were used for measurement of superoxide dismutase (SOD) [21], catalase (CAT) [22], glutathione peroxidase (GPx) [23], reduced glutathione (GSH) [24] and lipid peroxidation (LPO) [25] were evaluated for antioxidant activity. 2.9. Statistical analysis The data are expressed as mean ± standard error of the mean (S.E.M.). Statistical comparisons were performed by one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test (DMRT). The results were considered statistically significant if the P values were 0.05 or less.

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Table 1 Effect of aqueous extract of Cynodon dactylon leaves on total cholesterol (TC), triglyceride (TG), HDL, LDL, VLDL and AI in alloxan induced diabetic rats. Values are expressed mean ± S.E.M. of six animals. Groups

Normal Diabetic induced Diabetic treateda a

Plasma lipid profile (mg/dl) TC

TG

HDL

LDL

VLDL

AI

31.8 ± 3.21 42.1 ± 2.75 30.9 ± 4.25

64.7 ± 4.24 105.7 ± 6.84 48.6 ± 5.96

25.6 ± 2.89 28.5 ± 3.24 35.6 ± 3.84

11.6 ± 3.98 19.5 ± 2.58 10.7 ± 2.94

13.0 ± 1.25 18.2 ± 2.84 9.1 ± 2.13

0.23 ± 0.05 0.49 ± 0.09 0.14 ± 0.03

P < 0.05, as compared to diabetic induced.

treatment, there was a decrease in LDL and VLDL level compared to diabetic control. The continuous treatment with aqueous extract of C. dactylon leaves brought down the lipid parameters in the diabetic rats to almost normal levels as indicated in Table 1. 3.4. Effect on plasma enzymes level (AST, ALT, ALP, AP LDH and CPK)

Fig. 1. Effect of aqueous extract of Cynodon dactylon leaves on blood glucose levels in alloxan induced diabetic rat after feeding glucose (10 g/kg). Values are expressed mean ± S.E.M. of six animals.

A significant elevation of plasma ALT and AST activity (P < 0.05) were observed in diabetic control rats as compared to normal group. Treatment with the aqueous extract of C. dactylon leaves to diabetic rats resulted in a significant decrease in the levels of ALT and AST when compared to diabetic control rats. A significant increase in the levels of ALP, AP, LDH and CPK (P < 0.05) were observed in diabetic control when compared to normal group. A significant reduction of plasma marker enzyme levels (ALP, AP, LDH and CPK) were noticed due to the effect of aqueous extract of leaves of C. dactylon as indicated in Table 2. 3.5. Effect on antioxidant activity

3. Results 3.1. Plasma glucose level Diabetic rats treated with crude aqueous extract at oral dose of 450 mg/kg for 15 days resulted in a decreased blood glucose level as compared to diabetic rats. Blood glucose levels of normal, diabetic induced and treated animals were 101 ± 5.2, 210 ± 6.9 and 125 ± 4.15 mg/dl, respectively (P < 0.05). A significant level of reduction of oral glucose tolerance was noticed due to the effect of aqueous extract of leaves of C. dactylon as indicated in Fig. 1.

Table 3 shows the activities of antioxidant enzymes in the three groups. The activities of SOD and GPx were significantly increased in diabetic treated rats compared to diabetic rats. No significant difference in CAT activity was observed among the various groups. The level of lipid peroxidation in diabetic rats was significantly higher than normal rats. The levels of lipid peroxidation in diabetic rats treated with the aqueous extract were significantly reduced than in normal and diabetic induced. The concentration of GSH in diabetic treated rats was increased compared to diabetic rats due to effect aqueous extract.

3.2. Effect on plasma total cholesterol and HDL-cholesterol level A significant increase in cholesterol level was observed in diabetic control when compared to normal groups. There was no much change in HDL-cholesterol. Treatment with the aqueous extract of C. dactylon leaves to diabetic rats resulted in a significant decrease in cholesterol level and enhanced HDL-cholesterol level when compared to diabetic control rats as indicated in Table 1. 3.3. Effect on plasma triglyceride, LDL and VLDL levels A significant increase in triglyceride, LDL and VLDL (P < 0.05) levels were observed in diabetic rats as compared to normal. After the

3.6. Gas chromatography coupled to mass spectrometry analysis GC–MS analysis of C. dactylon extract detected molecular peaks, with typical retention time of analyzed components, which are depicted in total ions chromatogram (TIC) obtained from MS analysis (Fig. 2a and b). We have reported the constituents of aqueous extract of C. dactylon and its chemical structure, which are presented in Fig. 3 and Table 4. The GC–MS analysis of plant extract revealed the presence of seven major compounds mainly phenylmethanol (t R;7.67), 2-propenoic acid (cinnamic acid) (t R;11.98), sesquiterpene (t R;15.45), 2-methoxy-4prop-2-enylphenyl) acetate (t R;16.1), 4’,5,7-trihydroxyisoflavone

Table 2 Effect of aqueous extract of Cynodon dactylon leaves on AST, ALT, ALP, AP, LDH and CPK in alloxan induced diabetic rats. Values are expressed mean ± S.E.M. of six animals. Group

Normal Diabetic induced Diabetic treateda a

Plasma enzyme profile (U/L) AST

ALT

ALP

AP

LDH

CPK

50.8 ± 5.2 88.5 ± 7.52 69.5 ± 6.35

24.5 ± 2.85 40.8 ± 4.28 28.0 ± 3.86

119.0 ± 9.35 158.0 ± 8.25 124.0 ± 8.52

33.7 ± 3.51 40.0 ± 5.84 34.0 ± 5.24

71.6 ± 7.25 112.8 ± 8.71 89.5 ± 10.24

350 ± 32.54 400 ± 35.21 372 ± 40.12

P < 0.05, as compared to diabetic induced.

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Table 3 Effect of aqueous extract of Cynodon dactylon leaves on SOD, CAT, GPx, GSH and LPO activity in alloxan induced diabetic rats. Values are expressed mean ± S.E.M. of six animals. Group

Normal Diabetic induced Diabetic treateda

Antioxidant activity SOD (U1 /mg protein)

CAT (U2 /mg protein)

GPx (U3 /mg protein)

GSH (mM of GSH/mg protein)

LPO (nM of MDA/mg protein)

8.80 ± 1.25 6.62 ± 0.96 14.7 ± 2.35

31.30 ± 3.21 29.70 ± 2.58 31.60 ± 4.21

455 ± 10.25 309 ± 11.58 400 ± 8.54

251 ± 8.24 211 ± 10.23 246 ± 13.21

56 ± 3.25 75 ± 6.35 49 ± 5.21

a P < 0.05, as compared to diabetic control. U1 /one unit of activity was taken as the enzyme reaction, which gave 50% ihibition of NBT reduction in 1 min. U2 /mmol of hydrogen peroxide consumed/minute. U3 /mg of glutathione consumed/minute.

(t R;16.27), procyanidin (t R; 16.9) and 3,7,11,15-tetramethyl-2hexadecen-1-ol (t R;17.6). 3.7. 2-DE protein patterns of Cynodon dactylon leaves The two-dimensional electrophoresis analysis of proteins pattern from C. dactylon leaves showed with relative molecular weight marker were between 10 and 170 kDa as indicated in Fig. 4. Isoelectric points were between 3 and 10 using range pH 3–10 pH ampholyte (17 cm), and most of them were in the range of 3.5–7. Totally about 95 protein spots could be visualized on the 2-DE maps by using a web based gel viewing and annotation system (http://www.gelscape.ualberta.ca). 4. Discussion Diabetes mellitus is a pathologic condition, resulting in severe metabolic imbalances and non-physiologic changes in many tissues, where oxidative stress plays an important role in the etiology [26]. Alloxan causes diabetes by rapid depletion of ␤-cells, which leads to reduction in insulin release [27]. Hyperglycemia causes oxidative damage by generation of ROS [28] and development of diabetic complications [29]. The administration of C. dactylon leaves extract to alloxan diabetic rats reduced blood glucose levels, is in accordance with our previous study [30]. These effects may be attributed to the insulin mimicking effect of plant extract [31]. Lowering the plasma lipid levels through dietary or drug therapy appears to be associated with a decrease in the risk of vascular disease [32]. The increase in the serum lipids on the diabetic subject is mainly due to the increased mobilization of free fatty acids from peripheral deposits, since insulin inhibits the hormone sensitive lipase [33]. On the other hand, glucagon, catecholamines and other hormones enhance lipolysis. Liver is an insulin dependent tissue that plays a pivotal role in glucose and lipid homeostasis and it is severely affected during diabetes [34]. Diabetes results show a decrease in glucose utilization and an increase in glucose production in insulin-dependent tissues, such as liver. A marked increase in hepatic lipid concentration has been earlier observed during diabetes [35]. Cholesterol is a powerful risk factor for many coronary heart diseases. The degree of hypercholesterolemia is directly proportional to severity in diabetes. In the present study, we have observed higher levels of cholesterol in plasma of diabetic rats. The increased level of cholesterol in plasma is due to the decreased

level of HDL-cholesterol. This in turn results in decreased removal of cholesterol from extrahepatic tissues by the HDL-cholesterol [36]. Administration of C. dactylon leaves extract to diabetic rats normalizes plasma levels of cholesterol. The underlying mechanism by which plant extract exerts its cholesterol lowering effect seems to be a decrease in cholesterol absorption from the intestine, by binding with bile acids within the intestine and increasing bile acids excretion [37]. Plant extract may acts by decreasing the cholesterol biosynthesis especially by decreasing the HMG-CoA reductase activity, a key enzyme of cholesterol biosynthesis and/or by reducing the NADPH required for fatty acids and cholesterol biosynthesis [38]. In addition, plant extract may improve hypercholesterolemia by modifying lipoprotein metabolism: enhanced uptake of LDL by increasing LDL receptors [39] and/or by increasing the lecitin:cholesterol acyl transferase activity [40], which may contribute to the regulation of blood lipids. HDL cholesterol is recognized as a factor that protects against development of atherosclerotic disease, and low HDL-cholesterol is associated with an increased risk of CHD in individuals both with and without diabetes. Total cholesterol/HDL and LDL/HDL cholesterol ratio are also predictor of coronary risk [41]. In the present study, rats treated with C. dactylon leaves extract had markedly reduced ratios. These results indicated that C. dactylon leaves extract might have some protective effects against hypercholesterolemia risks in diabetes. In our study, diabetic rats exhibited clear-cut abnormalities in lipid metabolism as evidenced from the significant elevation of VLDLcholesterol level and atherogenic index. Treatment with C. dactylon leaves extract for 15 days significantly and greatly reduced VLDLcholesterol level and decreased in atherogenic index in diabetic rats indicating its potent antihyperlipidemic and antiatherogenic activities. The marked increase in plasma triglyceride observed in diabetic rats is in agreement with the finding of the some researcher [42]. The increased level of triglycerides in alloxan diabetes observed, in our study, may be due to lack of insulin, which normally activates the enzyme lipoprotein lipase. Lopez-Virella et al. reported that treatment with insulin served to lower plasma triglyceride levels, by returning lipoprotein lipase levels to normal [43]. Oral administration of C. dactylon leaves extract to diabetic rats reduced plasma triglycerides levels, thus our finding is a very desirable biochemical state for prevention of atherosclerosis. Oxidative stress in diabetes coexists with a reduction in the antioxidant capacity, which can increase the deleterious effects of free radicals. Increased oxidative stress is believed to play an

Table 4 Compounds present in aqueous extract of Cynodon dactylon leaves. S No.

Retention time (t R)

Formula

Molecular weight

Component name

1 2 3 4 5 6 7

7.67 11.98 15.45 16.1 16.27 16.9 17.6

C 7 H8 O C9 H8 O2 C15 H24 C12 H14 O C15 H10 O5 C15 H11 O6 C20 H40 O

108.14 148.16 204.11 206.23 270.24 281.12 296.54

Phenylmethanol 2-Propenoic acid (cinnamic acid) Sesquiterpene 2-Methoxy-4-prop-2-enylphenyl acetate 4 ,5,7-Trihydroxyisoflavone Procyanidin 3,7,11,15-Tetramethyl-2-hexadecen-1-ol

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Fig. 2. a and b: Secondary metabolites detected using GC-MS, with relative retention time (t R) form Cynodon dactylon leaves extract.

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Fig. 3. Molecular structure of secondary metabolites analyzed in Cynodon dactylon leaves extract.

important role in the aetiology and pathogenesis of chronic complications of diabetes. This study analyzed the effect of an aqueous extract from C. dactylon leaves on biomarkers of oxidative stress in plasma antioxidant of diabetic rats. In our study, the activity of SOD was found to be lower in diabetic rats. This decrease in SOD activity could result from inactivation by H2 O2 or by glycation of the enzyme, which are known to occur during diabetes [44]. Reduced activities of CAT in plasma have been observed during diabetes. CAT is a hemeprotein that catalyses the reduction of hydrogen

peroxides and protects the tissues from highly reactive hydroxyl radicals [45]. Therefore, reduced in the activity of this enzymes may result in a number of deleterious effects due to the accumulation of superoxide anion radicals and hydrogen peroxide. Administration of C. dactylon leaves extract resulted in the elevation of the SOD and CAT level, which protects oxidative damage in tissues. Under in vivo conditions, GSH acts as an antioxidant and its level was reduced in diabetes mellitus [29]. During diabetes, we also observed a significant decreased in GSH levels in plasma. In a sim-

Fig. 4. Two-dimensional electrophoresis patterns of protein from Cynodon dactylon leaves (pH 3–10).

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ilar manner, Matkovics et al. showed decreased amounts of GSH in the skeletal muscle, liver and lung homogenates of mice with diabetes [46]. The decrease in GSH levels represents increased utilization due to oxidative stress [47]. The depletion of GSH content also may lower glutathione-S-transferase (GST) activity, because GSH is required as a substrate for glutathione-S-transferase (GST) activity [48]. Depression in glutathione peroxidase activity was also observed in plasma during diabetes. GPx has been shown to be an important adaptive response to conditions of increased peroxidative stress [49]. Administration of C. dactylon leaves extract resulted in the elevation of the GSH level, which protects the cell membrane against oxidative damage by regulating the redox status of protein in the cell membrane. The increase in the GSH content may protect the tissues against diabetes-associated tissue injury by reducing the susceptibility to toxic radicals. Lipid peroxidation is a marker of cellular oxidative damage initiated by reactive oxygen species [50]. The increase in oxygen free radicals in diabetes could be due to an increase in levels of blood glucose, which upon auto-oxidation, generates free radicals. Insulin secretion is also closely associated with lipoxygenase-derived peroxides [51]. Low levels of lipoxygenase peroxides stimulate the secretion of insulin, but when the concentration of endogenous peroxides increases, it may initiate uncontrolled lipid peroxidation leading to cellular infiltration and islet cell damage in type 1 diabetes [52]. Increased lipid peroxidation impairs membrane functions by decreasing membrane fluidity and changing the activity of membrane-bound enzymes and receptors. Its products (lipid radical and lipid peroxide) are harmful to the cells in the body and associated with atherosclerosis, brain, kidney and other tissue damage [53]. In the present study, the concentrations of lipid peroxidation, indicating an increased in the generation of free radicals were increased in the stomach and spleen tissues of diabetic rats. After administration of C. dactylon leaves extract, the levels of lipid peroxidation declined significantly and thus prevent tissue damage. Some transition metals, such as nickel, chromium, and vanadium might act as catalysts of the oxidative deterioration of biological molecules [54]. This might occur via formation of reactive oxygen species and enhanced lipid peroxidation, depletion of sulfhydryls, and oxidative tissue injury [55]. Vanadium compounds may behave as antioxidants and prooxidants, depending on experimental conditions and the dose of vanadium. In our results, induced diabetes by alloxan caused a significant decrease in plasma GSH and increase in plasma LPO. The administration of C. dactylon leaves extract to diabetic rats reduced LPO and turned GSH towards its normal values. These results suggest that the administration of C. dactylon leaves extract may have a protective effect against tissues damage in pro-oxidant conditions during diabetes. Administration of aqueous extract of C. dactylon leaves improved the liver function by decreasing the plasma AST, ALT and ALP levels in diabetic treated rats. The increase of AST and ALT will increase the incidence of heart and liver diseases. AST is an enzyme found primarily in the cells of the liver, heart, skeletal muscles, kidneys, pancreas and to a lesser extent, in red blood cells. Its plasma concentration is in proportion to the amount of cellular leakage or damage. It is released into plasma in larger quantities when any one of these tissues is damaged. Its increased levels are usually associated with heart attacks or liver disease [56]. The aqueous extract of C. dactylon leaves were decreased the AST level, which is an indication of the protective effect on liver and heart. ALT an enzyme found primarily in the liver is far greater. Its enhanced release into the bloodstream is the result of liver abnormality. It therefore serves as a specific indicator of liver status and its elevated levels in plasma indicate liver damage. Increased levels of ALP indicate bone disease, liver disease or bile tract blockage [57]. The aqueous extract of C. dactylon leaves were reduced, the

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ALT and ALP levels too, indicating its protective effect over liver and improvement in liver functional efficiency. Measurement of enzymic activities of acid phosphatases (AP) is of clinical and toxocological importance as changes in their activities are indicative of tissue damage by toxicants or in disease conditions. Acid phosphatase activity of plasma of diabetic control rats was found to be increased. At the dose of 450 mg/kg for 15 days, aqueous extract is reported to inhibit acid phosphatase activity and reduced the AP level in plasma [58]. The significant increases of LDH are mainly due to leakage of these enzymes in to the blood because of alloxan toxicity in liver. Higher activity of glucose 6-phosphatase provides H+, which binds with NADP+ to form NADPH, which is helpful in the synthesis of fats from carbohydrates. When glycolysis slows down because of cellular activity, pentose phosphate pathway that is still active in liver provides NADPH, which converts acetyl radicals in to long chain fatty acids during diabetes mellitus. Similar results were reported by other researchers in experimental diabetes [59]. However, treatment of alloxan diabetic rats with aqueous extract of C. dactylon leaves for 15 consecutive days could restore the normal metabolism by shifting the balance from lipids metabolism to carbohydrate metabolism [60]. It is well known that diagnosis of cardiac enzymes is important. Plasma CPK activity is a more sensitive indicator in early stage of myocardial ischemia, while peak rises in myocardial tissue [61]. The results in diabetic treated animals in this experiment shows a protective effect of aqueous extract of C. dactylon leaves on the heart of experimental animals. Moreover, the significantly lowered activities of CPK scientifically suggest that the leaf extract of C. dactylon have the potential of reducing the factors that produce infarction in the myocardium. This is so because the metabolism of alloxaninduced infarct myocardium may be studied by assessing the level of marker enzyme in the plasma [62]. Two-dimensional electrophoresis analysis of proteins extracted from C. dactylon was performed using pH range 3–10. It implied the potential application of the comparative proteomics study of C. dactylon in understanding the proteon expression and their function. 5. Conclusion In conclusion, the administration of C. dactylon leaves extract may be able to reduce hyperglycemia and hyperlipidemia related to the risk of diabetes. This has clinical implications that the relatively nontoxic C. dactylon extract, if used as a hypoglycemic agent, may also reverse dyslipidemia associated with diabetes, and prevent the CV complications, which are very prevalent in diabetic patients. GC–MS analyses of the extracts of chemical constituents are active antidiabetic and antioxidant as well as hypolipidemic principles in C. dactylon. The present investigation has also opened avenues for further research especially with reference to the development of potent phytomedicine for diabetes mellitus from C. dactylon leaves. Disclosure of interest This research paper was written independently by both authors; no company or institution supported it financially. Acknowledgement The authors are thankful to PRIST University, Thanjavur, Tamil Nadu, India for providing research facility. We would like to extend our thanks to Sophisticated Analytical Instrument Facility, Indian Institute of Technology (IIT), Chennai, Tamil Nadu, India for providing GC–MS facility.

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