Hepatoprotective effect of the aqueous extract of the roots of Decalepis hamiltonii against ethanol-induced oxidative stress in rats

Hepatology Research 35 (2006) 267–275

Hepatoprotective effect of the aqueous extract of the roots of Decalepis hamiltonii against ethanol-induced oxidative stress in rats Anup Srivastava, T. Shivanandappa ∗ Department of Food Protectants and Infestation Control, Central Food Technological Research Institute, Mysore 570020, Karnataka, India Received 28 November 2005; received in revised form 11 April 2006; accepted 27 April 2006 Available online 14 June 2006

Abstract The hepatoprotective activity of the aqueous extract of the roots of Decalepis hamiltonii was investigated against ethanol-induced oxidative stress and liver damage. Pretreatment of rats with aqueous extract of the roots of D. hamiltonii, single (50, 100 and 200 mg/kg b.w.) and multiple doses (50 and 100 mg/kg b.w. for 7 days) significantly prevented the ethanol (5 g/kg b.w.) induced increases in the activities of the serum enzymes, aspartate and alanine transaminases, alkaline phosphatase and lactate dehydrogenase in a dose dependent manner. Parallel to these changes, the root extract inhibited the ethanol-induced oxidative stress in the liver by suppressing lipid peroxidation and protein carbonylation and maintaining the levels of antioxidant enzymes and glutathione. The biochemical changes were consistent with histopathological observations suggesting marked hepatoprotective effect of the root extract. The protective effect of the root extract against hepatotoxicity of alcohol was more pronounced by the multiple dose pretreatment. Hepatoprotective activity of the aqueous extract of the roots of D. hamiltonii could be attributed to the antioxidant effect of the constituents and enhanced antioxidant defenses. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Decalepis hamiltonii; Hepatoprotective; Antioxidant system; Lipid peroxidation

1. Introduction Chronic liver damage is a widespread pathology characterized by a progressive evolution from steatosis to chronic hepatitis, fibrosis, cirrhosis and hepatocellular carcinoma. The ability of ethanol to increase the production of reactive oxygen species (ROS) and enhance peroxidation of lipids, protein and DNA has been demonstrated in a variety of systems, cells and species including humans [1]. The mechanism of ethanol-induced oxidative stress and cell injury appear to involve redox state changes (decrease in the NAD+ /NADH redox ratio) produced as a result of ethanol oxidation by alcohol and acetaldehyde dehydrogenases, production of the reactive metabolite acetaldehyde, one electron oxidation of ethanol to the 1-hydroxy ethyl radical [2–4]. Many of these pathways are not exclusive of one another and it is likely that ∗

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several systems contribute to the ability of ethanol to induce a state of oxidative stress. As the oxidative stress plays a central role in liver pathologies and progression, the use of antioxidants has been proposed as therapeutic agents, as well as drug coadjuvants, to counteract liver damage [5]. A number of studies have shown that antioxidants including the plant extracts protect against ethanol hepatotoxicity by inhibiting lipid peroxidation and enhancing antioxidant enzyme activity [6,7]. Decalepis hamiltonii (family: Asclepiadaceae), a climbing shrub, grows in the forests of peninsular India. Its tuberous roots are consumed as pickles and juice for its alleged health promoting properties. The roots of D. hamiltonii are used in folk medicine and as a substitute for Hemidesmus indicus in ayurvedic preparations [8]. We have earlier shown that the roots of D. hamiltonii possess potent antioxidant properties, which could be associated with their alleged health benefits [9]. Earlier work indicated that the roots of D. hamiltonii contain aldehydes, inositols, saponins, amyrins,

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lupeols and volatile flavor compounds such as 2-hydroxy4-methoxybenzaldehyde, vanillin, 2-phenyl ethyl alcohol, benzaldehyde and others. Our recent work has shown that the aqueous extract of the roots of D. hamiltonii is a cocktail of novel antioxidants namely, 4-hydroxy isophthalic acid, 14aminotetradecanoic acid, 4-(1-hydroxy-1-methylethyl)-1methyl-1,2-cyclohexane diol, 2-hydroxymethyl-3-methoxybenzaldehyde and 2,4,8-trihydroxybicyclo[3.2.1]octan-3one [10]. We have also reported that the methanolic extract contains several antioxidant compounds viz., 2-hydroxy4-methoxybenzaldehyde, p-anisaldehyde, vanillin, borneol, salicylaldehyde and decalepin [11]. The extract as well as the root powder are not toxic to rats in acute and subacute studies [12]. In order to evaluate the health promoting potential of the roots of D. hamiltonii, the present study was undertaken to investigate the hepatoprotective potential of the aqueous extract of the roots of D. hamiltonii against ethanol hepatotoxicity in rats. 2. Experimental 2.1. Chemicals Nicotinamide adenine dinucleotide phosphate reduced (NADPH), 1-chloro-2,4-dinitrobenzene (CDNB), thiobarbituric acid (TBA), glutathione (GSH), oxidized glutathione (GSSG), glutathione reductase (GR), cumene hydroperoxide (CHP), pryogallol, bovine serum albumin (BSA), 2,4dinitrophenyl hydrazine (DNPH) and tetraethoxypropane were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Trichloroacetic acid (TCA), hydrogen peroxide (H2 O2 ), 5,5 dithiobis(2-nitrobenzoic acid) (DTNB) and other chemicals were purchased from Sisco Research Laboratories, Mumbai, India. All the chemicals used were of highest purity grade available. 2.2. Preparation of the root powder and extraction Roots of D. hamiltonii (10 kg), procured from the local suppliers, were washed with water, followed by crushing with a roller to separate the inner woody core from the outer fleshy layer. The fleshy portions were pooled, dried at 40 ◦ C in a hot air oven and fine powdered. The powder (1.9 kg) was used for extraction. The aqueous extract was prepared by homogenizing the root powder (200 g) in warm water (50 ◦ C) and allowed to stand for 24 h, filtered with Whatman paper No. 1 and the filtrate was lyophilized and weighed (34.75 g). We have earlier reported that aqueous extract of D. hamiltonii shows high antioxidant activity among the different solvent extracts [9].

animal experiments. In single dose pretreatment (oral) experiment, administration of aqueous extract of the roots of D. hamiltonii at 50, 100 and 200 mg/kg b.w. was followed, after 1 h, by oral administration of ethanol (1/2 LD50 —5 g/kg b.w.). In multiple dose pretreatment experiment, aqueous extract of the roots of D. hamiltonii was administered for 7 consecutive days at 50 and 100 mg/kg b.w. followed by a single oral dose of ethanol (5 g/kg b.w.) on the 7th day. Animals were sacrificed by diethyl ether anesthesia 16 h after the ethanol administration, the liver perfused with saline were dissected out and processed immediately for biochemical assays. 2.4. Experimental groupings 2.4.1. Single dose Group I—control; Group II—ethanol (5 g/kg b.w.) (physiological saline was used as the vehicle); Group III—D. hamiltonii aqueous extract (DHA) (50 mg/kg b.w.) + ethanol; Group IV—DHA (100 mg/kg b.w.) + ethanol; Group V—DHA (200 mg/kg b.w.) + ethanol; Group VI—DHA (200 mg/kg b.w.). 2.4.2. Multiple dose Group I—control; Group II—ethanol; Group III—DHA (50 mg/kg b.w.) + ethanol; Group IV—DHA (100 mg/kg b.w.) + ethanol; Group V—DHA (100 mg/kg b.w.). 2.5. Serum enzymes Blood samples were collected in tubes, allowed to clot and the serum was collected by centrifugation at 2000 × g for 10 min and stored at 4 ◦ C for biochemical analysis. Serum transaminases (ALT and AST) were determined by the method of Reitman and Frankel [13]. The reaction mixture containing the substrates (l-alanine (200 mM) or l-aspartate (200 mM) with ␣-ketoglutarate) and enzyme in phosphate buffer (0.1 M, pH 7.4) was incubated for 30 and 60 min for ALT and AST, respectively. After incubation, DNPH (1 mM) was added and kept for another 30 min at room temperature. The color was developed by the addition of NaOH (0.4N) and read at 505 nm in a spectrophotometer. Lactate dehydrogenase activity was assayed by the method of Kornberg [14]. The reaction mixture consisted of NADH (0.02 M), sodium pyruvate (0.01 M) in sodium phosphate buffer (0.1 M, pH 7.4). The change in the absorbance was recorded at 340 nm at 30 s interval for 3 min. Alkaline phosphatase activity was assayed by the method of Walter and Schutt [15] with p-nitrophenyl phosphate (1.25 mM) as the substrate. The enzyme activity was calculated using the extinction coefficient, 1.85 × 10−3 M−1 cm−1 for p-nitrophenol.

2.3. Animals and treatments 2.6. Lipid peroxidation Sixty-day-old adult male Wistar rats (180–200 g) were divided into groups of eight each. Appropriate guidelines of the local animal ethics committee were followed for the

Lipid peroxidation (LPO) in the tissue homogenate was measured by estimating the formation of thiobarbituric acid

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reactive substances (TBARS) [16]. Tissue homogenate (10%, w/v, in 50 mM phosphate buffer, pH 7.4) was boiled in TCA (10%) and TBA (0.34%) for 15 min, cooled and centrifuged. Absorbance of the supernatant was read at 535 nm. TBARS was calculated using tetraethoxypropane as the standard. 2.7. Antioxidant enzymes Liver tissue was homogenized (10%, w/v) in icecold 50 mM phosphate buffer (pH 7.4), centrifuged at 10,000 × g for 20 min at 4 ◦ C and the supernatant was used to assay the enzyme activities. Superoxide dismutase (SOD) activity was measured using pyrogallol (2 mM) autoxidation in tris buffer [17]. Catalase (CAT) activity

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was measured using H2 O2 (3%) as the substrate in phosphate buffer [18]. Glutathione peroxidase (GPx) activity was measured by the indirect assay method using glutathione reductase. Cumene hydroperoxide (1 mM) and glutathione (0.25 mM) were used as substrates and oxidation of NADPH by glutathione reductase (0.25 U) in tris buffer (0.05 M, pH 7.4) was monitored at 340 nm [19]. Glutathione reductase activity was estimated using oxidized glutathione (0.5 mM) and NADPH (2 mM) in potassium phosphate buffer (0.1 M, pH 7.4) [19]. GlutathioneS-transferase (GST) activity was assayed in phosphate buffer (0.1 M, pH 7.6) containing glutathione (0.5 mM) and CDNB (0.5 mM) and change in the absorbance at 344 nm was monitored in a UV–vis spectrophotometer [19].

Fig. 1. Protective effect of the aqueous extract of the roots of D. hamiltonii (pretreatment—single dose) on ethanol hepatotoxicity: serum enzymes. Group I—control; Group II—ethanol (5 g/kg b.w.); Group III—DHA (50 mg/kg b.w.) + ethanol (5 g/kg b.w.); Group IV—DHA (100 mg/kg b.w.) + ethanol (5 g/kg b.w.); Group V—DHA (200 mg/kg b.w.) + ethanol (5 g/kg b.w.); Group VI—DHA (200 mg/kg b.w.). LDH: lactate dehydrogenase; ALT: alanine aminotransferase; AST: aspartate aminotransferase; ALP: alkaline phosphatase. Each bar represents the mean ± S.E., n = 8; bars with different alphabets differ significantly at p < 0.05 level (DMRT).

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2.8. Glutathione A 10% (w/v) liver homogenate was prepared in 5% (w/v) trichloroacetic acid, centrifuged at 2000× g for 20 min and the glutathione content in the deproteinized supernatant was estimated by Ellman’s reagent with a standard curve [20]. 2.9. Protein carbonyls Liver homogenate (10%, w/v) was prepared in 20 mM tris–HCl buffer, pH 7.4, with 0.14 M NaCl, centrifuged at 10,000× g for 10 min at 4 ◦ C. 1.0 ml of the supernatant was precipitated with an equal volume of 20% TCA and centrifuged. The pellet was resuspended in 1.0 ml of DNPH (10 mM in 2 M HCl) and allowed to stand at room temperature for 60 min with occasional vortexing. 0.5 ml of 20%

TCA was added to the reaction mixture and centrifuged, the pellet obtained was washed three times with acetone and 1.0 ml of 2% of SDS (in 20 mM tris–HCl, 0.1 M NaCl, pH 7.4) was added to solublize the pellet. The absorbance of the solution was read at 360 nm and the carbonyl content was calculated using a molar extinction coefficient of 22,000 M−1 cm−1 [21]. Protein content was estimated by the method of Lowry et al. [22] with bovine serum albumin as the standard. 2.10. Histopathological examination Pieces of liver from the same lobe were fixed in Bouin’s fluid for 24 h and processed for paraffin embedding. Sections (6 ␮m thick) were stained with hematoxylin and eosin and observed under the microscope.

Fig. 2. Protective effect of the aqueous extract of the roots of D. hamiltonii (pretreatment—multiple dose) on ethanol hepatotoxicity: serum enzymes. Group I—control; Group II—ethanol (5 g/kg b.w.); Group III—DHA (50 mg/kg b.w.) + ethanol (5 g/kg b.w.); Group IV—DHA (100 mg/kg b.w.) + ethanol (5 g/kg b.w.); Group V—DHA (100 mg/kg b.w.). LDH: lactate dehydrogenase; ALT: alanine aminotransferase; AST: aspartate aminotransferase; ALP: alkaline phosphatase. Each bar represents the mean ± S.E., n = 8; bars with different alphabets differ significantly at p < 0.05 level (DMRT).

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Table 1 Effect of D. hamiltonii aqueous extract pretreatment (single dose) on ethanol-induced changes in hepatic lipid peroxidation, antioxidant profile and protein carbonylation of rats Group

LPOa

SODb

CATc

GPxd

GRd

GSTe

GSHf

PCg

I II III IV V VI

3.97a 5.43c 4.60b 4.05a 3.94a 3.82a

2.36a 0.33d 0.61d 1.41c 2.04b 2.52a

5.92e 4.15a 4.71b 5.25c 5.60d 6.01e

103.41e 68.58a 77.87b 86.44c 98.94d 106.80e

276.89d 170.82a 211.02b 248.98c 276.89d 292.52d

157.73c 127.88a 133.32a 148.13b 156.58c 180.42d

16.24c 10.04a 12.87b 14.96c 16.10c 16.79c

36.28a 47.82b 47.34b 42.21ab 37.47a 36.33a

Means with different suffix letters differ significantly (p < 0.05). Group I—control; Group II—ethanol (5 g/kg b.w.); Group III—DHA (50 mg/kg b.w.) + ethanol (5 g/kg b.w.); Group IV—DHA (100 mg/kg b.w.) + ethanol (5 g/kg b.w.); Group V—DHA (200 mg/kg b.w.) + ethanol (5 g/kg b.w.); Group VI—DHA (200 mg/kg b.w.). a LPO—lipid peroxidation (nmol MDA/mg protein). b SOD—superoxide dismutase (U/mg protein). c CAT—catalase (␮mol H O /min/mg protein). 2 2 d GPx—glutathione peroxidase, GR—glutathione reductase (nmol NADPH/min/mg protein). e GST—glutathione-S-transferase (␮mol CDNB conjugate/min/mg protein). f GSH—glutathione (␮g/mg protein). g PC—protein carbonyl (␮mol/mg protein).

2.11. Statistics

3.2. Lipid peroxidation

Data were expressed as mean ± S.E. (n = 8) and significant difference between the groups was statistically analyzed by Duncan’s multiple range test (Statistica Software, 1999). A difference was considered significant at p < 0.05.

3. Results

The effect of DHA on ethanol-induced lipid peroxidation measured as TBARS in the liver is shown in Tables 1 and 2. Ethanol increased the hepatic TBARS concentration significantly which was inhibited by DHA pretreatment. Multiple dose pretreatment with a lower dose of DHA (50 mg/kg b.w.) was more effective than the single doses (100 and 200 mg/kg b.w.) in inhibiting the hepatic lipid peroxidation.

3.1. Serum enzymes

3.3. Antioxidant enzymes

Levels of the serum marker enzymes of hepatic damage, AST, ALT, LDH and ALP, increased significantly in ethanol treated rats compared to the control group. In single dose experiment, DHA at 100 and 200 mg/kg b.w. prevented the liver damage as judged by the restored enzyme levels (Fig. 1). Pretreatment with multiple treatment of DHA at a lower dose (50 mg/kg b.w.) was more effective than that of a single higher dose (100 and 200 mg/kg b.w.) (Fig. 2).

The hepatic antioxidant enzyme activities were decreased in the liver of rats after administration of ethanol. Activities of SOD, CAT, GPx, GR and GST were restored by DHA pretreatment. Multiple dose pretreatment of the root extract was more effective in the restoration of biochemical changes than a single dose. Further, DHA multiple dose pretreatment, by itself, boosted the antioxidant enzyme activities in the liver (Tables 1 and 2).

Table 2 Effect of D. hamiltonii aqueous extract pretreatment (multiple dose) on ethanol-induced changes in hepatic lipid peroxidation, antioxidant profile and protein carbonylation of rats Group

LPOa

SODb

CATc

GPxd

GRe

GSTf

GSHg

PCh

I II III IV V

3.72b 5.31c 4.62b 4.09b 3.30a

2.30a 0.84c 1.77b 2.26a 2.59a

5.17c 3.58a 4.13b 5.03c 5.49d

110.55d 73.94a 93.05b 105.20c 112.88d

290.29c 177.52a 234.47b 289.17c 332.72d

143.50c 121.97a 132.27b 140.72c 155.88d

16.62cd 10.50a 14.03b 16.26c 17.85d

31.16a 41.41b 36.99ab 33.94a 31.28a

Means with different suffix letters differ significantly (p < 0.05). Group I—control; Group II—ethanol (5 g/kg b.w.); Group III—DHA (50 mg/kg b.w.) + ethanol (5 g/kg b.w.); Group IV—DHA (100 mg/kg b.w.) + ethanol (5 g/kg b.w.); Group V—DHA (100 mg/kg b.w.). a LPO—lipid peroxidation (nmol MDA/mg protein). b SOD—superoxide dismutase (U/mg protein). c CAT—catalase (␮mol H O /min/mg protein). 2 2 d GPx—glutathione peroxidase, GR—glutathione reductase (nmol NADPH/min/mg protein). e GST—glutathione-S-transferase (␮mol CDNB conjugate/min/mg protein). f GSH—glutathione (␮g/mg protein). g PC—protein carbonyl (␮mol/mg protein).

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Plate 1. Effects of the aqueous extract of the roots of D. hamiltonii pretreatment (single dose) on ethanol-induced liver damage. H&E staining; magnification, ×400. Group I—control; Group II—DHA (200 mg/kg b.w.); Group III—DHA (50 mg/kg b.w.) + ethanol (5 g/kg b.w.); Group IV—DHA (100 mg/kg b.w.) + ethanol (5 g/kg b.w.); Group V—DHA (200 mg/kg b.w.) + ethanol (5 g/kg b.w.); Group VI—ethanol (5 g/kg b.w.).

3.4. Glutathione Administration of ethanol decreased the hepatic GSH level which was restored to normal level by DHA pretreatment (Tables 1 and 2). Pretreatment of DHA raised the hepatic GSH levels which was significant in multiple dose pretreatment. 3.5. Protein carbonyls Ethanol treatment increased the protein carbonyl content in the rat liver. DHA pretreatment prevented ethanol-

induced protein carbonyl formation in dose dependent manner (Tables 1 and 2). 3.6. Histopathology Histopathological examination of the liver of ethanol administered rats revealed mild degenerative changes such as vacuolization and pycnotic nuclei in the parenchymal cells in the centrilobular areas. Histological picture of the liver of rats pretreated with DHA did not reveal degenerative signs but was comparable to that of control (Plates 1 and 2).

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Plate 2. Effects of the aqueous extract of the roots of D. hamiltonii pretreatment (multiple dose) on ethanol-induced liver damage. H&E staining; magnification, ×400. Group I—control; Group II—DHA (100 mg/kg b.w.); Group III—DHA (50 mg/kg b.w.) + ethanol (5 g/kg b.w.); Group IV—DHA (100 mg/kg b.w.) + ethanol (5 g/kg b.w.); Group V—ethanol (5 g/kg b.w.).

4. Discussion Alcohol-induced oxidative stress in the liver cells plays a major role in the development of alcoholic liver disease. This condition results from several processes related to alcohol metabolism: (a) changes in the NAD/NADH ratio resulting from alcohol breakdown by alcohol dehydrogenase, (b) production of ROS during alcohol metabolism by the microsomal ethanol-oxidizing system, (c) depletion of GSH and (d) decreased activity of antioxidant enzymes [3,23]. Increased ROS production and decreased antioxidant potential, among other harmful effects, causes lipid peroxidation which leads to damage to liver cells.

Lipid peroxidation has been implicated in the pathogenesis of hepatic injury by ethanol and which leads to membrane dysfunction [24]. In the present study, increased malondialdehyde (MDA), a product of lipid peroxidation, observed in the liver of ethanol administered rats indicated excessive formation of free radicals resulting in hepatic damage. The hepatoprotective effect of certain plant extracts against ethanol-induced liver injury possibly involves mechanisms related to free radical scavenging effects [25]. Pretreatment of D. hamiltonii extract prevented lipid peroxidation which could be attributed to its free radical scavenging activity [9]. Ethanol-induced hepatic damage is characterized by the raised levels of serum AST, ALT, ALP and LDH levels which

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reflects the severity of liver injury [26]. In our study, substantial increase in AST, ALT, ALP and LDH in the serum were observed after administration of ethanol. The leakage of the enzymes into the blood stream is attributed to the hepatic damage. However, ethanol-induced increase of these enzymes was considerably reduced by pretreatment with D. hamiltonii aqueous extract, implying that the extract protected the liver against ethanol-induced damage. However, the histopathological damage by a single dose of ethanol appeared less severe as judged by the changes in the liver. It is known that ethanol at sublethal acute doses is not as damaging as, for example, CCl4 [26]. Chronic consumption of ethanol, however, is known to cause liver cirrhosis [23]. Hepatoprotective actions of other plant extracts have also been reported [6,27]. It has been shown that antioxidants or plant extracts having antioxidant activity exhibit hepatoprotective activity [28,29]. Ethanol administration to rats lowered the antioxidant capacity of the rat liver as reflected in the decreased activity of the antioxidant enzymes which is in agreement with earlier reports [30]. Pretreatment with D. hamiltonii extract restored the antioxidant enzyme profile in the liver. Further, multiple dose pretreatment of the D. hamiltonii extract significantly boosted the antioxidant enzyme activities in the liver. Induction of antioxidant enzymes in the liver by plant-derived polyphenols has been reported [31,32]. GSH, the ubiquitous tripeptide, regulates intracellular redox status and directly scavenges free radicals or acts as a substrate for GPx and GST during the detoxification of peroxides and electrophilic compounds. Ethanol administration led to a significant depletion of glutathione level, an important factor in the ethanol toxicity [33,34]. The mechanism of hepatoprotection by D. hamiltonii against ethanol toxicity could be mediated by increased GSH level since multiple dose pretreatment increased hepatic GSH level. Plant-derived antioxidants have been shown to elevate GSH level by acting on ␥-glutamylcysteine synthetase, a key enzyme in the biosynthesis of GSH [35]. It remains to be shown whether the extract of D. hamiltonii induces the enzyme that would explain increased GSH levels. One of the major consequences of oxidative stress is irreversible protein modification such as generation of carbonyls or loss of thiol residues [36]. These oxidative modifications alter the biological properties of proteins leading to their fragmentation, increased aggregation and enzyme dysfunction [37]. Increasing evidence suggests that irreversible oxidative modifications of proteins are important in the pathophysiology of several degenerative diseases [38]. Radical mediated modification of protein thiol groups, specifically cystein residues, can be repaired by cellular antioxidants such as the GSH or thioredoxin [39]. In our study, a significant increase in the protein carbonyl content of the liver was observed in ethanol intoxicated rats which is consistent with earlier reports [40]. Ethanol-induced protein carbonylation in the liver was prevented by pretreatment with D. hamiltonii extract which could be attributed to its antioxidant activity. Recently

we have shown that the aqueous extract and its active principles inhibit oxidative damage to human LDL in vitro [10]. In conclusion, the root extract of D. hamiltonii extract effectively protected against ethanol-induced oxidative damage to the liver. The hepatoprotective activity of the extract could, at least partly, be due to the free radical scavenging property or enhanced antioxidant capacity of the liver. The bioactive antioxidant principles of the aqueous extract that have been identified could be responsible for the observed hepatoprotective effect [10]. This study, therefore, provides a scientific basis for the alleged health promoting potential of D. hamiltonii roots. Further studies with the antioxidant compounds from D. hamiltonii on hepatocytes are underway in our laboratory which will enable us to understand the mechanism of hepatoprotective action. Acknowledgements The authors wish to thank the Director of the institute for his support in this study. The first author acknowledges Council for Scientific and Industrial Research, New Delhi, for awarding the research fellowship. References [1] Cederbaum AI. Introduction—serial review: alcohol, oxidative stress and cell injury. Free Radic Biol Med 2001;31:1524–6. [2] Bondy SC. Ethanol toxicity and oxidative stress. Toxicol Lett 1992;63:231–42. [3] Nordmann R. Alcohol and antioxidant systems. Alcohol Alcohol 1994;5:513–22. [4] Tsukamoto H, Lu SC. Current concepts in the pathogenesis of alcoholic liver injury. FASEB J 2001;15:1335–49. [5] Vitaglione P, Morisco F, Caporaso N, Fogliano V. Dietary antioxidant compounds and liver health. Crit Rev Food Sci Nutr 2004;44:575–86. [6] Rajagopal SK, Manickam P, Periyasamy V, Namasivayam N. Activity of Cassia auriculata leaf extract in rats with alcoholic liver injury. J Nutr Biochem 2003;14:452–8. [7] Naik RS, Mujumdar AM, Ghaskabdi S. Protection of liver cells from ethanol cytotoxicity by curcumin in liver slice culture in vitro. J Ethnopharmacol 2004;95:31–7. [8] Nayar RC, Shetty JKP, Mary Z, Yoganarshimhan SN. Pharmacognostical studies on the root of Decalepis hamiltonii Wt. and Arn. and comparison with Hemidesmus indicus (L.) R.Br. Proc Indian Acad Sci 1978;87:37–48. [9] Srivastava A, Shereen, Harish R, Shivanandappa T. Antioxidant activity of the roots of Decalepis hamiltonii (Wight & Arn.). Lebensm-Wiss Technol, 2006, in press. [10] Srivastava A, Harish R, Shivanandappa T. Novel antioxidant compounds from the aqueous extract of the roots of Decalepis hamiltonii and their inhibitory effect on LDL oxidation. J Agric Food Chem 2006;54:790–5. [11] Harish R, Divakar S, Srivastava A, Shivanandappa T. Isolation of antioxidant compounds from the methanolic extract of the roots of Decalepis hamiltonii (Wight & Arn.). J Agric Food Chem 2005;53:7709–14. [12] Shereen. Mammalian toxicity assessment and nutraceutical properties of the swallow root, Decalepis hamiltonii. Ph.D. Thesis. University of Mysore, Mysore; 2005.

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