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Potential hepatoprotective activity of ononitol monohydrate isolated from Cassia Tora L. on carbon tetrachloride induced hepatotoxicity in wistar rats.

Abstract

Ononitol monohydrate, structurally similar to glycoside was isolated from Cassia tora L. leaves. Fifty Male rats were divided into five groups. Group I served as normal control. Group II, III and IV rats were induced hepatotoxicity by [CCI.sub.4] administering single dose of [CCI.sub.4] on 8th day only. Group III was treated with ononitol monohydrate (20 mg/kg body weight) and (group IV) was treated with reference drug silymarin (20 mg/kg body weight) both dissolved in corn oil and administering for 8 days. Ononitol monohydrate with corn oil alone was given for 8 days (group V). At the end of the experimental period all the animals were sacrificed and analyzed for biochemical parameters to assess the effect of ononitol monohydrate treatment in [CCI.sub.4] induced hepatotoxicity. In in vivo study, ononitol monohydrate decreased the levels of serum transaminase, lipid peroxidation and TNF-[alpha] but increased the levels of antioxidant and hepatic glutathione enzyme activities. Compared with reference drug silymarin ononitol monohydrate possessesed high hepatoprotective activity. Histopathological results also suggested the hepatoprotective activity of ononitol monohydrate with no adverse effect. Hence we conclude that ononitol monohydrate is a potent hepatoprotective agent.

[c] 2009 Elsevier GmbH. All rights reserved.

Keywords: Ononitol monohydrate; Cassia tora; Carbon tetrachloride; Hepatoprotective

Introduction

Carbon tetrachloride ([CCI.sub.4]) has proved to be highly useful as an experimental agent for the induction of acute liver injury. Cytokines play a major role in the process of acute liver injury and repair. [CCI.sub.4] produces chemical hepatic injury. It is biotransformed by hepatic microsomal cytochrome p450 to trichloromethyl-free radical ([CCI.sub.3] * or [CCI.sub.3OO] *) (Ashok et al. 2001). Generally these metabolites react with antioxidant enzymes such as catalase and superoxide dismutase (SOD). However, over production of trichloromethyl-free radicals is considered the initial step in a chain of events that eventually lead to membrane lipid peroxidation (Basu 2003). Antioxidant and radical scavengers have been used to study the mechanism of [CCI.sub.4] toxicity as well as to protect liver cells from [CCI.sub.4] induced damage by breaking the chain of lipid peroxidation (Weber et al. 2003).

Cassia tora Linn. (Caesalpiniaceae) is a shrub widely used as traditional medicine in Africa and India for the treatment of ulcers. The fermented leaves are pounded and added to food or local gin and taken orally for anthelmintic and purgative effects (Daiziel 1995). In in vivo model. Cassia tora leaves methanol extract was effective in protecting liver against [CCl.sub.4]-induced liver damage (Choi et al. 2002).

Ononitol monohydrate is a class of glycoside isolated from Cassia tora leaves. Many experiments showed that glycosides have hepatoprotective effect in various experimental models. Glycosides which are antioxidants from natural origin have generated considerable interest as potential therapeutic agents against a wide variety of chronic diseases (Cesquini et al. 2003). The present work was carried out to study the hepatoprotective effect of ononitol monohydrate isolated from C. tora on [CC1.sub.4] induced hepatotoxicity in rats.

Materials and methods

AST, ALP and LDH kits were procured from Futura chemicals (Italy). TNF-[alpha] kit was obtained from Genei (Chennai, India). Silymarin was purchased from Sigma chemicals (Bangalore, India). Silymarin consists of a mixture of 70-80% Silybin (2 diastereomers), Silydianin and Silychristin with a small amount of Dehydrosilybin. The rest contains flavanolignans and non identified phenolics. The composition of Silymarin is equivalent to Legalon [R] (firm Madaus, Germany). All the other chemicals were of highest quality and analytical grade marketed by Himedia Chemicals, Mumbai, India.

Cassia tora leaves were collected from Padappai near Chennai. Collected leaves were shade-dried at room temperature and powdered by an expeller. One kilogram of leaf powder was soaked in sequential solvent extraction of 31 of Hexane, ethyl acetate and methanol in a 51 conical flask for about 48 h with intermittent shaking, and filtered using Buchner funnel with Whatmann No.l paper and then condensed using rotary evaporator at 40 [degrees]C The crude extract was collected in clean borosil vials and kept at 4 [degrees]C.

The crude ethyl acetate extract (25 g) was adsorbed on to a silica gel (Acmae's 60-120 mesh) and subjected to column chromatography over silica gel (100-200 mesh) and eluted with hexane followed by the combination of hexane: ethylacetate ranging from 95:5 to 0:100. The fractions were collected in a 200 ml conical flask. The eluted fractions were combined to give major fractions by comparing the [R.sub.f] values of the collected fractions when run on TLC [F.sub.254] plates with similar solvent systems. The separated components on the silica plate were visualized in UV light and then to Iodine; later after the iodine evaporated the plates were sprayed with Vanillin sulphuric acid and heated for 2 min. Based upon the TLC pattern the fractions were pooled together to get major fractions. Totally 8 fractions were collected; from that fraction 4 formed crystal (2.4 g). The total % yield of ononitol monohydrate was 0.24% from 1 kg of Cassia tora leaves. The crystal was subjected to X-ray crystallography analysis (Brukcr instrument, IIT, Chennai, India) to determine its structure.

Male Albino Wistar strain rats were procured from Tamil Nadu university of Animal and Veterinary Science (Chennai, India) and the animal experiments were performed in accordance with legislation on animal welfare (CPCSEA). They were fed with normal rat chow marketed by Kings Institute, Chennai and were provided with clean drinking water ad libitum. All the rats were allowed to acclimatize for 10 days prior to experimentation.

The animals were randomly divided into five groups containing ten rats in each. Liver toxicity was induced by oral administration of 2 ml/kg [CCl.sub.4] (20%; diluted in corn oil) (Simile et al. 2001). Group 1 served as normal control and received vehicle corn oil on 8th day. Group II, III and IV were induced liver toxicity by [CCl.sub.4] administration by a single dose of [CCl.sub.4] on 8th day only. Group III animals were treated with ononitol monohydrate (20 mg/kg body weight) dissolved in corn oil for 8 days. Silymarin (20 mg/kg body weight) was dissolved in corn oil and then administered to rats in Group IV for 8 days. Ononitol monohydrate with corn oil alone was given to Group V for 8 days. Administration was done orally using the intra gastric tubes. The dose was fixed based on the preliminary studies (data not shown). At the end of the experiment, the rats were fasted overnight and all the animals were sacrificed by administering ketamine (30 mg/kg body weight) intramuscularly. All samples were used to analyze the biochemical parameters and histopathological analysis.

Serum level of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) were estimated according to the manufacturer's kits (Futura chemicals, Italy). The activities of superoxide dismutase (SOD) were assayed by the method of Kakkar et al. (1984). Catalases were determined by the method of Sinha (1972). Ascorbic acid was estimated by the method of Omaye et al. (1979).Glutathione reductase (GR) was measured by the method of Carlberg and Mannervik (1985). Glutathione peroxidase (GPx) was estimated by the method of Rotruck et al. (1973). GST was determined by the method of Moron et al. (1979). The levels of MDA were estimated by the method of Ohkawa et al. (1979). Liver lysates were analyzed for TNF-[degrees] levels with the ELISA kit (Genei., India), according to the manufacturer's instructions. Protein content in all the samples was estimated by the method of Lowry et al using bovine serum albumin as standard (1951). For histopathological examination liver sections were stained with haema-toxylin and eosin using standard protocol and then analyzed by light microscope.

The results were expressed as means [+ or -]SD. Statistical comparisons were made by means of one-way analysis of variance (ANOVA) followed by a Duncan multiple range comparison test (DMRT). Differences were considered significant when the P-values were <0.05.

Results

Fig. 1 shows the structure of ononitol monohydrate and the molecular formula of cystal us [C.sub.7][H.sub.16][O.sub.7]. The hepatoprptective efficacy of the ononitol monohydrate against [CC1 sub 4] induced hapatotoxieity was evaluated in male wistar rats. The levels of AST, ALT and ALP increased significantly (p <0.05) in [CCI sub 4] alone treated animals. In ononitol monohydrate (group III) and silymarin (group IV), there was a significant (p <0.05) decrease in the levels of AST, ALT and LDH (Table 1). The levels of SOD, CAT, ascorbic acid, GR, [GP sub x] and GST decreased significantly (p<0.05) in [CC1 sub 4] alone animals when compared with normal control animals. In rats which received [CCI sub 4] with ononitol monohydrate (group III) and silymarin (group IV), there was a significant (p<0.05) increase in the levels of SOD, CAT and ascorbic acid, GR, GPx and GST in [CC1 sub 4] alone treated animals (group II) which were near to normal (Tables 1 and 2).

[FIGURE 1 OMITTED]
Table 1. Effect of ononitol monohydrate on serum transaminase and
antioxidant enzymes in control and treated animals.

Particulars                   Group I               Group II

AST (IU/L)                    85.82[+ or -]8.59     181.17[+ or -]14.49
                              (a)                   (c)

ALT (IU/L)                    30.25[+ or -]5.8 (a)  78.26[+ or -]7.73
                                                    (d)

ALP (IU/L)                    89.97[+ or -]7.68     166.75[+ or -]13.84
                              (a)                   (d)

SOD (U/mg protein)             9.75[+ or -]0.92     4.03[+ or -]0.45
                              (a), (b)              (d)

Catalase (U/mg protein)       27.37[+ or -]4.65     10.67[+ or -]2.81
                              (d)                   (a)

Ascorbic acid (U/mg protein)   0.79[+ or -]0.05     0.49[+ or -]0.04
                              (a), (b)              (c)

Particulars                   Group III              Group IV

AST (IU/L)                    89.96[+ or -]9.35      104.42[+ or -]11.3
                              (a)                    (b)

ALT (IU/L)                    39.43[+ or -]5.89      47.95[+ or -]6.89
                              (b)                    (c)

ALP (IU/L)                    97.77[+ or -]7.63      109.58[+ or -]8.48
                              (b)                    (c)

SOD (U/mg protein)             8.48[+ or -]0.76      7.34[+ or -]0.71
                               (b), (c)              (b), (c)

Catalase (U/mg protein)       22.34[+ or -]4.33      17.92[+ or -]3.49
                              (b)                    (c)

Ascorbic acid (U/mg protein)   0.77[+ or -]0.07 (b)  0.74[+ or -]0.05
                                                     (b)

Particulars                   Group V

AST (IU/L)                    79.65[+ or -]11.3 (a)

ALT (IU/L)                    29.78[+ or -]5.91 (a)

ALP (IU/L)                    85.38[+ or -]8.42 (a)

SOD (U/mg protein)             9.86[+ or -]0.94 (a)

Catalase (U/mg protein)       28.95[+ or -]3.24 (d)

Ascorbic acid (U/mg protein)   0.85[+ or -]0.08 (a)

Values are expressed as means [+ or -] S.D.
Values not sharing a common superscript differ significantly at p<0.05
(DMRT).
Table 2. Effect of ononitol monohydrate on Glutathione related enzymes
in control and treated animals.

Particulars      Group I              Group II          Group III

GR (nmol/min mg  9.40[+ or -]0.33     6.60[+ or -]0.28  9.05[+ or -]
                                                        0.34

protein)         (c)                  (a)               (b)
GPx ([mu]mol/mg  5.89[+ or -]0.20(c)  4.30[+ or -]0.16  5.46[+ or -]
                                                        0.21

protein)                              (a)               (b)
GST (U/mg        0.97[+ or -]0.18     1.35[+ or -]0.18  1.03[+ or -]
                                                        0.16

protein)         (a)                  (c)               (b)

Particulars      Group IV          Group V

GR (nmol/min mg  8.78[+ or -]0.28  9.47[+ or -]0.30
protein)         (b)               (c)

GPx ([mu]mol/mg  5.35[+ or -]0.17  5.92[+ or -]0.20
protein)         (b)               (c)

GST (U/mg        1.13[+ or -]0.14  0.93[+ or -]0.14
protein)         (b)               (a)

Values are expressed as means [+ or -] S.D.
Values not sharing a common superscript differ significantly at p<0.05
(DMRT).


Elevated levels of MDA was found in [CCI sub 4] alone rats when compared to normal control animals (p < 0.05). Treatment with ononitol monohydrate and silymarin decreased the levels significantly (p < 0.05). The levels of TNF-[alpha] were significantly increased in [CCI sub 4] treated animals. In the case of treated groups (group III & IV) the levels were significantly decreased when compared with group II animals (Table 3).
TABLE 3
Table 3. Effect of ononitol monohydrate on lipid peroxidation (MDA) and
TNF-[alpha] levels in control and treated animals.

Particulars                    Group I             Group II

MDA (nmol/100g tissue)  0.79[+ or -]0.09 (a)   2.06[+ or -]0.18 (d)
TNF-[alpha] (pg/ml)     119.5[+ or -]8.45(a)   270.5[+ or -]15.23 (d)

Particulars             Group III              Group IV

MDA (nmol/100g tissue)   0.98[+ or -]0.09 (b)    1.25[+ or -]0.11 (c)
TNF-[alpha] (pg/ml)     174.2[+ or -]12.3 (b)  195.12[+ or -]13.47 (c)

Particulars             Group V

MDA (nmol/100g tissue)   0.74[+ or -]0.08 (a)
TNF-[alpha] (pg/ml)     114.8[+ or -]9.12 (a)

Values are expressed as means [+ or -] S.D.
Values not sharing a common superscript differ significantly at p<0.05
(DMRT).


M. Dhanasekaran ft al. / Phytomedicine 16 (2009) 891 895

Histopathological analysis of group I (Fig. 2a) animals showed normal architecture. In rats treated with [CCl.sub.4] alone the normal architecture of liver was completely lost with the appearance of centrilobular necrosis; scattered masses of necrotic tissues were detected in most of the areas (Fig. 2b); enlarged nuclei were also observed in [CC1.sub.4] alone groups. In both ononitol monohydrate (Fig. 2c) and silymarin (Fig. 2d) treated animals (group III & IV) the hepatocytes showed near normal architecture with uniform sinusoids. Rats treated with ononitol monohydrate alone (Fig. 2e) showed normal architecture of hepatocytes (group V).

[FIGURE 2 OMITTED]

Discussion

In the present study, we isolated an active compound ononitol monohydrate from Cassia tora leaves which was tested against the [CC1.sub.4] induced hepatotoxicity. The hepatotoxicity of [CCl.sub.4] has been reported to be due to the formation of the highly reactive trichioro free radical, which attacks polyunsaturated fatty acids. It produces hepatotoxicity by altering liver microsomal membranes in experimental animals. The extent of hepatic damage is normally assessed by histopathological evaluation and the levels of cytoplasmic enzymes ALT, AST and ALP in circulation (Plaa and Charbon-neau 1994). ALP, AST and ALP are reliable markers for liver function. The increased levels of serum enzyme such as AST and ALT have been observed in [CC1.sub.4] administered rats which indicated increased permeability, damage and necrosis of hepatocytes. ALP is membrane bound enzyme which is excreted by liver via bile and hence when liver is affected, the serum enzyme level increases due to defective excretion (Nemesanszky 1996). Elevated levels of the enzyme indicated severe damage to tissue membrane during [CCl.sub.4] toxicity. Ononitol monohydrate and silymarin protected the liver thereby decreasing the levels of ALT, AST and ALP.

The antioxidant activity and the inhibition of free radical generation are important in terms of protecting the liver from [CCl.sub.4]-induced damage. Generally antioxidant enzymes such as catalase, SOD and ascorbic acid are easily inactivated by lipid peroxides or reactive oxygen species, which results in decreased activities of these enzymes in [CC1.sub.4] toxicity. SOD is an extremely effective antioxidant enzyme and is responsible for catalytic dismutation of highly reactive and potentially toxic superoxide radicals to H202 (Reiter et al. 2000). Catalase is a major antioxidant enzyme with hematin as the prosthetic group and it is ubiquitously present in all aerobic cells containing a cytochrome system. It is most abundant in the liver and is responsible for the catalytic decomposition of H202 to oxygen and water (Baudrimont el al. 1997: Reiter et al. 2000). Ononitol monohydrate significantly increased the levels of catalase, SOD and ascorbic acid in treated rats when compared with [CC1.sub.4] alone treated rats. Ononitol monohydrate exhibits preventive effect in [CCl.sub.4] induced hepatotoxicity by reducing the levels of oxidative injuries on hepatocytes.

GR and GPx are important markers in protecting the liver from damage of lipid peroxidation. GR and GPx are glutathione related enzymes, which can catalyze the synthesis of GST to ease the burden of lipid peroxidation. When trichloromethyl appeared as a highly reactive metabolite of [CC1.sub.4], GST, a phase II enzyme, would immediately increase in high level in blood, promoting the combination of free radicals and cell proteins (Sugiyama et al. 2006). Ononitol monohydrate treated animals increased the hepatic glutathione related enzymes which protected the liver against hepatotoxicity.

Measurement of MDA levels is the most commonly used method for lipid peroxidation (Kalender et al. 2005). MDA levels with antioxidant capacity have been important biochemical components to detect tissue damage after [CC1.sub.4] toxicity in liver tissue. Ononitol monohydrate treatment decreased these accumulations and prevented the inflammatory reactions in liver. Tumor necrosis factor-[alpha] (TNF-([alpha]) is an important cytokine in the development of various liver diseases. TNF-[alpha] recruits inflammatory cells and triggers the production of other cytokines which inhibit the healing process leading to hepatocytes damage. The proinflammatory cytokines induced by TNF-[alpha] were thought to be responsible for the pathogenesis of liver diseases (Youssef and McCullough 2002). Our present study indicates that the decreased level of TNF-[alpha] is due to the treatment with ononitol monohydrate which stimulates immune response against cytokines which protects liver.

The above results, the serum transaminase levels, level of lipid peroxidation, antioxidants and hepatic glutathione related enzymes support the highly potent hepatoprotectivc and antioxidant activity of ononitol monohydrate. Histopathological results also suggest that ononitol monohydrate has hepatoprotective activity with no adverse effects. Level of TNF-[alpha] also indicated that ononitol monohydrate has potential immunostimulatory effect which protects the liver. From this we conclude that ononitol monohydrate is a potent hepatoprotective agent.

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Muniyappan Dhanasekaran (a), Savarimuthu Ignacimuthu (a) *, Paul Agastian (b)

(a) Division of Ethnoph.armaeology, Entomology Research Institute, Loyola College, Chennai 600 034, Tamil Nadu, India

(b) Department of Plant Biology and Biotechnology, Loyola College, Chennai 600 034, Tamil Nadu, India

* Corresponding author. Tel.: +91 4428178348; fax: -91442817 4644.

E-mail address: entolc@hotmail.com (S. Ignacimuthu).

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doi:10.1016/j.phymed.2009.02.006
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Author:Dhanasekaran, Muniyappan; Ignacimuthu, Savarimuthu; Agastian, Paul
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
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Date:Sep 1, 2009
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