Evaluation of hepatoprotective effects of Helminthostachys zeylanica (L.) Hook against carbon tetrachloride-induced liver damage in Wistar rats

Journal of Ethnopharmacology 92 (2004) 61–66

Evaluation of hepatoprotective effects of Helminthostachys zeylanica (L.) Hook against carbon tetrachloride-induced liver damage in Wistar rats S.R. Suja a , P.G. Latha a,∗ , P. Pushpangadan b , S. Rajasekharan a a

Tropical Botanic Garden and Research Institute, Palode, Thiruvananthapuram 695562, India b National Botanical Research Institute, Lucknow 226001, India

Received 22 September 2003; received in revised form 13 November 2003; accepted 26 January 2004

Abstract The rhizomes of Helminthostachys zeylanica (L.) are used by the Kattunaikan tribe of Kerala, for the treatment of various hepatic disorders. In the present study, the effect of the methanolic extract of Helminthostachys zeylanica rhizomes on carbon tetrachloride (CCl4 )-induced liver damage in Wistar rats was studied. The results showed that significant hepatoprotective effect was obtained against CCl4 -induced liver damage, by oral administration of Helminthostachys zeylanica methanolic extract as evident from decreased levels of serum enzymes and an almost normal architecture of the liver, in the treated groups, compared to the controls. The extract was effective in increasing the choleretic activity of anaesthetised normal rats. It also shortened hexobarbitone-induced sleeping time in mice, which was increased by CCl4 treatment, besides showing significant antilipid peroxidant effect in vitro. Thus, the present study provides a scientific rationale for the traditional use of this plant in the management of liver diseases. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Helminthostachys zeylanica; Carbon tetrachloride; Hepatoprotecive activity; Histopathology; Choleretic activity; Hexobarbitone; Lipid peroxidation

1. Introduction In the plant world, pteridophytes are said to be primitive vascular plants. Like other plant groups, pteridophytes also show medicinal utility. Helminthostachys zeylanica (L.) Hook (Ophioglossaceae) is an endangered medicinal pteridophyte, growing in swampy places in the Western Ghats of South India, up to an altitude of 3000 ft. It is locally known as “Pazhutharakali”, due to the resemblance of its rhizome to the feet of the centipede (Pazhuthara—centipede, Kal—feet). The plant possesses an array of medicinal properties. It is used as an aperiant, febrifuge, intoxicant, anodyne and to treat sciatica, boils, ulcers and to cure malaria. The Kattunaikan tribe of Malappuram district, Kerala uses the rhizome of this plant to treat snake bites (Dan and Shanavaskhan, 1991). The aboriginals of Nicobar Islands are known to use the rhizome of this plant for treating impotency (Dagar and Dagar, 1987). It is reported to possess antiviral properties (Pandey and Bhargavan, 1980). The plant is used traditionally for the treatment of jaundice (Prakash, ∗ Corresponding author. Tel.: +91-472-2869226/2869626; fax: +91-472-2869646. E-mail address: [email protected] (P.G. Latha).

1998). We have already reported the aphrodisiac properties of Helminthostachys zeylanica (Suja et al., 2002). Herein, we report the hepatoprotective properties of the rhizome of Helminthostachys zeylanica against CCl4 -induced liver damage in Wistar rats.

2. Materials and methods 2.1. Plant material and preparation of extract Helminthostachys zeylanica plants were collected from Kulathupuzha in Kollam district of Kerala, India and authenticated by Dr Mathew Dan, plant taxonomist of the institute. A voucher specimen (TBGT 41608 dated 27 June 2001) had been deposited at the herbarium of the Institute. The rhizomes were washed thoroughly in tap water, shade-dried and powdered. The powder (100 g) was extracted with 1000 ml methanol overnight, with constant stirring. The extract was filtered and the filtrate, concentrated under reduced pressure to yield 800 mg of the crude extract (0.8%, w/w, with respect to the dried plant material). This crude extract was referred to as HZ. The crude extract was suspended in 0.5% Tween-80, to required concentrations and used for the experiments.

0378-8741/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2004.01.019

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S.R. Suja et al. / Journal of Ethnopharmacology 92 (2004) 61–66

2.2. Animals Wistar albino rats, males (150–200 g) and Swiss albino mice, males (20–25 g) obtained from the Institute animal house of the institute were used for the present study. They were housed under standard conditions and fed commercial rat feed (Lipton India Ltd, Mumbai, India) and boiled water ad libitum. All experiments involving animals were done according to NIH guidelines, after getting the approval of the Institute’s Animal Ethics Committee. 2.3. CCl4 -induced hepatotoxicity studies Rats were divided into five groups (six per group). Group I, the normal control group animals were administered p.o., a single daily dose of 0.5% Tween-80 (1 ml) on all 5 days and olive oil (1 ml/kg) s.c., on days 2 and 3. Group II, the CCl4 control group animals were administered a single daily dose of 0.5% Tween-80 (1 ml) p.o., on all 5 days and on the second and third day, they were administered s.c., CCl4 : olive oil (1:1). Group III animals were administered HZ (100 mg/kg) p.o., on all 5 days and a single dose of CCl4 (2 ml/kg) s.c., on days 2 and 3, 30 min after HZ administration. Group IV animals were administered HZ (200 mg/kg) p.o., on all 5 days and a single dose of CCl4 (2 ml/kg) s.c., on days 2 and 3, 30 min after HZ administration. Group V animals were administered Silymarin, the known hepatoprotective compound (Sigma Chemical Company, USA), at a dose of 100 mg/kg p.o., on all 5 days and a single dose of CCl4 (2 ml/kg) s.c., on days 2 and 3, 30 min after Silymarin administration. On the fifth day, all the animals were sacrificed by mild ether anaesthesia. Blood samples were collected for evaluating the biochemical parameters and liver tissue samples were collected for histological studies. 2.4. Biochemical estimations Biochemical parameters like serum enzymes: serum aspartate aminotransferase (AST), serum alanine aminotransferase (ALT), serum alkaline phosphatase (SAKP) and serum bilirubin (SB) were assayed according to standard methods (Malloy and Evelyn, 1937; Reitman and Frankel, 1957; King and Armstrong, 1980). 2.5. Histopathological studies Seven micrometre thick paraffin sections of buffered formalin-fixed liver samples were stained with haematoxylin–eosin for photomicroscopic observations of the liver histological architecture of the control and treated rats.

50 ␮l/kg, p.o.) or vehicle (olive oil) was given to Groups I and II, whereas Group III and Group IV animals received CCl4 (50 ␮l/kg,) p.o., and also the plant extract, HZ (100 and 200 mg/kg, p.o.), 1 h after CCl4 administration. All the four groups of animals were given hexobarbitone (60 mg/kg, i.p.) 2 h after CCl4 /vehicle treatment. The time between loss of righting reflex and its recovery was recorded as previously reported (Puyvelde et al., 1989). 2.7. Choleretic activity Overnight fasted rats (three per group, males, 400–500 g) were anaesthetised by i.p. injection of pentobarbitone (30 mg/kg), the common bile duct was surgically exposed by middle line laprotomy and cannulated with polyethylene tubing. Bile collected for the first 10 min was discarded and then it was collected in graduated tubes. The plant extract HZ (100 and 200 mg/kg) was given intraduodenally after 1 h bile collection and then the bile was collected hourly, for 4 h. 2.8. Assessment of lipid peroxidation The antilipid peroxidant effect of HZ was studied in vitro, following the modified method of Yoshiyuki et al. (1981) and Masao et al. (1993). Protein content was determined by the method of Lowry et al. (1951). Briefly, 2 g of rat liver tissue was sliced and homogenised with 150 mM KCl–Tris–HCl buffer (pH 7.2). The reaction mixture was composed of 0.25 ml liver homogenate, Tris–HCl buffer (pH 7.2), 0.1 mM ascorbic acid (AA), 4 mM FeCl2 and 0.05 ml of various concentrations of HZ extract. The mixture was incubated at 37 ◦ C for 1 h in capped tubes. Then, 0.1 N HCl, sodium dodecyl sulphate (SDS: 9.8%), 0.9 ml distilled water and 2 ml of thiobarbituric acid (TBA: 0.6%) were added to each tube and vigorously shaken. The tubes were placed in a boiling water bath at 100 ◦ C for 30 min. After cooling, 5 ml of butanol was added and centrifuged at 3000 rpm for 25 min. The absorbance of the supernatant was measured at 532 nm. The experiment was repeated twice. 2.9. Behavioural and toxic effects Three groups of 10 mice were administered p.o., 100, 200 and 400 mg/kg of the HZ extract. They were observed continuously for 1 h for any gross behavioural changes, symptoms of toxicity and mortality if any and intermittently for the next 6 h and then again, 24 h after dosing with HZ extract.

2.6. Hexobarbitone-induced sleeping time studies 2.10. Statistical analysis Four groups of Swiss albino mice were used for this study (six per group). Food was withdrawn on the preceding night of the experiment. Carbon tetrachloride (CCl4 :

This was carried out using the Student’s t-test (Snedecor and Cochran, 1980).

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Table 1 Effect of Helminthostachys zeylanica (HZ) rhizome methanolic extract on rat serum enzymes after CCl4 administrationa Groups

AST (IU/l)

Normal control CCl4 control CCl4 + HZ (100 mg/kg) CCl4 + HZ (200 mg/kg) CCl4 + HZ (300 mg/kg) CCl4 + Silymarin (100 mg/kg)

85.6 255.2 200.1 90.0 91.0 89.3

a

± ± ± ± ± ±

ALT (IU/l)

10.30 18.50 12.00 9.23∗∗ 8.21∗∗ 10.20∗∗

34.2 180.6 120.3 41.2 40.3 39.3

± ± ± ± ± ±

SAKP (KA units/100 ml)

4.30 10.20 9.80 7.91∗∗ 6.26∗∗ 9.70∗∗

49.3 95.3 60.3 55.2 53.1 52.3

± ± ± ± ± ±

SB (IU/l)

2.71 10.31 6.30 7.26∗∗ 6.21∗∗ 4.72∗∗

0.25 5.00 3.50 1.70 1.68 1.30

± ± ± ± ± ±

0.01 1.50 1.30 1.20∗∗ 0.99∗∗ 0.30∗∗

Values are the mean ± S.D., n = 6. Significance P ≤ 0.001, compared to CCl4 control.

∗∗

Table 2 Effect of Helminthostachys zeylanica methanolic extract (HZ) on hexobarbitone-induced sleeping time in CCl4 -intoxicated micea

Table 4 Inhibitory effect of HZ extract on FeCl2 –ascorbic acid-induced lipid peroxidation in rat liver homogenate in vitroa

Groups

Sleeping time (min)

Groups

Normal control CCl4 control CCl4 + HZ (100 mg/kg) CCl4 + HZ (200 mg/kg)

19.0 100.1 50.6 51.3

HZ concentration (␮g/ml)

MDA (n mol/mg protein)

Normal control FeCl2 –AA control FeCl2 –AA + HZ FeCl2 –AA + HZ FeCl2 –AA + HZ

– – 1 5 10

1.35 2.31 2.07 1.52 1.30

a

± ± ± ±

2.10 3.70 4.76∗∗ 3.68∗∗

Hepatoprotective activity (%) – – 49.4 48.7

Values are the mean ± S.D., n = 6. Significance P < 0.01, compared to CCl4 control.

∗∗

a

± ± ± ± ±

0.60 0.02 0.03 0.07∗∗ 0.01∗∗

MDA inhibition (%) – – 10.38 34.20 43–0.72

Values are the mean ± S.D., n = 3. Significance P ≤ 0.01, compared to FeCl2 –AA group.

∗∗

3. Results Administration of CCl4 to rats caused significant increase in serum enzymes like AST, ALT, SAKP and SB, compared to normal control rats. Treatment with HZ caused significant reduction of these values (Table 1), dose-dependently, almost comparable to the Silymarin treated group. Histological observations basically support the results obtained from serum enzyme assays. The liver of CCl4 -intoxicated rats showed massive fatty changes, gross necrosis, broad infiltration of lymphocytes and Kupffer cells around the central vein and loss of cellular boundaries. The histological pattern of liver of rats pretreated with HZ and subsequently given CCl4 showed a more or less normal lobular pattern with a mild degree of fatty change, necrosis and lymphocyte infiltration almost comparable to the normal control and Silymarin groups (Figs. 1–4). The time of loss of righting reflex induced by 60 mg/kg of hexobarbitone was prolonged significantly by CCl4 ad-

ministration. The administration of a single dose of HZ (100 mg/kg) significantly shortened the “sleeping time”, compared to CCl4 group (Table 2) indicating its hepatoprotective potential. HZ treatment resulted in a significant stimulation of bile output in normal rats indicating the choleretic activity of HZ, dose-dependently at the doses, 100 and 200 mg/kg. The highest dose used did not enhance bile output any further (Table 3). HZ showed very potent inhibition of FeCl2 –AA stimulated rat liver lipid peroxidation in vitro. There was a significant increase of malondialdehyde (MDA) in FeCl2 –AA treated rat liver homogenate, compared to normal control without FeCl2 –AA (Table 4). In the toxicity study, no mortality occurred within 24 h with the three doses of HZ tested. The LD50 , was therefore, greater than 400 mg/kg p.o., in mice (data not shown).

Table 3 Effect of Helminthostachys zeylanica rhizome methanolic extract (HZ) on bile flow in normal anaesthetised ratsa Bile flow (ml/100 g)

Control

HZ (100 mg/kg)

HZ (200 mg/kg)

1 h before HZ treatment (A) 2–6 h after HZ treatment (B) B/A

0.31 ± 0.01 0.83 ± 0.05 2.7

0.30 ± 0.02 1.80 ± 0.02∗∗ 6.0

0.33 ± 0.01 1.96 ± 0.02∗∗ 6.0

a

Values are the mean ± S.D., n = 3. Significance P ≤ 0.01, compared to control (0.5% Tween-80).

∗∗

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Fig. 1. Section of liver of normal control rat showing hepatic cells with nuclei, cytoplasm, central vein and portal triad (×350). Fig. 2. Section of carbon tetrachloride-treated (CCl4 control group) rat liver, showing marked necrosis, severe fatty degeneration and extensive vacoulisation with disappearance of nuclei (×350). Fig. 3. Section of HZ (200 mg/kg) + CCl4 -treated rat liver, showing marked improvement over CCl4 control group (×350). Fig. 4. Section of Silymarin (100 mg/kg) + CCl4 showing normalcy of hepatic cells, central vein and portal triad (×350).

4. Discussion Preventive action of liver damage by CCl4 has been widely used as an indicator of liver protective activity of drugs in general (Clauson, 1989). Since the changes associated with CCl4 -induced liver damage are similar to that of acute viral hepatitis (Rubinstein, 1962), CCl4 -mediated hepatotoxicity was chosen as the experimental model. It has been established that CCl4 is accumulated in hepatic parenchyma cells and metabolically activated by cytochrome P450 -dependent monooxygenases to form a trichloromethyl radical (CCl3 ). The CCl3 radical alkylates cellular proteins and other macromolecules with a simultaneous attack on polyunsaturated fatty acids, in the presence of oxygen, to produce lipid peroxides, leading to liver damage (Bishayee et al., 1995). Thus, antioxidant or free radical generation inhibition is important in protection against CCl4 -induced liver lesions (Castro et al., 1974). AST, ALT, SAKP and SB are the most sensitive tests for diagnosis of liver diseases (Mahendale et al., 1985). Hepatotoxic compounds such as CCl4 are known to cause marked elevation in serum enzymes and bilirubin levels. The present study revealed a significant increase in the activities of AST,

ALT, SAKP and SB within 48 h of exposure to CCl4 , indicating considerable hepatocellular injury. Administration of HZ extract attenuated the increased levels of the serum enzymes, produced by CCl4 , and caused a subsequent recovery towards normalization almost like that of Silymarin treatment as reported by (Morazzoni and Bombardelli, 1995). This suggested the possibility that HZ extract is able to condition the hepatocytes, so as to cause accelerated regeneration of parenchyma cells, thus protecting against membrane fragility and decrease of leakage of the marker enzymes into the circulation. Silymarin is a known hepatoprotective compound. It is reported to have a protective effect on the plasma membrane of hepatocytes (Ramellini and Meldolesi, 1976). In histopathological assessment, it was found that treatment with HZ showed an obvious depression of centrilobular necrosis. Thus, histological architecture of the HZ treated liver samples showed the ability of the HZ to prevent hepatocellular necrosis or mononuclear infiltration as reported by Dhuley and Naik (1997). The Kattunaikan tribal healers administer Helminthostachys zeylanica rhizome to treat jaundice. The dose, according to their jargon is the size of a gooseberry, four times a day, that is approximately 100 g/day, for 5 days. This is

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equivalent to the effective dose used in the present study (200 mg/kg in rat) as per the conversion factor suggested by Dhawan (1984). The present finding that treatment with 200 mg/kg of HZ for 5 days significantly reduced CCl4 -induced damage in rat liver, therefore, justifies the therapeutic dosage of the drug employed by the tribe. HZ caused significant increase in choleretic activity of treated normal rats. This is an indication of the healthy status and strong stimulating action on the secretory activity of the liver, as reported by Shukla et al. (1991). Our results indicate that HZ was able to significantly shorten CCl4 -induced prolongation of hexobarbitone-induced “sleeping time”. The fact that hepatic damage can significantly prolong the loss of righting reflex induced by short acting barbiturates has been well established (Sharma et al., 1989). The amount of hypnotic compound broken down per unit time in liver damaged mice is less, thereby resulting in a prolonged sleeping time (Vogel, 1977). The protective effect exhibited by HZ could be due to the protection of hepatic drug metabolizing enzymes. The hepatic injury caused by CCl4 is associated with damage to the endoplasmic reticulum and any compound capable of preventing the toxicity of CCl4 must have some direct or indirect effect on the liver (Maurice et al., 1987). FeCl2 –ascorbic acid mixture is known to stimulate lipid peroxidation in rat liver in vivo and microsomes and mitochondria in rat liver in vitro (Chun et al., 1998). In the present study, HZ was found to be effective in decreasing MDA production in liver of rats treated with FeCl2 –ascorbic acid mixture, showing its antilipid peroxidant effects. Our results also revealed the toxic effects of HZ in mice. This is not surprising, as it is extensively used in the medical practice of the Kattunaikans of Kerala. Helminthostachys zeylanica is reported to be rich in minerals like calcium, phosphorous, iron, carotene and ascorbic acid (Anonymous, 1959; Dan and Shanavaskhan, 1991), which promote strength and general well being. Carotene and ascorbic acid have the potential of minimizing deleterious effects of free radicals including peroxyradicals (Burton, 1989) and thereby can be ranked as hepatoprotective agents (Bishayee et al., 1995). Perhaps they may be responsible for the observed hepatoprotective effects of HZ. However, which of these compounds is (are) actually responsible for the antihepatotoxic properties is not clearly known and warrants further investigation on their chemical nature. The present findings, therefore, substantiate the traditional claim of the hepatoprotective effect of HZ.

Acknowledgements The authors thank Dr G.M. Nair, the Director of the Institute for facilities, Mr S. Radhakrishna Pillai for technical assistance and Mr S. Suresh Kumar for photography. Financial assistance from the Kerala State Planning Board, Trivandrum is gratefully acknowledged.

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