Hepatocytes are protected by herb Phyllanthus ... - Pathophysiology

Pathophysiology 14 (2007) 113–120

Hepatocytes are protected by herb Phyllanthus niruri protein isolate against thioacetamide toxicity Mrinal K. Sarkar, Parames C. Sil ∗ Department of Chemistry, Bose Institute, 93/1, Acharya Prafulla Chandra Road, Kolkata 700009, West Bengal, India Received 26 June 2007; accepted 23 August 2007

Abstract The herb, Phyllanthus niruri has been known to possess protective activity against various drugs and toxins induced hepatic disorders. Present study was conducted to evaluate the role of the protein isolate of the herb against thioacetamide (TAA)-induced cytotoxicity in mice hepatocytes. In vitro cell viability, lactate dehydrogenase (LDH) and alanine amino transferase (ALT) leakage were measured as the indicators of cell damage. In addition, measurement of the level of non-protein thiol, glutathione (GSH); activities of the antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT) and glutathione S-transferase (GST) as well as the extent of lipid peroxidation were carried out to evaluate the prooxidant-antioxidant status of the cell. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay was performed to determine the radical scavenging activity of the protein isolate. Results showed that the administration of the protein isolate prior to TAA exposure significantly reduced the release of LDH and ALT leakage and enhanced the cell viability in a dose-dependent manner in hepatocytes. Besides, the protein isolate appeared to prevent the alterations in GSH levels and activities of the anti-oxidant enzymes related to prooxidant-antioxidant status of hepatocytes. It also reduced the TAA-induced lipid peroxidation significantly as demonstrated by the reduction of malondialdehyde (MDA) production. DPPH radical scavenging assay showed that the protein isolate possessed radical scavenging activity. Combining, the data suggest that the protein isolate could protect hepatocytes from TAA-induced cellular injury probably by its antioxidative and radical scavenging properties. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Hepatocyte; Thioacetamide; Cytotoxicity; Phyllanthus niruri; Protein isolate; Hepatoprotection

1. Introduction Thioacetamide (TAA) is an experimental hepatotoxin [1] which is a thiono-sulfur-containing compound endowed with liver-damaging and carcinogenic activity. TAA undergoes metabolism to acetamide and thioacetamide-S-oxide by the mixed function oxidase system [2]. Studies have Abbreviations: ALT, alanine aminotranferase; CAT, catalase; DMEM, Dulbecco’s modified Eagle’s medium; DTNB, 5,5 dithiobis-2-nitrobenzoic acid; GSH, glutathione; GST, glutathione S-transferase; LDH, lactate dehydrogenase; MDA, malonaldehyde; ROS, reactive oxygen species; TAA, thioacetamide; TCA, trichloroacetic acid. ∗ Corresponding author. Tel.: +91 33 23506619/2402/3x412/3; fax: +91 33 2350 6790. E-mail addresses: [email protected], parames [email protected] (P.C. Sil). 0928-4680/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.pathophys.2007.08.001

shown that this compound is bioactivated by CYP450 and/or flavin-containing monooxygenase (FMO) systems to sulfine (sulfoxide) and sulfene (sulfone) metabolites, which cause centrilobular necrosis [3]. Studies suggest that thioacetamide sulfoxide, a reactive oxygen species (ROS), is obligatory for the hepatotoxic effects of this compound, indicating that it is the penultimate reactive metabolite [4]. The ROS produced can cause a variety of pathophysiological conditions by enhancing lipid peroxidation in biomembranes. Subsequently it can cause structural and functional degeneration of different enzymes and DNA inside the cell and might lead to death of the cell. Although several free radical scavengers and antioxidants have been used to protect liver against drug and toxin-induced oxidative stress and hepatotoxicity [1,5–7], in most cases, the exact mechanisms of their action are not clearly known and some have been found

114

M.K. Sarkar, P.C. Sil / Pathophysiology 14 (2007) 113–120

to reduce cell viability during hepatoprotection. Alternatively, scientists are in search of the herbal plants possessing hepatoprotective action with very little or no side effect. Cajanus indicus [8,9], Phyllanthus species [10–12], Silybum marianum [13–15], etc., are some of the well-known herbs belonging to that particular class. The herb, Phyllanthus niruri is in use in herbal medicine systems and in clinical research over the years. The plant has been demonstrated as liver protective [16,17], anti-lithic [18,19], pain-relieving [20], anti-hypotensive, anti-spasmodic [21], anti-viral [22], anti-parasitic [23] anti-diuretic, anti-mutagenic [24], etc. In all studies, the active components of the herb are mainly presented as alkaloids obtained from organic extracts of different compositions. In our laboratory, we were eager to know the role of the aqueous extract of the herb in hepatic disorders. We found that the water-soluble fraction of the herb possesses potent radical scavenging as well as hepatoprotective activities [25–27]. We also found that administration routes of the aqueous extract made significant difference in the hepatoprotective activity of the herb. Intraperitoneal administration of the aqueous extract was more effective than the oral administration [28]. Based on these results we started working on the protein isolate of the herb and found that it also possessed strong protective activity against a number of toxins and drugs both in vivo and in vitro [29–32]. In the present study, we further extended our work and designed various experiments to understand the role of the protein isolate of the herb against TAA-induced cytotoxicity in mice hepatocytes. The objective of the present study was, therefore, to investigate the in vitro protective ability of the protein isolate against TAA-induced cytotoxicity in freshly isolated hepatocytes from normal Swiss albino mice. The cellular damage was assessed by determining the cell viability and the leakage of ALT and LDH in hepatocytes. The prooxidantantioxidant status of the cells was assessed by determining the level of GSH and extent of lipid peroxidation as well as the activities of the anti-oxidant enzymes, SOD, CAT and GST. The results have been compared with a positive control, vitamin C, in which hepatocytes were pre-incubated with it instead of the protein isolate and then exposed to TAA.

2. Materials and methods 2.1. Animals and chemicals Male Swiss albino mice of body weight 20 ± 2 g were acclimatized in the laboratory for a fortnight before starting experiment. They were fed ad libitum with food and water. The mice were fasted for 16–18 h before sacrifice. TAA was obtained from Sisco Research Laboratory, India. Collagenase IV and Dulbecco’s modified Eagle’s medium (DMEM) were obtained from Sigma Chemical Co., St. Louis, USA. ALT and LDH measurement kits were purchased from

Span Diagnostic India. All chemicals were reagent grade of the highest laboratory purity. 2.2. Preparation of the protein isolate The leaves from P. niruri were homogenized in 50 mM phosphate buffer, pH 7.4 and the soup was brought to 60% ammonium sulphate saturation. The pellet was reconstituted and dialysed against 50 mM phosphate buffer, passed through DEAE cellulose column and eluted in linear gradient of 0–1 M NaCl in the same phosphate buffer. Two major peaks were observed. The protein fractions from first peak were collected and concentrated and dialyzed in 50 mM phosphate buffer. Biological activity of this fraction was checked and the protein of the active fraction was used for experiments. 2.3. Hepatocyte isolation Hepatocytes were isolated from mice liver according to the method of Sarkar and Sil [33]. The livers were isolated under aseptic condition and placed in phosphate buffer saline. The livers were irrigated in buffer A (10 mM HEPES, 3 mM KCl, 130 mM NaCl, 1 mM NaH2 PO4 –H2 O and 10 mM glucose, pH 7.4) and then incubated with buffer B (5 mM CaCl2 , 0.03% collagenase type IV) for about 45 min at 37 ◦ C. The liver was then passed through wide bore syringe and 80 ␮m decron mesh, respectively. The dissociated cells were centrifuged at 500 × g and the pellet was suspended in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal calf serum and 5 ␮g/ml insulin, 5 ␮M hydrocortisone, 100 U/ml penicillin and 100 ␮g/ml streptomycin. The suspension was adjusted to obtain ∼2 × 106 cells/ml. 2.4. In vitro experiments About 1 ml of hepatocyte suspension (∼2 × 106 cells, ∼90% viability) was incubated with the protein in varying concentrations (10, 25, 50 and 100 ␮g/ml) for different sets of experiment for 10 min. TAA (2 mM) was then added and incubated for another 60 min. The normal control cells were incubated in the medium only. The toxin control was done by incubating the hepatocytes with TAA (2 mM) for 60 min. For positive control, hepatocytes were incubated with vitamin C (100 mg/ml) and then exposed to TAA as mentioned above. For all the experiments, incubation was executed at 37 ◦ C with gentle shaking. 2.5. Measurement of cytotoxicity 2.5.1. Cell viability assay Cell viability was evaluated by trypan blue dye exclusion test [34]. Aliquot of cell suspension (2 ml) was combined with 0.08% trypan blue (2 ml) for 3 min. Then, 400 ␮l of the mixture were counted for cells using a hemocytometer. Cell


viability was defined by the following formula: cells excluding trypan blue Cell viability = × 100. total cells 2.5.2. Cell leakage Hepatocyte suspensions were centrifuged at 60 × g. The leakage of the enzymes, ALT and LDH secreted outside the cells were determined from the supernatant following the manufacturer’s protocol [35,36]. 2.6. Determination of the parameters related to oxidative stress

115

2.7. Determination of radical scavenging activity 2.7.1. Determination of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity in vitro To determine the radical scavenging activity of the protein isolate, different concentrations of the protein isolate were incubated with DPPH (2 ml 100 ␮M in ethanol) and kept at room temperature for 30 min [43]. The decrease of absorbance of DPPH was measured at 517 nm. DPPH in ethanol was used as control and percentage inhibition was calculated by comparing the absorbance values of the control and the protein isolate. 2.8. Protein estimation

The assay of lipid peroxidation was performed by a colorimetric reaction using thiobarbituric acid (TBA) [37]. Hepatocytes containing about 1 mg protein were mixed with TCA–TBA mixture and heated at 100 ◦ C for 30 min. The flocculent precipitate was removed by centrifugation and the absorbance of thiobarbituric acid reactive substance (TBARS) formed was measured at 532 nm. TBARS concentration of the samples was calculated using the extinction coefficient of MDA which is 1.56 × 10−5 mmol−1 cm−1 . GSH concentration in hepatocytes was determined by the method of Tietze [38]. Hepatocytes were deproteinated with trichloroacetic acid (TCA) by centrifugation and GSH released in the supernatant were derivatized with 5,5 dithiobis-2-nitrobenzoic acid (DTNB). The development of colour was measured at 412 nm. 2.6.1. Determination of antioxidant enzyme activity CAT activity was measured in sonicated hepatocytes by the method of Bonaventura et al. [39]. For the assay, the sonicated hepatocytes containing 5 ␮g total protein was mixed separately with 700 ␮l, 5 mM hydrogen peroxide and incubated at 25 ◦ C. The disappearance of peroxide was observed at 240 nm for 15 min. One unit of CAT activity is that which reduces 1 ␮mol of hydrogen peroxide per minute. SOD was determined following the method originally developed by Nishikimi et al. [40] and later modified by Kakkar et al. [41]. Briefly, to 1.2 ml sodium pyrophosphate buffer (pH 8.3, 0.052 M), 0.1 ml 186 ␮M phenazine methosulphate, 0.3 ml 300 ␮M NBT, 0.2 ml NADH (780 ␮M), 1.1 ml water and hepatocytes (containing 2 ␮g protein) was added and incubated exactly for 90 s at 30 ◦ C. The reaction was started by adding NADH. After 90 s the reaction was stopped by the addition of 1 ml of glacial acetic acid and chromogen was read at 560 nm. A control set was carried out simultaneously containing all the components except hepatocytes. GST activity was measured by the method of Habig et al. [42]. Briefly the sonicated hepatocytes containing 5 ␮g total protein was mixed with 400 ␮l EDTA, 200 ␮l GSH, 100 ␮l CDNB, 1000 ␮l phosphate buffer and 500 ␮l double distilled water. The appearance of the conjugated compound was observed at 340 nm for 15 min.

Protein concentration was estimated according to the method of Bradford [44]. Bovine serum albumin (BSA) was taken as standard protein. 2.9. Statistical analysis All the experiments were done in triplicates under the same conditions. The values are represented as mean ± S.D. Differences between the groups were analyzed by one-way ANOVA, using the Epistat package. P values of 0.05 or less were considered significant.

3. Results 3.1. Effect of the protein isolate on the TAA-induced cytotoxicity 3.1.1. Cell viability Fig. 1(a) shows that 2 mM TAA treatment for 60 min caused a loss of about 50% of the cell viability. When hepatocytes were treated with the protein just before the addition of TAA, a significant increase in cell viability was observed in a dose-dependent manner. About 97% cell viability was restored at a protein concentration of 100 ␮g/ml for 60 min incubation. When vitamin C was used instead of the protein isolate, the cell viability was restored almost completely. 3.1.2. Cellular leakage Fig. 1(b) demonstrates the ALT leakage from hepatocytes treated with TAA and the protein isolate. ALT leakage was elevated to almost 170% with respect to normal hepatocytes upon TAA treatment indicating severe cellular damage. Protein treatment before TAA intoxication significantly inhibited the damages as evident from less ALT leakage. ALT leakage was decreased linearly with increase in protein concentration and at the concentration of 100 ␮g/ml, the level was reduced to almost normal. Vitamin C administration also showed similar result. The LDH leakage obtained upon similar treatment also showed that severe cytotoxicity has been caused by TAA

116


Control TAA P1+TAA P2+TAA P3+TAA P4+TAA Vitamin C+TAA

(a) 100


a

180

(b)

b

160

75

ALT(% over control)

Cell viability (% over control)

b

b

a 50

25

b

140

b

120

b

100 80 60 40

0

LDH (% over control)

(c) 160


a

b 120

b

b

80

40

Fig. 1. (a). Effect of the protein isolate on cell viability in isolated hepatocytes treated with TAA as well as (b) on ALT and (c) LDH leakages. Results have been given as percentage of control. Control: hepatocytes were treated with DMEM, TAA: hepatocytes exposed to TAA (2 mM) for 60 min. P1 + TAA, P2 + TAA, P3 + TAA and P4 + TAA: hepatocytes incubated with protein isolate (10, 25, 50 and 100 ␮g/ml) prior to TAA administration for 60 min, vitamin C + TAA: hepatocytes first incubated with vitamin C and then with TAA. Each value represents mean ± S.D. (Pa < 0.05; Pb < 0.05).

(150%) in hepatocytes (Fig. 1(c)). Protein treatment effectively inhibited the membrane disruption caused by TAA as revealed from the less LDH level outside the cells. The significant decrease in LDH level outside the cell was observed at a protein concentration 25 ␮g/ml and almost 100% recovery in the leakage takes place at a concentration 100 ␮g/ml. 3.2. Effect of the protein isolate on the TAA-induced lipid peroxidation MDA level indicates the extent of lipid peroxidation. Fig. 2 shows the TAA-induced lipid peroxidation, an indicator of membrane damage. TAA administration (2 mM) increased the lipid peroxidation to 160% with respect to the normal cells. Protein treatment prior to TAA administration caused

inhibition in the lipid peroxidation in a linear fashion. Significant membrane damage recovery has been observed at a protein concentration of 25 ␮g/ml and at a concentration 100 ␮g/ml, the damage was recovered almost to its normal level. 3.3. Effect of the protein isolate on the TAA-induced oxidative stress Fig. 3 demonstrates that TAA treatment decreased GSH level to about 40%. Protein treatment prior to TAA administration increased the GSH level in a dose-dependent manner. GSH level was significantly increased at a concentration of 25 ␮g/ml protein. About 100% GSH level restoration was taken place at the protein concentration of 100 ␮g/ml.



a 160

MDA (% over control)

b b 120

b

117

effect of the protein isolate on SOD activity and GST activities, respectively. The results are almost similar to CAT activity. 3.5. In vitro free radical scavenging activity of the protein Fig. 5 shows the free radical scavenging activity of the protein. The plot shows that with increase in concentration of the protein from 20 to 160 ␮g/ml, the colour of the DPPH radical vanishes more rapidly at 517 nm indicating strong radical scavenging activity of the protein.

80

40

Fig. 2. Effect of the protein isolate on MDA levels in hepatocytes treated with TAA. Results have been given as percentage over control. Control: hepatocytes treated with DMEM, TAA: hepatocytes exposed to TAA (2 mM) for 60 min. P1 + TAA, P2 + TAA, P3 + TAA and P4 + TAA: hepatocytes incubated with protein isolate (10, 25, 50 and 100 ␮g/ml) prior to TAA administration for 60 min, vitamin C + TAA: hepatocytes first incubated with vitamin C and then with TAA. Each value represents mean ± S.D. (Pa < 0.05; Pb < 0.05).

3.4. Effect of the protein isolate on the TAA-induced antioxidant enzyme activity Fig. 4(a) shows that TAA treatment decreased the CAT activity by about 40%. Significant increase in CAT activity was observed at a protein concentration 25 ␮g/ml and at the protein concentration 100 ␮g/ml the CAT activity was reached to almost normal. Fig. 4(b and c) demonstrate the Control TAA P1+TAA P2+TAA P3+TAA P4+TAA Vitamin C+TAA

100

b

b

GSH (% over control)

b 75 a

50

25

Fig. 3. Effect of the protein isolate on GSH levels in hepatocytes. Results have been given as percentage of control. Control: hepatocytes treated with DMEM, TAA: hepatocytes exposed to TAA (2 mM) for 60 min. P1 + TAA, P2 + TAA, P3 + TAA and P4 + TAA: hepatocytes incubated with protein isolate (10, 25, 50 and 100 ␮g/ml) prior to TAA administration for 60 min, vitamin C + TAA: hepatocytes first incubated with vitamin C and then with TAA. Each value represents mean ± S.D. (Pa < 0.05; Pb < 0.05).

4. Discussion The objective of this investigation was to evaluate the protective effect of the protein isolate from the herb, P. niruri, against TAA-induced cytotoxicity in hepatocytes. Results suggest that the cytotoxic effect was imposed by the oxidative insult. TAA caused severe damages to cells by reacting with cellular macromolecules and causing increased production of ROS in hepatocytes as evidenced from the decreased cell viability, enhanced ALT and LDH leakage, decreased levels of GSH content and antioxidant enzyme activities and enhanced levels of lipid peroxidation. With increase in TAA concentration cell viability was decreased; on the other hand, ALT and LDH levels were increased in the extracellular part. It has been shown that the leakage of LDH correlates with cellular viability and hence a useful indicator of membrane damages [45]. At a certain concentration of TAA (2 mM) cell viability was minimal and did not change further with the increased concentration of TAA. Treatment with the protein isolate prior to TAA treatment not only increased the cell viability gradually to about 90% with respect to the control but also significantly reduced the enzyme leakage. TAA-induced liver injury is believed to be mediated through the action of IL1␤ and TNF␣ on hepatocytes [46]. Although the mechanism is not fully understood, various studies in rats and cultured hepatocytes indicated the involvement of oxidative stress in TAA-induced toxicity [1,47,48]. In these studies, TAA has been found to stimulate lipid peroxidation by generation of ROS [49]. TAA also decreased the GSH/GSSG ratio [49], altered the levels of different antioxidant enzymes and increased the susceptibility of hepatocytes to in vitro lipid peroxidation. Similar results have also been observed in our studies. Protein treatment prior to TAA administration showed practically no enhancement of MDA content in hepatocytes suggesting that the protein might have a protective effect against the ROS-induced membrane damages. TAA also produced oxidative stress by depleting the GSH level suggesting the presence of free radicals generated by TAA. When the hepatocytes were incubated with the protein prior to TAA administration, the consumption of GSH by the hepatocytes was reduced. Result suggests that the pro-

118



(a) 100

b

(b) 100

b

b

b

b

SOD Activity (% over control)

CAT Activity (% over control)


75 a

50

b 75 a

50

25

25

GST Activity (% Over control)

(c)


100

b

b b 75 a

50

25

Fig. 4. (a). Effect of the protein isolate on catalase, (b) SOD and (c) GST activity in hepatocytes. Results have been given as percentage of control. Control: hepatocytes treated with DMEM, TAA: hepatocytes exposed to TAA (2 mM) for 60 min. P1 + TAA, P2 + TAA, P3 + TAA and P4 + TAA: hepatocytes incubated with protein isolate (10, 25, 50 and 100 ␮g/ml) prior to TAA administration for 60 min, vitamin C + TAA: hepatocytes first incubated with vitamin C and then with TAA. Each value represents mean ± S.D. (Pa < 0.05; Pb < 0.05).

tein increased the amount of hepatic GSH and maintained its normal level in presence of TAA. The DPPH scavenging activity of the protein also showed that the protein can scavenge free radicals. So the results suggest that the restoration of GSH level was taken place as the protein scavenges the free radicals produced by TAA. The antioxidant enzymes (CAT, SOD, GST) assays showed that TAA treatment caused the depletion of these enzymes; protein treatment prior to the TAA administration kept the activity of the enzymes almost unaltered. It could, therefore, be said that TAA caused the cellular damage by inhibiting the activity of the antioxidant enzymes and that could be prevented by the protein pretreatment.

In all experiments vitamin C was used as a reference antioxidant, and it showed similar results but the protein can be a far better antioxidant than vitamin C because for similar results protein concentration was much lower than vitamin C. In conclusion, the cytoprotective activity of the protein isolate against TAA-induced injury in hepatocytes may be due to its radical scavenging and antioxidative properties. The protein isolate seems to protect hepatocytes from TAA-induced injury by maintaining the level of GSH and by inhibiting the production of MDA. Further studies are needed to elucidate its mechanism of action in detail and currently are in progress.

M.K. Sarkar, P.C. Sil / Pathophysiology 14 (2007) 113–120 125

% Inhibition of DPPH

100

75

50

25

0 0

50

100

150

200

Protein concentration (µg/ml) Fig. 5. DPPH scavenging activity of the protein isolate of Phyllanthus niruri with increasing concentrations.

Acknowledgements The work has been supported by the Council of Scientific and Industrial Research, Government of India (a Grant-InAid to PCS, Scheme Number: 01(1788)/02/EMR-II). MKS acknowledges the receipt of CSIR (partly) fellowship as well as the fellowship from Bose Institute. The authors are grateful to the Director of Bose Institute for providing the laboratory facilities and Mr. Prasanta Pal for excellent technical assistance for the study.

References [1] A. Akbay, K. Cinar, O. Uzunalimoglu, S. Eranil, C. Yurdaydin, H. Bozkaya, M. Bozdayi, Serum cytotoxin and oxidant stress markers in N-acetylcysteine treated thioacetamide hepatotoxicity of rats, Hum. Exp. Toxicol. 18 (1999) 669–676. [2] E. Chieli, G. Malvaldi, Role of the microsomal FAD-containing monooxygenase in the liver toxicity of thioacetamide S-oxide, Toxicology 31 (1984) 41–51. [3] A.L. Hunter, M.A. Holscher, R.A. Neal, Thioacetamide induced hepatic necrosis. I. Involvement of the mixed-function oxidase enzyme system, J. Pharmacol. Exp. Ther. 200 (1977) 439–448. [4] W.R. Porter, R.A. Neal, Metabolism of thioacetamide and thiacetamide S-oxide by rat liver microsomes, Drug Metab. Dispos. 6 (1978) 379–388. [5] C. Diez-Fernandez, N. Sanz, A.M. Alvarez, A. Zaragoza, M. Cascales, Influence of aminoguanidine on parameters of liver injury and regeneration induced in rats by a necrosis dose of thioacetamide, Br. J. Pharmacol. 125 (1998) 102–108. [6] R. Bruck, H. Aeed, Y. Avni, H. Shirin, Z. Matas, M. Shahmurov, I. Avinoach, G. Zozulya, N. Weizman, A. Hochman, Melatonin inhibits nuclear factor kappa B activation and oxidative stress and protects against thioacetamide induced liver damage in rats, J. Hepatol. 40 (2004) 86–93. [7] R. Bruck, H. Aeed, R. Schey, Z. Matas, R. Raifen, G. Zaiger, A. Hochman, Y. Avni, Pyrrolidine dithiocarbamate protects against thioacetamide-induced fulminant hepatic failure in rats, J. Hepatol. 36 (2002) 370–377.

119

[8] K.R. Kirtikar, B.D. Basu, in: E. Blatter, J.F. Caius, R.S. Mhaskarr (Eds.), Indian Medicinal Plants, vol. I, Probasi Press, Calcutta, 1935, pp. 809–1122. [9] R.N. Chopra, S.L. Nayar, I.C. Chopra, Glossary of Indian Medicinal Plants, Publication and Information Directorate, CSIR, New Delhi, 1986. [10] C.K. Chauhan, S.A. Nanivadekar, F.R. Billimoria, Effect of a herbal hepatoprotective product on drug metabolism in patients of cirrhosis and hepatic enzyme function in experimental liver damage, Ind. J. Pharmacol. 24 (1992) 107–110. [11] S.P. Thyarajan, S. Subramanian, T. Thirunalasundari, P.S. Venkateswaran, B.S. Blumberg, Effect of Phyllanthus amarus on chronic carriers of hepatitis B virus, Lancet 2 (1998) 764–766. [12] P. Padma, O.H. Setty, Protective effect of Phyllanthus against CCl4 induced mitochondrial dysfunction, Life Sci. 64 (1999) 2411–2417. [13] A. Pietrangelo, F. Borella, G. Casalgrandi, Antioxidant activity of silybin in vivo during long term iron overload in rats, Gastroenterology 109 (1995) 1941–1949. [14] H. Farghali, L. Kamenikova, S. Hynie, E. Kmonickova, Silymarin effects on intracellular calcium and cytotoxicity: a study in perfused rat hepatocytes after oxidative stress injury, Pharmacol. Res. 41 (2000) 231–237. [15] J.S. Kang, Y.J. Jeon, S.K. Park, K.H. Yang, H.M. Kim, Protection against lipopolysaccharide induced sepsis and inhibition of interleukin-1beta and prostaglandin E2 synthesis by silymarin, Biochem. Pharmacol. 67 (2004) 175–181. [16] K.V. Syamasundar, B. Singh, R.S. Thakur, A. Husain, Y. Kiso, H. Hikino, Antihepatotoxic principles of Phyllanthus niruri herbs, J. Ethnopharmacol. 14 (1985) 41–44. [17] S. Khatoon, V. Rai, A.K. Rawat, S. Mehrotra, Comparative pharmacognostic studies of three Phyllanthus species, J. Ethnopharmacol. 104 (2005) 79–86. [18] R. Campos, A. Gorrido, R. Guerra, A. Valenznela, Silybin dihemisuccinate protects against glutathione depletion and lipid peroxidation induced by acetaminophen on rat liver, Planta Med. 55 (1989) 417– 419. [19] J.L. Nishiura, A.H. Campos, M.A. Boim, I.P. Heilberg, N. Schor, Phyllanthus niruri normalizes elevated urinary calcium levels in calcium stone forming (CSF) patients, Urol. Res. 32 (2004) 362–366. [20] T. Iizuka, H. Moriyama, M. Nagai, Vasorelaxant effects of methyl brevifolincarboxylate from the leaves of Phyllanthus niruri, Biol. Pharm. Bull. 29 (2006) 177–179. [21] A.R. Santos, R.O. De Campos, O.G. Miguel, V.C. Filho, A.C. Siani, R.A. Yunes, J.B. Calixto, Antinociceptive properties of extracts of new species of plants of the genus Phyllanthus (Euphorbiaceae), J. Ethnopharmacol. 72 (2000) 229–238. [22] R.L. Huang, Y.L. Huang, J.C. Ou, C.C. Chen, F.L. Hsu, C. Chang, Screening of 25 compounds isolated from Phyllanthus species for anti-human hepatitis B virus in vitro, Phytother. Res. 17 (2003) 449– 453. [23] V.V. Asha, S. Akhila, P.J. Wills, A. Subramoniam, Further studies on the antihepatotoxic activity of Phyllanthus maderaspatensis Linn., J. Ethnopharmacol. 92 (2004) 67–70. [24] C.G. Mellinger, E.R. Carbonero, G.R. Noleto, T.R. Cipriani, M.B. Oliveira, P.A. Gorin, M. Iacomini, Chemical and biological properties of an arabinogalactan from Phyllanthus niruri, J. Nat. Prod. 68 (2005) 1479–1483. [25] M.K. Sarkar, K. Sarkar, R. Bhattacharjee, M. Chatterjee, P.C. Sil, Curative role of the aqueous extract of the herb, Phyllanthus niruri, against nimesulide induced oxidative stress in murine liver, Biomed. Res. 16 (2005) 171–176. [26] M. Chatterjee, P.C. Sil, Hepatoprotective effect of aqueous extract of Phyllanthus niruri on nimesulide-induced oxidative stress in vivo, Indian J. Biochem. Biophys. 43 (2006) 299–305. [27] M. Chatterjee, P.C. Sil, Protective role of Phyllanthus niruri against nimesulide-induced hepatic damage, Indian J. Clin. Biochem. 22 (2007) 109–116.

120


[28] M. Chatterjee, K. Sarkar, P.C. Sil, Herbal (Phyllanthus niruri) protein isolate protects liver from nimesulide induced oxidative stress, Pathophysiology 13 (2006) 95–102. [29] R. Bhattacharjee, P.C. Sil, The protein fraction of Phyllanthus niruri plays a protective role against acetaminophen induced hepatic disorder via its antioxidant properties, Phytother. Res. 20 (2006) 595– 601. [30] R. Bhattacharjee, P.C. Sil, Protein isolate from the herb, Phyllanthus niruri, protects liver from acetaminophen induced toxicity, Biomed. Res. 17 (2006) 75–79. [31] R. Bhattacharjee, P.C. Sil, Protein isolate from the herb, Phyllanthus niruri L. (Euphorbiaceae), plays hepatoprotective role against carbon tetrachloride induced liver damage via its antioxidant properties, Food Chem. Toxicol. 45 (2007) 817–826. [32] R. Bhattacharjee, P.C. Sil, Protein isolate from the herb, Phyllanthus niruri, modulates carbon tetrachloride-induced cytotoxicity in hepatocytes, Toxicol. Mech. Methods 17 (2006) 41–47. [33] K. Sarkar, P.C. Sil, A 43 kDa protein from the herb Cajanus indicus L. protects thioacetamide induced cytotoxicity in hepatocytes, Toxicol. In Vitro 20 (2006) 634–640. [34] K. Bove, P. Neumann, N. Gertzberg, A. Johnson, Role of eNOS-derived NO in mediating TNF-induced endothelial barrier dysfunction, Am. J. Physiol. Lung Cell Mol. Physiol. 280 (2001) 914– 922. [35] S. Reitman, S. Frankel, A colorimetric method for the determination of serum glutamic oxaloacetic and glutamic pyruvic transaminases, Am. J. Clin. Pathol. 28 (1957) 56–63. [36] R.L. Searey, Diagonistic Biochemistry, McGraw-Hill, New York, NT, 1969. [37] H. Esterbauer, K.H. Cheeseman, Determination of aldehydic lipid peroxidation products: malonaldehyde and 4-hydroxynonenal, Methods Enzymol. 186 (1990) 407–421. [38] F. Tietze, Enzymic method for quantitative determination of nanogram amount of total and oxidizes glutathione: applications to mammalian blood and other tissues, Anal. Biochem. 27 (1969) 502– 522. [39] J. Bonaventura, W.A. Schroeder, S. Fang, Human erythrocyte catalase: an improved method of isolation and a revaluation of

[40]

[41]

[42]

[43] [44]

[45]

[46]

[47]

[48]

[49]

reported properties, Arch. Biochem. Biophys. 150 (1972) 606– 617. M. Nishikimi, N.A. Rao, K. Yagi, The occurrence of superoxide anion in the reaction of reduced phenazine methosulfate and molecular oxygen, Biochem. Biophys. Res. Commun. 46 (1972) 849–854. P. Kakkar, B. Das, P.N. Viswanathan, A modified spectrophotometric assay of superoxide dismutase, Ind. J. Biochem. Biophys. 21 (1984) 130–132. W.H. Habig, M.J. Pabst, W.B. Jakoby, Glutathione S-transferases. The first enzymatic step in mercapturic acid formation, J. Biol. Chem. 249 (1974) 7130–7139. M.S. Blois, Antioxidant determination by use of a stable free radical, Nature 29 (1958) 248–254. M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding, Anal. Biochem. 72 (1976) 248–254. N. Del Raso, In vitro methods for assessing chemical or drug toxicity and metabolism in primary hepatocytes, in: R.R. Watson (Ed.), In Vitro Methods of Toxicology, CRC Press Inc., Boca Raton, FL, 1992, pp. 176–201. S.L. Liu, S. Degli Esposti, T. Yao, A.M. Diehl, M.A. Zern, Vitamin E therapy of acute CCl4 -induced hepatic injury in mice is associated with inhibition of nuclear factor kappa B binding, Hepatology 22 (1995) 1474–1481. N. Sanz, C. Diez-Fernandez, L. Fernandez-Simon, A. Alvarez, M. Cascales, Necrogenic and regenerative responses of liver of newly weaned rats against a sublethal dose of thioacetamide, Biochim. Biophys. Acta 1384 (1998) 66–78. S.C. Lu, Z.Z. Huang, H. Yang, H. Tsukamoto, Effect of thioacetamide on the hepatic expression of gamma-glutamylcysteine synthetase subunits in the rat, Toxicol. Appl. Pharmacol. 159 (1999) 161–168. R. Bruck, H. Aeed, H. Shirin, et al., The hydroxyl radical scavengers dimethylsulfoxide and dimethylthiourea protect rats against thioacetamide-induced fulminant hepatic failure, J. Hepatol. 31 (1999) 27–38.