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Hepatoprotective and antioxidant activity of Amaranthus spinosus against CCl4 induced toxicity
Journal of Ethnopharmacology 125 (2009) 364–366
Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
Ethnopharmacological communication
Hepatoprotective and antioxidant activity of Amaranthus spinosus against CCl4 induced toxicity Hussain Zeashan a , G. Amresh a,∗ , Satyawan Singh b , Chandana Venkateswara Rao a a b
Pharmacognosy and Ethnopharmacology Division, National Botanical Research Institute, Lucknow, Uttar Pradesh 226001, India Department of Pharmacy, Saroj Institute of Technology & Management, Lucknow, Uttar Pradesh, India
a r t i c l e
i n f o
Article history: Received 17 May 2008 Received in revised form 25 April 2009 Accepted 11 May 2009 Available online 22 May 2009 Keywords: Amaranthus spinosus Antioxidant Hepatoprotective HepG2
a b s t r a c t Aim: 50% ethanolic extract (ASE) of Amaranthus spinosus (whole plant) was evaluated for in vitro antioxidant and hepatoprotective activity. Methods: The total phenolics and reducing capacity of ASE was determined using standard curve of gallic acid (0–1.0 mg/ml) and butylated hydroxy anisole. In vitro antioxidant activity was determined by DPPH, superoxide, hydroxyl radicals, hydrogen peroxide and nitric oxide scavenging methods. The hepatoprotective activity of ASE was evaluated at 6, 7, 8, 9 and 10 g/ml concentration against CCl4 (1%) induced toxicity in freshly isolated rat hepatocytes and HepG2 cells. Results: ASE was found to contain 336 ± 14.3 mg/g total polyphenolics expressed as gallic acid equivalent while the reducing capacity was 2.26 times of BHA. ASE showed significant antioxidant activity in DPPH assay (IC50 29 g/ml), scavenges superoxide (IC50 ∼ 66–70 g/ml), hydrogen peroxide (IC50 ∼120–125 g/ml), hydroxyl radicals (IC50 ∼140–145 g/ml) and nitric oxide (IC50 ∼ 135–140 g/ml). ASE (6, 7, 8, 9 and 10 g/ml) was able to normalise the levels of biochemical parameters in isolated rat hepatocytes intoxicated with CCl4 . A dose dependent increase in percentage viability was observed in CCl4 intoxicated HepG2 cells. Conclusions: ASE possesses significant hepatoprotective activity which might be due to antioxidant defence factors and phenolics might be the main constituents responsible for activity. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Plant Amaranthus spinosus Linn. (Family: Amaranthaceae), commonly known as “Kate Wali Chaulai (Kanatabhajii)” in “Hindi”, is used as vegetable and cultivated throughout in India, Sri Lanka and many tropical countries (Kirtikar and Basu, 2001). Plants were freshly collected, identified and authenticated at National Botanical Research Institute, India and a voucher specimen (NAB 75006) for the collected samples has been deposited in the institute for future references.
ity, aqueous extract has shown significant immunostimulating and stem extract has been credited with antimalarial activity (Zeashan et al., 2008). 3. Previously isolated constituents Amaranthus spinosus contains amaranthine, isoamaranthine, hydroxycinnamates, quercetin, kaempferol glycosides, amaranthoside, amaricin and stigmasterol glycoside (Zeashan et al., 2008).
2. Uses in traditional medicine and other reported activities 4. Materials and methods The juice of Amaranthus spinosus is used by tribals of Kerala, India to prevent swelling around stomach while the leaves are boiled without salt and consumed for 2–3 days to cure jaundice (Hema et al., 2006). The plant possess hepatoprotective, antioxidant activ-
∗ Corresponding author. Tel.: +91 941 5520130/522 2391902/522 2205831 35; fax: +91 522 2205836/2205839. E-mail address: [email protected] (G. Amresh). 0378-8741/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2009.05.010
4.1. Preparation of the sample The coarsely powdered plant material (1 kg) was extracted with petroleum ether thrice to remove the fatty material and further marc was extracted thrice with ethanol (50%, v/v) and concentrated under reduced pressure to yield 6.12%, w/w (ASE). For in vitro hepatoprotective studies ASE was dissolved in DMSO and the volume was made up to 10 ml with Ham’s F12/MEM. Further dilutions were
H. Zeashan et al. / Journal of Ethnopharmacology 125 (2009) 364–366
made to obtain different concentrations and were used for in vitro investigations. 4.2. Determination of total phenolics and reducing power The total phenolics in the ASE were determined using Folin–Ciocalteau reagent (Taga et al., 1984) and reducing power was evaluated according to the method of Oyaizu (1986). 4.3. Free radical scavenging assays Free radical scavenging activity was determined by different methods such as DPPH assay (Rao et al., 2006), superoxide (Yen and Chen, 1995), hydroxyl radicals (Aruoma and Halliwell, 1987), hydrogen peroxide (Ruch et al., 1989) and nitric oxide (Sedlak and Lindsay, 1968). 4.4. In vitro hepatoprotective studies 4.4.1. Hepatoprotective effect of ASE on freshly isolated rat hepatocytes Liver cells were isolated by the procedure of Seglen (1994). The isolated hepatocytes were cultured in Ham’s F12 medium, supplemented with 10% newborn calf serum, antibiotics, 10−6 M dexamethasone and 10−8 bovine insulin. The cell suspension was incubated at 37 ◦ C for 30 min in a humidified incubator under 5% CO2 . After an incubation of 24 h, the hepatocytes were exposed to the fresh medium containing CCl4 (1%) along with/without various concentrations of the ASE or the medium alone (as normal). After 60 min of CCl4 challenge, concentrations of ASAT, ALAT, ALP, TGL, total proteins, albumin, total bilirubin and direct bilirubin in the medium were measured as an indication of hepatocytes necrosis using diagnostic kits (Yoshinobu et al., 1983). Cell viability was determined by the Trypan blue dye exclusion method (Freshney, 2000). 4.4.2. Hepatoprotective effect in HepG2 cell line Human liver HepG2 cells were exposed to a medium containing 1% CCl4 along with/without various concentrations of the ASE. At the end of the period, cytotoxicity was assessed by estimating the viability of HepG2 cells by MTT reduction assay (Ke et al., 1999). 4.5. Statistical methods All the data are presented as mean ± SEM and analysed by SPSS for Windows, version 9 (SPSS, Chicago, IL, USA) for the possible significant interrelation between the various groups, the independent samples t test. A value of P < 0.05 was considered statistically significant.
365
5. Results 5.1. Total phenolics, reducing power and in vitro antioxidant activity of ASE ASE was found to contain 336 ± 14.3 mg/g total polyphenolics expressed as gallic acid equivalent (GAE, mg/g of GAE). The reducing capacity of extract in vitro, another significant indicator of antioxidant activity was also found to be highly significant, 2.26 times of that of BHA at higher dose, used as a standard. ASE scavenged as much as 81.7% of DPPH radicals at a concentration of 50 g/ml. The IC50 value was 29 g/ml. ASE significantly and dose dependently scavenged O2 •− as well (Fig. 1). As much as 54.4% of O2 •− was be scavenged by ASE at a concentration of 400 g/ml (IC50 ∼ 66–70 g/ml). Ability of ASE to scavenge H2 O2 was determined and the extract was found to dose dependently scavenge H2 O2 as well (IC50 ∼ 120–125 g/ml). As compared to the other ROS/RNS scavenged by the extract, scavenging of H2 O2 was the weakest (Fig. 1). ASE also scavenged • OH radicals significantly (Fig. 1). The • OH radicals were generated by Fenton type reaction and measured by their ability to degrade deoxyribose sugar into the fragments that react with thiobarbituric acid to form a pink chromogen. At a concentration of 400 g/ml, the extract could scavenge 38% of • OH radicals, IC50 being 140–145 g/ml. Control experiments showed that the extract did not interfere with deoxyribose assay. NO scavenging activity of ASE was determined by using a NO donor, SNP. Incubation of solution of SNP in PBS at 25 ◦ C for 120 min resulted in the release of significant amount of NO. The extract effectively and dose dependently decreased NO release (Fig. 1) with the IC50 value of 135–140 g/ml. Control experiments showed that even at high concentrations, the extract did not interfere with the reaction between nitrite and Griess reagent. 5.2. Hepatoprotective effect of ASE in freshly isolated rat hepatocytes A significant (P < 0.001) increase in the levels of ALAT, ASAT, ALP, total bilirubin and direct bilirubin and a significant (P < 0.001) reduction in the levels of TGL, total protein and albumin was observed in the hepatocytes exposed to CCl4 compared to control. These cells, when treated with different concentrations (6, 7, 8, 9 and 10 g/ml) of ASE showed a significant (P < 0.001) restoration of the altered biochemical parameters towards the normal compared to CCl4 treated group and is dose dependent (Table 1). 5.3. Hepatoprotective effect in HepG2 cell line The CCl4 exposed HepG2 cells showed a percentage viability of (P < 0.001) 24.38% compared to normal cell. The significant percent-
Fig. 1. Free radical compared with control of hydrogen peroxide, superoxide, hydroxyl radical and nitric oxide free radicals in vitro by ASE. Values are mean ± SEM; n = 6. Significance level: a P < 0.05, b P < 0.001, and c P < 0.001 vs. control group. The free radical (percentage) compared with control is expressed in different methods at different dose levels. The doses of the ASE and BHA were as follows: ASE 50 g/ml, ASE 100 g/ml, ASE 200 g/ml, ASE 400 g/ml, BHA 200 mol/ml. Control is the 100% free radical without any reducing agent treatment.
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H. Zeashan et al. / Journal of Ethnopharmacology 125 (2009) 364–366
Table 1 Effect of treatment of ASE on the biochemical parameters of CCl4 intoxicated freshly isolated rat hepatocytes. Treatments
ALAT (U/l)
Control CCl4 (1%) ASE (6 g/ml) ASE (7 g/ml) ASE (8 g/ml) ASE (9 g/ml) ASE (10 g/ml)
16.00 57.00 30.02 26.7 25.80 26.70 19.00
± ± ± ± ± ± ±
0.01 0.52a 1.62b 0.98b 1.03b 0.67b 1.23b
ASAT (U/l) 11.00 69.00 24.80 21.60 19.40 13.40 13.00
± ± ± ± ± ± ±
0.39 0.49a 0.57b 0.52b 0.5b 0.72b 0.93b
ALP (U/l) 27.00 78.00 33.00 30.20 29.20 26.00 28.80
± ± ± ± ± ± ±
0.45 2.87a 1.80b 1.75b 1.33b 1.38b 0.99b
TGL (mg/dl)
Total protein (g/dl)
180.00 ± 9.07 97.00 ± 3.05a 180.00 ± 6.72b 184.40 ± 5.53b 186.60 ± 9.80b 193.20 ± 8.90b 197.60 ± 10.07b
0.761 0.107 0.544 0.584 0.62 0.705 0.74
± ± ± ± ± ± ±
0.04 0.06a 0.03b 0.02b 0.03b 0.03b 0.02b
Albumin (g/dl) 1.213 0.30 0.902 0.912 0.993 1.13 1.20
± ± ± ± ± ± ±
0.006 0.01a 0.04b 0.05b 0.05b 0.07b 0.04b
Total bilirubin (mg/dl) 0.204 0.518 0.358 0.304 0.298 0.27 0.255
± ± ± ± ± ± ±
0.005 0.01a 0.02b 0.02b 0.02b 0.01b 0.04b
Direct bilirubin (mg/dl) 0.034 0.174 0.079 0.072 0.06 0.054 0.044
± ± ± ± ± ± ±
0.002 0.03a 0.003b 0.003b 0.001b 0.002b 0.002b
Values are mean ± SEM; no. of independent experiments = 3, 5 replicates. a Significance level: P < 0.001, compared to normal group. b Significance level: P < 0.001, compared to CCl4 group.
age viability were (P < 0.001) 77.31, 82.24, 84.31, 88.84, 93.72, and at the dose 6, 7, 8, 9 and 10 g/ml of the ASE respectively compared to CCl4 intoxicated group. 6. Discussion The present study reveals the hepatoprotective effect of ASE against CCl4 induced toxicity in isolated rat hepatocytes and HepG2 cells in culture. Treatment with the ASE at different concentrations (6, 7, 8, 9 and 10 g/ml) exhibited significant restoration of the altered biochemical parameters towards normal in CCl4 intoxicated rat hepatocytes. The effect of the ASE at 10 g/ml was found to be comparable to control group. The investigation carried out in human liver derived HepG2 cells against CCl4 induced damage also confirmed the hepatoprotective nature of the ASE. ASE possessing potent free radical scavenging activity, may ameliorate the levels of H2 O2 and O2 •− , consequently restoring enzyme activity. The components of ASE may also induce the de novo synthesis of the antioxidant enzymes. Overall, it could be concluded that ASE protects against oxidative injury induced by CCl4 in vitro and it is also capable of enhancing the activities of hepatic enzymes implicated in combating ROS however further investigation should be carried out on the extract to identify the active constituents responsible for hepatoprotection. References Aruoma, O.I., Halliwell, B., 1987. Action of hypochlorous acid on the antioxidant protective enzymes superoxide dismutase, catalase and glutathione peroxidase. Biochemical Journal 248, 973–976.
Freshney, I., 2000. Cytotoxicity. Culture of Animal Cells: A Manual of Basic Technique, 4th ed. Wiley-Liss, New York, pp. 331–332. Hema, E.S., Sivadasan, M., Anil, K.N., 2006. Studies on edible species of Amaranthacea and Araceae used by Kuruma and Paniya tribes in Wayanad district, Kerala, India. Ethnobotany 18, 122–126. Ke, H., Hisayoshi, K., Aijun, D., Yongkui, J., Shigeo, I., Xinsheng, Y., 1999. Antineoplastic agents III: steroidal glycosides from Solanum nigrum. Planta Medica 65, 35–38. Kirtikar, K.R., Basu, B.D., 2001. Indian Medicinal Plants, vol. 9., 2nd ed. Oriental Enterprises, Rajpur, Dehradun, Uttranchal, India, pp. 2832–2836. Oyaizu, M., 1986. Studies on products of browning reaction prepared from glucosamine. Japanese Journal of Nutrition 44, 307–315. Rao, A.R., Sarada, R., Baskaran, V., Ravishankar, G.A., 2006. Antioxidant activity of Botryococcus braunii extract elucidated in vitro models. Journal of Agricultural and Food Chemistry 54, 4593–4599. Ruch, R.J., Cheng, S.J., Klaunig, J.E., 1989. Prevention of cytotoxicity and inhibition of intercellular communication by antioxidant catechins isolated from Chinese green tea. Carcinogenesis 10, 1003–1008. Sedlak, J., Lindsay, R.H., 1968. Estimation of total, protein bound, and nonprotein sulfhydryl groups in tissue with Ellman’s reagent. Analytical Biochemistry 25, 192–205. Seglen, P.O., 1994. Culture of specific cell types: epithelial cells isolation of hepatocytes. In: Celis, J.E. (Ed.), Cell Biology: A Laboratory Handbook, vol. 1. Academic Press, pp. 96–102. Taga, M.S., Miller, E.E., Pratt, D.E., 1984. Chia seeds as a source of natural lipid antioxidant. Journal of the American Oil Chemist Society 61, 928–931. Yen, G., Chen, H., 1995. Antioxidant activity of various tea extract in relation to their antimutagenicity. Journal of Agricultural and Food Chemistry 43, 27–32. Yoshinobu, K., Masahiro, T., Hiroshi, H., 1983. Assay method for antihepatotoxic activity using carbon tetrachloride induced cytotoxicity in primary cultured hepatocytes. Planta Medica 49, 222–225. Zeashan, H., Amresh, G., Singh, S., Rao, C.V., 2008. Hepatoprotective activity of Amaranthus spinosus in experimental animals. Food and Chemical Toxicology 46, 3417–3421.
Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
Ethnopharmacological communication
Hepatoprotective and antioxidant activity of Amaranthus spinosus against CCl4 induced toxicity Hussain Zeashan a , G. Amresh a,∗ , Satyawan Singh b , Chandana Venkateswara Rao a a b
Pharmacognosy and Ethnopharmacology Division, National Botanical Research Institute, Lucknow, Uttar Pradesh 226001, India Department of Pharmacy, Saroj Institute of Technology & Management, Lucknow, Uttar Pradesh, India
a r t i c l e
i n f o
Article history: Received 17 May 2008 Received in revised form 25 April 2009 Accepted 11 May 2009 Available online 22 May 2009 Keywords: Amaranthus spinosus Antioxidant Hepatoprotective HepG2
a b s t r a c t Aim: 50% ethanolic extract (ASE) of Amaranthus spinosus (whole plant) was evaluated for in vitro antioxidant and hepatoprotective activity. Methods: The total phenolics and reducing capacity of ASE was determined using standard curve of gallic acid (0–1.0 mg/ml) and butylated hydroxy anisole. In vitro antioxidant activity was determined by DPPH, superoxide, hydroxyl radicals, hydrogen peroxide and nitric oxide scavenging methods. The hepatoprotective activity of ASE was evaluated at 6, 7, 8, 9 and 10 g/ml concentration against CCl4 (1%) induced toxicity in freshly isolated rat hepatocytes and HepG2 cells. Results: ASE was found to contain 336 ± 14.3 mg/g total polyphenolics expressed as gallic acid equivalent while the reducing capacity was 2.26 times of BHA. ASE showed significant antioxidant activity in DPPH assay (IC50 29 g/ml), scavenges superoxide (IC50 ∼ 66–70 g/ml), hydrogen peroxide (IC50 ∼120–125 g/ml), hydroxyl radicals (IC50 ∼140–145 g/ml) and nitric oxide (IC50 ∼ 135–140 g/ml). ASE (6, 7, 8, 9 and 10 g/ml) was able to normalise the levels of biochemical parameters in isolated rat hepatocytes intoxicated with CCl4 . A dose dependent increase in percentage viability was observed in CCl4 intoxicated HepG2 cells. Conclusions: ASE possesses significant hepatoprotective activity which might be due to antioxidant defence factors and phenolics might be the main constituents responsible for activity. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Plant Amaranthus spinosus Linn. (Family: Amaranthaceae), commonly known as “Kate Wali Chaulai (Kanatabhajii)” in “Hindi”, is used as vegetable and cultivated throughout in India, Sri Lanka and many tropical countries (Kirtikar and Basu, 2001). Plants were freshly collected, identified and authenticated at National Botanical Research Institute, India and a voucher specimen (NAB 75006) for the collected samples has been deposited in the institute for future references.
ity, aqueous extract has shown significant immunostimulating and stem extract has been credited with antimalarial activity (Zeashan et al., 2008). 3. Previously isolated constituents Amaranthus spinosus contains amaranthine, isoamaranthine, hydroxycinnamates, quercetin, kaempferol glycosides, amaranthoside, amaricin and stigmasterol glycoside (Zeashan et al., 2008).
2. Uses in traditional medicine and other reported activities 4. Materials and methods The juice of Amaranthus spinosus is used by tribals of Kerala, India to prevent swelling around stomach while the leaves are boiled without salt and consumed for 2–3 days to cure jaundice (Hema et al., 2006). The plant possess hepatoprotective, antioxidant activ-
∗ Corresponding author. Tel.: +91 941 5520130/522 2391902/522 2205831 35; fax: +91 522 2205836/2205839. E-mail address: [email protected] (G. Amresh). 0378-8741/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2009.05.010
4.1. Preparation of the sample The coarsely powdered plant material (1 kg) was extracted with petroleum ether thrice to remove the fatty material and further marc was extracted thrice with ethanol (50%, v/v) and concentrated under reduced pressure to yield 6.12%, w/w (ASE). For in vitro hepatoprotective studies ASE was dissolved in DMSO and the volume was made up to 10 ml with Ham’s F12/MEM. Further dilutions were
H. Zeashan et al. / Journal of Ethnopharmacology 125 (2009) 364–366
made to obtain different concentrations and were used for in vitro investigations. 4.2. Determination of total phenolics and reducing power The total phenolics in the ASE were determined using Folin–Ciocalteau reagent (Taga et al., 1984) and reducing power was evaluated according to the method of Oyaizu (1986). 4.3. Free radical scavenging assays Free radical scavenging activity was determined by different methods such as DPPH assay (Rao et al., 2006), superoxide (Yen and Chen, 1995), hydroxyl radicals (Aruoma and Halliwell, 1987), hydrogen peroxide (Ruch et al., 1989) and nitric oxide (Sedlak and Lindsay, 1968). 4.4. In vitro hepatoprotective studies 4.4.1. Hepatoprotective effect of ASE on freshly isolated rat hepatocytes Liver cells were isolated by the procedure of Seglen (1994). The isolated hepatocytes were cultured in Ham’s F12 medium, supplemented with 10% newborn calf serum, antibiotics, 10−6 M dexamethasone and 10−8 bovine insulin. The cell suspension was incubated at 37 ◦ C for 30 min in a humidified incubator under 5% CO2 . After an incubation of 24 h, the hepatocytes were exposed to the fresh medium containing CCl4 (1%) along with/without various concentrations of the ASE or the medium alone (as normal). After 60 min of CCl4 challenge, concentrations of ASAT, ALAT, ALP, TGL, total proteins, albumin, total bilirubin and direct bilirubin in the medium were measured as an indication of hepatocytes necrosis using diagnostic kits (Yoshinobu et al., 1983). Cell viability was determined by the Trypan blue dye exclusion method (Freshney, 2000). 4.4.2. Hepatoprotective effect in HepG2 cell line Human liver HepG2 cells were exposed to a medium containing 1% CCl4 along with/without various concentrations of the ASE. At the end of the period, cytotoxicity was assessed by estimating the viability of HepG2 cells by MTT reduction assay (Ke et al., 1999). 4.5. Statistical methods All the data are presented as mean ± SEM and analysed by SPSS for Windows, version 9 (SPSS, Chicago, IL, USA) for the possible significant interrelation between the various groups, the independent samples t test. A value of P < 0.05 was considered statistically significant.
365
5. Results 5.1. Total phenolics, reducing power and in vitro antioxidant activity of ASE ASE was found to contain 336 ± 14.3 mg/g total polyphenolics expressed as gallic acid equivalent (GAE, mg/g of GAE). The reducing capacity of extract in vitro, another significant indicator of antioxidant activity was also found to be highly significant, 2.26 times of that of BHA at higher dose, used as a standard. ASE scavenged as much as 81.7% of DPPH radicals at a concentration of 50 g/ml. The IC50 value was 29 g/ml. ASE significantly and dose dependently scavenged O2 •− as well (Fig. 1). As much as 54.4% of O2 •− was be scavenged by ASE at a concentration of 400 g/ml (IC50 ∼ 66–70 g/ml). Ability of ASE to scavenge H2 O2 was determined and the extract was found to dose dependently scavenge H2 O2 as well (IC50 ∼ 120–125 g/ml). As compared to the other ROS/RNS scavenged by the extract, scavenging of H2 O2 was the weakest (Fig. 1). ASE also scavenged • OH radicals significantly (Fig. 1). The • OH radicals were generated by Fenton type reaction and measured by their ability to degrade deoxyribose sugar into the fragments that react with thiobarbituric acid to form a pink chromogen. At a concentration of 400 g/ml, the extract could scavenge 38% of • OH radicals, IC50 being 140–145 g/ml. Control experiments showed that the extract did not interfere with deoxyribose assay. NO scavenging activity of ASE was determined by using a NO donor, SNP. Incubation of solution of SNP in PBS at 25 ◦ C for 120 min resulted in the release of significant amount of NO. The extract effectively and dose dependently decreased NO release (Fig. 1) with the IC50 value of 135–140 g/ml. Control experiments showed that even at high concentrations, the extract did not interfere with the reaction between nitrite and Griess reagent. 5.2. Hepatoprotective effect of ASE in freshly isolated rat hepatocytes A significant (P < 0.001) increase in the levels of ALAT, ASAT, ALP, total bilirubin and direct bilirubin and a significant (P < 0.001) reduction in the levels of TGL, total protein and albumin was observed in the hepatocytes exposed to CCl4 compared to control. These cells, when treated with different concentrations (6, 7, 8, 9 and 10 g/ml) of ASE showed a significant (P < 0.001) restoration of the altered biochemical parameters towards the normal compared to CCl4 treated group and is dose dependent (Table 1). 5.3. Hepatoprotective effect in HepG2 cell line The CCl4 exposed HepG2 cells showed a percentage viability of (P < 0.001) 24.38% compared to normal cell. The significant percent-
Fig. 1. Free radical compared with control of hydrogen peroxide, superoxide, hydroxyl radical and nitric oxide free radicals in vitro by ASE. Values are mean ± SEM; n = 6. Significance level: a P < 0.05, b P < 0.001, and c P < 0.001 vs. control group. The free radical (percentage) compared with control is expressed in different methods at different dose levels. The doses of the ASE and BHA were as follows: ASE 50 g/ml, ASE 100 g/ml, ASE 200 g/ml, ASE 400 g/ml, BHA 200 mol/ml. Control is the 100% free radical without any reducing agent treatment.
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H. Zeashan et al. / Journal of Ethnopharmacology 125 (2009) 364–366
Table 1 Effect of treatment of ASE on the biochemical parameters of CCl4 intoxicated freshly isolated rat hepatocytes. Treatments
ALAT (U/l)
Control CCl4 (1%) ASE (6 g/ml) ASE (7 g/ml) ASE (8 g/ml) ASE (9 g/ml) ASE (10 g/ml)
16.00 57.00 30.02 26.7 25.80 26.70 19.00
± ± ± ± ± ± ±
0.01 0.52a 1.62b 0.98b 1.03b 0.67b 1.23b
ASAT (U/l) 11.00 69.00 24.80 21.60 19.40 13.40 13.00
± ± ± ± ± ± ±
0.39 0.49a 0.57b 0.52b 0.5b 0.72b 0.93b
ALP (U/l) 27.00 78.00 33.00 30.20 29.20 26.00 28.80
± ± ± ± ± ± ±
0.45 2.87a 1.80b 1.75b 1.33b 1.38b 0.99b
TGL (mg/dl)
Total protein (g/dl)
180.00 ± 9.07 97.00 ± 3.05a 180.00 ± 6.72b 184.40 ± 5.53b 186.60 ± 9.80b 193.20 ± 8.90b 197.60 ± 10.07b
0.761 0.107 0.544 0.584 0.62 0.705 0.74
± ± ± ± ± ± ±
0.04 0.06a 0.03b 0.02b 0.03b 0.03b 0.02b
Albumin (g/dl) 1.213 0.30 0.902 0.912 0.993 1.13 1.20
± ± ± ± ± ± ±
0.006 0.01a 0.04b 0.05b 0.05b 0.07b 0.04b
Total bilirubin (mg/dl) 0.204 0.518 0.358 0.304 0.298 0.27 0.255
± ± ± ± ± ± ±
0.005 0.01a 0.02b 0.02b 0.02b 0.01b 0.04b
Direct bilirubin (mg/dl) 0.034 0.174 0.079 0.072 0.06 0.054 0.044
± ± ± ± ± ± ±
0.002 0.03a 0.003b 0.003b 0.001b 0.002b 0.002b
Values are mean ± SEM; no. of independent experiments = 3, 5 replicates. a Significance level: P < 0.001, compared to normal group. b Significance level: P < 0.001, compared to CCl4 group.
age viability were (P < 0.001) 77.31, 82.24, 84.31, 88.84, 93.72, and at the dose 6, 7, 8, 9 and 10 g/ml of the ASE respectively compared to CCl4 intoxicated group. 6. Discussion The present study reveals the hepatoprotective effect of ASE against CCl4 induced toxicity in isolated rat hepatocytes and HepG2 cells in culture. Treatment with the ASE at different concentrations (6, 7, 8, 9 and 10 g/ml) exhibited significant restoration of the altered biochemical parameters towards normal in CCl4 intoxicated rat hepatocytes. The effect of the ASE at 10 g/ml was found to be comparable to control group. The investigation carried out in human liver derived HepG2 cells against CCl4 induced damage also confirmed the hepatoprotective nature of the ASE. ASE possessing potent free radical scavenging activity, may ameliorate the levels of H2 O2 and O2 •− , consequently restoring enzyme activity. The components of ASE may also induce the de novo synthesis of the antioxidant enzymes. Overall, it could be concluded that ASE protects against oxidative injury induced by CCl4 in vitro and it is also capable of enhancing the activities of hepatic enzymes implicated in combating ROS however further investigation should be carried out on the extract to identify the active constituents responsible for hepatoprotection. References Aruoma, O.I., Halliwell, B., 1987. Action of hypochlorous acid on the antioxidant protective enzymes superoxide dismutase, catalase and glutathione peroxidase. Biochemical Journal 248, 973–976.
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