Journal of Ethnopharmacology 158 (2014) 442–446
Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
Hepatoprotective activity of ginsenosides from Panax ginseng adventitious roots against carbon tetrachloride treated hepatic injury in rats Hosakatte Niranjana Murthy a,b,n, Vijayalaxmi S. Dandin b, Kee Yoeup Paek a,n a b
Research Center for the Development of Advanced Horticultural Technology, Chungbuk National University, Cheongju 361-763, Republic of Korea Department of Botany, Karnatak University, Dharwad 580003, India
art ic l e i nf o
a b s t r a c t
Article history: Received 8 July 2014 Received in revised form 21 September 2014 Accepted 17 October 2014 Available online 31 October 2014
Ethnopharmacological relevance: Ginseng (Panax ginseng C. A. Meyer) has a beneficial role in the treatment of various diseases including liver disorders like acute/chronic hepatotoxicity, hepatitis, hepatic fibrosis/ cirrhosis and hepatocellular carcinoma. Materials and methods: Tissue culture raised mountain ginseng adventitious root (TCMGARs) extract with ginsenosides in abundance was used as an experimental material. ‘Sprague–Dawley’ male rats were used as experimental systems and were fed with TCMGARs extracts at doses of 30, 100, 300 mg/kg body weight for two weeks to test the effect on carbon tetrachloride (CCl4) induced acute liver damage. Field cultivated Korean ginseng root extract fed rats (100 mg/kg) were used as positive control. Plasma enzyme levels, alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) were assessed. Glutathione (GSH) and malondialdehyde (MDA) concentrations were also evaluated. Results: TCMGARs extracts remarkably prevented the elevation of ALT, AST, ALP and liver peroxides in CCl4treated rats. Hepatic glutathione levels were significantly increased by the treatment with the extracts in experimental groups. Conclusion: The TCMGARs rich in varied ginsenosides can afford protection against CCl4-induced hepatocellular injury. & 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Ginsenosides Glutathione Hepatoprotective activity Lipid peroxidation Mountain ginseng
1. Introduction Panax ginseng C. A. Meyer is an important herbal medicine in East Asian countries. It is used in traditional medicine to promote health and healing, as an adaptogen and a stimulant. The major components of ginseng are triterpenoidal dammarane glycosides called ginsenosides. Pharmacological effects of ginseng have been demonstrated in cancer, diabetes mellitus, cardiovascular system, immune system and central nervous system including anti-stress and anti-oxidant activities (Park et al., 2005). Wild ginseng is a scanty and expensive commodity. To cultivate ginseng in fields, it needs 5–7 years till harvesting stage during which the plant may suffer from various diseases and pests. Adventitious roots have been induced from 100 year old ginseng and the roots have been cultivated in vitro in large scale bioreactors for the use of pharmaceutical industry (Paek et al., 2009). Toxicological evaluation has proved that the tissue cultured mountain ginseng adventitious roots n Corresponding authors at: Research Center for the Development of Advanced Horticultural Technology, Chungbuk National University, Cheongju 361-763, Republic of Korea. Tel.: þ 82 43 266 3245; fax: þ 82 43 266 3246. E-mail addresses: [email protected] (H. Niranjana Murthy), [email protected] (K. Yoeup Paek).
http://dx.doi.org/10.1016/j.jep.2014.10.047 0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.
(TCMGARs) are biosafe and possess higher amounts of ginsenosides when compared to cultivated ginseng (Sivakumar et al., 2006). Various products such as ginseng powder, sirup, wine, tablets, ginseng-based cosmetics and dietary supplements made out of TCMGARs are available in the market (Murthy et al., 2014a). Presently, our research work is focused on the analysis of efficacy tests of TCMGARs and recently, the anti-glycemic effect of TCMGARs is reported (Murthy et al., 2014b). In the present study, we aimed to investigate the efficacy of TCMGARs on liver function and its hepatoprotective activity in mice against the carbon tetrachloride stress on hepatocytes.
2. Materials and methods 2.1. Material and preparation of extracts Panax ginseng C. A. Meyer (Araliaceae) plant material was collected from Mt. Odaesan, Gangwon province, Republic of Korea and the herbarium is deposited with voucher number-2002-5761 in Korean Simmani Society, South Korea. The adventitious roots (tissue cultured mountain ginseng adventitious roots) and cultivated Korean ginseng roots (positive control) were powdered and soaked in 70% aqueous ethanol for 10 days at 25 1C and filtered.
H. Niranjana Murthy et al. / Journal of Ethnopharmacology 158 (2014) 442–446
The solution evaporated in vacuo gave a semi-gelatinous extract and the yield of crude ginsenosides was 128.51 mg/g and 100.08 mg/g from ginseng adventitious roots and cultivated ginseng roots respectively.
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(Bergmeyer et al., 1976), alkaline phosphatase (ALP) (Bergmeyer, 1980) assays were carried out. Glutathione (GSH) level was estimated as per the protocol of Moron et al. (1979). Lipid peroxidation in liver was measured by the formation of malondialdehyde (MDA) and it was estimated by using the procedures of Ohakawa et al. (1979).
2.2. Determination of ginsenoside content The chemical profiling of ginsenosides in these two materials was carried out by high pressure liquid chromatography (HPLC) in Korean Food Research Institute, Sungnam-Si, Republic of Korea (File no. AO2012-06-26-200). Extraction and analysis of ginsenosides were carried out by the method of Yu et al. (2002). The ginsenoside fraction was analyzed using an HPLC system (Shimadzu, Kyoto) consisting of 10AT pump, 10AXL autosampler, SPDM10A photodiode array detector, and CTO-10A column oven, 5 mM Lichrosorb column (250 4.6 mm2) (Altech, Deerfild, IL), and a C18 guard column, at 40 1C. The eluted peaks were detected at 203 nm and quantified against external standards of ginsenosides Rf, Rb2, Rd, (Karl Roth, Germany), Re, Rg1, Rg2, Rh1, Rh2, Rb1, Rb3, Rc and Rg2 (Wako, Osaka, Japan). The mobile phase was a gradient elution of water (A) and acetonitrile (B), commencing with 20% B, rising to 22% B after 20 min then to 46% after 45 min and 55% B after 50 min. 2.3. Experimental animals Hepatotoxicity experiments were conducted in Biotoxtech Laboratories, South Korea on Sprague–Dawley male rats. All the animals involved in the experiment were maintained on a standard diet and kept in a room maintained under controlled conditions of 2471 1C temperature and 12 h light: 12 h dark cycles. The animals had free access to water and a standard diet [a normal laboratory commercial stock diet containing 16% protein, 56% carbohydrate, and 8% fat (w/w)]. All animal procedures were conducted in accordance with legal requirements appropriate to the species and with the approval by the local Ethical Committee with an ethical clearance number, CBNUR-188-1001. 2.4. Treatment of animals In the treatment studies, animals were divided into six groups of seven animals each (G). G1 was served normal control; G2 was CCl4 control and G3, G4 and G5 were treated with tissue cultured mountain ginseng extract at a dose of 30, 100 and 300 mg/kg body weight, respectively. G6 animals were treated with cultivated ginseng extract of 100 mg/kg body weight. All the grouped animals were maintained with above feeding schedule for 14 days. Further, the rats were administered with CCl4, which is a model agent to induce hepatic lesions. A single dose of CCl4 leads to centrizonal necrosis and steatosis (Pierce et al., 1987), while prolonged administration leads to liver fibrosis, cirrhosis, and hepato-cellular carcinoma (Perez, 1983). Acute oral dose of administration consisted of 0.5 ml/kg of 1:1 (v/v) mixture of CCl4 and corn oil (Smile et al., 2001). Group 2–6 received single dose of CCl4 on day 14. Normal control received equal amount of corn oil instead of CCl4. All the rats were sacrificed after 24 h of CCl4 administration. 2.5. Assessment of liver functions Blood was collected from the blood vessels of neck region under mild ether anesthesia and kept for 30 min at 4 1C. Serum was separated by centrifugation at 2500 rpm at 4 1C for 15 min. The liver was removed rapidly and cut into separate portions for hepatic glutathione, lipid peroxidation estimation. Alanine aminotransferase (ALT) (Amador et al., 1963), aspartate aminotransferase (AST)
2.6. Statistical analysis All values are expressed as mean with standard error. The results were statistically analyzed by using Analysis of variance (ANOVA) to note the significance among different groups and further Student's t-test was carried out to find out the significant differences between two groups.
3. Results 3.1. Standardization of ginsenosides present in the extract The high pressure liquid chromatography (HPLC) analysis data on ginsenosides of TCMGARs and Korean ginseng is presented in Table 1. The results revealed that Rb1, Rb2, Rb3, Rc, Rd, Re, Rg1, Rg2, Rg3, Rh1, Rh2 and Rf ginsenosides were present in the extract of TCMGARs. The ginsenosides present in the Korean cultivated ginseng root extract were Rb1, Rb2, Rc, Rd, Re, Rg1, and Rf. Ginsenosides such as Rb3, Rg2, Rg3, Rh1 and Rh2 which were not present in the Korean ginseng roots were abundant in the TCMGARs, the content of other ginsenosides are also at higher levels in TCMGARs when compared to Korean cultivated ginseng (Table 1; Fig. 1).
3.2. Evaluation of AST, ALT and ALP In the rats, which were administered with CCl4 showed an elevated level of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) levels. Treatment with the extract of TCMGARs at a dose of 100 and 300 mg/kg as well as the treatment with the extract of cultivated ginseng at a dose of 100 mg/kg significantly (p r0.05) provided the resistance against the CCl4 stress in rats. Korean ginseng extract which was used as positive control also showed a remarkable protection towards CCl4 intoxication (Table 2). Table 1 Ginsenoside content of tissue cultured mountain ginseng adventitious roots in comparison with cultivated Korean ginseng.a Ginsenoside
Rb1 Rb2 Rb3 Rc Rd Re Rg1 Rg2 Rg3 Rh1 Rh2 Rf
Content (mg/g dry weight) TCMGARs
Korean ginseng
4.1 2.3 0.9 2.0 4.2 0.2 0.19 0.38 11.2 0.49 3.8 1.64
0.05 0.06 – 0.03 0.06 0.24 0.17 – – – – 0.03
a Analysis was carried out by Korea Food Research Institute, Sungnam-si, Republic of Korea.
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H. Niranjana Murthy et al. / Journal of Ethnopharmacology 158 (2014) 442–446
Fig. 1. HPLC chromatogram of field cultivated ginseng (above) and tissue cultured mountain ginseng adventitious roots (below).
Table 2 Effect of ginseng adventitious root extract on CCl4-induced liver damage in rats. Treatment groups G1 Normal control
G2 CCl4 control, (0.5 ml/kg),
G3 TCMGARa (30 mg/kg)þ CCl4
G4 TCMGAR (100 mg/kg) þCCl4
G5 TCMGAR (300 mg/kg)þ CCl4
G6 Cultivated ginseng, 100 mg/ kgþ CCl4
ALT (U/L) Mean 24.2 SEM 1.1 N 7 Mean 118.3n SEM 30.7 N 7 Mean 110.3 SEM 32.3 N 7 Mean 40.2† SEM 6.6 N 7 Mean 40.7† SEM 8.7 N 7 Mean 57.1† SEM 6.5 N 7
AST (U/L)
ALP (U/L)
173.7 9.9 7 397.1n 52.5 7 261.8 53.7 7 216.0† 29.5 7 186.5† 10.9 7 187.0† 20.9 7
221.2 19.9 7 287.3n 14.5 7 234.9 27.4 7 237.8† 20.7 7 223.5† 19.3 7 211.4† 22.3 7
N ¼Number of animals. Values are expressed as mean7 S.E. a n
†
TCMGAR – Tissue cultured mountain ginseng adventitious root extract. po 0.05 vs normal control. po 0.05 vs CCl4 control.
3.3. Estimation of reduced lipid peroxides and glutathione (GSH) levels Rats administered with CCl4 alone were found to have significantly (pr0.05) lower hepatic glutathione (GSH level). Treatment with ethanol extract of TCMGARs as well as with Korean ginseng extracts significantly increased the hepatic glutathione levels (Table 3). In the various preventive treatments, rats treated with 300 mg/kg ethanolic extract showed an increase in glutathione levels in liver. The significant increase (p r0.05) in tissue MDA level was observed in CCl4 alone treated rats. However, the CCl4-induced elevation of tissue malondialdehyde (MDA) concentration was lowered significantly (p r0.05) with the administration of the extract to rats. In the ethanolic extract-treated rats, MDA levels remarkably reduced which are comparable to the Korean ginseng treatment (Table 3).
4. Discussion Carbon tetrachloride has been one of the most intensively studied hepatotoxic agents and the effect of CCl4 on hepatocytes has been attributed to its metabolism by cytochrome P450 to yield toxic trichloromethyl radicals that act as free radical indicators (Clawson, 1989; Dhum and Jones, 1996; Weber et al., 2003). Elevation of serum AST, ALT and ALP indicates the toxicity which specifically affects the liver (Anand et al., 1992) and activities of AST and ALT are most commonly used as biochemical markers for liver damage (Sturgill and Lambert, 1997). Since these enzymes are cytoplasmic in nature, upon liver injury these enzymes enter into the circulatory system due to altered permeability of membrane (Zimmerman and Seeff, 1970). Administration of butanolic extract of TCMGARs as well as the cultivated ginseng significantly prevented CCl4 induced elevation of AST, ALT and ALP indicating the hepatoprotective activity of TCMGARs and also the cultivated ginseng. The administration of 300 mg/kg TCMGARs and 100 mg/kg cultivated ginseng extract significantly lowered the AST, ALT and ALP levels (Table 2). The HPLC analysis demonstrated that TCMGARs possessed novel ginsenosides such as Rb3, Rg2, Rg3, Rh1 and Rh2. TCMGARs also had varied other ginsenosides in very high concentrations (Table 1). Over accumulation of ginsenosides in TCMGARs was due to methyl jasmonate elicitation during the production of tissue cultured mountain ginseng adventitious roots. It is reported that treatment of cultures with 100 mM methyl jasmonate has boosted the accumulation of ginsenosides by 5–6 folds in the cultures (Yu et al., 2002; Kim et al., 2004). Tissue cultured mountain ginseng adventitious roots were not only rich in ginsenosides but also they contained biophenols and enzymatic and non-enzymatic antioxidant enzymes due to methyl jasmonate elicitation (Sivakumar and Paek, 2005; Ali et al., 2006). In the current results, due to richness of varied ginsenosides, biophenols and antioxidant enzymes, the tissue cultured mountain ginseng adventitious root extracts have been useful in providing the resistance against the CCl4 stress in liver cells. Korean cultivated ginseng extract was also efficient in combating the toxic effects of CCl4 on hepatocytes of rats (Table 2). Similarly, hepatoprotective effects of cultivated Korean ginseng were on records (Jeong et al., 1977; Kang et al., 2007; Shukla and Kumar, 2009). It was reported that ginsenoside Rb1 was known for hepatoprotective function (Lee et al., 2005) and the content of Rb1 was 0.05 mg/g dry weight in Korean ginseng, its level was 4.1 mg/g dry weight in tissue cultured
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Table 3 Effect of ginseng adventitious root extracts on GSH and MDA in CCl4-induced liver damage in rats. Treatment groups
Dose (mg/kg)
G1 Normal control
0
G2 CCl4 control
0.5 ml/kg
G3 TCMGARa þCCl4
30
G4 TCMGAR þ CCl4
100
G5 TCMGAR þ CCl4
300
G6 Cultivated ginsengþ CCl4
100
Mean SEM N Mean SEM N Mean SEM N Mean SEM N Mean SEM N Mean SEM N
GSH-peroxidase activity (m Unit/min/mg protein)
Malondialdehyde (MDA, nmol/g liver)
204.0 5.4 7 185.7n 3.9 7 191.0† 9.1 7 196.1† 10.3 7 201.5† 12.7 7 198.1† 8.1 7
150.7 8.6 7 207.4n 6.6 7 160.5† 8.4 7 173.6† 7.3 7 150.5† 8.4 7 156.3† 6.6 7
N ¼Number of animals. Values are expressed as mean7 S.E. a n
†
TCMGAR – Tissue cultured mountain ginseng adventitious root extract. po 0.05 vs normal control. p o0.05 vs CCl4 control.
mountain ginseng adventitious roots (Table 1); it is obvious that the hepatoprotective activity of tissue cultured mountain ginseng adventitious roots extract is as significant as Korean cultivated ginseng extracts (Table 2). Reduction in liver GSH in CCl4 control rats as observed in this study indicates the damage to hepatic cells. Rats administered with CCl4 significantly lowered the hepatic glutathione (GSH) level. The biochemical function of glutathione peroxidase is to reduce lipid hydroperoxides to their corresponding alcohols and to reduce free hydrogen peroxide into water for the protection of cells from oxidative damage (Ali et al., 2005). Treatment of ethanolic ginseng extract exhibited significant increase in hepatic glutathione levels (Table 3). Rats treated with cultivated Korean ginseng extract prevented the lowering of hepatic GSH. In various treatments in the current study, rats treated with 300 mg/kg methanolic extract showed the increased glutathione levels in the liver. Increase in GSH activity with the treatment of plant extracts are in accordance with the reports of Raja et al. (2007) and Wills and Asha (2006). Increase in tissue MDA was observed in CCl4 alone treated rats. However, CCl4 induced elevation of tissue MDA concentration was lowered by pretreatment of rats with the ginseng extract (Table 3). Decrease in lipid peroxidation i.e., decrease in MDA levels with administration of ethanolic extracts of 100 mg/kg cultivated ginseng and 300 mg/kg tissue cultured mountain ginseng adventitious roots and increase in antioxidant level as indicated by the increase in GSH level specifies the hepatoprotective nature of tissue cultured mountain ginseng adventitious roots as well as cultivated ginseng. The results of serum biochemical parameters, levels of hepatic lipid peroxides, and hepatic glutathione studies in rats which were treated with TCMGARs extract together support the hepatoprotective and antioxidant activities of tissue cultured mountain ginseng adventitious roots. Most pharmacological actions of ginseng are attributed to ginsenosides, which can act in a wide range of tissues (Park et al., 2005) and tissue cultured mountain ginseng adventitious roots contain higher concentrations of ginsenosides especially Rb1 ginsenoside (4.1 mg/g dry weight) which is reported to be hepatoprotective compound (Lee et al., 2005) and these results showed the efficacy of tissue cultured mountain ginseng adventitious roots.
Acknowledgments This study was supported by a grant of Korea Healthcare Technology R&D project, Ministry of Health and Welfare, Republic of Korea (Grant no. A103017). Dr. H. N. Murthy is thankful to Ministry of Education, Science and Technology, Republic of Korea for the award of Brain Pool Fellowship (131S-4-3-0523) and this paper was studied with the support of Ministry of Science, ICT and Planning (MSIP). We are thankful to Prof. Yerivanthelimath, Department of English, Karnatak University, Dharwad, India for language editing of the manuscript.
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Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
Hepatoprotective activity of ginsenosides from Panax ginseng adventitious roots against carbon tetrachloride treated hepatic injury in rats Hosakatte Niranjana Murthy a,b,n, Vijayalaxmi S. Dandin b, Kee Yoeup Paek a,n a b
Research Center for the Development of Advanced Horticultural Technology, Chungbuk National University, Cheongju 361-763, Republic of Korea Department of Botany, Karnatak University, Dharwad 580003, India
art ic l e i nf o
a b s t r a c t
Article history: Received 8 July 2014 Received in revised form 21 September 2014 Accepted 17 October 2014 Available online 31 October 2014
Ethnopharmacological relevance: Ginseng (Panax ginseng C. A. Meyer) has a beneficial role in the treatment of various diseases including liver disorders like acute/chronic hepatotoxicity, hepatitis, hepatic fibrosis/ cirrhosis and hepatocellular carcinoma. Materials and methods: Tissue culture raised mountain ginseng adventitious root (TCMGARs) extract with ginsenosides in abundance was used as an experimental material. ‘Sprague–Dawley’ male rats were used as experimental systems and were fed with TCMGARs extracts at doses of 30, 100, 300 mg/kg body weight for two weeks to test the effect on carbon tetrachloride (CCl4) induced acute liver damage. Field cultivated Korean ginseng root extract fed rats (100 mg/kg) were used as positive control. Plasma enzyme levels, alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) were assessed. Glutathione (GSH) and malondialdehyde (MDA) concentrations were also evaluated. Results: TCMGARs extracts remarkably prevented the elevation of ALT, AST, ALP and liver peroxides in CCl4treated rats. Hepatic glutathione levels were significantly increased by the treatment with the extracts in experimental groups. Conclusion: The TCMGARs rich in varied ginsenosides can afford protection against CCl4-induced hepatocellular injury. & 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Ginsenosides Glutathione Hepatoprotective activity Lipid peroxidation Mountain ginseng
1. Introduction Panax ginseng C. A. Meyer is an important herbal medicine in East Asian countries. It is used in traditional medicine to promote health and healing, as an adaptogen and a stimulant. The major components of ginseng are triterpenoidal dammarane glycosides called ginsenosides. Pharmacological effects of ginseng have been demonstrated in cancer, diabetes mellitus, cardiovascular system, immune system and central nervous system including anti-stress and anti-oxidant activities (Park et al., 2005). Wild ginseng is a scanty and expensive commodity. To cultivate ginseng in fields, it needs 5–7 years till harvesting stage during which the plant may suffer from various diseases and pests. Adventitious roots have been induced from 100 year old ginseng and the roots have been cultivated in vitro in large scale bioreactors for the use of pharmaceutical industry (Paek et al., 2009). Toxicological evaluation has proved that the tissue cultured mountain ginseng adventitious roots n Corresponding authors at: Research Center for the Development of Advanced Horticultural Technology, Chungbuk National University, Cheongju 361-763, Republic of Korea. Tel.: þ 82 43 266 3245; fax: þ 82 43 266 3246. E-mail addresses: [email protected] (H. Niranjana Murthy), [email protected] (K. Yoeup Paek).
http://dx.doi.org/10.1016/j.jep.2014.10.047 0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.
(TCMGARs) are biosafe and possess higher amounts of ginsenosides when compared to cultivated ginseng (Sivakumar et al., 2006). Various products such as ginseng powder, sirup, wine, tablets, ginseng-based cosmetics and dietary supplements made out of TCMGARs are available in the market (Murthy et al., 2014a). Presently, our research work is focused on the analysis of efficacy tests of TCMGARs and recently, the anti-glycemic effect of TCMGARs is reported (Murthy et al., 2014b). In the present study, we aimed to investigate the efficacy of TCMGARs on liver function and its hepatoprotective activity in mice against the carbon tetrachloride stress on hepatocytes.
2. Materials and methods 2.1. Material and preparation of extracts Panax ginseng C. A. Meyer (Araliaceae) plant material was collected from Mt. Odaesan, Gangwon province, Republic of Korea and the herbarium is deposited with voucher number-2002-5761 in Korean Simmani Society, South Korea. The adventitious roots (tissue cultured mountain ginseng adventitious roots) and cultivated Korean ginseng roots (positive control) were powdered and soaked in 70% aqueous ethanol for 10 days at 25 1C and filtered.
H. Niranjana Murthy et al. / Journal of Ethnopharmacology 158 (2014) 442–446
The solution evaporated in vacuo gave a semi-gelatinous extract and the yield of crude ginsenosides was 128.51 mg/g and 100.08 mg/g from ginseng adventitious roots and cultivated ginseng roots respectively.
443
(Bergmeyer et al., 1976), alkaline phosphatase (ALP) (Bergmeyer, 1980) assays were carried out. Glutathione (GSH) level was estimated as per the protocol of Moron et al. (1979). Lipid peroxidation in liver was measured by the formation of malondialdehyde (MDA) and it was estimated by using the procedures of Ohakawa et al. (1979).
2.2. Determination of ginsenoside content The chemical profiling of ginsenosides in these two materials was carried out by high pressure liquid chromatography (HPLC) in Korean Food Research Institute, Sungnam-Si, Republic of Korea (File no. AO2012-06-26-200). Extraction and analysis of ginsenosides were carried out by the method of Yu et al. (2002). The ginsenoside fraction was analyzed using an HPLC system (Shimadzu, Kyoto) consisting of 10AT pump, 10AXL autosampler, SPDM10A photodiode array detector, and CTO-10A column oven, 5 mM Lichrosorb column (250 4.6 mm2) (Altech, Deerfild, IL), and a C18 guard column, at 40 1C. The eluted peaks were detected at 203 nm and quantified against external standards of ginsenosides Rf, Rb2, Rd, (Karl Roth, Germany), Re, Rg1, Rg2, Rh1, Rh2, Rb1, Rb3, Rc and Rg2 (Wako, Osaka, Japan). The mobile phase was a gradient elution of water (A) and acetonitrile (B), commencing with 20% B, rising to 22% B after 20 min then to 46% after 45 min and 55% B after 50 min. 2.3. Experimental animals Hepatotoxicity experiments were conducted in Biotoxtech Laboratories, South Korea on Sprague–Dawley male rats. All the animals involved in the experiment were maintained on a standard diet and kept in a room maintained under controlled conditions of 2471 1C temperature and 12 h light: 12 h dark cycles. The animals had free access to water and a standard diet [a normal laboratory commercial stock diet containing 16% protein, 56% carbohydrate, and 8% fat (w/w)]. All animal procedures were conducted in accordance with legal requirements appropriate to the species and with the approval by the local Ethical Committee with an ethical clearance number, CBNUR-188-1001. 2.4. Treatment of animals In the treatment studies, animals were divided into six groups of seven animals each (G). G1 was served normal control; G2 was CCl4 control and G3, G4 and G5 were treated with tissue cultured mountain ginseng extract at a dose of 30, 100 and 300 mg/kg body weight, respectively. G6 animals were treated with cultivated ginseng extract of 100 mg/kg body weight. All the grouped animals were maintained with above feeding schedule for 14 days. Further, the rats were administered with CCl4, which is a model agent to induce hepatic lesions. A single dose of CCl4 leads to centrizonal necrosis and steatosis (Pierce et al., 1987), while prolonged administration leads to liver fibrosis, cirrhosis, and hepato-cellular carcinoma (Perez, 1983). Acute oral dose of administration consisted of 0.5 ml/kg of 1:1 (v/v) mixture of CCl4 and corn oil (Smile et al., 2001). Group 2–6 received single dose of CCl4 on day 14. Normal control received equal amount of corn oil instead of CCl4. All the rats were sacrificed after 24 h of CCl4 administration. 2.5. Assessment of liver functions Blood was collected from the blood vessels of neck region under mild ether anesthesia and kept for 30 min at 4 1C. Serum was separated by centrifugation at 2500 rpm at 4 1C for 15 min. The liver was removed rapidly and cut into separate portions for hepatic glutathione, lipid peroxidation estimation. Alanine aminotransferase (ALT) (Amador et al., 1963), aspartate aminotransferase (AST)
2.6. Statistical analysis All values are expressed as mean with standard error. The results were statistically analyzed by using Analysis of variance (ANOVA) to note the significance among different groups and further Student's t-test was carried out to find out the significant differences between two groups.
3. Results 3.1. Standardization of ginsenosides present in the extract The high pressure liquid chromatography (HPLC) analysis data on ginsenosides of TCMGARs and Korean ginseng is presented in Table 1. The results revealed that Rb1, Rb2, Rb3, Rc, Rd, Re, Rg1, Rg2, Rg3, Rh1, Rh2 and Rf ginsenosides were present in the extract of TCMGARs. The ginsenosides present in the Korean cultivated ginseng root extract were Rb1, Rb2, Rc, Rd, Re, Rg1, and Rf. Ginsenosides such as Rb3, Rg2, Rg3, Rh1 and Rh2 which were not present in the Korean ginseng roots were abundant in the TCMGARs, the content of other ginsenosides are also at higher levels in TCMGARs when compared to Korean cultivated ginseng (Table 1; Fig. 1).
3.2. Evaluation of AST, ALT and ALP In the rats, which were administered with CCl4 showed an elevated level of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) levels. Treatment with the extract of TCMGARs at a dose of 100 and 300 mg/kg as well as the treatment with the extract of cultivated ginseng at a dose of 100 mg/kg significantly (p r0.05) provided the resistance against the CCl4 stress in rats. Korean ginseng extract which was used as positive control also showed a remarkable protection towards CCl4 intoxication (Table 2). Table 1 Ginsenoside content of tissue cultured mountain ginseng adventitious roots in comparison with cultivated Korean ginseng.a Ginsenoside
Rb1 Rb2 Rb3 Rc Rd Re Rg1 Rg2 Rg3 Rh1 Rh2 Rf
Content (mg/g dry weight) TCMGARs
Korean ginseng
4.1 2.3 0.9 2.0 4.2 0.2 0.19 0.38 11.2 0.49 3.8 1.64
0.05 0.06 – 0.03 0.06 0.24 0.17 – – – – 0.03
a Analysis was carried out by Korea Food Research Institute, Sungnam-si, Republic of Korea.
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Fig. 1. HPLC chromatogram of field cultivated ginseng (above) and tissue cultured mountain ginseng adventitious roots (below).
Table 2 Effect of ginseng adventitious root extract on CCl4-induced liver damage in rats. Treatment groups G1 Normal control
G2 CCl4 control, (0.5 ml/kg),
G3 TCMGARa (30 mg/kg)þ CCl4
G4 TCMGAR (100 mg/kg) þCCl4
G5 TCMGAR (300 mg/kg)þ CCl4
G6 Cultivated ginseng, 100 mg/ kgþ CCl4
ALT (U/L) Mean 24.2 SEM 1.1 N 7 Mean 118.3n SEM 30.7 N 7 Mean 110.3 SEM 32.3 N 7 Mean 40.2† SEM 6.6 N 7 Mean 40.7† SEM 8.7 N 7 Mean 57.1† SEM 6.5 N 7
AST (U/L)
ALP (U/L)
173.7 9.9 7 397.1n 52.5 7 261.8 53.7 7 216.0† 29.5 7 186.5† 10.9 7 187.0† 20.9 7
221.2 19.9 7 287.3n 14.5 7 234.9 27.4 7 237.8† 20.7 7 223.5† 19.3 7 211.4† 22.3 7
N ¼Number of animals. Values are expressed as mean7 S.E. a n
†
TCMGAR – Tissue cultured mountain ginseng adventitious root extract. po 0.05 vs normal control. po 0.05 vs CCl4 control.
3.3. Estimation of reduced lipid peroxides and glutathione (GSH) levels Rats administered with CCl4 alone were found to have significantly (pr0.05) lower hepatic glutathione (GSH level). Treatment with ethanol extract of TCMGARs as well as with Korean ginseng extracts significantly increased the hepatic glutathione levels (Table 3). In the various preventive treatments, rats treated with 300 mg/kg ethanolic extract showed an increase in glutathione levels in liver. The significant increase (p r0.05) in tissue MDA level was observed in CCl4 alone treated rats. However, the CCl4-induced elevation of tissue malondialdehyde (MDA) concentration was lowered significantly (p r0.05) with the administration of the extract to rats. In the ethanolic extract-treated rats, MDA levels remarkably reduced which are comparable to the Korean ginseng treatment (Table 3).
4. Discussion Carbon tetrachloride has been one of the most intensively studied hepatotoxic agents and the effect of CCl4 on hepatocytes has been attributed to its metabolism by cytochrome P450 to yield toxic trichloromethyl radicals that act as free radical indicators (Clawson, 1989; Dhum and Jones, 1996; Weber et al., 2003). Elevation of serum AST, ALT and ALP indicates the toxicity which specifically affects the liver (Anand et al., 1992) and activities of AST and ALT are most commonly used as biochemical markers for liver damage (Sturgill and Lambert, 1997). Since these enzymes are cytoplasmic in nature, upon liver injury these enzymes enter into the circulatory system due to altered permeability of membrane (Zimmerman and Seeff, 1970). Administration of butanolic extract of TCMGARs as well as the cultivated ginseng significantly prevented CCl4 induced elevation of AST, ALT and ALP indicating the hepatoprotective activity of TCMGARs and also the cultivated ginseng. The administration of 300 mg/kg TCMGARs and 100 mg/kg cultivated ginseng extract significantly lowered the AST, ALT and ALP levels (Table 2). The HPLC analysis demonstrated that TCMGARs possessed novel ginsenosides such as Rb3, Rg2, Rg3, Rh1 and Rh2. TCMGARs also had varied other ginsenosides in very high concentrations (Table 1). Over accumulation of ginsenosides in TCMGARs was due to methyl jasmonate elicitation during the production of tissue cultured mountain ginseng adventitious roots. It is reported that treatment of cultures with 100 mM methyl jasmonate has boosted the accumulation of ginsenosides by 5–6 folds in the cultures (Yu et al., 2002; Kim et al., 2004). Tissue cultured mountain ginseng adventitious roots were not only rich in ginsenosides but also they contained biophenols and enzymatic and non-enzymatic antioxidant enzymes due to methyl jasmonate elicitation (Sivakumar and Paek, 2005; Ali et al., 2006). In the current results, due to richness of varied ginsenosides, biophenols and antioxidant enzymes, the tissue cultured mountain ginseng adventitious root extracts have been useful in providing the resistance against the CCl4 stress in liver cells. Korean cultivated ginseng extract was also efficient in combating the toxic effects of CCl4 on hepatocytes of rats (Table 2). Similarly, hepatoprotective effects of cultivated Korean ginseng were on records (Jeong et al., 1977; Kang et al., 2007; Shukla and Kumar, 2009). It was reported that ginsenoside Rb1 was known for hepatoprotective function (Lee et al., 2005) and the content of Rb1 was 0.05 mg/g dry weight in Korean ginseng, its level was 4.1 mg/g dry weight in tissue cultured
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Table 3 Effect of ginseng adventitious root extracts on GSH and MDA in CCl4-induced liver damage in rats. Treatment groups
Dose (mg/kg)
G1 Normal control
0
G2 CCl4 control
0.5 ml/kg
G3 TCMGARa þCCl4
30
G4 TCMGAR þ CCl4
100
G5 TCMGAR þ CCl4
300
G6 Cultivated ginsengþ CCl4
100
Mean SEM N Mean SEM N Mean SEM N Mean SEM N Mean SEM N Mean SEM N
GSH-peroxidase activity (m Unit/min/mg protein)
Malondialdehyde (MDA, nmol/g liver)
204.0 5.4 7 185.7n 3.9 7 191.0† 9.1 7 196.1† 10.3 7 201.5† 12.7 7 198.1† 8.1 7
150.7 8.6 7 207.4n 6.6 7 160.5† 8.4 7 173.6† 7.3 7 150.5† 8.4 7 156.3† 6.6 7
N ¼Number of animals. Values are expressed as mean7 S.E. a n
†
TCMGAR – Tissue cultured mountain ginseng adventitious root extract. po 0.05 vs normal control. p o0.05 vs CCl4 control.
mountain ginseng adventitious roots (Table 1); it is obvious that the hepatoprotective activity of tissue cultured mountain ginseng adventitious roots extract is as significant as Korean cultivated ginseng extracts (Table 2). Reduction in liver GSH in CCl4 control rats as observed in this study indicates the damage to hepatic cells. Rats administered with CCl4 significantly lowered the hepatic glutathione (GSH) level. The biochemical function of glutathione peroxidase is to reduce lipid hydroperoxides to their corresponding alcohols and to reduce free hydrogen peroxide into water for the protection of cells from oxidative damage (Ali et al., 2005). Treatment of ethanolic ginseng extract exhibited significant increase in hepatic glutathione levels (Table 3). Rats treated with cultivated Korean ginseng extract prevented the lowering of hepatic GSH. In various treatments in the current study, rats treated with 300 mg/kg methanolic extract showed the increased glutathione levels in the liver. Increase in GSH activity with the treatment of plant extracts are in accordance with the reports of Raja et al. (2007) and Wills and Asha (2006). Increase in tissue MDA was observed in CCl4 alone treated rats. However, CCl4 induced elevation of tissue MDA concentration was lowered by pretreatment of rats with the ginseng extract (Table 3). Decrease in lipid peroxidation i.e., decrease in MDA levels with administration of ethanolic extracts of 100 mg/kg cultivated ginseng and 300 mg/kg tissue cultured mountain ginseng adventitious roots and increase in antioxidant level as indicated by the increase in GSH level specifies the hepatoprotective nature of tissue cultured mountain ginseng adventitious roots as well as cultivated ginseng. The results of serum biochemical parameters, levels of hepatic lipid peroxides, and hepatic glutathione studies in rats which were treated with TCMGARs extract together support the hepatoprotective and antioxidant activities of tissue cultured mountain ginseng adventitious roots. Most pharmacological actions of ginseng are attributed to ginsenosides, which can act in a wide range of tissues (Park et al., 2005) and tissue cultured mountain ginseng adventitious roots contain higher concentrations of ginsenosides especially Rb1 ginsenoside (4.1 mg/g dry weight) which is reported to be hepatoprotective compound (Lee et al., 2005) and these results showed the efficacy of tissue cultured mountain ginseng adventitious roots.
Acknowledgments This study was supported by a grant of Korea Healthcare Technology R&D project, Ministry of Health and Welfare, Republic of Korea (Grant no. A103017). Dr. H. N. Murthy is thankful to Ministry of Education, Science and Technology, Republic of Korea for the award of Brain Pool Fellowship (131S-4-3-0523) and this paper was studied with the support of Ministry of Science, ICT and Planning (MSIP). We are thankful to Prof. Yerivanthelimath, Department of English, Karnatak University, Dharwad, India for language editing of the manuscript.
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