Hepatoprotective activity of ginsenosides from Panax ginseng adventitiousroots against carbon tetrachloride treated hepatic injury in rats

Hosakatte Niranjana Murthy a,b,n, Vijayalaxmi S. Dandin b, Kee Yoeup Paek a,n

a Research Center for the Development of Advanced Horticultural Technology, Chungbuk National University, Cheongju 361-763, Republic of Koreab Department of Botany, Karnatak University, Dharwad 580003, India

a r t i c l e i n f o

Article history:Received 8 July 2014Received in revised form21 September 2014Accepted 17 October 2014Available online 31 October 2014

Keywords:GinsenosidesGlutathioneHepatoprotective activityLipid peroxidationMountain ginseng

a b s t r a c t

Ethnopharmacological relevance: Ginseng (Panax ginseng C. A. Meyer) has a beneficial role in the treatmentof 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 withginsenosides in abundance was used as an experimental material. ‘Sprague–Dawley’ male rats were used asexperimental systems and were fed with TCMGARs extracts at doses of 30, 100, 300 mg/kg body weight fortwo weeks to test the effect on carbon tetrachloride (CCl4) induced acute liver damage. Field cultivatedKorean 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) wereassessed. Glutathione (GSH) and malondialdehyde (MDA) concentrations were also evaluated.Results: TCMGARs extracts remarkably prevented the elevation of ALT, AST, ALP and liver peroxides in CCl4-treated rats. Hepatic glutathione levels were significantly increased by the treatment with the extracts inexperimental groups.Conclusion: The TCMGARs rich in varied ginsenosides can afford protection against CCl4-induced hepatocel-lular injury.

& 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Panax ginseng C. A. Meyer is an important herbal medicine inEast Asian countries. It is used in traditional medicine to promotehealth and healing, as an adaptogen and a stimulant. The majorcomponents of ginseng are triterpenoidal dammarane glycosidescalled ginsenosides. Pharmacological effects of ginseng have beendemonstrated in cancer, diabetes mellitus, cardiovascular system,immune system and central nervous system including anti-stressand anti-oxidant activities (Park et al., 2005). Wild ginseng is ascanty and expensive commodity. To cultivate ginseng in fields, itneeds 5–7 years till harvesting stage during which the plant maysuffer from various diseases and pests. Adventitious roots havebeen induced from 100 year old ginseng and the roots have beencultivated in vitro in large scale bioreactors for the use of pharmaceu-tical industry (Paek et al., 2009). Toxicological evaluation has provedthat the tissue cultured mountain ginseng adventitious roots

(TCMGARs) are biosafe and possess higher amounts of ginsenosideswhen compared to cultivated ginseng (Sivakumar et al., 2006). Variousproducts such as ginseng powder, sirup, wine, tablets, ginseng-basedcosmetics and dietary supplements made out of TCMGARs are avail-able in the market (Murthy et al., 2014a). Presently, our research workis 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 ofTCMGARs on liver function and its hepatoprotective activity inmice 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 wascollected from Mt. Odaesan, Gangwon province, Republic of Koreaand the herbarium is deposited with voucher number-2002-5761in Korean Simmani Society, South Korea. The adventitious roots(tissue cultured mountain ginseng adventitious roots) and culti-vated Korean ginseng roots (positive control) were powdered andsoaked in 70% aqueous ethanol for 10 days at 25 1C and filtered.

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Journal of Ethnopharmacology

http://dx.doi.org/10.1016/j.jep.2014.10.0470378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.

n Corresponding authors at: Research Center for the Development of AdvancedHorticultural Technology, Chungbuk National University, Cheongju 361-763,Republic of Korea. Tel.: þ82 43 266 3245; fax: þ82 43 266 3246.

E-mail addresses: nmurthy60@yahoo.co.in (H. Niranjana Murthy),paekky@chungbuk.ac.kr (K. Yoeup Paek).

Journal of Ethnopharmacology 158 (2014) 442–446

The solution evaporated in vacuo gave a semi-gelatinous extractand the yield of crude ginsenosides was 128.51 mg/g and100.08 mg/g from ginseng adventitious roots and cultivated gin-seng roots respectively.

2.2. Determination of ginsenoside content

The chemical profiling of ginsenosides in these two materialswas carried out by high pressure liquid chromatography (HPLC) inKorean Food Research Institute, Sungnam-Si, Republic of Korea(File no. AO2012-06-26-200). Extraction and analysis of ginseno-sides were carried out by the method of Yu et al. (2002). Theginsenoside fraction was analyzed using an HPLC system (Shi-madzu, 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), anda C18 guard column, at 40 1C. The eluted peaks were detected at203 nm and quantified against external standards of ginsenosidesRf, Rb2, Rd, (Karl Roth, Germany), Re, Rg1, Rg2, Rh1, Rh2, Rb1, Rb3,Rc and Rg2 (Wako, Osaka, Japan). The mobile phase was a gradientelution 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% Bafter 50 min.

2.3. Experimental animals

Hepatotoxicity experiments were conducted in BiotoxtechLaboratories, South Korea on Sprague–Dawley male rats. All theanimals involved in the experiment were maintained on a stan-dard diet and kept in a room maintained under controlledconditions of 2471 1C temperature and 12 h light: 12 h darkcycles. The animals had free access to water and a standard diet[a normal laboratory commercial stock diet containing 16% pro-tein, 56% carbohydrate, and 8% fat (w/w)]. All animal procedureswere conducted in accordance with legal requirements appropri-ate to the species and with the approval by the local EthicalCommittee with an ethical clearance number, CBNUR-188-1001.

2.4. Treatment of animals

In the treatment studies, animals were divided into six groupsof seven animals each (G). G1 was served normal control; G2 wasCCl4 control and G3, G4 and G5 were treated with tissue culturedmountain ginseng extract at a dose of 30, 100 and 300 mg/kg bodyweight, respectively. G6 animals were treated with cultivatedginseng extract of 100 mg/kg body weight. All the groupedanimals were maintained with above feeding schedule for 14 days.Further, the rats were administered with CCl4, which is a modelagent to induce hepatic lesions. A single dose of CCl4 leads tocentrizonal necrosis and steatosis (Pierce et al., 1987), whileprolonged administration leads to liver fibrosis, cirrhosis, andhepato-cellular carcinoma (Perez, 1983). Acute oral dose of admin-istration consisted of 0.5 ml/kg of 1:1 (v/v) mixture of CCl4 andcorn oil (Smile et al., 2001). Group 2–6 received single dose of CCl4on day 14. Normal control received equal amount of corn oilinstead of CCl4. All the rats were sacrificed after 24 h of CCl4administration.

2.5. Assessment of liver functions

Blood was collected from the blood vessels of neck region undermild ether anesthesia and kept for 30 min at 4 1C. Serum wasseparated by centrifugation at 2500 rpm at 4 1C for 15 min. The liverwas removed rapidly and cut into separate portions for hepaticglutathione, lipid peroxidation estimation. Alanine aminotransfer-ase (ALT) (Amador et al., 1963), aspartate aminotransferase (AST)

(Bergmeyer et al., 1976), alkaline phosphatase (ALP) (Bergmeyer, 1980)assays were carried out. Glutathione (GSH) level was estimated as perthe protocol of Moron et al. (1979). Lipid peroxidation in liver wasmeasured by the formation of malondialdehyde (MDA) and it wasestimated by using the procedures of Ohakawa et al. (1979).

2.6. Statistical analysis

All values are expressed as mean with standard error. Theresults were statistically analyzed by using Analysis of variance(ANOVA) to note the significance among different groups andfurther Student's t-test was carried out to find out the significantdifferences between two groups.

3. Results

3.1. Standardization of ginsenosides present in the extract

The high pressure liquid chromatography (HPLC) analysis data onginsenosides of TCMGARs and Korean ginseng is presented inTable 1. The results revealed that Rb1, Rb2, Rb3, Rc, Rd, Re, Rg1,Rg2, Rg3, Rh1, Rh2 and Rf ginsenosides were present in the extract ofTCMGARs. The ginsenosides present in the Korean cultivated ginsengroot extract were Rb1, Rb2, Rc, Rd, Re, Rg1, and Rf. Ginsenosides suchas Rb3, Rg2, Rg3, Rh1 and Rh2 which were not present in the Koreanginseng roots were abundant in the TCMGARs, the content of otherginsenosides are also at higher levels in TCMGARs when compared toKorean cultivated ginseng (Table 1; Fig. 1).

3.2. Evaluation of AST, ALT and ALP

In the rats, which were administered with CCl4 showed anelevated level of serum alanine aminotransferase (ALT), aspartateaminotransferase (AST) and alkaline phosphatase (ALP) levels.Treatment with the extract of TCMGARs at a dose of 100 and300 mg/kg as well as the treatment with the extract of cultivatedginseng at a dose of 100 mg/kg significantly (pr0.05) providedthe resistance against the CCl4 stress in rats. Korean ginsengextract which was used as positive control also showed a remark-able protection towards CCl4 intoxication (Table 2).

Table 1Ginsenoside content of tissue cultured mountain ginseng adventitious roots incomparison with cultivated Korean ginseng.a

Ginsenoside Content (mg/g dry weight)

TCMGARs Korean ginseng

Rb1 4.1 0.05Rb2 2.3 0.06Rb3 0.9 –

Rc 2.0 0.03Rd 4.2 0.06Re 0.2 0.24Rg1 0.19 0.17Rg2 0.38 –

Rg3 11.2 –

Rh1 0.49 –

Rh2 3.8 –

Rf 1.64 0.03

a Analysis was carried out by Korea Food Research Institute, Sungnam-si,Republic of Korea.

H. Niranjana Murthy et al. / Journal of Ethnopharmacology 158 (2014) 442–446 443

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 withethanol extract of TCMGARs as well as with Korean ginseng extractssignificantly increased the hepatic glutathione levels (Table 3). In thevarious preventive treatments, rats treated with 300 mg/kg ethanolicextract showed an increase in glutathione levels in liver.

The significant increase (pr0.05) in tissue MDA level wasobserved in CCl4 alone treated rats. However, the CCl4-inducedelevation of tissue malondialdehyde (MDA) concentration waslowered significantly (pr0.05) with the administration of theextract to rats. In the ethanolic extract-treated rats, MDA levelsremarkably reduced which are comparable to the Korean ginsengtreatment (Table 3).

4. Discussion

Carbon tetrachloride has been one of the most intensively studiedhepatotoxic agents and the effect of CCl4 on hepatocytes has beenattributed to its metabolism by cytochrome P450 to yield toxictrichloromethyl radicals that act as free radical indicators (Clawson,1989; Dhum and Jones, 1996; Weber et al., 2003). Elevation of serumAST, ALT and ALP indicates the toxicity which specifically affects theliver (Anand et al., 1992) and activities of AST and ALT are mostcommonly used as biochemical markers for liver damage (Sturgilland Lambert, 1997). Since these enzymes are cytoplasmic in nature,upon liver injury these enzymes enter into the circulatory system dueto altered permeability of membrane (Zimmerman and Seeff, 1970).Administration of butanolic extract of TCMGARs as well as thecultivated ginseng significantly prevented CCl4 induced elevation ofAST, ALT and ALP indicating the hepatoprotective activity ofTCMGARs and also the cultivated ginseng. The administration of300 mg/kg TCMGARs and 100 mg/kg cultivated ginseng extractsignificantly lowered the AST, ALT and ALP levels (Table 2). The HPLCanalysis demonstrated that TCMGARs possessed novel ginsenosidessuch as Rb3, Rg2, Rg3, Rh1 and Rh2. TCMGARs also had varied otherginsenosides in very high concentrations (Table 1). Over accumula-tion of ginsenosides in TCMGARs was due to methyl jasmonateelicitation during the production of tissue cultured mountain ginsengadventitious roots. It is reported that treatment of cultures with100 mM methyl jasmonate has boosted the accumulation of ginseno-sides by 5–6 folds in the cultures (Yu et al., 2002; Kim et al., 2004).Tissue cultured mountain ginseng adventitious roots were not onlyrich in ginsenosides but also they contained biophenols and enzy-matic and non-enzymatic antioxidant enzymes due to methyljasmonate 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 mountainginseng adventitious root extracts have been useful in providing theresistance against the CCl4 stress in liver cells. Korean cultivatedginseng extract was also efficient in combating the toxic effects ofCCl4 on hepatocytes of rats (Table 2). Similarly, hepatoprotectiveeffects of cultivated Korean ginseng were on records (Jeong et al.,1977; Kang et al., 2007; Shukla and Kumar, 2009). It was reportedthat ginsenoside Rb1 was known for hepatoprotective function (Leeet al., 2005) and the content of Rb1 was 0.05 mg/g dry weight inKorean ginseng, its level was 4.1 mg/g dry weight in tissue cultured

Fig. 1. HPLC chromatogram of field cultivated ginseng (above) and tissue cultured mountain ginseng adventitious roots (below).

Table 2Effect of ginseng adventitious root extract on CCl4-induced liver damage in rats.

Treatment groups ALT (U/L) AST (U/L) ALP (U/L)

G1 Normal control Mean 24.2 173.7 221.2SEM 1.1 9.9 19.9N 7 7 7

G2 CCl4 control, (0.5 ml/kg), Mean 118.3n 397.1n 287.3n

SEM 30.7 52.5 14.5N 7 7 7

G3 TCMGARa (30 mg/kg)þCCl4 Mean 110.3 261.8 234.9SEM 32.3 53.7 27.4N 7 7 7

G4 TCMGAR (100 mg/kg)þCCl4 Mean 40.2† 216.0† 237.8†

SEM 6.6 29.5 20.7N 7 7 7

G5 TCMGAR (300 mg/kg)þCCl4 Mean 40.7† 186.5† 223.5†

SEM 8.7 10.9 19.3N 7 7 7

G6 Cultivated ginseng, 100 mg/kgþCCl4

Mean 57.1† 187.0† 211.4†

SEM 6.5 20.9 22.3N 7 7 7

N¼Number of animals.Values are expressed as mean7S.E.

a TCMGAR – Tissue cultured mountain ginseng adventitious root extract.n po0.05 vs normal control.† po0.05 vs CCl4 control.

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mountain ginseng adventitious roots (Table 1); it is obvious that thehepatoprotective activity of tissue cultured mountain ginseng adven-titious roots extract is as significant as Korean cultivated ginsengextracts (Table 2).

Reduction in liver GSH in CCl4 control rats as observed in thisstudy indicates the damage to hepatic cells. Rats administeredwith CCl4 significantly lowered the hepatic glutathione (GSH)level. The biochemical function of glutathione peroxidase is toreduce lipid hydroperoxides to their corresponding alcohols and toreduce free hydrogen peroxide into water for the protection ofcells from oxidative damage (Ali et al., 2005). Treatment ofethanolic ginseng extract exhibited significant increase in hepaticglutathione levels (Table 3). Rats treated with cultivated Koreanginseng extract prevented the lowering of hepatic GSH. In varioustreatments in the current study, rats treated with 300 mg/kgmethanolic extract showed the increased glutathione levels inthe liver. Increase in GSH activity with the treatment of plantextracts are in accordance with the reports of Raja et al. (2007) andWills and Asha (2006). Increase in tissue MDA was observed inCCl4 alone treated rats. However, CCl4 induced elevation of tissueMDA concentration was lowered by pretreatment of rats with theginseng extract (Table 3). Decrease in lipid peroxidation i.e.,decrease in MDA levels with administration of ethanolic extractsof 100 mg/kg cultivated ginseng and 300 mg/kg tissue culturedmountain ginseng adventitious roots and increase in antioxidantlevel as indicated by the increase in GSH level specifies thehepatoprotective nature of tissue cultured mountain ginsengadventitious roots as well as cultivated ginseng.

The results of serum biochemical parameters, levels of hepaticlipid peroxides, and hepatic glutathione studies in rats which weretreated with TCMGARs extract together support the hepatoprotectiveand antioxidant activities of tissue cultured mountain ginsengadventitious roots. Most pharmacological actions of ginseng areattributed to ginsenosides, which can act in a wide range of tissues(Park et al., 2005) and tissue cultured mountain ginseng adventitiousroots contain higher concentrations of ginsenosides especially Rb1ginsenoside (4.1 mg/g dry weight) which is reported to be hepato-protective compound (Lee et al., 2005) and these results showed theefficacy of tissue cultured mountain ginseng adventitious roots.

Acknowledgments

This study was supported by a grant of Korea HealthcareTechnology R&D project, Ministry of Health and Welfare, Republicof Korea (Grant no. A103017). Dr. H. N. Murthy is thankful to Ministryof Education, Science and Technology, Republic of Korea for theaward of Brain Pool Fellowship (131S-4-3-0523) and this paper wasstudied with the support of Ministry of Science, ICT and Planning(MSIP). We are thankful to Prof. Yerivanthelimath, Department ofEnglish, Karnatak University, Dharwad, India for language editing ofthe manuscript.

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G6 Cultivated ginsengþCCl4 100 Mean 198.1† 156.3†

SEM 8.1 6.6N 7 7

N¼Number of animals.Values are expressed as mean7S.E.

a TCMGAR – Tissue cultured mountain ginseng adventitious root extract.n po0.05 vs normal control.† po0.05 vs CCl4 control.

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