Saudi Journal of Biological Sciences (2014) 21, 409–416

King Saud University

Saudi Journal of Biological Sciences

www.ksu.edu.sawww.sciencedirect.com

ORIGINAL ARTICLE

Hepatoprotective effect of Rhodiola imbricatarhizome against paracetamol-induced liver toxicity

in rats

* Corresponding author. Tel.: +91 04222428305; fax: +91

04222425706.

E-mail address: drparimel@gmail.com (T. Parimelazhagan).

Peer review under responsibility of King Saud University.

Production and hosting by Elsevier

http://dx.doi.org/10.1016/j.sjbs.2014.04.001

1319-562X ª 2014 Production and hosting by Elsevier B.V. on behalf of King Saud University.

Ravichandran Senthilkumar, Rahul Chandran, Thangaraj Parimelazhagan *

Department of Botany, Bioprospecting Laboratory, Bharathiar University, Coimbatore, Tamil Nadu, India

Received 3 February 2014; revised 8 April 2014; accepted 9 April 2014Available online 24 April 2014

KEYWORDS

Hepatotoxicity;

In vivo antioxidants;

Histopathology;

Hematology;

Biochemical markers

Abstract Rhodiola imbricata is a perennial herb of the family Crassulaceae, which has significant

traditional usage as medicine and is also known to biosynthesize phytochemicals such as flavonoids,

coumarins and phenyl glycosides. The present investigation was aimed to estimate the hepatopro-

tective activity of R. imbricata rhizome acetone extract against paracetamol (2 g/kg) induced liver

toxicity. Paracetamol was administered to induce hepatic damage in Wistar rats. 200 and

400 mg/kg doses of rhizome acetone extract and silymarin (25 mg/kg) were used as treatment

groups. The blood samples were analyzed for biochemical markers of hepatic injury and tissue sam-

ples were subjected for estimation of liver antioxidants and histopathological studies. Analysis of

the extract treated rats (400 mg/kg) showed an elevation of superoxide dismutase (0.326 units/

min/mg protein), catalase (185.03 lmole of H2O2 consumed/min/mg protein), glutothione peroxi-

dase (19.26 mg GSH consumed/min/mg protein) and reduced glutathione (16.2 lmole of GSH/mg

protein). Moreover, the biochemical parameters in serum like alkaline phosphatase, serum glutamic

oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT) and lipid profiles

were also improved in treated groups compared to the control. The oral administration of different

doses of rhizome acetone extract significantly protected the hepatic cells from damage. The hema-

tological and biochemical parameters were also normal in extract treated rats compared to the con-

trol and standard (silymarin) groups. The HPLC analysis revealed the presence of some important

phenolic compounds which could be responsible for the hepatoprotective activity. This study

proved that R. imbricata could be taken as a good natural source of the hepatoprotective agent.ª 2014 Production and hosting by Elsevier B.V. on behalf of King Saud University.

1. Introduction

The liver as a vital organ in the body is primarily responsible

for the metabolism of endogenous and exogenous agents. Itplays an important role in drug elimination and detoxificationand liver damage may be caused by xenobiotics, alcohol

410 R. Senthilkumar et al.

consumption, malnutrition, infection, anemia and medications(Mroueh et al., 2004). Paracetamol is widely used an analgesicand antipyretic, but produces acute liver damage at higher

doses. The hepatotoxicity of paracetamol has been attributedto the formation of toxic highly reactive metabolite n-acetylparabenzoquineimine (NAPQI). Despite the fact that hepatic

problems are responsible for a significant number of livertransplantations and deaths recorded worldwide, availablepharmacotherapeutic options for liver diseases are very limited

and there is a great demand for the development of new effec-tive drugs (Akindele et al., 2010). Conventional or syntheticdrugs used in the treatment of liver diseases are inadequateand can have serious adverse effects. So there is a worldwide

trend to go back to traditional medicinal plants. Many naturalproducts of herbal origin are in use for the treatment of liverailments (Mitra et al., 2000).

Rhodiola imbricata Edgew is a perennial herb of the familyCrassulaceae, commonly known as golden or arctic root,grows on rocky slopes, common in drier areas of the western

Himalaya at an altitude of 4000–5000 m. Rhodiola root hasbeen used extensively since time immemorial for its medicinalproperties in traditional folk medicine in China, Tibet, Mon-

golia and the former Soviet Republics to increase physicalendurance, work productivity, longevity and to treat fatigue,asthma, hemorrhage, impotence and gastrointestinal ailments(Kelly, 2001). R. imbricata is the major constituent of herbal

tea and is rich in antioxidant value. The aqueous extract ofR. imbricata root was found to contain gallic acid, p-tyrosol,rosavin and rosin (Mishra et al., 2008). Recently, rhizome of

R. imbricata, was found to possess radioprotective (Aroraet al., 2005), cytoprotective and antioxidant (KanupriyaPrasad et al., 2005), wound healing (Gupta et al., 2007), immu-

nomodulatory (Mishra et al., 2006), adaptogenic (Spasovet al., 2000), anti-fatigue (Darbinyan et al., 2000), neuroprotec-tive (Mook-Jung et al., 2002) and antiproliferative activities in

HT-29 human colon cancer cells (Senthilkumar et al., 2013).Increased concerns on the side effects of current therapeuticmodalities, plants are being valued because of their efficientcurative properties and least or no side effects. Hence an

attempt has been made to assess the hepatoprotective role ofR. imbricata.

2. Materials and methods

2.1. Collection and identification of plant material

The fresh rhizomes of R. imbricata were collected from theWestern Himalayas, Leh-Ladakh, India during the month of

August to September, 2011. The plant material was identifiedand authenticated by Dr. O.P. Chaurasia, an Ethnobotanistat the Defence Institute of High Altitude Research (DIHAR),

Leh-Ladakh. A voucher specimen of the same is available atthe Field Research Laboratory (FRL), Leh.

2.2. Preparation of plant extracts

Freshly collected plant materials were washed under runningtap water and distilled water to remove adhering dust and thendried under shade. The dried samples were powdered in a

mechanical grinder and used for solvent extraction.

2.3. Animals and management

Experiments were conducted using Wistar albino rats (male,150–200 g) and Swiss albino mice (male, 25–30 g), procuredfrom Small Animal Breeding Station (SABS), College of

Veterinary and Animal Sciences, Mannuthy, Thrissur, Kerala.The animals were housed in groups for a minimum of 7 daysprior to pharmacological experiments. Animal quarters weremaintained at a temperature of 22 ± 2 �C and with a 12 h

light/12 h dark cycle. The animals had free access to commer-cial food pellets and clean drinking water. The study receivedapproval from the Institutes Animal Ethics Committee (IAEC)

for the Committee (CPCSEA) for the Purpose of Control andSupervision of Experiments on Animals (Reg. No.KMCRET/Ph.D/04/2011).

2.4. Acute toxicity

An acute oral toxicity study was performed as per OECD

guidelines for the testing of chemicals, Test No. 423 (OECD;acute oral toxicity-acute toxic class method). Swiss albino mice(n = 6) were used for the acute toxicity study. The animalswere kept overnight with access to water but not food, after

which the R. imbricata acetone extract was administered orallyat a dose level of 500, 1000 and 2000 mg/kg body weight andthe animals were observed for 24 h. Further, they were

observed continuously for the first 2 h for morbidity and upto 24 h for mortality. If mortality was observed in 2 out of 3animals, then the dose administered was identified as a toxic

dose. If mortality was observed in one animal, then the samedose was repeated again to confirm the toxic dose. If mortalitywas observed again, the procedure was repeated for lowerdoses (300, 50 and 5 mg/kg body weight).

2.5. Hepatoprotective property of R. imbricata

Hepatotoxicity was induced by paracetamol induced liver

damage model by Sreedevi et al. (2009). Paracetamol (Acet-aminophen, Sigma Chemical Company, USA) was suspendedin 0.5% Tween-80 and administered p.o., at a dose of 2 g/kg.

Wistar albino rats (male) weighing between 150 and 200 g weredivided into 5 groups of 6 animals each. The weight range ofthe animals was equally distributed throughout the groups.

Group-I served as control and received water, Group-II servedas negative control, administered with paracetamol (2 g/kg,p.o.), Group-III standard, silymarin received (Sigma ChemicalCompany, USA) at a dose of 25 mg/kg p.o., Group-IV and V

received acetone extract (200 and 400 mg/kg, p.o., respec-tively), once daily for 14 days.

On the 14th day, blood samples were collected from all ani-

mals by puncturing retro-orbital plexus under mild ether anes-thesia, later animals were sacrificed and liver tissues werecollected. The blood samples were analyzed for biochemical

markers of hepatic injury and tissue samples were subjectedfor estimation of liver antioxidants and histopathologicalstudies.

2.5.1. Preparation of serum from blood

Blood was drawn by puncturing the retro-orbital plexus underdiethyl ether anesthesia. Whole blood for hematogram was

Hepatoprotective effect of Rhodiola imbricata 411

collected in bottles containing the anticoagulant, ethylene dia-mine tetra-acetic acid (EDTA) while samples for biochemicalanalysis were collected in plain sample bottles. Serum was sep-

arated by centrifugation at 600·g for 15 min. and analyzed forvarious biochemical parameters. Sera were stored in the�80 �C freezer before they were analyzed. The erythrocytes,

leucocytes and platelets were determined with an Auto Hema-tology Analyzer (Sysmex F-800, Japan). Alkaline phosphatase(ALP), serum glutamic oxaloacetic transaminase (SGOT),

serum glutamic pyruvic transaminase (SGPT) and lipid pro-files like total cholesterol (TC), triglycerides (TG) and bio-chemical parameters such as creatinine and bilirubin werealso determined using Span Diagnostics Limited kit, India.

2.5.2. Preparation of liver homogenate

Hepatic tissues were homogenized in 10% w/v 0.1 M phos-

phate buffer or 0.1 M tris buffer (pH 7.0) and centrifugedat 12,000·g for 10 min. The supernatant was used for themeasurement of liver enzymatic and non enzymaticantioxidants.

2.5.3. Determination of antioxidant enzymes

Total Protein content of the tissues was estimated by the

method of Lowry et al. (1951). Enzymatic antioxidants weredetermined by estimating superoxide dismutase (SOD)(Kakkar et al., 1984), catalase (CAT) (Sinha, 1972), glutothi-one peroxidase (GPx) activity (Rotruck et al., 1973) and

non-enzymatic antioxidants by reduced glutathione (GSH)(Ellman, 1959) and lipid peroxidation (LPO) (Hogberg et al.,1974).

2.5.4. Histopathology

After the experimental period animals were sacrificed, liverremoved immediately, sliced and washed in saline. Liver pieces

were preserved in 10% formalin for histopathological studies.Pieces of the liver were processed and embedded in paraffinwax. Sections were taken and stained with hematoxylin and

eosin and photographed.

Table 1 Effect of acetone extract of R. imbricata on hematological

Group-I

(control)

Group-II

(paraceta

Erythrocytes

RBC count (mill/cmm) 7.1 ± 0.2 3.1 ± 0

Hemoglobin (gm%) 13.5 ± 0.3 7.2 ± 1

Hematocrit (%) 32.1 ± 1.3 17.1 ± 2

MCV (fl) 47.6 ± 0.1 54.7 ± 0

MCH (pg) 19.1 ± 0.6 23.1 ± 0

MCHC (%) 38.3 ± 1.1 42.1 ± 2

RDW (%) 11.3 ± 1.4 13.6 ± 3

Leucocytes

Total WBC count (cells/cmm) 7300 ± 1.0 2600 ± 2

Polymorphs (%) 8 ± 1.1 10 ± 0

Lymphocytes (%) 88 ± 0.1 90 ± 0

Platelets (thou/cmm) 524 ± 1.5 84 ± 2

Data represent mean ± S.E.M (n= 6).* p < 0.05 compared to corresponding control.

2.6. High performance liquid chromatography (HPLC) analysis

HPLC analysis of acetone extract of R. imbricata was performedusing the ShimadzuHPLC system. TheHPLC system (Shimadzu,Japan) consisted of a LC-10AT VP pump, a SPD-10AVP, PDA

detector, a Phenomenex Luna C18 (250 mm · 4.6 mm, 5 lm) col-umn, a Phenomenex, HPLC guard cartridge system and a classcbm-20A/20Alite software. Separationwas performed in a reversephase column bymaintaining the isocratic flow rate (1 mL/min) of

themobile phase (methanol:water, 70:30) and peaks were detectedat 226 nm. The standard substances gallic acid and rutin fractionswere analyzed in the same conditions. Peakswere assigned by spik-

ing the samples with standard substances and comparison of theretention time and UV spectrum.

2.7. Statistical analysis

The results were statistically analyzed and expressed as mean(n= 6) ± standard error. Values are analyzed by the Dunnet’s

test (SPSS, ANNOVA statistical software 17.0, TULSA, USA).

3. Results

3.1. Acute toxicity

The acetone extract of R. imbricata was subjected to acute tox-

icity testing in Swiss albino mice and animals were monitoredfor 24 h. The acetone extract of R. imbricata rhizome did notcause any mortality up to 2000 mg/kg, and hence 1/10th and

1/5th of the maximum dose administered (i.e. 200 and400 mg/kg, p.o.) were selected for the present study.

3.2. Effect of acetone extract of R. imbricata on hematologicalparameters in paracetamol intoxicated rats

The effect of acetone extract of R. imbricata at two dose levels

(200 mg/kg and 400 mg/kg, p.o.) on hematological parametersin paracetamol induced hepatic damage is shown in Table 1.

parameters in rats treated with paracetamol.

mol)

Group-III

(silymarin

25 mg/kg)

Group-IV

(200 mg/kg)

Group-V

(400 mg/kg)

.1* 6.9 ± 1.2 5.4 ± 0.2* 6.5 ± 0.5

.1* 12.3 ± 2.1 11.6 ± 2.1 11.8 ± 1.2

.1* 31.4 ± 1.4 25.1 ± 0.3* 29.5 ± 1.1

.3* 46.3 ± 1.6 30.4 ± 0.4* 35.2 ± 2.1*

.3 17.1 ± 2.0 17.3 ± 0.2 16.2 ± 1.2

.2* 36.3 ± 2.3 30.1 ± 0.5* 32.1 ± 2.1*

.0 10.1 ± 2.1 8.1 ± 1.0 9.2 ± 1.0

.0* 6000 ± 1.0* 5000 ± 1.0* 5800 ± 2.0*

.4 7 ± 1.1 6 ± 2.1 6.2 ± 1.2

.3* 78 ± 1.1* 68 ± 0.4* 70 ± 0.3*

.1* 404 ± 0.2* 377 ± 1.0* 396 ± 2.0*

Table 2 Effect of acetone extract of R. imbricata on serum biochemical parameters in rats treated with paracetamol.

Biochemical markers Group-I

(control)

Group-II

(only paracetamol)

Group-III

(silymarin, 25 mg/kg)

Group-IV

(200 mg/kg)

Group-V

(400 mg/kg)

Creatinine (mg/dl) 0.68 ± 0.04 0.73 ± 0.01 0.69 ± 0.02 0.71 ± 0.01 0.70 ± 0.03

Bilirubin (mg/dl) 0.16 ± 0.01 0.17 ± 0.08 0.16 ± 0.01 0.17 ± 0.01 0.17 ± 0.02

00SGOT (U/l) 34.74 ± 0.1 157.36 ± 0.3* 53.63 ± 0.3* 121.82 ± 0.6* 88.43 ± 0.3*

SGPT (U/l) 32.71 ± 0.6 148.52 ± 0.4* 47.35 ± 0.2* 117.37 ± 0.5* 79.56 ± 0.3*

ALP (U/l) 158.48 ± 0.4 261.56 ± 0.3* 167.68 ± 0.4* 224.53 ± 0.3* 193.53 ± 0.3*

Cholesterol (mg/dl) 54.45 ± 0.1 53.59 ± 0.2* 54.68 ± 0.2* 53.49 ± 0.2* 54.64 ± 0.5*

Triglycerides (mg/dl) 53.45 ± 0.1 51.39 ± 0.4* 53.71 ± 0.3* 53.42 ± 0.3 53.56 ± 0.3*

Data represent mean ± S.E.M (n= 6).

SGOT – serum glutamic oxaloacetic transaminase, ALP – alkaline phosphatase, SGPT – serum glutamic pyruvic transaminase.* p < 0.05 compared to the corresponding control.

Table 3 Effect of acetone extract of R. imbricata on total protein, enzymatic and non-enzymatic antioxidants in rats treated with

paracetamol.

Parameters Group-I

(control)

Group-II

(only paracetamol)

Group-III

(silymarin, 25 mg/kg)

Group-IV

(200 mg/kg)

Group-V

(400 mg/kg)

Groups

Total protein (lg/10 mg of tissue) 0.251 0.085 0.236 0.105 0.185

SOD (units/min/mg protein) 0.473 0.191 0.501 0.242 0.326

CAT (lmole of H2O2 consumed/min/mg protein) 234.27 98.4 229.38 145.71 185.03

GPx (mg GSH consumed/min/ mg protein) 21.64 12.73 23.53 14.76 19.26

GSH (lmole of GSH/mg protein) 18.84 11.52 17.98 14.26 16.2

SOD – superoxide dismutase, CAT – catalase, GPx – glutothione peroxidase, GSH – reduced glutathion.

Figure 1 Histopathological examination for the hepatoprotective activity of R. imbricata in paracetamol treated Wistar rats. (A)

paracetamol treated rat liver showing portal tract inflammation with lymphocyst (original magnification 10·); (B) paracetamol treated rat

liver showing early fibrosis of the perivenular region; (C) protective effect of the R. imbricata (400 mg/kg) in the liver of the tested rats.

Liver showing mild dilatation of sinusoids (original magnification 40·).

412 R. Senthilkumar et al.

Figure 3 HPLC chromatogram for acetone extract of R.

imbricata.

Hepatoprotective effect of Rhodiola imbricata 413

Hepatic injury induced by paracetamol caused a decrease inerythrocytes (RBC count, hemoglobin, hematocrit, MCV,MCH, MCHC, RDW), leucocytes (total WBC count, Poly-

morphs, Lymphocytes) and platelets. Silymarin (25 mg/kg)was used as reference standard. Administration of acetoneextract (400 mg/kg) resulted in increased levels of hemato-

logical (RBC count 6.5±0.5 mill/cmm, total WBC count5800±2.0 cells/cmm and platelets 396 ± 2.0 thou/cmm)parameters.

3.3. Effect of acetone extract of R. imbricata on serum

biochemical markers in paracetamol intoxicated rats

Table 2 shows the effect of acetone extract (200 mg/kg and 400 mg/kg) on serum biochemical markers in paracetamol induced hepaticdamage. Hepatic injury causes elevated level of liver enzymes suchas SGOT (serumglutamic oxaloacetic transaminase), SGPT (serum

glutamic pyruvic transaminase) and ALP (alkaline phosphatase).Treatment with R. imbricata at 400 mg/kg revealed comparableactivity with reference standard silymarin (25 mg/kg). Acetone

extract decreased the livermarkers SGPT (88.43 ± 0.3 U/l), SGOT(79.56 ± 0.3 U/l) and ALP (193.53 ± 0.3 U/l).

3.4. Effect of acetone extract of R. imbricata on in vivoantioxidant activity in paracetamol intoxicated rats

Administration of paracetamol at the concentration of 2 g/kgto the Wistar rats caused an elevation of lipid peroxidation

and decreased enzymatic antioxidants. The acetone extract of

Figure 2 HPLC chromatogram for rutin and gallic acid.

R. imbricata (400 mg/kg, p.o.) rhizome increased the total pro-tein (0.185 lg/10 mg of liver tissue), enzymatic antioxidants

SOD (0.326 units/min/mg protein), CAT (185.03 lmole ofH2O2 consumed/min/mg protein) and GPx (19.26 mg GSHconsumed/min/mg protein). The results are shown in Table 3.

3.5. Histopathological examination

The hepatoprotective effect of the R. imbricata acetone extract

against paracetamol induced injury was confirmed by histopa-thological examination. The hepatocyte damage caused byparacetamol is shown in Fig. 1. Paracetamol treated rat livershowed portal tract inflammation with lymphocysts and

showed early fibrosis of the perivenular region but the R.imbricata acetone extract protects the liver hepatocytes by pre-venting oxidation in liver cells.

3.6. HPLC analysis of R. imbricata acetone extract

Qualitative analysis of data obtained using HPLC confirms the

presence of gallic acid and rutin in the acetone extract of R.imbricata. The retention times of the compounds (Figs. 2and 3) present in the acetone extract (4.881 and 2.545) were

eluted very close to the standards in the chromatograms ofrutin (5.299) and gallic acid (2.855).

4. Discussion

The primary function of the liver is to maintain body homeo-stasis, besides it plays a key role in metabolism, detoxification,

414 R. Senthilkumar et al.

and inflammatory response (Tacke et al., 2009). The liver is aversatile organ in the body concerned with regulation of inter-nal chemical environment. Therefore, damage to the liver

inflicted by a hepatotoxic agent is of grave consequence. Acet-aminophen, also called as paracetamol or APAP (N-acetyl-p-aminophenol), is often considered as a safe painkiller drug,

eventhough, overdoses of this drug lead to acute liver failureand hepatic cytolysis (Michaut et al., 2014). Hepatotoxic drugssuch as D-galactosamine and acetaminophen reduces liver

functional capacity, which leads to an accumulation of wasteproducts such as ammonia in the blood (Mao et al., 2014). Par-acetamol is a common analgesic and antipyretic agent, knownto possess hepatotoxic effects in humans at very high doses. It

has been used as a successful experimental model to evaluatethe efficacy of hepatoprotective agents (Sreedevi et al., 2009).The mode of action of paracetamol on the liver is by covalent

binding of its toxic metabolite, n-acetyl-p-benzoquinone-amineto the sulfhydryl group of protein resulting in cell necrosis andlipid peroxidation (Kapur et al., 1994). Due to liver injury

caused by paracetamol overdose, the transport function ofthe hepatocytes gets disturbed resulting in the leakage of theplasma membrane (Zimmerman and Seeff, 1970), thus causing

an increase in serum enzyme levels.Administration of R. imbricata acetone extract at concen-

trations of 200 and 400 mg/kg, p.o., for 14 days resulted in asignificant reduction of paracetamol induced elevation of

serum enzyme markers, comparable to the effect of silymarin,the positive control used. Silymarin is a known hepatoprotec-tive compound obtained from Silybum marianum. It is

reported to have a protective effect on plasma membrane ofhepatocytes (Ramellini and Meldolesi, 1976). Hepatic injuryinduced by paracetamol caused a decrease in erythrocytes

(RBC count, hemoglobin, hematocrit, MCV, MCH, MCHC,RDW), leucocytes (total WBC count, Polymorphs, Lympho-cytes) and platelets.

It is known that many toxic compounds accumulate in theliver where they are detoxified (Clarke and Clarke, 1977). Livertransaminases such as AST (aspartate transaminase) or SGOT(serum glutamic oxaloacetic transaminase), and ALT (alanine

transaminase) or SGPT (serum glutamic pyruvic transaminase)have still remained the gold standards for the assessment ofliver injury, and have been used as biomarkers of choice for

decades (Howell et al., 2014). A study of liver function testsmay therefore prove useful in assessing especially the toxiceffects of medicinal plants on the liver. These tests involve

mainly the determination of AST and ALT (Tilkian, 1979)and any marked necrosis of the liver cells leads to a significantrise of these enzymes in the blood serum. The lack of this effecton these liver enzymes shows that the extract is non-toxic to the

hepatocytes. The results of enzymatic antioxidants such asSOD (superoxide dismutase), CAT (catalase), GPx (glutathi-one peroxidase) and non-enzymatic antioxidants such as

GSH (reduced glutathione) and LPO (lipid peroxidation) indi-cate that the extract is non-toxic and safe. SGOT, SGPT andALP (alkaline phosphatase) are important serum enzymes in

the human liver and usually help to detect chronic liver diseasesby monitoring their concentrations. The values were well com-parable to the control group. Hepatic injury causes elevated

levels of liver enzymes such as SGOT, SGPT and ALP. Treat-ment with R. imbricata at the concentration of 400 mg/kgrevealed a comparable activity with the reference standardsilymarin, a potent hepatoprotective drug. Administration of

paracetamol at the concentration of 2 g/kg to the Wistar ratscaused an elevation of lipid peroxidation and decreased enzy-matic antioxidants. The acetone extract of R. imbricata rhi-

zome increased the total protein and enzymatic antioxidants.In this experiment, the R. imbricata acetone extract appears

to be effective in reducing the paracetamol caused injury as

observed from a significant reduction of paracetamol inducedelevated serum enzyme levels. It was also noted that the histo-pathological damage induced by paracetamol was improved in

rat liver treated with plant extract. This implies that concomi-tant administration of R. imbricata prevented hepatonecroticchanges induced by the toxic dose of paracetamol.

Hepatoprotective effect of R. imbricata was further con-

firmed by histopathological studies of the liver, which basicallysupported the results from the serum assays. Histopathologicalstudies of the liver showed fatty changes, swelling and necrosis

with loss of hepatocytes in paracetamol treated rats. R. imbricatatreated groups showed regeneration of hepatocytes, normaliza-tion of fatty changes and necrosis of the liver. The maximum

protection against hepatic damage was achieved with the ace-tone extract at a dose of 400 mg/kg. The histopathologicalobservations of the liver of rats treated with R. imbricata

showed a more or less normal architecture of the liver havingreversed to a large extent, the hepatic lesions produced by par-acetamol, almost comparable to the normal control groups.

R. imbricata species is reported to be a potent adaptogen

(Gupta et al., 2008; Perfumi and Mattioli, 2007). A high con-tent of bioactive compounds such as rosavin, rosin, p-tyrosoland gallic acid was reported in aqueous extract of R. imbricata

species (Mishra et al., 2008). Qualitative analysis of dataobtained using HPLC confirms the presence of gallic acidand rutin or its analogs in the acetone extract of R. imbricata.

GC/MS analysis revealed that the methanolic extract ofR. imbricata contains phytosterols, alkyl halide, phenols, andfatty acid esters, and is responsible for its antiradical properties

(Tayade et al., 2013). Salidroside [2-(4-hydroxyphenyl) ethylb-D-glucopyranoside (1)], was treated under oxidative stressinduced by hydrogen peroxide in human erythrocytes. Flowcytometric analysis revealed that Salidroside protects human

erythrocytes by its antioxidative activity, and caspase-3 inhibi-tion in a dose-dependent fashion (Qian et al., 2012). Rhodiolapossesses Salidroside and its metabolite p-tyrosol as a major

phenolic compound. These compounds were determined byLC–MS/MS based method in rat liver tissues using paraceta-mol as the internal standard (Guo et al., 2014). Poly (ADP-

ribose) polymerase (PARP) is a DNA repair enzyme, and isactively involved in cell apoptosis. Salidroside protects hema-topoietic stem cells from oxidative stress by activating PARP1(Li et al., 2014). These studies reveal that the extract is rich in

these antioxidant compounds, which clearly support the anti-oxidant and other related pharmacological properties includ-ing hepatoprotective activity of Rhodiola. Hence, the

phenolic compounds present in R. imbricata rhizome mightbe responsible for its observed hepatoprotective activity. Fur-ther experiments are underway to discern the molecular mech-

anism of phytoconstituents.

5. Conclusion

From the present study it is evident that the acetone extract ofR. imbricata rhizome has no toxicity even at a higher dose of

Hepatoprotective effect of Rhodiola imbricata 415

2000 mg/kg. Liver antioxidant markers were elevated signifi-cantly, while the serum and lipid parameters were maintainedat normal levels compared to control groups. Histopatholo-

gical examinations of the liver showed that extract and silym-arin have a protective role over the toxicity of paracetamol.The HPLC profiles of rhizome showed the presence of some

important phenolic compounds which could be responsiblefor imparting protection to the liver of rats. Hence R. imbricatacould be one of the best sources of natural hepatoprotective

agents.

Acknowledgement

The authors are thankful to the DRDO-DIHAR, Govt. ofIndia for the financial support.

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