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Journal of Traditional and Complementary Medicine 6 (2016) 160e167

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Journal of Traditional and Complementary Medicine

journal homepage: http: / /www.elsevier .com/locate/ j tcme

Original article

Extract of a polyherbal formulation ameliorates experimentalnonalcoholic steatohepatitis

Mohammed Azeemuddin a, Mohamed Rafiq a, *, Suryakanth Dattatraya Anturlikar a,Lakkavalli Mohan Sharath Kumar b, Pralhad Sadashiv Patki a, Uddagiri Venkanna Babu b,Ramakrishnan Shyam c

a Department of Pharmacology, R&D Center, The Himalaya Drug Company, Bangalore, Indiab Department of Phytochemistry, R&D Center, The Himalaya Drug Company, Bangalore, Indiac Chief Scientific Officer, R&D Center, The Himalaya Drug Company, Bangalore, India

a r t i c l e i n f o

Article history:Received 17 September 2014Received in revised form24 November 2014Accepted 4 December 2014Available online 22 January 2015

Keywords:antifibrotichigh-fat dietinsulin resistanceLiv.52nonalcoholic steatohepatitis

* Corresponding author. Department of PharmacoloDrug Company, Makali, Bangalore-562 162, Karnataka

E-mail address: dr.rafiq@himalayawellness.com (MPeer review under responsibility of The Center

National Taiwan University.

http://dx.doi.org/10.1016/j.jtcme.2014.12.0022225-4110/Copyright © 2014, Center for Food and Bio

a b s t r a c t

The objective of the present study is to evaluate the effect of the extract of a well-known hepatospecificpolyherbal formulation, Liv.52, in an experimental model of high-fat diet (HFD)-induced nonalcoholicsteatohepatitis (NASH) in rats. Feeding a HFD for 15 weeks resulted in significant impairment of the lipidprofile, elevation of hepatic enzyme markers, and insulin resistance in rats. The histological examinationof the liver furthermore indicated fibrotic changes and fat deposition in hepatic tissues. The treatmentwith Liv.52 extract [125 mg/kg body weight per os (b.wt. p.o.)], which was administered from week 9onward, reversed the HFD-induced changes to a statistically significant extent, compared to the un-treated positive control animals. The effect observed with Liv.52 extract was comparable to that ofpioglitazone (4 mg/kg b.wt.), a standard drug that is useful in the management of NASH. The treatmentwith Liv.52 extract significantly reduced steatosis, collagen deposition, and necrosis in hepatic tissues,which indicates its antifibrotic and antinecrotic properties. The results obtained in the present set ofexperiments indicate that Liv.52 extract effectively reverses metabolic and histological changes associ-ated with HFD-induced NASH.Copyright © 2014, Center for Food and Biomolecules, National Taiwan University. Production and hosting

by Elsevier Taiwan LLC. All rights reserved.

1. Introduction

The liver, which is the key organ of metabolism and excretion, isconstantly endowed with the task of detoxifying xenobiotics,environmental pollutants, and chemotherapeutic agents. Disordersassociated with this organ are numerous and varied. Nonalcoholicfatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH)are the most common liver diseases in the world. The mechanisminvolved in the pathogenesis of NAFLD/NASH has not been thor-oughly investigated. Some studies show that insulin resistance hasa key role in their pathogenesis. The exact mechanisms thatmediate the transition from steatosis to NASH remain unknown,although oxidative stress and cytokine-mediated injuries may have

gy, R&D Center, The Himalaya, India.. Rafiq).for Food and Biomolecules,

molecules, National Taiwan Unive

a key role in NASH pathogenesis.1,2 Under oxidative stress condi-tions, reactive oxygen species (ROS) lead to membrane lipid per-oxidation, inflammatory responses, stimulation of stellate cells, andfinally fibrosis.3

Modern medicine has not yet found a promising curative agent.Hence, the current use of peroxisome proliferator-activated re-ceptor gamma (PPAR-g), corticosteroids, and immunosuppressiveagents only produce symptomatic relief. Furthermore, their usageis associated with the risks of relapse and adverse effects.4e6

The indigenous system of medicine in India has a long traditionof treating liver disorders with plant drugs. Liv.52 is one suchpolyherbal proprietary formulations. It is approved by the Gov-ernment of India's Drug Regulatory Authority, the department ofAyurveda, Yoga, Naturopathy, Unani, Siddha and Homoeopathy(AYUSH) of the Ministry of Health and Family Welfare (New Delhi,India). The usefulness of Liv.52 in liver disorders of various etiol-ogies is confirmed by a substantial number of clinical trials con-ducted across the globe. It is also beneficial in treating viralhepatitis, drug-induced hepatic damage, and alcoholic liver

rsity. Production and hosting by Elsevier Taiwan LLC. All rights reserved.

M. Azeemuddin et al. / Journal of Traditional and Complementary Medicine 6 (2016) 160e167 161

disorders.7e10 The present study aimed to evaluate the effect ofLiv.52 extract in an experimental model of high-fat diet (HFD)-induced nonalcoholic steatohepatitis (NASH) in rats. In the presentstudy, pioglitazone, a standard reference drug that further validatesthe experimental model/procedure, was used for comparativestudy. The HFD model is a very appropriate model to induce NASH,obesity, and insulin resistance. “Overnutrition” with carbohydratesor fats or bothmay lead to obesity-related NAFLD. Insulin resistanceand obese diabetes do not solely initiate steatohepatitis, but they docontribute to the progression of the entire spectrum of pathology ofsteatohepatitis, at least partly, via the upregulation of genesinvolved in lipogenesis, inflammation, and fibrogenesis. Agonists ofPPAR-g reportedly prevent the progression of NASH in a dietarymodel of NASH associated with obese diabetes.11

2. Composition and preparation of Liv.52 extract

The Liv.52 extract constitutes a mixture of roots of Capparisspinosa (續隨子根 xù suí zǐ), seeds of Cichorium intybus (菊苣 jú jù),whole plant of Solanum nigrum (龍葵 l�ong kuí), Terminalia arjuna(bark), seed of Cassia occidentalis (望江南 w�ang ji�ang n�an), Achilleamillefolium (洋蓍草 y�ang sh�ı cǎo; aerial plant), and Tamarix gallica(whole plant). These crude herbal materials were subjected topulverization to obtain their coarse powders, and then blended.They were then soaked in pure water and boiled in a steam-jacketed stainless steel reactor until the extraction was complete.The liquid extract thus obtained was filtered through a muslin clothand concentrated in the reactors to attain 30% total solids. The softextract was then subjected to spray drying to obtain the dry extractpowder. It was tested for its quality and consistency at eachmanufacturing step per the accepted principles of goodmanufacturing practice (GMP) and good laboratory practice (GLP).Its botanical identification, quality parameters, and Ayurvediccriteria complied with the international guidelines and pharma-copoeial standards.12,13

3. High-performance liquid chromatography and liquidchromatographyemass spectrometry/mass spectroscopyfingerprinting of Liv.52 extract

3.1. Sample preparation and liquid chromatographic conditions

The concentration of the Liv.52 extract (DD-100) was 10 mg/mLin methanol. Liquid chromatography was performed by the Shi-madzu LC-20AD series pump (Shimadzu Corporation, Kyoto, Japan)and DUG-20A3 series Shimadzu degasser (Shimadzu Corporation,Kyoto, Japan). Chromatographic separation was performed on theLuna C18 column (250 mm � 4.6 mm, 5 mm; Phenomenex, Tor-rance, CA, USA). For the separation, the mobile phase gradientconsisted of water (J.T. Baker brand; Avantor Performance Mate-rials, Inc., Center Valley, PA, USA) with 10mM ammonium acetateand 0.1% formic acid in pump A and acetonitrile (J.T. Baker brand;Avantor PerformanceMaterials, Inc.) in pump B. The linear gradientprogramwas set as follows: 0e27 minutes of 20% of acetonitrile to80% acetonitrile (linear); 27e30 minutes of 80% acetonitrile to 20%acetonitrile (linear); followed by 30 minutes of 20% acetonitrile(isocratic) equilibration period for 2 minutes, delivered at a flowrate of 0.6 mL/min and a run time of approximately 32 minutes.Peak elution was monitored at 245 nm and 360 nm through theShimadzu SPD-20A UV/VIS detector (Shimadzu Corporation). Theinjection volume of 20 mL was injected through the SIL-HTC Shi-madzu Autosampler (Shimadzu Corporation). The ambient tem-perature was achieved through a CTO-10 AS VP column oven(Shimadzu Corporation) at 25 �C.

3.2. Mass spectrometric conditions

The API 2000 mass spectrometer (Applied Biosystems/MDSSCIEX, Ontario, Canada) was coupled with an electron spray ioni-zation source and a chromatographic system. Batch acquisition anddata processing were controlled by Analyst 1.5 version software.

Optimization of the mass spectroscopy (MS) parameters wasperformed with 2 mg/mL of test solutions that were preparedseparately and diluted in methanol (J.T. Baker brand; Avantor Per-formance Materials, Inc.). The intensity response was checked inthe positive ionization mode and negative ionization mode. Theintense response was good in the positive mode. Interface condi-tions such as the declustering potential (40 V), ion spray voltage(3500 V), nebulizing gas [GS1 (55 psi) and GS2 (65 psi)], curtain gas(25 psi), focusing potential (400 V), entrance potential (10 V), andsource temperature (420 �C) were optimized by multiple runsthrough liquid chromatography. Acquisition was performed bysetting the mass of the analytes with the appropriate scan range.(Fig. 1).

4. Materials and methods

4.1. Drugs and chemicals

Liv.52 extract was procured from the Phytochemistry Depart-ment of The Himalaya Drug Company (Bangalore, India) and pio-glitazone was procured from Sun Pharmaceuticals Industries Ltd.(Mumbai, India). Cholic acid and cholesterol were procured fromHiMedia Laboratories Ltd. (Mumbai, India). All other chemicalsused in the study were of reagent/analytical grade from reputedsuppliers. Biochemical parameters such as glucose, cholesterol,triglycerides (TGs), serum glutamic oxaloacetic transaminase(SGOT), serum glutamate pyruvate transaminase (SGPT), andalkaline phosphatase (ALP) were estimated in a biochemistryautoanalyzer (EM-360; Erba Diagnostics Mannheim GmbH, Man-nheim, Germany) using ready-made assay kits (Erba kits; Erba Di-agnostics Mannheim GmbH). Insulin was estimated using aradioimmunoassay kit (RIAK-1) supplied by Bhabha AtomicResearch Centre/Board of Radiation and Isotope Technology (BARC/BRIT; Mumbai, India).

4.2. Experimental animals

Inbred male Wistar rats (260e280 g) were housed under stan-dard conditions of temperature (22 ± 3 �C) at a relative humidity of55 ± 5% and 12-hour light/dark cycle prior to and during the study.The normal group animals were fed a standard pellet diet (ProvimiAnimal Nutrition India Pvt. Ltd., Bangalore, Karnataka, India), andwater ad libitum. The experimental protocols were approved by theInstitutional Animal Ethics Committee (IAEC) of The Himalaya DrugCompany (Bangalore, India). The animals received humane care asprescribed by the guidelines of the Committee for the Purpose ofControl and Supervision on Experiments on Animals (CPCSEA),Ministry of Environment and Forests, Government of India (NewDelhi, India).

4.3. Composition of HFD

The HFD consisted of 87.5% normal laboratory rodent feed, 10%animal fat, 0.5% cholic acid, and 2% cholesterol.14

4.4. HFD-induced nonalcoholic steatohepatitis

Male Wistar rats (n ¼ 36) that weighed 260e280 g wererandomly divided into four groups consisting of nine animals each.

Fig. 1. The chromatograms of Liv.52 extract by (A) high-performance liquid chromatography (LC) and (B) mass spectroscopy (MS).

M. Azeemuddin et al. / Journal of Traditional and Complementary Medicine 6 (2016) 160e167162

M. Azeemuddin et al. / Journal of Traditional and Complementary Medicine 6 (2016) 160e167 163

Group I rats received a normal diet and all other groups receivedHFD for 60 days. After 60 days, blood was collected from the retro-orbital plexus under mild ether anesthesia to determine the lipidprofile and to ensure that all rats in the HFD-fed groups werehyperlipidemic. After confirmation, the rats were segregated. Allrats received an equal amount of water (10 mL/kg b.wt.) as avehicle. Group I (i.e., animals fed a normal diet) served as normalcontrol, Group II (i.e., animals fed a HFD) served as the positivecontrol, and Group III rats received Liv.52 extract (125 mg/kg b.wt.)along with HFD. The dose was selected based on dose range findingstudies performed in the laboratory to evaluate its hep-atoprotective activity in rats; the dosing volumewas determined bythe weekly mean group body weight of the animals. Group IV ratsingested a HFD and received pioglitazone (4 mg/kg b.wt.).

Each group received its respective assigned treatment orallyonce daily for 42 days. Daily clinical observations and the weeklybody weights of the animals were recorded for all groups. At theend of the treatment period, the animals underwent 12-hourfasting. Their blood was collected from the retro-orbital plexusunder mild ether anesthesia to estimate biochemical parameterssuch as glucose, insulin, cholesterol, TG, SGPT, SGOT, and ALP. Aftercollecting their blood, the rats were euthanized by deep etheranesthesia. A small portion of their liver was fixed in formalin,subjected to routine histological procedures, and stained with he-matoxylin and eosin (H&E) and Masson trichrome.15e17

The serum insulin was estimated by standard radioimmuno-assay technique using a kit (RIAK-1) supplied by BARIC/BRIT(Mumbai, India). The degree of insulin resistance was estimated byusing the homeostasis model assessment (HOMA) as the index ofinsulin resistance.18 The formula for the HOMA index is:

HOMA index¼ InsulinðmU per literÞ�glucoseðmmol per literÞ22:5

4.5. Statistical analysis

All values are expressed as the mean ± standard error of themean (SEM). The results were statistically analyzed by one-wayanalysis of variance (ANOVA), followed by Dunnett's post test, us-ing Prism GraphPad 4.03 software, Windows version (GraphPadSoftware Inc., San Diego, CA, USA). A p value <0.05 was consideredstatistically significant.

5. Results and discussion

The present study evaluated the effect of Liv.52 extract in anexperimental model of HFD-induced NASH in rats. Feeding ani-mals a HFD significantly impaired the lipid profile and elevated

Table 1The serum biochemical parameters of the rats.

SGOT (IU/L) SGPT (IU/L) ALP (IU/L) Cho(m

Normal control 152.3 ± 5.7 69.8 ± 1.7 164.3 ± 10.3 97.Positive control (untreated) 199.6 ± 6.7b 88.5 ± 5.1a 272.4 ± 25.4b 1Liv.52 extract (125 mg/kg p.o.) 163.6 ± 7.5c 71.7 ± 6.2c 190.5 ± 12.6d 113Pioglitazone (4 mg/kg p.o.) 163.0 ± 18.0c 69.4 ± 2.6d 201.2 ± 13.2c 116

All values are expressed as the mean ± the standard error of the mean (SEM).ALP ¼ alkaline phosphatase; HOMA ¼ homeostasis model assessment; p.o. ¼ per os; SGtransaminase; TG ¼ triglyceride.

a Indicates significance at p < 0.05, compared to the normal control.b Indicates significance at p < 0.01, compared to the normal control.c Indicates significance at p < 0.05, compared to the positive control.d Indicates significance at p < 0.01, compared to the positive control.

the level of serum insulin, glucose, and liver function marker en-zymes SGPT, SGOT, and ALP. The changes were statistically sig-nificant, compared to the normal controls (i.e., animals fed anormal diet). Treatment with the Liv.52 extract was initiated at theend of 9th week of HFD supplementation. Group IV animals weretreated with pioglitazone, which was used as the referencestandard.

Forty-two days of treatment orally with Liv.52 extract (125 mg/kg) reversed the elevated serum markers SGOT, SGPT, ALP, andreversed the cholesterol and TG levels to a statistically significantextent, compared to the untreated HFD control animals. The oraladministration of pioglitazone (4 mg/kg) also decreased theaforementioned serum parameters to a statistically significantlevel, compared to the positive untreated control (Table 1).

Insulin resistance that developed because of the intake of a HFDwas also decreased in animals treated with Liv.52 extract or pio-glitazone. This difference was statistically significant, compared tothe positive untreated HFD animals. Liv.52 extract and pioglitazoneboth decreased the fasting serum glucose, the insulin level, and theHOMA index, which are measures of insulin resistance (Table 1).

The histopathological examination and scoring of the liversections (stained by H&E and Masson trichome) also revealed thebeneficial effect of Liv.52 extract and pioglitazone in reversingmany of the pathological changes associated with a HFD in rats.Most notably, the drugs significantly reduced collagen deposition,necrosis, and steatosis in hepatic tissues, which are hallmarks ofNASH (Table 2 and Figs. 2 and 3). Nonalcoholic steatohepatitis re-fers to a spectrum of hepatic pathology that resembles alcoholicliver disease, but appears in individuals who have low or negligiblealcohol consumption. Nonalcoholic fatty liver disease ranges from afatty liver alone to NASH. Increasing evidence suggests that NASH isassociated with progressive fibrosis, cirrhosis, and eventually he-patocellular cancer.19 It has been suggested that the term “nonal-coholic fatty liver disease” should be used only for the more severeforms of NAFLD that correspond to types 3 and 4with alcoholic-likehistological findings. The medical conditions most frequentlyassociated with NAFLD and NASH are obesity, diabetes mellitus,and dyslipidemia.20

The HFD model is useful in reproducing the human conditionbecause it contains predominantly saturated fat intake, which ispositively correlated with the insulin sensitivity index, and alanineaminotransferase levels in patients with NASH. Peripheral insulinresistance can induce hepatic steatosis, which then causes insulinresistance in the liver and is characterized by a reduced insulin-suppressing effect in hepatic glucose production; this aggravatesperipheral insulin resistance and contributes to hepaticlipogenesis.21

Insulin resistance is a complex metabolic abnormality that af-fects the ability of peripheral tissues to respond to insulin. It is aprominent feature of metabolic syndrome and type 2 diabetes, and

lesterolg/dL)

TG (mg/dL) Fasting glucose(mg/dL)

Insulin (mU/mL) HOMA index

88 ± 3.7 117.4 ± 14.3 64.33 ± 3.3 27.93 ± 3.5 3.857 ± 0.4446 ± 10a 174.3 ± 12.4a 78.5 ± 5.2a 52.71 ± 3.6b 7.678 ± 0.44a

.1 ± 8d 106.9 ± 12d 48.43 ± 2.8d 35.33 ± 2.5c 4.686 ± 0.43d

.8 ± 6.3c 144.5 ± 21.6c 48 ± 3.1d 34.00 ± 6.23c 4.671 ± 0.43d

OT ¼ serum glutamic oxaloacetic transaminase; SGPT ¼ serum glutamate pyruvate

Table 2Histopathological findings of the rat liver samples among the four groups.

Normal control Positive control(untreated)

Liv.52 extract(125 mg/kg b.wt.)

Pioglitazone(4 mg/kg b.wt.)

H&E stainInflammatory reaction 0.778 ± 0.32 2.000 ± 0.29a 1.444 ± 0.24 1.333 ± 0.33Biliary hyperplasia 0.556 ± 0.29 2.444 ± 0.34b 0.222 ± 0.22d 0.667 ± 0.33d

Fat vacuolating accumulation in parenchyma 0.000 ± 0.00 3.333 ± 0.21b 2.857 ± 0.50 1.714 ± 0.47c

Necrosis in parenchyma 0.000 ± 0.00 1.571 ± 0.28b 0.428 ± 0.29d 0.222 ± 0.15d

Masson’s trichrome stainCollagen deposition 0.667 ± 0.29 3.500 ± 0.18b 1.714 ± 0.29d 1.875 ± 0.44d

Fat vacuolating accumulation 0.000 ± 0.00 3.333 ± 0.21b 2.857 ± 0.50 1.714 ± 0.47c

H&E Stain (score):Inflammatory reaction (score of 1e3)Biliary hyperplasia (score of 1e3)Fat vacuolating accumulation (score of 1e4)Necrosis in parenchyma [score-focal (1) and multi-focal (2)]Masson's trichrome Stain (score):Collagen deposition (score of 1e4)Fat vacuolating accumulation in the parenchyma (score of 1e4)All values are expressed as the mean ± the standard error of the mean (SEM).b.wt. ¼ body weight; H&E ¼ hematoxylin and eosin.

a Indicates significance at p < 0.05, compared to the normal control.b Indicates significance at p < 0.01, compared to the normal control.c Indicates significance at p < 0.05, compared to the positive control.d Indicates significance at p < 0.01, compared to the positive control.

M. Azeemuddin et al. / Journal of Traditional and Complementary Medicine 6 (2016) 160e167164

is a major risk factor for cardiovascular disease. There may be aclose interplay between increased tumor necrosis factor (TNF)-aexpression, insulin resistance, and lipolysis in adipose tissue thatresults in the increased delivery of free fatty acids, TNF-a, andcortisol to the liver, which initially lead to the development ofsteatosis. In patients with NAFLD, increased fat accumulationeventually results in hepatic insulin resistance. The insulin resis-tance increases fat oxidation, which in association with TNF-a,

Fig. 2. Photomicrographs of the liver sections show (A) normal structure and architectumacrovesicular steatosis in the positive control animals fed a high-fat diet; (C) Grade 2 steakg body weight; and (D) Grade 2 steatosis in animals fed a high-fat diet and treated with100�).

generates oxidative stress and mitochondrial uncoupling. Theexpression of TNF-a and mitochondrial dysfunction leads to he-patocyte death, inflammation, and ultimately fibrosis.22,23

In the present study, the effect of Liv.52 extract was comparedwith pioglitazone, whichwas used as the reference standard in a ratmodel of NASH. Previous reports indicate the usefulness of piogli-tazone in an experimental model of NASH in rats.24 Pioglitazone isalso a reasonable option for treating NASH, particularly in patients

re in the normal control animals fed a normal diet; (B) severe microvesicular andtosis in animals fed a high-fat diet and treated with Liv.52 extract at a dose of 125 mg/piglitazone at a dose of 4 mg/kg body weight (hematoxylin and eosin; magnification,

Fig. 3. Photomicrographs of liver sections show(A) no collagen deposition in the normal control animals fed a normal diet; (B) severe perivascular edema and collagen deposition inthe positive control animals fed a high-fat diet; (C) Grade 2 collagen deposition around the periportal zone and perivascular necrosis and edema in animals treated with Liv.52extract (12 mg/kg body weight); and (D) mild steatosis and collagen deposition in animals treated with picoglitazone (4 mg/kg body weight) (Masson's trichome; magnification,100�).

M. Azeemuddin et al. / Journal of Traditional and Complementary Medicine 6 (2016) 160e167 165

with type 2 diabetes mellitus or impaired glucose tolerance. Pio-glitazone therapy was associated with reduced hepatocellularinjury and fibrosis in patients with NASH. However, the cardio-vascular benefiterisk ratio of this drug must at least beconsidered.25

Oxidative stress may contribute to the development of severalage-related and chronic diseases such as cancer, diabetes, neuro-degenerative diseases, and cardiovascular diseases.26e28 In partic-ular, ROS and reactive nitrogen species have a crucial role in diseaseinduction and in the progression of liver diseases such as hepato-cellular carcinoma, viral and alcoholic hepatitis, and nonalcoholicsteatosis.29,30 In our previous studies, Liv.52 extract exhibited apotent beneficial effect in regulating oxidative stress induced byvarious liver toxicants. It abrogates copper (II)-induced cytotoxicityin HepG2 cells by inhibiting lipid peroxidation, increasing theglutathione content, and increasing endogenous antioxidantenzyme activities.31 By preventing intracellular glutathionedepletion and lipid peroxidation, it also reportedly inhibits hepaticcell death evoked by tertiary-butyl hydroperoxide.32

There are other reports that indicate the usefulness of plant-based antioxidants in the management of NASH. Genistein is astrong antioxidant agent that is capable of decreasing the plasmaTNF-a level and attenuating the incidence of NASH33. A Chinesepolyherbal blend comprising Panax pseudoginseng (三七 s�an q�ı),Eucommia ulmoides (杜仲 dù zh�ong), Polygonati rhizoma (黃精

hu�ang j�ıng), and licorice root (甘草根 g�an cǎo g�en) reportedlymarkedly reduces macrosteatosis while significantly improvinghistological markers of inflammation in HFD-fed experimentalanimals. These effects have also been associated with a markedreduction in serum aminotransferases and hepatic lipoperoxideand glutathione content, which implies that the herbal complex hasnoticeable antioxidant activity.34

Nonalcoholic steatohepatitis is histologically characterized bydiffuse fatty infiltration, lobular inflammation, ballooning degen-eration, and fibrosis in the liver.35,36 In the present study, many liverhistopathological features of animals fed a HFD were similar to thefindings of other published reports, and the treatment by Liv.52extract or pioglitazone significantly reversed these histopatholog-ical anomalies (Figs. 2 and 3). The Liv.52 extract treatment signifi-cantly reduced steatosis, collagen deposition, and necrosis ofhepatic tissues, which indicate its antifibrotic and antinecroticproperties. Increased mean body weight occurred in the animalstreated a HFD, compared to the normal control group; however,this difference was not statistically significant. The treated groupsshowed amarginal decrease in mean body weight in comparison tothe positive untreated controls; however, this finding was similarlyinsignificant (Fig. 4).

The results of the present study showed that the treatment withthe Liv.52 extract was comparable to pioglitazone at the selecteddose, by the method employed, and by the mode of administration.The mechanism behind the beneficial action of Liv.52 extract in thepresent experimental model of NASH could be because of its potentantioxidant and other hepatospecific actions, as reportedpreviously.

6. Conclusion

From the biochemical and histopathological data obtained in thepresent study, the treatment by Liv.52 extract administered orallyat a daily dose of 125 mg/kg b.wt. exhibited a beneficial effect byreversing serum biochemical parameters in rats with NASHinduced by a HFD. The Liv.52 extract treatment also significantlyreduced steatosis, collagen deposition, and necrosis in hepatic tis-sues, which indicate its antifibrotic and antinecrotic properties. The

Fig. 4. The mean body weight change in the different groups.

M. Azeemuddin et al. / Journal of Traditional and Complementary Medicine 6 (2016) 160e167166

effect of Liv.52 extract was comparable to that of pioglitazone at theselected dose, by the method employed, and by the mode ofadministration. The mechanism behind the beneficial action ofLiv.52 extract in the present experimental model of NASH could bebecause of its potent antioxidant and other hepatospecific actions,as previously reported in the literature.

Conflicts of interest

The authors declare no conflicts of interest and guarantee noethical conflicts among the authors or the experimental method-ology. The test drug (i.e., Liv.52 extract) was received in a codedform from the Phytochemistry Department of The Himalaya DrugCompany (Bangalore, India) and all experiments were performed inthe Department of Pharmacology at the Research & DevelopmentCenter of The Himalaya Drug Company (Bangalore, India). Noformal funding from any agency was used for this project.

Acknowledgments

The authors acknowledge the work of Dr. Jayashree B Keshavand her team (Scientific Publications Division, The Himalaya DrugCompany, Bangalore, India), in terms of editing and proofreadingthe paper.

References

1. Pinto HC, Moura DEMC, Day CP. Nonalcoholic steatohepatitis: from cell biologyto clinical practice. J Hepatol. 2006;44:197e208.

2. Day CP, James OF. Steatohepatitis: a tale of two hits. Gastroenterology.1998;114:842e845.

3. Chitturi S, Farrell GC. Etiopathogenesis of nonalcoholic steatohepatitis. SeminLiver Dis. 2001;21:27e41.

4. Farrell GC, George J, Hall P, McCullough AJ. Fatty Liver Disease: NASH and RelatedDisorders. Oxford, England: Blackwell; 2005:1e12.

5. Oneta CM, Dufour JF. Nonalcoholic fatty liver disease: treatment options basedon pathogenic considerations. Swiss Med Wkly. 2002;132:493e505.

6. Aguilar FP, Benlloch S, Berengnes M, Beltram B, Berengner J. Nonalcoholicsteatohepatitis: physiopathological, clinical and therapeutic implications. RevEsp Enferm Dig. 2004;96:628e648.

7. Kalab M, Krechler T. Effect of the hepatoprotective drug Liv.52 on liver damage[in Czechoslovakian] J Czech Physicians. 1997;24:758e760.

8. Chauhan BL, Kulkarni RD. Dose response of ethanol after chronic administra-tion of alcohol and effect of Liv.52 on blood, liver ethanol and acetaldehydelevels in rats. Eur J Pharmacol. 1990;5:1864.

9. Huseini HF, Alavian SM, Heshmat R, Heydari MR, Abolmaali K. The efficacy ofLiv-52 on liver cirrhotic patients: a randomized, double-blind, placebo-controlled first approach. Phytomedicine. 2005;12:619e624.

10. Chauhan BL, Kulkarni RD. Effect of liv52, a herbal preparation, on absorptionand metabolism of ethanol in humans. Eur J Clin Pharmacol. 1991;40:189e191.

11. Ota T, Takamura T, Kurita S, et al. Insulin resistance accelerates a dietary ratmodel of nonalcoholic steatohepatitis. Gastroenterology. 2007;132:282e293.

12. Anonymous. World Health Organization: General Guidelines for MethodologiesResearch and Evaluation of Traditional Medicine. vol. 1. Geneva, Switzerland:World Health Organization; 2000:1e74.

13. Ministry of Health and Family Welfare. Department of Ayurveda, Yoga, Natu-ropathy, Unani, Siddha and Homeopathy. The Ayurvedic Pharmacopoeia of IndiaGovernment of Indiavol. 2. New Delhi, India: Ministry of Health and FamilyWelfare; 2007:133e246.

14. Dai DL, Shen W, Yu HF, Guan XQ, Yi YF. Effect of Cordyceps sinensis onuncoupling protein 2 in experimental rats with nonalcoholic fatty liver. J HealthSci. 2006;52:390e396.

15. Xu P, Zhang XG, Li YM, Yu CH, Xu L, Xu GY. Research on the protection effect ofpioglitazone for nonalcoholic fatty liver disease (NAFLD) in rats. J Zhejiang UnivSci. 2006;7:627e633.

16. Hong XZ, Li LD, Wu LM. Effects of finofibrate and xuezhikang on high-fat diet-induced non-alcoholic fatty liver disease. Clin Exp Pharmacol Physiol. 2007;34:27e35.

17. Altunkaynak BZ. Effects of high fat diet induced obesity on female rat livers (ahistochemical study). Eur J Gen Med. 2005;2:100e109.

18. Mlinar B, Marc J, Janez PM, Pfeifer M. Molecular mechanisms of insulin resis-tance and associated diseases. Clin Chim Acta. 2007;375:20e35.

19. Nanji AA. Another animal model for nonalcoholic steatohepatitis: how close tothe human condition? Am J Clin Nutr. 2004;79:350e351.

20. Malnick SDH, Beergabel M, Knobler H. Nonalcoholic fatty liver: a commonmanifestation of a metabolic disorder. QJM. 2003;96:699e709.

21. Svegliati-Baroni G, Candelaresi C, Saccomanno S, et al. A model of insulinresistance and nonalcoholic steatohepatitis in rats: role of peroxisomeproliferator-activated receptor-and n-3 polyunsaturated fatty acid treatmenton liver injury. Am J Cardiovasc Pathol. 2006;169:846e860.

22. Chen CC, Liu IM, Cheng JT. Improvement of insulin resistance by miracle fruit(Synsepalum dulcificum) in fructose-rich chow-fed rats. Phytother Res. 2006;20:987e992.

23. Day CP. Pathogenesis of steatohepatitis. Best Pract Res Clin Gastroenterol.2002;16:663e678.

24. Aithal GP, Thomas JA, Kaye PV, et al. Randomized, placebo-controlled trial ofpioglitazone in nondiabetic subjects with nonalcoholic steatohepatitis.Gastroenterology. 2008;135:1176e1184.

25. Eslami L, Merat S, Moghaddam SN. Treatment of nonalcoholic fatty liver dis-ease (NAFLD): a systematic review. Middle East J Dig Dis. 2009;1:89e99.

26. Tiwari AK. Antioxidants: new-generation therapeutic base for treatment ofpolygenic disorders. Curr Sci. 2004;86:1092e1102.

27. Willcox JK, Ash SL, Catignani GL. Antioxidants and prevention of chronic dis-ease. Crit Rev Food Sci Nutr. 2004;44:275e295.

28. Ballinger SW. Mitochondrial dysfunction in cardiovascular disease. Free RadicBiol Med. 2005;28:1278e1295.

29. Adachi M, Ishii H. Role of mitochondria in alcoholic liver injury. Free Radic BiolMed. 2002;32:487e491.

30. Vitaglione P, Morisco F, Caporaso N, Fogliano V. Dietary antioxidant com-pounds and liver health. Crit Rev Food Sci Nutr. 2004;44:575e586.

M. Azeemuddin et al. / Journal of Traditional and Complementary Medicine 6 (2016) 160e167 167

31. Vidyashankar S, Patki PS. Liv.52 attenuate copper induced toxicity by inhibitingglutathione depletion and increased antioxidant enzyme activity in HepG2cells. Food Chem Toxicol. 2010;48:1863e1868.

32. Vidyashankar S, Mitra SK, Nandakumar KS. Liv.52 protects HepG2 cells fromoxidative damage induced by tert-butyl hydroperoxide. Mol Cell Biochem.2010;333:41e48.

33. Yalniz M, Bahcecioglu IH, Kuzu N, et al. Preventive role of genistein in anexperimental nonalcoholic steatohepatitis model. J Gastroenterol Hepatol.2007;22:2009e2014.

34. Lima VM, Oliveira CP, Sawada LY, et al. Yo jyo hen shi ko, a novel Chineseherbal prevents nonalcoholic steatohepatitis in ob/ob mice fed a high fat ormethionine-choline-deficient diet. Liver Int. 2007;27:227.

35. Harrison SA, Kadakia S, Lang KA, Schenker S. Nonalcoholic steatohepatitis:what we know in the new millennium. Am J Gastroenterol. 2002;97:2714.

36. Rivera CA. Risk factors and mechanisms of nonalcoholic steatohepatitis. Path-ophysiology. 2008;15:109.