Original article

Design and evaluation of herbal hepatoprotective formulation againstparacetamol induced liver toxicity

Arti Gupta a Navin R Sheth b Sonia Pandey a Dinesh R Shah a Jitendra S Yadav c

aMaliba Pharmacy College Bardoli Gujarat IndiabDepartment of Pharmacognosy Saurashtra University Rajkot Gujarat IndiacDepartment of Pharmaceutics Vidya Bharti Trust College of Pharmacy Bardoli Gujarat India

a r t i c l e i n f o

Article historyReceived 19 August 2013Accepted 11 December 2013Available online 7 January 2014

KeywordsButea monospermaBauhinia variegataOcimum gratissimumPolyherbal formulationHepatoprotective activity

a b s t r a c t

Aim To isolate and identify the quercetin from polyherbal hepatoprotective formulation Polyherbalformulations were developed by using five bioactive fractionated extracts of Butea monosperma Bauhiniavariegata and Ocimum gratissimum for treatment of liver disorders by exploiting the knowledge oftraditional system of medicine and evaluated for hepatoprotective activity using acute liver toxicitymodel of paracetamol induced liver damage in ratsMethods Major active fractions were isolated by solvent fractionation and quantified by HPTLC methodTwo polyherbal tablet formulations were developed by the wet granulation method using microcrys-talline cellulose aerosil and other excipients and subjected for physicochemical evaluation to assessphysical stability followed by pharmacological screening The prepared tablets were finally subjected tostability testing to assess its shelf-life The rats were monitored for change in liver morphologybiochemical parameters like serum glutamate pyruvate transaminase (SGPT) serum glutamate oxalo-acetate transaminase (SGOT) alkaline phosphatase (ALP) and total bilirubin for polyherbal tabletformulation at 50 mgkg and polyherbal tablet formulation at 100 mgkgResults Active principle was isolated quantified by HPTLC and characterized with IR Both formulationsshowed significant hepatoprotective activity The histological studies were also support the biochemicalparameters From the results of biochemical analysis and histopathological studies it can be accom-plished that polyherbal tablet formulation at 100 mgkg can be effectively formulated into a suitabledosage form with added benefit of no side effects for control and cure of chronic ailments like liverdisorders A comparative histopathological study of liver exhibited almost normal architecture ascompared to toxicant groupConclusion Biochemical marker showed improved results for polyherbal tablet formulation at 100 mgkg Polyherbal tablet formulation contains a potent hepatoprotective agent suggested to be a flavoneconcentrated in polyherbal formulation which may find clinical application in amelioration of paracet-amol induced liver damageCopyright 2013 InPharm Association Published by Reed Elsevier India Pvt Ltd All rights reserved

1 Introduction

In the last few decades there has been an exponential growth inthe field of herbal medicine It is getting popularized in developingand developed countries owing to its natural origin and lesser sideeffects1 Herbal remedies provide rational means for the treatmentof many internal diseases which are considered to be stubborn andincurable in other system of medicines It lays a great deal of

emphasis upon the maintenance of positive health of an individualIt thus aims at both the prevention and cure of diseases2 An ancientsystem of traditional medicines like Siddha Ayurveda Chinese andJapanese have been adopted for the diagnosis prevention andtreatment of liver disorders This attempts to prove scientificinsight behind the traditional adaption Less toxicity better thera-peutic effect good patient compliance and cost effectiveness arethe reasons for choosing drug from natural origin3

Ayurvedic and herbal medicinal products contain a combinationof number of chemical compounds that may give the anticipatedactivity in combination4

The development of a stable polyherbal formulation is a chal-lenging task because of the large number of varied chemical

Corresponding author Maliba Pharmacy College Gopal Vidhya Nagar BardoliGujarat India

E-mail address artiguptautuacin (A Gupta)

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0975-1483$ e see front matter Copyright 2013 InPharm Association Published by Reed Elsevier India Pvt Ltd All rights reservedhttpdxdoiorg101016jjyp201312003

Journal of Young Pharmacists 5 (2013) 180e187

compounds present in the different medicinal plants Hence theentire herbal drug or herbal drug preparation is regarded as activedrug substance regardless of whether constituents with definedtherapeutic activity are known and unfortunately the quality of amajority of them remains uncontrolled All these issues have beenacknowledged in the draft of the Strategic Plan for RegionalTraditional Medicine of the World Health Organization5

A great deal of research has been carried out to evaluate sci-entific basis for the claimed hepatoprotective activity of herbalagents as in the form of polyherbal formulation Butea monosperma(Fabaceae) is commonly known as palas and is beneficial in con-ditions of liver disease and inflammation6 The plant mainly con-tains flavones (quercetin)7 kino-tannic acid and gallic acid8

Bauhinia variegata (Leguminosae) commonly known as Kachnaris found to be beneficial in Ayurveda as tonic to the liver and anti-inflammatory healing activity antioxidant activity9 B variegatahas been reported to contain quercetin rutin apigenin and apige-nin 7-O-glucoside Flavonoids and quercetin in particular arepotent antioxidants and are known to modulate the activities ofvarious enzyme systems due to their interaction with variousbiomolecules10

Ocimum gratissimum Linn (Lamiaceae) is known traditionallyfor its effect in liver diseases inflammation and antibacterial ac-tivity11 The active constituents flavonoids quercetin 3-O-gluco-side rutin kaempferol 3-O-rutinoside and vicenin-2 wereidentified12

Quercetin (flavonoid) and ursolic acid (triterpenic acid) wellknown for its hepatoprotective effects in both acute chemicallyinduced liver injury and chronic liver fibrosis and cirrhosis13

Ursolic acid was previously determined in dichloromethaneand ethyl acetate fractions of methanolic extract of O gratissimumand in developed herbal hepatoprotective tablet in my previousarticle8

The present study was designed to develop polyherbal formu-lation taking advantage of ayurvedic principles where herbal in-gredients present in plants are known to have specific activity inmodulation of liver disease condition so the present study wasundertaken to evaluate the hepatoprotective effect of developedpolyherbal formulation in which acute hepatotoxicity was inducedby paracetamol treatment

2 Material and methods

21 Material

The fresh bark of B monosperma B variegata and fresh leaves ofO gratissimum were collected from Maliba Pharmacy CollegeCampus Voucher specimen (No MPC1303201001 02 and 03) hasbeen deposited in the Department of Bioscience Veer NarmadSouth Gujarat University Surat India Rats were used for hep-atoprotective study with prior approval from the InstitutionalAnimal Ethical Committee (Registration No 71702aCPCSEA30Jan 2010) of Maliba Pharmacy College Uka Tarsadia University

22 Extraction

All the three crude drugs were extracted with alcohol and thenalcoholic extract of each plant was subjected to solventfractionation

B monosperma Ethanolic extract obtained was fractionatedwith petroleum ether benzene chloroform and acetone (AcO) inthe order of increasing polarity to obtain respective fractions14

B variegata Methanolic extract was fractionated with hexaneethyl acetate (EtOAc) and n-butanol (n-ButOH) in the order of theirincreasing polarity to obtain respective fractions15

O gratissimum Methanolic extract of O gratissimum wasfractionated with hexane dichloromethane(DCM) ethyl acetate(EtOAc) and methanol in the order of their increasing polarity toobtain respective fractions16

23 Establishment of qualitative phytoprofile of fractionatedextracts

231 Qualitative phytochemical analysisEach fractionwas subjected to various qualitative chemical tests

using reported methods to determine the presence or absence ofmetabolites viz alkaloids tannins flavonoid steroid terpenoidsand phenolic compounds etc17

2311 Chemical test for flavonoids in each fraction

i Dilute ammonia solution 5 mL was added to a portion of eachextract fraction followed by addition of concentrated H2SO4 ayellow coloration observed indicated the presence of flavonoids

ii The yellow coloration disappeared on standing Few drops of 1aluminum solution were added to a portion of each fraction Ayellow coloration was observed indicating the presence offlavonoid18

24 Qualitative phytochemical analysis

241 Determination of total phenolsAcetone fraction of B monosperma ethyl acetate and n-butanol

fractions of B variegata and dichloromethane and ethyl acetatefractions ofO gratissimum (100 mL) of each fractionweremixedwith1 mL FolineCiocalteu reagent and 08 mL of 75 Na2CO3 Theresultant mixture of each fractionwasmeasured at 765 nm after 2 hat room temperature The mean of three readings was used and thetotal phenolic content was expressed in milligram of gallic acidequivalents1 g extract The coefficient of determination was foundto be r2 frac14 099219

242 Determination of total flavonoidsStandard quercetinwas used tomake the calibration curve [004

002 00025 and 000125mgmL in 80 ethanol (vv)] The standardsolutions and acetone fraction of B monosperma ethyl acetate andn-butanol fractions of B variegata and dichloromethane and ethylacetate fractions of O gratissimum (05 mL) of each fraction wasmixed with 15 mL of 95 ethanol (vv) 01 mL of 10 aluminumchloride (wv) 01 mL of 1 molL sodium acetate and 28 mL waterThe volume of 10 aluminum chloride was substituted by the samevolume of distilled water in blank After incubation at room tem-perature for 30 min the absorbance of the reaction mixture of eachfraction and standard solutionwasmeasured at 415nm Themeanofthree readings was used and the total flavonoid content wasexpressed in milligram of quercetin equivalents1 g extract Thecoefficient of determination was r2 frac14 099020

25 Preparation of formulations

Polyherbal tablet formulations (PTF) contain the crude rawmaterials of B monosperma B variegata and O gratissimum pre-pared by wet granulation method using suitable excipients likemicrocrystalline cellulose starch crospovidone aerosil and mag-nesium stearate21 The composition of tablet formulations is shownin Table 1

A Gupta et al Journal of Young Pharmacists 5 (2013) 180e187 181

26 Quantification of quercetin by HPTLC

A Camag HPTLC system was used with beam size of04 04 mm and wavelength of 380 nm Silica gel 60 F 254(20 20 cm 025-mm thick Merck) plates were used for HPTLCSamples were applied (15mm from the bottom edge of the plate) inthe form of 6 mm bandwidth with Hamilton syringes using Lino-mat V applicator A constant application rate of 150 nls was sup-plied and a space of 10 mm was kept between two bands Themobile phase constituted of tolueneethyl acetateformic acid5401 (vvv) Linear ascending development was carried out in atwin trough glass chamber saturated with mobile phase Theoptimized saturation time for mobile phase was 30 min at roomtemperature (25 C 2 C) with relative humidity of 55 5 Afterdeveloping TLC plate a current of air was used to dry it Densito-metric scanning was performed on Camag TLC scanner III inabsorbance mode at 380 nm The source of light was deuteriumbeam The quantitative determination was performed by using theexternal calibration method A stock solution of quercetin (500 mgmL) was prepared in methanol Different volume of stock solutionie 05 10 15 20 25 in mgspot were spotted on TLC plate of 200400 800 1600 and 3200 ngspot of quercetin Calibration curverange (05e25 mgspot) for quercetin was found to be linear Thedata of peak areas plotted against the corresponding concentra-tions were collected To determine the content of quercetin inextract 10 mg of each fraction was transferred in 10 mL volumetricflask containing 8 mL methanol vortexed for 10 min and diluted to10 mL with methanol The resulting solution was filtered throughWhatman filter paper The 15 mL of the filtered solutionwas appliedon TLC plate subsequently developed and scanned as describedabove

27 Evaluation of power blends

The polyherbal tablets were prepared by wet granulationmethod hence micromeritic studies (angle of repose bulk densitytapped density compressibility) were evaluated prior to punch-ing of tablets

28 Physical characterization of polyherbal tablets

Polyherbal tablets systems were characterized for thicknessdiameter disintegration time weight variation and friabilityCrushing strength thickness and friability were determined byMonsanto hardness tester digital vernier calipers and friabilatorrespectively

29 Isolation and identification of quercetin from polyherbal tablet

Weight accurately 500 mg equivalent of polyherbal tablet wastransferred to the 10 mL volumetric flask and dissolved in 10 mLmethanol This solution was sonicated for 10 min and filteredthroughWhatman No1 paper to get solution containing 10mgmLConcentrated solution obtained from polyherbal tablet was sub-jected to preparative TLC Preparative TLC were performed on20 cm 10 cm TLC aluminum plate coated with 200 mm layerthickness of silica gel 60 F 254 using tolueneethyl acetateformicacid 5401 (vvv) as mobile phase The quantificationwas carriedout by densitometric scanning in absorbance mode at wavelength380 nm The silica in the respective marked area was scraped offand collected carefully in a test tube The scraped silica whichcontained the standard quercetin was extracted in methanol andfiltered individually The filtrates containing quercetin were evap-orated under reduced pressure to obtain quercetin

Melting point of quercetin was determined by open capillarymethod Structural confirmation of the isolated quercetinwas doneby IR Spectroscopy and compared with that of standard

210 In-vivo hepatoprotective activity of polyherbal tabletformulations against paracetamol induced toxicity

2101 Test animalsMale Wistar rats (125e175 g) were used for determination of

maximum tolerable dose (MTD) and evaluation of hepatoprotectiveactivity The animals were housed in polypropylene cages at25 C 1 C with the relative humidity of 55 5 under 12 h12 hlightdark cycle They were received a standard chow and water adlibitum during experimentation The food was with-drawn on theday before the experiment but free access of water was allowed Aminimum of six animals was used in each group Throughout theexperiments animals were processed according to the suggestedinternational ethical guidelines for the care of laboratory animalsThe study protocol was approved by the Institutional Animal EthicsCommittee according to the regulation of Committee for the Pur-pose of Control and Supervision of Experiments on Animals (MPC1007 dated 30012010)

2102 Preparation and administration of test samplesApproved IAEC method was adopted for screening of hep-

atoprotective activity Dried fractions were suspended in 1 CMCsolution before oral administration to animals Animals weredivided into five groups of six rats each were used for the studyGroups 1 and 2 received normal saline (1 mLkg orally) for 7 daysGroups 3 4 and 5 received 100 mgkg bw doses of silymarin PTF1(50 mgkg bw doses) and PTF2 (100 mgkg bw doses) orally once aday for 7 days On the fifth day after the administration of therespective treatments all the animals in groups II III IV and V wereadministered paracetamol (PCM) 2 gkg orally On the seventh daythe blood samples were collected via orbital sinus puncture for theestimation of biochemical marker enzymes Then the liver wascarefully isolated and cleaned off extraneous tissue and preservedin 10 neutral formalin and then subjected to histopathologicalstudies2223

Table 1Composition of polyherbal tablets formulations (PTF)

Sr no Ingredients PTF-1 PTF-2

Quantity(mgtablet)

Quantity(mgtablet)

Dry mix1 Acetone fraction of

Butea monosperma10 20

2 Ethyl acetate fractionof Bauhinia variegata

10 20

3 n-Butanol fraction ofBauhinia variegata

10 20

4 Dichloromethane ofOcimum gratissimum

10 20

5 Ethyl acetate fractionof Ocimum gratissimum

10 20

6 Microcrystallinecellulose

325 275

7 Starch 50 508 Crospovidone 20 20

Granulation9 Water qs qs

Prelubrication10 Starch 25 2511 Aerosil 10 1012 Talc 15 15

Lubrication13 Magnesium stearate 5 5Tablet weight (mg) 500 500

A Gupta et al Journal of Young Pharmacists 5 (2013) 180e187182

2103 Assessment of biochemical parameters of liverThe enzymatic parameters of serums like serum glutamate py-

ruvate transaminase (SGPT) serum glutamate oxaloacetate trans-aminase (SGOT) and serum alkaline phosphatase (ALP) and non-enzymatic parameter like total bilirubin (TB) were assayed ac-cording to standard methods2425

2104 Statistical analysisThe results are expressed as mean SEM (N frac14 6) Statistical

significance was determined by one-way analysis of variance withP lt 001 and P lt 005 considered significant followed by DunnettMultiple Comparisons Test The analysis was performed by GraphPad InStat software

2105 Stability studyStability studies were carried out at 25 C 2 C60 RH 5

and 40 C 2 C75 RH 5 for a specific time period of 30 daysfor selected (PF2) formulation The selected samples were evalu-ated for physical appearance hardness and disintegration time inevery 10 days interval

3 Results and discussion

31 Phytochemical screening

Preliminary phytochemical screening of alcoholic extract and itsfractions showed the presence of flavonoids steroids terpenoidstannins and phenolic compounds The chemical tests and TLCanalysis demonstrated that AcO fraction of B monosperma EtOAcfraction and n-ButOH fraction of B variegata DCM and EtOAcfractions of O gratissimum were rich in phenolic compounds Thephenolic content in B monosperma (acetone fraction) B variegata(ethyl acetate and n-butanol fractions) and O gratissimum(dichloromethane and ethyl acetate fractions) were found to be452 16 7124 24 4425 11 735 21 and 1365 14 mggallic acid1 g fraction respectively The flavonoid content in Bmonosperma (acetone fraction) B variegata (ethyl acetate and n-butanol fractions) and O gratissimum (dichloromethane and ethylacetate fractions) were found to be 251 18 417 22 227 323945 24 and 717 52 mg quercetin1 g fraction respectivelyThe phenol and flavonoid contents are responsible for hep-atoprotective activity hence these solvent fractions were selectedfor further study

32 HPTLC analysis

In HPTLC chromatogram all tracks for standard quercetin atwavelength 380 nmwere shown in Fig 1A The Rf value of standard

quercetin was found to be 038 and peak area was 9726 (Fig 1B)Acetone fraction of B monosperma showed twelve peaks the sixthpeak Rf value (039) was coinciding with standard Rf value and itspeak area was 9909 (Fig 2A) ethyl acetate fraction of B variegatashowed eight peaks the fourth peak Rf value (038) was coincidingwith standard Rf value of quercetin and its peak area was 7898(Fig 2B) n-butanol fraction of B variegata showed six peaks thethird peak Rf value (038) was coinciding with standard Rf valueand its peak area was 7570 (Fig 2C) dichloromethane fraction of Ogratissimum showed nine peaks the third peak Rf value (038) wascoinciding with standard Rf value and its peak area was 9246(Fig 2D) ethyl acetate fraction of O gratissimum showed sevenpeaks the third peak Rf value (039) was coinciding with standardRf value and its peak areawas 12427 (Fig 2E) The concentrations ofquercetin in acetone fraction of B monosperma ethyl acetate and n-butanol fractions of B variegata dichloromethane and ethyl acetatefractions of O gratissimum were found to be 0395 01744 0138203229 06734 (mg10 mg) respectively

33 Pre- and post-compression evaluation parameters

Pre-compression parameters (angle of repose bulk densitytapped density and compressibility) of powder blends and tabletsshowed satisfactory micromeritics properties as angle of repose ofwas less than 30 and compressibility was in the range of 15e17The physical properties (weight variation thickness diametercrushing strength friability) of polyherbal tablets shown in Table 2Tablets are circular biconvex in shape light greenish with fewwhite spots and slightly dark greenish spots color for PTF-1 andlight greenish with slightly dark greenish spots for PTF-2 tabletsodor and taste are characteristics for the formulations They pro-duced average weight between (506 377 and 5006 002)uniform thickness crushing strength and acceptable friability (lessthan 1) Tablets were disintegrated between 2 and 45 min

34 Identification of quercetin by FTIR

The IR spectra of isolated compound (quercetin) from poly-herbal tablet were displayed in Fig 3 The spectrumwas typical of aflavonoid structure The spectra were recorded in the range from4000 to 600 cm1 and showed identical absorption bands withmaxima at the following frequencies (max) 339235 238076165894 144645 137865 cm1 The peaks at 3392 and 2919 cm1

are characteristic of the aromatic ring structure The absorptionmaxima at 1610 and 1557 cm1 indicate the presence of a quinoidstructure and eC]Ce bonds respectively The absorptionmaximum at 1375 cm1 is due to phenolic OH groups Meltingpoint of isolated compound was found to be 314 C

Fig 1 3D-chromatogram (A) and densitogram (B) of standard quercetin (05e25 mgspot)

A Gupta et al Journal of Young Pharmacists 5 (2013) 180e187 183

Fig 2 HPTLC densitogram of acetone fraction of Butea monosperma (A) ethyl acetate fraction of Bauhinia variegata (B) n-butanol fraction of Bauhinia variegata (C) dichloromethanefraction of Ocimum gratissimum (D) ethyl acetate fraction of Ocimum gratissimum (E) showing quercetin

Table 2Pre- and post-compression evaluation of polyherbal tablet formulations PF1 PF2

SN Quality control tests PF1 PF2

Pre-compression parameters of evaluation of polyherbal tablet formulations PF1 PF21 Angle of repose (mean SD) (n frac14 3) 293 153 287 0582 Bulk density (mean SD) (n frac14 3) (gcc) 0427 0003 0450 00033 Tapped density (mean SD) (n frac14 3) (gcc) 0515 0005 0523 00084 Compressibility index () (mean SD) (n frac14 3) 1715 02 1531 012Post-compression evaluation of tablets5 Thickness (mm) (mean SD) (n frac14 10) 55 002 56 0036 Diameter (mm) (mean SD) (n frac14 10) 11 004 11 0057 Hardness (kgcm2) (mean SD) (n frac14 10) 31 019 33 0128 Disintegration time (min) (mean SD) (n frac14 6) 210 042 425 1129 Average weight (mg) (mean SD) (n frac14 10) 506 377 5006 00210 Friability () (mean SD) (n frac14 3) 03 023 02 016Physical parameters (appearance) of tablet formulations11 Color Light greenish with few white spots

slightly dark greenish spotsLight greenish with slightlydark greenish spots

12 Odor Characteristic Characteristic13 Shape Circular biconvex tablet Circular biconvex tablet14 Taste Characteristic Characteristic

Fig 3 IR spectrum of isolated compound from the mixture of fractions of Butea monosperma Bauhinia variegata and Ocimum gratissimum

A Gupta et al Journal of Young Pharmacists 5 (2013) 180e187184

26 Quantification of quercetin by HPTLC

A Camag HPTLC system was used with beam size of04 04 mm and wavelength of 380 nm Silica gel 60 F 254(20 20 cm 025-mm thick Merck) plates were used for HPTLCSamples were applied (15mm from the bottom edge of the plate) inthe form of 6 mm bandwidth with Hamilton syringes using Lino-mat V applicator A constant application rate of 150 nls was sup-plied and a space of 10 mm was kept between two bands Themobile phase constituted of tolueneethyl acetateformic acid5401 (vvv) Linear ascending development was carried out in atwin trough glass chamber saturated with mobile phase Theoptimized saturation time for mobile phase was 30 min at roomtemperature (25 C 2 C) with relative humidity of 55 5 Afterdeveloping TLC plate a current of air was used to dry it Densito-metric scanning was performed on Camag TLC scanner III inabsorbance mode at 380 nm The source of light was deuteriumbeam The quantitative determination was performed by using theexternal calibration method A stock solution of quercetin (500 mgmL) was prepared in methanol Different volume of stock solutionie 05 10 15 20 25 in mgspot were spotted on TLC plate of 200400 800 1600 and 3200 ngspot of quercetin Calibration curverange (05e25 mgspot) for quercetin was found to be linear Thedata of peak areas plotted against the corresponding concentra-tions were collected To determine the content of quercetin inextract 10 mg of each fraction was transferred in 10 mL volumetricflask containing 8 mL methanol vortexed for 10 min and diluted to10 mL with methanol The resulting solution was filtered throughWhatman filter paper The 15 mL of the filtered solutionwas appliedon TLC plate subsequently developed and scanned as describedabove

27 Evaluation of power blends

The polyherbal tablets were prepared by wet granulationmethod hence micromeritic studies (angle of repose bulk densitytapped density compressibility) were evaluated prior to punch-ing of tablets

28 Physical characterization of polyherbal tablets

Polyherbal tablets systems were characterized for thicknessdiameter disintegration time weight variation and friabilityCrushing strength thickness and friability were determined byMonsanto hardness tester digital vernier calipers and friabilatorrespectively

29 Isolation and identification of quercetin from polyherbal tablet

Weight accurately 500 mg equivalent of polyherbal tablet wastransferred to the 10 mL volumetric flask and dissolved in 10 mLmethanol This solution was sonicated for 10 min and filteredthroughWhatman No1 paper to get solution containing 10mgmLConcentrated solution obtained from polyherbal tablet was sub-jected to preparative TLC Preparative TLC were performed on20 cm 10 cm TLC aluminum plate coated with 200 mm layerthickness of silica gel 60 F 254 using tolueneethyl acetateformicacid 5401 (vvv) as mobile phase The quantificationwas carriedout by densitometric scanning in absorbance mode at wavelength380 nm The silica in the respective marked area was scraped offand collected carefully in a test tube The scraped silica whichcontained the standard quercetin was extracted in methanol andfiltered individually The filtrates containing quercetin were evap-orated under reduced pressure to obtain quercetin

Melting point of quercetin was determined by open capillarymethod Structural confirmation of the isolated quercetinwas doneby IR Spectroscopy and compared with that of standard

210 In-vivo hepatoprotective activity of polyherbal tabletformulations against paracetamol induced toxicity

2101 Test animalsMale Wistar rats (125e175 g) were used for determination of

maximum tolerable dose (MTD) and evaluation of hepatoprotectiveactivity The animals were housed in polypropylene cages at25 C 1 C with the relative humidity of 55 5 under 12 h12 hlightdark cycle They were received a standard chow and water adlibitum during experimentation The food was with-drawn on theday before the experiment but free access of water was allowed Aminimum of six animals was used in each group Throughout theexperiments animals were processed according to the suggestedinternational ethical guidelines for the care of laboratory animalsThe study protocol was approved by the Institutional Animal EthicsCommittee according to the regulation of Committee for the Pur-pose of Control and Supervision of Experiments on Animals (MPC1007 dated 30012010)

2102 Preparation and administration of test samplesApproved IAEC method was adopted for screening of hep-

atoprotective activity Dried fractions were suspended in 1 CMCsolution before oral administration to animals Animals weredivided into five groups of six rats each were used for the studyGroups 1 and 2 received normal saline (1 mLkg orally) for 7 daysGroups 3 4 and 5 received 100 mgkg bw doses of silymarin PTF1(50 mgkg bw doses) and PTF2 (100 mgkg bw doses) orally once aday for 7 days On the fifth day after the administration of therespective treatments all the animals in groups II III IV and V wereadministered paracetamol (PCM) 2 gkg orally On the seventh daythe blood samples were collected via orbital sinus puncture for theestimation of biochemical marker enzymes Then the liver wascarefully isolated and cleaned off extraneous tissue and preservedin 10 neutral formalin and then subjected to histopathologicalstudies2223

Table 1Composition of polyherbal tablets formulations (PTF)

Sr no Ingredients PTF-1 PTF-2

Quantity(mgtablet)

Quantity(mgtablet)

Dry mix1 Acetone fraction of

Butea monosperma10 20

2 Ethyl acetate fractionof Bauhinia variegata

10 20

3 n-Butanol fraction ofBauhinia variegata

10 20

4 Dichloromethane ofOcimum gratissimum

10 20

5 Ethyl acetate fractionof Ocimum gratissimum

10 20

6 Microcrystallinecellulose

325 275

7 Starch 50 508 Crospovidone 20 20

Granulation9 Water qs qs

Prelubrication10 Starch 25 2511 Aerosil 10 1012 Talc 15 15

Lubrication13 Magnesium stearate 5 5Tablet weight (mg) 500 500

A Gupta et al Journal of Young Pharmacists 5 (2013) 180e187182

2103 Assessment of biochemical parameters of liverThe enzymatic parameters of serums like serum glutamate py-

ruvate transaminase (SGPT) serum glutamate oxaloacetate trans-aminase (SGOT) and serum alkaline phosphatase (ALP) and non-enzymatic parameter like total bilirubin (TB) were assayed ac-cording to standard methods2425

2104 Statistical analysisThe results are expressed as mean SEM (N frac14 6) Statistical

significance was determined by one-way analysis of variance withP lt 001 and P lt 005 considered significant followed by DunnettMultiple Comparisons Test The analysis was performed by GraphPad InStat software

2105 Stability studyStability studies were carried out at 25 C 2 C60 RH 5

and 40 C 2 C75 RH 5 for a specific time period of 30 daysfor selected (PF2) formulation The selected samples were evalu-ated for physical appearance hardness and disintegration time inevery 10 days interval

3 Results and discussion

31 Phytochemical screening

Preliminary phytochemical screening of alcoholic extract and itsfractions showed the presence of flavonoids steroids terpenoidstannins and phenolic compounds The chemical tests and TLCanalysis demonstrated that AcO fraction of B monosperma EtOAcfraction and n-ButOH fraction of B variegata DCM and EtOAcfractions of O gratissimum were rich in phenolic compounds Thephenolic content in B monosperma (acetone fraction) B variegata(ethyl acetate and n-butanol fractions) and O gratissimum(dichloromethane and ethyl acetate fractions) were found to be452 16 7124 24 4425 11 735 21 and 1365 14 mggallic acid1 g fraction respectively The flavonoid content in Bmonosperma (acetone fraction) B variegata (ethyl acetate and n-butanol fractions) and O gratissimum (dichloromethane and ethylacetate fractions) were found to be 251 18 417 22 227 323945 24 and 717 52 mg quercetin1 g fraction respectivelyThe phenol and flavonoid contents are responsible for hep-atoprotective activity hence these solvent fractions were selectedfor further study

32 HPTLC analysis

In HPTLC chromatogram all tracks for standard quercetin atwavelength 380 nmwere shown in Fig 1A The Rf value of standard

quercetin was found to be 038 and peak area was 9726 (Fig 1B)Acetone fraction of B monosperma showed twelve peaks the sixthpeak Rf value (039) was coinciding with standard Rf value and itspeak area was 9909 (Fig 2A) ethyl acetate fraction of B variegatashowed eight peaks the fourth peak Rf value (038) was coincidingwith standard Rf value of quercetin and its peak area was 7898(Fig 2B) n-butanol fraction of B variegata showed six peaks thethird peak Rf value (038) was coinciding with standard Rf valueand its peak area was 7570 (Fig 2C) dichloromethane fraction of Ogratissimum showed nine peaks the third peak Rf value (038) wascoinciding with standard Rf value and its peak area was 9246(Fig 2D) ethyl acetate fraction of O gratissimum showed sevenpeaks the third peak Rf value (039) was coinciding with standardRf value and its peak areawas 12427 (Fig 2E) The concentrations ofquercetin in acetone fraction of B monosperma ethyl acetate and n-butanol fractions of B variegata dichloromethane and ethyl acetatefractions of O gratissimum were found to be 0395 01744 0138203229 06734 (mg10 mg) respectively

33 Pre- and post-compression evaluation parameters

Pre-compression parameters (angle of repose bulk densitytapped density and compressibility) of powder blends and tabletsshowed satisfactory micromeritics properties as angle of repose ofwas less than 30 and compressibility was in the range of 15e17The physical properties (weight variation thickness diametercrushing strength friability) of polyherbal tablets shown in Table 2Tablets are circular biconvex in shape light greenish with fewwhite spots and slightly dark greenish spots color for PTF-1 andlight greenish with slightly dark greenish spots for PTF-2 tabletsodor and taste are characteristics for the formulations They pro-duced average weight between (506 377 and 5006 002)uniform thickness crushing strength and acceptable friability (lessthan 1) Tablets were disintegrated between 2 and 45 min

34 Identification of quercetin by FTIR

The IR spectra of isolated compound (quercetin) from poly-herbal tablet were displayed in Fig 3 The spectrumwas typical of aflavonoid structure The spectra were recorded in the range from4000 to 600 cm1 and showed identical absorption bands withmaxima at the following frequencies (max) 339235 238076165894 144645 137865 cm1 The peaks at 3392 and 2919 cm1

are characteristic of the aromatic ring structure The absorptionmaxima at 1610 and 1557 cm1 indicate the presence of a quinoidstructure and eC]Ce bonds respectively The absorptionmaximum at 1375 cm1 is due to phenolic OH groups Meltingpoint of isolated compound was found to be 314 C

Fig 1 3D-chromatogram (A) and densitogram (B) of standard quercetin (05e25 mgspot)

A Gupta et al Journal of Young Pharmacists 5 (2013) 180e187 183

Fig 2 HPTLC densitogram of acetone fraction of Butea monosperma (A) ethyl acetate fraction of Bauhinia variegata (B) n-butanol fraction of Bauhinia variegata (C) dichloromethanefraction of Ocimum gratissimum (D) ethyl acetate fraction of Ocimum gratissimum (E) showing quercetin

Table 2Pre- and post-compression evaluation of polyherbal tablet formulations PF1 PF2

SN Quality control tests PF1 PF2

Pre-compression parameters of evaluation of polyherbal tablet formulations PF1 PF21 Angle of repose (mean SD) (n frac14 3) 293 153 287 0582 Bulk density (mean SD) (n frac14 3) (gcc) 0427 0003 0450 00033 Tapped density (mean SD) (n frac14 3) (gcc) 0515 0005 0523 00084 Compressibility index () (mean SD) (n frac14 3) 1715 02 1531 012Post-compression evaluation of tablets5 Thickness (mm) (mean SD) (n frac14 10) 55 002 56 0036 Diameter (mm) (mean SD) (n frac14 10) 11 004 11 0057 Hardness (kgcm2) (mean SD) (n frac14 10) 31 019 33 0128 Disintegration time (min) (mean SD) (n frac14 6) 210 042 425 1129 Average weight (mg) (mean SD) (n frac14 10) 506 377 5006 00210 Friability () (mean SD) (n frac14 3) 03 023 02 016Physical parameters (appearance) of tablet formulations11 Color Light greenish with few white spots

slightly dark greenish spotsLight greenish with slightlydark greenish spots

12 Odor Characteristic Characteristic13 Shape Circular biconvex tablet Circular biconvex tablet14 Taste Characteristic Characteristic

Fig 3 IR spectrum of isolated compound from the mixture of fractions of Butea monosperma Bauhinia variegata and Ocimum gratissimum

A Gupta et al Journal of Young Pharmacists 5 (2013) 180e187184

2103 Assessment of biochemical parameters of liverThe enzymatic parameters of serums like serum glutamate py-

ruvate transaminase (SGPT) serum glutamate oxaloacetate trans-aminase (SGOT) and serum alkaline phosphatase (ALP) and non-enzymatic parameter like total bilirubin (TB) were assayed ac-cording to standard methods2425

2104 Statistical analysisThe results are expressed as mean SEM (N frac14 6) Statistical

significance was determined by one-way analysis of variance withP lt 001 and P lt 005 considered significant followed by DunnettMultiple Comparisons Test The analysis was performed by GraphPad InStat software

2105 Stability studyStability studies were carried out at 25 C 2 C60 RH 5

and 40 C 2 C75 RH 5 for a specific time period of 30 daysfor selected (PF2) formulation The selected samples were evalu-ated for physical appearance hardness and disintegration time inevery 10 days interval

3 Results and discussion

31 Phytochemical screening

Preliminary phytochemical screening of alcoholic extract and itsfractions showed the presence of flavonoids steroids terpenoidstannins and phenolic compounds The chemical tests and TLCanalysis demonstrated that AcO fraction of B monosperma EtOAcfraction and n-ButOH fraction of B variegata DCM and EtOAcfractions of O gratissimum were rich in phenolic compounds Thephenolic content in B monosperma (acetone fraction) B variegata(ethyl acetate and n-butanol fractions) and O gratissimum(dichloromethane and ethyl acetate fractions) were found to be452 16 7124 24 4425 11 735 21 and 1365 14 mggallic acid1 g fraction respectively The flavonoid content in Bmonosperma (acetone fraction) B variegata (ethyl acetate and n-butanol fractions) and O gratissimum (dichloromethane and ethylacetate fractions) were found to be 251 18 417 22 227 323945 24 and 717 52 mg quercetin1 g fraction respectivelyThe phenol and flavonoid contents are responsible for hep-atoprotective activity hence these solvent fractions were selectedfor further study

32 HPTLC analysis

In HPTLC chromatogram all tracks for standard quercetin atwavelength 380 nmwere shown in Fig 1A The Rf value of standard

quercetin was found to be 038 and peak area was 9726 (Fig 1B)Acetone fraction of B monosperma showed twelve peaks the sixthpeak Rf value (039) was coinciding with standard Rf value and itspeak area was 9909 (Fig 2A) ethyl acetate fraction of B variegatashowed eight peaks the fourth peak Rf value (038) was coincidingwith standard Rf value of quercetin and its peak area was 7898(Fig 2B) n-butanol fraction of B variegata showed six peaks thethird peak Rf value (038) was coinciding with standard Rf valueand its peak area was 7570 (Fig 2C) dichloromethane fraction of Ogratissimum showed nine peaks the third peak Rf value (038) wascoinciding with standard Rf value and its peak area was 9246(Fig 2D) ethyl acetate fraction of O gratissimum showed sevenpeaks the third peak Rf value (039) was coinciding with standardRf value and its peak areawas 12427 (Fig 2E) The concentrations ofquercetin in acetone fraction of B monosperma ethyl acetate and n-butanol fractions of B variegata dichloromethane and ethyl acetatefractions of O gratissimum were found to be 0395 01744 0138203229 06734 (mg10 mg) respectively

33 Pre- and post-compression evaluation parameters

Pre-compression parameters (angle of repose bulk densitytapped density and compressibility) of powder blends and tabletsshowed satisfactory micromeritics properties as angle of repose ofwas less than 30 and compressibility was in the range of 15e17The physical properties (weight variation thickness diametercrushing strength friability) of polyherbal tablets shown in Table 2Tablets are circular biconvex in shape light greenish with fewwhite spots and slightly dark greenish spots color for PTF-1 andlight greenish with slightly dark greenish spots for PTF-2 tabletsodor and taste are characteristics for the formulations They pro-duced average weight between (506 377 and 5006 002)uniform thickness crushing strength and acceptable friability (lessthan 1) Tablets were disintegrated between 2 and 45 min

34 Identification of quercetin by FTIR

The IR spectra of isolated compound (quercetin) from poly-herbal tablet were displayed in Fig 3 The spectrumwas typical of aflavonoid structure The spectra were recorded in the range from4000 to 600 cm1 and showed identical absorption bands withmaxima at the following frequencies (max) 339235 238076165894 144645 137865 cm1 The peaks at 3392 and 2919 cm1

are characteristic of the aromatic ring structure The absorptionmaxima at 1610 and 1557 cm1 indicate the presence of a quinoidstructure and eC]Ce bonds respectively The absorptionmaximum at 1375 cm1 is due to phenolic OH groups Meltingpoint of isolated compound was found to be 314 C

Fig 1 3D-chromatogram (A) and densitogram (B) of standard quercetin (05e25 mgspot)

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Fig 2 HPTLC densitogram of acetone fraction of Butea monosperma (A) ethyl acetate fraction of Bauhinia variegata (B) n-butanol fraction of Bauhinia variegata (C) dichloromethanefraction of Ocimum gratissimum (D) ethyl acetate fraction of Ocimum gratissimum (E) showing quercetin

Table 2Pre- and post-compression evaluation of polyherbal tablet formulations PF1 PF2

SN Quality control tests PF1 PF2

Pre-compression parameters of evaluation of polyherbal tablet formulations PF1 PF21 Angle of repose (mean SD) (n frac14 3) 293 153 287 0582 Bulk density (mean SD) (n frac14 3) (gcc) 0427 0003 0450 00033 Tapped density (mean SD) (n frac14 3) (gcc) 0515 0005 0523 00084 Compressibility index () (mean SD) (n frac14 3) 1715 02 1531 012Post-compression evaluation of tablets5 Thickness (mm) (mean SD) (n frac14 10) 55 002 56 0036 Diameter (mm) (mean SD) (n frac14 10) 11 004 11 0057 Hardness (kgcm2) (mean SD) (n frac14 10) 31 019 33 0128 Disintegration time (min) (mean SD) (n frac14 6) 210 042 425 1129 Average weight (mg) (mean SD) (n frac14 10) 506 377 5006 00210 Friability () (mean SD) (n frac14 3) 03 023 02 016Physical parameters (appearance) of tablet formulations11 Color Light greenish with few white spots

slightly dark greenish spotsLight greenish with slightlydark greenish spots

12 Odor Characteristic Characteristic13 Shape Circular biconvex tablet Circular biconvex tablet14 Taste Characteristic Characteristic

Fig 3 IR spectrum of isolated compound from the mixture of fractions of Butea monosperma Bauhinia variegata and Ocimum gratissimum

A Gupta et al Journal of Young Pharmacists 5 (2013) 180e187184

Fig 2 HPTLC densitogram of acetone fraction of Butea monosperma (A) ethyl acetate fraction of Bauhinia variegata (B) n-butanol fraction of Bauhinia variegata (C) dichloromethanefraction of Ocimum gratissimum (D) ethyl acetate fraction of Ocimum gratissimum (E) showing quercetin

Table 2Pre- and post-compression evaluation of polyherbal tablet formulations PF1 PF2

SN Quality control tests PF1 PF2

Pre-compression parameters of evaluation of polyherbal tablet formulations PF1 PF21 Angle of repose (mean SD) (n frac14 3) 293 153 287 0582 Bulk density (mean SD) (n frac14 3) (gcc) 0427 0003 0450 00033 Tapped density (mean SD) (n frac14 3) (gcc) 0515 0005 0523 00084 Compressibility index () (mean SD) (n frac14 3) 1715 02 1531 012Post-compression evaluation of tablets5 Thickness (mm) (mean SD) (n frac14 10) 55 002 56 0036 Diameter (mm) (mean SD) (n frac14 10) 11 004 11 0057 Hardness (kgcm2) (mean SD) (n frac14 10) 31 019 33 0128 Disintegration time (min) (mean SD) (n frac14 6) 210 042 425 1129 Average weight (mg) (mean SD) (n frac14 10) 506 377 5006 00210 Friability () (mean SD) (n frac14 3) 03 023 02 016Physical parameters (appearance) of tablet formulations11 Color Light greenish with few white spots

slightly dark greenish spotsLight greenish with slightlydark greenish spots

12 Odor Characteristic Characteristic13 Shape Circular biconvex tablet Circular biconvex tablet14 Taste Characteristic Characteristic

Fig 3 IR spectrum of isolated compound from the mixture of fractions of Butea monosperma Bauhinia variegata and Ocimum gratissimum

A Gupta et al Journal of Young Pharmacists 5 (2013) 180e187184

35 Evaluation of hepatoprotective activity

From the results of acute toxicity study by previously publishedliterature 2000 mgkg was considered as maximum tolerable dose(MTD) of alcoholic extract of B monosperma14 B variegata26 and Ogratissimum27 120th of this dose was taken as experimental dose

for subsequent hepatoprotective studies The effective dose of eachfractionwas decided on the basis of previously published report ie100 mgkg of body weight

Paracetamol has enhanced the levels of SGPT SGOT bilirubin(both total and direct bilirubin levels) alkaline phosphatase level(ALP) The results indicated that the polyherbal tablet formulations

Table 3Effect of polyherbal formulation PTF-1 and PTF-2 on biochemical parameters in paracetamol induced hepatic injury in rats

Treatment SGPT UL SGOT UL ALP UL Total bilirubin (mgdl) Direct bilirubin (mgdl)

Control 6975 196 13567 163 14547 373 089 007 023 001Paracetamol treated 28425 138 42118 112 46443 136 374 092 065 003Silymarin treated 7146 081 14263 082 17473 124 125 083 029 005

PTF-1 treated 7585 135 14963 173 18064 125NS 148 042NS 032 003NS

PTF-2 treated 6556 168 15298 148 16787 178 132 078 033 010

P lt 0001 vs control P lt 005 P lt 001 vs PCM treated NS non significant

Fig 4 Graphs showing effect of polyherbal formulation PTF-1 and PTF-2 on biochemical parameters in paracetamol induced hepatic injury in rats

Fig 5 The photomicrographs of liver section from rats (A) received saline 05 mL as normal control group (10 10) (B) received paracetamol (2 gkg) (10 10) (C) receivedsilymarin (100 mgkg bw) thorn PCM (10 10) (D) received PF1 (50 mgkg bw) thorn PCM and (E) received PF2 (100 mgkg bw)

A Gupta et al Journal of Young Pharmacists 5 (2013) 180e187 185

(PTF-1 and PTF-2) significantly reduced the elevated levels of SGPTSGOT ALP and bilirubin when compared to paracetamol treatedgroup The formulation PTF-1 has reduced the increased SGPT levelfrom 28425 UL to 7585 UL SGOT level from 42118 UL to14963 UL ALP level from 46443 UL to 18064 UL and bilirubinlevel from 374 mgdl to 148 mgdl The formulation PTF-2 hasreduced the increased SGPT level from 28425 UL to 6556 ULSGOT level from 42118 UL to 15298 UL ALP level from 46443 ULto 16787 UL and bilirubin level from 374 mgdl to 132 mgdl

Hepatic cells participate in metabolic activities and contain hostof enzymes In tissue SGOT and SGPT exist in mitochondria In liverinjury transport function of the hepatocytes gets disturbedresulting in the leakage of plasma membrane and thereby causingan increased enzyme level in serum The elevated activities of theseenzymes are indicative of cellular leakage and the functionalintegrity of the cell membranes in liver ALP is excreted by liver viabile in the liver injury due to hepatotoxins which results in adefective excretion of bile by the liver and is reflected in theirincreased levels in serum In drug-induced liver toxicity the level oftotal and direct bilirubin gets elevated Both formulations showedmarked decrease in the elevated levels of SGOT SGPT ALP andbilirubin (total and direct) which is nearer to the levels of controlgroup28 The results are shown in Table 3 Fig 4

Histopathological examination of the liver sections confirmedthat the normal liver (Fig 5A) architecture was damaged withparacetamol administration (Table 4 Fig 5B) However pretreat-ment of the PTF1 (50 mgkg) and PTF2 (100 mgkg) dose signifi-cantly reduced the severity of histopathological injury (comparedwith the paracetamol group) The results of the biochemical testsand histopathological observations suggest that PTF2 formulationis better than PTF1

Rats treated with silymarin PTF-1 and PTF-2 formulationsshowed noticeable improvement in histopathological parameters(Table 4 and Fig 5CeE) Therefore both the formulations have

prominent action on paracetamol-induced liver damage Moreoverat necropsy livers of rats treated with paracetamol appeareddegeneration in hepatocytes hepatic cell injury focal necrosiscongestion in central vein vascular swelling and Kupffer cell pro-liferation Furthermore no gross pathological findings were notedin the livers of the other groups of rats

36 Stability studies

Selected polyherbal tablet formulation PTF-2 was found to bestable when subjected to stability studies at 25 C60 RH and40 C75 RH for a time period of 30 days There was no significantchange in the physicochemical properties of tablets ie appearancedisintegration time and hardness of the tablets The results areshown in Table 5

4 Conclusion

Biochemical marker showed improved results for polyherbaltablet formulation at 100 mgkg Polyherbal tablet formulationcontains a potent hepatoprotective agent suggested to be a flavoneconcentrated in polyherbal formulation which may find clinicalapplication in amelioration of paracetamol induced liver damage

Authors contribution

Principle investigator and project leader Mrs Arti GuptaConcept development Dr Navin R ShethAnalysis and written the manuscript Mrs Sonia PandeyStatistical analysis Mr Jitendra S Yadav and Dr Dinesh Shah

Conflicts of interest

All authors have none to declare

Acknowledgment

Dr Minoo H Parabia Professor Department of Bioscience VeerNarmad South Gujarat University Surat and Dr Renu ChauhanQuality Assurance Department are acknowledged for their help

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Table 4Histopathological changes in the liver of rats treated with polyherbal formulationPTF-1 and PTF-2

Microscopic observation Control PCM Silymarin PTF-1 PTF-2

Degeneration in hepatocytes thorn thornthornthorn thornthorn thornthorn thornthornHepatic cell injury Oslash thornthornthorn thornthorn thorn thornthornFocal necrosis Oslash thornthornthorn Oslash thornt thornCongestion in central vein thorn thornthornthorn thorn thornt thornt

Vascular swelling thorn thornthornthorn thorn thorn thornt

Kupffer cell proliferation Oslash thornthorn thorn thornthorn thornOslash absent t few thorn mild thornthorn moderate thornthornthorn severe thornthornthornthorn extremely severePCM e paracetamol

Table 5Stability studies data for PTF-2 formulation at 25 C60 RH

Parameter PTF-2

0 day 10 days 20 days 30 days

Stability studies data for PTF-2 formulation at 25 C60 RHAppearance Hardness (kgcm2)

(n frac14 3) (mean SD)33 012 33 015 33 012 36 006

Disintegration time(min) (n frac14 3)(mean SD)

212 012 332 015 233 012 220 006

Stability studies data for PTF-2 formulation 40 C75 RHAppearance Hardness (kgcm2)

(n frac14 3)34 012 33 015 35 023 38 015

Disintegration time(min) (n frac14 3)

400 058 430 083 436 086 530 169

Light greenish color with slightly dark greenish spots No change Lightgreenish color with dark greenish spots

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