Rev. bras. farmacogn. vol.27 número1

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w ww . e l s e v i e r . c o m / l o c a t e / b j p

Original

Article

Metabolic

profile

and

␤

-glucuronidase

inhibitory

property

of

three

species

of

Swertia

Swagata

Karak,

Gargi

Nag,

Bratati

De

∗

PhytochemistryandPharmacognosyResearchLaboratory,DepartmentofBotany,UniversityofCalcutta,Kolkata,India

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received29February2016 Accepted18July2016 Availableonline3October2016

Keywords: Swertia

␤-Glucuronidase Xanthones Hepatoprotective

a

b

s

t

r

a

c

t

␤-Glucuronidaseinhibitorsaresuggestedaspotentialhepatoprotectiveagents.Swertiachirayita(Roxb.)

Buch.-Ham.exC.B.Clarke,Gentianaceae,isknownforitshepatoprotectiveandanti-hepatotoxic

activ-ityinAyurvedicsystemofmedicineforages.ThisplantissubstitutedbyotherspecieslikeS.decussata

NimmoexC.B.ClarkeandS.bimaculata(Siebold&Zucc.)Hook.f.&ThomsonexC.B.Clarke.Theaimofthe

studywastocomparemetaboliteprofileand␤-glucuronidaseinhibitoryactivityofthesethreeimportant

speciesofSwertiaandtoidentifytheactiveconstituents.S.chirayita(IC50210.97␮g/ml)andS.decussata

(IC50269.7␮g/ml)showed␤-glucuronidaseinhibitoryactivitysignificantlyhigherthanthatofsilymarin,

theknowninhibitoroftheenzyme.TheactivityofS.bimaculatawaslow.Themetabolitespresentinthe

threespecieswereanalyzedbyHPLCandGC-MSbasedmetabolomicsapproach.Fiveaminoacids,twenty

oneorganicacids,oneinorganicacid,eightfattyacids,twentyonephenolsincludingxanthones,eight

sugars,sevensugaralcohols,fiveterpenoidsandamarogentinwereidentified.Activitiesofthe

xan-thonesmangiferin(IC5016.06␮g/ml),swerchirin(IC50162.84␮g/ml),decussatin(IC50195.11␮g/ml),

1-hydroxy-3,5,8-trimethoxyxanthone(IC50245.97␮g/ml),bellidifolin(IC50390.26␮g/ml)were

signifi-cantlyhigherthanthatofsilymarin(IC50794.62␮g/ml).Quinicacid(IC502.91mg/ml),O-acetylsalicylic

acid(IC5048.4mg/ml),citricacid(IC501.77mg/ml),d_-malic_acid_(IC₅₀_14.82_mg/ml)_and_succinic_acid

(IC5038.86mg/ml)alsoinhibitedtheenzyme␤-glucuronidase.Thefindingssuggestthatconstituents,

inadditiontothexanthones,probablyalsocontributetothebioactivityofdifferentSwertiaspeciesby

synergisticeffect.Furtherinvivostudyisrequiredtosupporttheclaim.

accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Liverdiseasehasbecomeamajorhealthissueglobally(Byass,

2014). The liver is a vital organ that is involved in

mainte-nance of metabolic functions and helps in the detoxification

processbycountering severalexogenousand endogenous

chal-lenges(Kshirsagaretal.,2011).Glucuronidationisamajorpathway

ofphase IIxenobiotic biotransformation(deGraafet al.,2002).

Conjugationoftoxinswithglucuronicaciddeactivatespotentially

damagingcompoundsandsubsequentlyeliminatesthemfromthe

body.However,thisprocessbecomeslimitedbytherateof

deglu-curonidationby␤-glucuronidase.Hydrolysis oftheglucuronide

moietycanbecarriedoutby␤-glucuronidasepresentinmostof

thetissues,inendocrineandreproductiveorgans(Dutton,1980).

∗ Correspondingauthor.

E-mail:bdbot@caluniv.ac.in(B.De).

Liverdamagecausesanincreaseinthelevelof␤-glucuronidasein

blood(Pinedaetal.,1959),andlivercancercouldberelatedtothis

enzyme(MillsandSmith,1951).

␤-Glucuronidaseinhibitorsreducethecarcinogenicpotentialof

toxiccompoundsnormallyexcretedinbileafterglucuronidation

(Walaszeketal.,1984).Duetothiscorrelation,␤-glucuronidase

inhibitorsaresuggestedaspotentialhepatoprotectiveagents(Shim

et al., 2000). Certain hepatoprotective plant extracts and their

constituentsare knowntoinhibittheenzyme, ␤-glucuronidase

(JoshiandSanmugapriya,2007).Silymarin(amixtureof

flavono-lignans),thecommercialplantderived␤-glucuronidaseinhibitor

(Kimetal.,1994),isusedtotreatliverdisordersandalsocertain

cancers(Dixitetal.,2007).Butithaspoorbioavailability(Dixitetal.,

2007).Silymarinhascertainotherlimitationsrelatedto

gastroin-testinaltractlikebloating,dyspepsia,nausea,irregularstooland

diarrhoea.Italsoproducedpruritus,headache,exanthema,malaise,

asthenia,andvertigo(PradhanandGirish,2006).Hence,searchfor

glucuronidaseinhibitorycompoundsfrommedicinallyimportant

traditionalplantsthatareearlierreportedtobehepatoprotective

isnecessary.

http://dx.doi.org/10.1016/j.bjp.2016.07.007

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PlantsofthegenusSwertia,Gentianaceae,arewellrecognizedin

literatureasimportantmedicinalherbhavinganarrayof

biologi-calandtherapeuticproperties(Negietal.,2011).Hepatoprotective

andanti-hepatotoxicactivityofSwertiasp.havealreadybeen

estab-lishedinAyurvedicmedicalsystemandvalidatedscientificallyin

animalsystem(Mukherjeeetal.,1997;Karanetal.,1999;Reen

etal.,2001).Swertiachirayita(Roxb.)Buch.-Hamex C.B.Clarke,

consideredtobethemostimportantspeciesofSwertiareported

fromIndia,foritsmedicinalproperties,hasbeenconsideredas

crit-icallyendangeredplant(Pantetal.,2000;JoshiandDhawan,2005;

Bhargavaetal.,2009).Thisplantissubstitutedbyotherspecies likeS.decussataNimmoexC.B.ClarkeandS.bimaculata(Siebold&

Zucc.)Hook.f.&ThomsonexC.B.Clarke(Chopraetal.,1956;Phoboo

etal., 2010).Metabolitessuchas terpenoids,flavonoids, iridoid

glycosidesandxanthonesareconsideredasactiveconstituentsof

Swertiasp.,xanthonesbeingthemainactivesecondarymetabolite

(Brahmacharietal.,2004;Nagetal.,2015).

In a previous study antioxidant, anti-glycosidase and

anti-acetylcholinesterase properties of S. chirayita and the two

substituteswerereportedfromthelaboratory(Nagetal.,2015).

Although the hepatoprotective property of S. chirayita is well

known,themodeofactionforhepatoprotectionhasnotyetbeen

studied.Theactive principlesfor hepatoprotection arealso not

known.␤-Glucuronidaseinhibitorypropertiesoftheextractsof

theseplantswouldfurthervalidatetheirhepatoprotective

prop-erty.So,theaimofthestudywastocomparemetaboliteprofile

and␤-glucuronidaseinhibitoryactivityofthreeimportantspecies

ofSwertiai.e.S.chirayita,S.decussataandS.bimaculatainorderto

identifytheactiveconstituents.

Materialsandmethods

Plantmaterial

LeafyshootsofthreespeciesofSwertia,Gentianaceae,namely

Swertiachirayita(Roxb.) Buch.-HamexC.B.Clarke (Voucherno.

Bot332S-1),S.bimaculata(Siebold&Zucc.)Hook.f.&Thomson

exC.B.Clarke(VoucherNo.Bot332S-2)werecollectedfrom

Dar-jeelingHimalayas.Thethird speciesS.decussata NimmoexC.B.

Clarke(VoucherNo.Bot332S-3)wascollectedfromtheWestern

Ghats,India.VoucherspecimensareavailableintheDepartment

ofBotany,UniversityofCalcutta.ThetwonamesS.chirayitaand

S.decussataareunresolvedasperIPNI(InternationalPlantNames Index).

Chemicalsandreagents

␤-Glucuronidase (ex. bovine liver), 4-nitrophenyl-␤-d

-glucuronide; methoxyamine hydrochloride, N-methyl-N

-(trimethylsilyl)trifluoroacetamidewith1%trimethylchlorosilane

(MSTFA),adonitoland FAME(Fatty Acid MethylEster) markers

were obtainedfrom Sigma–Aldrich (St. Louis, MO, USA); HPLC

gradeacetonitrile,formicacid,water,methanol,chloroformand

pyridinefromMerckSpecialitiesPrivateLimited(Mumbai,India).

Six standard compounds: mangiferin, amarogentin, bellidifolin,

swerchirin/methylbellidifolin, decussatin and 1-hydroxy-3, 5,

8-trimethoxyxanthonewereavailableinthelaboratory.

Samplepreparation

MethanolicextractsoftheleafyshootsofSwertiasp.were

pre-paredbyrefluxingdried,groundmaterialswithmethanolfor5h.

Foreachplantmaterial,thefiltrate,afterextraction,was

evapo-ratedtodrynessunderreducedpressure.Differentconcentrations

ofthemethanolicextractandthatofreferencecompoundswere

usedforstudyingtheenzymeinhibitionactivityinvitroaswellas

forHPLCandGC/MSanalysis.

Assayforˇ-glucuronidaseinhibition

␤-Glucuronidaseinhibition assaywascarried outas perthe

methodofKimetal.(1999)withmodification.Inbrief,100␮lof

␤-glucuronidase(986.4units/mlin0.1Mphosphatebuffer,pH7.0)

and340␮loftestsolution/referencestandardofvarious

concentra-tionsin0.1Mphosphatebuffer(pH7.0)werepre-incubatedat37◦_C

for15min.Followingthepre-incubation,60␮lofp-nitrophenyl-␤

-d-glucuronide(3.15mg/mlin0.1Mphosphatebuffer,pH7.0)was

addedandincubatedat37◦_C_for₅₀_min._The_colour_developed_was

readat405nminspectrophotometer.Controlsweredevoidoftest

samples.Thepercentinhibitionwascalculatedasfollows:

Inhibition (%)=

_Control_OD₋_Sample_OD

ControlOD

×100.

HPLCanalysis

TheHPLCanalysiswasperformedonanAgilent1260(Agilent

Technologies,USA)HPLCsystemconsistingofaquaternarypump,

acolumntemperaturecontrollerandadiode-arraydetector(DAD).

The analytical column (Agilent Eclipse plus C18, 100×4.6mm,

3.5␮m)wasusedfortheanalysis.Themobilephasewascomposed

ofsolventA(acetonitrile)andsolventB(0.1%formicacidaqueous,

v/v).Thelineargradientprogrammefollowedwas:10%Aat0min,

30%Aat20min,60%Aat35minand80%Aat45min(Duetal.,

2012).Theflowratewas0.7ml/min.20␮laliquotswereinjected.

UVspectraofthepeakswererecordedfrom190–400nmovera

rangeof8differentUVwavelengths(210,214,230,250,254,260,

273,and280nmrespectively).

GC/MSanalysis

GC–MSanalysiswasperformedusingAgilent7890AGC

[soft-waredriverversion4.01(054)]equippedwith5795CinertMSD

withTripleAxisDetector.Thecolumnusedforquantification

anal-ysiswasHP-5MScapillary column[AgilentJ&W;GC Columns

(USA)]of dimensions30m×0.25mm×0.25␮m. Themethodof

Kind et al. (2009) was followed after modification (Das et al., 2016).Theanalysiswasperformedunderthefollowingoven

tem-peratureprogramme:ovenramp60◦_C₍₁_min_hold),_to₃₂₅◦_C_at

10◦_C/min,_held_for₁₀_min_before_cool-down_producing_a_run_time

of37.5min.Theinjectiontemperaturewassetat250◦_C,_the_MSD

transferlineat290◦_C_and_the_ion_source_at₂₃₀◦_C. _Helium_was

usedasthecarriergas(flowrate0.723ml/min;carrierlinear

veloc-ity 31.141cm/s). Thedried crude extract wasderivatized using

methoxyaminehydrochlorideandMSTFAtoincreasethe

volatil-ityofthemetabolites.AmixtureofinternalRetentionIndex(RI)

markers(methylestersofC8,C10,C12,C14,C16,C18,C20,C22,

C24andC26linearchainlengthfattyacids)(2␮l)wasaddedto

each sample. Derivatizedsamples wereinjected viasplit mode

(splitratio10:1)ontothecolumn.Massspectraranging30–500m/z

wererecorded.Automatedmassspectraldeconvolutionand

iden-tificationsystem(AMDIS)wasusedtodeconvoluteand identify

chromatographicpeaks.Themetaboliteswereidentifiedby

com-paringthefragmentationpatternsofthemassspectra,retention

times(RT)and retentionindices(RI)withentriesofmass

spec-tra,RTandRIinAgilentGC-MSMetabolomicsRTLLibrary(2008)

(AgilentTechnologies,USA).Therelativeresponseratiosofallthe

metaboliteswerecalculatedafternormalizingthepeakareasofthe

metabolitesbyextractdryweightandthepeakareaoftheinternal

(3)

Statisticalanalysis

Each experiment was repeated four–five times. Percentage

inhibitionin activityispresentedasmean±standarddeviation.

Regressionequationswerepreparedfromtheconcentrationsofthe

extractsandpercentageinhibitionofenzymeactivity.IC50

(concen-trationofsamplerequiredtoinhibit50%enzymeactivity)values

werecalculatedfromtheseregressionequations.Thedifferences

inactivitywerecalculatedbyTukey’sandBonferroni’stests.

Resultsanddiscussions

The entire plant of S. chirayita is used in traditional

sys-temof medicine(Joshi andDhawan, 2005).During thepresent

studyleafyshoots couldbecollected. Somethanolicextractsof

theleafy shoots of three species of Swertiawere tested for ␤

-glucuronidaseinhibitoryactivity.Concentrationrequiredfor50%

inhibitionofenzymeactivity(IC50value)forsilymarinwasdetected

tobe794.62±10.01␮g/ml.So, initially,␤-glucuronidase

inhibi-tionactivitiesofallthreespeciesweremeasuredat500␮g/ml.It

wasobservedthatS.bimaculatahadlowestactivityatthetested

concentration(Fig.1).TheactivitiesofS.chirayitaandS.

decus-satawerehigherwithnosignificantdifferencesinactivitybetween

them. Sothe IC50 values of the two species S. chirayita and S.

decussataweredetermined.Theextractsinhibitedtheenzymein

adosedependentmanner.ItwasobservedthatS.chirayitahad

lowerIC50valuebetweenthetwospeciesindicatingstronger

activ-ity (Fig. 2). IC50 values of both the extracts were significantly

0

S. chirayita S. decussata S. bimaculata

Concentration (500 µg/ml)

% inhibition

98.19 ± 0.77 _{95.95 ± 1.36}

30.89 ± 4.02

20 40 60 80 100 120

Fig.1.Comparisonof␤-glucuronidaseinhibitoryactivityofSwertiasp.

0.00

S. chirayita

210.97 ± 0.04

IC

50

v

alue (

µ

g/ml

±

sd)

269.70 ± 2.11

794.62 ± 10.01

S. decussata Silymarin

100.00 200.00 300.00 400.00 500.00 600.00 700.00 800.00 900.00

Fig.2.Comparisonofenzymeactivity.

mAU 50 0 –50 –100 –150 (A)

(B)

(C) –200 –250 –300 –350

10

1 2

3,4 5

6

5 3,4 2

1

1 2

3,4

5 6

20 30 40 50 min

10 20 30 40 50 min

10 –600

–600 –400 –200 0 200

–500 –400 –300 –200 –100 0 mAU mAU

*DAD1 A, sig=254.4 ref=360 100 (21-10-2014\SB000003.D) *DAD1 A, sig=254.4 ref=360 100 (21-10-2014\Authentics00006.D)

*DAD1 A, sig=254.4 ref=360 100 (20-10-2014\SDNEW000004.D) *DAD1 A, sig=254.4 ref=360 100 (20-10-2014\Authentics00007.D) *DAD1 A, sig=254.4 ref=360 100 (17-10-2014\SC000002.D) *DAD1 A, sig=254.4 ref=360 100 (17-10-2014\Authentics00006.D)

20 30 40 50 min

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Table1

ComparisonofHPLCidentifiedmetabolitesamongthreespeciesofSwertiainelutionorder.

Standard/referencecompounds S.chirayita S.decussata S.bimaculata

Relativeresponseratiopergextract

Mangiferin 1.084±0.39 0.758±0.17 0.209±0.02

Amarogentin 0.376±0.07 – 0.040±0.01

Bellidifolin+1-Hydroxy-3,5,8-trimethoxyxanthone 0.661±0.22 1.463±0.31 0.043±0.01

Decussatin 0.159±0.02 1.130±0.21 0.066±0.01

Swerchirin 0.233±0.06 0.452±0.17 –

100.00 80.00 60.00 40.00 20.00 0.00

0.00

0 100 200 300 400 100.00 200.00 300.00

Concentration (µg/ml)

Bellidifolin

Decussatin Mangiferin

1-hydroxy-3.5.8-trimethoxy xanthone

y=0.2004x+0.8437

R2_=0.9448

y=0.499x−31 174

R2_=0.9879

y=0.3225x−75 669

R2_=0.9597

y=0.2868x−5.9433

R2_=0.9876 y=–0.1502x2+9.2781x– 60 279

R2_=0.9827

Swerchirin

Concentration (µg/ml)

% inhibition

100.00 80.00 60.00 40.00 20.00 0.00

% inhibition

120.00

80.00 100.00

60.00 40.00 20.00 0.00 0

0 10 20 30 40

100 200 300

90 80 70 60 50 40 30 20 10

80.00 70.00 60.00 50.00 40.00 30.00 20.00 10.00 0.00

0 100 200 300 400 0

% inhibition

400.00 500.00 600.00

Fig.4. ␤-Glucuronidaseinhibitionbythexanthones.

lower than that of silymarin. The bioactivity of a plant is due tothephytoconstituentspresent in it.For a comparativestudy ofthemetaboliteprofilein thethree species,identificationand semiquantitativeanalysesofthemetaboliteswereperformedby HPLC withphotodiode array detectionand GC–MSfollowing a metabolomicsapproach.HPLCprofileofthethreespeciesof

Swer-tia(Fig.3) showedthe confirmedpresence of three xanthones

(swerchirin,decussatin,mangiferin)andtheiridoidamarogentin.

Bellidifolinand1-hydroxy-3,5,8-trimethoxyxanthonecouldnotbe

separatedfromeach otherbyHPLCastheirretentiontime(RT)

andabsorbanceweresame.Semiquantitativecomparisonofthe

normalizedpeakarearevealedthatmangiferinwaspresentin

max-imumconcentrationinS.chirayita,followedbyS.decussataandS.

bimaculata.AmarogentinandswerchirinwerenotdetectedinS. decussataandS.bimaculatarespectively.Decussatinwasfoundto

beinmaximumamountinS.decussata.Acomparativeaccountof

theHPLCidentifiedmetaboliteshasbeenrepresentedinTable1.

GC–MSbasedmetabolomicsapproachhelpedinidentificationof

72compoundsfromthemethanolextractofthreespeciesof

Swer-tia.Fiveaminoacids,twentyoneorganicacids,oneinorganicacid,

eightfattyacids,sixteenphenols,eightsugars,sevensugaralcohols,

fiveterpenoidsandoneotherorganiccompound(porphine)were

identified.Asemiquantitativecomparison,basedontherelative

responseratiopergextract,oftheidentifiedmetaboliteshasbeen

representedinTable2.S.chirayitapresentedmaximumnumberof

metabolitesfollowedbyS.bimaculataandS.decussatarespectively.

Five xanthones and the iridoid amarogentin, isolated from

S. chirayita previously (Nag et al., 2015), were tested for their

␤-glucuronidaseinhibitory property. The activitiesof the

com-pounds were compared with that of silymarin. The activities

of the xanthones tested were proportional to their

concentra-tions (Fig. 4). All the xanthones showed inhibitory activities

significantly higher than that of the commercial drug (Fig. 5).

0.00

3 6

IC

50

v

alue (

µ

g/ml

±

sd)

5 1 4 Silymarin

390.26

±

2.92

162.84

±

3.72

195.11

±

5.03

16.06

±

0.05 245.97

±

4.19

794.62

±

10.01

100.00 200.00 300.00 400.00 500.00 600.00 700.00 800.00 900.00

(5)

Table2

ComparativemetabolicprofileofthreespeciesofSwertiausingGC/MS.

Metabolites Relativeresponseratiopergextract

Swertiachirayita Swertiadecussata Swertiabimaculata

Aminoacids

N-Ethylglycine 1.13±1.09 – –

N-Acetyl-l-glutamicacid 15.73±2.85 – 21.72±9.41

l-Glutamicacid(dehydrated) 14.43±1.50 1.57±0.71b _–

l-Pyroglutamicacid – – 17.12±3.46

Allantoin 37.14±8.73 – –

Organicacids

l-(+)Lacticacid 28.97±2.06 25.03±7.83 137.05±14.21a

Glycolicacid 6.83±0.31 2.74±3.95 12.21±0.83a

Oxalicacid 0.98±0.31 – 4.00±2.27a

Malonicacid 3.82±0.91 – 2.88±1.66

Maleicacid 2.59±0.29 2.21±0.45 1.75±1.00

Succinicacid 86.36±2.13 1.62±0.08b _98.39_±_7.51a

Glycericacid 90.34±2.72 4.77±0.82b _67.42_±_3.79b

Fumaricacid 103.78±7.32 7.43±1.57b _18.97_±_3.81b

Citraconicacid 1.03±0.22 – 0.34±0.25b

Citramalicacid 3.79±0.92 – –

Mandelicacid 1.05±0.38 – 0.84±0.19

d-Malicacid 705.76±25.78 6.08±0.58b _296.55

±44.42b

Adipicacid – – 0.76±0.66

Citricacid 19.33±4.44 – 17.68±11.19

Gluconicacidlactone 144.13±14.48 41.30±5.64b _76.96_±_8.32b

Gluconicacid 15.29±7.38 – –

2-Isopropylmalicacid 1.51±0.19 – –

3-Hydroxy-3-methylglutaricacid(dicrotalicacid) 3.29±0.13 – –

Ribonicacid-gamma-lactone 22.24±35.46 – –

4-Guanidinobutyricacid 0.93±0.30 – –

Nicotinicacid 2.04±0.87 – 2.74±0.81

Inorganicacid

Phosphoricacid 14.97±2.36 3.99±0.45b _5.66_±_1.15b

Fattyacids

Lauricacid 1.26±0.13 1.44±0.73 6.16±2.69a

Behenicacid 8.71±1.06 – 14.63±3.04a

Myristicacid – – 6.79±1.82

Palmiticacid 109.52±11.08 94.42±15.02 247.32±10.48a

Linoleicacid 26.08±5.82 3.69±2.31b _27.39_±_7.70c

Oleicacid 32.02±9.41 4.93±3.89b _20.48

±16.95

Stearicacid 64.63±7.34 64.87±10.13 116.75±13.21a

Arachidicacid 4.13±0.99 – 20.27±4.24a

Phenols

Benzoicacid – – 1.97±0.22

O-Acetylsalicylicacid – – 3.60±0.35

4-Hydroxybenzoicacid 9.44±0.72 – 4.75±0.12b

2,3-Dihydroxybenzoicacid 41.22±0.14 22.78±1.75b _112.23_±_2.24a

4-Hydroxy-3-methoxybenzoicacid 56.08±1.65 1.48±0.40b _47.02_±_0.72b

Gentisicacid – 3.58±1.76 33.83±6.61c

Shikimicacid 1.47±0.21 – –

3,4-Dihydroxybenzoicacid 43.59±16.66 – 8.83±0.67b

Quinicacid 2.06±0.33 5.29±2.96 26.20±0.77a

Coniferylalcohol 6.90±1.95 – 22.27±9.60a

4-Hydroxycinnamicacid – 11.30±5.60 –

Ferulicacid – 7.76±6.08 –

Sinapylalcohol – – 3.46±1.96

Caffeicacid 0.86±0.09 1.44±0.72 –

Neohesperidin 0.95±0.34 –

Isoquercitrin 59.38±8.10 – –

Sugars

Methyl-␤-d-galactopyranoside 105.99±1.29 26.71±14.48b _45.95

±40.50b

Sucrose 1482.49±114.56 27.04±4.29b _1682.28

±1055.76c

Lactose 46.34±43.84 – –

d-(+)-Trehalose 248.92±66.71 22.69±7.72b _86.93_±_15.65b

Raffinose 2.46±1.91 – 6.39±5.06

Melezitose 3.70±1.99 3.19±0.28 4.55±3.22

d-(+)-Melezitose 1.98±0.83 86.62±100.91 –

Adenosine 9.66±7.90 – 6.95±1.24

Sugaralcohols

Glycerol 108.61±1.07 58.94±1.09b _563.47

±10.97a

Glycerol1-phosphate 2.98±0.26 – –

D-Threitol 58.63±4.42 7.61±1.60b _4.60

±0.93b

(6)

Table2(Continued)

Metabolites Relativeresponseratiopergextract

Swertiachirayita Swertiadecussata Swertiabimaculata

d-Mannitol 533.42±19.80 12.45±1.06b _66.75_±_1.01b

d-Sorbitol 199.74±2.90 115.11±18.17b _323.37

±47.73a

Galactinol 37.99±6.29 – –

Terpenoids

Phytol – – 3.44±0.66

Loganin – 105.87±12.81c _34.05

±7.11

Palatinitol 28.05±15.45 – 8.68±4.87

Stigmasterol 39.26±5.79 3.15±2.76b _42.25_±_7.60

Lanosterol – – 35.94±7.56

Macrocycleorganiccompound

Porphine 6.41±4.38 – –

–,notdetected.

a_{Significantly}_higher_than_S._chirayita_.

b _{Significantly}_lower_than_S._chirayita_.

c _{Significantly}_higher_between_S._decussata_and_S._bimaculata_.

60.00

50.00

40.00

30.00

20.00

10.00

0.00 D-malic

acid

Succinic acid

IC

50

v

a

lue (mg/ml

±

sd)

14.82

±

0.36

38.86

±

1.24

1.77

±

0.02

48.40

±

1.31

2.91

±

0.02

0.79

±

0.01

Citric acid

O-acetyl salicylic acid

Quinic acid

Silymarin

Fig.6.Comparisonof␤-glucuronidaseinhibitoryactivityoforganicandphenolic acidswithrespecttosilymarin.

Mangiferinshowedthebest␤-glucuronidaseinhibitionwithan

IC50valueof16.06±0.05␮g/mlor0.038mMfollowedby

swer-chirin(IC50 162.84±3.72␮g/ml or 0.565mM), decussatin(IC50

195.11±5.03␮g/ml or 0.646mM), 1-hydroxy-3,5,8-trimethoxy

xanthone(IC50245.97±4.19␮g/mlor0.814mM)andbellidifolin

(IC50390.26±2.92␮g/mlor1.424mM).However,thebitteriridoid

compound,amarogentindidnotshowanyenzymeinhibitory

activ-ity.Outof72metabolitesidentifiedbyGC–MS,ninecompounds,

availableinthelaboratory,weretestedfortheir␤-glucuronidase

inhibitory activity. These were succinic acid, d-malic acid,

cit-ricacid,O-acetylsalicylicacid,4-hydroxybenzoicacid,quinicacid,

4-hydroxycinnamicacid,sucroseandglycerol.4-Hydroxybenzoic

acid,4-hydroxycinnamicacid,sucroseandglyceroldidnothaveany

␤-glucuronidase inhibitory activity. Remaining five compounds

inhibited␤-glucuronidaseinadose-dependentmanner.

Compari-sonoftheiractivitieswithrespecttosilymarinhadbeenillustrated

in Fig.6.Among these, citric acid (IC50 1.77±0.02mg/ml) and

quinicacid(IC502.91±0.02mg/ml)showedactivitycloseto

sily-marin(IC500.79±0.01mg/ml).

Xanthoneshad already beenreported topossess a range of

pharmacologicalactions(Peresetal.,2000).S.chirayita,S.

decus-sata and S. bimaculata are considered to be a natural source

oftetraoxygenatedxanthones(Pereset al.,2000).Mangiferin, a

xanthone-C-glycoside,had previously beenreported to possess

antioxidant(Nagetal.,2015);anti-diabeticandantitumour

activ-itiesto namea few (Suryawanshiet al., 2006).Bellidifolin and

swerchirinhadbeenfoundtobepotenthypoglycemicagent(Bajpai

etal.,1991;Basnetetal.,1995).Inaddition,swerchirin,hadalso

beenreportedtobehepatoprotective(Hajimehdipooretal.,2006)

onparacetamol-inducedhepatotoxicity inmice models.Several

studiesthathadbeencarriedouttoadvocatethehepatoprotective

andanti-hepatotoxicpropertyofthisgenuscreditsthisattribute

tothexanthonecontentpresentinextractoftheplant.Thefive

xanthonesthathadbeenconsideredinourstudyshowedgood␤

-glucuronidaseinhibition incomparisontothecommercialdrug,

silymarin,therebyproposingapossiblemechanismof

hepatopro-tectiveactionvia␤-glucuronidaseinhibition.Thesefindingshad

not been reported earlier in literature. The present study also

revealsthatsomeorganicacidsviz.,succinicacid,d_-malic_acid,_citric

acidandphenoliccompoundsviz.,O-acetylsalicylicacidandquinic

acidhave␤-glucuronidaseinhibitoryproperties.Citricacidwas

reportedearliertoreducelipopolysaccharideinducedliverinjury

andoxidativestress(Abdel-Salametal.,2014).So,␤-glucuronidase

inhibitionpropertyoftheconstituentspresentinSwertiasp.maybe

amechanismforhepatoprotectiveactivityoftheseplants.Further

invivostudyisrequiredinthisregard.

Conclusion

Theproperties of three Swertiasp. e.g. S.chirayita, S.

decus-sataandS.bimaculatatoinhibit␤-glucuronidase,amechanismfor

hepatoprotection,wereassessedandthecontributoryconstituents

hadbeenidentified.Severalxanthoneswereidentifiedtobemajor

componentstohavesignificantlyhigher␤-glucuronidase

inhibi-tionpropertiesthanthatofsilymarin.Metabolitesotherthanthe

xanthones,probablyalsocontributetothebioactivityofdifferent

Swertiaspeciesbysynergisticeffect.Thepresentfindingssuggest

that␤-glucuronidaseinhibitionmaybeoneofthemechanismsfor

thehepatoprotectivepropertyofSwertiasp.Furtherinvivostudy

isrequiredtosupporttheclaim.

Ethicaldisclosures

Protectionofhumanandanimalsubjects. Theauthorsdeclare

thatnoexperimentswereperformedonhumansoranimalsduring

thestudy.

Confidentialityofdata. Theauthorsdeclarethatnopatientdata

appearinthisarticle.

Righttoprivacyandinformedconsent. Theauthorsdeclarethat

(7)

Authors’contributions

SKperformedextraction, chromatographicanalysis,

bioactiv-ity studies and preparation of the manuscript. GN performed

extractionandsomeanalysis.BDprovidedidea,projectplanand

subsequentlypreparationofthemanuscript.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

TheauthorsacknowledgefinancialsupportfromDepartmentof

ScienceandTechnology(GovernmentofWestBengal),Department

ofScienceandTechnologyFISTProgramme(GovernmentofIndia),

UniversityGrantsCommission.

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