Industrial Crops and Products 70 (2015) 238244

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Industrial Crops and Products

jo u r n al homep age: www.elsev ier .com/ locate / indcrop

Chrom axanthin r exreferen

Shivraj HDepartment of niver

a r t i c l

Article history:Received 10 DReceived in reAccepted 12 M

Keywords:GingerAntioxidantAnti-inammatoryXanthine oxidHPTLCHPLC

pice uract ae oxielect

they revealed the difference in bioactivity against studied parameters. The ethyl acetate extract (EAE)showed signicant antioxidant activity studied by DPPH, FRAP, and H2O2 assay (IC50 SEM [g/mL]:6.8 0.6, 12 0.2, and 20 2.5, respectively). In the xanthine/xanthine oxidase system, the antioxidantpotentials of EAE and the water extract (WE) (% inhibition: 76% and 74%, respectively) were higher than

1. Introdu

Ginger (spice food an importamedicines fas asthma, betes, and rSeveral stucommonly properties was reportefungal, antactivities (Bexhibits cha

CorresponE-mail ad

(S.W. Park).

http://dx.doi.o0926-6690/ ase those of the ethanol extract (EE), diethyl ether extract (DEE), and n-butanol extract (NBE). Regardinganti-inammatory activity, EAE exhibited greater inhibition of lipoxidase (80%), and -glucuronidase(78%) compared to hyaluronidase (46%) and diene-conjugates (37%). Chromatographic analysis revealedthat several principal substances including 6-gingerol, 6-shogaol, and 6-paradol were responsible for thebiological activities for ginger. Compound 6-gingerol revealed high FRAP-reducing activity (IC50 SEM[M]: 5 0.4). 6-Gingerol also signicantly inhibited the activities of xanthine oxidase (85%), lipoxidase(87%), -glucuronidase (85%), and hyaluronidase (56%), respectively. These results indicated that gingerrhizome fractions and its active constituents having promising antioxidant, anti-inammatory, and anti-gout properties and might be used as potential natural drug against oxidative stress and inammatoryrelated diseases after successful in vivo study and clinical trials.

2015 Elsevier B.V. All rights reserved.

ction

Zingiber ofcinale, Zingiberacae) is commonly used asand dietary supplement and has been considered asnt ingredient in Ayurvedic, Unani and Chinese herbalor the treatment of various diseases and disorders suchgingivitis, catarrh, toothache, stroke, constipation, dia-heumatism (Wang and Wang, 2005; Tapsell et al., 2006).dies have examined and reected that the ginger isused as medicinal spice as it reects various medicinal(Chrubasik et al., 2005; Badreldin et al., 2008). Gingerd to have medicinal properties like antimicrobial, anti-iviral, antioxidant, anti-inammatory, and anticancerartley and Jacobs, 2000; Dugasani et al., 2009), andracteristic odors and avors with a pungent taste (Jolad

ding author. Tel.: +82 24503739; fax: +8224503739.dresses: nileshivraj@gmail.com (S.H. Nile), sewpark@konkuk.ac.kr

et al., 2005). As ginger is known to be having antioxidant and anti-inammatory agent; it also exhibits cancer prevention properties,and is used as a postoperative antiemetic (Grzanna et al., 2005;Chaiyakunapruk et al., 2006; Shukla and Singh, 2007). The odor ofginger depends mainly on its volatile oil, the yield of which variesfrom 1% to 3%. Over 50 components of the oil have been charac-terized, including monoterpenoids and sesquiterpenoids (Janick,2012). The homologous series of phenols called gingerols responsi-ble for pungency and odor in fresh ginger. Ginger rhizome extractscontain specic phenolic compounds gingerol and its derivativeswith various biological activities specically; antioxidant and anti-cancer (Yeh et al., 2014). Curcumin, another active componentpresent in ginger, has wide range of activities like antimicrobial,anticancer, antioxidant and an anti-inammatory activity, also acti-vate the heme oxygenase-1 activity, and protects endothelial cellsagainst oxidative stress occurred due to free radicals (Motterliniet al., 2000). In ginger, the widely distributed compounds is 6-gingerol and its derivatives, although smaller quantities of othercompounds are also present like shogaol, paradol and other phe-

rg/10.1016/j.indcrop.2015.03.0332015 Elsevier B.V. All rights reserved.atographic analysis, antioxidant, anti-ine oxidase inhibitory activities of gingece compounds

ariram Nile , Se Won Park Bio-Resources and Food Sciences, College of Life and Environmental Sciences, Konkuk U

e i n f o

ecember 2014vised form 11 March 2015arch 2015

a b s t r a c t

Ginger, Zingiber ofcinale Roscoe, is a sreport the HPTLC analysis of ginger extidant, anti-inammatory, and xanthinusing different polarity solvents with smmatory, andtracts and its

sity, Seoul 143-701, South Korea

sed as a medicinal plant in many countries. We are the rst tond analysis of their active principles with comparative antiox-dase inhibitory activities. The ve fractions were obtained byive extraction procedure from ginger rhizomes and found that

S.H. Nile, S.W. Park / Industrial Crops and Products 70 (2015) 238244 239

nolic acids with different chain (Badreldin et al., 2008). As the odorand pungency of the fresh and dry ginger mainly results from dehy-drated forms of gingerols but in many preparations using ginger thethermal processing can produce shogaols which may lead to pro-duction of oet al., 2005)natural souin the foodpreparationThus, the dthe growinfor developagainst varvey, it was phenolic anderivatives diseases liktric ulcer, h(Chrubasik many benethis study. and HPLC aantioxidantproperties. chemical cowere evaluhydrazine)-ties, and ineffects of gwere assesshyaluronidamost activewere determ

2. Materia

2.1. Chemic

The chemin (BSA) Chemicals, thiobarbitutestis hyaluphosphate lipoxidase fblue tetrazooxidase weUSA). 6-Gincured fromThe chemicunless state

2.2. Plant m

Ginger rchased fromwere storedginger was 1 h. Unpeeginger loses

2.3. Extract

Various (EE), diethywere used

ginger (10 gm) was separately extracted with distilled water(5 200 mL) in an ultrasonicator bath (Wise-Clean, Korea) at40 5 C, for 1 h. The collected supernatants were ltered throughfunnel using glass wool and washed with 10 mL of extraction

t. Thvacuto suum cns pe wce wh (W. To olutedl etheredud ev

C unto co

a c

PTLC

LC ca mo) andth anled ), anen h

For rds luallyplatehe Ced ws: 10etwe frwereundele coversition//s anngthrk, 2

PLC a

C ans 6-glent

A) wty pe

ed ). Al

usinhe sacetoe o

atogrrtnerdor and pungency in fresh and dry ginger (Wohlmuth. The antioxidant compounds or phytochemicals fromrces like plants, fruits, crops and spices are important

industry because of their usefulness in various foods and health promoting effects (Ibanez et al., 2003).emand for natural antioxidants has increased due tog interest in the food and pharmaceutical industriesment of drug which has less side effects and potentious diseases (Yeh et al., 2014). From literature sur-found that the ginger contains a number of bioactived non phenolic constituents, which in pure form or itsmight be potentially useful in the treatment of variouse oxidative stress, diabetes, cancer, arthritis, gout, gas-ypercholesterolemia, pain, microbial or viral infectionet al., 2005; Badreldin et al., 2008), here we presentedts of ginger from reviewed literature and formulatedTherefore, in this study, we investigated the HPTLCnalysis of ginger extract for bioactive constituents with, anti-inammatory, and xanthine oxidase inhibitoryAntioxidant activities of the ginger extracts with itsnstituents like 6-gingerol, 6-shogaol, and 6-paradolated by assaying their DPPH (2,2-diphenyl-1-picryl

and OH-radical-scavenging activities, reducing abili-hibition of xanthine oxidase (XO). Anti-inammatoryinger extracts, 6-gingerol, 6-shogaol, and 6-paradoled using in vitro diene-conjugate, -glucuronidase, andse lipoxidase inhibition assays. The compositions of the

sub-fractions of 6-gingerol, 6-shogaol, and 6-paradolined by HPLC and HPTLC.

ls and methods

als

micals used in this study were bovine serum albu-and quercetin which was purchased from DaejungKorea. 2-Diphenyl-1-picrylhydrazine radical (DPPH),ric acid (TBA), trichloroacetic acid (TCA), bovineronidase (BTH), nicotinamide adenine dinucleotide(NADPH), 5,5-dithiobis-(2-nitrobenzoicacid) (DTNB),rom glycine max (LOX), luminol, linoleic acid (LA), nitrolium (NBT), xanthine, allopurinol (AL), and xanthinere purchased from SigmaAldrich Co. (St. Louis, MO,gerol (6G), 6-shogaol (6S), and 6-paradol (6P) were pro-

Chromadex (Santa Ana, CA, USA). Glutathione (GSH).als and solvents used for this study were of HPLC grade,d otherwise.

aterial

hizomes (5 kg) (Zingiber ofcinale Roscoe) were pur- a local supermarket, Seoul, Korea. The ginger rhizomes

at 4 C and washed thoroughly prior to use. Washedsliced into small pieces and dried in an oven at 60 C forled ginger was used for solvent extraction since peeled

much of its essential oil content.

and sub fractions preparation

solvent systems [distilled water (DWE), 70% ethanoll ether (DEE), ethyl acetate (EAE) and n-butanol (NBE)]to prepare the extracts and sub-fractions. Powdered

solvenunder jected a vacufractiorhizomand ontor batfor 1 hther didiethyunder tion an40 5jected to gain2013)

2.4. H

HPTusing [v/v/v]ing wiice-coo(10 mLwas thature).standaindividto the using tequippsettingtance bdistancplates alized availabware (absorp20 mmwaveleand Pa

2.5. H

HPLpoundan AgiCA, UScapaciperform1.8 mtrolledUSA). Ting of run timchromSchwee ltered residue was then concentrated to drynessum (Buchi System, Switzerland) at 40 5 C and sub-bsequent cooling at 80 C, further lyophilized usingoncentrator until to get a constant weight. For sub-reparations, the accurately weighed 5 gm of gingeras separately extracted using methanol (5 100 mL)ith 100 mL of 80% (v/v) methanol using ultrasonica-ise-Clean, Korea) at controlled temperatures (40 5 C)btain subtractions, the methanolic extracts were fur-

with water and successively partitioned using ethanol,r, ethyl acetate and n-butanol after solvent evaporation

ced pressure. All extracts obtained by successive extrac-aporation of solvent were concentrated to dryness atder a vacuum (Bchi System, Switzerland), further sub-oling at 80 C, and lyophilized using a vacuum dryeronstant weight (Tomczyk et al., 2011; Bazylko et al.,

analysis

hromatograms for ginger rhizome extracts developedbile phase (ethyl acetate:water:formic acid, 85:10:5

the plates (Silica gel 60, F254) were subjected to spray-isaldehyde reagent (the reagent was prepared using

methanol (170 mL), acetic acid (170 mL), sulfuric acidd 1 mL of anisaldehyde compound. The added mixtureeated at 10 C for 3 min and cooled to room temper-the qualitative analysis of reference compounds, theike 6-gingerol, 6-shogaol, and 6-paradol (1 mg) were

dissolved in 10 mL methanol, and each was applieds as 10 mm bands. Sample application was performedAMAG-Linomat IV automated spray on band applicatorith a 10 mL syringe and operated with the following

mm band length, 10 mL/s application rate, 4 mm dis-een, 1.5 cm distance from the plate side edge, and 2 cmom the bottom of the plate. After development, the

air-dried for 15 min, and the chromatograph was visu-r CAMAG TLC Scanner 3 to quantify the bands of thesempounds in ginger extract using the WIN CATS soft-on 4X). The scanner operating parameters were: Mode:reection; slit dimensions; 5 0.1 mm; scanning rate:d monochromatic band width: 20 nm at an optimized

of 254 nm and in the visible range (Rai et al., 2006; Nile014a).

nalysis of ginger extract

alysis of ginger extract, along with reference com-ingerol, 6-shogaol, and 6-paradol was performed using1100 LC System (Agilent Technologies Inc., Palo Alto,ith an auto-injector sampler programmed at 5 L

r injection. HPLC chromatographic separations wereon Zorbax Stable Bond, C18 column (4.6 mm 50 mm,l operations, acquisitions, and data analysis were con-g the Chemstation software (Agilent Technologies,

eparation was performed with a mobile phase consist-nitrile and water (85:15, v/v) and a chromatographic

f 20 min at 30 C with a ow-rate of 1.0 mL/min, andams were monitored at 280 nm (Wang et al., 2009;

et al., 2007).

240 S.H. Nile, S.W. Park / Industrial Crops and Products 70 (2015) 238244

2.6. DPPH radical-scavenging assay

The DPPH-radical-scavenging potential of ginger extracts(10 mg) and 6-gingerol, 6-shogaol, and 6-paradol (2 mM) wasexamined b(0.18 mL) ousing ultraobtained musing spectrecorded wments wereas a standarthe formulaabsorbanceantioxidant

2.7. FRAP a

The Fe3+

and 6-paradmodicatioextract (10in 0.5% v/v solution (1 water bath acid (TCA) portion of thand 0.1 mL bated at romeasured ution. All testhe reaction(2 mM) was

2.8. OH-rad

This assdensation wobtained rethe OH-radisystem (thenal volum(20 mM, pHascorbic aciger extract solution wesamples th1 mL of 2% mixture waAfter coolinsolution. Alwas used ascomparing

2.9. Xanthi

Bovine mmation at 5reaction mdase (500 8.75, 30 L)100 M nreaction miand other te(100 L, 10formation o

ture was incubated for 2 h, at 37 C in a water bath and nally thereaction product absorbance was recorded at 550 nm. For blank,xanthine was omitted from samples and allopurinol was used asa positive control for this assay. All values were expressed as the

of thol, 6-ibito

Nile

nti-i

all aed w

(EAf 6-gs anrds.

Dienjugaerol,

des mMeters

T/Ct and

-Gibitiol, aned bce fo

Hya hyaol, 6-ell

n wa

Lipoibitioaol, ed bted cntrol

tatis

resur of es bec tesance

ults

PTLC

h-peo ideamram fyl acwereum as of y mixing with absolute ethanol and 1 103 mol/Lf DPPH. The obtained samples were properly mixedsonication and incubated for 45 min, thereafter theixture was then monitored at 515 nm against a blankrophotometer (UVvis Shimadzu) and absorbance werehen the reaction reached a steady state. All measure-

performed in triplicate. Quercetin (2 mM) was usedd. The radical scavenging activity was calculated using: % inhibition = (Ac (o) AA (t)/Ac (o) 100 (Ac (o) is the

of control at t = 0 min and AA (t) is the absorbance of at t = 1 h.) (Nile et al., 2013).

ssay (reducing power assay)

reducing power of ginger extract, 6-gingerol, 6-shogaol,ol was determined as described previously with slightns. The reaction mixture contained 10 mL of ginger

mg) and 6-gingerol, 6-shogaol, and 6-paradol (5 mMdimethyl sulfoxide) in 3 mL of potassium ferricyanidemM). The obtained mixture then incubated at 50 C in afor 20 min and after incubation, 0.5 mL of trichloroacetic(10%) was added to terminate the reaction. The uppere solution (1 mL) was mixed with distilled water (1 mL)

FeCl3 solution (0.01%). The reaction mixture was incu-om temperature for 10 min, and the absorbance wassing spectrophotometer at 700 nm using blank solu-ts were performed in triplicate. A higher absorbance of

mixture indicated greater reducing power. Glutathione used as a reference compound (Nile and Park, 2014b).

ical scavenging assay

ay is based on the degradation 2-deoxyribose by con-ith thiobarbituric acid (TBA) and quantication of

action product using spectrophotometer. In this assay,cal was generated using the Fe3+-ascorbate-EDTA-H2O2

Fenton reaction). This reaction mixture contained, in ae 2-deoxy-2-ribose (1 mL, 2.8 mM), KH2PO4-KOH buffer

7.4), FeCl3 (100 M), EDTA (100 M), H2O2 (1.0 mM),d (100 M). To this each test sample like 0.3 mL of gin-(10 mg), 6-gingerol, 6-shogaol, and 6-paradol (2 mM)re added respectively. The obtained reaction with testen incubated for 1 h at 37 C, to which 1 mL 3% TCA,aqueous TBA was added. After addition this reactions incubated for 15 min at 90 C for color development.g, the absorbance was recorded at 532 nm using blankl tests were performed three times. Glutathione (2 mM)

a positive control. Percent inhibition was calculated bythe test and blank solutions (Nile and Park, 2014b).

ne oxidase inhibition

ilk XO activity was measured based on formazan for-50 nm using a UVvis spectrophotometer at 25 C. Theixture in the sample wells consisted of xanthine oxi-M nal concentration) in phosphate buffer (0.01 M, pH, NBT (50 L, 100 M, nal concentration), PMS (50 L,al concentration), Triton X-100 (20 L, 0.5%). To thisxture the respective sample like ginger extracts (10 mg)st compounds like 6-gingerol, 6-shogaol, and 6-paradol

g/mL) were added to inhibit the xanthine oxidase byf formazan. After addition of test samples the total mix-

meansgingerthe inh2011;

2.10. A

For[distillacetateeach osamplestanda

2.10.1.Con

6-gingviouslyacid (1paramity = (1the tes

2.10.2.Inh

shogaodescribreferen

2.10.3.The

gingerto 96 wHepari

2.10.4.Inh

6-shogdescribcalculative co

2.11. S

Thenumbeferencpost hosignic

3. Res

3.1. H

Higused tanti-inmatogof ethplates maximtogramree experiments. The IC50 values of ginger extract, 6-shogaol, 6-paradol and allopurinol were obtained fromr concentration-activity curve (Nile and Khobragade,

and Park, 2013).

nammatory activity

nti-inammatory assays, 50 mg of each ginger extractater (DWE), 70% ethanol (EE), diethyl ether (DEE), ethylE) and n-butanol (NBE)] was used along with 1 mM ofingerol (6G), 6-shogaol (6S), and 6-paradol (6P) as testd salicylic acid (SA), heparin (HE) and quercetin (QR) as

e-conjugate assayted diene assays for ginger extract, along with6-shogaol, and 6-paradol were performed using pre-cribed method by Nile and Park (2014a). Salicylic) was used as a standard. The percent activity for all

was calculated using the standard formula: % activ-) 100; where, T and C represent the absorbances of

control samples, respectively.

lucuronidase inhibition assayn of -glucuronidase by ginger extract, 6-gingerol, 6-d 6-paradol was determined using previous method asy Nile and Park (2014a). Salicylic acid was used as ar comparison.

luronidase activity inhibitionluronidase inhibitory activities of ginger extract, 6-shogaol, and 6-paradol were determined by modiedmicrotiter plates method by Piwowarski et al. (2011).s used as a positive control.

xidase activity inhibitionn of lipoxidase (LOX) by ginger extract, 6-gingerol,and 6-paradol was determined using the methody Bazylko et al. (2013). Percentage LOX inhibition wasompared to the control. Quercetin was used as a posi-.

tical analysis

lts were expressed as the means SEM of the indicatedexperiments (n 3). The statistical signicance of dif-tween means was established by ANOVA with Duncansts. P values

S.H. Nile, S.W. Park / Industrial Crops and Products 70 (2015) 238244 241

gingerol (6G), 6-shogaol (6S), and 6-paradol (6P) on HPTLC platesunder CAMAG TLC Scanner. The bands were densitometrically stud-ied using the WIN CATS software (version 4X) at an optimizedwavelength of 254 nm and in the visible range. 6-Gingerol showeda violet zone at Rf 0.36, 6-paradol showed a dark brown zoneat Rf 0.74, and 6-shogaol showed a violet zone at Rf 0.53. Thengerprint of the test solution was similar to that of the corre-sponding botanical reference samples (Fig. 1). Use of these solventsystems provides good separation of the phytochemicals as pre-sented by Fig. 1 (Amin et al., 2006; Lai et al., 2009). This mobilephase offers an improvement over that described earlier and facil-itates the separation of avonoids, lectins, and saponins (Zou et al.,2004). In addition, 6-gingerol, 6-shogaol, and 6-paradol was deter-mined by HPLC using external standards for quantication and aC-18 reverse-phase column with acetonitrile and water (85:15, v/v)as the mobile phase. Peaks ac showed UV spectral characteris-tics similar to those of 6-gingerol, 6-shogaol, and 6-paradol; i.e., aUV absorption maximum wavelength of 230 nm (Figs. 2 and 3). InHPLC analysis of ginger extracts during quantication of 6-gingerol,6-shogaol, and 6-paradol, we did not identify any substance thatmay interfere with the analysis ginger extracts. The quanticationand peak identications for 6-gingerol, 6-paradol, and 6-shogaolin ginger extract were based on the retention times of the stan-dards and conrmed by comparing their photodiode array spectrato those of the individual standards. To the best of our knowledge,so far there was very limited reports on HPTLC analysis of Zingiber

Fig. 1. HPTLC chromatograms of the tested ginger rhizome extracts, lane assign-ments, from left to right: standards 1: 6-shogaol, 2: 6-gingerol, 3: 6-paradol, 4:water extract, 5: ethanol extract, 6: ethyl acetate extract, 7: diethyl ether extract, 8:n-butanol extract.

ofcinale extract, only few scientist studied the qualitative analysisgingerol, shogaol and zerumbone (Alam, 2013; Salmon et al., 2012;Rout et al., 2009), in our study we qualitatively analyzed presenceall major components in ginger by HPTLC analysis by using differ-ent solvent system [acetonitrile and water (85:15, v/v)] rather than

6-sho

rol, b: 6-shogaol, and c: 6-paradol.Fig. 2. HPLC analysis of standards (a: 6-gingerol, b:

Fig. 3. HPLC analysis of ginger extract showing a: 6-gingegaol, and c: 6-paradol).

242 S.H. Nile, S.W. Park / Industrial Crops and Products 70 (2015) 238244

Table 1Antioxidant activity of tested ginger extracts, sub fractions, and bioactivecomponents.

Extracts/fractions/compounds IC50 SEM(extracts/fractions[g/mL];bioactive components [mM])

DPPH FRAP H2O2

Distilled water extract (DWE) 08 0.5 20 0.8 32 1.1Ethanol extracts (70%) (EE) 10 0.1 16 1.1 34 2.4Diethyl ether extract (DEE) 14 0.5 22 0.6 40 3.2Ethyl acetate extract (EAE) 6.8 0.6 12 0.2 20 2.5n-Butanol extract (NBE) 17 0.8 28 0.4 45 1.86-Gingerol (6G) 8 0.1 5 0.4 10 0.46-Shogaol (6S) 12 0.3 8 0.6 13 0.56-Paradol (6P) 10 0.3 9 1.4 15 0.8Quercetin (QR) 5 0.2 Glutathione (GT) 6 0.1 8 0.5

Values are mean SD (n = 3).

previously described methods and also the results were conrmedby HPLC method. Thus, the developed HPTLC and HPLC methods forquantitation of ginger compounds was found to be simple, accurate,reproduciblof other gin

3.2. Antioxi

The anttogether wiand 6-paraagainst DPPobtained uethanol 70used; resulits componantioxidantconcentratiwas found cals in ethy12 0.2 anoxidase sys(% inhibitioother extramation ranTherefore, Dactivity, whactivity. Thoxidative dcardiovascu

Fig. 4. Xanthi6-gingerol, 6-s

variety of other disorders (Noguchi and Nikki, 2000). Antioxidantsare the molecules which scavenge free radicals are now known topossess preventive as well as therapeutic potential in free radical-mediated disease conditions (Visioli et al., 2000). The antioxidantaction of ginger has been proposed as one of the major possiblemechanisms for the protective actions against toxicity (Ali et al.,2008). Recently, it has been shown that 6-gingerol is endowedwith strong antioxidant action both in vivo and in vitro, in addi-tion to strong anti-inammatory and anti-apoptotic actions (Kimet al., 2007). The data presented in this study revealed that the ethylacetate (EAE) and distilled water (DWE) extracts of ginger are withsignicant free radical inhibitors and acted as primary antioxidantsthat react with free radicals. The several authors have shown thatginger is endowed with strong in vitro antioxidant properties andto this our results are comparable with previous studies (Stoilovaet al., 2007; Ali. et al., 2008; Dugasani et al., 2009).

3.3. Xanthine oxidase inhibition

All extracts from ginger rhizome and their bioactive compoundsshowed good to excellent inhibition of xanthine oxidase activity.

d WE exhibited the highest XO inhibitory activity of thets. Rer XOalues0.5

(Fig. dy o

ne oe inhobrat in t

asssuchive-s

such relativs reeasin

in of foafooors ent of chotives

are e and sensitive and might be applicable to the analysisger varieties.

dant activity

ioxidant activities of the ginger rhizome fractions,th the bioactive components like 6-gingerol, 6-shogaol,dol, were investigated in vitro antioxidant activityH, FRAP, and H2O2 radicals. Ginger rhizome fractionssing different solvents of different polarities (water,%, diethyl ether, ethyl acetate and n-butanol) werets are presented in Table 1. The ginger extract andents like 6-gingerol, 6-shogaol, and 6-paradol showed

activity against all examined reactive species in aon-dependent manner. The highest scavenging activityagainst DPPH as compared to FRAP and H2O2 radi-l acetate extract (EAE) (IC50 SEM [g/mL]: 6.8 0.6,d 20 2.5, respectively). Only the xanthine/xanthinetem was the antioxidant potential of EAE and DWEn: 76% and 74%, respectively) higher than that of thects. Depending on the concentration, formazan for-ged from 55% to 34% for EE, DEE, and NBE (Table 1).WE showed greater DPPH- and ferric-ion-scavenging

ile EAE exhibited greater inhibition of xanthine oxidaseere is considerable evidence that free radicals induceamage to biomolecules and play an important role inlar disease, aging, cancer, inammatory diseases and a

EAE anextracgreateIC50 vwere 1tively any stuxanthioxidasand Khinteresdue tofoods, oxidataction,equallyin oxidndingis incrchangeintakeand seinhibittreatmdrug oalternaeffectsne oxidase inhibition by ginger extracts and its bioactive compounds (100 M). The datahogaol, 6-paradol and allopurinol are 10.5 0.5 M, 15.2 0.3 M, 12.4 0.6 M, and 8. are expressed as the means SD, n = 3. The IC50 values of compound4 0.4 M, respectively (short forms explained in Tables 1 and 2).

garding the bioactive compounds, 6-gingerol (6G) had inhibitory activity than 6-shogaol and 6-paradol. The

of 6-gingerol, 6-shogaol, 6-paradol and allopurinol 0.5, 15.2 0.3, 12.4 0.6, and 8.4 0.4 M, respec-

4). To the best of our knowledge, so far there was nor report on activity of Zingiber ofcinale extract againstxidase but only few scientists studied the xanthineibition using plant, vegetable and spice extracts (Nilegade, 2011; Nile and Park, 2013). There is signicanthe direct antioxidant activities of dietary polyphenols,ociations between consumption of polyphenol-rich

as fruits and vegetables, and decreased incidence oftress related disease. However, indirect antioxidant

as the inhibition of ROS-producing enzymes, may beevant to health benets through a general reductione stress (Dew et al., 2005; Shan et al., 2005). Recentvealed that the incidence of gout and hyperuricemiag worldwide drastically and the possible reason mayunusual habits of food, smoking and drinking also,ods which rich in nucleic acids, including meat, pork,d. Hypouricemic agents, including xanthine oxidaseand uricosuric agents, are commonly used for thef chronic gouty arthritis. In general, allopurinol is theice; however, it has serious side effects. Thus, novel

with increased therapeutic activity and fewer sidedesired (Nile et al., 2013; Nile and Park, 2014b). Thus,

S.H. Nile, S.W. Park / Industrial Crops and Products 70 (2015) 238244 243

Table 2Anti-inammatory activity of tested ginger extracts, sub fractions, and bioactive components.

Extracts/fractions/compounds Inhibition (%) (mean S.E from three experiments)

-Glucuronidase Diene-conjugate Hyaluronidase Lipoxidase

Distilled waEthanol extrDiethyl etheEthyl acetaten-Butanol ex6-Gingerol (6-Shogaol (66-Paradol (6Salicylic acidHeparin (HEQuercetin (Q

Values are me

we attempinhibitors fr

3.4. Anti-in

Anti-instituents wlipoxidase, This study tested extraexhibited ttested (Tabmore stronfor -glucu35% for hytively). At 1the activitytive compo(SA), heparevidence hels of inamhuman cellsolated thereet al., 2005;anti-inamdiene-conjuinhibition. paradol are(Shukla anpharmacoloanti-inamthis study, compoundsoxidase inhthine oxidaand depositacts as a souPark, 2014bextract andoxide prodxanthine oxthat phenolsuperoxideand shogoabition of xacompoundsimportant production pharmacolo

clus

ress aloaol (mato

in v as ated his nal utract-shog or rgh itty ofstudfor aovidiundsmato

wled

s resm-20

nces

, 2013act anms. As

Blundmacooe): a

Norazent ofn, H.Ater extract (DWE) 64 2.4 32 0.9 acts (70%) (EE) 46 1.8 28 0.5 r extract (DEE) 38 1.5 22 0.4

extract (EAE) 78 3.6 37 0.1 tract (NBE) 30 4.1 17 0.5 6G) 85 3.1 48 0.3 S) 70 1.6 40 0.7 P) 63 1.3 35 0.4

(SA) 82 2.1 50 1.4) R)

an SD (n = 3).

ted to identify phytochemicals as xanthine oxidaseom various foods and food products, including ginger.

ammatory activity

ammatory activity for ginger extracts and active con-as determined using diene-conjugate, -glucuronidase,and hyaluronidase in vitro inhibition assay (Table 2).revealed signicant anti-inammatory activity of thects and active constituents. EAE, WE, and 6-gingerol

he greatest anti-inammatory activity of all samplesle 2). At 50 mg, EAE and WE inhibited all enzymesgly than did the remaining samples (78 and 64%ronidase, 37 and 32% for diene-conjugate, 46 andaluronidase, and 80 and 70% for lipoxidase, respec-

mM, 6-gingerol (6G) exhibited greater inhibition of of all enzymes compared to both the other bioac-unds 6-shogaol (6S), 6-paradol (6P); and salicylic acidin (HE), and quercetin (QR) (Table 2). Several lines ofave been provided, mostly in different animal mod-

mation, and to a much lesser extent in humans or, of the effectiveness of either ginger or of compounds is

from against inammation and its mediators (Grzanna Lantz et al., 2007). Here, in this study, we studied thematory properties of ginger by using in vitro assay viz.;gate, -glucuronidase, lipoxidase, and hyaluronidase6-Gingerol, 8-gingerol, 10-gingerol, 6-shogaol, and 6-

the important and major compounds found in gingerd Singh, 2007), which are identied with variousgical and biological properties such as antioxidant,matory, anticancer, and antiulcer activities. Here, inwe did comparative analysis of ginger extracts and its

for the antioxidant, anti-inammatory, and xanthineibitory activity. During the metabolism of purine xan-se convert xanthine to uric acid and excess productionion of uric acid leads to gout, so here xanthine oxidase

4. Con

Ourextract6-shoginamfurtherizationassociaicals. Tmediciger ex(6G), 6ventinAlthoudiversiin this value the prcompoinam

Ackno

ThiProgra

Refere

Alam, P.extrcrea

Ali, B.H.,pharRosc

Amin, I.,cont

Badreldi

rce of oxygen free radicals (Nile and Park, 2013; Nile and). In this study, we demonstrated that ginger rhizome

6-gingerol, 6-shogaol and 6-paradol inhibited super-uction and exhibited the strongest inhibition againstidase. Based on a previous report (Dugasani et al., 2009)ic compounds inhibit xanthine oxidase and/or scavenge, the inhibition of superoxide production by gingerolsl may be due to both superoxide scavenging and inhi-nthine oxidase activity. The identication of natural

for the prevention of ROS-associated disorders is anstrategy for antioxidant therapy. Suppression of ROSby gingerols and shogaol may be attributable to variousgical actions of ginger (Badreldin et al., 2008).

phytochem(Zingiber o46, 4094

Bartley, J., JacoAustralian

Bazylko, A., Piantioxidanand its ma

ChaiyakunaprLeelasettapostopera194, 959

Chrubasik, S., comprehe684701.

Dew, T.P., Dayof food ex35 1.0 70 1.328 0.3 60 0.423 0.5 43 0.346 0.7 80 0.720 0.3 38 0.356 0.3 87 1.651 0.4 75 1.248 0.6 72 1.4 55 1.6 80 2.1

ions

ults suggest that ginger ethyl acetate and waterng with active constituents such as 6-gingerol (6G),6S), and 6-paradol (6P) have strong antioxidant, anti-ry, and xanthine oxidase inhibitory activities. However,ivo and clinical studies are required for their character-n agent for use against various diseases and disorderswith oxidative stress, xanthine oxidase, and free rad-study provides a scientic support for some of theses; hence suggesting that increasing the intake of gin-

and utilization of their active compounds; 6-gingerolgaol (6S), and 6-paradol (6P) may be helpful in pre-educing the progress of some lifestyle related diseases.

is not simple to extrapolate from in vitro results, the the pharmacological activities of the ginger observedy suggests that extracts of this ginger species may be ofpplication in human and animal health. Furthermore,ng evidence that the ginger extract and its bioactive

might be potential sources of new antioxidant, anti-ry and anti-gout drugs.

gment

earch paper was supported by KU-Research Professor14, Konkuk University, Seoul, Republic of Korea.

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Chromatographic analysis, antioxidant, anti-inflammatory, and xanthine oxidase inhibitory activities of ginger extracts an...1 Introduction2 Materials and methods2.1 Chemicals2.2 Plant material2.3 Extract and sub fractions preparation2.4 HPTLC analysis2.5 HPLC analysis of ginger extract2.6 DPPH radical-scavenging assay2.7 FRAP assay (reducing power assay)2.8 OH-radical scavenging assay2.9 Xanthine oxidase inhibition2.10 Anti-inflammatory activity2.10.1 Diene-conjugate assay2.10.2 -Glucuronidase inhibition assay2.10.3 Hyaluronidase activity inhibition2.10.4 Lipoxidase activity inhibition

2.11 Statistical analysis

3 Results and discussion3.1 HPTLC and HPLC analysis3.2 Antioxidant activity3.3 Xanthine oxidase inhibition3.4 Anti-inflammatory activity

4 ConclusionsAcknowledgmentReferences