Industrial Crops and Products 70 (2015) 238244

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Industrial Crops and Products

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Chromatographic analysis, antioxidant, anti-inammatory, and

xanthine oxidase inhibitory activities of ginger extracts and its
reference compounds
Shivraj Hariram Nile , Se Won Park
Department of Bio-Resources and Food Sciences, College of Life and Environmental Sciences, Konkuk University, Seoul 143-701, South Korea

a r t i c l e

i n f o

Article history:
Received 10 December 2014
Received in revised form 11 March 2015
Accepted 12 March 2015
Keywords:
Ginger
Antioxidant
Anti-inammatory
Xanthine oxidase
HPTLC
HPLC

a b s t r a c t
Ginger, Zingiber ofcinale Roscoe, is a spice used as a medicinal plant in many countries. We are the rst to
report the HPTLC analysis of ginger extract and analysis of their active principles with comparative antioxidant, anti-inammatory, and xanthine oxidase inhibitory activities. The ve fractions were obtained by
using different polarity solvents with selective extraction procedure from ginger rhizomes and found that
they revealed the difference in bioactivity against studied parameters. The ethyl acetate extract (EAE)
showed signicant antioxidant activity studied by DPPH, FRAP, and H2 O2 assay (IC50 SEM [g/mL]:
6.8 0.6, 12 0.2, and 20 2.5, respectively). In the xanthine/xanthine oxidase system, the antioxidant
potentials of EAE and the water extract (WE) (% inhibition: 76% and 74%, respectively) were higher than
those of the ethanol extract (EE), diethyl ether extract (DEE), and n-butanol extract (NBE). Regarding
anti-inammatory activity, EAE exhibited greater inhibition of lipoxidase (80%), and -glucuronidase
(78%) compared to hyaluronidase (46%) and diene-conjugates (37%). Chromatographic analysis revealed
that several principal substances including 6-gingerol, 6-shogaol, and 6-paradol were responsible for the
biological 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 ginger
rhizome fractions and its active constituents having promising antioxidant, anti-inammatory, and antigout properties and might be used as potential natural drug against oxidative stress and inammatory
related diseases after successful in vivo study and clinical trials.
2015 Elsevier B.V. All rights reserved.

1. Introduction
Ginger (Zingiber ofcinale, Zingiberacae) is commonly used as
spice food and dietary supplement and has been considered as
an important ingredient in Ayurvedic, Unani and Chinese herbal
medicines for the treatment of various diseases and disorders such
as asthma, gingivitis, catarrh, toothache, stroke, constipation, diabetes, and rheumatism (Wang and Wang, 2005; Tapsell et al., 2006).
Several studies have examined and reected that the ginger is
commonly used as medicinal spice as it reects various medicinal
properties (Chrubasik et al., 2005; Badreldin et al., 2008). Ginger
was reported to have medicinal properties like antimicrobial, antifungal, antiviral, antioxidant, anti-inammatory, and anticancer
activities (Bartley and Jacobs, 2000; Dugasani et al., 2009), and
exhibits characteristic odors and avors with a pungent taste (Jolad

Corresponding author. Tel.: +82 24503739; fax: +8224503739.

E-mail addresses: nileshivraj@gmail.com (S.H. Nile), sewpark@konkuk.ac.kr
(S.W. Park).
http://dx.doi.org/10.1016/j.indcrop.2015.03.033
0926-6690/ 2015 Elsevier B.V. All rights reserved.

et al., 2005). As ginger is known to be having antioxidant and antiinammatory 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 of
ginger depends mainly on its volatile oil, the yield of which varies
from 1% to 3%. Over 50 components of the oil have been characterized, including monoterpenoids and sesquiterpenoids (Janick,
2012). The homologous series of phenols called gingerols responsible for pungency and odor in fresh ginger. Ginger rhizome extracts
contain specic phenolic compounds gingerol and its derivatives
with various biological activities specically; antioxidant and anticancer (Yeh et al., 2014). Curcumin, another active component
present in ginger, has wide range of activities like antimicrobial,
anticancer, antioxidant and an anti-inammatory activity, also activate the heme oxygenase-1 activity, and protects endothelial cells
against oxidative stress occurred due to free radicals (Motterlini
et al., 2000). In ginger, the widely distributed compounds is 6gingerol and its derivatives, although smaller quantities of other
compounds are also present like shogaol, paradol and other phe-

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

nolic acids with different chain (Badreldin et al., 2008). As the odor
and pungency of the fresh and dry ginger mainly results from dehydrated forms of gingerols but in many preparations using ginger the
thermal processing can produce shogaols which may lead to production of odor and pungency in fresh and dry ginger (Wohlmuth
et al., 2005). The antioxidant compounds or phytochemicals from
natural sources like plants, fruits, crops and spices are important
in the food industry because of their usefulness in various food

preparations and health promoting effects (Ibanez

et al., 2003).
Thus, the demand for natural antioxidants has increased due to
the growing interest in the food and pharmaceutical industries
for development of drug which has less side effects and potent
against various diseases (Yeh et al., 2014). From literature survey, it was found that the ginger contains a number of bioactive
phenolic and non phenolic constituents, which in pure form or its
derivatives might be potentially useful in the treatment of various
diseases like oxidative stress, diabetes, cancer, arthritis, gout, gastric ulcer, hypercholesterolemia, pain, microbial or viral infection
(Chrubasik et al., 2005; Badreldin et al., 2008), here we presented
many benets of ginger from reviewed literature and formulated
this study. Therefore, in this study, we investigated the HPTLC
and HPLC analysis of ginger extract for bioactive constituents with
antioxidant, anti-inammatory, and xanthine oxidase inhibitory
properties. Antioxidant activities of the ginger extracts with its
chemical constituents like 6-gingerol, 6-shogaol, and 6-paradol
were evaluated by assaying their DPPH (2,2-diphenyl-1-picryl
hydrazine)- and OH-radical-scavenging activities, reducing abilities, and inhibition of xanthine oxidase (XO). Anti-inammatory
effects of ginger extracts, 6-gingerol, 6-shogaol, and 6-paradol
were assessed using in vitro diene-conjugate, -glucuronidase, and
hyaluronidase lipoxidase inhibition assays. The compositions of the
most active sub-fractions of 6-gingerol, 6-shogaol, and 6-paradol
were determined by HPLC and HPTLC.
2. Materials and methods
2.1. Chemicals
The chemicals used in this study were bovine serum albumin (BSA) and quercetin which was purchased from Daejung
Chemicals, Korea. 2-Diphenyl-1-picrylhydrazine radical (DPPH),
thiobarbituric acid (TBA), trichloroacetic acid (TCA), bovine
testis hyaluronidase (BTH), nicotinamide adenine dinucleotide
phosphate (NADPH), 5,5-dithiobis-(2-nitrobenzoicacid) (DTNB),
lipoxidase from glycine max (LOX), luminol, linoleic acid (LA), nitro
blue tetrazolium (NBT), xanthine, allopurinol (AL), and xanthine
oxidase were purchased from SigmaAldrich Co. (St. Louis, MO,
USA). 6-Gingerol (6G), 6-shogaol (6S), and 6-paradol (6P) were procured from Chromadex (Santa Ana, CA, USA). Glutathione (GSH).
The chemicals and solvents used for this study were of HPLC grade,
unless stated otherwise.

239

ginger (10 gm) was separately extracted with distilled water

(5 200 mL) in an ultrasonicator bath (Wise-Clean, Korea) at
40 5 C, for 1 h. The collected supernatants were ltered through
funnel using glass wool and washed with 10 mL of extraction
solvent. The ltered residue was then concentrated to dryness
under vacuum (Buchi System, Switzerland) at 40 5 C and subjected to subsequent cooling at 80 C, further lyophilized using
a vacuum concentrator until to get a constant weight. For subfractions preparations, the accurately weighed 5 gm of ginger
rhizome was separately extracted using methanol (5 100 mL)
and once with 100 mL of 80% (v/v) methanol using ultrasonicator bath (Wise-Clean, Korea) at controlled temperatures (40 5 C)
for 1 h. To obtain subtractions, the methanolic extracts were further diluted with water and successively partitioned using ethanol,
diethyl ether, ethyl acetate and n-butanol after solvent evaporation
under reduced pressure. All extracts obtained by successive extraction and evaporation of solvent were concentrated to dryness at
40 5 C under a vacuum (Bchi System, Switzerland), further subjected to cooling at 80 C, and lyophilized using a vacuum dryer
to gain a constant weight (Tomczyk et al., 2011; Bazylko et al.,
2013)

2.4. HPTLC analysis

HPTLC chromatograms for ginger rhizome extracts developed
using a mobile phase (ethyl acetate:water:formic acid, 85:10:5
[v/v/v]) and the plates (Silica gel 60, F254) were subjected to spraying with anisaldehyde reagent (the reagent was prepared using
ice-cooled methanol (170 mL), acetic acid (170 mL), sulfuric acid
(10 mL), and 1 mL of anisaldehyde compound. The added mixture
was then heated at 10 C for 3 min and cooled to room temperature). For the qualitative analysis of reference compounds, the
standards like 6-gingerol, 6-shogaol, and 6-paradol (1 mg) were
individually dissolved in 10 mL methanol, and each was applied
to the plates as 10 mm bands. Sample application was performed
using the CAMAG-Linomat IV automated spray on band applicator
equipped with a 10 mL syringe and operated with the following
settings: 10 mm band length, 10 mL/s application rate, 4 mm distance between, 1.5 cm distance from the plate side edge, and 2 cm
distance from the bottom of the plate. After development, the
plates were air-dried for 15 min, and the chromatograph was visualized under CAMAG TLC Scanner 3 to quantify the bands of these
available compounds in ginger extract using the WIN CATS software (version 4X). The scanner operating parameters were: Mode:
absorption/reection; slit dimensions; 5 0.1 mm; scanning rate:
20 mm/s and monochromatic band width: 20 nm at an optimized
wavelength of 254 nm and in the visible range (Rai et al., 2006; Nile
and Park, 2014a).

2.5. HPLC analysis of ginger extract

2.2. Plant material
Ginger rhizomes (5 kg) (Zingiber ofcinale Roscoe) were purchased from a local supermarket, Seoul, Korea. The ginger rhizomes
were stored at 4 C and washed thoroughly prior to use. Washed
ginger was sliced into small pieces and dried in an oven at 60 C for
1 h. Unpeeled ginger was used for solvent extraction since peeled
ginger loses much of its essential oil content.
2.3. Extract and sub fractions preparation
Various solvent systems [distilled water (DWE), 70% ethanol
(EE), diethyl ether (DEE), ethyl acetate (EAE) and n-butanol (NBE)]
were used to prepare the extracts and sub-fractions. Powdered

HPLC analysis of ginger extract, along with reference compounds 6-gingerol, 6-shogaol, and 6-paradol was performed using
an Agilent 1100 LC System (Agilent Technologies Inc., Palo Alto,
CA, USA) with an auto-injector sampler programmed at 5 L
capacity per injection. HPLC chromatographic separations were
performed on Zorbax Stable Bond, C18 column (4.6 mm 50 mm,
1.8 m). All operations, acquisitions, and data analysis were controlled using the Chemstation software (Agilent Technologies,
USA). The separation was performed with a mobile phase consisting of acetonitrile and water (85:15, v/v) and a chromatographic
run time of 20 min at 30 C with a ow-rate of 1.0 mL/min, and
chromatograms were monitored at 280 nm (Wang et al., 2009;
Schwertner et al., 2007).

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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) was
examined by mixing with absolute ethanol and 1 103 mol/L
(0.18 mL) of DPPH. The obtained samples were properly mixed
using ultrasonication and incubated for 45 min, thereafter the
obtained mixture was then monitored at 515 nm against a blank
using spectrophotometer (UVvis Shimadzu) and absorbance were
recorded when the reaction reached a steady state. All measurements were performed in triplicate. Quercetin (2 mM) was used
as a standard. The radical scavenging activity was calculated using
the formula: % inhibition = (Ac (o) AA (t)/Ac (o) 100 (Ac (o) is the
absorbance of control at t = 0 min and AA (t) is the absorbance of
antioxidant at t = 1 h.) (Nile et al., 2013).
2.7. FRAP assay (reducing power assay)
The Fe3+ reducing power of ginger extract, 6-gingerol, 6-shogaol,
and 6-paradol was determined as described previously with slight
modications. The reaction mixture contained 10 mL of ginger
extract (10 mg) and 6-gingerol, 6-shogaol, and 6-paradol (5 mM
in 0.5% v/v dimethyl sulfoxide) in 3 mL of potassium ferricyanide
solution (1 mM). The obtained mixture then incubated at 50 C in a
water bath for 20 min and after incubation, 0.5 mL of trichloroacetic
acid (TCA) (10%) was added to terminate the reaction. The upper
portion of the solution (1 mL) was mixed with distilled water (1 mL)
and 0.1 mL FeCl3 solution (0.01%). The reaction mixture was incubated at room temperature for 10 min, and the absorbance was
measured using spectrophotometer at 700 nm using blank solution. All tests were performed in triplicate. A higher absorbance of
the reaction mixture indicated greater reducing power. Glutathione
(2 mM) was used as a reference compound (Nile and Park, 2014b).
2.8. OH-radical scavenging assay
This assay is based on the degradation 2-deoxyribose by condensation with thiobarbituric acid (TBA) and quantication of
obtained reaction product using spectrophotometer. In this assay,
the OH-radical was generated using the Fe3+ -ascorbate-EDTA-H2 O2
system (the Fenton reaction). This reaction mixture contained, in a
nal volume 2-deoxy-2-ribose (1 mL, 2.8 mM), KH2 PO4 -KOH buffer
(20 mM, pH 7.4), FeCl3 (100 M), EDTA (100 M), H2 O2 (1.0 mM),
ascorbic acid (100 M). To this each test sample like 0.3 mL of ginger extract (10 mg), 6-gingerol, 6-shogaol, and 6-paradol (2 mM)
solution were added respectively. The obtained reaction with test
samples then incubated for 1 h at 37 C, to which 1 mL 3% TCA,
1 mL of 2% aqueous TBA was added. After addition this reaction
mixture was incubated for 15 min at 90 C for color development.
After cooling, the absorbance was recorded at 532 nm using blank
solution. All tests were performed three times. Glutathione (2 mM)
was used as a positive control. Percent inhibition was calculated by
comparing the test and blank solutions (Nile and Park, 2014b).

ture was incubated for 2 h, at 37 C in a water bath and nally the

reaction product absorbance was recorded at 550 nm. For blank,
xanthine was omitted from samples and allopurinol was used as
a positive control for this assay. All values were expressed as the
means of three experiments. The IC50 values of ginger extract, 6gingerol, 6-shogaol, 6-paradol and allopurinol were obtained from
the inhibitor concentration-activity curve (Nile and Khobragade,
2011; Nile and Park, 2013).
2.10. Anti-inammatory activity
For all anti-inammatory assays, 50 mg of each ginger extract
[distilled water (DWE), 70% ethanol (EE), diethyl ether (DEE), ethyl
acetate (EAE) and n-butanol (NBE)] was used along with 1 mM of
each of 6-gingerol (6G), 6-shogaol (6S), and 6-paradol (6P) as test
samples and salicylic acid (SA), heparin (HE) and quercetin (QR) as
standards.
2.10.1. Diene-conjugate assay
Conjugated diene assays for ginger extract, along with
6-gingerol, 6-shogaol, and 6-paradol were performed using previously described method by Nile and Park (2014a). Salicylic
acid (1 mM) was used as a standard. The percent activity for all
parameters was calculated using the standard formula: % activity = (1 T/C) 100; where, T and C represent the absorbances of
the test and control samples, respectively.
2.10.2. -Glucuronidase inhibition assay
Inhibition of -glucuronidase by ginger extract, 6-gingerol, 6shogaol, and 6-paradol was determined using previous method as
described by Nile and Park (2014a). Salicylic acid was used as a
reference for comparison.
2.10.3. Hyaluronidase activity inhibition
The hyaluronidase inhibitory activities of ginger extract, 6gingerol, 6-shogaol, and 6-paradol were determined by modied
to 96 well microtiter plates method by Piwowarski et al. (2011).
Heparin was used as a positive control.
2.10.4. Lipoxidase activity inhibition
Inhibition of lipoxidase (LOX) by ginger extract, 6-gingerol,
6-shogaol, and 6-paradol was determined using the method
described by Bazylko et al. (2013). Percentage LOX inhibition was
calculated compared to the control. Quercetin was used as a positive control.
2.11. Statistical analysis
The results were expressed as the means SEM of the indicated
number of experiments (n 3). The statistical signicance of differences between means was established by ANOVA with Duncans
post hoc tests. P values <0.05 were considered to indicate statistical
signicance.

2.9. Xanthine oxidase inhibition

3. Results and discussion

Bovine milk XO activity was measured based on formazan formation at 550 nm using a UVvis spectrophotometer at 25 C. The
reaction mixture in the sample wells consisted of xanthine oxidase (500 M nal concentration) in phosphate buffer (0.01 M, pH
8.75, 30 L), NBT (50 L, 100 M, nal concentration), PMS (50 L,
100 M nal concentration), Triton X-100 (20 L, 0.5%). To this
reaction mixture the respective sample like ginger extracts (10 mg)
and other test compounds like 6-gingerol, 6-shogaol, and 6-paradol
(100 L, 10 g/mL) were added to inhibit the xanthine oxidase by
formation of formazan. After addition of test samples the total mix-

3.1. HPTLC and HPLC analysis

High-performance thin-layer chromatography (HPLTC) was
used to identify the compounds responsible for the antioxidant,
anti-inammatory, and xanthine oxidase activities. HPTLC chromatogram for ginger rhizome was developed using a mobile phase
of ethyl acetate: formic acid: water, 85:5:10 (v/v/v), and the
plates were sprayed with anisaldehyde reagent. UV spectra showed
maximum absorbance at approximately 254 nm. The HPTLC densitograms of garlic extract qualitatively revealed of the presence of

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

gingerol (6G), 6-shogaol (6S), and 6-paradol (6P) on HPTLC plates

under CAMAG TLC Scanner. The bands were densitometrically studied using the WIN CATS software (version 4X) at an optimized
wavelength of 254 nm and in the visible range. 6-Gingerol showed
a violet zone at Rf 0.36, 6-paradol showed a dark brown zone
at Rf 0.74, and 6-shogaol showed a violet zone at Rf 0.53. The
ngerprint of the test solution was similar to that of the corresponding botanical reference samples (Fig. 1). Use of these solvent
systems provides good separation of the phytochemicals as presented by Fig. 1 (Amin et al., 2006; Lai et al., 2009). This mobile
phase offers an improvement over that described earlier and facilitates the separation of avonoids, lectins, and saponins (Zou et al.,
2004). In addition, 6-gingerol, 6-shogaol, and 6-paradol was determined by HPLC using external standards for quantication and a
C-18 reverse-phase column with acetonitrile and water (85:15, v/v)
as the mobile phase. Peaks ac showed UV spectral characteristics similar to those of 6-gingerol, 6-shogaol, and 6-paradol; i.e., a
UV absorption maximum wavelength of 230 nm (Figs. 2 and 3). In
HPLC analysis of ginger extracts during quantication of 6-gingerol,
6-shogaol, and 6-paradol, we did not identify any substance that
may interfere with the analysis ginger extracts. The quantication
and peak identications for 6-gingerol, 6-paradol, and 6-shogaol
in ginger extract were based on the retention times of the standards and conrmed by comparing their photodiode array spectra
to those of the individual standards. To the best of our knowledge,
so far there was very limited reports on HPTLC analysis of Zingiber

241

Fig. 1. HPTLC chromatograms of the tested ginger rhizome extracts, lane assignments, 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 analysis

gingerol, shogaol and zerumbone (Alam, 2013; Salmon et al., 2012;
Rout et al., 2009), in our study we qualitatively analyzed presence
all major components in ginger by HPTLC analysis by using different solvent system [acetonitrile and water (85:15, v/v)] rather than

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

Fig. 3. HPLC analysis of ginger extract showing a: 6-gingerol, b: 6-shogaol, and c: 6-paradol.

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

Table 1
Antioxidant activity of tested ginger extracts, sub fractions, and bioactive
components.
Extracts/fractions/compounds

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

Distilled water extract (DWE)

Ethanol extracts (70%) (EE)
Diethyl ether extract (DEE)
Ethyl acetate extract (EAE)
n-Butanol extract (NBE)
6-Gingerol (6G)
6-Shogaol (6S)
6-Paradol (6P)
Quercetin (QR)
Glutathione (GT)

08 0.5
10 0.1
14 0.5
6.8 0.6
17 0.8
8 0.1
12 0.3
10 0.3
5 0.2

FRAP

H2 O2

20 0.8
16 1.1
22 0.6
12 0.2
28 0.4
5 0.4
8 0.6
9 1.4

6 0.1

32 1.1
34 2.4
40 3.2
20 2.5
45 1.8
10 0.4
13 0.5
15 0.8

8 0.5

Values are mean SD (n = 3).

variety of other disorders (Noguchi and Nikki, 2000). Antioxidants

are the molecules which scavenge free radicals are now known to
possess preventive as well as therapeutic potential in free radicalmediated disease conditions (Visioli et al., 2000). The antioxidant
action of ginger has been proposed as one of the major possible
mechanisms for the protective actions against toxicity (Ali et al.,
2008). Recently, it has been shown that 6-gingerol is endowed
with strong antioxidant action both in vivo and in vitro, in addition to strong anti-inammatory and anti-apoptotic actions (Kim
et al., 2007). The data presented in this study revealed that the ethyl
acetate (EAE) and distilled water (DWE) extracts of ginger are with
signicant free radical inhibitors and acted as primary antioxidants
that react with free radicals. The several authors have shown that
ginger is endowed with strong in vitro antioxidant properties and
to this our results are comparable with previous studies (Stoilova
et al., 2007; Ali. et al., 2008; Dugasani et al., 2009).
3.3. Xanthine oxidase inhibition

previously described methods and also the results were conrmed

by HPLC method. Thus, the developed HPTLC and HPLC methods for
quantitation of ginger compounds was found to be simple, accurate,
reproducible and sensitive and might be applicable to the analysis
of other ginger varieties.
3.2. Antioxidant activity
The antioxidant activities of the ginger rhizome fractions,
together with the bioactive components like 6-gingerol, 6-shogaol,
and 6-paradol, were investigated in vitro antioxidant activity
against DPPH, FRAP, and H2 O2 radicals. Ginger rhizome fractions
obtained using different solvents of different polarities (water,
ethanol 70%, diethyl ether, ethyl acetate and n-butanol) were
used; results are presented in Table 1. The ginger extract and
its components like 6-gingerol, 6-shogaol, and 6-paradol showed
antioxidant activity against all examined reactive species in a
concentration-dependent manner. The highest scavenging activity
was found against DPPH as compared to FRAP and H2 O2 radicals in ethyl acetate extract (EAE) (IC50 SEM [g/mL]: 6.8 0.6,
12 0.2 and 20 2.5, respectively). Only the xanthine/xanthine
oxidase system was the antioxidant potential of EAE and DWE
(% inhibition: 76% and 74%, respectively) higher than that of the
other extracts. Depending on the concentration, formazan formation ranged from 55% to 34% for EE, DEE, and NBE (Table 1).
Therefore, DWE showed greater DPPH- and ferric-ion-scavenging
activity, while EAE exhibited greater inhibition of xanthine oxidase
activity. There is considerable evidence that free radicals induce
oxidative damage to biomolecules and play an important role in
cardiovascular disease, aging, cancer, inammatory diseases and a

All extracts from ginger rhizome and their bioactive compounds

showed good to excellent inhibition of xanthine oxidase activity.
EAE and WE exhibited the highest XO inhibitory activity of the
extracts. Regarding the bioactive compounds, 6-gingerol (6G) had
greater XO inhibitory activity than 6-shogaol and 6-paradol. The
IC50 values of 6-gingerol, 6-shogaol, 6-paradol and allopurinol
were 10.5 0.5, 15.2 0.3, 12.4 0.6, and 8.4 0.4 M, respectively (Fig. 4). To the best of our knowledge, so far there was no
any study or report on activity of Zingiber ofcinale extract against
xanthine oxidase but only few scientists studied the xanthine
oxidase inhibition using plant, vegetable and spice extracts (Nile
and Khobragade, 2011; Nile and Park, 2013). There is signicant
interest in the direct antioxidant activities of dietary polyphenols,
due to associations between consumption of polyphenol-rich
foods, such as fruits and vegetables, and decreased incidence of
oxidative-stress related disease. However, indirect antioxidant
action, such as the inhibition of ROS-producing enzymes, may be
equally relevant to health benets through a general reduction
in oxidative stress (Dew et al., 2005; Shan et al., 2005). Recent
ndings revealed that the incidence of gout and hyperuricemia
is increasing worldwide drastically and the possible reason may
change in unusual habits of food, smoking and drinking also,
intake of foods which rich in nucleic acids, including meat, pork,
and seafood. Hypouricemic agents, including xanthine oxidase
inhibitors and uricosuric agents, are commonly used for the
treatment of chronic gouty arthritis. In general, allopurinol is the
drug of choice; however, it has serious side effects. Thus, novel
alternatives with increased therapeutic activity and fewer side
effects are desired (Nile et al., 2013; Nile and Park, 2014b). Thus,

Fig. 4. Xanthine oxidase inhibition by ginger extracts and its bioactive compounds (100 M). The data are expressed as the means SD, n = 3. The IC50 values of compound
6-gingerol, 6-shogaol, 6-paradol and allopurinol are 10.5 0.5 M, 15.2 0.3 M, 12.4 0.6 M, and 8.4 0.4 M, respectively (short forms explained in Tables 1 and 2).

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

243

Table 2
Anti-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 water extract (DWE)

Ethanol extracts (70%) (EE)
Diethyl ether extract (DEE)
Ethyl acetate extract (EAE)
n-Butanol extract (NBE)
6-Gingerol (6G)
6-Shogaol (6S)
6-Paradol (6P)
Salicylic acid (SA)
Heparin (HE)
Quercetin (QR)

64 2.4
46 1.8
38 1.5
78 3.6
30 4.1
85 3.1
70 1.6
63 1.3
82 2.1

32 0.9
28 0.5
22 0.4
37 0.1
17 0.5
48 0.3
40 0.7
35 0.4
50 1.4

35 1.0
28 0.3
23 0.5
46 0.7
20 0.3
56 0.3
51 0.4
48 0.6

55 1.6

70 1.3
60 0.4
43 0.3
80 0.7
38 0.3
87 1.6
75 1.2
72 1.4

80 2.1

Values are mean SD (n = 3).

we attempted to identify phytochemicals as xanthine oxidase

inhibitors from various foods and food products, including ginger.
3.4. Anti-inammatory activity
Anti-inammatory activity for ginger extracts and active constituents was determined using diene-conjugate, -glucuronidase,
lipoxidase, and hyaluronidase in vitro inhibition assay (Table 2).
This study revealed signicant anti-inammatory activity of the
tested extracts and active constituents. EAE, WE, and 6-gingerol
exhibited the greatest anti-inammatory activity of all samples
tested (Table 2). At 50 mg, EAE and WE inhibited all enzymes
more strongly than did the remaining samples (78 and 64%
for -glucuronidase, 37 and 32% for diene-conjugate, 46 and
35% for hyaluronidase, and 80 and 70% for lipoxidase, respectively). At 1 mM, 6-gingerol (6G) exhibited greater inhibition of
the activity of all enzymes compared to both the other bioactive compounds 6-shogaol (6S), 6-paradol (6P); and salicylic acid
(SA), heparin (HE), and quercetin (QR) (Table 2). Several lines of
evidence have been provided, mostly in different animal models of inammation, and to a much lesser extent in humans or
human cells, of the effectiveness of either ginger or of compounds is
olated there from against inammation and its mediators (Grzanna
et al., 2005; Lantz et al., 2007). Here, in this study, we studied the
anti-inammatory properties of ginger by using in vitro assay viz.;
diene-conjugate, -glucuronidase, lipoxidase, and hyaluronidase
inhibition. 6-Gingerol, 8-gingerol, 10-gingerol, 6-shogaol, and 6paradol are the important and major compounds found in ginger
(Shukla and Singh, 2007), which are identied with various
pharmacological and biological properties such as antioxidant,
anti-inammatory, anticancer, and antiulcer activities. Here, in
this study, we did comparative analysis of ginger extracts and its
compounds for the antioxidant, anti-inammatory, and xanthine
oxidase inhibitory activity. During the metabolism of purine xanthine oxidase convert xanthine to uric acid and excess production
and deposition of uric acid leads to gout, so here xanthine oxidase
acts as a source of oxygen free radicals (Nile and Park, 2013; Nile and
Park, 2014b). In this study, we demonstrated that ginger rhizome
extract and 6-gingerol, 6-shogaol and 6-paradol inhibited superoxide production and exhibited the strongest inhibition against
xanthine oxidase. Based on a previous report (Dugasani et al., 2009)
that phenolic compounds inhibit xanthine oxidase and/or scavenge
superoxide, the inhibition of superoxide production by gingerols
and shogoal may be due to both superoxide scavenging and inhibition of xanthine oxidase activity. The identication of natural
compounds for the prevention of ROS-associated disorders is an
important strategy for antioxidant therapy. Suppression of ROS
production by gingerols and shogaol may be attributable to various
pharmacological actions of ginger (Badreldin et al., 2008).

4. Conclusions
Our results suggest that ginger ethyl acetate and water
extracts along with active constituents such as 6-gingerol (6G),
6-shogaol (6S), and 6-paradol (6P) have strong antioxidant, antiinammatory, and xanthine oxidase inhibitory activities. However,
further in vivo and clinical studies are required for their characterization as an agent for use against various diseases and disorders
associated with oxidative stress, xanthine oxidase, and free radicals. This study provides a scientic support for some of the
medicinal uses; hence suggesting that increasing the intake of ginger extract and utilization of their active compounds; 6-gingerol
(6G), 6-shogaol (6S), and 6-paradol (6P) may be helpful in preventing or reducing the progress of some lifestyle related diseases.
Although it is not simple to extrapolate from in vitro results, the
diversity of the pharmacological activities of the ginger observed
in this study suggests that extracts of this ginger species may be of
value for application in human and animal health. Furthermore,
the providing evidence that the ginger extract and its bioactive
compounds might be potential sources of new antioxidant, antiinammatory and anti-gout drugs.
Acknowledgment
This research paper was supported by KU-Research Professor
Program-2014, Konkuk University, Seoul, Republic of Korea.
References
Alam, P., 2013. Densitometric HPTLC analysis of 8-gingerol in Zingiber ofcinale
extract and ginger-containing dietary supplements, teas and commercial
creams. Asian Pac. J. Trop. Biomed. 3, 634638.
Ali, B.H., Blunden, G., Tanira, M.O., Nemmar, A., 2008. Some phytochemical,
pharmacological and toxicological properties of ginger (Zingiber ofcinale
Roscoe): a review of recent research. Food Chem. Toxicol. 46 (2), 409420.
Amin, I., Norazaidah, Y., Hainida, K.I.M., 2006. Antioxidant activity and phenolic
content of raw and blanched Amaranthus species. Food Chem. 94, 4752.
Badreldin, H.A., Gerald, B., Musbah, O.T., Abderrahim, N., 2008. Some
phytochemical: pharmacological and toxicological properties of ginger
(Zingiber ofcinale Roscoe): a review of recent research. Food Chem. Toxicol.
46, 409420.
Bartley, J., Jacobs, A., 2000. Effects of drying on avour compounds in
Australian-grown ginger (Zingiber ofcinale). J. Sci. Food Agric. 80, 209215.
Bazylko, A., Piwowarski, J.P., Filipek, A., Bonarewicz, J., Tomczyk, M., 2013. In vitro
antioxidant and anti-inammatory activities of extracts from Potentilla recta
and its main ellagitannin, agrimoniin. J. Ethnopharmacol. 149, 222227.
Chaiyakunapruk, N., Kitikannakorn, N., Nathisuwan, S., Leeprakobboon, K.,
Leelasettagool, C., 2006. The efcacy of ginger for the prevention of
postoperative nausea and vomiting: a meta-analysis. Am. J. Obstet. Gynecol.
194, 9599.
Chrubasik, S., Pittler, M.H., Roufogalis, B.D., 2005. Zingiberis rhizoma: a
comprehensive review on the ginger effect and efcacy proles. Phytomed 12,
684701.
Dew, T.P., Day, A.J., Morgan, M.R., 2005. Xanthine oxidase activity in vitro: effects
of food extracts and components. J. Agric. Food Chem. 53, 65106515.

244

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

Dugasani, S., Pichika, M.R., Nadarajah, V.D., Balijepalli, M.K., Tandra, S.,
Korlakuntab, J.N., 2009. Comparative antioxidant and anti-inammatory
effects of [6]-gingerol,[8]-gingerol, [10]-gingerol and [6]-shogaol. J.
Ethnopharmacol. 127, 515520.
Grzanna, R., Lindmark, L., Frondoza, C.G., 2005. Ginger: an herbal medicinal
product with broad anti-inammatory actions. J. Med. Food 8, 125132.

Ibanez,
E., Kubtov, A., Senorans,
F.J., Cavero, S., Reglero, G., Hawthorne, S.B., 2003.
Subcritical water extraction of antioxidant compounds from rosemary plants.
J. Agric. Food Chem. 51, 375382.
Janick, J., 2012. Horticultural Reviews, 39. Wiley-Blackwell, pp. 480.
Jolad, S.D., Lantz, R.C., Chen, G.J., Bates, R.B., Timmermann, B.N., 2005.
Commercially processed dry ginger (Zingiber ofcinale): composition and
effects on LPS-stimulated PGE2 production. Phytochemistry 66, 16141635.
Kim, J.K., Kim, Y., Na, K.M., Surh, Y.J., Kim, T.Y., 2007. [6]-Gingerol prevents
UVB-induced ROS production and COX-2 expression in vitro and in vivo. Free
Radical Res. 41, 603614.
Lai, S.C., Peng, W.H., Huang, S.C., Ho, Y.L., Huang, T.H., Lai, Z.R., Chang, Y.S., 2009.
Analgesic and anti-inammatory activities of methanol extract from
Desmodium triorum DC in mice. Am. J. Clin. Med. 37, 573588.
Lantz, R.C., Chen, G.J., Sarihan, M., Solyom, A.M., Jolad, S.D., Timmermann, B.N.,
2007. The effect of extracts from ginger rhizome on inammatory mediator
production. Phytomedicine 14, 123128.
Motterlini, R., Foresti, R., Bassi, R., Green, C.J., 2000. Curcumin, an antioxidant and
anti-inammatory agent: induces heme oxygenase-1 and protects endothelial
cells against oxidative stress. Free Radical. Biol. Med. 28, 13031312.
Nile, S.H., Khobragade, C.N., 2011. In vitro anti-inammatory and xanthine oxidase
inhibitory activity of Tephrosia purpurea shoot extract. Nat. Prod. Commun. 6,
14371440.
Nile, S.H., Kumar, B., Park, S.W., 2013. In vitro evaluation of selected benzimidazole
derivatives as an antioxidant and xanthine oxidase inhibitors. Chem. Biol. Drug
Des. 82, 290295.
Nile, S.H., Park, S.W., 2013. Total phenolics: antioxidant and xanthine oxidase
inhibitory activity of three colored onions (Allium cepa L.). Front. Life Sci. 7,
224228.
Nile, S.H., Park, S.W., 2014a. HPTLC analysis antioxidant, anti-inammatory and
antiproliferative activities of Arisaema tortuosum tuber extract. Pharma Biol.
52, 221227.
Nile, S.H., Park, S.W., 2014b. Antioxidant: -glucosidase and xanthine oxidase
inhibitory activity of bioactive compounds from maize (Zea mays L.). Chem.
Biol. Drug Des. 83, 119125.
Noguchi, C., Nikki, E., 2000. Phenolic antioxidants: a rationale for design and
evaluation of novel antioxidant drugs for atherosclerosis. Free Radical Biol.
Med. 28, 15381546.
Piwowarski, J.P., Kiss, A.K., Kozowska-Wojciechowska, M., 2011.
Anti-hyaluronidase, anti-elastase activity screening of tannin-rich plant

materials used in traditional Polish medicine for external treatment of diseases

with inammatory background. J. Ethnopharmacol. 137, 937941.
Rai, S., Mukherjee, K., Mal, M., Wahile, A., Saha, B.P., Mukherjee, P.K., 2006.
Determination of 6-gingerol in ginger (Zingiber ofcinale) using
high-performance thin-layer chromatography. J. Sep. Sci. 29, 22922295.
Rout, K.K., Mishra, S.K., Sherma, J., 2009. Development and validation of an HPTLC
method for analysis of Zerumbone, the anticancer marker from Zingiber
zerumbe. Acta Chromatogr. 3, 443452.
Salmon, C.N.A., Bailey-Shaw, Y.A., Hibbert, S., Green, C., Smith, A.M., Williams,
L.A.D., 2012. Characterisation of cultivars of Jamaican ginger (Zingiber ofcinale
Roscoe) by HPTLC and HPLC. Food Chem. 131, 15171522.
Schwertner, H.A., Deborah, C., Rios, D.C., 2007. High-performance liquid
chromatographic analysis of 6-gingerol, 8-gingerol, 10-gingerol, and 6-shogaol
in ginger-containing dietary supplements, spices, teas, and beverages. J.
Chromatogr. B 856, 4147.
Shan, B., Cai, Y.Z., Sun, M., Corke, H., 2005. Antioxidant capacity of 26 spice extracts
and characterization of their phenolic constituents. J. Agric. Food Chem. 53,
77497759.
Shukla, Y., Singh, M., 2007. Cancer preventive properties of ginger: a brief review.
Food Chem. Toxicol. 45, 683690.
Stoilova, I., Krastanov, A., Stoyanova, A., Denev, P., Gargova, S., 2007. Antioxidant
activity of a ginger extracts (Zingiber ofcinale). Food Chem. 102, 764770.
Tapsell, L.C., Hemphill, I., Cobiac, L., Patch, C.S., Sullivan, D.R., Fenech, M.,
Roodenrys, S., Keogh, J.B., Clifton, P.M., Williams, P.G., Fazio, V.A., Inge, K.E.,
2006. Health benets of herbs and spices: the past the present, the future.
Med. J. Aust. 185, S4S24.

Tomczyk, M., Wiater, A., Pleszczynska,

M., 2011. In vitro anticariogenic effects of
aerial parts of Potentilla recta and its phytochemical prole. Phytother. Res. 25,
343350.
Visioli, F., Borsani, L., Galli, C., 2000. Diet and prevention of coronary heart disease:
the potential role of phytochemicals. Cardiovasc. Res. 47, 419425.
Wang, W., Li, C.Y., Wen, X.D., Li, P., Qi, L.W., 2009. Simultaneous determination of
6-gingerol, 8-gingerol, 10-gingerol and 6-shogaol in rat plasma by liquid
chromatography-mass spectrometry: application to pharmacokinetics. J.
Chromatogr. B. 877, 671679.
Wang, W.H., Wang, Z.M., 2005. Studies of commonly used traditional
medicine-ginger. Zhongguo Zhong Yao Za Zhi 30, 15691573.
Wohlmuth, H., Leach, D.N., Smith, M.K., Myers, S.P., 2005. Gingerol content of
diploid and tetraploid clones of ginger (Zingiber ofcinale Roscoe). J. Agric. Food
Chem. 53, 57725778.
Yeh, H.Y., Chuang, C.H., Chen, H.C., Wan, C.J., Chen, T.L., Lin, L.Y., 2014. Bioactive
components analysis of two various gingers (Zingiber ofcinale Roscoe) and
antioxidant effect of ginger extracts. LWT- Food Sci. Technol. 55, 329334.
Zou, Y., Lu, Y., Wei, D., 2004. Antioxidant activity of avonoid rich extract of
Hypericum perforatum L. in vitro. J. Agric. Food Chem. 52, 50325039.