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Inhibitory potential of ginger extracts against enzymes linked to type 2 diabetes, inflammation and induced oxidative stress
International Journal of Food Sciences and Nutrition,March 2011; 62(2): 106–110
Inhibitory potential of ginger extracts against enzymes linkedto type 2 diabetes, inflammation and induced oxidative stress
M. PRIYA RANI, K. P. PADMAKUMARI, B. SANKARIKUTTY, O. LIJO CHERIAN, V. M. NISHA &K. G. RAGHU
Agroprocessing & Natural Products Division, National Institute for Interdisciplinary Science and Technology, CSIR, Trivandrum,Kerala, India
AbstractGinger (Zingiber officinale Roscoe) continues to be used as an important cooking spice and herbal medicine around the world. Gingerols, the major pungent components of ginger, are known to improve diabetes, including the effect of enhancement againstinsulin sensitivity. In the current study, ginger sequentially extracted with different solvents—namely, hexane, ethyl acetate,methanol, 70% methanol – water and water—were screened to determine the variations in phenolic-linked active constituents. The potential of these extracts to inhibit key enzymes relevant to type 2 diabetes and inflammation was studied. Phenoliccompounds—namely, gingerols and shoagols—were quantified using high-performance liquid chromatography. Ethyl acetateextract showed higher activity compared with other extracts. These studies indicate that ginger has very good potential fora-glucosidase and a-amylase inhibition relevant for type 2 diabetes management and cyclooxygenase inhibition for inflammation.
Keywords: a-Glucosidase, a-amylase, cyclooxygenase, reactive oxygen species, flow cytometer, C2C12
Ginger, the rhizome of Zingiber officinale Roscoe
anti-inflammatory activity, anti-fungal property, be a
(Zingiberaceae), is a perennial herbaceous plant native
most potent cyclooxygenase (COX) inhibitor and are
to Southern Asia. It is the underground stem or
anti-platelet agents (Nurtjahja et al. 2003), and also
rhizome of the plant and continues to be used as an
lower blood pressure and reduce blood clotting
important cooking spice and is valued for its pungency.
(Thomson et al. 2002, Ghayur and Gilani 2005).
It has been widely used in traditional system of
Reactive oxygen species (ROS) are typically generated
medicines all over the world, for a wide array of
as the byproduct of cellular metabolic processes and
unrelated ailments that include arthritis, rheumatism,
are carefully controlled by cellular antioxidants or
sprains, muscular aches, pains, sore throats, cramps,
scavengers. Oxidative stress is considered the main
constipation, indigestion, vomiting, hypertension,
cause for several chronic diseases, including diabetes
dementia, fever and helminthiasis (Badreldin et al.
(Wei et al. 2009). Oxidative stress occurs in the cell
2008). Ginger represents a rich source of biologically
when the generation of ROS overwhelms the cells’
active constituents. It is a strong antioxidant substanceand may either mitigate or prevent generation of free
Int J Food Sci Nutr Downloaded from informahealthcare.com by Cape Peninsula University of Technology on 08/22/11
radicals (Haksar et al. 2006, Kim et al. 2007). It is
The present study involves the inhibitory effect
considered a safe herbal medicine with only few and
of sequentially extracted Indian ginger on a-amylase,
insignificant adverse/side effects. Several reviews have
a-glucosidase and cyclooxygenase for anti-diabetic
appeared in the literature about this plant as a spice
and anti-inflammatory properties and its reaction of
and a medicinal plant (Afzal et al. 2001, Chrubasik
ROS. High-performance liquid chromatography
et al. 2005). The pungent constituents of ginger,
(HPLC) quantification of the active constituents
gingerols, have been reported to possess strong
present in the extracts was also carried out.
Correspondence: K. P. Padmakumari, Agroprocessing & Natural Products Division, National Institute for Interdisciplinary Science andTechnology (NIIST), CSIR, Industrial Estate P O, Trivandrum 695019, Kerala, India. Tel: 91 471 2515347. Fax: 91 471 495050. E-mail: kppad@yahoo.co.in
ISSN 0963-7486 print/ISSN 1465-3478 online q 2011 Informa UK, Ltd. DOI: 10.3109/09637486.2010.515565
a-Glucosidase inhibition assay. a-Glucosidase inhibitionwas assayed using different concentrations of sample
Fresh ginger rhizomes (5 kg) procured from a local
stock solution (100 – 250 mg/ml), 100 ml of 0.1 M
market at Thiruvananthapuram, Kerala, India were
phosphate buffer (pH 6.9) containing a-glucosidase
used. Voucher specimens have been kept in the
solution (1.0 U/ml), and was incubated in 96-well
Herbarium of the Institute. The rhizomes were
plates at 258C for 10 min. After pre-incubation, 50 ml
chipped and dried at 508C in a drier for 8 h to a 10%
of 5 mM p-nitrophenyl-a-D-glucopyranoside solution
moisture level and were powdered for further studies.
in 0.1 M phosphate buffer (pH 6.9) was added to eachwell at timed intervals. The reaction mixtures were
incubated at 258C for 5 min. Before and afterincubation, absorbance readings were recorded at
a-Amylase from Aspergillus oryzae, a-glucosidase type
405 nm by a Synergy 4 Biotek multiplate reader (Biotek
1 from baker’s yeast, N,N,N 0,N 0-tetramethyl-p-phe-
Instruments Inc., Highland Park, PO Box 998,
nylenediamine (TMPD), Hematin, Tris – HCl, dini-
Winooski, Vermont-0504-0998, USA) and compared
tro-salicylic acid, p-nitrophenyl-a-D-glucopyranoside,
with a control that had an adequate amount of buffer
arachidonic acid, 20,70-dichlorodihydrofluorescein-
solution in place of the extract (Apostolidis et al. 2007).
diacetate (DCFH-DA), indomethacin and N-vanillyl-
Acarbose was used as the standard. The a-glucosidase
nonanamide were purchased from Sigma-Aldrich
inhibitory activity was expressed as the inhibition
(St Louis, MO, USA). The COX inhibitor assay
percentage and was calculated as follows:
screening kit was obtained from Cayman (Ann Arbor,MI, USA). Acarbose was obtained from ServaElectrophoresis GmbH (Mannheim, Germany). % inhibition ¼ ðAcontrol 2 AsampleÞ=Acontrol £ 100 ð1Þ
Sodium chloride, hydrogen peroxide, anhydroussodium phosphate monobasic and anhydrous sodium
where Acontrol is the absorbance of control without
phosphate dibasic were bought from Sisco Research
sample and Asample is the absorbance of the sample.
Laboratories Pvt Ltd (Mumbai, India).
The concentration of the extract having 50% inhibition(IC50) was calculated from the concentration-inhibition response curve.
C2C12 cells purchased from the National Centre for
a-Amylase inhibition assay. The a-amylase inhibition
Cell Science (Pune, India) were cultured in Dulbec-
assay was carried out by the method of Apostolidis
co’s modified Eagle’s medium supplemented with
et al. (2007). Briefly, 500 ml of 0.02 M sodium
10% fetal calf serum, 100 U/ml penicillin and
phosphate buffer (pH 6.9 with 0.006 M sodium
100 mg/ml streptomycin. Cultures were maintained at
chloride) containing a-amylase solution (0.5 mg/ml)
378C in a 5% carbon dioxide incubator. When the cells
and different concentrations of the stock solution of
were about to cover 80% of the flask area, they were
extracts (500 – 1,250 mg/ml) were incubated at 258C
disrupted and seeded on 24-well plates. After attaining
for 10 min. After pre-incubation, 500 ml of 1% starch
, 70 – 80% confluency, the cells were rinsed twice with
solution in 0.02 M sodium phosphate buffer (pH 6.9
phosphate-buffered saline (PBS) and changed with
with 0.006 M sodium chloride) was added to each
medium containing extracts at different concen-
tube at timed intervals. The reaction mixtures were
trations. After 24 h incubation, the cells were washed
then incubated at 258C for 10 min. The reaction was
twice with PBS and 50 mM H2O2 was maintained in
stopped with 1.0 ml dinitro-salicylic acid color
individual wells for 1 h at 378C. These cells were
reagent. The test tubes were then incubated in a
detached by trypsin to assay by flow cytometry.
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boiling-water bath for 5 min and cooled to roomtemperature. The reaction mixture was then diluted
after adding 10 ml distilled water and absorbance wasmeasured at 540 nm using a Synergy 4 Biotek
Dried ginger powder (500 g having moisture content
multiplate reader. Acarbose was used as the positive
10%) was successively extracted with 1 litre of each of
control. The percentage inhibition was calculated
the solvents at room temperature (278C) with hexane
using Equation (1). A graph was plotted with
(yield: 3.87%), ethyl acetate (yield: 1.93%), methanol
concentration along the x axis and percentage
(yield: 3.75%), 70% methanol – water (yield: 4.82%)
inhibition along the y axis to obtain the IC
and water (yield: 5.68%). The solvent was evaporated
using a rotavapor under reduced pressure. Sample
stock solution (5 mg/ml) was prepared for all theextracts in methanol and was used for diabetic studies.
In vitro evaluation of COX inhibitory activity. Enzymatic
Different concentrations of the successive extracts (38,
activity of COX was measured according to the method
95 and 190 mg/ml) were used for inflammatory studies.
of Copeland et al. (1994) with slight modifications using
a chromogenic assay based on the oxidation of TMPD
during the reduction of PGG2 to PGH2. Briefly, various
In the present study, potential anti-diabetic effect
concentrations of the sample solution contained
of ginger extracts obtained by sequential extraction
(30 – 200 mg/ml) Tris – HCl buffer (100 mM, pH 8.0),
of dried ginger powder with different solvents was
Hematin (15 mM), ethylenediamine tetraacetic acid(3 mM), enzyme (100 mg COX). The mixture was pre-
investigated. The ability of ginger extracts to inhibit
incubated at 258C for 15 min and then the reaction was
a-glucosidase and a-amylase was measured using four
initiated by the addition of arachidonic acid and
different dosages. a-Glucosidase inhibitory activity
TMPD, in total volume of 1 ml. The enzymatic
was measured using concentrations of 100, 150, 200
activity was determined by estimating the rate of
and 250 mg/ml. The ethyl acetate extract of ginger
TMPD oxidation for the first 25 sec of the reaction by
showed the highest activity (Figure 1) among the
following the increase in absorbance at 603 nm.
extracts and expressed in terms of IC50 value. A lower
Indomethacin was used as the standard. A low rate of
IC50 value indicates higher inhibition. The IC50 value
non-enzymatic oxidation observed in the absence of
of standard acarbose was 36 mg/ml and that of ethyl
COX was subtracted from the experimental value while
acetate extract was 180.13 mg/ml. The inhibitory
calculating the percentage inhibition (see Equation (1)).
potential of a-glucosidase on ginger extracts was
The IC50 value was calculated from the concentration-
related to its phenolic content, gingerol and shoagol.
The results of HPLC quantification of differentextracts presented in Table I indicate that ethyl acetate
Evaluation of oxidative stress inhibition
extract contains a higher amount of active compounds,gingerol and shoagol.
Cytoprotective effect against the oxidative stress
a-Amylase inhibitory activity was measured at
induced by H2O2 was measured by determining the
various dosages (500, 750, 1,000 and 1,250 mg/ml).
intracellular content of ROS. Intracellular ROS levels
As in the case of a-glucosidase inhibitory activity,
were measured employing DCFH-DA. DCFH-DA is
cleaved intracellularly by non-specific esterase and
a-amylase inhibitory activity (Figure 2) compared
turn to high-fluorescent 2,7-dichlorofluorescein upon
oxidation by ROS, which were analyzed with FACS
Other extracts would not respond to the tests. Earlier
Aria II (BD Bioscience, San Jose, CA, USA). C2C12cells pretreated with ethyl acetate extract of ginger
reports show that phenolics play a role in mediating
were incubated with DCFH-DA at 378C for 1 h and
amylase inhibition and therefore have potential to
contribute to the management of type 2 diabetes
(McCue and Shetty 2004). Ginger extract gavemild a-amylase inhibitory activity compared with
Quantification of gingerols and shoagols in ginger extracts
a-glucosidase. Previous reports had also indicated that
Sample preparation. A sample of 5 mg/ml stock solution
excessive inhibition of a-amylase could result in the
of ginger extracts was prepared in methanol.
abnormal bacterial fermentation of undigested carbo-
N-Vanillylnonanamide dissolved in methanol was
hydrates in the colon, and therefore mild a-amylase
used as the standard at a concentration of 0.2 mg/ml.
inhibition activity is useful (Horii et al. 1986).
Chromatographic conditions. The analytical HPLC was
performed on a Waters liquid chromatographequipped with a Rheodyne injector and a Waters
2487 (M/s. Waters Ges.m.b.H, Hietzinger Haupstasse
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145, A 1130, Vienna, Austria, Europe) UV detector
(150 £ 4.6 mm, 5.0 mm). The mobile phase consistedof solvent A 1% acetic acid in water and solvent B
acetonitrile (40:60). The flow rate was 1.5 ml/min.
The experimental results are expressed as the mean ^standard deviation of three parallel measurements.
20 40 60 80 100 120 140 160 180 200 220 240 260
The results were subjected to one way analysis of
variance and the significance of differences betweensample means was calculated. P # 0.05 was considered
a-Glucosidase inhibition of ethyl acetate extract of ginger
HPLC quantification of gingerols and shoagols in successive extracts.
Anti-inflammatory effects of the extracts were
the conversion of arachidonic acid into prostaglandins,
evaluated by percentage inhibition of three different
which play a significant role in health, and in disease
dosages (38, 95 and 190 mg/ml) of extracts. Of these,
in the gastrointestinal tract and in the renal, skeletal
ethyl acetate extract showed the highest activity, with
and ocular systems (Raju et al. 2010). The effective-
an IC50 value of 145.04 mg/ml compared with
ness of the ginger extract used in folk medicine
to suppress inflammatory responses may thus be due
COX, as shown in Figure 3. COX was the key marker
to their capacity to reduce oxidative stress and to their
enzyme for the diseases with impaired arachidonic
acid metabolism. Non-steroidal anti-inflammatory
The biological significance of increased formation of
drugs bind to COX and inhibit the production of
ROS may occur in diabetes for reasons possibly related
prostaglandins from arachidonic acid. COX catalyzes
to an increase in glucose level concentration in plasmaand tissue. Interestingly, accumulation of intracellularROS in H2O2-treated cells were decreased when cells
were pretreated with ethyl acetate extract of gingerat 10, 20, 40, 80 and 100 mg/ml (P , 0.05) in a dose-
dependent manner, as shown in Figure 4 (average meanfluorescence from triplicates is expressed per each
group of cells). This could be explained as gingerinhibits intracellular oxidative stress and protects
C2C12 cells from oxidative damage. No evidence ofany increase or decrease in 2,7-dichlorofluorescein
fluorescence was observed in cells incubated withextracts alone. Many experimental and clinical obser-
vations indicate oxidative stress to be an important
a-Amylase inhibition of ethyl acetate extract of ginger
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Evaluation of oxidative stress in C2C12 cell lines by flow
cytometry. A, blank cells without any treatment; B, control cells
treated with hydrogen peroxide; C, cells treated with hydrogenperoxide and ascorbic acid (25 mg/ml); D, cells treated with
hydrogen peroxide þ ethyl acetate extract of ginger (10 mg/ml); E,
cells treated with hydrogen peroxide þ ethyl acetate extract of ginger
(20 mg/ml); F, cells treated with hydrogen peroxide þ ethyl acetateextract of ginger (40 mg/ml); G, cells treated with hydrogen peroxide
COX inhibition of ethyl acetate extract of ginger with
þ ethyl acetate extract of ginger (80 mg/ml); H, cells treated with
hydrogen peroxide þ ethyl acetate extract of ginger (100 mg/ml).
mechanism in obesity-associated metabolic syndrome,
linked to type 2 diabetes and hypertension. Innov Food Sci
in development of diabetes and its complications, heart
disease and many satellite conditions.
Badreldin HA, Gerald B, Musbah OT, Abderrahim N. 2008.
Some phytochemical, pharmacological and toxicological proper-
The goal of the present study was to provide in vitro
ties of ginger (Zingiber Officinale Roscoe): A review of recent
evidence for potential a-glucosidase, a-amylase and
research. Food Chem Toxicol 46:409 – 420.
COX inhibitors to generate stronger biochemical
Chrubasik S, Pittler MH, Roufogalis BD. 2005. Zingiberis rhizoma:
rationale for further clinical studies. Among the five
A comprehensive review on the ginger effect and efficacy profiles.
extracts, ethyl acetate extract showed the highest activity.
The content of gingerol and shoagol, the pungent
Copeland RA, Williams JM, Giannaras J, Nurnberg S, Covington M,
Pinto D, Pick S, Tizaskos JM. 1994. Mechanism of selective
compounds in ginger, was determined by HPLC in the
inhibition of the inducible isoform of prostaglandin G/H
extracts (Table I). Owing to the insoluble nature of 70%
synthase. Proc Natl Acad Sci USA 91:11202 – 11206.
methanol – water and water extracts, gingerol quantifi-
Ghayur MN, Gilani AH. 2005. Ginger lowers blood pressure
cation was not done in these extracts. Even though the
through blockade of voltage-dependent calcium channels.
total pungency was almost comparable in ethyl acetate
J Cardiovasc Pharmacol 45:74 – 80.
Haksar A, Sharma A, Chawla R, Kumar R, Arora R, Singh S,
and hexane extracts, the reduced activity observed in
Prasad J, Gupta M, Tripathi RP, Arora MP, Islam F, Sharma RK.
hexane extract may be due to its dilution with other
2006. Zingiber officinale exhibits behavioral radioprotection
inactive or retarding constituents as is shown by its
against radiation-induced CTA in a gender-specific manner.
higher yield. Thus the anti-diabetic and anti-inflamma-
Pharmacol Biochem Behav 84:179 – 188.
tory activity of ginger revealed in the present study can be
Horii S, Fukase H, Matsuo T, Kameda Y, Asano N, Matsui K. 1986.
utilized to develop a suitable herbal formulation.
Synthesis and alpha-D glucosidase inhibitory activity ofN-substituted valiolamine derivatives as potential oral antidia-betic agents. J Med Chem 29:1038 – 1046.
Kim JK, Kim Y, Na KM, Surh YJ, Kim TY. 2007. [6]-Gingerol
The authors are thankful to the Director, National
prevents UVB-induced ROS production and COX-2 expressionin vitro and in vivo. Free Radic Res 41:603 – 614.
Institute for Interdisciplinary Science and Technology
McCue P, Shetty K. 2004. Inhibitory effects of rosmarinic acid
(NIIST), CSIR for providing all the facilities for
extracts on porcinepancreatic amylase in vitro. Asia Pacific J Clin
Nurtjahja TE, Ammit AJ, Roufogalis BD, Tran VH, Duke CC.
Declaration of interest: The authors report no
2003. Effective anti-platelet and COX-1 enzyme inhibitors from
conflicts of interest. The authors alone are responsible
pungent constituents of ginger. Thromb Res 111:259 – 265.
for the content and writing of the paper.
Raju G, Amit S, Sanjay MJ, Aravind S. 2010. Anti-inflammatory,
cyclooxygenase (COX)-2, COX-1 inhibitory and antioxidanteffects of Dysophylla stellata Benth. Fitoterapia 81:45 – 49.
Thomson M, Al-Qattan KK, Al-Sawan SM, Alnaqeeb MA, Khan I,
Ali M. 2002. The use of ginger (Zingiber officinale Rosc.) as a
Afzal M, Al-Hadidi D, Menon M, Pesek J, Dhami MS. 2001.
potential anti-inflammatory and antithrombotic agent. Prosta-
Ginger: An ethnomedical, chemical and pharmacological review.
glandins Leukot Essent Fatty Acids 67:475 – 478.
Drug Metabol Drug Interact 18:159 – 190.
Wei W, Qiuju L, Yi T, Lucheng L, Xiaokun L, Lu C. 2009. Oxidative
Apostolidis E, Kwon YI, Shetty K. 2007. Inhibitory potential of
stress, diabetes, and diabetic complications. Hemoglobin 33:
herb, fruit, and fungal-enriched cheese against key enzymes
Int J Food Sci Nutr Downloaded from informahealthcare.com by Cape Peninsula University of Technology on 08/22/11