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: [email protected] 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.
Int J Food Sci Nutr Downloaded from informahealthcare.com by Cape Peninsula University of Technology on 08/22/11 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 Int J Food Sci Nutr Downloaded from informahealthcare.com by Cape Peninsula University of Technology on 08/22/11 145, 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 Int J Food Sci Nutr Downloaded from informahealthcare.com by Cape Peninsula University of Technology on 08/22/11 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).
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