POLYPHENOL lá và CỌNG r SATIVUS

Plant Foods Hum Nutr (2010) 65:8–17 DOI 10.1007/s11130-009-0148-6 ORIGINAL PAPER Polyphenolics Profile, Antioxidant and Radical Scavenging Activity of Leaves and Stem of Raphanus sativus L Syed Sultan Beevi & Mangamoori Lakshmi Narasu & Bandi Boje Gowda Published online: 14 January 2010 # Springer Science+Business Media, LLC 2010 Abstract Aerial parts (leaves and stem) of Raphanus sativus, which are usually discarded were found to possess potent antioxidant and radical scavenging activity, as measured by standard antioxidant assays Methanolic and acetone extracts of R sativus leaves had total polyphenolic content of 86.16 and 78.77 mg/g dry extract, which were comparable to the traditional rich sources such as green tea and black tea.HPLCidentificationofpolyphenolics indicated the presence of catechin, protocatechuic acid, syringic acid, vanillic acid, ferulic acid, sinapic acid, o-coumaric acid, myricetin, and quercetin in leaves and stem Among the different extraction solvents, methanolic extract of leaves and stem showed potent reductive capacity, significantly inhibited linoleic acid peroxidation and displayed metal chelating activity Further, they scavenged free radicals effectively with IC50 (half maximal inhibitory concentration) of 31 and 42 µg/ml for DPPH radical, 23 and 52 µg/ml for superoxide radical, 67 and 197 µg/ml for hydrogen peroxide, and 56 and 62 µg/ml for nitric oxide, respectively Leaves showed most potent antioxidant and radical scavenging activity as compared to stem, which may be accounted for the high polyphenolic content Leaves and stem of R sativus, often under-utilized part of this vegetable, thus possessed considerable amount of polyphenolics Hence, it should be regarded as a potential source of natural antioxidants and could be effectively employed as an ingredient in health or in functional food Keywords Raphanus sativus Polyphenolics.Antioxidant S S Beevi : M L Narasu (*) : B B Gowda Centre for Biotechnology, Institute of Science and Technology, Jawaharlal Nehru Technological University, Kukatpally, Hyderabad – 500 085, Andhra Pradesh, India e-mail: mangamoori@jntuh.ac.in e-mail: mangamoori@rediffmail.com activity.Radicalscavengingactivity Introduction Epidemiological studies have established an inverse relationship between intake of fruits and vegetables and mortality from age-related degenerative diseases such, as cancer, diabetes, emphysema, cardiovascular diseases, brain dysfunction, etc [1], which may be attributed to their antioxidant property These researches have augmented the consumer awareness of the potential health benefits of naturally occurring phytochemicals from plants Free radicals such as reactive oxygen species (ROS) and reactive nitrogen species (RNS) have been widely implicated as mediators in the development of these chronic diseases [2] Among the dietary constituents, polyphenolics appear to a play a significant role as antioxidants in the protective effect of plant derived foods [3] Phenolics have become the focus of current nutritional and therapeutic interest The antioxidant activity of the dietary phenolics is considered to be superior to that of the essential vitamins and is ascribed to its high redox potential which allows them to interrupt free radical mediated reactions by donating hydrogen from the phenolic hydroxyl groups [4] More- over, these natural antioxidants have easy and unlimited access to metabolic processes in the body, and produce virtually none of the side effects associated with synthetic antioxidants [5] Raphanus sativus, Linn, belonging to a large family of Cruciferae, is widely consumed as important components in traditional Indian cuisine The most popular part for consumption is the napiform taproot, although the entire plant is edible and the aerial part can be used as a leaf vegetable Plant Foods Hum Nutr (2010) 65:8–17 It is widely grown in India for its culinary and medicinal purposes Roots are used in Indian traditional medicine to support a healthy liver and to promote digestion [6, 7] It is also a reputed medicine for gastrodynic pains and urinary complaints Leaves and stem have been used as laxative, stimulant and appetizer in herbal medicine [6, 7] Pharmacological studies have shown that R sativus root extracts alleviated hyperlipidemic changes in the colon mucosa of rats fed with fat rich diet [8] and prevented the formation of calculus in the urinary tract in experimental animals [9] R sativus sprout extracts have shown inhibition of cell proliferation and induce cancer cells to undergo apoptosis [10] Root, stem and leaf of R sativus showed broad spectrum of antibacterial activity against food-borne pathogens and drug resistant strains [11, 12] Recently, R sativus is gaining renewed interest as an ingredient for the production of healthy functional foods owing to the presence of glucosinolates and their degradation products, isothiocyanates However, robust health promoting properties of R sativus have also been attributed to phenolic compounds Previous studies have characterized the radical scavenging activity of crude methanolic extracts of several culinary plants including R sativus sprouts, which displayed one of the highest potency among the studied common vegetables and found to contain sinapic acid esters and flavonoids as main phenolic components [13] However, information regarding the polyphenolics and the antioxidant property of leaves and stem of R sativus is almost lacking Hence, we felt that a well documented and comprehensive study on antioxidant activity of leaves and stem of R sativus, often under-utilized part of this vegetable would substantiate their value in the human nutrition as well as food and pharmaceutical supplements In the present study, polyphenolics were extracted from leaves and stem of R sativus by solvents of increasing polarity (hexane, chloroform, ethyl acetate, acetone, methanol and water) and screened by HPLC-DAD The antioxidant activities of these extracts were evaluated by four different model systems such as FRAP assay, reducing power, inhibition of linoleic acid peroxidation, and metal chelating activity The radical scavenging potential was assessed against DPPH, reactive oxygen species (superoxide radical and hydrogen peroxide), and reactive nitrogen species (nitric oxide) Materials and Methods Chemicals and Reagents All chemicals and reagents used in the experiments were of analytical grade and obtained from Sigma-Aldrich (St Louis, MO, USA) and Merck (Mumbai, India) Solvents used for extraction were of HPLC grade and were purchased from HiMedia (Mumbai, India) Ultra pure water used for bioassays and HPLC analysis was prepared inhouse using a Milli-Q system (Millipore, USA) Plant Materials and Preparation of Extracts Raphanus sativus L aerial part was purchased fresh from the local supermarket in Hyderabad city and was processed on the same day itself It was separated into leaves and stem, washed thoroughly with distilled water and freeze dried The dried samples were ground to powder and stored air tight at −20 °C until further analysis The powdered leaves and stem were extracted three times with solvents of varying polarity such as methanol, acetone, ethyl acetate, chloroform and hexane at room temperature for 24 h with a mass to volume ratio of 1:40 (g/ml), and were, evaporated to dryness under vacuum on a rotary evaporator (HeidolphRotacool, Germany) at 40 °C Dried residues were subsequently redissolved in methanol for HPLC analysis and to measure their total polyphenolics and antioxidant activities Water extract of R sativus was prepared as above by soaking dried powder into distilled water and mixed with magnetic stirrer at lesser rpm for 24 h Then, the ex- tract was filtered over Whatman No paper and was sub- sequently lyophilized in a lyophilizer at µm Hg pressure at −50 °C (ScanVac-CoolsafeTM, Denmark) All the extracts were filtered through a 0.45 µm membrane filters (Millipore, Bedford, MA) and were stored at −80 °C until use Total Polyphenolics Content Total polyphenolics content of R sativus extracts was determined using Folin–Ciocalteau reagent as described by Kim et al [14] The total concentration of polyphenolic compounds in R sativus extracts was measured as catechin equivalent and expressed as mg/g of dry extract HPLC-DAD Analysis Polyphenolics in the different parts of R sativus were analyzed by Shimadzu LC10 HPLC system equipped with diode array detector (DAD) Separation of polyphenolics was carried out using a Luna C18 column (250 mm× 4.6 mm i.d.; particle size, µm) with a C18 guard column The mobile phase consisted of 6% acetic acid in mM sodium acetate (solvent A) and acetonitrile (solvent B) A solvent gradient was maintained varying the proportion of solvent A to solvent B in the following manner: a linear gradient of 0–15% of B for 45 min, 15–30% B for 15 min, 30–50% B for min, and 50–100% B for The set time of recording chromatograms and spectra was 70 min, 10 while the total running time was 80 including 10 post run at initial conditions for equilibration of column The column temperature was set at 30 °C The flow rate was 1.0 ml/min and the injection volume was 20 µl The detector was set at 280 nm for catechin, protocatechuic acid, vanillic acid, syringic acid, and o-coumaric acid; 320 nm for sinapic acid and ferulic acid, and 360 nm for myricetin and quercetin The standard polyphenolics selected for the identification of compounds in R sativus were catechin, protocatechuic acid, vanillic acid, syringic acid, ocoumaric acid, sinapic acid, ferulic acid, myricetin, and quercetin Polyphenolics in the R sativus extracts were quantified using linear regression equations derived from authentic standards The polypheno- lic compounds were identified on the basis of their comparison of their retention time and spectral matching with that of authentic standards, and also by co-elution to further confirm the identity of the compound Antioxidant Activity of R sativus The FRAP assay was done according to Benzie and Strain [15] and is based on the reduction of TPTZ-Fe3+ complex to TPTZ-Fe 2+ form in the presence of antioxidants The antioxidant capacity was expressed as µM FeSO4/ g of dried extract Quercetin and butylated hydroxyl toluene (BHT) were used as positive controls The reducing power of the extracts was evaluated according to the method described by Yen and Chen [16] and expressed as absorbance at 700 nm The chelating capacity of R sativus extracts on Fe2+ ions was determined according to the method of Dinis et al [17], wherein the Fe2+ chelating potential of the extracts was monitored by measuring the ferrous iron—ferrozine complex at 562 nm [18], and was expressed as percent chelation of Fe2+ ions relative to negative control without R sativus extracts or standards (EDTA, quercetin and BHT) The capacity of R sativus extracts to inhibit the formation of peroxides in linoleic acid system was determined according to the thiocyanate method [19] The method is based on the capacity of peroxides to catalyze the oxidation of Fe 2+ to Fe 3+ The Fe3+ produced is linked to the thiocyanate anion, yielding a red complex, which is measured spectrophotometrically at 500 nm [20] Radical-Scavenging Activity of R sativus Extracts DPPH radical-scavenging activity of R sativus extracts and standards (quercetin and BHT) were determined as previously described [21] The capacity of extracts to scavenge the lipid-soluble 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical, which results in the bleaching of the purple color exhibited by the stable DPPH radical, is monitored at an Plant Foods Hum Nutr (2010) 65:8–17 absorbance of 517 nm The ability of R sativus extracts, quercetin, and BHT to quench the generation of superoxide radicals was determined according to the method of Nishikimi et al [22] Superoxide radicals were generated in PMS-NADH system by oxidation of NADH and analyzed by NBT reduction at 560 nm The method of Sinha [23] originally designed for the estimation of the antioxidant enzyme, catalase, was adopted to evaluate the hydrogen peroxide scavenging effect of R sativus extracts and standards (quercetin and BHT) This method is based on the coupling of excess H 2O2 with dichromate in acetic acid to produce a green color which was measured at 620 nm The method of Rai et al [24] based on the spontaneous generation of nitric oxide (NO·) from the sodium nitroprusside (SNP) buffered solution was used to assess the NO scavenging ability of R sativus extracts and standards (quercetin and BHT) Radical scavenging activity of extracts and standard was expressed as IC50, which was interpolated from graph constructed using percent inhibition (Y-axis) against concentration (X-axis) of the extracts and standards Statistical Analysis Results calculated from triplicate data were expressed as means ± standard deviations The data were compared by least significant difference test using Statistical Analysis System (SAS, ver 9.1) Graphing, curve fitting, and IC 50 were performed using GraphPad Prism (ver.5.0a) Results and Discussion Effect of Solvent on the Yield of Total Soluble Solids and Polyphenolics The yield of soluble substances, expressed as mg/g dry weight of leaves and stem of R sativus and the total extractable polyphenolics, expressed as catechin equivalents, are closely dependent on the solvent, as shown in Table No correlation was found between the extraction yield and the polyphenolics content The highest yield was obtained when extraction of leaves and stem was performed with water, followed by that obtained with methanol, ethyl acetate, acetone, chloroform, and hexane The contents of polyphenolics varied among different extracting solvents and parts of the plant used Leaves were found to contain high amounts of polyphenolics Methanol was the solvent which rendered more polyphenolics (86.16 mg/g) followed by that obtained with acetone (78.77 mg/g) The amount of total polyphenolics in water and ethyl acetate extract was in the range 34–37 mg/g, which was markedly less as compared to that of methanol and acetone extracts Plant Foods Hum Nutr (2010) 65:8–17 11 Table Extraction yields, total polyphenolics content, ferric reducing activity (FRAP assay), and percentage inhibition of linoleic acid peroxidation of R sativus leaves and stem extractsa Extraction solvent Parts of R sativus Yield (mg/g dry weight) Total phenolicsb Water Leaves Stem Leaves Stem Leaves Stem Leaves Stem Leaves Stem Leaves Stem _ _ 106.10±14.58 94.60±8.24 61.20±8.44 61.20±5.26 21.60±4.06 20.80±3.69 18.50±1.63 21.80±1.84 14.40±0.98 14.40±1.50 21.60±3.47 15.20±1.26 _ _ 34.16±3.44 23.55±2.20 86.16±4.51 56.69±1.84 78.77±5.32 56.84±1.59 36.81±1.70 30.43±2.31 22.37±1.51 16.78±1.20 4.97±0.19 1.92±0.08 _ _ Methanol Acetone Ethyl acetate Chloroform Hexane Quercetin BHT FRAP (mM FeSO4/g) Percent inhibition of linoleic acid peroxidation 1.71±0.031 1.68±0.090 2.83±0.083 1.87±0.034 1.78±0.012 1.44±0.022 0.89±0.001 0.59±0.000 0.57±0.005 0.48±0.006 0.06±0.000 0.05±0.000 15.61±1.42 1.28±0.065 79.68±1.62 67.12±6.33 81.99±4.50 76.55±2.95 73.32±3.68 72.60±6.71 77.42±6.81 62.54±2.70 67.12±5.30 60.66±2.77 53.59±4.59 57.01±1.87 81.64±1.73 82.98±1.05 a Values are expressed as means ± SD (n =3) b Expressed as mg catechin/g dry extract However, chloroform and hexane yielded a least amount of polyphenolics which were 22.37 mg and 4.97 mg/g Total polyphenolics of stem were less than that of leaves and were found in the following order; methanol extract (56.69 mg/g), water extract (23.55 mg/g), acetone extract (32.77 mg/g), ethyl acetate extract (30.43 mg/g), chloroform extract (16.78 mg/g), and hexane extract (1.92 mg/g) The colorimetric method for the determination of polyphenolics based on the use of Folin–Ciocalteu reagent is not specific for phenolic compounds as other reducing compounds can interfere [25] and its reactivity is different for different polyphenolics [26] Although, this method is generally used and preferred as it is straightforward to obtain comparative results with other plant materials accounted in the literature From our result, it was apparent that the recovery of polyphenolics was dependent on the extraction solvents and their polarity With polar solvents, we could be able to extract significant amounts of phenolics from R sativus Previous studies indicated a similar trend whereby the most typical polyphenolics were significantly extracted into the polar solvents [27] The total polyphenolic content reported for black kale leaves (1366 ng/g) [28] and ginger rhizomes (0.05–0.98 mg/g) [29] appeared to be much lesser than that of leaves and stem of R sativus In addition, the polyphenolic content of leaves was almost comparable to the phenolic content of traditionally rich sources such as black tea (81–135 mg/g) and green tea (66– 106 mg/g), respectively [30] Our findings thus suggested the potential of leaves and stem of R sativus to be exploited as a source of nutritional polyphenolics HPLC-DAD Analysis of Polyphenolics in Leaves and Stem of R sativus HPLC-DAD analysis has the advantage over total phenolics content determined by the Folin Ciocalteu method, as it provides more precise information of individual compounds Several polyphenolic compounds were identified in the leaves and stem of R sativus These included catechin, protocatechuic acid, vanillic acid, syringic acid, ferulic acid, sinapic acid, o-coumaric acid, myricetin, and quercetin (Table 2) Representative HPLC profiles recorded at 280 nm for standard polyphenolics mixture and methanolic extract of leaves and stem are presented as Fig 1a–c HPLC profile of leaves and stem was almost similar, except that leaf extracts contained more amounts of polyphenolics than that of the stem extract Catechin (4.88 mg/g and 1.13 mg/g) was the most abundant phenolic acid in the water extract of leaves and stem Vanillic acid, ferulic acid, sinapic acid, and o-coumaric acid were the predominant phenolic acids in the methanolic extract of leaves and stem Catechin and vanillic acid were detected as major phenolics in acetone extract Although, flavonols such as myricetin and quercetin were not detected in any of these extracts While myricetin (6.41 mg/g) was the main flavonol identified in the ethyl acetate extract of leaves Chloroform extract contained moderate amounts of protocatechuic acid and Quercetin, and none of the polyphenolics (standards used for analysis) were detected in the hexane extracts of leaves and stem Previous studies reported the presence of sinapic acid esters and kaempferol as major phenolics in Japanese R 12 Plant Foods Hum Nutr (2010) 65:8–17 Table Polyphenolics content of leaves and stem of R sativus (mg/g dry weight)a Extraction solvent Catechin Parts of R sativus Proto-catechuic acid Syringic acid Vanillic acid Ferulic acid Sinapic acid Water Leaves 4.88±0.13 NDb ND ND ND 0.62±0.008 1.05±0.005 ND ND Stem 1.13±0.034 ND 0.70±0.006 0.69±0.004 ND ND 0.57±0.006 ND ND Leaves 0.36±0.005 ND ND 4.13±0.34 1.29±0.046 3.21±0.19 2.13±0.049 ND ND Stem 0.22±0.002 ND ND 1.76±0.25 0.32±0.003 1.32±0.092 1.64±0.061 ND ND Leaves 2.11±0.097 ND ND 1.96±0.096 ND ND 0.19±0.001 ND Stem 1.03±0.083 ND ND 1.64±0.083 ND ND 0.86±0.007 ND Leaves ND ND 0.53±0.003 1.39±0.072 ND 1.09±0.057 ND Stem Chloroform Leaves ND ND 0.08±0.000 ND 0.19±0.000 ND ND ND ND ND 0.75±0.005 0.36±0.003 0.41±0.002 ND ND ND ND 0.79±0.004 Stem ND 0.33±0.002 ND ND ND ND ND ND ND Leaves ND ND ND ND ND ND ND ND ND Stem ND ND ND ND ND ND ND ND ND Methanol Acetone Ethyl acetate Hexane o-Coumaric acid Myricetin 6.41±0.23 Quercetin ND ND 0.49±0.000 a Values b Not are means ± SD (n =3) detected sativus sprouts [13] However, we detected an assortment of phenolics in leaves and stem of R sativus Catechin, vanillic acid, sinapic acid, ferulic acid, ocoumaric acid, and myricetin seemed to be the most abundant phenolics in them The significant findings of this study was that the catechin content of the water extract (4.88 mg/g) and acetone extract (2.11 mg/g) of leaves were much higher than that accounted for green tea (1.3 mg/g) and black tea (1.7 mg/g) [30] Sinapic acid, the most predominant phenolic acid in most cruciferous vegetables was to be higher than those reported for cauliflower [31] and black cabbage [28] Similarly, ferulic acid content of methanolic extract (1.29 mg/g) of leaves was significantly higher than that present in bitter cumin (0.376 mg/g) [32] Likewise, myricetin content of ethyl acetate extract of leaves (6.41 mg/g) was also considerably higher compared to that present in the red grapes skin (44 µg/g) [33] Antioxidant Properties of R sativus Several mechanisms have been proposed to be involved in the antioxidant activity such as hydrogen donation, termination of free radical mediated chain reaction, prevention of hydrogen abstraction, chelation of catalytic ions, and elimination of peroxides [34] Antioxidant activity is a dependent system and the characteristic of a particular system can influence the outcome of the analysis Hence, a single assay would not be representative of the antioxidant potential of plant extracts In the present study, we employed different models of antioxidant assays which could provide a more reliable approach to assess the antioxidant and radical scavenging potential of leaves and stem of R sativus The ferric reducing ability (FRAP) of the leaves and stem of R sativus is shown in Table The water, methanol, and acetone extracts were able to reduce ferric ions efficiently and had reducing activity in the range of 1.68–2.83 mM/g, which was greater than synthetic antioxidant BHT (1.28 mM/g) Ethyl acetate and chloroform extracts showed moderate reducing activity in the range of 0.48–0.89 mM/g However, hexane extract of leaves and stem had negligible reducing activity All the extracts were less effective when compared with the reducing activity of quercetin (15.61 mM/g) The reducing power of leaves and stem of R sativus and standard antioxidants such as quercetin and BHT at a concentration of 250 µg/ml is presented in Fig Leaves and stem extracts showed variable reducing power with leaves displaying the higher reducing power than stem extracts The reducing ability of methanolic extracts of leaves and stem was relatively more pronounced than the other extracts and presented a reducing power of 0.698 and 0.497, respectively However, hexane extracts of leaves and stem showed the least reducing power Quercetin and BHT revealed the most potent reducing power of 0.974 and 0.928, respectively, which were distinctly higher than that of any of the R sativus extracts Antioxidant activity has been reported to be concomitant with the reducing power of the plant extract Results from this study suggested that the distinct reducing ability of leaves and stem of R sativus appeared to be the result of their antioxidant activity Hence, it can be assumed that the polyphenolics present in the leaves and stem could act as reductones by donating electrons to free radicals and terminating the free radical mediated chain reactions [34] Plant Foods Hum Nutr (2010) 65:8–17 13 a) 600 500 400 300 200 100 b) 140 120 100 80 60 40 20 10 20 10 20 30 40 50 60 c) 200 150 100 50 30 40 50 60 Fig Representative HPLC chromatograms recorded at 280 nm a) Mixture of standard polyphenolics; b) Methanolic extract of stem; c) Methanolic extract of leaves Peaks: (1) catechin; (2) protocatechuic acid; (3) syringic acid; (4) vanillic acid; (5) ferulic acid; (6) sinapic acid; (7) o-coumaric acid; (8) myricetin; (9) quercetin Leaves and stem of R sativus were able to chelate ferrous ion in a concentration dependent manner However, the estimated IC50 value was very high (more than 2.0 mg/ml), especially in comparison with EDTA (7.75 µg/ml) Quercetin and BHT showed moderate metal chelating activity when compared with EDTA with an IC50 value of 134 µg/ml and 86 µg/ml, respectively Figure shows the metal chelating activity of leaves and stem of R sativus at a concentration of 1.0 mg/ml, and EDTA, Quercetin, and BHT at a concentra- tion of 250 µg/ml The metal chelating capacity of R sativus extracts varied from 2.15% to 30.83% Methanolic extracts were the highest, followed by water, acetone, and ethyl acetate extracts Chloroform and hexane extracts of leaves and stem displayed least activity and there were no significant differences, among them EDTA, quercetin, and BHT exhibited 99.23%, 60.54% and 71.36% of metal chelating activity respectively, which were significantly higher than that of the R sativus extracts Transition metal ions gain utmost significance in the biological systems due to their ability to generate reactive free radicals They can initiate Fenton type reaction with production of hydroxyl radicals or Haber-Weiss reactions with superoxide radicals These active oxygen free radicals can in turn perpetuate the chain reaction of lipid peroxidation which is implicated in various chronic diseases [35] Metal chelating agents reduce the concentration of metal ions in the Fenton type reaction and thus would protect the system from oxidative damage through inhibition of metal dependent processes [36] Even though, none of the R sativus extracts showed metal chelating ability as significant as EDTA, they could still chelate iron at higher concentrations This result is not surprising as non-phenolic compounds are supposed to be the better chelators of transition metal ions rather than polyphenols [37] Antioxidative activity of the leaves and stem extracts in the linoleic acid system (250 µg/ml) is shown in Fig 4a and b In the absence of extracts, linoleic acid was autooxidized, which was followed by a rapid increase of peroxides started at the 2nd day of testing, reached maximum on the 6th day and decreased on the 7th day probably due to lack of linoleic acid in the reaction system All the extracts displayed strong to moderate antioxidant activity in the linoleic acid system and significantly delayed the peroxidation of linoleic acid at a concentration of 250 µg/ml, as compared to the control Among the different extracts tested, methanolic extracts were most effective in the inhibition of linoleic acid peroxidation (Table 1) The percentage inhibition of oxidation in linoleic acid system by 250 µg/ml of methanolic extract of leaves and stem at the 6th day of analysis was found to be 81.99% and 76.55%, respectively, which were comparable to the reference antioxidants, such as quercetin (81.64%) and BHT (82.98%) Water, acetone, and ethyl acetate extracts showed inhibitory activities in the range of 63–80% The other extracts of R sativus were less effective in comparison with quercetin and BHT, but showed inhibitory activity at the range of 54–67% Lipid peroxidation is a free radical mediated chain reaction that can inactivate cellular components and are purportedly associated with various chronic disorders including carcinogenesis [35] It is a known fact that transition metal ions may either initiate lipid peroxidation through generation of hydroxyl radicals or propagate chain 14 Plant Foods Hum Nutr (2010) 65:8–17 0.5 Fig Reducing power of R sativus leaves and stem extracts, quercetin and BHT at a concentration of 250 µg/ml Results are means ± standard deviation of three parallel measurements 0.4 0.3 0.8 0.2 0.7 0.1 0.6 Quercetin BHT reaction by way of decomposition of lipid hydroperoxides Hence, reduction or removal of metal ions through chelating agents is supposed to be one of the mechanism by which initiation of lipid peroxidation can be inhibited In this study, R sativus extracts significantly inhibited peroxyl radical induced oxidation of linoleic acid However, removal of metal ions alone could not be the mechanism responsible for the observed activity as R sativus extracts were found to possess low metal chelating activity Hence, we assumed that polyphenolics present in the extracts could scavenge peroxyl radicals by donating hydrogen atom before it can react with linoleic acid and thus inhibited lipid peroxidation Free Radical Scavenging Ability of R sativus Fig Metal chelating ability of R sativus leaves and stem extracts (1.0 mg/ml), EDTA, quercetin and BHT (250 µg/ml) Results are means ± SD (n =3) Water 40 100 20 80 Water Methanol Acetone Ethyl acetate Chloroform Ethyl acetate Chloroform Hexane Basic information on the efficacy of compounds in R sativus extracts to quench free radicals can be deduced from the DPPH· assay (Table 3) R sativus extracts revealed a concentration-dependent scavenging of DPPH radicals, with leaves presenting the strongest effects Among the different extracts, methanolic extract showed the strongest effect (IC50 at 31 µg/ml for leaves and 42 µg/ml for stem), followed by water, acetone, and ethyl acetate extracts Chloroform and hexane extracts displayed the weakest activity with IC50 of over 1.0 mg/ml Comparison of DPPH radical scavenging activity with the standard antioxidants EDTA BHT Acetone 60 120 Quercetin Methanol Hexane Plant Foods Hum Nutr (2010) 65:8–17 15 Ethyl acetate a 2.5 0.5 Ethyl acetate b Chloroform Hexane 2.5 Chloroform 0.5 1.5 Water Acetone Water Methan Methan ol Control Control 1.5 Hexane ol Acetone Days Days Fig a Antioxidative activity of R sativus leaves extracts in the linoleic acid system (250 µg/ml) Results are the means of duplicate analysis b Antioxidative activity of R sativus stem extracts in the linoleic acid system (250 µg/ml) Results are the means of duplicate analysis showed that the most potent R sativus extracts had scavenging ability higher than BHT (IC 50 at 493 µg/ml), but lower than the quercetin (IC50 at 11 µg/ml) The scavenging activities of the R sativus extracts on superoxide radicals, H2O2, and nitric oxide radicals are presented in Table Extracts from leaves and stem of R sativus displayed concentration dependent protective activity against the reactive species, of which, leaves were the most effective material Methanolic extracts of leaves (IC50 at the range of 23–56µg/ml) and stem (IC50 at the range of 60–197µg/ml) showed the most potent scavenging activity Water, acetone and ethyl acetate extracts showed strong to moderate activity with IC50 in the range of 46–682 µg/ml Chloroform extracts of leaves and stem exhibited significant scavenging activity against nitric oxide, moderate activity against hydrogen peroxide and the least activity against superoxide radicals Hexane extracts showed the least activity against any of the reactive species generated in vitro When radical scavenging activity of R sativus extracts compared to the IC50 values calculated for the reference antioxidants, methanolic extract of leaf was as active as quercetin and more effective than BHT Table Scavenging ability of leaves and stem of R sativus and standard antioxidants on DPPH·, superoxide radical (O2·), hydrogen peroxide (H2O2), and nitric oxide (NO·) as determined by their IC50, expressed as mg/g dry weighta Extraction solvent Parts of R sativus IC50 (expressed as mg/ml) DPPH· O 2· H 2O2 NO· Water Leaves Stem 0.216±0.025 0.877±0.049 0.328±0.008 0.418±0.020 0.628±0.037 0.652±0.028 0.490±0.044 0.682±0.009 Methanol Leaves Stem Leaves Stem Leaves Stem Leaves Stem Leaves Stem Quercetin BHT 0.031±0.000 0.042±0.001 0.215±0.030 0.225±0.001 0.628±0.022 0.606±0.034 1.48±0.075 1.21±0.031 1.86±0.13 >2.0 0.011±0.002 0.493±0.057 0.023±0.002 0.052±0.002 0.046±0.007 0.189±0.023 0.061±0.002 0.131±0.013 0.806±0.034 0.965±0.051 1.85±0.059 >2.0 0.010±0.000 0.019±0.002 0.067±0.003 0.197±0.032 0.457±0.041 0.680±0.009 0.488±0.029 0.644±0.013 1.86±0.022 1.97±0.019 >2.0 >2.0 0.034±0.001 0.089±0.003 0.056±0.002 0.062±0.007 0.091±0.001 0.198±0.002 0.316±0.009 0.424±0.035 0.699±0.037 0.551±0.015 1.59±0.038 1.69±0.009 0.036±0.002 0.047±0.000 Acetone Ethyl acetate Chloroform Hexane Standard antioxidants a All data were average (± SD) of three replicates 16 Superoxide radical, hydrogen peroxide, and nitric oxide could generate potentially reactive oxygen and nitrogen species, such as singlet oxygen, hydroxyl radicals, and peroxynitrite These reactive species are believed to act as inducers of cellular injury through initiation of lipid peroxidation, oxidation of proteins, and induction of DNA strand breaks R sativus leaves and stem have demonstrated their ability to scavenge free radicals such as DPPH·, superoxide anion, hydrogen peroxide and nitric oxide effectively by acting as chain-breaking antioxidants Anti- oxidant and radical scavenging properties of phenolic acids and flavonoids detected in leaves and stem of R sativus were reported previously in various model systems [38] Several studies have reported the relationship between polyphenolics structure and antioxidant activity, demon- strating that polyphenolics possessing hydroxyl groups on the phenyl rings effectively contribute to the chain-breaking antioxidant activity by stabilizing the radical form in electron delocation [39] Among the polyphenolics found in the leaves and stem, many have hydroxyl groups in their structure which could make possible the inhibition of the free radical-induced chain reactions, and thus could contribute significantly to the antioxidant activity of R sativus A comparison between the DPPH radical scavenging activities of R sativus and some common culinary spices such as ginger, basil, and parsley showed that the leaves and stem of R sativus were more potent in terms of radical scavenging activity whereby their IC50 were comparatively much lower than these culinary spices [29, 40], thus further demonstrating the effectiveness of R sativus leaves and stem as natural antioxidants Polyphenolics of cruciferous vegetables are reported to have preventive and curative properties against various chronic diseases However, polyphenolics seemed to be more concentrated in the discarded parts of the vegetables such as leaves and stem than in their edible parts Hollman and Arts [41] demonstrated higher flavonoid contents in the leaves of cauliflower than in their edible parts, where trace amounts of flavonoids were identified Ayaz et al [28] likewise detected abundant phenolic acid and excellent antioxidant properties in the leaves and seed of black cabbage Conclusions Our studies have shown for the first time the presence of an array of polyphenolics such as catechin, protecatechuic acid, syringic acid, vanillic acid, ferulic acid, sinapic acid, o-coumaric acid, myricetin, and quercetin in the leaves and stem of R sativus Among the different solvents used, methanol extract possessed significant amounts of polyphenolics and showed potent antioxidant and radical scavenging activity and their potency was in the order of Plant Foods Hum Nutr (2010) 65:8–17 methanol>acetone>ethyl acetate>water>chloroform>hexane Leaves showed significant antioxidant and radical scavenging activity as compared to stem, which may be accounted for the high polyphenolic content Aerial 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