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Antiradical efficacy of phytochemical extracts from defatted rice bran
Food and Chemical Toxicology 45 (2007) 2014–2021 www.elsevier.com/locate/foodchemtox
Antiradical efficacy of phytochemical extracts from defatted rice bran R. Renuka Devi, C. Arumughan
*
Agroprocessing and Natural Products Division, Regional Research Laboratory (CSIR), Thiruvananthapuram 695 019, Kerala, India Received 16 May 2006; accepted 26 April 2007
Abstract The phytochemical compounds oryzanols, tocopherols, tocotrienols and ferulic acid were identified in the crude methanolic extracts (CME) of defatted rice bran (DRB) by HPLC. Enrichment of antioxidants in CME was achieved by sequential extraction and fractionation resulting in three enriched fractions viz. acetone extract (AE), acetone extract-lipophilic fraction (AE-LP) and acetone extract-polar fraction (AE-PP). The scavenging effects of DRB extracts and their phytochemical constituents against DPPH and superoxide radicals were investigated. The EC50 (g antioxidant/kg DPPH) values of CME, AE, AE-LP, AE-PP, oryzanols, ferulic acid, tocols (Tmix), tricin, b-sitosterol, BHT and TBHQ were 1977, 1945, 7985, 1072, 972, 174, 164, 3947, 21416, 1120 and 61, respectively. Tricin and b-sitosterol isolated from CME were identified with the help of spectral data. The DRB extracts and their phytochemical constituents when assayed by cytochrome c and NBT methods showed positive superoxide radical scavenging effects. The order of efficacies of the extracts was AE-PP > AE > CME > AE-LP in both assays, but the activities were higher for the former method. The DPPH as well as superoxide scavenging activities of AE, AE-LP and AE-PP could largely be attributed to the levels of total phenols (TPC) and ferulic acid in it. 2007 Published by Elsevier Ltd. Keywords: Defatted rice bran; Phytochemicals; HPLC; DPPH; Superoxide radicals
1. Introduction Reactive oxygen species (ROS) such as superoxide O 2 , H2O2 and hydroxyl radicals (OH) are byproducts of normal metabolism and attack certain biological molecules, leading to destabilisation and disintegration of cell membranes and many age related diseases (Babior, 2000). Lipid peroxidation which has a profound role both in quality deterioration of lipid containing foods as well as in a number of destructive biological processes is also mediated by free radicals (Cosgrove et al., 1987). In the human body,
Abbreviations: AE, acetone extract; AE-LP, acetone extract-lipophilic fraction; AE-PP, acetone extract-polar fraction; CME, crude methanolic extract; DPPH, 1,1-diphenyl-2-picrylhydrazyl radical; DRB, defatted rice bran; HPLC, high performance liquid chromatography; NBT, nitro blue tetrazolium; ROS, reactive oxygen species; TPC, total phenolic content; XOD, xanthine oxidase. * Corresponding author. Tel.: +91 471 492901; fax: +91 471 491712. E-mail address: [email protected] (C. Arumughan). 0278-6915/$ - see front matter 2007 Published by Elsevier Ltd. doi:10.1016/j.fct.2007.04.020
the toxic effects of ROS are combated regularly by a number of endogenous defense and protective mechanisms which include various enzymes and non-enzymatic antioxidants. These self-defense systems may also be supported by antioxidative compounds taken as foods, cosmetics and medicine particularly in the elderly (Ramarathnam et al., 1997). Though many synthetic compounds like BHA, BHT, TBHQ, etc. are efficient antioxidants, their use is being restricted because of their possible toxic and carcinogenic effects (Branen, 1975). Thus, the health concern of synthetic food additives and evidence in phytonutrients as chemopreventive agents led to resurgence in bio- and chemoprospecting of plant materials for potential therapeutic properties. Agro-industrial byproducts like oilseed meals are also re-examined from this perspective and a few deoiled meals from sesame, soybean, canola, peanut, cotton seeds, mustard, etc. have been phytochemically characterized (Wettasinghe et al., 2002). However, rice bran meal (defatted rice bran) has not been studied in detail. Defatted rice bran (DRB), the predominant byproduct of rice bran oil (RBO)
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extraction is a good source of insoluble dietary fiber, protein, phytic acid, inositol and vitamin B (Hargrove, 1994). Though the antioxidant potential of DRB was also known (Shin et al., 1992), it still remained a relatively unexplored source material, which demanded further investigation especially with regard to its phytochemical composition related to possible health benefits as antioxidants. Free radical scavenging is generally the accepted mechanism for antioxidants inhibiting lipid oxidation and radical scavenging studies have emerged as a convenient methodology for its measurement (Brand-Williams et al., 1995). The specific radicals used could be stable ones like DPPH or those generated in chemical model systems like ABTS+, superoxide radical, etc. (Davalos et al., 2004). Measurement of radical scavenging activity using discoloration of 1,1diphenyl-2-picrylhydrazyl radicals (DPPH) has been widely used due to its stability, simplicity and reproducibility (Kitts et al., 2000). In the present study, the radical scavenging effects of DRB extracts and their various phytochemical constituents were studied using the stable DPPH radical and superoxide radicals generated in situ by the xanthine– xanthine oxidase system, one of the main enzymatic sources of reactive oxygen species in vivo (Unno et al., 2000).
2. Materials and methods 2.1. Materials Defatted rice bran samples were obtained from rice bran oil industry (M/s. Chakkiyathumooda Solvent Extractions, Ankamali, Kerala, India). Tocopherol standards were obtained from E-Merck, Darmstadt, Germany and tocotrienol standards from Calbiochem (Calbiochem–Novabiochem Co., San Diego, CA, USA). Ferulic acid, 1,1-diphenyl-2-picrylhydrazyl radical (DPPH), nitro blue tetrazolium (NBT) salt, xanthine, xanthine oxidase and ferricytochrome c were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Standard compounds of oryzanols, cycloartenyl ferulate and 24-methylene cycloartanyl ferulate were a generous gift from M/s. Tsuno Rice Fine Chemicals, Wakayama, Japan. High performance liquid chromatography (HPLC) grade solvents were purchased from Merck (India) Ltd. All other reagents were of analytical grade.
2.2. Extraction and enrichment of antioxidants from DRB Defatted rice bran (10 g) was extracted with methanol (150 ml) for 10 h in a soxhlet extractor. The extract was filtered through a Whatman No. 1 filter paper and the solvent was removed under vacuum/N2 flow to dryness. The weighed residue was redissolved in methanol to get an antioxidant solution of known concentration. The crude methanolic extract (CME) thus obtained was partially purified by re-extraction with acetone to give an acetone extract (AE). For further purification of the acetone extract, sequential extraction was employed yielding a lipophilic phase (AE-LP) with hexane and a polar phase (AE-PP) with acetone. All the extracts/fractions were evaporated to dryness under reduced pressure on a rotary evaporator at 40 C using amber colored round bottom flasks. The dried extracts were weighed and stored at 4 C under nitrogen atmosphere and were dissolved in methanol for routine analysis within 4 weeks.
2.3. Silica gel column chromatography The extract (20 g) was chromatographed on a glass column (5 cm · 120 cm) packed with 400 g of silica gel (100–200 mesh particle size,
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Merck). The column was eluted with gradients of petroleum ether : ethyl acetate (100% petroleum ether to 100% ethyl acetate). Eight hundred milliliters fractions were collected and fractions similar in TLC profiles were pooled. Pure compounds 1 and 2 were obtained from fractions eluted with 20% ethyl acetate and 50% ethyl acetate, respectively. Compound 1 was recrystallised from petroleum ether and compound 2 from ethyl acetate and were identified with the help of UV, IR, NMR and mass spectral (MS) data.
2.4. Compositional analysis of DRB extracts The extracts were analysed for fat, protein, sugar and ash according to AOAC methods (1984). The total phenolic content (TPC) of the extracts was determined using Folin–Ciocalteau reagent (AOAC, 1984).
2.5. Identification of active compounds in rice bran extracts 2.5.1. Analysis of oryzanols The analysis was performed using a Shimadzu make HPLC binary system (Kyoto, Japan) with a LC-10 AD model pump, a 7125 model Rheodyne injector (Cotati, CA, USA) fitted with a 20 ll sample loop, a SPD-10A UV–vis detector, with a C-R7 Ae plus integrator for data acquisition and display. A Waters l-bondapak TM C18 column (4.6 mm i.d. · 25 cm) (Millford, MA) was used in the reversed-phase with the solvent system of acetonitrile, dichloromethane, and acetic acid (88:6:6, by vol):methanol, n-butyl alcohol, water (90:2:8, by vol) in the ratio of 75:25 (v/v) at a flow rate of 1 ml/min. The UV detector was set at 325 nm. All the extracts were filtered through PTFE membrane before injection into the HPLC column. Samples were diluted with the mobile phase and analysed. Peak identification was based on comparison of RT values with authentic standards of cycloartenyl ferulate, 24-methylene cycloartanyl ferulate and a mixture of oryzanol standards. The total oryzanols were quantified based upon the peak area of 24-methylene cycloartanyl ferulate, the major oryzanol component in rice bran (Renuka Devi and Arumughan, 2007). 2.5.2. Analysis of ferulic acid Preliminary trials were done using isocratic elution with 20% acetonitrile in water adjusted to pH 2 with trifluoroacetic acid and at a wavelength of 280 nm (Adom and Liu, 2002). But later on, the same HPLC conditions used for oryzanols was followed. The main advantage was the simultaneous detection of both ferulic acid and oryzanols in a single run. Peak identification was based on comparison of RT values with authentic standard of trans-ferulic acid and was further confirmed by spiking studies. The ferulic acid concentration of sample extracts was extrapolated from the pure trans-ferulic acid standard curve. 2.5.3. Analysis of tocopherols and tocotrienols The same HPLC system and a Shim-pack (LC-NH2(M)) column (4.6 mm i.d. · 25 cm) was used in the normal-phase with the solvent system, n-hexane:isopropanol (96:4, v/v) and at a flow rate of 1 ml/min. The UV detector was set at 297 nm (Renuka Devi et al., 2000). Peak identification was based on comparison of RT values with authentic standards of tocopherols and tocotrienols. The total vitamin E forms were quantitated based upon the peak areas relative to standard calibration plots by external standard method. Samples were redissolved in n-hexane and 20 ll of the solutions were injected into the HPLC column.
2.6. Determination of DPPH radical scavenging capacity The method of Brand-Williams et al. (1995) modified by Sa´nchezMoreno et al. (1998) was followed. 0.025 g/l DPPH solution was prepared in methanol. Various concentrations of samples (extracts and pure compounds) based on respective dry weights were also prepared in methanol. To 3.9 ml of DPPH solution, 0.1 ml of appropriately diluted sample solution was added and absorbance at 515 nm was measured at different time intervals in a UV–vis spectrophotometer (UV-160A, Shimadzu) for
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30 min. The DPPH concentration in the reaction medium was calculated using the following equation obtained by linear regression: A 515 nm = 2936.68 [DPPH]T 2.18 · 103. The percentage of remaining DPPH was calculated as % DPPHREM ¼ ½DPPH T ¼T =½DPPH T ¼0 100, where [DPPH]T=T is the concentration of DPPH at 30 min time and [DPPH]T=0 is the concentration at zero time (initial concentration). The percentage of remaining DPPH against the standard/sample concentration was plotted to obtain the EC50 concentration (the amount of antioxidant required to decrease the initial DPPH concentration by 50%).
2.7. Determination of superoxide radical scavenging activity Superoxide radicals generated in situ by the xanthine–xanthine oxidase system was monitored using two different probes viz. ferricytochrome c and nitro blue tetrazolium. 2.7.1. Superoxide scavenging by ferricytochrome c method The superoxide scavenging ability of DRB extracts was studied by xanthine/xanthine oxidase/ferricytochrome c method according to Khanom et al. (2000). Briefly, the assay was performed in a 3.0 ml of a reaction mixture in a 1.0 cm cuvette. The assay mixture consisted of 0.3 ml of a sample solution, 0.5 ml of ferricytochrome c (1 · 105 M), 0.5 ml of xanthine (5 · 106 M), 1.0 ml distilled water and 0.5 ml of a KH2PO4/ K2HPO4 buffer (50 mM; pH 7.8) containing EDTA (1 · 104 M). A solution of 0.2 ml of xanthine oxidase (0.5 · 108 M) was added to start the reaction and the rate of reduction of ferricytochrome c was determined by using UV–vis spectrophotometer at 550 nm at 0 s and thereafter every 30 s for up to 900 s. Superoxide radical scavenging capacities of the additives were calculated as follows: % ferricytochrome reduction = [rate of change of absorbance of sample]/[rate of change of absorbance of control] · 100. Radical scavenging ability = 100 % ferricytochrome reduction.
CME and tocols of CME are given in Figs. 1 and 2, respectively. Compositional analysis of CME showed 7.3% fat, 24.0% sugar, 22.5% protein and 3.7% minerals. The total phenolic content was 5.3%. Oryzanols, ferulic acid and tocol contents of CME in ppm were 7812, 5756 and 138, respectively. Re-extraction of CME with acetone yielded an acetone extract (AE) which was enriched more than 1.5fold in TPC, oryzanols, ferulic acid and tocols compared to CME (Table 1). Further purification of the acetone extract employing sequential extraction yielded a lipophilic phase (AE-LP) and a polar phase (AE-PP). AE-LP was enriched in oryzanols and tocols and their quantities were increased by two fold or more compared to those in CME. However, the contents of total phenolics, and ferulic acid were decreased significantly (Table 1). On the other hand, AE-PP fraction was enriched in TPC and ferulic acid by three-fold compared to those in CME with a consequent decrease in the contents of oryzanols and tocols (Table 1). Pure compounds 1 and 2 isolated from CME by column chromatography were identified to b-sitosterol and tricin with the help of UV, IR, NMR and MS data (Renuka Devi, 2005). 3.2. DPPH radical scavenging activity In the present study, various concentrations (based on respective dry weights) of DRB extracts viz. CME, AE,
2.7.2. Superoxide scavenging by NBT method The superoxide scavenging ability of DRB extracts was studied by xanthine/xanthine oxidase/NBT method according to Sur et al. (2001). The assay mixture consisted of 1 ml of sodium carbonate/ethylene diamine tetraacetic acid buffer, (carbonate buffer 50 mM, pH 10.2 containing EDTA), 50 ll of xanthine (8.7 mg ml1), 10 ll of NBT (6 mg ml1) and 20 ll of test sample. Twenty millilitres of xanthine oxidase (10 mg ml1) was added to start the reaction and the time course for the reduction of NBT into formazan complex was followed for about 15 min at 560 nm. The percentage of NBT reduction was calculated as follows: % NBT reduction = [rate of change of absorbance of sample]/[rate of change of absorbance of control] · 100. Radical scavenging ability = 100 % NBT reduction.
2.8. Statistical analysis All measurements were duplicated on duplicate samples (2 · 2). The results were statistically analysed by ANOVA and DMRT. Statistical significance was accepted at a level of P < 0.05 (Duncan, 1955).
3. Results and discussion 3.1. Chemical and phytochemical composition of DRB extracts The optimization of the process parameters for the preparation and enrichment of an antioxidant extract from DRB was based on the yields of TPC and also on the yields of oryzanols, tocols and ferulic acid, the phytochemicals identified in DRB extracts (Renuka Devi and Arumughan, 2007). The HPLC profiles of oryzanols and ferulic acid of
Fig. 1. Reverse- phase HPLC/UV–vis detection analysis of oryzanols and ferulic acid of defatted rice bran. Peak identification: (1) ferulic acid; (2) unidentified; (3) stigmasteryl ferulate; (4) unidentified; (5) cycloartenyl ferulate; (6) 24-methylene cycloartanyl ferulate; (7) campesteryl ferulate; (8) b-sitosteryl ferulate; (9) cycloartanyl ferulate.
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idant/kg DPPH) were taken as a measure of antiradical activity. The lower the EC50, higher the antioxidant potential (Brand-Williams et al., 1995). The EC50 (g antioxidant/kg DPPH) values of CME, AE, AE-LP, AE-PP, oryzanols (OYL), ferulic acid (FA), Tmix, tricin, b-sitosterol, BHT and TBHQ were 1977, 1945, 7985, 1072, 972, 174, 164, 3947, 21416, 1120 and 61, respectively (Table 2). The order of antiradical activity was TBHQ > FA = Tmix > OYL > AE-PP = BHT > CME = AE > tricin > AE-LP > b-sitosterol. The activity of the phytochemical constituents followed the order FA = Tmix > OYL > tricin > sterol, whereas for the extracts the order was AE-PP > CME = AE > AELP. The activity of AE-PP equalled that of BHT and for ferulic acid and Tmix it was several fold higher than that of BHT. The number of hydroxyl groups on an aromatic ring is the major factor contributing to the efficacy of phenolic antioxidants (Chen et al., 1996). This may explain the higher activity of TBHQ (a diphenolic antioxidant) compared to that of BHT, a monophenolic antioxidant. The Table 2 Free radical scavenging capacitiesA of defatted rice bran extracts TreatmentsB
CME AE AE-LP AE-PP BHT TBHQ OYL FA Tmix Sterol Tricin
Fig. 2. Normal-phase HPLC/UV–vis detection analysis of tocopherols (T) and tocotrienols (T3) of defatted rice bran. Peak identification: (1) a-T; (2) a-T3; (3) c-T3; (4) d-T; (5) d-T3.
AE-LP and AE-PP, its phytochemical constituents oryzanols, ferulic acid, Tmix, tricin and b-sitosterol and synthetic antioxidants viz. BHT and TBHQ were allowed to react with DPPH. Tmix constituted a 1:1 mixture of a-T and cT3, the predominant tocopherol and tocotrienol homologs identified in DRB extracts. At each concentration, a graph was plotted with time versus % DPPHREM (DPPH remaining) for 30 min. The % DPPHREM was then plotted against the standard concentration to obtain the amount of antioxidant necessary to decrease the initial DPPH concentration by 50% and is denoted as EC50. The EC50 values (g antiox-
Radical scavenging capacity in various models DPPH, EC50 values (g/kg DPPH)
Superoxide/ cytochrome, IC50 values (lg)
1977 (±39)e 1945 (±34)e 7985 (±76)g 1072 (±23)d 1120 (±26)d 61 (± 3)a 972 (±24)c 174 (±8)b 164 (±7)b 21,416 (±165)h 3947 (±59)f
47 38 57 28 17 5 – 34 38 – 35
(±2.6)f (±2.2)e (±2.5)g (±1.6)c (±1.0)b (±0.3)a (±1.7)d (±2.1)e (±1.7)d
X/XO/NBT, IC50 values (lg) 125 97 171 77 – – – 39 – – 41
(±6.8)d (±6.1)c (±7.8)e (±3.8)b
(±2.6)a
(±2.5)a
a–h
Means within a column with different letters are significantly different (P < 0.05) according to DMRT. A Each value in the table represents the mean (±standard deviation) of four analyses from two replications. B CME, crude methanolic extract; AE, acetone extract; AE-LP, acetone extract-lipophilic phase; AE-PP, acetone extract-polar phase; OYL, oryzanol; FA, ferulic acid.
Table 1 Phytochemical compositionA of defatted rice bran extracts ExtractsB
Recovery (%)C
Phytochemical constituents in the extract TPC (wt%)
CME AE AE-LP AE-PP a–d
– 47.7c (±1.8) 26.6b (±0.9) 19.9a (±0.8)
b
5.3 (±0.2) 10.5c (±0.3) 4.7a (±0.2) 17.5d (±0.4)
Oryzanols (ppm) b
7812 (±57) 14,680c (±102) 20,445d (±129) 960a (±9)
Ferulic acid (ppm) b
5756 (±33) 9188c (±66) 932a (±8) 15,820d (±113)
Tocols (ppm) b
138 (±2) 253c (±3) 335d (±4) 45a (±1)
Tricin (wt%)
Sterol (wt%)
0.4 – – –
1.1 – – –
Means within a column with different letters are significantly different (P < 0.05) according to DMRT. Each value in the table represents the mean (±standard deviation) of four analyses from two replications. B CME, crude methanolic extract; AE, acetone extract; AE-LP, acetone extract-lipophilic phase; AE-PP, acetone extract-polar phase. C Recovery percentages are based on CME; for AE-LP and AE-PP, recovery percentages based on AE are 55.8 (±1.9) and 41.8 (±1.8), respectively.
A
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antiradical activity of BHT, ferulic acid and tocols is due to their phenolic hydrogen atoms. In BHT, both the ortho positions to the hydroxy function are substituted by tertiary-butyl groups with strong inductive effects which can enhance the stability of the phenoxyl radical. More over, the steric hindrance exerted by the bulky t-butyl groups reduces the rate of possible propagation reactions involving antioxidant radicals thus increasing its stability (Shahidi and Wanasundara, 1992). The hydroxyl group in ferulic acid is para-substituted on an aromatic ring which is connected to a highly conjugated side chain which allows the phenoxyl radical to be delocalised across the entire molecule and therefore stabilised (Graf, 1992). The ortho substitution with the electron donor methoxy group is another factor that enhances the stability and hence the antioxidant and antiradical activities of ferulic acid (McMurry, 1984). The chromanoxyl radicals (TO) formed in the case of tocopherols and tocotrienols (chromanols) also are stabilised by methyl groups in ortho positions. In addition to forming harmless adducts with other radicals, TO can also undergo self-coupling to form dimers which too possess antioxidant activity (Burton et al., 1985). Apart from these structural aspects, kinetic factors also contribute to the observed activity order of FA = Tmix > BHT. Tocopherols are reported to have intermediate kinetic behaviour reaching the steady state within 5–30 min (Sa´nchez-Moreno et al., 1998), whereas BHT and ferulic acid showed slow kinetic behaviour taking more than 30 min to reach the steady state (Sa´nchez-Moreno et al., 1998; Bondet et al., 1997). The reported TEC50 values (time to reach the steady state at the concentration corresponding to EC50) for a-tocopherol, ferulic acid and BHT were 10, 50 and 300 min, respectively (Sa´nchez-Moreno et al., 1998; Bondet et al., 1997). In the present study, the EC50 values were determined after 30 min of the reaction time and not at the steady state in order to provide identical experimental conditions for all the test compounds. The higher activity of ferulic acid and Tmix compared to that of BHT could readily be followed when the above-mentioned structural and kinetic factors were taken into consideration. It is also reported by Chen and Ho (1997) and by Nenadis and Tsimidou (2002) that the DPPH activities of ferulic acid and atocopherol were greater than that of BHT. The observed order FA > OYL is supported by other literature reports too. For example, Kikuzaki et al. (2002) have found that at 20 lM levels, the DPPH radical scavenging effect of ferulic acid was more than that of oryzanols and Xu and Godber (2001) have attributed the antioxidant activity of oryzanols to the phenolic hydroxyl group in the ferulate portion of their structure. Also, the EC50 value of 163 for ferulic acid as reported by Sa´nchez-Moreno et al. (1998) was in close agreement with the values obtained in the present study. The lower activity of tricin could be attributed to the methylation of the hydroxyl groups (3 and 5) in ring B as the 3 0 , 4 0 -dihydroxy structure (catechol structure) in flavonoids is important for the expression of radial scavenging activity (Watanabe, 1999).
For the fractions AE, AE-LP and AE-PP, it could be seen that both the order of DPPH radical scavenging activity and their respective phenolic contents (TPC) could be correlated and TPC content followed the order AEPP > AE > AE-LP. Further, the ferulic acid content of these fractions could be responsible for the lower EC50 values for these fractions. The DPPH scavenging activities therefore could be largely attributed to the levels of TPC and FA in these fractions. However, other constituents of the extracts such as proteins, sugars, and unidentified phenolics could also contribute to the higher antioxidant efficacy observed here. DPPH radical scavenging is considered a good in vitro model widely used to assess antioxidant efficacy in relatively short time. In its radical form, DPPH has an absorbance at 515 nm which disappears on reduction by an antioxidant compound (AH) or a radical species R to become a stable diamagnetic molecule with the result the colour changes from purple to yellow (Brand-Williams et al., 1995), which could be taken as an indication of the hydrogen donating ability of the tested compounds. Larrauri et al. (1999) have reported that the EC50 values of Spanish red wines ranged from 1030 to 2450 g/kg DPPH. Davalos et al. (2003) have reported EC50 values of 147,626 for strawberry extracts. The EC50 values of moringa leaf extracts have been shown to be in the range of 1050–3070 g/kg DPPH (Siddhuraju and Becker, 2003). DPPH activity is also reported for coloured rices (Oki et al., 2002) and rice hulls (Lee et al., 2003) and procyanidins have been attributed to the DPPH activity of red hulled rice (Oki et al., 2002) while dihydro ferulic acid and dihydro sinapic acid have been suggested as the major DPPH active compounds of Kurosu (Japanese unpolished rice vinegar) (Shimoji et al., 2002). The results of the present study indicated that the various phytochemical constituents of DRB extracts viz. oryzanols, ferulic acid, tricin and Tmix exhibited considerable DPPH activity and their synergistic effects could largely be responsible for the observed antiradical efficacies of DRB extracts. The DPPH activities of some of the DRB extracts (AE-PP) was equal to that of BHT and the activities of the phytochemical constituents viz. ferulic acid and Tmix was several fold higher that of BHT. Moreover, Tmix and ferulic acid possessed about one third the activity of TBHQ, a synthetic compound with very high antioxidant potentials. 3.3. Superoxide radical scavenging activity 3.3.1. Superoxide radical scavenging activity by cytochrome c method The superoxide scavenging effects of DRB extracts and their various phytochemical constituents at 20lg levels is shown in Fig. 3. In the control, superoxide radicals generated with a xanthine/xanthine oxidase system reduce ferricytochrome c with consequent increase in absorbance at 550 nm. After the addition of superoxide scavengers, the reduction rate of cytochrome c decreases. The IC50 values
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Fig. 3. Superoxide radical scavenging effects of DRB extracts and its phytochemical constituents at 20 lg levels by xanthine–xanthine oxidase/ cytochrome method.
interpolated from dose response curves for CME, AE, AELP, AE-PP, FA, Tmix, tricin, BHT and TBHQ were 47, 38, 57, 28, 34, 38, 35, 17 and 5 lg, respectively (Table 2). Thus, the activity of the experimental sets followed the order TBHQ > BHT > AE-PP > FA = tricin > AE = Tmix > CME > AE-LP. The IC50 values of oryzanols and b-sitosterol could not be determined by this method owing to their low solubility and consequent turbidity that affected the spectrophotometric measurement. The activity of ferulic acid and tricin were similar and was more than that of Tmix and the activity order for the extracts was AE-PP > AE > CME > AE-LP. 3.3.2. Superoxide radical scavenging by NBT method Xanthine/xanthine oxidase generated superoxide radicals reduce tetrazolium blue into formazan blue (kmax 560 nm), but in presence of radical scavengers, the formation of formazan blue is inhibited and therefore absorption at 560 nm decreases. Antiradical activity was defined as the amount of antioxidant necessary to decrease initial O 2 concentration by 50% (Gaulejac et al., 1999). The superoxide scavenging effects of DRB extracts and pure compounds at 40 lg levels is shown in Fig. 4. The IC50 values interpolated from dose response curves for CME, AE, AE-LP, AE-PP, FA and tricin were 125, 97, 171, 77, 39 and 41 lg, respectively (Table 2). The activity of the experimental sets followed the order FA = tricin > AE-PP > AE > CME > AE-LP. The IC50 values of BHT, TBHQ and Tmix could not be determined by this method as these compounds interfered with measurement by reducing NBT directly. Oryzanols and sterols were poorly soluble in this assay system too and hence their IC50 values could not be determined by this method also. Activities of ferulic acid and tricin were similar and the activity of the extracts followed the order AE-PP > AE > CME > AE-LP.
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Fig. 4. Superoxide radical scavenging effects of defatted rice bran extracts and its phytochemical constituents at 40 lg levels by xanthine–XOD/NBT method.
Xanthine oxidase (XOD) is one of the main enzymatic sources of reactive oxygen species (ROS) in vivo. Xanthine oxidase oxidizes xanthine or hypoxanthine to uric acid, superoxide anions and H2O2 (Unno et al., 2000). Though O 2 cannot directly initiate lipid oxidation, in the presence of metal ions, the highly reactive hydroxyl radical (OH) can be generated by the Fenton reaction (Kanner et al., 1987). Xanthine oxidase-derived superoxide radical has been linked to the postischemic tissue injury and generation of neutrophil chemotoxins (Cos et al., 1998). Under normal physiological conditions, the endogenous superoxide scavengers in the system protect tissues by neutralizing these radicals. This in vivo reaction is simulated in the in vitro model so as to use it as an analytical tool to evaluate the ROS scavenging abilities of natural products. The DRB extracts when assayed by cytochrome c method and NBT method showed positive radical scavenging effects. But the activities were found to be higher for the former method. The order of antioxidant efficacies by the two methods were found to be similar for the extracts and it was in the order AE-PP > AE > CME > AE-LP, which also followed the order of their TPC and ferulic acid contents. According to Khanom et al. (2000), the superoxide scavenging activity of Bangladeshi medicinal plants determined by the cytochrome method were mostly higher than that of the NBT method and they suggested that the former method measured not only the superoxide scavenging activity but also other biochemical properties. In the present study, the IC50 values for the DRB extracts by the cytochrome c method were in the range of 28–57 lg and those by the NBT method were in the range of 77–171 lg. These assays make use of the competition kinetics of reduction of cytochrome c/NBT (probe) and O 2 scavenger (antioxidant compound) by superoxide. But many antioxidants are capable of reducing the probes directly (Huang et al., 2005). In the present study, such a problem was not
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encountered with the cytochrome c method, whereas many tested antioxidants viz. BHT, TBHQ and Tmix was found to reduce NBT directly. This can in turn result in a lower estimate of the true superoxide scavenging activity of the tested compounds as only lesser amounts of the added compounds would now be available for scavenging O 2 . Complications can also arise in determining the extent to which the probe is reduced by the antioxidant and by O 2 . The situation will be more complex in the case of multicomponent natural extracts like DRB especially when it is not fully characterized. These methods were used by various authors for natural extracts. Hot water extracts of budrock were required at 1 mg levels to obtain 65% inhibition (Duh, 1998) and the IC50 value of tomato pulp was reported to be 1640 lg on dry weight basis (Lavelli and Giovanelli, 2003). Superoxide scavenging effects of fermented rice bran extracts were attributed to proteins by Miyamoto et al. (2000). Itani et al. (2002) have attributed the superior scavenging effects of red and purple black hulled rices compared to that of white hulled rices to the tannins and anthocyanins of the former. Kim et al. (1995) have studied the SOD-like activity of 24 lipophilic antioxidants including c-oryzanol by measuring the inhibition of pyrogallol autoxidation catalysed by superoxide radicals. The superoxide scavenging activity of ferulic acid was reported by Graf (1992). The reduction rate constant (K 102–106 M1 S1) of superoxide radicals by flavonoids is reported to be the highest among biological compounds (Jovanovic et al., 1998). The results of the present study demonstrated that the various phytochemical constituents of DRB extracts viz. ferulic acid, tricin and Tmix exhibited excellent superoxide radical scavenging activity thus directly supporting the superior antiradical efficacies of DRB extracts. However, the presence of proteins, sugars and other unidentified phenolics that constituted the bulk of the extracts would also have affected their antioxidant efficacy either synergistically or antagonistically. Acknowledgements The first author is grateful to CSIR for the senior research fellowship granted during the period of this work. The authors acknowledge the financial support from Technology Mission on Oil Seeds and Pulses (TMO&P), Government of India. The authors thank Director, Regional Research Laboratory, Thiruvananthapuram for his support and constant encouragement. References Adom, K.K., Liu, R.H., 2002. Antioxidant activity of grains. Journal of Agricultural and Food Chemistry 50, 6182–6187. Association of Official Analytical Chemsits (AOAC), 1984. Official Methods of Analysis, 11th ed. Association of Official Analytical Chemsits, Washington, DC.
Babior, B.M., 2000. Phagocytes and oxidative stress. American Journal of Medicine 109, 33–44. Bondet, V., Brand-Williams, W., Berset, C., 1997. Kinetics and mechanisms of antioxidant activity using the DPPH free radical method. Food Science and Technology 30, 609–615. Brand-Williams, W., Cuvelier, M.E., Berset, C., 1995. Use of free radical method to evaluate antioxidant activity. Lebensmittel-Wissenchaft und Technologie 28, 25–30. Branen, A.C., 1975. Toxicology and biochemistry of butylated hydroxy anisole and butylated hydroxyl toluene. Journal of the American Oil Chemists Society 52, 59–63. Burton, G.W., Doba, T., Gabe, E.J., Hughes, L., Lee, F.L., Prasad, L., Ingold, K.U., 1985. Autoxidation of biological molecules. 4. Maximising the antioxidant activity of phenols. Journal of American Chemical Society 107, 7053–7065. Chen, J.H., Ho, C.-T., 1997. Antioxidant activities of caffeic acid and its related hydroxycinnamic acid compounds. Journal of Agricultural and Food Chemistry 45, 2374–2378. Chen, Z.Y., Chan, P.T., Ho, K.Y., Fung, K.P., Wang, J., 1996. Antioxidant activity of natural flavonoids is governed by number and location of their aromatic hydroxyl groups. Chemistry and Physics of Lipids 79, 157–163. Cos, P., Ying, L., Calomme, M., Hu, J.P., Cimanga, K., Van Poel, B., Pieters, L., Vlietinck, A.J., Berghe, D.V., 1998. Structure–activity relationship and classification of flavonoids as inhibitors of xanthine oxidase and superoxide scavengers. Journal of Natural Products 61, 71–76. Cosgrove, J.P., Church, D.F., Pryor, W.A., 1987. The kinetics of the autoxidation of polyunsaturated fatty acids. Lipids 22, 299–304. Davalos, A., Gomez-Cordoves, C., Bartolome, B., 2003. Commercial dietary antioxidant supplements assayed for their antioxidant activity by different methodologies. Journal of Agricultural and Food Chemistry 51, 2512–2519. Davalos, A., Bartolome, B., Gomez-Cordoves, C., 2004. Inhibition of methyl linoleate autoxidation by phenolics and other related compounds under mild oxidative conditions. Journal of the Science of Food and Agriculture 84, 631–638. Duh, P.-D., 1998. Antioxidant activity of burdock (Arctium lappa Linne´): its scavenging effect on free-radical and active oxygen. Journal of the American Oil Chemists society 75, 455–461. Duncan, D.B., 1955. Multiple range and multiple F tests. Biometrics 11, 1– 42. Gaulejac, N.S.-C., Vivas, N., Freitas, V., Bourgeois, G., 1999. The influence of various phenolic compounds on scavenging activity assessed by an enzymatic method. Journal of the Science of Food and Agriculture 79, 1081–1090. Graf, E., 1992. Antioxidant potential of ferulic acid. Free Radical Biology and Medicine 13, 435–448. Hargrove Jr., K.L., 1994. Processing and utilization of rice bran in the United States. In: Marshall, W.E., Wadsworth, J.I. (Eds.), Rice Science and Technology. Marcel Decker Inc., New York, pp. 381–404. Huang, D., Ou, B., Prior, R.L., 2005. The chemistry behind antioxidant capacity assays. Journal of Agricultural and Food Chemistry 53, 1841– 1856. Itani, T., Tatemoto, H., Okamoto, M., Fujii, K., Muto, N., 2002. A comparative study on antioxidative activity and polyphenol content of colored kernel rice. Nippon Shokuhin Kagaku Kogaku Kaishi 49, 540–543. Jovanovic, S.V., Steenken, S., Simic, M.G., Hara, Y., 1998. In: RiceEvans, C.A., Packer, C. (Eds.), Flavonoids in Health and Disease. Marcel Decker, New York, pp. 137–161. Kanner, J., German, J.B., Kinsella, J.E., 1987. Initiation of lipid peroxidation in biological systems. Critical Reviews in Food Science and Nutrition 25, 317–364. Khanom, F., Kayahara, H., Tadasa, K., 2000. Superoxide-scavenging and prolyl endopeptidase inhibitory activities of Bangladeshi indigenous medicinal plants. Bioscience, Biotechnology and Biochemistry 64, 837– 840.
R. Renuka Devi, C. Arumughan / Food and Chemical Toxicology 45 (2007) 2014–2021 Kikuzaki, H., Hisamoto, M., Hirose, K., Akiyama, K., Taniguchi, H., 2002. Antioxidant properties of ferulic acid and its related compounds. Journal of Agricultural and Food Chemistry 50, 2161–2168. Kim, S.J., Han, D., Moon, K.D., Rhee, J.S., 1995. Measurement of superoxide dismutase-like activity of natural antioxidants. Bioscience, Biotechnology and Biochemistry 59, 822–826. Kitts, D.D., Wijewickreme, A.N., Hu, C., 2000. Antioxidant properties of a North American ginseng extract. Molecular and Cellular Biochemistry 203, 1–10. Larrauri, J.A., Sanchez-Moreno, C., Ruperez, P., Saura-Calixto, F., 1999. Free radical scavenging capacity in the aging of selected red Spanish wines. Journal of Agricultural and Food Chemistry 47, 1603–1606. Lavelli, V., Giovanelli, G., 2003. Evaluation of heat and oxidative damage during storage of processed tomato products. II. Study of oxidative damage indices. Journal of the Science of Food and Agriculture 83, 966–971. Lee, S.-C., Kim, J.-H., Jeong, S.-M., Kim, D.-R., Ha, J.-U., Nam, K.C., Ahn, D.U., 2003. Effect of far-infrared radiation on the antioxidant activity of rice hulls. Journal of Agricultural and Food Chemistry 51, 4400–4403. McMurry, J., 1984. Organic Chemistry. Brooks/Cole Publishing Company, Belmont, pp. 478–515. Miyamoto, Y., Noda, H., Ohya, H., Kamada, H., 2000. SOD-like activities of rice bran extracts after fermentation. Nippon Shokuhin Kagaku Kogaku Kaishi 47, 214–219. Nenadis, N., Tsimidou, M., 2002. Observations on the estimation of scavenging activity of phenolic compounds using rapid 1, 1-diphenyl2-picrylhydrazyl (DPPH) tests. Journal of the American Oil Chemists Society 79, 1191–1195. Oki, T., Masuda, M., Kobayashi, M., Nishiba, Y., Furuta, S., Suda, I., Sato, T., 2002. Polymeric procyanidins as radical-scavenging components in red-hulled rice. Journal of Agricultural and Food Chemistry 50, 7524–7529. Ramarathnam, N., Ochi, H., Takeuchi, M., 1997. Antioxidative defense system in vegetable extracts. In: Shahidi, F. (Ed.), Natural Antioxidants, Chemistry, Health Effects, and Applications. AOCS Press, Illinois, pp. 76–77. Renuka Devi, R., 2005. Studies on the bioactive natural antioxidants from oilseeds. Ph.D. Thesis. Cochin University of Science and Technology, pp. 99–104.
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Renuka Devi, R., Arumughan, C., 2007. Phytochemical characterization of defatted rice bran and optimization of a process for their extraction and enrichment. Bioresource Technology 98, 3037–3043. Renuka Devi, R., Suja, K.P., Jayalekshmy, A., Arumughan, C., 2000. Tocopherol and tocotrienol profiles of some vegetable oils by HPLC. Journal of Oil Technologists Association of India 32, 176–182. Sa´nchez-Moreno, C., Larrauri, J.A., Saura-Calixto, F., 1998. A procedure to measure the antiradical efficiency of polyphenols. Journal of the Science of Food and Agriculture 76, 270–276. Shahidi, F., Wanasundara, P.K., 1992. Phenolic antioxidants. Critical Reviews in Food Science and Nutrition 32, 67–103. Shimoji, Y., Tamura, Y., Nakamura, Y., Nanda, K., Nishidai, S., Nishikawa, Y., Ishihara, N., Uenakai, K., Ohigashi, H., 2002. Isolation and identification of DPPH radical scavenging compounds in Kurosu (Japanese unpolished rice vinegar). Journal of Agricultural and Food Chemistry 50, 6501–6503. Shin, Z.I., Chang, Y.S., Kang, W.S., Jung, S.U., 1992. Antioxidant extracted from the defatted rice bran. US Patent 5,175,012. Siddhuraju, P., Becker, K.J., 2003. Antioxidant properties of various solvent extracts of total phenolic constituents from three different agroclimatic origins of drumstick tree (Moringa oleiferaLam.) leaves. Journal of Agricultural and Food Chemistry 51, 2144–2155. Sur, P., Chaudhuri, T., Vedasiromani, J.R., Gomez, A., Ganguli, D.K., 2001. Anti-inflammatory and antioxidant property of saponins of tea [Camellia sinensis (L.) O. Kuntze] root extract. Phytotherapy Research 15, 174–176. Unno, T., Sugimoto, A., Kakuda, T., 2000. Scavenging effect of tea catechins and their epimers on superoxide anion radicals generated by a hypoxanthine and xanthine oxidase system. Journal of the Science of Food and Agriculture 80, 601–606. Watanabe, M., 1999. Antioxidative phenolic compounds from Japanese barnyard millet (Echinochloa utilis) grains. Journal of Agricultural and Food Chemistry 47, 4500–4505. Wettasinghe, M., Shahidi, F., Amarowics, R., 2002. Identification and quantification of low molecular weight phenolic antioxidants in seeds of evening primrose (Oenothera biennis L.). Journal of Agricultural and Food Chemistry 50, 1267–1271. Xu, Z., Godber, J.S., 2001. Antioxidant activities of major components of c-oryzanol from rice bran using a linoleic acid model. Journal of the American Oil Chemists Society 78, 645–649.
Antiradical efficacy of phytochemical extracts from defatted rice bran R. Renuka Devi, C. Arumughan
*
Agroprocessing and Natural Products Division, Regional Research Laboratory (CSIR), Thiruvananthapuram 695 019, Kerala, India Received 16 May 2006; accepted 26 April 2007
Abstract The phytochemical compounds oryzanols, tocopherols, tocotrienols and ferulic acid were identified in the crude methanolic extracts (CME) of defatted rice bran (DRB) by HPLC. Enrichment of antioxidants in CME was achieved by sequential extraction and fractionation resulting in three enriched fractions viz. acetone extract (AE), acetone extract-lipophilic fraction (AE-LP) and acetone extract-polar fraction (AE-PP). The scavenging effects of DRB extracts and their phytochemical constituents against DPPH and superoxide radicals were investigated. The EC50 (g antioxidant/kg DPPH) values of CME, AE, AE-LP, AE-PP, oryzanols, ferulic acid, tocols (Tmix), tricin, b-sitosterol, BHT and TBHQ were 1977, 1945, 7985, 1072, 972, 174, 164, 3947, 21416, 1120 and 61, respectively. Tricin and b-sitosterol isolated from CME were identified with the help of spectral data. The DRB extracts and their phytochemical constituents when assayed by cytochrome c and NBT methods showed positive superoxide radical scavenging effects. The order of efficacies of the extracts was AE-PP > AE > CME > AE-LP in both assays, but the activities were higher for the former method. The DPPH as well as superoxide scavenging activities of AE, AE-LP and AE-PP could largely be attributed to the levels of total phenols (TPC) and ferulic acid in it. 2007 Published by Elsevier Ltd. Keywords: Defatted rice bran; Phytochemicals; HPLC; DPPH; Superoxide radicals
1. Introduction Reactive oxygen species (ROS) such as superoxide O 2 , H2O2 and hydroxyl radicals (OH) are byproducts of normal metabolism and attack certain biological molecules, leading to destabilisation and disintegration of cell membranes and many age related diseases (Babior, 2000). Lipid peroxidation which has a profound role both in quality deterioration of lipid containing foods as well as in a number of destructive biological processes is also mediated by free radicals (Cosgrove et al., 1987). In the human body,
Abbreviations: AE, acetone extract; AE-LP, acetone extract-lipophilic fraction; AE-PP, acetone extract-polar fraction; CME, crude methanolic extract; DPPH, 1,1-diphenyl-2-picrylhydrazyl radical; DRB, defatted rice bran; HPLC, high performance liquid chromatography; NBT, nitro blue tetrazolium; ROS, reactive oxygen species; TPC, total phenolic content; XOD, xanthine oxidase. * Corresponding author. Tel.: +91 471 492901; fax: +91 471 491712. E-mail address: [email protected] (C. Arumughan). 0278-6915/$ - see front matter 2007 Published by Elsevier Ltd. doi:10.1016/j.fct.2007.04.020
the toxic effects of ROS are combated regularly by a number of endogenous defense and protective mechanisms which include various enzymes and non-enzymatic antioxidants. These self-defense systems may also be supported by antioxidative compounds taken as foods, cosmetics and medicine particularly in the elderly (Ramarathnam et al., 1997). Though many synthetic compounds like BHA, BHT, TBHQ, etc. are efficient antioxidants, their use is being restricted because of their possible toxic and carcinogenic effects (Branen, 1975). Thus, the health concern of synthetic food additives and evidence in phytonutrients as chemopreventive agents led to resurgence in bio- and chemoprospecting of plant materials for potential therapeutic properties. Agro-industrial byproducts like oilseed meals are also re-examined from this perspective and a few deoiled meals from sesame, soybean, canola, peanut, cotton seeds, mustard, etc. have been phytochemically characterized (Wettasinghe et al., 2002). However, rice bran meal (defatted rice bran) has not been studied in detail. Defatted rice bran (DRB), the predominant byproduct of rice bran oil (RBO)
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extraction is a good source of insoluble dietary fiber, protein, phytic acid, inositol and vitamin B (Hargrove, 1994). Though the antioxidant potential of DRB was also known (Shin et al., 1992), it still remained a relatively unexplored source material, which demanded further investigation especially with regard to its phytochemical composition related to possible health benefits as antioxidants. Free radical scavenging is generally the accepted mechanism for antioxidants inhibiting lipid oxidation and radical scavenging studies have emerged as a convenient methodology for its measurement (Brand-Williams et al., 1995). The specific radicals used could be stable ones like DPPH or those generated in chemical model systems like ABTS+, superoxide radical, etc. (Davalos et al., 2004). Measurement of radical scavenging activity using discoloration of 1,1diphenyl-2-picrylhydrazyl radicals (DPPH) has been widely used due to its stability, simplicity and reproducibility (Kitts et al., 2000). In the present study, the radical scavenging effects of DRB extracts and their various phytochemical constituents were studied using the stable DPPH radical and superoxide radicals generated in situ by the xanthine– xanthine oxidase system, one of the main enzymatic sources of reactive oxygen species in vivo (Unno et al., 2000).
2. Materials and methods 2.1. Materials Defatted rice bran samples were obtained from rice bran oil industry (M/s. Chakkiyathumooda Solvent Extractions, Ankamali, Kerala, India). Tocopherol standards were obtained from E-Merck, Darmstadt, Germany and tocotrienol standards from Calbiochem (Calbiochem–Novabiochem Co., San Diego, CA, USA). Ferulic acid, 1,1-diphenyl-2-picrylhydrazyl radical (DPPH), nitro blue tetrazolium (NBT) salt, xanthine, xanthine oxidase and ferricytochrome c were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Standard compounds of oryzanols, cycloartenyl ferulate and 24-methylene cycloartanyl ferulate were a generous gift from M/s. Tsuno Rice Fine Chemicals, Wakayama, Japan. High performance liquid chromatography (HPLC) grade solvents were purchased from Merck (India) Ltd. All other reagents were of analytical grade.
2.2. Extraction and enrichment of antioxidants from DRB Defatted rice bran (10 g) was extracted with methanol (150 ml) for 10 h in a soxhlet extractor. The extract was filtered through a Whatman No. 1 filter paper and the solvent was removed under vacuum/N2 flow to dryness. The weighed residue was redissolved in methanol to get an antioxidant solution of known concentration. The crude methanolic extract (CME) thus obtained was partially purified by re-extraction with acetone to give an acetone extract (AE). For further purification of the acetone extract, sequential extraction was employed yielding a lipophilic phase (AE-LP) with hexane and a polar phase (AE-PP) with acetone. All the extracts/fractions were evaporated to dryness under reduced pressure on a rotary evaporator at 40 C using amber colored round bottom flasks. The dried extracts were weighed and stored at 4 C under nitrogen atmosphere and were dissolved in methanol for routine analysis within 4 weeks.
2.3. Silica gel column chromatography The extract (20 g) was chromatographed on a glass column (5 cm · 120 cm) packed with 400 g of silica gel (100–200 mesh particle size,
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Merck). The column was eluted with gradients of petroleum ether : ethyl acetate (100% petroleum ether to 100% ethyl acetate). Eight hundred milliliters fractions were collected and fractions similar in TLC profiles were pooled. Pure compounds 1 and 2 were obtained from fractions eluted with 20% ethyl acetate and 50% ethyl acetate, respectively. Compound 1 was recrystallised from petroleum ether and compound 2 from ethyl acetate and were identified with the help of UV, IR, NMR and mass spectral (MS) data.
2.4. Compositional analysis of DRB extracts The extracts were analysed for fat, protein, sugar and ash according to AOAC methods (1984). The total phenolic content (TPC) of the extracts was determined using Folin–Ciocalteau reagent (AOAC, 1984).
2.5. Identification of active compounds in rice bran extracts 2.5.1. Analysis of oryzanols The analysis was performed using a Shimadzu make HPLC binary system (Kyoto, Japan) with a LC-10 AD model pump, a 7125 model Rheodyne injector (Cotati, CA, USA) fitted with a 20 ll sample loop, a SPD-10A UV–vis detector, with a C-R7 Ae plus integrator for data acquisition and display. A Waters l-bondapak TM C18 column (4.6 mm i.d. · 25 cm) (Millford, MA) was used in the reversed-phase with the solvent system of acetonitrile, dichloromethane, and acetic acid (88:6:6, by vol):methanol, n-butyl alcohol, water (90:2:8, by vol) in the ratio of 75:25 (v/v) at a flow rate of 1 ml/min. The UV detector was set at 325 nm. All the extracts were filtered through PTFE membrane before injection into the HPLC column. Samples were diluted with the mobile phase and analysed. Peak identification was based on comparison of RT values with authentic standards of cycloartenyl ferulate, 24-methylene cycloartanyl ferulate and a mixture of oryzanol standards. The total oryzanols were quantified based upon the peak area of 24-methylene cycloartanyl ferulate, the major oryzanol component in rice bran (Renuka Devi and Arumughan, 2007). 2.5.2. Analysis of ferulic acid Preliminary trials were done using isocratic elution with 20% acetonitrile in water adjusted to pH 2 with trifluoroacetic acid and at a wavelength of 280 nm (Adom and Liu, 2002). But later on, the same HPLC conditions used for oryzanols was followed. The main advantage was the simultaneous detection of both ferulic acid and oryzanols in a single run. Peak identification was based on comparison of RT values with authentic standard of trans-ferulic acid and was further confirmed by spiking studies. The ferulic acid concentration of sample extracts was extrapolated from the pure trans-ferulic acid standard curve. 2.5.3. Analysis of tocopherols and tocotrienols The same HPLC system and a Shim-pack (LC-NH2(M)) column (4.6 mm i.d. · 25 cm) was used in the normal-phase with the solvent system, n-hexane:isopropanol (96:4, v/v) and at a flow rate of 1 ml/min. The UV detector was set at 297 nm (Renuka Devi et al., 2000). Peak identification was based on comparison of RT values with authentic standards of tocopherols and tocotrienols. The total vitamin E forms were quantitated based upon the peak areas relative to standard calibration plots by external standard method. Samples were redissolved in n-hexane and 20 ll of the solutions were injected into the HPLC column.
2.6. Determination of DPPH radical scavenging capacity The method of Brand-Williams et al. (1995) modified by Sa´nchezMoreno et al. (1998) was followed. 0.025 g/l DPPH solution was prepared in methanol. Various concentrations of samples (extracts and pure compounds) based on respective dry weights were also prepared in methanol. To 3.9 ml of DPPH solution, 0.1 ml of appropriately diluted sample solution was added and absorbance at 515 nm was measured at different time intervals in a UV–vis spectrophotometer (UV-160A, Shimadzu) for
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30 min. The DPPH concentration in the reaction medium was calculated using the following equation obtained by linear regression: A 515 nm = 2936.68 [DPPH]T 2.18 · 103. The percentage of remaining DPPH was calculated as % DPPHREM ¼ ½DPPH T ¼T =½DPPH T ¼0 100, where [DPPH]T=T is the concentration of DPPH at 30 min time and [DPPH]T=0 is the concentration at zero time (initial concentration). The percentage of remaining DPPH against the standard/sample concentration was plotted to obtain the EC50 concentration (the amount of antioxidant required to decrease the initial DPPH concentration by 50%).
2.7. Determination of superoxide radical scavenging activity Superoxide radicals generated in situ by the xanthine–xanthine oxidase system was monitored using two different probes viz. ferricytochrome c and nitro blue tetrazolium. 2.7.1. Superoxide scavenging by ferricytochrome c method The superoxide scavenging ability of DRB extracts was studied by xanthine/xanthine oxidase/ferricytochrome c method according to Khanom et al. (2000). Briefly, the assay was performed in a 3.0 ml of a reaction mixture in a 1.0 cm cuvette. The assay mixture consisted of 0.3 ml of a sample solution, 0.5 ml of ferricytochrome c (1 · 105 M), 0.5 ml of xanthine (5 · 106 M), 1.0 ml distilled water and 0.5 ml of a KH2PO4/ K2HPO4 buffer (50 mM; pH 7.8) containing EDTA (1 · 104 M). A solution of 0.2 ml of xanthine oxidase (0.5 · 108 M) was added to start the reaction and the rate of reduction of ferricytochrome c was determined by using UV–vis spectrophotometer at 550 nm at 0 s and thereafter every 30 s for up to 900 s. Superoxide radical scavenging capacities of the additives were calculated as follows: % ferricytochrome reduction = [rate of change of absorbance of sample]/[rate of change of absorbance of control] · 100. Radical scavenging ability = 100 % ferricytochrome reduction.
CME and tocols of CME are given in Figs. 1 and 2, respectively. Compositional analysis of CME showed 7.3% fat, 24.0% sugar, 22.5% protein and 3.7% minerals. The total phenolic content was 5.3%. Oryzanols, ferulic acid and tocol contents of CME in ppm were 7812, 5756 and 138, respectively. Re-extraction of CME with acetone yielded an acetone extract (AE) which was enriched more than 1.5fold in TPC, oryzanols, ferulic acid and tocols compared to CME (Table 1). Further purification of the acetone extract employing sequential extraction yielded a lipophilic phase (AE-LP) and a polar phase (AE-PP). AE-LP was enriched in oryzanols and tocols and their quantities were increased by two fold or more compared to those in CME. However, the contents of total phenolics, and ferulic acid were decreased significantly (Table 1). On the other hand, AE-PP fraction was enriched in TPC and ferulic acid by three-fold compared to those in CME with a consequent decrease in the contents of oryzanols and tocols (Table 1). Pure compounds 1 and 2 isolated from CME by column chromatography were identified to b-sitosterol and tricin with the help of UV, IR, NMR and MS data (Renuka Devi, 2005). 3.2. DPPH radical scavenging activity In the present study, various concentrations (based on respective dry weights) of DRB extracts viz. CME, AE,
2.7.2. Superoxide scavenging by NBT method The superoxide scavenging ability of DRB extracts was studied by xanthine/xanthine oxidase/NBT method according to Sur et al. (2001). The assay mixture consisted of 1 ml of sodium carbonate/ethylene diamine tetraacetic acid buffer, (carbonate buffer 50 mM, pH 10.2 containing EDTA), 50 ll of xanthine (8.7 mg ml1), 10 ll of NBT (6 mg ml1) and 20 ll of test sample. Twenty millilitres of xanthine oxidase (10 mg ml1) was added to start the reaction and the time course for the reduction of NBT into formazan complex was followed for about 15 min at 560 nm. The percentage of NBT reduction was calculated as follows: % NBT reduction = [rate of change of absorbance of sample]/[rate of change of absorbance of control] · 100. Radical scavenging ability = 100 % NBT reduction.
2.8. Statistical analysis All measurements were duplicated on duplicate samples (2 · 2). The results were statistically analysed by ANOVA and DMRT. Statistical significance was accepted at a level of P < 0.05 (Duncan, 1955).
3. Results and discussion 3.1. Chemical and phytochemical composition of DRB extracts The optimization of the process parameters for the preparation and enrichment of an antioxidant extract from DRB was based on the yields of TPC and also on the yields of oryzanols, tocols and ferulic acid, the phytochemicals identified in DRB extracts (Renuka Devi and Arumughan, 2007). The HPLC profiles of oryzanols and ferulic acid of
Fig. 1. Reverse- phase HPLC/UV–vis detection analysis of oryzanols and ferulic acid of defatted rice bran. Peak identification: (1) ferulic acid; (2) unidentified; (3) stigmasteryl ferulate; (4) unidentified; (5) cycloartenyl ferulate; (6) 24-methylene cycloartanyl ferulate; (7) campesteryl ferulate; (8) b-sitosteryl ferulate; (9) cycloartanyl ferulate.
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2017
idant/kg DPPH) were taken as a measure of antiradical activity. The lower the EC50, higher the antioxidant potential (Brand-Williams et al., 1995). The EC50 (g antioxidant/kg DPPH) values of CME, AE, AE-LP, AE-PP, oryzanols (OYL), ferulic acid (FA), Tmix, tricin, b-sitosterol, BHT and TBHQ were 1977, 1945, 7985, 1072, 972, 174, 164, 3947, 21416, 1120 and 61, respectively (Table 2). The order of antiradical activity was TBHQ > FA = Tmix > OYL > AE-PP = BHT > CME = AE > tricin > AE-LP > b-sitosterol. The activity of the phytochemical constituents followed the order FA = Tmix > OYL > tricin > sterol, whereas for the extracts the order was AE-PP > CME = AE > AELP. The activity of AE-PP equalled that of BHT and for ferulic acid and Tmix it was several fold higher than that of BHT. The number of hydroxyl groups on an aromatic ring is the major factor contributing to the efficacy of phenolic antioxidants (Chen et al., 1996). This may explain the higher activity of TBHQ (a diphenolic antioxidant) compared to that of BHT, a monophenolic antioxidant. The Table 2 Free radical scavenging capacitiesA of defatted rice bran extracts TreatmentsB
CME AE AE-LP AE-PP BHT TBHQ OYL FA Tmix Sterol Tricin
Fig. 2. Normal-phase HPLC/UV–vis detection analysis of tocopherols (T) and tocotrienols (T3) of defatted rice bran. Peak identification: (1) a-T; (2) a-T3; (3) c-T3; (4) d-T; (5) d-T3.
AE-LP and AE-PP, its phytochemical constituents oryzanols, ferulic acid, Tmix, tricin and b-sitosterol and synthetic antioxidants viz. BHT and TBHQ were allowed to react with DPPH. Tmix constituted a 1:1 mixture of a-T and cT3, the predominant tocopherol and tocotrienol homologs identified in DRB extracts. At each concentration, a graph was plotted with time versus % DPPHREM (DPPH remaining) for 30 min. The % DPPHREM was then plotted against the standard concentration to obtain the amount of antioxidant necessary to decrease the initial DPPH concentration by 50% and is denoted as EC50. The EC50 values (g antiox-
Radical scavenging capacity in various models DPPH, EC50 values (g/kg DPPH)
Superoxide/ cytochrome, IC50 values (lg)
1977 (±39)e 1945 (±34)e 7985 (±76)g 1072 (±23)d 1120 (±26)d 61 (± 3)a 972 (±24)c 174 (±8)b 164 (±7)b 21,416 (±165)h 3947 (±59)f
47 38 57 28 17 5 – 34 38 – 35
(±2.6)f (±2.2)e (±2.5)g (±1.6)c (±1.0)b (±0.3)a (±1.7)d (±2.1)e (±1.7)d
X/XO/NBT, IC50 values (lg) 125 97 171 77 – – – 39 – – 41
(±6.8)d (±6.1)c (±7.8)e (±3.8)b
(±2.6)a
(±2.5)a
a–h
Means within a column with different letters are significantly different (P < 0.05) according to DMRT. A Each value in the table represents the mean (±standard deviation) of four analyses from two replications. B CME, crude methanolic extract; AE, acetone extract; AE-LP, acetone extract-lipophilic phase; AE-PP, acetone extract-polar phase; OYL, oryzanol; FA, ferulic acid.
Table 1 Phytochemical compositionA of defatted rice bran extracts ExtractsB
Recovery (%)C
Phytochemical constituents in the extract TPC (wt%)
CME AE AE-LP AE-PP a–d
– 47.7c (±1.8) 26.6b (±0.9) 19.9a (±0.8)
b
5.3 (±0.2) 10.5c (±0.3) 4.7a (±0.2) 17.5d (±0.4)
Oryzanols (ppm) b
7812 (±57) 14,680c (±102) 20,445d (±129) 960a (±9)
Ferulic acid (ppm) b
5756 (±33) 9188c (±66) 932a (±8) 15,820d (±113)
Tocols (ppm) b
138 (±2) 253c (±3) 335d (±4) 45a (±1)
Tricin (wt%)
Sterol (wt%)
0.4 – – –
1.1 – – –
Means within a column with different letters are significantly different (P < 0.05) according to DMRT. Each value in the table represents the mean (±standard deviation) of four analyses from two replications. B CME, crude methanolic extract; AE, acetone extract; AE-LP, acetone extract-lipophilic phase; AE-PP, acetone extract-polar phase. C Recovery percentages are based on CME; for AE-LP and AE-PP, recovery percentages based on AE are 55.8 (±1.9) and 41.8 (±1.8), respectively.
A
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antiradical activity of BHT, ferulic acid and tocols is due to their phenolic hydrogen atoms. In BHT, both the ortho positions to the hydroxy function are substituted by tertiary-butyl groups with strong inductive effects which can enhance the stability of the phenoxyl radical. More over, the steric hindrance exerted by the bulky t-butyl groups reduces the rate of possible propagation reactions involving antioxidant radicals thus increasing its stability (Shahidi and Wanasundara, 1992). The hydroxyl group in ferulic acid is para-substituted on an aromatic ring which is connected to a highly conjugated side chain which allows the phenoxyl radical to be delocalised across the entire molecule and therefore stabilised (Graf, 1992). The ortho substitution with the electron donor methoxy group is another factor that enhances the stability and hence the antioxidant and antiradical activities of ferulic acid (McMurry, 1984). The chromanoxyl radicals (TO) formed in the case of tocopherols and tocotrienols (chromanols) also are stabilised by methyl groups in ortho positions. In addition to forming harmless adducts with other radicals, TO can also undergo self-coupling to form dimers which too possess antioxidant activity (Burton et al., 1985). Apart from these structural aspects, kinetic factors also contribute to the observed activity order of FA = Tmix > BHT. Tocopherols are reported to have intermediate kinetic behaviour reaching the steady state within 5–30 min (Sa´nchez-Moreno et al., 1998), whereas BHT and ferulic acid showed slow kinetic behaviour taking more than 30 min to reach the steady state (Sa´nchez-Moreno et al., 1998; Bondet et al., 1997). The reported TEC50 values (time to reach the steady state at the concentration corresponding to EC50) for a-tocopherol, ferulic acid and BHT were 10, 50 and 300 min, respectively (Sa´nchez-Moreno et al., 1998; Bondet et al., 1997). In the present study, the EC50 values were determined after 30 min of the reaction time and not at the steady state in order to provide identical experimental conditions for all the test compounds. The higher activity of ferulic acid and Tmix compared to that of BHT could readily be followed when the above-mentioned structural and kinetic factors were taken into consideration. It is also reported by Chen and Ho (1997) and by Nenadis and Tsimidou (2002) that the DPPH activities of ferulic acid and atocopherol were greater than that of BHT. The observed order FA > OYL is supported by other literature reports too. For example, Kikuzaki et al. (2002) have found that at 20 lM levels, the DPPH radical scavenging effect of ferulic acid was more than that of oryzanols and Xu and Godber (2001) have attributed the antioxidant activity of oryzanols to the phenolic hydroxyl group in the ferulate portion of their structure. Also, the EC50 value of 163 for ferulic acid as reported by Sa´nchez-Moreno et al. (1998) was in close agreement with the values obtained in the present study. The lower activity of tricin could be attributed to the methylation of the hydroxyl groups (3 and 5) in ring B as the 3 0 , 4 0 -dihydroxy structure (catechol structure) in flavonoids is important for the expression of radial scavenging activity (Watanabe, 1999).
For the fractions AE, AE-LP and AE-PP, it could be seen that both the order of DPPH radical scavenging activity and their respective phenolic contents (TPC) could be correlated and TPC content followed the order AEPP > AE > AE-LP. Further, the ferulic acid content of these fractions could be responsible for the lower EC50 values for these fractions. The DPPH scavenging activities therefore could be largely attributed to the levels of TPC and FA in these fractions. However, other constituents of the extracts such as proteins, sugars, and unidentified phenolics could also contribute to the higher antioxidant efficacy observed here. DPPH radical scavenging is considered a good in vitro model widely used to assess antioxidant efficacy in relatively short time. In its radical form, DPPH has an absorbance at 515 nm which disappears on reduction by an antioxidant compound (AH) or a radical species R to become a stable diamagnetic molecule with the result the colour changes from purple to yellow (Brand-Williams et al., 1995), which could be taken as an indication of the hydrogen donating ability of the tested compounds. Larrauri et al. (1999) have reported that the EC50 values of Spanish red wines ranged from 1030 to 2450 g/kg DPPH. Davalos et al. (2003) have reported EC50 values of 147,626 for strawberry extracts. The EC50 values of moringa leaf extracts have been shown to be in the range of 1050–3070 g/kg DPPH (Siddhuraju and Becker, 2003). DPPH activity is also reported for coloured rices (Oki et al., 2002) and rice hulls (Lee et al., 2003) and procyanidins have been attributed to the DPPH activity of red hulled rice (Oki et al., 2002) while dihydro ferulic acid and dihydro sinapic acid have been suggested as the major DPPH active compounds of Kurosu (Japanese unpolished rice vinegar) (Shimoji et al., 2002). The results of the present study indicated that the various phytochemical constituents of DRB extracts viz. oryzanols, ferulic acid, tricin and Tmix exhibited considerable DPPH activity and their synergistic effects could largely be responsible for the observed antiradical efficacies of DRB extracts. The DPPH activities of some of the DRB extracts (AE-PP) was equal to that of BHT and the activities of the phytochemical constituents viz. ferulic acid and Tmix was several fold higher that of BHT. Moreover, Tmix and ferulic acid possessed about one third the activity of TBHQ, a synthetic compound with very high antioxidant potentials. 3.3. Superoxide radical scavenging activity 3.3.1. Superoxide radical scavenging activity by cytochrome c method The superoxide scavenging effects of DRB extracts and their various phytochemical constituents at 20lg levels is shown in Fig. 3. In the control, superoxide radicals generated with a xanthine/xanthine oxidase system reduce ferricytochrome c with consequent increase in absorbance at 550 nm. After the addition of superoxide scavengers, the reduction rate of cytochrome c decreases. The IC50 values
R. Renuka Devi, C. Arumughan / Food and Chemical Toxicology 45 (2007) 2014–2021
Fig. 3. Superoxide radical scavenging effects of DRB extracts and its phytochemical constituents at 20 lg levels by xanthine–xanthine oxidase/ cytochrome method.
interpolated from dose response curves for CME, AE, AELP, AE-PP, FA, Tmix, tricin, BHT and TBHQ were 47, 38, 57, 28, 34, 38, 35, 17 and 5 lg, respectively (Table 2). Thus, the activity of the experimental sets followed the order TBHQ > BHT > AE-PP > FA = tricin > AE = Tmix > CME > AE-LP. The IC50 values of oryzanols and b-sitosterol could not be determined by this method owing to their low solubility and consequent turbidity that affected the spectrophotometric measurement. The activity of ferulic acid and tricin were similar and was more than that of Tmix and the activity order for the extracts was AE-PP > AE > CME > AE-LP. 3.3.2. Superoxide radical scavenging by NBT method Xanthine/xanthine oxidase generated superoxide radicals reduce tetrazolium blue into formazan blue (kmax 560 nm), but in presence of radical scavengers, the formation of formazan blue is inhibited and therefore absorption at 560 nm decreases. Antiradical activity was defined as the amount of antioxidant necessary to decrease initial O 2 concentration by 50% (Gaulejac et al., 1999). The superoxide scavenging effects of DRB extracts and pure compounds at 40 lg levels is shown in Fig. 4. The IC50 values interpolated from dose response curves for CME, AE, AE-LP, AE-PP, FA and tricin were 125, 97, 171, 77, 39 and 41 lg, respectively (Table 2). The activity of the experimental sets followed the order FA = tricin > AE-PP > AE > CME > AE-LP. The IC50 values of BHT, TBHQ and Tmix could not be determined by this method as these compounds interfered with measurement by reducing NBT directly. Oryzanols and sterols were poorly soluble in this assay system too and hence their IC50 values could not be determined by this method also. Activities of ferulic acid and tricin were similar and the activity of the extracts followed the order AE-PP > AE > CME > AE-LP.
2019
Fig. 4. Superoxide radical scavenging effects of defatted rice bran extracts and its phytochemical constituents at 40 lg levels by xanthine–XOD/NBT method.
Xanthine oxidase (XOD) is one of the main enzymatic sources of reactive oxygen species (ROS) in vivo. Xanthine oxidase oxidizes xanthine or hypoxanthine to uric acid, superoxide anions and H2O2 (Unno et al., 2000). Though O 2 cannot directly initiate lipid oxidation, in the presence of metal ions, the highly reactive hydroxyl radical (OH) can be generated by the Fenton reaction (Kanner et al., 1987). Xanthine oxidase-derived superoxide radical has been linked to the postischemic tissue injury and generation of neutrophil chemotoxins (Cos et al., 1998). Under normal physiological conditions, the endogenous superoxide scavengers in the system protect tissues by neutralizing these radicals. This in vivo reaction is simulated in the in vitro model so as to use it as an analytical tool to evaluate the ROS scavenging abilities of natural products. The DRB extracts when assayed by cytochrome c method and NBT method showed positive radical scavenging effects. But the activities were found to be higher for the former method. The order of antioxidant efficacies by the two methods were found to be similar for the extracts and it was in the order AE-PP > AE > CME > AE-LP, which also followed the order of their TPC and ferulic acid contents. According to Khanom et al. (2000), the superoxide scavenging activity of Bangladeshi medicinal plants determined by the cytochrome method were mostly higher than that of the NBT method and they suggested that the former method measured not only the superoxide scavenging activity but also other biochemical properties. In the present study, the IC50 values for the DRB extracts by the cytochrome c method were in the range of 28–57 lg and those by the NBT method were in the range of 77–171 lg. These assays make use of the competition kinetics of reduction of cytochrome c/NBT (probe) and O 2 scavenger (antioxidant compound) by superoxide. But many antioxidants are capable of reducing the probes directly (Huang et al., 2005). In the present study, such a problem was not
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encountered with the cytochrome c method, whereas many tested antioxidants viz. BHT, TBHQ and Tmix was found to reduce NBT directly. This can in turn result in a lower estimate of the true superoxide scavenging activity of the tested compounds as only lesser amounts of the added compounds would now be available for scavenging O 2 . Complications can also arise in determining the extent to which the probe is reduced by the antioxidant and by O 2 . The situation will be more complex in the case of multicomponent natural extracts like DRB especially when it is not fully characterized. These methods were used by various authors for natural extracts. Hot water extracts of budrock were required at 1 mg levels to obtain 65% inhibition (Duh, 1998) and the IC50 value of tomato pulp was reported to be 1640 lg on dry weight basis (Lavelli and Giovanelli, 2003). Superoxide scavenging effects of fermented rice bran extracts were attributed to proteins by Miyamoto et al. (2000). Itani et al. (2002) have attributed the superior scavenging effects of red and purple black hulled rices compared to that of white hulled rices to the tannins and anthocyanins of the former. Kim et al. (1995) have studied the SOD-like activity of 24 lipophilic antioxidants including c-oryzanol by measuring the inhibition of pyrogallol autoxidation catalysed by superoxide radicals. The superoxide scavenging activity of ferulic acid was reported by Graf (1992). The reduction rate constant (K 102–106 M1 S1) of superoxide radicals by flavonoids is reported to be the highest among biological compounds (Jovanovic et al., 1998). The results of the present study demonstrated that the various phytochemical constituents of DRB extracts viz. ferulic acid, tricin and Tmix exhibited excellent superoxide radical scavenging activity thus directly supporting the superior antiradical efficacies of DRB extracts. However, the presence of proteins, sugars and other unidentified phenolics that constituted the bulk of the extracts would also have affected their antioxidant efficacy either synergistically or antagonistically. Acknowledgements The first author is grateful to CSIR for the senior research fellowship granted during the period of this work. The authors acknowledge the financial support from Technology Mission on Oil Seeds and Pulses (TMO&P), Government of India. The authors thank Director, Regional Research Laboratory, Thiruvananthapuram for his support and constant encouragement. References Adom, K.K., Liu, R.H., 2002. Antioxidant activity of grains. Journal of Agricultural and Food Chemistry 50, 6182–6187. Association of Official Analytical Chemsits (AOAC), 1984. Official Methods of Analysis, 11th ed. Association of Official Analytical Chemsits, Washington, DC.
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