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Effects of Various Solvents on the Extraction of Antioxidant Phenolics
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  Effects of various solvents on the extraction of antioxidant phenolicsfrom the leaves, seeds, veins and skins of   Tamarindus indica  L. Nurhanani Razali, Sarni Mat-Junit, Amirah Faizah Abdul-Muthalib, Senthilkumar Subramaniam,Azlina Abdul-Aziz ⇑ Department of Molecular Medicine, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia a r t i c l e i n f o  Article history: Received 12 August 2010Received in revised form 20 June 2011Accepted 1 September 2011Available online 5 September 2011 Keywords:Tamarindus indica TamarindAntioxidant activityPhenolicsSolvent extractionDPPHABTS a b s t r a c t The effects of solvents, of varying polarities, on the extraction of antioxidant phenolics from the leaves,seeds, veins and skins of   Tamarindus indica  ( T. indica ) were studied. The efficiencies of the solvents forextraction of the antioxidant phenolics were in the order: methanol > ethyl acetate > hexane. Phenoliccontent ranged from 3.17 to 309 mg of gallic acid equivalents/g. Methanol leaf extract (MEL) had thehighest phenolic content and was the most potent scavenger of DPPH and superoxide radicals. Methanolvein extract had the highest ferric reducing activity whereas methanol seed extract was the most potentABTS radical-scavenger. A positive correlation existed between phenolic content and antioxidant activi-ties of the plant parts. HPLC analyses of MEL revealed the presence of catechin, epicatechin, quercetin andisorhamnetin. Overall, methanol was the most effective solvent for extraction of antioxidant phenolicsfrom  T. indica .  T. indica , particularly the leaf, can be a useful source of natural antioxidants.   2011 Elsevier Ltd. All rights reserved. 1. Introduction Oxidative damage by free radicals is implicated in the aetiologyof many diseases, cancer and heart diseases being the more com-mon ones (Azad, Rojanasakul, & Vallyathan, 2008; Heitzer, Schlin-zig, Krohn, Meinertz, & Münzel, 2001; Madamanchi, Vendrov, &Runge, 2005).Antioxidants are useful for providing protection against oxida-tive damage. Antioxidants, such as glutathione, ubiquinone, uricacid andthe antioxidantenzymesglutathioneperoxidase,superox-ide dismutaseand catalase, can be generated in the body; however,the amounts maybe inadequate, particularly under conditions of oxidative stress or inflammation where production of free radicalsis increased. Hence, adequate amounts of antioxidants are impor-tant to prevent build up of free radicals and oxidative damage inthe body.Plants are rich alternative sources of natural antioxidants whichcan complement the antioxidants produced by the human body.Various studies have shown plants to be a rich source of antioxi-dants. Compounds with antioxidant properties found in plantsinclude the vitamins A, E and C and phenolic compounds, includingflavonoids, tannins and lignins (Boots, Haenen, & Bast, 2008; Valko,Rhodes,Moncol,Izakovic,&Mazur,2006).Flavonoidsareoneofthemain phenolics studied, due to their documented potent antioxi-dantactivities(Rice-Evans,Miller,&Paganga,1996),somearemorepotent than the well known antioxidant vitamins. In addition, cor-relation studies have demonstrated a link between antioxidantactivities in plants and their phenolic content, underlining the sig-nificant contribution which phenolics can make to antioxidantactivities (Cai, Luo, Sun, & Corke, 2004; Kaur & Kapoor, 2002; Razab& Abdul Aziz, 2010). In view of the potential of plants to provide anatural source of antioxidants, studies are on-going in search of plants with extracts of high phenolic content and antioxidantactivities. Tamarindus indica  ( T. indica ), commonly known as tamarind, isubiquitouslyfoundintropicalcountriesalthoughitsrcinatedfromAfrica. It is a tree from the family Fabaceae. The pulp of this plant isused in cooking due to its sour taste and particularly to impart fla-vour to savoury dishes.  T. indica  is also used medicinally for gastricand digestion . problems. An animal study, using hamsters, demon-stratedthehypolipidaemiceffectoftheseedsof  T.Indica (Martinelloetal.,2006).Thefruitsandseedsofthisplantshowedanti-bacterial,anti-inflammatoryandanti-diabetogeniceffects(Maiti,Jana,Das,&Ghosh, 2004; Paula et al., 2009). Most research on  T. indica  has con-centrated on the fruits and seeds of this plant, mainly extractedusing polar solvents (Luengthanaphol et al., 2004; Razali, AbdulAziz, & Mat Junit, 2010; Siddhuraju, 2007; Soong & Barlow, 2004;Sudjaroen et al., 2005). However,not much informationis availableon the antioxidant potential of the other parts of   T. indica  or the 0308-8146/$ - see front matter    2011 Elsevier Ltd. All rights reserved.doi:10.1016/j.foodchem.2011.09.001 ⇑ Corresponding author. Tel.: +60 3 79674915; fax: +60 3 79674957. E-mail address:  azlina_aziz@um.edu.my (A. Abdul-Aziz).Food Chemistry 131 (2012) 441–448 Contents lists available at SciVerse ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem  effectofvarioustypesofsolventsonextractionofantioxidantsfromthis plant. Hence,the aim of this study was to analyse the effective-ness of methanol, ethyl acetate and hexane for the extraction of antioxidant phenolics from the leaves, seeds, skins and veins of   T.indica . Such study can provide complete information on the in vitro  antioxidant potential of the other parts of this plant thatare less well researched but nonetheless important. 2. Materials and methods  2.1. Chemicals All reagents used in the experiments were of analytical gradeand obtained mostly from Fluka and Sigma. Solvents used forextraction of plant samples were purchased from Fisher Scientific.The phenolicstandards were obtainedfrom Sigma. Waterused wasof Millipore quality.  2.2. Plant material Leaves, veins, skins and seeds of   T. indica  were collected fromKedah, northern part of Malaysia. The material was identified bya taxonomist of Rimba Ilmu Herbarium, University of Malaya,Malaysia. A voucher specimen was deposited in Rimba Ilmu Her-barium, having the voucher specimen number KLU 45976.  2.3. Preparation of plant extracts The various parts of   T. Indica  were thoroughly cleaned and air-dried and, once their weight was stable, the dried parts wereground into powder forms, using a commercial blender. The pow-ders were extracted, using methanol, ethyl acetate and hexane atroom temperature for 24 h (recording 0.05 g/ml). The solventswere thenremoved usinga rotary evaporatorand the resulting res-idues were re-dissolved in 10% DMSO. The extracts were stored at  20   C prior to further analyses.  2.4. Assay for phenolic content  Total phenolic contents of the extracts were measured using theFolin–Ciocalteu assay developed by Singleton and Rossi (1965).This is a colorimetric assay, involving production of a blue molyb-denum–tungsten complex in the presence of phenolics which canbe measured relative to gallic acid as the standard.Aqueous Folin–Ciocalteu reagent (1:10) was added to the plantextract or standard, incubated for 5 min before addition of 0.115 mg/ml of Na 2 CO 3 . After a 2 h incubation period, absorbancewas read at 765 nm. Gallic acid was used as a standard and a cal-ibration curve was plotted in a concentration range of 50–200 mg/l. All analyses were performed in triplicate and results were ex-pressed as mg of gallic acid equivalents/g dried plant.  2.5. Ferric reducing activity (FRAP) The ferric reducing ability of plasma (FRAP) assay uses antioxi-dants as reductants in a redox-linked colorimetric reaction, reduc-ing a ferric-tripyridyltriazine (Fe (III)-TPTZ) complex to the ferrous,Fe (II) form (Benzie & Strain, 1996), forming an intense blue colourcomplex which can be measured colorimetrically.Reagents for this assay consisted of 300 mM acetate buffer,10 mM TPTZ in 40 mM of HCl and 20 mM FeCl 3 .6H 2 O. The respec-tive solutions were mixed in a ratio of 10:1:1 as needed. The freshsolution was warmed (37   C) for 5 min. After taking a blank read-ing, plant extract or standard and water were added to the FRAPreagent. Absorbances were taken at 0 and 4 min after the start of the reaction. The differences between the two absorbance readingswere measured and compared to the standard graph. FeSO 4  wasused as the standard and analysed as above.  2.6. DPPH radical-scavenging activity The scavenging activity of the stable free radical; 2,2-diphenyl-1-picrylhydrazyl (DPPH) was determined by the method describedby Cos et al. (2002). The principle of DPPH assay involves reactionof the antioxidants with the stable DPPH radical, converting thecomplex from a deep violet colour to a colourless complex. Thedegree of discolouration indicates the scavenging potentials of the samples.DPPH  solution (0.04 mg/ml) was added to varying concentra-tions of the plant extracts and absorbance was read at 715 nm fol-lowing a 20 min incubation period. Results were expressed asmmol of Trolox equivalent antioxidant capacity (TEAC) per g of dried plant material. Percentage inhibition of the DPPH radicalswas calculated using the formula below: Percentage of inhibition ð % Þ¼ð OD control  OD sample Þ = OD control  100 % where OD = optical density.  2.7. ABTS  +  radical-scavenging activity This assay is based on the inhibition, by antioxidants, of theabsorbance of the radical cation of 2,2 0 -azinobis(3-ethylbenzo-thiazoline 6-sulphonate) (ABTS +  ) at a wavelength of 734 nm (Reet al., 1998). This assay involves generation of the ABTS radicalchromophore by the oxidation of ABTS +  with potassium persul-phate, which was conducted in the dark for 12–16 h followed byadjustment of the absorbance of the reactant to 0.700 ± 0.02.Briefly, appropriate amounts of the radicals were mixed withthe plant extracts to give a 20–80% inhibition of the absorbance.Readings were taken at 1 and 15 min after the start of the reaction.Trolox was used as the standard and a standard curve was plotted.All analyses were done in triplicate and results were expressed asTEAC values.  2.8. Superoxide anion radical-scavenging activity The superoxide anion radicals were generated by the NADH–phenazine methosulphate (PMS) system (Siddhuraju & Becker,2007). Scavenging activity was assessed by monitoring the abilityof antioxidants in the plant extracts to inhibit reduction of nitro-blue tetrazolim (NBT) by the superoxide anion radicals.The reaction mixture consisted of 0.1 M phosphate buffer (pH7.4), 150 l M NBT, 60 l M PMS, 468 l M NADH and varying concen-trations of the plant extracts. Absorbance was read at 560 nm aftera 10 min incubation period and percentage inhibition of the super-oxide anion by the plant extracts was calculated. Quercetin and ru-tin were used as positive controls and trolox was used as thestandard. All results were of triplicate analyses and expressed asTEAC values.  2.9. Analyses of flavonoid content using high performance liquidchromatography (HPLC) 2.9.1. Acid hydrolysis of leaves Acid hydrolysis of the dried leaf powder was performed accord-ingtothemethoddescribedbyAziz,Edwards,Lean,&Crozier,1998.Briefly, 20 mg of dried leaves were hydrolysed at 90   C for 2 h in a3 ml glass V-vial containing 1.2 M HCl in 50% aqueous methanoland 20 mM sodium diethyldithiocarbamate as an antioxidant. The 442  N. Razali et al./Food Chemistry 131 (2012) 441–448  vialswereplacedinaReacti-Thermheatingblock(Pierce,Rockford,USA) with a stirring capacity. Aliquots of the samples were takenafterthehydrolysis,centrifugedfor5 minat5000  g  anddilutedwithdistilled water (pH 2.5) prior to analysis by HPLC. The hydrolysedsamplescontainedbothfreeflavonoidsandaglyconesreleasedfromconjugated flavonoids, following acid hydrolysis.  2.9.2. High performance liquid chromatography Samples were analysed using a Shimadzu HPLC system com-prising a system controller, a binary pump (LC 20AC), a manualinjector (Rheodyne 7725i manual injector with a 50 l l sampleloop), a column oven (CTO-10AS VP) and a dual channel (SPD-20A UV–VIS) UV detector. Separation of flavonoids was achievedusing a NovaPak (end-capped) C 18  reversed-phase column(150  3.0 mm i.d, 4 l m) (Waters, USA) fitted with a guard car-tridge column, under controlled temperature (40   C) in a columnoven. The mobile phase consisted of water with trifluoroacetic acid(TFA) (pH 2.5) and acetonitrile, eluted at a flow rate of 0.5 ml/min.Shimadzu Corporation LC solution software (version 1.23) wasused for the data acquisition through the dedicated personalcomputer.Stock standard solutions were prepared at a concentration of 1 mg/ml and stored at  20   C. Standard solutions, containing cate-chin, epicatechin, rutin, genistin, myricetin, morin, quercetin, gen-istein, kaempferol and isorhamnetin, were mixed together andinjected at a volume of 50 l l.Separation of flavonoids was achieved, based on the method de-scribed by Aziz et al. (1998) with modifications. Mobile phase Aconsisted of water with TFA, adjusted to pH 2.5 and mobile phaseB was 100% acetonitrile. A linear gradient system, starting with 7%B, increasing to 40% in 20 min, at a flow rate of 0.5 ml/min, was uti-lised and absorbance was measured at a wavelength of 260 nm.  2.10. Statistical analyses All analyses were done in triplicate and results were expressedas means ± S.D. Student’s  t  -test (Microsoft Excel 97–2003 Work-book) was performed to analyse for statistically significant results.Regression analyses (R) were performed to confirm correlation of two data sets (Microsoft Excel 97–2003 Workbook). 3. Results and discussion  3.1. Extraction yield and phenolic content  Table 1 shows the extraction yield of the various plant partswhich range from 20 to 320 mg/g dried weight. The extractionyields, in descending order, were: methanol > ethyl acetate > hex-ane. This shows that methanol was the best solvent for extractionof compounds from the various parts of the  T. Indica  plant.Phenolic compounds are secondary plant metabolites with ben-eficial biological effects, e.g. as anti-bacterial, anti-inflammatoryand anti-allergic agents (Koshihara et al., 1983; Schramm & Ger-man, 1998). Most important is their documented action as potentantioxidants (Formica & Regelson, 1995; Rice-Evans et al., 1996).Hence, it is common practice to measure both phenolic contentand antioxidant activities when investigating the antioxidant po-tential of plants as various studies have shown that plants rich inphenolics are also potent antioxidants (Maisuthisakul, Suttajit, &Pongsawatmanit, 2007; Razali, Razab, Mat Junit, & Abdul Aziz,2008).There were varying phenolic contents in the tamarind extracts,ranging from 3.17 to 308 mg GAE/g dried weight (Table 1) but,generally, the methanol extracts had higher phenolic levels thanhad the ethyl acetate and hexane extracts of the same plant part.Overall, the methanol extract of the leaf (MEL) had the highestphenolic content and the hexane extract of the skin (HESK) thelowest. The methanol extracts of the seed (MES) and of the vein(MEV) also contained considerable amounts of phenolics(>200 mg GAE/g dried weight). The ethyl acetate extract of the leaf (EAEL) and the methanol extract of the skin (MESK) had phenoliccontents in the range of 100–200 mg GAE/g dried weight. Theremainder of the extracts had phenolic contents of less than100 mg GAE/g dried weight.The phenolic content of the  T. Indica  leaves, veins and skins hasnot previously been reported. Most studies have concentrated onthe seeds and fruits (Luengthanaphol et al., 2004; Siddhuraju,2007; Soong & Barlow, 2004). Soong and Barlow (2004) reportedthe seeds, extracted with 50% ethanol to contain 94.5 mg GAE/g.The phenolic content of the methanol seed extract in our studywas much higher.  3.2. Ferric reducing activity FRAP assay provides a simple and effective method for measur-ing the ability of antioxidants in plant samples to act as reducingagents.We foundMEVto containthe highestferricreducingcapac-ity, followed by MEL and MES (Table 1). When compared with thepositive controls, quercetin was considerably more potent; how-ever, the reducing activity was comparable to rutin.In most instances, the methanol extracts of the different plantparts contained substantial ferric reducing activities compared tothe ethyl acetate and hexane extracts. Among the plant parts, theveins possessed the most ferric reducing activities, followed bythe leaves whereas the skins had the lowest activities. MEV andMEL were found to have higher ferric reducing activities than sev-eral Chinese medicinal plants, such as  Scutellaria baicalensis  and Fraxinus rhynchophylla  (Li, Wong, Cheng, & Chen, 2008). This sug-gests the potency of the tamarind plant for medicinal use.On the other hand, the ferric reducing activity of MES in thisstudy was lower than the  T. Indica  seed extract reported by Soongand Barlow (2004) (1460 vs 2468 l mol/g, respectively). This couldbe due to differences in the variety of plant, as well as locationwhere the plant is grown.  3.3. DPPH radical-scavenging activity The three extracts with the highest DPPH radical-scavengingcapacity were in the order: MEL (3.17 ± 0.00 mmol/g driedweight) > MES (2.94 ± 0.14 mmol/g dried weight) > EAEL (2.76 ±0.03 mmol/g dried weight) (Table 1). Radical-scavenging activitiesoftheseextractswerecomparabletoquercetinandrutin,suggestingtheir potency.The DPPH radical-scavenging activities of the extracts depicteda dose–response relationship (Fig. 1). The inhibition reactions ap-peared to be still occurring, even at the highest concentration(100 l g/ml) tested. At this concentration, MEL achieved more than80% inhibition of the DPPH radicals, almost matching that of rutin,whereas quercetin was slightly higher, at 95%.A weaker pattern of scavenging capacities was observed for theethyl acetate extracts of the various plant parts (compared to themethanol extracts). Highest inhibition was demonstrated by theEAEL (72%). The ethyl acetate extract of seed (EAES) was almostnon-reactive, with inhibition not exceeding 25%.The hexane extracts of the various plant parts were generallythe least reactive with highest inhibition at 35% (HEL). The scav-enging activities of the hexane extracts in most cases showed ahyperbolic curve. This observation is fairly common in plants, asnot many non-polar compounds are able to act as potent antioxi-dants (Kulkarni, Aradhya, & Divakar, 2004). N. Razali et al./Food Chemistry 131 (2012) 441–448  443   Table 1 Phenolic content and antioxidant activities of methanol, ethyl acetate and hexane extracts of various parts of   T. Indica A . Yield (mg/gdriedweight)Phenoliccontent (GAEmg/g) B Ferric reducingactivity (mmol/g driedweight)DPPH  radical-scavengingactivity (mmol Trolox/g driedweight) C ABTS +  radical-scavengingactivity (mmol Trolox/g driedweight) C Superoxide anion-scavengingactivity (mmol trolox/g driedweight) C Leaves(M)320 309 ± 3.78 a 1.87 ± 0.09 a 3.17 ± 0.00 a 1.65 ± 0.04 a 4.64 ± 0.003 a Leaves(E)40 101 ± 12.2 b 0.57 ± 0.92 b 2.76 ± 0.03 b 0.70 ± 0.01 b 4.54 ± 0.14 a Leaves(H)40 31.8 ± 3.70 c 0.12 ± 0.07 c,i 1.35 ± 0.04 c,d 0.51 ± 0.03 c 3.99 ± 0.01 c Seeds(M)280 272 ± 18.2 a 1.46 ± 0.001 d 2.94 ± 0.14 a,b 2.88 ± 0.02 d 3.96 ± 0.35 c,b Seeds(E)80 26.6 ± 13.6 c,e 0.12 ± 0.24 c 0.98 ± 0.00 c,e,f,g,h,i 0.77 ± 0.01 e 0.76 ± 0.02 d Seeds(H)40 95.3 ± 14.9 b,d 0.31 ± 0.25 e 1.13 ± 0.01 e,j,k 0.90 ± 0.04 f  3.14 ± 0.10 e Veins(M)240 230 ± 3.33 f  2.05 ± 0.22 f  2.54 ± 0.10 l 1.41 ± 0.003 g 3.85 ± 0.01 b Veins(E)40 65.6 ± 10.0 d 0.31 ± 0.47 e 1.41 ± 0.03 d,f  0.30 ± 0.01 h 2.53 ± 0.01 f  Veins(H)40 30.4 ± 2.60 c 0.01 ± 0.49 g 1.20 ± 0.08 g,j,m 0.16 ± 0.01 i 1.31 ± 0.01 g Skins(M)240 116 ± 1.08 b 0.92 ± 0.01 h 2.34 ± 0.0l 1 1.32 ± 0.004  j 4.54 ± 0.06 a Skins(E)40 17.2 ± 2.89 e 0.10 ± 0.01 i 1.19 ± 0.04 h,k,m 0.89 ± 0.02 f  2.93 ± 0.02 e Skins(H)20 3.17 ± 1.11 l ND 0.73 ± 0.01 i 0.43 ± 0.01 k 1.50 ± 0.03 h Rutin 3.36 ± 0.003  j 3.32 ± 0.00 n 1.72 ± 0.01 a,l 5.47 ± 0.01 i Quercetin 13.3 ± 0.002 k 3.60 ± 0.00 o 4.18 ± 0.03 m 5.67 ± 0.004  j Values not sharing the same letter within the same column were significantly different at  p  < 0.01.M – Methanol, E – Ethyl acetate, H – Hexane, ND – not detected. A Results were expressed as the averages of triplicates ± S.D. B Phenolic content was expressed as mg gallic acid equivalents (GAE) in 1 g of dry weight material ± S.D. C The antioxidant activities of the samples were compared with a trolox standard curve and results were expressed as trolox equivalent antioxidant capacity (TEAC). Fig. 1.  DPPH radical-scavenging capacity of various parts of   T. indica  extracted with methanol, ethyl acetate and hexane. All analyses were performed in triplicate and resultsexpressed as % inhibition of the absorbance of radicals ± S.D. MEL: methanol extract-leaves; EAL: ethyl acetate extract-leaves; HEL: hexane extract-leaves; MES: methanolextract-seeds; EAES: ethyl acetate extract-seeds; HES: hexane extract-seeds; MEV: methanol extract-veins; EAEV: ethyl acetate extract-veins; HEV: hexane extract-veins;MESK: methanol extract-skins; EAESK: ethyl acetate extract-skins; HESK: hexane extract-skins.444  N. Razali et al./Food Chemistry 131 (2012) 441–448
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