American Jounal of Food Technology 2 (5): 414-420, 21 ISSN 1557-4571 © 2007 Academie Journals Ine. Pakistan Phenolic Composition and Antioxidant Properties of Some Spices M. Muchuweti, E. Kativu, CH. Mupure, C. Chidewe, A.R. Nahlala and MAN. Benhura Department of Biochemistry, University of Zimbabwe, MP. 167, Mount Pleasant, Harare, Zimbabwe Abstract: Aquocus methanolic extracts of 9 spices wore investigated for their phenclie ‘compounds compesition and antioxidant properties. The spices investigated were, Laurel noble (bay leaves), Rosimarimus officinalis (osemry), Salvia ofiinalts (sags), Origanum ‘marjorana (wasjotam). Origaran valgare(ceezane), Cinmamomn zelanicum (cinnamon). Penroselium crtspum (patsley), Oct hasiltcum (et basil ancl Mentha peperita (sin), ‘The phenolic compound eantants were detormined by the Folin Cicealteu, tannin binding, assay and High Performance Liquid Chromatography (HPLC). The antioxidant properties were determined by the reducing power assay, radical seavenging assay and the P-carotene linoleic acid model system, Oregano had the highest total phencie compound concentration 0f 1583 mg GAE g~ and ciamon had the highest polyphenolic compound concentration 0f 13.60 mg GAE g”. Marjoram had the highest proportion of simple phenolic compounds of 95.57%, Ascorbic acid was used as a contol in ll the antioxidant assays. At 25 mg mi. cinnamon and oregano recorded a high redacing power activity with absorbance of 0.12, ‘hile parsley had the lowest activity with absorbance of 0.075 at 655 nm. Cinnamon and ‘marjoram had the highest radia scavenging aetivites of 92.0 and 91.3% respectively while sta concentration of 5 mg mL~, prsley had the les radical scavenging activity of 47.90%. Cinmamoe and oregano had the highest antioxidant activities of 61.76 and 58.28%, respectively while sweet basil had the lowest activity of 667%. Mest ofthe spices showed better antioxidant properties than ascorbic acid, HPLC analysis detected gallie acid, protochatechnic acid, phytirexybenaoie aid, p-hydroxy benzaldehyde, vanillic acid, calfeie acid, p-coumurie acid and ferllic acid in the studied spices, Key words: Phenolic acids, anticxican, spies, tannin an fice naticals INTRODUCTION Phenolic compounds are sovondary plant metabolites that possess in common an eromatic sing bearing one or more hyoxyl substituents. Phenolic compouncls are water soluble and may ccour combined with a sugar melocole, as plyeesdes (Harboe, 1998). Phenolic cormpoxmds are divided into sub-proups and these include, phenols, phenolic aids, phenvipropunoids, flavonoids, avones, alyeoflavonenesand biflavonsls, minor Nevo, aurones, Navonones,dlydochaleanes,isollvones, ‘anthones and stlbenes, hydrolysable and condensed (proanthooyanidins) tanains and quinines (arbone, 1998; Strack, 1997), Phenolic compounds have diverse biclgical activities ranging foe toxicity to hormonal mimiery and act as cell wall material, colorful atwuctants for birds and insects helping seed dispersal and prllination. Phenolic compounds also act as defense mechanisms of plants under different Cenvizcemental stess conditions such as wounding, infection, excessive light or UV iaradition ‘Carrespaning Author: 5 Munve Depainest of Bincheniay,Lnivasiy wf Zinbabwe, SUP 16 Mourt Piva, Harare, Zsa Tel 00358 (0) AS08017 FX: 0026 0) 908085 44Am. J Food Teclmol., 2 (5): 414-420, 2007 (ashone, 1998), The biological potency of phenolic compounds includes possible pharmacological value (Ingold, 1960), Phenolic compounds have long been recognized to possess anialergenic ati- inflammatory, antiviral and antiprlierative activities. Phenolic empounds have some antioxidant activity, They ae able to tomminate fre radicals and chelate metal ions that are capable of catalyzing formation of oxygen reactive species that promeke lipid peroxidation. Phenolic compounds interfere with oxidation of lipids and other free radicals by sapid donation of « hydrogen atom or electors to the oxidized molecule of radicals, The resultant ‘adioal ftom the reaction of phenol with lipid radio is stabilized by the delocaisatcn of unpaired letrons around the aromatic rng (Ingold, 1960). Stability ofthe phenoxy radical reduces the rate of propagation of auto-cxidation cain reactions because the propagation reaction is slow and the bulky groups of ? and 6 positions offers steric hindrances in the region ofthe radical and reduces the rate of ‘propagation (Hudson, 1990), Phenolic compounds ae understood to induoe the cellular antexidant system: quetootn and flavonoids were found to increase the intra calular concentration of glutathione bby approximately 50%, Flavonoids are important in the modulation of y-glutamyleysteine synthase ‘nboth celular antioxidant defenses and dete ticaion of xenobiotics. Glutathione is impoetantin redex regulation of transripton factors and enzymes for signal transduction. Iti therefore likely that polyphenols mediated segulation of ghutaione alters cellar processes (Hudson, 1990; Yoshida er al, 1997) Spices are plant produets added to food to contribute towards arcma, test, Haver, calor and ppungency. These atibutes are believed to be due to the presence of phenolic compounds in spives. Spices are derived from tt bark, ower, leaves, rhizomes, rots, seed, oe ere plant ongans, The use of spices is widely spread in Asian countries. Historically spices were exploited fer ther ati- _mjerobial properties to preserve mest products, "There aze no repor' on the antioxidant capacities of Laurel noblis (bay leaves), Rasimarinas officinalis (rosemary), Salvia officals (sg), Origartum marjorana (marjoram, Origanum valgare (oregano), Cinnamorum zeslanicum (cinnamon), Petroselium crispum (parsley), Octum basiicum (sweet basil) and Mentha peperia (aint) cultivated in Zimbabwe. The aim of the study was to determine he umount of phenolic compounds content and antioxidant activites of the selected spices. ‘All the studied spices are native in the Meditersanean and coo climates but they can all be gsovin in arden in oer climates n Zimbabwe they are gronm ata smal scale in isolated farms in the eastem highveld andthe watershed regions. MATERIALS AND METHODS: Reagents ‘The chemical standards used were all of aulytical grade. Folin-Ciocalteau gall aid, eatechin, ‘vari aid, caffeic acid, p-zoumare acd, protocatechuic acid, ferulie acid, p-hycony-bervoic acid, pehydroxybenzaldelyde, Nittoblue tetrazolium salt (NBT), 1, -tiphenyl-2 pierylhydrazy radical (DPPH), pherazine methosuipluate (PMS), ascorbic aid, trichloroacetic eeid (TCA) and potsssium Ferroyanide were cblainad fiom Sigm-Aldtich Chemie (Stainheim, Germany) Reduced nicotinamide adenine dinucleotide (NADH) was obtained from Boehringer, Manheim, Germany. Sodium carbonate, ‘methanol (HPLC grack), ascorbic acid, HCl, sthyt acctate, diethyl ether, ankydrous Ne,S0,, acetonitrile Super purity solvent), acetic acid and fete ammonium sulphate were obtained locally. Folin-Ciocalteu reagent (1 N), 20% sodium carborat, standard gallic acid (0.5 mg mL), 50% ‘methanol in distilled water (1:1 iv) and Feri reagent (2% Ferre ammonium sulfate in 2M HCI) were usd for analysis, Procurement of Samples All samples were obtained fom the local spennaskets and they were all processed in Zimbabwe. ‘The spies were packaged by local companies numely New Seasons, Mr Tasties Foods and Spice 41sAm. J Food Teclmol., 2 (5): 414-420, 2007 Foods. The spices obtained were analyzed before the expiry date, They were manufactured for food ‘avoring purposes. Samples were Kept at room temperature before extraction. The spices investigated included the Libéatae family, Rosmarinus officinalis (rosemary), Ociun baslcwn sweet basil), Salvia officinalis (sage), Origanum valgare (cxeganc), rigaram marjorana (majorar) ane Mentha peperia (ui, the Laure! tay, Laure nobilis tay leaves) and Cinnamon eylanicum (cinnasnce) ad the ellifreae faily, Petroselium erispum (pastes), Extraction of Phenolic Compounds ‘Toval phenolic compounds were extracted from the ground material as described by Makar (1999), Phenolic acids for HPLC analysis were extracted following the method described by Pena-Neira ea, (2000), ‘Quantification of Phenolic Compounds ‘Total phenolic compounds were determined following the methed by Makar (1999). Amount of tannins were determined using the method of Makar and Goodshild (1999), Antioxidant Activity Assays “The DPPH radical scavenging activity and the reducing power effets wore determined following the mettod by Kuda ef a, (2005). The inkubition of lipid oxidation was assayed using the method of Amin and Tan (2002), Detection of phenolic acids was euied out by measuring absorbance at 280 nm according to PenscNeira eral. (2000). Aer each ran, the system was reecnditioned for 15 min before analysis of thenext sample RESULTS AND DISCUSSION ‘Total Phenolic Compounds Determination. ‘The concentration of the phenolie compounds in the spices ranged fiom 6:90 to 1583 mg GAE g and they were in the order eregano™cinnamon>sweet basi>bay leaves>mint> soge>rosemary-pasley>marjoram (Fig. 1). The results are comparable to those obvained by Miliauskas er af, (2003), who studied some culinary plants and obtained ranges of 430 to 37.90 mg GAE e~. Wagensteen ef a, (2004) obtained 19 mg GAE g~ total phenolic eompounds in some coriander plans, Ismail eral. (2004) detected ranges of 11.07 to 71.67 mg GAE gin selected vegetables. Capecka ef af (2003) detected between 11.07 and 14.06 mg GAE g~ total phenclic ‘compound contents in some herbs. Vatiation of phenolic compounds content arises due to several actors, which include the area of cultivation ane other environmental stresses (Makar, 1999) ‘The Tannin-Binding Assay ‘Most phenolic compounds in plants occur as polyphenolic compounds (Fig. 2). Polyvinyl polypyrolidone (PVPP) binds eficienty to tannins (Makar, 1999). The concentration of tannins after ‘treating the sample with (PVP) was found to ange frm 031 to 13.66 mg. GAB g’. The order of the ‘concentration of tannins in the studied spices was a follows: cinnamon oregano>sweethasil>mint> bay leaves> parsley>sage>rosemary>maroram. ‘The Reducing Power Assay Inoreasein absorbance with increasing concentration indicated an increase in edueing power, The ‘phenolic compounds in methanalie extracts of spices were able to reduce potassium ferrieyaride toa 46Am. J Food Teclmol., 2 (5): 414-420, 2007 ‘Sepee Fig. 1: The total phenolie compounds concentration exp Comerton (ng CAB ‘Sales ‘Samples Fig. 2: The tins concentration expressed as mg GAE g~ of sannples (a) and the simple phenolic ‘compounds concentration expressed as mg GAE 2+ sample () Ferrous state. Ascorbic acids a known seducing a Itcan be deduced that phenolic compounds in the spices are more potent reducing agents compared to ascorbic acid (Fig 3), Ascorbie acid fad an absorbance of 0.03 at 0.5 mg mL~' and at25 mg mL.~'it had an absorbance of 0.04. For al the spices Aa Sig ml." the absorbencies ranged from 0,05 to 0.07 and at 25 mg ml.“ the absorbencies ranged from 0.08 to 0.12, At25 mg g” the reducing power of the spices investigated was decreasing 2s follows: cinnamon>oregano>marjeransage>rosemary>mintsweet _basi>bayleaves>parsley. “Marjoram exhitited high reducing power activity though t had ow levels of total phenolic compounds. ‘The hgh reducing power of marjoram may be due tte high levels of simple phenolic compounds, The five phenols cun easily donate ther electtons to any eleeton-deficient substance ‘The DPPH Radical Scavenging Assay The raical scavenging activity of spices increases with ineease in concentration (Fig. 4). At 5 mg e* bay leaves had 91.1% radical scavenging activity, rosemary 88.5%, marjoram 91%, sage 88.35%, organo 89.8%, cinnamon 92%, parsley 47.5%, sweet bil 90.1 and mint $3% Most of the samples managed to scavenge the DPPH radical to above 75% with the exception of parsley. The results ae comparable to those found by Mau ef a. (2004) who obtained 78 8, 79.4 and 94.1% DPPH scavenging activites from methanelic extracts of Terminotemnscesalbuminosus, Grifla frondosa and Marchella esculenta, xespestively (Wagensteen et al 2004), B-Carotene Linoleic Acid Model System ‘The antowan activity was measured by comparing the Meaching capacity of sung control, which contained no antioxidant component, Cinnamon and oregano hed the highest activity with the 417Am. J Food Teclmol., 2 (5): 414-420, 2007 oo Connon (x) Fig, 3: The reducing povrer activity ofthe studied spices kr: ik g 1 Beyer ie Seo Be ke oF Teese + Assorbie acid en eTtsis 3 TT (Concentration (mg ml") ‘Concentration (mg mL. ") 1 Fe ; ete is IE Rie satst is ixeconson tne Fig. 4: The percentage scavenging activity on the free radical DPPH of the nine spices ‘with sweet basil having the least antioxidant percentage activity of 6.67% (Table 1), The activities of cinnamon and oregano are comparable to these obtained by Ismail ef al. (2004) fice extracts of shallots, spinach, swamp cabbage, cabbage and kale, which were 69.1, 66, 60.3, 59.3 and 50.2%, sespoetvely. The differences in the aationidunt activity of the spices could be due to the method of cultivation used forthe diferent spices and differences in environmental factors they were exposed to, such as climatic growth eonditions and duration of storage, Characteristics of the phenolic ‘compounds may affect the antioxidant activity. Rioo-Evans ot af. (1995) suggests that the onto. substitation with electron donating ally] or methoxy groups of flavonoids, inereases the stability oF fice radical and hence its anoxiclant potential. The postion and degree of hydroxylation of phenclic ‘compounds is of primary importanee in determining the antioxidant activity of phenelic compounds The ortho and para positions of hydroxyl groups contribute markedly to the antioxidant activity witle the meta pestion as litte or no effect on the antioidant HPLC Analysts [HPLC was used to identify the phenolic compounds that were present in the sidied spices. The ‘etention times of the standards were used to identify th individual phenolic compounds What are found in the spives. Gallic acid, protochatechuic acid, p-hydroxybenacic acid, p-hydsoxy benzaldehyde, vanillic acid, caffeic acid, p-ccumaric acid and ferullic acid were pesitvely identified in the studied spiees though they didnot all our in the sume sample. Feullieweid was common in all the spices. (Caffee seid was found inal the spices expect in mint but the peaks it produced were very small 41gAm. J Food Teclmol., 2 (5): 414-420, 2007 ‘able The percentage satioxida actiy of the sued pics showing the capacity of mies extracts to iit eching of ferote soliton Sane Tscanage of ena at gy lees Ln6e128 evamy 21321006 Sage 22308152 reso sam033 Camaen euses07 Panky 1s:065 See os 661020 Mat aasmaa7 Manoa sera Aerie sid Table 2: HPLC els showin ihe nic seid cmposton tenn ges Sample Phenolic compound dated CCimamen Vani siete afer ad Parley Calis, proochteinie acl eae cil. p-couei ac fic ab By ewes Vili ai ete mi feria rovemay Valli acid elec id precumai acd ‘MajreainPectocalechse ai, vnc ad eulfieal eis Sage Pllyroeybencadee, vile nei cafe ai cour i fic aid Gregan Pyroxybezalehye pldrestezoi ac poorer ai eli aid Sweats! tdatedave ait plgdeaglenzak ai pydrosberalddyde calle aid -coumaric cl fle seit Mint__Gallie ai, prtachtecnie ni, vail 3 p-ecsmaaidferlie aed suggesting tat itis found in very stall quantities in all the samples. Vanilic acid was found in all samples except in sweet basil Only oregano and sweet basil contained p-hytlroxybenzoie acid and phydroxybenzaldehyide (Table 2), The HPLC results obtained are comparable to those we obtained in Ziciplaus martiana and Vapaca Kriana fits (Mucluovet et al.2005, Muchuweti et af, 2006) ‘The phenolic compounds identified are widely found in plants. Schindler er af (2005) detected ‘-hydroxybenzaldehyde, p-coumatic acd, feullie acid and vanllie acid in tomatoes. CONCLUSION ‘The nine spices studied are important sources of potent phenolic compounds, Phenolic compounds from spices are valuable sources of antioxidants and they have shown to lave generally ‘etter antioxidant properties than ascorbic acid, The spices exhibited strong antioxidant capacity im vitro and they may be potential sources of natural antioxidants. REFERENCES. Amin, I, and SH. Tan, 2002. Antioxidant activity of selected seaweeds, Malaysian, J. Nutr, 8: 167-177. (Capocks, E., A. Mareeezek and M. Leja, 2004. Antioxidant activity of fresh and dry herbs of some Lamiaceae species. Food Chem , 93: 223-226 Barbone, JB., 1998, Phytochemical Methods Guide to Modern Techniques in Plant Analysis, 3rd Edn, Chapman and Hall, London, pp: 42-58. Hudson, BF, 1990, Food antioxidants, Elsevier applied science, London, 1-18 and 193-200. Ingold, K.U., 1960. Inibition of vil oxidation by 2,6 di-t-butyld-substtuted phenols. J. Phys. (Chem, 64: 1636-162. Ismail, A, AZ. Marjan and W-C. Fong, 2004, Total antioxidant activity and phenolic content of selected vegotables, Food Chem, 87; 81-586 419Am. J Food Teclmol., 2 (5): 414-420, 2007 Kuda, T., M. Tsunekawa, H. Goto and Y. Arak, 2005. Antioxidant properties of four edible algae harvested in the Noto Peninsul, Japan. J. Food Composition Ana, 18: 625-633. Makkar, HLP.S. and A.V. Goodetild, 1996. Quantification of tannins: A laboratory: manna International Center for Agricultural Research in Dry Areas, Aleppo, pp: 4-6. Makar, HLPS,, 1999, Quantification of tannins in tee foliage: A laboratory manual for FAQILABA. coordinated research project on the Use of nuclear and related techniques to devalop simple tannin assay for predicting and improving the safety and efficiency of feeding ruminants om the tanniniferous tre foliage. Joint FAOMAEA Division of Nuclear Techniques in Food and Agriculture, Vienna, pp: 1-28, Mau, LJ, CN. Chang. S.J. Hung and C.C. Chen, 2004. Antioxidant properties of methanolic ‘extacs fiom Gra, frondose, Marchella esculenta and Termioremyces albuminosusrayeain ood Chem, 94: 415-419. Miliauskas, G, RR. Venskutonis and vanT.A, Beck, 2004. Screening of radical scavenging activity ‘of some medicinal and aromatic plant extracts. Food Chem, 85: 231-237 Muchuwet, M. G. Zenda, AR Nablala and A. Kasivamfura, 2005. Sugars, ompanie acids and phenolic compounds of Zisjpus mauritian fiat, Eur, Food Res. Technol., 221: 570-574 Muclasyst, M,, AR. Nailala and. Kasiyamiuns, 2006. Analysis of phenolic compourds including tannins, gallotannins and flavonols, Food Chem, 94; 415-419, Pena-Neita, A. I, Estelle, C, Gareia-Vallgjo, T. Hemandez and AJ. Suarez, 2000, A survey of phenolic compounds in Spanish wines of different geographical origin. Food Res. Techno, 210: 445-448, Rice-Evans, C., NJ. Millet, PG. Bolwell, PM. Bramley and J.B. Pridham, 1995, The relative antioxidant of plant derived polyphenolic flavonoids. Free Radic. Res, 22: 375.383. Schindler, M.S. Solar and G. Saniog, 2005, Phenolic compounds in tomatoes: Natural variation and offeets of garnma-radiaton, Eur. Food Res, Technol, 21: 439-444 Strack, D, 1997, Phenolic Metabolism. In: Plant Biochemistry. Dey, PM. end J.B, Harberne (Eds), London, UR: Academie Press, pp: 387-416. ‘Wagensteen, H., B.A. Samuelsen and E.K, Malterude, 2004, Antioxidant aetvity in extracts fom cariander, Food Chem, 88: 23-297, Yoshida, T, H. Ito, M. Kut, T.Nakish, A. Inada, H. Maruta, N. Matsiura, K. Ono, M.Nods, FA Lang and J. Murata, 1997, New hycolysable tannins, shephigenis A and B fiom Sherpadtia argentea os HIV-1 reverse transicriptase inhibitors. Chem. Pharmnaco. Bull, 44: 1436-1439. 420