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1 CENTRE FOR RESEARCH Centre for Reserh nd Development (CERAD) The Federl University of Tehnology, Akure, Nigeri JoST 212 AND DEVE LOPMENT nd Effet of ooking durtion on the phenoli ontent ntioxidnt properties of Blk umin ( N igell stiv L) 1 2 2* SHODEHINDE, S.A., ADEMILUYI,A.O. nd OBOH, G. Deprtment of Biohemistry, Adekunle Ajsin University Akung Akoko, Ondo Stte, Nigeri 2Deprtment of Biohemistry, Federl University of Tehnology, Akure, Ondo Stte, Nigeri 1 A BSTRACT: B lk umin ( Nigell stiv L.) seeds re normlly used s spies in food prepr- tions ndinfolklore for the tretment nd mngement of severl diseses. This present study sought to ssess the effet of ooking durtion (5 15 min) on the phenoli ontent nd ntioxidnt properties of lk umin seed. The results of the study reveled tht ooking for 5 1 min used signifint (P<.5) inrese in the phenoli ontent (totl phenol nd flvonoid) with signifint derese ( P<.5) t 15 min of ooking. This sme pttern ws oserved for the ntioxi- dnt properties of the seed. Hene, miniml het tretment durtion (5 1 min) through ooking is reommended to prevent the mjor loss of ntioxidnt properties nd phenoli onstituents of lk umin seed. K eywords: Blk umin seed ( Nigell stiv); phenolis; ntioxidnt; ooking; reduing power B lk umin ( Nigell stiv L.) is n nnul flowering plnt elonging to Rnunulee fmily, widely distriuted in ountries ordering the Mediterrnen Se, Centrl Europe nd Western Asi (Hshem nd El-Kiey, 1982). The seeds re lk nd tringulr in shpe with pungent itter tste nd fint smell of strwerries. Cumin is one of the ommonly used spies in food preprtions. Trditionlly Cumin is used in mediine s stimulnt, rmintive, n stringent nd s remedy ginst indigestion, fltulene nd dirrhoe (Normn, 199). Blk umin is widely used s spie/ ondiments in vegetrin nd non-vegetrin preprtions long with other spies in Indi nd Ari (Thippeswmy nd Nidu, 25). The seed n e powdered nd pplied to food preprtions during or fter ooking. INTRODUCTION JoST (2): Aepted for Pulition, Novemer 13, 212 Thymoquinone, dithymoquinone, thymohydroquinone nd thymol re the phrmologilly tive quinones of lk umin oil (Ghosheh et l., 1999). Like other spies, umin hs een reported s good soure of phenoli phytohemils with strong ntioxidnt tivity (Thippeswmy nd Nidu, 25). Phytohemils from spies, re very good ntioxidnts whih offer protetion ginst lipid peroxidtion. Antioxidnt tivities of mny plnt foods hve een found to e funtion of the quntity nd qulity of phenoli phytohemils present (Chu et l., 22). The ntioxidnt properties of phenolis re relted to their hemil struture whih mkes them ple of neutrlizing retive oxygen speies, svenge free rdils, helte metl tlyst nd t to protet the ell. Free rdils nd retive 2 * Correspondene to: Ooh, G., gooh21@yhoo.o m Journl of Sustinle Tehnology, Vol. 3, No. 2 (Novemer 212), ISSN: Pp 89

2 S.A. Shodehinde, et l. oxygen speies (ROS) re entire lss of highly retive moleules ple of reting with lmost every known moleule in the iologil system in their viinity. Free rdils dmge proteins, use rekdown of DNA strnds nd initite the peroxidtion of vrious ompounds. Almost ll the vitl omponents of ells re suseptile to dmge y free rdils. Free rdils lso uses lipid peroxidtion whih is n utotlyti mehnism leding to oxidtive destrution of ellulr memrnes (Cheesemn nd Slter, 1993). Although extensive studies hve een onduted on phrmologil properties of the essentil oil of lk umin nd thymoquinone suh s; ntioxidnt tivity (Burits nd Bur, 2), ntihypertensive effet ( Zoui et l., 2) nd heptoprotetive properties (Mnsour et l., 21); nevertheless there is still derth of informtion on the effet of durtion of ooking on the ntioxidnt properties of lk umin seed. Also, sine spies re used s wter-extrted pste or dry powder in food preprtions (ooked nd unooked), this work ws designed to further investigte the effet of ooking on the phenoli ontent nd ntioxidnt properties of lk umin seed. Mterils B lk umin seeds ( Nigell stiv L) were soured lolly from the entrl mrket in Kno, Nigeri. The identifition nd uthentition ws done t the Deprtment of Biology, Federl University of Tehnology, Akure, Nigeri. All the hemils used were of nlytil grde, while distilled wter ws used. Methods Smple preprtion nd queous extrtion The seeds were hnd-piked to remove dirt nd inedile portions. The edile portions were rinsed, sun dried nd ground into powder. One grmme of the powdered rw lk umin seed ws soked overnight in 1 ml distilled wter. The superntnt ws olleted nd treted s reported in this work. For the ooked smples; 1g of the powdered rw lk umin seed ws ooked in 1 ml distilled wter in onil flsk with stopper for 5, 1 nd 15 min respetively. Therefter the mixtures were entrifuged t 358g for 1 min nd superntnts olleted were mde upto 1ml, nd lter used for further nlysis. MATERIALSAND METHODS Determintion of totl phenol ontent The totl phenol ontent ws determined on the extrts using the method reported y Singleton et l. (1999). Approprite dilution of the extrts ( 1 μl ) were oxidised with 2.5 ml of 1% Folin Ciolteu s regent (v/v) nd neutrlised with 2. ml of 7.5 % sodium ronte. The retion o mixture ws inuted for 4 min t 45 C nd the sorne ws mesured t 765 nm (UV Spetrophotometer JENWAY 635). The totl phenol ontent ws susequently lulted s Glli id equivlent. Determintion of totl flvonoid ontent The totl flvonoid ontent of the extrts were determined using slightly modified method reported y Med et l. (25). Briefly,.5 ml of ppropritely diluted smple ws mixed with.5 ml methnol, 5 μl f 1 % AlCl, 5 μl of 1 o 3 M potssium ette nd 1.4 ml wter. The mixture ws llowed to inute t room temperture for 3 min. Therefter, the sorne of the retion mixture ws Journl of Sustinle Tehnology, Vol. 3, No. 2 (Novemer 212), ISSN: Pp 9

3 Cooking ffets Blk umin phenolis susequently mesured t 415 nm (UV Spetrophotometer JENWAY 635). The totl flvonoid ontent ws lulted s Queretin equivlent. F e 2 heltion ssy T he Fe 2 helting ility of oth extrts wer e determined using modified method of Minotti nd Aust (1987) with slight modifition. Freshly prepred 25 μl FeSO ( 15 4 μl ) ws dded t o retion mixture ontining 168 μl of. 1 M Tris-HCl (ph 7.4), 218 μl sline nd the extrts ( 25 μl ). The retion mixture ws inuted for 5 min, efore the ddition of 13 μl o f.25 % 1,1-phennthroline (w/v). The sorne ws susequently mesured t 51 nm (UV Spetrophotometer JENWAY 635). P erentge Fe 2 helting ility w s susequently lulted.. OH rdil svenging ility 2 T he ility of the extrts to prevent Fe / H O indued deomposition of deoxyriose ws rried out using the method of Hlliwell nd Gutteridge (1981). Briefly, freshly prepred queous extrt ( 1 μl ) ws dded to retion mixture ontining 12 μl 2 mm deoxyriose, 4 μl.1 M phosphte uffer, 4 μl 2 mm hydrogen peroxide nd 4 μl 5 μm FeSO nd the volume ws mde up to 4 8 μl with distilled wter. The retion mixtur e o ws inuted t 37 C for 3 min nd the retion ws then stopped y the ddition of.5 ml of 2.8% trihloroeti id (TCA), this ws followed y the ddition of.4 ml of.6% thiorituri id (TBA) solution. The tues were susequently inuted in oiling wter for 2 min. The sornews red t 532 nm (UV Spetrophotometer JENWAY 635). Perentge ÿoh svenging ility ws susequently lulted. 2,2-zinois(3-ethylenzo-thizoline-6- sulfonte) ABTS. svenging ility The ABTSÿ svenging ility of the extrt s ws determined ording to the method. desried y Re et l., (1999). ABTS ws generted y reting n ABTS queous solution (7 mm) with K S O (2.45 mm, finl onentrtion) in the drk for 16 h nd djusting the Asorne t 734 nm to.7 with ethnol. Approprite dilution (.2 ml) of the extrt ws dded to 2. ml ABTSÿ solution nd th e sorne were red t 734 nm (UV Spetrophotometer JENWAY 635) fter 15 min. Trolox ws used s the stndrd nd susequently, totl ntioxidnt pity ws lulted s trolox equivlent (TEAC). Determintion of reduing power The reduing property of the extrts ws determined y ssessing the ility of the extrt to redue FeCl solution s desried y Oyizu 3 (1986). A2.5 ml liquot ws mixed with 2.5 ml of 2 mm sodium phosphte uffer (ph 6.6) nd 2.5 ml of 1% potssium ferriynide. The mixture o ws inuted t 5 C for 2 min nd then 2.5 ml of 1 % trihloroeti id ws dded. This m ixture ws entrifuged t 358 g for 1 min. Five milliliter of the superntnt ws mixed with n equl volume of wter nd 1 ml of.1% ferri hloride ws dded. The sorne ws red t 7 nm (UV-Spetrophotometer JENWAY 635). Asori id ws used s stndrd reduing gent nd ferri reduing power (AAE) ws susequently lulted s sori id equivlent. Dt nlysis The results of the three replite experiments were pooled nd expressed s men ± stndrd devition. One-wy nlysis of vrine (ANOVA) ws rried out nd signifine ws epted t P <. 5. Journl of Sustinle Tehnology, Vol. 3, No. 2 (Novemer 212), ISSN: Pp 91

4 S.A. Shodehinde, et l. RESULTS AND Blk umin seed is populr spie used s flvour enhner nd in folk mediine for the mngement of severl degenertive diseses. Its role in humn nutrition nd helth ould e euse of its iologil tive phytohemils tht my hve ntioxidnt tivities. The totl extrtle phenoli ompound of lk umin seed is presented in tle 1. The vlues rnged from mg/1g (rw extrt) to mg/ 1g (1 min ooked extrt). The totl phenoli ontent of the lk umin seed inresed in the order rw < 5 min ooked < 1 min ooked. This is n indition tht ooking used signifint inrese in the totl phenol ontent of lk umin seed nd it is in greement with the report of Ooh (25) where inrese in the phenoli ontent of vegetles were oserved fter lnhing. However, there ws signifint derese in the totl phenol ontent of the seed fter 1 minutes of ooking (15 min ooked). This finding is onsistent with tht of Amin et l. (26) on phenoli ontent of rw nd lnhed Amrnthus speie. Nevertheless, the vlues reorded re lower thn tht of queous extrts of umin nd itter umin (Thippeswmy nd Nidu, 25). Also, the totl flvonoid ontent of the seed (Tle 1) reveled tht ooking used DISCUSSIONS signifint inrese in the flvonoid ontent from 1.96 mg/1g (rw extrt) to mg/1g (1 min ooked extrt) nd signifint ( P <.5) derese (21.23 mg/1g) fter 1 minutes of ooking (15 min ooked). The pttern oserved for the totl flvonoid ontent of the seed is in greement with tht of totl phenoli ontent where signifint ( P<.5) derese ws oserved fter 1 minutes of ooking. Food proessing tehniques hve een reported to influene phenoli ontent of plnt foods (Ooh, 25). Ismil et l. (24) reported tht het tretment used mrked redution in the totl phenoli ontents of some vegetles. Phenoli ompounds hve een shown to e responsile for the ntioxidnt tivity of plnt mterils ( Rie-Evns e t l., 1996). Prevention of the hin initition step y svenging vrious retive speies suh s free rdils is onsidered n importnt ntioxidnt mode of tion (Dstmlhi e t l., 27). Therefore, the free rdil svenging ility of the lk umin extrts ws studied using modertely stle nitrogen-entered rdil s peies; ABTS ( Re et l., 1999). The ABTS rdil sed model of free rdil svenging ility hs the dvntge of eing more verstile Tle 1: Effet of ooking time (5 15 min) on the totl phenol nd totl flvonoid ontent of lk umin seed Time period Rw 5 min ooked 1 min ooked 15 min ooked Totl phenol d ± ± ± ± 6.5 (mg/1g) Totl flvonoid d 1.96 ± ± ± ± 1.4 (mg/1g) Vlues represent men ± stndrd devition of triplite experiments Vlues with the sme supersript letters long the sme olumn re not signifintly (P<.5) different. Journl of Sustinle Tehnology, Vol. 3, No. 2 (Novemer 212), ISSN: Pp 92

5 Cooking ffets Blk umin phenolis 3 Totl Antioxidnt pity (mmol TEAC/1g) Rw extrt 5 min ooked extrt due to miniml spetrl interferene s the sorption mximum used is 76 nm, wvelength not normlly enountered with nturl produts (Re et l., 1999). ABTS svenging ility reported s TROLOX equivlent ntioxidnt pity (TEAC) is presented in Figure 1. The result reveled tht ooking for 5 nd 1 min used no signifint inrese in the rdil svenging ility (2.29 nd 2.24 mmol/1g respetively) when ompred with the rw seed (2.16 mmol/1g). However, signifint ( P<.5) deresein the rdil svenging ility ws oserved fter 15 min. The trend in the ABTS svenging ility of extrts lso greed with the phenoli ontents. Findings hve shown tht totl ntioxidnt tivity of plnt foods is funtion of their phenoli ontent, hene, the derese i n the ABTS svenging ility of the extrt fter ooking for 1 minutes suggests redution in the phenoli ontents. The ility to helte nd detivte trnsition metls, prevent suh metls from prtiipting in the initition of lipid peroxidtion nd 1 min ooked extrt 15 min ooked extrt Blk Cumin seed extrts Figure 1: Effet of ooking time (5 15 min) on the totl ntioxidnt pity of Blk Cumin seed oxidtive stress through metl tlysed retion is onsidered one of the mehnism through whih phenolis exhiit their ntioxidnt tivity ( Ooh et l., 27). The Fe 2 helting ilit y (Figure 2) of the lk umin seed extrts inresed from 38.7% (rw) to 6.65% (1 min ooked) t the onentrtion tested (2.17 mg/ ml), however t 15 min, signifint ( P <.5) d erese in the Fe 2 helting ility w s oserved. This is in greement with the phenoli ontent (totl phenol nd flvonoid) of the lk umin seed s influened y the ooking time. Findings hve shown tht ntioxidnt pity of plnt food is funtion of their phenoli ontent (Chu et l., 22), hene, the oserved d erese in the Fe 2 helting ility of the l k seed extrts ooked t 15 minutes of ooking. N evertheless, the Fe 2 helting ility of th e extrts fll within tht reported for hot pepper ( Ooh et l., 27). Iron, n essentil metl for norml ellulr physiology, hd een implited in ellulr injury (when in exess), euse it plys tlyti role in the initition of free rdil retions. The mehnism y whih iron Journl of Sustinle Tehnology, Vol. 3, No. 2 (Novemer 212), ISSN: Pp 93

6 S.A. Shodehinde, et l. 2 h e l tin g ility (% ) F e d Rw extrt 5 min ooked extrt 1 min ooked extrt 15 min ooked extrt Conentrtion of Blk Cumin seed extrts (2.17 mg/ml) F igure 2: Effet of ooking time (5 15 min) on the Fe helting ility of Blk Cumin see 2 d 2 n use this deleterious effet is tht Fe n ret with hydrogen peroxide (H O ) to produe 2 2 the hydroxyl rdil ( OH) vi the Fenton retion (Frg nd Oteiz, 22). Ooh et l. ( 27) reported tht Fe 2 heltion my e pr t of the mehnism y whih plnt polyphenols p revents Fe 2 indued lipid peroxidtion i n tissues nd oxidtive stress medited y iron o verlods. The high Fe 2 helting ility o f lk umin ould e of immense importne in the protetion ginst oxidtive stress, euse it is usully too lte to ttempt to use OH svengers for therpeuti purposes, due to extreme high retivity of hydroxyl rdils towrds most iomoleules nd would require unresonly high onentrtions of interepting svengers to neutrlize the iomoleules of interest. These very high onentrtions of svengers re diffiult to hieve in vivo. Consequently, they re not likely to e used for therpeuti purposes, t herey mking Fe 2 heltors ette r t herpeuti lterntive (Byir e t l., 26). The O H svenging ility of the lk umin seed extrts is s presented in Figure 3. The results reveled tht ooking for oth 5 nd 1 min used signifint ( P <.5) inrese in OH svenging ility of the seed rnging from 78.24% to 95.34% respetively, with signifint (P<.5) redution t 15 min of ooking. This lso greed with the phenoli ontent of the seed s ffeted y ooking. Nevertheless, lk umin seed extrts hs stronger OH svenging ility ompred with wht ws reported for tree pepper (Ooh nd Roh, 28), nd red nd green hot pepper (Ooh e t l., 27). O H is highly retive ROS usully produed s result of Fe tlysed retion (Fenton) (Frg nd Oteiz, 22). It is ple of ttking most iomoleules e.g.; rohydrtes, DNA, polyunsturted ftty ids nd proteins. The prevention of suh deleterious retions is highly signifint in humn helth. Journl of Sustinle Tehnology, Vol. 3, No. 2 (Novemer 212), ISSN: Pp 94

7 Cooking ffets Blk umin phenolis svenging ility (%) OHrdil Rw extrt 5 min ooked extrt Conentrtion of Blk Figure 3: Effet of ooking time (5 15 min) on the 1 min ooked extrt 15 min ooked extrt Cumin seed extrts (2.17 mg/ml). OH svenging ility of Blk Cumin seed As represented in Figure 4, the reduing power of the lk umin seed extrts s sori id equivlent (AAE) inresed from.74 mmol.aae/1g (rw) to 4.11 mmol.aae/1g (1 min oiled). However, signifint derese in the reduing power ws oserved t 15 min of ooking (.89 mmol.aae/1g) nd the trend of the reduing power lso followed the pttern of the phenoli ontent where ooking (5 nd 1 min) used signifint inrese ( P<.5) nd derese t 15 min of ooking time. Reduing power is potent ntioxidnt defense mehnism, whih explores theeletron nd/or hydrogen tom trnsfer potentil of ntioxidnt moleules. This is sed on the ility to redue F e 3 t o F e2, euse, the ferri-to-ferrous iro n redution ours rpidly with ll redutnts with hlf retion redution potentils ove tht of F e 3 / Fe 2 ; the vlues in the ferri reduin g ntioxidnt properties (FRAP) ssy will express the orresponding onentrtion of eletron- donting ntioxidnts (Hlvorsen e t l., 22). The reduing power of the extrts my e due to their phytohemil onstituents. Antioxidnt tivity of phenoli ompounds hve een linked to its redox properties whih llowed them to t s reduing gents, hydrogen donors nd singlet oxygen quenhers (Rie-Evns e t l., 1996). Antioxidnt tivities of plnt food hve een shown to orrelte with the phenoli ontent of the food (Chu et l., 22). Any proess or tivities tht led to the redution of the phenoli ontent of the food my hve reduing effet on the totl ntioxidnt properties of the food. Different ooking times might hve resulted in some loss of ntioxidnts suh s sori id, -toopherol nd phenoli ompounds suh s flvonoids. On the other hnd, het lso tends to degrde ertin ompounds with ntioxidnt properties (Amin nd Lee, 25). The loss in the ntioxidnt pity of lk umin seed my e due to losses or degrdtion of ertin types of phenoli ompounds or other free rdil-svenger omponents fter ooking for more thn 1 minutes. Journl of Sustinle Tehnology, Vol. 3, No. 2 (Novemer 212), ISSN: Pp 95

8 S.A. Shodehinde, et l. Ferri reduing power (mmol AAE/1g) Rw extrt 5 min ooked extrt 1 min ooked extrt Blk Cumin seed extrts d 15 min ooked extrt Figure 4: Effet of ooking time (5 15 min) on the Reduing power of Blk Cumin seed Cooking for few minutes (5 1 min) onsiderly inresed the phenoli ontent (totl phenol nd totl flvonoid ontent) nd ntioxidnt properties of lk umin seed. However, ooking for 15 min used negtive effet on the phenoli ontent nd redution in the ntioxi- CONCLUSION REFERENCES dnt properties. Thus, miniml het tretment durtion (5 1 min) through ooking is reom- mended to prevent the mjor loss of ntioxidnt properties nd phenoli ontent of lk umin seed. AMIN, I. ndlee, W.Y. (25). Effet of differen t lnhing times on ntioxidnt properties in seleted ruiferous vegetles. Journl o f the Siene of Food nd Agriulture, 85: AMIN, I., NORAZAIDAH, Y. nd EMMY HAINIDA, K.I. (26). Antioxidnt tivit y nd phenoli ontent of rw nd lnhed A mrnthus speies. Food Chemistry, 94: BAYIR,H.,KOCHANEK,P.M. ndkaganv.e. ( 26). Oxidtive stress in immture rin fter trumti rin injury. Developmentl N eurosienes, 28: BURITS, M. nd BUCAR, F. (2). Antioxidn t tivity of Nigell stiv essentil oil. P hytotherpy Reserh, 14: CHEESEMAN, K.H. nd SLATER, T.F. (1993). An introdution to free rdil iohemistry. British Medil Bulletin, 49: CHU,Y., SUN, J., WU, X. nd LIU, R.H. (22). Antioxidnt nd ntiprolifertive tivity of ommon vegetles. Journl of Agriulturl nd Food Chemistry, 5: DASTMALCHI, K., DORMAN, H.J.D., KOSAR, M. nd HILTUNEN, R. (27). Chemil omposition nd in vitro ntioxidnt evlution of wter solule Moldvin Journl of Sustinle Tehnology, Vol. 3, No. 2 (Novemer 212), ISSN: Pp 96

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