Antioxidant Activities and Phytochemicals of Tagetes Erecta Flowers As Affected by Drying Methods

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1 J. Appl. Environ. Biol. Si., 4(9S) , , TextRod Pulition ISSN: Journl of Applied Environmentl nd Biologil Sienes Antioxidnt Ativities nd Phytohemils of Tgetes Eret Flowers As Affeted y Drying Methods Khnzdi Ftim Khttk 1,2* 1 Adul Wli Khn University, Mrdn, Pkistn 2 Nuler Institute for Food nd Agriulture, P.O. Box 446, Peshwr, Pkistn Reeived: Septemer 12, 2014 Aepted: Novemer 23, 2014 ABSTRACT The flowers extrts of Tgetes eret Linn. Hve extensive usges in industries like food, everges, osmetis, feed, frgrnes nd dyeing. In this study, the influene of four different drying tehniques nmely spry drying, shde drying, oven drying nd sun drying on the levels of seleted phytohemils nd svenging tivity of the flowers ws studied. The dt showed signifint effet of drying tretments on the onentrtions of the phytohemils. Asori id, totl phenoli nd totl flvonoid ontents rnged from 57.3 to 91.0 mg/100 g, to mg/100 g nd to mg/g, respetively. Highest onentrtion of sori id ws found for shde dried smple, while lowest for sun dried smple. Similrly, the shde dried smple showed the highest phenoli ontent. The shde, oven nd spry dried smples showed sttistilly similr vlues for flvonoid ontents, wheres lowest vlue ws oserved for sun dried smple. The spry dried smple showed the highest vlues for oth DPPH nd hydrogen peroxide svenging tivities. KEYWORDS: Tgetes eret; Drying Methods; Svenging Ativities; Phytohemils 1 INTRODUCTION Tgetes speies elong to fmily Asteree nd re of high demnd in dyeing, frgrnes, osmetis, everges, food, feed nd phrmeutil industries. These plnts hve een widely used s folk mediine for treting vrious ilments nd exploited s nturl insetiides, inset repellents, heriides, teriides nd fungiides (Btish et l., 2007; Fizi et l., 2008; Vsudevn et l., 1997). The flower pigments re ommerilly onsumed s nturl olouring gents in food, feed nd everge industries (Vsudevn et l., 1997). Tgetes eret Linn. is very populr in Pkistn s n ornmentl plnt. It is lolly known s Gend Phul. The English nme of the plnt is Afrin mrigold. Different prts of this plnt re used in folk mediines to ure severl diseses. The whole her is used for mediinl properties suh s nthelminti, stringent, romti, diureti, sedtive nd ntisepti. Leves re used to tret kidney troules, musulr pin, ulers, piles nd pplied externlly to oils, wounds nd runles (Gopi et l., 2012; Kumr et l., 2006). The flowers lso hve numerous uses. These re used in slds, urrys, te, ondiments nd s flvoring nd oloring gents (Kisoon et l., 2011). Mediinlly, the flowers re useful for the tretment of fevers, epilepti fits, sore throts, indigestion, oughs, dysentery, sies, liver omplints nd eye diseses (Kirtikr nd Bsu, 1987; Ghni, 1998). Externlly, the flowers re utilized to tret runles, ezem, sore eyes, erhes nd rheumtism. The flowers produe romti oil tht is lrgely used in superior qulity perfumes (Jothi, 2008; Prsd et l., 2010). The flower pigments re extensively used in food industry, textile olourtion nd to olour egg yolks (Jothi, 2008; Vnkr et l., 2009). Numerous sientifi investigtions showed tht the plnt hve nemtoidl, ntifungl, insetiidl, heriidl, ntimlril, ntioxidnt, ntimutgeni, nthelminti, ntinoieptive, heptoprotetive nd nti-inflmmtory tivities (Giri et l., 2011; Gopi et l., 2012; Gupt nd Vsudev, 2010; Li-Wei et l., 2012; Shinde et l., 2009). Phytohemil nlysis of different prts of the plnt hve resulted in the findings of vrious hemil onstituents suh s phenolis, lkloids, thiophenes, steroids, terpenoids, flvonoids nd rotenoids (Fizi nd Nz, 2004; Li-Wei et l., 2012). The plnt hs een shown to ontin lutein, queretgetin, syringi id, queretin, thienyl gllte, ethyl gllte nd methyl 3,5-dihydroxy-4- methoxy-enzote (Ghni, 1998; Jothi, 2008).The petls extrts prepred y using different solvents led to the isoltion of dodenoi id, myristi id, plmiti id, steri id, oteiosne-8-one nd triontne-1-ol (Prsd et l., 2010). * Corresponding Author: Dr. Khnzdi Ftim Khttk, Adul Wli Khn University, Mrdn, Pkistn. & Nuler Institute for Food nd Agriulture, P.O. Box 446, Peshwr, Pkistn. khttkkf@yhoo.om 253

2 Khttk, 2014 Reent worldwide penhnt for the use of nturl ompounds hs extremely ugmented the importne of plnt mterils for usge in industries like phrmeutil, nutreutil, food, osmeti nd pestiides et. Correspondingly, there is n inresing interest in the ssurne of qulity, sfety nd effiy of rw mterils nd plnt sed produts (WHO, 2000, 2003). Plnt mterils re now undntly ville in rw, semi-proessed nd produt forms ll over the world mrkets. Though, plnt extrt powders nd dried mterils (rks, roots, stems, seeds, flowers nd leves) show numer of dvntges over the onventionl fluids nd unproessed forms euse they hve higher mounts of tive ompounds whih inrese the therpeuti effiy nd produt vlue (Clixto, 2000; Roh et l., 2011: Runh et l.,2001). Rw plnt mterils re lso diffiult to trnsport euse of their ulkiness nd high trnsporttion hrges. Drying redues the moisture ontents nd therefore virtuous for reduing storge nd trnsporttion osts (Clixto, 2000). It lso dereses the hnes of miroil ontmintion (Hssnin, 2011). Drying n ffet oth the quntity nd qulity of the tive onstituents, therefore the seletion of the pproprite drying method for plnt mterils nd their extrts remins serious riterion (Roh et l., 2011). The present study ws intended to ssess the effet of different methods of drying (sun, shde, oven nd spry drying) on the phenoli, flvonoid nd sori id ontents s well s svenging tivities of Tgetes eret flowers. 2. MATERIALS AND METHODS Colletion of plnt smples: Tgetes eret flowers were olleted from NIFA Trn, Peshwr, in the month of Deemer nd immeditely trnsferred to the lortory. The plnt mteril ws uthentited y PCSIR Ls., Peshwr, Pkistn. Smple preprtion: The flowers were lened from foreign prtiles, ut into smll piees nd divided into three equl prts. One prt ws shde dried with nturl ir irultion. The verge room temperture ws 19 C. The seond portion ws sun dried in len ple. The third prt ws dried in lortory oven t 45 C. Afterwrd, the dried plnt mterils were ground in grinder (Reth Muhle-Germny) nd pssed through the sieves of mesh size 30. The powdered smples were pked in ler polyethylene pouhes nd seled with eletri Seler PFS 300, Jpn. For spry drying, out 500 g of fresh flowers of Tgetes eret were properly wshed with tp wter nd then rinsed with distilled wter. The flowers were mixed with 1.5 litre of distilled wter nd homogenized in lender. Lter, the solution ws filtered through Whtmn No. 1 filter pper. The filtrte ws onentrted to 50 % in rotry evportor t 45 C. The smple ws then dried using spry-drier model Pmio L. 01/08 (Pmio Tehnologies, Fisld, Pkistn) ontining o-urrent nozzle nd ylone with powder olleting ottle. The dryer hs the pity of drying up to 10 litres per hour. The opertionl onditions employed during spry-drying re shown s follows: Inlet temperture: 175 ± 10 C Outlet temperture: 65 ± 5 C Atomiztion pressure: 1.5 rs Preprtion of extrts: The dried flower smples (50 g, eh) were extrted seprtely using distilled wter. The mixtures were kept in n oritl shker t room temperture for 8 hours. The extrts were seprted from the residues y filtering through Whtmn No. 1 filter pper. The residues were extrted thrie with fresh distilled wter nd finlly the extrts were omined. The pooled extrts were onentrted under redued pressure t 45 C using rotry evportor nd stored t 4 C until further proessing. Estimtion of sori id: Asori id ws mesured y titrtion method (AOAC, 1984). Four grms of the dried smples of Tgetes eret flowers were extrted in 3% metphosphori id (50 ml), filtered nd the extrts were titrted with dye solution (2,6-dihlorophenol indophenol). The end point is the pperne of light pink olour, whih persists for few minutes. Asori id ontent ws then lulted sed on the following formul: Asori id (mg/100 g) = (titre dye ftor volume mde up 100)/(liquot of extrt volume of smple). Estimtion of phenoli ontent: The totl phenoli ontents of plnt smples were estimted y the Folin- Ciolteu olorimetri method (Khttk, 2012). The filtered extrts of different onentrtions were tken in 10 ml glss tues nd totl volumes were mde to 2 ml with distilled wter. 4 ml of 2% queous sodium ronte solution ws dded to these nd mixed thoroughly. Then 500 µl of Folin-Ciolteu regent ws dded to the mixtures. The mixtures were llowed to stnd for 1 hour with intermittent shking 254

3 J. Appl. Environ. Biol. Si., 4(9S) , 2014 nd the sorne of the green-lue omplexes were mesured t 750 nm in spetrophotometer ginst lnk. The stndrd urve ws prepred using known onentrtions of glli id. The totl phenol ontent in the test smples were lulted from the stndrd urve nd the results were expressed s milligrm of glli id equivlents per 100 grms (mg/100 g) of the dry extrt. Estimtion of totl flvonoids: Flvonoids ontents were determined y following the method of Aiyegroro nd Okoh (2010). One ml of the different extrts of the plnt were mixed with 3ml of methnol, 0.2 ml of 10% luminum hloride, 0.2 ml of 1 M potssium ette nd 5.6 ml of distilled wter. The mixtures were kept t room temperture for 30 minutes. The sorne of the mixtures ws mesured t 420 nm with UV visile spetrophotometer. Queretin ws used s stndrd (0 1 mg/ml). Flvonoid ontents were determined from the stndrd urve nd expressed s queretin equivlent (mg/g). Determintion of DPPH rdil svenging tivity: The ntioxidnt tivity of the smples ws ssessed using 1,1-diphenyl-2-pirylhydrzyl (DPPH) rdil (Khttk, 2012). Vrious onentrtions of the dried extrts were dded to 2 ml of DPPH in methnol solution (100 µm) in test tue nd shken vigorously. After inution t 37 C for 50 minutes in the drk, the sorne of eh solution ws estimted t 517 nm. The perentge of free rdil svenging effet ws lulted s follows: Svenging effet (%) = [(A 517 ontrol A 517 test smple)/a 517 ontrol] X 100 Where A 517 ontrol = Asorne of the ontrol t 517 nm A 517 test smple = Asorne of the test smple t 517 nm. The results otined from the rdil svenging experiments were expressed s EC 50 vlues. EC 50 vlue is the extrt onentrtion t whih DPPH rdils were redued y 50% nd lulted from the liner regression nlysis. Hydrogen peroxide ssy: The ility of plnt extrts to svenge hydrogen peroxide ws determined ording to the method of Ruh et l. (1989). A solution of hydrogen peroxide (40 mm) is prepred in phosphte uffer (50 mm, ph 7.4). The onentrtion of hydrogen peroxide is determined y using spetrophotometer. Extrts ( µg/ml) ws dded to hydrogen peroxide nd mixed. After 10 minutes the sorne ws determined t 230 nm ginst lnk solution ontining phosphte uffer without hydrogen peroxide. Alph toopherol ws used s positive ontrol. The perentge of hydrogen peroxide svenging is lulted s follows: % Svenged (H 2 O 2 ) = (A 0 A 1 / A 0 ) X 100 Where; A 0 is the sorne of ontrol nd A 1 is the sorne of test smple. Dt Anlysis: All determintions were otined from triplite mesurements nd results were expressed s mens ± stndrd devitions. The dt were nlyzed using one-wy ANOVA nd lest signifint differene tests for the men differenes. Sttistil signifine ws delred t p < RESULTS AND DISCUSSION The effet of four drying tretments nmely, sun drying, shde drying, oven drying nd spry drying ws heked on the onentrtions of flvonoids, sori id nd totl phenolis s well s svenging tivity using DPPH rdil nd hydrogen peroxide of Tgetes eret flowers. Vitmin C tkes prt in numerous iologil funtions suh s ollgen formtion, sorption of inorgni iron, redution of plsm holesterol level, inhiition of nitrosomine formtion, oosting of immune system nd svenging of free rdils, nd thus plys vitl role in the mintenne of helthy skin, gums nd lood vessels (Lee nd Kder, 2000). The sori id ontents of the flowers of Tgetes eret were determined on dry weight sis nd results re presented in Figure 1. The ontent of the tested smples rnged from 57.3 to 91.0 mg/100 g. The results showed tht drying tretments signifintly ffeted (p< 0.05) the ontents of sori id in Tgetes eret flowers. The shde dried smple showed the highest vlue (91.0 mg/100 g), followed y spry dried (90.0 mg/100 g) nd oven dried (84.3 mg/100 g). The sun dried (57.3 mg/100 g) smple showed the minimum onentrtion of sori id. The reson might e the omprtively high rte of oxidtion in the presene of diret sunlight. No informtion is ville on the effet of drying onditions on vitmin C ontent of the plnt. In omprison to our findings, study onduted y Mishr et l. (2009) on different dehydrtion tehniques (freeze drying, sun drying, spry drying, hot ir drying nd vuum drying) showed thelowest onentrtion of sori id in the sun dried powder of Aml (Emli offiinlis).similrly, sientifi investigtion rried out y Sohil et l. (2011) on the physiohemil nd miroiologil properties of sun dried tomtoes reveled tht the nutrient whih is highly ffeted y sun drying ws vitmin C. In fresh tomtoes, the sori id 255

4 Khttk, 2014 ontent ws 32.5 mg/100 g whih ws redued to 24.6 mg/100 g fter sun drying. Joshi nd Meht (2010) reported tht fter dehydrtion the mximum mount of vitmin C ws noted in shde dried smple, s in this tehnique the leves were not exposed to diret het nd ir. Plnt derived phenoli ompounds hve reeived onsiderle ttention due to their ntioxidnt tivities nd free rdil svenging ilities, whih hve enefiil implitions in humn helth nd usges in food nd osmeti industries (Oksn et l., 2012).The totl phenoli ontent of the flower extrts were determined y Folin-Ciolteu olorimetri method nd the results re expressed s milligrms of glli id equivlents (GAE) per grm of dry weight of the extrts (Figure 2). The phenoli ontents rnged from to mg/100 g. Previously, study onduted y Kisoon et l. (2012) on seleted edile flowers showed mg/100 g of phenoli ompounds in the dry weight of the extrts of Tgetes eret. Gong et l. (2012) worked on the optimiztion of extrtion prmeters of iotive omponents in the petls of Tgetes eret nd reported mg GAE/g of totl phenolis. It ws oserved tht drying methods signifintly (p < 0.05) ffeted the totl phenolis in the queous extrts of the flowers. The shde dried powder hd the highest phenoli ontent, followed y sun dried powder while spry dried smple exhiited the lowest onentrtion of phenolis. Eerlier reserh studies showed diverse results. Comprle findings were reported y Rmmoorthy nd Bono (2007). Their investigtions reveled lowest phenoli ontent in spry dried while highest in vuum dried smples of the fruit extrts of Morind itrifoli. Hossin et l. (2010) heked the effet of effet of drying methods (shde, freeze nd oven drying) on the totl phenoli ontent of rosemry, oregno, mrjorm, sge, sil nd thyme. They reported tht the shde dried smples hd signifintly higher phenolis ontents thn freeze- nd oven-dried smples throughout the storge period of 60 dys. Chovnlikit et l. (2012) reveled tht spry drying n preserve totl phenoli in mngosteen powder etter thn vuum drying. Novkovi et l. (2011) investigted the influene of different drying tretments (onvetive, osmoti nd freeze) on the ntioxidnt tivity nd phenoli ontent of rsperry fruits. The onvetive nd osmoti drying showed signifint derese in phenoli ontent of the fruit smples, when ompred with freeze drying tretment. Hung nd Duy (2012) ompred the effet of freeze nd oven drying on the phytohemils of rrot, tro, tomto, red eetroot nd eggplnt, grown in Vietnm. Their results indited tht rise of temperture in het-drying method signifintly redued the totl phenolis. Flvonoids re low moleulr weight ompounds tht hve wide rnge of iologil tivities inluding nti-inflmmtory, ntiteril, ntivirl, ntioxidnt, ntillergi, ytotoxi, ntitumour nd vsodiltory et. (Sndhr et l., 2011). The flvonoid ontents of the powdered smples of Tgetes eret were determined nd presented in Figure 3. The levels of flvonoids rnged etween to mg/g. Previously, Kisoon et l. (2011) disovered tht the flowers of Tgetes eret hd 68.9 mg/g of flvonoid ontent. Comprle results were desried y Gong et l. (2012). They reveled tht fermented petls of deftted Tgetes eret hd mg RE/g of totl flvonoid ontent. The present study showed tht the flvonoid ontents oserved for shde dried (161.3 mg/g), oven dried (155.7 mg/g) nd spry dried (158.0 mg/g) re sttistilly sme (p> 0.05). While, signifint derese (p < 0.05) in flvonoid ontent ws oserved for sun dried smple (137.0 mg/g). Erlier, Rmmoorthy nd Bono (2007) reported highest flvonoid ontent in the ethyl ette extrt of spry dried smples of Morind itrifoli fruit. A sientifi investigtion onduted y Hung nd Duy (2012) showed signifintly redued flvonoid ontents in rrot, tro, tomto, red eetroot nd eggplnt fter het-drying method. Novkovi et l. (2011) reported tht onvetive nd osmoti drying resulted in signifint dereses in the onentrtion levels of flvonoids in rsperry fruits when ompred with freeze drying. The free rdil svenging tivity of the extrts of the plnt ws nlyzed y using DPPH rdil. The results otined from these experiments re expressed s EC 50 vlues (µg/ml) nd presented in Figure 4. A low EC 50 vlue is the sign of high DPPH svenging tivity. The EC 50 vlue of Tgeteseret smples rnged etween 13.9 to 25.3 µg/ml. Previously, Chivde et l. (2011) reported tht the IC 50 vlues ws 3.4 µg/ml for the ethnoli extrt of flowers of Tgetes eret. Wheres, Gutierrezet l. (2011) worked on the oil ontent of Tgetes eret nd reported Lower DPPH svenging tivity s EC 50 vlue ws 71.5 µg/ml. The urrent dt showed strong orreltion (p < 0.05) of drying tretments with DPPH svenging tivity. Highest DPPH svenging tivity ws reorded for spry dried flowers, while lowest for sun dried smples. Exposure to diret sunlight my led to initition of elerted oxidtion. The results of the present study me in onsistene with the findings of Rmmoorthy nd Bono (2007). They noted the highest DPPH svenging tivity in spry dried extrts of Morind itrifoli fruit. Likewise, Hossin et l. (2010) reported signifintly higher ntioxidnt tivity for shde-dried smples of rosemry, oregno, mrjorm, sge, sil nd thyme, when ompred with freeze-dried nd oven-dried smples. 256

5 Asori id (mg/100 g) J. Appl. Environ. Biol. Si., 4(9S) , 2014 Conversely, Annmli et l. (2011) reported higher DPPH svenging tivity for mirowve dried smple of Crdiospermum hlium s ompred to tht of the shde nd sun dried smples. Hung nd Duy (2012) ompred the effet of freeze drying nd oven drying on DPPH svenging tivity of rrot, tro, tomto, red eetroot nd eggplnt. Their results indited signifint losses in the ntioxidnt pity of oven dried smples. The Figure 5. showed the effet of sun, shde, oven nd spry drying tretments on the hydrogen peroxide svenging ssy of the flowers of Tgetes eret. It ws ler from the dt tht drying tretment hs signifintly (p < 0.05) ffeted the hydrogen peroxide svenging tivity of Tgetes eret flowers extrts. The exhiited EC 50 vlues were 94.9, 96.8, 85.4 nd 79.9 µg/ml for sun dried, shde dried, oven dried, nd spry dried smples, respetively. There is no informtion ville in the literture on the effet of drying methods on the svenging tivity of the plnt. However, erlier Novkovi et l. (2011) investigted the influene of different drying tretments (onvetive, osmoti nd freeze) on the ntioxidnt tivity of rsperry fruits nd reported tht the onvetive nd osmoti drying showed signifint redutions in hydrogen peroxide svenging tivity, when ompred with freeze drying Sun Shde Oven Spry Drying method Figure 1. Effet of drying methods on the sori id ontent of Tgetes eret flower. Vlues re mens of triplite determintions (n=3) ± stndrd devitions. The vertil rs represent the stndrd devition for eh dt point. Vlues with different supersript letters re signifintly different (p < 0.05). 257

6 Phenoli ontent (mg/100 g) Phenoli ontent (mg/100 g) Khttk, d Sun Shde Oven Spry Drying methods Figure 2. Effet of drying methods on the phenoli ontent yields of Tgetes eret flower. Vlues re mens of triplite determintions (n=3) ± stndrd devitions. The vertil rs represent the stndrd devition for eh dt point. Vlues with different supersript letters re signifintly different (p < 0.05). 700 d Sun Shde Oven Spry Drying methods Figure 2. Effet of drying methods on the phenoli ontent yields of Tgetes eret flower. Vlues re mens of triplite determintions (n=3) ± stndrd devitions. The vertil rs represent the stndrd devition for eh dt point. Vlues with different supersript letters re signifintly different (p < 0.05). 258

7 EC 50 (DPPH) Flvonoids (mg/g) J. Appl. Environ. Biol. Si., 4(9S) , Sun Shde Oven Spry Drying methods Figure 3. Effet of drying methods on the flvonoid ontent yields of Tgetes eret flower. Vlues re mens of triplite determintions (n=3) ± stndrd devitions. The vertil rs represent the stndrd devition for eh dt point. Vlues with different supersript letters re signifintly different (p < 0.05) d 10 0 Sun Shde Oven Spry Drying methods Figure 4. Effet of drying methods on the EC 50 vlue (µg/ml) of the DPPH svenging tivity of Tgetes eret flower. Vlues re mens of triplite determintions (n=3) ± stndrd devitions. The vertil rs represent the stndrd devition for eh dt point. Vlues with different supersript letters re signifintly different (p < 0.05). 259

8 EC 50 (Hydrogen peroxide) Khttk, Sun Shde Oven Spry Drying methods Figure 5. Effet of drying methods on the EC 50 vlue (µg/ml) of the hydrogen peroxide svenging tivity of Tgetes eret flower. Vlues re mens of triplite determintions (n=3) ± stndrd devitions. The vertil rs represent the stndrd devition for eh dt point. Vlues with different supersript letters re signifintly different (p < 0.05). 4. CONCLUSION With the inresed onsumer interest in plnt sed vlue-dded produts, there is growing sientifi emphsis in proessing tehnologies nd onsequent evlution of the influene of these tehniques on the hemil onstituents, iologil tivities nd physil hrteristis of the plnt mterils. Proessing nd vlue ddition tivities re impertive for getting onentrted nd qulity produts. The present study showed tht spry drying is the est tehnique to hve mximum svenging tivity nd etter retention of enefiil phytohemils in the dried extrts of Tgetes eret, followed y shde drying. Aknowledgement: The uthor is thnkful to the Diretor, NIFA for providing ll the filities to ondut the present reserh work. The uthor lso grtefully knowledges the ssistne of Mr. Mushtq Khn in rrnging the plnt smples. REFERENCES 1. Aiyegroro, O. A. nd Okoh, A. I Preliminry phytohemil sreening nd in vitro ntioxidnt tivities of queous extrt of Helihrysum longifolium DC. BMC Complementry nd Alterntive Mediine 10: Annmli, A., Ponmri, G., Sthishkumr, R. nd Lkshmi, P. T. V Effet of drying tretment on the ontents of ntioxidnts in Crdiospermum hlium Linn. Interntionl Journl of Phrm nd Bio Sienes 2: B304 B AOAC Offiil Methods of Anlysis. 14 th edn. Assoition of Offiil Anlytil Chemists. Virgini. 4. Btish, D. R., Aror, K., Singh, H. P. nd Kohli, R. K Potentil utiliztion of dried powder of Tgetes minut s nturl heriide for mnging rie weeds. Crop Protetion26:

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