Sugar and anthocyanin characterization of four Iranian pomegranate (Punica granatum L.) varieties using HPLC System

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1 Journl of Biodiversity nd Environmentl Sciences (JBES) ISSN: (Print) (Online) Vol. 4, No. 4, p RESEARCH PAPER OPEN ACCESS Sugr nd nthocynin chrcteriztion of four Irnin pomegrnte (Punic grntum L.) vrieties using HPLC System Somyeh Mirzee Food Sciences, University of Tehrn, Ars Interntionl Cmpus, Irn Article published on April 18, 2014 Key words: Anthocynin, sugr, cultivr, pomegrnte. Abstrct Among ll types of fruit, pomegrnte hs been known s n excellent fruit due to its high vlued nutrceuticl components; i.e. nthocynins, phenolic compounds nd orgnic cids. This reserch imed to mesure nthocynins nd sugrs in different vrieties of pomegrnte. Four cultivrs were Vhshe Kne Tehrn, Gorche Shhvr Yzdi, Shirin Shhvre Yzd nd Pust sefid. High performnce liquid chromtogrphy (HPLC) ws used in order to nlyze the smples for mesuring their components. Cynidin 3-glucoside, cynidin 3,5-diglucoside, delphinidin 3-glucoside, pelrgonidin 3-glucoside nd pelrgonidin 3,5-diglucoside were detected in ll smples s mjor nthocynins. However, their quntities were different such tht cynidin 3,5-diglucoside ws the min pigment, followed by cynidin 3-glucoside nd delphinidin 3-glucoside, while pelrgonidin derivtives were lwys present in smll mounts. Pust Sefid nd Shirin Shhvre Yzd cultivrs contined more nthocynin compred to Gorche Shhvr Yzdi nd Vhshe Kne Tehrn. Results showed Fructose, glucose nd sucrose were mjor sugrs of the cultivrs. Vhshe Kne Tehrn hd the highest fructose nd sucrose content, while Gorche Shhvr Yzdi hd the highest glucose content. Sugr content in Gorche Shhvr Yzdi nd Vhshe Kne Tehrn ws more thn tht in Pust sefid nd Shirin Shhvre Yzd cultivrs. * Corresponding Author: Somyeh Mirzee mzirheidri1364@gmil.com 248 Mirzee et l

2 Introduction Irn commercilly covers lrge number types of fruit especilly pomegrnte tht its totl production is 650, ,000 tons per nnum (Mousvinejd et l., 2009). Pomegrnte is rich source of functionl ingredients involving polyphenols, crotenoids, diet fibers, pectin nd sugrs (Mousvinejd et l., 2000; Gil MI et l, 2000), Its juice is known to be mjor source of phenolic compounds, where nthocynins re the most importnt, especilly 3-glucosides nd 3,5- diglucosides of delphinidin, cynidin, nd pelrgonidin (Du et l., 1975). Anthocynins (ACs) re phenolic compounds tht re widely distributed in fruit nd vegetbles. Anthocynins hve single circulr structure tht is clled cynidin nd ll of them re derived from this molecule through the ddition nd removl of hydroxyl groups, methyltion of hydroxyl groups or sugr concentrtions (Hulme, 1970). Anthocynins re imprting color to plnt (Hulme, 1970), nd hve n rry of helth promoting benefits (Mertens-Tlcott et l., 2006). Anthocynin cts s phytochemicl ntioxidnt with nticncer nd nti therosclerotic effects (Kong et l., 2003). Some positive therpeutic effects of nthocynins re somewht relted to the ntioxidnt properties (Miguel et l., 2004). Pomegrnte juice is nutritionlly n importnt beverge due to its phenolic compounds such s ACs, ellgic cid nd tnnins (Mousvinejd et l., 2009). Three types of Anthocynins; Cynidin (cusing red or drk red), Delphinidin (cusing purple or violet) nd Pelrgonidin (cusing bright red to ornge red), nd their derivtives were the dominnt pigment in vrious prts of pomegrnte, especilly its juice (Hulme, 1970). Gil et l (2000) mesured the concentrtion of nthocynins in pomegrnte vriety nmed Wonderful by mens of high performnce liquid chromtogrphy (Gil et l., 2000). Miguel et l (2004) evluted the nthocynin concentrtion in pomegrnte cultivr nmed Assri (Sepúlved et l., 2010). Sepúlved et l (2010) evluted the type nd mount of sugr, type nd mount of nthocynins, ntioxidnt ctivity nd color of pomegrnte juice of cultivr nmed Chilen. Sugrs hd n importnt role in creting vrious tstes of pomegrnte juice through interction with orgnic cids. Hulme s results show tht Glucose ws the most common sugr in pomegrnte juice, nd other sugrs were Arbinose, Glctose, Fructose, Sucrose nd Rhmnose (Hulme, 1970). This study ws conducted for identifiction nd quntittive determintion of sugrs nd nthocynins compounds in four commercil cultivrs of pomegrnte juice by HPLC nlysis. Juices mde from fruits of Irnin pomegrnte (Punic grntum L.) ccessions of pomegrnte were studied for their orgnic cids, sugrs, nd nthocynin contents, using high performnce liquid chromtogrphy. The im of this reserch is study of sugr nd nthocynin chrcteriztion of four Irnin pomegrnte (Punic grntum L.) vrieties using HPLC System. Mteril nd methods Smple preprtion Four Commercil pomegrnte cultivrs including Shirin Shhvre Yzd (SSY), Vhshe Kne Tehrn (VKT), Pust Sefid (PS) nd Gorche Shhvr Yzdi (GSY) were selected in November 2012 of the Reserch Center of Yzd Province, Irn. Thirty Kg of ech vriety ws purchsed nd ll of them trnsferred to 4 C cold storge on the sme dy s they were hrvested. Defected pomegrntes (scrtched, crcked, cut nd sunburnt) were discrded before the experiments nd the helthy ones selected for extrcting juice. After Pomegrnte vrieties were mnully peeled by hnd-held device; juice ws extrcted under pressure ffect nd pulp nd seeds remined on the screen mesh. Pomegrnte juice ws pssed through the plstic filter to chieve the lowest impurities, then it ws 249 Mirzee et l

3 stored in drk glsses in freezer (24 C) in order to protect ginst the light nd biochemicl chnges. Chemicls Five stndrd nthocynins (Cynidin 3-glucoside (Cy3), Cynidin 3,5-di glucoside (Cy3,5), Delphynidin 3-glucoside (Dp3), Pelrgonidin 3-glucoside (Pg3) nd Pelrgonidin 3,5-diglucoside (Pg3,5)) were purchsed from Apin Chemicls Ltd. (Abingdon, Englnd). Also three sugr Stndrds which found in pomegrnte juice (sucrose, glucose nd fructose) were purchsed from Sigm Aldrich (Steinheim, Germny). All the stndrds were HPLC-grde. Other chemicls used in this study were obtined from Merck Compny (Drmstdt, Germny). HPLC nlysis Anthocynin nd sugr nlyses were performed ccording to the method of Gil et l. (2000). Knuer HPLC system (Berlin, Germny) equipped with UV vis detector (K-2600), nd Trithlon uto smpler nd K-1001 pump used for chromtogrphic qulittive nd quntittive nlysis. Column used for seprtion of nthocynins ws RP C18 Nucleosil 100 (12.5 cm 5.0 mm 5.0 µm). After centrifuging in n eppendorf tube (4 min t 5000 rpm) nd llowing superntnt to pss through 0.45µm PTFE filter (Chromfil CA-45/25 S, Duren, Germny) juice ws injected into the column HPLC system. The mobile phse ws included in: A solvent (2.5% v/v, cetic cid in wter) nd B solvent (2.5% v/v, cetic cid in methnol). Grdient profile used for these two solvents were considered s: 100% A solvent t 0-5 min, 90% A for 15 min, 50% A for 45 min, 100% A in 55 minutes. Flow rte ws 1 mm/min nd chromtogrms were recorded t 510 nm. Column used for seprtion of sugrs ws Eurokt H (300mm 8 mm 10 µm). Pomegrnte juices were prepred s mentioned conditions; Chromtogrms were recorded with the RI detector (Refrct Index). Mobile phse included in wter with constnt flow rte of 0.5 mm/min. Injection volumes were 50 µl for nthocynins nd 20 µl for sugrs. In order to drw stndrd curves of the nthocynins nd sugrs, vrious concentrtions of pure smples of ech compound were injected in column similr to originl smple injection conditions in the HPLC system. Prepred concentrtions were in the rnge of mg/100µl for nthocynins (Cy3, Cy3,5, Dp3, Dp3,5, Pg3 nd Pg3,5). These rnges were % for glucose nd fructose, nd % for sucrose. Ech compound ws quntified by compring its pek re with stndrd curve of tht. Sttisticl nlysis Experiments were performed in Triplicte; Dt nlysed using SPSS sttisticl softwre (SPSS version ; SPSS Inc., Chicgo, IL). Anlysis of vrince (ANOVA) were performed using Duncn s multiple rnge test with α set t 0.05 for compring mens t 95% confidence level (P 0.05). Results nd discussion Anthocynins Anthocynins in juices were mesured by HPLC using Knuer HPLC system. Elution of compounds in reversed phse HPLC depends on their polrity; more polr compounds re eluted erlier. HPLC chromtogrm recorded t 510 nm for getting shrp peks. Figure 1 shows chromtogrm obtined by HPLC for nlysis of nthocynins in PS cultivr. Polrity levels of compounds re s follows: Cy3,5 > Dp3 > Cy3 > Pg3. Pg3 ws the lst pek recorded on the chromtogrm, so it ws nonpolr compound mong mentioned Anthocynins. Anthocynin profiles nd their elution order were similr for ll cultivrs, but the pek res vried significntly mong studied cultivrs which were in coincidence with results obtined by Mousvinejd et l (2009). As shown in figure 1, the most polr nthocynin mong the four cultivrs ws Cy3,5 nd the others were respectively Dp3, Cy3, nd Pg3. As it ws showed in tble1, P3,5 ws not exist in ny of the pomegrnte juice nd this result ws similr to findings of Gill et l (2000). The most nthocynins for SSY were cynidin 3,5-diglucoside ( mg/l), followed by cynidin 3-glucoside ( mg/l) nd for PS it ws cynidin 3-glucoside ( mg/l). 250 Mirzee et l

4 Diglucoside type of Anthocynins for ll cultivrs were found t higher concentrtion levels thn monoglucosides except GSY vriety which ws similr to results obtined by Mousvinejd et l. (2009). The identified nthocynins were Cy3, Cy3,5, Dp3, Pg3 nd Pg3,5 (trce) which their mounts were mg/l, mg/l, mg/l nd mg/l, respectively. SSY ( mg/l) hd the most mount of nthocynin in pomegrnte juice, followed by PS ( mg/l). As it showed in tble2, the difference between mount of nthocynins in ll vrieties were significnt sttisticlly with p<0.05. Fig.1. Typicl HPLC chromtogrm recorded t 510 nm showing the nthocynins Peks of pomegrnte juice (SSY cultivr) using HPLC column RP C18 Nucleosil 100 (12.5 cm 5.0 mm 5.0 lm). : Cy 3,5 b: Dp3, c: Cy3, d: Pg3 Tble 1. Anthocynin composition of 4 Irnin pomegrnte vrieties (mg/l) vriety Cy3 Cy3,5 Pg3 Pg3,5 Dp3 Totl PS ± ± ± ± ±55.64 SSY ±8.39b ±27.47b ±1.37b ±2.87b ±40.04b GSY ±12.84c ±12.84c VKT ±6.68d ±11.63d 11.44±1.08d ±17.25d Different letters in the sme column present significnt difference t p<0.05 Sugrs Sugrs in juices were mesured by HPLC system equipped with column nmed Eurokt H. Mens for significnt difference for mount of sugr between the VKT nd GSY vrieties but the difference between them with SSY nd PS cultivrs ws significnt totl sugr content in four cultivrs were sttisticlly (p<0.05). Observed significnt g/100g. Figure 2 shows typicl HPLC differences for mount of sugr between vrieties chromtogrm for nlysis of sugrs in PS. Sugr profiles nd their elution order were similr for ll cultivrs, but the pek res vried significntly mong studied cultivrs which were in coincidence with results obtined by Hulme, A. C. (1970). Figure 3 shows concentrtion of glucose, fructose nd sucrose in four commercil cultivrs of pomegrnte juice. Fructose ( g/100g) nd sucrose ( g/100g) hd highest nd lowest vlues in the pomegrnte juice; this finding were similr to results could be due to the differences in hrvesting time, rinfll in growing seson, geogrphicl condition nd the type of cultivrs. VKT nd GSY were the best vrieties in terms of sugr percentge in pomegrnte juice. Results found by Fdvi et l (2005), shows higher levels of sugrs in compre with present findings (Fdvi et l., 2005). These differences my be result from different times of hrvesting due to the fct tht the mount of sugr is incresed by the time of fruit mturity. obtined by Melgrejo et l (2000). VKT hd the highest sucrose (5.3 g/100g) nd fructose (9.16 g/100g) while the highest glucose (7.14 g/100g) ws found for the GSY cultivr. There ws not found 251 Mirzee et l

5 sugr content (g/100gr) J. Bio. & Env. Sci References Mousvinejd G, Emm-Djomeh Z, Rezei K, Khodprst MHH Identifiction nd quntifiction of phenolic compounds nd their effects on ntioxidnt ctivity in pomegrnte juices of eight Irnin cultivrs, Journl of Food Chemistry 115, Fig.2. Typicl HPLC chromtogrm recorded t 365 nm showing the sugrs peks of pomegrnte juice (SSY cultivr) using HPLC column Eurokt H (300mm 8 mm 10 µm), : sucrose, b : glucose, c : fructose Gil MI, Toms-Brbern FA, Hess-Pierce B, Holcroft DM, Kder AA Antioxidnt ctivity of pomegrnte juice nd its reltionship with phenolic composition nd processing, Journl of Agriculturl nd Food Chemistry 48, /jf Glucose fructose sucrose Totl b b Du C, Wng P, Frncis FJ Anthocynins of pomegrnte, Punic grntum. Journl of Food Science 40, b b b c b Hulme AC The biochemistry of fruits nd their products, Vol. 1. London nd New York: Acdemic Press PS SSY GSY VKT Cultivrs Fig. 3. Sugr profiles of different pomegrnte cultivrs, -b: Different letters present significnt difference t p < 0.05 Conclusion Results indicted significnt differences mong different cultivrs of pomegrnte in terms of their nthocynins nd their sugr contents. PS nd SSY cultivrs contined higher mounts of nthocynins compred to GSY nd VKT. While, in contrst, GSY nd VKT hd higher content of sugrs. Fructose ws detected s the most common sugr nd Cy3,5 s the most common nthocynin in pomegrnte juice. It is therefore recommended to use juices produced from mixture of juices from ll cultivrs to get functionl beverge of pomegrnte. Mertens-Tlcott SU, Jilm-Stohlwetz P, Rios J, Hingorni L, Derendorf H Absorption, metbolism nd ntioxidnt effects of pomegrnte (Punic grntum L.) Polyphenols fter ingestion of stndrdized extrct in helthy humn volunteers, Journl of Agriculturl nd Food Chemistry 54, Kong JM, Chi LS, Goh NK, Chi TF, Brouillrd R Anlysis nd biologicl ctivities of nthocynins. Journl of Phytochemistry 64, Miguel G, Fontes C, Antunes D, Neves A, Mrtins D Anthocynin concentrtion of Assri pomegrnte fruits during different cold storge conditions, Journl of Biomedicine nd Biotechnology 5, Mirzee et l

6 Sepúlved E, Sáenz C, Peñ Á, Robert P, Brtolomé B, Gómez-Cordovés C Influence of the genotype on the nthocynin composition, ntioxidnt cpcity nd color of Chilen pomegrnte (Punic grntum L.) juices, Chilen Journl of Agriculturl Reserch 70(1), Fdvi A, Brzegr M, Azizi MH, Byt M Physicochemicl composition of ten pomegrnte cultivrs (Punic grntum L.) grown in Irn, Food Science nd Technology Interntionl 11, / Melgrejo P, Slzr DM, Artes F Orgnic cids nd Sugrs composition of hrvested pomegrnte fruits. Eur Food Res Technol 211, /s Mirzee et l