J Pood Sox Nutr Vol 2, No.2, p.83~B81997) Isolation and Identification of Anthocyanins from Purple Sweet Potatoes Lan-Sook Lee*, Eun-Ju Chang, Jong-Whan Rhim*, Byoung-Seob Ko"* and Sang Won Choi” Depi. of Food Science and Nutrition, Catholie University of Taegu-Hyosung, Kyungsan 713-702, Korea “Dept. of Food Engineering, Mokpo National University, Chonnam 534-729, Korea “‘Korea institule of Oriental Medicine, Seoul 135-100, Korea Abstract Anthocyanin pigments of purple sweet potato roots(Ipomoea batatas L.) were extracted with 0556 TFA (rifluoroacetic acid) in 95% EtOH, and further isolated and purified by Amberlite XAD~7 and ODS column chromatography, and final preparative HPLC. Among nine anthocyanins isolated, the structure of three major anthocyanins were identified as 3-O-(6-O-trans~caffey!)-2-O-(6-O-trans-caifeylglucopyravosyl)-8-D~ slucopyranosyl-5-O-(8-D-glucopyranosyl)-peonidin, 3-O-(6-O-trans~caffeyl)~2-O-(6-O-trans-feruloyl~ glucopyranosyl)-f-D-gucopyranosyl-5-O-(B-D-glucopyranosyl)-peonidin, and 3~O-(6-O-trans-catfeyl)— 2-0-(6-O-p-hydroxylbenzoylglucopyranosyl)~) D-glucopyranosyl-5-O-~(S—D-ghucopyranosyl)—peoniclin, bby using UV-visible absorption spectra, 'H-NMR and FAB-MS analysis. Key words: sweet potatoes, anthocyanins, chromtogrepitic and spectral analyses INTRODUCTION ‘There is much attention in the development of food colorants from natural sources to replace synthetic food colorants. In particular, anthocyanins have considerable potential in the food industry as safe and effective food additives due to apparent harmiessness to human(1), Compared to the synthetic colorants, anthocyanins have been less extensively used because of their in~ stability towards a variety of chemical and physical fac~ tors(2,3). However, since the novel polyacylated an~ thoeyanins displaying marked stability were recently found in several plants(4~6), further studies on the pro- duction of these stable anthocyanins by cell cultures, and their epplication in foods and beverages have been performed 7-9). Recently, anthocyanins have been recognized as bio logically active substances(10-12) and nataral colorants. ‘Tsuda et al.(13,14) suggested that anthocyanins may play an important role as dietary antioxidants for pre~ vention of oxidative damage caused by active oxygen radicals in living systems. Anthocyanins together with other flavonoids are also known to have an important impact on inhibiting the lipid peroxidation of human low-density lipoprotein( 15}. "Comesponding author Many researches on the isolation and structural elu~ cidation of anthocyanins from sweet potatoes are avail~ able. The presence of several cyanidin and peonicin gly~ cosides, as well as several hydroxybenzoy] and hydroxy- cinnamoyl acylated anthocyanins was reported in seve ral cultivars and tissues of sweet potatoes( 16-20), These anthocyanin pigments were found to be mone stable than ther anthocyanins in a neutral or wealty acide aqueous solution(21,22). Especially, much interest has recently received on the acylated anthocyanins in sweet potato as natural food colorants due to high stability and anti- oxidative activity(23), ‘The purpose of this stndy was to isolate and charac~ terize anthocyanin pigments of newly bred purple swoet potatoes by using several chromatographic, and "H-NMR. and FABMS techniaues. MATERIALS AND METHODS Materials and reagents ‘The roots of purple sweet potatoes, Yamagawa Mu~ rasaki, ([pomoea batatas 1.) came from Kusbui in Japan, ‘These were grown on the farm of Mokpo Experisnent Station in Honam Agricultural Experiment Station, Fresh roots of sweet potatoes were harvested, washed anda Lan-Sook Lee. Eun-Ju Chang, Jong-Whan Rhim, Byoung-Seob Ko and Sang-Won Choi stored at incubator(HB-1065, Hanbaek, RH. 8596, 19°C) unl used, Triflucroacetic acid TFA), CFsCOOD, DMSO~ e, were obtained from Sigma Chem. Co.(St. Louis, MO, USA), Acetonitrile and other solvents for HPLC analysis, ‘were supplied by Merck(Darmstadt, Germany). All abo~ reatory chemicals used in this study were of reagent grade, Isolation and purification of anthocyanin pigments ‘The schematic procedure for isolation and purification of anthocyanins from purple sweet potato roots is shown in Fig. 1. The sliced purple sweet potato reots(100g) were twice extracted with IL of 0596 TFA in 9526 BiOH over night. filtered and then concentrated to @ small volume below 40°C in vacua. The aqueous solution was extracted with n-hexane to remove the lipid, and then applied on the Amberlite XAD-7(Organo Co. Ltd., Tokyo, Japan) column(25mm 500mm) with 100ml of 0.5% TFA so lution, and final anthocyanin was eluted with 1.5L of 0.1% TRA in MeOH-H,0(70 : 30} solution. The pigment. fraction was evaporated under vacuum to dryness, and, 100rnl of 19 HCl in MeOH and 300m! of ethylether were Sweet potato mants(i00g) Extracted with 05% TIA m 95% E}OH for overmght Filteres Evaporated ta dryness in vacuo EXOH extOFg) | Solubilized in 0596 TFA Amberhte XAD-7 column cliromatogranhy 120 i, MOH 01% TPA in MeOH-H:O (90:70) fr (7030) $41.25) I Precipitate of anthocyanin with 2x0 (aang! 0S eon ersten HOACICHACN/HO/TEA {292839508 f (ne) Preparctive HPLC Anthocyanin Pi(2hme), PxGSing) i Pa(Sima) SHeNMR & FAB-MS —— Fig. 1, Schematic procedure for extraction, separation ‘and purification of three major anthocyanins from purple sweet potato roots. further acided stepwise to the residue to precipitate the anthocyanin pigments. The precipitates were collected by centrifugation(Z,500mm, 20min) and then dried in a ‘vacuum desiccator. These partially purified anthocyanins were adsorbed on the ODS(Sigma Chem. Co. 40~63u) ‘open eohumn(20rnm x 20mm) and then fractionated into 4 fractions using following solvent systems; solvent A (HOAc-CH:CN-H0=20 : 30 50) + solvent B(0.196 TRA) (151,21, 3:1 and 4:1, w/v). The last fraction was further purilied by preparative HPLC (Delta Prep-4000, (Waters) using ODS-5 colurnn(20mm x 250m, Nomura Chem. Co, Led, Seto, Japan) at 40°C with a flow rate of Sml/min monitoring at 525nm, Solvent systems used ‘were as follows; a linear gradient elution for 40min from 40) to 85% solvent BU.5% HaPO,, 2026HOAc, 25% CHACN in HxO) in solvent (159% HaPOs in HO). Three major anthocyanin pigments, Py(2img), Py(65mg), Ps(6lmg) were obtzined, ‘Thin-layer chromatography(TLC) analyses ‘TLC of three purified anthocyenins from sweet potato roots was castied out an cellulose plate(20% 20cm, 0.2mm, ‘Merck, Darmstadt, Germany) by using following solvent systems: BAW(n- BuOH-HOAc ~IEO, 4: 1 : §), BuHIC} (n-BuOH-2M HCI, 1 : 1), AHW(HOAc-HCI-H.O, 15 23282), and 196 HCIHCIH:O, 3 : 97) Instrumental analyses (a) UV-visible spectrometry: UV-visible absorption spectra of purified anthocyanins were recortied on a spec trophotometer(Spectronic Genesys, Milton Roy, US.A) in 0.1% HCl in MeOH. (b) "H-NMR and FAB-MS spectrometry: 'H-NMR (5Q0MFf2) was measured on a Varian Unity Plus spec~ trometer(California, US.A.) in CR:COsD-DMSO-ds(1 + 9) containing tetramethylsilaneCTMS) as an internal stan dard, and the chemical shifts were given as 6 values. The {ast atom bombardment mass spectra(FAB-MS) was recorded on « JEOL JMS-AX-506 WAUapan Electron and Optics Laboratory Co, Ltd,, Tokyo, Japan) with gly cerol as the mounting matrix. RESULTS AND DISCUSSION Isolation and purification of anthocyanins ‘The typical HPLC chromatogram of erude anthocyaninIsolation and Ientificon of Anthoevanins fiom Purple Sweet Potatoes % Pe Rg Fig. 2, HPLC chromatogram of crude anthocyanins ex— tracted from purple sweet potato roots. HPLC(Waters Delta Prea 4000) conditions: column, ODS-5(20%250mm): flow tate, 1Omi/mmni detection at Sténm: column Temp., 40°C. Solvent systems used ‘were as follows’ a Linear grachent: elution for d0min from 49 to 9096 solvent BU.5% EsPO,, 20% HOAc, 25% CHSC in H:0) in solvent A(.5% HsPOs in HA). pigments from sweet potato roots extracted with 0.5% ‘TFA in 95% ethanol is shown in Fig, 2. Three major anthocyanin pigments. P;~Ps, which represented 10.7 ~12.2%, 33.8~35.5%, and 24.5 ~26.8% of the total peak area, respectively, and six minor rigments were observed. Among these anthocyanin peaks, three major anthocy- anins were isolated, purified and identified, as descri~ bed in Materials and Methods. Chromatographic and spectral data of three anthocy~ anins are showa in Table 1. The strong intensities of UV absorption at 330nm suggest that three anthocyanin pig- ments are acylated by cinnamate derivatives such as caffexe and ferulic acids, The ratio oF Bas Brsnox of three anthocyanins was higher than that of simple anthocyanin glycosides, indicating that the presence of one or two eylated anthocyanins(19). In addition, the UV-visible absorption mnaxima of three anthocyanins were shifted tovvards lower wavelengths compared to that of cyani~ clin anthocyanidin, and they did not display bathochromic shifts at $29nm on the addition of AICk, indicating that the presence of peonidin nucieus in three anthocyanins, ‘Meanwhile, from ‘H-NMR spectra, benzene A ring of three anthocyanins has substituted group at H-6 and H-7 position, which was deduced fram the 3 part of pro~ ton nucleus at 6S5~697(H-6), 7.05~7 08(ET-8) and 89. ~894(H-4)ppm, Benzene B ring was identified as phenol with one methoxyl group at H-3" position and one hy~ croxyl group at H-1’ position, which was deduced from the 3 part of proton nucleus at 704~7.09(H-S"), 795— 790(H-2') and 830~836(H-6')ppm, and the 3H part at 3.90~3.93ppm representing ~OCHs group. Other spec trai data and Ry values of three major pigments were given in Table 1. Meanwhile, the precise composition of three major anthocyanins was identified by FABMS and ‘H-NMR spectroscopy, as well as by analogy with previous studies. FABMS of P; gave its molecular ion pealkIM'] at 11m/2, Its "H-NMR spectrum, as measured in triflu- ‘orcacetic-di-DMSO- dell : 9), showed the presence of a peonidin nucleus, two caffeic acids, and three hexoses. ‘Three hexoses must be B=glicopyranoside forms based on the vicinal coupking constants with J values\ Jia Jas Jsaand Jis) of 70-1000 Hz, By application of the nega tive NOB difference spectroseopy(24.25), anomeric pro~ tons at 8566, 5.13 and 476 were assigned as glucose ‘A, B and C, respectively. Two caffeic acids were de termined to be bonded to C-6 hydroxyl groups of glu~ cose A and C by the observation of low-field shifts of both methylene protons(H-a & H-6a) of glucose ‘A and C at 8429, 435 and 402, 403, Moreover, the ‘Table 1. Ri values, spectral properties, and FAB-MS spectra of tree major anthocyanins isolated from purple sweet potato roots ‘Antho Re values"( 100) Spertral data” FAB-MS” ssanin BAW BuHiCl ic AHW Arn) — xy Ent) AIC EMI Pi ® 15 5 re er 0 1m PB 20 8 6 m4 523.295 80S 0 1125 Py 8 % B B55 IHL 0 1049 "Developed in cellulose plate by descending method at 25°C using following solvent systems; BAW a- BuOH-HOAe-H.0. 41:9), BuHClin-BuOH: HCI, 1:1) 1%SHICKHCI-H20, 397); AHW(HOAc-HCI-HO, 15382) Determined in 01% HCl-MeOH, and glycerol as a matrix, repectivelyLan-Sook Lee, Eun-Ju Chang, Jong-Whan Rum, Bycung-Seob Ko and Sang-Won Che ‘Table 2. 'H-NMR spectra data of three major anthocyanins isolated from purple sweet potato roots in CRxCOsD~ DMSO-ds, 1:9 at 25°C) Proton SEEESESEEESETEGISEESESEGESSEESED/ C7 EOEESOEOSOEAEEEEISENanEE OEE PB. Pa, Peonidin 1 as 5 Bots ass 6 695 br s 607 bs 655 4 15) 8 705 & 0 708 6) 708 br 5 2 i 4.08) 797 418) 736 4 22) 3s 708 d (9) 704 4 (3) 79 4 3) & 835 dd (1.3, 88) 34 dd (18, 88) 830 dd 7, 93) 3'-OMe 301 390, 393 Catfec acid (D 2 895 brs aga as) 636 br 5 5 878 brs a7 dO 677d Gr 8 585 dd (8, 90) 684 dd 8 90) a5 ad (18 8) He 62 4 053) 612 4.058) 61 é 82) He 7.32 d (15.3) Tal d (15.6) 731 4 (162) Calfeic acid (ID Ferulic acid (If) prHydroxybenzoic acid (ID 2 687 brs ‘6.85 brs 5 575.4 (901 65,4 0) Fa 5'- 661d @t) 8 578 dd (20, 900 677 ad (20, 90) 2a e744 03) i 590 d (155) 605 6.053) i 716 4 (155) 72 6 053) 3-OMe 20 Giucose ‘Sophorose-glucose A 1 585 4 (70) 5 4 (0) sald 7 2 385 t i) 35 t 396 t (5) 3 35 t 3) 38 « 69) 2791 G9) 4 300 ¢ 68) 38.1 (89) 354 t Go) 5 390? m 389° m 383" m Ga 429 dd (88, 11.0) (4.28 dd (89, 110) 429 dd (88, 1.0) & 435 6 10) 458 4 GL) 439 8 G10) Sopitrose-ghicose C 1 4786 G7) ana uo 476 6 78) 2 318 319 ¢ 5) a18¢ GS) 3 325" m 32 m 327 m 4 30103 321 (5) aa2t (5) 3 328" m 341 m 340" m fa ‘oe da ¢60, 114) 44 ad 0, 114 408 dd (80, 115) ® 400 ad (114) 49 aa a. a3 aa) ‘5-Glucose B 1 513 d (77) 512 d (75) ‘BS d (78) 2 357 t (84) ‘B58 t (85) 357 t (85) 3 342 + GL) 2a 010) BA ¢ (il) 4 3.30 t (10.0) 3.30 1 (10.0) 33Lt (10.2) 5 350° m 3.50" m 850° m 6a ‘359° dd (7.8, 11.7) 0" dd (7.8, LL7) & ge2auin ae 6aLa a2 d <7) Assigned by ‘HH COSY Coupling constants(/ in Hz) in parentheses glycosidic linkage of sophorose(glucose A and C) was deduced from a low-field shift(3.96ppm) of glucose A H-2, suggesting that C-1 of glucose C was bonded to C-2 of glucose A. Anomeric protons of glucose A and were finally related to the two acylated methylene protons(6429 and 4.35) and (6402 and 4.09), respectively, by 'H-'H COSY spectrum(4). Other protons of Ps were similar to those of acylated anthocyanin isolated fromIsolation and Hlentiication of Anthocyanins from Purple Sweet Potatoes Ea Pharbitis nil(26). From the above data, P, was deter- rnined to be 3-O-(6-O-trans-catteyl)-2-O-(6-O- trans caéleylglucopyranosyl}-6-D-glucopyranosyl-5~O-(8 ~D-ghacopyranosyl)-peonidin, This anthocyenin pig ‘ment was frst isolated and identified from purple sweet potato roots, although the pigment has already heen reported to be present in the stems of sweet potato(16) ‘The FABMS of P2 gave its molecular ion peak (M'] at 1125m/2, Its H-NMR spectrum exhibited six protons of a peonidin nucleus and three protons of a methoxyl roup assignable to ferulic acid. Three anomeric protons of glucose and the glycosidic linkage of sophorose(ghi- cose A and C) were in full agreement with those of P, Additionally, the bonding site of caffeic and ferulic acids to glucose A and C, as well as four olefinic protons of two acids were almost consistent with that of Pi, al~ though the acid bonded to each glucose was not es~ tablished firmly, Thus, Po was identified as 3-O-(6- (O-trans-caffey!)-2-O-(6-O-trans-feruloylglucopyta~ nosyl)-B-D-glvcopyranosy!-5-O-(8-D-glucopyranosyl) ~peonidin. ‘The FABMS of Ps gave its molecular ion peak (M'] at 1066/2. Its "H-NMR spectrum exhibited the presence of a peonidin, one mole of caffeic and p-hydroxybenzoic acids, Three anomeric protons of glucose and the gly- cosidic linkage of sophorose(giucose A and C) were si ilar to those of P, and Pe, However, two olefinic pro- tons at 8y 664d, J=84H2) and by 7494d, J-93#2} assig ome on on aucecer Spon ot aeeconncen Fon ox ra necoctot F-08004 on Fig. 3. Chemical structures of three major anthocyanins isolated from purple sweet. potato roots. rable to p-hydroxybenzoie acid were specifically ob- served. Although we cid not confirm correctly where the acid was bonded to the C-6 position of glucose A and C, the p-hydroxybenzoic acid could likely be bonded to the C-6 position of glucose C due to low-field shift of C-Eia and C-6b position of glucose C in comparison with those of glucose C of P) and Pz, Thus, Ps was identified 25 3-O-(6-O-trans-calfeyl)-2-O-(6-O-p- hydroxylbenzoylalucopyranosy!)-B-D-glucopyrano— syl-5-O-(G-D~glucopyranosy!)-peonidin, The assig~ nments of 'H-NMR and chemical structures of three anthocyanins are given in Table 2 and Fig. 3 ‘Thus, the complete structural assignments of three ‘major diacylated anthocyanins from purple sweet potato roots are reported here forthe first time, although those anthocyanins were already found in several cultivars and tissues of sweet potato( 16-20). It is very interesting to note that the newly bred purple sweet potato con tained a fairly large amount of acylated peonidin glu~ cosidestin fresh periderms around 70~80% against total anthocyanin contents). Therefore, future successful bio technological processes using cell cultures for the pro~ uetion of these stable anthocyanins as potential source of natural food colorants is promising. Further isolation and identification of minor anthocyanin pigments from purple sweet potatoes are needed ACKNOWLEDGEMENTS ‘This study was supported by a non-directed research funding from the Food Industrial Technology Research Cemter(97-15-08-02-A-3). REFERENCES 1. Markakis, P, - Food colorants. In “Anthocyanins as food colors” Markakis, Ped), Academic Press, New Yorks, 1.245(1982) 2 Strack, D. and Weay, V. The anthocyanins. [n “The fia- vvonoids” Harborne, J. BAed.), Chapman & Hall, Landa, pti) 3, Francis, F. J.! Food colorants : Anthocyanins(review). rit Rew, Food Sci, Nutr. 28, 273(1989) 4, Kondo, T, Teomura, H, Yoshida, K. and Goto T. : Structure fof maloaylshisonin, a genuine pigment ia pamie leaves of Perilla ocimcides L. var. crispa Benth, Agric. Biol Quem, 153, 797(1999) 5, Tesahara, N. andl Suz, H.* A diacylated anthocyanin from Tibouchina uruttega flowers. J. Narural Products, 96, 335(1993)10, LL. 13, 4 iB 6. lLay-So0k Lee, Eut-Ju Chang, Jong-Whan Rhim, Byoung-Seob Ko and Sang-Won Chot Toki, K, Takeuchi, M, Seito, N. and Honda, T. : Two ‘alonylated anthocyenin glvcosides in Ranunculus asiatcus, Photocher., 42, 1055(1996) Callebaur, A. Hendticks, G., Voets. A, M. and Mate, J.C, Anthocyanins in cll cultures of Ajuga reptans. Phytochan, 29, 2153(1900) Glabgen, W. E,, Wray, Vi, Strack, D, Metzger, J. W. and Seitz, HU. = Anthooy-nins fram cell suspensions cultures oF Daucus carota. Prytockem., 31, 15851352) Megard, D.” Apalication of anthocyanms in foods and beverages, Paper presented at 2nd Intemational Congress & Symposium on Natural Colorants, Mexico(1997) Gabor, E. : Possible biological role of some anthocyanins in food. Bull Liaison-groupe Polyphenols, 14, 130(1988) Drenske, D, Bantutove, and Oveharoy, R,: Anti-convul- sant effect of anthocyanins and antioxidants. Farmatsiva (Sofia), 39, 330989) Meunier, M.'T., Durows, B. and Bastide, P, : Antioxidant activity of procvanidol oligomers and anthocyanins with regard to superoxide anion and lipid peroxication. Plant Med. Phytother,, 23, 267.1989) Tsuda, T., Watanabe, M,, Ohshima, K, Norinoby, S. Choi, S. W, Kawakishi, S. ané Osawa, T, : Antioxidative activity of the anthceyaran pigments cyanidim 3-O-6-D- slucoside and eyanidin, J. Agri Foce! Chem, 42, 2407 go) ‘Touda, T,, Ohshima, K., Kawakishi, S, and Osawa, T. Inhibition of lipid peroxidarion and radical scavenging ef~ fect of anthocyanin pigments isolated rom the seeds of | Phaseolus vulgaris L,* International Confereace on Food Factors : Chemistry and Cancer Prevention, Hamamatsu, Japan(1996) Frankel, E, N.. Kanner, J, German, J.B, Parks, B. and ‘Kinsella... Inhibition of exidaton of human low-density lipoprotein by phenolic substances in red wine, Lancet, 341, 454(1993) Innbert, MP, Seaforth, C. E. and Williams, D. B+ An ‘hocyamn piginents of the sweet potatolporsea batatas) J Am. Hort, Sci, 88, 48111965) 17 Tsui, A. Kuwano, K. and Mitarmura, A: Anthoeyanin pigment isolated from purple root of sweet potato, Kaise- ssalca Zasshi, 34, 1531983) 18, Miyazaki, T., Tsuzcki, W. and Suzuki, : Composition and structure of anthocyanins un the veriderm and flesh of sweet potatoes. L. Jon. Sac Hort Sci, 60, 21701991) 19, Zurin, S, Bassa, L A., Gabriel, S. L. and Francis, &. J. Anthecyanin pigments of sweet potatoes ~Zpemoea batatas J: Food Sci, 51, TE5K982} 20, Odake, I, Teratara, N., Saito, N, Toki, K. ated Hooda, T= Chemical structures of two anthocyanins from purple sswieet potato, Jpomoea batatas, Phytochem, 31, 2127192) 21, Bassa, LA. and Francis, F. J. Stability of anthocyanin from sweet potatoes in a model beverage. J. Food Sci, ‘82, 1753 (1967) 2, Lee, LS, Rhim, JW. Kir, S.J and Chung, B.C. ? Study fon the stability of anthocyanin pigment extracted {rom purple sweet potato, Korean J Food Sci. Tectnol, 28, 352(1998), 2. Chang, E. J, Choi, S. W., Lee, L$. and Rhim, J, W, © An~ tioxicative activity oF daeylated anthocyanins érom sweet potatoes, Paper presented at 4lth Korean Soe, Food Sei & Nutr, Teejeon, Korea( 1997) 2%. La, TS, Saito, N, Yokoi, M, Stigihara, A. and Honda, T.? An acylated peonicin glycoside in the violet~blue lowers of Pharbitis nil, Phytoohem, 30, 2387(1981) 25, Kondo, T,, Kawai, T, Tamura, Hand Goto, T, . Structure determunation of heavenly blue anthocyanin, a complex ‘monomeric anthocyanin from the morning glory Ipomoea tricolor, by wean of the negative NOE method, Tetrahedron Letters, 28, 227300987) %, Lu, T.S. Saito, N,, Yokoi, M., Shigihara, A. and Honda, ‘T. + Acylated peonidin glycosides in the violet-biue cul lavars of Pharbitis nit Phytoohem, 31, 6251980) (Received April 17, 1997)