Studies on Simultaneous Determination of Chlorophyll a and b, Pheophorbide a, and β-carotene in Chlorella and Spirulina Products

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1 J. Fd Hyg. Sfety 20(3), (2005) j v ù t w, b, r s β-e p w Á½ Á½ Áw Á w Á z Á t t Studies on Simultneous Determintion of nd b, Pheophorbide, nd β-crotene in Chlorell nd Spirulin Products Young J Lee, So Hee Kim, Jin-Sook Kim, Jeong A Hn, He Jeom Seo, Hyo Jeong Lim, nd Soo Young Choi 4FPVM3FHJPOBM'PPEBOE%SVH"ENJOJTUSBUJPO 3FDFJWFE.BSDI"DDFQUFE4FQUFNCFS ABSTRACT A simple nd sensitive nlysis method bsed on reverse phse (RP) HPLC with UV detector ws developed for simultneous determintion of chlorophyll nd b, pheophorbide nd β-crotene in Chlorell nd Spirulin products. For dded concentrtion (50 µg/ml) of chlorophyll nd b, pheophorbide nd β-crotene, recoveries of those were 70.3, 71.6, 60.1 nd 90.5%, respectively, with reltive stndrd devitions of 2.8, 6.0, 10.6 nd 10.4%. Limit of detection nd quntifiction hd rnges of 0.1~1.0 µg/ml nd 0.2~2.0 µg/ml, respectively. Clibrtion curve ws liner with correltion coefficient of for chlorophyll nd b, pheophorbide nd β- crotene. Results of simultneous determintion in Chlorell nd Spirulin products were showed rnges of 121.9~543.0 µg/ml for chlorophyll, 0.6~160.0 µg/ml for chlorophyll b, 19.2~60.3 µg/ml for pheophorbide nd 383.6~ µg/ml for β-crotene, respectively. b contents in Chlorell products were detected bove 30 times level to those in Spirulin products. β-crotene contents in Spirulin products were detected 2.7 times level to those in Chlorell products. Key words: chlorophyll nd b, pheophorbide, β-crotene, Chlorell, Spirulin» t w w, 90% w» w ƒ w, e š ùkû 1). š» t w x, 68%ƒ» t z ƒ ƒ š y 2).» t 1980 t» w» w w» Food for Specific Helth Use(FOSHU) Actƒ, 1994 Dietry Supplement Helth nd Eduction Act ù 1989 (DSHEA)ƒ ƒƒ 3). z tœ ü t ƒw š, 2002» t 4), 2 ³ w» t w w» Author to whom correspondence should be ddressed. ƒ w Áe Á Á Á Áy xk Áƒœw t wš. w, xw» tœ» t x 32 t w» Á³ ƒƒ ù y w xw» Á³ x w w š rák Á w w š. p,» t t j v ù t š w w sƒ. t x 5,6) w ƒ ü ey w w y w yw w y e p t w w yw w ƒwš 7). 10) ƒœ g8), j 9) š l w yw e p w ƒƒ HPLC 141

2 142 :PVOH+B-FFFUBM š š ù, xw j v ù t Á³, b r s w y x w x Ÿ TLC ³ ƒƒ ƒ wš HPLC»» w y v w.,» t j v ù t, b, r s β-e p w HPLC y wš w. x w l ¾ ü y l v m j v ù t ƒƒ 4 w w. w j t 97~100% w w t, v ù t 3 90~100% w t 1 51% w t w. w Wko p w š, HPLC w w Fisher HPLC w. t t w (97%), b(95%), r s (93%) Wko t, β-crotene(95%) Sigm t w. t, b, r s β-e p t t y e wš m ƒw ƒƒ 500 µg/ml w z, x w w. Á w z m š, b, r s β-e p t 0.1, 0.2, 1, 5, 10, 50, 100, 200, 400 µg/ml, b 0.08, 0.15, 0.75, 1.5, 7.5, 15, 75, 150 µg/ml, r s 0.1, 0.5, 1.0, 5.0, 10, 50, 100 µg/ml, š β-e p 1, 2, 5, 10, 50, 100, 200 µg/ml ƒƒ w š w. j v ù t w ƒ yw š w w. w w ƒ yw 5.0~7.5 µg/ml t 11) w j m» Morrison w. z w 50 µg/ml yw t 1 ml e p w w t 100 mg ƒw, 10 ml m ƒw t ƒ w d w. e p m j v ù t w w š, 100 mg ƒƒ w 10 ml m ƒw 20 q» wš (4 C) o w ew. ew w (3,000 rpm, 5 min, 4 C)w d w z e o 10 ml m š yww w w d w. d ww ƒ w m w. 1 ml m ƒw 0.45 µm Nylon filter(technochrom, Indi) w HPLC x w. HPLC m HPLC HP1100(Agilent, USA) w š, HPLC d q, b, r s β-e p d w 440 nm d w Tble 1 12). Tble 1. HPLC nlyticl condition of chlorophyll, chlorophyll b, pheophorbide, nd β-crotene Column Flow rte Detector Injection vol. Mobile phse Grdient progrm Cpcellpk C Å, 5 µm 4.6 mm I.D. 250 mm 0.8 ml/min UV 440 nm 20 µl A-Methnol : 0.5 M Ammonium cette (80:20) B-Methnol : Acetone (70:30) Time(min) A(%) B(%) Initil š š m w t RP-HPLC w Ÿ» w yw t w y w (Fig. 1). y

3 4UVEJFTPO4JNVMUBOFPVT%FUFSNJOBUJPOPG$IMPSPQIZMMBBOEC1IFPQIPSCJEFBBOEβ$BSPUFOFÃ 143 Fig. 1. Clibrtion curves of chlorophyll (A), chlorophyll b (B), pheophorbide (C) nd β-crotene (D)., b, r s, š β-e p š Fig. 2 ùkü. š ƒ yw ü ƒ, yw j ùkü w w. Á w z m w ƒ ƒ yw ƒ ùkû. b β-e p š r s. Morrison w w yw β-e p w (limits of detection; LOD) 0.1~1.0 µg/ml, w (limits of quntittion; LOQ) 0.2~2.0 µg/ml ùkû. ƒ t (clibrtion rnge) Tble 2. e p w wš yw t ƒw w z Tble 2. Clibrtion rnge, detection limit, quntifiction limit of chlorophyll, chlorophyll b, pheophorbide, nd β- crotene Substnce Clibrtion rnge (µg/ml) Detection limit (µg/ml) Quntifiction limit (µg/ml) b Pheophorbide β-crotene d w s³ 73% z ùkü, β- e p 90.5% ƒ š, r s ƒ ƒ û 60.1% y. t r (reltive stndrd devition; RSD) 2.8~10.6%, w yw ƒ ƒ û t r (Tble 3). j v ù t m» t ü m š j

4 144 :PVOH+B-FFFUBM Tble 3. Recovery of chlorophyll, chlorophyll b, pheophorbide nd β-crotene in known concentrtion MenÛSD RSD(%) 70.3Û b 71.6Û Pheophorbide 60.1Û β-crotene 90.5Û v ù t w, b, r s β- e p HPLC w ƒ y w ƒ û w r s» w 100 mg w. wš w e p yw» sww yw, Ÿw w w. w ƒ y yw» w w š, m, k, k. m w s q w» z» šƒ 13). ù» t j t s n w w œ sw m w w 14), x j v ù t m kw w. 8 Fig. 2. HPLC profile of cetone extrct from stndrd mixture (chlorophyll, chlorophyll b, pheophorbide nd β-crotene) (A), Chlorell (B) nd Spirulin (C) products. Detection ws crried out t 440 nm. Chromtogrphic conditions re described in Tble 1.

5 4UVEJFTPO4JNVMUBOFPVT%FUFSNJOBUJPOPG$IMPSPQIZMMBBOEC1IFPQIPSCJEFBBOEβ$BSPUFOFà 145 xw m z HPLC/PDA w yw e p w w m w w xw y w, w y yw m w š 10). j v ù t RP-HPLC j v ù t m w HPLC j m t j m w y ù, b β-e p w ƒ j (Fig. 2). HPLC j m l ƒ yw y w ù, e š w t l ƒ vj HPLC/ PDA w t ù j m w, ƒƒ w vj y w (not shown). e p yw HPLC w 1980 z l w ù15), yw 8,10,16) w x š š. HPLC j t 232.1~543.0, b 75.8~160.0, r s 37.2~60.3, š β-e p 383.6~709.4 mg/100g ùkû. š 90~100% v ù t 121.9~503.6, b 0.6~15.5, r s 19.2~30.3 š β-e p 865.2~ mg/100 g ùkû. 4 j t(a~d) w 4w w t 1.6~2.3 ƒ ùkù ù, v ù t(e~h) 2~25 v ù t w j t t ƒ ùkû. w j t, b ƒ 3:1 ùkù s³ w w. b w j t s³ mg/100 g, v ù t 10.5 mg/100 g 30 w wš ùk û, β-e p w v ù t s³ mg/100 g j t s³ mg/100 g s³ w 2.7. t r s w j y. š v ù Tble 4. Comprison of the quntity of chlorophyll, chlorophyll b, pheophorbide nd β-crotene contined in cetone extrcts of Chlorell nd Spirulin products. Products b Pheophorbide β-crotene A B Chlorell C D E F Spirulin G H % w w H 100% w w t ƒ s³ w w w ƒ ùkû (Tble 4). j v ù t» z wš w, b r s x» tœ» j v ù t» Á³ β-e p wz v ù» Á³ ƒƒ š w.» Á³ e v w x Ÿ d w w w. ù ww v w w. b y x 85% m w z dq sw k z y w w. r s x diethyl ether, w š x w. w ƒ wš z s³ 70.3% ùkû ù» Á³ x Ÿ ƒ x» Á³ w». b ³ y x ù x e š y ƒ w. r s z 60.1% ùkû ù» 2~4.» Á³ y x Ÿ w ƒ w, x j w».

6 146 :PVOH+B-FFFUBM f w» t j v ù t w, b, r s β-e p HPLC y w, ƒ 50 µg/ml, b, r s β-e p w z x, ƒƒ 2.8, 6.0, % t r 70.3, 71.6, % z ƒƒ ùkþ. w 0.1~1.0 µg/ml, w 0.2~2.0 µg/ml ü m j v ù t w, b, r s β-e p w w 121.9~543.0, b 0.6~160.0, r s β-e p 383.6~ mg/100 g ùkû. b w j t s³ mg/100 g v ù t s³ 10.5 mg/100 g 30 w wš y. ù β-e p w v ù t s³ mg/100 g j t s³ mg/100 g s³ w 2.7 ùkû. ü» tœ j v ù t, b, r s w ³ w ƒ w, x j w». š x 1. Lee, E. J., Ro, S. O. nd Lee, C. H. : A survey of consumer ttitude towrd helth food in Kore (1) Consumer perceptions of helth nd food hbits. Koren J. Diet Culture, 11, (1996). 2. Lee, E. J., Ro, S. O. nd Lee, C. H. : A survey of consumer ttitude towrd helth food in Kore (2) Consumer perceptions of helth food. Koren J. Diet. Culture, 11, (1996). 3. Lee, C. H. : Functionl food of interest to ASEAN : from trditionl experience to modern production nd trding. Food Sci. Biotechnol., 13, (2004). 4.» t w Piorreck, M., Bsch, K. H. nd Pohl, P. : Biomss production, totl protein, chlorophylls, lipids nd cids of freshwter green nd blue-green lge under different nitrogen regimers. Phytochem., 23, (1984). 6. Cost, J. A. V., Coll, L. M. nd Filho, P. F. D. : Improving Spirulin pltensis biomss yield using fed-btch process. Bioresource Technol (2003). 7.,, :» j. w œwz, 19, pp (2004). 8. Edelenbos, M., Christensen, L. P. nd Grevsen, K. : HPLC determintion of vhlorophyll nd crotenoid pigments in processed green pe cultivrs (Pisum stivum L.). J. Agric. Food Chem., 49, (2001). 9. McGhie, T. K. nd Ainge, G. D. : Color in fruit of the genus Actinidi : crotenoid nd chlorophyll compositions. J. Agric. Food Chem., 50, (2002). 10. Suzuki, Y. nd Shioi, Y. : Identifiction of chlorophylls nd crotenoids in mjor tes by high-performnce liquid chromtogrphy with photodiode rry detection. J. Agric. Food Chem., 51, (2003). 11. Morrison, G. H. : Guidelines for dt cquisition nd dt qulity evlution in environmentl chemistry. Anl. Chem., 52, (1980). 12. Furuy, K., Hyshi, M. nd Ybushit, Y. : HPLC determintion of phytoplnkton pigments using N, N- dimethylformmide. J. Ocenogrphy, 54, (1998). 13. Ergun, E., Demirt, B., Gumus, G. nd Apk, R. : Simultneous determintion of chlorophyll nd chlorophyll b by derivtive spectrophotometry. Anl. Bionl. Chem., 379, (2004). 14. Atsushi, M. : Technology on qulity control in Chlorell production. Food J., 9, (1999). 15. Grrido, J. L. nd Zpt, M. : Ion-pir reversed-phse highperformnce liquid chromtogrphy of lgl chlorophylls. J. Chromtogrphy A, 738, (1996). 16. Gndul-Rojs, B., Cepero, M. Roc-L. nd Minguez- Mosquer, M. I. : nd crotenoid ptterns in olive fruits, Ole europe Cv. Arbequin. J. Agric. Food Chem., 47, (1999).