265-271. 1985. Chlorophyll Degradation by Peroxidase In

J. Japan. Soc. Hort. Sci. 54(2) : 265-271. 1985.

Chlorophyll Degradation by Peroxidase in Parsley Leaves'

Naok1 YAMAUCHI2and Takahlsa MINAMIDE College of Agriculture, University of Osaka Prefecture, Sakai, Osaka 591

Summary The mechanism of chlorophyll degradation by peroxidase in parsley leaves was studied. Chlorophylls present in the ethyl alcohol (EtOH) extract of parsley leaves were degraded by a peroxidase-hydrogen peroxide system. Purified chlorophylls, however, were not affected by the system, suggesting the existence of a substance which acts together with peroxidase to degrade chlorophyll in parsley leaves. The substance was isolated from the EtOH extract by the methods of solvent fractionation and column chromatography. After hydrolysis, it was identified as an apigenin, which is a major flavonoid contained in parsley, by measuring Rf value on thin layer chromatogram and spectral characteristics. From the results obtained, it is concluded that in parsley leaves chlorophylls are not degraded by peroxidasedirectly, but that peroxidase oxidizes the apigenin and then the oxidizedapigenin degrades chlorophylls.

Introduction also been postulated(8,10, 24). The bleaching of chlorophyll seems to involve lipoxy- Leaf vegetables deteriorate rapidly after genase and peroxidase (9, 11, 14, 17, 28, 30). harvest as compared with other fruits and Orthoef er and Dugan Jr. observed that chlo- vegetables. One of the main factors causing rophyll a was bleached in a system which deterioration of freshness is yellowing of consisted of linoleic acid and lipoxygenase leaves. Eskin summarized that the yellow- (17). Holden also noted that chlorophyll ing phenomenon, the degradation of chloro- was bleached when leaves of various species phylls, appears to involve one or more of of plants, which were rich in lipoxygenase, the following reactions :1) pheophytin for- were incubated in an aqueous acetone solu- mation from chlorophyll by organic acids, 2) tion at pH 6.0 in the dark(9). Matile(14) chlorophyllide formation by chlorophyllase, and Huff (11) reported that chlorophylls were and 3) bleaching reaction of chlorophyll by degraded by a peroxidase-hydrogen peroxide oxidative reactions (4). The formation of system in the presence of phenolic com- pheophytin is particularly found in processed pounds, such as 2, 4-dichlorophenol and re- foodstuffs, like brined cucumber(12) and fro- sorcinol. In a previous paper we also report- zen spinach(3). Although the chlorophyl- ed that chlorophyll in parsley was degraded, lase has been thought to catalyze the degra- not by chlorophyllase but by peroxidase, and dation of chlorophylls, a function for the that the degradation was retarded by L-as- enzyme in the synthesis of chlorophylls has corbic acid(30). 1 Received for publication February 17 This report deals with the mechanism of , 1985. chlorophyll degradation by peroxidase in pars- Mechanism of chlorophyll degradation in harvested leaf vegetables. I. ley leaves. 2 Present address : Himeji College of Hyogo Materials and Methods Prefecture, Shinzaike-honcho, Himeji, Hyogo 670 Materials

265 266 YAMAUCHI, N. AND T. MINAMID,E

Parsley (Petroselium sat ivum Hoffm., cv a) or 644 nm(chlorophyll b), and degraded Paramount) used in this study was purchas- chlorophylls were calculated subtracting this ed at a local market. value from the blank (distilled water instead Purification of chlorophylls a and b of hydrogen peroxide) one. Chlorophylls a and b were purified by the Isolation of unknown substances involved in method of Yoshiura and Iriyama(31). The chlorophyll degradation pigments were extracted with 80 ml acetone The unknown substances involved in chlo- from 20 g of parsley leaves. Into the ex- rophyll degradation were extracted with boil- tract, 1, 4-dioxane and distilled water were ing EtOH from parsley leaves(10 g). Frac- added and the mixture was allowed to stand tionation with different solvents was carried untill a precipitate was formed. The precip- out as shown in Fig. l and each fraction was itate was collected by centrifugation at isolated by column chromatography(Sephadex 10, 000 g for 15 min., and dissolved in a LH-20, 1.8x100 cm, solvent : 80% EtOH, small quantity of acetone. Chlorophylls a flow rate : 0.4 ml/min., fraction : 5 ml). and b were then separated by thin layer Isolation of flavonoid aglycones chromatography (silica gel, solvent ; n-pen- The mixture of fractions I, II, and III tane : acetone : t-butyl alcohol=90 : 5 : 5). contained in butyl alcohol(ButOH) and aque- Chlorophyll a and b fractions also contained ous fractions shown in Fig. 2 were hydro- chlorophylls a' and b', respectively. lyzed with 2.0 M HCl for 40 min. at 100°C and Determination of chlorophyll degradation the aglycones were then extracted with ethyl The reaction mixture contained 0.2 ml of acetate. The extract was evaporated to dry- 80% ethyl alcohol (EtOH) extracts of parsley ness under reduced pressure and dissolved in leaves(or 30 pg purified chlorophyll plus fla- a small volume of EtOH. The aglycones in vonoid), 0.04% Triton X-100, 0.012% hydro- the EtOH solution were isolated by thin gen peroxide, 20 pg peroxidase(Sigma chemical, horseradish), and 80 mM phosphate buff- Table 1. Chlorophyll degradation by peroxidase- er (pH 6.0) in a total volume of 2.5 ml. hydrogen peroxide system. After 10 min. at 25°C, the reaction was stop- ped by the addition of EtOH(2.5 ml). The remaining (non-degraded) chlorophylls were extracted with hexane and were assayed by reading the absorbance at 663 nm(chlorophyll

Table 2. Chlorophyll degradation by peroxidase- hydrogen peroxide system in the presence of the each fraction obtained by solvent fractionation from parsley extract.

Fig. 1. Procedure of the extraction of substances which relate to chlorophyll degradation from parsley leaves. * Each fraction was evaporated to dryness under reduced pressure and dissolved in a small volume of 80% EtOH. CHLOROPHYLL DEGRADATION BY PEROXIDASE IN PARSLEY LEAVES 267

Fig. 2. Isolation of the substances having chlorophyll a degradation activity from ButOH and aqueous fractions by using of column chromatography. Column : Sephadex LH-20 (1.8 x 100 cm), Solvent : 80% EtOH, Flow rate : 0.04 ml/ min., Fraction : 5 ml. Upper : Optical density at 250 nm (substances including flavonoids) and 450 nm (substances including carotenoids). Lower : Chlorophyll degradation activity The reaction mixture contained 0.3 ml of each fraction, 30,ug chlorophyll a, 0.04% Triton X-100, 0.012% hydrogen peroxide, 20,ug peroxidase, and 68 mM phosphate buffer (pH 6.0) in a total volume of 2.5 ml. Chlorophyll degradation was assayed spectrophotometrically by reading the fall of absorbance at 668 nm, the spectral maximum of chlorophyll a in this reaction solution, as chlorophyll a was degraded. layer chromatography (silica gel, solvent ; parsley extract were degraded, and the degree toluene : ethyl formate : formic acid=5 : 4 of degradation of chlorophyll a was apprecia- 1). bly higher than that of chlorophyll b. This fact seemed to indicate that the chlo- Results rophylls were not degraded directly by the Table 1 shows the results of chlorophyll peroxidase-hydrogen peroxide system, but degradation by the peroxidase-hydrogen per- that peroxidase oxidized some unknown sub- oxide system. Purified chlorophylls a and b stances in the parsley extract which then were not degraded by the system. On the degraded the chlorophylls. Consequently, other hand, chlorophylls contained in the the identification of the unknown substances 268 YAMAUCIII, N. AND T. MINAMIDE

T able 3. Spectral maxima of I, II, and III fractions.

Fig. 3. Isolation and identification of aglycone from flavonoids included in I, IT, and III fraction shown in Fig.2. The 3 fractions were mixed. Detection :1-2% FeC13(brown), 2-2% AIC13 (yellow in UV), 3-2% FeCl3 (brown), 4-2 AICl3 (yellow in UV). was carried out as follows. because the fractions exhibited yellowing with By using different solvents shown in Fig. AlC13 and H2SO4, browning with FeC13, and 1, the unknown substances were fractionated blueing with FeC13 plus K3Fe(CN)6. As is from the EtOH extracts of parsley leaves. apparent in Table 3, fraction I contained not The chlorophyll degradation activities of each only flavonoid but also carotenoid pigments, fraction were determined. As shown in showing spectral maxima in the region of Table 2, chlorophyll a was degraded by the 420-470 nm. The carotenoids were isolated peroxidase-hydrogen peroxide system in the by thin layer chromatography (silica gel, sol- presence of some substances in the ButOH vent ; benzene : ethyl acetate : McOH=75 : and aqueous fractions, and the degradation 20:5), and the chlorophyll degradation by by the aqueous fraction was more significant the peroxidase-hydrogen peroxide system in than that by ButOH fraction. Fig. 2 shows the presence of carotenoids was studied. It the isolation by column chromatography of was found that the carotenoids were not in- the substances involved in chlorophyll degra- volved in the reaction of chlorophyll degradation. The substances in the ButOH frac- dation (data not shown). tion showed activity in 2 fractions(I and II) As the substances involved in chlorophyll of the chromatogram, while those in the degradation seemed to be flavonoid glycosi- aqueous fraction, chiefly, in one fraction des having sugars at various position(26), (III). The substances in fraction III mark- fractions I, II, and III were hydrolyzed by edly degraded chlorophyll as compared with HC1 solution and the resultant aglycones others. The spectral maxima of the frac- were identified. Fig. 3 shows the thin layer tions(I, II, and III) in methyl alcohol (MeOH) chromatogram of the aglycones ; the Rf value solution are shown in Table 3. The maxima of the main aglycone was about 0. 63, agree- of UV absorption spectrum occurred at 268 ing well with that of apigenin. The absorp- and 330-333 nm in all fractions, correspond- tion spectrum of the aglycone was also deter- ing well to that of apigenin glycosides(26). mined ; as shown in Table 5, the spectral The color reactions of the 3 fractions with maxima of the aglycone in McOH solution diagnostic reagents are shown in Table 4. as well as the shift of the maxima by the These results also indicated that flavonoid addition of sodium methylate(NaOMe) and pigments were contained in the 3 fractions AiC13 coincided completely with those of api- CHLOROPHYLL DEGRADATION BY PEROXIDASE IN PARSLEY LEAVES 269

Table 5. Spectral maxima in UV of the isolated phytols to form chlorophylls(24). Purvis ob- aglycone, and their shift by addition of served that chlorophyllase in green calamon- sodium methylate (NaOMe) and AIC13. din fruits showed higher activity than that in calamondin fruits which have lost approx- imately half of their chlorophyll(21). In addition, Aljuburi et al. suggested that chlorophyllase was closely related to chlorophyll biosynthesis since chlorophyllase activity increased following the increase of chlorophyll content during the regreening of valencia oranges(1). In detached leaves of tobacco and radish, chlorophyllase activity decreased Table 6. Chlorophyll degradation by peroxidase- hydrogen peroxide system in the presence with yellowing(20, 23). We reported in the of apigenin. previous paper that chlorophyllase activity showed a sharp decline with yellowing in stored parsley leaves (30), suggesting that chlorophyllase may not be involved in chlorophyll degradation associated with leaf yellowing during storage. It has been further reported that peroxidase activity increased with the progress of senescence in many species of plants(6,19). We also found that peroxidase activity increased genin. with yellowing of stored parsley leaves and Degradation of chlorophyll by the peroxi- that the enzyme was supposed to relate to dase-hydrogen peroxide system in the pre- chlorophyll degradation(30). In this study, sence of apigenin was also determined. As it is concluded that peroxidase degrades chlo- expected, chlorophyll was degraded in the rophylls : peroxidase first oxidizes an api- system, while no change occurred without genin, a major flavonoid of parsley leaves apigenin, as shown in Table 6. (29), and then chlorophylls are degraded by From these results, the mechanism of chlo- the oxidized apigenin. It was also recognized rophyll degradation by the peroxidase-hydro- by Matile(14) and Huff(11) that chlorophylls gen peroxide system in parsley leaves is as- were degraded by a peroxidase-hydrogen per- sumed to be that peroxidase first oxidizes an oxide system in the presence of phenolic com- apigenin, and then chlorophylls are degraded pounds, such as 2, 4-dichlorophenol and re- by the oxidized apigenin. sorcinol. Ferguson et al. demonstrated that the hydrogen peroxide content increased in Discussion parallel with chlorophyll degradation when It has been reported that chlorophyllase discs of cucumber cotyledon were floated(5). catalyzes the removal of phytols from chlo- Frenkel and Eskin observed that the hydro- rophylls to form chlorophyllides(8,10). Lo- gen peroxide content increased in the course oney and Patterson observed that chlorophyl- of ripening of tomato fruits after harvest lase activity in apple and banana fruits in- (7). In addition, it was noticed that the creased following climacteric rise(13). It chlorophyll content declined in rice seedlings was also reported that in ethylene-treated treated with hydrogen peroxide as compared citrus fruits, chlorophyll in peels was de- with non-treated ones (18). These facts graded with the enhancement of chlorophyl- seem to indicate that the chlorophyll degra- lase activity(2, 25). dation by the peroxidase-hydrogen peroxide On the other hand, it was thought that system would occur in many fruits and veg- chlorophyllase combines chlorophyllides and etables as well as in parsley leaves. 270 YAMAUCHI,N. AND T. MINAMIDE

While we have recognized the effect of L- texture. p.4. Academic Press, New York. ascorbic acid in retarding the chlorophyll de- 5. FERGUSON,I. B., C. B. WATKINS and J. E. HAR- gradation by peroxidase(30), it is thought MAN. 1983. Inhibition by calcium of senes- that L-ascorbic acid would reduce the amount cence of detached cucumber cotyledons. Plant of oxidized apigenin produced by peroxidase. Physiol. 71 : 182-186. 6. FORD,T. W. and E. W. SIMON. 1972. Peroxi- Martinoia et al. demonstrated that chloro- dase and glucose-6-phosphate dehydrogenase plast thylakoids showed a chlorophyll-bleach- levels in cotyledons of Cucumis sativus L. ing activity in the presence of hydrogen per- J. Exp. Bot. 23:423-431. oxide and monophenol, such as a 2, 4-dichlo- 7. FRENKEL,C. and M. ESKIN. 1977. Ethylene rophenol (15). Takahama also found that fla- evolution as related to changes in hydro- vonols, such as quercetin and quercitrin, peroxides in ripening tomato fruit. HortSci- were oxidized by peroxidase-like activity in ence 12 : 552-553. spinach chloroplasts in the presence of hy~ 8. HOLDEN,M. 1961. The breakdown of chloro- drogen peroxide(27). Nakano and Asada ob- phyll by chlorophyllase. Biochem. J. 78: served that hydrogen peroxide was generated 359-364. 9. HOLDEN,M. 1970. Chlorophyll bleaching sys- from thylakoids in illuminated spinach tems in leaves. Phytochem. 9 : 1771-1777. leaves (16). Sanuders and McClure found 10. HOLDEN,M. 1976. Chlorophylls. p.1-37. In that many kinds of flavonoid were contained T. W. Goodwin. (eds) Chemistry and bio- in the chloroplasts of many species of vas- chemistry of plant pigments. Vol. 2. Academ- cular plants(22). Consequently, it is inferred ic Press, New York. that both peroxidase and apigenin are 11. HUFF, A. 1982. Peroxidase-catalyzed oxida- contained in parsley chloroplast, and that tion of chlorophyll by hydrogen peroxide. the oxidation of apigenin by the peroxidase- Phytochem. 21: 261-265. hydrogen peroxide system occurs in the chlo- 12. JONES,I. D., R. C. WHITE and E. GIBES. 1963. roplast. Conformation of this is the subject Influence of blanching or brining treatment on the formation of chlorophyllides, pheo- for a future study. phytins, and pheophorbides in green plant Acknowledgement tissue. J. Food Sci. 28 : 437-439. 13. LOONEY,N. E. and M. E. PATTERSON. 1967. The authors are grateful to Dr. Takashi Chlorophyllase activity in apples and bananas Iwata, Professor of University of Osaka Pre- during the climacteric phase. Nature 214 fecture, for a critical reading of this manu- 1245-1246. script. 14. MATILE, Ph. 1980. Catabolism of chloro- This study was supported in part by a re- phyll : Involvement of peroxidase ? Z. Pflan- search scholarship from the Japan Society for zenphysiol. 99 : 475-478. the Promotion of Science. 15. MARTINOIA,E., M. J. DALLINGand Ph. MATILE. 1982. Catabolism of chlorophyll : Demonstra- Literature Cited tion of chloroplast-localized peroxidative and 1. ALJUBURI,H., A. HUFF and M. HSHIEH. 1979. oxidative activities. Z. Pflanzenphysiol. 107: Enzymes of chlorophyll catabolism in orange 269-279. flavedo. Plant Physiol. 63 : S-73. 16. NAKANO,Y. and K. ASADA. 1980. Spinach 2. BARMORE,C. R. 1975. Effect of ethylene on chloroplasts scavenge hydrogen peroxide on chlorophyllase activity and chlorophyll cont- illumination. Plant Cell Physiol. 21 :1295- ent in calamondin rind tissue. HortScience 1307. 10 : 595-596. 17. ORTHOEFER,F. T. and L. R. DUGAN Jr. 1973. 3. DIETRICH,W. C., M. D. NUTTING,R. L. OLSON, The coupled oxidation of chlorophyll a with F. E. LINDQUIST,M. M. BOGGS,G. S. BOHART, linoleic acid catalyzed by lipoxidase. J. Sci. H. J. NEUMANNand H. J. MORRIS.1959. Time- Fd Agric. 24 : 357-365. temperature tolerance of frozen foods. XVI. 18. PARIDA,P. K., M. KAR and D. MISHRA. 1978. Quality retention of frozen green snap beans Enhancement of senescence in excised rice in retail packages. Food Technol. 13: 136- leaves by hydrogen peroxide. Can. J. Bot. 145. 56 : 2937-2941. 4. ESKIN,M. 1979. Plant pigments, flavors and 19. PARISH, R. W. 1968. Studies on senescing CHLOROPHYLL DEGRADATION BY PEROXIDASE IN PARSLEY LEAVES 271

tobacco leaf disks with special reference to In : T. W. Goodwin. (eds) Chemistry and peroxidase. Planta 82 : 1-13. biochemistry of plant pigments. Vol. 2. 20. PHILLIPS, D. R., R. F. HORTON and R . A. FLET- Academic press, New York. CHER. 1969. Ribonuclease and chlorophyllase 27. TAKAHAMA, U. 1984. Hydrogen peroxide- activities in senescing leaves. Physiol. Plant. dependent oxidation of flavonols by intact 22 : 1050-1054. spinach chloroplasts. Plant Physiol. 74: 21. PURVIS, A. C. 1980. Sequence of chloroplast 852-855. degreening in calamondin fruit as influenced 28. WALKER,G. C. 1964. Color determination in by ethylene and AgNO3. Plant Physiol. 66 frozen french beans (Phaseolus vulgaris). 624-627. 2. The effect of blanching. J. Food Sci. 22. SAUNDERS, J. A. and J. W. MCCLURE. 1976. 29 : 389-392. The distribution of flavonoids in chloroplasts 29. WILDANGER,W. and K. HERRMANN. 1973. of twenty five species of vascular plants. Flavonole and flavone der gemusearten. I . Phytochem. 15 : 809-810. Flavonole der kohlarten. Z. Lebensm. Un- 23. SHIMIZU, S. and E. TAMAKI. 1962. Chlorophyl- ters.-Forsch. 152 : 134-137. lase of tobacco plants. I. Preparation and 30. YAMAUCHI,N., S. HAMAGUCHIand K. OGATA. properties of water soluble enzyme. Bot. 1980. Physiological and chemical studies on Mag. 75 : 462-467. ascorbic acid of fruits and vegetables. VII. 24. SHIMIZU, S. and E. TAMAKI. 1963. Chlorophyl- Mechanism of chlorophyll degradation and lase of tobacco plants. II. Enzymatic phytyla- action of ascorbic acid in the inhibition of tion of chlorophyllade and pheophorbide in yellowing in harvested parsley leaves. J. vitro. Arch. Biochem. Biophys. 102:152- Japan. Soc. Hort. Sci. 49:414-420. (In 158. Japanese, with English summary) 25. SHIMOKAWA, K., S. SHIMADA and K. YAEO. 31. YOSHIURA,M, and K. IRIYAMA. 1979. Methods 1978. Ethylene-enhanced chlorophyllase acti- for the preparation and qualitative and quan- vity during degreening of Citrus unshiu titative analysis of chlorophylls. Protein, Marc. Scientia Hortic. 8 : 129-135. nucleic acid and enzyme 24:612-620. (In 26. SWAIN, T. 1976. Flavonoids, p. 166-206. Japanese)

パセリーにおけるペルオキシダーゼによるクロロフィルの分解1

山内直樹2・南出隆久大阪府立大学農学部591堺市百舌鳥梅町

摘要

本研究は,パセリーにおけるペルオキシダーゼによる配糖体であると推定した.さらに,塩酸による加水分解クロロフィルの分解について追究した.パセリー葉身のによりアグリコンを抽出し,薄層クロマトグラフィーでエタノール抽出物にペルオキシダーゼ並びに過酸化水素のRf値並びにスペクトル特性の検討により,未知物質を添加すると,クロロフィルの分解が認められた.しかはパセリーの主要フラボノイドのアピゲニンであることしながら,精製したクロロフィルはペルオキシダーゼ・を同定した. 過酸化水素系で分解されなかった.この結果から,パセ以上の結果から,パセリーにおけるクロロフィルの分リーのエタノー・ル抽出物中に含有される未知物質がペル解は,ペルオキシダーゼがアピゲニンを酸化し,生成しオキシダーゼ・過酸化水素系によって酸化され,その酸たアピゲニンの酸化物がクロロフィルを分解することを化生成物がクロロフィルを分解しているものと思われた認め,収穫後におけるパセリーの黄化にペルオキシダーので,以下未知物質の検討を行った. ゼが関与しているものと推察した. 1収穫された葉菜類のクロロフィル分解機構未知物質は溶媒分画並びにカラムクロマトグラフィー .第1報. によって分離され,紫外部吸収極大位置からアピゲニン 2現在兵庫県立姫路短期大学