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 chloro- phylls, 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 bleach- ing 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 ha- rvested 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 chemi- cal, 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 degra- dation. 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).