FoodSci. Techno!. Re,~'., 6 (1 ), 59-61, 2000 Note

Variation of Total Polyphenol and Polyphenol Oxidase Activity during Maturation of Fruit (Manglfera indica L. 'Irwin' ) Cultured in a Plastic House

Motoko UEDA,1 Katsuaki SASAKI,2 Kazunari INABAl and Yoshihide SHIMABAYASHI 1

!Faculty' ofAgriculture, Kinki University~ 3327-204 Nakamachi, Nara-shi, Nara, 63]~505, Japan 2The Experimenta/ Farm, Kinki University~ 2355-2 Yuasa, Yuasa-cho, Wakay'ama 643~004, Japan

Received August 1 1 , 1999; Accepted December 18, 1999

Variation of total polyphenol (TPP) and polyphenol oxidase activity (PPO) was investigated in 'Irwin' mango fruit in various color stages of development: green, purple, purplish red and red. Fruits used in this experiment were cultured in a plastic house. From each sample, six compounds in the skin and four compounds in the flesh were detected as polyphenol by thin-layer chromatography; The TPP content in the skin was 1 1-29 times higher than that in the flesh at various maturity stages varying during nraturation. For PPO activity, nruch higher levels were detected in the skin and increased during maturation. The activity in the skin was 4-12 times higher than that in the flesh at various stages.

Keywords: 'Irwin' mango fruit, maturation of mango, variation of total polyphenol, variation of polyphenol oxidase activity

The mango (Manglfera indica L.) is one of the most important Materials and Methods fruit crops in the world and is produced mainly in tropical Plant material Mango fruits of the 'Irwin' variety grown regions. Trade in mango is currently restricted, primarily because in a plastic house at the Yuasa Experimental Farm of Kinki Uni- the lack of established techniques regarding handling, transport, versity were harvested on the 4th and 18th of August 1997. The storage and the after-ripening of the fruit is beset with a number harvested fruits were separated into four stages of maturity based of problems. To establish recommended conditions for storage on the skin color, green, purple, purplish red and red. Only and after-ripening, a number of studies have looked into the undamaged fruits free from apparent infection were chosen for effects of various factors of mango cultivar, temperature, time, the study. In the fastest possible time, the weight of each fruit and relative humidity and ethylene use (Krishnamurthy & Subraman- the soluble solids content of its juice were measured. yam, 1970; Lakshminarayana, 1973; Kalra & Tandon, 1 983; Assay' oftotal polyphenol The skin and flesh of each man- Tandon & Kalra, 1 983; Thomas & Oke, 1983; Vazquez-Salinas go were sampled for the assay. & Lakshminarayana, 1985; Medlicott et al., 1 986; Seymour et Preparation of extract The skin and fiesh from each of five al., 1990). There have been numerous reports on variations in fruits were poo]ed. Then, the skin or the fiesh was refluxed with fruit color, fiesh firmness, respiratory rate and principal com- five volumes of methanol for I O min, and then homogenized. pounds, such as pigments, vitamins, sugars and organic acids for The homogenate was refluxed again for 30 min and was filtered maturation, after-ripening and storage. by sucking. This procedure was repeated two times and the total 'Iiwin' mangoes have recently been cultivated in plastic filtrate was concentrated in vacuo to remove the methanol . The houses in Okinawa and Kagoshima, Japan, and studies have been extract was filled to a volume with water. Clear filtrate of the made on physiological and biochemical changes with growth extract was applied for assay of the total polyphenol. and ripening of the fruit (Ito et al., 1 997; Ueda et al., 1999). In Detection Thin-layer chromatography was employed to comparison with other major types of fruit, however, relatively detect and investigate the polyphenol in the filtrate. The chroma- little research has been made on polyphenol and polyphenol oxi- tography was performed with a silica gel plate 60F.54 (Merck dase, which affect the quality and storage life of mangoes Co.) by the ascending technique at room temperature. The sol- (Saeed, 1976; Brown et al., 1986; Loveys et al., 1992; Robinson vent employed to develop the chromatogram was toluene-ethyl et al., 1993). formate-formic acid (5:4:1) (Zweig & Sherma, 1972). As stan- In this study, variation in total polyphenol content and dard markers, a total of 1 8 polyphenols (containing chlorogenic polyphenol oxidase activity during maturation of mango fruits acid, gentisic acid, catechin, catechol, pyrocatechol, 3,4-dihy- cultured in a plastlc house was determined in the physiological droxyphenylalanine) were used. and biochemical evaluation of fruit development. Another aim Determination Five milliliters of the extract was mixed with was to obtain fundamental data on preventing quality deteriora- 5 ml of I N Folin reagent. Af~ter keeping this reaction mixture for tion and preserving the mango fruit. 3 min at room temperature, 5 ml of lO% Na)CO~ was added, and this mixture was then allowed to stand for I h at room tempera- ture. The absorbance of the reaction was then measured at 700

E-mail: ueda@ nara.kindai.ac.jp nm and the total polyphenol content was determined using the 60 M. UEDA et a/ standard curve with chlorogenic acid. tion. Table I shows the average weight and average soluble sol- Polyphenol oxidase activity The skin and fiesh of the ids content of the fruits used for the assay. mangoes were sampled for the assay. Detection ofpolyphenol Aliquots of each extract prepared Preparation of crude enzyme solution All operations were from the skin and the flesh were applied on a silica gel plate and carried out at 4'C. The skin or the flesh was homogenized with developed by the ascending technique. The developed chromato- 12 volumes of cold (-20'C) acetone, and the residue was col- grams were examined under ultraviolet light at 254 nm. Six spots lected by sucking filtration. The residue on the filter was washed as polyphenol were detected from the skin, and four spots from with excess volumes of cold acetone and then a little ethyl ether. the fiesh. The residue was dried in vacuo. Five-tenths of a gram of the ace- Of the six compounds in the skin, compounds with Rf Values tone powder was suspended in 1 5 ml of O. I M phosphate buffer, of 0.36 and 0.5 1 may be compounds which exist only in the skin. pH 7.2, and stirred gently overnight at 4'C. The aqueous extract The chromatographic patterns on the extracts suggested no quali- was centrifuged at 10,000xg for 15 min, and the supernatant tative difference of polyphenol compounds in the mangoes at retained; the supernatant is hereafter referred to as crude enzyme various maturity stages. Of the compounds detected, those with solution . Rf Values of 0.5 1 , 0.06 and 0.03 coincided with authentic gentisic The enzyme activity Polyphenol oxidase activity was meas- acid, chlorogenic acid and 3 ,4-dihydroxyphenylalanine, respec- ured according to the method reported by Robinson et al. (1993). tively, developed at the same time as the standard markers. That is, one ml of the enzyme solution was incubated with 2.5 ml Variation oftotal polyphenol during maturation Variation of 0.1 M phosphate buffer, pH 5.5, which contained 0.01 M 4- of total polyphenol content during maturation is shown in Fig. l. methylcatechol as the substrate for 10 min at 25'C. The enzyme It can be seen that the total polyphenol content of the skin was activity was determined by the increase in absorbance at 400 nm. higher than that of the flesh in the various maturity stages of the One unit of the enzyme activity was defined as a 0.001 absor- mangoes. The ratio of the total polyphenol content of the skin to bance increase per min under the employed assay conditions. that of the whole fruit was 95.8% for green, 94.2% for purple, 91.7% for purplish red and 96.7% for red. In other words, the Results and Discussion skin content was about 23 times that of the flesh for green, 1 6 Weight and soluble solids content of mango fruits at various times for purple, 1 1 times for purplish red and 29 times for red. color stages The weight of each fruit and the soluble solids Although the total polyphenol content of the flesh appeared to content of its juice were measured as soon as possible after selec- vary slightly during maturation, the content of the skin varied, increasing remarkably for red after decreasing once for purple and/or purplish red. Table 1. Weight and soluble solids content of mango fruit at various color Kalra and Tandon ( 1 983) reported that the content of tannins, stages used for assay. in whole fruit of 'Dashehari' mangoes appeared to vary slightly Color stage Weight (g) Soluble solids content ('Brix) with the stage of fruit development stage, for instance, 1 28.6 Green 192.67d:57.75 6.30~0. 1 7 Purple 1 40.99+42.43 7.50~0.30 mg%, 101.7 mg% and 126.4 mg% for mangoes harvested at 85 Purplish red 163.82+45.65 7.70+0.48 days, 90 days and 95 days after fruit set, respectively. Their ex- 292.49~122.5 1 5 .0+ I .6 l Red periments, however, differed from those of the present authors, in Each value is an average ~ standard deviation of five measurements. the cultivar and maturity of the mangoes used for study, in the

~~o 3 o bb 8 o '~ ~ J:I v' ~$,, ,L, (, ~:: '~ ~ v' J~~ G' v' Lt $, 1~ 6 ~ oh ~~ 2 Fl o ~,l I" ~ JE:~ ~'!'* v' a, ,,, ,HL d~ t" ,L, p\' 4 ~ ~'h ~uo '~ - 1 ~ o u,~, 1~ ID a, v' ~:1 :' 1' 2 ~ ~ h O ~o -1 c~e ~ G, c:' ;~ ~ el o h O green purple Purplish red eL, green purplc purplish red Color Stage Color stage Fig. 1. Variation of total polyphenol content in 'Irwin' mangoes during Fig. 2. Variation of polyphenol oxidase in activity 'lrwin' mangoes during maturation. Each value is an averaged:standard deviation of five estimations. maturation. Each value is an averaged:standard deviation of five estimations. II: Skin, ~Z]: Flesh. I: Skin, E~: Flesh. Polyphenol and Its Oxidase Activity of Mango Fruit 61 sampling for determination of polyphenolic compounds, and in References other factors. Consequently, it is somewhat difficult to directly Brown, B.1.,Wells, I.A. and Murray. C.F. (1986). Factors affecting the discuss the experimental results of one in comparison with those incidence and severity of mango sapburn and its control. ASEAN Food J., 2, 127-132. of the other. It may nonetheless be useful to consider the srnall Hamauzu, Y., Chachin, K., Ding, C.K. and Kurooka, H. (1997). Differ- number of similarities in the tendency of the variation on poly- ences in surface color, fiesh firmness, physiological activity, and phenols with fruit development. some components of 'Loquat' fruits picked at various stages of On the other hand. Hamauzu et a/. ( 1997) showed that the maturity. Engei Gakkaishi, 65, 859-865 (in Japanese). Ito, T., Sasaki, K. and Yoshida, Y. (1997). Changes in respiration rate, content of phenols and the ratio of ortho-diphenol to total pheno- saccharide and organic acid content during the development and rip- lics for varieties of loquat fruit increase during fruit maturation. ening of mango fruit ( indica L. 'Irwin') cultured in a In addition, Sannomaru et al. (1998) found that in the skin of all plastic house. Engei Gakkaishi, 66, 629-635 (in Japanese). varieties of the apples they sampled, the total polyphenol content Kalra, S.K, and Tandon, D.K. (1983). Ripening-behaviour of 'Dashe- was four or five times that of the fiesh and core, and also that the hari' mango in relation to harvest period. Sci. Hortic., 19, 263-269. Krishnamurthy, S. and Subramanyam, H. (1970). Respiratory climac- content of total polyphenol, chlorogenic acid and epicatechin de- teric and chemical changes in the mango fruit, Manglfera indica L.. creased during the development of maturation. J. Am. Soc. Hort. Sci., 95. 33_ 3-337. These results suggest that the content of total polyphenol in Lakshminarayana, S. (1973). Respiration and ripening patterns in the different types of fruit varies with the development of maturation, life cycle of the mango fruit. J. Hort. Sci., 48, 227-233. Loveys, B.R., Robinson, S.P., Brophy, J.J. and Chacko, E.K. (1992). even if the varieties in the different types of fruit differ. Mango sapburn: Components of fruit sap and their role in causing Variation of polyphenol oxidase activity Variation of skin damage. Aust. J. Plant Physiol., 19, 449~}59. polyphenol oxidase activity during maturation is shown in Fig. 2. Medlicott, A.P., Reynolds. S.B. and Thompson, A.K. (1986). Effects of Enzyme activity was detected on the skin and flesh at various temperature on the ripening of mango fruit (Manglfera indica L. maturity stages, and there was an increase in the activity during var. ). J. Sci. Food. Agric., 37, 469~,74. Robinson, S.P., Loveys, B.R. and Chacho, E.K. (1993). Polyphenol maturation. In the skin, the activity of the red fruit increased by oxidase enzymes in the sap and skin of mango fruits. Aust. J. Plant about 7 fold over that of the green fruit and the activity in the Physiol., 20, 99-1 07. flesh increased by about I .8 fold. Much higher levels of activity Saeed, A.R. (1976). Polyphenolic compounds in the pulp of Man- were detected in the skin: about 3.6 fold higher than in the fiesh gnfera indica L. J. Food Sci., 41, 959-960. for the green fruit, about 1 2 fold for the purple fruit and about 7 Sannomaru, Y., Katayama, O., Kashimura, Y, and Kaneko, K. (1998). Changes in polyphenol content and polyphenol oxidase activity of fold for the purplish red or red fruit. apple fruits during ripening process. Nihon Shokuhin Kagaku The above results are in accord with the results of Robinson et Kogaku Kaishi., 45, 37~3 (in Japanese). al. (1993) on polyphenol oxidase activity in the flesh, skin and Seymour, G.B., N'Diaye, M., Wainwright, H. and Tucker, G.A. sap of green and ripe 'Kensington' and ripe 'Iiwin' mangoes. (1990). Effects of cultivar and harvest maturity on ripening of man- On variation of the enzyme activity during maturation of dif- goes during storage. J. Hort. Sci., 65, 479~83. Tandon, D.K. and Kalra, S.K. (1983). Changes in sugars, starch, and ferent types of fruit, Sannomaru et al. (1998) reported that the amylase activity during development of mango fruit cv Dashehari. J. enzyme activity in the flesh of all varieties of the apples they Hort. Sci., 58, 449~53. sampled decreased rapidly during the early stages of maturity Thomas, P. and Oke, M.S. ( 1983). Improvement in quality and storage development and thereafter increased gradually. of '' mangoes by cold adaptation. Sci. Hortic., 19, 257- The results therefore indicate that variation of polyphenol oxi- 262. Ueda, M., Sasaki, K., Utsunomiya, N., Inaba, K. and Shimabayashi, Y. dase activity varies with fruit maturation. ( 1999). Effects of storage on fruit color, flesh firmness, respiratory rate and several principal compounds in fully matured mango fruit Acknowledgement We thank Mr. Hiroyuki Nozaki for his technical assis- (Mangl[fera indica L. 'Irwin' ) cultured in plastic house. Nihon tance. Shokuhin Kagaku Kogaku Kaishi., 46, 16-23 (in Japanese). Vazquez-Salinas, C. and Lakshminarayana. S. (1985). Compositional changes in mango fruit during ripening at different storage tempera- ture. J. Food Sci., 50, 1646-1648. Zweig, G. & Sherma, J. (Editors) ( 1972). "Handbook of Chromatogra- phy" Volume I CRC Press Cleveland, p. 591.