† To whom correspondence should be addressed. Tel: +81-3-5981-3430; Fax: +81-3-5981-3426; Email: nvc＠fc.jwu.ac.jp

Fig. 1. Gac Fruit.1, Fruit meat (yellow part); 2, Seed membrane (red part)

Biosci. Biotechnol. Biochem., 66 (11), 2479–2482, 2002

Note

Carotenoid Pigments in GAC Fruit (Momordica cochinchinensis SPRENG)

Hiromitsu AOKI,1 Nguyen Thi Minh KIEU,2 Noriko KUZE,1 Kazue TOMISAKA,2

and Nguyen Van CHUYEN2,†

1Food Color Laboratory Division, San-Ei Gen F.F.I. Inc., 1-1-11 Sanwa-cho, Toyonaka,Osaka 561-8588, Japan2Department of Food and Nutrition, Japan Women's University, 2-8-1 Mejirodai, Bunkyo-ku,Tokyo 112-8681, Japan

Received April 24, 2002; Accepted June 6, 2002

The carotenoids in Gac fruit (Momordica Cochin-chinensis spreng) were analysed by high-performanceliquid chromatography (HPLC), and the concentrationsof b-carotene, lycopene, zeaxanthin and b-cryptoxan-thin were measured. Lycopene was found to bepredominantly present in the Gac seed membrane at aconcentration of up to 380 mgWg of seed membrane. Theconcentration of lycopene in the Gac seed membranewas about ten-fold higher than that in known lycopene-rich fruit and vegetables, indicating that Gac fruit couldbe a new and potentially valuable source of lycopene.

Key words: carotenoid; momordica; b-carotene; lyco-pene; Gac

The protective eŠects of carotenoids against dis-ease are assumed to be through such antioxidative ac-tivities as quenching of singlet oxygen and scavengingof peroxyl radicals.1) Carotenoids in nature areresponsible for the characteristic colors of variouskinds of fruit, vegetables and shellˆsh. Tomatoes andwatermelon with the red color of lycopene, carrotswith the orange color of b-carotene, and dark greenvegetables are important sources of carotenoid com-pounds. In Vietnam, a subtropical region, among thewide variety of fruit and vegetables, Momordicacochinchinensis called ``Gac'' is very popular be-cause of its perfect combination of a natural redcolor and high b-carotene content.2) Gac is used inVietnam as a colorant for cooking red glutinous rice,a popular dish in this country, similar to Sekihan(rice boiled together with red beans) in Japan.However, carotenoid data for Gac fruit are limited,2)

and a quantitative analysis of the carotenoid pig-ments in Gac is therefore necessary. In addition, Gacfruit are cheap and easy to grow. Quantitative meas-urement of the carotenoid compounds in Gac willprovide information to support the use of Gac as auseful and economical carotenoid food source.

The inside of Gac contains two parts: (1) fruit meat

(yellow part) and (2) red membrane surrounding theseeds (seed membrane), as shown in Fig. 1. In orderto obtain data on the whole Gac fruit, we measuredthe carotenoid pigments in both the fruit meat andseed membrane. Five samples of Gac fruit were pur-chased from diŠerent markets in Ho Chi Minh Cityand separately analysed. The fruit meat and seedmembrane from each sample were separately re-moved and frozen at „209C until needed.

The carotenoids were analysed after being extract-ed and saponiˆed. Brie‰y, 56 g of fruit meat or 10 gof seed membrane was homogenized and then ex-tracted with a four-fold volume of acetone until com-plete exhaustion of the color. The extract was ˆlteredand transferred to a separating funnel, together withan equal volume of diethyl ether. The obtained upperphase was washed three times with a saturated NaClsolution, dehydrated with Na2SO4 anhydride, anddried under reduced pressure at 259C. The driedsample of fruit meat was dissolved in 4 ml ofdichloromethane, that of the seed membrane was dis-solved in 4 ml of diethyl ether, and acetonitrile wasthen added to each sample to make up to 20 ml.

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Fig. 2. Carotenoids in Gac Fruit Meat and Seed Membrane.(a) and (b) were analysed under diŠerent HPLC conditions. (c) and (d) were analysed under the same HPLC conditions.

2480 H. AOKI et al.

These solutions were analysed by HPLC.The saponiˆcation process was carried out for an

analysis of the carotenoid esters. To the dried sampleof fruit meat or seed membrane were added 50 ml ofethanol and 5 ml of 60z KOH, and the solutionsaponiˆed in N2 gas for 24 h in the dark at 59C. Thesolution was then adjusted to pH 4 with HCl andacetic acid, before being transferred to a separatingfunnel together with 50 ml of petroleum ether. Thisprocedure was repeated once, and the combined up-per phase was washed four times with water, de-hydrated with Na2SO4 anhydride, dried underreduced pressure at 259C, and analysed by HPLC.

The HPLC analysis was performed with an AS-950autosampler (Jasco), two PU-980 pumps (Jasco), aDG-980-50 degasser (Jasco), a UV-970 UVWVIS de-tector (Jasco), a CS-300B column casket (ChromatoScience), and a Symmetry C18 column (250×4.6 mmi.d., 5 mm; Waters) at 409C. To analyze the extractedfruit meat samples, the mobile phase consisted of amixed solution of (A) acetonitrile:dichloromethane:methanol (vWv 7:2:1) and (B) dichloromethane. Thecolumn was eluted by using consecutive linear

gradients 100z of solution A for 30 minutes, 0–50zof solution B for 5 minutes, and 50z of solution Bfor 5 minutes. The ‰ow rate was 1 mlWmin, detectionwas at 450 nm, and the injection volume was 25 ml.To analyze the saponiˆed fruit meat, the mobilephase was made up of A acetonitrile and mixed solu-tion B of acetonitrile:dichloromethane:methanol(7:2:1 vWv). The column was eluted by using consecu-tive linear gradients of 0–100z of solution B for 20minutes and 100z of solution B for 30 minutes. The‰ow rate was 1 mlWmin, detection was at 450 nm, andthe injection volume was 25 ml. The HPLC condi-tions for the analysis of the extracted and saponiˆedseed membrane were similar to those used for theanalysis of the extracted fruit meat. The results of theHPLC analysis were completely reproducible.

The carotenoid pigments were identiˆed by com-paring their HPLC retention times (Fig. 2) with thoseof authentic samples as standards. The content of anauthentic sample solution for the HPLC analysis wasconˆrmed from the extinction coe‹cent of E (1z,1 cm) in the petroleum ether of lycopene (3450, lmax

472 nm), b-carotene (2592, lmax 453 nm), zeaxanthin

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Table 1. Contents of Carotenoid Pigments in Gac Fruit (mgWg offresh fruit)

Content

HPLC method

Fruit meat(yellow part)

Seed membrane(red part)

Extracted sampleb-carotene 22.1±15.2 101±38Lycopene 0.9±0.7 380±71Total 23.0±17.3 481±89

Saponiˆed sampleb-carotene 14.7±10.9 81±31Lycopene 0.4±0.3 348±54Zeaxanthin 1.6±0.4 9±4b-cryptoxanthin 3.5±1.8 2±2Total 20.2±12.7 440±72

Each value is presented as the mean±SD of 5 Gac samples purchasedfrom diŠerent markets.

2481Carotenoid Pigments in Gac Fruit

(2350, lmax 452 nm) and b-cryptoxanthin (2386, lmax

452 nm).3)

The analytical results for the fruit meat and seedmembrane are summarized in Table 1. One gram ofextracted fruit meat contained 7–37 mg of b-caroteneand 0.2–1.6 mg of lycopene, the total content ofcarotenoid pigments being 6–40 mg. The analysis ofthe saponiˆed samples detected lycopene, b-carotene,zeaxanthin and b-cryptoxanthin (Fig. 2b). One g ofsaponiˆed fruit meat contained 4–26 mg of b-caro-tene, 0.1–0.7 mg of lycopene, 1–2 mg of zeaxanthin,and 2–5 mg of b-cryptoxanthin, giving a total of8–33 mg of carotenoid pigments. The red color wasreduced about 40z by saponiˆcation.

The lycopene and b-carotene content in the seedmembrane were found to be much higher than that inthe fruit meat, both in the extracted and saponiˆedsamples (Table 1). As an average, 1 g of extractedseed membrane contained 310–460 mg of lycopeneand 60–140 mg of b-carotene, compared to 0.2–1.6 mgof lycopene and 7–37 mg of b-carotene in the extract-ed fruit meat. In the saponiˆed samples, 1 g of theseed membrane contained 300–400 mg of lycopeneand 50–110 mg of b-carotene, compared to 0.1–0.7 mgof lycopene and 4–26 mg of b-carotene in the fruitmeat. Zeaxanthin and b-cryptoxanthin were also de-tected in the saponiˆed seed membrane (Fig. 2d), buttheir contents were very low (Table 1). About 10z ofthe red color was reduced by the saponiˆcationprocedure.

Carotenoids are usually found in fruit and vegeta-bles in the free form or as fatty acid esters. Zeaxan-thin and b-cryptoxanthin have hydroxyl groups in the6-membered ring, and these hydroxyl groups canbind with fatty acids to form carotenoid esters. Thisis the reason why the carotenoid esters, zeaxanthinand b-cryptoxanthin, were found in the saponiˆedsamples, but not in the extracted samples. The degreeof esteriˆcation of carotenoids in some fruit increasesduring ripening.4,5) Breithaupt et al.6) have analysed

the carotenoid esters in 64 types of fruit and vegeta-bles, and found the highest carotenoid ester concen-tration in red chili (171 mgWg), while most of theinvestigated fruit and vegetables showed concentra-tions up to 15 mgWg.

Many plants are known to contain b-carotene, butfew plants containing lycopene are known, except fortomato (31 mgWg of lycopene), watermelon (41 mgWg),guava (54 mgWg) and pink grapefruit (33.6 mgWg)(Finnish food data).7) Among them, guava had thehighest concentration of lycopene. However, theconcentration of lycopene in the Gac seed membranewas seven-fold higher than that (380 mgWg). The edi-ble seed membrane in Gac fruit made up an averageof 30z (30.2±4.9z, n＝5) and the inedible fruitmeat averaged 50z (52.1±7.7z, n＝5). Lycopeneexhibits the highest singlet oxygen-quenchingcapability among carotenoids.8) The antioxidativeeŠect of lycopene in lowering the risk of developingmajor chronic disorders such as coronary heart dis-ease and cancer has been reported through many stu-dies.9–12) In a clinical trial at Hanoi University in Viet-nam,13) an oil extract of Gac was found to be eŠectivein the treatment of liver cancer.

Our study has revealed that Gac contained a higherlycopene quantity than that in other fruits known tocontain lycopene. Gac fruit are easily grown in a sub-tropical climate; therefore, this new ˆnding is expect-ed to contribute a new promising source of lycopeneworldwide.

References

1) Sies, H., Stahl, W., and Sundquist, A. R., Antiox-idant functions of vitamins. Vitamin E and C, b-caro-tene, and other carotenoids. Ann. N. Y. Acad. Sci.,669, 7–20 (1992).

2) Nguyen, T. L., Le, D. D., and Phan, Q. K., Agingpreventive foods. In ``Medicine and functional foodsin Vietnam'' (in Vietnamese), Agriculture Publica-tion, Hanoi, pp. 150–156 (1999).

3) Davies, B. H., In ``Chemistry and Biochemistry ofPlant Pigment'', ed. Goodwin, T. W., Acad. Press.,2, pp. 38–165 (1976).

4) Minguez-Mosquera, M. I., and Hornero-Mendez, D.,Formation and transformation of pigments duringthe fruit ripening of Capsicum annuum Cv. Bola andAgridulce. J. Agric. Food Chem., 42, 38–44 (1994).

5) Subagio, A., and Morita, N., Changes in carotenoidsand their fatty acid esters in banana peel during ripen-ing. Food Sci. Technol. Int. Tokyo, 3, 264–268(1997).

6) Breithaupt, D. E., and Bamedi, A., Carotenoid estersin vegetables and fruits: A screening with emphasis onb-cryptoxanthin esters. J. Agric. Food Chem., 49,2064–2070 (2001).

7) Mangels, A. R., Holden, J. M., Beecher, G. R.,Forman, M. R., and Lanza, E., Carotenoid contentof fruits and vegetables: an evaluation of analyticdata. J. Am. Diet. Assoc., 93, 284–296 (1993).

24822482 H. AOKI et al.

8) Di Paolo, M., Stephan, K., and Helmut, S., Lyco-pene as the most e‹cient biological carotenoid singletoxygen quencher. Arch. Biochem. Biophys., 274,532–538 (1989).

9) Khachik, F., Beecher, G. R., and Smith, J. C.,Lutein, lycopene, and their oxidative metabolites inchemoprevention of cancer. J. Cell. Biochem., 22,236–246 (1995).

10) Nagasawa, H., Mitamura, T., Sakamoto, S., andYamamoto, K., EŠects of lycopene on spontaneousmammary tumor development in SHN virgin mice.Anticancer Res., 15, 1173–1178 (1995).

11) Kobayashi, T., Iijima, K., Mitamura, T., Toriizuka,K., Cyong, J. C., and Nagasawa, H., EŠects of lyco-pene, a carotenoid, on intrathymic T-cell diŠerentia-

tion and peripheral CD4WCD8 ratio in a high mam-mary tumor strain of SHN retired mice. AnticancerDrugs, 7, 195–198 (1996).

12) Narisawa, T., Fukaura, Y., Hasebe, M., Ito, M.,Aizawa, R., Murakoshi, M., Memura, S., Khachik,F., and Nishino, H., Inhibitory eŠects of naturalcarotenoids, a-carotene, b-carotene, lycopene, andlutein, on colonic aberrant crypt formation in rats.Cancer Lett., 107, 137–149 (1996).

13) Trinh, V. B., Study on anti-toxic eŠect of Gacavit indioxin infected mice. In ``Dioxin in the war, a longterm harmful chemical to humans and nature'' (inVietnamese), eds. Hoang, D. C., Le, C. D., Dinh, Q.M., and Le, B. T., Cong Doan Publication, Hanoi,Vietnam, pp. 452–457 (1993).