Flavonol Changes in Seedlings of Vigna mungo During Growth

J. Plant Physiol.

Vol. 142. pp. 647-650 (1993)

Flavonol Changes in Seedlings of Vigna mungo During Growth MASAMI MATO

and N ARIYUKI ISHIKURA *

Department of Biological Science, Faculty of Science, Kumamoto University, Kurokami, Kumamoto 860, Japan Received March 24,1993 . Accepted June 21,1993

Summary

The accumulation of three glycosides of each of kaempferol and quercetin in seedlings of Vigna mungo was demonstrated by using high performance liquid chromatography. The identity of these compounds was determined in comparison to standards by paper chromatographic and UV spectral analyses; they include kaempferol 3-0-robinobioside-7-0-rhamnoside (robinin) (K1), kaempferol 3-O-rutinoside (K2), kaempferol 7-0-rhamnoside (K3), quercetin 3-0-robinobioside-7-0-rhamnoside (Q 1), quercetin 3-0-rutinoside (rutin) (Q2) and quercetin 3-0-glucoside (isoquercitrin) (Q3). K1, K2, and K3 (trace) were found in young leaves of 4 to 20-day-old seedlings, while Q1, Q2, Q3 and K1 were found in the hypocotyls and stems. At the age of 2 to 6 months, both leaves and stems produced all six flavonol glycosides.

Key words: Flavonol formation, Leguminosae (Fabaceae), Vigna mungo, black gram.

Introduction

It is well known that quercetin and kaempferol are widely distributed in leaves and flowers of legumes and that they are useful as phylogenetic markers (Harborne, 1971). According to a previous study on the distribution of flavonoids in Vigna species (Ishikura et ai., 1981), twelve kinds of flavonoids including five flavonol glycosides are found in the hypocotyls, seed-coats, and mature leaves. The leaves of V. mungo (black gram) contain only kaempferol glycosides, with kaempferol 3-O-robinobioside-7-0-rhamnoside (robinin) as the predominant flavonol glycoside, while those of V. radiata (green gram) contain the glycosides (mainly quercetin 3-O-rutinoside) of both quercetin and kaempferoi. However, the present work by means of high performance liquid chromatography of the extracts of hypocotyls and stems of black gram reveals that there are many more presumptive flavonoid peaks occurring than have been reported in the literature (Ishikura et ai., 1981). The present paper describes the identification of flavonol glycosides and changes in their composition in various parts of black gram seedlings during growth.

* Correspondence should be addressed to Prof. N. Ishikura. © 1993 by Gustav Fischer Verlag, Stuttgart

Materials and Methods

Plant materials Seeds of black gram, Vigna mungo (L.) Hepper (NIAS no. R-30; cf. Ishikura et a!., 1981), were germinated at 25°C on cotton wetted with tap water, and seedlings were illuminated at 10 klux for 18h per day with cool-white fluorescent lamps prior to harvest 4 to 20 days after cultivation was started. Some of the seedlings were cultivated at the experimental farm of Kumamoto University for 2 to 6 months from middle March to middle September. The seedlings, which were harvested at various growth stages, were divided into parts as follows: first pair (opposite) of simple leaves, stem and hypocotyl from 4 to 8-day-old seedlings (Fig. 1 a); first simple leaves, hypocotyl and stem first internode, trifoliate leaves and stem from 14 to 20-day-old seedlings, and trifoliate leaves and stem from 2 to 6-month-old plants. All parts were weighed while fresh and subsequently stored in the dark below 0 °C until use.

Extraction and identification 0/flavonol glycosides in various parts 0/ black gram seedlings Extraction in acetone and identification of extracted flavonol glycosides in various parts of the seedlings were carried out as described previously (Ishikura et a!., 1981). The solvent systems for

648

a

MASAMI MATO and NARIYUKI ISHIKURA

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values were directly compared with that of authentic samples. Flavonoids identified were quantified by measuring the absorbance (A, peak area) at 360 nm of the column effluent.

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Results

Fig. 1: 8- (a) and 20-day-old (b) seedlings of black gram. Vigna mungo (L.) Hepper (NIAS no. R-30).

Table 1: PC and HPLC behavior of flavonol glycosides found in the seedlings of black gram. FlavonoP Kl K2 K3 Ql Q2 Q3

Rf values x 100 in 2 II 81 66 32 79 61 46

73 60 18 68 54 41

tR4

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III

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N

(min)

40 57 80 30 48 64

Bk Bk LY Bk Bk Bk

LY LY LY Y Y Y

YBr YBr YBr YBr YBr YBr

14.4 42.1 13.9*

Six kinds of flavonol glycosides were found in the seedlings of black gram (Table 1). Five were identified as kaempferol 3-0-robinobioside-7-0-rhamnoside (K1, robinin), kaempferol 3-0-rutinoside (K2), kaempferol 7-0-rhamnoside (K3), quercetin 3-0-robinobioside-7-0-rhamnoside (Q 1), quercetin 3-0-rutinoside (Q 2, rutin) and quercetin 3-0-glucoside (Q3, isoquercitrin) by comparison with standards (Ishikura et aI., 1981). Q1, quercetin 3-0-robinobioside7-0-rhamnoside, is reported here for the first time from seedlings of the genus Vigna. UV spectral analysis of Q 1 indicated that it has glycosidic linkages at the 3 and 7 positions of quercetin. On complete hydrolysis with dilute HCl, Q1, isolated from 2-month-old seedlings by means of mass PC, gave quercetin, galactose, and rhamnose; Q 1 also yielded ro-

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and 2 See text for composition. 3 Fluorescent color under UV light at 257 nm (UV), with ammonia vapor ( + NH3), and color reaction with the reagent diazotized pnitroaniline (N). Bk, black; LY, lemon-yellow; Y, yellow; YBr, yellow-brown. 4 HPLC tR values in solvent A, except tR value of K3 in solvent B is marked with an asterisk. 1

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paper chromatography (PC) were as follows; acetic acid/conc. HClIwater (3 : 1 : 8, v/v) (solvent I), 15 % acetic acid (II) and n-butanol/acetic acid/water (4: 1: 5, v/v, upper layer) (III). Paper chromatograms were examined under UV light (256 nm) before and after exposure to ammonia vapor. The detecting reagent for flav01lOids was p-nitroaniline (Ishikura and Teramoto, 1983). Eluted flavonols were subjected to UV spectral analysis before and after acid hydrolysis using the diagnostic reagents (d. Mabry et ai., 1970). Each compound was identified by UV spectral and chromatographic comparison with authentic samples. For high performance liquid chromatography (HPLC), fresh tissue (ca. 2.0 g) was extracted with 70 % methanol (20 ml, 90 min) under refluxing. The extraction was repeated twice. Combined extracts were concentrated under reduced pressure. The aqueous concentrate was repeatedly washed with petroleum ether and then diethyl ether. After removal of the remaining ether by evaporation, the mother liquor was filtered through a cellulose acetate filter (0.45 11m, Dismic-13 cp, Advantec, Tokyo, Japan) and applied to a C18 reversed-phase column (4x250mm, liChrospher 100 RP-18, Merck, Darmstadt, Germany) in a Tri Rotar-V HPLC Gapan Spectroscopic Co., Ltd., Tokyo, Japan) using the isocratic solvent systems acetonitrile/water/phosphoric acid (17: 83: 0.2, v/v) (solvent A) and acetonitrile/water/phosphoric acid (30: 70: 0.2, v/v) (solvent B), at a flow rate of 0.7 ml/ min. Each peak in a HPlC elution profile of the mother liquor was monitored at 360 nm and their tR

Retention time (min)

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d

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60

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Retention time (min)

Fig. 2: HPlC elution profiles of the extract of various tissues, which were separated from 14-day-old seedlings of black gram. a, first simple leaf; b, trifoliate leaf; c, hypocotyl and first internode and d, stem.

Flavonols in Vigna mungo Seedlings binobiose, 6-0-a-L-rhamnopyranosyl-D-galactose, upon hydrogen peroxide degradation, indicating a linkage of the disaccharide to the 3 position of quercetin as in robinin (Kl) (Ishikura et ai., 1981). HPLC elution profiles of the extracts from the first simple leaves (Fig. 2 a) and young trifoliate leaves (Fig. 2 b) of 14day-old seedlings show the presence of K1 (maximum content; 1.006 g/kg fresh mass) as the predominant pigment and K2 as the minor pigment, while those of hypocotyls and stem first internodes (Fig. 2 c) and stems (Fig. 2 d) show the presence of Q1, Q2 and Q3; K1 as a minor pigment and a trace of K3 were also detected. K3 could be detected from the HPLC elution profile of the extracts in solvent B rather than A. Fig. 3 shows the amounts of flavonol glycosides in seedlings at various growth stages as measured by HPLC. First simple leaf and trifoliate leaf of 8 and 14-day-old seedlings contained similar amount of K1, 0.8 ± 1.2 g/kg fresh mass (Figs. 3 a, b). K2 was present at much lower levels, 0.02 0.03 g/kg fresh mass, and K3 was found in only trace amounts. No quercetin glycoside was detected. K2 was completely absent from the hypocotyl and stem of 8 and 14-dayold seedlings, but K1, Q1, Q2 and Q3 were found in low amounts, the quercetin glycosides increasing with age (Figs. 3 a, b). Flavonol components in 20-day-old seedlings were

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Fig. 3: The flavonoid content (g/kg fro mass) of seedlings of black gram at various growth stages; a = 8-day-old seedlings, b = 14-dayold seedlings, c = 2-month-old seedlings and d = 6-month-old seedlings. Flavonoids are Kl = robonin, K2 = kaempferoI3-0-rutinoside, K3 = kaempferoI7-O-rhamnoside, Ql = quercetin 3-O-robinobioside-7-O-rhamnoside, Q2 = rutin and Q3 = isoquercitrin. Tr = trace.

649

very similar to those in 14-day-old seedlings (data not shown). On the other hand, the leaves and the stems of 2 to 6-month-old plants contained all six flavonol glycosides with K 1 the main pigment (maximum content 1.314 g/kg fresh mass) in the leaves and Ql (0.076g/kg fresh mass) the main pigment in the stems (Figs. 3 c, d). Discussion

In the present study, quercetin 3-0-robinobioside-7-0rhamnoside (Q 1) was found for the first time in plants belonging to the genus Vigna. Q 1 has so far been found only in leaves of Vinca minor (= Catharanthus minor) (Harborne and Williams, 1975). Q1 was formed in both leaves and stems of 2 to 6-month-old seedlings, but it was not detected in leaves of 8 to 20-day-old seedlings. As a result of surveying the flavonoids of black gram with HPLC, it beame evident that three kaempferol glycosides and three quercetin glycosides were formed in the seedlings, and also that the composition of flavonols in seedlings changes depending on the kind of tissues and their age. In a previous paper (Ishikura et aI., 1981), it was reported that mature leaves of black gram contain only kaempferol glycosides, while those of green gram contain the glycosides of both quercetin and kaempferoI. However, the present study discloses that mature leaves of black gram are also able to form quercetin glycosides although seedling leaves do not. Hypocotyl and stem tissues of black gram form all three quercetin glycosides, thus their flavonol composition is quite different from that of young leaves. These results seem to point to higher 3' -hydroxylase activity for flavonoid in stem and hypocotyl tissues. It is likely that the initiation of quercetin formation in mature leaves is due to an acquired ability of quercetin formation during growth rather than the translocation of quercetin glycosides from stems into leaves since there is no evidence supporting the translocation of endogenous flavonoids within the vascular system of plants. Drastic changes in the spectrum of flavonoids, especially anthocyanin, during growth and development of leaves have been reported in several plants (Ishikura, 1972; Barz and Hosel, 1975 - also refer to Harborne and Williams (1988) for within plant variations in distribution}. Del Rio et ai. (1992) have also pointed out that morphological changes in cells of Citrus aurantium are correlated with expression of flavonoids. Furthermore, in tree-grown fruit of Citrus limon cv. Eureka, the total amount of hesperidin per lemon accumulates rapidly at a young stage, after which eriocitrin begins to increase rapidly, which continues until the fruit reaches full size (Vandercook and Tisserat, 1989). Thus, it is evident that there are significant differences in tissue and/or cell parameters relating to the formation of flavonoids, and also that flavonoid components change during different growth stages of tissues. Therefore, these facts must be considered when flavonoid components are used as phylogenetic markers. Acknowledgements

We thank Dr. S. Teramoto (Department of Biological Science, Kumamoto University) for helpful discussions.

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HARBORNE, J. B.: Distribution of flavonoids in the Leguminose. In: HARBORNE, J. B., D. BOULTER, and B. L. TURNER (eds.): Chemotaxonomy of the Leguminosae, 31-71. Academic Press, London (1971).

HARBORNE, J. B. and C. A. WILLIAMS: Flavone and flavonol glycosides. In: HARBORNE, J. B., T. J. MABRY, and H. MABRY (eds.): The Flavonoids, 376-441. Chapman and Hall, London (1975).

- - Flavone and flavonol glycosides. In: HARBORNE, J. B. (ed.): The Flavonoids, Advances in Research Since 1980, 303-328. Chapman and Hall, London (1988). ISHIKURA, N.: Anthocyanins and other phenolics in autumn leaves. Phytochemistry 11, 2555-2558 (1972). ISHIKURA, N., M. IWATA, and S. MIYAZAKI: Flavonoids of some Vig· na-plants in Leguminosae. Bot. Mag. Tokyo 94, 197 - 205 (1981). ISHIKURA, N. and S. TERAMOTO: Procyanidins and catechin from callus and cell suspension cultures of Cryptomeria japonica. Agric. bioI. Chern. 47, 421-423 (1983). MABRY, T. J., K. R. MARKHAM, and M. B. THOMAS: The Systematic Identification of Flavonoids. Springer-Verlag, Berlin (1970). VANDERCOOK, C. E. and B. TISSERAT: Flavonoid changes in developing lemons grown in vivo and in vitro. Phytochemistry 28,799803 (1989).