J. Plant Res.

107 : 275-282, 1994

Journal of Plant Research (~ by The Botanical Society of Japan 1994

Flavonoid Variation and Evolution in Asplenium normale and Related Species (Aspleniaceae)
Tsukasa Iwashina and Sadamu Matsumoto
Tsukuba Botanical Garden, National Science Museum, Tsukuba, 305 Japan

Flavonoid profiles of 132 populations (472 individuals) of Asplenium normale, and related species, A. boreale, A. shimurae, and A. oligophlebium var. oligophlebium and var. iezimaense in Japan were surveyed by HPLC and 2D-PC. Of the five taxa, each of Asplenium boreale, A. shimurae and A. oligophlebium including var. iezimaense had consistent flavonoid composition : apigenin 7, 4'-di-O-rhamnoside (9) in Asplenium boreale, 7-O-glucosylrhamnosides of apigenin and luteolin (6 and 7) in A. shimurae and genkwanin 4'-O-glucosylrhamnoside (5) in two A. oligophlebium varieties. On the other hand, Asplenium normale was divided into seven chemotypes A - G : A-type has 7-O-dirhamnosides of apigenin and luteolin (1 and 2) and genkwanin 4'-O-glucosylrhamnoside (5) ; B-type, 5 alone ; C-type, apigenin 7-O-rhamnoside-4'-O-glucosylrhamnoside (8) ; D-type, 1 and 2 ; E-type, 1, 2 and 8 ; F-type, 1, 2, 5 and 8 ; and G-type, 5 and 8. Among them, the most frequent types were A, B and C, and A-type was mainly distributed in inland of Honshu, Shikoku and Kyushu, while B- and C-types extended their distribution areas southwards in general and occur along the Pacific coast with several exception. Chemical and evolutionary relationships among Asplenium boreale, A. shimurae, A. oligophlebium, and the chemotypes of A. normale were discussed on the basis of general biosynthetic pathway. Key words: Apigenin 7,4'-dirhamnoside--Aspleniaceae - - Asplenium normale - - Chemotaxonomy Flavonoids m Geographic Variations

i.e. 7-O-dirhamnosides of apigenin and luteolin, and genkwanin 4'-O-glucosytrhamnoside in A. normale, apigenin 7 , 4 ' - d i - O - r h a m n o s i d e , which has been misidentified as genkwanin 4'-O-glucosylrhamnoside on account of their very similar chromatographic and UV spectral properties, in A. boreale, and 7-O-glucosylrhamnosides of apigenin and luteolin in A. shimurae. In the two varieties of Asplenium oligophlebium, genkwanin 4'O-glucosylrhamnoside alone has been reported. In addition, two C-glycosylflavones, vicenin-2 and 6, 8 - d i - C glycosylluteolin, were found in all five taxa (Iwashina et al. 1990). In contrast to our finding, however, one earlier study (Harada et al. 1958) has reported the occurrence of flavonol, kaempferol but not flavones such as apigenin, luteolin or genkwanin, from Asplenium normale. Available data suggest, thus, the occurrence of distinct chemical races in this species. In this paper, we describe the presence of chemotypes in Japanese Asplenium normale and discuss the chemical and evolutionary relationships among the three related species, A. boreale, A. shimurae and A. oligophlebium, and among chemotypes of A. normale.

Materials and Methods Plant materials Asplenium normale (343 individuals from 79 populations), A. boreale (67 individuals from 27 populations), A. shimurae (28 individuals from 9 populations), and A. oligophlebium var. oligophlebium (33 individuals from 16 populations) and var. iezimaense (1 individual from 1 population) which were used in this experiment. They were collected by S. Mitsuta, members of Nippon Fernist Club (see, Tables 1 and 2), and S. Matsumoto from various places in Japan (Tables 1 and 2). Voucher specimens are deposited in Tsukuba Botanical Garden, National Science Museum (TNS). Isolation of flavonoids Apigenin 7-O-dirhamnoside (1), luteolin 7-O-dirhamnoside (2), vicenin-2 (3), 6, 8-di-C-glycosylluteolin (4), genkwanin 4'-O-glucosylrhamnoside (5), apigenin 7 - 0 glucosylrhamnoside (6), luteolin 7-O-glucosylrhamnoside (7), and apigenin 7-O-rhamnoside-4'-O-glucosylrhamnoside (8) were isolated from Asplenium normale, A.

Asplenium normale D. Don (Aspleniaceae) is broadly distributed throughout East and Southeast Asia, Africa and Hawaii (Nakaike 1992), and two related species, A. boreale (Ohwi ex Kurata) Nakaike and A. shimurae (H. Ito) Nakaike occur in the Sino-Japanese area (Ching and Iwatsuki 1982). Recently, the latter two were recognized at the species rank (Nakaike 1992). Another related Asplenium oligophlebium Bak. including var. iezimaense (Tagawa) Tagawa, which is restricted to lejima island, is endemic to Japan (Nakaike 1992). In previous paper (Iwashina et al. 1990), we have presented that Asplenium normale, A. boreale, A. shimurae and A. oligophlebium have distinct flavonoid patterns,

276

T. Iwashina and S. Matsumoto Table 1. Sample numbers, collected places, collectors and chemotypes of Asplenium normale 1-6 (4: np*), Nanakai-mura, Ibaraki Pref., S. Matsumoto, A; 7-12, Tenryu-shi, Shizuoka Pref., S. Matsumoto, A; 13-7'6, Shimoda-shi, Shizuoka Pref., S. Matsumoto, A (15)**, 8 (42) and G (6) ; 77-80, Nishi-izu-cho, Shizuoka Pref., S. Matsumoto, B ; 81-83, Oogusu. Kamo-mura, Shizuoka Pref., S. Matsumoto, B ; 84-86, Ookusa-zamo, Kawo-mura, Shizuoka Pref., S. Matsumoto, E ; 87-88, One, Hosoe-cho, Shizuoka Pref., T. Nakura, A ; 89-91, Iwane, Hosoe-cho, Shizuoka Pref., T. Nakura, B ; 92-94, Mikkabi-cho, Shizuoka Pref., T. Nakura, A ; 95-96, Kosai-shi, Shizuoka Pref., T. Nakura, A ; 97, Hachijojima Isl., Tokyo Pref., S. Matsumoto, C; 98, Inuyama-shi, Aichi Pref., K. Inukai, A; 99-113 (111-113: np*), Gifu-shi, Gifu Pref., S. Matsumoto, A ; 114-115, Miyama-cho, Gifu Pref., H. Miyazaki, A ; 116-122, Ooi-cho, Fukui Pref., S. Matsumoto, C ; 123-125, Kouno-mura, Fukui Pref., Y. Saito, A ; 126-127, Imajou-cho, Fukui Pref., Y. Saito, A ; 128, Kyoto-shi, Kyoto Pref., S. Mitsuta, A ; 129-131, Miyama-cho, Kyoto Pref., S. Matsumoto, A ; 132-134. Ise-shi, Mie Pref., S. Yamauchi, A ; 135-137, Seki-cho, Mie Pref., S. Yamauchi, A (1) and B (2); 138-141, Toba-shi, Mie Pref., M. Uniya, C; 142-196, Owase-shi, Mie Pref. (13 populations), S. Matsumoto, M. Kawazoe and R. Ito, A (17), B (1), C (22), E (8), F (2) and G (4) ; 197-199, Tado-cho, Mie Pref., M. Kawazoe, A (2) and E (1) ; 200-205, Miyama-cho, Mie Pref., R. Ito, A (3), B (1) and E (2) ; 206-222, Oouchiyama-mura, Mie Pref., Y. Higuchi, A (14) and B (3) ; 223-224, Kihou-cho, Mie Pref., S. Iwanaka, E ; 225-227, Kumano-shi, Mie Pref., S. Iwanaka, B ; 228-231, Shingu-shi, Wakayama Pref., K. Oohora, A (2) and E (2) ; 232-234, Nachi-katsuura-cho, Wakayama Pref., K. Oohora, B; 235-239 (238-239: rip*), Shimizu-cho, Wakayama Pref., S. Mitsuta, A; 240-242, Koza-cho, Wakayama Pref., H. Manago, B (1) and C (2) ; 243-245, Kozagawa-cho, Wakayama Pref., H. Manago, B (2) and E (1) ; 246, Kumanogawa-cho, Wakayama Pref., H. Manago, A; 247-250, Ootou-mura, Wakayama Pref., H. Manago, B; 251-257, Susami-cho, Wakayama Pref., (2 popurations), H. Manago, B (6) and C (1) ; 258-259, Hikigawa-cho, Wakayama Pref., H. Manago, (3 ; 260-263, Tanabe-shi, Wakayama Pref. (2 populations), H. Manago, A (2) and C (2) ; 264-268, Kanaya-cho, Wakayama Pref. (2 populations), H. Manago, B (2) and E (3); 269-271, Yura-cho, Wakayama Pref., H. Manago, A; 272, Ookawachi-cho, Hyogo Pref., N. Iwaya, B ; 273-274, Hattou-cho, Tottori Pref., A. Tanaka, A ; 275-277, Kawakami-cho, Okayama Pref., T. Watanabe, A ; 278-279, Fuchu-shi, Hiroshima Pref., T. Takeda, A ; 280-286 (284-286 : np*), Yamagu chi-shi, Yamaguchi Pref., S. Miyake, A ; 287-295 (294 : rip*), Toyota-cho, Yamaguchi Pref., G. Imada, A ; 296-297, Nang oku-shi, Kochi Pref., S. Matsumoto, A; 298-302, Kochi-shi, Kochi Pref., S. Matsumoto, C (3) and E (2); 303-304, Nakamura-shi, Kochi Pref., S. Matsumoto, C; 305-307, Yasuda-cho, Kochi Pref., S. Matsumoto, B; 308-309, Oozu-shi, Ehime Pref., M. Hyodo, G ; 310-314 (pp*), Nomura-cho, Ehime Pref., M. Hyodo, A ; 315-317, Nakagawa-cho, Fukuoka Pref., S. Tsutsui, D ; 318-320, Tachibana-cho, Fukuoka Pref., S. Tsutsui, C ; 321-322, Saiki-shi, Ooita Pref., M. Hadano, A ; 323325, Yayoi-cho, Ooita Pref., M. Hadano, G; 326-32"7, Hasami-cho,Nagasaki Pref., S. Koga, B; 328-330, Hondo-shi, Kumamoto Pref., Y. Kobayashi, A (2) and 8 (1) ; 331-333, Yatsushiro-shi, Kumamoto Pref., K. Watanabe, G ; 334, Tsunocho, Miyazaki Pref., S. Matsumoto, A; 335-337, Kitagawa-cho, Miyazaki Pref., M. Hyodo, G; 338-340, Kamou-cho, Kagoshima Pref., K. Takesako, A; 341-342, Yakushima Isl., Kagoshima Pref., S. Mitsuta, C; 343, Tokunoshima Isl., Kagoshima Pref., S. Mitsuta, C. * n p : non-proliferous form like Asplenium boreale. pp : pinnae-proliferous form with one proliferous bud on a rachis. ** The numeral in parenthesis indicates the numbers collection. A=apigenin 7-O-dirhamnoside (1), luteolin 7-O-dirhamnoside (2) and genkwanin 4'-O-glucosylrhamnoside (5); B= flavonoid 5 ; C--apigenin 7-O-rhamnoside-4'-O-glucosylrhamnoside (8) ; D=flavonoids 1 and 2 ; E=flavonoids 1, 2 and 8 ; F--flavonoids 1, 2, 5 and 8 ; and G-flavonoids 5 and 8. Vicenin-2 (3) and 6, 8-di-C-glyoosylluteolin (4) were detected in all chemotypes.

shimurae and A. oligophlebium by methods as those of described by Iwashina et al. (1990, 1993). Flavonoid (9),
w h i c h has been confused with genkwanin 4 ' - 0 glucosylrhamnoside on a c c o u n t of extremely similar chromatographic and UV spectral properties (Iwashina et al. 1990), were isolated from Asplenium boreale as follows. Fresh fronds were extracted with MeOH, filtrated and evaporated to an aqueous residue. After washing with petroleum ether, the residue was shaken with ethyl acetate. T h e organic layer was concentrated, applied to polyamide column and eluted with 70% MeOH. T h e eluent was evaporated to dryness, dissolved in 70% MeOH, and isolated on Sephadex LH-20 column (solvent system: 70% MeOH). Flavonoid (9) was obtained as pale yellow needles from 70% MeOH in the cold.

(co-PC) with authentic specimens using four solvent systems (see Table 3), UV spectral analysis according to Mabry et al. (1970), acid hydrolysis, HPLC comparisons, and then by 1H-NMR and F A B - M S analysis. 1H-Nuclear magnetic resonance (NMR) spectra were recorded in dimethylsulfoxide-d6 (DMSO-d6) using tetramethyl silane (TMS) as an internal standard, and fast atom bombardment mass spectra (FAB-MS) using nitrobenzyl alcohol (NBA).

Two-dimensional paper chromatography (2D-PC)

Mature fresh fronds (0.1-2.0 g / e a c h i n d i v i d u a l ) w e r e extracted with MeOH, filtrated and evaporated. Crops were t w o - d i m e n s i o n a l l y chromatographed using BAW and then 15% AcOH.

Identification of flavonoids
Flavonoids were identified by co-paper chromatography

F l a v o n o i d V a r i a t i o n s of Asplenium normale Table 2. Sample numbers, collected places and collectors of Asplenium boreale, A. shimurae and A. oligophlebium var. oligophlebium and var. iezimaense A. boreale 344-345, Kanuma-shi, Tochigi Pref., S. Tanuma and T. Tashiro; 346, Sano-shi, Tochigi Pref., T. Tashiro; 347-348, Utsunomiya-shi, Tochigi Pref., T. Tashiro ; 349-354, Tenryu-shi, Shizuoka Pref., S. Matsumoto ; 355-358, Shimoda-shi, Shizuoka Pref., S. Matsumoto ; 359-361, Hamakita-shi, Shizuoka Pref., T. Nakura ; 362-363, Hosoe-cho, Shizuoka Pref., T. Nakura; 364-365, Mikkabi-cho, Shizuoka Pref., T. Nakura; 366-357, Kosai-shi, Shizuoka Pref., T. Nakura; 368-369, Toyohashi-shi, Aichi Pref., T. Nakura; 370-377, Gifu-shi, Gifu Pref., S. Matsumoto; 378, Kyoto-shi, Kyoto Pref., S. Mitsuta; 379-380, Nachi-katsuura-cho, Mie Pref., K. Oohora; 381-382, Tado-cho, Mie Pref., M. Kawazoe; 383-385, Ise-shi, Mie Pref., S. Yamauchi ; 386-388, Watarai-cho, Mie Pref., S. Yamauchi; 389, Oouchiyama-mura, Mie Pref., Y. Higuchi ; 390-392, Toba-shi, Mie Pref., M. Uniya ; 393-394, Kanaya-cho, Wakayama Pref., M. Manago ; 395, Yura-cho, Wakayama Pref., H. Manago ; 396-398, Shimizu-cho, Wakayama Pref., S. Mitsuta ; 399-400, Youka-cho, Hyogo Pref., Y. Hayashi ; 401, Kochi-shi, Kochi Pref., S. Matsumoto ; 402-403, Uwa-cho, Ehime Pref., M. Hyodo ; 404-405, Nomura-cho, Ehime Pref., M. Hyodo ; 405, Yayoi-cho, Ooita Pref., M. Hadano ; 407-410, Ashikita-cho, Kumamoto Pref., M. Kido. A. shimurae 411-415, Tenryu-shi, Shizuoka Pref., S. Matsumoto ; 416, Sakuma-cho, Shizuoka Pref., S. Matsumoto ; 417-426, Owaseshi, Mie Pref. (2 populations), S. Matsumoto and M. Kawazoe ; 42"/'-429, Oouchiyama-mura, Mie Pref., Y. Higuchi ; 430432, Kozagawa-cho, Wakayama Pref., H. Manago ; 433-434, Kumanogawa-cho, Wakayama Pref., H. Manago ; 435-437, Tosa-yamada-cho, Kochi Pref., S. Matsumoto ; 438, Nangoku-shi, Kochi Pref., S. Matsumoto. A. oligophlebium var. oligophlebium 439, Inuyama-shi, Aichi Pref., K. Inukai; 440, Miyama-cho, Gifu Pref., H. Miyazaki; 441-442, Ooi-cho, Fukui Pref., S. Matsumoto; 443-444, Mikata-cho, Fukui Pref., S. Matsumoto; 445-446, Kyoto-shi, Kyoto Pref., S. Mitsuta; 447-450, Nabari-shi, Mie Pref., S. Yamakawa ; 451-452, Shimizu-cho, Wakayama Pref., S. Mitsuta ; 453-454, Youka-cho, Hyogo Pref., Y. Hayashi ; 455-456, Wakasa-cho, Tottori Pref., T. Kiyosue ; 457-459, Toyota-cho, Yamaguchi Pref., G. Imada; 460, Kochi-shi, Kochi Pref., S. Matsumoto ; 461-462, Uwa-cho, Ehime Pref., M. Hyodo ; 463-465, Nakagawa-cho, Fukuoka Pref., S. Tsutsui ; 466-468, Kurume-shi, Fukuoka Pref., S. Tsutsui ; 469, Makurazaki-shi, Kagoshima Pref., K. Takesako ; 470-471, Yakushima Isl., Kagoshima Pref., S. Matsumoto. A. oligophlebium var. iezimaense 472, lejima Isl., Okinawa Pref., S. Mitsuta.

277

Table 3. Paper-chromatographic properties of flavonoids in Asplenium normale, A. boreale, A. shimurae and A. oligophlenium Rf values Flavonoids BAW 1 2 3 4 5 6 7 8 9 0.71 0.61 0.27 0.18 0.68 0.65 0.52 0.46 0.64 BEW 0.64 0.51 0.31 0.18 0.53 0.61 0.47 0.33 0.53 5% AcOH 0.08 0.04 0.34 0.20 0.10 0.13 0.09 0.48 0.12 15% AcOH 0.28 0.20 0.53 0.39 0.36 0.39 0.31 0.63 0.35 UV dp dp dp dp dp dp dp dp dp UV/NH3 dgy y dgy dy dp dgy dy dp dp A. A. all all A. A. A. A. A. A. normale normale taxa taxa nomale and oligophlebium shimurae shimurae normale boreale Colors Occurrence

BAW=n-BuOH/AcOH/H20 (4 : 1 : 5, upper phase), BEW=n-BuOH/AcOH/H=O (4 : 1 : 2.2), 5% AcOH=AcOH/H=O (5 : 95) and 15% AcOH=AcOH/H20 (15 : 85). dp=dark purple, dgy=dark greenish yellow, y----yellow and dy=dark yellow. l=apigenin 7-dirhamnoside, 2=luteolin 7-dirhamnoside, 3=vicenin-2, 4 = 6 , 8-di-C-glycosylluteolin, 5=genkwanin 4'-glucosylrhamnoside, 6=apigenin 7-glucosylrhamnoside, 7=luteolin 7-glucosylrhamnoside, 8 = apigenin 7-rhamnoside-4'-glucosylrhamnoside and 9=apigenin 7, 4'-dirhamnoside.

278
Table 4. Flavonoids in 1 2 3 4 5 6 7 8 9 MeOH 267, 336 259sh, 266, 349 273, 331 261sh, 270, 357 269, 320 268, 335 256, 264sh, 350 270,316 269, 318

T. Iwashina and S. M a t s u m o t o UV spectral properties of flavonoids in Asplenium nermale, A. boreale, A. shimurae and A. oligophlebium ;[ max (nm) +NaOMe 272, 379 (inc) 264, 385 (inc) 283, 333, 400 (inc) 268, 407 (inc) 287, 371 (dec) 274, 378 (inc) 267, 388 (inc) 293, 378 (dec) 287, 369 (dec) +AICI3 274, 299, 347, 386 274, 428 280, 305, 351, 380 278, 427 279, 340, 275, 347, 273, 278, 338, 278, 339, 300, 376 299, 384 428 298, 380 299, 379 +AICIJHCI 276, 342, 274, 357, 297, 381 294, 386 +NaOAc 266, 389 263, 395 282, 374 270, 282, 400 272, 316 267, 388 261,400 270,~6 270, 318 +NaOAc/H3B03 267, 341 262, 373 283, 322, 404sh 271, 287sh, 393 270, 321 267, 339 260, 374 270, 320 269, 320

279, 304, 344, 376 279, 297sh, 362, 384 281, 298, 333, 378 276, 298, 341,381 272, 293sh, 358, 387 279, 298, 334, 379 279, 298, 332, 378

inc=remarkable increase in intensity relative to the spectrum of MeOH solution. dec=remarkable decrease in intensity, sh-shoulder.

12

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Sample No. 296

sample No. 307

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Sample No. 97

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Fig. 1. HPLC chromatograms of three chemotypes A, B and C, in Asplenium normale, l=apigenin 7-O-dirhamnoside (Rt 6.67), 2=luteolin 7-O-dirhamnoside (Rt 4.67), 3=vicenin-2 (Rt 3.16), 4-6, 8-di-C-glycosylluteolin (Rt 2.34), 5=genkwanin 4'-O-glucosylrhamnoside (Rt 12.49) and 8-apigenin 7-O-rhamnoside-4"-O-glucosylrhamnoside (Rt 3.53). It was showed by UV spectra that other peaks were not flavonoid except two peaks (Rt 10.92 in B-type and 4.30 in C-type) which may be genkwanin 4'-O-glycoside and apigenin 7, 4'-O-glycoside, respectively.

Sample No. 435

Sample No. 368

Sample No. 457 5

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A. oligophlebium

Fig. 2. HPLC chromatograms of Aspenium shimurae, A. boreale and A. oligophlebium. Eluent: Acetonitrile-H20-H3P03 (35 : 65 : 0.2). Flow-rate : 1.0 ml/min. Injection : 10#1. Detection : 345 nm. 3=vicenin-2 (Rt 3.16), 4=6, 8-di-C-glycosylluteolin (Rt 2.34), 5=genkwanin 4 ' - 0 glucosylrhamnoside (Rt 12.49), 6=apigenin 7-O-glucosylrhamnoside (Rt 4.65), 7=luteolin 7 - 0 glucosylrhamnoside (Rt 3.61) and 9=apigenin 7, 4'-di-O-rhamnoside (Rt 4.96).

Flavonoid Variations of Asplenium normale

279

High performance liquid chromatography (HPLC) HPLC separations were performed with JASCO HPLC systems including an 880-PU pump, 880-51 2-line degasser and Syringe loading sample injector 25 model 7125 (Rheodyne Inc.). Multi-channel UV-visible detector Multi-330 (JASCO) coupled with a PC-9801 VX personal computer (NEC), was used for recording chromatograms and UV spectra. Finepak SIL CmS, 5 # m (4.6 mmX150 mm) column equipped with a Finepak SIL C18T-P precolumn was used. Crude MeOH extracts or authentic flavonoid solutions were filtrated through Toyopak ODS M (Tosoh) and then Chromatodisc 13N, 0.45 #m, and eluted with CH3CN/H20/H3P04 (35 : 65 : 0.2). Detection was at 345 nm, and flow-rate was 1.0 ml/min.

Results and Discussion

Identification of apigenin 7, 4'-di-O-rhamnoside (9) Flavonoid (9), which was isolated from Asplenium boreale, had not been able to be distinguished from genkwanin 4'-O-glucosylrhamnoside (5) from A. normale and A. oligophlebium (Iwashina et al. 1990), because both show very similar UV spectra (the presence of free 5-OH and substituted 7, 4'-diOH) (Table 4) and chromatographic properties (Table 3). In the present study, it was proved that the retention time of HPLC of flavonoid (9) was clearly different from that of flavonoid (5) (Fig. 1). Acid hydrolysis of flavonoid (9) gave apigenin and rhamnose which were identified by direct PC comparisons with authentic specimens. FAB-MS indicated [ M - H ] - at m/z 561 calcd C2z

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Fig. 3. Two dimensional chromatograms of Asplenium normale, A. boreale, A. shimurae and A. oligophlebium, l=apigenin 7-O-dirhamnoside, 2----luteolin 7-O-dirhamnoside, 3=vicenin-2, 4=6, 8-di-C-glycosylluteolin, 5=genkwanin 4'-O-glucosylrhamnoside, 6=apigenin 7-0glucosylrhamnoside, 7=luteolin 7-O-glucosylrhamnoside, 8=apigenin 7-O-rhamnoside-4'-Oglucosylrhamnoside, 9=apigenin 7, 4'-di-O-rhamnoside and U=unknown flavonoids which may be apigenin 7, 4'-O-glycosides,

280

T. Iwashina and S. Matsumoto (flavonoids 6, 7, 3 and 4), and A. oligophlebium including var. iezimaense (flavonoids 5, 3 and 4) were completely identical, and those differed among the species (Table 2, Figs. 2 and 3). It was confirmed that three species are chemically definable taxa, respectively. Recently, Nakaike (1992) raised Asplenium normale var. boreale and var. shimurae as independent species, A. boreale (Ohwi ex Kurata) Nakaike and A. shimurae (H. Ito) Nakaike, respectively. Our flavonoid evidence supports Nakaike's treatment.

H29013 showing the attachment of 2 mol rhamnose to apigenin. 1H-NMR spectrum indicated seven aromatic protons, two rhamnosyl methyl protons, and two rhamnosyl anomeric protons(s). Consequently, the flavonoid (9) was identified as apigenin 7, 4'-di-O-cL-L-rhamnoside, which was found in nature for the first time. The chemical data of the flavonoid (g) are shown as follows : FAB-MS (NBA) Found: m/z 561 [ M - H ] - , 415 EMmonorhamnosyI-H]- and 269 J-M-dirhamnosyI-H]-. 1H-NMR(270MHz, DMSO-d6) : 68.07 (2H, d, J = 8 . 8 Hz, H-2', 6'), 7.21 (2H, d, J = 8 . 8 Hz, H-3', 5'), 6.93 (1H, s, H-3), 6.86 (1H, d, J = 2 . 4 Hz, H-8), 6.45 (1H, d, J = 2 . 5 Hz, H-6), 5.55 and 5.54 (each 1H, s, rhamnosyl anomers), 5.2-3.7 (8H, m, rhamnosyl H-2, 3, 4 and 5X2), 1.13 and 1.11 (each 3H, d, rhamnosyl CH3 X2).
Flavonoid variations among Asplenium normale, A. boreale, A. shimurae and A. oligophlebium Iwashina et al. (1990) described that Asplenium normale, A. boreale, A. shimurae and A. oligophlebium have their own diagnostic flavonoid patterns. The present HPLC and PC survey for many individuals and populations proved that the flavonoid profiles of all individuals in each of A. boreale (flavonoids 9, 3 and 4), A. shimurae

Intra-specific flavonoid variation of Asplenium normale in Japan In contrast with Asplenium boreale, A. shimurae and A. oligophlebium, A. normale was subdivided into seven chemotypes (A-G) by HPLC and PC surveys (Figs. 1 and 3). The most frequent types were A , B and C. A-type contained apigenin 7-O-dirhamnoside (1) and luteolin 7O-dirhamnoside (2) in addition to genkwanin 4 ' - O glucosylrhamnoside (5). It was found in many inland populations of Honshu, Shikoku and Kyushu (Fig. 4). We recognized three morphological forms in this chemotype, i.e., 1) a non-proliferous form as in Asplenium boreale, 2) a pinnae-proliferous form with one proliferous bud on a rachis, and 3) typical form with one proliferous bud on a

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A A

. :. ~..q

! ~

s ~.......... AAi:y

84 ::AB

o ........'-, 9....~ 9 ...,. ~ : c~A ..,. 9 o

~ ~o.

~--/G
1 : / .. ...... .::

.... G S
A

.... GAC

Fig. 4. A geographic distribution of seven chemotypes of Asplenium normale. A=flavonoids 1, 2 and 5, B=5 alone, C=8 alone, D=I and 2, E=I, 2 and 8, F=I, 2, 5 and 8, and G=5 and 8.

Flavonoid Variations of Asplenium normale rachis. The former two are marked "np" and "pp" in Table1, respectively. The non-proliferous form differs from Asplenium boreale by not only the shape of lateral pinna and the habitat, but also flavonoid pattern. The non-proliferous form of the A-type in Asplenium normale and that of A. boreale may be represent resemblance by parallel evolution. B-type has flavonoid 5 alone and is distributed mostly along the Pacific coast from Izu Peninsula to Hondo-shi, Kumamoto Pref., Kyushu. C-type, which contained apigenin 7-O-rhamnoside-4'-O-glucosylrhamnoside (8), was also found along the Pacific coast, exceptionally in a population of Fukui Pref. (sample no. 116-122). Its distribution areas extend to southern islands such as Hachijojima, Yakushima, and Tokunoshima. Of the four less common chemotypes, D-type characterized by flavonoids 1 and 2 was found only in one population in Fukuoka Pref. (sample no. 315-317). Many of the individuals of E- (flavonoids 1,2 and 8), F(flavonoids 1, 2, 5 and 8), and G-types (flavonoids 5 and 8) coexisted with A-, B- and/or C-types (Table1, Fig. 4). The co-occurrence is prominent in Kii and Izu Peninsulae. Two C-glycosylflavones (3 and 4) were present in all

28!

individuals examined of the five taxa. The flavonoid pattern of B-type was identical with that of Asplenium oligophlebium. However, differences in morphological characters (Nakaike 1992) as well as in chromosome number (2n=144, 4X in all chemotypes of Asplenium normale in Japan; 2n=72, 2X in A. oligophlebium [Matsumoto, unpublished data-I) suggest that Btype of A. normale and A. oligophlebium are different entities. Asplenium oligophlebium is morphologically rather similar to C-type of A. normale in having prominently toothed pinnae and sometimes lobed segments at the base of pinna. Among the three species and their chemotypes of Asplenium normale, B- and C-types, A. boreale and A. oligophlebium differ from D-type and A. shimurae by absence of luteolin glycosides (2 and 7). Moreover, Dtype and A. shimurae are divided by further rhamnosylation or glucosylation of apigenin and luteolin 7-O-rhamnosides (Fig. 5). Iwashina and Matsumoto (1990) have reported that the diploid Asplenium normale from Nepal (Matsumoto and Nakaike 1988) had more glycosylated apigenin 7, 4'-Oglycosides, but did not have O-methylated flavonoid. If

H O

O 0

OR -

fimplification

A. boreale (qX) .................................. RO ORG !

i ......................... ~ i i
] il II
I

-luteolin methylation

HO o C-type ................................
................... ~-; .........

G-type(5and8)
E-type(I, 2 and 8)

O-R-G

lebium(2
A. normale (2X)

F-type(I, 2, 5and8)
I,)j ,,", ,
I t

(hypothetic ancestral type)

RO RO

~
HO

i
O

OH

A-type(I,2 and5)

OH ................................. 2 'ill R-O-R-o'~O~~ "OH rhamnosylation

Intraspecific

hybrids

...............................

H O

"~ 0

D-type

A normale (4X) .
] L

+luteolin glucosylation

G - O - R - O ~ o H HO O

C~glucosyl HO r , , / o ~ OH H c-gluc~ ~ J ~ HO c-giycosyl ~ O H ~'~ 3 O ~OH

G - O - R - O ~ o HO O A. shimurae (4X)

H
c-glycosyl HO

O
O

O H

Fig. 5. Presumedflavonoid evolution in Asplenium boreale, A. shimurae, A. oligophlebium, and seven chemotypes of A. normale. R=rhamnosyl,G--glucosyl.

282

T. Iwashina and S. Matsumoto

an evolutionary trend of flavonoid modification in ferns has proceeded toward the simplification (Hiraoka 1978) and O-methylation (Harborne 1966), C-type may be considered a more primitive chemotype than any other types, and B-type was derived from C-type by O-methylation in Japan (Fig. 5). As regards the distribution patterns, C-type may be considered as a subtropical element, compared to the other types (Fig. 4). Flavonoid comparison suggests that A-type was formed by intraspecific hybridization between chemotypes B and D. D-type, which was found only in three individuals from one population in northern Kyushu (Fig. 4), may be a Sino-Japanese element for the two following reasons : 1) A-type is not found from southern islands of Japan, and 2) Asplenium shimurae has flavonoids 6 and 7 which are chemically related with flavonoids 1 and 2 present in A- and D-types, and is distributed in the Sino-Japanese area (Ching and Iwatsuki 1982). In order to clarify the systematic relationships of Asplenium normale and the related taxa, flavonoid surveys should be extended to populations outside Japan, in particular those of China and tropical Asia. In an earlier study, Harada et al. (1958) have reported the presence of kaempferol glycoside but not apigenin in Asplenium normale (collected place was not cited). However, kaempferol was not found in all samples in this experiment. The authors thank Dr. Shigeyuki Mitsuta, Doshisha Univ. for his useful information and collection of samples. Authors' thanks are also due to members of the Nippon Fernist Club for providing plant materials, to Dr. Yuichi Yoshida, Tokyo Research Lab., Wako Pure Chemical Ind. for 1H-NMR measurements, and to Miss Kazuko Akuzawa, Tsukuba Research Lab., Nippon Oil & Fats Co., Ltd. for FAB-MS measurements.

References Ching, R.-C. and Iwatsuki, K. 1982. Annotations and corrigenda on the Sino-Japanese Pteridophytes (1). J. Jap. Bot. 57: 129-132. Harada, T., Kishimoto, Y., Saiki, Y., Ueno, A. and Amano, Y. 1958. Pharmaceutical studies on ferns. Memorial Rep. Shizuoka Coll. Pharm. 76-95 (in Japanese). Harborne, J.B. 1966. The evolution of flavonoid pigments in plants. In T. Swain ed., Comparative Phytochemistry. Academic Press, London. p. 271-295. Hiraoka, A. 1978. Flavonoid patterns in Athyriaceae and Dryopteridaceae. Biochem. Syst. Ecol. 6: 171-175. Iwashina, T. and Matsumoto, S. 1990. Flavonoid profiles of Asplenium normale in Nepal with taxonomic comments. In M. Watanabe and S.B. Malla, eds., Cryptogams of the Himalayas Vol. 2, Central and Eastern Nepal. National Science Museum, Tsukuba. p. 179-185. Iwashina, T., Matsumoto, S., Ozawa, K. and Akuzawa, K. 1990. Flavone glycosides from Asplenium normale. Phytochemistry 29 : 3543-3546. Iwashina, T., Matsumoto, S. and Yoshida, Y. 1993. Apigenin 7-rhamnoside-4'-glucosylrhamnoside from Asplenium normale. Phytochemistry 32 : 16291630. Kurata, S. and Nakaike, S. 1981. Illustrations of Pteridophytes of Japan. Vol. 2. Univ. of Tokyo Press, Tokyo (in Japanese). Mabry, T.J., Markham, K.R. and Thomas, M.B. 1970. The Systematic Identification of Flavonoids. SpringerVerlag, London. p. 3-184. Matsumoto, S. and Nakaike, T. 1988. Chromosome numbers of some ferns in Kathmandu, Nepal. In M. Watanabe and S.B. Malla, eds., Cryptogams of the Himalayas Vol. 1, The Kathmandu Valley, National Science Museum, Tsukuba. p. 177-185. Nakaike, T. 1992. New Flora of Japan. Pteridophyta. Revised & Enlarged. Shibundo, Tokyo (in Japanese). (Received September 8, 1993 : Accepted July 8, 1994)