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 bor- eale, A. shimurae and A. oligophlebium including var. iezimaense had consistent flavonoid composition : apigenin 7, 4'-di-O-rhamnoside (9) in Asplenium bor- eale, 7-O-glucosylrhamnosides of apigenin and luteolin (6 and 7) in A. shimurae and genkwanin 4'-O-glucosyl- rhamnoside (5) in two A. oligophlebium varieties. On the other hand, Asplenium normale was divided into seven chemotypes A-G: A-type has 7-O-dirham- nosides 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 rela- tionships 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--Asple- niaceae - - Asplenium normale - - Chemotaxonomy Flavonoids m Geographic Variations

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. shimur- ae and A. oligophlebium have distinct flavonoid patterns,

i.e. 7-O-dirhamnosides of apigenin and luteolin, and genkwanin 4'-O-glucosytrhamnoside in A. normale, apigenin 7 ,4 ' -d i -O-rhamnoside, 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-glucosylrham- nosides of apigenin and luteolin in A. shimurae. In the two varieties of Asplenium oligophlebium, genkwanin 4'- O-glucosylrhamnoside alone has been reported. In addi- tion, 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. Avail- able data suggest, thus, the occurrence of distinct chemi- cal 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 popula-

tions), 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-dirham-

noside (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-glucosylrham- noside (8) were isolated from Asplenium normale, A.

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 ; 323- 325, 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, Tsuno- cho, 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), wh ich has been confused wi th genkwanin 4 ' - 0 - g lucosylrhamnoside on account of extremely similar chromatographic and UV spectral properties ( Iwashina et al. 1990), were isolated from Asplenium boreale as fol lows. Fresh fronds were extracted wi th MeOH, fi l trated and evaporated to an aqueous residue. After washing wi th petroleum ether, the residue was shaken wi th ethyl ace- tate. The organic layer was concentrated, appl ied to polyamide column and eluted wi th 70% MeOH. The 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 ye l low needles from 70% MeOH in the cold.

Identification of flavonoids Flavonoids were identi f ied by co-paper chromatography

(co-PC) wi th authent ic 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 FAB-MS analysis. 1H-Nuclear magnet ic resonance (NMR) spectra were recorded in d imethylsul foxide-d6 (DMSO-d6) using tetramethyl si lane (TMS) as an internal standard, and fast atom bombard- ment mass spectra (FAB-MS) using nitrobenzyl alcohol (NBA).

Two-dimensional paper chromatography (2D-PC) Mature fresh fronds (0.1-2.0 g /each i nd i v i dua l )we re

extracted with MeOH, f i l trated and evaporated. Crops were two-d imens iona l ly chromatographed using BAW and then 15% AcOH.

Flavono id Var ia t i ons of Asplenium normale

Table 2. Sample numbers, collected places and collectors of Asplenium boreale, A. shimurae and A. oligophlebium var. oligophlebium and var. iezimaense

277

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, Owase- shi, Mie Pref. (2 populations), S. Matsumoto and M. Kawazoe ; 42"/'-429, Oouchiyama-mura, Mie Pref., Y. Higuchi ; 430- 432, 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, Fukuo- ka Pref., S. Tsutsui ; 466-468, Kurume-shi, Fukuoka Pref., S. Tsutsui ; 469, Makurazaki-shi, Kagoshima Pref., K. Takesa- ko ; 470-471, Yakushima Isl., Kagoshima Pref., S. Matsumoto.

A. oligophlebium var. iezimaense 472, lejima Isl., Okinawa Pref., S. Mitsuta.

Table 3. Paper-chromatographic properties of flavonoids in Asplenium normale, A. boreale, A. shimurae and A. oligophlenium

Rf values Colors Flavonoids Occurrence

BAW BEW 5% AcOH 15% AcOH UV UV/NH3

1 0.71 0.64 0.08 0.28 dp dgy A. normale

2 0.61 0.51 0.04 0.20 dp y A. normale

3 0.27 0.31 0.34 0.53 dp dgy all taxa

4 0.18 0.18 0.20 0.39 dp dy all taxa

5 0.68 0.53 0.10 0.36 dp dp A. nomale and A. oligophlebium

6 0.65 0.61 0.13 0.39 dp dgy A. shimurae

7 0.52 0.47 0.09 0.31 dp dy A. shimurae

8 0.46 0.33 0.48 0.63 dp dp A. normale

9 0.64 0.53 0.12 0.35 dp dp A. boreale

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=luteol in 7-dirhamnoside, 3=vicenin-2, 4=6, 8-di-C-glycosylluteolin, 5=gen- kwanin 4'-glucosylrhamnoside, 6=apigenin 7-glucosylrhamnoside, 7=luteol in 7-glucosylrhamnoside, 8 = apigenin 7-rhamnoside-4'-glucosylrhamnoside and 9=apigenin 7, 4'-dirhamnoside.

278 T. Iwashina and S. Matsumoto

Table 4. UV spectral properties of flavonoids in Asplenium nermale, A. boreale, A. shimurae and A. oligophlebium

;[ max (nm) Flavonoids

in MeOH +NaOMe +AICI3 +AICIJHCI +NaOAc +NaOAc/H3B03

1 267, 336 272, 379 274, 299, 276, 297, 266, 389 267, 341 (inc) 347, 386 342, 381

2 259sh, 266, 264, 385 274, 428 274, 294, 263, 395 262, 373 349 (inc) 357, 386

3 273, 331 283, 333, 280, 305, 279, 304, 282, 374 283, 322, 400 (inc) 351, 380 344, 376 404sh

4 261sh, 270, 268, 407 278, 427 279, 297sh, 270, 282, 271, 287sh, 357 (inc) 362, 384 400 393

5 269, 320 287, 371 279, 300, 281, 298, 272, 316 270, 321 (dec) 340, 376 333, 378

6 268, 335 274, 378 275, 299, 276, 298, 267, 388 267, 339 (inc) 347, 384 341,381

7 256, 264sh, 267, 388 273, 428 272, 293sh, 261,400 260, 374 350 (inc) 358, 387

8 270,316 293, 378 278, 298, 279, 298, 270 ,~6 270, 320 (dec) 338, 380 334, 379

9 269, 318 287, 369 278, 299, 279, 298, 270, 318 269, 320 (dec) 339, 379 332, 378

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

12 Sample No. 296 5 i ! sample No. 307

1 ..... I ................. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I

I 8 . . . . i 5 . . . . . : t l . . . . !15 El 5 11LZI i 5

A-type B- type

•qll 3 Sample No. 97

C-type

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.

~ 3 L I

8 '

Sample No. 435

8

8 !8 ! 5

h. shimurae

ABU

3 Sample No. 368

1 8 1 5

A. boreal e

~ B U

Sample No. 457

5

...... ................... I ........

8 . . . . 5 1 8 1 5

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-l ine degas- ser and Syringe loading sample injector 25 model 7125 (Rheodyne Inc.). Multi-channel UV-visible detector Mult i-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 speci- mens. FAB-MS indicated [ M - H ] - at m/z 561 calcd C2z

<

OD | 1 7 4

(~)

rm

5 % A c O H

A. normale (A-type)

Q |

| | <

m-

5 % A c O H

A. normale (D-type)

G <

r ~

5 %A ~ O H A. normale (B-type) and A. oligophlebium

Q CD

1 5 % A c O H A. normale (E-type)

5 ~ A c O H A. normale (C-type)

(9 @0 |174

| | |

5 % A c O H A. normale (F - type )

O @

| |

t 5 % A c O " 5 % A c OH A. normMe (G-type) A. shimurae

(9 |

5 % A c O H

A. boreale

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 7-0- glucosylrhamnoside, 7=luteolin 7-O-glucosylrhamnoside, 8=apigenin 7-O-rhamnoside-4'-O- glucosylrhamnoside, 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

H29013 showing the attachment of 2 mol rhamnose to apigenin. 1H-NMR spectrum indicated seven aromatic protons, two rhamnosyl methyl protons, and two rham- nosyl 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 EM- monorhamnosyI-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 nor- male, 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

(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, Na- kaike (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.

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 7- O-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

~ ~o.

A A

. .~ . . ! ~.......... A A i : y �84 ;A ~ s ::A B

�9 ...,. ~ ....~ �9

~--/G GAC .... .... G S

1 : / .. ...... .:: A

. . . . . . . . ' - , ..,.

: c ~ A �9

f.J /

: ~I A /

; ~ ...if? .... A i:. : 1 . . .

�9 .A ........ i: . :. ~ . . q

o o

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 28!

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 distri- bution areas extend to southern islands such as Hachijo- jima, Yakushima, and Tokunoshima.

Of the four less common chemotypes, D-type char- acterized 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

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. oligoph- lebium [Matsumoto, unpublished data-I) suggest that B- type of A. normale and A. oligophlebium are different entities. Asplenium oligophlebium is morphologically rather similar to C-type of A. normale in having prominent- ly 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, D- type and A. shimurae are divided by further rhamnosyla- tion or glucosylation of apigenin and luteolin 7-O-rham- nosides (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'-O- glycosides, but did not have O-methylated flavonoid. If

fimplification

-luteolin

methylation

A. normale (2X) (hypothetic ancestral type)

rhamnosylation

+luteolin

glucosylation

R - O ~ O-R

HO 0 A. boreale (qX)

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i . . . . . . . . . . . . . . . . . . . . . . . . .

R O O R G ! ~

I HO o C- type i i

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G-type(5 and 8) . . . . . . . . . . . . . . . . . . . ~ - ; . . . . . . . . . ] i l I I

O-R-G E-type(I, 2 and 8)

lebium (2• F-type(I, 2, 5 and 8)

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

R O R O ~ i / OH I,,,, )"j, A-type (I, 2 and 5)

HO O I t OH R - O - R - o ' ~ O ~ ~ "OH ' i l l Intraspecific hybrids

" ~ 2 D-type A. normale (4X) HO 0 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ] L

G - O - R - O ~ o H

HO O OH

G - O - R - O ~ o H

HO O A. shimurae (4X)

C~glucosyl

H O ~ r , , / o ~ O H

c-gluc~ ~ J ~ ~'~ 3 HO O

c-giycosyl ~ O H H O ~ OH

c-glycosyl HO O

Fig. 5. Presumed flavonoid 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 con- sidered 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 sur- veys 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 Aku- zawa, 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. Memo- rial Rep. Shizuoka Coll. Pharm. 76-95 (in Japanese).

Harborne, J.B. 1966. The evolution of flavonoid pig- ments in plants. In T. Swain ed., Comparative Phyto- chemistry. 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 com- ments. 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 : 1629- 1630.

Kurata, S. and Nakaike, S. 1981. Illustrations of Pterido- phytes 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. Springer- Verlag, London. p. 3-184.

Matsumoto, S. and Nakaike, T. 1988. Chromosome num- bers 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)