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Studies on Epimedium Species: Flavonol glycosides and isozymes
BiochemicalSystematicsand Ecology,Vol. 19, No. 4, pp. 315-318, 1991. Printed in Great Britain.
0305-1978/91 $3.00+ 0.00 © 1991 PergamonPressplc.
Studies on EpimediumSpecies: Flavonol Glycosides and Isozymes S. KOGA, Y. SHOYAMA and I. NISHIOKA Faculty of Pharmaceutical Sciences, Kyushu University 3-1-I Maidashi, Higashi-ku, Fukuoka 812, Japan
Key Word Index--Epimedium species; Berberidaceae; chemotaxonomy; icariin; seasonal variation; isozyme; electrophoretic analysis. Abstract--Flavonol glycosides, icariin, epimedin B and C, contained in six Epimedium species were qualitatively and quantitatively analysed by HPLC. Significant differences in the flavonol glycoside content were found between species. The highest accumulation of flavonol glycoside occurred about two months after germination. Electmphoretic analyses of isozymes, phosphoglucomutase, phosphoglucose isomerase, glutamate oxaloacetate transaminase, 6-phosphogluconate dehydrogenase, isocitrate dehydrogenase and glutamate dehydrogenase, were performed. E. diphyllum and E. sagittatum can be distinguished from other species. It is evident from this analysis that E. setosum and E. grandiflorum vat. higoense are closely related to E. diphyllum, E. sempervirens and E. grandiflorum. These results show that the electrophoretic analysis of isozymes supports the morphological classification of Epimedium species in Japan.
Introduction The aerial parts of Epimedium species are used as a tonic in the Chinese drug "YinYang-huo" in Japan and China. Previously, the authors have reported on the micropropagation of Epimedium species by embryogenesis [1]. In that study, some differences in ability to regenerate were noted. For this reason a phytochemical study of Epimedium species was undertaken. Japanese Epimedium species are classified into three types by spur length of flowers, which can be used as a significant characteristic for species diagnosis [2]: spurless flower--E, diphyllum; short spurred flowers--E, trifoliatobinaturn, E. setosum ; long spurred flowers--E, grandiflorum, E. sernpervirens, E. grandiflorum vat. higoense. Histological characteristics and types of ramification of leaf have also been investigated previously [2-4]. From these results, it is evident that at least seven species of Epirnedium occur in Japan [5]. However, it is known that hybridization between species occurs, resulting in the diversification of populations of Epimedium species [6]. Morphological classification of Epimedium species is therefore still somewhat fluid. It is well-known that Epimedium species contain a range of flavonol glycosides like icariin [7-13]. Mizuno et aL [14, 15] have previously reported phytochemical studies. However, the relation between phytochemistry and morphology is still unclear. Although chromosome numbers are frequently used for the characterization of plant species or in arguments concerning plant taxonomy, the chromosome number in all Epimedium species is 2n = 12. Therefore, the analysis of chromosomes is not helpful for studying their taxonomy [16]. For these reasons, the study of chemotaxonomy including isozymes is needed. In this communication we wish to discuss the value, in the taxonomy of Epimedium species, of the chemical analysis of flavonol glycosides and several isozymes. Materials and Methods Plant materials. Epimedium diphyllum, E. sempervirens and E. setosum were collected from Shobara city in Hiroshima prefecture. E. grandiflorum var. higoense was collected from Sujiyu-machi in Oita prefecture. E. (Received3 December 1990) 315
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koreanum was donated from Mr S. Katsuki, herbal garden at Kitasato University. E. sagittatum and E. grandiflorum were donated from the herbal garden of Takeda Chemical Industries, Ltd. All species were identified morphologically [5], and cultivated in the herbal garden of the Faculty of Pharmaceutical Sciences, Kyushu University. Extraction and isolation of flavonoid glycoside. The aerial parts of E. diphyllum (594 g) were extracted with MeOH. The MeOH extract (79 g) was suspended in H20 and filtered. The filtrate was partitioned between EtOAC and n-BuOH, respectively. The n-BuOH extract (15 g) was repeatedly purified by column chromatography on Sephadex LH-20, MCI gel CHP-20 P and Fujigel ODS-G3 using a MeOH-H20 mixture to give icariin (450 rag), epimedin B (100 rag) and epimedin C (20 mg). Icariin [7], amorphous powder, mp 255-257=C, [0~]~9 = - 8 7 ° (MeOH, c = 1.0), UV ~'maxMeOH rim: 274, 315, 350. Negative FABMS (m/z): 677 (M--H)=, 513 (M--hexose) , 367 (M-hexose × 2) . 1H NMR (pyr-ds) 6:1.39 (3H, d, J = 4 Hz, rha-6), 1.60, 1.77 (each 3H, s, 14, 15-H), 3.71 (3H, s, OMe), 4.66 (1H, d, J = 7 . 5 Hz, glc-1), 5.09 (1H, bs, rha-1), 6.28 (1H, s, 6-H), 7.13, 8.17 (each 2H, d, J = 9 Hz, 3% 5"-, 2'-, 6'-H). 13C NMR (pyr-d 5) 6:18.1 (rha-1), 18.2 (C-15), 22.5 (C-11, 14), 55.4 (OMe), 62.2 (glc-6), 71.1 (glc-4), 71.9 (rha-5), 72.0 (rha-2), 72.4 (rha-3), 73.1 (rha-4), 74.8 (glc-2), 78.6 (glc-3), 78.9 (glc-5), 99.2 (C-6), 102.6 (rha-1), 103.8 (glc-1), 107.0 (C-8), 109.3 (C-10), 114.4 (C-3', 5'), 122.4 (C-12), 123.2 (C-1'), 131.2 (C-2', 6'), 131.4 (C-13), 135.8 (C-3), 153.9 (C-2), 157.7 (C-9), 160.6 (C-7), 161.8 (C-5), 162.2 (C-4"), 179.4 (C-4). Epimedin B [8, 11], amorphous powder, [¢]~9 = _72.5 ° (MeOH, c = 1.2), UV ~max M,OHrim.. 274, 315, 350. Negative FABMS (m/z): 809 (M--H)=. ~H NMR (pyr-ds) 6:1.39 (3H, d, J = 4 Hz, rha-6), 1.61, 1.78 (each 3H, s, C-14, 15-H), 3.75 (3H, s, OMe), 4.62 (1 H, d, J = 8 Hz, glc-1 ), 4.95 (1 H, bs, rha-1 ), 5.17 (1 H, d, J = 7.5 Hz, xyl-1 ), 6.27 (1 H, s, 6-H), 7.12, 8.17 (each 2H, d, J = 9 Hz, 3"-, 5% 2'-, 6'-Hi. ~3CNMR (pyr-ds) 6:18.1 (rha-1), 18.6 (C-15), 22.5 (C-11, 14), 55.5 (OMe), 62.2 (glc-6), 67.4 (xyl-5), 70.4 (xyl-4), 70.9 (glc-4), 71.7 (rha-5), 72.2 (rha-3), 73.4 (rha-4), 74.8 (glc-2), 75.5 (xyl-2), 78.3 (xyl-3), 78.6 (glc-3), 78.9 (glc-5), 81.8 (rha-2), 99.3 (C-6), 102.5 (rha-1), 103.5 (glc-1), 106.9 (C-8), 107.8 (xyl-1), 109.4 (C-10), 114.5 (C-3', 5'), 122.6 (C-12), 123.2 (C-1'), 131.2 (C-2', 6'), 131.5 (C-13), 135.7 (C-3), 153.9 (C-2), 157.7 (C-9), 160.6 (C-7), 161.8 (C-5), 162.3 (C-4'), 179.4 (C.4). Epimedin C [8, 11], amorphous powder, [¢]~9 = - 1 0 9 . 5 ° (MeOH, c=0.19). Negative FABMS (m/z): 823 (M-H) . ~H NMR (pyr-d~) 6: 1.39, 1.49 (each, 3H, d, J = 4 Hz, rha-6, 6'), 1.61, 1.75 (each 3H, s, C-14, 15-H), 3.78 (3H, s, OMe), 4.62 (1H, d, J = 7.5 Hz, glc-1), 4.95 (1H, bs, rha-l"), 5.57 (1H, bs, rha-1), 6.28 (1H, s, 6-H), 7.12, 8.18 (each, 2H, d, J = 9 Hz, 3'-, 5'-, 2'-, 6'-H). ~C NMR (pyr-ds) 6:18.1 (rha-1, 1'), 18.6 (C-15), 22.5 (C-11, 14), 55.5 (OMe), 62.2 (glc-6), 70.3 (rha-5'), 70.9 (glc-4), 71.7 (rha-5), 72.0 (rha-3"), 72.1 (rha-2'), 72.2 (rha-3), 73.2 (rha-4'), 73.4 (rha-4), 74.8 (glc-2), 78.6 (glc-3), 78.9 (glc-5), 81.8 (rha-2), 99.3 (C-6), 102.5 (rha-1), 103.4 (rha-1 '), 103.5 (glc-1), 106.9 (C-8), 109.4 (C-10), 114.5 (C-3', 5'), 122.6 (C-12), 123.2 (C-1'), 131.2 (C-2', 6'), 131.5 (C-13), 135.7 (C-3), 153.9 (C-2), 157.7 (C~9), 160.6 (C-7), 161.8 (C-5), 162.2 (C-4'), 179.4 (C-4). Analysis of flavonoid glycosides. The aerial parts of Epirnedium diphyllum, E. sagittatum, E. koreanum, E. grandiflorum var. higoense, E. sempervirens and E. setosum, grown in the herbal garden of the Faculty of Pharmaceutical Sciences, Kyushu University, were collected in August. The aerial parts of E. diphyllum were collected in April, May, June, August and November. Individual aerial parts were dried and powdered. The dried powder (100 mg) was extracted with H=O-MeCN (73:27; 5 ml) under sonication for 1 h. The residue was further extracted twice with H20-MeCN (73:27; 5 ml) under a 5-min sonication. 5-10 ~1 of the combined solution was injected onto the HPLC. Analyses of icariin, epimedin B and C were performed by the following methods: HPLC: Model 576 (Gasukuro Kogyo), detector: spectro detector 502T (detection; 270 nm, Gasukuro Kogyo) and photodiodearray (Shimadzu SPD-MIA), column: TSK-gel ODS 80T (4.6 × 250 mm), solvent: H20MeCN (73:27; 0.5 ml min-~). Analysis of isozyme. Two leaves of individual Epimedium species, grown in Khyushu University's herbal garden, were collected, washed with tap water and then with distilled water. The leaves were homogenized with the extraction buffer and sea sand, and PVP was added and mixed. The crude enzyme solution was absorbed on filter paper (2.5 × 11 mm). Gels I and II were prepared as previously reported [17, 18] and stored in a refrigerator until use. Gel I was run at 65 mA at 0°C for 5.2 h. Gel II was run at 52 mA, 200 V at 0°C for 6 h. Individual gels were sliced into 2-mm sections. Gel I containing glutamate dehydrogenase and glutamate oxaloacetate transaminase (GDH and GOT) and gel II containing isocitrate dehydrogenase, 6-phosphogluconate dehydrogenase, phosphoglucose isomerase and phosphoglucomutase (IDH, 6-PGD, PGI and PGM) were stained by previously reported methods [17, 18] at 37°C for 1 h, respectively.
Results and Discussion The relative contents of icariin, epimedin B and C contained in the six Epimedium species are shown in Table 1. These flavonol glycosides were absent from E. sagittatum, and E. sempervirens, E. setosum and E. diphyllum contained only small amounts. The highest content was found in E. koreanum and E. grandiflorum vat.
higoense. The seasonal variation of these flavonol glycosides in E. diphyllum is shown in Table 2. From these results, the content of flavonol glycoside was found to be highest in April and May, decreasing in June and August. Epimedin B and icariin almost disappeared in November. This phenomenon suggests that Epimedium species should be
F L A V O N O L G L Y C O S I D E S OF EPIMEDIUM
317
T A B L E 1. RELATIVE C O N T E N T S OF F L A V O N O L G L Y C O S I D E S IN EPIMEDIUM SPECIES*
E. diphyllyum E. sagittatum E. koreanum E. grandiflorum var. higoense E. sempervirens E. setosum
Epimedin B
Epimedin C
Icariin
+ +++ ++ + +
+ ++ + +_ +_
+ +++ +++ ± ±
*Compared to the flavonol glycoside contents of E. diphyllum.
T A B L E 2. S E A S O N A L VARIATION OF F L A V O N O L GLYCOSIDE C O N T E N T S IN E. DIPHYLLUM
Epimedin B April May June August November
Epimedin C
Icariin
+++
+4-+
+++
+++
+++
+++
+
+
+
+
+
+
±
+
_+
harvested within two months of germination. Although the relationship of flavonol glycosides in Epimedium species and the pharmacological activity is obscure, it seems that other components may also be much higher in the younger leaves. Previously icariin, epimedin A, B and C have been reported [15] from E. sagittatum, contrary to this investigation. These results suggest that there are quantitative and qualitative variations in Epimedium species. Therefore, it may be difficult to employ these flavonoids in the chemotaxonomy of Epimedium species. Isozyme analysis is widely employed for the classification and/or identification of plant species as well as ribulose 1,6-diphosphate decarboxylase (R1 protein) [19-21]. Figure 1 shows the electrophoretic patterns of isozymes of Epimedium species. Epimedium diphyllum was easily distinguished from the other species by the comparisons of IDH, GDH, PGI and PGM. E. sagittatum, which is indigenous to China, also had a unique electrophoretic pattern. Therefore, these two species can easily be distinguished from other species. This is in good agreement with the morphological classification [5]. E. setosum is, in part of its range, sympatric with E. diphyllurn and E. sempervirens, in the narrow part of Hiroshima prefecture in Japan. E. setosurn seems to be a hybrid of E. diphyllum and E. sempervirens [5]. This suggestion was supported by the electrophoretic patterns of 6-PGD and GOT indicating that E. setosum is closely related to E. diphyllum and E. sempervirens. This agrees with the finding of Suzuki [6] who found that they crossed easily with each other. The authors also found the same
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1 E d~phyllum 2. ~ ~t~um 3 E. s ~ e ~ ~ E.gr~i~ru~
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FIG. 1. ELECTROPHORETIC PATTERNS OF LEAF S A M P L E S OF EPIMEDIUMSPECIES A F T E R S T A I N I N G BY V A R I O U S E N Z Y M E S .
318
S. KOGA ETAL.
result in the field e x p e r i m e n t s regarding the crossing of E. diphyllum and E. sempervirens [Shoyama, Y., unpublished results]. It is also suggested that E. grandiflorum var. higoense is a hybrid of E. diphyllum and E. grandiflorum as E. setosum is of E. diphyllum and E. sempervirens, because E. grandiflorum grows t o g e t h e r with E. diphyllum in the same area in Kyushu as pointed out by Suzuki [22]. This a r g u m e n t was s u p p o r t e d by the c o m p a r i s o n of GDH and PGM patterns w h i c h w e r e almost the same as those of E. grandiflorum var. higoense and E. grandiflorum. Furthermore, the patterns of PGI and 6-PGD proved to be intermediate between those of E. grandiflorum and E. diphyllum. E. grandiflorum var. higoense is m o r p h o l o g i c a l l y similar to E. setosum and E. trifoliatobinatum (not discussed in this study) except that E. grand/florum var. higoense has persistent hairs on the upper surface of the leaves [3]. As Suzuki pointed out [6], since Epirnedium species easily hybridized, these three species m a y be intermediate. Therefore, E. grandiflorum var. higoense should be regarded as a species at the same level if E. setosum and E. trifoliatobinatum are treated as individual species. Since the classification and identification of E p i m e d i u m species by f l a v o n o i d c o m p o n e n t s is unclear, as already noted, the electrophoretic analysis m a y be a useful means for the c h e m o t a x o n o m i c a l classification of Epirnedium species.
References 1. Koga, S., Shoyama,Y. and Nishioka, I. (1991) PlantCell. Tiss. Organ Cult, (in press). 2. Suzuki, K. (1978)J. Jap. Bot. 53, 203. 3. Shimizu,T. (1960)Acta Phytotax. Geobot. 18, 117. 4. Suzuki, K. (1981)J. Jap. Bot. 56, 9. 5. Kitamura, S. and Murata, G. (1979) Coloured Illustration of Herbaceous Plants of Japan II (Choripetalae), p. 201. Hoikusha,Osaka. 6. Suzuki, K. (1983) Bot. Mag. Tokyo96, 343. 7. Takemoto,T., Daigo, K. and Tokuoka,Y. (1975) YakugakuZasshi95, 312. 8. Oshima,Y., Okamoto, M. and Hikino, H. (1987) Heterocycles26, 935. 9. Miyase,T., Ueno, A., Takizawa,N., Kobayashi,H. and Karasawa,H. (1987) Chem. Pharm. Bull. 35, 1109. 10. Mizuno, M., Hanioka, S., Suzuki, N., linuma, M., Tanaka, T., Xin-shun, L. and Zhi-da, M. (1987) Phytochemistry 26, 861. 11. Ito, Y., Hirayama,F., Suto, K., Sagara,K. and Yoshida,T. (1988) Phytochemistry27, 911. 12. Fukai,T. and Nomura, T. (1988) Phytochemistry27, 259. 13. Mizuno, M., linuma, M., Tanaka, T., Sakakibara, N., Nakanishi,T., Inada, A. and Nishi, M. (1989) Chem. Pharm. Bull. 37, 2241. 14. Mizuno, M. (1987)The 1st InternationalSymposium of Chinese Drugs, Abstract, p. 11. 15. Mizuno, M., Sakakibara,N., linuma, M., Tanaka,T., Xin-shun, L., Hanioka,H. and Tomoda,Y. (1987)The 34th Annual Meeting of the JapaneseSociety of Pharmacognosy,Abstract, p. 83. 16. Koyama,H. (1965)Acta Phytotax. Geobot. 21, 69. 17. Shaw, C. R. and Prasad,R. (1970) Biochem. Genet. 4, 297. 18. Vallejos, E. (1983) in Isozymes in Plant Genetics and Breeding (Tanksley,S. D. and Orton, T. J., eds), pp. 469-516. Part A. Elsevier,Oxford. 19. Chert, K., Kung, S. D., Gray, J. C. and Wildman, S. G. (1976) Plant Sci. Le~ 7, 429. 20. Strobaek,S., Gibbons,G. S., Haslett, B., Bouiter,D. and Wildman, S. G. (1976) Garlsberg Res. Commun. 41, 335. 21. Rick,C. M., Kesicki, E., Fober,J. F. and Holle, M. (1976) Theor App/. Genet. 47, 55. 22. Suzuki, K. (1981)J. Jap. Bot. 56, 33.
0305-1978/91 $3.00+ 0.00 © 1991 PergamonPressplc.
Studies on EpimediumSpecies: Flavonol Glycosides and Isozymes S. KOGA, Y. SHOYAMA and I. NISHIOKA Faculty of Pharmaceutical Sciences, Kyushu University 3-1-I Maidashi, Higashi-ku, Fukuoka 812, Japan
Key Word Index--Epimedium species; Berberidaceae; chemotaxonomy; icariin; seasonal variation; isozyme; electrophoretic analysis. Abstract--Flavonol glycosides, icariin, epimedin B and C, contained in six Epimedium species were qualitatively and quantitatively analysed by HPLC. Significant differences in the flavonol glycoside content were found between species. The highest accumulation of flavonol glycoside occurred about two months after germination. Electmphoretic analyses of isozymes, phosphoglucomutase, phosphoglucose isomerase, glutamate oxaloacetate transaminase, 6-phosphogluconate dehydrogenase, isocitrate dehydrogenase and glutamate dehydrogenase, were performed. E. diphyllum and E. sagittatum can be distinguished from other species. It is evident from this analysis that E. setosum and E. grandiflorum vat. higoense are closely related to E. diphyllum, E. sempervirens and E. grandiflorum. These results show that the electrophoretic analysis of isozymes supports the morphological classification of Epimedium species in Japan.
Introduction The aerial parts of Epimedium species are used as a tonic in the Chinese drug "YinYang-huo" in Japan and China. Previously, the authors have reported on the micropropagation of Epimedium species by embryogenesis [1]. In that study, some differences in ability to regenerate were noted. For this reason a phytochemical study of Epimedium species was undertaken. Japanese Epimedium species are classified into three types by spur length of flowers, which can be used as a significant characteristic for species diagnosis [2]: spurless flower--E, diphyllum; short spurred flowers--E, trifoliatobinaturn, E. setosum ; long spurred flowers--E, grandiflorum, E. sernpervirens, E. grandiflorum vat. higoense. Histological characteristics and types of ramification of leaf have also been investigated previously [2-4]. From these results, it is evident that at least seven species of Epirnedium occur in Japan [5]. However, it is known that hybridization between species occurs, resulting in the diversification of populations of Epimedium species [6]. Morphological classification of Epimedium species is therefore still somewhat fluid. It is well-known that Epimedium species contain a range of flavonol glycosides like icariin [7-13]. Mizuno et aL [14, 15] have previously reported phytochemical studies. However, the relation between phytochemistry and morphology is still unclear. Although chromosome numbers are frequently used for the characterization of plant species or in arguments concerning plant taxonomy, the chromosome number in all Epimedium species is 2n = 12. Therefore, the analysis of chromosomes is not helpful for studying their taxonomy [16]. For these reasons, the study of chemotaxonomy including isozymes is needed. In this communication we wish to discuss the value, in the taxonomy of Epimedium species, of the chemical analysis of flavonol glycosides and several isozymes. Materials and Methods Plant materials. Epimedium diphyllum, E. sempervirens and E. setosum were collected from Shobara city in Hiroshima prefecture. E. grandiflorum var. higoense was collected from Sujiyu-machi in Oita prefecture. E. (Received3 December 1990) 315
316
S. KOGA ETAL.
koreanum was donated from Mr S. Katsuki, herbal garden at Kitasato University. E. sagittatum and E. grandiflorum were donated from the herbal garden of Takeda Chemical Industries, Ltd. All species were identified morphologically [5], and cultivated in the herbal garden of the Faculty of Pharmaceutical Sciences, Kyushu University. Extraction and isolation of flavonoid glycoside. The aerial parts of E. diphyllum (594 g) were extracted with MeOH. The MeOH extract (79 g) was suspended in H20 and filtered. The filtrate was partitioned between EtOAC and n-BuOH, respectively. The n-BuOH extract (15 g) was repeatedly purified by column chromatography on Sephadex LH-20, MCI gel CHP-20 P and Fujigel ODS-G3 using a MeOH-H20 mixture to give icariin (450 rag), epimedin B (100 rag) and epimedin C (20 mg). Icariin [7], amorphous powder, mp 255-257=C, [0~]~9 = - 8 7 ° (MeOH, c = 1.0), UV ~'maxMeOH rim: 274, 315, 350. Negative FABMS (m/z): 677 (M--H)=, 513 (M--hexose) , 367 (M-hexose × 2) . 1H NMR (pyr-ds) 6:1.39 (3H, d, J = 4 Hz, rha-6), 1.60, 1.77 (each 3H, s, 14, 15-H), 3.71 (3H, s, OMe), 4.66 (1H, d, J = 7 . 5 Hz, glc-1), 5.09 (1H, bs, rha-1), 6.28 (1H, s, 6-H), 7.13, 8.17 (each 2H, d, J = 9 Hz, 3% 5"-, 2'-, 6'-H). 13C NMR (pyr-d 5) 6:18.1 (rha-1), 18.2 (C-15), 22.5 (C-11, 14), 55.4 (OMe), 62.2 (glc-6), 71.1 (glc-4), 71.9 (rha-5), 72.0 (rha-2), 72.4 (rha-3), 73.1 (rha-4), 74.8 (glc-2), 78.6 (glc-3), 78.9 (glc-5), 99.2 (C-6), 102.6 (rha-1), 103.8 (glc-1), 107.0 (C-8), 109.3 (C-10), 114.4 (C-3', 5'), 122.4 (C-12), 123.2 (C-1'), 131.2 (C-2', 6'), 131.4 (C-13), 135.8 (C-3), 153.9 (C-2), 157.7 (C-9), 160.6 (C-7), 161.8 (C-5), 162.2 (C-4"), 179.4 (C-4). Epimedin B [8, 11], amorphous powder, [¢]~9 = _72.5 ° (MeOH, c = 1.2), UV ~max M,OHrim.. 274, 315, 350. Negative FABMS (m/z): 809 (M--H)=. ~H NMR (pyr-ds) 6:1.39 (3H, d, J = 4 Hz, rha-6), 1.61, 1.78 (each 3H, s, C-14, 15-H), 3.75 (3H, s, OMe), 4.62 (1 H, d, J = 8 Hz, glc-1 ), 4.95 (1 H, bs, rha-1 ), 5.17 (1 H, d, J = 7.5 Hz, xyl-1 ), 6.27 (1 H, s, 6-H), 7.12, 8.17 (each 2H, d, J = 9 Hz, 3"-, 5% 2'-, 6'-Hi. ~3CNMR (pyr-ds) 6:18.1 (rha-1), 18.6 (C-15), 22.5 (C-11, 14), 55.5 (OMe), 62.2 (glc-6), 67.4 (xyl-5), 70.4 (xyl-4), 70.9 (glc-4), 71.7 (rha-5), 72.2 (rha-3), 73.4 (rha-4), 74.8 (glc-2), 75.5 (xyl-2), 78.3 (xyl-3), 78.6 (glc-3), 78.9 (glc-5), 81.8 (rha-2), 99.3 (C-6), 102.5 (rha-1), 103.5 (glc-1), 106.9 (C-8), 107.8 (xyl-1), 109.4 (C-10), 114.5 (C-3', 5'), 122.6 (C-12), 123.2 (C-1'), 131.2 (C-2', 6'), 131.5 (C-13), 135.7 (C-3), 153.9 (C-2), 157.7 (C-9), 160.6 (C-7), 161.8 (C-5), 162.3 (C-4'), 179.4 (C.4). Epimedin C [8, 11], amorphous powder, [¢]~9 = - 1 0 9 . 5 ° (MeOH, c=0.19). Negative FABMS (m/z): 823 (M-H) . ~H NMR (pyr-d~) 6: 1.39, 1.49 (each, 3H, d, J = 4 Hz, rha-6, 6'), 1.61, 1.75 (each 3H, s, C-14, 15-H), 3.78 (3H, s, OMe), 4.62 (1H, d, J = 7.5 Hz, glc-1), 4.95 (1H, bs, rha-l"), 5.57 (1H, bs, rha-1), 6.28 (1H, s, 6-H), 7.12, 8.18 (each, 2H, d, J = 9 Hz, 3'-, 5'-, 2'-, 6'-H). ~C NMR (pyr-ds) 6:18.1 (rha-1, 1'), 18.6 (C-15), 22.5 (C-11, 14), 55.5 (OMe), 62.2 (glc-6), 70.3 (rha-5'), 70.9 (glc-4), 71.7 (rha-5), 72.0 (rha-3"), 72.1 (rha-2'), 72.2 (rha-3), 73.2 (rha-4'), 73.4 (rha-4), 74.8 (glc-2), 78.6 (glc-3), 78.9 (glc-5), 81.8 (rha-2), 99.3 (C-6), 102.5 (rha-1), 103.4 (rha-1 '), 103.5 (glc-1), 106.9 (C-8), 109.4 (C-10), 114.5 (C-3', 5'), 122.6 (C-12), 123.2 (C-1'), 131.2 (C-2', 6'), 131.5 (C-13), 135.7 (C-3), 153.9 (C-2), 157.7 (C~9), 160.6 (C-7), 161.8 (C-5), 162.2 (C-4'), 179.4 (C-4). Analysis of flavonoid glycosides. The aerial parts of Epirnedium diphyllum, E. sagittatum, E. koreanum, E. grandiflorum var. higoense, E. sempervirens and E. setosum, grown in the herbal garden of the Faculty of Pharmaceutical Sciences, Kyushu University, were collected in August. The aerial parts of E. diphyllum were collected in April, May, June, August and November. Individual aerial parts were dried and powdered. The dried powder (100 mg) was extracted with H=O-MeCN (73:27; 5 ml) under sonication for 1 h. The residue was further extracted twice with H20-MeCN (73:27; 5 ml) under a 5-min sonication. 5-10 ~1 of the combined solution was injected onto the HPLC. Analyses of icariin, epimedin B and C were performed by the following methods: HPLC: Model 576 (Gasukuro Kogyo), detector: spectro detector 502T (detection; 270 nm, Gasukuro Kogyo) and photodiodearray (Shimadzu SPD-MIA), column: TSK-gel ODS 80T (4.6 × 250 mm), solvent: H20MeCN (73:27; 0.5 ml min-~). Analysis of isozyme. Two leaves of individual Epimedium species, grown in Khyushu University's herbal garden, were collected, washed with tap water and then with distilled water. The leaves were homogenized with the extraction buffer and sea sand, and PVP was added and mixed. The crude enzyme solution was absorbed on filter paper (2.5 × 11 mm). Gels I and II were prepared as previously reported [17, 18] and stored in a refrigerator until use. Gel I was run at 65 mA at 0°C for 5.2 h. Gel II was run at 52 mA, 200 V at 0°C for 6 h. Individual gels were sliced into 2-mm sections. Gel I containing glutamate dehydrogenase and glutamate oxaloacetate transaminase (GDH and GOT) and gel II containing isocitrate dehydrogenase, 6-phosphogluconate dehydrogenase, phosphoglucose isomerase and phosphoglucomutase (IDH, 6-PGD, PGI and PGM) were stained by previously reported methods [17, 18] at 37°C for 1 h, respectively.
Results and Discussion The relative contents of icariin, epimedin B and C contained in the six Epimedium species are shown in Table 1. These flavonol glycosides were absent from E. sagittatum, and E. sempervirens, E. setosum and E. diphyllum contained only small amounts. The highest content was found in E. koreanum and E. grandiflorum vat.
higoense. The seasonal variation of these flavonol glycosides in E. diphyllum is shown in Table 2. From these results, the content of flavonol glycoside was found to be highest in April and May, decreasing in June and August. Epimedin B and icariin almost disappeared in November. This phenomenon suggests that Epimedium species should be
F L A V O N O L G L Y C O S I D E S OF EPIMEDIUM
317
T A B L E 1. RELATIVE C O N T E N T S OF F L A V O N O L G L Y C O S I D E S IN EPIMEDIUM SPECIES*
E. diphyllyum E. sagittatum E. koreanum E. grandiflorum var. higoense E. sempervirens E. setosum
Epimedin B
Epimedin C
Icariin
+ +++ ++ + +
+ ++ + +_ +_
+ +++ +++ ± ±
*Compared to the flavonol glycoside contents of E. diphyllum.
T A B L E 2. S E A S O N A L VARIATION OF F L A V O N O L GLYCOSIDE C O N T E N T S IN E. DIPHYLLUM
Epimedin B April May June August November
Epimedin C
Icariin
+++
+4-+
+++
+++
+++
+++
+
+
+
+
+
+
±
+
_+
harvested within two months of germination. Although the relationship of flavonol glycosides in Epimedium species and the pharmacological activity is obscure, it seems that other components may also be much higher in the younger leaves. Previously icariin, epimedin A, B and C have been reported [15] from E. sagittatum, contrary to this investigation. These results suggest that there are quantitative and qualitative variations in Epimedium species. Therefore, it may be difficult to employ these flavonoids in the chemotaxonomy of Epimedium species. Isozyme analysis is widely employed for the classification and/or identification of plant species as well as ribulose 1,6-diphosphate decarboxylase (R1 protein) [19-21]. Figure 1 shows the electrophoretic patterns of isozymes of Epimedium species. Epimedium diphyllum was easily distinguished from the other species by the comparisons of IDH, GDH, PGI and PGM. E. sagittatum, which is indigenous to China, also had a unique electrophoretic pattern. Therefore, these two species can easily be distinguished from other species. This is in good agreement with the morphological classification [5]. E. setosum is, in part of its range, sympatric with E. diphyllurn and E. sempervirens, in the narrow part of Hiroshima prefecture in Japan. E. setosurn seems to be a hybrid of E. diphyllum and E. sempervirens [5]. This suggestion was supported by the electrophoretic patterns of 6-PGD and GOT indicating that E. setosum is closely related to E. diphyllum and E. sempervirens. This agrees with the finding of Suzuki [6] who found that they crossed easily with each other. The authors also found the same
PGI _-.... . . . . . .
_
GOT ~ D
E~-
~
mc
. . . . . . .
IDH
so
--
PGM (Phospltoglu~ornutase) PG[ [~f~;~ohogluco~e~ e ~ e ) GOT (Glu=~te o ~ l ~ t e tr ~ i n ~ e ) ~D { ~ t e d~r~en~e) IDH [Is~rate d e ~ r ~ n ~ ) GDH {Glutam=ed ~ r ~ e )
1 E d~phyllum 2. ~ ~t~um 3 E. s ~ e ~ ~ E.gr~i~ru~
_.. _
.........
E 4°
~
E 30 -
-
-~ ~ ~ ~_
•=:: -- = __-.= .
.
.
.
.
;
~ ~ GDH ~ E
.
~ ~ : I0
--
. . . .
s~. t he~~~m , ~ S. ~ ~ , ~ t , m
-- O ~ r ~
Uncl~r ~
Br~d
t 3
I ~ 5 O
~
I
I
I
~
k
2 3 4 ~ e
I
~
~
~
~
I
1 2 3 4 S e
FIG. 1. ELECTROPHORETIC PATTERNS OF LEAF S A M P L E S OF EPIMEDIUMSPECIES A F T E R S T A I N I N G BY V A R I O U S E N Z Y M E S .
318
S. KOGA ETAL.
result in the field e x p e r i m e n t s regarding the crossing of E. diphyllum and E. sempervirens [Shoyama, Y., unpublished results]. It is also suggested that E. grandiflorum var. higoense is a hybrid of E. diphyllum and E. grandiflorum as E. setosum is of E. diphyllum and E. sempervirens, because E. grandiflorum grows t o g e t h e r with E. diphyllum in the same area in Kyushu as pointed out by Suzuki [22]. This a r g u m e n t was s u p p o r t e d by the c o m p a r i s o n of GDH and PGM patterns w h i c h w e r e almost the same as those of E. grandiflorum var. higoense and E. grandiflorum. Furthermore, the patterns of PGI and 6-PGD proved to be intermediate between those of E. grandiflorum and E. diphyllum. E. grandiflorum var. higoense is m o r p h o l o g i c a l l y similar to E. setosum and E. trifoliatobinatum (not discussed in this study) except that E. grand/florum var. higoense has persistent hairs on the upper surface of the leaves [3]. As Suzuki pointed out [6], since Epirnedium species easily hybridized, these three species m a y be intermediate. Therefore, E. grandiflorum var. higoense should be regarded as a species at the same level if E. setosum and E. trifoliatobinatum are treated as individual species. Since the classification and identification of E p i m e d i u m species by f l a v o n o i d c o m p o n e n t s is unclear, as already noted, the electrophoretic analysis m a y be a useful means for the c h e m o t a x o n o m i c a l classification of Epirnedium species.
References 1. Koga, S., Shoyama,Y. and Nishioka, I. (1991) PlantCell. Tiss. Organ Cult, (in press). 2. Suzuki, K. (1978)J. Jap. Bot. 53, 203. 3. Shimizu,T. (1960)Acta Phytotax. Geobot. 18, 117. 4. Suzuki, K. (1981)J. Jap. Bot. 56, 9. 5. Kitamura, S. and Murata, G. (1979) Coloured Illustration of Herbaceous Plants of Japan II (Choripetalae), p. 201. Hoikusha,Osaka. 6. Suzuki, K. (1983) Bot. Mag. Tokyo96, 343. 7. Takemoto,T., Daigo, K. and Tokuoka,Y. (1975) YakugakuZasshi95, 312. 8. Oshima,Y., Okamoto, M. and Hikino, H. (1987) Heterocycles26, 935. 9. Miyase,T., Ueno, A., Takizawa,N., Kobayashi,H. and Karasawa,H. (1987) Chem. Pharm. Bull. 35, 1109. 10. Mizuno, M., Hanioka, S., Suzuki, N., linuma, M., Tanaka, T., Xin-shun, L. and Zhi-da, M. (1987) Phytochemistry 26, 861. 11. Ito, Y., Hirayama,F., Suto, K., Sagara,K. and Yoshida,T. (1988) Phytochemistry27, 911. 12. Fukai,T. and Nomura, T. (1988) Phytochemistry27, 259. 13. Mizuno, M., linuma, M., Tanaka, T., Sakakibara, N., Nakanishi,T., Inada, A. and Nishi, M. (1989) Chem. Pharm. Bull. 37, 2241. 14. Mizuno, M. (1987)The 1st InternationalSymposium of Chinese Drugs, Abstract, p. 11. 15. Mizuno, M., Sakakibara,N., linuma, M., Tanaka,T., Xin-shun, L., Hanioka,H. and Tomoda,Y. (1987)The 34th Annual Meeting of the JapaneseSociety of Pharmacognosy,Abstract, p. 83. 16. Koyama,H. (1965)Acta Phytotax. Geobot. 21, 69. 17. Shaw, C. R. and Prasad,R. (1970) Biochem. Genet. 4, 297. 18. Vallejos, E. (1983) in Isozymes in Plant Genetics and Breeding (Tanksley,S. D. and Orton, T. J., eds), pp. 469-516. Part A. Elsevier,Oxford. 19. Chert, K., Kung, S. D., Gray, J. C. and Wildman, S. G. (1976) Plant Sci. Le~ 7, 429. 20. Strobaek,S., Gibbons,G. S., Haslett, B., Bouiter,D. and Wildman, S. G. (1976) Garlsberg Res. Commun. 41, 335. 21. Rick,C. M., Kesicki, E., Fober,J. F. and Holle, M. (1976) Theor App/. Genet. 47, 55. 22. Suzuki, K. (1981)J. Jap. Bot. 56, 33.