Flavonol glycosides from
Asplenium foreziense
and
its
"
ve related taxa and
A. incisum
Tsukasa Iwashina
!,
*
, Jose
H
A. Lo
H
pez-Sa
H
ez
"
, Alberto Herrero
#
,
Junichi Kitajima
$
, Sadamu Matsumoto
!
!Tsukuba Botanical Garden, National Science Museum, Tsukuba, Ibaraki 305-0005, Japan
"Laboratorio de Arqueobotanica, Centro de Estudios Historicos, CSIC, Duque de Medinaceli, 8, Madrid 28014, Spain
#Departamento de Biologia Vegetal I, Facultad de Biologia, Universidad Complutense, Madrid 28040, Spain
$Laboratory of Pharmacognosy, Showa College of Pharmaceutical Sciences, Machida, Tokyo 194-8543, Japan
Received 22 June 1999; accepted 11 August 1999
Abstract
The#avonoids ofAsplenium foreziense,A. fontanumsubsp.fontanumand subsp. pseudofon-tanum,A. obovatumsubsp.obovatumvar.obovatumand var.protobillotii,A. obovatumsubsp.
lanceolatum, andA. incisumwere isolated and identi"ed for chemotaxonomic survey. A major constituent of all taxa was kaempferol 3-O-gentiobioside. As minor compounds, kaempferol 3,7-O-glycoside and/or kaempferol 3-O-glycoside were found inA. fontanum,A. obovatumand
A. foreziense, and kaempferol 3-O-gentiobioside-4@-O-glucoside, kaempferol 3-O-glucoside and quercetin 3-O-diglucoside inA. incisum. It was suggested thatA. foreziense,A. fontanumincluding subsp.pseudofontanumandA. obovatumincluding subsp.lanceolatumare not only
morphologi-cally but also chemotaxonomimorphologi-cally related. The East Asian A. incisumwas chemically and
geographically di!erent from these taxa. ( 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Asplenium fontanum; Asplenium foreziense; Asplenium incisum; Asplenium obovatum; Aspleniaceae; kaempferol 3-O-gentiobioside; kaempferol 3-O-gentiobioside-4@-O-glucoside
1. Introduction
Asplenium forezienseLegrand ex HeHrib. is an allotetraploid derived by polyploidiz-ation after hybridizpolyploidiz-ation of two diploids, A. obovatum Viv. subsp. obovatum and
*Corresponding author. Tel.:#81-298-51-5159; fax:#81-298-53-8998.
E-mail address:iwashina@kahaku.go.jp (T. Iwashina)
Table 1
The ploidies and distributions ofAspleniumtaxa examined
Taxa Ploidies Distributions
A. obovatumsubsp.obovatum Diploid (n"36) The Mediterranean region, Canary Islands
A. obovatumsubsp.lanceolatum Tetraploid (n"72) derived from
A. obovatumsubsp.obovatum
Atlantic Europe, the west Mediterranean region, Macaronesian Islands
A. obovatumvar.protobillotii ? Turkey, Greece, Spain
A. foreziense Allotetraploid (n"72) fromA. obovatum
subsp.obovatumandA. fontanum
Southwestern Europe, Morocco
A. fontanumsubsp.fontanum Diploid (n"36) South and central Europe
A. fontanumsubsp.
pseudofontanum
Diploid (n"36) West Asia
A. incisum Diploid (n"36) East Asia, Japan
A. fontanum(L.) Bernh. (Sleep, 1967,1983).A. obovatumViv. subsp.lanceolatum(Fiori) P. Silva is a tetraploid derived from the diploidA. obovatumsubsp.obovatum(Table 1). A. foreziense is distributed in southwestern Europe and Morocco (Marchetti, 1995; Rumsey and Vogel, 1996) while A. obovatumsubsp. obovatum var. obovatum is native to the Mediterranean region and Canary Islands (Pichi Sermolli and Bizarri, 1992). A. obovatum var. protobillotii Demiriz, Viane & Reichst. is native to Turkey, Greece and Spain (Demiriz et al., 1990; Rasbach et al., 1990; Viane et al., 1996) while A. obovatum subsp. lanceolatum is distributed mainly in Atlantic Europe, the west Mediterranean region and Macaronesian Islands. A. fontanum (L.) Bernh. is widespread from south and central Europe to west Asia (Tutin et al., 1993), and is distinguishable as subsp. pseudofontanum (Koss.) Reichst. and Schneller in west Asia (Reichstein and Schneller, 1982; Nakaike and Malik, 1992). A. incisum Thunb. is native to east Asia including Japan (Nakaike, 1992). In previous papers, kaempferol 3-O-gentiobioside and kaemp-ferol 3-O-(gentiobioside 6@-sulfate) have been reported from A. fontanum (Imperato, 1980). The#avonoid compositions of other taxa have not previously been surveyed.
In a series of chemotaxonomic studies of the genusAsplenium, we have surveyed the
#avonoid compositions ofA. normaleD. Don and four related species (Iwashina and Matsumoto, 1990,1994; Iwashina et al., 1990,1993b),A. dalhousiae Hook. and two related species (Iwashina et al., 1993a) andA. trichomanes-ramosumL. ("A. viride Huds.) (Iwashina et al., 1995).
2. Materials and methods
2.1. Plant materials
Asplenium fontanum(L.) Bernh. subsp.fontanum(diploid,n"36) was collected in Hoz de Beteta, Cuenca, Spain, at an elevation of 1100 m, A. fontanum subsp. pseudofontanum (Koss.) Reichst. & Schneller in Shogran, Mansehra Dist., NWFP Prov., Pakistan, at an elevation of 2300 m (TNS 9507321), Kund, near Sharan Forest, Mansehra Dist., NWFP Prov., Pakistan, at an elevation of 1800 m (TNS 9507322) and Sikyan, near Nadi, Mansehra Dist., NWFP Prov., Pakistan, at an elevation of 2000 m (TNS 9507323) during the botanical expedition to Pakistan in 1991, A. foreziense Legrand ex HeHrib. (allotetraploid, n"72) in Sierra de los Filabres, GeHrgal, AlmermHa, Spain, at an elevation of 1700 m, A. incisumThunb. in Mt. Esan, Oshima, Hokkaido, Japan, Miyaji, Ichinomiya, Kumamoto Pref., Japan (TNS 9508062) and Higashibara, Ichinomiya, Yamanashi Pref., Japan, A. obovatum Viv. subsp.lanceolatum(Fiori) P. Silva ("A. billotiiF. W. Schultz) (tetraploid,n"72) in Miel Valley, Algeciras, CaHdiz, Spain, A. obovatum subsp. obovatum var. obovatum (diploid,n"36) in Cap du Dramont, Le Dramont, Dept. Var, France, at an elevation of 30 m, andA. obovatumsubsp.obovatumvar.protobillotiiDemiriz, Viane & Reichst. in Miel Valley, Algeciras, CaHdiz, Spain, at an elevation of ca. 200 m (TNS 9508557). Voucher specimens were deposited in the National Science Museum (Japan), Tsukuba (TNS) and in the MACB Herbarium (Faculty of Biology, Complutense University, Madrid).
2.2. Isolation ofyavonoids
Fresh or dry fronds ofAsplenium foreziense,A. fontanumsubsp.fontanumand subsp. pseudofontanum(3 populations),A. obovatumsubsp.obovatumvar.obovatumand var. protobillotii, A. obovatum subsp. lanceolatum, and A. incisum (3 populations) were extracted with MeOH. All extracts, except those ofA. fontanumsubsp.pseudofontanum andA. incisum, were found to have the same#avonoid pro"les by 2D-PC and HPLC. These extracts were combined, concentrated and applied to preparative paper chromatography (PPC) using solvent systems: BAW (n-BuOH/HOAc/H
2O"
4 : 1 : 5, upper phase), 15% HOAc and BEW (n-BuOH/EtOH/H
2O"4 : 1 : 2.2).
Extracts ofA. fontanumsubsp.pseudofontanum andA. incisumwere also applied to PPC. Each isolated#avonoid was puri"ed by Sephadex LH-20 column chromatogra-phy (solvent system: 70% MeOH).
2.3. Identixcation ofyavonoids
The#avonoids were identi"ed by UV,1H and13C NMR, and FAB-mass spectra, complete and mild acid hydrolysis, and direct PC and HPLC comparisons with authentic specimens.
Kaempferol 3-O-gentiobioside. PC: Rf 0.36 (BAW), 0.49 (BEW), 0.52 (15% HOAc), 0.39 (5% HOAc); UV-dark purple, UV/NH
nm: MeOH 266, 296sh, 348;#NaOMe 275, 326, 400 (inc.);#AlCl J"7.9 Hz, glucosyl anomeric H).13C NMR (125 MHz, pyridine-d
5):d(kaempferol)
Kaempferol 3-O-gentiobioside-4@-O-glucoside. PC: Rf 0.11 (BAW), 0.32 (BEW), 0.65 (15% HOAc), 0.54 (5% HOAc); UV and UV/NH
3-dark purple. UV j max nm:
MeOH 267, 305sh, 345;#NaOMe 277, 293sh, 368 (dec.);#AlCl
3276, 298, 345, 394;
#AlCl
3/HCl 277, 294sh, 341, 392;#NaOAc 276, 295sh, 371;#NaOAc/H3BO3
268, 325sh, 344.
Kaempferol 3-O-glucoside. PC: Rf 0.72 (BAW), 0.76 (BEW), 0.43 (15% HOAc), 0.23 (5% HOAc); UV-dark purple, UV/NH
3-dark greenish yellow. UVjmax nm: MeOH
266, 295sh, 350; #NaOMe 275, 326, 400 (inc.); #AlCl
3 274, 304, 351, 396;
#AlCl
3/HCl 275, 301, 346, 396;#NaOAc 274, 311, 389;#NaOAc/H3BO3 266,
296sh, 353.
Quercetin 3-O-diglucoside. PC: Rf 0.24 (BAW), 0.37 (BEW), 0.44 (15% HOAc), 0.28 (5% HOAc); UV-dark purple, UV/NH
3-dark yellow. UV j max nm: MeOH 257,
266sh, 296sh, 357;#NaOMe 274, 327, 414 (inc.);#AlCl
3 273, 433;#AlCl3/HCl
269, 300, 364, 398;#NaOAc 273, 325, 398;#NaOAc/H
3BO3261, 299, 377.
3. Results
A major#avonoid was isolated from the fronds of all Aspleniumtaxa which were surveyed in this experiment and was identi"ed as kaempferol 3-O-gentiobioside (see Section 2). 2D-PC and HPLC surveys of A. fontanum subsp. fontanum, A. foreziense,A. obovatumsubsp.obovatumvar.obovatumand var.protobillotiiandA. obovatum subsp. lanceolatum revealed the same #avonoid pro"les, i.e., kaempferol 3-O-gentiobioside together with small amount of kaempferol 3-O-polyglycoside and 3,7-O-glycoside (Table 2).
Table 2
Flavonoid pro"les ofAsplenium foreziense, A. fontanum,A. obovatumandA. incisum
Taxa Flavonoids
A. fontanumsubsp.fontanum Kaempferol 3-gentiobioside, kaempferol 3-polyglycoside, kaem-pferol 3,7-glycoside
A. foreziense Kaempferol 3-gentiobioside, kaempferol 3-polyglycoside, kaem-pferol 3,7-glycoside
A. obovatum Kaempferol 3-gentiobioside, kaempferol 3-polyglycoside, kaem-pferol 3,7-glycoside
A. fontanumsubsp.pseudofontanum Kaempferol 3-gentiobioside, kaempferol 3,7-glycoside
A. incisum Kaempferol 3-gentiobioside, kaempferol 3-glucoside, kaempferol 3-gentiobioside-4@-glucoside, quercetin 3-diglucoside
produced and were identi"ed as kaempferol 3-O-gentiobioside, kaempferol 3-O -glucoside and kaempferol 4@-O-glucoside by HPLC comparison with authentic sam-ples. Thus, the #avonoid was characterized as kaempferol 3-O-gentiobioside-4@-O -glucoside. Although kaempferol 3-O-glucosylglucoside-4@-O-glucoside has been iso-lated as kaempferol 3-O-sophoroside-4@-O-glucoside from the fronds of Asplenium
septentrionale(L.) Ho!m. (Imperato, 1990), kaempferol 3-O-gentiobioside-4@-O -gluco-side is reported here for the"rst time in nature.
InA. incisumandA. fontanumsubsp.pseudofontanum, the materials of each of the three di!erent sources in Japan and Pakistan were surveyed by 2D-PC and HPLC. These#avonoid compositions were the same for each taxa from all sources.
4. Discussion
In a previous study, kaempferol 3-O-gentiobioside and kaempferol 3-O -(gentiobio-side 6@-sulfate) were reported from an Italian sample ofAsplenium fontanum(Imperato, 1980). In this survey, kaempferol 3-O-gentiobioside was found, but sulfated glycoside could not be detected in a Spanish sample ofA. fontanum. The#avonoid compositions of A. foreziense, A. obovatum subsp.lanceolatum, A. obovatumsubsp. obovatumvar. obovatumand var.protobillotiiwhich were collected in Spain or France were identical with that of Spanish A. fontanum subsp. fontanum. Moreover, it was shown that a major#avonoid of PakistaniA. fontanumsubsp.pseudofontanumis also kaempferol 3-O-gentiobioside, and not a sulfated kaempferol glycoside. The#avonoid composi-tion of this last taxon was only slightly di!erent (absence of kaempferol 3-O-glycoside) from those of European taxa. This di!erence may be attributable to their geographical distance.
A. fontanum(the occurrence of a sulfated#avonol in an Italian sample and its absence in a Spanish sample).
Japanese Asplenium incisum also contained kaempferol 3-O-gentiobioside, but it was accompanied by kaempferol 3-O-gentiobioside-4@-O-glucoside, kaempferol 3-O -glucoside and quercetin 3-O-diglucoside which could not be detected from other Aspleniumtaxa examined in this survey.
A. incisum is morphologically related to Asplenium normale (Nakaike, private communication) which is widely distributed in Asia, Africa and Hawaii. However, the
#avonoid characters of A. incisum (#avonols) were clearly di!erent from those of A. normale (#avones) which have previously been reported by us (Iwashina and Matsumoto, 1990,1994; Iwashina et al., 1990, 1993a,b).
Acknowledgements
Collection of plant materials from Pakistan in 1991 was partially supported by a grant from the Monbusho Scienti"c Research Program (No. 02041091), the Ministry of Education, Science, Sports and Culture, Japan.
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