Phytoalexins from Dolichos Biflorus Noel T
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Phytoalexins from Dolichos biflorus Noel T. Keen Department of Plant Pathology, University of California, Riverside, California 92521, USA and John L. Ingham Phytochemical Unit, Department of Botany, University of Reading, Reading RG 6 2AS, England Z. Naturforsch. 35 c, 923—926 (1980); received June 30, 1980 Leguminosae, Dolichos, Isoflavonoids, Phytoalexins, Structure Elucidation Genistein, 2'-hydroxygenistein, dalbergioidin, kievitone and phaseollidin have been found to accumulate in leaves and stems of Dolichos biflorus (horsegram) following inoculation with the non-pathogens Pseudomonas pisi and Phytophthora megasperma f. sp. glycinea, respectively. They are accompanied by isoferreirin (5,7,4'-trihydroxy-2'-methoxyisoflavanone), a compound not previously reported as a natural product. Coumestrol and psoralidin occur constitutively in Dolichos leaves and stems. Introduction TLC bioassays against Cladosporium cucumerinum; only B3 and B4 were antibacterial when similarly A variety of isoflavonoid phytoalexins have been bioassayed using P. pisi as the test organism. All isolated from papilionate grain legumes such as four zones were eluted, and after further TLC Lablab purpureus (syn. L. niger), Phaseolus vulgaris, purification (see Experimental) yielded isoferreirin Psophocarpus tetragonolobus and Vigna unguiculata (5,7,4/-trihydroxy-2/-m ethoxyisoflavanone, 1) to [1-3], all of which belong to the widely distributed gether with the known isoflavonoids, genistein and taxonomically advanced tribe Phaseoleae (sub (5,7,4'-trihydroxyisoflavone, 2), 2'-hydroxygenis- tribe Phaseolinae) [4]. As part of a continuing tein (5,7,2',4'-tetrahydroxyisoflavone, 3), dalber phytochemical study of the Leguminosae we have gioidin (5,7,2',4'-tetrahydroxyisoflavanone, 4), kievi examined the phytoalexins produced by horsegram, tone (5,7,2',4'-tetrahydroxy-8-isopentenylisoflava- Dolichos biflorus L. (Phaseoleae, subtribe Phase none, 5), phaseollidin (3,9-dihydroxy-10-isopentenyl olinae), a pulse extensively grown in parts of south pterocarpan, 6), coumestrol (3,9-dihydroxycoumes- ern India. This paper describes the isolation and tan, 7) and psoralidin (3,9-dihydroxy-2-isopentenyl- identification of eight induced and constitutive Do coumestan, 8 ). Compounds 2 -8 were identified by lichos isoflavonoids including a previously unknown UV, MS and TLC (4 or 5 solvent systems) com isoflavanone (5,7,4'-trihydroxy-2'-methoxyisoflava- parison with authentic material. Only coumestrol none) for which we suggest the trivial name iso and psoralidin were isolated from H 20-infiltrated ferreirin. (control) leaves. These pre-infectional coumestans, as well as compounds 1 - 6 were also obtained from Results and Discussion Extracts of Pseudomonas /?w/-infiltrated Dolichos leaves were chromatographed (see Experimental) to afford four fluorescence-quenching zones (B 1- B4), three of which (Bl, B3 and B4) were associated with pronounced antifungal activity as judged by Reprint requests to Professor N. T. Keen or Dr. J. L. Ingham. 0341-0382/80/1100-0923 $01.00/0 Isoferreirin (1) 924 N. T. Keen and J. L. Ingham • Phytoalexins from Dolichos biflorus Dolichos stems inoculated with the fungus Phyto P. pisi ([10] and S. S. G nanam anickam , per s. com phthora megasperma f. sp. glycinea. mun.). Although coumestrol is known to inhibit P. MS analysis of the new isoflavanone (isoferreirin, phaseolicola and P. glycinea [11, 12] neither it nor 1) revealed a low intensity parent ion ( m /e 302; psoralidin have yet been tested against P. pisi. Isoflavonoids 2—6 have all previously been isolat C 16H 140 6) and associated major fragments at m /e 153 (base peak), 150, 135 and 107. Together with ed as phytoalexins from species belonging to the UV data (see Experimental), the MS fragmentation Phaseoleae [1-3, 5, 13] and as such their formation by D. biflorus - a member of the same legume tribe pattern strongly suggested that 1 was an isoflavanone derivative [5], this possibility being subsequently - is not particularly surprising. It is noteworthy, however, that isoflavan derivatives are apparently confirmed by CH 2N 2 methylation [ 6 ] to yield a phenolic diMe ether (M+330) identical (UV, MS, not produced by D. biflorus despite the fact that TLC) with 5-hydroxy-7,2',4'-trimethoxyisoflavanone several compounds of this type have been obtained prepared from authentic ferreirin (5,7,2'-trihydroxy- from Phaseolus vulgaris, Lablab purpureus and 4/-methoxyisoflavanone, 9). As 1 separated from 9 Vigna unguiculata [1,2]. Similarly, the present study upon Si gel TLC in CHCl3:MeOH (25:1) (1, RF failed to reveal any phaseollin (a well-known phyto 0.46; 9, R f 0.54), it was provisionally identified as a alexin o f P. vulgaris) although its presumed bio ferreirin isomer. Two OH groups were readily as synthetic precursor, phaseollidin accumulated in signed to ring A (C-5 and C-7) on the basis of significant amounts. Although isoferreirin has been bathochromic shifts (see Experimental) observed provisionally identified as a phytoalexin in Helmin- thosporium carbonum- inoculated hypocotyls of Flor when A1C1 3 in MeOH, or solid anhyd. NaOAc were added to methanolic solutions of 1 [7]. A-ring di- ida velvet bean, Stizolobium deeringianum (Pha hydroxylation and a B-ring substituted with single seoleae, subtribe Erythrininae), it could not be OH and OMe groups could also be deduced from isolated from this source in amounts sufficient for the major MS ions at m /e 153 and 150 respectively [5]. comprehensive structural analysis ([ 8 ] and J. L. Ingham, unpublished data). Direct comparison (UV, The B-ring substitution pattern (C-2' OMe and MS, TLC) of the Stizolobium compound and Doli C-4' OH) of 1 was proved unequivocally in the chos isoferreirin has now confirmed that they are following manner. Ethylation with diethyl sulphate identical. gave 7,4'-di-0-ethylisoferreirin (10) admixed with a small quantity of the corresponding 7-O-ethyl deriv ative (11). After TLC purification, the diethylated Experimental isoflavanone was degraded using the alkali-peroxide procedure [ 8 ] to yield a B-ring derived product Plant and fungal material. Seeds o f Dolichos bi indistinguishable (UV, MS, TLC) from 2-methoxy- florus L. (obtained from J. L. Hudson, Seedsman, 4-ethoxybenzoic acid. Formation of this compound Redwood City, California) were glasshouse grown allows the structure of isoferreirin to be firmly (22°-30°C without supplemental lighting) in defined as 5,7,4'-trihydroxy-2'-methoxyisoflavanone. 1 0 cm pots containing a sand/peat moss mixture. 2-Ethoxy-4-methoxybenzoic acid would have been Cultures of Pseudomonas pisi (supplied by R. Good obtained if the C-2' and C-4' substituents of 1 were man, Dept, of Plant Pathology, University of Mis reversed as in ferreirin [ 8 ]. souri, Columbia, Missouri, USA) and Phytophthora TLC bioassays ( Cladosporium cucumerinum) of megasperma f. sp. glycinea (race 1, isolate P 1139) the induced Dolichos isoflavonoids indicated that [14] were maintained on nutrient agar and V - 8 juice phaseollidin was the most fungitoxic being followed agar, respectively [ 1 1 , 15]. by kievitone, dalbergioidin and isoferreirin respec Induction and extraction of Dolichos isoflavonoids. tively. Genistein and 2'-hydroxygenistein were in Leaves from 3-4 week-old Dolichos plants were in active, thereby supporting the results of an earlier filtrated with H20 suspensions of P. pisi ( 8 x l0 7 study [9]. A gainst P. pisi, only kievitone and dal cells/ml) using a hand sprayer [16]. After a further bergioidin exhibited strong antibacterial properties. 2 -4 days in the glasshouse, the leaves were harvest Other workers have also found that kievitone is in ed and immediately extracted (approx. 7 h) with hibitory to various Pseudomonas species including 40% EtOH as described elsewhere [16]. T he extracts N. T. Keen and J. L. Ingham ■ Phytoalexins from Dolichos biflorus 925 were concentrated in vacuo (40 °C) and then shaken 331 (2), 330 (M +; 14), 165 (11), 164(100), 151 (4), (x 2) with approx. equal vols of EtOAc. The pooled 150 (5), 149 (56), 121 (30). organic fractions were dried (anhyd. MgSOJ, evap Ethylation of isoferreirin. Pure isoferreirin (1.5 mg) orated, and the residue transferred to vials with was ethylated with diethyl sulphate [ 8 ] and the peroxide-free Et 20. Following removal of solvent products chromatographed (Si gel TLC, layer and re-dissolution in EtOAc, the extracts were chro thickness 0.25 mm) in CHC1 3 :CC14 (3:1) to give matographed (Si gel TLC, GF-254, layer thickness 7,4'-di-0-ethylisoferreirin 10 (R f 0.72; approx. 1.2 0.375mm) in hexanes *: EtOAc: MeOH (HEM, 60:40:1) mg) and 7-O-ethylisoferreirin 11 (/?/?• 0.05; approx. to afford fluorescence-quenching bands at RF 0.49 0.2 mg). Both compounds were eluted (MeOH) and (B l), 0.41 (B 2), 0.31 (B3) and 0.21 (B4). These rechromatographed in either «-pentane:Et 20 : glacial zones were eluted (Me 2CO) and their components HOAc (PEA, 75:25:3) (11, RF 0.37) or PEA + CC1 4 further purified (Si gel TLC) as follows, i) B1/B2, (75:25:3:25) (10, RF 0.71) prior to UV and mass CHC13 :M e 2CO:NH4OH (65:35:1) gave 6 (RF 0.58) spectroscopy. and 8 (RF 0.13) respectively; ii) B3, CHCl3:MeOH 5-Hydroxy-7,4'-diethoxy-2'-methoxyisoflavanone (10). (CM, 95:5, x3) gave 5 (lower zone), 7 (intermediate Diazotised /j-nitroaniline, orange; Gibbs reagent, zone) and 1 +2 (upper zone), the latter partially Prussian blue. Amax(nm) MeOH 213 (100%), 229 resolved isoflavonoids being eluted together prior to (81%), 287 (70%), 330 sh (13%); NaOH 218, 245 sh, complete separation by multiple development (x3- 288, 357; A1C1 3 274, 284 sh, 311, 364; the MeOH 5) in CM (1, upper zone; 2, lower zone); and iii) B4, spectrum was unaffected by addition of NaOAc. MS CM (95:5, x3) gave 3 (upper zone) and 4 (lower (rel. int.) 359 (3), 358 (M+; 10), 179(13), 178 (100), zone).