Tectorigenin, a Phytoalexin of Centrosema Haitiense and Other Centrosema Species
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Tectorigenin, a Phytoalexin of Centrosema haitiense and Other Centrosema Species Kenneth R. Markham * and John L. Ingham Phytochemical Unit, Department of Botany, University of Reading, Reading RG 6 2AS, England Z. Naturforsch. 35 c, 919-922 (1980); received June 30, 1980 Centrosema, Phytoalexins, Isoflavones, Tectorigenin, Aesculetin, Chemotaxonomy A phytoalexin isolated from the fungus-inoculated leaflets of Centrosema haitiense, C. pubescens (3 accessions) and C. virginianum has been identified as 5,7,4'-trihydroxy-6-methoxy- isoflavone (tectorigenin). This compound is variously accompanied by other isoflavones includ ing the previously described phytoalexin, cajanin (5,2',4'-trihydroxy-7-methoxyisoflavone). Sub stantial quantities of aesculetin were obtained when C. haitiense leaves were submerged for 48 h in water or fungal spore suspensions. The taxonomic position of Centrosema within the legume tribe Phaseoleae is discussed. Introduction material extracted from the diffusates after 48 h in cubation. With the exception of C. brasilianum, all Although isoflavonoid phytoalexins (particularly the species produced simple phenolic isoflavones pterocarpans and isoflavans) have been isolated (Table I), in particular a non-fluorescent substance from the fungus-treated tissues of many Leguminosae which exhibited UV/visible absorption maxima (subfamily Papilionoideae), comparatively few spe characteristic of those compounds having a 5,7-di- cies appear to produce fungitoxic isoflavones in hydroxy- 6 -m ethoxy substitution pattern [7]. MS more than trace amounts [1]. Notable amongst these analysis defined the mol. wt. as 300, and associated are members of the tribe Genisteae ( e. g. Laburnum fragments at m /e 285 (M+-Me), 257 (M+-M e- anagyroides) as well as certain Phaseoleae (e. g. CO), 167/139 (loss of Me and Me + CO respectively Cajanus cajan and Neonotonia wightii ) where simple from a dihydroxy-monomethoxy substituted A-ring and/or complex isoflavones frequently represent a fragm ent [ 8 ]) and 118 (B-ring fragm ent w ith mono- major component of the phytoalexin response [2-4]. hydroxylation [ 8 ]) suggested that the compound was In other legumes, e.g. Phaseolus vulgaris (Phaseoleae, probably tectorigenin (5,7,4'-trihydroxy-6-methoxy- subtribe Phaseolinae), isoflavones generally co isoflavone, 1). This provisional identification was occur with substantially greater quantities of ptero- confirmed by direct UV, MS and TLC (5 solvent carpan, isoflavan or isoflavone derivatives [1]. As systems on Si gel and cellulose) comparison with yet there are no reports of phytoalexin formation in authentic material. The fungal induction of tec Centrosema (Phaseoleae, subtribe Phaseolinae), a torigenin has not previously been described; how genus of approx. 35 species endemic to the American ever, this isoflavone occurs uncombined or as its tropics and subtropics [5]. Several members of the 7-O-glucoside (tectoridin) in the healthy tissues of group, e. g. C. pubescens (“centro”) are widely grown several legumes, notably species of Baptisia (tribe as fodder crops. We report here the results of a Thermopsideae) and Dalbergia (tribe Dalbergieae) recent examination of selected species of this genus. [9-11], where it may possibly confer some degree of protection (either as a prohibitin or post-inhibitin Results and Discussion [ 1 2 ]) against microbial invasion. In addition to tectorigenin, one accession of C. The excised leaflets of 4 Centrosema species (Ta pubescens also accumulated significant quantities of ble I) were treated with conidial suspensions of cajanin (5,2',4/-trihydroxy-7-methoxyisoflavone, 2), Helminthosporium carbonum [6 ] and EtOAc soluble a phytoalexin previously obtained from the H. carbonum-inoculated stems of pigeon pea, Cajanus * Permanent address: Chemistry Division, D. S. I. R , cajan (Phaseoleae, subtribe Cajaninae) [3]. Although Private Bag, Petone, New Zealand. cajanin was absent from leaf diffusates of the two Reprint requests to Dr. J. L. Ingham. other C. pubescens accessions, it should be noted 0341-0382/80/1100-0919 $01.00/0 that intraspecific variation in phytoalexin produc- 920 K. R. Markham and J. L. Ingham • Tectorigenin, a Phytoalexin of Centrosema haitiense Table I. Isoflavones present in diffusates from Helmintho- sporium carbonum- inoculated leaflets of Centrosema spe cies ** b’ c. Species Seed Isoflavone(s) Con Source Detected centra tion Hg/ml C. brasilianum HI None - Benth. (CPI 40061) C. haitiense HI Tectorigenin 38 Urb. & Ekman (CPI 46540) Afrormosin 2 Glycitein < 2 6,7,4'-Tri- TR hydroxyisoflavone C. pubescens HI Tectorigenin 16 Benth. Afrormosin 2 Formononetin 2 C. pubescens Gm Tectorigenin 13 Benth. Afrormosin 1 Formononetin < 1 Glycitein TR C. pubescens Jn Tectorigenin 14 Benth. Cajanin 23 C. virginianum HI Tectorigenin 26 (L.) Benth. (CPI 40054) Formononetin TR TR = trace (insufficient for UV quantification); identified by TLC comparison with authentic material. a Isoflavone concentrations were determined spectropho- tometrically using the following extinction coefficients: i) £=30000 for tectorigenin (at 268 nm) and cajanin (260 nm); and ii) £ = 22400 for afrormosin, glycitein (both at approx. 258 nm) and formononetin (250 nm) [21, 22], b Seed sources: G m = S. S. Gnanamanickam, Centre for upon TLC biassay against Cladosporium herbarum [16] Advanced Studies in Botany, University of Madras, India; only tectorigenin and cajanin were associated with Hl = E. F. Henzell, The Cunningham Laboratory, C. S. I. regions of antifungal activity. None of the above R. O., St. Lucia, Queensland, Australia (plant introduction (CPI) numbers are in parentheses); Jn = D. H. Janzen, isoflavones was detected in control (H 20) diffusates. Department of Biology, University of Pennsylvania, Phil All attempts to isolate tectorigenin, cajanin or other adelphia, Pennsylvania, USA (collected in the Santa Rosa antifungal material from diffusates of C. brasilian- National Park, Costa Rica). c C. haitiense = C. schottii (R. J. Clements and R. J. um were unsuccessful. Williams, pers. commun.). W ith C. haitiense, an attempt was also made to increase the yield of tectorigenin by immersing leaflets in a dense spore suspension of H. carbonum. tion has recently been described in legumes such as However, examination of the resulting diffusate Arachis hypogaea [13, 14] and Vigna unguiculata revealed not tectorigenin, but large quantities (5 mg/ [15]. As misidentification of the Costa Rican acces 0.75 g dry wt. leaves) of a colourless, crystalline sion of C. pubescens has been excluded (D. H. com pound (M + 178; D iacetate, M + 262) indistin Janzen, pers. commun), its differing phytoalexin guishable (UV, TLC) from authentic aesculetin response presumably reflects genuine chemical vari (6,7-dihydroxycoumarin, 3). The same compound ability in this very widely distributed species (D. H. was obtained in comparable amounts when C. Janzen and R. J. Clements, pers. commun). haitiense leaflets were similarly submerged in de Apart from compounds 1 and 2, leaf diffusates ionised H 20. Although aesculetin does not occur variously contained other known isoflavones with naturally in the leaves of C. haitiense, its 7-0- 7,4'(formononetin) or 6,7,4'(afrormosin, glycitein and glucoside (aesculin) can be isolated in high yield 6,7,4'-trihydroxyisoflavone) oxygenation patterns. (6.5% on a dry wt. basis) by extraction with aqueous All were present at low concentrations (Table I), and MeOH. Hydrolysis of aesculin to aesculetin occurs K. R. Markham and J. L. Ingham • Tectorigenin, a Phytoalexin of Centrosema haitiense 921 readily upon tissue damage, and formation of the Centrosema accessions have been deposited in the latter compound as described above may well reflect University of Reading Herbarium. a stress response of the immersed leaflets. TLC Isolation of Centrosema isoflavones. Si gel TLC bioassays ( C. herbarum ) indicated that neither aes- (CH Cl3 :MeOH, 50:3 layer thickness 0.25 mm) of culin nor aesculetin possessed antifungal activity. diffusate extracts (EtOAc) variously afforded iso- The phytoalexin data presented in Table I are in flavone bands at RF 0.70 (afrormosin), 0.46 (formo- teresting because of the uncertain allocation of nonetin), 0.36 (cajanin), 0.35 (tectorigenin), 0.23 Centrosema to the subtribe Phaseolinae [17]. Studies (glycitein) and 0 . 1 0 (6,7,4'-trihydroxyisoflavone). undertaken at Reading University (J. L. Ingham, un For the Costa Rican accession of C. pubescens, published data) indicate that subtribes of the Pha- cajanin and tectorigenin were eluted (MeOH) seoleae fall into two broad categories depending on together, and subsequently resolved by TLC in whether their constituent genera are characterised either C 6H 6 :MeOH, 9:1 (1, R F0A3-, 2, /?F0.34) or by the accumulation of simple or predominantly «-pentane:Et 20: glacial HO Ac, 75:25:3, x3 (1, up complex isoflavonoid phytoalexins. Thus, in the per zone; 2, lower zone). Isoflavones from all other Phaseolinae, Centrosema is clearly atypical as spe sources were quantified without additional purifica cies belonging to other genera examined (e.g. Do - tion. lichos, Lablab, Phaseolus, Psophocarpus and Vigna) Isolation of aesculin and aesculetin. One dimen [1 , 18] invariably produce phytoalexins with prenyl sional PC (H 20 : glacial HOAc, 20:3) of leaf extracts or dimethylchromen attachments. Although simple (70% MeOH) gave aesculin as a purple/white flu isoflavones (e.g. 2 '-hydroxygenistein) do occur in orescent (UV) band at high R F. Freshly