Journal of Chromatography, 409 (1987) 353.59 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands CHROM. 19 974 REVERSED-PHASE HIGH-PERFORMANCE LIQUID CHROMATO- GRAPHY OF CAJANUS CAJAN PHYTOALEXINS* _ JAGROOP S. DAHIYA Department of Botany & Microbiology, University College, Gower Street, London WCIE GBT (U.K.) and *Department of Plant Science, University of Manitoba, Winnipeg, Manitoba R3T 2N2 (Canada) (First received April 21st, 1987; revised manuscript received August 19th, 1987) SUMMARY Seedlings of Cajunus cujan accumulated four phytoalexins when treated with silver nitrate solution. The phytoalexins mixture was fractionated by using reversed- phase high-performance liquid chromatography into four active fractions when bioassayed for antifungal activity. These fractions were identified as cajanol, cajanin, isoprenylated genistein and an isoflavonoid phytoalexin. Cajanol was the prominent phytoalexin accumulated in the stressed seedlings. No phytoalexin accumulated in the control seedlings treated with sterile distilled water. INTRODUCTION Phytoalexins are a chemically heterogeneous group of low-molecular-weight compounds with antimicrobial properties. These secondary metabolites are not pres- ent in healthy plant tissues but they appear at the site of infection. Phytoalexin syn- thesis can be induced by molecules of abiotic or biotic origin called elicitors. How- ever, their precise role in disease resistance is not fully understood’. Accumulation of cajanol, cajanin, genistein and formononetin in stressed stem tissues of Cujunus cajun has been reported2v3 but their role in disease resistance has been hampered due to lack of chromatographic procedure which can easily resolve and quantify complex metabolite mixtures. The present study was aimed at elucidation of a chromatograph- ic method for quantification of cajanol and other phytoalexins in seedlings stressed with silver nitrate. MATERIALS AND METHODS Seed Pigeonpea (Cajunus cajun) cv UPAS- used in the present studies was ob- tained from the Plant Breeding Department, Haryana Agricultural University, His- ar- 125004, India. l Contribution No. 790 from the UCL, London, U.K. 0021-9673/87/$03.50 0 1987 Elsevier Science Publishers B.V. 356 J. S. DAHIYA Extraction of phytoalexins Seedlings (20 days old) were treated with 0.1 M sdlver nitrat ;e solultion and incubated for 6 days at 25°C in the dark. Ccmtrols were tlreated with sterile distiilled - Cajanol, RF 0.66 - Cajanin , RF 0.59 - Isopwnylatc !d genistein I.49 - lsof favonoid phytoarex 0.34 Fig. 1. CIadosparimsilica gel gel TLC bioassay. A, Silver nitrate-stressed seedlings extract; B, unstressed seedlings extract (control). Solvent: chloroform-methanol (85: 15). RP-HPLC OF CAJANUS CAJAN PHYTOALEXINS 351 water. Treated seedlings were homogenized in methanol-water (70:30) with a Sorvall omnimixer and filtered. The filtrate was dried in vacua at 4o”C, the residue redissolved in 50 ml of distilled water and partitioned first against petroleum ether and then with ethyl acetate. Ethyl acetate fractions were pooled and dried in vacua, the residue redissolved in acetonitrile-water (1: 1). Fractionation of extracts The sample (1 ml) was injected into high-performance liquid chromatography (HPLC) instrument consisting of Altex pump and injection valve, a column (25 x 1.0 cm I.D.) of Hypersil ODS, a Pye Unicam LC-UV detector set at 287 nm, and a Tekman potentiometer chart recorder. The HPLC solvent system used was acetonitrile-water-O.l M acetic acid (49:50:1). Active fractions were defined origi- nally by their ability to inhibit Cladosporium cucumerinum in the thin-layer chro- matographic (TLC) assay 1,4 (Fig. 1) and subsequently by their retention time and absorption of light at 287 nm. Identification of the active fractions was established through UV, MS and NMR spectral analysis reported elsewhere4. Time course experiment In order to study at what stage phytoalexin synthesis is triggered, samples were taken after a regular interval of 12 h, extracted and a quantitative estimation was done as reported earlier4s5 using a modified solvent system of acetonitrile-water-O. 1 A4 acetic acid. RESULTS HPLC analysis of Cajanus cajun phytoalexins on semi-preparative Hypersil ODS column gave a retention time of 12.0 min for cajanol, the major phytoalexin, whereas the retention times for cajanin, isoprenylated genistein and isoflavonoid phy- toalexins were 7.0, 15.0 and 14.0 min, respectively. An HPLC trace of the de novo accumulation of phytoalexins by silver nitrate- stressed seedlings of Cajanus cajun is illustrated in Fig. 2. Fig. 2A shows that the unstressed seedlings do not accumulate significant quantities of phytoalexins. More- over, hydrolytic extraction for the detection of isoflavonoid glycosides showed there was not a reservoir of isoflavones in the unstressed seedlings either as aglycones or glycosides. Fig. 2B shows that silver nitrate stress will cause the accumulation of a number of compounds which were not present in the unstressed plant tissues. Vari- ation of the acetonitrile content of the HPLC solvent mixture confirmed the absence of additional phytoalexins which may have been obscured by the solvent front peak or by long retention times. Cajanus cajan seedlings accumulated four phytoalexins, the most prominent being cajanol. Fig. 3 shows that cajanol accumulated in the stressed seedlings in appreciable amounts after 48 h whereas accumulation of other phytoalexins started after 60 h of incubation. Moreover, no phytoalexin was found to accumulate when silver nitrate treated seedlings were exposed to light. The identification of the major peaks on the basis of UV, MS and NMR4 spectral analysis data are given in Fig. 4. J. S. DAHIYA 400- Sf A I J ,b, 0 24 46 72 26 120 144 3 6 9 12 15 16 21 Retention Time (min ) Time (h ) Fig. 2. HPLC trace of Cajunus cajun phytoalexins. A, Unstressed seedlings extract; B, silver nitrate-stressed seedlings extract. Peaks: Sf = Solvent front (solvent peak); 1 = cajanin; 2 = cajanol; 3 = isotlavonoid; 4 = isoprenylated genistein. Solvent: Acetonitrile-water-O.1 M acetic acid (495O:l). Fig. 3. Time course experiment. A-A = cajanol; 0-0 = cajanin; O-‘--O = isoprenylated genistein; &--A = isoflavonoid phytoalexin. A Fig. 4. Chemical structures of pigeonpea phytoalexins. A = Cajanin; B = cajanol; C = isoprenylated genistein; D = isoflavonoid phytoalexin. RP-HPLC OF CAJANUS CAJAN PHYTOALEXINS 359 DISCUSSION Phytoalexin surveys in the Papillionoideae, a subfamily of the Leguminosae, suggest that cajanol is of exceptionally rare occurrence. Indeed, apart from CU@WS cu$z~, this isoflavanone has only been obtained from the hypocotyl of Stizobbium deeringianum (tribe Erythrineae) challenged with Helminthosporium carbonum2*3. There it occurs with various other isoflavonoids including genistein, 2-hydroxygeni- stein and dalbergiodin. The chromatographic system used in the present study facilitates a rapid and efficient separation of cajanol and related isoflavonoid phytoalexins from pigeonpea. Addition of 0.1 M acetic acid to the acetonitrile-water mixture increased the reso- lution of peaks 3 (isoflavonoid) and 4 (isoprenylated genistein). The results presented here are further supported by the earlier workers who used an identical solvent system although different proportions to separate isoflavonoid compounds from Cicer arietinum6. Thus, the present study provides an efficient chromatographic procedure to quantify the phytoalexins from pigeonpea and study their role in plant defense mech- anisms. ‘. ACKNOWLEDGEMENTS I wish to thank SERC (U.K.) for financial assistance and Dr. M. N. Mruzek, Chemistry Department, University College, London (U.K.) for helpin scanning the HPLC peaks by MS. REFERENCES 1 J. A. Bailey and J. W. Mansfield, in J. A. Bailey and J. W. Mansfield (Editors), f%~coulexins, Blackie & Son, Bishopsbriggs, Glasgow, London, 1982, pp. 24 and 253. 2 J. L. Ingham, 2. Nuturforsch. C, 31 (1976) 504508. 3 J. L. Ingham, 2. Naturforsch. C, 34 (1979) 159-161. 4 J. S. Dahiya, R. N. Strange, K. G. Bilyard, C. J. Cooksey and P. J. Garratt, Phytochemistry, 23 (1984) 871-873. 5 J. S. Dahiya and D. Singh, Cur. Sci., 55 (1986) 3940. 6 J. Kaster, A. Zmok and W. Barz, J. Chromatogr., 270 (1983) 392-395.