Phytduvwistry. 1978. Vol. 17. pp. 179%1800 @ Pergamon zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Press Ltd Printedm England 0031-9422,/78/1W1~ 1799 $02.00,&3 TRITERPENOIDS IN EPICUTICULAR WAXES OF DUDLEYA SPECIES BRUCE S. MANHEIM JR. and THOMAS W. MULROY Botany Department, Pomona College, Claremont, CA 91711. U.S.A. (Redsed receiced 30 March 1978) Key Word Index--Dud&z; Crassulaceae; epicuticular wax; triterpenoids; /?-amyrin acetate; taraxerone. Abstract-The pentacyclic ttiterpenoids, /I-amyrin acetate and tataxerone, are major components in the epicuticular wax of the genus Dudleyu. Heavily glaucous, non-glaucous and intermediately glaucous populations are found in many species of Dudleya Experimental crosses and field observations suggest that glaucescence is a polygenic trait [l]. The constituents of both non-glaucous and glaucous epi- cuticular waxes are investigated here to determine the chemical compounds possibly responsible for glauces- cence in Dudleyu. Single wax constituents have been shown to cause glauccscencc in a variety of plants. Glaucous peas have a C,, hydrocarbon as the major constituent 141, while a C ag alkane is the predominant component of glaucous ca bage [S]. Major fi-diketones cause glaucescence in Eucalyptus, [63, POU colinsoi [73, barley [S] and durum wheat [9-J. Recrystallization experiments and ultra- microscopical examination of the crystal structure of the epicuticular waxes is currently being performed to determine whether pentacyclic triterpenoids are res- ponsible for glaucescence in Dudleya. RESULTS AND DISCUSSION IR and TLC indicated that the epicuticular wax of the two Dudleya species examined here consisted of long chain alkanes, wax esters, primary alcohols and car- boxylic acids. The glaucous waxes gave a positive reac- tion to the Lieberman-Burchard test indicating the presence of triterpenoids. By MS, two pentacyclic triter- penoids, /3-amyrin acetate and taraxerone, were detected. Both of these compounds have been previously reported in plant waxes. /?-amyrin acetate by Horn and Lamber- ton [2] and taraxerone by Tulloch [3]. Chromatographic analysis of conspecific green and glaucous plants of many zyxwvutsrqponmlkjihgfedcbaZYXW Dudley species indicates that the glaucous plants differ from one another in the dominance of one of the two triterpenoids, fl-amyrin acetate and taraxerone. The pattern of occurrence of these triterpenoids in glaucous species may be of systematic value in Dudleya [JO]. EXPERIMENTAL The approximate percent composition of the waxes of the green and glaucous forms of two Dudfeya species is listed in Table 1. These figures were determined by weighing fractions isolated by silicic acid column chromatography. It is of interest to note the dominance of triterpenoids zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA in these conspecific forms. The D. brittonii glaucous wax is 40-45 72 /3-amyrin acetate while the non-glaucous form contains only trace amounts of this compound. Similarly, the D.farinosa glaucous wax is 3942% tacaxerone while the green form contains trace amounts of this triterpenoid. Plant material. Mature plants grown in a greenhouse in Claremont, California were analysed for their wax constituents. D. ~~~cf~n~i green and glaucous form were h-year-old plants grown from seed collected near Ihe type locality at La Misi6n. Northwestern Baja California del Norte, Mexico. The green and glaucous forms of D.farimxu were malure plants collected from population samples of Point Arena, Mendocino County, California. They were grown for 3 yr under the same conditions as the D. britronii plants. These plants represent the 4 major types of wax constituent patterns in the genus. Table 1. Approximate percent composition of components in waxes of the green and glaucous forms of D. faritwsa and D. brittanii Exfracfim. TLC showed that the qualitative composition of the wax was the same for both young and old leaves of a parti- cular apzcics, Thus, the older leaves closer to the base of the caudex were removed, leaving a rosette of younger leaves for plant survival. The surface wax was extracted by immersion and agitation of the leaves in Et,0 for 5-IOsec. To avoid contamination the cut ends of the leaves were not immersed. The soln was then vacuum liltered to remove dirt and on evapora- tion the surface lipid was obtained. D./arirwsa D. brittonii Components green glaucous green glaucous Alkanes 50 28 20 30 Esters 21 II 35 9 B-Amyria acetate tr tr tr W5 Taraxerone tr 3942 tr tr Primary alcohols 6 4 8 2 Carboxylic acids 10 IS 22 3 tr = less than I”/,. 1799 fractionation The epicuticular wax (D. brirronii glau. 229 mg, D. hrirrnnii green 148 rng, D. jarinosa glau. 531 mg. D. farinosa green 178mg) was dissolved in Et,0 and dried into Silic AR-CC-4 100 mesh (Malinckrodt, St. Louis). The siIicAR and wax was then placed on a column of silicAR. The column was eluted with n-hexane (16Oml), C,H, (160ml), CHCl, (160 ml) and MeOH (20I)ml). Fractions(20 ml)werecollected and evapor- ated to dryness. Chromatography. TLC was carried out with commercial 20 Y 20 cm plates (Kieselgel60 F-254) precoated with a 0.25 mm thick layer of Si gel G. C,H, and CHCI, were used as develop-