Suppressors of Cancer Cell Proliferation from Fig (Ficus carica) Resin: Isolation and Structure Elucidation Shai Rubnov, † Yoel Kashman, ‡ Ruth Rabinowitz, § Michael Schlesinger, § and Raphael Mechoulam* ,† Department of Medicinal Chemistry and Natural Products, School of Pharmacy, The Hebrew University, Jerusalem 91120, Israel, School of Chemistry, Tel-Aviv University, Ramat Aviv, Tel-Aviv 69978, Israel, and Hubert H. Humphrey Center for Experimental Medicine & Cancer Research, Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel Received December 8, 2000 A mixture of 6-O-acyl--D-glucosyl--sitosterols, the acyl moeity being primarily palmitoyl and linoleyl with minor amounts of stearyl and oleyl, has been isolated as a potent cytotoxic agent from fig (Ficus carica) latex and soybeans. Identity was established by spectroscopic methods (NMR, MS) and confirmed by chemical synthesis. Both the natural and the synthetic compounds showed in vitro inhibitory effects on proliferation of various cancer cell lines. Fig (Ficus carica, Moraceae) products are widely used both as a food and as medicine in the Middle East. The latex released on picking the fruits is used to treat skin tumors and warts. 1 The first scientific investigation of the activity of fig latex was done by Ullman et al. in the 1940s. 2-4 High doses of fig latex injected into rats were found to be lethal. Smaller doses injected into mice bearing a benzpyrene-induced sarcoma caused inhibition of the growth of the tumor and even the disappearance of small tumors. 2 The dialysate of the latex contained the active ingredient. Although isola- tion of the active components was not pursued further, some pharmacological work was reported by the same group. 3,4 Fig latex has also been tested for its antihelmintic activity, but was found to cause acute toxicity with hemor- rhagic enteritis. 5 The leaf decoction affected lipid catabo- lism in hypertriglyceridemic rats 6 and had hypoglycemic action in type-I diabetic patients. 7 Several phytochemical investigations of Ficus carica leaves have been published, but with no biological data. Athnasios et al. 8 have isolated psoralen, -sitosterol, ber- gapten, and taraxasterol from the petroleum ether extract of the leaves. Others have isolated triterpenoids. 9-10 In this paper we describe the isolation and identification of a potent cytotoxic agent from fig latex, which is also present in soy products. Fig latex is present in the plant in extremely small quantities and must be collected drop by drop. Many procedures were examined in order to obtain a maximum yield of the active cytotxic compounds from the fig resin. The optimal procedure (see Experimental Section) involved removal of the inactive polymeric material by filtration, followed by extraction of the aqueous filtrate with various solvents. An extract that was found to inhibit Raji cell proliferation was further chromatographed by several methods, and the activity was monitored with an assay based on the proliferation of Raji cells. Ultimately a small amount of solid active material was isolated (0.002% overall yield from 500 mL of resin, 0.008% of the nonaqueous material in the resin). The compound migrated as a single band (R f 0.7) on thin- layer chromatography (TLC) silica plates developed with chloroform-methanol (9:1). Purity was determined as >99% by normal-phase HPLC analysis. The cytotoxic agent was identified by spectroscopic methods and confirmed by chemical synthesis as a mixture of 6-O-acyl--D-glucosyl--sitosterols (6-AGS). Acylated si- tosteryl glucosides have been isolated from soybeans, 11 millet seeds, 12 wheat flour, 13 and other plants 14 and have also been synthesized. 15 The major problem with the structure elucidation of the active material was the variability of its composition and the small amounts available. Nevertheless, from the NMR data (Supporting Information), it was clear that 6-AGS always contains a C 29 -sterol (m/z 397, see below, and δ H 0.629 s, Me-18, and 1.003 s, Me-19), a hexose unit (δ C 99.6 d, an anomeric C-atom, and 70.3 d, 73.6 d, 75.7 d, 76.4 d, and 62.1 t, indicative of C adjacent to oxygen), and a long aliphatic chain, which differed in its NMR profile from batch to batch. In certain isolation batches this was a saturated chain, while in others there were double bonds (δ C 126.0-130.2 ppm) and a -CHdCH-CH 2 -CHdCH- group (δ H 2.76 t and δ C 25.6 t, a double allylic methylene). FABMS gave a major molecular ion at m/z 837 (MH + ), accompanied by weak higher peaks, as well as two strong fragments at m/z 696 (M - 141, M - C 10 H 21 ) and m/z 397 (100%). The molecular ion was confirmed by obtaining 22 * To whom correspondence should be addressed. Tel: 972-2-6758634. Fax: 972-2-6757076. E-mail: mechou@cc.huji.ac.il. R.M. is affiliated with the David R. Bloom Center for Pharmacy of the Hebrew University. † School of Pharmacy, The Hebrew University. ‡ School of Chemistry, Tel-Aviv University. § Center for Experimental Medicine & Cancer Research, Hebrew Uni- versity-Hadassah Medical School. 993 J. Nat. Prod. 2001, 64, 993-996 10.1021/np000592z CCC: $20.00 © 2001 American Chemical Society and American Society of Pharmacognosy Published on Web 07/07/2001