J Hattori Bot. Lab. No. 81 : 257- 262 (Feb. 1997) CHEMICAL CONSTITUENTS OF JAPANESE RICCIOCARPOS NATANS 1 TATSUHIKO YOSHIDA , MASAO TOYOTA1, TOSHIHIRO 1 1 HASHIMOT0 and YOSHINORl ASAKAWA ABSTRACT. From the methanol extract of Japanese Ricciocarpos natans (Ricciaceae), stigmast-4-en- 3-one, sitost-4-en-3-one, lunularic acid and phytol have been isolated, together with the mixture of stigmasterol, sitosterol and fatty acids. This is the first example of the isolation of the steroid ketones from the bryophytes. The chemical constituents of Japanese R. natans are collected in the field are quite different from those of R. natans grown in axenic culture. INTRODUCTION Liverworts are rich sources of mono-, sesqui- and diterpenoids and lipophilic aromatic compounds several of which show interesting biological activity (Asakawa 1982, 1990, 1993, 1996). Ricciocarpos natans which lives on the surface of pond and paddy field be­ longs to the Ricciaceae. Previously, Huneck et al. (1972) reported the presence of sitosterol (3) in the field-collected R. natans. Recently, the chemical constituents of European R. natans grown in axenic culture was analyzed and showed to have cuparane- (6) and mono­ cyclofarnesane-type sesquiterpenoids (7) (Wurzel & Becker 1989, 1990), bibenzyl and benzyl derivative including their glucosides (5, 8-10) and cyclic bis(bibenzyl) dimer, pusi­ latin B (12) (Kunz & Becker 1992; Siegmund and Becker 1994), cyclic bis(bibenzyl), ric­ cardin C (11) (Asakawa & Matsuda 1982; Kunz & Becker 1992), fatty acids, palmitic, stearic, oleic, linoleic, linolenic, arachidonic, 5,8,l l,14,l 7-eicosapentaenoic acids (Kohn et al. 1988) and flavonoids (Markham & Porter 1975). We are continuing to study the chemi­ cal constituents of bryophytes not only from the isolation and the structure elucidation of biologically active components but also from chemosystematics. Recently, four structurally unique and biologically active bis(bibenzyl) dimers named pusilatins A, B (12), C and D were isolated from the liverwort Blasia pusilla (Metzgeriales) (Asakawa 1994, 1995; Hashimoto et al. 1994). As pusilatin B (12) and its monomer, riccardin C (11) have been found in axenic cultured R. natans (Kunz & Becker 1994) we studied the chemical con­ stituents of Japanese field collected R. natans in order to isolate or detect pusilatin-type cyclic bis(bibenzyl) dimers. In place of cyclic bis(bibenzyl) and its dimer, steroid ketones and lunularic acid (5) were isolated from Japanese R. natans together with phytosterols and fatty acid mixtures. Here we report the distribution of steroids and fatty acids in Japanese R. natans and comparison of the chemical constituents of field collected Japanese R. natans and European R. natans grown in axenic culture. 1 Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho, Tokushima 770, Japan. 258 J. Hattori Bot. Lab. No. 81 I 9 9 7 EXPERIMENTAL Thin Layer Chromatography (TLC) TLC was carried out on precoated plates (Kieselgel 60 F254, 0.25 mm thick, Merck) using n-hexane-EtOAc (4: l); spots were detected by illumination with UV lamp (254nm) or by spraying Godin reagent followed by heating at 120°C. Gas Chromatography-Mass Spectrometry (GC-MS) GC-MS analysis of the EtOAc extract, each fraction of EtOAc extracts, silyl ethers and methyl esters of fatty acids was carried out using Hewlett-Packard 5890 Series II gas chromatograph coupled with a mass selective detector (MSD 5971), at 70 eV; the GC col­ umn was a fused-silica column coated with DB-17 (30 mX0.25 mm i.d., film thickness 0.25 µm), and helium was used as carrier gas (lml/min). The oven temperature was pro­ grammed from 50°C to 250°C at 5°C/min; the injection temperature was 250°C. Plant Material Ricciocarpos natans (L.) Corda was collected in Mikicho, Kagawa Prefecture, in Nov. 1994 by Mr. S. Mitani and identified by Dr. M. Mizutani and deposited at the Institute of Pharmacognosy, TBU. Extraction and Isolation The air-dried R. natans (226 g) was mechanically ground and the resulting powder was extracted with methanol (MeOH) for 2 weeks to give the green extract (9.85 g) after being filtered and evaporated the solvent. This extract was partitioned between ethyl acetate (EtOAc) and water. The organic layer was concentrated in vacua to afford the viscous oil (5.42 g) which was chromatographed on Sephadex LH-20 using MeOH and chloroform (CHC1 3) (1 : 1) to divide into 7 fractions. Fraction A-4 (l 19.4mg) contained precipitate which was filtered off through a short column packed with cotton and evaporated the sol­ vent to give lunularic acid (5) (36.9 mg). Fraction A-2 (2.380 g) was further chro­ matographed on Sephadex LH-20 using the same solvent system described above to divide into 5 fractions (B). Fraction B-3 ( 1. 719 g) was further chromatographed on silica gel using n-hexane and EtOAc (9: 1) to give 5 fractions (C).Fraction C-2 was purified by HPLC (sil­ ica gel/n-hexane-EtOAc 9 : l) to yield stigmast-4-en-3-one (1) (2.1 mg) and sitost-4-en-3- one (2) (2. 7 mg) whose spectral data were identical with those of the synthetic products prepared from stigmasterol (3) and sitosterol (4) by Oppenauer oxidation (see later). Frac­ tion C-3 contained phytol (55.2 mg). Fraction C-4 contained a mixture of phytosterols, stig­ masterol (3) and sitosterol (4) (173.4mg). The presence of compounds (3) and (4) was con­ firmed by comparison of mass spectra of their acetates with those of authentic samples. Fraction C-5 (293.6 mg) contained a mixture of fatty acids which was treated with N,0- bis(trimethylsilyl)acetamide in acetonitrile to give the mixture of silyl ethers. GC-MS analysis of the resulting silyl esters showed the presence of caprylic acid, palmitic acid, stearic acid and linoleic acid of which the major component was palmitic acid. Fraction B- 4 (722.9 mg) was methylated with diazomethane to give a mixture of methyl esters. GC­ MS analysis showed that the resulting methyl esters contained methyl esters of palmitoleic, T . YOSHIDA et al. : Chemical constituents of Japanese Ricciocarpos 259 7, 10, 13-hexadecatrienoic, 11, 14-octadecadienoic or 10, 13-octadecadienoic, linoleic acid and linolenic acid of which 11,14-octadecadienoic acid was the major component. Oppenauer Oxidation of Stigmasterol (3) and Sitosterol ( 4). To stigmasterol (3) (Tama Biochem. Co. Ltd.) (115.3 mg) in cyclohexanone (1.2 ml) was added aluminium isopropoxide (125 mg) in dry toluene and refluxed for 1 hr at 135°C. Work-up as usual gave stigmast-4-en-3-one (1) (88.9 mg, 77.5% yield). Sitosterol (4) (Nakarai Tesque. Inc.) (158 mg) was treated in the same manner as described above to af­ ford sitost-4-en-3-one (2) (127.1 mg, 80.8% yield). RESULTS AND D ISCUSSION It has been known that European in vitro cultured R. natans produces not only cu­ parane- (for example 6) and monocyclofamesane-type sesquiterpenoids (for example 7) (Wurzel & Becker 1989, 1990), but also phenolic glucosides (9, 10) (Kunz & Becker 1992, 1994) and cyclic bis(bibenzyl), riccardin C (11) and its dimer (12) (Kunz & Becker 1994). However, neither sesquiterpenoids nor phenolic compounds including cyclic bis(bibenzyl) dimer, pusilatin B (12), have been detected in the methanol extract of Japanese R. natans except for the presence of bibenzyl derivative, lunularic acid (5). On the other hand, the 0 (1) (2) (3) (4) OH (5) Fig. 1. Steroid ketones and sterols found in field collected R. natans. 260 J. Hattori Bot. Lab. No. 81 I 9 9 7 ~o-{, --{fijJ (6) (7) (10) (8) (9) OH HO OH HO OH OH (12) (11) Fig. 2. Representative compounds found in R. natans grown m axemc culture. (Wurzel & Becker 1989, 1990; Kunz & Becker 1992; Siegmund & Becker 1994) steroid ketones (1) and (2) found in Japanese R. natans have not been detected in the cul­ tured R. natans. Although the presence of phytosterols, such as campesterol, stigmasterol (3) and sitosterol (4) have been isolated or detected in a great number of bryophytes (Asakawa 1982, 1995; Huneck 1983), the presence of steroid ketones, stigrnast-4-en-3-one (1) and sitost-4-en-3-one (2) has not been found in bryophytes. These steroid ketones have been found in some sponges (Sheikh & Djerassi 1974) and the heartwood of Tabebuia rosea. (Joshi et al. 1974). The fatty acid components of R. natans are very similar to those detected in the other liverworts (Huneck 1983). It seems that there are at least two chemo­ types of Ricciocarpas natans, although the chemical constituents of the type I originated from axenic cultured R. natans; the type I (European population) produces sesquiter­ penoids and phenolic compounds including their glucosides and the type II (Japanese pop­ ulation) does not elaborate the above components. There are two genera of the Ricciaceae, the Ricciocaropos (L.) Corda and Riccia L. We are currently studying the chemical con- T. YOSHIDA et al.: Chemical constituents of Japanese Ricciocarpos 261 stituents of R. natans collected in the different location and a few Riccia species in order to look for the different chemo-type of the species of each genus and to compare the chemical constituents between the Ricciocarpos and Riccia species. A CKNOWLEDGMENTS The authors thank Mr. S. Mitani for his collection of Ricciocarpos natans and to Dr. M. Mizutani (The Hattori Botanical Laboratory, Nichinan, Japan) for his identification of the liverwort. A part of this work was supported by a Grant-in-Aid of Cancer Research from the Ministry of Education, Japan. LITERATURES CITED Asakawa, Y. 1982. Chemical Constituents of Hepaticae. In W. Herz, H. Grisebach & G. W. Kirby (eds.), Progress in the Chemistry of Organic Natural Products: vol.