Chemistry and Bioactivity of Physciaceae Lichens Pyxine consocians. and Heterodermia leucomelos.

Abstract

Cabraleadiol monoacetate, atranorin, methyl-β.-orcinolcarboxylate, zeorin, 4-O.-methylcryptochlorophaeic acid, and lichexanthone were isolated from the lichen Pyxine consocians Vainio... Heterodermia leucomelos (L)... Poelt contained a new triterpenoid, 6α-hydroxyhop-21β.H-22(29)-en, along with atranorin, zeorin, glyceryl trilinolate, and 3,6-dimethyl-2-hydroxy-4-methoxybenzoic acid. Cabraleadiol monoacetate, 4-O.-methylcryptochlorophaeic acid, lichexanthone, and 3,6-dimethyl-2-hydroxy-4-methoxybenzoic acid showed mosquito larvicidal activity against the second instar larvae of Aedes aegypti.. Lichexanthone exhibited human sperm motility enhancing activity.

Keywords:

• Bioactivity
• Heterodermia leucomelos
• lichen
• lichen metabolites
• new triterpenoid
• Physciaceae
• Pyxine consocians

Introduction

Lichens are distributed worldwide and grow on rocks and poorly developed soils or as epiphytes on trees and shrubs. They produce large concentrations of lichen substances, which are bioactive (Huneck, 1999). In our continuing search for biologically active compounds from tropical lichens, we have investigated Pyxine consocians. Vainio and Heterodermia leucomelos (L)... Poelt, which are foliose type lichens belonging to the family Physciaceae. In this paper, we report the isolation and structural elucidation of several metabolites, including a new triterpenoid 6α-hydroxyhop-21β.H-22(29)-en (7). A number of these constituents have been evaluated for their mosquito larvicidal and spermicidal/sperm motility enhancing activity.

Materials and Methods

General experimental procedures

Melting points (uncorrected) were determined by using a Kofler hot-stage apparatus (UK). UV absorptions were measured with a Shimadzu 1601 UV spectrophotometer (Japan). ¹H and ¹³C NMR, correlation spectroscopy (COSY), distortionless enhanced polarization transfer (DEPT), heteronuclear correlation spectroscopy (HETCOR), heteronuclear multiple quantum correlation (HMQC), and heteronuclear multiple bond correlation (HMBC) spectra were recorded on a VARIAN (¹H 300 and ¹³C 75.45 MHz) in CDCl₃ with TMS as the internal standard (USA). Low- and high-resolution electron impact mass spectra were recorded on a Kratos/AEI MS-902 spectrometer (USA). Silica gel used was Merck Kieselgel (230–400 mesh ASTM) (Germany).

Lichen collection

P. consocians. was collected from the campus of the University of Peradeniya growing on the stem bark of the palm tree Roystonia regia., and H. leucomelos. was collected from the mossy rocks in the Horton Plains, Central Province, Sri Lanka. Specimens were identified by Pat Wolseley of the British Natural History Museum, and voucher specimens have been deposited at the Royal Botanic Gardens, Peradeniya, Sri Lanka.

Extraction and isolation

Cleaned and air-dried P. consocians. lichen (100 g) was ground into a powder and sequentially extracted into hexane, CH₂Cl₂, and MeOH (1 l each) in a Soxhlet extractor for 2 h. The solvent was removed under reduced pressure (<40°C) using a rotavapor. Cleaned and air-dried H. leucomelos. lichen (40 g) was ground into a powder.

Pyxine consocians

The hexane extract of P. consocians. was subjected to MPLC (eluent: hexane to ethyl acetate to MeOH) yielded atranorin (2) (13 mg) (crystallized CH₂Cl₂). Further separation by MPLC (eluent: ethyl acetate:hexane (1:19) to ethyl acetate) yielded methyl-β.-orcinolcarboxylate (3) (5.4 mg) and zeorin (4) (24.6 mg). Further separation by MPLC [eluent: CH₂Cl₂: hexane (1:1) to MeOH: CH₂Cl₂ (1:39)] to give cabraleadiol monoacetate (1) (50 mg) and 4-O.-methylcryptochlorophaeic acid (5) (12 mg). MPLC of the methanol extract (eluent: hexane to CH₂Cl₂ to MeOH) yielded lichexanthone (6) (51.2 mg).

Heterodermia leucomelos

The finely powdered lichen of H. leucomelos. was ground together with the silica gel (40 g) and subjected to MPLC [eluent: CH₂Cl₂:hex (1:9) to MeOH] to yield glyceryl trilinolate (8) (4 mg) and atranorin (2). Further separation by MPLC [eluent: CH₂Cl₂:hexane (3:2) to CH₂Cl₂] gave 3,6-dimethyl-2-hydroxy-4-methoxybenzoic acid, 9 (1.6 mg), the 6α-hydroxyhop-21β.H-22(29)-en, 7 (4.2 mg), and zeorin, 4 (130 mg).

Cabraleadiol monoacetate (.1)..: Pale yellow needles (CH₂Cl₂); m.p. 158–160°C (lit. m.p. 148–149°C) (Qui et al., 2001); Cabraleadiol (.1′)..: Pale yellow needles (CH₂Cl₂); m.p. 180–182°C (lit. m.p. 176–177°C) (Qui et al., 2001); [α]_D^25.7 + 13.3° (CH₂Cl₂, C 0.3); lit. [α]_D + 18.0° (C 1.0) (Rao et al., 1975). Atranorin (.2)..: Colorless needles (CH₂Cl₂) (Brassy et al., 1982). Methyl-β.-orcinolcarboxylate (.3)..: Colorless small needles (CH₂Cl₂) (Caccamese et al., 1986). Zeorin (.4).:. Colorless needles (CH₂Cl₂) (Yosioka et al., 1972). 4-O-Methylcryptochlorophaeic acid (.5)..: Colorless needles (CH₂Cl₂) (Shibuya et al., 1983). Lichexanthone (.6).:. Straw yellow needles (CH₂Cl₂) (Hay & Harris, 1972).

6α-Hydroxyhop-21β.H-22(29)-en (.7)..: Colorless powder; m.p. 153–155°C; ¹H NMR (500 MHz, CDCl₃): δ 0.60 (3H, s, Me-28), 0.75 (3H, s, Me-25), 0.85 (3H, s, Me-24), 0.88 (1H, s, H-1β.), 0.91 (3H, s, Me-27), 0.98 (3H, s, Me-26), 1.03 (3H, s, Me-23), 1.37 (2H, m, H-19), 1.47 (1H, s, H-7), 1.58 (1H, s, H-1α), 1.78 (3H, s, Me-30), 2.20 (1H, m, H-21), 2.70 (2H, m, H-20), 3.98 (1H, dt, J. = 16.8, 3.9 Hz, H-6), 4.80 (2H, bs, =CH₂, H-29) and 1.10–2.00 (16H); ¹³C NMR (125 MHz, CDCl₃): C-1: 40.5, C-2, C-26: 18.7, C-3, C-19: 44.0, C-4, C-15: 33.8, C-5: 61.3, C-6: 69.5, C-7: 45.7, C-8, C-14, C-18: 42.0, C-9: 49.2, C-10: 39.5, C-11: 21.2, C-12: 24.0, C-13: 50.0, C-16: 21.8, C-17: 55.1, C-20: 25.2, C-21: 46.6, C-22: 148.8, C-23: 36.9, C-24: 22.3, C-25: 17.3, C-27: 17.0, C-28: 16.2, C-29: 110.3, C-30: 27.5; EIMS m/z (rel. int. %) 409, 205, 189, 154, 135, 121, 107 (100); HREIMS (M-H)⁺, 425.3756; calcd. for C₃₀H₄₉O, 425.3783.

Glyceryl trilinolate (.8).:. Yellow oil (Yueh–Hsiung Kuo & Pei–Hungchu, 2002). 3,6-Dimethyl-2-hydroxy-4-methoxybenzoic acid (.9)..: Pale yellow needles; m.p. 195–196°C (lit. m.p. 196°C) (Tsuda et al., 1980).

Bioassays

The mosquito larvicidal assay was carried out on cabraleadiol monoacetate (1), atranorin (2), methyl-β.-orcinolcarboxylate (3), zeorin (4), 4-O-. methylcryptochlorophaeic acid (5), lichexanthone (6), and 3,6-dimethyl-2-hydroxy-4-methoxybenzoic acid (9) against the second instar larvae of Aedes aegypti. (Bandara et al., 2000).

Human sperm motility stimulating activity was measured against ejaculated human spermatozoa (Belsy et al., 1980) at 10, 100, 1000 µg/ml of test compound following 5, 15, 30 and 60 min of incubation. Evaluation was carried out on atranorin (2), zeorin (4), and lichexanthone (6).

Results and Discussion

The hexane extract of P. consocians. yielded cabraleadiol monoacetate (1). Treatment of compound (1) in 6% MeOH/KOH resulted in the loss of the acetyl moiety, yielding the corresponding alcohol (1′). Spectral and physical data of the hydrolyzed product were identical to those reported for cabraleadiol (1′) reported from the plants Dysoxylum malabaricum. (stem bark) (Hisham et al., 1996), Cabralea eichleriana. (wood) (Rao et al., 1975), Cabralea polytricha. (fruits) (Cascon & Brown, 1972), Aglaia lawaii. (Qui et al., 2001), and Nothofagus dombeyi. (Thoison et al., 2004). That our study constitutes the first report of a triterpenoid of the cabraleadiol family from a lichen is of chemotaxonomic significance.

The lichen powder of H. leucomelos. furnished compound 7 whose high resolution electron ionization mass spectrometry (HREIMS) showed a self-deprotonated molecular ion at 425.3756 suggesting an elemental composition of C₃₀H₅₀O. The ¹³C NMR spectrum showed 26 signals of which some were overlapping. The ¹H/¹³C NMR spectra of compound 7 exhibited seven quarternary methyl singlets. HMBC and COSY data (Fig. 1) readily identified the A–E ring system of a triterpenoid with an α-hydroxyl functionality at C-6 of ring B (δ 3.98, dt, J. = 16.8, 3.9 Hz, H-6) and an α-isopropylidene moiety attached to C-21 of ring E. A comparison of the ¹H NMR chemical shift values for H-21 in zeorin (4) (δ 2.28, dt, J. = 14.7, 3.3 Hz), acetyl zeorin (δ 2.19, dt, J. = 12.4, 3.9 Hz), zeorinone (δ 2.19, dt, J. = 12.3, 1.8 Hz) with the triterpenoid 7 (δ 2.20, m), lent credence to the position and stereochemistry of the isopropylidine group. Furthermore, the NMR spectral data of compound 7 differed from those of lupeol (isopropylidene group at C-19) [compound 7: H-21 (¹H δ 2.20, m; ¹³C δ 44.0); lupeol: H-19 (¹H δ 2.38, dt, J. = 16.5, 5.7 Hz; ¹³C δ 48.3)]. To the best of our knowledge, the triterpenoid 7 is a new natural product.

Figure 1 HMBC and COSY correlations of the triterpenoid 7.

The following compounds showed significant mosquito larvicidal activity against the second instar larvae of Aedes aegypti. at 10 ppm: Cabraleadiol monoacetate (1) (90% moribund after 24 h), 4-O.-methylcryptochlorophaeic acid (5) (60% dead after 24 h), lichexanthone (6) (80% moribund after 24 h), and 3,6-dimethyl-2-hydroxy-4-methoxybenzoic acid (9) (90% moribund after 24 h). 4-O.-Methylcryptochlorophaeic acid (5) is a known inhibitor of prostaglandin synthetase (Shibuya et al., 1983). Lichexanthone (6) exhibited sperm motility enhancing activity when incubated with human spermatozoa; the increment in motility was significant from the fifth min and lasted throughout the incubation period of 60 min. Currently there are only a few known sperm motility stimulants, including caffeine (Aitken et al., 1983), and there is a need and demand for new sperm stimulants to be used in asthenozoospermia and in some assisted reproductive program.

Acknowledgments

The authors thank the NSF and NRC, Sri Lanka, the International Foundation for Science, Stockholm, Sweden, and the Organisation for the Prohibition of Chemical Weapons (OPCW), The Hague, The Netherlands, through a grant to Dr. Veranja Karunaratne.