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ont matter # 2004 Elsevier Ltd. All rights reserved.

03.08.010

Biochemical Systematics and Ecology 32 (2004) 449–453

www.elsevier.com/locate/biochemsyseco

Chemical study of Ramalina africana(Ramalinaceae) from the Garhwal Himalayas

Vertika Shukla a, Sandeep Negi a, M.S.M. Rawat a,�, Geeta Panta, Akito Nagatsu b

a Department of Chemistry, H.N.B. Garhwal University, Srinagar, Garhwal 246 174, Uttaranchal, Indiab Faculty of Pharmaceutical Sciences, Nagoya City University, Nagoya, Tanabe-dori, Mizuho-ku, Japan

Received 28 January 2003; accepted 18 August 2003

Keywords: Ramalina africana; Usnic acid; Divaric acid; Ethyl divaricatinate; Sterols

1. Subject and source

Ramalina africana (Stein) Dodge, Ramalinaceae, is a fruticose lichen highly

sensitive to air pollution found in the remote forests of the Garhwal Himalayas at

an altitude of 1800 m. It was collected in October 2000 from Khirsu, Pauri Garh-

wal, Uttaranchal, India and identified by Dr. D.K. Upreti, National Botanical

Research Institute, Lucknow, Uttar Pradesh, India. Voucher specimen 0-219568/C

(LWG) is deposited in the lichen herbarium of N.B.R.I., Lucknow.

2. Previous study

(+)-Usnic, evernic, obtusatic acids were isolated from R. farnacea (L.) Ach.

(Rastogi and Mehrotra, 1970–1979). d-Protolichesterinic and nephrosterinic acids

were obtained from R. almquisii (Hirayama et al., 1980). Amino acid constituents

of R. sinensis Jatta have been studied (Ramakrishnan and Subramanian, 1966). So

far, no phytochemical work has been reported on R. africana.

V. Shukla et al. / Biochemical Systematics and Ecology 32 (2004) 449–453450

3. Present study

Dried and powdered plant material (1 kg) was extracted (Soxhlet) with ethanol(95%; 40–50

vC). The ethanolic residue was further extracted by light petroleum

(60–80vC). The petroleum extract (100 g) was column chromatographed over Si-

gel using gradient elution with light petroleum (60–80vC) and acetone (99.5:0.5–

86:14), affording divaric acid (4 mg), ethyl divaricatinate (8 mg), (+)-usnic acid (25mg), and a mixture of sterols. The separation of sterols was done by repeated col-umn chromatography on silica gel using light petroleum (60–80

vC) and acetone

(90:10) as eluting solvents, which gave b-sitosterol (4 mg), 24-methylcholesta-5,22-dien-3b-ol (brassicasterol; 10 mg) and ergosta-5,8,22-trien-3b–ol (lichesterol; 8 mg).The compounds were characterized by EI-mass spectrum (MS), IR, 1H NMR, 13CNMR, preparation of their derivatives and by comparing the data with those in theliterature (Chamy et al., 1985; Culberson, 1967; Thompson et al., 1972; Lentonet al., 1973; Huneck et al., 1968).

4. Result and discussion

EI-MS was recorded on JMS-SX102A 5890 series II spectrometer. Opticalrotation was measured on a JASCO DIP 140 digital polarimeter, and IR on anFTIR 8100 Shimadzu spectrophotometer. The 1H and 13C NMR spectra wererecorded on a JEOL JNM-A600 spectrometer with TMS as an internal standardand chemical shifts were expressed on the d (ppm) scale. TLC was performed onsilica gel using CHCl3–MeOH–H2O (64:32:10, lower layer) and the spots weredetected by spraying with 5% H2SO4 reagent followed by heating.

4.1. Divaric acid

The EI-MS exhibited molecular ion at m/z 196 (C10H12O4). M.p. 158–160vC

[lit. m.p. 157–159vC (Chamy et al., 1985)]. In the 1H NMR spectrum, multiplet at

d 1.56 (2H), triplet at 2.85 (2H) and a triplet at 0.96 (3H) suggested an n-propylgroup linked to an aromatic ring. Signals were exhibited corresponding to two aro-matic meta-coupled protons, a doublet at d 6.30 (J ¼ 2:5 Hz) and a doublet atd 6.37 (J ¼ 2:5 Hz). The 1H NMR also exhibited a broadened singlet at d 9.58(3H) due to two phenolic and one acidic protons. The nature of these groups wasalso evident from the IR spectrum (3380, 1630 cm�1). On the basis of these datatypical of a substituted divarinol nucleus (Chamy et al., 1985), compound 1 wasidentified as divaric acid.

4.2. Ethyl divaricatinate

The EI-MS revealed ion peak at m/z 238 corresponding to the molecular for-mula C13H18O4 (M+, 238). M.p. 40–42

vC [lit. m.p. 41–42

vC (Chamy et al.,

1985)]. The 1H NMR spectrum exhibited a downfield one-proton singlet at d 11.8ppm assignable to the chelated hydrogen of the phenolic group. The meta coupledone-proton doublets at d 6.34 (3-H) and 6.28 (5-H) ppm with J values 2.5 Hz indi-

451V. Shukla et al. / Biochemical Systematics and Ecology 32 (2004) 449–453

cated the presence of a tetra substituted benzene nucleus. The other signalsrevealed the presence of an ethyl ester group; d 4.40 (2H, q, J ¼ 7:0 Hz) and 1.41(3H, t, J ¼ 7:0 Hz) and an n-propyl attached to an aromatic ring: d 2.85 (2H, t),1.50 (2H, m) and 0.96 (3H, t). Additionally, a sharp three-proton singlet at d 3.80indicated the presence of a methoxy group. These observations were affirmed byCOSY spectrum. The long range correlations were shown by HMBC spectrum,aromatic proton (5-H) showed correlation with C-1, C-3, C-4 and C-7, while meth-oxyl proton (8-H) displayed its correlation with only C-4 carbon affirming its directlinkage at C-4 position. From the above spectral data, 2 was identified as ethyldivaricatinate.

4.3. (+)-Usnic acid

Obtained as yellow crystals. TLC showed single spot. The EI-MS revealed apeak at m/z 344 corresponding to the molecular formula C18H16O7 and ions at m/z329, 260, 233 displayed the fragmentation pattern of the parent and fragmentions as reported (Huneck et al., 1968). 1H NMR spectrum showed signal of an aro-matic proton at d 6.2 (1H, s). The three phenolic protons gave the downfield sig-nals at d 11.3 (1H, s, 9-OH), 13.30 (1H, s, 7-OH) and 18.80 (1H, s, 3-OH) showingthe intramolecular hydrogen bonding (chelation). The structure was finally estab-lished by the 13C NMR spectrum using DEPT mode with flip angles at 45

v, 90

v,

and 135vto establish multiplicities. 3 was identified as usnic acid by the study of its

IR, MS, HMQC, HMBC and COSY. Circular dichroism established compound 3

to have positive rotation. Final identification of the compound 3 was established as(+)-usnic acid by comparison of m.p. 203–204

vC [lit. 203–204

vC (Culberson,

1967)].

4.4. b-Sitosterol

Crystallized from methanol as white needles. M.p. 139–140vC [lit. m.p. 139–140

vC

(Thompson et al., 1972)]. IR (mmax KBr cm�1) gave bands at 3340, 2970, 2959and 1640. The ion peak at m/e 414 suggested its molecular formula C29H50O. The1H NMR spectrum revealed the presence of signals at d 0.68, 0.78, 0.88 (each 3H),d 0.97 (6H, d, two methyl groups) and d 1.0 (3H, s). A signal at d 3.7 (31H, s)observed for 31 protons of methylene and methine type. A signal for OH groupproton was observed at d 4.5. Co-TLC, and comparative study of its IR, MSand NMR established its structure to be b-sitosterol. The structure was furtherconfirmed by preparation of its acetate m.p. 125–126

vC [lit. m.p. 125–126

vC

(Gupta and Gang, 1968)].

4.5. Brassicasterol

The EI-MS exhibited peak at m/z 398 corresponding to the molecular formulaC28H46O. The

1H NMR spectrum revealed the presence of trans-olefinic proton atd 5.19 (2H, m, J ¼ 7 Hz, 22H and 23H). The side chain methyl resonances were atd 1.01 (3H, d, J ¼ 6 Hz, 21-CH3), 0.82 (3H, d, J ¼ 7 Hz, 26-CH3), 0.83 (3H, d,J ¼ 7 Hz, 27-CH3), 0.90 (3H, d, J ¼ 7 Hz, 28-CH3). The two methyls of the sterol

V. Shukla et al. / Biochemical Systematics and Ecology 32 (2004) 449–453452

ring occurred at d 0.69 (3H, s, 18-CH3) and 1.01(3H, s, 18-CH3). The13C signals at

121.7,131.7,135.8 and 140.7 ppm indicated the presence of two double bonds. 13Csignal at 71.8 ppm confirmed the b-linkage of -OH group to the ring at C-3 posi-tion. The above spectral data established the structure of compound 5 to be 24-

methylcholesta-5,22-dien-3b-ol (brassicasterol) m.p. 149–151vC [lit. m.p. 149–151

vC

(Thompson et al., 1972)], and by comparison of the mass spectral data (Lentonet al., 1973).

4.6. Lichesterol

Crystallized as plates from acetone:methanol. IR (mmax KBr cm�1) showed peaks

at 805, 965, 1235, 1265. m.p. 124–125vC [lit. m.p. 125–126

vC (Lenton et al.,

1973)]. The mass spectral data of acetate showed ions at m/e 438, 378, 363, 353,351, 337, 313, 253 and 211 confirming two nuclear double bonds and the presenceof an ion peak at m/e 337 suggested a ring B diene. The 1H NMR data showedtrans-olefinic protons at d 5.19 (2H, m, J ¼ 6:0 Hz, H-22/H-23) and the proton ofa trisubstituted double bond at d 5.34 (1H, m, 6H). The second nuclear doublebond was tetrasubstituted and was assigned at 8(9) due to the methyl proton reso-nances at d 0.67 (3H, s, 18-CH3), and 1.20 (3H, s, 19-CH3), lower than in sterolswith 8(14) double bond. The side chain methyl resonances d 1.01 (3H, d, J ¼ 6 Hz,21-CH3), 0.82 (3H, d, J ¼ 7 Hz, 26-CH3), 0.84 (3H, d, J ¼ 7 Hz, 27-CH3), 0.91(3H, d, J ¼ 7 Hz, 28-CH3) were all consistent with the reported data (Lenton et al.,1973) of lichesteryl acetate.

5. Chemotaxonomic significance

Usnic acid is reported as the chemotaxonomic marker of the family Usneaceae.Divaric acid and ethyl divaricatinate were previously reported from Protusneamalacea (Chamy et al., 1985). Also divaric acid and ethyl divaricatinate and sterolsas b-sitosterol, brassicasterol, and lichesterol are being reported in Ramalina spe-cies for the first time.

Acknowledgements

We thank Dr. D.K. Upreti, N.B.R.I., Lucknow for identification of the lichensample.

References

Chamy, M.C., Gambaro, V., Garbarino, J.A., 1985. Journal of Natural Products 48 (2), 307.

Culberson, C.F., 1967. Phytochemistry 6, 719.

Gupta, D.R., Gang, S.K., 1968. Journal of Indian Chemical Society 45, 256.

Hirayama, T., Fujikawa, F., Kasahara, T., Otsuka, M., Nishida, N., Mizuno, D., 1980. Yakugaku Zas-

shi 100 (7), 755, (MAPA 1981,02, 0865).

453V. Shukla et al. / Biochemical Systematics and Ecology 32 (2004) 449–453

Huneck, S., Djerassi, C., Becher, D., Barber, M., 1968. Tetrahedron 24, 2707.

Lenton, J.R., Goad, L.J., Goodwin, T.W., 1973. Phytochemistry 12, 1135.

Ramakrishnan, S., Subramanian, S.S., 1966. Current Science 5, 124.

Rastogi, R.P., Mehrotra, B.N., 1970–1979. Compendium of Indian Medicinal Plants vol. 2. Central

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Thompson, M.J., Dutky, S.R., Patterson, G.W., Gooden, E.L., 1972. Phytochemistry 11, 1781.