Abietane diterpenoids from Salvia rubescens ssp. truxillensis

PHARMACEUTICA ACTA HELVETIAE ELSEVIER

Phamraceutica Acta Helvetiae 72 (1997) 233-238

Abietane diterpenoids from Salvia rubescens

truxillensis

ssp.

’

J. Manuel Amaro-Luis Grupo de Productos Naturales, Departamento de Quhica,

Far&ad

de Ciencias, Uniuersidad de Los Andes (ULA), Estado M&da, Merida 5101, Venezuela

Received 8 October 1996; revised 29 November 1996; accepted 18 December 1996

Abstract From aerial parts of Salvia rubescens ssp. tru.xilZensis the abietane diterpenes camosol, 11,12-di-0-methylcamosol, 16-hydroxycamosol, rosmanol and 16-hydroxy-7-methoxyrosmanol have been isolated and identified on the basis of spectral data. An exhaustive allowed the revision of analysis of the uni- and two-dimensional ‘H- and 13C-NMR spectra of 16-hydroxy-7-methoxyrosmanol, previously assignment on some ‘H- and 13C-NMR signals. The above refereed 16-hydroxylated diterpenes are reported for the second

time as natural products. 0 1997 Elsevier Science B.V. Keywords: Saluia rubescens ssp. truxillensis; Lubiatae; Diterpenes; Abietane; Bidimensional RMN

1. Introduction The large Salvia genus (Labiatue) with over 900 species is found throughout most of the world and particularly in tropical and temperate regions (Hedge, 1992). Since ancient times, many species of this genus have been used as culinary herbs or as medicinal plants for the treatment of microbial infections, viral hepatitis, heart disease, menstrual disorders, hemorrhages and other blood abnormalities. (Chen, 1984; Heinrich, 1992; Rivera Ntifiez and Ob6n de Castro, 1992). The phytochemical studies of several Sulviu species have led to the isolation of flavonoids, pentacyclic triterpenoids and mainly abietane and neoclerodane diterpenoids, which show antioxidant properties (Cuvelier et al., 1994) and antitumoral, antimicrobial and anti-feedant activity (Blaney et al., 1988; Gonzalez et al., 1989a; Luis, 1991; Rodriguez-Hahn et al., 1992a,b). In continuation of our chemical studies on species from the flora of Venezuela used in popular medicine, I have

’ Part 12 in the series ‘Phytochemical Studies on the Venezuela Andean Flora’. For Part 11 see J.M. Amaro-Luis and M. A&i&n R. (1997) Pharmazie 52, 162-163. 0031-6865/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved. PII SOO31-6865(97)00012-5

now investigated the aerial parts of Suluiu truxillensis Briq an endemic species to the Andean region, recently reclassified as S. rubescens Kunth in H.B.K. ssp. truxillensis (Briq.) Wood and Harley (1989). To the best of my knowledge, there are no reports in the literature referring to chemical or bioactivity studies of this subspecies.

2. Experimental

procedures

2.1. General experimentul

procedures

Melting points were determined with a Fisher-Johns apparatus and they have not been corrected. Optical activities were measured in CHCl, on a Rudolph Research Autopol III polarimeter. IR spectra were taken on a Perkin-Elmer FT-1725X spectrophotometer and UV spectra on a Varian Stand 3 instrument. ‘H-, 13C- and two-dimensional NMR spectra were recorded on Bruker AMX400 spectrometer. Mass spectra were run on a HewlettPackard 5930A at 70 eV. TLC was carried out on 0.25 mm layers of silica gel PF 254 (Merck). Vacuum column chromatography (VCC) was performed with silica gel 60 (70-230 mesh).

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Pharmaceutics

2.2. Collection and identification

of plant material

Saluia rubescens ssp. truxillensis (Briq.) Wood and Harley was collected at Quebrada de Gavidia, Mucuchies, (Estado M&da, Venezuela) in August 1989. A voucher specimen (JMA 1609) was deposited in the National Herbarium, UCV, Caracas, Venezuela. 2.3. Extraction

and isolation of compounds

Dried and finely powdered stems and leaves (1.15 kg) were extracted with distilled acetone at room temperature. The remaining residue (48 g), after evaporation of solvent under reduced pressure, was preadsorbed on celite and separated by VCC over silica gel (Co11 and Bowden, 1986) using hexane with increasing amounts of EtOAc as eluent. Fractions of 1 1 were collected and combined based upon TLC monitoring. The different fractions were purified by chromatography on Sephadex LH-20 with 2: 1:l n-hexane/CHCl,/MeOH as eluent and then by repeated flash chromatography or TLC. The compounds were obtained in the following sequence: 11,12-di-o-methylcamosol (18 mg), oleanolic and ursolic acid mixture (420 mg), carnosol (22 mg), rosmanol (16 mg), 16-hydroxycamosol (35 mg) and 16-hydroxy-7-methoxyrosmanol (30 mg). 2.4. Identification

of compounds

l-5

(1)

R,= R2= H

(4)

R,=R2=H

(2)

R,=CH,

(5)

R, = CH, R2 = CH,OH

(3)

R, = H RZ = CH,OH

R,=H

2.4.1. Carnosol (1) Crystalline solid (EtOH), mp: 232-235°C; [ a ID = - 58” (c, 0.5 in CHCl,); UV (MeOH) hmax: 210, 285 nm; IR (KBr) vmax: 3500, 3302, 171.5, 1588, 1450, 1355, 1128, 1030, 992, 923, cm-‘; ‘H-NMR (CDCl,) 6: 0.87 (3H, s, H-19) 0.90 (3H, S, H-18,) 1.19 (6H d, J= 7.0 HZ, H-16 and H-17) 1.30 (lH, dd, J, = 3.5 J,= 14.5 HZ, H-3o), 1.52-1.60 (2H, m, H-2(-y and H-3/3) 1.75 (lH, q. J= 7.0 Hz, H-5) 1.92 (lH, td, J, = 7.0, J, = 1.5 Hz, H-6P),

Acta Heluetiae 72 (1997) 233-238

2.00 (lH, dt, J, =4.0, J, = 14.0 H-2P), 221(1H, m, H-6cu), 2.40 (lH, td, J, =4.3, J2 = 14.0, H-lcr), 2.89 (lH, bd, J= 10.5 Hz, H-l p), 3.05 (lH, sept, J= 7.0 Hz, H-15) 4.25 (lH, bs, Ar-OH), 5.37 (lH, dd, J, = 1.5, J2 = 4.5 Hz, H-7), 5.75 (IH, bs, Ar-OH), 6.65 (lH, s, ~-14); ‘?c-NMR (CD,OD) 6: 19.82 (c-2), 20.06 (c-1 7), 23.22 (C-16)*, 23.31 (C-19)“, 27,83 (C-15) 30.16 (C-l)+, 30.61 (C-6)+, 32.34 (C-18), 35.31 (C-4), 42.06 (C-3), 46.50 (C-5), 49.54 (C-lo), 78.42 (C-7), 112.62 (C-14) 123.24 (C-9) 133.51 (C-8), 135.22 (C-13) 143.67 (C-l2)#, 143.80 (C-l l)#, 176.15 (C-20). (* + # Assignments interchangeable). MS, m/z (%): 330 (4, M+), 287 (8) 286 (41) 271 (8), 215 (23) 201 (16), 128 (30), 115 (43), 69 (55) 55 (100). 2.4.2. 11,12-di-0-Methylcarnosol (2) Crystalline plates (EtOH), mp: 155-157°C; [ alD = - 76” (c, 0.5 in CHCl,); UV (MeOH) h,,,: 239, 275 nm; IR (KBr) v,:,,,: 2945, 1738, 1270, 852, cm-‘; ‘H-NMR (CDCl,) 6: 0.86 (3H, s, H-19), 0.90 (3H, s, H-18), 1.18 (6H, d, J = 7.0 Hz, H-16 and H-17), 1.33 (lH, bd, J = 14.0 Hz, H-3a), 1.62-1.64 (2H, m, H-2& and H-3P) 1.70 (IH, q, H-5), 1.90 (IH, td, J, = 7.0, J, = 1.5 Hz, H-6@), 2.10 (lH, dt, J, = 4.0, .I, = 14.0 H-2@), 2.21(1H, m, H-6~) 2.43 (lH, td, J, = 4.3, J, = 14.0, H-la), 2.80 (lH, bd, J= 10.0 Hz, H-1/3), 3.28 (IH, sept, J= 7.0 Hz, H-15) 3.81 (3H, s, Ar-OMe), 3.83 (3H, s, Ar-OMe), 5.39 (lH, dd, J, = 1.5, J, =4.5 Hz, H-7), 6.84 (lH, s, H-14); “C-NMR (CDCl,) 6: 19.45 (C-2), 20.23 (C-17) 23.81 (C-19)*, 23.98 (C-16)*, 27.22 (C-15), 28.71 (C-l), 30.00 (C-6), 32.26 (C-18) 35.98 (C-4), 41.57 (C-3), 45.76 (C-5), 49.12 (C-lo), 61.10 (Ar-OMe), 61.43 (Ar-OMe) 77.92 (C-7), 116.45 (C-14) 129.35 (C-9), 135.92 (C-8), 142.45 (C-13) 151.26 (C-12)+, 151.99 (C-11)+, 176.22 MS, m/z (%I: (C-20). ( * + Assignments interchangeable). 358 (11, M+), 315 (22) 314 (loo), 299 (30) 271 (9) 245 (lo), 243 (13), 232 (231, 229 (21), 201 (191, 141 (231, 128 (32), 115 (33) 69 (66) 55 (93). 2.4.3. l&Hydroxicarnosol(3) White crystalline plates (MeOH), mp: 2 16-218°C; [ a ID = - 69.5” (c, 0.42 in CHCl,); UV (MeOH) Amax: 285 nm; IR (KBr) v,,,: 3470, 2960, 1720, 1455, 1391, 1300, 1250, 1217, 1161, 1122, 1073, 1029, 986, 920, 865, 832 cm-‘; ‘H-NMR and 13C-NMR see Table 1; MS, m/z (%Io):346 (46, M+), 315 (3) 302 (100) 287 (15) 284 (55), 271 (24) 231(17), 220 (31) 213 (52) 201 (22) 199 (30) 187 (12) 173 (14), 155 (14) 141 (19), 128 (27), 115 (28) 91 (23) 83(19), 77 (23) 69 (45) 57 (30) 55 (81). 2.4.4. Rosmanol (4) Isolated as an amorphous solid, mp: 237-239°C; [al, = - 38” (c, 0.5 in MeOH); UV (MeOH) h,,,: 212, 286

J.M. Ammo-Luis Table 1 ‘H and 13C-NMR spectral data of compounds

/ Phannaceutica

3 and 5 (S-values

Acta Heluetiae 72 (1997) 233-238

(ppm), 400 and 100 MHz, CDCl,,

3

235

TMS as int. standard)

5 13

C

‘H

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 OCH, OH

2.50, 1.761.76_ 1.762.21, 5.35, _ _ _ _ _

td; 2.90, br d (13.5) 1.95, complex 1.95, complex

6.50, 3.12, 3.75, 1.32, 0.90, 0.85, _

bs sx, (7.2) dd; 4.02, t, (9.3; 3.0) d, (7.2) s s

C

1.95,complex m dd, (4.3; 1.O)

3. 45; 6.20; 8.80 s

29.15 19.26 41.50 34.9 1 45.85 30.15 78.18 132.65 122.28 48.80 144.05 143.27 129.40 113.35 37.89 70.47 15.49 32.12 20.08 176.69 _ _

nm.; IR (KBr) vmax: 3480, 3298, 2950, 1743, 14.50, 1276, 1120, 1078, 1005, 950, 900, 855, 805 cm-‘; ‘H-NMR (CD,OD] 6: 0.89 (3H, S, H-19) 1.01 (3H s, H-18,), 1.16, 1.18 (each 3H, d, J = 7 Hz, H-16 and H-17) 2.26 (1I-I s, H-5), 3.28 (lH, sept, H-15) 3.30 (lH, bd, J= 10.5 Hz, H-lp), 4.50 (IH, d, J= 3.3 Hz, H-6), 4.57(1I-I, d, J= 3.3 Hz, H-7) 6.83 (IH, S, H-14); 13C-NMR (CD@) 6: 20.16 (C-2), 22.39 (C-17), 22.51 (C-19) 22.96 (C-16) 27.90 (C-15) 28.61 (C-l), 31.90 (C-18) 32.26 (C-4) 39.35 (C-3), 48.36 (C-lo), 51.62 (C-5), 69.17 (C-7), 79.19 (C-6), 120.46 (C-14), 124.95 (C-9), 129.20 (C-g), 137.45 (c-13), 143.17 (C-12) 145.04 (C-11) 180.02 (C-20). MS, m/z (%): 346 (48, M+), 330 (26) 314 (11) 300 (34), 287 (44), 285 (42) 284 (loo), 273 (21) 271 (44) 269 (80, 257 (26) 239 (28) 231 (46) 215 (99) 165 (25) 149 (23) 128 (24) 121 (44) 115 (25) 91 (20) 77 (18) 71 (29) 69 (37) 65 (26). 2.4.5. 16-Hydroxy-7-methoxyrosmanolf5) Pale yellow oil; UV (MeOH) A,,,: 282 nm; IR (film) 3410, 3305, 1752, 1592, 1345, 1250, 1125, 1073, V max: 930, 860, 835 cm-‘; ‘H-NMR and 13C-NMR see Table 1; MS, m/z (%): 376 (2, M+), 360 (16) 316 (6) 314 (18) 301 (5) 285 (3) 273 (4) 269 (5) 259 (8) 245 (25) 231 (12) 215 (26) 203 (lo), 187 (11), 171 (11), 165 (ll), 152

‘H

13C

3.2, dd; 1.96, td (13.4; 4) 1.62, m; 1.51, m 1.44, dd; 1.25, td 2.23, s 4.69, d, (3.3) 4.25, d, (3.3) _ _ _ _ 6.60, 3.01, 3.71, 1.30, 1.01, 0.92, _ 3.64, 3.46;

bs sx, (7.0) t; 3.90, dd, (9.3; 3.0) d, (7.0) s s s 6.38: 9.15, s

27.01 19.01 38.18 31.57 50.73 74.50 71.5 1 126.25 123.33 47.03 144.36 142.58 130.98 121.50 38.18 69.19 15.01 31.33 22.04 179.02 58.17 _

(12), 141 (15), 128 (23), 115 (28), 91 (X2), 86 (59), 84 (88), 77 (25), 71 (30), 69 (58), 57 (43), 55 (100).

3. Results and discussion Exhaustive chromatography, over silica gel and Sephadex LH-20, of the acetone extract of the air dried stems and leaves of S. truxillensis produced a mixture of oleanoic and ursolic acids and the abietatriene diterpenes camosol (l), 11,12-di-0-methylcarnosol (2), 16-hydroxycarnosol (3), rosmanol (4) and 16-hydroxy-7methoxyrosmanol (5). Compounds 1, 2 and 4 were identified by comparing their physical and spectral data with those reported in the literature. Camosol (1) has previously been isolated from Saluia carnosa, S. ojfkinalis, S. triloba and rosemary (Rosmarinus oficinalis) (Brieskorn et al., 1964; Narayanan and Linde, 1965; Wenker et al., 1965; Wu et al., 1982). Rosmanol(4) recently reported from several Suluia species (Gonzalez et al., 1987, 1989b; Luis and Grillo, 1993; Luis and San And&s, 1993; Luis et al., 1994; Cuvelier et al., 1994), was first isolated from rosemary by Nakatani and Inatani (1981) who on the basis of spectroscopic evidence proposed a /3-orientation for the C-7 hydroxyl group, but

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Acta Heluetiae 72 (1997) 233-238

and S. munzii, respectively, (Luis et al., 1993; Luis and Grille, 1993;) are here studied in detail using the advanced NMR instrumentation, in order to have a complete picture of the assignments of all C and H atoms present in the respective molecules. The “C-NMR spectra of 3 (Table 1) indicated the presence of 20 carbons (discriminated at the DEPT spectra as three methyl, four methylene, four aliphatic and one aromatic methine carbons, and eight quatemary carbons) and its ‘H-NMR spectrum showed signals for 26 hydrogens. These data, in conjunction with a molecular ion at m/z:346 in its mass spectrum, indicated the molecular

later it was revised and assigned an a-orientation by means of an X-ray crystal structural analysis of the correspondent triacetylderivative (Nakatani and Inatani, 1984; Fraga et al., 1985). Diterpenes 1 and 4 exhibit strong antioxidative properties (Cuvelier et al., 1994) and possess a moderate antibacterial and antiviral activity (Gonzalez et al., 1989a; Paris et al., 1993). Compound 2, known only as a synthetic product (Shew and Meyer, 1968) has recently been reported as a natural constituent of S. columbariae (Luis et al., 1994). Diterpenes 3 and 5, which have previously been described only once as natural constituents of S. mellifera

OCH, I

H-14

H-7

H-6

1

I

j I P

(PDm)

20

40

HI/G

[

:.

.- 60

H7/0cHs ....I

100

aa

,. H7lC9

J 01

[

(_

.;

__,

B?

0

..-.

Fig.

1.HMBC spectrum of 16hydroxy-7-methoxyrosmanol(5).

-160

0

J.M. Amaro-Luis / Pharmaceutics Table 2 i3C-iH long-range

coupling

for 3 and 5, determined

3

by HMBC

5

‘H

i3C

H-l (Y H-2 (Y H-5 H-6P H-7 H-14 H-16

C-3, C-4, C-l, c-7, c-5, C-7, c-13,

C-9, C-10, C-20 C-10 C-4, C-9, C-18, C-19 C-8, c-10 c-9, c-14 C-9, C-12, C-15 c-17

‘H

t3C

H-lo H-5 H-7 H-14 H-15 H-16 H-17 H-18 -0C H,

c-2, c-9, c-10, c-20 c-9, C-18, c-19, c-20 C-5, C-6, C-9, -OCH, c-7, c-9, c-12, c-15 c-12, c-13, c-14, c-17 c-13, c-17 C-13, C-15, C-16 c-5 C-7

formula C,,H,,O,, which corresponds to eight units of unsaturation. Its IR spectrum exhibited the characteristic absorptions due to alcoholic and phenolic hydroxyl groups (3.470, 2960 cm-‘), a saturated Glactone (1.720 cm-’ ) and aromatic groups (920, 865 cm- ’ ). The presence in the ’ H-NMR spectrum of a 2-hydroxyisopropyl group (6 1.32, 3H, d, J= 7.2 Hz; 6 3.12, lH, sext, J = 7.2 Hz; S 3.75, lH, dd, J’ = 9.3 Hz J, = 3.0 Hz; 6 4.02, lH, t, J= 9.3 Hz), an aromatic proton (6 6.50) and two typical one-proton singlets exchangeable with D,O (6 6.20 and 6 8.801, was clearly indicative of an abietatriene skeleton with two phenolic hydroxyl groups. In addition, the existence of two singlets at 6 0.85 and 6 0.90, characteristics of a gem-dimethyl group and a double doublet at 6 5.35 (J, = 4.3, Hz J, = 1.0 Hz), typical of a benzylic hydrogen geminal to an oxygen function, agree with the structure of 16-hydroxycamosol. Confirmation of the proposed structure (3), came from analysis of ‘H-COSY, HMQC and HMBC spectra. The 13C-’ H connectivities (Table 2) revealed by these two-dimensional NMR techniques, allowed the assignments shown in Table 1. The structure of 5 was established as 16-hydroxy-7methoxyrosmanol from the following data. Low resolution mass spectrum showed a molecular ion at m/z 376 and the 13C-NMR spectrum displayed signals for 21 carbon atoms, which were completely characterized (hybridization and substitution grade) by its DEPT orientation, suggesting for compound 5 the molecular formula C 2’H 280,. The ’ H-NMR spectrum showed signals due to a methoxy group (6 3.641, two tertiary methyls (6 0.92, 6 1.011, an aromatic proton (6 6.601, a monohydroxyisopropyl group on an aromatic ring (6 1.30, 3H, d, J = 7.0 Hz; 6 3.01, lH, sext, J = 7.0 Hz; S 3.71, t, lH, J= 9.3 Hz; S 3.90, lH, dd, J, = 9.3 Hz J2 = 3.0 Hz) and two deuterium oxide exchangeable phenolic hydroxyl protons (S 6.38, 6

Acta Helvetiae 72 (1997) 233-238

237

9.15). The above data suggest a dehydroabietane diterpene structure for 5. The appearance in the ‘H-NMR spectrum of a doublet at 6 4.25 (J = 3.3 Hz), typical of a benzylic proton geminal to the methoxy group, and another intercoupled doublet (6 4.69, J = 3.3 Hz) corresponding to the proton closure of a y-lactone (IR, v,,,~~: 1752 cm-’ : 13C-NMR, 6: 74.50, 179.021, suggest that 5 is a C-7 methoxyrosmanol derivative; the downfield shift of the H-5 singlet at 6 2.23 indicated an a-orientation of the methoxy group (Nakatani and Inatani, 1984) and the upfield shift of the doublet attributed to the proton H-6 geminal to the lactone closure (6 4.69), exclude a possible isorosmanol structure for 5 (Nakatani and Inatani, 1984; Luis and San And&, 1993). The ‘H- and 13C-NMR spectral assignment reported by Luis and Grill0 (1993) for 5 are in general correct, but leaves a space for some reasonable doubts, particularly on ring B proton and carbon signals. Therefore, in our study, unambiguous assignment for H-6, H-7, C-6 and C-7 were made on the basis of the HMQC and HMBC spectral analysis. In fact, the long-range coupling observed in the HMBC spectrum of 5 (Fig. 1; Table 2), between the doublet centered at 6 4.25 and the signals attributed to C-5, C-6, C-9 and OCH,, indicate that this doublet corresponds to H-7 and not to H-6, as have previously reported Luis and Grill0 (1993); equally the C-7/H-14 and C7/OCH, correlations confirm this assignment. On the other hand, Gonzalez et al. (1989b) have unequivocally established that in rosmanol(4), the lower chemical shift is that of H-7, therefore it is not possible to postulate a general rule, which allows a correct assignment of the H-6 and H-7 NMR signals in the rosmanol derivatives.

Acknowledgements I am grateful to CDCHT-ULA for financial support (Grant C-46%90B). Sincere thanks are due to technician staff of Instituto Universitario de BioOrganica. Universidad de La Laguna, Tenerife (Spain), for recording de MS and NMR spectra.

References Blaney, W.M., Simmonds, M.S.J., Ley, S.V., Jones, P.S., 1988. Insect antifeedants: A behavioural and electrophysiological investigation of natural and synthetically derived clerodane diterpenoids. Entomol. Exp. Appl. 46, 261-214. Brieskom, C.H., Fuchs, A., Bredenberg, J.B., McChesney, J.D., Wenkert, E., 1964. The structure of carnosol. J. Org. Chem. 29, 2293-2298. Coll, J.C., Bowden, B.F., 1986. The application of vacuum liquid chromatography to the separation of terpene mixtures. J. Nat. Prod. 49, 934-936.

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J.M. Amaro-Luis / Pharmaceutics

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