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Constituents of the latex of Euphorbia antiquorum
Phytochemistry,
Vol. 29, No. Printed in Great Britain.
5, pp. 1625-1628, 1990.
CONSTITUENTS
0031-9422/90 $3.00+0.00 ~0 1990 Pergamon Press pk.
OF THE LATEX OF EUPHORBIA
AM’IQUORUM
MOHAN B. GEWALI,* MASAO HATTORI,? YASUHIRO TEZUKA, TOHRU KIKUCHI and TSUNEO NAMBA Research Institute for Wakan-Yaku (Traditional Sino-Japanese Medicines), Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-01, Japan (Received 22 June 1989) Key Word Index-Euphorbia
antiquorum;Euphorbiaceae; latex; euphol 3-O-cinnamate; antiquol A; antiquol B.
Abstract-From the latex of Euphorbia anfiquorum, three new triterpenes, euphol 3-0-cinnamate, antiquol A and antiquol B, together with known triterpenes, euphol, 24-methylenecycloartanol and cycloeucalenol, were isolated. (Z)-9-Nonacosene, sitosterol and p-acetoxyphenol were also isolated from the latex.
INTRODUCTION Euphorbiu antiquorum L., a plant native to India, Sri Lanka and southeast Asia, has various medicinal uses. Its latex has been described as having emetic, purgative, diuretic and poisonous properties [l]. It is used in relieving toothache and earache. It has also found uses in the treatment of nervous diseases, dropsy, palsy, deafness and amaurosis [2]. The latex is also said to be an aphrodisiac [3]. However, the most widespread use of the latex is in the preparation of surgical threads for use in the Kshara Sutra treatment, a surgical procedure of the Ayurveda for the treatment of fistula wounds. In this treatment, the surgical thread is prepared by inpregnating it with E. antiquorum latex, alkaline ash of Achyranthes aspera L. (Amaranthaceae) and powder of Curcuma longa L. (Zingiberaceae). After the thread has dried up, it is used to suture the fistula wound. The Kshara Sutra treatment is credited with outstanding recovery of the wound. Two phytochemical studies of the latex of E. antiquorum have been reported; one dealing with isolation of some triterpenes [4] and the second [3] reporting the isolation of a diterpene, 3-0-angeloylingenol. In order to isolate the biologically active compounds from E. antiquorum latex, we undertook chemical and biochemical studies of the latex. To date we have reported on the isolation of four macrocyclic diterpenes and on the mitogenic activity of the latex on mouse spleen cells [S, 61. In this paper, we describe the isolation and characterization of three new triterpenes and three known triterpenes, together with a hydrocarbon, a sterol and a phenolic compound. RESULTS AND
-CH-S-Me]+, which were derived from the cleavage of the carbon-carbon bond between the two methyl sulphide substituents [7]. This finding indicated that the original double bond was located at the 9,10-position. The double bond was assigned to have Z-stereochemistry from the 13C chemical shift values of cc-carbons (6, 27.3 for both C-8 and C-11) which appear at higher field in (Z)-alkenes than in (Qalkenes, due to the y-effect [8]. Compound 1 was thus concluded to be (Z)-9-nonacosene. Compound 2 contained a conjugated ester group (17OOcm-‘) but no hydroxyl group. The molecular formula was determined to be C,,H,,O, by its HR mass spectrum. The ‘H NMR spectrum of 2 showed signals for five tert-methyl groups (60.77, 0.89, 0.93, 0.96 and l.Ol), one set-methyl group (60.86) and two vinylic methyl groups (6 1.61 and 1.69) suggesting a lanostane or an euphane/ tricullane skelton. Furthermore, the ‘H NMR spectrum also showed characteristic two doublet signals at 66.45 and 7.67 with a coupling constant of 16.1 Hz, corresponding to protons at Q-unsaturated carbons. This observation along with the presence of mass fragment ions at m/z 131 [Ph-CH=CH-CO]+ and 147 [Ph-CH =CH-CO-O]’ strongly suggested that the hydroxyl group was esterified with cinnamic acid. The coupling
DISCUSSION
Compound 1 was assigned the molecular formula C,,H,, by its HR mass spectrum. The GC-MS of a dimethyl disulphide adduct of 1 showed a molecular ion at m/z 500 and major fragment ions at m/z 173 [Me
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M. B.
GEWALI
constant of r&unsaturated protons (J= 16.1 Hz) demonstrated that the double bond had E-stereochemistry. Hydrolysis of 2 gave euphol and cinnamic acid, which led us to conclude that 2 is euphol 3-0-cinnamate. The ‘3C NMR signals of 2 supported this structure (Table 1). This is the first report of a naturally occurring cinnamate ester of euphol. Compound 3 was identified as euphol by comparing its spectral data with published data [9: 101. Euphol was isolated as the major compound of the ethyl acetate fraction (ca 70%) and has already been reported from E. rLfltiquorum latex [SJ. Complete assignments of 13C NMR signals of 3 were performed on the basis of ‘H-‘H COSY, 13G’H COSY, long-range 13Cm’H COSY and NOE experiments. Some of the 13C chemical shifts
Table 1. ‘jC NMR spectral data for compounds 2,3,8 and 9 (100 MHz. CDCl,) C
2 (22.5 MHz)
1
36.5 1 26.3 I X2.6 d 39.7 s 52.7 d 20.4 t 29.1 1 136.2 s 135.2 s” 38.8 \ 23.0 t 32.4 1 45.6 s 51.6 \ 31.9 1 31.3 t 51.3 d 17.1 q 21.X q 37.4 d 20.4 y 37.0 r
2 3 4 5 6 I 8 9 10 11 I2 13 14 15 16 17 18 19 20 21 22 23 24 75 26 27 28 29 30 1’ 2’ 3’ I, 1 2”. 6” 3”. 5” 4”
25.9I 126.X d 131.6s IX.3 q 21.2 q 29.6 q 17.0 q 26.1 q 168.3 s 120.5 d 145.8 d 135.6 ha 129.5 d 130.4 d l31.6d
3 35.2 t 27.8 t 7X.Xd 38.8 s 50.9 d 18.89 t 27.6 t 134.0 s 133.4 S 37.2 s 21.5 t
30.8f 44.0 s 4Y.9 s 29.7 t
2x.1r 49.6 d 15.6 y 20.1 q 35.X d 18.86 q 35.3 I 24.1 t 125.1 d 130.6 .s 17.6 q 26.0 q 28.0 q 15.5 q 24.4 q
I 31.2 I 23.X t Bl.Od 38.2 s 56.0 d 18.2 I 27.5 1 42.3 d 50.6 d 37.1 s 21.6 t 35.2 t 40.4 s 50.3 s 3X.8 r 24.X t 49.9 d 15.5 y 16.5 y 75.4 ., 25.4 q 40.6 I 22.6 I 124.7 d 130.1 s 17.7 q 25.8 y 28.0 q 16.7 q 16.3 q 166.8 S 11X.9 d 144.3 d 134.6 s 12X.0 d 12X.9 d 130.1 d
9 18.9 t 28.0t 76.4 d 41.0 s 142.0 s 121.9 s 25.3 t 44.7 d 35.2 s 49.5 d 35.8 t 30.5 t 47.5 S 46.2 s 34.2 t 28.2 t 50.4 d 15.4 q 19.5 y 35.3 d 19.1 q 35.5 t 24.7 t 124.5 d 131.0s 17.7 q 25.X q 29.0 q 25.6 q 19.0 q
The assignments were performed on the basis of distortion&s enhancement by polarization transfer WEPT) and ‘H-‘H, ‘H-l%. long-range “C-‘H shift correlated spectroscopic experiments. “The assignments may he exchanged.
reported in the literature [9. 101 have been revised as shown in Table 1. Compounds 4 and 5 were identified as 24-methylenecycloartanol and cycloeucalenol, respectively. by comparison of their spectral data with the reported data [ll. 121. Compound 6 was found to be sitosterol by comparing it with an authentic sample. Compound 7, named antiquol A, contained a hydroxyl ester group group (3500 cm ’ ) and a conjugated (1690cm-I). Both EIMS and FABMS did not show a molecular ion peak but showed a dehydrated molecular ion peak at m/z 556. In the 17C NMR spectrum. besides the signal of a methine carbon hearing an acyloxy group (6 81.0, C-3), an additional signal of a quarternary carbon bearing a hydroxy group (0 75.4, C-20) was also seen. In the ‘H NMR spectrum. the signals ascribable to the cinnamic ester moiety and the methine proton attached to the acyloxy-bearing carbon (~54.64.dd like. J = 15.3 and 6.8 Hz, H-3) were almost identical with those of 2. indicating a /I-hydroxy group at C-3 was esterified with cinnamic acid. Its ‘H NMR spectrum also revealed that the usual sec.methyl (H,-21) of the side chain in the tetracyclic triterpenes was missing, which suggested that the additional hydroxyl group was probably located at C-20. These findings indicated that 7 was a 3-0-cinnamoyl-20hydroxy derivative of lanostane or euphane. This was further confirmed by ‘HP’H COSY. 1.7C ‘H COSY and long-range 13C-‘H COSY experiments. Long-range shift correlations were observed between the following methyl protons and neighbouring carbons: H,- 18 vs C- 12. C-l 3 and C-14; HA-19 vs C-5, C-9 and C-10; H,-21 \s C-17. C-20 and C-22; HI-26 vs C-27: H,-27 vs C-26: H,-28 vs C-29: H,-29 vs C-4, C-S and C-28: H,-30 vs C-R. C-13 and C-14. In addition, appreciable NOES were observed between H,-19 and H,-18 or H,-29 and between H,-18 and H,-19, thus indicating that 7 possesses the stcreochemistry of a lanostane-type rather than that of an euphane-type compound. Cornpound 8 had spectral properties identical with those of p-acetoxyphenol. Compound 9, named antiquol B. was assigned the molecular formula CJoH,,O. Its ‘H NMR spectrum showed the presence of five rrrt-methyl groups (60.8 I, 0.84, 0.88, 1.06 and 1.12). one src-methyl group (50.86. J = 6.2 Hz), two vinylic methyl groups (b 1.61 and 1.6’)). one methine substituted by oxygen (63.47. hr 5) and two vinylic protons (S5.09, hr f, .I = 7.0 Hz; 6 5.63, d. J =5.8 Hz). In the EIMS, 9 exhibited a characteristic fragment peak at m/z 3 13 [M -side chain - 2H] ’ . resulting from the cleavage of the side chain. The presence of the 2-methyl-hept-2-en-6-yl sidr chain was also confirmed by ‘H NMR and “C NMR. ‘I-1 ~‘tl COSY. ‘“C’H COSY and ‘3Cm’H long-range COSY experiments revealed a cucurhitan-type triterpene skeleton for 9 (Fig. 1); appreciable long-range shift correlations were observed between the following protons and carbons: I+2 (6, ca 1.7, overlapped with the HA-27 signal) vs C-4 and C-10; H-l 1 (6, ca 1.55) vs C-X, C-9. C-12. C-13 and C-19: H,-28/H,-29 vs C-3, C-4 and C-5; H,-26/H,-27 vs C-24. C-25 and C-27/C-26; HA-21 vs C- 17: H,-1 8 vs C- 12, C- 13, C-14 and C-17; H,-30 vs C-X. C-13, C-14 and C-15. The presence of an equatorial proton (H-3) with a broad singlet signal at 6, 3.47 was indicative of the 3rconfiguration of the hydroxy group. These findings led us to conclude the structure of9 is lOr-cucurbita-S.Wdiene3x-01. The structure was also supported by the MS fragmentation pattern which has almost identical with
Constituents
of the latex of Euphorbia antiquorum
Fig. 1. Shift correlations of compound 9 determined by a combination of long-range i3C’H COSY, ‘H-‘H COSY, and i3C-‘H COSY experiments. The values in parentheses indicate ‘H-chemical shifts of methyl protons, the others indicate 13C-chemical shifts.
that of the 3/I-epimer (anhydrolitsomentol) [13]. Though the latter compound has already been isolated from the seeds of the ground Lagennria leucantha var. Gourda (Cucurbitaceae) [ 131 and some other cucurbitaceous plants [ 141, and synthesized by dehydration of litsomentyl acetate with fused potassium hydrogen sulphate [lS] and by isomerization of cycloartenol with a catalytic amount of hydrogen sulphate [ 161, the 3cc-derivative was obtained for the first time from natura1 sources. EXPERIMENTAL
Mps: uncorr. ‘H and i3C NMR: 270 and 22.5 MHz, respectively. EIMS: 70 eV, FABMS: 6 kV; Xe and glycerol as a neutral gas and a matrix, respectively. Chemicals. Wakogel C-200 (Wako, Osaka) was used for CC. Merck Kieselgel 60 F,,, (Merck, F.R.G.) was used for TLC. Plant material. Euphorhia antiquorum was growing in the wild in Sri Lanka. It was identified by Dr U. Pilapitiya (Bandara-
nayake Memorial Ayurvedic Research Institute, Sri Lanka), who also collected the latex. Isofation. The latex (1675 g) was mixed with 4 I of Hz0 and extracted (x 3) with EtzO, EtOAc and BuOH (3.6 1each). The
EtOAc fraction (30 g) was chromatographed on silica gel (800 g) with hexane, hexane-EtOAc (I, 3, 5, 10, 20 and 50% EtOAc in hexane) and EtOAc. Repeated CC and prep. TLC gave nine compounds (l-9). Compound 3 was purified by prep. HPLC (Chemosorb 5 Si, Chemco Co.; column size, 10mm i.d. x 500 mm; solvent system, hexane-C,H,Cl,-EtOH, 50: 15: 1). The nine compounds were identified as (Z)-9-nonacosene (1, 15 mg), euphol 3-0-cinnamate (2, 30 mg), euphol(3, crude 22 g), 24-methylenecycloartanol (4, 12 mg), cycloeucalenol (5, 15 mg), b-sitosterol(6,20 mg), antiquol A (7,15 mg), p-acetoxyphenol(8, 20 mg) and antiquol B (9, 10 mg), respectively. Z-9-Nonacosene, (1). HR MS m/z: 406.4560 [Ml’, Calcd for Cz9H5s: 406.4539; ‘H NMR (270 MHz, CDCI,): 60.874.89 (6H, 2 x Me), 1.25-1.27 (46H, m, 23 x -CH,-), 2.02 (4H, 2 x =CH-CHz-), 5.36 (2H, br t, J=4.7 Hz, 2 x =CH-); “C NMR (22.4 MHz, CDCI,): 6 14.2, 22.8, 27.3, 29.4, 29.8, 32.8, 130.0. Derivatization of 1, and G&MS analysis of the derivative. Compound 1 (1 mg) was dissolved in hexane (0.5 ml). MezS, (0.5 ml) and I, soln (0.025 ml; 60 mg I, in 1 ml Et,O) were added. The reaction mixt. was kept for 24 hr at 50” and dild with hexane (1 ml). The excess of I, was removed by treating with 5% Na,S,O, soln (2 ml). The hexane layer was removed and the remaining aq. layer extracted with hexane. The combined organic layers were coned and an aliquot was immediately analysed by GC-MS under the following conditions: 3% silicon OV-
1627
1 column, 2.5 mm x 2 m; temp, 15&310” at a rate of 32”/min. MS m/z (rel. int.): 500 [M]’ (33), 327 [Me- (CH,),sCH-S-Me]+ (lOO), 173 [Me- (CH,),-CH-S-Me]+ (77). Euphol3-0-cinnamate (2). Crystals, mp 1 l&l 11”; HRMS m/z: 556.4319 [Ml’, Calcd for C,,H,,O,: 556.4280; FABMS m/z: 557 [M+ 11’; EIMS m/z (rel. int.): 556 [M]’ (25) 541 [M - 151’ (20), 147 (Ph-CH=CH-CO-O]+ (20), 131 [Ph-CH =CH-CO]+ (100); IR vzf: cm-‘: 1700, 1630, 1442, 1162, 994; ‘H NMR (270 MHz, CDCl,): 6 0.77 (3H, s, Me), 0.86 (3H, d, J =6.4 Hz, Ha-21), 0.89 (3H, s, Me), 0.93 (3H, s, Me), 0.96 (3H, s, Me), l.O1(3H, s, Me), 1.61(3H, s, H,-26), 1.69 (3H, s, H,-27), 4.66 (lH, m, H-3), 5.11 (lH, br t, J=7.5Hz, H-24), 6.45 (lH, d, J = 16.1 Hz, H-2’), 7.3-7.6(5H, m, H-2”, 3”, 4”, S”, 6”) 7.67 (lH, d, J= 16.1 Hz, H-3’). Hydrolysis of2. Compound 2 (15 mg) was stirred with 10% KOH (2 ml) in MeOH for 4 hr. Hz0 was added to the reaction mixture, which was extracted with EtzO. The Et,0 layer was dried with NazSO, and coned under red. pres. The Et,0 extract after purification gave euphol (3, 7 mg). The aq. layer after acidification with dil HCI was extracted with Et,O. After drying with NazSO,, it was coned under red. pres. Purification afforded pure cinnamic acid (3 mg). Euphol(3). Crystals; EIMS m/z (rel. int.): 426 [Ml+ (50); The ‘H and ‘sC NMR spectra agreed with those reported [9, lo]. 24-Methylenecycfoartanol(4). Crystals; HRMS m/z: 440.4031 [Ml’. Calcd for C,,Hs,O: 440.4018. The ‘H and i3C NMR spectra were identical with those described in the literature [ll]. Cycloeucalenol (5). Crystals; EIMS m/z (rel. int.): 426 [M]’ (15). The ‘HNMR and i3CNMR spectra were identical with those described in the literature [12]. j-Sitosterol, (6). Crystals. EIMS m/z (rel. int.): 414 [M]’ (40); The ‘H and i3CNMR spectra agreed with those of an authentic sample. Antiquol A (7). Powder, mp 151-152”; [a];’ = +23.2” (CHCI,; ~0.81); FABMS m/z: 557 [M - 18 $11’; EIMS m/z (rel. int.): 556 [M-H,O]+ (50), 131 [Ph-CH=CH-CO]+ (100); IR v:f; cm-i: 3500(OH), 1690 (conjugated C=O), 1630 (C=C); ‘H NMR (270 MHz, CDCl,): 60.89 (3H, s, Hs-30), 0.90 (6H, s, H,19,28), 0.94(3H, s, H,-29) 0.98 (3H, s, H,-18), 1.15 (3H, s, H,-21), 1.63(3H, s, H,-26), 1.70(3H,s, H,-27),4.64(1H, ddlike,J= 15.3, 6.8 Hz, H-3), 5.12 (lH, br t, 5=7.1 Hz, H-24), 6.44 (lH, d, J = 16.1 Hz, H-2’), 7.367.56 (5H, m, H-2”, 3”, 4”, 5”, 6”), 7.67 (lH, d, .I= 16.1 Hz, H-3’). 4-Acetoxyphenol(8). Powder; EIMS m/z (rel. int.): 152 [M]’ (25) 151 [M-l]+ (lOO), 109 [M-43]+ (100). ‘HNMR (270MHz, CD,OD): 6 2.09 (3H, s, AcO-4), 6.74 (ZH, d, J =8.9 Hz, H-2, 6), 7.30 (2H, d, Jz8.9 Hz, H-3, 5). Antiquol B (3- epi-anhydrohtsomentof) (9). [a];” = + 13.0” (CHCl,; c 1.58); EIMS m/z (rel. int.): 426 [M] + (1 I), 411 (7), 408 (11),393(4),313(22),288(40),274(73),259(58),231(11),205(14), 163 (44), 161 (44), 150(34), 134 (lOO), 123 (62), 121 (54) 119 (39) 107 (36); ‘H NMR (400 MHz, CDCl,): 6 0.81(3H, s, Hs-18), 0.84 (3H, s, H,-19), 0.86 (3H, d, J = 5.8 Hz, Hs-21), 0.88 (3H, s, H,-30), 1.06 (3H, s, H,-29), 1.12 (3H, s, H,-28), 1.61 (3H, s, H,-26), 1.69 (3H, s, H,-27), 3.47 (lH, br s, H-3), 5.09 (lH, br t, J=7.0 Hz, H24), 5.63 (lH, d, J=5.8 Hz, H-6). Acknowledgement-This study was funded in part by Akiyama Sangyo Co. (Tokyo). We are grateful to Dr Upali Pilapitiya (Bandaranayake Memorial Ayurvedic Research Institute, Nawinna, Sri Lanka), for collecting the latex of E. antiquorum.
REFERENCES
1. Perry, L. M. (1980) in Medicinal Plants of East and Southeast Asia p. 141. MIT Press, Cambridge, Massachusetts.
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M. B. G~:WAI.I <‘I(I/.
2. Jayaweera,
D. M. A. (1980) in Medicinul Plants Used in Natural Science Council of Sri Lanka, Colombo, Sri Lanka. Adolf, W., Chanai, S. and Hecker, E. (1983) J. Sci. Sock. Thailand 9, 8 1. Anjaneyulu. V. and Ramachandra, R. L. (1967) Curr. Sci. 8, 204. Gewali, M. II., Hattori, M., Tezuka, Y., Kikuchi, T. and Namba, T. (1989) Chem. Pharm. Bull. 37, 1547. Namba, T., Sawa, K.. Gewali, M. B., Hattori, M., Naruse, Y. and Kagamimori, S. (1989) Shoyukuqaku Zusshi 43, 250. Scribe, P., Guezennec, J., Dagaut, J.. Pepe, C. and Saliot, A. (1988) Anal. Chem. 60, 928. Breitmaier, E. and Voelter, W. (1987) in Carbon-13 NMR Ceylon,
3. 4. 5. 6. 7.
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p. 203.
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High-Resolution
Chemistry
Weinheim.
Methods
and Biochemistry.
and Applications
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3rd Edn, p, 192. VCH,
9. Teresa, J. De P.. Urones, J. G., Marcos, I. S., Basabe, P.. Cuadrado, M. J. S. and Moro. R. F. (19X7) Phy~ochm~isrr~~ 26, 1767.
10. Knight, S. A. (1973) Tt~rruhrdron f.cfrers, X3. II. Kikuchi. T.. Kadota, S. and Tsubono. K. (1986) Client. Phurm. Bull. 34, 2479. 12. Anjaneyulu, V., Prasad. K. H.. Ravi. K. and Connolly, J. D (1985) Ph~loc,hrmistr~ 24, 2359. 13. Itoh, T.. Tamura. T., Jeong, T. M.. Tamura. T. and Matsutnoto, T. (1980) Lipids 15, 122. 14. Kikuchi, M., Ishisawa. T., Iida, T., Seto, S., Tamura, T. and Matsumoto. T. (1986) Agric. Biol. Chem. 50. 2921. 15. Govindachari, T. R., Viswanathan. N. and Mohamed, P. A. (1971) Tetrahedron 27, 4991. 16. Shimizu, N., Itoh, T.. Saito, M. and Matsumoto. T. (1984) J. Orq. C‘heitt. 49. 709 (1984).
Vol. 29, No. Printed in Great Britain.
5, pp. 1625-1628, 1990.
CONSTITUENTS
0031-9422/90 $3.00+0.00 ~0 1990 Pergamon Press pk.
OF THE LATEX OF EUPHORBIA
AM’IQUORUM
MOHAN B. GEWALI,* MASAO HATTORI,? YASUHIRO TEZUKA, TOHRU KIKUCHI and TSUNEO NAMBA Research Institute for Wakan-Yaku (Traditional Sino-Japanese Medicines), Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-01, Japan (Received 22 June 1989) Key Word Index-Euphorbia
antiquorum;Euphorbiaceae; latex; euphol 3-O-cinnamate; antiquol A; antiquol B.
Abstract-From the latex of Euphorbia anfiquorum, three new triterpenes, euphol 3-0-cinnamate, antiquol A and antiquol B, together with known triterpenes, euphol, 24-methylenecycloartanol and cycloeucalenol, were isolated. (Z)-9-Nonacosene, sitosterol and p-acetoxyphenol were also isolated from the latex.
INTRODUCTION Euphorbiu antiquorum L., a plant native to India, Sri Lanka and southeast Asia, has various medicinal uses. Its latex has been described as having emetic, purgative, diuretic and poisonous properties [l]. It is used in relieving toothache and earache. It has also found uses in the treatment of nervous diseases, dropsy, palsy, deafness and amaurosis [2]. The latex is also said to be an aphrodisiac [3]. However, the most widespread use of the latex is in the preparation of surgical threads for use in the Kshara Sutra treatment, a surgical procedure of the Ayurveda for the treatment of fistula wounds. In this treatment, the surgical thread is prepared by inpregnating it with E. antiquorum latex, alkaline ash of Achyranthes aspera L. (Amaranthaceae) and powder of Curcuma longa L. (Zingiberaceae). After the thread has dried up, it is used to suture the fistula wound. The Kshara Sutra treatment is credited with outstanding recovery of the wound. Two phytochemical studies of the latex of E. antiquorum have been reported; one dealing with isolation of some triterpenes [4] and the second [3] reporting the isolation of a diterpene, 3-0-angeloylingenol. In order to isolate the biologically active compounds from E. antiquorum latex, we undertook chemical and biochemical studies of the latex. To date we have reported on the isolation of four macrocyclic diterpenes and on the mitogenic activity of the latex on mouse spleen cells [S, 61. In this paper, we describe the isolation and characterization of three new triterpenes and three known triterpenes, together with a hydrocarbon, a sterol and a phenolic compound. RESULTS AND
-CH-S-Me]+, which were derived from the cleavage of the carbon-carbon bond between the two methyl sulphide substituents [7]. This finding indicated that the original double bond was located at the 9,10-position. The double bond was assigned to have Z-stereochemistry from the 13C chemical shift values of cc-carbons (6, 27.3 for both C-8 and C-11) which appear at higher field in (Z)-alkenes than in (Qalkenes, due to the y-effect [8]. Compound 1 was thus concluded to be (Z)-9-nonacosene. Compound 2 contained a conjugated ester group (17OOcm-‘) but no hydroxyl group. The molecular formula was determined to be C,,H,,O, by its HR mass spectrum. The ‘H NMR spectrum of 2 showed signals for five tert-methyl groups (60.77, 0.89, 0.93, 0.96 and l.Ol), one set-methyl group (60.86) and two vinylic methyl groups (6 1.61 and 1.69) suggesting a lanostane or an euphane/ tricullane skelton. Furthermore, the ‘H NMR spectrum also showed characteristic two doublet signals at 66.45 and 7.67 with a coupling constant of 16.1 Hz, corresponding to protons at Q-unsaturated carbons. This observation along with the presence of mass fragment ions at m/z 131 [Ph-CH=CH-CO]+ and 147 [Ph-CH =CH-CO-O]’ strongly suggested that the hydroxyl group was esterified with cinnamic acid. The coupling
DISCUSSION
Compound 1 was assigned the molecular formula C,,H,, by its HR mass spectrum. The GC-MS of a dimethyl disulphide adduct of 1 showed a molecular ion at m/z 500 and major fragment ions at m/z 173 [Me
1626
M. B.
GEWALI
constant of r&unsaturated protons (J= 16.1 Hz) demonstrated that the double bond had E-stereochemistry. Hydrolysis of 2 gave euphol and cinnamic acid, which led us to conclude that 2 is euphol 3-0-cinnamate. The ‘3C NMR signals of 2 supported this structure (Table 1). This is the first report of a naturally occurring cinnamate ester of euphol. Compound 3 was identified as euphol by comparing its spectral data with published data [9: 101. Euphol was isolated as the major compound of the ethyl acetate fraction (ca 70%) and has already been reported from E. rLfltiquorum latex [SJ. Complete assignments of 13C NMR signals of 3 were performed on the basis of ‘H-‘H COSY, 13G’H COSY, long-range 13Cm’H COSY and NOE experiments. Some of the 13C chemical shifts
Table 1. ‘jC NMR spectral data for compounds 2,3,8 and 9 (100 MHz. CDCl,) C
2 (22.5 MHz)
1
36.5 1 26.3 I X2.6 d 39.7 s 52.7 d 20.4 t 29.1 1 136.2 s 135.2 s” 38.8 \ 23.0 t 32.4 1 45.6 s 51.6 \ 31.9 1 31.3 t 51.3 d 17.1 q 21.X q 37.4 d 20.4 y 37.0 r
2 3 4 5 6 I 8 9 10 11 I2 13 14 15 16 17 18 19 20 21 22 23 24 75 26 27 28 29 30 1’ 2’ 3’ I, 1 2”. 6” 3”. 5” 4”
25.9I 126.X d 131.6s IX.3 q 21.2 q 29.6 q 17.0 q 26.1 q 168.3 s 120.5 d 145.8 d 135.6 ha 129.5 d 130.4 d l31.6d
3 35.2 t 27.8 t 7X.Xd 38.8 s 50.9 d 18.89 t 27.6 t 134.0 s 133.4 S 37.2 s 21.5 t
30.8f 44.0 s 4Y.9 s 29.7 t
2x.1r 49.6 d 15.6 y 20.1 q 35.X d 18.86 q 35.3 I 24.1 t 125.1 d 130.6 .s 17.6 q 26.0 q 28.0 q 15.5 q 24.4 q
I 31.2 I 23.X t Bl.Od 38.2 s 56.0 d 18.2 I 27.5 1 42.3 d 50.6 d 37.1 s 21.6 t 35.2 t 40.4 s 50.3 s 3X.8 r 24.X t 49.9 d 15.5 y 16.5 y 75.4 ., 25.4 q 40.6 I 22.6 I 124.7 d 130.1 s 17.7 q 25.8 y 28.0 q 16.7 q 16.3 q 166.8 S 11X.9 d 144.3 d 134.6 s 12X.0 d 12X.9 d 130.1 d
9 18.9 t 28.0t 76.4 d 41.0 s 142.0 s 121.9 s 25.3 t 44.7 d 35.2 s 49.5 d 35.8 t 30.5 t 47.5 S 46.2 s 34.2 t 28.2 t 50.4 d 15.4 q 19.5 y 35.3 d 19.1 q 35.5 t 24.7 t 124.5 d 131.0s 17.7 q 25.X q 29.0 q 25.6 q 19.0 q
The assignments were performed on the basis of distortion&s enhancement by polarization transfer WEPT) and ‘H-‘H, ‘H-l%. long-range “C-‘H shift correlated spectroscopic experiments. “The assignments may he exchanged.
reported in the literature [9. 101 have been revised as shown in Table 1. Compounds 4 and 5 were identified as 24-methylenecycloartanol and cycloeucalenol, respectively. by comparison of their spectral data with the reported data [ll. 121. Compound 6 was found to be sitosterol by comparing it with an authentic sample. Compound 7, named antiquol A, contained a hydroxyl ester group group (3500 cm ’ ) and a conjugated (1690cm-I). Both EIMS and FABMS did not show a molecular ion peak but showed a dehydrated molecular ion peak at m/z 556. In the 17C NMR spectrum. besides the signal of a methine carbon hearing an acyloxy group (6 81.0, C-3), an additional signal of a quarternary carbon bearing a hydroxy group (0 75.4, C-20) was also seen. In the ‘H NMR spectrum. the signals ascribable to the cinnamic ester moiety and the methine proton attached to the acyloxy-bearing carbon (~54.64.dd like. J = 15.3 and 6.8 Hz, H-3) were almost identical with those of 2. indicating a /I-hydroxy group at C-3 was esterified with cinnamic acid. Its ‘H NMR spectrum also revealed that the usual sec.methyl (H,-21) of the side chain in the tetracyclic triterpenes was missing, which suggested that the additional hydroxyl group was probably located at C-20. These findings indicated that 7 was a 3-0-cinnamoyl-20hydroxy derivative of lanostane or euphane. This was further confirmed by ‘HP’H COSY. 1.7C ‘H COSY and long-range 13C-‘H COSY experiments. Long-range shift correlations were observed between the following methyl protons and neighbouring carbons: H,- 18 vs C- 12. C-l 3 and C-14; HA-19 vs C-5, C-9 and C-10; H,-21 \s C-17. C-20 and C-22; HI-26 vs C-27: H,-27 vs C-26: H,-28 vs C-29: H,-29 vs C-4, C-S and C-28: H,-30 vs C-R. C-13 and C-14. In addition, appreciable NOES were observed between H,-19 and H,-18 or H,-29 and between H,-18 and H,-19, thus indicating that 7 possesses the stcreochemistry of a lanostane-type rather than that of an euphane-type compound. Cornpound 8 had spectral properties identical with those of p-acetoxyphenol. Compound 9, named antiquol B. was assigned the molecular formula CJoH,,O. Its ‘H NMR spectrum showed the presence of five rrrt-methyl groups (60.8 I, 0.84, 0.88, 1.06 and 1.12). one src-methyl group (50.86. J = 6.2 Hz), two vinylic methyl groups (b 1.61 and 1.6’)). one methine substituted by oxygen (63.47. hr 5) and two vinylic protons (S5.09, hr f, .I = 7.0 Hz; 6 5.63, d. J =5.8 Hz). In the EIMS, 9 exhibited a characteristic fragment peak at m/z 3 13 [M -side chain - 2H] ’ . resulting from the cleavage of the side chain. The presence of the 2-methyl-hept-2-en-6-yl sidr chain was also confirmed by ‘H NMR and “C NMR. ‘I-1 ~‘tl COSY. ‘“C’H COSY and ‘3Cm’H long-range COSY experiments revealed a cucurhitan-type triterpene skeleton for 9 (Fig. 1); appreciable long-range shift correlations were observed between the following protons and carbons: I+2 (6, ca 1.7, overlapped with the HA-27 signal) vs C-4 and C-10; H-l 1 (6, ca 1.55) vs C-X, C-9. C-12. C-13 and C-19: H,-28/H,-29 vs C-3, C-4 and C-5; H,-26/H,-27 vs C-24. C-25 and C-27/C-26; HA-21 vs C- 17: H,-1 8 vs C- 12, C- 13, C-14 and C-17; H,-30 vs C-X. C-13, C-14 and C-15. The presence of an equatorial proton (H-3) with a broad singlet signal at 6, 3.47 was indicative of the 3rconfiguration of the hydroxy group. These findings led us to conclude the structure of9 is lOr-cucurbita-S.Wdiene3x-01. The structure was also supported by the MS fragmentation pattern which has almost identical with
Constituents
of the latex of Euphorbia antiquorum
Fig. 1. Shift correlations of compound 9 determined by a combination of long-range i3C’H COSY, ‘H-‘H COSY, and i3C-‘H COSY experiments. The values in parentheses indicate ‘H-chemical shifts of methyl protons, the others indicate 13C-chemical shifts.
that of the 3/I-epimer (anhydrolitsomentol) [13]. Though the latter compound has already been isolated from the seeds of the ground Lagennria leucantha var. Gourda (Cucurbitaceae) [ 131 and some other cucurbitaceous plants [ 141, and synthesized by dehydration of litsomentyl acetate with fused potassium hydrogen sulphate [lS] and by isomerization of cycloartenol with a catalytic amount of hydrogen sulphate [ 161, the 3cc-derivative was obtained for the first time from natura1 sources. EXPERIMENTAL
Mps: uncorr. ‘H and i3C NMR: 270 and 22.5 MHz, respectively. EIMS: 70 eV, FABMS: 6 kV; Xe and glycerol as a neutral gas and a matrix, respectively. Chemicals. Wakogel C-200 (Wako, Osaka) was used for CC. Merck Kieselgel 60 F,,, (Merck, F.R.G.) was used for TLC. Plant material. Euphorhia antiquorum was growing in the wild in Sri Lanka. It was identified by Dr U. Pilapitiya (Bandara-
nayake Memorial Ayurvedic Research Institute, Sri Lanka), who also collected the latex. Isofation. The latex (1675 g) was mixed with 4 I of Hz0 and extracted (x 3) with EtzO, EtOAc and BuOH (3.6 1each). The
EtOAc fraction (30 g) was chromatographed on silica gel (800 g) with hexane, hexane-EtOAc (I, 3, 5, 10, 20 and 50% EtOAc in hexane) and EtOAc. Repeated CC and prep. TLC gave nine compounds (l-9). Compound 3 was purified by prep. HPLC (Chemosorb 5 Si, Chemco Co.; column size, 10mm i.d. x 500 mm; solvent system, hexane-C,H,Cl,-EtOH, 50: 15: 1). The nine compounds were identified as (Z)-9-nonacosene (1, 15 mg), euphol 3-0-cinnamate (2, 30 mg), euphol(3, crude 22 g), 24-methylenecycloartanol (4, 12 mg), cycloeucalenol (5, 15 mg), b-sitosterol(6,20 mg), antiquol A (7,15 mg), p-acetoxyphenol(8, 20 mg) and antiquol B (9, 10 mg), respectively. Z-9-Nonacosene, (1). HR MS m/z: 406.4560 [Ml’, Calcd for Cz9H5s: 406.4539; ‘H NMR (270 MHz, CDCI,): 60.874.89 (6H, 2 x Me), 1.25-1.27 (46H, m, 23 x -CH,-), 2.02 (4H, 2 x =CH-CHz-), 5.36 (2H, br t, J=4.7 Hz, 2 x =CH-); “C NMR (22.4 MHz, CDCI,): 6 14.2, 22.8, 27.3, 29.4, 29.8, 32.8, 130.0. Derivatization of 1, and G&MS analysis of the derivative. Compound 1 (1 mg) was dissolved in hexane (0.5 ml). MezS, (0.5 ml) and I, soln (0.025 ml; 60 mg I, in 1 ml Et,O) were added. The reaction mixt. was kept for 24 hr at 50” and dild with hexane (1 ml). The excess of I, was removed by treating with 5% Na,S,O, soln (2 ml). The hexane layer was removed and the remaining aq. layer extracted with hexane. The combined organic layers were coned and an aliquot was immediately analysed by GC-MS under the following conditions: 3% silicon OV-
1627
1 column, 2.5 mm x 2 m; temp, 15&310” at a rate of 32”/min. MS m/z (rel. int.): 500 [M]’ (33), 327 [Me- (CH,),sCH-S-Me]+ (lOO), 173 [Me- (CH,),-CH-S-Me]+ (77). Euphol3-0-cinnamate (2). Crystals, mp 1 l&l 11”; HRMS m/z: 556.4319 [Ml’, Calcd for C,,H,,O,: 556.4280; FABMS m/z: 557 [M+ 11’; EIMS m/z (rel. int.): 556 [M]’ (25) 541 [M - 151’ (20), 147 (Ph-CH=CH-CO-O]+ (20), 131 [Ph-CH =CH-CO]+ (100); IR vzf: cm-‘: 1700, 1630, 1442, 1162, 994; ‘H NMR (270 MHz, CDCl,): 6 0.77 (3H, s, Me), 0.86 (3H, d, J =6.4 Hz, Ha-21), 0.89 (3H, s, Me), 0.93 (3H, s, Me), 0.96 (3H, s, Me), l.O1(3H, s, Me), 1.61(3H, s, H,-26), 1.69 (3H, s, H,-27), 4.66 (lH, m, H-3), 5.11 (lH, br t, J=7.5Hz, H-24), 6.45 (lH, d, J = 16.1 Hz, H-2’), 7.3-7.6(5H, m, H-2”, 3”, 4”, S”, 6”) 7.67 (lH, d, J= 16.1 Hz, H-3’). Hydrolysis of2. Compound 2 (15 mg) was stirred with 10% KOH (2 ml) in MeOH for 4 hr. Hz0 was added to the reaction mixture, which was extracted with EtzO. The Et,0 layer was dried with NazSO, and coned under red. pres. The Et,0 extract after purification gave euphol (3, 7 mg). The aq. layer after acidification with dil HCI was extracted with Et,O. After drying with NazSO,, it was coned under red. pres. Purification afforded pure cinnamic acid (3 mg). Euphol(3). Crystals; EIMS m/z (rel. int.): 426 [Ml+ (50); The ‘H and ‘sC NMR spectra agreed with those reported [9, lo]. 24-Methylenecycfoartanol(4). Crystals; HRMS m/z: 440.4031 [Ml’. Calcd for C,,Hs,O: 440.4018. The ‘H and i3C NMR spectra were identical with those described in the literature [ll]. Cycloeucalenol (5). Crystals; EIMS m/z (rel. int.): 426 [M]’ (15). The ‘HNMR and i3CNMR spectra were identical with those described in the literature [12]. j-Sitosterol, (6). Crystals. EIMS m/z (rel. int.): 414 [M]’ (40); The ‘H and i3CNMR spectra agreed with those of an authentic sample. Antiquol A (7). Powder, mp 151-152”; [a];’ = +23.2” (CHCI,; ~0.81); FABMS m/z: 557 [M - 18 $11’; EIMS m/z (rel. int.): 556 [M-H,O]+ (50), 131 [Ph-CH=CH-CO]+ (100); IR v:f; cm-i: 3500(OH), 1690 (conjugated C=O), 1630 (C=C); ‘H NMR (270 MHz, CDCl,): 60.89 (3H, s, Hs-30), 0.90 (6H, s, H,19,28), 0.94(3H, s, H,-29) 0.98 (3H, s, H,-18), 1.15 (3H, s, H,-21), 1.63(3H, s, H,-26), 1.70(3H,s, H,-27),4.64(1H, ddlike,J= 15.3, 6.8 Hz, H-3), 5.12 (lH, br t, 5=7.1 Hz, H-24), 6.44 (lH, d, J = 16.1 Hz, H-2’), 7.367.56 (5H, m, H-2”, 3”, 4”, 5”, 6”), 7.67 (lH, d, .I= 16.1 Hz, H-3’). 4-Acetoxyphenol(8). Powder; EIMS m/z (rel. int.): 152 [M]’ (25) 151 [M-l]+ (lOO), 109 [M-43]+ (100). ‘HNMR (270MHz, CD,OD): 6 2.09 (3H, s, AcO-4), 6.74 (ZH, d, J =8.9 Hz, H-2, 6), 7.30 (2H, d, Jz8.9 Hz, H-3, 5). Antiquol B (3- epi-anhydrohtsomentof) (9). [a];” = + 13.0” (CHCl,; c 1.58); EIMS m/z (rel. int.): 426 [M] + (1 I), 411 (7), 408 (11),393(4),313(22),288(40),274(73),259(58),231(11),205(14), 163 (44), 161 (44), 150(34), 134 (lOO), 123 (62), 121 (54) 119 (39) 107 (36); ‘H NMR (400 MHz, CDCl,): 6 0.81(3H, s, Hs-18), 0.84 (3H, s, H,-19), 0.86 (3H, d, J = 5.8 Hz, Hs-21), 0.88 (3H, s, H,-30), 1.06 (3H, s, H,-29), 1.12 (3H, s, H,-28), 1.61 (3H, s, H,-26), 1.69 (3H, s, H,-27), 3.47 (lH, br s, H-3), 5.09 (lH, br t, J=7.0 Hz, H24), 5.63 (lH, d, J=5.8 Hz, H-6). Acknowledgement-This study was funded in part by Akiyama Sangyo Co. (Tokyo). We are grateful to Dr Upali Pilapitiya (Bandaranayake Memorial Ayurvedic Research Institute, Nawinna, Sri Lanka), for collecting the latex of E. antiquorum.
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