- Email: [email protected]
Dammarane derivatives from the dried fruits of Forsythia suspensa
Phytochemistry 56 (2001) 815–818 www.elsevier.com/locate/phytochem
Dammarane derivatives from the dried fruits of Forsythia suspensa A.S. Shamsur Rouf a,*, Yukihiro Ozaki b, Mohammad A. Rashid a,1, Jing Rui c a Department of Pharmacy, University of Dhaka, Dhaka-1000, Bangladesh Division of Pharmacognosy and Phytochemistry, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya Ku, Tokyo 158-0851, Japan c Tianjin Municipal Institute for Drug Control, 98 Guizhou Road, Tianjin 00070, China
b
Received 23 February 2000; accepted 1 December 2000
Abstract Anti-inflammatory activity guided fractionation of the n-hexane soluble fraction of a 70% aqueous methanolic extract of the dried fruits of Forsythia suspensa afforded two new triterpenes. The structures of these compounds were elucidated as 3b-acetyl20,25-epoxydammarane-24a-ol (1) and 3b-acetyl-20,25-epoxydammarane-24b-ol (2) on the basis of spectral data interpretation as well as by comparison with those of structurally similar compounds. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Forsythia suspense; Oleaceae; Damaranes; Triterpenoids
1. Introduction Forsythia suspensa Vahl (Oleaceae) is a climbing plant, which is widely distributed in China, North and South Korea and Japan (Namba, 1993). This plant is also cultivated in Japan for its beautiful yellow flowers. The extract of the dried fruits has long been used in the Chinese and Japanese folk medicines to treat gonorrhea, erysipelas, inflammation, pharyngitis, pyrexia, tonsillitis, and ulcer (Jinasu, 1977; Tagaki et al., 1982). Moreover, the extract of the dried fruits showed potential antibacterial, antiviral, choleretic and antipyretic effects (Hikino, 1982; Ishizuka et al., 1992; Nishibe et al., 1992; Miura et al., 1987). Previous studies with the fruits have shown them to contain lignans, including a few with platelet activating factor (PAF) inhibitory effects (Iwakami et al., 1992; Nishibe et al., 1989). The 70% methanolic crude extract of F. suspensa fruits and its n-hexane soluble fraction showed anti-inflammatory activity in Wistar rats (Ozaki et al., 1997). In this paper, we report the isolation and characterization of two triterpenes (1,2)
* Corresponding author. Tel.: +880-2-59661900-59 extn. 4837; fax: +880-2-8615583. E-mail addresses: [email protected] (A.S.S. Rouf), [email protected] (Y. Ozaki). 1 Present address: SAIC Frederick, NCI-Frederick Cancer Research and Development Center, Bldg. 560, Rm. 32-63B, Post Box B, Frederick, MD 21702, USA.
from the n-hexane soluble fraction of a 70% methanolic extract of fruits of same species. Although triterpenoids 1 and 2 have been synthesized from a hydroxydammaren-one-II (Biellmann, 1966), and 20,25-epoxydammarane-3b,24b-diol, and the deacetyl derivative of 2 has been prepared by reduction of an isolate (20,25epoxy-24b-hydroxy-3-dammaranone) of Citrus libanotis (Teresa et al., 1982), this is the first report of their occurrence from a natural source. The complete 1H and 13 C NMR spectral data for these compounds are also reported here for the first time.
2. Results and discussion Compound 1 was the major isolate obtained in this investigation. HRFABMS showed a protonated ion peak at 503.4076 which corresponded to the molecular formula C32H54O4, suggesting six double-bond equivalents in the molecule. The mass spectral fragments at m/z 443 due to loss of acetic acid from the pseudo molecular ion suggested the presence of an acetyl moiety in 1. Additionally, it showed strong fragment ion peaks at m/z 485 (C32H52O3) and 425 (C30H49O) due to the loss of water and water plus acetic acid, respectively from the molecular ion, [M+H]+. The 13C NMR spectrum of 1 displayed 32 carbon resonances, while the HSQC experiment confirmed that 25 out of the 32 carbons were attached to protons. The multiplicities of the carbon signals were
0031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0031-9422(01)00028-0
816
A.S.S. Rouf et al. / Phytochemistry 56 (2001) 815–818
determined by performing DEPT experiments using last pulse angles () 45, 90 and 135 , which revealed the presence of nine methyls, ten methylenes, six methines, and seven quaternary carbons. The 13C NMR spectral data of 1 showed close correspondence to that of cabraleadiol monoacetate (3a-acetyl-20S,24S-epoxydammarane-25-ol) (Hisham et al., 1996), ocotillol-II (20S, 24R-epoxydammarane-3b,24-diol) (Ohmoto et al., 1978), 20S,25-epoxydammarane-3b,6a,12b,24a-tetrol (Duc et al., 1994), 20S,25-epoxy-3b,12b,23x,24x-tetrahydroxy dammarane (Fujita et al., 1995), and (20R)- and (20S)panaxadiols (Duc et al., 1994; Fujita et al., 1995). The 1 H NMR spectrum of 1 (Table 1) showed eight tertiary
methyl singlets, an acetyl group at 2.01, two oxymethine protons at 3.69 (1H, t,=7.0 Hz) and 4.44 (1H, dd,=10.5, 5.5 Hz) and a series of resolved and unresolved multiplets extending from 0.79 to 1.83. These structural features revealed that the compound 1 was a triterpenoid. Therefore, the unsaturation index exhibited by the molecular formula (C32H54O4) of 1 was satisfied by the four rings of a dammarane skeleton, the cyclized side chain at C-17 and an ester carbonyl group (C 170.9).
Table 1 NMR assignments for compounds 1 and 2 in CDCl3 Position
1 13
C
2
1
H
38.7 1.00 1.65 2 23.7 1.60 1.62 3 80.9 4.44 4 37.9 – 5 55.9 0.79 6 18.1 1.38 1.47 7 35.2 1.23 1.48 8 40.4 – 9 50.7 1.32 10 37.1 – 11 21.5 1.15 1.46 12 27.3 1.18 1.73 13 42.9 1.52 14 50.0 – 15 31.4 1.03 1.42 16 25.7 1.43 1.75 17 49.5 1.76 18 15.4 0.92 19 16.4 0.81 20 86.3 – 21 23.5 1.09 22 35.7 1.58 1.68 23 26.1 1.74 1.83 24 83.3 3.69 25 71.4 – 26 24.3 1.08 27 27.4 1.17 28 16.2 0.83 29 27.9 0.81 30 16.4 0.83 CH3CO 21.3 2.01 CH3CO 170.9 –
mult J (Hz)
1
(3H) (3H) (3H)
(3H) (3H) (3H) (3H) (3H) (3H)
m m m m dd – m m m m m – m – m m m m m – m m m m m s s – m m m m m t – s s s s s s –
13
C
1
H
38.7 1.03 1.67 23.7 1.60 1.62 10.5, 5.5 80.9 4.46 37.9 – 55.9 0.80 18.2 1.42 1.47 35.2 1.24 1.50 40.4 – 50.7 1.31 37.1 – 21.8 1.18 1.48 26.9 1.18 1.75 42.8 1.60 50.0 – 31.4 1.03 1.42 25.8 1.28 1.70 49.8 1.83 15.5 0.93 16.4 0.81 86.5 – 24.1 1.09 34.9 1.63 1.81 26.3 1.73 1.82 7.0 86.3 3.62 70.2 – 27.1 1.12 27.8 1.17 16.3 0.84 27.9 0.82 16.5 0.85 21.3 2.02 171.0 2.00
mult J (Hz) m m m m dd
(3H) (3H) (3H)
m m m m m – m – m m m m m – m m m m m s s – s m m m m dd – s s s s s
(3H) (3H) (3H) (3H) (3H) (3H) (3H) s
10.3, 5.5
Table 2 Key HMBC correlations observed for 1
10.5, 5.3
Proton
2
3
H-3 H-5 Ha-7 H-9 H-13 Ha-15 H-17 H-18 H-19 H-21 Hb-23 H-24 H-26 H-27 H-28 H-29 H-30 C-32
C-2 C-6 C-6, C-8 C-8, C-10 C-14 C-14, C-16 C-13, C-16, C-20 C-8 C-10 C-20 C-22 C-23, C-25 C-25 C-25 C-4 C-4 C-14 C-31
C-1, C-5, C-28, C-29, C-31 C-3, C-7, C-9, C-19, C-29 C-5, C-9, C-18 C-5, C-11, C-12, C-14, C-19 C-8, C-20, C-30 C-13, C-17, C-30 C12, C-14, C-15, C-21 C-7, C-9, C-14 C-1, C-5, C-9 C-17, C-22 C-20, C-25 C-22, C-26, C-27 C-24, C-27 C-24, C-26 C-3, C-5, C-29 C-3, C-5, C-28 C-13, C-15 –
J
J
A.S.S. Rouf et al. / Phytochemistry 56 (2001) 815–818
The downfield resonance (H 4.44) of one of the oxymethine protons in 1, which was attached to a carbon at 80.9 ppm suggested it to be esterified. The large couplings (10.5 and 5.5 Hz) of this proton demonstrated the equatorial (b-) orientation of the acetyl moiety. In the HMBC spectrum (Table 2), the proton at 4.44 showed two bond correlations to C 23.7 (C-2) and 37.9 (C-4), and connectivities over to 3J 38.7 (C-1), 55.9 (C-5) and the C-4 methyl groups at C 16.2 and 27.9. These HMBC correlations defined the site of esterification at C-3. The chemical shift and splitting pattern of the upfield oxymethine proton (identical to those observed for the synthetic 3b-acetyl-20,25-epoxydammarane-24aol) (Biellmann, 1966) in the side chain implied that the secondary hydroxyl group must be situated next to a quaternary carbon, namely C-22 or C-24. The location of this functionality at C-24 was confirmed by HMBC correlations from this proton to C 71.4 (C-25), 24.3 (C26) and 27.4 (C-27). The axial (a-) orientation of the hydroxyl function at C-24 was determined from the coupling constant (7.0 Hz), between H-24b (equatorial) and H2-23. The assignments of the remaining carbon signals in 1 were based on HMBC experiments using 2J and 3J correlations from the methine and methyl group protons to carbons, while the 1J C–H interactions observed in the HSQC spectrum allowed unambiguous assignments of the methylene protons in 1. The relative stereochemistry of the C-17 side chain and the hydroxyl substituent at C-24 in 1 was finally determined by 1D NOESY experiments as shown on the structure. Irradiation at the resonance frequency of H24 produced strong enhancements of the methyl signals, H3-21 and H3-27 and weak enhancement of H3-26. On the other hand, similar irradiations of the H3-21 and H3-27 signals showed significant enhancement of H-24, while the former one also showed enhancement of H-13, suggesting their close proximity. On the basis of above spectral data, the structure of compound 1 was elucidated as 3b-acetyl-20,25-epoxydammarane-24a-ol. HRFABMS of 2 showed the pseudomolecular ion peak at m/z 635.3104 [M+Cs]+, compatible with the molecular formula, C32H54O4, identical to that for 1. The mass spectrum showed a major fragment ion and the base peak at m/z 485 and 425 attributable to the loss of water and water plus acetic acid from the molecular ion, [M+H]+ at m/z 503. The 1H and 13C NMR spectra of compound 2 (Table 1) were almost identical to those recorded for 3b-acetyl-20,25-epoxydammarane-24a-ol (1). However, the upfield oxymethine proton appeared as a double doublets (J=10.2, 5.3 Hz) at 3.62, which was attached to C 86.3 as evident from the HSQC experiment. The splitting pattern of this proton as well as the downfield shift of the carbon signal suggested the b-orientation of the C-24 hydroxyl group, as opposed to its a-orientation in 1. A similar 3.3 ppm difference in the 13 C signal was observed between the C-24 epimeric tri-
817
terpenoids, ocotillone and cabraleone (Tanaka and Yahara, 1970). The assignments of the 1H and 13C resonances in 2 were made by comparison with those for compound 1 as well as on the basis of HSQC data. The a-configuration of H-24 was substantiated by 1D NOESY experiments as depicted on the structure 2. Irradiation of the H-24 signal produced strong enhancements of the C-24 methyl signals, suggesting their close proximity. Similar irradiation at the resonance frequency of H3-21 caused a significant enhancement of H-13, indicative of a cis relationship between these. Thus compound 2 was identified as 3b-acetyl-20,25-epoxydammarane-24b-ol. Although triterpenoids 1 and 2 were isolated from the fraction exhibiting anti inflammatory activity, the purified compounds have not been tested yet.
3. Experimental 3.1. General experimental procedures Optical rotations were measured with a Perkin-Elmer 241 polarimeter. Ultraviolet (UV) and infrared (IR) spectra were obtained on a Beckman DU-640 and PerkinElmer 1600 FTIR spectrometer, respectively. The 1H and 13C NMR spectra were recorded in CDCl3 on a Varian VXR-300 or Varian 500S spectrometer and the chemical shifts are reported in ppm relative to the residual undeuterated solvent. The number of attached protons for 13C signals was determined using the DEPT pulse sequence. Inverse detected heteronuclear correlations were measured using HSQC (optimized for 1 JCH=140 Hz) and HMBC (optimized for nJCH=8.3 Hz) pulse sequences. Mass spectra and accurate mass measurements were performed on a VG Micromass ZAB mass spectrometer. 3.2. Plant materials The dried fruits of F. suspensa Vahl used in this study were purchased from the Tianjin market, China in 1996 and identified by Jing Rui, Tianjin Municipal Institute for Drug Control, Tianjin 300070, China. Voucher specimen are on deposit at the Tianjin Municipal Institute for Drug Control, China. 3.3. Extraction and isolation The dried and powdered fruits (500 g) were refluxed twice with 70% methanol in H2O. The combined extract was concentrated under reduced temperature and pressure, and freeze dried to give a powdered mass (84.5 g). The crude extract was then re-dissolved in water and extracted twice with n-hexane to yield 4.53 g of n-hexane soluble fraction. An aliquot (2.0 g) of the hexane soluble
818
A.S.S. Rouf et al. / Phytochemistry 56 (2001) 815–818
material was subjected to column chromatography over silica gel 60 eluted with CHCl3. Based on TLC characteristics, four major fractions (FS-I to FS-IV) were made. Assay for in vivo anti-inflammatory activity (RE) on Wister male rats revealed FS-IV as the active fraction. The active fraction FS-IV (200 mg), predominantly consisting of triterpenoids and fat, was further purified by silica gel 60 column chromatography using n-hexaneethyl acetate (9:1) as the developing solvents to afford triterpenes 1 (40 mg) and 2 (15 mg). 3.4. 3 -Acetyl-20,25-epoxydammarane-24 -ol (1) White amorphous; [a]D +22.7 (c 0.022, CHCl3) [lit. (Biellmann, 1966)+52 ]. IR (film) max cm 1: 3492, 2949, 1714, 1451, 1378, 1250, 1143, 1042, 980. 1H (500 MHz) and 13C (125 MHz) NMR data — see Table 1. HRFABMS m/z 503.4076 (MH)+, calc. for [C32H55O4]+ 503.4100. FABMS: m/z 503 [M+H]+, 485 [503-H2O]+, 443 [503-CH3COOH]+, 425 [503-(H2O+CH3COOH)]+. 3.5. 3 -Acetyl-20,25-epoxydammarane-24 -ol (2) White gum; [a]D +81 (c 0.05, CHCl3) [lit. (Biellmann, 1966) +50 ]. IR (film) max cm 1: 3446, 2948, 2875, 1730, 1451, 1368, 1250, 1o89, 1025, 980. HRFABMS m/z 635.3104 (M+Cs)+, calc. for [C32H54O4Cs]+ 635.3076. FABMS m/z: 503 [M+H]+, 485, 543, 425. 1H (500 MHz) and 13C (125 MHz) NMR data — see Table 1.
Acknowledgements We wish to thank the Natural Products Chemistry Section of the Laboratory of Drug Discovery Research and Development, Frederick Cancer Research and Development Center, National Cancer Institute, Frederick, Maryland 21702, USA for assisting with the NMR studies and Dr. L.K. Pannell, National Institute of Diabetic Disorders and Kidney Diseases (NIDDK), Bethesda, Maryland, USA for mass spectral measurements.
References Biellmann, J.F., 1966. Chiralite du dipterocarpol. Tetrahedron Letters 40, 4803–4805. Duc, N.M., Kasai, R., Ohtani, K., Ito, A., Nham, N.T., Yamasaki, K., Tanaka, O., 1994. Saponins from Vietnamese Ginseng, Panax vietnamensis Ha et Grushv. collected in central Vietnam. III. Chemical and Pharmaceutical Bulletin 42, 634–640. Fujita, S., Kasai, R., Ohtani, K., Yamasaki, K., Ming-Hua, C., RuiLin, N., Tanaka, O., 1995. Dammarane glycosides from aerial part of Neoalsomitra integrifolia. Phytochemistry 39, 591–602. Hikino, H., 1982. Constituents and physiological actions of Schizonepeta and Forsythia. Journal of Traditional Sino Japanese Medicines 3, 41–45. Hisham, A., Bai, M.D.A., Fujimoto, Y., Hara, N., Shimada, H., 1996. Complete 1H and 13C NMR spectral assignment of cabraleadiol, a dammarane triterpene from Dosoxylum malabaricum Bedd. Magnetic Resonance in Chemistry 34, 146–150. Ishizuka, O., Kumazawa, N., Ohta, S., Kamogawa, A., Shinoda, M., 1992. Effects of various methanol extracts of crude drugs on experimental subacute and chronic hepatic injury. Yakugaku Zasshi 112, 174–182. Iwakami, S., Wu, J.B., Ebizuka, Y., Sankawa, U., 1992. Platelet activating factor (PAF) antagonists contained in medicinal plants: lignans and sesquiterpenes. Chemical and Pharmaceutical Bulletin 40, 1196–1198. Jiansu, X.Y.X.Y., 1977. Zhong Yao Da Ci Dian. Shanghai Kexue Jishu Pub, Shanghai 1111–1113. Miura, M., Ohta, S., Kamogawa, A., Shinoda, M., 1987. Basic study of assay method of choleretic effect and the screening of crude drugs. Yakugaku Zasshi 107, 992–1000. Namba, T., 1993. The Encyclopedia of Wakan Yaku. Hoikusha, Osaka 296–298. Nishibe, S., Okabe, K., Tsukamoto, H., Sakushima, A., Hisada, S., Baba, H., Akisada, T., 1992. Studies on the Chinese crude drug ‘‘Forsythiae Fructus’’. VI. The structure and antibacterial activity of suspensaside isolated from Forsythia suspensa. Chemical and Pharmaceutical Bulletin 30, 4548–4553. Ohmoto, T., Nikaido, T., Ikuse, M., 1978. Constituents of pollen. V. Constituents of Betula platyphylla var. Japonica. Chemical and Pharmaceutical Bulletin 26, 1437–1442. Ozaki, Y., Rui, J., Tang, Y., Satake, M., 1997. Anti inflammatory effect of Forsythia suspensa Vahl and its active fraction. Biological and Pharmaceutical Bulletin 20, 861–864. Tanaka, O., Yahara, S., 1970. Dammarane saponins of leaves of Panax pseudo ginseng subsp. himalaicus. Phytochemistry 17, 1353–1358. Takagi, K., Kimura, M., Harada, M., Otsuka, Y., 1982. Pharmacology of Medicinal Herbs in East Asia. Nanzando, Tokyo 187–188. Teresa, J.D.P., Urones, J.G., Basabe, P., Marcos, I.S., Granell, F., 1982. Componentes minoritarios de Cistus libanotis L. Anales De Quimica 76, 324–327.
Dammarane derivatives from the dried fruits of Forsythia suspensa A.S. Shamsur Rouf a,*, Yukihiro Ozaki b, Mohammad A. Rashid a,1, Jing Rui c a Department of Pharmacy, University of Dhaka, Dhaka-1000, Bangladesh Division of Pharmacognosy and Phytochemistry, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya Ku, Tokyo 158-0851, Japan c Tianjin Municipal Institute for Drug Control, 98 Guizhou Road, Tianjin 00070, China
b
Received 23 February 2000; accepted 1 December 2000
Abstract Anti-inflammatory activity guided fractionation of the n-hexane soluble fraction of a 70% aqueous methanolic extract of the dried fruits of Forsythia suspensa afforded two new triterpenes. The structures of these compounds were elucidated as 3b-acetyl20,25-epoxydammarane-24a-ol (1) and 3b-acetyl-20,25-epoxydammarane-24b-ol (2) on the basis of spectral data interpretation as well as by comparison with those of structurally similar compounds. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Forsythia suspense; Oleaceae; Damaranes; Triterpenoids
1. Introduction Forsythia suspensa Vahl (Oleaceae) is a climbing plant, which is widely distributed in China, North and South Korea and Japan (Namba, 1993). This plant is also cultivated in Japan for its beautiful yellow flowers. The extract of the dried fruits has long been used in the Chinese and Japanese folk medicines to treat gonorrhea, erysipelas, inflammation, pharyngitis, pyrexia, tonsillitis, and ulcer (Jinasu, 1977; Tagaki et al., 1982). Moreover, the extract of the dried fruits showed potential antibacterial, antiviral, choleretic and antipyretic effects (Hikino, 1982; Ishizuka et al., 1992; Nishibe et al., 1992; Miura et al., 1987). Previous studies with the fruits have shown them to contain lignans, including a few with platelet activating factor (PAF) inhibitory effects (Iwakami et al., 1992; Nishibe et al., 1989). The 70% methanolic crude extract of F. suspensa fruits and its n-hexane soluble fraction showed anti-inflammatory activity in Wistar rats (Ozaki et al., 1997). In this paper, we report the isolation and characterization of two triterpenes (1,2)
* Corresponding author. Tel.: +880-2-59661900-59 extn. 4837; fax: +880-2-8615583. E-mail addresses: [email protected] (A.S.S. Rouf), [email protected] (Y. Ozaki). 1 Present address: SAIC Frederick, NCI-Frederick Cancer Research and Development Center, Bldg. 560, Rm. 32-63B, Post Box B, Frederick, MD 21702, USA.
from the n-hexane soluble fraction of a 70% methanolic extract of fruits of same species. Although triterpenoids 1 and 2 have been synthesized from a hydroxydammaren-one-II (Biellmann, 1966), and 20,25-epoxydammarane-3b,24b-diol, and the deacetyl derivative of 2 has been prepared by reduction of an isolate (20,25epoxy-24b-hydroxy-3-dammaranone) of Citrus libanotis (Teresa et al., 1982), this is the first report of their occurrence from a natural source. The complete 1H and 13 C NMR spectral data for these compounds are also reported here for the first time.
2. Results and discussion Compound 1 was the major isolate obtained in this investigation. HRFABMS showed a protonated ion peak at 503.4076 which corresponded to the molecular formula C32H54O4, suggesting six double-bond equivalents in the molecule. The mass spectral fragments at m/z 443 due to loss of acetic acid from the pseudo molecular ion suggested the presence of an acetyl moiety in 1. Additionally, it showed strong fragment ion peaks at m/z 485 (C32H52O3) and 425 (C30H49O) due to the loss of water and water plus acetic acid, respectively from the molecular ion, [M+H]+. The 13C NMR spectrum of 1 displayed 32 carbon resonances, while the HSQC experiment confirmed that 25 out of the 32 carbons were attached to protons. The multiplicities of the carbon signals were
0031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0031-9422(01)00028-0
816
A.S.S. Rouf et al. / Phytochemistry 56 (2001) 815–818
determined by performing DEPT experiments using last pulse angles () 45, 90 and 135 , which revealed the presence of nine methyls, ten methylenes, six methines, and seven quaternary carbons. The 13C NMR spectral data of 1 showed close correspondence to that of cabraleadiol monoacetate (3a-acetyl-20S,24S-epoxydammarane-25-ol) (Hisham et al., 1996), ocotillol-II (20S, 24R-epoxydammarane-3b,24-diol) (Ohmoto et al., 1978), 20S,25-epoxydammarane-3b,6a,12b,24a-tetrol (Duc et al., 1994), 20S,25-epoxy-3b,12b,23x,24x-tetrahydroxy dammarane (Fujita et al., 1995), and (20R)- and (20S)panaxadiols (Duc et al., 1994; Fujita et al., 1995). The 1 H NMR spectrum of 1 (Table 1) showed eight tertiary
methyl singlets, an acetyl group at 2.01, two oxymethine protons at 3.69 (1H, t,=7.0 Hz) and 4.44 (1H, dd,=10.5, 5.5 Hz) and a series of resolved and unresolved multiplets extending from 0.79 to 1.83. These structural features revealed that the compound 1 was a triterpenoid. Therefore, the unsaturation index exhibited by the molecular formula (C32H54O4) of 1 was satisfied by the four rings of a dammarane skeleton, the cyclized side chain at C-17 and an ester carbonyl group (C 170.9).
Table 1 NMR assignments for compounds 1 and 2 in CDCl3 Position
1 13
C
2
1
H
38.7 1.00 1.65 2 23.7 1.60 1.62 3 80.9 4.44 4 37.9 – 5 55.9 0.79 6 18.1 1.38 1.47 7 35.2 1.23 1.48 8 40.4 – 9 50.7 1.32 10 37.1 – 11 21.5 1.15 1.46 12 27.3 1.18 1.73 13 42.9 1.52 14 50.0 – 15 31.4 1.03 1.42 16 25.7 1.43 1.75 17 49.5 1.76 18 15.4 0.92 19 16.4 0.81 20 86.3 – 21 23.5 1.09 22 35.7 1.58 1.68 23 26.1 1.74 1.83 24 83.3 3.69 25 71.4 – 26 24.3 1.08 27 27.4 1.17 28 16.2 0.83 29 27.9 0.81 30 16.4 0.83 CH3CO 21.3 2.01 CH3CO 170.9 –
mult J (Hz)
1
(3H) (3H) (3H)
(3H) (3H) (3H) (3H) (3H) (3H)
m m m m dd – m m m m m – m – m m m m m – m m m m m s s – m m m m m t – s s s s s s –
13
C
1
H
38.7 1.03 1.67 23.7 1.60 1.62 10.5, 5.5 80.9 4.46 37.9 – 55.9 0.80 18.2 1.42 1.47 35.2 1.24 1.50 40.4 – 50.7 1.31 37.1 – 21.8 1.18 1.48 26.9 1.18 1.75 42.8 1.60 50.0 – 31.4 1.03 1.42 25.8 1.28 1.70 49.8 1.83 15.5 0.93 16.4 0.81 86.5 – 24.1 1.09 34.9 1.63 1.81 26.3 1.73 1.82 7.0 86.3 3.62 70.2 – 27.1 1.12 27.8 1.17 16.3 0.84 27.9 0.82 16.5 0.85 21.3 2.02 171.0 2.00
mult J (Hz) m m m m dd
(3H) (3H) (3H)
m m m m m – m – m m m m m – m m m m m s s – s m m m m dd – s s s s s
(3H) (3H) (3H) (3H) (3H) (3H) (3H) s
10.3, 5.5
Table 2 Key HMBC correlations observed for 1
10.5, 5.3
Proton
2
3
H-3 H-5 Ha-7 H-9 H-13 Ha-15 H-17 H-18 H-19 H-21 Hb-23 H-24 H-26 H-27 H-28 H-29 H-30 C-32
C-2 C-6 C-6, C-8 C-8, C-10 C-14 C-14, C-16 C-13, C-16, C-20 C-8 C-10 C-20 C-22 C-23, C-25 C-25 C-25 C-4 C-4 C-14 C-31
C-1, C-5, C-28, C-29, C-31 C-3, C-7, C-9, C-19, C-29 C-5, C-9, C-18 C-5, C-11, C-12, C-14, C-19 C-8, C-20, C-30 C-13, C-17, C-30 C12, C-14, C-15, C-21 C-7, C-9, C-14 C-1, C-5, C-9 C-17, C-22 C-20, C-25 C-22, C-26, C-27 C-24, C-27 C-24, C-26 C-3, C-5, C-29 C-3, C-5, C-28 C-13, C-15 –
J
J
A.S.S. Rouf et al. / Phytochemistry 56 (2001) 815–818
The downfield resonance (H 4.44) of one of the oxymethine protons in 1, which was attached to a carbon at 80.9 ppm suggested it to be esterified. The large couplings (10.5 and 5.5 Hz) of this proton demonstrated the equatorial (b-) orientation of the acetyl moiety. In the HMBC spectrum (Table 2), the proton at 4.44 showed two bond correlations to C 23.7 (C-2) and 37.9 (C-4), and connectivities over to 3J 38.7 (C-1), 55.9 (C-5) and the C-4 methyl groups at C 16.2 and 27.9. These HMBC correlations defined the site of esterification at C-3. The chemical shift and splitting pattern of the upfield oxymethine proton (identical to those observed for the synthetic 3b-acetyl-20,25-epoxydammarane-24aol) (Biellmann, 1966) in the side chain implied that the secondary hydroxyl group must be situated next to a quaternary carbon, namely C-22 or C-24. The location of this functionality at C-24 was confirmed by HMBC correlations from this proton to C 71.4 (C-25), 24.3 (C26) and 27.4 (C-27). The axial (a-) orientation of the hydroxyl function at C-24 was determined from the coupling constant (7.0 Hz), between H-24b (equatorial) and H2-23. The assignments of the remaining carbon signals in 1 were based on HMBC experiments using 2J and 3J correlations from the methine and methyl group protons to carbons, while the 1J C–H interactions observed in the HSQC spectrum allowed unambiguous assignments of the methylene protons in 1. The relative stereochemistry of the C-17 side chain and the hydroxyl substituent at C-24 in 1 was finally determined by 1D NOESY experiments as shown on the structure. Irradiation at the resonance frequency of H24 produced strong enhancements of the methyl signals, H3-21 and H3-27 and weak enhancement of H3-26. On the other hand, similar irradiations of the H3-21 and H3-27 signals showed significant enhancement of H-24, while the former one also showed enhancement of H-13, suggesting their close proximity. On the basis of above spectral data, the structure of compound 1 was elucidated as 3b-acetyl-20,25-epoxydammarane-24a-ol. HRFABMS of 2 showed the pseudomolecular ion peak at m/z 635.3104 [M+Cs]+, compatible with the molecular formula, C32H54O4, identical to that for 1. The mass spectrum showed a major fragment ion and the base peak at m/z 485 and 425 attributable to the loss of water and water plus acetic acid from the molecular ion, [M+H]+ at m/z 503. The 1H and 13C NMR spectra of compound 2 (Table 1) were almost identical to those recorded for 3b-acetyl-20,25-epoxydammarane-24a-ol (1). However, the upfield oxymethine proton appeared as a double doublets (J=10.2, 5.3 Hz) at 3.62, which was attached to C 86.3 as evident from the HSQC experiment. The splitting pattern of this proton as well as the downfield shift of the carbon signal suggested the b-orientation of the C-24 hydroxyl group, as opposed to its a-orientation in 1. A similar 3.3 ppm difference in the 13 C signal was observed between the C-24 epimeric tri-
817
terpenoids, ocotillone and cabraleone (Tanaka and Yahara, 1970). The assignments of the 1H and 13C resonances in 2 were made by comparison with those for compound 1 as well as on the basis of HSQC data. The a-configuration of H-24 was substantiated by 1D NOESY experiments as depicted on the structure 2. Irradiation of the H-24 signal produced strong enhancements of the C-24 methyl signals, suggesting their close proximity. Similar irradiation at the resonance frequency of H3-21 caused a significant enhancement of H-13, indicative of a cis relationship between these. Thus compound 2 was identified as 3b-acetyl-20,25-epoxydammarane-24b-ol. Although triterpenoids 1 and 2 were isolated from the fraction exhibiting anti inflammatory activity, the purified compounds have not been tested yet.
3. Experimental 3.1. General experimental procedures Optical rotations were measured with a Perkin-Elmer 241 polarimeter. Ultraviolet (UV) and infrared (IR) spectra were obtained on a Beckman DU-640 and PerkinElmer 1600 FTIR spectrometer, respectively. The 1H and 13C NMR spectra were recorded in CDCl3 on a Varian VXR-300 or Varian 500S spectrometer and the chemical shifts are reported in ppm relative to the residual undeuterated solvent. The number of attached protons for 13C signals was determined using the DEPT pulse sequence. Inverse detected heteronuclear correlations were measured using HSQC (optimized for 1 JCH=140 Hz) and HMBC (optimized for nJCH=8.3 Hz) pulse sequences. Mass spectra and accurate mass measurements were performed on a VG Micromass ZAB mass spectrometer. 3.2. Plant materials The dried fruits of F. suspensa Vahl used in this study were purchased from the Tianjin market, China in 1996 and identified by Jing Rui, Tianjin Municipal Institute for Drug Control, Tianjin 300070, China. Voucher specimen are on deposit at the Tianjin Municipal Institute for Drug Control, China. 3.3. Extraction and isolation The dried and powdered fruits (500 g) were refluxed twice with 70% methanol in H2O. The combined extract was concentrated under reduced temperature and pressure, and freeze dried to give a powdered mass (84.5 g). The crude extract was then re-dissolved in water and extracted twice with n-hexane to yield 4.53 g of n-hexane soluble fraction. An aliquot (2.0 g) of the hexane soluble
818
A.S.S. Rouf et al. / Phytochemistry 56 (2001) 815–818
material was subjected to column chromatography over silica gel 60 eluted with CHCl3. Based on TLC characteristics, four major fractions (FS-I to FS-IV) were made. Assay for in vivo anti-inflammatory activity (RE) on Wister male rats revealed FS-IV as the active fraction. The active fraction FS-IV (200 mg), predominantly consisting of triterpenoids and fat, was further purified by silica gel 60 column chromatography using n-hexaneethyl acetate (9:1) as the developing solvents to afford triterpenes 1 (40 mg) and 2 (15 mg). 3.4. 3 -Acetyl-20,25-epoxydammarane-24 -ol (1) White amorphous; [a]D +22.7 (c 0.022, CHCl3) [lit. (Biellmann, 1966)+52 ]. IR (film) max cm 1: 3492, 2949, 1714, 1451, 1378, 1250, 1143, 1042, 980. 1H (500 MHz) and 13C (125 MHz) NMR data — see Table 1. HRFABMS m/z 503.4076 (MH)+, calc. for [C32H55O4]+ 503.4100. FABMS: m/z 503 [M+H]+, 485 [503-H2O]+, 443 [503-CH3COOH]+, 425 [503-(H2O+CH3COOH)]+. 3.5. 3 -Acetyl-20,25-epoxydammarane-24 -ol (2) White gum; [a]D +81 (c 0.05, CHCl3) [lit. (Biellmann, 1966) +50 ]. IR (film) max cm 1: 3446, 2948, 2875, 1730, 1451, 1368, 1250, 1o89, 1025, 980. HRFABMS m/z 635.3104 (M+Cs)+, calc. for [C32H54O4Cs]+ 635.3076. FABMS m/z: 503 [M+H]+, 485, 543, 425. 1H (500 MHz) and 13C (125 MHz) NMR data — see Table 1.
Acknowledgements We wish to thank the Natural Products Chemistry Section of the Laboratory of Drug Discovery Research and Development, Frederick Cancer Research and Development Center, National Cancer Institute, Frederick, Maryland 21702, USA for assisting with the NMR studies and Dr. L.K. Pannell, National Institute of Diabetic Disorders and Kidney Diseases (NIDDK), Bethesda, Maryland, USA for mass spectral measurements.
References Biellmann, J.F., 1966. Chiralite du dipterocarpol. Tetrahedron Letters 40, 4803–4805. Duc, N.M., Kasai, R., Ohtani, K., Ito, A., Nham, N.T., Yamasaki, K., Tanaka, O., 1994. Saponins from Vietnamese Ginseng, Panax vietnamensis Ha et Grushv. collected in central Vietnam. III. Chemical and Pharmaceutical Bulletin 42, 634–640. Fujita, S., Kasai, R., Ohtani, K., Yamasaki, K., Ming-Hua, C., RuiLin, N., Tanaka, O., 1995. Dammarane glycosides from aerial part of Neoalsomitra integrifolia. Phytochemistry 39, 591–602. Hikino, H., 1982. Constituents and physiological actions of Schizonepeta and Forsythia. Journal of Traditional Sino Japanese Medicines 3, 41–45. Hisham, A., Bai, M.D.A., Fujimoto, Y., Hara, N., Shimada, H., 1996. Complete 1H and 13C NMR spectral assignment of cabraleadiol, a dammarane triterpene from Dosoxylum malabaricum Bedd. Magnetic Resonance in Chemistry 34, 146–150. Ishizuka, O., Kumazawa, N., Ohta, S., Kamogawa, A., Shinoda, M., 1992. Effects of various methanol extracts of crude drugs on experimental subacute and chronic hepatic injury. Yakugaku Zasshi 112, 174–182. Iwakami, S., Wu, J.B., Ebizuka, Y., Sankawa, U., 1992. Platelet activating factor (PAF) antagonists contained in medicinal plants: lignans and sesquiterpenes. Chemical and Pharmaceutical Bulletin 40, 1196–1198. Jiansu, X.Y.X.Y., 1977. Zhong Yao Da Ci Dian. Shanghai Kexue Jishu Pub, Shanghai 1111–1113. Miura, M., Ohta, S., Kamogawa, A., Shinoda, M., 1987. Basic study of assay method of choleretic effect and the screening of crude drugs. Yakugaku Zasshi 107, 992–1000. Namba, T., 1993. The Encyclopedia of Wakan Yaku. Hoikusha, Osaka 296–298. Nishibe, S., Okabe, K., Tsukamoto, H., Sakushima, A., Hisada, S., Baba, H., Akisada, T., 1992. Studies on the Chinese crude drug ‘‘Forsythiae Fructus’’. VI. The structure and antibacterial activity of suspensaside isolated from Forsythia suspensa. Chemical and Pharmaceutical Bulletin 30, 4548–4553. Ohmoto, T., Nikaido, T., Ikuse, M., 1978. Constituents of pollen. V. Constituents of Betula platyphylla var. Japonica. Chemical and Pharmaceutical Bulletin 26, 1437–1442. Ozaki, Y., Rui, J., Tang, Y., Satake, M., 1997. Anti inflammatory effect of Forsythia suspensa Vahl and its active fraction. Biological and Pharmaceutical Bulletin 20, 861–864. Tanaka, O., Yahara, S., 1970. Dammarane saponins of leaves of Panax pseudo ginseng subsp. himalaicus. Phytochemistry 17, 1353–1358. Takagi, K., Kimura, M., Harada, M., Otsuka, Y., 1982. Pharmacology of Medicinal Herbs in East Asia. Nanzando, Tokyo 187–188. Teresa, J.D.P., Urones, J.G., Basabe, P., Marcos, I.S., Granell, F., 1982. Componentes minoritarios de Cistus libanotis L. Anales De Quimica 76, 324–327.