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Triterpene and flavanone glycoside from Rhododendron simsii
Phytochemistry 56 (2001) 875±879
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Triterpene and ¯avanone glycoside from Rhododendron simsii Hironobu Takahashi, Sachiyo Hirata, Hiroyuki Minami, Yoshiyasu Fukuyama * Institute of Pharmacognosy, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho, Tokushima, 770-8514, Japan Received 7 July 2000; received in revised form 7 November 2000
Abstract Antioxidative substances were isolated from the leaves of Rhododendron simsii. These were a triterpene and ¯avanone glycoside, together with the known matteucinol and two known benzoic acid derivatives. Their structures were characterized as 19,24-dihydroxyurs-12-en-3-one-28-oic acid and 7-O-b-d-apiofuranosyl-(1!6)-b-d-glucopyranosylmatteucinol by spectroscopic analysis. # 2001 Published by Elsevier Science Ltd. Keywords: Rhododendron simsii; Ericaceae; Flavanone glycoside; Apiose; Ursane-type triterpene; Antioxidant
1. Introduction As a part of our ongoing studies on antioxidant natural products, we have already reported a variety of antioxidant compounds isolated from Garcinia subelliptica (Fukuyama et al., 1991; Minami et al., 1994) and Casuarina equisetifolia (Takahashi et al., 1999). Our screening program for antioxidants in natural products uses three in vitro assay systems, namely anti-lipid peroxidation activity (ALPO) (Stocks et al., 1974), free radical scavenging activity of the a,a-diphenyl-b-picrylhydrazyl radical (DPPH) (Blois, 1958) and superoxidation anion scavenging activity (O2 ) (McCord and Fridovich, 1969). This indicated that the methanol extract of Rhododendron simsii contained potent antioxidative compounds. Rhododendron simsii belongs to the Ericaceae and is an evergreen short tree distributed in China, Taiwan, the Ogasawara islands and Japan, and no chemical studies on this plant have been documented. In this report, we describe chemical components of the methanol extract of R. simsii, the purpose being, aimed at the isolation of its antioxidative compounds. Antioxidant activity was concentrated in the resulting methylene chloride±ethyl acetate (1:1) and ethyl acetate soluble fractions, from which were isolated a new triterpene 1, ¯avanone glycoside 2 and compounds 7 and 8 as antioxidative principles. This paper describes the characterization of two new compounds and the identi®cation of antioxidative substances. * Corresponding author. Tel.: +81-88-622-9611 ext 5911; fax: +8188-655-3051. E-mail address: [email protected] u.ac.jp (Y. Fukuyama). 0031-9422/01/$ - see front matter # 2001 Published by Elsevier Science Ltd. PII: S0031-9422(00)00493-3
876
H. Takahashi et al. / Phytochemistry 56 (2001) 875±879
2. Results and discussion The leaves of R. simsii were extracted with methanol and the methanol extract was absorbed on celite and eluted through a glass column with n-hexane, methylene chloride, methylene chloride±ethyl acetate (1:1), ethyl acetate and methanol. Each fraction was tested for antilipidperoxidation activity, free radical scavenging activity and superoxide anion scavenging activity (Table 1). The active fractions that eluted with methylene chloride±ethyl acetate (1:1) and ethyl acetate were combined and further subjected to silica gel and Sephadex LH-20 chromatography to aord a new triterpene 1 and ¯avanone glycoside 2 in addition to 3,4-dihydroxybenzoic acid (7) and methyl 3,4-dihydroxybenzoate (8). Compound 1 had a molecular formula of C30H46O5 (HR-EIMS, m/z 486.3366 [M]+). Its IR spectrum revealed the presence of hydroxyl groups at 3572, 3457 cm 1 and a carbonyl group at 1688 cm 1. The 1H NMR spectrum of 1 had signals due to ®ve tertiary methyl groups [H 1.11 (6 H), 1.45 (3 H), 1.49 (3 H), 1.69 (3 H)], one secondary methyl group [H 1.13 (d, J=6.9 Hz)], an oxymethylene group [H 3.83, 4.34 (each d, J=11.0 Hz)] and an ole®n proton (H 5.59). The 13C NMR and DEPT spectra of 1 displayed 30 carbon signals which consisted of one carbonyl group (C 214.6), one trisubstituted double bond (C 127.6, 140.1), one carboxylic group (C 180.6), an oxygen-bearing quaternary carbon (C 72.6), one oxymethylene (C 65.1), six methyl groups, nine methylenes, three methines and ®ve quaternary carbons. These spectral data indicated that compound 1 was an ursane-type triterpene. In fact, the 13C NMR spectral data of 1 were very similar to those of pomolic acid (4) (Mahato and Kundu, 1994) except for signals due to the A-ring (Table 2). We presumed that the C-3 hydroxyl group in 4 was oxidized to a ketone (C 214.6) and either the C-23 or the C-24 methyl group was hydroxylated to an oxymethylene group [H 3.83, 4.34; C 65.1] in 1, respectively. In order to con®rm this structure, HMBC experiments (Fig. 1) were carried out. The C-24 oxymethylene signals showed cross-peaks to the C-3 ketone and C-23, and the H3-23 (H 1.49) signal showed cross-
peaks to the C-3 ketone and the C-24 methylene. These results suggested that 1 might be rotundanoic acid (3) (Wen and Chen, 1996), but its spectral data (1H NMR, [a]D and mp) were dierent from those of 3. In the 2DNOESY spectrum (Fig. 2), the oxymethylene signals at C24 correlated to the H3-25, and the H3-23 proton signal correlated to the H-5 signal. Thus, compound 1 was established as 19,24-dihydroxyurs-12-en-3-one-28-oic acid. Compound 2 showed IR (3383, 1628 cm 1) and UV absorptions (212, 247, 253, 259 and 279 nm) characteristic of a ¯avanone. The FAB-MS of 2 showed a quasimolecular ion peak at m/z 631 [M+Na] and the molecular formula of 2 was determined as C29H36O14 by high-resolution FAB±MS. The 1H NMR spectrum of 2 showed signals due to two methyl groups at 2.57 (3H, s) and 2.65 (3H, s) which should be bonded to a benzene ring, a 40 -methoxyphenyl moiety at 3.66 (3H, s), 7.02 (2H, d, J=7.0 Hz) and 7.58 (2H, d, J=7.0 Hz), a hydrogen-bonded hydroxyl group at 12.6 (1H, s), an ABX-system [ 2.89 (1H, dd, J=3.0, 17.0 Hz), 3.25 (1H, dd, J=13.0, 17.0 Hz) and 5.42 (1H, dd, J=3.0, 13.0 Hz)] corresponding to a C-ring of a ¯avanone. These data indicated that the ¯avanone moiety was matteucinol (5) (Tanaka et al., 1985; Basnet et al., 1993). Further, the 13 C NMR spectra data (Table 3) showed 27 carbon signals, of which 22 were similar to those of 7-O-b-d-glucopyranosyl matteucinol (6) (Tanaka et al., 1985) and the remaining ®ve carbon signals could be assigned to an apiose unit (C 111.1, 80.4, 77.8, 75.0 and 65.7). The b con®guration on C-1 anomeric orientation of apiose was con®rmed by comparing the 13C NMR spectra data of 2 with those of a-d- (C 104.5) and b-d-apiofuranosides (C 111.5), respectively (Kitagawa et al., 1993). The HMBC experiment of 2 showed long-range correlations (Fig. 3) between the H-1 (H 5.66) of apiose and the C-6 (C
Fig. 2. NOESY correlations of 1.
Fig. 1. Partial structures (bold lines) obtained by HMBC correlations from methyl proton signals and representatives HMBC correlations (arrows) from particular proton signals of 1.
Fig. 3. HMBC correlations of 2.
H. Takahashi et al. / Phytochemistry 56 (2001) 875±879
68.6) of glucose as well as between the H-6 (H 4.58) of glucose and the C-1 (C 111.1) of apiose, thus suggesting the linkage of apiose-(1!6)-glucose. Additionally, the HMBC correlation between the H-1 (H 5.41) of glucose and the C-7 (C 162.6) suggested that the glucose was located at the C-7 position. Finally, the CD spectrum of 2 observed a positive Cotton eect ([]285 37,600), thereby indicating that the absolute con®guration on C-2 is S in the light of the literature (Gaeld, 1970). Thus, 2 was established as 7-O-b-d-apiofuranosyl-(1!6)-b-d-glucopyranosyl matteucinol.
877
Compounds 7 and 8 were isolated as antioxidant principles in the title plants, and tested by using three in vitro assay systems such as anti-lipid peroxidation activity (ALPO), free radical scavenging activity of the a,adiphenyl-b-picrylhydrazyl radical (DPPH) and superoxidation anion scavenging activity (O2 ). As shown in Table 1, protocatechuic acid (7) and protocatechuic acid methyl ester (8) showed inhibitory activities in the three assays whose degree are higher than those of methanol extracts and catechin. However, compounds 1 and 2 had no antioxidant properties.
Table 1 Antioxidant activity of compounds 7 and 8 ALPO a (%)
DPPH b
Concentration (mg/ml)
10
5
1
MeOH extract 7 8 Catechin
93.3 100 71.8 40.3
46.5 100 44.6
49.2
a b c
(%)
100
10
1
150
15
94.8 96.3 95.8 88.5
54.2 95.7 86.0 54.5
7.2 42.3 19.8 16.4
100 93.1 96.6 57.1
47.8 48.3 27.6 14.3
1
Anti-lipid peroxidation activity (per cent inhibition in rat homogenates). Chemically stable radical scavenging activity (per cent inhibition of a,a-diphenyl-b-picrylhydrazyl (DPPH) radical). Superoxidation anion scavenging activity (per cent inhibition in xanthine and xanthine oxidase system).
Table 2 13 C NMR spectral data of compounds 1 and 4 in py-d5a C
1
4a
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
40.3 35.6 214.6 55.2 58.1 20.1 33.7 40.4 47.3 37.2 24.3 127.6 140.1 42.1 29.3 26.3 48.3 54.6 72.6 42.4 26.9 38.5 20.8 65.1 15.6 17.1 24.6 180.6 27.1 16.8
38.7 28.0 78.2 39.3 55.8 18.9 33.6 40.3 47.7 37.3 24.0 128.1 139.9 42.1 29.2 26.6 48.2 54.5 72.7 42.3 27.0 37.4 15.5 28.7 16.7 17.1 24.6 180.6 26.8 16.4
a
c
O2
Values taken from Mahato and Kundu 1994.
Table 3 13 C NMR spectral data of compounds 2, 5 and 6 in py-d5 5a
6b
78.5 43.3 196.6 159.3 102.9 160.8 102.0 157.8 102.9 131.0 127.5 114.1 159.8 6.9 7.6 55.4
78.7 43.3 198.3 159.4 112.3 162.7 111.1 158.1 105.7 131.5 128.2 114.5 160.2 9.4 10.0 55.2 105.8 75.7 78.4 71.5 78.7 62.6
2 C-2 3 4 5 6 7 8 9 10 10 20 30 40 6-Me 8-Me O-Me Glc-1 2 3 4 5 6 Api-1 2 3 4 5 a b
78.8 43.6 198.4 159.5 112.3 162.6 111.3 158.3 105.8 131.7 128.3 114.5 160.3 9.4 10.0 55.2 105.8 75.7 78.3 71.5 77.4 68.6 111.1 77.8 80.4 75.0 65.7
In CDCl3, 100 MHz. Values taken from Tanaka et al., 1985.
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H. Takahashi et al. / Phytochemistry 56 (2001) 875±879
3. Experimental 3.1. General Mps: uncorr. 1H (600 MHz) and 13C (150 MHz) NMR spectra were recorded on a Varian Unity 600 instrument. Optical rotations were measured on a Jasco DIP-1000 digital polarimeter. IR spectra were measured on a Jasco FT-IR 5300 infrared spectrophotometer. The MS were recorded on a JEOL AX-500 instrument. CC: Silica gel (Merck, 230400 mesh and Wakogel C-300) and Sephadex LH-20 (25100 mm, Pharmacia). TLC: precoated silica gel 60 F254 (Merck, 0.25 mm) and RP-8 F254 (Merck, 0.25 mm). The spots were visualized by UV (254 nm) and CeSO4±H2SO4. 3.2. Plant material Rhododendron simsii was collected in Ishigaki islands, Japan and identi®ed by Dr. Hiroyuki Murata (Kagoshima, Japan). A voucher specimen has been deposited in this institute. 3.3. Extraction and isolation The dried and powdered leaves were immersed in MeOH at room temperature for 1 month. The MeOH extract was evaporated in vacuo to give a gummy extract (250 g). The MeOH extract was mixed with celite (250 g) and evapd in vacuo to give the solids. The resultant powder was packed in a glass column and then eluted with nhexane (1L), CH2Cl2 (1L), CH2Cl2±EtOAc (1:1) (1L), EtOAc (1L), EtOAc±MeOH (9:1) (1L) and MeOH (1L) to give 6 frs (1-6). Frs. 3 and 4 (4.6 g) were subjected to CC on Sephadex LH-20 with CHCl3±MeOH (1:4) to give 5 frs (11±15). Fr.12 (1.97 g) was puri®ed by CC on silica gel (n-hexane±EtOAc, 5:1) to give 1 (15.5 mg) and 2 (11.8 mg). Fr. 13 (687 mg) was again subjected to repeated CC on silica gel (CH2Cl2±EtOAc) to give protocatechuic acid (7) (20.2 mg) and protocatechuic acid methyl ester (8) (15.1 mg). Fr 14 (424 mg) was puri®ed by CC on silica gel (CHCl3±MeOH) to give matteucinol (5) (7.9 mg), 7 (5.2 mg) and 8 (200 mg). 3.4. 19,24-Dihydroxyurs-12-en-3-one-28-oic acid (1) Colorless prisms, mp 189.5±191.0 C, 17:6 D +27.4 (c 1.55, acetone). EI-MS m/z (rel. int.): 486.3366 [M]+ (calc. 486.3346 for C30H46O5), 440 (27), 410 (71), 338 1 (42), 146 (199); IR FT film cm : 3572, 3457 (OH), 1688 1 (CO); H NMR (600 MHz, Py-d5) : 1.11 (6H, s, CH325, 26), 1.13 (3H, d, J=6.9 Hz, CH3-30), 1.39 (1H, m, H-5), 1.45 (3H, s, CH3-29), 1.49 (3H, s, CH3-23), 1.51 (1H, m, H-19), 1.69 (3H, s, CH3-27), 1.76 (1H, ddd, J=2.7, 6.0, 13.2 Hz, H-1), 1.86 (1H, dd, J=8.8, 9.1 Hz, H-9), 2.79 (1H, ddd, J=2.8, 6.0, 14.3 Hz, H-2), 3.06
(1H, s, H-18), 3.13 (1H, ddd, J=8.5, 12.9, 13.2 Hz, H16), 3.83 (1H, d, J=11.0 Hz, H-24), 4.34 (1H, d, J=11.0 Hz, H-24), 5.59 (1H, dd, J=3.4, 3.4 Hz, H-12); 13C NMR (150 MHz, Py-d5) : Table 1. 3.5. 7-O- -d -Apiofuranosyl-(1!6)- -d -glucopyranosyl matteucinol (2) 22:6 16.8 (c 0.37, acetone). HR-FABMS m/z: D Found 631.2003 [M+Na]+, calc. 631.2003 for C29H36 EtOH 1 O14Na; IR FT film cm : 3383 (OH), 1628; UV lmax nm (E):212 (36600), 247 (15800), 253 (17900), 259 (16500), 279 (19400); CD (MeOH, c 0.017): []249 +18700, []285 37600, []354 +13000; 1H NMR (600 MHz, Py-d5) : 2.57 (3H, s, C8-CH3), 2.65 (3H, s, C6-CH3), 2.89 (1H, dd, J=17.0, 3.0 Hz, H-3eq), 3.25 (1H, dd, J=17.0, 13.0 Hz, H-3ax), 3.66 (3H, s), 4.06 (1H, ddd, J=9.9, 6.0, 1.6 Hz, glc-5), 4.08 (2H, s, api-5), 4.16 (1H, dd, J=9.9, 8.5 Hz, glc-4), 4.18 (1H, dd, J=11.3, 6.0 Hz, glc-6), 4.32 (1H, dd, J=9.1, 8.5 Hz, glc-3), 4.29 (1H, d, J=9.3 Hz, api-4), 4.34 (1H, dd, J=9.1, 7.4 Hz, glc-2), 4.45 (1H, d, J=9.3 Hz, api-4), 4.58 (1H, d, J=11.3, 1.6 Hz, glc-6), 4.61 (1H, d, J=2.2 Hz, api-2), 5.41 (1H, d, J=7.4 Hz, glc-1), 5.42 (1H, dd, J=3.0, 13.0 Hz, H-2), 5.66 (1H, d, J=2.2 Hz, api-1), 7.02 (2H, d, J=7.0 Hz, H-30 and 50 ), 7.58 (2H, d, J=7.0 Hz, H-20 and 60 ) 12.6 (1H, s, 5-OH); 13 C NMR (150 MHz, Py-d5): Table 2. Acknowledgements The authors thank Mr. Toyokichi Yoshizawa, Seiwa Pharmaceutical Co. LTD for carrying out the antioxidant assays, and Dr Masami Tanaka and Miss Ikuko Okamoto (TBU) for 600 MHz NMR and MS measurements. References Basnet, P., Kadota, S., Shimizu, M., Xu, H.-X., Namba, T., 1993. 20 Hydroxymatteucinol, a new C-methyl ¯avanone derivative from Matteccia orientalis; potent hypoglycemic activity in streptozotocin (STZ)-induced diabetic rat. Chemical and Pharmaceutical Bulletin 41 (10), 1790±1795. Blois, M.S., 1958. Antioxidant determinations by the use of a stable free radical. Nature 181, 1199±1200. Fukuyama, Y., Kamiyama, A., Mima, Y., Kodama, M., 1991. Prenylated xanthones from Garcinia subelliptica. Phytochemistry 30, 3433±3436. Gaeld, W., 1970. Circular dichroism, optical rotatory dispersion and absolute con®guration of ¯avanones, 3-hydroxy¯avanones and their glycosides. Tetrahedron 26, 4093±4108. Kitagawa, I., Hori, K., Sakagami, M., Hashiuchi, F., Yoshikawa, M., Ren, J., 1993. Saponin and sapogenol. XLIX. On the constituents of the roots of Glycyrrhiza in¯ata BATALIN from Xinjiang, China. Characterization of two sweet oleanane-type triterpene oligoglycosides, apioglycyrrhizin and araboglycyrrhizin. Chemical and Pharmaceutical Bulletin 41, 1350±1357. Mahato, B.S., Kundu, P.A., 1994. 13C NMR spectra of pentacyclic triterpenoids Ð A compilation and some salient features. Phytochemistry 37, 1517±1575.
H. Takahashi et al. / Phytochemistry 56 (2001) 875±879 McCord, J.M., Fridovich, I., 1969. Superoxide dismutase. Journal of Biological Chemistry 244, 6049±6055. Minami, H., Kinoshita, M., Fukuyama, Y., Kodama, M., Yoshizawa, T., Sugiura, M., Nakagawa, K., Tago, H., 1994. Antioxidant xanthones from Garcinia subelliptica. Phytochemistry 36, 501±506. Stocks, J., Gutterige, J.M.C., Sharp, R.J., Dormandy, T.L., 1974. Inhibition of lipid autoxidation by human serum and its relation to serum proteins and a-tocopherol. Clinical Science Molecular Medicine 47 (3), 223±233.
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Takahashi, H., Iuchi, M., Fujita, Y., Minami, H., Fukuyama, Y., 1999. Coumaroyl triterpenes from Casuarina equisetifolia. Phytochemistry 51, 543±550. Tanaka, N., Murakami, T., Wada, H., Gutierrez, A.B., Saiki, Y., Chen, C.-M., 1985. Chemical and chemotaxonomical studies of ®lices. LXI. Chemical studies on the constituents of Pronephrium triphyllum HOLLT. Chemical and Pharmaceutical Bulletin 33 (12), 5231±5238. Wen, D-.X., Chen, Z.-L., 1996. A dimeric sinapaldehyde glucoside from Ilex rotunda. Phytochemistry 41, 657±659.
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Triterpene and ¯avanone glycoside from Rhododendron simsii Hironobu Takahashi, Sachiyo Hirata, Hiroyuki Minami, Yoshiyasu Fukuyama * Institute of Pharmacognosy, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho, Tokushima, 770-8514, Japan Received 7 July 2000; received in revised form 7 November 2000
Abstract Antioxidative substances were isolated from the leaves of Rhododendron simsii. These were a triterpene and ¯avanone glycoside, together with the known matteucinol and two known benzoic acid derivatives. Their structures were characterized as 19,24-dihydroxyurs-12-en-3-one-28-oic acid and 7-O-b-d-apiofuranosyl-(1!6)-b-d-glucopyranosylmatteucinol by spectroscopic analysis. # 2001 Published by Elsevier Science Ltd. Keywords: Rhododendron simsii; Ericaceae; Flavanone glycoside; Apiose; Ursane-type triterpene; Antioxidant
1. Introduction As a part of our ongoing studies on antioxidant natural products, we have already reported a variety of antioxidant compounds isolated from Garcinia subelliptica (Fukuyama et al., 1991; Minami et al., 1994) and Casuarina equisetifolia (Takahashi et al., 1999). Our screening program for antioxidants in natural products uses three in vitro assay systems, namely anti-lipid peroxidation activity (ALPO) (Stocks et al., 1974), free radical scavenging activity of the a,a-diphenyl-b-picrylhydrazyl radical (DPPH) (Blois, 1958) and superoxidation anion scavenging activity (O2 ) (McCord and Fridovich, 1969). This indicated that the methanol extract of Rhododendron simsii contained potent antioxidative compounds. Rhododendron simsii belongs to the Ericaceae and is an evergreen short tree distributed in China, Taiwan, the Ogasawara islands and Japan, and no chemical studies on this plant have been documented. In this report, we describe chemical components of the methanol extract of R. simsii, the purpose being, aimed at the isolation of its antioxidative compounds. Antioxidant activity was concentrated in the resulting methylene chloride±ethyl acetate (1:1) and ethyl acetate soluble fractions, from which were isolated a new triterpene 1, ¯avanone glycoside 2 and compounds 7 and 8 as antioxidative principles. This paper describes the characterization of two new compounds and the identi®cation of antioxidative substances. * Corresponding author. Tel.: +81-88-622-9611 ext 5911; fax: +8188-655-3051. E-mail address: [email protected] u.ac.jp (Y. Fukuyama). 0031-9422/01/$ - see front matter # 2001 Published by Elsevier Science Ltd. PII: S0031-9422(00)00493-3
876
H. Takahashi et al. / Phytochemistry 56 (2001) 875±879
2. Results and discussion The leaves of R. simsii were extracted with methanol and the methanol extract was absorbed on celite and eluted through a glass column with n-hexane, methylene chloride, methylene chloride±ethyl acetate (1:1), ethyl acetate and methanol. Each fraction was tested for antilipidperoxidation activity, free radical scavenging activity and superoxide anion scavenging activity (Table 1). The active fractions that eluted with methylene chloride±ethyl acetate (1:1) and ethyl acetate were combined and further subjected to silica gel and Sephadex LH-20 chromatography to aord a new triterpene 1 and ¯avanone glycoside 2 in addition to 3,4-dihydroxybenzoic acid (7) and methyl 3,4-dihydroxybenzoate (8). Compound 1 had a molecular formula of C30H46O5 (HR-EIMS, m/z 486.3366 [M]+). Its IR spectrum revealed the presence of hydroxyl groups at 3572, 3457 cm 1 and a carbonyl group at 1688 cm 1. The 1H NMR spectrum of 1 had signals due to ®ve tertiary methyl groups [H 1.11 (6 H), 1.45 (3 H), 1.49 (3 H), 1.69 (3 H)], one secondary methyl group [H 1.13 (d, J=6.9 Hz)], an oxymethylene group [H 3.83, 4.34 (each d, J=11.0 Hz)] and an ole®n proton (H 5.59). The 13C NMR and DEPT spectra of 1 displayed 30 carbon signals which consisted of one carbonyl group (C 214.6), one trisubstituted double bond (C 127.6, 140.1), one carboxylic group (C 180.6), an oxygen-bearing quaternary carbon (C 72.6), one oxymethylene (C 65.1), six methyl groups, nine methylenes, three methines and ®ve quaternary carbons. These spectral data indicated that compound 1 was an ursane-type triterpene. In fact, the 13C NMR spectral data of 1 were very similar to those of pomolic acid (4) (Mahato and Kundu, 1994) except for signals due to the A-ring (Table 2). We presumed that the C-3 hydroxyl group in 4 was oxidized to a ketone (C 214.6) and either the C-23 or the C-24 methyl group was hydroxylated to an oxymethylene group [H 3.83, 4.34; C 65.1] in 1, respectively. In order to con®rm this structure, HMBC experiments (Fig. 1) were carried out. The C-24 oxymethylene signals showed cross-peaks to the C-3 ketone and C-23, and the H3-23 (H 1.49) signal showed cross-
peaks to the C-3 ketone and the C-24 methylene. These results suggested that 1 might be rotundanoic acid (3) (Wen and Chen, 1996), but its spectral data (1H NMR, [a]D and mp) were dierent from those of 3. In the 2DNOESY spectrum (Fig. 2), the oxymethylene signals at C24 correlated to the H3-25, and the H3-23 proton signal correlated to the H-5 signal. Thus, compound 1 was established as 19,24-dihydroxyurs-12-en-3-one-28-oic acid. Compound 2 showed IR (3383, 1628 cm 1) and UV absorptions (212, 247, 253, 259 and 279 nm) characteristic of a ¯avanone. The FAB-MS of 2 showed a quasimolecular ion peak at m/z 631 [M+Na] and the molecular formula of 2 was determined as C29H36O14 by high-resolution FAB±MS. The 1H NMR spectrum of 2 showed signals due to two methyl groups at 2.57 (3H, s) and 2.65 (3H, s) which should be bonded to a benzene ring, a 40 -methoxyphenyl moiety at 3.66 (3H, s), 7.02 (2H, d, J=7.0 Hz) and 7.58 (2H, d, J=7.0 Hz), a hydrogen-bonded hydroxyl group at 12.6 (1H, s), an ABX-system [ 2.89 (1H, dd, J=3.0, 17.0 Hz), 3.25 (1H, dd, J=13.0, 17.0 Hz) and 5.42 (1H, dd, J=3.0, 13.0 Hz)] corresponding to a C-ring of a ¯avanone. These data indicated that the ¯avanone moiety was matteucinol (5) (Tanaka et al., 1985; Basnet et al., 1993). Further, the 13 C NMR spectra data (Table 3) showed 27 carbon signals, of which 22 were similar to those of 7-O-b-d-glucopyranosyl matteucinol (6) (Tanaka et al., 1985) and the remaining ®ve carbon signals could be assigned to an apiose unit (C 111.1, 80.4, 77.8, 75.0 and 65.7). The b con®guration on C-1 anomeric orientation of apiose was con®rmed by comparing the 13C NMR spectra data of 2 with those of a-d- (C 104.5) and b-d-apiofuranosides (C 111.5), respectively (Kitagawa et al., 1993). The HMBC experiment of 2 showed long-range correlations (Fig. 3) between the H-1 (H 5.66) of apiose and the C-6 (C
Fig. 2. NOESY correlations of 1.
Fig. 1. Partial structures (bold lines) obtained by HMBC correlations from methyl proton signals and representatives HMBC correlations (arrows) from particular proton signals of 1.
Fig. 3. HMBC correlations of 2.
H. Takahashi et al. / Phytochemistry 56 (2001) 875±879
68.6) of glucose as well as between the H-6 (H 4.58) of glucose and the C-1 (C 111.1) of apiose, thus suggesting the linkage of apiose-(1!6)-glucose. Additionally, the HMBC correlation between the H-1 (H 5.41) of glucose and the C-7 (C 162.6) suggested that the glucose was located at the C-7 position. Finally, the CD spectrum of 2 observed a positive Cotton eect ([]285 37,600), thereby indicating that the absolute con®guration on C-2 is S in the light of the literature (Gaeld, 1970). Thus, 2 was established as 7-O-b-d-apiofuranosyl-(1!6)-b-d-glucopyranosyl matteucinol.
877
Compounds 7 and 8 were isolated as antioxidant principles in the title plants, and tested by using three in vitro assay systems such as anti-lipid peroxidation activity (ALPO), free radical scavenging activity of the a,adiphenyl-b-picrylhydrazyl radical (DPPH) and superoxidation anion scavenging activity (O2 ). As shown in Table 1, protocatechuic acid (7) and protocatechuic acid methyl ester (8) showed inhibitory activities in the three assays whose degree are higher than those of methanol extracts and catechin. However, compounds 1 and 2 had no antioxidant properties.
Table 1 Antioxidant activity of compounds 7 and 8 ALPO a (%)
DPPH b
Concentration (mg/ml)
10
5
1
MeOH extract 7 8 Catechin
93.3 100 71.8 40.3
46.5 100 44.6
49.2
a b c
(%)
100
10
1
150
15
94.8 96.3 95.8 88.5
54.2 95.7 86.0 54.5
7.2 42.3 19.8 16.4
100 93.1 96.6 57.1
47.8 48.3 27.6 14.3
1
Anti-lipid peroxidation activity (per cent inhibition in rat homogenates). Chemically stable radical scavenging activity (per cent inhibition of a,a-diphenyl-b-picrylhydrazyl (DPPH) radical). Superoxidation anion scavenging activity (per cent inhibition in xanthine and xanthine oxidase system).
Table 2 13 C NMR spectral data of compounds 1 and 4 in py-d5a C
1
4a
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
40.3 35.6 214.6 55.2 58.1 20.1 33.7 40.4 47.3 37.2 24.3 127.6 140.1 42.1 29.3 26.3 48.3 54.6 72.6 42.4 26.9 38.5 20.8 65.1 15.6 17.1 24.6 180.6 27.1 16.8
38.7 28.0 78.2 39.3 55.8 18.9 33.6 40.3 47.7 37.3 24.0 128.1 139.9 42.1 29.2 26.6 48.2 54.5 72.7 42.3 27.0 37.4 15.5 28.7 16.7 17.1 24.6 180.6 26.8 16.4
a
c
O2
Values taken from Mahato and Kundu 1994.
Table 3 13 C NMR spectral data of compounds 2, 5 and 6 in py-d5 5a
6b
78.5 43.3 196.6 159.3 102.9 160.8 102.0 157.8 102.9 131.0 127.5 114.1 159.8 6.9 7.6 55.4
78.7 43.3 198.3 159.4 112.3 162.7 111.1 158.1 105.7 131.5 128.2 114.5 160.2 9.4 10.0 55.2 105.8 75.7 78.4 71.5 78.7 62.6
2 C-2 3 4 5 6 7 8 9 10 10 20 30 40 6-Me 8-Me O-Me Glc-1 2 3 4 5 6 Api-1 2 3 4 5 a b
78.8 43.6 198.4 159.5 112.3 162.6 111.3 158.3 105.8 131.7 128.3 114.5 160.3 9.4 10.0 55.2 105.8 75.7 78.3 71.5 77.4 68.6 111.1 77.8 80.4 75.0 65.7
In CDCl3, 100 MHz. Values taken from Tanaka et al., 1985.
878
H. Takahashi et al. / Phytochemistry 56 (2001) 875±879
3. Experimental 3.1. General Mps: uncorr. 1H (600 MHz) and 13C (150 MHz) NMR spectra were recorded on a Varian Unity 600 instrument. Optical rotations were measured on a Jasco DIP-1000 digital polarimeter. IR spectra were measured on a Jasco FT-IR 5300 infrared spectrophotometer. The MS were recorded on a JEOL AX-500 instrument. CC: Silica gel (Merck, 230400 mesh and Wakogel C-300) and Sephadex LH-20 (25100 mm, Pharmacia). TLC: precoated silica gel 60 F254 (Merck, 0.25 mm) and RP-8 F254 (Merck, 0.25 mm). The spots were visualized by UV (254 nm) and CeSO4±H2SO4. 3.2. Plant material Rhododendron simsii was collected in Ishigaki islands, Japan and identi®ed by Dr. Hiroyuki Murata (Kagoshima, Japan). A voucher specimen has been deposited in this institute. 3.3. Extraction and isolation The dried and powdered leaves were immersed in MeOH at room temperature for 1 month. The MeOH extract was evaporated in vacuo to give a gummy extract (250 g). The MeOH extract was mixed with celite (250 g) and evapd in vacuo to give the solids. The resultant powder was packed in a glass column and then eluted with nhexane (1L), CH2Cl2 (1L), CH2Cl2±EtOAc (1:1) (1L), EtOAc (1L), EtOAc±MeOH (9:1) (1L) and MeOH (1L) to give 6 frs (1-6). Frs. 3 and 4 (4.6 g) were subjected to CC on Sephadex LH-20 with CHCl3±MeOH (1:4) to give 5 frs (11±15). Fr.12 (1.97 g) was puri®ed by CC on silica gel (n-hexane±EtOAc, 5:1) to give 1 (15.5 mg) and 2 (11.8 mg). Fr. 13 (687 mg) was again subjected to repeated CC on silica gel (CH2Cl2±EtOAc) to give protocatechuic acid (7) (20.2 mg) and protocatechuic acid methyl ester (8) (15.1 mg). Fr 14 (424 mg) was puri®ed by CC on silica gel (CHCl3±MeOH) to give matteucinol (5) (7.9 mg), 7 (5.2 mg) and 8 (200 mg). 3.4. 19,24-Dihydroxyurs-12-en-3-one-28-oic acid (1) Colorless prisms, mp 189.5±191.0 C, 17:6 D +27.4 (c 1.55, acetone). EI-MS m/z (rel. int.): 486.3366 [M]+ (calc. 486.3346 for C30H46O5), 440 (27), 410 (71), 338 1 (42), 146 (199); IR FT film cm : 3572, 3457 (OH), 1688 1 (CO); H NMR (600 MHz, Py-d5) : 1.11 (6H, s, CH325, 26), 1.13 (3H, d, J=6.9 Hz, CH3-30), 1.39 (1H, m, H-5), 1.45 (3H, s, CH3-29), 1.49 (3H, s, CH3-23), 1.51 (1H, m, H-19), 1.69 (3H, s, CH3-27), 1.76 (1H, ddd, J=2.7, 6.0, 13.2 Hz, H-1), 1.86 (1H, dd, J=8.8, 9.1 Hz, H-9), 2.79 (1H, ddd, J=2.8, 6.0, 14.3 Hz, H-2), 3.06
(1H, s, H-18), 3.13 (1H, ddd, J=8.5, 12.9, 13.2 Hz, H16), 3.83 (1H, d, J=11.0 Hz, H-24), 4.34 (1H, d, J=11.0 Hz, H-24), 5.59 (1H, dd, J=3.4, 3.4 Hz, H-12); 13C NMR (150 MHz, Py-d5) : Table 1. 3.5. 7-O- -d -Apiofuranosyl-(1!6)- -d -glucopyranosyl matteucinol (2) 22:6 16.8 (c 0.37, acetone). HR-FABMS m/z: D Found 631.2003 [M+Na]+, calc. 631.2003 for C29H36 EtOH 1 O14Na; IR FT film cm : 3383 (OH), 1628; UV lmax nm (E):212 (36600), 247 (15800), 253 (17900), 259 (16500), 279 (19400); CD (MeOH, c 0.017): []249 +18700, []285 37600, []354 +13000; 1H NMR (600 MHz, Py-d5) : 2.57 (3H, s, C8-CH3), 2.65 (3H, s, C6-CH3), 2.89 (1H, dd, J=17.0, 3.0 Hz, H-3eq), 3.25 (1H, dd, J=17.0, 13.0 Hz, H-3ax), 3.66 (3H, s), 4.06 (1H, ddd, J=9.9, 6.0, 1.6 Hz, glc-5), 4.08 (2H, s, api-5), 4.16 (1H, dd, J=9.9, 8.5 Hz, glc-4), 4.18 (1H, dd, J=11.3, 6.0 Hz, glc-6), 4.32 (1H, dd, J=9.1, 8.5 Hz, glc-3), 4.29 (1H, d, J=9.3 Hz, api-4), 4.34 (1H, dd, J=9.1, 7.4 Hz, glc-2), 4.45 (1H, d, J=9.3 Hz, api-4), 4.58 (1H, d, J=11.3, 1.6 Hz, glc-6), 4.61 (1H, d, J=2.2 Hz, api-2), 5.41 (1H, d, J=7.4 Hz, glc-1), 5.42 (1H, dd, J=3.0, 13.0 Hz, H-2), 5.66 (1H, d, J=2.2 Hz, api-1), 7.02 (2H, d, J=7.0 Hz, H-30 and 50 ), 7.58 (2H, d, J=7.0 Hz, H-20 and 60 ) 12.6 (1H, s, 5-OH); 13 C NMR (150 MHz, Py-d5): Table 2. Acknowledgements The authors thank Mr. Toyokichi Yoshizawa, Seiwa Pharmaceutical Co. LTD for carrying out the antioxidant assays, and Dr Masami Tanaka and Miss Ikuko Okamoto (TBU) for 600 MHz NMR and MS measurements. References Basnet, P., Kadota, S., Shimizu, M., Xu, H.-X., Namba, T., 1993. 20 Hydroxymatteucinol, a new C-methyl ¯avanone derivative from Matteccia orientalis; potent hypoglycemic activity in streptozotocin (STZ)-induced diabetic rat. Chemical and Pharmaceutical Bulletin 41 (10), 1790±1795. Blois, M.S., 1958. Antioxidant determinations by the use of a stable free radical. Nature 181, 1199±1200. Fukuyama, Y., Kamiyama, A., Mima, Y., Kodama, M., 1991. Prenylated xanthones from Garcinia subelliptica. Phytochemistry 30, 3433±3436. Gaeld, W., 1970. Circular dichroism, optical rotatory dispersion and absolute con®guration of ¯avanones, 3-hydroxy¯avanones and their glycosides. Tetrahedron 26, 4093±4108. Kitagawa, I., Hori, K., Sakagami, M., Hashiuchi, F., Yoshikawa, M., Ren, J., 1993. Saponin and sapogenol. XLIX. On the constituents of the roots of Glycyrrhiza in¯ata BATALIN from Xinjiang, China. Characterization of two sweet oleanane-type triterpene oligoglycosides, apioglycyrrhizin and araboglycyrrhizin. Chemical and Pharmaceutical Bulletin 41, 1350±1357. Mahato, B.S., Kundu, P.A., 1994. 13C NMR spectra of pentacyclic triterpenoids Ð A compilation and some salient features. Phytochemistry 37, 1517±1575.
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