Studies on chemical constituents of Camellia oleifera Abel

Received on Oct. 25,  2002; Supported by the National Natural Science Foundation of China (No.29862002)

Abstracts In this paper,  the chemical constituents in seeds cake of Camellia oleifera Abel were well studied,  and seven monocases were firstly isolated,  on the basis of its spectral and physicochemical evidence,  the structures of these compounds were elucidated to be: Dimethylterephthalate (I),   p-Hydroxybenzoic acid (II),  Kaemferol (III), Kaemferol-3-O-a-L-rhamnopyranosyl(1→6)-b-D-glucopyranoside(IV),   Kaemferol-3-O-[2-O-b-D-glucopyranosyl-6- O-a-L-rhamnopyranosyl]-b-D-glucopyranoside(V),  Kaemferol-3-O-[2-O-b-D-xylopyranosyl-6-O-a-L- rhamnopyranosyl]-b-D-glucopyranoside (VI),   Kaemferol-3-O-[2-O-a-L- rhamnopyranosyl-6-O-b-D-xylopyranosyl]-b-D-glucopyranoside (VII). These compounds were first isolated and obtained from this plant.
Keywords Camellia oleifera; chemical constituents ; elucidation

Camellia oleifera Abel is a plant of Theaceae,  it is an important oil material in China. Its oil is used to treat intestinal disorders;its roots can be used to treat stomach-ache,  facial and external injury;and its defatted seeds cake can treat injury,   kill four pests and field snails. While it is prescribed,  there are many chemical constituents in the defatted seeds of Camellia oleifera Abel, such as saponins,  flavonols. Yet,   up to now there has no monocase been reported.For the sake of elucidating its bioactive constituents, exploiting its medical values and advancing its integrative usage,   defatted seeds cake of Camellia oleifera Abel had been well investigatied,  and, seven compounds were isolated ,  their structures were identified on the basis of its chemical and spectral evidence,  which are: Dimethylterephthalate(I),  p-Hydroxybenzoic acid(II) ,  Kaemferol (III),   Kaemferol-3-O-rhamno-pyranosyl (1→6) glucopyranoside (IV),   Kaemferol-3-O-(2-O-glucopyranosyl-6-O-rhamnopyranosyl)-glucopyranoside (V),   Kaemferol-3-O-(2-O-xylopyranosyl-6-O-rham-nopyranosyl)-glucopyranoside (VI),   Kaemferol-3-O-(2-O-rhamnopyranosyl-6-O-xylopyranosyl)-glucopyranoside(VII). These compounds were first obtained from this plant.

1 INSTRUMENTS AND MATERIALS
General UV and IR spectra values were measured with a DU-650 and a FT-IR 2000 spectrophotometer, ¹H- and ¹³C-NMR spectra were recorded on a INOVA-500 spectrometers.Chemical shifts are expressed as the value,  using TMS as an internal standard. RSI-MS data were measured with VG ZAB-HS mass spectrometer, Preparative HPLC was done with Dionex system (Pump 580, 340SDAD diode arrays, YWG-C18column: F=10um,  10×20mm,   detection,  UV 250nm); defatted seeds cake of Camellia oleifera were bought from Jiangxi Haitian Co.

2 EXTRACTION AND ISOLATION
The defatted seeds cake of Camellia oleifera Abel were broken into pieces,  and extracted with boiling water for 3 times.After filtration , the water extract was concentrated, and the concentrate was extracted with EtOAc and BuOH in turn. The EtOAc layer was evaporated,   and the extract was fractionated by silica gel column chromatography(eluation system: CHCl₃-EtOAc),  2 white needle crystals and 1 yellow crystals were obtained from the eluate.After recrystallization, compound I (80mg), II(130mg) and III(500 mg) were obtained.The BuOH layer was also evaporated,   and the extract was subjected to macroreticular resin column chromatography first(eluation system:EtOH-Water), and 40% EtOH eluate was collected and fractionated by column chromatography(eluation system:EtOH-Water),  4 crude yellow crystals were obtained , which were repurified by preparative HPLC(eluation system:BuOH-Water) to give 4 constituents , namely compound IV(70mg),  V(6mg),  VI(10mg),  VII(5mg).

3 ELUCIDATION
Compound I Compound Iwas isolated as white needle crystals with a mp of 141-143ºC, and the molecular formula C₁₀H₁₀O₄ .UV l_maxnm(MeOH): 201,  241,   285,  314. IR(KBr) n_maxcm^-1 :3017, 2960, 1961, 1719 (C=O), 1537(benzene ring), 1504(benzene ring), 1434, 1280,   1263, 1107，1017,  954, 731. EI-MSm/z(%):194(M⁺, 37), 179(M-CH₃, 10), 163(M-OCH₃, 100),  135 (M-COOCH₃, 30), 120(M-COOCH₃-CH₃, 15), 103(M-COOCH₃-OCH₃, 22), 92(7), 76(C₆H₄⁺, 21), 66(9). ¹H-NMR(CDCl₃) dppm:8.1(4H，S), 3.9(6H, S).On the basis of its spectral data,  compound I was: Dimethylterephthalate.
Compound II Compound II was a white needle crystal with a mp of 216-218℃, and the molecular formula C₇H₆O₃. UV l_maxnm(EtOH):252. IR(KBr)n_maxcm^-1:3435(OH), 2936,  2866, 1736(C=O), 1462(benzene ring), 1380 (C-H), 1059 (C-O-C), 959,  801. EI-MS m/z(%)：138(M⁺, 90), 121 (M-OH,   100), 93(M-COOH,   35), 77(C₆H₅⁺, 15), 65(C₅H₅⁺, 35), 45(-COOH,   100), 39(38).¹H-NMR(CD₃OD)dppm:6.8(2H,  m,  J=14Hz,  3Hz,  2.5Hz,  H-3,   H-5),  7.8(2H,  m,  J=14Hz,  3Hz,  2.5Hz,  H-2, H-6). ¹³C-NMR(CD₃OD)dppm: 116.0(C-3, C-5), 122.7(C-1), 133.0(C-2, C-6), 163.4(C-4), 170.1(-COOH). Upon its spectral evidence , its structure should be p-Hydroxybenzoic acid.
Compound III Compound III was isolated as yellow crystals with a mp of 275-277ºC, and the molecular formula C₁₅H₁₀O₆. UV l_maxnm (MeOH)365, 327sh,  294sh, 265, 253sh. IR(KBr) n_maxcm^-1 :3747(-OH, sharp band),   3321(-OH, broad band),  2463,  2364,  1654(C=O), 1607(benzene ring), 1503(benzene ring), 1378, 1180, 1101, 985, 820. EI-MS m/z(%): 286(M⁺, 100), 270, 258(M-CO, 27), 229(M-CO-CO, 25), 213(18), 153(A₁+H, 21), 121(B₂⁺, 60), 93(C₆H₅O⁺, 22), 77(C₆H₅⁺, 18), 69(30).¹H-NMR (CD₃COCD₃) dppm:6.2(1H, d, J=2Hz, C₆-H), 6.5(1H, d, J=2Hz, C₈-H), 7.0(2H, dd,  J=9Hz, 3Hz, H-3, H-5'),  8.1(2H, dd,   J=9Hz, 3Hz, H-2',  H-6'). Its data of ¹³C-NMR(CD₃OD) were in the table 1 .On the basis of relevant reference^[1]and its spectral data,  compound III is 3, 5, 7, 4'-Tetrahydroxyflavone,  namely Kaemferol.
Compound IV Compound IV was a yellow crystal with a mp of 223-224ºC, and the molecular formula C₂₇H₃₀O₁₅ . UV l_maxnm (MeOH):351(band I),  319sh,  265(band II); l_maxnm (MeOH+NaOAc):383(band I),  352,  305sh,  273(band II). IR(KBr)n_maxcm^-1 :3425(OH), 2930, 2361, 1684(C=O), 1563(benzene ring), 1507(benzene ring).1180(C-O), 887, 831, 582. FAB-MS m/z(%): 595(M+H, 27), 449(M-rha+H, 12),  287(Kaemferol+H, 44),   113(100),  87(14),  59(8). ¹H-NMR (CD₃OD) dppm:8.1(2H, d, J=7.5Hz,  H-2',   H-6'),  6.9(2H, d, J=7.5Hz, H-3',  H-5'),  6.4(1H, s, H-8),   6.2(1H, s, H-6),  5.2(1H, d, J=7Hz,  glc-H-1),  4.5(1H, s, rha-H-1),   3.2-3.8(10H，m，rha- and glc-H), 1.3-1.7(3H，m，rha-CH₃). Its data of ¹³C-NMR(CD₃OD) are in the Table 1 and Table 3. Because 6-C of glucose in ¹³C-NMR dropped from 60.8 to 68.6ppm, due to the glycosylation shift,  rhamnose should link to 6-C of glucose, so it was Kaemferol-3-O-a-L-rhamnopyranosyl(1→6)-b-D-glucopyranoside^{[1, 2]}.
Compound V Compound V was isolated as light-yellow crystals, and the molecular formula C₃₃H₄₀O₂₀. UV l_maxnm (MeOH):346(band I),  265(band II);l_maxnm (MeOH+NaOAc);358(band I), 335, 271(band II).IR(KBr)_maxcm^-1: 3401(-OH, broad band), 2929(CH₂), 1712(C=O), 1654(benzene ring), 1608(benzene ring), 1364, 1178, 1075(C-O), 842. RSI-MSm/z(%): 757(M+H, 70), 595(M-glc+H, 30), 449(M-glc-rha+H, 35), 287(Kaemferol+H, 100). ¹H-NMR(DMSO-d₆) dppm:12.6(1H, s, -OH), 9.5(1H, s, -OH), 8.0(2H,  d,  J=8.7Hz,  H-2',  H-6'),  6.9 (2H, d, J=8.7Hz, H-3',   H-5'),  6.4(1H, s, H-8), 6.2(1H, s, H-6),  5.6(1H, d, J=7Hz, glc-H-1''),   5.4(1H, m, glc-H-1''''),  4.6(1H, m, rha-H-1'''),  3.0-4.3(13H, m, 2glc-and rha-H), 0.8-1.4(3H, m, rha-CH₃). Its data of ¹³C-NMR(CD₃OD) are in the Table1 and Table3. Acid hydrolysis of Compound V with 5% H₂SO₄ provided L-rhamnose,  and D-glucose. According to relevant reference^{[3, 4]},   compound V was elucidated as: Kaemferol-3-O-[2-O-b-D-glucopyranosyl-6-O-a-L-rhamnopyranosyl]-b-D-glucopyranoside.
Compound VI Compound VI was isolated as yellow crystals,  and the molecular formula C₃₂H₃₈O₁₉. UV l_maxnm (MeOH):347(band I),  266(band II); l_maxnm (MeOH+NaOAc):353(band I), 339(sh),  273(band II). IR n_maxcm^-1 :3402(-OH, broad band),  2931,   1718(-CO),  1610(benzene ring), 1263, 1077(C-O-C),  892. RSI-MSm/z(%):727(M+H, 39), 595(M-xyl+H, 30),  449(M-xyl-rha+H, 10),   287(Kaemferol+H, 100). ¹H-NMR(DMSO-d₆) dppm: 9.5(1H, s, -OH),  8.0(2H, d, J=8.5Hz, H-2',  H-6'),  6.9(2H, d,  J=8.5Hz,  H-3',  H-5'),   6.4(1H, s, H-8),  6.2(1H, s, H-6),  5.3(1H, d, J=7Hz,  glc-H-1''),   5.0(1H, m, xyl- H-1'''),  4.6(1H, m, rha-H-1''''), 3.1-4.4(13H, m, glc-, xyl- and rha-H), 0.8-1.8(3H, m, rha-CH₃). Its data of ¹³C-NMR(CD₃OD) are in the Table 1 and Table 3.Acid hydrolysis of Compound VI with 5% H₂SO₄ provided L-rhamnose,   D-glucose and D-xylose. Compared relevant reference^[3-6] with its spectral evidence, compound VI is Kaemferol-3-O-[2-O-b-D-xylopyranosyl-6-O-a-L- rhamno-pyranosyl]-b-D-glucopyranoside.
Compound VII Compound VII was got as yellow crystals, and the molecular formula C₃₂H₃₈O₁₉. UV l_maxnm (MeOH):349(band I),  266(band II); l_maxnm (MeOH+NaOAc):358(band I), 334(sh), 271(band II). IRn_maxcm^-1: 3413(-OH, broad band),  2929, 1718(C=O),  1610(benzene ring), 1376, 1263, 1077(C-O-C), 893. RSI-MS m/z(%):727(M+H, 7), 581(M-rha+H),  449(M-rha-xyl+H, 10),  287(Kaemferol+H, 100). ¹H-NMR(DMSO-d₆) dppm:9.5(1H, s, -OH),  8.0(2H, d, J=8.3Hz, H-2',  H-6'),  6.9(2H, d, J=8.3Hz, H-3',  H-5'),  6.4(1H, s, H-8),  6.2(1H, s, H-6),  5.3 (1H, d, J=7Hz,  glc-H-1''),  5.0(1H, m, xyl-H-1''''), 4.6(1H, m, rha-H-1'''), 3.1-4.4(13H, m, glc-, xyl- and rha-H), 0.8-1.8(3H, m, rha-CH₃). The data of ¹³C-NMR(CD₃OD)were in the Table 1 and Table 3. Acid hydrolysis of Compound VI with 5% H₂SO₄ provided L-rhamnose and D-glucose, D-xylose. Compared its spectral evidence to relevant reference^[3-6] , its structrue is Kaemferol-3-O-[2-O-a-L- rhamnopyranosyl-6-O-b-D-xylopyranosyl]-b-D-glucopyranoside .

Table 1 Comparing exerperimental data of ¹³C-NMR of aglycone in compound III, IV, V, VI, VII with the references.

Table 2 The data of ¹³C NMR of three sugar in the references. (K means kaemferol)

Table 3 Experimental data of ¹³C NMR of sugars in compound IV,  V,  VI,  VII

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