Li Bin, Luo Yongming
(Jiangxi College of Traditional Chinese Medicine, pharmacy department, Nanchang 330006)
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–L-rhamnopyranosyl→-D-glucopyranoside(IV), Kaemferol-3-O-[2-O–D-glucopyranosyl-6- O–L-rhamnopyranosyl]–D-glucopyranoside(V), Kaemferol-3-O-[2-O–D-xylopyranosyl-6-O–L- rhamnopyranosyl]–D-glucopyranoside (VI), Kaemferol-3-O-[2-O–L- rhamnopyranosyl-6-O–D-xylopyranosyl]–D-glucopyranoside (VII). These compounds were first isolated and obtained from this plant.
KeywordsCamellia 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, H- 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:=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: CHCl-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
CompoundCompound Iwas isolated as white needle crystals with a mp of 141143ºC, and the molecular formula C1010 .UVmaxnmMeOH: 201, 241, 285, 314. IR(KBr)maxcm-1 :3017, 2960, 1961, 1719 (C=O, 1537benzene ring, 1504benzene ring, 1434, 1280, 1263, 1107，1017, 954, 731. EI-MSm/z(%):194(M, 37), 179(M-CH, 10), 163(M-OCH, 100), 135 (M-COOCH, 30), 120(M-COOCH-CH, 15), 103(M-COOCH-OCH, 22), 92(7), 76(C, 21), 66(9). H-NMR(CDClppm:8.1(4H，S), 3.9(6H, S).On the basis of its spectral data, compound I was: Dimethylterephthalate.
CompoundIICompound II was a white needle crystal with a mp of 216218℃, and the molecular formula C. UVmaxnmEtOH:252. IRKBrmaxcm-1:3435OH, 2936, 2866, 1736C=O, 1462benzene ring, 1380 (C-H), 1059 (C-O-C), 959, 801. EI-MS m/z(%)：138, 90, 121 (M-OH, 100), 93M-COOH, 35, 77, 15, 65, 35, 45-COOH, 100, 3938H-NMRCDODppm:6.82H, m, J=14Hz, 3Hz, 2.5Hz, H-3, H-5, 7.82H, m, J=14Hz, 3Hz, 2.5Hz, H-2, H-6. 13C-NMRCDODppm: 116.0C-3, C-5, 122.7C-1, 133.0C-2, C-6, 163.4C-4, 170.1-COOH. Upon its spectral evidence , its structure should be p-Hydroxybenzoic acid.
CompoundIIICompound III was isolated as yellow crystals with a mp of 275277ºC, and the molecular formula C1510. UVmaxnm (MeOH)365, 327sh, 294sh, 265, 253sh. IR(KBr)maxcm-1 :3747(-OH, sharp band), 3321(-OH, broad band), 2463, 2364, 1654(C=O), 1607benzene 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(A+H, 21), 121(B, 60), 93(C, 22), 77(C, 18), 69(30).H-NMR (CDCOCDppm:6.2(1H, d, J=2Hz, C-H), 6.5(1H, d, J=2Hz, C-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(CDOD) were in the table 1 .On the basis of relevant reference[1]and its spectral data, compoundIIIis 3, 5, 7, 4′-Tetrahydroxyflavone, namely Kaemferol.
CompoundIVCompound IV was a yellow crystal with a mp of 223224ºC, and the molecular formula C273015 . UVmaxnm (MeOH):351(band I), 319sh, 265(bandII);maxnm (MeOH+NaOAc):383(bandI), 352, 305sh, 273(band II). IR(KBr)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). H-NMR (CDOD)ppm: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), 3.23.810H，，rha- and glc-H, 1.31.73H，，rha-CH. Its data of 13C-NMR(CDOD) 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–L-rhamnopyranosyl(1→6)–D-glucopyranoside[1, 2]
CompoundCompound V was isolated as light-yellow crystals, and the molecular formula C334020. UVmaxnm (MeOH):346(band I), 265(band II);maxnm (MeOH+NaOAc);358(band I), 335, 271(band II).IR(KBr)maxcm-1: 3401(-OH, broad band), 2929(CH), 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). H-NMR(DMSO-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), 0.81.4(3H, m, rha-CH). Its data of 13C-NMR(CDOD) are in the Table1 and Table3. Acid hydrolysis of Compound V with 5% HSO provided L-rhamnose, and D-glucose. According to relevant reference[3, 4], compound was elucidated as: Kaemferol-3-O-[2-O–D-glucopyranosyl-6-O–L-rhamnopyranosyl]–D-glucopyranoside.
CompoundVICompound VI was isolated as yellow crystals, and the molecular formula C323819. UVmaxnm (MeOH):347(band I), 266(band II);maxnm (MeOH+NaOAc):353(band I), 339(sh), 273(band II). IRmaxcm-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). H-NMR(DMSO-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””), 3.14.4(13H, m, glc-, xyl- and rha-H), 0.81.8(3H, m, rha-CH). Its data of 13C-NMR(CDOD) are in the Table 1 and Table 3.Acid hydrolysis of Compound VI with 5% HSO provided L-rhamnose, D-glucose and D-xylose. Compared relevant reference[3-6] with its spectral evidence, compoundVIis Kaemferol-3-O-[2-O–D-xylopyranosyl-6-O–L- rhamno-pyranosyl]–D-glucopyranoside.
CompoundVIICompound VII was got as yellow crystals, and the molecular formula C323819. UVmaxnm (MeOH):349(band I), 266(band II);maxnm (MeOH+NaOAc):358(band I), 334sh, 271(band II). IRmaxcm-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). H-NMR(DMSO-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”’), 3.14.4(13H, m, glc-, xyl- and rha-H), 0.81.8(3H, m, rha-CH). The data of 13C-NMR(CDOD)were in the Table 1 and Table 3. Acid hydrolysis of Compound VI with 5% HSO 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–L- rhamnopyranosyl-6-O–D-xylopyranosyl]–D-glucopyranoside .
Table 1 Comparing exerperimental data of 13C-NMR of aglycone in compound III, IV, V, VI, VII with the references.
| Kaempferol | Compound IV | Compound V | Compound VI | Compound VII | ||
| Reference (DMSO-d |
Experiment (CDOD) |
Experiment (CDOD) |
Experiment (DMSO-d |
Experiment (DMSO-d | Experimen (DMSO-d |
|
| 10 | 146 135 175 156 98 163 93 160 103 121 129 115 159 115 129 |
147.0 136.4 176.4 157.6 99.0 165.1 94.3 161.8 103.9 123.0 130.3 116.2 160.2 116.2 130.3 |
158.6 135.5 179.4 159.4 99.9 166.1 95.0 163.0 105.7 122.7 132.4 116.1 161.5 116.1 132.4 |
149.5 133.5 178.1 160.4 100.6 167.1 95.4 165.1 107.2 123.7 132.5 116.7 162.5 116.7 132.5 |
149.8 134.0 180.4 158.1 100.9 168.0 96.2 164.3 107.0 124.2 133.1 117.0 162.0 117.0 133.1 |
149.1 134.8 180.4 158.4 100.7 168.1 96.2 165.0 106.7 124.7 133.6 117.2 161.5 117.2 133.6 |
Table 2 The data of 13NMR of three sugar in the references. (K means kaemferol)
| K-3-O-glc | K-3-O-rha | -D-glc | -L- rha | -D-xyl | |
| 100.8 74.1 77.3 69.8 76.3 60.8 |
101.9 70.0 70.6 71.2 69.8 17.3 |
96.8 75.2 76.7 70.1 76.7 61.8 |
94.8 71.8 71.0 73.2 69.1 17.7 |
97.5 75.1 76.8 70.2 66.1 |
Table 3 Experimental data of 13C NMR of sugars in compound IV, V, VI, VII
| Compound IV (CDOD) |
Compound V (DMSO-d |
Compound VI (DMSO-d |
Compound VII (DMSO-d |
||||||||
| -D-glc | -L- rha | glc 1 | rha | glc 2 | glc | rha | xyl | glc | xyl | rha | |
| 104.6 75.8 77.2 71.4 78.1 68.6 |
102.4 72.1 72.3 73.9 69.7 17.9 |
104.8 76.8 78.1 72.5 79.2 68.0 |
103.4 79.4 70.3 71.9 69.7 17.9 |
105.2 73.7 76.1 69.4 76.1 60.6 |
104.6 76.5 77.8 72.2 78.5 68.0 |
102.7 79.1 71.3 71.9 70.6 17.7 |
105.5 76.8 78.8 73.4 66.2 |
105.6 76.2 77.0 72.6 78.9 68.4 |
107.0 82.0 77.8 73.8 66.9 |
101.9 70.1 70.3 72.9 68.3 18.0 |