The systematic study on the chemical constituents of Rubus plants began in the late 1970s and early 1980s. Chemists at home and abroad have studied the chemical constituents of more than 20 species of Rubus, and obtained more than 50 new compounds, most of which are triterpenoids (glycosides) and diterpenoids (glycosides). Diterpenoids and triterpenoids are considered to be the characteristic components of Rubus. However, there are still many deficiencies in the study of chemical constituents of raspberries, so we systematically isolated them and obtained four compounds, namely (3- sitosterol (1), carotene (1) and arsine (1kloc-0/658).
1 Instruments and materials
BrukerAM-400 and BiukerDRX-500 used for NMR use TMS as internal standard; VGAutospec-3000； for MS; UV757CRT external ultraviolet spectrometer; The melting point instrument is XRC- 1 microscope melting point instrument, and the thermometer is not calibrated, which is produced by Yike Factory of Sichuan University. Both thin-layer silica gel and column chromatography silica gel are produced by Qingdao Ocean Chemical Plant. Other reagents are analytically pure.
Raspberry samples were purchased from anguo city market in Anhui Province in 2003, and identified by Associate Professor Fang Chengwu of Anhui College of Traditional Chinese Medicine.
2 Extraction and separation
Crushing 48kg raspberry, extracting with 80% ethanol, and filtering the recovered ethanol without alcohol smell. Dissolve the filter residue in water to prepare suspension, extract with ethyl acetate to obtain 15g extract, separate with silica gel dry column, and sequentially elute with CHCl3-MeOH gradient, and collect the eluent, with each 100ml as a fraction. In the CHCVMeOH(8: 1) section, fraction 5 was concentrated and left to separate white needle-like crystals. In the part of ghq 3-MeOH(5∶ 1), the fraction 17 was repeatedly subjected to silica gel column chromatography to obtain compound Li (20 mg); The filtrate was passed through a D 10 1 resin column, and eluted with H2O and ethanol with different concentrations in turn. Taking 1000ml as the first fraction, the 29303 1 fractions were combined and concentrated in the elution part of 60% ethanol to obtain 14g extract. Silica gel was mixed and separated by dry column. Sequentially eluting with ghcl3-MeOH (5:1-1) gradient, finally washing with pure methanol, collecting eluent, eluting with CHOA-MeOH (5: 1) with1ml as the fraction. Combine fractions 26-28, concentrate, extract with ethyl acetate to obtain extract 108g, mix with silica gel, dry and separate, sequentially elute with CHO 3-Me0h (20:1-1), and finally wash with pure methanol. Fractions111-180 were combined and concentrated to obtain 700 mg of solid. The solid was separated by silica gel mixed dry column and eluted with ethyl acetate-acetone -H2O- acetic acid (5∶ 1∶0.5∶02) to obtain compound iV(45mg).
3 recognition
3. 1 compound 1 colorless needle crystal (CHCljEtOAC), MP136 ~138 qliebeimanri-burdiard was positive, and ei-MSM/z (%): 414. 38 1(45)329(75), 303(64), 255(7 1), 2 13(95),145 (100).
3.2 Compound U is white amorphous powder (methanol), MP 285 ~ 287°C, Liebermariri-Burchard reaction is positive, EI-MSM/Z (%) 414 (m-GLC) (7) 396 (100), 382. 145(60), 12 1(46) and 69(73) were hydrolyzed in the usual way. After TLC development, the Rf values of aglycone and sugar are consistent with those of the accompanying sitosterol and glucose standards, respectively, and are consistent with the literature values, so the compound 1.
3.3 compound HI yellow powder (methanol), MP 2 14 ~ 2 16 C, magnesium hydrochloride powder and Morris all showed positive reaction and yellow fluorescence under ultraviolet light. UVXnm:348266 is a typical ultraviolet absorption of flavonoids. Acid hydrolysis, by thin layer chromatography, compared with the control, showed glucose; The comparison of cis -R, C-NMR and literature shows that there are kaempferol in 13'4| and a group of AB coupling systems in' H-NMR spectrum. 7.3 1( 1H, DJ =15.9 Hz) 6.11(1h, d, J = 15.9hz)a2 B2 system. 7.98(2H, dJ=8.8Hz)685(2H, dJ=8.8Hz)7.36(2H, (iJ=8.6Hz), 6.78(2H, (iJ=8.6Hz) and the element coupling system: 637( 1H, DJ = DJ = 1.7 Hz) has groups with the same chemical shift in L3C- NMR spectrum. Signals: 1 15.0(X2), 13Q0(X2), 130.7.
(X2) and ester-based signal S 166. 1, HSQC extension 1 1 (1h, DJ = 8 ") and HMBC expression A5.1/c. 7.3 1C 1H, dJ= 15.6Hz) and1(in 071660 (09/65436e-ms). The signal of sugar in this compound is 00 ... 74 ... 76 ... 70 ... 74.1,62.9. It is worth noting that 00.6 (5.44, 1 HDJ = 7.3 Hz) shows (three-dimensional glucose). According to HMBC, 5.44 (1 HDJ = 7. 1 Hz) is correlated with 133(C-3), indicating that glucose is linked to the C-3 position. In HBMC spectrum, it can be seen that 1660 cc-9〃-9〃) and d-C methylene 4.02( 1H, dJ= 12.0Hz) and 426C 1H of sugar.
In the previous literature [130 1 belongs to C-2,6! While 1308 is classified as C2 < 6 >, but combining with 1H, W-COSY and HMBC and HSQG in 2D~NMR, we have reason to classify 130. 1 as C2 < 6 > 65438. See tables 1H-HMR, 13 NMR and HMBC for data attribution.
3.4 compound IV yellow powder (methanol) MP171~173c, magnesium hydrochloride powder reacted positively with Morris, and showed yellow fluorescence under ultraviolet light, and UVXim:348266 was a typical ultraviolet absorption of flavonoids; Acid hydrolysis, through thin layer chromatography, showed glucose and rhamnose compared with the control; The comparison of 1h-NMR and 13C-NMR with the compound 1 shows that kaempferol exists, and there is a group of A2B2 systems in the 1H-NMR spectrum. 7.97 (2 HDJ = 8.7 Hz) 6.87 (2 HDJ = 8.7 Hz) and element coupling system; 639( 1H, dJ= 1.7Hz), 6. 19( 1H, dJ= 1.7Hz)BC-NMR(DEPT) showed that the signal of sugar in this compound was 0/. 669 and s10Q7 (4.351HDJ =1.2hz) 70.3, 70.67 1.868.2, 17.7, respectively, belong to 6-position substitution (3-D). 1.2Hz) is related to 66.9(C-6), which shows that rhamnose is linked to the C-6 position of glucose in one configuration, and is related to1.3 (5.301HDJ = 7.5hz) and133.2 (0/33.2). It can be inferred that the compounds W are Kaempferol-3-13-D (RHA)-GLCCV)1HMR, 13C-NMR and HMBC. The data attribution of compounds W(DMSO)H-HMR and 65438+ are shown in Table 2.
4 discussion
4. 1 In the previous literature, C2 and 6 were classified as 130 1, while 1308 was classified as C2 < 6 > but combined with 2&in NMR,1h "hcosy, HMBC and HSQC.
4.2 Quercetin and Kaempferol are the main flavonol compounds in Rubus, and the glycoside position is mostly "-3", and the monosaccharide glycoside, disaccharide glycoside and triglycoside are all 17], but compound IV has not been reported in this species.