Triterpenoid compound and preparation method and application thereof
阅读说明：本技术 一种三萜类化合物及其制备方法和应用 (Triterpenoid compound and preparation method and application thereof ) 是由 魏文文 于 2021-07-15 设计创作，主要内容包括：本发明公开了一种三萜类化合物及其制备方法和应用。三萜类化合物,结构式如式I所示。本发明从多穗稠分离出一个具有抗肿瘤活性的三萜类化合物,能够用于制备抗肿瘤药物,尤其是抗肺癌、宫颈癌、肝癌的药物,具有较好的应用价值。(The invention discloses a triterpenoid compound and a preparation method and application thereof. The structural formula of the triterpenoid is shown as a formula I. The triterpenoid with the anti-tumor activity is separated from the thick panicle, can be used for preparing anti-tumor medicines, particularly medicines for resisting lung cancer, cervical cancer and liver cancer, and has a good application value.)
1. Triterpenes have a structural formula shown in formula I:
2. a method of preparing the triterpenoid of claim 1, which is isolated from thick tassel plants.
3. The preparation method according to claim 2, characterized by comprising the following steps:
extracting the thick ear with 95% ethanol solution, concentrating the extractive solution to obtain ethanol extract, precipitating with 20% ethanol solution, centrifuging to obtain precipitate, subjecting to normal phase silica gel column chromatography, gradient elution is carried out by trichloromethane-methanol at the ratio of 10: 0-70: 30v/v, fraction St-10 with the developing agent of chloroform-methanol at the ratio of 8:2v/v and the sulfuric acid vanillin developing color and the Rf value of 0.65 is collected and checked by TLC, then reversed phase gel column chromatography is carried out, gradient elution is carried out by methanol-water at a ratio of 40: 60-80: 20v/v, a fraction St-10-p with a developing agent of chloroform-methanol at a ratio of 8:2v/v and a sulfuric acid vanillin developing color and an Rf value of 0.62 is collected and checked by TLC, and then the fraction is eluted by methanol through Sephadex LH-20 column chromatography and repeatedly purified to obtain the triterpenoid.
4. Use of a thick tassel for the preparation of the triterpenoid of claim 1.
5. Use of the triterpenoid of claim 1 in the preparation of an anti-tumor medicament.
6. The use of claim 5, wherein the anti-tumor drug is a drug against lung cancer, cervical cancer or liver cancer.
7. An antitumor agent characterized by comprising the triterpenoid compound according to claim 1 as an active ingredient.
8. The antitumor drug as claimed in claim 7, wherein the antitumor drug is a drug against lung cancer, cervical cancer or liver cancer.
The technical field is as follows:
the invention belongs to the field of natural products, and particularly relates to a triterpenoid and a preparation method and application thereof.
Background art:
the sweet taste drink is a folk nonsugar sweet drink with a long eating history, and is a eupolyphaga (Fagaceae) and Lithocarpus (Lithocarpus) evergreen plant. The thick ears are mostly distributed in the south provinces of the Yangtze river in a wild state, and are abundant and concentrated in the provinces of Hunan, Fujian, Jiangxi, Guangxi, Anhui and the like. The product can be used as a medicine for preventing and treating warm dysentery, skin pruritus, carbuncle, cellulitis, and malignant boil, and also has effects of protecting secondary antibody, protecting liver, and reducing blood sugar and blood lipid. According to the literature, the flavonoid compound with thick ears is the main component and the functional substance, and other chemical components are less reported.
The invention content is as follows:
the first purpose of the invention is to provide a triterpenoid lithocarpic acid O with anti-tumor activity, the structural formula of which is shown in formula I:
the second purpose of the invention is to provide a preparation method of the triterpenoid, which is obtained by separating from thick tassel plants.
The method specifically comprises the following steps:
leaching the thick polycarps with 95% ethanol solution in volume fraction, concentrating the extracting solution to obtain ethanol extract, precipitating the ethanol extract with 20% ethanol solution in volume fraction, centrifuging to obtain a precipitate, performing normal phase silica gel column chromatography on the precipitate, performing gradient elution with chloroform-methanol 10: 0-70: 30v/v, collecting the fraction subjected to TLC inspection, wherein a developing agent is chloroform-methanol 8:2v/v, vanillin sulfate is developed, a fraction St-10 with an Rf value of 0.65, performing reverse phase gel column chromatography, performing gradient elution with methanol-water 40: 60-80: 20v/v, collecting the fraction subjected to TLC inspection, a developing agent is chloroform-methanol 8:2v/v, vanillin sulfate is developed, a fraction St-10-p with an Rf value of 0.62, and performing Sephadex LH-20 column chromatography, and performing methanol elution and repeated purification to obtain lithocarpic acid O.
The third purpose of the invention is to provide the application of the polyporus thick in the preparation of the triterpenoid.
The fourth purpose of the invention is to provide the application of the triterpenoid in preparing anti-tumor drugs.
The fifth object of the present invention is to provide an antitumor agent comprising the above triterpenoid as an active ingredient.
The anti-tumor drug is a drug for resisting lung cancer, cervical cancer or liver cancer.
The fifth purpose of the invention is to provide the application of the polyporus thick in the preparation of the triterpenoid.
The triterpenoid with the anti-tumor activity is separated from the thick panicle, can be used for preparing anti-tumor medicines, particularly medicines for resisting lung cancer, cervical cancer and liver cancer, and has a good application value.
Description of the drawings:
FIG. 1 is a schematic representation of Compound 11H-1H COSY (bold line) and HMBC (arrow) related signals.
The specific implementation mode is as follows:
the following examples are further illustrative of the present invention and are not intended to be limiting thereof.
Example 1:
1 Material Instrument
Silica gel for column chromatography (Qingdao ocean chemical Co., Ltd.); column chromatography on reversed phase silica gel Develosil ODS (particle size 75 μm, Nippon Fuji chemical Co.); sephadex LH-20 (Amersham Biosciences, Sweden); MCI for column chromatography (Merck, Germany); thin layer chromatography plate TLC (tiaojiangyou silica gel development ltd); reversed phase (RP-18) TLC plates (Merck, Germany); ESI-MS (Applied Bio-systems, USA);1h NMR and13c NMR (Bruker DRX-500 model, Bruker, Switzerland); semi-preparative HPLC (Shimadzu corporation, japan); four cell lines (kunming institute of chinese academy of sciences); the reagent used is analytically pure or chromatographically pure.
The thick panicle is collected from the Robooshan of Huizhou city in Guangdong province in 2013 months, identified by a Renhuang investigator in the plant garden of south China, China academy of sciences, and the whole plant of the thick panicle is dried and crushed for later use.
2 extraction and separation
Soaking 2.77kg of soft dry powder with 95% ethanol solution at room temperature for 4 times (48 hr each time), mixing extractive solutions, concentrating under reduced pressure to obtain 0.97kg of ethanol extract, precipitating with 20% ethanol solution at volume fraction overnight, centrifuging to obtain precipitate (259g) and supernatant (463 g). And (3) carrying out normal-phase silica gel column chromatography on the precipitate part, eluting by a chloroform-methanol gradient (10: 0-70: 30v/v), collecting fractions, and combining the fractions into 15 components (St-1-St-15) after TLC inspection. St-10(8.2g) (8: 2v/v chloroform-methanol as developing solvent, vanillin sulfate color, Rf value of 0.65) is subjected to reversed phase gel column chromatography, eluted by methanol-water (40: 60-80: 20v/v), and the same components are combined through TLC inspection to obtain 17 sub-components (St-10-a-St-10-q). St-10-p (chloroform-methanol 8:2v/v as developing solvent, vanillin sulfate color, Rf value 0.62) was subjected to Sephadex LH-20 column chromatography and repeated purification with methanol elution to give compound 1(6 mg).
3 structural characterization
Compound 1, white powder, readily soluble in pyridine;HR-ESI-MS:m/z 515.3707[M+Na]+(calcd for C30H52O5Na+,515.3693,error 2.7ppm)；1H-NMR(500Hz)and 13C-NMR (125Hz) is shown in Table 1 below
TABLE 1 Hydrogen and carbon Spectroscopy data (pyridine-d)5)
Determining the molecular weight of the compound 1 to be 492 according to the peak of HR-ESI-MS molecular ions; the unsaturation of the compound was 5 as calculated from the molecular formula.1High field of H-NMR spectrum has 7 groups of methyl proton signals deltaH1.05(3H, s),1.02(3H, s),1.58(3H, s),1.56(3H, s),0.94(3H, s),1.46(3H, s),1.48(3H, s); characteristic AB coupled proton signal deltaH0.77(1H, d, J ═ 4.1Hz),0.44(1H, d, J ═ 4.1 Hz); low field area signal deltaH3.81(1H, d, J ═ 8.5Hz) is the proton signal on the carbon to the hydroxyl group;13C-NMR and DEPT spectra gave 7 methyl-carbon signals deltaC(19.3,19.0,26.4,26.6,20.3,27.4,32.4), 7 quaternary carbon signals δC(177.6,75.7,23.3,27.9,45.7,49.7,73.3), and one carbonyl carbon, δC(177.6) the signal, which is combined with the above information, can be concluded that the compound is a cyclonorrane-type triterpene, and the hydrogen spectrum and carbon spectrum data are assigned by combining two-dimensional nuclear magnetic resonance spectrum (COSY and HSQC) (see Table 1). HMBC remote correlation data deltaH1.46(3H, s, H-29),1.48(3H, s, H-30) and δC46.4(C-5) correlation, δH1.46(3H, s, H-29),1.48(3H, s, H-30) and δC75.7(C-4) related (FIG. 1), it can be deduced that the two methyl groups are linked at the 4-carbon, and that the 4-carbon is an oxygen-containing quaternary carbon; deltaH1.05(3H, s, H-18),1.02(3H, s, H-21) and δC53.6(C-17) correlation, it can be concluded that these two methyl groups are attached to the C-17 side chain; by deltaH3.81(1H, d, J ═ 8.5Hz, H-24) and δC26.4(C-26),26.6(C-27), ΔH1.58(3H, s, H-26),1.56(3H, s, H-27) and δC73.3(C-25) correlation, δH1.58(3H, s, H-26),1.56(3H, s, H-27) and δC79.6(C-24) related, a chain diol fragment can be deduced; by deltaH1.02(3H, s, H-21) and δC53.6(C-17) correlation, it can be deduced that the chain diol fragment is linked to C-17. The structure of the compound 1 is identified as a new compound, namely lithacic acid O, and the structural formula is shown as a formula I.
4 Activity assay
The test sample is new compound 1, the positive control is Adriamycin (ADM), and the DMSO solution is 10mmol/L for standby. Logarithmic growth of A549, HeLa, HepG2, MCF-7 cells were added to 96-well plates containing 5000 cells per 100. mu.L well. New Compound 1 was added and ADM was used as a control. Setting 6 concentration gradients of 50, 10, 2, 0.4, 0.08 and 0.016 mu mol/L for each sample, repeating 3 times of treatment, culturing at 37 ℃ for 72h in a carbon dioxide incubator, adding 20 mu L MTT (5mg/mL) solution 4h before the experiment is ended, culturing for 4h, discarding the culture solution, adding 0.15mL DMSO, dissolving the crystal, and detecting each sample on an enzyme-linked detector under the wavelength of 570nmOD of the wells. The growth inhibition rate was determined according to the following formula, and IC was determined by SPSS (17.0) software50The value is obtained.
The Inhibition Ratio (IR) is (1-medicinal OD/control OD) × 100%
The results of the experiment are shown in table 2 below:
TABLE 2 cytotoxic Activity (IC) of novel Compound 150,μM)
IC50Expressed as Mean ± SD.
The results show that the new compound 1 has inhibitory activity on lung cancer cells (A549), cervical cancer cells (HeLa) and liver cancer cells (HepG 2).