CN110407907B - Rhododendron simsii diol and preparation method and application thereof - Google Patents
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Abstract
The invention provides a triterpenoid, namely, Ximi rhododendron diol for short, which has a structural formula as follows:
. The triterpenes are extracted from euphorbia milii: the euphorbia pekinensis whole plant dry powder is obtained by firstly extracting and concentrating by 95% ethanol to obtain a crude extract, then extracting and concentrating by ethyl acetate, then enabling ethyl acetate extract to pass through macroporous resin by ethanol-water, enabling components to pass through normal phase silica gel by petroleum ether-ethyl acetate, enabling components to pass through gel column chromatography by dichloromethane-methanol, and finally separating and purifying by liquid chromatography. The triterpene compounds can be used for preparing antitumor and anticancer drugs. The extracted compound provides a material basis for the research and development of novel antitumor drugs, and is beneficial to the further development of the medicinal value of the euphorbia pekinensis.
Description
Technical Field
The invention belongs to the field of natural medicinal chemistry, and relates to a preparation method of a triterpenoid and application of the triterpenoid in preparation of antitumor drugs.
Background
Cancer becomes the leading cause of death of diseases of residents in China, and the morbidity and mortality rate rise year by year, thus posing a great threat to public health. Most of traditional antitumor drugs are cytotoxic drugs, and although the traditional antitumor drugs have a certain clinical curative effect, the defects of strong toxic and side effects, poor selectivity, easy generation of drug resistance and the like are difficult to overcome, so that novel antitumor drugs with high efficiency, low toxicity and strong specificity need to be continuously developed, and the living quality of cancer patients is improved. The structural diversity and the characteristic of easy combination with biological macromolecules of the natural product determine incomparable advantages of the natural product in the process of participating in life physiology, and the natural product has important position irreplaceable in the research and development of new drugs and is an important source for finding drug lead structures and candidate drugs. Euphorbia peplus is an important plant resource, and secondary metabolites thereof have been reported to have various structural types and diverse biological activities, particularly in terms of antitumor activity. Therefore, the discovery of metabolites with antitumor activity from euphorbia milii is of great significance for the development of novel antitumor drugs.
Disclosure of Invention
In order to further develop the medicinal value of Euphorbia peplus, the invention provides a triterpenoid extracted from Euphorbia peplus, which has anti-tumor activity.
The invention also aims to provide a preparation method of the triterpenoid.
The invention further aims to provide the application of the triterpenoid in treating tumors.
In order to achieve the purpose, the invention adopts the following technical scheme.
A triterpene compound, named as simmer cuckoo diol for short, has a structural formula shown as a formula (I):
formula (I).
The structural type of the triterpenoid belongs to variant hopane-type triterpenoids (E: B-friedo-hopane type triterpenoids) and has double hydroxyl substitutions at the 3-and 22-positions.
The preparation method of the triterpenoid comprises the following steps:
(1) drying Euphorbia peplus L.and pulverizing, extracting with ethanol, and concentrating the extractive solution to obtain crude extract;
(2) suspending the crude extract in water, extracting with ethyl acetate, and concentrating the ethyl acetate extract phase to obtain ethyl acetate extract;
(3) subjecting the ethyl acetate extract to D101 macroporous resin column chromatography, and sequentially eluting with 30%, 50%, 80% and 95% v/v ethanol-water to obtain 4 components A-D;
(4) subjecting the component D to normal phase silica gel column chromatography, sequentially eluting with petroleum ether-ethyl acetate at ratio of 20:1, 15:1, 10:1, 7:1, and 5:1 v/v to obtain components D1-D8;
(5) subjecting the component D8 to Sephadex LH-20 gel column chromatography, eluting with 1:1 v/v dichloromethane-methanol to obtain components D-8-1 and D-8-2;
(6) subjecting the fraction D-8-2 to YMC-Pack ODS-A chromatography column, gradient eluting with 95% -100% v/v methanol water solution, detecting wavelength to 210 nm, collecting fraction with retention time of 27.0 min, and removing solvent to obtain pure EuphorbiA peplus extract.
In the step (1), the feed-liquid ratio of the euphorbia pekinensis to the ethanol is 1:2.5 (w/v).
In the step (1), the euphorbia pekinensis is crushed to the particle size diameter of less than 3 mm.
In the step (1), the leaching times are 4 times, and each time is 7 days.
In the step (1), the mixture is concentrated to 1/50-1/70 in original volume.
In the step (2), the volume ratio of the crude extract to water is 1: 1-2.
In the step (2), the volume ratio of the ethyl acetate to the water is 1: 1.
In the step (2), the extraction times of the ethyl acetate are 1-3 times.
In step (6), the flow rate was 3.0 mL/min.
An application of the triterpene compounds in preparing antitumor and anticancer medicines is provided. The tumor is selected from, but not limited to, cervical cancer, breast cancer and lung cancer.
The invention has the following advantages:
the invention provides a triterpenoid extracted from euphorbia pekinensis, a preparation method thereof and application thereof in preparing anti-tumor drugs. The triterpenoid belongs to variant hopane triterpenes, and has double hydroxyl substitution at 3-position and 22-position. Compared with the reported sitaxasterol, the compound has one more hydroxyl substitution at the C-22 position. The triterpenoid can remarkably inhibit the growth of tumor cells A549, HeLa, MCF-7 and MDA-MB-231 and cause the death of the tumor cells, and the action mechanism of the triterpenoid is to cause the death of the tumor cells by inducing apoptosis and causing cell cycle arrest. The extracted compound provides a material basis for the research and development of novel antitumor drugs, and is beneficial to the further development of the medicinal value of the euphorbia pekinensis.
Drawings
FIG. 1 is a high resolution mass spectrum of Rhododendron simsii diol;
FIG. 2 is a hydrogen spectrum of Rhododendron simsii diol;
FIG. 3 is a carbon spectrum of Rhododendron simsii diol;
FIG. 4 is a two-dimensional COSY spectrum of Rhododendron simsii diol;
FIG. 5 is a two-dimensional HSQC spectrum of Rhododendron simsii diol;
FIG. 6 is a two-dimensional HMBC spectrum of Rhododendron simsii diol;
FIG. 7 is a two-dimensional ROESY spectrum of Rhododendron simsii diol;
FIG. 8 is a graph of Rhododendron simsii diol-induced changes in HeLa cell morphology;
FIG. 9 shows that Semipriol significantly induces HeLa apoptosis;
FIG. 10 is that Semipril diol causes HeLa cell cycle arrest.
Detailed Description
The present invention will be further described with reference to the following examples and drawings, but the present invention is not limited to the following examples.
EXAMPLE 1 preparation of Rhododendron simsii diol
Drying Euphorbiae radix 10 kg, pulverizing to particle size less than 3 mm, soaking in 95% ethanol for extraction for 7 days, 25L each time, 4 times. Mixing the ethanol extractive solutions, and concentrating under reduced pressure to obtain 1.5 kg crude extract;
the crude extract was suspended in 2.0L of water and then extracted three times with 2.0L of ethyl acetate each time. Mixing ethyl acetate extract phases, and concentrating under reduced pressure to obtain 470 g of ethyl acetate extract;
subjecting the extract to D101 macroporous resin column chromatography, eluting with ethanol-water (v/v, 30%, 50%, 80% and 95%) to obtain 4 components A-D in sequence;
subjecting the component D (190 g) to normal phase silica gel column chromatography, eluting with petroleum ether-ethyl acetate (v/v, 20:1, 15:1, 10:1, 7:1, 5: 1), analyzing eluent components according to thin layer chromatography, and collecting to obtain components D1-D8;
fraction D8 was subjected to gel (Sephadex LH-20) column chromatography and eluted with methylene chloride-methanol (v/v, 1: 1) to give 2 fractions D-8-1 and D-8-2.
The fraction D-8-2 was purified by preparative liquid chromatography (column: YMC-Pack ODS-A, 10X 250 mm, flow rate: 3.0 mL/min, detection wavelength: 210 nm), gradient-eluted with methanol-water (v/v, 95% -100%), and fractions with retention time of 27.0 min were collected, and the solvent was removed to give 7.2 mg of A pure new fraction extracted from EuphorbiA peplus L.var.John.
The physical properties of the new ingredients extracted from the euphorbia pekinensis are as follows: white amorphous powder, easily soluble in acetonitrile, dimethyl sulfoxide and chloroform, insoluble in water; specific rotation angle [ alpha ]α] D 25 +54.4 (c 0.72, CHCl3); ECD (c 0.04, MeCN) λ(Δε) 210 (+2.94) nm。
Example 2 structural identification of Rhododendron simsii diol
Obtaining the dehydrated molecular ion peaks by resolution of high resolution Mass Spectrometry (FIG. 1)m/z 425.3774（[M+H–H2O]+Theoretical value 425.3778), and the molecular formula C of the extracted new component of Euphorbia peplus is determined by combining the data of carbon spectrum (figure 3)30H50O2. By further analysis of the hydrogen (FIG. 2) and carbon spectra and comparison with rhododendrol nuclear magnetic data, the planar structure of the compound was found to be a variant hopane-type triterpene (E: B-friedo-hopane type triterpene).
TABLE 1 Hydrogen spectra of extracted constituents (600 MHz, CDCl)3) And carbon spectrum (150 MHz, CDCl)3) Data of
The structure of the new component extracted from Euphorbia peplus L.is finally determined by analyzing two-dimensional nuclear magnetic spectrum including COSY, HSQC, HMBC and ROESY (FIGS. 4-7). Compared with the simethiol, the component has one more hydroxyl substitution at the C-22 position. Through comparison experiments and theoretical calculation of ECD data, the absolute configuration of a new component extracted from Euphorbia pulcherrima as shown in the following formula is determined, and the absolute configuration is abbreviated as ximidadiol:
example 3 antitumor Activity of Rhododendron simsii diol
1. Inhibiting different in vitro tumor cells
The method adopts an MTT method to test the survival rate of the tumor cells in vitro of the Rhododendron simsii diol, and the screened tumor cell strains are HeLa, A549, MCF-7 and MDA-MB-231. The specific process is as follows: rhododendron simsii diol was prepared as a 20 mM stock in DMSO, and then diluted to 0.50-45.0. mu.M with the culture medium. HeLa, MCF-7 and MDA-MB-231 cells were cultured in DMEM (Gibco) medium and A549 cells in RPMI-1640 (Gibco) medium at 37 ℃ with 5% CO2In a cell culture chamber. Cells in logarithmic growth phase were seeded in 96-well plates (100. mu.L/well, 0.4-0.6X 10)4Cells/well) and then incubated at 37 ℃ for 24 hours. The old medium was discarded, and then fresh medium containing varying concentrations of azalea diol was added and incubated at 37 ℃ for 48 hours. After completion of the incubation, 10. mu.L of 3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyl-triazolium bromide (MTT) solution (5 mg/mL, Sigma) was added to each well, followed by incubation at 37 ℃ for 4 hours. After discarding the supernatant, 100. mu.L of DMSO solution was added to each well to dissolve the crystals sufficiently. Finally, the absorbance of each well was measured at 490 nm wavelength using a SPARK microplate reader. Doxorubicin was used as a positive control.
TABLE 2 Experimental data for in vitro antitumor Activity of Rhododendron simsii diol
The results show that the simethiol shown in the formula (I) has obvious inhibition on four tumor cellsThe activity is particularly remarkable for HeLa and A549 cells, and the IC of the HeLa and A549 cells50The values are shown in Table 2. Therefore, the azalea diol shown in the formula (I) can be used for preparing antitumor drugs.
2. Induction of apoptosis in HeLa cells
DAPI staining experiment: HeLa cells were seeded on a confocal culture dish and cultured for 24 hours with the addition of DMSO (0.1%) and cetrimide diol at final concentrations of 3.0, 6.0 and 12.0. mu.M, respectively, for a total of 48 hours. After the completion of the culture, 4% paraformaldehyde was added, reacted for 10 min, and then washed 3 times with PBS. Finally, the cells were reacted with DAPI dye (bi yun, jiangsu, china) for 15 min in the dark, washed 3 times and photographed with an inverted fluorescence microscope (Leica DMi8, germany). As shown in fig. 1, HeLa cells treated with rhododendron diol showed significant changes in cell morphology, such as cell shrinkage, nuclear compaction and nuclear fragmentation, compared to the negative control, especially at 6.0 and 12.0 μ M drug concentrations. DAPI experiment shows that the Rhododendron simsii diol can effectively induce HeLa cell apoptosis.
The proportion of apoptosis was investigated using flow cytometry in combination with Annexin V/PI double staining experiments: HeLa cells in logarithmic growth phase were seeded in 6-well plates at about 20 ten thousand cells per well and cultured at 37 ℃ for 24 hours, followed by the addition of simethicone at final concentrations of 3.0, 6.0 and 12.0. mu.M, and negative control DMSO (0.1%) for a total of 48 hours. Cells were collected, washed 2 times with PBS solution, and then gently suspended in binding solution. Finally, HeLa cells were reacted with Annexin-V/FITC (5. mu.L) and PI (5. mu.L) (BD Biosciences, USA) at room temperature and in the dark for 15 min, and then apoptosis information was analyzed by flow cytometry (ACEA Biosciences, USA). The proportion of apoptotic cells was obtained after processing by NovoExpress analysis software. As shown in fig. 2, the total apoptosis ratio (including early and late withers) of negative control DMSO and 3.0 μ M azalea diol was 7.98% and 9.83%, respectively, whereas when the concentration of azalea diol was increased to 6.0 and 12.0 μ M, the total apoptosis ratio of HeLa cells was increased to 27.27% and 49.94%, respectively. The experiment shows that the Rhododendron simsii diol can induce HeLa cell apoptosis in a concentration-dependent manner.
The phase of simmer diol blocking HeLa cells was further verified by flow cytometry: the specific experiment is as follows: HeLa cells were seeded in 6-well plates at about 20 ten thousand cells per well and cultured at 37 ℃ for 24 hours. Rhododendron simsii diol was added at final concentrations of 3.0, 6.0 and 12.0. mu.M, respectively, and a negative control DMSO, and co-incubated for 36 hours. The cells were harvested, centrifuged to remove the medium, washed 2 times with PBS, and then fixed with 70% ice-cold ethanol overnight at 4 ℃. The ethanol was removed, the cells were resuspended in ice-cold wash solution and centrifuged. Cells were reacted with PI dye (Genview, USA) at 37 ℃ for 30 min in dark environment, cells at different cycle stages were detected with flow cytometry, and then data were processed with NovoExpress software. As shown in FIG. 3, Semipriol was able to significantly concentration-dependently arrest HeLa cells in the S and G2 growth phases.
Claims (6)
1. The preparation method of the triterpenoid is characterized by comprising the following steps:
(1) drying Euphorbia peplus L.and pulverizing, extracting with ethanol, and concentrating the extractive solution to obtain crude extract;
(2) suspending the crude extract in water, extracting with ethyl acetate, and concentrating the ethyl acetate extract phase to obtain ethyl acetate extract;
(3) subjecting the ethyl acetate extract to D101 macroporous resin column chromatography, and sequentially eluting with 30%, 50%, 80% and 95% v/v ethanol-water to obtain 4 components A-D;
(4) subjecting the component D to normal phase silica gel column chromatography, sequentially eluting with petroleum ether-ethyl acetate at ratio of 20:1, 15:1, 10:1, 7:1, and 5:1 v/v to obtain components D1-D8;
(5) subjecting the component D8 to Sephadex LH-20 gel column chromatography, eluting with 1:1 v/v dichloromethane-methanol to obtain components D-8-1 and D-8-2;
(6) subjecting the component D-8-2 to YMC-Pack ODS-A chromatography column, gradient eluting with 95% -100% v/v methanol water solution, detecting wavelength at 210 nm, collecting component with retention time of 27.0 min, and removing solvent to obtain triterpene compounds;
the structural formula of the triterpenoid is as follows:
in the step (1), the leaching times are 4 times, and each time is 7 days.
2. The method according to claim 1, wherein in step (1), the ratio of the euphorbia milii to the ethanol is 1:2.5 w/v.
3. The method of claim 1, wherein in step (1), the Euphorbia peplus is ground to a particle size of less than 3 mm in diameter.
4. The method of claim 1, wherein in step (1), the concentrate is 1/50-1/70.
5. The preparation method according to claim 1, wherein in the step (2), the volume ratio of the crude extract to water is 1: 1-2.
6. The method according to claim 1, wherein in the step (2), the volume ratio of the ethyl acetate to the water is 1: 1; the extraction times of the ethyl acetate are 1-3 times.
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Citations (4)
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| JPH04187641A (en) * | 1990-11-20 | 1992-07-06 | Katakura Kogyo Kk | New carcinostatic substance |
| CN101880308A (en) * | 2010-07-01 | 2010-11-10 | 沈阳药科大学 | New hopane-type triterpene and preparation method and application thereof |
| CN105943540A (en) * | 2016-04-26 | 2016-09-21 | 江苏康缘药业股份有限公司 | Application of simiarenol |
| CN106117328A (en) * | 2016-07-01 | 2016-11-16 | 中国科学院植物研究所 | Oryza sativa L. sheath alcohol synthesis associated protein and encoding gene thereof and application |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH04187641A (en) * | 1990-11-20 | 1992-07-06 | Katakura Kogyo Kk | New carcinostatic substance |
| CN101880308A (en) * | 2010-07-01 | 2010-11-10 | 沈阳药科大学 | New hopane-type triterpene and preparation method and application thereof |
| CN105943540A (en) * | 2016-04-26 | 2016-09-21 | 江苏康缘药业股份有限公司 | Application of simiarenol |
| CN106117328A (en) * | 2016-07-01 | 2016-11-16 | 中国科学院植物研究所 | Oryza sativa L. sheath alcohol synthesis associated protein and encoding gene thereof and application |
Non-Patent Citations (2)
| Title |
|---|
| New e:b-Friedo-Hopane Type Triterpenoids from Euphorbia peplus with Simiarendiol Possessing Significant Cytostatic Activity against HeLa Cells by Induction of Apoptosis and S/G2 Cell Cycle Arrest;Jin-Hai Yu et al.;《Molecules》;20190827;第24卷;第3106页 * |
| Triterpenoids from Ainsliaea yunnanensis Franch.and Their Biological Activities;Jinjie Li et al.;《Molecules》;20161107;第21卷;第1481页 * |
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