Preparation method and application of a dammaranetype diterpenoid compound

Abstract

The present invention provides a euphane-type diterpenoid compound, which is extracted from the traditional Chinese medicine Euphorbiae Semen: The seeds of Euphorbiae Semen are first extracted with 95% ethanol and concentrated to obtain a crude extract, then extracted with ethyl acetate, and the extract is subjected to macroporous resin column chromatography with an ethanol-water system. Among them, the components are subjected to normal-phase silica gel column chromatography with a petroleum ether-ethyl acetate system, and finally obtained after separation and purification by high-performance liquid chromatography. The above-mentioned diterpenoid compound can be used for the preparation of anti-tumor and anti-cancer drugs. The present invention lays a material foundation for effectively inhibiting the proliferation of cancer cells and developing new anti-tumor drugs, and is conducive to the further development of the medicinal value of Euphorbiae Semen.

Description

Technical Field

[0001] The present invention belongs to the field of natural medicine chemistry, and relates to a preparation method of diterpenoid compounds and their application in the preparation of anti-tumor and anti-cancer drugs. Background Art

[0002] Phytomedicine has always been an important source of natural medicine and also the main source for humans to prevent and treat diseases. Cancer has become the leading cause of death among Chinese residents, and its incidence and mortality are still increasing year by year, posing a huge threat to public health. Traditional anti-tumor drugs are mostly cytotoxic drugs. Although they have certain curative effects clinically, it is difficult to overcome the defects of strong toxic side effects, poor selectivity, and easy generation of drug resistance. Therefore, it is necessary to continuously develop new anti-tumor drugs with high efficiency, low toxicity, and strong specificity to improve the quality of life of cancer patients. The structural diversity of natural products and their characteristics of being easily combined with biological macromolecules determine their incomparable advantages in participating in life physiological processes. All these endow natural products with an irreplaceable important position in new drug research and development and are an important source for discovering drug lead structures and candidate drugs.

[0003] As an important medicinal plant, Euphorbia lathyris has a long medicinal history in China, and its secondary metabolites have been reported to have various structural types and diverse biological activities. Therefore, excavating metabolites with anti-tumor activity from Euphorbia lathyris is of great significance for further developing the medicinal value of Euphorbia lathyris and the research and development of new anti-tumor drugs, and can lay a foundation for the research and development of new anti-tumor drugs. In view of this, we propose a preparation method and application of a new diterpenoid compound. Summary of the Invention

[0004] In order to further explore the medicinal value of Euphorbia lathyris, the present invention provides a euphane-type diterpenoid compound extracted from Euphorbia lathyris, which has anti-tumor and anti-cancer activities.

[0005] Another object of the present invention is to provide a preparation method of the above-mentioned diterpenoid compound.

[0006] Another object of the present invention is to provide an application of the above-mentioned diterpenoid compound in anti-tumor and anti-cancer aspects.

[0007] To achieve the above objects, the present invention adopts the following technical solutions:

[0008] A euphane-type diterpenoid compound, whose structure is shown in formula (Ⅰ):

[0009]

[0010] Formula (Ⅰ)

[0011] The preparation method of the above-mentioned euphane-type diterpenoid compound includes the following steps:

[0012] (1) The seeds of Euphorbia lathyris are pulverized and extracted with 95% ethanol, and the extract is concentrated to obtain a crude extract.

[0013] (2) The crude extract is suspended in water and then extracted with ethyl acetate. After concentration of the organic phase, an ethyl acetate extract is obtained.

[0014] (3) The ethyl acetate extract is subjected to column chromatography on a D101 macroporous resin column and eluted with 80% ethanol to obtain the main eluted component.

[0015] (4) The eluted component from the above macroporous column chromatography is subjected to column chromatography on a normal-phase silica gel column and eluted successively with petroleum ether - ethyl acetate at a volume ratio of 50:1 - 1:5 v / v to obtain 10 fractions, Fr.1 - Fr.10.

[0016] (5) Fraction Fr.6 is subjected to column chromatography on a normal-phase silica gel column and eluted successively with petroleum ether - ethyl acetate at a volume ratio of 50:1 - 1:3 v / v to obtain fractions Fr.6.1 - Fr.6.3.

[0017] (6) Fraction Fr.6.3 is passed through a YMC-Pack ODS-A chromatographic column and isocratically eluted with 80% v / v methanol - water. The detection wavelength is 210 nm, and the fraction with a retention time of 15 min is collected to obtain the pure product of the above diterpenoid compound.

[0018] Preferably, in step (1), the material-liquid ratio of Euphorbia lathyris to 95% ethanol is 1:2 - 1:3 (w / v).

[0019] Preferably, in step (1), the Euphorbia lathyris is pulverized to a particle size diameter of less than 3 mm.

[0020] Preferably, in step (1), the extraction is carried out 3 times, and each time is for 7 days.

[0021] Preferably, in step (1), the concentration is to 1 / 50 - 1 / 100 of the original volume.

[0022] Preferably, in step (2), the ratio of the crude extract to water is 1:1 - 1:3 (w / v).

[0023] Preferably, in step (2), the volume ratio of ethyl acetate to water is 1:1 - 1.5:1; the number of ethyl acetate extractions is 2 - 4 times.

[0024] Use of a euphane-type diterpenoid compound in the preparation of anti-tumor and anti-cancer drugs. The tumor is selected from, but not limited to, breast cancer.

[0025] The beneficial effects of the present invention are as follows: The present invention provides a euphane - type diterpenoid compound extracted from Euphorbia lathyris, its preparation method, and its application in anti - tumor and anti - cancer drugs. The above - mentioned diterpenoid can effectively inhibit the growth of tumor cells MDA - MB - 231. The active compound extracted by the present invention provides a material basis for the research and development of anti - tumor drugs, which is conducive to the further development of the medicinal value of Euphorbia lathyris. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is the 1 1H NMR spectrum of the compound;

[0027] Figure 2 is the 13 13C NMR spectrum of the compound;

[0028] Figure 3 is the DEPT spectrum of the compound;

[0029] Figure 4 is the HSQC spectrum of the compound;

[0030] Figure 5 is the HMBC spectrum of the compound;

[0031] Figure 6 is the 1 1H - 1 HCOSY spectrum of the compound;

[0032] Figure 7 is the NOESY spectrum of the compound;

[0033] Figure 8 is the HR - ESIMS spectrum of the compound;

[0034] Figure 9 is the ECD spectrum of the compound. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0035] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0036] Example 1 Preparation of a Diterpenoid Compound

[0037] 10 kg of Euphorbia lathyris seeds are crushed to a particle size diameter less than 3 mm, soaked and extracted 3 times with 95% ethanol, 30 L each time, for 7 days each time. The ethanol extracts are combined and concentrated under reduced pressure to obtain 2.8 kg of a crude extract;

[0038] The crude extract was suspended in 3 L of water and then extracted three times with ethyl acetate, 4 L each time. The ethyl acetate extracts were combined and concentrated under reduced pressure to obtain 1.98 kg of the ethyl acetate extract;

[0039] The extract was subjected to column chromatography on D101 macroporous resin, eluted with 80% ethanol, and the main eluted samples were detected by thin-layer chromatography and combined to obtain 800 g;

[0040] The eluted sample from the macroporous resin column chromatography was subjected to normal-phase silica gel column chromatography and eluted with a petroleum ether-ethyl acetate system (v / v, 50:1 - 1:5). According to the analysis of the components of the eluate by thin-layer chromatography, fractions Fr.1 - Fr.10 were collected;

[0041] Fraction Fr.6 was subjected to normal-phase silica gel column chromatography and gradient eluted with a petroleum ether-ethyl acetate system (v / v, 50:1 - 1:3). According to the analysis of the components of the eluate by thin-layer chromatography, fractions Fr.6.1 - Fr.6.3 were collected;

[0042] Fraction Fr.6.3 was purified by high-performance liquid chromatography (column: YMC-Pack ODS-A, flow rate 2 mL / min, detection wavelength 210 nm), isocratically eluted with methanol-water (v / v, 80%), and the fraction with a retention time of 15 min was collected to obtain the euphane-type diterpenoid compound.

[0043] Physical properties of the new component extracted from Euphorbiae Semen: Euphane-type diterpenoid, colorless oily, soluble in methanol and chloroform, insoluble in water. Specific rotation [α] 20 D +38.0 (c 0.1, MeOH); Ultraviolet (MeOH) λ max (logε) 274(4.58) nm; Circular dichroism ECD (MeOH) λ (Δε) 287(+26.90), 261 ( −15.23), 217 ( −14.95)nm.

[0044] Example 2 Structure identification of the compound

[0045] By analyzing its high-resolution mass spectrum ( Figure 8 ), the quasi-molecular ion [M + Na + peak at m / z 587.2613 can be obtained. Combining with the nuclear magnetic data analysis, the molecular formula of the compound can be deduced as C 33 H 40 O8 (the calculated value is 587.2615). By further using modern nuclear magnetic spectroscopy techniques, analyzing its 1 H spectrum ( Figure 1 ) and 13 C spectrum ( Figure 2), the compound was determined to be a euphane - type diterpenoid. By analyzing its two - dimensional nuclear magnetic resonance spectra, including COSY, HSQC, HMBC, and NOESY ( Figures 4 - 7 ), its planar structure and relative configuration were determined. Further, by comparing its circular dichroism ECD spectrum ( Figure 9 ), and considering the biogenesis of other euphane - type diterpenoids isolated from the Chinese medicine Euphorbiae Semen, the absolute configuration of this diterpenoid was determined. The structure of this compound was determined as shown in formula (I), belonging to the euphane - type diterpenoid:

[0046]

[0047] Formula (I)

[0048] The chemical name of this compound is: (2S, 3S, 4S, 5R, 9S, 11R, 15R)-15,17 - diacetoxy - 3 - cinnamoyloxy - 5 - hydroxy - 14 - oxolathyra - 6Z,12E - diene, and it is named Euphorbia factor L 35 .

[0049] Example 3 In vitro antitumor activity of the compound

[0050] The SRB method was used to test the in vitro inhibitory activity of the compound against tumor cells. The selected tumor cell line was MDA - MB - 231. The specific process was as follows: MDA - MB - 231 cells were cultured in DMEM (Gibco) medium and placed in a cell culture incubator at 37°C with 5% CO2. Cells in the logarithmic growth phase were seeded into 96 - well plates (100 μL / well, 0.6 - 0.8×10 4 cells / well), and then incubated at 37°C for 24 h. The old medium was discarded, and then fresh medium containing different concentrations of the compound was added and cultured at 37°C for 48 h. The old culture solution was discarded, 150 μL of 10% TCA was added to each well, and fixed in a 4°C refrigerator for 1 h. After 1 h, the 96 - well plate was taken out, the fixing solution was discarded, each well was washed 5 times with PBS, the excess PBS was shaken off and the plate was inverted, and after natural drying, 50 μL of SRB was added to each well and stained on a shaker for 5 min. The SRB staining solution was discarded, and then washed 5 times with 4% acetic acid solution, the excess solution was shaken off and the plate was inverted, and after natural drying, 100 μL of Tris - HCl was added to each well to dissolve. The absorbance value of each well was detected at 540 nm with an enzyme - linked immunosorbent assay reader. The cell survival rate was calculated according to the formula: TNBC cell survival rate = (average absorbance of the experimental group - average absorbance of the blank group) / (average absorbance of the control group - average absorbance of the blank group)×100%, with doxorubicin as the positive control.

[0051] In this study, the cytotoxic activity of this clerodane diterpenoid against breast cancer MDA-MB-231 cell line was determined. The determination results showed that this compound could effectively inhibit the growth of MDA-MB-231 cells, and the IC 50 value was 21.3 μM.

[0052] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A euphane - type diterpenoid compound, whose structure is shown in formula (Ⅰ): Formula (Ⅰ).

2. A method for preparing the clerodane diterpenoid compound as described in claim 1, characterized in that: It includes the following steps: (1) Euphorbia seeds are crushed and extracted with 95% ethanol. The extract is concentrated to obtain a crude extract; (2) The crude extract is suspended in water and then extracted with ethyl acetate. The organic phase is concentrated to obtain an ethyl acetate extract; (3) The ethyl acetate extract is subjected to column chromatography on a D101 macroporous resin column and eluted with 80% ethanol to obtain the main eluted component; (4) The main eluted component obtained from the above macroporous resin column chromatography is subjected to normal - phase silica gel column chromatography and eluted successively with petroleum ether - ethyl acetate with a volume ratio of 50:1 - 1:5 v / v to obtain 10 fractions Fr.1 - Fr.10; (5) Fraction Fr.6 is subjected to normal - phase silica gel column chromatography and eluted successively with petroleum ether - ethyl acetate with a volume ratio of 50:1 - 1:3 v / v to obtain fractions Fr.6.1 - Fr.6.3; (6) Fraction Fr.6.3 is passed through a YMC - Pack ODS - A chromatographic column and eluted with 80% v / v methanol - water. The detection wavelength is 210 nm. The fraction with a retention time of 15 min is collected, and the solvent is removed to obtain the pure product of the above - mentioned euphane - type diterpenoid compound.

3. The preparation method according to claim 2, characterized in that: In step (1), the material - liquid ratio of the Euphorbia seeds to 95% ethanol is 1:2 - 1:3 according to w / v.

4. The preparation method according to claim 2, wherein: In step (1), the Euphorbia seeds are crushed to a particle size diameter of less than 3 mm.

5. The preparation method according to claim 2, characterized in that: In step (1), the extraction times are 3 times, and each time is 7 days.

6. The preparation method according to claim 2, wherein: In step (1), the concentration is 1 / 50 - 1 / 100 of the original volume.

7. The preparation method according to claim 2, characterized in that: In step (2), the ratio of the crude extract to water is 1:1 - 1:3 according to w / v.

8. The preparation method according to claim 2, characterized in that: In step (2), the volume ratio of ethyl acetate to water is 1:1 - 1.5:1; the number of ethyl acetate extraction times is 2 - 4 times.

9. Use of the clerodane diterpenoid compound as described in claim 1 in the preparation of an anti-tumor drug, characterized in that: The tumor is breast cancer.

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