Euphorbia fischeriana terpene A as well as preparation method and application thereof

Abstract

The invention discloses euphorbia fischeriana terpene A as well as a preparation method and application thereof, and relates to a novel substance capable of resisting breast cancer in vitro as well as a method and application thereof. The invention provides a novel substance with in-vitro anti-breast cancer activity and a preparation method and application thereof. The molecular formula of the euphorbia fischeriana terpene A is C21H34O13. The preparation method comprises the following steps: 1, carrying out cold-soaking extraction on euphorbia fischeriana by using ethanol; 2, dispersing the extract from which the ethanol is recovered with water, and extracting; and 3, carrying out repeated silica gel column chromatography separation on the ethyl acetate extraction layer, and then utilizing high performance liquid chromatography to obtain a monomeric compound which is identified as a new compound through spectroscopy and named Euphorbia fischeriana terpene A. The invention also discloses a preparation method of the Euphorbia fischeriana terpene A. The euphorbia fischeriana terpene A is applied to preparation of anti-breast cancer drugs. The IC50 of the euphorbia fischeriana terpene A to breast cancer MCF-7 cells, the IC50 of the euphorbia fischeriana terpene A to ZR-75-1 cells and the IC50 of the euphorbia fischeriana terpene A to MDA-MB-231 cells are 25.4 [mu] mol / L, 19.2 [mu] mol / L and 21.9 [mu] mol / L respectively, and it is indicated that the euphorbia fischeriana terpene A has The invention belongs to the field of research on effective components of natural medicines.

Description

Technical Field

[0001] The present invention relates to a new substance having in vitro anti-breast cancer effect and a method and application thereof. Background Art

[0002] Euphorbia chamaejasma is included in the 2020 edition of the Chinese Pharmacopoeia and is a traditional Chinese medicinal material. It tastes bitter and pungent, is neutral in nature, and is toxic. It has the effects of draining water and removing fluid, breaking up accumulation and killing insects. It is mainly used to treat edema, abdominal distension, phlegm, food and insect accumulation, heart and abdominal pain, tuberculosis, scabies, etc.

[0003] Modern pharmacological research shows that the main active ingredients of Euphorbia chamaejasme are diterpenes, which have anti-tumor, anti-bacterial, anti-fungal, anti-viral and anti-epileptic effects, and are a very valuable source of plant anti-tumor drugs.

[0004] At present, there are many preparations of Euphorbia stellariae on the market, but as a medicinal plant with a long application, it is of great significance to carry out systematic chemical separation of Euphorbia stellariae in order to improve its quality standards and discover more index components with anti-breast cancer activity.

[0005] Breast cancer is a malignant tumor that occurs in the mammary epithelium or ductal epithelium. It ranks first in the incidence of female malignant tumors. Comprehensive therapy is the most common treatment for breast cancer. Common methods include surgery combined with radiotherapy and chemotherapy, molecular targeted therapy, immunotherapy, endocrine therapy, etc. In my country, advanced breast cancer still accounts for the majority of patients. Most of them use chemotherapy, which will produce a variety of adverse reactions, and there is a certain probability of metastasis and recurrence. Therefore, the research work of finding new drugs for the treatment of breast cancer is arduous and long-term. Traditional Chinese medicine has the advantages of multiple targets, low toxicity and side effects, and is not easy to produce drug resistance in the treatment of cancer. It not only conforms to the multi-factor and multi-link pathogenic mechanism of tumors, but also has significant effects on improving the treatment success rate of tumor patients, improving the quality of life of tumor patients, and prolonging life. Therefore, screening out anti-tumor active ingredients with strong biological activity and low toxicity and side effects from traditional Chinese medicine has broad research prospects. Summary of the invention

[0006] The purpose of the present invention is to provide a new compound with in vitro anti-breast cancer activity and a preparation method and application thereof.

[0007] The molecular formula of Euphorbia chamaejasme Terpene A is C 23 H 34 O5, molecular structure formula is:

[0008]

[0009] The preparation method of the Euphorbia chamaejasme terpene A is carried out according to the following steps:

[0010] 1. placing the Euphorbia chamaejasme in an extraction tank of a Soxhlet dynamic extraction and concentration unit, extracting with ethanol to obtain an ethanol extract;

[0011] 2. The extract obtained in step 1 is concentrated and dispersed with water, and extracted with petroleum ether and ethyl acetate respectively;

[0012] 3. Subject the ethyl acetate extract of step 2 to silica gel column chromatography, and elute with a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 100:1, 80:1, 50:1, and 30:1 in sequence; take the eluate with a volume ratio of petroleum ether to ethyl acetate of 30:1 for secondary silica gel column chromatography, and elute with a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 50:1, 40:1, 30:1, and 20:1 in sequence; collect the eluate to prepare a high performance liquid chromatography sample, using a mixed solvent of methanol and water in a volume ratio of 85:15 as the mobile phase, at a flow rate of 3 mL / min, and collect the absorption peak that appears at the 21st minute of the elution time to obtain wolfsbane euphorbia terpene A.

[0013] The pressure of the Soxhlet dynamic extraction and concentration unit in step 1 is set to extraction at normal temperature and pressure.

[0014] The high performance liquid chromatogram in step 3 was collected by using a Waters 2535 semi-preparative high performance liquid chromatograph. 18 Reverse phase silica gel was used as the filler of the chromatographic column and a Waters 2414 differential detector was used as the detector.

[0015] The stellera chamaejasme terpene A is used for preparing anti-breast cancer drugs.

[0016] The dosage forms of the anti-breast cancer drug include injection, lyophilized powder injection and oral preparation.

[0017] The oral preparations include tablets, granules, soft capsules, hard capsules, oral liquids, and sustained-release preparations.

[0018] The Euphorbia chamaejasme terpenoid A is used as an active ingredient of an anti-breast cancer drug for inhibiting the proliferation of breast cancer MCF-7, ZR-75-1 and MDA-MB-231 cells.

[0019] The chemical name of the Euphorbia chamaejasme terpenoid A of the present invention is Methyl-12-hydroxy-14-methoxy-11-(9-methoxy-18,19,20-trimethyldecahydronaphthalen-8-yl)benzoate, and the molecular formula is C 23 H 34 O5.

[0020] The compound of the present invention can be added with pharmaceutically acceptable excipients to prepare pharmaceutically acceptable solid preparations or liquid preparations.

[0021] The auxiliary materials described in the present invention are not limited as long as they are pharmaceutically acceptable.

[0022] Compared with the prior art, the present invention has the following advantages:

[0023] The present invention provides a new compound derived from Euphorbia chamaejasme and a method for extracting, separating and purifying the new compound of the present invention. The new compound is successfully obtained by separation and purification using methods such as ethanol extraction, extraction, silica gel column chromatography, and preparative high performance liquid chromatography. The operation method is simple and rapid, and the compound separated by the method has a high purity. In addition, studies have shown that the above compound has anti-breast cancer activity.

[0024] The new compound can inhibit the proliferation of breast cancer MCF-7, ZR-75-1 and MDA-MB-231 cells. When the compound is at a concentration of 100 μM, 50 μM, 25 μM, 12.5 μM and 6.25 μM, the inhibition rates on MCF-7 cells are 80.2%, 70.7%, 57.1%, 33.8% and 6.4%, respectively, and the inhibition rates on ZR-75-1 cells are 90.6%, 77.8%, 62.2%, 44.2% and 8.1%, respectively. The inhibition rates of the Euphorbia chamaejasme terpenoid A of the present invention on MDA-MB-231 cells are 86.4%, 79.6%, 64.6%, 24.8% and 11.2%. The IC values ​​for inhibition of breast cancer MCF-7, ZR-75-1 and MDA-MB-231 cells are 1. 50 The results show that the terpenoid A of the present invention can be prepared into anti-breast cancer drugs such as injection, lyophilized powder injection, infusion or oral preparation (including tablets, granules, soft capsules, hard capsules, oral liquid and sustained-release preparations). DETAILED DESCRIPTION

[0025] The technical solution of the present invention is not limited to the specific implementation modes listed below, but also includes any combination of the specific implementation modes.

[0026] Specific implementation method 1: In this implementation method, the molecular formula of Euphorbia chamaejasme terpenoid A is C 23 H 34 O5, molecular structure formula is:

[0027]

[0028] Specific implementation method 2: The preparation method of Euphorbia chamaejasme terpene A described in specific implementation method 1 is carried out according to the following steps:

[0029] 1. placing the Euphorbia chamaejasme in an extraction tank of a Soxhlet dynamic extraction and concentration unit, extracting with ethanol to obtain an ethanol extract;

[0030] 2. The extract obtained in step 1 is concentrated and dispersed with water, and extracted with petroleum ether and ethyl acetate respectively;

[0031] 3. Subject the ethyl acetate extract of step 2 to silica gel column chromatography, and elute with a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 100:1, 80:1, 50:1, and 30:1 in sequence; take the eluate with a volume ratio of petroleum ether to ethyl acetate of 30:1 for secondary silica gel column chromatography, and elute with a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 50:1, 40:1, 30:1, and 20:1 in sequence; collect the eluate to prepare a high performance liquid chromatography sample, using a mixed solvent of methanol and water in a volume ratio of 85:15 as the mobile phase, at a flow rate of 3 mL / min, and collect the absorption peak that appears at the 21st minute of the elution time to obtain wolfsbane euphorbia terpene A.

[0032] Specific implementation method three: The difference between this implementation method and specific implementation method two is that the pressure of the Soxhlet dynamic extraction and concentration unit in step one is set to extraction at normal temperature and pressure.

[0033] Specific embodiment 4: This embodiment is different from specific embodiment 2 or 3 in that the high performance liquid chromatography spectrum in step 3 is collected by using a Waters 2535 semi-preparative high performance liquid chromatograph. 18 Reverse phase silica gel is used as the filler of the chromatographic column, and Waters 2414 differential detector is used as the detector. Other aspects are the same as those of the second or third specific implementation.

[0034] Specific embodiment 5: The Euphorbia chamaejasme terpenoid A described in this embodiment and specific embodiments 1 to 4 is used for preparing anti-breast cancer drugs.

[0035] Specific embodiment 6: This embodiment is different from specific embodiment 5 in that the dosage form of the anti-breast cancer drug includes injection, lyophilized powder injection, and oral preparation. The rest is the same as specific embodiment 5.

[0036] Specific embodiment 7: This embodiment is different from specific embodiment 6 in that the oral preparation includes tablets, granules, soft capsules, hard capsules, oral liquids, and sustained-release preparations. The rest is the same as specific embodiment 6.

[0037] Specific embodiment eight: This embodiment is different from specific embodiments one to seven in that the Euphorbia chamaejasme terpenoid A is used as an active ingredient of an anti-breast cancer drug to inhibit the proliferation of breast cancer MCF-7, ZR-75-1 and MDA-MB-231 cells. The rest is the same as specific embodiments one to seven.

[0038] The following experiments were used to verify the effect of the present invention:

[0039] Experiment 1:

[0040] Euphorbia chamaejasminoides terpenoid A, wherein the molecular formula of Euphorbia chamaejasminoides terpenoid A is C 23 H 34 O5, molecular structure formula is:

[0041]

[0042] The preparation method of the Euphorbia chamaejasme terpene A is carried out according to the following steps:

[0043] 1. placing the Euphorbia chamaejasme in an extraction tank of a Soxhlet dynamic extraction and concentration unit, extracting with ethanol to obtain an ethanol extract;

[0044] 2. The extract obtained in step 1 is concentrated and dispersed with water, and extracted with petroleum ether and ethyl acetate respectively;

[0045] 3. Subject the ethyl acetate extract of step 2 to silica gel column chromatography, and elute with a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 100:1, 80:1, 50:1, and 30:1 in sequence; take the eluate with a volume ratio of petroleum ether to ethyl acetate of 30:1 for secondary silica gel column chromatography, and elute with a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 50:1, 40:1, 30:1, and 20:1 in sequence; collect the eluate to prepare a high performance liquid chromatography sample, using a mixed solvent of methanol and water in a volume ratio of 85:15 as the mobile phase, at a flow rate of 3 mL / min, and collect the absorption peak that appears at the 21st minute of the elution time to obtain wolfsbane euphorbia terpene A.

[0046] The pressure of the Soxhlet dynamic extraction and concentration unit in step 1 is set to extraction at normal temperature and pressure.

[0047] The high performance liquid chromatogram in step 3 was collected by using a Waters 2535 semi-preparative high performance liquid chromatograph. 18 Reverse phase silica gel was used as the filler of the chromatographic column and a Waters 2414 differential detector was used as the detector.

[0048] The stellera chamaejasme terpene A is used for preparing anti-breast cancer drugs.

[0049] The dosage forms of the anti-breast cancer drug include injection, lyophilized powder injection and oral preparation.

[0050] The oral preparations include tablets, granules, soft capsules, hard capsules, oral liquids, and sustained-release preparations.

[0051] This experiment used 30 kg of dried Euphorbia chamaejasme, and finally obtained 6.34 mg of Euphorbia chamaejasme terpenoid A. The new compound was named Methyl-12-hydroxy-14-methoxy-11-(9-methoxy-18,19,20-trimethyldecahydronaphthalen-8-yl)benzoate according to its structure. Table 1 shows the NMR data of the new compound: 1 H-NMR and 13 C-NMR in DMSO.

[0052] Table 1: NMR data of Euphorbia chamaejasme Terpene A of the present invention

[0053]

[0054] Experiment 2:

[0055] The proliferation inhibition activity of Euphorbia chamaejasme terpenoid A prepared in Experiment 1 on breast cancer MCF-7, ZR-75-1 and MDA-MB-231 cells was tested in the following steps:

[0056] 1. Drug preparation

[0057] Euphorbia chamaejasme terpenoid A was dissolved in DMSO and diluted with PBS to test concentrations of 100 μmol / L, 50 μmol / L, 25 μmol / L, 12.5 μmol / L, and 6.25 μmol / L.

[0058] 2. Cell culture

[0059] MCF-7 and ZR-75-1 cells were grown in DMEM medium containing 10% fetal bovine serum and cultured in an incubator at 5% CO2, saturated humidity and 37°C. Cells in the logarithmic growth phase were used for the experiment.

[0060] MDA-MB-231 cells were grown in DMEM medium containing 10% fetal bovine serum and cultured in an incubator at 5% CO2, saturated humidity and 37°C. Cells in the logarithmic growth phase were used for the experiment.

[0061] 3. CCK-8 test of the effect of drugs on cell proliferation

[0062] MCF-7, ZR-75-1 and MDA-MB-231 cells were grown at 2×10 5The cells were inoculated at a density of 100 / mL in a 96-well plate. After the cells adhered to the plate, they were treated with different concentrations of Euphorbia chamaejasme Terpene A and 5-fluorouracil (positive control drug) for 24 hours. The cells were removed and the supernatant was discarded. 10 μL CCK-8 solution and 90 μL complete medium were added to each well. The culture plate was incubated in an incubator for 45 minutes, and the absorbance (OD value) at 450 nm was measured with a microplate reader. The wells with only CCK-8 solution and complete medium were recorded as complete blank groups. All groups had three replicate wells, and the cell IC was calculated. 50 value.

[0063] IC of Euphorbia chamaejasma terpenoid A of the present invention on breast cancer MCF-7, ZR-75-1 and MDA-MB-231 cells 50 The results are as follows: 25.4 μmol / L, 19.2 μmol / L and 21.9 μmol / L respectively, indicating that it has good in vitro anti-breast cancer activity. The invention belongs to the research field of effective ingredients of natural medicines.

Claims

1. Euphorbia chamaejasme terpenoid A, characterized in that The molecular formula of the Euphorbia chamaejasme terpene A is C 21 H 34 O5, molecular structure formula is:

2. The method for preparing the Euphorbia chamaejasme terpene A according to claim 1, characterized in that The preparation method of the Euphorbia chamaejasme terpene A is carried out according to the following steps:

1. placing the Euphorbia chamaejasme in an extraction tank of a Soxhlet dynamic extraction and concentration unit, extracting with ethanol to obtain an ethanol extract; 2. The extract obtained in step 1 is concentrated and dispersed with water, and extracted with petroleum ether and ethyl acetate respectively; 3. Subject the ethyl acetate extract of step 2 to silica gel column chromatography, and elute with a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 100:1, 80:1, 50:1, and 30:1 in sequence; take the eluate with a volume ratio of petroleum ether to ethyl acetate of 30:1 for secondary silica gel column chromatography, and elute with a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 50:1, 40:1, 30:1, and 20:1 in sequence; collect the eluate to prepare a high performance liquid chromatography sample, using a mixed solvent of methanol and water in a volume ratio of 85:15 as the mobile phase at a flow rate of 3 mL / min; collect the absorption peak that appears at the elution time of the 21st minute in the high performance liquid chromatography, and obtain wolfsbane euphorbia terpene A.

3. The method for preparing Euphorbia chamaejasme terpene A according to claim 2, characterized in that The pressure of the Soxhlet dynamic extraction and concentration unit in step 1 is set to extraction at normal temperature and pressure.

4. The method for preparing Euphorbia chamaejasme terpene A according to claim 2, characterized in that The high performance liquid chromatography spectrum in step 3 was collected by using a Waters 2535 semi-preparative high performance liquid chromatograph. 18 Reverse phase silica gel was used as the filler of the chromatographic column and a Waters 2414 differential detector was used as the detector.

5. The use of Euphorbia chamaejasme Terpene A according to claim 1, characterized in that The stellera chamaejasme terpene A is used for preparing anti-breast cancer drugs.

6. The use of Euphorbia chamaejasme Terpene A according to claim 5, characterized in that The dosage forms of the anti-breast cancer drug include injection, lyophilized powder injection and oral preparation.

7. The use of Euphorbia chamaejasme Terpene A according to claim 6, characterized in that The oral preparations include tablets, granules, soft capsules, hard capsules, oral liquids, and sustained-release preparations.

8. The use of Euphorbia chamaejasme Terpene A according to claim 5, characterized in that The Euphorbia chamaejasme terpenoid A is used as an active ingredient of an anti-breast cancer drug for inhibiting the proliferation of MCF-7, ZR-75-1 and MDA-MB-231 cells.

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