Euphorbia fischeriana steud terpenes a and preparation method and application thereof

Abstract

Euphorbia fischeriana steud ter A, preparation method and application thereof, relate to a new substance with in-vitro anti-breast cancer and its method and application.The present application provides a new substance with in-vitro anti-breast cancer activity and its preparation method and application.Euphorbia fischeriana steud ter A has a molecular formula of C 21 H 34 O 13 The preparation method is as follows: I, Euphorbia fischeriana steud is extracted by cold soaking with ethanol; II, the extract of recovered ethanol is dispersed with water and extracted; III, the ethyl acetate extraction layer is separated by repeated silica gel column chromatography, and then a single compound is obtained by high performance liquid chromatography, which is identified as a new compound by spectroscopy and named as Euphorbia fischeriana steud ter A.Euphorbia fischeriana steud ter A is applied to preparation of anti-breast cancer drugs.The IC 50 of Euphorbia fischeriana steud ter A on breast cancer MCF-7, ZR-75-1 and MDA-MB-231 cells are 25.4 μmol / L, 19.2 μmol / L and 21.9 μmol / L respectively, which indicates that it has good in-vitro anti-breast cancer activity.The present application belongs to the field of natural medicine effective component research.

Description

TECHNICAL FIELD

[0001] The present application relates to a new substance with in vitro anti-breast cancer activity and its method and application. BACKGROUND

[0002] Radix Jatrophae Curcas is a traditional Chinese medicinal material recorded in Chinese Pharmacopoeia 2020 edition. It has bitter and pungent taste, neutral property and toxicity, and has the effects of draining water, reducing fluid, resolving accumulation and killing insects, and is used to treat edema, abdominal distension, phlegm and food accumulation, heart and abdominal pain, tuberculosis, and tinea, etc.

[0003] Modern pharmacological studies have shown that the main active ingredients of Radix Jatrophae Curcas are diterpenoids, etc., which have the effects of anti-tumor, anti-bacterial, anti-fungal, anti-viral and anti-epilepsy, etc., and are a valuable plant anti-tumor drug source.

[0004] At present, there are many preparations of Radix Jatrophae Curcas on the market, but Radix Jatrophae Curcas has been used as a medicinal plant for a long time. In order to improve its quality standard and find more index components with anti-breast cancer activity, it is of great significance to carry out systematic chemical separation.

[0005] Breast cancer is a malignant tumor occurring in the epithelium or duct epithelium of the breast, which ranks first in the incidence of female malignant tumors. Comprehensive therapy is the most common treatment for breast cancer, and common methods include surgery combined with radiotherapy and chemotherapy, molecular targeted therapy, immunotherapy, endocrine therapy, etc. In China, advanced breast cancer still accounts for the majority of patients, and most of them use chemotherapy, which can produce various adverse reactions and has a certain probability of metastasis and recurrence. Therefore, the research work of finding new drugs for treating breast cancer is arduous and long-term. Traditional Chinese medicine has the advantages of multi-target, low toxicity and side effects, and is not easy to produce drug resistance, which not only meets the mechanism of multi-factor and multi-link of tumor, but also has significant effects on improving the success rate of tumor treatment, improving the quality of life of tumor patients and prolonging life. Therefore, it has broad research prospects to screen out effective anti-tumor components with strong biological activity and low toxicity from traditional Chinese medicine. SUMMARY

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

[0007] The molecular formula of Radix Jatrophae Curcas terpene A is C 23 H 34 The molecular structure formula is:

[0008]

[0009] The preparation method of the Radix Jatrophae Curcas terpene A is carried out according to the following steps:

[0010] I. Wolf Euphorbia is placed in the Soxhlet dynamic extraction concentrator extraction tank, using ethanol extraction, ethanol extract;

[0011] II, the extract obtained in step one is concentrated and dispersed with water, and petroleum ether and ethyl acetate are used for extraction respectively;

[0012] III, the ethyl acetate extract of step two is subjected to silica gel column chromatography separation, and the mixed solvent of petroleum ether and ethyl acetate with a volume ratio of 100:1, 80:1, 50:1 and 30:1 is eluted in turn, and the eluate with a volume ratio of 30:1 of petroleum ether and ethyl acetate is subjected to secondary silica gel column chromatography separation, and the mixed solvent of petroleum ether and ethyl acetate with a volume ratio of 50:1, 40:1, 30:1 and 20:1 is eluted in turn, and the eluate is collected to prepare high performance liquid chromatography sample, and the mixed solvent of methanol and water with a volume ratio of 85:15 is used as mobile phase, the flow rate is 3mL / min, and the absorption peak appearing at the elution time of 21min is collected, i.e. wolf euphorbia terpene A is obtained.

[0013] The pressure of the Soxhlet dynamic extraction concentrator set in step I is normal temperature and normal pressure extraction.

[0014] The high performance liquid chromatogram in step III is collected by Waters 2535 semi-preparative high performance liquid chromatograph, and C 18 The reverse phase silica gel is the filler of the chromatographic column, and the Waters 2414 type differential detector is the detector.

[0015] The wolf euphorbia terpene A is used for preparing an anti-breast cancer drug.

[0016] The dosage form of the anti-breast cancer drug includes injection, freeze-dried powder injection, oral preparation.

[0017] The oral preparation includes tablets, granules, soft capsules, hard capsules, oral liquids and controlled release preparations.

[0018] The wolf euphorbia terpene A is used as an active ingredient of the anti-breast cancer drug to inhibit the proliferation of breast cancer MCF-7, ZR-75-1 and MDA-MB-231 cells.

[0019] The wolf euphorbia terpene A 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 application can be added to pharmaceutically acceptable adjuvants to prepare pharmaceutically acceptable solid preparations or liquid preparations.

[0021] The adjuvant of the present application is not limited as long as it is pharmaceutically acceptable.

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

[0023] The present application provides a new compound derived from Euphorbia Fischeriana and an extraction, separation and purification method for the new compound, which is separated and purified by ethanol extraction, extraction, silica gel column chromatography, and preparative high performance liquid chromatography, and successfully obtains the new compound, which has high purity and is simple and fast to operate, and 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 concentration of the compound is 100 μM, 50 μM, 25 μM, 12.5 μM and 6.25 μM, the inhibition rates of the compound on MCF-7 cells are 80.2%, 70.7%, 57.1%, 33.8% and 6.4%, respectively, and the inhibition rates of the compound on ZR-75-1 cells are 90.6%, 77.8%, 62.2%, 44.2% and 8.1%, respectively. The inhibition rates of the Euphorbia Fischeriana terpene A on MDA-MB-231 cells are 86.4%, 79.6%, 64.6%, 24.8% and 11.2%, respectively. The IC50 of the breast cancer MCF-7, ZR-75-1 and MDA-MB-231 cells is 25.4 μM, 19.2 μM and 21.9 μM, respectively, which indicates that the Euphorbia Fischeriana terpene A has good in-vitro anti-breast cancer activity. The Euphorbia Fischeriana terpene A can be prepared into an injection, a freeze-dried powder, a transfusion agent or an oral preparation (including tablets, granules, soft capsules, hard capsules, oral liquids and controlled-release preparations) for treating breast cancer. 50 DETAILED DESCRIPTION

[0025] The technical solution of the present application is not limited to the following specific embodiments, and any combination of the specific embodiments is also included.

[0026] Specific embodiment one: the molecular formula of the Euphorbia Fischeriana terpene A in the present embodiment is C 23 H 34 O5, and the molecular structure formula is:

[0027]

[0028] Specific embodiment two: the preparation method of the Euphorbia Fischeriana terpene A in the specific embodiment one is performed according to the following steps: ​

[0029] I. Wolf Euphorbia is placed in the Soxhlet dynamic extraction concentrator extraction tank, using ethanol extraction, ethanol extract;

[0030] II, the extract obtained in step one is concentrated and dispersed with water, and petroleum ether and ethyl acetate are used for extraction, respectively;

[0031] III, the ethyl acetate extract of step two is subjected to silica gel column chromatography separation, and the mixed solvent of petroleum ether and ethyl acetate with a volume ratio of 100:1, 80:1, 50:1, 30:1 is eluted in turn, and the eluent of petroleum ether and ethyl acetate with a volume ratio of 30:1 is subjected to secondary silica gel column chromatography separation, and the mixed solvent of petroleum ether and ethyl acetate with a volume ratio of 50:1, 40:1, 30:1, 20:1 is eluted in turn, and the eluent is collected to prepare high performance liquid chromatography sample, and the mixed solvent of methanol and water with a volume ratio of 85:15 is used as mobile phase, and the flow rate is 3mL / min, and the absorption peak appearing at the elution time of 21min is collected, that is, wolf euphorbia terpene A is obtained.

[0032] Specific embodiment three: the difference between this embodiment and specific embodiment two is that the pressure of the Soxhlet dynamic extraction concentrator in step one is set to normal temperature and normal pressure extraction.

[0033] Specific embodiment four: the difference between this embodiment and specific embodiment two or three is that the high performance liquid chromatogram in step three is collected by Waters 2535 semi-preparative high performance liquid chromatograph, and C 18 The reverse phase silica gel is the filler of the chromatographic column, and the Waters 2414 type differential detector is the detector. The others are the same as specific embodiment two or three.

[0034] Specific embodiment five: wolf euphorbia terpene A in specific embodiments one to four is used for preparing anti-breast cancer drugs.

[0035] Specific embodiment six: the difference between this embodiment and specific embodiment five is that the dosage form of the anti-breast cancer drug includes injection, freeze-dried powder injection, oral preparation. The others are the same as specific embodiment five.

[0036] Specific embodiment seven: the difference between this embodiment and specific embodiment six is that the oral preparation includes tablets, granules, soft capsules, hard capsules, oral liquids, and controlled release preparations. The others are the same as specific embodiment six.

[0037] Specific embodiment eight: the difference between this embodiment and one of specific embodiments one to seven is that wolf euphorbia terpene A is used as the active ingredient of anti-breast cancer drugs to inhibit the proliferation of breast cancer MCF-7, ZR-75-1 and MDA-MB-231 cells. The others are the same as one of specific embodiments one to seven.

[0038] The effect of the present application is verified by the following experiments:

[0039] Experiment 1:

[0040] The wolf-toxic euphorbia ter A has a molecular formula of C 23 H 34 O5, and a molecular structure formula:

[0041]

[0042] The preparation method of the wolf-toxic euphorbia ter A is carried out according to the following steps:

[0043] I. The wolf-toxic euphorbia is placed in the extraction tank of the Soxhlet dynamic extraction concentrator, and extracted with ethanol to obtain an ethanol extract;

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

[0045] III. The ethyl acetate extract of step II is subjected to silica gel column chromatography separation, and the mixed solvent of petroleum ether and ethyl acetate with a volume ratio of 100:1, 80:1, 50:1 and 30:1 is eluted in turn, and the eluate with a volume ratio of petroleum ether to ethyl acetate of 30:1 is subjected to secondary silica gel column chromatography separation, and the mixed solvent of petroleum ether and ethyl acetate with a volume ratio of 50:1, 40:1, 30:1 and 20:1 is eluted in turn, and the eluate is collected to prepare high performance liquid chromatography sample, and the mixed solvent of methanol and water with a volume ratio of 85:15 is used as mobile phase, and the flow rate is 3mL / min, and the absorption peak appearing at the elution time of 21min is collected, and wolf-toxic euphorbia ter A is obtained.

[0046] The pressure of the Soxhlet dynamic extraction concentrator in step I is set to normal temperature and normal pressure extraction.

[0047] The high performance liquid chromatogram in step III is collected by Waters 2535 semi-preparative high performance liquid chromatograph, and the packing of the chromatographic column is reversed phase silica gel, and the detector is Waters 2414 type differential detector. 18 The packing of the chromatographic column is reversed phase silica gel, and the detector is Waters 2414 type differential detector.

[0048] The wolf-toxic euphorbia ter A is used for preparing anti-breast cancer drugs.

[0049] The dosage form of the anti-breast cancer drug includes injection, lyophilized powder injection, oral preparation.

[0050] The oral preparation includes tablets, granules, soft capsules, hard capsules, oral liquids and controlled release preparations.

[0051] The experiment adopts 30 kg dry Wolfberry Euphorbia, and finally obtains Wolfberry Euphorbia terpene A 6.34 mg, the new compound is named as Methyl-12-hydroxy-14-methoxy-11-(9-methoxy-18,19,20-trimethyldecahydronaphthalen-8-yl)benzoate according to the structure, and Table 1 is the nuclear magnetic data of the new compound: 1 H-NMR and 13 C-NMR in DMSO.

[0052] Table 1: Nuclear magnetic data of Wolfberry Euphorbia terpene A of the application

[0053]

[0054] Experiment two:

[0055] The proliferation inhibition activity of Wolfberry Euphorbia terpene A prepared in experiment one on breast cancer MCF-7, ZR-75-1 and MDA-MB-231 cells is tested, and the steps are as follows:

[0056] 1. Drug preparation

[0057] Wolfberry Euphorbia terpene A is dissolved with DMSO. Diluted with PBS, and the test concentration is 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 grow in DMEM culture medium containing 10% fetal bovine serum, and are cultured in a 5% CO2, saturated humidity and 37℃ incubator, and logarithmic growth period cells are used for experiment.

[0060] MDA-MB-231 cells grow in DMEM culture medium containing 10% fetal bovine serum, and are cultured in a 5% CO2, saturated humidity and 37℃ incubator, and logarithmic growth period cells are used for experiment.

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

[0062] MCF-7, ZR-75-1 and MDA-MB-231 cells are 2×10 5The density of 1x104 / mL was inoculated in 96-well plates, and after adhering, different concentrations of Euphorbia tirucalli terpenoid A and 5-fluorouracil (positive control drug) were used for treatment for 24 hours, then removed, discarded supernatant, 10 μL CCK-8 solution and 90 μL complete medium were added to each well, the culture plate was incubated in the incubator for 45 min, and then the absorbance (OD value) at 450 nm was determined by using an enzyme-labeled instrument. Only the well with CCK-8 solution and complete medium was recorded as a complete blank group, all groups had three duplicate wells, and the cell IC 50 values were calculated.

[0063] The IC 50 of Euphorbia tirucalli terpenoid A on breast cancer MCF-7, ZR-75-1 and MDA-MB-231 cells were 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 present application belongs to the field of natural medicine effective component research.

Claims

1. Euphorbia fischeriana terpenoid A, characterized in that... The molecular formula of the euphorbia pekinensis A is C. 23 H 34 O5, the molecular structural formula is: 。 2. The method for preparing Euphorbia lathyris terpene A according to claim 1, characterized in that... The preparation method of the euphorbia pekinensis A is carried out according to the following steps:

1. Place Euphorbia fischeriana in the extraction tank of a Soxhlet dynamic extraction and concentration unit, and extract with ethanol to obtain ethanol extract; 2. After concentrating the extract obtained in step 1, disperse it in water and extract it with petroleum ether and ethyl acetate respectively.

3. Separate the ethyl acetate extract from step 2 by silica gel column chromatography, eluting sequentially with mixed solvents of petroleum ether and ethyl acetate at volume ratios of 100:1, 80:1, 50:1, and 30:

1. Take the eluent with a petroleum ether to ethyl acetate volume ratio of 30:1 and perform secondary silica gel column chromatography, eluting sequentially with mixed solvents of petroleum ether and ethyl acetate at volume ratios of 50:1, 40:1, 30:1, and 20:

1. Collect the eluent to prepare a high-performance liquid chromatography (HPLC) sample. Use a mixed solvent of methanol and water at a volume ratio of 85:15 as the mobile phase and a flow rate of 3 mL / min. Collect the absorption peak that appears at the elution time of 21 min in the HPLC, which is the euphorbia pekinensis A.

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

4. The method for preparing Euphorbia lathyris terpene A according to claim 2, characterized in that... Step 3, the high-performance liquid chromatography (HPLC) data were collected using a Waters 2535 semi-preparative HPLC system, with C0.05... 18 The reversed-phase silica gel column was used as the packing material, and the Waters 2414 differential detector was used as the detector.

5. The application of the Euphorbia fischeriana terpenoid A according to claim 1, characterized in that... The euphorbia triterpenoid A is used to prepare an anti-breast cancer drug.

6. The application of Euphorbia fischeriana terpenoid A according to claim 5, characterized in that... The dosage form of the anti-breast cancer drug is selected from injections, lyophilized powder injections, and oral preparations.

7. The application of Euphorbia fischeriana terpenoid A according to claim 6, characterized in that... The oral formulation is selected from tablets, granules, soft capsules, hard capsules, oral liquids, and sustained-release formulations.

8. The application of Euphorbia fischeriana terpenoid A according to claim 5, characterized in that... The euphorbia terpene A mentioned above is used as the active ingredient in an anti-breast cancer drug to inhibit the proliferation of MCF-7, ZR-75-1, and MDA-MB-231 cells.

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