阅读说明：本技术 一种黄槿中抗肿瘤有效部位的制备方法及应用 (It is a kind of Huang the rose of Sharon in anti-tumor effective component preparation method and application ) 是由 陈德力 马国需 刘洋洋 李光 李宜航 许旭东 孙晓波 于 2019-09-26 设计创作，主要内容包括：本发明涉及一种黄槿中抗肿瘤有效部位的制备方法及应用,本发明方法包括粉碎、有机溶剂提取、浓缩、硅胶柱层析、大孔吸附树脂洗脱以及干燥等步骤。该方法简单、易于操作,所得有效部位中杂质明显减少,UPLC检测结果显示药效物质的含量达70％以上,符合国家五类新药规定的50％的含量标准。本发明还对所得有效部位进行了药效学研究,结果显示该有效部位对多种肿瘤细胞均具有抗增殖活性,提示其在抗肿瘤药物或保健品中具有良好的应用前景。(The present invention relates to a kind of preparation method and application of anti-tumor effective component in yellow rose of Sharon, the method for the present invention includes crushing, organic solvent extraction, concentration, silica gel column chromatography, macroporous absorbent resin elution and drying and other steps.This method is simple, easily operated, and impurity significantly reduces in gained active component, and UPLC testing result shows that the content of effective substance up to 70% or more, meets as defined in national five kind new medicines 50% content standard.The present invention has also carried out pharmacodynamic study to gained active component, and the active component all has antiproliferative activity to kinds of tumor cells as the result is shown, it is prompted to have a good application prospect in anti-tumor drug or health care product.)

1. the preparation method of anti-tumor effective component in a kind of Huang rose of Sharon, which comprises the following steps:

(1) yellow rose of Sharon medicinal material trunk is taken, is crushed；

(2) it is heated to reflux using organic solvent or cold soaking extracts；The organic solvent is methylene chloride or acetone；

(3) yellow rose of Sharon total extract is obtained by filtration, being concentrated under reduced pressure into fluid paste density is 0.9~1.2g/ml, obtains total medicinal extract；

(4) the total medicinal extract of gained is taken, mixes sample using silica gel dry method, it is levigate after organic solvent volatilization is dry, using silica gel column chromatography, Using petroleum ether as eluant, eluent, petroleum ether part is obtained, reusing methylene chloride-methanol is eluant, eluent, elutes 3~10 column volumes Until red shoals, eluent is collected；

(5) it is eluted using macroporous absorbent resin, using the ethanol solution of volume fraction 70~90% as eluant, eluent, collects eluent, It is concentrated under reduced pressure, vacuum freeze drying, obtains dry powder-shaped active component.

2. the preparation method of anti-tumor effective component in the Huang rose of Sharon according to claim 1, which is characterized in that methylene chloride- The volume ratio of methanol is (38~42): 1.

3. the preparation method of anti-tumor effective component in the Huang rose of Sharon according to claim 1, which is characterized in that the macropore is inhaled Attached resin is D101, D201 and D411 type macroporous absorbent resin, and three's mass ratio is (4~5): 1:(1~2).

4. the preparation method of anti-tumor effective component in the Huang rose of Sharon according to claim 1, which is characterized in that return time is 6~12h, refluxing extraction 3 times, cold soaking extraction time is that for 24 hours, cold soaking extracts 3 times.

5. the preparation method of anti-tumor effective component in the Huang rose of Sharon according to claim 1, which is characterized in that in step (5), Eluent after removing the elution of 70% ethanol solution of volume fraction, washing after the elution of 80~90% ethanol solution of collected volume score De- liquid；Elution speed is 1.8~2mL/min, 1.5~2h of elution time.

6. anti-tumor effective component in the yellow rose of Sharon, which is characterized in that pass through the described in any item preparation method systems of claim 1-5 , the main component of active component is Hibiscone A, Hibiscone B and Hibiscone C, the compound structure of three It is as follows:

7. anti-tumor effective component in the Huang rose of Sharon according to claim 6, which is characterized in that Hibiscone in active component A, the mass ratio of Hibiscone B and Hibiscone C are (4.2~5.8): (2.1~3.7): (70.3~77.6).

8. anti-tumor effective component answering in the drug of preparation prevention or treatment tumor disease in the Huang rose of Sharon as claimed in claim 6 With, or the application in the health care product of preparation prevention tumor disease.

9. application according to claim 8, which is characterized in that tumour cell is liver cancer cells, Ascitic Tumor Cells, adenocarcinoma of lung Cell, cervical cancer cell or gastric adenocarcinoma cells.

10. application according to claim 9, which is characterized in that tumour cell is murine hepatocarcinoma cell H22.

Technical field

The present invention relates to a kind of preparation method and applications of anti-tumor effective component in yellow rose of Sharon, belong to medical biotechnology neck Domain.

Background technique

The yellow rose of Sharon (Hibiscus tiliaceus) belongs to Malvaceae (Malvaceae) Hibiscus (Hibiscus), is a kind of tool There are the mangrove associates of important ecology, medicinal and ornamental value, is distributed widely in China, Vietnam, Cambodia, Burma, India, print The torrid areas such as Nicaea, Malaysia, Philippine and Laos are spent, China's main product is in Hainan, Guangxi, Taiwan, Guangdong, Fujian etc. It saves, extensive growing and cultivation.It is recorded according to " national Chinese herbal medicine compilation ", the yellow rose of Sharon has clearing and antitussive, removing toxicity for detumescence and other effects, In The coastal resident area in Hainan is as drug for venomous snake bite, ulcerative carbuncle pyogenic infections, bronchitis etc..Because it can be raw in intertidal zone It deposits, and sociales can be become on the beach, and can have important ecology in the amphibious xylophyta of terrestrial environment natural propagation Function and unique environmental adaptability, cause the yellow rose of Sharon to contain a large amount of structure novel, and high specificity has significant medicinal activity Molecule.

1980, foreign scholar had found to report 12 Furanosteroid race sesquiterpenoids from the yellow rose of Sharon.Closely Between 40 years, foreign scholar is less to the report of such compound in the yellow rose of Sharon.And domestic scholars to the chemical constitution study of the yellow rose of Sharon compared with To be shallow, the chemical components such as a small amount of triterpene, steroid, Phenylpropanoid Glycosides, flavones, amides are only therefrom obtained.In pharmacology activity research Aspect, external research report is less, and the domestic only simple Huang rose of Sharon extract that is directed to is screened, it is found that the yellow rose of Sharon has and resisted The pharmacological activity such as scorching, analgesia, antitumor, anti-oxidant.In terms of the extraction process of effective component, even lacking one kind can mention simultaneously The method for taking plurality of active ingredients.

Summary of the invention

The present invention provides a kind of preparation method and application of anti-tumor effective component in the yellow rose of Sharon.

The technical solution adopted by the present invention is as follows:

It is a kind of Huang the rose of Sharon in anti-tumor effective component preparation method, comprising the following steps:

(1) yellow rose of Sharon medicinal material trunk is taken, is crushed；

(2) it is heated to reflux using organic solvent or cold soaking extracts；The organic solvent is methylene chloride or acetone；

(3) yellow rose of Sharon total extract is obtained by filtration, being concentrated under reduced pressure into fluid paste density is 0.9~1.2g/ml, obtains and always soaks Cream；

(4) the total medicinal extract of gained is taken, mixes sample using silica gel dry method, it is levigate after organic solvent volatilization is dry, use silica gel column layer Analysis, using petroleum ether as eluant, eluent, obtains petroleum ether part, and reusing methylene chloride-methanol is eluant, eluent, elutes 3~10 columns Volume collects eluent until red shoals；

(5) it is eluted using macroporous absorbent resin, using the ethanol solution of volume fraction 70~90% as eluant, eluent, collects elution Liquid is concentrated under reduced pressure, vacuum freeze drying, obtains dry powder-shaped active component.

Preferably, the volume ratio of methylene chloride-methanol is (38~42): 1.

Preferably, the macroporous absorbent resin is D101, D201 and D411 type macroporous absorbent resin, and three's mass ratio is (4 ~5): 1:(1~2).

Preferably, return time is 6~12h, and refluxing extraction 3 times, cold soaking extraction time is that for 24 hours, cold soaking extracts 3 times.

Preferably, in step (5), the eluent after 70% ethanol solution of volume fraction elutes, collected volume score are removed Eluent after the elution of 80~90% ethanol solutions；Elution speed is 1.8~2mL/min, 1.5~2h of elution time.

The present invention, which analyzes, to be determined, the main component of anti-tumor effective component is in the yellow rose of Sharon of gained by the method for the invention Hibiscone A, Hibiscone B and Hibiscone C, the compound structure of three are as follows:

Testing result is shown: the mass ratio of Hibiscone A, Hibiscone B and Hibiscone C are in active component (4.2~5.8): (2.1~3.7): (70.3~77.6).

The present invention also carries out pharmacodynamic study to gained Huang rose of Sharon active component, and the active component is thin to liver cancer as the result is shown Born of the same parents, Ascitic Tumor Cells, lung adenocarcinoma cell, cervical cancer cell or gastric adenocarcinoma cells etc. are inhibited, especially to Mouse Liver Cancer cell H22 has obvious inhibiting effect, and active component of the present invention is prompted to have anti-tumor activity.

Based on pharmacodynamic study as a result, present invention gained active component can be in preparation prevention or the drug for the treatment of tumor disease Middle application, or the application in the health care product of preparation prevention tumor disease.

Compared with prior art, the beneficial effects of the present invention are:

A. the method for the present invention is used, obtains the effective of significant anti-tumor activity by a series of Extraction and enrichment processing step Position.Impurity significantly reduces in gained active component, and UPLC testing result shows that the content of effective substance up to 70% or more, meets 50% content standard as defined in national five kind new medicines.

B. yellow rose of Sharon active component raw material sources of the invention are abundant, inexpensive, and preparation method is simple, is filled using low polar solvent Divide extraction, is enriched with the anti-tumor active substance in the yellow rose of Sharon, it is easily operated, it is low in cost.

C. the active component is preventing or is treating have important use in tumor disease drug or health care product.

Detailed description of the invention

Fig. 1: the UPLC testing result figure of yellow rose of Sharon active component and yellow rose of Sharon extract.

Fig. 2: influence result figure of each group sample to H22 tumor-bearing mice shape of tumor volume.

Fig. 3: influence result figure of each group sample to H22 tumor weight.

Fig. 4: influence result figure of each group sample to H22 tumor-bearing mice tumor proliferation rate.

Specific embodiment

Below by specific embodiment combination attached drawing, invention is further described in detail.

Experimental method used in the embodiment of the present invention is conventional method unless otherwise specified.

Material used in the embodiment of the present invention, reagent etc., are commercially available unless otherwise specified.