阅读说明：本技术 一种鸡骨香根提取物及其超临近流体提取方法、应用 (Chicken bone vetiver extract and super-near fluid extraction method and application thereof ) 是由 孙金月 刘超 陈英凯 程安玮 王新坤 王青 于 2019-05-29 设计创作，主要内容包括：本发明属于药物提取技术领域,具体涉及一种鸡骨香根提取物及其超临近流体提取方法及其应用。该提取物的结构式如下：<Image he="151" wi="153" file="DEST_PATH_IMAGE001.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>。本发明提取的化合物药物原料易得,纯度高,且抗肿瘤活性高,具有抗增殖和诱导细胞凋亡的作用,具有作为抗癌疗法候选物的潜力,易于产业化,可以根据需要制备成不同的剂型,具有巨大的社会效益。(The invention belongs to the technical field of medicine extraction, and particularly relates to a chicken bone vetiver extract, a super-near fluid extraction method and application thereof. The structural formula of the extract is as follows: . The compound extracted by the invention has the advantages of easily obtained raw materials, high purity, high anti-tumor activity, antiproliferative and apoptosis-inducing effects, potential as a candidate for anti-cancer therapy, easy industrialization, capability of being prepared into different formulations according to requirements and huge social benefits.)

1. The chicken bone vetiver extract is characterized in that the structural formula of the extract is as follows:

。

2. the method for extracting a super-near fluid of an extract of a chicken bone in accordance with claim 1, comprising the steps of:

(1) taking the dry root of the chicken bone, crushing, sieving by a 40-mesh sieve, weighing a sample, putting the sample into a reaction kettle of a supercritical CO2 fluid extraction apparatus, firstly carrying out static extraction, then carrying out dynamic extraction, taking methanol as an entrainer to obtain a dark yellow extract, dispersing the extract by warm water, and fully extracting by dichloromethane to obtain an extract;

(2) preparing an eluent, eluting 5.0L of the eluent at each proportion by using a polar gradient of petroleum ether/acetone (50: 1-1: 2), analyzing the extract by using thin-layer chromatography, selecting a petroleum ether-acetone separation system, and performing coarse separation by using a 100-mesh 200-mesh silica gel dry method for column packing; detecting and merging by thin-layer chromatography to obtain 5 components A1-A5;

(3) Subdividing A4 by using 200-300 mesh silica gel; preparing petroleum ether-ethyl acetate system eluent for gradient elution, wherein the polarity gradient is 8:1-5:1, precipitating colorless blocky crystals at the 8:1 part, further purifying by using a semi-preparative liquid phase after dissolving methanol, performing gradient elution by using a C18 column and 65-85% CH3OH/H2O 2 mL/min for 20 min), and detecting by using an ultraviolet detector at the 210 nm position to obtain a compound J-11.

3. The extraction process according to claim 2, characterized in that the static extraction is an extraction for 30min at a pressure of 25MPa and a temperature of 35 ℃.

4. The extraction method according to claim 2 or 3, wherein the flow rate of CO2 is 5.0L/min, methanol is used as entrainer, and the flow rate of methanol is 5.4 mL/min.

5. The extraction process according to claim 2, wherein said a1 is a petroleum ether/acetone =50:1-30:1 fraction; a2 is a petroleum ether/acetone =15:1 fraction; a3 is a petroleum ether/acetone =10:1-8:1, 8.5 g fraction; a4 is a petroleum ether/acetone =6:1-2:1 fraction; a5 is a petroleum ether/acetone =1:1-1:2 fraction.

6. The extraction process according to claim 2 or 5, wherein in step (3), the volume ratio of dichloromethane to methanol is 1: 1.

7. use of a compound extracted by the ultra-close extraction method according to any one of claims 2 to 6 in the preparation of an anti-tumor medicament.

Technical Field

The invention belongs to the technical field of medicine extraction, and particularly relates to a chicken bone vetiver extract, and a supercritical fluid extraction method and application thereof.

Background

the genus croton is an euphorbiaceae family, with over 1300 genera worldwide, a large genus widely distributed in tropical and subtropical regions. About 21 chicken bone spices have grown in china, and some of them have long been used for relieving dysmenorrhea and for the traditional Chinese treatment of dyspepsia and malaria. Plants of the genus croton are known for their various terpenoids and possess a wide range of biological activities. In addition, alkaloids, flavanones, lignans, polyphenolic compounds, phenylbutyric acid and peptides are obtained.

Croton crassifolius C. crassifolius C. crassifoliuscroton crassifolius Geisel is a species of Croton genus, and is mainly distributed in the southern China and other regions of southeast Asia. As a traditional Chinese medicine named "chicken bone fragrant", its root has been used for the treatment of cancer, stomach ache, sore throat and rheumatism. Current phytochemical studies have shown that the major phytochemicals in c. crassifolius are terpenoids, including sesquiterpenes, diterpenes and triterpenes. Diterpenes are considered to be the main and characteristic compounds in c. crassifolius, having various biological activities, such as antiviral and antiangiogenic activities.

C. crassifoliusCurrently, most studies on c. crassifolius focus on the use of its root extract in medicine, and the utilization of its low-polar part is rarely studied. In the prior art, the low polarity portion of the herb is reported to be rich in active compounds, but specific studies have been less. There is no description about the extraction of the compound of the present invention.

Disclosure of Invention

The invention aims to provide a novel Chinese eaglewood root extract which has high anti-tumor activity and has the functions of resisting proliferation and inducing apoptosis.

The invention also provides a supercritical extraction method of the chicken bone vetiver extract.

The invention also aims to provide application of the chicken bone vetiver extract.

The technical scheme adopted by the invention for realizing the purpose is as follows:

The invention provides a super-near extraction method of a chicken bone vetiver extract, which comprises the following steps:

(1) Taking the dry root of the chicken bone, crushing, sieving by a 40-mesh sieve, weighing a sample, putting the sample into a reaction kettle of a supercritical CO2 fluid extraction apparatus, firstly carrying out static extraction, then carrying out dynamic extraction, taking methanol as an entrainer to obtain a dark yellow extract, dispersing the extract by warm water, and fully extracting by dichloromethane to obtain an extract;

(2) Preparing an eluent, eluting 5.0L of the eluent at each proportion by using a polar gradient of petroleum ether/acetone (50: 1-1: 2), analyzing the extract by using thin-layer chromatography, selecting a petroleum ether-acetone separation system, and performing coarse separation by using a 100-mesh 200-mesh silica gel dry method for column packing; detecting and merging by thin-layer chromatography to obtain 5 components A1-A5;

(3) Subdividing A4 by using 200-300 mesh silica gel; preparing petroleum ether-ethyl acetate system eluent for gradient elution, wherein the polarity gradient is 8:1-5:1, precipitating colorless blocky crystals at the 8:1 part, further purifying by using a semi-preparative liquid phase after dissolving methanol, performing gradient elution by using a C18 column and 65-85% CH3OH/H2O 2 mL/min for 20 min), and detecting by using an ultraviolet detector at the 210 nm position to obtain a compound J-11.

The structural formula of the compound J-11 of the chicken bone vetiver extract provided by the invention is as follows:

。

Further, the static extraction is carried out for 30min under the conditions of 25MPa of pressure and 35 ℃.

Further, during the dynamic extraction, the flow rate of CO2 is 5.0L/min, methanol is used as an entrainer, and the flow rate of the methanol is 5.4 mL/min.

Further, said a1 is a petroleum ether/acetone =50:1-30:1 fraction; a2 is a petroleum ether/acetone =15:1 fraction; a3 is a petroleum ether/acetone =10:1-8:1, 8.5 g fraction; a4 is a petroleum ether/acetone =6:1-2:1 fraction; a5 is a petroleum ether/acetone =1:1-1:2 fraction.

Further, in the step (3), the volume ratio of the dichloromethane to the methanol is 1: 1.

the invention also provides application of the compound extracted by the ultra-near extraction method in preparing an anti-tumor medicament.

The compound extracted from the chicken bone incense can inhibit the proliferation of A549 cells and block the A549 cell cycle in the G2/M phase. The compounds increase the Bax/Bcl-2 ratio in A549 cells, leading to a breakdown of mitochondrial membrane potential, followed by cytochrome C release and activation of the caspase cascade, leading to cleavage of PARP and ultimately apoptosis.

The invention has the beneficial effects that:

(1) the compound extracted by the invention has easily obtained raw materials, is easy to industrialize, can be prepared into different formulations according to requirements, and has great social benefit.

(2) The compound extracted by the invention has high purity and high anti-tumor activity, has the effects of resisting proliferation and inducing apoptosis, and has the potential of being used as a candidate of anti-cancer therapy.

Drawings

FIG. 1 is a spectrum of HMBC and RORSY, and ECD spectrum of compound J-11.

FIG. 2 is a graph of the effect of varying concentrations of compound J-11 on migration of A549 cells.

FIG. 3 is a graph of the induction of autophagy in A549 cells by Compound J-11.

Detailed Description

The technical solution of the present invention is further explained and illustrated by the following specific examples.