CN101028322A - Use of Maoliefengdou extract for preparing anti-cancer medicine - Google Patents

Abstract

The invention relates to the technical field of medicine, and relates to a butterbur pilaris extract, a preparation method thereof, and a use of the butterbur pilaris extract in preparing medicines for treating tumor diseases. The extract is obtained by thermally extracting the medicinal material of Butterbur trichomes with water-containing ethanol, cold soaking or percolation, and is separated by macroporous adsorption resin to refine the extract of Butterbur trichomes. The main active ingredients of butterbur extract are butterbur lactone D (bakkenolide-D), butterbur lactone B (bakkenolide-B), butterbur lactone-IVa (bakkenolide-IIIa), honey Bakkenolide-IIIa, the total content of the four components is as high as 50%. After biological activity screening tests, it can effectively prevent and treat tumors, especially liver cancer, lung cancer, cervical cancer, intestinal cancer, kidney cancer, and oral epidermoid cancer , breast cancer, so the butterbur pilaris extract can be used to prepare anti-tumor drugs.

Description

Application of Butterbur pilaris Extract in the Preparation of Drugs for Preventing and Treating Tumor Diseases

technical field

The present invention relates to the technical field of medicine, in particular, to an extract of the rhizome of Butterbur pilaris and its preparation method and the use of the extract for preparing medicines for treating and preventing tumor diseases

Background technique

Petasites tricholobus Franch. belongs to the plant of the genus Butterbur in the Asteraceae family. Its rhizomes are used in the folk for detoxification and blood stasis, and for external application to treat bruises, fractures and snakebites. In order to develop and utilize this plant, the present inventors have carried out systematic chemical composition and pharmacological research on this plant, and have reported four new butterbur lactone compounds and butterbur lactone compounds in Butterbur pilaris It is used to treat diseases that may be related to histamine and/or leukotrienes (patent publication number: CN1844114), but so far, there has been no report that Butterbur pilaris and its extracts have anti-tumor activity.

Contents of the invention

The object of the present invention is to provide a kind of Butterbur pilaris extract that extracts from Butterbur pilaris and preparation method thereof, through in vitro test, the Butterbur pilaris extract that makes has the effect on liver cancer, lung cancer, Cervical cancer, intestinal cancer, kidney cancer, oral epidermoid cancer, breast cancer tumor cell lines have obvious inhibitory effect, therefore, the butterbur extract of the present invention can be used to treat tumor diseases. These tumor diseases preferably include liver cancer, lung cancer, cervical cancer, colon cancer, kidney cancer, oral epidermoid cancer, and breast cancer. The butterbur pilaris extract prepared by the invention has the application of preparing anti-tumor disease medicines.

The present invention is accomplished through the following technical solutions, and the specific content includes: the preparation of the extract of the butterbur pilaris and the experiment of antitumor pharmacological activity.

(1) Preparation of Butterbur pilaris Extract

The preparation method of the butterbur extract of the present invention consists of the following steps successively:

(1) extraction

After the rhizome of Butterbur pilaris is crushed, it is extracted with 20-95% aqueous ethanol to obtain an extract, the extract is concentrated under reduced pressure, centrifuged or filtered, the precipitate is discarded, and the supernatant or filtrate is used for later use;

The method of aqueous ethanol extraction is hot extraction, cold soaking or percolation; when using the above method for extraction, the volume of ethanol used is 5-15 times the mass of crude drug;

(2) Adsorption and elution

After the above supernatant or filtrate is subjected to polystyrene-type porous adsorption resin adsorption, and water-50% water-containing lower alcohol to remove impurities, use 60-95% of the volume of 4-12 times the mass of the sample crude drug The aqueous C ₁ ~ C ₅ lower alcohol elution;

(3) Concentration and drying

The eluate is concentrated under reduced pressure and dried to obtain the Butterbur pilaris extract of the present invention.

Heat the dried rhizomes of Butterbur pilaris with 20-95% ethanol or soak them coldly, concentrate the extract, centrifuge or filter, take the supernatant or filtrate for macroporous resin adsorption, first use -50% water-containing low-grade Wash with alcohol to remove impurities, then elute with 60-95% water-containing lower alcohol, concentrate the eluent to no alcohol, and dry it to obtain the extract of Butterbur pilaris of the present invention; or pass the concentrated solution through polyamide chromatography , sequentially eluted with water and 50% ethanol, collected the 50% ethanol eluate, concentrated, and dried to obtain the extract of Butterbur pilaris.

Component analysis of the obtained Butterbur pilaris extract: After repeated silica gel column chromatography, 4 compounds with large amounts were obtained. The general structural formulas of these 4 compounds are as follows:

Namely, butterburide D, butterburide B, butterburide IVa, and butterburide IIIa.

in:

Compound name R ₁ R ₂

Butterbur lactone-D OCOC(CH ₃ )=CHCH ₃ OCOCH ₃

Butterbur lactone-B OCOC(CH ₃ )=CHCH ₃ OCOCH ₃

Butterbur lactone IVa OCOCH＝CHSCH ₃ OCOCH＝C(CH ₃ ) ₂

Butterbur lactone IIIa OCOCH=CHSCH ₃ OCOC(CH ₃ )=CHCH ₃

Therefore, the butterbur extract is mainly a mixture of butterbur lactone D, butterbur lactone B, butterbur lactone IVa and butterbur lactone IIIa. Quantitatively determined by HPLC, these 4 compounds The sum of the content of butterbur lactone accounts for more than 50% of the total extract, and the content ratio of butterbur lactone D, butterbur lactone B, butterbur lactone IVa and butterbur lactone IIIa is: 0.1-5:0.1 -5:1-9:1-9.

(2) Biological activity test: the inhibitory effect of the test drug on the growth of tumor cells in vitro

Drugs tested: Butterbur pilaris extract (number 1), butterbur lactone D (number 2), butterbur lactone B (number 3), butterbur lactone IVa (number 2 4), butterbur lactone IIIa (No. 5) drug preparation: dissolve the test drug in dimethyl sulfoxide (DMSO) to make a stock solution, use RPMI1640 cell culture medium containing 10% fetal bovine serum Diluted 100 times. The positive control drug is 5-fluorouracil (5-FU), and the preparation method is the same as that of the test drug.

Tumor cell lines: human lung cancer cell A549, human cervical cancer cell Hela, human colon cancer cell HCT116, human liver cancer cell HepG2, human liver cancer cell BEL-7402, human kidney cancer cell RXF-631L, human oral epidermoid cancer cell KB, human Breast adenocarcinoma MCF-7.

Determination method: Digest each cell line grown to the logarithmic phase with trypsin, make cell suspension with RPMI1640 medium containing 10% fetal bovine serum, inoculate 96-well cells at a concentration of ^10-4 cells per well 200 μl per well on the culture plate. After 24 hours, the medium was removed, and the medium containing the test drug was added. The final concentration of the test drug was 1×10 ^-3 -10 μg/ml, and the final concentration of 5-FU was 0.01-10 μg/ml. No test drug was added to the control group. After continuing to culture for 3 days, the culture was terminated, and the cell viability was detected by MTT method. Before terminating the culture, 10 μl of MTT solution (5 mg/ml) was added to each well to make the final concentration of MTT 0.5 mg/ml. After continuing to incubate for 4 h at 37° C. in an incubator with 5% CO ₂ , the culture was terminated and the supernatant was sucked off. Add 100 μl of dissolving agent (10% SDS, 5% isopropanol, 0.01M HCl) to each well, fully dissolve at 37 ° C, observe under a microscope that after the crystals are completely dissolved, measure the absorbance of each well at 550 nm on a microplate reader ( OD) value to calculate cell viability. Cell viability (%)=OD value of the tested drug/OD value of the control group×100%. The cell survival rate was plotted against the dose logarithm, and the half inhibitory concentration (IC ₅₀ ) of the test drug on each tumor cell was calculated. Results: See Table 1

Table 1 Inhibitory effect of the tested drugs on tumor cell growth in vitro cell line Test drug IC50(μM) 1 2 3 4 5 5-FU Human cervical cancer cells Hela 2.7 2.7 2.7 2.7 2.7 12.5 Human Oral Epidermoid Carcinoma KB 8.2 8.2 8.2 8.2 8.2 7.1 Human hepatoma cell HepG2 2.13×10 ^-4 2.85×10 ^-4 3.5×10 ^-4 2.15×10 ^-4 2.31×10 ^-4 8.3 Human liver cancer cell BEL-7402 6.14×10 ^-3 7.88×10 ^-3 8.21×10 ^-3 7.15×10 ^-3 7.26×10 ^-3 6.4 Human colorectal cancer cell HCT116 0.7 0.8 0.8 0.8 0.8 4 Human lung cancer cell A549 1.3 1.5 1.7 1.5 1.5 4 Human breast adenocarcinoma MCF-7 3.6 3.6 3.6 3.6 3.6 5 Human Renal Cancer Cell RXF-631L 0.61 0.61 0.61 0.61 0.61 6

The results showed that the tested drugs had obvious inhibitory effects on the tested tumor cells, especially for the two kinds of liver cancer cells, the inhibitory effects were very obvious, and the effect was better than that of 5-FU. The results also showed that butterbur extract, butterbur lactone IVa, butterbur lactone IIIa were more effective than butterbur lactone D and butterbur lactone B.

Detailed ways

The following examples are given to further illustrate the present invention but are not intended to limit the present invention.

Example 1

Heat the dried rhizomes of Butterbur trichomes with 20-95% ethanol for heating and extraction, and the amount of solvent used for the first time is about 5-10 times of the crude drug amount, or extract the rhizomes of Butterbur trichomes by percolation with 20-95% ethanol, and the dosage of solvents is 5-15 times of the amount of crude drug, combined percolate, and concentrated under reduced pressure to a certain volume to obtain a concentrated solution, the centrifuged supernatant or filtrate of the concentrated solution is adsorbed by a macroporous resin, (the macroporous resin is a frame with styrene Polystyrene type porous adsorption resin of bridge material, such as: D101, D201, ZTC-1, AB-8, D4020, 860021, HP20, SP825 type macroporous resin, etc.), rinse with water-50% aqueous ethanol to remove Impurities; then elute with a water-containing lower alcohol with a concentration of 60-95%. The lower alcohol is C1-C5 alcohols such as methanol, ethanol, or propanol, and its dosage is 4-12 times the crude drug amount. liquid, concentrated to no alcohol, and dried, which is the extract of Butterbur pilaris of the present invention.

Example 2

Put 50 kg of dried rhizomes of Butterbur trichomes into a percolation tank, add 250 liters of 80% ethanol, soak for 12 hours, percolate, combine the percolation liquid, concentrate it under reduced pressure to 30 liters, centrifuge, and then pass through the macroporous adsorption resin SP825 (Beijing Lvbaicao Co.), rinsed with 300 liters of 40% ethanol to remove impurities; then eluted with 300 liters of 70% ethanol, collected the eluate, concentrated under reduced pressure and dried to obtain the extract of Butterbur pilaris of the present invention 0.25 kg.

The main active ingredients of the butterbur extract of the present invention are butterbur lactone D (bakkenolide-D), butterbur lactone B (bakkenolide-B), butterbur lactone-IVa (bakkenolide-IIIa) , butterbur lactone (bakkenolide-IIIa), the total content of the four is as high as more than 50%. Through in vitro tests, it can effectively prevent and treat tumors, especially liver cancer, lung cancer, cervical cancer, intestinal cancer, kidney cancer, oral epidermoid cancer , breast cancer, so the butterbur pilaris extract can be used to prepare anti-tumor drugs.

Claims (5)

1. A butterbur pilaris extract for the treatment of tumor diseases, characterized in that butterbur lactone D, butterbur lactone B, butterbur lactone-IIIa, butterbur lactone in the butterbur pilaris extract The sum of the contents of acuminolactone-IVa is greater than 50%. 2. a kind of butterbur extract for the treatment of tumor diseases according to claim 1, characterized in that: butterbur lactone-D, butterbur lactone-B, butterbur lactone IVa and The content ratio of butterbur lactone IIIa is 0.1-5:0.1-5:1-9:1-9. 3. a kind of preparation method of the Butterbur pilaris extract for the treatment of tumor diseases described in claim 1, is characterized in that being made up of following steps successively: (1) extraction After the rhizome of Butterbur pilaris is crushed, it is extracted with 20-95% aqueous ethanol to obtain an extract, the extract is concentrated under reduced pressure, then centrifuged or filtered, the precipitate is discarded, and the supernatant or filtrate is used for later use; (2) Adsorption and elution After the above supernatant or filtrate is subjected to polystyrene-type porous adsorption resin adsorption, and water-50% water-containing lower alcohol to remove impurities, use 60-95% of the volume of 4-12 times the mass of the sample crude drug The aqueous C ₁ ~ C ₅ lower alcohol elution; (3) Concentration and drying The eluate is concentrated under reduced pressure and dried to obtain the Butterbur pilaris extract of the present invention. 4. according to the preparation method of the said butterbur extract of claim 3, it is characterized in that the method for extracting with 20-95% hydrous ethanol in the step (1) is heat extraction, cold soaking or percolation, adopts The volume consumption of ethanol during extraction by the above method is 5-15 times of the crude drug quality. 5. the application of a kind of Butterbur pilaris extract for treating tumor diseases described in claim 1 in the preparation of anti-liver cancer, lung cancer, cervical cancer, colon cancer, kidney cancer, oral cavity epidermoid cancer, breast cancer medicine.