CN105982934A - Preparation method and medicinal use of Asarum europaeum L. antitumor extract and composition thereof - Google Patents
Preparation method and medicinal use of Asarum europaeum L. antitumor extract and composition thereof Download PDFInfo
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- Medicines Containing Plant Substances (AREA)
Abstract
The invention belongs to the technical field of medicines, and concretely relates to a preparation method of a Uygur medicine Asarum europaeum L. antitumor extract and a composition thereof, and an application of the extract in the preparation of medicines for preventing and treating breast cancer, lung cancer, stomach cancer, colorectal carcinoma, pancreas cancer, cervical carcinoma, brain glioma, liver cancer and other malignant tumors. The preparation method of the extract comprises the following steps: carrying out ultrasonic extraction on the Uygur medicine Asarum europaeum L. with a solvent, concentrating the above obtained extract liquid, carrying out silica gel separation, eluting a sample introduced silica gel column with a petroleum ether-acetone mixed solvent with the volume ratio of petroleum ether to acetone of 100:0-100:6, continuously eluting the silica gel column with a petroleum ether-acetone mixed solvent with the volume ratio of petroleum ether to acetone of 100:2-100:50, and drying an eluate obtained through elution with the petroleum ether-acetone mixed solvent with the volume ratio of petroleum ether to acetone of 100:2-100:50 in order to obtain the extract.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a Uygur medicine asarum anti-tumor extract, a preparation method of a composition of the Uygur medicine asarum anti-tumor extract, and application of the Uygur medicine asarum anti-tumor extract in preparation of medicines for preventing and treating malignant tumors such as liver cancer, lung cancer, stomach cancer, colon cancer, pancreatic cancer, breast cancer, cervical cancer, brain glioma and the like.
Background
The product is derived from Asarum europaeum L of Asarum of Aristolochiaceae, and is an exterior-releasing medicine for clinical use in Chinese medicine, and has effects in dispelling pathogenic wind and cold, relieving pain and obstruction, relieving cough and asthma, and promoting blood circulation; since Shen nong Ben Cao Jing (Shen nong's herbal), there are two thousand years of history of application, and there are Wu Mei Wan, Xiao Qing Long Tang, Dang Gui Si Ni Tang and Ma Huang Fu Zi xi Xin Tang, etc. in Zhang Zhongjing (Shang Han Lun); it can be used for treating wind-cold exterior syndrome, various pain syndromes, obstruction of orifices, and cough and asthma due to lung cold. Asarum europaeum is a good drug, and the rhizome of Asarum europaeum is used as a drug for relieving fever, promoting urination, relieving pain and tranquilizing. For headache, it has the actions of inducing sweat and eliminating phlegm, so it is a former one with pungent taste due to its thin root and strong fragrance.
Disclosure of Invention
The technical problem to be solved by the invention is to provide an asarum europaea extract.
The invention also provides a composition taking the asarum europaea extract as a main active ingredient.
The invention also provides the preparation of the asarum europaea extract and the composition thereof and the application of the asarum europaea extract and the composition thereof in preparing anti-tumor drugs.
The invention is realized by the following technical scheme:
ultrasonic extracting Uygur medicine asarum sieboldii with methanol, concentrating the extract, separating with silica gel column, loading the sample on silica gel column, gradient eluting with petroleum ether-acetone mixed solvent (100:0-0:00) (petroleum ether: acetone, the specific ratio is shown in table 1), eluting with each petroleum ether-acetone mixed solvent, concentrating, drying, testing the anti-tumor activity with in vitro anti-tumor activity screening system, and unexpectedly finding that the anti-tumor activity is tested with petroleum ether-acetone mixed solvent (petroleum ether: acetone)100:0 to 100:5, volume ratio, preferably 100:0 to 100:1 or 100:3), followed by elution with a mixed solvent of petroleum ether and acetone (petroleum ether: acetone, 100:6 to 100:100, preferably 100:6 or 100:10, volume ratio), petroleum ether: after the acetone-100: 2-100:50 eluent is dried, the acetone-100: 50 eluent has remarkable anti-tumor activity. Preferably, elution is carried out with a mixed solvent of petroleum ether and acetone (petroleum ether: acetone, 100:2, volume ratio) and then with a mixed solvent of petroleum ether and acetone (petroleum ether: acetone, 100:3, volume ratio), the ratio of petroleum ether: and drying the eluent with the ratio of acetone to acetone being 100:3 to obtain the product. The compound with antitumor activity can be enriched by the method, so as to be separated from other impurity components. The active site has never been reported in the literature and its 100:3 eluent TLC analysis is characterized as shown in figure 1. The chromatographic strip is GF254Silica gel board, development system: and (3) developing by using petroleum ether, ethyl acetate and acetone in a ratio of 6:1:1, and performing vanillin concentrated sulfuric acid.
Or eluting with mixed solvent of petroleum ether and acetone (petroleum ether: acetone, 100:6, volume ratio), and eluting with mixed solvent of petroleum ether and acetone (petroleum ether: acetone, 100:10, volume ratio) sequentially, wherein the ratio of petroleum ether: the eluent was dried at 100:10 acetone. More preferably, the elution is carried out with a mixed solvent of petroleum ether and acetone (petroleum ether: acetone, 100:6, volume ratio) and then with a mixed solvent of petroleum ether and acetone (petroleum ether: acetone, 100:10, volume ratio), the ratio of petroleum ether: and drying the eluent with the ratio of acetone to acetone being 100:10 to obtain the product. The compound with antitumor activity can be enriched by the method, so that the compound is separated from other impurity components. The active site has never been reported in the literature and its TLC analysis of 100:10 eluate is characterized as shown in FIG. 2. Chromatographic conditions are GF254Silica gel board, development system: and (3) developing by using petroleum ether, ethyl acetate and acetone in a ratio of 6:1:1, wherein vanillin-concentrated sulfuric acid is used for developing color.
Specifically, the discovery process is as follows:
1. preparation of Asarum europaeum ultrasonic extract
Taking 20g of Uygur medicine asarum, ultrasonically extracting for 15min by 100ml of methanol, and concentrating the extracting solution to obtain an extract SN0075A4.5751g; extracting with 100ml water for 15min to obtain SN0075B 0.3555g, mixing 4.9306g, 3.0286g, and keeping 1.9020 g.
2. Petroleum ether-acetone mixed solvent gradient elution method and results
Taking 20g of Uygur medicine asarum, ultrasonically extracting for 15min by 100ml of methanol, and concentrating the extracting solution to obtain an extract SN0075A4.5751g; extracting with 100ml water for 15min to obtain SN0075B 0.3555g, mixing 4.9306g, and collecting 3.0286 g. The remaining 1.9020g were applied to the column and dissolved in methanol to give a total of 15ml methanol. Performing silica gel column chromatography, mixing 2g of silica gel, 10g of blank silica gel, the inner diameter of a glass column is 2cm, and petroleum ether: gradient elution is carried out by an acetone system (the conditions are shown in table 1), the column volume is 20ml, each gradient elution is 200ml, the eluate of the eluate is extracted at each concentration, and the solvent is recovered, so that the petroleum ether: the extract was eluted with an acetone gradient in yields as shown in Table 1 and TLC analysis results as shown in FIG. 3. Chromatographic conditions are GF254The silica gel plate is developed by petroleum ether, ethyl acetate and acetone in a ratio of 6:1:1 and vanillin-concentrated sulfuric acid.
TABLE 1
3. Screening method and results for antitumor Activity
1) Conditions of the experiment
a) Information on cell lines
Human breast cancer cell line MCF7 cells were purchased from the China center for type culture Collection cell Bank.
b) Medicine and reagent
Under the aseptic condition of the sample, DMSO is dissolved to prepare a mother solution with the concentration of 100mg/mL, and when the sample is used, the mother solution is diluted to the required concentration (DMSO is less than or equal to 1 per thousand) by using an RPMI-1640 culture solution.
RPMI Medium 1640 Medium was purchased as dry powder from GIBCO, USA. Fetal bovine serum was purchased from the institute of biotechnology, yohima, beijing. Trypsin 1:250 and dimethyl diphenyl tetrazole bromide (MTT).
c) Instrument for measuring the position of a moving object
CO2Incubator (SANYO, Japan)
Vertical laminar flow clean bench (Shanghai clean and clean equipment Co., Ltd.)
Inverted biological microscope (Chongqing photoelectric instrument Co., Ltd.)
Low/high speed desk centrifuge (Shanghai' an pavilion scientific instrument factory)
Electronic balance (Aohaus instrument Co., Ltd.)
Enzyme mark instrument (Bole life medical products Co., Ltd.)
Vertical pressure steam sterilizer (Shanghai Shenan medical equipment factory)
Air-blast drying cabinet (Shanghai Yuejin medical equipment factory)
PH meter (SARTORIUS, Germany)
2) Experimental methods and procedures
The cells of the human breast cancer cell line MCF7 are paved on a 96-well plate at the density of 4000/well, each well is 100ul, and the cells are used after 12 h. The components are dissolved in DMSO to be 100mg/mL stock solution and stored at-20 ℃ for later use. 12h after plating, the cell confluence rate was observed to be about 40%, and the drugs were added at concentrations of 100. mu.g/mL, 3 duplicate wells per fraction. Two control groups (medium + cells; medium + cells with 0.1% DMSO), 3 blank wells (medium only) were set up per plate of cells. After 24 hours of drug addition, 20. mu.l of tetramethylazozole salt chemically named 3- (4, 5-dimethylthiazole) -2, 5-diphenyltetrazolium bromide (3- (4, 5-dimethyl-2-thiazolyl) -2,5-diphenyl-tetrazolium bromide, MTT) was added and the culture was continued for 4 hours. The supernatant was completely discarded, 100. mu.l DMSO was added, and MTT was dissolved and bubbled. OD was measured at 490 nm.
3) Results of the experiment
TABLE 2
As a result, it was found that:
the invention preferably adopts the following technical scheme: ultrasonic extracting Uygur medicine asarum herb with organic solvent, concentrating the extracting solution, separating with silica gel column, eluting with petroleum ether-acetone mixed solvent (petroleum ether: acetone, 100:2, volume ratio), eluting with petroleum ether-acetone mixed solvent (petroleum ether: acetone, 100:3, volume ratio), recovering organic solvent from the eluent of 100:3, and drying to obtain high-activity extract, i.e. the anti-tumor extract. The active site has never been reported in the literature, and the TLC analysis characterization is shown in FIG. 1.
Or ultrasonic extracting Uygur medicine asarum herb with organic solvent, concentrating the extracting solution, separating with silica gel column, eluting with petroleum ether-acetone mixed solvent (100: 6 by volume ratio) and then with petroleum ether-acetone mixed solvent (100: 10 by volume ratio), recovering organic solvent from the eluent of 100:10 by using petroleum ether and acetone, and drying to obtain high-activity extract, i.e. the anti-tumor extract. The active site has never been reported in the literature and its TLC analysis is characterized as shown in FIG. 2.
4. Research on extraction method of anti-tumor effective extract
1) Species study of extraction solvent
Extracting with organic solvent such as methanol, ethanol, acetone, chloroform, ethyl acetate, methanol-water mixed solvent, and ethanol-water mixed solvent, respectively, and testing the extract for anti-tumor activity on MCF7 cells, the results are as follows:
TABLE 3
The research result shows that: the organic solvent for extraction may be methanol, ethanol, acetone, chloroform, ethyl acetate, methanol-water mixed solvent, or ethanol-water mixed solvent.
2) Research on the amount of extraction solvent
The extracts were extracted with 1, 2,5, 10, 20, 30, 40, 50-fold (weight/volume ratio) organic solvent, respectively, and tested for anti-tumor activity against C6 cells, with the following results:
TABLE 4
The organic solvent for extracting the extract accounts for 2-50 times of the weight of the medicinal materials (weight/volume ratio).
3) Examination of drying method
The obtained antitumor active site is dried by methods such as a vacuum drying method, a freeze drying method, an air-blast drying method, a centrifugal drying method, a rotary evaporation drying method and the like respectively, and the vacuum drying method, the freeze drying method, the air-blast drying method, the centrifugal drying method and the rotary evaporation drying method are found to be suitable for drying the MCF7 cell extract by taking the water content, the TLC and the activity test as indexes, wherein the vacuum drying method and the freeze drying method are most preferred.
TABLE 5
| Sample 20g | Vacuum drying method | Freeze drying method | Air-blast drying method | Centrifugal drying method | Rotary evaporation drying method |
| Water content (%) | 8.9 | 8.7 | 10.6 | 11.5 | 10.0 |
| Inhibition ratio (%) | 46.6 | 49.9 | 45.5 | 39.6 | 40.6 |
Therefore, the preparation method of the asarum antitumor extract comprises the following steps:
preferably, the asarum sieboldii, an Uyghur medicine, is subjected to ultrasonic extraction by a solvent, an extracting solution is concentrated, then is separated by a silica gel column, and the loaded silica gel column is eluted by a petroleum ether-acetone mixed solvent (petroleum ether: acetone, 100:2, volume ratio) for 2-50 times of column volume, then is eluted by a petroleum ether: acetone mixed solvent (petroleum ether: acetone, 100:3, volume ratio) for 2-50 times of column volume, and the petroleum ether: acetone-100: 2 eluent is subjected to organic solvent recovery and drying to obtain the extract. The optimal conditions are that 10 times of column volume is eluted by petroleum ether-acetone mixed solvent (petroleum ether: acetone, 100:2, volume ratio), and 15 times of column volume is eluted by petroleum ether-acetone mixed solvent (petroleum ether: acetone, 100:3, volume ratio).
1) The solvent for extracting extract can be methanol, ethanol, acetone, chloroform, ethyl acetate, methanol-water mixed solvent, or ethanol-water mixed solvent, preferably methanol and 95% ethanol. The organic solvent for extracting the extract accounts for 2-50 times of the weight of the medicinal materials (weight/volume ratio).
2) The extract drying method can be vacuum drying method, freeze drying method, forced air drying, centrifugal drying, rotary evaporation drying method, etc., preferably vacuum drying method and freeze drying method.
Ultrasonic extracting Uyghur medicine asarum herb with solvent, concentrating the extracting solution, separating with silica gel column, eluting with petroleum ether-acetone mixed solvent (100: 6 vol.%) for 2-50 times of column volume, eluting with petroleum ether-acetone mixed solvent (100: 10 vol.%) for 2-50 times of column volume, recovering organic solvent from the eluent of 100:10 (100: 10), and drying. The optimal conditions are that firstly a petroleum ether-acetone mixed solvent (petroleum ether: acetone, 100:10, volume ratio) is used for eluting by 15 times of the column volume, and then a petroleum ether-acetone mixed solvent (petroleum ether: acetone, 100:10, volume ratio) is used for eluting by 15 times of the column volume. Wherein,
1) the solvent for extracting extract can be methanol, ethanol, acetone, chloroform, ethyl acetate, methanol-water mixed solvent, or ethanol-water mixed solvent, preferably methanol and 95% ethanol. The organic solvent for extracting the extract accounts for 2-50 times of the weight of the medicinal materials (weight/volume ratio).
2) The extract drying method can be vacuum drying method, freeze drying method, forced air drying, centrifugal drying, rotary evaporation drying method, etc., preferably vacuum drying method and freeze drying method.
5. Research on antitumor activity of Asarum europaeum antitumor effective extract
Respectively testing antitumor activity and IC of Asarum europaeum antitumor extract by using various tumor cells, such as brain glioma, breast cancer, lung cancer, gastric cancer, colon cancer, pancreatic cancer, cervical cancer, and hepatocarcinoma50The results are as follows:
TABLE 6
Therefore, the asarum herb antitumor extract has antitumor activity, and is expected to be used for preventing and treating various tumors such as brain glioma, lung cancer, gastric cancer, intestinal cancer, pancreatic cancer, colon cancer, cervical cancer, liver cancer and other malignant tumors.
6. Asarum europaeum antitumor effective extract composition and preparation method thereof
1) Solid dispersion
Prescription
The preparation method comprises the following steps:
weighing Asarum europaeum extract and polyvinylpyrrolidone (PVP) as carrier at a weight ratio of 1:2, 1:4 and 1:6, respectively placing into a beaker, adding anhydrous ethanol, stirring with a magnetic stirrer until Asarum europaeum extract and carrier are completely dissolved, transferring into a rotary steaming instrument to remove organic solvent, drying, pulverizing, and sieving with 80 mesh sieve to obtain Asarum europaeum extract PVP clathrate.
2) Cyclodextrin inclusion compounds
Prescription:
the preparation method comprises the following steps:
grinding beta-cyclodextrin and 1-5 times of water uniformly, adding asarum europaea extract (the water-insoluble one should be dissolved in a small amount of organic solvent) and fully grinding to paste, and drying at low temperature to obtain the final product.
3) The prescription of the dispersible tablet is as follows:
the preparation method comprises the following steps:
1. preparing starch dispersion of Asarum europaeum extract, precisely weighing Asarum europaeum extract, adding appropriate amount of sodium lauryl sulfate, dissolving with 70% ethanol, adding soluble starch at equal ratio, mixing, evaporating at 70 deg.C, pulverizing, and sieving with 100 mesh sieve;
2. weighing the prescription amount, the asarum europaea extract starch dispersoid, the crospovidone and the pregelatinized starch, using 70% ethanol as a wetting agent, adding while stirring, preparing wet granules, sieving with a 14-mesh sieve, standing at room temperature for 15min, oven-drying at 60 ℃ for 45min, grading with a 16-mesh sieve, adding talcum powder and differential silica gel, mixing uniformly, and tabletting.
7. Research on detection method of effective anti-tumor extract of Asarum europaeum L
We have found that the anti-tumor effective extract of Asarum europaeum can be well characterized and characterized by thin layer chromatography. See fig. 1, fig. 2.
Chromatographic conditions are as follows: GF254Silica gel board, development system: ethyl acetate and acetone in a ratio of 6:1:1, ethyl ether and acetone in a ratio of 1:1:1, and a color development method: and (4) developing the color by vanillin-concentrated sulfuric acid.
Description of the drawings:
FIG. 1 TLC chromatogram of Asarum sieboldii MCF7 inhibitory extract;
FIG. 2 TLC chromatogram of Asarum sieboldii MCF7 inhibitory extract;
FIG. 3 TLC chromatogram of the chemical composition of Asarum europaeum extract;
Detailed Description
The following examples illustrate the utility of the invention, which is not limited thereby.
Example 1:
ultrasonic extracting 20g Uygur medicine Asarum europaeum L with 100ml methanol for 15min, ultrasonic extracting with 100ml water for 15min, concentrating the extractive solution, separating with silica gel column, loading on silica gel column (2.0cm inner diameter column), adding petroleum ether: acetone mixed solvent (petroleum ether: acetone, 100:2, volume ratio) was eluted 10 column volumes, followed by petroleum ether: acetone mixed solvent (petroleum ether: acetone, 100:3, volume ratio) elutes 15 times column volume, petroleum ether: recovering organic solvent from the eluate of acetone mixed solvent (petroleum ether: acetone, 100:3, volume ratio), and vacuum drying to obtain the extract with yield of 0.045%.
Example 2:
ultrasonic extracting 20g Uygur medicine Asarum europaeum L with 100ml ethanol for 15min, then ultrasonic extracting with 100ml water for 15min, concentrating the extractive solution, separating with silica gel column, loading on silica gel column (2.0cm inner diameter small column), adding petroleum ether: acetone mixed solvent (petroleum ether: acetone, 100:2, volume ratio) was eluted 10 column volumes, followed by petroleum ether: acetone mixed solvent (petroleum ether: acetone, 100:3, volume ratio) elutes 15 times column volume, petroleum ether: recovering organic solvent from the eluate of acetone mixed solvent (petroleum ether: acetone, 100:3, volume ratio), and freeze drying to obtain the extract with yield of 0.043%.
Example 3:
ultrasonic extracting 20g Uygur medicine Asarum europaeum L with 100ml acetone for 15min, ultrasonic extracting with 100ml water for 15min, concentrating the extractive solution, separating with silica gel column, loading on silica gel column (2.0cm inner diameter column), adding petroleum ether: acetone mixed solvent (petroleum ether: acetone, 100:2, volume ratio) was eluted 10 column volumes, followed by petroleum ether: acetone mixed solvent (petroleum ether: acetone, 100:3, volume ratio) elutes 15 times column volume, petroleum ether: recovering organic solvent from acetone mixed solvent (100: 3 by volume) eluate, and air drying to obtain the extract with yield of 0.033%.
Example 4:
taking 20g of Uygur medicine asarum, ultrasonically extracting for 15min by 100ml of chloroform, ultrasonically extracting for 15min by 100ml of water, concentrating the extracting solution, separating by using a silica gel column, loading the sample on a silica gel column (a small column with the inner diameter of 2.0 cm), and firstly using petroleum ether: acetone mixed solvent (petroleum ether: acetone, 100:2, volume ratio) was eluted 10 column volumes, followed by petroleum ether: acetone mixed solvent (petroleum ether: acetone, 100:3, volume ratio) elutes 15 times column volume, petroleum ether: recovering organic solvent from the eluate of acetone mixed solvent (petroleum ether: acetone, 100:3, volume ratio), and centrifuging to dry to obtain the extract with yield of 0.039%.
Example 5:
taking 20g of Uygur medicine asarum, ultrasonically extracting for 15min by 100ml of ethyl acetate, ultrasonically extracting for 15min by 100ml of water, concentrating the extracting solution, separating by using a silica gel column, loading the sample on a silica gel column (a small column with the inner diameter of 2.0 cm), and firstly using petroleum ether: acetone mixed solvent (petroleum ether: acetone, 100:2, volume ratio) was eluted 10 column volumes, followed by petroleum ether: acetone mixed solvent (petroleum ether: acetone, 100:3, volume ratio) elutes 15 times column volume, petroleum ether: recovering organic solvent from the eluent of acetone mixed solvent (petroleum ether: acetone, 100:3, volume ratio), and rotary steaming and drying to obtain the extract with yield of 0.037%.
Example 6:
taking 20g of Uygur medicine asarum, ultrasonically extracting for 15min by 100ml of methanol-water mixed solvent (methanol: water is 1:1), then ultrasonically extracting for 15min by 100ml of water, concentrating the extract, separating by using a silica gel column, loading the sample on a silica gel column (a small column with the inner diameter of 2.0 cm), and firstly using petroleum ether: acetone mixed solvent (petroleum ether: acetone, 100:2, volume ratio) was eluted 10 column volumes, followed by petroleum ether: acetone mixed solvent (petroleum ether: acetone, 100:3, volume ratio) elutes 15 times column volume, petroleum ether: recovering organic solvent from the eluate of acetone mixed solvent (petroleum ether: acetone, 100:3, volume ratio), and drying to obtain the extract with yield of 0.038%.
Example 7:
taking 20g of Uygur medicine asarum, ultrasonically extracting for 15min by 100ml of ethanol-water mixed solvent (ethanol: water is 1:1), then ultrasonically extracting for 15min by 100ml of water, concentrating the extract, separating by using a silica gel column, loading the sample on a silica gel column (a small column with the inner diameter of 2.0 cm), and firstly using petroleum ether: acetone mixed solvent (petroleum ether: acetone, 100:2, volume ratio) was eluted 10 column volumes, followed by petroleum ether: acetone mixed solvent (petroleum ether: acetone, 100:3, volume ratio) elutes 15 times column volume, petroleum ether: recovering organic solvent from the eluate of acetone mixed solvent (petroleum ether: acetone, 100:3, volume ratio), and drying to obtain the extract with yield of 0.038%.
Example 8:
ultrasonic extracting 20g of Uygur medicine Asarum europaeum L with 100ml of ethanol-water mixed solvent (ethanol: water: 90:10) for 15min, then ultrasonic extracting with 100ml of water for 15min, concentrating the extract, separating with silica gel column, loading on silica gel column (2.0cm inner diameter small column), extracting with petroleum ether: acetone mixed solvent (petroleum ether: acetone, 100:2, volume ratio) was eluted 10 column volumes, followed by petroleum ether: acetone mixed solvent (petroleum ether: acetone, 100:3, volume ratio) elutes 15 times column volume, petroleum ether: recovering organic solvent from the eluate of acetone mixed solvent (petroleum ether: acetone, 100:3, volume ratio), and drying to obtain the extract with yield of 0.042%.
Example 9:
ultrasonic extracting 20g Uygur medicine Asarum europaeum L with 100ml methanol for 15min, ultrasonic extracting with 100ml water for 15min, concentrating the extractive solution, separating with silica gel column, loading on silica gel column (2.0cm inner diameter column), adding petroleum ether: acetone mixed solvent (petroleum ether: acetone, 100:6, volume ratio) was eluted 20 column volumes, followed by petroleum ether: acetone mixed solvent (petroleum ether: acetone, 100:10, volume ratio) elutes 30 column volumes, petroleum ether: 100 parts of acetone: 10 the organic solvent is recovered from the eluent, and the extract is obtained after vacuum drying, with the yield of 0.22 percent.
Example 10:
ultrasonic extracting 20g Uygur medicine Asarum europaeum L with 100ml ethanol for 15min, then ultrasonic extracting with 100ml water for 15min, concentrating the extractive solution, separating with silica gel column, loading on silica gel column (2.0cm inner diameter small column), adding petroleum ether: acetone mixed solvent (petroleum ether: acetone, 100:6, volume ratio) was eluted 20 column volumes, followed by petroleum ether: acetone mixed solvent (petroleum ether: acetone, 100:10, volume ratio) elutes 30 column volumes, petroleum ether: 100 parts of acetone: 10 the eluent is subject to organic solvent recovery and freeze drying, thus obtaining the extract with the yield of 0.2%.
Example 11:
ultrasonic extracting 20g Uygur medicine Asarum europaeum L with 100ml acetone for 15min, ultrasonic extracting with 100ml water for 15min, concentrating the extractive solution, separating with silica gel column, loading on silica gel column (2.0cm inner diameter column), adding petroleum ether: acetone mixed solvent (petroleum ether: acetone, 100:6, volume ratio) was eluted 20 column volumes, followed by petroleum ether: acetone mixed solvent (petroleum ether: acetone, 100:10, volume ratio) elutes 30 column volumes, petroleum ether: 100 parts of acetone: 10 the organic solvent is recovered from the eluent, and the extract is obtained after forced air drying, with the yield of 0.14 percent.
Example 12:
taking 20g of Uygur medicine asarum, ultrasonically extracting for 15min by 100ml of chloroform, ultrasonically extracting for 15min by 100ml of water, concentrating the extracting solution, separating by using a silica gel column, loading the sample on a silica gel column (a small column with the inner diameter of 2.0 cm), and firstly using petroleum ether: acetone mixed solvent (petroleum ether: acetone, 100:6, volume ratio) was eluted 20 column volumes, followed by petroleum ether: acetone mixed solvent (petroleum ether: acetone, 100:10, volume ratio) elutes 30 column volumes, petroleum ether: 100 parts of acetone: 10 the eluent is recovered with organic solvent, and is centrifugally dried to obtain the extract, and the yield is 0.17 percent.
Example 13:
taking 20g of Uygur medicine asarum, ultrasonically extracting for 15min by 100ml of ethyl acetate, ultrasonically extracting for 15min by 100ml of water, concentrating the extracting solution, separating by using a silica gel column, loading the sample on a silica gel column (a small column with the inner diameter of 2.0 cm), and firstly using petroleum ether: acetone mixed solvent (petroleum ether: acetone, 100:6, volume ratio) was eluted 20 column volumes, followed by petroleum ether: acetone mixed solvent (petroleum ether: acetone, 100:10, volume ratio) elutes 30 column volumes, petroleum ether: 100 parts of acetone: 10 the organic solvent is recovered from the eluent, and the extract is obtained after rotary evaporation and drying, with the yield of 0.18 percent.
Example 14:
taking 20g of Uygur medicine asarum, ultrasonically extracting for 15min by 100ml of methanol-water mixed solvent (methanol: water is 1:1), then ultrasonically extracting for 15min by 100ml of water, concentrating the extract, separating by using a silica gel column, loading the sample on a silica gel column (a small column with the inner diameter of 2.0 cm), and firstly using petroleum ether: acetone mixed solvent (petroleum ether: acetone, 100:6, volume ratio) was eluted 20 column volumes, followed by petroleum ether: acetone mixed solvent (petroleum ether: acetone, 100:10, volume ratio) elutes 30 column volumes, petroleum ether: 100 parts of acetone: 10 the eluent is recovered with organic solvent, and is frozen and dried to obtain the extract, the yield is 0.21 percent.
Example 15:
taking 20g of Uygur medicine asarum, ultrasonically extracting for 15min by 100ml of ethanol-water mixed solvent (ethanol: water is 1:1), then ultrasonically extracting for 15min by 100ml of water, concentrating the extract, separating by using a silica gel column, loading the sample on a silica gel column (a small column with the inner diameter of 2.0 cm), and firstly using petroleum ether: acetone mixed solvent (petroleum ether: acetone, 100:6, volume ratio) was eluted 20 column volumes, followed by petroleum ether: acetone mixed solvent (petroleum ether: acetone, 100:10, volume ratio) elutes 30 column volumes, petroleum ether: 100 parts of acetone: 10 the organic solvent is recovered from the eluent, and the extract is obtained after vacuum drying, with the yield of 0.2 percent.
Example 16:
ultrasonic extracting 20g of Uygur medicine Asarum europaeum L with 100ml of ethanol-water mixed solvent (ethanol: water: 90:10) for 15min, then ultrasonic extracting with 100ml of water for 15min, concentrating the extract, separating with silica gel column, loading on silica gel column (2.0cm inner diameter small column), extracting with petroleum ether: acetone mixed solvent (petroleum ether: acetone, 100:6, volume ratio) was eluted 20 column volumes, followed by petroleum ether: acetone mixed solvent (petroleum ether: acetone, 100:10, volume ratio) elutes 30 column volumes, petroleum ether: 100 parts of acetone: 10 the eluent is subject to organic solvent recovery and freeze drying, thus obtaining the extract with the yield of 0.17%.
Example 17:
ultrasonic extracting 20g Uygur medicine Asarum europaeum L with 100ml methanol for 15min, ultrasonic extracting with 100ml water for 15min, concentrating the extractive solution, separating with silica gel column, loading on silica gel column (2.0cm inner diameter column), adding petroleum ether: acetone mixed solvent (petroleum ether: acetone, 100:2, volume ratio) was eluted 20 column volumes, followed by petroleum ether: acetone mixed solvent (petroleum ether: acetone, 100:3, volume ratio) elutes 30 column volumes, petroleum ether: recovering organic solvent from acetone mixed solvent (100: 3 by volume) eluate, and freeze drying to obtain the extract with yield of 0.05%.
Example 18:
ultrasonic extracting 20g Uygur medicine Asarum europaeum L with 100ml ethanol for 15min, then ultrasonic extracting with 100ml water for 15min, concentrating the extractive solution, separating with silica gel column, loading on silica gel column (2.0cm inner diameter small column), adding petroleum ether: acetone mixed solvent (petroleum ether: acetone, 100:2, volume ratio) was eluted 20 column volumes, followed by petroleum ether: acetone mixed solvent (petroleum ether: acetone, 100:3, volume ratio) elutes 30 column volumes, petroleum ether: recovering organic solvent from the eluate of acetone mixed solvent (petroleum ether: acetone, 100:3, volume ratio), and vacuum drying to obtain the extract with yield of 0.04%.
Example 19:
ultrasonic extracting 20g Uygur medicine Asarum europaeum L with 100ml acetone for 15min, ultrasonic extracting with 100ml water for 15min, concentrating the extractive solution, separating with silica gel column, loading on silica gel column (2.0cm inner diameter column), adding petroleum ether: acetone mixed solvent (petroleum ether: acetone, 100:2, volume ratio) was eluted 20 column volumes, followed by petroleum ether: acetone mixed solvent (petroleum ether: acetone, 100:3, volume ratio) elutes 30 column volumes, petroleum ether: recovering organic solvent from eluate of acetone mixed solvent (petroleum ether: acetone, 100:3, volume ratio), and air-blast drying to obtain the extract with yield of 0.034%.
Example 20:
taking 20g of Uygur medicine asarum, ultrasonically extracting for 15min by 100ml of chloroform, ultrasonically extracting for 15min by 100ml of water, concentrating the extracting solution, separating by using a silica gel column, loading the sample on a silica gel column (a small column with the inner diameter of 2.0 cm), and firstly using petroleum ether: acetone mixed solvent (petroleum ether: acetone, 100:2, volume ratio) was eluted 20 column volumes, followed by petroleum ether: acetone mixed solvent (petroleum ether: acetone, 100:3, volume ratio) elutes 30 column volumes, petroleum ether: recovering organic solvent from acetone mixed solvent (100: 3 by volume) eluate, and centrifuging and drying to obtain the extract with yield of 0.045%.
Example 21:
taking 20g of Uygur medicine asarum, ultrasonically extracting for 15min by 100ml of ethyl acetate, ultrasonically extracting for 15min by 100ml of water, concentrating the extracting solution, separating by using a silica gel column, loading the sample on a silica gel column (a small column with the inner diameter of 2.0 cm), and firstly using petroleum ether: acetone mixed solvent (petroleum ether: acetone, 100:2, volume ratio) was eluted 20 column volumes, followed by petroleum ether: acetone mixed solvent (petroleum ether: acetone, 100:3, volume ratio) elutes 30 column volumes, petroleum ether: recovering organic solvent from the eluate of acetone mixed solvent (petroleum ether: acetone, 100:3, volume ratio), and rotary steaming and drying to obtain the extract with yield of 0.044%.
Example 22:
taking 20g of Uygur medicine asarum, ultrasonically extracting for 15min by 100ml of methanol-water mixed solvent (methanol: water is 1:1), then ultrasonically extracting for 15min by 100ml of water, concentrating the extract, separating by using a silica gel column, loading the sample on a silica gel column (a small column with the inner diameter of 2.0 cm), and firstly using petroleum ether: acetone mixed solvent (petroleum ether: acetone, 100:2, volume ratio) was eluted 20 column volumes, followed by petroleum ether: acetone mixed solvent (petroleum ether: acetone, 100:3, volume ratio) elutes 30 column volumes, petroleum ether: recovering organic solvent from the eluate of acetone mixed solvent (petroleum ether: acetone, 100:3, volume ratio), and freeze drying to obtain the extract with yield of 0.047%.
Example 23:
taking 20g of Uygur medicine asarum, ultrasonically extracting for 15min by 100ml of ethanol-water mixed solvent (ethanol: water is 1:1), then ultrasonically extracting for 15min by 100ml of water, concentrating the extract, separating by using a silica gel column, loading the sample on a silica gel column (a small column with the inner diameter of 2.0 cm), and firstly using petroleum ether: acetone mixed solvent (petroleum ether: acetone, 100:2, volume ratio) was eluted 20 column volumes, followed by petroleum ether: acetone mixed solvent (petroleum ether: acetone, 100:3, volume ratio) elutes 30 column volumes, petroleum ether: recovering organic solvent from the eluate of acetone mixed solvent (petroleum ether: acetone, 100:3, volume ratio), and vacuum drying to obtain the extract with yield of 0.045%.
Example 24:
ultrasonic extracting 20g of Uygur medicine Asarum europaeum L with 100ml of ethanol-water mixed solvent (ethanol: water: 90:10) for 15min, then ultrasonic extracting with 100ml of water for 15min, concentrating the extract, separating with silica gel column, loading on silica gel column (2.0cm inner diameter small column), extracting with petroleum ether: acetone mixed solvent (petroleum ether: acetone, 100:2, volume ratio) was eluted 20 column volumes, followed by petroleum ether: acetone mixed solvent (petroleum ether: acetone, 100:3, volume ratio) elutes 30 column volumes, petroleum ether: recovering organic solvent from the eluate of acetone mixed solvent (petroleum ether: acetone, 100:3, volume ratio), and freeze drying to obtain the extract with yield of 0.04%.
Example 25: solid dispersion
Weighing Asarum europaeum extract and polyvinylpyrrolidone (PVP) as carrier at a weight ratio of 1:2, 1:4 and 1:6, respectively placing into a beaker, adding anhydrous ethanol, stirring with a magnetic stirrer until Asarum europaeum extract and carrier are completely dissolved, transferring into a rotary steaming instrument to remove organic solvent, drying, pulverizing, and sieving with 80 mesh sieve to obtain Asarum europaeum extract PVP clathrate.
Example 26: cyclodextrin inclusion compounds
Grinding beta-cyclodextrin and 1-5 times of water uniformly, adding asarum europaea extract (the water-insoluble one should be dissolved in a small amount of organic solvent) and fully grinding to paste, and drying at low temperature to obtain the final product.
Example 27: dispersible tablet
1. Weighing dried Asarum europaeum extract, adding appropriate amount of sodium lauryl sulfate, dissolving with 70% ethanol, adding water soluble starch and Asarum europaeum extract (1: 1), stirring, mixing, drying at 70 deg.C, pulverizing, and sieving with 100 mesh sieve to obtain Asarum europaeum extract dispersoid.
2. Precisely weighing the prescription amount, the Asarum europaeum extract dispersoid, the crospovidone and the compressible starch, using 70 ethanol as a wetting agent, stirring while adding, preparing wet granules by a 14-mesh sieve, standing for 15min at room temperature, drying for 45min in an oven at 70 ℃, granulating by a 16-mesh sieve, adding talcum powder and differential silica gel, mixing uniformly, and tabletting.
Claims (10)
1. An Asarum sieboldii extract as Uygur medicine is characterized in that Asarum sieboldii as Uygur medicine is subjected to ultrasonic extraction by a solvent, an extracting solution is concentrated and then is separated by a silica gel column, the silica gel column after sample loading is firstly eluted by a petroleum ether-acetone mixed solvent with the volume ratio of 100:0-100:5, and then is eluted by a petroleum ether-acetone mixed solvent with the volume ratio of 100:6-100:100, and the petroleum ether: and (3) drying the eluent with acetone =100:6-100:100 to obtain the product.
2. An extract of asarum europaea as an Uygur medicine as claimed in claim 1, wherein the extract is prepared by mixing the following components in a volume ratio of 100: eluting with 0-100:2 petroleum ether-acetone mixed solvent, and eluting with 100:3 petroleum ether-acetone mixed solvent, wherein the ratio of petroleum ether: drying the eluent with acetone =100:3 to obtain the product;
or eluting with a petroleum ether-acetone mixed solvent with the volume ratio of 100:6, and continuously eluting with a petroleum ether-acetone mixed solvent with the volume ratio of 100:10, wherein the petroleum ether: and (3) drying the eluent with acetone =100:10 to obtain the product.
3. The Asarum sieboldii extract as claimed in claim 1 or 2, wherein the extract is obtained by eluting with a mixed solvent of petroleum ether and acetone at a volume ratio of 100:2, and further eluting with a mixed solvent of petroleum ether and acetone at a volume ratio of 100:3, recovering the organic solvent from the eluate of petroleum ether and acetone =100:3, and drying;
or eluting with petroleum ether-acetone mixed solvent at volume ratio of 100:6, eluting with petroleum ether-acetone mixed solvent at volume ratio of 100:10, recovering organic solvent from petroleum ether-acetone =100:10 eluate, and drying to obtain the extract.
4. The Asarum sieboldii extract as claimed in any one of claims 1 to 3, wherein the organic solvent used for the extraction is methanol, ethanol, acetone, chloroform, ethyl acetate, a mixed solvent of methanol and water, or a mixed solvent of ethanol and water.
5. An Asarum sieboldii extract as claimed in any one of claims 1 to 4, wherein the elution volume of the petroleum ether-acetone mixed solvent is 2 to 50 column volumes.
6. The Asarum sieboldii extract as claimed in any one of claims 1 to 4, wherein 10 column volumes are eluted with a mixed solvent of petroleum ether and acetone in a volume ratio of 100:2, and 15 column volumes are eluted with a mixed solvent of petroleum ether and acetone in a volume ratio of 100: 3;
or eluting with petroleum ether-acetone mixed solvent with volume ratio of 100:6 for 15 times of column volume, and eluting with petroleum ether-acetone mixed solvent with volume ratio of 100:10 for 15 times of column volume.
7. A pharmaceutical composition comprising the asiasarum sieboldii extract of any one of claims 1-5 and a pharmaceutically acceptable carrier.
8. A pharmaceutical preparation comprising the Asarum europaeum extract of any one of claims 1-5 or the pharmaceutical composition of claim 6.
9. Use of the Asarum europaeum extract according to any one of claims 1-6 or the composition according to claim 7 or the pharmaceutical preparation according to claim 8 for the preparation of an anti-tumor medicament, wherein the tumor is brain glioma, lung cancer, stomach cancer, colon cancer, pancreatic cancer, cervical cancer, breast cancer or liver cancer.
10. The Asarum europaeum extract of any one of claims 1-6, characterized by chromatography conditions, as determined by TLC, of: GF254Silica gel board, development system: petroleum ether, ethyl acetate and acetone =6:1:1, and the color development method comprises the following steps: heating vanillin concentrated sulfuric acid to develop color.
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| 陈泉生: "细辛与其同属植物A.kooyanum Makino var.nipponicum Kitamura和A.hexalobum F.Maekawa根的水溶性成分", 《国外药学(植物药分册)》 * |
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| CN113759023A (en) * | 2021-03-10 | 2021-12-07 | 北京康仁堂药业有限公司 | Preparation process and evaluation method of angelica sinensis Sini decoction |
| CN113759023B (en) * | 2021-03-10 | 2023-04-18 | 北京康仁堂药业有限公司 | Preparation process and evaluation method of angelica sinensis Sini decoction |
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