CN114191459B - Artemisia rupestris L alcohol extract and extraction method and application thereof - Google Patents
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Abstract
The invention provides a artemisia anomala alcohol extract, an extraction method and application thereof, belongs to the technical field of biological medicine, and particularly relates to a component collected after 80-90% of ethanol extract of artemisia anomala is eluted by 30% ethanol of macroporous adsorption resin. Experiments show that the artemisia scoparia ethanol extract (AAEM) promotes the expression of DC surface co-stimulatory molecules CD40 and CD86, and can improve the level of IL-6 and TNF-alpha secretion of DC. In vitro experiments, the AAEM shows the capability of promoting DC maturation, shows that the AAEM has an immune enhancement effect, can provide candidate materials for screening safe and efficient immune regulators, and can be used as candidate drugs for diseases related to tumors and inflammatory infection.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a artemisia rupestris L alcohol extract and an extraction method and application thereof.
Background
Dendritic Cells (DCs) are powerful professional antigen presenting cells that link the innate and adaptive immune systems, balance the adaptive response between self and non-self, and function to present antigen and induce T cell responses. With the research of people, the immune function regulation of dendritic cells can be used for treating diseases such as autoimmune diseases, tumors and the like. In recent years, DC can induce anti-tumor immunity, and DC vaccines enhance the anti-tumor activity of the body. In modern medical technology, chemotherapy and radiotherapy are the current conventional means for treating cancer, but they cannot solve the problems of late-stage cancer treatment effect, large influence on the immune system of a patient and the like, and the active ingredients of Chinese herbal medicines are attracted to people as a novel treatment mode. The Chinese herbal medicine has the advantages of multiple targets, small toxic and side effects and the like, can be used as an immunologic adjuvant, and can also promote DC maturation, so that the aim of immunotherapy is fulfilled. The prior art discloses: the Pleurotus ferulae water extract can up-regulate the expression of CD40, CD80, CD86 and MHC II in mouse bone marrow derived DC, and increase the production of IL-12; the celastrus orbiculatus rattan ethyl acetate alcohol extract promotes the expression of DC surface costimulatory molecules (CD 40, CD80 and CD 86) and MHC molecules, increases the expression level of chemokine receptor CCR7, enhances the capability of DC to activate T lymphocytes, and is dose-dependent. In addition, the Chinese herbal medicine can effectively prevent and treat various immune diseases and is a source of a novel tolerance adjuvant.
The main active component of Artemisia L plant is sesquiterpene, and has antiinflammatory, immunity regulating, antitumor, antimalarial, analgesic, and antibacterial effects. Artemisia absinthium L of Artemisia is perennial herb of Artemisia of Compositae, and the herb is bitter and cold in taste, and the medicinal parts are leaves and stems. The Artemisia princeps Pampanini mainly contains terpenoid derivatives, volatile oil, flavonoids, phenols, lignans and other compounds, has effects of resisting inflammation, relieving pain, protecting liver, resisting oxidation, resisting bacteria and tumor, and can be used for treating diseases such as joint swelling and pain, tumor and acute bacillary dysentery.
The prior art CN 112089743A discloses an immunosuppressive drug, the main active component of which comprises a Artemisia sieboldii extract. However, there is no report on the use of Artemisia rupestris extract in the preparation of immunopotentiating drugs.
Disclosure of Invention
The invention aims to provide a artemisia scoparia ethanol extract, an extraction method and application thereof.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides an extraction method of artemisia scoparia ethanol extract, which comprises the following steps:
1) Mixing the overground part of the artemisia scoparia with an extraction solvent for alcohol extraction, and removing an alcohol solvent in an obtained alcohol extraction solution to obtain an alcohol extract, wherein the extraction solvent is an ethanol water solution with the volume concentration of 80-90%, and the alcohol extraction temperature is 55-65 ℃;
2) And (3) passing the alcohol extract through an AB-8 macroporous resin column, sequentially carrying out gradient elution by using water and an ethanol aqueous solution with the volume concentration of 30%, and collecting an eluent, wherein the eluent contains the artemisia rupestris L alcohol extract.
Preferably, the ratio of the mass of the aerial parts of the artemisia scoparia to the volume of the extraction solvent in the step 1) is 1:8 to 12; the extraction times are 2-4.
The invention also provides the artemisia scoparia ethanol extract extracted by the extraction method in the scheme, wherein the mass percent of polysaccharide, flavone and terpenes in the artemisia scoparia ethanol extract is 24.0-24.5%, 22.5-23.0% and 28.0-28.5%.
The invention also provides the artemisia scoparia ethanol extract extracted by the extraction method in the scheme or the application of the artemisia scoparia ethanol extract in preparing the immunopotentiation medicine.
Preferably, the immune enhancing drug comprises a drug that promotes dendritic cell maturation and/or modulates dendritic cell function.
Preferably, the modulation of dendritic cell function comprises: increasing one or more of the expression of surface costimulatory molecules of dendritic cells and enhancing the secretion level of inflammatory cytokines.
Preferably, the surface co-stimulatory molecules comprise CD40 and/or CD86.
Preferably, the inflammatory cytokines include IL-6 and/or TNF- α.
The invention also provides the artemisia scoparia ethanol extract extracted by the extraction method in the scheme or the application of the artemisia scoparia ethanol extract in preparing a medicine for treating tumor and/or inflammatory infection related diseases.
The invention provides a preparation method of artemisia anomala alcohol extract, and particularly relates to a component collected after 80-90% of ethanol extract of artemisia anomala is eluted by 30% ethanol of macroporous adsorption resin. Experiments show that the DC surface co-stimulatory molecules CD40 and CD86 expressed by the Artemisia scoparia ethanol extract (AAEM) of the invention can improve the level of IL-6 and TNF-alpha secreted by DC, and the AAEM has an immune enhancement effect, thereby providing candidates for the preparation of immune enhancement drugs.
Drawings
FIG. 1 is a graph showing the results of AAEM in promoting the expression level of DC surface co-stimulatory molecule CD40, after AAEM treatment of DC for 12h, the expression level of DC surface molecules CD40, CD86 is analyzed by flow cytometry, the data is analyzed by one-way variance analysis, a P<0.001, comparing the processing group with the Control group to obtain;
FIG. 2 is a graph showing the results of AAEM in promoting the expression level of DC surface co-stimulatory molecule CD86, after AAEM treatment of DC for 12h, the expression level of DC surface molecule CD86 is analyzed by flow cytometry, data is analyzed by one-way analysis of variance, a P<0.001, comparing the processing group with the Control group to obtain;
FIG. 3 is a diagram showing the results of IL-6 levels in the DC secretion promotion by AAEM, after the DC is treated by AAEM for 12 hours, the cell supernatant is collected, and the content of IL-6 is detected by ELISA kit; the data were analyzed using a one-way analysis of variance, a P<0.001, comparing the processing group with the Control group to obtain;
FIG. 4 is a result chart of TNF- α level at which AAEM promotes DC to secrete inflammatory cytokines, after the DC is treated by AAEM for 12 hours, cell supernatants are collected, and an ELISA kit is used for detecting the content of TNF- α; the data were analyzed using a one-way analysis of variance, a P<0.001, comparing the processing group with the Control group.
Detailed Description
The invention provides an extraction method of artemisia scoparia ethanol extract, which comprises the following steps:
1) Mixing the overground part of the artemisia scoparia with an extraction solvent for alcohol extraction, and removing an alcohol solvent in an obtained alcohol extraction solution to obtain an alcohol extract, wherein the extraction solvent is an ethanol water solution with the volume concentration of 80-90%, and the alcohol extraction temperature is 55-65 ℃;
2) And (3) passing the alcohol extract through an AB-8 macroporous resin column, sequentially carrying out gradient elution by using water and an ethanol water solution with the volume concentration of 30%, and collecting an eluent, wherein the eluent contains the artemisia rupestris alcohol extract.
The invention firstly mixes the overground part of the artemisia rupestris with an extraction solvent for alcohol extraction, removes an alcohol solvent in the obtained alcohol extract to obtain an alcohol extract, wherein the extraction solvent is an ethanol water solution with the volume concentration of 80-90%, and the temperature of the alcohol extraction is 55-65 ℃.
In the invention, the overground part of the artemisia anomala is preferably dried, crushed and sieved, and undersize products are collected to obtain powder; the drying method comprises the steps of drying and naturally airing; the screened aperture is preferably 30 to 50 mesh, more preferably 40 mesh. The source of the overground part of the middle-Asia artemisia selengensis is not particularly limited, and the overground part of the middle-Asia artemisia selengensis which is well known in the field can be adopted.
In the present invention, the ratio of the mass of the aerial parts of artemisia anomala to the volume of the extraction solvent is preferably 1:8 to 12, more preferably 1:10; the volume concentration of the ethanol water solution is preferably 85%; the concentration of the ethanol aqueous solution with the volume concentration of 80-90% is beneficial to extracting high-content active substances with the immune enhancement effect compared with the ethanol aqueous solution with other concentrations. In the present invention, the temperature of the extraction is preferably 60 ℃; preferably, the water bath extraction is carried out in the extraction process so as to ensure constant-temperature extraction; the number of times of extraction is preferably 2 to 4 times, more preferably 3 times; the time for each extraction is preferably 2 to 3 hours.
In the present invention, after the extraction, the extracts are combined, and preferably ethanol is removed by rotary evaporation concentration.
In order to accurately quantify the concentration of the alcohol extract passing through the AB-8 macroporous resin column, it is preferable to include removing water and then dissolving in water again to accurately obtain the concentration of the alcohol extract as a sample solution. The concentration of the ethanol extract loading solution is 60mg/mL.
After the alcohol extract is obtained, the alcohol extract is processed by AB-8 macroporous resin column, water and 30% ethanol water solution with volume concentration are sequentially used for gradient elution, and eluent is collected, wherein the eluent contains the artemisia rupestris alcohol extract.
In the present invention, each gradient preferably elutes 4 column volumes. In the present invention, after collecting the fraction eluted with 30 vol% aqueous ethanol, it is preferable to perform rotary evaporation concentration and then freeze-drying to obtain ethanol-eluted fraction powder. In the present invention, after collecting the components eluted with the mixture of petroleum ether and ethyl acetate in a volume ratio of 1.
The invention also provides the artemisia rupestris L alcohol extract extracted by the extraction method in the scheme. In the invention, the content of polysaccharide in the alcohol extract obtained by the method is determined by adopting a phenol-sulfuric acid method, the content of polysaccharide in the Artemisia scoparia alcohol extract is preferably 24.0-24.5% by mass, the content of flavone is determined by adopting an alkaline method, the content of flavone is preferably 22.5-23.0% by mass, the content of terpenes is determined by adopting a vanillin-glacial acetic acid colorimetric method, and the content of terpenes is preferably 28.0-28.5% by mass.
The invention also provides the artemisia scoparia ethanol extract extracted by the extraction method in the scheme or the application of the artemisia scoparia ethanol extract in preparing the immunopotentiation medicine.
In the invention, the dosage form of the immunity enhancing medicine is preferably an oral dosage form, more preferably a liquid oral dosage form, and the content of the artemisia scoparia ethanol extract in the immunity enhancing medicine is preferably 50-100 mg/mL.
In the present invention, the active substance of the immunopotentiating drug preferably further includes other substances having an enhancer effect. The method for preparing the immunopotentiating drug is not particularly limited, and the method for preparing the immunopotentiating drug known in the art can be adopted.
In the present invention, the immunopotentiating agent preferably includes an agent that promotes dendritic cell maturation and/or regulates dendritic cell function.
In the present invention, the modulation of the dendritic cell function preferably includes: increasing one or more of the expression of surface costimulatory molecules of the dendritic cells, enhancing the expression level of inflammatory cytokines and enhancing the secretion level of inflammatory cytokines. In the present invention, the surface co-stimulatory molecules preferably comprise CD40 and/or CD86. In the present invention, the inflammatory cytokine preferably includes IL-6 and/or TNF-alpha. AAEM obviously improves the expression level of CD40 and CD86 and can improve the level of DC secreting IL-6 and TNF-alpha. The above results indicate that AAEM can promote maturation of DCs.
The invention also provides the application of the artemisia scoparia ethanol extract extracted by the extraction method in the scheme or the application of the artemisia scoparia ethanol extract in preparing a medicine for treating diseases related to tumor and/or inflammatory infection.
In the invention, the tumor preferably comprises human cervical cancer cells, murine cervical cancer cells and liver cancer HepG2 cells. In the present invention, the inflammatory infection-related disease preferably includes immunodeficiency.
In the invention, the treatment of the tumor and/or immunodeficiency related diseases is realized by promoting the maturation of dendritic cells and/or regulating the functions of the dendritic cells by using the artemisia scoparia ethanol extract.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
Example 1
Pulverizing aerial parts of Artemisia scoparia, sieving with a 40-mesh sieve, collecting undersize powder, and adding into 85% ethanol water solution at a material-liquid ratio of 1. Mixing them, placing in 60 deg.C water bath, and leaching at constant temperature for 3 times, each for 2 hr. After extraction, carrying out solid-liquid separation, combining supernatants, carrying out rotary evaporation and concentration until no ethanol smell exists, and freeze-drying to obtain the 85% ethanol extract of the artemisia scoparia. Dissolving 85% ethanol extract of Artemisia scoparia with distilled water, adjusting to obtain 60mg/mL ethanol extract solution, passing through AB-8 macroporous resin column, sequentially eluting with water, 30%, 50%, 70%, 85% ethanol water solution, anhydrous ethanol, etc. in gradient manner, eluting 4 column volumes each, collecting the eluted components with 30% ethanol water solution, concentrating by rotary evaporation, lyophilizing to obtain AAEM, and dissolving with dimethyl sulfoxide (DMSO).
The content of polysaccharide in AAEM is measured by adopting a phenol-sulfuric acid method, the content of polysaccharide is 24.30%, the content of flavone is measured by adopting an alkaline method, the content of flavone is 22.50%, and the content of terpene is measured by adopting a vanillin-glacial acetic acid colorimetric method, and the content of terpene is 28.19%.
Example 2
Pulverizing aerial parts of Artemisia scoparia, sieving with a 50-mesh sieve, collecting undersize powder, adding into 80% ethanol water solution, and mixing at a ratio of material to liquid of 1. Mixing them, placing in 65 deg.C water bath, and leaching at constant temperature for 3 hr for 2 times. After extraction, carrying out solid-liquid separation, combining supernatants, carrying out rotary evaporation and concentration until no ethanol smell exists, and freeze-drying to obtain the artemisia scoparia 80% ethanol extract. Dissolving 80% ethanol extract of Artemisia scoparia with distilled water, adjusting to obtain ethanol extract solution with concentration of 60mg/mL, passing through AB-8 macroporous resin column, sequentially performing gradient elution with water, 30% ethanol aqueous solution with volume concentration, 50%, 70%, 85% ethanol aqueous solution and absolute ethanol, eluting 3 column volumes per gradient, screening to obtain the optimal component of 30% ethanol elution component, collecting the component eluted from 30% ethanol aqueous solution, performing rotary evaporation concentration, lyophilizing to obtain AAEM, and dissolving with dimethyl sulfoxide (DMSO).
The content of polysaccharide in AAEM is measured by adopting a phenol-sulfuric acid method, the content of polysaccharide is 24.26%, the content of flavone is measured by adopting an alkaline method, the content of flavone is 22.42%, and the content of terpene is measured by adopting a vanillin-glacial acetic acid colorimetric method, and the content of terpene is 28.20%.
Example 3
Pulverizing aerial parts of Artemisia scoparia, sieving with a 30-mesh sieve, collecting undersize powder, and adding into 90% ethanol water solution at a ratio of 1. Mixing completely, placing in 55 deg.C water bath, leaching at constant temperature for 3 times, each time for 3 hr. After extraction, carrying out solid-liquid separation, combining supernatants, carrying out rotary evaporation and concentration until no ethanol smell exists, and freeze-drying to obtain the 90% ethanol extract of the artemisia scoparia. Dissolving 90% alcohol extract of Artemisia adamsii with distilled water, adjusting to obtain 60mg/mL alcohol extract solution, passing through AB-8 macroporous resin column, sequentially performing gradient elution with water, 30%, 50%, 70%, 85% ethanol water solution, anhydrous ethanol, etc., each gradient eluting for 4 column volumes, collecting component eluted from 30% ethanol water solution, rotary evaporating for concentration, lyophilizing to obtain AAEM, and dissolving with dimethyl sulfoxide (DMSO).
The content of polysaccharide in AAEM is measured by adopting a phenol-sulfuric acid method, the content of polysaccharide is 24.36%, the content of flavone is measured by adopting an alkaline method, the content of flavone is 22.81%, and the content of terpene is measured by adopting a vanillin-glacial acetic acid colorimetric method, and the content of terpene is 28.26%.
Example 4
After maturation of DCs, the expression of CD40 and CD86 was up-regulated, the DCs cultured until the sixth day (DC positive rate > 80%) were treated with three AAEM-30% of different concentrations of 50, 100, 150. Mu.g/mL, three replicates per set-up, 12h after treatment, cells were collected, centrifuged, and antibodies (FITC-CD 11c, PE-CD40, APC-CD80, PE-CD 86) were added to the pellet, stained, and analyzed using a flow cytometer. After AAEM of the artemisia scoparia alcohol extract prepared in the embodiments 1 to 3 is respectively adopted to treat the DC, the DC is detected by using flow cytometry and a conventional ELISA method, and the promotion effect of the DC on the maturation after the DC is treated by the AAEM is verified. A blank Control group (Control group), an LPS group and a solvent Control group (DMSO group) are also set.
The results show that AAEM significantly enhances the expression of CD40 and CD86 (P < 0.001) (Table 1) and significantly increases the secretion level of inflammatory cytokines IL-6 and TNF-alpha (P < 0.001) (Table 2) compared with the Control group. The above results indicate that AAEM can promote expression of DC surface molecules.
TABLE 1 Effect of AAEM treatment on CD40 and CD86 expression
TABLE 2 Effect of AAEM treatment on the levels of secretion of the inflammatory cytokines IL-6 and TNF- α
The embodiment proves that the artemisia scoparia ethanol extract AAEM prepared by the method can effectively enhance the secretion level of DC surface molecules and inflammatory cytokines. The AAEM can be used as a candidate drug to be applied to the treatment of tumor and inflammatory infection.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and amendments can be made without departing from the principle of the present invention, and these modifications and amendments should also be considered as the protection scope of the present invention.
Claims (8)
1. The artemisia scoparia ethanol extract for enhancing immunity is characterized in that the mass percent of polysaccharide, flavone and terpenes in the artemisia scoparia ethanol extract is 24.0-24.5%, 22.5-23.0% and 28.0-28.5%;
the extraction method of the artemisia anomala ethanol extract comprises the following steps:
1) Mixing the overground part of the artemisia scoparia with an extraction solvent for alcohol extraction, and removing an alcohol solvent in an obtained alcohol extraction solution to obtain an alcohol extract, wherein the extraction solvent is an ethanol water solution with the volume concentration of 80-90%, and the alcohol extraction temperature is 55-65 ℃;
2) Passing the alcohol extract through an AB8 macroporous resin column, sequentially carrying out gradient elution by using water and an ethanol aqueous solution with the volume concentration of 30%, and collecting an eluent which contains the artemisia rupestris alcohol extract; each of the gradients eluted 4 column volumes.
2. The immune enhancing artemisia scoparia alcoholic extract according to claim 1, wherein the ratio of the mass of the aerial parts of the artemisia scoparia to the volume of the extraction solvent in the step 1) of the extraction step is 1:8 to 12; the extraction times are 2-4.
3. Use of an immune enhancing artemisia scoparia ethanol extract as claimed in claim 1 or 2 in the preparation of an immune enhancing medicament.
4. The use of claim 3, wherein the immune enhancing drug comprises a drug that promotes dendritic cell maturation and/or modulates dendritic cell function.
5. The use of claim 4, wherein said modulating dendritic cell function comprises: increasing one or more of the expression of surface costimulatory molecules of dendritic cells and enhancing the secretion level of inflammatory cytokines.
6. The use of claim 5, wherein the surface co-stimulatory molecules comprise CD40 and/or CD86.
7. The use according to claim 5, wherein said inflammatory cytokines comprise IL-6 and/or TNF- α.
8. Use of an immune enhancing Artemisia scoparia alcoholic extract of claim 1 or 2 in the preparation of a medicament for treating diseases associated with tumor and/or inflammatory infection.
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| Title |
|---|
| "Effects of Artemisia Absinthium L. Extract on the Maturation and Function of Dendritic Cells";Mojtaba Shahnazi et al;《Jundishapur J Nat Pharm Prod》;20150520;第10卷(第2期);1-6 * |
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| CN114191459A (en) | 2022-03-18 |
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