CN115813984A - Extraction method of alkaloid in Xuanhuanglian, alkaloid extract and application and pharmaceutical composition thereof - Google Patents
Extraction method of alkaloid in Xuanhuanglian, alkaloid extract and application and pharmaceutical composition thereof Download PDFInfo
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- CN115813984A CN115813984A CN202211719115.7A CN202211719115A CN115813984A CN 115813984 A CN115813984 A CN 115813984A CN 202211719115 A CN202211719115 A CN 202211719115A CN 115813984 A CN115813984 A CN 115813984A
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- alkaloid
- ethyl acetate
- extraction
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Medicines Containing Plant Substances (AREA)
Abstract
The invention provides an extraction method of alkaloid in Xuan Huang Lian, an alkaloid extract, application thereof and a pharmaceutical composition, and relates to the technical field of medicines. The invention carries out alcohol extraction on the rhizome of the Coptis chinensis Franch, concentrates and dries the obtained extracting solution to obtain a crude extract; mixing the crude extract with water, adjusting the pH value of the obtained aqueous solution of the crude extract to 2-4, and then extracting by using petroleum ether to obtain a petroleum ether extract and a water layer; adjusting the pH value of the water layer to 8-10, and extracting by using ethyl acetate to obtain an ethyl acetate extract and a water layer; and concentrating the ethyl acetate extract to obtain the total alkaloid extract of the Chinese goldthread. Further, dissolving the total alkaloid extract of the radix et rhizoma Rhei Lian, and separating by high performance liquid chromatography to obtain six-stage alkaloid extract. The invention can realize the rapid and efficient enrichment of the alkaloid in the Xuan Huang Lian, and the enriched alkaloid has strong anti-inflammatory activity, and can be used for developing the medicine for preventing or treating inflammatory diseases.
Description
Technical Field
The invention relates to the technical field of medicines, and particularly relates to an extraction method of alkaloid in Xuan Huang Lian, an alkaloid extract, application thereof and a pharmaceutical composition.
Background
The Coptis chinensis Franch is a medicinal plant of Coptis, the corresponding variety is Coptis brevifolia, and the medicinal part is a rhizome; because it takes the Xuan Zhou area of Anhui province as the genuine producing area, it is called Xuan Huang Lian and mainly distributed in the southern Anhui mountainous area. The rhizome of Xuan Huang Lian is "like Lian Zhu" in shape, and is good for hard and solid food, and has the actions of cold and cool without stagnation, cold and dry nature, and can reduce fire and eliminate dampness to check diarrhea and dysentery, so it is the most important herb for treating dysentery. At present, the research on the chemical components and the pharmacodynamic substance basis of the Xuan Huang Lian is lacked, the effective extraction of the pharmacodynamic components of the Xuan Huang Lian is not realized, and the problems of poor enrichment effect, few effective components, incapability of fully exerting the medicinal value and the like of the existing extraction of other Coptis plants generally exist.
Disclosure of Invention
In view of the above, the present invention aims to provide an extraction method of alkaloids in rhizoma coptidis from rhizoma coptidis, an alkaloid extract, and applications and pharmaceutical compositions thereof. The extraction method provided by the invention can efficiently enrich the alkaloid in the Xuan Huang Lian, and the enriched alkaloid has obvious anti-inflammatory activity.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for extracting alkaloid from Xuan Huang Lian, which comprises the following steps:
extracting the rhizome of the Coptis chinensis Franch with alcohol, and concentrating the obtained extract to obtain a crude extract;
mixing the crude extract with water, adjusting the pH value of the obtained aqueous solution of the crude extract to 2-4, and extracting by adopting petroleum ether to respectively obtain a petroleum ether extract and a water layer;
adjusting the pH value of the water layer to 8-10, and extracting by using ethyl acetate to obtain an ethyl acetate extract and a water layer respectively;
and concentrating the ethyl acetate extract to obtain the total alkaloid extract of the Chinese goldthread.
Preferably, the method further comprises the following steps:
dissolving the Xuanhuanglian total alkaloid extract to obtain a Xuanhuanglian total alkaloid solution;
and (2) carrying out high performance liquid chromatography separation on the Xuanhuang total alkaloid solution, wherein the conditions of the high performance liquid chromatography separation comprise: the mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is methanol, the mobile phase B is an acetic acid aqueous solution, and the volume fraction of acetic acid in the acetic acid aqueous solution is 0.2-0.5%; gradient elution; the chromatographic column is a semi-preparative phenethyl chromatographic column;
the elution procedure of the gradient elution is 0 to 60min, the volume ratio of the methanol to the acetic acid aqueous solution is from 40 to 100, and the separation liquid of the methanol to the acetic acid aqueous solution is from 40 to 45;
preferably, the alcohol reagent adopted by the alcohol extraction is methanol or ethanol water solution, the volume fraction of ethanol in the ethanol water solution is 70-95%, and the dosage ratio of the rhizome of the coptis chinensis to the alcohol reagent is 1g.
Preferably, the alcohol extraction is performed under the conditions of heating reflux or ultrasound; the times of alcohol extraction are 1-3, and the time of single alcohol extraction is 20-30 min.
Preferably, the volume ratio of the crude extract aqueous solution to petroleum ether is 1; the number of times of petroleum ether extraction is 2-5.
Preferably, the volume ratio of the water layer to ethyl acetate is 1; the extraction times of the ethyl acetate are 2-5 times.
Preferably, the column temperature of the high performance liquid chromatography is 25 ℃, and the flow rate of the mobile phase is 3mL/min.
The invention provides the alkaloid extract obtained by the extraction method in the technical scheme.
The invention provides application of the alkaloid extract in the technical scheme in preparing a medicament for preventing or treating inflammatory diseases.
The invention also provides a pharmaceutical composition, which comprises alkaloid, pharmaceutically acceptable salt thereof, auxiliary materials, diluents and carriers; the alkaloid is the alkaloid extract in the technical scheme.
The invention provides a method for extracting alkaloid from Xuan Huang Lian, which comprises the steps of carrying out alcohol extraction on rhizome of Xuan Huang Lian, concentrating and drying the obtained extract to obtain a crude extract; mixing the crude extract with water, adjusting the pH value of the obtained aqueous solution of the crude extract to 2-4, and extracting by using petroleum ether to respectively obtain a petroleum ether extract and a water layer; adjusting the pH value of the water layer to 8-10, and extracting by using ethyl acetate to obtain an ethyl acetate extract and a water layer respectively; and concentrating the ethyl acetate extract to obtain the total alkaloid extract of the Chinese goldthread. Further, dissolving the total alkaloid extract of the radix et rhizoma Rhei Lian to obtain a total alkaloid solution of the radix et rhizoma Rhei Lian; and (3) carrying out high performance liquid chromatography separation on the total alkaloid solution of the Chinese goldthread to obtain six sections of alkaloid extracts which are respectively marked as a Fr.1 alkaloid extract, a Fr.2 alkaloid extract, a Fr.3 alkaloid extract, a Fr.4 alkaloid extract, a Fr.5 alkaloid extract and a Fr.6 alkaloid extract. The method adopts the acid-extraction-alkali-precipitation method to extract the Xuan coptis chinensis, can realize the rapid and efficient enrichment of alkaloids in the Xuan coptis chinensis, and the types of the enriched alkaloids are 31.
The alkaloid provided by the invention has strong anti-inflammatory activity, and especially has strong anti-inflammatory activity of Fr.1 alkaloid extract, fr.4 alkaloid extract, fr.5 alkaloid extract and Fr.6 alkaloid extract. The alkaloid extract provided by the invention can be used for developing a medicament for preventing or treating inflammatory diseases.
Drawings
FIG. 1 is a TIC diagram of Fr.1 corresponding UPLC-QTOF-MS/MS positive ion mode;
FIG. 2 is a TIC chart of UPLC-QTOF-MS/MS positive ion mode corresponding to Fr.2;
FIG. 3 is a TIC diagram of a UPLC-QTOF-MS/MS positive ion mode corresponding to Fr.3;
FIG. 4 is a TIC chart of UPLC-QTOF-MS/MS positive ion mode corresponding to Fr.4;
FIG. 5 is a TIC chart of Fr.5 corresponding UPLC-QTOF-MS/MS positive ion mode;
FIG. 6 is a TIC chart of UPLC-QTOF-MS/MS positive ion mode corresponding to Fr.6;
FIG. 7 is a diagram of the effect of berberine at different concentrations on proteins ERK1/2, TLR4 and NLRP3, in FIG. 7, (a) is a diagram of Westernblot effect of berberine (BBR) on proteins ERK1/2, TLR4 and NLRP3, (b) is a diagram of the effect of berberine at different concentrations on proteins ERK1/2, (c) is a diagram of the effect of berberine at different concentrations on protein TLR4, and (d) is a diagram of the effect of berberine at different concentrations on protein NLRP 3;
FIG. 8 is a graph showing the effects of sanguinarine and berberine oxide on NF-. Kappa.B and NLRP3 at different concentrations, and in FIG. 8, (a) is a graph showing the effects of sanguinarine and berberine oxide on NF-. Kappa.B and (B) is a graph showing the effects of sanguinarine and berberine oxide on NLRP3 at different concentrations;
FIG. 9 is a graph showing the effects of sanguinarine and berberine oxide on protein ERK1/2 and TLR4 at different concentrations, in FIG. 9, (a) is a graph showing the effects of sanguinarine and berberine oxide on protein ERK1/2 at different concentrations, and (b) is a graph showing the effects of sanguinarine and berberine oxide on protein TLR4 at different concentrations.
Detailed Description
The invention provides a method for extracting alkaloid from Xuan Huang Lian, which comprises the following steps:
extracting the rhizome of the Coptis chinensis Franch with alcohol, and concentrating the obtained extract to obtain a crude extract;
mixing the crude extract with water, adjusting the pH value of the obtained aqueous solution of the crude extract to 2-4, and extracting by using petroleum ether to respectively obtain a petroleum ether extract and a water layer;
adjusting the pH value of the water layer to 8-10, and extracting by using ethyl acetate to respectively obtain an ethyl acetate extract and a water layer;
and concentrating the ethyl acetate extract to obtain the total alkaloid extract of the Chinese goldthread.
The invention carries out alcohol extraction on the rhizome of the Xuan Huang Lian, and concentrates the obtained extract to obtain a crude extract. In advance of the alcohol, the rhizome of the coptis chinensis is preferably dried in the invention. In the invention, the alcohol reagent adopted by the alcohol extraction is preferably methanol or ethanol water solution, and the mass fraction of ethanol in the ethanol water solution is preferably 70-95%, and more preferably 75-85%; the dosage ratio of the rhizome of the Coptis chinensis Franch to the alcohol reagent is 1g. In the present invention, the alcohol extraction is preferably performed under heating reflux or ultrasonic conditions; the number of times of alcohol extraction is preferably 1-3, more preferably 2, and the time of single alcohol extraction is preferably 20-30 min, more preferably 30min; in the invention, the rhizome of the coptis chinensis is preferably soaked in the alcohol reagent for 20-60 min, and then the alcohol extraction is carried out. In the present invention, the concentration method is preferably distillation under reduced pressure, and the distillation under reduced pressure is based on concentration to dryness.
After the crude extract is obtained, the crude extract is mixed with water, the pH value of the aqueous solution of the crude extract is adjusted to 2-4, and petroleum ether is used for extraction to respectively obtain a petroleum ether extract and an aqueous layer. The invention has no special requirement on the dosage of the water, and the crude extract can be dissolved. In the present invention, the reagent used for adjusting the pH is preferably hydrochloric acid, and the pH of the aqueous solution of the crude extract is preferably adjusted to 2.5 to 3.5. In the present invention, the volume ratio of the crude extract aqueous solution to petroleum ether is preferably 1; the number of times of the petroleum ether extraction is preferably 2 to 5 times, and more preferably 2 to 3 times. The invention adjusts the pH value of the crude extract aqueous solution to 2-4, and then adopts petroleum ether to extract (namely, an acid extraction method), so that the components with fat solubility and smaller polarity in the crude extract can be removed. In the invention, the petroleum ether extract can recover the petroleum ether by vacuum distillation.
After the water layer is obtained, the pH value of the water layer is adjusted to 8-10, and ethyl acetate is adopted for extraction to respectively obtain an ethyl acetate extract and the water layer. In the invention, the reagent for adjusting the pH value is preferably ammonia water; the pH of the aqueous layer is preferably adjusted to 8.5 to 9.5. In the present invention, the volume ratio of the water layer to ethyl acetate is preferably 1; the number of times of ethyl acetate extraction is preferably 2 to 5 times, more preferably 2 to 3 times, and ethyl acetate extraction layers obtained by the multiple times of ethyl acetate extraction are combined. According to the invention, after the pH value of the water layer is adjusted to 8-10, ethyl acetate is adopted for extraction (namely an alkali precipitation method), so that isoquinoline alkaloid components with strong alkalinity can be effectively enriched.
After an ethyl acetate extract is obtained, the ethyl acetate extract is concentrated to obtain the total alkaloid extract of the Chinese goldthread. In the present invention, the concentration method is preferably distillation under reduced pressure, and ethyl acetate can be recovered by the distillation under reduced pressure.
After the total alkaloid extract of the Chinese goldthread is obtained, the invention also preferably selects: dissolving the total alkaloid extract of the Chinese goldthread to obtain a total alkaloid solution of the Chinese goldthread;
and (2) carrying out high performance liquid chromatography separation on the Xuanhuang total alkaloid solution, wherein the conditions of the high performance liquid chromatography separation comprise: the mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is methanol, the mobile phase B is an acetic acid aqueous solution, and the volume fraction of acetic acid in the acetic acid aqueous solution is 0.2-0.5%; gradient elution; the chromatographic column is a semi-preparative phenethyl chromatographic column;
the elution procedure of the gradient elution is 0 to 60min, the volume ratio of the methanol to the acetic acid aqueous solution is from 40 to 100, and the separation liquid of the methanol to the acetic acid aqueous solution is from 40 to 45.
In the invention, the reagent adopted for dissolving is preferably methanol, and the invention has no special requirement on the dosage of the methanol and can dissolve the total alkaloid extract of the radix et rhizoma Rhei Lian. After the rhizoma coptidis total alkaloid extract is dissolved, the invention also preferably filters the obtained rhizoma coptidis total alkaloid solution with a filter membrane.
In the present invention, the high performance liquid chromatography separation is preferably performed using a preparative HPLC-DAD instrument; the semi-preparative phenethyl chromatography column preferably has a specification of 10X 250mm. In the present invention, the volume fraction of acetic acid in the aqueous acetic acid solution is preferably 0.2 to 0.3%; during the gradient elution, the column temperature of the high performance liquid chromatography is preferably 25 ℃, and the flow rate of the mobile phase is preferably 3mL/min.
The extraction method provided by the invention can realize rapid and efficient enrichment of alkaloid in the Xuanhuanglian, and has strong operability.
The invention provides an alkaloid extract obtained by the extraction method in the technical scheme, wherein the alkaloid extract is the total alkaloid extract of the Chinese goldthread, and further comprises a Fr.1 alkaloid extract, a Fr.2 alkaloid extract, a Fr.3 alkaloid extract, a Fr.4 alkaloid extract, a Fr.5 alkaloid extract and a Fr.6 alkaloid extract. The invention adopts UPLC-QTOF-MS/MS technology to perform qualitative analysis on Fr.1-Fr.6 alkaloid extracts, and the analysis results Fr.1-Fr.6 alkaloid extracts totally contain 31 kinds of isoquinoline type alkaloids, wherein the alkaloid contained in the Fr.1 alkaloid extracts is compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, compound 7, compound 8, compound 9, compound 10, compound 11, compound 13, compound 14, compound 15, compound 16, compound 17 and compound 18; fr.2 alkaloid extracts include alkaloids of compound 3, compound 4, compound 5, compound 6, compound 7, compound 8, compound 9, compound 10, compound 16, compound 17, compound 18, compound 21, compound 22, and compound 24; fr.3 alkaloid extract comprises alkaloids of compound 5, compound 6, compound 8, compound 9, compound 16, compound 17, compound 18, compound 21, compound 22, and compound 26; fr.4 alkaloid extracts include alkaloids of compound 9, compound 14, compound 16, compound 17, compound 18, compound 26, compound 27, compound 28, and compound 29; fr.5 alkaloid extract includingThe base is compound 6, compound 8, compound 9, compound 14, compound 16, compound 17, compound 18, compound 24, compound 26, compound 27, compound 29, and compound 31; fr.6 alkaloid extracts include alkaloids of compound 10, compound 12, compound 19, compound 20, compound 23, compound 24, compound 25, compound 26, and compound 30. The chemical formulas of the compounds 1 to 31 are C in sequence 20 H 24 NO 4 、C 19 H 18 NO 4 、C 20 H 17 NO 6 、C 19 H 16 NO 4 、C 20 H 17 NO 7 、C 20 H 20 NO 4 、C 20 H 18 NO 4 、C 20 H 20 NO 4 、C 19 H 14 NO 4 、C 20 H 17 NO 5 、C 19 H 16 N 3 O、C 21 H 21 NO 6 、C 20 H 21 NO 4 、C 20 H 17 NO 5 、C 20 H 14 NO 4 、C 20 H 16 NO 4 、C 21 H 22 NO 4 、C 20 H 18 NO 4 、C 20 H 17 NO 5 、C 19 H 15 NO 5 、C 19 H 13 NO 5 、C 18 H 19 NO 4 、C 18 H 19 NO 4 、C 19 H 12 NO 6 、C 19 H 14 NO 5 、C 19 H 17 NO 5 、C 21 H 20 NO 4 、C 18 H 15 NO 6 、C 19 H 16 NO 4 、C 21 H 17 NO 7 、C 10 H 9 NO 3 . The invention evaluates the anti-inflammatory activity of total alkaloid extract of the Chinese goldthread, fr.1 alkaloid extract, fr.2 alkaloid extract, fr.3 alkaloid extract, fr.4 alkaloid extract, fr.5 alkaloid extract and Fr.6 alkaloid extract, and evaluates 14 isoquinoline alkaloids in the total alkaloid extract, fr.1 alkaloid extract, fr.2 alkaloid extract, fr.3 alkaloid extract, fr.4 alkaloid extract, fr.5 alkaloid extract and Fr.6 alkaloid extractSeparate anti-inflammatory activity evaluations were performed for compound 1 (magnoflorine), compound 4 (gladioxin), compound 6 (african tetrandrine), compound 7 (epiberberine), compound 8 (jateorhizine), compound 9 (coptisine), compound 11 (dehydroevodiamine), compound 13 (tetrahydroberberine), compound 15 (sanguinarine), compound 16 (methyl coptisine), compound 17 (palmatine), compound 18 (berberine), compound 19 (oxidized berberine), and compound 29 (berrubine), respectively. The anti-inflammatory activity comprises the inhibition of the expression levels of NO, IL-6 and TNF-alpha, and the down regulation of the expression of ERK1/2, TLR4, NF-kappa B and NLRP3 induced by LPS (lipopolysaccharide), and the evaluation shows that the alkaloid extract has good anti-inflammatory activity. According to the invention, LPS is used for stimulating a RAW264.7 cell inflammation model for the first time, and the anti-inflammatory effects of the total alkaloid extract of the Chinese goldthread, the alkaloid extracts at Fr.1-Fr.6 stages and the 14 isoquinoline alkaloids are evaluated; the result shows that the alkaloid extract of the Chinese goldthread has strong anti-inflammatory activity, the components with strong anti-inflammatory activity are Fr.1, fr.4, fr.5 and Fr.6, and the components with strong anti-inflammatory activity in 14 kinds of isoquinoline alkaloids are sanguinarine, berberine oxide, tetrahydroberberine, methyl coptisine, berberine, dehydroevodiamine and coptisine. The invention adopts UPLC-QTOF-MS/MS technology to carry out qualitative analysis on alkaloid components in the Fr.1-Fr.6 sections, and is beneficial to searching medicinal resources capable of replacing Xuanhuanglian according to the analysis result.
The invention provides application of the alkaloid in the technical scheme in preparing a medicament for preventing or treating inflammatory diseases.
The invention also provides a pharmaceutical composition, which comprises alkaloid, pharmaceutically acceptable salt thereof, auxiliary materials, diluent and carrier; the alkaloid is the alkaloid extract in the technical scheme. The invention has no special requirements on the auxiliary materials, the diluents and the carriers, and can adopt corresponding materials well known by the technical personnel in the field; the dosage form of the pharmaceutical composition of the present invention is not particularly limited, and may be those well known to those skilled in the art.
The following examples are provided to illustrate the methods for extracting alkaloids from xuan coptidis rhizoma, the alkaloid extracts, the application and the pharmaceutical compositions thereof in detail, but they should not be construed as limiting the scope of the present invention.
Example 1
The method for extracting the Xuan Huang Lian comprises the following steps:
(1) Soaking dried rhizome 25g of radix et rhizoma Rhei Coptis in 75% (volume fraction) ethanol water solution (250 mL) for 20min, heating and reflux-extracting for 2 times, and recovering extractive solution under reduced pressure to obtain crude extract of radix et rhizoma Rhei Coptis;
(2) Dissolving the crude extract obtained in the step (1) with water, adjusting the pH value to 2.5 with hydrochloric acid, and extracting for 2 times with petroleum ether of the same volume to obtain a petroleum ether extract and a water layer;
(3) Adjusting the pH value of the water layer obtained in the step (2) to 8.5 by using ammonia water, and extracting for 2 times by using ethyl acetate with the same volume to obtain an ethyl acetate extract and a water layer;
(4) Distilling the ethyl acetate extract obtained in the step (3) under reduced pressure, concentrating and recovering ethyl acetate to obtain a total alkaloid extract of the radix et rhizoma Rhei; dissolving the total alkaloid extract of the Coptis chinensis Franch with appropriate amount of methanol, separating with preparative HPLC-DAD instrument, wherein the chromatographic column is semi-preparative phenethyl chromatographic column, the mobile phase is methanol and acetic acid aqueous solution (acetic acid volume fraction in acetic acid aqueous solution is 0.2%), gradient eluting, and eluting procedure: 0-60 min, the volume ratio of methanol to acetic acid aqueous solution is 40-100, and the detection is carried out at 345nm, the column temperature is 25 ℃, and the flow rate is 3mL/min. According to chromatographic peaks, the rhizoma coptidis phenylethyl column segmented samples Fr.1-Fr.6 alkaloid extracts are obtained (the volume ratio of the Fr.1-Fr.6 alkaloid extracts to methanol and acetic acid aqueous solution is respectively from 40 to 55, 45.
(5) Qualitative analysis is carried out on the alkaloids in the phenylethyl column segmented sample Fr.1-Fr.6 obtained in the step (4) by using an UPLC-QTOF-MS/MS technology, the TIC graphs of UPLC-QTOF-MS/MS positive ion modes corresponding to the Fr.1-Fr.6 alkaloid extracts are respectively shown in figures 1-6, and 31 alkaloid compounds (shown in Table 1) are identified, wherein 14 components are verified by a reference substance.
TABLE 1UPLC-QTOF-MS method for identifying alkaloid information (Fr.1-Fr.6) in Xuan Huang Lian
Note: compounds marked with an x are confirmed in comparison to the control.
(6) The comparison of the reference substances proves that each component separated by the phenylethyl column in the step (5) has 14 isoquinoline alkaloid components which are palmatine (17), coptisine (9), epiberberine (7), jateorhizine (8), african tetrandrine (6), gladioxin (4), magnoflorine (1), berberine (18), methyl coptisine (16), berberberrubine (29), tetrahydroberberine (13), dehydroevodiamine (11), sanguinarine (15) and berberine oxide (19) in Fr.1-Fr.6.
Example 2
The method for extracting the Xuan Huang Lian comprises the following steps:
(1) Soaking dried rhizome of Coptis chinensis Franch 20g in 70% (volume fraction) ethanol (300 mL) for 30min, heating and reflux-extracting for 2 times, and recovering extractive solution under reduced pressure to obtain crude extract of Coptis chinensis Franch;
(2) Dissolving the crude extract obtained in the step (1) with water, adjusting the pH value to 3 with hydrochloric acid, and extracting for 2 times by using petroleum ether with the same volume to obtain a petroleum ether extract and a water layer;
(3) Adjusting the pH value of the water layer obtained in the step (2) to 9 by using ammonia water, and extracting for 2 times by using ethyl acetate with the same volume to obtain an ethyl acetate extract and a water layer;
(4) Separating the ethyl acetate extract (the total alkaloid extract of the Chinese goldthread) obtained in the step (3) by a preparative HPLC-DAD instrument, wherein a chromatographic column is a semi-preparative phenethyl chromatographic column, a mobile phase is methanol and acetic acid aqueous solution (the volume fraction of acetic acid in the acetic acid aqueous solution is 0.3 percent), and performing gradient elution and an elution program: 0-60 min, the volume ratio of methanol to acetic acid aqueous solution is 45-90, the detection is carried out at 345nm, the column temperature is 25 ℃, and the flow rate is 3mL/min. Obtaining alkaloid extracts of segmented samples Fr.1-Fr.6 of the Xuan Huang Lian phenylethyl column according to chromatographic peaks;
(5) And (4) qualitatively analyzing the alkaloid in the phenylethyl column segmented sample Fr.1-Fr.6 extract obtained in the step (4) by using an UPLC-QTOF-MS/MS technology, wherein the TIC graphs of positive ion modes corresponding to Fr.1-Fr.6 are the same as those of example 1.
(6) And (3) in the step (5), the phenethyl column segments samples, and the 14 identified isoquinoline alkaloid components are the same as in example 1 through comparison and verification of a reference substance.
Example 3
The method for extracting the Xuanhuanglian comprises the following steps:
(1) Soaking dried rhizome of Coptis chinensis Franch 30g in 80% (volume fraction) ethanol (dosage is 450 mL) for 45min, ultrasonically extracting for 3 times, and recovering extractive solution under reduced pressure to obtain crude extract of Coptis chinensis Franch;
(2) Dissolving the crude extract obtained in the step (1) with water, adjusting the pH value to 3.5 with hydrochloric acid, and extracting for 3 times with isovolumetric petroleum ether to obtain a petroleum ether extract and a water layer;
(3) Adjusting the pH value of the water layer obtained in the step (2) to 9.5 by using ammonia water, and extracting for 3 times by using ethyl acetate with the same volume to obtain an ethyl acetate extract and a water layer;
(4) Separating the ethyl acetate extract (the total alkaloids of the Chinese goldthread) obtained in the step (3) by a preparative HPLC-DAD instrument, wherein a chromatographic column is a semi-preparative phenethyl chromatographic column, a mobile phase is methanol and acetic acid aqueous solution (the volume fraction of acetic acid in the acetic acid aqueous solution is 0.4 percent), and performing gradient elution and an elution program: 0-60 min, the volume ratio of methanol to acetic acid aqueous solution is 40-80, the detection is carried out at 345nm, the column temperature is 25 ℃, and the flow rate is 3mL/min. And obtaining alkaloid extracts of segmented samples Fr.1-Fr.6 of the Xuan Huang Lian phenylethyl column according to chromatographic peaks.
(5) And (5) qualitatively analyzing the alkaloid in the phenylethyl column segmented sample Fr.1-Fr.6 extract obtained in the step (4) by using an UPLC-QTOF-MS/MS technology, wherein the TIC graphs of positive ion modes corresponding to Fr.1-Fr.6 are the same as those in example 1.
(6) In each segmented sample of the phenylethyl column in the step (5), 14 isoquinoline alkaloid components are identified by comparison and verification of a reference substance, which is the same as that in the example 1.
Example 4
The method for extracting the Xuan Huang Lian comprises the following steps:
(1) Soaking dried rhizome of Coptis chinensis Franch 15g in 95% (volume fraction) ethanol (dosage is 450 mL) for 1h, heating and reflux-extracting for 2 times, and recovering extractive solution under reduced pressure to obtain crude extract of Coptis chinensis Franch;
(2) Dissolving the crude extract obtained in the step (1) with water, adjusting the pH value to 2 with hydrochloric acid, and extracting for 2 times by using petroleum ether with the same volume to obtain a petroleum ether extract and a water layer;
(3) Adjusting the pH value of the water layer obtained in the step (2) to 9 by using ammonia water, and extracting for 2 times by using ethyl acetate with the same volume to obtain an ethyl acetate extract and a water layer;
(4) And (3) carrying out HPLC analysis and separation on the ethyl acetate extract (the total alkaloid extract of the Chinese goldthread) obtained in the step (3), and carrying out separation by using a preparative HPLC-DAD instrument, wherein a chromatographic column is a semi-preparative phenethyl chromatographic column, a mobile phase is methanol and acetic acid aqueous solution (the volume fraction of acetic acid in the acetic acid aqueous solution is 0.5%), gradient elution is carried out, and an elution program is as follows: 0-60 min, the volume ratio of methanol to acetic acid aqueous solution is 40-95, the detection is carried out at 345nm, the column temperature is 25 ℃, and the flow rate is 3mL/min. And obtaining segmented samples Fr.1-Fr.6 of the Xuan Huanglian phenylethyl column according to chromatographic peaks.
(5) And (4) qualitatively analyzing the alkaloid in the phenylethyl column segmented sample Fr.1-Fr.6 extract obtained in the step (4) by using an UPLC-QTOF-MS/MS technology, wherein the TIC graphs of positive ion modes corresponding to Fr.1-Fr.6 are the same as those of example 1.
(6) And (3) comparing and verifying the sectional samples of the phenethyl column in the step (5) by using a reference substance, and identifying 14 isoquinoline alkaloid components as in example 1.
The anti-inflammatory action and mechanism of the total alkaloid extract of the Chinese goldthread from example 1 to 4, the Fr.1 to Fr.6 alkaloid extract separated from the phenethyl column and the 14 confirmed isoquinoline alkaloids are characterized by the following steps:
(1) RAW264.7 cell culture
Cells were assayed in DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin (diabody) at 37 ℃ with 5% CO 2 Under the environment of。
(2) MTT method for detecting influence of Xuanhuang alkaloid on cell proliferation
The culture medium for RAW264.7 cells was prepared to 5X 10 5 Single cell suspension per mL, cultured in 96-well plates at 200 μ Ι _ per well, 2 replicates per concentration; after incubation for 24h, respectively adding 6.25, 12.5, 25, 50 and 100 mug/mL final concentration total alkaloid samples of the Xuan Huang Lian, xuan Huang Lian phenethyl column segmented samples (Fr.1-Fr.6) and 14 kinds of isoquinoline alkaloids contained in the Xuan Huang Lian such as berberine and berberine oxide and taking resveratrol as positive control drugs. After further incubation for 24h, 20. Mu.L MTT (5 mg/mL) was added to each well, after 4h, 150. Mu.L supernatant was removed, 150. Mu.L DMSO was added, shaking was carried out for 5min, and OD was measured at a wavelength of 570nm in a microplate reader to reflect cell viability.
Cell viability (%) = (test well OD mean/blank well OD mean) × 100%
(3) Determination of levels of NO, IL-6 and TNF-alpha in supernatants
Subculturing RAW264.7 cells, and adjusting cell concentration to 1 × 10 with DMEM high sugar medium 5 Cells per well. The cells are inoculated in a 96-well culture plate, 200 mu L of the cells are cultured and incubated for 24h, then the drugs are added into the culture plate, 1 mu L of the drugs are added after being dissolved by DMSO, (the final concentration is 100, 50, 25, 12.5 and 6.25 mu g/mL), 1 mu L of LPS (the final concentration is 1 mu g/mL) is added after 1h, administration wells, blank wells (only adding culture medium), model wells (only adding LPS without adding drugs for stimulation culture) and resveratrol positive control wells are arranged, and the cells are cultured for 24h at 37 ℃ in an incubator. After further incubation for 24h, the supernatant was taken. NO is measured by detecting its stable oxidative metabolite nitrite. mu.L of the medium was mixed with an equal volume of Griess reagent, left at room temperature for 10min, and the absorbance was measured at 570 nm. Nitrite concentration was determined using a calibration curve of sodium nitrite standard solution. The concentrations of IL-6 and TNF- α in the cell culture supernatants were determined using a mouse IL-6ELISA kit and a mouse TNF- α ELISA kit, respectively, according to the instructions.
(4) Western Blot method for detecting effects of isoquinoline alkaloid compounds berberine, oxidized berberine, methyl coptisine, sanguinarine and dehydroevodiamine on NLRP3, TLR4, NF-kappa B and ERK1/2 four proteins
According to a ratio of 3.2X 10 per culture dish 6 The RAW264.7 cells were seeded at individual density in a culture dish and incubated overnight, followed by addition of 1, 2.5, and 5. Mu. Mol. L, respectively -1 Berberine was pre-treated for 1h, dmso was used as a blank control. Adding 1. Mu.g/mL -1 LPS stimulation 24h, westernblot assay, cells were washed with cold PBS, blotted clean of PBS, placed on ice, and lysed in RIPA lysis buffer with protease inhibitors added for 30min. Protein concentration was determined for each sample using the BCA protein assay kit. Equivalent amounts of denatured proteins were electrophoresed through 7.5% SDS-PAGE, transferred to PVDF membrane, blocked with 5% skimmed milk powder in TBST for 2h, and after blocking, incubated with antibodies to ERK1/2, TLR-4, NF-. Kappa.B, NLRP3 and GAPDH (1. The TBST washed membrane was incubated with HRP-labeled goat anti-rabbit or goat anti-mouse antibody for 2h at room temperature. TBST washing was followed by detection using a chemiluminescent detection system according to the instructions and quantitation was performed using GAPDH as an internal control protein.
(5) Statistical analysis
All statistical calculations were performed using SPSS 25.0 software and data are presented as mean ± standard error of three independent experiments. Data analysis employed one-way analysis of variance followed by post hoc testing using Ponflorentb. P<0.05 was considered to have a significant difference. Making histograms and computing ICs 50 GraphPad Prism 8 software was used.
(6) The experimental results are as follows:
(1) cell proliferation experimental results (shown in table 2) show that, under the test concentration, the total alkaloid extract of the Chinese goldthread, the components (Fr.1-Fr.6 alkaloid extract) of the Chinese goldthread, and isoquinoline alkaloids and resveratrol (positive control) in 14 Chinese goldthread such as berberine and berberine oxide have no cytotoxicity to RAW264.7 cells.
TABLE 2 safe dosing concentration range
(2) According to the result of an NO release inhibition experiment (shown in table 3), the NO inhibition effect of the total alkaloid extract of the Chinese goldthread is found to be remarkable for the first time, the 6 components of the Fr.1-Fr.6 alkaloid extract have stronger effects of Fr.1, fr.4, fr.5 and Fr.6, isoquinoline alkaloids exist in the components, and sanguinarine, berberine oxide, tetrahydroberberine, methylprednisolide, berberine, dehydroevodiamine and coptisine have stronger NO inhibition effects, and the inhibition effect of the sanguinarine is stronger than that of resveratrol (positive control).
TABLE 3 inhibition of NO in LPS-induced RAW264.7 cells by Xuan Huang Lian alkaloid IC 50 Value of
Note: "-" indicates that the inhibition ratio is too low to calculate IC 50 Value, or IC 50 The value is too large.
(3) The effect of the coptis alkaloid on the release of IL-6 and TNF-alpha in LPS-induced RAW264.7 cells is shown in Table 4. Compared with a model group, the contents of TNF-alpha and IL-6 in the rhizoma coptidis total alkaloid extract, fr.4, fr.5, fr.6, berberine, methyl coptisine and sanguinarine in each administration group are obviously reduced, and the anti-inflammatory effect of the compound sanguinarine is found to be superior to that of a positive medicine resveratrol for the first time.
TABLE 4 Effect of Coptis alkaloid on LPS-induced IL-6 and TNF- α release in RAW264.7 cells ((S))
n＝3)
Note: p <0.05, P <0.01, compared to model group
(4) Berberine (BBR) is added at 2.5 mu M,At the concentration of 5 mu M, the upregulation of ERK1/2, TLR4 and NLRP3 induced by LPS can be remarkably reversed, and the dose dependence is realized; while berberine with concentration of 1 μ M has no influence on protein expression of ERK1/2, TLR4 and NLRP3 induced by LPS. Berberine shows an inhibitory effect on LPS-induced RAW264.7 cell inflammation by down-regulating ERK1/2, TLR4 and NLRP3 levels. FIG. 7 is a graph of the effect of berberine at different concentrations on proteins ERK1/2, TLR4 and NLRP3, in FIG. 7, (a) is a graph of the Western blot effect of berberine (BBR) on proteins ERK1/2, TLR4 and NLRP3, (b) is a graph of the effect of berberine at different concentrations on proteins ERK1/2, (c) is a graph of the effect of berberine at different concentrations on protein TLR4, and (d) is a graph of the effect of berberine at different concentrations on protein NLRP 3. Wherein, GAPDH is used as an internal control; for quantitative results
And (4) showing. Comparison with blank control, # P<0.05，##P<0.01; comparison with LPS group<0.05，**P<0.01。
In addition, sanguinarine at concentrations of 0.039. Mu.M, 0.078. Mu.M, 0.156. Mu.M, and berberine oxide at concentrations of 5. Mu.M, 10. Mu.M, 20. Mu.M, all significantly reversed LPS-induced upregulation of ERK1/2, TLR4, NF-. Kappa.B, and NLRP3, and were dose-dependent. FIG. 8 is a graph of the effect of sanguinarine and oxidized berberine on proteins NF-. Kappa.B and NLRP3 at different concentrations, and in FIG. 8 (a) is a graph of the effect of sanguinarine (corresponding to abscissa numbers 3, 4, 5) and oxidized berberine (corresponding to abscissa numbers 6, 7, 8) on protein NF-. Kappa.B at different concentrations, (B) is a graph of the effect of sanguinarine (corresponding to abscissa numbers 3, 4, 5) and oxidized berberine (corresponding to abscissa numbers 6, 7, 8) on protein NLRP3 at different concentrations (1: blank; 2 GAPDH 3; 0.039. Mu.M; 4.078. Mu.M; 5.156. Mu.M; 6. FIG. 9 is a graph of the effect of sanguinarine and oxidized berberine on protein ERK1/2 and TLR4 at different concentrations, and in FIG. 9 (a) is a graph of the effect of sanguinarine (corresponding to abscissa numbers 3, 4, 5) and oxidized berberine (corresponding to abscissa numbers 6, 7, 8) on protein ERK1/2 at different concentrations, (b) is a graph of the effect of sanguinarine (corresponding to abscissa numbers 3, 4, 5) and oxidized berberine (corresponding to abscissa numbers 6, 7, 8) on protein TLR4 at different concentrations (1: blank; 2 GAPDH 3; 0.039. Mu.M; 4.078. Mu.M; 5.156. Mu.M; 6.
The embodiments show that the extraction method provided by the invention can efficiently enrich the alkaloid in the Xuan Coptis chinensis Franch, and the enriched alkaloid has obvious anti-inflammatory activity.
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 decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A method for extracting alkaloid from Xuan Huang Lian is characterized by comprising the following steps:
extracting the rhizome of the Coptis chinensis Franch with alcohol, and concentrating the obtained extract to obtain a crude extract;
mixing the crude extract with water, adjusting the pH value of the obtained aqueous solution of the crude extract to 2-4, and extracting by using petroleum ether to respectively obtain a petroleum ether extract and a water layer;
adjusting the pH value of the water layer to 8-10, and extracting by using ethyl acetate to respectively obtain an ethyl acetate extract and a water layer;
and concentrating the ethyl acetate extract to obtain the total alkaloid extract of the Chinese goldthread.
2. The extraction method according to claim 1, further comprising:
dissolving the total alkaloid extract of the Chinese goldthread to obtain a total alkaloid solution of the Chinese goldthread;
and (2) carrying out high performance liquid chromatography separation on the Xuanhuang total alkaloid solution, wherein the conditions of the high performance liquid chromatography separation comprise: the mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is methanol, the mobile phase B is an acetic acid aqueous solution, and the volume fraction of acetic acid in the acetic acid aqueous solution is 0.2-0.5%; gradient elution; the chromatographic column is a semi-preparative phenethyl chromatographic column;
the elution procedure of the gradient elution is 0 to 60min, the volume ratio of the methanol to the acetic acid aqueous solution is from 40 to 100, and the separation liquid of the methanol to the acetic acid aqueous solution is from 40 to 45.
3. The extraction method according to claim 1, wherein the alcohol reagent adopted in the alcohol extraction is methanol or ethanol water solution, and the volume fraction of ethanol in the ethanol water solution is 70-95%; the dosage ratio of the rhizome of the Xuanhuanglian to the alcohol reagent is 1g.
4. The extraction method according to claim 1 or 3, wherein the alcohol extraction is performed under the conditions of heating reflux or ultrasound; the times of alcohol extraction are 1-3, and the time of single alcohol extraction is 20-30 min.
5. The extraction method according to claim 1, wherein the volume ratio of the crude extract aqueous solution to petroleum ether is 1; the number of times of petroleum ether extraction is 2-5.
6. The extraction process according to claim 1, wherein the volume ratio of the water layer to ethyl acetate is 1; the extraction times of the ethyl acetate are 2-5 times.
7. The extraction method according to claim 1, wherein the column temperature of the high performance liquid chromatography is 25 ℃ and the flow rate of the mobile phase is 3mL/min.
8. An alkaloid extract obtained by the extraction method according to any one of claims 1 to 7.
9. Use of the alkaloid extract of claim 8 in the manufacture of a medicament for the prevention or treatment of inflammatory diseases.
10. A pharmaceutical composition, comprising an alkaloid and a pharmaceutically acceptable salt thereof, an adjuvant, a diluent, and a carrier; the alkaloid is the alkaloid extract of claim 8.
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