Method for separating anti-inflammatory factor bergenin from bergenia purpurascens and application

Abstract

The invention relates to the technical field of separation and extraction, in particular to a method for separating anti-inflammatory factor bergenin from bergenia purpurascens and application of the anti-inflammatory factor bergenin from the bergenia purpurascens. The separation and extraction method comprises the steps that firstly, a natural plant bergenia purpurascens extract is selected, and the separation and extraction method of the technical scheme comprises secondary circulating filtration, purification is conducted through 85% ethyl alcohol, then the bergenin is fed into a macroporous resin chromatographic column XDA-8, and the bergenin is obtained through purification. According to the technical scheme, the content of bergenin in the initial raw material is increased to 5.13-7.47% from 1.27-4.3% of the rhizome of the initial bergenia, the content of the bergenin in the initial raw material is increased by 2-4 times, meanwhile, three-step separation and purification are adopted, and the purity of the bergenin is improved. In conclusion, the separation and purification procedures of the technical scheme are simple, the method is suitable for large-scale production, the yield is high, and the monomer purity is high.

Description

Technical Field

[0001] The present invention relates to the technical field of separation and extraction, and in particular to a method for isolating the anti-inflammatory factor bergenin from Bergenia bergenia and its application. Background Art

[0002] Bergenia is the dried rhizome of the Saxifragaceae plant Bergenia. The bergenin and arbutin in Bergenia are the highest content in natural medicinal plants recorded in the literature. It has multiple biologically active ingredients. Bergenin has antitussive and expectorant, antibacterial and anti-inflammatory, arthritis, anti-ulcer, and treatment of chronic bronchitis effects.

[0003] However, the existing separation and purification process of bergenin is relatively complicated, not suitable for large-scale industrial production, and has a low recovery rate and low monomer purity, which is far from meeting market demand. Summary of the invention

[0004] The purpose of the present invention is to provide a method and application of isolating the anti-inflammatory factor bergenin from Bergenia, so as to solve the problems that the existing separation and purification process of bergenin is relatively complicated, not suitable for large-scale industrial production, and has a low recovery rate and low monomer purity, which is far from meeting market demand.

[0005] To achieve the above object, the present invention provides a method for isolating the anti-inflammatory factor bergenin from Bergenia, and the method for isolating the anti-inflammatory factor bergenin from Bergenia comprises the following steps: S1: Take the roots of Bergenia bergensis, physically crush and cut them into segments to obtain short and broken roots of Bergenia bergensis; S2: The short roots of Bergenia were crushed and extracted twice with 85% ethanol; S3: collecting the two extracts, concentrating the extracts under reduced pressure and vacuum, and recovering ethanol until the ethanol concentration is less than 10%, thereby obtaining a concentrated extract; S4: The concentrated extract is loaded onto an XDA-8 macroporous resin column by a wet method. After loading, impurities are removed by washing with purified water until the extract is colorless and odorless. Then, the extract is washed with 20% ethanol aqueous solution and then with 60% ethanol, and the eluate is collected. S5: concentrating the eluate under reduced pressure and vacuum, and recovering ethanol to obtain a concentrated extract; S6: The concentrated extract was mixed with 200 mesh silica gel, dried in the shade at low temperature, and then chromatographed on a pressurized silica gel column by a dry method. The air in the silica gel was evacuated by vacuum, and acetone and ethyl acetate were added as eluents. The eluent was collected and the solvent was recovered under reduced pressure to obtain an off-white powder. S7: Dissolve the obtained off-white powder by heating it with a high alcohol solvent, filter it, cool it and let it stand for 24 hours to crystallize, and then filter it to obtain bergenin crystals.

[0006] Among them, in step S2, the 85% ethanol used to extract the Bergenia short root pieces is specifically 85% ethanol with a weak acid alcohol of pH=4-5. The specific steps of extracting the Bergenia short root pieces twice using 85% ethanol are: The first extraction was carried out at a solid-liquid ratio of 1:10, an extraction temperature of 90°C, a Soxhlet reflux extraction time of 2 hours, and the extract was collected; the second extraction was carried out at a solid-liquid ratio of 1:8, an extraction temperature of 85°C, a Soxhlet reflux extraction time of 1.5 hours, and the extract was collected.

[0007] Wherein, in step S3, when the extract is concentrated under reduced pressure and vacuum to recover ethanol, the reduced pressure and vacuum conditions are a temperature of 75° C. and a vacuum of 0.07-0.08 MPa.

[0008] Wherein, in step S4, when elution is performed with pure water, 5 column volumes of pure water are required, and the elution flow rate is 1VB / H.

[0009] Wherein, in step S4, when eluting with 20% ethanol aqueous solution on the column, 4-5 column volumes of 20% ethanol aqueous solution are required, and the elution flow rate is V=1.0VB / H.

[0010] Wherein, in step S4, when eluting with 60% ethanol on the column, 4-6 column volumes of 60% ethanol are required, and the elution flow rate is V=0.5VB / H.

[0011] Wherein, in step S5, the eluent is concentrated under reduced pressure and vacuum, and the conditions for recovering ethanol are: temperature T≤70°C, pressure≥0.1kpa.

[0012] Wherein, in step S6, the conditions for dry pressurized silica gel column chromatography are: 100 mesh silica gel, diameter-to-height ratio ≥ 10:1; When using acetone ethyl acetate as eluent, 6-10 column volumes of 60% ethanol are required, the elution flow rate is V=1VB / H, the pressure p=0.008kpa, and the ratio of methanol to acetone in the acetone ethyl acetate eluent is 3:1.

[0013] The present invention also provides a method for isolating the anti-inflammatory factor bergenin from bergenia, and its application in the preparation of drugs for assisting the treatment of respiratory inflammation. The bergenin crystals extracted by the method for isolating the anti-inflammatory factor bergenin from bergenia can be used for the preparation of drugs for assisting the treatment of respiratory inflammation.

[0014] The present invention discloses a method for isolating the anti-inflammatory factor bergenin from bergenia and its application. The material selected in the technical scheme is an extract of the natural plant bergenia, and the separation and extraction method of the technical scheme is secondary circulation filtration, firstly purifying with 85% ethanol, then subjecting to a macroporous resin chromatography column XDA-8, and then subjecting to silica gel column chromatography, and finally crystallizing to white, and obtaining a crystalline monomer compound after standing and cooling. The technical scheme is adopted to increase the content of bergenin in the initial raw material from 1.27-4.3% in the initial rhizomes of bergenia to 5.13-7.47%, so that the content in the starting raw material bergenia is increased by 2-4 times. At the same time, by using three-step separation and purification, a monomer compound bergenin with a monomer purity of 90.42-99.18% can be obtained. In summary, the separation and purification process of the technical scheme is simple, suitable for large-scale production, with high yield and high monomer purity. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0016] Figure 1 It is a flow chart of the steps of the method for isolating the anti-inflammatory factor bergenin from Bergenia provided by the present invention. DETAILED DESCRIPTION

[0017] Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and should not be construed as limiting the present invention.

[0018] See also Figure 1 The present invention provides a method for isolating the anti-inflammatory factor bergenin from Bergenia, and the method for isolating the anti-inflammatory factor bergenin from Bergenia comprises the following steps: S1: Take the roots of Bergenia bergensis, physically crush and cut them into segments to obtain short and broken roots of Bergenia bergensis; S2: The short roots of Bergenia were crushed and extracted twice with 85% ethanol; S3: collecting the two extracts, concentrating the extracts under reduced pressure and vacuum, and recovering ethanol until the ethanol concentration is less than 10%, thereby obtaining a concentrated extract; S4: The concentrated extract is loaded onto an XDA-8 macroporous resin column by a wet method. After loading, impurities are removed by washing with purified water until the extract is colorless and odorless. Then, the extract is washed with 20% ethanol aqueous solution and then with 60% ethanol, and the eluate is collected. S5: concentrating the eluate under reduced pressure and vacuum, and recovering ethanol to obtain a concentrated extract; S6: The concentrated extract was mixed with 200 mesh silica gel, dried in the shade at low temperature, and then chromatographed on a pressurized silica gel column by a dry method. The air in the silica gel was evacuated by vacuum, and acetone and ethyl acetate were added as eluents. The eluent was collected and the solvent was recovered under reduced pressure to obtain an off-white powder. S7: Dissolve the obtained off-white powder by heating it with a high alcohol solvent, filter it, cool it and let it stand for 24 hours to crystallize, and then filter it to obtain bergenin crystals.

[0019] In this embodiment, the material selected by this technical scheme is an extract of the natural plant Bergenia, and the separation and extraction method of this technical scheme is secondary circulation filtration, first purified with 85% ethanol, then subjected to macroporous resin chromatography column XDA-8, and then subjected to silica gel column chromatography, and finally the crystals turn white, and after standing and cooling, a crystalline monomer compound is obtained. The use of this technical scheme increases the content of bergenin in the initial raw material from the initial 1.27-4.3% of the rhizomes of Bergenia to 5.13-7.47%, increasing the content in the starting raw material Bergenia by 2-4 times. At the same time, by using three-step separation and purification, a monomer compound bergenin with a monomer purity of 90.42-99.18% can be obtained. In summary, the separation and purification process of this technical scheme is simple, suitable for large-scale production, with high yield and high monomer purity.

[0020] Further, in step S2, the 85% ethanol used to extract the Bergenia short root pieces is specifically 85% ethanol with a weak acid alcohol having a pH of 4-5. The specific steps of extracting the Bergenia short root pieces twice using 85% ethanol are as follows: The first extraction was carried out at a solid-liquid ratio of 1:10, an extraction temperature of 90°C, a Soxhlet reflux extraction time of 2 hours, and the extract was collected; the second extraction was carried out at a solid-liquid ratio of 1:8, an extraction temperature of 85°C, a Soxhlet reflux extraction time of 1.5 hours, and the extract was collected.

[0021] Furthermore, in step S3, when the extract is concentrated under reduced pressure and vacuum to recover ethanol, the reduced pressure and vacuum conditions are a temperature of 75° C. and a vacuum of 0.07-0.08 MPa.

[0022] Furthermore, in step S4, when elution is performed with pure water, 5 column volumes of pure water are required, and the elution flow rate is 1VB / H.

[0023] Furthermore, in step S4, when eluting with 20% ethanol aqueous solution on the column, 4-5 column volumes of 20% ethanol aqueous solution are required, and the elution flow rate is V=1.0VB / H.

[0024] Furthermore, in step S4, when eluting with 60% ethanol, 4-6 column volumes of 60% ethanol are required, and the elution flow rate is V=0.5VB / H.

[0025] Furthermore, in step S5, the eluent is concentrated under reduced pressure and vacuum, and the conditions for recovering ethanol are: temperature T≤70°C, pressure≥0.1 kPa.

[0026] Further, in step S6, the conditions for dry pressurized silica gel column chromatography are: 100 mesh silica gel, diameter-to-height ratio ≥ 10:1; When using acetone ethyl acetate as eluent, 6-10 column volumes of 60% ethanol are required, the elution flow rate is V=1VB / H, the pressure p=0.008kpa, and the ratio of methanol to acetone in the acetone ethyl acetate eluent is 3:1.

[0027] The present invention also provides a method for isolating the anti-inflammatory factor bergenin from bergenia, and its application in the preparation of drugs for assisting the treatment of respiratory inflammation. The bergenin crystals extracted by the method for isolating the anti-inflammatory factor bergenin from bergenia can be used for the preparation of drugs for assisting the treatment of respiratory inflammation. Example

[0028] Take 1.5kg of Bergenia root, use physical crushing and cut into 3-5mm segments to obtain 1.47kg of Bergenia short roots, with a loss of 2%. The Bergenia short roots are extracted twice with pH=4-5 weak acid alcohol 85% ethanol. The first extraction is based on a solid-liquid ratio of 1:10, an extraction temperature of 90°C, and a Soxhlet reflux extraction time of 2h, and the extract is collected; the second extraction is based on a solid-liquid ratio of 1:8, an extraction temperature of 85°C, and a Soxhlet reflux extraction time of 1.5h. Collect the two extracts, reduce the pressure and vacuum (temperature 75°C, vacuum 0.07-0.08MPa) to recover ethanol, and dilute with water to an ethanol concentration of less than 10%.

[0029] The extract was loaded onto the XDA-8 macroporous resin column by wet loading. After loading, saturated adsorption was performed for 50 minutes. The impurities were eluted with pure water until it was colorless and odorless, about 5 column volumes of pure water, and the flow rate was 1VB / H. Then 20% ethanol aqueous solution was used for elution for 4-5 column volumes, V=1.0VB / H, and then 60% ethanol was used for elution for 4-6 column volumes, with a flow rate of V=0.5VB / H, and the eluate was collected. The eluate was concentrated under reduced pressure and vacuum, and ethanol was recovered (temperature T≤70℃, pressure≥0.1kpa), and 379g of concentrated extract was obtained, with a water content of 60% and a purity of 70.92%. The above extract was mixed with 200 mesh silica gel, dried in the shade at low temperature, and then subjected to dry method pressurized silica gel column chromatography (100 mesh silica gel, diameter-to-height ratio ≥10:1). The air in the silica gel was evacuated by vacuum, and acetone ethyl acetate eluent (methanol:acetone=3:1) was used for elution for 6-10 column volumes, with a flow rate of V=1.0VB / H and a pressure of p=0.008kpa. The eluate was collected and the solvent was recovered under reduced pressure to obtain an off-white powder. The above mixed crystalline sample was dissolved by heating with a high alcohol solvent (95% CH3CH2OH), filtered, cooled, and allowed to stand for crystallization for 24 hours, and then filtered to obtain 23 g of crystals with a content purity of 95.44%. Example

[0030] Take 1.5kg of Bergenia root, use physical crushing and cut into 3-5mm segments to obtain 1.45kg of Bergenia short roots, with a loss of 2%. The Bergenia short roots are extracted twice with pH=4-5 weak acid alcohol 85% ethanol. The first extraction is based on a solid-liquid ratio of 1:2, an extraction temperature of 90°C, and a Soxhlet reflux extraction time of 2h, and the extract is collected; the second extraction is based on a solid-liquid ratio of 1:8, an extraction temperature of 85°C, and a Soxhlet reflux extraction time of 1.5h. Collect the two extracts, recover ethanol under reduced pressure vacuum (temperature 75°C, vacuum 0.07-0.08MPa), and dilute with water to an ethanol concentration of less than 10%.

[0031] The extract was loaded onto the XDA-8 macroporous resin column by wet loading. After loading, saturated adsorption was performed for 50 min. Impurities were removed with purified water until colorless and odorless, approximately 5 column volumes of purified water, flow rate 1VB / hours. Then 20% ethanol aqueous solution was used for elution for 4-5 column volumes, V=1.0VB / H, and then 60% ethanol was used for elution for 4-6 column volumes, flow rate V=0.5VB / H, and the eluate was collected. The eluate was concentrated under reduced pressure and vacuum, and ethanol was recovered (temperature T≤70℃, pressure≥0.1kpa), and 709g of concentrated extract was obtained, with a water content of 60% and a purity yield of 79.61%. The above extract was mixed with 200 mesh silica gel, dried in the shade at low temperature, and then subjected to dry method pressurized silica gel column chromatography (100 mesh silica gel, diameter-to-height ratio ≥10:1). The air in the silica gel was evacuated by vacuum, and acetone ethyl acetate eluent (methanol:acetone=3:1) was used for elution for 6-10 column volumes, with a flow rate of V=1.0VB / H and a pressure of p=0.008kpa. The eluate was collected and the solvent was recovered under reduced pressure to obtain an off-white powder. The above crystalline sample was dissolved by heating with a high alcohol solvent (95% CH3CH2OH), filtered, cooled, and allowed to stand for crystallization for 24 hours, and then filtered to obtain 50 g of crystals with a content purity of 99.54%. Comparative Example 1: Take 1.5kg of Bergenia root, use physical crushing and cut into 3-5mm segments to obtain 1.47kg of Bergenia short roots, with a loss of 2%. The Bergenia short roots are extracted twice with pH=4-5 weak acid alcohol 85% ethanol. The first extraction is based on a solid-liquid ratio of 1:12, an extraction temperature of 90°C, and a Soxhlet reflux extraction time of 2h, and the extract is collected; the second extraction is based on a solid-liquid ratio of 1:8, an extraction temperature of 85°C, and a Soxhlet reflux extraction time of 1.5h. Collect the two extracts, reduce the pressure and vacuum (temperature 75°C, vacuum 0.07-0.08MPa) to recover ethanol, and dilute with water to an ethanol concentration of less than 10%.

[0032] The extract was loaded onto the XDA-8 macroporous resin column by wet loading. After loading, saturated adsorption was performed for 50 minutes. The impurities were eluted with pure water until it was colorless and odorless, about 5 column volumes of pure water, and the flow rate was 1VB / hours. Then 20% ethanol aqueous solution was used for elution for 4-5 column volumes, V=1.0VB / H, and then 60% ethanol was used for elution for 4-6 column volumes, with a flow rate of V=0.5VB / H, and the eluate was collected. The eluate was concentrated under reduced pressure and vacuum, and ethanol was recovered (temperature T≤70℃, pressure≥0.1kpa), and 427g of concentrated extract was obtained, with a water content of 60% and a purity of 79.91%. The above extract was mixed with 200 mesh silica gel, dried in the shade at low temperature, and then subjected to dry method pressurized silica gel column chromatography (100 mesh silica gel, diameter-to-height ratio ≥10:1). The air in the silica gel was evacuated by vacuum, and acetone ethyl acetate eluent (acetone: ethyl acetate = 3:1) was used for elution for 6-10 column volumes, with a flow rate of V = 1.0VB / H and a pressure of p = 0.008 kpa. The eluate was collected and the solvent was recovered under reduced pressure to obtain an off-white powder. The above mixed crystalline sample was dissolved by heating with a high alcohol solvent (95% CH3CH2OH), filtered, cooled, and allowed to stand for crystallization for 24 hours, and then filtered to obtain 27 g of crystals with a content purity of 77.44%.

[0033] In summary, the technical solution used to extract bergenin crystals has a higher yield and higher monomer purity.

[0034] What is disclosed above is only a preferred embodiment of the present invention, and it certainly cannot be used to limit the scope of rights of the present invention. Ordinary technicians in this field can understand that all or part of the processes of the above embodiment and equivalent changes made according to the claims of the present invention still fall within the scope of the invention.

Claims

1. A method for isolating the anti-inflammatory factor bergenin from Bergenia, characterized in that: The steps include: S1: Take the roots of Bergenia bergensis, physically crush and cut them into segments to obtain short and broken roots of Bergenia bergensis; S2: The short roots of Bergenia were crushed and extracted twice with 85% ethanol; S3: collecting the two extracts, concentrating the extracts under reduced pressure and vacuum, and recovering ethanol until the ethanol concentration is less than 10%, thereby obtaining a concentrated extract; S4: The concentrated extract is loaded onto an XDA-8 macroporous resin column by a wet method. After loading, impurities are removed by washing with purified water until the extract is colorless and odorless. Then, the extract is washed with 20% ethanol aqueous solution and then with 60% ethanol, and the eluate is collected. S5: concentrating the eluate under reduced pressure and vacuum, and recovering ethanol to obtain a concentrated extract; S6: The concentrated extract was mixed with 200 mesh silica gel, dried in the shade at low temperature, and then chromatographed on a pressurized silica gel column by a dry method. The air in the silica gel was evacuated by vacuum, and acetone and ethyl acetate were added as eluents. The eluent was collected and the solvent was recovered under reduced pressure to obtain an off-white powder. S7: Dissolve the obtained off-white powder by heating it with a high alcohol solvent, filter it, cool it and let it stand for 24 hours to crystallize, and then filter it to obtain bergenin crystals.

2. The method for isolating the anti-inflammatory factor bergenin from Bergenia as claimed in claim 1, characterized in that: In step S2, the 85% ethanol used to extract the Bergenia short root pieces is specifically 85% ethanol with a weak acid alcohol pH of 4-5. The specific steps of extracting the Bergenia short root pieces twice using 85% ethanol are as follows: The first extraction was carried out at a solid-liquid ratio of 1:10, an extraction temperature of 90°C, a Soxhlet reflux extraction time of 2 hours, and the extract was collected; the second extraction was carried out at a solid-liquid ratio of 1:8, an extraction temperature of 85°C, a Soxhlet reflux extraction time of 1.5 hours, and the extract was collected.

3. The method for isolating the anti-inflammatory factor bergenin from Bergenia as claimed in claim 2, characterized in that: In step S3, when the extract is concentrated under reduced pressure and vacuum to recover ethanol, the reduced pressure and vacuum conditions are a temperature of 75° C. and a vacuum of 0.07-0.08 MPa.

4. The method for isolating the anti-inflammatory factor bergenin from Bergenia as claimed in claim 3, characterized in that: In step S4, when elution is performed with pure water, 5 column volumes of pure water are required, and the elution flow rate is 1VB / H.

5. The method for isolating the anti-inflammatory factor bergenin from Bergenia as claimed in claim 4, characterized in that: In step S4, when eluting with 20% ethanol aqueous solution, 4-5 column volumes of 20% ethanol aqueous solution are required, and the elution flow rate is V=1.0VB / H.

6. The method and use of isolating the anti-inflammatory factor bergenin from Bergenia as claimed in claim 5, characterized in that: In step S4, when eluting with 60% ethanol, 4-6 column volumes of 60% ethanol are required, and the elution flow rate is V=0.5VB / H.

7. The method for isolating the anti-inflammatory factor bergenin from Bergenia as claimed in claim 6, characterized in that: In step S5, the eluent is concentrated under reduced pressure and vacuum, and the conditions for recovering ethanol are: temperature T≤70°C, pressure≥0.1 kPa.

8. The method for isolating the anti-inflammatory factor bergenin from Bergenia as claimed in claim 7, characterized in that: In step S6, the conditions for dry pressurized silica gel column chromatography are: 100 mesh silica gel, diameter-to-height ratio ≥ 10:1; When using acetone ethyl acetate as eluent, 6-10 column volumes of 60% ethanol are required, the elution flow rate is V=1VB / H, the pressure p=0.008kpa, and the ratio of methanol to acetone in the acetone ethyl acetate eluent is 3:

1.

9. The method for isolating the anti-inflammatory factor bergenin from Bergenia as claimed in claim 8, characterized in that: Used in the preparation of drugs for auxiliary treatment of respiratory inflammation.

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