CN116019847A - Preparation method of drynaria extract - Google Patents
Preparation method of drynaria extract Download PDFInfo
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- CN116019847A CN116019847A CN202211707888.3A CN202211707888A CN116019847A CN 116019847 A CN116019847 A CN 116019847A CN 202211707888 A CN202211707888 A CN 202211707888A CN 116019847 A CN116019847 A CN 116019847A
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- 239000000284 extract Substances 0.000 title claims abstract description 69
- 238000002360 preparation method Methods 0.000 title claims abstract description 24
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- 238000000605 extraction Methods 0.000 claims abstract description 94
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- 238000001035 drying Methods 0.000 claims description 4
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- 239000003814 drug Substances 0.000 claims 2
- 239000001606 7-[(2S,3R,4S,5S,6R)-4,5-dihydroxy-6-(hydroxymethyl)-3-[(2S,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxyoxan-2-yl]oxy-5-hydroxy-2-(4-hydroxyphenyl)chroman-4-one Substances 0.000 abstract description 45
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- 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
Abstract
The invention belongs to the technical field of plant extraction processes, and particularly provides a preparation method of a drynaria extract, which solves the problems that in the prior art, the extraction efficiency is low, the naringin content of a final product is low, potential safety hazards exist during production, and the risk of exceeding the standard of solvent residues of the product is caused; during extraction, complex enzyme is added, and ethanol solution is adopted for extraction after water extraction, so that both water-soluble substances and alcohol-soluble substances in the drynaria rhizome raw material can be effectively extracted, the water-soluble substances and the alcohol-soluble substances can be fully extracted, and the extraction efficiency is further improved; the method adopts polyamide to enrich and purify flavone in the drynaria extract to prepare the drynaria extract with high naringin content, has high naringin transfer rate, fills up the technical blank in the field of polyamide purification of drynaria, does not use toxic or harmful organic solvents, is environment-friendly, safe and efficient, is easy to operate, and can realize large-scale production.
Description
Technical Field
The invention belongs to the technical field of plant extraction processes, and particularly relates to a preparation method of a drynaria extract.
Background
Rhizoma drynariae is a dry rhizome of a water dragon orthopaedics plant, namely drynaria fortunei Drynaria fortunei (Kunze) J.Sm, and the Chinese pharmacopoeia records that the rhizoma drynariae has the effects of treating injury and pain, tonifying kidney and strengthening bones, is used for treating diseases such as traumatic injury, bone fracture, kidney deficiency and lumbago, loosening teeth and the like, and is a Chinese medicinal material with wide application. Modern pharmacological studies have shown that flavone is a key substance of drynaria rhizome for its efficacy, and naringin is a representative flavone compound of drynaria rhizome. Therefore, the extraction and enrichment of naringin in rhizoma drynariae are key points for preparing the rhizoma drynariae extract.
Patent document publication No. CN110279725A discloses a preparation method of rhizoma Drynariae extract, which comprises extracting rhizoma Drynariae with boiling water, continuously extracting at low temperature for 5-7 h, continuously concentrating, and spray drying. The method saves water and energy, but can not completely extract alcohol-soluble substances in the raw materials, so that the extraction rate is reduced; meanwhile, the long-time water extraction can inevitably lead to the entry of polysaccharide, protein, inorganic salt and other impurities in the raw materials into the extracting solution, and can influence the subsequent procedures of filtration, purification and the like.
The publication No. CN113952367A discloses a process for producing rhizoma drynariae extract, which adopts mixed ion exchange resin in the impurity removal step to reduce ash content in the extract, but the extraction temperature of the process is only 50+/-3 ℃, and the lower extraction temperature can not lead the effective components to be completely extracted, so that the yield is too low. In addition, the mixed ion exchange resin can only reduce the heavy metal, ash and other impurities of the product, but cannot improve the naringin content, so that the naringin content of the final product is finally too low, and the application range of the product is limited to a certain extent.
Patent document publication No. CN101897734A discloses a method for preparing an active ingredient of drynaria rhizome, which comprises concentrating and drying an ethanol extract of drynaria rhizome, refining the extract by using macroporous adsorption resin treatment, and crystallizing the extract by using ethyl acetate. Although the drynaria extract with high naringin content can be obtained, ethyl acetate with strong toxicity and combustibility is used in the final refining process, a certain potential safety hazard exists in production, and the risk of exceeding the standard of solvent residue is brought to the product, so that the large-scale production of the drynaria extract is restricted.
Disclosure of Invention
The preparation method of the drynaria extract provided by the invention aims to solve the problems of low extraction efficiency, low naringin content of the final product, potential safety hazard in production and risk of exceeding the standard of solvent residue of the product in the prior art.
For this reason, the invention provides a preparation method of rhizoma drynariae extract, which comprises the following steps:
1) Pulverizing rhizoma Drynariae into coarse powder, adding water and complex enzyme into the coarse powder for enzymolysis, heating and refluxing for extraction after enzymolysis, and filtering for the first time to obtain a solution which is the first extraction solution;
2) Adding water into the residues after the first filtering, heating, refluxing and extracting, and performing the second filtering, wherein the solution obtained after the second filtering is the second extracting solution;
3) Adding ethanol solution into the residue obtained after the second filtration, heating and refluxing for extraction, and filtering for the third time, wherein the solution obtained after the third filtration is the third extraction solution;
4) Mixing the first extracting solution, the second extracting solution and the third extracting solution, and then filtering for the fourth time, wherein the solution obtained after the fourth filtering is the fourth extracting solution;
5) Adjusting the pH value of the fourth extracting solution to a target pH value to obtain a sample loading solution;
6) Adsorbing and loading the sample liquid through a chromatographic column filled with polyamide, washing the mixture in the chromatographic column with water after loading, discarding the washing liquid and the effluent liquid during loading, eluting the mixture in the chromatographic column after washing with ethanol solution, and collecting the eluent;
7) Concentrating and drying the eluent to obtain the drynaria extract.
Preferably, the coarse powder has a particle size of 2 to 3mm.
Preferably, in the step 1), the mass ratio of coarse powder, water and complex enzyme is 1 (10-15): (0.001-0.002).
Preferably, the enzymolysis temperature in the step 1) is 35-45 ℃, and the enzymolysis time is 30-60 minutes.
Preferably, the heating reflux extraction time in the steps 1), 2) and 3) is 1-2 hours.
Preferably, the mass ratio of the residues after the first filtering to water is 1 (7-10), and the mass ratio of the residues after the second filtering to the ethanol solution is 1 (7-10).
Preferably, the compound enzyme consists of cellulase and pectase according to the mass ratio of 1:1.
Preferably, the mass concentration of the ethanol solution in the step 3) is 50% -90%.
Preferably, the polyamide has a particle size of 60 to 90 mesh.
Preferably, the loading flow rate, the washing flow rate and the eluting flow rate in the step 6) are all 1-2 BV/h.
The invention has the beneficial effects that:
1. according to the preparation method of the drynaria extract, the drynaria is a rhizome medicinal material, the texture is hard, the penetration of solvents into the plant cell tissues is prevented during extraction, active ingredients are difficult to extract under mild conditions, and the compound enzyme is added during extraction, so that the tissue structure of the plant cell walls and the cell interstitials can be damaged by utilizing the specificity and the high efficiency of the compound enzyme, and the extraction of active ingredients is promoted.
2. According to the preparation method of the drynaria extract, complex enzyme consisting of cellulase and pectase is added during extraction, so that the tissue structures such as plant cell walls and cell interstitium are fully destroyed, and the intracellular active ingredients are promoted to be more easily dissolved and diffused, so that the extraction efficiency is improved.
3. The preparation method of the drynaria extract provided by the invention adopts a step-by-step extraction method, and ethanol solution is adopted for extraction after water extraction, so that both water-soluble substances and alcohol-soluble substances in drynaria raw material can be effectively extracted, the water-soluble substances and the alcohol-soluble substances can be fully extracted, and the extraction efficiency is further improved.
4. According to the preparation method of the drynaria extract, provided by the invention, the polyamide is adopted to enrich and purify flavone in the drynaria extract, so that the drynaria extract with high naringin content is prepared, the naringin transfer rate is high, and the technical blank in the field of polyamide purification of drynaria is filled.
5. The preparation method of the drynaria extract provided by the invention does not use toxic or harmful organic solvents, has the characteristics of environment friendliness, safety, high efficiency and easiness in operation, and can realize large-scale production.
Drawings
The present invention will be described in further detail with reference to the accompanying drawings.
FIG. 1 is an HPLC chromatogram of the rhizoma Drynariae raw material used in all of the following examples and comparative examples, wherein the chromatographic peak with retention time of 7.048 minutes is naringin.
FIG. 2 is an HPLC chart of the rhizoma Drynariae extract prepared in example 1, wherein the chromatographic peak with retention time of 7.188 min is naringin.
FIG. 3 is an HPLC chart of the rhizoma Drynariae extract prepared in example 2, wherein the chromatographic peak with retention time of 7.141 min is naringin.
FIG. 4 is an HPLC chart of the rhizoma Drynariae extract prepared in example 3, wherein the chromatographic peak with retention time of 7.179 min is naringin.
Detailed Description
The principles and features of the present invention are described below with reference to the drawings, the examples are illustrated for the purpose of illustrating the invention and are not to be construed as limiting the scope of the invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
A preparation method of rhizoma Drynariae extract comprises the following steps:
1) Pulverizing rhizoma Drynariae into coarse powder, adding water and complex enzyme into the coarse powder for enzymolysis, heating and refluxing for extraction after enzymolysis, and filtering for the first time to obtain a solution which is the first extraction solution;
2) Adding water into the residues after the first filtering, heating, refluxing and extracting, and performing the second filtering, wherein the solution obtained after the second filtering is the second extracting solution;
3) Adding ethanol solution into the residue obtained after the second filtration, heating and refluxing for extraction, and filtering for the third time, wherein the solution obtained after the third filtration is the third extraction solution;
4) Mixing the first extracting solution, the second extracting solution and the third extracting solution, and then filtering for the fourth time, wherein the solution obtained after the fourth filtering is the fourth extracting solution;
5) Adjusting the pH value of the fourth extracting solution to a target pH value to obtain a sample loading solution;
6) Adsorbing and loading the sample liquid through a chromatographic column filled with polyamide, washing the mixture in the chromatographic column with water after loading, discarding the washing liquid and the effluent liquid during loading, eluting the mixture in the chromatographic column after washing with ethanol solution, and collecting the eluent;
7) Concentrating and drying the eluent to obtain the drynaria extract.
Because the drynaria rhizome belongs to rhizome medicinal materials and has hard texture, the solvent can be prevented from penetrating into the inside of plant cell tissues during extraction, and active ingredients are difficult to extract under mild conditions.
The preparation method of the drynaria extract provided by the invention adopts a step-by-step extraction method, and ethanol solution is adopted for extraction after water extraction, so that both water-soluble substances and alcohol-soluble substances in drynaria raw material can be effectively extracted, the water-soluble substances and the alcohol-soluble substances can be fully extracted, and the extraction efficiency is further improved. The method has the advantages that the polyamide is adopted to enrich and purify the flavone in the drynaria extract, the drynaria extract with high naringin content is prepared, the naringin transfer rate is high, and the technical blank in the field of polyamide purification of drynaria is filled. Compared with macroporous adsorption resin, the polyamide has stronger and more exclusive adsorption performance on flavonoid compounds, and can play a better role in separation and decolorization. The amide bond in the polyamide is easy to form a hydrogen bond with the hydroxyl in the flavonoid compound so as to be adsorbed, and the substances which cannot form the hydrogen bond flow out along with effluent. The polyamide is treated by water and ethanol solution with proper concentration respectively, and the drynaria flavone can be purified while avoiding the use of toxic and flammable ethyl acetate, so as to prepare the drynaria extract with high naringin content.
The preparation method of the drynaria extract provided by the invention does not use toxic or harmful organic solvents, has the characteristics of environment friendliness, safety, high efficiency and easiness in operation, and can realize large-scale production.
Preferably, the coarse powder has a particle size of 2 to 3mm.
The extraction efficiency can be improved by crushing the drynaria raw material into coarse powder with the particle size of 2-3 mm.
Preferably, in the step 1), the mass ratio of coarse powder, water and complex enzyme is 1 (10-15): (0.001-0.002).
If the addition amount of the complex enzyme is too small, the catalytic activity of the complex enzyme is too low, and the drynaria raw material cannot be fully treated; when the addition amount of the complex enzyme reaches saturation, the addition of the additional complex enzyme does not greatly help to improve the activity, and the material cost is increased. The ratio reduces the material cost on the basis of ensuring high catalytic activity. When in use, the complex enzyme should be dissolved in water in advance and then added into the coarse powder, so as to ensure the catalytic activity of the complex enzyme.
Preferably, the enzymolysis temperature in the step 1) is 35-45 ℃, and the enzymolysis time is 30-60 minutes.
The temperature is 35-45 ℃ to ensure that the complex enzyme can destroy cell walls, thereby being beneficial to fully releasing active ingredients during subsequent decoction and improving the extraction efficiency. The enzymolysis time is 30-60 minutes, so that the cell walls can be fully destroyed.
Preferably, the heating reflux extraction time in the steps 1), 2) and 3) is 1-2 hours.
The extraction efficiency is better.
Preferably, the mass ratio of the residues after the first filtering to water is 1 (7-10), and the mass ratio of the residues after the second filtering to the ethanol solution is 1 (7-10).
The extraction efficiency and the extraction effect are better.
Preferably, the compound enzyme consists of cellulase and pectase according to the mass ratio of 1:1.
The plant cell wall of drynaria is rich in cellulose and pectin, and if only a single kind of enzyme is used, the cell tissue cannot be thoroughly destroyed, and the cell wall can still maintain a certain strength, so that the extraction is incomplete. Therefore, during extraction, the composite enzyme formed by combining cellulase and pectase according to the proportion of 1:1 is added to carry out enzymolysis treatment on the raw materials, the cell wall is destroyed in a proper temperature range catalyzed by the enzyme in advance, the full release of active ingredients is facilitated during subsequent decoction, and the extraction efficiency is improved.
Preferably, the mass concentration of the ethanol solution in the step 3) is 50% -90%.
The main active components of rhizoma Drynariae comprise polysaccharides, phenolic acids, triterpenes, etc. besides flavone. Only a part of the water-soluble active ingredient can be extracted by using water alone as an extraction solvent. In order to ensure complete extraction of the active ingredients of the drynaria rhizome, ethanol solution with a certain concentration is also required to be used for extraction. The method for measuring the extract under the condition of the rhizoma drynariae recorded in the 2020 edition of Chinese pharmacopoeia uses dilute ethanol with the concentration of 49.5-50.5% as an extraction solvent, and indicates that the active substances in the rhizoma drynariae can be extracted by using an ethanol solution with the concentration of 50%. In order to improve the extraction efficiency, ethanol solution with higher concentration can be selected so as to reduce the extraction time. Therefore, the invention adopts a step-by-step extraction mode, namely, water is firstly used for extracting the water-soluble components of the drynaria raw material, then 50% -90% ethanol solution is used for extracting the alcohol-soluble components, so that the effective components in the drynaria are obtained to the maximum extent, and the extraction efficiency is further improved.
Preferably, the polyamide has a particle size of 60 to 90 mesh.
When the polyamide is used for purification, 60-90 meshes of polyamide is selected to achieve both the separation effect of the polyamide on the flavone and the separation speed. Too low mesh number reduces the adsorption capacity to flavonoid compounds, too high mesh number can make chromatographic column packing too compact, and the liquid outflow speed is greatly slowed down, which is unfavorable for improving purification efficiency.
Preferably, the loading flow rate, the washing flow rate and the eluting flow rate in the step 6) are all 1-2 BV/h.
The flow rate should not exceed 2BV/h throughout the purification process of loading, washing and desorption. When the flow rate is too high, the acting time of the liquid and the polyamide is shortened, and the adsorption rate, the impurity removal rate and the desorption rate are reduced; too low a flow rate can greatly extend purification time, affecting efficiency. During washing, substances flowing out along with the washing liquid are mainly enzymes added during extraction and impurities such as inorganic salts in the drynaria raw materials, and the substances cannot be adsorbed by polyamide, so that the impurities are washed and removed by using enough water, and the specific water quantity is positively related to the water quantity used during raw material extraction.
Preferably, the mass concentration of the ethanol solution in the elution in the step 6) is 70-80%.
Because naringin belongs to a dihydroflavonoid compound, has smaller polarity and better solubility in high-concentration ethanol, nonpolar impurities can be eluted due to the too high concentration of the ethanol, and the purity of a product is reduced, so that 70-80% of ethanol is selected as an eluent.
Preferably, the washing liquid is water with the water content of 2-4 BV, and the eluent dosage is 3-5 BV. Meeting the washing and eluting requirements.
Preferably, the target PH value is 4-5; specifically, dilute hydrochloric acid or sodium hydroxide solution is adopted to adjust the pH value of the fourth extracting solution to 4-5.
The used drynaria rhizome raw material is purchased from the medicinal material market, and the naringin content is 0.95% through HPLC detection.
The BV described in all examples and comparative examples below refers to the column volume of the column packing.
Example 1:
a preparation method of rhizoma Drynariae extract comprises the following steps:
1) Crushing 100g of drynaria raw material into coarse powder with the particle size of 2-3 mm, and putting the coarse powder into an extraction tank; dissolving 0.2g of complex enzyme in 1500mL of water, putting into an extraction tank, heating to 35 ℃ and performing enzymolysis for 60 minutes; heating and reflux extracting for 1 hour, and filtering for the first time, wherein the solution obtained after the first filtering is the first extracting solution; the compound enzyme consists of cellulase and pectase according to the mass ratio of 1:1;
2) Adding 1000mL of water into the residues after the first filtration, heating and refluxing for extraction for 1 hour, and performing the second filtration, wherein the solution obtained after the second filtration is the second extraction solution;
3) Adding 1000mL of 50% ethanol solution into the residue obtained after the second filtration, heating and refluxing for 2 hours, and filtering for the third time, wherein the solution obtained after the third filtration is the third extraction solution;
4) Mixing the first extracting solution, the second extracting solution and the third extracting solution, and then filtering for the fourth time, wherein the solution obtained after the fourth filtering is the fourth extracting solution;
5) Adjusting the pH value of the fourth extracting solution to 4-5 by using dilute hydrochloric acid or sodium hydroxide solution to obtain a sample loading solution;
6) Adsorbing and loading the sample liquid through a chromatographic column filled with 60-90 meshes of polyamide, adding water for 4BV after loading, washing a mixture in the chromatographic column by using water, controlling the flow rates of the sample liquid and the washing liquid to be 2BV/h, discarding all the washing liquid and effluent liquid during loading, eluting the mixture in the chromatographic column after washing by using an ethanol solution with the mass concentration of 80% for 4BV, controlling the flow rate of the eluent to be 1.5 BV/h, and collecting the eluent;
7) After the elution is finished, the eluent is concentrated under reduced pressure below 75 ℃, ethanol is recovered, dried and crushed to obtain 2.24g of drynaria extract, and the naringin content is 40.2 percent and the naringin transfer rate is 94.8 percent through HPLC detection.
Example 2:
a preparation method of rhizoma Drynariae extract comprises the following steps:
1) Crushing 100g of drynaria raw material into coarse powder with the particle size of 2-3 mm, and putting the coarse powder into an extraction tank; dissolving 0.15g of complex enzyme in 1200mL of water, putting into an extraction tank, heating to 40 ℃ and performing enzymolysis for 50 minutes under temperature control; heating and reflux extracting for 1 hour, and filtering for the first time, wherein the solution obtained after the first filtering is the first extracting solution; the compound enzyme consists of cellulase and pectase according to the mass ratio of 1:1;
2) Adding 800mL of water into the residues after the first filtration, heating and refluxing for extraction for 1 hour, and performing the second filtration, wherein the solution obtained after the second filtration is the second extraction solution;
3) Adding 800mL of 70% ethanol solution into the residue obtained after the second filtration, heating and refluxing for 1.5 hours, and filtering for the third time, wherein the solution obtained after the third filtration is the third extraction solution;
4) Mixing the first extracting solution, the second extracting solution and the third extracting solution, and then filtering for the fourth time, wherein the solution obtained after the fourth filtering is the fourth extracting solution;
5) Adjusting the pH value of the fourth extracting solution to 4-5 by using dilute hydrochloric acid or sodium hydroxide solution to obtain a sample loading solution;
6) Adsorbing and loading the sample liquid through a chromatographic column filled with 60-90 meshes of polyamide, adding water for 3BV after loading, washing the mixture in the chromatographic column by using water, controlling the flow rate of the sample liquid and the flow rate of the washing liquid to be 1.5 BV/h, discarding all the washing liquid and the effluent liquid during loading, eluting the mixture in the chromatographic column after washing by using 4.5BV of ethanol solution with the mass concentration of 75%, controlling the flow rate of the eluent to be 1.5 BV/h, and collecting the eluent;
7) After the elution is finished, the eluent is concentrated under reduced pressure below 75 ℃, ethanol is recovered, dried and crushed to obtain 2.30g of drynaria extract, and the naringin content is 39.3 percent and the naringin transfer rate is 95.1 percent through HPLC detection.
Example 3:
a preparation method of rhizoma Drynariae extract comprises the following steps:
1) Crushing 100g of drynaria raw material into coarse powder with the particle size of 2-3 mm, and putting the coarse powder into an extraction tank; dissolving 0.15g of complex enzyme in 1000mL of water, putting into an extraction tank, heating to 45 ℃ and performing enzymolysis for 45 minutes; heating and reflux extracting for 1 hour, and filtering for the first time, wherein the solution obtained after the first filtering is the first extracting solution; the compound enzyme consists of cellulase and pectase according to the mass ratio of 1:1;
2) Adding 700mL of water into the residues after the first filtration, heating and refluxing for extraction for 1 hour, and performing the second filtration, wherein the solution obtained after the second filtration is the second extraction solution;
3) Adding 700mL of 90% ethanol solution into the residue obtained after the second filtration, heating and refluxing for 1 hour, and filtering for the third time, wherein the solution obtained after the third filtration is the third extraction solution;
4) Mixing the first extracting solution, the second extracting solution and the third extracting solution, and then filtering for the fourth time, wherein the solution obtained after the fourth filtering is the fourth extracting solution;
5) Adjusting the pH value of the fourth extracting solution to 4-5 by using dilute hydrochloric acid or sodium hydroxide solution to obtain a sample loading solution;
6) Adsorbing and loading the sample liquid through a chromatographic column filled with 60-90 meshes of polyamide, adding water 2BV after loading, washing the mixture in the chromatographic column with water, controlling the flow rates of the sample liquid and the washing liquid to be 1.3 BV/h, discarding all the washing liquid and the effluent liquid during loading, eluting the mixture in the chromatographic column after washing with an ethanol solution 5BV with the mass concentration of 70%, controlling the flow rate of the eluent to be 1.5 BV/h, and collecting the eluent;
7) After the elution is finished, the eluent is concentrated under reduced pressure below 75 ℃, ethanol is recovered, dried and crushed to obtain 2.40g of drynaria extract, and the naringin content is 38% and the naringin transfer rate is 96% by HPLC detection.
Example 4:
a preparation method of rhizoma Drynariae extract comprises the following steps:
1) Crushing 100g of drynaria raw material into coarse powder with the particle size of 2-3 mm, and putting the coarse powder into an extraction tank; dissolving 0.10g of complex enzyme in 1000mL of water, putting into an extraction tank, heating to 35 ℃ and performing enzymolysis for 60 minutes; heating and reflux-extracting for 2 hours, and filtering for the first time, wherein the solution obtained after the first filtering is the first extracting solution; the compound enzyme consists of cellulase and pectase according to the mass ratio of 1:1;
2) Adding 700mL of water into the residues after the first filtration, heating and refluxing for extraction for 2 hours, and performing the second filtration, wherein the solution obtained after the second filtration is the second extraction solution;
3) Adding 700mL of 90% ethanol solution into the residue obtained after the second filtration, heating and refluxing for 2 hours, and filtering for the third time, wherein the solution obtained after the third filtration is the third extraction solution;
4) Mixing the first extracting solution, the second extracting solution and the third extracting solution, and then filtering for the fourth time, wherein the solution obtained after the fourth filtering is the fourth extracting solution;
5) Adjusting the pH value of the fourth extracting solution to 4-5 by using dilute hydrochloric acid or sodium hydroxide solution to obtain a sample loading solution;
6) Adsorbing and loading the sample liquid through a chromatographic column filled with 60-90 meshes of polyamide, adding water for 4BV after loading, washing a mixture in the chromatographic column by using water, controlling the flow rates of the sample liquid and the washing liquid to be 1 BV/h, discarding all the washing liquid and effluent liquid during loading, eluting the mixture in the chromatographic column after washing by using an ethanol solution 3BV with the mass concentration of 80%, controlling the flow rate of the eluent to be 1 BV/h, and collecting the eluent;
7) After the elution is finished, the eluent is concentrated under reduced pressure below 75 ℃, ethanol is recovered, dried and crushed to obtain 2.20g of drynaria extract, and the naringin content is 40.7 percent and the naringin transfer rate is 94.3 percent through HPLC detection.
Example 5:
a preparation method of rhizoma Drynariae extract comprises the following steps:
1) Crushing 100g of drynaria raw material into coarse powder with the particle size of 2-3 mm, and putting the coarse powder into an extraction tank; dissolving 0.2g of complex enzyme in 1500mL of water, putting into an extraction tank, heating to 45 ℃ and performing enzymolysis for 30 minutes; heating and reflux extracting for 1.5 hours, and filtering for the first time, wherein the solution obtained after the first filtering is the first extracting solution; the compound enzyme consists of cellulase and pectase according to the mass ratio of 1:1;
2) Adding 1000mL of water into the residues after the first filtration, heating and refluxing for extraction for 1.5 hours, and performing the second filtration, wherein the solution obtained after the second filtration is the second extraction solution;
3) Adding 1000mL of 50% ethanol solution into the residue obtained after the second filtration, heating and refluxing for 2 hours, and filtering for the third time, wherein the solution obtained after the third filtration is the third extraction solution;
4) Mixing the first extracting solution, the second extracting solution and the third extracting solution, and then filtering for the fourth time, wherein the solution obtained after the fourth filtering is the fourth extracting solution;
5) Adjusting the pH value of the fourth extracting solution to 4-5 by using dilute hydrochloric acid or sodium hydroxide solution to obtain a sample loading solution;
6) Adsorbing and loading the sample liquid through a chromatographic column filled with 60-90 meshes of polyamide, adding water for 4BV after loading, washing a mixture in the chromatographic column by using water, controlling the flow rates of the sample liquid and the washing liquid to be 2BV/h, discarding all the washing liquid and effluent liquid during loading, eluting the mixture in the chromatographic column after washing by using an ethanol solution with the mass concentration of 75% for 4BV, controlling the flow rate of the eluent to be 1.5 BV/h, and collecting the eluent;
7) After the elution is finished, the eluent is concentrated under reduced pressure below 75 ℃, ethanol is recovered, dried and crushed to obtain 2.35g of drynaria extract, and the naringin content is 38.0 percent and the naringin transfer rate is 94.0 percent through HPLC detection.
Comparative example 1
A preparation method of rhizoma Drynariae extract comprises the following steps:
(1) Crushing 100g of drynaria raw material into coarse powder with the particle size of 2-3 mm, and putting the coarse powder into an extraction tank; heating and refluxing for 1 hour, and filtering to obtain a first extracting solution; adding 1000mL of water into the raw materials, heating, refluxing and extracting for 1 hour, and filtering to obtain a second extracting solution; adding 1000mL of ethanol solution with the mass concentration of 50% into the raw materials, heating, refluxing and extracting for 2 hours, and filtering to obtain a third extracting solution;
(2) Mixing the first extracting solution, the second extracting solution and the third extracting solution, filtering to obtain clear extracting solution, and regulating the pH value of the extracting solution to 4-5 by using dilute hydrochloric acid or sodium hydroxide solution to obtain a sample loading solution;
(3) Loading the sample liquid through a chromatographic column filled with 60-90 meshes of polyamide, adding water for 4BV washing after loading, controlling the flow rate of the sample liquid and the washing liquid to be 2BV/h, and discarding the effluent liquid of loading and washing;
(4) After the water washing is finished, adding an ethanol solution with the mass concentration of 80% for 4BV for eluting, controlling the flow rate of the eluent to be 1.5 BV/hour, and collecting the eluent;
(5) After the elution is finished, the eluent is concentrated under reduced pressure below 75 ℃, ethanol is recovered, dried and crushed to obtain 1.65g of drynaria extract, and the naringin content is 35.1 percent and the naringin transfer rate is 61.0 percent through HPLC detection.
Comparative example 2
A preparation method of rhizoma Drynariae extract comprises the following steps:
(1) Crushing 100g of drynaria raw material into coarse powder with the particle size of 2-3 mm, and putting the coarse powder into an extraction tank; dissolving 0.2g of complex enzyme in 1500mL of water, putting into an extraction tank, heating to 35 ℃ and performing enzymolysis for 60 minutes; heating and refluxing for 1 hour, and filtering to obtain a first extracting solution; adding 1000mL of water into the raw materials, heating, refluxing and extracting for 1 hour, and filtering to obtain a second extracting solution; adding 1000mL of water into the raw materials, heating, refluxing and extracting for 2 hours, and filtering to obtain a third extracting solution; the compound enzyme consists of cellulase and pectase according to the mass ratio of 1:1;
(2) Mixing the first extracting solution, the second extracting solution and the third extracting solution, filtering to obtain clear extracting solution, and regulating the pH value of the extracting solution to 4-5 by using dilute hydrochloric acid or sodium hydroxide solution to obtain a sample loading solution;
(3) Loading the sample liquid through a chromatographic column filled with 60-90 meshes of polyamide, adding water for 4BV washing after loading, controlling the flow rate of the sample liquid and the washing liquid to be 2BV/h, and discarding the effluent liquid of loading and washing;
(4) After the water washing is finished, adding an ethanol solution with the mass concentration of 80% for 4BV for eluting, controlling the flow rate of the eluent to be 1.5 BV/hour, and collecting the eluent;
(5) After the elution is finished, the eluent is concentrated under reduced pressure below 75 ℃, ethanol is recovered, dried and crushed to obtain 1.61g of drynaria extract, and the naringin content is 27.4 percent and the naringin transfer rate is 46.4 percent by HPLC detection.
Comparative example 3
A preparation method of rhizoma Drynariae extract comprises the following steps:
(1) Crushing 100g of drynaria raw material into coarse powder with the particle size of 2-3 mm, and putting the coarse powder into an extraction tank; dissolving 0.2g of complex enzyme in 1500mL of water, putting into an extraction tank, heating to 35 ℃ and performing enzymolysis for 60 minutes; heating and refluxing for 1 hour, and filtering to obtain a first extracting solution; adding 1000mL of water into the raw materials, heating, refluxing and extracting for 1 hour, and filtering to obtain a second extracting solution; adding 1000mL of ethanol solution with the mass concentration of 50% into the raw materials, heating, refluxing and extracting for 2 hours, and filtering to obtain a third extracting solution;
(2) Mixing the first extracting solution, the second extracting solution and the third extracting solution, filtering to obtain clear extracting solution, and regulating the pH value of the extracting solution to 4-5 by using dilute hydrochloric acid or sodium hydroxide solution to obtain a sample loading solution;
(3) Loading the sample liquid through a chromatographic column filled with D-101 type macroporous adsorption resin, adding water for 4BV washing after loading, controlling the flow rate of the sample liquid and the washing liquid to be 2BV/h, and discarding the effluent liquid of loading and washing;
(4) After the water washing is finished, adding an ethanol solution with the mass concentration of 80% for 4BV for eluting, controlling the flow rate of the eluent to be 1.5 BV/hour, and collecting the eluent;
(5) After the elution is finished, the eluent is concentrated under reduced pressure below 75 ℃, ethanol is recovered, dried and crushed to obtain 2.13g of drynaria extract, and the naringin content is 34.6 percent and the naringin transfer rate is 81.2 percent through HPLC detection.
The experimental data of examples 1 to 5 and comparative examples 1 to 3 are shown in Table 1.
Table 1 experimental data for examples 1 to 5 and comparative examples 1 to 3
In comparative example 1, compared with examples 1 to 5, the raw materials are not pretreated by adding complex enzyme during extraction, plant tissues are not fully destroyed, and the extraction solvent is not easy to penetrate into the inside of plant cell tissues, so that the extraction of active ingredients is incomplete, and the quality of the extract and the transfer rate of naringin are lower.
Comparative example 2 uses water of strong polarity as an extraction solvent in all extraction, and according to the principle of "similar miscibility", naringin of low polarity has limited solubility in water, and only water is used as an extraction solvent, so that alcohol-soluble substances cannot be extracted completely, resulting in incomplete extraction.
In comparative example 3, D-101 macroporous adsorption resin is used to replace polyamide to separate and purify rhizoma drynariae extract, however, the adsorption specificity of macroporous adsorption resin to flavonoid compounds is weaker than that of polyamide, namely, naringin which can be adsorbed by macroporous resin with the same column volume is less than that of polyamide, so that part of naringin cannot be fully adsorbed by macroporous resin, and flows out along with effluent during loading, so that part of product is lost, and the quality of extract and naringin transfer rate are reduced. The increase of the column volume of the macroporous adsorption resin can improve the adsorption capacity of naringin, but can also increase the use amount of washing liquid and eluent, greatly prolong the purification process time, and have inferior economic benefit and time cost compared with the polyamide purification process.
As can be seen from Table 1, under the combination of different extraction and purification process parameters, the rhizoma drynariae extract with naringin content not less than 38% can be prepared in examples 1 to 5, and the naringin transfer rate is over 94%. The whole process does not use toxic and harmful organic solvents, and is simple to operate, green and efficient. The invention can fully extract active substances in the drynaria raw material, effectively improve the naringin content of the extract and has remarkable effect.
The foregoing examples are merely illustrative of the present invention and are not intended to limit the scope of the present invention, and all designs that are the same or similar to the present invention are within the scope of the present invention.
Claims (10)
1. A preparation method of drynaria extract is characterized in that: the method comprises the following steps:
1) Pulverizing rhizoma Drynariae into coarse powder, adding water and complex enzyme into the coarse powder for enzymolysis, heating and refluxing for extraction after enzymolysis, and filtering for the first time to obtain a solution which is the first extraction solution;
2) Adding water into the residues after the first filtering, heating, refluxing and extracting, and performing the second filtering, wherein the solution obtained after the second filtering is the second extracting solution;
3) Adding ethanol solution into the residue obtained after the second filtration, heating and refluxing for extraction, and filtering for the third time, wherein the solution obtained after the third filtration is the third extraction solution;
4) Mixing the first extracting solution, the second extracting solution and the third extracting solution, and then filtering for the fourth time, wherein the solution obtained after the fourth filtering is the fourth extracting solution;
5) Adjusting the pH value of the fourth extracting solution to a target pH value to obtain a sample loading solution;
6) Adsorbing and loading the sample liquid through a chromatographic column filled with polyamide, washing the mixture in the chromatographic column with water after loading, discarding the washing liquid and the effluent liquid during loading, eluting the mixture in the chromatographic column after washing with ethanol solution, and collecting the eluent;
7) Concentrating and drying the eluent to obtain the drynaria extract.
2. The method for preparing the drynaria extract as claimed in claim 1, wherein: the grain diameter of the coarse powder is 2-3 mm.
3. The method for preparing the drynaria extract as claimed in claim 1, wherein: the mass ratio of coarse powder to water to complex enzyme in the step 1) is 1 (10-15): (0.001-0.002).
4. The method for preparing the drynaria extract as claimed in claim 1, wherein: the enzymolysis temperature in the step 1) is 35-45 ℃, and the enzymolysis time is 30-60 minutes.
5. The method for preparing the drynaria extract as claimed in claim 1, wherein: the heating reflux extraction time in the steps 1), 2) and 3) is 1-2 hours.
6. The method for preparing the drynaria extract as claimed in claim 1, wherein: the mass ratio of the medicine residue after the first filtering to the water is 1 (7-10), and the mass ratio of the medicine residue after the second filtering to the ethanol solution is 1 (7-10).
7. The method for preparing the drynaria extract as claimed in claim 1, wherein: the compound enzyme consists of cellulase and pectase according to the mass ratio of 1:1.
8. The method for preparing the drynaria extract as claimed in claim 1, wherein: the mass concentration of the ethanol solution in the step 3) is 50-90%.
9. The method for preparing the drynaria extract as claimed in claim 1, wherein: the particle size of the polyamide is 60-90 meshes.
10. The method for preparing the drynaria extract as claimed in claim 1, wherein: the sample loading flow rate, the washing flow rate and the elution flow rate in the step 6) are all 1-2 BV/h.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040048811A1 (en) * | 2000-12-29 | 2004-03-11 | Yanming Xie | Drynaria extractions for treating osteoporosis and their extraction process |
| CN101897734A (en) * | 2009-05-25 | 2010-12-01 | 北京卓越同创药物研究院 | Method for preparing fortune's drynaria rhizome active ingredient |
| CN113952367A (en) * | 2021-10-28 | 2022-01-21 | 华茂(湖州)保健品有限公司 | Production process of drynaria extract |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040048811A1 (en) * | 2000-12-29 | 2004-03-11 | Yanming Xie | Drynaria extractions for treating osteoporosis and their extraction process |
| CN101897734A (en) * | 2009-05-25 | 2010-12-01 | 北京卓越同创药物研究院 | Method for preparing fortune's drynaria rhizome active ingredient |
| CN113952367A (en) * | 2021-10-28 | 2022-01-21 | 华茂(湖州)保健品有限公司 | Production process of drynaria extract |
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