CN108484557B - Method for extracting isofraxidin from acanthopanax senticosus - Google Patents
Method for extracting isofraxidin from acanthopanax senticosus Download PDFInfo
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Abstract
The invention provides a method for extracting isofraxidin from acanthopanax senticosus. According to the invention, through the hydrolysis in-situ extraction, a glycoside compound formed by combining isofraxidin and glucose, namely the combined isofraxidin, is completely converted into free isofraxidin, so that the isofraxidin in the extraction liquid is increasedThe content of (A); wherein, the H+The ion exchange resin is used as a catalyst to promote the hydrolysis of combined isofraxidin, and the chlorine-containing solvent is used as an extractant to extract free isofraxidin. Thus, in the hydrolysis in situ extraction process, the hydrolysis of the bound isofraxidin and the extraction of the free isofraxidin are accomplished simultaneously. Meanwhile, the hydrolysis in-situ extraction method provided by the invention effectively avoids the redundancy of two-step operation of hydrolysis and extraction, saves the operation procedures and the usage amount of the solvent, and has low production cost and high isofraxidin yield.
Description
Technical Field
The invention relates to the technical field of compound extraction, and particularly relates to a method for extracting isofraxidin from acanthopanax senticosus.
Background
Acanthopanax senticosus is widely distributed in a plurality of provinces and areas in China, and is one of the important Chinese herbal medicines in China. Acanthopanax senticosus has the effects of benefiting qi, invigorating spleen, tonifying kidney, tranquilizing mind, enhancing nonspecific defense capability of organism, regulating immunity, resisting tumor, aging, radiation injury and fatigue, and can be used for treating cardiovascular diseases, diabetes, neurasthenia, etc. The root and stem parts of Acanthopanax senticosus contain various active chemical components, and particularly isofraxidin and phenolic glycoside compounds. The isofraxidin is one of important active ingredients in acanthopanax, has various pharmacological activities of sedation, anti-inflammation, antibiosis, anti-tumor, cardiovascular prevention and treatment and the like, and the content of the isofraxidin is one of indexes for evaluating the quality of the acanthopanax.
At present, the method for extracting isofraxidin from acanthopanax senticosus generally comprises the steps of extracting acanthopanax senticosus, directly extracting and concentrating an extracting solution, and separating by using macroporous adsorption resin or silica gel. Such methods have the following drawbacks: in addition to free isofraxidin, combined isofraxidin also exists in acanthopanax senticosus; if the combined isofraxidin is not extracted, about 30% of isofraxidin is lost, and meanwhile, the organic solvent for separating the resin or the silica gel has a large recovery load and cannot be recycled, so that the waste of cost and energy is caused.
Disclosure of Invention
The invention aims to provide a method for extracting isofraxidin from acanthopanax senticosus, which has high yield and low cost.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for extracting isofraxidin from acanthopanax senticosus, which comprises the following steps:
extracting acanthopanax senticosus by adopting a first ethanol water solution to obtain an extracting solution;
mixing the extract with H+Mixing the type ion exchange resin and a chlorine-containing solvent, and then carrying out hydrolysis in-situ extraction to obtain an extract liquid;
separating out an organic phase in the extraction liquid, concentrating the organic phase to be dry, and dissolving the obtained material by adopting a second ethanol aqueous solution to obtain a dissolved solution;
and enriching the dissolved solution by using macroporous adsorption resin, eluting the enriched macroporous adsorption resin by using a third ethanol aqueous solution, and removing the solvent in the obtained eluent to obtain the isofraxidin.
Preferably, the volume percentage content of the first ethanol aqueous solution is 60-90%; the volume percentage of the second ethanol aqueous solution is 20-30%; the third ethanol aqueous solution is 75-90% by volume.
Preferably, the mass ratio of the acanthopanax senticosus to the first ethanol aqueous solution is 1: (7-15).
Preferably, the extraction temperature is 70-90 ℃ and the extraction time is 1-5 h.
Preferably, said H+The ion exchange resin in the form of a resin is pretreated before use, and the pretreatment is carried outThe method comprises the following steps:
(1) by ethanol on H+Soaking the type ion exchange resin to obtain H+Washing the type ion exchange resin with water;
(2) using an alkaline reagent to treat H obtained in the step (1)+Soaking the type ion exchange resin to obtain H+Washing the type ion exchange resin with water to be neutral;
(3) using an acid reagent to treat H obtained in the step (2)+Soaking the type ion exchange resin to obtain H+Washing the type ion exchange resin with water to be neutral;
(4) h obtained in the step (3)+Sequentially carrying out the treatments in the step (2) and the step (3) on the type ion exchange resin to obtain pretreated H+The type ion exchange resin is ready for use.
Preferably, the H is calculated according to the absolute dry mass of the acanthopanax+The type ion exchange resin accounts for 0.25-5% of the mass of the acanthopanax senticosus.
Preferably, the chlorine-containing solvent comprises dichloromethane, trichloromethane, 1-dichloroethane or 1, 2-dichloroethane.
Preferably, the volume ratio of the chlorine-containing solvent to the extracting solution is (0.4-2.5): 1.
preferably, the hydrolysis in situ extraction is performed under closed conditions; the temperature of the hydrolysis in-situ extraction is 75-95 ℃, and the time is 1-4 h.
Preferably, the enrichment and elution are performed under closed conditions; the sample loading concentration in the enrichment process is 0.2-1.0 mg/mL.
The invention provides a method for extracting isofraxidin from acanthopanax senticosus, which comprises the steps of extracting acanthopanax senticosus by adopting a first ethanol water solution to obtain an extracting solution; mixing the extract with H+Mixing the type ion exchange resin and a chlorine-containing solvent, and then carrying out hydrolysis in-situ extraction to obtain an extract liquid; separating out an organic phase in the extraction liquid, concentrating the organic phase to be dry, and dissolving the obtained material by adopting a second ethanol aqueous solution to obtain a dissolved solution; enriching the dissolved solution by macroporous adsorption resinAnd eluting the enriched macroporous adsorption resin by using a triethanolamine aqueous solution, and removing the solvent in the obtained eluent to obtain the isofraxidin. According to the method, through the hydrolysis in-situ extraction, a glycoside compound formed by combining isofraxidin and glucose, namely the combined isofraxidin, is completely converted into free isofraxidin, so that the content of isofraxidin in the extraction liquid is increased; wherein, the H+The ion exchange resin is used as a catalyst to promote the hydrolysis of combined isofraxidin, and the chlorine-containing solvent is used as an extractant to extract free isofraxidin. Thus, in the hydrolysis in situ extraction process, the hydrolysis of the bound isofraxidin and the extraction of the free isofraxidin are accomplished simultaneously. Meanwhile, the hydrolysis in-situ extraction method provided by the invention effectively avoids the redundancy of two-step operation of hydrolysis and extraction, saves the operation procedures and the usage amount of the solvent, and has low production cost and high isofraxidin yield. The experimental results of the examples show that the yield of the isofraxidin obtained by the method provided by the invention is 142.74% and the purity is 34.2% based on 100% of the yield of the isofraxidin in the extracting solution.
Furthermore, the hydrolysis in-situ extraction, enrichment and elution processes are carried out under a closed condition, so that the method is pollution-free, and the solvent can be recycled; and said H+The ion exchange resin can be recycled after being regenerated. The production cost is further reduced, and the industrial large-scale production is facilitated.
Drawings
FIG. 1 is a high performance liquid chromatogram of isofraxidin (a) and isofraxidin standard (b) obtained in example 1.
Detailed Description
The invention provides a method for extracting isofraxidin from acanthopanax senticosus, which comprises the following steps:
extracting acanthopanax senticosus by adopting a first ethanol water solution to obtain an extracting solution;
mixing the extract with H+Mixing the type ion exchange resin and a chlorine-containing solvent, and then carrying out hydrolysis in-situ extraction to obtain an extract liquid;
separating out an organic phase in the extraction liquid, concentrating the organic phase to be dry, and dissolving the obtained material by adopting a second ethanol aqueous solution to obtain a dissolved solution;
and enriching the dissolved solution by using macroporous adsorption resin, eluting the enriched macroporous adsorption resin by using a third ethanol aqueous solution, and removing the solvent in the obtained eluent to obtain the isofraxidin.
The invention adopts a first ethanol aqueous solution to extract acanthopanax senticosus to obtain an extracting solution. In the invention, the volume percentage content of the first ethanol aqueous solution is preferably 60-90%, and more preferably 70-80%. In the present invention, the mass ratio of acanthopanax senticosus to the first ethanol aqueous solution is preferably 1: (7-15), more preferably 1: (9-12).
In the invention, the acanthopanax is preferably natural air-dried acanthopanax root; in the invention, the water content of the acanthopanax root is below 20% after natural air drying, and the acanthopanax root is not required to be treated by any drying process before use. In the present invention, the acanthopanax senticosus is preferably pulverized before use to obtain a powder of acanthopanax senticosus having a particle size of 60 to 100 mesh.
In the invention, the extraction temperature is preferably 70-90 ℃, and more preferably 75-85 ℃; in an embodiment of the present invention, the temperature of the extraction is specifically the reflux temperature of the first aqueous ethanol solution. In the invention, the extraction time is preferably 1-5 h, and more preferably 2-4 h. In the invention, in order to ensure that the isofraxidin is sufficiently extracted, the number of times of extraction is preferably 2-4 times, and more preferably 3 times.
After the extraction is completed, the invention preferably performs solid-liquid separation on the obtained system, and uses the obtained liquid material as an extracting solution. The solid-liquid separation method is not particularly limited, and a method known to those skilled in the art, such as filtration, may be used.
The acanthopanax senticosus is extracted by the first ethanol aqueous solution, the raw materials are directly used without extra drying, and the energy consumption is low; the isofraxidin yield is high, and the fire safety hidden danger is low; meanwhile, the first ethanol aqueous solution can remove more polar components in the acanthopanax senticosus, such as polysaccharide, protein and the like.
After the extracting solution is obtained, the extracting solution is mixed with H+Mixing the type ion exchange resin and a chlorine-containing solvent, and then carrying out hydrolysis in-situ extraction to obtain an extract. In the present invention, the volume ratio of the chlorine-containing solvent to the extraction solution is preferably (0.4 to 2.5): 1, more preferably (1-2): 1. in the present invention, the chlorine-containing solvent preferably includes dichloromethane, trichloromethane, 1-dichloroethane or 1, 2-dichloroethane.
In the invention, the H is calculated by the absolute dry mass of the acanthopanax+The type ion exchange resin is preferably 0.25 to 5% by mass, more preferably 0.5 to 3% by mass, most preferably 1 to 2% by mass of Acanthopanax senticosus. The invention is directed to said H+The type and source of the type ion exchange resin are not particularly limited, and H of the type well known to those skilled in the art is used+The ion exchange resin may be a commercially available product. In the present invention, said H+The type of type ion exchange resin preferably comprises SK1B, 001 × 7, D061, D072, 110, D113, D151, D152 or WK 11.
In the present invention, said H+The type and properties of the type ion exchange resin are shown in the following table:
H+type and properties of type ion exchange resin
In the present invention, said H+The ion exchange resin of type (ii) is preferably pretreated before use, said pretreatment preferably comprising the steps of:
(1) by ethanol on H+Soaking the type ion exchange resin to obtain H+Washing the type ion exchange resin with water;
(2) using an alkaline reagent to treat H obtained in the step (1)+Soaking the type ion exchange resin to obtain H+Washing the type ion exchange resin with water to be neutral;
(3) using an acid reagent toH obtained in step (2)+Soaking the type ion exchange resin to obtain H+Washing the type ion exchange resin with water to be neutral;
(4) h obtained in the step (3)+Sequentially carrying out the treatments in the step (2) and the step (3) on the type ion exchange resin to obtain pretreated H+The type ion exchange resin is ready for use.
Ethanol is preferably adopted for H in the invention+Soaking the type ion exchange resin to obtain H+The type ion exchange resin is washed with water. In the invention, the soaking time is preferably 10-14 h, and more preferably 12 h. In the present invention, the water used for the water washing is preferably distilled water; in the present invention, the washing with water is not particularly limited, and H obtained after the soaking treatment can be treated+Washing with ethanol in the type ion exchange resin.
After ethanol is adopted for soaking treatment and water washing is finished, the invention preferably adopts an alkali reagent to treat the obtained H+Soaking the type ion exchange resin to obtain H+The type ion exchange resin is washed to be neutral by water. In the present invention, the alkali agent is preferably a NaOH solution. The concentration of the NaOH solution is not particularly limited in the invention, and the concentration well known to those skilled in the art can be adopted; in the embodiment of the invention, the concentration of the NaOH solution is specifically 0.1 mol/L. In the invention, the soaking time is preferably 10-14 h, and more preferably 12 h. In the present invention, the water used for the water washing is preferably distilled water; in the present invention, the washing with water is not particularly limited, and H obtained after the soaking treatment can be treated+Washing the type ion exchange resin to be neutral.
After the soaking treatment is carried out by adopting an alkali reagent and the water washing is finished, the invention preferably adopts an acid reagent to treat the obtained H+Soaking the type ion exchange resin to obtain H+The type ion exchange resin is washed to be neutral by water. In the present invention, the acid reagent is preferably a HCl solution. The concentration of the HCl solution is not particularly limited in the present invention, and may be a concentration well known to those skilled in the art; in the examples of the invention, the HCl solution isThe concentration is specifically 0.1 mol/L. In the invention, the soaking time is preferably 10-14 h, and more preferably 12 h. In the present invention, the water used for the water washing is preferably distilled water; in the present invention, the washing with water is not particularly limited, and H obtained after the soaking treatment can be treated+Washing the type ion exchange resin to be neutral.
After the soaking treatment is carried out by adopting the acid reagent and the water washing is finished, the invention preferably adopts the alkali reagent to treat the obtained H according to the technical scheme+Soaking the type ion exchange resin to obtain H+Washing the type ion exchange resin with water to be neutral; then adopting acid reagent to react with the obtained H according to the technical scheme+Soaking the type ion exchange resin to obtain H+Washing the molded ion exchange resin with water to neutrality to obtain pretreated H+The type ion exchange resin is ready for use.
In the present invention, said H+Type ion exchange resins are commercially available containing small amounts of oligomers and unreacted monomers, as well as inorganic impurities such as iron, lead, copper, etc., which are transferred to the solution when they come into contact with other solutions, affecting the quality of the final product. Thus, for the H+The pretreatment of the ion exchange resin can prevent soluble impurities in the ion exchange resin from transferring into the solution and polluting the water quality of effluent water at the initial stage of use.
In the present invention, the hydrolysis in situ extraction is preferably carried out under closed conditions; the temperature of the hydrolysis in-situ extraction is preferably 75-95 ℃, more preferably 80-90 ℃, and most preferably 85 ℃; the time is preferably 1 to 4 hours, and more preferably 2 to 3 hours. According to the method, through the hydrolysis in-situ extraction, a glycoside compound formed by combining isofraxidin and glucose, namely the combined isofraxidin, is completely converted into free isofraxidin, so that the content of isofraxidin in the extraction liquid is increased; wherein, the H+The ion exchange resin is used as a catalyst to promote the hydrolysis of combined isofraxidin, and the chlorine-containing solvent is used as an extractant to extract free isofraxidin. Therefore, in the hydrolysis in-situ extraction process, the hydrolysis of the combined isofraxidin and the extraction of the free isofraxidin are simultaneously completedIn (1). Meanwhile, the hydrolysis in-situ extraction method provided by the invention effectively avoids the redundancy of two-step operation of hydrolysis and extraction, saves the operation procedures and the usage amount of the solvent, and has low production cost and high isofraxidin yield. In addition, the hydrolysis in-situ extraction is carried out under closed conditions, no pollution is caused, the solvent can be recycled, and the H+The type ion exchange resin is regenerated after being used (and the H+The pretreatment carried out before the use of the type ion exchange resin is consistent and is not described herein any more) can be recycled, thus being convenient for industrial large-scale production.
After the extraction liquid is obtained, the organic phase in the extraction liquid is separated, the organic phase is concentrated to be dry, and the obtained material is dissolved by adopting a second ethanol aqueous solution to obtain a dissolved solution. According to the invention, the organic phase is preferably separated off by allowing the extracts to stand for demixing. The concentration in the present invention is not particularly limited, and a concentration technique known to those skilled in the art may be used. In the invention, the volume percentage of the second ethanol aqueous solution is preferably 20-30%, and more preferably 23-27%. In the present invention, the mass ratio of the material obtained after concentrating the organic phase to dryness to the second aqueous ethanol solution is preferably 1: (25-35), more preferably 1: 30.
after the solution is obtained, the invention adopts macroporous adsorption resin to enrich the solution, adopts a third ethanol water solution to elute the enriched macroporous adsorption resin, and removes the solvent in the obtained eluent to obtain the isofraxidin. The kind and source of the macroporous adsorbent resin are not particularly limited in the present invention, and commercially available macroporous adsorbent resins of the kind well known to those skilled in the art may be used. In the present invention, the macroporous adsorbent resin is preferably selected from the group consisting of HPD80, HPD100, HPD300, HPD5000, AB-8, D101, HPD-D, DM-130, ADS-17, HPD826, ADS-7 and DA-201.
In the present invention, the types and properties of the macroporous adsorbent resin are shown in the following table:
type and properties of macroporous adsorbent resin
In the present invention, the macroporous adsorbent resin is pretreated before use, and the pretreatment method preferably comprises the following steps:
soaking the macroporous adsorption resin in 90-95% ethanol by volume for 8-12 h, washing the soaked macroporous adsorption resin with distilled water to remove residual ethanol, filtering with a funnel, sealing and storing for 22-26 h to balance moisture, and thus obtaining the pretreated macroporous adsorption resin for later use.
In the invention, the concentration of the sample in the enrichment process is preferably 0.2-1.0 mg/mL, and more preferably 0.3-0.8 mg/mL. In the invention, the volume percentage of the third ethanol aqueous solution is preferably 75-90%, and more preferably 80-85%.
In the invention, the enrichment and elution are preferably carried out under a closed condition, no pollution is caused, the solvent can be recycled, and the industrial large-scale production is convenient. The present invention is not limited to the specific operation method of enrichment and elution, and the technical scheme of enrichment and elution well known to those skilled in the art can be adopted. The invention preferably pours the macroporous absorption resin into a column, uses the obtained closed column to enrich the dissolved solution under the closed condition, and then uses the third ethanol water solution to elute the enriched closed column. In the present invention, the diameter-height ratio of the closed column is preferably 1: (18-22), more preferably 1: 20; the volume of the dissolving solution is preferably 3-4 times of the volume of the closed column, and more preferably 3.5 times; the volume of the third ethanol aqueous solution is preferably 15-25 times, and more preferably 20 times of the volume of the closed column.
The method for removing the solvent in the eluent obtained after elution is not particularly limited in the invention, and the technical scheme for removing the solvent, which is well known to those skilled in the art, can be adopted, such as reduced pressure distillation. In the present invention, the solvent in the obtained eluate is preferably recovered and reused to reduce the production cost.
After the solvent in the obtained eluent is removed, the obtained material is preferably dried to obtain the isofraxidin. In the present invention, the drying is preferably vacuum drying; in the invention, the temperature of the vacuum drying is preferably 35-45 ℃, and more preferably 40 ℃; the time is preferably 5-7 h, and more preferably 6 h.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
Crushing the naturally air-dried acanthopanax root to obtain acanthopanax powder with the particle size of 70 meshes, weighing 10.0g of acanthopanax powder (calculated by absolute dry mass) and putting the acanthopanax powder into a round-bottom flask, wherein the mass ratio of the acanthopanax powder to an ethanol water solution is 1: adding 80% ethanol water solution by volume percentage, reflux-extracting in 85 deg.C water bath for 2 hr, repeating the extraction for 2 times, mixing the extracted materials, filtering, and collecting the liquid material as extractive solution;
to H+Pretreating type ion exchange resin (001 × 7) with ethanol+Soaking type ion exchange resin for 12H to obtain H+Washing the type ion exchange resin with distilled water; the obtained H is treated with 0.1mol/L NaOH solution+Soaking type ion exchange resin for 12H to obtain H+Washing the type ion exchange resin to be neutral by using distilled water; the H obtained is reacted with a 0.1mol/L HCl solution+Soaking type ion exchange resin for 12H to obtain H+Washing the type ion exchange resin to be neutral by using distilled water; repeating the steps of soaking treatment by using 0.1mol/L NaOH solution and washing by distilled water to be neutral, and soaking treatment by using 0.1mol/L HCl solution and washing by distilled water to be neutral to obtain pretreated H+A type ion exchange resin;
mixing the extractive solution with pretreated H+Type ion exchangeMixing resin 1.0g and 30mL of 1, 2-dichloroethane, refluxing in a water bath at 90 ℃ under a closed condition, and performing hydrolysis in-situ extraction for 2 hours to obtain an extract;
standing and layering the extract, separating out an organic phase, concentrating the organic phase to dryness, and then mixing the obtained material (in absolute dry mass) and an ethanol water solution according to the mass ratio of 1: adding 25 volume percent of ethanol water solution into the mixture, concentrating the organic phase until the organic phase is dried, and dissolving the obtained material to obtain a dissolved solution;
soaking macroporous adsorption resin (type AB-8) in 95% ethanol for 10h, washing the soaked macroporous adsorption resin with distilled water to remove residual ethanol, filtering with a funnel, grinding the mouth of the bottle, sealing and storing for 24h to balance moisture, filling the obtained pretreated macroporous adsorption resin into a column, and enriching the dissolved solution by using the obtained closed column (diameter-height ratio of 1:20, volume of 11.8mL) under a closed condition, wherein the sample loading concentration in the enrichment process is 1.0 mg/mL; and then eluting the enriched closed column by using an ethanol water solution (the volume of which is 20 times of the volume of the closed column) with the volume percentage content of 80%, and removing the solvent in the obtained eluent by reduced pressure distillation to obtain the isofraxidin.
Measuring the content of isofraxidin in the extracting solution, and calculating the yield; meanwhile, the yield of isofraxidin obtained according to the method in example 1 was calculated. The results showed that the yield of isofraxidin obtained by the method of example 1 was 128.68% based on 100% of the yield of isofraxidin in the extract. This shows that in the hydrolysis in-situ extraction process, the glycoside compound formed by combining isofraxidin and glucose, namely the combined isofraxidin, is converted into free isofraxidin, thereby improving the yield of isofraxidin. The purity analysis of the isofraxidin obtained according to the method of example 1 showed that the purity of the obtained isofraxidin was 33.5%. Wherein, the content and purity of the isofraxidin are detected and calculated by high performance liquid chromatography, and are specifically shown in figure 1.
According to pair H+The pretreatment method of the ion exchange resin comprises the step of hydrolyzing H subjected to in-situ extraction+Type ion exchangeAnd regenerating the resin. The results show that the regenerated H+The type ion exchange resin is repeatedly used for 4 times, and the hydrolysis efficiency is 86.52% of the primary hydrolysis efficiency.
Example 2
Pulverizing the naturally air-dried acanthopanax root to obtain acanthopanax powder with the particle size of 60 meshes, weighing 100.0g of acanthopanax powder (calculated by absolute dry mass) and putting the acanthopanax powder into a round-bottom flask, wherein the mass ratio of the acanthopanax powder to an ethanol water solution is 1: adding 85% ethanol water solution by volume, reflux-extracting in 75 deg.C water bath for 2.5 hr, repeating the extraction for 2 times, mixing the extractive materials, filtering, and collecting the liquid material as extractive solution;
to H+Pretreating type ion exchange resin (001 × 7) with ethanol+Soaking type ion exchange resin for 12H to obtain H+Washing the type ion exchange resin with distilled water; the obtained H is treated with 0.1mol/L NaOH solution+Soaking type ion exchange resin for 12H to obtain H+Washing the type ion exchange resin to be neutral by using distilled water; the H obtained is reacted with a 0.1mol/L HCl solution+Soaking type ion exchange resin for 12H to obtain H+Washing the type ion exchange resin to be neutral by using distilled water; repeating the steps of soaking treatment by using 0.1mol/L NaOH solution and washing by distilled water to be neutral, and soaking treatment by using 0.1mol/L HCl solution and washing by distilled water to be neutral to obtain pretreated H+A type ion exchange resin;
mixing the extractive solution with pretreated H+Mixing 1.0g of type ion exchange resin with 30mL of 1, 2-dichloroethane, refluxing in a water bath at 90 ℃ under a closed condition, and performing hydrolysis in-situ extraction for 3.5 hours to obtain an extract;
standing and layering the extract, separating out an organic phase, concentrating the organic phase to dryness, and then mixing the obtained material (in absolute dry mass) and an ethanol water solution according to the mass ratio of 1: 25, adding 30 volume percent of ethanol aqueous solution, concentrating the organic phase to dryness, and dissolving the obtained material to obtain a dissolved solution;
soaking macroporous adsorption resin (type AB-8) in 95% ethanol for 10h, washing the soaked macroporous adsorption resin with distilled water to remove residual ethanol, filtering with a funnel, grinding the mouth of the bottle, sealing and storing for 24h to balance moisture, filling the obtained pretreated macroporous adsorption resin into a column, and enriching the dissolved solution by using the obtained closed column (diameter-height ratio of 1:20, volume of 11.8mL) under a closed condition, wherein the sample loading concentration in the enrichment process is 1.0 mg/mL; and then eluting the enriched closed column by adopting an ethanol water solution with the volume percentage content of 90% (the volume is 20 times of the volume of the closed column), and removing the solvent in the obtained eluent by reduced pressure distillation to obtain the isofraxidin.
Measuring the content of isofraxidin in the extracting solution, and calculating the yield; meanwhile, the yield of isofraxidin obtained according to the method in example 2 was calculated. The results showed that the yield of isofraxidin obtained by the method of example 2 was 142.74% based on 100% of the yield of isofraxidin in the extract. This shows that in the hydrolysis in-situ extraction process, the glycoside compound formed by combining isofraxidin and glucose, namely the combined isofraxidin, is converted into free isofraxidin, thereby improving the yield of isofraxidin.
The purity analysis of the isofraxidin obtained according to the method in example 2 showed that the purity of the obtained isofraxidin was 34.2%.
According to pair H+The pretreatment method of the ion exchange resin comprises the step of hydrolyzing H subjected to in-situ extraction+Regenerating the type ion exchange resin. The results show that the regenerated H+The type ion exchange resin is repeatedly used for 4 times, and the hydrolysis efficiency is 88.22% of the primary hydrolysis efficiency.
Example 3
Crushing the naturally air-dried acanthopanax root to obtain acanthopanax powder with the particle size of 70 meshes, weighing 1000.0g of acanthopanax powder (calculated by absolute dry mass) and putting the acanthopanax powder into a pilot-plant round-bottom flask, wherein the mass ratio of the acanthopanax powder to an ethanol water solution is 1: adding 85% ethanol water solution by volume, reflux-extracting in 90 deg.C water bath for 2.5 hr, repeating the extraction for 2 times, mixing the extracted materials, and filtering to obtain liquid material as extractive solution;
to H+Pretreating type ion exchange resin (001 × 7) with ethanol+Soaking type ion exchange resin for 12H to obtain H+Washing the type ion exchange resin with distilled water; the obtained H is treated with 0.1mol/L NaOH solution+Soaking type ion exchange resin for 12H to obtain H+Washing the type ion exchange resin to be neutral by using distilled water; the H obtained is reacted with a 0.1mol/L HCl solution+Soaking type ion exchange resin for 12H to obtain H+Washing the type ion exchange resin to be neutral by using distilled water; repeating the steps of soaking treatment by using 0.1mol/L NaOH solution and washing by distilled water to be neutral, and soaking treatment by using 0.1mol/L HCl solution and washing by distilled water to be neutral to obtain pretreated H+A type ion exchange resin;
mixing the extractive solution with pretreated H+Mixing 1.0g of type ion exchange resin with 30mL of 1, 2-dichloroethane, refluxing in a water bath at 80 ℃ under a closed condition, and performing hydrolysis in-situ extraction for 2 hours to obtain an extract;
standing and layering the extract, separating out an organic phase, concentrating the organic phase to dryness, and then mixing the obtained material (in absolute dry mass) and an ethanol water solution according to the mass ratio of 1: adding 20 volume percent ethanol water solution into the mixture, concentrating the organic phase until the organic phase is dried, and dissolving the obtained material to obtain a dissolved solution;
soaking macroporous adsorption resin (type AB-8) in 95% ethanol for 10h, washing the soaked macroporous adsorption resin with distilled water to remove residual ethanol, filtering with a funnel, grinding the mouth of the bottle, sealing and storing for 24h to balance moisture, filling the obtained pretreated macroporous adsorption resin into a column, and enriching the dissolved solution by using the obtained closed column (diameter-height ratio of 1:20, volume of 11.8mL) under a closed condition, wherein the sample loading concentration in the enrichment process is 1.0 mg/mL; and then eluting the enriched closed column by using an ethanol water solution (the volume of which is 20 times of the volume of the closed column) with the volume percentage content of 80%, and removing the solvent in the obtained eluent by reduced pressure distillation to obtain the isofraxidin.
Measuring the content of isofraxidin in the extracting solution, and calculating the yield; meanwhile, the yield of isofraxidin obtained according to the method in example 3 was calculated. The results showed that the yield of isofraxidin obtained by the method of example 3 was 134.56% based on 100% yield of isofraxidin in the extract. This shows that in the hydrolysis in-situ extraction process, the glycoside compound formed by combining isofraxidin and glucose, namely the combined isofraxidin, is converted into free isofraxidin, thereby improving the yield of isofraxidin.
The purity analysis of the isofraxidin obtained according to the method in example 3 showed that the purity of the obtained isofraxidin was 32.6%.
According to pair H+The pretreatment method of the ion exchange resin comprises the step of hydrolyzing H subjected to in-situ extraction+Regenerating the type ion exchange resin. The results show that the regenerated H+The type ion exchange resin is repeatedly used for 4 times, and the hydrolysis efficiency is 85.44 percent of the primary hydrolysis efficiency.
As can be seen from the above examples, the glycoside compound formed by combining isofraxidin and glucose, i.e., the combined isofraxidin, can be completely converted into the free isofraxidin by the hydrolysis in-situ extraction, so as to increase the content of isofraxidin in the extract liquid; wherein, the H+The ion exchange resin is used as a catalyst to promote the hydrolysis of combined isofraxidin, and the chlorine-containing solvent is used as an extractant to extract free isofraxidin. Thus, in the hydrolysis in situ extraction process, the hydrolysis of the bound isofraxidin and the extraction of the free isofraxidin are accomplished simultaneously. Meanwhile, the hydrolysis in-situ extraction method provided by the invention effectively avoids the redundancy of two-step operation of hydrolysis and extraction, saves the operation procedures and the usage amount of the solvent, and has low production cost and high isofraxidin yield.
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 (5)
1. A method for extracting isofraxidin from acanthopanax senticosus comprises the following steps:
extracting acanthopanax senticosus by adopting a first ethanol water solution to obtain an extracting solution; the first ethanol water solution is 60-90% in percentage by volume; the mass ratio of the acanthopanax senticosus to the first ethanol water solution is 1: (7-15);
mixing the extract with H+Mixing the type ion exchange resin and a chlorine-containing solvent, and then carrying out hydrolysis in-situ extraction to obtain an extract liquid; based on the absolute dry mass of the acanthopanax, the H+The type ion exchange resin is 0.25-5% of the acanthopanax senticosus by mass, and the volume ratio of the chlorine-containing solvent to the extracting solution is (0.4-2.5): 1;
separating out an organic phase in the extraction liquid, concentrating the organic phase to be dry, and dissolving the obtained material by adopting a second ethanol aqueous solution to obtain a dissolved solution; the volume percentage of the second ethanol aqueous solution is 20-30%; according to the absolute dry mass, the mass ratio of the material obtained after the organic phase is concentrated to be dry to the second ethanol aqueous solution is 1: (25-35);
filling macroporous adsorption resin into a column, enriching the dissolved solution by using the obtained closed column under a closed condition, eluting the enriched closed column by using the third ethanol aqueous solution, and removing the solvent in the obtained eluent to obtain isofraxidin; the third ethanol aqueous solution is 75-90% in volume percentage; the diameter-height ratio of the closed column is 1: (18-22); the volume of the dissolving solution is 3-4 times of the volume of the closed column; the volume of the third ethanol aqueous solution is 15-25 times of the volume of the closed column; the sample loading concentration in the enrichment process is 0.2-1.0 mg/mL.
2. The method according to claim 1, wherein the temperature of the extraction is 70-90 ℃ and the time is 1-5 h.
3. The method of claim 1, wherein the H is+The type ion exchange resin is pretreated before use, and the pretreatment method comprises the following steps:
(1) by ethanol on H+Soaking the type ion exchange resin to obtain H+Washing the type ion exchange resin with water;
(2) using an alkaline reagent to treat H obtained in the step (1)+Soaking the type ion exchange resin to obtain H+Washing the type ion exchange resin with water to be neutral;
(3) using an acid reagent to treat H obtained in the step (2)+Soaking the type ion exchange resin to obtain H+Washing the type ion exchange resin with water to be neutral;
(4) h obtained in the step (3)+Sequentially carrying out the treatments in the step (2) and the step (3) on the type ion exchange resin to obtain pretreated H+The type ion exchange resin is ready for use.
4. The method of claim 1, wherein the chlorine-containing solvent comprises dichloromethane, trichloromethane, 1-dichloroethane, or 1, 2-dichloroethane.
5. The process according to claim 1, characterized in that the hydrolysis in situ extraction is carried out under closed conditions; the temperature of the hydrolysis in-situ extraction is 75-95 ℃, and the time is 1-4 h.
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