阅读说明：本技术 一种五味子乙素提取物的提取方法 (Extraction method of schisandrin B extract ) 是由 周有财 于 2020-06-04 设计创作，主要内容包括：本发明提供了一种五味子乙素的提取方法,并以五味子乙素为指标制订了质量标准。它的提取工艺是通过CO<Sub>2</Sub>超临界提取、硅胶柱层析、己烷纯化三步得到,经过HPLC法测得含量为25％～95％。本工艺完整、成本低,适宜工业推广使用。(The invention provides a method for extracting schisandrin B, and establishes quality standard by taking schisandrin B as an index. It is extracted by CO 2 The extract is obtained by three steps of supercritical extraction, silica gel column chromatography and hexane purification, and the content is 25-95 percent by HPLC. The process is complete, low in cost and suitable for industrial popularization and use.)

1. A method for extracting schisandrin B extract is characterized by comprising the following steps:

s1) crushing the schisandra chinensis to obtain schisandra chinensis powder;

s2) carrying out supercritical carbon dioxide extraction on the schisandra chinensis powder to obtain a supercritical extract;

s3) dissolving the supercritical extract with an organic solvent, eluting and separating by silica gel column chromatography, and concentrating to obtain Schisandrin B concentrated fraction;

s4) dissolving the Schisandrin B concentrated fraction in a mixed solvent of hexane and isopropanol, and carrying out low-temperature crystallization to obtain a Schisandrin B crude product;

s5) purifying the crude schisandrin B product by hexane to obtain a schisandrin B extract.

2. The extraction method according to claim 1, wherein the mesh number of the Schisandra chinensis Baill powder is 30-50 meshes.

3. The extraction method according to claim 1, wherein the supercritical carbon dioxide extraction pressure is 20 to 30 MPa; the extraction temperature is 30-50 ℃; the extraction time is 1.5-2.5 h.

4. The extraction method according to claim 1, wherein the step S3) is specifically:

A) dissolving the supercritical extract with an organic solvent, performing silica gel column chromatography, eluting and separating with a first eluent, and concentrating to obtain a lignan concentrated fraction; the organic solvent is petroleum ether; the first eluent is petroleum ether;

B) dissolving the lignan concentrated fraction with a second organic solvent, performing silica gel column chromatography, eluting and separating with a second eluent, and concentrating to obtain schisandrin B concentrated fraction; the second organic solvent is a mixed solvent of petroleum ether and acetone; the volume ratio of the petroleum ether to the acetone in the second organic solvent is (90-97): (10-3); the second eluent is a mixed solvent of petroleum ether and acetone; the volume ratio of the petroleum ether to the acetone in the second eluent is (90-97): (10-3).

5. The extraction method according to claim 4, wherein the flow rate of the first eluent in the step A) is 3-7 ml/min; the flow rate of the second eluent in the step B) is 3-7 ml/min.

6. The method according to claim 1, wherein the volume ratio of hexane to isopropanol in the step S4) is 1: (1.5-2.5); the temperature of the low-temperature recrystallization is-10 ℃ to-30 ℃.

7. The method according to claim 1, wherein the volume ratio of hexane to isopropanol in the step S4) is 1: 2; the temperature of the low-temperature recrystallization is-20 ℃.

8. The extraction method according to claim 1, wherein the mass-to-volume ratio of the schisandrin B concentrated fraction to the mixed solvent in the step S4) is 1 g: (1-3) ml.

9. The extraction method of claim 1, wherein the crude schisandrin B is eluted and purified with 0-8 ℃ hexane.

10. The extraction method according to claim 1, wherein the schizandrin-b extract contains schizandrin-b as an index component in an amount of 25-95 wt%.

Technical Field

The invention belongs to the technical field of plant extraction, and particularly relates to a method for extracting a schisandrin B extract.

Background

Fructus Schisandrae chinensis is dried mature fruit of Schisandra chinensis of Magnoliaceae, and has astringent, qi invigorating, salivation promoting, kidney invigorating, and heart calming effects. Lignans compounds with biphenyl cyclooctadiene structure in schisandra are main bioactive components, mainly comprise schisandrin B, deoxyschizandrin, schizandrol A and the like, wherein the content of the schisandrin B is generally 0.1-0.4%.

According to records of book keeping, the schisandra chinensis has the effects of regulating skin, beautifying, prolonging life and the like. "Shen nong Ben Cao Jing" (Shen nong's herbal Jing) listed in Schisandra chinensis is the top grade; "Ben Cao gang mu" calls that Wu Wei Zi "is good for human body, and has rich nutrition, and has the functions of ruddy complexion and white skin. Li shizhen is said to be: wu Wei Zi is good for nourishing because it is difficult to be carried on the back. In recent years, researches show that schisandrin B not only has various biological activities of resisting tumor, protecting liver, resisting ulcer, resisting virus and the like, but also has better effect of eliminating free radicals. Scientific experiments prove that: schisandrin B can completely remove free radicals generated by aerobic metabolism, effectively remove free radicals generated by anaerobic metabolism, directly promote a human body to automatically activate antioxidant substances while removing the free radicals and inhibiting the formation of lipid peroxide, improve the self-oxidation resistance of the human body, and have more comprehensive, more lasting and more effective oxidation resistance effect. Therefore, the extract of the schisandra chinensis can be widely applied to various component preparations, health-care foods, medicines and skin care products.

The development and application of schisandrin B have important research values, but the source of schisandrin B is few, and the schisandrin B is mainly prepared by extracting and purifying from a schisandra chinensis medicinal material, and the schisandrin B obtained from the schisandra chinensis medicinal material has low content and more complex structural analogues, so that the research and development of a preparation process capable of industrially preparing and separating the schisandrin B have important research values.

Disclosure of Invention

In view of the above, the technical problem to be solved by the present invention is to provide a method for extracting schizandrin b extract, which has the advantages of simple process, mild operation conditions and good retention of active ingredients.

The invention provides a method for extracting a schisandrin B extract, which comprises the following steps:

s1) crushing the schisandra chinensis to obtain schisandra chinensis powder;

s2) carrying out supercritical carbon dioxide extraction on the schisandra chinensis powder to obtain a supercritical extract;

s3) dissolving the supercritical extract with an organic solvent, eluting and separating by silica gel column chromatography, and concentrating to obtain Schisandrin B concentrated fraction;

s4) dissolving the Schisandrin B concentrated fraction in a mixed solvent of hexane and isopropanol, and carrying out low-temperature crystallization to obtain a Schisandrin B crude product;

s5) purifying the crude schisandrin B product by hexane to obtain a schisandrin B extract.

Preferably, the mesh number of the schisandra chinensis powder is 30-50 meshes.

Preferably, the extraction pressure of the supercritical carbon dioxide extraction is 20-30 MPa; the extraction temperature is 30-50 ℃; the extraction time is 1.5-2.5 h.

Preferably, the step S3) is specifically:

A) dissolving the supercritical extract with an organic solvent, performing silica gel column chromatography, eluting and separating with a first eluent, and concentrating to obtain a lignan concentrated fraction; the organic solvent is petroleum ether; the first eluent is petroleum ether;

B) dissolving the lignan concentrated fraction with a second organic solvent, performing silica gel column chromatography, eluting and separating with a second eluent, and concentrating to obtain schisandrin B concentrated fraction; the second organic solvent is a mixed solvent of petroleum ether and acetone; the volume ratio of the petroleum ether to the acetone in the second organic solvent is (90-97): (10-3); the second eluent is a mixed solvent of petroleum ether and acetone; the volume ratio of the petroleum ether to the acetone in the second eluent is (90-97): (10-3).

Preferably, the flow rate of the first eluent in the step A) is 3-7 ml/min; the flow rate of the second eluent in the step B) is 3-7 ml/min.

Preferably, the volume ratio of hexane to isopropanol in the step S4) is 1: (1.5-2.5); the temperature of the low-temperature recrystallization is-10 ℃ to-30 ℃.

Preferably, the volume ratio of hexane to isopropanol in the step S4) is 1: 2; the temperature of the low-temperature recrystallization is-20 ℃.

Preferably, the mass-to-volume ratio of the schisandrin b concentrated fraction to the mixed solvent in the step S4) is 1 g: (1-3) ml.

Preferably, the crude schisandrin B is eluted and purified by using hexane at 0-8 ℃.

Preferably, the schisandrin B extract takes schisandrin B as an index component, and the content of the schisandrin B is 25-95 wt%.

The invention provides a method for extracting schisandrin B, which comprises the following steps: s1) crushing the schisandra chinensis to obtain schisandra chinensis powder; s2) carrying out supercritical carbon dioxide extraction on the schisandra chinensis powder to obtain a supercritical extract; s3) dissolving the supercritical extract with an organic solvent, eluting and separating by silica gel column chromatography, and concentrating to obtain Schisandrin B concentrated fraction; s4) dissolving the Schisandrin B concentrated fraction in a mixed solvent of hexane and isopropanol, and carrying out low-temperature recrystallization to obtain a Schisandrin B crude product; s5) purifying the crude schisandrin B with hexane to obtain schisandrin B. Compared with the prior art, the schisandrin B extract is obtained by sequentially performing supercritical carbon dioxide extraction technology, silica gel column chromatography and hexane purification, the preparation process is simple, the operation condition is mild, the active ingredient retention effect is good, the method is suitable for extracting natural products, is particularly suitable for extracting schisandrin B with low molecular weight, low polarity, lipophilicity and low boiling point, and is suitable for industrial production, and the prepared schisandrin B extract has high schisandrin B content.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. 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.

The invention provides a method for extracting a schisandrin B extract, which comprises the following steps: s1) crushing the schisandra chinensis to obtain schisandra chinensis powder; s2) carrying out supercritical carbon dioxide extraction on the schisandra chinensis powder to obtain a supercritical extract; s3) dissolving the supercritical extract with an organic solvent, eluting and separating by silica gel column chromatography, and concentrating to obtain Schisandrin B concentrated fraction; s4) dissolving the Schisandrin B concentrated fraction in a mixed solvent of hexane and isopropanol, and carrying out low-temperature recrystallization to obtain a Schisandrin B crude product; s5) purifying the crude schisandrin B product by hexane to obtain a schisandrin B extract.

The present invention is not particularly limited in terms of the source of all raw materials, and may be commercially available.

In the invention, preferably, the schisandra chinensis is aired and dried; the airing is preferably carried out outdoors; the airing time is preferably 1 to 3 days, and more preferably 2 days.

After impurities of the dried schisandra chinensis are preferably removed, crushing is carried out; the crushing is preferably carried out by a crusher; after crushing, screening by using a screen preferably to obtain schisandra chinensis powder; in order to reduce the scattering distance between the fluid and the solute in the natural mother body, the natural raw material should be subjected to crushing pretreatment. The raw material pretreatment requires uniform crushing, the mass transfer effect can be influenced by the excessive coarse or fine crushing, and the larger the granularity is, the smaller the material particles are. In the invention, the mesh number of the schisandra chinensis powder is preferably 30-50 meshes, and more preferably 40 meshes.

Performing supercritical carbon dioxide extraction on the schisandra chinensis powder to obtain a supercritical extract; extraction pressure is an important operating parameter in supercritical extraction processes. The pressure of the supercritical fluid is the main factor affecting the solubility of the extracted substance in the fluid, which directly affects the extraction efficiency, and therefore it is necessary to find the most suitable pressure for the extraction of a specific product. Supercritical fluids have a viscosity similar to gases, a density and a solvency similar to liquids. Generally, the diffusion coefficient of a solute in a solvent is proportional to the density of the solvent and inversely proportional to the viscosity of the solvent. The fluid density increases and the dissolving capacity increases with the increase of the pressure, and after a certain pressure is reached, the solvent density reaches a certain value, the influence of the pressure on the density becomes insignificant, and the increase of the extraction rate becomes gentle. Meanwhile, after the pressure reaches a certain value, the requirements on the pressure resistance and the sealing property of equipment are very strict, so that the extraction cost is obviously increased under the condition that the product yield is not changed greatly. Therefore, in the SFE process, when the extraction pressure satisfies a certain extraction rate, the extraction rate is not increased by the pressure increasing method. In the invention, the pressure of the supercritical carbon dioxide extraction is preferably 20-30 MPa; temperature is another very important and active parameter of the supercritical extraction process. On one hand, the influence of the extraction temperature on the solute extraction rate is divided into two aspects, namely that the solute thermal motion is accelerated due to the increase of the temperature, the vapor pressure is increased, and the supercritical CO causes₂Extraction is also a mass transfer process as other extraction processes, so that the increase of the vapor pressure of solute inevitably leads to the increase of the extraction rate; on the other hand, the solvent CO is caused by the increase of the temperature₂The density of (a) becomes smaller and the size of the density determines the dissolving capacity of the solute, so that the temperature is increased and the dissolving capacity is reduced. Thus, the temperature increase may cause an increase, a constant or a decrease in the yield of the extract(ii) a It is determined by the CO reduced by the temperature rise₂The two competing effects of density and increased diffusion coefficient are the result. In the invention, the extraction temperature is preferably 30-50 ℃; the extraction time is preferably 1.5-2.5 h. The obtained supercritical extract is preferably preserved in a dark place at a low temperature, and more preferably preserved in a brown bottle with a bottle stopper at a low temperature; the temperature of the low temperature is preferably 2 to 10 ℃.

The supercritical extraction has the obvious advantages of high efficiency and high yield, can selectively separate various substances, and can effectively reduce impurities and control the content. In addition, the method has the obvious advantages of short extraction time, short production period, convenient operation, low power consumption and high efficiency, and is very suitable for preparing the traditional Chinese medicinal materials in an industrialized and large scale.

Dissolving the supercritical extract with organic solvent, and eluting and separating by silica gel column chromatography; in order to increase the purity of elution, it is preferable to specifically: A) dissolving the supercritical extract with an organic solvent, performing silica gel column chromatography, eluting and separating with a first eluent, and concentrating to obtain a lignan concentrated fraction; b) Dissolving the lignan concentrated fraction with a second organic solvent, performing silica gel column chromatography, eluting and separating with a second eluent, and concentrating to obtain schisandrin B concentrated fraction; the mass-volume ratio of the supercritical extract to the organic solvent is preferably 1 g: (1-3) ml, more preferably 1 g: (1.5-2.5) ml, preferably 1 g: 2 ml; the organic solvent is preferably petroleum ether; the mass ratio of the supercritical extract to the silica gel column in the silica gel column chromatography is preferably 1: (1-3), more preferably 1: (1.5-2.5), and more preferably 1: 2; the first eluent is preferably petroleum ether; the flow rate of the first eluent is preferably 3-7 ml/min; in the present invention, when the first eluent is used for elution, TLC is preferably used for monitoring the effluent, and before schizandrin b is eluted, the effluent is preferably collected and concentrated to obtain a fatty oil concentrated fraction; concentrating the eluate containing schisandrin B to obtain lignanoid concentrated fraction; dissolving the concentrated lignan fraction in a second organic solvent; the mass-to-volume ratio of the lignan concentrated fraction to the second organic solvent is preferably 1 g: (0.5-1.5) ml, more preferably 1 g: 1 ml; the second organic solvent is preferably a mixed solvent of petroleum ether and acetone; the volume ratio of the petroleum ether to the acetone in the second organic solvent is preferably (90-97): (10-3), more preferably (90-95): (10-5), and more preferably (92-95): (8-5), most preferably 95: 5; after dissolution, the mixture is subjected to silica gel column chromatography and elution separation by using a second eluent; the mass ratio of the lignan-concentrated fraction to the silica gel column is preferably 1: (5-15), more preferably 1: (8-12), and more preferably 1: 10; the second eluent is preferably a mixed solvent of petroleum ether and acetone; the volume ratio of the petroleum ether to the acetone in the second eluent is preferably (90-97): (10-3), more preferably (90-95): (10-5), and more preferably (92-95): (8-5), most preferably 95: 5; the dosage of the second eluent is preferably 6-10 times of the volume of the silica gel column; the flow rate of the second eluent is preferably 3-7 ml/min; in the present invention, TLC is preferably used to monitor the effluent, which shows that single-point schizandrin B has been collected and elution is complete; concentrating the eluate to obtain Schisandrin B concentrated fraction.

The silica gel column chromatography has good adsorption function and separation function, convenient time control and strong pressure resistance. The column chromatography silica gel has good thermal stability and chemical stability, and can selectively adsorb and purify isomer components from multi-component solution.

Dissolving the Schisandrin B concentrated fraction in a mixed solvent of hexane and isopropanol, and performing low-temperature recrystallization to obtain a Schisandrin B crude product; wherein, the volume ratio of the hexane to the isopropanol is preferably 1: (1.5-2.5), more preferably 1: (1.8-2.2), and more preferably 1: 2; the mass volume ratio of the schisandrin B concentrated fraction to the mixed solvent is preferably 1 g: (1-3) ml, more preferably 1 g: (1.5-2.5) ml, preferably 1 g: 2 ml; the temperature of the low-temperature recrystallization is preferably-10 ℃ to-30 ℃, more preferably-15 ℃ to-25 ℃, and further preferably-20 ℃; the time for low-temperature recrystallization is preferably 8-15 hours, and is further preferably 10-12 hours; recrystallizing at low temperature, preferably vacuum filtering, and separating from the mother liquor to obtain crude schisandrin B; more preferably, the crude schisandrin B is obtained after filtration and washing by cold hexane.

Purifying the crude schisandrin B product by using hexane; the purification method is preferably leaching; more preferably, the Schizandrin B extract is obtained by eluting and purifying with 0-8 deg.C hexane, further preferably 0-5 deg.C hexane, further preferably 2-4 deg.C hexane, and most preferably 3 deg.C hexane.

According to the invention, Schisandrin B is preferably used as a reference substance, and the content of Schisandrin B in the Schisandrin B extract is detected by HPLC analysis; the schisandrin B extract takes schisandrin B as an index component, and the content of the schisandrin B is preferably 25-95 wt%; the HPLC column is preferably a Nova-PakC184 μm (3.9 mm. times.300 mm) column; the mobile phase is preferably a mixed solution of acetonitrile and water; the volume ratio of the acetonitrile to the water is preferably (50-60): (50-40), more preferably (53-57): (47-47), preferably 55:45, a first step of; the fluidity of the mobile phase is preferably 0.5-2 ml/min, more preferably 0.5-1.5 ml/min, and still more preferably 1 ml/min; the elution is preferably monitored during HPLC using UV absorbance at 254 nm.

The schisandrin B extract is obtained by sequentially performing supercritical carbon dioxide extraction technology, silica gel column chromatography and hexane purification, the preparation process is simple, the operation condition is mild, the active ingredient retention effect is good, the method is suitable for extracting natural products, is particularly suitable for extracting schisandrin B with low molecular weight, low polarity, lipophilicity and low boiling point, and is suitable for industrial production, and the prepared schisandrin B extract has high schisandrin B content.

In order to further illustrate the present invention, the following will describe the extraction method of schizandrin b extract provided by the present invention in detail with reference to the examples.

The reagents used in the following examples are all commercially available.