CN113512084B - Method for extracting stigmasterol from coix seed stems and leaves by combining ultrasonic countercurrent with biological enzyme - Google Patents

Abstract

The invention discloses a method for extracting stigmasterol from coix stems and leaves by combining ultrasonic countercurrent with biological enzyme, which comprises the steps of adding coix seed powder into a solution containing quicklime and sodium thiosulfate for soaking, filtering, putting coix seed powder and water into an ultrasonic countercurrent extraction tank, adding biological enzyme, carrying out ultrasonic countercurrent extraction at different temperatures, and filtering to obtain leaching liquor; concentrating the leaching solution under reduced pressure, dissolving the extract in an organic solvent containing sodium hydroxide and sodium tripolyphosphate, and performing saponification reaction; extracting the saponified product with PBS buffer solution and acetonitrile, discarding the water phase, concentrating under reduced pressure, and recovering acetonitrile to obtain crude extract; and filling the crude extract into polyacrylamide gel column chromatography, eluting with an eluent, collecting stigmasterol eluent, concentrating and recovering the eluent, filtering and crystallizing the obtained stigmasterol product to obtain the stigmasterol product. The method has the advantages of high extraction rate, high extraction efficiency, simpler process, low production cost, product purity reaching more than 99 percent, and the like.

Description

A method for extracting stigmasterol from coix stems and leaves using ultrasonic countercurrent combined with biological enzymes

Technical field

The invention relates to the technical field of extracting effective plant ingredients, specifically a method for extracting stigmasterol from Coix stems and leaves using ultrasonic countercurrent combined with biological enzymes.

Background technique

Coixlachryma-jobi L. belongs to the genus Coix of the Gramineae family. It has a sweet taste and a slightly cold nature. It has the effects of strengthening the spleen, nourishing the lungs, clearing away heat and diuresis, and is known as the "King of Nourishing and Health Care". At present, there are relatively few studies on the chemical components of coix, and most of them focus on coix kernel. There are not many literature reports on its pharmacological activity, and the basis of bioactive substances is unclear, resulting in its clinical application being limited. Research has proven that Coix leaves also contain chemical components such as steroids, polysaccharides, flavonoids, organic acids, sheaths, and triterpenes. Therefore, studying how to extract stigmasterol from Coix leaves can turn waste into treasure and increase the value of the whole Coix plant.

Stigmasterol (stigmaster01), C29H450. The molecular weight is 412, also known as stigmaster-5,22-diene-3B-ol or 24R-ethylcholesteryl-5,22-diene-3B-ol. It is a plant sterol that is widely present in a variety of plants. middle. Stigasterol has significant pharmacological activities such as antioxidant, anti-tumor, and anti-cardiovascular diseases. It is also the starting material for the synthesis of steroid drugs and is used in pharmaceutical, food, chemical and other industries. In recent years, with the discovery of new steroid drugs, the development of production, and the expansion of production scale, the demand for raw materials has gradually increased; on the other hand, due to unreasonable mining, resource shortages have led to a decline in saponin content in plants, which has made the international market There is a contradiction between supply and demand. How to obtain more and higher-purity stigmasterol is an important direction in stigmasterol research.

The applicant has previously provided a method for extracting stigmasterol from Coix stems, which includes steps such as enzymatic hydrolysis, ultrasonic extraction, saponification, urea column chromatography and crystallization. However, during the test process, the applicant found that using this method to extract stigmasterol from Coix leaves still has shortcomings such as more impurities, low product purity, low extraction rate, and low production efficiency. Therefore, the present invention makes better improvements to the original process.

Contents of the invention

The object of the present invention is to provide a method for extracting stigmasterol from Coix stems and leaves using ultrasonic countercurrent combined with biological enzymes. The method of the present invention uses ultrasonic countercurrent combined with biological enzymes to extract stigmasterol, which has the advantages of high extraction rate, high product purity, high extraction efficiency, relatively simple process, and low production cost.

In order to achieve the above objects, the technical solutions adopted by the present invention are as follows:

A method for extracting stigmasterol from coix stems and leaves using ultrasonic countercurrent combined with biological enzymes, including the following steps:

(1) Clean the coix stems or leaves, crush them to 40-100 mesh, and sift to obtain coix powder. First, add the coix powder to a solution containing quicklime and sodium thiosulfate and soak it for 30-60 minutes, filter, and then sieve the coix powder. and water, then put it into an ultrasonic counter-current extraction tank, add biological enzymes, conduct ultrasonic counter-current extraction at different temperatures, and filter to obtain a leach liquid; the biological enzymes are composed of cellulase, pectinase and xylanase;

(2) The extract is concentrated under reduced pressure to obtain an extract, which is then dissolved in an organic solvent with sodium hydroxide and sodium tripolyphosphate, and heated to 50-70°C for a saponification reaction of 60-90 minutes to obtain saponification. product;

(3) Extract the saponified product with PBS buffer and acetonitrile, discard the water phase, and concentrate under reduced pressure to recover acetonitrile to obtain a crude extract;

(4) Fill the crude extract into polyacrylamide gel column chromatography, elute with an eluent, collect the stigmasterol eluate, concentrate and recover the eluate, and the obtained stigmasterol product is filtered and crystallized, that is Stigmesterol products are available.

Further, the mass ratio of quicklime and sodium thiosulfate is 2-4:1; the amount added is such that the mass concentration of the soaking solution is 3-5%.

Further, the ultrasonic counter-current extraction is performed at a temperature of 50-60°C for 1-2 hours, then the temperature is raised to 65-70°C for 1.0-1.5 hours, and finally the temperature is raised to 75-80°C for 0.5-1.0 hours.

Further, the added amount of the biological enzyme is 0.5-1.5% of the weight of coix seed powder.

Further, the mass ratio of cellulase, pectinase and xylanase in the biological enzyme is 3-5:1-3:0.5-1.

Further, the addition amounts of sodium hydroxide and sodium tripolyphosphate described in step (2) are 10-15% and 3-5% of the mass of the organic solvent respectively.

Further, the organic solvent consists of tetrahydrofuran and ethanol in a volume ratio of 1:3-5.

Further, the volume ratio of the PBS buffer and acetonitrile is 1:5-10.

Further, the pH of the PBS buffer is 5.8.

Further, the eluent consists of phosphoric acid solution and ethyl acetate with a volume ratio of 1:10-15.

Compared with the existing technology, the advantages and beneficial effects of the present invention are:

1. The present invention uses Coix peel stems or leaves as raw materials, first soaks them in quicklime and sodium thiosulfate, adds an ultrasonic countercurrent extraction tank and combines biological enzymes to extract the extract, and then extracts stigmasterol through saponification reaction, column chromatography and other steps. The method has the advantages of high extraction rate, high extraction efficiency, relatively simple process, low production cost, and product purity of more than 99%.

2. The method of the present invention adds coix powder to quicklime and sodium thiosulfate solution and soaks it before ultrasonic counterflow, which can promote the rupture of the cell wall of coix stems or leaves, making it easier to extract stigmasterol and improve the extraction rate.

3. This method carries out saponification reaction in sodium hydroxide and sodium tripolyphosphate solution, and then extracts it with PBS buffer and acetonitrile. The added sodium tripolyphosphate can not only promote the saponification reaction, but also play a role in adsorbing and purifying impurities; Extraction with PBS buffer can neutralize the alkali in the saponification reaction and makes it easier to separate stigmasterol.

4. The present invention uses polyacrylamide gel column chromatography, which makes it easier to separate stigmasterol, and can play a role in adsorbing impurities and purifying it, and improving the purity of stigmasterol.

Detailed ways

The present invention will be further described in detail below in conjunction with specific embodiments. It should be emphasized that the following description is exemplary only and is not intended to limit the scope of the invention and its applications.

Example 1

A method for extracting stigmasterol from coix stems and leaves using ultrasonic countercurrent combined with biological enzymes, including the following steps:

(1) Clean the stems or leaves of Coix, crush them to 60 mesh, and sift to obtain coix powder. First, add the coix powder to a solution containing quicklime and sodium thiosulfate with a mass ratio of 3:1 (the mass concentration of the solution is 4%), soak for 60 minutes, filter, then put the coix powder and water into the ultrasonic countercurrent extraction tank, add cellulase, pectinase and xylanase biological enzymes with a mass ratio of 4:1:1, the biological enzymes The added amount is 1.2% of the weight of coix powder. Ultrasonic countercurrent extraction was carried out at a temperature of 60°C for 2 hours, then the temperature was raised to 70°C for 1.0 hour, and finally the temperature was raised to 75°C for 1.0 hour, and filtered to obtain the extract.

(2) The extract liquid is concentrated under reduced pressure at a pressure of -0.06MPa and a temperature of 80°C to obtain an extract, which is then dissolved in an organic solvent containing sodium hydroxide and sodium tripolyphosphate. The addition amounts of sodium and sodium tripolyphosphate are 13% and 3% of the mass of the organic solvent respectively. The organic solvent is composed of tetrahydrofuran and ethanol with a volume ratio of 1:3. The saponification reaction is heated to 60°C for 80 minutes to obtain a saponification product;

(3) Extract the saponified product with PBS buffer (pH 5.8) and acetonitrile with a volume ratio of 1:8, discard the water phase, and concentrate under reduced pressure to recover acetonitrile to obtain a crude extract;

(4) Fill the crude extract into polyacrylamide gel column chromatography, elute with an eluent consisting of a phosphoric acid solution and ethyl acetate with a volume ratio of 1:12, collect the stigmasterol eluate, concentrate and recover The eluent and the obtained stigmasterol product are filtered and crystallized to obtain the stigmasterol product.

After testing, the yield of stigmasterol in this example was 93.18%, and the purity of stigmasterol was 99.48%.

Example 2

A method for extracting stigmasterol from coix stems and leaves using ultrasonic countercurrent combined with biological enzymes, including the following steps:

(1) Clean the stems or leaves of Coix, crush them to 40 mesh, and sift to obtain coix powder. First, add the coix powder to a solution containing quicklime and sodium thiosulfate with a mass ratio of 2:1 (the mass concentration of the solution is 4.5%), soak for 50 minutes, filter, then put the coix powder and water into the ultrasonic countercurrent extraction tank, add cellulase, pectinase and xylanase biological enzymes with a mass ratio of 5:1.5:0.5, the biological enzymes The added amount is 1.4% of the weight of coix powder. Ultrasonic countercurrent extraction at a temperature of 55°C for 1.5h, then raising the temperature to 65°C for 1.5h, and finally raising the temperature to 75°C for 0.5 hour, filtering to obtain the extract.

(2) The extract is concentrated under reduced pressure at a pressure of -0.07MPa and a temperature of 75°C to obtain an extract, which is then dissolved in an organic solvent containing sodium hydroxide and sodium tripolyphosphate. The addition amounts of sodium and sodium tripolyphosphate are 15% and 3% of the mass of the organic solvent respectively. The organic solvent is composed of tetrahydrofuran and ethanol with a volume ratio of 1:4. The saponification reaction is heated to 70°C for 90 minutes to obtain a saponification product;

(3) Extract the saponified product with PBS buffer (pH 5.8) and acetonitrile with a volume ratio of 1:5, discard the water phase, and concentrate under reduced pressure to recover acetonitrile to obtain a crude extract;

(4) Fill the crude extract into polyacrylamide gel column chromatography, elute with an eluent consisting of a phosphoric acid solution and ethyl acetate with a volume ratio of 1:15, collect the stigmasterol eluate, concentrate and recover The eluent and the obtained stigmasterol product are filtered and crystallized to obtain the stigmasterol product.

After testing, the yield of stigmasterol in this example was 90.47%, and the purity of stigmasterol was 99.32%.

Example 3

A method for extracting stigmasterol from coix stems and leaves using ultrasonic countercurrent combined with biological enzymes, including the following steps:

(1) Clean the stems or leaves of Coix, crush them to 100 mesh, and sift to obtain coix powder. First, add the coix powder to a solution containing quicklime and sodium thiosulfate with a mass ratio of 3:1 (the mass concentration of the solution is 5%), soak for 40 minutes, filter, then put the coix powder and water into the ultrasonic countercurrent extraction tank, add cellulase, pectinase and xylanase biological enzymes with a mass ratio of 5:2:1, the biological enzymes The added amount is 1.2% of the weight of coix powder. Ultrasonic counter-current extraction at a temperature of 60°C for 1.5h, raising the temperature to 70°C for 1.5h, and finally raising the temperature to 75°C for 1.0 hour, filtering to obtain the extract.

(2) The extract liquid is concentrated under reduced pressure at a pressure of -0.06MPa and a temperature of 80°C to obtain an extract, which is then dissolved in an organic solvent containing sodium hydroxide and sodium tripolyphosphate. The addition amounts of sodium and sodium tripolyphosphate are 10% and 5% of the mass of the organic solvent respectively. The organic solvent is composed of tetrahydrofuran and ethanol with a volume ratio of 1:4. The saponification reaction is heated to 70°C for 70 minutes to obtain a saponification product;

(3) Extract the saponified product with PBS buffer (pH 5.8) and acetonitrile with a volume ratio of 1:10, discard the water phase, and concentrate under reduced pressure to recover acetonitrile to obtain a crude extract;

(4) Fill the crude extract into polyacrylamide gel column chromatography, elute with an eluent consisting of a phosphoric acid solution and ethyl acetate with a volume ratio of 1:10, collect the stigmasterol eluate, concentrate and recover The eluent and the obtained stigmasterol product are filtered and crystallized to obtain the stigmasterol product.

After testing, the yield of stigmasterol in this example was 89.65%, and the purity of stigmasterol was 99.38%.

Example 4

A method for extracting stigmasterol from coix stems and leaves using ultrasonic countercurrent combined with biological enzymes, including the following steps:

(1) Clean the stems or leaves of Coix, crush them to 60 mesh, and sift to obtain coix powder. First, add the coix powder to a solution containing quicklime and sodium thiosulfate with a mass ratio of 4:1 (the mass concentration of the solution is 4.0%), soak for 45 minutes, filter, then put the coix powder and water into the ultrasonic countercurrent extraction tank, add cellulase, pectinase and xylanase biological enzymes with a mass ratio of 4:1:1, the biological enzymes The added amount is 0.9% of the weight of coix powder. Ultrasonic countercurrent extraction was carried out at a temperature of 60°C for 2 hours, then the temperature was raised to 70°C for 1.0 hour, and finally the temperature was raised to 75°C for 1.0 hour, and filtered to obtain the extract.

(2) The extract is concentrated under reduced pressure at a pressure of -0.07MPa and a temperature of 75°C to obtain an extract, which is then dissolved in an organic solvent containing sodium hydroxide and sodium tripolyphosphate. The addition amounts of sodium and sodium tripolyphosphate are 12% and 3% of the mass of the organic solvent respectively. The organic solvent is composed of tetrahydrofuran and ethanol with a volume ratio of 1:4. The saponification reaction is heated to 70°C for 60 minutes to obtain a saponification product;

(3) Extract the saponified product with PBS buffer (pH 5.8) and acetonitrile with a volume ratio of 1:10, discard the water phase, and concentrate under reduced pressure to recover acetonitrile to obtain a crude extract;

(4) Fill the crude extract into polyacrylamide gel column chromatography, elute with an eluent consisting of a phosphoric acid solution and ethyl acetate with a volume ratio of 1:12, collect the stigmasterol eluate, concentrate and recover The eluent and the obtained stigmasterol product are filtered and crystallized to obtain the stigmasterol product.

After testing, the yield of stigmasterol in this example was 89.65%, and the purity of stigmasterol was 99.19%.

Example 5

A method for extracting stigmasterol from coix stems and leaves using ultrasonic countercurrent combined with biological enzymes, including the following steps:

(1) Clean the stems or leaves of Coix, crush them to 80 mesh, and sift to obtain coix powder. First, add the coix powder to a solution containing quicklime and sodium thiosulfate with a mass ratio of 2:1 (the mass concentration of the solution is 3.6%), soak for 40 minutes, filter, then put the coix powder and water into the ultrasonic countercurrent extraction tank, add cellulase, pectinase and xylanase biological enzymes with a mass ratio of 3:1:1, the biological enzymes The added amount is 1.5% of the weight of coix powder. Ultrasonic countercurrent extraction at a temperature of 55°C for 2 hours, raising the temperature to 70°C for 1.5 hours, finally raising the temperature to 75°C for 0.5 hours, and filtering to obtain the extract.

(2) The extract liquid is concentrated under reduced pressure at a pressure of -0.06MPa and a temperature of 80°C to obtain an extract, which is then dissolved in an organic solvent containing sodium hydroxide and sodium tripolyphosphate. The addition amounts of sodium and sodium tripolyphosphate are 13% and 4% of the mass of the organic solvent respectively. The organic solvent is composed of tetrahydrofuran and ethanol with a volume ratio of 1:4. The saponification reaction is heated to 70°C for 60 minutes to obtain a saponification product;

(3) Extract the saponified product with PBS buffer (pH 5.8) and acetonitrile in a volume ratio of 1:7, discard the water phase, and concentrate under reduced pressure to recover acetonitrile to obtain a crude extract;

(4) Fill the crude extract into polyacrylamide gel column chromatography, elute with an eluent consisting of a phosphoric acid solution and ethyl acetate with a volume ratio of 1:15, collect the stigmasterol eluate, concentrate and recover The eluent and the obtained stigmasterol product are filtered and crystallized to obtain the stigmasterol product.

After testing, the yield of stigmasterol in this example was 91.57%, and the purity of stigmasterol was 99.27%.

The above content is a further detailed description of the present invention in combination with specific/preferred embodiments, and it cannot be concluded that the specific implementation of the present invention is limited to these descriptions. For those of ordinary skill in the technical field to which the present invention belongs, they can also make several substitutions or modifications to the described embodiments without departing from the concept of the present invention, and these substitutions or modifications should be regarded as belong to the protection scope of the present invention.

Claims (4)

1. A method for extracting stigmasterol from coix stems and leaves using ultrasonic countercurrent combined with biological enzymes, which is characterized in that it includes the following steps: (1) Clean the coix stems or leaves, crush them to 40-100 mesh, and sift to obtain coix powder. First, add the coix powder to a solution containing quicklime and sodium thiosulfate and soak it for 30-60 minutes, filter, and then sieve the coix powder. and water, then put it into an ultrasonic counter-current extraction tank, add biological enzymes, conduct ultrasonic counter-current extraction at different temperatures, and filter to obtain a leach liquid; the biological enzymes are composed of cellulase, pectinase and xylanase; (2) The extract is concentrated under reduced pressure to obtain an extract, which is then dissolved in an organic solvent with sodium hydroxide and sodium tripolyphosphate, and heated to 50-70°C for a saponification reaction of 60-90 minutes to obtain saponification. product; (3) Extract the saponified product with PBS buffer and acetonitrile, discard the water phase, and concentrate under reduced pressure to recover acetonitrile to obtain a crude extract; (4) Fill the crude extract into polyacrylamide gel column chromatography, elute with an eluent, collect the stigmasterol eluate, concentrate and recover the eluate, and the obtained stigmasterol product is filtered and crystallized, that is Stigasterol products are available; The mass ratio of quicklime and sodium thiosulfate is 2-4:1; the amount added is such that the mass concentration of the soaking solution is 3-5%; The ultrasonic countercurrent extraction is performed at a temperature of 50-60°C for 1-2 hours, then the temperature is raised to 65-70°C for 1.0-1.5 hours, and finally the temperature is raised to 75-80°C for 0.5-1.0 hours; The addition amount of the biological enzyme is 0.5-1.5% of the weight of coix powder; The mass ratio of cellulase, pectinase and xylanase in the biological enzyme is 3-5:1-3:0.5-1; The addition amounts of sodium hydroxide and sodium tripolyphosphate described in step (2) are 10-15% and 3-5% of the mass of the organic solvent respectively; The organic solvent is composed of tetrahydrofuran and ethanol with a volume ratio of 1:3-5. 2. A method for extracting stigmasterol from coix stems and leaves using ultrasonic countercurrent combined with biological enzymes according to claim 1, characterized in that: the volume ratio of the PBS buffer and acetonitrile is 1:5-10. 3. A method for extracting stigmasterol from Coix stems and leaves using ultrasonic countercurrent combined with biological enzymes according to claim 1, characterized in that: the pH of the PBS buffer is 5.8. 4. A method for extracting stigmasterol from Coix stems and leaves using ultrasonic countercurrent combined with biological enzymes according to claim 1, characterized in that: the eluent is composed of a phosphoric acid solution and ethyl acetate with a volume ratio of 1:10-15. Composed of esters.