Extraction process of polysaccharide in cistanche

Abstract

The invention discloses an extraction process of polysaccharide in cistanche, and belongs to the technical field of separation and purification, the extraction process provided by the invention comprises the following steps: taking fleshy stems of cistanche as a raw material, degreasing, and further extracting the polysaccharide in the cistanche through high-low-frequency ultrasonic circulating treatment under specific conditions in the presence of compound enzyme. Further adding a composite solvent for multiple times and performing precipitation treatment for multiple times to finally obtain the cistanche deserticola polysaccharide. The extraction process is beneficial to reduction of the loss of the cistanche polysaccharide and further removal of impurities, so that the yield of the cistanche polysaccharide and the purity of the cistanche polysaccharide are improved.

Description

Technical Field

[0001] The invention belongs to the technical field of separation and purification, and particularly relates to a process for extracting polysaccharides from Cistanche deserticola. Background Art

[0002] Cistanche deserticola is also known as Cistanche deserticola and Cunyun. Cistanche deserticola has always been known as the "desert ginseng". As a traditional precious Chinese medicinal material, Cistanche deserticola has high medicinal value.

[0003] Cistanche polysaccharide is an effective component in Cistanche deserticola. It can enhance the activity of immune cells, improve the body's defense against pathogens, and regulate the balance of the immune system. It can also protect cells from aging by removing free radicals and reducing oxidative damage, and delay the aging process of skin and organs. Cistanche polysaccharide also has the effects of anti-virus and anti-cancer, lowering blood sugar, moisturizing the intestines and promoting bowel movements, protecting cardiovascular and cerebrovascular vessels, and resisting gastric ulcers.

[0004] In the prior art, water extraction, alcohol extraction or ultrasound-assisted extraction is often used to extract Cistanche deserticola polysaccharide from Cistanche deserticola. The corresponding yield of Cistanche deserticola polysaccharide is generally lower than 45%, and the purity of Cistanche deserticola polysaccharide is relatively low.

[0005] Therefore, there is an urgent need to provide a new Cistanche polysaccharide extraction process, which has a high yield of Cistanche polysaccharide and further improves the purity of Cistanche polysaccharide. Summary of the invention

[0006] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention proposes a process for extracting polysaccharides from Cistanche deserticola. The yield of Cistanche deserticola polysaccharides corresponding to the extraction process of the present invention is high, for example, it can exceed 52%, and further, the purity of Cistanche deserticola polysaccharides is improved, and the mass fraction is greater than 55%.

[0007] In order to achieve the above object, the present invention is implemented through the following technical solutions.

[0008] The process provided by the present invention is to use the fleshy stem of Cistanche deserticola as raw material, and has been subjected to defatting treatment. In the presence of a composite enzyme, the polysaccharide in Cistanche deserticola is further extracted by high-low frequency ultrasonic wave circulation treatment. High-frequency ultrasonic wave is conducive to the rupture of cell wall, and low-frequency ultrasonic wave is conducive to the release of Cistanche deserticola polysaccharide after the cell wall is ruptured, so that multiple cycles are carried out, and the effect of the composite enzyme is coordinated, which is conducive to fully obtaining the polysaccharide in Cistanche deserticola, thereby improving the Cistanche deserticola polysaccharide yield. The obtained concentrated solution is further conducive to reducing the loss of Cistanche deserticola polysaccharide by adding a composite solvent of a specific volume fraction and multiple precipitation processes, and is conducive to the further removal of impurities, thereby improving the purity of Cistanche deserticola polysaccharide.

[0009] A process for extracting polysaccharides from Cistanche deserticola comprises the following steps: (1) taking the fleshy stem of Cistanche deserticola, drying and crushing it, defatting it with a composite solvent, filtering it, obtaining a filter residue, and setting it aside; (2) mixing the filter residue, complex enzyme, water and ethanol, treating them under pulsed ultrasound, and then centrifuging to remove the residue to obtain a supernatant, and concentrating the supernatant under reduced pressure to obtain a concentrated solution; (3) adding the composite solvent to the concentrated solution for the first time to perform a first precipitation to obtain a first precipitate, then adding the composite solvent to the first precipitate for the second time to perform a second precipitation to obtain a second precipitate, then adding the composite solvent to the second precipitate for the third time to perform a third precipitation to obtain a third precipitate, freeze-drying the precipitate, washing the precipitate, and obtaining Cistanche deserticola polysaccharide; In step (1), the composite solvent includes ethyl acetate, ethanol and propanol; In step (2), the process of treating under pulsed ultrasound is to first treat under 30-40 kHz ultrasound for 5-10 minutes, then treat under 85-95 kHz ultrasound for 1-3 minutes, and then treat under 30-40 kHz ultrasound for 5-10 minutes, and repeat this process for more than 2 times; In step (3), the volume fractions of ethanol and ethyl acetate in the composite solvent used three times gradually increase.

[0010] Preferably, in step (1), the drying temperature is 80-100° C., and the drying time is 8-24 hours.

[0011] Preferably, in step (1), the pulverization is performed to a mesh size of 100-150 mesh, more preferably 120-140 mesh.

[0012] Preferably, in step (1), the volume ratio of ethyl acetate, ethanol and propanol is 1: (0.5-1.5): (0.2-1.0), and more preferably 1: (0.8-1.2): (0.4-0.8).

[0013] Preferably, in step (1), the degreasing treatment is carried out at a temperature of 65-80° C. and for a time of 2-5 hours.

[0014] Preferably, in step (2), the ratio of the mass of the filter residue to the total volume of water and ethanol is 1 g: (20-60) mL, more preferably 1 g: (30-50) mL.

[0015] Preferably, in step (2), the complex enzyme includes at least one of papain, cellulase and pectinase.

[0016] Further preferably, the complex enzyme comprises papain, cellulase and pectinase, and the mass ratio of papain, cellulase and pectinase is 1:(0.1-0.8):(0.2-1.2).

[0017] Preferably, in step (2), after the filter residue, the composite enzyme, water and ethanol are mixed, hydrochloric acid is added to adjust the pH of the mixture to 6-7. This pH is conducive to the enzymatic hydrolysis of the composite enzyme.

[0018] Preferably, in step (2), the mass of the composite enzyme is 0.01% to 0.1% of the mass of the filter residue, and more preferably 0.1% to 0.8%.

[0019] Preferably, in step (2), the supernatant is concentrated under reduced pressure to 1 / 4-1 / 5 of the initial volume of the supernatant.

[0020] Preferably, in step (2), the temperature for treatment under pulsed ultrasound is 35-45°C, more preferably 35-40°C.

[0021] Preferably, in step (2), when the ultrasonic wave is treated at 30-40 kHz, the corresponding ultrasonic power is 800-1000 W, and when the ultrasonic wave is treated at 85-95 kHz, the corresponding ultrasonic power is 400-500 W. Ultrasonic waves of different frequencies corresponding to different ultrasonic powers are beneficial to reducing the breakage of polysaccharide chains in the polysaccharide, thereby contributing to the improvement of the final polysaccharide yield.

[0022] Preferably, in step (3), the first precipitation time is 6-10 hours, the second precipitation time is 4-6 hours, and the third precipitation time is 1-3 hours.

[0023] Preferably, in step (3), the composite solvent added for the first time is composed of ethyl acetate, ethanol and propanol in a volume ratio of 1: (0.8-1.2): (0.8-1.0).

[0024] Preferably, in step (3), the composite solvent added for the second time is composed of ethyl acetate, ethanol and propanol in a volume ratio of 1: (0.8-1.2): (0.5-0.7).

[0025] Preferably, in step (3), the composite solvent added for the third time is composed of ethyl acetate, ethanol and propanol in a volume ratio of 1: (0.8-1.2): (0.2-0.3).

[0026] Preferably, in step (3), the washing is performed with acetone and anhydrous ether respectively for 1 to 3 times.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows: The process provided by the present invention uses the fleshy stem of Cistanche deserticola as raw material, undergoes a defatting treatment, and further extracts polysaccharides in Cistanche deserticola by high- and low-frequency ultrasonic cyclic treatment in the presence of a composite enzyme. High-frequency ultrasound is conducive to the rupture of cell walls, and low-frequency ultrasound is conducive to the release of Cistanche deserticola polysaccharides after the cell walls are ruptured. The obtained concentrated solution is further added with a composite solvent of a specific volume fraction and subjected to multiple precipitation treatments, which is conducive to reducing the loss of Cistanche deserticola polysaccharides and further removing impurities, thereby improving the yield of Cistanche deserticola polysaccharides and improving the purity of Cistanche deserticola polysaccharides. DETAILED DESCRIPTION

[0028] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] Unless otherwise specified, the raw materials, reagents or devices used in the following examples can be obtained from conventional commercial sources or by existing known methods.

[0030] The Cistanche deserticola used in the following examples is Inner Mongolian Cistanche deserticola.

[0031] Example 1

[0032] A process for extracting polysaccharides from Cistanche deserticola comprises the following steps: (1) taking the fleshy stem of Cistanche deserticola, drying it (drying temperature is 90° C., drying time is 10 hours), crushing it to a mesh size of 100-120, degreasing it with a composite solvent, the degreasing temperature is 70° C., the time is 3 hours, filtering, obtaining a filter residue, and setting it aside; (2) Mixing the filter residue, composite enzyme (the composite enzyme is composed of papain, cellulase and pectinase, the mass ratio of papain, cellulase and pectinase is 1:0.5:0.5, and the mass of the composite enzyme is 0.05% of the mass of the filter residue), water and ethanol, adding hydrochloric acid to the obtained mixture to adjust the pH of the mixture to 6.5, the mass ratio of the filter residue to the total volume of water and ethanol is 1 g:45 mL, treating the mixture under pulsed ultrasonic wave, and then centrifuging to remove the residue to obtain a supernatant, and concentrating the supernatant under reduced pressure to 1 / 5 of the initial volume of the supernatant to obtain a concentrated solution; (3) adding the composite solvent to the concentrated solution for the first time to perform a first precipitation to obtain a first precipitate, then adding the composite solvent to the first precipitate for the second time to perform a second precipitation to obtain a second precipitate, then adding the composite solvent to the second precipitate for the third time to perform a third precipitation to obtain a third precipitate, and after freeze-drying, washing with acetone and anhydrous ether once respectively, and then drying at 60° C. for 4 hours to obtain Cistanche deserticola polysaccharide; In step (1), the composite solvent includes ethyl acetate, ethanol and propanol, and the volume ratio of ethyl acetate, ethanol and propanol is 1:1:0.4; In step (2), the process of treating under pulsed ultrasound is to first treat under 40 kHz ultrasound for 5 minutes, then treat under 85 kHz ultrasound for 2 minutes, and then treat under 40 kHz ultrasound for 5 minutes, and repeat this process twice; The temperature for treatment under pulsed ultrasound was 40°C; When the ultrasonic wave is 40kHz or 85KHz, the corresponding ultrasonic power is 1000W. The first precipitation time was 8 hours, the second precipitation time was 5 hours, and the third precipitation time was 2 hours; The composite solvent added for the first time is composed of ethyl acetate, ethanol and propanol in a volume ratio of 1:1:0.8; The composite solvent added for the second time is composed of ethyl acetate, ethanol and propanol in a volume ratio of 1:1:0.6; The composite solvent added for the third time consists of ethyl acetate, ethanol and propanol in a volume ratio of 1:1:0.2.

[0033] Example 2

[0034] A process for extracting polysaccharides from Cistanche deserticola comprises the following steps: (1) taking the fleshy stem of Cistanche deserticola, drying it (drying temperature is 100° C., drying time is 8 hours), crushing it to a mesh size of 120-130, degreasing it with a composite solvent, degreasing it at a temperature of 75° C., time is 3 hours, filtering it, obtaining a filter residue, and setting it aside; (2) Mixing the filter residue, composite enzyme (the composite enzyme is composed of papain, cellulase and pectinase, the mass ratio of papain, cellulase and pectinase is 1:0.6:0.6, and the mass of the composite enzyme is 0.1% of the mass of the filter residue), water and ethanol, adding hydrochloric acid to the obtained mixture to adjust the pH of the mixture to 6, the mass ratio of the filter residue to the total volume of water and ethanol is 1 g:50 mL, treating the mixture under pulsed ultrasonic wave, and then centrifuging to remove the residue to obtain a supernatant, and concentrating the supernatant under reduced pressure to 1 / 5 of the initial volume of the supernatant to obtain a concentrated solution; (3) adding the composite solvent to the concentrated solution for the first time to perform a first precipitation to obtain a first precipitate, then adding the composite solvent to the first precipitate for the second time to perform a second precipitation to obtain a second precipitate, then adding the composite solvent to the second precipitate for the third time to perform a third precipitation to obtain a third precipitate, and after freeze-drying, washing with acetone and anhydrous ether once respectively, and then drying at 60° C. for 4 hours to obtain Cistanche deserticola polysaccharide; In step (1), the composite solvent includes ethyl acetate, ethanol and propanol, and the volume ratio of ethyl acetate, ethanol and propanol is 1:1:0.4; In step (2), the process of treating under pulsed ultrasound is to first treat under 35 kHz ultrasound for 8 minutes, then treat under 90 kHz ultrasound for 1 minute, and then treat under 35 kHz ultrasound for 8 minutes, and repeat this process three times; The temperature for treatment under pulsed ultrasound was 40°C; When the ultrasonic wave is 35kHz or 90KHz, the corresponding ultrasonic power is 1000W. The first precipitation time was 9 hours, the second precipitation time was 4 hours, and the third precipitation time was 2 hours; The composite solvent added for the first time is composed of ethyl acetate, ethanol and propanol in a volume ratio of 1:0.9:0.8; The composite solvent added for the second time is composed of ethyl acetate, ethanol and propanol in a volume ratio of 1:0.8:0.6; The composite solvent added for the third time is composed of ethyl acetate, ethanol and propanol in a volume ratio of 1:0.8:0.2.

[0035] Example 3

[0036] A process for extracting polysaccharides from Cistanche deserticola comprises the following steps: (1) taking the fleshy stem of Cistanche deserticola, drying it (drying temperature is 90° C., drying time is 10 hours), crushing it to a mesh size of 100-120, degreasing it with a composite solvent, the degreasing temperature is 70° C., the time is 3 hours, filtering, obtaining a filter residue, and setting it aside; (2) Mixing the filter residue, composite enzyme (the composite enzyme is composed of papain, cellulase and pectinase, the mass ratio of papain, cellulase and pectinase is 1:0.5:0.5, and the mass of the composite enzyme is 0.05% of the mass of the filter residue), water and ethanol, adding hydrochloric acid to the obtained mixture to adjust the pH of the mixture to 6.5, the mass ratio of the filter residue to the total volume of water and ethanol is 1 g:45 mL, treating the mixture under pulsed ultrasonic wave, and then centrifuging to remove the residue to obtain a supernatant, and concentrating the supernatant under reduced pressure to 1 / 5 of the initial volume of the supernatant to obtain a concentrated solution; (3) adding the composite solvent to the concentrated solution for the first time to perform a first precipitation to obtain a first precipitate, then adding the composite solvent to the first precipitate for the second time to perform a second precipitation to obtain a second precipitate, then adding the composite solvent to the second precipitate for the third time to perform a third precipitation to obtain a third precipitate, and after freeze-drying, washing with acetone and anhydrous ether once respectively, and then drying at 60° C. for 4 hours to obtain Cistanche deserticola polysaccharide; In step (1), the composite solvent includes ethyl acetate, ethanol and propanol, and the volume ratio of ethyl acetate, ethanol and propanol is 1:1:0.4; In step (2), the process of treating under pulsed ultrasound is to first treat under 40 kHz ultrasound for 5 minutes, then treat under 85 kHz ultrasound for 2 minutes, and then treat under 40 kHz ultrasound for 5 minutes, and repeat this process twice; The temperature for treatment under pulsed ultrasound was 40°C; When the ultrasonic wave is used for treatment at 40kHz, the corresponding ultrasonic power is 800W; when the ultrasonic wave is used for treatment at 85kHz, the corresponding ultrasonic power is 400W. The first precipitation time was 8 hours, the second precipitation time was 5 hours, and the third precipitation time was 2 hours; The composite solvent added for the first time is composed of ethyl acetate, ethanol and propanol in a volume ratio of 1:1:0.8; The composite solvent added for the second time is composed of ethyl acetate, ethanol and propanol in a volume ratio of 1:1:0.6; The composite solvent added for the third time consists of ethyl acetate, ethanol and propanol in a volume ratio of 1:1:0.2.

[0037] Comparative Example 1 Compared with Example 1, the difference of Comparative Example 1 is that in step (2) of Comparative Example 1, the process of treating under pulsed ultrasound is to first treat under 60 kHz ultrasound for 5 minutes, then treat under 70 kHz ultrasound for 2 minutes, and then treat under 60 kHz ultrasound for 5 minutes, and repeat this process twice; the other processes are the same as those of Example 1.

[0038] Comparative Example 2 Compared with Example 1, the difference of Comparative Example 2 is that in step (1) of Comparative Example 2, the composite solvent is composed of ethanol only, and in step (2), the composite enzyme is composed of papain and cellulase, and the mass ratio of papain to cellulase is 1:1.2.

[0039] Comparative Example 3 Compared with Example 1, the difference in Comparative Example 3 is only that step (3) of Comparative Example 3 is different from that of Example 1, that is, only one precipitation is carried out in step (3) of Comparative Example 3. The content of step (3) of Comparative Example 3 is as follows: The composite solvent was added to the concentrated solution for the first time for the first precipitation to obtain the first precipitate. After freeze-drying, it was washed once with acetone and anhydrous ether in turn, and then dried at 60 °C for 4 hours to obtain cistanche polysaccharide.

[0040] Product effect test Calculated according to the cistanche polysaccharide yield = mass of dried cistanche polysaccharide / mass of dried cistanche fleshy stem * 100%, the cistanche polysaccharide yields corresponding to the above examples and comparative examples are shown in Table 1:

[0041] As can be seen from Table 1, the extraction processes of Examples 1-3 can significantly improve the yield of cistanche polysaccharide compared with Comparative Examples 1-3. From the results of Example 1 and Comparative Examples 1-3, it can be seen that when changing the pulsed ultrasonic conditions or the composition of the composite solvent and composite enzyme in the extraction process, the yield of cistanche polysaccharide is likely to decrease.

[0042] Using the conventional spectrophotometry combined with chromatographic techniques, the purities (mass fractions) of the cistanche polysaccharides obtained in Example 1 and Comparative Example 3 were 58.6% and 49.7% respectively. Thus, it can be seen that the process of three precipitation treatments in step (3) of the extraction process of the present invention can improve the purity of cistanche polysaccharide.

[0043] It should be noted that in this article, terms such as "including", "comprising" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements not only includes those elements, but also includes other elements not explicitly listed, or also includes elements inherent to such a process, method, article or device.

[0044] Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A process for extracting polysaccharides from Cistanche deserticola, characterized in that: The following steps are involved: (1) taking the fleshy stem of Cistanche deserticola, drying and crushing it, defatting it with a composite solvent, filtering it, obtaining a filter residue, and setting it aside; (2) mixing the filter residue, complex enzyme, water and ethanol, treating them under pulsed ultrasound, and then centrifuging to remove the residue to obtain a supernatant, and concentrating the supernatant under reduced pressure to obtain a concentrated solution; (3) adding the composite solvent to the concentrated solution for the first time to perform a first precipitation to obtain a first precipitate, then adding the composite solvent to the first precipitate for the second time to perform a second precipitation to obtain a second precipitate, then adding the composite solvent to the second precipitate for the third time to perform a third precipitation to obtain a third precipitate, freeze-drying the precipitate, washing the precipitate, and obtaining Cistanche deserticola polysaccharide; In step (1), the composite solvent includes ethyl acetate, ethanol and propanol; In step (2), the process of treating under pulsed ultrasound is to first treat under 30-40 kHz ultrasound for 5-10 minutes, then treat under 85-95 kHz ultrasound for 1-3 minutes, and then treat under 30-40 kHz ultrasound for 5-10 minutes, and repeat this process for more than 2 times; In step (3), the volume fractions of ethanol and ethyl acetate in the composite solvent used three times gradually increase.

2. The extraction process according to claim 1, characterized in that In step (1), the drying temperature is 80-100° C. and the drying time is 8-24 hours; and / or the pulverization is performed to a mesh size of 100-150 meshes.

3. The extraction process according to claim 1, characterized in that In step (1), the volume ratio of ethyl acetate, ethanol and propanol is 1: (0.5-1.5): (0.2-1.0).

4. The extraction process according to claim 1, characterized in that In step (1), the degreasing treatment is carried out at a temperature of 65-80°C and for a time of 2-5 hours.

5. The extraction process according to claim 1, characterized in that In step (2), the ratio of the mass of the filter residue to the total volume of water and ethanol is 1 g: (20-60) mL.

6. The extraction process according to claim 1, characterized in that In step (2), the complex enzyme includes at least one of papain, cellulase and pectinase.

7. The extraction process according to claim 1, characterized in that In step (2), the mass of the complex enzyme is 0.01% to 0.1% of the mass of the filter residue.

8. The extraction process according to any one of claims 1 to 7, characterized in that: In step (2), the temperature for treatment under pulsed ultrasonic wave is 35-45°C.

9. The extraction process according to claim 8, characterized in that In step (3), the first precipitation time is 6-10 hours, the second precipitation time is 4-6 hours, and the third precipitation time is 1-3 hours.

10. The extraction process according to claim 8, characterized in that In step (3), the composite solvent added for the first time is composed of ethyl acetate, ethanol and propanol in a volume ratio of 1: (0.8-1.2): (0.8-1.0); and / or, the composite solvent added for the second time is composed of ethyl acetate, ethanol and propanol in a volume ratio of 1: (0.8-1.2): (0.5-0.7); and / or, the composite solvent added for the third time is composed of ethyl acetate, ethanol and propanol in a volume ratio of 1: (0.8-1.2): (0.2-0.3).

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