A Lycium barbarum polysaccharide, its preparation method and application in preparing a product with anti-aging effect

Abstract

The present invention discloses a wolfberry polysaccharide, a preparation method thereof, and an application thereof in the preparation of a product with anti-aging effect. The preparation method of the wolfberry polysaccharide comprises the following steps: S1. Take wolfberries, and then perform heating reflux extraction with water to obtain a wolfberry water extract, and concentrate the wolfberry water extract to obtain a wolfberry concentrated solution; S2. Add ethanol to the wolfberry concentrated solution, stand and filter, and take the precipitate to obtain crude wolfberry polysaccharide; S3. Mix the crude wolfberry polysaccharide with water to obtain a mixed solution, and perform ultrafiltration; Take the filtrate, concentrate and dry it to obtain a solid, and take the solid to obtain the wolfberry polysaccharide. Further discovery shows that the anti-aging effect of the wolfberry polysaccharide obtained by further eluting the wolfberry polysaccharide passing through the ultrafiltration membrane with a macroporous resin column has been further greatly improved. Research shows that the wolfberry polysaccharide prepared by the present invention has a much higher anti-aging effect than the crude wolfberry polysaccharide prepared by the water extraction and alcohol precipitation method, and has a very excellent anti-aging effect.

Description

Technical Field

[0001] The present invention relates to the technical field of natural products, and particularly relates to a wolfberry polysaccharide, a preparation method thereof, and an application thereof in the preparation of a product with anti-aging effect. Background Art

[0002] Aging is one of the basic characteristics of life, a degenerative change that inevitably occurs in the life cycle, and the result of the combined action of many factors inside and outside the body. In the process of exploring the mechanism of aging, many scholars have successively proposed various theories of aging, including the oxidative stress theory, the immune theory, the free radical theory, the neuroendocrine theory, etc. Since the 20th century, with the development of social economy and the progress of science and technology, the average life expectancy of humans has gradually increased, and the aging trend of the world's population structure has become increasingly obvious. An era of population aging has arrived. With the increase in age, a series of systemic and multi-faceted degenerative changes will inevitably occur in the human body in terms of morphological structure and physiological function, such as skin atrophy, bone loss, atherosclerosis, senile dementia, etc. At that time, the huge medical expenses of the elderly will become a heavy burden on each country. Improving the health level of the elderly is the main way to solve this problem.

[0003] Wolfberry is a deciduous shrub plant of the genus Lycium in the Solanaceae family. Wolfberry fruit is the mature fruit of wolfberry and is a traditional anti-aging tonic, being listed as one of the top anti-aging foods. Research shows that wolfberry polysaccharide is the main active ingredient in wolfberry for anti-aging. However, the anti-aging effect of the crude wolfberry polysaccharide prepared by the water extraction and alcohol precipitation method needs to be further improved. Therefore, it has important application value to further prepare wolfberry polysaccharide with better anti-aging effect on the basis of crude wolfberry polysaccharide. Summary of the Invention

[0004] In order to overcome at least one of the technical problems existing in the prior art, the present invention first provides a preparation method of wolfberry polysaccharide.

[0005] The above technical problems to be solved by the present invention are realized through the following technical solutions:

[0006] The present invention first provides a preparation method of wolfberry polysaccharide, which comprises the following steps:

[0007] S1. Take wolfberry, and then perform heating and reflux extraction with water to obtain a wolfberry water extract, and concentrate the wolfberry water extract to obtain a wolfberry concentrate;

[0008] S2. Add ethanol to the wolfberry concentrate so that the final volume concentration of ethanol is 70-80%, let it stand and then filter, and take the precipitate to obtain crude wolfberry polysaccharide;

[0009] S3. Mix the crude wolfberry polysaccharide with water to obtain a mixed solution, and then perform ultrafiltration using an ultrafiltration membrane with a molecular weight cut-off of 100-120 kDa; take the filtrate, concentrate and dry it to obtain a solid, and take the solid to obtain the wolfberry polysaccharide.

[0010] The present invention provides a brand-new method for preparing wolfberry polysaccharide; in the research of the present invention, it is found that on the basis of the crude wolfberry polysaccharide prepared by the water extraction and alcohol precipitation method, the wolfberry polysaccharide prepared by further ultrafiltration using an ultrafiltration membrane with a molecular weight cut-off of 100-120 kDa has a significantly improved anti-aging effect compared with the crude wolfberry polysaccharide.

[0011] Preferably, in step S1, the weight dosage of the water is 4-8 times that of the wolfberry.

[0012] Most preferably, in step S1, the weight dosage of the water is 5 times that of the wolfberry.

[0013] Preferably, in step S1, the temperature of the heating reflux extraction is 70-85 °C, and the time of the heating reflux extraction is 1-3 h.

[0014] Most preferably, in step S1, the temperature of the heating reflux extraction is 80 °C, and the time of the heating reflux extraction is 2 h.

[0015] Preferably, in step S1, the concentration is to concentrate the wolfberry water extract to 1 / 2-1 / 4 of the volume of the water.

[0016] Most preferably, in step S1, the concentration is to concentrate the wolfberry water extract to 1 / 3 of the volume of the water.

[0017] Preferably, in step S2, the standing time is 8-16 h.

[0018] Preferably, in step S2, the final volume concentration of the ethanol is 75%.

[0019] Preferably, in step S3, the molecular weight cut-off of the ultrafiltration membrane is 110 kDa.

[0020] Preferably, in step S3, the weight dosage of the water is 6-10 times that of the crude wolfberry polysaccharide.

[0021] Most preferably, in step S3, the weight dosage of the water is 8 times that of the crude wolfberry polysaccharide.

[0022] Preferably, the method for preparing wolfberry polysaccharide further includes a step of eluting with a macroporous resin column, specifically:

[0023] S4. Load the solid obtained in step S3 onto a macroporous resin column, first elute and remove impurities with an ethanol aqueous solution; then elute with an ethanol aqueous solution having a volume concentration of 13-15%, collect the eluate eluted by the ethanol aqueous solution having a volume concentration of 13-15%, and after concentration and drying, the wolfberry polysaccharide is obtained.

[0024] More preferably, in step S4, in the elution and impurity removal with the ethanol aqueous solution, the volume concentration of the ethanol aqueous solution is 2-4%, and the amount of the ethanol aqueous solution used is 2-4 times the column volume.

[0025] More preferably, in step S4, in the elution with the ethanol aqueous solution having a volume concentration of 13-15%, the amount of the ethanol aqueous solution used is 5-8 times the column volume.

[0026] Most preferably, the macroporous resin column elution step in step S4 is specifically as follows:

[0027] S4. Load the solid obtained in step S3 onto a macroporous resin column, first elute and remove impurities with 3 times the column volume of an ethanol aqueous solution having a volume concentration of 3%; then elute with 6 times the column volume of an ethanol aqueous solution having a volume concentration of 14%, collect the eluate eluted by the ethanol aqueous solution having a volume concentration of 14%, and after concentration and drying, the wolfberry polysaccharide is obtained.

[0028] In further research of the present invention, it is found that the anti-aging effect of the wolfberry polysaccharide prepared by ultrafiltration through an ultrafiltration membrane and further eluted by the macroporous resin column of the present invention is further greatly improved.

[0029] The inventor also found in the research that the elution conditions of the macroporous resin column of the present invention are very crucial. Research shows that only the wolfberry polysaccharide prepared under the macroporous resin elution conditions of the present invention can its anti-aging effect be further greatly improved; while the wolfberry polysaccharide prepared under other macroporous resin elution conditions cannot have its anti-aging effect further greatly improved.

[0030] Preferably, the macroporous resin column is an AB-8 type macroporous resin column.

[0031] The present invention provides a wolfberry polysaccharide prepared by the preparation method of any one of the above.

[0032] The present invention also provides the application of the wolfberry polysaccharide in the preparation of a product having an anti-aging effect.

[0033] Preferably, the product is a health product or a medicine.

[0034] Beneficial effects: The present invention provides a brand-new method for preparing wolfberry polysaccharide; research shows that the wolfberry polysaccharide prepared by the method of the present invention has a significantly higher anti-aging effect than the crude wolfberry polysaccharide prepared by the water extraction and alcohol precipitation method, and has an extremely excellent anti-aging effect; therefore, using the wolfberry polysaccharide prepared by the method of the present invention as an active ingredient to prepare health products or drugs with anti-aging effects has important application prospects. Detailed implementation manners

[0035] The following specific examples are used to further explain the present invention, but the examples do not limit the present invention in any form.

[0036] Example 1 Preparation of wolfberry polysaccharide

[0037] S1. Take wolfberries, and then perform heating reflux extraction with 5 times the weight of water at 80 °C for 2 h to obtain a wolfberry water extract. Concentrate the wolfberry water extract to 1 / 3 of the volume of water to obtain a wolfberry concentrate;

[0038] S2. Add ethanol to the wolfberry concentrate so that the final volume concentration of ethanol is 75%. After standing for 12 h, filter and take the precipitate to obtain crude wolfberry polysaccharide;

[0039] S3. Mix the crude wolfberry polysaccharide with 8 times the weight of water to obtain a mixed solution, and then perform ultrafiltration with a ultrafiltration membrane with a molecular weight of 110 kDa; take the filtrate, concentrate and dry it to obtain a solid, and take the solid to obtain the wolfberry polysaccharide described above.

[0040] Example 2 Preparation of wolfberry polysaccharide

[0041] S1. Take wolfberries, and then perform heating reflux extraction with 5 times the weight of water at 80 °C for 2 h to obtain a wolfberry water extract. Concentrate the wolfberry water extract to 1 / 3 of the volume of water to obtain a wolfberry concentrate;

[0042] S2. Add ethanol to the wolfberry concentrate so that the final volume concentration of ethanol is 75%. After standing for 12 h, filter and take the precipitate to obtain crude wolfberry polysaccharide;

[0043] S3. Mix the crude wolfberry polysaccharide with 8 times the weight of water to obtain a mixed solution, and then perform ultrafiltration with a ultrafiltration membrane with a molecular weight of 110 kDa; take the filtrate, concentrate and dry it to obtain a solid;

[0044] S4. Load the solid obtained in step S3 onto an AB-8 macroporous resin column (the macroporous resin column is filled with AB-8 macroporous resin with 30 times the weight of the solid obtained in step S3). First, elute and remove impurities with 3 column volumes of an ethanol aqueous solution with a volume concentration of 3%; then elute with 6 column volumes of an ethanol aqueous solution with a volume concentration of 14%, collect the eluate eluted by the ethanol aqueous solution with a volume concentration of 14%, and concentrate and dry it to obtain the wolfberry polysaccharide described above.

[0045] Example 3 Preparation of Lycium barbarum polysaccharide

[0046] S1. Take Lycium barbarum, and then carry out heating reflux extraction with 4 times the weight of water at 85 °C for 1 h to obtain the aqueous extract of Lycium barbarum. Concentrate the aqueous extract of Lycium barbarum to 1 / 2 of the volume of water to obtain the concentrated extract of Lycium barbarum;

[0047] S2. Add ethanol to the concentrated extract of Lycium barbarum to make the final volume concentration of ethanol 80%. Let it stand for 8 h and then filter. Take the precipitate to obtain the crude polysaccharide of Lycium barbarum;

[0048] S3. Mix the crude polysaccharide of Lycium barbarum with 6 times the weight of water to obtain a mixed solution, and then carry out ultrafiltration with a ultrafiltration membrane with a molecular weight of 100 kDa; Take the filtrate, concentrate and dry it to obtain a solid;

[0049] S4. Load the solid obtained in step S3 onto an AB-8 macroporous resin column (the macroporous resin column is filled with AB-8 macroporous resin with 30 times the weight of the solid obtained in step S3). First, elute and remove impurities with 3 column volumes of an aqueous ethanol solution with a volume concentration of 2%; Then, elute with 6 column volumes of an aqueous ethanol solution with a volume concentration of 13%. Collect the eluate eluted by the aqueous ethanol solution with a volume concentration of 13%, concentrate and dry it to obtain the Lycium barbarum polysaccharide.

[0050] Example 4 Preparation of Lycium barbarum polysaccharide

[0051] S1. Take Lycium barbarum, and then carry out heating reflux extraction with 8 times the weight of water at 70 °C for 3 h to obtain the aqueous extract of Lycium barbarum. Concentrate the aqueous extract of Lycium barbarum to 1 / 4 of the volume of water to obtain the concentrated extract of Lycium barbarum;

[0052] S2. Add ethanol to the concentrated extract of Lycium barbarum to make the final volume concentration of ethanol 70%. Let it stand for 16 h and then filter. Take the precipitate to obtain the crude polysaccharide of Lycium barbarum;

[0053] S3. Mix the crude polysaccharide of Lycium barbarum with 10 times the weight of water to obtain a mixed solution, and then carry out ultrafiltration with a ultrafiltration membrane with a molecular weight of 120 kDa; Take the filtrate, concentrate and dry it to obtain a solid;

[0054] S4. Load the solid obtained in step S3 onto an AB-8 macroporous resin column (the macroporous resin column is filled with AB-8 macroporous resin with 30 times the weight of the solid obtained in step S3). First, elute and remove impurities with 3 column volumes of an aqueous ethanol solution with a volume concentration of 4%; Then, elute with 6 column volumes of an aqueous ethanol solution with a volume concentration of 15%. Collect the eluate eluted by the aqueous ethanol solution with a volume concentration of 15%, concentrate and dry it to obtain the Lycium barbarum polysaccharide.

[0055] Comparative Example 1 Preparation of crude polysaccharide of Lycium barbarum

[0056] S1. Take wolfberries, then perform heat reflux extraction with 5 times the weight of water at 80 °C for 2 h to obtain the water extract of wolfberries. Concentrate the water extract of wolfberries to 1 / 3 of the original volume of water to obtain the concentrated wolfberry extract;

[0057] S2. Add ethanol to the concentrated wolfberry extract to make the final volume concentration of ethanol 75%. Let it stand for 12 h and then filter. Take the precipitate to obtain crude wolfberry polysaccharide.

[0058] Comparative Example 2 Preparation of Wolfberry Polysaccharide

[0059] S1. Take wolfberries, then perform heat reflux extraction with 5 times the weight of water at 80 °C for 2 h to obtain the water extract of wolfberries. Concentrate the water extract of wolfberries to 1 / 3 of the original volume of water to obtain the concentrated wolfberry extract;

[0060] S2. Add ethanol to the concentrated wolfberry extract to make the final volume concentration of ethanol 75%. Let it stand for 12 h and then filter. Take the precipitate to obtain crude wolfberry polysaccharide;

[0061] S3. Mix the crude wolfberry polysaccharide with 8 times the weight of water to obtain a mixed solution, and then perform ultrafiltration with a ultrafiltration membrane with a molecular weight cut-off of 110 kDa; Take the filtrate, concentrate and dry it to obtain a solid;

[0062] S4. Load the solid obtained in step S3 onto an AB-8 macroporous resin column (the macroporous resin column is filled with AB-8 macroporous resin with 30 times the weight of the solid obtained in step S3). Elute with an aqueous ethanol solution with a volume concentration of 14% for 6 times the column volume. Collect the eluate eluted by the aqueous ethanol solution with a volume concentration of 14%, concentrate and dry it to obtain the wolfberry polysaccharide.

[0063] Comparative Example 3 Preparation of Wolfberry Polysaccharide

[0064] S1. Take wolfberries, then perform heat reflux extraction with 5 times the weight of water at 80 °C for 2 h to obtain the water extract of wolfberries. Concentrate the water extract of wolfberries to 1 / 3 of the original volume of water to obtain the concentrated wolfberry extract;

[0065] S2. Add ethanol to the concentrated wolfberry extract to make the final volume concentration of ethanol 75%. Let it stand for 12 h and then filter. Take the precipitate to obtain crude wolfberry polysaccharide;

[0066] S3. Mix the crude wolfberry polysaccharide with 8 times the weight of water to obtain a mixed solution, and then perform ultrafiltration with a ultrafiltration membrane with a molecular weight cut-off of 110 kDa; Take the filtrate, concentrate and dry it to obtain a solid;

[0067] S4. Load the solid obtained in step S3 onto an AB-8 macroporous resin column (the macroporous resin column is filled with AB-8 macroporous resin with a weight 30 times that of the solid obtained in step S3). First, wash it with 3 column volumes of water to remove impurities. Then, elute it with 6 column volumes of an ethanol aqueous solution with a volume concentration of 20%. Collect the eluate eluted by the ethanol aqueous solution with a volume concentration of 20%. After concentration and drying, the wolfberry polysaccharide is obtained.

[0068] Preparation of Lycium barbarum polysaccharide in Comparative Example 4

[0069] S1. Take Lycium barbarum, and then perform heating reflux extraction with 5 times the weight of water at 80 °C for 2 h to obtain a water extract of Lycium barbarum. Concentrate the water extract of Lycium barbarum to 1 / 3 of the volume of water to obtain a concentrated Lycium barbarum solution.

[0070] S2. Add ethanol to the concentrated Lycium barbarum solution to make the final volume concentration of ethanol 75%. Let it stand for 12 h and then filter. Take the precipitate to obtain crude Lycium barbarum polysaccharide.

[0071] S3. Mix the crude Lycium barbarum polysaccharide with 8 times the weight of water to obtain a mixed solution, and then perform ultrafiltration with a ultrafiltration membrane with a molecular weight of 110 kDa. Take the filtrate, concentrate it and dry it to obtain a solid.

[0072] S4. Load the solid obtained in step S3 onto an AB-8 macroporous resin column (the macroporous resin column is filled with AB-8 macroporous resin with a weight 30 times that of the solid obtained in step S3). First, elute it with 3 column volumes of an ethanol aqueous solution with a volume concentration of 15% to remove impurities. Then, elute it with 6 column volumes of an ethanol aqueous solution with a volume concentration of 30%. Collect the eluate eluted by the ethanol aqueous solution with a volume concentration of 30%. After concentration and drying, the wolfberry polysaccharide is obtained.

[0073] Experimental Example Anti-aging experiment

[0074] Animal grouping: After acclimating SD female rats for one week, weigh them. Select rats with a body weight of 200 g ± 20 g and divide them into 10 groups (10 rats in each group).

[0075] Administration method: The blank control group is intraperitoneally injected with normal saline every day. The model group and the experimental groups are intraperitoneally injected with a 25% D-galactose normal saline solution at a dose of 500 mg / kg / d. While each group is intraperitoneally injected with normal saline or D-galactose normal saline solution, the experimental groups are intragastrically administered the wolfberry polysaccharides prepared in Examples 1 - 4 and Comparative Examples 1 - 4 at a dose of 10 mg / kg / d every day. The blank group, the model group are intragastrically administered an equal amount of normal saline every day.

[0076] Each experimental group was continuously administered the drug for 6 weeks. After the administration ended, the rats were anesthetized with 10% chloral hydrate. The abdominal cavity was opened, blood was taken from the artery, and it was left standing at room temperature for 30 min, then centrifuged at 4°C at 3000 rpm for 15 min. The supernatant was taken to detect the content of SOD (superoxide dismutase) according to the kit instructions. The test results are shown in Table 1.

[0077] Table 1

[0078] Group Sample to be tested SOD (U / mL) Blank control group - 61.00±7.75 Model group - 55.98±4.28 Experimental group 1 Lycium barbarum polysaccharide prepared in Example 1 62.78±5.57 Experimental group 2 Lycium barbarum polysaccharide prepared in Example 2 78.71±6.36 Experimental group 3 Lycium barbarum polysaccharide prepared in Example 3 75.12±6.09 Experimental group 4 Lycium barbarum polysaccharide prepared in Example 4 77.09±6.36 Experimental group 5 Crude Lycium barbarum polysaccharide prepared in Comparative Example 1 58.21±5.04 Experimental group 6 Lycium barbarum polysaccharide prepared in Comparative Example 2 63.54±5.72 Experimental group 7 Lycium barbarum polysaccharide prepared in Comparative Example 3 64.12±5.98 Experimental group 8 Lycium barbarum polysaccharide prepared in Comparative Example 4 60.33±5.68

[0079] It can be seen from the experimental data in Table 1 that the content of SOD in the blood of the rats intragastrically administered with the wolfberry polysaccharide prepared in Example 1 was significantly higher than that of the crude wolfberry polysaccharide prepared in Comparative Example 1; this indicates that: on the basis of the crude wolfberry polysaccharide prepared by the water extraction and alcohol precipitation method, the wolfberry polysaccharide prepared by further ultrafiltration with an ultrafiltration membrane with a molecular weight of 100-120 kDa has a significantly improved anti-aging effect compared with the crude wolfberry polysaccharide.

[0080] It can be seen from the experimental data in Table 1 that the content of SOD in the blood of the rats intragastrically administered with the wolfberry polysaccharide prepared in Example 2 was further significantly higher than that of the wolfberry polysaccharide prepared in Example 1; this indicates that: the wolfberry polysaccharide prepared by further eluting the wolfberry polysaccharide prepared by ultrafiltration with the macroporous resin column described in the present invention has a further significantly improved anti-aging effect.

[0081] It can be seen from the experimental data in Table 1 that the content of SOD in the blood of the rats intragastrically administered with the wolfberry polysaccharide prepared in Comparative Examples 2-4 was not higher than or significantly higher than that of the wolfberry polysaccharide prepared in Example 1; this indicates that: the elution conditions of the macroporous resin column of the present invention are very crucial, and only the wolfberry polysaccharide prepared under the elution conditions of the macroporous resin described in the present invention can have a further significantly improved anti-aging effect; while the wolfberry polysaccharide prepared under other macroporous resin elution conditions cannot have a further significantly improved anti-aging effect.

[0082] The above embodiments are only the preferred embodiments of the present invention, which are only used to explain the present invention and not to limit the present invention. Any changes, substitutions, modifications, etc. made by those skilled in the art without departing from the spirit and essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. A preparation method of wolfberry polysaccharide, characterized in that, It comprises the following steps: S1. Take wolfberries, and then perform heat reflux extraction with water to obtain a water extract of wolfberries. Concentrate the water extract of wolfberries to obtain a concentrated wolfberry extract; S2. Add ethanol to the concentrated wolfberry extract so that the final volume concentration of ethanol is 70-80%. Let it stand and then filter. Take the precipitate to obtain crude wolfberry polysaccharide; S3. Mix the crude wolfberry polysaccharide with water to obtain a mixed solution, and then perform ultrafiltration with an ultrafiltration membrane having a molecular weight of 100-120 kDa; Take the filtrate, concentrate and dry it to obtain a solid; Take the solid to obtain the wolfberry polysaccharide as described; S4. Load the solid obtained in step S3 onto a macroporous resin column, first elute and remove impurities with an ethanol aqueous solution; Then elute with an ethanol aqueous solution having a volume concentration of 13-15%. Collect the eluate eluted by the ethanol aqueous solution having a volume concentration of 13-15%, concentrate and dry it to obtain the wolfberry polysaccharide as described; In the elution and impurity removal with the ethanol aqueous solution first, the volume concentration of the ethanol aqueous solution is 2-4%, and the amount of the ethanol aqueous solution used is 2-4 times the column volume.

2. The preparation method of a wolfberry polysaccharide according to claim 1, wherein In step S1, the weight of the water used is 4-8 times that of the wolfberries.

3. The preparation method of a wolfberry polysaccharide according to claim 1, characterized in that, In step S1, the temperature of the heat reflux extraction is 70-85°C, and the time of the heat reflux extraction is 1-3 h; The concentration is to concentrate the water extract of wolfberries to 1 / 2-1 / 4 of the volume of water.

4. The preparation method of a wolfberry polysaccharide according to claim 1, characterized in that, In step S2, the standing time is 8-16 h.

5. The preparation method of a wolfberry polysaccharide according to claim 1, characterized in that, In step S3, the weight of the water used is 6-10 times the weight of the crude wolfberry polysaccharide.

6. The preparation method of a wolfberry polysaccharide according to claim 1, characterized in that, In step S4, in the elution with the ethanol aqueous solution having a volume concentration of 13-15% again, the amount of the ethanol aqueous solution used is 5-8 times the column volume.

7. A wolfberry polysaccharide prepared by the preparation method according to any one of claims 1-6.

8. Use of the wolfberry polysaccharide according to claim 7 in the preparation of a product having an anti-aging effect.