Extraction method and application of momordica grosvenori polysaccharide

Abstract

The invention relates to the technical field of extraction of effective components of plants, in particular to an extraction method and application of momordica grosvenori polysaccharide, the method comprises the steps that an ultrasonic-assisted extraction solvent is adopted for extracting momordica grosvenori, in addition, extraction conditions of momordica grosvenori crude polysaccharide are optimized, and a new method is provided for efficient and green extraction of momordica grosvenori polysaccharide. An important technical reference is provided for industrial development of the momordica grosvenori polysaccharide.

Description

Technical Field

[0001] The present invention belongs to the technical field of extraction of effective components from plants, and particularly relates to a method for extracting Momordica grosvenori polysaccharide and its application.

Background Art

[0002] Momordica grosvenori, as a natural plant with both medicinal and edible properties, has attracted much attention at home and abroad due to its unique medicinal and edible values. In terms of extraction methods, the research on Momordica grosvenori polysaccharide mainly focuses on hot water extraction method, and some studies are supplemented with strengthening means such as ultrasonic and microwave. Although the traditional hot water extraction technology is simple to operate, it has the disadvantages of wasting time, polluting the environment and high cost.

[0003] An aqueous two-phase system (ATPS) is a mixture composed of a polymer (or short-chain ethanol) and an aqueous salt solution, which can extract two or more polysaccharides simultaneously, and has the advantages of low viscosity, high mass transfer efficiency, easy phase separation, high extraction rate and easy amplification, etc., and is suitable for industrial applications.

[0004] However, there are few reports on the application of the aqueous two-phase system in the extraction of Momordica grosvenori polysaccharide at present.

Summary of the Invention

[0005] In view of the above, it is necessary to provide a method for extracting Momordica grosvenori polysaccharide and its application. The present invention first proposes the application of a self-made extractant in the extraction of Momordica grosvenori polysaccharide, providing a new method for efficient and green extraction of Momordica grosvenori polysaccharide.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] A method for extracting Momordica grosvenori polysaccharide, the method includes extracting Momordica grosvenori with an ultrasonic-assisted extraction solvent, and the extraction solvent is obtained by dissolving ammonium sulfate in deionized water, then adding dextran and mixing evenly, then adding ethanol to the solution, and then using a vortex oscillator to mix well and standing for 4 - 10 h.

[0008] In the present invention, further, in the extraction solvent, the mass ratio of ammonium sulfate, dextran, ethanol and deionized water is 3 - 4:0.1 - 0.2:4 - 5:15.

[0009] In the present invention, further, the specific extraction method of the Momordica grosvenori polysaccharide includes the following steps:

[0010] (1) Weigh Momordica grosvenori powder, mix it with the extraction solvent, place it in an ultrasonic cleaner for extraction, and then perform centrifugal separation to fully separate the supernatant phase. Subsequently, repeat the above operation once with the Momordica grosvenori residue, and combine the upper phases of the filtrates from the two extractions, and remove the lower phase;

[0011] (2) The upper phase extract is centrifuged to collect the supernatant, and then 1 / 4 volume of Sevag reagent (chloroform: n-butanol = 4:1) is added to the supernatant, and the mixture is placed on a magnetic stirrer and stirred for 25-35 minutes to remove protein, and this step is repeated until no protein precipitation appears in the middle layer. After deproteinization is completed, the supernatant is collected, and anhydrous ethanol is used for overnight alcohol precipitation at 3-6°C. The precipitate is redissolved in deionized water and dialyzed for 24 hours using a dialysis membrane to remove small molecular impurities. Finally, the dialyzed solution is placed in a freeze dryer for freeze drying to obtain the upper phase polysaccharide, which is the Momordica grosvenori polysaccharide.

[0012] In the present invention, further, in the step (1), the liquid-to-solid ratio of the monk fruit powder to the extraction solvent is 10-50 mL / g, the extraction conditions are as follows: the ultrasonic power is 200-600 W, the extraction temperature is 30-70° C., and the extraction time is 10-50 min.

[0013] In the present invention, further, the liquid-to-solid ratio of the monk fruit powder and the extraction solvent is 32 mL / g, the extraction conditions are as follows: the ultrasonic power is 300 W, the extraction temperature is 60° C., and the extraction time is 30 min.

[0014] The present invention also provides a method for preparing a skin care product by extracting Momordica grosvenori polysaccharide using the method as described above, wherein the skin care product has the effects of moisturizing, anti-aging and the like.

[0015] The present invention also provides a skin care product, which is composed of the Momordica grosvenori polysaccharide prepared by the method.

[0016] The present invention has the following beneficial effects:

[0017] 1. The present invention proposes a method for extracting Momordica polysaccharide, wherein Momordica polysaccharide is obtained by extracting Momordica polysaccharide with an ultrasonic-assisted extraction solvent, wherein the extraction solvent is obtained by dissolving ammonium sulfate in deionized water, then adding dextran, then adding ethanol, and then using a vortex oscillator to fully mix, and then standing until the solution spontaneously separates to obtain an extraction solvent. The system can effectively achieve the extraction and separation of target components through the two-phase separation characteristics, and provides stable phase separation conditions for subsequent experiments. In addition, the applicant further optimized the extraction conditions and finally obtained a Momordica polysaccharide extraction method with a high extraction yield. The method is green and safe, has mild reaction conditions, is easy to operate, and has a great cost advantage over conventional extractants. In addition, the polysaccharide extracted by the improved extraction solvent of the present application has a higher purity.

[0018] 2. The Momordica grosvenori polysaccharide extracted by the extraction method of the present invention can be applied to the preparation of skin care products. We selected the upper-phase extract to obtain the polysaccharide and found that it showed significant advantages in scavenging hydroxyl radicals and superoxide anions, which may be related to its similar structural characteristics. In addition, the experiment also showed that the polysaccharide of the present application also has good hygroscopic and moisturizing properties. The polysaccharide combines with the three-dimensional network structure formed by its molecular chain to prevent the loss of water molecules. Moreover, with the increase of age, the endogenous level gradually decreases. Therefore, supplementing exogenous antioxidants can delay the damage of free radicals to the skin. The experiment shows that compared with glycerol, the Momordica grosvenori polysaccharide of the present application has good antioxidant capacity and hygroscopic and retention properties. Therefore, the application of the Momordica grosvenori polysaccharide in skin care products and skin care products has great development potential.

Specific Embodiments

[0019] In order to make the above objects, features, and advantages of the present invention more obvious and understandable, the specific embodiments of the present invention will be described in detail below. Many specific details are set forth in the following description in order to fully understand the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without departing from the connotation of the present invention. Therefore, the present invention is not limited by the specific embodiments disclosed below.

[0020] Example 1:

[0021] This example provides an extraction method for Momordica grosvenori polysaccharide, which includes the following steps:

[0022] (1) Weigh Momordica grosvenori powder and mix it with an extraction solvent. The liquid-solid ratio is 40 mL / g. The extraction solvent is obtained by dissolving ammonium sulfate in deionized water, then adding dextran and mixing evenly, and then adding ethanol to the solution. After that, use a vortex oscillator to mix well and let it stand for 4 h to obtain the extraction solvent. In the extraction solvent, the mass ratio of ammonium sulfate, dextran, ethanol, and deionized water is 3:0.1:4:15. Then place it in an ultrasonic cleaner for extraction. The extraction conditions are ultrasonic power of 600 W, extraction temperature of 40 °C, and extraction time of 50 min. Then perform centrifugal separation to fully separate the supernatant. Subsequently, repeat the above operation once with the Momordica grosvenori residue and combine the upper phases of the filtrates from the two extractions, and discard the lower phase;

[0023] (2) Centrifuge the upper-phase extract to collect the supernatant. Subsequently, add 1 / 4 volume of Sevag reagent (chloroform: n-butanol = 4:1) to the supernatant, place it on a magnetic stirrer and continuously stir for 35 min to remove proteins, and repeat this step until no protein precipitate appears in the intermediate layer. After protein removal is completed, collect the supernatant, perform overnight alcohol precipitation treatment with absolute ethanol at 3°C, dissolve the precipitate in deionized water, and use a dialysis membrane to dialyze for 24 h to remove small molecule impurities. Finally, place the dialyzed solution in a freeze dryer for freeze-drying treatment to obtain the upper-phase polysaccharide, which is the Siraitia grosvenorii polysaccharide mentioned above.

[0024] Example 2:

[0025] This example provides a method for extracting Siraitia grosvenorii polysaccharide, which includes the following steps:

[0026] (1) Weigh Siraitia grosvenorii powder and mix it with an extraction solvent. The liquid-solid ratio is 32 mL / g. The extraction solvent is obtained by dissolving ammonium sulfate in deionized water, then adding dextran and mixing evenly, and then adding ethanol to the solution. Subsequently, use a vortex oscillator to mix well and let it stand for 6 h to obtain the extraction solvent. In the extraction solvent, the mass ratio of ammonium sulfate, dextran, ethanol, and deionized water is 3:0.2:4:15. Then place it in an ultrasonic cleaner for extraction. The extraction conditions are an ultrasonic power of 300 W, an extraction temperature of 50°C, and an extraction time of 30 min. Then perform centrifugal separation to fully separate the supernatant. Subsequently, repeat the above operation once with the Siraitia grosvenorii residue, and combine the upper phases of the filtrates from the two extractions and discard the lower phase;

[0027] (2) Centrifuge the upper-phase extract to collect the supernatant. Subsequently, add 1 / 4 volume of Sevag reagent (chloroform: n-butanol = 4:1) to the supernatant, place it on a magnetic stirrer and continuously stir for 30 min to remove proteins, and repeat this step until no protein precipitate appears in the intermediate layer. After protein removal is completed, collect the supernatant, perform overnight alcohol precipitation treatment with absolute ethanol at 4°C, dissolve the precipitate in deionized water, and use a dialysis membrane to dialyze for 24 h to remove small molecule impurities. Finally, place the dialyzed solution in a freeze dryer for freeze-drying treatment to obtain the upper-phase polysaccharide, which is the Siraitia grosvenorii polysaccharide mentioned above.

[0028] Example 3:

[0029] This example provides a method for extracting Siraitia grosvenorii polysaccharide, which includes the following steps:

[0030] (1) Weigh the Momordica grosvenori powder and mix it with the extraction solvent at a liquid-solid ratio of 20 mL / g. The extraction solvent is prepared by dissolving ammonium sulfate in deionized water, then adding dextran and mixing evenly, followed by adding ethanol to the solution. After thoroughly mixing with a vortex oscillator, let it stand for 10 h to obtain the extraction solvent. In the extraction solvent, the mass ratio of ammonium sulfate, dextran, ethanol, and deionized water is 4:0.2:5:15. Then place it in an ultrasonic cleaner for extraction. The extraction conditions are an ultrasonic power of 200 W, an extraction temperature of 60 °C, and an extraction time of 10 min. Then perform centrifugal separation to fully separate the supernatant. Subsequently, repeat the above operation once with the Momordica grosvenori residue, and combine the upper phases of the filtrates from the two extractions, removing the lower phase.

[0031] (2) Centrifuge the upper-phase extract to collect the supernatant. Then add 1 / 4 volume of Sevag reagent (chloroform: n-butanol = 4:1) to the supernatant, place it on a magnetic stirrer and continuously stir for 25 min to remove proteins, and repeat this step until no protein precipitate appears in the middle layer. After protein removal is completed, collect the supernatant, perform overnight alcohol precipitation treatment with absolute ethanol at 6 °C, dissolve the precipitate in deionized water, and use a dialysis membrane to dialyze for 24 h to remove small-molecule impurities. Finally, place the dialyzed solution in a freeze dryer for freeze-drying treatment to obtain the upper-phase polysaccharide, which is the Momordica grosvenori polysaccharide.

[0032] Example 4:

[0033] This example provides a skin care product, and the ingredients of the skin care product include the Momordica grosvenori polysaccharide in Example 2.

[0034] Test example:

[0035] Test 1: Compare the extraction rates of Momordica grosvenori polysaccharide under different ultrasonic conditions, as shown in Table 1:

[0036] Table 1 Effects of ultrasonic extraction parameters on the polysaccharide extraction rate

[0037]

[0038]

[0039] It can be seen from Table 1 that when the ultrasonic power is 300 W, the extraction temperature is 50 °C, and the extraction time is 30 min, the extraction rate of polysaccharide is the highest. When the extraction parameters are lower or higher than the above parameters respectively, the extraction rate is lower than the extraction parameters in Example 2 of the present invention (i.e., ultrasonic power is 300 W, extraction temperature is 50 °C, and extraction time is 30 min), indicating that the ultrasonic extraction parameters described in Example 2 of the present invention are the optimal extraction parameters for polysaccharide extraction.

[0040] Experiment 2: Comparison of the yield and purity of Momordica grosvenori under different extraction methods:

[0041] This experiment studied the extraction rate and purity of Momordica grosvenori polysaccharide under different extraction methods, and the groups were as follows:

[0042] Group 1: Extract Momordica grosvenori polysaccharide by the method of Example 2 of this application;

[0043] Group 2: The liquid-solid ratio of the extraction solvent to Momordica grosvenori powder was 10 mL / g, and other methods were the same as those in Group 1;

[0044] Group 3: The liquid-solid ratio of the extraction solvent to Momordica grosvenori powder was 50 mL / g, and other methods were the same as those in Group 1;

[0045] Group 4: Remove dextran, and other methods were the same as those in Group 1;

[0046] Group 5: Replace ammonium sulfate with sodium carbonate, and other methods were the same as those in Group 1;

[0047] Group 6: Replace ethanol with methanol, and other methods were the same as those in Group 1.

[0048] Compare the extraction rate and purity of the polysaccharides obtained by extraction in the above groups. The results are shown in Table 2:

[0049] Table 2 Comparison of the extraction rate and purity of polysaccharides in each group

[0050] Extraction rate (%) Purity (%) The first group 25.87％ 89.9％ The second group 15.31％ 86.7％ The third group 16.26％ 88.2％ The fourth group 25.77％ 79.3％ The fifth group 14.32％ 81.1％ The sixth group 21.57％ 82.36％

[0051] According to the results in Table 2, it can be seen that the yield and purity of Momordica grosvenori polysaccharide obtained by the method of this application are high. The extractant and liquid-solid ratio of this application have certain effects on the extraction rate and purity of polysaccharides. It is particularly important to select appropriate extraction parameters and components.

[0052] Experiment 3: Application of Momordica grosvenori polysaccharide:

[0053] Apply the Momordica grosvenori polysaccharide prepared by the method of this application to the preparation of skin care products and skin care products.

[0054] Further test the biological activities of Momordica grosvenori polysaccharide, including hydroxyl radical scavenging activity, superoxide anion scavenging activity and moisture absorption and retention performance.

[0055] Among them, the test method for antioxidant activity is:

[0056] Using ascorbic acid (Vc) as the positive control, the scavenging ability of polysaccharide superoxide anion radical and hydroxyl radical was measured by a hydroxyl radical determination kit and an inhibition and production determination kit for superoxide anion respectively.

[0057] The grouping is as follows:

[0058] The Momordica grosvenori polysaccharide obtained in the manner of Example 2 was taken as the first group;

[0059] Weigh Momordica grosvenori powder and mix it with an extraction solvent. The liquid-solid ratio is 32 mL / g. The extraction solvent is obtained by dissolving ammonium sulfate in deionized water, then adding dextran and mixing evenly, and then adding ethanol to the solution. Subsequently, it is fully mixed using a vortex oscillator and then left to stand for 6 h to obtain the extraction solvent. In the extraction solvent, the mass ratio of ammonium sulfate, dextran, ethanol, and deionized water is 3:0.2:4:15. Then it is placed in an ultrasonic cleaner for extraction. The extraction conditions are an ultrasonic power of 300 W, an extraction temperature of 50 °C, and an extraction time of 30 min. Then centrifugal separation is carried out to fully separate the supernatant. Subsequently, the above operation is repeated once using the Momordica grosvenori residue, and the lower phase is combined. The lower-phase extract is centrifuged to collect the supernatant. Then, 1 / 4 volume of Sevag reagent (chloroform: n-butanol = 4:1) is added to the supernatant, and it is continuously stirred on a magnetic stirrer for 30 min to remove proteins, and this step is repeated until no protein precipitate appears in the middle layer. After protein removal is completed, the supernatant is collected, and overnight alcohol precipitation treatment is carried out with absolute ethanol at 4 °C. The precipitate is redissolved in deionized water, and dialysis is carried out using a dialysis membrane for 24 h to remove small molecule impurities. Finally, the dialyzed solution is placed in a freeze dryer for freeze-drying treatment to obtain the lower-phase polysaccharide as the second group.

[0060] Mix 0.5 g of fruit powder with 17.5 mL of deionized water and extract at 75 °C for 2.5 h, and then repeat this operation once. First, centrifuge the extract at 10,000 rpm for 10 min to collect the supernatant. Then, 1 / 4 volume of Sevag reagent (chloroform: n-butanol = 4:1) is added to the supernatant, and it is continuously stirred on a magnetic stirrer for 30 min to remove proteins, and this step is repeated until no protein precipitate appears in the middle layer. After protein removal is completed, the supernatant is collected, and overnight alcohol precipitation treatment is carried out with absolute ethanol at 4 °C. The precipitate is redissolved in deionized water, and dialysis is carried out using a dialysis membrane for 24 h to remove small molecule impurities. Finally, the dialyzed solution is placed in a freeze dryer for freeze-drying treatment to obtain the crude Momordica grosvenori polysaccharide finished product as the third group;

[0061] Using a eutectic solvent composed of betaine and malic acid, betaine and malic acid (molar ratio 1:3) and deionized water (30%) were added to a beaker respectively, and heated in a water bath at 80 °C for 2 h until the solution became clear and transparent, thus obtaining the eutectic solution. Accurately weigh 0.5 g of Momordica grosvenori powder, according to the liquid-solid ratio of 34 mL / g, temperature of 52 °C, ultrasonic power of 325 W, extraction time of 39 min. Then centrifuge to obtain the supernatant, repeat the operation on the residue once, combine the two filtrates, and dilute to 50 mL with deionized water. Centrifuge the extract at 10,000 rpm for 10 min, collect the supernatant, then add 1 / 4 volume of Sevag reagent (chloroform: n-butanol = 4:1) to the supernatant, place it on a magnetic stirrer and stir continuously for 30 min to remove proteins, and repeat this step until no protein precipitate appears in the middle layer. After deproteinization, collect the supernatant, perform overnight alcohol precipitation treatment with absolute ethanol at 4 °C, dissolve the precipitate in deionized water, and use a dialysis membrane to dialyze for 24 h to remove small molecule impurities. Finally, place the dialyzed solution in a freeze dryer for freeze-drying treatment, and the obtained polysaccharide is the fourth group;

[0062] The polysaccharide concentrations of the above groups are all 0.8 mg / mL. Using Vc as a control, the test results are shown in Table 1:

[0063] Table 3 Superoxide anion radical scavenging ability and hydroxyl radical scavenging ability of various polysaccharides

[0064] Superoxide anion radical scavenging ability Hydroxyl radical scavenging ability The first group 38.59±0.87 98.42±0.05％ The second group 8.12±0.55％ 87.15±0.41％ The third group 37.72±0.20％ 91.89±0.16％ The fourth group 25.19±0.20％ 27.46±2.85％ Ascorbic acid (Vc) 95.36±0.41％ 98.15±0.41％

[0065] According to the results in Table 3, it can be seen that the polysaccharides prepared by the method of this application show relatively good hydroxyl radical and superoxide anion scavenging activities, and have great potential in the application in fields such as skin care products.

[0066] The above embodiments only represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the present invention.

Claims

1. A method for extracting Momordica grosvenori polysaccharide, characterized in that, The method includes extracting Siraitia grosvenorii with an ultrasonic-assisted extraction solvent, which is obtained by dissolving ammonium sulfate in deionized water, then adding dextran and mixing evenly, subsequently adding ethanol to the solution, fully mixing with a vortex oscillator, and then standing for 4 - 10 h.

2. The method according to claim 1, characterized in that, In the extraction solvent, the mass ratio of ammonium sulfate, dextran, ethanol and deionized water is 3 - 4:0.1 - 0.2:4 - 5:

15.

3. The method according to claim 1, wherein The specific extraction method of the Siraitia grosvenorii polysaccharide includes the following steps: (1) Weigh Siraitia grosvenorii powder, mix it with the extraction solvent, place it in an ultrasonic cleaner for extraction, and then perform centrifugal separation to fully separate the supernatant. Subsequently, repeat the above operation once with the Siraitia grosvenorii residue, and combine the upper phases of the filtrates from the two extractions, removing the lower phase; (2) Centrifuge the upper-phase extract to collect the supernatant. Then add 1 / 4 volume of Sevag reagent to the supernatant, place it on a magnetic stirrer and stir continuously for 25 - 35 min to remove proteins, and repeat this step until no protein precipitate appears in the middle layer. After protein removal is completed, collect the supernatant, perform overnight alcohol precipitation treatment with absolute ethanol at 3 - 6 °C, redissolve the precipitate in deionized water, and dialyze with a dialysis membrane for 24 h to remove small molecule impurities. Finally, place the dialyzed solution in a freeze dryer for freeze-drying treatment to obtain the upper-phase polysaccharide, which is the Siraitia grosvenorii polysaccharide.

4. The method according to claim 3, characterized in that, In step (1), the liquid-solid ratio of the Siraitia grosvenorii powder to the extraction solvent is 10 - 50 mL / g, and the extraction conditions are an ultrasonic power of 200 - 600 W, an extraction temperature of 30 - 70 °C, and an extraction time of 10 - 50 min.

5. The method according to claim 4, wherein The liquid-solid ratio of the Siraitia grosvenorii powder to the extraction solvent is 32 mL / g, the extraction conditions are an ultrasonic power of 300 W, an extraction temperature of 60 °C, and an extraction time of 30 min.

6. A method for preparing skin care products using the Siraitia grosvenorii polysaccharide extracted by the method according to any one of claims 1 - 5.

7. A skin care product, characterized in that The skin care product is composed of the Siraitia grosvenorii polysaccharide prepared by the method according to any one of claims 1 - 5.

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