A method for extracting and purifying polysaccharides from golden sunflower seeds and its application

Abstract

The present invention relates to a method for extracting and purifying polysaccharides from golden sunflower seeds and their application, and belongs to the technical field of plant polysaccharide extraction and preparation. The polysaccharide preparation method of the present invention mainly comprises the following steps: microbial fermentation and enzymatic hydrolysis followed by distillation to remove volatile components and then extracting, enzymatic hydrolysis of the extract to convert macromolecules into small molecules, and then purification through a macroporous resin to obtain a polysaccharide component with antioxidant and anti-inflammatory effects. The polysaccharide component obtained by the present invention has a good moisturizing effect, can effectively remove oxygen free radicals, etc., and inhibit inflammatory factors, and has a significant effect. The polysaccharide can be used to prepare cosmetics with moisturizing, antioxidant and anti-inflammatory effects.

Description

Technical Field

[0001] The invention belongs to the technical field of plant polysaccharide extraction and preparation, and relates to a method for extracting and purifying polysaccharides from helianthus annuus seeds and application thereof. Background Art

[0002] In the prior art, there are many studies on the roots, stems, and flowers of Helianthus annuus. Its main components are flavonoids, unsaturated fatty acids, polysaccharides, trace elements, and alkaloids, which have the effects of anti-oxidation, lipid-lowering, anti-inflammatory, antipyretic, analgesic, and immunity-enhancing. In addition, the content of the above components in the flower ranks first, especially the total flavonoids, which are significantly richer than those in the roots, stems, and peels, making it an ideal part for medicinal use.

[0003] However, there are few reports on the research of Helianthus annuus seeds, especially the extraction, preparation and use of polysaccharides in Helianthus annuus seeds. Summary of the invention

[0004] The main purpose of the present invention is to provide a polysaccharide in Helianthus annuus seeds and a method for extracting and purifying the polysaccharide. The Helianthus annuus seed polysaccharide obtained by the method of the present invention has good moisturizing, anti-oxidation and anti-inflammatory effects, and the effect is remarkable.

[0005] The present invention adopts the following technical solutions to achieve the above purpose:

[0006] A method for extracting and purifying polysaccharides from golden sunflower seeds, specifically comprising the following steps:

[0007] Step A, crushing golden sunflower seeds, adding 2-3 times of water by weight, mixing and inoculating Aspergillus niger, fermenting at 25-30° C. for 10-12 hours, adding a complex enzyme aqueous solution containing lysozyme and β-galactosidase after fermentation, performing enzymolysis at 35-40° C. for 6-8 hours, performing ultrasonic treatment after enzymolysis, adjusting the pH to 7.5-8.0, adding taurine and distilling until no volatile components are produced, filtering the solution after distillation, and using the residue I and filtrate I for later use;

[0008] Step B, adding petroleum ether to the filter residue I obtained in step A for reflux extraction, filtering to obtain filter residue II, volatilizing to remove residual petroleum ether, and setting aside; adding 20-25% by mass ethanol solution to the obtained filter residue II, reflux extraction, filtering after the extraction is completed, obtaining filtrate II, and setting aside;

[0009] Step C, combining the filtrate I obtained in step A and the filtrate II obtained in step C into a mixed solution, adding β-mannanase, xylanase and glucose oxidase to the mixed solution, performing enzymolysis at 35-40° C. for 8-10 hours, heating to 70-80° C. to inactivate the enzyme, adjusting the pH to 5.5-6.0, the alcohol content to 80-85%, standing, and filtering to obtain a precipitate;

[0010] Step D, dissolving the precipitate obtained in step C in water, loading the sample onto a macroporous resin, eluting the sample with a lower layer solution of ethyl acetate-ethanol-water as an eluent, collecting the eluate, and drying the eluate to obtain Helianthus annuus seed polysaccharide.

[0011] Preferably, the amount of Aspergillus niger added in the above step A is 5-6% of the weight of the golden sunflower seeds.

[0012] Preferably, the amount of Aspergillus niger spores in the above step A is 20 billion / g.

[0013] Preferably, the amount of the complex enzyme aqueous solution added in the above step A is 3-5 times the weight of the golden sunflower seeds.

[0014] Preferably, in the above step A, the enzyme activity of lysozyme is 20,000 U / mg, and the enzyme activity of β-galactosidase is 10,000 U / g.

[0015] Preferably, the content of lysozyme in the aqueous solution of complex enzyme in step A is 10-15 g / L, and the content of β-galactosidase is 3-5 g / L.

[0016] Preferably, the amount of taurine added in the above step A is 10-15% of the weight of the sunflower seeds.

[0017] Preferably, in the above step B, the mass-volume ratio of the filter residue I to petroleum ether is 1:(5-8) in g / mL.

[0018] Preferably, in the above step B, the mass-volume ratio of the filter residue II to the ethanol solution is 1:(8-10) in g / mL.

[0019] Preferably, in the above step C, the enzyme activity of β-mannanase is 100,000 U / g, the enzyme activity of xylanase is 100,000 U / g, and the enzyme activity of glucose oxidase is 10,000 U / g.

[0020] Preferably, in the above step C, the amount of β-mannanase added is 3-5 g / L, the amount of xylanase added is 8-10 g / L, and the amount of glucose oxidase added is 1-1.5 g / L.

[0021] Preferably, the macroporous resin in the above step D is D101 macroporous adsorption resin.

[0022] Preferably, the volume ratio of ethyl acetate-ethanol-water in the above step D is 1:(0.3-0.35):(1-1.5).

[0023] The present invention also provides a specific use of the helianthus annuus seed polysaccharide prepared by the method, that is, the polysaccharide can be used in cosmetics, specifically, can be used in cosmetics with moisturizing, anti-inflammatory and antioxidant effects.

[0024] The present invention has the following beneficial effects:

[0025] 1. In the preparation method provided by the present invention, the volatile components in the golden sunflower seeds are first removed. The presence of the volatile components will affect the dissolution of polysaccharides and reduce the extraction rate. In the process of removing volatile components, Aspergillus niger fermentation is adopted, and the decomposition effect of microorganisms is utilized to destroy the tissue structure and cell structure of golden sunflower seeds, and the volatile components and sugar components inside and outside the cells and in the tissues are fully released; at the same time, Aspergillus niger will produce abundant enzymes during the fermentation process, and after the enzymatic hydrolysis of lysozyme and glycosidase, the enzymes in the Aspergillus niger spores are fully released, and the golden sunflower seeds are further enzymatically hydrolyzed to destroy their structure, which is conducive to the release or dissolution of volatile components and sugar components; the golden sunflower seeds after fermentation and enzymatic hydrolysis are distilled to remove volatile components. The temperature during the distillation process is relatively high, and some polysaccharide components will be broken or curled under high temperature conditions, turning into monosaccharides and oligosaccharides, resulting in the inability to obtain the active polysaccharide components of the present invention. Therefore, after trying multiple methods, the inventors found that adding taurine during the distillation process can effectively maintain the stability of the polysaccharide, avoid the destruction of the polysaccharide by high temperature, and obtain a polysaccharide component with significant activity. After distillation, there are still a small amount of volatile components in the golden sunflower seeds that cannot be distilled out. The petroleum ether extraction method is used to remove the residual volatile components as much as possible, and then an ethanol solution with a low alcohol content is used to extract the polysaccharides in the golden sunflower seeds.

[0026] 2. The polysaccharide-containing solution extracted with water and ethanol solution is enzymatically hydrolyzed to decompose the large molecular polysaccharide substance into small molecular sugars, and then the sugar components are separated out by acid precipitation plus alcohol precipitation. After purification by macroporous resin, the inventors found that the activities of the polysaccharide components obtained by traditional purification by elution with ethanol solutions of different mass fractions were quite different. Therefore, the inventors further screened the elution solution and found that the polysaccharide components obtained by purifying the polysaccharide in the lower layer solution after ethyl acetate, ethanol and water were the most active, and the yield of the polysaccharide was higher.

[0027] 3. The polysaccharide component of golden sunflower seeds obtained by the method of the present invention was subjected to moisture absorption, moisturizing, anti-inflammatory and antioxidant tests in vitro. The results showed that the polysaccharide component obtained by the present invention has high moisturizing, anti-inflammatory and antioxidant effects, and has a good moisturizing effect, and can effectively remove DPPH free radicals, O2 - Free radicals and inflammatory factors can be inhibited, and can be used to prepare cosmetics with moisturizing, antioxidant and anti-inflammatory effects, providing a direction for the further development and utilization of sunflower seed polysaccharides. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 : Comparison of moisture absorption rates of different polysaccharides;

[0029] Figure 2 : Comparison of moisture retention rates of different polysaccharides;

[0030] Figure 3 :Comparison of scavenging ability of different polysaccharides on DPPH· free radicals;

[0031] Figure 4 :Effects of different polysaccharides on ·O2 - Comparison of cleaning capabilities;

[0032] Figure 5 : Comparison of anti-inflammatory effects of different polysaccharides. DETAILED DESCRIPTION

[0033] The present invention is further explained below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. After reading the present invention, various equivalent modifications to the present invention by those skilled in the art all fall within the scope of protection of the claims of this application.

[0034] Example 1

[0035] Step A, crushing 1 kg of golden sunflower seeds, adding 3 kg of water, mixing and inoculating 60 mg of Aspergillus niger (the Aspergillus niger with the collection number of CGMCC 3.13901 purchased from the China General Microbiological Culture Collection Center is activated according to the instructions and cultured to a spore quantity of 20 billion / g), fermenting at 30° C. for 10 hours, adding 3 kg of a composite enzyme aqueous solution containing 15 g / L lysozyme (enzyme activity of 20,000 U / mg) and 3 g / L β-galactosidase (enzyme activity of 10,000 U / g) after fermentation, performing enzymolysis at 35° C. for 8 hours, ultrasonically treating for 30 minutes after enzymolysis, adjusting the pH to 8.0, adding 150 mg of taurine and distilling until no volatile components are produced, filtering the solution after distillation, and using the residue I and filtrate I for standby use;

[0036] Step B, adding petroleum ether to the residue I obtained in step A at a solid-liquid ratio of 1:8, g / mL, and reflux extraction, filtering after extraction to obtain residue II, volatilizing to remove residual petroleum ether, and setting aside; adding 25% ethanol solution by mass to the obtained residue II at a solid-liquid ratio of 1:8, g / mL, reflux extraction, filtering after extraction to obtain filtrate II, and setting aside;

[0037] Step C, combining the filtrate I obtained in step A and the filtrate II obtained in step C into a mixed solution, adding 5 g / L β-mannanase (enzyme activity of 100,000 U / g), 8 g / L xylanase (enzyme activity of 100,000 U / g), and 1.5 g / L glucose oxidase (enzyme activity of 10,000 U / g) to the mixed solution, performing enzymolysis at 40°C for 8 hours, heating to 80°C to inactivate the enzyme, adjusting the pH to 6.0, the alcohol content to 85%, standing, and filtering to obtain a precipitate;

[0038] Step D, dissolving the precipitate obtained in step C with an appropriate amount of water, loading the precipitate onto D101 macroporous adsorption resin, eluting the precipitate with a lower layer solution of ethyl acetate:ethanol:water=1:0.35:1.5 as an eluent, collecting the eluate, and drying the eluate to obtain Helianthus annuus seed polysaccharide.

[0039] Example 2

[0040] Step A, crushing 1 kg of golden sunflower seeds, adding 2 kg of water, mixing and inoculating 50 mg of Aspergillus niger (the Aspergillus niger with the collection number of CGMCC 3.13901 purchased from the China General Microbiological Culture Collection Center is activated according to the instructions and cultured to a spore quantity of 20 billion / g), fermenting at 25° C. for 12 hours, adding 5 kg of a composite enzyme aqueous solution containing 10 g / L lysozyme (enzyme activity of 20,000 U / mg) and 5 g / L β-galactosidase (enzyme activity of 10,000 U / g) after fermentation, performing enzymolysis at 40° C. for 6 hours, ultrasonically treating for 30 minutes after enzymolysis, adjusting the pH to 7.5, adding 100 mg of taurine and distilling until no volatile components are produced, filtering the solution after distillation, and using the residue I and filtrate I for standby use;

[0041] Step B, adding petroleum ether to the residue I obtained in step A at a solid-liquid ratio of 1:5, g / mL, and reflux extraction, filtering after extraction to obtain residue II, volatilizing to remove residual petroleum ether, and setting aside; adding 20% ​​ethanol solution by mass to the obtained residue II at a solid-liquid ratio of 1:10, g / mL, reflux extraction, filtering after extraction to obtain filtrate II, and setting aside;

[0042] Step C, combining the filtrate I obtained in step A and the filtrate II obtained in step C into a mixed solution, adding 3 g / L β-mannanase (enzyme activity of 100,000 U / g), 10 g / L xylanase (enzyme activity of 100,000 U / g), and 1 g / L glucose oxidase (enzyme activity of 10,000 U / g) to the mixed solution, performing enzymolysis at 35° C. for 10 h, heating to 70° C. to inactivate the enzyme, adjusting the pH to 5.5, the alcohol content to 80%, standing, and filtering to obtain a precipitate;

[0043] Step D, dissolving the precipitate obtained in step C with an appropriate amount of water, loading the precipitate onto D101 macroporous adsorption resin, eluting the precipitate with a lower layer solution of ethyl acetate:ethanol:water=1:0.3:1 as an eluent, collecting the eluate, and drying the eluate to obtain Helianthus annuus seed polysaccharide.

[0044] Example 3

[0045] Step A, crushing 1 kg of golden sunflower seeds, adding 2.5 kg of water, mixing and inoculating 55 mg of Aspergillus niger (the Aspergillus niger with the collection number of CGMCC 3.13901 purchased from the China General Microbiological Culture Collection Center is activated according to the instructions and cultured to a spore quantity of 20 billion / g), fermenting at 30° C. for 11 hours, adding 4 kg of a composite enzyme aqueous solution containing 13 g / L lysozyme (enzyme activity of 20,000 U / mg) and 4 g / L β-galactosidase (enzyme activity of 10,000 U / g) after fermentation, performing enzymolysis at 40° C. for 7 hours, ultrasonically treating for 30 minutes after enzymolysis, adjusting the pH to 7.5, adding 130 mg of taurine and distilling until no volatile components are produced, filtering the solution after distillation, and using the residue I and filtrate I for standby use;

[0046] Step B, adding petroleum ether to the residue I obtained in step A at a solid-liquid ratio of 1:6, g / mL, reflux extraction, filtering after extraction to obtain residue II, volatilizing to remove residual petroleum ether, and setting aside; adding 23% ethanol solution by mass to the obtained residue II at a solid-liquid ratio of 1:9, g / mL, reflux extraction, filtering after extraction to obtain filtrate II, and setting aside;

[0047] Step C, combining the filtrate I obtained in step A and the filtrate II obtained in step C into a mixed solution, adding 4 g / L β-mannanase (enzyme activity of 100,000 U / g), 9 g / L xylanase (enzyme activity of 100,000 U / g), and 1.2 g / L glucose oxidase (enzyme activity of 10,000 U / g) to the mixed solution, performing enzymolysis at 40°C for 9 hours, heating to 70°C to inactivate the enzyme, adjusting the pH to 6.0, the alcohol content to 83%, standing, and filtering to obtain a precipitate;

[0048] Step D, dissolving the precipitate obtained in step C with an appropriate amount of water, loading the precipitate onto D101 macroporous adsorption resin, eluting the precipitate with a lower layer solution of ethyl acetate:ethanol:water=1:0.3:1.3 as an eluent, collecting the eluate, and drying the eluate to obtain Helianthus annuus seed polysaccharide.

[0049] Comparative Example 1

[0050] 1 kg of Helianthus annuus seeds were crushed, 5 kg of water was added for reflux extraction, and after the extraction was completed, the filtrate was filtered to obtain a filtrate; the alcohol content of the filtrate was adjusted to 85%, and the filtrate was allowed to stand, and suction was filtered to obtain a precipitate, which was Helianthus annuus seed polysaccharide.

[0051] Comparative Example 2

[0052] Step A, crushing 1 kg of golden sunflower seeds, adding 5 kg of water, distilling until no volatile components are produced, adjusting the alcohol content of the solution to 25%, refluxing extraction, filtering after the extraction is completed, and using the filtrate for later use;

[0053] Step B, adding 4 g / L β-mannanase (enzyme activity of 100,000 U / g), 9 g / L xylanase (enzyme activity of 100,000 U / g), and 1.2 g / L glucose oxidase (enzyme activity of 10,000 U / g) to the filtrate of step A, performing enzymolysis at 40° C. for 9 hours, heating to 70° C. to inactivate the enzyme, adjusting the pH to 6.0, the alcohol content to 83%, standing, and filtering to obtain a precipitate;

[0054] Step C, adding an appropriate amount of water to dissolve the precipitate obtained in step C, loading the precipitate onto D101 macroporous adsorption resin, eluting the precipitate with a lower layer solution of ethyl acetate:ethanol:water=1:0.3:1.3 as an eluent, collecting the eluate, and drying the eluate to obtain Helianthus annuus seed polysaccharide.

[0055] Comparative Example 3

[0056] Step A, crushing 1 kg of golden sunflower seeds, adding 5 kg of water for reflux extraction, filtering after extraction, and obtaining a filtrate for later use;

[0057] Step B, adding 4g / L β-mannanase (enzyme activity of 100,000 U / g), 9g / L xylanase (enzyme activity of 100,000 U / g), and 1.2g / L glucose oxidase (enzyme activity of 10,000 U / g) to the filtrate obtained in step A, performing enzymolysis at 40°C for 9h, heating to 70°C to inactivate the enzyme, adjusting the pH to 6.0, the alcohol content to 83%, standing, and filtering to obtain a precipitate, which is Helianthus annuus seed polysaccharide.

[0058] Comparative Example 4

[0059] Step A, crushing 1 kg of golden sunflower seeds, adding 2.5 kg of water, mixing and inoculating 55 mg of Aspergillus niger (the Aspergillus niger with the deposit number of CGMCC 3.13901 purchased from the China General Microbiological Culture Collection Center is activated according to the instructions and cultured to a spore quantity of 20 billion / g), fermenting at 30° C. for 11 hours, ultrasonically treating for 30 minutes after the fermentation is completed, adjusting the pH to 7.5, distilling until no volatile components are produced, filtering the solution after distillation, and using the residue I and the filtrate I for later use;

[0060] Step B, adding the residue I obtained in step A to an ethanol solution with a mass fraction of 23% at a solid-liquid ratio of 1:9, g / mL, reflux extraction, and filtering after the extraction to obtain filtrate II for standby use;

[0061] Step C, combining the filtrate I obtained in step A and the filtrate II obtained in step C into a mixed solution, adding 4 g / L β-mannanase (enzyme activity of 100,000 U / g) and 9 g / L xylanase (enzyme activity of 100,000 U / g) to the mixed solution, performing enzymolysis at 40°C for 9 hours, heating to 70°C to inactivate the enzyme, adjusting the pH to 6.0, the alcohol content to 83%, standing, and filtering to obtain a precipitate;

[0062] Step D, adding an appropriate amount of water to dissolve the precipitate obtained in step C, loading it onto D101 macroporous adsorption resin, eluting it with 10% by mass ethanol solution as an eluent, collecting the eluate, and drying it to obtain Helianthus annuus seed polysaccharide.

[0063] 1. Effects of different extraction methods on the extraction rate of polysaccharides from golden sunflower seeds

[0064] 1. Calculation of crude polysaccharide extraction rate from golden sunflower seeds

[0065] The precipitates obtained in step C of Examples 1 to 3, the precipitate obtained in Comparative Example 1, the precipitate obtained in step B of Comparative Example 2, the precipitate obtained in step B of Comparative Example 3, and the precipitate obtained in step C of Comparative Example 4 were weighed to calculate the yield of crude polysaccharides.

[0066] Crude polysaccharide yield (%) = M1 / M0 × 100%

[0067] In the formula, M1 is the sedimentation mass, and M0 is the weight of golden sunflower seeds.

[0068] 2. Calculation of the yield of polysaccharides from golden sunflower seeds

[0069] The polysaccharides obtained in step D of Examples 1 to 3, the polysaccharides obtained in step C of Comparative Example 2, and the polysaccharides obtained in step D of Comparative Example 4 were weighed to calculate the yield of helianthus annuus seed polysaccharides.

[0070] Crude polysaccharide yield (%) = M2 / M1×100%

[0071] Wherein, M2 is the mass of polysaccharide, and M1 is the weight of the above-mentioned precipitate.

[0072] 3. Results and Analysis

[0073] As shown in the results in Table 1, the content of crude polysaccharides in the golden sunflower seeds obtained by the preparation method of the present invention is relatively high, above 20%, which is significantly higher than the preparation methods described in Comparative Examples 1 to Comparative Examples 4, and the yield of polysaccharides in the golden sunflower seeds after purification is also relatively high. In Comparative Example 1, polysaccharides are extracted by conventional water extraction and alcohol precipitation methods, and the yield is relatively low; in Comparative Example 2, the golden sunflower seeds are subjected to conventional distillation to remove volatile components, and taurine is not added during the distillation process, and the volatile components are not extracted again by petroleum ether, and the removal of volatile components is incomplete, which affects the extraction of polysaccharides; in Comparative Example 3, the crude polysaccharides are enzymatically hydrolyzed after extraction by conventional water extraction and alcohol precipitation methods, and the extraction rate is relatively low; in Comparative Example 4, some necessary enzymatic hydrolysis steps are missing compared to the method of the present invention, and although the polysaccharide yield is higher than that of other comparative examples, it is still lower than that of the embodiment. It can be seen that the preparation method of the present invention is conducive to improving the extraction rate of polysaccharides.

[0074] Table 1 Effects of different preparation methods on the yield of polysaccharides from Helianthus annuus seeds

[0075]

[0076] 2. Moisturizing, antioxidant and anti-inflammatory effects of golden sunflower seed polysaccharides

[0077] 1. Determination of moisture absorption and moisture retention capacity of Helianthus annuus polysaccharide

[0078] 1.1 Experimental process

[0079] Weigh 10g of each of the polysaccharides of Helianthus annuus seeds prepared by the methods of Examples 1 to 3 and Comparative Examples 1 to 4, keep them in an environment of 20±2°C and 80±2% humidity for 50h, measure the weight every 5h, and the difference between the weight weighed each time and the initial weight (W0) is the moisture absorption, and calculate the moisture absorption rate. Sodium alginate is used as a reference.

[0080] Moisture absorption rate (%) = moisture absorption / initial weight × 100%

[0081] The polysaccharides and sodium alginate kept for 50 hours were placed in a 20±2°C, 40±2% humidity environment for 50 hours, and the weight was measured every 5 hours to calculate the moisture retention rate. The weight of the polysaccharide kept in the hygroscopic environment for 50 hours was taken as the starting weight.

[0082] Moisture retention rate (%) = weight per weighing / starting weight × 100%

[0083] 1.2 Test results and analysis

[0084] since Figure 1 The results show that the moisture absorption rate of the golden sunflower seed polysaccharide prepared by the present invention increases slowly, and the moisture absorption rate is lower than that of comparative examples 1 to 4, and the amplitude of the change is small, without obvious fluctuation. Figure 2The results show that the moisturizing performance of the golden sunflower seed polysaccharide prepared by the present invention is better, although it shows a downward trend, its moisturizing rate is significantly higher than that of other groups. In Comparative Example 1, the polysaccharide is obtained by the traditional water extraction and alcohol precipitation method, and its polysaccharide composition is complex, its hygroscopic performance is strong, but its moisturizing performance is poor; the extraction methods of polysaccharides in Comparative Examples 2-4 are different from those of the present invention, and although the hygroscopic performance of the polysaccharides is good, the moisturizing performance is poor.

[0085] 2. Determination of the antioxidant effect of Helianthus annuus polysaccharide

[0086] 2.1 Experimental process

[0087] 2.1.1 Scavenging ability of DPPH free radicals

[0088] Referring to existing literature, the polysaccharides prepared by the methods of Examples 1 to 3 and Comparative Examples 1 to 4 were used to prepare a 1.0 g / L polysaccharide solution; a 0.20 mmol / L DPPH solution was prepared, and 2.0 mL of different polysaccharide solutions and 2.0 mL of DPPH solution were taken respectively, mixed and reacted at 37°C in the dark for 30 minutes, and then the absorbance was measured. C The positive control group was distilled water, and the negative control group was distilled water. The scavenging rate of polysaccharide on DPPH· was calculated. Each group was tested in parallel 3 times and the average value was taken.

[0089] Clearance rate (%) = (A0-A1) / A0×100%

[0090] Where A0 is the absorbance of the negative control group, and A1 is the absorbance of the sample solution.

[0091] 2.1.2 Superoxide anion free radical (·O2 - )

[0092] Referring to existing literature, the polysaccharides prepared from Helianthus annuus seeds according to the methods of Examples 1 to 3 and Comparative Examples 1 to 4 were prepared into 1.0 g / L polysaccharide solutions; 2.5 mL PBS (50.0 mmol / L) and 0.3 mL pyrogallol solution (3.0 mmol / L) were added to 1 mL of different polysaccharide solutions, mixed and reacted at 25°C for 20 min, and then 0.2 mL hydrochloric acid solution (8.0 mmol / L) was added to terminate the reaction, and the absorbance was measured. V C The positive control group and distilled water were used as negative control group. The effect of polysaccharide on ·O2 - Each group was tested 3 times in parallel and the average value was taken.

[0093] Clearance rate (%) = (A0-A1) / A0×100%

[0094] Where A0 is the absorbance of the negative control group, and A1 is the absorbance of the sample solution.

[0095] 2.2 Results and Analysis

[0096] since Figure 3 , Figure 4 The results show that the polysaccharide prepared by the present invention has good antioxidant properties, and its antioxidant properties are better than those of V C The polysaccharides prepared in Comparative Examples 1 to 4 have significantly weaker antioxidant properties. Considering that the polysaccharide components are complex and there are sugar components without antioxidant properties, the overall effect is poor.

[0097] 3. Determination of the anti-inflammatory effect of Helianthus annuus seed polysaccharide

[0098] 3.1 Experimental process

[0099] The polysaccharide of Helianthus annuus seed prepared by the methods of Examples 1 to 3 and Comparative Examples 1 to 4 was prepared into a 200 μg / mL polysaccharide solution. Referring to the existing literature, RAW264.7 cells in the logarithmic growth phase were added to a DMEM medium containing 10% fetal bovine serum by volume and diluted to 5×10 5 / mL, and inoculated into a 96-well plate. Culture in a constant temperature incubator for 24 hours, discard the supernatant, add 200μL of complete culture medium to the blank group, add 200μL of complete culture medium containing 100μg / mL dexamethasone to the control group, and add 200μL of culture medium containing 200μg / mL polysaccharide to the test group. Continue to culture in the incubator for 16 hours, take the supernatant and test the TNF-α content according to the instructions of the ELISA kit. Take the average value of 5 parallel wells in each group.

[0100] 3.2 Results and Analysis

[0101] since Figure 5 It can be seen from the results that the polysaccharide component prepared in the present invention has a good anti-inflammatory effect, and the effect is equivalent to that of dexamethasone, and is better than the polysaccharides obtained in Comparative Examples 1 to 4. The reason is probably that the content of the polysaccharide components with anti-inflammatory effects in Comparative Examples 1 to 4 is relatively small, and some polysaccharides have no anti-inflammatory effect.

Claims

1. A method for extracting and purifying polysaccharides from golden sunflower seeds, characterized in that: The following steps are involved: Step A, crushing golden sunflower seeds, adding 2-3 times of water by weight, inoculating Aspergillus niger after mixing, fermenting at 25-30° C. for 10-12 hours, adding a composite enzyme aqueous solution containing lysozyme and β-galactosidase after fermentation, performing enzymolysis at 35-40° C. for 6-8 hours, ultrasonic treatment after enzymolysis, adjusting the pH to 7.5-8.0, adding taurine and distilling until no volatile components are produced, filtering the solution after distillation, and using the filter residue I and the filtrate I for standby use; wherein, the amount of Aspergillus niger added is 5-6% of the weight of golden sunflower seeds, the spore amount of Aspergillus niger is 20 billion / g, and the Aspergillus niger is the Aspergillus niger with a preservation number of CGMCC 3.13901 of the China General Microbiological Culture Collection Center; the amount of the composite enzyme aqueous solution added is 3-5 times the weight of the golden sunflower seeds, and the content of lysozyme in the composite enzyme aqueous solution is 10-15 g / L, The content of β-galactosidase is 3-5g / L, the enzyme activity of lysozyme is 20000U / mg, and the enzyme activity of β-galactosidase is 10,000U / g; Step B, adding petroleum ether to the filter residue I obtained in step A for reflux extraction, filtering to obtain filter residue II, volatilizing to remove residual petroleum ether, and setting aside; adding 20-25% by mass ethanol solution to the obtained filter residue II, reflux extraction, filtering after the extraction is completed, obtaining filtrate II, and setting aside; Step C, combining the filtrate I obtained in step A and the filtrate II obtained in step B into a mixed solution, adding β-mannanase, xylanase and glucose oxidase to the mixed solution, performing enzymolysis at 35-40° C. for 8-10 hours, heating to 70-80° C. to inactivate the enzymes, adjusting the pH to 5.5-6.0, the alcohol content to 80-85%, standing, and filtering to obtain a precipitate; wherein the amount of β-mannanase added is 3-5 g / L, the amount of xylanase added is 8-10 g / L, and the amount of glucose oxidase added is 1-1.5 g / L; the enzyme activity of the β-mannanase is 100,000 U / g, the enzyme activity of the xylanase is 100,000 U / g, and the enzyme activity of the glucose oxidase is 10,000 U / g; Step D, dissolving the precipitate obtained in step C in water, loading the sample onto a macroporous resin, eluting with a lower layer solution of ethyl acetate-ethanol-water as an eluent, collecting the eluate, and drying to obtain Helianthus annuus seed polysaccharide; wherein the volume ratio of ethyl acetate-ethanol-water is 1:(0.3-0.35):(1-1.5).

2. The method according to claim 1, characterized in that The mass-volume ratio of the filter residue I to petroleum ether in step B is 1:(5-8) in g / mL.

3. The method according to claim 1, characterized in that The mass-volume ratio of the filter residue II to the ethanol solution in step B is 1:(8-10) in g / mL.

4. The method according to claim 1, characterized in that The macroporous resin in step D is D101 macroporous adsorption resin.

5. Use of the Helianthus annuus seed polysaccharide prepared according to the method according to any one of claims 1 to 4 in cosmetics.

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