A kind of polysaccharide extracted from sunflower receptacle and its preparation method and application

Abstract

The invention discloses a polysaccharide extracted from a sunflower receptacle, a preparation method and an application thereof. The technical peak molecular weight (Mp), weight average molecular weight (Mw) and number average molecular weight (Mn) of the polysaccharide from the sunflower receptacle are 6.8×10 4 Da, 1.6×10 5 Da and 3.1×10 4 Da, the content of each component in the polysaccharide extracted from sunflower receptacle is: the content of reducing sugar is 3.0-5.1wt%, the content of polysaccharide is 60.1-63.1wt%, and the content of total sugar is 63.1-68.2wt%; the preparation method is simple, efficient and safe. The sunflower receptacle polysaccharide prepared by the method has no toxicity to skin cells, and has the effects of resisting ultraviolet radiation damage, moisturizing and anti-oxidation, and can be used in cosmetics and external-use medicines for resisting ultraviolet radiation damage.

Description

Technical Field

[0001] The invention relates to the technical field of polysaccharide extracted from sunflower receptacle, and in particular to polysaccharide extracted from sunflower receptacle, a preparation method and application thereof. Background Art

[0002] Sunflower (Helianthus annuus L.) is a plant of the genus Helianthus in the Asteraceae family. It is an important oil crop in my country, with a wide planting area and production all over the country. Sunflower seed meal is a byproduct of oil extraction from sunflower seeds. The annual output of sunflower seed meal in my country exceeds 400,000 tons (Zhang Weinong, Liu Dachuan. Comprehensive development of sunflower seed resources [J]. Journal of Wuhan Polytechnic Institute, 2002 (1): 29-30+35.). Sunflower flower is a head inflorescence, consisting of ligulate flowers, tubular flowers, involucre and receptacle. Since the head inflorescence is about 10 to 30 cm in diameter, as big as a disk, it is commonly known as the sunflower disk. The receptacle of sunflower seeds can be used as medicine. According to existing literature reports, the receptacle of sunflower contains chemical components such as polysaccharides, diterpenes, flavonoids and chlorogenic acid (Zhao Ping, Saeed Hamid Saeed Omer, Yang Heng, Liu Jintao, Zhang Ziliang, Liu Yuhui, Wang Huan, Wei Mingguang. Functional components of sunflower disc and research status [J]. Biological Resources, 2018, 40(3): 203-207). At present, the receptacle of sunflower has not been fully developed and utilized. It is generally used as animal feed or abandoned as crop waste.

[0003] Patent document (CN201710159728.2) discloses a method for extracting and separating sunflower stem core polysaccharides with anti-tumor activity. To obtain sunflower stem core polysaccharides with anti-tumor activity, the preparation method includes the steps of removing monosaccharides and oligosaccharides, separating through a DEAE cellulose DE-52 ion exchange column, gradient elution, etc. The preparation method is complicated, the yield is low, the production cost is high, and it is difficult to promote and apply.

[0004] Patent document (CN201210466655.9) discloses sunflower polysaccharide and a preparation method thereof, which includes hot water soaking, enzymatic hydrolysis and other steps. The obtained polysaccharide has a high yield and has certain immunomodulatory activity.

[0005] It can be seen that there is currently no method for preparing sunflower receptacle polysaccharide that is non-toxic to skin cells and has the effects of resisting ultraviolet radiation damage, moisturizing and anti-oxidation. Summary of the invention

[0006] The purpose of the present invention is to solve the above-mentioned problems and provide a polysaccharide extracted from sunflower receptacle and a preparation method and application thereof. The technical peak molecular weight (Mp), weight average molecular weight (Mw) and number average molecular weight (Mn) of the polysaccharide from sunflower receptacle are 6.8×10 4 Da, 1.6×10 5 Da and 3.1×10 4 Da, and establish a safe, simple and efficient preparation method. The sunflower receptacle polysaccharide prepared by this method is non-toxic to skin cells, and has the effects of resisting ultraviolet radiation damage, moisturizing and anti-oxidation, and can be used in cosmetics and topical medicines that resist ultraviolet radiation damage.

[0007] The present invention is achieved through the following technical solutions:

[0008] The invention provides: a polysaccharide extracted from a sunflower receptacle, wherein the technical peak molecular weight, weight average molecular weight and number average molecular weight of the polysaccharide extracted from the sunflower receptacle are 6.8×10 4 Da, 1.6×10 5 Da and 3.1×10 4 Da.

[0009] Furthermore, a polysaccharide extracted from a sunflower receptacle is provided, wherein the contents of the components in the polysaccharide extracted from the sunflower receptacle are as follows: the content of reducing sugar is 3.0-5.1wt%, the content of polysaccharide is 60.1-63.1wt%, and the content of total sugar is 63.1-68.2wt%.

[0010] The present invention also provides a method for preparing polysaccharides extracted from sunflower receptacles, comprising the following steps:

[0011] (1) taking dried sunflower receptacles and crushing them into 200-300 meshes to obtain sunflower receptacle powder;

[0012] (2) adding water to the sunflower receptacle powder for 2 to 4 times of heat extraction, combining the extracts obtained from the multiple heat extractions to obtain a total extract;

[0013] (3) concentrating the total extract to obtain a thick paste with a density of 0.2 to 0.02 g / mL;

[0014] (4) adjusting the alcohol content of the thick paste to 68-72 wt % with ethanol, allowing the thick paste to stand, filtering, taking a precipitate, and freeze-drying the precipitate to obtain a crude polysaccharide;

[0015] (5) re-dissolving the crude polysaccharide with water, centrifuging, removing insoluble impurities, taking the supernatant, and obtaining a crude polysaccharide solution; adjusting the alcohol content of the crude polysaccharide solution to 68-72 wt % with ethanol, performing secondary alcohol precipitation, standing, filtering, taking the precipitate, and freeze-drying to obtain a secondary alcohol-precipitated polysaccharide;

[0016] (6) ultrasonically cleaning the polysaccharide obtained by secondary alcohol precipitation with 88-92 Vol% ethanol for 2-4 times, filtering, and evaporating the ethanol to obtain the sunflower receptacle extracted polysaccharide.

[0017] Furthermore, in the step (1), dried sunflower receptacles with a moisture content of less than 8 wt % are taken and crushed to 250-300 meshes to obtain sunflower receptacle powder.

[0018] Furthermore, in the step (2), water is added to the sunflower receptacle powder for heat extraction 3 to 4 times, and the extracts obtained from multiple heat extractions are combined to obtain a total extract; during the heat extraction, the amount of water used for each heat extraction is 15 to 20 times the weight of the sunflower receptacle powder, and each heat extraction takes 0.8 to 1.2 hours.

[0019] Furthermore, in the step (3), the total extract is concentrated at 48-52° C. to obtain a thick paste with a density of 0.2-0.02 g / mL.

[0020] Furthermore, in the step (4), the alcohol content of the thick paste is adjusted to 70-72 wt % with anhydrous ethanol at 8-12° C., and the mixture is allowed to stand at 8-12° C. for 10-15 h, filtered, and the precipitate is taken and freeze-dried to obtain crude polysaccharide.

[0021] Furthermore, in step (5), three-stage freeze-drying is performed to obtain a polysaccharide subjected to secondary alcohol precipitation; the process of the three-stage freeze-drying is as follows: the temperatures of the three-stage freeze-drying are -80 to -75°C, -65 to -60°C, and -75 to -70°C, respectively; and the times of the three-stage freeze-drying are 4 to 5 hours, 8 to 10 hours, and 23 to 25 hours, respectively.

[0022] Furthermore, in step (5), three-stage freeze-drying is performed to obtain a polysaccharide subjected to secondary alcohol precipitation; the process of the three-stage freeze-drying is as follows: the temperatures of the three-stage freeze-drying are -78 to -75°C, -63 to -60°C, and -72 to -75°C, respectively; and the times of the three-stage freeze-drying are 4.5 to 5h, 9 to 10h, and 24 to 25h, respectively.

[0023] The present invention also provides an application of polysaccharides extracted from sunflower receptacles, which is applied to cosmetics or external medicines for resisting damage caused by ultraviolet radiation.

[0024] The invention discloses a polysaccharide extracted from a sunflower receptacle, a preparation method thereof and an application thereof. The technical peak molecular weight (Mp), weight average molecular weight (Mw) and number average molecular weight (Mn) of the polysaccharide from the sunflower receptacle are 6.8×10 4 Da, 1.6×10 5 Da and 3.1×10 4Da, the content of each component in the polysaccharide extracted from the sunflower receptacle is: the content of reducing sugar is 3.0-5.1wt%, the content of polysaccharide is 60.1-63.1wt%, and the content of total sugar is 63.1-68.2wt%; the preparation method is simple, efficient and safe, the sunflower receptacle polysaccharide prepared by the method has no toxicity to skin cells, has the effects of resisting ultraviolet radiation damage, moisturizing and anti-oxidation, and can be applied to cosmetics and external medicines for resisting ultraviolet radiation damage.

[0025] The invention discloses a polysaccharide extracted from a sunflower receptacle, a preparation method thereof and an application thereof. When preparing a crude polysaccharide, the temperature of the alcohol precipitation ethanol and the standing temperature are reasonably controlled, which can effectively remove impurities and effectively improve the yield of the crude polysaccharide, thereby improving the yield of the polysaccharide extracted from the sunflower receptacle. In step (5), three-stage freeze-drying is sequentially performed, which can affect the microstructure of the polysaccharide precipitated by the secondary alcohol precipitation. When the polysaccharide precipitated by the secondary alcohol precipitation is subsequently cleaned by ultrasonic, less polysaccharide is washed off, thereby effectively improving the yield of the polysaccharide extracted from the sunflower receptacle. DETAILED DESCRIPTION

[0026] Any feature disclosed in this specification (including any additional claims and abstract), unless otherwise stated, may be replaced by other equivalent or alternative features having similar purposes. That is, unless otherwise stated, each feature is only an example of a series of equivalent or similar features.

[0027] The invention discloses a polysaccharide extracted from a sunflower receptacle. The technical peak molecular weight, weight average molecular weight and number average molecular weight of the polysaccharide extracted from the sunflower receptacle are 6.8×10 4 Da, 1.6×10 5 Da and 3.1×10 4 Da; the content of each component in the polysaccharide extracted from the sunflower receptacle is: the content of reducing sugar is 3.0-5.1wt%, the content of polysaccharide is 60.1-63.1wt%, and the content of total sugar is 63.1-68.2wt%.

[0028] The present invention discloses a polysaccharide extracted from a sunflower receptacle. The method for determining the content of total sugar in the following examples and comparative examples is as follows:

[0029] (1) Accurately weigh an appropriate amount of D-anhydrous glucose reference substance and prepare a 10 mg / mL reference substance solution with pure water for later use;

[0030] (2) Accurately weigh 0.1 g of polysaccharide extract into a conical flask, add 25 mL of pure water, ultrasonicate at 80°C for 40 min, reweight, centrifuge, accurately pipette 1 mL of supernatant into a 25 mL volumetric flask, add pure water to the mark, and shake well to obtain the test solution;

[0031] (3) Accurately pipette 1 mL of reference solution and test solution of different concentrations into different test tubes, add 5 mL of anthrone sulfate solution (accurately weigh 0.1 g of anthrone, add 100 mL of 80% concentrated sulfuric acid to dissolve, and shake well to obtain the solution). Shake well, heat in a 95°C water bath for 15 min, cool in an ice water bath, take out, measure the absorbance at 621 nm, and calculate the total sugar content.

[0032] The present invention discloses a polysaccharide extracted from a sunflower receptacle. The method for determining the content of reducing sugar in the following examples and comparative examples is as follows:

[0033] (1) Accurately weigh an appropriate amount of D-anhydrous glucose reference substance, add water to make up to volume, and prepare a reference substance solution with a concentration of 1 mg / mL;

[0034] (2) The preparation of the test solution is consistent with the total sugar content determination;

[0035] (3) Accurately pipette 1 mL of reference solution and test solution of different concentrations into a 25 mL stoppered test tube, add 1.5 mL of DNS test solution, shake well, and place in a boiling water bath for 5 min. Take out and cool to room temperature with running water, make up to 25 mL mark, shake well, measure the absorbance at 540 nm, and calculate the total sugar content.

[0036] The present invention discloses a polysaccharide extracted from a sunflower receptacle. The molecular weight determination method of the polysaccharide extracted from a sunflower receptacle in the following embodiment is as follows:

[0037] (1) The relative molecular weight of sunflower receptacle polysaccharide was determined by high performance liquid chromatography-differential refractometer detection;

[0038] (2) Different molecular weight dextran reference substances and test substances were dissolved in the mobile phase and the retention time t of the reference substance chromatographic peak was used. R The standard curve was prepared with 1gMw as the horizontal axis and 1gMw as the vertical axis. The results were processed using GPC-specific software to calculate various molecular weights. The technical peak molecular weight (Mp), weight average molecular weight (Mw) and number average molecular weight (Mn) of the polysaccharide were 6.8×10 4 Da, 1.6×10 5 Da and 3.1×10 4 Da.

[0039] Example 1: Preparation of the sunflower receptacle polysaccharide extraction

[0040] A method for preparing polysaccharides extracted from sunflower receptacles, comprising the following steps:

[0041] (1) taking 100 g of dried sunflower receptacles with a moisture content of less than 8 wt %, and crushing them into 200 meshes to obtain sunflower receptacle powder;

[0042] (2) adding water to the sunflower receptacle powder for heat extraction twice, combining the extracts obtained from multiple heat extractions to obtain a total extract; during the heat extraction, the amount of water used for each heat extraction is 15 times the weight of the sunflower receptacle powder, and each heat extraction takes 0.8 h;

[0043] (3) concentrating the total extract at 48° C. to obtain a thick paste with a density of 0.2 g / mL;

[0044] (4) adjusting the alcohol content of the thick paste to 68 wt % with 8° C. ethanol, standing at 8° C. for 10 h, filtering, taking the precipitate, and freeze-drying the precipitate at -60° C. for 72 h to obtain crude polysaccharide;

[0045] (5) re-dissolving the crude polysaccharide with water, centrifuging, removing insoluble impurities, taking the supernatant, and obtaining a crude polysaccharide solution; adjusting the alcohol content of the crude polysaccharide solution to 68 wt% with ethanol for secondary alcohol precipitation, standing, filtering, taking the precipitate, and performing three-stage freeze-drying to obtain a polysaccharide with secondary alcohol precipitation; the three-stage freeze-drying process is: the temperatures of the three-stage freeze-drying are -80°C, -65°C, and -75°C, respectively, and the times of the three-stage freeze-drying are 4h, 8h, and 23h, respectively; specifically, performing three-stage freeze-drying is explained as follows: first pre-freezing the precipitate, placing it in a -80°C low-temperature refrigerator for pre-freezing for 4h, then quickly transferring the precipitate to a freeze dryer, evacuating the freeze dryer and adjusting the internal temperature to -65°C, freezing it at -65°C for 8h, and finally adjusting the temperature in the freeze dryer to -75°C, freezing it at -75°C for 23h, and the total freezing time is 35h; the following embodiments are the same as embodiment 1, so they are not repeated here;

[0046] (6) ultrasonically cleaning the polysaccharide precipitated by the secondary alcohol precipitation twice with 88 Vol% ethanol, filtering, and evaporating the ethanol to obtain the sunflower receptacle extracted polysaccharide; the yield of the sunflower receptacle extracted polysaccharide was 36.28%; the results of the determination of the total sugar and reducing sugar contents in the sunflower receptacle polysaccharide are shown in Table 1 below;

[0047] Table 1 Test results of Example 1

[0048]

[0049] Example 2: Preparation of the sunflower receptacle polysaccharide extraction

[0050] A method for preparing polysaccharides extracted from sunflower receptacles, comprising the following steps:

[0051] (1) taking 100 g of dried sunflower receptacles with a moisture content of less than 8 wt %, and crushing them into 300 meshes to obtain sunflower receptacle powder;

[0052] (2) adding water to the sunflower receptacle powder for heat extraction 4 times, combining the extracts obtained from multiple heat extractions to obtain a total extract; during the heat extraction, the amount of water used for each heat extraction is 20 times the weight of the sunflower receptacle powder, and each heat extraction takes 1.2 hours;

[0053] (3) concentrating the total extract at 52° C. to obtain a thick paste with a density of 0.02 g / mL;

[0054] (4) adjusting the alcohol content of the thick paste to 72 wt % with 12° C. ethanol, standing at 12° C. for 15 h, filtering, taking the precipitate, and freeze-drying the precipitate at -60° C. for 72 h to obtain crude polysaccharide;

[0055] (5) re-dissolving the crude polysaccharide with water, centrifuging, removing insoluble impurities, taking the supernatant, and obtaining a crude polysaccharide solution; adjusting the alcohol content of the crude polysaccharide solution to 72 wt% with ethanol for secondary alcohol precipitation, standing, filtering, taking the precipitate, and performing three-stage freeze-drying to obtain a polysaccharide precipitated with secondary alcohol; the three-stage freeze-drying process is as follows: the temperatures of the three-stage freeze-drying are -75°C, -60°C, and -70°C, respectively, and the times of the three-stage freeze-drying are 5 h, 10 h, and 25 h, respectively;

[0056] (6) The polysaccharide obtained by secondary alcohol precipitation was ultrasonically cleaned 4 times with 92 Vol% ethanol, filtered, and evaporated to obtain the sunflower receptacle extracted polysaccharide; the yield of the sunflower receptacle extracted polysaccharide was 37.35%; the results of the determination of the total sugar and reducing sugar contents in the sunflower receptacle polysaccharide are shown in Table 2 below;

[0057] Table 2 Test results of Example 2

[0058]

[0059] Example 3: Preparation of the sunflower receptacle polysaccharide extraction

[0060] A method for preparing polysaccharides extracted from sunflower receptacles, comprising the following steps:

[0061] (1) taking 100 g of dried sunflower receptacles with a moisture content of less than 8 wt %, and crushing them into 250 meshes to obtain sunflower receptacle powder;

[0062] (2) adding water to the sunflower receptacle powder for heat extraction three times, combining the extracts obtained from multiple heat extractions to obtain a total extract; during the heat extraction, the amount of water used for each heat extraction was 16 times the weight of the sunflower receptacle powder, and each heat extraction took 1.1 h;

[0063] (3) concentrating the total extract at 50° C. to obtain a thick paste with a density of 0.1 g / mL;

[0064] (4) adjusting the alcohol content of the thick paste to 70 wt % with 10° C. ethanol, standing at 10° C. for 12 h, filtering, taking the precipitate, and freeze-drying the precipitate at -60° C. for 72 h to obtain crude polysaccharide;

[0065] (5) re-dissolving the crude polysaccharide with water, centrifuging, removing insoluble impurities, taking the supernatant, and obtaining a crude polysaccharide solution; adjusting the alcohol content of the crude polysaccharide solution to 70 wt% with ethanol for secondary alcohol precipitation, standing, filtering, taking the precipitate, and performing three-stage freeze-drying to obtain a polysaccharide precipitated with secondary alcohol; the three-stage freeze-drying process is as follows: the temperatures of the three-stage freeze-drying are -78°C, -63°C, and -72°C, respectively, and the times of the three-stage freeze-drying are 4.5 h, 9 h, and 24 h, respectively;

[0066] (6) The polysaccharide obtained by secondary alcohol precipitation was ultrasonically cleaned three times with 90 Vol% ethanol, filtered, and evaporated to obtain the sunflower receptacle extracted polysaccharide; the yield of the sunflower receptacle extracted polysaccharide was 38.26%; the results of the determination of the total sugar and reducing sugar contents in the sunflower receptacle polysaccharide are shown in Table 3 below;

[0067] Table 3 Test results of Example 3

[0068]

[0069] Example 4: Preparation of the sunflower receptacle polysaccharide extraction

[0070] A method for preparing polysaccharides extracted from sunflower receptacles, comprising the following steps:

[0071] (1) taking 100 g of dried sunflower receptacles with a moisture content of less than 8 wt %, and crushing them into 250 meshes to obtain sunflower receptacle powder;

[0072] (2) adding water to the sunflower receptacle powder for heat extraction three times, combining the extracts obtained from multiple heat extractions to obtain a total extract; during the heat extraction, the amount of water used for each heat extraction was 16 times the weight of the sunflower receptacle powder, and each heat extraction took 1.1 h;

[0073] (3) concentrating the total extract at 50° C. to obtain a thick paste with a density of 0.05 g / mL;

[0074] (4) adjusting the alcohol content of the thick paste to 70 wt % with ethanol, allowing it to stand for 12 h, filtering, taking a precipitate, and freeze-drying the precipitate at -60 ° C for 72 h to obtain a crude polysaccharide;

[0075] (5) re-dissolving the crude polysaccharide with water, centrifuging, removing insoluble impurities, taking the supernatant, and obtaining a crude polysaccharide solution; adjusting the alcohol content of the crude polysaccharide solution to 70 wt% with ethanol for secondary alcohol precipitation, standing, filtering, taking the precipitate, and performing three-stage freeze-drying to obtain a polysaccharide precipitated with secondary alcohol; the three-stage freeze-drying process is as follows: the temperatures of the three-stage freeze-drying are -78°C, -63°C, and -72°C, respectively, and the times of the three-stage freeze-drying are 4.5 h, 9 h, and 24 h, respectively;

[0076] (6) The polysaccharide obtained by secondary alcohol precipitation was ultrasonically cleaned three times with 90 Vol% ethanol, filtered, and evaporated to obtain the sunflower receptacle extracted polysaccharide; the yield of the sunflower receptacle extracted polysaccharide was 31.36%; the results of the determination of the total sugar and reducing sugar contents in the sunflower receptacle polysaccharide are shown in Table 4 below;

[0077] Table 4 Test results of Example 4

[0078]

[0079] Example 5: Preparation of the sunflower receptacle polysaccharide extraction

[0080] A method for preparing polysaccharides extracted from sunflower receptacles, comprising the following steps:

[0081] (1) taking 100 g of dried sunflower receptacles with a moisture content of less than 8 wt %, and crushing them into 250 meshes to obtain sunflower receptacle powder;

[0082] (2) adding water to the sunflower receptacle powder for heat extraction three times, combining the extracts obtained from multiple heat extractions to obtain a total extract; during the heat extraction, the amount of water used for each heat extraction was 16 times the weight of the sunflower receptacle powder, and each heat extraction took 1.1 h;

[0083] (3) concentrating the total extract at 50° C. to obtain a thick paste with a density of 0.07 g / mL;

[0084] (4) adjusting the alcohol content of the thick paste to 70 wt % with 10° C. ethanol, standing at 10° C. for 12 h, filtering, taking the precipitate, and freeze-drying the precipitate at -60° C. for 72 h to obtain crude polysaccharide;

[0085] (5) re-dissolving the crude polysaccharide with water, centrifuging, removing insoluble impurities, taking the supernatant, and obtaining a crude polysaccharide solution; adjusting the alcohol content of the crude polysaccharide solution to 70 wt % with ethanol, performing secondary alcohol precipitation, standing, filtering, taking the precipitate, and freeze-drying at -60°C for 72 h to obtain a polysaccharide precipitated with secondary alcohol;

[0086] (6) The polysaccharide obtained by secondary alcohol precipitation was ultrasonically cleaned three times with 90 Vol% ethanol, filtered, and evaporated to obtain the sunflower receptacle extracted polysaccharide; the yield of the sunflower receptacle extracted polysaccharide was 30.28%; the results of the determination of the total sugar and reducing sugar contents in the sunflower receptacle polysaccharide are shown in Table 5 below;

[0087] Table 5 Test results of Example 5

[0088]

[0089] Example 6: Preparation of the sunflower receptacle polysaccharide extraction

[0090] A method for preparing polysaccharides extracted from sunflower receptacles, comprising the following steps:

[0091] (1) taking 100 g of dried sunflower receptacles with a moisture content of less than 8 wt %, and crushing them into 250 meshes to obtain sunflower receptacle powder;

[0092] (2) adding water to the sunflower receptacle powder for heat extraction three times, combining the extracts obtained from multiple heat extractions to obtain a total extract; during the heat extraction, the amount of water used for each heat extraction was 16 times the weight of the sunflower receptacle powder, and each heat extraction took 1.1 h;

[0093] (3) concentrating the total extract at 50° C. to obtain a thick paste with a density of 0.08 g / mL;

[0094] (4) adjusting the alcohol content of the thick paste to 70 wt % with ethanol, allowing it to stand for 12 h, filtering, taking a precipitate, and freeze-drying the precipitate at -60 ° C for 72 h to obtain a crude polysaccharide;

[0095] (5) re-dissolving the crude polysaccharide with water, centrifuging, removing insoluble impurities, taking the supernatant, and obtaining a crude polysaccharide solution; adjusting the alcohol content of the crude polysaccharide solution to 70 wt % with ethanol, performing secondary alcohol precipitation, standing, filtering, taking the precipitate, and freeze-drying at -60°C for 72 h to obtain a polysaccharide precipitated with secondary alcohol;

[0096] (6) The polysaccharide obtained by secondary alcohol precipitation was ultrasonically cleaned three times with 90 Vol% ethanol, filtered, and evaporated to obtain the sunflower receptacle extracted polysaccharide; the yield of the sunflower receptacle extracted polysaccharide was 28.75%; the results of the determination of the total sugar and reducing sugar contents in the sunflower receptacle polysaccharide are shown in Table 6 below;

[0097] Table 6 Test results of Example 6

[0098]

[0099] Comparative Example 1

[0100] A method for preparing polysaccharides extracted from sunflower receptacles, comprising the following steps:

[0101] (1) taking 100 g of dried sunflower receptacles with a moisture content of less than 8 wt %, and crushing them into 250 meshes to obtain sunflower receptacle powder;

[0102] (2) adding water to the sunflower receptacle powder for heat extraction three times, combining the extracts obtained from multiple heat extractions to obtain a total extract; during the heat extraction, the amount of water used for each heat extraction was 16 times the weight of the sunflower receptacle powder, and each heat extraction took 1.1 h;

[0103] (3) concentrating the total extract at 50° C. to obtain a thick paste with a density of 0.06 g / mL;

[0104] (4) adjusting the alcohol content of the thick paste to 70 wt % with 10° C. ethanol, standing at 10° C. for 12 h, filtering, taking the precipitate, and freeze-drying the precipitate at -60° C. for 72 h to obtain the sunflower receptacle extracted polysaccharide; the yield of the sunflower receptacle extracted polysaccharide is 45.58%.

[0105] Experimental study on the prevention of ultraviolet radiation damage by the polysaccharide extracted from sunflower receptacle prepared by the method of Example 1:

[0106] (1) Cytotoxicity of sunflower receptacle polysaccharide on human immortalized keratinocytes (HaCaT)

[0107] Take HaCaT cells with a confluency of more than 80%, digest them, and adjust the cell concentration to 4-5×10 cells with culture medium (DMEM high-glucose medium containing 10% FBS and 1% double antibody). 5 / mL, inoculated in a 96-well plate, 100μL of cell suspension was added to each well, and the plate was placed in a 5% CO2, 37°C incubator for 24h. Sunflower receptacle polysaccharide and hyaluronic acid were prepared into 50mg / mL solutions with PBS, diluted with culture medium and added to the 96-well plate. The added volume of the sample was 100μL. The final concentrations of each sample after addition were 4000, 3000, 2000, 1000, 800, 400, 200 and 100μg / mL, respectively. Three replicate wells were set for each concentration, and a cell control and solvent blank group were also set up. After adding the sample, the culture was continued for 24h, the culture medium was aspirated, 100μL of 10% CCK-8 solution (prepared with serum-free culture medium) was added to each well, and after culturing for 2h, the OD value was measured at 450nm, the inhibition rate was calculated, and the half inhibition rate (IC) was calculated using statistical software. 50 ), the inhibition rate calculation formula is as follows, and the results are shown in Table 7.

[0108] Inhibition rate % = [1-(OD 给药 -OD空白 ) / (OD 细胞对照 -OD 空白 )]×100%

[0109] The results showed that the IC value of sunflower receptacle polysaccharide on HaCaT 50 Greater than 4000 μg / mL, proving that sunflower receptacle polysaccharides have low toxicity to HaCaT cells.

[0110] (2) Protective effect of sunflower receptacle polysaccharide on HaCaT radiation damage caused by UVB and UVA

[0111] Take HaCaT cells with a confluency of more than 80%, digest them, and adjust the cell concentration to 4-5×10 with culture medium (DMEM high-glucose medium containing 10% FBS and 1% double antibody). 5 / mL, inoculated in a 96-well plate, and 100μL of cell suspension was added to each well. A model group that received radiation, a sunflower receptacle polysaccharide administration group (with high, medium and low doses) and a hyaluronic acid positive control group were set up, and a cell control group that did not receive radiation was set up, with 3 replicates in each group. After the cells were plated and cultured for 24 hours, the supernatant was aspirated, and 100μL of each sample solution diluted with PBS to an appropriate concentration was added to the administration group, and 100μL of PBS was added to both the model group and the control group. The final concentrations of each administration group were: 2000μg / mL for the hyaluronic acid (300,000) positive control group, and 2000, 1000 and 500μg / mL for the high, medium and low dose groups of sunflower receptacle polysaccharides, respectively. The lids of the cells in each group that received radiation were opened and irradiated under a certain energy of UVB and UVA, and the radiation energy UVB was 5mJ / cm 2 , UVA is 150mJ / cm 2 , the radiation energy calculation formula is:

[0112] Radiation dose (mJ / cm 2 ) = radiation intensity (mw / cm 2 )×irradiation time (sec)

[0113] After the irradiation was completed, the supernatant was aspirated, each well was washed once with PBS, and 100 μL of culture medium was added to each well and cultured for 24 hours. The cell control group was not irradiated, and other operation steps were the same. After 24 hours, the CCK-8 method was used to develop color and calculate the survival rate. The survival rate calculation formula is as follows, and the results are shown in Table 8.

[0114] Survival rate % = (OD 辐射组 -OD 空白 ) / (OD 细胞对照 -OD 空白 )×100%

[0115] The results showed that the high, medium and low dose groups of sunflower receptacle polysaccharide had a preventive effect on HaCaT radiation damage caused by UVB and UVA, and the survival rate was significantly different from that of the model group (P < 0.01); among them, the preventive effect of the high and medium dose groups was significantly better than that of the hyaluronic acid group (P < 0.01).

[0116] Table 7 Cytotoxicity test results ( n=3)

[0117] Sample name Effector Cells <![CDATA[IC 50 (μg / mL)]]> Hyaluronic acid (300,000) HaC ＞4000 Sunflower Receptacle Polysaccharide HaC ＞4000

[0118] Table 8 Protective effect of sunflower receptacle polysaccharide on HaCaT radiation damage caused by UVB and UVA ( n=3)

[0119] Group Survival rate (%) Model Group 45.99±1.37 Hyaluronic acid (300,000) 50.90±2.42* Sunflower receptacle polysaccharide high dose group <![CDATA[56.48±1.52** ## ]]> Sunflower receptacle polysaccharide medium dose group <![CDATA[55.89±2.40** ## ]]> Sunflower receptacle polysaccharide low dose group 51.63±1.45**

[0120] Note: * indicates P < 0.05 compared with the model group, ** indicates P < 0.01 compared with the model group; ## It indicates P<0.01 compared with the hyaluronic acid group.

[0121] The water absorption rate of the sunflower receptacle extracted polysaccharide prepared by the method of Example 1 was measured

[0122] Take a number of clean weighing bottles with a diameter, mark them and weigh them with a balance, and record the mass of the weighing bottle m0. Weigh appropriate amounts of hyaluronic acid and sunflower receptacle polysaccharide (0.03-0.05g) respectively, place them in weighing bottles, record the mass of the weighing bottle and the sample m1, cover the bottle cap immediately after weighing, place the weighing bottle in a dryer with a relative humidity of RH=42.8% and 84.3% respectively, open the weighing bottle cap, cover the dryer lid, and weigh the sample after placing it in the dryer for 3h, 6h, 10h, 24h, 30h, 35h, 48h, 56h, and 72h, and record the mass of the weighing bottle and sample m at each time point. x The ambient temperature was kept at 25°C, and three parallel samples were set for each group of experiments. The moisture absorption rate was calculated as follows:

[0123]

[0124] The results are as follows:

[0125] Table 9 Moisture absorption rate of hyaluronic acid with different molecular weight and sunflower receptacle polysaccharide (%) (RH = 42.8%, n=3)

[0126]

[0127]

[0128] Note: * indicates P < 0.05 compared with hyaluronic acid (1.2 million). # It indicates that P < 0.05 compared with hyaluronic acid (300,000).

[0129] Table 10 Moisture absorption rate of hyaluronic acid with different molecular weight and sunflower receptacle polysaccharide (%) (RH=84.3%, n=3)

[0130]

[0131] Note: * indicates P < 0.05 compared with hyaluronic acid (1.2 million). # It indicates that P < 0.05 compared with hyaluronic acid (300,000).

[0132] From the experimental results, we can see that the water absorption rate of sunflower receptacle polysaccharide is between 11-34%, which has a certain moisturizing effect.

[0133] Experimental study on the antioxidant effect of the polysaccharide extracted from sunflower receptacle prepared by the method of Example 1:

[0134] (1) Determination of DPPH free radical scavenging ability

[0135] The DPPH free radical scavenging ability was determined according to the reference method (Zhao L, Kim JC, Hur J S. 7-Hydroxy-2-octenoic acid-ethylester mixture as an UV protectant secondary metabolite of an endolichenic fungus isolated from Menegazziaterebrata [J]. Archives of Microbiology, 2022, 204 (7): 395.) and the half clearance rate (IC 50 ), the results are shown in Table 11.

[0136] (2)ABTS + Free radical scavenging assay

[0137] Reference method (Li Qi, Wu Chaoquan, Pei Hehuan. Effect of salt roasting on the spectrum-effect relationship of Guangxi Emblica emblica scavenging two free radicals [J]. Chinese Journal of Hospital Pharmacy, 2023, 43(2): 168-174.) ABTS + The free radical scavenging ability was determined by using vitamin C as a positive control and calculating the half clearance rate (IC 50 ), the results are shown in Table 11.

[0138] (3) Hydroxyl radical scavenging assay

[0139] The hydroxyl free radical scavenging ability was determined by the reference method (Huang Shangfeng, Lu Feiyan, Huang Yuanhe, Pan Qiaodan, Zhang Xiangui, Huang Suoyi. Study on the antioxidant activity of Panax notoginseng polysaccharide [J]. Trace Elements and Health Research, 2018, 35(1): 30-32.) and the half clearance rate (IC 50 ), the results are shown in Table 11.

[0140] Table 11 Antioxidant test results ( n=3)

[0141]

[0142] Note: * indicates significant difference compared with the vitamin C group, P < 0.05; # = indicates P < 0.05 compared with the hyaluronic acid (300,000) group; - indicates no obvious free radical scavenging activity.

[0143] (4) Experimental summary of antioxidant effect:

[0144] The experimental results show that sunflower receptacle polysaccharide has certain antioxidant activity. Although its antioxidant activity is not as strong as vitamin C, its ability to scavenge three free radicals is significantly better than hyaluronic acid.

[0145] The sunflower receptacle extracted polysaccharide prepared in Comparative Example 1 was not subjected to multiple separation and purification. The sunflower receptacle extracted polysaccharide prepared in Comparative Example 1 was used to test the cytotoxicity of the sunflower receptacle polysaccharide to human epidermal keratinocytes (HaCaT). The results showed that the sunflower receptacle polysaccharide was highly toxic to HaCaT cells, and the sunflower receptacle polysaccharide had a weak protective effect against HaCaT radiation damage caused by UVB and UVA. The water absorption rate of the sunflower receptacle polysaccharide was about 10%, and the moisturizing effect was not strong. The sunflower receptacle extracted polysaccharide prepared in Comparative Example 1 was used to perform an antioxidant effect experiment. The experiment showed that the sunflower receptacle polysaccharide had a poor antioxidant effect. It can be seen from this that the present invention provides a sunflower receptacle extracted polysaccharide and a preparation method and application thereof. The technical peak molecular weight (Mp), weight average molecular weight (Mw) and number average molecular weight (Mn) of the sunflower receptacle polysaccharide are 6.8×10 4 Da, 1.6×10 5 Da and 3.1×10 4Da, the content of each component in the polysaccharide extracted from the sunflower receptacle is: the content of reducing sugar is 3.0-5.1wt%, the content of polysaccharide is 60.1-63.1wt%, and the content of total sugar is 63.1-68.2wt%; the preparation method is simple, efficient and safe, the sunflower receptacle polysaccharide prepared by the method has no toxicity to skin cells, has the effects of resisting ultraviolet radiation damage, moisturizing and anti-oxidation, and can be applied to cosmetics and external medicines for resisting ultraviolet radiation damage.

[0146] The invention discloses a polysaccharide extracted from a sunflower receptacle, a preparation method thereof and an application thereof. When preparing crude polysaccharide, the temperature of anhydrous ethanol used for alcohol precipitation and the standing temperature are reasonably controlled, which can effectively remove impurities and effectively improve the yield of crude polysaccharide, thereby improving the yield of polysaccharide extracted from the sunflower receptacle. In step (5), three-stage freeze-drying is sequentially performed, which can affect the microstructure of the polysaccharide precipitated by secondary alcohol. When the polysaccharide precipitated by secondary alcohol is subsequently cleaned by ultrasonic, less polysaccharide is washed off, thereby effectively improving the yield of polysaccharide extracted from the sunflower receptacle.

[0147] The foregoing description of specific exemplary embodiments of the present invention is for the purpose of illustration and demonstration. These descriptions are not intended to limit the present invention to the precise form disclosed, and it is clear that many changes and variations can be made based on the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present invention and its practical application, so that those skilled in the art can realize and utilize various different exemplary embodiments of the present invention and various different selections and changes. The scope of the present invention is intended to be limited by the claims and their equivalents.

Claims

1. A polysaccharide extracted from sunflower receptacle, characterized in that: The technical peak molecular weight, weight average molecular weight and number average molecular weight of the polysaccharide extracted from the sunflower receptacle were 6.8×10 4 Da, 1.6×10 5 Da and 3.1×10 4 Da; The contents of the components in the polysaccharide extracted from the sunflower receptacle are as follows: the content of reducing sugar is 3.0-5.1wt%, the content of polysaccharide is 60.1-63.1wt%, and the content of total sugar is 63.1-68.2wt%; The method for preparing polysaccharides extracted from sunflower receptacles comprises the following steps: (1) taking dried sunflower receptacles and crushing them into 200-300 meshes to obtain sunflower receptacle powder; (2) adding water to the sunflower receptacle powder for 2 to 4 times of heat extraction, combining the extracts obtained from the multiple heat extractions to obtain a total extract; (3) concentrating the total extract to obtain a thick paste with a density of 0.2 to 0.02 g / mL; (4) adjusting the alcohol content of the thick paste to 68-72 wt % with ethanol, allowing the thick paste to stand, filtering, taking a precipitate, and freeze-drying the precipitate to obtain a crude polysaccharide; (5) re-dissolving the crude polysaccharide with water, centrifuging, removing insoluble impurities, taking the supernatant, and obtaining a crude polysaccharide solution; adjusting the alcohol content of the crude polysaccharide solution to 68-72 wt % with ethanol, performing secondary alcohol precipitation, standing, filtering, taking the precipitate, and freeze-drying to obtain a secondary alcohol-precipitated polysaccharide; (6) ultrasonically cleaning the polysaccharide precipitated from the secondary alcohol with 88-92 vol% ethanol for 2-4 times, filtering, and evaporating the ethanol to obtain the sunflower receptacle extracted polysaccharide; In the step (5), three stages of freeze drying are performed to obtain a polysaccharide precipitated with secondary alcohol; the three stages of freeze drying process are: -80 to -75°C, -65 to -60°C, and -75 to -70°C, and the time for the three stages of freeze drying is 4 to 5 hours, 8 to 10 hours, and 23 to 25 hours, respectively; The polysaccharide extracted from the sunflower receptacle is applied to cosmetics or external medicines for resisting damage caused by ultraviolet radiation.

2. The polysaccharide extracted from sunflower receptacle according to claim 1, characterized in that: In step (1) of the preparation method, dried sunflower receptacles with a moisture content of less than 8 wt % are taken and crushed to 250-300 meshes to obtain sunflower receptacle powder.

3. The polysaccharide extracted from sunflower receptacle according to claim 1, characterized in that: In step (2) of the preparation method, water is added to the sunflower receptacle powder for heat extraction 3 to 4 times, and the extracts obtained from multiple heat extractions are combined to obtain a total extract; during the heat extraction, the amount of water used for each heat extraction is 15 to 20 times the weight of the sunflower receptacle powder, and each heat extraction takes 0.8 to 1.2 hours.

4. The polysaccharide extracted from sunflower receptacle according to claim 1, characterized in that: In step (3) of the preparation method, the total extract is concentrated at 48-52° C. to obtain a thick paste with a density of 0.2-0.02 g / mL.

5. The polysaccharide extracted from sunflower receptacle according to claim 1, characterized in that: In step (4) of the preparation method, the alcohol content of the thick paste is adjusted to 70-72 wt % with anhydrous ethanol at 8-12° C., and the thick paste is allowed to stand at 8-12° C. for 10-15 hours, filtered, and the precipitate is taken and freeze-dried to obtain crude polysaccharide.

6. The polysaccharide extracted from sunflower receptacle according to claim 1, characterized in that: In step (5) of the preparation method, three-stage freeze-drying is performed to obtain a polysaccharide subjected to secondary alcohol precipitation; the process of the three-stage freeze-drying is as follows: the temperatures of the three-stage freeze-drying are -78 to -75°C, -63 to -60°C, and -72 to -75°C, respectively; and the times of the three-stage freeze-drying are 4.5 to 5h, 9 to 10h, and 24 to 25h, respectively.

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