阅读说明：本技术 一种香水莲花多糖及其制备方法 (Nymphaea hybrid polysaccharide and preparation method thereof ) 是由 谭诗敏 程建华 罗志刚 周心慧 于 2020-12-09 设计创作，主要内容包括：本发明公开了一种香水莲花多糖及其制备方法,包括如下步骤：步骤一：将香水莲花粉碎后过筛,得到香水莲花粗粉；向香水莲花粗粉中加入乙醇,加热回流,滤渣烘干得香水莲花粉末；步骤二：向香水莲花粉末中加入水,超声处理,加热搅拌提取,过滤,减压浓缩,得到浓缩的香水莲花粗多糖溶液；步骤三：向香水莲花粗多糖溶液中加入经过预处理的大孔树脂,加热搅拌吸附,抽滤得到脱色脱蛋白的多糖溶液,减压浓缩,得到浓缩糖液；步骤四：向浓缩糖液中加入乙醇进行沉淀,离心,收集沉淀物并真空干燥,得到香水莲花多糖。本发明提取率高,所得多糖分子量高,约1100Kda,其外观淡黄色,纯度高、色泽均匀稳定,具有良好的保湿、抗氧化性、抗炎活性。(The invention discloses a nymphaea hybrid polysaccharide and a preparation method thereof, and the method comprises the following steps: the method comprises the following steps: pulverizing nymphaea hybrid, and sieving to obtain nymphaea hybrid coarse powder; adding ethanol into the nymphaea hybrid coarse powder, heating and refluxing, and drying filter residues to obtain nymphaea hybrid powder; step two: adding water into the nymphaea hybrid powder, performing ultrasonic treatment, heating, stirring, extracting, filtering, and concentrating under reduced pressure to obtain a concentrated nymphaea hybrid crude polysaccharide solution; step three: adding pretreated macroporous resin into the crude polysaccharide solution of the nymphaea hybrid, heating, stirring, adsorbing, performing suction filtration to obtain decolorized and deproteinized polysaccharide solution, and performing reduced pressure concentration to obtain concentrated sugar solution; step four: and adding ethanol into the concentrated sugar solution for precipitation, centrifuging, collecting precipitate, and vacuum drying to obtain the nymphaea hybrid polysaccharide. The invention has high extraction rate, the obtained polysaccharide has high molecular weight of about 1100Kda, and has faint yellow appearance, high purity, uniform and stable color, and good moisture-keeping, oxidation resistance and anti-inflammatory activity.)

1. The preparation method of the nymphaea hybrid polysaccharide is characterized by comprising the following steps:

the method comprises the following steps: pulverizing nymphaea hybrid, and sieving to obtain nymphaea hybrid coarse powder; adding ethanol into the nymphaea hybrid coarse powder, heating and refluxing, and drying filter residues to obtain nymphaea hybrid powder;

step two: adding water into the nymphaea hybrid powder, performing ultrasonic treatment, heating, stirring, extracting, filtering, and concentrating under reduced pressure to obtain a concentrated nymphaea hybrid crude polysaccharide solution;

step three: adding pretreated macroporous resin into the crude polysaccharide solution of the nymphaea hybrid, heating, stirring, adsorbing, performing suction filtration to obtain decolorized and deproteinized polysaccharide solution, and performing reduced pressure concentration to obtain concentrated sugar solution;

step four: and adding ethanol into the concentrated sugar solution for precipitation, centrifuging, collecting precipitate, and vacuum drying to obtain the nymphaea hybrid polysaccharide.

2. The preparation method of claim 1, wherein the heating reflux temperature in the first step is 60-80 ℃, the heating reflux time is 3-5h, the volume fraction of the ethanol is 90 +/-5%, and the mass-volume ratio of the nymphaea hybrid coarse powder to the ethanol is 1:3-1:6 g/mL.

3. The preparation method according to claim 1, wherein the mass ratio of the nymphaea hybrid powder to water in the step two is 1:30-1:60, and the ultrasonic time is 10-40 min; the ultrasonic power is 60-400W.

4. The method as claimed in claim 3, wherein the heating temperature in step two is 55-85 ℃, the time is 2-4h, and the stirring speed is 100-300 rpm; the reduced pressure concentration temperature in the second step is 40-70 ℃.

5. The preparation method according to claim 4, wherein the mass-to-volume ratio of the added macroporous resin to the crude polysaccharide solution of the nymphaea hybrid is 0.5:1-1:1g/mL in step three, the heating temperature is 30-50 ℃, the stirring time is 3-6 h, and the stirring speed is 100-300 rpm; the decompression concentration temperature in the third step is 40-70 ℃.

6. The preparation method according to any one of claims 1 to 5, wherein the macroporous resin in the third step is AB-8 weak polar resin.

7. The preparation method of claim 6, wherein the pretreatment method of the macroporous resin in the third step is as follows: sequentially soaking the macroporous resin in water and ethanol, cleaning until no alcohol smell exists, soaking in hydrochloric acid, cleaning until the pH value is neutral, soaking in a sodium hydroxide solution, and cleaning until the pH value is neutral to obtain the pretreated AB-8 weak polar resin.

8. The preparation method according to claim 7, wherein the water soaking time is 6-12h, the ethanol volume fraction is 70-100%, the hydrochloric acid soaking time is 2-4h, the hydrochloric acid concentration is 2-5%, the sodium hydroxide solution soaking time is 2-4h, and the sodium hydroxide solution concentration is 2-5%.

9. The preparation method according to any one of claims 1 to 5, wherein the ethanol in the fourth step is absolute ethanol or an ethanol solution with a volume fraction of 90-95%, and the volume ratio of the added ethanol to the concentrated sugar solution is 3:1-5: 1; the precipitation temperature is 0-4 ℃, and the precipitation time is 6-12 h; the centrifugal rotating speed is 4000-10000r/min, and the centrifugal time is 5-20 min; the vacuum drying temperature is 40-60 ℃, and the drying time is 3-5 h; the drying temperature in the step one is 35-60 ℃, and the drying time is 10-24 h.

10. The nymphaea hybrid polysaccharide prepared by the method of any one of claims 1 to 9.

Technical Field

The invention relates to extraction of natural active substances, and provides a nymphaea hybrid polysaccharide and a preparation method thereof.

Background

Nymphaea hybrid is a herbal plant of aquatic perennial root of Nymphaeaceae family Nymphaea. At present, nine colors such as gold, purple, blue, yellow, white, red and the like exist, so the nine-grade nymphaea hybrid is also named. At present, planting bases are available in Guangdong Zhuhai, Guangxi Liuzhou, Fujian, Sichuan and the like, and resources are rich. However, besides being used as an ornamental, the main product of the nymphaea hybrid is the nymphaea hybrid tea. Researches show that the nymphaea hybrid has the functions of reducing blood fat, resisting oxidation, whitening skin, protecting liver and the like. The edible safety of the nymphaea hybrid is also proved. The effective components are deeply excavated, and the additional value of the nymphaea hybrid can be improved.

The nymphaea hybrid has rich polysaccharide content, but is not widely and deeply developed. The nymphaea hybrid polysaccharide is a green and natural plant polysaccharide, and meets the pursuit of modern people for non-additive, non-irritant and non-toxic extracts. The nymphaea hybrid polysaccharide has good moisturizing, antioxidant and anti-inflammatory effects and can be used in the field of cosmetics; has the effects of resisting tumor and virus, reducing blood lipid, enhancing immunity, etc., and can be applied in the fields of food, medicine, functional food, etc.

At present, the preparation method of the nymphaea hybrid polysaccharide mainly comprises the steps of extracting with boiling water and then precipitating with ethanol, and the method has the advantages of high extraction temperature, low extraction efficiency, great damage to the polysaccharide structure, low purity and uneven color, and cannot meet the requirement of people on wide application of the polysaccharide. In the existing research, Sevage method and trichloroacetic acid method are adopted for removing protein of lotus polysaccharide, which results in residue of highly toxic reagents such as chloroform and trichloroacetic acid, and the loss amount of polysaccharide is high. Researches show that the hydrogen peroxide method has obvious polysaccharide decoloring effect, can degrade the molecular weight of polysaccharide while oxidizing pigment, and is not suitable for preparing high-molecular-weight nymphaea hybrid polysaccharide. Currently, the research on the nymphaea hybrid polysaccharide is mainly focused on middle-low molecular weight 200-400kDa, the research on the high molecular weight polysaccharide is less, and DEAE-52 and Sephadex G-150 chromatographic columns are applied in separation and purification, so that the obtained polysaccharide has high purity, but the yield is low, the production cost is high, the steps are complicated, and the nymphaea hybrid polysaccharide is not suitable for industrial production. The polysaccharide activity is related to the molecular weight, and the high, medium and low molecular weight polysaccharides can be combined to design a formula in cosmetics to achieve the moisturizing effect of different levels. The high molecular weight polysaccharide has the functions of immunoregulation, anti-inflammation, antioxidation, whitening, moisture retention, film formation and the like.

Disclosure of Invention

The invention aims to provide a preparation method of high molecular weight nymphaea hybrid polysaccharide with moisturizing, antioxidant and anti-inflammatory activities. The in vitro weighing method is adopted to determine the in vitro moisture retention and hygroscopicity of polysaccharide in saturated sodium carbonate solution, saturated ammonium sulfate solution and dried allochroic silica gel, and a body surface instrument is adopted to determine the moisture content of the horny layer of the skin and the moisture loss of the epidermis. The antioxidant activity of the polysaccharide was evaluated by using DPPH.radical, ABTS.radical, hydroxyl radical, and total reducing power as indices. The anti-inflammatory activity of the polysaccharide is determined by the hyaluronidase activity inhibition rate. Provides basis for the development and application of high molecular weight nymphaea hybrid polysaccharide. The method has high extraction efficiency and high polysaccharide yield.

The invention also aims to provide the high-molecular-weight nymphaea hybrid polysaccharide, and the obtained polysaccharide has high purity and uniform color.

The purpose of the invention is realized by the following technical scheme:

a preparation method of nymphaea hybrid polysaccharide comprises the following steps:

the method comprises the following steps: pulverizing nymphaea hybrid, and sieving to obtain nymphaea hybrid coarse powder; adding ethanol into the nymphaea hybrid coarse powder, heating and refluxing, and drying filter residues to obtain nymphaea hybrid powder;

step two: adding water into the nymphaea hybrid powder, performing ultrasonic treatment, heating, stirring, extracting, filtering, and concentrating under reduced pressure to obtain a concentrated nymphaea hybrid crude polysaccharide solution;

step three: adding pretreated macroporous resin into the crude polysaccharide solution of the nymphaea hybrid, heating, stirring, adsorbing, performing suction filtration to obtain decolorized and deproteinized polysaccharide solution, and performing reduced pressure concentration to obtain concentrated sugar solution;

step four: and adding ethanol into the concentrated sugar solution for precipitation, centrifuging, collecting precipitate, and vacuum drying to obtain the nymphaea hybrid polysaccharide.

Preferably, in the step one, the heating reflux temperature is 60-80 ℃, the time is 3-5h, the volume fraction of the ethanol is 90 +/-5%, and the mass-volume ratio of the nymphaea hybrid coarse powder to the ethanol is 1:3-1:6 g/mL.

Preferably, the mass ratio of the nymphaea hybrid powder to water in the step two is 1:30-1:60, and the ultrasonic time is 10-40 min; the ultrasonic power is 60-400W.

Preferably, the heating temperature in the second step is 55-85 ℃, the time is 2-4h, and the stirring speed is 100-300 rpm; the reduced pressure concentration temperature in the second step is 40-70 ℃.

Preferably, the mass volume ratio of the macroporous resin to the crude polysaccharide solution of the nymphaea hybrid is 0.5:1-1:1g/mL in the third step, the heating temperature is 30-50 ℃, the stirring time is 3-6 h, and the stirring speed is 300 rpm; the decompression concentration temperature in the third step is 40-70 ℃.

Preferably, the macroporous resin in step three is AB-8 weak polar resin, which is purchased from Shanghai-derived leaf Biotech, Inc.

Preferably, the pretreatment method of the macroporous resin in the third step is as follows: sequentially soaking the macroporous resin in water and ethanol, cleaning until no alcohol smell exists, soaking in hydrochloric acid, cleaning until the pH value is neutral, soaking in a sodium hydroxide solution, and cleaning until the pH value is neutral to obtain the pretreated AB-8 weak polar resin.

Preferably, the water soaking time is 6-12h, the ethanol volume fraction is 70-100%, the hydrochloric acid soaking time is 2-4h, the hydrochloric acid concentration is 2-5%, the sodium hydroxide solution soaking time is 2-4h, and the sodium hydroxide solution concentration is 2-5%.

Preferably, the ethanol in the fourth step is absolute ethanol or ethanol solution with volume fraction of 90-95%, and the volume ratio of the added ethanol to the concentrated sugar solution is 3:1-5: 1;

the precipitation temperature is 0-4 ℃, and the precipitation time is 6-12 h;

the centrifugal rotating speed is 4000-10000r/min, and the centrifugal time is 5-20 min;

the vacuum drying temperature is 40-60 ℃, and the drying time is 3-5 h.

Preferably, the drying temperature in the step one is 35-60 ℃, and the drying time is 10-24 h.

The method adopts ultrasonic-assisted extraction of the nymphaea hybrid polysaccharide, utilizes high-speed vibration and cavitation effect generated by ultrasonic waves to accelerate the rupture of plant cell walls, is beneficial to mixing of a solvent and the polysaccharide, and achieves the aims of improving the extraction rate of the polysaccharide and shortening the extraction time. Before extraction, most of lipid, micromolecular substances, pigment and the like are removed by heating and refluxing ethanol. Most of protein and pigment in the extracting solution are removed by adsorption of AB-8 macroporous resin, the purity and color of the polysaccharide are improved, and the residue of toxic reagents such as chloroform and the like is avoided. Vacuum drying is adopted to protect the structure and functional activity of the polysaccharide and improve the water solubility of the polysaccharide. GPC measurements indicated that the polysaccharide had a relatively high molecular weight of about 1100 kDa. Experiments of in vitro moisture absorption, moisture retention and body surface moisture retention show that the high molecular weight nymphaea hybrid polysaccharide can be used as a substitute of hyaluronic acid. The four antioxidant activity indexes show that the high molecular weight nymphaea hybrid polysaccharide has good antioxidant activity. The hyaluronidase activity inhibition experiment shows that the high molecular weight nymphaea hybrid polysaccharide has skin anti-inflammatory activity.

The invention solves the problem of low extraction rate of the traditional boiling water extraction method. Except protein and pigment, the green natural recyclable macroporous resin is adopted. The high molecular weight nymphaea hybrid polysaccharide extracted by the method has the advantages of light yellow appearance, high purity, uniform and stable color, is a good natural plant extract, and has good moisturizing, oxidation resistance and anti-inflammatory activity.

Drawings

FIG. 1 is a GPC chart of example 1.

FIG. 2 is a UV scan of example 1.

FIG. 3 is an infrared spectrum of example 1.

Fig. 4 is an ion chromatogram of example 1.

FIG. 5 is the in vitro hygroscopicity and moisture retention of the trollius chinensis polysaccharides, glycerin and hyaluronic acid under different humidity environments in example 1; a is a moisture absorption map at 43% humidity, b is a moisture absorption map at 81% humidity, c is a moisture absorption map at 0% humidity, and d is a moisture absorption map at 81% humidity.

FIG. 6 shows the change rate (a) of body surface skin moisture content and the change rate (b) of percutaneous moisture loss in example 1.

FIG. 7 shows the scavenging activity and total reducing power (d) for DPPH (a), ABTS (b), OH (c) in example 1.

FIG. 8 is a graph showing the inhibition of hyaluronidase activity by the nymphaea hybrid polysaccharide and dipotassium glycyrrhizinate in example 1.

Detailed Description

The technical solution of the present invention is clearly and completely described below with reference to specific embodiments.

Example 1 preparation of high molecular weight nymphaea hybrid polysaccharide from nymphaea hybrid:

(1) pulverizing nymphaea hybrid, sieving with a 40-mesh sieve, weighing 200g of nymphaea hybrid coarse powder, placing in 600mL of 90% ethanol by volume, heating and refluxing at 70 ℃ for 3h, filtering with filter paper, and drying the filter residue in a 40 ℃ oven for 12h to obtain nymphaea hybrid powder.

(2) Accurately weighing 100g of nymphaea hybrid powder, performing ultrasonic treatment at the mass ratio of the nymphaea hybrid powder to deionized water of 1:30 for 30min under 200W, heating and extracting at 70 ℃ for 4h in a water bath kettle, performing filtration with a stirring paddle speed of 200rpm, performing vacuum concentration to 300mL with a rotary evaporator at 40 ℃, and thus obtaining a concentrated nymphaea hybrid crude polysaccharide extracting solution.

(3) Soaking the AB-8 macroporous resin in deionized water for 6h, then soaking in ethanol with the volume fraction of 70% for 6h, and washing with deionized water until no alcohol smell exists. 3% hydrochloric acid solution is soaked for 3h, deionized water is washed to be neutral, 5% sodium hydroxide solution is soaked for 3h, and deionized water is washed to be neutral for later use. Adding 300g of pretreated AB-8 macroporous resin into 300mL of concentrated nymphaea hybrid crude polysaccharide solution, heating at 35 ℃, stirring and adsorbing at the rotating speed of 200rpm for 6h, performing suction filtration to obtain a decolorized polysaccharide solution, and performing reduced pressure concentration to 100mL at 40 ℃ by using a rotary evaporator to obtain concentrated and decolorized nymphaea hybrid polysaccharide solution.

(4) Adding 4 times volume of absolute ethyl alcohol into 100mL of concentrated sugar solution, precipitating in a refrigerator at 4 ℃ for 12h, centrifuging at 4000r/min for 20min, collecting precipitate, and vacuum drying at 60 ℃ for 3h to obtain the nymphaea hybrid polysaccharide. The extraction rate is 10.45% and the content of the nymphaea hybrid polysaccharide is 62.36% by measuring with a phenol-sulfuric acid method.

Example 2 preparation of nymphaea hybrid polysaccharides from nymphaea hybrid:

(1) pulverizing nymphaea hybrid, sieving with a 40-mesh sieve, weighing 200g of nymphaea hybrid coarse powder, placing in 600mL of 90% ethanol by volume, heating and refluxing at 80 ℃ for 3h, filtering with filter paper, and drying the filter residue in a 40 ℃ oven for 12h to obtain nymphaea hybrid powder.

(2) Accurately weighing 100g of nymphaea hybrid powder, heating and extracting in a water bath kettle at 80 ℃ for 3h after 250W ultrasonic treatment for 25min with the mass ratio of the nymphaea hybrid powder to deionized water being 1:40, wherein the stirring speed is 200rpm, filtering by using gauze, and concentrating to 300mL at 40 ℃ under reduced pressure by using a rotary evaporator to obtain a concentrated nymphaea hybrid crude polysaccharide extracting solution.

(3) Soaking the AB-8 macroporous resin in deionized water for 6h, then soaking in ethanol with the volume fraction of 70% for 6h, and washing with deionized water until no alcohol smell exists. 3% hydrochloric acid solution is soaked for 3h, deionized water is washed to be neutral, 5% sodium hydroxide solution is soaked for 3h, and deionized water is washed to be neutral for later use. Adding 200g of pretreated AB-8 macroporous resin into 300mL of concentrated nymphaea hybrid crude polysaccharide solution, heating at 40 ℃, stirring and adsorbing at the rotating speed of 150rpm for 5h, performing suction filtration to obtain a decolorized polysaccharide solution, and performing reduced pressure concentration to 100mL at 40 ℃ by using a rotary evaporator to obtain concentrated and decolorized nymphaea hybrid polysaccharide solution.

(4) Adding 4 times volume of absolute ethyl alcohol into 100mL of concentrated sugar solution, precipitating at 0 ℃ for 6h, centrifuging at 6000r/min for 15min, collecting precipitate, and vacuum drying at 60 ℃ for 3h to obtain the nymphaea hybrid polysaccharide. The extraction rate is 10.2% and the content of the nymphaea hybrid polysaccharide is 63.44% by measuring with a phenol-sulfuric acid method.

Example 3 preparation of nymphaea hybrid polysaccharides from nymphaea hybrid:

(1) the method comprises the following steps of crushing nymphaea hybrid, sieving with a 40-mesh sieve, weighing 400g of nymphaea hybrid coarse powder, placing the nymphaea hybrid coarse powder into 1600mL of 90% ethanol by volume, heating and refluxing for 3h at 70 ℃, filtering with filter paper, and placing filter residues in a 40 ℃ oven for drying for 10h to obtain nymphaea hybrid powder.

(2) Accurately weighing 200g of nymphaea hybrid powder, performing ultrasonic treatment at 300W for 25min, heating and extracting at 70 ℃ for 3h in a water bath kettle at the stirring paddle speed of 200rpm for 25min, filtering with gauze, and concentrating at 60 ℃ to 400mL by a rotary evaporator to obtain a concentrated nymphaea hybrid crude polysaccharide extracting solution.

(3) Soaking the AB-8 macroporous resin in deionized water for 6h, then soaking in ethanol with the volume fraction of 70% for 6h, and washing with deionized water until no alcohol smell exists. 3% hydrochloric acid solution is soaked for 3h, deionized water is washed to be neutral, 5% sodium hydroxide solution is soaked for 3h, and deionized water is washed to be neutral for later use. Adding 200g of pretreated AB-8 macroporous resin into 400mL of concentrated nymphaea hybrid crude polysaccharide solution, heating at 45 ℃, stirring and adsorbing at 150rpm for 4h, performing suction filtration to obtain decolorized polysaccharide solution, and performing reduced pressure concentration to 200mL at 60 ℃ by using a rotary evaporator to obtain concentrated and decolorized nymphaea hybrid polysaccharide solution.

(4) Adding 90% ethanol with 4 times volume of 200mL of concentrated sugar solution, precipitating at 0 deg.C for 6h, centrifuging at 8000r/min for 10min, collecting precipitate, and vacuum drying at 60 deg.C for 3h to obtain the final product. The extraction rate is 11.07% and the content of the nymphaea hybrid polysaccharide is 60.25% by measuring with a phenol-sulfuric acid method.

Control group 1: accurately weighing 100g of nymphaea hybrid powder, wherein the mass ratio of the nymphaea hybrid powder to deionized water is 1:30, heating and extracting for 4h at 70 ℃ in a water bath kettle, the stirring paddle speed is 200rpm, filtering by using gauze, and concentrating to 300mL at 40 ℃ under reduced pressure by using a rotary evaporator to obtain a concentrated nymphaea hybrid crude polysaccharide extracting solution. Adding 300g of pretreated AB-8 macroporous resin into 300mL of concentrated nymphaea hybrid crude polysaccharide solution, heating at 35 ℃, stirring and adsorbing at the rotating speed of 200rpm for 6h, performing suction filtration to obtain a decolorized polysaccharide solution, and performing reduced pressure concentration to 100mL at 40 ℃ by using a rotary evaporator to obtain concentrated and decolorized nymphaea hybrid polysaccharide solution. Adding 4 times volume of absolute ethyl alcohol into 100mL of concentrated sugar solution, precipitating in a refrigerator at 4 ℃ for 12h, centrifuging at 4000r/min for 20min, collecting precipitate, and vacuum drying at 60 ℃ for 3h to obtain the nymphaea hybrid polysaccharide.

Control group 2: accurately weighing 100g of nymphaea hybrid powder, wherein the mass ratio of the nymphaea hybrid powder to deionized water is 1:30, heating and extracting for 4h at 70 ℃ in a water bath kettle, the stirring paddle speed is 200rpm, filtering by using gauze, and concentrating to 300mL at 40 ℃ under reduced pressure by using a rotary evaporator to obtain a concentrated nymphaea hybrid crude polysaccharide extracting solution. Adding 300g of pretreated AB-8 macroporous resin into 300mL of concentrated nymphaea hybrid crude polysaccharide solution, heating at 35 ℃, stirring and adsorbing at the rotating speed of 200rpm for 6h, performing suction filtration to obtain a decolorized polysaccharide solution, and performing reduced pressure concentration to 100mL at 40 ℃ by using a rotary evaporator to obtain concentrated and decolorized nymphaea hybrid polysaccharide solution. Adding Sevage reagent into the concentrated sugar solution, wherein the volume ratio of the polysaccharide solution to the Sevage reagent is 4:1, oscillating for 30min, centrifuging at 4000r/min for 10min, separating the sugar solution, and repeating for 5 times. Adding 4 times volume of anhydrous ethanol into the sugar solution, precipitating in a refrigerator at 4 deg.C for 12h, centrifuging at 4000r/min for 20min, collecting precipitate, and vacuum drying at 60 deg.C for 3h to obtain the nymphaea hybrid polysaccharide.

Control group 3: accurately weighing 100g of nymphaea hybrid powder, performing ultrasonic treatment at the mass ratio of the nymphaea hybrid powder to deionized water of 1:30 for 30min under 200W, heating and extracting at 70 ℃ for 4h in a water bath kettle, performing filtration with a stirring paddle speed of 200rpm, performing vacuum concentration to 300mL with a rotary evaporator at 40 ℃, and thus obtaining a concentrated nymphaea hybrid crude polysaccharide extracting solution. Adding 300g of pretreated AB-8 macroporous resin into 300mL of concentrated nymphaea hybrid crude polysaccharide solution, heating at 35 ℃, stirring and adsorbing at the rotating speed of 200rpm for 6h, performing suction filtration to obtain a decolorized polysaccharide solution, and performing reduced pressure concentration to 100mL at 40 ℃ by using a rotary evaporator to obtain concentrated and decolorized nymphaea hybrid polysaccharide solution. Adding Sevage reagent into the concentrated sugar solution, wherein the volume ratio of the polysaccharide solution to the Sevage reagent is 4:1, oscillating for 30min, centrifuging at 4000r/min for 10min, separating the sugar solution, and repeating for 5 times. Adding 4 times volume of anhydrous ethanol into the sugar solution, precipitating in a refrigerator at 4 deg.C for 12h, centrifuging at 4000r/min for 20min, collecting precipitate, and vacuum drying at 60 deg.C for 3h to obtain the nymphaea hybrid polysaccharide.

The protein content, polysaccharide extraction rate and polysaccharide content of the polysaccharide prepared in example 1 and the polysaccharides prepared in the control groups 1 to 3 were measured. The extraction rate and content of polysaccharide are determined by phenol-sulfuric acid method. Protein content was determined by the Coomassie Brilliant blue method. As shown in Table 1, the extraction rate of the nymphaea hybrid polysaccharide prepared by the invention is 10.45% which is far higher than that of the extraction group with boiling water, and the Sevage method can remove a small amount of protein, but the loss amount of the polysaccharide is extremely high. The polysaccharide content is higher than that of a control group 1-2 and slightly lower than that of a control group 3, and the protein content is lower than that of a boiling water extraction group and is close to that of a Sevage-treated control group 3 after ultrasonic treatment. Therefore, the method has the advantage of high extraction rate.

TABLE 1 protein, total sugar content and polysaccharide extraction rate of different nymphaea hybrid polysaccharides

	Example 1	Control group 1	Control group 2	Control group 3
					Protein content (%)	2.41	3.63	3.04	2.15
Total sugar content (%)	62.36	58.12	60.82	64.61
					Polysaccharide extraction (%)	10.45	4.74	2.95	6.80

The following performance tests were performed using the nymphaea hybrid polysaccharide prepared in example 1 as a sample:

determination of molecular weight:

the molecular weight of the trollius chinensis polysaccharide is determined by Gel Permeation Chromatography (GPC). GPC chromatographic conditions: waters chromatograph, chromatography columns Ultrahydrogel 120, Ultrahydrogel 250, Ultrahydrogel 500 of Waters corporation are connected in series, column temperature: the mobile phase is 0.02mol/L KH at 35 DEG C₂PO₄The solution, pH 6.0, flow rate 0.80 mL/min. Preparing dextran standard substances with different molecular weights and flos Trollii polysaccharide into 2mg/mL solution with mobile phase, and filtering with 0.22 μm filter membrane to obtain 20 μ L sample. As shown in FIG. 1, the molecular weight of the obtained nymphaea hybrid polysaccharide is about 1100Kda according to the standard curve, and the nymphaea hybrid polysaccharide is high molecular weight polysaccharide.

Ultraviolet spectrum scanning:

the 3 kinds of polysaccharides which are not treated by AB-8 resin, AB-8 resin and 5 times Sevage method are respectively prepared into 1mg/mL solution, and are subjected to ultraviolet scanning within the range of 200-500nm by using distilled water as a contrast.

As shown in figure 2, the polysaccharide which is not treated by the AB-8 resin has ultraviolet absorption near 260nm, while the polysaccharide treated by the AB-8 resin has no obvious ultraviolet absorption at 260nm and 280nm, which shows that the protein and nucleic acid content in the nymphaea hybrid polysaccharide is very small after the treatment by the AB-8 resin, and the treatment effect of the AB-8 resin is similar to that of the Sevage method treatment for 5 times, which shows that the AB-8 resin effectively adsorbs the protein in the nymphaea hybrid polysaccharide, does not need chloroform, and is green and environment-friendly.

Infrared spectrum scanning:

precisely weighing 2mg of sample and 200mg of potassium bromide, pressing into tablets, and pressing the blank control by potassium bromide powder into tablets. Respectively arranged at 500-4000cm of a Fourier transform infrared spectrometer^-1Scanning is performed over the range.

As shown in FIG. 3, the absorption band is in 3600-^-1Is a stretching vibration absorption peak of-OH, and the absorption peak in this region is a characteristic peak of the glucide. 3421cm^-1Is the absorption peak of stretching vibration of O-H and is the characteristic peak of saccharide. At 2927cm^-1Has an absorption peak which is the C-H stretching vibration of the polysaccharide. At 1747cm^-1There is an absorption peak, attributed to the stretching vibration of C ═ O. At 1650cm^-1There is an absorption peak, attributed to the stretching vibration of C ═ O. 1440cm^-1Absorption peaks due to stretching vibration attributed to C-O. At 1328cm^-1There is an absorption peak, which is attributed to the symmetric stretching vibration of C ═ O. 1230cm^-1And belongs to the absorption peak caused by the variable angle vibration of O-H. 1020cm^-1And belongs to the absorption peak caused by the variable angle vibration of O-H. At 917cm^-1There is an absorption peak attributed to asymmetric ring stretching vibration.

Monosaccharide composition and uronic acid content determination:

16 monosaccharide standards (fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, glucuronic acid, galactosamine hydrochloride, glucosamine hydrochloride, N-acetyl-D glucosamine, guluronic acid, mannuronic acid) are prepared by ion chromatography to form about 10mg/ml standard solution.

About 5mg of sample was taken in an ampoule, 10ml of 2M TFA was added and hydrolyzed at 120 ℃ for 3 h. Accurately absorbing the acid hydrolysis solution, transferring the acid hydrolysis solution into a tube, blowing and drying the acid hydrolysis solution by nitrogen, adding 5ml of water, uniformly mixing the acid hydrolysis solution and the tube by vortex, absorbing 100uL of the acid hydrolysis solution, adding 900uL of deionized water, and centrifuging the mixture at 12000rpm for 5 min. The supernatant was taken for IC analysis.

A chromatographic column: dionex Carbopac^TMPA20(3 × 150); mobile phase: a: H2O; 250mM NaOH; c50 mM NaOH&500mM NaOAC; flow rate: 0.3 ml/min; sample introduction amount: 5 mu L of the solution; column temperature: 30 ℃; a detector: an electrochemical detector.

Precisely preparing the monosaccharide Standard solutions into gradient concentration Standard products of 0.1, 0.5, 1, 5, 10, 20 and 50mg/L as Standard 1-7. According to the absolute quantitative method, the mass of different monosaccharides is determined, and the molar ratio is calculated according to the molar mass of the monosaccharides.

TABLE 2 monosaccharide composition of nymphaea hybrid polysaccharides

As shown in fig. 4, it was confirmed that the nymphaea hybrid polysaccharide was composed of galacturonic acid, glucuronic acid, galactose, rhamnose, mannose, xylose, arabinose, glucosamine hydrochloride by comparing the retention time with each monosaccharide standard. As can be seen from Table 2, the polysaccharides contained relatively high amounts of galacturonic acid and glucuronic acid, in molar ratios of 36.3% and 11.2%, respectively. The mol ratio of galactose, xylose and rhamnose is 16.5%, 11.5% and 11.0%, respectively.

In vitro hygroscopicity of the nymphaea hybrid polysaccharide:

accurately weighing 100mg of dry polysaccharide in a weighing bottle, respectively placing the weighing bottle in a dryer with relative humidity of 43% (saturated sodium carbonate solution) and 81% (saturated ammonium sulfate solution), respectively taking out for 0h, 12h, 24h, 36h, 48h and 60h, and weighing the polysaccharide. Glycerol and hyaluronic acid were used as controls.

The nymphaea hybrid polysaccharide has in vitro moisture retention:

accurately weighing 100mg of dry polysaccharide in a weighing bottle, adding 40% of deionized water, respectively placing the weighing bottle in a dryer with the relative humidity of 43% (saturated sodium carbonate solution) and silica gel for dehydration, respectively taking out the bottles in 0h, 12h, 24h, 36h, 48h and 60h, and weighing the polysaccharide. Glycerol and hyaluronic acid were used as controls.

Moisture retention activity results analysis:

as shown in figures 5-a and 5-d, the hygroscopicity of the high molecular weight nymphaea hybrid polysaccharide is similar to that of hyaluronic acid under the environment of 43% humidity and 81% humidity, and the moisture retention of the high molecular weight nymphaea hybrid polysaccharide is similar to that of hyaluronic acid under the environment of 43% humidity and dry silica gel as shown in figures 5-b and 5-c, which indicates that the high molecular weight nymphaea hybrid polysaccharide prepared by the method can be used as a substitute of hyaluronic acid.

Determination of skin moisture content MMV and transdermal water dispersion loss TEWL:

30 qualified volunteers were tested with random gender between 18-65 years of age. The test environment temperature is 21 ℃ and the humidity is 50%. After cleaning the inside of the arm of the subject, a 3cm × 3cm test area was marked, and the subject was rested for 20min in a standard-compliant room before testing, and the skin was relaxed. The subject measured at 2mg/cm²The dosage of the test solution is smeared with 1 percent of the trollius chinensis bunge polysaccharide solution, 1 percent of hyaluronic acid solution is used as a positive control, deionized water is used as a blank control, and the test time is 4 hours.

Before the product is used, after the product is used for 1h, 2h and 4h, the moisture content of the stratum corneum of the skin and the moisture loss of the skin through skin are measured.

Analyzing the body surface moisturizing result:

as shown in figure 6, after the application of the 1% nymphaea hybrid polysaccharide solution, the moisture content of the skin is increased, and at 2h, the change rate of the hydration degree is the largest, and the moisture content of the skin is the highest. The skin hydration rate of the polysaccharide solution was higher than that of the hyaluronic acid solution at 1h, and similar to that of the hyaluronic acid solution at 2h and 4 h. Within 4h, the percutaneous moisture loss change rate of the polysaccharide solution is negative and is obviously lower than that of a blank group. The transdermal water loss rate of the polysaccharide solution was lower than that of the hyaluronic acid solution at 1h and 2h, and slightly worse than that at 4 h. The preparation method of the polymer nymphaea hybrid polysaccharide can replenish water to the skin and lock the water of the skin, and the effect is similar to that of hyaluronic acid.

The eliminating activity of the nymphaea hybrid polysaccharide on DPPH & free radical:

prepare the polysaccharide solution with the concentration of 2mg/mL and dilute the solution into different concentration gradients. 2.0mL of the polysaccharide sample solution was added to 2mL of DPPH solution, mixed well and reacted in the dark for 30min, and the absorbance at 517nm was measured.

The eliminating activity of the nymphaea hybrid polysaccharide on ABTS & free radicals:

polysaccharide solutions were prepared at 4mg/mL and diluted to different concentration gradients. Mixing 7mM ABTS solution with 2.45mM potassium persulfate solution in equal volume, standing at room temperature in dark place for 16h, and diluting the generated ABTS +. solution until the absorbance value at 734nm is 0.7 +/-0.02. And (3) oscillating and uniformly mixing 0.4mL of polysaccharide solution and 3mL of ABTS solution, standing for 30min at room temperature in a dark place, and then measuring the light absorption value at 734 nm.

The eliminating activity of the nymphaea hybrid polysaccharide on OH & free radical:

prepare polysaccharide solution with concentration of 5mg/mL and dilute to different concentration gradients. Sequentially adding 2.0mL of FeSO into 1.0mL of sample solution₄(9.0mmol/L) solution, 2.0mL salicylic acid (9.0mmol/L) solution and then 2.0mL H₂O₂(9.0mmol/L) the reaction was started, and after a water bath at 37 ℃ for 60min, the absorbance was measured at 510 nm.

And (3) measuring the total reducing power of the nymphaea hybrid:

a5 mg/mL polysaccharide solution was prepared and diluted. 1.0mL of each of the solutions with different concentrations was added with 2.5mL of a buffer (0.2mol/L, pH 6.6) and 1mL of 1% potassium ferricyanide, heated in a water bath at 50 ℃ for 30min, cooled, added with 2.5mL of trichloroacetic acid, centrifuged for 10min, and 2mL of the supernatant was added with 2mL of distilled water and 1mL of 0.1% FeCl₃Standing the solution for 10min, and measuring light absorption value at 700nm with distilled water as reference.

And (4) antioxidant activity result analysis:

as shown in FIG. 7, the DPPH, ABTS, and OH clearance rates are increased with the increase of the concentration of the trollius chinensis polysaccharides, and a remarkable dosage effect is shown. The DPPH-clearance increases from 30% to 75% with increasing polysaccharide concentration in the range of 0.2mg/mL to 0.6mg/mL, the rate of increase slows down in the range of 0.6mg/mL to 1mg/mL, and the clearance reaches the highest at 0.8 mg/mL. ABTS clearance increased from 10% to 72% with increasing polysaccharide concentration in the range of 0.1mg/mL to 1mg/mL, the rate of increase slowed in the range of 1mg/mL to 4mg/mL, and the curve tended to equilibrate at 2 mg/mL. In the range of 0.1mg/mL to 1mg/mL, the OH.multidot.clearance increased from 5% to 30% with the increase in polysaccharide concentration, and in the range of 1mg/mL to 5mg/mL, the rate of increase slowed down, and at 5mg/mL, the clearance reached the highest. The absorbance increased from 0.008 to 1.08 with the increase in the polysaccharide concentration in the range of 0.1mg/mL to 1mg/mL, and the rate of increase decreased in the range of 1mg/mL to 5mg/mL, and the absorbance reached the highest value at 5 mg/mL.

Determination of hyaluronidase Activity inhibition:

mixing the calcium chloride solution, the hyaluronidase solution and the acetic acid buffer solution, keeping the temperature at 37 ℃ for 20min, adding 5mg/mL nymphaea hybrid polysaccharide solution and the acetic acid buffer solution, and keeping the temperature at 37 ℃ for 20 min. Adding sodium hyaluronate solution, keeping the temperature at 37 deg.C for 30min, standing at room temperature for 5min, and heating at 80 deg.C for 20 min. Adding sodium hydroxide solution and acetylacetone solution, heating in boiling water bath for 15min, immediately cooling on ice for 5min, adding Ellisib reagent and anhydrous ethanol, and standing for 20min for color development. Dipotassium glycyrrhizinate was used as a positive control. The hyaluronidase activity is related to skin inflammation, after 0.5% of the trollius chinensis bunge polysaccharide solution is added, the inhibition rate of the hyaluronidase activity reaches 57.69%, and the inhibition effect of 0.3% of dipotassium glycyrrhizinate on the hyaluronidase activity is close, so that the nymphaea chinensis bunge polysaccharide has a good anti-inflammatory effect.