An acidic polysaccharide from Prinsepia utilis Royle and its preparation method and application

Abstract

The present invention relates to a pricklyash fruit acidic polysaccharide, a preparation method thereof and an application. The preparation method comprises the following steps: (1) mixing pricklyash fruit residue with a solvent, heating and extracting, and concentrating the obtained extract by an organic membrane to obtain a first purified solution; (2) subjecting the first purified solution to adsorption and elution by a cation exchange resin to obtain a second purified solution; (3) subjecting the second purified solution to adsorption and elution by an anion exchange resin to obtain a third purified solution; (4) concentrating, dialyzing and drying the third purified solution to obtain the pricklyash fruit acidic polysaccharide. By adopting an organic membrane, a cation exchange resin and an anion exchange resin for multiple adsorption and separation, the obtained acidic polysaccharide has a high content of total polysaccharide and uronic acid, a low protein content, a strong DPPH free radical scavenging ability, and can effectively inhibit the oxidative damage of HaCat cells caused by UVB irradiation. Moreover, the preparation method is simple to operate and economical in cost, and has good industrial production prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and more specifically, relates to a Prinsepia utilis Royle acidic polysaccharide, a preparation method thereof, and an application thereof. Background Art

[0002] Polysaccharides are a class of high-molecular-weight carbohydrates formed by the condensation of multiple monosaccharide (including aldose and ketose) molecules. Acidic polysaccharides refer to polysaccharides containing acidic groups such as carboxyl groups or sulfate groups. Research shows that acidic polysaccharides have a complex structure and possess various biological activities, such as antioxidant, immunomodulatory, hypoglycemic, and anti-tumor activities. Acidic polysaccharides have no toxic side effects and have high potential application value.

[0003] Prinsepia utilis Royle is the mature fruit of Prinsepia utilis Royle of the genus Prinsepia in the family Rosaceae, and grows in areas such as Yunnan, Guizhou, and Sichuan. "Compendium of Materia Medica in Yunnan" records: "Prinsepia utilis Royle is slightly cold in nature and bitter in taste. It can cure all kinds of sores and boils. If there is pus, it can make the pus come out; if there is no pus, it can disappear immediately; it can disperse tuberculosis, and chew it finely and take it with wine." Prinsepia utilis Royle is rich in oil and is an oil plant that can be used both as medicine and food. People in places such as Lijiang and Diqing in Yunnan often extract Prinsepia utilis Royle oil from Prinsepia utilis Royle for consumption. Prinsepia utilis Royle oil is rich in compounds such as flavonoids, triterpenes, and fatty acids, and is widely used as a skin care oil in cosmetics, with effects such as moisturizing, nourishing, anti-inflammatory, and repair.

[0004] At present, the research on Prinsepia utilis Royle polysaccharides mainly focuses on the treatment of diabetes and the repair of skin barriers. Chinese Patent Application CN104887881A discloses a pure traditional Chinese medicine preparation containing Prinsepia utilis Royle polysaccharides, which can be used for the prevention and treatment of diabetes; Chinese Patent Application CN117959224A discloses a composition containing an extract of Prinsepia utilis Royle, and this extract is used for the repair of the skin barrier.

[0005] Although there are currently a small number of research reports on the activity of Prinsepia utilis Royle polysaccharides, there are no reports on the preparation method of Prinsepia utilis Royle acidic polysaccharide extract and the large-scale application of Prinsepia utilis Royle acidic polysaccharide. Therefore, it has great practical significance to study and develop it.

[0006] Therefore, there is an urgent need to provide a preparation method of Prinsepia utilis Royle acidic polysaccharide with excellent biological activity, good quality stability, and suitable for large-scale production. Summary of the Invention

[0007] Aiming at the deficiencies of the prior art, the main object of the present invention is to provide a Prinsepia utilis Royle acidic polysaccharide, a preparation method thereof and an application thereof. The preparation method of the Prinsepia utilis Royle acidic polysaccharide aims to fill the technical gap of the preparation method of Prinsepia utilis Royle acidic polysaccharide suitable for industrial application at the present stage; and based on the preparation method of the present invention, Prinsepia utilis Royle acidic polysaccharide with good biological activity and good quality stability can be prepared, as well as the application of the Prinsepia utilis Royle acidic polysaccharide in antioxidant and UV damage repair skin care products.

[0008] To achieve this purpose, the present invention adopts the following technical solutions:

[0009] In the first aspect, the present invention provides a preparation method of Prinsepia utilis Royle acidic polysaccharide, and the preparation method includes the following steps:

[0010] Step S1: Mix Prinsepia utilis Royle residue with a solvent and heat for extraction to obtain a crude extract, and concentrate the crude extract through an organic membrane to obtain a first purified extract;

[0011] Step Ss: Adsorb and elute the first purified extract obtained in Step S1 through a cation exchange resin to obtain a second purified extract;

[0012] Step S3: Adsorb and elute the second purified extract obtained in Step S2 through an anion exchange resin to obtain a third purified extract;

[0013] Step S4: Concentrate, dialyze and dry the third purified extract obtained in Step S3 to obtain the Prinsepia utilis Royle acidic polysaccharide.

[0014] The Prinsepia utilis Royle acidic polysaccharide and the preparation method thereof involved in the present invention adopt an organic membrane, a cation exchange resin and an anion exchange resin for multiple adsorption and separation. The content of total polysaccharide and uronic acid in the obtained acidic polysaccharide is relatively high, the protein content is low, and the obtained acidic polysaccharide has excellent DPPH free radical scavenging ability and the performance of effectively inhibiting the oxidative damage of HaCat cells by UVB irradiation.

[0015] In Step S1, on the one hand, the adoption of the organic membrane can remove small molecules such as monosaccharides and oligosaccharides in the Prinsepia utilis Royle extract to achieve the purpose of enriching Prinsepia utilis Royle acidic polysaccharide; on the other hand, the organic membrane can concentrate the extract, so there is no need to use ethanol to precipitate the extract to enrich the polysaccharide, avoiding the use of organic reagents in the above preparation method, saving production costs, and greatly improving the environmental protection performance of the process.

[0016] Preferably, in step S1, the solvent is an alkaline solution, and the alkaline solution is selected from NaOH solution or KOH solution. The molar concentration of the NaOH solution or KOH solution includes but is not limited to 0.3 mol / L, 0.4 mol / L, and 0.5 mol / L. Preferably, the molar concentration of the NaOH solution or KOH solution is 0.3 mol / L to 0.5 mol / L. Controlling the molar concentration of the alkaline solution in the crude extraction process in step S1 helps to synergistically maintain the contents of total polysaccharides and uronic acids in the acidic polysaccharides of Prinsepia utilis Royle at a relatively high level in subsequent operation steps.

[0017] Preferably, in step S1, the mass ratio of the Prinsepia utilis Royle residue to the solvent is 1:(10 - 20), and the value of the mass ratio can be 1:11, 1:13, 1:15, 1:17, 1:19, etc.

[0018] Preferably, in step S1, the number of extractions is 1 - 3 times; preferably, the number of extractions is 1 time; preferably, the number of extractions is 2 times; preferably, the number of extractions is 3 times.

[0019] Preferably, in step S1, the single extraction duration is 1 - 2 h; preferably, the single extraction duration is 1.1 h, 1.2 h, 1.4 h, 1.6 h, 1.8 h, 1.9 h, etc.

[0020] Preferably, in step S1, the heating temperature is 80 - 95 °C; preferably, the temperature values can be 82 °C, 84 °C, 86 °C, 88 °C, 90 °C, 92 °C, 94 °C, 95 °C, etc.

[0021] Preferably, in step S1, after the crude extract is concentrated by an organic membrane, the pH value of the concentrated solution is adjusted with acetic acid until pH = 7, thereby obtaining the first purified extract.

[0022] Preferably, in step S1, the pore size of the organic membrane for retaining effective substances is 800 - 5000 Da; preferably, the organic membrane includes ultrafiltration membrane and nanofiltration membrane, and the pore size of the organic membrane for retaining effective substances is 900 Da, 1000 Da, 1500 Da, 2000 Da, 2500 Da, 3000 Da, 3500 Da, 4000 Da, 4500 Da.

[0023] Among them, according to the monosaccharide category and molecular weight distribution of the condensed Prinsepia utilis Royle acidic polysaccharide, the pore size of the effective substance intercepted by the organic membrane is specifically designed, which is beneficial to simultaneously achieving the technical effects of impurity separation and purification and effective substance enrichment and concentration of the crude extract. Further, the organic membrane with a specific filtration accuracy is well adapted to the size of the Prinsepia utilis Royle acidic polysaccharide target molecule. If the intercepted pore size of the organic membrane is too large, the purpose of enriching Prinsepia utilis Royle acidic polysaccharide cannot be achieved; if the intercepted pore size is too small, small molecule substances such as monosaccharides cannot be effectively removed, and the concentration time will be prolonged.

[0024] Preferably, in step S2, the cation exchange resin is selected from any one of a uniform pore strongly acidic styrene-based cation exchange resin, a sulfonic acid group polystyrene-based macroporous strongly acidic cation exchange resin, or a gel-type strongly acidic styrene-based cation exchange resin. Among them, the cation exchange resin can adopt cation exchange resins of models JK008, LSD001, and 001×7.

[0025] Preferably, in step S2, the solvent used for elution includes water.

[0026] The above cation exchange resin selected in the present invention can effectively adsorb charged proteins, but has poor adsorption to acidic polysaccharides, and pure water can elute Prinsepia utilis Royle acidic polysaccharide.

[0027] Preferably, in step S2, after the elution, there is also a concentration step.

[0028] Preferably, the solid content of the second purified liquid obtained after concentration is 20-30%, and preferably, the solid content is 21%, 22%, 24%, 26%, 28%, 29%, etc.

[0029] Preferably, in step S3, the anion exchange resin is selected from any one of a macroporous weakly basic free amine type anion exchange resin, a highly porous weakly basic styrene-based dimethylamine type anion exchange resin, a macroporous weakly basic styrene-based anion exchange resin, or a macroporous multi-amino weakly basic anion exchange resin.

[0030] The anion exchange resin selected in the present invention has good selectivity and adsorption capacity, large particle size, and high efficiency, and can quickly separate and purify Prinsepia utilis Royle acidic polysaccharide. Among them, the anion exchange resin can adopt anion exchange resins of models D900, WA30, D315, and 201×7.

[0031] Preferably, in step S3, the solvent used for elution includes water and a salt solution.

[0032] Preferably, in step S3, the elution method includes: sequentially eluting with water and a salt solution. Thus, the effect of enriching acidic polysaccharides while removing neutral polysaccharides is achieved.

[0033] Preferably, the salt solution comprises an aqueous solution with any one or more of NaCl, KCl, MgCl2, and Na2SO4 as the solute.

[0034] Preferably, the concentration of the salt solution is 0.3 - 0.5 mol / L; preferably, the concentration of the salt solution is 0.32 mol / L, 0.34 mol / L, 0.36 mol / L, 0.38 mol / L, 0.40 mol / L, 0.42 mol / L, 0.44 mol / L, 0.45 mol / L, 0.46 mol / L, 0.48 mol / L, etc. It should be noted that the applicant found that by controlling the molar concentration of the salt solution used for elution in step S3 above at a relatively low level, i.e., 0.3 - 0.5 mol / L, not only can the acidic polysaccharide of Prinsepia utilis Royle containing less impurities be efficiently eluted and enriched, but also the efficiency of subsequent dialysis is improved.

[0035] Preferably, the dosage of the salt solution is 4 - 8 BV; preferably, the dosage of the salt solution is 4.5 BV, 5 BV, 5.5 BV, 6 BV, 6.5 BV, 7 BV, 7.5 BV, etc.

[0036] Preferably, in step S4, the pore size of the dialysis bag used for dialysis is 8000 - 20000 Da; preferably, the pore size of the dialysis bag used for dialysis is 10000 Da, 12000 Da, 14000 Da, 16000 Da, 18000 Da, etc.

[0037] Preferably, in step S4, the drying includes any one or more of freeze - drying, drying, or spray - drying.

[0038] In a second aspect, the present invention also provides an acidic polysaccharide of Prinsepia utilis Royle prepared by the preparation method of the acidic polysaccharide of Prinsepia utilis Royle described above.

[0039] Preferably, based on the mass of galacturonic acid as the measurement benchmark, the mass percentage content of galacturonic acid in the acidic polysaccharide of Prinsepia utilis Royle is 20% to 32%, for example, it can be 22%, 24%, 26%, 28%, 30%, etc.

[0040] Preferably, the protein mass percentage content in the acidic polysaccharide of Prinsepia utilis Royle is not higher than 4%, for example, it can be 0.5%, 1%, 1.5%, 2%, 2.5%, etc.

[0041] Preferably, the weight - average molecular weight of the acidic polysaccharide of Prinsepia utilis Royle is 20 - 40 kDa, for example, it can be 22 kDa, 25 kDa, 28 kDa, 30 kDa, 32 kDa, 35 kDa, 38 kDa, etc.

[0042] Preferably, the monosaccharides in the acidic polysaccharide from Prinsepia utilis Royle include arabinose, galacturonic acid, galactose, glucose, rhamnose, xylose, glucuronic acid, mannose, and fucose.

[0043] Preferably, the concentration of the acidic polysaccharide from Prinsepia utilis Royle is 0.125 - 2 mg / mL, its scavenging rate for DPPH free radicals is 40% - 98%, and its protective effect on UVB-induced photodamage of HaCat cells results in a survival rate of HaCat cells of 60% - 95%.

[0044] In a third aspect, the present invention provides an application of the aforementioned acidic polysaccharide from Prinsepia utilis Royle in efficacy skin care products for antioxidant and prevention of skin ultraviolet damage.

[0045] Compared with the prior art, the present invention has at least the following beneficial effects:

[0046] (1) The present invention uses an organic membrane to remove small molecules such as monosaccharides and oligosaccharides, while achieving the enrichment and concentration of polysaccharides, and sequentially uses cation exchange resin and anion exchange resin to achieve the removal of pigments and proteins, for the purpose of separating and purifying acidic polysaccharides. Through the coordinated compounding effect among the organic membrane, cation exchange resin and anion exchange resin, multiple adsorption and separation are carried out, and the contents of total polysaccharides and uronic acid in the obtained product are relatively high, and the protein content is low. In the preferred scheme, the total sugar content in the obtained acidic polysaccharide is as high as 80.47 - 82.51%, the uronic acid content is as high as 29.16 - 31.25%, the protein content is as low as 3.46 - 3.83%, and it has good solubility, strong DPPH free radical scavenging ability and effectively inhibits the oxidative damage of HaCat cells by UVB irradiation.

[0047] (2) The monosaccharide composition and weight average molecular weight of the acidic polysaccharide from Prinsepia utilis Royle prepared in the present invention are clear, and it has high biological activity.

[0048] (3) The present invention uses Prinsepia utilis Royle residue as the raw material, improving the comprehensive utilization rate of Prinsepia utilis Royle, and avoiding waste of resources to a certain extent. At the same time, the preparation method and equipment of the acidic polysaccharide from Prinsepia utilis Royle adopted in the present invention are simple, without special requirements, and industrial production can be achieved, with the characteristics of simple operation, economical cost, and environmental protection process. Description of the Drawings

[0049] Figure 1 is the absolute molecular weight analysis chart of the acidic polysaccharide from Prinsepia utilis Royle prepared in Example 1.

[0050] Figure 2A is the liquid chromatography chart of the monosaccharide composition analysis of the acidic polysaccharide from Prinsepia utilis Royle prepared in Example 1.

[0051] Figure 2B is the liquid chromatography chart of the monosaccharide standard mixture.

[0052] Figure 3 It is the infrared spectrogram of the pricklyash fruit acidic polysaccharide prepared in Example 1.

[0053] Figure 4 It is the DPPH free radical scavenging rate graph of Vc, the pricklyash fruit acidic polysaccharides prepared in Example 1, Examples 4 - 8, 11 and Comparative Examples 1 - 5 at different concentrations. Detailed implementation manners

[0054] The technical solution of the present invention will be further described below in conjunction with the accompanying drawings and through specific implementation manners. However, the following examples are only simple examples of the present invention and do not represent or limit the scope of the protection of the rights of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0055] The source information of the relevant raw materials and materials involved in the following examples or comparative examples is as follows:

[0056] The pricklyash fruit residue is the remaining fruit residue obtained after physical pressing, supercritical, and subcritical extraction of pricklyash fruit oil;

[0057] The sulfonic acid group polystyrene type macroporous strong acidic cation exchange resin is purchased from Xi'an Lanhxiao New Materials Co., Ltd., and the model is LSD001;

[0058] The uniform pore strong acidic styrene type cation exchange resin is purchased from Xi'an Lanhxiao New Materials Co., Ltd., and the model is JK008;

[0059] The gel type strong acidic styrene type cation exchange resin is purchased from Xi'an Lanhxiao New Materials Co., Ltd., and the model is 001×7;

[0060] The highly porous weak basic styrene type dimethylamine type anion exchange resin is purchased from Beijing Greengrass Science & Technology Development Co., Ltd., and the model is WA30;

[0061] The macroporous weak basic styrene type anion exchange resin is purchased from Beijing Solarbio Science & Technology Co., Ltd., and the model is D900;

[0062] The macroporous multi - amino weak basic anion exchange resin is purchased from Tianjin Xinyue Huamei Environmental Protection Technology Co., Ltd., and the model is D315;

[0063] The gel type strong basic styrene type anion exchange resin is purchased from Beijing Solarbio Science & Technology Co., Ltd., and the model is 201×7.

[0064] Example 1

[0065] This example provides a preparation method of the pricklyash fruit acidic polysaccharide, and the specific steps are as follows:

[0066] Step S1: Mix 50 g of pricklyash fruit residue with 0.4 mol / L NaOH aqueous solution and heat for extraction at 90 °C. The solid-liquid ratio (i.e., the mass ratio of pricklyash fruit residue to solvent) is 1:15. Extract twice, 2 h each time. Filter the extraction solution while it is hot under reduced pressure, and combine the filtrates. Concentrate the filtrate through a 2500 Da organic membrane to obtain the first purified solution of pricklyash fruit residue;

[0067] Step S2: After adsorbing the first purified solution of pricklyash fruit residue with cation exchange resin LSD001, perform separation and elution with 5 BV of water, collect the water eluate, and concentrate it under reduced pressure at 60 °C to obtain a second purified solution with a solid content of 25%;

[0068] Step S3: After adsorbing the second purified solution with anion exchange resin WA30, perform separation and elution successively with 5 BV of water and 5 BV of 0.4 mol / L NaCl aqueous solution to obtain a third purified solution;

[0069] Step S4: Collect the third purified solution, concentrate it under reduced pressure at 60 °C, dialyze it through a 10000 Da dialysis bag, and freeze-dry it to obtain pricklyash fruit acidic polysaccharide.

[0070] Example 2

[0071] This example provides a method for preparing pricklyash fruit acidic polysaccharide, and the specific steps are as follows:

[0072] Step S1: Mix 50 g of pricklyash fruit residue with 0.4 mol / L KOH aqueous solution and heat for extraction at 85 °C. The solid-liquid ratio (i.e., the mass ratio of pricklyash fruit residue to solvent) is 1:10. Extract three times, 2.5 h each time. Filter the extraction solution while it is hot under reduced pressure, and combine the filtrates. Concentrate the filtrate through a 2500 Da organic membrane to obtain the first purified solution of pricklyash fruit residue;

[0073] Step S2: After adsorbing the first purified solution of pricklyash fruit residue with cation exchange resin LSD001, perform separation and elution with 5 BV of water, collect the water eluate, and concentrate it under reduced pressure at 60 °C to obtain a second purified solution with a solid content of 25%;

[0074] Step S3: After adsorbing the second purified solution with anion exchange resin WA30, perform separation and elution successively with 5 BV of water and 5 BV of 0.3 mol / L KCl aqueous solution to obtain a third purified solution;

[0075] Step S4: Collect the 0.3 mol / L KCl eluate, concentrate it under reduced pressure at 60 °C, dialyze it through a 20000 Da dialysis bag, and freeze-dry it to obtain pricklyash fruit acidic polysaccharide.

[0076] Example 3

[0077] This example provides a method for preparing acanthopanax senticosus acidic polysaccharide, which is only different from Example 1 in that in step S1, extraction is carried out by mixing and heating with 0.35 mol / L NaOH aqueous solution at 90 °C, and other operations remain unchanged.

[0078] Example 4

[0079] This example provides a method for preparing acanthopanax senticosus acidic polysaccharide, which is only different from Example 1 in that in step S1, extraction is carried out by mixing and heating with 0.8 mol / L NaOH aqueous solution at 90 °C, and other operations remain unchanged.

[0080] Example 5

[0081] This example provides a method for preparing acanthopanax senticosus acidic polysaccharide, which is only different from Example 1 in that in step S1, extraction is carried out by mixing and heating with 0.1 mol / L NaOH aqueous solution at 90 °C, and other operations remain unchanged.

[0082] Example 6

[0083] This example provides a method for preparing acanthopanax senticosus acidic polysaccharide, which is only different from Example 1 in that in step S1, the filtrate is concentrated through an 8000 Da organic membrane to obtain acanthopanax senticosus residue concentrated solution, and other operations remain unchanged.

[0084] Example 7

[0085] This example provides a method for preparing acanthopanax senticosus acidic polysaccharide, which is only different from Example 1 in that in step S4, after the water elution concentrated solution is adsorbed by anion exchange resin WA30, it is sequentially separated and eluted with 5 BV water and 5 BV 0.7 mol / L NaCl aqueous solution, and other operations remain unchanged.

[0086] Example 8

[0087] This example provides a method for preparing acanthopanax senticosus acidic polysaccharide, which is only different from Example 1 in that in step S4, after the water elution concentrated solution is adsorbed by anion exchange resin WA30, it is sequentially separated and eluted with 5 BV water and 5 BV 0.1 mol / L NaCl aqueous solution, and other operations remain unchanged.

[0088] Example 9

[0089] This example provides a method for preparing acanthopanax senticosus acidic polysaccharide, which is only different from Example 1 in that in step S3, cation exchange resin JK008 is used to replace cation exchange resin LSD001 in Example 1, and other operations remain unchanged.

[0090] Example 10

[0091] This embodiment provides a method for preparing acidic polysaccharides from Principia utilis. The only difference between this method and Example 1 is that, in step S4, anion exchange resin D315 is used to replace the anion exchange resin WA30 in Example 1, and other operations remain unchanged.

[0092] Example 11

[0093] This embodiment provides a method for preparing acidic polysaccharides from Principia utilis. The only difference from Example 1 is that in step S4, after the water elution concentrate is adsorbed on anion exchange resin WA30, only 5BV 0.4mol / L NaCl aqueous solution is used for separation and elution, and other operations remain unchanged.

[0094] Comparative Example 1

[0095] Step S1: 50 g of thorn prunes pomace and an aqueous solution were mixed and heated at 90° C. for extraction, with a material-liquid ratio (i.e., the mass ratio of thorn prunes pomace to solvent) of 1:15, and the extraction was performed twice, each time for 2 hours. The extract was filtered under reduced pressure while hot, and the filtrates were combined; the filtrates were concentrated through a 2500 Da organic membrane to obtain a first purified solution of thorn prunes pomace;

[0096] Step S2: After the first purified liquid of the pomace of the thorn tree is adsorbed on a cation exchange resin LSD001, it is separated and eluted with 5BV water, the water eluate is collected, and concentrated under reduced pressure at 60° C. to obtain a second purified liquid with a solid content of 25%;

[0097] Step S3: After the second purified liquid is adsorbed on anion exchange resin WA30, it is separated and eluted with 5BV of water and 5BV of 0.4 mol / L NaCl aqueous solution in sequence to obtain a third purified liquid;

[0098] Step S4: collecting the third purified liquid, concentrating under reduced pressure at 60° C., dialyzing with a 10,000 Da dialysis bag and freeze-drying to obtain the utilitarian fruit acidic polysaccharide.

[0099] Comparative Example 2

[0100] Step S1: 50 g of thorn prunes pomace and 0.4 mol / L NaOH aqueous solution were mixed and heated at 90° C. for extraction, with a material-liquid ratio of 1:15 (i.e., the mass ratio of thorn prunes pomace to solvent), and the extraction was performed twice, each time for 2 hours. The extract was filtered under reduced pressure while hot, and the filtrates were combined; the filtrates were concentrated under vacuum to obtain a first purified solution of thorn prunes pomace;

[0101] Step S2: After the first purified liquid of the pomace of the thorn tree is adsorbed on a cation exchange resin LSD001, it is separated and eluted with 5BV water, the water eluate is collected, and concentrated under reduced pressure at 60° C. to obtain a second purified liquid with a solid content of 25%;

[0102] Step S3: After adsorbing the water-washed and concentrated solution with anion exchange resin WA30, separate and elute it successively with 5 BV of water and 5 BV of 0.4 mol / L NaCl aqueous solution to obtain the third purified solution;

[0103] Step S4: Collect the third purified solution, concentrate it under reduced pressure at 60°C, dialyze it with a 10000 Da dialysis bag, and freeze-dry it to obtain the acidic polysaccharide from Prinsepia utilis Royle.

[0104] Comparative Example 3

[0105] Step S1: Mix 50 g of Prinsepia utilis Royle residue with 0.4 mol / L NaOH aqueous solution, heat and extract at 90°C, with a material-liquid ratio of 1:15 (i.e., the mass ratio of Prinsepia utilis Royle residue to the solvent), extract 2 times, 2 h each time. Filter the extract while it is hot under reduced pressure, and combine the filtrates; Concentrate the filtrate through a 2500 Da organic membrane to obtain the first purified solution of Prinsepia utilis Royle residue;

[0106] Step S2: After deproteinizing the first purified solution of Prinsepia utilis Royle residue with sevage reagent, adsorb it with anion exchange resin WA30, and separate and elute it successively with 5 BV of water and 5 BV of 0.4 mol / L NaCl aqueous solution;

[0107] Step S3: Collect the 0.4 mol / L NaCl eluate, concentrate it under reduced pressure at 60°C, dialyze it with a 10000 Da dialysis bag, and freeze-dry it to obtain the acidic polysaccharide from Prinsepia utilis Royle.

[0108] Comparative Example 4

[0109] Step S1: Mix 50 g of Prinsepia utilis Royle residue with 0.4 mol / L NaOH aqueous solution, heat and extract at 90°C, with a material-liquid ratio of 1:15 (i.e., the mass ratio of Prinsepia utilis Royle residue to the solvent), extract 2 times, 2 h each time. Filter the extract while it is hot under reduced pressure, and combine the filtrates; Concentrate the filtrate through a 2500 Da organic membrane to obtain the first purified solution of Prinsepia utilis Royle residue;

[0110] Step S2: After adsorbing the first purified solution of Prinsepia utilis Royle residue with anion exchange resin WA30, separate and elute it successively with 5 BV of water and 5 BV of 0.4 mol / L NaCl aqueous solution;

[0111] Step S3: Collect the 0.4 mol / L NaCl eluate, concentrate it under reduced pressure at 60°C, dialyze it with a 10000 Da dialysis bag, and freeze-dry it to obtain the acidic polysaccharide from Prinsepia utilis Royle.

[0112] Comparative Example 5

[0113] Step S1: Mix 50 g of prickly pear pomace with 0.4 mol / L NaOH aqueous solution and heat for extraction at 90 °C. The solid-liquid ratio is 1:15 (i.e., the mass ratio of prickly pear pomace to solvent), extract twice, 2 h each time. Filter the extract while it is hot under reduced pressure, and combine the filtrates. Concentrate the filtrate through a 2500 Da organic membrane to obtain the first purified solution of prickly pear pomace.

[0114] Step S2: Subject the first purified solution of prickly pear pomace to ethanol precipitation with 85% ethanol overnight. Collect the precipitate, dissolve it in water, load it onto an anion exchange resin WA30 for adsorption, and then separate and elute it successively with 5 BV of water and 5 BV of 0.4 mol / L NaCl aqueous solution.

[0115] Step S3: Collect the 0.4 mol / L NaCl eluate, concentrate it under reduced pressure at 60 °C, dialyze it through a 10000 Da dialysis bag, and freeze-dry it to obtain prickly pear acidic polysaccharide.

[0116] Test Example 1

[0117] Detection of total polysaccharide, uronic acid, and protein contents in acidic polysaccharide

[0118] The method for detecting the total polysaccharide content in the prickly pear acidic polysaccharide involved below is as follows:

[0119] (1) Solution preparation: a. Preparation of standard solution: Prepare a 0.1 mg / mL glucose standard solution. Respectively pipette 0 mL, 0.1 mL, 0.2 mL, 0.4 mL, 0.8 mL, 1.6 mL, and 2 mL into test tubes, and then add distilled water to each test tube to make up to 2 mL for use. b. Preparation of sample solution: Accurately weigh the sample powder, dissolve it with distilled water to prepare a 0.1 mg / mL sample solution for testing. c. Preparation of 5% phenol solution: Accurately weigh 2.5 g of phenol powder, add 47.5 mL of distilled water for dissolution, and store it in the dark for standby.

[0120] (2) Determination of total polysaccharide content: Sequentially pipette 1 mL of the prepared standard solution and sample solution into test tubes, add 0.5 mL of 5% phenol solution and 2.5 mL of concentrated sulfuric acid solution for reaction, gently shake. Using distilled water as the blank control, measure its absorbance at 490 nm with an enzyme-linked immunosorbent assay (ELISA) reader. Plot a standard curve with concentration as the abscissa and absorbance as the ordinate. Substitute the absorbance of the sample solution into the standard curve y = 7.5233x - 0.057, (R 2 = 0.9992) to calculate the content of total polysaccharide in the sample.

[0121] The method for detecting the uronic acid content in the prickly pear acidic polysaccharide involved below is as follows:

[0122] (1) Solution preparation: a. Preparation of borate solution: Weigh 2.39 g of sodium tetraborate and completely dissolve it in 250 mL of concentrated sulfuric acid; b. Preparation of 4 mol / mL sulfamate solution: Weigh 38.90 g of sulfamic acid, dissolve it in an appropriate amount of distilled water, and gradually add saturated NaOH solution to fully dissolve the sulfamic acid. Adjust the pH value of the solution to 1.6 and then make up the volume to 100 mL; c. Preparation of m-hydroxybiphenyl solution: Weigh 0.15 g of m-hydroxybiphenyl, dissolve it in 5 mg / mL NaOH solution, and make up the volume to 100 mL, with a mass concentration of 1.5 mg / mL; d. Preparation of standard solution: Accurately weigh 10.04 mg of galacturonic acid, make up the volume to 25 mL with distilled water, divide it into aliquots and store at 20 °C for later use; Take 1 mL of the stock solution and make up the volume to 10 mL with water to prepare a standard working solution; e. Preparation of sample solution: Weigh 5 mg of the sample respectively, dissolve it in distilled water and make up the volume to 5 mL to prepare a 1 mg / mL sample solution.

[0123] (2) Plotting of the standard curve: Take 0 mL, 0.1 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL and 1 mL of the working standard solution into test tubes respectively, make up the volume to 1 mL with distilled water. Add 10 μL of sulfamate to each tube, shake well, slowly add 5 mL of borate sulfuric acid solution, mix well by shaking and then place it in a boiling water bath for 15 min. After taking it out, cool it to room temperature. Slowly add 80 μL of m-hydroxybiphenyl solution to each tube, fully shake until the color is uniform, and then let it stand at room temperature for 30 min. Take 200 μL and transfer it to a 96-well plate, measure its absorbance at 525 nm, use the uronic acid concentration as the abscissa and the absorbance value as the ordinate to plot the standard curve.

[0124] (3) Determination of the uronic acid content in the sample: Take 1 mL of the sample solution respectively and determine the uronic acid content according to the above method.

[0125] The detection method for the protein content in the acidic polysaccharide of Prinsepia utilis Royle involved below is as follows:

[0126] (1) Preparation of protein standard: Add 1.2 mL of protein standard preparation solution to a tube of protein standard (30 mg BSA), fully dissolve it to prepare a 25 mg / mL protein standard solution; Take an appropriate amount of 25 mg / mL protein standard and dilute it to a final concentration of 0.5 mg / mL.

[0127] (2) Preparation of BCA working solution: Mix 5 mL of BCA reagent A and 100 μL of BCA reagent B to prepare 5.1 mL of BCA working solution.

[0128] (3) Protein concentration detection: Add the standard products in volumes of 0 μL, 1 μL, 2 μL, 4 μL, 8 μL, 12 μL, 16 μL, and 20 μL to the standard product wells of a 96-well plate, and make up to 20 μL with the standard product diluent, corresponding to standard product concentrations of 0 mg / mL, 0.025 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, and 0.5 mg / mL respectively; add 20 μL of the sample to the sample wells of the 96-well plate; add 200 μL of the BCA working solution to each well, and incubate at 37 °C for 30 min; measure the absorbance at a wavelength of 562 nm using a microplate reader; according to the standard curve y = 1.2523x + 0.0195, (R 2 = 0.9992) calculate the protein concentration of the sample.

[0129] The total polysaccharide content, uronic acid content, and protein content of the prickly pear acidic polysaccharides prepared in Examples 1-11 and Comparative Examples 1-5 were measured, and the results are shown in Table 1:

[0130] Table 1.

[0131] Group Yield % Total polysaccharide content % Uronic acid content % Protein content % Example 1 2.45 82.51 31.25 3.46 Example 2 2.31 81.23 29.63 3.61 Example 3 2.39 81.73 29.58 3.64 Example 4 2.59 71.24 22.74 7.75 Example 5 2.14 73.48 23.43 7.14 Example 6 2.21 72.68 21.58 8.78 Example 7 2.85 71.26 22.85 7.55 Example 8 1.78 74.85 23.82 6.93 Example 9 2.25 80.68 29.91 3.83 Example 10 2.31 80.47 29.16 3.79 Example 11 3.64 75.57 21.39 6.68 Comparative Example 1 3.06 72.36 15.34 6.04 Comparative Example 2 3.54 75.51 18.96 7.45 Comparative Example 3 2.13 71.62 18.05 12.46 Comparative Example 4 4.17 70.32 11.33 23.35 Comparative Example 5 5.45 71.35 10.23 25.61

[0132] It can be seen from the test results that:

[0133] (1) By analyzing the total polysaccharide content, uronic acid content, and protein content of the prickly pear acidic polysaccharides prepared in Examples 1-11 and Comparative Examples 1-5, it can be seen that among Examples 1-11, the total polysaccharide content and uronic acid content of the prickly pear acidic polysaccharides prepared in Examples 1-3 and Examples 9-10 are optimal (the total polysaccharide content is not less than 80.47 wt%, the uronic acid content is not less than 29.16 wt%, and the protein content does not exceed 3.83 wt%). On the contrary, the total polysaccharide content and uronic acid content of the prickly pear acidic polysaccharides prepared in Examples 4-8 and Comparative Examples 1-5 are significantly inferior to those of the prickly pear acidic polysaccharides prepared in Examples 1-3 and Examples 9-10. Specifically:

[0134] In Examples 9-10, the cation exchange resin and anion exchange resin in Example ① were equivalently replaced respectively, and from the perspective of the total polysaccharide content and uronic acid content of the prickly pear acidic polysaccharides prepared in Examples 9-10, their total polysaccharide content and uronic acid content are similar to those in Example 1. It can be seen that in step S3 of the preparation method of the prickly pear acidic polysaccharide of the present invention, the cation exchange resin JK008 or the cation exchange resin LSD001, and in step S4, the anion exchange resin D315 or the anion exchange resin WA30 can both be used.

[0135] Example 4-5 The molar concentration of the NaOH aqueous solution in step S1 of Example 1 was adjusted to 0.8 mol / L and 0.1 mol / L respectively. The molar concentration values of the adjusted NaOH aqueous solution exceeded the upper and lower limit value ranges of 0.3 mol / L to 0.5 mol / L in the preparation method described in the present invention. Moreover, the total polysaccharide content and uronic acid content of the acanthopanax senticosus acidic polysaccharide prepared in Examples 4-5 decreased significantly. Thus, it can be seen that the value range of 0.3 mol / L to 0.5 mol / L in step S1 of the preparation method described in the present invention is an ideal molar concentration value range of the NaOH aqueous solution, which can cooperate with other steps and operation parameters in the preparation method to keep the total polysaccharide content and uronic acid content of the acanthopanax senticosus acidic polysaccharide prepared at an ideal level.

[0136] Example 6 The pore size of the organic membrane for retaining effective substances in step S1 of Example 1 was adjusted to 8000 Da. This pore size exceeded the upper limit value of the value range of 800-5000 Da in the preparation method described in the present invention, resulting in too large a retention pore size and poor effect of enriching acanthopanax senticosus acidic polysaccharide.

[0137] Examples 7-8 The molar concentration of the NaCl aqueous solution in the eluent in step S4 of Example 1 was adjusted to 0.7 mol / L and 0.1 mol / L respectively. It can be seen that when the molar concentration of the NaCl aqueous solution in the eluent was 0.7 mol / L, exceeding the upper limit value of 0.3-0.5 mol / L in the preparation method described in the invention, the total polysaccharide content and uronic acid content of the acanthopanax senticosus acidic polysaccharide prepared decreased significantly compared with those in Examples 1-3; while when the molar concentration of the NaCl aqueous solution in the eluent was 0.1 mol / L, lower than the lower limit value of 0.3-0.5 mol / L in the preparation method described in the invention, the total polysaccharide content and uronic acid content of the acanthopanax senticosus acidic polysaccharide prepared still decreased significantly compared with those in Examples 1-3.

[0138] Example 11 When “after the water elution concentrate was adsorbed by the anion exchange resin WA30, it was separated and eluted successively with 5 BV of water and 5 BV of 0.4 mol / L NaCl aqueous solution” in step S4 was replaced with “after the water elution concentrate was adsorbed by the anion exchange resin WA30, it was only separated and eluted with 5 BV of 0.4 mol / L NaCl aqueous solution”, the total polysaccharide content and uronic acid content of the acanthopanax senticosus acidic polysaccharide prepared decreased significantly compared with those in Examples 1-3.

[0139] Comparing Examples 1-3, 9-10 with Comparative Examples 1-5, it can be seen that in this application, alkaline solution extraction is adopted, an organic membrane is used to concentrate the extract, and cation exchange resin is used to remove proteins. Compared with pure water extraction, alcohol precipitation method and organic reagent sevage method, it can effectively increase the contents of total polysaccharide and uronic acid in the acidic polysaccharide of Prinsepia utilis Royle, and the obtained protein content is relatively low.

[0140] Test Example 2

[0141] Determination of the molecular weight range of the acidic polysaccharide of Prinsepia utilis Royle

[0142] The molecular weight of the acidic polysaccharide of Prinsepia utilis Royle prepared in Example 1 was determined, and the test method was as follows:

[0143] The sample of the acidic polysaccharide of Prinsepia utilis Royle prepared in Example 1 was dissolved in an aqueous solution of 0.1 mol / L NaNO3 (containing 0.02% NaN3, w / w), with a final concentration of 1 mg / mL, and filtered through a filter membrane with a pore size of 0.45 μm for inspection.

[0144] The chromatographic system used was a gel chromatography - differential - multi - angle laser light scattering system. The liquid phase system was U3000 (Thermo, USA), the differential detector was Optilab T - rEX (Wyatt technology, CA, USA), and the laser light scattering detector was DAWN HELEOSⅡ (Wyatt technology, CA, USA). A gel exclusion chromatography column OhpakSB - 805HQ (300×8 mm) and OhpakSB - 803HQ (300×8 mm) were connected in series. The column temperature was 45°C, the injection volume was 100 μL, the mobile phase A was (0.02% NaN3, 0.1 M NaNO3), the flow rate was 0.6 mL / min, and isocratic elution was carried out for 75 min.

[0145] The chromatographic data was processed using software ASTRA6.1, as Figure 1 shown. The absolute molecular weight analysis graph had the detected retention time (Time, min) as the abscissa and the molar mass (Molar Mass, g / mol) as the ordinate. The red line represented the multi - angle laser light scattering signal (i.e., LS, V), the blue line represented the differential signal (i.e., RI, RIU), and the black line was the molecular weight fitted by the two signals. Through analysis, the Mw molecular weight of the acidic polysaccharide prepared in Example 1 was 29.39 kDa.

[0146] Test Example 3

[0147] Determination of the monosaccharide composition of the acidic polysaccharide of Prinsepia utilis Royle

[0148] The monosaccharide composition of the acidic polysaccharide of Prinsepia utilis Royle prepared in Example 1 was determined, and the test method was as follows:

[0149] (1) Hydrolysis reaction: The acidic polysaccharide of Prinsepia utilis Royle fruit obtained in Example 1 was hydrolyzed with trifluoroacetic acid (TFA). 5 mg of the acidic polysaccharide sample of Prinsepia utilis Royle fruit was accurately weighed, 2 mL of TFA (2 mol / L) was added, and hydrolysis was carried out at 110 °C for 2 h. After complete hydrolysis, the sample was dried with a nitrogen blower to completely remove TFA.

[0150] (2) Derivatization reaction: Derivatization was carried out with 1-phenyl-3-methyl-5-pyrazolone (PMP) reagent. Quantitatively weigh the standard products of D-glucose, D-galactose, D-mannose, D-xylose, L-rhamnose, L-arabinose, D-fructose, D-galacturonic acid, D-glucuronic acid and the hydrolyzed sample of acidic polysaccharide (Example 1), add 0.5 mL of PMP (0.5 mol / L) reagent and 0.5 mL of NaOH (0.3 mol / L) solution, react at 70 °C for 30 min, after cooling, add 0.5 mL of HCl (0.3 mol / L) and 0.5 mL of distilled water for neutralization, add an equal volume of chloroform solution, shake and extract, collect the upper layer liquid, and filter through a 0.22 μM filter membrane for testing.

[0151] (3) High performance liquid chromatography test: Qualitative and quantitative analysis of the derivatized sample was carried out by high performance liquid chromatography (HPLC). The chromatographic conditions were as follows: Phase A was phosphate buffer (pH = 7.2), and Phase B was acetonitrile; gradient elution was carried out, 0 - 3 min: 15% B, 3 - 4 min: 15 - 18% B, 4 - 10 min: 18% B, 10 - 25 min: 18 - 40% B; column model: Agilent ZORBAX SB-C18, 4.6×150 mm; flow rate 0.8 mL / min; column temperature 30 °C; DAD: 250 nm; injection volume 5 μL.

[0152] As Figure 2A and Figure 2B shown, from the corresponding monosaccharide mixed standard product, the proportion of monosaccharide composition of the acidic polysaccharide of Prinsepia utilis Royle fruit in Example 1 was arabinose (31.25%), galacturonic acid (27.61%), galactose (21.43%), glucose (6.08%), rhamnose (4.28%), xylose (3.52%), glucuronic acid (2.94%), mannose (2%), fucose (0.05%).

[0153] Test Example 4

[0154] Infrared spectrum test

[0155] The acidic polysaccharide of Prinsepia utilis Royle fruit prepared in Example 1 was tested by infrared spectrum. The test method was as follows:

[0156] Take 5 mg of the pricklyash fruit acidic polysaccharide prepared in Example 1, make a KBr tablet, and perform infrared scanning with a Nicolet 5700 infrared spectrometer in the range of 4000 - 400 cm -1 in the United States. As Figure 3 shown, in the IR spectrum, a strong and broad absorption peak appears at 3417 cm -1 , which is the strong absorption peak of the O - H stretching vibration on the polysaccharide, indicating that hydrogen bonds exist both within and between the polysaccharide molecules; the absorption peak at 1651 cm -1 is the carbonyl vibration peak of uronic acid, further indicating that the polysaccharide sample in Example 1 contains uronic acid. See Table 2.

[0157] Table 2.

[0158]

[0159]

[0160] Test Example 5

[0161] Test for the scavenging rate of DPPH free radicals [[ID=2�]]

[0162] The DPPH free radical scavenging rates of the pricklyash fruit acidic polysaccharides prepared in Example 1 and Examples 4 - 8, 11, and Comparative Examples 1 - 5 were tested. The test method is as follows:

[0163] Take 0.5 mL of the pricklyash fruit acidic polysaccharide test samples with different concentrations in a test tube, add 1.5 mL of distilled water and 2 mL of DPPH (0.1 mmol / L) ethanol solution, mix well, react at room temperature in the dark for 30 min, and measure the absorbance value at a wavelength of 517 nm. Calculate the scavenging rate of the test samples for DPPH free radicals according to the following formula.

[0164] Scavenging rate = [1 - (A 样品 - A 空白样品 ) / (A 对照 - A 空白对照 )] × 100%

[0165] In the formula: A 样品 : The absorbance value measured for 150 μL of the sample solution + 150 μL of the DPPH alcohol solution; A 空白样品 : The absorbance value measured for 150 μL of the sample solution + 150 μL of absolute ethanol; A 对照 : The absorbance value measured for 150 μL of the DPPH solution + 150 μL of the sample solvent; A 空白对照 : The absorbance value measured for 150 μL of absolute ethanol + 150 μL of the sample solvent.

[0166] The IC of the DPPH free radical scavenging rates of the pricklyash fruit acidic polysaccharides prepared in Example 1 and Examples 4 - 8, 11, and Comparative Examples 1 - 550 The value is shown in Table 3. It is easy to obtain that the IC 50 value of the DPPH free radical scavenging rate of the acidic polysaccharide from Prinsepia utilis Royle prepared in Example 1 is significantly better than that of the acidic polysaccharide from Prinsepia utilis Royle prepared in Examples 4-8, 11 and Comparative Examples 1-5. 50 value. In the present invention, multiple adsorption and separation are carried out by using an organic membrane, a cation exchange resin and an anion exchange resin, and the prepared acidic polysaccharide from Prinsepia utilis Royle has stronger DPPH free radical scavenging ability.

[0167] Table 3.

[0168] Group <![CDATA[IC 50 (mg / mL)]]> Vitamin C 0.027 Example 1 0.164 Example 4 0.512 Example 5 0.497 Example 6 0.614 Example 7 0.503 Example 8 0.483 Example 11 0.625 Comparative Example 1 0.863 Comparative Example 2 0.726 Comparative Example 3 0.731 Comparative Example 4 0.875 Comparative Example 5 0.882

[0169] Test Example 6

[0170] Test on the protective effect against UVB photodamage of HaCat cells

[0171] The protective effect against UVB photodamage of the acidic polysaccharide from Prinsepia utilis Royle prepared in Example 1 and Examples 4-8, 11 and Comparative Examples 1-5 was tested, and the test method is as follows:

[0172] This test was divided into a blank control group, a UVB control group and an experimental group. Among them, the test method of the experimental group is as follows: Take HaCat cells in the logarithmic growth phase and seed 96-well plates with 1.0×10 4 cells per well, culture them in 10% FBS DMEM medium at 37°C and 5% CO2 for 24 h. After the cells are completely adherent, wash them 1-2 times with PBS, and then change to 10% FBS DMEM medium containing the acidic polysaccharide from Prinsepia utilis Royle, and culture them at 37°C and 5% CO2 for 2 h. Then change the culture medium to PBS containing the acidic polysaccharide from Prinsepia utilis Royle, irradiate with UVB ultraviolet light at a dose of 30 mJ / cm 2 , and then change to 10% FBS DMEM medium and culture in an environment of 37°C and 5% CO2 for 24 h. Then, the survival rate of HaCat cells was detected by the CCK8 method. The preparation method of the blank control group is as follows: Take HaCat cells in the logarithmic growth phase and seed 96-well plates with 1.0×10 4 cells per well, culture them in 10% FBS DMEM medium at 37°C and 5% CO2 for 24 h. After the cells are completely adherent, wash them 1-2 times with PBS, and then change to 10% FBS DMEM medium and culture in an environment of 37°C and 5% CO2 for 24 h. Then, the survival rate of HaCat cells was detected by the CCK8 method. The preparation method of the UVB control group is as follows: Take HaCat cells in the logarithmic growth phase and seed 96-well plates with 1.0×10 4A 96-well plate was cultured with 10% FBS DMEM medium at 37°C and 5% CO2 for 24 h. After the cells were completely adherent, they were washed 1-2 times with PBS, and then replaced with fresh 10% FBS DMEM medium and cultured at 37°C and 5% CO2 for 2 h. Then the culture medium was replaced with PBS, and UVB ultraviolet light was irradiated at a dose of 30 mJ / cm 2 After that, it was replaced with fresh 10% FBS DMEM medium and cultured in an environment of 37°C and 5% CO2 for 24 h. Then the viability of HaCat cells was detected by the CCK8 method. The test results are shown in Table 4.

[0173] Table 4.

[0174]

[0175]

[0176] It is easy to conclude that the protective effect of the Rosa roxburghii tratt acidic polysaccharide prepared in Example 1 on the UVB photodamage of HaCat cells is significantly better than that of the Rosa roxburghii tratt acidic polysaccharides prepared in Examples 4-8, 11 and Comparative Examples 1-5 on the UVB photodamage of HaCat cells.

[0177] The applicant declares that the above description is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed by the present invention fall within the protection scope and the disclosure scope of the present invention.

Claims

1. A preparation method of pricklyash fruit acidic polysaccharide, characterized in that, The preparation method includes the following steps: Step S1: Mix the prickly pear pomace with a solvent and heat for extraction to obtain a crude extract, and concentrate the crude extract through an organic membrane to obtain a first purified extract; the solvent is an alkaline solution, and the alkaline solution is selected from NaOH solution or KOH solution, and the molar concentration of the NaOH solution or KOH solution ranges from 0.3 mol / L to 0.5 mol / L; the pore size of the organic membrane for retaining effective substances is 2500 - 5000 Da; Step S2: Adsorb and elute the first purified extract obtained in Step S1 through a cation exchange resin to obtain a second purified extract; Step S3: Adsorb and elute the second purified extract obtained in Step S2 through an anion exchange resin to obtain a third purified extract; the elution in this step is sequentially carried out with water and a salt solution; the salt solution includes an aqueous solution with any one or more solutes selected from NaCl, KCl, MgCl2, and Na2SO4; wherein, the concentration of the salt solution is 0.3 - 0.5 mol / L; Step S4: Collect the eluate of the salt solution obtained in Step S3, and obtain the prickly pear acidic polysaccharide through concentration, dialysis, and drying.

2. The preparation method according to claim 1, characterized in that, In Step S1, the mass ratio of the prickly pear pomace to the solvent is 1:(10 - 20); the number of extractions is 1 - 3 times; after the crude extract is concentrated through the organic membrane, the pH value of the concentrated solution is adjusted with acetic acid until pH = 7, thereby obtaining the first purified extract.

3. The preparation method according to claim 1 or 2, characterized in that, In Step S1, the organic membrane includes an ultrafiltration membrane and a nanofiltration membrane.

4. The preparation method according to claim 1 or 2, characterized in that, In Step S2, the cation exchange resin is selected from any one of a uniform pore strongly acidic styrene-based cation exchange resin, a sulfonic acid group polystyrene-based macroporous strongly acidic cation exchange resin, or a gel-type strongly acidic styrene-based cation exchange resin; the solvent used for elution in Step S2 is water, and a concentration step is further included after elution; the solid content of the second purified extract obtained after concentration is 20 - 30 wt%.

5. The preparation method according to claim 1 or 2, characterized in that, In Step S3, the anion exchange resin is selected from any one of a macroporous weakly basic free amine-type anion exchange resin, a highly porous weakly basic styrene-based dimethylamine-type anion exchange resin, a macroporous weakly basic styrene-based anion exchange resin, or a macroporous multi-amino weakly basic anion exchange resin.

6. The preparation method according to claim 1 or 2, characterized in that In Step S4, the pore size of the dialysis bag used for dialysis is 8000 - 20000 Da; the drying includes any one or more of freeze-drying, drying, or spray-drying.

7. A prickly pear acidic polysaccharide prepared by the preparation method of the prickly pear acidic polysaccharide according to any one of claims 1 - 6; based on the mass of galacturonic acid as the measurement benchmark, the mass percentage content of galacturonic acid in the prickly pear acidic polysaccharide is 20% to 32%; the protein mass percentage content in the prickly pear acidic polysaccharide is not higher than 4%; the weight-average molecular weight of the prickly pear acidic polysaccharide is 20 - 40 kDa; the monosaccharides in the prickly pear acidic polysaccharide include arabinose, galacturonic acid, galactose, glucose, rhamnose, xylose, glucuronic acid, mannose, and fucose.

8. Use of the pricklyash fruit acidic polysaccharide according to claim 7 in the preparation of skin care products for antioxidant and prevention of skin ultraviolet damage.

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