Prinsepia utilis royle acidic polysaccharide, and preparation method therefor and use thereof

Abstract

The present invention relates to a Prinsepia utilis Royle acidic polysaccharide, and a preparation method therefor and the use thereof. The preparation method comprises the following steps: (1) mixing a pomace of Prinsepia utilis Royle with a solvent, heating and extracting same, and concentrating the obtained extract via an organic membrane to obtain a first purified liquid; (2) subjecting the first purified liquid to adsorption and elution with cation exchange resin to obtain a second purified liquid; (3) subjecting the second purified liquid to adsorption and elution with anion exchange resin to obtain a third purified liquid; and (4) subjecting the third purified liquid to concentration, dialysis, and drying to obtain the Prinsepia utilis Royle acidic polysaccharide. According to the present invention, the organic membrane, cation exchange resin and anion exchange resin are used for multiple adsorption and separation, so that the obtained acidic polysaccharide has a high content of total polysaccharide and uronic acid, a low protein content, and a strong DPPH radical scavenging ability, and effectively inhibits oxidative damage to HaCat cells caused by UVB irradiation. In addition, the preparation method is simple and convenient to operate, and cost-effective, and has good industrial production prospects.

Description

An acidic polysaccharide from *Prickly pear* fruit, its preparation method, and its application. Technical Field

[0001] This invention belongs to the field of biomedical technology, and more specifically, relates to an acidic polysaccharide from *Cypripedium spp.*, its preparation method, and its application. Background Technology

[0002] Polysaccharides are a class of high-molecular-weight carbohydrates composed of the condensation of multiple monosaccharide molecules (including aldoses and ketoses). Acidic polysaccharides refer to polysaccharides containing acidic groups such as carboxyl or sulfate groups. Studies have shown that acidic polysaccharides have complex structures and possess various biological activities, such as antioxidant, immunomodulatory, hypoglycemic, and antitumor effects. Acidic polysaccharides have no toxic side effects and have high potential application value.

[0003] The fruit of *Prinsepia utilis* Royle, belonging to the genus *Prinsepia* in the family Rosaceae, grows in Yunnan, Guizhou, and Sichuan provinces. The *Diannan Materia Medica* records that "the fruit of *Prinsepia utilis* is slightly cold in nature and bitter in taste. It is effective for all kinds of sores and toxins; if there is pus, it will drain; if there is no pus, it will disappear immediately; it disperses nodules, and can be chewed and taken with wine." Rich in oil, *Prinsepia utilis* is an oilseed plant used both as food and medicine. People in Lijiang and Diqing, Yunnan, often extract *Prinsepia utilis* oil for consumption. This oil is rich in flavonoids, triterpenes, and fatty acids, and is widely used in cosmetics as a skincare oil, possessing moisturizing, anti-inflammatory, and repairing effects.

[0004] At present, research on the polysaccharide of *Cyprinus burmannii* mainly focuses on the treatment of diabetes and the repair of the skin barrier. Chinese patent application CN104887881A discloses a pure traditional Chinese medicine preparation containing polysaccharide of *Cyprinus burmannii*, which can be used for the prevention and treatment of diabetes. Chinese patent application CN117959224A discloses a composition containing extract of *Cyprinus burmannii*, and the extract is used for the repair of the skin barrier.

[0005] Although there have been a few research reports on the activity of Prickly pear polysaccharides, there are no reports on the preparation methods of Prickly pear acidic polysaccharide extracts or the large-scale application of Prickly pear acidic polysaccharides. Therefore, research and development of these polysaccharides is of great practical significance.

[0006] Therefore, there is an urgent need to provide a method for preparing acidic polysaccharides from *Cynanchum paniculatum* that has excellent bioactivity, good quality stability, and is suitable for large-scale production. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the main objective of this invention is to propose an acidic polysaccharide from *Prickly pear* fruit, its preparation method, and its applications. The preparation method of the acidic polysaccharide from *Prickly pear* fruit aims to fill the technological gap in the current stage of preparation methods suitable for industrial application. Furthermore, based on the preparation method of this invention, acidic polysaccharides from *Prickly pear* fruit with excellent bioactivity and good quality stability can be prepared, and the acidic polysaccharide from *Prickly pear* fruit can be applied in anti-oxidation and UV damage repair skin care products.

[0008] To achieve this objective, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention provides a method for preparing acidic polysaccharides from *Prickly pear* fruit, the method comprising the following steps:

[0010] Step S1: Mix the pomace of the sea buckthorn fruit with a solvent and heat to extract crude extract. Concentrate the crude extract through an organic membrane to obtain the first purified extract.

[0011] Step S2: The first purified solution obtained in step S1 is adsorbed and eluted by a cation exchange resin to obtain the second purified solution;

[0012] Step S3: The second purified solution obtained in step S2 is adsorbed and eluted by anion exchange resin to obtain the third purified solution;

[0013] Step S4: The third purified liquid obtained in step S3 is concentrated, dialyzed, and dried to obtain the acidic polysaccharide of *Cyprinus pungens*.

[0014] The acidic polysaccharide from *Cyprinus sativus* and its preparation method involved in this invention utilizes multiple adsorption and separation processes using organic membranes, cation exchange resins, and anion exchange resins. The resulting acidic polysaccharide has a high content of total polysaccharides and uronic acids, a low protein content, and exhibits excellent DPPH free radical scavenging ability and effective inhibition of oxidative damage to HaCat cells caused by UVB irradiation.

[0015] In step S1, the use of organic membranes can remove small molecules such as monosaccharides and oligosaccharides from the extract of sea buckthorn fruit, thereby enriching the acidic polysaccharides of sea buckthorn fruit. On the other hand, the organic membrane can concentrate the extract, thus eliminating the need to use ethanol for alcohol precipitation to enrich polysaccharides. This avoids the use of organic reagents in the above preparation method, saves production costs, and greatly improves the environmental performance of the process.

[0016] Preferably, in step S1, the solvent is an alkaline solution, selected from NaOH or KOH solution, and the molar concentration of the NaOH 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 or KOH solution is between 0.3 mol / L and 0.5 mol / L. Controlling the molar concentration of the alkaline solution in the crude extraction process of step S1 helps to coordinate with subsequent operations to maintain a high level of total polysaccharide and uronic acid content in the acidic polysaccharide of *Semen Cirsium japonicum*.

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

[0018] Preferably, in step S1, the extraction is performed 1-3 times; preferably, the extraction is performed once; preferably, the extraction is performed twice; preferably, the extraction is performed three times.

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

[0020] Preferably, in step S1, the heating temperature is 80-95℃; preferably, the temperature value can be 82℃, 84℃, 86℃, 88℃, 90℃, 92℃, 94℃, 95℃, 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 solution.

[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 an ultrafiltration membrane and a 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, or 4500 Da.

[0023] Specifically, based on the monosaccharide type and molecular weight distribution of the acidic polysaccharides formed by condensation from *Prickly pear*, the pore size of the organic membrane for retaining effective substances is specifically designed. This facilitates the simultaneous achievement of impurity separation and purification as well as the enrichment and concentration of effective substances in the crude extract. Furthermore, the organic membrane with a specific filtration precision is well-matched to the size of the target molecules of the acidic polysaccharides from *Prickly pear*. If the pore size of the organic membrane is too large, the enrichment of the acidic polysaccharides from *Prickly pear* cannot be achieved; if the pore size is too small, small molecules 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 the following: uniformly porous strong acid styrene-based cation exchange resin, sulfonic acid-based polystyrene-based macroporous strong acid cation exchange resin, or gel-type strong acid styrene-based cation exchange resin. The cation exchange resin may be of type JK008, LSD001, or 001×7.

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

[0026] The cation exchange resin selected in this invention can effectively adsorb charged proteins, but it has poor adsorption capacity for acidic polysaccharides. Pure water is sufficient to wash away the acidic polysaccharides from the Chinese prickly pear fruit.

[0027] Preferably, in step S2, the elution process further includes a concentration step.

[0028] Preferably, the solid content of the second purified liquid obtained after concentration is 20-30%, and more 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 macroporous weakly basic free amine anion exchange resin, highly porous weakly basic styrene-based dimethylamine anion exchange resin, macroporous weakly basic styrene-based anion exchange resin, or macroporous polyamino weakly basic anion exchange resin.

[0030] The anion exchange resin selected in this invention has good selectivity and adsorption capacity, large particle size, and high efficiency, enabling rapid separation and purification of acidic polysaccharides from *Semen Cirsium japonicum*. The anion exchange resin can be of type D900, WA30, D315, or 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 salt solution. This achieves the effect of enriching acidic polysaccharides while removing neutral polysaccharides.

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

[0034] Preferably, the concentration of the salt solution is 0.3-0.5 mol / L; more 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 is worth noting that the applicant has found that by controlling the molar concentration of the elution salt solution in step S3 to a low level, i.e., 0.3-0.5 mol / L, not only can the acidic polysaccharides of *Cynanchum paniculatum* containing fewer impurities be eluted and enriched efficiently, but the efficiency of subsequent dialysis is also improved.

[0035] Preferably, the amount of salt solution used is 4-8 BV; preferably, the amount of salt solution used 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 dialysis bag used for dialysis has a pore size of 8000-20000 Da; preferably, the dialysis bag used for dialysis has a pore size of 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, oven drying, or spray drying.

[0038] Secondly, the present invention also provides an acidic polysaccharide of *Rhizophora stylosa* prepared according to the aforementioned method for preparing acidic polysaccharides of *Rhizophora stylosa*.

[0039] Preferably, the mass percentage of galacturonic acid in the acidic polysaccharide of *Cynanchum paniculatum* is 20% to 32%, for example, 22%, 24%, 26%, 28%, 30%, etc.

[0040] Preferably, the protein content in the acidic polysaccharide of *Cynanchum paniculatum* 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 from *Prickly pear* fruit 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 of *Cynanchum paniculatum* include arabinose, galacturonic acid, galactose, glucose, rhamnose, xylose, glucuronic acid, mannose, and fucose.

[0043] Preferably, the concentration of the acidic polysaccharide from the prickly pear fruit is 0.125-2 mg / mL, its DPPH free radical scavenging rate is 40%-98%, and its protective effect against UVB photodamage to HaCat cells results in a HaCat cell survival rate of 60%-95%.

[0044] Thirdly, the present invention provides an application of the aforementioned acidic polysaccharide from *Prickly pear* fruit in skincare products that have antioxidant and anti-ultraviolet damage effects.

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

[0046] (1) This invention utilizes an organic membrane to remove small molecules such as monosaccharides and oligosaccharides, while simultaneously enriching and concentrating polysaccharides. Cation exchange resins and anion exchange resins are then used sequentially to remove pigments and proteins, thus purifying acidic polysaccharides. Through the coordinated action of the organic membrane, cation exchange resin, and anion exchange resin, multiple adsorption and separation are achieved, resulting in a product with high total polysaccharide and uronic acid content and low protein content. In the preferred embodiment, the obtained acidic polysaccharide has a total sugar content as high as 80.47-82.51%, a uronic acid content as high as 29.16-31.25%, and a protein content as low as 3.46-3.83%. It also exhibits good solubility, strong DPPH free radical scavenging ability, and effective inhibition of oxidative damage to HaCat cells from UVB irradiation.

[0047] (2) The monosaccharide composition and weight-average molecular weight of the acidic polysaccharide of *Cyprinus sativus* prepared by this invention are well-defined, and it has high biological activity.

[0048] (3) This invention uses the pomace of the prickly pear as raw material, which improves the comprehensive utilization rate of the prickly pear and avoids the waste of resources to a certain extent. At the same time, the preparation method and equipment of the acidic polysaccharide of the prickly pear used in this invention are simple and have no special requirements, which can realize industrial production. It has the characteristics of simple operation, economical cost and environmentally friendly process. Attached Figure Description

[0049] Figure 1 is an absolute molecular weight analysis diagram of the acidic polysaccharide from *Cypripedium spp.* prepared in Example 1.

[0050] Figure 2A is a liquid chromatogram of the monosaccharide composition analysis of the acidic polysaccharide from *Rhizophora stylosa* prepared in Example 1.

[0051] Figure 2B is a liquid chromatogram of a mixture of monosaccharide standards.

[0052] Figure 3 is the infrared spectrum of the acidic polysaccharide from *Cypripedium spp.* prepared in Example 1.

[0053] Figure 4 shows the DPPH free radical scavenging rate of the acidic polysaccharides from *Rhizophora stylosa* prepared with Vc, Examples 1, 4-8, 11, and Comparative Examples 1-5 at different concentrations. Detailed Implementation

[0054] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.

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

[0056] The pomace of the prickly pear is the residue obtained after physical pressing, supercritical or subcritical extraction of prickly pear oil;

[0057] The sulfonic acid-based polystyrene macroporous strong acid cation exchange resin was purchased from Xi'an Lanxiao Technology New Material Co., Ltd., model number LSD001.

[0058] The uniformly porous, strongly acidic styrene-based cation exchange resin was purchased from Xi'an Lanxiao New Material Technology Co., Ltd., model number JK008.

[0059] The gel-type strong acid styrene-based cation exchange resin was purchased from Xi'an Lanxiao Technology New Material Co., Ltd., model number 001×7.

[0060] The highly porous, weakly basic styrene-based dimethylamine anion exchange resin was purchased from Beijing Lvbaicao Technology Development Co., Ltd., model number WA30.

[0061] The macroporous weakly basic styrene-based anion exchange resin, model D900, was purchased from Beijing Solarbio Technology Co., Ltd.

[0062] The macroporous polyamino weakly basic anion exchange resin was purchased from Tianjin Xinyue Huamei Environmental Protection Technology Co., Ltd., model number D315.

[0063] The gel-type strong base styrene-based anion exchange resin was purchased from Beijing Solarbio Technology Co., Ltd., model number 201×7.

[0064] Example 1

[0065] This embodiment provides a method for preparing acidic polysaccharides from *Prickly pear* fruit, the specific steps of which are as follows:

[0066] Step S1: Mix 50g of *Cyprinus sarmentosus* pomace with 0.4mol / L NaOH aqueous solution and heat to 90℃ for extraction. The material-to-liquid ratio (i.e., the mass ratio of *Cyprinus sarmentosus* pomace to solvent) is 1:15. Extract twice, 2 hours each time. Filter the extract under reduced pressure while hot and combine the filtrates. Concentrate the filtrate through a 2500Da organic membrane to obtain the first purified extract of *Cyprinus sarmentosus* pomace.

[0067] Step S2: After the first purified liquid of the prickly pear pomace is adsorbed by the cation exchange resin LSD001, it is separated and eluted with 5BV water. The water eluent is collected and concentrated under reduced pressure at 60°C to a second purified liquid with a solid content of 25%.

[0068] Step S3: After the second purified solution is adsorbed by anion exchange resin WA30, it is separated and eluted sequentially with 5 BV water and 5 BV 0.4 mol / L NaCl aqueous solution to obtain the third purified solution.

[0069] Step S4: Collect the third purified liquid, concentrate it under reduced pressure at 60℃, dialyze it using a 10000Da dialysis bag, and freeze-dry it to obtain acidic polysaccharide from the Chinese prickly pear fruit.

[0070] Example 2

[0071] This embodiment provides a method for preparing acidic polysaccharides from *Prickly pear* fruit, the specific steps of which are as follows:

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

[0073] Step S2: After the first purified liquid of the prickly pear pomace is adsorbed by the cation exchange resin LSD001, it is separated and eluted with 5BV water. The water eluent is collected and concentrated under reduced pressure at 60°C to a second purified liquid with a solid content of 25%.

[0074] Step S3: After the second purified solution is adsorbed by anion exchange resin WA30, it is separated and eluted sequentially with 5 BV water and 5 BV 0.3 mol / L KCl aqueous solution to obtain the third purified solution;

[0075] Step S4: Collect 0.3 mol / L KCl eluent, concentrate under reduced pressure at 60℃, dialyze using a 20000 Da dialysis bag, and freeze-dry to obtain acidic polysaccharides from *Cypripedium spp.*

[0076] Example 3

[0077] This embodiment provides a method for preparing acidic polysaccharides from *Cynanchum paniculatum*. The only difference between this method and Example 1 is that in step S1, a 0.35 mol / L NaOH aqueous solution is mixed and heated to 90°C for extraction, while other operations remain unchanged.

[0078] Example 4

[0079] This embodiment provides a method for preparing acidic polysaccharides from *Cypripedium spp.*, which differs from Example 1 only in that, in step S1, a 0.8 mol / L NaOH aqueous solution is mixed and heated to 90°C for extraction, while other operations remain unchanged.

[0080] Example 5

[0081] This embodiment provides a method for preparing acidic polysaccharides from *Cynanchum paniculatum*. The only difference between this method and Example 1 is that in step S1, a 0.1 mol / L NaOH aqueous solution is mixed and heated to 90°C for extraction, while other operations remain unchanged.

[0082] Example 6

[0083] This embodiment provides a method for preparing acidic polysaccharides from *Cyprinus pungens*. The only difference between this method and Example 1 is that in step S1, the filtrate is concentrated through an 8000 Da organic membrane to obtain a concentrated *Cyprinus pungens* pomace solution, while other operations remain unchanged.

[0084] Example 7

[0085] This embodiment provides a method for preparing acidic polysaccharides from *Cynanchum paniculatum*. The only difference between this method and Example 1 is that in step S4, the concentrated water eluent is adsorbed onto anion exchange resin WA30 and then separated and eluted sequentially with 5 BV of water and 5 BV of 0.7 mol / L NaCl aqueous solution. All other operations remain unchanged.

[0086] Example 8

[0087] This embodiment provides a method for preparing acidic polysaccharides from *Cynanchum paniculatum*. The only difference between this method and Example 1 is that in step S4, the concentrated water eluent is adsorbed onto anion exchange resin WA30 and then separated and eluted sequentially with 5 BV of water and 5 BV of 0.1 mol / L NaCl aqueous solution. All other operations remain unchanged.

[0088] Example 9

[0089] This embodiment provides a method for preparing acidic polysaccharides from *Cynanchum paniculatum*. The only difference between this method and Example 1 is that in step S3, cation exchange resin JK008 is used instead of cation exchange resin LSD001 in Example 1, while other operations remain unchanged.

[0090] Example 10

[0091] This embodiment provides a method for preparing acidic polysaccharides from *Cypripedium spp.*. The only difference between this method and Example 1 is that in step S4, anion exchange resin D315 is used instead of anion exchange resin WA30 in Example 1, while other operations remain unchanged.

[0092] Example 11

[0093] This embodiment provides a method for preparing acidic polysaccharides from *Cynanchum paniculatum*. The only difference between this method and Example 1 is that in step S4, after the concentrated water eluent is adsorbed by anion exchange resin WA30, it is separated and eluted only with 5 BV 0.4 mol / L NaCl aqueous solution. All other operations remain unchanged.

[0094] Comparative Example 1

[0095] Step S1: Mix 50g of prickly pear pomace with an aqueous solution and heat to 90℃ for extraction. The material-to-liquid ratio (i.e., the mass ratio of prickly pear pomace to solvent) is 1:15. Extract twice, 2 hours each time. Filter the extract under reduced pressure while hot and combine the filtrates. Concentrate the filtrate through a 2500Da organic membrane to obtain the first purified prickly pear pomace extract.

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

[0097] Step S3: After the second purified solution is adsorbed by anion exchange resin WA30, it is separated and eluted sequentially with 5 BV water and 5 BV 0.4 mol / L NaCl aqueous solution to obtain the third purified solution.

[0098] Step S4: Collect the third purified liquid, concentrate it under reduced pressure at 60℃, dialyze it using a 10000Da dialysis bag, and freeze-dry it to obtain acidic polysaccharide from the Chinese prickly pear fruit.

[0099] Comparative Example 2

[0100] Step S1: Mix 50g of *Cyprinus sarmentosus* pomace with 0.4mol / L NaOH aqueous solution and heat to 90℃ for extraction. The material-to-liquid ratio is 1:15 (i.e., the mass ratio of *Cyprinus sarmentosus* pomace to solvent). Extract twice, 2 hours each time. Filter the extract under reduced pressure while hot and combine the filtrates. Concentrate the filtrate under reduced pressure to obtain the first purified extract of *Cyprinus sarmentosus* pomace.

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

[0102] Step S3: After the concentrated water eluent is adsorbed by anion exchange resin WA30, it is separated and eluted sequentially with 5 BV water and 5 BV 0.4 mol / L NaCl aqueous solution to obtain the third purified solution.

[0103] Step S4: Collect the third purified liquid, concentrate it under reduced pressure at 60℃, dialyze it using a 10000Da dialysis bag, and freeze-dry it to obtain acidic polysaccharide from the Chinese prickly pear fruit.

[0104] Comparative Example 3

[0105] Step S1: Mix 50g of *Cyprinus sarmentosus* pomace with 0.4mol / L NaOH aqueous solution and heat to 90℃ for extraction. The material-to-liquid ratio is 1:15 (i.e., the mass ratio of *Cyprinus sarmentosus* pomace to solvent). Extract twice, 2 hours each time. Filter the extract under reduced pressure while hot and combine the filtrates. Concentrate the filtrate through a 2500Da organic membrane to obtain the first purified extract of *Cyprinus sarmentosus* pomace.

[0106] Step S2: After the first purified liquid of the prickly pear pomace is deproteinized with Sevage reagent, it is adsorbed by anion exchange resin WA30, and then separated and eluted with 5 BV water and 5 BV 0.4 mol / L NaCl aqueous solution in sequence.

[0107] Step S3: Collect the 0.4 mol / L NaCl eluent, concentrate it under reduced pressure at 60℃, dialyze it using a 10000 Da dialysis bag, and freeze-dry it to obtain acidic polysaccharides from the fruit of *Cypripedium spp.*

[0108] Comparative Example 4

[0109] Step S1: Mix 50g of *Cyprinus sarmentosus* pomace with 0.4mol / L NaOH aqueous solution and heat to 90℃ for extraction. The material-to-liquid ratio is 1:15 (i.e., the mass ratio of *Cyprinus sarmentosus* pomace to solvent). Extract twice, 2 hours each time. Filter the extract under reduced pressure while hot and combine the filtrates. Concentrate the filtrate through a 2500Da organic membrane to obtain the first purified extract of *Cyprinus sarmentosus* pomace.

[0110] Step S2: After the first purified liquid of the prickly pear pomace is adsorbed by anion exchange resin WA30, it is separated and eluted sequentially with 5 BV water and 5 BV 0.4 mol / L NaCl aqueous solution.

[0111] Step S3: Collect the 0.4 mol / L NaCl eluent, concentrate it under reduced pressure at 60℃, dialyze it using a 10000 Da dialysis bag, and freeze-dry it to obtain acidic polysaccharides from the fruit of *Cypripedium spp.*

[0112] Comparative Example 5

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

[0114] Step S2: The first purified liquid of the prickly pear pomace was precipitated with 85% ethanol overnight. The precipitate was collected, dissolved in water, and then loaded onto anion exchange resin WA30 for adsorption. Separation and elution were carried out sequentially with 5 BV water and 5 BV 0.4 mol / L NaCl aqueous solution.

[0115] Step S3: Collect the 0.4 mol / L NaCl eluent, concentrate it under reduced pressure at 60℃, dialyze it using a 10000 Da dialysis bag, and freeze-dry it to obtain acidic polysaccharides from the fruit of *Cypripedium spp.*

[0116] Test Example 1

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

[0118] The following are the methods for determining the total polysaccharide content in acidic polysaccharides from *Cypripedium spp.*

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

[0120] (2) Determination of total polysaccharide content: 1 mL of the prepared standard solution and sample solution were sequentially added to a test tube. 0.5 mL of 5% phenol solution and 2.5 mL of concentrated sulfuric acid solution were added to initiate the reaction. The tube was gently shaken, with distilled water as a blank control. The absorbance was measured at 490 nm using an ELISA reader. A standard curve was plotted with concentration on the x-axis and absorbance on the y-axis. The absorbance of the sample solution was substituted into the standard curve y = 7.5233x - 0.057 (R² = 0.9992) to calculate the total polysaccharide content in the sample.

[0121] The following are the methods for detecting the uronic acid content in acidic polysaccharides from *Citrus reticulata*:

[0122] (1) Solution preparation: a. Preparation of borate solution: Dissolve 2.39g sodium tetraborate completely in 250mL concentrated sulfuric acid; b. Preparation of 4mol / mL aminosulfonate solution: Dissolve 38.90g aminosulfonic acid in an appropriate amount of distilled water, add saturated NaOH solution dropwise to fully dissolve the aminosulfonic acid, adjust the pH of the solution to 1.6, and then bring the volume to 100mL; c. Preparation of m-hydroxybiphenyl solution: Dissolve 0.15g m-hydroxybiphenyl in... d. Preparation of standard solution: Accurately weigh 10.04 mg of galacturonic acid, dilute to 25 mL with distilled water, dispense into portions, and store at 20℃ for later use; take 1 mL of the stock solution and dilute to 10 mL with water to prepare the standard working solution; e. Preparation of sample solution: Weigh 5 mg of the sample, dissolve in distilled water and dilute to 5 mL to prepare a 1 mg / mL sample solution.

[0123] (2) Construction 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 working standard solution into test tubes, respectively, and add distilled water to 1 mL. Add 10 μL of aminosulfonate to each tube, shake well, slowly add 5 mL of borate-sulfuric acid solution, shake well, and place in a boiling water bath for 15 min. After removal, cool to room temperature. Slowly add 80 μL of m-hydroxybiphenyl solution to each tube, shake thoroughly until the color is uniform, and place at room temperature for 30 min. Take 200 μL to a 96-well plate and measure its absorbance at 525 nm. Plot the standard curve with uronic acid concentration as the abscissa and absorbance value as the ordinate.

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

[0125] The following are the methods for detecting the protein content in acidic polysaccharides from *Cypripedium spp.*:

[0126] (1) Preparation of protein standards: Take 1.2 mL of protein standard preparation solution and add it to a tube of protein standard (30 mg BSA). After dissolving it completely, prepare a protein standard solution of 25 mg / mL. 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: Add 100μL of BCA reagent B to 5mL of BCA reagent A, mix well, and prepare 5.1mL of BCA working solution.

[0128] (3) Protein concentration detection: Add 0 μL, 1 μL, 2 μL, 4 μL, 8 μL, 12 μL, 16 μL, and 20 μL of standard to the standard wells of a 96-well plate, and add standard diluent to bring the total to 20 μL. This corresponds to standard 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 sample to the sample wells of the 96-well plate. Add 200 μL of LCA working solution to each well and incubate at 37°C for 30 min. Measure the absorbance at 562 nm using a microplate reader. Calculate the protein concentration of the sample based on the standard curve y = 1.2523x + 0.0195 (R² = 0.9992).

[0129] The total polysaccharide content, uronic acid content, and protein content of the acidic polysaccharides obtained from Examples 1-11 and Comparative Examples 1-5 were determined, and the results are shown in Table 1.

[0130] Table 1.

[0131] The test results show that:

[0132] (1) Analysis of the total polysaccharide content, uronic acid content, and protein content of the acidic polysaccharides from *Prickly pear* prepared in Examples 1-11 and Comparative Examples 1-5 shows that, in Examples 1-11, the acidic polysaccharides from Examples 1-3 and 9-10 have the best total polysaccharide and uronic acid content (total polysaccharide content not less than 80.47 wt%, uronic acid content not less than 29.16 wt%, and protein content not exceeding 3.83 wt%). Conversely, the acidic polysaccharides from Examples 4-8 and Comparative Examples 1-5 have significantly lower total polysaccharide and uronic acid content than those from Examples 1-3 and 9-10. Specifically:

[0133] Examples 9-10 respectively replace the cation exchange resin and anion exchange resin in Example 1 with equivalent substitutions. From the total polysaccharide content and uronic acid content of the acidic polysaccharides of *Cynanchum paniculatum* obtained 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 acidic polysaccharides of *Cynanchum paniculatum* of the present invention, cation exchange resin JK008 or cation exchange resin LSD001, and in step S4, anion exchange resin D315 or anion exchange resin WA30 can all be used.

[0134] In Examples 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 all exceeded the upper and lower limits of 0.3 mol / L to 0.5 mol / L in the preparation method described in this invention. Furthermore, the total polysaccharide content and uronic acid content of the acidic polysaccharide from *Cypripedium spp.* obtained in Examples 4-5 were significantly reduced. Therefore, it can be seen that the range of 0.3 mol / L to 0.5 mol / L in step S1 of the preparation method described in this invention is a relatively ideal range for the molar concentration of the NaOH aqueous solution. It can work in conjunction with other steps and operating parameters in the preparation method to maintain the total polysaccharide content and uronic acid content of the obtained acidic polysaccharide from *Cypripedium spp.* at an ideal level.

[0135] In Example 6, the pore size of the organic membrane used in step S1 of Example 1 was adjusted to 8000 Da. This pore size exceeds the upper limit of the numerical range of 800-5000 Da in the preparation method described in this invention, resulting in a pore size that is too large and poor enrichment of acidic polysaccharides from the fruit of *Rhizophora stylosa*.

[0136] In Examples 7-8, the molar concentration of the NaCl aqueous solution in the eluent of step S4 in 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 is 0.7 mol / L, which exceeds the upper limit 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 obtained *Cynanchum paniculatum* acidic polysaccharide are significantly lower than those in Examples 1-3. When the molar concentration of the NaCl aqueous solution in the eluent is 0.1 mol / L, which is lower than the lower limit 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 obtained *Cynanchum paniculatum* acidic polysaccharide are still significantly lower than those in Examples 1-3.

[0137] In Example 11, the total polysaccharide content and uronic acid content of the acidic polysaccharide from *Rhizophora stylosa* were significantly lower than those in Examples 1-3, except that the step S4, in which "the concentrated water eluent was adsorbed by anion exchange resin WA30 and then separated and eluted sequentially with 5 BV water and 5 BV 0.4 mol / L NaCl aqueous solution", was replaced with "the concentrated water eluent was adsorbed by anion exchange resin WA30 and then separated and eluted only with 5 BV 0.4 mol / L NaCl aqueous solution".

[0138] As can be seen from the comparison of Examples 1-3, 9-10 with Comparative Examples 1-5, the present application uses alkaline solution extraction, organic membrane to concentrate the extract, and cation exchange resin to remove protein. Compared with pure water extraction, alcohol precipitation and organic reagent sevage method, it can effectively increase the content of total polysaccharides and uronic acid in the acidic polysaccharides of *Cynanchum paniculatum*, and the obtained protein content is lower.

[0139] Test Example 2

[0140] Determination of the molecular weight range of acidic polysaccharides from *Cypripedium spp.*

[0141] The molecular weight of the acidic polysaccharide from *Cyprinus sativus* prepared in Example 1 was determined using the following method:

[0142] The acidic polysaccharide sample of *Cynanchum paniculatum* prepared in Example 1 was dissolved in a 0.1 mol / L NaNO3 aqueous solution (containing 0.02% NaN3, w / w) to a final concentration of 1 mg / mL, and then filtered through a 0.45 μm pore size filter membrane before testing.

[0143] The chromatographic system employed a gel chromatography-differential chromatography-multi-angle laser light scattering system. The liquid chromatography system was a U3000 (Thermo, USA), the differential detector was an Optilab T-rEX (Wyatt Technology, CA, USA), and the laser light scattering detector was a DAWN HELEOS II (Wyatt Technology, CA, USA). Size exclusion columns Ohpak SB-805HQ (300×8 mm) and Ohpak SB-803HQ (300×8 mm) were used in series. The column temperature was 45℃, the injection volume was 100 μL, the mobile phase was A (0.02% NaN3, 0.1 M NaNO3), the flow rate was 0.6 mL / min, and isocratic elution was performed for 75 min.

[0144] Chromatographic data were processed using ASTRA 6.1 software, as shown in Figure 1. The absolute molecular weight analysis chromatogram uses retention time (time, min) as the x-axis and molar mass (g / mol) as the y-axis. The red line represents the multi-angle laser light scattering signal (LS, V), the blue line represents the difference signal (RI, RIU), and the black line is the molecular weight obtained by fitting the two signals. Analysis showed that the molecular weight (Mw) of the acidic polysaccharide prepared in Example 1 was 29.39 kDa.

[0145] Test Example 3

[0146] Determination of monosaccharide composition of acidic polysaccharides from *Cypripedium spp.*

[0147] The monosaccharide composition of the acidic polysaccharide from *Cyprinus sativus* prepared in Example 1 was determined using the following method:

[0148] (1) Hydrolysis reaction: Trifluoroacetic acid (TFA) was used to hydrolyze the acidic polysaccharide of Prickly pear obtained in Example 1. 5 mg of Prickly pear acidic polysaccharide sample was accurately weighed, 2 mTFA (2 mol / L) was added, and hydrolysis was carried out at 110 °C for 2 h. After complete hydrolysis, the sample was dried by a nitrogen blower to completely remove TFA.

[0149] (2) Derivatization reaction: Derivatization was performed using 1-phenyl-3-methyl-5-pyrazolone (PMP) reagent. Quantitative amounts of D-glucose, D-galactose, D-mannose, D-xylose, L-rhamnose, L-arabinose, D-fructose, D-galacturonic acid, D-glucuronic acid standards and acidic polysaccharide (Example 1) hydrolyzed samples were added to 0.5 mL of PMP (0.5 mol / L) reagent and 0.5 mL of NaOH (0.3 mol / L) solution. The mixture was reacted at 70 °C for 30 min. After cooling, 0.5 mL of HCl (0.3 mol / L) and 0.5 mL of distilled water were added for neutralization. An equal volume of chloroform solution was added for extraction by shaking. The supernatant was collected and filtered through a 0.22 μM filter membrane for testing.

[0150] (3) High-performance liquid chromatography (HPLC): The derivatized samples were qualitatively and quantitatively analyzed 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 performed: 0-3 min: 15% B, 3-4 min: 15-18% B, 4-10 min: 18% B, 10-25 min: 18-40% B; column type: Agilent ZORBAX SB-C18, 4.6×150 mm; flow rate: 0.8 mL / min; column temperature: 30℃; DAD: 250 nm; injection volume: 5 μL.

[0151] As shown in Figures 2A and 2B, the monosaccharide composition of the acidic polysaccharide from *Cynanchum paniculatum* in Example 1 is as follows: 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%), and fucose (0.05%).

[0152] Test Example 4

[0153] Infrared spectroscopy test

[0154] The acidic polysaccharide from *Prickly pear* fruit prepared in Example 1 was subjected to infrared spectroscopy testing. The testing method is as follows:

[0155] Take 5 mg of the acidic polysaccharide from *Cypripedium spp.* prepared in Example 1, compress it into tablets using KBr, and analyze the results using a Nicolet 5700 infrared spectrometer (4000-400 cm⁻¹). -1 Infrared scanning. As shown in Figure 3, the IR spectrum is at 3417 cm⁻¹. -1 A strong and broad absorption peak appears, which is the strong absorption peak of the OH stretching vibration on the polysaccharide, indicating that hydrogen bonds exist both intramolecularly and intermolecularly in the polysaccharide; 1651 cm⁻¹ -1 The absorption peak at [location] is the carbonyl vibration peak of uronic acid, further indicating that the polysaccharide sample of Example 1 contains uronic acid. See Table 2.

[0156] Table 2.

[0157] Test Example 5

[0158] DPPH radical scavenging rate test

[0159] The DPPH free radical scavenging rate of the acidic polysaccharides from *Prickly pear* prepared in Examples 1, 4-8, 11, and Comparative Examples 1-5 was tested using the following methods:

[0160] Take 0.5 mL of different concentrations of acidic polysaccharides from *Cynanchum aizoon* in a test tube, add 1.5 mL of distilled water and 2 mL of DPPH (0.1 mmol / L) ethanol solution, mix well, and react at room temperature in the dark for 30 min. Measure the absorbance at 517 nm. Calculate the DPPH free radical scavenging rate of the test sample according to the following formula: Scavenging rate = [1 - (A...] 样品 -A 空白样品 ) / (A 对照 -A 空白对照 )]×100%

[0161] In the formula: A 样品 The absorbance value was measured by adding 150 μL of sample solution and 150 μL of DPPH alcohol solution; A 空白样品 The absorbance value measured by adding 150 μL of sample solution and 150 μL of anhydrous ethanol; A 对照 The absorbance value was measured by adding 150 μL of DPPH solution and 150 μL of sample solvent; A 空白对照 The absorbance value was measured by adding 150 μL of anhydrous ethanol and 150 μL of sample solvent.

[0162] IC50 of DPPH free radical scavenging rate of acidic polysaccharides from *Rhizophora stylosa* prepared in Examples 1, 4-8, 11, and Comparative Examples 1-5 50 The values ​​are shown in Table 3. The IC50 value of the DPPH free radical scavenging rate of the acidic polysaccharide from *Prickly pear* prepared in Example 1 can be easily obtained. 50The IC50 value of the DPPH free radical scavenging rate of the acidic polysaccharide from *Rhizopus chinensis* prepared in Examples 4-8, 11 and Comparative Examples 1-5 was significantly better than that of the polysaccharide prepared in Examples 4-8 and 11 and Comparative Examples 1-5. 50 Value. This invention employs multiple adsorption and separation processes using organic membranes, cation exchange resins, and anion exchange resins to prepare acidic polysaccharides from *Rhizophora stylosa*, which exhibit stronger DPPH free radical scavenging capabilities.

[0163] Table 3.

[0164] Test Example 6

[0165] Test of the protective effect of HaCat cells against UVB photodamage

[0166] The protective effect of the acidic polysaccharides from *Rhizophora stylosa* prepared in Examples 1, 4-8, 11, and Comparative Examples 1-5 against UVB photodamage in HaCat cells was tested. The test methods are as follows:

[0167] This test was divided into a blank control group, a UVB control group, and an experimental group. The test method for the experimental group was as follows: HaCat cells in the logarithmic growth phase were used at a density of 1.0 × 10⁶ cells per well. 4 Cells were cultured in 96-well plates with 10% FBSDMEM medium at 37°C and 5% CO2 for 24 hours. After complete cell adhesion, the cells were washed 1-2 times with PBS, then the medium was replaced with 10% FBSDMEM medium containing *Cynanchum paniculatum* acidic polysaccharide, and cultured at 37°C and 5% CO2 for 2 hours. The medium was then replaced with PBS containing *Cynanchum paniculatum* acidic polysaccharide, and the cells were irradiated with UVB at 30 mJ / cm². 2 After the initial dose, the medium was replaced with 10% FBSDMEM and cultured at 37℃ with 5% CO2 for 24 hours. The viability of HaCat cells was then detected using the CCK8 assay. The blank control group was prepared as follows: HaCat cells in logarithmic growth phase were used at a density of 1.0 × 10⁻⁶ cells per well. 4 HaCat cells were cultured in 96-well plates with 10% FBSDMEM medium at 37°C and 5% CO2 for 24 hours. After complete cell attachment, the cells were washed 1-2 times with PBS, replaced with 10% FBSDMEM medium, and cultured at 37°C and 5% CO2 for another 24 hours. The viability of HaCat cells was then assessed using the CCK8 assay. The UVB control group was prepared as follows: HaCat cells in logarithmic growth phase were cultured at a density of 1.0 × 10⁶ cells per well. 4 Cells were cultured in 96-well plates with 10% FBSDMEM medium at 37°C and 5% CO2 for 24 hours. After complete cell adhesion, the cells were washed 1-2 times with PBS, and then the medium was replaced with fresh 10% FBSDMEM medium. The cells were then cultured at 37°C and 5% CO2 for 2 hours. Finally, the medium was replaced with PBS, and the cells were irradiated with UVB light at 30 mJ / cm². 2After the initial dose, the medium was replaced with fresh 10% FBSDMEM medium and cultured at 37°C with 5% CO2 for 24 hours. HaCat cell viability was then assessed using the CCK8 assay. The results are shown in Table 4.

[0168] Table 4.

[0169] It is readily apparent that the protective effect of the acidic polysaccharide of *Cyprinus sativus* prepared in Example 1 against UVB light damage in HaCat cells is significantly better than that of the acidic polysaccharides of *Cyprinus sativus* prepared in Examples 4-8, 11 and Comparative Examples 1-5 against UVB light damage in HaCat cells.

[0170] The applicant declares that the above description is only a specific embodiment 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 in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A method for preparing acidic polysaccharides from *Prickly pear* fruit, characterized in that, The preparation method includes the following steps: Step S1: Mix the pomace of the sea buckthorn fruit with a solvent and heat to extract crude extract. Concentrate the crude extract through an organic membrane to obtain the first purified extract. Step S2: The first purified solution obtained in step S1 is adsorbed and eluted by a cation exchange resin to obtain the second purified solution; Step S3: The second purified solution obtained in step S2 is adsorbed and eluted by anion exchange resin to obtain the third purified solution; Step S4: The third purified liquid obtained in step S3 is concentrated, dialyzed, and dried to obtain the acidic polysaccharide of *Cyprinus pungens*.

2. The preparation method according to claim 1, characterized in that, In step S1, the mass ratio of the prickly ash fruit residue to the solvent is 1:(10-20); the solvent is an alkaline solution, which is selected from NaOH solution or KOH solution, and the molar concentration of the NaOH solution or KOH solution is 0.3mol / L to 0.5mol / L; the extraction is performed 1-3 times. The crude extract is concentrated using an organic membrane, and the pH of the concentrated solution is adjusted with acetic acid until pH=7, thus obtaining the first purified solution.

3. The preparation method according to claim 1 or 2, characterized in that, In step S1, the pore size of the organic membrane for retaining effective substances is 800-5000 Da; the organic membrane includes ultrafiltration membranes and nanofiltration membranes.

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 uniformly porous strong acid styrene-based cation exchange resin, sulfonic acid polystyrene-based macroporous strong acid cation exchange resin, or gel-type strong acid styrene-based cation exchange resin. The solvent used for elution includes water; wherein, in step S2, a concentration step is further included after elution; The solid content of the second purified solution 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 macroporous weakly basic free amine anion exchange resin, highly porous weakly basic styrene-based dimethylamine anion exchange resin, macroporous weakly basic styrene-based anion exchange resin, or macroporous polyamino weakly basic anion exchange resin. The solvents used for elution include water and salt solutions; The elution method includes sequential elution with water and salt solution.

6. The preparation method according to claim 5, characterized in that, The salt solution comprises an aqueous solution with one or more of NaCl, KCl, MgCl2, and Na2SO4 as solutes; wherein, The concentration of the salt solution is 0.3-0.5 mol / L; the amount of the salt solution used is 4-8 BV.

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

8. An acidic polysaccharide of *Prickly pear* fruit prepared by the method according to any one of claims 1-7; Based on the mass of galacturonic acid, the mass percentage of galacturonic acid in the acidic polysaccharide of *Cynanchum paniculatum* is 20% to 32%. The protein content of the acidic polysaccharide from the prickly pear fruit is no higher than 4%. The weight-average molecular weight of the acidic polysaccharide from *Cyprinus sativus* is 20-40 kDa. The monosaccharides in the acidic polysaccharide of *Corydalis yanhusuo* include arabinose, galacturonic acid, galactose, glucose, rhamnose, xylose, glucuronic acid, mannose, and fucose.

9. The acidic polysaccharide from *Prickly pear* fruit according to claim 8, characterized in that, The concentration of the acidic polysaccharide from the prickly pear fruit is 0.125-2 mg / mL. Its DPPH free radical scavenging rate is 40%-98%, and its protective effect against UVB photodamage to HaCat cells results in a HaCat cell survival rate of 60%-95%.

10. The application of the acidic polysaccharide of *Prickly pear* fruit according to claim 8 or 9 in a skin care product with antioxidant and anti-ultraviolet damage effects.

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