Rattan tea selenium polysaccharide IV, extraction method thereof and application thereof

Abstract

The present application relates to the field of biological extraction, and particularly to a kind of ampelopsis grossedentata selenium polysaccharide IV and its extraction method and its application. The present application is extracted by boiling water, impurities are removed by ethanol precipitation, lipid is removed by petroleum ether, and is redissolved by ammonia water. The supernatant is subjected to polyamide column chromatography to remove color, the eluate is subjected to gel column chromatography to remove impurities, and finally, the refined ampelopsis grossedentata selenium polysaccharide IV is obtained by concentration and freeze-drying. The present application has the advantages of simple process, low production cost, high yield and purity of ampelopsis grossedentata selenium polysaccharide IV. The ampelopsis grossedentata selenium polysaccharide IV prepared can be used to prepare functional food for preventing high uric acid, has good liver and kidney protection effect, has no damage to normal cells, and can be taken for a long time.

Description

Technical Field

[0001] This invention relates to the field of bio-extraction, specifically to a vine tea selenium polysaccharide IV, its extraction method, and its applications. Background Technology

[0002] According to the "Gout Report White Paper (2021)," the overall incidence of hyperuricemia (HUA) in my country has reached 13.3%, representing approximately 177 million people. HUA has become a global public health problem, ranking as the "fourth highest" after hypertension, diabetes, and hyperlipidemia, and its incidence is trending towards younger ages. Some existing chemically synthesized uric acid-lowering drugs on the market have been proven to have toxic side effects, such as abnormal liver function, diarrhea and abdominal pain, epidermal telangiectasia, rashes, cardiovascular problems, and allergic reactions; in severe cases, it can even lead to death. Therefore, the need to develop highly effective and safe functional foods for lowering uric acid is urgent. Plant-derived natural selenium polysaccharides possess diverse and bioactive pharmacophores with specific spatial and electronic properties, making them suitable for the research and development of functional food factors for preventing and lowering uric acid. The following are reports on the effects of plant-derived selenium polysaccharides and polysaccharides on lowering uric acid: Li Tingting et al. [2] found that crude polysaccharide of the rare medicinal fungus Sanghuangporus can promote uric acid excretion and alleviate kidney pathological damage by activating the ABCG2 transporter protein in the kidney; Wang Shiqi [3] found that polysaccharide of Inonotus obliquus can reduce the uric acid level in mice and can significantly alleviate the damage to the kidney caused by high uric acid; Guo Min [4] found that licorice polysaccharide can reduce the concentration of uric acid in the body and alleviate liver and kidney pathological damage by inhibiting XOD activity and hindering uric acid synthesis.

[0003] Vine tea belongs to the genus Ampelopsis in the family Vitaceae. The plant name is Ampelopsis grossedentata (Hand-Mazz) WTWang. It is a vine plant and is a new food resource approved by the National Health Commission of the People's Republic of China in 2013. Vine tea is an important characteristic economic crop in Southwest my country, mainly produced in mountainous provinces such as Hubei, Hunan, Guangdong, Guangxi, and Jiangxi. Vine tea is rich in flavonoids and selenium polysaccharides. Modern pharmacological studies have proven that its flavonoid components have the effect of lowering uric acid. However, there are currently no reports on the uric acid-lowering effect of selenium polysaccharides from the new food resource vine tea. There are only studies on the in vitro antibacterial effect of vine tea selenium polysaccharides (Zhang Haiqing, Zeng Hong, Yang Hongtao, et al. Study on the in vitro antibacterial effect of Enshi vine tea selenium polysaccharides [J]. Trace Elements and Health Research, 2009, 26(02):12-14.), but no process technology for preparing selenium polysaccharides from vine tea is involved. Currently, there is no application of selenium polysaccharide IV, derived from vine tea, which has high uric acid-lowering efficacy and low toxicity, in the preparation of functional food factors for preventing hyperuricemia.

[0004] On the other hand, existing technologies have disclosed some studies on extracting polysaccharides from traditional Chinese medicinal herbs such as *Vanilla rotundifolia*, *Inonotus obliquus*, and licorice for uric acid reduction, but the following problems still exist: First, the target population for polysaccharides derived from traditional Chinese medicinal herbs is limited; second, the extracted polysaccharides do not contain selenium. Selenium, as one of the essential trace elements for the human body, has preventative and health-promoting effects such as lowering blood sugar, anti-aging, and improving human immunity. Therefore, selenium-enriched polysaccharides from food plant resources have unique advantages. Third, when traditional purification processes are used to extract polysaccharides from vine tea, they are subject to interference from impurities such as plant polyphenols, flavonoids, pigments, and nucleic acids, resulting in low yields and purity of vine tea selenium polysaccharide IV compounds, and even the potential for extracting other substances that could cause toxic side effects on normal cells. Summary of the Invention

[0005] The purpose of this invention is to solve the above-mentioned technical problems and provide a method for extracting selenium polysaccharide IV from selenium-rich vine tea that is simple in process, low in production cost, and has a high yield and purity of selenium polysaccharide IV compound.

[0006] Another objective of this invention is to provide a vine tea selenium polysaccharide IV extracted by the above method, which has significant in vivo uric acid-lowering activity and no toxic side effects on normal cells.

[0007] Another object of the present invention is to provide an application of vine tea selenium polysaccharide IV.

[0008] To achieve the above objectives, the technical solution provided by this invention is as follows:

[0009] A method for extracting selenium polysaccharide IV from selenium-enriched vine tea includes the following steps:

[0010] 1) Boiling water extraction: Grind the selenium-enriched vine tea into powder using a pulverizer; extract the selenium-enriched vine tea powder by reflux in a boiling water bath, remove impurities using a 0.45-2μm hollow fiber ultrafiltration membrane, and collect the filtrate.

[0011] 2) Ethanol precipitation for impurity removal: Add ethanol solution to the filtrate and stir, control the final concentration to 80-85% (volume percentage) of ethanol solution, then let it stand to precipitate, centrifuge to collect the precipitate and freeze dry to obtain resin;

[0012] 3) Petroleum ether degreasing: The dried resin was degreased and eluted by adding petroleum ether, and then vacuum dried to obtain crude selenium polysaccharide;

[0013] 4) Redissolution with ammonia: Redissolve the crude selenium polysaccharide with 10% (w / w) ammonia and stir until homogeneous. Then allow it to stand to precipitate and collect the supernatant solution by centrifugation.

[0014] 5) Decolorize the supernatant by polyamide column chromatography: elute with deionized water, 1-5% ammonia, and 10-15% ammonia by mass, and collect the 15% ammonia eluent as the target eluent.

[0015] 6) The eluent was purified by gel column chromatography: eluted with 10-15% ammonia water, the volume ratio of the target eluent to ammonia water was 1:100, and 1 / 5 of the total elution volume was collected as a fraction. The fourth fraction collected was the target eluent. It was concentrated and freeze-dried to obtain the purified vine tea selenium polysaccharide IV.

[0016] Preferably, in step 1), the organic selenium content in the selenium-enriched vine tea is ≥2 mg / kg.

[0017] In step 1), the boiling water bath reflux extraction temperature is 98℃ and the time is 1.5-2.0 h.

[0018] Preferably, in step 2), the centrifugation speed is 5000-6000 rpm / min.

[0019] Preferably, in step 3), the ratio of petroleum ether to liquid in the elution is 1 / 100 to 2 / 100.

[0020] Preferably, in step 4), the ratio of ammonia to liquid for redissolving is 1 / 400 to 2.5 / 400, and the centrifugation speed is 5000-6000 rpm / min.

[0021] A type of selenium polysaccharide IV from vine tea was prepared by the above method.

[0022] Preferably, the vine tea selenium polysaccharide IV is obtained by linking glucose, fructose, galactose, mannose, fucose, xylose, rhamnose, glucuronic acid, and ribose through glycosidic bonds, with a molecular weight range of 14,000-16,000; wherein, the content of monosaccharides after hydrolysis accounts for the following mass percentage of the total sugar content of vine tea selenium polysaccharide IV: glucose 50-55.47%, fructose 10-12.48%, galactose 4-4.81%, mannose 2-3.17%, fucose 2-3.49%, xylose 1.58-2.58%, rhamnose 0.98-1.37%, glucuronic acid 1.07-1.27%, and ribose 0.56-1.12%.

[0023] Se is bound to monosaccharide molecules via Se-OC and Se-SC, with the preferred organic selenium content being 0.4-1 mg / kg.

[0024] To address the problems existing in the background technology, the inventors made the following improvements:

[0025] 1) Boiling water extraction is employed, allowing for direct application in large-scale production. High-temperature denaturation of biomolecules such as proteins facilitates subsequent impurity removal, offering advantages such as simple operation and suitability for industrial production. Hollow fiber ultrafiltration membranes are selected for impurity removal, effectively removing biomolecules such as DNA and proteins, preventing their precipitation during subsequent ethanol precipitation of polysaccharides. The pore size of the hollow fiber ultrafiltration membrane should be controlled between 0.45 and 2 μm to effectively filter solid particulate impurities, macromolecular proteins, and polymers formed by nucleic acid denaturation. A pore size that is too large will reduce filtration efficiency, causing these impurities to permeate the membrane, affecting subsequent purification processes. A pore size that is too small will increase production costs, easily clog the membrane, and reduce production efficiency.

[0026] 2) After filtration to remove impurities, select 80-85% ethanol for precipitation. The polarity of the selected solvent is between that of ethanol and water. This is the polarity range of most polyphenols, flavonoids, pigments and other impurities. Based on the principle of like dissolves like, it can effectively dissolve most polyphenols, flavonoids, pigments and other impurities. Then, it can be removed in the supernatant solution by centrifugation.

[0027] 3) In the petroleum ether degreasing step, petroleum ether is creatively selected to extract the freeze-dried crude selenium polysaccharide. This is because petroleum ether has weak polarity and penetrability, which allows it to effectively dissolve lipids within the crude selenium polysaccharide. Simultaneously, petroleum ether exhibits good inertness, does not react with polysaccharides, has a low boiling point, and good volatility, facilitating subsequent removal. When used for degreasing crude selenium polysaccharide, it specifically dissolves lipids but does not dissolve highly polar substances within the polysaccharide. Therefore, it requires a smaller amount compared to other traditional degreasing solvents, effectively eliminating interference from water molecules, ensuring more thorough contact between the solvent and the substance, and effectively dissolving lipids, denatured proteins, and other large molecules, thus facilitating subsequent impurity removal. The preferred material-to-liquid ratio (g / ml) is 1 / 100 to 2 / 100. A ratio that is too high will lead to insufficient removal of lipid impurities, affecting the purity of the polysaccharide; a ratio that is too low will waste solvent and increase production costs.

[0028] 4) In the ammonia resolution step, 10% (by mass) of ammonia is initially selected to resolute the crude polysaccharide. At this point, some proteins denature in the strongly alkaline solution, becoming insoluble in water and precipitating out. Secondly, ammonia treatment alters the solution's pH and ionic strength, while avoiding the cumbersome desalination process in traditional methods. The preferred material-to-liquid ratio (g / ml) is 1 / 400 to 2.5 / 400. A ratio that is too high will prevent sufficient resolution of the polysaccharide, leaving it in the precipitate and affecting the final polysaccharide yield. A ratio that is too low will waste solvent, resulting in a low polysaccharide concentration in the solution, which is detrimental to subsequent concentration processing and increases production costs.

[0029] 5) During the decolorization and impurity removal process of polyamide (63428-83-1, 300-400 mesh) column chromatography, deionized water elution, 1-5% ammonia elution, and 10-15% ammonia elution are selected sequentially. Among them, deionized water elution can effectively remove the neutral polysaccharide components of inactive ingredients, and the strong alkalinity of ammonia can effectively elute and purify the target acidic polysaccharide components. DNA and some proteins undergo denaturation and cleavage in the strong alkaline solution to form small molecules, which are convenient for removal by gel chromatography in the later stage. Secondly, ammonia treatment can change the solution's pH and ionic strength, and also avoid the cumbersome desalting steps in the later stage of the traditional process.

[0030] 6) Finally, during the gel chromatography (Sephadex G-25) process, the fourth component was collected as the target eluent. It was concentrated and freeze-dried, and the yield of the final tea selenium polysaccharide IV was ≥5%, with a purity of ≥98%. Se element was bound to the monosaccharide molecules through Se-OC and Se-SC. Under the premise of effective impurity removal, organic selenium was retained to the maximum extent, and the organic selenium content was 0.4-1 mg / kg.

[0031] Further research revealed that the extracted vine tea selenium polysaccharide IV is composed of glucose, fructose, galactose, mannose, fucose, xylose, rhamnose, glucuronic acid, and ribose linked by glycosidic bonds, with a molecular weight ranging from 14,000 to 16,000. This vine tea selenium polysaccharide IV compound exhibits significant effects in lowering blood uric acid and alleviating kidney damage caused by hyperuricemia. It also possesses certain kidney-protective effects during the uric acid-lowering process and has no toxic side effects on the proximal convoluted tubule epithelial cells (HK-2) of normal human renal cortex, making it suitable for long-term use.

[0032] Based on the above findings, the daily intake of the vine tea selenium polysaccharide IV prepared by this invention, after conversion, is only 4.81-19.23g for a 70kg adult. It can be added with other excipients to make any dosage form that is acceptable in food and pharmaceutical terms, such as solid beverages, instant tea, canned liquid beverages, powders, etc.

[0033] Beneficial effects:

[0034] The present invention has a simple process, low production cost, and high yield and purity of vine tea selenium polysaccharide IV. The prepared vine tea selenium polysaccharide IV has good liver and kidney protection effects, does not harm normal cells, and can be taken for a long time. Attached Figure Description

[0035] Figure 1 This is a morphological diagram of selenium polysaccharide IV from vine tea.

[0036] Figure 2 IVLC-MS particle flow map of selenium polysaccharide from vine tea;

[0037] Figure 3This is a bar chart comparing the effect of *Dendrobium nobile* selenium polysaccharide IV (DMY) prepared in Example 1 on serum uric acid levels (mg / L) in mice. Values ​​are the mean ± SD (n=8), *, p < 0.05, compared with the control group.

[0038] Figure 4 This is a bar chart comparing the effect of selenium polysaccharide IV from *Tea japonica* prepared in Example 1 on the relative expression level of the OAT1 gene in mouse kidney tissue; where, values ​​are mean ± SD (n=8), *, p < 0.05, compared with blank.

[0039] Figure 5 The image below shows the effect of selenium polysaccharide IV from vine tea prepared in Example 1 on mouse kidney tissue (HE staining). Detailed Implementation

[0040] Example 1

[0041] Boiling water extraction: Take selenium-rich vine tea (from Tongcheba Vine Tea Professional Cooperative, Xianfeng County, with organic selenium content ≥2 mg / kg), pulverize it with a grinder, and pass it through a 60-mesh sieve to obtain selenium-rich vine tea powder. Take 100 g of selenium-rich vine tea powder and extract it by reflux in a boiling water bath at 98℃ for 1.5 h, with a pure water-to-liquid ratio (g / ml) of 1 / 50; remove impurities using a 0.45~2μm hollow fiber ultrafiltration membrane, with the flow rate controlled at 0.5 L / min, and collect the filtrate.

[0042] Ethanol precipitation for impurity removal: Add ethanol solution to the filtrate, stir at 1000 rpm / min, control the final concentration to 85% (v / v) ethanol solution, and let it stand for precipitation for 4 h; collect the precipitate by centrifugation at 6000 rpm / min and freeze-dry to obtain resin.

[0043] Petroleum ether degreasing: The dried resin was degreased and eluted with petroleum ether (analytical grade) at a mass-volume ratio (g / ml) of 1 / 100, and then vacuum dried to obtain crude selenium polysaccharide.

[0044] Redissolve the crude selenium polysaccharide in 10% ammonia water at a material-to-liquid ratio of 1 / 400 (g / ml), stir at 1000 rpm for 2 h, then let it stand to precipitate for 4 h; centrifuge at 6000 rpm to collect the supernatant solution.

[0045] Polyamide column chromatography decolorization: elution was performed using deionized water, 5% (w / w) ammonia solution, and 15% (w / w) ammonia solution, respectively. The 15% (w / w) ammonia solution eluent was collected as the target eluent.

[0046] Gel column chromatography for impurity removal: Elution with 15% ammonia water at a volume ratio of 1:100. One fraction was collected as 1 / 5 of the total elution volume. The fourth fraction collected was the target eluent. The eluent was concentrated and freeze-dried to obtain purified vine tea selenium polysaccharide IV.

[0047] See Figure 1 As can be seen, the selenium polysaccharide IV from vine tea is an opaque milky white color and has no odor. Figure 2 The nine main ion peaks of selenium polysaccharide IV from *Tea vine* were observed as follows: glucose (Rt 1.046 min), fructose (Rt 7.616 min), galactose (Rt 8.384 min), mannose (Rt 9.723 min), fucose (Rt 10.816 min), xylose (Rt 11.232 min), rhamnose (Rt 19.692 min), glucuronic acid (Rt 24.997 min), and ribose (Rt 27.668 min).

[0048] The yield of *Selenium tsao-ko* polysaccharide IV obtained by this process was 5.2%, with a purity of 98.4%. *Selenium tsao-ko* polysaccharide IV is a heterozygous polysaccharide IV with a molecular weight range of 14,000-16,000. The monosaccharide content (mass percentage) after hydrolysis, as a percentage of the total sugar content of *Selenium tsao-ko* polysaccharide IV, is as follows: glucose 55.47%, fructose 11.28%, galactose 4.17%, mannose 2.67%, fucose 2.81%, xylose 2.08%, rhamnose 0.99%, glucuronic acid 1.12%, and ribose 0.81%, linked by glycosidic bonds. The organic selenium content of selenium polysaccharide IV is 0.56 mg / kg.

[0049] Example 2

[0050] Boiling water extraction: Take selenium-rich vine tea (from Tongcheba Vine Tea Professional Cooperative, Xianfeng County, with organic selenium content ≥2 mg / kg), pulverize it with a grinder, and pass it through a 60-mesh sieve to obtain selenium-rich vine tea powder. Take 100 g of selenium-rich vine tea powder and extract it by reflux in a boiling water bath at 98℃ for 1.5 h, with a pure water-to-liquid ratio of 1 / 50; remove impurities using a 0.45–2 μm hollow fiber ultrafiltration membrane, with the flow rate controlled at 0.5 L / min, and collect the filtrate.

[0051] Ethanol precipitation for impurity removal: Add ethanol solution to the filtrate, stir at 1000 rpm / min, control the final concentration to 85% ethanol solution, and let it stand for precipitation for 4 h; collect the precipitate by centrifugation at 5000 rpm / min and freeze-dry to obtain resin.

[0052] Petroleum ether degreasing: The dried resin was degreased and eluted with petroleum ether (analytical grade) at a mass-volume ratio (g / ml) of 2 / 100, and then vacuum dried to obtain crude selenium polysaccharide.

[0053] Redissolve the crude selenium polysaccharide with 10% ammonia at a material-to-liquid ratio of 1 / 400, stirring at 1000 rpm for 2 h; then allow it to settle for 4 h; finally, centrifuge at 5000 rpm to collect the supernatant.

[0054] Polyamide column chromatography decolorization: elution was performed with deionized water, 1% ammonia, and 10% ammonia, respectively. The 15% ammonia eluent was collected as the target eluent.

[0055] Gel column chromatography for impurity removal: elution with 15% ammonia water, with a volume ratio of eluent to ammonia water of 1:100. One fraction was collected as 1 / 5 of the total elution volume. The fourth fraction collected was the target eluent, which was concentrated and freeze-dried to obtain purified vine tea selenium polysaccharide IV.

[0056] The yield of *Selenium tsao-ko* polysaccharide IV obtained by this process was 5.5%, with a purity of 98.7%. *Selenium tsao-ko* polysaccharide IV is a heterozygous polysaccharide with a molecular weight range of 14,000-16,000. The monosaccharide content (mass percentage) after hydrolysis, as a percentage of the total sugar content of *Selenium tsao-ko* polysaccharide IV, is as follows: glucose 52.37%, fructose 11.21%, galactose 4.56%, mannose 2.99%, fucose 3.11%, xylose 1.69%, rhamnose 1.22%, glucuronic acid 1.15%, and ribose 0.71%, linked by glycosidic bonds. The organic selenium content of selenium polysaccharide IV is 0.57 mg / kg.

[0057] Example 3

[0058] Boiling water extraction: Take selenium-rich vine tea (from Tongcheba Vine Tea Professional Cooperative, Xianfeng County, with organic selenium content ≥2 mg / kg), pulverize it with a grinder, and pass it through a 60-mesh sieve to obtain selenium-rich vine tea powder. Take 100 g of selenium-rich vine tea powder and extract it by reflux in a boiling water bath at 98℃ for 2 hours, with a pure water-to-liquid ratio of 1 / 50; remove impurities using a 0.45–2 μm hollow fiber ultrafiltration membrane, with the flow rate controlled at 0.5 L / min, and collect the filtrate.

[0059] Ethanol precipitation for impurity removal: Add ethanol solution to the filtrate, stir at 1000 rpm / min, control the final concentration to 80% ethanol solution, and let it stand for precipitation for 4 h; collect the precipitate by centrifugation at 6000 rpm / min and freeze-dry to obtain resin.

[0060] Petroleum ether degreasing: The dried resin was degreased and eluted with petroleum ether (analytical grade) at a mass-volume ratio of 1.5 / 100, and then vacuum dried to obtain crude selenium polysaccharide.

[0061] Redissolve the crude selenium polysaccharide with 10% ammonia at a material-to-liquid ratio of 1 / 400, stirring at 1000 rpm for 2 h; then allow it to settle for 4 h; centrifuge at 5000-6000 rpm to collect the supernatant.

[0062] Polyamide column chromatography decolorization: elution was performed with deionized water, 3% ammonia, and 12% ammonia, respectively. The 15% ammonia eluent was collected as the target eluent.

[0063] Gel column chromatography for impurity removal: elution with 10% ammonia water, with a volume ratio of eluent to ammonia water of 1:100. One fraction was collected as 1 / 5 of the total elution volume. The fourth fraction collected was the target eluent, which was concentrated and freeze-dried to obtain purified vine tea selenium polysaccharide IV.

[0064] The yield of *Selenium tsao-ko* polysaccharide IV obtained by this process was 5.8%, with a purity of 99.1%. *Selenium tsao-ko* polysaccharide IV is a heterozygous polysaccharide with a molecular weight range of 14,000-16,000. The monosaccharide content (mass percentage) after hydrolysis, as a percentage of the total sugar content of *Selenium tsao-ko* polysaccharide IV, is as follows: glucose 52.67%, fructose 11.58%, galactose 4.25%, mannose 3.05%, fucose 2.81%, xylose 2.49%, rhamnose 1.15%, glucuronic acid 1.09%, and ribose 0.88%, linked by glycosidic bonds. The organic selenium content of *Selenium tsao-ko* polysaccharide IV is 0.45 mg / kg.

[0065] Example 4

[0066] Boiling water extraction: Take selenium-rich vine tea (from Tongcheba Vine Tea Professional Cooperative, Xianfeng County, with organic selenium content ≥2 mg / kg), pulverize it with a grinder, and pass it through a 60-mesh sieve to obtain selenium-rich vine tea powder. Take 100 g of selenium-rich vine tea powder and extract it by reflux in a boiling water bath at 98℃ for 1.5 h, with a pure water-to-liquid ratio of 1 / 50; remove impurities using a 0.45–2 μm hollow fiber ultrafiltration membrane, with the flow rate controlled at 0.5 L / min, and collect the filtrate.

[0067] Ethanol precipitation for impurity removal: Add ethanol solution to the filtrate, stir at 1000 rpm / min, control the final concentration to 85% ethanol solution, and let it stand for precipitation for 4 h; collect the precipitate by centrifugation at 5000 rpm / min and freeze-dry to obtain resin.

[0068] Petroleum ether degreasing: The dried resin was degreased and eluted with petroleum ether (analytical grade) at a mass-volume ratio of 1.5 / 100, and then vacuum dried to obtain crude selenium polysaccharide.

[0069] Redissolve the crude selenium polysaccharide with 10% ammonia at a material-to-liquid ratio of 2 / 400, stirring at 1000 rpm for 2 h; then allow it to settle for 4 h; finally, centrifuge at 6000 rpm to collect the supernatant.

[0070] Polyamide column chromatography decolorization: elution was performed using deionized water, 5% ammonia, and 15% ammonia, respectively. The 15% ammonia eluent was collected as the target eluent.

[0071] Gel column chromatography for impurity removal: elution with 10% ammonia water, with a volume ratio of eluent to ammonia water of 1:100. One fraction was collected as 1 / 5 of the total elution volume. The fourth fraction collected was the target eluent, which was concentrated and freeze-dried to obtain purified vine tea selenium polysaccharide IV.

[0072] The yield of *Caulis Chinensis* selenium polysaccharide IV obtained by this process was 5.45%, with a purity of 96%. *Caulis Chinensis* selenium polysaccharide IV is a heterozygous polysaccharide IV with a molecular weight range of 14,000-16,000. The monosaccharide content (mass percentage) after hydrolysis, as a percentage of the total sugar content of *Caulis Chinensis* selenium polysaccharide IV, is as follows: glucose 52.68%, fructose 10.39%, galactose 4.18%, mannose 2.91%, fucose 3.55%, xylose 2.19%, rhamnose 1.28%, glucuronic acid 1.26%, and ribose 1.00%, linked by glycosidic bonds. The organic selenium content of selenium polysaccharide IV is 0.52 mg / kg.

[0073] Example 5

[0074] Boiling water extraction: Take selenium-rich vine tea (from Tongcheba Vine Tea Professional Cooperative, Xianfeng County, with organic selenium content ≥2 mg / kg), pulverize it with a grinder, and pass it through a 60-mesh sieve to obtain selenium-rich vine tea powder. Take 100 g of selenium-rich vine tea powder and extract it by reflux in a boiling water bath at 98℃ for 1.5 h, with a pure water-to-liquid ratio of 1 / 50; remove impurities using a 0.45–2 μm hollow fiber ultrafiltration membrane, with the flow rate controlled at 0.5 L / min, and collect the filtrate.

[0075] Ethanol precipitation for impurity removal: Add ethanol solution to the filtrate, stir at 1000 rpm / min, control the final concentration to 80% ethanol solution, and let it stand for 4 hours to precipitate; collect the precipitate by centrifugation at 6000 rpm / min and freeze-dry to obtain resin.

[0076] Petroleum ether degreasing: The dried resin was degreased and eluted with petroleum ether (analytical grade) at a mass-volume ratio of 1.5 / 100, and then vacuum dried to obtain crude selenium polysaccharide.

[0077] Redissolve the crude selenium polysaccharide with 10% ammonia water: Redissolve the crude selenium polysaccharide with 10% ammonia water at a material-to-liquid ratio of 2.5 / 400, stir at 1000 rpm / min for 2 h; then let it stand to precipitate for 4 h; centrifuge at 5000-6000 rpm / min to collect the supernatant solution.

[0078] Polyamide column chromatography decolorization: elution was performed using deionized water, 5% ammonia, and 15% ammonia, respectively. The 15% ammonia eluent was collected as the target eluent.

[0079] Gel column chromatography for impurity removal: elution with 15% ammonia water, with a volume ratio of eluent to ammonia water of 1:100. One fraction was collected as 1 / 5 of the total elution volume. The fourth fraction collected was the target eluent, which was concentrated and freeze-dried to obtain purified vine tea selenium polysaccharide IV.

[0080] The yield of *Caulis Chinensis* selenium polysaccharide IV obtained by this process was 5.4%, with a purity of 98.15%. *Caulis Chinensis* selenium polysaccharide IV is a heterozygous polysaccharide IV with a molecular weight range of 14,000-16,000. The monosaccharide content (mass percentage) after hydrolysis, as a percentage of the total sugar content of *Caulis Chinensis* selenium polysaccharide IV, is as follows: glucose 53.84%, fructose 10.61%, galactose 4.33%, mannose 2.82%, fucose 3.11%, xylose 1.97%, rhamnose 1.16%, glucuronic acid 1.05%, and ribose 1.02%, linked by glycosidic bonds. The organic selenium content of selenium polysaccharide IV is 0.81 mg / kg.

[0081] Example 6

[0082] Take 3.0 g of the vine tea selenium polysaccharide IV obtained in Example 1, and add 0.10 g each of kudzu root, papaya, broccoli, green tea, polygonatum, and tomato extracts (the extracts are polysaccharides and flavonoids), 6.50 g of sucrose, 0.20 g of DL-malic acid, and 3.00 g of β-cyclodextrin. After mixing, spray dry to obtain a dry powder, bag and irradiate sterilize to obtain a selenium-enriched solid beverage product. Under this process, based on the fuzzy mathematical sensory evaluation method and response surface methodology, the sensory score of the selenium-enriched solid beverage product reached 91.85±0.35 points. Instructions for use: Pour 2 g of this product into a cup, add 100-150 mL of boiling water, stir thoroughly to dissolve, and drink.

[0083] Experimental Example 1

[0084] Efficacy test of refined vine tea selenium polysaccharide IV (obtained in Example 1) in lowering uric acid in mice with hyperuricemia.

[0085] Forty-eight male Kunming mice, weighing 18 - 22 g, were purchased from Hubei Provincial Center for Experimental Animals, with the license number: SCXK (E) 2020 - 0018. The animal house was kept well-ventilated, with 12 h of lighting per day, a temperature of 23 - 27 °C, and a humidity of 60% - 70%. After 1 week of adaptive feeding of the mice, potassium cyanate was administered by gavage and yeast extract was injected to establish a hyperuricemia mouse model. Blood was collected from the tail vein 1 h after the first modeling to detect the blood uric acid level of the mice. The mice were evenly divided into six groups according to their uric acid levels. The experiment was carried out according to Table 1 for 21 days. During the experiment, potassium oxonate and hypoxanthine were administered by gavage to the mice every day to build the HUA model. After 1 h interval, allopurinol was administered by gavage to the positive group, and extracts at different concentrations were administered by gavage to the three sample groups. The blank and HUA groups were administered sodium carboxymethyl cellulose to exclude the influence of the solvent. On the 28th day, the mice were fasted for 12 h before the last administration, and then the modeling and drug administration treatment operations were carried out. 1 h after administration, the mice were weighed, blood was collected by eye puncture, and gross dissection was performed to collect the liver and kidneys.

[0086] Table 1 Experimental design of modeling and drug treatment

[0087]

[0088] Pathohistological analysis was performed on the liver and kidney tissues of the above mice to judge the protective effect of the samples on the liver and kidney tissues. The liver of the mice was weighed and homogenized with 0.9% normal saline (tissue: normal saline = 1:9) in an ice bath. The qPCR method was used to analyze the up-regulation or down-regulation of the expression of related genes OAT1 and to detect and identify them. Total RNA was extracted from kidney tissues using Trizol reagent. The total RNA samples were treated with DNase I to remove genomic DNA contamination. To quantify OAT1 the expression level of genes in tissues, primers were designed for them and reverse transcription was performed to synthesize cDNA, and then real-time quantitative polymerase chain reaction (qRT-PCR) was carried out. The formula for the relative expression ratio is: n-fold transcription = 2 -△△Ct , △△Ct = △Ct (drug treatment) / △Ct (untreated), where △Ct represents the difference between the cycle threshold (Ct) of the gene under study and the Ct of the internal control GAPDH gene, and the t-test was used to analyze the results and the influence of the transcriptional expression of related target genes.

[0089] After 21 days of modeling, HPLC was used to detect various physiological indexes of the mice. The experimental results showed that the serum uric acid content of the mice in the refined Ampelopsis grossedentata selenium polysaccharide IV treatment groups (12.5, 25, 50 mg / kg·d) was significantly lower than that of the model group ( Figure 3Furthermore, the dosage of selenium polysaccharide IV (SPHIV) showed a positive correlation with its uric acid-lowering effect, preliminarily demonstrating that SPHIV has a uric acid-lowering effect in vivo. Organic anion transporter 1 (OAT1) is a transporter protein containing 12 transmembrane domains. OAT1 is mainly expressed on the basement membrane of the proximal convoluted tubule of the kidney, responsible for transporting uric acid from the renal tubular capillaries to the renal tubular epithelial cells, where it plays a role in uric acid secretion. OAT1 Functional defects and decreased expression of the OAT1 gene can lead to elevated serum uric acid levels. Studies have demonstrated a link between OAT1 gene mutations and familial gouty nephropathy in young adults. Compared to the control group, the hyperuricemia model group (HUA) showed... OAT1 Gene expression levels were significantly reduced. Figure 4 This indicates that the kidney damage in the hyperuricemia model group (HUA) resulted in poor renal uric acid secretion, leading to its accumulation in serum and the formation of hyperuricemia. Selenium polysaccharide IV can significantly promote [the development of] ... OAT1 The transcriptional expression of the gene promotes the renal secretion of uric acid and reduces serum uric acid levels. Selenium polysaccharide IV exhibits a dose-response relationship with the expression of the OAT1 gene; the highest concentration dose, group H (50 mg / kg·d), showed a significantly greater effect on [the expression of the OAT1 gene]. OAT1 Gene expression has returned to normal levels. Furthermore, the positive control drug allopurinol significantly inhibited [gene expression]. OAT1 Gene expression indicates that, unlike the positive control drug allopurinol, selenium polysaccharide IV can reduce uric acid levels in the body by promoting uric acid excretion.

[0090] Experimental Example 2:

[0091] Anatomical analysis of organs, serum parameters, and pathological examination of liver tissue sections in mice with hyperuricemia treated with drugs (obtained in Example 1).

[0092] A biochemical analyzer was used to detect alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine, and urea; liver and kidneys were dissected, embedded in paraffin, stained with hematoxylin and eosin (HE), sectioned, and subjected to panoramic scanning to observe tissue condition.

[0093] The liver tissue of mice in the hyperuricemia model group treated with refined vine tea selenium polysaccharide IV appeared reddish-brown, soft and elastic, with neat edges. The effects of refined vine tea selenium polysaccharide IV on the body weight and liver weight of the hyperuricemia model mice are shown in Table 2. There were no significant differences in body weight, liver size, and liver / body weight ratio between the control group and the refined vine tea selenium polysaccharide IV treatment group, preliminarily indicating that refined vine tea selenium polysaccharide IV had no significant effect on the liver. However, the positive drug treatment group (AP) showed a significant reduction in body weight, indicating that it has strong toxic side effects; therefore, its use should be carefully considered. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are mainly found in hepatocytes. When hepatocytes are damaged due to inflammation, necrosis, or poisoning, transaminases are released into the bloodstream, causing elevated serum transaminase levels. Therefore, they are important indicators for detecting liver health in medicine. The ALT and AST values ​​in the hyperuricemia model group (HUA) were significantly higher than those in the control group, indicating that hyperuricemia causes liver damage. The mean ALT and AST values ​​of mice treated with refined vine tea selenium polysaccharide IV were significantly lower than those of the blank group, model group, and AP group, which fully demonstrates that refined vine tea selenium polysaccharide IV can alleviate liver damage caused by high uric acid and has a significant liver-protective effect.

[0094] See Figure 5 In the group treated with refined vine tea selenium polysaccharide IV, the renal tissues (glomeruli and tubules) of hyperuricemia model mice showed intact structure and clear cell staining. The corticomedullary boundary was distinct, with glomeruli concentrated in the cortex and fewer in the medulla. The glomerular boundaries were clear, and the direction of capillaries within the glomeruli could be observed. The tubular lumens were clearly defined, with no inflammatory cell infiltration or mesangial stromal cell proliferation, and no atrophy or dilation. The proximal and distal convoluted tubules were clearly distinguishable; the proximal tubules exhibited a star-shaped radial pattern, while the distal convoluted tubules were mostly elliptical or round. The effects of refined vine tea selenium polysaccharide IV on body weight and kidney weight in hyperuricemia model mice are shown in Table 2. There were no significant differences in body weight, kidney size, and kidney / body weight ratio between the control group and the group treated with refined vine tea selenium polysaccharide IV, suggesting that dihydromyricetin has no significant effect on the kidneys. An abnormally high creatinine level indicates impaired kidney filtration function, while an abnormally high urea level suggests potential kidney damage, excessive protein breakdown, or other diseases. Therefore, these two parameters are commonly used in medicine to reflect kidney health. There were no significant differences in serum creatinine and urea levels between the hyperuricemia model group (HUA) and the blank control group, indicating that hyperuricemia has a relatively small impact on kidney function. The serum creatinine and urea levels of mice treated with different doses of refined vine tea selenium polysaccharide IV were not significantly different from those in the blank and model groups, indicating that refined vine tea selenium polysaccharide IV does not have nephrotoxicity. The serum creatinine and urea levels in the positive control group (allopurine) were ≥200% higher than other experimental groups, demonstrating its strong nephrotoxicity. It is speculated that the significant weight loss in the positive control group is directly related to kidney damage.

[0095] Table 2. Effects of the active ingredient, selenium polysaccharide IV from vine tea, on liver and kidney indicators in hyperuricemia model mice.

[0096]

[0097] Note: Lowercase letters in the same column indicate significant differences.

[0098] Experimental Example 3

[0099] Efficacy test of refined vine tea selenium polysaccharide IV (obtained in Example 1) against HK-2 cytotoxic activity in normal human renal cortical proximal tubular epithelial cells.

[0100] When the HK-2 cell density reached 85%, the cells were digested with 1% trypsin until they became rounded, and then cell culture medium was added to stop the digestion. The cell suspension was appropriately diluted and seeded into 96-well plates at a density of 100 μL per well, with 5000 cells per well. The cells were incubated at 37 ℃ and 5% CO2 for 24 h. The discarded culture medium was discarded, and 100 μL of fresh culture medium containing the test drug was added. The final concentrations of each drug were 12.5, 25.0, 50.0, 100.0, 200.0, 400.0, and 800.0 μg / mL, respectively. The control group consisted of an equal volume of fresh culture medium, and cell-free cell culture medium served as the negative control group. Each concentration was tested in triplicate. The cells were incubated again under the same conditions for 24 h. Cell growth was observed and photographed every 6 h using an inverted microscope.

[0101] Cells were then treated with CCK-8 reagent, with 10 μL of CCK-8 reagent added to each well and incubated at 37 °C for 1 hour. Cell viability was calculated by reading the absorbance at 450 nm using a microplate reader. The calculation formula is shown below:

[0102]

[0103] In the formula: A0 - negative group: cellless, with culture medium and CCK-8 added; A1 - sample group: containing cells, with sample, culture medium and CCK-8 added; A2 - control group: containing cells, with culture medium and CCK-8 added.

[0104] After incubating HK-2 cells with purified vine tea selenium polysaccharide IV for 24 h, the inhibition rate of normal HK-2 cells was 0.18% at a concentration of 400 μg / mL and -0.48% at a concentration of 800 μg / mL.

[0105] Comparative Example 1:

[0106] Compared with Example 1, the difference is that only crude selenium polysaccharide was prepared, and it was used to conduct uric acid-lowering efficacy tests in hyperuricemic mice and cytotoxicity tests in normal HK-2 cells.

[0107] As shown in Table 3, after feeding mice with high uric acid with crude selenium polysaccharide for 21 days, there was no significant decrease in serum uric acid levels as the concentration of the drug increased.

[0108] As shown in Table 4, after incubating normal HK-2 cells with crude selenium polysaccharide for 24 hours, the cell survival rate decreased significantly with increasing drug concentration, proving that the crude selenium polysaccharide has significant toxic side effects on normal HK-2 cells.

[0109] Comparative Example 2:

[0110] Compared with Example 1, the difference is that in Example 1, after boiling water reflux, the extract was "purified by a 0.45-2μm hollow fiber ultrafiltration membrane, with the flow rate controlled at 0.5 L / min, and the filtrate was collected" was changed to centrifugation at 5000-6000 rpm / min to collect the supernatant solution. The rest is the same as in Example 1, and vine tea selenium polysaccharide IV was obtained.

[0111] The yield of selenium polysaccharide IV compound obtained by this process was only 4.17%, and the purity was only 85.12%, which was significantly lower than the yield and purity of selenium polysaccharide obtained by the process of this invention.

[0112] It was then used in a uric acid-lowering efficacy test in mice with high uric acid levels.

[0113] As shown in Table 3, after feeding mice with high uric acid with crude selenium polysaccharide for 21 days, there was no significant decrease in serum uric acid levels as the concentration of the drug increased.

[0114] It was then used in cytotoxicity assays on normal HK-2 cells.

[0115] As shown in Table 4, after incubating normal HK-2 cells with crude selenium polysaccharide for 24 hours, the cell survival rate decreased significantly with increasing drug concentration, proving that the crude selenium polysaccharide has significant toxic side effects on normal HK-2 cells.

[0116] Comparative Example 3:

[0117] Compared with Example 1, the difference is that "the crude selenium polysaccharide was re-dissolved in 10% ammonia water at a material-to-liquid ratio of 1 / 400" was changed to re-dissolved in deionized water.

[0118] The yield of selenium polysaccharide IV compound obtained by this process was only 4.27%, and the purity was only 84.78%, which was significantly lower than the yield and purity of selenium polysaccharide obtained by the process of this invention.

[0119] Comparative Example 4:

[0120] Compared with Example 1, the difference is that "gel column chromatography for impurity removal: elution with 15% ammonia water, collection of 1 / 5 of the total elution volume as a fraction, and the fourth fraction collected is the target eluent" is changed to elution with deionized water.

[0121] The yield of selenium polysaccharide IV compound obtained by this process is only 3.48%, and the purity is only 87.18%, which is significantly lower than the yield and purity of selenium polysaccharide obtained by the process of this invention.

[0122] It was then used in a uric acid-lowering efficacy test in mice with high uric acid levels.

[0123] As shown in Table 3, after feeding mice with high uric acid with crude selenium polysaccharide for 21 days, there was no significant decrease in serum uric acid levels as the concentration of the drug increased.

[0124] It was then used in cytotoxicity assays on normal HK-2 cells.

[0125] As shown in Table 4, after incubating normal HK-2 cells with crude selenium polysaccharide for 24 hours, the cell survival rate decreased significantly with increasing drug concentration, proving that the crude selenium polysaccharide has significant toxic side effects on normal HK-2 cells.

[0126]

[0127] Note: Lowercase letters in the same column indicate significant differences.

[0128] Table 4. Cytotoxic activity of crude selenium polysaccharide extract

[0129]

[0130] Note: Lowercase letters in the same column indicate significant differences.

Claims

1. A method for extracting selenium polysaccharide IV from selenium-enriched vine tea, characterized in that, Includes the following steps: 1) Boiling water extraction: Grind the selenium-enriched vine tea into powder using a pulverizer; extract the selenium-enriched vine tea powder by reflux in a boiling water bath, remove impurities using a 0.45-2μm hollow fiber ultrafiltration membrane, and collect the filtrate. 2) Ethanol precipitation for impurity removal: Add ethanol solution to the filtrate and stir, control the final concentration to 80-85% (volume percentage) of ethanol solution, then let it stand to precipitate, centrifuge to collect the precipitate and freeze dry to obtain resin; 3) Petroleum ether degreasing: The dried resin was degreased and eluted by adding petroleum ether, and then vacuum dried to obtain crude selenium polysaccharide; 4) Redissolution with ammonia: Redissolve the crude selenium polysaccharide with 10% (w / w) ammonia and stir until homogeneous. Then allow it to stand to precipitate and collect the supernatant solution by centrifugation. 5) Decolorize the supernatant by polyamide column chromatography: elute with deionized water, 1-5% ammonia solution, and 10-15% ammonia solution respectively, and collect the 15% ammonia solution eluent as the target eluent. 6) The eluent was purified by gel column chromatography: eluted with 10-15% ammonia water, the volume ratio of the target eluent to ammonia water was 1:100, and 1 / 5 of the total elution volume was collected as a fraction. The fourth fraction collected was the target eluent. It was concentrated and freeze-dried to obtain the purified vine tea selenium polysaccharide IV.

2. The method for extracting selenium polysaccharide IV from selenium-enriched vine tea as described in claim 1, characterized in that, In step 1), the organic selenium content in the selenium-enriched vine tea is ≥2 mg / kg.

3. The method for extracting selenium polysaccharide IV from selenium-enriched vine tea as described in claim 1 or 2, characterized in that, In step 1), the boiling water bath reflux extraction temperature is 98℃ and the time is 1.5-2.0 h.

4. The method for extracting selenium polysaccharide IV from selenium-enriched vine tea as described in claim 1, characterized in that, In step 2), the centrifugation speed is 5000-6000 rpm / min.

5. The method for extracting selenium polysaccharide IV from selenium-enriched vine tea as described in claim 1, characterized in that, In step 3), the ratio of petroleum ether to liquid in the elution solution is 1 / 100 to 2 / 100.

6. The method for extracting selenium polysaccharide IV from selenium-enriched vine tea as described in claim 1, characterized in that, In step 4), the ratio of ammonia to liquid for redissolving is 1 / 400 to 2.5 / 400, and the centrifugation speed is 5000-6000 rpm / min.

7. A vine tea selenium polysaccharide IV, characterized in that, It is prepared by any one of the methods of claims 1-6.

8. The vine tea selenium polysaccharide IV as described in claim 7, characterized in that, It is composed of glucose, fructose, galactose, mannose, fucose, xylose, rhamnose, glucuronic acid, and ribose linked by glycosidic bonds, with a molecular weight range of 14,000-16,000. The mass percentage of monosaccharides in the total sugar content of vine tea selenium polysaccharide IV after hydrolysis is as follows: glucose 50-55.47%, fructose 10-12.48%, galactose 4-4.81%, mannose 2-3.17%, fucose 2-3.49%, xylose 1.58-2.58%, rhamnose 0.98-1.37%, glucuronic acid 1.07-1.27%, and ribose 0.56-1.12%.

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