A selenium flavone iii of a vine tea and an extraction method and application thereof

Abstract

The present application relates to the field of biological extraction, and particularly relates to a kind of rattan tea selenium flavone III and its extraction method and application, the method includes boiling water extraction, macroporous resin purification, normal phase chromatography impurity removal, gel chromatography column chromatography impurity removal process, finally concentrated, spray drying treatment, as refined rattan tea selenium flavone III.The present application has simple process, low production cost, high rattan tea selenium polysaccharide IV compound yield and purity, the rattan tea selenium flavone III prepared has excellent anticancer activity to cancer cell, and also has no toxic side effects to normal lung epithelial cells, can be made into any dosage form acceptable in food and pharmacy.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological extraction, in particular to a kind of rattan tea selenium flavone III and its extraction method and application. BACKGROUND

[0002] Natural selenium flavone of plant origin has diversity and bioactive pharmacophore, with specific space and electronic characteristics, which can be used for the development of anticancer drugs and functional food factors.

[0003] The prior art discloses some processes and process equipment inventions for extracting selenium flavone from medicinal plants such as rhizoma polygonati, broccoli and kudzu root, as well as the role in inhibiting oxidative stress inflammatory response. The patent "Method for inhibiting oxidative stress inflammatory response by selenium flavone" (CN202211714760.X) only relates to a method for inhibiting oxidative stress inflammatory response by selenium flavone; the patent "Quantitative packaging device for selenium flavone composition" (CN202220175398.2) only relates to a quantitative packaging device for selenium flavone composition; the patent "Processing structure for extracting selenium flavone from fruits and vegetables" (CN202021355154.X) only relates to a processing structure for extracting selenium flavone from fruits and vegetables; the patent "Selenium flavone composition and preparation method thereof" (CN202010665880.X) only relates to a selenium flavone composition and a preparation method thereof; the patent "Leaching equipment and process for leaching selenium flavone from rhizoma polygonati" (CN201911057712.6) only relates to a leaching equipment and process for leaching selenium flavone from rhizoma polygonati. When traditional process is used to extract selenium flavone, the extraction of flavone is interfered by impurities such as polyphenol compounds, polysaccharides, pigments and lipids in plants, resulting in low yield and purity, and even the problem of toxic and side effects on normal cells caused by the extraction of other substances.

[0004] In addition, current chemotherapy is the main means for treating lung cancer, but the specific recognition of existing chemotherapy drugs needs to be improved, and it is difficult to avoid killing normal cells under the premise of killing cancer cells, so the industry needs an anticancer functional food factor that can specifically kill cancer cells without damaging normal cells.

[0005] However, there is no report on the research of selenium flavone from new food resource rattan tea, and there is no application of plant-derived selenium flavone with high anticancer efficacy and low toxicity in the preparation of functional food factors for lung cancer SUMMARY

[0006] The purpose of the present application is to solve the above technical problems, and to provide an extraction method of selenium flavone III in selenium-rich rattan tea with simple process, low production cost, high yield and purity of rattan tea selenium polysaccharide IV compound.

[0007] Another object of the present application is to provide a selenium flavone III extracted from the above method, which has no toxic side effects on normal cells.

[0008] Another object of the present application is to provide an application of the selenium flavone III.

[0009] To achieve the above object, the technical scheme provided by the present application is as follows:

[0010] A method for extracting selenium flavone III from selenium-rich Ampelopsis grossedentata, comprising the following steps:

[0011] 1) Boiling water extraction: The selenium-rich Ampelopsis grossedentata is ground by a powder machine to obtain selenium-rich Ampelopsis grossedentata powder. The selenium-rich Ampelopsis grossedentata powder is extracted by boiling water bath reflux. The impurities are removed by a 0.22-0.45 μm hollow fiber ultrafiltration membrane, and the filtrate is collected.

[0012] 2) Macroporous resin purification: The ADS-17 macroporous adsorption resin is mixed with the collected filtrate for resin enrichment. The macroporous adsorption resin after enrichment is filtered and collected, and then dried until the weight becomes constant. The dried resin is washed and eluted with petroleum ether, followed by elution with 80-85% ethanol solution and collection of the eluate. The eluate is centrifuged, and the supernatant is concentrated to obtain an extraction infusion, i.e., crude selenium flavone of Ampelopsis grossedentata.

[0013] 3) Impurity removal by normal phase chromatography: H normal phase silica gel is used to purify the extraction infusion, followed by defatting elution with dichloromethane, and then elution with ethanol solution and collection of the eluate.

[0014] 4) Impurity removal by gel chromatography column chromatography: The eluate is purified by passing through a gel chromatography column using an ethanol solution as the target eluent. The eluent is eluted with 80% volume percentage ethanol solution, and the eluate is collected in groups at a ratio of 1:100 of eluate to ethanol solution, with 1 / 4 of the total elution volume collected in each group. The third group is the target eluate, which is concentrated, spray-dried, and processed to obtain refined selenium flavone III of Ampelopsis grossedentata.

[0015] In step 1), the organic selenium content in the selenium-rich Ampelopsis grossedentata is ≥1 mg / kg.

[0016] Preferably, in step 1), the boiling water reflux extraction is performed at atmospheric pressure, a temperature ≥98℃, a time of 1.5-2.0 h, and a pure water to feed liquid ratio of 1 / 50-2 / 50 g / mL.

[0017] Preferably, the flow rate of the hollow fiber ultrafiltration membrane for impurity removal is 0.3-0.5 L / min.

[0018] Preferably, in step 2), the feed liquid ratio for resin enrichment is 1 / 100-2.5 / 100, and the hot air drying temperature is 35-40℃.

[0019] Preferably, in the step 2), the petroleum ether degreasing elution liquid ratio is 1 / 50-2.5 / 50, and the ethanol solution elution liquid ratio is 1 / 100-2.5 / 100.

[0020] Preferably, in the step 3), the purification is carried out by 300-400 mesh H normal phase silica gel, the dichloromethane degreasing elution liquid ratio is 1 / 100-2.5 / 100, and the ethanol solution elution liquid ratio is 1 / 100-2.5 / 100.

[0021] The fuzhuan selenium flavone III is prepared by the above method.

[0022] Preferably, the fuzhuan selenium flavone III contains, by mass percentage, at least 86-89% of dihydromyricetin, 1-1.5% of myricetin, 0.5-0.9% of taxifolin, 0.3-0.6% of myricitrin, 0.1-0.4% of quercetin, and ≥0.30 mg / g of organic selenium.

[0023] A fuzhuan selenium flavone III for use in the preparation of a functional food for resisting lung cancer.

[0024] In view of the problems in the background art, the inventors have made the following improvements:

[0025] 1) Boiling water extraction can be directly used in large-scale production practice, and high-temperature variable proteins and other biological macromolecules are beneficial to later impurity removal, having the advantages of simple operation and being conducive to industrialized production. The use of boiling water as the extraction solvent has the following advantages: (1) the main flavonoid active ingredients in fuzhuan tea, such as dihydromyricetin and myricetin, contain a large number of hydroxyl groups and have high polarity, and have high solubility in water; (2) the higher the temperature of boiling water, the more effective the dissolution of flavonoids, and the higher the extraction efficiency; (3) water as the extraction solvent is cheaper, more environmentally friendly, and has lower production cost than other organic solvents. A liquid ratio of 1 / 50-2 / 50 g / mL of pure water is too high, which can cause insufficient dissolution of flavonoids, the active ingredients of selenium-rich fuzhuan tea, and waste of raw materials; and a too low ratio can make it difficult to recover flavonoids, the active ingredients, from the aqueous solution, increasing the production cost.

[0026] The hollow fiber ultrafiltration membrane is selected to remove impurities, and solid impurity particles, denatured and insoluble biological macromolecular DNA, protein and other impurities are effectively removed. Compared with the prior art, the pollution of solid impurity particles in the extraction liquid to the macroporous resin filler is reduced, the efficiency of the macroporous resin in adsorbing and enriching organic selenium flavones is improved, the service life of the macroporous resin is greatly improved, and the production cost is effectively reduced. The pore size of the hollow fiber ultrafiltration membrane is controlled to be 0.22-0.45 μm, so that the impurities in the backflow extraction liquid of the vine tea can be effectively removed. The impurities in the backflow extraction liquid of the vine tea mainly include vine tea tissue fragments, cell fragments, DNA, denatured and insoluble polymers of protein macromolecules, insoluble polysaccharides, starch and the like. The diameters of more than 90% of the impurities are greater than 0.45 μm. If the pore size of the ultrafiltration membrane is too large, many impurities cannot be effectively filtered out, the membrane separation efficiency is reduced, and if the pore size is too small, a larger pressure needs to be provided to filter out the required flavone active ingredients, and the filter membrane is easily blocked, which seriously affects the service life of the hollow fiber ultrafiltration membrane and increases the production cost. If the flow rate is too high, a larger pressure needs to be provided, which seriously affects the service life of the hollow fiber ultrafiltration membrane and increases the production cost; and if the flow rate is too low, the production efficiency is reduced, and the production cost is also increased.

[0027] 2) The ADS-17 resin is enriched by selecting a liquid-solid ratio of 1 / 100-2.5 / 100. The liquid-solid ratio of the macroporous resin for enrichment is accurately controlled. If the proportion of the macroporous resin is too small, the enrichment and recovery of the organic selenium flavones are insufficient, the raw materials are wasted, and the total flavone yield is not high; and if the proportion of the macroporous resin is too high, the impurities in the enrichment are also increased, and the production cost is increased.

[0028] 3) In the prior art, the enriched resin is first eluted and then defatted. However, the elution solvent has a great interference with the petroleum ether defatting, and therefore, the dried resin is defatted and eluted with the petroleum ether, so that the defatting solvent and the target flavone substance are more fully contacted, the lipid substances, denatured proteins and other macromolecular substances are more effectively dissolved, and the later impurity removal is more facilitated. Meanwhile, the solvent consumption is less than that in the traditional defatting, and the interference of the elution solvent molecules is effectively eliminated. The liquid-solid ratio of the petroleum ether defatting and elution is preferably 1 / 50-2.5 / 50. Too much liquid-solid ratio leads to insufficient removal of lipid impurities, affects the purity of the selenium flavones, and too little liquid-solid ratio wastes the solvent and increases the difficulty of the solvent removal and the production cost. The elution is performed with 80-85% ethanol, and the selected solvent polarity is between that of ethanol and methanol. The polarity of most flavone compounds is within the range, and the design is based on the principle of similar dissolves similar. The selected solvent can effectively dissolve most flavone compounds, elute the flavone compounds adsorbed on the resin, and improve the selenium flavone yield.

[0029] 4) 300-400 mesh H normal phase silica gel has a strong adsorption capacity for highly polar compounds such as polysaccharides, exhibiting a very good separation effect. Moreover, the separation material is more affordable and applicable than other materials such as reversed phase silica gel and gel, which can effectively reduce its production cost.

[0030] 5) Using dichloromethane with a material-to-liquid ratio of 1 / 100 to 2.5 / 100 as elution can effectively remove lipid-soluble pigments of inactive ingredients. Using ethanol solution with a material-to-liquid ratio of 1 / 100 to 2.5 / 100 can efficiently elute the target substance of selenium flavonoids. This allows highly polar polysaccharides and other impurities to remain on the silica gel for separation, effectively removing both highly polar and weakly polar compounds such as lipids and polysaccharides, which facilitates the removal of large and small molecule impurities by gel chromatography in the later stages.

[0031] The vine tea selenium flavonoid III compound of this invention exhibits anticancer active ingredient concentrations of 38-43% against lung cancer cells A549 and LA-795 at 400 μg / mL, and 50-55% against lung cancer cells A549 and LA-795 at 800 μg / mL. Furthermore, the vine tea selenium flavonoid III compound exhibits 0% inhibition rate against normal lung cells BEAS-2B at concentrations of 0-800 μg / mL.

[0032] Beneficial effects:

[0033] This invention employs multiple methods to effectively remove small molecules such as tannins, polyphenols, and lipids, as well as large molecular impurities such as polysaccharides, pigments, and nucleic acids. It utilizes both polarity and molecular weight as indicators to effectively remove impurities from the target selenium flavonoids, while simultaneously maximizing the retention of organic selenium elements. This results in the effective enrichment of selenium in the obtained selenium flavonoid III, with a yield of ≥30% and a total flavonoid content of ≥95%. The described selenium flavonoid III compound exhibits excellent anticancer activity against human and mouse lung cancer cells, while simultaneously showing no toxic side effects on normal lung epithelial cells. It can be formulated into any dosage form acceptable to food and pharmaceutical manufacturers, such as solid beverages, instant tea, canned liquid beverages, and powders. Attached Figure Description

[0034] Figure 1 This is a diagram showing the properties of flavonoid III;

[0035] Figure 2 Here is the HPLC chromatogram of selenoflavone III;

[0036] Figure 3 A thermographic comparative analysis of the anticancer activity of selenium flavonoids in vine tea;

[0037] Figure 4 A schematic diagram illustrating the anticancer activity of selenium flavonoids in vine tea;

[0038] Figure 5 Figure 1 shows the anti-cancer activity of selenium-enriched selenium-flavonoids from the microscopical view of the tea. DETAILED DESCRIPTION

[0039] Example 1

[0040] Boiling water extraction: Take selenium-enriched tea (Chuanfeng County tube car tea professional cooperative, organic selenium content ≥ 1 mg / kg), powder machine crushing, 60 mesh sieve to obtain selenium-enriched tea powder. Take 20 g of selenium-enriched tea powder 100 ℃ boiling water bath reflux extraction 1.5 h, pure water liquid ratio 1 / 50. 0.22 μm hollow fiber ultrafiltration membrane impurity, the flow rate control at 0.4 L / min, collection of filtrate.

[0041] Macroporous resin purification: ADS-17 macroporous adsorption resin and extraction liquid according to 1 / 100 of the liquid ratio (g / ml) resin enrichment, stirring speed 100 rmp / min, temperature control 35 ℃, time control 1 h. Filter collection after enrichment of macroporous adsorption resin, 40 ℃ hot air drying until become constant weight. Dried resin according to the mass volume ratio (g / ml) for 1 / 50 of the petroleum ether (analytical pure) degreasing elution, followed by using liquid ratio (g / ml) 1 / 100 of 80% ethanol solution elution and collection of eluate. Centrifugation of eluate, take the supernatant, get extraction extract, namely crude selenium flavonoids from tea.

[0042] Normal phase chromatography impurity removal: 400 mesh H normal phase silica gel (pore size 120A, particle size 50 μm) and extraction extract according to the mass ratio of 5 / 1 purification, select liquid ratio (g / ml) 1 / 100 of dichloromethane (analytical pure) degreasing elution. Again using liquid ratio (g / ml) 1 / 100 of ethanol elution and collection of eluate.

[0043] Gel (Sephadex G-25) chromatography column chromatography impurity removal process, select ethanol solution target elution through gel chromatography column purification, select concentration 80% volume percent ethanol elution, eluate and ethanol solution volume ratio is 1:100, according to the total elution volume 1 / 4 collection as a component, collection to the third component is the target eluent, concentrated and spray drying treatment, for refined selenium flavonoids III from tea.

[0044] Content detection and drying: spectrophotometer detection of total flavonoids content, LC-MS detection of flavonoids purity and content, ICP-MS detection of organic selenium content, spray drying, get refined selenium flavonoids III.

[0045] The preparation of selenium flavonoids III from tea material morphology see Figure 1 , the preparation of selenium flavonoids flavonoids III component species and content see Figure 2 and table 1. See Figure 1 It can be seen that the purified selenium flavonoids III is a white cream-like solid powder. Figure 2It can be seen that purified selenium flavones III have 7 main flavone ion peaks in the ESI(-), Scan Frag 80.0V condition: dihydromyricetin (Rt 22.52 min), myricetin (Rt 15.12 min), taxifolin (Rt 11.29 min), myricitrin (Rt 10.36 min), quercetin (Rt 23.47 min).

[0046] Table 1 Content of selenium flavones III and organic selenium in tea

[0047]

[0048] Example 2

[0049] Boiling water extraction: Take selenium-rich tea (Xianfeng County Tongchabam tea professional cooperative, organic selenium content≥1mg / kg), crush with a powder machine, and pass through a 60-mesh sieve to obtain selenium-rich tea powder. Take 20g of selenium-rich tea powder and extract it in a boiling water bath at 100°C for 2h, with a pure water liquid-to-solid ratio (g / ml) of 1.5 / 50. Remove impurities with a 0.45μm hollow fiber ultrafiltration membrane, and control the flow rate at 0.3L / min to collect the filtrate.

[0050] Macroporous resin purification: ADS-17 macroporous adsorption resin is enriched with the extract at a solid-to-liquid ratio of 1.5 / 100, the stirring speed is 100rmp / min, the temperature is controlled at 35°C, and the time is controlled for 1h. Collect the macroporous adsorption resin after enrichment by filtration, and dry it with hot air at 40°C until it becomes constant in weight. The dried resin is eluted with petroleum ether (analytical pure) at a mass-to-volume ratio of 1.5 / 50 for degreasing, followed by elution with 85% ethanol solution at a ratio of 1 / 100 and collection of the eluate. Centrifuge the eluate, take the supernatant, and concentrate to obtain the extraction infusion, i.e., crude selenium flavones of tea.

[0051] Normal phase chromatography impurity removal: 400-mesh H normal phase silica gel (pore size 120A, particle size 50μm) is used to purify the extraction infusion at a mass ratio of 5 / 1, and dichloromethane (analytical pure) is used for degreasing elution at a solid-to-liquid ratio (g / ml) of 1.5 / 100. Then, ethanol solution is used for elution at a ratio of 2.5 / 100 and the eluate is collected.

[0052] Gel (Sephadex G-25) chromatography column chromatography impurity removal process: ethanol solution is used for elution through the gel chromatography column, 80% volume percentage ethanol solution is used for elution, the eluate is collected at a volume ratio of 1:100 with ethanol solution, and 1 / 4 of the total elution volume is collected as one component. The third component collected is the target eluate, which is concentrated, spray-dried, and processed to obtain refined selenium flavones III of tea.

[0053] Content detection and drying: spectrophotometer detects total flavonoid content, LC-MS detects flavonoid purity and content, ICP-MS detects organic selenium content, spray drying, and get refined selenium flavonoids III.

[0054] The yield of the obtained kudzu vine tea selenium flavonoids III compound is 35.71%, the total flavonoid content is 95.13%, the dihydromyricetin content is 86.49%, the myricetin content is 1.28%, the taxifolin content is 0.64%, the myricitrin content is 0.32%, the quercetin content is 0.26%, and the organic selenium content is 0.34 mg / g.

[0055] Example 3

[0056] Boiling water extraction: take selenium-rich kudzu vine tea (Xianfeng County Tongchabam Kudzu Vine Tea Professional Cooperative, organic selenium content≥1 mg / kg), crush with a powder machine, and pass through a 60-mesh sieve to obtain selenium-rich kudzu vine tea powder. Take 20 g of selenium-rich kudzu vine tea powder, and extract for 1.5 h in a 100℃ boiling water bath, with a pure water to feed liquid ratio of 2 / 50. Remove impurities with a 0.45μm hollow fiber ultrafiltration membrane, and control the flow rate at 0.5 L / min. Collect the filtrate.

[0057] Macroporous resin purification: ADS-17 macroporous adsorption resin is enriched with the extract at a feed liquid ratio of 1.5 / 100, the stirring speed is 100 rmp / min, the temperature is controlled at 35℃, and the time is controlled for 1 h. Collect the macroporous adsorption resin after enrichment by filtration, and dry it with hot air at 40℃ until it becomes constant weight. The dried resin is eluted with petroleum ether (analytical pure) at a mass volume ratio of 1.5 / 50 for degreasing, followed by elution with 80% ethanol solution at a mass volume ratio of 1.5 / 100, and the eluate is collected. Centrifuge the eluate, take the supernatant, and concentrate to obtain the extraction infusion, i.e. kudzu vine tea crude selenium flavonoids.

[0058] Normal phase chromatography impurity removal: 400-mesh H normal phase silica gel (pore size 120A, particle size 50μm) is used to purify the extraction infusion at a mass ratio of 5 / 1, and 2.5 / 100 dichloromethane (analytical pure) is selected for degreasing elution. Then, 1.5 / 100 ethanol solution is used for elution, and the eluate is collected.

[0059] Gel (Sephadex G-25) chromatography column chromatography impurity removal process, select ethanol solution target elution through gel chromatography column purification, select 80% concentration volume percentage ethanol solution for elution, eluate to ethanol solution volume ratio is 1:100, according to the total elution volume 1 / 4 collected as a component, collected to the third component is the target eluate, concentrated and, spray drying treatment, for refined kudzu vine tea selenium flavonoids III.

[0060] Content detection and drying: spectrophotometer detects total flavonoid content, LC-MS detects flavonoid purity and content, ICP-MS detects organic selenium content, spray drying, and get refined selenium flavonoids III.

[0061] The yield of the obtained selenium flavonoids III compound of the tea vine was 31.48%, the total flavonoid content was 96.55%, the dihydromyricetin content was 88.69%, the myricetin content was 1.45%, the taxifolin content was 0.78%, the myricitrin content was 0.54%, the quercetin content was 0.33%, and the organic selenium content was 0.40 mg / g.

[0062] Example 4

[0063] Boiling water extraction: Take selenium-rich tea vine (Xianfeng County Tongchabam tea vine professional cooperative, organic selenium content≥1 mg / kg), crush with a powder machine, and pass through a 60-mesh sieve to obtain selenium-rich tea vine powder. Take 20 g of selenium-rich tea vine powder, and extract for 2 h in a 100℃ boiling water bath, with a pure water to feed liquid ratio of 1 / 50. Remove impurities with a 0.45 μm hollow fiber ultrafiltration membrane, and control the flow rate at 0.5 L / min. Collect the filtrate.

[0064] Macroporous resin purification: ADS-17 macroporous adsorption resin is enriched with the extract at a feed liquid ratio of 2.5 / 100, the stirring speed is 100 rmp / min, the temperature is controlled at 35℃, and the time is controlled for 1 h. Collect the macroporous adsorption resin after enrichment by filtration, and dry it with hot air at 40℃ until it becomes constant weight. The dried resin is eluted with petroleum ether (analytical pure) at a mass volume ratio of 2.5 / 50 for degreasing, followed by elution with 85% ethanol solution at a mass volume ratio of 2.5 / 100, and the eluate is collected. Centrifuge the eluate, take the supernatant, and concentrate to obtain the extraction infusion, i.e. crude selenium flavonoids of tea vine.

[0065] Normal phase chromatography impurity removal: 400-mesh H normal phase silica gel (pore size 120A, particle size 50 μm) is used to purify the extraction infusion at a mass ratio of 5 / 1, and 1.5 / 100 dichloromethane (analytical pure) is selected for degreasing elution. Then, 1.5 / 100 ethanol solution is used for elution, and the eluate is collected.

[0066] Gel (Sephadex G-25) chromatography column chromatography impurity removal process: ethanol solution is selected for elution through gel chromatography column purification, 80% volume percentage ethanol solution is selected for elution, the eluate and the ethanol solution are at a volume ratio of 1:100, and 1 / 4 of the total elution volume is collected as a component. The third component collected is the target eluate, which is concentrated and spray dried to obtain refined selenium flavonoids III of tea vine.

[0067] Content detection and drying: the total flavonoid content is detected by spectrophotometry, the purity and content of flavonoids are detected by LC-MS, the organic selenium content is detected by ICP-MS, and spray drying is performed to obtain refined selenium flavonoids III.

[0068] The yield of the obtained selenium flavonoids III of the tea vine was 33.20%, the total flavonoid content was 95.65%, the dihydromyricetin content was 87.05%, the myricetin content was 1.34%, the taxifolin content was 0.52%, the myricitrin content was 0.44%, the quercetin content was 0.27%, and the organic selenium content was 0.49 mg / g.

[0069] Example 5

[0070] Boiling water extraction: The selenium-rich tea vine (Tongchabam tea professional cooperative in Xianfeng County, organic selenium content≥1 mg / kg) was crushed by a powder machine and sieved through a 60-mesh sieve to obtain selenium-rich tea vine powder. 20 g of the selenium-rich tea vine powder was extracted in a boiling water bath at 100°C for 1.5 h, and the pure water to liquid ratio was 1.5 / 50. The impurities were removed by a 0.45 μm hollow fiber ultrafiltration membrane, and the flow rate was controlled at 0.4 L / min. The filtrate was collected.

[0071] Macroporous resin purification: The ADS-17 macroporous adsorption resin was enriched with the extract at a liquid to resin ratio of 2.5 / 100, the stirring speed was 100 rmp / min, the temperature was controlled at 35°C, and the time was controlled at 1 h. The macroporous adsorption resin after enrichment was filtered and collected, and was dried by hot air at 40°C until it became constant in weight. The dried resin was eluted with petroleum ether (analytical pure) at a mass to volume ratio of 2.5 / 50 for degreasing, and then was eluted with 80% ethanol solution at a mass to volume ratio of 2.5 / 100, and the eluate was collected. The eluate was centrifuged, the supernatant was concentrated, and the extraction infusion, i.e., the crude selenium flavonoids of the tea vine, was obtained.

[0072] Normal phase chromatography impurity removal: 300-mesh H normal phase silica gel (pore size 120A, particle size 50 μm) was used to purify the extraction infusion at a mass ratio of 5 / 1, and dichloromethane (analytical pure) was used for degreasing elution at a ratio of 1.5 / 100. Then, ethanol solution was used for elution at a ratio of 1 / 100, and the eluate was collected.

[0073] Gel (Sephadex G-25) chromatography column chromatography impurity removal process: ethanol solution was used for elution through the gel chromatography column, 80% volume percentage ethanol solution was used for elution, the eluate to ethanol solution volume ratio was 1:100, and 1 / 4 of the total elution volume was collected as one component. The third component collected was the target eluate, which was concentrated, spray dried, and treated to obtain the refined selenium flavonoids III of the tea vine.

[0074] Content detection and drying: the total flavonoid content was detected by spectrophotometry, the purity and content of the flavonoids were detected by LC-MS, the organic selenium content was detected by ICP-MS, and spray drying was performed to obtain the refined selenium flavonoids III.

[0075] The obtained selenium flavonoids III of the vine tea has a yield of 36.73%, a total flavonoid content of 96.08%, a dihydromyricetin content of 86.57%, a myricetin content of 1.39%, a taxifolin content of 0.69%, a myricitrin content of 0.48%, a quercetin content of 0.29%, and an organic selenium content of 0.37 mg / g.

[0076] Example 6

[0077] The selenium flavonoids III of 3.0 g obtained in Example 1, 0.5 g of licorice flavonoids, 0.5 g of green tea polyphenols, 0.50 g of DL-malic acid, and 6 g of β-cyclodextrin are added and uniformly mixed, and then freeze-dried to obtain a dry powder. The selenium-rich flavonoid solid beverage is obtained by sterilizing the dry powder in a bag. In this process, based on the fuzzy mathematical sensory evaluation method and the response surface method, the selenium-rich solid beverage product has a unique flavor and rich and harmonious aroma, and the sensory score reaches 92.33±5.81. The drinking method is as follows: 2 g of the product is poured into a cup, 100-150 mL of boiling water is added, and the mixture is stirred to dissolve, and then it can be drunk.

[0078] The selenium flavonoids III obtained in Example 1 are used to study the anticancer and normal cell toxicity as follows.

[0079] Experimental Example 1

[0080] The anti-cancer activity and cell toxicity activity of the vine tea selenium flavonoids III (obtained in Example 1) on human lung cancer cells A549 are tested.

[0081] When the density of A549 cells reaches 85%, the cells are digested with 1% trypsin until the cells become round, and then the digestion is terminated by adding cell culture medium. The cell suspension is appropriately diluted, and then 100 μL of the cell suspension is inoculated into each well of a 96-well plate at a density of 5000 cells per well. The plate is cultured in a cell incubator at 37°C and 5% CO2 for 24 h. The culture medium is discarded, and 100 μL of fresh culture medium containing the drug to be tested is added. The final concentration of each drug is 1.0, 2.0, 4.0, 8.0, 16.0, 32.0, 64.0, 128.0, 256.0, 512.0, and 1024.0 μg / mL, respectively. The control group is added with an equal amount of fresh culture medium, and the negative control group is added with cell-free cell culture medium. Each concentration is tested in triplicate. The plate is cultured again under the same conditions for 24 h. The growth of the cells is observed and photographed every 6 h by an inverted microscope.

[0082] Then, the cells are treated with CCK-8 reagent, and 10 μL of CCK-8 reagent is added to each well. The plate is incubated at 37°C for 1 h. The absorbance value at 450 nm is read by a microplate reader to calculate the cell viability. The calculation formula is as follows:

[0083]

[0084] 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.

[0085] After incubating A549 cells with vine tea selenium flavonoid III for 24 hours, the inhibition rate against A549 lung cancer cells was 39.45% at a concentration of 256 μg / mL; and 50.57% at a concentration of 512 μg / mL. Figure 2 , Figure 5 B, 5E). For example... Figure 3 As shown, with increasing drug concentration, the inhibitory effect of vine tea selenium flavonoid III on lung cancer cells A549 was enhanced, while the cell viability decreased.

[0086] Experiment Example 2

[0087] Efficacy test of anticancer and cytotoxic activity of vine tea selenium flavonoid III (obtained in Example 1) against mouse lung cancer cells.

[0088] When the LA-795 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°C 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 1.0, 2.0, 4.0, 8.0, 16.0, 32.0, 64.0, 128.0, 256.0, 512.0, and 1024.0 μg / mL. 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 performed 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.

[0089] 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:

[0090]

[0091] 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.

[0092] After incubating LA-795 cells with the selenium flavone III of the vine tea for 24 hours, the inhibition rate of the lung cancer cells LA-795 was 42.19% when the drug concentration was 256.0 μg / mL; the inhibition rate of the lung cancer cells LA-795 was 52.49% when the drug concentration was 512.0 μg / mL (as shown in Figure 2 、 Figure 5 C, 5F). As shown in Figure 3 , the inhibition effect of the selenium flavone III of the vine tea on the lung cancer cells LA-795 was enhanced with the increase of the drug concentration (as shown in Figure 3 ).

[0093] Experimental Example 3

[0094] Anticancer activity and cytotoxic activity pharmacological test of the selenium flavone III of the vine tea (obtained in Example 1) on normal human lung epithelial cells.

[0095] When the density of the BEAS-2B cells reached 85%, the cells were digested with 1% trypsin until the cells became round, and then the digestion was terminated by adding cell culture solution. The cell suspension was appropriately diluted, and then inoculated in the 96-well plate at a density of 100 μL per well and 5000 cells per well, and cultured in the cell culture box at 37°C and 5% CO2 for 24 hours. The culture solution was discarded, 100 μL of fresh culture solution containing the drug to be tested was added, the final concentration of each drug was 1.0, 2.0, 4.0, 8.0, 16.0, 32.0, 64.0, 128.0, 256.0, 512.0 and 1024.0 μg / mL respectively, the control group was added with the same amount of fresh culture medium, and the negative control group was added with cell-free cell culture solution, and three parallel samples were prepared for each concentration. The cells were cultured again under the same conditions for 24 hours. The growth of the cells was observed and photographed through the inverted microscope every 6 hours.

[0096] Then the cells were treated with the CCK-8 reagent, 10 μL of CCK-8 reagent was added to each well, and incubated at 37°C for 1 hour. The absorbance value at 450 nm was read by the enzyme marker to calculate the cell viability. The calculation formula is as follows:

[0097]

[0098] In the formula: A0 is the negative group: no cells, add culture medium and CCK-8; A1 is the sample group: with cells, add sample, culture medium and CCK-8; A2 is the control group: with cells, add culture medium and CCK-8.

[0099] After incubating BEAS-2B cells with the selenium flavone III of the vine tea for 24 hours, the inhibition rate of the normal lung cells BEAS-2B was 0.48% when the drug concentration was 256.0 μg / mL; the inhibition rate of the normal lung cells BEAS-2B was -0.59% when the drug concentration was 512.0 μg / mL (as shown in Figure 2, Figure 5 A, 5D). As shown in Figure 5D, RSVF III had no significant inhibitory effect on lung normal epithelial cells BEAS-2B, and the cell survival rate was not affected (as shown in Figure 5C). Figure 3 Figure 3

[0100] Comparative Example 1

[0101] In comparison with Example 1, only crude RSVF was prepared, and the anti-cancer activity test was performed on human lung cancer cells A549 using the crude RSVF.

[0102] As shown in Table 2, after the human lung cancer cells A549 were incubated with the crude RSVF for 24 h, the inhibition rate of the lung cancer cells A549 was 23.48% at a concentration of 256 μg / mL, and the inhibition rate of the lung cancer cells A549 was 34.27% at a concentration of 512 μg / mL. With the increase of the concentration, the cell survival rate did not change significantly, which proved that the crude RSVF had weak inhibitory effect on human lung cancer cells A549.

[0103] The anti-cancer activity test was performed on mouse lung cancer cells LA-795, and as shown in Table 2, after the mouse lung cancer cells LA-795 were incubated with the crude RSVF for 24 h, the inhibition rate of the lung cancer cells LA-795 was 18.97% at a concentration of 256.0 μg / mL, and the inhibition rate of the lung cancer cells LA-795 was 29.68% at a concentration of 512.0 μg / mL. With the increase of the concentration, the cell survival rate did not change significantly, which proved that the crude RSVF had weak inhibitory effect on mouse lung cancer cells LA-795.

[0104] The anti-cancer activity test was performed on normal human lung epithelial cells BEAS-2B, and as shown in Table 2, after the normal human lung epithelial cells BEAS-2B were incubated with the crude RSVF for 24 h, the inhibition rate of the normal lung cells BEAS-2B was 14.79% at a concentration of 256.0 μg / mL, and the inhibition rate of the normal lung cells BEAS-2B was 28.95% at a concentration of 512.0 μg / mL. With the increase of the concentration, the cell survival rate changed significantly, which proved that the crude RSVF had significant toxic side effects on normal human lung epithelial cells BEAS-2B.

[0105] Comparative Example 2

[0106] In comparison with Example 1, the difference was that the "Sephadex G-25 column chromatography impurity removal process" was omitted, and the target elution of the ethanol solution obtained in the previous step was directly concentrated and spray-dried to obtain refined RSVF. The anti-cancer activity test was performed on human lung cancer cells A549 using the refined RSVF.

[0107] ​​As shown in Table 2, after the human lung cancer cell A549 cells were incubated with the crude selenium flavonoids of the tea plant for 24 h, the inhibition rate of the lung cancer cell A549 was 28.63% when the concentration of the drug was 256 μg / mL; the inhibition rate of the lung cancer cell A549 was 34.29% when the concentration of the drug was 512 μg / mL. With the increase of the concentration of the drug, the cell survival rate did not change significantly, which proved that the crude selenium flavonoids of the tea plant had weak inhibitory effect on the human lung cancer cell A549.

[0108] The anti-cancer activity test was performed on the mouse lung cancer cell LA-795.

[0109] As shown in Table 2, after the mouse lung cancer cell LA-795 was incubated with the crude selenium flavonoids of the tea plant for 24 h, the inhibition rate of the lung cancer cell LA-795 was 23.97% when the concentration of the drug was 256.0 μg / mL; the inhibition rate of the lung cancer cell LA-795 was 30.73% when the concentration of the drug was 512.0 μg / mL. With the increase of the concentration of the drug, the cell survival rate did not change significantly, which proved that the crude selenium flavonoids of the tea plant had weak inhibitory effect on the mouse lung cancer cell LA-795.

[0110] The anti-cancer activity test was performed on the normal human lung epithelial cell BEAS-2B.

[0111] As shown in Table 2, after the normal human lung epithelial cell BEAS-2B was incubated with the crude selenium flavonoids of the tea plant for 24 h, the inhibition rate of the normal lung cell BEAS-2B was 13.68% when the concentration of the drug was 256.0 μg / mL; the inhibition rate of the normal lung cell BEAS-2B was 21.09% when the concentration of the drug was 512.0 μg / mL. With the increase of the concentration of the drug, the cell survival rate changed significantly, which proved that the crude selenium flavonoids of the tea plant had significant toxic side effects on the normal human lung epithelial cell BEAS-2B.

[0112] Table 2 Anti-cancer activity of the crude selenium flavonoids extract of the tea plant

[0113]

[0114]

[0115] Comparative Example 3

[0116] Compared with Example 1, the difference lies in that the "ADS-17 macroporous adsorption resin and the extract liquid are enriched at a solid-liquid ratio of 1 / 100" in Example 1 is changed to "ADS-17 macroporous adsorption resin and the extract liquid are enriched at a solid-liquid ratio of 0.5 / 100", and the obtained crude selenium flavonoids are further purified.

[0117] As shown in Table 3, the yield of selenium flavone III compound of the obtained rattan tea through the process is 34.27%, but the purity is only 84.62%, and the organic selenium content is only 0.28 mg / g, which is obviously less than the yield, purity and organic selenium content of the rattan tea selenium flavone III of Example 1.

[0118] Comparative Example 4:

[0119] Compared with Example 1, the difference is that the "ADS-17 macroporous adsorption resin and the extract liquid are enriched at a ratio of 1 / 100 of the liquid to resin" in Example 1 is changed to "ADS-17 macroporous adsorption resin and the extract liquid are enriched at a ratio of 3 / 100 of the liquid to resin", and the obtained crude selenium flavone is further purified and refined.

[0120] As shown in Table 3, the yield of selenium flavone III compound of the obtained rattan tea through the process is 28.61%, but the purity is only 88.79%, and the organic selenium content is only 0.26 mg / g, which is obviously less than the yield, purity and organic selenium content of the rattan tea selenium flavone III of Example 1.

[0121] Comparative Example 5:

[0122] Compared with Example 1, the difference is that the "ADS-17 macroporous adsorption resin and the extract liquid are enriched at a ratio of 1 / 100 of the liquid to resin" in Example 1 is changed to "ADS-17 macroporous adsorption resin and the extract liquid are enriched at a ratio of 3 / 100 of the liquid to resin", and the obtained crude selenium flavone is further purified and refined.

[0123] As shown in Table 3, the yield of selenium flavone III compound of the obtained rattan tea through the process is 26.13%, but the purity is only 80.38%, and the organic selenium content is only 020 mg / g, which is obviously less than the yield, purity and organic selenium content of the rattan tea selenium flavone III of Example 1.

[0124] Comparative Example 6:

[0125] Compared with Example 1, the difference is that the "ADS-17 macroporous adsorption resin and the extract liquid are enriched at a ratio of 1 / 100 of the liquid to resin" in Example 1 is changed to "ADS-17 macroporous adsorption resin and the extract liquid are enriched at a ratio of 3 / 100 of the liquid to resin", and the obtained crude selenium flavone is further purified and refined.

[0126] As shown in Table 3, the yield of selenium flavone III compound of the obtained rattan tea through the process is 31.37%, but the purity is only 84.68%, and the organic selenium content is only 0.15 mg / g, which is obviously less than the yield, purity and organic selenium content of the rattan tea selenium flavone III of Example 1.

[0127] Comparative Example 7:

[0128] Compared with Example 1, the difference lies in that the "normal phase chromatography impurity removal" in Example 1 is replaced by a direct gel (Sephadex G-25) chromatography column chromatography impurity removal process on the ethanol solution target eluent obtained in the previous step, followed by concentration, spray drying treatment, and then refined selenium flavone of kudzu vine tea.

[0129] As shown in Table 3, the yield of kudzu vine tea selenium flavone III compound obtained by the process is 28.91%, but the purity is only 74.59%, and the organic selenium content is only 0.24 mg / g, which is significantly less than the yield, purity, and organic selenium content of kudzu vine tea selenium flavone III of the process of the present application.

[0130] Table 3: Effect of extraction process parameters on crude selenium flavone indicators

[0131]

[0132]

[0133] Note: The same column lowercase letters represent a significant difference.

Claims

1. A method for extracting selenium flavonoid III 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 0.22-0.45μm hollow fiber ultrafiltration membrane, and collect the filtrate. 2) Macroporous resin purification: ADS-17 macroporous adsorption resin was mixed with the collected filtrate for resin enrichment. The enriched macroporous adsorption resin was collected by filtration and dried until constant weight was achieved. The dried resin was defatted and eluted with petroleum ether, followed by elution with 80-85% (v / v) ethanol solution and collection of the eluent was performed. The eluent was centrifuged, and the supernatant was concentrated to obtain the extract, namely, crude selenium flavonoids from vine tea. The material-to-liquid ratio for resin enrichment was 1 / 100 to 2.5 / 100, and the hot air drying temperature was 35-40 °C. 3) Purification by normal phase chromatography: The extract was purified by normal phase silica gel with H, then defatted and eluted with dichloromethane, and then eluted with ethanol solution and the eluent was collected. 4) Gel chromatography column purification process: The target is purified by elution with ethanol solution through gel chromatography column. The elution is carried out with 80% volume percentage ethanol solution. The volume ratio of eluent to ethanol solution is 1:

100. 1 / 4 of the total elution volume is collected as a fraction. The third fraction collected is the target eluent. After concentration and spray drying, it is the purified vine tea selenium flavonoid III.

2. The method for extracting selenium flavonoid III 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 ≥1 mg / kg.

3. The method for extracting selenium flavonoid III from selenium-enriched vine tea as described in claim 1 or 2, characterized in that, In step 1), boiling water reflux extraction is performed at atmospheric pressure, temperature ≥98 ℃, time 1.5-2.0 h, and pure water to liquid ratio 1 / 50-2 / 50.

4. The method for extracting selenium flavonoid III from selenium-enriched vine tea as described in claim 1 or 2, characterized in that, The flow rate for impurity removal using the hollow fiber ultrafiltration membrane is 0.3-0.5 L / min.

5. The method for extracting selenium flavonoid III from selenium-enriched vine tea as described in claim 1, characterized in that, In step 2), the material-to-liquid ratio for petroleum ether degreasing and elution is 1 / 50 to 2.5 / 50, and the material-to-liquid ratio for ethanol elution is 1 / 100 to 2.5 / 100.

6. The method for extracting selenium flavonoid III from selenium-enriched vine tea as described in claim 1, characterized in that, In step 3), the material is purified by 300-400 mesh H normal phase silica gel. The material-to-liquid ratio for degreasing and elution with dichloromethane is 1 / 100-2.5 / 100, and the material-to-liquid ratio for elution with ethanol solution is 1 / 100-2.5 / 100.

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

8. The vine tea selenium flavonoid III as described in claim 7, characterized in that, Calculated by mass percentage, it contains at least 86-89% dihydromyricetin, 1-1.5% myricetin, 0.5-0.9% piperidin, 0.3-0.6% myricetin, and 0.1-0.4% quercetin.

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