Preparation and application of organic selenium flavone compounds with high antioxidant activity and low toxic side effects

Abstract

This invention discloses the preparation and application of organoselenium flavonoids with high antioxidant activity and low toxicity, relating to the field of functional food technology. The method of this invention includes pulverizing selenium-enriched vine tea, selenium-enriched kudzu root, selenium-enriched gastrodia elata, and selenium-enriched papaya, then mixing them according to a specified ratio, low-temperature reflux extraction, hollow fiber ultrafiltration membrane purification, activated carbon depigmentation, reversed-phase silica gel degreasing and polysaccharide removal, gel chromatography purification, and freeze-drying or spray drying. This invention uses a novel food resource combination of vine tea, kudzu root, gastrodia elata, and papaya—medicinal and edible herbs—as raw materials to extract organoselenium flavonoids with a high yield. Furthermore, the extracted selenium flavonoids exhibit extremely strong DPPH free radical scavenging activity and show no toxic side effects on normal human renal tubular epithelial cells (HKC) and normal lung epithelial cells (BEAS-2B). Therefore, the content of this invention is of great significance to the development of the field of antioxidant functional foods.

Description

Technical Field

[0001] This invention relates to the field of functional food technology, specifically to the preparation and application of organic selenium flavonoids with high antioxidant activity and low toxicity, particularly the application of these organic selenium flavonoids as antioxidant active ingredients in functional food factors. Background Technology

[0002] Reactive oxygen species (ROS) play a crucial role in regulating various physiological functions of organisms. When the body is exposed to harmful stimuli from the external environment, the secretion of various proteases in the body becomes uncontrolled, leading to an imbalance between ROS and the antioxidant defense system in the biological system, which in turn induces pathological changes at the cellular and tissue levels. Atherosclerosis, neurodegenerative diseases, age-related diseases, and carcinogenesis are all closely related to ROS accumulation and oxidative stress.

[0003] Therefore, antioxidants can play a very effective role in preventing and treating these diseases by reducing excessive free radicals in the body. Many synthetic antioxidants are available on the market, but they all have certain side effects and are not suitable for long-term use. Natural antioxidants are much safer than chemically synthesized antioxidants, and some of their antioxidant efficiencies may even be higher than those of synthetic drugs. Natural flavonoids derived from food resources and medicinal plants such as vine tea and kudzu root possess diverse and bioactive pharmacophores with specific spatial and electronic properties. They can prevent lipid peroxidation, maintain the balance of free radicals in the body, and thus improve the body's ability to resist a series of diseases caused by oxidative stress.

[0004] The following are reports on flavonoids derived from vine tea: Patent application CN202310937129.4 only relates to a method for separating total flavonoids from vine tea stems or leaves and the preparation and application of a cream; patent application CN202210725262.9 only relates to a vine tea flavonoid extract and its application in the preparation of a compound vine tea flavonoid oral liquid; patent application CN201811211063.6 only relates to a vine tea flavonoid composition liposome and its preparation method and application; patent application CN201210039472.9 also only relates to a vine tea facial mask capsule and its preparation method. It can be seen that currently there are no reports on the application of extracting selenium flavonoids from the new food resource vine tea and various medicinal and edible plants such as kudzu root, gastrodia elata, and wrinkled papaya, and their use in preparing functional food factors with high antioxidant activity and low toxicity in the food field. Therefore, the industry needs a functional food factor with high antioxidant activity and low toxicity to prevent lipid peroxidation, maintain the balance of free radicals in the body, and thus improve the body's ability to resist a series of diseases caused by oxidative stress.

[0005] Vine tea, belonging to the genus *Ampelopsis* of the Vitaceae family, is a vine plant. Its plant name is *Ampelopsis grossedentata* (Hand-Mazz) WT Wang. Approved as a new food ingredient in 2013, it is considered a new food resource. Vine tea is an important specialty economic crop in Southwest China, boasting a flavonoid content as high as 30%, earning it the nickname "King of Flavonoids." In Enshi alone, the artificial cultivation area exceeds 100,000 mu (approximately 6,667 hectares), with an annual output value exceeding 2 billion yuan. Vine tea is rich in flavonoids and selenium polysaccharides. Modern pharmacological studies have proven that its flavonoid components have the effects of clearing heat and detoxifying, relieving sore throat and swelling, and calming the liver and lowering blood pressure. It has been widely used in folk medicine for the prevention and treatment of conditions such as lowering blood lipids, lowering blood pressure, and fighting inflammation and infection. However, there are currently no reports on the antioxidant activity of selenium flavonoids from vine tea, a new food resource, or on the processing technology for preparing selenium flavonoids from vine tea. Existing technologies also lack information on the application of high-antioxidant-activity, low-toxicity selenium flavonoids from vine tea in the preparation of functional food factors with in vitro antioxidant activity.

[0006] Based on the above reasons, this application is hereby submitted. Summary of the Invention

[0007] Based on the above reasons, and in view of the problems or defects existing in the prior art, the purpose of this invention is to provide a method for preparing an organic selenium flavonoid compound with high antioxidant activity and low toxicity and side effects, and its application in functional food factors, thereby solving or at least partially solving the above-mentioned technical defects existing in the prior art.

[0008] To achieve the first objective of this invention, the technical solution adopted by this invention is as follows:

[0009] A method for preparing an organoselenium flavonoid compound with high antioxidant activity and low toxicity includes the following steps: pulverizing selenium-enriched vine tea, selenium-enriched kudzu root, selenium-enriched gastrodia elata, and selenium-enriched papaya; then mixing them according to a certain ratio; low-temperature reflux extraction; removal of impurities using a hollow fiber ultrafiltration membrane; removal of pigments using activated carbon; removal of fats and polysaccharides using reversed-phase silica gel; removal of impurities using gel chromatography; and freeze-drying or spray drying.

[0010] Furthermore, in the above technical solution, the organic selenium flavonoid compound contains active ingredients such as dihydromyricetin, myricetin, puerarin, daidzein, gastrodin, p-hydroxybenzyl alcohol, oleanolic acid, ursolic acid, and organic selenium.

[0011] Furthermore, in the above technical solution, the yield of the obtained organic selenium flavonoids is ≥10%. The total flavonoid content of the organic selenium flavonoids is ≥85%; the content of dihydromyricetin is 60-70%, myricetin is 0.5-1.0%, puerarin is 0.5-0.9%, daidzein is 0.3-0.6%, gastrodin is 1-1.5%, p-hydroxybenzyl alcohol is 0.1-0.3%, oleanolic acid is 0.2-0.8%, and ursolic acid is 0.2-0.8%.

[0012] Furthermore, in the above technical solution, the obtained organic selenium flavonoid compound contains ≥0.15 mg / kg of organic selenium, clarifying that the Se element contained in the organic selenium flavonoid compound is bound to the flavonoid monomer molecule through Se-OC and Se-SC mechanisms. Many existing technologies for selenium flavonoids do not specify the form in which selenium exists (only describing it as organic or inorganic selenium), and the inorganic form of selenium has toxic side effects on the human body.

[0013] Furthermore, in the above-mentioned technical solution, the organic selenium content in the raw materials such as selenium-enriched vine tea, selenium-enriched kudzu root, selenium-enriched gastrodia elata, and selenium-enriched papaya is ≥1.2mg / kg. In a preferred embodiment of the present invention, the present invention selects selenium-enriched vine tea, selenium-enriched kudzu root, selenium-enriched gastrodia elata, and selenium-enriched papaya grown in selenium-enriched soil in Enshi. Through the absorption of selenium element and its metabolism into organic selenium element, which is combined with its own flavonoids, polysaccharides, proteins, and other macromolecules, the bioconversion efficiency of selenium element is improved. The organic selenium flavonoid compounds obtained from the above-mentioned selenium-enriched raw materials are safe and non-toxic.

[0014] Furthermore, in the aforementioned technical solution, the dry weight ratio of the raw materials selected for the blending of selenium-enriched vine tea, selenium-enriched kudzu root, selenium-enriched gastrodia elata, and selenium-enriched papaya (especially selenium-enriched wrinkled papaya) is 4.5–5.0: 1.0–1.5: 1.0–1.5: 1.0–1.5. Among the selected raw materials, the flavonoid content of vine tea reaches over 20% (w / w), and the flavonoid content, types, and antioxidant effects of various raw materials are also different. Arbitrarily changing the proportion of raw materials will affect the final yield of selenium flavonoids, the state properties of flavonoids, and antioxidant activity, and may also lead to toxicity to normal cells.

[0015] Furthermore, in a preferred embodiment of the present invention, the low-temperature reflux extraction specifically uses a 65wt%–70wt% ethanol solution as the extraction solvent, and the extraction temperature is ≤65℃, for example, 55–65℃. The reflux extraction time is 1.0–1.5 h, and the solid-liquid ratio is 1 / 50–2.5 / 50 g / mL.

[0016] Specifically, the above-mentioned technical solution employs low-temperature reflux extraction, which can be directly applied to large-scale production practices. Low temperature maximizes the protection of flavonoid bioactive components, offering advantages such as simple operation and suitability for industrial production. This invention selects a 65wt%–70wt% ethanol solution as the extraction solvent because the polarity range of the active components in the raw material is similar, making it the optimal solvent for solubility and extraction efficiency. The extraction-to-liquid ratio is 1 / 50–2.5 / 50 g / mL. An excessively high ratio leads to insufficient dissolution of the flavonoid components in the raw material, wasting raw materials; an excessively low ratio makes it difficult to enrich and recover the flavonoid bioactive components from the ethanol solution, increasing production costs.

[0017] Furthermore, in the above-mentioned technical solution, since most of the impurities such as tissue fragments and cell fragments in the plant raw materials are larger than 0.45 μm, the hollow fiber ultrafiltration membrane impurity removal method described in this invention specifically involves directly passing the extract obtained from low-temperature reflux extraction through a 0.22–0.45 μm hollow fiber ultrafiltration membrane to remove impurities, effectively removing solid impurity particles, denatured and insoluble biological macromolecules such as DNA and proteins.

[0018] Furthermore, in the above technical solution, regarding the removal of pigments by activated carbon, this invention selects a feed-to-liquid ratio of 1 / 100 to 2 / 100 g / mL for granular activated carbon adsorption and pigment removal. This invention precisely controls the feed-to-liquid ratio for enriching granular activated carbon. Adding too high a proportion of granular activated carbon will cause organic selenium flavonoids to also be adsorbed onto the surface of the activated carbon, resulting in insufficient recovery of these compounds, leading to waste of raw materials and a low final yield of total flavonoids. Conversely, adding too low a proportion of granular activated carbon will result in insufficient adsorption of pigments and other impurities, leading to excessively high impurity content in the final organic selenium flavonoid compounds and increased production costs.

[0019] Furthermore, in the above technical solution, the step of removing fats and polysaccharides with reversed-phase silica gel specifically involves further passing the filtrate obtained after removing pigments from activated carbon through a reversed-phase silica gel column to remove impurities such as fats and polysaccharides. The impurity removal process includes sequentially eluting polysaccharides with ultrapure water at a feed-to-liquid ratio of 1 / 500 to 5 / 500 g / mL, and eluting with a 70 wt% to 80 wt% ethanol solution at a feed-to-liquid ratio of 1 / 500 to 2 / 500 g / mL.

[0020] Furthermore, in a preferred embodiment of the present invention, the above technical solution selects C. 18 Reversed-phase silica gel (pore size 100 Å, particle size 40–60 μm) is used to purify polysaccharides and lipids. C atoms are bonded to the surface of the reversed-phase silica gel. 18 Carbon chains have a strong adsorption capacity for weakly polar lipids and a weak adsorption capacity for strongly polar compounds such as polysaccharides and phenols, exhibiting a very good separation effect and achieving rapid and effective impurity removal.

[0021] Specifically, in the preferred embodiment of the present invention, the above technical solution involves eluting polysaccharides with ultrapure water at a feed-to-liquid ratio of 1 / 500 to 5 / 500 g / mL, and eluting C with a 70 to 80 wt% ethanol solution at a feed-to-liquid ratio of 1 / 500 to 2 / 500 g / mL. 18 Reversed-phase silica gel chromatography was used, with the ethanol eluent collected as the target eluent. Specifically, ultrapure water at a feed-to-liquid ratio of 1 / 500 to 5 / 500 g / mL effectively removed highly polar active ingredients such as polysaccharides, organic acids, and polyphenols; a 70-80 wt% ethanol solution at a feed-to-liquid ratio of 1 / 500 to 2 / 500 g / mL efficiently eluted the target substance, selenium flavonoids, while retaining weakly polar impurities such as lipids on the silica gel, effectively removing both highly and weakly polar compounds such as lipids and polysaccharides. This facilitates subsequent removal of large and small molecule impurities via gel chromatography.

[0022] Furthermore, in the preferred embodiment of the above technical solution, the gel chromatography impurity removal step employs a Sephadex G-25 column chromatography. A 70-80 wt% ethanol solution is used to elute the gel column for purification. One-third of the total elution volume is collected as a fraction, and the second fraction is the target eluent. This eluent is concentrated and then freeze-dried or spray-dried to obtain purified organoselenium flavonoids with high antioxidant activity and low toxicity. This invention utilizes both polarity and molecular weight as indicators to effectively remove impurities from the target selenium flavonoids, resulting in purified organoselenium flavonoids with a total flavonoid content ≥85%, a purity unattainable by existing methods.

[0023] Furthermore, in the above technical solution, the freeze-drying temperature is -20 to -25°C, and the time is 6 to 8 hours.

[0024] The second objective of this invention is to provide an organoselenium flavonoid compound with high antioxidant activity and low toxicity and side effects prepared by the method described above.

[0025] The third objective of this invention is to provide an application of highly antioxidant and low-toxicity organic selenium flavonoids prepared by the method described above as antioxidant active ingredients in functional food factors.

[0026] This invention provides a functional food factor with antioxidant activity, including organic selenium flavonoids with high antioxidant activity and low toxicity and side effects prepared by the method described above.

[0027] This invention provides a functional food with antioxidant activity, comprising the aforementioned functional food factors with antioxidant activity and excipients in any dosage form acceptable to food pharmacology.

[0028] Furthermore, in the above technical solution, the functional foods include solid beverages, instant tea, canned liquid beverages, and powdered drinks.

[0029] This invention, based on the principles of traditional Chinese medicine compatibility, namely "four natures and five flavors," and according to the principles of compatibility based on the seven emotions and the roles of principal, assistant, adjuvant, and guide herbs, integrates kudzu root, gastrodia elata, papaya, and a new food resource, vine tea, along with their corresponding medicinal properties, into a formula with strong antioxidant effects. Modern pharmacological research has found that kudzu root is rich in isoflavone antioxidant active ingredients, possessing the effects of promoting yang and relieving rashes, generating fluids and quenching thirst, and relieving muscle tension and reducing fever; gastrodia elata is rich in flavonoids, polyphenols, polysaccharides, and other antioxidant active ingredients, possessing the effects of calming wind and relieving spasms, suppressing liver yang, dispelling wind and unblocking collaterals, and strengthening the brain and protecting nerves; papaya (especially wrinkled papaya) is rich in various natural antioxidant active ingredients such as polyphenols, organic acids, and flavonoids, possessing the effects of invigorating tendons and collaterals, resolving dampness in the stomach, and anti-inflammatory and analgesic effects. Therefore, combining kudzu root, gastrodia elata, and papaya with vine tea can obtain antioxidant active ingredients with even stronger antioxidant effects. Selenium, as one of the essential trace elements for the human body, has preventive and health-promoting effects such as lowering blood sugar, anti-aging, and improving human immunity, and enjoys the reputation of "the spark of life" and "the element of longevity." Therefore, selenium-rich flavonoids from food resources plants have unique advantages. However, the industry currently lacks information on the extraction of organic selenium flavonoids from new food resources such as vine tea and combinations with kudzu root, gastrodia elata, and papaya, as well as their antioxidant effects.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] (1) The present invention found that organic selenium flavonoids were extracted with a high yield by using a combination of kudzu root, gastrodia elata, papaya and other medicinal and edible Chinese medicinal materials as raw materials. Moreover, the extracted selenium flavonoids have extremely strong DPPH free radical scavenging activity and have no toxic side effects on normal human renal tubular epithelial cells (HKC).

[0032] (2) Existing technologies have not studied the form of selenium in the extracted selenium flavonoids or the flavonoid components. The present invention has found that we have clarified the structural characteristics of the prepared organic selenium flavonoid compounds, such as flavonoid purity, flavonoid monomer composition, and relative content.

[0033] (3) Many active ingredients in traditional Chinese medicine contain unsaturated bonds, which are what give them their antioxidant effects. However, this also makes the active substances themselves extremely unstable, easily reacting with oxygen at high temperatures and causing them to lose their activity. Therefore, the low-temperature reflux extraction method used in this invention differs from other methods in the prior art in that it can greatly protect the active ingredients from being destroyed. Secondly, this invention has the advantages of simple operation and suitability for industrial production, and can be directly applied to large-scale production practices.

[0034] (4) The present invention uses hollow fiber ultrafiltration membrane to remove impurities. Compared with the prior art, it reduces the contamination of solid impurity particles in the extract on the subsequent activated carbon and reverse silica gel filler, improves the extraction efficiency of pigment molecules by activated carbon, increases the service life of activated carbon, and effectively reduces production costs.

[0035] (5) This invention has found that the organoselenium flavonoids prepared in this invention have extremely strong DPPH free radical scavenging activity, and at the same time have no toxic side effects or damage to normal human renal tubular epithelial cells (HKC) and normal lung epithelial cell line BEAS-2B. Many existing synthetic antioxidants have certain side effects, which are not conducive to long-term use. The organoselenium flavonoids provided by this invention have been proven to have no effect on normal cell activity and are safe in origin, effectively solving the problem of toxic side effects of existing antioxidants. Therefore, the content of this invention is of great significance to the development of the field of antioxidant functional foods.

[0036] (6) When the concentration of the organic selenium flavonoid compound described in this invention in the functional food factor is 0.4 mg / mL, the DPPH free radical scavenging rate is 95-100%; when the concentration is 0.1 mg / mL, the DPPH free radical scavenging rate is 20-25%.

[0037] Furthermore, the organic selenium flavonoids described in this invention exhibit a 0% inhibition rate against normal human renal tubular epithelial cells (HKC) at concentrations ranging from 0 to 400 ug / mL.

[0038] When the concentration of the organic selenium flavonoids described in this invention is 0-400 ug / mL, the inhibition rate against the normal lung epithelial cell line BEAS-2B is also 0%. Attached Figure Description

[0039] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0040] Figure 1 Left image: Blank control DPPH (purple); Right image: Photograph of the product after DPPH was scavenged by the purified organic selenium flavonoids in Example 1.

[0041] Figure 2This is a comparison diagram of the antioxidant activities of the organoselenium flavonoids prepared in Example 1 and the positive control drug ascorbic acid; where: ascorbic acid represents the positive control drug ascorbic acid; Selenium-flavonoid represents the organoselenium flavonoids prepared in Example 1;

[0042] Figure 3 A photograph of the purified organic selenium flavonoids prepared in Example 1;

[0043] Figure 4 The diagram shows the toxic activity of organoselenium flavonoids against normal cells under a microscope in Example 1; where: A: toxic activity of organoselenium flavonoids at a concentration of 400 μg / mL against BEAS-2B cells; B: toxic activity of organoselenium flavonoids at a concentration of 400 μg / mL against HKC cells;

[0044] Figure 5 The image shows the HPLC chromatogram of the purified organic selenium flavonoids prepared in Example 1. Detailed Implementation

[0045] The purpose of this invention is to provide a method for preparing organic selenium flavonoids with high antioxidant activity and low toxicity, and their application in the preparation of antioxidant functional food factors. Existing technologies disclose methods for preparing flavonoids from the novel food resource *Zanthoxylum bungeanum* and their application in creams, oral flavonoid liquids, and facial masks; and inventions of processes and equipment for extracting selenium flavonoids from medicinal plants such as *Polygonatum sibiricum*, broccoli, and kudzu root, as well as their effects on inhibiting oxidative stress and inflammatory responses. However, there is currently no information in the industry regarding the extraction of selenium flavonoids from the novel food resource *Zanthoxylum bungeanum* and combinations of medicinal and edible herbs such as kudzu root, *Gastrodia elata*, and papaya, and their application in antioxidant functional foods.

[0046] The present invention will be further described in detail below through implementation examples. These implementation examples are carried out based on the technology of the present invention. Detailed implementation methods and specific operating procedures are provided to illustrate the inventiveness of the present invention, but the scope of protection of the present invention is not limited to the following implementation examples.

[0047] Based on the information contained in this application, various modifications to the precise description of the invention can be readily made by those skilled in the art. It should be understood that the scope of the invention is not limited to the defined processes, properties, or components, as these embodiments and other descriptions are merely illustrative of specific aspects of the invention.

[0048] The equipment and raw materials used in this invention are all commercially available or commonly used in the field. Unless otherwise specified, the methods in the following embodiments are conventional methods in the field.

[0049] The hollow fiber ultrafiltration membranes used in the following embodiments or comparative examples of the present invention were purchased from Hubei Huawei Scientific Instruments Co., Ltd., item number: ZYC-001.

[0050] The granular activated carbon used in the following embodiments or comparative examples of the present invention was purchased from Enshi Kangyong Trading Co., Ltd., item number: TY-15.

[0051] The present invention uses inductively coupled plasma mass spectrometry (ICP-MS) to detect the organic selenium content of the organic selenium flavonoids prepared in each example or comparative example according to the current national standard GB 5009.93-2017.

[0052] In the following application examples or comparative application examples of the present invention, the antioxidant activity is quantitatively analyzed by testing the DPPH free radical scavenging activity method. The DPPH free radical scavenging activity assay is a commonly used method for evaluating the antioxidant activity of drugs, and the present invention selects ascorbic acid as a positive control.

[0053] The normal cells described in the following application examples or comparative application examples of the present invention are preferably normal human renal tubular epithelial cells (HKC). The toxicity activity of the present invention was quantitatively analyzed by the CCK-8 method, which can distinguish between normally growing cells, apoptotic cells, and necrotic cells; the cell growth status and density were also visualized and analyzed by an inverted microscope; and cell quantification was performed by a hemocytometer.

[0054] Example 1

[0055] This embodiment describes a method for preparing refined organic selenium flavonoids, comprising the following steps:

[0056] Ingredients: Selenium-rich vine tea, selenium-rich kudzu root, selenium-rich gastrodia elata, and selenium-rich wrinkled papaya raw materials grown in selenium-rich soil in Enshi. The organic selenium content of each raw material is 1.0 mg / kg. The dry weight ratio of the raw materials is 5.0 / 1.5 / 1.5 / 1.0. Mix evenly, grind with a pulverizer, and pass through a 60-mesh sieve to obtain the raw material powder.

[0057] Low-temperature reflux extraction: Take 25g of mixed raw material powder and reflux extract at 55℃ with 65wt% ethanol solution for 1h, with a material-to-liquid ratio of 1 / 50g / mL.

[0058] Hollow fiber ultrafiltration membrane impurity removal: Collect the above low-temperature reflux extract and filter it through a hollow fiber ultrafiltration membrane with a pore size of 0.45 μm to remove impurities. The pressure is controlled at 1.5 atmospheres and the flow rate is controlled at 1 L / min. Collect the filtrate.

[0059] Activated carbon adsorption for pigment removal: Granular activated carbon was mixed with the filtrate obtained after impurity removal via hollow fiber ultrafiltration membrane at a feed-to-liquid ratio of 1 / 100 g / mL for activated carbon adsorption. The temperature was controlled at 35℃, and the time was controlled at 1 h. The filtrate with pigments removed was then filtered and collected.

[0060] Reverse-phase silica column for removing impurities such as fats and polysaccharides: utilizing C 18 The polysaccharides were purified by mixing reversed-phase silica gel (pore size 100 Å, particle size 40-60 μm) with the filtrate after removing pigments at a mass ratio of 5 / 1. Then, the polysaccharides were washed away with ultrapure water at a feed-to-liquid ratio of 1 / 500 g / mL. The eluent was then collected by eluting with 80 wt% ethanol solution at a feed-to-liquid ratio of 1 / 500 g / mL.

[0061] Gel chromatography for impurity removal: The eluent obtained by degreasing and polysaccharide treatment on a reversed-phase silica gel column was purified by gel chromatography (Sephadex G-25) using a 75wt% ethanol solution. The total elution volume was determined by a ratio of 1 / 500 (v / v) of the sample to the total elution volume. One-third of the total elution volume was collected as a fraction, and the second fraction collected was the target eluent. After concentration and freeze-drying at -25℃ for 7 hours to constant weight, the purified organoselenin flavonoids were obtained.

[0062] The purified organic selenium flavonoids obtained in this embodiment are in the form of a black paste-like solid. Figure 3 .

[0063] The purified organic selenium flavonoids prepared in this embodiment had a total flavonoid yield of 11.42%, a purity of 86.88%, and an organic selenium content of 0.16 mg / g. The types and contents of the purified organic selenium flavonoids are detailed below. Figure 5 See Table 1. Figure 5 It can be seen that the refined organic selenium flavonoids have 8 main absorption peaks at an absorption wavelength of 270 nm: dihydromyricetin (Rt15.44 min), myricetin (Rt16.78 min), puerarin (Rt13.26 min), daidzin (Rt10.33 min), gastrodin (Rt1.53 min), p-hydroxybenzyl alcohol (Rt7.69 min), oleanolic acid (Rt19.29 min), and ursolic acid (Rt23.62 min).

[0064] Table 1. Test results of the content of each component in the purified organic selenium flavonoids prepared in Example 1.

[0065]

[0066] Example 2

[0067] This embodiment describes a method for preparing refined organic selenium flavonoids, comprising the following steps:

[0068] Ingredients: Take selenium-enriched vine tea, selenium-enriched kudzu root, selenium-enriched gastrodia elata, and selenium-enriched wrinkled papaya as raw materials. The organic selenium content of each raw material is 1.0 mg / kg. The dry weight ratio of the raw materials is 4.75 / 1.25 / 1.25 / 1.25. Mix them evenly, grind them with a pulverizer, and pass them through a 60-mesh sieve to obtain the raw material powder.

[0069] Low-temperature reflux extraction: Take 25g of mixed raw material powder and reflux extract at 60℃ with 67.5wt% ethanol solution for 1.5h, with a material-to-liquid ratio of 2.5 / 50g / mL.

[0070] Hollow fiber ultrafiltration membrane impurity removal: Collect the above low-temperature reflux extract and filter it through a hollow fiber ultrafiltration membrane with a pore size of 0.22 μm to remove impurities. The pressure is controlled at 1.5 atmospheres and the flow rate is controlled at 1 L / min. Collect the filtrate.

[0071] Activated carbon adsorption for pigment removal: Granular activated carbon was mixed with the filtrate obtained after impurity removal via hollow fiber ultrafiltration membrane at a feed-to-liquid ratio of 2.0 / 100 g / mL for activated carbon adsorption. The temperature was controlled at 35℃ and the time was controlled at 1 h. After filtration, the filtrate with pigments removed was collected.

[0072] Reverse-phase silica column for removing impurities such as fats and polysaccharides: utilizing C 18 The polysaccharides were purified by mixing reversed-phase silica gel (pore size 100 Å, particle size 40–60 μm) with the filtrate after pigment removal at a mass ratio of 5 / 1. Then, the polysaccharides were removed by elution with ultrapure water at a feed-to-liquid ratio of 2.5 / 500 g / mL. The eluent was then collected by elution with 75 wt% ethanol solution at a feed-to-liquid ratio of 2 / 500 g / mL.

[0073] Gel chromatography for impurity removal: The eluent obtained by degreasing and polysaccharide treatment on a reversed-phase silica gel column was purified by gel chromatography (Sephadex G-25) using an 80wt% ethanol solution. The total elution volume was determined by a ratio of 1 / 500 (v / v) of the sample to the total elution volume. One-third of the total elution volume was collected as a fraction, and the second fraction collected was the target eluent. After concentration and freeze-drying at -20℃ for 8 hours to constant weight, the purified organoselenin flavonoids were obtained.

[0074] The purified organic selenium flavonoids prepared in this embodiment had a total flavonoid yield of 11.06%, a purity of 88.19%, and an organic selenium content of 0.21 mg / g.

[0075] Example 3

[0076] This embodiment describes a method for preparing refined organic selenium flavonoids, comprising the following steps:

[0077] Ingredients: Take selenium-enriched vine tea, selenium-enriched kudzu root, selenium-enriched gastrodia elata, and selenium-enriched wrinkled papaya as raw materials. The organic selenium content of each raw material is 1.0 mg / kg. The dry weight ratio of the raw materials is 4.5 / 1.0 / 1.0 / 1.5. Mix them evenly, grind them with a pulverizer, and pass them through a 60-mesh sieve to obtain the raw material powder.

[0078] Low-temperature reflux extraction: Take 25g of mixed raw material powder and reflux extract at 60℃ with 70wt% ethanol solution for 1.2h, with a material-to-liquid ratio of 1.5 / 50g / mL.

[0079] Hollow fiber ultrafiltration membrane impurity removal: Collect the above low-temperature reflux extract and filter it through a hollow fiber ultrafiltration membrane with a pore size of 0.45 μm to remove impurities. The pressure is controlled at 1.5 atmospheres and the flow rate is controlled at 1 L / min. Collect the filtrate.

[0080] Activated carbon adsorption for pigment removal: Granular activated carbon was mixed with the filtrate obtained after impurity removal via hollow fiber ultrafiltration membrane at a feed-to-liquid ratio of 1.5 / 100 g / mL for activated carbon adsorption. The temperature was controlled at 35℃, and the time was controlled at 1 h. The filtrate with pigments removed was then filtered and collected.

[0081] Reverse-phase silica column for removing impurities such as fats and polysaccharides: utilizing C 18 The polysaccharides were purified by mixing reversed-phase silica gel (pore size 100 Å, particle size 40-60 μm) with the filtrate after removing pigments at a mass ratio of 5 / 1. Then, the polysaccharides were washed away with ultrapure water at a ratio of 1 / 500 g / mL. The eluent was then collected by eluting with 75 wt% ethanol solution at a ratio of 1.5 / 500 g / mL.

[0082] Gel chromatography for impurity removal: The eluent obtained by degreasing and polysaccharide treatment on a reversed-phase silica gel column was purified by gel chromatography (Sephadex G-25) using a 70wt% ethanol solution. The total elution volume was determined by a ratio of 1 / 500 (v / v) of the sample to the total elution volume. One-third of the total elution volume was collected as a fraction, and the second fraction collected was the target eluent. After concentration and freeze-drying at -25℃ for 7 hours to constant weight, the purified organoselenin flavonoids were obtained.

[0083] The purified organic selenium flavonoids prepared in this embodiment have a total flavonoid yield of 12.67%, a purity of 87.28%, and an organic selenium content of 0.20 mg / g.

[0084] Example 4

[0085] This embodiment describes a method for preparing refined organic selenium flavonoids, comprising the following steps:

[0086] Ingredients: Take selenium-enriched vine tea, selenium-enriched kudzu root, selenium-enriched gastrodia elata, and selenium-enriched wrinkled papaya as raw materials. The organic selenium content of each raw material is 1.0 mg / kg. The dry weight ratio of the raw materials is 4.75 / 1.5 / 1.0 / 1.25. Mix them evenly, grind them with a pulverizer, and pass them through a 60-mesh sieve to obtain the raw material powder.

[0087] Low-temperature reflux extraction: Take 25g of mixed raw material powder and reflux extract at 65℃ with 67.5wt% ethanol solution for 1h, with a material-to-liquid ratio of 2.5 / 50g / mL.

[0088] Hollow fiber ultrafiltration membrane impurity removal: Collect the above low-temperature reflux extract and filter it through a hollow fiber ultrafiltration membrane with a pore size of 0.45 μm to remove impurities. The pressure is controlled at 1.5 atmospheres and the flow rate is controlled at 1 L / min. Collect the filtrate.

[0089] Activated carbon adsorption for pigment removal: Granular activated carbon was mixed with the filtrate obtained after impurity removal via hollow fiber ultrafiltration membrane at a feed-to-liquid ratio of 2 / 100 g / mL for activated carbon adsorption. The temperature was controlled at 35℃, and the time was controlled at 1 hour. The filtrate with pigments removed was then filtered and collected.

[0090] Reverse-phase silica column for removing impurities such as fats and polysaccharides: utilizing C 18 The polysaccharides were purified by mixing reversed-phase silica gel (pore size 100 Å, particle size 40-60 μm) with the filtrate after removing pigments at a mass ratio of 5 / 1. Then, the polysaccharides were washed away with ultrapure water at a feed-to-liquid ratio of 5 / 500 g / mL. Finally, the eluent was collected by eluting with 70 wt% ethanol solution at a feed-to-liquid ratio of 1 / 500 g / mL.

[0091] Gel chromatography for impurity removal: The eluent obtained by degreasing and polysaccharide treatment on a reversed-phase silica gel column was purified by gel chromatography (Sephadex G-25) using a 75wt% ethanol solution. The total elution volume was determined by a ratio of 1 / 500 (v / v) of the sample to the total elution volume. One-third of the total elution volume was collected as a fraction, and the second fraction collected was the target eluent. After concentration and freeze-drying at -25℃ for 7 hours to constant weight, the purified organoselenin flavonoids were obtained.

[0092] The purified organic selenium flavonoids prepared in this embodiment have a total flavonoid yield of 11.68%, a purity of 88.09%, and an organic selenium content of 0.18 mg / g.

[0093] Example 5

[0094] This embodiment describes a method for preparing refined organic selenium flavonoids, comprising the following steps:

[0095] Ingredients: Take selenium-enriched vine tea, selenium-enriched kudzu root, selenium-enriched gastrodia elata, and selenium-enriched wrinkled papaya as raw materials. The organic selenium content of each raw material is 1.0 mg / kg. The dry weight ratio of the raw materials is 5.0 / 1.25 / 1.5 / 1.5. Mix them evenly, grind them with a pulverizer, and pass them through a 60-mesh sieve to obtain the raw material powder.

[0096] Low-temperature reflux extraction: Take 25g of mixed raw material powder and reflux extract at 65℃ with 65wt% ethanol solution for 1h, with a material-to-liquid ratio of 1 / 50g / mL.

[0097] Hollow fiber ultrafiltration membrane impurity removal: Collect the above low-temperature reflux extract and filter it through a hollow fiber ultrafiltration membrane with a pore size of 0.45 μm to remove impurities. The pressure is controlled at 1.5 atmospheres and the flow rate is controlled at 1 L / min. Collect the filtrate.

[0098] Activated carbon adsorption for pigment removal: Granular activated carbon was mixed with the filtrate obtained after impurity removal via hollow fiber ultrafiltration membrane at a feed-to-liquid ratio of 1.5 / 100 g / mL for activated carbon adsorption. The temperature was controlled at 35℃, and the time was controlled at 1 h. The filtrate with pigments removed was then filtered and collected.

[0099] Reverse-phase silica column for removing impurities such as fats and polysaccharides: utilizing C 18 The polysaccharides were purified by mixing reversed-phase silica gel (pore size 100 Å, particle size 40–60 μm) with the filtrate after pigment removal at a mass ratio of 5 / 1. Then, the polysaccharides were removed by elution with ultrapure water at a feed-to-liquid ratio of 2.5 / 500 g / mL. The eluent was then collected by elution with 75 wt% ethanol solution at a feed-to-liquid ratio of 2 / 500 g / mL.

[0100] Gel chromatography for impurity removal: The eluent obtained by degreasing and polysaccharide treatment on a reversed-phase silica gel column was purified by gel chromatography (Sephadex G-25) using an 80wt% ethanol solution. The total elution volume was determined by a ratio of 1 / 500 (v / v) of the sample to the total elution volume. One-third of the total elution volume was collected as a fraction, and the second fraction collected was the target eluent. After concentration and freeze-drying at -22.5℃ for 6 hours to constant weight, the purified organic selenium flavonoids were obtained.

[0101] The purified organic selenium flavonoids prepared in this embodiment have a total flavonoid yield of 13.45%, a purity of 85.67%, and an organic selenium content of 0.19 mg / g.

[0102] Comparative Example 1

[0103] The preparation method of a crude selenium flavonoid compound in this comparative example includes the following steps:

[0104] Ingredients: Take selenium-enriched vine tea, selenium-enriched kudzu root, selenium-enriched gastrodia elata, and selenium-enriched wrinkled papaya as raw materials. The organic selenium content of each raw material is 1.0 mg / kg. The dry weight ratio of the raw materials is 5.0 / 1.5 / 1.5 / 1.0. Mix them evenly, pulverize them with an ICP grinder, and pass them through a 60-mesh sieve to obtain the raw material powder.

[0105] Low-temperature reflux extraction: Take 25g of mixed raw material powder and reflux extract at 55℃ with 65wt% ethanol solution for 1h, with a material-to-liquid ratio of 1 / 50g / mL.

[0106] Hollow fiber ultrafiltration membrane impurity removal: Collect the above low-temperature reflux extract and filter it through a hollow fiber ultrafiltration membrane with a pore size of 0.45 μm to remove impurities. The pressure is controlled at 1.5 atmospheres and the flow rate is controlled at 1 L / min. Collect the filtrate.

[0107] Activated carbon adsorption for pigment removal: Granular activated carbon was mixed with the filtrate obtained after impurity removal via hollow fiber ultrafiltration membrane at a feed-to-liquid ratio of 1 / 100 g / mL for activated carbon adsorption. The temperature was controlled at 35℃, and the time was controlled at 1 h. The filtrate with pigments removed was then collected by filtration.

[0108] The filtrate was concentrated and freeze-dried at -25°C for 7 hours until it reached constant weight, yielding crude selenium flavonoids. Spectrophotometry showed that the yield of crude selenium flavonoids in the filtrate after pigment removal was 11.45%, and LC-MS analysis showed that the purity of the obtained crude selenium flavonoids was 79.88%.

[0109] Comparative Example 2

[0110] The preparation method of a selenium flavonoid compound in this comparative example is basically the same as that in Example 1, except that the gel chromatography column chromatography process is omitted. Instead, the eluent obtained by removing impurities such as fats and polysaccharides by reversed-phase silica gel column chromatography is directly concentrated and freeze-dried. All other steps and processes are the same.

[0111] The yield of selenium flavonoids obtained by this process was 9.17%, but the purity was only 67.64% and the organic selenium content was only 0.10 mg / g, which was significantly lower than the yield, purity and organic selenium content of selenium flavonoids obtained by the process in Example 1 of this invention.

[0112] Comparative Example 3

[0113] The preparation method of refined vine tea selenium flavonoids in this comparative example is basically the same as that in Example 1, except that this comparative example only uses selenium-rich vine tea as raw material, and the other steps and processes are the same.

[0114] Comparative Example 4

[0115] The preparation method of a selenium flavonoid compound in this comparative example is basically the same as that in Example 1, except that this comparative example did not undergo column purification, while the other steps and processes are the same.

[0116] Comparative Example 5

[0117] The preparation method of a selenium flavonoid compound in this comparative example is basically the same as that in Example 1, except that the low-temperature reflux extraction process in this comparative example is as follows: take 25g of mixed raw material powder and reflux extract with 65wt% ethanol solution at 80℃ for 1h. Other steps and processes are the same.

[0118] Comparative Example 6

[0119] The preparation method of a selenium flavonoid compound in this comparative example is basically the same as that in Example 1, except that the specific process of activated carbon adsorption for pigment removal in this comparative example is as follows: granular activated carbon and filtrate obtained by removing impurities through hollow fiber ultrafiltration membrane are subjected to activated carbon adsorption at a material-to-liquid ratio of 0.5 / 100g / mL. All other steps and processes are the same.

[0120] The yield of selenium flavonoids obtained by this process was 9.14%, but the purity was only 74.36% and the organic selenium content was only 0.14 mg / g, which was significantly lower than the yield, purity and organic selenium content of the refined selenium flavonoids obtained by the process in Example 1 of this invention.

[0121] Comparative Example 7

[0122] The preparation method of a selenium flavonoid compound in this comparative example is basically the same as that in Example 1, except that the specific process of activated carbon adsorption for pigment removal in this comparative example is as follows: granular activated carbon and filtrate obtained by removing impurities through hollow fiber ultrafiltration membrane are subjected to activated carbon adsorption at a material-to-liquid ratio of 3 / 100 g / mL. All other steps and processes are the same.

[0123] The yield of selenium flavonoids obtained by this process was 6.46%, but the purity was only 64.10% and the organic selenium content was only 0.08 mg / g, which was significantly lower than the yield, purity and organic selenium content of the selenium flavonoids obtained by the process in Example 1 of this invention.

[0124] Comparative Example 8

[0125] The preparation method of a selenium flavonoid compound in this comparative example is basically the same as that in Example 1, except that the specific process of gel chromatography for impurity removal in this comparative example is as follows: the eluent obtained by removing impurities such as fat and polysaccharides by reversed-phase silica gel column chromatography is directly subjected to gel chromatography (Sephadex G-25) for impurity removal, then concentrated and freeze-dried. Other steps and processes are the same.

[0126] The yield of selenium flavonoids obtained by this process was 8.29%, but the purity was only 68.99% and the organic selenium content was only 0.11 mg / g, which was significantly lower than the yield, purity and organic selenium content of selenium flavonoids obtained by the process in Example 1 of this invention.

[0127] Comparative Example 9

[0128] The preparation method of refined vine tea selenium flavonoids in this comparative example is basically the same as that in Example 1, except that the dry weight ratio of selenium-enriched vine tea, selenium-enriched kudzu root, selenium-enriched gastrodia elata, and selenium-enriched wrinkled papaya raw materials is changed to 4.0 / 2.0 / 2.0 / 2.0. All other steps and processes are the same.

[0129] The yield of selenium flavonoids obtained by this process was only 6.29%, the purity was only 83.74%, and the organic selenium content was only 0.12 mg / g, which were significantly lower than the yield, purity, and organic selenium content of selenium flavonoids obtained by the process in Example 1 of this invention.

[0130] Comparative Example 10

[0131] The preparation method of refined vine tea selenium flavonoids in this comparative example is basically the same as that in Example 1, except that the dry weight ratio of selenium-enriched vine tea, selenium-enriched kudzu root, selenium-enriched gastrodia elata, and selenium-enriched wrinkled papaya raw materials is changed to 6.0 / 0.5 / 0.5 / 0.5. All other steps and processes are the same.

[0132] The yield of selenium flavonoids obtained by this process was only 9.47%, the purity was only 84.35%, and the organic selenium content was only 0.13 mg / g, which were significantly lower than the yield, purity, and organic selenium content of selenium flavonoids obtained by the process in Example 1 of this invention.

[0133] Application Example 1

[0134] The purified organic selenium flavonoids obtained in Example 1 were used to conduct the following studies on their antioxidant and normotoxic effects. The specific steps are as follows:

[0135] The DPPH free radical scavenging activity of purified organic selenium flavonoids (obtained in Example 1) was tested. A stock solution of the purified organic selenium flavonoids was prepared at a concentration of 20 mg / mL and diluted to a series of sample concentrations: 0.1, 0.5, 1.0, 2.0, and 4.0 mg / mL, with ascorbic acid as a control. Experimental groups were set up: sample + DPPH solution; control group: sample + anhydrous ethanol; and blank group: sterile water + DPPH. The microplate reader was set to 37℃ and in the dark for 30 min. The absorbance was measured at 517 nm. The blank was recorded as A0, the experimental group as A1, and the control group as A2. The color change of DPPH was observed and photographed. The DPPH free radical scavenging rate of the purified organic selenium flavonoids was calculated using the following formula:

[0136]

[0137] Half-maximal effect concentration (EC50) 50 DPPH free radical scavenging rate (%) was calculated for each concentration (0.1, 0.2, 0.3, 0.4 mg / mL) within the concentration range of 0.1-0.4 mg / mL. A linear fit was performed between the drug concentration (X) and the free radical scavenging rate (Y) to obtain the fitted curve Y = aX + / - b; the fitted curve r... 2 A value ≥0.99 represents an effective fitted curve. Under the condition of this effective fitted curve, the drug concentration X corresponding to a DPPH free radical scavenging rate Y = 50% is calculated as the EC50 of the drug for DPPH free radical scavenging. 50 .

[0138] The DPPH free radical scavenging activity of refined organic selenium flavonoids and ascorbic acid, such as Figure 1 , Figure 2 As shown in Table 2, within the concentration range of 0.1-0.4 mg / mL, the DPPH radical scavenging rate of selenium flavonoids increased with increasing sample concentration. The EC50 values ​​of the DPPH radical scavenging activities of selenium flavonoids and ascorbic acid were also discussed. 50 The values ​​were 0.12 mg / mL and 0.03 mg / mL, respectively.

[0139] Application Example 2

[0140] The cytotoxic activity of the purified organic selenium flavonoids obtained in Example 1 against normal human renal tubular epithelial cells (HKC) was tested.

[0141] When the HKC 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 (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 100, 200, 400, and 800 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 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.

[0142] 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:

[0143]

[0144] 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.

[0145] As shown in Table 2, after incubating HKC cells with purified organic selenium flavonoids for 24 hours, the inhibition rate of normal renal HKC cells was -0.17% at a concentration of 200 μg / mL, and -2.17% at a concentration of 400 μg / mL (e.g., ...). Figure 4 B). Therefore, refined organic selenium flavonoids have no significant inhibitory effect on HKC in normal renal cells, and cell viability is unaffected.

[0146] Application Example 3

[0147] The cytotoxic activity of the purified organic selenium flavonoids obtained in Example 1 against the normal human lung epithelial cell line BEAS-2B was tested.

[0148] When the BEAS-2B 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 (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 100, 200, 400, and 800 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 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.

[0149] 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:

[0150]

[0151] 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.

[0152] As shown in Table 2, after incubating BEAS-2B cells with purified organic selenium flavonoids for 24 hours, the inhibition rate of BEAS-2B cells was 0.78% at a concentration of 200 μg / mL, and 3.13% at a concentration of 400 μg / mL (e.g., ...). Figure 4 A). Therefore, selenium flavonoid II has no significant inhibitory effect on normal BEAS-2B, and cell viability is unaffected.

[0153] Table 2. Cytotoxic activity test results of the purified organic selenium flavonoids obtained in Example 2.

[0154]

[0155] Note: Lowercase letters in the same column indicate significant differences.

[0156] Application Example 4

[0157] This application example demonstrates the use of the refined organic selenium flavonoids prepared in Example 1 to prepare functional foods. The specific preparation method of the functional foods described in this application example is as follows, including the following steps:

[0158] Take 5.0g of the refined organic selenium flavonoids prepared in Example 1, add 44.0g of soluble starch, 0.5g of DL-malic acid, and 0.5g of β-cyclodextrin. Mix well, then package in 2g bags and sterilize by irradiation with 60Co for 1.5h to obtain the selenium-enriched solid beverage product. Directions for use: Pour one bag of this product into a cup, add 100mL of boiling water, stir thoroughly to dissolve, and then drink.

[0159] Application Comparative Example 1

[0160] Compared with Application Example 2, the difference lies in the cytotoxic activity efficacy test of crude selenium flavonoids (which were not purified by column and were obtained using crude selenium flavonoid compounds from Comparative Example 1) against normal human renal tubular epithelial cells (HKC).

[0161] After incubating normal human renal tubular epithelial cells (HKCs) with crude selenium flavonoids for 24 hours, the inhibition rate of normal HKC cells was 25.43% at a concentration of 200 μg / mL and 34.19% at a concentration of 400 μg / mL. The cell viability changed significantly with increasing concentration, demonstrating that the crude selenium flavonoids have significant toxic side effects on normal human renal tubular epithelial cells (HKCs).

[0162] Application Comparative Example 2

[0163] Compared with Application Example 3, the difference lies in the cytotoxic activity efficacy test of crude selenium flavonoids (which were not purified by column and were obtained using the crude selenium flavonoids obtained in Comparative Example 1) against normal human lung epithelial cells (BEAS-2B).

[0164] After incubating normal human lung epithelial cells BEAS-2B with crude selenium flavonoids for 24 hours, the inhibition rate of BEAS-2B cells was 8.27% at a concentration of 200 μg / mL and 19.38% at a concentration of 400 μg / mL. The cell viability changed significantly with increasing concentration, demonstrating that the crude selenium flavonoids have significant toxic side effects on normal human lung epithelial cells BEAS-2B.

[0165] Application Comparative Example 3

[0166] Compared with Application Example 2, the difference is that the gel (Sephadex G-25) column chromatography purification process in Example 1 is omitted, and the cytotoxic activity efficacy test of the selenium flavonoids obtained in Comparative Example 2 on normal human renal tubular epithelial cells (HKC) is conducted.

[0167] After incubating normal human renal tubular epithelial cells (HKCs) with the selenium flavonoids obtained in Comparative Example 2 for 24 h, the inhibition rate of HKCs was 15.49% at a concentration of 200 μg / mL and 27.91% at a concentration of 400 μg / mL. The cell survival rate changed significantly with increasing concentration, demonstrating that the selenium flavonoids obtained in Comparative Example 2 have strong cytotoxic activity against normal human renal tubular epithelial cells (HKCs).

[0168] Application Comparative Example 4

[0169] Compared with Application Example 3, the difference is that the gel (Sephadex G-25) column chromatography purification process in Example 1 is omitted, that is, the selenium flavonoids obtained in Comparative Example 2 are used to conduct anticancer activity tests on normal human lung epithelial cells BEAS-2B.

[0170] After incubating normal human lung epithelial cells BEAS-2B with the selenium flavonoids obtained in Comparative Example 2 for 24 h, the inhibition rate of BEAS-2B cells was 21.83% at a concentration of 200 μg / mL and 36.09% at a concentration of 400 μg / mL. The cell viability changed significantly with increasing concentration, demonstrating that the selenium flavonoids obtained in Comparative Example 2 exhibited strong cytotoxic activity against normal human lung epithelial cells BEAS-2B.

[0171] Application Comparative Example 5

[0172] The difference between this example and application example 2 lies in the use of the cytotoxic activity efficacy test of the selenium flavonoids obtained in comparative example 9 against normal human renal tubular epithelial cells (HKC).

[0173] After incubating normal human renal tubular epithelial cells (HKCs) with selenium flavonoids for 24 hours, the inhibition rate of normal HKCs was 12.12% at a concentration of 200 μg / mL and 25.06% at a concentration of 400 μg / mL. The cell viability changed significantly with increasing concentration, demonstrating that the selenium flavonoids have significant toxic side effects on normal human renal tubular epithelial cells (HKCs).

[0174] Application Comparative Example 6

[0175] Compared with Application Example 1, the difference is that the gel (Sephadex G-25) column chromatography purification process in Example 1 is omitted, and the DPPH free radical scavenging activity experiment of the selenium flavonoids obtained in Comparative Example 2 is used instead.

[0176] Within the concentration range of 0.1–0.4 mg / mL, the DPPH radical scavenging rate of the selenium flavonoids obtained in Comparative Example 2 did not increase significantly with increasing sample concentration. The EC50 of the DPPH radical scavenging activity of the selenium flavonoids obtained in Comparative Example 2 was [not specified in the original text]. 50 The value was 12.71 mg / mL.

[0177] Application Comparative Example 7

[0178] The difference between this example and Example 1 lies in the use of the refined vine tea selenium flavonoids obtained in Comparative Example 3 to conduct experiments on the DPPH free radical scavenging activity.

[0179] Within the concentration range of 0.1–0.4 mg / mL, the DPPH radical scavenging rate of the purified vine tea selenium flavonoids obtained in Comparative Example 3 did not increase significantly with increasing sample concentration. The EC50 of the DPPH radical scavenging activity of the purified vine tea selenium flavonoids obtained in Comparative Example 3 was [not specified in the original text]. 50 The value was 23.48 mg / mL.

[0180] Application Comparative Example 8

[0181] The difference compared to Application Example 1 is that the selenium flavonoids obtained in Comparative Example 4 were used in the DPPH free radical scavenging activity experiment.

[0182] In the activity test of the selenium flavonoids obtained in Comparative Example 4, within the concentration range of 0.1-0.4 mg / mL, the DPPH radical scavenging rate of the selenium flavonoids obtained in Comparative Example 4 did not increase significantly with increasing sample concentration. The EC50 of the DPPH radical scavenging activity of the selenium flavonoids obtained in Comparative Example 4 was... 50 The value was 15.4 mg / mL.

[0183] Application Comparison Example 9

[0184] The difference compared to Application Example 1 is that the selenium flavonoids obtained in Comparative Example 5 were used in the DPPH free radical scavenging activity experiment.

[0185] In the activity test of the selenium flavonoids obtained in Comparative Example 5, within the concentration range of 0.1-0.4 mg / mL, the DPPH radical scavenging rate of the selenium flavonoids obtained in Comparative Example 5 did not increase significantly with increasing sample concentration. The EC50 of the DPPH radical scavenging activity of the selenium flavonoids obtained in Comparative Example 5 was... 50 The value was 25.17 mg / mL.

[0186] Application Comparison Example 10

[0187] The difference compared to Application Example 1 is that the selenium flavonoids obtained in Comparative Example 10 were used in the DPPH free radical scavenging activity experiment.

[0188] In the activity test of the selenium flavonoids obtained using Comparative Example 10, within the concentration range of 0.1-0.4 mg / mL, the DPPH radical scavenging rate of the selenium flavonoids obtained in Comparative Example 10 did not increase significantly with increasing sample concentration. The EC50 of the DPPH radical scavenging activity of the selenium flavonoids obtained in Comparative Example 10 was... 50 The value was 6.28 mg / mL.

Claims

1. A method for preparing an organoselenium flavonoid compound with high antioxidant activity and low toxicity, characterized in that: The method comprises the steps of crushing selenium-rich tea, selenium-rich kudzu root, selenium-rich Gastrodia elata, and selenium-rich papaya, then mixing, low-temperature reflux extraction, removing impurities by hollow fiber ultrafiltration membrane, removing pigment by activated carbon, removing fat and polysaccharide by reverse phase silica gel, removing impurities by gel chromatography, and freezing or spray drying; the obtained organic selenium flavonoid compound has a yield of greater than or equal to 10%; the obtained organic selenium flavonoid compound has an organic selenium content of greater than or equal to 0.15 mg / kg; the dry weight proportion of the selenium-rich tea, the selenium-rich kudzu root, the selenium-rich Gastrodia elata, and the selenium-rich papaya is 4.5-5.0:1.0-1.5:1.0-1.5:1.0-1.5; the low-temperature reflux extraction specifically uses 65wt%-70wt% ethanol solution as the extraction solvent, the extraction temperature is less than or equal to 65°C, the reflux extraction time is 1.0-1.5h, and the solid-liquid ratio is 1 / 50-2.5 / 50 g / mL; the hollow fiber ultrafiltration membrane has a size of 0.22-0.45μm; the activated carbon used for removing pigment has a solid-liquid ratio of 1 / 100-2 / 100 g / mL; the step of removing fat and polysaccharide by reverse phase silica gel specifically comprises the following steps: further removing fat and polysaccharide impurities from the filtrate obtained after the activated carbon is used to remove pigment by passing the filtrate through a reverse phase silica gel column, and the removing impurities process comprises the following steps performed in sequence: washing and removing polysaccharide by ultrapure water with a solid-liquid ratio of 1 / 500-5 / 500 g / mL, and washing and removing fat by 70wt%-80wt% ethanol solution with a solid-liquid ratio of 1 / 500-2 / 500 g / mL.

2. The method of claim 1, wherein: The organic selenium flavonoid compound comprises dihydromyricetin, myricetin, puerarin, daidzin, gastrodin, p-hydroxybenzyl alcohol, oleanolic acid, ursolic acid, and organic selenium.

3. The method of claim 2, wherein: The total flavonoid content of the organic selenium flavonoid compound is greater than or equal to 85%; the dihydromyricetin content is 60-70%, the myricetin content is 0.5-1.0%, the puerarin content is 0.5-0.9%, the daidzin content is 0.3-0.6%, the gastrodin content is 1-1.5%, the p-hydroxybenzyl alcohol content is 0.1-0.3%, the oleanolic acid content is 0.2-0.8%, and the ursolic acid content is 0.2-0.8%.

4. The organic selenium flavonoid compound prepared by the method of any one of claims 1-3 has high antioxidant activity and low toxic side effects.

5. The organic selenium flavonoid compound prepared by the method of any one of claims 1-3 is used as an antioxidant active ingredient in a functional food factor.

6. A functional food factor having antioxidant activity, characterized by comprising: The organic selenium flavonoid compound prepared by the method of any one of claims 1-3.

7. A functional food having antioxidant activity, characterized by comprising: The functional food factor with antioxidant activity and the food and pharmaceutical acceptable auxiliary in any dosage form of claim 6.

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