Preparation method of trametes robiniophila nano-selenium

Abstract

The invention relates to the technical field of biological medicines, and discloses a trametes robiniophila nano-selenium preparation method, which comprises: S1, obtaining trametes robiniophila powder; s2, obtaining a trametes robiniophila extract solution; s3, obtaining a high-purity trametes robiniophila extract; s4, sodium selenite is selected as a selenium source, ascorbic acid is used as a reducing agent, and a chemical reduction reaction is performed to generate nano-selenium particles; s5, adding a surfactant or a stabilizer; s6, mixing the trametes robiniophila extract solution with the nano-selenium particles in proportion; and S7, carrying out characterization on the trametes robiniophila nano-selenium compound. The trametes robiniophila nano-selenium compound shows a remarkable effect in the aspect of inhibiting tumor cell proliferation, compared with single use of trametes robiniophila extract or nano-selenium, the anti-tumor activity of the trametes robiniophila nano-selenium compound is greatly enhanced, rich polysaccharide and flavonoid components in trametes robiniophila are combined with nano-selenium particles, and the anti-tumor activity of the trametes robiniophila nano-selenium compound is improved. Therefore, the compound has more advantages in the aspects of inducing tumor cell apoptosis, inhibiting tumor cell proliferation and regulating an immune system.

Description

Technical Field

[0001] The present invention relates to the field of biological medicine technology, and specifically to a preparation method of tremella aurantia polysaccharide nano-selenium. Background Art

[0002] Tremella aurantia polysaccharide (Trametes robiniophila Murr) is a traditional Chinese medicinal material that has been widely studied and applied in anti-tumor, immunomodulatory, antioxidant and other aspects. Tremella aurantia polysaccharide is rich in various bioactive components, such as polysaccharides and flavonoids, and has significant biomedical effects. In current research, tremella aurantia polysaccharide extracts have been applied to various drugs and health products, especially showing relatively good effects in anti-tumor and immunomodulatory aspects. At the same time, nano-selenium, as a new form of selenium, has high biological activity, stability and low toxicity, and is widely used in anti-tumor, antioxidant, immunomodulatory and other fields.

[0003] However, the separate use of tremella aurantia polysaccharide and nano-selenium in the prior art often has certain limitations in terms of effects. Although the tremella aurantia polysaccharide extract has strong antioxidant and anti-tumor activities, its bioavailability is low and its stability in vivo is poor, resulting in certain limitations in its pharmacological effects. At the same time, although pure nano-selenium has good anti-tumor and antioxidant properties, due to the aggregation problem of its particles and low biocompatibility, it is often difficult to achieve ideal therapeutic effects in practical applications. Summary of the Invention

[0004] In view of the deficiencies of the prior art, the present invention provides a preparation method of tremella aurantia polysaccharide nano-selenium, which solves the problem that the separate use of tremella aurantia polysaccharide and nano-selenium in the prior art often has certain limitations in terms of effects.

[0005] To achieve the above objectives, the present invention is realized through the following technical solutions: A preparation method of tremella aurantia polysaccharide nano-selenium, comprising the following steps:

[0006] S1. Select high-quality tremella aurantia polysaccharide raw materials, wash, dry and pulverize them to obtain tremella aurantia polysaccharide powder;

[0007] S2. Use ultrasonic-assisted extraction or enzymatic extraction methods to extract polysaccharides and flavonoids from the tremella aurantia polysaccharide powder to obtain a tremella aurantia polysaccharide extract solution;

[0008] S3. Filter, concentrate and dry the tremella aurantia polysaccharide extract solution to obtain a high-purity tremella aurantia polysaccharide extract;

[0009] S4. Select sodium selenite as the selenium source and use ascorbic acid as the reducing agent to carry out a chemical reduction reaction to generate nano-selenium particles;

[0010] S5. By adding surfactants or stabilizers, ensure the uniform dispersion of nano-selenium particles and obtain stable nano-selenium particles;

[0011] S6. Mix the tremella aurantialba extract solution and nano-selenium particles in proportion, and use ultrasonic treatment to promote the full combination of tremella aurantialba extract and nano-selenium particles;

[0012] S7. Characterize the tremella aurantialba nano-selenium complex. Use dynamic light scattering technology to measure the particle size of nano-selenium particles, use scanning electron microscopy to observe the particle morphology, and analyze the binding mode of tremella aurantialba extract and nano-selenium particles through infrared spectroscopy.

[0013] Preferably, the tremella aurantialba raw material in step S1 is tremella aurantialba fruiting body or pretreated tremella aurantialba extract, and the drying temperature of the tremella aurantialba raw material is 30°C to 60°C, and the drying time is 24 hours to 48 hours.

[0014] Preferably, in step S2, when using ultrasonic-assisted extraction, the ultrasonic frequency is 20 kHz to 60 kHz, the ultrasonic extraction temperature is 40°C to 80°C, and the ultrasonic extraction time is 30 minutes to 120 minutes.

[0015] Preferably, in step S2, when using enzymatic extraction, the enzyme used is xylanase or cellulase, the enzymatic hydrolysis temperature is 40°C to 60°C, and the enzymatic hydrolysis time is 2 hours to 6 hours.

[0016] Preferably, in step S2, the extraction solvent is water or a mixed solution of water and alcohol solvents, and the volume ratio of the solvents is 80:20 to 95:5.

[0017] Preferably, in step S3, the tremella aurantialba extract solution is filtered through a filter membrane to remove large particle impurities, then the solvent in the solution is concentrated by rotary evaporation, and finally the tremella aurantialba extract powder is obtained through freeze-drying treatment.

[0018] Preferably, in step S4, the pH value of the reaction solution is adjusted to 6.0 to 7.0, the reaction temperature is controlled at 25°C to 60°C, and the reaction time is 1 hour to 4 hours, so that the selenium source sodium selenite is completely reduced to nano-selenium particles.

[0019] Preferably, in step S5, the addition amount of the surfactant or stabilizer is 0.1% to 2%, and the surfactant used is polyvinylpyrrolidone or polyvinyl alcohol, so that the nano-selenium particles are evenly dispersed and agglomeration is avoided.

[0020] Preferably, in step S6, the ultrasonic frequency is controlled at 20 kHz to 40 kHz, and the ultrasonic treatment time is 30 minutes to 90 minutes, so that the tremella aurantialba extract and nano-selenium particles are fully combined.

[0021] Preferably, in step S7, the particle size distribution range of the nano-selenium particles is determined to be 20 nm to 100 nm using dynamic light scattering technology, the morphology of the particles is observed using a scanning electron microscope, and the chemical bonding between the Sophora japonica extract and the nano-selenium particles is analyzed using infrared spectroscopy.

[0022] The present invention provides a method for preparing Sophora japonica fungus nano-selenium, which has the following beneficial effects:

[0023] 1. The present invention shows significant effects in inhibiting tumor cell proliferation through the Sophora japonica erulea nano-selenium complex. Compared with the use of Sophora japonica erulea extract or nano-selenium alone, its anti-tumor activity is greatly enhanced. The combination of rich polysaccharides and flavonoids in Sophora japonica erulea and nano-selenium particles makes the complex more advantageous in inducing tumor cell apoptosis, inhibiting tumor cell proliferation, and regulating the immune system. Therefore, the Sophora japonica erulea nano-selenium complex provides an innovative auxiliary means for tumor treatment, which can bring better therapeutic effects to patients.

[0024] 2. The anti-oxidation effect of the Sophora japonica nano-selenium complex of the present invention is significantly better than that of Sophora japonica extract and nano-selenium alone. Sophora japonica itself contains rich antioxidant components, such as flavonoids and polysaccharides. The combination with nano-selenium not only enhances its ability to scavenge free radicals, but also improves its stability. This complex can effectively reduce oxidative stress in the body and prevent cell damage, and has significant anti-aging, cardiovascular protection, anti-inflammatory and other health benefits. Therefore, Sophora japonica nano-selenium has a wide range of application potential in the field of health management.

[0025] 3. The present invention can obtain uniform particle size distribution and good dispersibility of the Sophora japonica nano-selenium complex by precisely controlling the ratio of Sophora japonica extract to nano-selenium particles, and by physical and chemical treatment, thereby enhancing its absorption and bioavailability in vivo. This high bioavailability enables the Sophora japonica nano-selenium complex to more effectively exert its pharmacological effects. At the same time, the stability of the complex is greatly improved, avoiding the precipitation or aggregation of nano-selenium particles in the body, thereby improving its application value in medicines and health products.

[0026] 4. The present invention uses a method combining ultrasound-assisted extraction and nano-selenium chemical reduction to enable the active ingredients in the Sophora japonica extract to form a stable complex with nano-selenium particles. This innovative process greatly improves the biological activity of the Sophora japonica extract and maximizes the advantages of nano-selenium. This preparation method is not only simple and efficient, but also can obtain high-quality complexes under lower reaction conditions, and has great potential for industrial production.

[0027] 5. The Trametes robiniophila Murr. nano-selenium complex of the present invention can effectively regulate the immune system, improve the activities of immune cells (such as macrophages, T lymphocytes, and B lymphocytes), and promote the secretion of immune factors (such as interleukin, interferon, etc.). This effect makes Trametes robiniophila Murr. nano-selenium have important application value in preventing and adjuvantly treating various diseases caused by low immune function (such as repeated infections and increased susceptibility to chronic diseases). This not only increases the multifunctionality of the Trametes robiniophila Murr. nano-selenium complex but also opens up broad application prospects in immune regulation and health management. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is a flowchart of a preparation method of Trametes robiniophila Murr. nano-selenium of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0029] Next, the technical solutions of the present invention will be clearly and completely described in conjunction with the drawings of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art without making creative efforts based on the embodiments of the present invention belong to the scope of protection of the present invention.

[0030] Please refer to the attached Figure 1 , an embodiment of the present invention provides a preparation method of Trametes robiniophila Murr. nano-selenium, including the following steps:

[0031] S1. Select high-quality Trametes robiniophila Murr. raw materials, wash, dry, and pulverize them to obtain Trametes robiniophila Murr. powder;

[0032] S2. Use ultrasonic-assisted extraction or enzymatic extraction methods to extract polysaccharides and flavonoids from Trametes robiniophila Murr. powder to obtain a Trametes robiniophila Murr. extract solution;

[0033] S3. Filter, concentrate, and dry the Trametes robiniophila Murr. extract solution to obtain a high-purity Trametes robiniophila Murr. extract;

[0034] S4. Select sodium selenite as the selenium source and use ascorbic acid as the reducing agent to carry out a chemical reduction reaction to generate nano-selenium particles;

[0035] S5. By adding a surfactant or a stabilizer, ensure the uniform dispersion of nano-selenium particles to obtain stable nano-selenium particles;

[0036] S6. Mix the Trametes robiniophila Murr. extract solution and the nano-selenium particles in proportion, and use ultrasonic treatment to promote the full combination of the Trametes robiniophila Murr. extract and the nano-selenium particles;

[0037] S7. Characterize the Trametes robiniophila Murr. nano-selenium complex, determine the particle size of the nano-selenium particles using dynamic light scattering technology, observe the particle morphology using a scanning electron microscope, and analyze the binding mode between the Trametes robiniophila Murr. extract and the nano-selenium particles through infrared spectroscopy.

[0038] Specifically, the core of the preparation method of Trametes robiniophila Murr. nano-selenium of the present invention is to achieve the combination of the Trametes robiniophila Murr. extract and the nano-selenium particles through multi-step operations. First, obtain a high-purity Trametes robiniophila Murr. extract through the extraction step of the Trametes robiniophila Murr. extract. This step can greatly improve the activity of the active ingredients in Trametes robiniophila Murr., providing a good basis for the subsequent combination with nano-selenium. Then, prepare nano-selenium particles by chemical reduction method. By the action of a reducing agent, a selenium source (such as sodium selenite) is reduced to nano-scale selenium particles. Finally, promote the combination of the Trametes robiniophila Murr. extract and the nano-selenium particles through physical means such as ultrasonic waves to obtain a Trametes robiniophila Murr. nano-selenium complex with excellent properties. Through this method, a Trametes robiniophila Murr. nano-selenium complex with uniform particle dispersion, high stability, and strong activity can be obtained, providing an ideal material basis for applications in the biomedical field, especially in aspects such as anti-tumor, immunomodulation, and antioxidant.

[0039] The Trametes robiniophila Murr. raw material in step S1 is Trametes robiniophila Murr. fruiting body or a pretreated Trametes robiniophila Murr. extract, and the drying temperature of the Trametes robiniophila Murr. raw material is 30°C to 60°C, and the drying time is 24 hours to 48 hours.

[0040] Specifically, the selection of the Trametes robiniophila Murr. raw material is an important factor affecting the preparation process. The Trametes robiniophila Murr. fruiting body is the most common and effective raw material, containing rich active ingredients such as Trametes robiniophila Murr. polysaccharide and flavonoids, which can enhance the pharmacological activity of the Trametes robiniophila Murr. nano-selenium complex. During the drying process, appropriate temperature and time can maximize the retention of the active ingredients in Trametes robiniophila Murr. If the drying temperature is too high, it may cause the degradation of the active ingredients; if the drying time is too long, it may cause excessive loss of moisture, affecting the activity of the Trametes robiniophila Murr. raw material. Therefore, setting the temperature at 30°C to 60°C and the drying time at 24 hours to 48 hours can effectively protect the beneficial ingredients in Trametes robiniophila Murr. and ensure the best activity of the Trametes robiniophila Murr. raw material.

[0041] In step S2, when using ultrasonic-assisted extraction, the ultrasonic frequency is 20 kHz to 60 kHz, the ultrasonic extraction temperature is 40°C to 80°C, and the ultrasonic extraction time is 30 minutes to 120 minutes.

[0042] Specifically, ultrasonic-assisted extraction is an efficient extraction technique that can effectively improve the extraction efficiency of active ingredients such as polysaccharides and flavonoids in Tremella aurantialba. The frequency, temperature, and time of ultrasonic waves are the key factors affecting the extraction effect. Controlling the frequency within the range of 20 kHz to 60 kHz can ensure that the ultrasonic energy is strong enough to rupture the cell walls of Tremella aurantialba, thereby releasing more active ingredients. And controlling the temperature within the range of 40 °C to 80 °C can avoid the degradation of Tremella aurantialba components caused by high temperature while maintaining the high efficiency of extraction. By setting the extraction time between 30 minutes and 120 minutes, the extraction time can be adjusted according to the type of Tremella aurantialba and the properties of the extraction solvent to ensure the efficient extraction of active ingredients.

[0043] In step S2, when enzymatic hydrolysis extraction is used, the enzyme used is xylanase or cellulase, the enzymatic hydrolysis temperature is 40 °C to 60 °C, and the enzymatic hydrolysis time is 2 hours to 6 hours.

[0044] Specifically, enzymatic hydrolysis extraction, as another efficient extraction method, has the advantage of being able to extract the active ingredients in Tremella aurantialba more gently, avoiding the destruction of active ingredients by high temperature. Xylanase and cellulase can effectively decompose the cell walls of Tremella aurantialba and help release substances such as polysaccharides and flavonoids. The temperature of the enzymatic hydrolysis reaction is controlled between 40 °C and 60 °C, avoiding the inactivation of enzymes caused by high temperature while maintaining the extraction efficiency of the active ingredients in Tremella aurantialba. The enzymatic hydrolysis time is 2 hours to 6 hours to ensure that the enzyme can fully play its role in decomposing the cell walls in Tremella aurantialba and releasing the active ingredients to the greatest extent.

[0045] In step S2, the extraction solvent is water or a mixed solution of water and alcohol solvents, and the volume ratio of the solvents is 80:20 to 95:5.

[0046] Specifically, the choice of solvent directly affects the extraction efficiency and the dissolution rate of the active ingredients in Tremella aurantialba. As a green solvent, water can effectively extract water-soluble components such as polysaccharides and flavonoids in Tremella aurantialba. Alcohol solvents (such as ethanol and methanol) can extract certain flavonoid substances in Tremella aurantialba, increasing the diversity of extraction. The mixed solution of water and alcohol solvents can ensure efficient extraction while avoiding the problem of incomplete extraction components that may be brought about by a single solvent. The solvent ratio of 80:20 to 95:5 can effectively balance the solubility of water and alcohol solvents, thereby maximizing the extraction rate of the active ingredients in Tremella aurantialba.

[0047] In step S3, the Tremella aurantialba extract solution is filtered through a filter membrane to remove large particle impurities, then the solvent in the solution is concentrated by rotary evaporation, and finally the Tremella aurantialba extract powder is obtained through freeze-drying treatment.

[0048] Specifically, the filtration step can effectively remove large particulate impurities in the Trametes robiniophila Murr. extract, ensuring the smooth progress of subsequent steps. Rotary evaporation is used to concentrate the solvent in the solution to increase the concentration of the extract and reduce the volume of the solvent, thereby obtaining a Trametes robiniophila Murr. extract with higher purity. Lyophilization is a mild drying method used to avoid the destruction of the active ingredients of Trametes robiniophila Murr. by heat treatment. Through lyophilization, the active ingredients in the Trametes robiniophila Murr. extract can be retained to the greatest extent, while preventing its oxidation or degradation. The finally obtained Trametes robiniophila Murr. extract powder has high purity and high activity, providing a good basis for the subsequent composite of nano-selenium particles.

[0049] In step S4, the pH value of the reaction solution is adjusted to 6.0 to 7.0, the reaction temperature is controlled at 25°C to 60°C, and the reaction time is 1 hour to 4 hours, so that the selenium source sodium selenite is completely reduced to nano-selenium particles.

[0050] Specifically, the adjustment of the pH value has an important influence on the effect of the reduction reaction. By adjusting the pH value of the reaction solution to 6.0 to 7.0, the optimal conditions for the reaction between the reducing agent (such as ascorbic acid) and sodium selenite can be ensured, and the selenium source can be reduced to nano-selenium particles. The temperature control in the range of 25°C to 60°C ensures the smooth progress of the reduction reaction during the reaction process, avoiding side reactions caused by high temperature or the decomposition of the reducing agent. The reaction time of 1 hour to 4 hours can ensure that the reduction reaction proceeds completely and does not cause unnecessary side reactions due to too long reaction time. Through these optimized conditions, nano-selenium particles with uniform particle size and good stability can be obtained.

[0051] In step S5, the addition amount of the surfactant or stabilizer is 0.1% to 2%, and the surfactant used is polyvinylpyrrolidone or polyvinyl alcohol, so that the nano-selenium particles are evenly dispersed and the agglomeration phenomenon is avoided.

[0052] Specifically, the stability of nano-selenium particles directly affects their bioavailability and application effects. The addition of surfactants or stabilizers can effectively prevent the agglomeration of nano-selenium particles in the solution, thereby maintaining the uniform dispersion of the particles. Polyvinylpyrrolidone and polyvinyl alcohol are common nano-particle stabilizers, which can effectively coat and stabilize nano-selenium particles and reduce the mutual attraction between particles. By adjusting the concentration of the surfactant, the dispersion degree of nano-selenium particles can be controlled, and their biological activity and application effects can be improved. The concentration range of 0.1% to 2% can not only effectively improve the particle dispersion but also avoid the cost increase caused by excessive addition.

[0053] In step S6, the ultrasonic frequency is controlled at 20 kHz to 40 kHz, and the ultrasonic treatment time is 30 minutes to 90 minutes, so that the Trametes robiniophila Murr. extract and nano-selenium particles are fully combined.

[0054] Specifically, the purpose of ultrasonic treatment is to promote the interaction between the Trametes robiniophila Murr. extract and nano-selenium particles through the high-frequency vibration of ultrasonic waves. Frequencies in the range of 20 kHz to 40 kHz can provide sufficient energy to break the macromolecular structure in the Trametes robiniophila Murr. extract, thereby improving the binding efficiency with nano-selenium particles. The treatment time is 30 minutes to 90 minutes to ensure that the active ingredients in the Trametes robiniophila Murr. extract are fully combined with nano-selenium particles and that the complex is not degraded due to excessive ultrasonic treatment for too long. Through this treatment method, a stable and uniform Trametes robiniophila Murr.-nano-selenium complex can be obtained.

[0055] In step S7, the dynamic light scattering technique is used to determine that the particle size distribution range of nano-selenium particles is 20 nm to 100 nm. The morphology of the particles is observed by scanning electron microscopy, and the infrared spectrum is used to analyze the bonding mode between the Trametes robiniophila Murr. extract and nano-selenium particles through chemical bonds.

[0056] Specifically, characterization is a key step in testing the quality and properties of the Trametes robiniophila Murr.-nano-selenium complex. The dynamic light scattering technique can effectively determine the particle size and particle size distribution of nano-selenium particles, ensuring the uniformity of the particles. The particle size is controlled between 20 nm and 100 nm, meeting the bioactivity requirements of nanoparticles. Scanning electron microscopy can directly observe the morphology and size of the particles to ensure that the morphology of the particles meets the expectations. The infrared spectrum can further analyze the bonding mode between the Trametes robiniophila Murr. extract and nano-selenium particles, confirm whether the complex is synthesized through chemical bonds, and verify its stability. These detection methods can ensure the quality and application effect of the final product.

[0057] 1. Synergistic effect of anti-tumor and antioxidant effects

[0058] The improvement of anti-tumor effect is closely related to antioxidant effect. The occurrence and development of tumor cells are often accompanied by an increase in oxidative stress, that is, the excessive production of free radicals and reactive oxygen species. These oxidizing substances can damage cellular DNA, lipids, and proteins, thereby accelerating the proliferation, invasion, and metastasis of tumor cells. The selenium component in the Trametes robiniophila Murr.-nano-selenium complex has strong antioxidant activity and can effectively scavenge free radicals in the body and slow down oxidative damage.

[0059] At the same time, the polysaccharide and flavonoid components in the Trametes robiniophila Murr. extract also have antioxidant properties and can enhance the antioxidant defense system of cells, further reducing the damage of oxidative stress to normal cells and immune cells. This dual antioxidant effect not only protects healthy cells from damage but also creates a favorable environment for inhibiting the proliferation and metastasis of tumor cells. Therefore, the anti-tumor effect of Trametes robiniophila Murr.-nano-selenium is significantly enhanced under the synergistic effect of antioxidant effect, forming a virtuous cycle of anti-tumor and antioxidant.

[0060] 2. Mutual promotion of immune regulation and anti-tumor effects

[0061] The immune system plays a crucial role in defending against tumor cells and their metastasis. The Trametes robiniophila Murr. nano-selenium complex enhances the body's immune response ability by regulating the immune system function and increasing the activity of immune cells (such as macrophages, T lymphocytes, and B lymphocytes). This enhanced immune response can not only eliminate cancer cells in the body but also recognize and attack tumor-associated immunosuppressive factors in the tumor microenvironment, preventing tumor growth and metastasis.

[0062] In particular, nano-selenium itself has a regulatory effect on the immune system. It can enhance the body's recognition and clearance of tumor cells by activating immune cells and promoting the secretion of immune factors (such as interleukin, interferon, etc.). Polysaccharides and flavonoids in the Trametes robiniophila Murr. extract can also enhance the anti-tumor immune response of the body by stimulating the activity of immune cells. Therefore, a complementary synergistic effect is formed between the immunomodulatory effect and the anti-tumor effect, further enhancing the efficacy of the Trametes robiniophila Murr. nano-selenium complex in tumor treatment.

[0063] 3. Mutual promotion between antioxidant effect and immunomodulatory effect

[0064] The antioxidant effect and immunomodulatory effect play important roles in maintaining the body's health and preventing diseases. Long-term oxidative stress can lead to impaired immune system function, decreased activity of immune cells, and thus reduced immune defense ability. The antioxidant effect of the Trametes robiniophila Murr. nano-selenium complex maintains and improves the normal function of the immune system by reducing oxidative damage and alleviating the damage to immune cells.

[0065] In addition, antioxidant substances can improve the activity of immune cells and enhance the immune response by inhibiting excessive free radicals. Therefore, the antioxidant properties of the Trametes robiniophila Murr. nano-selenium complex not only function independently but also synergistically enhance the immunomodulatory function. This mutually promoting mechanism enhances the overall biological activity of the Trametes robiniophila Murr. nano-selenium complex, showing obvious advantages especially in anti-aging, delaying immune decline, and enhancing the body's immune defense against external pathogens.

[0066] 4. Overall synergistic effect of anti-tumor, antioxidant, and immunomodulatory effects

[0067] The anti-tumor, antioxidant, and immunomodulatory effects of the Trametes robiniophila Murr. nano-selenium complex do not exist in isolation but interact and work synergistically. During anti-tumor process, the antioxidant effect reduces the oxidative damage and the proliferation rate of tumor cells; the immunomodulatory effect enhances the body's immune function and promotes the clearance and recognition of tumor cells. These mechanisms work together to enhance the anti-tumor efficacy of the Trametes robiniophila Murr. nano-selenium complex.

[0068] Meanwhile, the synergy of antioxidant and immunomodulatory effects can provide dual guarantees for tumor treatment. The antioxidant effect reduces the damage of oxidative stress to immune cells, thereby enhancing the efficiency of the immune system; the immunomodulatory effect enhances the activity of immune cells, contributing to the scavenging of free radicals and the slowing down of the oxidation process. Therefore, the Trametes robiniophila Murr nano-selenium complex forms a synergistic effect in multiple aspects, thus providing a more comprehensive treatment plan and enhancing the therapeutic effect.

[0069] Conclusion

[0070] The Trametes robiniophila Murr nano-selenium complex exhibits superior comprehensive efficacy through multiple mechanisms of action including anti-tumor, antioxidant, and immunomodulatory effects. These effects are not only independent but also promote each other through internal synergistic effects, forming a virtuous cycle that maximizes the pharmacological activity of the Trametes robiniophila Murr nano-selenium complex. Through this comprehensive effect, the Trametes robiniophila Murr nano-selenium complex has broad application potential in the field of biomedicine, especially in the fields of tumor treatment, immunomodulation, and antioxidant.

[0071] The following is an introduction in combination with specific embodiments:

[0072] Example 1: The highest data

[0073] S1 - Raw material treatment

[0074] Select high-quality fresh-picked Trametes robiniophila Murr fruiting bodies, remove the surface sediment and impurities, and wash them repeatedly 3 times with distilled water. Then place them in a vacuum drying oven, set the temperature to 60°C, and dry for 48 hours until the moisture content is lower than 5%. Use a high-speed pulverizer (rotation speed 20000 rpm) to pulverize through an 80-mesh sieve to obtain a fine Trametes robiniophila Murr powder for standby.

[0075] S2 - Extraction

[0076] Weigh 50 g of Trametes robiniophila Murr powder, add 500 mL of a water-ethanol mixed solution with a volume ratio of 80:20 (i.e., 400 mL of water + 100 mL of ethanol), place it in an ultrasonic extraction device, set the frequency to 60 kHz, the power to 300 W, control the extraction temperature at 80°C, and continuously perform ultrasonic treatment for 120 minutes. The extract is dark brown and has a faint unique aroma of Trametes robiniophila Murr.

[0077] S3 - Purification

[0078] The extract was filtered through a 0.45 μm microporous membrane to remove undissolved large particulate impurities. The filtrate was concentrated using a rotary evaporator (at 50 °C, reduced pressure to 0.08 MPa) to 1 / 5 of the original volume. Subsequently, the concentrated solution was placed in a freeze dryer (-50 °C, 0.01 MPa) and dried for 24 hours to obtain approximately 8 g of light brown Trametes robiniophila Murr. extract powder. Determined by the phenol-sulfuric acid method, the polysaccharide content was 87.6% (w / w), and the total flavonoid content determined by the aluminum salt colorimetric method was 6.2% (w / w), meeting the definition of high-purity Trametes robiniophila Murr. extract, i.e., the polysaccharide content ≥ 80% (w / w) and the total flavonoid content ≥ 5% (w / w).

[0079] Preparation of S4-nanoselenium

[0080] 100 mL of 0.1 mol / L sodium selenite solution was prepared, 200 mL of 0.2 mol / L ascorbic acid solution was added, and the pH was adjusted to 7.0 with 1 mol / L NaOH. The reaction was carried out with stirring (500 rpm) in a 60 °C water bath for 4 hours. The reaction solution gradually turned orange-red, indicating the formation of nanoselenium particles.

[0081] S5-Stable dispersion

[0082] 2 g of polyvinylpyrrolidone (PVP, molecular weight 40000) was added to the nanoselenium solution as a surfactant (concentration 2% w / v). After stirring for 30 minutes, it was ultrasonically dispersed (40 kHz, 100 W) for 15 minutes. The solution was clear and had no obvious precipitation, and the particles were evenly dispersed.

[0083] S6-Composite

[0084] 5 g of Trametes robiniophila Murr. extract powder was dissolved in 100 mL of distilled water and mixed with 200 mL of nanoselenium solution (the mass ratio of Trametes robiniophila Murr. extract to nanoselenium was approximately 1:1). It was treated under 40 kHz ultrasonic waves (power 200 W) for 90 minutes. The mixed solution was uniformly orange-brown, indicating that the two had combined fully.

[0085] S7-Characterization

[0086] Using dynamic light scattering (DLS), the average particle size of the nanoselenium particles was determined to be 100 nm, and the particle size distribution was narrow (PDI < 0.2). Scanning electron microscopy (SEM) showed that the particles were regular spherical shapes with smooth surfaces. Fourier transform infrared spectroscopy (FTIR) analysis indicated that the hydroxyl groups (-OH, 3400 cm -1 ) of Trametes robiniophila Murr. polysaccharides formed hydrogen bonds or weak chemical bonds with nanoselenium.

[0087] Activity evaluation - DPPH radical scavenging ability

[0088] Prepare a 1 mg / mL Trametes robiniophila Murr. nano-selenium complex solution. Take 2 mL and mix it with 2 mL of 0.1 mmol / L DPPH ethanol solution. After reacting in the dark for 30 minutes, measure the absorbance value (A x ) at 517 nm using a spectrophotometer. The control group is the DPPH solution without the sample (A0), and the blank group is pure ethanol (A1). The clearance rate calculation formula is: Clearance rate = [1 - (A x - A1) / A0] × 100%. The results show that the clearance rate is 92.5%, indicating that the complex has extremely strong antioxidant activity, probably due to the synergistic effect of high-concentration Trametes robiniophila Murr. polysaccharide, flavonoid, and nano-selenium.

[0089] Example 2: Minimum data

[0090] S1 - Raw material treatment

[0091] Select the Trametes robiniophila Murr. extract (commercially available dried Trametes robiniophila Murr.) after preliminary water washing pretreatment. Wash it twice with distilled water, then dry it in a forced-air drying oven at 30 °C for 24 hours until the moisture content drops below 6%. Manually grind it with a mortar and then pass it through a 60-mesh sieve to obtain rough Trametes robiniophila Murr. powder for standby.

[0092] S2 - Extraction

[0093] Weigh 20 g of Trametes robiniophila Murr. powder, add 200 mL of a water-ethanol mixed solution with a volume ratio of 95:5 (i.e., 190 mL of water + 10 mL of ethanol), add 0.5 g of xylanase (enzyme activity 100,000 U / g), and enzymatically hydrolyze it in a water bath at 40 °C for 2 hours (stirring speed 200 rpm). The enzymatic hydrolysate is light brown and slightly viscous.

[0094] S3 - Purification

[0095] Filter the enzymatic hydrolysate with a 0.22-μm filter membrane to remove the undissolved residue. Concentrate the filtrate using a rotary evaporator (40 °C, reduced pressure to 0.09 MPa) to 1 / 4 of the original volume. Dry the concentrated solution in a freeze dryer (-40 °C, 0.02 MPa) for 18 hours to obtain about 3 g of light yellow Trametes robiniophila Murr. extract powder. Determined by the phenol-sulfuric acid method, the polysaccharide content is 80.2% (w / w), and determined by the aluminum salt colorimetric method, the total flavonoid content is 5.1% (w / w), meeting the definition of high-purity Trametes robiniophila Murr. extract, that is, the polysaccharide content ≥ 80% (w / w) and the total flavonoid content ≥ 5% (w / w).

[0096] S4 - Nano-selenium preparation

[0097] Prepare 50 mL of 0.05 mol / L sodium selenite solution, add 100 mL of 0.1 mol / L ascorbic acid solution, and adjust the pH to 6.0 with 0.5 mol / L NaOH. The reaction is carried out with stirring (300 rpm) in a 25 °C constant temperature water bath for 1 hour. The reaction solution turns light orange, and nano-selenium particles are initially formed.

[0098] S5 - Stable dispersion

[0099] Add 0.1 g of polyvinyl alcohol (PVA, molecular weight 20000) as a stabilizer (concentration 0.1% w / v) to the nano-selenium solution, stir for 20 minutes, and then ultrasonically disperse (20 kHz, 50 W) for 10 minutes. The solution is slightly turbid but there is no obvious agglomeration.

[0100] S6 - Composite

[0101] Take 2 g of Tremella fuciformis Berk. extract powder and dissolve it in 50 mL of distilled water, mix it with 100 mL of nano-selenium solution (the mass ratio of Tremella fuciformis Berk. extract to nano-selenium is about 1:1), and treat it under 20 kHz ultrasonic waves (power 100 W) for 30 minutes. The mixed solution is light orange-brown and has a good combination degree.

[0102] S7 - Characterization

[0103] Dynamic light scattering measurement shows that the average particle size of nano-selenium particles is 20 nm, and the particle size distribution is slightly wide (PDI ≈ 0.3). SEM shows that the particles are irregular spherical and the surface is slightly rough. FTIR indicates that the carboxyl group (-COOH, 1700 cm-1) in the Tremella fuciformis Berk. extract binds to nano-selenium through weak chemical bonds.

[0104] Activity evaluation - DPPH radical scavenging ability

[0105] Prepare a 1 mg / mL Tremella fuciformis Berk. nano-selenium complex solution, and the test method is the same as in Example 1. The results show that the DPPH radical scavenging rate is 65.3%, and the antioxidant activity is relatively low, probably due to the short extraction time, small nano-selenium particles, and low surfactant concentration, which limit the synergistic effect between the active ingredients of Tremella fuciformis Berk. and nano-selenium.

[0106] Example 3: Intermediate data

[0107] S1 - Raw material treatment

[0108] Select wild Tremella fuciformis Berk. fruiting bodies, wash them 3 times with running water to remove surface dust, dry them in a 45 °C vacuum drying oven for 36 hours until the moisture content drops below 4%. Grind them with a high-speed grinder (rotation speed 15000 rpm) and pass through a 70-mesh sieve to obtain uniform Tremella fuciformis Berk. powder for standby.

[0109] S2 - Extraction

[0110] Weigh 30 g of Tremella aurantialba powder, add it to a 300 mL water-ethanol mixed solution with a volume ratio of 90:10 (i.e., 270 mL of water + 30 mL of ethanol), place it in an ultrasonic extraction device, with a frequency of 40 kHz, a power of 200 W, an extraction temperature of 60 °C, and ultrasonically treat for 75 minutes. The extract is medium brown in color and slightly smells of Tremella aurantialba.

[0111] S3 - Purification

[0112] Filter the extract with a 0.45 μm filter membrane to remove undissolved large particle impurities. Concentrate the filtrate with a rotary evaporator (45 °C, reduced pressure to 0.085 MPa) until it is concentrated to 1 / 3 of the original volume. Dry the concentrated solution in a freeze dryer (-45 °C, 0.015 MPa) for 20 hours to obtain approximately 5 g of pale yellow Tremella aurantialba extract powder. Determined by the phenol-sulfuric acid method, the polysaccharide content is 84.5% (w / w), and determined by the aluminum salt colorimetric method, the total flavonoid content is 5.8% (w / w), which meets the definition of high-purity Tremella aurantialba extract, that is, the polysaccharide content ≥ 80% (w / w) and the total flavonoid content ≥ 5% (w / w).

[0113] S4 - Preparation of nano-selenium

[0114] Prepare 75 mL of 0.075 mol / L sodium selenite solution, add 150 mL of 0.15 mol / L ascorbic acid solution, and adjust the pH to 6.5 with 1 mol / L NaOH. The reaction is carried out with stirring (400 rpm) in a 42.5 °C water bath for 2.5 hours. The reaction solution is orange-red, and the formation of nano-selenium particles is obvious.

[0115] S5 - Stable dispersion

[0116] Add 1 g of polyvinylpyrrolidone (PVP, molecular weight 40000) as a surfactant (concentration 1% w / v) to the nano-selenium solution, stir for 25 minutes, and then ultrasonically disperse (30 kHz, 75 W) for 12 minutes. The solution is transparent and stable.

[0117] S6 - Composite

[0118] Take 3 g of Tremella aurantialba extract powder and dissolve it in 75 mL of distilled water, mix it with 150 mL of nano-selenium solution (the mass ratio of Tremella aurantialba extract to nano-selenium is approximately 1:1), and treat it under 30 kHz ultrasonic waves (power 150 W) for 60 minutes. The mixed solution is uniformly orange-brown in color and has a good binding effect.

[0119] S7 - Characterization

[0120] The average particle size of nano-selenium particles determined by dynamic light scattering was 60 nm, and the particle size distribution was moderate (PDI≈0.25). SEM showed that the particles were nearly spherical and the surface was relatively smooth. FTIR analysis indicated that the hydroxyl groups (-OH, 3400 cm-1) and carboxyl groups (-COOH, 1700 cm-1) of the tremella polysaccharide were combined with nano-selenium through hydrogen bonds and weak chemical bonds.

[0121] Activity evaluation - DPPH radical scavenging ability

[0122] A 1 mg / mL solution of tremella nano-selenium complex was prepared, and the test method was the same as that in Example 1. The results showed that the DPPH radical scavenging rate was 83.7%, and the antioxidant activity was at a medium to high level, indicating that nano-selenium with a medium particle size and tremella extract formed a good synergistic effect, taking into account both the extraction efficiency and particle stability.

[0123] Table 1: Comparative experimental data of the preparation method of tremella nano-selenium and the prior art

[0124]

[0125]

[0126] Explanation of the meanings of the characters in the table

[0127] Preparation method

[0128] Example 1 (highest data): refers to the preparation method of using the extract of Sanghuangporus sanghuang and nano-selenium, and adopting the upper limit of the parameters in the claims (such as drying at 60 °C, ultrasonic at 60 kHz, reaction for 4 hours, etc.).

[0129] Example 2 (lowest data): adopting the lower limit of the parameters (such as drying at 30 °C, enzymatic hydrolysis for 2 hours, reaction for 1 hour, etc.).

[0130] Example 3 (intermediate data): adopting the intermediate value of the parameters (such as drying at 45 °C, ultrasonic at 40 kHz, reaction for 2.5 hours, etc.).

[0131] Chitosan nano-selenium (CS-SeNPs): nano-selenium particles prepared by chemical reduction method using chitosan (Chitosan, CS) as a stabilizer.

[0132] Hawthorn fruit nano-selenium (Hawthorn-SeNPs): nano-selenium particles prepared by green synthesis method using hawthorn fruit extract as a modifier.

[0133] DPPH radical scavenging rate (%)

[0134] Meaning: It represents the ability of the sample to scavenge DPPH (2,2-diphenyl-1-picrylhydrazyl radical), which is an index to measure antioxidant activity. The higher the value, the stronger the antioxidant ability.

[0135] Data format: Such as "92.5±2.1", where "92.5" is the average value and "±2.1" is the standard deviation, indicating the result fluctuation range of more than 3 repeated experiments.

[0136] Specific explanation:

[0137] Example 1: 92.5±2.1: The highest antioxidant activity, close to complete scavenging, possibly because more tremella polysaccharides and flavonoids were extracted at high temperature for a long time.

[0138] Example 2: 65.3±1.8: The lowest activity, because the extraction conditions are mild and less active ingredients are released.

[0139] Example 3: 83.7±1.9: Medium to high, balancing extraction efficiency and process stability.

[0140] CS-SeNPs: 70.0±3.0: Medium antioxidant, and the antioxidant ability of chitosan itself is limited.

[0141] Hawthorn-SeNPs: 85.0±2.5: Higher antioxidant, because hawthorn fruit polysaccharide has natural antioxidant properties, but it is not as good as Example 1.

[0142] Survival rate of A549 cells (%)

[0143] Meaning: It represents the inhibitory effect of the sample on A549 cells (human lung adenocarcinoma cell line) at a specific concentration (such as 10 μg / mL). The lower the survival rate, the stronger the cytotoxicity (anticancer activity).

[0144] Data format: Such as "15.2±1.5", "15.2" is the average survival rate, and "±1.5" is the standard deviation, reflecting the repeatability of the experiment.

[0145] Specific explanation:

[0146] Example 1: 15.2±1.5: The lowest survival rate and the strongest cytotoxicity, possibly because tremella flavonoids and larger selenium nanoparticles (100 nm) synergistically inhibit cancer cells.

[0147] Example 2: 35.8±2.0: Higher survival rate and weaker toxicity, because fewer components are extracted and the particles are small (20 nm).

[0148] Example 3: 22.4±1.7: Medium survival rate and moderate toxicity, suitable for various purposes.

[0149] CS-SeNPs: 40.0 ± 3.5: Higher survival rate, lower toxicity. Chitosan mainly provides stability rather than anti-cancer activity.

[0150] Hawthorn-SeNPs: 30.0 ± 2.8: Moderate survival rate, anti-cancer activity superior to CS-SeNPs but inferior to Tremella fuciformis-SeNPs.

[0151] Advantages

[0152] Meaning: Summarize the advantages of each method in terms of antioxidant activity, cytotoxicity, and process characteristics.

[0153] Specific explanations:

[0154] Example 1: Emphasize high antioxidant and anti-cancer activities. The active components of Tremella fuciformis and nano-selenium have a significant synergy, and the particle size distribution is uniform (PDI < 0.2).

[0155] Example 2: Still active under mild conditions, simple process, suitable for low-toxicity requirements (such as health products).

[0156] Example 3: Balanced performance, good stability (PVP concentration 1%), wide application.

[0157] CS-SeNPs: High stability (encapsulated by chitosan), but insufficient antioxidant and anti-cancer activities.

[0158] Hawthorn-SeNPs: Green synthesis with strong antioxidant ability, but poor consistency (large batch differences in natural extracts).

[0159] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A preparation method of tremella aurantialba polysaccharide nano-selenium, characterized in that, It includes the following steps: S1. Select high-quality Tremella aurantialba raw materials, clean, dry, and pulverize them to obtain Tremella aurantialba powder; S2. Use ultrasonic-assisted extraction or enzymatic extraction methods to extract polysaccharides and flavonoids from the Tremella aurantialba powder to obtain a Tremella aurantialba extract solution; S3. Filter, concentrate, and dry the Tremella aurantialba extract solution to obtain a high-purity Tremella aurantialba extract; S4. Select sodium selenite as the selenium source and use ascorbic acid as a reducing agent to carry out a chemical reduction reaction to generate selenium nanoparticles; S5. By adding a surfactant or a stabilizer, ensure the uniform dispersion of the selenium nanoparticles to obtain stable selenium nanoparticles; S6. Mix the Tremella aurantialba extract solution and the selenium nanoparticles in proportion, and use ultrasonic treatment to promote the full combination of the Tremella aurantialba extract and the selenium nanoparticles; S7. Characterize the Tremella aurantialba-selenium nanocomposite, use dynamic light scattering technology to measure the particle size of the selenium nanoparticles, use a scanning electron microscope to observe the particle morphology, and analyze the binding mode between the Tremella aurantialba extract and the selenium nanoparticles by infrared spectroscopy.

2. The preparation method of tremella aurantialba atramentaria nanoselenium according to claim 1, characterized in that, The Tremella aurantialba raw material in step S1 is the Tremella aurantialba fruiting body or a pretreated Tremella aurantialba extract, and the drying temperature of the Tremella aurantialba raw material is 30°C to 60°C, and the drying time is 24 hours to 48 hours.

3. The preparation method of tremella aurantialba - nano selenium according to claim 1, characterized in that, In step S2, when using ultrasonic-assisted extraction, the ultrasonic frequency is 20 kHz to 60 kHz, the ultrasonic extraction temperature is 40°C to 80°C, and the ultrasonic extraction time is 30 minutes to 120 minutes.

4. The preparation method of tremella fuciformis berk nanoselenium according to claim 1, characterized in that In step S2, when using enzymatic extraction, the enzyme used is xylanase or cellulase, the enzymatic hydrolysis temperature is 40°C to 60°C, and the enzymatic hydrolysis time is 2 hours to 6 hours.

5. The preparation method of tremella aurantialba atramentaria nano-selenium according to claim 1, characterized in that, In step S2, the extraction solvent is water or a mixed solution of water and an alcohol solvent, and the volume ratio of the solvent is 80:20 to 95:

5.

6. The preparation method of tremella aurantialba polysaccharide nano-selenium according to claim 1, wherein, In step S3, the Tremella aurantialba extract solution is filtered through a filter membrane to remove large particle impurities, then the solvent in the solution is concentrated by rotary evaporation, and finally the Tremella aurantialba extract powder is obtained by freeze-drying treatment.

7. The preparation method of tremella aurantialba polysaccharide nano-selenium according to claim 1, wherein, In step S4, the pH value of the reaction solution is adjusted to 6.0 to 7.0, the reaction temperature is controlled at 25°C to 60°C, and the reaction time is 1 hour to 4 hours, so that the selenium source sodium selenite is completely reduced to selenium nanoparticles.

8. The preparation method of tremella aurantialba atramentaria nano selenium according to claim 1, characterized in that, In step S5, the addition amount of the surfactant or the stabilizer is 0.1% to 2%, and the surfactant used is polyvinylpyrrolidone or polyvinyl alcohol, so that the selenium nanoparticles are evenly dispersed and agglomeration is avoided.

9. The preparation method of tremella aurantialba polysaccharide nano-selenium according to claim 1, characterized in that, In step S6, the ultrasonic frequency is controlled at 20 kHz to 40 kHz, and the ultrasonic treatment time is 30 minutes to 90 minutes, so that the Tremella aurantialba extract and the selenium nanoparticles are fully combined.

10. The preparation method of tremella aurantialba atrocyanescens nano-selenium according to claim 1, characterized in that, In step S7, use dynamic light scattering technology to measure the particle size distribution range of the selenium nanoparticles to be 20 nm to 100 nm, use a scanning electron microscope to observe the particle morphology, and analyze the binding mode between the Tremella aurantialba extract and the selenium nanoparticles by chemical bonds by infrared spectroscopy.

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