Composite nutrient for triple dynamic immune defense as well as preparation method and application of composite nutrient

Abstract

The invention discloses a triple dynamic immune defense compound nutrient as well as a preparation method and application thereof, and relates to the technical field of immunity. The composition consists of red algae sulfated polysaccharide, beta-glucan, an elderberry extract, an echinacea purpurea extract, lactoferrin, nano-selenium, nano-zinc, citric acid and high fructose corn syrup according to a specific ratio. The core of the invention lies in that a triple dynamic immune defense shield theory of'oropharyngeal mucosa barrier (immediate defense)-system immune activation (short-term mobilization)-specific immune regulation (long-term consolidation) 'is proposed and constructed in the field of food for the first time, and through the cooperation of multiple mechanisms of physical blocking, biological inactivation, immune training and long-term regulation, the immunity of the oral pharyngeal mucosa is enhanced. And broad-spectrum prevention and auxiliary intervention on various viruses are realized. All the raw materials with homology of medicine and food and the food nutrition enhancer are adopted, so that the safety is high, and the applicable crowd is wide.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of immunology, and more particularly to a triple dynamic immune defense composite nutrient, as well as a preparation method and application thereof. BACKGROUND

[0002] Although modern medicine has made some progress in the field of antiviral, the existing mainstream technical means still has obvious limitations, and it is difficult to comprehensively cope with complex and variable viral threats. For example, the widely used chemical synthetic antiviral drugs, the mechanism of action often targets a specific replication step or key protein of the virus. This highly specific target design makes the drug particularly vulnerable when facing rapidly evolving viruses - viruses can easily develop drug resistance through genetic mutation, leading to reduced or even ineffective drug effects. In addition, chemical drugs often cause a series of side effects during metabolism in the human body, such as liver and kidney toxicity, digestive tract reactions, or abnormal activation of the immune system, limiting their safety for long-term or large-scale application.

[0003] In terms of immunological prevention, vaccines are undoubtedly the core means to control the spread of infectious diseases, but their development and deployment also have inherent lags and limitations. Vaccine development is usually based on known viral strains, and it takes a long period of time from pathogen identification, antigen screening to clinical verification, which cannot respond to new or emerging viral variants in a timely manner. When the vaccine is finally put into use, the virus may have undergone antigenic drift or antigenic shift, greatly reducing the protective effect of the original vaccine. Therefore, the epidemic prevention strategy relying on a single technical path often falls into the dilemma of "passive response" in actual combat.

[0004] In the field of nutritional health products, although there have been many studies on the supporting effect of composite nutritional compositions on the immune system, the design concept of most related products still stays at the level of simple component superposition or general nutritional supplementation. For example, some compositions emphasize the single effect of a certain type of nutrients, ignoring the multi-stage and multi-level characteristics of the immune system in response to pathogen invasion; other products only provide nutritional support as the basis, lacking a deep understanding of the timing and integrity of the immune defense mechanism. Due to the single mechanism and unclear targeting of such composite nutritional compositions, they cannot systematically simulate the precise coordination and dynamic response process exhibited by the human natural immune system when responding to pathogen invasion, and thus the supporting effect provided by them as daily nutritional supplements in the complex infection environment of the real world is often not comprehensive enough, and the actual application value is also very limited.

[0005] In recent years, multidisciplinary research has provided new scientific insights for the development of complex nutritional compositions with comprehensive immune support. Marine biology research has revealed that red algal-derived sulfated polysaccharides can act as highly effective "virus decoys" by competitively binding and blocking viral adhesion and invasion of host cells due to their structural similarity to host cell surface heparin sulfate. Immunology research has confirmed that beta-glucan derived from Saccharomyces cerevisiae can train innate immune cells and induce a "trained immunity" effect, enabling the body to mount a more rapid and stronger defense response upon re-exposure to pathogens. Nutritional research has further discovered that anthocyanins rich in elderberry can directly destroy viral envelope structures, while alkylamide components in echinacea can rapidly activate macrophages and natural killer cells. These findings reveal the potential value of specific nutritional ingredients in supporting immune defense and regulation from different perspectives.

[0006] However, despite these encouraging scientific findings, how to integrate these nutritional ingredients, which are scattered across different disciplines and act on different immune processes, into a unified and synergistic theoretical framework and composition design remains a technical challenge that has not yet been overcome. Current research mostly focuses on the mechanism exploration of single ingredients, lacking systematic simulation and timing control thinking of the entire immune defense process. Without fundamentally understanding and simulating the complete dynamic process of "immediate barrier - rapid mobilization - long-term memory" exhibited by the human immune system in response to viral invasion, it is difficult to design a truly efficient, broad-spectrum, and daily nutritional support complex nutritional composition. This is the core challenge and key bottleneck currently faced by the field. SUMMARY

[0007] In view of the above, the present application provides a triple dynamic immune defense complex nutrient and a preparation method and application thereof.

[0008] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows: A triple dynamic immune defense complex nutrient, the complex nutrient comprises the following components in parts by weight: Red algal sulfated polysaccharide 15-25 parts, beta-glucan 10-20 parts, elderberry extract 8-15 parts, echinacea extract 5-12 parts, lactoferrin 3-8 parts, nano-selenium 0.1-0.5 parts, nano-zinc 0.5-2 parts, citric acid 3-8 parts, fructose syrup 10-30 parts.

[0009] Core theory: triple dynamic immune defense shield The specific connotation and corresponding technical implementation of "triple dynamic immune defense shield" are as follows: First defense shield: oropharyngeal mucosal barrier (immediate defense) Viruses mainly invade the human body through the mucous membranes of the oral and nasal pharynx. This super defense aims to establish a chemical and physical barrier at the moment of entry, intercepting the virus at the "starting point" of infection.

[0010] Virus trap mechanism: Red algae sulfate polysaccharide can mimic the molecular structure of human cell surface heparin sulfate, acting as a "decoy" to competitively bind to the spike protein of the virus (such as the S protein of SARS-CoV-2), neutralizing the virus and expelling it with swallowing.

[0011] Bio-iron deprivation mechanism: Adding lactoferrin, by depriving the virus and bacteria of the iron ions necessary for their growth, inhibits their early colonization and reproduction in the throat.

[0012] Environmental acidification mechanism: Citric acid creates a local weak acid environment in the mouth, which is not conducive to the maintenance of the activity of acid-sensitive viruses (such as most enveloped viruses). Second defense shield: Systemic immune activation (short-term mobilization) If the virus breaks through the first line of defense, the body needs to quickly activate the non-specific immune response to clear the virus before it replicates in large quantities. This stage focuses on "quick response" and "powerful clearance".

[0013] Direct virus inactivation: Elderberry extract can effectively destroy the hemagglutinin spines and membrane integrity of enveloped viruses such as influenza virus and coronavirus, rendering them inactive.

[0014] Activation of innate immunity: Echinacea extract can quickly activate macrophages and natural killer cells, enhancing their ability to phagocytose and clear infected cells.

[0015] Key enzyme inhibition: Nano-zinc enters cells in the form of ions, directly inhibiting the RNA-dependent RNA polymerase activity of RNA viruses such as the new coronavirus, blocking viral replication. Third defense shield: Specific immune regulation (long-term consolidation) Provide long-term, intelligent immune protection, regulate specific immunity and induce "trained immunity" to enable the immune system to maintain a high level of alertness and response capability for a longer period of time.

[0016] Immune training effect: β-glucan can bind to the Dectin-1 receptor on immune cells such as macrophages, without causing excessive inflammation, but "training" them to produce a stronger and faster response when encountering real pathogens in the future.

[0017] Antioxidation and immune balance: Nano-selenium, as a core component of glutathione peroxidase, not only helps to reduce oxidative damage during the immune response, but also promotes T lymphocyte proliferation, optimizing the balance and efficiency of immune response. Further, the composition comprises the following components in parts by weight: Red algae sulfate polysaccharide 20 parts, beta-glucan 15 parts, elderberry extract 12 parts, guayule extract 8 parts, lactoferrin 5 parts, nano selenium 0.3 parts, nano zinc 1.2 parts, citric acid 5 parts, fructose syrup 20 parts.

[0018] Further, comprising the following steps: (1) Raw material pretreatment: Take the red algae sulfate polysaccharide raw material (molecular weight 50-200kDa, sulfate content ≥25%) or beta-glucan (β-1,3 and β-1,6 structure ratio ≥85%) that meets the quality standard, crush it by a super micro pulverizer, then pass through a 100 mesh sieve, and place it in a cool and dry place for standby; (2) Nanometer component pretreatment: According to the formula amount, weigh the nano selenium powder (particle size 40-60nm) or nano zinc powder (particle size 60-80nm), add 5 times its weight of pure water, and use an ultrasonic cell crusher to disperse for 15 minutes at a power of 400W to obtain a uniform nanodispersion; Mix the nanodispersion with malt dextrin (wall material) at a mass ratio of 1:3, and use a spray dryer to perform microencapsulation treatment, with an inlet temperature of 160±5℃ and an outlet temperature of 80±5℃, to obtain well-embedded microencapsulated nanoparticles; (3) Extract preparation: Prepare the elderberry extract and guayule extract; (4) Dosing and mixing According to the formula in claim 1, weigh the raw materials and mix them in a low-temperature and low-humidity environment (temperature <20℃, humidity <30%RH) using the equal increment method: First, mix the red algae sulfate polysaccharide and beta-glucan base material evenly, then add the elderberry extract and guayule extract in turn and mix evenly, add the microencapsulated nanoparticle mixture and mix evenly, finally add lactoferrin, citric acid, and 50% of the formula amount of fructose syrup, and mix evenly to obtain the premixed raw material; the mixing time for each step is not less than 15 minutes; (5) Granulation process: Mix the remaining 50% of the fructose syrup with pure water to prepare a 50% concentration solution, and add 0.1% hydroxypropyl methylcellulose as a binding enhancer; put the premixed raw material into a wet granulator, slowly add the binder, control the stirring speed at 200rpm, and the chopping speed at 1000rpm, to make suitable soft material; pass through a 20 mesh sieve to obtain uniform wet granules; (6) Drying and granulating: The wet granules are laid on the freeze-drying tray with a thickness controlled within 1 cm, frozen at -40℃ for 4 hours, and then gradually heated to 25℃ under a vacuum degree of <10 Pa until the moisture content is ≤4%. The dried granules are sized through a 24-mesh screen to remove fine powder and oversized granules.

[0019] Further, The red seaweed sulfated polysaccharide has a molecular weight of 50-200 kDa and a sulfate content of ≥25%; The β-glucan has a structure ratio of β-1, 3 to β-1, 6 of ≥85%; The lactoferrin has a purity of ≥95%; The high-fructose syrup has a fructose content of ≥55%; The nano-selenium powder has a particle size of 40-60 nm, and the nano-zinc powder has a particle size of 60-80 nm. Further, the preparation method of the elderberry extract in step (3) is as follows: S1-1 raw material pretreatment: Mature and non-rotten black elderberry fruits are selected, rinsed with clean water, and then drained; then a fruit and vegetable special crushing and pulping machine is used to crush and pulp the fruits to obtain fruit pulp; S1-2 enzymatic hydrolysis: 0.3% by mass of pectinase is added to the elderberry fruit pulp, and enzymatic hydrolysis is carried out at 50℃ for 1-2 hours; this step is aimed at destroying the pectin structure and improving the dissolution rate and extraction efficiency of active substances such as anthocyanins; S1-3 ethanol extraction: The enzymatically hydrolyzed fruit pulp is transferred to an extraction tank, 5 times the mass of 60%-70% ethanol solution is added, and hot reflux extraction is carried out at 60℃ for 2 times, 1.5 hours each time; S1-4 concentration and drying: The two extraction solutions are combined, filtered through a 100-mesh screen, and then the ethanol is recovered using a vacuum concentration unit at a temperature of ≤60℃ to obtain a concentrated extract. Finally, the extract is dried through a spray drying tower (inlet air temperature 160±5℃, outlet air temperature 80±5℃) to obtain a deep purple red elderberry extract powder.

[0020] In the extraction process of the active ingredients of the Aronia melanocarpa, the structural stability of the heat-sensitive anthocyanins is particularly concerned. By introducing pectinase for mild enzymatic treatment, the plant cell wall barrier is effectively broken, and the dissolution of the target ingredients is promoted, while avoiding the destruction of active ingredients under high temperature, strong acid and strong base conditions. In the extraction stage, multi-stage countercurrent extraction is carried out at a controlled temperature using an appropriate concentration of ethanol solution, which ensures the full release of anthocyanins while minimizing thermal degradation. In the subsequent concentration process, vacuum low-temperature evaporation technology is used, and the temperature is strictly controlled below 60 degrees Celsius to prevent anthocyanins from being inactivated due to high temperature. In the final drying step, spray drying process is selected, and by optimizing the inlet and outlet air temperature parameters, the retention rate of anthocyanins is significantly improved under the premise of ensuring the moisture content of the product, and the biological activity of anthocyanins is fully maintained. Further, the preparation method of the Echinacea purpurea extract in step (3) is: S2-1 raw material pretreatment: Take the dried Echinacea purpurea aerial part (including flowers, leaves, stems), cut into about 2-3 cm small pieces, and reserve; S2-2 extraction: Put the pretreated Echinacea purpurea raw material into a multifunctional extraction tank, add 10-12 times the mass of 70% ethanol, heat to 70-75℃, and perform hot reflux extraction for 3 times, each time for 1 hour; S2-3 concentration: Combine the three times of ethanol extraction liquid, and perform precision filtration with a plate and frame filter; under the conditions of ≤60℃, vacuum degree-0.08 to-0.10 MPa, the filtrate is concentrated to no alcohol taste by a double-effect concentrator, to obtain a flow extract with a relative density of about 1.10-1.15 (60℃); S2-4 extraction and enrichment: Dilute the flow extract with an equal amount of purified water, then perform liquid-liquid extraction with 80℃ petroleum ether to remove chlorophyll and other fat-soluble impurities; after discarding the petroleum ether layer, the water layer is extracted with an equal volume of ethyl acetate for 3 times; combine the ethyl acetate extraction liquid, and this step aims to enrich active ingredients such as alkyl amides; S2-5 drying: Vacuum reduce-pressure recovery of the ethyl acetate layer under the condition of ≤50℃ to obtain a paste; finally, dry it by a vacuum belt dryer, and crush it through a 100 mesh sieve to obtain a brownish to brownish yellow Echinacea purpurea extract powder.

[0021] This invention focuses on the efficient enrichment of alkylamide lipid-soluble components for the preparation of echinacea active ingredients. Hot reflux extraction with a specific concentration of ethanol solution ensures extraction efficiency and lays the foundation for subsequent purification. After preliminary concentration, a systematic liquid-liquid extraction separation strategy is employed, using petroleum ether to remove lipid-soluble impurities such as chlorophyll, followed by selective enrichment of the target alkylamide components with ethyl acetate. This refined purification process effectively removes water-soluble impurities such as polysaccharides and proteins, significantly increasing the content of active ingredients in the product. Finally, low-temperature vacuum drying technology is used to obtain the final product, ensuring the chemical stability and biological activity of the alkylamide components, providing a material basis for the product to achieve its expected immunomodulatory function. Application of a complex nutrient with triple dynamic immune defense in the preparation of antiviral drugs. As can be seen from the above technical solution, compared with the prior art, the beneficial effects of the present invention are as follows: This invention proposes a novel theoretical framework of "triple dynamic immune defense shield," enabling the design of antiviral foods to move from "listing ingredients" to "system simulation," achieving a revolutionary breakthrough in concepts.

[0022] Through a triple defense mechanism, it covers the entire process of viral invasion, replication, and pathogenesis, providing comprehensive protection from immediate to long-term. Each component works at different stages and targets different points, producing significant synergistic effects. The role, dosage, and compatibility of each component are based on solid modern virological and immunological research, with a clear mechanism of action and robust theoretical support. All ingredients are derived from the list of food and medicine homologous substances and legally permitted nutritional fortifiers, and have undergone rigorous toxicological testing, demonstrating no toxic side effects and making them suitable for long-term consumption by various population groups. The low-temperature freeze-drying flash-release process and microencapsulation technology employed maximize the efficacy of the active ingredients in the final product and optimize user experience and speed of action.

[0023] This invention constructs a multi-layered immune defense system with sequential coordination and complementary functions, achieving effective intervention in the entire process of viral invasion. In the initial stage of viral invasion, the molecular structural characteristics of red algae sulfated polysaccharide mimic host cell surface receptors, acting as a molecular decoy to competitively bind to the viral spike protein, forming a viral trap mechanism. Simultaneously, the iron-depriving effect of lactoferrin inhibits the early colonization of pathogens on the mucosal surface. Combined with the local weakly acidic environment created by citric acid, these three elements synergistically establish the first chemical and physical barrier in the oropharynx, achieving immediate interception and neutralization clearance.

[0024] Once the virus breaches the first line of defense, the system can rapidly initiate a non-specific immune response. The high concentration of anthocyanins in elderberry extract can directly disrupt the structural integrity of enveloped viruses, while echinacea extract, through its alkylamide components, quickly activates innate immune effector cells such as macrophages and natural killer cells. Simultaneously, nano-zinc ions can penetrate deep into cells, specifically inhibiting the activity of key enzymes involved in viral replication. These three actions together constitute a rapid-response second line of defense, completing the clearance task before the virus replicates on a large scale.

[0025] More importantly, this system also establishes a long-term immune regulation mechanism. β-glucan activates the training immune effect of innate immune cells through specific receptors, enabling the immune system to have a stronger memory response capacity; while nano-selenium, as a core component of key antioxidant enzymes, promotes lymphocyte functional balance while alleviating immune stress damage. The two work synergistically to achieve sustained and intelligent regulation of the immune system.

[0026] This technological solution breaks through the traditional single-target design approach, integrating dispersed antiviral mechanisms into a unified, systematic solution. The dosage and compatibility of each component are based on a solid foundation of modern virology and immunology research, with clear mechanisms of action and synergistic effects. All raw materials are sourced from the legally mandated food and drug homology list and the category of nutritional fortifiers, undergoing rigorous safety verification to ensure long-term safety for consumption. Through innovative low-temperature freeze-drying flash-release technology and microencapsulation technology, the biological efficacy of each active ingredient is preserved to the maximum extent, and the user experience and efficacy of the product are optimized, achieving comprehensive innovation from theoretical framework to product realization. Detailed Implementation

[0027] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Example 1

[0028] A complex nutrient for triple dynamic immune defense, the composition comprising, by weight, the following components: 20 parts of red algae sulfated polysaccharide, 15 parts of β-glucan, 12 parts of elderberry extract, 8 parts of echinacea extract, 5 parts of lactoferrin, 0.3 parts of nano selenium, 1.2 parts of nano zinc, 5 parts of citric acid, and 20 parts of fructose syrup. The preparation method of compound nutrients includes the following steps: (1) Raw material pretreatment: Take red algae sulfate polysaccharide raw materials that meet the quality standards (molecular weight 50-200kDa, sulfate content ≥25%) or β-glucan (β-1,3 and β-1,6 structural ratio ≥85%), pulverize them with an ultra-fine pulverizer, pass them through a 100-mesh sieve, and store them in a cool and dry place for later use. (2) Pretreatment of nano-components: Weigh out the nano-selenium powder (particle size 40-60nm) or nano-zinc powder (particle size 60-80nm) according to the formula, add 5 times their weight of purified water, and disperse them for 15 minutes using an ultrasonic cell disruptor at 400W power to obtain a uniform nano-dispersion. The nano-dispersion was mixed with maltodextrin (wall material) at a mass ratio of 1:3, and microencapsulation was performed using a spray dryer with an inlet air temperature of 160±5℃ and an outlet air temperature of 80±5℃ to obtain well-encapsulated microencapsulated nanoparticles. (3) Preparation of extract: Preparation of elderberry extract and echinacea extract; (4) Ingredients and mixing Weigh the raw materials according to the proportions described in claim 1, and mix them using an equal-incremental mixing method in a low-temperature and low-humidity environment (temperature <20℃, humidity <30%RH): First, mix red algae sulfate polysaccharide and β-glucan base evenly. Then, add elderberry extract and echinacea extract in sequence and mix evenly. Add microencapsulated nanoparticle mixture and mix evenly. Finally, add lactoferrin, citric acid and 50% of the formula weight of fructose syrup and mix evenly to obtain premixed raw materials. The mixing time for each step shall not be less than 15 minutes. (5) Granulation process: Mix the remaining 50% by weight of fructose syrup with purified water to prepare a 50% concentration solution, and add 0.1% hydroxypropyl methylcellulose as a binder; put the premixed raw materials into a wet granulator, slowly add the binder, control the stirring speed at 200 rpm and the chopping speed at 1000 rpm to make a suitable soft material; granulate through a 20-mesh sieve to obtain uniform wet granules. (6) Drying and granulation: Vacuum freeze-drying technology was used to spread the wet granules evenly on a freeze-drying tray with a thickness controlled within 1 cm. The tray was then placed at -40℃ for 4 hours until completely frozen. Under vacuum conditions of <10 Pa, the temperature was gradually increased to 25℃ and dried until the moisture content was ≤4%. The dried granules are granulated through a 24-mesh sieve to remove fine powder and oversized particles. The molecular weight of the red algae sulfated polysaccharide raw material is 50-200 kDa, and the sulfate content is ≥25%. The β-glucan (β-1,3 and β-1,6 structural ratio ≥85%). The purity of the lactoferrin is ≥95%; The fructose content of the fructose syrup is ≥55%; The nano-selenium powder has a particle size of 40-60 nm, and the nano-zinc powder has a particle size of 60-80 nm. The preparation method of the elderberry extract in step (3) is as follows: S1-1 Raw Material Pretreatment: Select ripe, unrotten black elderberry fruits, rinse them with clean water and drain them; then crush and pulp them using a fruit and vegetable crusher to obtain fruit pulp. S1-2 enzymatic hydrolysis: Add 0.3% pectinase to elderberry pulp and enzymatically hydrolyze it at 50°C for 1-2 hours; this step aims to break down the pectin structure and improve the dissolution rate and extraction efficiency of anthocyanins and other active substances. S1-3 Ethanol Extraction: The enzymatically hydrolyzed fruit pulp was transferred to an extraction tank, and 5 times its weight of 60%-70% ethanol solution was added. The mixture was then subjected to hot reflux extraction twice at 60°C, for 1.5 hours each time. S1-4 Concentration and Drying: The two extracts were combined, filtered through a 100-mesh sieve, and then the ethanol was recovered using a vacuum concentrator at ≤60℃ to obtain a concentrated extract. Finally, the extract was dried in a spray drying tower (inlet air temperature 160±5℃, outlet air temperature 80±5℃) to obtain a deep purplish-red elderberry extract powder. The preparation method of the echinacea extract in step (3) is as follows: S2-1 Raw Material Pretreatment: Take the dried above-ground parts of Echinacea purpurea (including flowers, leaves, and stems), cut them into small sections of about 2-3 cm, and set aside. S2-2 Extraction: Put the pretreated echinacea raw material into a multi-functional extraction tank, add 10-12 times the mass of 70% ethanol, heat to 70-75℃, and perform hot reflux extraction 3 times, 1 hour each time; S2-3 Concentration: The three ethanol extracts were combined and finely filtered using a plate and frame filter press. The filtrate was concentrated in a double-effect concentrator at ≤60℃ and a vacuum of -0.08 to -0.10 MPa until no alcohol odor was detected, yielding a fluid extract with a relative density of approximately 1.10-1.15 (60℃). S2-4 extraction and enrichment: The fluid extract was diluted with an equal volume of purified water, and then liquid-liquid extraction was performed with petroleum ether at 80°C to remove lipid-soluble impurities such as chlorophyll. After discarding the petroleum ether layer, the aqueous layer was extracted three times with an equal volume of ethyl acetate. The ethyl acetate extracts were combined. This step was intended to enrich active ingredients such as alkylamides. S2-5 drying: The ethyl acetate layer was subjected to vacuum decompression at ≤50℃ to recover the solvent, resulting in a paste. Finally, it was dried using a vacuum belt dryer and pulverized through a 100-mesh sieve to obtain a brownish-yellow echinacea extract powder. Example 2

[0029] A complex nutrient for triple dynamic immune defense. The complex nutrient comprises the following components by weight: 15 parts of red algae sulfated polysaccharide, 10 parts of β-glucan, 8 parts of elderberry extract, 5 parts of echinacea extract, 3 parts of lactoferrin, 0.1 parts of nano selenium, 0.5 parts of nano zinc, 3 parts of citric acid, and 30 parts of fructose syrup.

[0030] The preparation method of the compound nutrients is the same as in Example 1. Example 3

[0031] A complex nutrient for triple dynamic immune defense, comprising the following components by weight: 25 parts red algae sulfated polysaccharide, 20 parts β-glucan, 15 parts elderberry extract, 12 parts echinacea extract, 8 parts lactoferrin, 0.5 parts nano selenium, 2 parts nano zinc, 8 parts citric acid, and 10 parts fructose syrup.

[0032] The preparation method of the compound nutrients is the same as in Example 1. Example 4

[0033] In vitro toxicity test of compound nutrients 1. Materials and reagents: (1) Cells: Low passage canine kidney cell line (MDCK cells) and human laryngeal carcinoma epithelial cells (Hep-2 cells) were purchased from Shanghai Bosheng Biotechnology Co., Ltd.

[0034] (2) Reagents: DMEM culture medium, fetal bovine serum (FBS), and antibiotics were all purchased from Gibco, USA; phosphate-buffered saline (PBS) and 0.25% trypsin-0.02% EDTA were all purchased from Thermo Fisher Scientific, USA; CCK-8 assay kit (Dojindo, Japan). (3) Instruments and environment: clean bench, CO2 constant temperature incubator, 96-well micro cell culture plate, pipette, biological inverted microscope, centrifuge, vacuum freeze dryer (ScientZ), enzyme reader.

[0035] 2. Experimental Methods: Composition: The lyophilized compound nutrient powders obtained in Examples 1-5 were serially diluted with cell maintenance solution to obtain sample solutions with concentrations of 0, 0.5, 1, 2, 4, 6, 8, and 10 mg / mL.

[0036] Cell passage and plating were performed. When MDCK and Hep-2 cells in 96-well plates reached 80% dense monolayer and were in good condition, culture was stopped. Various concentrations of the compound nutrient sample were added to the cultured cells, with four replicates per concentration and 100 μL per well. The cells were incubated at 37°C with 5% CO2 for 24 h. Cell morphology was observed under an inverted microscope, and cell viability was assessed using a CCK-8 assay kit.

[0037] Cell proliferation activity (%) = [A(drug-treated) - A(blank)] / [A(0-drug-treated) - A(blank)] × 100%; Note: A (drug addition): Absorbance of the pores containing cells, CCK solution, and drug solution; A (blank): Absorbance of pores containing culture medium and CCK solution but without cells; A(0 drug): Absorbance of the pore containing cells and CCK solution but no drug solution.

[0038] Safety was evaluated based on cell viability levels and the allocation ratios of each group. Table 1. Effects of different concentrations of compound nutrients on cell viability (24h)

[0039] The results showed that the compound nutrients had no significant toxic effects on either cell type at concentrations ≤4 mg / mL, and cell viability remained above 95%. At concentrations of 1-2 mg / mL, cell proliferation was slightly promoted, reaching 10²-10⁶%. When the concentration was ≥6 mg / mL, cells showed shrinkage and shedding, and cell viability decreased significantly. Example 5

[0040] Determination of TCID50 of H1N1 influenza virus 1. Experimental materials 1.1 Experimental Cells and Viruses Low-passage canine kidney cell line (MDCK cells) was purchased from Shanghai Boson Biotechnology Co., Ltd. H1N1 influenza A virus strain, respiratory syncytial virus (RSV), and human coronavirus (HCoV-229E) were purchased from Signalway Antibody, Inc., USA, and stored at -80℃ for future use.

[0041] 1.2 Experimental Reagents DMEM medium, fetal bovine serum (FBS), antibiotics, virus culture medium, and stock solutions containing penicillin and streptomycin were all purchased from Gibco, USA; phosphate-buffered saline (PBS) and 0.25% trypsin-0.02% EDTA were all purchased from Thermo Fisher Scientific, USA.

[0042] 1.3 Experimental Apparatus Clean bench, CO2 incubator, 96-well microcell culture plate, pipette, inverted biological microscope, centrifuge, BSL-2 laboratory, biosafety cabinet.

[0043] 2. Experimental Methods 2.1 Cell Preparation (1) After cell culture, take well-grown MDCK cells and Hep-2 cells and plate them, and incubate them in an incubator for 24 hours.

[0044] 2.2 Virus inoculation (1) Virus dilution was performed using a semi-logarithmic dilution method, diluting the virus with viral culture medium to a concentration of 10-1. -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 10 -8 The serially diluted virus solution was inoculated into cell culture plates and incubated for 1 hour, with the plates shaken every half hour. Cells were washed once with 100 μL of PBS in each well, followed by the addition of virus culture medium and incubation. Cytopathic effects were observed daily.

[0045] 2.3 Virus titer determination Once the cytopathic effect stopped developing, the viral titer was calculated using the Reed and Muench methods.

[0046] 3. Experimental Results 3.1 Cell morphology observation The cytopathic effect was observed under an inverted microscope. The endpoint was set at the highest dilution where no new lesions appeared. When the virus infection lasted for 72 hours, the cytopathic effect stopped developing, and the phenomenon of cells becoming round, wrinkled, and detached could be clearly observed.

[0047] 3.2 Virus titration results Based on the Reed and Muench methods, the TCID50 values ​​of the three viruses for cell infection are shown in Table 2. Table 2. Median infection rate of three viruses on cells. Example 6

[0048] Antiviral effects of compound nutrients 1. Experimental materials Same as Example 5.

[0049] 2. Experimental Methods Cell plating: Same as in Example 5.

[0050] Dilution of compound nutrients: The compound nutrients obtained in Example 1 were diluted sequentially from the maximum non-toxic concentration to different concentrations to obtain composition concentrations of 0, 0.5, 1, 2, and 4 mg / mL.

[0051] Virus inoculation: After culture, the cell surface was washed three times with PBS. Then, 100 TCID50 of virus diluted in cell maintenance medium was added to each well (50 μL / well) for 1-2 hours, gently agitating the cells every 30 minutes. After adsorption, the supernatant virus solution was aspirated, and the cells were washed twice with PBS. Different concentrations of compound nutrients were added, and the cells were cultured in an incubator. Cell pathogenesis was observed every 8 hours. When the CPE in the virus control group reached 75%, cell viability in each group was assessed using CCK8 assay.

[0052] 3. Experimental Results 3.1 Cell morphology observation Observation of cytopathic effects after viral inoculation revealed obvious cell shrinkage, detachment, and death. Following drug intervention, the cytopathic characteristics weakened with increasing drug concentration.

[0053] 3.2 Antiviral Results of the Composition The control group, with a compound nutrient concentration of 0 mg / mL, showed low cell survival. However, after intervention with different concentrations of the compound nutrient, cell survival significantly increased. This indicates that the compound nutrient, within a safe concentration range, can effectively improve the survival rate of virus-infected cells and has a significant antiviral effect. The results are shown in Table 3.

[0054] Table 3. Antiviral effects of compound nutrients against three viruses. Example 7

[0055] Verification test of compound nutrient ratio To analyze the importance of each component in the composition, the optimization results of the compound ratio were compared and verified by removing a certain component or increasing its dosage, and the inhibitory effect of the composition on H1N1 influenza virus was also compared.

[0056] 1. Experimental materials The experiment on the effect of differences in the content of individual components in the compound nutrients on cytotoxicity was the same as in Example 29, and the experiment on the inhibitory effect of differences in the content of individual components in the compound nutrients on H1N1 influenza virus was the same as in Example 5.

[0057] 2. Experimental Methods Cell plating: Example 5.

[0058] Drug: Within the maximum non-toxic concentration range, the optimal concentration of the compound nutrient with the greatest antiviral effect was determined to be 2 mg / mL. The synergistic effects of each component were investigated separately. Exploration process (1): Referring to Example 1, the weight parts of red algae sulfated polysaccharide in the composition were replaced with 0 parts, 20 parts, and 30 parts, respectively, while other aspects remained unchanged, to obtain the corresponding compositions. The cytotoxicity and antiviral effects of each composition were tested, and the results are shown in Table 4 below.

[0059] Table 4. Toxicity and antiviral effects of compositions obtained with different amounts of red algae sulfated polysaccharides

[0060] Exploration process (2): Referring to Example 1, the weight parts of β-glucan in the composition were replaced with 0 parts, 15 parts, and 30 parts, respectively, while other aspects remained unchanged, to obtain the corresponding compositions. The cytotoxicity and antiviral effects of each composition were tested, and the results are shown in Table 5 below.

[0061] Table 5. Toxicity and antiviral effects of compositions with different β-glucan fractions.

[0062] Exploration process (3): Referring to Example 1, the weight parts of elderberry extract in the compositions were replaced with 0 parts, 12 parts, and 25 parts, respectively, while other aspects remained unchanged, to obtain the corresponding compositions. The cytotoxicity and antiviral effects of each composition were tested, and the results are shown in Table 6 below. Table 6. Toxicity and antiviral effects of compositions obtained from different amounts of elderberry extract.

[0063] Exploration process (4): Referring to Example 1, the weight parts of Echinacea extract in the compositions were replaced with 0 parts, 8 parts, and 12 parts, respectively, while other aspects remained unchanged, to obtain the corresponding compositions. The cytotoxicity and antiviral effects of each composition were tested, and the results are shown in Table 7 below.

[0064] Table 7. Toxicity and antiviral effects of compositions obtained from different amounts of elderberry extract.

[0065] Experimental results The results showed that excessively high or low weights of one or more of the following components in the composition—red algae sulfate polysaccharide, β-glucan, elderberry extract, echinacea extract, lactoferrin, nano selenium, and nano zinc—significantly affected cell viability and antiviral levels.

[0066] The combination with good cell viability promoting and antiviral effects is as follows: 15-25 parts by weight of red algae sulfated polysaccharide, 10-20 parts by weight of β-glucan, 8-15 parts by weight of elderberry extract, 5-12 parts by weight of echinacea extract, 3-8 parts by weight of lactoferrin, 0.1-0.5 parts by weight of nano selenium, 0.5-2 parts by weight of nano zinc, 3-8 parts by weight of citric acid, and 10-30 parts by weight of fructose syrup; The optimal formula consists of a complex of nutrients including 20 parts by weight of red algae sulfated polysaccharide, 15 parts by weight of β-glucan, 12 parts by weight of elderberry extract, 8 parts by weight of echinacea extract, 5 parts by weight of lactoferrin, 0.3 parts by weight of nano selenium, 1.2 parts by weight of nano zinc, 5 parts by weight of citric acid, and 20 parts by weight of fructose syrup.

[0067] In summary, the compound nutrients of this invention are non-toxic to cells and promote cell proliferation within a safe concentration range, making them the optimal concentration for use. After optimization, the formulation can be considered the best composition ratio. In vitro studies show that the compound nutrients have a significant protective effect against virus-infected cells, exhibiting a broad-spectrum antiviral effect. Furthermore, this compound nutrient formulation confirms that the optimal ratio of its components provides excellent antiviral activity, laying a theoretical foundation for subsequent in vivo and clinical studies. The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A complex nutrient for triple dynamic immune defense, characterized in that, The complex nutrient comprises the following components by weight: 15-25 parts of red algae sulfated polysaccharide, 10-20 parts of β-glucan, 8-15 parts of elderberry extract, 5-12 parts of echinacea extract, 3-8 parts of lactoferrin, 0.1-0.5 parts of nano selenium, 0.5-2 parts of nano zinc, 3-8 parts of citric acid, and 10-30 parts of fructose syrup.

2. The compound nutrient according to claim 1, characterized in that, The composition comprises the following components in parts by weight: 20 parts of red algae sulfated polysaccharide, 15 parts of β-glucan, 12 parts of elderberry extract, 8 parts of echinacea extract, 5 parts of lactoferrin, 0.3 parts of nano selenium, 1.2 parts of nano zinc, 5 parts of citric acid, and 20 parts of fructose syrup.

3. The method for preparing the compound nutrient according to claim 1, characterized in that, Includes the following steps: (1) Raw material pretreatment: Take red algae sulfated polysaccharide raw material or β-glucan that meets the quality standards, pulverize it with an ultra-fine pulverizer, pass it through a 100-mesh sieve, and store it in a cool and dry place for later use; (2) Pretreatment of nano-components: Weigh out the nano-selenium powder or nano-zinc powder according to the formula, add 5 times its weight of purified water, and disperse it for 15 minutes using an ultrasonic cell disruptor at 400W power to obtain a uniform nano-dispersion. The nano-dispersion was mixed with maltodextrin at a mass ratio of 1:3, and microencapsulation was performed using a spray dryer with an inlet air temperature of 160±5℃ and an outlet air temperature of 80±5℃ to obtain well-encapsulated nanoparticles. (3) Preparation of extract: Preparation of elderberry extract and echinacea extract; (4) Ingredients and mixing Weigh the raw materials according to the proportions described in claim 1, and mix them using an equal-incremental mixing method in a low-temperature and low-humidity environment (temperature <20°C, humidity <30%RH). First, mix red algae sulfate polysaccharide and β-glucan base evenly. Then, add elderberry extract and echinacea extract in sequence and mix evenly. Add microencapsulated nanoparticle mixture and mix evenly. Finally, add lactoferrin, citric acid and 50% of the formula weight of fructose syrup and mix evenly to obtain premixed raw materials. The mixing time for each step shall not be less than 15 minutes. (5) Granulation process: Mix the remaining 50% by weight of fructose syrup with purified water to prepare a 50% concentration solution, and add 0.1% hydroxypropyl methylcellulose as a binder; put the premixed raw materials into a wet granulator, slowly add the binder, control the stirring speed at 200 rpm and the chopping speed at 1000 rpm to make a suitable soft material; granulate through a 20-mesh sieve to obtain uniform wet granules. (6) Drying and granulation: Vacuum freeze-drying technology was used to spread the wet granules evenly on a freeze-drying tray with a thickness controlled within 1 cm. The tray was then placed at -40℃ for 4 hours until completely frozen. Under vacuum conditions of <10 Pa, the temperature was gradually increased to 25℃ and dried until the moisture content was ≤4%. The dried granules are granulated through a 24-mesh sieve to remove fine powder and oversized particles.

4. The preparation method according to claim 2, characterized in that, The molecular weight of the red algae sulfated polysaccharide raw material is 50-200 kDa, and the sulfate content is ≥25%. The ratio of β-1,3 to β-1,6 structures in the β-glucan is ≥85%; The purity of the lactoferrin is ≥95%; The fructose content of the fructose syrup is ≥55%; The nano-selenium powder has a particle size of 40-60 nm, and the nano-zinc powder has a particle size of 60-80 nm.

5. The preparation method according to claim 2, characterized in that, The preparation method of the elderberry extract in step (3) is as follows: S1-1 Raw Material Pretreatment: Select ripe, unrotten black elderberry fruits, rinse them with clean water and drain them; then crush and pulp them using a fruit and vegetable crusher to obtain fruit pulp. S1-2 enzymatic hydrolysis: Add 0.3% pectinase to elderberry pulp and hydrolyze at 50℃ for 1-2 hours; S1-3 ethanol extraction: The enzymatically hydrolyzed fruit pulp was transferred to an extraction tank, and 5 times its weight of 60%-70% ethanol solution was added. The mixture was then subjected to hot reflux extraction twice at 60°C, for 1.5 hours each time. S1-4 Concentration and Drying: The extracts from the two extractions were combined, filtered through a 100-mesh sieve, and then the ethanol was recovered using a vacuum concentrator at ≤60℃ to obtain a concentrated extract. Finally, the extract was dried in a spray drying tower with an inlet air temperature of 160±5℃ and an outlet air temperature of 80±5℃ to obtain a deep purplish-red elderberry extract powder.

6. The preparation method according to claim 2, characterized in that, The preparation method of the echinacea extract in step (3) is as follows: S2-1 Raw Material Pretreatment: Take the dried aerial parts of Echinacea and cut them into small sections of about 2-3 cm for later use; S2-2 Extraction: The pre-treated echinacea raw material was put into a multi-functional extraction tank and added... 70% of 10-12 times the mass Ethanol, heated to 70-75℃, was extracted by hot reflux three times, one hour each time; S2-3 Concentration: The three ethanol extracts were combined and finely filtered using a plate and frame filter press. The filtrate was concentrated in a double-effect concentrator at ≤60℃ and a vacuum of -0.08 to -0.10 MPa until no alcohol odor was detected, yielding a fluid extract with a relative density of approximately 1.10-1.

15. S2-4 extraction and enrichment: The fluid extract was diluted with an equal volume of purified water, and then liquid-liquid extraction was performed with petroleum ether at 80°C to remove lipid-soluble impurities such as chlorophyll. After discarding the petroleum ether layer, the aqueous layer was extracted three times with an equal volume of ethyl acetate. The ethyl acetate extracts were then combined. S2-5 drying: The ethyl acetate layer was subjected to vacuum decompression at ≤50℃ to recover the solvent, resulting in a paste. Finally, it was dried using a vacuum belt dryer and pulverized through a 100-mesh sieve to obtain a brownish-yellow echinacea extract powder.

7. The use of the compound nutrients according to claim 1 in the preparation of antiviral drugs.

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