Cellular immunity anti-aging activator based on medicinal and edible plant extract and preparation method and application thereof

Abstract

The present application relates to a kind of cell immune anti-aging activator based on medicinal food homologous plant extract and its preparation method and application, the present application with Huangqi extract, huangjiu extract, sambucus nigra anthocyanin, olea europaea fruit polyphenol, tremella polysaccharide as core raw material, by scientific proportioning form synergic system, the synergic system combines double target cell immune activation and delays immune aging mechanism, regulates macrophage, T lymphocyte inflammatory factor secretion level, improves immune aging, inflammatory aging from source, repairs cell immune function, expands its application in health food, and preparation method adopts low-temperature segmented extraction process, maximum retains the active ingredient of each raw material, successfully prepared immune regulation effect clear, safe and compliant, the cell immune activator of industrial production.

Description

Technical Field

[0001] This invention relates to a cell immune anti-aging activator based on extracts from medicinal and edible plants, its preparation method and application, belonging to the field of food processing and immune regulation technology. Background Technology

[0002] Cellular immunity is the core defense line of the body against the invasion of external pathogens, the elimination of abnormal cells, and the maintenance of homeostasis. Its functional decline is not only an important marker of aging and immunosenescence, but also a key contributing factor to recurrent infections, fatigue, immune disorders, and the decline of age-related physiological functions. Aging is closely related to immunosenescence (decreased immune function) and inflammatory senescence (a chronic, low-grade inflammatory state), and there is an interaction between the two: inflammatory senescence can further exacerbate immunosenescence. In 2000, Franceschi et al. established "inflammatory senescence" as a hallmark of immunosenescence, namely a chronic, low-grade, systemic inflammatory state in the elderly, characterized by persistently elevated levels of various pro-inflammatory cytokines, such as interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and C-reactive protein (CRP). In the process of immunosenescence, aging-associated macrophages exhibit enhanced pro-inflammatory activity, secreting more pro-inflammatory cytokines, especially IL-6 and TNF-α; senescent T cells highly express perforin and interferon-γ (IFN-γ). With the accelerated pace of modern life, increased work pressure, unhealthy dietary habits, and environmental factors, the number of people experiencing decreased cellular immune function and premature immunosenescence is increasing. Therefore, developing safe, effective, and compliant cellular immune modulation products and anti-aging products has significant market value and broad application prospects.

[0003] Currently, there are reports on cell immunomodulators based on natural plant extracts, but existing technologies still have the following shortcomings: First, the raw material formulations lack innovation, often using single plant extracts or simple combinations of traditional medicinal and edible raw materials such as ginseng, Ganoderma lucidum, and wolfberry, without forming a clear synergistic effect system, resulting in limited immunomodulatory and anti-aging effects; second, the mechanisms of action are relatively simple, with most schemes targeting only a certain type of immune cell or a single signaling pathway, making it difficult to achieve multi-target synergistic intervention in immune aging and failing to meet the comprehensive needs of the body for comprehensive immune regulation and delaying aging; third, the preparation process is unreasonable, generally using high-temperature extraction or mixed extraction methods, which easily destroys heat-sensitive active ingredients such as polysaccharides, anthocyanins, and polyphenols, resulting in low activity retention rates, thus limiting the immunomodulatory and anti-aging efficacy of the products.

[0004] Chinese patent document CN110105426A discloses a method for preparing *Syngonium spp.* glycoprotein. The method specifically includes: extracting *Syngonium spp.* glycoprotein using processes such as acetone defatting, hot water extraction, and ethanol precipitation; and verifying its immunomodulatory effects through RAW264.7 cell experiments. The immunomodulatory effect is mainly achieved by promoting cell proliferation, enhancing phagocytic capacity, and promoting the secretion of cytokines such as NO and IL-6. However, this patent uses only *Syngonium spp.* as a single raw material to prepare the immunomodulatory component, and its mechanism of action targets only macrophages, a single immune cell, failing to achieve multi-target synergistic activation of cellular immune function, thus limiting the immunomodulatory effect.

[0005] For example, Chinese patent document CN118717793A discloses the application of Dendrobium officinale polysaccharide in microglia immunomodulation. This application extracts and purifies DOPN2 from Dendrobium officinale, and in vitro cell experiments verify that it can activate microglia and regulate the expression of inflammatory factors. However, this application uses a single Dendrobium officinale polysaccharide as the immunomodulatory component, and its target is mainly concentrated on microglia, making it difficult to meet the needs of comprehensive immune regulation in the human body. Furthermore, this patent does not cover optimized processes suitable for large-scale production of health food products, thus limiting its application scope.

[0006] For example, Chinese patent document CN106727799A discloses a traditional Chinese medicine composition for health preservation, which uses traditional medicinal and edible raw materials such as cordyceps, ginseng, and Panax notoginseng in a simple compounding process, lacking a systematic formulation design based on the synergistic effect of multiple components. This composition only provides a preliminary evaluation of conventional immune indicators, without exploring its regulatory role in key mechanisms such as immunosenescence and inflammatory senescence, nor does it involve multi-target combined intervention strategies. Therefore, its anti-aging and immune-regulating functions are limited.

[0007] Astragalus and Polygonatum, as classic medicinal and edible ingredients, have traditional effects of tonifying Qi and strengthening the body, and regulating immunity. Their active ingredients (Astragalus polysaccharide and Polygonatum polysaccharide) have been proven to regulate immune cell activity and delay immune cell aging. Elderberry anthocyanins and olive fruit polyphenols, derived from elderberry and olive fruit respectively, are rich in anthocyanins and polyphenols, and have antioxidant, antiviral, anti-inflammatory, and immune-regulating effects, as well as delaying cell aging. Tremella polysaccharide, as a natural active polysaccharide, can protect immune cells, enhance immune cell proliferation, and reduce oxidative damage to cells. However, in the current technology, there are no reports on the scientific formulation of these five raw materials and the use of low-temperature segmented extraction processes to prepare an immune-stimulating and anti-aging composition. This makes it impossible to fully utilize the synergistic effects of each raw material in enhancing immunity and delaying aging, and it is also difficult to solve the technical pain points of low activity, insufficient innovation, and lack of anti-aging function in existing products.

[0008] Therefore, developing an immune-antiaging composition based on medicinal and edible plant extracts with innovative formulation, clear mechanism, and reasonable process has become an urgent technical problem to be solved in this field. Summary of the Invention

[0009] In view of the shortcomings of existing technologies, especially the lack of innovation in the formulation of cellular immune modulators, the single mechanism of action, the low retention rate of active ingredients, and the lack of anti-aging and immune-aging function improvement, this invention provides a cellular immune anti-aging activator based on extracts of medicinal and edible plants, its preparation method and application.

[0010] This invention uses Astragalus membranaceus extract, Polygonatum sibiricum extract, anthocyanins from elderberry, olive fruit polyphenols, and Tremella fuciformis polysaccharides as core raw materials, and forms a synergistic system through scientific formulation. This synergistic system combines a dual-target cellular immune activation and immune aging delay mechanism, regulates the secretion level of inflammatory factors in macrophages and T lymphocytes, improves immune aging and inflammatory aging from the source, repairs cellular immune function, and expands its application in health foods.

[0011] The preparation method of this invention adopts a low-temperature segmented extraction process to retain the active ingredients of each raw material to the maximum extent, and successfully prepares a cell immune activator with clear immunomodulatory effects, safety and compliance, and industrial production capability.

[0012] The technical solution of the present invention is as follows:

[0013] A cell-mediated immune-boosting and anti-aging activator based on extracts from medicinal and edible plants, comprising a main ingredient and excipients, wherein the main ingredient is composed of the following raw materials in parts by weight:

[0014] Astragalus extract 15-25 parts, Polygonatum extract 15-25 parts, elderberry anthocyanin 8-12 parts, olive fruit polyphenols 5-8 parts, Tremella polysaccharide 5-10 parts.

[0015] According to a preferred embodiment of the present invention, a cell immune anti-aging activator based on extracts of medicinal and edible plants includes a main ingredient and excipients. The main ingredient is composed of the following raw materials in parts by weight: 18-22 parts of Astragalus membranaceus extract, 18-22 parts of Polygonatum sibiricum extract, 9-11 parts of anthocyanins from elderberry, 6-7 parts of olive fruit polyphenols, and 7-9 parts of Tremella fuciformis polysaccharide.

[0016] According to a preferred embodiment of the present invention, a cell immune anti-aging activator based on extracts of medicinal and edible plants includes a main ingredient and excipients. The main ingredient is composed of the following raw materials in parts by weight: 20 parts of Astragalus membranaceus extract, 20 parts of Polygonatum sibiricum extract, 10 parts of anthocyanins from elderberry, 6.5 parts of olive fruit polyphenols, and 8 parts of Tremella fuciformis polysaccharide.

[0017] According to a preferred embodiment of the present invention, the excipient is selected from one or more of microcrystalline cellulose, pregelatinized starch, crospovidone, and magnesium stearate, and the weight of the excipient is 10-40% of the weight of the main ingredient.

[0018] According to a preferred embodiment of the present invention, the Astragalus extract is prepared by the following method:

[0019] a. Raw material pretreatment: Remove impurities, wash, dry and pulverize the dried rhizomes of Astragalus membranaceus to obtain Astragalus membranaceus powder; use petroleum ether for low-temperature soaking and degreasing, filter to remove petroleum ether after soaking; then perform pre-freezing treatment and micro-nano pulverization to obtain degreased and cell wall broken Astragalus membranaceus powder;

[0020] b. Pressure permeation extraction: Add cellulase to the compound extraction solvent, then add defatted and cell wall-breaking Astragalus powder. First, permeate under high pressure, then extract under low pressure. Combine the two extracts to obtain a mixture.

[0021] c. Purification and separation: The mixed liquid is filtered through a membrane. Macroporous adsorption resin is added to the filtered extract and stirred for adsorption. Then, liquid-liquid extraction is performed. After standing and separating, the aqueous phase is collected and concentrated under reduced pressure to obtain a high-purity Astragalus extract concentrate.

[0022] d. Recrystallization and drying: Dissolve the concentrated Astragalus extract in a crystallization solvent, stir and filter to remove insoluble impurities, cool the filtrate to room temperature and let it stand for crystallization for 4-6 hours, filter and collect the crystals, wash the crystals, dissolve the washed crystals in purified water, add maltodextrin and glucanase, stir evenly and then enzymatically hydrolyze, inactivate the enzyme after enzymatic hydrolysis, and then spray dry to obtain Astragalus extract.

[0023] According to a preferred embodiment of the present invention, in step a, drying and pulverizing are performed by drying in a forced-air drying oven at 45~55℃ until the moisture content is ≤8%, and then passing the pulverized material through an 80~100 mesh sieve.

[0024] According to a preferred embodiment of the present invention, in step a, the weight-to-volume ratio of Astragalus powder to petroleum ether is 1g:20~30ml.

[0025] According to a preferred embodiment of the present invention, in step a, the low-temperature soaking degreasing is performed by soaking at 0-6°C for 8-10 hours.

[0026] According to a preferred embodiment of the present invention, in step a, the pre-freezing temperature of the pre-freezing treatment is -35~-45℃, and the pre-freezing time is 2~3h.

[0027] According to a preferred embodiment of the present invention, in step b, the composite extraction solvent is a mixture of 1,2-propanediol and ethanolamine in a volume ratio of 3 to 4:1, and the amount of cellulase added is 0.5 to 1% of the weight of Astragalus powder.

[0028] According to a preferred embodiment of the present invention, in step b, the defatted and cell wall-breaking Astragalus powder is added at a ratio of 1g:6~10ml.

[0029] According to a preferred embodiment of the present invention, in step b, the process of first pressurizing and then extracting under low pressure specifically involves: placing the mixture in a high-pressure extraction vessel, controlling the pressure at 8-10 MPa and the temperature at 30-35°C, and pressurizing and soaking for 30-40 minutes; subsequently reducing the pressure to 0.03-0.05 MPa, raising the temperature to 50-60°C, and extracting under reduced pressure for 30-40 minutes.

[0030] In step b, magnetic stirring is used during the pressure soaking and depressurization extraction processes, with a stirring speed of 300~500 r / min; after each extraction, the mixture is allowed to stand for 10~15 min before filtration; the amount of ethanolamine is controlled at 20~25% of the total mass of the compound solvent.

[0031] According to a preferred embodiment of the present invention, in step c, the amount of macroporous adsorption resin used is 2% to 4% of the weight of Astragalus powder. After stirring and adsorbing for 1 to 2 hours, the adsorbent is removed by filtration. Liquid-liquid extraction is performed using n-butanol. After standing and separating, the aqueous phase is collected. The aqueous phase is concentrated under reduced pressure of 0.06 to 0.08 MPa and 45 to 55°C to a relative density of 1.10 to 1.15 at 25°C.

[0032] According to a preferred embodiment of the present invention, in step d, the crystallization solvent is a mixture of ethanol and diethyl succinate in a volume ratio of 3-8:2, the stirring is carried out at 100-200 rpm for 10-20 min at 60-70°C, and the crystals are washed with ethanol 1-2 times.

[0033] According to a preferred embodiment of the present invention, in step d, the amount of maltodextrin added is 20-30% of the mass of the crystals, the amount of dextranase added is 0.3-0.5% of the mass of the crystals, the enzymatic hydrolysis is carried out at 40-45℃ for 30-40 min, the enzyme inactivation is carried out by heating to 90-100℃, and the spray drying parameters are: inlet air temperature 160-170℃, outlet air temperature 70-75℃, feed rate 20-25 mL / min, and drying is carried out until the moisture content of the extract is ≤5%.

[0034] According to a preferred embodiment of the present invention, in step d, the DE value of the maltodextrin is 15-20; the atomization pressure of the spray drying is 0.2-0.25 MPa; and the crystals collected after recrystallization.

[0035] According to a preferred embodiment of the present invention, the Polygonatum extract is prepared by the following method:

[0036] 1) Raw material pretreatment: Remove impurities from the dried rhizomes of Polygonatum, wash them, slice them, dry them, pulverize them and sieve them to obtain Polygonatum powder; subject the Polygonatum powder to low-temperature pre-freezing treatment to obtain pre-frozen Polygonatum powder;

[0037] 2) Enzymatic-ultrasonic extraction: Add extraction solvent and cellulase to pre-frozen Polygonatum powder, and extract using an enzymatic-ultrasonic synergistic extraction method to obtain the extract;

[0038] 3) Purification and concentration: The extract is filtered through a membrane, and nitrogen gas is introduced into the filtered extract to change the hydrophilic solvent in the extraction solvent from hydrophilic to hydrophobic. After standing and separating the layers, the solvent is separated and recovered, and the aqueous phase is collected. The aqueous phase is concentrated under reduced pressure to obtain the concentrated extract of Polygonatum sibiricum.

[0039] 4) Encapsulation and drying: Add β-cyclodextrin to the concentrated extract of Polygonatum sibiricum, stir and mix evenly, and spray dry to obtain Polygonatum sibiricum extract.

[0040] According to a preferred embodiment of the present invention, in step 1), the slice thickness is 2~3mm, the drying is carried out in a 50~60℃ forced-air drying oven until the moisture content is ≤8%, the slices are crushed and passed through an 80~100 mesh sieve, the low-temperature pre-freezing treatment temperature is -30~-40℃, and the pre-freezing time is 1.5~2.5h.

[0041] According to a preferred embodiment of the present invention, in step 2), the extraction solvent is a mixture of deionized water and a carbon dioxide-responsive switchable hydrophilic solvent at a volume ratio of 7-8:2. The carbon dioxide-responsive switchable hydrophilic solvent is prepared by mixing food-grade ammonia and deionized water at a volume ratio of 1:8-10. The pre-frozen Polygonatum powder is added to the extraction solvent at a material-to-liquid ratio of 1g:(8-12)mL, the pH is adjusted to 6.8-7.2, and cellulase is added at a weight of 0.5-0.8% of the Polygonatum powder. The enzymatic-ultrasonic synergistic extraction method involves extraction twice at an ultrasonic power of 280-320W and an extraction temperature of 50-60℃, each time for 1.5-2.5h, and all extracts are combined.

[0042] According to a preferred embodiment of the present invention, in step 2), during the enzymatic-ultrasonic synergistic extraction process, the mixture is stirred once every 30 minutes, with each stirring lasting 5-10 minutes; during ultrasonic extraction, the ultrasonic interval is controlled to be 30 seconds of operation followed by 10 seconds of rest to avoid overheating and damage to the active ingredients; the cellulase hydrolysis time is synchronized with the ultrasonic extraction time, and after the hydrolysis is completed, the enzyme activity is inactivated by heating to 65-70°C and holding for 10-15 minutes to ensure the purity of the subsequent extract.

[0043] According to a preferred embodiment of the present invention, in step 3), the vacuum concentration is carried out at a vacuum pressure of 0.06~0.08MPa and a temperature of 50~60°C until the relative density at 25°C is 1.12~1.18.

[0044] According to a preferred embodiment of the present invention, in step 4), the amount of β-cyclodextrin added is 15-25% of the solid mass of the concentrate, and the spray drying parameters are: inlet air temperature 165-175℃, outlet air temperature 72-78℃, feed rate 22-28mL / min, drying until the moisture content of the extract is ≤5%; after adding β-cyclodextrin, magnetic stirring is performed at 300-400r / min for 15-20min to ensure uniform encapsulation.

[0045] According to a preferred embodiment of the present invention, in step 4), the purity of the β-cyclodextrin is ≥98%; the atomization pressure of the spray drying is 0.22~0.28MPa; and the recovered switchable hydrophilic solvent is distilled under reduced pressure at a temperature of 45~55℃ and a pressure of 0.05~0.07MPa and reused for extraction in step b.

[0046] According to a preferred embodiment of the present invention, the elderberry anthocyanins are prepared according to the following method:

[0047] (1) Freeze-drying and anti-oxidation treatment: clean and remove impurities from fresh elderberries, process them using low-temperature gradient freeze-drying, and then pulverize them into micro-nano powder; add ascorbic acid-citric acid composite antioxidant to elderberry powder, mix evenly to obtain a mixture;

[0048] (2) Ultrasonic-microwave synergistic extraction: Add extraction solvent to the mixture and extract using ultrasonic-microwave synergistic extraction method. Repeat the extraction 1 to 2 times and combine all extracts.

[0049] (3) Centrifugation, filtration and purification: Centrifuge the combined extracts, take the supernatant, and filter the supernatant through a membrane to remove suspended impurities; add chitosan to the filtered supernatant to adsorb and remove protein impurities, filter again and load the sample onto an AB-8 macroporous resin column for purification;

[0050] (4) Gradient elution and drying: The gradient elution process was used for elution, the eluent was concentrated under reduced pressure, and then freeze-dried under vacuum to obtain elderberry anthocyanins.

[0051] According to a preferred embodiment of the present invention, in step (1), the low-temperature gradient freeze-drying process specifically includes: pre-freezing temperature -40~-50℃, pre-freezing time 2~3h, sublimation drying temperature -15~-25℃, desorption drying temperature 30~40℃, micro-nano pulverization at 0~5℃ to 60~80 mesh, ascorbic acid-citric acid composite antioxidant in which the mass ratio of ascorbic acid to citric acid is 2:1, and the amount of ascorbic acid-citric acid composite antioxidant added is 0.3~0.5% of the mass of elderberry powder.

[0052] According to a preferred embodiment of the present invention, in step (2), the extraction solvent is a mixture of 30-40% edible alcohol and choline chloride / glycerol (1:2) DES at a volume ratio of 8:2, the pH is 3.0-3.5, the material-liquid ratio is 1g:(12-18)mL, the ultrasonic-microwave synergistic extraction is performed with an ultrasonic power of 250-350W, a microwave power of 150-200W, an extraction temperature of 35-45℃, and an extraction time of 20-40min.

[0053] According to a preferred embodiment of the present invention, in step (2), during the ultrasonic-microwave synergistic extraction process, the ultrasonic interval is controlled to be 20s working and 10s resting, and the microwave interval is controlled to be 30s working and 20s resting; the mixture is stirred once every 10 minutes during the extraction process to ensure uniform extraction.

[0054] According to a preferred embodiment of the present invention, in step (3), centrifugation is performed at 3000~5000 r / min for 10~20 min, membrane filtration is performed by filtering through a 0.22 μm filter membrane to remove suspended impurities, the amount of chitosan added is 0.1~0.2% of the volume of the supernatant, after stirring evenly, it is allowed to stand for 15~20 min, the resin column is pretreated with citrate buffer at pH 3.0~3.5 before loading the sample, and the loading flow rate is controlled at 1~3 BV / h.

[0055] According to a preferred embodiment of the present invention, in step (4), the gradient elution process is as follows: first, impurities are eluted with deionized water at pH 3.0 to 3.5, with an elution volume of 3 to 5 BV; then, anthocyanin active ingredients are eluted with 50 to 60% edible alcohol (containing 0.1% ascorbic acid) in a gradient elution process. The edible alcohol eluent is collected and concentrated under reduced pressure to a solid content of 20 to 30% under reduced pressure conditions of 45 to 55°C and -0.07 to -0.09 MPa. Nitrogen gas is introduced during the concentration process.

[0056] According to a preferred embodiment of the present invention, in step (4), the deionized water elution flow rate is 2~4 BV / h; the edible alcohol elution flow rate is 1~2 BV / h, and the elution volume is 4~6 BV; the vacuum freeze-drying parameters are: pre-freezing temperature -40~-50℃, pre-freezing time 3~4h, sublimation drying temperature -20~-30℃, desorption drying temperature 35~45℃, vacuum degree 0.08~0.095MPa, and drying to a moisture content ≤5%; the ascorbic acid added to the eluent can be removed by vacuum distillation in the later stage of concentration.

[0057] According to a preferred embodiment of the present invention, the olive fruit polyphenols are prepared according to the following method:

[0058] I. Raw material pretreatment and color protection / antioxidant

[0059] Fresh olives are pre-treated, washed, drained, and dried at a low temperature. The dried olives are then pitted, retaining the pulp, which is then crushed and sieved to obtain a uniform olive pulp powder.

[0060] Food-grade tocopherol-rosemary extract complex antioxidant was added to olive pulp powder and thoroughly mixed. The mixed pulp powder was then pre-frozen at low temperature and then pulverized at low temperature to obtain pre-treated olive pulp powder.

[0061] II. Synergistic extraction using ultrasound and enzyme methods

[0062] Add extraction solvent to the pretreated olive fruit pulp powder, and use ultrasound-enzyme synergistic extraction method to extract at a constant temperature for 1-2 hours. Combine all extracts.

[0063] III. Degreasing, centrifugal purification and concentration

[0064] The combined extracts were first degreased at low temperature, allowed to stand and separate into layers, and the lower aqueous phase was retained. The degreased extracts were centrifuged, and the clear supernatant was taken, filtered through a membrane, and polyamide resin was added to the filtered supernatant and stirred at room temperature to adsorb, thus obtaining a purified polyphenol extract. The purified polyphenol extract was concentrated under reduced pressure until the system had no alcohol odor, thus obtaining a concentrated olive fruit polyphenol extract.

[0065] IV. Adding a carrier and freeze-drying

[0066] Maltodextrin was added to the concentrated olive fruit polyphenol solution as a carrier, and the mixture was stirred at low speed until homogeneous. Then, the mixture was freeze-dried under vacuum to obtain olive fruit polyphenols.

[0067] According to a preferred embodiment of the present invention, in step I, the fresh olive fruit is a mature fresh olive fruit that is free from rot, mold, and pests and diseases. The pretreatment involves removing the fruit stem, branches, leaves, impurities, and foreign objects. The washing involves washing the fruit with purified water at room temperature 2-3 times to thoroughly remove dust, residual dirt, and pesticide residues from the surface of the fruit. The low-temperature drying involves placing the fruit in a forced-air drying oven at 45-55℃ and drying it at a low temperature until the overall moisture content of the fruit is ≤8%. After crushing, the fruit is passed through a 60-80 mesh sieve.

[0068] According to a preferred embodiment of the present invention, in step I, the amount of food-grade tocopherol-rosemary extract composite antioxidant added is 0.2-0.4% of the weight of olive fruit pulp powder, and the mass ratio of tocopherol to rosemary extract in the food-grade tocopherol-rosemary extract composite antioxidant is (0.8-1.2):1. The mixture is thoroughly stirred and homogeneous to effectively inhibit the oxidative degradation of fruit polyphenols. The pre-freezing temperature for the low-temperature pre-freezing treatment is -35 to -45°C, and the pre-freezing time is 1-2 hours. The low-temperature pulverization temperature is maintained at 0-4°C, and the final pulverization is carried out to a particle size of 40-60 μm.

[0069] According to a preferred embodiment of the present invention, in step II, an extraction solvent is added to the pretreated olive fruit pulp powder at a ratio of 1g:(15~20)mL; the extraction solvent is a mixture of 40~50% edible alcohol and a deep eutectic solvent at a volume ratio of 7:3, adapted to the dissolution characteristics of fruit polyphenols, and the pH of the system is adjusted to 2.5~3.0 using a food-grade weak acid, and the deep eutectic solvent is a mixture of food-grade lactic acid and fructose at a molar ratio of 2:1.

[0070] According to a preferred embodiment of the present invention, in step II, the ultrasonic-enzymatic synergistic extraction is performed with an ultrasonic power of 300-400W and an enzymatic hydrolysis temperature of 40-50℃. Pectinase is added during the extraction process, and the amount of pectinase added is 0.4-0.6% of the weight of the olive fruit pulp powder. The extraction is carried out at a constant temperature for 1-2 hours, and the extraction is repeated once with the same process parameters to fully dissolve the bound and free polyphenols in the fruit.

[0071] According to a preferred embodiment of the present invention, in step II, during the ultrasonic-enzymatic synergistic extraction process, the ultrasonic intermittent mode is controlled to work for 30 seconds and pause for 10 seconds, with stirring once every 20 minutes, and each stirring lasting 8-12 minutes, to avoid the sedimentation of fruit pulp powder and improve mass transfer efficiency; after enzymatic hydrolysis, the temperature is rapidly raised to 65-70°C and kept at that temperature for 10-15 minutes to completely inactivate pectinase activity and prevent excessive enzymatic hydrolysis from affecting the purity and stability of polyphenols.

[0072] According to a preferred embodiment of the present invention, in step III, the low-temperature defatting treatment temperature is 10~15℃, the standing layer separation time is 30~40min to remove the upper floating oil and fat-soluble impurities, the extract is centrifuged at 4000~6000r / min for 15~25min, the membrane filtration is performed through a 0.22μm organic filter membrane to thoroughly remove fine suspended impurities and fruit pulp residue, the polyamide resin is added at 0.3~0.5% of the supernatant volume, the mixture is stirred and adsorbed at room temperature for 20~30min to specifically adsorb pigments and impurities, the resin is removed by filtration, and the polyphenol extract is purified.

[0073] According to a preferred embodiment of the present invention, in step III, vacuum concentration is performed under reduced pressure at 40~50℃ and -0.08~-0.095MPa, with the concentration rate controlled at 1~2mL / min, until the system has no alcohol odor. High-purity nitrogen is continuously introduced throughout the concentration process to prevent polyphenol oxidation at high temperatures, thereby obtaining concentrated olive fruit polyphenol solution.

[0074] According to a preferred embodiment of the present invention, in step IV, the amount of maltodextrin added is 10-20% of the solid mass of the concentrate, and the mixture is stirred at low speed for 15-20 minutes until it is completely mixed and homogeneous. The vacuum freeze-drying parameters are: pre-freezing temperature -40 to -50°C, pre-freezing time 3-5 hours; drying temperature -55 to -45°C, vacuum degree 0.05-0.15 Pa, continuous drying for 20-28 hours, and after drying, the product is lightly pulverized and sieved.

[0075] The preparation method of the above-mentioned cell immune anti-aging activator based on extracts of medicinal and edible plants includes the following steps:

[0076] S1. Raw material pretreatment: Astragalus extract, Polygonatum extract, Elderberry anthocyanins, and olive fruit polyphenols are pulverized by low-temperature airflow at 0~5℃ and then passed through a 100~120 mesh sieve. Argon gas is introduced for protection during the pulverization process. Tremella polysaccharide is pulverized by low-temperature micro-nano pulverization at 0~4℃ to a particle size of 50~80μm, passed through an 80~100 mesh sieve, and placed in a dry and sealed container for later use. The relative humidity of the environment is controlled at 30~40% and the temperature is 10~15℃.

[0077] S2. Premixing: According to the formula, first put the Astragalus extract and Polygonatum extract into the intelligent temperature-controlled three-dimensional mixer, introduce argon gas to remove air from the equipment, reduce the oxygen content to ≤0.5%, control the mixing temperature at 15~20℃ and the speed at 18~22r / min, mix for 12~18min, and reverse stirring once every 4~6min during the period, at a speed of 25~30r / min for 30s. Simultaneously, online particle size monitoring technology is used to monitor the mixing uniformity in real time (uniformity ≥98%). After the standard is met, premix A is obtained.

[0078] S3. Co-mixing: Add elderberry anthocyanins and olive fruit polyphenols to premix A, maintain argon protection, mix at 15~20℃, adjust the speed to 20~25r / min, mix for 18~22min, use segmented stirring mode, combined with low-frequency ultrasonic-assisted dispersion, and monitor uniformity online simultaneously. After meeting the standard, premix B is obtained.

[0079] S4. Formulation and molding: Add tremella polysaccharide to premix B, stir and mix evenly, add excipients according to the ratio and mix evenly, adjust the pH of the system to 6.8~7.2 with phosphate buffer to obtain a mixture; dry the mixture using low temperature ultrasonic atomization spray drying process, and after drying, pulverize at low temperature and pass through a 100~120 mesh sieve to obtain a cell immune anti-aging activator.

[0080] According to a preferred embodiment of the present invention, in step S3, the segmented stirring is performed by rotating forward for 5 minutes and then rotating backward for 3 minutes, with the ultrasonic power of the low-frequency ultrasonic-assisted dispersion being 100~150W, and the operation being intermittent for 20 seconds and then pausing for 40 seconds.

[0081] According to a preferred embodiment of the present invention, in step S4, the excipient is selected from one or more of microcrystalline cellulose, pregelatinized starch, crospovidone, and magnesium stearate.

[0082] According to a preferred embodiment of the present invention, in step S4, the parameters for low-temperature ultrasonic atomization spray drying are: inlet air temperature 165~175℃, outlet air temperature 72~78℃, feed rate 25~30mL / min, atomization pressure 0.25~0.3MPa, drying chamber temperature controlled at 45~55℃, and ultrasonic atomization frequency 20~30kHz.

[0083] According to a preferred embodiment of the present invention, in step S4, argon gas is introduced for protection throughout the drying process, and an intelligent temperature control system is used to regulate the temperature of the drying chamber in real time to avoid local overheating.

[0084] The above-mentioned cell immune anti-aging composition based on medicinal and edible compound plant extracts is used in the preparation of health foods and special dietary foods that enhance cell immune function.

[0085] According to a preferred embodiment of the present invention, the cellular immune-anti-aging composition of the medicinal and edible compound plant extract is in the form of powder, granules, capsules or tablets.

[0086] Technical features of the present invention:

[0087] The Astragalus extract of this invention is a natural immunomodulatory component rich in Astragalus polysaccharides and Astragalus saponins (including Astragaloside A), belonging to the category of medicinal and edible functional extracts, possessing the core functions of regulating immune responses and delaying the aging of immune cells. As a core functional component of the immunomodulatory and anti-aging composition, the Astragalus polysaccharides (AMPs) exhibit various pharmacological effects, including immunomodulatory, antiviral, anti-aging, and antioxidant effects. They can stimulate the phagocytic activity of macrophages, and their effects on specific immune cells are mainly manifested in promoting the proliferation and differentiation of B lymphocytes and T lymphocytes, enhancing the secretory function of plasma cells, increasing serum antibody concentration, and regulating T lymphocyte subsets (such as CD4+). + and CD8 + It balances the immune system and possesses antioxidant and immunomodulatory activities; astragaloside A in it can intervene in the activation of NF-κB and MAPK signaling pathways, inhibit the production of inflammatory factors such as TNF-α and IL-6, and at the same time reduce oxidative stress and aging damage of immune cells, bringing disordered immune function back to homeostasis, providing core support for inhibiting inflammatory aging and repairing cellular immunity.

[0088] The Polygonatum extract of this invention is a type of natural immune-active component rich in Polygonatum polysaccharides, Polygonatum saponins, flavonoids, etc. It belongs to the category of medicinal and edible functional extracts and has the core functions of anti-inflammatory, antioxidant, immune regulation, and delaying the aging of immune cells. As a key component of the immune homeostasis regulation and anti-aging composition, Polygonatum polysaccharide (PSP) can exert anti-inflammatory effects through the TLR4 / MyD88 / NF-κB and AMPK signaling pathways, significantly reducing the levels of pro-inflammatory factors such as IL-6, TNF-α, and IL-1β. Simultaneously, it can increase the activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), reduce malondialdehyde (MDA) content, scavenge free radicals, alleviate oxidative stress, and significantly increase the thymus and spleen indices in mice, promoting the proliferation and differentiation of T lymphocytes and B lymphocytes, enhancing the phagocytic capacity of macrophages, and regulating the body's immune function. The β-carboline compounds can inhibit the expression of nitric oxide (NO) and pro-inflammatory factors (TNF-α, IL-6, and IL-1β) in the inflammatory environment, synergistically reducing the damage of chronic low-grade inflammation to immune cells, restoring disordered immune function to homeostasis, and providing crucial support for inhibiting inflammatory aging and delaying immune cell aging.

[0089] Elderberry berries are rich in various bioactive components, mainly including anthocyanins (such as cyanidin-3-glucoside and cyanidin-3-morula disaccharide), phenolic acids (mainly chlorogenic acid), and flavonoids (mainly rutin). Furthermore, its cyanogenic glycoside content is much lower than that of the leaves and flowers, making it considered the safest and most nutritious part of the plant. In terms of efficacy, the berry extract exhibits clear anti-inflammatory activity, the mechanism of which lies in its regulatory effect on immunosenescence: in a lipopolysaccharide-stimulated macrophage model, it can significantly inhibit nitric oxide production and effectively downregulate pro-inflammatory cytokines (including IL-1β, IL-6, IL-8, and TNF-α), while upregulating the level of the anti-inflammatory cytokine IL-10, thereby exerting anti-inflammatory and anti-influenza virus effects (especially in the later stages of infection) from an immunomodulatory perspective.

[0090] The olive fruit polyphenols of this invention are functional extracts derived from olive fruit, rich in natural active ingredients such as hydroxytyrosol, oleuropein (and its aglycones), and olive thorns. They possess core functions including inhibiting excessive inflammatory activation, reducing oxidative stress damage, and regulating immune homeostasis. As a key functional component of the immune-anti-aging composition, hydroxytyrosol can inhibit the activation of pro-inflammatory transcription factors such as NF-κB, downregulate the levels of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6, and inhibit the production of inflammatory mediators such as COX-2, iNOS, NO, and PGE2. Oleuropein also inhibits NF-κB and TNF-α, IL-1β, IL-6, IL-8, and IL-17A. Olive thorns can dose-dependently inhibit COX-1 and COX-2, with efficacy superior to ibuprofen. At the antioxidant level, hydroxytyrosol reduces oxidative stress-induced cell damage by scavenging free radicals, activating the Nrf2-ARE pathway and AMPK signaling, and enhancing the activity of antioxidant enzymes such as SOD and catalase. In addition, olive fruit polyphenols also have neuroprotective, cardiovascular protective, anti-diabetic, cognitive function-improving, and gut microbiota-regulating effects. In summary, olive polyphenols achieve a dual "anti-inflammatory" and "antioxidant" effect by inhibiting NF-κB-mediated inflammatory pathways and activating Nrf2-mediated antioxidant defense, effectively regulating immune homeostasis and providing core support for inhibiting inflammatory aging and repairing cellular immunity.

[0091] The Tremella polysaccharide of this invention is a natural active polysaccharide derived from the fruiting body, mycelium, and fermentation broth of Tremella. Regarding its anti-inflammatory mechanism, Tremella polysaccharide exhibits anti-oxidative stress and anti-inflammatory properties in LPS-treated macrophages by inhibiting the miR-155 and NF-κB pathways. It also participates in life processes such as aging and inflammation by regulating SIRT1 protein expression. In terms of immunomodulation, Tremella polysaccharide enhances non-specific immunity, humoral immunity, and cellular immunity by activating macrophages, T lymphocytes, and B lymphocytes. In cyclophosphamide-induced immunosuppressed mice, it significantly increases thymus and spleen indices, elevates serum IL-2, IL-12, and IgG levels, reduces TGF-β levels, promotes IL-1β, IL-4, and IL-12 mRNA expression, and exerts immunomodulatory effects by inhibiting abnormally high-expressing CD4⁺CD25⁺ regulatory T cells. In summary, Tremella polysaccharides achieve a dual mechanism of anti-inflammation and immune regulation by inhibiting the NF-κB pathway and regulating SIRT1 expression, providing core support for inhibiting inflammatory senescence and repairing cellular immunity.

[0092] The beneficial effects of this invention are as follows:

[0093] 1. Scientifically formulated, with synergistic effects from multiple components.

[0094] This invention uses Astragalus membranaceus extract, Polygonatum sibiricum extract, elderberry anthocyanins, olive fruit polyphenols, and Tremella fuciformis polysaccharides as core raw materials, scientifically formulated to form a synergistic system. Astragalus membranaceus and Polygonatum sibiricum work synergistically to replenish Qi and strengthen the body, activating macrophages and lymphocytes as dual targets; elderberry anthocyanins and olive fruit polyphenols work synergistically to fight inflammation, oxidation, and viruses; Tremella fuciformis polysaccharides protect immune cells and enhance system stability. These five components form a complete chain of action: "replenishing Qi and strengthening the body + antiviral immunity + immune cell protection + delaying immune aging," achieving highly efficient dual-target activation of cellular immunity and improving immune aging and inflammatory aging, with effects significantly superior to single-ingredient or simple combination products.

[0095] 2. The mechanism of action is clear, and it regulates immune aging through dual targets.

[0096] The cellular immune anti-aging activator of this invention can simultaneously target macrophages and T lymphocytes, regulate key signaling pathways such as NF-κB, MAPK, TLR4 / MyD88, and Nrf2-ARE, significantly reduce the levels of pro-inflammatory factors such as TNF-α, IL-6, IL-1β, and IFN-γ, upregulate anti-inflammatory factors such as IL-10, alleviate chronic low-grade inflammation and oxidative stress damage, regulate the balance of CD4⁺ / CD8⁺ T cell subsets, promote the proliferation, differentiation, and phagocytic function of immune cells, improve immune aging and inflammatory aging from the source, and repair cellular immune function. Its mechanism of action is clear and comprehensive.

[0097] 3. Advanced technology preserves active ingredients to the greatest extent.

[0098] This invention employs optimized processes such as low-temperature segmented extraction, pressurized permeation extraction, enzymatic-ultrasonic synergistic extraction, ultrasonic-microwave synergistic extraction, low-temperature gradient freeze-drying, vacuum freeze-drying, recrystallization, β-cyclodextrin encapsulation, and inert gas protection. These processes effectively avoid the damage to active ingredients such as polysaccharides, anthocyanins, polyphenols, and saponins caused by high temperatures and oxidation, significantly improving the dissolution rate, purity, and stability of active ingredients, and ensuring the stable performance of the composition's immunomodulatory and anti-aging effects.

[0099] 4. Safe and compliant, with a wide range of applications.

[0100] The raw materials used in this invention include medicinal and edible ingredients or novel food ingredients, with no toxic components. The preparation process uses food-grade reagents and excipients, complying with relevant food processing standards. The composition has been verified through in vitro cell and in vivo animal experiments, showing no cytotoxicity or pathological damage to the body at effective doses, demonstrating high safety. It can be directly used in the production of health foods and special dietary foods related to enhancing cellular immunity, improving immune aging, and delaying aging, with broad application prospects.

[0101] 5. Addresses industry pain points with outstanding technological advantages.

[0102] This invention overcomes the shortcomings of existing technologies, such as outdated raw material formulations, single mechanisms of action, unreasonable preparation processes, low retention rates of active ingredients, and lack of anti-aging and immune-aging-improving functions. It achieves a unified approach of innovative formulation, multi-target synergy, high-efficiency process, clear efficacy, and safety compliance, providing an innovative technical solution for the field of cellular immune regulation and anti-aging. Detailed Implementation

[0103] The present invention will be described in detail below with reference to specific embodiments. The following embodiments are only for the purpose of enabling those skilled in the art to understand the technical solutions of the present invention, implement or use the present invention, and are not intended to limit the scope of protection of the present invention.

[0104] The Astragalus membranaceus, Polygonatum sibiricum, fresh elderberry fruit, olive leaves, Tremella fuciformis polysaccharide and other food-grade reagents (1,2-propanediol, ethanolamine, cellulase, etc.) used in this invention are all commercially available; all detection methods are performed in accordance with the standard methods described in the invention.

[0105] In Example 1, the preparation of Astragalus extract was carried out as follows:

[0106] a. Raw material pretreatment: Remove impurities and wash the dried rhizomes of Astragalus membranaceus, dry them at 50℃ with forced air until the moisture content is 7.2%, pulverize them and pass them through a 90-mesh sieve; degrease them by soaking in food-grade petroleum ether at low temperature (material-liquid ratio 1g:25ml), soak them at 5℃ for 9h, filter to remove petroleum ether; pre-freeze them at -40℃ for 2.5h, and pulverize them into micro-nano powder to 4μm to obtain degreased and cell wall broken Astragalus membranaceus powder.

[0107] b. Pressure permeation extraction: A compound extraction solvent (1,2-propanediol:ethanolamine = 3.5:1, volume ratio) was used, with 0.8% of cellulase by weight of Astragalus powder added. The material-to-liquid ratio was 1g:8ml. The mixture was permeated under pressure at 8.5MPa and 32℃ for 35min; then extracted under reduced pressure at 0.04MPa and 55℃ for 35min, with magnetic stirring (400r / min). After each extraction, the mixture was allowed to stand for 12min and then filtered. The extracts were combined.

[0108] c. Purification and separation: Filter through a 0.22μm filter membrane, add 3% by weight of Astragalus powder to AB-8 macroporous adsorption resin, stir and adsorb for 1.5h, filter to remove resin; extract with n-butanol, collect the aqueous phase, concentrate under reduced pressure of 0.07MPa and 50℃ to a relative density of 1.12 (25℃).

[0109] d. Recrystallization and drying: Crystallization solvent (ethanol: diethyl succinate = 5:2, volume ratio), stirred at 65℃ and 150 rpm for 15 min, filtered, cooled and allowed to stand for crystallization for 5 h, collected the crystals and washed twice with a small amount of ethanol; added 25% maltodextrin (DE value 18) by weight of the crystals, added 0.4% dextranase by weight of the crystals, enzymatically hydrolyzed at 42℃ for 35 min, spray dried (inlet air 165℃, outlet air 72℃, feed 22 mL / min, atomization pressure 0.22 MPa) until the moisture content was 4.2%, to obtain Astragalus extract.

[0110] In Example 1, the preparation of Polygonatum extract was as follows:

[0111] 1) Raw material pretreatment: Remove impurities and wash the dried rhizomes of Polygonatum odoratum, slice (2.5mm thick), dry at 55℃ with forced air until the moisture content is 7.5%, pulverize and pass through a 90-mesh sieve; pre-freeze at -35℃ for 2 hours;

[0112] 2) Enzymatic-ultrasonic extraction: Extraction solvent (deionized water: switchable hydrophilic solvent = 7.5:2, volume ratio; for carbon dioxide responsive switchable hydrophilic solvent, food-grade ammonia and deionized water are mixed at a volume ratio of 1:9), adjust pH to 7.0, add 0.6% cellulase by weight of Polygonatum powder, extract twice at 300W and 55℃, 2 hours each time, stirring for 8 minutes every 30 minutes, with ultrasonic intervals (30 seconds working, 10 seconds rest), incubate at 68℃ for 12 minutes after enzymatic hydrolysis to inactivate, and combine the extracts;

[0113] 3) Purification and concentration: Filter through a 0.22μm filter membrane, purge with nitrogen to separate the solvent into layers, recover the solvent, and collect the aqueous phase; concentrate under reduced pressure at 0.07MPa and 55℃ to a relative density of 1.15 (25℃).

[0114] 4) Encapsulation and drying: Add 20% by weight of β-cyclodextrin (purity 98.5%) of the concentrated solids, stir at 350 r / min for 18 min, spray dry (inlet air 170℃, outlet air 75℃, feed 25 mL / min, atomization pressure 0.25 MPa) until the moisture content is 4.1% to obtain Polygonatum extract.

[0115] In Example 1, the preparation of elderberry anthocyanins was carried out as follows:

[0116] (1) Freeze-drying and anti-oxidation treatment: Elderberry fresh fruit was cleaned and washed, pre-frozen at -45℃ for 2.5h, sublimation drying temperature was -20℃, and desorption drying temperature was 35℃; micro-nano pulverization was carried out at 0~5℃ to 70 mesh, and 0.4% of ascorbic acid-citric acid composite antioxidant (mass ratio 2:1) was added.

[0117] (2) Ultrasonic-microwave synergistic extraction: Extraction solvent (35% edible alcohol: DES = 8:2, volume ratio), adjust pH to 3.2, ultrasonic power 300W, microwave power 180W, 40℃ for 30min, repeat extraction once, ultrasonic interval (working for 20s, resting for 10s), microwave interval (working for 30s, resting for 20s), stir once every 10min;

[0118] (3) Centrifugation, filtration and purification: Centrifuge at 4000 r / min for 15 min, take the supernatant and filter it through a 0.22 μm filter membrane; add 0.15% of the supernatant volume of chitosan, let stand for 18 min, filter and load the sample into an AB-8 macroporous resin column (loading flow rate 2 BV / h, resin column pretreated with pH 3.2 citrate buffer).

[0119] (4) Gradient elution and drying: Impurities were eluted with 4 BV of deionized water at pH 3.2 (flow rate 3 BV / h), and 5 BV of 55% edible alcohol (containing 0.1% ascorbic acid) was eluted (flow rate 1.5 BV / h). The eluent was collected and concentrated under reduced pressure at 50℃ and -0.08 MPa to a solid content of 25% (nitrogen gas was introduced). The solution was then freeze-dried under vacuum (pre-frozen at -45℃ for 3.5 h, sublimated at -25℃, desorption at 40℃, vacuum degree 0.09 MPa) to obtain elderberry anthocyanins.

[0120] In Example 1, the preparation of olive fruit polyphenols was carried out as follows:

[0121] I. Raw material pretreatment and color protection and anti-oxidation: Select mature, fresh olives that are free from rot, mold, and pests. Manually remove the fruit stems, branches, leaves, and impurities. Wash twice with purified water at room temperature. After draining, dry in a 50℃ forced-air drying oven until the moisture content is 7.3%. After pitting, pulverize through a 70-mesh sieve to obtain olive pulp powder. Add 0.3% by weight of food-grade tocopherol-rosemary extract compound antioxidant (mass ratio 1:1) to the powder and mix evenly. Pre-freeze at -40℃ for 1.5 hours, then pulverize at 0~4℃ to 50μm and seal for low-temperature storage.

[0122] II. Ultrasonic-Enzymatic Co-extraction: Add extraction solvent (45% edible alcohol: food-grade deep eutectic solvent DES = 7:3, DES prepared from lactic acid and fructose in a molar ratio of 2:1) to the pretreated fruit pulp powder at a material-to-liquid ratio of 1g:18mL, and adjust the pH to 2.8. Add 0.5% by weight of food-grade pectinase and perform ultrasonic-enzymatic co-extraction. Use an ultrasonic power of 350W and an extraction temperature of 45℃ for 1.5 hours. Repeat the extraction once with the same parameters. The ultrasonic interval is 30 seconds of operation followed by a 10-second pause. Stir for 10 minutes every 20 minutes. After enzymatic hydrolysis, incubate at 68℃ for 12 minutes to inactivate the pectinase. Combine the two extracts.

[0123] III. Degreasing, Centrifugation, Purification, and Concentration: The combined extracts were defatted at 12℃ for 35 min to remove the upper layer of floating oil and fat-soluble impurities; the defatted extracts were centrifuged at 5000 r / min for 20 min, and the supernatant was filtered through a 0.22 μm organic filter membrane; 0.4% by volume of food-grade polyamide resin was added to the filtered supernatant, and the mixture was stirred and adsorbed at room temperature for 25 min, and the resin was removed by filtration; the purified supernatant was concentrated under reduced pressure at 45℃ and -0.085 MPa at a concentration rate of 1.5 mL / min, with high-purity nitrogen gas purging throughout the process, until no alcohol odor was detected, yielding a concentrated olive fruit polyphenol solution.

[0124] IV. Adding a carrier and freeze-drying: Add 15% food-grade maltodextrin by solids to the concentrate and stir at low speed for 18 minutes until uniformly mixed; use vacuum freeze-drying with a pre-freezing temperature of -45℃ and a pre-freezing time of 4 hours, a drying temperature of -50℃ and a vacuum degree of 0.1 Pa, and continue drying for 24 hours; after drying, lightly pulverize and sieve to obtain olive fruit polyphenols.

[0125] Example 1

[0126] A cell-mediated immune-boosting and anti-aging activator based on extracts from medicinal and edible plants, comprising a main ingredient and excipients. The main ingredient consists of the following raw materials in parts by weight:

[0127] Astragalus extract 20 parts, Polygonatum extract 20 parts, Elderberry anthocyanin 10 parts, Olive fruit polyphenols 6.5 parts, Tremella polysaccharide 8 parts.

[0128] The excipients are a mixture of microcrystalline cellulose and pregelatinized starch, and the total weight of the excipients is 25% of the weight of the main ingredients.

[0129] The preparation method of the above-mentioned cell-mediated immune anti-aging activator includes the following steps:

[0130] S1 Raw Material Pretreatment: Astragalus extract, Polygonatum extract, elderberry anthocyanins, and olive fruit polyphenols were pulverized at 3°C ​​using a low-temperature airflow and passed through a 110-mesh sieve. Food-grade argon gas (99.99% purity) was introduced during the pulverization process. Tremella polysaccharide was pulverized at 3°C ​​using micro-nano technology to 65μm, passed through a 90-mesh sieve, and placed in a dry, sealed container at 35% relative humidity and 12°C for later use.

[0131] S2 Premix: Astragalus extract and Polygonatum extract are added to an intelligent temperature-controlled three-dimensional mixer according to the formula. Argon gas is introduced to reduce the oxygen content to 0.4%. The mixing temperature is controlled at 18℃ and the speed is 20r / min. The mixture is mixed for 15min. The mixture is stirred in reverse every 5min (28r / min, for 30s). The online monitoring shows that the mixing uniformity reaches 98.5%, and the premix A is obtained.

[0132] S3 Co-mixing: Elderberry anthocyanins and olive fruit polyphenols were added to premix A. Argon protection was maintained, the mixing temperature was 18℃, the speed was adjusted to 22r / min, and the mixture was mixed for 20min. A segmented stirring mode of 5min forward and 3min reverse was adopted, combined with 120W low-frequency ultrasonic-assisted dispersion (intermittent work for 20s, pause for 40s). Online monitoring showed that the uniformity reached 99.1%, and premix B was obtained.

[0133] S4 Formulation and Molding: Add Tremella polysaccharide to premix B, stir evenly, add excipients according to the ratio, mix evenly, and adjust the pH to 7.0 with food-grade phosphate buffer to obtain a mixture; low-temperature ultrasonic atomization spray drying (inlet air 170℃, outlet air 75℃, feed 28mL / min, atomization pressure 0.28MPa, drying chamber temperature 50℃, ultrasonic atomization frequency 25kHz), after drying, pulverize at low temperature and pass through a 110-mesh sieve to obtain the cell immune activator product; argon gas protection is introduced throughout the drying process.

[0134] Example 2

[0135] A cell-mediated immune-boosting and anti-aging activator based on extracts from medicinal and edible plants, comprising a main ingredient and excipients. The main ingredient consists of the following raw materials in parts by weight:

[0136] Astragalus extract 15 parts, Polygonatum extract 15 parts, elderberry anthocyanin 8 parts, olive fruit polyphenols 5 parts, and Tremella polysaccharide 5 parts.

[0137] The excipients are a mixture of crospovidone and magnesium stearate, and the total weight of the excipients is 11% of the weight of the main ingredient.

[0138] The preparation method of the above-mentioned cell-mediated immune anti-aging activator includes the following steps:

[0139] S1 Raw Material Pretreatment: Astragalus extract, Polygonatum extract, elderberry anthocyanins, and olive fruit polyphenols were pulverized at 4°C using low-temperature airflow and passed through a 100-mesh sieve. Food-grade argon gas (99.99% purity) was introduced during the pulverization process. Tremella polysaccharide was pulverized at 4°C using micro-nano technology to 55μm, passed through an 80-mesh sieve, and placed in a dry, sealed container at 32% relative humidity and 11°C for later use.

[0140] S2 Premix: Astragalus extract and Polygonatum extract are added to an intelligent temperature-controlled three-dimensional mixer according to the formula. Argon gas is introduced to reduce the oxygen content to 0.45%. The mixing temperature is controlled at 16℃ and the speed is 19r / min. The mixture is mixed for 13min, and then stirred in reverse every 4min (26r / min, for 30s). The online monitoring shows that the mixing uniformity reaches 98.2%, and premix A is obtained.

[0141] S3 Co-mixing: Elderberry anthocyanins and olive fruit polyphenols were added to premix A. Argon protection was maintained, the mixing temperature was 16℃, the speed was adjusted to 21r / min, and the mixture was mixed for 19min. A segmented stirring mode of 5min forward and 3min reverse was adopted, combined with 110W low-frequency ultrasonic-assisted dispersion (intermittent work for 20s, pause for 40s). Online monitoring showed that the uniformity reached 98.8%, and premix B was obtained.

[0142] S4 Formulation and Molding: Add Tremella polysaccharide to premix B, stir evenly, add excipients according to the ratio, mix evenly, and adjust the pH to 6.9 with food-grade phosphate buffer to obtain a mixture; low-temperature ultrasonic atomization spray drying (inlet air 168℃, outlet air 73℃, feed 26mL / min, atomization pressure 0.26MPa, drying chamber temperature 48℃, ultrasonic atomization frequency 22kHz), after drying, pulverize at low temperature and pass through a 100-mesh sieve to obtain the cell immune activator product; argon gas protection is introduced throughout the drying process.

[0143] Example 3

[0144] A cell-mediated immune-boosting and anti-aging activator based on extracts from medicinal and edible plants, comprising a main ingredient and excipients. The main ingredient consists of the following raw materials in parts by weight:

[0145] 25 parts of Astragalus membranaceus extract, 25 parts of Polygonatum sibiricum extract, 12 parts of anthocyanins from elderberry, 8 parts of olive fruit polyphenols, and 10 parts of Tremella fuciformis polysaccharide.

[0146] The excipients are a mixture of microcrystalline cellulose, pregelatinized starch, crospovidone, and magnesium stearate, and the total weight of the excipients is 39% of the weight of the main ingredients.

[0147] The preparation method of the above-mentioned cell-mediated immune anti-aging activator includes the following steps:

[0148] S1 Raw Material Pretreatment: Astragalus extract, Polygonatum extract, elderberry anthocyanins, and olive fruit polyphenols were pulverized at 2°C using a low-temperature airflow and passed through a 120-mesh sieve. Food-grade argon gas (99.99% purity) was introduced during the pulverization process. Tremella polysaccharide was pulverized at 2°C using micro-nano technology to 75μm, passed through a 100-mesh sieve, and placed in a dry, sealed container at 38% relative humidity and 14°C for later use.

[0149] S2 Premix: Astragalus extract and Polygonatum extract are added to an intelligent temperature-controlled three-dimensional mixer according to the formula. Food-grade argon gas is introduced to reduce the oxygen content to 0.35%. The mixing temperature is controlled at 19℃ and the speed is 21r / min. The mixture is mixed for 17min, and then stirred in reverse every 6min (29r / min, for 30s). The online monitoring shows that the mixing uniformity reaches 98.7%, and the premix A is obtained.

[0150] S3 Co-mixing: Elderberry anthocyanins and olive fruit polyphenols were added to premix A. Under food-grade argon protection and a mixing temperature of 19°C, the mixing speed was adjusted to 24 r / min and mixed for 21 min. A segmented stirring mode of 5 min forward and 3 min reverse was adopted, combined with 140W low-frequency ultrasonic-assisted dispersion (intermittent operation for 20 s and pause for 40 s). The uniformity was monitored online and found to be 99.3%, resulting in premix B.

[0151] S4 Formulation and Molding: Add Tremella polysaccharide to premix B, stir evenly, add excipients according to the ratio, mix evenly, adjust pH to 7.1 with food-grade phosphate buffer to obtain a mixture; low-temperature ultrasonic atomization spray drying (inlet air 172℃, outlet air 76℃, feed 29mL / min, atomization pressure 0.29MPa, drying chamber temperature 52℃, ultrasonic atomization frequency 28kHz), after drying, pulverize at low temperature and pass through a 120-mesh sieve to obtain the cell immune activator product; food-grade argon gas is introduced for protection throughout the drying process.

[0152] Comparative Example 1

[0153] The difference between this and the cellular immune anti-aging activator described in Example 1 is that:

[0154] No astragalus extract was added to the raw materials; otherwise, the process was carried out as described in Example 1.

[0155] The specific preparation method is the same as in Example 1, except that Astragalus extract is not added in this comparative example, while the rest is carried out in accordance with Example 1.

[0156] Comparative Example 2

[0157] The difference between this and the cellular immune anti-aging activator described in Example 1 is that:

[0158] No Polygonatum extract was added to the raw materials; otherwise, the process was carried out as described in Example 1.

[0159] The specific preparation method is the same as in Example 1, except that no Polygonatum extract is added in this comparative example, while the rest is carried out in accordance with Example 1.

[0160] Comparative Example 3

[0161] The difference between this and the cellular immune anti-aging activator described in Example 1 is that:

[0162] Elderberry anthocyanins were not added to the raw materials; otherwise, the process was carried out as described in Example 1.

[0163] The specific preparation method is the same as in Example 1, except that elderberry anthocyanins are not added in this comparative example, while the rest is carried out in accordance with Example 1.

[0164] Comparative Example 4

[0165] A cell-mediated immune anti-aging activator, as in Example 1.

[0166] The preparation method of the cell immune anti-aging activator is the same as in Example 1, except that:

[0167] In the preparation of Astragalus membranaceus extract, the pressure immersion temperature was increased from 32°C to 60°C, while the pressure extraction temperature remained unchanged at 55°C. In the preparation of Polygonatum sibiricum extract, the enzymatic-ultrasonic extraction temperature was increased from 55°C to 80°C, and the rest were consistent with Example 1.

[0168] Comparative Example 5

[0169] A cell-mediated immune anti-aging activator, as in Example 1.

[0170] The preparation method of the cell immune anti-aging activator is the same as in Example 1, except that:

[0171] In the preparation of elderberry anthocyanins, the solvent for ultrasonic-microwave synergistic extraction (35% edible alcohol: DES = 8:2, volume ratio) was replaced with pure distilled water, and the rest was consistent with Example 1.

[0172] Comparative Example 6

[0173] A cell-mediated immune anti-aging activator, as in Example 1.

[0174] The preparation method of the cell immune anti-aging activator is the same as in Example 1, except that:

[0175] In step S4, the low-temperature ultrasonic atomization spray drying is replaced with traditional hot air oven drying at a temperature of 80°C until the moisture content is ≤5%. All other steps are the same as in Example 1.

[0176] Comparative Example 7

[0177] A cell-mediated immune anti-aging activator, as in Example 1.

[0178] The preparation method of the cell immune anti-aging activator is the same as in Example 1, except that the food-grade argon protection in all steps is replaced with ordinary nitrogen protection, and the rest is the same as in Example 1.

[0179] Comparative Example 8

[0180] A cell-mediated immune anti-aging activator, as in Example 1.

[0181] The preparation method of the cell immune anti-aging activator adopts the existing conventional process. The difference from Example 1 is that: the preparation of each extract adopts conventional high-temperature decoction extraction. The extraction temperature of Astragalus membranaceus extract is 90℃ and the extraction time is 2h. The extraction temperature of Polygonatum sibiricum extract is 90℃ and the extraction time is 2h. Enzyme-assisted, ultrasonic-microwave synergistic and other optimized processes are not used. The preparation of the finished product does not adopt graded mixing, ultrasonic atomization drying and argon protection. Only ordinary mixing and hot air drying are used. The rest is the same as in Example 1.

[0182] Comparative Example 9

[0183] A cell-mediated immune anti-aging activator, as in Example 1.

[0184] The preparation method of the cell immune anti-aging activator is the same as in Example 1, except that:

[0185] In step IV of the preparation of olive fruit polyphenols and step d of the preparation of astragalus extract, the food-grade maltodextrin used was replaced with food-grade lactose, and the rest of the originals were the same as in Example 1.

[0186] Comparative Example 10

[0187] A cell-mediated immune anti-aging activator, as in Example 1.

[0188] The preparation method of the cell immune anti-aging activator is the same as in Example 1, except that in step 4) of the preparation of Polygonatum extract, the β-cyclodextrin embedding step is omitted, and the concentrated Polygonatum extract is directly spray-dried. The rest is the same as in Example 1.

[0189] Experimental Example 1: In vitro cytotoxicity experiment

[0190] Using in vitro cell models, this study verifies the cellular safety of the cell-mediated immune-anti-aging activator (hereinafter referred to as "immuno-anti-aging composition") based on medicinal and edible plant extracts and the extracts of each raw material. It clarifies the concentration range within which the composition has no toxic effect on cells, providing a scientific basis for subsequent in vitro cell-mediated immune-activating function experiments and in vivo animal experiments in terms of dosage setting. At the same time, it verifies the impact of different preparation processes (such as argon protection vs. ordinary nitrogen protection, conventional process vs. optimized low-temperature process) on the cellular safety of the immune-anti-aging composition, thus demonstrating the rationality of the process optimization of this invention.

[0191] Experimental group design:

[0192] Blank control group / cell control group: to establish the basic cell survival rate as a benchmark for toxicity assessment.

[0193] Examples 1–3: Verifying the cell safety of each example of the present invention.

[0194] Comparative Examples 1–3: Verify whether formulations lacking a single ingredient produce cytotoxicity and test the safety of the formulation.

[0195] Comparative Groups 4–10: to verify the effects of different preparation processes (temperature, solvent, protective gas, drying, embedding, etc.) on cell safety.

[0196] Single ingredient group: Verify the cytotoxicity of each ingredient when used alone, and confirm the biocompatibility of the formulation components.

[0197] 1. Experimental Materials

[0198] (1) Cell lines: mouse macrophages RAW264.7 and human T lymphocytes Jurkat, both of which were selected in the logarithmic growth phase.

[0199] (2) Test samples: immune anti-aging compositions of Examples 1-3, immune anti-aging compositions of Comparative Examples 1-10, and extracts of each single main ingredient (Astragalus membranaceus extract, Polygonatum sibiricum extract, anthocyanins from elderberry, olive fruit polyphenols, and Tremella fuciformis polysaccharides, all prepared according to the corresponding preparation methods of this invention);

[0200] (3) Cell culture medium: DMEM high glucose medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin solution).

[0201] (4) Reagents: CCK-8 assay kit, phosphate buffered saline (PBS, pH 7.2-7.4);

[0202] (5) Consumables: 96-well cell culture plate, sterile pipette tip, centrifuge tube.

[0203] 2 Experimental Methods

[0204] 2.1 Preparation of test solution

[0205] Accurately weigh 100 mg of each test sample, dissolve it in sterile PBS to prepare a 10 mg / mL stock solution, and filter it through a 0.22 μm filter membrane for sterilization. Before the experiment, dilute with culture medium to prepare working solutions of 10, 50, 100, 200, 500, and 1000 μg / mL, and prepare fresh before use.

[0206] 2.2 Cell Seeding and Treatment

[0207] RAW264.7 and Jurkat cells in logarithmic growth phase were selected and their concentration adjusted to 1×10⁻⁶. 5 Cells / mL were seeded into 96-well plates and incubated at 37°C with 5% CO2 for 24 h. The culture medium was discarded, and different concentrations of the test sample were added, with 6 replicates per group; blank control and cell control were also included. The plates were incubated for another 24 h and 48 h.

[0208] 2.3 Cytotoxicity Detection

[0209] 2.3.1 CCK-8 assay for cell viability

[0210] The CCK-8 assay was used. 10 μL of CCK-8 reagent was added to each well, and the cells were incubated for 2 hours. Absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated. A viability rate ≥70% was considered the criterion for no cytotoxicity.

[0211] 2.4 Data Calculation and Analysis

[0212] (1) Formula for calculating cell viability:

[0213] Cell viability (%) = (Experimental group OD) 450 Value - Blank control group OD 450 (value) / (cell control group OD) 450 Value - Blank control group OD 450 Value) × 100%

[0214] (2) Data analysis was performed using SPSS 26.0 statistical software. Quantitative data were expressed as mean ± standard deviation (x ± s). One-way ANOVA was used for comparisons between groups. P < 0.05 was considered statistically significant.

[0215] (3) The cell viability rate is the core indicator to determine the cytotoxicity of each test sample. The relevant technical specifications / industry guidelines for in vitro cytotoxicity evaluation are referenced, and the cell viability rate ≥70% is used as the criterion for determining that the test sample has no cytotoxicity.

[0216] (4) Determine the non-toxic concentration range of each test sample to RAW264.7 cells and Jurkat cells, focusing on the core effective and safe concentration of the immune anti-aging composition in Example 1.

[0217] 3. Experimental Results and Analysis

[0218] 3.1 Cytotoxicity Results of the Immunoantiaging Compositions in Examples 1-3

[0219] Table 1. Results of RAW264.7 cytotoxicity assay

[0220]

[0221] Table 2 Results of Jurkat cytotoxicity assay

[0222]

[0223] Experimental Results: The immunomodulatory anti-aging compositions of Examples 1-3, within a concentration range of 10-200 μg / mL, showed a survival rate of ≥85% for both RAW264.7 and Jurkat cells, meeting the criteria for non-cytotoxicity. There was no significant difference in cell survival rate between Examples 1 (intermediate ratio), 2 (lowest ratio), and 3 (highest ratio) (P>0.05), indicating that the safety of the immunomodulatory anti-aging compositions was consistent across different ratios within the range of raw material ratios. When the concentration was ≥500 μg / mL, the cell survival rate after treatment with the immunomodulatory anti-aging compositions of all three examples was significantly lower than 70%, and the toxicity gradually increased with increasing concentration and culture time, showing a consistent trend.

[0224] Therefore, 200 μg / mL was selected as the core effective and safe concentration of the immune anti-aging compositions in Examples 1-3. This concentration will be used preferentially in subsequent experimental cases to verify the in vitro cell immune activation function, representing all the reasonable ratios of the immune anti-aging compositions of the present invention for subsequent functional verification.

[0225] 3.2 Comparison of cytotoxicity of different formulations of immunomodulatory anti-aging compositions (Example 1 vs. Comparative Examples 1-3)

[0226] Table 3 Comparison of cell viability at 24 h (RAW264.7 cells, 200 μg / mL concentration)

[0227]

[0228] Results Analysis: As shown in Table 3, the immune anti-aging compositions of Comparative Examples 1-3, which lacked a single ingredient, showed no significant difference in the survival rate of RAW264.7 cells at a concentration of 200 μg / mL compared with Example 1 (P > 0.05), and the cell morphology was not significantly abnormal. This indicates that the absence of a single ingredient in the five-component synergistic formulation of the present invention does not lead to cytotoxicity of the immune anti-aging composition, and the overall safety redundancy of the formulation is good.

[0229] 3.3 Comparison of cytotoxicity of immunomodulatory anti-aging compositions prepared using different processes (Example 1 vs. Comparative Examples 4-10)

[0230] Table 4 Comparison of cell viability at 24h (RAW264.7 cells, 200 μg / mL concentration)

[0231]

[0232] Results Analysis: As shown in Table 4, the anti-aging compositions of Comparative Examples 1-10 with different processes showed no significant difference in the survival rate of RAW264.7 cells at a concentration of 200 μg / mL compared with Example 1 (P > 0.05), and all met the criteria for non-cytotoxicity. This indicates that the main function of the process optimization of the present invention (low-temperature extraction, food-grade argon protection, embedding, etc.) is to improve the retention rate of active ingredients and immune activation efficacy, rather than affecting cell safety, further verifying the rationality and safety of the process of the present invention.

[0233] Table 5. Cytotoxicity results of single main ingredient extracts (200 μg / mL concentration, 24 h)

[0234]

[0235] All the single main ingredient extracts in the table showed no cytotoxicity at a concentration of 200 μg / mL, which is consistent with the safety of the immune anti-aging composition of the present invention and also confirms the good biocompatibility of each raw material in the five-component formulation.

[0236] 4. Experimental Conclusions

[0237] (1) The immune anti-aging compositions of Examples 1-3 of the present invention, the extracts of each single main ingredient, and the immune anti-aging compositions of Comparative Examples 1-10 all showed no cytotoxicity in the concentration range of 10-200 μg / mL, and the cell morphology was not obviously abnormal.

[0238] (2) The core effective safe concentration of the immune anti-aging compositions in Examples 1-3 is 200 μg / mL. This concentration not only meets the requirement of non-toxicity, but also reserves sufficient activity space for subsequent immune activation function experiments. Among them, Example 1 (intermediate ratio) can be used as the preferred concentration for in vitro cell experiments. The safety of Example 2 (minimum ratio) and Example 3 (maximum ratio) is consistent with that of Example 1.

[0239] (3) The cell safety of the immune anti-aging composition lacking a single raw material is not significantly different from that of the five-component complete formulation activator, and the safety redundancy of the formulation of the present invention is good; the immune anti-aging compositions with different preparation processes are all free of cytotoxicity, and the process optimization of the present invention does not affect the product safety.

[0240] (4) This experimental example provides a scientific basis for the dosage setting of subsequent experimental example 2 (in vitro cell immune activation function verification) and experimental example 3 (in vivo animal experiment), ensuring that the dosage of subsequent experiments is reasonable and compliant, and avoiding toxicity risks.

[0241] Experiment Example 2: In Vitro Cellular Immunotherapy and Anti-aging Function Experiment

[0242] This study validated the immunomodulatory and anti-aging function of the cell-based immune-immune anti-aging composition (hereinafter referred to as "immunomodulatory anti-aging composition") based on medicinal and edible plant extracts using in vitro cell models. It clarified the effects of this composition on the secretion of immune-related cytokines by macrophages (RAW264.7) and lymphocytes (Jurkat), thus verifying the effectiveness of the dual-target cell-based immune-immune anti-aging mechanism. Simultaneously, it compared the effects of different formulations and preparation processes on the immunomodulatory and anti-aging efficacy of the composition, demonstrating the advantages of the synergistic effect of the formulation and the optimized process. Combined with the core effective and safe concentration of 200 μg / mL determined in Experiment 1, the in vitro functional validation was completed, providing a scientific basis for subsequent in vivo animal experiments and product application.

[0243] Experimental group design:

[0244] Blank control: contains no cells and only culture medium.

[0245] Normal cell control group (basal group): RAW264.7 / Jurkat cells, without senescence induction or sample application, only cultured in culture medium.

[0246] Immune aging model group:

[0247] ① RAW264.7 macrophage immune senescence model: Logarithmically growing RAW264.7 cells were placed in DMEM medium containing 10% fetal bovine serum, and deoxynivalenol (DON) was added to a final concentration of 2 μM. The cells were cultured at 37℃ and 5% CO2 for 24 h to induce a macrophage immune senescence model.

[0248] ② Jurkat T lymphocyte immune aging model: Jurkat cells in the logarithmic growth phase were used to establish a T lymphocyte immune aging model by continuously inducing Tat protein expression in RPMI 1640 medium containing 10% fetal bovine serum for 72 h.

[0249] Positive control group (aging model + levamisole): Aging model cells were treated with a predetermined concentration of levamisole (a positive drug for improving immune aging).

[0250] Examples 1-3 (Aging Model + Sample of the Invention): Aging model cells were treated with a predetermined concentration of the immune anti-aging composition of the present invention.

[0251] Comparative Examples 1-3 (Aging Model + Composition Lacking Any One of the Ingredients): Aging model cells were treated with a set concentration of the composition lacking any one of the ingredients.

[0252] Comparative Examples 4-10 (Aging Model + Process-Modified Composition): Aging model cells were subjected to a set concentration of a process-modified composition (high temperature / solvent replacement / no argon / no embedding, etc.).

[0253] Single ingredient group (aging model + single ingredient): Aging model cells are treated with a set concentration of a single ingredient extract.

[0254] 1. Experimental Materials and Instruments

[0255] 1.1 Experimental Materials

[0256] (1) Cell lines: mouse macrophages RAW264.7 and human T lymphocytes Jurkat (consistent with Experiment 1, logarithmic growth phase cells were selected).

[0257] (2) Test samples: Immunoantiaging compositions of Examples 1-3, immunoantiaging compositions of Comparative Examples 1-10, and extracts of each single raw material (consistent with those of Experimental Example 1) were all prepared into working solutions of 200 μg / mL (the core effective safe concentration determined in Experimental Example 1).

[0258] (3) Positive control: Levamisole (immune activation positive control, purity ≥98%, purchased from Sigma), prepared into a working solution of 200 μg / mL;

[0259] (4) Cell culture medium: DMEM medium and RPMI-1640 medium (both containing 10% fetal bovine serum and 1% penicillin-streptomycin solution) were purchased from Gibco.

[0260] (5) Reagents: Mouse TNF-α ELISA kit and human IFN-γ ELISA kit (both purchased from Solarbio).

[0261] (6) Consumables: 96-well cell culture plate, sterile pipette tips, centrifuge tubes, and enzyme-labeled plates (all medical sterile grade).

[0262] 2 Experimental Methods

[0263] All test samples were tested at a concentration of 200 μg / mL, with 6 replicates per group, strictly following the kit instructions and standard cell experiment procedures.

[0264] 2.1 Detection of immune-related cytokine secretion (ELISA method)

[0265] Cell supernatant was collected, and the contents of TNF-α (macrophages) and IFN-γ (lymphocytes) were detected. A standard curve was plotted and the results were quantified.

[0266] Results: TNF-α is a core pro-inflammatory immune factor secreted by activated macrophages and a key indicator reflecting the level of inflammatory senescence and chronic low-grade inflammation in the body. Higher TNF-α expression concentrations indicate a more significant pro-inflammatory activation effect of macrophage senescence and a more pronounced inflammatory senescence process in the body. IFN-γ is a core immunomodulatory factor secreted by activated T lymphocytes. In the context of aging, T cell function is imbalanced, with some senescent T cells abnormally overexpressing IFN-γ, resulting in a pro-inflammatory effect, accelerating the inflammatory senescence process, and leading to dysregulation of cellular immune responses and anti-infection defense functions.

[0267] 2.2 Data Calculation and Analysis

[0268] Data analysis was performed using SPSS 26.0 statistical software. Quantitative data were expressed as mean ± standard deviation (x ± s). One-way ANOVA was used for comparisons between groups. P < 0.05 was considered statistically significant, and P < 0.01 was considered extremely statistically significant. Using the cell control group as a baseline, the phagocytic activity, proliferation rate, and cytokine concentration of each experimental group and the positive control group were compared to clarify the immune activation effect of the immune-antiaging composition and the influence of formulation and process.

[0269] 3. Experimental Results and Analysis

[0270] 3.1 Effects of the anti-aging compositions in Examples 1-3 on the secretion of immune-related cytokines

[0271] Table 6 shows the effect on TNF-α secretion in RAW264.7 cells.

[0272]

[0273] Note:

[0274] Compared with the normal cell control group, P < 0.01.

[0275] b Compared with the immune aging model group, P < 0.01,

[0276] Compared with the positive control group, c, P<0.05.

[0277] Table 7 Effects on IFN-γ secretion in Jurkat cells

[0278]

[0279] Note:

[0280] Compared with the normal cell control group, P < 0.01.

[0281] b Compared with the immune aging model group, P < 0.01,

[0282] Compared with the positive control group, c, P<0.05.

[0283] Results analysis: The levels of TNF-α secretion in RAW264.7 cells and IFN-γ secretion in Jurkat T lymphocytes in the immunosenescence model group were significantly higher than those in the normal cell control group (P<0.01), indicating that the constructed macrophage and T lymphocyte immunosenescence model was successfully established and the secretion of cellular inflammatory factors showed a significant abnormal state.

[0284] In Examples 1-3, after treatment with the immune-antiaging composition, the secretion levels of TNF-α and IFN-γ in aging model cells were significantly lower than those in the model group (P < 0.01), and the reduction effect was significantly better than that of the positive control levamisole (P < 0.05). This indicates that the composition of the present invention can simultaneously target macrophages and T lymphocytes, effectively improve the excessive secretion of inflammatory factors in the two types of cells under aging conditions, restore the normal immune regulatory function of cells, and is completely consistent with the dual-target cell immune activation mechanism. Among them, Example 3 (highest ratio) showed the best effect in reducing inflammatory factors, followed by Example 1 (intermediate ratio), and Example 2 (lowest ratio) was slightly lower, but there was no significant difference among the three (P>0.05). This indicates that within the range of raw material ratios of the present invention, the compositions with different ratios have a stable effect on the inflammatory regulation of senescent macrophages and T lymphocytes. They can all effectively restore the secretion levels of TNF-α and IFN-γ to near normal levels, which is consistent with the trend of the lymphocyte proliferation capacity experiment. Together, they verify the immune anti-aging mechanism of the composition of the present invention targeting both macrophages and lymphocytes, and further confirm the rationality of the formulation ratio and the stability of the process.

[0285] 3.2 Formulation synergy validation (Example 1 vs. Comparative Examples 1-3 vs. Single ingredient extracts)

[0286] Based on Example 1, the immune activation effects (at a concentration of 200 μg / mL, with macrophage TNF-α secretion level and lymphocyte IFN-γ secretion level as the core indicators) of Comparative Examples 1-3 (lacking a single ingredient) and the extracts of each single main ingredient were compared. The results are shown in Table 8 below:

[0287] Table 8

[0288]

[0289] Note:

[0290] Compared with the normal cell control group, P < 0.01.

[0291] b Compared with the immune aging model group, P < 0.01,

[0292] c Compared with Example 1, P < 0.01.

[0293] Results analysis: Compared with Example 1 (complete five-component formulation), the levels of macrophage inflammatory factors and lymphocyte functional factors in the control group lacking any ingredient were significantly increased. Moreover, the immunomodulatory effects of all single main ingredient extract groups were significantly weaker than those of the complete formulation group. This indicates that the ingredients in the composition of the present invention are not simply superimposed, but rather exert a significantly better immune anti-aging effect than a single component through the synergistic effect of multiple components.

[0294] Among them, the control group lacking Astragalus membranaceus extract and Polygonatum sibiricum extract showed the greatest increase in the levels of inflammatory factors and functional factors, suggesting that Astragalus membranaceus extract and Polygonatum sibiricum extract are core components for maintaining immune cell homeostasis and inhibiting excessive inflammatory response. The control group lacking anthocyanins from Sambucus chinensis showed the second greatest increase, indicating that anthocyanins from Sambucus chinensis play a key role in alleviating inflammatory damage and protecting immune cell function. Although the single raw material groups of olive fruit polyphenols and Tremella fuciformis polysaccharides could improve cell function in the immune aging model to a certain extent, the improvement effect was far lower than that of the complete formula group, further verifying the synergistic effect mechanism among the components.

[0295] In summary, the five-component formula of this invention achieves significant immune-boosting and anti-aging effects through the synergistic effect of macrophage inflammation suppression and lymphocyte function repair. The absence of any ingredient will lead to a significant decrease in immune-regulating efficacy, which fully demonstrates the scientific nature and completeness of the formula composition.

[0296] 3.3 Validation of the advantages of process optimization (Example 1 vs. Comparative Examples 4-10)

[0297] Based on Example 1, the immune activation effects of Comparative Examples 4-10 with different processes were compared (at a concentration of 200 μg / mL, with macrophage TNF-α secretion level and lymphocyte IFN-γ secretion level as the core indicators). The results are shown in Table 9 below:

[0298] Table 9

[0299]

[0300] Note:

[0301] Compared with the normal cell control group, P < 0.01.

[0302] b Compared with the immune aging model group, P < 0.01,

[0303] c Compared with Example 1, P < 0.01,

[0304] Compared with Example 1, P < 0.05.

[0305] Results analysis: The levels of pro-inflammatory cytokines TNF-α and lymphocyte functional factor IFN-γ in RAW264.7 cells of the immunosenescence model group were significantly higher than those in the normal cell control group (P < 0.01), indicating a significant inflammatory imbalance in the immunosenescence model cells, and the model was successfully constructed. Compared with Example 1, the levels of TNF-α and IFN-γ in each comparative example were significantly higher than those in Example 1 (P < 0.05), indicating that the preparation process of the present invention can significantly improve the inflammatory imbalance in immunosenescence model cells, and any deviation from any process step will lead to a significant decrease in the inflammatory regulatory effect of the composition.

[0306] Among them, Comparative Example 8 (conventional high-temperature decoction process + no protective gas) showed the largest increase in TNF-α and IFN-γ levels, approaching the levels of the immune aging model group. This indicates that the traditional high-temperature unprotected process leads to a large degradation of the active ingredients in the composition, completely losing its ability to regulate inflammatory factors. Comparative Example 4 (increased extraction temperature of Astragalus / Polygonatum), Comparative Example 5 (the solvent for elderberry anthocyanin extraction was changed to distilled water), and Comparative Example 10 (omitted β- of Polygonatum extract) showed similar increases. The levels of inflammatory factors in the cyclodextrin-encapsulated formulation were the second lowest. In Comparative Example 4, the high temperature damaged the glycosidic bond structure of Astragalus membranaceus and Polygonatum polysaccharides. In Comparative Example 5, the insufficient solvent dissolution capacity led to a decrease in the anthocyanin dissolution rate. In Comparative Example 10, the lack of encapsulation protection for Polygonatum polysaccharides reduced stability. All three factors resulted in a significant decrease in the content of anti-inflammatory active ingredients in the composition, failing to effectively inhibit the release of pro-inflammatory factors. In Comparative Example 6 (hot air oven drying), the high temperature drying caused oxidative degradation of the active ingredients. In Comparative Example 7 (nitrogen protection instead of argon protection), the antioxidant capacity of nitrogen was weaker than that of argon, exacerbating the oxidative deterioration of the active ingredients. Both factors significantly weakened the inflammatory regulation effect of the composition. Although the levels of TNF-α and IFN-γ in Comparative Example 9 (lactose instead of maltodextrin) were significantly higher than those in Example 1, they were significantly lower than those in other process defect groups. This indicates that although lactose can be used as a carrier excipient, maltodextrin has a better encapsulation protection effect on the active ingredients and can better maintain the anti-inflammatory activity of the composition.

[0307] In summary, the process optimizations of this invention (low-temperature extraction, application of compound solvents, argon protection, β-cyclodextrin encapsulation, selection of maltodextrin carriers, etc.) can significantly improve the inflammatory imbalance of immune aging model cells by increasing the retention rate and stability of active ingredients, and reduce the excessive release of pro-inflammatory factors TNF-α and IFN-γ. The process optimizations are clearly necessary and have significant advantages in ensuring the synergistic immunomodulatory and anti-inflammatory effects of the composition.

[0308] Experiment Example 3: In vivo animal immune function verification experiment

[0309] Using in vivo animal models, the immunomodulatory function of the immune-anti-aging composition based on medicinal and edible plant extracts (hereinafter referred to as "immuno-anti-aging composition") of this invention was further verified, clarifying its effect on mouse serum immune cytokines. Combined with in vitro experimental results, the in vivo immune activation effect of the immune-anti-aging composition was comprehensively verified. At the same time, the effects of different formulations and different preparation processes on in vivo immune efficacy were verified, clarifying the advantages of the formulation synergy and process optimization of this invention, providing in vivo experimental basis for the practical application of the product, and evaluating the in vivo safety of the product.

[0310] Experimental group design:

[0311] Blank control group: Establishes the baseline of normal cytokines in mice.

[0312] Immunoaging model group: Mice were subcutaneously injected with 500 mg / kg of D-galactose daily for 9 weeks to induce accelerated aging.

[0313] Positive control group (aging model + levamisole): Mice with an immune aging model were given a set dose of levamisole (a positive drug for improving immune aging) as a reference standard for improving immune aging in vivo.

[0314] Examples 1–3 (aging model + sample of the present invention): Mice with an immune aging model were given a set dose of the immune anti-aging composition of Examples 1–3 (different ratios) to verify the effects of different ratios on in vivo cytokine secretion and immune improvement.

[0315] Comparative Examples 1–3 (Aging Model + Compositions Lacking Any One Ingredient): Immunoaging model mice were given a set dose of the Comparative Examples 1–3 composition to verify the decrease in in vivo immune efficacy after the absence of a single ingredient, demonstrating the synergistic core effect of the five components.

[0316] Comparative Examples 4–10 (Aging Model + Process-Modified Compositions): Immunosensitive aging model mice were given a set dose of the Comparative Examples 4–10 compositions to verify the reduced in vivo efficacy after process degradation, demonstrating the key value of processes such as low temperature, argon, encapsulation, and composite solvents.

[0317] Single ingredient group: In an immune aging model mouse, a set dose of a single ingredient extract was administered, verifying that the in vivo effect of the single component was much weaker than that of the compound formulation, further demonstrating the synergistic effect of the formulation.

[0318] Safety observation throughout the process: For all intervention group mice, it was confirmed that there was no in vivo toxicity or organ damage at the effective dose.

[0319] 1. Experimental Materials and Instruments

[0320] 1.1 Experimental Materials

[0321] (1) Experimental animals: SPF grade Kunming mice, weighing 18-22g, half male and half female, purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd.; breeding environment: temperature 22-25℃, relative humidity 50-60%, 12h light / 12h dark alternation, free access to water and food, and the experiment began after 3 days of acclimatization.

[0322] (2) Test samples: the immune anti-aging compositions of Examples 1-3, the immune anti-aging compositions of Comparative Examples 1-10, and the extracts of each single raw material (consistent with those of Experimental Examples 1 and 2) were all finely ground and prepared into suspensions of the corresponding concentrations with physiological saline.

[0323] (3) Positive control: Levamisole tablets (purity ≥98%, purchased from Sigma), finely ground and prepared into a 40 mg / kg suspension with physiological saline (dosage calculated based on mouse weight).

[0324] (4) Reagents: Mouse TNF-α and IFN-γ ELISA kits (both purchased from Solarbio), physiological saline, 4% paraformaldehyde solution;

[0325] (5) Consumables: sterile centrifuge tubes, pipette tips, and ELISA plates (all medical sterile grade).

[0326] 2 Experimental Methods

[0327] This experiment employed a randomized controlled group design, dividing mice into a blank control group, an immune aging model group, a positive control group, Example 1-3 groups, Comparative Example 1-10 groups, and a single raw material extract group. Each group contained 10 mice (5 males and 5 females). The specific procedures were as follows:

[0328] 2.1 Grouping and administration of test samples

[0329] Kunming mice that had been acclimatized for 3 days were randomly divided into 18 groups of 10 mice each, as follows:

[0330] (1) Blank control group: Normal feeding, without D-galactose modeling; given an equal volume of physiological saline by gavage once a day;

[0331] (2) Immunoaging model group: D-galactose modeling was used as a negative control for the aging model.

[0332] (3) Positive control group (aging model + levamisole): levamisole suspension was administered at a dose of 40 mg / kg by gavage once daily;

[0333] (4) Examples 1-3 (aging model + sample of the present invention): The immune anti-aging composition suspension of Examples 1-3 was administered to each group at a dose of 200 mg / kg (calculated based on the core effective concentration in the in vitro experiment and the mouse weight, corresponding to the in vitro concentration of 200 μg / mL), by gavage, once a day;

[0334] (5) Comparative Examples 1-3 (aging model + composition lacking any raw material): The immune anti-aging composition suspension of Comparative Examples 1-3 was administered by gavage at a dose of 200 mg / kg once a day.

[0335] (6) Comparative Examples 4-10 (aging model + process-modified composition): The comparative examples 4-10 were administered the immune anti-aging composition suspension at a dose of 200 mg / kg by gavage once a day.

[0336] (7) Single raw material extract group (aging model + single raw material extract): Each single raw material extract suspension was administered by gavage at a dose of 200 mg / kg once a day.

[0337] All groups were administered the drug continuously for 14 days, with a drug volume of 0.2 mL / 10 g body weight. During the administration period, the mice's mental state, diet, water intake, activity, and mortality were closely observed, and changes in mouse body weight were recorded.

[0338] 2.2 Sample Collection and Processing

[0339] 14 days after administration, the patient was kept on a fasting period but allowed free access to water for 12 hours before sample collection.

[0340] (1) Serum collection: Blood was collected from the orbital cavity of mice, placed in a sterile centrifuge tube, and centrifuged at 3000 r / min for 15 min after standing at room temperature for 2 h. The supernatant (serum) was collected and stored at -20℃ for the purpose of detecting the content of TNF-α and IFN-γ in serum.

[0341] 2.3 Indicator Testing

[0342] (1) Safety observation: Record the abnormal reactions of mice during the experiment (such as lethargy, loss of appetite, diarrhea, death, etc.). After the mice are euthanized, observe the morphology of organs such as thymus, spleen, liver, and kidneys to determine whether there is any pathological damage.

[0343] (2) Serum immune cytokine detection: The levels of TNF-α and IFN-γ in serum (pg / mL) were detected according to the ELISA detection method.

[0344] 2.4 Data Calculation and Analysis

[0345] Data analysis was performed using SPSS 26.0 statistical software. Quantitative data were expressed as mean ± standard deviation (x ± s). One-way ANOVA was used for comparisons between groups. P < 0.05 was considered statistically significant, and P < 0.01 was considered extremely statistically significant. Using a blank control group as a baseline and a negative control group for the aging model as a baseline, the cytokine concentrations of each experimental group and the positive control group were compared to clarify the in vivo immune activation effect of the immune-antiaging composition and the influence of formulation and process.

[0346] 3. Experimental Results and Analysis

[0347] 3.1 Observation of the general condition of mice during the experiment

[0348] During the experiment, no mice in any group died, and all mice were in good mental condition, with normal diet, water intake, and activity. After euthanasia, no pathological damage such as swelling, atrophy, congestion, or necrosis was observed in organs such as the thymus, spleen, liver, and kidneys. This indicates that the immune-antiaging composition and extracts of each single raw material of the present invention have no in vivo toxicity to mice at a dose of 200 mg / kg and have good safety.

[0349] Table 10 Effects of the anti-aging compositions in Examples 1-3 on serum immune cytokines in mice

[0350]

[0351] Note:

[0352] Compared with the blank control group, P < 0.01.

[0353] b Compared with the immune aging model group, P < 0.01,

[0354] Compared with the positive control group, c, P<0.05.

[0355] Results analysis: Compared with the blank control group, the serum concentrations of TNF-α (100.01±2.03 pg / mL) and IFN-γ (135.23±1.14 pg / mL) in the immunosenescence model group mice were significantly increased (P<0.01), indicating that the immunosenescence model successfully induced a pro-inflammatory state in mice, which is consistent with the characteristics of excessive activation of macrophages and lymphocytes and hypersecretion of pro-inflammatory factors during aging.

[0356] Compared with the immunosenescence model group, the serum TNF-α and IFN-γ concentrations in the positive control group and the groups treated with the compositions of Examples 1-3 were significantly reduced (P < 0.01), indicating that both the positive control drug and the compositions of the present invention can effectively alleviate the abnormal increase of pro-inflammatory factors induced by immunosenescence. Specifically, the TNF-α concentration in the groups of Examples 1-3 was significantly lower than that in the positive control group (P < 0.05), indicating that the compositions of the present invention are superior to the positive control levamisole in reducing pro-inflammatory factor levels.

[0357] TNF-α is a core pro-inflammatory immune factor secreted after macrophage activation, which can promote the inflammatory cascade response; IFN-γ is a key immunomodulatory factor secreted after lymphocyte activation, and excessive elevation during aging can exacerbate chronic inflammation. The test results showed that the compositions in each embodiment significantly reduced the abnormally elevated levels of TNF-α and IFN-γ in the serum of immunosenescent mice, correcting age-related inflammatory imbalances. This was completely consistent with the in vitro cytokine detection results, fully validating the effectiveness of the dual-target cellular immune activation mechanism. This indicates that the composition can improve the pro-inflammatory state induced by immunosenescence and strengthen the body's immune homeostasis by regulating the function of macrophages and lymphocytes.

[0358] 3.4 Formulation synergy validation (Example 1 vs. Comparative Examples 1-3 vs. Single ingredient extracts)

[0359] Based on Example 1, the effects of Comparative Examples 1-3 (without a single ingredient) and extracts of each single ingredient on the immune function of mice were compared (with serum TNF-α and IFN-γ concentrations as the core indicators). The results are shown in Table 11 below:

[0360] Table 11

[0361]

[0362] Note:

[0363] Compared with the normal cell control group, P < 0.01.

[0364] b Compared with the immune aging model group, P < 0.01,

[0365] c Compared with Example 1, P < 0.01.

[0366] Results analysis: The concentrations of macrophage inflammatory factor TNF-α and lymphocyte functional factor IFN-γ in the serum of mice in the immunosenescence model group were significantly higher than those in the blank control group (P < 0.01), indicating that the in vivo immunosenescence model was successfully established. Compared with Example 1 (complete five-component formulation), the serum TNF-α and IFN-γ levels in the control group lacking any ingredient were significantly restored, and the immunomodulatory effects of all single main ingredient extract groups were significantly weaker than those of the complete formulation group. This indicates that the ingredients in the composition of the present invention are not simply superimposed, but rather exert a significantly better in vivo immunosenescence and anti-aging effect than a single component through the synergistic effect of multiple components.

[0367] Among them, the control group lacking Astragalus membranaceus extract and Polygonatum sibiricum extract showed the greatest increase in serum TNF-α and IFN-γ levels, suggesting that Astragalus membranaceus and Polygonatum sibiricum are core components for maintaining immune homeostasis and inhibiting excessive inflammatory responses. The control group lacking anthocyanins from elderberry showed the second greatest increase, indicating that anthocyanins play a key role in alleviating inflammatory damage and protecting lymphocyte function. Although the single-ingredient groups of olive fruit polyphenols and Tremella fuciformis polysaccharides could reduce the serum pro-inflammatory factor levels in model mice to some extent, the improvement effect was far lower than that of the complete formula group, further verifying the synergistic effect mechanism among the components.

[0368] In summary, the formulation of this invention achieves significant in vivo immune anti-aging effects through the synergistic effect of macrophage inflammation inhibition and lymphocyte function repair. The absence of any ingredient will lead to a significant decrease in immune regulation efficacy, which fully confirms the scientific nature and completeness of the formulation composition. Moreover, it is completely consistent with the results of in vitro experiments, forming a dual verification closed loop of in vitro cell experiments and in vivo animal experiments.

[0369] 3.5 Validation of the advantages of process optimization (Example 1 vs. Comparative Examples 4-10)

[0370] Based on Example 1, the effects of comparative examples 4-10 with different processes on the immune function of mice were compared (serum TNF-α and IFN-γ concentrations were the core indicators). The results are shown in Table 12 below:

[0371] Table 12

[0372]

[0373] Note:

[0374] Compared with the normal cell control group, P < 0.01.

[0375] b Compared with the immune aging model group, P < 0.01,

[0376] c Compared with Example 1, P < 0.01,

[0377] Compared with Example 1, P < 0.05.

[0378] Results analysis: The serum levels of macrophage pro-inflammatory factor TNF-α and lymphocyte functional factor IFN-γ in mice of the immunosenescence model group were significantly higher than those in the blank control group (P < 0.01), indicating a significant inflammatory imbalance in the in vivo immunosenescence model, and the model was successfully constructed. Compared with Example 1, the levels of TNF-α and IFN-γ in each comparative example were significantly higher than those in Example 1 (P < 0.05 or P < 0.01), indicating that the preparation process of the present invention can significantly improve the inflammatory imbalance in immunosenescence model mice, and any deviation from any process step will lead to a significant decrease in the inflammatory regulatory effect of the composition.

[0379] Among them, Comparative Example 8 (conventional high-temperature decoction process + no protective gas) showed the largest increase in TNF-α and IFN-γ levels, approaching the levels of the immune aging model group. This indicates that the traditional high-temperature unprotected process leads to a large degradation of active ingredients such as polysaccharides, anthocyanins, and polyphenols in the composition, completely losing its ability to regulate inflammatory factors in the body. Comparative Example 4 (increased extraction temperature of Astragalus / Polygonatum), Comparative Example 5 (the solvent for elderberry anthocyanin extraction was changed to distilled water), and Comparative Example 10 (omitted β- of Polygonatum extract) showed similar increases. The levels of inflammatory factors in the cyclodextrin-encapsulated formulation were the second lowest. In Comparative Example 4, the high temperature damaged the glycosidic bond structure of Astragalus polysaccharide and Polygonatum polysaccharide. In Comparative Example 5, the anthocyanin dissolution rate decreased due to insufficient solubility of distilled water alone. In Comparative Example 10, the stability of Polygonatum polysaccharide decreased due to lack of encapsulation protection. All three factors led to a significant decrease in the content of anti-inflammatory active ingredients in the composition, which could not effectively inhibit the release of pro-inflammatory factors in vivo. In Comparative Example 6 (hot air oven drying), the active ingredients were oxidatively degraded due to high-temperature drying. In Comparative Example 7 (nitrogen protection instead of argon protection), the antioxidant capacity of nitrogen was weaker than that of argon, which aggravated the oxidation and deterioration of the active ingredients. Both factors led to a significant weakening of the in vivo inflammatory regulation effect of the composition. Although the TNF-α and IFN-γ levels in Comparative Example 9 (lactose instead of maltodextrin) were significantly higher than those in Example 1, they were significantly lower than those in other process defect groups. This indicates that although lactose can be used as a carrier excipient, maltodextrin has a better encapsulation protection effect on the active ingredients and can better maintain the in vivo anti-inflammatory activity of the composition.

[0380] In summary, the process optimization of this invention (low-temperature extraction, application of compound solvents, argon protection, β-cyclodextrin encapsulation, selection of maltodextrin carrier, etc.) can significantly improve the inflammatory imbalance in the immune aging model mice by increasing the retention rate and stability of active ingredients, and reduce the excessive release of pro-inflammatory factors TNF-α and IFN-γ. The process optimization has a clear necessity and significant advantages in ensuring the synergistic effects of in vivo immunomodulation and anti-inflammatory efficacy of the composition, and is completely consistent with the in vitro experimental results, forming a double-validation closed loop of process advantages.

[0381] Based on the results of in vitro cell experiments and in vivo animal experiments, it can be seen that:

[0382] (1) Efficacy verification: The cell-immune anti-aging compositions based on medicinal and edible compound plant extracts in Examples 1-3 of this invention can significantly reduce the secretion of pro-inflammatory cytokines. Their in vitro and in vivo immune anti-aging effects are significantly better than those of the positive control levamisole, successfully verifying the effectiveness of the "dual-target cell-immune anti-aging mechanism" and the "five-component synergistic formula".

[0383] (2) Synergistic effect of formulation: The absence of any raw material (Comparative Examples 1-3) or the use of a single raw material extract will lead to a significant decrease in the immune anti-aging effect, proving that the components in the formulation of this invention have irreplaceable and significant synergistic effects, which is the core basis for achieving efficient immune regulation.

[0384] (3) Process advantages: The immune anti-aging composition prepared by the specific process parameters of the present invention (low temperature segmented extraction, deep eutectic solvent + edible alcohol composite extraction, argon protection, β-cyclodextrin encapsulation, ultrasonic atomization drying, maltodextrin carrier) has the highest retention rate of active ingredients and the best stability. The final immune anti-aging effect is significantly better than that of conventional and comparative processes (comparative examples 4-10), which fully proves the scientific nature, advanced nature and practical value of the preparation process of the present invention.

[0385] (4) Safety assessment: Both in vitro cytotoxicity experiments and in vivo animal experiments show that the immune anti-aging composition of the present invention has no cytotoxicity within the effective dose range, and does not cause pathological damage to the growth and development of mice or their major organs (liver, spleen, lungs and kidneys). It has high safety and is suitable for long-term consumption.

[0386] (5) Application prospects: The immune anti-aging composition of the present invention has a novel formula, green and environmentally friendly process, high safety and excellent immune activity. It can be widely used in health food, special dietary food, functional food and pharmaceutical fields, providing high-quality raw materials and technical support for the development of new immune regulation products.

Claims

1. A cell immune anti-aging activator based on extracts from medicinal and edible plants, comprising a main ingredient and excipients, wherein the main ingredient is composed of the following raw materials in parts by weight: Astragalus extract 15-25 parts, Polygonatum extract 15-25 parts, elderberry anthocyanin 8-12 parts, olive fruit polyphenols 5-8 parts, Tremella polysaccharide 5-10 parts.

2. The cell immune anti-aging activator based on extracts of medicinal and edible plants according to claim 1, characterized in that, A cell-mediated immune-boosting and anti-aging activator based on extracts from medicinal and edible plants, comprising main ingredients and excipients. The main ingredients consist of the following raw materials in parts by weight: 18-22 parts Astragalus membranaceus extract, 18-22 parts Polygonatum sibiricum extract, 9-11 parts Elderberry anthocyanins, 6-7 parts Olive fruit polyphenols, and 7-9 parts Tremella fuciformis polysaccharides. The excipients are selected from one or more of microcrystalline cellulose, pregelatinized starch, crospovidone, and magnesium stearate, and the weight of the excipients is 10-40% of the weight of the main material.

3. The cell immune anti-aging activator based on extracts of medicinal and edible plants according to claim 1, characterized in that, The Astragalus extract was prepared by the following method: a. Raw material pretreatment: Remove impurities, wash, dry and pulverize the dried rhizomes of Astragalus membranaceus to obtain Astragalus membranaceus powder; use petroleum ether for low-temperature soaking and degreasing, filter to remove petroleum ether after soaking; then perform pre-freezing treatment and micro-nano pulverization to obtain degreased and cell wall broken Astragalus membranaceus powder; b. Pressure permeation extraction: Add cellulase to the compound extraction solvent, then add defatted and cell wall-breaking Astragalus powder. First, permeate under high pressure, then extract under low pressure. Combine the two extracts to obtain a mixture. c. Purification and separation: The mixed liquid is filtered through a membrane. Macroporous adsorption resin is added to the filtered extract and stirred for adsorption. Then, liquid-liquid extraction is performed. After standing and separating, the aqueous phase is collected and concentrated under reduced pressure to obtain a high-purity Astragalus extract concentrate. d. Recrystallization and drying: Dissolve the concentrated Astragalus extract in a crystallization solvent, stir and filter to remove insoluble impurities, cool the filtrate to room temperature and let it stand for crystallization for 4-6 hours, filter and collect the crystals, wash the crystals, dissolve the washed crystals in purified water, add maltodextrin and glucanase, stir evenly and then enzymatically hydrolyze, inactivate the enzyme after enzymatic hydrolysis, and then spray dry to obtain Astragalus extract.

4. The cell immune anti-aging activator based on extracts of medicinal and edible plants according to claim 1, characterized in that, The Polygonatum extract was prepared by the following method: 1) Raw material pretreatment: Remove impurities from the dried rhizomes of Polygonatum, wash them, slice them, dry them, pulverize them and sieve them to obtain Polygonatum powder; subject the Polygonatum powder to low-temperature pre-freezing treatment to obtain pre-frozen Polygonatum powder; 2) Enzymatic-ultrasonic extraction: Add extraction solvent and cellulase to pre-frozen Polygonatum powder, and extract using an enzymatic-ultrasonic synergistic extraction method to obtain the extract; 3) Purification and concentration: The extract is filtered through a membrane, and nitrogen gas is introduced into the filtered extract to change the hydrophilic solvent in the extraction solvent from hydrophilic to hydrophobic. After standing and separating the layers, the solvent is separated and recovered, and the aqueous phase is collected. The aqueous phase is concentrated under reduced pressure to obtain the concentrated extract of Polygonatum sibiricum. 4) Encapsulation and drying: Add β-cyclodextrin to the concentrated extract of Polygonatum sibiricum, stir and mix evenly, and spray dry to obtain Polygonatum sibiricum extract.

5. The cell immune anti-aging activator based on extracts of medicinal and edible plants according to claim 1, characterized in that, The elderberry anthocyanins were prepared according to the following method: (1) Freeze-drying and anti-oxidation treatment: clean and remove impurities from fresh elderberries, process them using low-temperature gradient freeze-drying, and then pulverize them into micro-nano powder; add ascorbic acid-citric acid composite antioxidant to elderberry powder, mix evenly to obtain a mixture; (2) Ultrasonic-microwave synergistic extraction: Add extraction solvent to the mixture and extract using ultrasonic-microwave synergistic extraction method. Repeat the extraction 1 to 2 times and combine all extracts. (3) Centrifugation, filtration and purification: Centrifuge the combined extracts, take the supernatant and remove suspended impurities by membrane filtration; Chitosan was added to the filtered supernatant to adsorb and remove protein impurities. After filtration again, the sample was loaded onto an AB-8 macroporous resin column for purification. (4) Gradient elution and drying: The gradient elution process was used for elution, the eluent was concentrated under reduced pressure, and then freeze-dried under vacuum to obtain elderberry anthocyanins.

6. The cell immune anti-aging activator based on extracts of medicinal and edible plants according to claim 1, characterized in that, The olive fruit polyphenols were prepared according to the following method: I. Raw material pretreatment and color protection / antioxidant Fresh olives are pre-treated, washed, drained, and dried at a low temperature. The dried olives are then pitted, retaining the pulp, which is then crushed and sieved to obtain a uniform olive pulp powder. Food-grade tocopherol-rosemary extract complex antioxidant was added to olive pulp powder and thoroughly mixed. The mixed pulp powder was then pre-frozen at low temperature and then pulverized at low temperature to obtain pre-treated olive pulp powder. II. Synergistic extraction using ultrasound and enzyme methods Add extraction solvent to the pretreated olive fruit pulp powder, and use ultrasound-enzyme synergistic extraction method to extract at a constant temperature for 1-2 hours. Combine all extracts. III. Degreasing, centrifugal purification and concentration The combined extracts were first degreased at low temperature, allowed to stand and separate into layers, and the lower aqueous phase was retained. The degreased extracts were centrifuged, and the clear supernatant was taken, filtered through a membrane, and polyamide resin was added to the filtered supernatant and stirred at room temperature to adsorb, thus obtaining a purified polyphenol extract. The purified polyphenol extract was concentrated under reduced pressure until the system had no alcohol odor, thus obtaining a concentrated olive fruit polyphenol extract. IV. Adding a carrier and freeze-drying Maltodextrin was added to the concentrated olive fruit polyphenol solution as a carrier, and the mixture was stirred at low speed until homogeneous. Then, the mixture was freeze-dried under vacuum to obtain olive fruit polyphenols.

7. A method for preparing the cell immune anti-aging activator based on extracts of medicinal and edible plants according to any one of claims 1-6, comprising the following steps: S1. Raw material pretreatment: Astragalus extract, Polygonatum extract, Elderberry anthocyanins, and olive fruit polyphenols are pulverized by low-temperature airflow at 0~5℃ and then passed through a 100~120 mesh sieve. Argon gas is introduced for protection during the pulverization process. Tremella polysaccharide is pulverized by low-temperature micro-nano pulverization at 0~4℃ to a particle size of 50~80μm, passed through an 80~100 mesh sieve, and placed in a dry and sealed container for later use. The relative humidity of the environment is controlled at 30~40% and the temperature is 10~15℃. S2. Premixing: According to the formula, first put the Astragalus extract and Polygonatum extract into the intelligent temperature-controlled three-dimensional mixer, introduce argon gas to remove air from the equipment, reduce the oxygen content to ≤0.5%, control the mixing temperature at 15~20℃ and the speed at 18~22r / min, mix for 12~18min, and reverse stirring once every 4~6min during the period, at a speed of 25~30r / min for 30s. Simultaneously, online particle size monitoring technology is used to monitor the mixing uniformity in real time (uniformity ≥98%). After the standard is met, premix A is obtained. S3. Co-mixing: Add elderberry anthocyanins and olive fruit polyphenols to premix A, maintain argon protection, mix at 15~20℃, adjust the speed to 20~25r / min, mix for 18~22min, use segmented stirring mode, combined with low-frequency ultrasonic-assisted dispersion, and monitor uniformity online simultaneously. After meeting the standard, premix B is obtained. S4. Formulation and molding: Add tremella polysaccharide to premix B, stir and mix evenly, add excipients according to the ratio and mix evenly, adjust the pH of the system to 6.8~7.2 with phosphate buffer solution to obtain a mixture; dry the mixture using low temperature ultrasonic atomization spray drying process, and after drying, pulverize at low temperature and pass through a 100~120 mesh sieve to obtain a cell immune anti-aging activator.

8. The preparation method according to claim 7, characterized in that, In step S3, the segmented stirring is performed by rotating forward for 5 minutes and then rotating backward for 3 minutes. The ultrasonic power for low-frequency ultrasonic-assisted dispersion is 100-150W, with intermittent operation for 20 seconds and a pause for 40 seconds. In step S4, the excipients are selected from one or more of microcrystalline cellulose, pregelatinized starch, cross-linked polyvinylpyrrolidone, and magnesium stearate. In step S4, the low-temperature ultrasonic atomization spray drying parameters are: inlet air temperature 165-175℃, outlet air temperature 72-78℃, feed rate 25-30mL / min, atomization pressure 0.25-0.3MPa, drying chamber temperature controlled at 45-55℃, and ultrasonic atomization frequency 20-30kHz.

9. The application of the cell-immune anti-aging composition based on medicinal and edible compound plant extracts as described in claim 1 in the preparation of health foods and special dietary foods that enhance cell-immune function.

10. The application according to claim 9, characterized in that, The cell-immune anti-aging composition of the medicinal and edible compound plant extracts is in the form of powder, granules, capsules or tablets.

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