Production process and application of compound preparation of cordyceps sinensis, ganoderma lucidum, dendrobium nobile, astragalus membranaceus and anthocyanin for preventing aging

Abstract

The invention belongs to the field of health-care products and biological medicines, and particularly discloses a production process and application of a compound preparation of cordyceps militaris, lucid ganoderma, dendrobium officinale, astragalus membranaceus and anthocyanin for preventing aging, and the compound preparation is prepared from a cordyceps militaris extract, a lucid ganoderma extract, a dendrobium officinale extract, an astragalus membranaceus extract and procyanidine through scientific compatibility. Through a modern extraction technology and a precise preparation process, the compound preparation constructs an anti-aging system integrating oxidation resistance, collagen promotion, inflammation resistance and immunity regulation. Experiments show that the compound preparation has remarkable antioxidant capacity, can promote skin collagen synthesis and structural reconstruction and reverse skin aging phenotypes, and can enhance the skin repair effect when being combined with hyaluronic acid. The invention provides a safe, effective and multi-target synergistic new product for the anti-aging field, and has a wide market application prospect.

Description

Technical Field

[0001] The invention belongs to the field of health care products and biomedicine, and specifically discloses a production process and application of a cordyceps, ganoderma, dendrobium, astragalus and anthocyanin compound for preventing aging. Background Art

[0002] With the aging of the population and the accelerated pace of life, people's demand for delaying aging and maintaining a healthy state is growing. Aging is a complex biological process involving the decline of functions of multiple organs and systems, among which skin aging is particularly obvious, manifested as sagging skin, increased wrinkles, and decreased elasticity. Traditional anti-aging methods mostly rely on single-ingredient or simple combination of skin care products and health care products, but these products often have limited effects and are difficult to fully address the various problems brought about by aging.

[0003] In the field of research and development of anti-aging drugs and health products, existing technical solutions have many shortcomings. On the one hand, many anti-aging products rely on a single active ingredient, such as antioxidants such as vitamin C and vitamin E. Although these ingredients can delay cell oxidative damage to a certain extent, their single function makes it difficult to fully meet the complex needs of anti-aging. On the other hand, although some compound preparations combine multiple ingredients, due to the lack of scientific compatibility and precise extraction technology, the active ingredients are unstable and have low bioavailability, which affects the actual effect of the product.

[0004] In addition, as people's awareness of health improves, the requirements for the safety and effectiveness of anti-aging products are getting higher and higher. Traditional anti-aging drugs and health products may contain potential side effects, and long-term use may have adverse effects on physical health. Therefore, it is particularly important to develop a safe, effective, multi-target synergistic anti-aging compound. Summary of the invention

[0005] In view of the above problems, the present invention proposes a Cordyceps, Ganoderma, Dendrobium, Astragalus anthocyanidin compound for preventing aging, which is scientifically compatible with Cordyceps militaris extract, Ganoderma lucidum extract, Dendrobium officinale extract, Astragalus extract and proanthocyanidins and other active ingredients, combined with modern extraction technology and precise preparation technology, aims to comprehensively improve the anti-aging effect, promote the skin to restore smoothness and elasticity, and ensure the safety and stability of the product. The present invention provides an innovative solution for the anti-aging field, which has important application value and market prospects.

[0006] The purpose of the present invention is achieved through the following technical solutions.

[0007] A cordyceps, ganoderma, dendrobium and astragalus anthocyanidin compound for preventing aging, comprising the following active ingredients in parts by weight:

[0008] 8-25 parts of Cordyceps militaris extract

[0009] Ganoderma lucidum extract 15-45 parts

[0010] 10-35 parts of Dendrobium candidum extract

[0011] Astragalus extract 3-18 parts

[0012] Proanthocyanidins 0.5-4 parts.

[0013] Furthermore, in the above-mentioned Cordyceps, Ganoderma Lucidum, Dendrobium, Astragalus and Anthocyanin compound for preventing aging, the preparation method of the Cordyceps militaris extract comprises the following steps:

[0014] a. The dried Cordyceps sinensis was crushed through a 60-100 mesh sieve and extracted 2-4 times with 50-70% ethanol at a solid-liquid ratio of 1:6-10, each for 1-2 hours;

[0015] b. The combined extracts were concentrated under reduced pressure until there was no alcohol taste, and loaded onto a D201 macroporous resin column, sequentially eluted with 10-20%, 20-30%, 35-45% ethanol gradient, and the 20-35% ethanol eluate was collected;

[0016] c. The eluate is pre-frozen at -40 to -60°C, and then freeze-dried at a vacuum degree of 2-10 Pa and -50 to -70°C for 24 to 48 hours to obtain an extract with a Cordyceps polysaccharide content of ≥5%.

[0017] Furthermore, in the above-mentioned Cordyceps, Ganoderma lucidum, Dendrobium, Astragalus anthocyanin compound for preventing aging, the preparation method of the Ganoderma lucidum extract comprises:

[0018] a. Grind the dried Ganoderma lucidum through a 40-80 mesh sieve, add 6-10 times the amount of pure water, and extract in a 60-80℃ water bath for 2-4 hours;

[0019] b. The extract was adsorbed on D101 macroporous resin at a dynamic adsorption flow rate of 1-2 BV / h and eluted with 50-70% ethanol at 0.5-2BV / h;

[0020] c. The eluent is concentrated under reduced pressure to a relative density of 1.15-1.25, and then dried under vacuum at 50-60°C, -0.08 to -0.10 MPa until the water content is ≤5%.

[0021] Furthermore, in the above-mentioned Cordyceps, Ganoderma Lucidum, Dendrobium, Astragalus and Anthocyanin compound for preventing aging, the preparation method of the Dendrobium officinale extract comprises:

[0022] a. Crush the whole herb of Dendrobium officinale to 20-40 mesh, add 4-8 times amount of pure water, and extract in a water bath at 70-90℃ for 1-3 hours;

[0023] b. The extract was filtered through an ultrafiltration membrane (molecular weight cutoff 10-30 kDa), the filtrate was collected and concentrated under reduced pressure to 1 / 4-1 / 2 of the original volume;

[0024] c. Spray drying: Inlet air temperature 160-180℃, outlet air temperature 85-95℃, to obtain Dendrobium officinale extract powder with a moisture content of ≤5%.

[0025] Furthermore, in the above-mentioned Cordyceps, Ganoderma Lucidum, Dendrobium, Astragalus Anthocyanidin Compound for preventing aging, the preparation method of the Astragalus extract comprises:

[0026] a. The dried Astragalus was crushed through a 80-120 mesh sieve and refluxed with 40-60% ethanol at a solid-liquid ratio of 1:10-14 for 1-3 times, each time for 1-2 hours;

[0027] b. Combine the extracts, concentrate under reduced pressure to a relative density of 1.05-1.15, add an equal volume of water, mix well and let stand for 36-60 hours;

[0028] c. Centrifuge and separate, take the supernatant, and spray-dry to obtain the astragalus extract.

[0029] The invention also discloses the application of the compound in preparing tablets, capsules, pills or granules.

[0030] The present invention also discloses a method for preparing the above compound into tablets or capsules, comprising the following steps:

[0031] a. Dissolve the Cordyceps militaris extract and proanthocyanidins in an appropriate amount of water or a suitable solvent, and stir at a constant temperature of 50-70°C until completely dissolved to obtain a mixed solution;

[0032] b. Ganoderma lucidum extract, Dendrobium candidum extract, Astragalus extract, microcrystalline cellulose and lactose were mixed and passed through a 80-200 mesh sieve to obtain a uniform powder mixture;

[0033] c. Using an efficient wet granulator, the mixed solution of step a is mixed with the powder mixture of step b, and the water activity is controlled to be ≤0.15-0.25, and a 3-7% ethanol solution of hydroxypropyl methylcellulose is used as a binder;

[0034] d. The wet granules are dried in a fluidized bed at 40-60℃ until the moisture content is ≤2-4%. After granulation, they are compressed into tablets or filled into capsules.

[0035] Furthermore, in the above preparation method, the disintegration time of the tablet is ≤10-15 minutes, and the particle size of the capsule content is ≤100-150 μm.

[0036] The present invention also discloses a method for preparing the compound into pills or granules, comprising the following steps:

[0037] Preparation method of pills:

[0038] 1) Raw material preparation:

[0039] Dissolve the Cordyceps militaris extract and proanthocyanidins in an appropriate amount of water or a suitable solvent, and stir at a constant temperature of 50-70°C until they are completely dissolved to obtain a mixed solution;

[0040] Mixing the ganoderma lucidum extract, the dendrobium officinale extract, the astragalus extract with appropriate amounts of microcrystalline cellulose and lactose, and passing through a 80-200 mesh sieve to obtain a uniform powder mixture;

[0041] 2) Mixing and pelleting:

[0042] The mixed solution obtained in step 1 is thoroughly mixed with the powder mixture;

[0043] According to the needs, you can add an appropriate amount of honey or refined honey as a binder to adjust the hardness to the appropriate softness;

[0044] The mixture is made into pills using a pill making machine, and the size and shape of the pills can be adjusted as needed;

[0045] 3) Drying and packaging:

[0046] The prepared pills are placed in a ventilated and dry place for drying until the specified moisture content is reached, and then packaged;

[0047] Preparation method of granules:

[0048] 4) Wet granulation:

[0049] Add the mixed liquid obtained in step 1) and the powder mixture into a high-efficiency wet granulator, and control the water activity to ≤ 0.15-0.25;

[0050] Using 3-7% hydroxypropyl methylcellulose ethanol solution as a binder, start the granulator for wet granulation;

[0051] During the granulation process, the amount of binder and the parameters of the granulator can be adjusted in time according to the formation of the granules;

[0052] 5) Drying and granulation:

[0053] Dry the wet granules in a fluidized bed dryer at 40-60°C until the moisture content is ≤2-4%;

[0054] After drying, the granules are sized using a granulator to remove oversized or undersized particles to obtain uniform granules.

[0055] The invention also discloses the use of the compound in combination with hyaluronic acid in preparing a pharmaceutical composition for promoting the restoration of smoothness and elasticity of the skin.

[0056] Compared with the existing technology, the present invention has the following advantages and beneficial effects:

[0057] The present invention discloses a cordyceps, ganoderma, dendrobium and astragalus anthocyanidin compound for preventing aging, which constructs a powerful antioxidant defense system. Experiments show that the DPPH free radical scavenging rate of Example 3 reaches 92.3%, and the SOD activity reaches 85.6 U / mg. A multi-level antioxidant network is formed by multiple components such as cordyceps polysaccharides and ganoderma triterpenes, which effectively blocks cell damage caused by oxidative stress; secondly, the components of the present invention can promote skin collagen synthesis and structural reconstruction, increase the secretion of type I collagen in UVB-induced aging fibroblasts by 131%, and achieve full-chain regulation by activating TGF-β / Smad signaling pathways; further, the components of the present invention can reverse the skin aging phenotype, increase the elastic recovery rate of UV-irradiated mouse skin by 49%, and achieve triple repair by inhibiting MMPs activity, regulating the expression of inflammatory factors, etc.; finally, the present invention can also have synergistic expansibility with functional components, for example, the skin scratch repair efficiency is increased by 16.7% after combined with hyaluronic acid, revealing a complementary mechanism, and can be embedded in cosmetic formulas or developed anti-aging combination products. This compound formulation constructs an anti-aging system that integrates antioxidant, collagen-promoting, anti-inflammatory, and immune regulation through precise proportions and synergistic effects of multiple ingredients. It provides a scientific basis and technical support for the development of a new generation of anti-aging products and has broad market application prospects. DETAILED DESCRIPTION

[0058] To make the purpose, technical solutions and advantages of the present invention clearer, the present invention is further described in detail below. However, it should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the scope of the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present invention. All raw materials in the embodiments of the present invention can be obtained through commercial channels.

[0059] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0060] Example 1

[0061] A cordyceps, ganoderma, dendrobium and astragalus anthocyanidin compound for preventing aging, comprising the following active ingredients in parts by weight:

[0062] 8 parts of Cordyceps militaris extract

[0063] Ganoderma Lucidum Extract 15 parts

[0064] 10 servings of Dendrobium candidum extract

[0065] Astragalus Extract 3 parts

[0066] Proanthocyanidins 0.5 parts

[0067] The above raw materials were purchased commercially.

[0068] Cordyceps militaris extract (powder) was purchased from Hubei Haijia Biotechnology Co., Ltd.

[0069] Ganoderma lucidum extract (powder) was purchased from Shaanxi New Horizon Biotechnology Co., Ltd.

[0070] Dendrobium officinale extract (powder) was purchased from Xi'an Anao Biotechnology Co., Ltd.

[0071] Astragalus extract (powder) was purchased from Hebei Jiuyu Biotechnology Co., Ltd.

[0072] Proanthocyanidins (powder) were purchased from Shaanxi Xintianyu Biotechnology Co., Ltd. (grape seed extract proanthocyanidins 95% OPC).

[0073] Example 2

[0074] A cordyceps, ganoderma, dendrobium and astragalus anthocyanidin compound for preventing aging, comprising the following active ingredients in parts by weight:

[0075] 8 parts of Cordyceps militaris extract

[0076] Ganoderma Lucidum Extract 15 parts

[0077] 10 servings of Dendrobium candidum extract

[0078] Astragalus Extract 3 parts

[0079] Proanthocyanidins 0.5 parts

[0080] The preparation method of the Cordyceps militaris extract comprises the following steps:

[0081] a. Grind the dried Cordyceps militaris through an 80-mesh sieve, and reflux extract with 60% ethanol at a solid-liquid ratio of 1:8 for 3 times, each time for 1.5 hours;

[0082] b. The combined extracts were concentrated under reduced pressure until there was no alcohol taste, and loaded onto a D201 macroporous resin column, sequentially eluted with 15%, 25%, and 40% ethanol gradients, and 20 - 35% ethanol eluate was collected;

[0083] c. After the eluate was pre-frozen at -50°C, it was freeze-dried at -60°C for 36 hours under a vacuum degree of 6 Pa to obtain an extract with a Cordyceps polysaccharide content of ≥5%.

[0084] The preparation method of the ganoderma lucidum extract comprises:

[0085] a. Grind the dried Ganoderma lucidum through a 60-mesh sieve, add 8 times the amount of pure water, and extract in a 70℃ water bath for 3 hours;

[0086] b. The extract was adsorbed by D101 macroporous resin at a dynamic adsorption flow rate of 1.5 BV / h and eluted with 60% ethanol at 1.5 BV / h;

[0087] c. The eluate was concentrated under reduced pressure to a relative density of 1.20 and dried in vacuum at 55°C, -0.09 MPa until the water content was ≤5%.

[0088] The preparation method of the Dendrobium officinale extract comprises:

[0089] a. Grind the whole herb of Dendrobium officinale into 30 mesh, add 6 times amount of pure water, and extract in a water bath at 80℃ for 2 hours;

[0090] b. The extract was filtered through an ultrafiltration membrane (molecular weight cutoff 20 kDa), the filtrate was collected and concentrated under reduced pressure to 1 / 3 of the original volume;

[0091] c. Spray drying: inlet air temperature 170℃, outlet air temperature 90℃, to obtain Dendrobium officinale extract powder with a moisture content of ≤5%.

[0092] The preparation method of the astragalus extract comprises:

[0093] a. Grind the dried Astragalus membranaceus through a 100-mesh sieve, and reflux extract with 50% ethanol at a solid-liquid ratio of 1:12 for 2 times, each time for 1.5 hours;

[0094] b. Combine the extracts, concentrate under reduced pressure to a relative density of 1.10, add an equal volume of water, mix well and let stand for 48 hours;

[0095] c. Centrifuge and separate, take the supernatant, and spray-dry to obtain the astragalus extract.

[0096] Example 3

[0097] A cordyceps, ganoderma, dendrobium and astragalus anthocyanidin compound for preventing aging, comprising the following active ingredients in parts by weight:

[0098] 15 parts of Cordyceps militaris extract

[0099] Ganoderma Lucidum Extract 25 parts

[0100] Dendrobium officinale extract 20 parts

[0101] Astragalus Extract 10 parts

[0102] 2 parts of proanthocyanidins

[0103] The preparation method of the Cordyceps militaris extract comprises the following steps:

[0104] a. Grind the dried Cordyceps militaris through an 80-mesh sieve, and reflux extract with 60% ethanol at a solid-liquid ratio of 1:8 for 3 times, each time for 1.5 hours;

[0105] b. The combined extracts were concentrated under reduced pressure until there was no alcohol taste, and loaded onto a D201 macroporous resin column, sequentially eluted with 15%, 25%, and 40% ethanol gradients, and 20 - 35% ethanol eluate was collected;

[0106] c. After the eluate was pre-frozen at -50°C, it was freeze-dried at -60°C for 36 hours under a vacuum degree of 6 Pa to obtain an extract with a Cordyceps polysaccharide content of ≥5%.

[0107] The preparation method of the ganoderma lucidum extract comprises:

[0108] a. Grind the dried Ganoderma lucidum through a 60-mesh sieve, add 8 times the amount of pure water, and extract in a 70℃ water bath for 3 hours;

[0109] b. The extract was adsorbed by D101 macroporous resin at a dynamic adsorption flow rate of 1.5 BV / h and eluted with 60% ethanol at 1.5 BV / h;

[0110] c. The eluate was concentrated under reduced pressure to a relative density of 1.20 and dried in vacuum at 55°C, -0.09 MPa until the water content was ≤5%.

[0111] The preparation method of the Dendrobium officinale extract comprises:

[0112] a. Grind the whole herb of Dendrobium officinale into 30 mesh, add 6 times amount of pure water, and extract in a water bath at 80℃ for 2 hours;

[0113] b. The extract was filtered through an ultrafiltration membrane (molecular weight cutoff 20 kDa), the filtrate was collected and concentrated under reduced pressure to 1 / 3 of the original volume;

[0114] c. Spray drying: inlet air temperature 170℃, outlet air temperature 90℃, to obtain Dendrobium officinale extract powder with a moisture content of ≤5%.

[0115] The preparation method of the astragalus extract comprises:

[0116] a. Grind the dried Astragalus membranaceus through a 100-mesh sieve, and reflux extract with 50% ethanol at a solid-liquid ratio of 1:12 for 2 times, each time for 1.5 hours;

[0117] b. Combine the extracts, concentrate under reduced pressure to a relative density of 1.10, add an equal volume of water, mix well and let stand for 48 hours;

[0118] c. Centrifuge and separate, take the supernatant, and spray-dry to obtain the astragalus extract.

[0119] Example 4

[0120] A cordyceps, ganoderma, dendrobium and astragalus anthocyanidin compound for preventing aging, comprising the following active ingredients in parts by weight:

[0121] Cordyceps militaris extract 25 parts

[0122] Ganoderma Lucidum Extract 45 parts

[0123] 35 servings of Dendrobium candidum extract

[0124] Astragalus Extract 18 parts

[0125] 4 parts of proanthocyanidins

[0126] The preparation method of the Cordyceps militaris extract comprises the following steps:

[0127] a. Grind the dried Cordyceps militaris through an 80-mesh sieve, and reflux extract with 60% ethanol at a solid-liquid ratio of 1:8 for 3 times, each time for 1.5 hours;

[0128] b. The combined extracts were concentrated under reduced pressure until there was no alcohol taste, and loaded onto a D201 macroporous resin column, sequentially eluted with 15%, 25%, and 40% ethanol gradients, and 20 - 35% ethanol eluate was collected;

[0129] c. After the eluate was pre-frozen at -50°C, it was freeze-dried at -60°C for 36 hours under a vacuum degree of 6 Pa to obtain an extract with a Cordyceps polysaccharide content of ≥5%.

[0130] The preparation method of the ganoderma lucidum extract comprises:

[0131] a. Grind the dried Ganoderma lucidum through a 60-mesh sieve, add 8 times the amount of pure water, and extract in a 70℃ water bath for 3 hours;

[0132] b. The extract was adsorbed by D101 macroporous resin at a dynamic adsorption flow rate of 1.5 BV / h and eluted with 60% ethanol at 1.5 BV / h;

[0133] c. The eluate was concentrated under reduced pressure to a relative density of 1.20 and dried in vacuum at 55°C, -0.09 MPa until the water content was ≤5%.

[0134] The preparation method of the Dendrobium officinale extract comprises:

[0135] a. Grind the whole herb of Dendrobium officinale into 30 mesh, add 6 times amount of pure water, and extract in a water bath at 80℃ for 2 hours;

[0136] b. The extract was filtered through an ultrafiltration membrane (molecular weight cutoff 20 kDa), the filtrate was collected and concentrated under reduced pressure to 1 / 3 of the original volume;

[0137] c. Spray drying: inlet air temperature 170℃, outlet air temperature 90℃, to obtain Dendrobium officinale extract powder with a moisture content of ≤5%.

[0138] The preparation method of the astragalus extract comprises:

[0139] a. Grind the dried Astragalus membranaceus through a 100-mesh sieve, and reflux extract with 50% ethanol at a solid-liquid ratio of 1:12 for 2 times, each time for 1.5 hours;

[0140] b. Combine the extracts, concentrate under reduced pressure to a relative density of 1.10, add an equal volume of water, mix well and let stand for 48 hours;

[0141] c. Centrifuge and separate, take the supernatant, and spray-dry to obtain the astragalus extract.

[0142] Comparative Example 1

[0143] No Cordyceps militaris extract added.

[0144] The rest is the same as Example 3.

[0145] Comparative Example 2

[0146] No Cordyceps militaris extract or Ganoderma lucidum extract is added.

[0147] The rest is the same as Example 3.

[0148] Comparative Example 3

[0149] No proanthocyanidin was added, and the rest was the same as in Example 3.

[0150] Comparative Example 4

[0151] The astragalus extract and proanthocyanidins were not added, and the other steps were the same as those in Example 3.

[0152] Comparative Example 5

[0153] The other steps were the same as in Example 3 except that the Dendrobium officinale extract was not added.

[0154] Preparation Example 1

[0155] The preparation method of the compound preparation only requires that the Cordyceps militaris extract, the Ganoderma lucidum extract, the Dendrobium candidum extract, the Astragalus extract and the proanthocyanidins are uniformly mixed at room temperature, then ground and passed through a 200-mesh sieve.

[0156] Test Example 1

[0157] In vitro antioxidant capacity evaluation

[0158] Methods: DPPH free radical scavenging experiment and SOD activity detection were used.

[0159] Sample treatment: The compound was dissolved in PBS to prepare a 1 mg / mL solution.

[0160] Positive control group: vitamin C (1 mg / mL), superoxide dismutase (SOD standard, 100 U / mL).

[0161] DPPH free radical scavenging experiment:

[0162] Take 100 μL of sample and mix it with 100 μL of DPPH ethanol solution (0.1 mM), react for 30 min in the dark, and measure the absorbance at 517 nm.

[0163] Clearance calculation: [(A_control - A_sample) / A_control] × 100%

[0164] SOD activity detection:

[0165] The WST-8 kit was used to measure the SOD activity in HaCaT cell lysate (pretreated for 24 h) according to the instructions.

[0166] MDA content determination:

[0167] The lipid peroxidation products in cell lysate were detected by thiobarbituric acid method at 532 nm.

[0168] The results are shown in Table 1

[0169]

[0170] Conclusion: The antioxidant capacity of Example 3 is significantly better than that of each comparative example and is close to the positive control level, which proves that the compound enhances the free radical scavenging ability through the synergistic effect of multiple components.

[0171] Test Example 2

[0172] Human skin fibroblast collagen synthesis experiment

[0173] method:

[0174] Cell culture: UVB-induced senescent HSF cells (HSF cell senescence induced by UVB irradiation (30 mJ / cm², once a day for 5 consecutive days)) were treated with Example 3 (100 μg / mL) and the comparative example (100 μg / mL) for 48 hours.

[0175] Detection indicators: ELISA method was used to determine the content of type I collagen (COL1A1), and qPCR was used to detect the expression of COL1A1 mRNA. The results are shown in Table 2.

[0176]

[0177] Conclusion: Example 3 significantly promotes collagen synthesis, while the effect is significantly reduced when the key components (such as Cordyceps militaris and Dendrobium) are missing in the comparative example.

[0178] Test Example 3

[0179] Mouse skin aging model experiment

[0180] method:

[0181] Animal model: UV irradiation-induced skin aging in BALB / c mice, BALB / c female mice (8 weeks old, n=8 / group)

[0182] Treatment group: Oral administration of Example 3 (100 mg / kg / d) for 8 consecutive weeks

[0183] Testing indicators: Skin elasticity meter was used to measure R2 value (elastic recovery rate), and HE staining was used to analyze epidermal thickness.

[0184] The results are shown in Table 3.

[0185]

[0186] Conclusion: Example 3 significantly reversed UV-induced skin aging, while the effect of Comparative Example 4 (lacking astragalus and anthocyanins) was weakened.

[0187] Test Example 4

[0188] Safety evaluation

[0189] method:

[0190] Acute toxicity test: Mice were gavaged once with 5000 mg / kg of the compound (Example 3) and observed for 14 days.

[0191] Long-term toxicity experiment: Rats were gavaged with 1000 mg / kg / d (Example 3) for 90 consecutive days, and blood routine, liver and kidney function were tested.

[0192] result:

[0193] Acute toxicity test: No death or abnormal behavior occurred, LD50>5000 mg / kg

[0194] Long-term toxicity experiment: There was no significant difference between the biochemical indicators (ALT, Cr, etc.) and the control group (p>0.05)

[0195] Conclusion: The compound medicine has good safety at the experimental dose and meets the application standards of health products.

[0196] Test Example 5

[0197] Evaluation of the skin repair effect of combined hyaluronic acid (in vitro scratch test)

[0198] Experimental purpose: To evaluate the synergistic effect of the compound alone and in combination with hyaluronic acid on the migration and repair of keratinocytes (HaCaT).

[0199] 1. Materials and Methods

[0200] 1.1 Sample preparation

[0201] Compound solution: The compound of Example 3 was dissolved in DMEM medium with a final concentration of 100 μg / mL (no cytotoxicity was confirmed by preliminary experiments).

[0202] Hyaluronic acid (HA) solution: Medical grade sodium hyaluronate (molecular weight 1.5 MDa) was dissolved in DMEM with a final concentration of 50 μg / mL (refer to the commonly used dose for skin repair).

[0203] Combined group: the compound (100 μg / mL) and HA (50 μg / mL) were mixed at a volume ratio of 1:1.

[0204] 1.2 Cell culture and grouping

[0205] Cell line: Human immortalized keratinocytes (HaCaT) cultured in DMEM medium containing 10% FBS (37°C, 5% CO2).

[0206] Experimental groups:

[0207] Control group: DMEM medium only

[0208] Compound group: 100 μg / mL compound

[0209] HA group: 50 μg / mL hyaluronic acid

[0210] Compound + HA group: 100 μg / mL compound + 50 μg / mL HA

[0211] 1.3 Scratch test steps

[0212] Cell plating: HaCaT cells were plated at 5×10 5 Cells / well were seeded in 6-well plates and cultured until 90% confluence.

[0213] Scratch preparation: Use a 200 μL sterile pipette tip to vertically scrape the cell layer and wash with PBS to remove detached cells.

[0214] Drug treatment: Corresponding treatment solution (2 mL / well) was added to each group, and 3 replicate wells were set up for each group.

[0215] Observation and record: The scratch area was photographed under an inverted microscope (10× objective lens) at 0 h, 12 h, and 24 h, and the scratch area healing rate was calculated using ImageJ software.

[0216] 1.4 Data Analysis

[0217] Healing rate (%) = [(initial area - remaining area) / initial area] × 100

[0218] The results were expressed as mean ± SD. One-way ANOVA was used for comparison among the groups, and p < 0.05 was considered significant.

[0219] The results are shown in Table 4.

[0220]

[0221] in conclusion

[0222] Single effect:

[0223] The healing rate of the compound group (76.4% at 24 h) was significantly better than that of the HA group (63.8%), indicating that the compound itself has a strong activity in promoting repair.

[0224] The effect of the HA group was better than that of the control group, but weaker than that of the compound, suggesting that hyaluronic acid promotes repair mainly by moisturizing, while the compound may activate pathways related to cell migration.

[0225] Verification of synergistic effect:

[0226] The healing rate of the compound + HA group reached 89.2% within 24 hours, which was 16.7% higher than that of the compound alone (p<0.05), confirming that the combination of the two can synergistically accelerate wound healing through different mechanisms (such as antioxidant + physical barrier).

[0227] It can be seen from the above test examples that the compound of the present invention exhibits significant multi-target synergistic effects in the field of anti-aging, and its core advantages can be refined as follows: 1. Strong antioxidant: The DPPH free radical scavenging rate of Example 3 reaches 92.3%, and the SOD activity reaches 85.6 U / mg, blocking oxidative damage through a multi-component network; 2. Promote collagen regeneration: Increase the secretion of type I collagen in aging fibroblasts by 131%, activate the signal pathway to achieve full-chain regulation; 3. Reverse aging phenotype: The elastic recovery rate of mouse skin is increased by 49%, inhibiting collagen degradation, regulating inflammation and promoting angiogenesis; 4. Formula synergy: The effect is greatly reduced after the key ingredients are missing, and increasing the dose can strengthen the intervention; 5. Safety and controllability: Toxicology experiments show safety, and the process guarantees quality; 6. Strong scalability: The repair efficiency is increased by 16.7% when used in combination with hyaluronic acid, and anti-aging products can be developed. The compound constructs an anti-aging system such as antioxidant and collagen promotion through precise proportioning, and has broad application prospects.

[0228] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Therefore, based on the innovative concept of the present invention, changes and modifications to the embodiments described herein, or equivalent structures or equivalent process transformations made using the contents of the present invention specification, directly or indirectly applying the above technical solutions to other related technical fields are all included in the protection scope of the patent of the present invention.

Claims

1. A Cordyceps, Ganoderma Lucidum, Dendrobium, Astragalus Anthocyanidin compound for preventing aging, characterized in that: The active ingredients are as follows: 8-25 parts of Cordyceps militaris extract Ganoderma lucidum extract 15-45 parts 10-35 parts of Dendrobium candidum extract Astragalus extract 3-18 parts Proanthocyanidins 0.5-4 parts.

2. The compound according to claim 1, characterized in that The preparation method of the Cordyceps militaris extract comprises the following steps: a. The dried Cordyceps sinensis was crushed through a 60-100 mesh sieve and extracted 2-4 times with 50-70% ethanol at a solid-liquid ratio of 1:6-10, each for 1-2 hours; b. The combined extracts were concentrated under reduced pressure until there was no alcohol taste, and loaded onto a D201 macroporous resin column, sequentially eluted with 10-20%, 20-30%, 35-45% ethanol gradient, and the 20-35% ethanol eluate was collected; c. The eluate is pre-frozen at -40 to -60°C, and then freeze-dried at a vacuum degree of 2-10 Pa and -50 to -70°C for 24 to 48 hours to obtain an extract with a Cordyceps polysaccharide content of ≥5%.

3. The compound according to claim 1, characterized in that The preparation method of the ganoderma lucidum extract comprises: a. Grind the dried Ganoderma lucidum through a 40-80 mesh sieve, add 6-10 times the amount of pure water, and extract in a 60-80℃ water bath for 2-4 hours; b. The extract was adsorbed on D101 macroporous resin at a dynamic adsorption flow rate of 1-2 BV / h and eluted with 50-70% ethanol at 0.5-2 BV / h; c. The eluate is concentrated under reduced pressure to a relative density of 1.15-1.25, and then dried under vacuum at 50-60°C, -0.08 to -0.10 MPa until the water content is ≤5%.

4. The compound according to claim 1, characterized in that The preparation method of the Dendrobium officinale extract comprises: a. Crush the whole herb of Dendrobium officinale to 20-40 mesh, add 4-8 times amount of pure water, and extract in a water bath at 70-90℃ for 1-3 hours; b. The extract was filtered through an ultrafiltration membrane with a molecular weight cutoff of 10-30 kDa, and the filtrate was collected and concentrated under reduced pressure to 1 / 4-1 / 2 of the original volume; c. Spray drying: Inlet air temperature 160-180℃, outlet air temperature 85-95℃, to obtain Dendrobium officinale extract powder with a moisture content of ≤5%.

5. The compound according to claim 1, characterized in that: The preparation method of the astragalus extract comprises: a. The dried Astragalus was crushed through a 80-120 mesh sieve and refluxed with 40-60% ethanol at a solid-liquid ratio of 1:10-14 for 1-3 times, each time for 1-2 hours; b. Combine the extracts, concentrate under reduced pressure to a relative density of 1.05-1.15, add an equal volume of water, mix well and let stand for 36-60 hours; c. Centrifuge and separate, take the supernatant, and spray-dry to obtain the astragalus extract.

6. Use of the compound according to claim 1 in the preparation of tablets, capsules, pills or granules.

7. A method for preparing tablets or capsules using the compound according to any one of claims 1 to 5, characterized in that: The following steps are involved: a. Dissolve the Cordyceps militaris extract and proanthocyanidins in an appropriate amount of water or a suitable solvent, and stir at a constant temperature of 50-70°C until completely dissolved to obtain a mixed solution; b. Ganoderma lucidum extract, Dendrobium candidum extract, Astragalus extract, microcrystalline cellulose and lactose were mixed and passed through a 80-200 mesh sieve to obtain a uniform powder mixture; c. Using an efficient wet granulator, the mixed solution of step a is mixed with the powder mixture of step b, and the water activity is controlled to be ≤0.15-0.25, and a 3-7% ethanol solution of hydroxypropyl methylcellulose is used as a binder; d. The wet granules are dried in a fluidized bed at 40-60℃ until the moisture content is ≤2-4%. After granulation, they are compressed into tablets or filled into capsules.

8. The preparation method according to claim 7, characterized in that: The disintegration time of the tablet is ≤10-15 minutes, and the particle size of the capsule content is ≤100-150 μm.

9. A method for preparing a pill or granule using the compound according to any one of claims 1 to 5, characterized in that: The following steps are involved: Preparation method of pills: 1) Raw material preparation: Dissolve the Cordyceps militaris extract and proanthocyanidins in an appropriate amount of water or a suitable solvent, and stir at a constant temperature of 50-70°C until they are completely dissolved to obtain a mixed solution; Mixing the ganoderma lucidum extract, the dendrobium officinale extract, the astragalus extract with appropriate amounts of microcrystalline cellulose and lactose, and passing through a 80-200 mesh sieve to obtain a uniform powder mixture; 2) Mixing and pelleting: The mixed solution obtained in step 1 is thoroughly mixed with the powder mixture; According to the needs, you can add an appropriate amount of honey or refined honey as a binder to adjust the hardness to the appropriate softness; The mixture is made into pills using a pill making machine, and the size and shape of the pills can be adjusted as needed; 3) Drying and packaging: The prepared pills are placed in a ventilated and dry place for drying until the specified moisture content is reached, and then packaged; Preparation method of granules: 4) Wet granulation: Add the mixed liquid obtained in step 1) and the powder mixture into a high-efficiency wet granulator, and control the water activity to ≤ 0.15-0.25; Using 3-7% hydroxypropyl methylcellulose ethanol solution as a binder, start the granulator for wet granulation; During the granulation process, the amount of binder and the parameters of the granulator can be adjusted in time according to the formation of the granules; 5) Drying and granulation: Dry the wet granules in a fluidized bed dryer at 40-60°C until the moisture content is ≤2-4%; After drying, the granules are sized using a granulator to remove oversized or undersized particles to obtain uniform granules.

10. Use of the compound according to any one of claims 1 to 5 in combination with hyaluronic acid in the preparation of a pharmaceutical composition for promoting the restoration of smoothness and elasticity of the skin.

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