Composition and preparation for preventing and treating Alzheimer disease and skin photoaging and application of composition and preparation

Abstract

The invention provides a composition for preventing and treating Alzheimer disease and skin photoaging, a preparation and application of the composition and the preparation, and relates to the technical field of medicines. The composition comprises the following components in parts by weight: 50-80 parts of a ginseng extract, 50-80 parts of a ginkgo leaf extract and 0.1-1 part of a huperzia serrata extract. Through the synergistic effect of the ginseng extract, the ginkgo leaf extract and the huperzia serrata extract, the obtained composition is high in safety, small in dosage, simple in medicine composition and better in curative effect, and is used for preventing and treating the Alzheimer disease and skin photoaging. The action effect of the composition is obviously better than that of each contrast group and a blank contrast group.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to a composition, a preparation and application thereof for preventing and treating Alzheimer's disease and skin photoaging. Background Art

[0002] Alzheimer's disease (AD) is a chronic neurodegenerative disease that mainly affects neurons in the brain, especially areas related to memory, thinking and learning functions. Clinical treatment drugs are usually based on cholinesterase inhibitors and antibody drugs, including donepezil, rivastigmine and galantamine. This type of drug only improves disease symptoms and has a limited course of treatment. It is only for patients with stage 4-6 AD. In addition, this type of drug usually has the side effect of damaging the liver and kidneys, and the current clinical needs are not met. In traditional Chinese medicine, Alzheimer's disease is caused by kidney deficiency due to old age, internal injuries caused by the seven emotions, long-term illness and depletion, which leads to insufficient qi and blood, or kidney essence deficiency, or phlegm and blood stasis, resulting in malnutrition of the brain marrow. In clinic, Chinese medicines that invigorate the brain and replenish qi, activate blood circulation and remove blood stasis, and resolve phlegm and dredge collaterals are often used for conditioning, such as ginseng, Polygonum multiflorum, Astragalus, Panax notoginseng, Acorus calamus, etc.; there are also Chinese medicine compound prescriptions for treatment, such as Xixin Decoction. Chinese invention patent application CN107007784A discloses a Chinese medicine composition for reversing Aβ amyloid protein damage to nerve cells, and its preparation method and application. The Chinese medicine composition includes the following parts by weight of extracts of the original medicine prescription: 3-9 parts of Gastrodia elata, 3-9 parts of Chu Shi, 3-9 parts of Ginseng, 5-12 parts of Poria cocos, 3-9 parts of Cistanche deserticola, 3-9 parts of Alpinia oxyphylla, 3-9 parts of Polygala tenuifolia, 3-9 parts of Acorus calamus, and 5-15 parts of Ginkgo biloba. The composition is complex, the quality is difficult to control, and the effect needs to be further improved.

[0003] Skin photoaging is mainly caused by repeated exposure to ultraviolet rays. Traditional Chinese medicine summarizes the causes of skin photoaging as external attack of phototoxicity, intolerance to endowment, weak skin, uncontrolled diet, etc. The causes are complex and diverse, but its pathological changes are mainly at the two ends of deficiency and excess. Deficiency is qi and blood deficiency and skin malnutrition; excess is phlegm and blood stasis, and blood vessels are blocked. Rhodiola rosea, ginseng, Eucommia ulmoides, Yupingfeng powder, etc. are often used for prevention and treatment. For example, Chinese invention patent application CN118615365A discloses a preparation for improving skin photoaging, which is made of the following three raw materials: raw rehmannia, Poria cocos and ginseng, wherein the ratio of raw rehmannia, Poria cocos and ginseng can be 6.5-7:1.5-2:1 by weight. However, the effect of the preparation needs to be further improved.

[0004] Therefore, there is an urgent need to develop a new Chinese medicine composition for preventing and treating Alzheimer's disease and skin photoaging that has high safety, low dosage, simple drug composition and better efficacy. Summary of the invention

[0005] The present invention aims at the problems existing in the prior art and provides a composition, preparation and application thereof for preventing and treating Alzheimer's disease and skin photoaging. Through the synergistic effect of ginseng extract, ginkgo leaf extract and melaleuca extract, the obtained composition has high safety, small dosage, simple drug composition and better efficacy, and is used for preventing and treating Alzheimer's disease and skin photoaging.

[0006] One of the purposes of the present invention is to provide a composition for preventing and treating Alzheimer's disease and skin photoaging, wherein the composition comprises the following components by weight: 50-80 parts of ginseng extract, 50-80 parts of ginkgo leaf extract, and 0.1-1 part of melaleuca extract.

[0007] Preferably, the composition comprises the following components by weight: 60-75 parts of ginseng extract, 55-75 parts of ginkgo leaf extract, and 0.1-0.5 parts of melaleuca extract.

[0008] Preferably, the composition comprises the following components by weight: 70 parts of ginseng extract, 60 parts of ginkgo leaf extract, and 0.2 parts of melaleuca extract.

[0009] Preferably, the composition comprises the following components by weight: 60 parts of ginseng extract, 75 parts of ginkgo leaf extract, and 0.5 parts of melaleuca extract.

[0010] Preferably, the composition comprises the following components by weight: 75 parts of ginseng extract, 55 parts of ginkgo leaf extract, and 0.1 parts of melaleuca extract.

[0011] Preferably, the ginseng extract is total saponins from ginseng stems and leaves, purchased from Jilin Hongjiu Biotechnology Co., Ltd.

[0012] Preferably, the ginkgo leaf extract is purchased from Jiangsu Bestcon Pharmaceutical Co., Ltd.

[0013] Preferably, the Melaleuca extract is prepared by the following steps: The original medicinal material Thousand Layer Tower is percolated with hydrochloric acid with a volume concentration of 1-1.5%, the pH is adjusted to 5-6, adsorbed with a macroporous resin, washed with water, eluted with 50% ethanol, concentrated, dried, and crushed to obtain a dry powder.

[0014] Further preferably, the amount of hydrochloric acid used is 3-6 times the weight of the lasagna.

[0015] The second aspect of the present invention is to provide a preparation comprising the above composition, wherein the preparation further comprises a pharmaceutically acceptable excipient.

[0016] Preferably, the auxiliary material is selected from any one or more of polyethylene glycol, dextrin, maltodextrin, soluble starch, lactose, citric acid, sodium citrate, mannitol, lecithin and sucralose.

[0017] More preferably, the auxiliary material is polyethylene glycol.

[0018] Preferably, the preparation is in the form of pills, tablets, capsules, granules, mixtures or syrups.

[0019] More preferably, the preparation is in the form of pellets.

[0020] Further preferably, the preparation method of the drop pills is: (1) Put polyethylene glycol into a melting pot, heat it to 80-110°C, melt it, stir it, and when the polyethylene glycol is completely in a transparent liquid state, lower the temperature of the melting pot to 70-100°C; (2) Slowly add the melaleuca extract into the melting pot while stirring continuously. Continue stirring for more than 10 minutes after all the extract is added. (3) Slowly add the ginkgo leaf extract and ginseng stem and leaf extract into the melting pot while stirring continuously for more than 2 hours; (4) Using dimethyl silicone oil as the condensate, starting the refrigeration and heating systems, and after the heating and refrigeration reach the set values, starting the condensate circulation pump, setting a suitable speed, maintaining a proper amount of overflow at the pipe mouth, filling the feed liquid, stirring, introducing clean compressed air, keeping the storage tank at a suitable pressure, and starting dripping to obtain the dripping pills.

[0021] The third aspect of the present invention relates to the use of the above composition or a preparation comprising the above composition in the preparation of a product for preventing and treating Alzheimer's disease and / or preventing and treating skin photoaging.

[0022] Compared with the prior art, the present invention has the following beneficial effects: through the synergistic effect of ginseng extract, ginkgo leaf extract and melaleuca extract, the obtained composition has high safety, small dosage, simple drug composition and better efficacy, and is used for preventing and treating Alzheimer's disease and skin photoaging. The effect of the composition of the present invention is significantly better than that of each comparative example and the blank control group. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 :The effect of SGS-01 sample on the activity of HT-22 cells; Figure 2 :Dose-effect relationship of cell activity of SGS-01 samples (IC 50 )curve; Figure 3 :Dose-effect relationship of SGS-01 in Aβ model (EC 50 )curve; Figure 4 :Dose-effect relationship of SGS-04 in Aβ model (EC 50 )curve; Figure 5 :Dose-effect relationship of SGS-05 in Aβ model (EC 50 )curve; Figure 6 :Dose-effect relationship of SGS-06 in Aβ model (EC 50 )curve; Figure 7 : Statistical results of fluorescence intensity of apoptotic cells in zebrafish tail after sample treatment; Figure 8 : Dose-effect curve of SGS-07. DETAILED DESCRIPTION

[0024] The following non-limiting examples can enable those skilled in the art to more fully understand the present invention, but do not limit the present invention in any way. The following content is merely an exemplary description of the scope of the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, which should also fall within the scope of the present invention.

[0025] In the embodiments of the present invention, the sources and batch numbers of the raw materials are: Ginseng extract (total saponins from ginseng stems and leaves): Jilin Hongjiu Biotechnology Co., Ltd., batch number 240201; Ginkgo biloba leaf extract: Jiangsu Beisikang Pharmaceutical Co., Ltd., batch number A0124030012; Melaleuca extract: Wanbangde Pharmaceutical Group Co., Ltd., batch number 501S240401.

[0026] Experimental Example 1: Cytotoxicity Experiment The cytotoxicity of the samples was evaluated by measuring their effects on the activity of HT-22 mouse hippocampal neurons.

[0027] 1. Experimental materials and reagents HT-22 mouse hippocampal neuronal cells, DMEM medium, trypsin, fetal bovine serum, antibiotics, DPBS buffer, cell culture plates, CCK-8 kit.

[0028] 2. Main equipment See Table 1 for details.

[0029] Table 1: Main equipment for cytotoxicity experiments

[0030] 3. Sample preparation A composition for preventing and treating Alzheimer's disease and skin photoaging, the composition comprising the following components by weight: 70 parts of ginseng extract, 60 parts of ginkgo leaf extract, and 0.2 parts of melaleuca extract. Specifically: Component 1 (mother solution, ginseng extract): yellow-brown solution, solvent: DMSO, concentration: 210 mg / mL; Component 2 (mother solution, Ginkgo biloba extract): brown solution, solvent: DMSO, concentration: 180 mg / mL; Component 3 (mother solution, Melaleuca extract): transparent solution, solvent: DMSO, concentration is 0.6 mg / mL.

[0031] Take 0.1 mL of component 1, component 2, and component 3, add 0.2 mL of DMSO and 4.5 mL of water to obtain SGS-01. See Table 2 for details.

[0032] Table 2: Composition of SGS-01 sample

[0033] 4. Experimental steps Cell viability test The revived cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum and antibiotics at 37°C and 5% CO2. When the cells grew to 80% confluence, they were digested with trypsin and inoculated in 96-well plates. After the cells adhered for 24 h, the test substance SGS-01 was added to 9 concentrations of 8%, 4%, 2%, 1%, 0.5%, 0.25%, 0.125%, 0.062%, and 0.031% diluted in culture medium, and 6 replicates were used for each concentration to test cell activity.

[0034] A blank control group was set up, and the cell activity was measured after 24 h of incubation (CCK-8 method), and the relative cell activity was calculated according to the following formula: Relative cell activity% = absorbance of test group / absorbance of blank control group × 100% If the cell viability of the test sample is less than 80% and there is a significant difference compared with the blank control group, it is considered to be cytotoxic.

[0035] Through the cell activity test, the safe concentration of samples that has no effect on cell activity (cell activity greater than 80%) is selected for the next stage of efficacy testing.

[0036] 5. Cell activity test results Compared with the blank control group, the cell activity of sample SGS-01 in the concentration range of 2%-8% was significantly less than 80%, which had an impact on cell activity; the cell activity of sample SGS-01 in the concentration range of 0.031%-1% was greater than 80%, which had no impact on cell activity, as shown in Table 3 and Figure 1 .

[0037] Table 3: SGS-01 sample cell activity experiment and difference significance analysis

[0038] Dose-effect curve Figure 2 Based on the cell activity data, the IC of the samples was calculated using GraphPad software. 50 (The sample concentration at which the cell activity is 50%) is 5.47%. The relevant data are shown in Table 4.

[0039] Table 4: SGS-01 sample cell activity IC 50 Concentration data of each component

[0040] 6. Conclusion Mouse hippocampal neuron cells HT-22 were used to measure the cell activity of the samples. The cell activity of the samples in the concentration range of 2%-8% was significantly less than 80%, which had an impact on the cell activity. The cell activity of the samples in the concentration range of 0.031%-1% was greater than 80%, which had no effect on the cell activity.

[0041] Experimental Example 2: Cell Damage Experiment (Aβ Model) HT-22 neuronal cells were damaged by Aβ1-42, and then the test substances were administered to evaluate the neuroprotective effect of the samples in restoring cell activity.

[0042] 1. Experimental materials and reagents HT-22 mouse hippocampal neuronal cells, DMEM medium, trypsin, fetal bovine serum, antibiotics, DPBS buffer, cell culture plates, CCK-8 kit.

[0043] 2. Main equipment Clean workbench, carbon dioxide incubator, analytical balance, pipette, multifunctional microplate reader.

[0044] 3. Aβ drug treatment Modeling agent information: β-amyloid protein 1-42 (Aβ1-42), from PONY Biopharmaceuticals, CAS: 166090-74-0, Aβ1-42 is a polypeptide composed of 42 amino acids, toxic to hippocampal slices, used in the study of Alzheimer's disease. Physical properties: powder, storage conditions: -20℃, preparation method: This product is unstable in solution state, it is recommended to prepare it immediately before use.

[0045] 4. Specific experimental methods: ① Take 1 mg of the bottled amyloid-β protein, add 45 µL of DMSO, mix well and transfer all to a centrifuge tube to obtain a 5 mM DMSO solution of Aβ protein. Then add 950 µL of serum-free culture medium to the solution to obtain a 250 µM working solution.

[0046] ② Before use, transfer 1 mL of 250 µM Aβ working solution to a 50 mL centrifuge tube, add 19 mL of complete culture medium containing 10% FBS and 1% P / S to make a 12.5 µM Aβ solution, and preheat it in a 37°C incubator.

[0047] ③Full medium replacement, adding culture medium containing 12.5 µM Aβ to 96-well plates, drug treatment for 24 hours, then discarding the culture medium (2nd day), adding culture medium containing the test drug, treatment for 24 hours, and detecting cell viability with CCK-8 on the 3rd day.

[0048] 5. Sample preparation ①The preparation method of SGS-01 is the same as that in Experimental Example 1, section “3. Sample preparation”.

[0049] ②SGS-04 is prepared from component 1 and component 2, SGS-05 is prepared from component 2 and component 3, and SGS-06 is prepared from component 1 and component 3. The specific composition is shown in the following table, where: Component 1 (mother solution, ginseng extract): yellow-brown solution, solvent: DMSO, concentration: 210 mg / mL; Component 2 (mother solution, Ginkgo biloba extract): brown solution, solvent: DMSO, concentration: 180 mg / mL; Component 3 (mother solution, Melaleuca extract): transparent solution, solvent: DMSO, concentration is 0.6 mg / mL.

[0050] Table 5: SGS-04, SGS-05, SGS-06 sample composition

[0051] SGS-01, SGS-04, SGS-05, and SGS-06 were diluted with culture medium to the test concentration.

[0052] 6. Experimental steps Cell viability test The revived cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum and antibiotics at 37°C and 5% CO 2 . When the cells grew to 80% confluence, they were digested with trypsin and inoculated in a 96-well plate.

[0053] A culture medium containing 12.5 µM Aβ was added to a 96-well plate and treated with drugs for 24 hours. The culture medium containing the modeling agent was then discarded and the test substance (SGS-01) was added to test the neuroprotective effect of SGS-01 at different concentrations to restore cell activity. The concentrations were 1%, 0.5%, 0.25%, 0.125%, 0.062%, and 0.031%, for a total of 6 concentrations.

[0054] A blank control group and a model group were also set up; the blank control group did not use the modeling agent Aβ, but added DMSO solvent equal to the highest drug dose (1%) as a solvent control; the model group used the modeling agent Aβ to damage cells, and did not receive drug treatment.

[0055] Determine cell activity (CCK-8 method) and calculate the relative cell activity according to the following formula: Cell survival rate % = absorbance of the experimental group / absorbance of the blank control group × 100%; Cell recovery rate % = (absorbance value of the experimental group – absorbance value of the model group) / (absorbance value of the blank control – absorbance value of the model group) × 100%.

[0056] The absorbance value of the blank well in the experimental platform (96-well plate) was subtracted from the absorbance value of each group to reduce the system error.

[0057] For example, when the cell survival rate of the blank control group is 100%, and the cell survival rate of the model group after modeling injury is 60%, the cell survival rate of the drug treatment group is expected to fall within the range of 60%-100%. If the cell survival rate of the drug treatment group is 80%, it means that the cell survival rate has increased from 60% to 80%, and the cell recovery rate is 50%. 50 Curve fitting: the data point on the curve where the cell recovery rate reaches 50% is the EC 50 Drug concentration. The above examples are used to illustrate cell viability, cell recovery rate and EC 50 The values ​​do not represent any actual experimental data.

[0058] The experimental steps for detecting the cell activity of SGS-04, SGS-05 and SGS-06 are the same as above.

[0059] 7. Statistical methods The experimental data were statistically analyzed using Graphpad Prism 10.1.2. The logistic curve in the nonlinear fitting “[Agonist] vs. normalized response – Variable slope” mode was used for regression analysis and EC was calculated. 50 value.

[0060] 8. Cell activity detection test results ①Cell recovery rate analysis: The specific results are shown in Table 6.

[0061] Table 6: Recovery rate of damaged cells by different concentrations of SGS-01, SGS-04, SGS-05, and SGS-06 samples

[0062] ②EC 50 Value: The efficacy relationship curves of SGS-01, SGS-04, SGS-05, and SGS-06 samples are shown in Figure 3-Figure 6 Based on the cell activity data, the EC of the samples was calculated using GraphPad software. 50 , the specific data results are shown in Table 7.

[0063] Table 7: EC values ​​of SGS-01, SGS-04, SGS-05 and SGS-06 samples for damaged cell recovery 50 Concentration of each component

[0064] According to the table above, the EC of SGS-01 50 The value is 0.29%, which is equivalent to SGS-01 being a combined preparation of component 1 of 12.21 mg / L, component 2 of 10.47 mg / L, and component 3 of 0.035 mg / L. The EC of SGS-04 50 The value is 0.77%, which is equivalent to SGS-04 being a combined preparation of component 1 of 32.34 mg / L and component 2 of 27.72 mg / L; the EC of SGS-05 50 The value is 1.17%, which is equivalent to SGS-05 being a combined preparation of component 2 at 42.12 mg / L and component 3 at 0.140 mg / L; the EC of SGS-06 50 The value is 0.70%, which is equivalent to SGS-06 being a combined preparation of component 1 at 29.40 mg / L and component 3 at 0.084 mg / L. 50 The experimental results show that when the three components of SGS-01 are used together, EC 50 The concentration value is higher than the EC when any of the components is removed.50 The concentration value of has been significantly reduced, and the combination of the three components can achieve a synergistic effect on the recovery of cell activity in the Aβ-damaged cell model.

[0065] Experimental Example 3: Anti-photoaging efficacy evaluation experiment 1. Sample preparation information A composition for preventing and treating Alzheimer's disease and skin photoaging, the composition comprising the following components by weight: 70 parts of ginseng extract, 60 parts of ginkgo leaf extract, and 0.2 parts of melaleuca extract. Specifically: Component 1 (mother solution, ginseng extract): yellow-brown solution, solvent: DMSO, concentration: 210 mg / mL; Component 2 (mother solution, Ginkgo biloba extract): brown solution, solvent: DMSO, concentration: 180 mg / mL; Component 3 (mother solution, Melaleuca extract): transparent solution, solvent: DMSO, concentration is 0.6 mg / mL.

[0066] Take 1 mL each of component 1, component 2, and component 3, mix them evenly, and the concentration of the mother liquor after mixing is 130.2 mg / mL to obtain SGS-07 (component 1+component 2+component 3) for use.

[0067] Take 3 mL of component 1, the concentration of the mother solution is 210 mg / mL, and set aside.

[0068] Positive control: tea polyphenols, yellow-brown powder, batch number Y12J11B115615, Shanghai Yuanye Biotechnology Co., Ltd., solvent is standard dilution water.

[0069] 2. Experimental Animals Zebrafish were raised in fish farming water at 28°C (water quality: 200 mg instant sea salt was added to every 1 L of reverse osmosis water, conductivity was 450-550 μS / cm; pH was 6.5-8.5; hardness was 50-100 mg / L CaCO3), bred and provided by our company's fish farming center, the experimental animal use license number is: SYXK (Zhejiang) 2022-0004, and the breeding management complies with the requirements of the international AAALAC certification (certification number: 001458), IACUC ethics review number: IACUC-2024-9517-01.

[0070] 3. Instruments, consumables and reagents Dissecting microscope (SZX7, OLYMPUS, Japan); CCD camera (VertA1, Shanghai Tusen Vision Technology Co., Ltd., China); precision electronic balance (CP214, OHAUS, USA); 6-well plate (Zhejiang Bellanbo Biotechnology Co., Ltd., China); simulated sunlight (SOL500, Hoenle, Germany); motorized focus continuous zoom fluorescence microscope (AZ100, Nikon, Japan). Methylcellulose (Batch No. C2004046, Shanghai Aladdin Biochemical Technology Co., Ltd., China); acridine orange (AO, Batch No. C12894919, Shanghai McLean Biochemical Technology Co., Ltd., China); dimethyl sulfoxide (DMSO, Batch No. BCCD8942, Sigma, Switzerland).

[0071] 4. Detection method 4.1. Determination of maximum detectable concentration (MTC) Wild-type AB strains at 3 days post-fertilization (3 dpf) were randomly selected in 6-well plates, and 30 zebrafish were treated in each well. Samples of different concentrations diluted with deionized water were given, and the concentrations are shown in Table 8. A normal control group and a model control group were set up at the same time, and the volume of each well was 3 mL. After treatment at 28℃ for 3 h, all experimental groups except the normal control group were irradiated with ultraviolet rays to establish a zebrafish light damage model. After treatment at 28℃ for 1 day, the MTC of the samples on the model zebrafish was measured.

[0072] 4.2. Evaluation of anti-photoaging efficacy 3 dpf wild-type AB strains were randomly selected in 6-well plates, and 30 zebrafish were treated in each well. Samples of different concentrations diluted with deionized water were given, and the concentrations are shown in Table 9; the positive control tea polyphenol concentration was 10.0 μg / mL, and a normal control group and a model control group were set up at the same time, with a capacity of 3 mL per well. After treatment at 28℃ for 3 h, except for the normal control group, the remaining experimental groups were irradiated with ultraviolet light to establish a zebrafish light damage model. After treatment at 28℃ for 1 day, each experimental group was first stained with AO. After completion, 10 zebrafish were randomly selected from each group and placed under a fluorescence microscope for photography. NIS-Elements D 3.20 advanced image processing software was used to analyze and collect data, and the fluorescence intensity (S) of apoptotic cells in the tail of zebrafish was analyzed. The statistical analysis results of this indicator were used to evaluate the anti-photoaging efficacy of the samples. The anti-photoaging efficacy was calculated as follows: Anti-photoaging effect (%)

[0073] The statistical results were expressed as mean ± SE. SPSS 26.0 software was used for statistical analysis, and p < 0.05 indicated that the difference was statistically significant.

[0074] 5. Test results MTC determination The results are shown in Table 8.

[0075] Table 8: Results of the concentration test on the anti-photoaging effect of samples (n = 30)

[0076] Under the experimental conditions, the maximum detection concentration (MTC) of SGS-07 (component 1+component 2+component 3) and component 1 for anti-photoaging efficacy was 81.4 μg / mL.

[0077] 5.2. Evaluation of anti-photoaging efficacy Under the experimental conditions, SGS-07 (component 1 + component 2 + component 3) has anti-photoaging effect, which is specifically manifested by reducing the fluorescence intensity of apoptotic cells in the tail; component 1 has no significant anti-photoaging effect. The anti-photoaging effect of the samples is detailed in Table 9, Figure 7 (Compared with the model control group, *p<0.05, **p<0.01, ***p<0.001). The dose-effect curve of SGS-07 is shown in Figure 8 .

[0078] Table 9: Sample anti-light aging efficacy evaluation test results (n = 10)

[0079] Compared with the model control group, *p<0.05, **p<0.01, ***p<0.001.

[0080] According to the experimental results in the table above, the SGS-07 (component 1 + component 2 + component 3) sample has a good anti-photoaging effect at a concentration of 5.09-81.4µg / mL, which is significantly better than the single component component 1. At a concentration of 20.4-81.4µg / mL, the effect is better than the positive control drug tea polyphenols.

[0081] Experimental Example 4: Effects of samples SGS-08 and SGS-09 on cell damage recovery rate 1. Experimental materials and reagents HT-22 mouse hippocampal neuronal cells, DMEM medium, trypsin, fetal bovine serum, antibiotics, DPBS buffer, cell culture plates, CCK-8 kit.

[0082] 2. Main equipment Clean workbench, carbon dioxide incubator, analytical balance, pipette, multifunctional microplate reader.

[0083] 3. Aβ drug treatment Modeling agent information: β-amyloid protein 1-42 (Aβ1-42), from PONY Biopharmaceuticals, CAS: 166090-74-0, Aβ1-42 is a polypeptide composed of 42 amino acids, toxic to hippocampal slices, used in the study of Alzheimer's disease. Physical properties: powder, storage conditions: -20℃, preparation method: This product is unstable in solution state, it is recommended to prepare it immediately before use.

[0084] 4. Specific experimental methods: ① Take 1 mg of the bottled amyloid-β protein, add 45 µL of DMSO, mix well and transfer all to a centrifuge tube to obtain a 5 mM DMSO solution of Aβ protein. Then add 950 µL of serum-free culture medium to the solution to obtain a 250 µM working solution.

[0085] ② Before use, transfer 1 mL of 250 µM Aβ working solution to a 50 mL centrifuge tube, add 19 mL of complete culture medium containing 10% FBS and 1% P / S to make a 12.5 µM Aβ solution, and preheat it in a 37°C incubator.

[0086] ③Full medium replacement, adding culture medium containing 12.5 µM Aβ to 96-well plates, drug treatment for 24 hours, then discarding the culture medium (2nd day), adding culture medium containing the test drug, treatment for 24 hours, and detecting cell viability with CCK-8 on the 3rd day.

[0087] 5. Preparation of test drugs SGS-08 and SGS-09 1) A composition for preventing and treating Alzheimer's disease and skin photoaging, comprising the following components by weight: 60 parts of ginseng extract, 75 parts of ginkgo leaf extract, and 0.5 parts of melaleuca extract. Specifically: Component 1: an appropriate amount of ginseng extract, dissolved in DMSO, and prepared into a yellow-brown solution with a concentration of 180 mg / mL; Component 2: appropriate amount of Ginkgo biloba extract, dissolved in DMSO, and prepared into a brown solution with a concentration of 225 mg / mL; Component 3: an appropriate amount of Melaleuca extract, dissolved in DMSO, and prepared into a transparent solution with a concentration of 1.5 mg / mL; Take 0.1 mL of component 1, component 2, and component 3, add 0.2 mL of DMSO and 4.5 mL of water to obtain SGS-08.

[0088] 2) A composition for preventing and treating Alzheimer's disease and skin photoaging, the composition comprising the following components by weight: 75 parts of ginseng extract, 55 parts of ginkgo leaf extract, and 0.1 parts of melaleuca extract. Specifically: Component 1: appropriate amount of ginseng extract, dissolved in DMSO, and prepared into a yellow-brown solution with a concentration of 225 mg / mL; Component 2: appropriate amount of Ginkgo biloba leaf extract, dissolved in DMSO, and prepared into a brown solution with a concentration of 165 mg / mL; Component 3: an appropriate amount of Melaleuca extract, dissolved in DMSO, and prepared into a transparent solution with a concentration of 0.3 mg / mL; Take 0.1 mL of component 1, component 2, and component 3, add 0.2 mL of DMSO and 4.5 mL of water to obtain SGS-09.

[0089] SGS-08 and SGS-09 were diluted with culture medium to the test concentration.

[0090] 6. Experimental steps The revived cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum and antibiotics at 37°C and 5% CO 2 . When the cells grew to 80% confluence, they were digested with trypsin and inoculated in a 96-well plate.

[0091] A culture medium containing 12.5 µM Aβ was added to a 96-well plate and treated with drugs for 24 hours. The culture medium containing the modeling agent was then discarded and the test drug was added to test the neuroprotective effect of different concentrations of the test drug in restoring cell activity. The concentrations were 1%, 0.5%, 0.25%, 0.125%, 0.062%, and 0.031%, for a total of 6 concentrations.

[0092] A blank control group and a model group were also set up; the blank control group did not use the modeling agent Aβ, but added DMSO solvent equal to the highest drug dose (1%) as a solvent control; the model group used the modeling agent Aβ to damage cells, and did not receive drug treatment.

[0093] Determine cell activity (CCK-8 method) and calculate the relative cell activity according to the following formula: Cell survival rate % = absorbance of the experimental group / absorbance of the blank control group × 100%; Cell recovery rate % = (absorbance value of the experimental group – absorbance value of the model group) / (absorbance value of the blank control – absorbance value of the model group) × 100%.

[0094] The absorbance value of the blank well in the experimental platform (96-well plate) was subtracted from the absorbance value of each group to reduce the system error.

[0095] The experimental data were statistically analyzed using Graphpad Prism 10.1.2. The specific results of cell recovery rate are shown in the table below.

[0096] Table 10: Effects of different concentrations of SGS-08 and SGS-09 on the recovery rate of damaged cells

[0097] According to the data in the table, SGS-08 and SGS-09 have a good recovery effect on the cell damage model.

[0098] Experimental Example 5: Anti-light aging efficacy evaluation test of samples SGS-10 and SGS-11 1. Sample preparation information: 1) A composition for preventing and treating Alzheimer's disease and skin photoaging, comprising the following components by weight: 60 parts of ginseng extract, 75 parts of ginkgo leaf extract, and 0.5 parts of melaleuca extract. Specifically: Component 1: an appropriate amount of ginseng extract, dissolved in DMSO, and prepared into a yellow-brown solution with a concentration of 180 mg / mL; Component 2: appropriate amount of Ginkgo biloba extract, dissolved in DMSO, and prepared into a brown solution with a concentration of 225 mg / mL; Component 3: an appropriate amount of Melaleuca extract, dissolved in DMSO, and prepared into a transparent solution with a concentration of 1.5 mg / mL; Take 1 mL each of component 1, component 2, and component 3, mix them evenly, and the concentration of the mother liquor after mixing is 135.5 mg / mL to obtain SGS-10 for use.

[0099] 2) A composition for preventing and treating Alzheimer's disease and skin photoaging, the composition comprising the following components by weight: 75 parts of ginseng extract, 55 parts of ginkgo leaf extract, and 0.1 parts of melaleuca extract. Specifically: Component 1: appropriate amount of ginseng extract, dissolved in DMSO, and prepared into a yellow-brown solution with a concentration of 225 mg / mL; Component 2: appropriate amount of Ginkgo biloba leaf extract, dissolved in DMSO, and prepared into a brown solution with a concentration of 165 mg / mL; Component 3: an appropriate amount of Melaleuca extract, dissolved in DMSO, and prepared into a transparent solution with a concentration of 0.3 mg / mL; Take 1 mL each of component 1, component 2, and component 3, mix them evenly, and the concentration of the mother liquor after mixing is 130.1 mg / mL to obtain SGS-11 for use.

[0100] 2. Experimental animals: Zebrafish were raised in fish farming water at 28°C (water quality: 200 mg of instant sea salt was added to every 1 L of reverse osmosis water, conductivity was 450-550 μS / cm; pH was 6.5-8.5; hardness was 50-100 mg / LCaCO3), bred and provided by our company's fish farming center, the experimental animal use license number is: SYXK (Zhejiang) 2022-0004, and the breeding management complies with the requirements of the international AAALAC certification (certification number: 001458), IACUC ethics review number: IACUC-2024-9517-01.

[0101] 3. Instruments, consumables and reagents: dissecting microscope (SZX7, OLYMPUS, Japan); CCD camera (VertA1, Shanghai Tusen Vision Technology Co., Ltd., China); precision electronic balance (CP214, OHAUS, USA); 6-well plate (Zhejiang Bellanbo Biotechnology Co., Ltd., China); simulated sunlight (SOL500, Hoenle, Germany); motorized focus continuous zoom fluorescence microscope (AZ100, Nikon, Japan). Methylcellulose (lot number C2004046, Shanghai Aladdin Biochemical Technology Co., Ltd., China); acridine orange (AO, lot number C12894919, Shanghai McLean Biochemical Technology Co., Ltd., China); dimethyl sulfoxide (DMSO, lot number BCCD8942, Sigma, Switzerland).

[0102] 4. Evaluation of anti-photoaging efficacy 3 dpf wild-type AB strains were randomly selected in 6-well plates, and 30 zebrafish were treated in each well. Samples of different concentrations diluted with deionized water were given, and the concentrations are shown in Table 11. The positive control tea polyphenol concentration was 10.0 μg / mL. At the same time, a normal control group and a model control group were set up, and the volume of each well was 3 mL. After treatment at 28℃ for 3 h, except for the normal control group, the other experimental groups were irradiated with ultraviolet light to establish a zebrafish light damage model. After treatment at 28℃ for 1 day, each experimental group was first stained with AO. After completion, 10 zebrafish were randomly selected from each group and placed under a fluorescence microscope for photography. NIS-Elements D 3.20 advanced image processing software was used to analyze and collect data. The fluorescence intensity (S) of apoptotic cells in the tail of zebrafish was analyzed. The statistical analysis results of this indicator were used to evaluate the anti-photoaging efficacy of the samples. The anti-photoaging efficacy was calculated as follows: Anti-photoaging effect (%)

[0103] The statistical results were expressed as mean ± SE. SPSS 26.0 software was used for statistical analysis, and p < 0.05 indicated that the difference was statistically significant. The specific results are shown in the table below.

[0104] Table 11: Sample anti-light aging efficacy evaluation test results (n = 10)

[0105] Compared with the model control group, *P<0.05, **P<0.01, ***P<0.001 in the drug-treated group.

[0106] According to the data in the table, it can be seen that SGS-10 and SGS-11 provided by the present invention have good anti-photoaging effects, especially the 40µg / mL and 80µg / mL dosage groups of SGS-10 and the 20µg / mL, 40µg / mL and 80µg / mL dosage groups of SGS-11 have better effects than those of tea polyphenols.

[0107] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A composition for preventing and treating Alzheimer's disease and skin photoaging, the composition comprising the following components by weight: 50-80 parts of ginseng extract, 50-80 parts of ginkgo leaf extract, and 0.1-1 part of melaleuca extract.

2. The composition according to claim 1, characterized in that The composition comprises the following components by weight: 60-75 parts of ginseng extract, 55-75 parts of ginkgo leaf extract, and 0.1-0.5 parts of melaleuca extract.

3. The composition according to claim 1, characterized in that The composition comprises the following components by weight: 70 parts of ginseng extract, 60 parts of ginkgo leaf extract and 0.2 parts of melaleuca extract.

4. A preparation containing the composition according to any one of claims 1 to 3, characterized in that: The preparation also includes pharmaceutically acceptable excipients.

5. The preparation according to claim 4, characterized in that The auxiliary material is selected from any one or more of polyethylene glycol, dextrin, maltodextrin, soluble starch, lactose, citric acid, sodium citrate, mannitol, lecithin and sucralose.

6. The preparation according to claim 4, characterized in that The dosage form of the preparation is drop pills, tablets, capsules, granules, mixtures or syrups.

7. The preparation according to claim 4, characterized in that The auxiliary material is polyethylene glycol, and the dosage form of the preparation is a drop pill.

8. The preparation according to claim 7, characterized in that The preparation method of the drop pill is: (1) Put polyethylene glycol into a melting pot, heat it to 80-110°C, melt it, stir it, and when the polyethylene glycol is completely in a transparent liquid state, lower the temperature of the melting pot to 70-100°C; (2) Slowly add the melaleuca extract into the melting pot while stirring continuously. Continue stirring for more than 10 minutes after all the extract is added. (3) Slowly add the ginkgo leaf extract and ginseng extract into the melting pot while stirring continuously for more than 2 hours; (4) Using dimethyl silicone oil as the condensate, starting the refrigeration and heating systems, and after the heating and refrigeration reach the set values, starting the condensate circulation pump, setting a suitable speed, maintaining a proper amount of overflow at the pipe mouth, filling the feed liquid, stirring, introducing clean compressed air, keeping the storage tank at a suitable pressure, and starting dripping to obtain the dripping pills.

9. Use of the composition according to any one of claims 1 to 3 or the preparation according to any one of claims 4 to 8 in the preparation of products for preventing and treating Alzheimer's disease and / or preventing and treating skin photoaging.

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