A composition, preparation and application thereof for preventing and treating Alzheimer's disease and skin photoaging

Abstract

The present invention provides a composition, a preparation and their applications for preventing and treating Alzheimer's disease and skin photoaging, relating to the technical field of medicine. The composition comprises the following components by weight: 50-80 parts of ginseng extract, 50-80 parts of ginkgo biloba extract, and 0.1-1 part of Huperzia serrata extract. Through the synergistic effect of ginseng extract, ginkgo biloba extract and Huperzia serrata extract, the obtained composition has high safety, small dosage, simple drug composition and better curative effect, 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.

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] In view of the problems existing in the prior art, the present invention provides a composition, a preparation and their applications for preventing and treating Alzheimer's disease and skin photoaging. Through the synergistic effect of ginseng extract, ginkgo biloba extract and Huperzia serrata extract, the obtained composition has high safety, small dosage, simple drug composition and better curative effect, 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, and the composition comprises the following components in parts by weight: 50-80 parts of ginseng extract, 50-80 parts of ginkgo biloba extract, and 0.1-1 part of Huperzia serrata extract.

[0007] Preferably, the composition comprises the following components in parts by weight: 60-75 parts of ginseng extract, 55-75 parts of ginkgo biloba extract, and 0.1-0.5 part of Huperzia serrata extract.

[0008] Preferably, the composition comprises the following components in parts by weight: 70 parts of ginseng extract, 60 parts of ginkgo biloba extract, and 0.2 part of Huperzia serrata extract.

[0009] Preferably, the composition comprises the following components in parts by weight: 60 parts of ginseng extract, 75 parts of ginkgo biloba extract, and 0.5 part of Huperzia serrata extract.

[0010] Preferably, the composition comprises the following components in parts by weight: 75 parts of ginseng extract, 55 parts of ginkgo biloba extract, and 0.1 part of Huperzia serrata extract.

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

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

[0013] Preferably, the Huperzia serrata extract is prepared by the following steps:

[0014] The original medicinal material Huperzia serrata is percolated with hydrochloric acid with a volume concentration of 1-1.5%, the pH is adjusted to 5-6, macroporous resin adsorption is used, after washing with water, elution is carried out with 50% ethanol, and then concentrated, dried and pulverized to obtain a dry powder.

[0015] Further preferably, the dosage of hydrochloric acid is 3-6 times the weight of Huperzia serrata.

[0016] The second aspect of the present invention lies in providing a preparation containing the above composition, and the preparation further comprises pharmaceutically acceptable excipients.

[0017] Preferably, the adjuvant is selected from any one or more of polyethylene glycol, dextrin, maltodextrin, soluble starch, lactose, citric acid, sodium citrate, mannitol, lecithin, and sucralose.

[0018] More preferably, the adjuvant is polyethylene glycol.

[0019] Preferably, the dosage form of the preparation is a dropping pill, tablet, capsule, granule, mixture or syrup.

[0020] More preferably, the dosage form of the preparation is a dropping pill.

[0021] More preferably, the preparation method of the dropping pill is as follows:

[0022] (1) Put polyethylene glycol into a melting pot, heat it to 80 - 110°C, melt it, stir it, and when all the polyethylene glycol becomes a transparent liquid, lower the heating temperature of the melting pot to 70 - 100°C;

[0023] (2) Slowly add the extract of Huperzia serrata into the melting pot while stirring continuously, and continue to stir for more than 10 minutes after adding all;

[0024] (3) Slowly add the extracts of Ginkgo biloba and Panax ginseng leaf into the melting pot while stirring continuously, and stir for more than 2 hours;

[0025] (4) Use dimethyl silicone oil as the condensate, turn on the refrigeration and heating systems, after the heating and refrigeration reach the set values, turn on the condensate circulation pump, set an appropriate rotation speed, keep an appropriate overflow at the nozzle, fill the material liquid, stir, introduce clean compressed air, keep the storage tank at an appropriate pressure, and start dropping to obtain the dropping pills.

[0026] The third aspect of the present invention relates to the use of the above - mentioned composition or a preparation containing the above - mentioned composition in the preparation of products for preventing and treating Alzheimer's disease and / or preventing and treating skin photo - aging.

[0027] Compared with the prior art, the present invention has the following beneficial effects: Through the synergistic effect of ginseng extract, Ginkgo biloba extract, and Huperzia serrata extract, the obtained composition has high safety, a small dosage, a simple drug composition, and better curative effects, and is used for preventing and treating Alzheimer's disease and skin photo - aging. The action effect of the composition of the present invention is significantly better than that of each comparative example and the blank control group. Description of the Drawings

[0028] Figure 1 : The effect of SGS - 01 sample on the activity of HT - 22 cells;

[0029] Figure 2 : The dose - effect relationship (IC 50 ) curve of the SGS - 01 sample cell activity;

[0030] Figure 3 : Dose-effect relationship (EC 50 ) curve of SGS-01 on the Aβ model;

[0031] Figure 4 : Dose-effect relationship (EC 50 ) curve of SGS-04 on the Aβ model;

[0032] Figure 5 : Dose-effect relationship (EC 50 ) curve of SGS-05 on the Aβ model;

[0033] Figure 6 : Dose-effect relationship (EC 50 ) curve of SGS-06 on the Aβ model;

[0034] Figure 7 : Statistical result graph of the fluorescence intensity of apoptotic cells at the tail of zebrafish after sample treatment;

[0035] Figure 8 : Dose-effect relationship curve of SGS-07. Detailed implementation manners

[0036] The following non-limiting examples can enable those of ordinary skill in the art to understand the present invention more comprehensively, but do not limit the present invention in any way. The following content is only an exemplary illustration of the scope claimed by the present invention. Those skilled in the art can make various changes and modifications to the invention of the present invention based on the disclosed content, and it should also fall within the scope claimed by the present invention.

[0037] In the embodiments of the present invention, the sources and batch numbers of the raw materials are as follows:

[0038] Ginseng extract (total ginsenosides from ginseng stems and leaves): Jilin Hongjiu Biotechnology Co., Ltd., batch number 240201;

[0039] Ginkgo biloba extract: Jiangsu Bescon Pharmaceutical Co., Ltd., batch number A0124030012;

[0040] Huperzia serrata extract: Wanbond Pharmaceutical Group Co., Ltd., batch number 501S240401.

[0041] Experimental example 1: Cytotoxicity experiment

[0042] The cytotoxicity of the sample was evaluated by measuring the effect of the sample on the activity of HT-22 mouse hippocampal neuronal cells.

[0043] 1. Test materials and reagents

[0044] HT-22 mouse hippocampal neuronal cells, DMEM medium, trypsin, fetal bovine serum, antibiotics, DPBS buffer, cell culture plates, CCK-8 kit.

[0045] 2. Main equipment

[0046] See Table 1 for details.

[0047] Table 1: Main equipment for cytotoxicity experiments

[0048]

[0049] 3. Sample preparation

[0050] 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 biloba extract, and 0.2 part of Huperzia serrata extract. Specifically:

[0051] Component 1 (stock solution, ginseng extract): Tan solution, solvent: DMSO, concentration: 210 mg / mL;

[0052] Component 2 (stock solution, ginkgo biloba extract): Brown solution, solvent: DMSO, concentration: 180 mg / mL;

[0053] Component 3 (stock solution, Huperzia serrata extract): Clear solution, solvent: DMSO, concentration: 0.6 mg / mL.

[0054] Take 0.1 mL each 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.

[0055] Table 2: Composition table of SGS-01 sample

[0056]

[0057] 4. Experimental procedures

[0058] Cell viability test

[0059] Resuscitate the cells and culture them in DMEM high-glucose medium containing 10% fetal bovine serum and antibiotics under the culture conditions of 37 °C and 5% CO2. When the cells grow to 80% confluence, digest them with trypsin and inoculate them into 96-well plates. After the cells adhere for 24 h, add the test substance SGS-01 diluted to 9 concentrations of 8%, 4%, 2%, 1%, 0.5%, 0.25%, 0.125%, 0.062%, and 0.031% with the medium, with 6 replicates for each concentration, and test the cell viability.

[0060] Another blank control group was set up. After incubation for 24 h, cell viability was measured (CCK-8 method), and the relative cell viability was calculated according to the following formula:

[0061] Relative cell viability % = absorbance value of the test group / absorbance value of the blank control group × 100%

[0062] 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 have cytotoxicity.

[0063] Through the cell viability test, the safe concentration of the sample that has no effect on cell viability (cell viability > 80%) was selected for the next-stage efficacy test.

[0064] 5. Results of the cell viability test

[0065] Compared with the blank control group, the cell viability of sample SGS-01 was significantly less than 80% in the concentration range of 2% - 8%, indicating an effect on cell viability; the cell viability of sample SGS-01 was greater than 80% in the concentration range of 0.031% - 1%, indicating no effect on cell viability. See Table 3 and Figure 1 .

[0066] Table 3: Cell viability experiment and analysis of significant differences of SGS-01 sample

[0067]

[0068] The dose-effect curve is shown in Figure 2 , and according to the cell viability data, the IC 50 (sample concentration with 50% cell viability) of the sample was calculated to be 5.47% using GraphPad software. The relevant data are shown in Table 4.

[0069] Table 4: IC 50 Concentration data of each component

[0070]

[0071] 6. Conclusion

[0072] The cell viability of the sample was measured using mouse hippocampal neuron cells HT-22. The cell viability of the sample was significantly less than 80% in the concentration range of 2% - 8%, indicating an effect on cell viability; the cell viability of the sample was greater than 80% in the concentration range of 0.031% - 1%, indicating no effect on cell viability.

[0073] Experimental Example 2: Cell damage experiment (Aβ model)

[0074] HT-22 neurons were damaged by Aβ1-42, and then the test substance was administered to evaluate the neuroprotective effect of the sample in restoring cell viability.

[0075] 1. Test materials and reagents

[0076] HT-22 mouse hippocampal neuron cells, DMEM medium, trypsin, fetal bovine serum, antibiotics, DPBS buffer, cell culture plates, CCK-8 kit.

[0077] 2. Main equipment

[0078] Clean bench, carbon dioxide incubator, analytical balance, pipette, multi-functional microplate reader.

[0079] 3. Aβ drug treatment

[0080] Information of the modeling agent: β-amyloid 1-42 (Aβ1-42), sourced from PONY Biopharmaceuticals, CAS: 166090-74-0. Aβ1-42 is a polypeptide composed of 42 amino acids, which is toxic to hippocampal slices and is used in the research of Alzheimer's disease. Physical properties: powder. Storage conditions: -20°C. Preparation method: This product is unstable in solution state. It is recommended to prepare and use it immediately.

[0081] 4. Specific experimental methods:

[0082] ① Take 1 mg of bottled β-amyloid and add 45 µL of DMSO. After mixing, transfer all to a centrifuge tube to obtain a 5 mM DMSO solution of Aβ protein. Subsequently, add 950 µL of serum-free medium to the solution to obtain a 250 µM working solution.

[0083] ② Immediately before use, transfer 1 mL of the 250 µM Aβ working solution to a 50 mL centrifuge tube, add 19 mL of complete medium containing 10% FBS and 1% P / S to prepare a 12.5 µM Aβ solution, and preheat it in a 37°C incubator.

[0084] ③ Replace all the medium. Add the medium containing 12.5 µM Aβ to a 96-well plate, perform drug treatment for 24 hours, then discard the medium (on the second day), add the medium containing the test drug, and perform treatment for 24 hours. On the third day, use CCK-8 to detect the cell survival rate.

[0085] 5. Sample preparation

[0086] ① The preparation method of SGS-01 is the same as that in the "3. Sample preparation" section of Experimental Example 1.

[0087] ② SGS-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 compositions are shown in the following table, where:

[0088] Component 1 (mother solution, ginseng extract): yellow-brown solution, solvent: DMSO, concentration: 210 mg / mL;

[0089] Component 2 (mother solution, Ginkgo biloba extract): brown solution, solvent: DMSO, concentration: 180 mg / mL;

[0090] Component 3 (mother solution, Melaleuca extract): transparent solution, solvent: DMSO, concentration: 0.6 mg / mL.

[0091] Table 5: SGS-04, SGS-05, SGS-06 sample composition

[0092]

[0093] SGS-01, SGS-04, SGS-05, and SGS-06 were diluted with culture medium to the test concentration.

[0094] 6. Experimental steps

[0095] Cell viability test

[0096] 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.

[0097] 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.

[0098] 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.

[0099] Determine cell activity (CCK-8 method) and calculate the relative cell activity according to the following formula:

[0100] Cell survival rate % = absorbance of the experimental group / absorbance of the blank control group × 100%;

[0101] 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%.

[0102] The absorbance values of each group were subtracted by the absorbance value of the blank well of the experimental platform (96-well plate) to reduce the systematic error.

[0103] For example: When the cell survival rate of the blank control group is 100%, after modeling injury, if the cell survival rate of the model group is 60%, then 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%, indicating that the cell survival rate has increased from 60% to 80%, then the cell recovery rate is 50%. Using the EC 50 curve fitting, the data point on the curve where the cell recovery rate reaches 50% is the EC 50 drug concentration. The above example is used to illustrate the cell survival rate, cell recovery rate, and EC 50 value, and does not represent any measured experimental data.

[0104] The experimental procedures for detecting the cell viability of SGS-04, SGS-05, and SGS-06 are the same as above.

[0105] 7. Statistical methods

[0106] The experimental data were statistically analyzed using Graphpad Prism 10.1.2. A logistic curve in the " [Agonist] vs. normalized response – Variable slope" mode of non-linear fitting was used for regression analysis to calculate the EC 50 value.

[0107] 8. Results of the cell viability detection test

[0108] ① Analysis of cell recovery rate:

[0109] The specific results are shown in Table 6.

[0110] Table 6: Recovery rates of damaged cells by SGS-01, SGS-04, SGS-05, and SGS-06 samples at different concentrations

[0111]

[0112] ② EC 50 value: The efficacy relationship curves of SGS-01, SGS-04, SGS-05, and SGS-06 samples are shown in Figures 3 - 6 . According to the cell viability data, the EC 50 of the samples was calculated using GraphPad software, and the specific data results are shown in Table 7.

[0113] Table 7: EC 50 concentrations of each component for the recovery of damaged cells by SGS-01, SGS-04, SGS-05, and SGS-06 samples

[0114]

[0115] According to the above table, it can be known that the EC 50 value of SGS-01 is 0.29%, which is equivalent to that SGS-01 is a combined preparation with component 1 at 12.21 mg / L, component 2 at 10.47 mg / L, and component 3 at 0.035 mg / L. And the EC 50 value of SGS-04 is 0.77%, which is equivalent to that SGS-04 is a combined preparation with component 1 at 32.34 mg / L and component 2 at 27.72 mg / L; the EC 50 value of SGS-05 is 1.17%, which is equivalent to that SGS-05 is a combined preparation with component 2 at 42.12 mg / L and component 3 at 0.140 mg / L; the EC 50 value of SGS-06 is 0.70%, which is equivalent to that SGS-06 is a combined preparation with component 1 at 29.40 mg / L and component 3 at 0.084 mg / L. From the experimental results of EC 50 , it can be known that the concentration value of EC 50 when the three components in SGS-01 are used in combination is significantly lower than the concentration value of EC 50 when any one of the components is removed. The combination of the three components can achieve a synergistic effect on the recovery of cell viability in the Aβ-damaged cell model.

[0116] Experimental Example 3: Anti-photoaging efficacy evaluation experiment

[0117] 1. Sample preparation information

[0118] A composition for preventing and treating Alzheimer's disease and skin photoaging, the composition comprises the following components by weight: 70 parts of ginseng extract, 60 parts of ginkgo biloba extract, and 0.2 part of Huperzia serrata extract. Specifically:

[0119] Component 1 (stock solution, ginseng extract): yellowish-brown solution, solvent: DMSO, concentration: 210 mg / mL;

[0120] Component 2 (stock solution, ginkgo biloba extract): brown solution, solvent: DMSO, concentration: 180 mg / mL;

[0121] Component 3 (stock solution, Huperzia serrata extract): clear solution, solvent: DMSO, concentration: 0.6 mg / mL.

[0122] Take 1 mL each of component 1, component 2, and component 3, mix well. After mixing, the concentration of the stock solution is 130.2 mg / mL, and SGS-07 (component 1 + component 2 + component 3) is obtained for standby.

[0123] Take 3 mL of Component 1 with a stock solution concentration of 210 mg / mL and set aside.

[0124] Positive control: Tea polyphenols, yellowish-brown powder, batch number Y12J11B115615, Shanghai Yuanye Bio-Technology Co., Ltd., and the solvent is standard dilution water.

[0125] 2. Experimental animals

[0126] Zebrafish are all raised in fish-raising water at 28 °C (water quality: add 200 mg of instant sea salt to every 1 L of reverse osmosis water, with a conductivity of 450 - 550 μS / cm; pH of 6.5 - 8.5; hardness of 50 - 100 mg / L CaCO3). They are provided by the fish-raising center of our company. The experimental animal use license number is: SYXK (Zhe) 2022 - 0004. The feeding management meets the requirements of international AAALAC accreditation (accreditation number: 001458), and the IACUC ethical review number is: IACUC - 2024 - 9517 - 01.

[0127] 3. Instruments, consumables and reagents

[0128] 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 Beilanbo Biotechnology Co., Ltd., China); Simulated sunlight (SOL500, Hoenle, Germany); Electrically focused continuous zoom fluorescence microscope (AZ100, Nikon, Japan). Methyl cellulose (batch number C2004046, Shanghai Aladdin Biochemical Technology Co., Ltd., China); Acridine orange (AO, batch number C12894919, Shanghai Macklin Biochemical Co., Ltd., China); Dimethyl sulfoxide (DMSO, batch number BCCD8942, Sigma, Switzerland).

[0129] 4. Detection methods

[0130] 4.1. Determination of the maximum test concentration (MTC)

[0131] Randomly select wild-type AB strains at 3 days post-fertilization (3 dpf) into 6-well plates, with 30 zebrafish in each well. Different concentrations of the sample diluted with deionized water are given, and the concentrations are shown in Table 8. At the same time, a normal control group and a model control group are set up, and the volume of each well is 3 mL. After treatment at 28 °C for 3 h, except for the normal control group, the other experimental groups are irradiated with ultraviolet light to establish a zebrafish light damage model. After treatment at 28 °C for 1 day, the MTC of the sample on the model zebrafish is measured.

[0132] 4.2. Evaluation of anti-photoaging efficacy

[0133] Randomly select wild-type AB strains at 3 dpf and place them in 6-well plates, with 30 zebrafish treated in each well. Different concentrations of the sample diluted with deionized water are given, and the concentrations are shown in Table 9; the positive control is tea polyphenol at a concentration of 10.0 μg / mL. At the same time, a normal control group and a model control group are set up, and the volume of each well is 3 mL. After treatment at 28 °C for 3 h, except for the normal control group, the rest of the experimental groups are irradiated with ultraviolet light to establish a zebrafish photo-damage model. After treatment at 28 °C for 1 day, each experimental group is first stained with AO. After completion, 10 zebrafish are randomly selected from each group and placed under a fluorescence microscope for photography. The NIS-Elements D 3.20 advanced image processing software is used to analyze and collect data, and the fluorescence intensity (S) of apoptotic cells in the zebrafish tail is analyzed. The anti-photoaging efficacy of the sample is evaluated based on the statistical analysis results of this index. The anti-photoaging efficacy is calculated as follows:

[0134] Anti-photoaging efficacy (%)

[0135] The results of statistical processing are expressed as mean ± SE. SPSS 26.0 software is used for statistical analysis, and p < 0.05 indicates that the difference is statistically significant.

[0136] 5. Detection results

[0137] 5.1. Determination of MTC

[0138] The results are shown in Table 8 for details.

[0139] Table 8: Results of the experiment for exploring the concentration of the anti-photoaging efficacy of the sample (n = 30)

[0140]

[0141] Under the conditions of this experiment, the maximum test concentration (MTC) of the anti-photoaging efficacy of SGS-07 (Component 1 + Component 2 + Component 3) and Component 1 is both 81.4 μg / mL.

[0142] 5.2. Evaluation of anti-photoaging efficacy

[0143] Under the conditions of this experiment, SGS-07 (Component 1 + Component 2 + Component 3) has anti-photoaging efficacy, specifically manifested as reducing the fluorescence intensity of apoptotic cells in the tail; the anti-photoaging efficacy of Component 1 is not significant. The anti-photoaging efficacy of the sample is shown in Table 9 Figure 7 (where, 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 .

[0144] Table 9: Experimental results of the evaluation of the anti-photoaging efficacy of the samples (n = 10)

[0145]

[0146] Compared with the model control group, *p < 0.05, **p < 0.01, ***p < 0.001.

[0147] According to the experimental results in the above table, it can be seen that the SGS-07 (Component 1 + Component 2 + Component 3) sample has good anti-photoaging efficacy at a concentration of 5.09 - 81.4 μg / mL, significantly better than the single-component Component 1. At a concentration of 20.4 - 81.4 μg / mL, the effect is better than the positive control drug tea polyphenols.

[0148] Experimental Example 4: Effects of Samples SGS-08 and SGS-09 on the Cell Damage Recovery Rate

[0149] 1. Test materials and reagents

[0150] HT-22 mouse hippocampal neuron cells, DMEM medium, trypsin, fetal bovine serum, antibiotics, DPBS buffer, cell culture plates, CCK-8 kit.

[0151] 2. Main equipment

[0152] Clean bench, carbon dioxide incubator, analytical balance, pipette, multi-functional microplate reader.

[0153] 3. Aβ drug treatment

[0154] Information of the modeling agent: β-amyloid 1-42 (Aβ1-42), sourced from PONY Biopharmaceuticals, CAS: 166090-74-0, Aβ1-42 is a polypeptide composed of 42 amino acids, which is toxic to hippocampal slices and is used in the research of Alzheimer's disease. Physical properties: powder, storage conditions: -20°C, preparation method: This product is unstable in solution state, it is recommended to prepare and use it immediately.

[0155] 4. Specific experimental method:

[0156] ① Take 1 mg of bottled β-amyloid 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. Subsequently, add 950 μL of serum-free medium to the solution to obtain a 250 μM working solution.

[0157] ② Immediately before use, transfer 1 mL of the 250 μM Aβ working solution to a 50 mL centrifuge tube, add 19 mL of complete medium containing 10% FBS and 1% P / S to prepare a 12.5 μM Aβ solution, and preheat it in an incubator at 37°C.

[0158] ③ Replace the medium completely. Add the medium containing 12.5 μM Aβ to a 96-well plate, treat with the drug for 24 hours, then discard the medium (on the second day), add the medium containing the test drug, and treat for 24 hours. On the third day, detect the cell viability using CCK-8.

[0159] 5. Preparation of the test drugs SGS-08 and SGS-09

[0160] 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 biloba extract, and 0.5 part of Huperzia serrata extract. Specifically:

[0161] Component 1: An appropriate amount of ginseng extract is dissolved in DMSO to prepare a yellowish-brown solution with a concentration of 180 mg / mL;

[0162] Component 2: An appropriate amount of ginkgo biloba extract is dissolved in DMSO to prepare a brown solution with a concentration of 225 mg / mL;

[0163] Component 3: An appropriate amount of Huperzia serrata extract is dissolved in DMSO to prepare a transparent solution with a concentration of 1.5 mg / mL;

[0164] Take 0.1 mL each of Component 1, Component 2, and Component 3, add 0.2 mL of DMSO and 4.5 mL of water to obtain SGS-08.

[0165] 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 biloba extract, and 0.1 part of Huperzia serrata extract. Specifically:

[0166] Component 1: An appropriate amount of ginseng extract is dissolved in DMSO to prepare a yellowish-brown solution with a concentration of 225 mg / mL;

[0167] Component 2: An appropriate amount of ginkgo biloba extract is dissolved in DMSO to prepare a brown solution with a concentration of 165 mg / mL;

[0168] Component 3: An appropriate amount of Huperzia serrata extract is dissolved in DMSO to prepare a transparent solution with a concentration of 0.3 mg / mL;

[0169] Take 0.1 mL each of Component 1, Component 2, and Component 3, add 0.2 mL of DMSO and 4.5 mL of water to obtain SGS-09.

[0170] Dilute SGS-08 and SGS-09 to the test concentration with the culture medium.

[0171] 6. Experimental Procedures

[0172] Resuscitate the cells and culture them in DMEM high-glucose medium containing 10% fetal bovine serum and antibiotics under the culture conditions of 37 °C and 5% CO2. When the cells grow to 80% confluence, digest them with trypsin and inoculate them into a 96-well plate.

[0173] Add the culture medium containing 12.5 µM Aβ to the 96-well plate and treat the cells with the drug for 24 hours; then discard the culture medium containing the modeling agent, add the test drug, and test the neuroprotective effect of the test drug at different concentrations in restoring cell activity. The concentrations are 1%, 0.5%, 0.25%, 0.125%, 0.062%, and 0.031%, a total of 6 concentrations.

[0174] Set up a blank control group and a model group separately; in the blank control group, do not use the modeling agent Aβ, add the same DMSO solvent as the highest drug dose (1%) as the solvent control; in the model group, use the modeling agent Aβ to damage the cells and do not give drug treatment.

[0175] Measure the cell activity (CCK-8 method) and calculate the relative cell activity according to the following formula:

[0176] Cell survival rate % = absorbance value of the test group / absorbance value of the blank control group × 100%;

[0177] Cell recovery rate % = (absorbance value of the test group – absorbance value of the model group) / (absorbance value of the blank control – absorbance value of the model group) × 100%.

[0178] Subtract the absorbance value of the blank well of the experimental platform (96-well plate) from the absorbance values of each group to reduce the systematic error.

[0179] The experimental data were statistically analyzed using Graphpad Prism 10.1.2. The specific results of the cell recovery rate are shown in the following table.

[0180] Table 10: Effects of different concentrations of SGS-08 and SGS-09 on the recovery rate of damaged cells

[0181]

[0182] According to the data in the table, it can be seen that SGS-08 and SGS-09 have a good recovery effect on the cell damage model.

[0183] Experimental Example 5: Anti-photoaging Efficacy Evaluation Experiment of Samples SGS-10 and SGS-11

[0184] 1. Sample preparation information:

[0185] 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 biloba extract, and 0.5 part of Huperzia serrata extract. Specifically:

[0186] Component 1: An appropriate amount of ginseng extract, dissolved in DMSO to prepare a yellow-brown solution with a concentration of 180 mg / mL;

[0187] Component 2: An appropriate amount of ginkgo biloba extract, dissolved in DMSO to prepare a brown solution with a concentration of 225 mg / mL;

[0188] Component 3: An appropriate amount of Huperzia serrata extract, dissolved in DMSO to prepare a transparent solution with a concentration of 1.5 mg / mL;

[0189] Take 1 mL of each of Component 1, Component 2, and Component 3, mix well. After mixing, the concentration of the mother liquor is 135.5 mg / mL to obtain SGS-10 for standby.

[0190] 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 biloba extract, and 0.1 part of Huperzia serrata extract. Specifically:

[0191] Component 1: An appropriate amount of ginseng extract, dissolved in DMSO to prepare a yellow-brown solution with a concentration of 225 mg / mL;

[0192] Component 2: An appropriate amount of ginkgo biloba extract, dissolved in DMSO to prepare a brown solution with a concentration of 165 mg / mL;

[0193] Component 3: An appropriate amount of Huperzia serrata extract, dissolved in DMSO to prepare a transparent solution with a concentration of 0.3 mg / mL;

[0194] Take 1 mL of each of Component 1, Component 2, and Component 3, mix well. After mixing, the concentration of the mother liquor is 130.1 mg / mL to obtain SGS-11 for standby.

[0195] 2. Experimental animals: Zebrafish were all reared in fish culture water at 28 °C (Water quality: 200 mg of instant sea salt was added to every 1 L of reverse osmosis water, with a conductivity of 450 - 550 μS / cm; pH of 6.5 - 8.5; hardness of 50 - 100 mg / L CaCO3). They were provided by the fish culture center of our company. The license number for the use of experimental animals is: SYXK (Zhe) 2022 - 0004. The feeding management met the requirements of international AAALAC accreditation (Accreditation number: 001458), and the IACUC ethical review number is: IACUC - 2024 - 9517 - 01.

[0196] 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 Beilanbo Biotechnology Co., Ltd., China); Simulated sunlight (SOL500, Hoenle, Germany); Electric focusing continuously variable fluorescence microscope (AZ100, Nikon, Japan). Methyl cellulose (Lot number C2004046, Shanghai Aladdin Biochemical Technology Co., Ltd., China); Acridine orange (AO, Lot number C12894919, Shanghai Macklin Biochemical Co., Ltd., China); Dimethyl sulfoxide (DMSO, Lot number BCCD8942, Sigma, Switzerland).

[0197] 4. Evaluation of anti - photoaging efficacy

[0198] Wild - type AB strains at 3 dpf were randomly selected and placed in 6 - well plates, with 30 zebrafish treated in each well. Different concentrations of the sample diluted with deionized water were given, and the concentrations are shown in Table 11. The positive control was tea polyphenol at a concentration of 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 °C for 3 h, except for the normal control group, the other experimental groups were irradiated with ultraviolet light to establish a zebrafish photo - damage model. One day after treatment at 28 °C, 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 photographing. The 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 zebrafish tails was analyzed. The anti - photoaging efficacy of the sample was evaluated based on the statistical analysis results of this index. The calculation of anti - photoaging efficacy is as follows:

[0199] Anti - photoaging efficacy (%)

[0200] The results of statistical analysis were expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software. A p < 0.05 indicated a statistically significant difference. The specific results are shown in the following table.

[0201] Table 11: Experimental results of the anti-photoaging efficacy evaluation of the samples (n = 10)

[0202]

[0203] Compared with the model control group, *P < 0.05, **P < 0.01, ***P < 0.001 in the dosing group.

[0204] 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 efficacy. In particular, the effects of the 40 µg / mL and 80 µg / mL dose groups of SGS-10 and the 20 µg / mL, 40 µg / mL, and 80 µg / mL dose groups of SGS-11 are better than those of tea polyphenols.

[0205] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than limiting the protection scope of the present invention. Any simple modification or equivalent replacement of the technical solution of the present invention by those of ordinary skill in the art shall not depart 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: 60-75 parts of ginseng extract, 55-75 parts of ginkgo leaf extract, and 0.1-0.5 parts of melaleuca extract.

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

3. A preparation containing the composition according to any one of claims 1 to 2, characterized in that: The preparation also includes pharmaceutically acceptable excipients.

4. The preparation according to claim 3, 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.

5. The preparation according to claim 3, characterized in that The dosage form of the preparation is drop pills, tablets, capsules, granules, mixtures or syrups.

6. The preparation according to claim 3, characterized in that The auxiliary material is polyethylene glycol, and the dosage form of the preparation is a drop pill.

7. The preparation according to claim 6, 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.

8. Use of the composition according to any one of claims 1 to 2 or the preparation according to any one of claims 3 to 7 in the preparation of products for preventing and treating Alzheimer's disease and / or preventing and treating skin photoaging.

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