Application of Janthinedine A in the preparation of anti-aging and cardiovascular protective drugs

Abstract

The present invention belongs to the field of biomedicine technology, and specifically provides the use of Janthinedine A in the preparation of anti-aging and cardiovascular protection drugs. The present invention uses Janthinedine A to prepare drugs for delaying, preventing and / or treating aging and cardiovascular diseases, providing a new drug option for anti-aging and cardiovascular protection, and has important practical application value in the fields of medicine, anti-aging and cardiovascular protection.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to the application of Janthinedine A in the preparation of anti-aging and cardiovascular protection drugs. Background Art

[0002] In recent years, with the rapid development of economy and society, people's living conditions have been continuously improved, the level of medical and health care has been continuously improved, the life expectancy of the population has been continuously extended, and the proportion of the middle-aged and elderly population in the population age structure has continued to rise. Population aging has become a major social problem in the world in the next few decades. The increasingly severe situation of population aging has brought huge challenges to the medical expenses and medical care of countries around the world. Therefore, exploring the biological nature of aging and finding safe and effective intervention strategies are crucial to global healthy aging.

[0003] Janthinedine A ((5-(Hydroxymethyl)thiazolo[5,4-b]pyridin-2-yl)(4-hydroxyphenyl)methanone) is a novel active alkaloid monomer component discovered from Penicillium janthinellum MPT25. Studying its activity and application is of great significance. Summary of the invention

[0004] The invention discloses application of Janthinedine A in preparing anti-aging and / or cardiovascular protection drugs. Janthinedine A is an active alkaloid compound with a novel structure and can be applied to preparing anti-aging and / or cardiovascular protection drugs, or to preparing anti-aging skin care products.

[0005] The present invention adopts the following technical solution:

[0006] Use of Janthinedine A in the preparation of anti-aging drugs; and / or

[0007] Application of Janthinedine A in the preparation of cardiovascular protection drugs;

[0008] The structural formula of Janthinedine A is as follows:

[0009]

[0010] Furthermore, the Janthinedine A can also be used to prepare anti-aging skin care products.

[0011] Furthermore, the dosage of Janthinedine A used in the preparation of anti-aging drugs is 11.0 mg / kg / d (the dosage is calculated based on the human 60kg standard corresponding to the high dose of Janthinedine A 100 mg / kg mouse dosage).

[0012] Furthermore, the dosage of Janthinedine A used in the preparation of cardiovascular protection drugs is 11.0 mg / kg / d (conversion of the mouse dosage of high-dose JA and human dosage).

[0013] Furthermore, the concentration of Janthinedine A when used in the preparation of anti-aging skin care products is 2 to 20 μM.

[0014] Furthermore, the dosage forms of the medicine include tablets, capsules, and oral liquids, and the skin care products include skin care lotions, facial masks, and skin care creams.

[0015] Furthermore, the Janthinedine A is used as a raw material for a preparation for reducing the activity of β-galactosidase in senescent cells.

[0016] Furthermore, the Janthinedine A is used as a raw material for a preparation for restoring the proliferation ability of senescent cells.

[0017] Furthermore, the Janthinedine A is used as a raw material for a preparation for restoring the migration ability of senescent cells.

[0018] Furthermore, the Janthinedine A is used as a raw material for a preparation for improving sports endurance.

[0019] Furthermore, the Janthinedine A is used as a raw material for a preparation for improving blood circulation in lower limbs.

[0020] Furthermore, the Janthinedine A is used as a raw material for a blood pressure lowering preparation.

[0021] Furthermore, the Janthinedine A is used to inhibit TNF-α, IL-1β, IL-8, IL-18 and / or MMP-2 inflammatory factors produced by cell aging.

[0022] Furthermore, the Janthinedine A is used to inhibit the expression of p21 and p53 proteins.

[0023] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0024] The present invention provides a new use of an alkaloid compound, Janthinedine A: Janthinedine A is used to prepare drugs for anti-aging and cardiovascular protection. Experimental results show that Janthinedine A can significantly inhibit the production of inflammatory factors such as TNF-α, IL-1β, IL-8, IL-18, and MMP-2 produced by D-gal-induced senescence of human umbilical vein endothelial cells (HUVEC), and can reduce the expression of β-galactosidase, p21, and p53 in D-gal-HUVEC senescent cells. Therefore, its anti-aging and cardiovascular protection effects are worthy of further study.

[0025] The present invention not only provides more optional drugs for preventing and treating aging and vascular-related diseases, but also contributes to human exploration of the health field and has great significance for the development of the global health industry. It helps to explore more effective drugs for delaying aging and protecting cardiovascular diseases, so as to better cope with the challenges of the aging of the global population. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Schematic diagram of the cell survival rate of D-gal-induced senescent HUVEC cells after incubation with Janthinedine A in the anti-aging application of Janthinedine A in the present invention (compared with the model group at each effective concentration: P < 0.05 * , P<0.01 ** );

[0027] Figure 2 Schematic diagram of the changes in cell proliferation ability (A), cell migration ability (B), in vitro angiogenesis (C), cell scratch area (D), and cell invasion number (E) of D-gal-induced senescent HUVEC cells after incubation with Janthinedine A in the anti-aging application of the present invention (each group compared with the model group: P < 0.05 * , P<0.01 ** );

[0028] Figure 3 It is a schematic diagram of the expression of β-galactosidase activity staining of D-gal-induced senescent HUVEC cells after Janthinedine A administration and incubation in the application of Janthinedine A in the anti-aging of the present invention;

[0029] Figure 4Schematic diagram of the changes of inflammatory factors such as TNF-α, IL-1β, IL-8, IL-18, and MMP-2 in senescent HUVEC cells induced by D-gal in the anti-aging application of Janthinedine A of the present invention (compared with the model group at each dose: P < 0.05 * , P<0.01 ** );

[0030] Figure 5 It is a schematic diagram of the changes in the survival weight of D-gal-induced aged mice after oral administration of high and low doses of Janthinedine A in the application of Janthinedine A in the anti-aging of the present invention;

[0031] Figure 6 It is a schematic diagram of the blood pressure changes of D-gal-induced aged mice after oral administration of high and low doses of Janthinedine A in the application of Janthinedine A in the anti-aging of the present invention;

[0032] Figure 7 Schematic diagram of the changes in exercise endurance of D-gal-induced aged mice after oral administration of high and low doses of Janthinedine A in the anti-aging application of the present invention (compared with the Model group at each dose: P < 0.05 a , P<0.01 b , each dose compared with Nature aging group: P<0.05 c , P<0.01 d );

[0033] Figure 8 It is a schematic diagram of ultrasound imaging of the changes in blood flow in the lower limbs of D-gal-induced aged mice after high and low doses of Janthinedine A were intragastrically administered in the application of Janthinedine A in the present invention in anti-aging;

[0034] Fig. 9 , 10 11 is a schematic diagram of the changes in the aortic endothelial function of D-gal-induced aged mice after high and low doses of Janthinedine A were intragastrically administered in the application of Janthinedine A in the present invention in anti-aging;

[0035] Fig.12 Schematic diagram of the electron microscope photograph of the elastic structure of the aorta of D-gal-induced aged mice after high and low doses of Janthinedine A were intragastrically administered in the application of Janthinedine A in the present invention in anti-aging;

[0036] Fig.13 It is a schematic diagram of HE staining of the liver morphology of D-gal-induced aged mice after high and low doses of Janthinedine A were intragastrically administered in the application of Janthinedine A in the present invention in anti-aging;

[0037] Fig.14 The figure is a schematic diagram of the expression of p21 and p53 proteins in D-gal-induced aged mice after high and low doses of Janthinedine A were intragastrically administered in the application of Janthinedine A in the anti-aging process of the present invention. DETAILED DESCRIPTION

[0038] In order to better understand the technical solution of the present invention, the embodiments of the present invention are further described in detail and completely in conjunction with the accompanying drawings. It should be clear that the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0039] Janthinedine A used in the following examples was prepared according to the following literature: Wen-Jing Wang, Liang-Xiu Liao, et al, Thiazolo[5,4-b]pyridine Alkaloid and Seven ar-BisabolSesquiterpenes Produced by the Endophytic Fungus Penicillium janthinellum[J], ACS Omega 2022, 7:35280-35287, and its structural formula is:

[0040]

[0041] Example 1 In vitro pharmacodynamics experiment of Janthinedine A in protecting cardiovascular system

[0042] 1 Materials

[0043] 1.1 Model organisms

[0044] Human umbilical vein endothelial cells (HUVEC) were purchased from Wuhan Huaerna Biotechnology Co., Ltd. (Cat. No.: WN-10112).

[0045] 1.2 Drugs and reagents

[0046] β-Nicotinamide mononucleotide was purchased from Shanghai Titan Technology Co., Ltd.; Cell senescence β-galactosidase staining kit was purchased from Shanghai Biotech Biotechnology Co., Ltd.; CCK8 kit was purchased from Wuhan Abotek Biotechnology Co., Ltd.; D-galactose was purchased from Sinopharm Chemical Reagent Co., Ltd.; ECM complete medium was purchased from Wuhan Warner Biotechnology Co., Ltd. (Cat. No. 1001), and other inorganic reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. The ECM complete medium for HUVEC cells was prepared from 500 mL of basal medium, 25 mL of fetal bovine serum, 5 mL of endothelial cell growth supplement (ECGS) and 5 mL of penicillin / streptomycin solution (P / S).

[0047] 1.3 Instruments

[0048] Carbon dioxide constant temperature incubator (Shanghai Kelifu Instrument Co., Ltd.), Multiskan FC multifunctional microplate reader (Thermo Fisher Shanghai Instrument Co., Ltd.), inverted fluorescence microscope (Guangzhou Haokang Biotechnology Co., Ltd., model HIF2000), small high-speed centrifuge (Denmark LaboGene Company), FN-6 constant temperature water bath (Beijing Liuyi Biological Co., Ltd.), SPX-150B-Z biochemical incubator (Beijing Liuyi Biological Co., Ltd.).

[0049] 2 Methods

[0050] 2.1 Experimental Grouping

[0051] It was divided into a model group (Model, 40 mg / mL D-galactose D-gal), a blank control group (Control), a positive drug group (Positive drugs, 300 μM β-nicotinamide mononucleotide, NMN) and 7 Janthinedine A treatment groups. The concentrations of Janthinedine A in the ECM complete culture medium of the Janthinedine A treatment groups were 75.0 μM, 50.0 μM, 25.0 μM, 20.0 μM, 10.0 μM, 5.0 μM and 1.0 μM, respectively.

[0052] 2.2 HUVEC cell culture

[0053] (1) Take out the HUVEC cell cryopreservation tube from the liquid nitrogen tank, thaw it quickly in a 37°C water bath, wipe the surface of the cryopreservation tube dry and disinfect it with 75% alcohol, then quickly take it back to the clean bench. Prepare a T25 cell culture flask and mark it, add 8 mL of preheated ECM complete medium (preheated in a 37°C constant temperature water bath). Transfer the cell suspension in the cell cryopreservation tube to the above-mentioned T25 culture flask, spread the cells evenly, and place them in carbon dioxide (5% carbon dioxide conditions in the present invention) and a 37°C constant temperature incubator for culture.

[0054] (2) When the cells grow to a density of 95%, the cells are subcultured. After confirming the cell growth status, the cell density is roughly calculated. ECM complete medium is added to dilute the cell solution as needed, and the cells are inoculated into 96-well plates for culture. 100 μL of cell solution is added to each well (each well contains no less than 10 cells). 4 At the same time, a layer of PBS (phosphate buffered saline to prevent evaporation error, the PBS used in the present invention is 1×PBS buffer of Biosharp / White Shark, item number: BL302A) is placed around the 96-well plate, the volume of which is the same as that of the cell solution. After the cells are inoculated, they are placed in a 37°C constant temperature carbon dioxide incubator for 24 hours.

[0055] 2.3D-gal construction of aging model

[0056] Preparation of D-gal: Weigh an appropriate amount of D-gal, dissolve it in DMEM high-glucose liquid culture medium (containing sodium pyruvate, without HEPES, purchased from Biosharp, product number: BL304A), prepare a mother solution with a concentration of 40.0 mg / mL, filter it with a disposable sterile filter on a clean bench, and store it in a refrigerator at 4°C for later use after aliquoting.

[0057] D-gal aging model: The prepared D-gal was preheated in a 37°C constant temperature water bath, and the original culture medium in the 96-well plate was discarded. Except for the blank control group, the preheated D-gal was evenly added to each well using a spray gun at a volume of 100 μL, and then placed in a 37°C, carbon dioxide constant temperature cell culture incubator for 4 hours. The cells in the blank control group were cultured with an equal amount of ECM complete culture medium.

[0058] 2.4 Effect of Janthinedine A on the survival rate of D-gal-induced senescent HUVEC cells

[0059] The experiment was divided into a model group (Model, 40 mg / mL D-gal), a blank control group (Control), a positive drug group (Positive drugs, 300 μM NMN) and 7 drug administration groups. Janthinedine A was prepared using ECM complete medium as a solvent at concentrations of 75.0 μM, 50.0 μM, 25.0 μM, 20.0 μM, 10.0 μM, 5.0 μM, and 1.0 μM, and 300 μM NMN was prepared as a positive drug for standby use.

[0060] The D-gal-containing culture medium used to establish the aging model was discarded, and 5 replicate wells were set up for each drug group. 100 μL of the drug of the corresponding drug concentration was added to each group. The blank control group was still given ECM complete culture medium and placed in a carbon dioxide constant temperature cell culture incubator for 12 hours before being taken out for observation (the cell culture incubator in the present invention was kept at a constant temperature of 37°C). The cell viability of each group was detected by CCK-8 method. The survival rate of HUVEC cells was analyzed using GraphPad Prism 10 software, and the survival curve was drawn, as shown in FIG. Figure 1 As shown, the results showed that Janthinedine A (ie, JA) at a concentration of 20.0 μM had the best effect, with a significant difference in cell survival rate compared to the model group, and better anti-aging effect than the positive drug, that is, the anti-aging effect was obvious.

[0061] Therefore, the HUVEC cells cultured at the above-mentioned 20.0 μM dosage concentration were used to perform cell scratch assays, Transwell cell migration assays, cell invasion assays, in vitro angiogenesis assays, etc., to compare and evaluate the cell proliferation and cell migration ability of HUVEC cells before and after Janthinedine A administration. The above experiments were all performed according to conventional means in the art.

[0062] The morphology of HUVEC cells in the above different experiments was photographed using an inverted fluorescence microscope to observe the changes in the corresponding cell morphology before and after JA administration, such as Figure 2 As shown, the results showed that after the administration of Janthinedine A, the proliferation and migration abilities of HUVEC cells had a significant positive effect compared with the cells in the model group, and the effect was stronger than that in the positive drug group.

[0063] At the same time, the HUVEC cells cultured at the above-mentioned 20.0 μM dosing concentration were used to carry out the cell senescence-related β-galactosidase (SA-β-Gal) staining activity experiment. The cell senescence β-galactosidase staining kit was used to detect the SA-β-Gal activity level in the target cells with X-Gal as the substrate. The target cells were inoculated into 6-well cell culture plates, and the cell density per well was not less than 8×10 5 / well, add no less than 3mL of ECM complete medium to each well, place it in a 37℃ constant temperature incubator, and conduct the experiment when the degree of fusion is about 60%. During the experiment, take the kit out of the refrigerated and frozen combination low-temperature preservation box in advance, balance it to room temperature (25℃, the same for others), and open the water bath in advance, preheat it to 37℃ before starting the experiment. Use a Pasteur pipette to remove the cell culture fluid, wash the cells twice with PBS, add 1mL of β-galactosidase staining fixative, and fix it at room temperature for 15min. After the fixation is completed, discard the fixative to obtain the staining working solution, add 1mL of staining working solution to each well, use parafilm sealing film to seal the edge of the 6-well cell culture plate, place it in a water bath, and incubate it at 37℃ overnight (12h). After the incubation is completed, discard the staining solution, add 1mL of PBS, and observe and take pictures using an ordinary optical microscope.

[0064] Since senescent cells have highly active β-galactosidase, in situ staining was performed with X-Gal as the substrate. Under the catalysis of β-galactosidase, a dark blue product was generated, which can be observed under an optical microscope. The results are as follows: Figure 3 As shown, after the D-gal aging model was established, the dark blue staining product of β-galactosidase was obvious, indicating that the aging model was successfully established; after the positive drugs NMN and Janthinedine A were administered, the dark blue staining product was significantly reduced, and the effect of the JA administration group was better than that of the positive drug NMN, indicating that Janthinedine A has good anti-aging activity.

[0065] 2.5 Effects of Janthinedine A on the changes of inflammatory factors such as TNF-α, IL-1β, IL-8, IL-18, and MMP-2 in D-gal-induced senescent HUVEC cells

[0066] Referring to the HUVEC cell culture described in step (1) of 2.2 above, when the HUVEC cells are in good growth condition (judged by common sense of personnel in this field), the cells are evenly seeded into a 6-well plate, and the number of cells per well in the 6-well plate is ensured to be no less than 10 8 After the cells were inoculated, they were placed in a 37°C constant temperature carbon dioxide incubator and cultured for 24 h.

[0067] Based on the above 2.4 Figure 1The experimental results of Janthinedine A on the survival rate of D-gal-induced senescent HUVEC cells are shown in Figure 2. Janthinedine A has a significant effect on HUVEC cells at 20.0μM. Therefore, in addition to the blank control group (Control), 40mg / mL D-gal was used to establish a cell senescence model in this experiment. The experimental groups were model group (D-gal), blank control group (Control), positive drug action group (D-gal+NMN, 40mg / mL D-gal+300μM NMN) and Janthinedine A administration group (D-gal+Janthinedine A, 40mg / mL D-gal+20.0μM JanthinedineA). ECM complete culture medium was used as the solvent for modeling and drug preparation. The concentration of Janthinedine A was prepared to be 20.0μM. At the same time, 300μM NMN was prepared as a positive drug for standby use. The specific construction methods of each group were the same as those in 2.3 and 2.4 above.

[0068] ELISA kits for inflammatory factors such as TNF-α, IL-1β, IL-8, IL-18, and MMP-2 were purchased from Wuhan Abotek Biotechnology Co., Ltd., with the catalog numbers RK00030, RK00001, RK00011, RK00176, and RK00309. The specific experimental steps are as follows:

[0069] 2.5.1 Reagent preparation

[0070] ① Before use, equilibrate the reagents to room temperature. If crystals are precipitated in the concentrate, place the reagents at room temperature and shake gently until the crystals are completely dissolved.

[0071] ② Preparation of standard: Add 1.0 mL of standard / sample diluent (R1) to the lyophilized standard and mix gently for at least 15 minutes to completely dissolve the lyophilized product to a concentration of 1000 pg / mL. Prepare EP tubes containing R1 diluent and perform gradient dilutions according to the prompts (the standard curve in this example uses the following concentrations: 1000, 500, 250, 125, 62.5, 31.2, 15.6, 0 pg / mL).

[0072] ③ Biotinylated antibody (100x) working solution: Dilute the concentrated biotinylated antibody at a ratio of 1:100 with biotinylated antibody diluent (R2) before use. Note: The diluted working solution should be used within 30 minutes.

[0073] ④ Streptavidin-HRP (100x) working solution: Dilute the concentrated streptavidin-HRP with streptavidin-HRP diluent (R3) at a ratio of 1:100 before use. Note: The diluted working solution should be used within 30 minutes.

[0074] ⑤ Preparation of 1x washing buffer: Dilute 20x concentrated washing buffer at a ratio of 1:20 with double distilled water or deionized water before use.

[0075] 2.5.2 HUVEC cell sample preparation

[0076] Preparation of cell culture supernatant: Centrifuge at 1000xg for 10 minutes and test in time; store the sample in aliquots at -80℃ (store at 2-8℃ for testing within 24 hours) to avoid repeated freezing and thawing. If the dilution of the cell culture supernatant sample is relatively large, use culture medium for intermediate dilution first, and finally dilute with standard / sample diluent (R1).

[0077] 2.5.3 Double antibody sandwich ELISA method to detect the expression of inflammatory factors

[0078] ① Remove the microwell strips to be used from the plate frame in the kit for washing, put the remaining strips back into the aluminum foil bag containing desiccant, and then reseal for storage. The washing method is: add 350μL of 1x washing buffer to each well, let it stand for 40s and then discard the liquid. This step is washed 3 times in total. Add 100μL of standard / sample diluent (R1) to the blank well. Add 100μL of standards or samples of different concentrations to the other wells, seal the wells with the provided sealing film, and incubate at 37℃ for 2h.

[0079] ② Prepare the biotinylated antibody (100x) working solution 15 minutes before use. Discard the liquid in the wells and wash according to the washing method in ① above. Add the biotinylated antibody working solution (100μL / well) to each well, cover with a new sealing film, and incubate at 37℃ for 1h. Prepare the streptavidin-HRP (100x) working solution 15 minutes before use. Discard the liquid in the wells and wash according to the washing method in ① above. Add the streptavidin-HRP working solution (100μL / well) to each well, cover with a new sealing film, and incubate at 37℃ for 30 minutes.

[0080] ③After completing the above steps, preheat the microplate reader. Discard the liquid in the wells, wash according to the washing method in ① above, and add TMB substrate (100μL / well) to the wells. Incubate at 37℃ in the dark for 15-20min. Finally, add the stop solution (50μL / well) and immediately put it into the microplate reader to measure the OD value of each well at 450nm within 5min.

[0081] ④ Select the correction wavelength, set it to 570nm, and subtract the 570nm reading from the 450nm reading to correct and remove the OD value of non-coloring substances, so as to obtain more accurate detection results and calculate the results.

[0082] The schematic diagram of changes in inflammatory factors such as TNF-α, IL-1β, IL-8, IL-18, and MMP-2 is shown in Figure 4 With the aging process, the expression of inflammatory factors such as TNF-α, IL-1β, IL-8, IL-18, and MMP-2 increases. In this experiment, D-gal was used to establish an aging model. Compared with the Control group, the expression of inflammatory factors such as TNF-α, IL-1β, IL-8, IL-18, and MMP-2 in the D-gal group was significantly increased, indicating that the D-gal aging model was successfully established in this experiment; after JA administration, the expression of inflammatory factors such as TNF-α, IL-1β, IL-8, IL-18, and MMP-2 was reduced compared with the D-gal group, indicating that JA administration can effectively reduce inflammatory factors such as TNF-α, IL-1β, IL-8, IL-18, and MMP-2 in the body, which can indicate that JA has the effect of delaying the inflammatory response in the body caused by aging.

[0083] Example 2 In vivo pharmacodynamics experiment of Janthinedine A in delaying aging and protecting cardiovascular system

[0084] 1 Materials

[0085] 1.1 Model organisms

[0086] C57BL / 6J mice were purchased from Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd., license number: SCXK(Su)2020-0009. Experimental Animal License of South-Central University for Nationalities, number: SYXK(E)2021-0089.

[0087] 1.2 Drugs and reagents

[0088] β-Nicotinamide mononucleotide (NMN) was purchased from Shanghai Titan Technology Co., Ltd., D-galactose (D-gal) was purchased from Sinopharm Chemical Reagent Co., Ltd., and normal saline was purchased from Shanghai Haohong Biotechnology Co., Ltd.

[0089] 1.3 Instruments

[0090] Multi-channel non-invasive blood pressure measuring instrument for mice and rats (Nanjing Calvin Biotechnology Co., Ltd., model: Medlab-3DX), rotarod fatigue apparatus for mice (Beijing Zhishu Duobao Biotechnology Co., Ltd., model: DB09-3X), ultra-high resolution small animal ultrasound imaging system (Visualsonics, USA, model: vevo2100), whole slice imaging system WSI (3DHISTECH Co., Ltd., Hungary), etc.

[0091] 2 Methods

[0092] 2.1 Animal grouping and dosing

[0093] In this experiment, 30 7-week-old C57BL / 6J mice were weighed and randomly divided into 5 groups: blank group (Control, normal saline), model group (Model, D-gal, 300 mg / kg), positive drug group (Positive Drug, D-gal+NMN, 300 mg / kg+300 mg / kg), Janthinedine A high-dose group

[0094] (D-gal+JA high dose, 300mg / kg+100mg / kg), Janthinedine A low dose group (D-gal+JA low dose, 300mg / kg+50mg / kg), except for the normal saline group, the drugs in other groups were dissolved in normal saline and prepared to the corresponding concentrations.

[0095] Six 16-month-old C57BL / 6J mice were used as the natural aging group (Natural Aging, saline). Six groups of C57BL / 6J mice were given drugs by gavage and raised in the same SPF-grade breeding room for 8 weeks (the drugs were given every other day after the first administration, and continued until the end of the animal experiment). In the 7th week, six 6-week-old C57BL / 6J mice were purchased as the young group (Young, saline) and raised in the same SPF-grade breeding room for one week with other groups of mice.

[0096] The in vitro animal experiment was divided into 7 groups. The weight of mice was weighed and recorded at the same time every day (the average value was used for each group). Figure 5 , i.e., the weight change graph of mice, shows that the volume of mice increased steadily throughout the experiment, and the drug administration had no significant effect on the normal growth state of mice; the microvascular density of the hind limbs of mice was measured 3 days before the autopsy, as shown in Figure 8 After dissection, the thoracic aorta and serum were obtained and placed at -80℃ for later use.

[0097] 2.2 Blood pressure measurement with multi-channel non-invasive blood pressure meter for mice and rats

[0098] The blood pressure of mice was measured using a multi-channel non-invasive blood pressure meter for mice and rats. The incubator was first set to 37°C. After preheating for 30 minutes, the mice were placed in a fixed tube with their backs facing up and their tails passed through a rubber band. The blood pressure of mice was measured after the mice adapted. This was repeated 3 times, and the average value was taken as the blood pressure value of the mice. Blood pressure was measured once every 6 days during the administration period.

[0099] The results are as follows Figure 6 As shown, oral administration of Janthinedine A to D-gal-induced aging model mice can significantly reduce the increase in blood pressure associated with aging.

[0100] 2.3 Testing exercise endurance with the mouse rotarod fatigue apparatus

[0101] In order to accurately evaluate the differences in the motor abilities of mice in the drug-treated group, natural aging group, juvenile group, positive drug group, and model group, the mice were subjected to a rotating rod fatigue test. The motor coordination and balance abilities of the mice were judged based on the time the mice moved on the rotating rod until they fell and the distance recorded by the rotating rod fatigue tester. A rotating rod with a diameter of 30 mm and a 6-channel mouse rotating rod fatigue tester were selected for the mouse hind limb exercise endurance test experiment. The ventilation, noise intensity, temperature, humidity, and lighting intensity of the environment were strictly kept consistent for each mouse during the experiment. Two days before the start of the experiment, the mice were first trained with a rotating rod, and the rotating rod speed that most mice could complete was monitored and recorded, and the rotating rod speed was determined as the final rotation speed. On the day of the experiment, the experiment was carried out in sequence according to the mouse group and number. The mice were placed on a rotating rod rotating at a uniform speed, and the movement time of each mouse when it fell was recorded. Each mouse was required to complete 3 rotating rod fatigue tests, with an interval of 1 hour between each experiment.

[0102] During the administration, the mice were continuously monitored and their exercise endurance was recorded every 8 days. The average exercise distance and exercise time were then calculated. The results are shown in Figure 7 As shown, both A and B graphs indicate the exercise endurance of mice, the ordinate of A is the exercise distance, and B is the exercise duration. The greater the exercise distance and the longer the duration, the better the exercise endurance of the hind limbs of the mice. After Janthinedine A was administered intragastricly to establish an aging model with D-gal, the exercise endurance of the mice was significantly improved compared with the model group and the natural aging group, and the phenomenon of decreased hind limb exercise ability of mice accompanied by aging was effectively reduced.

[0103] 2.4 2100 High-resolution Small Animal Ultrasound Imaging System for Detecting Microvascular Density

[0104] Anesthetize the mouse with 1.5% isoflurane. After the mouse is fully anesthetized (this process takes about 2-3 minutes), gently shake the induction box to check whether the animal is fully anesthetized. If the mouse falls to its side and does not try to return to its lying position, it indicates that the animal is fully anesthetized. Switch the three-way valve to ensure that the airflow from the anesthesia machine evaporator is connected to the anesthesia mask. After the concentration is adjusted, take the mouse out of the induction box, place its head / nose in the anesthesia mask and fix it, and confirm that the mouse is fully anesthetized. Place the mouse in the tray of the imaging device to ensure the safety and comfort of the animal. The 2100 high-resolution small animal ultrasound imaging system was used for ultrasound imaging to obtain blood oxygen characterization of the blood flow and vascular density of the mouse hind limbs, and then analyzed and concluded based on the imaging. After the animal experiment was completed, the evaporator was turned off and the animal was kept breathing in pure oxygen for about 5-10 minutes to facilitate rapid awakening of the animal.

[0105] The results are as follows Figure 8 As shown in the figure, after oral administration of Janthinedine A and feeding for 8 weeks, the microvascular density of the mouse hind limbs was significantly higher than that of the model group and the natural aging group. The control showed that Janthinedine A can significantly improve the blood oxygen concentration of the mouse hind limbs, and the effects of the high and low doses of Janthinedine A were better than those of the positive drug group, further indicating that Janthinedine A can improve the microvascular density of the mouse hind limbs.

[0106] 2.5 Vascular pathology (HE staining, EVG staining, Masson staining, transmission electron microscopy) observation

[0107] HE staining: The removed mouse aorta tissue was placed in the prepared 10% formalin fixative to maintain the original morphological structure of the tissue and cells. Then, the tissue block was washed with anhydrous alcohol gradient to remove the water in the tissue block. The tissue block was then placed in a transparent agent xylene for transparency, embedded in wax, cut into 4 μm slices with a slicer, baked and waxed, dewaxed, hydrated with alcohol gradient, stained with hematoxylin for 3 minutes, washed with running water for 3 minutes, differentiated, washed with water, blued, washed with running water for 10 minutes, stained with eosin, dehydrated with alcohol gradient, sealed for observation under a microscope, and photographed.

[0108] EVG staining: The removed mouse aorta tissue was placed in the prepared 10% formalin fixative to maintain the original morphology and structure of the tissue and cells. Then, the tissue block was gradient eluted with anhydrous alcohol to remove the water in the tissue block. The tissue block was then placed in a transparent agent xylene, embedded in wax, cut into 4 μm slices by a slicer, dewaxed by baking, stained with Verhoeff stain at room temperature for 2 to 3 minutes, washed with running water, differentiated with 2% ferric chloride for 15 to 20 seconds, washed with running water, counterstained with Van Gieson for 10 seconds, dehydrated, sealed for observation under a microscope, and photographed.

[0109] Masson staining: The tissue sections were dewaxed and hydrated, and the Masson staining kit (purchased from Wuhan Boerfu Biotechnology Co., Ltd., catalog number BH0002) was used to complete the staining experiment. The sections were placed in xylene I for 5 min, xylene II for 5 min, and xylene III for 5 min. 5min, anhydrous ethanol 1min, 95v / v% ethanol 1min, 80v / v% ethanol 1min, 75v / v% ethanol 1min, rinse with pure water for 8-10s, then stain with Weigert iron hematoxylin staining solution prepared in Masson staining kit for 8min, differentiate in acidic hydrochloric acid ethanol differentiation solution for 15s, wash with water; Masson blueing solution back to blue for 5min, wash with water; then stain with Ponceau red staining solution prepared in Masson staining kit for 5min, quickly rinse with distilled water; then wash with phosphomolybdic acid solution in the kit for 1min, weak acid working solution for 1min, counterstain with aniline blue staining solution for 2min, treat with 1% glacial acetic acid for 1min to complete differentiation; finally dehydrate quickly with 95v / v% ethanol for 2-3s, dehydrate with anhydrous ethanol 3 times, 5-10s each time, transparent with xylene 3 times, 1-2min each time, seal with neutral gum, and observe under a microscope after dehydration and transparency.

[0110] Transmission electron microscopy: Take fresh thoracic aorta tissue (section no more than 3mm 2 ), quickly washed with PBS, placed in electron microscope fixative at room temperature, and transferred to 4°C for storage after 2 hours. The fixed samples were rinsed with 0.1M, pH7.4 phosphate buffer for 3 times, 15 minutes each time, placed in 1% osmium acid for fixation at room temperature in the dark for 1-2 hours, rinsed again with 0.1M phosphate buffer for 3 times, dehydrated, dried in a dryer, and the samples were attached to a conductive carbon film for conductivity, observed with a scanning electron microscope, and photographed.

[0111] The above pathological sections are as follows Figure 9-12 As shown, oral administration of Janthinedine A to D-gal to establish an aging model mouse can significantly improve the growth of aortic endothelial cells and the elasticity of the aortic vascular wall of the mice, and the effects of the high and low doses of Janthinedine A were better than those of the positive drug group, further indicating that Janthinedine A can improve the functional morphology of the mouse aortic blood vessels.

[0112] 2.6 Observation of liver pathology (HE staining)

[0113] HE staining: After dissecting the mouse, the liver tissue was separated and each sample was fixed in 10% formalin solution, then dehydrated in anhydrous alcohol, and then the tissue block was placed in a transparent agent xylene for transparency, and finally embedded in paraffin and cut into 4μm thick slices with a slicer. Wax baking, dewaxing, alcohol gradient hydration, hematoxylin staining for 3min, washing with running water for 3min, differentiation, washing with water, anti-blue, washing with running water for 10min, eosin staining, alcohol gradient dehydration, sealing and waiting for observation under a microscope, taking pictures, the results are shown in Fig.13 .

[0114] The aging process is accompanied by fatty degeneration of the liver, and the liver tissue becomes loose and cavitated. Fig.13 In the experiment, the D-gal-established aging Model group and Nature aging group showed loose liver tissue and cavitation. After JA administration, it can be clearly seen that the liver tissue became tightly arranged and the cavitation phenomenon decreased, indicating that JA has a good effect on improving the cavitation phenomenon of liver tissue, and the effect of JA administration is better than that of the positive drug NMN.

[0115] 2.7 Western blot expression of p21 and p53 proteins in mouse aorta tissue

[0116] After dissecting the mouse, the thoracic aorta tissue was isolated and rinsed with pre-cooled PBS to remove residual blood and remove excess PBS; the tissue was divided into pieces of about 100 mg, wrapped in tin foil and stored in liquid nitrogen. p21 and p53 proteins in the aorta tissue were extracted, and the protein sample concentration was determined using a BCA protein assay kit. The protein samples were separated by SDS-PAGE electrophoresis, and the separated proteins were transferred from the gel to the NC membrane and stained with Ponceau red dye, and then the NC membrane was immediately placed in distilled water for rinsing. The electrotransferred NC membrane was soaked with 5% BSA TBST solution and slowly shaken on a shaker at room temperature for 1 hour. The blocking solution was removed, and the NC membrane was incubated with the diluted primary antibody (Jackson, Catalog No.: 111-035-003) at 4°C overnight. After washing with TBST buffer (10X, pH 7.4, Biosharp, Catalog No.: BL315B), the secondary antibody (1:2000, Mitaka, Catalog No.: 111-035-003) was added and incubated for 1 hour. Finally, the protein bands were observed using a chemiluminescence imager according to the instructions of the ECL luminescence kit. Image J was used to analyze and quantify Western blot images. The relative expression of the protein was normalized with the expression of β-actin. The results are shown in Fig.14As shown, since p21 and p53 proteins are aging indicator proteins, as the degree of aging accelerates, the p21 / p53 protein Model group shows high expression compared with the Control group, indicating that D-gal successfully established an aging model; along with the drug administration process, the expression of the two proteins in the JA drug administration group was lower than that in the Model group, indicating that high and low doses of JA have the effect of delaying aging.

[0117] In summary, Janthinedine A of the present invention has the activity of inhibiting D-gal-induced HUVEC cell senescence, delaying mouse senescence, protecting vascular endothelial function in aged mice, and affecting the expression of P21 and P53 proteins in mice.

Claims

1. Use of Janthinedine A in the preparation of anti-aging drugs; and / or Application of Janthinedine A in the preparation of cardiovascular protection drugs; The structural formula of Janthinedine A is as follows:

2. Application of Janthinedine A in the preparation of anti-aging skin care products, wherein the structural formula of Janthinedine A is as follows:

3. The use according to claim 2, characterized in that: The concentration of Janthinedine A when used in preparing anti-aging skin care products is 2 to 20 μM.

4. The use according to claim 1 or 2, characterized in that: The dosage form of the medicine is tablet, capsule or oral liquid, and the skin care product is skin care lotion, facial mask or skin care cream.

5. The use according to claim 1, characterized in that: The Janthinedine A is used as a raw material for a preparation for reducing the activity of β-galactosidase in senescent cells; and / or The Janthinedine A is used as a raw material for a preparation for restoring the proliferation ability of senescent cells; and / or The Janthinedine A is used as a raw material for a preparation for restoring the migration ability of senescent cells; and / or The Janthinedine A is used as a raw material for a preparation for improving sports endurance; and / or The Janthinedine A is used as a raw material for a preparation for improving blood circulation in lower limbs; and / or The Janthinedine A is used as a raw material for a blood pressure lowering preparation.

6. The use according to claim 1, characterized in that: The Janthinedine A is used to inhibit TNF-α, IL-1β, IL-8, IL-18 and / or MMP-2 inflammatory factors produced by cell aging.

7. The use according to claim 1, characterized in that: The Janthinedine A is used to inhibit the expression of p21 and p53 proteins.

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