New application of neem diol

Abstract

The invention provides a novel application of melia diol. The melia diol has a remarkable senescence cell removing capability in a bleomycin induced cell senescence model, an etoposide induced cell senescence model and a replicative cell senescence model; the activity of anti-apoptotic protein Bcl-2 can be obviously inhibited; the neem diol has no obvious liver and kidney toxicity while selectively removing senescent cells; the neem diol has the effects of remarkably lightening wrinkles on the back skin of a photoaged mouse, improving the transdermal moisture loss condition of the back skin of the photoaged mouse, increasing the moisture content of the back skin of the photoaged mouse, reducing the thickness of an epidermal layer and improving the fracture and content of collagenous fibers in a corium layer; the method has a wide application prospect in preparation of anti-aging products.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical biotechnology, and in particular to new uses of neem diol. Background Technology

[0002] With the increasing aging of the world's population, aging has gradually become a hot topic. One of the essential characteristics of cellular senescence is the permanent exit from the cell cycle (G1 phase or G2 / M phase arrest). All senescent cells exhibit irreversible cell cycle arrest, which is a hallmark event of aging. Cellular senescence is a complex phenotype involving multiple features such as cell cycle arrest, morphological changes, telomere shortening, and increased activity of senescence-related β-galactosidase (SA-β-gal). P16 and P21 are both cell cycle arrest proteins and belong to cyclin-dependent kinase inhibitors. In senescent cells, due to the permanent exit from the cell cycle, KI-67 (a proliferation marker) expression is significantly downregulated. KI-67 negativity can serve as auxiliary evidence of senescence-related proliferation arrest, and together with changes in P16 / P21 expression and SA-β-gal activity, it can be used to determine the senescence state.

[0003] Cellular senescence systematically drives degenerative changes in bodily functions through mechanisms such as senescence-associated secretory phenotype (SASP)-mediated chronic inflammation, stem cell depletion, and metabolic disorders. Targeted elimination of senescent cells has become an important direction in anti-aging research. SenoIytics are a class of drugs that selectively eliminate senescent cells by targeting the survival-dependent pathways specific to senescent cells, and are currently a hot topic in anti-aging research. SenoIytics provides a new strategy for anti-aging and the treatment of age-related diseases through precise elimination of senescent cells, and dozens of candidate drugs are currently under clinical evaluation, potentially becoming an important intervention for delaying aging in the future.

[0004] The evaluation of senolytic drugs needs to consider both cell clearance efficiency (therapeutic index TI value) and safety. The TI value is the drug's IC50 response to normal cells. 50 Value and IC50 value for senescent cells 50 The ratio of TI values ​​indicates the drug's ability to selectively eliminate senescent cells. ABT-737, a classic senolytic drug, works by selectively inducing apoptosis in senescent cells by targeting Bcl-2 family proteins, demonstrating clear efficacy in anti-aging and fibrotic diseases. However, existing senolytic drugs exhibit some cytotoxicity and are limited by specific cell types. Therefore, there is an urgent need to develop a new drug that delays aging by eliminating senescent cells, selectively eliminating a variety of senescent cells without significant hepatotoxicity or nephrotoxicity. Summary of the Invention

[0005] The present application aims to provide a new use of Cabralea diol.

[0006] The technical solution adopted by the present application is:

[0007] A use of Cabralea diol in the preparation of an anti-aging product, the structural formula of the Cabralea diol being as follows:

[0008]

[0009] Preferably, the use of the Cabralea diol described above, the anti-aging product can selectively remove a plurality of aging cells, and has no obvious hepatorenal toxicity.

[0010] Preferably, the use of the Cabralea diol described above, the aging cells are HEK or IMR-90 cells.

[0011] Preferably, the use of the Cabralea diol described above, the anti-aging product is an anti-wrinkle product.

[0012] Preferably, the use of the Cabralea diol described above, the anti-aging product is a health product, a pharmaceutical product or a skin care product.

[0013] An anti-aging product, comprising the Cabralea diol described above and an acceptable adjuvant.

[0014] The present application has the following beneficial effects:

[0015] The Cabralea diol has a significant ability to remove aging cells in a bleomycin-induced cell aging model, an etoposide-induced cell aging model and a replicative cell aging model; can significantly inhibit the activity of anti-apoptotic protein Bcl-2; the Cabralea diol can selectively remove aging cells without obvious hepatorenal toxicity; the Cabralea diol has a significant effect of lightening wrinkles on the back skin of a light-aging mouse, improving the transdermal water loss of the back skin of the light-aging mouse, increasing the water content of the back skin of the light-aging mouse, reducing the thickness of the epidermis layer and improving the breaking and content of collagen fibers in the dermis layer, thereby providing a theoretical basis, an experimental basis and a new strategy for understanding the aging mechanism and developing anti-aging drugs, and having a wide application prospect in the preparation of anti-aging products. Specifically:

[0016] Drug ability to eliminate senescent cells: TI value represents the ability of a drug to selectively eliminate senescent cells. In the bleomycin-induced senescence model of human skin keratinocytes (HEK cells), 10 μΜ jatrophane can significantly reduce the viability of HEK senescent cells without affecting the viability of control cells, indicating that it has the effect of eliminating HEK senescent cells. In the etoposide-induced senescence model of human embryonic lung cells (IMR-90 cells), jatrophane has a similar TI value to ABT-737, indicating that it has a similar ability to ABT-737 to eliminate senescent IMR-90 cells. 3.125 μΜ ABT-737 inhibits the growth of control cells when it exerts the ability to eliminate senescent IMR-90 cells, while 6.25 μΜ jatrophane does not inhibit the growth of control cells when it exerts a similar ability to 3.125 μΜ ABT-737 to eliminate senescent IMR-90 cells. This indicates that the toxicity of jatrophane to normal cells (non-senescent cells) is much lower than that of ABT-737. In the IMR-90 cell replicative senescence model, jatrophane has a higher TI value than ABT-737, indicating that jatrophane has a stronger ability to eliminate senescent cells than ABT-737 in the IMR-90 cell replicative senescence model.

[0017] Drug safety: jatrophane does not produce significant toxicity to liver and kidney cells when it exerts the effect of selectively eliminating senescent cells, indicating that jatrophane does not have significant liver and kidney toxicity when it selectively eliminates senescent cells. In addition, jatrophane has no hemolytic effect and no genotoxic effect. This indicates that jatrophane has high safety when it exerts the effect of eliminating senescent cells.

[0018] Drug ability to delay skin aging: In the photoaging mouse model, jatrophane reduces the transdermal water loss of the back skin of photoaging mice, increases the water content of the back skin of mice, and at the same time reduces the thickness of the epidermis layer of the back skin of photoaging mice, improves the state of collagen fiber fracture, disordered arrangement and loss in the dermis layer of the back skin of photoaging mice, and lightens the wrinkles of the back skin. This indicates that jatrophane can improve UV-induced skin aging. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 To verify the success of the bleomycin-induced HEK cell senescence model, wherein, Figure 1 A is the effect of 10, 25, 50 μg / mL bleomycin on HEK cell viability after 12 h, 24 h and 48 h of treatment, respectively; Figure 1 B is a histogram of SA-β-gal positive cells in HEK cells after 10, 25, 50 μg / mL treatment for 12 h, 24 h and 48 h, respectively; Figure 1C is the graph of HEK cell changes after 10, 25, 50 μg / mL bleomycin treatment for 12 h, 24 h and 48 h respectively; Figure 1 D is the graph of SA-β-gal positive cell area changes in HEK cells after 10, 25, 50 μg / mL bleomycin treatment for 12 h, 24 h and 48 h respectively. By Figure 1 It can be seen that compared with normal HEK cells, the SA-β-gal positive cell area of HEK cells treated with 50 μg / mL bleomycin for 48 h significantly increased, and the cell viability significantly decreased, indicating that the construction of HEK cell senescence model using 50 μg / mL bleomycin was successful (n = 3), wherein, ***P < 0.001 vs control group; **P < 0.01 vs control group; *P < 0.05 vs control group.

[0020] Figure 2 To verify the construction of IMR-90 cell senescence model induced by etoposide by SA-β-gal staining and P16, P21 mRNA expression level changes, wherein, Figure 2 A is to prove that IMR-90 cells are in a senescent state by SA-β-gal staining at two time points of the day of plating and the day before plating; Figure 2 B is to prove that IMR-90 cells are in a senescent state by P16, P21, KI-67 mRNA expression level changes. By Figure 2 It can be seen that compared with the control group (untreated control cells), the SA-β-gal positive cell area of IMR-90 cells treated with etoposide significantly increased, the P16, P21 mRNA expression level significantly increased, and the KI-67 mRNA expression level significantly decreased, indicating that the IMR-90 cells treated with etoposide were in a senescent state, and the construction of IMR-90 cell senescence model induced by etoposide was successful (n = 3), wherein, ***P < 0.001 vs control group; *P < 0.05 vs control group.

[0021] Figure 3 To verify the construction of late IMR-90 cell senescence model by continuous passage, wherein, Figure 3 A is to prove that the late IMR-90 cells by continuous passage are in a senescent state by SA-β-gal staining; Figure 3 B is to prove that the late IMR-90 cells are in a senescent state by detecting P16, P21, KI-67 mRNA expression level changes. By Figure 3It can be seen that compared with early IMR-90 cells, the area of SA-β-gal positive cells in late IMR-90 cells is significantly increased, the expression levels of P16 and P21 mRNA are significantly increased, and the expression level of KI-67 mRNA is significantly decreased, which proves that compared with early IMR-90 cells, late IMR-90 cells are in a senescent state, indicating that the construction of the replicative IMR-90 cell aging model caused by continuous passage is successful (n = 3), wherein ***P < 0.001 vs early passage IMR-90 cells; *P < 0.05 vs early passage IMR-90 cells.

[0022] Figure 4 To investigate the effect of Jatropha dioecula on removing bleomycin-induced senescent HEK cells. Figure 4 It can be seen that compared with the control group (untreated control cell group), the cell viability of the control group treated with Jatropha dioecula does not decrease, and compared with the untreated senescent cells, the cell viability of the senescent cells treated with Jatropha dioecula is significantly decreased. Jatropha dioecula can significantly reduce the viability of senescent cells without affecting the growth of control cells, indicating that Jatropha dioecula has the effect of selectively removing senescent HEK cells (n = 2), wherein ***P < 0.001 vs control group.

[0023] Figure 5 To determine the therapeutic index of Jatropha dioecula and ABT-737 on etoposide-induced senescent IMR-90 cells. Figure 5 It can be seen that the TI value of Jatropha dioecula (TI = 3.52) on senescent IMR-90 cells is similar to that of ABT-737 (TI = 3.92). It is shown that Jatropha dioecula and ABT-737 have similar abilities to remove senescent cells. When 6.25 μM of Jatropha dioecula and 3.125 μM of ABT-737 exert similar selective effects on removing senescent cells (ABT-737 inhibits the survival rate of senescent cells to 47.27%, and Jatropha dioecula inhibits the survival rate of senescent cells to 56.39%), compared with the control cells without drug treatment, ABT-737 treatment leads to a decrease in the survival rate of control cells to 56.5%, while Jatropha dioecula treatment does not significantly change the survival rate of control cells (the survival rate of control cells is 95.47%). It is shown that Jatropha dioecula selectively removes senescent cells without inhibiting the growth of control cells, and its toxicity to control cells is much lower than that of ABT-737 (n = 2).

[0024] Figure 6 To determine the therapeutic index of Jatropha dioecula and ABT-737 on early and late passage IMR-90 cells. Figure 6It can be seen that the South American genus diol, ABT-737 has a selective effect on the continuous passage of late IMR-90 cells, and the TI value of the South American genus diol (TI=3.58) is higher than that of ABT-737 (TI=1.7), indicating that the South American genus diol and ABT-737 can selectively remove senescent cells, which is consistent with the results of the South American genus diol in the etoposide-induced IMR-90 cell senescence model, and the South American genus diol has a better ability to remove senescent cells than ABT-737 (n=2).

[0025] Figure 7 The half-inhibitory concentration (IC 50 ) of the South American genus diol at different concentrations on human hepatoma cells HepG2 and embryonic kidney cells HEK293T was determined. The greater the half-inhibitory concentration, the greater the concentration of the drug required to produce the corresponding inhibitory effect, and the relatively lower the cytotoxicity. The IC 50 value of the South American genus diol on senescent IMR-90 cells was ≤21.88 μM. Figure 7 It can be seen that the IC 50 values of the South American genus diol on HepG2 and HEK293T cells were both ≥25 μM (91.13, 28.78 μM), which was greater than the IC 50 value of the South American genus diol on senescent IMR-90 cells, indicating that the South American genus diol did not damage liver and kidney cells when removing senescent cells, i.e. the South American genus diol did not have obvious hepatorenal toxicity when selectively removing senescent cells, and can be used for subsequent in vivo experiments to verify the effect of the South American genus diol on delaying skin aging (n=2).

[0026] Figure 8 The results of the hemolysis experiment of the South American genus diol are shown in the figure. Figure 8 It can be seen that compared with triton X-100 which has a hemolytic effect, the use of the South American genus diol at a maximum concentration of 100 μM (100 μM of the South American genus diol is about 5 times the concentration of the South American genus diol in the etoposide-induced senescence model and the replicative senescence model to remove senescent cells) did not cause significant hemolysis, indicating that the South American genus diol does not have a hemolytic effect (n=2).

[0027] Figure 9 The results of the South American genus diol-induced gene mutation are shown in the figure. The Ames test can determine whether a drug has an effect on inducing gene mutations in bacteria, i.e. the potential genetic toxicity of the drug, by measuring the change in the number of bacterial colonies after drug treatment. In this test, if the number of bacterial colonies does not change after administration, it indicates that the drug does not have a gene mutation effect on bacteria, i.e. there is no potential genetic toxicity. Figure 9It can be seen that compared with the positive control (2-amino fluorene, methyl methyl sulfonate), 100 μM Schinophrine did not change the number of bacterial colonies, indicating that Schinophrine had no potential genotoxicity, representing that Schinophrine had good safety and potential for further development (n=3).

[0028] Figure 10 To predict the binding target of Schinophrine by molecular docking and investigate whether it can target Bcl-2 protein by TR-FRET experiment, wherein, Figure 10 A is the PDB structure of apoptosis-related proteins Bcl-2, Hsp90, Bcl-XL, MDM2 and AKT1, protein function and related information of corresponding inhibitors; Figure 10 B is a schematic diagram of molecular docking method; Figure 10 C is a binding affinity heat map of Schinophrine and apoptosis-related proteins Bcl-2, Hsp90, Bcl-XL, MDM2 and AKT1; Figure 10 D is a molecular docking conformation diagram of ABT-737 and Schinophrine and Bcl-2 target; Figure 10 E is to determine whether Schinophrine can bind to Bcl-2 protein by TR-FRET experiment. The relative binding energy of each compound and protein is predicted by molecular docking, and the interaction of the ingredient and the target is evaluated by docking score. The lower the CDocker IntetactionEnergy value (binding affinity (kcal·mol -1 )) is, the more stable the docking system of the chemical ingredient and the protein receptor is, the more reliable the docking result is, and the higher the possibility of becoming an active ingredient is. For any given compound, the protein-ligand interaction with the lowest binding affinity (highest activity) is considered the most likely target for study, which accurately predicts the known binding interactions of ABT-737 with Bcl-2, ansamitocin with Hsp90, nutlin-3A with MDM2 and IQ0 with AKT1, proving the accuracy of molecular docking. Figure 10 C results show that the relative binding ability of Schinophrine and Bcl-2 protein is lower than that of Schinophrine and other apoptosis-related proteins Hsp90, Bcl-XL, MDM2 and AKT1, indicating that Schinophrine is most likely to target and bind to Bcl-2; Figure 10 D results show that Schinophrine has some amino acid residues (ARG107, TRY202 and GLY145) consistent with ABT-737 in binding to Bcl-2 protein, proving that Schinophrine can interact with residues in the common binding pocket of Bcl-2 like ABT-737. Figure 10EResults show that Schinus-terebinthifolius diol can bind to Bcl-2 protein and play a role in targeted inhibition of Bcl-2 protein. In summary, the results show that Schinus-terebinthifolius diol can inhibit Bcl-2 activity by targeting Bcl-2 protein, thereby playing a role in inducing apoptosis of senescent cells (n = 2), wherein the positive control is the relative Bcl-2 activity value produced by drug solvent treatment, and the relative Bcl-2 activity value produced by drug solvent treatment is defined as 1. ***P < 0.001 vs 1 (positive control); **P < 0.01 vs 1 (positive control).

[0029] Figure 11 The effect of Schinus-terebinthifolius diol on the water content of the back skin of photoaged mice. From Figure 11 It can be seen that the water content of the back skin of photoaged mice decreased significantly compared with that of normal mice; the water content of the back skin of photoaged mice treated with blank nanostructured lipid carrier gel showed no significant change compared with that of photoaged mice; and the water content of the back skin of photoaged mice treated with Schinus-terebinthifolius diol nanostructured lipid carrier gel increased significantly, indicating that Schinus-terebinthifolius diol has the effect of increasing the water content of the back skin of photoaged mice (n = 3), wherein ###P < 0.001 vs blank group; ##P < 0.01 vs blank group; ***P < 0.001 vs model group; **P < 0.01 vs model group; *P < 0.05 vs model group.

[0030] Figure 12 The effect of Schinus-terebinthifolius diol on the transdermal water loss of the back skin of photoaged mice. From Figure 12 It can be seen that the transdermal water loss value of the back skin of photoaged mice increased significantly compared with that of normal mice; the transdermal water loss value of the back skin of photoaged mice treated with blank nanostructured lipid carrier gel showed no significant change compared with that of photoaged mice; and the transdermal water loss value of the back skin of photoaged mice treated with Schinus-terebinthifolius diol nanostructured lipid carrier gel decreased significantly, indicating that Schinus-terebinthifolius diol has the effect of reducing the transdermal water loss of the back skin of photoaged mice (n = 3), wherein ###P < 0.001 vs blank group; ##P < 0.01 vs blank group; #P < 0.05 vs blank group; *P < 0.05 vs model group.

[0031] Figure 13The pathological section of the back skin of the photoaging mice was dyed. It was found from the H&E staining result that the epidermis of the back skin of the photoaging mice was significantly thicker than that of the normal mice. The thickness of the epidermis of the back skin of the photoaging mice treated with the blank nanostructured lipid carrier gel had no obvious change compared with the photoaging mice. The thickness of the epidermis of the back skin of the photoaging mice treated with the nanostructured lipid carrier gel containing the hydrolyzed extract of Jatropha dioica was significantly decreased. It was found from the Masson staining result that the collagen fibers in the dermis of the back skin of the photoaging mice were broken, arranged in disorder and reduced in content compared with the normal mice. The content of the collagen fibers in the dermis of the back skin of the photoaging mice treated with the blank nanostructured lipid carrier gel had no obvious change compared with the photoaging mice. The collagen fibers in the dermis of the back skin of the photoaging mice treated with the nanostructured lipid carrier gel containing the hydrolyzed extract of Jatropha dioica were arranged in order and significantly increased in content. It was shown that the hydrolyzed extract of Jatropha dioica had the effect of improving the aging skin of the back of the photoaging mice (n=3), wherein, ###P<0.001 vs the blank group; ***P<0.001 vs the model group.

[0032] Figure 14 The effects of the hydrolyzed extract of Jatropha dioica on the wrinkle depth and volume of the back skin of the photoaging mice were shown in FIG. 6. Figure 14 It was found that the wrinkle depth and volume of the back skin of the photoaging mice were significantly increased compared with the normal mice. The wrinkle depth and volume of the back skin of the photoaging mice treated with the blank nanostructured lipid carrier gel had no obvious change compared with the photoaging mice. The wrinkle depth and volume of the back skin of the photoaging mice treated with the nanostructured lipid carrier gel containing the hydrolyzed extract of Jatropha dioica were significantly decreased. It was shown that the hydrolyzed extract of Jatropha dioica had the effect of lightening the wrinkle depth and volume of the back skin of the photoaging mice (n=3), wherein, ##P<0.01 vs the blank group; **P<0.01 vs the model group; *P<0.05 vs the model group.

[0033] Figure 15 The effects of the hydrolyzed extract of Jatropha dioica on the compact softness of the back skin of the photoaging mice were shown in FIG. 7. Figure 15 It was found that the compact softness of the back skin of the photoaging mice was significantly decreased compared with the normal mice. The compact softness of the back skin of the photoaging mice treated with the blank nanostructured lipid carrier gel had no obvious change compared with the photoaging mice. The compact softness of the back skin of the photoaging mice treated with the nanostructured lipid carrier gel containing the hydrolyzed extract of Jatropha dioica was significantly increased. It was shown that the hydrolyzed extract of Jatropha dioica had the effect of improving the compact softness of the back skin of the photoaging mice (n=3), wherein, ##P<0.01 vs the blank group; #P<0.05 vs the blank group; *P<0.05 vs the model group. DETAILED DESCRIPTION

[0034] In order to make the technical personnel in the art better understand the technical solutions of the present application, the technical solutions of the present application are further described in detail below in combination with the drawings and specific embodiments.

[0035] Experimental materials and reagents

[0036] Cabral eadiol, the structural formula is as follows:

[0037] Purchased from Chengdu Lemaitian Pharmaceutical Technology Co., Ltd., content ≥98%.

[0038] IMR-90 cells were purchased from Aier Biotech (Shanghai) Co., Ltd.; HEK, HEK 293T cells were purchased from ATCC Cell Bank; HepG2 cells were purchased from Haixing Biotechnology Co., Ltd.

[0039] DMEM medium, penicillin-streptomycin, 0.25% trypsin + 0.02% EDTA, PBS phosphate buffer, all purchased from American Gibco Company.

[0040] Fetal bovine serum FBS, purchased from Shengong Bioengineering (Shanghai) Co., Ltd., American Gibco Company.

[0041] Cell culture plates were purchased from American Costar Company.

[0042] 4% paraformaldehyde, RNA Later, SA-β-gal staining kit, purchased from Beijing Solabio Technology Co., Ltd.

[0043] ABT-737 was purchased from Shanghai Yuan Ye Co., Ltd.

[0044] CCK-8 kit, RNA extraction kit, purchased from Beijing Quanshijin Biotechnology Co., Ltd.

[0045] CCK-8 solution preparation: CCK-8 mother liquor and 1×PBS were prepared according to the ratio of 1:9.

[0046] IMR-90 complete medium was prepared by adding 10% fetal bovine serum FBS (American Gibco Company) and 1% penicillin-streptomycin to DMEM medium and mixing evenly. HEK, HEK293T, HepG2 complete medium was prepared by adding 10% fetal bovine serum FBS (Shengong Bioengineering (Shanghai) Co., Ltd.) and 1% penicillin-streptomycin to DMEM medium and mixing evenly.

[0047] Etoposide was purchased from American MedChem Express Company.

[0048] Triton X-100 was purchased from Shanghai Melin Biochemical Technology Co., Ltd.

[0049] Ames kit was purchased from Beijing Hui Zhihe Yuan Biotechnology Co., Ltd.

[0050] β-actin, P16, P21, KI-67 primers were purchased from Shengong Bioengineering (Shanghai) Co., Ltd.

[0051] RNA reverse transcription kit, qRT-PCR Master Mix were purchased from Wuhan Aiboteke Biotechnology Co., Ltd.

[0052] T25 culture flask was purchased from Thermo Fisher Scientific company.

[0053] BCL-2 TR-FRET detection kit was purchased from Bps bioscience company.

[0054] SPF male ICR mice were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.

[0055] H&E staining kit, Masson staining kit were purchased from Wuhan Saivier Biological Technology Co., Ltd.

[0056] Mizolastine 52 was purchased from Nanjing Will Chemical Co., Ltd.

[0057] Soybean lecithin (SPC) was purchased from Shanghai Aivite Pharmaceutical Technology Co., Ltd.

[0058] Glyceryl behenate (AT0888) was purchased from Gattefosse, France.

[0059] Caprylic / capric triglyceride (Miglyol 812) was purchased from Beijing Fengli Jingqiu Business and Trade Co., Ltd.

[0060] Nipagin ethyl was purchased from Shanghai Yuan Ye Biological Technology Co., Ltd.

[0061] Carbomer 940 was purchased from Lubrizal company, USA.

[0062] Triethanolamine was purchased from Chengdu Huayi Pharmaceutical Auxiliary Material Manufacturing Co., Ltd.

[0063] Ethanol and chloroform were purchased from Tianjin Concord Biotech Co., Ltd.

[0064] Example 1

[0065] (1) Construction of senescence model:

[0066] The replicative senescence model was constructed using bleomycin (BLM) at a concentration of 50 μg / mL, etoposide at 15 μM, and continuous passage. The experimental method is as follows:

[0067] 1) Constructing bleomycin aging model:

[0068] Under the microscope, HEK cells (human skin keratinocytes) in T25 culture bottles were observed to have grown to 80-90%. Trypsin containing phenol red was used for 2 min of digestion at 37°C, and 1 x 10 5 Densely seeded in 96-well plates. After 24 h of cell culture, 10, 25, and 50 μg / mL bleomycin was added to the 96-well plates, and after 12, 24, and 48 h of 37°C culture, SA-β-gal (senescence-associated β-galactosidase) staining and cell viability detection were performed to verify aging.

[0069] 2) Constructing etoposide aging model:

[0070] Under the microscope, IMR-90 cells (human embryonic lung cells) in T25 vials were observed to have grown to 80-90%. Trypsin containing phenol red was used for 2 min of digestion at 37°C, and 1 x 10 5 Densely seeded in 6-well plates. After 24 h of cell culture, 15 μM etoposide was added to the 6-well plates, and after 2 days of 37°C culture, new IMR-90 complete medium was replaced to continue culture for 4 days to restore the cell state. The IMR-90 cells that had been restored on the 4th day were seeded in 6-well plates at 1 x 10 5 Densely seeded in 6-well plates, and SA-β-gal staining and qRT-PCR experiments were performed to verify aging.

[0071] 3) Constructing replicative aging model:

[0072] Early IMR-90 cells with <10 passages and late IMR-90 cells that had entered the aging state with >58 passages were used. The late IMR-90 cells that had entered the aging state were seeded in 6-well plates at 1 x 10 5 Densely seeded in 6-well plates, and SA-β-gal staining and qRT-PCR experiments were performed to verify aging.

[0073] (2) Model verification method:

[0074] Cell model verification:

[0075] 1) Cell viability verification:

[0076] The old medium in the HEK cells was aspirated, and 1 x PBS was used for washing. 100 μL CCK-8 solution was added to each well, and after 30 min of 37°C incubation, the value was measured at 450 nm on a microplate reader. The cell viability of the HEK cells treated with 50 μg / mL bleomycin for 48 h decreased significantly, indicating that the HEK cells had entered the aging state at this time.

[0077] 2) SA-β-gal staining verification:

[0078] The day before and on the day of the experiment, the HEK, IMR-90 complete medium, bleomycin induced HEK cell model, 15 μM etoposide induced IMR-90 cell model or early and late IMR-90 cells were seeded in 6-well plates at an initial density of 1 x 10 5 The old culture medium in the well plate was aspirated, and 1 x PBS was used for washing. 1 mL of β-Gal fixing solution was added, and the cells were fixed at room temperature for 15 min. The fixing solution was aspirated, and 1 x PBS was used for washing. 2 mL of staining working solution was added to each well, and the cells were incubated at 37 °C for 2 days. The staining working solution was aspirated, 1 x PBS was added, and the cells were observed under an inverted microscope and photographed. The SA-β-gal positive cell area was significantly increased in the HEK cells treated with 50 μg / mL bleomycin for 48 h, the IMR-90 cells induced with 15 μM etoposide, and the late IMR-90 cells, indicating that the HEK and IMR-90 cells had entered the senescent state.

[0079] 3) qRT-PCR verification:

[0080] The old culture medium in the senescent IMR-90 cells was aspirated, and 1 x PBS was used for washing. 1 mL of TransZol Up was added, and the cells were gently blown off using a pipette after being placed horizontally. The cell lysate was transferred to a 1.5 mL centrifuge tube, 0.2 mL of chloroform substitute (RNA Extraction Agent provided with the kit) was added, and vortexed for 5 min. Centrifugation was performed at 10,000 g for 15 min. The upper aqueous phase was carefully transferred to a CP3 centrifuge column, and an equal volume of anhydrous ethanol was added. After mixing, centrifugation was performed at 12,000 g for 30 s, and the effluent was discarded. RNA was eluted after the addition of a washing solution to remove impurities in the RNA. 200 μL of the centrifuge tube was added with 1 μL of MIX, 4 μL of RT Buffer, and deionized water to 10 μL. The RNA sample and deionized water were supplemented to make the total volume 20 μL, and reverse transcription was performed to obtain cDNA. Three replicate wells were set for each cDNA sample. The reaction system was added to an eight-row array for amplification to DNA to check the gene expression level.

[0081] The results are shown in Figures 1-3 After the HEK cells were treated with 50 μg / mL bleomycin for 48 h, the cell viability was significantly decreased, and the SA-β-gal positive cell area was significantly increased. In the IMR-90 cells induced with 15 μM etoposide and the late IMR-90 cells, the SA-β-gal positive cell area was significantly increased, and the P16 and P21 mRNA expression levels were significantly increased, indicating that the bleomycin-induced senescent HEK cell model, the etoposide-induced senescent IMR-90 cell model, and the replicative senescent IMR-90 cell model were successfully constructed.

[0082] In summary, from the above, Figures 1-3It can be seen that the HEK and IMR-90 cell senescence model has been successfully constructed.

[0083] Example 2

[0084] Validation of the ability of neem diol to clear senescent cells in three cell senescence models (bleomycin-induced HEK cell senescence model, etoposide-induced IMR-90 cell senescence model, and replicative IMR-90 cell senescence model):

[0085] Experimental methods: HEK and IMR-90 complete culture medium, bleomycin, and etoposide were used to induce senescence models or early and late passaged cells (replicative IMR-90 cell senescence models) at a concentration of 1×10⁻⁶. 5 The initial density was seeded in 96-well plates and incubated overnight at 37°C. Microscopic observation showed that the HEK and IMR-90 cell densities in the 96-well plates had reached 50%, indicating that drug administration was possible. The old culture medium in the 96-well plates was discarded, and a new medium with a concentration of 10... -1 After adding -100 μM of neem diol, the plates were incubated at 37°C in a 5% CO2 incubator for 3 days. The old culture medium in the plates was discarded, and the plates were washed with 1×PBS. 100 μL of LCK-8 solution was added to each well, and the plates were incubated at 37°C for 30 min before being measured at 450 nm using a microplate reader.

[0086] See results Figures 4-6 .Depend on Figure 4 It can be seen that neem diol significantly reduced the viability of senescent HEK cells without inhibiting the viability of control cells, indicating that neem diol has a selective scavenging effect on senescent HEK cells. Figure 5 It was found that ABT-737 and neem glycol selectively scavenged senescent IMR-90 cells. In the etoposide-induced IMR-90 cell senescence model, the TI value (therapeutic index) of neem glycol was similar to that of ABT-737. While both 6.25 μM and 3.125 μM ABT-737 exerted similar selective senescent cell scavenging effects, ABT-737 treatment significantly reduced the survival rate of control cells compared to untreated control cells, while neem glycol treatment did not show a significant difference in control cell survival. This indicates that while neem glycol exerts a similar senescent cell scavenging ability as ABT-737, its toxicity to control cells is far lower than that of ABT-737. Figure 6 It was found that in the replicative IMR-90 cell senescence model, the TI value (therapeutic index) of neem glycol was significantly higher than that of ABT-737. This indicates that neem glycol has the effect of clearing senescent cells, and its ability to clear senescent cells is superior to that of ABT-737.

[0087] In summary, ABT-737, a classic senolytic drug with good efficacy and safety in eliminating senescent cells, was followed by neem glycol in the etoposide-induced IMR-90 cell senescence model. Neem glycol exhibited similar senescent cell-eliminating effects to ABT-737, but its cytotoxicity to control cells was significantly lower than that of ABT-737. In the replicative IMR-90 cell senescence model, neem glycol demonstrated stronger senescent cell-eliminating ability than ABT-737. Furthermore, considering the limitations of traditional senolytic drugs in targeting specific cell models, the performance of neem glycol in multiple senescence cell models indicates that neem glycol has a broader selectivity for eliminating senescent cells.

[0088] Example 3

[0089] Safety evaluation of neem diols:

[0090] (1) Detection of liver and kidney toxicity of neem glycol from South American neem:

[0091] Experimental methods: HepG2 cells (human liver cancer cells) and HEK293T cells (human embryonic kidney cells) with fewer than 15 passages were used. HepG2 and HEK293T cells were cultured at a rate of 1×10⁻⁶ cells / cells. 5 The initial density was seeded in 96-well plates and incubated overnight at 37°C. Microscopic observation revealed that the HepG2 and HEK293T cell densities in the 96-well plates reached 50%. The old culture medium in the 96-well plates was discarded, and a new medium with a concentration ranging from 10... -1 After adding -100 μM neem glycol, the culture was incubated at 37°C in a 5% CO2 incubator for 3 days. The old culture medium was discarded, and the cells were washed with 1×PBS. 100 μL CCK-8 solution was added to each well, and the cells were incubated at 37°C for 30 min before measurement at 450 nm using a microplate reader. Results are shown below. Figure 7 .Depend on Figure 7 It is known that neem glycols, while selectively clearing senescent cells, did not produce significant toxicity to liver and kidney cells. This indicates that neem glycols do not exhibit significant hepatotoxicity or nephrotoxicity when exerting their selective senescent cell-clearing effect.

[0092] (2) Detection of the hemolytic effect of neem diol:

[0093] Experimental method: Take 2 mL of fresh mouse plasma, then dilute with 1 x PBS at a ratio of 1:10, shake well and centrifuge (1500 rpm, 10 min). After discarding the supernatant, the precipitated red blood cells are washed 3 times with 1 x PBS. Then the red blood cells are prepared into a 2% red blood cell suspension for testing. The positive control Triton X-100 is diluted with 1 x PBS to 1%, 0.1%, 0.01%. 50 μL of 2% red blood cell suspension is mixed with 50 μL of 1, 10, 100 μM Jatropha diol, 50 μL of 1, 10, 100 μM ABT-737 and 50 μL of 0.01%, 0.1%, 1% Triton X-100 in a 1.5 mL centrifuge tube, respectively, and incubated at 37°C for 1 h. After incubation, all samples to be tested are centrifuged (1500 rpm, 10 min), and 70 μL of supernatant from each group is taken to measure OD value at 545 nm wavelength on a microplate reader, and the hemolysis score is calculated according to the formula to quantify the content of hemoglobin released from red blood cells. The formula for calculating the hemolysis score (HR) is: Hemolysis score (HR) = (A test-A negative) / (A positive-A negative). The detection of hemoglobin indicates hemolytic effect. The results are shown in Figure 8 . It can be seen from Figure 8 that 1-100 μM Jatropha diol does not release hemoglobin from red blood cells, indicating that Jatropha diol does not have hemolytic effect.

[0094] (3) Evaluation of Jatropha diol-induced gene mutation experiment:

[0095] Experimental method: The Salmonella typhimurium TA100 strain and plate incorporation method were used. 2-aminofluorene and methyl methanesulfonate were used as positive controls, and the bottom culture medium, bacterial solution, top culture medium, S9 mixture or S9 blank solution (S9 mixture and S9 blank solution provided in the kit) and sterile test substance solution were mixed according to the preparation method and instructions provided in the kit. After the top culture medium was condensed, the experimental plates were inverted and placed in a 37°C incubator, and the experimental results were observed after 48 h of culture. The number of revertant colonies on the culture medium was directly counted, and the mean and standard deviation of the number of revertant colonies of 3 plates of each test drug were calculated. The results are shown in Figure 9 . It can be seen from Figure 9 that Jatropha diol does not have the effect of inducing gene mutation.

[0096] In summary, it can be seen from Figures 7-9 that Jatropha diol does not have obvious liver and kidney toxicity, hemolytic effect and the effect of inducing gene mutation when it selectively removes senescent cells.

[0097] Example 4

[0098] South American neem diols were used to predict binding targets via molecular docking and to investigate whether they could target the Bcl-2 protein using TR-FRET experiments.

[0099] Experimental Methods: Small molecule 2D / 3D structures were downloaded from Pubchem (https: / / pubchem.ncbi.nlm.nih.gov / ), and corresponding protein PDB structures were downloaded from PDB (https: / / www.rcsb.org / ). The active sites of each protein structure were determined based on the conformations of the corresponding binding inhibitors. Each compound was docked to the active site of each structure using the molecular docking software Discovery Studio, resulting in a series of predicted binding affinity values. The relative binding energy of each compound to the protein was predicted. Docking scores were used to evaluate the interaction between the component and the target. The CDocker Interaction Energy value (binding affinity (kcal·mol⁻¹)) was used. -1 The lower the value, the more stable the docking system between the chemical component and the protein receptor, the more reliable the docking result, and the higher the likelihood of it becoming an active ingredient. The docking posture of each compound was compared with that of ABT-737. Examination of each binding mode showed that neem glycol can interact with residues in the common binding pocket of Bcl-2, similar to ABT-737. The binding effect of neem glycol on Bcl-2 protein was determined using a BCL-2 TR-FRET assay kit.

[0100] See results Figure 10 ,Depend on Figure 10 Molecular docking analysis revealed that neem glycol is most likely to bind to Bcl-2, and it contains amino acid residues (ARG107, TRY202, and GLY145) that are identical to those in ABT-737 and bind to the Bcl-2 protein. TR-FRET experiments demonstrated that neem glycol can bind to the Bcl-2 protein and exert a targeted inhibitory effect. These results indicate that neem glycol can inhibit Bcl-2 protein activity by binding to it, suggesting that neem glycol can selectively eliminate senescent cells by targeting Bcl-2.

[0101] Example 5

[0102] Verification of the effect of neem diols on delaying photoaging of dorsal skin aging in mice:

[0103] Using a mouse model of photoaging, the effects of neem glycol on epidermal thickness, transepidermal water loss, water content, density and softness, wrinkle depth and volume, and collagen fibers in the dermis of the mouse dorsal skin were investigated. The experimental methods are as follows:

[0104] (1) Experimental grouping:

[0105] Twelve 4-week-old male ICR mice were weighed and then randomly assigned to 4 groups of 3 each, namely, a blank group, a model group, a blank nanostructured lipid carrier gel group, and a Schinopsis-biol nanostructured lipid carrier gel group. The blank group was not treated with UV (ultraviolet) irradiation and was normal mice; the model group was only subjected to UV irradiation and was photoaged mice; the blank nanostructured lipid carrier gel group and the Schinopsis-biol nanostructured lipid carrier gel group were subjected to UV irradiation and were topically applied with blank nanostructured lipid carrier gel and Schinopsis-biol nanostructured lipid carrier gel.

[0106] (2) Photoaging mouse modeling and administration method:

[0107] An SS-03AB ultraviolet phototherapy instrument was used to irradiate the skin of the mice to model. Before modeling, a mouse hair clipper was used to remove the hair on the back of the mice, and a depilatory cream was used to remove the remaining fluff, fully exposing the back skin (about 3x3 cm 2 ). A laboratory-made mouse fixing device was used to fix the mice, and an ultraviolet phototherapy instrument (UVA+UVB) was used to model. After 5 days of continuous irradiation, the mice were rested for 1 day, and a total of 9 cycles of irradiation were performed. The UV irradiation dose started from the minimum erythema dose (MED, UVA: 0.7 J / cm 2 , UVB: 0.07 J / cm 2 ), and the irradiation dose was exponentially increased for each cycle thereafter. The total UVA irradiation dose was 94.5 J / cm 2 , and the total UVB irradiation dose was 9.45 J / cm 2 .

[0108] During the experiment, the back skin of the mice in each group was photographed and recorded at the end of each experimental cycle.

[0109] (3) Preparation of blank gel:

[0110] Carbomer 940 was weighed and dispersed in 100 mL of deionized water, and stirred at room temperature at 500 rpm for 3 h until no obvious lumps were formed. Then, triethanolamine was used to adjust the pH of the carbomer 940 solution to 7 to make it into a gel state. Then, it was placed at 4°C for 12 h to swell, and a blank gel was obtained.

[0111] (4) Preparation of blank nanostructured lipid carrier gel and Schinopsis-biol nanostructured lipid carrier gel:

[0112] As a hydrophilic drug, jatrophadiene is encapsulated by nanostructured lipid carriers (NLC) to obtain jatrophadiene nanostructured lipid carriers, which are loaded into gels to assist transdermal administration.

[0113] a. Preparation of blank nanostructured lipid carriers and jatrophadiene nanostructured lipid carriers:

[0114] Preparation of blank nanostructured lipid carriers:

[0115] Take 100 mg of soybean lecithin and place it in a brown Schlenk flask. Dissolve it in ethanol at 75°C water bath. Take 100 mg of glyceryl behenate and 50 mg of caprylic triglyceride and dissolve them in chloroform at 75°C water bath. Mix the two to obtain the oil phase. Take 600 mg of Solutol HS 15 and dissolve it in 10 mL of 1×PBS at 75°C water bath to obtain the water phase. Slowly inject the oil phase into the water phase using a 2 mL syringe. Stir quickly at 75°C until the solution is clear. Then slowly stir to evaporate the organic phase. Cool to room temperature to obtain the blank nanostructured lipid carrier solution.

[0116] Preparation of jatrophadiene nanostructured lipid carriers:

[0117] Take 100 mg of soybean lecithin and place it in a brown Schlenk flask. Add jatrophadiene and dissolve it in ethanol at 75°C water bath. Take 100 mg of glyceryl behenate and 50 mg of caprylic triglyceride and dissolve them in chloroform at 75°C water bath. Mix the two to obtain the oil phase. Take 600 mg of Solutol HS 15 and dissolve it in 10 mL of 1×PBS at 75°C water bath to obtain the water phase. Slowly inject the oil phase into the water phase using a 2 mL syringe. Stir quickly at 75°C until the solution is clear. Then slowly stir to evaporate the organic phase. Cool to room temperature to obtain the jatrophadiene nanostructured lipid carrier solution.

[0118] b. Preparation of blank nanostructured lipid carrier gels and jatrophadiene nanostructured lipid carrier gels:

[0119] Preparation of blank nanostructured lipid carrier gels:

[0120] Take 10 mg of blank gel and add it to the blank nanostructured lipid carrier solution to prepare a blank nanostructured lipid carrier gel. Add an appropriate amount of nipagin ethyl to prevent mold growth of the gel. Place it at 4°C for subsequent animal experiments.

[0121] Preparation of jatrophadiene nanostructured lipid carrier gels:

[0122] Take 10 mg of blank gel and add it to the jatrophadiene nanostructured lipid carrier solution to prepare a jatrophadiene nanostructured lipid carrier gel with a concentration of 10 μg / 0.2 mg. Add an appropriate amount of nipagin ethyl to prevent mold growth of the gel. Place it at 4°C for subsequent animal experiments.

[0123] The amounts of each component in the preparation method are shown in Table 1, and the rest is ultrapure water.

[0124] Table 1 Composition of Jatropha gossypiifolia diol nanostructured lipid carrier gel formula

[0125]

[0126]

[0127] (5) Effect of Jatropha gossypiifolia diol on the epidermis layer of the back skin of photoaged mice:

[0128] a. Effect on the water content and transepidermal water loss value of the back skin of photoaged mice:

[0129] UV irradiation can damage the stratum corneum and lipid layer of the skin, leading to impaired skin barrier function, increased transepidermal water loss (TEWL), and decreased skin moisturizing ability, manifested as dryness, scaling, and other symptoms. Skin water content and TEWL are complementary indicators. The former reflects the water retention capacity of the stratum corneum, and the latter quantifies the rate of water loss through the epidermis to the external environment. The combination of the two can comprehensively evaluate the skin moisturizing ability and barrier integrity. At the end of each period, the changes in the water content of the mouse skin (Cornemeter CM825) and TEWL (Tewameter TM 300) were measured and recorded using a skin multi-probe detector.

[0130] The effects of Jatropha gossypiifolia diol on the water content and transepidermal water loss of the back skin of photoaged mice are shown in Figure 11 , Figure 12 . As can be seen from Figure 11 , compared with normal mice, the water content of the skin of photoaged mice decreased significantly, and compared with photoaged mice, the water content of the skin of photoaged mice treated with blank nanostructured lipid carrier gel showed no significant change. The water content of the skin of photoaged mice treated with Jatropha gossypiifolia diol nanostructured lipid carrier gel increased significantly, indicating that Jatropha gossypiifolia diol has the effect of improving skin water content. As can be seen from Figure 12 , compared with normal mice, the transepidermal water loss value of the skin of photoaged mice increased significantly, and compared with photoaged mice, the transepidermal water loss value of the skin of photoaged mice treated with blank nanostructured lipid carrier gel showed no significant change. The transepidermal water loss value of the skin of photoaged mice treated with Jatropha gossypiifolia diol nanostructured lipid carrier gel decreased significantly, indicating that Jatropha gossypiifolia diol has the effect of improving skin transepidermal water loss.

[0131] b. Effect on the thickness of the epidermis layer of the back skin of photoaged mice:

[0132] After fixing mouse skin tissue samples in 4% tissue fixative for 24 hours, subsequent embedding and sectioning were performed. The sections were then sequentially immersed in environmentally friendly dewaxing solution I (20 min), environmentally friendly dewaxing solution II (5 min), anhydrous ethanol, and 75% ethanol for 5 min each, and washed with ultrapure water.

[0133] H&E staining:

[0134] The sections were stained with hematoxylin for 5 min, rinsed with ultrapure water, differentiated with 1% hydrochloric acid ethanol for 10 s, rinsed again, and then blued with 0.2% ammonia solution, followed by rinsing with running water. The sections were then placed in 95% ethanol for 1 min to dehydrate, and stained with eosin for 15 s. After dehydration and hydration with anhydrous ethanol and other solutions, the sections were mounted with an appropriate amount of neutral resin. The sections were scanned using a slide scanner, and the changes in epidermal thickness were analyzed under 10× and 20× magnification.

[0135] The effect of neem diol on the thickness of the epidermal layer of the dorsal skin of photoaged mice is shown in [reference needed]. Figure 13 ,Depend on Figure 13 It was found that, compared with normal mice, the thickness of the epidermal layer of the dorsal skin of photoaged mice was significantly increased. Compared with photoaged mice, the thickness of the epidermal layer of the dorsal skin of photoaged mice treated with blank nanostructured lipid carrier gel did not change significantly. The thickness of the epidermal layer of the dorsal skin of photoaged mice treated with neem glycol nanostructured lipid carrier gel was significantly decreased, indicating that neem glycol has the effect of improving the thickness of the epidermal layer of the dorsal skin.

[0136] (6) Effects of neem diol on collagen fibers in the dermis of photoaged mice:

[0137] After fixing mouse skin tissue samples in 4% tissue fixative for 24 hours, subsequent embedding and sectioning were performed. The sections were then sequentially immersed in environmentally friendly dewaxing solution I (20 min), environmentally friendly dewaxing solution II (5 min), anhydrous ethanol, and 75% ethanol for 5 min each, and washed with ultrapure water.

[0138] Masson staining:

[0139] The slides were immersed in Masson A solution overnight, rinsed with ultrapure water, and then stained in a mixture of Masson B and Masson C solutions in equal proportions for 1 min. After rinsing with ultrapure water, they were differentiated with 1% hydrochloric acid and ethanol for a few seconds, rinsed with deionized water, immersed in Masson D solution for 6 min, rinsed with water, stained with Masson E solution for 1 min, slightly drained, and then stained with Masson F solution for 30 s. After rinsing with 1% acetic acid for differentiation, the slides were dehydrated and permeated successively with anhydrous ethanol and xylene, and mounted with neutral resin. The slides were scanned using a slide scanner, and structural changes were observed under 10× and 20× magnification. The collagen fiber content in the scanned images was analyzed using Image Pro Plus software.

[0140] Effects of neem diol on collagen fibers in the dermis of the back skin of photoaged mice (see below) Figure 13 .Depend on Figure 13 It was found that, compared with normal mice, the collagen fibers in the dermis of the dorsal skin of photoaged mice were disordered and significantly reduced in content. Compared with photoaged mice, the collagen fiber arrangement and content in the dorsal skin of photoaged mice treated with blank nanostructured lipid carrier gel did not change significantly; however, the collagen fibers in the dermis of the dorsal skin of photoaged mice treated with neem glycol nanostructured lipid carrier gel were well-arranged and significantly increased in content, indicating that neem glycol has the effect of improving the dorsal skin of photoaged mice.

[0141] (7) Effects of neem diols on skin wrinkles on the back of photoaged mice:

[0142] At the end of each cycle, images were captured using a tissue viability imaging system (Tissue Viability Imaging TiVi700, WheelsBridge AB, Sweden). The wrinkle analysis software Wrinkle Analyzer TiVi90 calculated the depth and volume of wrinkles based on wrinkle contour analysis. The effects of neem glycol on the depth and volume of wrinkles on the back skin of photoaged mice are shown below. Figure 14 .Depend on Figure 14 It was found that, compared with young mice, the wrinkles and volume of the skin on the back of photoaged mice were significantly increased. Compared with photoaged mice, the wrinkles and volume of the skin on the back of photoaged mice treated with blank nanostructured lipid carrier gel did not change significantly. The wrinkles and volume of the skin on the back of photoaged mice treated with neem glycol nanostructured lipid carrier gel were significantly reduced, indicating that neem glycol has the effect of reducing skin wrinkles.

[0143] (8) Effect of neem diols on the density and softness of dorsal skin in photoaged mice:

[0144] The changes in skin density and softness (Indentometer 1DM 800) in mice were measured and recorded at the end of each cycle using a multi-probe skin osmosis meter. The effect of neem diol on the skin density and softness of the back of photoaged mice is described in [reference needed]. Figure 15 .Depend on Figure 15 It was found that, compared with normal mice, the density and softness of the dorsal skin of photoaged mice decreased significantly. Compared with photoaged mice, the density and softness of the dorsal skin of photoaged mice treated with blank nanostructured lipid carrier gel did not change significantly. However, the density and softness of the dorsal skin of photoaged mice treated with neem glycol nanostructured lipid carrier gel increased significantly, indicating that neem glycol has the effect of improving the density and softness of the skin.

[0145] In summary, byFigures 11-15 It can be known that the Schinopsis balansae diol can reduce the skin tissue damage caused by UV irradiation, delay wrinkle production and improve the skin aging state.

[0146] It can be seen that the Schinopsis balansae diol can significantly reduce the activity of aging cells, has the effect of removing aging cells, can significantly inhibit the activity of Bcl-2 protein, can selectively remove aging cells without obvious liver and kidney toxicity, no hemolysis phenomenon and no phenomenon of causing bacterial colony gene mutation, can significantly reduce the thickness of the epidermis layer of the back skin of the photoaging mouse and the transdermal water loss, improve the water content and the dense softness of the back skin of the photoaging mouse, reduce the volume and depth of the wrinkle of the back skin of the photoaging mouse, and improve the fracture and loss of collagen fibers in the dermis layer of the back skin. Therefore, the Schinopsis balansae diol has important application value in the preparation of anti-aging products.

[0147] The above-described embodiments are only used to describe the preferred embodiments of the present application, and do not limit the scope of the present application, and various modifications and improvements to the technical solutions of the present application made by the ordinary engineering technical personnel in the art without departing from the design spirit of the present application shall fall within the protection scope of the present application.

Claims

1. Use of a baccatin in the manufacture of a product for delaying aging, characterized in that: The structural formula of the Schinol is as follows:

2. Use of a Schinopsis diol according to claim 1, characterized in that: The anti-aging product can selectively eliminate a plurality of senescent cells.

3. Use of a Schinopsis diol according to claim 2, characterized in that: The senescent cells are HEK or IMR-90 cells.

4. Use of a Schinopsis diol according to claim 1, characterized in that: The anti-aging product is an anti-wrinkle product.

5. Use of a Hevea brasiliensis diol according to claim 1 or 2 or 4, characterized in that: The anti-aging product is a health product, a pharmaceutical product or a skin care product.

6. A product for delaying aging, characterized by: The anti-aging product comprises the Schinol according to any one of claims 1-5 and an acceptable adjuvant.

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