Application of palm seed aqueous extract in preparation of anti-aging and cardiovascular protection drugs and / or skin care products

Abstract

The invention belongs to the technical field of new applications of medicines, and particularly discloses an application of a Palmetto seed water extract (WE) in preparation of anti-aging and cardiovascular protection medicines and / or skin care products. The effective component of the medicine and / or the skin care product is a pure water extract of a raw medicinal material palm seed. The palm seed aqueous extract is used for preparing the anti-aging and cardiovascular disease delaying, preventing and / or treating medicine, a new medicine choice is provided for anti-aging and cardiovascular protection, the curative effect is definite, safe and reliable, and the palm seed aqueous extract has important practical application value in the fields of anti-aging and cardiovascular protection.

Description

Technical Field

[0001] The present invention belongs to the technical field of new medical applications, and particularly relates to the application of a palm seed water extract in the preparation of anti-aging and cardiovascular protection drugs and / or skin care products. Background Art

[0002] Cardiovascular disease (CVD) leads the world in morbidity and mortality. In 2022, the American Heart Association listed vascular aging (VA) as the leading risk factor for CVD. Vascular aging poses a serious threat to human health and has become a global public health issue that demands urgent attention. Vascular aging is a fundamental physiological basis associated with age-related changes, an essential component of aging across all human organs, and a common pathogenesis of various chronic diseases in the elderly. In 2023, the Chinese Association of Aging Biomarkers (ABC) announced the characteristics of VA: increased vascular stiffness, decreased angiogenesis, increased secretion of inflammatory factors, intimal thickening, elastic fiber fragmentation and disorganization, and increased collagen fiber deposition. As the saying goes, "Vascular aging, human aging." Vascular aging leads to pathological remodeling, increasing the risk of cardiovascular diseases such as atherosclerosis and stroke. Therefore, delaying vascular aging is a feasible strategy for preventing and treating CVD. However, current clinical interventions for vascular aging are relatively limited, primarily focusing on managing risk factors that lead to further disease, and no effective treatments are available. Therefore, developing new, efficient and safe drugs to delay vascular aging and serve the strategic layout of "healthy aging" is crucial to contributing to the "Healthy China".

[0003] Palm seeds (ZLZ) are the dried, mature fruit of the palm Trachycarpus fortunei (Hook.) H. Wendl. of the family Palmaceae. They are a traditional Chinese medicinal ingredient, first mentioned in the Yellow Emperor's Classic of Internal Medicine and Compendium of Materia Medica. They are bitter and astringent, neutral in nature, and enter the liver and spleen meridians. According to the Supplement to the Compendium of Materia Medica and the Supplement to the Compendium of Materia Medica, palm seeds have hemostatic, astringent, antidiarrheal, and blood-nourishing properties, and are used to treat dysentery, intestinal bleeding, and metrorrhagia.

[0004] Palm seed decoctions and soluble powders are commonly used in folk medicine as anti-inflammatory and cardiovascular medications. Modern research indicates that the active ingredients in its extracts have anti-inflammatory properties and can also suppress blood cholesterol, contributing to cardiovascular health. Furthermore, palm seeds are used to treat a variety of conditions in ethnic minority prescriptions, such as Baiyao and Miao medicine, demonstrating their value in folk medicine. Summary of the Invention

[0005] In recent years, the inventors of this application have been engaged in research related to vascular aging and have achieved considerable results. The present preparation, prepared from a pure water extract of palm seeds, has the efficacy of delaying vascular aging and protecting the cardiovascular system, demonstrating significant anti-aging effects. Furthermore, to expand the indications for this extract, the inventors have used it in experimental studies of vascular aging (VA) in cells, Caenorhabditis elegans, and mice, finding significant results.

[0006] The present invention discloses the use of a palm seed aqueous extract (WE) in the preparation of anti-aging and cardiovascular protection drugs and / or skin care products. The palm seed aqueous extract is a pure water-extracted traditional Chinese medicine preparation that can be used in the preparation of anti-aging and cardiovascular protection drugs or anti-aging skin care products. The palm seed aqueous extract (WE) is obtained from palm seeds using pure water extraction.

[0007] Furthermore, the palm seed water extract WE is a pure water extract of the dried mature fruit of the palm Trachycarpus fortunei (Hook.) H. Wendl. of the family Palmaceae, a traditional Chinese medicinal material: the palm seeds are crushed, soaked and extracted with pure water for a period of time at a certain solid-liquid ratio (preferably a solid-liquid ratio of 1 g:10 mL, and soaked in pure water for 2 hours), then ultrasonically extracted for a period of time (preferably 600W ultrasonic extraction for 30 minutes), followed by oil bath reflux extraction for 1-4 times (preferably 80°C oil bath reflux twice, each time for 2 hours (starting from the first reflux drip)), and the filtrate is filtered and concentrated to obtain the palm seed water extract WE.

[0008] Furthermore, the dosage of the palm seed water extract WE for preparing anti-aging drugs is 11.0 mg / kg / d (this dosage is calculated based on the pharmacological test methodology and the corresponding high dose of WE 100 mg / kg / d for mice with a standard body weight of 70.0 kg for normal adults).

[0009] Furthermore, the dosage of the palm seed water extract WE for preparing cardiovascular protective drugs is 11.0 mg / kg / d (this dosage is calculated based on the pharmacological test methodology and the dose of 100 mg / kg / d WE given to mice corresponding to a normal adult body weight of 70.0 kg).

[0010] Furthermore, when the palm seed water extract WE is used to prepare anti-aging skin care products, the concentration of the palm seed water extract WE is 0.01-1.0 mg / mL.

[0011] Furthermore, the pharmaceutical dosage forms include tablets, capsules, and oral liquids, and the skin care products include skin care lotions, facial masks, and skin care creams.

[0012] Furthermore, the palm seed water extract WE is used as a raw material for a preparation for reducing β-galactosidase activity in senescent cells; and / or

[0013] The palm seed water extract WE is used as a raw material for a preparation for restoring the proliferation ability of senescent cells; and / or

[0014] The palm seed water extract WE is used as a raw material for a preparation for restoring the migration ability of senescent cells; and / or

[0015] The palm seed and the palm seed water extract WE are used as raw materials for a preparation for extending the life cycle of Caenorhabditis elegans and improving its survivability under heat stress; and / or

[0016] The palm seed water extract WE is used as a raw material for a preparation for improving exercise endurance; and / or

[0017] The palm seed water extract WE is used as a raw material for a preparation for improving blood circulation in lower limbs; and / or

[0018] The palm seed water extract WE is used as a raw material for a blood pressure lowering preparation.

[0019] Furthermore, the palm seed water extract WE is used to inhibit the pro-inflammatory factors IL-6, IL-1α, IL-1β, CCL2, CCL3, INF-γ, TNF-α produced by cell aging and upregulate the anti-inflammatory factor IL-10.

[0020] Furthermore, the palm seed water extract WE is used to inhibit the expression of p16 and p21 proteins in vivo and in vitro.

[0021] Furthermore, the palm seed water extract WE is used to upregulate the expression of IGF-1 and LaminB1 in the body and inhibit the expression of γ-H2A.X.

[0022] Furthermore, the palm seed water extract WE delays vascular aging and protects cardiovascular health by regulating the cGMP-PKG signaling pathway of Rhoa, VDAC1, VDAC2, and KNG2 protein targets.

[0023] This study focuses on the in-depth development of ethnic medicines and medicinal materials with edible and medicinal properties. By focusing on key pathological links and pathways of action, the study explores the synergistic integration mechanisms of multiple components, targets, and pathways, providing a reference for the creation of new formulations with clear ingredients, clear mechanisms, controlled quality, safety, and efficacy. This type of research is a key approach to the development of new Chinese medicines, aligning with the principles of translational medicine and the integration of traditional Chinese and Western medicine, and possesses significant theoretical value and application prospects.

[0024] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0025] The present invention provides a new use of a pure water extract of palm seeds, a traditional folk medicine, which is a mature fruit of palm. The palm seed water extract WE is used to prepare a drug for resisting aging and protecting cardiovascular function. Experimental results showed that palm seed aqueous extract (WE) restored the cell migration and proliferation capabilities of senescent cells, extended the lifespan of Caenorhabditis elegans, and improved its ability to survive heat stress. It significantly inhibited D-gal-induced senescence in human umbilical vein endothelial cells (HUVEC) by producing proinflammatory cytokines such as IL-6, IL-1α, IL-1β, CCL2, CCL3, INF-γ, and TNF-α, and upregulated the anti-inflammatory cytokine IL-10. Furthermore, it reduced the expression of β-galactosidase, p16, p21, and γ-H2A.X in D-gal-induced HUVEC senescent cells, while upregulating the expression of IFG-1 and LaminB1. Furthermore, it maintained the vascular epithelial barrier by regulating the cGMP-PKG signaling pathway through the Rhoa, VDAC1, VDAC2, and KNG2 protein targets, thereby delaying vascular aging. This demonstrates the systemic regulation and multi-component, multi-target synergistic therapeutic potential of Traditional Chinese Medicine (TCM) in treating complex diseases. Therefore, its anti-aging and cardiovascular protective effects warrant further investigation.

[0026] This invention not only provides more drug options for preventing and treating aging and vascular diseases, but also contributes to human exploration of health and has significant implications for the development of the global health industry. It will help develop more effective drugs for delaying aging and protecting cardiovascular health, thereby better addressing the challenges of an aging global population. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Figure 2 shows the safety test results of the non-toxic dosage concentration of palm seed water extract WE in Example 2 of the present invention (A) and the cell survival rate of HUVEC cells induced by D-gal after WE administration (B). Compared with the model group (Model), the groups treated with each effective concentration showed: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0028] Figure 2 Schematic diagram of the changes in cell migration (A), cell invasion ability (B), cell scratch area statistics (C), and cell invasion number statistics (D) of each group of D-gal-induced senescent HUVEC cells after incubation with palm fruit water extract WE in Example 2 of the present invention. Compared with the model group (Model), each group: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001;

[0029] Figure 3 This is a schematic diagram of the positive staining of β-galactosidase activity in D-gal-induced senescent HUVEC cells after incubation with palm fruit water extract WE in Example 2 of the present invention. Compared with the model group (Model), each group showed: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001;

[0030] Figure 4 Schematic diagram of the changes in the palm seed water extract WE in Example 3 of the present invention in extending the life cycle of Caenorhabditis elegans (A) and improving the heat stress survival ability (B);

[0031] Figure 5 This is a schematic diagram of the changes in the anti-aging application of the palm seed water extract WE in Example 4 of the present invention, which significantly inhibited the production of pro-inflammatory factors IL-6, IL-1α, IL-1β, CCL2, CCL3, INF-γ, and TNF-α in D-gal-induced aging C57BL / 6J mice and upregulated the anti-inflammatory factor IL-10. Compared with the model group (Model), each group: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; compared with the natural aging group (NatureAging), each group: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001;

[0032] Figure 6 Schematic diagram of the changes in the palm seed aqueous extract WE used to upregulate the expression of IGF-1 and LaminB1 and inhibit the expression of γ-H2A.X in vivo in Example 4 of the present invention. Compared with the model group (Model), each group: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; compared with the natural aging group (Nature Aging), each group: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001;

[0033] Figure 7 This is a schematic diagram showing the changes in body weight of D-gal-induced aged C57BL / 6J mice after oral administration of high and low doses of palm seed water extract WE in Example 3 of the present invention;

[0034] Figure 8 This is a schematic diagram of the changes in blood pressure in D-gal-induced aged C57BL / 6J mice after oral administration of high and low doses of palm seed water extract WE in Example 3 of the present invention;

[0035] Figure 9This is a schematic diagram of the changes in hind limb exercise endurance in D-gal-induced aging C57BL / 6J mice after oral administration of high and low doses of palm seed water extract WE in Example 3 of the present invention. Compared with the model group (Model), each group: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; compared with the natural aging group (Nature Aging), each group: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001;

[0036] Figure 10 This is a schematic diagram of ultrasound imaging of changes in lower limb blood flow in D-gal-induced aged C57BL / 6J mice after oral administration of high and low doses of palm seed water extract WE in Example 3 of the present invention;

[0037] Figure 11 Schematic diagram of HE staining (A) showing changes in thoracic aorta wall thickness in D-gal-induced aging C57BL / 6J mice after oral administration of high and low doses of palm seed water extract WE in Example 3 of the present invention; schematic diagram of EVG staining (B) and Masson staining (C) showing changes in elastic fiber / collagen fiber distribution in the thoracic aorta of mice; and schematic diagram of transmission electron microscopy of the internal elastic structure of the thoracic aorta of mice (D);

[0038] Figure 12 Schematic diagram of HE staining of the liver morphology (Liver) and the kidney morphology (Kidney) of D-gal-induced aging C57BL / 6J mice after oral administration of high and low doses of palm seed water extract WE in Example 3 of the present invention;

[0039] Figure 13 This is a schematic diagram of changes in liver function test indicators alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in D-gal-induced aging C57BL / 6J mice after oral administration of high and low doses of palm seed water extract WE in Example 3 of the present invention;

[0040] Figure 14 Schematic diagram of the changes in p16 and p21 levels in D-gal-induced senescent HUVEC cells after incubation with palm fruit water extract (WE) in Example 3 of the present invention (A); a schematic diagram of the changes in p16 and p21 levels in D-gal-induced senescent C57BL / 6J mice after oral administration of high and low doses of palm fruit water extract (WE) (B); and a schematic diagram of p16 and p21 protein expression in the thoracic aorta of mice (C);

[0041] Figure 15Schematic diagram of the verification of the cGMP-PKG signaling pathway in the anti-aging application of palm seed water extract WE in Example 5 of the present invention (A) and the expression of Rhoa, VDAC1, VDAC2, and KNG2 protein targets in the thoracic aorta of mice for delaying vascular aging and protecting cardiovascular health (B). DETAILED DESCRIPTION

[0042] In order to better understand the technical solutions of the present invention, the technical solutions in the embodiments of the present invention are clearly and completely described and illustrated below with reference to the accompanying drawings.

[0043] Example 1 Preparation method of palm seed aqueous extract WE

[0044] The dried palm seeds were crushed and extracted with pure water at a solid-liquid ratio of 1 g:10 mL. After soaking in pure water for 2 h, the seeds were extracted with 600 W ultrasound for 30 min, and refluxed in an 80°C oil bath twice for 2 h each time (starting from the first reflux dripping). The filtrate was filtered and concentrated to obtain the palm seed aqueous extract WE.

[0045] Example 2 In vitro pharmacodynamics of the palm seed aqueous extract WE prepared in Example 1 for cardiovascular protection

[0046] 1. Materials

[0047] 1.1 Model Organisms

[0048] Human umbilical vein endothelial cells (HUVEC) were purchased from Wuhan Saios Biotechnology Co., Ltd. (Product No.: CL-191h).

[0049] 1.2 Drugs and reagents

[0050] β-Nicotinamide mononucleotide was purchased from Shanghai Titan Technology Co., Ltd.; the β-galactosidase staining kit for cell senescence was purchased from Shanghai Beyotime Biotechnology Co., Ltd.; the 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 ScienCell Research Laboratories, Inc. (Cat. No. 1001); and all other inorganic reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. HUVEC cell-specific ECM complete medium consists of 500 mL of basal medium, 25 mL of fetal bovine serum (FBS), 5 mL of endothelial cell growth supplement (ECGS), and 5 mL of penicillin / streptomycin solution (P / S). Note: FBS, ECGS, and P / S are not premixed in the ECM basal medium and are added separately to prepare the ECM complete medium before use.

[0051] 1.3 Instruments

[0052] A carbon dioxide constant temperature incubator (Shanghai Kelifu Instrument Co., Ltd.), a Multiskan FC multifunctional microplate reader (Thermo Fisher Scientific Shanghai Instrument Co., Ltd.), an inverted fluorescence microscope (Guangzhou Haokang Biotechnology Co., Ltd., model HIF2000), a small high-speed centrifuge (LaboGene, Denmark), an FN-6 constant temperature water bath (Beijing Liuyi Biological Co., Ltd.), and an SPX-150B-Z biochemical incubator (Beijing Liuyi Biological Co., Ltd.) were used.

[0053] 2 Methods

[0054] 2.1 Experimental Grouping

[0055] The animals were divided into a model group (Model, 40 mg / mL D-galactose D-gal), a blank control group (Control), a positive drug group (Positive drugs, 40.0 μM β-nicotinamide mononucleotide, NMN) and 6 palm fruit water extract WE administration groups. The 6 palm fruit water extract WE administration groups were prepared with ECM complete culture medium to prepare palm fruit water extract WE at the following concentrations: 1.0 mg / mL, 0.5 mg / mL, 0.25 mg / mL, 0.1 mg / mL, 0.05 mg / mL and 0.01 mg / mL.

[0056] 2.2 HUVEC cell culture

[0057] (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 8mL 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 a carbon dioxide (5% carbon dioxide condition in the present invention) and 37°C constant temperature incubator for culture.

[0058] (2) When the cells grow to a density of 95%, the cells are subcultured. After confirming the cell growth status, the cell subculture is completed and 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 placed in each well. At the same time, a layer of PBS (phosphate buffered saline to prevent evaporation error, the PBS used in this invention is Biosharp-PBS buffer, product number: BL302A) is placed around the 96-well plate. The volume of PBS is the same as that of the cell solution. After the cell inoculation is completed, the cells are placed in a 37°C constant temperature carbon dioxide incubator and cultured for 24 hours.

[0059] 2.3D-gal construction of aging model

[0060] Preparation of D-gal: Weigh an appropriate amount of D-gal and dissolve it in DMEM high-glucose liquid medium (containing sodium pyruvate, without HEPES, purchased from Biosharp, catalog number: BL304A) to prepare a stock solution with a concentration of 40.0 mg / mL. Filter the solution through a disposable sterile filter on a clean bench, aliquot, and store in a refrigerator at 4°C until use.

[0061] D-gal was used to construct an aging model: the prepared D-gal was preheated in a 37°C constant temperature water bath. At the same time, the original culture medium in the above 96-well plate was discarded. 100 μL of preheated D-gal was evenly added to each well using a dispenser. The plate was then placed in a 37°C, 5% carbon dioxide constant temperature cell culture incubator and cultured for 4 hours.

[0062] 2.4 Effect of palm seed aqueous extract (WE) on the survival rate of D-gal-induced senescent HUVEC cells

[0063] First, the safe concentration range of palm seed water extract WE on HUVEC cells was detected. HUVEC cells were cultured in 96-well plates according to the above method. The concentration gradient of palm seed water extract WE was prepared using ECM complete medium: 0 / 0.01 / 0.05 / 0.1 / 0.25 / 0.5 / 1.0 / 5.0 / 10.0 / 15.0 mg / mL and added to the 96-well plate according to the prepared concentration gradient to incubate HUVEC cells. The cells were then incubated in a 37°C, 5% CO2 constant temperature cell culture incubator for 24 hours to explore the safe dosing range of palm seed water extract WE at the cell level. After incubation, the cell viability was detected using the CCK-8 kit. The survival rate of HUVEC cells was analyzed using GraphPad Prism10 software, and a survival curve was drawn, as shown in the figure. Figure 1 As shown in A, the safe concentration range of palm seed aqueous extract WE for HUVEC cells is 0.01-1.0 mg / mL.

[0064] Except for the blank control group, all other groups were treated with a dispenser to uniformly add 100 μL of preheated D-gal solution to each well, and then incubated in a 37°C, 5% CO2 constant temperature cell culture incubator for 4 hours. Cells in the blank control group were cultured with an equal amount of ECM complete medium.

[0065] The experimental groups were the same as in 2.1 above, and the HUVEC cell survival rates in different experimental groups were compared. The specific steps are as follows:

[0066] The D-gal-containing culture medium used to establish the aging model was aspirated and discarded. Five replicate wells were set up for each drug-dosing group, and 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. The cells were placed in a carbon dioxide constant temperature cell culture incubator for 24 hours and then taken out for observation (unless otherwise specified, the cell culture incubator in the present invention was kept at a constant temperature of 37°C and 5% carbon dioxide). The survival rate of HUVEC cells was analyzed and a survival curve was drawn, as shown in FIG. Figure 1 As shown in Figure B and Table 1, the results showed that the palm seed water extract WE had the best effect at a concentration of 0.5 mg / mL. At the same time, compared with the positive drug β-nicotinamide mononucleotide (NMN, 40.0 μM), the lowest effective concentration of palm seed water extract WE was 0.1 mg / mL. At this concentration, there was a significant difference in cell survival rate compared with the model group, and the anti-aging effect was better than the positive drug. The anti-aging effect was obvious, so the following HUVEC cell-level experiments were all carried out at a concentration of 0.1 mg / mL.

[0067] Table 1 Cell survival rate of HUVECs treated with different concentrations of WE (%)

[0068]

[0069] Therefore, the HUVEC cells cultured at the above-mentioned dosage concentration of 0.1 mg / mL were used to perform cell scratch assays, Transwell cell invasion assays, etc. to compare and evaluate the cell proliferation and cell migration ability of HUVEC cells before and after administration of the palm seed aqueous extract WE. The above experiments were all performed according to conventional methods in the art.

[0070] The morphology of HUVEC cells under the above different experiments was photographed using an inverted fluorescence microscope to observe the changes in the corresponding cell morphology before and after the administration of palm seed aqueous extract WE, such as Figure 2 As shown, the results showed that the proliferation and migration abilities of HUVEC cells after WE administration of palm seed water extract had a significant positive effect compared with the model group cells, and the effect was stronger than that of the positive drug group.

[0071] At the same time, the cell senescence-related β-galactosidase (SA-β-Gal) staining activity experiment was carried out using HUVEC cells cultured at the lowest effective concentration of 0.1 mg / mL. The cell senescence β-galactosidase staining kit was used to detect the SA-β-Gal activity level in the target cells using X-Gal as the substrate. The target cells were seeded into 6-well cell culture plates with a cell density of no less than 8×10 5Each well was filled with at least 3 mL of complete ECM medium. The cells were placed in a 37°C incubator and the experiment was performed when the confluency reached approximately 60%. During the experiment, the kit was removed from the refrigerated freezer combination low-temperature storage box in advance and equilibrated to room temperature (25°C, other conditions apply). The water bath was opened in advance and preheated to 37°C before the experiment began. The cell culture medium was aspirated using a Pasteur pipette, the cells were washed twice with PBS, and 1 mL of β-galactosidase staining fixative was added. The cells were fixed at room temperature for 15 minutes. After fixation, the fixative was discarded to obtain the staining working solution. 1 mL of staining working solution was added to each well. The edges of the 6-well cell culture plate were sealed with parafilm and placed in a water bath. The plate was incubated at 37°C overnight (12 hours). After incubation, the staining solution was discarded and 1 mL of PBS was added. Given that senescent cells have highly active β-galactosidase, in situ staining was performed using X-Gal as a substrate. Under the catalysis of β-galactosidase, a dark blue product was generated. The staining was observed under an ordinary 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 drug NMN and palm seed water extract WE were administered, the dark blue staining product was significantly reduced, and the effect of the palm seed water extract WE group was better than the positive drug NMN, indicating that palm seed water extract WE has good anti-aging activity.

[0072] Example 3 In vivo pharmacodynamics experiment on delaying aging and protecting cardiovascular system of the palm seed aqueous extract WE prepared in Example 1

[0073] 1. Materials

[0074] 1.1 Model Organisms

[0075] Caenorhabditis elegans (wild-type N2 nematode strain, product number: SYSK-JC-001) and Escherichia coli feed strain OP50 were purchased from SunyBiotech, Fuzhou, China. C57BL / 6J mice were purchased from Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd., license number: SCXK(Su)2020-0009. The experimental animal license of South-Central University for Nationalities is SYXK(E)2021-0089.

[0076] 1.2 Drugs and reagents

[0077] 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.

[0078] 1.3 Instruments

[0079] Low-temperature microbial incubator (Thermo, model: BK6160), multi-channel non-invasive blood pressure monitor for mice and rats (Nanjing Calvin Biotechnology Co., Ltd., model: Medlab-3DX), mouse rotarod fatigue apparatus (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.

[0080] 2 Methods

[0081] 2.1 Preparation of culture medium and related reagents

[0082] To prepare LA medium: Add 10g peptone, 5g yeast extract, and 10g sodium chloride to a 500mL glass Erlenmeyer flask. Add an appropriate amount of distilled water to a constant volume of 500mL and stir thoroughly. Autoclave at 120°C for 20 minutes to sterilize the medium. After sterilization, allow the medium to cool to room temperature before use.

[0083] LB medium was prepared by adding 2.0 g of sodium chloride, 2.0 g of tryptone, and 1.0 g of yeast extract. An appropriate amount of distilled water was added to adjust the volume to 200 mL. The mixture was sterilized by autoclaving at 120°C for 20 min and set aside.

[0084] Preparation of NGM culture medium: 2g of peptone, 16g of agar, and 2.4g of sodium chloride are placed in a clean 1000mL glass conical flask, 20mL of 1M potassium phosphate buffer is added, and the volume is made up to 800mL with distilled water (this mixture is the initial culture medium), sterilized by high-pressure steam at 120℃ for 30min, and then cooled in a 55℃ water bath; 0.8mL of 5mg / ml cholesterol (dissolved in ethanol, not sterilized), 0.8mL of high-pressure sterilized 1MMgSO4, and 0.8mL of 1M CaCl2 are added thereto in sequence, and shaken to obtain a complete culture medium.

[0085] Preparation of 1M potassium phosphate buffer: weigh 108.3g KH2PO4 and 35.6g K2HPO4, dissolve in 1000mL distilled water, and adjust the pH to 6.0;

[0086] Preparation of S-buffer: 0.22g K2HPO4, 1.2g KH2PO4, 1.2g sodium chloride, add distilled water and stir until completely dissolved, then dilute to 200mL, filter, autoclave at 120℃ for 15min, and set aside.

[0087] Preparation of M9 buffer: 42.26 mM Na2HPO4, 22.04 mM KH2PO4, 85.56 mM NaCl and 0.87 mM MgSO4.

[0088] 2.2 C. elegans lifespan experiment and high temperature heat stress experiment

[0089] 2.2.1 Cultivation and plating of Escherichia coli OP50

[0090] OP50 Culture: OP50 is the primary food source for E. coli. Streak a plate containing live OP50 bacteria in LB medium. Without sealing with parafilm, invert the plate and incubate overnight at 37°C. Use a pipette or inoculating loop to pick a single colony and inoculate it into 0.5 mL of LB medium (placed in a 1.5 mL centrifuge tube). Shake the medium at 37°C at 220 rpm for 1-2 days. Turbidity indicates bacterial growth. Store the resulting OP50 culture in a 4°C refrigerator until ready for use.

[0091] OP50 coating: Use a disposable sterile dropper to draw up an appropriate amount of OP50 bacterial solution and drop it in the center of a standard nematode growth medium (NGM) agar plate. Avoid moving the medium over which the OP50 bacterial solution has been dropped to prevent the OP50 bacterial solution from flowing around, which is not conducive to the later nematode counting. Place it in a clean bench for 2-3 days, let it dry, and then place it in a 4°C refrigerator until it is used. This will produce NGM agar plates (60 and 100 mm in size) coated with OP50.

[0092] 2.2.2 Cultivation of Caenorhabditis elegans

[0093] C. elegans culture: Use a sterile scalpel to cut the NGM agar plate containing C. elegans, place it on an equal volume of NGM agar plate coated with OP50 (60 mm size), and culture it in an incubator at 20°C until use.

[0094] 2.2.3 C. elegans expansion culture

[0095] Generally, one 60mm NGM plate can be used to scale up 8-10 100mm NGM plates. Use a clean scalpel blade to divide the NGM plate obtained in step 2.2.2 into eight equal pieces. Place each piece upside down on a 100mm NGM plate coated with OP50 to scale up the plate to obtain eight 100mm NGM plates.

[0096] 2.2.4 Synchronization of Caenorhabditis elegans includes the following steps:

[0097] Collect worm bodies and eggs: After 2-3 days, when there are a large number of adult nematodes on the NGM agar plate obtained in 2.2.3 or OP50 has been almost completely consumed, start nematode synchronization. Wash the nematodes on the culture dish into a 15mL centrifuge tube. Take 4 plates as a group, add 3mL of M9 buffer to each plate, shake or tap the culture plate to the extent that the liquid does not spill, so that the nematodes are washed by the liquid. Tilt the culture plate and use a glass pipette (plastic pipette tips will stick to some worms and cause losses) to transfer as many worms as possible to the centrifuge tube. Then, select 2 plates with more worm bodies in the group, add 2mL of M9 buffer to each plate, wash, transfer the liquid to the other two plates, and then merge them into the same 15mL centrifuge tube.

[0098] Lyse the nematodes to release the eggs: Centrifuge the tubes at 600 rpm for 30 seconds. Carefully remove the supernatant using a pipette or pump, taking care not to resuspend the nematodes. Prepare bleaching lysis buffer according to the following recipe (mass ratio of NaOH:NaClO:H₂O = 1:2:7). Start the timer and add 5 mL of lysis buffer to each tube. Cover the tube and shake and vortex continuously for 4 minutes. (This step requires strict attention and should generally not exceed 5 minutes.) Observe that most of the nematodes have begun to disappear from the solution (incomplete lysis will result in excessive impurities, while excessive lysis will damage the eggs and reduce the hatching rate. Weaker strains may even fail to hatch). After lysis is complete, immediately add 10 mL of M9 buffer to each tube, mix quickly, and proceed to the next step as soon as possible.

[0099] Wash eggs: Centrifuge at 4000 rpm for 2 minutes, discard the supernatant (directly pour out the liquid in the tube by inverting it). Add 10-15 mL of M9 buffer, resuspend, and centrifuge again to collect the eggs. Repeat this wash process at least three times. After each wash, combine the collected eggs to obtain an egg suspension.

[0100] Determine the egg concentration and plate the eggs: Prepare a 1.5 mL tube, add 90 μL of M9 buffer, and then add 10 μL of egg suspension. After mixing, pipette two 10 μL aliquots of the mixture onto the plate lid. Count the eggs under a microscope, aiming for 500-1000 eggs per 10 μL suspension. Using 3000 eggs as the base multiplier, plate the eggs onto the prepared NGM agar plates at 1x (3000), 2x (6000), and 3x (9000) for each experimental condition. Once the NGM agar plates are nearly dry, turn them upside down and place them in a 20°C incubator.

[0101] Nematode Collection and Synchronization: Observe the nematodes under a microscope. After culturing for two days, collect adult nematodes in the egg-laying stage. Synchronize the nematodes using the NaClO bleaching method: wash the egg-laying adult nematodes with M9 buffer solution into a sterile EP tube. Add 1 mL of lysis buffer to lyse the nematodes, shake repeatedly for 2 minutes, and then centrifuge at 3000 rpm for 1 minute. Discard the supernatant and wash twice with M9 buffer to remove the lysis buffer. Discard the supernatant and pipette the eggs from the nematodes after lysis. Place them dropwise onto NGM agar plates. After 2 days of culture in a 20°C incubator, the eggs will have essentially developed into L4 worms, completing synchronization.

[0102] 2.2.5 C. elegans lifespan determination

[0103] L4 worms, 30 per plate, were fed with 0 (control), 1.0, 0.5, or 0.25 mg / mL WE, or DMSO (1%) solvent control mixed with S-buffer. Worms were considered dead when they showed no response to repeated prodding with a pick and lacked a pharyngeal drum. Dead worms were counted daily, and any worms that crawled off the plate or bag were excluded.

[0104] In stress resistance tests, the number of surviving worms is monitored after exposure to a designated stressor. L4 worms are treated with 0 (control), 1.0, 0.5, or 0.25 mg / mL WE and then placed in a 37°C incubator for heat stress. Survival status is observed every hour.

[0105] The life span of Caenorhabditis elegans was prolonged after administration of palm seed aqueous extract WE and the changes in its survival ability under high temperature stress were as follows Figure 4 The changes in mean, median, and maximum lifespan of C. elegans under normal living conditions and high-temperature heat stress following administration of palm seed aqueous extract (WE) are shown in Tables 2 and 3. WE extended the lifespan of C. elegans in a dose-dependent manner. The 1.0 mg / mL WE concentration group extended the mean lifespan by 15.26% and the median lifespan by 11.11%. Furthermore, WE-treated C. elegans exhibited enhanced resistance to heat stress. Under high-temperature stimulation, the mean lifespan of mice treated with 1.0, 0.5, and 0.25 mg / mL WE increased by 20.29%, 27.50%, and 24.46%, respectively, and the median lifespan by 50.00%, 37.50%, and 50.00%, respectively. These results demonstrate that WE can effectively extend the lifespan of C. elegans and improve their resistance to high-temperature heat stress.

[0106] Table 2 Changes in the life cycle of Caenorhabditis elegans under different WE concentrations

[0107]

[0108] Table 3 Changes in the survival lifespan of Caenorhabditis elegans under heat stress under different WE concentrations

[0109]

[0110] 2.3 Animal experiments

[0111] 2.3.1 Animal grouping and drug administration

[0112] In this experiment, 30 7-week-old C57BL / 6J mice were weighed and randomly divided into 5 groups: blank group (normal saline), model group (D-gal, 300 mg / kg), positive drug group (D-gal+NMN, 300 mg / kg+300 mg / kg), palm seed water extract WE high-dose administration group (D-gal+WE-High, 300 mg / kg+100 mg / kg), palm seed water extract WE low-dose administration group (D-gal+WE-Low, 300 mg / kg+50 mg / kg). Except for the normal saline group, the drugs in other groups were dissolved in normal saline and prepared to the corresponding concentrations.

[0113] Six 16-month-old C57BL / 6J mice were used as the natural aging group (normal saline). Six groups of C57BL / 6J mice were administered the drug via gavage and housed in an equivalent SPF-grade housing facility for eight consecutive weeks (drug administration was continued every other day after the first dose until the end of the animal experiment).

[0114] In the 7th week, six 6-week-old C57BL / 6J mice were purchased as the juvenile group (Young, normal saline) and raised in the same SPF-grade breeding room as the other groups for one week.

[0115] The in vivo animal experiment was divided into 7 groups (blank group, model group, positive drug group, palm seed water extract WE high-dose group, palm seed water extract WE low-dose group, natural aging group and juvenile group). The weight of mice was weighed and recorded at the same time every day (the average value was used for each group). The weight changes of mice are shown in the figure below. Figure 7 As shown in Figure 2, the weight of mice increased steadily throughout the experiment, and the drug administration had no significant effect on the normal growth of mice. The microvascular density of the hind limbs of mice was measured 3 days before the autopsy. Figure 10 As shown, after dissection, the thoracic aorta and serum were obtained and stored at -80°C for later use.

[0116] 2.3.2 Blood pressure measurement using a multi-channel non-invasive blood pressure monitor for mice and rats

[0117] The blood pressure of mice was measured using a multi-channel non-invasive blood pressure monitor for rats and mice. 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 was measured after the mice adapted. This was repeated 3 times, and the average value was used as the blood pressure value of the mice. Blood pressure was measured once every 6 days during the administration period. The results are shown in the figure below. Figure 8 As shown, oral administration of D-gal to aging model mice using palm seed water extract WE can significantly reduce the increase in blood pressure caused by aging.

[0118] 2.3.3 Testing exercise endurance using the rotarod fatigue apparatus

[0119] To accurately assess differences in motor ability among the drug-treated, naturally aged, juvenile, positive drug, and model groups, a rotarod fatigue test was performed on the mice. The mice's motor coordination and balance abilities were assessed based on the time they spent on the rotarod until they fell and the distance they traveled as recorded by the rotarod fatigue tester. A 30 mm diameter rotarod and a 6-channel rotarod fatigue tester were used to test the mice's hindlimb exercise endurance. The ventilation, noise level, temperature, humidity, and lighting intensity were maintained consistent across all experiments. Two days before the experiment, the mice were trained on the rotarod. The speed at which the majority of mice could achieve the desired speed was monitored and recorded, and this speed was used as the final rotational speed. On the day of the experiment, the mice were placed on a uniformly rotating rotarod, and the time it took for each mouse to fall was recorded. Each mouse completed three rotarod fatigue tests, with a one-hour interval between each test.

[0120] During the administration process, the mice's exercise endurance was continuously monitored and recorded every 8 days. The average exercise distance and exercise time were then calculated. The results are shown in the figure. Figure 9 As shown, the indicators of mouse exercise endurance, Figure 9 The vertical coordinate of A is the movement distance, Figure 9 The vertical axis B is the duration of exercise. The greater the exercise distance, the longer the duration, indicating that the mouse has better hind limb exercise endurance. Figure 9 It can be seen that after the aging model was established by gavage of D-gal with palm seed water extract WE, the exercise endurance of mice was significantly improved compared with the model group and the natural aging group, and the decline in hind limb motor ability of mice associated with aging was effectively reduced.

[0121] 2.3.4 Detection of microvascular density using the 2100 high-resolution small animal ultrasound imaging system

[0122] Anesthetize the mouse with 1.5% isoflurane. Once 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 vaporizer is connected to the anesthesia mask. After maintaining the concentration, remove the mouse from the induction box, place its head / nose in the anesthesia mask, 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 to perform ultrasound imaging to obtain blood oxygen characterization of the mouse's hind limb blood flow and vascular density, and then analyzed and concluded based on the imaging. After the animal experiment was completed, the vaporizer was turned off and the animal was kept breathing in pure oxygen for about 5-10 minutes to facilitate rapid awakening of the animal. Figure 10 As shown in the results, after oral administration of palm seed water extract WE and feeding for 8 weeks, the microvascular density of the hind limbs of mice was significantly higher than that of the model group and the natural aging group. The control showed that palm seed water extract WE can significantly improve the blood oxygen concentration of the hind limbs of mice, and the effects of the high and low dose palm seed water extract WE groups were better than those of the positive drug group, further indicating that palm seed water extract WE can improve the microvascular density of the hind limbs of mice.

[0123] 2.3.5 Vascular pathology (HE staining, EVG staining, Masson staining, transmission electron microscopy) observation

[0124] HE staining: Removed mouse aorta tissue was fixed in a prepared 10% formalin fixative to preserve the original morphology and cellular structure. Then, the tissue blocks were dehydrated using anhydrous alcohol gradient elution. The tissue blocks were then cleared in xylene, embedded in wax, and sliced ​​into 4 μm thin sections using a microtome. The sections were then baked to dissolve the wax, dewaxed, and hydrated with a gradient of alcohol. The sections were then stained with hematoxylin for 3 minutes, rinsed with running water for 3 minutes, differentiated, washed with water, blued, rinsed with running water for 10 minutes, stained with eosin, dehydrated with a gradient of alcohol, and mounted for microscopic observation and photography.

[0125] EVG staining: Removed mouse aorta tissue was fixed in a prepared 10% formalin fixative to preserve the original morphology and cell structure. Then, the tissue blocks were washed with anhydrous alcohol gradient to remove moisture. The tissue blocks were then cleared in xylene, embedded in wax, and cut into 4 μm slices using a microtome. The sections were dewaxed and stained with Verhoeff stain at room temperature for 2–3 minutes. The sections were rinsed with running water, then differentiated with 2% ferric chloride for 15–20 seconds, rinsed with running water, and counterstained with Van Gieson stain for 10 seconds. The sections were dehydrated, mounted, and examined under a microscope and photographed.

[0126] Masson staining: The tissue sections were dewaxed and hydrated, and the Masson staining kit (purchased from Wuhan Boerfu Biotechnology Co., Ltd., product 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 purified water for 8-10s, then stain with Weigert iron hematoxylin staining solution prepared in the Masson staining kit for 8min, differentiate in 1% acidic hydrochloric acid ethanol differentiation solution for 15s, wash with water; Masson blueing solution to return to blue for 5min, wash with water; then stain with Ponceau red staining solution prepared in the Masson staining kit for 5min, quickly rinse with distilled water; then wash in 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 three times with anhydrous ethanol for 5-10s each time, clear with xylene three times for 1-2min each time, seal with neutral gum, and observe under a microscope after dehydration and clearing.

[0127] Transmission electron microscopy: Take fresh thoracic aorta tissue (section no more than 3mm 2 ), quickly rinsed with PBS, placed in electron microscopy fixative at room temperature, and after 2 hours, transferred to 4°C for storage until ready for use. Fixed samples were rinsed three times with 0.1M, pH 7.4 phosphate buffer for 15 minutes each, fixed in 1% osmium phosphate for 1-2 hours at room temperature in the dark, and rinsed again three times with 0.1M, pH 7.4 phosphate buffer. Dehydrated, dried in a desiccator, and the samples were attached to conductive carbon film for conductivity. Observed with a scanning electron microscope and photographed.

[0128] The above pathological sections are as follows Figure 11 As shown in Figures AD, oral administration of D-gal to aging mice established with palm seed water extract WE can significantly improve the growth of aortic endothelial cells and the elasticity of the aortic vascular wall in mice, and the effects of the high and low doses of palm seed water extract WE groups are better than those of the positive drug group, further demonstrating that palm seed water extract WE can improve the functional morphology of the aorta in mice.

[0129] 23.6 Observation of liver and kidney pathology (HE staining)

[0130] HE staining: After dissecting the mouse, the liver and kidney tissues were separated and each sample was fixed in 10% formalin solution, then dehydrated in anhydrous alcohol gradient, and then the tissue blocks were placed in xylene as a transparent agent to make them transparent. Finally, they were embedded in paraffin and cut into 4 μm thick slices using a microtome. The slices were waxed, dewaxed, hydrated in alcohol gradients, 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 in alcohol gradients, and sealed for observation under a microscope. The pictures were taken. The results are shown in the figure. Figure 12 .

[0131] The aging process is accompanied by fatty degeneration of the liver, loosening and vacuolation of the liver tissue, and loose arrangement of kidney tissue fibers. Figure 12 In the D-gal-established aging model group and the Nature aging group, the liver tissue loosened and vacuolated, and the fiber gaps in the kidney tissue became larger. After WE administration, it can be clearly seen that the liver tissue arrangement became tighter, the vacuolation phenomenon decreased, and the fiber gaps in the kidney tissue shrank; this shows that WE has a good effect on improving liver tissue vacuolation and maintaining the normal morphology of kidney fibers, and the effect of WE administration is better than that of the positive drug NMN.

[0132] 2.3.7 Hepatotoxicity index detection

[0133] Before dissection, blood was collected from the eyeballs, and the supernatant was collected and centrifuged at 12,000 rpm for 15 minutes at 4°C. The supernatant was then stored at -80°C for further experimental analysis. ELISA kits were used to detect serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. The aging process is accompanied by liver aging and impaired liver function. ALT and AST are highly expressed in the liver. However, after administration of palm seed water extract WE, the expression of liver ALT and AST indicators decreased. The results are as follows: Figure 13 shown.

[0134] 2.3.8 Western blot expression of p16 and p21 proteins in mouse thoracic aorta tissue

[0135] After mouse autopsy, thoracic aorta tissue was isolated and rinsed with pre-chilled PBS to remove any residual blood. Excess PBS was then aspirated. The tissue was then aliquoted into approximately 100 mg pieces, individually wrapped in foil, and stored in liquid nitrogen. p16 and p21 proteins were extracted from the aortic tissue, and protein concentrations were determined using a BCA protein assay kit. Protein samples were separated by SDS-PAGE electrophoresis, transferred from the gel to a NC membrane, and stained with Ponceau red. The membrane was then immediately rinsed in distilled water. The electrotransferred NC membrane was wetted with 5% BSA in TBST and gently 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 at 4°C overnight. After washing with TBST buffer (10×, pH 7.4, Biosharp, Catalog No.: BL315B), the membrane was incubated with the secondary antibody (1:5000, Mitaka, Catalog No.: SA00001-1) diluted solution 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 proteins was normalized to the expression of β-actin. The results are shown in Figure 2. Figure 14 As shown in Figure B, since p16 and p21 proteins are aging indicator proteins, their expression increases with accelerated aging, indicating that D-gal successfully established an aging model. Over the course of administration, the expression of these two proteins decreased in the WE-treated group compared to the Model and NatureAging groups, demonstrating that both high- and low-dose WE administrations have an aging-delaying effect. In summary, the palm seed aqueous extract (WE) of the present invention inhibits D-gal-induced HUVEC cell senescence, delays mouse aging, protects vascular endothelial function in aged mice, and affects the expression of p16 and p21 proteins in mice.

[0136] Example 4: The palm seed aqueous extract WE prepared in Example 1 affects aging-related phenotypes

[0137] 1. Materials

[0138] 1.1 Drugs and reagents

[0139] ELISA kits were used to detect IGF-1, LaminB1, γH2A.X, p16, and p21 in the HUVECs described in Section 2.1 of Example 2 and in the animal serum samples described in Section 2.3 of Example 3. The IGF-1 ELISA kit was purchased from ABclonal Biotechnology Co., Ltd.; the LaminB1 ELISA kit was purchased from Shanghai Fusheng Industrial Co., Ltd.; the p16 and p21 ELISA kits were both purchased from Ruixin Biotechnology Co., Ltd.; and the inflammatory factor multifactor ELISA kit was purchased from ABclonal Biotechnology Co., Ltd.

[0140] 2 Methods

[0141] 2.1 SASP aging-related secretory phenotype detection experiment

[0142] Flow cytometry is used to quantitatively detect IL-1α, IL-1β, IL-6, IL-10, TNF-α, IFN-γ, CCL2, and CCL3 in mouse biological specimens. First, the sample is mixed with a suspension of microspheres containing capture antibodies. The analytes in the sample bind to the specific capture antibodies on the microspheres. Next, the detection antibody is added, specifically binding to the reaction products from the previous step. Then, a fluorescein solution is added. The streptavidin on the fluorescein binds to the biotin on the detection antibody, forming a capture antibody-antigen-biotin complex on the microsphere surface. This "antigen-biotinylated antibody-streptavidin-fluorescein" complex forms on the microsphere surface. When detected by flow cytometry, the microsphere complexes differ. Different microsphere complexes can be identified and analyzed using flow cytometry to determine the specific analyte type and corresponding concentration, thereby enabling quantitative detection of SASP factors in the sample.

[0143] The changes of inflammatory factors such as IL-10, IL-6, IL-1α, IL-1β, CCL2, CCL3, INF-γ, and TNF-α are shown in the figure. Figure 5 With the aging process, the expression of pro-inflammatory factors such as IL-6, IL-1α, IL-1β, CCL2, CCL3, INF-γ, and TNF-α increases. In this experiment, D-gal was used to establish an aging model. Compared with the control group, the expression of proinflammatory factors such as IL-6, IL-1α, IL-1β, CCL2, CCL3, INF-γ, and TNF-α in the aging group was significantly increased, indicating that the D-gal aging model was successfully established in this experiment. After WE administration, the expression of proinflammatory factors such as IL-6, IL-1α, IL-1β, CCL2, CCL3, INF-γ, and TNF-α was reduced compared with the model group and the NatureAging group, indicating that WE administration can effectively reduce the expression of proinflammatory factors such as IL-6, IL-1α, IL-1β, CCL2, CCL3, INF-γ, and TNF-α in vivo. At the same time, the anti-inflammatory factor IL-10 was expressed at a low level in the model group and the NatureAging group, but its expression increased after WE administration compared with the model group and the NatureAging group. In summary, the palm seed aqueous extract WE has the effect of reducing and alleviating the inflammatory response in vivo caused by aging.

[0144] 2.2 Double-antibody enzyme-linked sandwich ELISA to detect the expression of p16, p21, IGF-1, LaminB1, and γ-H2A.X

[0145] ① Remove the unused microwell strips from the plate frame in the kit and wash them. Place the remaining strips back into the aluminum foil bag containing desiccant and reseal for storage. Wash by adding 350 μL of 1x PBS buffer to each well, letting it sit for 40 seconds, then discarding the liquid. This step should be repeated three times. Add 100 μL of standard / sample diluent (R1) to a blank well. Add 100 μL of standard or sample at different concentrations to each of the remaining wells. Seal the wells with the provided sealing film and incubate at 37°C for 2 hours.

[0146] 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°C for 1 hour. 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°C for 30 minutes.

[0147] ③ After completing the above steps, preheat the microplate reader. Discard the liquid in the wells and wash according to the washing method in ① above. Add TMB substrate (100 μL / well) to the wells. Incubate at 37°C in the dark for 15-20 minutes. Finally, add stop solution (50 μL / well) and immediately place the wells in the microplate reader. Measure the OD value of each well at 450 nm within 5 minutes.

[0148] ④ Select the calibration 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.

[0149] The schematic diagram of changes in inflammatory factors such as IGF-1, LaminB1, and γ-H2A.X is shown in Figure 6, the results showed that the IGF-1 content caused by aging was low in the model group and the natural aging group, and the IGF-1 content in the plasma increased after treatment with high and low doses of WE. This indicator also proves that WE has the effect of delaying vascular aging. In aging human and mouse cells, the expression of the cytoplasmic morphological marker LaminB1 is lost. The loss of LaminB1 and the increased accumulation of p21 and p16 are typical signs of aging. γ-H2A.X is a variant of histone H2A and a typical marker of the DDR pathway. When DNA is damaged, H2A.X is phosphorylated at Ser139 to form γ-H2A.X. As aging intensifies, the expression of γ-H2A.X in aging cells increases. Therefore, LaminB1 was lowly expressed in the aging model group and the natural aging group, and γ-H2A was highly expressed. After WE administration, the expression of LaminB1 in the high and low dose WE groups increased to varying degrees, and the expression of γ-H2.X decreased. Figure 6 In summary, there is evidence that WE can exhibit a positive effect in delaying aging by regulating the levels and expressions of several humoral markers of vascular aging in vivo before and after treatment, indicating that WE has the effects of delaying vascular aging and cardiovascular protection.

[0150] Example 5 Confirmation of the pathway and target of the palm seed aqueous extract WE prepared in Example 1 in delaying aging and protecting cardiovascular function

[0151] 1. Sample processing

[0152] 1.1 Protein extraction

[0153] The animal tissue samples from Section 2.3 of Example 3 were removed from a −80°C freezer and ground into a powder in liquid nitrogen. An appropriate amount of the powder was then transferred to a 1.5 ml centrifuge tube and lysis buffer (containing 8 M urea, 1 mM PMSF, and 2 mM EDTA) was added. The tube was sonicated on ice for 5 min and centrifuged at 15,000 g for 10 min at 4°C. The supernatant was collected. Finally, the protein concentration was determined using a BCA kit (Shanghai Biotech Co., Ltd.).

[0154] 1.2 Protein enzymatic desalination

[0155] According to the protein concentration, 100 μg of protein solution was taken and the volume was made up to 200 μL with 8 M urea. Then, DTT (final concentration 5 mM) was added for reduction at 37 ° C for 45 min, and iodoacetamide (final concentration 11 mM) was used for alkylation for 15 min in a dark room at room temperature. Then, 800 μL of 25 mM ammonium bicarbonate solution and 2 μL of trypsin (Promega, V5280) were added and digested at 37 ° C overnight. The enzymatic peptide fragments were adjusted to pH 2-3 with 20% TFA and desalted with C18 (Millipore, Billerica, MA) column material. Finally, PierceTM The peptide concentration was determined using a quantitative peptide detection reagent and standard kit (Thermo Fisher).

[0156] 2LC-MS / MS detection

[0157] 2.1 Nanoliter liquid chromatography detection

[0158] Samples were separated using a Vanquish Neo UHPLC nanoliter liquid phase system. Mobile phase A consisted of a 0.1% formic acid-water solution, and mobile phase B consisted of a 0.1% formic acid-acetonitrile solution (100% acetonitrile). The injection mode was a capture-analysis dual-column method, with the trap column being a PepMap Neo Trap Cartridge (300 μm × 5 mm, 5 μm) and the analytical column being an Easy-Spray TM PepMap TM Neo UHPLC column (150 μm × 15 cm, 2 μm). The temperature of the analytical column was controlled at 55°C by an integrated column oven, the sample load was 200 ng, the flow rate was 2.5 μL / min, the effective gradient was 6.9 min, and the total run time was 8 min.

[0159] 2.2 Orbitrap Astral mass spectrometer detection

[0160] DIA analysis was performed using a nanoliter-speed Vanquish Neo system (Thermo Fisher Scientific) for chromatographic separation. Samples separated by nanoliter-scale HPLC were analyzed by DIA (data-independent) mass spectrometry using an Orbitrap Astral high-resolution mass spectrometer (Thermo Scientific). Detection mode: positive ion, parent ion scan range: 380-980 m / z, primary mass spectrometry resolution: 240,000 at 200 m / z, Normalized AGC Target: 500%, Maximum Time Intensity: 5 ms. MS2 data acquisition mode: DIA, with 299 scan windows, Isolation Window: 2 Th, HCD Collision Energy: 25%, Normalized AGC Target: 500%, Maximum Time Intensity: 3 ms.

[0161] 3 Mass spectrometry data analysis

[0162] Mass spectrometry analysis can obtain the mass-to-charge ratio and signal intensity of peptides in the sample, as well as the mass-to-charge ratio and signal intensity of fragment ions after peptide fragmentation. Information at the peptide level is usually called primary spectrum, and peptide fragment ion information is called secondary spectrum. The information contained in the spectrum is very complex, and it requires the help of a database to correctly parse out the peptide sequences potentially contained in the spectrum. Before searching, a theoretical secondary spectrum database is constructed based on the protein sequences in the database. The secondary spectrum generated by the mass spectrometry is then searched and compared with the theoretical secondary spectrum, and the correctly matched theoretical peptide sequence is obtained after algorithm scoring and filtering. The protein information contained can be identified through the identified protein-specific peptides. Database retrieval is a complex computational process, and professional mass spectrometry data analysis software is required for data analysis. The library search software used for DIA mass spectrometry data in the present invention is DIA-NN (v1.8.1), and the library search is performed using the Libraryfree method. The library search parameters are as follows:

[0163] The database was uniprotkb_proteome_UP000000589_mouse_54910_20240528.fastadatabase (a total of 54,910 sequences). The deep learning-based parameters were checked to predict a spectral library. MBR (Match Between Runs) was checked to generate a spectral library using DIA data, and the DIA data were reanalyzed using this spectral library to obtain protein qualitative and quantitative results. The FDR (False Discovery Rate) for both precursor ion and protein level identification was filtered at 1%, and the filtered data could be used for subsequent bioinformatics analysis.

[0164] 4 Bioinformatics Analysis

[0165] To gain a thorough understanding of the functional properties of different proteins, the inventors performed comprehensive functional annotation on the identified proteins and the differentially expressed proteins in each comparison group, including aspects such as Gene Ontology (GO), KOG functional classification, KEGG pathway, protein domain, subcellular localization, and signal peptide (SignalP). The differentially expressed proteins in each comparison group were then enriched at four levels: GO classification, KOG functional classification, KEGG pathway, and protein domain. The enrichment analysis ultimately determined the significance of the enrichment using hypergeometric calculations using the P-value. The goal was to determine whether the differentially expressed proteins showed significant enrichment trends in certain functional categories relative to the background proteins (here, all identified proteins). This allowed for a more comprehensive assessment of the physiological functions in which the differentially expressed proteins were involved.

[0166] In order to determine the specific pathways and mechanisms by which WE delays vascular aging and regulates the expression levels of chronic inflammatory factors in the body, the inventors analyzed the proteomic results. First, the inventors analyzed the differences in the expression levels of differentially expressed proteins in the two groups of samples and their statistical significance. Figure 15 As shown. KEGG analysis showed that WE may delay vascular aging through the cGMP-PKG signaling pathway, and the related proteins Q60930 (VDAC2), Q60932 (VDAC1) and Q6S9I0 (KNG2) in this pathway were also among the differentially expressed proteins detected. ( Figure 15 A, B). According to the literature [Aburima, A., Walladbegi, K., Wake, JD, & Naseem, KM (2017). cGMP signaling inhibits platelet shape change through regulation of the RhoA-Rho Kinase-MLC phosphatase signaling pathway. Journal of thrombosis and haemostasis: JTH, 15(8), 1668–1678. https: / / doi.org / 10.1111 / jth.13738 ], Rhoa is a key protein in the cGMP-PKG signaling pathway. To further verify the effect of WE on the expression of these proteins, the inventors conducted WB experiments, and the results were as follows Figure 15 Figure C. In the thoracic aorta, Rhoa and KNG2 proteins are highly expressed in aging but decreased after WE treatment. VDAC1 and VDAC2 proteins are low in aging but increased after WE treatment. This suggests that WE's ability to slow vascular aging may be related to these four protein targets.

Claims

1. Use of palm seed aqueous extract WE in the preparation of anti-aging drugs and / or skin care products; and / or Application of palm seed water extract WE in the preparation of cardiovascular protection drugs; The palm seed water extract WE is obtained by using palm seeds as raw materials and extracting with pure water.

2. The use according to claim 1, characterized in that The preparation method of the palm seed aqueous extract WE comprises the following steps: The palm seeds are crushed, soaked and extracted with pure water for a period of time at a certain material-liquid ratio, then ultrasonically extracted for a period of time, and then subjected to oil bath reflux extraction for 1-4 times; the filtrate is filtered and concentrated to obtain the palm seed water extract WE.

3. The use according to claim 1 or 2, characterized in that The preparation method of the palm seed aqueous extract WE comprises the following steps: The palm seeds were crushed, soaked and extracted with pure water for a period of time at a certain material-liquid ratio, then extracted with 600W ultrasonic wave for 30min, refluxed in an 80℃ oil bath twice, each time for 2h, filtered and concentrated to obtain the palm seed aqueous extract WE.

4. The use according to claim 1, characterized in that The amount of the palm seed water extract WE used in the preparation of anti-aging drugs is 11.0 mg / kg / d; and / or The amount of the palm seed water extract WE used in the preparation of cardiovascular protection drugs is 11.0 mg / kg / d; and / or When the palm seed water extract WE is used to prepare anti-aging skin care products, the concentration of the palm seed water extract WE is 0.01-1.0 mg / mL.

5. The use according to claim 1, characterized in that The drug dosage form is tablets, capsules or oral liquid, and the skin care product is skin care lotion, facial mask or skin care cream.

6. The use according to claim 1, characterized in that: The palm seed aqueous extract WE is used as a raw material for a preparation for reducing β-galactosidase activity in senescent cells; and / or The palm seed water extract WE is used as a raw material for a preparation for restoring the proliferation ability of senescent cells; and / or The palm seed water extract WE is used as a raw material for a preparation for restoring the migration ability of senescent cells; and / or The palm seed aqueous extract WE is used as a raw material for a preparation for improving exercise endurance; and / or The palm seed water extract WE is used as a raw material for a preparation for improving blood circulation in lower limbs; and / or The palm seed water extract WE is used as a raw material for a blood pressure lowering preparation.

7. The use according to claim 1, characterized in that: The palm seed water extract WE is used to inhibit the pro-inflammatory factors IL-6, IL-1α, IL-1β, CCL2, CCL3, INF-γ, and TNF-α produced by cell aging and to upregulate the anti-inflammatory factor IL-10.

8. The use according to claim 1, characterized in that: The palm seed water extract WE is used to upregulate the expression of IGF-1 and LaminB1 in the body and inhibit the expression of γ-H2A.X.

9. The use according to claim 1, characterized in that: The palm seed water extract WE is used to inhibit the expression of p16 and p21 proteins in vivo and in vitro.

10. The use according to claim 1, characterized in that: The palm seed water extract WE delays vascular aging and protects cardiovascular system by regulating the cGMP-PKG signaling pathway of Rhoa, VDAC1, VDAC2 and KNG2 protein targets.

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