Application of N1, N10-dicaffeoyl spermidine in preparation of anti-aging drugs, health care products or functional food

Abstract

The invention discloses an application of N1, N10-dicaffeoyl spermidine in preparation of an anti-aging medicine, a health care product or a functional food. Belongs to the technical field of biological medicine. Animal experiments prove that the N1, N10-dicaffeoyl spermidine has an anti-aging effect, can significantly improve the ability of D-galactose to induce working memory and spatial memory of aged mice, significantly reduce the protein expression levels of aging markers P53, P21 and P16 of brain tissues of the mice, significantly reduce the contents of inflammatory factors 1L-1beta, 1L-6 and TNF-alpha of the aged mice, and significantly improve the anti-aging effect of the mice. The content of oxidative stress factors GSH and SOD is obviously improved. The N1, N10-dicaffeoyl spermidine is used for preparing the medicine, the health care product and / or the functional food for delaying, preventing and / or treating senescence, a new choice is provided for resisting senescence, and the N1, N10-dicaffeoyl spermidine has important practical application value in the fields of medicine and senescence resistance.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and more specifically relates to the application of N1,N10-dicaffeoyl spermidine in the preparation of anti-aging drugs, health products or functional foods. Background Art

[0002] Currently, the main anti-aging intervention methods include Senolytics (senescent cell scavengers), such as the combination of dasatinib + quercetin; NAD+ precursors, such as nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR); mTOR inhibitors, such as rapamycin derivatives, etc. There are also natural-source health products, such as resveratrol, curcumin, collagen peptides, etc. However, existing drugs generally have problems such as limited effects and significant side effects.

[0003] Due to their chemical diversity and low toxicity characteristics, natural products have become the core direction for the research and development of anti-aging drugs. Currently, it has been reported that natural compounds such as ginsenoside Rg1 and apigenin have anti-aging effects. Plant components (such as green tea polyphenols and salidroside) for long-term consumption or medicinal use have been historically verified, and the risk of toxic and side effects is much lower than that of synthetic drugs, making them suitable for long-term intervention.

[0004] N1,N10-dicaffeoyl spermidine is a monomeric active ingredient isolated by the inventor from medicinal and edible homologous traditional Chinese medicines such as wolfberry fruits. Currently, there are few research reports on the biological activities of N1,N10-dicaffeoyl spermidine, and there is no relevant record of N1,N10-dicaffeoyl spermidine being used for the prevention and treatment of aging. Summary of the Invention

[0005] In view of this, the present invention provides the application of N1,N10-dicaffeoyl spermidine in the preparation of drugs, health products or functional foods for the prevention and treatment of aging. Through preliminary animal experiments, the present invention found that N1,N10-dicaffeoyl spermidine has a significant anti-aging effect. Further developing it into a drug that can be used to delay, prevent and / or treat aging, or a health product or functional food that can delay, prevent and / or treat aging will have great clinical application value.

[0006] N1,N10-dicaffeoyl spermidine, with the molecular formula C 25 H 31 N 3 O 6, with a molecular weight of 469.22, and its full English name is (E)-3-(3,4-dihydroxyphenyl)-N-(3-((4-((E)-3-(3,4-dihydroxyphenyl)acrylamido)butyl)amino)propyl)acrylamide, abbreviated as N1,N10-bis(caffeoyl)spermidine or N1,N10-di-caffeoylspermidine. Its chemical structural formula is shown in Formula (1): Formula (1) To achieve the above object, the present invention adopts the following technical solutions: The application of N1,N10-dicaffeoylspermidine in the preparation of drugs for preventing and treating aging, or in the preparation of health care products or functional foods for preventing and treating aging.

[0007] Furthermore, N1,N10-dicaffeoylspermidine can significantly increase the spontaneous alternation response rate of aging mice in the Y-maze experiment, that is, improve their working memory ability.

[0008] Furthermore, N1,N10-dicaffeoylspermidine can significantly reduce the escape latency of aging mice in the Morris water maze experiment and increase the number of times of crossing the platform, that is, improve their spatial memory ability.

[0009] Furthermore, N1,N10-dicaffeoylspermidine can significantly reduce the protein expression levels of the aging markers P53, P21, and P16 in the brain tissue of aging mice.

[0010] Furthermore, N1,N10-dicaffeoylspermidine can significantly reduce the contents of the inflammatory factors 1L-1β, 1L-6, and TNF-α in the brain tissue of aging mice.

[0011] Furthermore, N1,N10-dicaffeoylspermidine can significantly increase the contents of the oxidative stress factors SOD and GSH in the brain tissue of aging mice.

[0012] An anti-aging drug, characterized in that it comprises N1,N10-dicaffeoylspermidine.

[0013] Furthermore, the dosage form is an oral dosage form, an external patch, or an injection dosage form permitted in pharmacy.

[0014] An anti-aging health care product comprising N1,N10-dicaffeoylspermidine.

[0015] An anti-aging functional food comprising N1,N10-dicaffeoylspermidine.

[0016] As can be seen from the above technical solutions, compared with the prior art, the beneficial effects achieved by the present invention are as follows: within the dose range of 10 - 25 mg / kg administered to experimental animals, N1,N10-dicaffeoyl spermidine can significantly improve the working memory and spatial memory abilities of D-galactose-induced aging mice, significantly reduce the protein expression levels of the aging markers P53, P21, and P16 in the mouse brain tissue, significantly reduce the contents of the inflammatory factors IL-1β, IL-6, and TNF-α in the aging mice, and significantly increase the contents of the oxidative stress factors SOD and GSH. The above all confirm that N1,N10-dicaffeoyl spermidine has an anti-aging effect and can be used to prepare drugs, health products, and / or functional foods for delaying, preventing, and / or treating aging. Description of the Drawings

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained according to the provided drawings.

[0018] Figure 1 Effect of N1,N10-dicaffeoyl spermidine on the body weight of aging mice (C is the normal group, M is the model group, P is the positive drug piracetam group, NNDCP-H is the high-dose group of N1,N10-dicaffeoyl spermidine, NNDCP-L is the low-dose group of N1,N10-dicaffeoyl spermidine; compared with the normal group, # p <0.05; compared with the model group, * p <0.05, ** p <0.01) Figure 2 Effect of N1,N10-dicaffeoyl spermidine on the spontaneous alternation response rate of aging mice investigated by the Y-maze experiment (C is the normal group, M is the model group, P is the positive drug piracetam group, NNDCP-H is the high-dose group of N1,N10-dicaffeoyl spermidine, NNDCP-L is the low-dose group of N1,N10-dicaffeoyl spermidine; compared with the normal group, # p <0.05, ## p <0.01, ### p <0.001; compared with the model group, ** p <0.01, *** p <0.001) Figure 3To investigate the effects of N1,N10-dicaffeoyl spermidine on the escape latency and the number of platform crossings in aging mice by Morris water maze experiment (C is the normal group, M is the model group, P is the positive drug piracetam group, NNDCP-H is the high-dose group of N1,N10-dicaffeoyl spermidine, NNDCP-L is the low-dose group of N1,N10-dicaffeoyl spermidine; compared with the normal group, ### p <0.001; compared with the model group, *** p <0.001) Figure 4 To investigate the effects of N1,N10-dicaffeoyl spermidine on the expression levels of senescence markers P53, P21 and P16 proteins in the brain tissues of aging mice (C is the normal group, M is the model group, NNDCP-H is the high-dose group of N1,N10-dicaffeoyl spermidine, NNDCP-L is the low-dose group of N1,N10-dicaffeoyl spermidine; compared with the normal group, # p <0.05, ## p <0.01; compared with the model group, * p <0.05, ** p <0.01, *** p <0.001) Figure 5 To investigate the effects of N1,N10-dicaffeoyl spermidine on the contents of inflammatory factors IL-1β, IL-6 and TNF-α in the brain tissues of aging mice (C is the normal group, M is the model group, P is the positive drug piracetam group, NNDCP-H is the high-dose group of N1,N10-dicaffeoyl spermidine, NNDCP-L is the low-dose group of N1,N10-dicaffeoyl spermidine; compared with the normal group, ## p <0.01, ### p <0.001; compared with the model group, * p <0.05, ** p <0.01, *** p <0.001) Figure 6 To investigate the effects of N1,N10-dicaffeoyl spermidine on the contents of oxidative stress factors SOD and GSH in the brain tissues of aging mice (C is the normal group, M is the model group, P is the positive drug piracetam group, NNDCP-H is the high-dose group of N1,N10-dicaffeoyl spermidine, NNDCP-L is the low-dose group of N1,N10-dicaffeoyl spermidine; compared with the model group, * p <0.05, *** p <0.001) Specific implementation manners

[0019] Next, in combination with the accompanying drawings in the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present invention.

[0020] N1,N10-Dicaffeoyl spermidine used in the following examples is the compound shown in the above formula (1), and can be obtained through commercial purchase or experimental self-preparation.

[0021] The medicaments required for the present invention are conventional experimental medicaments and are purchased from commercial channels; the experimental methods not mentioned are conventional experimental methods and will not be elaborated one by one here. Example 1

[0022] An anti-aging drug with an oral dosage form, comprising N1,N10-dicaffeoyl spermidine. Example 2

[0023] An anti-aging drug with an injection dosage form, comprising N1,N10-dicaffeoyl spermidine. Example 3

[0024] An anti-aging drug with an external patch dosage form, comprising N1,N10-dicaffeoyl spermidine. Example 4

[0025] An anti-aging health care product, comprising N1,N10-dicaffeoyl spermidine. Example 5

[0026] An anti-aging functional food, comprising N1,N10-dicaffeoyl spermidine.

[0027] The following animal experiments further illustrate the effects of the above Examples 1 to 5: I. Animal Experiment Design A D-galactose-induced aging mouse model was used. After intragastric administration of N1,N10-dicaffeoyl spermidine, the anti-aging effect of N1,N10-dicaffeoyl spermidine was evaluated by mouse body weight, Y-maze experiment, Morris water maze experiment, expression levels of aging markers P53, P21, and P16 proteins, contents of inflammatory factors IL-1β, IL-6, and TNF-α, and contents of oxidative stress factors SOD and GSH.

[0028] II. Experimental Procedure 1. Experimental Animals Healthy SPF-grade C57BL / 6 male mice weighing 20 - 30 g were provided by the Experimental Animal Center of Ningxia Medical University (Experimental Animal Use License SYSK (Ning) 2020 - 0001). They were housed in an SPF-grade barrier environment with a 12-hour light and 12-hour dark cycle and had free access to food and water.

[0029] 2. Experimental methods A D-galactose-induced aging mouse model was used. After 3 days of adaptive feeding of 40 mice, they were randomly divided into 5 groups according to body weight: a normal group (saline), a model group (saline), a piracetam positive drug group (468 mg / kg / d), a low-dose N1,N10-dicaffeoyl spermidine group (NNDCP-L, 10 mg / kg / d), and a high-dose N1,N10-dicaffeoyl spermidine group (NNDCP-H, 25 mg / kg / d), with 8 mice in each group. Piracetam and N1,N10-dicaffeoyl spermidine were both dissolved in saline and administered by gavage at a dose of 10 mL / kg. Except for the normal group, mice in other groups were subcutaneously injected with D-galactose (200 mg / kg) at a fixed time point every day to induce an aging model, and mice in the normal group were subcutaneously injected and gavaged with an equal volume of saline. The above subcutaneous injection and gavage were both once a day for 6 consecutive weeks of model establishment and drug administration.

[0030] 3. Y-maze experiment After 6 weeks of drug administration to mice in each group, the Y-maze experiment was used to investigate the effect of N1,N10-dicaffeoyl spermidine on the working memory ability of aging mice, and the spontaneous alternation response rate of each mouse was calculated for evaluation.

[0031] 4. Morris water maze experiment After 6 weeks of drug administration to mice in each group, the Morris experiment (continuously measured for 5 days) was used to investigate the effect of N1,N10-dicaffeoyl spermidine on the spatial memory ability of aging mice, and the escape latency and the number of times crossing the platform of each mouse were used for evaluation.

[0032] 5. Determination of the protein expression levels of aging markers P53, P21, and P16 After 6 weeks of drug administration and completion of behavioral tests in each group of mice, after taking serum, the mice were sacrificed by cervical dislocation. The whole brain was taken by decapitation on ice, immediately frozen in liquid nitrogen, and transferred to -80 °C for storage after the experiment for later use. An appropriate amount of mouse brain tissue was weighed and added to Lysis Buffer containing 0.1% protease inhibitor and 1% 100 mM PMSF. The glass homogenizer was manually homogenized up and down 30 - 50 times. The tissue homogenate was transferred to a pre-cooled 1.5 mL centrifuge tube and centrifuged at 12,000 xg / min at 4 °C for 5 min. The supernatant was transferred to a new pre-cooled centrifuge tube, which was the total protein extract. Protein quantification (BCA method) was performed. The total protein extract was added with 20% 5X SDS-PAGE protein loading buffer and pure water to prepare a protein loading solution with the same total protein concentration. The sample was loaded onto the SDS-PAGE gel and electrophoresed. The membrane was transferred by the wet transfer method. After membrane blocking, the primary antibodies against P53, P21, and P16 proteins were incubated overnight, followed by incubation with the secondary antibody. Finally, ECL imaging was performed, and the film was exposed and photographed.

[0033] 6. Determination of the Contents of IL-1β, IL-6, TNF-α, SOD, and GSH After 6 weeks of drug administration and completion of behavioral tests in each group of mice, after taking serum, the mice were sacrificed by cervical dislocation. The whole brain was taken by decapitation on ice, immediately frozen in liquid nitrogen, and transferred to -80 °C for storage after the experiment for later use. The mouse brain tissue was ground evenly with a tissue grinder to prepare a brain tissue homogenate, which was centrifuged at 5000 xg / min at 4 °C for 10 min. The supernatant was collected, and the contents of IL-1β, IL-6, TNF-α, SOD, and GSH in the supernatant were detected using a kit.

[0034] 7. Data Processing The data of this invention was processed using ImageJ and Graphpad Prism 9.5 for data analysis and statistics. ImageJ was used to analyze the protein bands, and the expression of the target protein content in each group was represented by the ratio of the gray value of the target protein band / the gray value of the internal reference protein band.

[0035] All the obtained results were expressed as mean ± standard deviation (X±S). One-way ANOVA was used for data processing of the inter-group difference comparison. The difference was considered statistically significant when p <0.05 was used as the standard.

[0036] III. Experimental Results 1. Effect on Mouse Body Weight The results of the effect of N1,N10-dicaffeoyl spermidine on mouse body weight are shown in Figure 1 the figure.

[0037] After 6 weeks of drug administration in each group of mice, compared with the control group, the body weight of the mice in the model group was significantly reduced ( p <0.05). Compared with the model group, the body weight in the piracetam positive drug group had an upward trend, but the difference was not statistically significant. However, the body weight of the mice in the N1,N10-dicaffeoyl spermidine group was significantly increased ( p <0.05 or p <0.01), indicating that N1,N10-dicaffeoyl spermidine had a significant improvement effect on the weight loss caused by the aging mouse model induced by D-galactose in mice.

[0038] 2. Effects on the spontaneous alternation response rate of mice The results of the Y maze experiment examining the effects of N1,N10-dicaffeoyl spermidine on the spontaneous alternation response rate of aging mice are shown in Figure 2 the figure.

[0039] The results showed that compared with the control group, the spontaneous alternation rate of the mice in the model group was significantly reduced ( p <0.001), indicating that the aging mouse model was successfully established and D-galactose could significantly damage the working memory ability of mice. Compared with the model group, the spontaneous alternation rates of the mice in the positive drug group and the N1,N10-dicaffeoyl spermidine group were significantly increased ( p <0.001 or p <0.01). This indicates that both the positive drug and N1,N10-dicaffeoyl spermidine have obvious protective effects on the damage of the working memory ability of aging mice.

[0040] 3. Effects on the escape latency and the number of platform crossings of mice The results of the Morris water maze experiment examining the effects of N1,N10-dicaffeoyl spermidine on the escape latency and the number of platform crossings of aging mice are shown in Figure 3 the figure.

[0041] The results showed that compared with the control group, the escape latency of the mice in the model group increased, and the number of platform crossings decreased significantly ( p <0.001). Compared with the model group, the escape latency of the mice in the positive drug group decreased, and the number of platform crossings increased significantly ( p <0.001). The escape latency of the mice in the N1,N10-dicaffeoyl spermidine group decreased, and the number of platform crossings of the mice in the high-dose N1,N10-dicaffeoyl spermidine group increased significantly ( p <0.001), while the number of platform crossings of the mice in the low-dose N1,N10-dicaffeoyl spermidine group had an upward trend. The above indicates that N1,N10-dicaffeoyl spermidine can improve the spatial memory ability of aging mice to a certain extent.

[0042] 4. Effects on the expression levels of senescence markers P53, P21, and P16 proteins in mouse brain tissue The results of the effects of N1,N10 - dicaffeoyl spermidine on the expression levels of senescence markers P53, P21, and P16 proteins in mouse brain tissue are shown in Figure 4 the figure below

[0043] The results showed that compared with the control group, the expression levels of senescence markers P53, P21, and P16 proteins in the brain tissue of mice in the model group were significantly increased ( p < 0.05 or p < 0.01), indicating that the D - galactose - induced mouse aging model was successfully established. Compared with the model group, the expression levels of P53, P21, and P16 in the brain tissue of mice in the N1,N10 - dicaffeoyl spermidine group were significantly decreased to varying degrees ( p < 0.05, p < 0.01 or p < 0.001). The results indicated that N1,N10 - dicaffeoyl spermidine could improve D - galactose - induced mouse aging.

[0044] 5. Effects on the contents of inflammatory factors IL - 1β, IL - 6, and TNF - α in mouse brain tissue The results of the effects of N1,N10 - dicaffeoyl spermidine on the contents of inflammatory factors IL - 1β, IL - 6, and TNF - α in the brain tissue of aging mice are shown in Figure 5 the figure below

[0045] The results showed that compared with the control group, the contents of IL - 1β and TNF - α in the brain tissue of mice in the model group were significantly increased ( p < 0.01 or p < 0.001), indicating that the mechanism of D - galactose - induced mouse aging may be related to the production of inflammation in the brain tissue. Compared with the model group, the contents of IL - 1β, IL - 6, and TNF - α in the brain tissue of mice in the positive drug group and the N1,N10 - dicaffeoyl spermidine group were significantly decreased ( p < 0.05, p < 0.01 or p < 0.001). The results indicated that the anti - aging mechanism of the positive drug and N1,N10 - dicaffeoyl spermidine may be related to their reduction of the contents of IL - 1β, IL - 6, and TNF - α in the brain tissue and the decrease of inflammation.

[0046] 6. Effects on the contents of oxidative stress factors SOD and GSH in mouse brain tissue The results of the effects of N1,N10 - dicaffeoyl spermidine on the contents of oxidative stress factors SOD and GSH in the brain tissue of aging mice are shown in Figure 6 the figure below

[0047] The results showed that compared with the control group, the contents of SOD and GSH in the brain tissues of the model group mice had a decreasing trend, suggesting that the mechanism of D-galactose-induced aging in mice might be related to oxidative stress in the brain tissues. Compared with the model group, the content of SOD in the brain tissues of the positive drug group mice had an increasing trend, while the content of SOD in the brain tissues of the N1,N10-dicaffeoyl spermidine group mice increased significantly ( p <0.05); the contents of GSH in the brain tissues of the positive drug group and N1,N10-dicaffeoyl spermidine group mice increased significantly ( p <0.05 or p <0.001). The results indicated that the mechanism of action of the positive drug and N1,N10-dicaffeoyl spermidine in anti-aging of mice might be related to their increasing the contents of SOD and GSH in the brain tissues, thereby reducing oxidative stress.

[0048] During the whole process of drug administration to animals, no mice died, and there were no abnormal conditions in the appearance and behavior of the mice.

[0049] The various embodiments in this specification are described in a progressive manner. Each embodiment focuses on the differences from other embodiments. The same or similar parts among the various embodiments can be referred to each other.

[0050] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Application of N1,N10-dicaffeoylspermidine in the preparation of medicines for preventing and treating aging, or in the preparation of health products or functional foods for preventing and treating aging.

2. The use according to claim 1, characterized in that: N1,N10-dicaffeoylspermidine can significantly increase the spontaneous alternation response rate of aged mice in the Y-maze test, that is, improve their working memory ability.

3. The use according to claim 1, characterized in that: N1,N10-dicaffeoylspermidine can significantly reduce the escape latency of aged mice in the Morris water maze test and increase the number of times they cross the platform, that is, improve their spatial memory ability.

4. The use according to claim 1, characterized in that: N1,N10-dicaffeoylspermidine can significantly reduce the expression levels of aging markers P53, P21 and P16 proteins in the brain tissue of aged mice.

5. The use according to claim 1, characterized in that: N1,N10-dicaffeoylspermidine can significantly reduce the levels of inflammatory factors 1L-1β, 1L-6 and TNF-α in the brain tissue of aging mice.

6. The use according to claim 1, characterized in that: N1,N10-dicaffeoylspermidine can significantly increase the levels of oxidative stress factors SOD and GSH in the brain tissue of aging mice.

7. An anti-aging drug, characterized in that: Including N1,N10-dicaffeoylspermidine.

8. The medicine according to claim 7, characterized in that The dosage form is a pharmaceutically acceptable oral dosage form, external patch or injection dosage form.

9. An anti-aging health product, characterized in that: Including N1,N10-dicaffeoylspermidine.

10. An anti-aging functional food, characterized in that: Including N1,N10-dicaffeoylspermidine.

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