Application of boxthorn leaf extract in preparation of anti-aging medicine, health care product or functional food
Through wolfberry leaf extract, improving memory ability and reducing aging markers in aged mice has been solved, and a new solution to the limited effect of existing anti-aging drugs is provided, and a new solution for low-cost and efficient anti-aging drugs, health products and functional foods is provided.
Patent Information
- Application Number
- CN202510558574.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-30
- Publication Date
- 2025-07-04
AI Technical Summary
The existing anti-aging drugs have limited effects, single targets, and high preparation costs. Most of the research on wolfberry fruits focuses on polysaccharide components, while the potential active parts of wolfberry leaves have not been fully developed.
The wolfberry leaf extract is used to soak the dried wolfberry leaves in ethanol aqueous solution, extract the active ingredients such as polysaccharides, flavonoids, alkaloids and polyphenols in the wolfberry leaves, and prepare them into drugs, health products or functional foods, significantly improve the working memory and spatial memory ability of aging mice, reduce aging markers and inflammatory factors, and improve the content of oxidative stress factors.
Lycium barbarum leaf extract significantly improves the memory ability of aging mice within the dose range of 100-250 mg/kg, reduces aging markers and inflammatory factors, and increases the content of oxidative stress factors, providing a new strategy for widely used anti-aging drugs, health products and functional foods.
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Figure CN120241883A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of traditional Chinese medicine, and more specifically relates to the application of wolfberry leaf extract in the preparation of anti-aging drugs, health products or functional foods. Background Art
[0002] The process of global population aging is accelerating. According to statistics of the World Health Organization (WHO), by 2050, the proportion of the global population aged 60 and above will double, reaching 22% (about 2.1 billion people). Aging is not only a natural process of the degradation of body functions, but also an independent driving factor for various chronic diseases.
[0003] At present, there are many anti-aging intervention means, including NAD+ enhancers (such as NMN) and mTOR inhibitors (such as rapamycin). Natural products such as resveratrol, ganoderma polysaccharide, and wolfberry polysaccharide have also been proven to have anti-aging effects. However, problems such as limited efficacy, single target, and high preparation cost of existing drugs limit their wide application. In addition, the research on traditional anti-aging plant resources (such as wolfberry fruits) mostly focuses on polysaccharide components, while other potential active parts (such as wolfberry leaves) have not been fully developed.
[0004] As a traditional medicine and food resource, wolfberry leaves are rich in active ingredients such as polysaccharides, flavonoids, alkaloids, and polyphenols. Existing research has confirmed that they have antioxidant, anti-inflammatory, immunomodulatory, and neuroprotective effects. However, there are no reports on the anti-aging research of wolfberry leaf extract. The present invention discovers for the first time that wolfberry leaf extract has anti-aging effects, providing an experimental basis for its use in the preparation of drugs, health products, and / or functional foods for delaying, preventing, and / or treating aging. Compared with other drugs, wolfberry leaf extract has a wide source, low cost, and high safety, providing a new strategy for the development of anti-aging drugs, health products, and / or functional foods with both high efficiency and wide applicability. Summary of the Invention
[0005] In view of this, the present invention provides the application of wolfberry leaf extract in the preparation of drugs, health products, or functional foods for preventing and treating aging. Through preliminary animal experiments, the present invention finds that wolfberry leaf extract 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] In order to achieve the above object, the present invention adopts the following technical scheme: The application of wolfberry leaf extract in the preparation of drugs for preventing and treating aging, or in the preparation of health products or functional foods for preventing and treating aging.
[0007] Further, the wolfberry leaves are derived from wild wolfberries and the genus Lycium of the Solanaceae familyLycium Lycium barbarum L. var. ningxiense Lycium barbarum The bud and leaf of a new wolfberry variety bred by interspecific hybridization of Lycium barbarum L., and later artificially domesticated and cultivated into a fruitless wolfberry that does not flower or bear fruit.
[0008] Further, the wolfberry leaf extract is prepared by the following method: using dried wolfberry leaves as raw materials, adding an ethanol aqueous solution for soaking, extracting, filtering, combining the filtrates, and concentrating to obtain the wolfberry leaf extract. Among them, the volume concentration of ethanol in the ethanol aqueous solution is 0-100%; the ratio of dried wolfberry leaves to the ethanol aqueous solution is mass (g): volume (mL) = 1:5-50; the soaking time is 0.5-1 h; the extraction method is heating reflux extraction or ultrasonic extraction, the extraction times are 1-3 times, and the extraction time is 0.5-3 h.
[0009] Further, the wolfberry leaf extract can significantly increase the spontaneous alternation response rate of aging mice in the Y-maze experiment, that is, improve their working memory ability.
[0010] Further, the wolfberry leaf extract 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.
[0011] Further, the wolfberry leaf extract can significantly reduce the protein expression levels of the aging markers P53, P21, and P16 in the brain tissue of aging mice.
[0012] Further, the wolfberry leaf extract can significantly reduce the contents of the inflammatory factors IL-1β, IL-6, and TNF-α in the brain tissue of aging mice.
[0013] Further, the wolfberry leaf extract can significantly increase the contents of the oxidative stress factors SOD and GSH in the brain tissue of aging mice.
[0014] An anti-aging drug, characterized in that it comprises a wolfberry leaf extract.
[0015] Further, the dosage form is an orally administrable dosage form, an external patch, or an injection dosage form permitted in pharmacy.
[0016] An anti-aging health product comprising a wolfberry leaf extract.
[0017] An anti-aging functional food comprising a wolfberry leaf extract.
[0018] 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 100-250 mg / kg administered to experimental animals, the wolfberry leaf extract can significantly improve the working memory and spatial memory abilities of D-galactose-induced senescent mice, significantly reduce the protein expression levels of senescence markers P53, P21, and P16 in the mouse brain tissue, significantly reduce the contents of inflammatory factors IL-1β, IL-6, and TNF-α in senescent mice, and significantly increase the contents of oxidative stress factors SOD and GSH. The above all confirm that the wolfberry leaf extract has an anti-aging effect and can be used to prepare drugs, health products, and / or functional foods for delaying, preventing, and / or treating senescence. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] 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 also be obtained according to the provided drawings.
[0020] Figure 1 Effect of wolfberry leaf extract on the body weight of senescent mice (C is the normal group, M is the model group, P is the positive drug piracetam group, LBE-H is the high-dose group of wolfberry leaf extract, LBE-L is the low-dose group of wolfberry leaf extract; compared with the normal group, # p <0.05) Figure 2 Effect of wolfberry leaf extract on the spontaneous alternation response rate of senescent mice investigated by the Y-maze test (C is the normal group, M is the model group, P is the positive drug piracetam group, LBE-H is the high-dose group of wolfberry leaf extract, LBE-L is the low-dose group of wolfberry leaf extract; compared with the normal group, ### p <0.001; compared with the model group, *** p <0.001) Figure 3 Effect of wolfberry leaf extract on the escape latency and the number of platform crossings of senescent mice investigated by the Morris water maze test (C is the normal group, M is the model group, P is the positive drug piracetam group, LBE-H is the high-dose group of wolfberry leaf extract, LBE-L is the low-dose group of wolfberry leaf extract; compared with the normal group, ### p <0.001; compared with the model group, ** p <0.01, *** p <0.001) Figure 4Effect of Lycium barbarum leaf extract on the expression levels of senescence markers P53, P21 and P16 proteins in the brain tissue of senescent mice (C is the normal group, M is the model group, LBE-H is the high-dose group of Lycium barbarum leaf extract, LBE-L is the low-dose group of Lycium barbarum leaf extract; compared with the normal group, # p <0.05, ## p <0.01, ### p <0.001; compared with the model group, * p <0.05, ** p <0.01, *** p <0.001) Figure 5 Effect of Lycium barbarum leaf extract on the contents of inflammatory factors IL-1β, IL-6 and TNF-α in the brain tissue of senescent mice (C is the normal group, M is the model group, P is the positive drug piracetam group, LBE-H is the high-dose group of Lycium barbarum leaf extract, LBE-L is the low-dose group of Lycium barbarum leaf extract; 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 Effect of Lycium barbarum leaf extract on the contents of oxidative stress factors SOD and GSH in the brain tissue of senescent mice (C is the normal group, M is the model group, P is the positive drug piracetam group, LBE-H is the high-dose group of Lycium barbarum leaf extract, LBE-L is the low-dose group of Lycium barbarum leaf extract; compared with the model group, * p <0.05) Detailed implementation manners
[0021] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.
[0022] The Lycium barbarum leaves used in the present invention are obtained from the bud leaves of the fruitless Lycium barbarum obtained by interspecific hybridization between wild Lycium barbarum and Lycium barbarum L. of the Solanaceae family Lycium L. Lycium barbarum Lycium barbarum L. after drying the bud leaves. Example 1
[0023] Take dry wolfberry leaves (2 kg), add anhydrous ethanol aqueous solution (10 L), with a solid-liquid ratio (mass g: volume mL = 1:5), soak for 0.5 h, heat under reflux for extraction, extract once for 3 h each time, filter while it is hot, combine the filtrates, and evaporate the filtrates to dryness under reduced pressure to obtain an extract (1079 g), thus obtaining the wolfberry leaf extract. Example 2
[0024] Take dry wolfberry leaves (50 g), add aqueous solution (2.5 L), with a solid-liquid ratio (mass g: volume mL = 1:50), soak for 1 h, decoct for extraction, extract 3 times for 0.5 h each time, filter while it is hot, combine the filtrates, and evaporate the filtrates to dryness under reduced pressure to obtain an extract (18 g), thus obtaining the wolfberry leaf extract. Example 3
[0025] Take dry wolfberry leaves (2 kg), add 60% ethanol aqueous solution (20 L), with a solid-liquid ratio (mass g: volume mL = 1:10), soak for 1 h, heat under reflux for extraction, extract 2 times for 1 h each time, filter while it is hot, combine the filtrates, and evaporate the filtrates to dryness under reduced pressure to obtain an extract (961 g), thus obtaining the wolfberry leaf extract. Example 4
[0026] An anti-aging drug, in an oral dosage form, comprising the wolfberry leaf extract. Example 5
[0027] An anti-aging drug, in an injection dosage form, comprising the wolfberry leaf extract. Example 6
[0028] An anti-aging drug, in an external patch dosage form, comprising the wolfberry leaf extract. Example 7
[0029] An anti-aging health product, comprising the wolfberry leaf extract. Example 8
[0030] An anti-aging functional food, comprising the wolfberry leaf extract.
[0031] The following animal experiments further illustrate the effects of the above Examples 1 to 8: I. Animal Experiment Design Use the D-galactose-induced aging mouse model. After intragastric administration of the wolfberry leaf extract, evaluate the anti-aging effect of the wolfberry leaf extract through the body weight of mice, 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.
[0032] II. Experimental Procedure 1. Experimental animals Healthy SPF-grade C57BL / 6 male mice weighing 20 - 30 g were obtained from the Experimental Animal Center of Ningxia Medical University (Experimental Animal Use License SYSK (Ning) 2020 - 0001). They were housed in a SPF-grade barrier environment with a 12 h light and 12 h dark cycle, and had free access to food and water.
[0033] 2. Experimental methods A D-galactose-induced aging mouse model was used. After 3 days of adaptive feeding, 40 mice were randomly divided into 5 groups: a normal group (saline), a model group (saline), a piracetam positive drug group (468 mg / kg / d), a low-dose Lycium barbarum leaf extract group (LBE-L, 100 mg / kg / d), and a high-dose Lycium barbarum leaf extract group (LBE-H, 250 mg / kg / d), with 8 mice in each group. Piracetam and Lycium barbarum leaf extract were 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 modeling and gavage were performed once a day for 6 consecutive weeks.
[0034] 3. Y-maze experiment After 6 weeks of drug administration in each group of mice, the Y-maze experiment was used to investigate the effect of Lycium barbarum leaf extract on the working memory ability of aging mice, and the spontaneous alternation response rate of each mouse was calculated for evaluation.
[0035] 4. Morris water maze experiment After 6 weeks of drug administration in each group of mice, the Morris experiment (continuously measured for 5 days) was used to investigate the effect of Lycium barbarum leaf extract on the spatial memory ability of aging mice, and the escape latency and the number of times of crossing the platform of each mouse were used for evaluation.
[0036] 5. Determination of the protein expression levels of aging markers P53, P21, and P16 After 6 weeks of drug administration to each group of mice and completion of the behavioral tests, serum was collected, and then the mice were sacrificed by cervical dislocation. The whole brain was removed 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 used to homogenize manually 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 was performed by the BCA method. The total protein extract was mixed 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 exposure and photography were carried out.
[0037] 6. Determination of the Contents of IL-1β, IL-6, TNF-α, SOD, and GSH After 6 weeks of drug administration to each group of mice and completion of the behavioral tests, serum was collected, and then the mice were sacrificed by cervical dislocation. The whole brain was removed 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 5,000 xg / min at 4 °C for 10 min. The supernatant was collected, and a kit was used to detect the contents of IL-1β, IL-6, TNF-α, SOD, and GSH in the supernatant.
[0038] 7. Data Processing For the data processing of this invention, ImageJ and Graphpad Prism 9.5 were used for data analysis and statistics. ImageJ was used to analyze the protein bands, and the expression levels of the target proteins in each group were represented by the ratio of the gray value of the target protein band to the gray value of the internal reference protein band.
[0039] 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, and the difference was considered statistically significant when p <0.05 was used as the standard.
[0040] III. Experimental Results 1. Effects on the Body Weight of Mice The results of the effects of the wolfberry leaf extract on the body weight of mice are shown in Figure 1 the figure below.
[0041] After 6 weeks of drug administration to 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 weights of mice in the piracetam positive drug group and the wolfberry leaf extract group showed an upward trend, but the difference was not statistically significant. The above indicates that the wolfberry leaf extract has a certain ameliorating effect on the body weight loss of mice caused by modeling.
[0042] 2. Effects on the spontaneous alternation response rate of mice The results of investigating the effects of wolfberry leaf extract on the spontaneous alternation response rate of aging mice by the Y-maze experiment are shown in Figure 2 as follows.
[0043] The results showed that compared with the control group, the spontaneous alternation rate of mice in the model group was significantly decreased ( p <0.001), indicating that the aging model of mice 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 mice in the positive drug group and the wolfberry leaf extract group were both significantly increased ( p <0.001). This indicates that both the positive drug and the wolfberry leaf extract have obvious protective effects on the damage of the working memory ability of aging mice.
[0044] 3. Effects on the escape latency and the number of platform crossings of mice The results of investigating the effects of wolfberry leaf extract on the escape latency and the number of platform crossings of aging mice by the Morris water maze experiment are shown in Figure 3 as follows.
[0045] The results showed that compared with the control group, the escape latency of 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 mice in the positive drug group decreased, and the number of platform crossings increased significantly ( p <0.001), and the escape latency of mice in the wolfberry leaf extract group decreased, and the number of platform crossings increased significantly ( p <0.01 or p <0.001). The above indicates that the wolfberry leaf extract can significantly improve the spatial memory ability of aging mice.
[0046] 4. Effects on the expression levels of aging markers P53, P21 and P16 proteins in the mouse brain tissue The results of the effects of wolfberry leaf extract on the expression levels of aging markers P53, P21 and P16 proteins in the mouse brain tissue are shown in Figure 4 as follows.
[0047] The results showed that compared with the control group, the expression levels of aging markers P53, P21 and P16 proteins in the brain tissue of mice in the model group were all significantly increased ( p <0.05, p <0.01 orp <0.001), 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 tissues of mice in the wolfberry leaf extract group were significantly decreased to varying degrees ( p <0.05, p <0.01 or p <0.001). The results showed that the wolfberry leaf extract could improve D-galactose-induced mouse aging.
[0048] 5. Effects on the contents of inflammatory factors IL-1β, IL-6, and TNF-α in mouse brain tissues The results of the effects of wolfberry leaf extract on the contents of inflammatory factors IL-1β, IL-6, and TNF-α in the brain tissues of aging mice are shown in Figure 5 as follows.
[0049] The results showed that compared with the control group, the contents of IL-1β and TNF-α in the brain tissues 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 tissues of mice in the positive drug group and the wolfberry leaf extract group were significantly decreased ( p <0.05, p <0.01 or p <0.001). The results showed that the anti-aging mechanism of the positive drug and the wolfberry leaf extract may be related to their reduction of the contents of IL-1β, IL-6, and TNF-α in the brain tissue and the reduction of inflammation.
[0050] 6. Effects on the contents of oxidative stress factors SOD and GSH in mouse brain tissues The results of the effects of wolfberry leaf extract on the contents of oxidative stress factors SOD and GSH in the brain tissues of aging mice are shown in Figure 6 as follows.
[0051] The results showed that compared with the control group, the contents of SOD and GSH in the brain tissues of mice in the model group tended to decrease, indicating that the mechanism of D-galactose-induced mouse aging may be related to oxidative stress in the brain tissue. Compared with the model group, the content of SOD in the brain tissues of mice in the positive drug group and the low-dose wolfberry leaf extract group tended to increase, while the content of SOD in the brain tissues of mice in the high-dose wolfberry leaf extract group was significantly increased ( p <0.05); the contents of GSH in the brain tissues of mice in the positive drug group and the wolfberry leaf extract group were significantly increased ( p<0.05). The results showed that the mechanisms of action of the positive drug and the wolfberry leaf extract in anti-aging of mice may be related to their increasing the contents of SOD and GSH in the brain tissue, thereby reducing oxidative stress.
[0052] During the whole process of drug administration to the animals, no mice died, and there were no abnormal conditions in the appearance and behavior of the mice.
[0053] The various embodiments in this specification are described in a progressive manner. Each embodiment focuses on the differences from other embodiments. For the same and similar parts among the various embodiments, reference can be made to each other.
[0054] 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 rather to the widest scope consistent with the principles and novel features disclosed herein.
Claims
1. Use of Lycium barbarum leaf extract in the preparation of drugs for preventing and treating senescence, or in the preparation of health care products or functional foods for preventing and treating senescence.
2. The application according to claim 1, characterized in that the wolfberry leaves are derived from the interspecific hybridization and breeding of wild wolfberry and Lycium barbarum L. of the genus Lycium in the Solanaceae family, and then through artificial domestication and cultivation, they become the bud leaves of the fruitless wolfberry that does not flower and does not bear fruit. Lycium L. Lycium barbarum Lycium barbarum L. The bud leaves of a new wolfberry variety bred through interspecific hybridization, and then through artificial domestication and cultivation, it becomes the fruitless wolfberry that does not flower and does not bear fruit.
3. The application according to claim 1, wherein, The Lycium barbarum leaf extract is prepared by the following method: using dried Lycium barbarum leaves as raw materials, adding an ethanol aqueous solution for soaking, extracting, filtering, combining the filtrates, and concentrating to obtain the Lycium barbarum leaf extract.
4. The preparation method of the wolfberry leaf extract according to claim 3, characterized in that, The volume concentration of ethanol in the ethanol aqueous solution is 0 to 100%; the ratio of the dried Lycium barbarum leaves to the ethanol aqueous solution is mass (g): volume (mL) = 1:5 to 50; the soaking time is 0.5 to 1 h; the extraction method is heating reflux extraction or ultrasonic extraction, the extraction times are 1 to 3 times, and the extraction time is 0.5 to 3 h.
5. The application according to claim 1, characterized in that, The Lycium barbarum leaf extract can significantly increase the spontaneous alternation response rate of senescent mice in the Y-maze experiment, that is, improve their working memory ability.
6. The application according to claim 1, wherein The Lycium barbarum leaf extract can significantly reduce the escape latency of senescent mice in the Morris water maze experiment and increase the number of times of crossing the platform, that is, improve their spatial memory ability.
7. The application according to claim 1, characterized in that, The Lycium barbarum leaf extract can significantly reduce the protein expression levels of senescence markers P53, P21 and P16 in the brain tissue of senescent mice.
8. An anti-aging drug, characterized in that, It includes Lycium barbarum leaf extract.
9. An anti-aging health care product, characterized in that, It includes Lycium barbarum leaf extract.
10. A functional food for anti-aging, characterized in that, It includes Lycium barbarum leaf extract.