Pharmaceutical composition with synergistic anti-aging effect as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicines, in particular to a pharmaceutical composition with a synergistic anti-aging effect as well as a preparation method and application of the pharmaceutical composition. The pharmaceutical composition specifically consists of dihydroquercetin and fisetin. According to the invention, two core anti-aging mechanisms of oxidation resistance and senescence cell removal are organically integrated for the first time, and multi-target collaborative intervention on the senescence process is realized. Compared with a single component, the composition not only can effectively repair oxidative damage, but also can selectively remove accumulated senescent cells, plays a role from the source and the result at the same time, overcomes the defects that a traditional single strategy is single in action path and limited in effect, and has a good application prospect. A more comprehensive and efficient new scheme is provided for delaying senescence and preventing and treating senescence-related diseases.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a pharmaceutical composition with synergistic anti-aging effects, its preparation method, and its application. Background Technology

[0002] Aging is a progressive biological process involving multiple factors and levels, characterized by genomic instability, telomere loss, epigenetic alterations, loss of protein homeostasis, mitochondrial dysfunction, cellular senescence, and chronic inflammation. These interconnected "aging markers" collectively drive the decline of bodily functions and significantly increase the risk of age-related chronic diseases such as cardiovascular disease, neurodegenerative diseases, metabolic syndrome, and malignant tumors. Therefore, exploring strategies to effectively intervene in the aging process and delay or improve age-related diseases has become an important direction in modern biomedical research. In recent years, the use of natural products for anti-aging interventions has attracted much attention due to their wide availability, multi-target effects, and relatively high safety.

[0003] Dihydroquercetin (also known as Taxifolin) is a dihydroflavonol compound primarily extracted from plants such as larch. Numerous studies have confirmed that dihydroquercetin possesses potent antioxidant activity, effectively scavenging free radicals, chelating metal ions, activating endogenous antioxidant enzymes, and protecting mitochondrial function. Furthermore, it has been found to have various pharmacological activities, including anti-inflammatory effects, improved microcirculation, cardiovascular protection, and regulation of lipid metabolism. Currently, dietary supplements and pharmaceuticals with dihydroquercetin as a main ingredient are widely used in many countries, primarily for the protection and improvement of the cardiovascular system. However, its anti-aging effects when used alone are mainly limited to the level of anti-oxidative stress, with limited effectiveness against the key anti-aging target of eliminating existing senescent cells.

[0004] Fisetin is a flavonol compound naturally found in fruits and vegetables such as strawberries, apples, and onions. Recent studies have shown that fisetin is a potential senolytic drug, meaning it can selectively induce apoptosis in senescent cells, thereby reducing tissue microenvironment damage caused by the aging-related secretory phenotype, which consists of pro-inflammatory factors, growth factors, and matrix remodeling enzymes secreted by senescent cells. By eliminating senescent cells, fisetin has been shown to alleviate pathological changes in various age-related disease models and improve the health of older individuals. However, when used alone, while fisetin can target senescent cells, its ability to repair damage from continuous oxidative stress in the body is relatively insufficient, making it difficult to fundamentally prevent the generation of new senescent cells.

[0005] Currently, although there are numerous reports on the application of dihydroquercetin in cardiovascular protection or the use of quercetin to clear senescent cells, aging is a complex process driven by both the accumulation of oxidative damage and the increased burden on senescent cells. Strategies targeting only one type of antioxidant or senescent cell clearance often fail to comprehensively block this process, resulting in limited anti-aging effects. In existing technologies, there are no reports or applications of combining dihydroquercetin and quercetin, leveraging their respective unique advantages in antioxidant protection and senescent cell clearance to achieve multi-target synergistic intervention in aging.

[0006] In summary, there is still an urgent need in this field to develop a novel composition that can simultaneously act on different key stages of the aging process and achieve a more comprehensive and efficient anti-aging effect through a multi-target synergistic mechanism. Summary of the Invention

[0007] The purpose of this invention is to provide a pharmaceutical composition with synergistic anti-aging effects, its preparation method, and its application.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a pharmaceutical composition with synergistic anti-aging effects, consisting of dihydroquercetin and quercetin.

[0009] Preferably, the mass ratio of dihydroquercetin to laccasein is 1~10:1~10.

[0010] Preferably, the mass ratio is 1~5:1~5.

[0011] Preferably, the mass ratio is 1:1.

[0012] Preferably, the dosage form of the pharmaceutical composition is an oral preparation, an injection, or a topical preparation.

[0013] Preferably, the oral preparation is a tablet, capsule, granule, powder, or oral liquid.

[0014] Preferably, it also includes pharmaceutically, food-grade, or cosmetically acceptable carriers, excipients, or diluents.

[0015] The present invention provides a method for preparing the pharmaceutical composition, wherein dihydroquercetin and quercetin are mixed, and pharmaceutically acceptable excipients are added to prepare the corresponding dosage form.

[0016] This invention provides the use of the pharmaceutical composition described herein in the preparation of medicaments for anti-aging, prevention or treatment of aging-related diseases.

[0017] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a pharmaceutical composition composed of dihydroquercetin and rutin, which for the first time organically integrates two core anti-aging mechanisms: antioxidation and scavenging of senescent cells, achieving multi-target synergistic intervention in the aging process. Compared with single components, this composition can not only effectively repair oxidative damage but also selectively clear accumulated senescent cells, acting simultaneously at both the source and the outcome levels. It overcomes the shortcomings of traditional single-strategy approaches, which have limited effects and limited pathways of action, providing a more comprehensive and efficient new approach for delaying aging and preventing age-related diseases.

[0018] At the cellular level, this composition exhibits a significant synergistic effect. By optimizing the ratio of the two components, its comprehensive regulatory capacity far exceeds that of a single component or simple superposition. It can effectively maintain mitochondrial functional homeostasis and promote cellular energy metabolism balance while reducing oxidative stress levels. This multi-dimensional synergistic effect lays a solid foundation for maintaining cellular health and delaying the cellular aging process.

[0019] At the tissue and organ level, this composition also exhibits sustained and superior anti-aging effects. Long-term application can effectively improve the structural state of tissues such as skin, promote the synthesis and maintenance of key structural proteins such as collagen, thereby delaying tissue degeneration. With its unique synergistic mechanism and broad applicability, this composition not only has great potential for developing anti-aging drugs in the pharmaceutical field, but also shows broad application prospects in functional foods, dietary supplements, and anti-aging cosmetics. Detailed Implementation

[0020] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0021] Example 1

[0022] This invention provides a pharmaceutical composition with synergistic anti-aging effects, consisting of dihydroquercetin and quercetin in a mass ratio of 1:10.

[0023] Example 2

[0024] This invention provides a pharmaceutical composition with synergistic anti-aging effects, consisting of dihydroquercetin and quercetin in a mass ratio of 10:1.

[0025] Example 3

[0026] This invention provides a pharmaceutical composition with synergistic anti-aging effects, consisting of dihydroquercetin and quercetin in a mass ratio of 2:8.

[0027] Example 4

[0028] This invention provides a pharmaceutical composition with synergistic anti-aging effects, consisting of dihydroquercetin and quercetin in a mass ratio of 5:6.

[0029] Example 5

[0030] This invention provides a pharmaceutical composition with synergistic anti-aging effects, consisting of dihydroquercetin and quercetin in a mass ratio of 1:1.

[0031] Example 6

[0032] A pharmaceutical composition granule with synergistic anti-aging effects is prepared as follows: (1) Raw material ratio: 50g of dihydroquercetin and 50g of quercetin (mass ratio 1:1).

[0033] (2) Excipients: 30g microcrystalline cellulose, 15g dextrin, 1g magnesium stearate.

[0034] (3) Preparation method: Dihydroquercetin, rosin, microcrystalline cellulose, and dextrin are mixed evenly and passed through an 80-mesh sieve. A soft mass is prepared using a wet granulation machine with 75% ethanol solution as a wetting agent (added at 20% of the total weight of raw materials), and then granulated through a 20-mesh sieve. The wet granules are dried at 60℃ until the moisture content is below 5.0%, and then granulated through a 20-mesh sieve. Magnesium stearate is added and mixed evenly to obtain the synergistic anti-aging granules.

[0035] Example 7

[0036] A method for preparing a pharmaceutical composition tablet with synergistic anti-aging effects, comprising the following steps: (1) Weigh dihydroquercetin and rutin raw materials precisely at a mass ratio of 1:1, pulverize them, pass them through a 100-mesh sieve, and mix them thoroughly to obtain a mixture of active ingredients. Then, add 30% microcrystalline cellulose as a filler, 5% croscarmellose sodium as a disintegrant, and 2% magnesium stearate as a lubricant to the above mixture, and mix thoroughly again. Use a wet granulation process, with 75% ethanol solution as a wetting agent (added at a ratio of 20% of the total weight of raw materials), and prepare soft material using a wet granulation machine, and granulate it through a 20-mesh sieve. Dry the wet granules at 60°C until the moisture content is below 3.0%, and granulate them through a 20-mesh sieve.

[0037] (2) The granules after granulation are fed into a rotary tablet press for tableting. The tablet weight and pressure are adjusted so that each tablet contains 100mg of dihydroquercetin and quercetin, that is, the total active ingredient content of each tablet is 200mg, thereby obtaining a tablet with synergistic anti-aging effect.

[0038] Example 8

[0039] A method for preparing a pharmaceutical composition emulsion with synergistic anti-aging effects, comprising the following steps: (1) Weigh 5g of dihydroquercetin and 5g of rutin at a mass ratio of 1:1, mix thoroughly, and use as active ingredients. Oil phase preparation: Take 80g of caprylic / capric triglyceride, 15g of polysorbate 80, and 5g of Span 80, heat to 70℃ and stir evenly, then add the above active ingredients to the oil phase, stir until completely dissolved, and maintain a constant temperature of 70℃. Aqueous phase preparation: Add purified water to a total volume of 1000ml, add 0.5g of ethylparaben as a preservative, and heat to 70℃. Under stirring in a high-speed shear emulsifier (speed 10000rpm), slowly add the aqueous phase to the oil phase, continue emulsification for 5 minutes to obtain the primary emulsion. Then homogenize the primary emulsion three times under a pressure of 50MPa using a high-pressure homogenizer, cool to room temperature, and fill to obtain the emulsion.

[0040] Experimental Example 1

[0041] Materials and Methods: Primary fibroblasts were obtained from normal human skin tissue remaining after hospital surgery (with informed consent from the patients). The tissue block adhesion method was used for isolation and culture, and cells in good growth status and in the logarithmic growth phase were used in this experiment.

[0042] Experimental grouping: Stably growing fibroblasts were randomly divided into experimental and control groups, with six replicates in each group. The average results were taken. The experimental group was further divided into four subgroups, each receiving the corresponding product, while the blank control group received only complete culture medium. Except for the added test substance, the culture conditions and procedures were kept consistent across all groups.

[0043] The composition obtained in Example 5 was used as Experimental Group 1; the final concentration of dihydroquercetin was 10 μg / mL and the final concentration of rosin was 10 μg / mL. Control group 1: Compared with Example 5, dihydroquercetin was replaced with an equal amount of quercetin (final concentration of quercetin was 20 μg / mL).

[0044] Control group 2: Compared with Example 5, the mass ratio of dihydroquercetin to quercetin was 1:15 (final concentration of dihydroquercetin: 1.25 μg / mL; final concentration of quercetin: 18.75 μg / mL).

[0045] The blank control group was prepared using complete culture medium.

[0046] Observation indicators: Intracellular reactive oxygen species (ROS) content was detected using the DCFH-DA fluorescent probe method. The specific procedure was as follows: After cell treatment, DCFH-DA probe working solution was added, and the cells were incubated at 37°C in the dark for 30 minutes. Then, the cells were washed three times with serum-free culture medium. Finally, the fluorescence intensity of each well was measured using a fluorescence microplate reader at an excitation wavelength of 480 nm and an emission wavelength of 525 nm. The fluorescence intensity was directly proportional to the intracellular ROS level.

[0047] Table 1 Fluorescence Intensity

[0048] (2) Mitochondrial SIRT3 activity assay: The assay was performed using a fluorescence method. The extracted mitochondria were used as the experimental subjects. The specific operation was performed in accordance with the instructions of the SIRT3 activity assay kit (Abcam, USA, product number: ab156067).

[0049] The extraction method for mitochondria is as follows: cells in a stable growth state are taken, homogenized under ice bath conditions, the cells are broken by mechanical means, and then excess residue and large organelles are removed by low-speed differential centrifugation. Finally, mitochondria are obtained by high-speed differential centrifugation. The entire process is carried out in an ice bath to ensure mitochondrial activity.

[0050] Table 2 shows the improvement rate of SIRT3 protein expression.

[0051] (3) Measurement of mitochondrial ATP production: The mitochondrial ATP production level of each group of cells after treatment was detected using a Seahorse XF 96 energy metabolism instrument (Agilent Technologies, USA). The specific operation steps were performed in accordance with the instructions of the Seahorse Bioscience Cell Mitochondrial Stress Detection Kit (product number: 103015-100).

[0052] Table 3 Improvement rate of ATP production in mitochondria

[0053] Tables 1 to 3 systematically evaluated the effects of different compositions on cellular oxidative stress, mitochondrial function, and energy metabolism. The results showed that when dihydroquercetin and rosin were used in a specific ratio, they exhibited superior effects on several key anti-aging indicators compared to single components or combinations in less than optimal ratios, suggesting a synergistic effect between the two.

[0054] In the determination of intracellular reactive oxygen species (ROS) levels, significant differences in fluorescence intensity were observed among the experimental groups, reflecting the varying effects of different ratios on cellular redox status. Compared to single components or combinations with significantly deviated ratios, the optimal combined application of these components more effectively reduced intracellular ROS levels, indicating a stronger comprehensive regulatory capacity in combating oxidative stress.

[0055] Regarding mitochondrial function, the experiment further examined changes in SIRT3 protein expression and ATP production rate. The results showed that the optimized combination group exhibited a more significant improvement trend in both SIRT3 protein expression rate and mitochondrial ATP synthesis rate. This indicates that the combination not only helps enhance the antioxidant defense capacity of mitochondria but also promotes energy metabolism efficiency and improves mitochondrial functional status.

[0056] In summary, the combined application of dihydroquercetin and rosin showed a synergistic enhancement trend in multiple anti-aging related indicators. This combination, by acting simultaneously on oxidative damage repair and mitochondrial function maintenance, may provide a more comprehensive intervention strategy for delaying aging and improving age-related metabolic disorders.

[0057] Experiment Example 2

[0058] Sixty Kunming mice were randomly assigned to each experimental group (15 mice per group). The mice were housed in a clean, well-ventilated, dry environment at a room temperature of 22±2℃ and fed the same diet. Before the experiment, hair was removed from the backs of the mice by 9 cm² using hair removal cream to facilitate drug application and skin sampling.

[0059] In this study, the emulsion prepared in Example 8 was used as the experimental group; control group 3 was prepared by replacing dihydroquercetin with an equal amount of lacquer xanthophyll compared to Example 8; control group 4 was prepared by a dihydroquercetin to lacquer xanthophyll mass ratio of 1:15; and a blank control group was prepared by applying an equal amount of petroleum jelly. Each experimental group was treated once daily, and five mice from each group were tested at 15, 30, and 45 days of treatment.

[0060] Mice were euthanized by cervical dislocation at various time points, and skin samples were collected from the drug-treated areas. The weight of the skin of equivalent area and the content of hydroxyproline were measured. The skin weight was measured as follows: the skin was cut along the edge of the test site, the skin was naturally peeled off, adhering adipose tissue was removed, and the skin was laid flat on a rubber plate with its natural tension. A 1.2 cm diameter punch was used to collect the sample, and the sample was precisely weighed using an electronic balance.

[0061] The method for determining hydroxyproline content is as follows: Weighed skin tissue was rinsed in cold physiological saline and dried with filter paper. 50 mg of the tissue was accurately weighed and placed in a 10 mL beaker. The tissue was minced using ophthalmic scissors, and cold physiological saline was added to a final volume of 5 mL. Homogenization was performed using an internal tissue homogenizer in an ice-water bath (homogenization time 10 s / time, 30 s interval, 5-6 times consecutively). The prepared homogenate was then processed according to the instructions of the skin hydroxyproline assay kit to determine its content. Specific results are shown in Tables 4 (skin weight) and 5 (hydroxyproline content).

[0062] Table 4 Skin Weight (mg)

[0063] Table 5 Hydroxyproline content (μg / mg)

[0064] Tables 4 and 5 show the dynamic changes in skin weight and hydroxyproline content in mice under different drug administration groups, reflecting the intervention effects of each composition on skin aging-related indicators. Overall, the experimental group showed a continuous increase in skin weight at 15, 30, and 45 days, and was superior to other control groups at each time point, demonstrating a good effect on maintaining or improving skin tissue structure. In contrast, while control groups 3 and 4 also showed some improvement, their increases and absolute values ​​were lower than those of the experimental group, indicating that the effects of single components or suboptimal combinations are limited. Furthermore, the skin weight of the blank control group even showed a decreasing trend in the later stages, further confirming the natural occurrence of the aging process in the model.

[0065] Regarding hydroxyproline content, the experimental group also showed a continuous upward trend, and its content was higher than that of other control groups at all time points, suggesting that it can more effectively promote collagen synthesis or inhibit its degradation. Hydroxyproline, as a characteristic amino acid of collagen, directly reflects the metabolic state of skin connective tissue through changes in its content. The superior performance of the experimental group indicates that this composition has a significant effect on delaying skin aging and maintaining skin elasticity. Although the hydroxyproline content in control groups 3 and 4 increased, the increase was relatively slow, indicating that the effect of a single component or a non-synergistic formulation in promoting collagen metabolism is relatively limited.

[0066] The combined data from both groups show that the experimental group exhibited a sustained and superior improvement compared to the control group in both key indicators: skin weight and hydroxyproline content. Furthermore, this advantage became increasingly pronounced with prolonged administration. This synergistic improvement in both indicators suggests that the composition may exert a comprehensive effect through a multi-target mechanism, promoting skin tissue regeneration and inhibiting the loss of age-related structural proteins, thereby delaying the overall skin aging process.

[0067] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A pharmaceutical composition with synergistic anti-aging effects, characterized in that, It is composed of dihydroquercetin and quercetin.

2. The pharmaceutical composition according to claim 1, characterized in that, The mass ratio of dihydroquercetin to quercetin is 1~10:1~10.

3. The pharmaceutical composition according to claim 2, characterized in that, The mass ratio is 1~5:1~5.

4. The pharmaceutical composition according to claim 3, characterized in that, The mass ratio is 1:

1.

5. The pharmaceutical composition according to any one of claims 1 to 4, characterized in that, The dosage form of the pharmaceutical composition is an oral preparation, an injection, or a topical preparation.

6. The pharmaceutical composition according to claim 5, characterized in that, The oral preparations are tablets, capsules, granules, powders, or oral liquids.

7. The pharmaceutical composition according to any one of claims 1 to 4, characterized in that, It also includes pharmaceutically, food-grade, or cosmetically acceptable carriers, excipients, or diluents.

8. A method for preparing the pharmaceutical composition according to any one of claims 1 to 7, characterized in that, Dihydroquercetin and quercetin are mixed and pharmaceutically acceptable excipients are added to prepare the corresponding dosage form.

9. Use of the pharmaceutical composition according to any one of claims 1 to 7 in the preparation of a medicament for anti-aging, prevention or treatment of aging-related diseases.

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