Agrimonia pilosa PDRN composition and application thereof

Abstract

The invention discloses a hairyvein agrimony PDRN composition and application thereof.The hairyvein agrimony PDRN composition is formed by ultrasonic mixing of a hairyvein agrimony extract and a PDRN solution, the ratio of fresh hairyvein agrimony whole herb from the hairyvein agrimony extract to the PDRN solution is 3 g: 8-12 mL, and the concentration of PDRN in the PDRN solution is 0.08-0.12%. Through the synergistic effect of the hairyvein agrimony extract and the PDRN, an enhanced effect is shown in the aspects of skin soothing and repairing, the effect is remarkably better than that of using of a single component, inflammatory response can be better inhibited, generation of active oxygen is reduced, fibroblast proliferation and cell factor release are promoted, and therefore the overall skin health level is improved.

Description

Technical Field

[0001] This invention belongs to the field of skin care technology, specifically relating to an Agrimonia pilosa PDRN composition and its application. Background Technology

[0002] Agrimony is a traditional Chinese medicinal herb, first recorded in the *Huainan Materia Medica*. It is slightly sweet and neutral in nature, with a bitter and astringent taste. It possesses astringent, hemostatic, antimalarial, antidiarrheal, detoxifying, and tonifying properties. In clinical applications, it is often combined with other medicinal herbs to treat gynecological metrorrhagia, various heart diseases caused by yin deficiency, epistaxis due to lung heat, gastrointestinal ulcers, and various cancers. According to the 2020 edition of the *Chinese Pharmacopoeia*, the medicinal part of Agrimony is its aerial parts, mainly including the stems and leaves. These parts contain abundant chemical components, such as flavonoids, tannins, and phenolic compounds, each exerting different pharmacological effects. For example, flavonoids help with antioxidation and anti-inflammation, tannins enhance astringent effects, and phenolic substances provide detoxification and protection. Studies have shown that the distribution of chemical components varies among different medicinal parts. Hong Wanmin et al. used HPLC fingerprinting combined with chemical pattern recognition methods to clarify that Agrimony leaves have a higher content of quercetin, while the stems have a higher content of ellagic acid. This difference in composition provides a basis for targeted extraction and application, making Agrimonia pilosa a potential value in the modern pharmaceutical and cosmetic fields.

[0003] Polydeoxyribonucleotides (PDRNs) are polymers composed of deoxyribonucleotides, obtainable from salmon or trout sperm cells and human placental extracts. PDRNs exhibit low immunogenicity and efficiently activate adenosine A2A receptors, thereby inhibiting the production of reactive oxygen species and promoting the release of cytokines such as vascular endothelial growth factor (VEGF), angiopoietin, and gamma-glutamyl transferase II. These mechanisms contribute to promoting fibroblast proliferation and regeneration, thus enhancing tissue repair and regeneration capabilities. Currently, PDRNs are widely used in medical aesthetics and skincare products, for example, in skin repair, anti-aging, and wound healing. However, due to their large molecular weight, PDRNs face absorption barriers in skincare products, requiring continuous exploration to overcome the limitations of transdermal absorption and fully realize their bioactivity.

[0004] In existing technologies, plant-based ingredients such as Agrimonia pilosa extract are often used alone in cosmetics to exert their anti-inflammatory, astringent, and skin-protecting effects, while PDRN is mainly added as an independent ingredient to promote cell regeneration. However, the combined application of the two still has shortcomings. First, the large molecular structure of PDRN makes it difficult to penetrate the skin barrier, affecting its actual efficacy. Second, current cosmetic formulations often use traditional mechanical mixing methods to combine plant-based ingredients with PDRN. This simple superposition makes it difficult to achieve synergistic interaction between the ingredients and cannot fully address their individual limitations. In addition, exposed PDRN is susceptible to degradation by nucleases in the environment, leading to a shortened action time and reduced stability. These problems limit the comprehensive utilization efficiency of the raw materials and make it difficult to meet the demands of modern skincare products for high efficiency, long-lasting effects, and multifunctionality. Summary of the Invention

[0005] The purpose of this invention is to overcome the defects of the prior art and provide an Agrimonia pilosa PDRN composition.

[0006] Another object of the present invention is to provide the application of the above-mentioned Agrimonia pilosa PDRN composition.

[0007] The technical solution of the present invention is as follows:

[0008] A Agrimonia pilosa PDRN composition, which is prepared by ultrasonic mixing of Agrimonia pilosa extract and PDRN solution, wherein the ratio of fresh Agrimonia pilosa whole herb from which the Agrimonia pilosa extract is derived to PDRN solution is 3 g: 8-12 mL, and the concentration of PDRN in PDRN solution is 0.08-0.12%.

[0009] In a preferred embodiment of the present invention, the preparation method of the Agrimonia pilosa extract includes the following steps:

[0010] (1) Wash the whole fresh Agrimonia pilosa with distilled water, dry the surface moisture with filter paper, chop it, soak it in 70-80% alcohol for 0.8-1.2 min, then wash away the alcohol residue with distilled water and wipe it dry.

[0011] (2) Add an enzyme solution with pH 5.7-5.9 containing cellulase and pectinase to the material obtained in step (1), and enzymatically hydrolyze it at 48-52 ℃ for 5-7 h;

[0012] (3) Centrifuge the material obtained in step (2) at 9500-11000 rpm for 0.8-1.2 h to remove the residue and retain the supernatant;

[0013] (4) Centrifuge the supernatant obtained in step (3) at 9000-11000×g and 2-4℃ for 40-50 min to remove the residue again and retain the supernatant;

[0014] (5) Filter the supernatant obtained in step (4) through a 0.4-0.5 μm filter membrane and collect the filtrate;

[0015] (6) Centrifuge the filtrate obtained in step (5) at 2-4 ℃ and 90000-100000×g for 65-75 min, collect the precipitate, and obtain Agrimonia pilosa extract.

[0016] More preferably, the enzyme solution also contains 0.5-0.7 mol / L of glycerol.

[0017] In a preferred embodiment of the present invention, the power of the ultrasonic mixing is 60%, and the mode is 2 seconds on, 6 seconds off, and 6-8 cycles.

[0018] The use of the above-mentioned Agrimonia pilosa PDRN composition in the preparation of cosmetic compositions.

[0019] In a preferred embodiment of the invention, the cosmetic composition includes toner, lotion, and cream.

[0020] In a preferred embodiment of the present invention, the content of the Agrimonia pilosa PDRN composition in the cosmetic composition is 1.5-4 wt%.

[0021] A cosmetic composition comprising the above-mentioned Agrimonia pilosa PDRN composition as its active ingredient.

[0022] In a preferred embodiment of the invention, it includes toner, lotion, and cream.

[0023] In a preferred embodiment of the present invention, the content of the Agrimonia pilosa PDRN composition is 1.5-4 wt%.

[0024] The beneficial effects of this invention are:

[0025] 1. This invention utilizes the synergistic effect of Agrimonia pilosa extract and PDRN to enhance skin soothing and repair, significantly outperforming the use of single ingredients. It can better inhibit inflammatory responses, reduce reactive oxygen species production, and promote fibroblast proliferation and cytokine release, thereby improving overall skin health.

[0026] 2. This invention improves the transdermal absorption performance of PDRN, overcomes the absorption barrier caused by its large molecular weight, and makes the active ingredients more likely to penetrate the skin barrier, giving full play to its role in activating adenosine A2A receptors and promoting angiogenesis. It is suitable for daily care of sensitive or damaged skin.

[0027] 3. This invention enhances the stability of PDRN and reduces the risk of nuclease degradation, thereby prolonging the duration of action of the composition on the skin, ensuring longer-lasting efficacy, avoiding the problem of diminishing effects in traditional applications, and providing long-lasting soothing and repairing effects.

[0028] 4. This invention is applicable to various cosmetic forms, such as toners, lotions, and creams, providing flexible application methods to meet different skin types and care needs, and improving the overall safety and effectiveness of the product while reducing potential irritation or allergy risks.

[0029] 5. The present invention achieves a non-simple mixing mode through ultrasound technology, which promotes molecular interaction between components and forms a more stable composite structure. This helps to reduce many problems in application, such as raw material degradation, uneven efficacy and poor storage stability, thereby optimizing the overall formulation design of cosmetics.

[0030] 6. This invention utilizes the complementary effects of components in Agrimonia pilosa extract and PDRN to achieve multi-target regulation, including anti-oxidation, astringent hemostasis and tissue regeneration, expanding its application potential in the fields of medical aesthetics and skin care, and is suitable for various scenarios such as anti-aging, wound healing and daily moisturizing.

[0031] 7. This invention reduces the limitations of using single ingredients, such as compensating for the shortcomings of Agrimonia pilosa extract in cell regeneration and the relative weakness of PDRN in anti-inflammation, thereby achieving a comprehensive improvement in efficacy and promoting the innovative utilization and sustainable development of cosmetic raw materials. Attached Figure Description

[0032] Figure 1 The figure shows the experimental results of Embodiment 5 of the present invention.

[0033] Figure 2 The figure shows the experimental results of Embodiment 6 of the present invention. Detailed Implementation

[0034] The technical solution of the present invention will be further explained and described below with reference to specific embodiments and accompanying drawings.

[0035] Example 1: Preparation of Agrimonia pilosa PDRN composition

[0036] (1) Take 3g of fresh Agrimonia pilosa whole herb, wash it with distilled water, dry the surface moisture with filter paper, chop it, soak it in 75% alcohol for 1 minute, then wash away the alcohol residue with distilled water and wipe it dry.

[0037] (2) Add 50 mL of enzyme solution (with cellulase concentration of 4%, pectinase concentration of 2%, mannitol concentration of 0.6 mol / L, and pH value of 5.8) and enzymatically hydrolyze for 6 h at 50 ℃;

[0038] (3) Centrifuge at 10,000 rpm for 1 h to remove the residue and retain the supernatant;

[0039] (4) Centrifuge at 10000×g and 4 ℃ for 45 min, remove the residue again, and retain the supernatant;

[0040] (5) Filter through a 0.45 μm filter membrane and collect the filtrate;

[0041] (6) Centrifuge at 4 ℃ and 100000×g for 70 min (this centrifugation step can be repeated depending on the clarity of the extract), collect the precipitate, and obtain Agrimonia pilosa extract;

[0042] (7) After thoroughly mixing the above Agrimonia pilosa extract with 10 mL of PDRN solution (the concentration of PDRN is 0.1%, PDRN is a commercially available product, and the solvent is ultrapure water), perform ultrasonic treatment (the equipment reference is VCX150, rated power 150W, 20kHz, the ultrasonic treatment power is 60% of the rated power, the treatment mode is 2 s on and 6 s off, and a total of 7 cycles) to obtain the final product.

[0043] Example 2: Toner

[0044] A toner with the Agrimonia pilosa PDRN composition prepared in Example 1 as the active ingredient comprises the following ingredients by weight percentage: 5 wt% glycerin, 3 wt% butylene glycol, and 88 wt% deionized water; active ingredient: 4 wt% Agrimonia pilosa PDRN composition prepared in Example 1.

[0045] Preparation method: Weigh out glycerin, butylene glycol and deionized water and add them to a water pot. Heat to 95 °C, stir and mix well, and then cool to room temperature. While stirring, add the Agrimonia pilosa PDRN composition prepared in Example 1 until it is stirred evenly to obtain toner.

[0046] Example 3: Emulsion

[0047] An emulsion with the Agrimonia pilosa PDRN composition prepared in Example 1 as the active ingredient comprises the following ingredients by weight percentage: 1,3-butanediol 3wt%, glycerin 6wt%, deionized water 77.3wt%, xanthan gum 0.2wt%, caprylic / capric triglyceride 5wt%, dioctyl carbonate 4wt%, cetearyl oleate / sorbitan oleate 3wt%; active ingredient: Agrimonia pilosa PDRN composition prepared in Example 1 1.5wt%.

[0048] Preparation method: Add phase A components (glycerol, 1,3-butanediol, deionized water, xanthan gum) to a water pot, heat to 95 °C, and stir until homogeneous; heat phase B components (caprylic / capric triglyceride, dioctyl carbonate, cetearyl oleate / sorbitan oleate) to 80 °C; add phase B to phase A and stir homogenously; cool to 45 °C using cooling water, and add the Agrimonia pilosa PDRN composition prepared in Example 1 while stirring until homogeneous, and fill to obtain the emulsion.

[0049] Example 4: Face Cream

[0050] A face cream with the Agrimonia pilosa PDRN composition prepared in Example 1 as the active ingredient comprises the following ingredients by weight percentage: 1,3-butanediol 3wt%, glycerin 6wt%, deionized water 75wt%, ammonium acryloyl dimethyl taurate / VP copolymer 0.5wt%, caprylic / capric triglyceride 5wt%, macadamia seed oil 4wt%, cetearyl alcohol oleate / sorbitan oleate 4wt%; active ingredient: Agrimonia pilosa PDRN composition prepared in Example 1 2.5wt%.

[0051] Preparation method: Add phase A components (glycerol, 1,3-butanediol, deionized water, ammonium acryloyldimethyl taurate / VP copolymer) to a water pot, heat to 95 °C, and stir to mix well; heat phase B components (caprylic / capric triglycerides, macadamia seed oil, cetearyl oleate / sorbitan oleate) to 80 °C; add phase B to phase A and homogenize and emulsify; cool to room temperature, and add the Agrimonia pilosa PDRN composition prepared in Example 1 while stirring until it is uniform, and the face cream is obtained.

[0052] Example 5: In vitro soothing experiment

[0053] This embodiment verifies the efficacy of the Agrimonia pilosa PDRN composition prepared in Example 1. RAW264.7 cells in the logarithmic growth phase and in good condition were digested, centrifuged, resuspended in the appropriate cell culture medium, and counted to prepare a cell suspension of a specific concentration. The experimental design included a blank group (PBS group), a control group (LPS group), and a sample group, with three parallel wells in each group. 200 μL of cell suspension was seeded into each well of a 48-well plate and cultured overnight at 37 ℃ and 5% CO2 for (14±2) h. The culture medium was discarded, and the cells were washed with 200 μL / well of physiological saline or PBS.

[0054] In the blank control group, 195 μL of culture medium and 5 μL of PBS were added to each well; in the control group, 191 μL of culture medium, 4 μL of LPS working solution (final LPS concentration of 1 μg / mL), and 5 μL of PBS were added to each well; in the sample group, 191 μL of culture medium, 4 μL of LPS working solution (final LPS concentration of 1 μg / mL), and 5 μL of the sample to be tested were added to each well. Cells were cultured at 37 ℃ and 5% CO2 for 24 h. Cell morphology was observed, cell supernatant was collected, and NO content was measured and calculated according to the kit instructions.

[0055] The samples to be tested include:

[0056] Sample 1 – Toner from Example 2

[0057] Sample 2 – as a blank control, specifically the toner of Example 2 with the removal of the active ingredient (deionized water to make up the weight).

[0058] Sample 3 – The active ingredient in the toner of Example 2 was replaced with Agrimonia pilosa extract obtained in step (6) of Example 1, with an addition amount of 4 wt%.

[0059] Sample 4—The active ingredient in the toner of Example 2 was replaced with the PDRN solution in step (7) of Example 1, and the amount added was 4wt%.

[0060] The results are as follows Figure 1 As shown, sample 1 showed the best NO inhibition effect, followed by sample 4, and then sample 3. The experiment confirmed that the Agrimonia pilosa PDRN composition prepared in Example 1 was more effective in soothing than the single component.

[0061] Example 6: Human Experiment

[0062] To verify the lasting efficacy of the composition, 40 healthy female volunteers aged 25 to 50 years were recruited and randomly divided into two groups of 20 each: Group A and Group B. Volunteers in Group A used the face cream of Example 4 (50g per person); volunteers in Group B used the control sample (the active ingredient in Example 4 was replaced with the PDRN solution in step (7) of Example 1, and 2.5wt% of the face cream was added, 50g per person). The facial skin condition of the volunteers was tested and recorded on day 0, day 28, and day 42.

[0063] Trial Requirements: Volunteers were required to discontinue the use of other face cream products during the trial period. Usage: Apply twice daily, morning and evening, for 28 consecutive days. Testing Instruments: Skin condition, including redness (inflammation area) and transepidermal water loss rate, was assessed using the transdermal moisture detection probes of the VISIA and CK skin analyzers.

[0064] Result: As Figure 2As shown, the test results on day 28 indicated that group A showed significantly better improvement in the skin redness a* value than group B (the redness a* value was obtained by image analysis of the redness images detected by VISIA, used to assess the improvement of facial skin redness and sensitivity. The lower the redness a* value and the higher the rate of change, the better the soothing effect). The test results on day 42 showed that group A showed better improvement in the skin redness than group B, and the rate of change in transepidermal water loss (the less transepidermal water loss and the higher the rate of change, the better the skin barrier and the better the product's repair effect) was also better than group B, with no significant change compared to day 28. This confirms that the Agrimonia pilosa PDRN composition prepared in Example 1 has a longer-lasting effect than a single ingredient.

[0065] The above description is merely a preferred embodiment of the present invention, and therefore should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made in accordance with the scope of the patent and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A Agrimonia pilosa PDRN composition, characterized in that: It is made by ultrasonically mixing Agrimonia pilosa extract and PDRN solution. The ratio of fresh Agrimonia pilosa whole herb from which the Agrimonia pilosa extract is derived to PDRN solution is 3 g: 8-12 mL, and the concentration of PDRN in the PDRN solution is 0.08-0.12%.

2. The Agrimonia pilosa PDRN composition as described in claim 1, characterized in that: The preparation method of the Agrimonia pilosa extract includes the following steps: (1) Wash the whole fresh Agrimonia pilosa with distilled water, dry the surface moisture with filter paper, chop it, soak it in 70-80% alcohol for 0.8-1.2 min, then wash away the alcohol residue with distilled water and wipe it dry. (2) Add an enzyme solution with pH 5.7-5.9 containing cellulase and pectinase to the material obtained in step (1), and enzymatically hydrolyze it at 48-52 ℃ for 5-7 h; (3) Centrifuge the material obtained in step (2) at 9500-11000 rpm for 0.8-1.2 h to remove the residue and retain the supernatant; (4) Centrifuge the supernatant obtained in step (3) at 9000-11000×g and 2-4℃ for 40-50 min to remove the residue again and retain the supernatant; (5) Filter the supernatant obtained in step (4) through a 0.4-0.5 μm filter membrane and collect the filtrate; (6) Centrifuge the filtrate obtained in step (5) at 2-4 ℃ and 90000-100000×g for 65-75 min, collect the precipitate, and obtain Agrimonia pilosa extract.

3. The Agrimonia pilosa PDRN composition as described in claim 2, characterized in that: The enzyme solution also contains 0.5-0.7 mol / L of glycerol.

4. A Agrimonia pilosa PDRN composition according to any one of claims 1 to 3, characterized in that: The ultrasonic mixing power is 60%, and the mode is 2 seconds on, 6 seconds off, and 6-8 cycles.

5. Use of the Agrimonia pilosa PDRN composition according to any one of claims 1 to 4 in the preparation of cosmetic compositions.

6. The use as described in claim 5, characterized in that: The cosmetic composition includes toner, lotion, and cream.

7. The use as described in claim 5 or 6, characterized in that: The content of the Agrimonia pilosa PDRN composition in the cosmetic composition is 1.5-4 wt%.

8. A cosmetic composition, characterized in that: Its active ingredients include the Agrimonia pilosa PDRN composition as described in any one of claims 1 to 4.

9. A cosmetic composition as described in claim 8, characterized in that: It includes toner, lotion, and cream.

10. A cosmetic composition as described in claim 8 or 9, characterized in that: The content of the Agrimonia pilosa PDRN composition is 1.5-4 wt%.

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