Use of matricaria o in the preparation of anti-aging products

Abstract

The application belongs to the technical field of medicine, and particularly relates to application of matricaria o in preparation of anti-aging products. The application provides, for the first time, application of matricaria o in preparation of anti-aging products. The application innovatively discovers that matricaria o (KO), an active ingredient of sophora flavescens, has an anti-oxidative stress effect, can reduce the content of beta-galactosidase, reduce the content of ROS, and increase the mitochondrial membrane potential, and can be applied to preparation of anti-oxidative and anti-aging related drugs and / or cosmetics, and has certain application prospect and social significance.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of matrine O in the preparation of anti-aging products. Background Technology

[0002] Sophora flavescens is a legume plant (Sophora flavescens). Sophora flavescens The dried root of *Sophora flavescens* (Ait.), first recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica), has a long history of clinical application and significant therapeutic effects. *Sophora flavescens* is bitter and cold in nature, and enters the heart, liver, stomach, large intestine, and bladder meridians. It has the effects of clearing heat and drying dampness, killing parasites, and promoting diuresis. It is used for dysentery, hematochezia, jaundice, urinary retention, leukorrhea, vulvar swelling and itching, eczema, damp sores, pruritus, scabies, and leprosy; externally, it is used to treat trichomonal vaginitis. According to modern pharmacological studies, *Sophora flavescens* extract has antioxidant and anti-inflammatory activities.

[0003] The mechanisms of cellular senescence are complex and interconnected, involving multiple aspects such as telomere shortening, oxidative stress damage, endoplasmic reticulum stress, oncogene activation, DNA damage, and mitochondrial dysfunction. These mechanisms work together to drive cells from a proliferative state to an irreversible growth arrest state.

[0004] Therefore, finding a new natural ingredient that can be used to prepare anti-aging products is a current key research direction. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides the application of matrine O in the preparation of anti-aging products.

[0006] Application of matrine O in the preparation of anti-aging products.

[0007] Furthermore, the matrine O has the molecular formula C0. 27 H 30 O 13 Its molecular weight is 562.52.

[0008] Furthermore, the specific structural formula of matrine O is as follows: .

[0009] Furthermore, the anti-aging product mentioned above is an anti-aging drug or cosmetic.

[0010] Furthermore, the anti-aging drug is a drug that reduces the content of β-galactosidase.

[0011] Furthermore, the anti-aging drug is a drug that reduces ROS levels.

[0012] Furthermore, the anti-aging drug is a drug that increases mitochondrial membrane potential.

[0013] Furthermore, the anti-aging drug includes oral or injectable formulations. The anti-aging drug includes at least one pharmaceutically acceptable conventional carrier and / or excipient, such as diluents, excipients, fillers, binders, humectants, disintegrants, absorption enhancers, surfactants, adsorbents, and lubricants.

[0014] The beneficial effects of this invention are as follows: This invention provides the first application of matrine O in the preparation of anti-aging products. This application innovatively discovers the antioxidant stress effect of matrine O (KO), an active ingredient of Sophora flavescens, which can reduce β-galactosidase content, reduce ROS content, and increase mitochondrial membrane potential. It can be applied to the preparation of drugs and / or cosmetics related to anti-oxidation and anti-aging, and has certain application prospects and social significance. Attached Figure Description

[0015] Figure 1 The image shows the cytotoxicity results of hydrogen peroxide (H2O2) as a modeling drug, using human embryonic lung fibroblasts (MRC-5) as an aging model in Example 1. Figure 2 Example 2: Human embryonic lung fibroblasts (MRC-5) were used as an aging model to detect the active ingredient matrine O (KO) in Sophora flavescens. Figure 3 This is a figure showing the effect of matrine O (KO), an active ingredient of Sophora flavescens, on the staining of β-galactosidase, a marker of oxidative stress, in a hydrogen peroxide (H2O2)-induced human embryonic lung fibroblast (MRC-5) model, as described in Example 3. Figure 4 This is a figure showing the effect of matrine O (KO), an active ingredient of Sophora flavescens, on the fluorescence staining of ROS, a marker of oxidative stress, in a hydrogen peroxide (H2O2)-induced human embryonic lung fibroblast (MRC-5) model, as described in Example 4. Figure 5 The figure shows the effect of fluorescence staining of matrine O (KO), an active ingredient of Sophora flavescens, on the mitochondrial membrane potential, a marker of oxidative stress, in a hydrogen peroxide (H2O2)-induced human embryonic lung fibroblast (MRC-5) model, as described in Example 5. Detailed Implementation

[0016] The specific embodiments of the present invention will be described in further detail below, but the present invention is not limited to these embodiments. Any improvements or substitutions based on the basic spirit of these embodiments shall still fall within the scope of protection claimed by the claims of the present invention.

[0017] Definition of noun The term "matrine O (KO)" in this article refers to a single chemical component obtained from the Chinese herb Sophora flavescens through specific extraction methods (commonly water, ethanol, or a water-alcohol mixture), filtration, concentration, purification, and drying.

[0018] The term “treatment” in this article includes preventing or alleviating a condition, slowing the onset or development of a condition, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or stopping symptoms associated with a condition, producing a complete or partial reversal of a condition, curing a condition, or a combination of the above.

[0019] In this article, the term "prevention" refers to preventing the onset, development, or worsening of a disease or health problem before or at an early stage, through proactive interventions (such as medication). Its core objective is to reduce the probability of disease occurrence, delay the onset time, and / or reduce the negative impact of disease on an individual.

[0020] The term "pharmaceutical acceptable" in this article means that the form of the compound must meet requirements such as safety, stability, and suitability for formulation.

[0021] This application does not impose any special limitations on the preparation process of matrine O (KO), an active substance of Sophora flavescens, and it can be extracted and prepared based on the existing publicly disclosed related processes for the preparation of matrine O (KO).

[0022] The preparation method of the Sophora flavescens extract includes: The traditional Chinese medicine Sophora flavescens powder was extracted by reflux at 75-85℃ using two solvents: 90%-99% (v / v) ethanol and 70%-80% (v / v) ethanol. Each extraction lasted 1-3 hours. The extracts were filtered and combined. The solvent was then recovered by vacuum concentration (temperature ≤40℃) to finally obtain a total ethanol extract with an alcohol content of less than 10% (w / w). Subsequently, liquid-liquid extraction was used to extract the obtained ethanol extract with ethyl acetate and water. The extracts were combined, concentrated under reduced pressure to recover the organic solvent, and then evaporated to dryness to obtain an extract. The extract was freeze-dried to obtain a powder, which was then separated and purified by polyamide column chromatography, gel column chromatography, and semi-preparative high-performance liquid chromatography. After drying, the structural formula of the compound was determined by 1H NMR and 1C NMR spectroscopy, and finally matrine O (KO), the active substance of Sophora flavescens, was obtained.

[0023] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0024] Material: The traditional Chinese medicine Sophora flavescens powder was extracted by reflux at 75-85℃ using two solvents: 90%-99% (v / v) ethanol and 70%-80% (v / v) ethanol. Each extraction lasted 1-3 hours. The extracts were filtered and combined. The solvent was then recovered by vacuum concentration (temperature ≤40℃) to finally obtain a total ethanol extract with an alcohol content of less than 10% (w / w). Subsequently, liquid-liquid extraction was used to extract the obtained ethanol extract with ethyl acetate and water. The extracts were combined, concentrated under reduced pressure to recover the organic solvent, and then evaporated to dryness to obtain an extract. The extract was freeze-dried to obtain a powder, which was then separated and purified by polyamide column chromatography, gel column chromatography, and semi-preparative high-performance liquid chromatography. After drying, the structural formula of the compound was determined by 1H NMR and 1C NMR spectroscopy, and finally matrine O (KO), the active substance of Sophora flavescens, was obtained.

[0025] Example 1 The effect of hydrogen peroxide (H2O2) on the viability of human embryonic lung fibroblasts (MRC-5) was tested using the CCK-8 assay kit.

[0026] (1) Experimental methods 1. Human embryonic lung fibroblasts (MRC-5) were passaged. After the cells adhered, target cells in the logarithmic growth phase were taken, digested with trypsin, resuspended in culture medium and the cell density was adjusted. 100 μL of each cell culture was seeded into 96-well cell culture plates (the edge wells were filled with an equal volume of PBS) and incubated in a 37°C, 5% CO2 incubator for 24 h-48 h to allow the cells to adhere and grow.

[0027] 2. Discard the old culture medium in the 96-well plate. Add 100 μL / well of culture medium containing hydrogen peroxide (H2O2) at gradient concentrations (20, 40, 50, 60, 80, 100 μmol / ml) to the experimental group to treat human embryonic lung fibroblasts (MRC-5). Add an equal volume of complete culture medium without H2O2 to the control group. Set up 3-5 replicates for each group and continue to incubate under the above culture conditions for 24 h. 3. Add 10 μL of CCK-8 reagent to each well for incubation. After gentle shaking to mix, incubate in the dark for 1–4 h. Then, perform absorbance measurement. Remove the 96-well plate after incubation and measure the absorbance at 450 nm using a microplate reader. Record the experimental data.

[0028] 4. Finally, cell viability was calculated. The absorbance of the blank wells was used as a baseline, and the net absorbance values ​​of the experimental and control groups were subtracted. Quantitative results were obtained using the formulas: "Cell viability (%) = (Net absorbance of experimental group - Net absorbance of blank group) / (Net absorbance of control group - Net absorbance of blank group) × 100%" and "Cell inhibition rate (%) = 100% - Cell viability". The above results, such as Figure 1 As shown.

[0029] (2) Experimental results The effect of hydrogen peroxide (H2O2) on the toxicity of human embryonic lung fibroblasts (MRC-5) was detected by the CCK-8 assay. The results were obtained from... Figure 1It can be seen that as the concentration of hydrogen peroxide (H2O2) increases, the cell survival rate decreases, while the dose of 60 μmol / ml and below has little effect on cell viability. Therefore, subsequent experiments determined 60 μmol / ml as the modeling dose.

[0030] Example 2 The effect of matrine O (KO), an active substance of Sophora flavescens, on the viability of human embryonic lung fibroblasts (MRC-5) was tested using the CCK-8 kit.

[0031] (1) Experimental methods 1. Passage human embryonic lung fibroblasts (MRC-5) one day in advance. After the cells adhere to the culture plate, take the target cells in the logarithmic growth phase, digest them with trypsin, resuspend them in culture medium and adjust the cell density. Seed 100 μL per well in a 96-well cell culture plate (with the edge wells filled with an equal volume of PBS), and incubate them in a 37°C, 5% CO2 incubator for 24 h-48 h to allow the cells to adhere and grow.

[0032] 2. Discard the old culture medium in the 96-well plate. Add 100 μL / well of culture medium containing matrine O (KO) at gradient concentrations (2.5, 5, 10, 20, 40 μmol / ml) to the experimental groups to treat human embryonic lung fibroblasts (MRC-5). Add an equal volume of 20 μmol / ml resveratrol culture medium to the positive control group. Set up 3-5 replicates for each group and continue to incubate under the above culture conditions for 24 h. After 24 h, discard the old culture medium and use 100 μL / well of 60 μmol / ml H2O2 culture medium to establish the model for 24 h.

[0033] 3. After 24 hours, add 10 μL of CCK-8 reagent to each well for incubation. After gentle shaking to mix, incubate in the dark for 1–4 hours. Then, perform absorbance measurement. Remove the 96-well plate after incubation and measure the absorbance at 450 nm using a microplate reader. Record the experimental data.

[0034] 4. Finally, cell viability was calculated. The absorbance of the blank wells was used as a baseline, and the net absorbance values ​​of the experimental and control groups were subtracted. Quantitative results were obtained using the formulas: "Cell viability (%) = (Net absorbance of experimental group - Net absorbance of blank group) / (Net absorbance of control group - Net absorbance of blank group) × 100%" and "Cell inhibition rate (%) = 100% - Cell viability". The above results, such as Figure 2 As shown.

[0035] (2) Experimental results The effect of matrine O (KO) on the toxicity of human embryonic lung fibroblasts (MRC-5) was detected by the CCK-8 assay. The results were obtained from... Figure 2It can be seen that the cell survival rate increases with the increase of matrine O (KO) concentration.

[0036] Example 3 Effects of matrine O (KO) on the expression level of β-galactosidase, a marker of aging, in a H2O2-induced oxidative stress model of human embryonic lung fibroblasts (MRC-5).

[0037] (1) Experimental methods 1. Human embryonic lung fibroblasts (MRC-5) in the logarithmic growth phase were seeded at an appropriate density in 12-well plates and cultured in an incubator for 24 hours until the cells adhered and covered approximately 50%–60% of the plate. After 24 hours, the culture medium was discarded. The experimental groups were given culture medium containing graded concentrations (5, 10, 20 μmol / ml) of matrine O (KO), and the positive control group was given 1 ml / well of 20 μmol / ml resveratrol culture medium. After 24 hours of culture, the original culture medium was discarded and 1 ml / well of 60 μmol / ml H2O2 culture medium was added to establish the model. The control group was given the same amount and concentration of complete culture medium throughout the process. Each group was divided into 3 replicates and incubated under the above culture conditions for another 24 hours.

[0038] 2. Prepare the SA-β-Gal staining working solution according to the kit instructions. Rinse the cells 2-3 times with PBS, each wash lasting 3-5 minutes. Then add an appropriate amount of cell fixation solution and fix at room temperature for 15-20 minutes. After fixation, wash the cells 3 times with PBS to remove residual fixation solution. After rinsing, add 1 ml of staining working solution to each well and incubate at 37°C in the dark for 12-24 hours. After incubation, discard the staining working solution, wash the cells 2-3 times with PBS, observe and photograph them under an optical microscope. Cells with blue-green staining in the cytoplasm are positive senescent cells.

[0039] (2) Experimental results Depend on Figure 3 It can be seen that matrine O (KO) can reduce the expression level of β-galactosidase in the model group in a dose-dependent manner, which further illustrates that matrine O (KO) reduces the content of β-galactosidase in a concentration-dependent manner and improves the degree of cellular oxidative stress to a certain extent.

[0040] Example 4 Effects of matrine O (KO) on the level of aging marker ROS in a H2O2-induced human embryonic lung fibroblast (MRC-5) oxidative stress model.

[0041] Experimental methods 1. Take human embryonic lung fibroblasts (MRC-5) in the logarithmic growth phase, seed them in six-well plates according to experimental requirements, and culture them until the cell confluence reaches 50%~60%. Then, treat them according to the experimental design. Discard the old culture medium, gently wash the cells twice with serum-free culture medium, and then add serum-free culture medium containing ROS fluorescent probe. Incubate at 37°C in the dark for 20~30 minutes.

[0042] 2. After incubation, discard the probe working solution and gently wash the cells three times with PBS to remove free probes that have not entered the cells. Add an appropriate amount of fresh serum-free culture medium to the culture dish and immediately place it under a fluorescence microscope. Select the corresponding excitation and emission wavelengths for observation and photography. The fluorescence intensity can reflect the level of ROS in the cells.

[0043] (2) Experimental results Depend on Figure 4 It can be seen that matrine O (KO) significantly reduced the ROS content compared to the model group, further demonstrating that matrine O (KO) has the activity of improving cellular oxidative stress.

[0044] Example 5 Effects of matrine O (KO) on mitochondrial membrane potential, a marker of aging, in a H2O2-induced oxidative stress model of human embryonic lung fibroblasts (MRC-5).

[0045] (1) Experimental methods 1. Take human embryonic lung fibroblasts in the logarithmic growth phase, seed them into six-well plates at an appropriate density, and incubate them in an incubator until the cell confluence reaches 60%~70%. Perform the corresponding treatments according to the experimental protocol. Discard the old culture medium, and gently wash the cells twice with PBS to remove residual culture medium and cell metabolic waste.

[0046] 2. Prepare JC-1 staining working solution according to the kit instructions. Add sufficient working solution to the culture dish to completely cover the cells. Incubate in a 37°C incubator in the dark for 20-30 minutes. After incubation, aspirate the staining solution, rinse the cells twice with JC-1 washing solution, add fresh culture medium, and immediately observe under a fluorescence microscope. Normal young cells have a high mitochondrial membrane potential and strong red fluorescence. As the membrane potential decreases after aging, the fluorescence weakens.

[0047] The above results, such as Figure 5 As shown.

[0048] (2) Experimental results The result is Figure 5 It can be seen that as matrine O (KO) increases mitochondrial membrane potential in a concentration-dependent manner, it improves the degree of cellular oxidative stress.

[0049] In summary, this invention provides for the first time the application of matrine O in the preparation of anti-aging products, which can be used to prepare drugs and / or cosmetics related to anti-oxidation and anti-aging, and has certain application prospects and social significance.

Claims

1. Application of matrine O in the preparation of anti-aging products.

2. The application of matrine O as described in claim 1 in the preparation of anti-aging products, characterized in that, The matrine O has the molecular formula C0. 27 H 30 O 13 Its molecular weight is 562.

52.

3. The application of matrine O as described in claim 1 in the preparation of anti-aging products, characterized in that, The specific structural formula of matrine O is as follows: 。 4. The application of matrine O as described in claim 1 in the preparation of anti-aging products, characterized in that, The anti-aging products mentioned are anti-aging drugs or cosmetics.

5. The application of matrine O as described in claim 4 in the preparation of anti-aging products, characterized in that, The anti-aging drug mentioned is one that reduces the content of β-galactosidase.

6. The application of matrine O as described in claim 4 in the preparation of anti-aging products, characterized in that, The anti-aging drug mentioned is one that reduces ROS levels.

7. The application of matrine O as described in claim 4 in the preparation of anti-aging products, characterized in that, The anti-aging drug mentioned is one that increases mitochondrial membrane potential.

8. The application of matrine O as described in claim 4 in the preparation of anti-aging products, characterized in that, The anti-aging drugs include oral or injectable formulations.

9. The application of matrine O as described in claim 8 in the preparation of anti-aging products, characterized in that, The anti-aging drug includes at least one of pharmaceutically acceptable conventional carriers and / or excipients, such as diluents, excipients, fillers, binders, humectants, disintegrants, absorption enhancers, surfactants, adsorbents, and lubricants.

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