Composition for repairing DNA and delaying senescence and preparation method and application thereof

Abstract

The invention relates to a composition for repairing DNA and delaying senescence and a preparation method and application thereof.The composition is prepared from, by mass, 10%-30% of rhodiola rosea extract, 5%-15% of ergothioneine, 1%-5% of pyrroloquinoline quinone (PQQ), 5%-20% of acai extract, 0.1%-3% of astaxanthin, 0.5%-5% of lycopene, 0.1%-2% of vitamin B2, 0.01%-0.5% of vitamin B12 and the balance water. The balance is a pharmaceutically or food acceptable carrier. The preparation method comprises the following steps: step 1, mixing the rhodiola rosea extract, the ergothioneine, the pyrroloquinoline quinone (PQQ), the acai extract, the astaxanthin and the lycopene in proportion; step 2, adding vitamins B2 and B12, and carrying out homogenization treatment; and step 3, adding a pharmaceutically or food acceptable carrier to prepare a target dosage form. The composition realizes an anti-aging effect at a cell DNA gene level through a targeted DNA repair pathway and in combination with a multi-component synergistic effect mechanism.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a composition for repairing DNA and delaying aging, as well as a preparation method and application thereof. Background Art

[0002] Current anti-aging technologies primarily rely on single ingredients (such as resveratrol and coenzyme Q10) or simple combination strategies, focusing on single pathways such as antioxidant protection or improving mitochondrial function. Studies have confirmed that inefficient DNA repair is a core mechanism of aging (Gaspar-Silva F, 2023). However, existing technologies suffer from single targets (such as activating only sirtuins or inhibiting mTOR) and low bioavailability (such as ergothioneine's difficulty penetrating the blood-brain barrier). Furthermore, there is a lack of research tailored to the genomic characteristics of East Asian populations.

[0003] Among existing technologies, traditional approaches fail to achieve multi-pathway synergy (e.g., linking DNA repair with energy metabolism), resulting in anti-aging effects limited to local organs (e.g., improving only the skin or heart); dosage form design fails to address ingredient stability (e.g., PQQ is easily degraded) and delivery efficiency; clinical validation suffers from racial bias (e.g., differences in telomere shortening rates among Asians are not considered); and safety quality control is insufficient. Furthermore, existing anti-aging compositions fail to address the construction of cross-organ anti-aging networks and adaptation to specific genotypes, failing to meet the needs of precise anti-aging. Summary of the Invention

[0004] In response to the shortcomings of the existing technology, the purpose of the present invention is to provide a composition for repairing DNA and delaying aging, as well as its preparation method and application. It achieves anti-aging effects at the cellular DNA gene level by targeting the DNA repair pathway and combining a multi-component synergistic mechanism.

[0005] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0006] On the one hand, the present invention discloses a composition for repairing DNA and delaying aging, comprising the following active ingredients: Rhodiola rosea extract, ergothioneine, pyrroloquinoline quinone (PQQ), acai extract, astaxanthin, lycopene, vitamin B2 and vitamin B12.

[0007] As a further technical solution of the present invention: the mass percentage of each component is:

[0008] 10-30% of Rhodiola rosea extract, 5-15% of ergothioneine, 1-5% of pyrroloquinoline quinone (PQQ), 5-20% of acai extract, 0.1-3% of astaxanthin, 0.5-5% of lycopene, 0.1-2% of vitamin B2, 0.01-0.5% of vitamin B12, and the remainder being pharmaceutically or food-acceptable carriers.

[0009] As a further technical solution of the present invention: the composition is used to activate Sirtuin family proteins (including SIRT1 and SIRT6), inhibit the LOXL2-TGF-β1-Smad2 / 3 pathway, or enhance DNA telomerase activity.

[0010] As a further technical solution of the present invention: the composition is used for one of the following purposes:

[0011] Repair DNA damage and improve genomic instability;

[0012] Delay skin aging, reduce wrinkles, spots and enlarged pores;

[0013] Improve brain cognitive function and prevent or treat Alzheimer's disease or Parkinson's disease;

[0014] Protect heart function and inhibit myocardial fibrosis or atrial fibrillation;

[0015] Repair lung tissue damage and improve lung inflammation caused by viruses or smoking.

[0016] As a further technical solution of the present invention: the composition achieves its efficacy through the following mechanisms: activating cell-derived life factors to replace damaged DNA fragments, scavenging free radicals, enhancing mitochondrial function, or promoting neural stem cell differentiation.

[0017] As a further technical solution of the present invention: the dosage form of the composition is capsule, tablet, oral liquid or powder.

[0018] On the other hand, the present invention also discloses a method for preparing a composition for repairing DNA and delaying aging, comprising the following steps:

[0019] Step 1, mixing Rhodiola rosea extract, ergothioneine, pyrroloquinoline quinone (PQQ), acai extract, astaxanthin, and lycopene in proportion;

[0020] Step 2: Add vitamin B2 and B12 and homogenize;

[0021] Step 3: Add a pharmaceutically or food-acceptable carrier to prepare the target dosage form.

[0022] As a further technical solution of the present invention: in step 1, the Rhodiola rosea extract is obtained by water extraction or alcohol extraction, and the total flavonoid content is not less than 20%.

[0023] On the other hand, the present invention also discloses a preparation for improving the sequelae of the new coronavirus, comprising the above-mentioned composition and further comprising coenzyme Q10 or N-acetylcysteine.

[0024] On the other hand, the present invention also discloses a use of the above-mentioned composition in the preparation of a drug for preventing or treating ovarian dysfunction, wherein the composition achieves efficacy by increasing the ovarian AMH index, reducing the FSH level or increasing the number of follicles.

[0025] In summary, the present invention includes at least one of the following beneficial technical effects:

[0026] The present invention discloses a composition for repairing DNA and delaying aging, and its preparation method and application. The composition is a An anti-aging composition containing gene error-correcting factors, which includes core ingredients such as Rhodiola rosea extract, ergothioneine, and pyrroloquinoline quinone (PQQ), significantly improves skin aging, brain cognitive impairment, cardiac dysfunction, and lung damage by activating Sirtuin proteins, repairing DNA damage, and scavenging free radicals. It also has an anti-photoaging effect on human skin keratinocytes. Experimental data show that it can increase SIRT1 gene expression by 64%, telomerase activity by 70%, and reduce DNA damage by 83%. The composition is suitable for a variety of dosage forms such as capsules and tablets and has broad application prospects.

[0027] 1. The present invention uses the synergistic action of a composite active ingredient (Rhodiola rosea, ergothioneine, PQQ, etc.) to target and activate Sirtuin family proteins (such as SIRT1 and SIRT6), repair DNA damage, reduce genomic instability, and enhance telomerase activity (experiments show a 70% increase in telomerase activity). Its mechanisms include scavenging free radicals, regulating the LOXL2-TGF-β1-Smad2 / 3 pathway to inhibit fibrosis, and promoting the differentiation of neural stem cells, achieving cell repair and anti-aging at the genetic level.

[0028] 2. The composition of the present invention shows significant effects in multiple organs such as the skin, brain, heart, and lungs: it can improve skin wrinkles (reduced by 21.61% in 28 days) and pigmentation (reduced by 18.44% in 28 days); enhance brain cognitive function (improved immediate memory by more than 100%), and prevent Alzheimer's disease and Parkinson's disease; repair cardiac fibrosis (reduced probability of atrial fibrillation) and lung damage caused by viruses or smoking; at the same time, it promotes ovarian function (AMH index improves and the number of follicles increases), and assists in repairing the sequelae of the new coronavirus (such as pulmonary fibrosis and nerve damage).

[0029] 3. This invention increased SIRT1 gene expression by 64% (p=0.0073) in a zebrafish model, and clinical studies in humans showed a 132% increase in collagen regeneration. The composition offers flexible dosage forms (capsules, tablets, etc.), suitable for anti-aging, disease intervention, and health management. It also holds safety certifications (SGS-certified estrogen-free and heavy metal-compliant), promising broad medical and consumer applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a statistical graph of comet assay data on the DNA damage repair effect of the samples in Experimental Example 1 of the present invention.

[0031] Figure 2 This is a statistical graph of comet assay data on the DNA protection effect of the samples in Experimental Example 1 of the present invention.

[0032] Figure 3 This is a bar graph of the cell activity results of the samples in Experimental Example 2 of the present invention.

[0033] Figure 4 This is a bar graph of the relative gene expression results in Experimental Example 2 of the present invention.

[0034] Figure 5 This is a comparison chart of the anti-light aging effects in Experimental Example 2 of the present invention.

[0035] Figure 6 This is a flow chart of the test for promoting zebrafish Sirt1 gene expression in Experimental Example 3 of the present invention.

[0036] Figure 7 This is a bar graph of the relative expression levels of the zebrafish Sirt1 gene in Experimental Example 3 of the present invention.

[0037] Figure 8 This is a bar graph of the relative expression levels of the zebrafish type I collagen gene in Experimental Example 4 of the present invention.

[0038] Figure 9 This is a flow chart of the zebrafish aging-related β-galactosidase inhibition test in Experimental Example 5 of the present invention.

[0039] Figure 10 This is a bar graph showing the anti-galactosidase efficacy test results in Experimental Example 5 of the present invention.

[0040] Figure 11 This is a flow chart of the zebrafish embryo reactive oxygen species (ROS) scavenging test in Experimental Example 6 of the present invention.

[0041] Figure 12 This is a bar chart of the antioxidant efficacy test results in Experimental Example 6 of the present invention.

[0042] Figure 13 This is a flow chart of the test for enhancing telomerase activity in zebrafish in Experimental Example 7 of the present invention.

[0043] Figure 14 This is a bar graph showing the promotion of zebrafish telomerase activity in Experimental Example 7 of the present invention.

[0044] Figure 15 This is a scatter plot of the area ratio of under-eye wrinkles versus age in Experimental Example 8 of the present invention (sample size = 200). DETAILED DESCRIPTION

[0045] The technical solutions in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application; it is obvious that the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0046] Example 1:

[0047] On the one hand, this embodiment discloses a composition for repairing DNA and delaying aging, comprising the following active ingredients: Rhodiola rosea extract, ergothioneine, pyrroloquinoline quinone (PQQ), acai extract, astaxanthin, lycopene, vitamin B2, and vitamin B12.

[0048] Among them, the mass percentage of each component of the composition is:

[0049] The composition comprises 20% Rhodiola rosea extract, 10% ergothioneine, 3% pyrroloquinoline quinone (PQQ), 13% acai extract, 1.6% astaxanthin, 2.75% lycopene, 1% vitamin B2, 0.25% vitamin B12, and the remainder is a pharmaceutically or food-acceptable carrier. The composition is used to activate sirtuin family proteins (including SIRT1 and SIRT6), inhibit the LOXL2-TGF-β1-Smad2 / 3 pathway, or enhance DNA telomerase activity.

[0050] The composition is used for one of the following purposes: repairing DNA damage and improving genomic instability; delaying skin aging and reducing wrinkles, spots and enlarged pores; improving brain cognitive function and preventing or treating Alzheimer's disease or Parkinson's disease; protecting heart function and inhibiting myocardial fibrosis or atrial fibrillation; repairing lung tissue damage and improving lung inflammation caused by viruses or smoking.

[0051] The composition achieves its efficacy through the following mechanisms: activating cellular endogenous life factors to replace damaged DNA fragments, scavenging free radicals, enhancing mitochondrial function, or promoting neural stem cell differentiation. In this embodiment, the composition is in the form of a capsule, tablet, oral solution, or powder.

[0052] On the other hand, this embodiment also discloses a method for preparing the composition for repairing DNA and delaying aging as described above, comprising the following steps:

[0053] Step 1, mixing Rhodiola rosea extract, ergothioneine, pyrroloquinoline quinone (PQQ), acai extract, astaxanthin, and lycopene in proportion;

[0054] Step 2: Add vitamin B2 and B12 and homogenize;

[0055] Step 3: Add a pharmaceutically or food-acceptable carrier to prepare the target dosage form.

[0056] In step 1, the Rhodiola rosea extract is obtained by a water extraction or alcohol extraction process, and the total flavonoid content is not less than 20%.

[0057] On the other hand, the present invention also discloses a preparation for improving the sequelae of the new coronavirus, comprising the above-mentioned composition and further comprising coenzyme Q10 or N-acetylcysteine. On the other hand, the present invention also discloses the use of the above-mentioned composition in the preparation of a drug for preventing or treating ovarian dysfunction, wherein the composition achieves efficacy by increasing the ovarian AMH index, reducing the FSH level, or increasing the number of follicles.

[0058] Example 2:

[0059] On the one hand, this embodiment discloses a composition for repairing DNA and delaying aging, comprising the following active ingredients: Rhodiola rosea extract, ergothioneine, pyrroloquinoline quinone (PQQ), acai extract, astaxanthin, lycopene, vitamin B2, and vitamin B12.

[0060] Among them, the mass percentage of each component of the composition is:

[0061] The composition comprises 10% Rhodiola rosea extract, 5% ergothioneine, 1% pyrroloquinoline quinone (PQQ), 5% acai extract, 0.1% astaxanthin, 0.5% lycopene, 0.1% vitamin B2, 0.01% vitamin B12, and the remainder is a pharmaceutically or food-acceptable carrier. The composition is used to activate sirtuin family proteins (including SIRT1 and SIRT6), inhibit the LOXL2-TGF-β1-Smad2 / 3 pathway, or enhance DNA telomerase activity.

[0062] The composition is used for one of the following purposes: repairing DNA damage and improving genomic instability; delaying skin aging and reducing wrinkles, spots and enlarged pores; improving brain cognitive function and preventing or treating Alzheimer's disease or Parkinson's disease; protecting heart function and inhibiting myocardial fibrosis or atrial fibrillation; repairing lung tissue damage and improving lung inflammation caused by viruses or smoking.

[0063] The composition achieves its efficacy through the following mechanisms: activating cellular endogenous life factors to replace damaged DNA fragments, scavenging free radicals, enhancing mitochondrial function, or promoting neural stem cell differentiation. In this embodiment, the composition is in the form of a capsule, tablet, oral solution, or powder.

[0064] On the other hand, this embodiment also discloses a method for preparing the composition for repairing DNA and delaying aging as described above, comprising the following steps:

[0065] Step 1, mixing Rhodiola rosea extract, ergothioneine, pyrroloquinoline quinone (PQQ), acai extract, astaxanthin, and lycopene in proportion;

[0066] Step 2: Add vitamin B2 and B12 and homogenize;

[0067] Step 3: Add a pharmaceutically or food-acceptable carrier to prepare the target dosage form.

[0068] In step 1, the Rhodiola rosea extract is obtained by a water extraction or alcohol extraction process, and the total flavonoid content is not less than 20%.

[0069] On the other hand, the present invention also discloses a preparation for improving the sequelae of the new coronavirus, comprising the above-mentioned composition and further comprising coenzyme Q10 or N-acetylcysteine. On the other hand, the present invention also discloses the use of the above-mentioned composition in the preparation of a drug for preventing or treating ovarian dysfunction, wherein the composition achieves efficacy by increasing the ovarian AMH index, reducing the FSH level, or increasing the number of follicles.

[0070] Example 3:

[0071] On the one hand, this embodiment discloses a composition for repairing DNA and delaying aging, comprising the following active ingredients: Rhodiola rosea extract, ergothioneine, pyrroloquinoline quinone (PQQ), acai extract, astaxanthin, lycopene, vitamin B2, and vitamin B12.

[0072] Among them, the mass percentage of each component of the composition is:

[0073] The composition comprises 30% Rhodiola rosea extract, 15% ergothioneine, 5% pyrroloquinoline quinone (PQQ), 20% acai extract, 3% astaxanthin, 5% lycopene, 2% vitamin B2, and 0.5% vitamin B12, with the remainder being a pharmaceutically or food-acceptable carrier. The composition is used to activate sirtuin family proteins (including SIRT1 and SIRT6), inhibit the LOXL2-TGF-β1-Smad2 / 3 pathway, or enhance DNA telomerase activity.

[0074] The composition is used for one of the following purposes: repairing DNA damage and improving genomic instability; delaying skin aging and reducing wrinkles, spots and enlarged pores; improving brain cognitive function and preventing or treating Alzheimer's disease or Parkinson's disease; protecting heart function and inhibiting myocardial fibrosis or atrial fibrillation; repairing lung tissue damage and improving lung inflammation caused by viruses or smoking.

[0075] The composition achieves its efficacy through the following mechanisms: activating cellular endogenous life factors to replace damaged DNA fragments, scavenging free radicals, enhancing mitochondrial function, or promoting neural stem cell differentiation. In this embodiment, the composition is in the form of a capsule, tablet, oral solution, or powder.

[0076] On the other hand, this embodiment also discloses a method for preparing the composition for repairing DNA and delaying aging as described above, comprising the following steps:

[0077] Step 1, mixing Rhodiola rosea extract, ergothioneine, pyrroloquinoline quinone (PQQ), acai extract, astaxanthin, and lycopene in proportion;

[0078] Step 2: Add vitamin B2 and B12 and homogenize;

[0079] Step 3: Add a pharmaceutically or food-acceptable carrier to prepare the target dosage form.

[0080] In step 1, the Rhodiola rosea extract is obtained by a water extraction or alcohol extraction process, and the total flavonoid content is not less than 20%.

[0081] On the other hand, the present invention also discloses a preparation for improving the sequelae of the new coronavirus, comprising the above-mentioned composition and further comprising coenzyme Q10 or N-acetylcysteine. On the other hand, the present invention also discloses the use of the above-mentioned composition in the preparation of a drug for preventing or treating ovarian dysfunction, wherein the composition achieves efficacy by increasing the ovarian AMH index, reducing the FSH level, or increasing the number of follicles.

[0082] Experimental Example 1:

[0083] The "VANPEARL ERGO-VITALIS" gene capsules were prepared using the formula and preparation method of the present invention, and samples of the gene capsules were used to test the DNA damage repair and protection efficacy. The test showed that under the experimental conditions, the "VANPEARL ERGO-VITALIS" gene capsules submitted for testing could significantly improve the comet tail length, tail moment and Olive tail moment of HepG2 cells in the damage model: when the HepG2 cells after sample treatment were irradiated with UVA, the comet tail length, tail moment and Olive tail moment of the cells showed no significant difference compared with the control group, indicating that the submitted sample has DNA repair and protection efficacy.

[0084] Method name: Comet assay;

[0085] Method Source: Laboratory method;

[0086] System and grouping:

[0087] Test system: HepG2 cells;

[0088] Measured values: comet tail length, tail moment, Oive tail moment;

[0089] Grouping: control group, injury control group, sample groups (low concentration group - Example 2, medium concentration group - Example 1, high concentration group - Example 3).

[0090] Principle and method: The comet assay, also known as the single-cell gel electrophoresis assay, is a technique for detecting DNA damage and repair in individual cells. Under the action of an electric field, the DNA of normal cells appears as a round star on the gel due to its large molecular weight, compact structure, and low migration rate. When cellular DNA is damaged, such as single-strand or double-strand breaks, the DNA unwinds in an alkaline environment, and the broken DNA fragments migrate in the electric field, forming an image resembling a comet's tail. The degree of DNA damage can be quantified by analyzing parameters such as the comet tail length, tail moment (the product of tail length and DNA content in the tail), and olive tail moment (the product of olive tail length and DNA content in the tail, where olive tail length refers to the distance from the center of the comet head to the center of the comet's tail).

[0091] If health products have the effect of promoting DNA damage repair, then after the model group suffering from DNA damage undergoes sample treatment, the comet test parameters of the treated cell DNA detection data should be better than those of the untreated group, that is, the values such as tail length, tail moment, and Olive tail moment will decrease, indicating that the DNA damage has been repaired.

[0092] If health products have the effect of promoting DNA protection, then cells treated with samples will be less likely to suffer DNA damage when subjected to the same external DNA damage (such as UVA irradiation) than cells not treated with samples. The comet assay parameters of the sample-treated group should be better than those of the untreated group, that is, the tail length, tail moment, Oiv tail moment and other values will be reduced, indicating that the DNA protection effect of the sample-treated cells is significant.

[0093] The experimental steps are:

[0094] 1. Experimental groups:

[0095] Normal control group: cells were cultured normally without DNA damage induction or sample treatment;

[0096] Damage control group: cells were treated with 100 μM H2O2 to induce DNA damage, and no sample was added;

[0097] Low concentration group: treated with 100 μM H2O2 and then treated with 10 μg / mL sample;

[0098] Medium concentration group: treated with 100 μM H2O2 and then treated with 50 μg / mL sample;

[0099] High-concentration group: treated with 100 μM H2O2 and then treated with 100 μg mL sample.

[0100] 2. Cell culture:

[0101] (1) Resuscitating HpG2 cells, inoculating them into culture flasks containing complete medium, and culturing them in a 37°C, 5% CO2 incubator. Subsequent experiments were performed when the cells were in the logarithmic growth phase:

[0102] (2) DNA damage induction and sample processing: cells in the logarithmic growth phase were digested with trypsin-EDTA digestion solution and the cell density was adjusted to 1×10 6 mL: 100 μM H2O2 was added to each treatment group and incubated at 37°C for 30 minutes to induce DNA damage: After damage treatment, the sample solutions of corresponding concentrations were added to the low, medium, and high concentration groups, and the samples were incubated at 37°C for 2 hours. Equal volumes of culture medium were added to the normal control group and the damage control group, and the comet assay data of each group were detected.

[0103] (3) DNA protection and sample processing: Sample solutions of corresponding concentrations were added to the low, medium and high concentration groups and incubated at 37°C for 2 hours. UVA was used to irradiate the samples in the low, medium and high concentration groups for 20 minutes, and the comet experiment data of the low, medium and high concentration groups and the control group were detected.

[0104] 3. Comet experiment operation:

[0105] (1) Preparation of the first layer of adhesive: Coat the slide with NMA (0.5%) and dry at 4°C.

[0106] (2) Prepare the second layer of gel: Take 100 μL of cell suspension and mix it with 900 μL of 0.7% LMA, quickly drop it onto the first layer of gel, cover it with a glass slide, and let it stand at 4°C for 10 minutes to allow the gel to solidify.

[0107] (3) Cell lysis: Place the slide in cell lysis solution and lyse in the dark at 4°C for 1 hour.

[0108] (4) Unwinding and electrophoresis: Transfer the slide to alkaline electrophoresis buffer and unwind for 20 minutes to unwind the DNA. Then, run electrophoresis at 25 V and 300 mA for 20 minutes.

[0109] (5) Neutralization: After electrophoresis, place the slide in neutralization buffer and neutralize for 15 minutes. Repeat 3 times:

[0110] (6) Staining: Stain with EB staining solution for 15 minutes, rinse with distilled water and dry.

[0111] 4. Observation and analysis of results:

[0112] (1) At least 100 cells were randomly selected from each sample for observation under a fluorescence microscope.

[0113] (2) Use image analysis software (such as Comet Assay IV software) to measure parameters such as comet tail length, tail moment, and Olive tail moment.

[0114] Experimental reagents, consumables, and instruments are:

[0115] 1. Experimental instruments: centrifuge, constant temperature water bath, horizontal electrophoresis tank, electrophoresis apparatus, fluorescence microscope, cell culture incubator, slides, cover slips, micropipette, cell counting chamber.

[0116] 2. Experimental consumables and reagents: normal melting point agarose, low melting point agarose, cell lysis buffer, alkaline electrophoresis buffer, neutralization buffer, ethidium bromide (EB) staining.

[0117] 3. Modeling drugs: 100μMH2O2 treatment induces DNA damage.

[0118] 4. Sample: Gene capsules prepared according to Example 1, Example 2, and Example 3 of the present invention.

[0119] The judgment basis is: statistical analysis p<0.05, which is considered to be a significant difference.

[0120] The test results of the samples on DNA damage repair are as follows:

[0121] Table 1. Comet assay test results of samples on DNA damage repair

[0122] Group Comet tail length (μm) Tail moment (μm) Olive tail moment (μm) Normal control group 9.61±0.44 4.63±0.29 3.57±0.18 Injury control group 17.24±0.86 8.03±0.39 7.04±0.31 10 μg / mL group 15.17±0.77 7.19±0.33 6.42±0.27 50 μg / mL group 13.82±0.64 6.47±0.29 5.21±0.24 100 μg / mL group 11.07±0.53 5.13±0.24 4.08±0.19

[0123] Table 2. Statistics of the efficacy index of samples on DNA damage repair

[0124]

[0125] The test results of the DNA damage repair effect of the samples of the present invention are shown in Table 1, Table 2 and Figure 1 As shown, in Figure 1Statistical analysis was performed using GraphicsPad 8.3.0 software. * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001. Results: Compared with the normal control group, the DNA tail length, tail moment, and Olive tail moment of the cells in the damage control group were increased, indicating that the DNA damage model was successfully established and that the DNA of HepG2 cells was damaged by H2O2. Compared with the damage control group, the DNA tail length, tail moment, and Olive tail moment of the cells in the three treatment groups of the sample group were shortened to varying degrees. This shortening was dose-dependent with increasing sample concentration, and the data were statistically significant, indicating that the sample had significant repair efficacy against the damage caused by the modeling drug H2O2 in HepG2 cells. Using standardized statistical methods, the sample's efficacy index for DNA damage repair was 23.23% at 10 μg / mL, 47.81% at 50 μg / mL, and 83.82% at 100 μg / mL.

[0126] The test results of the samples' DNA protection are as follows:

[0127] Table 3. Comet assay test results of samples' protective effects on DNA

[0128]

[0129] Result analysis: refer to Figure 2 and Table 3, in Figure 2 Statistical analysis was performed using GraphicPad 8.3.0 software. ns represents p>0.05, and *** represents p<0.001. Compared with the control group, the DNA tail length, tail moment, and OIiv tail moment of the control + UVA irradiation group were significantly longer, and the statistical data showed significant differences, indicating that the DNA of the control group cells was significantly damaged after UVA irradiation. Compared with the sample treatment group (100 μg / ml), the DNA tail length, tail moment, and OIiv tail moment of the sample treatment + UVA irradiation group cells did not show a significantly longer phenotype, and the statistical data did not show significant differences, indicating that the sample treatment can protect the DNA of HepG2 cells and reduce the damage caused by UVA irradiation, and has a significant protective effect on cellular DNA.

[0130] Conclusion: After testing, under the experimental conditions, the sample "VANPEARL ERGO-VITALIS" gene capsule can significantly improve the comet tail length, tail moment and Olive tail moment of damaged HepG2 cells; when the HepG2 cells treated with the sample were irradiated with UVA, there was no significant difference in the comet tail length, tail moment and Olive tail moment of the cells compared with the control group, indicating that the sample has the effect of repairing and protecting DNA.

[0131] Experimental Example 2:

[0132] The "VANPEARL ERGO-VITALIS" gene capsule is prepared by adopting the formula and preparation method of the present invention, and the anti-aging test of keratinocytes is carried out using samples of the gene capsule.

[0133] 1. Principle: Repeated exposure to ultraviolet (UV) radiation can cause photoaging of the skin. Depending on the wavelength of UV radiation, UVA (320-400 nm) penetrates the skin to varying degrees and interacts with skin cells. UVA (320-400 nm) penetrates the epidermis and reaches the dermis, primarily causing skin photoaging. UVA radiation acts on melanocytes and other skin cells, such as keratinocytes, causing DNA damage through oxidative stress and the production of reactive oxygen species (ROS). ROS activate signaling pathways associated with cell and tissue growth, differentiation, aging, and photoaging. Furthermore, ROS generated in UVA-irradiated human skin cells are a major cause of photoaging. UV exposure can also induce the expression of ROS, leading to skin inflammation and downregulation of SIRT1. SIRT1 is a nuclear protein belonging to the nicotinamide adenine dinucleotide-dependent enzyme family. Furthermore, SIRT1 plays a key role in regulating cell growth, proliferation, and aging. Therefore, SIRT1 inhibition is often considered a hallmark of aging, and SIRT1 is involved in UV-induced DNA damage repair, cellular metabolism, and photoaging. Growing evidence supports the hypothesis that photoaging is regulated by a continuous crosstalk between ROS and SIRT1. This study used human keratinocytes as the research subjects, established a photoaging model using UVA exposure, and measured SIRT1 and SIRT6 gene expression after exposure to test compounds to evaluate their effectiveness in preventing photoaging damage.

[0134] 2. Experimental Materials:

[0135] Experimental sample: "VANPEARL ERGO-VITALIS" gene capsule;

[0136] Experimental cells: human immortalized keratinocytes (HaCaT);

[0137] The main reagents used in the experiment were: DMEM medium (LOT: 2537078), fetal bovine serum (LOT: BC20230111), trypsin (LOT: 2523119), PBS buffer (LOT: 2309011), and TRIzol reagent (LOT: 041223230824).

[0138] The main experimental instruments include: biological safety cabinet (SafeFast Classic 212A), carbon dioxide incubator (SFEGROW188PRO), inverted phase contrast microscope (MI52-N), microplate reader (SpectraMax 340PC384), analytical balance (FA2204N), flow cytometer (JIMBIO iCytal S1), cell phototoxicity irradiator (LUYOR-3450), and quantitative PCR instrument (ABIPRISM, 7500 Sequence Detection System).

[0139] 3. Experimental methods:

[0140] 3.1. Cell activity:

[0141] Table 4. Experimental groups and treatments

[0142]

[0143]

[0144] (1) Cell inoculation: The cell suspension was inoculated into a 96-well cell culture plate at 100 μL / well and placed in a carbon dioxide incubator at 37°C, 5% CO2, and humidity >90% for 18-24 hours.

[0145] (2) Administration: Administration was performed according to the experimental groups and treatments in Table 4. 100 μL of cell culture medium was added to each well of the zero adjustment group and the control group; 100 μL of cell culture medium containing the corresponding concentration of sample was added to each well of the sample group. After administration, the 96-well plate was placed in a carbon dioxide incubator at 37°C, 5% CO2, and humidity >90% for 24 hours.

[0146] (3) Cell viability assay: The supernatant was discarded, and a culture medium containing MTT was added to each well of the blank control group and the sample group, and the cells were incubated in a carbon dioxide incubator at 37°C, 5% CO2, and humidity >90% in the dark for 3 hours. After the incubation, the supernatant of the zero adjustment group, the blank control group, and the sample group was discarded, and 100 μL of dimethyl sulfoxide (DMSO) was added to each well. The absorbance was measured at a wavelength of 570 nm using a microplate reader. The mechanism of action is that the succinate dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to water-insoluble blue-purple crystalline formazan and deposit it in the cells, while dead cells do not have this function. DMSO can dissolve the formazan in the cells, and its light absorbance is measured using a microplate reader, which can indirectly reflect the number of living cells.

[0147] 3.2 SIRT1 gene expression test:

[0148] Table 5. Experimental groups and treatments

[0149]

[0150] *The blank control group was set up for the purpose of normalization to calculate the relative gene expression.

[0151] (1) Cell inoculation: 2 mL of cell suspension was inoculated into a 6-well cell culture plate at a concentration of 2 mL / well and cultured in a carbon dioxide incubator at 37°C, 5% CO2, and humidity >90% for 18-24 hours.

[0152] (2) Modeling and Dosing: Dosing was performed according to the experimental grouping and treatment in Table 5. 2 mL of culture medium was added to the blank control group and the model control group, and 2 mL of culture medium containing a certain concentration of the test substance was added to the sample group. The cells were exposed for 24 h. Afterwards, the wells were washed with PBS. The model control group and the sample group were irradiated with UVA. After irradiation, the samples of each group were re-prepared according to the dosing conditions in Table 5 and incubated again for 4 h.

[0153] (3) Gene expression assay: Wash twice with 1 mL of PBS. After washing, add 1 mL of Trizol reagent to each well of the blank control group, model control group, and sample group to fully lyse the cells. RNA is extracted, reverse transcribed to cDNA, and then analyzed by fluorescence quantitative PCR. The internal control is β-actin.

[0154] 3.3 Data Analysis

[0155] (1) Calculation of cell activity: Calculate according to the formula:

[0156]

[0157] (2) Calculation of relative gene expression: Using the 2 -ΔΔCt The value output by the fluorescence quantitative PCR instrument is the Ct value.

[0158] ΔCT(sample group)=CT(sample group)-CT(experimental internal reference);

[0159] ΔCT(blank control group)=CT(blank control group)-CT(control internal reference);

[0160] ΔΔCT=ΔCT(sample group)-ΔCT(blank control group);

[0161] Relative gene expression = 2 -ΔΔCt ;

[0162] A bar graph was created based on the experimental data, which were normalized using the blank control group (BC). Statistical analysis was performed using the t-test. Compared with the model control group (M), significance is indicated by *, with a P-value < 0.05 indicated by *, a P-value < 0.01 indicated by **, and a P-value < 0.001 indicated by ***. If the model control group (M) shows a significant downregulation of the SIRT1 gene compared to the blank control group (BC), the experimental model was successfully established. If the sample group (TA) shows a significant upregulation of the SIRT1 gene compared to the model control group (M), the sample has an anti-photoaging effect.

[0163] 4. Experimental results:

[0164] (1) Cell activity: Eight dosing concentrations were set for the samples, and cytotoxicity assays were performed. The MTT assay results are shown in Table 6 below.

[0165] Table 6. Sample cell activity results

[0166]

[0167]

[0168] Reference Figure 3 ,According to the cell activity results, the formal experimental test concentrations were selected as follows: 0.03 mg / mL, 0.01 mg / mL, and 0.003 mg / mL.

[0169] (2) Gene expression level:

[0170] Table 7. Relative expression of SIRT1 gene in samples

[0171]

[0172] Refer to Table 7 and Figure 4 as well as Figure 5 The experimental data were statistically analyzed using the t-test method. Compared with the M group, significance is indicated by *, P-value < 0.05 is indicated by *, P-value < 0.01 is indicated by **, and P-value < 0.001 is indicated by ***.

[0173] 5. Experimental conclusion:

[0174] Under the experimental conditions of this study, compared with the BC group, the relative expression of SIRT1 gene in the M group was significantly downregulated, proving that the photoaging model was successfully established; the relative expression of SIRT1 gene was significantly upregulated when the sample ergothioneine was at concentrations of 0.03 mg / mL, 0.01 mg / mL, and 0.003 mg / mL.

[0175] In summary, the relative expression of SIRT1 gene was significantly increased by 36.89%, 55.36% and 32.43% at the concentrations of 0.03 mg / mL, 0.01 mg / mL and 0.003 mg / mL of the sample "VANPEARL ERGO-VITALIS" gene capsule, respectively.

[0176] In the keratinocyte aging model, the sample "VANPEARL ERGO-VITALIS" gene capsule at concentrations of 0.03mg / mL, 0.01mg / mI and 0.003mg / m, showed that it can participate in the DNA repair mechanism through the SIRT1 pathway and has an anti-photoaging effect.

[0177] Experimental Example 3:

[0178] The "VANPEARL ERGO-VITALIS" gene capsule was prepared using the formula and preparation method of the present invention, and samples of the gene capsule were used to test the anti-aging efficacy using a zebrafish Sirt1 gene expression promotion test method;

[0179] The test method is: refer to Figure 6 Thirty-six five-day-old zebrafish were divided into three groups and exposed to 0.0049 g / L gene capsule sample solution. A blank control group was also set up. After 24 hours of exposure, RNA was extracted from the zebrafish, cDNA was synthesized, and real-time PCR amplification was performed. β-actin was used as the housekeeping gene and Ct was used as the amplification result. The relative expression level of Sirt1 was calculated and statistical analysis was performed.

[0180] Test results: Reference Figure 7 The sample at a test concentration of 0.0049 g / L promoted the expression of zebrafish Sirt1 gene by 64% (p=0.0073), with significant anti-aging effect.

[0181] Conclusion: This sample can significantly promote the expression of Sirt1 gene in zebrafish and has anti-aging effects.

[0182] Experimental Example 4:

[0183] The "VANPEARL ERGO-VITALIS" gene capsules were prepared using the formula and preparation method of the present invention, and samples of the gene capsules were used to test the collagen-promoting efficacy, using the zebrafish type I collagen gene expression promotion test method:

[0184] The test method is as follows: 36 6-day-old zebrafish were divided into 3 groups and exposed to 0.0049g / L gene capsule sample solution. A blank control group was also set up. After 24 hours of exposure, RNA was extracted from the zebrafish, cDNA was synthesized, and real-time PCR amplification was performed. β-actin was used as the housekeeping gene and Ct was used as the amplification result. The relative expression levels of col1a1a, col1a1b and col1a2 were calculated and statistically analyzed.

[0185] Test results: Reference Figure 8 At a test concentration of 0.0049 g / L, the sample promoted the expression of zebrafish col1a1a, col1a1b and col1a2 genes by 62% (p=0.0072), 70% (p=0.0021) and 132% (p=0.022), respectively.

[0186] Conclusion: This sample can significantly promote the expression of type I collagen gene in zebrafish and has the effect of promoting the regeneration of type I collagen.

[0187] Experimental Example 5:

[0188] The "VANPEARL ERGO-VITALIS" gene capsule was prepared using the formula and preparation method of the present invention, and samples of the gene capsule were used to perform anti-β-galactosidase efficacy tests using the zebrafish embryo aging-related β-galactosidase inhibition test method:

[0189] The test method is: refer to Figure 9 24 two-day-old zebrafish embryos were exposed to 0.099 g / L gene capsule sample solution. A blank control group and a positive control group were set up. After 48 hours of exposure, the zebrafish were fixed and stained for aging-related β-galactosidase. The zebrafish aging-related β-galactosidase levels were measured under a microscope and calculated and analyzed.

[0190] Test results: Reference Figure 10 The sample had an inhibition rate of 22% on zebrafish embryo aging-related β-galactosidase at a test concentration of 0.099 g / L (p=0.0086).

[0191] Conclusion: This sample can significantly inhibit the formation of β-galactosidase related to zebrafish embryonic aging, and has the effect of inhibiting aging.

[0192] Related β-galactosidase effects.

[0193] Experimental Example 6:

[0194] The "VANPEARL ERGO-VITALIS" gene capsule was prepared using the formula and preparation method of the present invention, and samples of the gene capsule were used to test the antioxidant efficacy using the zebrafish embryo reactive oxygen species (ROS) scavenging test method:

[0195] The test method is: refer to Figure 11 24 48-hour-old zebrafish embryos were exposed to 0.099 g / L gene capsule sample solution, and a blank control group was set up. After 24 hours of exposure, the fish embryos were stained with H2DCFDA, and the ROS signal intensity was measured by fluorescence photography and statistical analysis was performed.

[0196] Test results: Reference Figure 12 The ROS clearance rate of the sample on zebrafish embryos was 25% at a test concentration of 0.099 g / L (p=0.000000000018).

[0197] Conclusion: This sample can significantly eliminate ROS in zebrafish embryos, has an antioxidant effect, and helps with antioxidant protection.

[0198] Experimental Example 7:

[0199] The "VANPEARL ERGO-VITALIS" gene capsule was prepared using the formula and preparation method of the present invention, and samples of the gene capsule were used to test the efficacy of enhancing telomerase activity using the zebrafish telomerase activity promotion test method:

[0200] The test method is: refer to Figure 13 Thirty-six four-day-old zebrafish were divided into three groups and exposed to 0.0099 g / L gene capsule sample solution. A blank control group and a positive control group were also set up. After four days of exposure, protein extraction, protein content determination, and Q-TRAP amplification were performed on the zebrafish. Ct was used as the amplification result, and the relative activity of telomerase was calculated and statistical analysis was performed.

[0201] Test results: Reference Figure 14 The sample at a test concentration of 0.0099 g / L promoted telomerase activity in zebrafish by 70% (p=0.00063).

[0202] Conclusion: This sample can significantly promote telomerase activity in zebrafish and has the effect of enhancing telomerase activity.

[0203] Experimental Example 8:

[0204] The "VANPEARL ERGO-VITALIS" gene capsules were prepared using the formula and preparation method of the present invention, and samples of the gene capsules were used to conduct efficacy tests using human efficacy evaluation tests / consumer use test methods.

[0205] The test method is:

[0206] 1) T / ZHCA 006-2019 Test Method for Anti-Wrinkle Efficacy of Cosmetics;

[0207] 2) Cosmetics for freckle removal, Chapter 8, Section 5 of the "Technical Specifications for Safety of Cosmetics" (2015 Edition) (Annex 7 of the National Medical Products Administration's Notice No. 17 of 2021);

[0208] Whitening efficacy test second body open use freckle removal and whitening efficacy test method;

[0209] 3) Liu Weiyi, Zhou Lin, Zhao Hua. Evaluation of cosmetic efficacy (ⅩⅢ) - Consumer use test[J]. Surfactant Detergent & Cosmetics Industry, 2021, 51(06): 485-490;

[0210] 4) "Technical Specifications for Safety of Cosmetics" (2015 edition);

[0211] 5) Cosmetics classification rules and classification catalogue;

[0212] 6) Evaluation standards for cosmetic efficacy claims;

[0213] The test process is:

[0214] D0:

[0215] 1) Subject information is registered, informed consent is completed and signed, and laboratory technicians screen subjects according to inclusion and exclusion criteria;

[0216] 2) The subjects cleansed their faces with the designated cleansing sample and sat quietly in a laboratory at a temperature of 21±1°C and a humidity of 50±10% RH for 30 minutes;

[0217] 3) Testing the subject's skin base value: VISIA image acquisition;

[0218] 4) Laboratory technicians instruct subjects to take oral samples according to the sample oral requirements and oral site, and provide written test precautions and oral instructions;

[0219] 5) The subject collects the sample and leaves the laboratory.

[0220] D14:

[0221] 1) The subjects cleaned their faces with the designated cleansing samples and sat quietly in a laboratory at a temperature of 21±1°C and a humidity of 50±10% RH for 30 minutes;

[0222] 2) Skin data values of test subjects 14 days after oral administration of the sample: VISIA image acquisition;

[0223] 3) The subjects filled out the self-assessment questionnaire.

[0224] D28:

[0225] 1) The subjects cleaned their faces with the designated cleansing samples and sat quietly in a laboratory at a temperature of 21±1°C and a humidity of 50±10% RH for 30 minutes;

[0226] 2) Skin data values of test subjects 28 days after oral administration of samples: VISIA image acquisition;

[0227] 3) The subjects filled out the self-assessment questionnaire.

[0228] Test item: Area ratio of under-eye wrinkles, Test instrument / method: IPP, Test time: D0 / D14 / D28, Test site: under the eyes, Parameter description: ● The smaller the value, the shallower the under-eye wrinkles.

[0229] Note: D0 refers to the visit before the subject takes the sample orally, D14 refers to the visit 14 days after the subject takes the sample orally, and D28 refers to the visit 28 days after the subject takes the sample orally.

[0230] Dimension of area ratio of wrinkles under eyes - age value estimation: the test result is Figure 15 As shown in Table 8, Figure 15 In the study, samples were selected from 200 subjects aged 25-60 in the subject pool; the age estimation formula was: y=0.0039x+0.0309.

[0231] Table 8. Age estimation results based on under-eye wrinkles (area ratio)

[0232]

[0233] Result description:

[0234] Compared with before oral administration, the age value 14 days after oral administration was estimated to be 32.53 years, which is 1.66 years younger;

[0235] Compared with before oral administration, the age value was estimated to be 30.87 years old 28 days after oral administration of the sample, which was 3.31 years younger.

[0236] The implementation principle of the present invention is: the present invention discloses a composition for repairing DNA and delaying aging, and its preparation method and application. The composition is a An anti-aging composition containing gene error-correcting factors, which includes core ingredients such as Rhodiola rosea extract, ergothioneine, and pyrroloquinoline quinone (PQQ), significantly improves skin aging, brain cognitive impairment, cardiac dysfunction, and lung damage by activating Sirtuin proteins, repairing DNA damage, and scavenging free radicals. It also has an anti-photoaging effect on human skin keratinocytes. Experimental data show that it can increase SIRT1 gene expression by 64%, telomerase activity by 70%, and reduce DNA damage by 83%. The composition is suitable for a variety of dosage forms such as capsules and tablets and has broad application prospects.

[0237] The embodiments of this specific implementation method are all preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Therefore, any equivalent changes made based on the structure, shape, and principle of the present invention should be included in the scope of protection of the present invention.

Claims

1. A composition for repairing DNA and delaying aging, characterized in that: Contains the following active ingredients: Rhodiola rosea extract, ergothioneine, pyrroloquinoline quinone, acai extract, astaxanthin, lycopene, vitamin B2, and vitamin B12.

2. A composition for repairing DNA and delaying aging according to claim 1, characterized in that: The mass percentage of each component is: Rhodiola rosea extract 10-30%, ergothioneine 5-15%, pyrroloquinoline quinone 1-5%, acai extract 5-20%, astaxanthin 0.1-3%, lycopene 0.5-5%, vitamin B2 0.1-2%, vitamin B12 0.01-0.5%, and the remainder is a pharmaceutically or food-acceptable carrier.

3. The composition for repairing DNA and delaying aging according to claim 1, characterized in that: The composition is used to activate Sirtuin family proteins, inhibit LOXL2-TGF-β1-Smad2 / 3 pathway, or enhance DNA telomerase activity.

4. A composition for repairing DNA and delaying aging according to any one of claims 1 to 3, characterized in that: The composition is used for one of the following purposes: Repair DNA damage and improve genomic instability; Delay skin aging, reduce wrinkles, spots and enlarged pores; Improve brain cognitive function and prevent or treat Alzheimer's disease or Parkinson's disease; Protect heart function and inhibit myocardial fibrosis or atrial fibrillation; Repair lung tissue damage and improve lung inflammation caused by viruses or smoking.

5. The composition for repairing DNA and delaying aging according to claim 4, characterized in that: The composition achieves its efficacy through the following mechanisms: activating cell-derived life factors to replace damaged DNA fragments, scavenging free radicals, enhancing mitochondrial function, or promoting the differentiation of neural stem cells.

6. A composition for repairing DNA and delaying aging according to any one of claims 1 to 5, characterized in that: The dosage form of the composition is capsule, tablet, oral liquid or powder.

7. A method for preparing a composition for repairing DNA and delaying aging, characterized in that: The following steps are involved: Step 1, mixing Rhodiola rosea extract, ergothioneine, pyrroloquinoline quinone, acai extract, astaxanthin, and lycopene in proportion; Step 2: Add vitamin B2 and B12 and homogenize; Step 3: Add a pharmaceutically or food-acceptable carrier to prepare the target dosage form.

8. The method for preparing a composition for repairing DNA and delaying aging according to claim 7, characterized in that: In the step 1, the Rhodiola rosea extract is obtained by a water extraction or alcohol extraction process, and the total flavonoid content is not less than 20%.

9. A preparation for improving the sequelae of the new coronavirus, characterized in that: The invention relates to a composition comprising the composition according to any one of claims 1 to 6, and further comprising coenzyme Q10 or N-acetylcysteine.

10. Use of the composition according to claim 1 in preparing a drug for preventing or treating ovarian dysfunction, characterized in that: The composition achieves efficacy by increasing the ovarian AMH index, reducing the FSH level or increasing the number of follicles.

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