Antioxidant traditional Chinese medicine monomer composition and application thereof

Abstract

The invention relates to the technical field of natural medicines, and particularly discloses an antioxidant traditional Chinese medicine monomer composition and application thereof, the active ingredients of the traditional Chinese medicine monomer composition comprise quercetin and caffeic acid, and the molar ratio of quercetin to caffeic acid is (1-2): 20. The quercetin and the caffeic acid in the composition are derived from natural products of traditional Chinese medicines, are low in toxicity and high in activity, contribute to the precise application of the related traditional Chinese medicines to oxidation resistance and aging resistance, and expand the clinical application range. The quercetin and the caffeic acid have the application prospect of being developed into the medicine for treating the premature ovarian failure in the form of the composition.

Description

Technical Field

[0001] The present invention relates to the technical field of natural medicines, in particular to an antioxidant Chinese medicine monomer composition and application thereof. Background Art

[0002] Premature ovarian failure refers to amenorrhea before the age of 40 due to ovarian failure, which can cause aging of ovarian granulosa cells. Granulosa cells are the main functional cells of the ovary, and their proliferation and differentiation processes play a decisive role in the overall functional activities of the ovary. Oxidative stress is closely related to the occurrence and development of premature ovarian failure and is one of the main causes of ovarian aging. H2O2, as one of the most well-known reactive oxygen species, is considered to be the main cause of the production of intracellular hydroxyl free radicals. The human ovarian granulosa cell line KGN has become an important tool for revealing the molecular mechanisms in the study of reproductive system diseases due to its ability to synthesize ovarian hormones. Therefore, an in vitro premature ovarian failure model was established by inducing KGN cells with H2O2.

[0003] Currently, premature ovarian failure is primarily treated with hormone replacement therapy, but prolonged hormone use can increase the risk of diseases such as endometrial cancer and breast cancer. In recent years, a growing number of studies have found that traditional Chinese medicine (TCM) treatments for premature ovarian failure offer minimal side effects and significant efficacy. Monomers are the active ingredients in TCM, each with a defined chemical structure. Therefore, identifying compounds within these monomers that may improve premature ovarian failure is of particular importance.

[0004] Cuscuta seeds are the dried, mature seeds of the Convolvulaceae plant Cuscuta chinensis. They are warm in nature, sweet in flavor, and enter the liver, kidney, and spleen meridians. They have the effects of tonifying yang and yin, consolidating essence and reducing urination, improving eyesight, and stopping diarrhea. As a key herb in Traditional Chinese Medicine for warming and tonifying kidney yang, they have advantages in treating female reproductive disorders and are commonly used to treat conditions such as premature ovarian failure. Cuscuta seeds contain a rich variety of active ingredients, including flavonoids, phenolic acids, alkaloids, and polysaccharides. Flavonoids and phenolic acids are found in high concentrations and possess excellent antioxidant properties. Quercetin, a natural flavonoid, exhibits significant anti-inflammatory, antioxidant, antiviral, and immunomodulatory effects. Studies have shown that quercetin can significantly improve ovarian pathological morphology and function in mice with premature ovarian failure. Caffeic acid, a phenolic acid compound widely found in traditional Chinese medicine, exhibits antioxidant and anti-inflammatory activities. Studies have shown that caffeic acid can significantly improve oxidative stress in rats with polycystic ovary syndrome and protect ovarian granulosa cells from apoptosis caused by oxidative stress. However, there are relatively few studies on the combined use of flavonoids and phenolic acid compounds. There are no reports on the combined use of quercetin and caffeic acid, nor is there any technology for combining the two to treat premature ovarian failure.

[0005] In recent years, domestic and international scholars have conducted extensive research on the material basis of Cuscuta seeds. Previous studies have identified 13 active ingredients in Cuscuta seeds with high content, but research on the combination and compatibility of these active ingredients is less thorough. The present invention screened the antioxidant activity of the active ingredients in Cuscuta seeds using DPPH and ABTS free radical scavenging experiments. It was found that quercetin and caffeic acid both have good antioxidant activity. Using quercetin and caffeic acid as the subjects, the optimal molar ratio of quercetin and caffeic acid combination was studied to explore its role and application in the treatment of premature ovarian failure. Summary of the Invention

[0006] The purpose of the present invention is to provide an antioxidant Chinese medicine monomer composition and its application in view of the defects of the prior art, so as to solve the problems raised by the above background technology.

[0007] To achieve the above object, the present invention provides the following technical solution: an antioxidant Chinese medicine monomer composition, wherein the active ingredients of the Chinese medicine monomer composition are composed of quercetin and caffeic acid, and the molar ratio of quercetin to caffeic acid is (1-2):20.

[0008] As a preferred technical solution of the present invention, the molar ratio of quercetin to caffeic acid is 1:20.

[0009] As a preferred technical solution of the present invention, the concentration of quercetin is 5-100 μM, and the concentration of caffeic acid is 5-100 μM.

[0010] As a preferred technical solution of the present invention, the concentration of quercetin is 5-10 μM, and the concentration of caffeic acid is 25-100 μM.

[0011] As a preferred technical solution of the present invention, the concentration of quercetin is 5 μM, and the concentration of caffeic acid is 100 μM.

[0012] An antioxidant composition for treating premature ovarian failure, comprising the above-mentioned antioxidant Chinese medicine monomer composition, can be prepared into at least one of pharmaceutically acceptable granules, tablets, injections, pills, capsules, aerosols or suppositories.

[0013] An antioxidant composition for treating premature ovarian failure comprises the antioxidant Chinese medicine monomer composition described above and can further be added with one or more of an excipient, a diluent, a binder or a stabilizer as a carrier.

[0014] A use of the above-mentioned antioxidant Chinese medicine monomer composition in the preparation of a medicine for treating premature ovarian failure.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] (1) Side effects caused by long-term hormone replacement therapy are the most common problem in clinical treatment. The use of the composition of the present invention can reduce the incidence of drug resistance.

[0017] (2) The quercetin and caffeic acid in the composition of the present invention are both natural products derived from traditional Chinese medicine, with low toxicity and high activity, which contributes to the precise application of the antioxidant and anti-aging effects of relevant traditional Chinese medicines and expands the scope of clinical application.

[0018] (3) The present invention provides quercetin and caffeic acid with the application prospect of being developed into a drug for treating premature ovarian failure in the form of a composition. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a schematic diagram of the process of the present invention;

[0020] Figure 2 This is the result of DPPH·scavenging experiment of Cuscuta seeds compounds;

[0021] Figure 3 ABTS ·+ Clear the experimental results graph;

[0022] Figure 4 This is a graph showing the effects of different concentrations of H2O2 on oxidative damage in KGN cells;

[0023] Figure 5 The graph shows the half-inhibitory concentration of H2O2 at different concentrations to induce oxidative damage in KGN cells;

[0024] Figure 6 This is the safe concentration range of quercetin and caffeic acid for KGN cells;

[0025] Figure 7 This is a graph showing the effects of quercetin and caffeic acid on the viability of oxidatively damaged granulosa cells;

[0026] Figure 8 This is the effect of quercetin combined with caffeic acid on the activity of oxidatively damaged granulosa cells;

[0027] Figure 9 This is a diagram showing the morphological changes of apoptosis in KGN cells induced by oxidative damage, inhibited by the combination of quercetin and caffeic acid;

[0028] Figure 10 The β-galactosidase staining image of cells showing that quercetin combined with caffeic acid inhibited KGN oxidative damage;

[0029] Figure 11 This is the SOD level graph of KGN cells treated with quercetin and caffeic acid;

[0030] Figure 12 This is the ROS fluorescence image of KGN cells treated with quercetin and caffeic acid;

[0031] Figure 13 This is the mean fluorescence intensity of ROS in KGN cells treated with quercetin and caffeic acid;

[0032] Figure 14 This is the fluorescence image of mitochondrial membrane potential in KGN cells treated with quercetin and caffeic acid;

[0033] Figure 15 This is the mean fluorescence intensity of CXMROS in KGN cells treated with quercetin and caffeic acid. DETAILED DESCRIPTION

[0034] The preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making a clearer and more precise definition of the protection scope of the present invention.

[0035] The anti-oxidation Chinese medicinal monomer composition of the present invention consists of quercetin and caffeic acid, and the molar ratio of quercetin to caffeic acid is (1-2):20, preferably 1:20.

[0036] In some embodiments, quercetin and caffeic acid are dissolved in physiological saline or other commonly used solvents for oral liquid preparations in the above ratios to prepare an oral liquid preparation, which can then be dissolved in an aqueous solution by conventional solubilization methods. The following experiments use DMSO as the solvent, and the effects of using other solvents are consistent.

[0037] In some embodiments, the concentration of quercetin is 5-100 μM, preferably 5-10 μM, and more preferably 5 μM. The concentration of caffeic acid is 5-100 μM, preferably 50-100 μM, and more preferably 100 μM. When quercetin and caffeic acid are administered at the aforementioned ratios and concentrations to KGN cells induced by H2O2, the two have an additive protective effect.

[0038] 1. Cell Lines

[0039] Human ovarian granulosa cell line KGN cells were purchased from Wuhan Punosai Life Science Technology Co., Ltd. KGN cells were cultured in DMEM / F12 medium containing 100 U / ml penicillin and streptomycin and 10% fetal bovine serum in an incubator at 37°C, 75% humidity, and 5% CO2 concentration.

[0040] 2. Materials and Methods

[0041] Quercetin and caffeic acid standards: Both were obtained from Chengdu Pusi Biotechnology Co., Ltd. and dissolved in DMSO to form a stock solution, which was stored in a refrigerator at 4°C until use. The composition was prepared by dissolving quercetin and caffeic acid in DMSO at a molar ratio of 1:20 to form a stock solution, which was stored in a refrigerator at 4°C until use. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-diazobis(3-ethylbenzothiazole-6-sulfonic acid)ammonium (ABTS) were purchased from Shanghai Yuanye Biotechnology Co., Ltd.; methanol was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; purified water was homemade in the laboratory; fetal bovine serum was purchased from Wuhan Punosai Life Science Technology Co., Ltd.; a cell senescence β-galactosidase staining kit was purchased from Beijing Lanjieke Technology Co., Ltd.; serum-free cell freezing medium, trypsin digestion solution, and Cell Counting Kit-8 (CCK-8) were all purchased from Suzhou Xinsaimei Biotechnology Co., Ltd.; DMEM / F12 medium and 1x PBS solution were purchased from Jiangsu Keygen Biotechnology Co., Ltd.; cell culture-grade DMSO and penicillin-streptomycin mixture were purchased from Xuzhou Bolida Biotechnology Co., Ltd.; BCA protein quantification kit, total SOD activity assay kit (NBT method), reactive oxygen species assay kit, and mitochondrial membrane potential and cell apoptosis assay kit were all purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0042] 3. Instruments and Equipment

[0043] The desktop high-speed centrifuge was purchased from Eppendorf, the ultra-clean bench was purchased from Airtech, the multifunctional microplate reader was purchased from Biotek, the electronic balance was purchased from G&G, the fully automatic sterilizer was purchased from Chitong Instrument (Shanghai) Co., Ltd., the constant temperature water bath was purchased from Changzhou Jintan Manufacturing Co., Ltd., the vertical ultra-low temperature refrigerator was purchased from Zhongke Meiling Low Temperature Technology Co., Ltd., the CO2 incubator was purchased from Wiggens, and the inverted biological microscope was purchased from Shanghai Caikang Optical Instrument Co., Ltd. The 60mm dishes and 96-well plates used for culturing cells were purchased from Corning.

[0044] Example 1: Determination of DPPH free radical scavenging ability of Cuscuta australis active ingredient

[0045] 1.1 Experimental Methods

[0046] (1) Preparation of stock solution: Weigh 0.002 g of DPPH powder into a 50 ml brown volumetric flask, add about 40 ml of methanol, sonicate to completely dissolve it, dilute to the mark, shake well, and prepare the 0.1 mM / L DPPH stock solution. Store at low temperature and away from light. Use immediately after preparation.

[0047] (2) Sample preparation: The active ingredients of Cuscuta seeds were diluted with methanol to form multi-level concentration sample working solutions (1, 5, 10, 20, 30 μg / mL -1 ), with the same concentration of ascorbic acid VC as a positive control, stored at low temperature and away from light.

[0048] (3) Sample spotting: 96-well plate, three groups, each group with 3 replicate wells, the amount added to each well and the distribution of the 96-well plate are as follows:

[0049] Sample group: 100 μL sample solution + 100 μL DPPH methanol solution (3 wells for each concentration);

[0050] Blank group: 100 μL of sample solution + 100 μL of methanol (3 wells for each concentration);

[0051] Control group: 100 μL of DPPH methanol solution + 100 μL of water (one plate can share one control group, 3 wells);

[0052] (4) Mix well, let stand in the dark at room temperature for 30 min, and measure the absorbance of each well at 517 nm using a microplate reader;

[0053] (5) Calculate the DPPH free radical scavenging rate:

[0054] (6) In the formula, AS is the absorbance of the experimental group, AN is the absorbance of the negative control group, and A0 is the absorbance of the blank control group;

[0055] (7) Statistical analysis and graphing: GraphPad Prism 9.5 software was used for statistical analysis and graphing.

[0056] 1.2 Experimental Results

[0057] Among the 13 compounds of Cuscuta seeds, the scavenging rates of quercetin and caffeic acid on DPPH· were greater than those of Vc, and the inhibition rates increased with the increase of concentration, 30-50μg / mL -1 When the inhibitory ability was quercetin>caffeic acid>isochlorogenic acid B>vitamin C>isochlorogenic acid A, the clearance rate of the five compounds was close to 90% within the concentration range tested. Combined with IC50 analysis, the IC50 of quercetin (5.877μg / mL -1 ) was the smallest, and the inhibition rate to DPPH· was the strongest, and the antioxidant activity was the strongest, while the clearance rate of astragalin was always lower than 10% and was not positively correlated with the concentration ( Figure 2 ).

[0058] Example 2: Determination of free radical scavenging ability of ABTS, an active ingredient of Cuscuta seeds

[0059] 2.1 Experimental Methods

[0060] (1) Preparation of stock solution: Weigh 96 mg of ABTS powder into a 25 ml brown volumetric flask, dilute to the mark with distilled water, sonicate to dissolve completely, shake well, and prepare a 7.4 mM / LDPPH stock solution. Store at low temperature and away from light. Prepare and use immediately. Weigh 378.4 mg of K2S2O8 powder into a 10 ml brown volumetric flask, dilute to the mark with distilled water, sonicate to dissolve completely, shake well, and prepare a 2.6 mM / LABTS stock solution.

[0061] (2) Preparation of working solution: Mix equal amounts of 7.4 mM / L ABTS solution and 2.6 mM / L potassium persulfate and place in the dark for 12 to 16 hours to prepare ABTS. ·+ Working solution.

[0062] (3) Sample preparation: The active ingredients of Cuscuta seeds were diluted with methanol to form multi-level concentration sample working solutions (1, 5, 10, 20, 30 μg / mL -1 ), with the same concentration of ascorbic acid VC as a positive control, stored at low temperature and away from light.

[0063] (4) Sample spotting: 96-well plate, three groups, each group with 3 replicate wells, the amount added to each well and the distribution of the 96-well plate are as follows:

[0064] Sample group: 100 μL sample solution + 100 μL DPPH methanol solution (3 wells for each concentration);

[0065] Blank group: 100 μL of sample solution + 100 μL of methanol (3 wells for each concentration);

[0066] Control group: 100 μL of DPPH methanol solution + 100 μL of water (one plate can share one control group, 3 wells);

[0067] (5) Mix well, let it stand in the dark at room temperature for 30 min, and then measure the absorbance of each well at 517 nm using a microplate reader.

[0068] (6) Calculate the ABTS free radical scavenging rate:

[0069] (7) In the formula, AS is the absorbance of the experimental group, AN is the absorbance of the negative control group, and A0 is the absorbance of the blank control group;

[0070] (8) Statistical analysis and graphing: GraphPad Prism 9.5 software was used for statistical analysis and graphing.

[0071] 2.2 Experimental Results

[0072] Among the 13 compounds of Cuscuta seeds, 30-50 μg / mL -1 When Vc is added to ABTS·+ The clearance rate of the five compounds was the largest, followed by quercetin > isorhamnetin > caffeic acid > neochlorogenic acid > isochlorogenic acid B. The maximum inhibition rate of the five compounds was close to 90% within the concentration range tested, and the inhibition rate increased with the increase of concentration. Combined with IC50 analysis, the IC50 of quercetin (5.575μg / mL) -1 ) is minimum, for ABTS ·+ The inhibition rate of astragalin was the strongest and the antioxidant activity was the strongest, while the clearance rate of astragalin was always lower than 30% and was not positively correlated with the concentration ( Figure 3 ).

[0073] Example 3: Establishment of a Cellular Oxidative Stress Injury Model (Human Ovarian Granulosa Cells KGN)

[0074] 3.1 Experimental Methods

[0075] (1) KGN cells in the logarithmic growth phase were digested with 0.25% trypsin and cultured at 4 × 10 4 The cells were seeded at a density of 100 μg / mL / well in a 96-well culture plate and cultured in a 37°C constant temperature and humidity incubator containing 5% CO2 for 24 h.

[0076] (2) Treat cells with different concentrations of H2O2, with three replicate wells for each concentration;

[0077] (3) After 2 h of drug treatment, the cells were taken out of the wells of the cell culture plate and placed in a clean bench away from light. 10 μl of CCK-8 reagent was added to each well. After thorough mixing, the 96-well plate was returned to the cell culture incubator and incubated for another 1 h.

[0078] (4) Place the 96-well plate on an ELISA plate shaker and shake for 30 seconds to mix thoroughly. Measure the absorbance (OD) at a wavelength of 450 nm using an ELISA reader.

[0079] The cell viability of each group was calculated using the following formula, where the cell viability of the control group was set to 100%. The calculation formula is as follows: Cell viability (%) = (OD value of the drug group - OD value of the blank group) / (OD value of the control group - OD value of the blank group) x 100%.

[0080] 3.2 Experimental Results

[0081] H2O2 was used to induce KGN cells to construct an oxidative damage model. As the concentration of H2O2 increased, the survival rate of KGN cells gradually decreased ( Figure 4). KGN cells were treated with H2O2 at different concentrations (0-500μM) to determine the H2O2 concentration that induced oxidative damage. The results showed that when the H2O2 concentration was 250μmol / L and the action time was 2h, the cell survival rate was 54.04±1.45%, and 250μM H2O2 reduced the viability of KGN cells by about 50%, with an IC50 value of 253.1μM ( Figure 3 、 Figure 4 Therefore, the present invention selected the H2O2 concentration and time of 250 μmol / L and 2 h as the subsequent experimental modeling conditions.

[0082] Example 4: Safe Concentration Ranges of Quercetin and Caffeic Acid

[0083] 4.1 Experimental Methods

[0084] (1) KGN cells in the logarithmic growth phase were digested with 0.25% trypsin and cultured at 4 × 10 4 The cells were seeded at a density of 100 μg / mL / well in a 96-well culture plate and cultured in a 37°C constant temperature and humidity incubator containing 5% CO2 for 24 h.

[0085] (2) Treat cells with different concentrations of quercetin and caffeic acid, with three replicate wells for each concentration;

[0086] (3) After 24 h of drug treatment, the cells were taken out of the wells of the cell culture plate and placed in a clean bench away from light. 10 μl of CCK-8 reagent was added to each well. After thorough mixing, the 96-well plate was returned to the cell culture incubator and incubated for another 3 h.

[0087] (4) Place the 96-well plate on an ELISA plate shaker and shake for 30 seconds to mix thoroughly. Measure the absorbance (OD) at a wavelength of 450 nm using an ELISA reader.

[0088] The cell viability of each group was calculated using the following formula, where the cell viability of the control group was set to 100%. The calculation formula is as follows: Cell viability (%) = (OD value of the drug group - OD value of the blank group) / (OD value of the control group - OD value of the blank group) x 100%.

[0089] 4.2 Experimental Results

[0090] The CCK-8 assay was used to examine the effects of quercetin and caffeic acid on KGN cell viability. Compared to the control group, quercetin and caffeic acid showed no cytotoxic effects on KGN cells within the concentration range of 25-50 μM. However, at a concentration of 100 μM, although caffeic acid still had no cytotoxicity, quercetin significantly reduced the viability of KGN cells ( Figure 5 ).

[0091] Example 5: Detection of the protective effects of quercetin and caffeic acid on oxidative damage

[0092] 5.1 Experimental Methods

[0093] (1) KGN cells in the logarithmic growth phase were digested with 0.25% trypsin and cultured at 4 × 10 4 The cells were seeded at a density of 100 μg / mL / well in a 96-well culture plate and cultured in a 37°C constant temperature and humidity incubator containing 5% CO2 for 24 h.

[0094] (2) Treat cells with different concentrations of quercetin and caffeic acid, with three replicate wells for each concentration;

[0095] (3) 24 hours after drug treatment, the cells were treated with DMEM / F12 medium containing 250 μM H₂O₂. After incubation in the incubator for another 2 hours, the cells were removed from the incubator to a clean bench in a dark place. 10 μl of CCK-8 reagent was added to each well. After thorough mixing, the 96-well plate was returned to the cell culture incubator and incubated for another 3 hours.

[0096] (4) Place the 96-well plate on an ELISA plate shaker and shake for 30 seconds to mix thoroughly. Measure the absorbance (OD) at a wavelength of 450 nm using an ELISA reader.

[0097] The cell viability of each group was calculated using the following formula, where the cell viability of the control group was set to 100%. The calculation formula is as follows: Cell viability (%) = (OD value of the drug group - OD value of the blank group) / (OD value of the control group - OD value of the blank group) x 100%.

[0098] 5.2 Experimental Results

[0099] Compared with the model group, different concentrations of low-concentration quercetin and high-concentration caffeic acid increased cell viability to varying degrees, confirming that quercetin and caffeic acid can protect granulosa cells from oxidative damage. Therefore, 5 μM quercetin and 100 μM caffeic acid were selected as the concentrations for subsequent experiments ( Figure 6 、 Figure 7 ).

[0100] Example 6: Detection of the protective effect of quercetin combined with caffeic acid on oxidative damage

[0101] 6.1 Experimental Methods

[0102] (1) KGN cells in the logarithmic growth phase were digested with 0.25% trypsin and cultured at 4 × 10 4 The cells were seeded at a density of 100 μg / mL / well in a 96-well culture plate and cultured in a 37°C constant temperature and humidity incubator containing 5% CO2 for 24 h.

[0103] (2) Treat cells with quercetin and caffeic acid at different concentrations and ratios (the final drug concentrations of the experimental group and the blank control group are listed in Table 1), with three replicate wells set for each concentration;

[0104] (3) 24 hours after drug treatment, the cells were treated with DMEM / F12 medium containing 250 μM H₂O₂. After incubation in the incubator for another 2 hours, the cells were removed from the incubator to a clean bench in a dark place. 10 μl of CCK-8 reagent was added to each well. After thorough mixing, the 96-well plate was returned to the cell culture incubator and incubated for another 3 hours.

[0105] (4) Place the 96-well plate on an ELISA plate shaker and shake for 30 seconds to mix thoroughly. Measure the absorbance (OD) at a wavelength of 450 nm using an ELISA reader.

[0106] The cell viability of each group was calculated using the following formula, where the cell viability of the control group was set to 100%. The calculation formula is as follows: Cell viability (%) = (OD value of the drug group - OD value of the blank group) / (OD value of the control group - OD value of the blank group) x 100%.

[0107] (5) The results were analyzed using Excel. The experimental operation was repeated three times, and the data were expressed as mean ± standard deviation. CompuSyn software was used to calculate the combination index (CI) value for synergistic discrimination. When CI = 1, the two were judged to have an additive effect; when CI < 1, the two were judged to have a synergistic effect, and the smaller the CI value, the stronger the synergistic effect; when CI > 1, the two were judged to have an antagonistic effect, and the larger the CI value, the stronger the antagonistic effect.

[0108] Table 1: Ratio of quercetin and caffeic acid at different concentrations

[0109]

[0110]

[0111] 6.2 Experimental Results

[0112] Compared with the model group, the combination of quercetin and caffeic acid at different ratios improved the cell viability to varying degrees compared with the single component, confirming that the combination of quercetin and caffeic acid can protect granulosa cells from oxidative damage (Table 2 and Figure 8Increasing the quercetin ratio decreased cell survival, while increasing the caffeic acid ratio increased cell survival, consistent with the protective effect of a single ingredient against oxidatively damaged cells. The results showed that when quercetin and caffeic acid were combined at a mass ratio of 1:20, the combined drug coefficient (CI) was 0.39, indicating a strong synergistic effect. At a mass ratio of 2:20, the combined drug coefficient (CI) was 0.87, indicating a weak synergistic effect. Therefore, the quercetin:caffeic acid (1:20) ratio, which exhibited a stronger synergistic effect, was selected as the mass ratio for subsequent experiments.

[0113] Table 2: CI values of quercetin and caffeic acid combined

[0114]

[0115]

[0116] Example 7: Effect of quercetin combined with caffeic acid on morphological changes of apoptosis in KGN cells induced by oxidative damage

[0117] 7.1 Experimental Methods

[0118] (1) KGN cells were seeded in a six-well culture plate and incubated in a culture incubator for 24 hours. The cells were then treated with DMEM / F12 medium containing 5 μM quercetin, 100 μM caffeic acid, and quercetin:caffeic acid (1:20).

[0119] (2) After administration, the cells were placed in an incubator and incubated for 24 hours, and then intervention was performed using a DMEM / F12 medium containing H2O2 with a concentration gradient interval of 250 μM.

[0120] (3) After adding H2O2, place the cells in the incubator and continue incubating for 2 hours. Then, take the cells out to the clean bench, dry the DMEM / F12 medium containing H2O2, add 1 ml of PBS to each well for rinsing, and observe and photograph directly under a microscope.

[0121] 7.2 Experimental Results

[0122] Morphological changes were observed under an inverted phase contrast microscope after 24 hours of drug treatment and 2 hours of H2O2 treatment in KGN cells. The cells in the control group, quercetin, caffeic acid, and quercetin + caffeic acid groups were uniform in size, with clear edges, tight connections between cells, and only a few normal apoptotic bodies. The KGN cells in the model group became round, and many abnormal apoptotic bodies appeared. The order of the groups with the most apoptotic bodies was: model group, quercetin group, caffeic acid group, and quercetin + caffeic acid group. It can be concluded that the quercetin + caffeic acid group had a stronger protective effect against oxidative damage to KGN cells than the single drug group ( Figure 9 ).

[0123] Example 8: Effect of quercetin combined with caffeic acid on β-galactosidase staining of KGN oxidatively damaged cells

[0124] 8.1 Experimental Methods

[0125] (1) KGN cells were seeded in six-well culture plates and incubated in an incubator for 24 hours. The cells were then treated with DMEM / F12 medium containing 5 μM quercetin, 100 μM caffeic acid, and 1:20 quercetin.

[0126] (2) After administration, the cells were placed in an incubator and incubated for 24 hours, and then intervention was performed using a DMEM / F12 medium containing H2O2 with a concentration gradient interval of 250 μM.

[0127] (3) After adding H2O2, place the plate in an incubator and continue incubating for 2 hours. Remove the plate to a clean bench and drain the DMEM / F12 medium containing H2O2. Add 1 ml of PBS to each well for rinsing. Add 500-1000 μl of fixative to each well and fix at room temperature for 7.5-10 minutes. (4) Aspirate the fixative and gently wash the plate three times with PBS, each for 3 minutes.

[0128] (5) Aspirate PBS and add about 500-1000 μl of staining working solution to each well.

[0129] (6) Incubate in a 37°C, CO2-free incubator in the dark overnight.

[0130] (7) Observe and take photos under an ordinary optical microscope.

[0131] 8.2 Experimental Results

[0132] During cell senescence, the activity level of β-galactosidase increases. It was possible to observe whether the combination of quercetin and caffeic acid has a protective effect on H2O2-induced KGN senescent cells. Under an inverted phase contrast microscope, KGN cells were observed after 24 hours of drug treatment and 2 hours of H2O2 treatment. The number of blue-stained senescent cells in the model group was significantly higher than that in the control group. However, after treatment with quercetin, caffeic acid, and the combination of quercetin and caffeic acid, the number of senescent cells was significantly reduced ( Figure 10 ).

[0133] Example 9: Detection of SOD Levels in Oxidatively Damaged KGN Cells by Combined Use of Quercetin and Caffeic Acid

[0134] 9.1 Experimental Methods

[0135] (1) Preparation of cell samples: KGN cells were collected and washed 1-2 times with ice-cold PBS. The precipitate was homogenized with ice-cold PBS at 4°C. The homogenate was then centrifuged at 4°C and the supernatant was used as the test sample.

[0136] (2) Preparation of the kit: Preparation of NBT / enzyme working solution: Prepare an appropriate amount of NBT / enzyme working solution according to the volume of 160μl per reaction. Evenly mix 158μl SOD detection buffer, 1μl NBT and 1μl enzyme solution to prepare 160μl NBT / enzyme working solution. Prepare an appropriate amount of NBT / enzyme working solution according to the number of samples to be tested (including standards). Specific preparation methods are shown in Table 3. Store the prepared NBT / enzyme working solution in an ice bath.

[0137] Table 3: SOD reaction configuration system

[0138] Number of samples to be tested 1 10 20 50 SOD assay buffer (μl) 158 1580 3160 7900 NBT (μl) 1 10 20 50 Enzyme solution (μl) 1 10 20 50 NBT / enzyme working solution (μl) 160 1600 3200 8000

[0139] Preparation of the Reaction Start Working Solution: Dissolve the Reaction Start Working Solution (40X) in the kit and mix thoroughly. Dilute 1 μl of Reaction Start Working Solution (40X) with 39 μl of SOD Assay Buffer and mix thoroughly. Prepare an appropriate amount of Reaction Start Working Solution based on the number of samples (including standards) to be tested. Store the prepared Reaction Start Working Solution on ice.

[0140] (3) Sample measurement: Use a 96-well plate to set up sample wells and various blank control wells. Add the sample to be tested and other solutions in sequence according to Table 4. Add the reaction starter solution and mix thoroughly. Incubate at 37°C for 30 minutes. Set 600nm as the reference wavelength. The absorbance reading at 560nm is deducted from the absorbance reading at the reference wavelength to obtain the actual measured reading.

[0141] Table 4: SOD sample measurement system

[0142]

[0143] (4) Measurement and calculation:

[0144] The SOD content was calculated according to the instructions of the detection kit.

[0145] 9.2 Experimental Results

[0146] SOD is an important oxidoreductase in cells. The test results showed that the SOD activity of the model group decreased significantly, and this phenomenon was significantly reversed after pretreatment with quercetin, caffeic acid, and the quercetin and caffeic acid combination group. Under oxidative stress, the SOD levels of the quercetin and caffeic acid groups showed an upward trend, and the SOD levels of the quercetin and caffeic acid combination group showed a significant upward trend. As can be seen from the figure, quercetin, caffeic acid, and the combination of quercetin and caffeic acid can improve the oxidoreductase system and enhance the protective effect on the cells against oxidative damage ( Figure 11 ).

[0147] Example 10: Detection of ROS levels in oxidatively damaged KGN cells by combined use of quercetin and caffeic acid

[0148] 10.1 Experimental Methods

[0149] (1) In situ probe loading: Dilute DCFH-DA with serum-free culture medium at a ratio of 1:1000 to a final concentration of 10 μmol / L. Remove the cell culture medium and add an appropriate volume of diluted DCFH-DA. The volume added should be sufficient to fully cover the cells. Add no less than 1 ml of diluted DCFH-DA to one well of a six-well plate. Incubate in a cell culture incubator at 37°C for 20 minutes. Wash the cells three times with serum-free cell culture medium to fully remove the DCFH-DA that has not entered the cells.

[0150] (2) The positive control can be used at a ratio of 1:1000. A very significant increase in the level of reactive oxygen species can be observed within 30 minutes after stimulation.

[0151] (3) Detection: Samples loaded with probes in situ can be directly observed using a laser confocal microscope.

[0152] 10.2 Experimental Results

[0153] Under a fluorescence microscope, morphological changes were observed in KGN cells after 24 hours of drug treatment and 2 hours of H2O2 treatment. The intracellular ROS level in the model group was significantly increased, while after pretreatment with quercetin, caffeic acid, and quercetin and caffeic acid combination groups, the intracellular ROS level was significantly decreased. The order of ROS release from the highest to the lowest was: model group, quercetin, caffeic acid, quercetin and caffeic acid combination group, normal control group ( Figure 12 、 Figure 13 ).

[0154] Example 11: Detection of mitochondrial membrane potential level in KGN cells with oxidative damage by combined use of quercetin and caffeic acid

[0155] 11.1 Experimental Methods

[0156] (1) Culture the cells in a 24-well plate. After apoptosis induction, centrifuge the plate at 1000 g for 5 minutes using a centrifuge that can centrifuge multi-well plates.

[0157] (2) Aspirate the cell culture medium and add PBS to wash once.

[0158] (3) Add 188 μl of Annexin V-FITC binding solution.

[0159] (4) Add 2 μl of Mito-Tracker Red CMXRos staining solution and 5 μl of Hoechst 33342 staining solution and mix gently.

[0160] (5) Incubate at room temperature (20-25°C) in the dark for 20-30 minutes, then place in an ice bath. Aluminum foil can be used to protect from light.

[0161] (6) Then observe under a fluorescence microscope. Mito-Tracker Red CMXRos shows red fluorescence, and Hoechst33342 shows blue fluorescence.

[0162] 11.2 Experimental Results

[0163] Under a fluorescence microscope, morphological changes were observed in KGN cells after 24 hours of drug treatment and 2 hours of H2O2 treatment. After quercetin and caffeic acid were used together, a gradual increase in red fluorescence was observed. This phenomenon indicates that the combination of quercetin and caffeic acid can more effectively prevent the loss of mitochondrial membrane potential than single drug use, thereby preventing the occurrence of cell apoptosis. The average fluorescence intensity of CMXROS in the groups from strong to weak is as follows: control group, quercetin and caffeic acid combination group, caffeic acid group, quercetin group, model group ( Figure 14 、 Figure 15 ).

[0164] The above embodiments merely illustrate the implementation methods of the present invention. Although the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that a person skilled in the art may make various modifications and improvements without departing from the scope of the present invention, and these modifications and improvements fall within the scope of protection of the present invention.

Claims

1. An antioxidant Chinese medicine monomer composition, characterized in that: The active ingredients of the traditional Chinese medicine monomer composition consist of quercetin and caffeic acid, and the molar ratio of quercetin to caffeic acid is (1-2):

20.

2. The antioxidant Chinese medicine monomer composition according to claim 1, characterized in that: The molar ratio of quercetin to caffeic acid is 1:

20.

3. The antioxidant Chinese medicine monomer composition according to claim 1 or 2, characterized in that: The concentration of quercetin was 5-100 μM, and the concentration of caffeic acid was 5-100 μM.

4. The antioxidant Chinese medicine monomer composition according to claim 3, characterized in that: The concentration of quercetin was 5-10 μM, and the concentration of caffeic acid was 25-100 μM.

5. The antioxidant Chinese medicine monomer composition according to claim 4, characterized in that: The concentration of quercetin was 5 μM, and the concentration of caffeic acid was 100 μM.

6. An antioxidant composition for treating premature ovarian failure, characterized in that: The antioxidant Chinese medicine monomer composition according to any one of claims 1 to 5 can be prepared into at least one of pharmaceutically acceptable granules, tablets, injections, dripping pills, capsules, aerosols or suppositories.

7. An antioxidant composition for treating premature ovarian failure, characterized in that: The antioxidant Chinese medicine monomer composition according to any one of claims 1 to 5 can also be added with one or more excipients, diluents, adhesives or stabilizers as carriers.

8. Use of the antioxidant Chinese medicine monomer composition according to any one of claims 1 to 5 in the preparation of a medicament for treating premature ovarian failure.

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