New application of raspberry extract

Abstract

The invention provides a novel application of a raspberry extract. It is found through tests that any one of isoquercitrin, ellagic acid, vanillic acid, oleanolic acid, kaempferol-3-O-rutinoside, kaempferol-3-O-sophoroside, tiliroside, tormentic acid and raspberry extract can remarkably improve the activity of the human ovarian granular cells or remarkably improve the oxidative stress level, and the activity of the human ovarian granular cells can be remarkably improved. According to the present invention, the ovarian function can be effectively protected by the eight components, the ovarian function can be effectively protected by the eight components, or the mitochondrial membrane potential level can be significantly regulated and controlled, or the estrogen level in the microenvironment can be effectively maintained, such that the ovarian function can be effectively protected by the eight components, and the raspberry extract containing the eight components. The invention provides a new choice for ovarian function protection products, and also provides a new application for the substances.

Description

[0001] This application is a divisional application of the invention patent application filed on February 27, 2023, with application number 2023101809807, entitled "Application of Raspberry Extract in the Preparation of Ovarian Function Protection Products". Technical Field

[0002] This invention belongs to the field of biomedical technology. More specifically, it relates to the application of raspberry extract in the preparation of products for the protection of ovarian function. Background Technology

[0003] Ovarian dysfunction (ODD) in women is a gradual process. In its early stages, it manifests as decreased ovarian reserve (DOR), characterized by symptoms that may affect female fertility, such as reproductive endocrine disorders, scanty menstruation, infrequent menstruation, and amenorrhea. The prevalence of DOR in the population is 10%, and the incidence is showing a trend of increasing year by year and affecting younger women. Mechanisms leading to ovarian dysfunction include abnormal ovarian microenvironment, accumulation of harmful substances, oxidative stress damage, and abnormal mitochondrial and telomerase function. However, the root cause is abnormal follicular growth and development, accelerated apoptosis of granulosa cells, leading to follicular atresia and the inability to ovulate normally.

[0004] Raspberries (Rubi fructus) are the dried, immature fruits of *Rubus chingii* Hu, a plant belonging to the genus *Rubus* in the Rosaceae family. They are harvested in early summer when the fruits turn from green to greenish-yellow. The stems and leaves are removed, and the fruits are briefly blanched or steamed in boiling water before drying. As a plant used for both food and medicine, raspberries are rich in flavonoids, terpenoids, steroidal compounds, and phenolic acids. Among these, flavonoids include linalool, kaempferol-3-O-rutin, and isoquercitrin; triterpenoids include potassium oleanolic acid and oleanolic acid; and phenolic acids include vanillic acid and ellagic acid. Due to its diverse array of active substances, raspberries exhibit effects such as improving memory, anti-oxidation, anti-aging, lowering blood sugar and lipids, and anti-cancer properties, and are widely used in traditional Chinese medicine and health foods. Raspberry, a commonly used traditional Chinese medicine listed in the Chinese Pharmacopoeia, can be combined with other traditional Chinese medicines such as kudzu root and angelica to make fertility-boosting decoctions, which have shown good efficacy in treating gynecological diseases such as infertility and polycystic ovary syndrome (Yang Wenwen. Clinical study on the treatment of ovarian dysfunction by tonifying the kidney and replenishing essence [D]. China Academy of Chinese Medical Sciences, 2021. DOI:10.27658 / d.cnki.gzzyy.2021.000208.). However, there are currently no reports on the protective effects of raspberry extract alone or its components such as isoquercitrin on ovarian function. Summary of the Invention

[0005] This invention addresses the shortcomings of existing technologies by providing the application of raspberry extract in the preparation of products for protecting ovarian function, as well as the application of its components, such as isoquercitrin, in the preparation of such products, thus offering new options for ovarian function protection products.

[0006] The primary objective of this invention is to provide the application of isoquercitrin in the preparation of products for the protection of ovarian function.

[0007] A second objective of this invention is to provide the application of ellagic acid in the preparation of products for the protection of ovarian function.

[0008] A third objective of this invention is to provide the application of vanillic acid in the preparation of products for the protection of ovarian function.

[0009] The fourth objective of this invention is to provide the application of oleanolic acid in the preparation of products for the protection of ovarian function.

[0010] The fifth objective of this invention is to provide the application of kaempferol-3-O-rutin in the preparation of products for protecting ovarian function.

[0011] The sixth objective of this invention is to provide the application of kaempferol-3-O-sophoroside in the preparation of products for protecting ovarian function.

[0012] The seventh objective of this invention is to provide the application of linden glycosides in the preparation of products for the protection of ovarian function.

[0013] The eighth objective of this invention is to provide the application of potassium tartrate in the preparation of products for protecting ovarian function.

[0014] The ninth objective of this invention is to provide the application of raspberry extract in the preparation of products for the protection of ovarian function.

[0015] The tenth objective of this invention is to provide a product for protecting ovarian function.

[0016] The above-mentioned objective of this invention is achieved through the following technical solution:

[0017] Any one of isoquercitrin, ellagic acid, vanillic acid, oleanolic acid, kaempferol-3-O-rutinoside, kaempferol-3-O-sophoroside, linaloside, potassium tartrate, and raspberry extract can significantly improve the viability of human ovarian granulosa cells, significantly improve oxidative stress levels, significantly regulate mitochondrial membrane potential levels, or effectively maintain estrogen levels in the microenvironment. This indicates that these eight components, as well as raspberry extract containing these eight components, can effectively protect ovarian function. Therefore, the application of any one of isoquercitrin, ellagic acid, vanillic acid, oleanolic acid, kaempferol-3-O-rutinoside, kaempferol-3-O-sophoroside, linaloside, potassium tartrate, and raspberry extract in the preparation of ovarian function protection products should be within the scope of protection of this invention.

[0018] This invention first constructed a human ovarian granulosa cell injury model by inducing damage with 2,2-azobis(2-methylpropylimidazolium) hydrochloride (AAPH) and triptolide (TP), respectively. Cell viability, intracellular reactive oxygen species (ROS) content, mitochondrial membrane potential, and cell supernatant estrogen content were measured after treatment with isoquercitrin, ellagic acid, vanillic acid, oleanolic acid, kaempferol-3-O-rutinoside, kaempferol-3-O-sophoroside, lindenyl glycoside, and potassium oleate. The results showed that these components can alleviate AAPH and triptolide damage by increasing the viability of human ovarian granulosa cells. The decreased cell viability induced by triptolide can be mitigated by reducing the production of reactive oxygen species in human ovarian granulosa cells, thus alleviating the oxidative damage caused by AAPH and triptolide. Alternatively, it can be mitigated by regulating the mitochondrial membrane potential level of human ovarian granulosa cells, thus alleviating the decrease in estrogen secretion caused by AAPH and triptolide. Or, it can be mitigated by increasing the estrogen secretion capacity of human ovarian granulosa cells, thus alleviating the decrease in estrogen secretion caused by AAPH and triptolide. This can effectively protect the function of ovarian granulosa cells, thus indicating that these monomeric components and raspberry extract containing these monomeric components can effectively protect ovarian function. Human ovarian granulosa cells (KGNs) are among the most important functional cells in follicles. Through the action of steroid hormones, follicle-stimulating hormone, luteinizing hormone, and cytokines, they maintain a microenvironment conducive to oocyte growth and maturation, participate in regulating oocyte maturation and fertilization, and play an important role in maintaining ovarian function. They are also a good cell model for evaluating ovarian function. AAPH is a free radical initiator that can trigger a free radical chain reaction after decomposition, inducing the production of various endogenous free radicals. It is a good modeling material for studying oxidative stress damage. Tripterygium wilfordii Hook.f. is an epoxy diterpene isolated from Tripterygium wilfordii. It can cause symptoms of decreased ovarian function in women, such as menstrual cycle disorders and amenorrhea. Applying it to ovarian granulosa cells can provide a suitable model for studying ovarian function.

[0019] The structural formula of isoquercitrin (IQ) is as follows: The structural formula of ellagic acid (EA) is: The structural formula of vanillic acid (VA) is: The structural formula of oleanolic acid (OA) is: The structural formula of kaempferol-3-O-rutinoside (KOR) is as follows: The structural formula of kaempferol-3-O-sophoroside (KS) is as follows: The structural formula of tiliroside (TS) is as follows: The structural formula of termentic acid (TA) is:

[0020] Preferably, the effective concentration of isoquercitrin in the body fluid at the site of action is 25–200 μM.

[0021] Preferably, the effective concentration of ellagic acid in the body fluid at the site of action is 5–20 μM.

[0022] Preferably, the effective concentration of vanillic acid in the body fluid at the site of action is 25–200 μM.

[0023] Preferably, the effective concentration of oleanolic acid in the body fluid at the site of action is 25–100 μM.

[0024] Preferably, the effective concentration of the kaempferol-3-O-rutin in the body fluid at the site of action is 25–200 μM.

[0025] Preferably, the effective concentration of kaempferol-3-O-sophoroside in the body fluid at the site of action is 25–200 μM.

[0026] Preferably, the effective concentration of linden glycoside in the body fluid at the site of action is 10–50 μM.

[0027] Preferably, the effective concentration of cinnamomea acid in the body fluid at the site of action is 10–25 μM.

[0028] Preferably, the effective concentration of the raspberry extract in the body fluid at the site of action is 0.03–6.5 mg / mL.

[0029] Preferably, the extraction method of the raspberry extract is as follows: raspberries are sequentially subjected to water extraction, filtration, and concentration.

[0030] More preferably, the water extraction is a stepwise water extraction.

[0031] More preferably, the stepwise water extraction is as follows: water is added to the raspberries for the first time, and the mixture is simmered for 0.8 to 1.2 hours; then water is added for the second time, and the mixture is simmered for another 0.8 to 1.2 hours.

[0032] More preferably, the ratio of the amount of water added in the first step to the amount of raspberries is 11-13 mL:1 g, and most preferably 12 mL:1 g.

[0033] More preferably, the ratio of the amount of water added in the second step to the amount of raspberries is 9-11 mL:1 g, and most preferably 10 mL:1 g.

[0034] More preferably, the concentration is carried out under vacuum of -0.09 to -0.07 MPa and at 75 to 85°C until the solid content is 28% to 32%.

[0035] Preferably, the ovarian function protection is one or more of the following: improving the viability of human ovarian granulosa cells, improving oxidative stress levels, regulating mitochondrial membrane potential levels, and maintaining estrogen levels in the microenvironment.

[0036] More preferably, the improvement in oxidative stress levels includes reducing the production of reactive oxygen species in human ovarian granulosa cells.

[0037] The present invention also provides an ovarian function protection product, which contains one or more of isoquercitrin, ellagic acid, vanillic acid, oleanolic acid, kaempferol-3-O-rutin, kaempferol-3-O-sophoroside, lindenin, and potassiolic acid.

[0038] Preferably, the product is food, health product, or medicine.

[0039] Preferably, the product further includes food-acceptable ingredients, health-product-acceptable excipients, and pharmaceutically acceptable excipients.

[0040] More preferably, the drug can be formulated into different dosage forms, such as tablets, powders, pills, capsules, granules, drop pills, oral liquids, etc.

[0041] The present invention has the following beneficial effects:

[0042] The present invention has found that any one of the following ingredients—isoquercetin, ellagic acid, vanillic acid, oleanolic acid, kaempferol-3-O-rutin, kaempferol-3-O-sophoroside, tiolinin, potassium oleate, and raspberry extract—can significantly enhance the viability of human ovarian granulosa cells, significantly improve oxidative stress levels, significantly regulate mitochondrial membrane potential, or effectively maintain estrogen levels in the microenvironment. This indicates that these eight ingredients, as well as raspberry extract containing these eight ingredients, can effectively protect ovarian function, providing a new option for ovarian function protection products and a new application for these substances. Attached Figure Description

[0043] Figure 1 The results are the KGN cell viability assay results from Example 2.

[0044] Figure 2 The results of the determination of reactive oxygen species in KGN cells in Example 2 are shown.

[0045] Figure 3 The results are the measurements of mitochondrial membrane potential in KGN cells in Example 2.

[0046] Figure 4 The results of estrogen determination in KGN cell supernatant in Example 2 are shown.

[0047] Figure 5 The results are the KGN cell viability assay results from Example 3.

[0048] Figure 6 The results of the determination of reactive oxygen species in KGN cells in Example 3 are shown.

[0049] Figure 7 The results of estrogen determination in KGN cell supernatant in Example 3. Detailed Implementation

[0050] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0051] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0052] Example 1: Determination of Indicators of Ovarian Function Protection

[0053] I. Extraction of the main functional components from raspberries

[0054] To 10 4 Add 120L of water to g of raspberries, boil gently for 1 hour, then add 100L of water and boil gently again for 1 hour. Filter, collect and combine the filtrates, and concentrate under vacuum at -0.08MPa and 80℃ until the solid content reaches 30±2% to obtain raspberry extract. After column chromatography separation and purification, high performance liquid chromatography analysis, and qualitative analysis with standard samples, it was determined that the raspberry extract contains vanillic acid, isoquercitrin, kaempferol-3-O-rutin, kaempferol-3-O-sophoroside, oleanolic acid, tiolinoside, potassium oleate, and ellagic acid.

[0055] II. Preparation of test drugs at different concentrations

[0056] (1) Vanillic acid solution: Weigh 3 mg of vanillic acid standard (purchased from Wuhan Tianzhi Biotechnology Co., Ltd.), dissolve it in DMSO, and prepare a 100 mM stock solution. Before use, dilute it with DMEM / F12 complete culture medium to prepare 25 μM, 50 μM, 100 μM and 200 μM vanillic acid solutions respectively.

[0057] (2) Isoquercetin solution: Weigh 3 mg of isoquercetin standard (purchased from Wuhan Tianzhi Biotechnology Co., Ltd.), dissolve it in DMSO, and prepare a 100 mM stock solution. Before use, dilute it with DMEM / F12 complete culture medium to prepare 25 μM, 50 μM, 100 μM and 200 μM isoquercetin solutions respectively.

[0058] (3) Kaempferol-3-O-rutin solution: Weigh 3 mg of kaempferol-3-O-rutin standard (purchased from Wuhan Tianzhi Biotechnology Co., Ltd.), dissolve it in DMSO, and prepare a 100 mM stock solution. Before use, dilute it with DMEM / F12 complete medium to prepare 25 μM, 50 μM, 100 μM and 200 μM kaempferol-3-O-rutin solutions respectively.

[0059] (4) Kaempferol-3-O-sophoroside solution: Weigh 3 mg of kaempferol-3-O-sophoroside standard (purchased from Wuhan Tianzhi Biotechnology Co., Ltd.), dissolve it in DMSO to prepare a 100 mM stock solution, and dilute it with DMEM / F12 complete medium to prepare 25 μM, 50 μM, 100 μM and 200 μM kaempferol-3-O-sophoroside solutions respectively before use;

[0060] (5) Oleanolic acid solution: Weigh 3 mg of oleanolic acid standard (purchased from Wuhan Tianzhi Biotechnology Co., Ltd.), dissolve it in DMSO, and prepare a 50 mM stock solution. Before use, dilute it with DMEM / F12 complete culture medium to prepare 25 μM, 50 μM and 100 μM oleanolic acid solutions respectively.

[0061] (6) Linden glycoside solution: Weigh 3 mg of linden glycoside standard (purchased from Wuhan Tianzhi Biotechnology Co., Ltd.), dissolve it in DMSO, and prepare a 100 mM stock solution. Before use, dilute it with DMEM / F12 complete medium to prepare 10 μM, 25 μM and 50 μM linden glycoside solutions respectively.

[0062] (7) Potentilla acid solution: Weigh 3 mg of potentilla acid standard (purchased from Wuhan Tianzhi Biotechnology Co., Ltd.), dissolve it in DMSO, and prepare a 100 mM stock solution. Before use, dilute it with DMEM / F12 complete culture medium to prepare 10 μM and 25 μM potentilla acid solutions respectively.

[0063] (8) Ellagic acid solution: Weigh 3 mg of ellagic acid standard (purchased from Wuhan Tianzhi Biotechnology Co., Ltd.), dissolve it in DMSO, and prepare a 50 mM stock solution. Before use, dilute it with DMEM / F12 complete culture medium to prepare 5 μM, 10 μM and 20 μM ellagic acid solutions respectively.

[0064] (9) AAPH solution: Weigh 1g of AAPH, dissolve it in ultrapure water to prepare a 400mM stock solution, and dilute it with DMEM / F12 complete medium to prepare 8mM, 10mM, 12mM, 14mM and 16mM AAPH solutions respectively before use.

[0065] (10) Tripterygium wilfordii solution: Weigh 3 mg of triptolide, dissolve it in DMSO to prepare a 400 μM stock solution, and dilute it with DMEM / F12 complete medium to prepare 25 nM, 50 nM, 70 nM, 100 nM and 140 nM triptolide solutions respectively before use.

[0066] III. Cell Culture

[0067] Human ovarian granulosa cells (KGN) were placed in DMEM / F12 complete medium and cultured in a cell culture incubator at 37°C and 5% CO2. The complete medium contained 10% fetal bovine serum (Gibco) and 1% triple antibodies (Beijing Solarbio, containing 100 IU / mL penicillin, 0.1 mg / mL streptomycin, and 0.25 μg / mL amphotericin B).

[0068] IV. Indicator Measurement

[0069] 1. Cell viability assay

[0070] (1) When KGN cells reach the logarithmic phase, KGN cells are seeded at a density of 10,000 cells / well in 96-well plates for culture; DMEM / F12 complete medium containing different concentrations of the test drug is added to each well and incubated at 37°C and 5% CO2 for 24 hours.

[0071] (2) Discard the old culture medium and add 100 μL of CCK8 solution to each well;

[0072] (3) Incubate the 96-well plate in a 37°C, 5% CO2 incubator for 2 hours;

[0073] (4) Measure the absorbance at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0074] 2. Intracellular reactive oxygen species (ROS) measurement

[0075] (1) When KGN cells reach the logarithmic phase, they are seeded into 96-well plates at a density of 10,000 cells / well for further culture.

[0076] (2) After 12 hours, the culture medium in the wells was aspirated and washed twice with serum-free and phenol red-free DMEM / F12 medium. 100 μL of DCFH-DA solution (diluted with serum-free and phenol red-free DMEM / F12 medium, DCFH-DA volume percentage was 0.1%) was added to each well and incubated in a cell culture incubator at 37°C in the dark for 20 minutes.

[0077] (3) Remove the culture medium in the wells, wash three times with serum-free and phenol red-free DMEM / F12 medium, and add serum-free and phenol red-free DMEM / F12 medium containing different concentrations of the test drug to each well.

[0078] (4) The fluorescence intensity was measured using a fluorescence microplate reader at an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

[0079] 3. Mitochondrial membrane potential (MMP) measurement

[0080] (1) When KGN cells reach the logarithmic growth phase, they are seeded into 6-well plates at a density of 300,000 cells / well. After 12 hours, the culture medium in the wells is aspirated, and serum containing different concentrations of the test drug is added to each well.

[0081] DMEM / F12 complete culture medium;

[0082] (2) After incubation for 24 hours, aspirate the culture medium from the well, wash once with PBS, and add 1 mL of DMEM / F12 complete culture medium;

[0083] (3) Add 1 mL of JC-1 staining solution (1X), mix thoroughly, and incubate at 37°C in the dark for 20 min;

[0084] (4) After incubation, discard the supernatant, wash twice with PBS, add 2 mL of DMEM / F12 complete culture medium, and observe under a fluorescence microscope.

[0085] 4. Assay of estrogen (E) in cell supernatant

[0086] (1) When KGN cells are cultured to the logarithmic phase, they are seeded into 6-well plates at a density of 300,000 cells / well. After 12 hours, the culture medium in the wells is aspirated, and DMEM / F12 complete medium containing different concentrations of the drug to be tested is added to each well.

[0087] (2) After incubation for 24 hours, the culture medium in each well was collected, and the estrogen in the KGN cell supernatant was measured according to the method of the human estrogen (E) enzyme-linked immunosorbent assay (ELISA) kit (Wuhan Huamei Biotechnology Co., Ltd.). The main reagents were all from the kit.

[0088] V. Data Processing and Statistical Analysis

[0089] The standard curve for ELISA assays was fitted using Origin 2019, fluorescence intensity was analyzed using Image J, and differences between groups were analyzed using GraphPad Prism 8.0. Statistical graphs were also plotted using GraphPad Prism 8.0. All results are expressed as mean ± standard error (Mean ± SEM), and one-way ANOVA was used for comparisons between groups. In the results, compared with the control group, *: p < 0.05 was considered statistically significant, and **: p < 0.01, ***: p < 0.001, and ****: p < 0.001 were considered highly significant. Compared with the model group, #: p < 0.05 was considered statistically significant, and ##: p < 0.01, ###: p < 0.001, and ####: p < 0.001 were considered highly significant.

[0090] Example 2: Protective effect on ovarian function (AAPH-induced KGN cell damage model)

[0091] I. Establishment of an AAPH-induced human ovarian granulosa cell injury model

[0092] A KGN cell damage model was established by inducing the cells with AAPH solution of different concentrations (specifically, the induction method involved incubating the cells with AAPH solution at 37°C in a 5% CO2 incubator for 24 hours). The viability of the KGN cells was then measured, and the results are as follows: Figure 1 As shown, cell viability gradually decreases with increasing AAPH concentration, and drops to approximately 50% when the AAPH concentration reaches 16 mM. Therefore, the following experiments used a KGN cell damage model induced by 16 mM AAPH solution. II. Regulatory Effect of AAPH on KGN Cell Viability

[0093] The regulatory effect of different concentrations of the test drug on the cell viability of an AAPH-induced human ovarian granulosa cell injury model was determined according to the KGN cell viability assay method in Example 1.

[0094] The results are as follows Figure 1 As shown, it can be seen that:

[0095] ① Compared with the control group (no modeling, no drug treatment), 16 mM AAPH significantly reduced cell viability (p<0.01);

[0096] ② Compared with the model group (16mM AAPH modeling but no drug treatment), 25, 50, and 200 μM vanillic acid (VA) significantly increased cell viability by 20% (p<0.01);

[0097] ③ Compared with the model group, isoquercitrin (IQ) at concentrations of 25–200 μM significantly improved cell viability (p<0.01), with 100 μM IQ increasing cell viability by approximately 50%.

[0098] ④ Compared with the model group, 25–200 μM kaempferol-3-O-rutin (KOR) significantly improved cell viability (p<0.01), with 200 μM KOR increasing cell viability by about 24%.

[0099] ⑤ Compared with the model group, 50–200 μM kaempferol-3-O-sophoroside (KS) significantly improved cell viability (p<0.01); among them, 200 μM KS increased cell viability by 17.6%.

[0100] ⑥ Compared with the model group, 25-50 μM oleanolic acid (OA), 25 μM potassium phosphate (TA), and 10 μM lindenyl glycoside (TS) significantly increased cell viability by 11-12%.

[0101] ⑦ Compared with the model group, 5-20 μM ellagic acid (EA) significantly improved cell viability (p<0.01), of which 10 μM ellagic acid increased cell viability by 28%.

[0102] In summary, this invention established an AAPH-induced human ovarian granulosa cell damage model, and then intervened with vanillic acid, isoquercitrin, kaempferol-3-O-rutin, kaempferol-3-O-sophoroside, ellagic acid, oleanolic acid, potassium oleate, and linaloside. The results showed that the reduced viability of human ovarian granulosa cells was alleviated, indicating that these drugs can effectively protect human ovarian granulosa cells and thus effectively protect ovarian function.

[0103] III. Its effect on improving AAPH-induced oxidative stress levels in KGN cells

[0104] The effect of different concentrations of the test drug on improving oxidative stress levels in an AAPH-induced human ovarian granulosa cell injury model was determined according to the method for measuring intracellular reactive oxygen species in KGN cells in Example 1.

[0105] The results are as follows Figure 2 As shown, it can be seen that:

[0106] ① After treatment of cells with 16mM AAPH, the intracellular reactive oxygen species level increased significantly to about 3 to 4 times that of the control group (without modeling or drug treatment) (p<0.01);

[0107] ② Compared with the model group (16mM AAPH modeling but no drug treatment), 10μM and 20μM ellagic acid intervention significantly reduced AAPH-induced intracellular ROS levels (p<0.01).

[0108] ③ Compared with the model group, 50 μM and 100 μM vanillic acid can significantly reduce the production of intracellular reactive oxygen species (p<0.05);

[0109] ④ Compared with the model group, 50 μM and 100 μM isoquercitrin can significantly scavenge AAPH-induced ROS (p<0.01) and alleviate oxidative damage to KGN cells caused by AAPH.

[0110] In summary, this invention first established an AAPH-induced human ovarian granulosa cell damage model, showing an increase in intracellular free radicals and a significant rise in reactive oxygen species (ROS) levels. After intervention with vanillic acid, isoquercitrin, and ellagic acid, it was found that these three drugs significantly reduced the production of ROS in human ovarian granulosa cells, alleviating AAPH-induced oxidative damage. This indicates that these drugs can effectively improve AAPH-induced oxidative stress levels in KGN cells, thereby effectively protecting ovarian function.

[0111] IV. Regulatory effect on AAPH-induced mitochondrial membrane potential in KGN cells

[0112] Based on the method for measuring mitochondrial membrane potential in KGN cells in Example 1, the regulatory effect of different concentrations of the test drug on the mitochondrial membrane potential level in an AAPH-induced human ovarian granulosa cell injury model was determined.

[0113] The results are as follows Figure 3 As shown, it can be seen that:

[0114] ① Compared with the control group (no modeling, no drug treatment), the red to green fluorescence ratio of the modeling group (16mM AAPH modeling but no drug treatment) was significantly reduced, indicating that after 16mM AAPH modeling, the mitochondrial membrane potential of cells decreased to about 40% of the control group level, and the green fluorescence was significantly enhanced (p<0.01).

[0115] ② Compared with the model group, 50 μM isoquercitrin increased the mitochondrial membrane potential level by 6%, and 100 μM isoquercitrin significantly increased the ratio of red to green fluorescence in cells (p<0.01), causing the mitochondrial membrane potential level to return to about 55% of the control group.

[0116] ③ Compared with the model group, 20 μM ellagic acid increased the mitochondrial membrane potential level by nearly 10%.

[0117] In summary, isoquercitrin and ellagic acid can significantly upregulate mitochondrial membrane potential, alleviate AAPH-induced mitochondrial damage, maintain the process of mitochondrial oxidative phosphorylation and ATP generation, ensure the normal growth and physiological function of ovarian granulosa cells, and effectively protect ovarian function.

[0118] V. Regulation of estrogen levels in the AAPH-induced KGN cell microenvironment

[0119] Based on the method for measuring estrogen in KGN cell supernatant in Example 1, the regulatory effect of different concentrations of the test drug on estrogen levels in the microenvironment of the AAPH-induced human ovarian granulosa cell injury model was determined.

[0120] The results are as follows Figure 4 As shown, it can be seen that:

[0121] ① Compared with the control group (no modeling, no drug treatment), the estrogen content in the supernatant of KGN cells in the modeling group (16mM AAPH modeling but no drug treatment) was significantly reduced (p<0.05);

[0122] ② Compared with the control group, without the addition of modeling substances, the use of 100 μM isoquercitrin or 20 μM ellagic acid to intervene in KGN cells alone significantly increased the estrogen level in the cell supernatant (p<0.01).

[0123] ③ Compared with the model group, 100 μM isoquercitrin significantly increased the cells' ability to secrete estrogen (p<0.05), and the estrogen in the cell supernatant returned to a level similar to that of the control group;

[0124] ④ Compared with the model group, 20 μM ellagic acid significantly increased the ability of cells to secrete estrogen (p<0.05);

[0125] In summary, this invention first established an AAPH-induced human ovarian granulosa cell damage model, simulating the decline in estrogen levels in vivo. Then, it used isoquercitrin and ellagic acid for intervention and found that these two drugs can significantly improve the cells' ability to secrete estrogen, effectively improve the estrogen secretion level of human ovarian granulosa cells, and achieve the effect of protecting ovarian granulosa cell function, thus effectively protecting ovarian function.

[0126] Example 3: Protective Effect on Ovarian Function (Tripterygium wilfordii-induced KGN Cell Injury Model) I. Establishment of a Tripterygium wilfordii-induced Human Ovarian Granulosa Cell Injury Model

[0127] A KGN cell injury model was established by inducing the cells with triptolide solution at concentrations of 25–140 nM (specifically, the induction method involved co-incubating the cells with triptolide solution at 37°C in a 5% CO2 incubator for 24 hours). The viability of the KGN cells was then measured, and the results are as follows: Figure 5 As shown, triptolide solutions of 25–140 nM all had a highly significant inhibitory effect on cell viability (p<0.01). When the concentration of triptolide reached 50 nM, cell viability decreased to about 50%. Therefore, the following experiments used a KGN cell damage model induced by 50 nM triptolide solution.

[0128] II. Regulatory effect on triptolide-induced KGN cell viability

[0129] According to the method for measuring KGN cell viability in Example 1, the regulatory effect of different concentrations of the test drug on cell viability in a triptolide-induced human ovarian granulosa cell injury model was determined.

[0130] The results are as follows Figure 5 As shown, it can be seen that:

[0131] ① Compared with the control group (no modeling, no drug treatment), the cell viability of the modeling group (50 nM TP modeling but no drug treatment) was significantly reduced (p<0.01);

[0132] ② Compared with the model group, vanillic acid at 50–200 μM significantly improved the cell viability of KGN cells (p<0.05), with the best efficacy observed at 200 μM;

[0133] ③ Compared with the model group, isoquercitrin at 50–200 μM significantly improved KGN cell viability (p<0.05), with the best efficacy observed at 100 μM;

[0134] ④ Compared with the model group, 100-200 μM kaempferol-3-O-rutin significantly improved KGN cell viability (p<0.05), with the best efficacy observed at 200 μM;

[0135] ⑤ Compared with the model group, 50–200 μM kaempferol-3-O-sophoroside significantly improved KGN cell viability (p<0.05), with the best efficacy observed at 200 μM;

[0136] ⑥ Compared with the model group, 25-100 μM oleanolic acid significantly improved KGN cell viability (p<0.01), with the best efficacy observed at 50 μM;

[0137] ⑦ Compared with the model group, 25 μM linden glycoside significantly enhanced KGN cell viability (p<0.05).

[0138] In summary, this invention established a human ovarian granulosa cell damage model induced by triptolide, and then intervened with vanillic acid, isoquercitrin, kaempferol-3-O-rutin, kaempferol-3-O-sophoroside, oleanolic acid, and linaloside. The results showed that the reduced viability of human ovarian granulosa cells was alleviated, indicating that these drugs can effectively protect human ovarian granulosa cells and thus effectively protect ovarian function.

[0139] III. The effect of triptolide on improving the level of oxidative stress in KGN cells

[0140] According to the method for measuring intracellular reactive oxygen species in KGN cells in Example 1, the effect of different concentrations of the test drug on improving the level of oxidative stress in a triptolide-induced human ovarian granulosa cell injury model was determined.

[0141] The results are as follows Figure 6 As shown, it can be seen that:

[0142] ① Compared with the control group (no modeling, no drug treatment), the reactive oxygen species level in KGN cells of the modeling group (50 nM TP modeling but no drug treatment) was slightly increased;

[0143] ② Compared with the model group, 200 μM kaempferol-3-O-sophoroside, 50–100 μM isoquercitrin, and 100 μM vanillic acid can all significantly reduce the ROS level in KGN cells (p<0.01);

[0144] ③ Compared with the model group, 25 μM oleanolic acid significantly reduced the production of intracellular ROS (p<0.05).

[0145] In summary, this invention first established a triptolide-induced human ovarian granulosa cell damage model, showing increased intracellular free radicals and significantly elevated reactive oxygen species (ROS) levels. Subsequent intervention with vanillic acid, isoquercitrin, kaempferol-3-O-sophoroside, and oleanolic acid revealed that these drugs significantly reduced ROS production in human ovarian granulosa cells, alleviating triptolide-induced oxidative damage. This indicates that these drugs can effectively improve triptolide-induced KGN cell oxidative stress levels, thereby effectively protecting ovarian function.

[0146] IV. Regulation of estrogen levels in the triptolide-induced KGN cell microenvironment

[0147] Based on the method for measuring estrogen in KGN cell supernatant in Example 1, the regulatory effect of different concentrations of the test drug on estrogen levels in the microenvironment of the triptolide-induced human ovarian granulosa cell injury model was determined.

[0148] The results are as follows Figure 7 As shown, it can be seen that:

[0149] ① Compared with the control group (no modeling, no drug treatment), the estrogen secretion level of KGN cells in the modeling group (50 nM TP modeling but no drug treatment) was slightly reduced;

[0150] ② Compared with the control group, without the addition of modeling substances, 100 μM isoquercitrin and 200 μM kaempferol-3-O-rutin both significantly increased the estrogen secretion level of KGN cells (p<0.05).

[0151] ③ Compared with the model group, when the modeling substances were added, 100 μM isoquercitrin and 200 μM kaempferol-3-O-rutin both significantly improved the ability of KGN cells to secrete estrogen (p<0.05).

[0152] In summary, this invention first established a human ovarian granulosa cell damage model induced by triptolide, simulating the decline in estrogen levels in vivo. Then, it used isoquercitrin and kaempferol-3-O-rutin for intervention and found that both drugs could significantly improve the cells' ability to secrete estrogen, effectively improve the estrogen secretion level of human ovarian granulosa cells, and achieve the effect of protecting ovarian granulosa cell function, thus effectively protecting ovarian function.

[0153] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. Application of linden glycosides in the preparation of products for protecting ovarian function.

2. The application according to claim 1, characterized in that, The effective concentration of linden glycoside in the body fluid at the site of action is 10–50 μM.

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