Ergothioneine composition capable of improving ovarian vitality and preparation method and application of ergothioneine composition

Abstract

The invention discloses an ergothioneine composition for improving ovarian vitality and a preparation method and application thereof, the composition comprises D-ribose and ergothioneine, the concentration of the D-ribose in the composition is 5-15 mM, and the concentration of the ergothioneine in the composition is 100-300 [mu] M. The pharmaceutical composition is used for regulating the redox balance of ovarian granular cells, inhibiting inflammatory response and up-regulating anti-mullerian hormone secretion so as to improve the ovarian activity.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical technology, specifically relating to an ergothioneine composition for improving ovarian vitality, its preparation method, and its application. Background Technology

[0002] Ovarian dysfunction is the core pathological manifestation of female reproductive system decline, essentially a syndrome characterized by a decrease in the number of follicles and a decline in oocyte quality. This process involves the interaction of multiple molecular mechanisms, among which energy metabolism disorders, oxidative stress damage, chronic inflammation, and abnormal secretion of anti-Müllerian hormone (AMH) constitute key pathological links. Under physiological conditions, oocyte maturation and development are highly dependent on adenosine triphosphate (ATP) synthesized by mitochondria, while D-ribose, as an important precursor for ATP synthesis in the body, is fundamental to maintaining cellular energy homeostasis. Simultaneously, ovarian tissue maintains a dynamic balance of reactive oxygen species (ROS) through endogenous antioxidant systems (such as glutathione and superoxide dismutase). However, when the body encounters environmental toxins, metabolic disorders, or aging, insufficient energy supply and excessive ROS accumulation jointly induce mitochondrial dysfunction, directly attacking oocyte DNA and damaging protein structural integrity, accelerating follicular atresia. Meanwhile, chronic low-grade inflammation activates signaling pathways such as NF-κB, prompting the massive release of pro-inflammatory factors (such as TNF-α and IL-6), further damaging the follicular microenvironment and inhibiting granulosa cell function, leading to a significant decrease in AMH secretion levels. As a key biomarker of follicular reserve, a decline in AMH levels indicates reduced ovarian responsiveness and fertility potential. Currently, approximately 12%–15% of women of reproductive age worldwide face premature ovarian failure or decreased ovarian reserve, necessitating safe and effective intervention strategies.

[0003] Currently, hormone replacement therapy (HRT) remains the preferred clinical intervention for hypoestrosis, which alleviates symptoms of low estrogen levels by exogenously supplementing steroid hormones such as estradiol and progesterone. However, HRT has significant limitations, such as long-term exposure to high doses of estrogen, which significantly increases the risk of endometrial hyperplasia, breast cancer, and thromboembolic events.

[0004] On the other hand, adjuvant antioxidant therapy is emerging in the field of ovarian protection. Antioxidants, including coenzyme Q10, vitamin E, and melatonin, are being used to improve oocyte quality, while D-ribose is being explored to enhance cellular energy metabolism. However, these approaches have significant limitations. For example, water-soluble antioxidants (such as vitamin C) have difficulty penetrating the cell membrane lipid bilayer, while lipid-soluble antioxidants (such as vitamin E) have limited intracellular transport efficiency, resulting in insufficient effective tissue concentrations. Furthermore, while D-ribose alone can provide an energy substrate, it cannot address the oxidative stress that accompanies energy metabolism and may even exacerbate ROS generation due to enhanced metabolism. In addition, these single-mechanism components cannot synergistically regulate the energy-oxidation balance, and their regulatory role in AMH secretion has not yet been clearly demonstrated.

[0005] L-Ergothioneine (EGT), a naturally occurring amino acid derivative, possesses potent antioxidant activity due to its unique thionyl group structure. Molecular mechanism studies show that after intestinal absorption, ergothioneine is transported via the organic cation transporter 1 (OCTN1) primarily targeting tissues highly expressing this transporter. Through mechanisms such as chelating transition metal ions, activating antioxidant pathways, and inhibiting inflammatory signaling, it demonstrates potential in neuroprotection and anti-photoaging of the skin.

[0006] However, current technologies regarding the application of ergothioneine in maintaining ovarian function are still very limited. Although ergothioneine has been shown to reduce oxidative damage to granulosa cells, its direct impact on key mechanisms such as AMH synthesis and secretion remains unclear. More importantly, current technologies have failed to organically combine the antioxidant advantages of ergothioneine with the energy-supplementing effects of D-ribose; therefore, the efficacy of existing formulations in improving ovarian function still needs further clarification.

[0007] Therefore, developing a formulation that can synergistically regulate cellular energy metabolism and oxidative stress balance, has significant therapeutic effects, a clear mechanism of action, and high safety to improve or treat ovarian dysfunction has become a key challenge that urgently needs to be addressed by those skilled in the art. Summary of the Invention

[0008] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0009] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0010] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide an ergothioneine composition for improving ovarian vitality, its preparation method, and its application.

[0011] To solve the above-mentioned technical problems, the present invention provides the following technical solution: an ergothioneine composition for improving ovarian vitality, characterized in that: the ergothioneine composition comprises D-ribose and ergothioneine, which is used to regulate the redox balance of ovarian granulosa cells, inhibit inflammatory response and upregulate the secretion of anti-Müllerian hormone, so as to improve ovarian vitality.

[0012] In a preferred embodiment of the ergothioneine composition of the present invention, the concentration of D-ribose in the ergothioneine composition is 5-15 mM, and the concentration of ergothioneine is 100-300 μM.

[0013] In a preferred embodiment of the ergothioneine composition of the present invention, the method for preparing the ergothioneine composition includes, D-ribose and ergothioneine were dissolved in serum-free cell culture medium to obtain D-ribose solution and EGT solution; the D-ribose solution and EGT solution were mixed and then sterilely filtered to obtain the final product.

[0014] As a preferred embodiment of the ergothioneine composition of the present invention, the dissolution process is carried out at room temperature of 20-25°C, and the stirring rate is 50-100 rpm until the solid is completely dissolved; the stirring rate during mixing is 100-200 rpm, and the stirring time is 5-10 min.

[0015] Another objective of this invention is to overcome the shortcomings of the prior art and provide an application of an ergothioneine composition in downregulating the level of reactive oxygen species in ovarian granulosa cells, wherein the application is to act the composition on KGN cells damaged by oxidative stress to reduce the accumulation of intracellular ROS.

[0016] As a preferred embodiment of the application described in this invention, the oxidative stress-damaged KGN cells are constructed by inducing with 300-500 μM hydrogen peroxide for 6 h, and after treatment with the composition, the ROS level of the KGN cells is downregulated by ≥50% compared with the model group.

[0017] In a preferred embodiment of the preparation method described in this invention, the application involves activating the antioxidant pathway of KGN cells and restoring the function of granulocytes in secreting AMH through the composition.

[0018] As a preferred embodiment of the preparation method described in this invention, compared with the oxidative stress model group, the Nrf2 level of KGN cells is upregulated by ≥50% and the AMH level is upregulated by ≥80% after treatment with the composition.

[0019] Another objective of this invention is to overcome the shortcomings of the prior art and provide an application of an ergothioneine composition in downregulating the levels of inflammatory factors and nuclear factor κB in ovarian granulosa cells, characterized in that: the inflammatory factors include interleukin-1β and tumor necrosis factor-α, and the application is to inhibit the inflammatory signaling pathway of KGN cells through the composition.

[0020] As a preferred embodiment of the application described in this invention, the inflammatory damage of the KGN cells is constructed by inducing 40-60 ng / mL lipopolysaccharide for 24 h. After treatment with the composition, the levels of IL-1β and TNF-α in the KGN cells are downregulated by ≥35% compared with the model group, and the level of NF-κB is downregulated by ≥40% compared with the model group.

[0021] Beneficial effects of this invention: This invention discovers that the composition of D-ribose and EGT can alleviate hydrogen peroxide-induced cellular oxidative stress and LPS-induced cellular inflammatory responses. It can downregulate the level of free radical ROS under KGN oxidative stress; increase the level of the antioxidant factor Nrf2 under KGN oxidative stress; restore cell viability under KGN oxidative stress; restore the level of the functional core biomarker AHM under KGN oxidative stress; downregulate the levels of inflammatory factors IL-1β and TNF-α under KGN inflammatory damage; inhibit the expression of the inflammatory core target NF-κB under KGN inflammatory damage; restore cell viability under KGN inflammatory damage; and restore the level of the functional core biomarker AHM under KGN inflammatory damage. Therefore, the composition used in this invention has great application potential in improving and repairing products that address ovarian dysfunction and reduced vitality caused by oxidative stress and inflammatory damage. Attached Figure Description

[0022] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein: Figure 1 The figure shows the effect of different concentrations of D-ribose and EGT on the viability of KGN cells in an embodiment of the present invention.

[0023] Figure 2 The figure shows the effect of D-ribose, EGT and their composition on the ROS level of H2O2-induced oxidation products in KGN cells according to embodiments of the present invention.

[0024] Figure 3The figure shows the effect of D-ribose, EGT and their composition on the level of the antioxidant factor Nrf2 induced by H2O2 in KGN cells according to the embodiments of the present invention.

[0025] Figure 4 The figure shows the effect of D-ribose, EGT and their composition on the cell viability of H2O2-induced KGN cells in the embodiments of the present invention.

[0026] Figure 5 The figure shows the results of the effects of D-ribose, EGT and their composition on the AMH level, a marker of ovarian function in KGN cells induced by H2O2, in an embodiment of the present invention.

[0027] Figure 6 This is a graph showing the effects of D-ribose, EGT, and their combinations on the levels of LPS-induced inflammatory factors IL-1β and TNF-α in KGN cells according to embodiments of the present invention. Figure 6 (A) represents IL-1β; Figure 6 (B) TNF-α.

[0028] Figure 7 The figure shows the effect of D-ribose, EGT and their composition on the level of NF-κB, a key inflammatory target in LPS-induced KGN cells, according to embodiments of the present invention.

[0029] Figure 8 The figure shows the effect of D-ribose, EGT and their composition on the cell viability of LPS-induced KGN cells in the embodiments of the present invention.

[0030] Figure 9 This is a graph showing the effects of D-ribose, EGT, and their combinations on the AMH level, a marker of ovarian function in LPS-induced KGN cells, according to embodiments of the present invention. Detailed Implementation

[0031] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0032] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0033] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0034] Unless otherwise specified, all raw materials used in the embodiments of this invention are commercially available. See Table 1 for details.

[0035] Table 1

[0036] The human ovarian granulosa cells (KGN) used in the experiment were purchased from Nanjing Senbeijia Biotechnology Co., Ltd.

[0037] In the attached figures, * indicates P < 0.05 compared to the model group; ** indicates P < 0.01 compared to the model group; *** indicates P < 0.001 compared to the model group; **** indicates P < 0.0001 compared to the model group.

[0038] Example 1 Effects of different concentrations of D-ribose and EGT on KGN cells In this embodiment, different concentrations of ribose or EGT were added to KGN cells for intervention, as follows: Frozen KGN cells were rapidly thawed at 37°C and resuspended in three times the volume of cell culture medium. After centrifugation at 500g for 5 min, the supernatant was discarded, and the cells were resuspended again in cell culture medium and seeded into T25 cell culture dishes. After three passages, cells were seeded into 96-well plates at a concentration of 5000 cells per well and incubated for 6 h to allow cell attachment. Cells were treated with 10, 25, 50, 100, 150, 200, and 250 μM EGT solution and 0.5, 1, 2, 4, 10, 20, and 50 mM D-ribose solution for 24 h, with six replicates per group and concentration. An additional 10 μL of LCK-8 solution was added, and the cells were incubated at 37°C for 2.5 h. The absorbance at 450 nm was then measured using a microplate reader, and cell viability was calculated as follows: Cell viability (%) = (Experimental group OD − Blank well OD) / (Control group OD − Blank well OD) × 100%. By detecting the effects of different concentrations of D-ribose and EGT on the viability of unmodeled cells, the safe concentration range for cells in normal physiological state was determined, and sample concentrations that do not interfere with the basic activity of cells were screened out.

[0039] Experimental results are as follows Figure 1 As shown, cell viability initially increases and then decreases with increasing sample concentration. Figure 1 In group A, when the concentration of D-ribose was between 0 and 10 mM, cell viability increased by 27.68% (P>0.0001). However, when the concentration was further increased to 20 mM, cell viability decreased by 1.69% compared to the group without added D-ribose, and decreased by 9.87% at 50 mM (P>0.05). Figure 1In B, when the EGT concentration was between 0 and 200 μM, cell viability increased by 35.17% (P>0.0001). However, when the concentration was further increased, cell viability began to decrease. When the concentration was increased to 300 μM, cell viability was only increased by 26.89%, at 400 μM it decreased by 0.95%, and at 500 μM it decreased by 8.21%.

[0040] The above results indicate that both D-ribose and EGT exhibit a "dose-window effect" in regulating the viability of unmodeled cells: the optimal viability-promoting concentration for D-ribose is 10 mM, and for EGT, it is 200 μM. When the concentration of either sample exceeds its optimal level, their viability-promoting effect gradually weakens, even showing a slight inhibitory effect at high concentrations, but this inhibitory effect is not statistically significant. These results provide direct evidence for selecting safe concentrations of the two samples in subsequent modeling experiments; that is, subsequent experiments should preferentially use 10 mM D-ribose and 200 μM EGT as intervention concentrations to avoid the non-specific effects of high concentrations on cell viability interfering with experimental conclusions.

[0041] Example 2 Induction of KGN and inflammatory cell models This embodiment induces KGN oxidative stress and inflammatory damage cell models, respectively, using the following methods: The frozen KGN cells were rapidly thawed at 37°C and resuspended in three times the volume of cell culture medium. The cells were centrifuged at 500g for 5 minutes, the supernatant was discarded, and the cells were resuspended again in cell culture medium and seeded into T25 cell culture dishes.

[0042] After resuscitation, cells were cultured at 37°C with 5% CO2. When approximately 80%-90% of the cell culture dish was covered by growing cells, the cells were passaged. Cells were digested with 1 mL of trypsin-EDTA (0.25%) and observed under a microscope. Once the cells were in suspension, digestion was immediately stopped with three times the volume of complete cell culture medium. After centrifugation, the cells were collected and passaged at a 1:2 ratio. The experimental groups are shown in Tables 2 and 3.

[0043] Table 2 Grouping Information for Oxidative Stress Models

[0044] Table 3 Grouping Information of Inflammation Injury Model

[0045] Example 3 Effects of D-ribose, EGT and their combinations on cellular oxidative stress Effects of D-ribose, EGT, and their combinations on cellular ROS levels After aspirating the supernatant and removing the culture medium, add an appropriate volume of DCFH-DA to a final concentration of 10 µM and incubate the cells at 37°C for 20 min. Aspirate the culture medium and repeatedly pipette the cells from the bottom of the flask with serum-free culture medium until the cell layer changes from translucent to clear. Then, pipette the cell layer with PBS until it detaches and collect it in a 1.5 mL EP tube. Incubate at 1000 rpm for 5 min, aspirate the supernatant, and resuspend the cells in PBS. Measure the absorbance at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. To correct for the degree of cell homogenization, determine the protein concentration in the supernatant using a BCA protein assay kit.

[0046] Reactive oxygen species (ROS) are natural byproducts of cellular aerobic metabolism, primarily originating from electron leakage in the mitochondrial respiratory chain and the oxidase system. Under physiological homeostasis, low concentrations of ROS act as key signaling molecules, participating in core physiological processes such as cell proliferation, differentiation, and apoptosis through the regulation of related pathways, and are crucial for maintaining cellular and tissue function. When ROS production is excessive (e.g., due to exogenous stimuli or endogenous metabolic disorders), or when the body's antioxidant defense system is impaired, the cellular redox balance is disrupted, inducing "oxidative stress." Experimental results are as follows... Figure 2 As shown, compared with the control group, the ROS level in the model group was significantly upregulated by 192.52 (fluorescence intensity / μg protein), while no statistically significant changes were observed after intervention with *Codonopsis pilosula* and D-ribose (P>0.05). Furthermore, both the EGT group and the D-ribose+EGT group significantly downregulated ROS levels (P<0.0001), with a 31.76% downregulation in the EGT group and a 54.07% downregulation in the D-ribose+EGT group.

[0047] Both the EGT group and the D-ribose + EGT group significantly downregulated ROS levels, with the D-ribose + EGT group showing a significantly better effect than the low-dose group. This indicates that D-ribose not only acts as an "accelerator" of energy metabolism but also produces a synergistic antioxidant effect with EGT.

[0048] Effects of D-ribose, EGT and their combinations on cellular Nrf2 levels After sample intervention, cell culture supernatant was collected from 12-well plates, centrifuged at 2000 rpm for 10 min, and the supernatant was used to detect Nrf-2 secretion levels. Standard solutions of different concentrations were prepared by diluting and loading them onto the enzyme-labeled plates, followed by the addition and dilution of the test samples. The plates were sealed and incubated at 37°C for 30 min, then washed five times with diluted washing buffer. Enzyme-labeled reagents (except for blank wells) were then added, followed by incubation and washing again. For color development, chromogenic agent A was added first, followed by chromogenic agent B, and development was performed in the dark for 15 min. After terminating the reaction, the absorbance of each well was measured at 450 nm. The entire measurement process must be completed within 15 minutes after the reaction is terminated.

[0049] Nuclear factor E2-associated factor 2 (Nrf2), a core regulator of oxidative stress, plays a crucial role in maintaining cellular redox homeostasis through its activation and regulation mechanisms. Under normal conditions, Nrf2 binds to Kelch-like ECH-associated protein 1 in the cytoplasm and remains inactive. When elevated ROS levels induce oxidative stress, ROS-mediated conformational changes in the binding protein cause Nrf2 to detach and translocate to the nucleus, where it binds to antioxidant response elements. This, in turn, regulates the expression of a series of downstream antioxidant enzymes and detoxification proteins, enhancing the cell's ability to scavenge ROS and attempting to restore redox balance. Experimental results are as follows: Figure 3 As shown, after H2O2 induction, the Nrf2 level in the model group was downregulated by 47.32% compared to the blank group (P<0.0001). After intervention, the Nrf2 level in the *Gynostemma pentaphyllum* group was upregulated by 14.68% (P<0.05); the Nrf2 level in the D-ribose group was upregulated by 14.56% (P<0.05); the Nrf2 level in the EGT group was upregulated by 35.05% (P<0.0001); and the Nrf2 level in the D-ribose + EGT group was upregulated by 61.11% (P<0.0001). These results indicate that H2O2 induction significantly reduces Nrf2 levels, while *Gynostemma pentaphyllum* and D-ribose, although able to upregulate Nrf2 levels, had weaker effects and lower statistical significance. The combination of EGT and D-ribose + EGT significantly upregulated Nrf2 levels, with D-ribose + EGT showing a greater upregulation.

[0050] This indicates that under oxidative stress, EGT can strongly activate the Nrf2 signaling pathway, and the combination of D-ribose and EGT can produce a synergistic effect, further amplifying the antioxidant response of Nrf2, thereby more effectively enhancing the antioxidant defense capacity of cells.

[0051] Effects of D-ribose, EGT and their combinations on cell viability After sample intervention, 6 replicates were set up for each concentration in each 96-well plate, and 10 μL of CCK-8 solution was added. The plates were then incubated at 37°C for 1.5 h. The absorbance at 450 nm was then measured using a microplate reader, and cell viability was calculated as follows: Cell viability (%) = (OD of experimental group − OD of blank well) / (OD of control group − OD of blank well) × 100%.

[0052] Cell viability and function play a crucial role in the occurrence, development, and regulation of oxidative reactions; the detection of cell viability can reflect the state of cells to a certain extent. Experimental results are as follows... Figure 4As shown, after H2O2 induction, cell viability in the model group decreased by 37.49% (P<0.0001). Compared with the model group, cell viability increased by 8.65% (P<0.05) after intervention with *Codonopsis pilosula*, by 8.71% (P<0.05) in the D-ribose group, and by 16.52% (P<0.0001) in the EGT group. The increase in cell viability was more significant in the D-ribose + EGT group, reaching 35.13% (P<0.0001).

[0053] The results of this experiment show that H2O2-induced oxidative damage can significantly inhibit KGN cell viability, while administration of *Codonopsis pilosula* or D-ribose alone only produces limited protective effects, with cell recovery of less than 9%, and the statistical significance is weak. In contrast, EGT alone can increase viability by nearly half of the reduction in the model group; when D-ribose is used in combination with EGT, cell viability recovers by 35.13%, which is close to the normal level and significantly better than each single-drug group (P < 0.0001). This suggests that D-ribose not only has a slight cytoprotective effect on its own, but can also form a synergistic mechanism with EGT to jointly reverse cell damage caused by oxidative stress.

[0054] Effects of D-ribose, EGT and their combinations on cellular AMH levels For experimental procedures, refer to Example 2 (2).

[0055] Granulosa cells are the "Supporting cells" for follicle development, and their functional integrity directly determines follicle growth, maturation, and ovulation. AMH is a specific molecular marker of granulosa cell function—only normally functioning granulosa cells can stably synthesize and secrete AMH. When KGN cells are damaged by oxidative stress (such as excessive ROS or H2O2-induced damage), the anabolic metabolic function of granulosa cells is the first to be affected. Therefore, detecting AMH levels can directly reflect whether oxidative stress has damaged the core function of KGN cells, and is a direct indicator of "functional impairment" of granulosa cells due to oxidative damage. Experimental results are as follows: Figure 5 As shown, compared with the blank group, the AMH level in the model group induced by H2O2 was significantly reduced, with a decrease of 58.10% (P<0.0001). After intervention with different samples, the AMH level in the *Codonopsis pilosula* group increased by 11.60% (P>0.05); the D-ribose group increased by 14.38% (P<0.05); the EGT group increased by 27.69% (P<0.0001); and the D-ribose + EGT group showed a more significant increase in cell viability, reaching 84.37% (P<0.0001).

[0056] Experimental results showed that H2O2-induced oxidative stress significantly reduced AMH levels in ovarian tissue, indicating severe impairment of granulosa cell function. In single-drug intervention, the recovery effect of Yangshen alone was not statistically significant. While both D-ribose and EGT could reverse this downward trend, the combined use of D-ribose and EGT resulted in a surge in AMH levels. AMH is a clinically recognized "gold standard" marker of ovarian reserve and vitality. D-ribose, by improving energy metabolism, can provide "fuel" for EGT-mediated antioxidant and cellular function repair. The two drugs synergistically activate pathways such as Nrf2 / ARE, jointly restoring the ability of granulosa cells to synthesize AMH, providing a new combined strategy for improving ovarian reserve function and intervening in early-onset ovarian insufficiency.

[0057] Example 4 The effects of D-ribose, EGT, and their combinations on cellular inflammatory damage (1) Effects of D-ribose, EGT and their combinations on cellular IL-1β and TNF-α levels For experimental procedures, refer to Example 2 (2).

[0058] IL-1β and TNF-α are important cytokines in the inflammatory response. Released along with other pro-inflammatory factors after tissue damage, they further contribute to the persistence of inflammation, promote collagen degradation and fibrous tissue damage. Experimental results are as follows... Figure 6 As shown. By Figure 6 A. The IL-1β level in the model group was 126.02 pg / mL, which was 2.07 times that of the blank group (P<0.0001). After intervention in different samples, the IL-1β level was downregulated by 9.47% in the Yangshen group (P<0.01), 13.84% in the D-ribose group (P<0.001), and 9.96% in the EGT group (P<0.01). The D-ribose + EGT group showed the largest downregulation, reaching 39.47% (P<0.0001). Figure 6 B. The TNF-α level in the model group was 293.84 pg / mL, which was upregulated by 141.98 pg / mL compared with the blank group (P<0.0001). After intervention in different samples, the TNF-α level was downregulated by 7.88% in the Yangshen group (P<0.001), 8.27% in the D-ribose group (P<0.001), and 11.49% in the EGT group (P<0.0001). The D-ribose + EGT group showed the largest downregulation, reaching 39.32% (P<0.0001).

[0059] Experimental results showed that LPS induction brought KGN cells into a typical inflammatory state: IL-1β and TNF-α levels surged to 2.07-fold and 1.93-fold, respectively, compared to the control group, suggesting that LPS induction can rapidly amplify the local inflammatory cascade in the ovary. In single-drug interventions, while Yangshen, D-ribose, and EGT all reduced the two pro-inflammatory factors, the reduction was only 7.88%–13.84%, failing to break through the "low-level inflammation" plateau. When D-ribose was combined with EGT, IL-1β and TNF-α were simultaneously reduced by nearly 40%, significantly superior to each single-drug group (P < 0.0001), and the inflammation level was brought back to near the baseline of the control group.

[0060] Effects of D-ribose, EGT and their combinations on cellular NF-κB levels After sample intervention, 500 μL of Trizol was added to the 12-well plate to extract total RNA. The OD260 / OD280 values ​​were detected. After reverse transcription was completed according to the reverse transcription kit instructions, the cDNA sample was diluted 20 times and the real-time fluorescence quantitative reaction was performed according to the q-PCR kit instructions. The primers are shown in Table 4.

[0061] Table 4 Primer sequences

[0062] NF-κB, as a nucleoprotein factor, regulates a wide range of gene expression. Overactivation induces the massive secretion of pro-inflammatory factors, making it a key target for inflammation activation. Figure 7 The results showed that LPS-induced NF-κB expression levels were significantly increased, reaching 1.97 times that of the control group (P<0.0001). After intervention, the expression levels were downregulated by 8.47% in the *Codonopsis pilosula* group (P<0.05), 15.17% in the D-ribose group (P<0.001), 12.49% in the EGT group (P<0.0001), and 43.92% in the D-ribose + EGT group (P<0.0001).

[0063] Experimental results showed that LPS significantly induced an increase in NF-κB expression levels in cells, confirming the successful establishment of the inflammation model. In single-drug interventions, *Codonopsis pilosula*, D-ribose, and EGT all decreased NF-κB expression, with D-ribose showing the best inhibitory effect, but still not exceeding 20%. In contrast, the combined use of D-ribose and EGT significantly reduced NF-κB expression, not only significantly better than each single-drug group (P < 0.0001), but also almost suppressing the inflammatory signal back to baseline levels. This result further confirms that D-ribose, by enhancing intracellular energy metabolism and reducing equivalent supply, can provide "synergistic fuel" for EGT, jointly blocking NF-κB nuclear translocation and its downstream inflammatory cascade, thereby efficiently reversing LPS-induced inflammatory damage to ovarian granulosa cells.

[0064] Effects of D-ribose, EGT and their combinations on cell viability For experimental procedures, refer to Example 2 (3).

[0065] In an LPS-induced KGN inflammation model, cell damage occurs through disruption of cell structure and interference with metabolic pathways. Assessing cell viability can directly determine the extent of inflammation-induced damage to KGN cell survival. Figure 8 The results showed that the cell viability of the LPS-induced model group was 67.68%, which was 30.15% lower than that of the control group (P<0.0001). After intervention, the cell viability of the *Codonopsis pilosula* group increased by 15.20% (P<0.01), the D-ribose group increased by 19.43% (P<0.001), the EGT group increased by 22.72% (P<0.0001), and the D-ribose + EGT group increased by 40.30% (P<0.0001).

[0066] Experimental results showed that LPS-induced inflammatory response significantly reduced KGN cell viability, indicating the successful establishment of the inflammatory damage model. In individual interventions, *Codonopsis pilosula*, D-ribose, and EGT all restored cell viability to some extent, with EGT showing the best effect alone. However, the combined use of D-ribose and EGT was significantly superior to each single-drug group (P<0.0001), almost restoring cell viability to near-normal levels. This result further confirms that D-ribose and EGT have a significant synergistic effect in anti-inflammation and cell protection, possibly by jointly inhibiting the NF-κB pathway, alleviating inflammatory damage, and enhancing cellular energy metabolism, thereby effectively reversing the LPS-induced decline in cell viability.

[0067] Effects of D-ribose, EGT and their combinations on cellular AMH levels For experimental procedures, refer to Example 2 (1).

[0068] When KGN cells are stimulated by LPS-induced inflammation, inflammatory signals interfere with the metabolic and synthetic pathways of granulocytes. A decrease in AMH levels directly indicates that inflammation has damaged the core functions of granulocytes; conversely, stable AMH levels suggest that inflammation has not significantly affected granulocyte function. Figure 9 The AMH level in the model group was 80.78 ng / mL, which was 50.22% lower than that in the blank group (162.27 ng / mL) (P<0.0001). After intervention, the AMH levels in the *Gynostemma pentaphyllum* group and the D-ribose group were increased by 13.98% and 13.30% respectively (P>0.05), the AMH level in the EGT group was increased by 29.23% (P<0.001), and the AMH level in the D-ribose + EGT group returned to 167.81 ng / mL (P<0.0001), which was basically the same as that in the blank group.

[0069] Experimental results showed that LPS-induced inflammatory stress caused KGN cells to lose almost half of their AMH secretion function. In single-drug intervention, yang-ginseng and D-ribose only brought about a 13% recovery, which was not statistically significant; EGT alone significantly increased AMH levels (P < 0.001). When D-ribose and EGT were used in combination, AMH levels reached 167.81 ng / mL, not only offsetting the damage caused by LPS, but also almost equivalent to the baseline of the control group (162.27 ng / mL). This result confirms that D-ribose, by providing sufficient energy and reducing equivalents, can synergistically inhibit NF-κB inflammatory signaling and reduce oxidative stress, thereby simultaneously restoring the ability of granulosa cells to synthesize and secrete AMH. This three-pronged strategy of "energy-antioxidant-anti-inflammatory" provides new experimental evidence and candidate combination solutions for intervening in clinical problems such as decreased ovarian reserve and reduced AMH caused by chronic inflammation.

[0070] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the present invention.

Claims

1. An ergothioneine composition for enhancing ovarian vitality, characterized in that: The composition includes D-ribose and ergothioneine, which are used to regulate the redox balance of ovarian granulosa cells, inhibit inflammatory responses, and upregulate the secretion of anti-Müllerian hormones to improve ovarian vitality.

2. The ergothioneine composition according to claim 1, characterized in that: The concentration of D-ribose in the composition is 5-15 mM, and the concentration of ergothioneine is 100-300 μM.

3. The method for preparing the ergothioneine composition according to claim 1 or 2, characterized in that: include, D-ribose and ergothioneine were dissolved in serum-free cell culture medium to obtain D-ribose solution and EGT solution; The solution was obtained by aseptic filtration after mixing D-ribose solution and EGT solution.

4. The preparation method according to claim 3, characterized in that: The dissolution process is carried out at room temperature of 20-25℃, with a stirring rate of 50-100 rpm until the solid is completely dissolved; during mixing, the stirring rate is 100-200 rpm and the stirring time is 5-10 min.

5. The use of the ergothioneine composition as described in claim 1 or 2 in downregulating reactive oxygen species levels in ovarian granulosa cells, characterized in that: The application involves applying the ergothioneine composition to KGN cells damaged by oxidative stress to reduce the accumulation of intracellular ROS.

6. The application as described in claim 5, characterized in that: The oxidative stress-damaged KGN cells were constructed by inducing with 300-500 μM hydrogen peroxide for 6 h. After treatment with the ergothioneine composition, the ROS level of the KGN cells was downregulated by ≥50% compared with the model group.

7. The application as described in claim 5, characterized in that: The application involves activating the antioxidant pathway of KGN cells and restoring the function of granulocytes in secreting AMH through the composition.

8. The application as described in claim 5, characterized in that: Compared to the oxidative stress model group, the Nrf2 level of KGN cells was upregulated by ≥50% and the AMH level was upregulated by ≥80% after treatment with the composition.

9. The use of the ergothioneine composition as described in claim 1 or 2 in downregulating the levels of inflammatory factors and nuclear factor κB in ovarian granulosa cells, characterized in that: The inflammatory factors include interleukin-1β and tumor necrosis factor-α, and the application is to inhibit the inflammatory signaling pathway of KGN cells through the composition.

10. The application as described in claim 9, characterized in that: The inflammatory damage of the KGN cells was constructed by inducing 40-60 ng / mL lipopolysaccharide for 24 h. After treatment with the composition, the levels of IL-1β and TNF-α in KGN cells were downregulated by ≥35% compared with the model group, and the level of NF-κB was downregulated by ≥40% compared with the model group.

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