Use of ginsenoside Rg4 as a sole active ingredient in the preparation of a drug for improving male delayed hypogonadism

Abstract

The application of ginsenoside Rg4 as the sole active ingredient in the preparation of drugs to improve late-onset hypogonadism (LOH) in men belongs to the field of biomedicine. This invention clarifies for the first time the mechanism by which Rg4 improves LOH: Rg4 can specifically upregulate the mRNA expression levels of StAR and StARD7 in testicular tissue, promoting cholesterol transport to the inner mitochondrial membrane of Leydig cells at the transcriptional level; Rg4 enhances the mRNA expression levels of P450scc and 3β-HSD in testicular tissue by promoting efficient cholesterol transport, thereby enhancing the conversion of cholesterol to pregnenolone and subsequent steroid synthesis, and improving endogenous testosterone synthesis; Rg4 can restore the negative feedback regulatory function of the HPG axis, regulating the secretion of pituitary LH and FSH to physiological homeostasis and avoiding abnormal fluctuations in hormone levels. This invention fills the technological gap in the field of Rg4 gene regulation of LOH.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of ginsenoside Rg4 as the sole active ingredient in the preparation of a drug to improve male late-onset hypogonadism. Background Technology

[0002] Late-onset hypogonadism (LOH) is a syndrome in middle-aged and elderly men caused by the decline of Leydig cell function in the testes. The core pathology involves reduced endogenous testosterone synthesis and dysfunction of the hypothalamus-pituitary-gonadal (HPG) axis. The mRNA expression levels of key genes involved in testosterone synthesis in Leydig cells are significantly downregulated. Clinical manifestations include decreased sexual function and metabolic disorders, severely impacting quality of life. Currently, clinical treatment primarily relies on testosterone replacement therapy. While it can rapidly increase endogenous testosterone synthesis, it has side effects such as benign prostatic hyperplasia and liver damage, and is contraindicated in some patients. Existing non-hormonal treatments are mostly adjunctive, generally suffering from unclear target points, unstable efficacy, and ambiguous mechanisms, making it difficult to meet the needs for safe clinical treatment.

[0003] Acute regulatory steroid protein (StAR), StAR-associated lipid transfer domain 7 (StARD7), cytochrome P450 cholesterol side chain lyase (P450scc / CYP11A1), and 3β-hydroxysteroid dehydrogenase (3β-HSD) are core genes for testosterone synthesis in Leydig cells of the testis. Downregulation of their mRNA expression levels is an important molecular mechanism in the development of late-onset hypogonadism in men. These core genes for testosterone synthesis in Leydig cells directly regulate cholesterol transport and conversion, and are the core links in the testosterone biosynthesis pathway.

[0004] Currently, no research has linked ginsenoside Rg4 to the core genes involved in testosterone synthesis in Leydig cells of the testes. Furthermore, no research has disclosed the role and molecular mechanism by which ginsenoside Rg4 improves male late-onset hypogonadism by regulating the transcriptional level of the core genes involved in testosterone synthesis in Leydig cells. Moreover, existing mechanism verification methods mostly rely on complex protein hybridization techniques, which are cumbersome and costly, hindering quality control and batch testing in subsequent product development.

[0005] In summary, clarifying the role and molecular mechanism of ginsenoside Rg4 in improving male late-onset hypogonadism by regulating the transcriptional level of key genes involved in testosterone synthesis in Leydig cells has significant practical implications for achieving non-hormonal targeted regulation of male late-onset hypogonadism. Summary of the Invention

[0006] To address the problems of significant side effects, unclear target of non-hormonal regulation, unstable efficacy, and ambiguous mechanism in existing treatments for late-onset hypogonadism (LOH), this invention provides the application of ginsenoside Rg4 as the sole active ingredient in the preparation of drugs to improve late-onset hypogonadism in men.

[0007] The technical solution adopted by this invention to solve the technical problem is as follows:

[0008] This invention provides the application of ginsenoside Rg4 as the sole active ingredient in the preparation of a drug to improve male late-onset hypogonadism.

[0009] In a preferred embodiment, the functions of ginsenoside Rg4 include:

[0010] (1) Increase serum levels of total and free testosterone;

[0011] (2) Inhibits abnormally elevated levels of luteinizing hormone and follicle-stimulating hormone in serum;

[0012] (3) Targeting the transcriptional level of key genes that regulate testosterone synthesis in Leydig cells of the testis;

[0013] (4) Promotes cholesterol transport and conversion;

[0014] (5) Promotes endogenous testosterone synthesis;

[0015] (6) Regulate the hormone secretion homeostasis of the HPG axis.

[0016] As a preferred embodiment, the preparation method of ginsenoside Rg4 is as follows:

[0017] (1) Preparation of Pediococcus lactis TH903 bacterial suspension;

[0018] After activation, P. ts. TH903 lactic acid bacteria was inoculated into liquid MRS medium, allowed to stand, centrifuged and the bacterial precipitate was collected. After suspension with sterile distilled water and adjustment of viable count, a P. ts. TH903 lactic acid bacteria suspension was obtained.

[0019] (2) Preparation of ginseng berry extract;

[0020] Weigh out ginseng berries, add deionized water, extract by hot reflux, filter, combine the filtrates, and concentrate under reduced pressure to obtain ginseng berry extract;

[0021] (3) Fermentation and conversion;

[0022] Ginseng berry extract was added to glucose, and hydrochloric acid was added to adjust the pH. Then, a suspension of Pietrococcus lactis TH903 was inoculated and cultured statically to obtain the fermentation product.

[0023] (4) The obtained fermentation product is concentrated under reduced pressure and freeze-dried under vacuum to obtain dry powder;

[0024] (5) The ginsenoside Rg4 contained in the dry powder was identified by high performance liquid chromatography.

[0025] As a preferred embodiment, the *Pediococcus lactis* TH903 was deposited at the China Center for Type Culture Collection on September 29, 2025, with accession number CCTCC NO: M20252155.

[0026] The beneficial effects of this invention are:

[0027] 1. This invention clarifies for the first time the core mechanism by which ginsenoside Rg4 promotes endogenous testosterone synthesis by regulating the core genes StAR, StARD7, P450scc, and 3β-HSD in Leydig cells of the testis at the transcriptional level. It targets the testosterone biosynthesis pathway specifically and directly, filling the technological gap in the field of gene regulation of male late-onset hypogonadism with ginsenoside Rg4.

[0028] 2. This invention utilizes ginsenoside Rg4 to regulate the transcriptional level of key genes involved in testosterone synthesis in Leydig cells of the testis, promoting endogenous testosterone synthesis without hormonal side effects. Animal experiments have verified that it does not damage liver and kidney function or the prostate. It can also synergistically regulate the HPG axis and the testicular tissue microenvironment, improving the symptoms of late-onset hypogonadism in men from multiple dimensions. The therapeutic effect is stable and long-lasting, making it suitable for long-term conditioning in middle-aged and elderly men.

[0029] 3. The entire process of this invention uses RT-qPCR technology to verify the mechanism of action. The operation is simple and highly reproducible, which is conducive to product development and quality control. Furthermore, the experiment uses a D-galactose-induced aging-related LOH animal model, which can accurately simulate the pathological characteristics of hypogonadism in middle-aged and elderly men. The model is stable, the results are reliable, and the clinical translation value is high.

[0030] 4. This invention prepares ginsenoside Rg4 through a one-step fermentation process, which has the advantages of being simple, low-cost, and using readily available raw materials. The prepared ginsenoside Rg4 has high purity, no chemical residues, and excellent safety. Attached Figure Description

[0031] Figure 1 shows the HPLC chromatograms of ginseng berry extract before and after fermentation with Pediococcus lactis TH903 in Example 1.

[0032] Figure 2 shows the effect of ginsenoside Rg4 on serum hormone levels in rats with D-galactose-induced aging-related LOH animal model in Example 2.

[0033] Figure 3 shows the effect of ginsenoside Rg4 on the expression of StAR, StARD7, P450scc and 3β-HSD gene mRNA in the testes of rats in the D-galactose-induced aging-related LOH animal model, as described in Example 2. Detailed Implementation

[0034] This invention demonstrates through experiments that ginsenoside Rg4 can increase serum total testosterone (TT) and free testosterone (FT) levels in middle-aged and elderly men, inhibit abnormal increases in serum luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels, regulate HPG axis homeostasis, and improve the core symptoms of late-onset hypogonadism in men, including decreased sexual function, abnormal mental and emotional state, metabolic disorders, and accompanying symptoms such as thinning hair and dry skin.

[0035] According to this invention, the core mechanism by which ginsenoside Rg4 improves D-galactose-induced late-onset hypogonadism in men is as follows: Ginsenoside Rg4 targets and regulates the transcriptional level of key genes involved in testosterone synthesis in Leydig cells of the testis, significantly upregulates the mRNA expression level of these key genes, promotes cholesterol transport and conversion, thereby enhancing endogenous testosterone synthesis, and simultaneously synergistically regulates the hormone homeostasis of the HPG axis.

[0036] This invention uses RT-qPCR technology to detect the relative mRNA expression levels of StAR, StARD7, P450scc, and 3β-HSD genes to verify their mechanisms of action. The specific mechanisms of action are as follows:

[0037] (1) Upregulation of transcriptional levels of key genes involved in testosterone synthesis in Leydig cells: Ginsenoside Rg4 specifically upregulates the relative mRNA expression levels of StAR and StARD7 in testicular tissue, promoting the transport of cholesterol to the inner mitochondrial membrane of Leydig cells at the transcriptional level, thus laying the material transport foundation for testosterone synthesis. This step is the rate-limiting step in testosterone biosynthesis. By promoting efficient cholesterol transport, it further significantly increases the relative mRNA expression levels of P450scc and 3β-HSD in testicular tissue, enhancing the conversion of cholesterol to pregnenolone and the subsequent catalytic synthesis of steroids, directly improving the endogenous testosterone synthesis capacity of Leydig cells.

[0038] (2) Synergistic effect of gene transcription regulation and HPG axis function repair: On the basis of significantly upregulating the mRNA expression of the core key gene for testosterone synthesis in Leydig cells of the testis and promoting endogenous testosterone synthesis, ginsenoside Rg4 can restore the negative feedback regulatory function of the HPG axis, regulate the secretion of pituitary LH and FSH to physiological homeostasis, and avoid abnormal fluctuations in hormone levels; at the same time, it improves the oxidative stress state of testicular tissue, protects the structural integrity of testicular Leydig cells and seminiferous tubules, and forms a multi-dimensional synergistic regulatory system of gene transcription activation-testosterone synthesis promotion-HPG axis function repair-testicular tissue protection, so as to achieve safe and long-term conditioning and improvement of male late-onset hypogonadism.

[0039] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0040] Unless otherwise specified, the experimental methods used in the following examples are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0041] The Pediococcus acidilactici TH903 used in Example 1 was deposited on September 29, 2025, at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province (Wuhan University Collection Center), with accession number CCTCC NO: M20252155.

[0042] In addition, other information about Pediococcus acidilactici TH903, including isolation, identification, and preservation, can be found in Chinese Patent No. CN121182704A (Invention title: A strain of Pediococcus acidilactici TH903 and its use in the directional preparation of rare ginsenoside Rg4 from fermented ginseng berries).

[0043] Example 1: Directed fermentation preparation process of ginsenoside Rg4

[0044] The directional fermentation preparation of ginsenoside Rg4 was carried out according to the preparation method disclosed in Chinese Patent No. CN121182704A (Invention title: A strain of *Pediococcus lactis* TH903 and its use in the directional preparation of rare ginsenoside Rg4 from fermented ginseng berries). The details are as follows:

[0045] 1. Preparation of Pediococcus lactis TH903 bacterial suspension;

[0046] Lyophilized *Pediococcus lactis* TH903 was streaked onto M17 agar plates and incubated at 30 °C for 24 h to activate it. A single colony was picked and inoculated into 50 mL of M17 liquid medium and incubated at 30 °C for 12 h to obtain primary seed culture. This seed culture was then transferred at a 2% (v / v) inoculum to 300 mL of M17-lactose liquid medium and incubated at 30 °C for 16 h until the viable count reached 1.0 × 10⁻⁶. 10 CFU·mL -1 Centrifuge at 4 ℃ and 7000 rpm for 8 min to collect the bacterial pellet. Wash twice with sterile 0.85% NaCl, then resuspend in an equal volume of sterile physiological saline and adjust the viable count to 1.0 × 10⁻⁶. 9 ~1.0×10 10 CFU·mL -1 This is a suspension of *Pediococcus lactis* TH903, stored at 4 ℃ for later use.

[0047] 2. Preparation of ginseng berry extract;

[0048] Weigh 500g of fresh ginseng berries and add deionized water at a ratio of 1:15 (w / v). Extract twice by hot reflux at 100 ℃ for 1.5 h each time. After filtration, combine the filtrates and concentrate under reduced pressure to 5000mL to obtain ginseng berry extract.

[0049] 3. Fermentation and conversion;

[0050] Glucose and yeast extract were added to the above-mentioned ginseng berry extract as a liquid fermentation medium (3 g / L glucose, 2 g / L yeast extract). After sterilization by filtration through 0.45 μm micropores, the medium was sterilized at 121 °C for 15 min. After cooling to room temperature, a suspension of *Pediococcus lactis* TH903 was inoculated at a rate of 3% (v / v) and cultured statically at 37 °C for 14 days to obtain the fermentation product. After fermentation, the obtained fermentation product was freeze-dried under vacuum (-80 °C, 5 Pa) to obtain a dry powder.

[0051] 4. Identification of rare ginsenosides contained in the dry powder;

[0052] (1) Sample preparation;

[0053] Accurately weigh 5.0 mg each of ginsenoside Re and Rg4 standards (purchased from Shanghai Yuanye Biotechnology Co., Ltd., purity ≥98%) and the dry powder obtained in step 3 of Example 2. At the same time, use the uninoculated ginseng berry extract obtained in step 2 of Example 2 as a blank control. Dissolve in chromatographic grade methanol by sonication (40 kHz, 25 ℃, 10 min) and make up to 5 mL. Filter through a 0.22 µm organic microporous membrane and keep the filtrate for later use.

[0054] (2) HPLC chromatographic conditions;

[0055] Instrument: Agilent 1260 Infinity II high performance liquid chromatograph with ultraviolet detector (UV).

[0056] Column: Agilent Pursuit 5 SB-C18 (4.6 × 250 mm, 5 µm).

[0057] Column temperature: 30 ℃; flow rate: 1.0 mL / min; detection wavelength: 203 nm; injection volume: 20 µL.

[0058] Mobile phase: Phase A is water, Phase B is acetonitrile; Gradient elution program: 0~40 min 18~21% (B); 40~42 min 21~26% (B); 42~46 min 26~32% (B); 46~66 min 32~34% (B); 66~71 min 34~38% (B); 71~77.70 min 38.0~49.1% (B); 77.70~82 min 49.1% (B); 82~83 min 49.1~50.6% (B); 83~88 min 50.6~59.6% (B); 88~89.80 min 59.6~65.0% (B); 89.80~97 min 65% (B); 97~102 min 65~75%(B); 102~110min 75~85%(B); 110~115min 85%(B); 115~125min 85~18%(B); 125~130min 18.0%(B).

[0059] (3) Result determination;

[0060] High-performance liquid chromatography (HPLC) analysis results are as follows Figure 1As shown, the retention times of the reference standards were as follows: ginsenoside Re was 36.76 min, and the retention time of rare ginsenoside Rg4 standard was 78.07 min. The dry powder obtained in Example 2 showed a single main peak at 78.08 min, consistent with the retention time of rare ginsenoside Rg4 standard (deviation < 0.2 min), with a peak purity factor > 990. The blank control (uninoculated ginseng berry extract) showed no significant absorption peak in the range of 75-80 min. The above results indicate that the present invention successfully prepared rare ginsenoside Rg4 by directionally converting ginseng berry extract into rare ginsenoside Rg4 through suspension fermentation of ginseng berry extract by Pediococcus acidilactici TH903. Furthermore, the content of rare ginsenoside Rg4 was calculated to be 97.16 mg / g using the standard curve.

[0061] In this embodiment, the biotransformation synthesis route of rare ginsenoside Rg4 is as follows: ginsenoside Re in ginseng berry extract is hydrolyzed by Pediococcus acidilactici TH903, and after dehydration to form a double bond, it is directionally converted into rare ginsenoside Rg4.

[0062] Example 2: Animal functional evaluation of ginsenoside Rg4 in improving male late-onset hypogonadism

[0063] 1. Experimental process;

[0064] Forty 12-month-old male SD rats, weighing 300g-350g, were randomly divided into a blank control group, a model group, a low-dose ginsenoside Rg4 group (25mg / kg / d), and a high-dose ginsenoside Rg4 group (50mg / kg / d), with 10 rats in each group. They were acclimatized for one week, kept in an environment with a temperature of 22±2℃, humidity of 50±5%, a 12h light / dark cycle, and free access to food and water. Except for the blank control group, the other three groups of rats were subcutaneously injected with D-galactose (120 mg / kg / d) once a day for 8 consecutive weeks to establish an aging-related LOH animal model. The blank control group was injected with an equal volume of physiological saline. After 8 weeks of modeling, blood was randomly collected from the orbital sinus of 3 rats in each group. The serum total testosterone (TT) and free testosterone (FT) levels in the model group were significantly lower than those in the blank control group (P < 0.01), and the model was considered to be successful when typical LOH-like manifestations such as lethargy, decreased libido, and increased body fat appeared.

[0065] After administration, rats were fasted for 12 hours, and blood was collected from the orbital cavity. Serum was separated by centrifugation at 3000 r / min for 10 min. The levels of total testosterone (TT), free testosterone (FT), luteinizing hormone (LH), follicle-stimulating hormone (FSH), and prostate-specific antigen (PSA) in serum were detected by ELISA. After euthanizing the rats, testicular tissue was quickly separated and stored at -80℃ for subsequent gene detection.

[0066] Table 1 Primers used for quantitative reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR)

[0067]

[0068] The relative mRNA expression levels of key genes involved in testosterone synthesis in Leydig cells of the testis were detected using RT-qPCR transcriptional analysis. The genes detected were StAR, StARD7, P450scc, and 3β-HSD, with GAPDH as the internal reference gene. Primers were designed and synthesized by a professional biotechnology company. Amplification was performed using qRT-qPCR on a real-time PCR instrument, and the relative mRNA expression levels of the genes were calculated using the 2^(-ΔΔCt) method.

[0069] 2. Experimental results;

[0070] (1) Serum hormone level test results;

[0071] like Figure 2 As shown, the serum total testosterone (TT) and free testosterone (FT) levels in the model group rats were significantly lower than those in the blank control group (P < 0.01), while the serum LH and FSH levels were significantly increased (P < 0.01). The low-dose and high-dose ginsenoside Rg4 groups significantly increased serum total testosterone (TT) and free testosterone (FT) levels and decreased serum luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels to physiological homeostasis (P < 0.01). The improvement in hormone levels was significantly positively correlated with the relative mRNA expression of key genes involved in testosterone synthesis in Leydig cells of the testes. There was no significant difference in prostate-specific antigen (PSA) levels among the groups (P > 0.05), indicating that ginsenoside Rg4 has no adverse effects on prostate function and has excellent safety.

[0072] (2) Results of RT-qPCR transcriptional level analysis;

[0073] like Figure 3As shown, the relative mRNA expression levels of StAR, StARD7, P450scc, and 3β-HSD in the testes of rats in the model group were significantly lower than those in the blank control group (P < 0.01), indicating that in the D-galactose-induced aging-related LOH animal model, the transcriptional level of key genes for testosterone synthesis in Leydig cells of the testes was significantly downregulated, which is the core reason for the reduction in endogenous testosterone synthesis. The low-dose and high-dose groups of ginsenoside Rg4 significantly upregulated the relative mRNA expression levels of key genes for testosterone synthesis in Leydig cells of the testes (P < 0.01), and the upregulation effect of the high-dose group of ginsenoside Rg4 was more significant, with no significant difference compared to the blank control group (P > 0.05). This confirms that ginsenoside Rg4 can directly activate the testosterone biosynthesis pathway in Leydig cells of the testes at the transcriptional level, promote cholesterol transport and catalytic conversion, and achieve a highly efficient increase in endogenous testosterone synthesis. This is the core molecular mechanism by which it improves male late-onset hypogonadism.

[0074] Based on a D-galactose-induced aging-related hypogonadism (LOH) animal model, this invention is the first to demonstrate that ginsenoside Rg4, prepared through directed fermentation according to this invention, can specifically upregulate the relative mRNA expression levels of key genes involved in testosterone synthesis, such as StAR, StARD7, P450scc, and 3β-HSD, in Leydig cells of the testis. This directly activates the testosterone biosynthesis pathway, promotes endogenous testosterone synthesis, and restores hormone homeostasis along the HPG axis, significantly improving the core symptoms of late-onset hypogonadism in men without significant toxic side effects. Furthermore, this invention utilizes RT-qPCR technology for molecular mechanism verification, which is simple to operate and yields reliable results.

[0075] This invention provides complete and scientifically sound experimental evidence for the development of products that improve male late-onset hypogonadism using ginsenoside Rg4, and also provides new candidate active ingredients and targets for non-hormonal targeted conditioning of male late-onset hypogonadism.

[0076] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. However, these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of ginsenoside Rg4 as the sole active ingredient in the preparation of drugs to improve male late-onset hypogonadism.

2. The use of ginsenoside Rg4 as the only active ingredient in the preparation of a drug for improving male late-onset hypogonadism according to claim 1, characterized in that, The functions of ginsenoside Rg4 include: (1) Increase serum levels of total and free testosterone; (2) Inhibits abnormally elevated levels of luteinizing hormone and follicle-stimulating hormone in serum; (3) Targeting the transcriptional level of key genes that regulate testosterone synthesis in Leydig cells of the testis; (4) Promotes cholesterol transport and conversion; (5) Promotes endogenous testosterone synthesis; (6) Regulate the hormone secretion homeostasis of the HPG axis.

3. The application of ginsenoside Rg4 as the sole active ingredient according to claim 1 in the preparation of a drug for improving late-onset male hypogonadism, characterized in that, The preparation method of the ginsenoside Rg4 is as follows: (1) Preparation of Pediococcus lactis TH903 bacterial suspension; After activation, P. ts. TH903 lactic acid bacteria was inoculated into liquid MRS medium, allowed to stand, centrifuged and the bacterial precipitate was collected. After suspension with sterile distilled water and adjustment of viable count, a P. ts. TH903 lactic acid bacteria suspension was obtained. (2) Preparation of ginseng berry extract; Weigh out ginseng berries, add deionized water, extract by hot reflux, filter, combine the filtrates, and concentrate under reduced pressure to obtain ginseng berry extract; (3) Fermentation and conversion; Ginseng berry extract was added to glucose, and hydrochloric acid was added to adjust the pH. Then, a suspension of Pietrococcus lactis TH903 was inoculated and cultured statically to obtain the fermentation product. (4) The obtained fermentation product is concentrated under reduced pressure and freeze-dried under vacuum to obtain dry powder; (5) The ginsenoside Rg4 contained in the dry powder was identified by high performance liquid chromatography.

4. The application of ginsenoside Rg4 as the sole active ingredient according to claim 3 in the preparation of a drug for improving late-onset male hypogonadism, characterized in that, The *Pediococcus lactis* TH903 was deposited at the China Center for Type Culture Collection (CCTCC) on September 29, 2025, with accession number CCTCC NO: M20252155.

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