Podospora leucotricha and its use in transforming rare ginsenosides

Abstract

The present application relates to the technical field of fermentation, in particular to irpex lacteus and its application in the conversion of rare ginsenosides. The present application discovers and cultivates a wild irpex lacteus which can convert rare ginsenosides in panax notoginseng, further optimizes the irpex lacteus-panax notoginseng bidirectional solid fermentation process through experiments, and confirms that the process can efficiently convert common ginsenosides in panax notoginseng into rare ginsenosides; through pharmacodynamic evaluation, it is further shown that fermented panax notoginseng can significantly improve the pathological state of lupus nephritis model mice through multiple pathways such as immune regulation, inflammation inhibition and anti-kidney fibrosis, and its overall curative effect is better than that of panax notoginseng. The present application not only provides a new technical path for the deep development and value improvement of panax notoginseng, but also provides a potential candidate drug for the treatment of lupus nephritis, and embodies the wisdom of traditional Chinese medicine processing "synergistic and detoxification".

Description

Technical Field

[0001] This invention relates to the field of fermentation technology, specifically to *Gynostemma pentaphyllum* and its application in the conversion of rare ginsenosides. Background Technology

[0002] Panax notoginseng (Burk.) FH Chen is a plant of the Araliaceae family. Its dried roots and rhizomes are used medicinally, possessing properties such as dispersing blood stasis, stopping bleeding, reducing swelling, and relieving pain. Studies have shown that saponins in Panax notoginseng can reduce glomerular filtration rate and proteinuria levels, thereby improving renal function in patients with nephritis. Saponins are one of the main active ingredients in Panax notoginseng roots. The three most abundant saponins are notoginsenoside R1, ginsenoside Re, and ginsenoside Rg1. These ginsenosides have poor bioavailability due to their large molecular weight and low cell membrane permeability, making them difficult for the human body to absorb. Deglycosylated ginsenosides, such as ginsenoside Rg3, derived from these ginsenosides, are more easily absorbed by the body. However, the content of rare saponins in Panax notoginseng is extremely low. Increasing the content of rare saponins and achieving the enrichment and extraction of the effective components of Panax notoginseng has always been a research hotspot. Therefore, it is urgent to develop a method for converting the original ginsenosides in Panax notoginseng into rare saponins and other bioactive components.

[0003] *Panax gracilistylus*, also known as white bud fungus or white bud fungus, belongs to the phylum Basidiomycota, class Agaricales, order Polyporaceae, family Porciniaceae, and genus *Panax gracilistylus*. It can be used to treat symptoms such as oliguria, edema, lower back pain, and high blood pressure, and also possesses anti-inflammatory activity. Currently, there are many studies on the structural transformation methods of saponins in Panax notoginseng roots, including acid hydrolysis and microwave-assisted degradation. However, these methods have low specificity, involve violent reactions, and cause significant environmental pollution, which is detrimental to industrial production. The core advantage of fungal fermentation of Panax notoginseng for saponin transformation lies in its ability to utilize the rich enzyme system of fungi, acting like a "biological scissors," to gently and directionally and efficiently transform the original saponins into rarer saponins (such as Rh1 and Rg3) that are more active, more water-soluble, and more easily absorbed by the human body. Simultaneously, this process is environmentally friendly and can produce complex active ingredients such as saponins and polysaccharides, achieving synergistic effects. This is a green and efficient strategy for enhancing the medicinal value of Panax notoginseng and developing high-value-added products. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide *Panax quinquefolius* and its application in the conversion of rare ginsenosides. This *Panax quinquefolius* can efficiently convert rare ginsenosides from Panax notoginseng.

[0005] The present invention provides *Tetranychus leucopus*, characterized in that its preservation number is CGMCC No. 42468.

[0006] This invention provides a method for culturing the aforementioned white rake tooth fungus, comprising the following steps:

[0007] Step 1: Inoculate the *Bacillus thunbergii* onto a slant culture medium for slant culture;

[0008] Step 2: Inoculate the white rake tooth fungus cultured on the slant culture medium and culture it.

[0009] In some embodiments, the slant culture medium is PDA solid culture medium, and the slant culture temperature is 24-28°C, and the time is 7-10 days.

[0010] In some embodiments, the liquid culture medium consists of 20-30g glucose, 10-20g yeast extract, 1-2g potassium dihydrogen phosphate, 1-2g magnesium sulfate, 1-2 vitamin B1 tablets and 800-1000mL distilled water. The culture temperature is 24-28℃, the rotation speed is 160-200rpm / min, and the culture time is 7-10 days.

[0011] This invention provides the application of the aforementioned *Gynostemma pentaphyllum* or *Gynostemma pentaphyllum* cultured by the aforementioned method in the conversion of rare ginsenosides.

[0012] In some embodiments, the rare ginsenosides include at least one of Rh1, Rg3, and Rg5.

[0013] This invention provides a two-way solid-state fermentation method for *Panax notoginseng*-*Bacillus thunbergii*, comprising the following steps:

[0014] S1. Mix Panax notoginseng powder with water, sterilize, and obtain Panax notoginseng fermentation substrate;

[0015] S2. Take the bacterial solution of the *Bacillus thunbergii* or the *Bacillus thunbergii* cultured by the above-mentioned culture method and inoculate it into the *Panax notoginseng* fermentation substrate for fermentation culture.

[0016] In some embodiments, in step S1, the particle size of the Panax notoginseng powder is 40-100 mesh, and the mass-to-volume ratio of the Panax notoginseng powder to water is 1g:(0.7-1)mL.

[0017] In some specific embodiments, the particle size of the Panax notoginseng powder is 60 mesh, and the mass-to-volume ratio of the Panax notoginseng powder to water is 1g:0.9mL.

[0018] In some embodiments, in step S2, the volume-to-mass ratio of the bacterial solution of *Bacillus thunbergii* to the fermentation substrate of *Panax notoginseng* is (0.4~0.8) mL:1g;

[0019] The fermentation culture is carried out at a temperature of 20-26°C for 8-14 days.

[0020] In some specific embodiments, the volume-to-mass ratio of the bacterial solution of *Bacillus thunbergii* to the fermentation substrate of *Panax notoginseng* is 0.6 mL: 1 g.

[0021] This invention provides a method for converting ginsenosides Rh1, Rg3 and / or Rg5, comprising the following steps:

[0022] Take Panax notoginseng powder and mix it with water, sterilize it, and obtain Panax notoginseng fermentation substrate;

[0023] The bacterial culture of *Bacillus thunbergii* or *Bacillus thunbergii* obtained by the culture method is inoculated into the *Panax notoginseng* fermentation substrate, and fermentation culture is carried out. After purification of saponin components according to the pharmacopoeia method, Rh1, Rd, Rg3 and / or Rg5 are obtained.

[0024] This invention provides the use of the product obtained by the fermentation method or the product obtained by the transformation method in the preparation of a medicament for the prevention and / or treatment of lupus nephritis.

[0025] The present invention provides a medicine comprising a product obtained by the fermentation method or a product obtained by the transformation method and pharmaceutically acceptable excipients.

[0026] This invention provides a method for preparing lupus nephritis, comprising administering the drug.

[0027] This invention provides *Gynostemma pentaphyllum* and its application in the transformation of rare ginsenosides. The preservation number of *Gynostemma pentaphyllum* is CGMCC No. 42468. This invention has the following beneficial effects:

[0028] This invention discovered and cultivated a wild-type *Pteris vittata* strain capable of converting rare ginsenosides from Panax notoginseng. Further experiments optimized the two-way solid-state fermentation process of *Pteris vittata*-Panax notoginseng, demonstrating that this process can efficiently convert ordinary ginsenosides from Panax notoginseng into rare ginsenosides. Pharmacological evaluation further showed that fermented Panax notoginseng significantly improved the pathological state of a mouse model of lupus nephritis through multiple pathways, including immune regulation, inflammation suppression, and anti-renal fibrosis, with overall efficacy superior to that of raw Panax notoginseng. This invention not only provides a feasible new technological path for the in-depth development and value enhancement of Panax notoginseng but also offers a potential candidate drug for the treatment of lupus nephritis, embodying the wisdom of "enhancing efficacy and reducing toxicity" in traditional Chinese medicine processing.

[0029] Biological Preservation Instructions

[0030] Biological material: CZ-2014; Classification and name: Irpex lacteus, deposited on January 8, 2026 at the China General Microbiological Culture Collection Center; Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing; Accession number: CGMCC No. 42468. Attached Figure Description

[0031] Figure 1 The changes in the content of saponins in Panax notoginseng before and after fermentation were detected by HPLC. Among them, 1. Panax notoginsenoside R1, 2. Ginsenoside Rg1, 3. Ginsenoside Rb1, 4. 20(S)-Ginsenoside Rh1, 5. Ginsenoside Rd, 6. 20(S)-Ginsenoside Rg3, and 7. Ginsenoside Rg5 were identified.

[0032] Figure 2 This example illustrates the effects of particle size, water content, inoculum quantity, and initial pH value on the overall score of fermented Panax notoginseng in Example 2.

[0033] Figure 3 The response surface plots of the interactions of various factors in Example 3 are shown.

[0034] Figure 4 The image shows the morphological observation of the spleens of mice in each group in Example 4. From left to right, the groups are K, M, Y, S, FSD, and FSG. The image includes: blank group (K); MRL / lpr mice, as a model animal of lupus nephritis (LN), were randomly divided into model group (M), positive drug group (Y), raw Panax notoginseng group (S), low-dose fermented Panax notoginseng group (FSD), and high-dose fermented Panax notoginseng group (FSG).

[0035] Figure 5 The results of HE staining of mouse kidney tissue from each group in Example 4 are shown.

[0036] Figure 6 The results of Masson staining of mouse kidney tissue in each group in Example 4 are shown.

[0037] Figure 7 The ELISA results of ANA, ds-DNA-Ab, Cr, BUN, TNF-α, and IL-6 in the serum of mice in Example 4 are shown. Compared with the blank group, # P < 0.05 ## P < 0.01; compared with the model group, * P < 0.05 ** P < 0.01;

[0038] Figure 8The expression results of key proteins in the PI3K / AKT / NF-κB signaling pathway in the kidney tissues of mice in Example 4, where, compared with the blank group, * P < 0.05 ** P < 0.01; compared with the model group, # P < 0.05 ## P < 0.01. Detailed Implementation

[0039] This invention provides *Gynostemma pentaphyllum* and its application in the conversion of rare ginsenosides. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0040] The Panax notoginseng medicinal material (origin: Yunnan, batch number: 23081001) was purchased from Hebei Kangyiqiang Pharmaceutical Co., Ltd., and identified by Professor Wang Shumin of Changchun University of Traditional Chinese Medicine as Panax notoginseng (Burk.) FH Chen, a plant of the Araliaceae family. The dried roots of the plant were used; prednisolone acetate (batch number: H12020689) was purchased from Tianjin Jinyao Group Co., Ltd.; ginsenoside R1 (batch number: DSTDS005003), ginsenoside Rg1 (batch number: DSTDR000902), ginsenoside Rb1 (batch number: DSTDR000603), 20(S)-ginsenoside Rh1 (batch number: DST221109-035), ginsenoside Rd (batch number: DSTDR001502), 20(S)-ginsenoside Rg3 (batch number: DSTDR001101), and ginsenoside Rg5 (batch number: DSTDR003202) were all purchased from Chengdu Desite Biotechnology Co., Ltd.; D-anhydrous glucose reference standard (batch number: 20230221) was purchased from Tianjin Guangfu Fine Chemicals Research Institute; ELISA... The test kits for mouse antinuclear antibody (ANA), mouse anti-double-stranded DNA antibody (ds-DNA-Ab), mouse creatinine (Cr), mouse blood urea nitrogen (BUN), mouse interleukin-6 (IL-6), and mouse tumor necrosis factor-α (TNF-α) were all purchased from Jiangsu Enzyme Immunoassay Co., Ltd., and the batch number of each kit was 202504.

[0041] The test materials used in this invention are all commercially available products. The invention will be further illustrated below with reference to specific embodiments.

[0042] Example 1: Species identification of *Tetranychus spp.*

[0043] The *Ipex Lacteus* strain provided in this invention was provided by the Medicinal Fungi Research Laboratory of Changchun University of Traditional Chinese Medicine and identified as *Ipex Lacteus* by Shanghai Sangon Biotech Co., Ltd. The identification process included:

[0044] 1. Sample preparation

[0045] The *Bacillus thunbergii* strain was provided by the Laboratory of Medicinal Fungi at Changchun University of Traditional Chinese Medicine as material for DNA extraction.

[0046] 2. DNA extraction

[0047] Use the Ezup column-based fungal genomic DNA extraction kit to extract genomic DNA from mycelia or spores, following the instructions.

[0048] 3. DNA quality testing

[0049] The extracted DNA was analyzed by 1.5% agarose gel electrophoresis to confirm its integrity and concentration.

[0050] 4. PCR amplification

[0051] PCR amplification was performed using universal primers for the fungal ribosomal ITS region: ITS1: TCCGTAGGTGAACCTGCGG (SEQ ID NO:1) and ITS4-R: TCCCCGCTTATTGATATGC (SEQ ID NO:2). The total reaction volume was 25 µL, containing template DNA, primers, Taq Plus DNA polymerase, etc. The PCR reaction conditions were: 95℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 57℃ annealing for 30 s, and 72℃ extension for 90 s, for a total of 30 cycles; and a final extension at 72℃ for 10 min.

[0052] 5. Detection of amplification products

[0053] The PCR product was verified by 1.5% agarose gel electrophoresis, confirming that the amplified fragment size was approximately 650 bp.

[0054] 6. Sequencing and Sequence Analysis

[0055] The amplified products were purified and sent to a sequencing company for bidirectional sequencing to obtain the ITS sequence.

[0056] (SEQ ID NO:3)

[0057] The obtained sequences were BLAST-aligned in the NCBI database.

[0058] 7. Result Comparison and Identification

[0059] The ITS sequences of all samples were 648 bp in length, and showed 99.85%–100% similarity to multiple strains of Irpex lacteus in the database, with an E value of 0.0 and a coverage rate of 100%. Based on the sequence alignment results, all strains were identified as Irpex lacteus.

[0060] 8. Conclusion

[0061] Through ITS sequence amplification, sequencing, and database comparison, the unknown fungus was successfully identified as Irpexlacteus, providing an accurate classification basis for subsequent research.

[0062] The strain was further preserved and named CZ-2014; its classification name is Irpexlacteus. It was deposited on January 8, 2026, at the China General Microbiological Culture Collection Center (CGMCC); address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing; accession number: CGMCCNo. 42468.

[0063] Example 2: Single-factor optimization method for increasing rare saponin content in Panax notoginseng fermented with *Pteris vittata*

[0064] This embodiment provides a method for increasing the content of rare saponins in Panax notoginseng by fermenting it with *Pteris vittata*, as detailed below:

[0065] (1) Solid culture: White stalk fungus was inoculated into PDA medium and cultured on slant until the hyphae covered the entire surface of the medium. The culture temperature was 25℃ and the culture time was 7 days. The composition of the slant medium was: 200g potato, 20g glucose, 10g peptone, 1.5g potassium dihydrogen phosphate, 0.75g magnesium sulfate, 20g agar, and 1000mL distilled water.

[0066] (2) Liquid culture of the strain: Prepare liquid culture medium and sterilize it at high temperature. Inoculate the white rake tooth bacteria on PDA medium into a wide-mouth bottle containing liquid culture medium and culture at a constant temperature of 25℃ and a shaking incubator at 180r / min for 7 days. The formula of liquid culture medium is: 27g glucose, 18g yeast extract, 1.8g potassium dihydrogen phosphate, 1.35g magnesium sulfate, 1 vitamin B1 tablet, and 900mL distilled water.

[0067] (3) Preparation of Panax notoginseng fermentation substrate: After crushing Panax notoginseng, pass it through a 60-mesh sieve. Take 10g of the sieved Panax notoginseng powder, add 9mL of distilled water, mix well, sterilize at 121℃ for 30 minutes, and then cool naturally to room temperature.

[0068] (4) Fermentation of Panax notoginseng by Mycorrhiza nigra: 6 mL of liquid Mycorrhiza nigra was inoculated into the Panax notoginseng fermentation substrate for two-way solid fermentation until the mycelium was fully grown. The fermentation temperature was 25℃ and the time was 10 days.

[0069] (5) HPLC analysis of saponin content in Panax notoginseng before and after fermentation, including the following specific methods:

[0070] ① Preparation of reference solution

[0071] Take appropriate amounts of notoginsenoside R1, ginsenosides Rg1, Rb1, 20(S)-Rh1, Rd, 20(S)-Rg3, and Rg5 reference standards respectively, and dissolve them in methanol to prepare mixed reference standard solutions with concentrations of 0.178, 0.74, 0.7, 0.15, 0.356, 0.068, and 0.078 mg / mL respectively.

[0072] ② Preparation of the test solution

[0073] Take 0.6g of Panax notoginseng powder (passed through a No. 4 sieve), weigh it accurately, add 50mL of methanol accurately, weigh it, let it stand overnight, place it in an 80℃ water bath and keep it gently boiling for 2 hours, cool it, weigh it again, make up the lost weight with methanol, shake it well, filter it, and take the filtrate to obtain the product.

[0074] ③ Chromatographic conditions

[0075] The chromatographic column was an X Bridge C18® column (250 mm × 4.6 mm, 5 μm), and the mobile phase was acetonitrile (A)-water (B) with gradient elution (0–25 min, 20% A; 25–30 min, 20%–25% A; 30–41 min, 25%–41% A; 41–55 min, 41%–45% A; 55–80 min, 45%–74% A; 80–82 min, 74%–100% A); the flow rate was 1.0 mL / min; the column temperature was 20℃; the detection wavelength was 203 nm; and the injection volume was 10 μL. The HPLC chromatograms of the mixed reference solution, fermented Panax notoginseng, and raw Panax notoginseng sample solutions are shown below. Figure 1 .

[0076] Further investigation was conducted on the single-factor experiments of the fermentation process of Panax notoginseng by *Bacillus thunbergii*, as detailed below:

[0077] 1. Particle size analysis

[0078] After pulverizing Panax notoginseng, it was passed through sieves of 20 mesh, 40 mesh, 60 mesh, 80 mesh, and 100 mesh. 10g of the sieved powder was taken, mixed with 6mL of water, and sterilized at high temperature. After cooling, 5mL of *Panax notoginseng* was inoculated, and the mixture was allowed to ferment naturally at pH until the mycelium had fully colonized. The fermentation time was recorded, and the optimal pulverization force was determined. The results are shown in [the table below]. Figure 2 Figure A shows that as the particle size increases, the overall score first increases and then decreases, reaching its maximum at a particle size of 60 mesh.

[0079] 2. Water addition test

[0080] Take 10g of Panax notoginseng powder that has passed through a 60-mesh sieve, and add 7mL, 8mL, 9mL, 10mL, and 11mL of water respectively. After mixing well, sterilize at high temperature, cool, and inoculate with 5mL of *Pteris vittata*. Allow to ferment naturally at pH until the mycelium has fully colonized. Record the number of fermentation days to determine the optimal amount of water. The results are shown in [the table below]. Figure 2 Figure B shows that as the amount of water added increases, the overall score first increases and then decreases, reaching its maximum when the amount of water added is 9 mL.

[0081] 3. Inoculation Quantity Assessment

[0082] Take 10g of Panax notoginseng powder that has passed through a 60-mesh sieve, add 9mL of water, mix well, and sterilize at high temperature. After cooling, inoculate with 4mL, 5mL, 6mL, 7mL, and 8mL of *Pteris vittata*, respectively. Allow to ferment naturally at pH until the mycelium has fully colonized the colony. Record the number of fermentation days to determine the optimal inoculum quantity. The results are shown in [the table below]. Figure 2 Figure C shows that as the inoculum quantity increases, the overall score first increases and then decreases, reaching its maximum when the inoculum quantity is 60%.

[0083] 4. pH value test

[0084] Take 10g of Panax notoginseng powder that has passed through a 60-mesh sieve, add 9mL of water, and adjust the initial pH values ​​to 6, 6.5, 7, 7.5, and 8 respectively. After mixing well, sterilize at high temperature, cool, and inoculate with 6mL of Mycorrhiza glabra. Ferment until the mycelium is fully grown, record the fermentation days, determine the optimal pH value, and the results are shown in the table below. Figure 2 Figure D shows that the overall score first increases and then decreases with increasing pH value, reaching its maximum at pH 7.5. Considering that pH value has a relatively small impact on the overall score, and the pH value of the water used in the experiment is around 7.5, the effect of pH value on the overall score will not be examined in the response surface methodology experiment.

[0085] Example 3: Optimization of Panax notoginseng fermentation process using Box-Behnken response surface methodology

[0086] 1. Experimental Design and Results

[0087] Based on the results of single-factor experiments, a three-factor, three-level Box-Behnken response surface methodology was designed using Design-Expert 12 software with particle size (A, mesh), water content (B, %), and inoculum content (C, %) as independent variables and the overall score (Y) as the response value to optimize the fermentation process of Panax notoginseng using *Bacillus thunbergii*. The parameters A were 40 mesh, 60 mesh, and 80 mesh; B was 8 mL, 9 mL, and 10 mL; and C was 50%, 60%, and 70%. The factor levels are shown in Table 1, and the response surface methodology and results are shown in Table 2.

[0088] Table 1 Factor Levels

[0089]

[0090] Table 2 Response Surface Experimental Design and Results

[0091]

[0092] 2. Comprehensive scoring model fitting and significance analysis

[0093] The data in Table 2 were subjected to analysis of variance and quadratic polynomial regression fitting using Design-Expert 12 software (see Table 3). The regression equation is Y = 95.69 + 1.16 A + 1.38 B + 1.35 C - 1.78 AB + 0.1 AC - 0.7825 BC - 7.91 A 2 -3.21 B 2 -8.54 C 2 The ANOVA table shows that the F-value of the binomial fitting model is 47.81, and the model P < 0.0001, indicating that the model is highly significant. The lack-of-fit term F = 3.84, P = 0.1134, indicating that the lack-of-fit term is not significant, and the regression equation fits well. The values ​​of particle size (A), water addition (B), and inoculum quantity (C) are all P < 0.05, indicating that all three have a significant impact on the process. The factors affecting the overall score are, in descending order: water addition (B) > inoculum quantity (C) > particle size (A).

[0094] Table 3 Regression Model and Analysis of Variance Results

[0095]

[0096] 3. Response Surface Diagram

[0097] Based on the regression equation and response surface fitted by CCD, the effects of particle size, water addition, and inoculum amount on the comprehensive score of the two-way solid-state fermentation of Panax notoginseng were analyzed. The results are shown in […]. Figure 1 Analysis of variance and response surface methodology show that the p-value of terms A and B is less than 0.05, indicating a significant interaction. The quadratic term A... 2 C 2 B 2 The p-value was less than 0.01, indicating a highly significant effect.

[0098] 4. Optimal process validation and comparison results with raw product

[0099] According to the Box-Behnken model, the optimal process predicted by response surface optimization is a crushing particle size of 61.059 mesh, a water addition of 9.192 mL, and an inoculum amount of 60.708%. Under these conditions, the predicted comprehensive score of the model is 95.90. Considering the actual situation, the final process is determined as a crushing particle size of 60 mesh, a water addition of 9 mL, and an inoculum amount of 60%. Three batches of Panax notoginseng samples were fermented with Irpex lacteus according to the above parameters for verification experiments, and the results are shown in Table 4. The average comprehensive score of the three verification experiments is 95.98, which is close to the predicted value of 95.90, and the RSD is 0.11%, indicating that the optimized best process has good stability.

[0100] Table 4 Results of the verification of the best process

[0101]

[0102] The average content of each component in fermented Panax notoginseng was compared with the average content of the raw product components, as shown in Table 5. The two-way solid fermentation of Panax notoginseng with Irpex lacteus induced profound component remodeling of the saponin components in Panax notoginseng, characterized by the effective degradation of ordinary ginsenosides (R1, Rg1, Rb1), while ginsenoside Rd and Rh1 were significantly accumulated, and new ginsenosides Rg3 and Rg5 were generated. This transformation result marks the success of achieving directional modification of saponins through microbial fermentation, providing an innovative research idea for enhancing the bioavailability and pharmacological activity of Panax notoginseng preparations. Those skilled in the art can use the method in the Chinese Pharmacopoeia to extract and purify the saponin components in Panax notoginseng to further improve the purity of rare ginsenosides.

[0103] Table 5 Comparison of the contents of saponin components before and after the fermentation of Panax notoginseng

[0104]

[0105] Example 4 Study on the therapeutic effect of Irpex lacteus-fermented Panax notoginseng on lupus nephritis mice

[0106] 1. Grouping and administration

[0107] Ten 7- to 8-week-old female BALB / c mice and forty 7- to 8-week-old female MRL / lpr mice were selected. They were all SPF grade, weighing 20-25 g, and purchased from Beijing Spey Foster Biotechnology Co., Ltd. The experimental animal production license number is: SCXK (Beijing) 2024-0001. They were raised in a fully enclosed SPF environment in the Experimental Animal Center of Changchun University of Chinese Medicine. The feeding conditions were: room temperature 22-25 °C, relative humidity 40%-60%, 12 h of daily light, freely obtaining sufficient feed and fresh drinking water, and regularly changing the clean bedding. The animal experiment was approved by the Animal Ethics Committee of Changchun University of Chinese Medicine (2025219), and the welfare and ethical principles of experimental animals were strictly observed.

[0108] BALB / c mice were used as healthy controls (K); MRL / lpr mice were used as models of lupus nephritis (LN) and randomly divided into four groups: model group (M), positive control group (Y), raw Panax notoginseng group (S), low-dose fermented Panax notoginseng group (FSD), and high-dose fermented Panax notoginseng group (FSG), with eight mice in each group. According to the Chinese Pharmacopoeia, 3g was the low-dose group and 9g was the high-dose group. Based on body surface area conversion and after removing the influence of water, the equivalent dose ratio for mice was 9.1. For a normal human body weighing 60kg, the low-dose fermented Panax notoginseng group was 0.455g / kg, and the raw and high-dose fermented Panax notoginseng groups were 1.365g / kg. The positive control group (prednisolone acetate) was 5mg / kg. Except for the control and model groups, which were administered an equal volume of distilled water by gavage, all other groups were administered the drug by gavage daily for 8 weeks.

[0109] 2. Obtain materials

[0110] After the last administration, blood samples were collected from mice by enucleation. The collected blood samples were centrifuged at 4000 r / min for 10 min to separate the serum. The serum was then properly stored at -80℃ for later use. After blood collection, the mice were euthanized by cervical dislocation according to experimental requirements. Subsequently, the kidneys, spleen, and liver were completely removed by laparotomy. The right kidney was fixed with 4% paraformaldehyde, and the left kidney was stored at -80℃ for tissue homogenization. The spleen was washed with physiological saline, weighed, and photographed. The liver was stored at -80℃. The contents of the rat cecum were taken on a clean bench and stored at -80℃ for 16S rRNA amplification and sequencing of the intestinal microbiota. Strict aseptic techniques were used during the sampling process to prevent contamination of the microbial samples.

[0111] 3. Observation of the external appearance of the spleen

[0112] Eight weeks after drug administration, spleen tissue was harvested from the abdominal cavity, washed with physiological saline to remove blood, and then subjected to gross morphological observation. Results are shown below. Figure 4 .

[0113] Depend on Figure 4 Morphological observation of the spleens of mice in each group showed that the spleens in group K were regular in shape, uniform in size, and without any lesions; the spleens in group M were enlarged, with an uneven surface, local dark congestion, inflammation, edema, and tissue necrosis; the spleens in group Y were similar in shape to the control group and had a uniform color; the congestion area in group S was reduced compared to the model group; the inflammation and edema in group FSD were partially improved; and the regularity of the shape and uniformity of the color in group FSG were similar to those in the control group.

[0114] 4. HE staining

[0115] The right kidney, fixed in 4% paraformaldehyde, was dehydrated via an ethanol gradient, embedded in paraffin, and sectioned into 4 μm thick sections using a microtome. Before staining, the sections were dewaxed in xylene, rehydrated with ethanol, washed with PBS, and then stained with hematoxylin and eosin (HE). Histopathological changes in the kidney tissue were observed using an optical microscope. Results are shown in [Figure number missing]. Figure 5 .

[0116] Depend on Figure 5 It can be seen that in group K, the glomeruli were evenly distributed, with uniform cell number and matrix within the glomeruli; the tubular epithelial cells were round and plump; the connective tissue between the urinary tubules was the renal interstitium, with no obvious proliferation; and no obvious inflammatory cell infiltration was observed. In group M, the glomeruli were evenly distributed; a large number of glomerular cells showed thickened matrix, a slight increase in the number of glomerular mesangial cells (yellow arrows), and unclear glomerular capillary structure; the tubular epithelial cells were round and plump; a very small amount of inflammatory cell infiltration, mainly lymphocytes, was observed around the blood vessels (orange arrows), and a very small amount of connective tissue proliferation was observed (green arrows). In group Y, the glomeruli were evenly distributed; a small number of glomerular cells showed mild matrix thickening (yellow arrows); the tubular epithelial cells were round and plump; the connective tissue between the urinary tubules was the renal interstitium, with no obvious proliferation; and no obvious inflammatory cell infiltration was observed. In Group S, the kidney tissue showed uniform glomerular distribution; thickened matrix in many glomerular cells and a slight increase in the number of glomerular mesangial cells (yellow arrows); unclear glomerular capillary structure; rounded and plump tubular epithelial cells; and minimal perivascular inflammatory cell infiltration, predominantly lymphocytes (orange arrows). In Group FSD, the kidney tissue showed uniform glomerular distribution; thickened matrix in many glomerular cells and a slight increase in the number of glomerular mesangial cells (yellow arrows); unclear glomerular capillary structure; rounded and plump tubular epithelial cells; the connective tissue between the urinary tubules was renal interstitium, with no significant proliferation; and no significant inflammatory cell infiltration. In Group FSG, the kidney tissue showed uniform glomerular distribution, uniform cell number and matrix within the glomeruli; rounded and plump tubular epithelial cells; the connective tissue between the urinary tubules was renal interstitium, with no significant proliferation; and no significant inflammatory cell infiltration.

[0117] 5. Masson staining

[0118] First, paraffin sections were dewaxed and treated with water. Then, cell nuclei were stained with hematoxylin, differentiated with hydrochloric acid and ethanol, and then blued again with running water. Next, muscle fibers and other components were stained with Ponceau S and acid fuchsin. Differentiation was then performed with phosphomolybdic acid. Collagen fibers were then counterstained with aniline blue (or light green). Finally, the sections were dehydrated, cleared, and mounted with neutral resin. Observation under an optical microscope revealed that collagen fibers appeared blue (or green), muscle fibers and cellulose fibers appeared red, and cell nuclei appeared blue-black, thus clearly showing the degree of tissue fibrosis and structural changes. The results are shown in the figure below. Figure 6 .

[0119] In group K, the renal tubules were regularly arranged, and no obvious fibrosis was observed in the interstitium. In group M, the renal tubules were irregularly arranged, with a small amount of fibrous connective tissue hyperplasia, which stained blue. In group Y, the renal tubules were regularly arranged, and no obvious fibrosis was observed in the interstitium. In group S, the renal tubules were irregularly arranged, with a small amount of fibrous connective tissue hyperplasia, which stained blue. In group FSD, the renal tubules were irregularly arranged, with a small amount of fibrous connective tissue hyperplasia, which stained blue. In group FSG, the renal tubules were regularly arranged, and no obvious fibrosis was observed in the interstitium.

[0120] 6. Following the kit instructions, the concentration levels of ANA, dsDNA-Ab, Cr, BUN, TNF-α, and IL-6 in the serum of mice in each group were determined using the ELISA method. The results are shown in the table below. Figure 7 .

[0121] ANA (antinuclear antibody) and ds-DNA-Ab (anti-double-stranded DNA antibody) are key immunological indicators for assessing lupus nephritis. Compared with the control group, the serum ANA and ds-DNA-Ab levels in the model group mice were significantly increased (P < 0.01). Compared with the model group, the serum ANA and ds-DNA-Ab levels in the Y, FSD, and FSG groups mice were significantly decreased (P < 0.05). Cr (serum creatinine) and BUN (blood urea nitrogen) are core indicators for assessing kidney function. Compared with the control group, the serum Cr and ds-DNA-Ab levels in the model group mice were significantly increased. BUN levels were significantly elevated (P < 0.01). Compared with the model group, serum Cr and BUN levels were significantly decreased in the Y, FSD, and FSG groups (P < 0.05). TNF-α (tumor necrosis factor-α) and IL-6 (interleukin-6) are key pro-inflammatory factors. Compared with the blank group, serum TNF-α and IL-6 levels were significantly elevated in the model group (P < 0.01), while serum TNF-α and IL-6 levels were significantly decreased in the Y, S, FSD, and FSG groups (P < 0.05). Combined with the histopathological improvements observed by HE and Masson staining and the changes in inflammatory factor levels detected by ELISA, comprehensive analysis showed that the FSG group had the most significant therapeutic effect on the mouse lupus nephritis model. Therefore, subsequent protein differential analysis was performed on FSG group mice to further explore the advantageous mechanism of this therapeutic effect.

[0122] 7. Western blot analysis of the differential expression of key proteins in the PI3K / AKT / NF-κB pathway in kidney tissue by fermented Panax notoginseng.

[0123] Kidney tissues from mice in each group were collected, total protein was extracted and quantified using the BCA method, then separated by SDS-PAGE gel electrophoresis and transferred to PVDF membranes. Subsequently, the membranes were blocked with 5% BSA and incubated sequentially with primary antibodies targeting PI3K, p-PI3K, AKT, p-AKT, NF-κB p65, p-NF-κB p65, and the internal control β-actin, as well as HRP-labeled secondary antibodies. Finally, the membranes were visualized by ECL chemiluminescence, and the grayscale values ​​of the target protein bands were analyzed using ImageJ software. After correction with the internal control, statistical comparisons were performed to determine whether fermented Panax notoginseng exerts its therapeutic effect by regulating this pathway. Results are shown in [Figure number missing]. Figure 8 .

[0124] Depend on Figure 8 It was found that, compared with the control group, the expression levels of phosphorylated proteins p-PI3K, p-AKT, and p-P65 in the kidneys of mice in the model group were significantly upregulated (P < 0.01), while the expression levels of the corresponding total proteins PI3K, AKT, and P65 did not change significantly among the groups. The ratios of p-PI3K / PI3K, p-AKT / AKT, and p-P65 / P65 were all significantly increased (P < 0.01), indicating that the PI3K / AKT / NF-κB signaling pathway in the kidneys is abnormally activated under lupus nephritis. Compared with the model group, the expression of p-PI3K, p-AKT, and p-P65 proteins was significantly downregulated in the fermented Panax notoginseng group (P < 0.01), and the ratios of p-PI3K / PI3K, p-AKT / AKT, and p-P65 / P65 were all reduced (P < 0.01). This indicates that fermented Panax notoginseng by *Pteris vittata* can inhibit the phosphorylation activation of PI3K, AKT, and NF-κB p65 subunits in the kidneys of mice with lupus nephritis.

[0125] Based on the above pharmacodynamic experimental results, fermented Panax notoginseng significantly alleviated splenomegaly in MRL / LPR model mice, suggesting that it effectively inhibited abnormal immune activation, thereby reducing the production of pathogenic autoantibodies (such as anti-dsDNA antibodies). Kidney histopathological results further indicated that fermented Panax notoginseng (especially the FSG group) significantly reduced glomerular mesangial cell proliferation, inflammatory cell infiltration, and tubulointerstitial fibrosis, demonstrating a superior renal protective effect compared to raw Panax notoginseng. This anti-fibrotic effect may be related to the downregulation of pro-fibrotic factors such as TGF-β1 by Panax notoginseng saponins, inhibiting epithelial-interstitial transition (EMT). At the molecular level, fermented Panax notoginseng significantly reduced serum ANA and dsDNA-Ab titers, confirming its immunomodulatory effects; simultaneously, it effectively improved renal function indicators (Cr, BUN) and inhibited the release of key pro-inflammatory factors (TNF-α, IL-6). The PI3K / AKT / NF-κB pathway is a core network regulating cellular inflammation, proliferation, and apoptosis. Its sustained activation is closely related to immune complex deposition, mesangial cell proliferation, and inflammatory factor production in lupus nephritis. In this study, fermented Panax notoginseng simultaneously inhibited the phosphorylation of both upstream (PI3K / AKT) and downstream key transcription factor (NF-κB) in this pathway. This inhibition implies that the transcription and release of a series of downstream pro-inflammatory factors (such as TNF-α, IL-6, and IL-1β) may be blocked, thereby alleviating renal inflammation. Therefore, fermented Panax notoginseng may exert its anti-inflammatory and renal protective effects by inhibiting the abnormal activation of the renal PI3K / AKT / NF-κB signaling pathway.

[0126] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. White-skinned tooth fungus, characterized in that, Its accession number is CGMCC No.42468.

2. The method for culturing *Tetranychus spp.* according to claim 1, characterized in that, Includes the following steps: Step 1: Inoculate the *Bacillus thunbergii* onto a slant culture medium for slant culture; Step 2: Inoculate the white rake tooth fungus cultured on the slant culture medium and culture it.

3. The cultivation method according to claim 2, characterized in that, The slant culture medium is PDA solid culture medium, and the slant culture temperature is 24-28℃, and the time is 7-10 days; The liquid culture medium consists of 20-30g glucose, 10-20g yeast extract, 1-2g potassium dihydrogen phosphate, 1-2g magnesium sulfate, 1-2 vitamin B1 tablets and 800-1000mL distilled water. The culture temperature is 24-28℃, the rotation speed is 160-200rpm / min, and the culture time is 7-10 days.

4. The application of *Gynostemma pentaphyllum* as described in claim 1 or *Gynostemma pentaphyllum* obtained by the cultivation method described in claim 2 or 3 in the conversion of rare ginsenosides.

5. The application according to claim 4, characterized in that, The rare ginsenosides include at least one of Rh1, Rg3 and Rg5.

6. A two-way solid-state fermentation method for *Panax notoginseng*-*Bacillus thunbergii*, characterized in that, Includes the following steps: S1. Mix Panax notoginseng powder with water, sterilize, and obtain Panax notoginseng fermentation substrate; S2. Take the bacterial solution of *Bacillus thunbergii* as described in claim 1 or *Bacillus thunbergii* obtained by the culture method described in claim 2 or 3, and inoculate it into the Panax notoginseng fermentation substrate for fermentation culture.

7. The fermentation method according to claim 6, characterized in that, In S1, the particle size of the Panax notoginseng powder is 40-100 mesh, and the mass-to-volume ratio of the Panax notoginseng powder to water is 1g:(0.7-1)mL; In step S2, the volume-to-mass ratio of the bacterial solution of *Bacillus thunbergii* to the fermentation substrate of *Panax notoginseng* is (0.4~0.8) mL:1g, and the fermentation culture temperature is 20~26℃ for 8~14 days.

8. A method for converting ginsenosides Rh1, Rd, Rg3 and / or Rg5, characterized in that, Includes the following steps: Take Panax notoginseng powder and mix it with water, sterilize it, and obtain Panax notoginseng fermentation substrate; The bacterial culture of *Bacillus thunbergii* as described in claim 1 or the *Bacillus thunbergii* cultured by the method described in claim 2 or 3 is inoculated into the *Panax notoginseng* fermentation substrate, and after fermentation and purification, Rh1, Rd, Rg3 and / or Rg5 are obtained.

9. The use of the product obtained by the fermentation method according to claim 6 or 7, or the product obtained by the transformation method according to claim 8, in the preparation of a medicament for the prevention and / or treatment of lupus nephritis.

10. A drug, characterized in that, It includes the product obtained by fermentation by the fermentation method of claim 6 or 7, or the product obtained by conversion by the method of claim 8, and pharmaceutically acceptable excipients.

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