Fermentation processing method for reducing toxicity of tripterygium wilfordii

Abstract

The invention belongs to the field of medicines, and particularly relates to a fermentation processing method for reducing toxicity of tripterygium wilfordii, which comprises the steps of tripterygium wilfordii matrix treatment, strain activation treatment and bidirectional solid fermentation. The method specifically comprises the following steps: crushing tripterygium wilfordii, and performing high-pressure steam sterilization to obtain a tripterygium And inoculating the fermentation seed liquid of the ganoderma lucidum strain into the tripterygium wilfordii matrix, and carrying out bidirectional solid fermentation. Starting from clinical application practice, the invention provides a more perfect, reasonable and systematic tripterygium wilfordii fermentation and toxicity reduction processing method. The method solves the problems that in the field of tripterygium wilfordii fermentation processing, ganoderma lucidum strains are not clear, most technical methods do not conform to actual clinical medication forms, the processing method has many uncertain factors, the non-druggability is increased after processing, and the like, and has popularization value.

Description

Technical Field

[0001] The invention relates to the field of fermentation and processing, and in particular to a fermentation and processing method for reducing the toxicity of Tripterygium wilfordii. Background Art

[0002] Tripterygium wilfordii Hook.f., also known as yellow rattan, yellow rattan grass, yellow rattan root, yellow rattan wood, yellow wax rattan, mangcao, water mang, water mang grass, water mangzi, water mangdou, broken intestines, etc., belongs to the genus Tripterygium of the Celastraceae family and is mainly distributed in southern my country. Tripterygium wilfordii has unique medicinal effects, and has multiple effects such as clearing away heat and detoxifying, dispelling wind and unblocking meridians, reducing swelling and relieving pain. In clinical practice, its peeled roots are mainly used as medicine, as an immunosuppressant with definite efficacy, mainly treating rheumatoid arthritis, glomerulonephritis, nephrotic syndrome, lupus erythematosus, oral and eye dryness syndrome, Behcet's disease, psoriasis, leprosy, eczema, scabies, stubborn ringworm and other arthritis or skin diseases. The Chinese patent medicines on the market with Tripterygium wilfordii as raw materials mainly include Tripterygium wilfordii polyglycosides tablets, Tripterygium wilfordii tablets, Tripterygium wilfordii double-layer tablets and Tripterygium wilfordii total terpenoid tablets. In recent years, the adverse reactions and toxic side effects of Tripterygium wilfordii have received more and more attention. Among them, the damage to the liver is particularly significant. Therefore, how to maintain the activity of Tripterygium wilfordii while reducing its toxic side effects has become an urgent problem to be solved.

[0003] The active ingredients in Tripterygium wilfordii are complex and diverse. Domestic and foreign scholars have successively isolated more than 400 monomer compounds such as diterpenes, triterpenes, alkaloids and glycosides from Tripterygium wilfordii. The specific compounds of Tripterygium wilfordii mainly include diterpenes such as triptolide, triptolide B, triptolide ketone and rafenolide; triterpenes such as triptolide, triptolide A and norzelaminaldehyde; and alkaloids such as triptolide, triptolide and triptolide.

[0004] The toxic side effects of Tripterygium wilfordii mainly come from diterpenoids, followed by alkaloids. Diterpenoids mainly damage the heart, liver, gastrointestinal tract and bone marrow; alkaloids mainly damage the liver and can destroy red blood cells, causing progressive anemia, and even induce hypoxic damage to the renal tubules. After absorption, they damage the central nervous system and can lead to severe malnutrition changes. Among them, triptolide is the most active epoxy diterpenoid lactone isolated from Tripterygium wilfordii, and it has also been confirmed to be one of the main hepatotoxic components of Tripterygium wilfordii; while triptolide A has strong anti-tumor and immunosuppressive activity, relatively mild toxicity, and is regarded as one of the main active ingredients. Therefore, the quality control of Tripterygium wilfordii products focuses more on the content of triptolide and triptolide A. For example, the content of triptolide in Tripterygium wilfordii polyglycosides tablets shall not exceed 0.1%, and the content of triptolide A shall not be less than 0.1%.

[0005] A series of important progress has also been made in reducing the toxicity of Tripterygium wilfordii. For monomers such as triptolide, the toxicity characteristics have given it new application value in anti-tumor. Chemical modification of its side chains and other functional groups can enhance its targeting and reduce its toxicity. However, the toxicity and activity of Tripterygium wilfordii are caused by multiple components, which have synergistic and complementary effects in physiological activity, and often have multi-target characteristics in terms of the scope of action. Therefore, how to change the multiple components of Tripterygium wilfordii from a holistic perspective to reduce toxicity and increase efficacy has become an important issue. There are traditional processing methods such as steaming, boiling, sheep blood processing, licorice processing, and conventional Chinese medicine compatibility methods to reduce the toxicity of Tripterygium wilfordii, but the gap between the effective dose and the toxic dose has not been significantly widened.

[0006] Modern Chinese medicine fermentation processing utilizes the physiological metabolism of microorganisms and the various active enzymes they produce to decompose and transform various organic matrices, thereby changing their components and the ratios between their components. It has the advantages of mild conditions and high biosafety, making it a very promising processing technology.

[0007] Zhuang Yi, Xie Xiaomei and others first used Ganoderma lucidum as the fermentation strain and the whole root of Tripterygium wilfordii with skin as the medicinal matrix. After 30 days of solid bidirectional fermentation under specific conditions, they obtained a specific Tripterygium wilfordii-Ganoderma lucidum complex, whose toxicity was greatly reduced. They also used ethyl acetate extraction to explore the change law of its chemical composition (Zhuang Yi, Xie Xiaomei. Preliminary study on the detoxification and sustained effect of Tripterygium wilfordii by a new (bidirectional) solid fermentation engineering of medicinal fungi [J]. China Journal of Traditional Chinese Medicine, 2009, 34(16): 2083-2087.; Xie Xiaomei, He Jing, Luo Hong Dan, et al. Detoxification and sustained effect of Ganoderma lucidum and Tripterygium wilfordii after bidirectional fermentation [J]. Chinese Herbal Medicine, 2009, 40(12): 1925-1929.; Zhang Puzhao, Shu Rengeng, Xie Xiaomei. Study on the chemical composition of Tripterygium wilfordii after bidirectional solid fermentation [J]. Chinese Edible Fungi, 2008, 27(1): 41-42.; Hou Zhifan, Liang Yonghong, He Libiao, et al. Preliminary study on the changes of chemical composition of Tripterygium wilfordii after bidirectional solid fermentation of Ganoderma lucidum [J]. Chinese Herbal Medicine, 2012, 43(2): 234-237.). The above series of studies only detected the death and lymphocyte proliferation of mice after administration, and only performed liquid chromatography detection, identifying the decrease in the content of triptolide and phthalate esters, and did not study the changes in the content of other main compounds in Tripterygium wilfordii.

[0008] He Luanying et al. used the medicinal fungus Ganoderma lucidum to ferment the medicinal matrix Tripterygium wilfordii (with skin unknown) using a two-way solid fermentation technique, and obtained the product Linglei fungus after 30 and 40 days of fermentation. They compared their effects on the release of inflammatory factors by LPS-induced M1 macrophages and the proliferation of normal human liver cells, and used liquid chromatography-mass spectrometry (LC-MS) to analyze the changes in the composition of Tripterygium wilfordii before and after fermentation (He Luanying, Lin Zichun, Lu Jiandong, et al. Study on the attenuation and persistence of Tripterygium wilfordii based on Ganoderma lucidum bidirectional solid fermentation [J]. Journal of Beijing University of Chemical Technology (Natural Science Edition), 2021, 48(4): 48-56.). It was found that when the inoculum size was 10mL / 100mL, at 27°C and 60% relative humidity, after 30 days of fermentation, the methanol extract product showed an attenuation effect in the experiment of human normal liver cells. In addition, LC-MS results showed that after fermentation, the contents of triptolide, triptolide, and triptolide increased, while the content of triptolide A decreased. The above results also only verified the toxicity of the fermentation products at the cellular level, and the increase in the content of the main toxic compounds increased the safety risk.

[0009] Chinese patent CN1911262A discloses the application of bidirectional solid fermentation engineering technology of medicinal fungi in the detoxification and sustained effect of Tripterygium wilfordii. The specific method of application is: activation treatment of fermented bacteria (Ganoderma lucidum, Versicolor, Sophora japonica, Red plug, Sulfur bacteria, Tree tongue), crushing and sterilization of fermentation matrix (Tripterygium wilfordii with skin), solid bidirectional fermentation, drying of fungal mass and extraction with 40% ethanol, and then toxicity and efficacy experiments. The results show that after fermentation of Tripterygium wilfordii, the polar toxicity of mice is reduced and it retains strong activity. The fermentation cycle described in the patent is 30-60 days, and the product is extracted with 40% ethanol, which is relatively long. The product extraction is inconsistent with the traditional clinical application form of Tripterygium wilfordii, and further clinical research is still difficult to apply.

[0010] Chinese patent CN118557620A discloses a fermentation processing method of Tripterygium wilfordii. The processing method comprises the following steps: adding radish to a solvent for the first heating, filtering for the first time, and collecting the first filtrate; after collecting the first filtrate, adding the radish to a solvent for the second heating, filtering for the second time, and collecting the second filtrate; after combining the first filtrate and the second filtrate, concentrating to obtain a concentrate; adding the concentrate and distiller's yeast to the crushed material of Tripterygium wilfordii (with skin), fermenting; drying after the fermentation is completed, obtaining a processed product of Tripterygium wilfordii, and subsequently detecting the contents of triptolide, triptolide A, triptolide red, and triptolide alcohol. The processing method of Tripterygium wilfordii described in the patent has a fermentation cycle of only 4 days, and the strain used is a distiller's yeast composite bacteria, and the microbial composition is not clear enough. The final product is extracted with methanol, and the toxicity is not verified. It only stays at the level of content detection of the target compound, and the evidence of attenuation is not sufficient.

[0011] The content of toxic components such as triptolide in the root bark of Tripterygium wilfordii is relatively high, and the solubility of substances such as triptolide in water is extremely low. Therefore, the clinical application of traditional Tripterygium wilfordii mainly adopts the method of peeling the root and decocting it in water, which not only ensures the efficacy but also reduces the toxicity as much as possible. Most of the existing literature and patent disclosures directly crush the whole root of Tripterygium wilfordii for processing, and most of the product extraction uses organic solvents, which increases the risk of toxicity and is out of touch with clinical application, making it difficult to carry out further in-depth research and application.

[0012] At present, in the field of Tripterygium wilfordii fermentation and processing, most of the focus is on the two-way fermentation of Tripterygium wilfordii and Ganoderma lucidum. The Ganoderma lucidum strain is unclear, most of the technical methods are inconsistent with the actual clinical medication form (the Tripterygium wilfordii used clinically is the peeled root), there is a lack of uniformity in the processing methods and product toxicity evaluation, and there are many uncertain factors. Summary of the invention

[0013] In view of the above problems, the present invention provides a more complete, reasonable and systematic fermentation and detoxification processing method of Tripterygium wilfordii based on the actual clinical application, which includes the treatment of Tripterygium wilfordii matrix, activation treatment of bacteria and two-way solid fermentation.

[0014] The fermentation processing method for reducing the toxicity of Tripterygium wilfordii of the present invention comprises the following specific steps:

[0015] (1) Preparation of Tripterygium wilfordii matrix: crushing Tripterygium wilfordii and sterilizing with high pressure steam to obtain Tripterygium wilfordii matrix;

[0016] (2) Bidirectional solid fermentation: The fermented seed liquid of the Ganoderma lucidum strain is inoculated into the Tripterygium wilfordii matrix of step (1), and after static fermentation, the fermentation product is taken out and dried.

[0017] Preferably, in step (1), the fermentation substrate of Tripterygium wilfordii is peeled slices of Tripterygium wilfordii root, which is safe and in line with the actual clinical application. In order to ensure sufficient fermentation, the slices are crushed. At the same time, in order to avoid contamination by foreign bacteria, the crushed powder is sterilized with high-pressure steam.

[0018] Preferably, in the step (1), the peeled Tripterygium wilfordii slices are mechanically coarsely crushed to ensure uniform crushing size, and the crushing size is less than 20 meshes, but not too fine, which will affect the air permeability of the fermentation process.

[0019] Preferably, the high-pressure steam sterilization in step (1) is carried out in a conical flask, and the sterilization conditions are 110-130° C., 10-40 min; preferably 121° C., 30 min.

[0020] Preferably, in step (2), two species of Ganoderma lucidum that are allowed to be artificially cultivated are selected, namely, Ganoderma lucidum sinensis G10 and Ganoderma purpurogenum, which have clear strain information and are safe and reliable. Ganoderma lucidum sinensis G10 is further preferred.

[0021] The method for preparing the fermented seed liquid of the Ganoderma lucidum species in step (2) is as follows:

[0022] (a) Strain activation treatment: Ganoderma lucidum strains were cultured on PDA plates to obtain seed plates;

[0023] (b) preparing the seed solution, taking the activated culture bacteria block on the plate in step (a), placing it in a PDB liquid culture medium for culture, and obtaining a fermentation seed solution;

[0024] More specifically, the operation in step (a) is carried out in a clean bench, a small piece of culture is scraped from the Ganoderma lucidum culture slant test tube and placed on a PDA plate, and the culture conditions are 28° C. and 60% humidity for 3-5 days, preferably 4 days.

[0025] Preferably, the step (b) is to take about 1 cm of the activated culture on the plate in step (a) 2 The bacterial blocks of different sizes are placed in 50 mL of PDB liquid culture medium and cultured at 28°C and 200 rpm for 5-7 days, preferably 6 days.

[0026] Preferably, the step (2) is performed in an ultra-clean working environment, using pure strains for fermentation, and inoculating 50 g of crushed Tripterygium wilfordii with 50 mL of seed liquid. After sufficient mixing, the mixture is allowed to ferment at 23-25° C. and 60-70% humidity. The fermentation conditions are as close to the natural state as possible, thus saving energy consumption.

[0027] Preferably, the fermentation period of step (2) is 18-22 days, preferably 21 days, to ensure that the active substance components are retained to the maximum extent and to achieve the purpose of reducing toxicity. The surface of the fermentation product is covered with white mycelium.

[0028] Preferably, the drying in step (2) is carried out at 50-70°C, preferably 60°C.

[0029] The fermentation product was post-processed and the quality evaluation was performed as follows:

[0030] (A) Liquid chromatography-mass spectrometry detection of active ingredients of fermentation products: Take a certain amount of fermentation drying product, add 10 times the volume (mass: volume) of 75% ethanol to fully soak and extract, repeat 3 times, then combine the extracts and concentrate to 1 times the volume equivalent of the product, and then perform liquid chromatography-mass spectrometry detection.

[0031] (B) Combined with the liquid chromatography-mass spectrometry detection results, a reasonable assessment of the fermentation quality was made.

[0032] (C) Toxicity testing of fermentation products: Take a certain amount of fermentation dried product, add 10 times the volume (mass: volume) of distilled water to fully soak and extract, repeat 3 times, then combine the extracts and concentrate to 10 times the volume equivalent of the product, and then spray dry; use the zebrafish animal model to observe its hatching rate and liver toxicity at a specific concentration.

[0033] Optionally, a subsequent detection step is also included.

[0034] Furthermore, the present invention also provides a fermentation product obtained by the method or a product prepared therefrom.

[0035] Beneficial effects of the present invention:

[0036] In the existing technology, the fermentation and processing of Tripterygium wilfordii mostly focuses on the two-way fermentation of Tripterygium wilfordii and Ganoderma lucidum. Most of the technical methods are inconsistent with the actual clinical medication form (the Tripterygium wilfordii used clinically is the peeled root). There is a lack of uniformity in the processing methods and product toxicity evaluation. The product extraction is not done by water extraction, and there are many risk factors. After processing, the product becomes undrugable.

[0037] The fermentation and processing process for attenuating the toxicity of Tripterygium wilfordii of the present invention uses peeled Tripterygium wilfordii slices as the Tripterygium wilfordii matrix to ensure consistency with clinical medication; the fermentation uses pure strains of Ganoderma lucidum for inoculation, the overall fermentation process is simple, the energy consumption is low, the fermentation process is safe and controllable, and contamination by foreign bacteria is avoided. The fermentation product detection of the present invention uses 75% ethanol to ensure the full dissolution of effective substances, thereby ensuring the accuracy of the detection of toxic compounds and active ingredients; the final product extraction can be extracted with distilled water, concentrated, and spray-dried, which is similar to the preparation process of Chinese medicine formula granules, ensuring safety and enhancing drugability. The attenuation effect of Tripterygium wilfordii and Ganoderma lucidum fermentation products has been confirmed in terms of toxic component content and zebrafish animal models, and has good systematicity and uniformity. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 It is a schematic diagram of the technical route of the present invention;

[0039] Figure 2 It is the fingerprint of liquid phase detection;

[0040] Figure 3 Liquid chromatography-mass spectrometry is used together to detect the content of target substances. DETAILED DESCRIPTION

[0041] The main instruments and materials involved in the following examples are as follows:

[0042] Tripterygium wilfordii slices, Ganoderma species (Ganoderma lucidum G10; Ganoderma sinensis; Ganoderma lucidum; Ganoderma balabacense; Ganoderma koreanese; Ganoderma oregonense; Ganoderma tsugae), zebrafish, incubator, liquid chromatograph, mass spectrometer, rotary evaporator, spray dryer, etc.

[0043] Example 1: Processing of Tripterygium wilfordii by fermentation

[0044] (1) Preparation of Tripterygium wilfordii matrix: Mechanically coarsely crush the peeled Tripterygium wilfordii slices to ensure uniform size and crush them to less than 20 mesh; place the crushed material in a conical bottle and sterilize it with high pressure steam at 121° C. for 30 minutes to obtain the Tripterygium wilfordii matrix;

[0045] (2) Activation of strains: In a clean bench, a small piece of culture was scraped from the slant test tube of Ganoderma lucidum culture and placed on a PDA plate at 28°C and 60% humidity for 4 days to obtain a seed plate;

[0046] (3) Seed solution preparation: Take about 1 cm of the activated culture on the seed plate. 2 The bacterial blocks of different sizes were placed in 50 mL of PDB liquid culture medium and cultured at 28°C, 200 rpm for 6 days to obtain the fermentation seed liquid;

[0047] (4) The fermented seed liquid was inoculated into the Tripterygium wilfordii matrix in an ultra-clean work environment at a ratio of 50 g of Tripterygium wilfordii crushed material to 50 mL of seed liquid. After being fully mixed, the mixture was allowed to ferment at 23-25° C. and 60-70% humidity for 21 days. The fermentation was then stopped and the fermentation product was taken out. The surface of the fermentation product was covered with white mycelium and dried at 60° C.

[0048] The fermentation product was post-processed and the quality evaluation was performed as follows:

[0049] Liquid chromatography-mass spectrometry detection of the active ingredients of the fermentation product: take a certain amount of fermentation drying product, add 10 times the volume (mass: volume) of 75% ethanol to fully soak and extract, repeat 3 times, then combine the extracts for concentration, concentrate to 1 times the volume equivalent of the product, and then perform liquid chromatography-mass spectrometry detection.

[0050] Combined with the liquid chromatography-mass spectrometry detection results, the fermentation quality can be reasonably evaluated.

[0051] Toxicity testing of fermentation products: Take a certain amount of fermentation dried product, add 10 times the volume (mass: volume) of distilled water to fully soak and extract, repeat 3 times, then combine the extracts and concentrate to 10 times the volume equivalent of the product, and then spray dry; use the zebrafish animal model to observe its hatching rate and liver toxicity at specific concentrations.

[0052] Example 2: Effect of Ganoderma lucidum strains on the attenuation of Tripterygium wilfordii

[0053] The process of Example 1 was adopted, 50 g of Tripterygium wilfordii substrate was taken, and different Ganoderma lucidum strains were inoculated respectively to carry out bidirectional solid fermentation, and the crude drug was inoculated with sterile water as a control group.

[0054] After the fermentation is completed, the toxicity comparison of the processed Tripterygium wilfordii is carried out. In order to be as consistent as possible with the clinical medication form, the toxicity evaluation is carried out by extracting the fermentation product with distilled water, concentrating it to a relative density of more than 1.05, and finally spray drying to obtain the fermented and processed Tripterygium wilfordii powder.

[0055] Wild-type AB zebrafish at 1-1.5 h post-fertilization (1-1.5 hpf) were randomly selected in a 6-well plate, and 30 zebrafish were treated in each well (experimental group). The samples were given water-soluble, and the concentration of water-soluble Tripterygium wilfordii powder was 500 μg / mL. A normal control group and a solvent control group were set up at the same time, and the volume of each well was 3 mL. The treatment was carried out at 28℃ for 5 days, and the number of zebrafish deaths in each experimental group was counted every day and the dead zebrafish were removed in time. Finally, statistical analysis was performed to calculate the LC of the samples. 50 , the results are shown in Table 1 below.

[0056] Table 1. Acute toxicity results of processed Tripterygium wilfordii on zebrafish

[0057]

[0058]

[0059] Note: TW is the crude drug control of Tripterygium wilfordii, TW-G10 is the fermentation group of Tripterygium wilfordii and Chinese Ganoderma lucidum G10, TW-ZZ is the fermentation group of Tripterygium wilfordii and purple Ganoderma lucidum, TW-PJ is the fermentation group of Tripterygium wilfordii and bonsai Ganoderma lucidum, TW-LJ is the fermentation group of Tripterygium wilfordii and antler Ganoderma lucidum, TW-HZ is the fermentation group of Tripterygium wilfordii and Korean Ganoderma lucidum, TW-MD is the fermentation group of Tripterygium wilfordii and American Ganoderma lucidum, TW-SS is the fermentation group of Tripterygium wilfordii and Pinus spruce Ganoderma lucidum.

[0060] The above results show that the median lethal concentration (LC50) of the control group of Tripterygium wilfordii 50 ) was 829 μg / mL, and the LC of TW-G10 and TW-ZZ 50The results showed that the levels of Tripterygium wilfordii fermented with Ganoderma lucidum G10 and Ganoderma lucidum showed obvious attenuation effect, among which Ganoderma lucidum G10 had the best attenuation effect.

[0061] Example 3: Effect of fermentation and processing on chemical components of Tripterygium wilfordii

[0062] Using the process of Example 1, 50 g of Tripterygium wilfordii matrix was taken and inoculated with Ganoderma lucidum G10 and Ganoderma lucidum in proportion for bidirectional solid fermentation, and the crude drug was inoculated with sterile water as a control group.

[0063] In order to ensure the accuracy of the test and to ensure that the effective substances can be fully extracted, the fermented product was extracted with 75% ethanol, concentrated, and the contents of triptolide, triptolide B, rafenolide, triptolide, and triptolide A were detected using a liquid chromatography-mass spectrometer. Figure 2 As shown in the figure, TW is a crude drug control of Tripterygium wilfordii, TW-G10 is a fermentation product of Tripterygium wilfordii and Ganoderma lucidum G10, and TW-Z is a fermentation product of Tripterygium wilfordii and Ganoderma lucidum. The ion peak intensity (i.e., relative content) of the mass spectrometry signal is shown in Figure 3 shown.

[0064] Figure 2 and 3 The results showed that after fermentation with Ganoderma lucidum, the contents of diterpenoids triptolide, triptolide B and rafenolide in the toxic components of Tripterygium wilfordii were significantly reduced, while the contents of triterpenoids triptolide and triptolide A did not decrease significantly. Among them, Ganoderma lucidum had the most significant effect on reducing the contents of triptolide and triptolide B, and Ganoderma lucidum G10 had the most significant effect on reducing the contents of rafenolide.

[0065] Example 4: Effect of fermentation and processing on target organ toxicity of Tripterygium wilfordii

[0066] Using the process of Example 1, 50 g of Tripterygium wilfordii matrix was taken and inoculated with Ganoderma lucidum G10 and Ganoderma lucidum in proportion for bidirectional solid fermentation, and the crude drug was inoculated with sterile water as a control group.

[0067] In order to be as consistent as possible with the clinical medication form, the toxicity evaluation used distilled water to extract the fermentation product, concentrate it to a relative density of the concentrate of more than 1.05, and finally spray-dried it to obtain the fermented and processed Tripterygium wilfordii powder.

[0068] Wild-type AB strain zebrafish at 1-1.5h post-fertilization (1-1.5hpf) were randomly selected in a 6-well plate, and 30 zebrafish were treated in each well (experimental group). The samples were given water-soluble (through preliminary exploration of the concentration, 500μg / mL of water-soluble Tripterygium wilfordii powder was determined), and a normal control group and a solvent control group were set up at the same time, with a capacity of 3mL per well. Treated at 28°C for 5 days, the number of zebrafish hatched and the number of deaths in each experimental group were counted every day, and the dead zebrafish were removed in time. After the experiment, the liver of zebrafish in each experimental group was observed under a dissecting microscope, and the incidence rate (%) was counted. The acute toxicity of the samples to zebrafish embryos was evaluated by the incidence of liver toxicity, and the results are shown in Table 2 below.

[0069] Table 2. Hepatotoxicity of different Tripterygium wilfordii samples after treatment at specific concentrations

[0070]

[0071] The above results show that the control group of Tripterygium wilfordii was incubated at 4% at a concentration of 500μg / mL, and induced obvious liver toxicity, which was specifically manifested as delayed yolk sac absorption and accompanied by liver loss; after Ganoderma lucidum fermentation, the overall hatching rate was significantly improved, and the liver loss rate was reduced. The hatching rate of the fermentation product of Ganoderma lucidum G10 increased by 15 times, and the liver loss rate was reduced by 2.6 times. The reduction in corresponding toxicity was the most significant.

Claims

1. A fermentation method for reducing the toxicity of Tripterygium wilfordii, characterized in that: The following steps are involved: (1) Preparation of Tripterygium wilfordii matrix: crush the Tripterygium wilfordii and sterilize with high pressure steam to obtain the Tripterygium wilfordii matrix; (2) Bidirectional solid fermentation: The fermented seed liquid of the Ganoderma lucidum strain is inoculated into the Tripterygium wilfordii matrix of step (1), and after standing for fermentation, the fermentation product is taken out and dried.

2. The method according to claim 1, characterized in that: In the step (1), peeled Tripterygium wilfordii root slices are selected and mechanically coarsely crushed to less than 20 meshes.

3. The method according to claim 1, characterized in that: The high pressure steam sterilization conditions of step (1) are 110-130° C., 10-40 min; preferably 121° C., 30 min.

4. The method according to claim 1, characterized in that: The Ganoderma lucidum strains selected in step (2) are Ganoderma lucidum G10 and Ganoderma lucidum.

5. The method according to claim 1, characterized in that: The preparation of the fermented seed liquid of the Ganoderma lucidum species in step (2) should be as follows: (a) Strain activation treatment: Ganoderma lucidum strains were cultured on PDA plates to obtain seed plates; (b) preparing the seed solution, taking the activated culture bacteria block on the plate in step (a), placing it in a PDB liquid culture medium for culture, and obtaining a fermentation seed solution; More specifically, in step (a), a small piece of culture is scraped from the Ganoderma lucidum culture slant test tube and placed on a PDA plate, and the culture conditions are 28° C. and 60% humidity for 3-5 days, preferably 4 days; The step (b) is to take the activated culture bacteria block on the plate in step (a), place it in PDB liquid culture medium, and culture it at 28° C. and 200 rpm for 5-7 days, preferably 6 days.

6. The method according to claim 1, characterized in that: The step (2) is carried out using pure strain fermentation.

7. The method according to claim 1, characterized in that: In the step (2), 50 g of the crushed Tripterygium wilfordii is inoculated with 50 mL of seed solution, the mixture is fully mixed, and then fermented at 23-25° C. and 60-70% humidity.

8. The method according to claim 1, characterized in that: The fermentation period of step (2) is 18-22 days, preferably 21 days.

9. The method according to claim 1, characterized in that: The drying in step (4) is performed at 50-70°C, preferably at 60°C; optionally, a subsequent detection step is also included.

10. The fermentation product obtained by the method according to any one of claims 1 to 9 or a product prepared therefrom.

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