Use of lachnospiraceae bacteria in preparation of medicine for reducing hepatotoxicity of saikosaponin D

Abstract

The application discloses application of Lachnospiraceae bacteria in preparation of a medicine for reducing hepatotoxicity of saikosaponin D, and relates to the field of medicines. The application identifies and applies an intestinal bacteria which has specific antagonism to hepatotoxicity of saikosaponin D for the first time. This breaks the limitation of traditional non-specific regulation through broad-spectrum probiotics or complex compounds, and realizes a leap from'macroscopic flora regulation' to 'key strain targeting'. The strategy directly aims at a key link of SSd toxicity, can effectively protect the liver while retaining the core efficacy of SSd to the greatest extent, and truly achieves the final goal of 'attenuation and efficacy reservation'.

Description

Technical Field

[0001] This invention relates to the field of pharmaceuticals, specifically to the application of bacteria from the family Trichophyceae in the preparation of drugs that reduce the hepatotoxicity of saikosaponin D. Background Technology

[0002] Bupleurum, a traditional Chinese medicine, possesses the effects of soothing the liver and relieving stagnation, as well as harmonizing the exterior and interior. It is widely used clinically to treat diseases such as liver qi stagnation, colds, and fever. Saikosaponin D (SSd) is the main active ingredient of Bupleurum and a key indicator component in the pharmacopoeia, exhibiting clear pharmacological activities such as hepatoprotection, anti-inflammation, anti-tumor, and immunomodulatory effects. However, SSd exhibits a significant "coexistence of efficacy and toxicity": on the one hand, it is the core substance for Bupleurum to exert its hepatoprotective and "soothing the liver and relieving stagnation" effects; on the other hand, it is the main toxic component causing "liver yin depletion" hepatotoxicity. This bidirectional regulatory effect severely restricts the safe clinical application of Bupleurum and SSd. Therefore, how to effectively reduce its hepatotoxicity without weakening the efficacy of SSd has become a crucial issue that urgently needs to be addressed in the modernization of traditional Chinese medicine and the rational use of drugs in clinical practice.

[0003] The gut microbiota is considered an important acquired "metabolic organ" in the human body, possessing a powerful capacity for drug metabolism. Through enzymatic reactions, the gut microbiota participates in the in vivo metabolism of exogenous drugs, directly influencing drug action processes such as in vivo processing and pharmacodynamic-toxicological responses by altering drug structure, bioavailability, bioactivity, or toxicity. This phenomenon is defined as "Pharmacomicrobiomics." When traditional Chinese medicine (TCM) is administered orally and reaches the intestines, its chemical components first come into contact with drug-metabolizing enzymes produced by the gut microbiota. Through metabolism by the gut microbiota, the chemical components of TCM are converted into corresponding active, inactive, or toxic metabolites. Simultaneously, the composition of the gut microbiota varies significantly depending on the body's syndrome state, directly affecting the body's ADMET / Act. (absorption, distribution, metabolism, excretion, toxicity, and activity) processes of drugs. This leads to greater differences in the body's sensitivity and tolerance to TCM components, thus influencing the presence or absence of TCM toxicity. Meanwhile, gut microbiota has a certain influence on the toxicity of Bupleurum, Bupleurum saponins and other traditional Chinese medicines that have both efficacy and toxicity. Differences in gut microbiota composition among different constitutions may be a potential reason for different people’s different tolerance to its toxicity.

[0004] Currently, the main strategies for reducing the hepatotoxicity of saikosaponin D include the following categories: (1) Structural modification and synthetic analogs: By chemically modifying the structure of SSd, its toxicity can be reduced while retaining its activity. For example, derivatives or analogs of saikosaponin D can be developed. However, this method is often complex and costly, and may change the natural properties and pharmacokinetics of the drug, resulting in limited clinical applicability. (2) Formulation technology optimization: By using delivery systems such as liposomes, nanoparticles, or cyclodextrin inclusion complexes, the solubility and targeting of SSd can be improved to reduce its accumulation in the liver. However, these technologies have problems such as poor stability and difficulty in large-scale production in practical applications, and the toxicity reduction effect is unstable. (3) Combination therapy and traditional Chinese medicine compound: By combining Bupleurum with other traditional Chinese medicines (such as Scutellaria baicalensis and Glycyrrhiza uralensis), the interaction between the traditional Chinese medicines can be used to antagonize toxicity. For example, the classic compound "Xiao Chai Hu Tang" is considered to be able to reduce the liver damage caused by Bupleurum. However, the toxicity reduction mechanism of this method is unclear, the effect is greatly affected by individual differences, and it lacks specificity. (4) Regulation of biological metabolic pathways: In recent years, studies have focused on the role of gut microbiota in the metabolism of traditional Chinese medicine. As the "acquired metabolic organ" of the human body, gut microbiota can alter the structure, bioavailability, and toxicity of exogenous drugs through enzymatic reactions. This field is known as "pharmaceutical microbiome." Studies have shown that gut microbiota participate in the metabolism of various traditional Chinese medicine components (such as rhubarb and Tripterygium wilfordii), affecting their toxicity and efficacy. Differences in the composition of gut microbiota among different individuals may lead to different tolerances to SSd toxicity, but specific attenuated strains against SSd have not yet been systematically discovered and applied. Existing literature mainly focuses on macroscopic microbiota regulation (such as fecal microbiota transplantation), lacking identification and mechanistic studies of specific key strains, thus failing to achieve precise attenuation.

[0005] In summary, existing attenuation methods have shortcomings in terms of specificity, stability, and clinical feasibility. In particular, there is a lack of SSd attenuation strategies based on targeted regulation of the gut microbiota, which fails to fundamentally solve the problem of "coexistence of efficacy and toxicity." Summary of the Invention

[0006] The purpose of this invention is to at least solve one of the technical problems existing in the prior art, and to provide the application of Trichophyceae bacteria in the preparation of drugs that reduce the hepatotoxicity of saikosaponin D.

[0007] The technical solution of the present invention is as follows:

[0008] Bacteria of the family Trichophyceae Lachnospiraceae bacterium Application in the preparation of drugs that reduce the hepatotoxicity of saikosaponin D.

[0009] Furthermore, the bacteria of the Trichophyceae family... Lachnospiraceae bacterium It can reduce serum AST, ALT and ALP levels induced by saikosaponin D.

[0010] Furthermore, the drug comprises saikosaponin D and bacteria from the Trichophyceae family. Lachnospiraceae bacterium .

[0011] Furthermore, the bacteria of the Trichophyceae family... Lachnospiraceae bacterium It accounts for 10-20% of the mass of saikosaponin D.

[0012] Furthermore, the drug also includes a pharmaceutically acceptable carrier.

[0013] Furthermore, the pharmaceutical preparation includes one of granules, tablets, capsules, oral liquids, injections, ointments, emulsions, and pills.

[0014] The beneficial effects of this invention are:

[0015] (1) The present invention provides a specific intestinal microbial strain that can specifically antagonize the hepatotoxicity induced by saikosaponin D.

[0016] (2) This invention provides a method for the application of this strain in preventing and / or reducing the hepatotoxicity of saikosaponin D, so as to effectively control the risk of liver damage while exerting the efficacy of SSd, and achieve the fundamental goal of "reducing toxicity while preserving efficacy".

[0017] (3) This invention provides a safe and efficient intervention program for the toxicity of traditional Chinese medicine based on the regulation of gut microbiota, which provides a new idea and practical tool for solving the clinical safety problem of traditional Chinese medicine with "coexistence of efficacy and toxicity" including Bupleurum, and develops a "microbiota-targeted toxicity reduction and efficacy preservation strategy". Attached Figure Description

[0018] Figure 1 The table shows the serum ALT, AST, and ALP activity levels in different groups of rats. A represents ALT activity, B represents AST activity, and C represents ALP activity. Compared with the normal control group, This indicates a significant difference (p < 0.001) in the SSd treatment group. This indicates a significant difference (p < 0.0001) in the SSd treatment group;

[0019] Figure 2 The results of hepatotoxicity index analysis in rats regrouped by phenotype are shown below. A represents ALT enzyme activity, B represents AST enzyme activity, and C represents ALP enzyme activity. These values ​​are compared with the hepatotoxicity group. This indicates a significant difference (p < 0.0001) between the normal control group and the non-hepatotoxic group.

[0020] Figure 3 This is the result of bioinformatics analysis, where A is... αDiversity analysis results; B represents NMDS analysis results based on Bray-Curtis distance; C represents the relative abundance of gut bacteria at the species level in the hepatotoxic group and the non-hepatotoxic group. Figure 4 The negative correlations between strains and hepatotoxicity indicators are shown, where A represents the negative correlation between strains and ALT; B represents the negative correlation between strains and AST; and C represents the negative correlation between strains and ALP.

[0021] Figure 5 The effect of bacterial strain intervention on the hepatotoxicity of SSd is shown in the figure. A represents the intervention flowchart; B represents the ALT activity level after intervention; C represents the AST activity level after intervention; and D represents the ALP activity level after intervention, compared with the SSd model group. This indicates a significant difference (p < 0.0001) between the normal control group and the bacterial strain intervention group. Detailed Implementation

[0022] The embodiments of the present invention are described in detail below. These embodiments are exemplary and are only used to explain the present invention, and should not be construed as limiting the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0023] The technical solution of the present invention will be further illustrated by specific experiments below.

[0024] Experiment 1: Establishment and Phenotypic Verification of a Saikosaponin D (SSd) Hepatotoxicity Animal Model: This invention first establishes an SSd hepatotoxicity model through animal experiments. Specifically, male SD rats were randomly divided into a normal control group and an SSd administration group after acclimatization. The SSd administration group was administered SSd at a dose of 60 mg / kg by gavage daily for 21 consecutive days to induce drug-induced liver injury. After modeling, serum was collected, and key hepatotoxicity indicators such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) were measured. The results showed ( Figure 1 Compared with the normal control group, the serum AST, ALT and ALP activities of rats in the SSd-treated group were significantly increased, successfully replicating the in vivo model of SSd-induced hepatotoxicity.

[0025] Experiment 2: Phenotypic Regrouping of Model Animals Based on Hepatotoxicity Indicators: Thirty SSd-treated rats were phenotypically regrouped according to the diagnostic criteria for drug-induced liver injury. The hepatotoxicity criteria were set as follows: ALT ≥ 3 times the mean of the normal control group or ALP ≥ 2 times the mean of the normal control group. Results showed that of the 30 SSd-treated rats, 11 were assigned to the non-hepatotoxic group and 19 to the hepatotoxic group. Analysis of hepatotoxicity indicators indicated (…).Figure 2 In the hepatotoxic group, the serum AST, ALT, and ALP enzyme activities of rats were significantly higher than those of the normal control group and the non-hepatotoxic group, while there were no significant differences in any of the indicators between the hepatotoxic group and the normal control group. This grouping result successfully obtained a comparative population with distinctly different hepatotoxic responses under the same drug administration background, laying an important foundation for subsequent mechanistic studies.

[0026] Experiment 3: Metagenomic Screening of Key Differential Strains Associated with SSd Hepatotoxicity: To explore the gut microbiological mechanisms underlying individual differences in SSd hepatotoxicity, this experiment collected colon contents from rats in both the non-hepatotoxic and hepatotoxic groups for metagenomic sequencing analysis. After total DNA extraction, quality control, library construction, and sequencing using the Illumina MiSeq platform, the data underwent systematic bioinformatics analysis. α-diversity analysis results showed ( Figure 3 In the non-hepatotoxic group (Group A), compared with the hepatotoxic group, the Chao1 index, Shannon index, and Simpson index were significantly increased, indicating that their gut microbiota had higher species richness and diversity. NMDS analysis based on Bray-Curtis distance showed (…). Figure 3 (B) The two groups of samples showed clear separation, confirming significant differences in their bacterial community structure (β-diversity). Further species composition analysis at the species level revealed that bacteria of the family Trichophyceae ( Lachnospiraceae bacterium The most significant difference in relative abundance was observed between the two groups, with the relative abundance in the non-hepatotoxic group (16.5%) being significantly higher than that in the hepatotoxic group (3.4%). Figure 3 The above results indicate differences in the structure and composition of the gut microbiota, particularly among bacteria of the Trichophyceae family (C). Lachnospiraceae bacterium The relative abundance of SSd is closely related to the occurrence of SSd-induced hepatotoxicity.

[0027] Experiment 4: Verification of the negative correlation between key strains and hepatotoxicity indicators: To confirm the direct association between the differentially expressed strains found in Experiment 3 and liver injury, this experiment further used the Pearson / Spearman correlation analysis method to analyze the bacteria of the Trichophyceae family ( Lachnospiraceae bacterium The relative abundance of [a specific substance] was correlated with hepatotoxicity indicators such as AST, ALT, and ALP. A correlation coefficient |r| > 0.6 and P < 0.05 was considered to indicate a significant correlation between the two. The results clearly showed that ([a specific substance] was associated with a specific hepatotoxicity indicator such as AST, ALT, and ALP). Figure 4 The relative abundance of this strain was significantly negatively correlated with all liver injury indicators (|r|>0.6, P<0.05), which statistically strongly suggests that it is a member of the Trichophyceae family (…). Lachnospiraceae bacterium () is a key potential functional strain for regulating SSd hepatotoxicity.

[0028] Experiment 5: Cultivation and Preparation of Key Strains: To achieve exogenous intervention of this strain, this experiment provides a specific cultivation and preparation method. Standard strains were purchased from the Beina Bio-Henan Provincial Industrial Microbial Strains Engineering Technology Research Center. Lachnospiraceae bacterium (BNCC354474) was revived and passaged in MRS solid medium at 37°C under anaerobic conditions. Cells in the logarithmic growth phase were harvested, centrifuged, washed, and resuspended in sterile PBS to prepare a 1×10⁻⁶ concentration. 9 A bacterial suspension of CFU / mL was prepared for use in subsequent animal intervention experiments.

[0029] Experiment 6: Verify its efficacy in reducing SSd hepatotoxicity through strain intervention experiments; to directly verify the efficacy of Trichophyton spp. ( Lachnospiraceae bacterium To assess the toxicity reduction effect of SSd, this invention designed a bacterial strain intervention experiment. Healthy SD rats were randomly divided into three groups (n=10 per group): a normal control group, an SSd model group, and a bacterial strain intervention group. The SSd model group was administered SSd (60 mg / kg) by gavage, while the bacterial strain intervention group was administered the same dose of SSd by gavage, combined with a bacterial suspension prepared in Experiment 5 (1 mL / 100 g) for 21 consecutive days to evaluate the antagonistic effect of this bacterial strain on the hepatotoxicity of SSd. In the bacterial strain intervention experiment, bacteria from the Trichophyceae family... Lachnospiraceae bacterium The calculation of the proportion of SSd mass is based on the following: the concentration of the bacterial suspension prepared in Experiment 5 is 1×10⁻⁶. 9 The concentration of the bacterial suspension was calculated to be 1 mg / mL based on standard microbiological parameters (bacterial dry weight approximately 1 pg / CFU). The experimental rats were administered SSd at a dose of 60 mg / kg via gavage, with a simultaneous gavage volume of 1 mL / 100 g body weight. The calculated ratio of the gavage mass of the bacterial strain (1 mg / 100 g) to the gavage mass of SSd (0.06 mg / g, i.e., 60 mg / kg) per unit body weight was (1 / 100) / 0.06 = 1 / 6, indicating that the bacterial strain accounted for 16.7% of the SSd mass. Therefore, the bacteria belong to the Trichophyceae family. Lachnospiraceae bacterium A concentration of 10-20% of the mass of saikosaponin D is expected to be feasible.

[0030] Experiment 7: Evaluation of the effect of bacterial strain intervention on SSd hepatotoxicity: After the intervention experiment, serum samples were collected from rats in each group, and the levels of AST, ALT, and ALP were measured again to evaluate the efficacy. The results showed ( Figure 5 Compared with the normal control group, the hepatotoxicity indicators in the SSd model group were significantly elevated; while compared with the SSd model group, the above indicators in the bacterial strain intervention group were significantly reduced to near-normal levels. These results ultimately confirm that the combined supplementation... Lachnospiraceae bacterium It can significantly improve the hepatotoxicity induced by SSd, thus completing a complete technical solution from discovery and identification to functional verification.

[0031] In summary, the present invention has achieved the following results:

[0032] (1) This invention is the first to identify and apply an intestinal bacterium that has a specific antagonistic effect on the hepatotoxicity of saikosaponin D. This breaks through the limitations of traditional non-specific regulation through broad-spectrum probiotics or complex compound preparations, and achieves a leap from "macro-microbial community regulation" to "targeting key strains". This strategy directly targets the key links in the production of SSd toxicity, and can effectively protect the liver while retaining the core efficacy of SSd to the greatest extent, truly achieving the ultimate goal of "reducing toxicity while preserving efficacy".

[0033] (2) The strains used in this invention are derived from the symbiotic bacteria inherent in the intestines of mammals (such as rats and humans), and have extremely high biocompatibility. Using them as an exogenous supplement avoids the additional toxic side effects that may result from chemical drugs or complex formulations. This method lays the foundation for developing a novel intestinal symbiotic-based biotherapy to address the toxicity issues of traditional Chinese medicine.

[0034] (3) This invention provides key modern biological evidence for understanding the classic theories of Bupleurum chinense's "no harm when there is a cause" (i.e., the drug does not damage the body when there is a disease) and "efficacy and toxicity coexist." It reveals the potential reason for the differences in tolerance to the toxicity of saikosaponin D among different individuals—that is, whether there are sufficient protective strains in the intestine.

[0035] (4) This invention has the potential for industrialization and can be derived into various product forms. For example:

[0036] Live biological therapy products: Develop this strain into a high-purity probiotic preparation, which can be used in combination with Bupleurum or SSd preparations as a "toxicity reduction companion" for clinical medication.

[0037] Microbiome regulators: Develop prebiotics or functional foods that can specifically promote the growth and proliferation of the protective strain in the body.

[0038] Diagnostic and prognostic tools: By detecting the abundance of this strain in an individual's gut, the hepatotoxicity of SSd can be predicted, providing guidance for achieving "personalized precision medicine".

[0039] (5) Through rigorous animal experiments, this invention has demonstrated that supplementation with this Trichophyceae bacterium can significantly reduce serum AST, ALT, and ALP levels induced by SSd. Metagenomic analysis further revealed that the relative abundance of this strain is significantly negatively correlated with liver damage indicators, providing solid scientific data and clear mechanistic support for the attenuation effect, making the effect quantifiable and reproducible.

[0040] The embodiments described above are merely preferred embodiments of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various other corresponding changes and modifications based on the technical solutions and concepts described above, and all such changes and modifications should fall within the protection scope of the claims of the present invention.

Claims

1. Bacteria of the family Trichophyceae Lachnospiraceae bacterium The application of BNCC354474 in the preparation of drugs to reduce the hepatotoxicity of saikosaponin D, wherein the bacteria belong to the family Trichophyceae. Lachnospiraceae bacterium BNCC354474 was purchased from Beina Biotechnology - Henan Provincial Industrial Microbial Strains Engineering Technology Research Center.

2. The application according to claim 1, characterized in that, The drug includes saikosaponin D and the aforementioned Trichophyton family bacteria. Lachnospiraceae bacterium BNCC354474.

3. The application according to claim 2, characterized in that, The bacteria of the family Trichophyceae Lachnospiraceae bacterium BNCC354474 accounts for 10-20% of the mass of saikosaponin D.

4. The application according to claim 1, characterized in that, The drug also includes a pharmaceutically acceptable carrier.

5. The application according to claim 1, characterized in that, The drug includes one of the following: granules, tablets, capsules, oral liquids, injections, ointments, emulsions, or pills.

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