Application of Qijia Shengbai Capsule in the preparation of drugs for regulating intestinal microbial flora disorder

Abstract

The application discloses application of Qijiao Shengbai capsules in preparation of medicines for regulating intestinal microorganism flora disorder. The Qijiao Shengbai capsule is composed of Xue Renshen, Huangqi, Ajiao, Danggui, Da Zao, Ku Shen and Yinyanghe. By using the Qijiao Shengbai capsule, the microorganism flora is increased in the level of Firmicutes, increased in the level of Proteobacteria, reduced in the level of Bacteroidetes, reduced in the level of Akkermansia muciphila, and reduced in the level of Actinobacteria; in the level of genus, the microorganism flora is increased in the level of Prevotella, increased in the level of Lactobacillus, increased in the level of Parabacteroides, reduced in the level of Bacteroides, reduced in the level of Enterobacter, and reduced in the level of Staphylococcus, so as to regulate the intestinal microorganism flora. The application provides a new idea for research and development of medicines for preventing and treating intestinal flora disorder.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and particularly relates to application of Qijiaoshengbai capsule in preparation of medicines for regulating intestinal microbial flora disorder. BACKGROUND

[0002] Intestinal flora is the most complex and largest number of microbial flora in human body, containing a large number of bacterial species, more than 1000, and the number is about 10 times the total number of human cells. These intestinal microorganisms participate in physiological activities such as material metabolism and nutrient absorption and synthesis of the body, maintain normal physiological activities such as immunity and metabolism of the human body, and establish a mutually beneficial symbiotic relationship with the body. The intestine contains the largest proportion of immune cells. It is indispensable to expose to new antigens and immune stimulations from diet, microbiota and intestinal pathogens. It is an indispensable “metabolic organ” that plays a crucial role in maintaining the homeostasis of the body, mainly including digestion, nutrient absorption, energy supply, immune regulation and the like. The intestinal microbiota, including bacteria, archaea, fungi, viruses and bacteriophages, inhabits in the gastrointestinal tract. This symbiotic microbial flora helps to regulate the host immune response and internal balance. And changes in intestinal flora are found in many immune-related diseases. The intestinal microbiota is a complex and highly dynamic microbial community. As the largest and most complex microecological system in the human body, intestinal microorganisms and their metabolites not only regulate human health, but also play an important role as a bridge between diet and host.

[0003] Qijiaoshengbai capsule (QJSB) is a famous Miao medicine composed of seven components, including Xueshen- shen, Huangqi, Ejiao, Danggui, Dazao, Kuqian and Yinyang- huo, with a ratio of 3:6:2:2:2:2:3. It is clinically used for treating leukopenia. According to the functional classification in modern Chinese medicine, Dazao and Huangqi belong to qi- tonifying drugs; Danggui and Ejiao belong to blood- tonifying drugs; Yinyang- huo belongs to yang- tonifying drugs; Kuqian has the effect of clearing heat and drying dampness; and Xueshen- shen has the effects of nourishing yin and invigorating deficiency and promoting blood circulation. The combination of the medicinal materials in the prescription has the effects of tonifying qi and invigorating blood. Related literatures report that Qijiaoshengbai capsule (QJSB) can regulate the immune system and treat leukopenia. However, the effect of Qijiaoshengbai capsule on regulating intestinal microbial flora has not been reported. SUMMARY

[0004] The purpose of the present application is to provide the application of Qijiaoshengbai capsule in preparation of medicines for regulating intestinal microbial flora disorder, and to provide a new idea for the research and development of medicines for preventing and treating intestinal flora disorder.

[0005] The technical scheme of the present application is as follows:

[0006] The application of Qijiaoshengbai capsule in preparation of medicines for regulating intestinal microbial flora disorder.

[0007] The application of Qishen Shengbai Capsules in the preparation of a medicine for regulating intestinal microbial flora disorder of patients with leukopenia.

[0008] The aforementioned microbial flora comprises, at the door level, Firmicutes microbial flora, Proteobacteria microbial flora, Bacteroidetes microbial flora, Akkermansia muciniphila microbial flora, and Actinobacteria microbial flora, and comprises, at the genus level, Bacteroides, Prevotella, Lactobacillus, Alistipes, Enterorhabdus, and Staphylococcus.

[0009] The aforementioned microbial flora comprises, at the door level, increased Firmicutes microbial flora, increased Proteobacteria microbial flora, decreased Bacteroidetes microbial flora, decreased Akkermansia muciniphila microbial flora, and decreased Actinobacteria microbial flora, and comprises, at the genus level, increased Prevotella, increased Lactobacillus, increased Alistipes, decreased Bacteroides, decreased Enterorhabdus, and decreased Staphylococcus.

[0010] A medicine for regulating intestinal microbial flora, comprising Qishen Shengbai Capsules.

[0011] A medicine for regulating intestinal microbial flora of patients with leukopenia, comprising Qishen Shengbai Capsules.

[0012] Compared with the prior art, the application has the following beneficial effects:

[0013] After the use of Qishen Shengbai Capsules, compared with the model group, at the door level, Firmicutes is increased by 28.08%, Proteobacteria is increased by 74.81%, Bacteroidetes is decreased by 7.84%, Akkermansia muciniphila is decreased by 96.00%, and Actinobacteria is decreased by 37.68%; compared with the model group, at the genus level, Prevotella is increased by 24.81%, Lactobacillus is increased by 124.54%, Alistipes is increased by 89.39%, Bacteroides is decreased by 20.87%, Enterorhabdus is decreased by 32.90%, and Staphylococcus is decreased by 56.08%, so as to regulate the intestinal microbial flora and make the overall flora distribution close to that of the normal group. Moreover, the content of acetic acid, butyric acid and valeric acid in the intestinal short-chain fatty acid is affected, so that the content of acetic acid, butyric acid and valeric acid is significantly higher than that of the model group of mice administered with cyclophosphamide (p<0.05). BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 NMDS plot of mice in the model group and the QJSB group (n=7);

[0015] Figure 2 Bray-curtis Anosim plot of mice in the model group and the QJSB group (n=7);

[0016] Figure 3QJSB on mice 16S rRNA-related indicators (n=7) (A ACE index; B chao1 index; C Shannon index; D observed index);

[0017] Figure 4 : The top ten colonies of mice 16S rRNA door level (n=7);

[0018] Figure 5 : UPGMA mice 16S rRNA door level clustering analysis chart (n=7);

[0019] Figure 6 : Mice 16S rRNA genus level flora (n=7);

[0020] Figure 7 : The concentration of SCFAs in cecal contents (***P<0.001, **P<0.01). DETAILED DESCRIPTION

[0021] Example 1

[0022] Prescription: Xueshen 375g, Huangqi 750g, Ejiao 250g, Danggui 250g, Dazao 250g, Kushen 250g and Yiyanghe 375g;

[0023] Preparation process:

[0024] (1) The above-mentioned medicinal materials are weighed according to the weight ratio and prepared for use;

[0025] (2) Take Ejiao and make it into a paste, and prepare for use;

[0026] (3) Take Danggui and crush it into fine powder, and prepare for use;

[0027] (4) Xueshen, Huangqi, Dazao, Kushen and Yiyanghe are decocted with water for three times, the first time for 2 hours, the second time for 1.5 hours and the third time for 1 hour. The decoction is combined and filtered. The filtered liquid is added to the pasted Ejiao, stirred evenly, concentrated to a thick paste with a relative density of 1.28-1.32 at 80°C, and then the Danggui fine powder is added and mixed evenly to form granules. The granules are dried, filled into capsules, and Qijiao Shengbai Capsules are obtained.

[0028] Usage and dosage: orally take 4 capsules at a time, 3 times a day.

[0029] Effect: Prevent or treat intestinal flora imbalance in patients with leukopenia.

[0030] The inventors have conducted a large number of experiments, and the following are some experimental studies

[0031] 1 16S rRNA sequencing experiment

[0032] 1.1 Animal modeling and grouping

[0033] A total of 30 male (ICR) mice (18±2 g) were provided by Guizhou Medical University (Animal License No. SCXK(Xiang)2022-0011), kept in a standard animal laboratory with temperature (25±1°C), humidity (60±5%), light / dark cycle 12 hours, and free access to water and food. All animal experiments were reviewed and approved by the Institutional Animal Ethics Committee, and were strictly in accordance with the "Guidelines for the Care and Use of Animals at Guizhou Medical University". After one week of adaptive feeding, 30 mice were randomly divided into three groups (n=10): normal group (Normal), model group (Model) and Qijiao Shengbai Capsule group (QJSB). After grouping, all mice were mixed and co-fed. Modeling: (1) The Normal group of mice was intraperitoneally injected with 0.1 mL of 0.9% normal saline for 3 consecutive days. (2) The Model group of mice was intraperitoneally injected with 100 mg / kg / d cyclophosphamide (CTX) 0.1 mL for 3 consecutive days. (3) Correspondingly, the QJSB group was consistent with the modeling method of the Model group, but from the 4th day after modeling, QJSB solution (1.0 g / kg / d, for 14 consecutive days) 0.3 ml was administered by gavage. The mice in the Normal and Model groups were administered with the same solvent (i.e.: distilled water) according to the method and dosage of the QJSB group.

[0034] 1.2 Description of 16S rRNA sequencing method

[0035] 1.2.1 Sequencing section

[0036] 1.2.1.1 Extraction and PCR amplification of genomic DNA

[0037] The genomic DNA of the sample was extracted by CTAB or SDS method, and then the purity and concentration of the DNA were detected by agarose gel electrophoresis. An appropriate amount of sample DNA was taken in a centrifuge tube, and the sample was diluted to 1 ng / μl with sterile water.

[0038] Using the diluted genomic DNA as a template, according to the selection of the sequencing region, specific primers with Barcode were used, New England Biolabs' High-Fidelity PCR Master Mix with GC Buffer, and high-efficiency high-fidelity enzyme were used for PCR to ensure amplification efficiency and accuracy.

[0039] Primer corresponding region:

[0040] 16S V4 region primers (515F and 806R): identify bacterial diversity;

[0041] ​18S V4 region primers (528F and 706R): identify eukaryotic microbial diversity;

[0042] ITS1 region primers (ITS5-1737F and ITS2-2043R): identify fungal diversity;

[0043] In addition, the amplification region also includes: 16S V3-V4 / 16S V4-V5 / 16SV5-V7; archaea 16S V4-V5 / archaea 16S V8; 18S V9 and ITS2 region.

[0044] 1.2.2.2 Mixing and purification of PCR products

[0045] The PCR products are detected by electrophoresis using an agarose gel with a concentration of 2%; the qualified PCR products are purified by magnetic beads, and the enzyme-labeled quantity is determined, and the PCR products are mixed in equal amounts according to the concentration of the PCR products, and then detected by electrophoresis using an agarose gel with a concentration of 2%; the target band is recovered by using the gel recovery kit provided by Qiagen.

[0046] 1.3 Library construction and sequencing

[0047] Using The library construction is performed using the DNA PCR-Free Sample Preparation Kit, and the constructed library is quantified by Qubit and Q-PCR. After the library is qualified, NovaSeq6000 is used for sequencing.

[0048] 1.3.1 16S rRNA sequencing

[0049] The results of 16S rRNA sequencing are as follows. Figure 1 The NMDS plot shows that the intestinal microbial composition curves of the Normal, Model and QJSB groups are obviously separated, indicating that the differences between the groups are obvious, and the QJSB group is sandwiched between the Normal group and the Model group, indicating that QJSB has a regulating effect on the intestinal microorganisms of mice, proving its therapeutic effect. From the Bray-curtis Anosim( Figure 2 ) can be seen that the longitudinal coordinate of the first box is higher than that of the other boxes, indicating that the sample difference between groups is greater than that within groups (R>0).

[0050] Figure 3 (A-D) are Ace, chao1, shannon, observed index, each index is improved compared with the Model group, and tends to be closer to the Normal group. It is proved that after taking QJSB, the intestinal microbial flora disorder caused by CTX can be adjusted, and the composition and richness of the community tend to be closer to the normal group.

[0051] Figure 4 Figure 11 shows the results of species annotation analysis of the three groups revealed by 16S rRNA sequencing at the phylum level. The figure shows the top ten most abundant bacteria at the phylum level, with the remaining species combined into "Others", and unclassified species representing species that were not annotated.

[0052] At the phylum level, the intestinal microorganisms of mice were mainly composed of p_Bacteroidetes, p_Fimicutes, p_Proteobacteria, p_verrucomicrobiota and p_Actinobacteria. Compared with the model group, the p_Fimicutes increased by 28.08%, the p_Proteobacteria increased by 74.81%, the p_Bacteroidetes decreased by 7.84%, the p_verrucomicrobiota decreased by 96.00%, and the p_Actinobacteria decreased by 37.68% in the QJSB group. Figure 5 Figure 12 is a UPGMA clustering analysis chart at the phylum level, and the results show that the OUT between the Normal group and the QJSB group is small, indicating that the difference between the two is relatively small and more similar.

[0053] Figure 6 At the genus level, QJSB mainly regulated the dominant genera of p_Bacteroides, p_Alloprevotella, p_Lactobacillus, p_Alistipes, etc., while down-regulating p_unidentified_Enterobacteriaceae and p_Staphylococcus. Among them, p_Alloprevotella increased by 24.81%, p_Lactobacillus increased by 124.54%, and p_Alistipes increased by 89.39%, while p_Bacteroides decreased by 20.87%, p_unidentified_Enterobacteriaceae decreased by 32.90%, and p_Staphylococcus decreased by 56.08%. This indicates that after taking QJSB, the composition and abundance of intestinal microorganisms can be changed by increasing a series of beneficial bacteria and reducing pathogenic bacteria, so that the overall bacterial community distribution tends to be close to the normal group, and the body develops in a healthy direction, thereby maintaining the intestinal homeostasis.

[0054] Table 1: Abundance of intestinal microbial flora in the normal group, model group and QJSB

[0055]

[0056] Table 2: Abundance of intestinal microbial flora in the normal group, model group and QJSB

[0057]

[0058]

[0059] 1.3.2 Determination of short-chain fatty acids

[0060] 1.3.3.1 Method

[0061] Take 200 mg of cecal contents into a 2 mL EP tube, add 1 mL of ultrapure water, vortex for 2 minutes to mix the cecal contents and ultrapure water thoroughly. Place in a centrifuge at 1000 rpm for 10 minutes, take the supernatant through a 0.45 μm water membrane, and the filtrate is placed in an EP tube. Add 7 μL of 50% sulfuric acid solution, 1 mL of ether solution to the filtrate, vortex for 2 minutes, centrifuge at 1000 rpm for 10 minutes, and place in a 4°C refrigerator for extraction for 30 minutes. Take the ether layer through a 0.45 μm oil membrane and place it in a sample bottle for GC detection.

[0062] GC analysis was performed using an Agilent gas chromatograph (Agilent 7980A, Agilent Technologies, Santa Clara, USA) and a flame ionization detector (FID), which was equipped with an InertCap WAX GC column (30 m x 0.53 mm i.d. film thickness 1 μm, Agilent) and high-purity nitrogen as the carrier gas. Non-split injection was used with an injection volume of 2 μL. The initial column temperature was maintained at 100° for 1 minute, then increased to 150° at a rate of 5° for 7 minutes. The FID detector was maintained at 280°C. The content of SCFA was determined using an external standard method.

[0063] 1.4 Results

[0064] To investigate the effect of QJSB on the mouse intestine, the concentrations of acetic acid, propionic acid, butyric acid and valeric acid in the cecal contents were determined (Table 3 and Table 4). The results showed that QJSB significantly increased the concentrations of acetic acid, butyric acid and valeric acid (p < 0.01), but there was no significant difference in the concentration of propionic acid compared with the model group. These data suggest that QJSB can promote the production of SCFAs in mice. Figure 7 Table 3 Concentrations of acetic acid, butyric acid and valeric acid in normal, model and QJSB groups (mg / L)

[0065]

[0066]

[0067]

[0068] Table 4 Ratios of acetic acid, butyric acid and valeric acid in normal, model and QJSB groups (mg / L) ​

[0069]

[0070] 1.5 Analysis

[0071] In terms of gut microbiota, the results of this study found that the diversity, richness, and evenness of gut microbiota in QJSB group mice were higher than those in model group mice. The abundance of bacteria also differed at the phylum and genus levels. Gut microbiota can significantly affect the function of the intestinal barrier. Some intestinal probiotics (such as lactobacilli, bifidobacteria, a few strains of escherichia coli, and a new generation of probiotics, including bacteroides and mucous akkermansia) can maintain intestinal epithelial homeostasis and promote health. The 16S rRNA results in this study showed that QJSB mainly mediated the effects of six intestinal microorganisms at the genus level, including: (g_Bacteroides), (g_Alloprevotella), (g_Lactobacillus), (g_Alistipes), (g_unidentified_Enterobacteriaceae), and (g_Staphylococcus). Among them, (g_Bacteroides) is one of the main lineages of bacteria, which appeared early in the evolutionary process. A recent study showed that the relationship between bacteroides and the human host is a process of mutualistic symbiosis, and the bacteroides genus is considered beneficial only at the appropriate abundance. The results of this study showed that QJSB can effectively regulate the cyclophosphamide-induced bacteroides disorder, restore it to normal levels, and thus have a positive effect on intestinal health. (g_Alloprevotella) is generally considered a bacterium associated with a healthy plant-based diet, which plays a "probiotic" role in humans, and the core role of this microorganism in human health and disease is to protect against invasive pathogens by shaping immune development, immune responses, and metabolism. (g_Alloprevotella) is also considered a producer of short-chain fatty acids. Lactobacilli have a relatively small proportion in the gut microbiota, but due to their wide application in intestinal health and disease, they maintain the stability of the intestinal microbiome by producing various trypsin-like metabolic products that reduce the permeability of the intestinal tract to glycolytic probiotics. They are mostly non-pathogenic and can produce antimicrobial substances such as hydrogen peroxide and bacteriocins. The probiotic lactobacillus rhamnosus GR-1 has been shown to induce macrophages to produce G-CSF, and IL-6 deficiency reduces lactobacilli. Since the early 20th century, lactobacilli have been considered to compete with pathogenic bacteria, including streptococci, escherichia coli, and staphylococcus aureus, and play a key inhibitory role in human health. It has also been reported that taurine can significantly regulate the intestinal flora and reverse the decrease in lactobacilli abundance, suggesting that lactobacilli are promoted by the increase in intestinal taurine content. Lactobacilli are a potential probiotic and are also effective producers of acetic acid and butyric acid.This is consistent with our research results, that the abundance of Enterobacteriaceae and Staphylococcus in the QJSB group was significantly lower than that in the model group, confirming the competitive effect of Lactobacillus on pathogenic bacteria and the reliability of the experimental results, and hypothesizing that it would increase the levels of colonic G-CSF and IL-6, and promote the increase in acetic acid and butyric acid. g_Alistipes belongs to the Gram-negative bacteria of the phylum Mycobacterium, and it is a relatively new genus of bacteria that is mainly isolated from medical and clinical samples. Due to the diversity of the gastrointestinal microbiota, there is a strong correlation between dysbiosis and inflammatory bowel disease (IBD). From an ecological point of view, g_Alistipes is mainly present in the intestines of healthy individuals. It is also a bacteria that produces butyric acid and valeric acid. The link between the gut microbiota and short-chain fatty acids is closely related. Therefore, the content of SCFAs in the cecum of mice was determined in this study. The results showed that QJSB can up-regulate the content of acetic acid, butyric acid and valeric acid in the cecum of mice. SCFAs have an impact on the integrity of the human intestinal epithelium and mucosal barrier, immune response and diversity of the microbiota. Acetate promotes the differentiation of Treg cells, stimulates FOXP3 transcription, and also positively affects the enhancement of mucosal immunity. Butyrate supports the integrity of the intestinal epithelial barrier by regulating the expression of tight junction proteins and supporting the production of intestinal mucus. It has been shown that butyrate helps intestinal motility by acting as a ligand and activator of SCFAs receptors. The mucosal barrier monolayer of intestinal epithelial cells and their adjacent mucosal layer are the first line of defense for the host's intestinal immunity, and butyrate has a direct beneficial effect on supporting the integrity of the intestinal epithelial barrier. QJSB changes the composition of the intestinal flora, increases the level of SCFA-producing bacteria, and affects the levels of acetic acid, valeric acid and butyric acid, thereby regulating the homeostasis of the intestinal immune system.

[0072] When the immune function of the body decreases, it leads to the proliferation of Escherichia coli, causing damage to the gastrointestinal mucosa and inducing an inflammatory response in the intestinal mucosa. Studies have shown that L-serine has a competitive relationship with Enterobacteriaceae in the intestine. In this study, QJSB can effectively reduce the content of Enterobacteriaceae and restore it to normal levels. It confirms that L-serine can reduce the content of pathogenic Enterobacteriaceae and restore the damaged intestinal mucosa and inflammation. It is hypothesized that after taking QJSB, the related intestinal metabolites may have a synergistic effect, working together to combat pathogenic bacteria and thus maintain intestinal health. The study confirms that QJSB treats diseases by regulating the dominant flora in the intestine, reducing pathogenic bacteria and moving the organism towards a more beneficial direction.

Claims

1. The application of Astragalus Capsules in the preparation of drugs for regulating intestinal microbiota dysbiosis in patients with leukopenia, characterized in that: The microbial flora comprises increased Firmicutes microbial flora, increased Proteobacteria microbial flora, reduced Bacteroidetes microbial flora, reduced Akkermansia muciniphila microbial flora, and reduced Actinobacteria microbial flora at the door level; and comprises increased Prevotella, increased Lactobacillus, and increased Parabacteroides at the genus level, and reduced Bacteroides, reduced Enterobacter, and reduced Staphylococcus. The medicinal effective components of the Qijia Shengbai capsule are prepared from 3 parts of blood ginseng, 6 parts of astragalus, 2 parts of donkey-hide gelatin, 2 parts of angelica, 2 parts of jujube, 2 parts of sophora, and 3 parts of epimedium.

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