Application of addition and subtraction polygonatum odoratum decoction in preparation of anti-Klebsiella products and in-vitro screening of anti-Klebsiella active components

Abstract

The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to application of radix polygonati officinalis addition and subtraction decoction in preparation of anti-Klebsiella products and in-vitro screening of anti-Klebsiella active ingredients. It is found for the first time that the addition and subtraction polygonatum odoratum decoction has remarkable bacteriostatic activity on Klebsiella, can remarkably lower the abundance of Klebsiella in intestinal tracts, can further remarkably inhibit formation of Klebsiella biofilms, promotes intestinal barrier repair, and relieves damage and inflammation of the intestinal tracts and lungs. Therefore, the polygonatum odoratum addition and subtraction decoction can be used for preparing products for resisting Klebsiella or preparing medicines for improving intestinal inflammation or lung inflammation.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of traditional Chinese medicine, and particularly relates to application of modified Hui Liu decoction in preparation of products against Klebsiella and in vitro screening of active ingredients against Klebsiella. BACKGROUND

[0002] Klebsiella is a kind of gram-negative bacillus, belonging to Enterobacteriaceae, and mainly divided into 7 species, among which Klebsiella pneumoniae and Klebsiella oxytoca are the main pathogenic bacteria. Klebsiella Klebsiella pneumoniae is an important pathogen in hospital-acquired and community infections, which can cause pneumonia, urinary tract infection, bloodstream infection, liver abscess, meningitis, enteritis and other diseases. Klebsiella pneumoniae In recent years, Klebsiella pneumoniae shows resistance to many antibiotics, especially carbapenem-resistant strains (CRKP), which poses a major threat to public health. Klebsiella oxytoca In order to adapt to the surrounding environment, Klebsiella pneumoniae forms a biofilm. Klebsiella pneumoniae forms a complex structure on the surface of the host body or internal organs through the biofilm, thereby enhancing its adhesion ability and protecting it from the host immune system. Therefore, Klebsiella pneumoniae in the biofilm state shows significant antibiotic resistance, and conventional antibiotics are difficult to eradicate the biofilm. In addition to the combination of multiple antibiotics, surgical debridement or new anti-biofilm strategies (such as phage therapy) are also needed. There is no specific antibiotic for Klebsiella.

[0003] However, the large use of antibiotics (especially broad-spectrum antibiotics) can destroy the balance of intestinal flora, and pathogenic bacteria (such as Escherichia coli, Klebsiella, Enterococcus, etc.) overproliferate, while probiotics (such as Bifidobacterium, Lactobacillus) decrease. The toxins (such as endotoxins, enterotoxins) secreted by pathogenic bacteria can directly damage the intestinal mucosal barrier, increase intestinal permeability, and make bacteria and their metabolites enter the mucosal lamina propria, activate the immune system and trigger an inflammatory response.

[0004] SUMMARY

[0005] To solve the above technical problems, the application provides the application of modified Hui Liu decoction in preparation of products against Klebsiella and in vitro screening of active ingredients against Klebsiella. The application first finds that modified Hui Liu decoction has significant bacteriostatic activity against Klebsiella, can significantly down-regulate the abundance of Klebsiella in the intestinal tract, and has a significant inhibitory effect on the formation of Klebsiella biofilm. Therefore, modified Hui Liu decoction can be used for preparing products against Klebsiella, or for in vitro screening of active ingredients against Klebsiella.

[0006] To achieve the above application purposes, the application adopts the following technical solutions: ​The application provides the use of the added and subtracted Hei Liu decoction in the preparation of a product for resisting Klebsiella, and the active ingredients of the product comprise the added and subtracted Hei Liu decoction and / or processed products thereof.

[0007] The added and subtracted Hei Liu decoction is from the Qing Dynasty “Revised Popular Treatise on Cold Pathogenic Diseases”, and is composed of raw Hei Liu (Polygonatum odoratum), Radix Oxytropis, Radix Sophorae Tonkinensis, Allium fistulosum, Platycodon grandiflorum, Radix Glycyrrhizae and Mentha haplocalyx, and is used for treating common cold, cough caused by body deficiency of yin or lung yin deficiency.

[0008] The application finds that the added and subtracted Hei Liu decoction has significant bacteriostatic activity on Klebsiella, and can significantly down-regulate the abundance of Klebsiella in the intestinal tract; in addition, the added and subtracted Hei Liu decoction can significantly inhibit the formation of Klebsiella biofilm, down-regulate the expression of group induction self-inducing molecule-2 (AI-2, Autoinducer-2), down-regulate the levels of type III pilus related genes mrkA and group induction related genes luxS , and also has a down-regulation effect on genes such as Capsular Polysaccharide (CPS, Capsular Polysaccharide) related genes galF , type I pilus related genes fimH , iron transport protein related genes feoB and the like, which affect the formation of biofilm, and the inhibition effect on the formation of Klebsiella biofilm can be achieved through multiple mechanisms, and the inhibition test of the biofilm phenotype proves that the added and subtracted Hei Liu decoction can actually play a role in inhibiting the formation of Klebsiella biofilm. In addition, the animal experiment proves that the added and subtracted Hei Liu decoction not only can down-regulate the abundance of Klebsiella in the intestinal tract, but also can help to improve the inflammation and damage of the intestinal tract and the lung and reduce the level of inflammatory factors in the blood plasma.

[0009] At present, there is no report on the use of the added and subtracted Hei Liu decoction for inhibiting the proliferation of Klebsiella and inhibiting the formation of Klebsiella biofilm, and the application provides a safe and effective new choice for inhibiting Klebsiella and the formation of Klebsiella biofilm and reducing drug resistance, and can avoid the adverse reactions such as colitis caused by the large use of antibiotics.

[0010] Preferably, the added and subtracted Hei Liu decoction is prepared from the following raw materials in parts by mass: 16-20 parts of Polygonatum odoratum, 11-13 parts of raw Allium fistulosum, 8-10 parts of Platycodon grandiflorum, 5.5-6.5 parts of Radix Oxytropis, 22-26 parts of Radix Sophorae Tonkinensis, 8-10 parts of Mentha haplocalyx, 2.5-3.5 parts of fried Radix Glycyrrhizae and 35-45 parts of Fructus Jujubae.

[0011] Further preferably, the added and subtracted Hei Liu decoction is prepared from the following raw materials in parts by mass: 18 parts of Polygonatum odoratum, 12 parts of raw Allium fistulosum, 9 parts of Platycodon grandiflorum, 6 parts of Radix Oxytropis, 24 parts of Radix Sophorae Tonkinensis, 9 parts of Mentha haplocalyx, 3 parts of fried Radix Glycyrrhizae and 40 parts of Fructus Jujubae.

[0012] Further preferably, the preparation method of the Jiajian Heiyu Decoction is as follows: the raw materials except for peppermint and white onion are soaked in water, and then extracted by refluxing twice, each for 100-140 minutes, and the peppermint and white onion are added at 13-17 minutes before the end of the second extraction; and the extractives obtained in each extraction are combined to obtain the Jiajian Heiyu Decoction.

[0013] Preferably, the preparation method of the processed product is as follows: the Jiajian Heiyu Decoction is concentrated and / or dried.

[0014] Preferably, the product is a product for inhibiting the formation of Klebsiella biofilm.

[0015] Preferably, the product is a pharmaceutical product.

[0016] Further preferably, the pharmaceutical product is a product for intestinal inflammation or lung inflammation.

[0017] The present application discloses a Jiajian Heiyu Decoction and a preparation method thereof.

[0018] Further preferably, the dosage form of the pharmaceutical product is an oral preparation.

[0019] Optionally, the oral preparation includes, but is not limited to, a powder, a granule, a capsule, a tablet, etc.

[0020] Preferably, the product is a product for inhibiting the formation of Klebsiella biofilm.

[0021] Further preferably, the product is a daily-use product for resisting Klebsiella, such as a spray, a cleaning solution for environmental sterilization, a leave-on gel, a wet wipe for skin sterilization, etc.

[0022] The second aspect of the present application provides an application of the Jiajian Heiyu Decoction or a processed product thereof in in-vitro screening of an active ingredient for resisting Klebsiella or an active ingredient for affecting the expression of Klebsiella biofilm-related genes, wherein the in-vitro screening method takes the Jiajian Heiyu Decoction or the processed product thereof as a positive control drug.

[0023] Compared with in-vivo drug screening methods, in-vitro screening has the advantages of convenience, economy and high efficiency, and is a common means for exploring new active substances at present.

[0024] Preferably, the Jiajian Huixie Decoction is made from the following raw materials in mass parts: 16-20 parts of Polygonatum odoratum, 11-13 parts of Allium fistulosum, 8-10 parts of Platycodon grandiflorum, 5.5-6.5 parts of Cynanchum paniculatum, 22-26 parts of Soybean Glycine max, 8-10 parts of Mentha haplocalyx, 2.5-3.5 parts of Radix Glycyrrhizae, and 35-45 parts of Fructus Jujubae.

[0025] Further preferably, the Jiajian Huixie Decoction is made from the following raw materials in mass parts: 18 parts of Polygonatum odoratum, 12 parts of Allium fistulosum, 9 parts of Platycodon grandiflorum, 6 parts of Cynanchum paniculatum, 24 parts of Soybean Glycine max, 9 parts of Mentha haplocalyx, 3 parts of Radix Glycyrrhizae, and 40 parts of Fructus Jujubae.

[0026] Preferably, the preparation method of the Jiajian Huixie Decoction is as follows: the raw materials except for Mentha haplocalyx and Allium fistulosum are soaked in water, and then extracted with water for 2 times, each for 100-140 minutes, and the Mentha haplocalyx and Allium fistulosum are added at 13-17 minutes before the end of the second extraction; and the extractives obtained in each extraction are combined, to obtain the Jiajian Huixie Decoction.

[0027] Preferably, the preparation method of the processed product is as follows: the Jiajian Huixie Decoction is concentrated and / or dried.

[0028] The present application has the advantages that the present application first discovers that the Jiajian Huixie Decoction has significant bacteriostatic activity on Klebsiella and can effectively inhibit the formation of Klebsiella biofilm, thus reducing the drug resistance of Klebsiella and avoiding adverse reactions such as colitis caused by the large use of antibiotics. At present, there is no specific antibiotic for Klebsiella, and the present application provides a safe and effective new choice for resisting Klebsiella and inhibiting the formation of Klebsiella biofilm, and improves the application value of the Jiajian Huixie Decoction. The present application also discovers that the Jiajian Huixie Decoction has the potential to reduce intestinal inflammation and lung inflammation and damage. Therefore, the Jiajian Huixie Decoction can reduce the drug resistance caused by Klebsiella biofilm in the treatment of intestinal inflammation and lung inflammation, thereby improving the therapeutic effect of antibiotics, reducing the amount and types of antibiotics, and at the same time reducing inflammation and damage.

[0029] In addition, since the Jiajian Huixie Decoction can significantly inhibit the proliferation of Klebsiella and the formation of Klebsiella biofilm, it can also be used as a positive drug for screening active substances that can inhibit the proliferation of Klebsiella and the formation of Klebsiella biofilm in vitro. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 The present application has the advantages that the present application first discovers that the Jiajian Huixie Decoction has significant bacteriostatic activity on Klebsiella and can effectively inhibit the formation of Klebsiella biofilm, thus reducing the drug resistance of Klebsiella and avoiding adverse reactions such as colitis caused by the large use of antibiotics. At present, there is no specific antibiotic for Klebsiella, and the present application provides a safe and effective new choice for resisting Klebsiella and inhibiting the formation of Klebsiella biofilm, and improves the application value of the Jiajian Huixie Decoction. The present application also discovers that the Jiajian Huixie Decoction has the potential to reduce intestinal inflammation and lung inflammation and damage. Therefore, the Jiajian Huixie Decoction can reduce the drug resistance caused by Klebsiella biofilm in the treatment of intestinal inflammation and lung inflammation, thereby improving the therapeutic effect of antibiotics, reducing the amount and types of antibiotics, and at the same time reducing inflammation and damage. K. pneumoniae The present application has the advantages that the present application first discovers that the Jiajian Huixie Decoction has significant bacteriostatic activity on Klebsiella and can effectively inhibit the formation of Klebsiella biofilm, thus reducing the drug resistance of Klebsiella and avoiding adverse reactions such as colitis caused by the large use of antibiotics. At present, there is no specific antibiotic for Klebsiella, and the present application provides a safe and effective new choice for resisting Klebsiella and inhibiting the formation of Klebsiella biofilm, and improves the application value of the Jiajian Huixie Decoction. The present application also discovers that the Jiajian Huixie Decoction has the potential to reduce intestinal inflammation and lung inflammation and damage. Therefore, the Jiajian Huixie Decoction can reduce the drug resistance caused by Klebsiella biofilm in the treatment of intestinal inflammation and lung inflammation, thereby improving the therapeutic effect of antibiotics, reducing the amount and types of antibiotics, and at the same time reducing inflammation and damage. Figure 2 For the change of intestinal bacteria abundance before and after JJ, JY and YZ of Example 1 of the present application were co-cultured with intestinal flora, * p<0.05, ** p<0.01, *** p<0.001; Figure 3 For the influence of JJ, JY and YZ of Example 1 of the present application on the formation of Klebsiella pneumoniae biofilm; Figure 4 For the influence of JJ, JY and YZ of Example 1 of the present application on the formation of Klebsiella pneumoniae biofilm, * p<0.05, ** p<0.01; Figure 5 For the general morphology of mice in Example 2 of the present application; Figure 6 For the change of body weight of mice in Example 2 of the present application; * p<0.05, ** p<0.01, *** p<0.001; Figure 7 For the colon pathology of mice in Example 2 of the present application; Figure 8 For the change of intestinal lamina propria immune cells of mice in Example 2 of the present application; compared with the control group, # p<0.05, ## p<0.01; compared with the model group, * p<0.05, ** p<0.01, *** p<0.001; Figure 9 For the change of lung immune cells of mice in Example 2 of the present application; compared with the control group, # p<0.05, ## p<0.01; compared with the model group, * p<0.05, ** p<0.01, *** p<0.001; Figure 10 For the expression level of inflammatory factors in the plasma of mice in Example 2 of the present application; compared with the control group, # p<0.05, ### p<0.001; compared with the model group, * p<0.05; Figure 11 For the abundance of Klebsiella in the feces of mice in Example 2 of the present application. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical scheme and advantages of the present application more clear, the present application will be further described in detail below in combination with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and not to limit the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the present application.

[0032] Klebsiella pneumoniae is an important pathogen in hospital-acquired and community infections, which can cause various diseases. Klebsiella pneumoniae has shown resistance to many antibiotics, and biofilm is a key factor for its antibiotic resistance. There is no specific antibiotic for Klebsiella alone, and it is difficult to eradicate biofilm with conventional antibiotics, so methods to deal with its drug resistance need to be combined with surgery debridement or new anti-biofilm strategies. However, the overuse of antibiotics can disrupt the balance of intestinal flora, causing excessive proliferation of pathogenic bacteria, which can directly damage the intestinal mucosal barrier and increase intestinal permeability, allowing bacteria and their metabolites to enter the mucosal lamina propria, activate the immune system and trigger an inflammatory response.

[0033] The present application first found that the modified Weiyu Decoction has significant antibacterial activity against Klebsiella, can significantly down-regulate the abundance of Klebsiella in the intestine, and has a significant inhibitory effect on Klebsiella biofilm formation. Therefore, the present application provides the application of the modified Weiyu Decoction in the preparation of products against Klebsiella.

[0034] The present application also provides the application of the modified Weiyu Decoction in screening active ingredients against Klebsiella or active ingredients affecting the expression of Klebsiella biofilm-related genes in vitro.

[0035] The scheme of the present application will be described below through specific embodiments.

[0036] The polygonatum, platycodon, halloysite, dasyochloa, mentha, and honeyed licorice used in the following embodiments all meet the respective provisions of the corresponding medicinal materials in the Chinese Pharmacopoeia 2020 Edition, and the fresh green onions are commercially available.

[0037] The composition of the anaerobic culture solution used in the following embodiments is as follows: dipotassium hydrogen phosphate 0.293 g / L, potassium dihydrogen phosphate 0.176 g / L, sodium chloride 0.443 g / L, ammonium sulfate 0.45 g / L, calcium chloride 0.045 g / L, magnesium sulfate 0.094 g / L, resazurin 0.001 g / L, L-cysteine 0.5 g / L, L-ascorbic acid 0.5 g / L, sodium carbonate 4.0 g / L, beef extract powder 1.0 g / L, protein peptone 1.0 g / L, agar 1.0 g / L, and pH value (25℃) 7.5-8.0.

[0038] K. pneumoniae used in the following examples was from BNCC with strain number BNCC102997 (equivalent number ATCC10031).

[0039] Other reagents and drugs used in the following examples were obtained from commercial sources unless otherwise specified. The experiments involved in the following examples used conventional experimental methods in the art except for the contents specified.

[0040] Example 1 This example investigated the effect of modified Heiyou Decoction on inhibiting K. pneumoniae and its biofilm formation.

[0041] 1. Experimental materials 1.1 Preparation of modified Heiyou Decoction sample (JJ) Raw materials: 180 g of Polygonatum, 120 g of raw green onion, 90 g of Platycodon, 60 g of Cynanchum, 240 g of Soybean, 90 g of Mentha, 30 g of Fried Licorice, and 400 g of Jujube.

[0042] Except for Mentha and raw green onion, the rest of the medicinal materials were soaked in water with a water amount of 10 times the weight of the medicinal materials for 30 minutes, and extracted by heating reflux for 2 hours. The medicinal liquid was filtered through gauze. The second extraction was performed by adding water with a water amount of 8 times the weight of the medicinal materials, and extracting for 2 hours. 15 minutes before the end of the second extraction, Mentha and raw green onion were added. After removing the medicinal residues, the two medicinal liquids were combined. After cooling, the medicinal liquid was evaporated to dryness using a rotary evaporator, and concentrated to the state of extract. The extract was poured into a culture dish, covered with plastic wrap, and small holes were punched on the surface to facilitate the evaporation of water vapor. After being frozen overnight in a refrigerator at -25°C, the freeze-drying machine was started, and the sample was freeze-dried until the water was completely removed, and a powder was obtained. The powder was transferred to a centrifuge tube, sealed, and placed in a desiccator for standby.

[0043] 1.2 Preparation of modified Heiyou Decoction sample without Polygonatum (JY) Raw materials: 120 g of raw green onion, 90 g of Platycodon, 60 g of Cynanchum, 240 g of Soybean, 90 g of Mentha, 30 g of Fried Licorice, and 400 g of Jujube.

[0044] The preparation method was the same as that of the modified Heiyou Decoction sample.

[0045] 1.3 Preparation of Polygonatum sample (YZ) P. multiflora was soaked in 10 times the weight of water for 30 minutes, and extracted by heating reflux for 2 hours. The liquid was filtered through gauze. The second extraction was performed by adding 8 times the weight of water to P. multiflora, and boiling for 2 hours. The residue was removed, and the two extracts were combined. After the liquid was cooled, the excess water was evaporated using a rotary evaporator. The liquid was concentrated to the extract state, poured into a petri dish, covered with plastic wrap, and small holes were punched in the surface to allow the water vapor to escape. The sample was placed in a -25°C freezer overnight, and then placed in a freeze dryer. The sample was freeze-dried until the water was completely removed, and the sample was in powder form. The P. multiflora sample was obtained.

[0046] 2. Experimental methods 2.1 Antibacterial activity of modified Huiyu Decoction on Klebsiella pneumoniae JJ and JY freeze-dried powder was added to sterilized LB medium to prepare drug-containing medium (liquid) with a freeze-dried powder concentration of 25, 50, 100, 200, and 400 mg / mL. YZ freeze-dried powder was added to sterilized LB medium to prepare drug-containing medium (liquid) with a freeze-dried powder concentration of 1.56, 3.125, 6.25, 12.5, 25, 50, 100, and 200 mg / mL. Klebsiella pneumoniae was activated and cultured to the logarithmic growth phase (bacterial solution concentration 1 x 10 7 CFU / mL), and the obtained bacterial suspension was added to LB agar medium (1:2500, v / v), mixed, and poured into a plate as a detection layer, with nutrient agar medium as a support layer. 50 μL of the liquid and 50 μL of sterile water for the negative control and 10 μL of 50 mg / mL kanamycin sulfate for the positive control were added to the wells. The Oxford cup method was used to detect the antibacterial ring size of JJ, JY, and YZ, and to evaluate the antibacterial activity.

[0047] 2.2 Effect of modified Huiyu Decoction and intestinal flora co-culture on intestinal flora abundance An appropriate amount of fresh mouse feces was placed in an anaerobic culture bag, transferred to an anaerobic incubator, mixed with sterile saline (feces: saline (m:v) = 1:5), and centrifuged at 12000 rpm for 10 min. The supernatant was discarded to obtain a fecal bacterial suspension.

[0048] The drug group: the fecal bacterial suspension was mixed with JJ, JY, and YZ anaerobic culture solution at a volume ratio of 1:9, and the final concentration of JJ, JY, and YZ was 5 mg / mL. The blank group (con) was a mixture of anaerobic culture solution without drugs and fecal bacterial suspension at a volume ratio of 1:9. The mixtures of the drug group and the blank group were cultured at 37°C under anaerobic conditions, and samples were taken at 0 and 24 h. The fecal genomic DNA of the samples was extracted. Using the extracted sample genomic DNA as a template, the mRNA expression levels of different bacterial genera were detected and calculated (using the con group as a control reference, relative expression = 2 -△△ct ).

[0049] 2.3 Effect of Jiajian Weirui Decoction on Klebsiella pneumoniae biofilm formation After activating the frozen bacterial suspension, subculture until bacterial growth stabilizes and viability is restored. Transfer 10 μL of the bacterial suspension to LB medium and LB medium containing JJ (5 mg / mL) and culture until the bacterial logarithmic growth phase. Transfer the activated bacterial suspension to a 24-well plate. Add LB medium alone and LB medium containing JJ as blank controls. Incubate in a constant temperature incubator at 37°C for 24 hours to promote biofilm formation. Biofilm formation in each group was examined using crystal violet staining. Finally, add 75% ethanol to each well, gently shake the plate, and completely dissolve the biofilm for 3-5 minutes at room temperature. Absorbance was measured at 570 nm using a microplate reader.

[0050] Frozen Klebsiella pneumoniae were activated by inoculating 10 μL of the frozen bacterial solution into 5 mL of LB medium and shaking at 200 rpm for 18 h. After 1-2 passages, 10 μL of the bacterial solution was transferred to LB medium and LB medium containing modified Weirui Decoction (5 mg / mL). After shaking and incubation for 24 h, the bacterial solution was centrifuged and the supernatant of the bacterial culture was collected. The concentration of bacterial autoinducer-2 (AI-2) was detected by ELISA kit.

[0051] 2.4 Effect of Jiajian Weirui Decoction on Klebsiella pneumoniae biofilm-related genes Drug-containing media (JJ, JY, and YZ) at a concentration of 5 mg / mL were prepared using LB medium. A blank control (con group) was inoculated with activated K. pneumoniae culture medium containing no drug and cultured on a shaking platform for 24 hours. Bacterial RNA was extracted and reverse transcribed, and the expression levels of biofilm genes were determined using fluorescent quantitative PCR.

[0052] 3. Experimental results 3.1 Antibacterial activity of JJ, JY, and YZ against Klebsiella pneumoniae According to the evaluation index: inhibition zone > 20 mm is extremely sensitive, 15-20 mm is highly sensitive, 10-15 mm is moderately sensitive, and <10 mm is resistant, the antibacterial activity of different concentrations of drug solution was judged. The antibacterial diameter of JJ at 200 mg / mL was 13.2±0.2 mm, which showed moderate sensitivity, and the antibacterial diameter of JJ at 400 mg / mL was 15.3±0.2 mm, which showed high sensitivity; the antibacterial diameter of JY at 200 mg / mL was 10.8±0.2 mm, and the antibacterial diameter of JY at 400 mg / mL was 14.3±0.2 mm, both showing moderate sensitivity. The other concentrations had no antibacterial activity. Figure 1 shown.

[0053] 3.2 Effects of JJ, JY and YZ on the abundance of intestinal flora in mice The abundance of intestinal flora at the phylum and genus levels was determined by in vitro co-culture of JJ, JY and YZ with intestinal microorganisms in mice and qPCR technique. At the phylum level, the abundance of Firmicutes was significantly increased in JJ and JY groups (p<0.05, p<0.01), and the abundance of Bacteroidetes, Proteobacteria and Actinobacteria tended to decrease in JJ and JY groups compared with the con24 group (blank group at 24 h). The abundance of Proteobacteria and Actinobacteria was also reduced in YZ group. At the genus level, the abundance of Bifidobacterium, Lactobacillus and Bacteroides tended to increase in JJ group compared with the con24 group, and the abundance of Akkermansia was significantly increased in JJ and JY groups compared with the con0 group (blank group at 0 h) (p<0.01, p<0.05). The abundance of Klebsiella was significantly reduced in JJ, JY and YZ groups compared with the con24 group (p<0.001, p<0.001, p<0.01). As shown in Figure 3. Figure 2

[0054] 3.3 Effects of JJ on the formation of Klebsiella pneumoniae biofilm Biofilm crystal violet staining showed that JJ and JY significantly inhibited the formation of Klebsiella pneumoniae biofilm. Semi-quantitative detection by enzyme-labeled instrument showed that LB medium containing 5 mg / mL JJ and JY could significantly inhibit the formation of Klebsiella pneumoniae biofilm compared with LB medium (p<0.001).

[0055] LB medium containing 5 mg / mL JJ and JY could significantly reduce the production of AI-2 compared with LB medium alone (p<0.001). As shown in Figure 4. Figure 3

[0056] 3.4 Effects of JJ, JY and YZ on the biofilm-related genes of Klebsiella pneumoniae Compared with the con group, the level of type III pilus-related gene mrkA was significantly reduced in the JJ group (p<0.05), and the level of quorum sensing-related gene luxS was significantly reduced in the JJ, JY and YZ groups (p<0.01, p<0.01, p<0.05). Compared with the con group, the level of CPS-related gene rcsA , wzm , wzi was significantly increased in the YZ group (p<0.01, p<0.01, p<0.05), the level of type I pilus-related gene fimH was significantly decreased in the JY group (p<0.05), and the level of secretion system-related gene gspE (p<0.05) and LPS-related gene was significantly increased in the YZ group.​​wbbm , rfbA , rfbB (p<0.01, p<0.05, p<0.01), biofilm synthesis-related genes pgaA (p<0.05), iron transport protein-related genes feoA , exbB , exbD , tonB (p<0.05, p<0.05, p<0.01, p<0.01). The JJ group also had an up-regulating effect on LPS-related genes (p<0.05). As shown in wbbm . Figure 4

[0057] Example 2 This example investigates the effect of Jiawei Heiyu Decoction on intestinal inflammation and lung inflammation.

[0058] 1. Experimental materials: same as in Example 1.

[0059] 2. Experimental method 2.1 Preparation of experimental animal model, grouping and administration scheme 6-week-old clean healthy C57BL / 6N male mice were used as research subjects.

[0060] Experimental grouping: C57BL / 6N mice were randomly divided into control (Control, hereinafter abbreviated as Con) group, model group (Model), administration group (respectively: Jiawei Heiyu Decoction (JJ) group, Jiawei Heiyu Decoction without Yu Zhu (JY) group, Yu Zhu (YZ) group, high-dose Yu Zhu (2YZ) group, positive drug (dexamethasone, DEX) group) by random number control method, a total of 7 groups, 18 mice in each group.

[0061] Preparation of animal model: Before modeling, the mice were weighed and placed in a fixed device, exposing the tail, and the tail was wiped with a 75% alcohol cotton ball to fully reveal the tail vein. A disposable 1 mL sterile insulin syringe was used to draw the LPS (1 mg / mL) solution prepared in advance, with a modeling dose of 5 mg / kg, and injected at a dose of 0.1 mL / 20g. During the drawing process, large air bubbles were avoided, the needle was slowly inserted into the vein, and the liquid was pushed at a uniform speed. Dry cotton balls were used to press the injection site to prevent bleeding. The model group and the administration group were injected into the tail vein for modeling for 3 consecutive days on the 3rd, 4th and 5th days of the experiment, and the blank group was injected with an equal amount of normal saline.

[0062] ​Dosing method: each group of mice was given intragastrically once a day for 7 consecutive days. According to the clinical drug dose and the conversion relationship between the mouse dose and humans, the mouse doses of the positive drug group, the modified Weiliu Decoction group, the modified Weiliu Decoction without Yu Zhu group, the Yu Zhu group, and the high-dose Yu Zhu group were 5 mg / kg, 7.8650 g / kg (clinical equivalent dose, calculated as crude drug), 6.6950 g / kg (calculated as crude drug), 1.56 g / kg (clinical equivalent dose, calculated as crude drug), and 3.12 g / kg, respectively. The intervention was performed at a dose of 0.1 mL per 10 g of body weight, and the blank group and the model group were given the same volume of ultrapure water. The mice were weighed once a day, the body weight was recorded, and the drug volume was adjusted in time according to the changes.

[0063] 2.2 Blood and organ sample collection Blood and colon samples were collected on the 8th day.

[0064] 2.3 Detection of pathological damage of mouse colon Hematoxylin-eosin staining (HE staining) was used for pathological detection of colon tissue.

[0065] 2.4 Detection of colon immune cell composition The intestinal lamina propria immune cells were collected after pretreatment of the colon samples. Flow cytometry was used to detect the intestinal immune and adaptive immune cells in the mouse colon samples, including monocytes, macrophages, neutrophils, CD4 + T cells, CD8 + T cells, B cells, ILC3 cells, IL-17 + ILC3 cells, IL-22 + ILC3 cells.

[0066] 2.5 Detection of lung immune cell composition The lung immune cells were collected after pretreatment of the lung samples. Flow cytometry was used to detect the intestinal immune and adaptive immune cells in the mouse colon samples, including monocytes, macrophages, neutrophils, CD4 + T cells, CD8 + T cells, B cells, ILC3 cells, IL-17 + ILC3 cells, IL-22 + ILC3 cells.

[0067] 2.6 Detection of mouse plasma inflammatory factors After standing for more than 30 min, the collected mouse whole blood was centrifuged to collect the upper plasma. Enzyme-linked immunosorbent assay (ELISA) was used to determine the inflammatory factors TNF-α, IL-6, and IL-1β in the mouse plasma.

[0068] 2.7 Detection of Klebsiella abundance in mouse feces Fresh fecal samples were collected from mice on the 7th day of the experiment, and fecal DNA was extracted using a fecal genomic extraction kit. The concentration of each sample was determined using a UV spectrophotometer, and the relative abundance of Klebsiella in the feces of mice in each group was detected by PCR.

[0069] 3. Experimental results 3.1 Mouse signs Intraperitoneal injection of LPS can significantly induce abnormal physiological state of mice. Compared with the control group, the model group mice showed fluffy and upright fur, reduced spontaneous activity, slow reaction, and typical symptoms such as loose stool or diarrhea. After administration of JJ and YZ, the spirit, hair and feces of mice were improved. As shown in the table. Figure 5 .

[0070] 3.2 Mouse weight The body weight of mice in the control group gradually increased over time, while the body weight of mice in the model group decreased continuously from the 4th day after modeling. Compared with the control group, the body weight of mice in the model group decreased significantly (p<0.001) at the end of the experiment. After administration, JJ group, YZ group and 2YZ group significantly improved the weight loss of mice caused by LPS modeling (p<0.05, p<0.01, p<0.001), and JY group had no effect on the weight of mice. As shown in the table. Figure 6 .

[0071] 3.3 Colonic pathological injury of mice The crypt structure of the mucosa layer of the control group mice was complete, compared with the control group, the crypt structure of the model group was distorted, the inflammation infiltration of the lamina propria and submucosa appeared, the submucosa edema, and the connective tissue hyperplasia, suggesting intestinal barrier dysfunction. Compared with the model group, the colonic pathological injury was reduced after treatment with JJ and YZ. As shown in the table. Figure 7 .

[0072] 3.4 Intestinal lamina propria immune cells of mice The flow cytometry results showed that Figure 8 .

[0073] Compared with the control group, the number of neutrophils in the model group mice increased significantly (p<0.05); compared with the model group, the number of neutrophils in JJ group mice and DEX group mice decreased significantly (p<0.01).

[0074] Compared with the control group, the number of macrophages in the model group increased significantly (p<0.05), and the number of macrophages in JJ group and DEX group mice decreased compared with the model group, but there was no significant difference.

[0075] The number of monocytes in the model group was not significantly different from that in the control group. Compared with the model group, the number of monocytes in each drug administration group showed a downward trend, but there was no significant difference.

[0076] Compared with the control group, the number of B cells in the model group decreased significantly (p<0.05), and the number of B cells in the DEX group decreased significantly compared with the model group (p<0.01), and there was no significant difference in the remaining groups.

[0077] Compared with the control group, the number of CD4 + T cells in the model group increased significantly (p<0.05), and the number of CD8 + T cells showed a trend of increase, and the ratio of CD4 + T cells to CD8 + T cells showed a downward trend, but there was no significant difference. Compared with the model group, the number of CD4 + T cells in the 2YZ group and the DEX group decreased significantly (p<0.01), and there was no significant change in the number of CD8 + T cells in each group, and the ratio of CD4 + T cells to CD8 + T cells in the 2YZ group and the DEX group decreased significantly (p<0.05).

[0078] Compared with the control group, the number of ILC3 cells in the model group increased significantly (p<0.01); compared with the model group, the number of ILC3 cells in the DEX group decreased significantly (p<0.01), and the remaining groups showed a downward trend but no significant difference.

[0079] Compared with the control group, the number of IL-22 + ILC3 cells in the model group and each drug administration group showed a downward trend but no significant difference. The secretion of IL-22 + ILC3 cells, a key cytokine, can maintain the integrity of the epithelial barrier, promote the expression of tight junction proteins, and inhibit the production of pro-inflammatory factors, thereby reducing LPS-induced intestinal barrier damage and systemic inflammatory response. The number of IL-22 + ILC3 cells in the drug administration groups was significantly higher than that in the model group, indicating that the drugs promoted the repair of the intestinal barrier and reduced damage and inflammation.

[0080] Compared with the control group, the number of IL-17 + ILC3 cells in the model group showed a downward trend but no significant difference; the number of IL-17 + ILC3 cells in the YZ group, the 2YZ group and the DEX group increased significantly (p<0.001).

[0081] 3.5 Mouse lung immune cells The flow cytometry results showed that Figure 9 .

[0082] Compared with the control group, the neutrophils in the model group decreased significantly (p<0.001).

[0083] Compared with the control group, the number of macrophages in the model group had a trend of increase, and the number of macrophages in each group after administration had a trend of decrease, but there was no significant difference. The number of monocytes in the model group decreased significantly compared with the control group (p<0.01).

[0084] Compared with the control group, the number of CD4 + T cells in the model group decreased significantly (p<0.05), and the number of CD8 + T cells had a trend of increase, and the ratio of CD4 + T cells to CD8 + T cells decreased significantly (p<0.05), and the number of CD4 + T cells in the JJ group, YZ group, 2YZ group and DEX group increased significantly after administration (p<0.01, p<0.05, p<0.01, p<0.05), and the number of CD8 + T cells in each group had no obvious change, and the JJ group, YZ group, 2YZ group and DEX group significantly increased the ratio of CD4 + T cells to CD8 + T cells (p<0.01, p<0.05, p<0.01, p<0.05).

[0085] There was no significant difference in ILC3 cells among each group, but compared with the number of IL-22 + ILC3 cells, the number of ILC3 cells in the model group decreased significantly compared with the blank group (p<0.001), and the JJ group, YZ group and 2YZ group had a trend of increase, and the number of IL-22 + ILC3 cells in the DEX group increased significantly (p<0.001).

[0086] Compared with the control group, the number of IL-17 + ILC3 cells in the model group increased significantly (p<0.01), and after administration, they all had a trend of decrease, but only the 2YZ group had a significant difference (p<0.05).

[0087] 3.6 Expression level of inflammatory factors in plasma Compared with the control group, the model group mice plasma IL-1β and IL-6 were significantly increased (p<0.05, p<0.001), TNF-α level had no obvious change. Compared with the model group, the 2YZ group and DEX group mice plasma TNF-α expression level was significantly decreased (p<0.05, p<0.05). Compared with the model group mice plasma IL-1β level, JJ, YZ and 2YZ administration had a downward trend, but no significant difference. Compared with the model group, JJ group and DEX group significantly reduced the mice plasma IL-6 (p<0.05, p<0.05), and the plasma IL-6 level had a downward trend after YZ and 2YZ administration, but no significant difference. As shown in Figure 10 .

[0088] 3.7 Klebsiella abundance in feces At the genus level, compared with the control group, the relative abundance of Klebsiella in the feces of the model group mice was significantly increased; compared with the model group, the relative abundance of Klebsiella in the JJ group was significantly reduced (p<0.001). As shown in Figure 11 .

[0089] Example 3 The present embodiment provides the application of Jiawei Heqi Decoction in screening active substances against Klebsiella pneumoniae in vitro.

[0090] JJ freeze-dried powder (preparation method same as example 1) and test substances were taken and added to sterilized LB agar to prepare drug-containing medium, the freeze-dried powder concentration in the drug-containing medium was 200, 400 mg / mL, and the test substance concentration was set in gradient, such as 1.56, 3.125, 6.25, 12.5, 25, 50, 100, 200 mg / mL. Klebsiella pneumoniae was activated and cultured to the logarithmic growth phase (bacterial solution concentration 1×10 7 CFU / mL), the obtained bacterial suspension was added to LB agar medium (1:2500, v / v), mixed and poured into plates as detection layer. Oxford cup method was used to detect the antibacterial activity of JJ and test substances, and active substances with anti-Klebsiella pneumoniae effect were screened according to the size of the inhibition zone.

[0091] Example 4 The present embodiment provides the application of Jiawei Heqi Decoction in screening active ingredients affecting Klebsiella biofilm-related gene expression in vitro.

[0092] LB medium was used to prepare culture medium containing different concentrations of test substances, JJ freeze-dried powder (preparation method same as example 1) concentration was 5 mg / mL, blank group (con group) was LB medium without drugs, and Klebsiella pneumoniae activation bacterial solution was inoculated and cultured on a shaking table for 24 h. Bacterial RNA was extracted, and after reverse transcription, the fluorescence quantitative PCR method was used to detect the effect of JJ and test substances on biofilm genesmrkA , luxS , wbbm expression levels of the genes listed in Table 1.

[0093] The above description is only the preferred embodiment of the application, not to limit the application, any modification, equivalent replacement or improvement made within the spirit and principle of the application should be included in the protection scope of the application.

Claims

1. The application of Jiajian Weirui Decoction in the preparation of anti-Klebsiella products, characterized in that: The active ingredients of the product include Jiajian Weirui Decoction and / or its processed products.

2. The use according to claim 1, characterized in that The modified Weirui decoction is prepared from the following raw materials in parts by mass: 16-20 parts of polygonatum, 11-13 parts of raw scallion white, 8-10 parts of platycodon grandiflorum, 5.5-6.5 parts of white mulberry, 22-26 parts of light fermented black beans, 8-10 parts of mint, 2.5-3.5 parts of roasted liquorice root, and 35-45 parts of jujube.

3. The use according to claim 2, characterized in that The modified Weirui decoction is prepared from the following raw materials in parts by mass: 18 parts of Polygonatum odoratum, 12 parts of raw scallion white, 9 parts of Platycodon grandiflorum, 6 parts of Bletilla striata, 24 parts of light fermented black beans, 9 parts of mint, 3 parts of roasted liquorice root, and 40 parts of jujube; and / or The preparation method of the modified Weirui decoction comprises: soaking the raw materials except mint and scallion white in water, extracting them twice with water reflux, each time for 100 to 140 minutes, adding the mint and scallion white 13 to 17 minutes after the end of the second extraction; combining the extracts obtained from each extraction, and / or The preparation method of the processed product is: concentrating and / or drying the modified Weirui decoction.

4. The use according to any one of claims 1 to 3, characterized in that The product is a product that inhibits the formation of Klebsiella biofilm.

5. The use according to any one of claims 1 to 3, characterized in that The product described is a pharmaceutical product.

6. The use according to claim 5, characterized in that The medicine is a medicine for intestinal inflammation or lung inflammation.

7. The use according to any one of claims 1 to 3, characterized in that The product is a product that resists Klebsiella colonization or inhibits Klebsiella biofilm formation in vitro.

8. Use of Jiajian Weirui Decoction or its processed products in in vitro screening of active ingredients against Klebsiella or active ingredients that affect the expression of Klebsiella biofilm-related genes, characterized in that: The in vitro screening method uses Jiajian Weirui Decoction or its processed products as a positive control drug.

9. The use according to claim 8, characterized in that The modified Weirui decoction is prepared from the following raw materials in parts by mass: 16-20 parts of polygonatum, 11-13 parts of raw scallion white, 8-10 parts of platycodon grandiflorum, 5.5-6.5 parts of white mulberry, 22-26 parts of light fermented black beans, 8-10 parts of mint, 2.5-3.5 parts of roasted liquorice root, and 35-45 parts of jujube.

10. The use according to claim 9, characterized in that The modified Weirui decoction is prepared from the following raw materials in parts by mass: 18 parts of Polygonatum odoratum, 12 parts of raw scallion white, 9 parts of Platycodon grandiflorum, 6 parts of Bletilla striata, 24 parts of light fermented black beans, 9 parts of mint, 3 parts of roasted liquorice root, and 40 parts of jujube; and / or The preparation method of the modified Weirui decoction comprises: soaking the raw materials except mint and scallion white in water, extracting them twice with water reflux, each time for 100 to 140 minutes, adding the mint and scallion white 13 to 17 minutes after the end of the second extraction; combining the extracts obtained from each extraction, and / or The preparation method of the processed product is: concentrating and / or drying the modified Weirui decoction.

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