CN111544535A - Traditional Chinese medicine compound preparation for inhibiting escherichia coli and biofilm thereof and application thereof - Google Patents
Traditional Chinese medicine compound preparation for inhibiting escherichia coli and biofilm thereof and application thereof Download PDFInfo
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Abstract
The invention provides a traditional Chinese medicine compound preparation for inhibiting escherichia coli and a biological membrane thereof and application thereof. The Chinese medicine compound preparation provided by the invention is an oral preparation which is prepared from active ingredients of Sijihong, hibiscus leaf, sargentgloryvine stem, root of Chinese barberry, hairyvein agrimony, lalang grass rhizome and forsythia fruit and pharmaceutic adjuvants. The Chinese medicinal compound preparation has the effects of clearing away heat and toxic materials, promoting diuresis, treating stranguria, and promoting blood circulation for removing blood stasis, and has significant inhibiting effect on Escherichia coli and its biomembrane at concentration of 5 mg/mL. By inhibiting the Escherichia coli from forming a bacterial biofilm, the traditional Chinese medicine compound preparation can effectively solve the problems that related diseases caused by the Escherichia coli are not cured for a long time and are repeatedly attacked, and has very obvious curative effect.
Description
Technical Field
The invention relates to a traditional Chinese medicine compound preparation, in particular to a traditional Chinese medicine compound preparation for inhibiting escherichia coli and a biological membrane thereof and application thereof.
Background
Under certain conditions, Escherichia coli can cause acute or chronic bacterial infection of gastrointestinal tract of human and various animals, and often causes acute or chronic diseases such as acute diarrhea, chronic diarrhea, hemorrhagic enteritis, peritonitis, appendicitis, cholecystitis, even septicemia and the like.
Currently, for diseases caused by escherichia coli, oral antibiotics are often used for treatment. However, about 85% of clinical strains isolated from patients have the capability of forming a biofilm, the bacterial biofilm can be adhered to the surface of epithelial cells and secrete extracellular matrixes to wrap the bacterial biofilm to form a special structure with group effect, the bacterial group structure has extremely strong drug resistance, and antibiotics are difficult to completely remove, so that chronic infection is not cured and has repeated attack.
Therefore, for diseases caused by escherichia coli, especially chronic diseases, a new treatment concept and method which can be used for a long time and has small side effect and can resist bacterial drug resistance and bacterial biofilm formation are needed to overcome the defects of antibiotic treatment.
Disclosure of Invention
The invention provides a traditional Chinese medicine compound preparation for inhibiting escherichia coli and a biological membrane thereof and application thereof. The preparation provided by the invention is a traditional Chinese medicine compound preparation, has the effects of clearing away heat and toxic materials, promoting diuresis for treating stranguria, and promoting blood circulation to remove blood stasis, and the inventor finds that the preparation has a remarkable inhibiting effect on escherichia coli and a biological membrane thereof, and has remarkable medicinal effect, safety and reliability. The specific technical scheme is as follows:
the invention provides a traditional Chinese medicine compound preparation for inhibiting escherichia coli and a biological membrane thereof, which is an oral preparation consisting of active ingredients and pharmaceutic adjuvants, wherein the active ingredients comprise the following components in percentage by weight: 20-40% of Sijihong, 0.5-3% of hibiscus leaf, 8-25% of sargentgloryvine stem, 4-19% of barberry root, 4-19% of hairyvein agrimonia herb and bud, 10-30% of cogongrass rhizome and 8-25% of fructus forsythiae.
The oral preparation comprises tablets, capsules, granules, pills, oral liquid and the like, wherein each unit dosage preparation contains 0.2-1000 mg of quarternary red in terms of gallic acid, and the unit dosage refers to the actual dosage unit of the preparation, such as each tablet, each bottle of oral liquid and the like.
The compound preparation can be prepared by the following method.
Step one, pretreatment: weighing 50-75% of the formula amount of the medicinal materials of hairyvein agrimony, weeping forsythia and rhizoma imperatae, sterilizing for 25 minutes at the temperature of 98 +/-2 ℃, drying and crushing into crude drug fine powder for later use; taking the rest agrimony, forsythia and cogongrass rhizome in the prescription amount, and the Sijihong, hibiscus leaf, sargentgloryvine stem and root of Chinese barberry in the prescription amount, sorting, washing with water, drying and preparing into pure medicinal materials for later use;
step two, preparation: decocting the pure medicinal materials with water for three times, adding 6-8 times of water for the first time, soaking for half an hour, decocting for 2 hours, filtering, adding 4-6 times of water for the second time, decocting for 1.5 hours, filtering, adding 3-5 times of water for the third time, decocting for 1.5 hours, filtering, discarding medicine residues, combining filtrates, concentrating under reduced pressure and evaporating to obtain an extract with the relative density of 1.15-1.21 and the temperature of 60-70 ℃; mixing the crude drug fine powder and medicinal adjuvants with the extract, granulating, and making into various oral preparations by conventional method.
The compound preparation can also be prepared by the following method.
Step one, preparing an extract: weighing herba Polygoni Capitati, folium Hibisci Mutabilis, caulis Sinomenii, radix Berberidis, herba et Gemma Agrimoniae, fructus forsythiae and lalang grass rhizome according to the prescription amount, soaking in 6-8 times of water, decocting, and filtering; adding 3-7 times of water into the decoction dregs, and decocting twice; discarding residues, mixing filtrates, and concentrating under reduced pressure to obtain extract; adding 80% ethanol in an amount which is 1-2 times that of the extract, uniformly stirring, standing for 24 hours, taking supernatant, filtering, recovering ethanol from filtrate, and concentrating to obtain an extract with the relative density of 1.15-1.2;
step two, preparation: spray drying 70% of the extract to obtain powder, mixing with the rest extract and medicinal adjuvants, granulating, drying, and making into various oral preparations such as tablet, capsule, granule, etc. by conventional method; or adding medicinal adjuvants into the extract, and making into oral liquid by conventional method.
The pharmaceutical excipients are commonly used in the preparation of pharmaceutical preparations, and are not particularly limited herein.
Particularly, the invention mainly provides application of the traditional Chinese medicine compound preparation in preparing medicines for treating diseases related to escherichia coli and biomembrane thereof. Furthermore, the invention also provides application of the active ingredients in the traditional Chinese medicine compound preparation, and the traditional Chinese medicine compound preparation is used for preparing medicines for treating diseases related to escherichia coli and biomembranes thereof. The inventor surprisingly finds that the traditional Chinese medicine compound preparation provided by the invention has the traditional effects of clearing away heat and toxic materials, removing dampness by diuresis, treating stranguria, promoting blood circulation and removing blood stasis, can show the inhibition effect on the proliferation of escherichia coli and the formation of bacterial biomembranes at 5mg/mL, almost completely inhibits the proliferation of the escherichia coli and the biomembrane forming capability at 10mg/mL, and thus has obvious curative effect on related diseases.
The invention provides a safe and reliable Chinese herbal compound preparation with remarkable drug effect aiming at the problems that the bacterial colony structure formed by Escherichia coli has extremely strong drug resistance, and antibiotics are difficult to completely remove, so that chronic infection is not cured for a long time and is recurrent, the preparation can be used for a long time, has small side effect, can resist the drug resistance of bacteria and the formation of bacterial biofilms, can be used singly or combined with the antibiotics, and provides a new treatment idea and method for the complete cure of related chronic diseases.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:
FIG. 1 is a comparison graph of the inhibition effect of the compound Chinese medicine preparation of the present invention on Escherichia coli at different concentrations.
Detailed Description
The preferred embodiments of the present invention will be described in conjunction with the accompanying drawings, and it will be understood that they are described herein for the purpose of illustration and explanation and not limitation.
EXAMPLE 1 preparation of extract 1
Taking 200g of Sijihong, 30g of hibiscus leaf, 200g of sargentgloryvine stem, 150g of root of Chinese barberry, 150g of hairyvein agrimony, 150g of lalang grass rhizome and 120g of weeping forsythia, adding 7 times of water, soaking for 0.5 hour, decocting for 2 hours, filtering, adding 5 times of water into medicine residues, decocting for 1.5 hours, filtering, and adding 4 times of water into the medicine residues, and decocting for 1.5 hours; filtering, removing the medicine residues, combining the filtrates, concentrating the filtrate under reduced pressure and evaporating to obtain an extract, adding 1.2 times of 80% ethanol into the extract, uniformly stirring, standing for 24 hours, taking supernatant, filtering the supernatant, recovering ethanol from the filtrate, and concentrating to obtain the extract with the relative density of 1.10-1.20.
EXAMPLE 2 preparation of extract 2
350g of Sijihong, 10g of hibiscus leaf, 150g of sargentgloryvine stem, 120g of berberis poiretii, 120g of hairyvein agrimony, 100g of lalang grass rhizome and 150g of weeping forsythia fruit are taken, the preparation method is the same as that of the example 1, and extractum is obtained.
Example 3 preparation of sugarless granules
Taking the extract 1 obtained in the example 1, taking 70% of the extract, spray-drying to prepare fog powder for later use, mixing the rest extract with the spray powder, a proper amount of stevioside and dextrin, granulating, drying, preparing sugar-free granules, and subpackaging (6 g/bag) to obtain the sugar-free granules.
EXAMPLE 4 preparation of oral liquid
Taking the extract 2 obtained in the example 2, adding cane sugar and sodium benzoate into the extract, heating to dissolve, adding distilled water to the required volume, refrigerating for 24 hours, filtering, filling (10 ml/bottle), sterilizing and packaging to obtain the product. Each bottle contains 3.9mg gallic acid.
EXAMPLE 5 preparation of Capsule formulations
Taking 400g of Sijihong, 5g of hibiscus leaf, 80g of sargentgloryvine stem, 50g of berberis poiretii, 50g of hairyvein agrimony, 200g of lalang grass rhizome and 215g of weeping forsythia, firstly taking 60% of the medicinal materials of the hairyvein agrimony, the weeping forsythia and the lalang grass rhizome according to the weight, sterilizing for 25 minutes at the temperature of 98 +/-2 ℃, drying, and crushing into crude drug fine powder for later use; taking the rest agrimony, forsythia and cogongrass rhizome in the prescription amount, and the Sijihong, hibiscus leaf, sargentgloryvine stem and root of Chinese barberry in the prescription amount, sorting, washing with water, drying and preparing into pure medicinal materials for later use;
decocting the pure medicinal materials with water for three times, adding 8 times of water for the first time, soaking for half an hour, decocting for 2 hours, filtering, adding 6 times of water for the second time, decocting for 1.5 hours, filtering, adding 5 times of water for the third time, decocting for 1.5 hours, filtering, discarding dregs, combining filtrates, concentrating under reduced pressure, and evaporating to obtain an extract with a relative density of 1.15-1.21 and a temperature of 60-70 ℃; mixing the fine powder of the crude drugs with the extract, granulating, drying, and encapsulating to obtain oral preparation.
Example 6 Escherichia coli inhibition experiment
MIC values were determined by the broth microdilution method according to the American society for Clinical and Laboratory Standards Institute (CLSI). The granular powder obtained in example 3 was dissolved in a culture medium to prepare a concentration of 40 mg/mL, mixed and filtered, 200. mu.L of the mixture was added to column 1 of a U-bottom 96-well plate, 100. mu.L of the culture medium was added to each of the remaining wells, and each row of the 96-well plate was diluted in a 1:2 gradient from left to right to form a drug concentration gradient. Bacteria were cultured to mid-log phase using sterile TSB or LB medium, the suspensions were calibrated to an absorbance OD600 of 0.5, i.e., about 108 Colony Forming Units (CFU)/mL, diluted 1:20 medium to a bacterial content of about 5 × 106 CFU/mL, and 10 μ L of suspension per well was added to the prepared microdilution plates to a final test bacterial concentration of about 5 × 105 CFU/mL. In the test process, 3 compound holes are arranged for each drug concentration, and a growth control hole and a sterile control hole are arranged at the same time. And (3) placing the micro dilution plate in an incubator at 37 ℃ for standing culture for 16-20 h, and observing the lowest concentration of the antibacterial agent which can completely inhibit the growth of bacteria in the micro dilution holes by naked eyes, wherein the lowest concentration is the MIC value. The experiment was repeated 3 times using E.coli ATCC25922 and CFT073 strains, with an MIC of 20 mg/mL.
The biofilms were observed using a scanning electron microscope. The granulated drug powder obtained in example 3 was added to the test group medium, and the concentration lower than the MIC was set as the final drug concentration, and the control group was replaced with deionized water. Using a 24-well culture plate, putting a cover glass (a circular cover glass with the diameter of 8 mm) into the culture plate, culturing and diluting the bacterial suspension as described above until the final bacterial content is about 5 × 105 CFU/mL, performing static culture at 37 ℃ for about 24 hours, removing the liquid culture medium by a pipette, and rinsing the liquid culture medium for 3 times by using a sterilized phosphate buffer solution; fixing: 2.5% glutaraldehyde was added and left to stand at 4 ℃ overnight. Removing glutaraldehyde with a pipettor, rinsing with 0.1 mol/L phosphate buffer for 10min for 3 times; fixing with 1% osmate fixing solution for 1h, rinsing with 0.1 mol/L phosphate buffer solution for 10min, 3 times; after dehydration, drying at critical point and spraying gold, observing with FEI QUANTA 250 scanning electron microscope. The experiment was repeated 3 times. As shown in figure 1, the control result shows that the traditional Chinese medicine compound preparation with the concentration of 5mg/mL shows the inhibition effect on the formation of the bacterial biofilm, the film forming capability of the CFT073 strain is almost completely inhibited when the concentration reaches 10mg/mL, and the film forming capability of the ATCC25922 strain is also severely limited. The results also show that the traditional Chinese medicine compound preparation can effectively inhibit the formation of Escherichia coli and a biological membrane thereof.
Example 7 toxicity test
Acute toxicity test: the maximum tolerance of the preparation (oral liquid) of the invention in the example 4 for the intragastric administration of mice is more than 16g/kg and is 167 times of the clinical dosage (50 kg for clinical adults, 4.8 g/day, 0.096g/kg), so the clinical administration is safe.
Long-term toxicity test: the daily dosage of 1 day of the two groups of rats subjected to gastric gavage by using the preparation (oral liquid) of the embodiment 4 of the invention is 4g/kg, 1.5g/kg is equivalent to 42 times and 15.6 times of the daily oral dosage (0.096g/kg) of an adult (50g/kg), the experimental period is 35 days (equivalent to 4 treatment courses of clinical medication), and the animals in each group subjected to drug administration are observed after stopping taking the preparation for 2 weeks, so that the general conditions of the animals in each group are good, and the weight is continuously increased, and the indexes of hemogram, blood biochemical index, organ coefficient and pathological examination are not obviously different from those in a control group, thereby prompting that the traditional Chinese medicine compound preparation of the invention has no long-term toxic effect on the rats and no delayed toxic effect.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (7)
1. A traditional Chinese medicine compound preparation for inhibiting Escherichia coli and a biological membrane thereof comprises active ingredients and pharmaceutic adjuvants, and is characterized in that the active ingredients comprise the following components in percentage by weight: 20-40% of Sijihong, 0.5-3% of hibiscus leaf, 8-25% of sargentgloryvine stem, 4-19% of barberry root, 4-19% of hairyvein agrimonia herb and bud, 10-30% of cogongrass rhizome and 8-25% of fructus forsythiae.
2. The compound traditional Chinese medicine preparation for inhibiting escherichia coli and biofilm thereof according to claim 1, wherein the compound traditional Chinese medicine preparation is tablets, capsules, granules, pills or oral liquid.
3. The compound traditional Chinese medicine preparation for inhibiting escherichia coli and biofilm thereof according to claim 2, wherein the content of quaternaries red in each unit dosage preparation of the compound traditional Chinese medicine preparation is 0.2-1000 mg in terms of gallic acid.
4. The method for preparing a compound Chinese medicinal preparation for inhibiting escherichia coli and biofilm thereof as claimed in any one of claims 1 to 3, comprising the steps of:
step one, pretreatment: weighing 50-75% of the formula amount of the medicinal materials of hairyvein agrimony, weeping forsythia and rhizoma imperatae, sterilizing for 25 minutes at the temperature of 98 +/-2 ℃, drying and crushing into crude drug fine powder for later use; taking the rest agrimony, forsythia and cogongrass rhizome in the prescription amount, and the Sijihong, hibiscus leaf, sargentgloryvine stem and root of Chinese barberry in the prescription amount, sorting, washing with water, drying and preparing into pure medicinal materials for later use;
step two, preparation: decocting the pure medicinal materials with water for three times, adding 6-8 times of water for the first time, soaking for half an hour, decocting for 2 hours, filtering, adding 4-6 times of water for the second time, decocting for 1.5 hours, filtering, adding 3-5 times of water for the third time, decocting for 1.5 hours, filtering, discarding medicine residues, combining filtrates, concentrating under reduced pressure and evaporating to obtain an extract with the relative density of 1.15-1.21 and the temperature of 60-70 ℃; mixing the fine powder of crude drug and medicinal adjuvants with the extract, granulating, and making into preparation.
5. The method for preparing a compound Chinese medicinal preparation for inhibiting escherichia coli and biofilm thereof as claimed in any one of claims 1 to 3, comprising the steps of:
step one, preparing an extract: weighing herba Polygoni Capitati, folium Hibisci Mutabilis, caulis Sinomenii, radix Berberidis, herba et Gemma Agrimoniae, fructus forsythiae and lalang grass rhizome according to the prescription amount, soaking in 6-8 times of water, decocting, and filtering; adding 3-7 times of water into the decoction dregs, and decocting twice; discarding residues, mixing filtrates, and concentrating under reduced pressure to obtain extract; adding 80% ethanol in an amount which is 1-2 times that of the extract, uniformly stirring, standing for 24 hours, taking supernatant, filtering, recovering ethanol from filtrate, and concentrating to obtain an extract with the relative density of 1.15-1.2;
step two, preparation: spray drying 70% of the extract to obtain powder, mixing with the rest extract and medicinal adjuvants, granulating, drying, and making into preparation.
6. The use of the compound traditional Chinese medicine preparation for inhibiting escherichia coli and biofilm thereof according to any one of claims 1 to 3, for preparing a medicament for treating diseases related to escherichia coli and biofilm thereof.
7. The use of the active ingredient in the compound traditional Chinese medicine preparation for inhibiting escherichia coli and biofilm according to any one of claims 1-3, for preparing a medicament for treating diseases related to escherichia coli and biofilm thereof.
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| 刘桂杰等: "宁泌泰胶囊对表皮葡萄球菌生物膜的抑制作用", 《中国中西医结合外科杂志》 * |
| 刘薇等: "宁泌泰胶囊对大鼠大肠杆菌感染性盆腔炎的改善作用研究", 《药物评价研究》 * |
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