CN114984055A - Periplaneta americana intestinal bacteria metabolite extract, pharmaceutical composition and application of pharmaceutical composition in preparation of drugs for treating cardiovascular and cerebrovascular diseases - Google Patents
Periplaneta americana intestinal bacteria metabolite extract, pharmaceutical composition and application of pharmaceutical composition in preparation of drugs for treating cardiovascular and cerebrovascular diseases Download PDFInfo
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- CN114984055A CN114984055A CN202210482479.1A CN202210482479A CN114984055A CN 114984055 A CN114984055 A CN 114984055A CN 202210482479 A CN202210482479 A CN 202210482479A CN 114984055 A CN114984055 A CN 114984055A
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- quinolinone
- hydroxy
- periplaneta americana
- intestinal bacteria
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Abstract
The invention belongs to the technical field of medicines, and particularly relates to an extract of periplaneta americana intestinal bacteria metabolites, a pharmaceutical composition and application thereof in preparation of medicines for treating cardiovascular and cerebrovascular diseases. The periplaneta americana intestinal bacteria metabolite extract and the pharmaceutical composition contain specific quinolinone compounds or quinolinic acid compounds, can relax mesenteric arteries and coronary arteries, increase vascular endothelial nitric oxide and enzyme (eNOS) phosphorylation, can be used for preventing and/or treating cardiovascular and cerebrovascular diseases, and can be used as active ingredients for preparing medicines for preventing and/or treating the cardiovascular and cerebrovascular diseases.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to an extract of periplaneta americana intestinal bacteria metabolites, a pharmaceutical composition and application thereof in preparation of medicines for treating cardiovascular and cerebrovascular diseases.
Background
Cardiovascular diseases are a major disease threatening human health, and the morbidity, disability rate and mortality of the cardiovascular diseases are high, and the incidence rate is continuously increased in recent years. With the development of science and technology and medicine, cardiovascular and cerebrovascular diseases are continuously researched and treated with considerable results, but a large number of patients still die every year because of the failure of effective treatment.
Cardiovascular diseases are caused by heart and blood vessel diseases, and researches show that endothelial dysfunction is often the cause and the pathological basis of each cardiovascular disease. Endothelial dysfunction refers to the damage of vascular endothelial cells due to ischemia, changes in blood flow shear forces, or the action of some drugs, resulting in endothelial dysfunction. Endothelial dysfunction directly contributes to the pathogenesis of a variety of cardiovascular diseases, including hypertension, atherosclerosis, stroke, diabetes, obesity, and renal failure. These major chronic diseases constitute the leading cause of death and disability worldwide. Healthy, elastic endothelial cells are not only critical for normal development, but are also essential for optimal functioning of the entire cardiovascular system. Improving the function of vascular endothelium is probably the main direction for preventing and treating cardiovascular diseases, so that the medicine with the functions of relaxing blood vessels and protecting vascular endothelial cells is searched for preventing and treating cardiovascular and cerebrovascular diseases, and has important social significance and economic significance.
Disclosure of Invention
Aiming at the technical problems, the invention provides the periplaneta Americana intestinal bacteria metabolite and the application thereof in preparing the medicines for treating the cardiovascular and cerebrovascular diseases.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a periplaneta americana metabolite extract in a first aspect, which comprises quinolinone compounds and/or quinolinic acid compounds, wherein the quinolinone compounds are selected from 6-Hydroxy-2-quinolinone (8-Hydroxy-2(1H) -quinolinone, CAS:19315-93-6), 8-Hydroxy-2-quinolinone (8-Hydroxy-2(1H) -quinolinone, CAS:15450-76-7), 2-quinolinone (quinolinone-2 (1H) -one, CAS:59-31-4), 6-Hydroxy-3, 4-dihydro-2-quinolinone (6-Hydroxy-3, 4-dihydro-1H-quinolinone, CAS:54197-66-9), At least one of 5-hydroxy-3, 4-dihydro-2-quinolinone (5-hydroxy-3, 4-dihydro-1H-quinon-2-one, CAS:30389-33-4) and 3, 4-dihydro-2-quinolinone (3, 4-dihydro-1H-quinon-2-one, CAS:553-03-7), wherein the quinolinic acid compound is 8-hydroxyquinon-2-carboxylic acid (8-hydroxyquinon-2-carboxylic acid, CAS: 1571-30-8).
Wherein the structural formula of the 6-hydroxy-2-quinolinone, the 8-hydroxy-2-quinolinone and the 2-quinolinone is shown as the formula (I):
6-hydroxy-2-quinolinone: r is 1 ＝OH，R 2 ＝H；
8-hydroxy-2-quinolinone: r 1 ＝H，R 2 ＝OH；
2-quinolinone: r is 1 ＝R 2 ＝H。
The structural formulas of 6-hydroxy-3, 4-dihydro-2-quinolinone, 5-hydroxy-3, 4-dihydro-2-quinolinone and 3, 4-dihydro-2-quinolinone are shown in formula (II):
6-hydroxy-3, 4-dihydro-2-quinolinone: r is 1 ＝R 3 ＝H,R 2 ＝OH
5-hydroxy-3, 4-dihydro-2-quinolinone: r 1 ＝OH,R 2 ＝R 3 ＝H
3, 4-dihydro-2-quinolinone: r 1 ＝R 2 ＝R 3 ＝H
The structural formula of the 8-hydroxyquinoline-2-carboxylic acid is shown as the formula (III):
the inventor finds that the quinolinone compounds and the quinolinic acid compounds can relax mesenteric arteries and coronary arteries, increase vascular endothelial nitric oxide and enzyme (eNOS) phosphorylation, protect vascular endothelial cells and reduce blood pressure, so that the quinolinone compounds and the quinolinic acid compounds can be used for preventing and/or treating cardiovascular and cerebrovascular diseases, and further can be used for preparing medicines for preventing and/or treating the cardiovascular and cerebrovascular diseases.
In combination with the first aspect, the periplaneta americana intestinal bacteria metabolite extract is an ethanol extract of periplaneta americana intestinal bacteria metabolite.
Optionally, the periplaneta americana intestinal bacteria metabolite extract is prepared by ultrasonically extracting periplaneta americana feces with 70% -80% v/v ethanol water solution, filtering, concentrating the obtained filtrate, suspending with 10 times of water, loading onto macroporous resin, sequentially washing with water and 95% v/v ethanol, collecting 95% v/v ethanol eluate, concentrating, and drying.
Preferably, the ultrasonic extraction is performed for 1-2 times, and the extraction time is 0.5-1.5 h.
In a second aspect, the present invention provides a pharmaceutical composition, which includes at least one of the quinolinone compounds and quinolinic acid compounds.
In combination with the second aspect, the pharmaceutical composition contains at least one of 8-hydroxy-2-quinolinone or 8-hydroxyquinoline-2-carboxylic acid. Experiments show that 8-hydroxy-2-quinolinone or 8-hydroxyquinoline-2-carboxylic acid has different vasodilation efficiency on the whole blood vessel and the endothelial desquamation blood vessel, which indicates that the whole blood vessel and the endothelial desquamation blood vessel relax through endothelium dependency, so the invention has the function of protecting the vascular endothelium.
With reference to the second aspect, the quinolinone compound and the quinolinic acid compound are provided in the form of a monomer, or in the form of a herbal extract containing the quinolinone compound and the quinolinic acid compound.
Optionally, the herbal extract is an alcohol extract (PAF) of intestinal metabolites of periplaneta americana.
The third aspect of the invention provides the application of the periplaneta americana intestinal bacteria metabolite extract or the pharmaceutical composition in preparing the medicines for treating cardiovascular and cerebrovascular diseases.
In combination with the third aspect, the cardiovascular and cerebrovascular diseases are diseases associated with vasodilation rate and/or vascular endothelial injury.
Preferably, the cardiovascular and cerebrovascular diseases comprise at least one of atherosclerosis, coronary heart disease, myocardial infarction, hypertension, hypertensive heart disease, diabetic vascular complications, restenosis after angioplasty, cerebral stroke, pulmonary hypertension, pulmonary heart disease, vasculitis, vascular headache and microangiopathy.
Optionally, the cardiovascular and cerebrovascular disease medicament further comprises a pharmaceutically acceptable carrier or excipient.
Optionally, the pharmaceutically acceptable carrier or excipient is selected from at least one of a solvent, diluent, disintegrant, precipitation inhibitor, surfactant, glidant, binder, lubricant, dispersant, suspending agent, isotonic agent, thickening agent, emulsifier, preservative, stabilizer, hydrating agent, emulsification accelerator, buffer, absorbent, colorant, flavoring agent, sweetener, ion exchanger, mold release agent, coating agent, flavoring agent, or antioxidant.
In the present invention, the term "treatment" has its ordinary meaning, and herein may refer to the treatment of a mammalian subject (preferably a human) already suffering from a cardiovascular or cerebrovascular disease with the medicament of the present invention in order to produce a therapeutic, curative, palliative, etc. effect on the disease. Similarly, as used herein, the term "prevention" has its ordinary meaning and may refer herein to the treatment of a mammalian subject who may suffer from or is at risk of suffering from a cardiovascular or cerebrovascular disease with an agent of the present invention in an effort to prevent, arrest, abrogate, etc. the disease.
Herein, "pharmaceutically acceptable" means having no substantial toxic effect when used in the usual dosage amounts, and thus being approved by the government or equivalent international organization or approved for use in animals, more particularly in humans, or registered in the pharmacopoeia.
The "pharmaceutically acceptable carrier or excipient" useful in the present invention may be any conventional carrier in the art of pharmaceutical formulation, and the selection of a particular carrier will depend on the mode of administration or the type and state of the disease used to treat a particular patient. The preparation of suitable cardiovascular and cerebrovascular disease drugs for a particular mode of administration is well within the knowledge of those skilled in the pharmaceutical arts.
In addition, the periplaneta americana intestinal bacteria metabolite extract and the pharmaceutical composition can also exist or be provided in the forms of health products, essential oil, functional food, food additives and the like.
Drawings
FIG. 1 shows the effect of alcoholic extracts of periplaneta americana intestinal bacteria metabolites at different concentrations on the expression of the p-eNOS, eNOS proteins in HUVEC cells in example 12 of the present invention;
FIG. 2 is the effect of alcoholic extracts of periplaneta americana intestinal bacteria metabolites at different concentrations on HUVEC cell p-eNOS protein expression in example 12 of the present invention;
FIG. 3-1 is a graph showing the effect of alcoholic extracts of the metabolite of periplaneta americana enterobacteria at different concentrations on the vasodilation rate of u46619 pre-contracted intact coronary artery blood vessel rings in example 13 of the present invention;
FIG. 3-2 is a graph showing the effect of alcoholic extracts of the metabolite of periplaneta americana enterobacteria at different concentrations on the vasodilation rate of u46619 pre-contracted endothelial coronary artery vessel rings in example 13 of the present invention;
FIGS. 3-3 are graphs showing the effect of alcoholic extracts of the metabolite of periplaneta americana enterobacteria at different concentrations on the vasodilation rate of the endothelial coronary artery vessel ring with or without preshrinking u46619 in example 13 of the present invention;
FIG. 4-1 is a graph showing the effect of alcoholic extracts of the metabolite of periplaneta americana enterobacteria at different concentrations on u46619 pre-systolic whole mesenteric artery vasodilator rate in example 13 of the present invention;
FIG. 4-2 is a graph showing the effect of alcoholic extracts of the metabolite of periplaneta americana enterobacteria at different concentrations on the vasodilation rate of u46619 pre-contracted endothelial mesenteric artery blood vessel rings in example 13 of the present invention;
FIGS. 4-3 are graphs showing the effect of alcoholic extracts of the metabolite of periplaneta americana enterobacteria at different concentrations on the vasodilation rate of the vascular ring of the endothelial mesenteric artery with or without preshrinking u46619 in example 13 of the present invention;
FIG. 5 is a graph showing the effect of different concentrations of 6-hydroxy-2-quinolinone on the vasodilation rate of u46619 pre-systolic coronary artery blood vessel rings in example 13 of the present invention;
FIG. 6 is a graph showing the effect of different concentrations of 6-hydroxy-2-quinolinone on the vasodilation rate of u46619 pre-systolic mesenteric artery blood vessel ring in example 13 of the present invention;
FIG. 7 is a graph showing the effect of different concentrations of 8-hydroxy-2-quinolinone on the vasodilation rate of u46619 pre-contracted coronary artery blood vessel rings in example 13 of the present invention;
FIG. 8-1 is a graph showing the effect of different concentrations of 8-hydroxy-2-quinolinone on u46619 pre-systolic whole mesenteric arterial vessel ring diastolic rate in example 13 of the present invention;
FIG. 8-2 is a graph showing the effect of different concentrations of 8-hydroxy-2-quinolinone on the vasodilation rate of u46619 pre-contracted endotheliai mesenteric artery blood vessel rings in example 13 of the present invention;
FIGS. 8-3 are graphs showing the effect of different concentrations of 8-hydroxy-2-quinolinone on the vasodilation rate of the endothelial mesenteric artery with or without presystoresis of u46619 in example 13 of the present invention;
FIG. 9 is a graph showing the effect of different concentrations of 2-quinolinone on u46619 pre-systolic coronary vasodilator ratio in example 13 of the present invention;
FIG. 10 is a graph showing the effect of different concentrations of 2-quinolinone on the vasodilation rate of u46619 pre-systolic mesenteric artery blood vessel rings in example 13 of the present invention;
FIG. 11 is a graph showing the effect of different concentrations of 6-hydroxy-3, 4-dihydro-2-quinolinone on u46619 pre-systolic coronary vasodilation rate in example 13 of the present invention;
FIG. 12 is a graph showing the effect of different concentrations of 6-hydroxy-3, 4-dihydro-2-quinolinone on u46619 pre-systolic mesenteric arterial vessel ring diastolic rate in example 13 of the present invention;
FIG. 13 is a graph showing the effect of different concentrations of 5-hydroxy-3, 4-dihydro-2-quinolinone on u46619 pre-systolic coronary vasodilation rate in example 13 of the present invention;
FIG. 14 is a graph showing the effect of different concentrations of 5-hydroxy-3, 4-dihydro-2-quinolinone on u46619 pre-systolic mesenteric arterial vessel ring diastolic rate in example 13 of the present invention;
FIG. 15 is a graph showing the effect of different concentrations of 3, 4-dihydro-2-quinolinone on the vasodilation rate of u46619 pre-systolic coronary blood vessel rings in example 13 of the present invention;
FIG. 16 is a graph showing the effect of different concentrations of 3, 4-dihydro-2-quinolinone on u46619 pre-systolic mesenteric arterial vessel vasodilation rate in example 13 of the present invention;
FIG. 17 is a graph showing the effect of different concentrations of 8-hydroxyquinoline-2-carboxylic acid on the vasodilation rate of u46619 pre-contracted coronary artery blood vessels in example 13 of the present invention;
FIG. 18-1 is a graph showing the effect of different concentrations of 8-hydroxyquinoline-2-carboxylic acid on the vasodilation rate of u46619 pre-contracted integral mesenteric arterial vessel rings in example 13 of the present invention;
FIG. 18-2 is a graph showing the effect of different concentrations of 8-hydroxyquinoline-2-carboxylic acid on the vasodilation rate of u46619 pre-contracted to endotheliai mesenteric artery blood vessels in example 13 of the present invention;
FIGS. 18-3 are graphs showing the effect of different concentrations of 8-hydroxyquinoline-2-carboxylic acid on the vasodilation rate of u46619 pre-systolic and non-endothelial mesenteric arterial vessel rings in example 13 of the present invention;
FIG. 19 is a graph showing the effect of 6-hydroxy-2-quinolinone, 8-hydroxy-2-quinolinone, 6-hydroxy-3, 4-dihydro-2-quinolinone, 5-hydroxy-3, 4-dihydro-2-quinolinone, 8-hydroxyquinoline-2-carboxylic acid on coronary artery diastolic rate in example 13 of the present invention;
FIG. 20 shows the effect of 6-hydroxy-2-quinolinone, 8-hydroxy-2-quinolinone, 6-hydroxy-3, 4-dihydro-2-quinolinone, 5-hydroxy-3, 4-dihydro-2-quinolinone, 8-hydroxyquinoline-2-carboxylic acid on mesenteric artery diastolic rate in example 13 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to specific embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. All other embodiments that can be derived by one of ordinary skill in the art from the disclosure are intended to be within the scope of the disclosure.
The american sickle is a dried body of Periplaneta americana (Linnaeus) which is an insect belonging to the family blattaceae (chinese herbal medicine single-page standard (2021 year), chinese herbal medicine standard (2010 version), and chinese herbal medicine standard (2009 version) in hunan province). At present, the periplaneta americana is a commercially available preparation with an active ingredient, such as a Xinmailong injection, and the product has the effects of tonifying qi and activating blood, activating yang and inducing diuresis, is an adjuvant drug for chronic congestive heart failure caused by chronic pulmonary heart disease, and can be used for improving symptoms such as palpitation, edema, short breath, gloomy complexion, cyanosis of lips and the like caused by chronic congestive heart failure with both deficiency of qi and yang and internal resistance of blood stasis.
The periplaneta americana intestinal bacteria metabolite is derived from periplaneta americana feces, is different from the periplaneta americana whole worm, and has the active ingredients with the types and the contents which are obviously different from the periplaneta americana whole worm dried body. Periplaneta americana feces are generally treated as waste at present.
The first aspect of the embodiments of the present invention provides an extract of periplaneta americana intestinal bacteria metabolites, which comprises quinolinone compounds and/or quinolinic acid compounds, wherein the quinolinone compounds are at least one selected from 6-hydroxy-2-quinolinone, 8-hydroxy-2-quinolinone, 6-hydroxy-3, 4-dihydro-2-quinolinone, 5-hydroxy-3, 4-dihydro-2-quinolinone, and the quinolinic acid compounds are 8-hydroxyquinoline-2-carboxylic acid.
The inventor of the invention discovers that quinolinone compounds 6-hydroxy-2-quinolinone, 8-hydroxy-2-quinolinone, 6-hydroxy-3, 4-dihydro-2-quinolinone, 5-hydroxy-3, 4-dihydro-2-quinolinone, 3, 4-dihydro-2-quinolinone and quinolinic acid compounds 8-hydroxyquinoline-2-carboxylic acid contained in alcohol extracts of the periplaneta americana enterobacteria metabolites have the functions of expanding coronary artery, mesenteric artery and protecting vascular endothelium and are helpful for protecting heart function through deep research on the periplaneta americana enterobacteria metabolites, thereby being used for preventing and/or treating cardiovascular and cerebrovascular diseases related to vasodilatation and blood vessel inner wall functions. At present, no relevant report exists on the research of the periplaneta americana fecal intestinal bacteria metabolite extract on the aspects of vasodilatation and vascular endothelial function protection. The invention provides data support for recycling the periplaneta americana feces and also provides a new application direction for the periplaneta americana in the aspect of cardiovascular and cerebrovascular diseases.
In the present invention, the extraction method of the extract is not particularly limited as long as the quinolinone compound and/or quinolinic acid compound can be obtained, and a person skilled in the art can obtain the alcoholic extract containing the quinolinone compound and quinolinic acid compound by the existing extraction method, and exemplarily, the alcoholic extract containing the metabolite of periplaneta americana intestinal bacteria containing the quinolinone compound and quinolinic acid compound can be obtained by performing ultrasonic extraction with 70% -80% v/v ethanol solution.
Since the quinolinone compounds and the quinolinic acid compounds have the functions of dilating blood vessels and protecting vascular endothelium, the second aspect of the embodiments of the present invention provides a pharmaceutical composition, which includes the quinolinone compounds and the quinolinic acid compounds.
In some embodiments of the second aspect of the invention, the pharmaceutical composition comprises at least one of 8-hydroxy-2-quinolinone or 8-hydroxyquinoline-2-carboxylic acid. Both can not only relax artery blood vessel, but also protect blood vessel endothelium.
In some embodiments of the second aspect of the present invention, the quinolinone compound and the quinolinic compound are provided in the form of a monomer, or in the form of a herbal extract containing the quinolinone compound and the quinolinic compound. Wherein the Chinese medicinal extract can be selected from alcohol extract of metabolite of periplaneta Americana intestinal bacteria, such as alcohol extract obtained by ultrasonic extraction with 70% -80% v/v ethanol water solution.
The third aspect of the embodiment of the invention provides application of the periplaneta americana intestinal bacteria metabolite extract or the pharmaceutical composition in preparation of drugs for treating cardiovascular and cerebrovascular diseases.
In some embodiments of the present invention, cardiovascular and cerebrovascular diseases suitable for use include, but are not limited to, atherosclerosis, coronary heart disease, myocardial infarction, hypertension, hypertensive heart disease, diabetic vascular complications, restenosis following angioplasty, stroke, pulmonary hypertension, pulmonary heart disease, vasculitis, vascular headache, microangiopathy, and the like.
In some embodiments of the third aspect of the present invention, the medicament for treating cardiovascular and cerebrovascular diseases further comprises a pharmaceutically acceptable carrier or excipient.
In some embodiments of the third aspect of the present invention, the pharmaceutically acceptable carrier or excipient is selected from at least one of a solvent, diluent, disintegrant, precipitation inhibitor, surfactant, glidant, adhesive, lubricant, dispersant, suspending agent, isotonizing agent, thickener, emulsifier, preservative, stabilizer, hydrating agent, emulsification accelerator, buffer, absorbent, colorant, flavoring agent, sweetener, ion exchanger, mold release agent, coating agent, flavoring agent, or antioxidant.
The periplaneta americana intestinal bacteria metabolite extract or the pharmaceutical composition of the present invention may be formulated with one or more pharmaceutically acceptable carriers or excipients into desired dosage forms using conventional techniques in the pharmaceutical field, particularly in the formulation field.
The dosage form of the cardiovascular and cerebrovascular disease drug of the present invention may be selected from a pharmaceutical preparation suitable for oral administration, a pharmaceutical preparation (e.g., solution) suitable for parenteral injection (e.g., intravenous injection, subcutaneous injection), a pharmaceutical preparation (e.g., ointment, patch or cream) suitable for surface administration, a pharmaceutical preparation (e.g., suppository) suitable for rectal administration, and the like. Dosage forms for oral administration may include, for example, tablets, pills, hard or soft capsules, solutions, suspensions, emulsions, syrups, powders, fine granules, pellets, elixirs and the like, without limitation. In addition to the active ingredient, these preparations may contain diluents (e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, and glycine), lubricants (e.g., silica, talc, stearic acid or its magnesium salt, calcium salt, and polyethylene glycol). Tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, and polyvinylpyrrolidone. If necessary, it may further contain pharmaceutically acceptable additives such as disintegrating agents (e.g., starch, agar, alginic acid or sodium salt thereof), absorbents, coloring agents, flavoring agents, sweetening agents, etc. Tablets may be prepared according to conventional mixing, granulating or coating methods.
Terms used herein, if not explicitly stated or defined, have their ordinary meanings as known to those skilled in the art.
Materials referred to in the following examples: human umbilical vein endothelial cells: ossels Biotechnology, Inc.; fetal bovine serum: gibco, USA; endothelial cell complete medium: oersel Biotechnology, Inc.; phosphate Buffered Saline (PBS): israel BI Inc.; RIPA lysate: beijing kang is a century Biotechnology Ltd; phenylmethylsulfonyl fluoride (PMSF): beijing kang is a century Biotechnology Ltd; BCA protein quantification kit: beijing kang is a century Biotechnology Ltd; SDS-PAGE protein loading buffer: beijing Solaibao science and technology, Inc.; SDS electrophoresis solution: beijing kang is a century Biotechnology Ltd; film transferring liquid: beijing kang is a century Biotechnology Ltd; methanol: tianjin Damao chemical reagent works; TBST buffer: beijing kang is a worldJi Biotechnology, Inc.; marker: american society for siemer and femier technologies; PVDF film: millipore, USA; and (3) skim milk powder: BD corporation, usa; hypersensitive ECL chemiluminescent kit: millipore, USA; the eNOS antibody: abcam corporation, USA; p-eNOS antibody, Abcam, USA; secondary antibody rabbit antibody: jackson, USA; 9, 11-dideoxy-11 α,9 α -methyleneepoxyprostaglandin F2 α (11-Didexy-11 α,9 α -epoxyethane prostaglandin F2 α, u46619), acetylcholine (Ach), Dimethylsulfoxide (DMSO): sigma corporation; potassium chloride (KCl): borott (Tianjin) chemical trade, Inc.; sodium chloride (NaCl): tianjin, Daimao chemical reagent plant; magnesium sulfate (MgSO) 4 ·7H 2 O): tianjin, Guang Compound science and technology development Co., Ltd; sodium bicarbonate (NaHCO) 3 ): tianjin, Daimao chemical reagent plant; glucose (D-glucose): tianjin, Daimao chemical reagent plant; anhydrous potassium dihydrogen phosphate instrument (KH) 2 PO 4 ): tianjin Bailuns Biotechnology Ltd; crystalline calcium chloride (CaCl) 2 ·2H 2 O): tianjin Bailuns Biotechnology Ltd;
the apparatus referred to in the following examples: pipette gun (Research plus): eppendorf, Germany; microplate reader: (
F50) The method comprises the following steps TECAN, switzerland; western electrophoresis and membrane transfer device (1658033): BIO-RAD, USA; ultrasensitive multifunctional imager (AI 600): GE, USA; vortex oscillator (Votex-5): lonbel instruments manufacturing, Inc., of Haiman; low speed centrifuge (LX-900): lorber instruments manufacturing, Inc. of Lomenea; multichannel isolated microvascular tonicity determinator 620M: DMT company; PowerLab biological signal acquisition and analysis system: ADInstructions Inc
Other experimental materials and methods used in the following examples are conventional materials and methods unless otherwise specified.
The following examples of periplaneta americana feces samples were taken from GAP culture base of the stone mountain periplaneta americana of Yunnan Teng pharmaceutical GmbH, wherein the feeding conditions of the periplaneta americana were strictly performed according to the GAP standard, and the main ingredient of the feed was bean. The stool samples of the sickle americana used in the following examples had a 6-hydroxy-3, 4-dihydro-2-quinolinone content of 0.11-0.15%, a 8-hydroxy-2-quinolinone content of 0.55-0.71%, and a 8-hydroxy-2-quinolinic acid content of 1.28-1.72%.
Example 1
The embodiment of the invention provides an American cockroach intestinal bacteria metabolite extract, and the preparation method comprises the following steps:
carrying out ultrasonic extraction on 1kg of periplaneta americana feces sample by using a 75% v/v ethanol water solution for 1h, filtering after extraction, merging obtained filtrate, concentrating under reduced pressure, suspending the concentrated solution by using 10 times of water, separating by using macroporous adsorption resin, eluting by using water and 95% v/v ethanol in sequence, collecting 95% v/v ethanol eluate, recovering a solvent under reduced pressure, and drying to obtain ethanol extract powder of the periplaneta americana intestinal bacteria metabolite.
Example 2
The embodiment of the invention provides an American cockroach intestinal bacteria metabolite extract, and the preparation method comprises the following steps:
carrying out ultrasonic extraction on 1kg of periplaneta americana fecal sample twice with 70% v/v ethanol water solution for 1.5h each time, wherein the using amount of the ethanol water solution is 5.5L each time, filtering after extraction, merging obtained filtrate, carrying out reduced pressure concentration, suspending the concentrated solution with 10 times of water, separating by adopting macroporous adsorption resin, eluting by using water and 95% v/v ethanol in sequence, collecting 95% v/v ethanol eluent, recycling the solvent under reduced pressure, and drying to obtain the ethanol extract powder of the periplaneta americana intestinal bacteria metabolite.
Example 3
The embodiment of the invention provides an American cockroach intestinal bacteria metabolite extract, and the preparation method comprises the following steps:
carrying out ultrasonic extraction on 1kg of periplaneta americana fecal sample twice with 80% v/v ethanol water solution for 0.5h each time, wherein the using amount of the ethanol water solution is 4.5L each time, filtering after extraction, merging obtained filtrate, carrying out reduced pressure concentration, suspending the concentrated solution with 10 times of water, separating by adopting macroporous adsorption resin, eluting by using water and 95% v/v ethanol in sequence, collecting 95% v/v ethanol eluent, recycling the solvent under reduced pressure, and drying to obtain the ethanol extract powder of the periplaneta americana intestinal bacteria metabolite.
Example 4
The embodiment of the invention provides application of the periplaneta americana intestinal bacteria metabolite extract in preparation of medicines for treating cardiovascular and cerebrovascular diseases.
The preparation method comprises the following steps: the alcohol extract powder of the periplaneta americana intestinal bacteria metabolite obtained in any one of the embodiments 1 to 3 and an auxiliary material such as a diluent are prepared into an oral preparation.
Example 5
The embodiment of the invention provides application of the periplaneta americana intestinal bacteria metabolite extract in preparation of medicines for treating cardiovascular and cerebrovascular diseases.
The preparation method comprises the following steps: sterilizing the alcohol extract powder of the periplaneta americana intestinal bacteria metabolite obtained in any one of embodiments 1 to 3, and preparing the sterilized powder for injection.
Example 6
The embodiment of the invention provides a pharmaceutical composition which consists of 8-hydroxy-2-quinolinone and 8-hydroxyquinoline-2-carboxylic acid.
Example 7
The embodiment of the invention provides a pharmaceutical composition which comprises 8-hydroxy-2-quinolinone, 8-hydroxyquinoline-2-carboxylic acid and at least one of 6-hydroxy-2-quinolinone, 6-hydroxy-3, 4-dihydro-2-quinolinone, 5-hydroxy-3, 4-dihydro-2-quinolinone and 3, 4-dihydro-2-quinolinone.
Example 8
The embodiment of the invention provides a pharmaceutical composition which comprises 8-hydroxy-2-quinolinone and at least one of 6-hydroxy-2-quinolinone, 6-hydroxy-3, 4-dihydro-2-quinolinone, 5-hydroxy-3, 4-dihydro-2-quinolinone and 3, 4-dihydro-2-quinolinone.
Example 9
The embodiment of the invention provides a pharmaceutical composition which comprises 8-hydroxyquinoline-2-carboxylic acid and at least one of 6-hydroxy-2-quinolinone, 6-hydroxy-3, 4-dihydro-2-quinolinone, 5-hydroxy-3, 4-dihydro-2-quinolinone and 3, 4-dihydro-2-quinolinone.
Example 10
The embodiment of the invention provides application of the pharmaceutical composition in preparing a medicament for treating cardiovascular and cerebrovascular diseases.
The preparation method comprises the following steps: the pharmaceutical composition of any one of embodiments 6 to 9 and an auxiliary material such as a diluent are prepared into an oral preparation.
Example 11
The embodiment of the invention provides application of the pharmaceutical composition in preparing a medicament for treating cardiovascular and cerebrovascular diseases.
The preparation method comprises the following steps: the pharmaceutical composition according to any one of embodiments 6 to 9 is pulverized and sterilized to prepare sterile powder for injection.
Example 12
The embodiment of the invention provides an experiment for the influence of alcohol extract of periplaneta americana intestinal bacteria metabolite on the expression of human umbilical vein endothelial cells p-eNOS and eNOS proteins.
1.1 cell plating
(1) When the human umbilical vein endothelial cells grow to adhere to the wall and fuse 90%, the cells are collected.
(2) Discarding the supernatant, adding 1mL of endothelial cell complete medium, repeatedly blowing the cells for about 7 times by the gun head against the bottom of the bottle to fully disperse the cells, adding 2mL of endothelial cell complete medium, and uniformly mixing the cell suspension.
(3) Cell counting: pipette 10. mu.L of the cell suspension into a 200. mu.L centrifuge tube using a 10. mu.L pipette, add 90. mu.L of human umbilical vein endothelial cell complete medium, and blow-beat uniformly. The counting plate and the cover glass are wiped clean, and the cover glass is covered on the counting plate. And sucking 10 mu L of cell suspension, dripping the cell suspension on the edge of a cover glass to ensure that the suspension is filled between the cover glass and the counting plate, standing for 3min, wherein bubbles can not exist under the cover glass, and the suspension can not flow into a side groove. The total number of the four big grids of the counting plate is calculated, the line pressing cells are only counted on the left side and the upper side, and the counting is carried out according to a formula: cell number/mL-total of four large cells/4 × 10 5 . If the cell mass is more than two cells, the cell mass is calculated according to a single cell, and if the cell mass is more than 10%, the dispersion is not good, and the cell suspension needs to be prepared again.
(4) Diluting the cell density to1×10 6 one/mL was seeded in six well plates, 2mL per well. The six-well plate needs to be coated with collagen, 670. mu.L of diluted coating solution is added into each well, so that the coating solution is uniformly distributed on the bottom surface of the culture bottle, and the culture bottle is placed in an incubator at 37 ℃ and is placed overnight. The concentration of the coating liquid in the six-hole plate is 5 mu g/cm 2 。
1.2 pharmaceutical intervention
1.2.1 preparation of reagents for experiments
Precisely weighing 15, 35, 75 and 115mg of the periplaneta americana intestinal bacteria metabolite prepared in example 1, and adding 1mL of DMSO respectively to prepare 15, 35, 75 and 115mg/mL of the periplaneta americana intestinal bacteria metabolite ethanol extract solution. Adding alcohol extract solution of periplaneta americana intestinal bacteria metabolites with various concentrations into a basic culture medium according to the proportion of 1:1000, and adding 0.4% fetal bovine serum to prepare a drug-containing culture medium with final concentrations of 15, 35, 75 and 115 mu g/mL. The DMSO group was added with DMSO in the corresponding ratio.
1.2.2 pharmaceutical intervention
(1) Grouping
When 90% of cells are fused, dividing the six-hole plate paved with human umbilical vein endothelial cells into 6 groups: blank group, DMSO group, ethanol extract of periplaneta americana intestinal bacteria metabolite 15 microgram/mL group, ethanol extract of periplaneta americana intestinal bacteria metabolite 35 microgram/mL group, ethanol extract of periplaneta americana intestinal bacteria metabolite 75 microgram/mL group, ethanol extract of periplaneta americana intestinal bacteria metabolite 115 microgram/mL group, and marking. And (3) discarding the endothelial cell complete culture medium in the six-well plate, adding 1mL of PBS into each well to uniformly distribute the PBS on the cell surface of the six-well plate, discarding the PBS after 1min, and repeating the steps twice.
(2) Cell starvation
The cell starvation treatment is carried out for 6 hours in a DMSO group, a periplaneta americana intestinal bacteria metabolite alcohol extract 15 microgram/mL group, a periplaneta americana intestinal bacteria metabolite alcohol extract 35 microgram/mL group, a periplaneta americana intestinal bacteria metabolite alcohol extract 75 microgram/mL group and a periplaneta americana intestinal bacteria metabolite alcohol extract 115 microgram/mL group, a basal medium containing 0.4% FBS is added into each air, and the blank group is not treated.
(3) After 6h of drug intervention, the culture medium is discarded, the drug-containing culture medium is added into each group, the blank group is not treated, and the cells are collected after 1h of incubation. The specific intervention method comprises the following steps:
blank group: endothelial cell complete culture medium culture;
DMSO group: 1/1000DMSO in 0.4% basal medium;
alcohol extracts of periplaneta americana intestinal bacteria metabolites 15 μ g/mL group: 0.4% basal medium with the final concentration of alcohol extract of the periplaneta americana intestinal bacteria metabolite of 15 mug/mL;
alcohol extracts of periplaneta americana intestinal bacteria metabolites 35 μ g/mL group: 0.4% basal medium with the final alcohol extract concentration of the periplaneta americana intestinal bacteria metabolite of 35 mug/mL;
alcohol extracts of periplaneta americana enterobacteria metabolites of 75 μ g/mL group: 0.4% basal medium with the final concentration of alcohol extract of the periplaneta americana intestinal bacteria metabolite of 75 mug/mL;
alcohol extract of periplaneta americana intestinal bacteria metabolite 115 μ g/mL group: the final alcohol extract concentration of the periplaneta americana intestinal bacteria metabolite is 115 mug/mL of 0.4% basal medium.
1.3 extraction of cell Total protein
1.3.1 protein extraction
(1) Preparing a lysis solution: 980 μ L RIPA lysate +10 μ L PMSF +10 μ L phosphatase inhibitor, i.e. protein lysate: protease inhibitor: phosphatase inhibitor: 100:1:1, placed on ice.
(2) Placing six-well plate on ice, discarding the culture medium containing drug, adding 2mL precooled PBS into each well to make PBS uniformly distributed on the cell surface, discarding PBS after 1min, and repeating the above steps for 3 times.
(3) The PBS in the six-well plate was aspirated off, 20. mu.L of protein lysate was added to each well, the cells at the bottom of the plate were scraped off with a cell scraper and dissolved in the protein lysate, the protein lysate was aspirated into a 1.5mL centrifuge tube and lysed on ice for 30min, and the mixture was mixed once every 10 min.
(4) After each set of centrifuge tubes was placed in a centrifuge, and after leveling, the tubes were centrifuged at 12000rpm for 15min at 4 ℃ and the supernatant was aspirated, placed in a corresponding new 1.5mL centrifuge tube, and the volume of the supernatant was recorded.
1.3.2 BCA protein quantification
(1) BSA standard gradient dilution
Marking A-G by 7 centrifugal tubes of 600 mu L respectively, adding 60 mu L PBS into the centrifugal tubes marked B-G by a 100 mu L pipette, adding 60 mu L standard substance into the tube B, and changing the gun head to blow and beat uniformly; sucking 60 mu L of the mixture from the tube B, adding the mixture into the tube C, and changing the gun head to blow and beat uniformly; the operations were performed sequentially, without any standard added to tube G, and undiluted standard added to tube A.
(2) Sample dilution
And (5) taking 600 mu L centrifuge tubes with the same number as the samples to be detected, and marking the centrifuge tubes with numbers. And adding 57 mu L of PBS into each centrifuge tube, and then adding 3 mu L of the sample to be detected corresponding to the serial number, namely diluting the sample to be detected by 20 times. PBS should be added first, then the sample to be tested should be added, the order cannot be reversed, and a new gun head should be replaced when different samples to be tested are added.
(3) Preparing a BCA working solution
Calculating the total amount of the BCA working solution: total BCA working solution ═ number of samples (BSA standard + number of samples) × number of replicate wells × volume of BCA working solution per sample.
And preparing the BCA working solution according to the calculated total amount of the BCA working solution required, and fully and uniformly mixing the BCA working solution and the BCA-B according to the volume ratio of 50: 1.
When preparing the BCA working solution, 1 more well is prepared due to possible errors in sample application.
(4) Quantitative determination (micropore determination method)
The diluted A-G BSA standard and the protein sample to be tested (dilution) were added to the labeled wells of a 96-well plate in an amount of 25. mu.L each. 2 parallel reactions were performed for each sample assayed.
Adding 200 μ L BCA working solution into each well, mixing well, covering 96-well plate, incubating at 37 deg.C for 30min, cooling to room temperature, and completing detection within 3-5 min.
And detecting the light absorption values of the standard protein and each sample at the wavelength of 560nm by using a microplate reader, and simultaneously recording.
And drawing a standard curve according to the absorbance value of the BSA standard substance gradient liquid, and calculating the protein concentration of the sample to be detected according to a curve formula (y ═ ax + b). The final concentration of the protein is set as 1 mu g/mu L, and the required lysate volume and Loading buffer volume of each group of protein are calculated according to the protein volume.
1.3.3 denaturation of protein
Adding the lysate and Loading buffer volume obtained by protein quantification into a protein sample in sequence, fully and uniformly mixing, performing metal bath denaturation at 100 ℃ for 5min, standing at room temperature, and storing at-80 ℃.
1.4 Western blot
1.4.1 preparation of SDS-PAGE gels
8% of the separation gel was selected according to the molecular weight size for experimental purposes:
(1) the electrophoresis slide should be cleaned before glue pouring. The glass plates are placed into a clamp for clamping after being aligned, and then are vertically clamped on a frame for glue pouring. The operation is to align the two glasses to avoid glue leakage.
(2) Different volumes of 30% Acr-Bis (29:1), gel buffer and ultrapure water were mixed in a 50mL centrifuge tube. 10% ammonium persulfate and TEMED were added and mixed well with gentle stirring to avoid the formation of bubbles. Adding TEMED, shaking to mix well, adding gel liquid to a distance of 1.5cm from the top of the front glass plate or 0.5cm from the comb teeth, and covering a layer of anhydrous ethanol of 1cm on the gel to keep the gel surface flat. Standing for 40-60min until a clear interface appears between the separation gel and the water layer, and polymerizing the surface gel. The aqueous layer overlaid on the separation gel was removed.
(3) Different volumes of 30% Acr-Bis (29:1), gel-concentrate buffer and ultrapure water were mixed in a 50mL centrifuge tube. 10% ammonium persulfate and TEMED were added and mixed by gentle stirring to avoid the formation of bubbles. Immediately after TEMED was added, the gel was poured by shaking, and the concentrated gel solution was added to the top of the separation gel until the gel solution reached the front end of the front glass plate. A comb was inserted into the gel to avoid air bubbles. Standing for 10-20min, and waiting for polymerization of the concentrated gel.
1.4.2 Loading and electrophoresis
(1) Preparation of electrophoresis solution (1 ×): electrophoresis solution (10X) 100mL + 900mL of ultrapure water.
(2) And (3) putting the glue into an electrophoresis tank, and if only one glue is run, padding a plastic plate at the other end of the electrophoresis tank. The electrophoresis liquid is filled between the two pieces of glue, and the two hands respectively hold the two sides of the comb and vertically and lightly pull out the comb.
(3) 2 mu L of marker is respectively injected into the first sample loading hole and the second sample loading hole, a blank group, a DMSO group, a American cockroach intestinal bacteria metabolite alcohol extract 15 mu g/mL group, a American cockroach intestinal bacteria metabolite alcohol extract 35 mu g/mL group, a American cockroach intestinal bacteria metabolite alcohol extract 75 mu g/mL group and an American cockroach intestinal bacteria metabolite alcohol extract 115 mu g/mL histone are sequentially injected into the sample loading holes, each hole is 10 mu L, and after the sample loading of the proteome is finished, 2 mu L of marker is injected into the last sample loading hole so as to record the sequence and the membrane shearing.
(4) Electrophoresis was performed in a constant voltage mode. Electrophoresis is carried out at 90V, when the strip runs out and the gel begins to be separated after the gel is concentrated, the voltage is raised to 120V, and the electrophoresis can be stopped when the marker strip reaches the position near the bottom end of the gel, or the electrophoresis can be stopped after the target protein is expected to be properly separated according to the standard electrophoresis condition of the marker protein molecular weight.
1.4.3 transfer film
(1) Preparing a membrane transferring liquid (1 x): spin-on solution (10X) 100mL + methanol 200mL + ultrapure water 700mL
(2) PVDF membranes with the same size are cut according to the size of the colloid and placed in a culture dish filled with methanol.
(3) Prying the glass plate open, and slightly scraping off the concentrated glue.
(4) And a clamp for transferring the membrane, two sponge pads, filter paper and the PVDF membrane soaked by methanol are placed in an enamel tray added with the membrane transferring liquid. The order of the planking is blackboard → sponge → filter paper → colloid → PVDF membrane → filter paper → sponge → whiteboard. No air bubbles can exist between the film and the colloid. The clamp is placed into the film rotating clamping groove, ice blocks with proper sizes are placed into the film rotating groove, and the film rotating groove is placed into the foam box filled with crushed ice.
(5) The film is rotated in a constant current mode, and the current is set to be 300mA for 90 min.
1.4.4 sealing
(1) Preparing a sealing liquid:
TBST (1 ×) solution: TBST (10X) 100mL + 900mL of ultrapure water
Confining liquid (5% skim milk powder): defatted milk powder 5g was dissolved in 100mL TBST (1X) solution.
(2) And taking out the PVDF membrane, putting the PVDF membrane into a TBST (1 x) solution, shaking the table for 5min, and washing off the residual membrane transferring solution on the PVDF membrane.
(3) The PVDF membrane is placed in the confining liquid and incubated on a shaker for 2 h.
1.4.5 incubation Primary antibody
(1) Preparing a primary antibody: primary antibodies were diluted to the corresponding concentrations with 1% BSA.
(2) After the sealing, the PVDF membrane was taken out, put into TBST (1X) solution, and shaken for 5min to wash off the residual sealing solution on the PVDF membrane.
(3) And (3) cutting the PVDF membrane according to the molecular weight of the target protein antibody, and respectively putting the PVDF membrane into different antibody incubation boxes. The corresponding primary antibody was added, the membrane was soaked thoroughly by bottoming, incubated on a shaker at 4 ℃ and left overnight.
(4) After overnight, the antibody incubation box was removed and allowed to rewarmed at room temperature for 30min, and primary antibody was recovered. The membranes were shaken three times with TBST (1X) solution for 15min each time, and the liquid was decanted off each time. The film can not be blown when TBST is added, and the film must be washed thoroughly when being washed, so that the black shadow is prevented from appearing during color development.
1.4.6 incubation Secondary antibodies
(1) Preparing a secondary antibody: the secondary antibody was diluted to 1:10000 with 5% skim milk powder.
(2) 3mL of secondary antibody diluent is added into each grid, and the mixture is placed on a shaking table to be incubated for 2 hours at room temperature.
(3) After recovery of the secondary antibody, the membrane was washed three times with TBST (1X) solution for 15min each. The film must be washed thoroughly to prevent the occurrence of black shadow during color development.
1.4.7 color development
(1) Preparing a developing solution: two bottles of Immobilon Western HRP substrate were added to a light shielding box at a ratio of 1:1 and mixed well for use, and the developer was prepared as it is.
(2) The PVDF membrane is completely soaked in the developing solution for 1 min.
(3) The PVDF membrane is placed in a gel imager, placed in parallel and automatically exposed.
1.4.8 elution of antibodies
(1) After completion of the luminescence detection, the PVDF membrane was rinsed for 5min with distilled water by a shaker.
(2) Discarding distilled water, adding 3mL primary-antibody and secondary-antibody scavenger per cell, and rinsing for 10min by a shaking table.
(3) Discard primary anti-secondary antibody scavenger, add 5ml of LTBST (1X) solution to each compartment to rinse PVDF membrane for 5min each time, repeat 4 times.
Thereafter, blocking, primary antibody incubation, secondary antibody incubation, development and the like are performed.
1.4.9 Western blot result quantitative analysis
And analyzing the chemiluminescence gray value of the measured protein by using Image J software, and comparing and analyzing the chemiluminescence gray value with the gray value of the internal reference protein.
1.5 data processing
The results are expressed as Mean + -SEM, and are bar charts using GraphPad Prism 7 software. Statistical analysis was performed using SPSS21.0 software, independent sample T test was used for data comparisons between two groups, and ANOVA analysis of variance was used for significance tests for data comparisons between groups, with P <0.05 considered significant differences.
The effect of alcohol extract of periplaneta americana intestinal bacteria metabolite on the expression of human umbilical vein endothelial cell p-eNOS, eNOS protein is shown in fig. 1 and fig. 2. Fig. 1 shows the effect of alcoholic extract of periplaneta americana intestinal bacteria metabolite on expression of HUVEC cell p-eNOS and eNOS protein (n ═ 4), wherein C is blank control group, D is DMSO group, and 15, 35, 75, 115 are respectively alcoholic extract of periplaneta americana intestinal bacteria metabolite 35, 75, 115 μ g/mL. FIG. 2 shows HUVEC cells from alcohol extracts of metabolites of periplaneta americanaEffect of p-eNOS protein expression (n-5), compared to the blank control group, * P<0.05， ** P<0.01
the alcohol extract of the periplaneta americana intestinal bacteria metabolite can obviously increase eNOS protein phosphorylation, and the 115 microgram/mL alcohol extract of the periplaneta americana intestinal bacteria metabolite can obviously improve the expression of p-eNOS in human umbilical vein endothelial cells.
Example 13
The embodiment of the invention provides an experiment for the influence of quinolinone compounds and quinolinic acid compounds on the relaxation of coronary arteries and mesenteric arteries.
Selecting 8-10 week-old SPF male C57BL/6 mice with weight of 18-25 g; male SD rats at 8 weeks of age, body weight 200-. The rat is anesthetized and sacrificed, and the coronary artery and the mesenteric artery of the C57BL/6 mouse of the SD rat are respectively taken, the fat and the connective tissue around the blood vessel are removed, and the blood vessel is cut into a blood vessel ring with the length of 3-4mm (the preparation of the ring for removing the endothelial artery: the rat beard with proper thickness is taken to pass through the complete blood vessel ring, and the inner wall of the blood vessel ring is lightly rubbed for 3-4 times). The vascular ring was placed in a solution containing Krebs-Hennseleit (K-H) (composition of K-H solution: NaCl 118mmol/L, KCl 4.7.7 mmol/L, NaHCO) 3 25mmol/L、KH 2 PO 4 1.2mmol/L、MgSO 4 1.2mmol/L、CaCl 2 1.3mmol/L, D-glucose 10mmol/L) bath, maintaining the temperature at 37 ℃ and continuously passing 95% O 2 And 5% CO 2 And (4) mixing the gases.
Under a microscope, a blood vessel ring penetrates into two parallel steel needles, one steel needle is connected with a pressure sensor, the other steel needle is connected with a tension adjusting knob, no action force is given to the blood vessel at the moment, the blood vessel adapts for 15min under the unstressed state, and an instrument is zeroed; rotating a tension adjusting knob, adjusting the initial tension of the blood vessel to be 2.8mN, replacing the K-H solution once every 15min, and if the tension is reduced, readjusting to be 2.8mN, and balancing the blood vessel for 45 min; adding 20nmol/L u46619 for stimulating vasoconstriction, and sequentially adding 10% of the final concentration after the maximum constriction value -9 、10 -8 、10 -7 、10 -6 、10 -5 measuring the contractility of the acetylcholine after the acetylcholine is added by adopting a multichannel in vitro vascular tension measuring system, and calculating the diastolic rate according to the following formula:
the diastolic rate = (1- (after addition of acetylcholine-initial tension)/u 46619 maximum contraction) × 100%.
The maximal relaxation rate of the blood vessel reaches 60-80%, which indicates that the blood vessel endothelium is intact (the vasodilation reaction is less than 20% and deendothelialization is successful), so that the next experiment can be carried out.
After the detection of vascular endothelium is finished, u46619 is respectively added into a bath tank in which mesenteric artery and coronary artery are incubated to stimulate vasoconstriction, 5, 10, 20, 40 and 40 mu g/mL of alcohol extract powder solution of the periplaneta americana intestinal bacteria metabolite prepared in example 1 (DMSO is used as a solvent) are sequentially added at an interval of 5min after the constriction is stable; after the vascular endothelium detection is finished, u46619 is added into a bath tank in which mesenteric artery and coronary artery are incubated to stimulate vasoconstriction, and after the constriction is stable, the final concentration of 10 is sequentially added at an interval of 5min -6 ，10 -5 ，10 -4 ，10 -3.3 ，10 -3 The mol/L of the monomers such as 6-hydroxy-2-quinolinone, 8-hydroxy-2-quinolinone, 6-hydroxy-3, 4-dihydro-2-quinolinone, 5-hydroxy-3, 4-dihydro-2-quinolinone, 8-hydroxyquinoline-2-carboxylic acid and the like are as described above (DMSO is used as a solvent). Meanwhile, setting the control group as an interval of 5min, sequentially adding equal volume of DMSO (dimethyl sulfoxide), respectively recording the relaxation rate, and drawing a relaxation curve to obtain the following data and graphs:
the results of the influence of the alcohol extract of the periplaneta americana intestinal bacteria metabolite on the coronary artery diastolic rate are shown in table 1 and fig. 3-1 to fig. 3-3, and compared with the control group, * P<0.05， ** P<0.01, n is 8, the de-endothelialization group compared to the endothelium integrity group, # P<0.05， ## P<0.01，n＝8。
TABLE 1 influence of alcoholic extracts of periplaneta americana enterobacteria metabolites on the tension of the u46619 precontracted rat coronary vascular ring ((R))
100％，n＝10)
Note: compared with the contrast group, the ratio of the composition, * P＜0.05， ** p is less than 0.01. Compared with the alcohol extract of the metabolite of the periplaneta americana intestinal bacteria, # P＜0.05， ## P＜0.01。
the influence of the alcohol extract of the periplaneta americana intestinal bacteria metabolite on the mesenteric artery relaxation rate is shown in table 2 and fig. 4-1 to 4-3, and compared with the control group * P<0.05， ** P<0.01, n is 8, the de-endothelialization group compared to the endothelium integrity group, # P<0.05， ## P<0.01，n＝8。
TABLE 2 Effect of alcohol extracts of the metabolites of Periplaneta americana on u46619 precontracted mouse mesenteric arterial vascular ring tone (II)
100％，n＝10)
Note: compared with the contrast group, the ratio of the composition, * P＜0.05， ** P＜0.01。
the results of the effect of 6-hydroxy-2-quinolinone on coronary artery diastolic rate are shown in Table 3 and FIG. 5.
TABLE 36 Effect of hydroxy-2-quinolinone on u46619 precontracted rat coronary vascular Ring tone: (
100％，n＝3)
Note: compared with the control group, the ratio of the compound to the control group, * P＜0.05， ** P＜0.01。
the results of the effect of 6-hydroxy-2-quinolinone on the mesenteric artery diastolic rate are shown in Table 4 and FIG. 6.
TABLE 46 Effect of hydroxy-2-quinolinone on u46619 precontracted mouse mesenteric arterial vascular Ring tension (II)
100％，n＝3)
Note: compared with the contrast group, the ratio of the composition, * P＜0.05， ** P＜0.01。
the results of the effect of 8-hydroxy-2-quinolinone on coronary artery diastolic rate are shown in Table 5 and FIG. 7.
TABLE 58 Effect of-hydroxy-2-quinolinone on u46619 precontracted rat coronary vascular Ring tension ((S))
100％，n＝3)
Note: compared with the contrast group, the ratio of the composition, * P＜0.05， ** P＜0.01。
the results of the effect of 8-hydroxy-2-quinolinone on mesenteric artery diastolic rate are shown in Table 6 and FIGS. 8-1 to 8-3, and compared with the control group, * P<0.05， ** P<0.01; the de-endothelialization group compared to the endothelium completion group, # P<0.05， ## P<0.01，n＝3。
TABLE 68 Effect of-hydroxy-2-quinolinone on u46619 precontracted mouse mesenteric arterial vascular Ring tone (II)
100％，n＝3)
Note: compared with the contrast group, the ratio of the composition, * P＜0.05， ** p is less than 0.01. The de-endothelialization group compared to the endothelium completion group, # P＜0.05， ## P＜0.01。
the results of the effect of 2-quinolinone on coronary artery diastolic Rate are shown in Table 7 and FIG. 9
TABLE 72 Effect of quinolinones on u46619 precontracted rat coronary vascular Ring tone ((C))
100％，n＝3)
Note: compared with the contrast group, the ratio of the composition, * P＜0.05， ** P＜0.01。
the results of the effect of 2-quinolinone on the mesenteric artery diastolic rate are shown in table 8 and figure 10,
TABLE 82 Effect of quinolinones on u46619 Pre-constriction of vascular Ring tension in mesenteric arteries of mice (S) ((S))
100％，n＝3)
Note: compared with the control group, the ratio of the compound to the control group, * P＜0.05， ** P＜0.01。
the results of the effect of 6-hydroxy-3, 4-dihydro-2-quinolinone on coronary artery diastolic rate are shown in Table 9 and FIG. 11.
TABLE 96 Effect of hydroxy-3, 4-dihydro-2-quinolinone on u46619 Pre-constriction of vascular tone in coronary arteries in rats: (
100％，n＝3)
Note: compared with the contrast group, the ratio of the composition, * P＜0.05， ** P＜0.01。
the results of the effect of 6-hydroxy-3, 4-dihydro-2-quinolinone on mesenteric artery diastolic rate are shown in Table 10 and FIG. 12.
TABLE 106 Effect of hydroxy-3, 4-dihydro-2-quinolinone on u46619 Pre-contraction mouse mesenteric arterial vascular Ring tension: (
100％，n＝3)
Note: compared with the contrast group, the ratio of the composition, * P＜0.05， ** P＜0.01。
the results of the effect of 5-hydroxy-3, 4-dihydro-2-quinolinone on coronary artery diastolic rate are shown in Table 11 and FIG. 13.
TABLE 115 Effect of-hydroxy-3, 4-dihydro-2-quinolinone on u46619 precontracted rat coronary vascular Ring tone: (
100％，n＝3)
Note: compared with the contrast group, the ratio of the composition, * P＜0.05， ** P＜0.01。
the results of the effect of 5-hydroxy-3, 4-dihydro-2-quinolinone on mesenteric artery diastolic rate are shown in Table 12 and FIG. 14.
TABLE 125 Effect of hydroxy-3, 4-dihydro-2-quinolinone on u46619 Pre-contraction mouse mesenteric arterial vascular Ring tension: (
100％，n＝3)
Note: compared with the contrast group, the ratio of the composition, * P＜0.05， ** P＜0.01。
the results of the effect of 3, 4-dihydro-2-quinolinone on coronary artery diastolic rate are shown in Table 13 and FIG. 15.
TABLE 133 Effect of 4-dihydro-2-quinolinone on u46619 precontracted rat coronary vascular Ring tension: (
100％，n＝3)
Note: compared with the contrast group, the ratio of the composition, * P＜0.05， ** P＜0.01。
the results of the effect of 3, 4-dihydro-2-quinolinone on the diastolic rate of mesenteric arteries are shown in Table 14 and FIG. 16, and the effect of table 143, 4-dihydro-2-quinolinone on the vascular ring tension of mesenteric arteries in u46619 preconstricted mice: (
100％，n＝3)
Note: compared with the contrast group, the ratio of the composition, * P＜0.05， ** P＜0.01。
the results of the effect of 8-hydroxyquinoline-2-carboxylic acid on coronary artery diastolic rate are shown in Table 15 and FIG. 17,
TABLE 158 Effect of Hydroxyquinoline-2-carboxylic acid on u46619 Pre-constriction of vascular Ring tension in coronary arteries in rats: (
100％，n＝3)
Note: compared with the control group, the ratio of the compound to the control group, * P＜0.05， ** P＜0.01。
the results of the effect of 8-hydroxyquinoline-2-carboxylic acid on the mesenteric artery diastolic rate are shown in Table 16 and FIGS. 18-1 to 18-3.
TABLE 168 Effect of Hydroxyquinoline-2-carboxylic acid on u46619 Pre-contracted mouse mesenteric arterial ring tone (II)
100％，n＝3)
Note: compared with the control group, the ratio of the compound to the control group, * P＜0.05， ** p is less than 0.01; the de-endothelialization group compared to the endothelium-intact group, # P＜0.05， ## P＜0.01。
the effects of 6-hydroxy-2-quinolinone, 8-hydroxy-2-quinolinone, 6-hydroxy-3, 4-dihydro-2-quinolinone, 5-hydroxy-3, 4-dihydro-2-quinolinone, and 8-hydroxyquinoline-2-carboxylic acid on coronary artery diastolic rate are shown in FIG. 19, and the effects on mesenteric artery diastolic rate are shown in FIG. 20.
According to the figures 3-1-3-2, the alcohol extract of the periplaneta americana intestinal bacteria metabolite has a relaxation effect on rat coronary artery vascular rings with complete endothelium, and has a significant difference compared with a control group; the alcohol extract of the periplaneta americana intestinal bacteria metabolite has a relaxation effect on the rat coronary artery vascular ring without endothelium, and has a significant difference compared with a control group; the rate of relaxation of the de-endothelialized vascular ring compared to the endothelial intact vascular ring was significantly different. As can be seen from figures 4-1 to 4-3, the experimental results show that the alcohol extract of the periplaneta americana intestinal bacteria metabolite has a relaxation effect on mouse mesenteric artery blood vessel rings with complete endothelium, and has a significant difference compared with a control group; the alcohol extract of the periplaneta americana intestinal bacteria metabolite has a relaxation effect on the rat coronary artery vascular ring without endothelium, and has a significant difference compared with a control group; the rate of relaxation of the de-endothelialized vascular ring compared to the endothelial intact vascular ring was significantly different. The results show that the alcohol extract of the periplaneta americana intestinal bacteria metabolite can remarkably relax coronary artery and mesenteric artery, has the effect of relaxing blood vessels, and has the effect of endothelium dependence.
As shown in FIG. 5, 6-hydroxy-2-quinolinone has a relaxation effect on the coronary artery blood vessel ring of rats with intact endothelium, and is significantly different from the control group. As can be seen from FIG. 6, the experimental results show that 6-hydroxy-2-quinolinone has a relaxation effect on mesenteric artery ring of mice with intact endothelium, and has a significant difference compared with the control group. The results show that the 6-hydroxy-2-quinolinone can remarkably relax coronary arteries and mesenteric arteries and has the function of relaxing blood vessels.
As shown in FIG. 7, 8-hydroxy-2-quinolinone has a relaxation effect on the coronary artery blood vessel ring of rats with intact endothelium, and has a significant difference compared with the control group. As can be seen from FIGS. 8-1 to 8-3, 8-hydroxy-2-quinolinone has a relaxation effect on mesenteric arterial loop of mice with intact endothelium, and has significant difference compared with the control group; 8-hydroxy-2-quinolinone has a relaxation effect on the mouse mesenteric artery ring with the endothelium removed, and has a significant difference compared with a control group; the rate of relaxation of the de-endothelialized vascular ring compared to the endothelial intact vascular ring was significantly different. The results show that 8-hydroxy-2-quinolinone can remarkably relax coronary arteries and mesenteric arteries and has the function of relaxing blood vessels, and 8-hydroxy-2-quinolinone relaxes mesenteric arteries and has endothelium dependence.
As shown in FIG. 9, 2-quinolinone has a relaxation effect on the coronary artery blood vessel ring of rats with intact endothelium, and has a significant difference compared with the control group. As can be seen from FIG. 10, 2-quinolinone has a relaxation effect on mesenteric arterial loop of mice with intact endothelium, and is significantly different from the control group. The results show that the 2-quinolinone can remarkably relax coronary arteries and mesenteric arteries and has the function of relaxing blood vessels.
As can be seen from FIG. 11, 6-hydroxy-3, 4-dihydro-2-quinolinone has a vasodilating effect on the coronary artery blood vessel rings of rats with intact endothelium, and is significantly different from the control group. As can be seen from FIG. 12, 6-hydroxy-3, 4-dihydro-2-quinolinone has a relaxation effect on the mesenteric arterial ring of mice with intact endothelium, and is significantly different from the control group. The results show that the 6-hydroxy-3, 4-dihydro-2-quinolinone can remarkably relax coronary arteries and mesenteric arteries and has the function of relaxing blood vessels.
As can be seen from FIG. 13, 5-hydroxy-3, 4-dihydro-2-quinolinone has a vasodilating effect on the coronary artery blood vessel rings of rats with intact endothelium, and is significantly different from the control group. As can be seen from FIG. 14, 5-hydroxy-3, 4-dihydro-2-quinolinone has a relaxation effect on the mesenteric arterial ring of mice with intact endothelium, and is significantly different from the control group. The results show that the 5-hydroxy-3, 4-dihydro-2-quinolinone can remarkably relax coronary arteries and mesenteric arteries and has the function of relaxing blood vessels.
As can be seen from FIG. 15, 3, 4-dihydro-2-quinolinone has a relaxation effect on the coronary artery blood vessel ring of rats with intact endothelium, and is significantly different from the control group. As can be seen from FIG. 16, 3, 4-dihydro-2-quinolinone has a relaxation effect on mesenteric arterial loop of mice with intact endothelium, and is significantly different from the control group. The results show that the 3, 4-dihydro-2-quinolinone can remarkably relax coronary arteries and mesenteric arteries and has the function of relaxing blood vessels.
As shown in FIG. 17, 8-hydroxyquinoline-2-carboxylic acid was found to relax the coronary artery blood vessel ring of rats with intact endothelium, which is significantly different from the control group. As can be seen from FIGS. 18-1 to 18-3, 8-hydroxyquinoline-2-carboxylic acid has a relaxing effect on the mesenteric arterial loop of mice with intact endothelium, and has significant difference compared with the control group; 8-hydroxyquinoline-2-carboxylic acid has a relaxation effect on the mouse mesenteric artery ring with the endothelium removed, and has a significant difference compared with a control group; compared with the endothelial intact mesenteric artery ring, the relaxation rate of the de-endothelial artery ring is significantly different from that of the endothelial intact mesenteric artery ring. The results show that 8-hydroxyquinoline-2-carboxylic acid can remarkably relax coronary arteries and mesenteric arteries and has the function of relaxing blood vessels, and 8-hydroxyquinoline-2-carboxylic acid relaxes mesenteric arteries and has endothelium dependence.
According to the results, the periplaneta americana intestinal bacteria metabolite extract, 6-hydroxy-2-quinolinone, 8-hydroxy-2-quinolinone, 6-hydroxy-3, 4-dihydro-2-quinolinone, 5-hydroxy-3, 4-dihydro-2-quinolinone, 3, 4-dihydro-2-quinolinone and 8-hydroxyquinoline-2-carboxylic acid provided by the invention can relax mesenteric and coronary arteries and increase vascular endothelial nitric oxide and enzyme (eNOS) phosphorylation, so that the periplaneta americana intestinal bacteria metabolite extract can protect vascular endothelial cells and reduce blood pressure, and can be used as an active ingredient for preparing a medicament for preventing and/or treating cardiovascular and cerebrovascular diseases.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents or improvements made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (10)
1. The periplaneta americana intestinal bacteria metabolite extract is characterized by comprising the following components in parts by weight: comprises quinolinone compounds and/or quinolinic acid compounds, the quinolinone compounds are selected from at least one of 6-hydroxy-2-quinolinone, 8-hydroxy-2-quinolinone, 6-hydroxy-3, 4-dihydro-2-quinolinone, 5-hydroxy-3, 4-dihydro-2-quinolinone and 3, 4-dihydro-2-quinolinone, and the quinolinic acid compounds are 8-hydroxyquinoline-2-carboxylic acid.
2. The periplaneta americana intestinal bacteria metabolite extract according to claim 1, characterized in that: the periplaneta americana intestinal bacteria metabolite extract is an alcohol extract of periplaneta americana intestinal bacteria metabolite.
3. The periplaneta americana intestinal bacteria metabolite extract according to claim 2, characterized in that: the preparation method of the periplaneta americana intestinal bacteria metabolite extract comprises the steps of carrying out ultrasonic extraction on periplaneta americana excrement by using 70-80% v/v ethanol water solution, filtering, concentrating the obtained filtrate and drying.
4. The pharmaceutical composition is characterized by comprising at least one of the quinolinone compounds and the quinolinic acid compounds, wherein the quinolinone compounds are selected from at least one of 6-hydroxy-2-quinolinone, 8-hydroxy-2-quinolinone, 6-hydroxy-3, 4-dihydro-2-quinolinone, 5-hydroxy-3, 4-dihydro-2-quinolinone and 3, 4-dihydro-2-quinolinone, and the quinolinic acid compounds are 8-hydroxyquinoline-2-carboxylic acid.
5. The pharmaceutical composition of claim 4, wherein the pharmaceutical composition comprises at least one of 8-hydroxy-2-quinolinone or 8-hydroxyquinoline-2-carboxylic acid.
6. The pharmaceutical composition of claim 4, wherein the quinolinone compound and the quinolinic compound are provided in the form of a monomer or a herbal extract containing the quinolinone compound and the quinolinic compound.
7. The pharmaceutical composition of claim 6, wherein the Chinese medicinal extract is an alcohol extract of intestinal bacteria metabolites of Periplaneta americana.
8. Use of the periplaneta americana intestinal bacteria metabolite extract of any one of claims 1 to 3 or the pharmaceutical composition of any one of claims 4 to 7 in the preparation of drugs for treating cardiovascular and cerebrovascular diseases.
9. Use according to claim 8, characterized in that said cardiovascular and cerebrovascular diseases are diseases associated with vasodilation rates and/or vascular endothelial damages.
10. The use of claim 9, wherein the cardiovascular and cerebrovascular diseases comprise at least one of atherosclerosis, coronary heart disease, myocardial infarction, hypertension, hypertensive heart disease, diabetic vascular complications, restenosis following angioplasty, stroke, pulmonary hypertension, pulmonary heart disease, vasculitis, vascular headache, microangiopathy.
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