CN111053803B - Composition with anti-staphylococcus aureus effect and preparation method and application thereof - Google Patents
Composition with anti-staphylococcus aureus effect and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a composition with an anti-staphylococcus aureus effect, which consists of lightyellow sophora root total flavonoids and liquorice total flavonoids. The total flavone part of the sophora flavescens and the liquorice is prepared by methods of extraction, enrichment and the like, and is compounded by the two. In vitro studies show that the composition not only directly inhibits the growth of gram-positive bacteria such as staphylococcus aureus, but also can reduce biofilm formation and the content of staphylococcal flavin; in vivo research proves that the mouse mammary gland acinus hyperplasia and acinus inflammatory cell infiltration caused by staphylococcus aureus infection can be obviously reduced, and the bacterial load of mammary tissue and the contents of IL-1 beta, IL-2 and TNF-alpha inflammatory factors of the mammary tissue of each model group of mice are obviously reduced. The method can realize comprehensive utilization of the sophora flavescens and liquorice medicinal materials and quality and efficiency improvement of the industry, changes waste into valuable, has good economic effect and social benefit, and accords with the strategy of sustainable development.
Description
Technical Field
The invention relates to a traditional Chinese medicine composition, in particular to a composition of active ingredients of radix sophorae flavescentis and liquorice for preventing and treating staphylococcus aureus infection, especially mastitis, and a preparation method and application thereof.
Background
Kuh-seng, which is listed as the middle-grade product from Shen nong Ben Cao Jing, is the dried root of Sophora flavescens ait, a plant of Sophora of Leguminosae, has the effects of clearing heat and drying dampness, killing parasites, inducing diuresis and the like, and is used for treating dysentery with fever, hematochezia, jaundice anuria, eczema, skin pruritus, scabies leprosy and the like. Licorice, which was listed as the top grade from Shennong Ben Cao Jing, is the dry root and rhizome of Glycyrrhiza uralensis Fisch, Glycyrrhiza inflata Bat, or Glycyrrhiza glabra L, belonging to Glycyrrhiza of Leguminosae, has the effects of invigorating spleen, replenishing qi, clearing away heat and toxic materials, eliminating phlegm, relieving cough, relieving spasm, alleviating pain, harmonizing the effects of other drugs, and is commonly used for weakness of spleen and stomach, palpitation, shortness of breath, cough with excessive phlegm, spasm and pain of limbs, carbuncle, sore, drug toxicity, fulness and the like.
As a large amount of traditional Chinese medicinal materials with long application history, the research on the modern chemical components and pharmacological action of the radix sophorae flavescentis and the liquorice is relatively deep. The chemical components reported in sophora flavescens mainly comprise alkaloids, flavonoids, lignans, triterpenoid saponins, phenolic acids, a small amount of phenylpropanoids and long-chain fatty acid components. Alkaloids and flavonoids are considered as the main bioactive substances, and the matrine components are mainly quinolizidine alkaloids such as matrine, oxymatrine, and cytisine; the radix Sophorae Flavescentis flavonoid component mainly comprises isopentenyl substituted flavonoid component such as kurarinone, and sophoraflavanone, and the main substitution positions of the side chain of isopentenyl are 6 and/or 8 of A ring. The antibacterial and antitumor activities of the matrine have better application prospects clinically, and the currently marketed preparations taking the matrine as a main component comprise kuh-seng tablets, compound kuh-seng injection, compound kuh-seng lotion and the like. In the development process of sophora flavescens pharmaceutical preparations, acid water extraction and alkali water precipitation are usually combined to extract the total alkaloid part or matrine and oxymatrine monomer components in sophora flavescens, and the extracted part is largely discarded as medicine residues.
The currently reported chemical components in liquorice mainly comprise components such as triterpenoid saponins, flavonoids, coumarins, alkaloids, neolignanoids, polysaccharides and the like. Wherein the triterpenoid saponin components and flavonoid components are representative active components thereof, and the saponin components mainly comprise glycyrrhizin, glycyrrhizic acid, glycyrrhetinic acid, uralenin, glycyrrhizin, etc.; the basic mother nucleus of the licorice flavonoids compound is flavanone, the licorice also contains isopentenyl flavonoids, and the main substitution position of an isopentenyl side chain is a B ring. At present, the liquorice preparation is mainly focused on the development of saponin components of the liquorice preparation, such as diammine glycyrrhizate, magnesium isoglycyrrhizinate injection, compound glycyrrhizin injection and the like, the liquorice preparation is one of the first-line medicines for anti-inflammatory and liver-protecting treatment in the field of liver diseases at present, and the market share and the demand are huge. In the process of preparing the glycyrrhizic acid preparation, a large amount of medicine residues are also generated, and no matter the medicine residues of the radix sophorae flavescentis or the glycyrrhizic acid preparation are rich in abundant flavonoid components, the medicine residues serve as medicine residues to be buried, so that the land and water sources are polluted, huge pressure is brought to the ecological environment, and serious resource waste is caused. Meanwhile, the sophora flavescens and the liquorice are medicinal plants of roots of leguminous, are mainly distributed in northwest areas with fragile ecological environment in China, and long-term root digging and medicine taking tend to further aggravate local ecological deterioration, so that the resources are comprehensively utilized, and the purpose of eating, drying and squeezing the resources is realized to meet the green and sustainable development war.
Staphylococcus aureus (Staphylococcus aureus) is a gram-positive bacterium representative, a common food-borne pathogenic microorganism causing infections in humans and animals. With the wide use of antibacterial drugs, especially the abuse of antibiotics by animals with higher economic value such as cows, staphylococcus aureus generates drug-resistant bacteria, and the drug-resistant bacteria become a serious problem which cannot be ignored all over the world. And staphylococcus aureus is more difficult to remove and more harmful by producing biofilm and staphyloxanthin. Mastitis is inflammation of mammary gland cells and mammary gland mesenchyme, is a highly-diseased disease in dairy farms, and is easy to relapse after being cured by antibiotics. Pathogenic bacteria causing mastitis are streptococcus, streptococcus pyogenes and the like in about 85 percent, staphylococcus in 5 percent, tubercle bacillus, brucella and the like in other parts. In clinical practice, long-term low-dose antibiotic perfusion and soaking are mostly adopted for prevention and treatment, so that antibiotic residues in milk products are caused, and further, the accumulation of antibiotics in human beings is caused. According to the regulations of veterinary drug administration regulations and feed additive administration regulations, some of the drug feed additives for stopping production, import, operation and use are published and mainly comprise 10 additives such as oxytetracycline premix, oxytetracycline calcium premix, methylene salicylic acid bacitracin premix, nosiheptide premix, bacitracin zinc premix, enramycin premix, quinocetone premix, flavomycin premix (fermentation) and virginiamycin premix. Therefore, the search for new substitute antibiotic feed additive components from traditional Chinese medicines and natural medicines is a consensus and must way of industry. Through the development of the invention, scientific basis is provided for the discovery of resource value of the radix sophorae flavescentis and the liquorice and the comprehensive development and utilization of the radix sophorae flavescentis and the liquorice, meanwhile, reference is provided for the development and research of daily chemicals, Chinese patent medicines and Chinese veterinary medicines infected by the dairy cow mastitis and other staphylococcus aureus, and support is provided for the research of replacing antibiotics in the animal husbandry and breeding industry.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to provide a composition of kuh-seng total flavonoids and licorice total flavonoids which has the functions of preventing and treating staphylococcus aureus infection, particularly good prevention and treatment effect on mastitis, and in-vitro research shows that the composition not only directly inhibits the growth of gram-positive bacteria such as staphylococcus aureus, but also can reduce the formation of biofilm and the content of staphylococcal flavin; in vivo research proves that the active composition can obviously reduce mouse mammary acinar hyperplasia and acinar inflammatory cell infiltration caused by staphylococcus aureus infection, obviously reduce the bacteria-carrying quantity of mammary tissue of mice of each model group and the contents of IL-1 beta, IL-2 and TNF-alpha inflammatory factors of the mammary tissue, is expected to prevent and treat the occurrence and the development of cow mastitis, and can prevent and treat human and livestock diseases caused by other clinical staphylococcus aureus infection. The invention also aims to provide a preparation method and application of the composition of the sophora flavescens total flavonoids and the liquorice total flavonoids.
The technical scheme is as follows: in order to achieve the above purpose, the invention adopts the technical scheme that:
a composition with anti-Staphylococcus aureus effect comprises radix Sophorae Flavescentis total flavonoids and Glycyrrhrizae radix total flavonoids.
Preferably, the composition with the anti-staphylococcus aureus effect is prepared by mixing the lightyellow sophora root total flavonoids and the liquorice total flavonoids in a mass ratio of 1:4-4: 1. Particularly preferred mass ratios are 1:1, 2:1 or 1: 2.
The preparation method of the composition with the anti-staphylococcus aureus effect comprises the following steps:
(1) pulverizing radix Sophorae Flavescentis, adding 5-20 times of ethyl acetate or 95% ethanol, soaking, extracting for 3 times, filtering, and concentrating the filtrate to obtain radix Sophorae Flavescentis extract;
(2) pulverizing Glycyrrhrizae radix, adding 5-20 times of ethyl acetate or 95% ethanol, soaking, extracting for 3 times, filtering, and concentrating the filtrate to obtain Glycyrrhrizae radix extract;
(3) enriching the sophora flavescens ait extract in the step (1) by adopting a sodium carbonate solution extraction-dilute hydrochloric acid precipitation method, a macroporous adsorption resin column chromatography method or a polyamide column chromatography method to obtain the sophora flavescens ait total flavonoids;
(4) enriching licorice total flavone from the licorice extractum in the step (2) by adopting a sodium carbonate solution extraction-dilute hydrochloric acid precipitation method, a macroporous adsorption resin column chromatography method or a polyamide column chromatography method;
(5) and (3) compounding the sophora flavescens total flavonoids in the step (3) with the liquorice total flavonoids in the step (4) according to the ratio of 1:4-4:1, and uniformly mixing to obtain the sophora flavescens-liquorice active composition.
Preferably, in the above preparation method of the composition with anti-staphylococcus aureus effect, the raw materials in step (1) and step (2) are medicinal decoction pieces of radix sophorae flavescentis and liquorice or waste decoction dregs in the production process of radix sophorae flavescentis injection and glycyrrhizic acid preparation.
Preferably, the above-mentioned method for preparing a composition having an anti-staphylococcus aureus effect, the sodium carbonate solution extraction-dilute hydrochloric acid precipitation method in steps (3) and (4), comprises the following steps: taking the extract, adding a dilute sodium carbonate solution with the volume of 2-10 times of the weight of the extract, uniformly stirring, standing for 6-24h, filtering to obtain a filtrate, repeating the operations, combining the filtrates for 2 times, adding dilute hydrochloric acid into the filtrate to adjust the pH value to 4, fully stirring, standing for 6-24h, filtering the filter residue, washing with water until the pH value is 5-6, and drying to obtain the total flavone.
Preferably, the preparation method of the composition with anti-staphylococcus aureus effect, the macroporous adsorbent resin column chromatography or polyamide column chromatography in the step (3) and the step (4) is as follows: dissolving the extract with 2-10 times of anhydrous ethanol, subjecting the supernatant to macroporous adsorbent resin or polyamide column chromatography, eluting with water or 10% ethanol, eluting with 30-95% ethanol, and concentrating the eluate under reduced pressure to obtain total flavonoids.
Preferably, in the above preparation method of the composition having the anti-staphylococcus aureus effect, the extraction method in step (1) and step (2) is one or more of heating reflux, ultrasonic-assisted extraction, cold soaking method, percolation method and the like. As a particular preference, the extraction process is a percolation process.
As a particularly preferred embodiment, the preparation method of the above-mentioned composition with anti-staphylococcus aureus effect comprises the following steps: taking lightyellow sophora root, crushing, placing in an open glass column, adding 10 times of ethyl acetate, soaking overnight, collecting percolate at the flow rate of 3ml/min, sequentially adding 6 times of ethyl acetate and 3 times of ethyl acetate, soaking for 12h, sequentially collecting percolate at the flow rates of 5ml/min and 8ml/min, combining the percolate, and concentrating under reduced pressure to obtain lightyellow sophora root extract for later use.
As a particularly preferred embodiment, the preparation method of the composition with anti-staphylococcus aureus effect described above, the preparation method of the glycyrrhiza extract in the step (2) is: taking lightyellow sophora root, crushing, placing in an open glass column, adding 10 times of ethyl acetate, soaking overnight, collecting percolate at the flow rate of 3ml/min, sequentially adding 6 times of ethyl acetate and 3 times of ethyl acetate, soaking for 12h, sequentially collecting percolate at the flow rates of 5ml/min and 8ml/min, combining the percolate, and concentrating under reduced pressure to obtain lightyellow sophora root extract for later use.
Preferably, the preparation method of the composition with anti-staphylococcus aureus effect described above, the preparation method of the total flavone part in step (3) and step (4) is one or more of a sodium carbonate solution extraction-dilute hydrochloric acid precipitation method, a macroporous adsorption resin column chromatography method, and a polyamide column chromatography method.
Preferably, in the above method for preparing the composition with anti-staphylococcus aureus effect, the concentration of the dilute sodium carbonate solution in the step (3) and the step (4) is 1-5%, preferably 2%, and the concentration of the dilute hydrochloric acid is 15-20%, preferably 18%.
Preferably, in the preparation method of the composition with the anti-staphylococcus aureus effect, the model number of the macroporous adsorption resin in the step (3) and the step (4) is one or more of AB-8, ADS-5, ADS-7, ADS-17, D101, HPD-100, HP20, HPD-300, HPD-400 or S-8. As a particularly preferred scheme, the ADS-17 with hydrogen bond type selectivity is preferred, and has the physical adsorption principle of macroporous adsorption resin and the chemical adsorption principle of hydrogen bonds, and is particularly suitable for enriching and purifying flavonoid components.
Preferably, in the preparation method of the composition with the anti-staphylococcus aureus effect, the particle size of the polyamide in the step (3) and the step (4) is 20-100 meshes. As a particularly preferred embodiment, 60 to 80 mesh is preferred.
As a particularly preferred embodiment, the preparation method of the above-mentioned composition with anti-staphylococcus aureus effect comprises the following steps of: adding ethanol with the volume amount of 5 times of the weight of the sophora flavescens extractum in the step (1), stirring and dissolving, taking supernatant, mixing the supernatant with polyamide, drying the mixture by a rotary evaporator under reduced pressure until no alcohol smell exists, placing the sample after mixing in an ADS-17 macroporous adsorption resin column, removing the components which are not fully adsorbed and have large polarity by using deionized water and 35% ethanol, eluting by using ethanol with the volume concentration of 75%, concentrating the eluent under reduced pressure, and concentrating the filtrate to obtain the sophora flavescens total flavone.
As a particularly preferred embodiment, the preparation method of the composition with anti-staphylococcus aureus effect described above, the preparation method of the licorice total flavonoids in step (4) is: adding ethanol with volume amount of 5 times of the weight of the extractum glycyrrhizae radix of the step (2), stirring and dissolving, taking supernatant, mixing the supernatant with polyamide, drying under reduced pressure by a rotary evaporator until no alcohol smell exists, placing the sample after mixing into an ADS-17 macroporous adsorption resin column, removing the insufficiently adsorbed and large polar components by deionized water and 35% ethanol, eluting with 75% ethanol, concentrating the eluent under reduced pressure, and concentrating the filtrate to obtain the licorice total flavonoids.
Preferably, in the preparation method of the composition with the anti-staphylococcus aureus effect, in the step (5), the weight percentage of the total flavonoids in the sophora flavescens is 50%, the weight percentage of the total flavonoids in the liquorice is 50%, and the two components are in a ratio of 1: 1.
The composition with the effect of resisting staphylococcus aureus can be used for preparing daily necessities, Chinese patent medicines or traditional Chinese veterinary medicines for treating diseases such as mastitis.
Has the advantages that: the composition with the effect of resisting staphylococcus aureus provided by the invention is researched for new application on the basis of the traditional application of radix sophorae flavescentis and liquorice, and is applied to the development of daily chemical products, Chinese patent medicines and veterinary medicines for preventing and treating staphylococcus aureus infection, and compared with the prior art, the composition has the following advantages:
(1) the method mainly focuses on flavonoid components of the radix sophorae flavescentis and the liquorice, but not traditional alkaloid components and triterpenoid components, so that waste residues generated in the production process of preparations such as radix sophorae flavescentis injection and glycyrrhizic acid can be used as raw materials to prepare active compositions, the active compositions are changed into valuable, the industrial chain of the radix sophorae flavescentis and the liquorice can be extended, the comprehensive utilization of radix sophorae flavescentis and liquorice resources and the quality improvement and synergy of industries are realized, and the method has good economic effect, social benefit, ecological benefit and environmental benefit and conforms to the strategy of sustainable development.
(2) The sophora flavescens-liquorice active composition capable of preventing and treating staphylococcus aureus infection, particularly mastitis, provided by the invention is obtained by screening a large number of experiments according to basic theories of traditional Chinese medicines and modern pharmacological research results, is prepared by compounding sophora flavescens total flavonoids and liquorice total flavonoids, and has good antibacterial and anti-inflammatory activities: in vitro studies show that the composition not only directly inhibits the growth of gram-positive bacteria such as staphylococcus aureus, but also can reduce biofilm formation and the content of staphylococcal flavin; in vivo research proves that the active composition can obviously reduce mouse mammary acinar hyperplasia and acinar inflammatory cell infiltration caused by staphylococcus aureus infection, obviously reduce the bacteria-carrying quantity of mammary tissue of mice of each model group and the contents of IL-1 beta, IL-2 and TNF-alpha inflammatory factors of the mammary tissue, is expected to prevent and treat the occurrence and the development of cow mastitis, and can prevent and treat human and livestock diseases caused by other clinical staphylococcus aureus infection.
(3) The preparation method provided by the invention has the advantages of simple and convenient process route, less use of organic reagents, environmental friendliness, high automation degree and no toxic or side effect, can be used for preparing human and animal diseases caused by staphylococcus aureus infection, is particularly used for preparing daily chemical products, Chinese patent medicines and veterinary medicines for preventing and treating staphylococcus aureus infection, and has important application prospects.
Detailed Description
The present invention is further illustrated by the following examples, which are intended to be purely exemplary and are not intended to limit the scope of the invention, as various equivalent modifications of the invention will occur to those skilled in the art upon reading the present disclosure and fall within the scope of the appended claims.
The medicinal materials and the ethanol are used in a weight-volume ratio. Ethanol is referred to as volume concentration. Measuring the content of radix Sophorae Flavescentis total flavone and Glycyrrhrizae radix total flavone by ultraviolet spectrophotometry, measuring ultraviolet absorption value at 287nm with Sophora flavanone G as reference substance; the content determination of total flavonoids in Glycyrrhrizae radix adopts rutin as reference substance, and adds 5% sodium nitrite solution, and determines absorbance at 500 nm.
Example 1
A method for preparing a composition having an anti-Staphylococcus aureus effect, comprising the steps of:
(1) pulverizing radix Sophorae Flavescentis, adding 10 times of 95% ethanol, heating and refluxing for 3 times, mixing extractive solutions, and concentrating under reduced pressure to obtain radix Sophorae Flavescentis extract;
(2) pulverizing Glycyrrhrizae radix, adding 10 times of 95% ethanol, heating and refluxing for 3 times, mixing extractive solutions, and concentrating under reduced pressure to obtain Glycyrrhrizae radix extract;
(3) adding absolute ethanol with volume amount of 5 times of the weight of the sophora flavescens extractum in the step (1), stirring and dissolving, then transferring supernatant into a D-101 macroporous adsorption resin column, washing off water-soluble and unadsorbed components by using water, then eluting by using ethanol with volume concentration of 80%, and concentrating eluent under reduced pressure to obtain sophora flavescens total flavonoids;
(4) adding absolute ethanol with volume amount of 5 times of the weight of the extractum glycyrrhizae radix of the step (2), stirring for dissolving, transferring the supernatant into a D-101 macroporous adsorption resin column, washing with water to remove water-soluble and unadsorbed components, eluting with ethanol with volume concentration of 80%, and concentrating the eluate under reduced pressure to obtain total flavonoids;
(5) and (4) compounding the sophora flavescens total flavonoids in the step (3) with the liquorice total flavonoids in the step (4) according to the weight ratio of 1:4, and uniformly mixing to obtain the compound preparation.
By adopting an ultraviolet spectrophotometry, the content of the radix sophorae flavescentis total flavonoids in the step (3) is 67.14%, and the content of the liquorice total flavonoids in the step (4) is 64.48%.
Example 2
A method for preparing a composition having an anti-Staphylococcus aureus effect, comprising the steps of:
(1) taking radix sophorae flavescentis dregs, placing the radix sophorae flavescentis dregs in an open glass column, adding 10 times of ethyl acetate to soak the radix sophorae flavescentis dregs overnight, collecting percolate at the flow rate of 3ml/min, sequentially adding 6 times of ethyl acetate and 3 times of ethyl acetate to soak the radix sophorae flavescentis dregs for 12 hours, sequentially collecting percolate at the flow rates of 5ml/min and 8ml/min, combining the percolate, and performing reduced pressure concentration to obtain radix sophorae flavescentis extract for later use;
(2) placing the licorice dregs in an open glass column, adding 10 times of ethyl acetate, soaking overnight, collecting percolate at the flow rate of 3ml/min, sequentially adding 6 times of ethyl acetate and 3 times of ethyl acetate, soaking for 12h, sequentially collecting percolate at the flow rates of 5ml/min and 8ml/min, combining the percolate, and concentrating under reduced pressure to obtain licorice extract for later use;
(3) adding absolute ethanol with the volume of 5 times of the weight of the lightyellow sophora root extract in the step (1), stirring and dissolving, taking supernatant, transferring the supernatant into a polyamide chromatographic column with 60-80 meshes, washing off unadsorbed components by using 10% ethanol, then eluting by using ethanol with the volume concentration of 80%, concentrating the eluent under reduced pressure, and concentrating the filtrate to obtain lightyellow sophora root total flavonoids;
(4) adding absolute ethanol with volume amount of 5 times of the weight of the extractum glycyrrhizae radix of the step (2), stirring for dissolving, transferring the supernatant into a polyamide chromatographic column, washing off unadsorbed components with 10% ethanol, eluting with 80% ethanol, concentrating the eluate under reduced pressure, and concentrating the filtrate to obtain total flavonoids;
(5) and (4) compounding the sophora flavescens total flavonoids in the step (3) with the liquorice total flavonoids in the step (4) according to the weight ratio of 1:2, and uniformly mixing to obtain the compound traditional Chinese medicine.
By adopting an ultraviolet spectrophotometry, the content of the sophora flavescens total flavone in the step (3) is 68.58%, and the content of the liquorice total flavone in the step (4) is 67.90%.
Example 3
A method for preparing a composition having an anti-Staphylococcus aureus effect, comprising the steps of:
(1) taking a sophora flavescens medicinal material, crushing, placing in an open glass column, adding 10 times of ethyl acetate, soaking overnight, collecting percolate at the flow rate of 3ml/min, sequentially adding 6 times of ethyl acetate and 3 times of ethyl acetate, soaking for 12h, sequentially collecting percolate at the flow rates of 5ml/min and 8ml/min, combining the percolate, and concentrating under reduced pressure to obtain a sophora flavescens extract for later use;
(2) taking a licorice root medicinal material, crushing, placing the crushed material in an open glass column, adding 10 times of ethyl acetate, soaking overnight, collecting percolate at the flow rate of 3ml/min, sequentially adding 6 times of ethyl acetate and 3 times of ethyl acetate, soaking for 12 hours, sequentially collecting percolate at the flow rates of 5ml/min and 8ml/min, combining the percolate, and concentrating under reduced pressure to obtain a licorice root extract for later use;
(3) adding ethanol with the volume amount of 5 times of the weight of the sophora flavescens extractum in the step (1), stirring and dissolving, taking supernatant, mixing the supernatant with polyamide, drying the mixture by a rotary evaporator under reduced pressure until no alcohol smell exists, placing the sample after mixing in an ADS-17 macroporous adsorption resin column, removing the components which are not fully adsorbed and have large polarity by using deionized water and 35% ethanol, eluting the sample by using ethanol with the volume concentration of 75%, concentrating the eluent under reduced pressure, and concentrating the filtrate to obtain the sophora flavescens total flavone;
(4) adding ethanol with volume amount of 5 times of the weight of the extractum glycyrrhizae radix of the step (2), stirring and dissolving, taking supernatant, mixing the supernatant with polyamide, drying the mixture by a rotary evaporator under reduced pressure until no alcohol smell exists, placing the sample after mixing the sample on an ADS-17 macroporous adsorption resin column, removing the components which are not fully adsorbed and have large polarity by using deionized water and 35% ethanol, eluting the sample by using ethanol with volume concentration of 75%, concentrating the eluent under reduced pressure, and concentrating the filtrate to obtain the total flavonoids of glycyrrhizae radix;
(5) and (4) compounding the sophora flavescens total flavonoids in the step (3) with the liquorice total flavonoids in the step (4) according to the weight ratio of 1:1, and uniformly mixing to obtain the compound preparation.
By adopting an ultraviolet spectrophotometry, the content of the radix sophorae flavescentis total flavonoids in the step (3) is 72.98%, and the content of the liquorice total flavonoids in the step (4) is 71.17%.
Example 4
A method for preparing a composition having an anti-Staphylococcus aureus effect, comprising the steps of:
(1) taking a sophora flavescens medicinal material, crushing, placing in an open glass column, adding 20 times of ethyl acetate, soaking overnight, collecting percolate at the flow rate of 5ml/min, sequentially adding 15 times of ethyl acetate and 10 times of ethyl acetate, soaking for 12h, sequentially collecting percolate at the flow rates of 10ml/min and 15ml/min, combining the percolate, and concentrating under reduced pressure to obtain a sophora flavescens extract for later use;
(2) taking a licorice root medicinal material, crushing, placing the crushed material in an open glass column, adding 20 times of ethyl acetate, soaking overnight, collecting percolate at the flow rate of 5ml/min, sequentially adding 15 times of ethyl acetate and 10 times of ethyl acetate, soaking for 12 hours, sequentially collecting percolate at the flow rates of 10ml/min and 15ml/min, combining the percolate, and carrying out reduced pressure concentration to obtain a licorice root extract for later use;
(3) adding ethanol with the volume of 8 times of the weight of the sophora flavescens extractum in the step (1), stirring and dissolving, taking supernatant, adding the supernatant into an AB-8 macroporous adsorption resin column, washing off unadsorbed components by using ethanol with the volume concentration of 10%, then eluting by using ethanol with the volume concentration of 80%, and concentrating the eluent under reduced pressure to obtain sophora flavescens total flavonoids;
(4) adding ethanol with volume amount of 8 times of the weight of the extractum glycyrrhizae radix of the step (2), stirring and dissolving, taking supernatant, adding the supernatant into an AB-8 macroporous adsorption resin column, washing off unadsorbed components by using ethanol with volume concentration of 10%, then eluting by using ethanol with volume concentration of 80%, and concentrating the eluent under reduced pressure to obtain total flavonoids;
(5) and (4) compounding the sophora flavescens total flavonoids in the step (3) with the liquorice total flavonoids in the step (4) according to the weight ratio of 2:1, and uniformly mixing to obtain the compound preparation.
By adopting an ultraviolet spectrophotometry, the content of the sophora flavescens total flavone in the step (3) is 65.26%, and the content of the liquorice total flavone in the step (4) is 68.25%.
Example 5
A method for preparing a composition having an anti-Staphylococcus aureus effect, comprising the steps of:
(1) taking radix Sophorae Flavescentis residue, placing in an extraction tank, adding 10 times, 8 times and 6 times of 95% ethanol, respectively, heating and reflux extracting for 3 times, filtering, and concentrating the filtrate to obtain radix Sophorae Flavescentis extract;
(2) placing the residues of radix Glycyrrhizae in an extraction tank, adding 10 times, 8 times and 6 times of 95% ethanol, respectively, heating and reflux-extracting for 3 times, filtering, and concentrating the filtrate to obtain radix Glycyrrhizae extract;
(3) adding a 2% sodium carbonate solution 3 times the weight of the medicinal materials into the sophora flavescens extractum in the step (1), uniformly stirring, standing for 12 hours, filtering the filtrate, continuously adding the 2% sodium carbonate solution into the filter residue, uniformly stirring, standing for 12 hours, filtering the filtrate, and combining the filtrates for 2 times; adjusting pH to 4 with 18% diluted hydrochloric acid, stirring, standing for 12 hr, filtering, washing with water to pH 5-6, and drying to obtain radix Sophorae Flavescentis total flavonoids;
(4) adding a 2% sodium carbonate solution 3 times the weight of the medicinal materials into the licorice extract obtained in the step (2), uniformly stirring, standing for 12 hours, filtering the filtrate, continuously adding the 2% sodium carbonate solution into the filter residue, uniformly stirring, standing for 12 hours, filtering the filtrate, and combining the filtrates for 2 times. Adjusting pH to 4 with 18% diluted hydrochloric acid, stirring, standing for 12 hr, filtering, washing with water to pH 5-6, and drying to obtain Glycyrrhrizae radix total flavone;
(5) and (3) compounding the sophora flavescens total flavonoids in the step (3) with the liquorice total flavonoids in the step (4) according to the weight ratio of 4:1, and uniformly mixing to obtain the sophora flavescens-liquorice composition.
By adopting an ultraviolet spectrophotometry, the content of the radix sophorae flavescentis total flavonoids in the step (3) is 67.42%, and the content of the liquorice total flavonoids in the step (4) is 65.21%.
EXAMPLE 6 Staphylococcus aureus growth inhibition assay
1. Medicine and reagent
LB liquid medium, LB solid medium, MH broth medium, TSB medium were purchased from Shanghai-derived leaf Biotech limited; chromatographic methanol (chromatographically pure), absolute ethanol (analytically pure), crystal violet (analytically pure) were purchased from the national pharmaceutical group chemical reagents ltd; shake flask, plate, Corning Total-white enzyme-labeled 96-well microplate purchased from Shanghai chemical laboratory instruments Ltd; ultrapure water was supplied from the laboratory Millipore laboratory ultrapure water system.
2. Experimental strains
Staphylococcus aureus (ATCC 25923) from Shanghai pharmaceutical industry research institute, Boss.
3. Experimental methods
3.1 recovery of strains and preparation of bacterial liquid
And (2) inoculating staphylococcus aureus to an LB plate, performing activation culture at 37 ℃ for 18-24h, and taking a single colony to perform subculture at 37 ℃ for 18-24h in an LB liquid culture medium to a logarithmic phase. The above-mentioned bacterial solution was adjusted to turbidity of 0.5 McLeod (about 1.5X 10) using sterile MH broth medium9CFU/mL), ready for use.
3.2 determination of MIC values
Staphylococcus aureus in logarithmic growth phase was diluted to a concentration of about 1.5X 10 by taking a broth dilution method using American society for clinical standards (CLSI) M07-A96And (3) determining the MIC value (namely the lowest haze-free drug concentration) of the samples in the examples to staphylococcus aureus by adopting a 96-well microplate method, wherein the positive control drug is penicillin sodium freeze-dried powder injection for injection, and the negative control group is a blank culture medium. Adopting a 4-time continuous dilution method for each sample to ensure that the final concentration of the composition is 0.39-100 mu g/ml; the penicillin sodium is obtained by 10-fold continuous dilution method, and the final concentration is 10-3About 10. mu.g/ml. Culturing in an incubator at 37 ℃ for 18-24h, detecting the OD value of each well at 600nm, repeating for 3 times, taking the mean value, and calculating the bacteriostatic rate and the minimum bacteriostatic concentration MIC value of the samples of each example.
The bacteriostasis rate is (blank control hole OD value-test hole OD value)/blank control hole OD value is multiplied by 100 percent
4. Results of the experiment
As can be seen from table 1: the concentration of 25 mu g/ml and above of each example can obviously inhibit the growth of staphylococcus aureus, the MIC value is less than 25 mu g/ml, particularly, the inhibiting effect of the example 3 is most obvious, and the inhibiting effect is only 1 order of magnitude smaller than that of penicillin.
TABLE 1 growth inhibition of Staphylococcus aureus and MIC values (n ═ 9)
The initial dose of penicillin was 10. mu.g/ml and was a 10-fold serial dilution
Example 7 Staphylococcus aureus biofilm inhibition assay
1. Drugs and reagents
The same as in example 6.
2. Experimental strains
The same as in example 6.
3. Experimental methods
Taking staphylococcus aureus liquid in logarithmic growth phase, diluting to 1.5 × 106CFU/mL, inoculated into 96-well plate, placed in 37 ℃ incubator for 36h to make the capsule fully grow. Then the culture medium is discarded, 200 mu l of PBS solution is sucked into each hole, the washing is carried out for three times, then 200 mu l of methanol is added into each hole, the methanol is discarded after the fixation for 15min and the air is dried for 10min, and 200 mu l of 0.5 percent ethanol solution of crystal violet is added into each hole. Shaking for 10min, discarding crystal violet solution, adding 200 μ l purified water into each well, washing for three times, air drying, and adding 33% glacial acetic acid solution into each well. Dissolving with shaking at 37 deg.C for 15min, measuring OD at 570nm, repeating for 3 times, taking average value, and calculating biofilm formation inhibition rate.
4. Results of the experiment
As can be seen from Table 2: in each of examples 3. mu.g/ml and above, the formation of a biofilm of Staphylococcus aureus was inhibited, thereby reducing the risk of infection with Staphylococcus aureus, and the inhibition effect of example 3 on biofilm formation was particularly excellent.
TABLE 2 Staphylococcus aureus biofilm inhibition (n ═ 9)
EXAMPLE 8 Staphylococcus aureus flavin production inhibition experiment
1. Drugs and reagents
The same as in example 6.
2. Experimental strains
The same as in example 6.
3. Experimental methods
Taking the logarithmic phase concentration as 1.5X 107CFU/mL of Staphylococcus aureus was added to the TSB medium, and each test sample solution was added at 1/16-1 MIC. After co-culturing for 16h at 37 ℃ with shaking, 10ml of culture medium was centrifuged at 16600g for 5min, the supernatant was discarded, and the precipitate was washed with 10ml of PBS solution and three times in total. Adding 2.5ml methanol into the precipitate, heating in water bath at 55 deg.C for 3min, centrifuging at 16600g for 10min, collecting supernatant, measuring OD at 465nm, repeating for 3 times, taking average value, and calculating flavin generation inhibition rate.
4. Results of the experiment
As can be seen from Table 3: the samples of each example can inhibit the generation of the staphylococcus aureus flavin so as to enhance the immune clearance sensitivity of the host when the concentration is 25 mug/ml or more, and particularly, the sample of example 3 has the best inhibition effect on the generation of the staphylococcus aureus flavin.
TABLE 3 inhibition of Staphylococcus aureus flavin formation (n ═ 9)
Example 9 mouse mastitis assay
1. Drugs and reagents
Mouse tumor necrosis factor (TNF-alpha) ELISA kit, mouse interleukin-2 (IL-2) ELISA kit, mouse interleukin-1 beta (IL-1 beta) ELISA kit purchased from Nanjing gold Yibai Biotech limited; the hematoxylin-eosin staining solution kit is purchased from Jiangsu Kai-based biotechnology, Inc.; sodium carboxymethylcellulose (CMC-Na, purity greater than or equal to 99%) and veterinary penicillin (GMP certificate 010) are available from carbofuran biotechnology Limited.
2. Laboratory animal
BALB/C pregnant mouse, female, purchased from Qinglongshan animal breeding farm in Jiangning district of Nanjing, SPF grade, and the number of the qualification certificate of the experimental animal: no.201905178, animal production license number: SCXK (su) 2017-; a barrier feeding environment system, wherein the temperature is 20-25 ℃, the relative humidity is 40-70%, the ventilation frequency is 10-20 times/h, and the illumination is performed alternately for 12h/12h every day; during the experiment, the ground, the table top and the wall surface are disinfected every day, and the disinfectant is new and clean, lysol and ethanol are alternately used.
3. Experimental methods
The pregnant mouse is suitable for feeding for one week and is delivered, and the pregnant mouse is randomly divided into a blank group, a model group and each administration group after 6-8 days of delivery. Centrifuging 1000g of activated Staphylococcus aureus liquid for 10min, discarding supernatant, and suspending precipitate with normal saline to make Staphylococcus aureus concentration 2.0 × l04CFU/ml. Model group and each administration group were injected with the aforementioned Staphylococcus aureus suspension at the 5 th breast of 50. mu.L each side, and the blank group was administered with the same volume of physiological saline at the same position. Dreaming for 24h, the administration groups were gavaged with the total flavone composition of Sophora flavescens and Glycyrrhiza uralensis (100mg/Kg body weight) prepared in examples 1-5 of the present invention, and the blank control group and the model group were gavaged with the same volume of CMC-Na solution and administered for 7 days continuously. After 7 days of administration, the mice were sacrificed by removing the neck, and the breast tissue between the fourth pair of papillae and the 5 th pair of papillae was taken by rapidly cutting the skin of the chest.
A portion of the breast tissue was taken for histopathological evaluation. And adding sterile normal saline (1ml/g) into another part of the mammary tissue, and grinding on ice to prepare a mammary tissue homogenate stock solution for later use. Preparing a high-salt mannitol agar powder plate, and taking the mammary tissue stock solution, and diluting the mammary tissue stock solution to a proper concentration by using sterile normal saline. Uniformly coating 100 mu l of the bacterial liquid on a flat plate, culturing in an incubator at 37 ℃ for 16-18h, selecting the flat plate with the CFU of 30-300 for counting, converting the bacterial load of mammary tissues of each mouse before dilution, and measuring the content of IL-1 beta, IL-2 and TNF-alpha in the residual mammary homogenate stock solution.
4. Results of the experiment
4.1 histopathological evaluation
Histopathological morphological observations show that: the mouse model group has obvious mammary gland acinus lesion, obvious acinus hyperplasia, a large amount of inflammatory cell fragments in acinus cavities, various inflammatory cell types and mainly neutrophils. Each example group was effective in reducing inflammatory cell infiltration with only a slight denudation of mammary acinar epithelial cells.
4.2 mammary gland tissue carrying capacity
As can be seen from Table 4: a large amount of staphylococcus aureus proliferates in mammary tissue of a model group mouse, the bacteria content of the mammary tissue can be effectively reduced by each embodiment group, the in-vivo antibacterial activity is obvious, and the inhibition effect of the embodiment 3 is optimal.
TABLE 4 mammary tissue loading and inflammatory factor content (n ═ 10)
4.3 Biochemical index of mammary tissue
As can be seen from Table 4: compared with a blank group, the contents of IL-1 beta, IL-2 and TNF-alpha inflammatory factors in mammary tissues of a model group mouse are obviously increased, and each embodiment group can effectively reduce the content of the inflammatory factors in the mammary tissues and shows obvious anti-inflammatory activity, particularly the most obvious effect of embodiment 3.
The experiment results show that the composition of the radix sophorae flavescentis and the liquorice provided by the invention has good antibacterial and anti-inflammatory activities: in vitro studies show that the composition not only directly inhibits the growth of gram-positive bacteria such as staphylococcus aureus, but also can reduce biofilm formation and the content of staphylococcal flavin; in vivo research proves that the active composition can obviously reduce mouse mammary acinar hyperplasia and acinar inflammatory cell infiltration caused by staphylococcus aureus infection, obviously reduce the bacteria-carrying quantity of mammary tissue of mice of each model group and the contents of IL-1 beta, IL-2 and TNF-alpha inflammatory factors of the mammary tissue, is expected to prevent and treat the occurrence and the development of cow mastitis, and can prevent and treat human and livestock diseases caused by other clinical staphylococcus aureus infection.
The preparation method provided by the invention has the advantages of simple and convenient process route, less use of organic reagents, environmental friendliness and high automation degree; the purity of the total flavone part obtained by the process is more than 64.48%, the purity of the active ingredient is high, and the total flavone part has no toxic or side effect, can be used for preparing daily chemical products, Chinese patent medicines and Chinese veterinary medicines for preventing and treating staphylococcus aureus infection, and has important application prospects.
The comparison of the activities of the sophora flavescens total flavonoids and the liquorice total flavonoids in different weight ratios for preventing and treating mastitis shows that the in-vitro antibacterial activity is optimal and the in-vivo mastitis preventing and treating activity is strongest, namely, the sophora flavescens and liquorice total flavonoids composition compounded by the sophora flavescens total flavonoids and the liquorice total flavonoids in a ratio of 1:1 has obvious inhibiting and reducing effects on the growth of staphylococcus aureus, the formation of biofilm and the content of staphylococcal flavin, and has optimal improvement effects on the hyperplasia of mammary glands, the infiltration of acinar inflammatory cells, the bacterial carrying capacity of mammary tissues and the content of inflammatory factors of mammary tissues IL-1 beta, IL-2 and TNF-alpha of a mammitis mouse, so that a good and unexpected technical effect is obtained.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (1)
1. The application of a composition with the effect of resisting staphylococcus aureus in preparing a medicament for preventing and treating mastitis comprises lightyellow sophora root total flavonoids and liquorice total flavonoids in a weight ratio of 1: 1;
the sophora flavescens total flavonoids and the liquorice total flavonoids are prepared by the following method:
(1) taking a sophora flavescens medicinal material, crushing, placing in an open glass column, adding 10 times of ethyl acetate, soaking overnight, collecting percolate at the flow rate of 3ml/min, sequentially adding 6 times of ethyl acetate and 3 times of ethyl acetate, soaking for 12h, sequentially collecting percolate at the flow rates of 5ml/min and 8ml/min, combining the percolates, and concentrating under reduced pressure to obtain a sophora flavescens extract for later use;
(2) crushing a licorice medicinal material, placing the crushed licorice medicinal material into an open glass column, adding 10 times of ethyl acetate, soaking the crushed licorice medicinal material overnight, collecting percolate at the flow rate of 3ml/min, sequentially adding 6 times of ethyl acetate and 3 times of ethyl acetate, soaking the soaked licorice medicinal material for 12 hours, sequentially collecting percolate at the flow rates of 5ml/min and 8ml/min, combining the percolate, and concentrating under reduced pressure to obtain a licorice extract for later use;
(3) adding ethanol with the volume amount of 5 times of the weight of the sophora flavescens extractum in the step (1), stirring and dissolving, taking supernatant, mixing the supernatant with polyamide, drying the mixture by a rotary evaporator under reduced pressure until no alcohol smell exists, placing the sample after mixing in an ADS-17 macroporous adsorption resin column, removing the components which are not fully adsorbed and have large polarity by using deionized water and 35% ethanol, eluting the sample by using ethanol with the volume concentration of 75%, concentrating the eluent under reduced pressure, and concentrating the filtrate to obtain the sophora flavescens total flavone;
(4) adding ethanol with volume amount of 5 times of the weight of the extractum glycyrrhizae radix of the step (2), stirring and dissolving, taking supernatant, mixing the supernatant with polyamide, drying under reduced pressure by a rotary evaporator until no alcohol smell exists, placing the sample after mixing into an ADS-17 macroporous adsorption resin column, removing the insufficiently adsorbed and large polar components by deionized water and 35% ethanol, eluting with 75% ethanol, concentrating the eluent under reduced pressure, and concentrating the filtrate to obtain the licorice total flavonoids.
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