CN108888685B - Application of Tetrastigma hemsleyanum Diels et Gilg extract TH-w part to staphylococcus aureus and action method thereof - Google Patents
Application of Tetrastigma hemsleyanum Diels et Gilg extract TH-w part to staphylococcus aureus and action method thereof Download PDFInfo
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Abstract
The invention discloses application of a radix tetrastigme extract TH-w part to staphylococcus aureus and an action method thereof, wherein the optimum part is found out as a TH-w part by respectively testing and comparing a TH-t part, a TH-p part, a TH-a part, a TH-b part, a TH-w1 part, a TH-w2 part, a TH-w3 part and a TH-w4 part; the invention cultures staphylococcus aureus at TH-w parts of the radix tetrastigme extract by using different culture medium dilution concentrations in a multiple ratio, and detects and contrasts the biofilm formation capability of bacteria by a crystal violet staining method to find out the optimal concentration.
Description
Technical Field
The invention relates to the field of bacteriostasis, in particular to application of a Tetrastigma hemsleyanum Diels et Gilg extract TH-w part to staphylococcus aureus and an action method thereof.
Background
Bacterial biofilm (bifilm) refers to an organized population of bacteria, attached to the surface of living or inanimate objects, encapsulated by extracellular macromolecules of bacteria, and is another common mode of survival distinct from free bacteria. Staphylococcus aureus (s. aureus) often secretes exopolysaccharides, DNA and proteins themselves to form a "mushroom-like" membrane polymer biofilm; after the staphylococcus aureus forms a biofilm, chronic persistent infection often occurs to a patient, the drug resistance of the staphylococcus aureus is 10-100 times stronger than that of planktonic bacteria, and great difficulty is brought to clinical anti-infection treatment. The main reason for the formation of the bacterial biofilm is antibiotic abuse, staphylococcus aureus which remains in human bodies for a long time is continuously stimulated by antibiotic with sub-bacteriostatic concentration, so that the bacterial gene mutation causes drug resistance, the traditional antibacterial drugs at present are difficult to effectively treat infection induced by the staphylococcus aureus biofilm, and the development of new antibiotics is difficult to keep pace with the bacterial drug resistance. Therefore, the development of drugs against microbial membranes has become a hot topic in the recent field of microbiology.
The radix tetrastigme is a dry root tuber of Tetrastigma hemsleyanum Diels et Gilg, also called golden silk hanging gourd and the like, is a special plant in China and is a commonly used medicine mainly distributed in folks in southeast China. The active ingredients of the radix tetrastigme mainly contain flavonoid chemical ingredients, are cool in nature, pungent and bitter in taste and non-toxic, have the effects of clearing heat and removing toxicity, promoting blood circulation to arrest pain, dispelling wind and reducing phlegm, promoting blood circulation to arrest pain and the like, and are commonly used for diseases such as febrile convulsion, infantile common cold and fever, itching and swelling and pain of throat, cough, pneumonia, hepatitis, enteritis, dysentery and the like. Modern pharmacological research shows that the radix tetrastigme extract and the antibiotic have obvious curative effect on treating bacterial infectious diseases. Therefore, the research and development of the effective components in the radix tetrastigme for resisting the bacterial microbial membrane have important clinical significance.
The invention observes the forming and dispersing effects of the radix tetrastigme extract on staphylococcus aureus biomembranes, discovers that the radix tetrastigme extract can effectively inhibit and disperse the generation of the staphylococcus aureus biomembranes, discovers that the radix tetrastigme extract has new medical application in combined antibacterial infection, and can provide experimental reference for the research and development of medicaments for treating some patients infected with drug-resistant bacteria. And then the TH-t part, the TH-p part, the TH-a part, the TH-b part, the TH-w1 part, the TH-w2 part, the TH-w3 part and the TH-w4 part are respectively tested, and the part with the best antibacterial effect is found.
Disclosure of Invention
In order to solve the defects of the prior art, the invention aims to provide the application of the Hevea hemsleyana extract TH-w part to staphylococcus aureus and an action method thereof, wherein the TH-t part, the TH-p part, the TH-a part, the TH-b part, the TH-w1 part, the TH-w2 part, the TH-w3 part and the TH-w4 part are respectively tested and compared, and the optimal part which is the TH-w part and the optimal action method are found out.
In order to achieve the above object, the present invention adopts the following technical solutions:
application of TH-w part of radix tetrastigme extract to Staphylococcus aureus, and TH-w part of radix tetrastigme extract can inhibit the growth of Staphylococcus aureus.
The sensitivity of the hemsley rockvine root extract TH-w part to staphylococcus aureus is as follows: the methicillin-sensitive staphylococcus aureus is larger than the standard staphylococcus aureus and larger than the methicillin-resistant staphylococcus aureus.
Application of TH-w part of radix tetrastigme extract on Staphylococcus aureus, and formation of biofilm of Staphylococcus aureus can be inhibited by TH-w part of radix tetrastigme extract.
An action method of a radix tetrastigme extract TH-w part on staphylococcus aureus is to dilute the radix tetrastigme extract TH-w part to 32.0mg/mL by LB broth multiple ratio to culture the staphylococcus aureus.
According to the acting method of the hemsley rockvine root extract TH-w part on staphylococcus aureus, after the hemsley rockvine root extract TH-w part is diluted to 32.0mg/mL by M-H broth multiple ratio and cultured for 16-20H at 35 ℃, the bacterial inhibition rate is 0.88.
According to the acting method of the hemsley rockvine root extract TH-w part on staphylococcus aureus, after the hemsley rockvine root extract TH-w part is diluted by M-H broth in a multiple ratio of 32.0mg/mL and cultured for 16-20H at 35 ℃, the bacterial inhibition rate is 0.34.
According to the acting method of the hemsley rockvine root extract TH-w part on staphylococcus aureus, after the hemsley rockvine root extract TH-w part is diluted by M-H broth multiple ratio to 32.0mg/mL, the staphylococcus aureus standard strain is cultured for 16-20H at 35 ℃, and the bacterial inhibition rate is 0.7.
According to the acting method of the radix tetrastigme extract TH-w part on the staphylococcus aureus, 32.0mg/mL of LB broth is diluted by multiple times, and after the methicillin-sensitive staphylococcus aureus and methicillin-resistant staphylococcus aureus are cultured for 24 hours at 37 ℃, a crystal violet staining method is adopted to detect that the value of the absorbance A570mm of the biological membrane is 3.1.
The invention has the advantages that:
the method comprises the steps of respectively testing and comparing a TH-t part, a TH-p part, a TH-a part, a TH-b part, a TH-w1 part, a TH-w2 part, a TH-w3 part and a TH-w4 part, and finding out the optimal part as the TH-w part;
the invention cultures staphylococcus aureus at TH-w parts of the radix tetrastigme extract by using different culture medium dilution concentrations in a multiple ratio, and detects and contrasts the biofilm formation capability of bacteria by a crystal violet staining method to find out that the optimal concentration is 32 mg/mL.
Drawings
FIG. 1 is a graph comparing the ability of Tetrastigma hemsleyanum Diels et Gilg extract of the present invention to inhibit MSSA-1 biofilm formation;
FIG. 2 is a graph comparing the ability of Tetrastigma hemsleyanum Diels et Gilg extract of the present invention to inhibit MSSA-2 biofilm formation;
FIG. 3 is a graph comparing the ability of Tetrastigma hemsleyanum Diels et Gilg extract of the present invention to inhibit MSSA-3 biofilm formation;
FIG. 4 is a graph comparing the ability of Tetrastigma hemsleyanum Diels et Gilg extract of the present invention to inhibit MSSA-4 biofilm formation;
FIG. 5 is a graph comparing the ability of Tetrastigma hemsleyanum Diels et Gilg extract of the present invention to inhibit MSSA-5 biofilm formation;
FIG. 6 is a graph comparing the ability of Tetrastigma hemsleyanum Diels et Gilg extract of the present invention to inhibit MRSA-1 biofilm formation;
FIG. 7 is a graph comparing the ability of Tetrastigma hemsleyanum Diels et Gilg extract of the present invention to inhibit MRSA-2 biofilm formation;
FIG. 8 is a graph comparing the ability of Tetrastigma hemsleyanum Diels et Gilg extract of the present invention to inhibit MRSA-3 biofilm formation;
FIG. 9 is a graph comparing the ability of Tetrastigma hemsleyanum Diels et Gilg extract of the present invention to inhibit MRSA-4 biofilm formation;
FIG. 10 is a graph comparing the ability of Tetrastigma hemsleyanum Diels et Gilg extract of the present invention to inhibit MRSA-5 biofilm formation;
FIG. 11 is a graph showing a comparison of absorbance (A570mm) measured by a crystal violet staining method after culturing the respective parts of the Tetrastigma hemsleyanum Diels et Gilg extract of the present invention in a culture medium of different concentrations;
FIG. 12 is a schematic view of the extraction method of the extract of Hemsleya amabilis of the present invention.
Detailed Description
The invention is described in detail below with reference to the figures and the embodiments.
Extracting and separating radix tetrastigme medicinal material
The radix tetrastigme extract is prepared by dividing an extracting solution into the following components according to a preparation selected during extraction: TH-t part, TH-p part, TH-a part, TH-b part, TH-w1 part, TH-w2 part, TH-w3 part, TH-w4 part.
The material comprises: the radix tetrastigme dry root tuber is purchased from state pharmaceutical and medicinal materials company of Ningbo city; 80% macroporous adsorbent resin tree chromatograph; yanaco micro melting point apparatus (thermometer uncorrected); a mass spectrometer of the Varian MAT-212 type; bruker-speckospin model AC-600P NMR spectrometer. The thin-layer chromatography silica gel plate and the column chromatography silica gel (100-200 meshes and 200-300 meshes) are both produced by Shandong Yangttaijiang friend silica gel development company; sephadex LH-20 (20-80 μm) is produced by Pharmacia; ODS C18 reverse phase silica gel, manufactured by Merck corporation; extracting with ethanol; petroleum ether, ethyl acetate and n-butanol are medicinal grade, water is distilled water, and other reagents are analytically pure.
As shown in fig. 12, the extraction method includes the steps of:
extracting radix Apioris Fortunei extract with reference to literature [ bright, etc.. Cluster analysis and main component analysis method to study chloroform part HPLC fingerprint [ J ] of radix Apioris Fortunei, Chinese patent medicine, 2016,38(3): 607-.
The extraction method of the TH-t part, the TH-p part, the TH-a part, the TH-b part and the TH-w part comprises the following steps: soaking 500g of radix tetrastigme dry medicinal material in 5 times of 80% ethanol water solution for 24h, heating and reflux-extracting for 3 times (40 min each time), mixing extractive solutions, filtering, recovering under reduced pressure, and evaporating to dryness to obtain total extract TH-t part with yield of 11.3%; mixing the total extracts with water, suspending, and respectively extracting with 10 times of petroleum ether (water saturated) step by step to obtain petroleum ether part TH-p with yield of 0.08%; ethyl acetate position TH-a, yield 0.31%; butanol position TH-b, yield 1.1%; and the water position TH-w, the yield is 8.76%.
The extraction method of the TH-w1 part comprises the following steps: carrying out macroporous adsorption resin chromatography on the water part TH-w, eluting with pure water to remove sugar, and sequentially eluting with 10%, 30%, 60% and 95% ethanol water solutions; wherein, 10% ethanol water eluent is decompressed, recovered and evaporated to dryness to obtain a water-position desugared fraction TH-w1, and the yield is 0.1%;
the extraction method of the TH-w2 part comprises the following steps: carrying out macroporous adsorption resin chromatography on the water part TH-w, eluting with pure water to remove sugar, and sequentially eluting with 10%, 30%, 60% and 95% ethanol water solutions; wherein 30% ethanol water elution solution is evaporated to dryness to obtain a water-position desugarized fraction TH-w 1g, and the yield is 0.17%;
the extraction method of the TH-w3 part comprises the following steps: decocting the medicinal material of radix tetrastigme extracted by 80% ethanol (TH-t) with water, recovering under reduced pressure, and evaporating to dryness to obtain 5.4 g of dehydrate;
the extraction method of the TH-w4 part comprises the following steps: decocting the radix Apioris Fortunei material after 80% ethanol extraction (TH-t) with water, recovering under reduced pressure, evaporating to dryness, and sequentially desugaring with 10%, 30%, 60%, and 95% ethanol water solution respectively to obtain desugarized fraction of 10% ethanol water fraction.
The results are shown in the following table:
TABLE 1 results of extraction of Tetrastigma hemsleyanum Diels et Gilg by different extraction methods
Test 1: verifying that the part of each radix tetrastigme extract with the strongest inhibitory capacity on the staphylococcus aureus biomembrane is a TH-w stroke section;
materials and methods
The strain is as follows: 5 methicillin-sensitive Staphylococcus aureus (MSSA) strains and 5 methicillin-resistant Staphylococcus aureus (MRSA) strains remained in the conventional detection of the microbial chamber.
The apparatus and reagents include: VITEK MS full-automatic microbial mass spectrometry detection system (Merria, France); 96-well cell culture plates (GIBCO, usa); a multifunctional microplate detection enzyme linked immunosorbent assay (BioTek, USA); M-H broth and LB broth (Hangzhou Binghe company);
the experimental method comprises the following steps:
establishing a staphylococcus aureus biofilm model; and (3) selecting a single staphylococcus aureus colony in 4mL of LB culture medium, transferring the single staphylococcus aureus colony to a 35 ℃ incubator, culturing the single staphylococcus aureus colony for 24 hours at a speed of 120r/min, and taking out the single staphylococcus aureus colony to adjust the concentration of the single staphylococcus aureus colony to 0.5 McLeod turbidity unit. After diluting the bacterial suspension with LB culture solution 1:100, 100 mul is added into a 96-well plate, 100 mul is added into the LB culture solution, 200 mul without adding bacterial solution is used as blank control, and after static culture at 35 ℃ for 24h, 48h and 72h, the biological membrane is respectively detected. All experiments were repeated 3 times, each time with 3 replicates.
The detection process of the staphylococcus aureus biomembrane forming capability comprises the following steps: the bacterial biofilm formation ability is detected by adopting a crystal violet staining method, a single colony which is freshly cultured on a Columbia blood agar plate overnight is selected to be cultured in 4ml of LB culture medium, and the culture is carried out for 18h at the temperature of 35 ℃ and the speed of 120 r/min. 10. mu.l of the culture broth was aspirated into 4mL of fresh LB medium for subculture. Repeat 3 times. The concentration of the bacterial liquid obtained after subculture was adjusted to 0.5 McLeod unit, 10. mu.l of the bacterial liquid was added to a 96-well cell culture plate containing 190. mu.l of LB medium, each strain was subjected to 3-fold wells, and 200. mu.l of LB medium without bacterial liquid was used as a blank. Standing and culturing for 6h at 35 ℃. After the biofilm was formed, the supernatant of the planktonic bacteria was aspirated, 200. mu.l of crystal violet solution was added for staining for 15min, the staining solution was discarded and washed with PBS 3 times to remove planktonic bacteria, 200. mu.l of 95% ethanol was added after drying to dissolve the crystal violet bound to the bacteria, and the value of absorbance at 570nm (A570nm) was measured with an enzyme-linked analyzer. The average absorbance value of the blank wells +3 times the standard deviation of the blank wells was taken as cut off value (Ac). If the A570nm of the experimental group is less than or equal to Ac, the film forming capability is negative; if A, A570nm > Ac, the film-forming ability was positive.
The biofilm formation inhibition test comprises the following steps: single colonies were picked and cultured in LB broth at 37 ℃ for 16h at 200r/min, centrifuged at 3000g for 20min and the concentration of the suspension was adjusted to 1 McLeod turbidity unit with physiological saline. Diluting TH-w segments of the radix tetrastigme extract to different concentrations (0.078-l 0mg/mL) by using LB broth multiple ratio, sucking 198 mul of the solution respectively, adding the solution into a 96-well plate, taking LB broth without the radix tetrastigme TH-w extract as a negative control, and adding 2 mul of the prepared bacterial suspension into each well respectively. After incubation at 37 ℃ for 24h, the cells were rinsed 3 times with physiological saline to remove planktonic bacteria. (1) Crystal violet dyeing: and (2) sucking residual liquid in the micropores by using a micropipette, adding 200 mu L (5g/L) of crystal violet solution into each hole, shaking for 15min on an oscillator, discarding dye solution, rinsing for 3 times by using normal saline to remove the unbound crystal violet solution, adding 200 mu L of 95% ethanol solution into each hole after air drying to elute the crystal violet bound with the biological membrane, and detecting the absorbance at 570nm by using a multifunctional enzyme-linked immunosorbent assay after standing for 20min (A570 mm). (2) Plate colony dilution counting is carried out on negative control wells and experimental wells added with different concentrations (0.078-l 0mg/mL) of the hemsley rockvine root TH-w extract: adding 200 mul of sterile normal saline into each micropore without planktonic bacteria, ultrasonically oscillating (frequency: 40kHz, power: 200W) for 15min to disperse the biological membrane adhered to the pore wall and the pore bottom into a planktonic state, diluting with multiple times of normal saline, uniformly coating 10 mul of diluted bacterial suspension on a sheep blood agar plate, culturing in an incubator overnight, and selecting a plate with 5-20 bacterial colonies to count the number of bacterial colonies. The experiment was repeated 3 times, each time with 3 replicate wells.
And (4) analyzing results:
and (3) analyzing the film forming capability of staphylococcus aureus: both the 5 S.aureus strains and the 5 standard strains were able to form biofilms. The value of the absorbance A570mm of the biological membrane reaches the peak value which is 1.63 plus or minus 0.47 when MSSA is cultured for 24 hours; the time for 48h and 72h is 1.18 +/-0.83 and 1.56 +/-0.44 respectively. The film forming time of the MRSA is long, and the A570mm value is 0.38 +/-0.15 at 24 hours; the absorbance A570mm at 48h is 1.27 +/-0.36, and the peak value is reached; the reduction is 1.11 +/-0.76 at 72 h.
As can be seen from the results of fig. 1-10: each radix tetrastigme extract can inhibit the formation of a staphylococcus aureus biomembrane, and TH-w sections of the radix tetrastigme extract have the most obvious inhibiting effect on the formation of the staphylococcus aureus biomembrane.
Adding radix tetrastigme extracts with different concentrations and different extraction sections simultaneously in the process of forming the biological membrane; biofilm formation of MSSA group and MRSA group after 24 TH-w section induction by radix tetrastigme extract is obviously lower than that of a control group, which indicates that TH-w inhibition is obvious, and statistical difference (P is less than 0.05) exists between the comparison groups; the A570 value of the strain with the same concentration and different strains acts for 24 hours is obviously reduced compared with a control group, and the groups with the same concentration have statistical difference (P < 0.05). The different radix tetrastigme extraction sections with the same drug concentration have obvious inhibiting effect difference on the biofilm formation of staphylococcus aureus, and the statistical difference (P <0.05) exists between groups. The tetrastigma hemsleyanum extract with the same drug concentration has obvious difference on the inhibition effect of different staphylococcus aureus biofilm formation, and the statistical difference (P <0.05) is relatively uniform among groups.
The results shown in FIGS. 1-10 may indicate that:
the concentrations (0.078-32mg/mL) of the Hevea triloba extract TH-t part, TH-p part, TH-a part, TH-b part, TH-w1 part, TH-w2 part, TH-w3 part and TH-w4 part have inhibition effect on 5 strains of methicillin-sensitive staphylococcus aureus and methicillin-resistant staphylococcus aureus biomembrane, and the inhibition effect on all strains is enhanced along with the increase of the drug concentration. There is a drug concentration dependence.
The most sensitive to TH-t parts, TH-p parts, TH-a parts, TH-b parts, TH-w1 parts, TH-w2 parts, TH-w3 parts and TH-w4 parts are methicillin-sensitive staphylococcus aureus, and the inhibition difference of two pairs of staphylococcus aureus with weaker effect on methicillin-resistant staphylococcus aureus reaches more than 30%.
The experiment shows that: after the Hevea triloba extract TH-t part, TH-p part, TH-a part, TH-b part, TH-w1 part, TH-w2 part, TH-w3 part and TH-w part are diluted to 32.0mg/mL by M-H broth multiple ratio, 5 strains of Staphylococcus aureus MSSA are cultured for 16-20H at 35 ℃, and the inhibition of bacterial membrane generation is most sensitive to TH-w part.
After the Hevea triloba extract TH-t part, TH-p part, TH-a part, TH-b part, TH-w1 part, TH-w2 part, TH-w3 part and TH-w part are diluted to 32.0mg/mL by M-H broth multiple ratio, 5 strains of Staphylococcus aureus MRSA are cultured for 16-20H at 35 ℃, and the inhibition of bacterial membrane generation is most sensitive to TH-w part.
From this, it is clear that different Hevea trifoliata extracts are sensitive to TH-w sites for inhibiting biofilm formation by Staphylococcus aureus MSSA and MRSA.
Test 2: performing a bacterial growth inhibition test on the radix tetrastigme extract, and finding out the optimal concentration of the TH-w part of the radix tetrastigme extract diluted by a culture medium in a multiple ratio;
materials and methods
The strain comprises: methicillin-sensitive Staphylococcus aureus (MSSA-29213) 1 strain, methicillin-resistant Staphylococcus aureus (MRSA-BAA 1029)1 strain and Staphylococcus aureus standard strain (ATCC 259231)1 strain.
The apparatus and reagents include: VITEK MS full-automatic microbial mass spectrometry detection system (Merria, France); 96-well cell culture plates (GIBCO, usa); a multifunctional microplate detection enzyme linked immunosorbent assay (BioTek, USA); M-H broth and LB broth (Hangzhou Binghe company);
the experimental method comprises the following steps:
the procedure of the inhibition of bacterial growth test was: in the experiments of inhibiting the growth of staphylococcus aureus and inhibiting bacterial biofilms by the radix tetrastigme extract, the part of each part of the radix tetrastigme extract with the strongest inhibitory capacity to the staphylococcus aureus biofilms is a TH-w stroke section. Therefore, the TH-w site was selected as the test drug in this study. Prepared Hemsley rockvine root extract TH-w part was diluted with M-H broth (32.0, 8.0, 2.0, 0.5, 0.125, 0.03, 0.0075mg/mL) and M.H broth without any drug was used as a blank. A microtiter plate was prepared by adding 100. mu.l to each well of a 96-well plate. Staphylococcus aureus MSSA-29213, MRSA-BAA1029 and ATCC259231 strain suspensions were adjusted to 0.5 McLeod turbidity units and diluted 1:20 to 5X 106CFU/mL, 10. mu.l of the above bacterial suspension was added to the prepared trace amountIn a dilution tray. Culturing at 35 deg.C for 16-20h, and determining the lowest concentration of the drug capable of completely inhibiting bacterial growth as the Minimum Inhibitory Concentration (MIC). The experiment was repeated 3 times, each time with 3 replicate wells.
And (4) analyzing results:
the results shown in fig. 11 may indicate that:
the concentrations (0.078-20mg/mL) of the TH-w parts of the radix tetrastigme extract have inhibition effects on the growth of three strains of staphylococcus aureus, and the inhibition effects on all strains are enhanced along with the increase of the drug concentration. There is a drug concentration dependence.
The most sensitive to TH-w site is methicillin-sensitive staphylococcus aureus, followed by a standard strain of staphylococcus aureus, which is the least effective against methicillin-resistant staphylococcus aureus.
The experiment shows that: the TH-w part of the radix tetrastigme extract is diluted to 32.0mg/mL by M-H broth multiple ratio, and after the staphylococcus aureus MSSA-29213 is cultured for 16-20H at 35 ℃, the bacterial inhibition rate is 0.88.
The bacterial inhibition rate of the Tetrastigma hemsleyanum Diels et Gilg extract TH-w part is 0.34 after the Staphylococcus aureus MRSA-BAA1029 is cultured for 16-20H at 35 ℃ by using M-H broth multiple dilution 32.0 mg/mL.
The bacterial inhibition rate of the Tetrastigma hemsleyanum Diels et Gilg extract TH-w part is 0.7 after 32.0mg/mL of M-H broth is diluted by multiple times and the Staphylococcus aureus ATCC259231 is cultured for 16-20H at 35 ℃.
From this, it was found that the optimum concentration of the Hevea hemsleyana extract TH-w diluted in the medium was 32.0 mg/mL.
The invention provides a use of radix tetrastigme extract TH-w on staphylococcus aureus and an action method thereof, which are used for respectively testing and comparing a TH-t part, a TH-p part, a TH-a part, a TH-b part, a TH-w1 part, a TH-w2 part, a TH-w3 part and a TH-w4 part to find out the optimal part which is the TH-w part; the invention cultures staphylococcus aureus at TH-w parts of the radix tetrastigme extract by using different culture medium dilution concentrations in a multiple ratio, and detects and contrasts the biofilm formation capability of bacteria by a crystal violet staining method to find out the optimal concentration.
The foregoing illustrates and describes the principles, general features, and advantages of the present invention. It should be understood by those skilled in the art that the above embodiments do not limit the present invention in any way, and all technical solutions obtained by using equivalent alternatives or equivalent variations fall within the scope of the present invention.
Claims (5)
1. The application of the TH-w part of the radix tetrastigme extract in preparing a medicament for resisting staphylococcus aureus is characterized in that the TH-w part of the radix tetrastigme extract inhibits the growth of staphylococcus aureus; the extraction method of the TH-w part of the radix tetrastigme extract comprises the following steps: soaking 500g of radix Apioris Fortunei dry medicinal material in 5 times of 80% ethanol water solution for 24 hr, heating and reflux extracting for 3 times, each for 40min, mixing extractive solutions, filtering, recovering under reduced pressure, and evaporating to dryness to obtain total extract TH-t part; suspending the total extract in water, and respectively extracting with 10 times volume of water saturated petroleum ether step by step to obtain petroleum ether part TH-p, ethyl acetate part TH-a, butanol part TH-b, and water part TH-w; the staphylococcus aureus is methicillin-resistant staphylococcus aureus.
2. The application of the TH-w part of the radix tetrastigme extract in preparing a medicament for resisting staphylococcus aureus is characterized in that the TH-w part of the radix tetrastigme extract inhibits the formation of a biofilm of the staphylococcus aureus; the extraction method of the TH-w part of the radix tetrastigme extract comprises the following steps: soaking 500g of radix Apioris Fortunei dry medicinal material in 5 times of 80% ethanol water solution for 24 hr, heating and reflux extracting for 3 times, each for 40min, mixing extractive solutions, filtering, recovering under reduced pressure, and evaporating to dryness to obtain total extract TH-t part; suspending the total extract in water, and respectively extracting with 10 times volume of water saturated petroleum ether step by step to obtain petroleum ether part TH-p, ethyl acetate part TH-a, butanol part TH-b, and water part TH-w; the staphylococcus aureus is methicillin-resistant staphylococcus aureus.
3. The application of the TH-w part of the radix tetrastigme extract in preparing a medicament for resisting staphylococcus aureus is characterized in that the TH-w part of the radix tetrastigme extract is diluted to 32.0mg/mL by using LB broth multiple ratio to culture the staphylococcus aureus; the extraction method of the TH-w part of the radix tetrastigme extract comprises the following steps: soaking 500g of radix Apioris Fortunei dry medicinal material in 5 times of 80% ethanol water solution for 24 hr, heating and reflux extracting for 3 times, each for 40min, mixing extractive solutions, filtering, recovering under reduced pressure, and evaporating to dryness to obtain total extract TH-t part; suspending the total extract in water, and respectively extracting with 10 times volume of water saturated petroleum ether step by step to obtain petroleum ether part TH-p, ethyl acetate part TH-a, butanol part TH-b, and water part TH-w; the staphylococcus aureus is methicillin-resistant staphylococcus aureus.
4. The use of a TH-w part of a radix tetrastigme extract in the preparation of a medicament for resisting staphylococcus aureus as claimed in claim 3, wherein the bacterial inhibition rate of the TH-w part of the radix tetrastigme extract is 0.34 after 32.0mg/mL of the TH-w part of the radix tetrastigme extract is diluted by M-H broth in a multiple ratio and cultured for 16-20H at 35 ℃.
5. The use of the TH-w part of the radix tetrastigme extract in the preparation of a medicament for resisting staphylococcus aureus as claimed in claim 3, wherein the absorbance A570mm value of the biological membrane is detected to be 3.1 by adopting a crystal violet staining method after the TH-w part of the radix tetrastigme extract is diluted by 32.0mg/mL LB broth in a multiple ratio to culture methicillin-resistant staphylococcus aureus at 37 ℃ for 24 h.
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