CN112516195B - Anti-staphylococcus aureus and drug-resistant strain traditional Chinese medicine composition thereof, and preparation method and application thereof - Google Patents
Anti-staphylococcus aureus and drug-resistant strain traditional Chinese medicine composition thereof, and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a traditional Chinese medicine composition for resisting staphylococcus aureus and drug-resistant strains thereof, and a preparation method and application thereof, wherein the traditional Chinese medicine composition comprises the following components in parts by volume: 10-40 parts of magnolia officinalis extract, 10-40 parts of gallnut extract, 10-40 parts of scutellaria baicalensis extract and 10-40 parts of benzoin extract. The traditional Chinese medicine composition takes the magnolia officinalis and the gallnut as main antibacterial components, and is matched with traditional Chinese medicines with the effects of diminishing inflammation, easing pain, inhibiting bacteria, detoxifying and the like based on the baical skullcap root and the benzoin as auxiliary medicinal materials, the best antibacterial effect is achieved by the synergy of staphylococcus aureus and drug-resistant strains thereof, and the medicinal materials used in the formula do not contain toxic medicinal materials, so that the traditional Chinese medicine composition has the characteristics of high safety and small toxic and side effects.
Description
Technical Field
The invention belongs to the technical field of antibacterial drugs, and particularly relates to a traditional Chinese medicine composition for resisting staphylococcus aureus and a drug-resistant strain thereof, and a preparation method and application thereof.
Background
Staphylococcus aureus (Staphylococcus aureus), also known as Staphylococcus aureus, is an important pathogenic bacterium belonging to the genus Staphylococcus. Staphylococcus aureus is ubiquitous in nature, found in the air, water, dust and human and animal excreta, the most common pathogen in human pyogenic infections, and can cause human and animal diseases including skin infections, pneumonia, enteritis, endocarditis, and more serious diseases such as sepsis and toxic shock. Antibiotics are commonly used in the clinical treatment of staphylococcus aureus infections and cause tremendous survival pressure on bacteria due to their role in killing and inhibiting their growth. However, with the widespread use and unjustified use of antibiotics, bacteria are increasingly developing resistance to antibiotics, even including multidrug resistance, such resistant strains we call methicillin-resistant staphylococcus aureus (MRSA). MRSA was first reported in 1961 shortly after methicillin was used in the clinic. Considered to be the major causative agent of community-acquired staphylococcus aureus infections worldwide, antibiotic-resistant strains have become one of the greatest threats to global public health and social development due to poor therapeutic efficacy of antibiotics in treating antibiotic-resistant bacterial infections, an increase in the number of serious infections, and an increase in mortality. Therefore, there is an urgent need to develop new antibiotics or drugs against drug-resistant bacteria to combat bacterial infections, particularly those caused by drug-resistant bacteria. The traditional Chinese medicinal materials with antibacterial activity and multiple plant sources are good new medicament sources for resisting bacteria and drug-resistant bacteria.
The traditional Chinese medicine has wide sources, and has the advantages of relatively small toxic and side effects, difficult generation of drug resistance and the like in the treatment of bacterial infection diseases. The traditional Chinese medicine is derived from the summary of the long-term struggle and experience between Chinese nationalities and diseases, has a long history, can be examined by a large amount of documents, and has an exact curative effect. The Chinese medicinal composition has a large number of properties and functions of Chinese medicaments and formulas thereof, is clinically used from ancient times to present, and is continuously developed. In clinical treatment of infectious diseases, although antibiotics have been widely used, their use is limited by drug resistance, iatrogenic complications, allergic reactions, and the like. The traditional Chinese medicine heat-clearing and detoxifying drugs have obvious treatment effects on infectious diseases, and are widely proved by a large number of clinical treatment researches and pharmacological experimental researches. Its anti-infective action is not a single antimicrobial, antiviral action, but rather is accomplished through the modulation of various mechanisms. And the drugs have little toxic and side effects, so that a wide way for treating acute infectious diseases is opened up. In recent years, the traditional Chinese medicine for clearing heat and removing toxicity has antibacterial effect in vitro on some multi-drug resistant 'super bacteria' including MRSA and has no drug resistance. Therefore, screening out safe and efficient antibacterial drugs which are not easy to generate drug resistance from the traditional Chinese medicines becomes a hotspot and a focus of the research field. At present, no report on the staphylococcus aureus resistance of a compound formula of magnolia officinalis, Chinese gall, scutellaria baicalensis and benzoin is searched, and no report on the compound staphylococcus aureus resistance MRSA is found.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a traditional Chinese medicine composition for resisting staphylococcus aureus and drug-resistant strains thereof, and a preparation method and application thereof.
In order to achieve the purpose, the technical scheme adopted by the invention for solving the technical problems is as follows:
a traditional Chinese medicine composition for resisting staphylococcus aureus and drug-resistant strains thereof comprises the following components in parts by volume: 10-40 parts of magnolia officinalis extract, 10-40 parts of gallnut extract, 10-40 parts of scutellaria baicalensis extract and 10-40 parts of benzoin extract.
Further, the anti-staphylococcus aureus and drug-resistant strain traditional Chinese medicine composition comprises the following components in parts by volume: 10-40 parts of magnolia officinalis extract, 10-40 parts of gallnut extract, 10-20 parts of scutellaria baicalensis extract and 10-20 parts of benzoin extract.
Further, the anti-staphylococcus aureus and drug-resistant strain traditional Chinese medicine composition comprises the following components in parts by volume: 10 parts of magnolia officinalis extract, 10 parts of gallnut extract, 20 parts of scutellaria baicalensis extract and 10 parts of benzoin extract.
Further, the anti-staphylococcus aureus and drug-resistant strain traditional Chinese medicine composition comprises the following components in parts by volume: 20 parts of magnolia officinalis extract, 40 parts of gallnut extract, 10 parts of scutellaria baicalensis extract and 20 parts of benzoin extract.
Further, the anti-staphylococcus aureus and drug-resistant strain traditional Chinese medicine composition comprises the following components in parts by volume: 40 parts of magnolia officinalis extract, 40 parts of gallnut extract, 20 parts of scutellaria baicalensis extract and 10 parts of benzoin extract.
Further, the anti-staphylococcus aureus and drug-resistant strain traditional Chinese medicine composition comprises the following components in parts by volume: 40 parts of magnolia officinalis extract, 40 parts of Chinese gall extract, 10 parts of scutellaria baicalensis extract and 20 parts of benzoin extract.
Further, the magnolia bark extract, the Chinese gall extract, the scutellaria baicalensis extract and the benzoin extract are prepared by the following methods: pulverizing cortex Magnolia officinalis, Galla chinensis, Scutellariae radix and Benzonum respectively, adding 70% ethanol, ultrasonic dispersing for 25-35min, extracting at 25-30 deg.C for 30-90 days, and filtering to obtain cortex Magnolia officinalis extract, Galla chinensis extract, Scutellariae radix extract and Benzonum extract; wherein the feed-liquid ratio is 1:3-6g/mL, and the leaching time is preferably 30-35 days.
Further, the anti-staphylococcus aureus and drug-resistant strain traditional Chinese medicine composition further comprises pharmaceutically acceptable auxiliary materials.
Such adjuvants include, but are not limited to, solvents, propellants, solubilizers, solubilizing agents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, tonicity adjusting agents, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adherents, sequestering agents, permeation enhancers, buffers, plasticizers, surfactants, foaming agents, antifoaming agents, thickening agents, encapsulating agents, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, release retardants, and the like.
Further, the anti-staphylococcus aureus and drug-resistant strain traditional Chinese medicine composition is tablets, tinctures, liniments, pastilles or pills.
The preparation method of the traditional Chinese medicine composition for resisting staphylococcus aureus and drug-resistant strains thereof comprises the following steps: mixing cortex Magnolia officinalis extract, Galla chinensis extract, Scutellariae radix extract and Benzonum extract according to formula.
The application of the anti-staphylococcus aureus and the drug-resistant strain traditional Chinese medicine composition thereof in preparing anti-staphylococcus aureus medicines.
The anti-staphylococcus aureus and drug-resistant strain traditional Chinese medicine composition provided by the invention, as well as the preparation method and the application thereof, have the following beneficial effects:
magnolia bark: bitter; pungent taste; and (4) temperature. Is a conventional medicinal material, has the effects of eliminating dampness and phlegm, resisting ulcer, has obvious central inhibition effect, and is resistant to pathogenic microorganisms and tumors. Magnolol and honokiol are two phenylpropanoid compounds with high content in magnolia officinalis, the activity of the magnolol is closely related to the traditional efficacy of the magnolia officinalis, more recent research reports that phenylethanoid glycosides compounds stably exist in the magnolia officinalis in different production places, and data show that the phenylethanoid glycosides compounds in the magnolia officinalis play a great role in resisting bacteria and diminishing inflammation.
Gallnut: astringent taste and cold nature. Galla chinensis has broad-spectrum antibacterial and anti-inflammatory effects, has strong inhibitory effect on staphylococcus, typhoid bacillus and pseudomonas aeruginosa, also has antioxidant and anti-aging effects, and also has remarkable effect in reducing blood sugar. The main chemical component of the gallnut is tannic acid, and the content of the gallnut can reach 70%.
Scutellaria baicalensis: is bitter in taste; is cold in nature. Has broad-spectrum antibacterial effect, and has effects in inhibiting Bacillus and coccus, treating influenza virus and dermatophyte, and treating upper respiratory infection. The main effective component in the scutellaria is flavonoid compound, mainly contains baicalin, baicalein, wogonin and the like, and researches show that the flavonoid compound is closely related to the effects of purging intense heat, detoxifying and resisting inflammation of the scutellaria.
Benzoin: pungent in flavor and bitter in property. The benzoin has effects of refreshing, activating qi-flowing, promoting blood circulation, relieving pain, eliminating phlegm, resisting bacteria and promoting healing of ulcer wound. The main component of benzoin is resin, the content is 70-80%.
The prescription takes mangnolia officinalis and gallnut as main antibacterial drugs, and obtains better anti-staphylococcus aureus effect under the synergistic effect of scutellaria baicalensis and benzoin. The magnolol with the most content in the magnolia officinalis has good antibacterial and anti-inflammatory effects, and the phenylethanoid glycosides compound in the magnolia officinalis has better treatment effect in the aspects of antibacterial and anti-inflammatory; galla chinensis has broad-spectrum antibacterial and anti-inflammatory effects, and especially has good antibacterial and bactericidal effects on Staphylococcus aureus and Pseudomonas aeruginosa. Therefore, the magnolia officinalis and the gallnut are selected as main antibacterial components in the formula, and are matched with traditional Chinese medicines with anti-inflammation, pain relieving, bacteriostasis, detoxification and other effects, such as scutellaria baicalensis and benzoin, as auxiliary medicinal materials, so that the best bacteriostasis effect is achieved in cooperation with staphylococcus aureus and resistant strains thereof, and the medicinal materials used in the formula do not contain toxic medicinal materials, and the magnolia officinalis and the gallnut have the characteristics of high safety and small toxic and side effects.
Drawings
FIG. 1 is a comparison of the bacteriostatic results of examples 1-8 at a 1:15 (6.25%) dosing concentration for each example;
FIG. 2 shows the bacteriostatic and bactericidal results of the compound MW1810 and a control group on Staphylococcus aureus at different times;
FIG. 3 shows the bacteriostatic and bactericidal results of the compound MW1810 and a control group on Staphylococcus aureus under different concentrations;
FIG. 4 is a graph of the relative conductivity change of Compound MW1810 and control groups after treatment with Staphylococcus aureus;
FIG. 5 shows the change in AKP activity in the culture broth after Staphylococcus aureus treatment with the control group and the combination MW 1810;
FIG. 6 shows the morphological structure of Staphylococcus aureus observed under scanning electron microscope after treating Staphylococcus aureus with compound MW1810 and control group;
FIG. 7 is an amplification curve and a dissolution curve of virulence genes and ABC transporter corresponding genes after the compound MW1810 treats staphylococcus aureus at different concentrations;
FIG. 8 shows the expression levels of virulence genes and ABC transporter genes in a compound MW1810 treated with Staphylococcus aureus at different concentrations;
FIG. 9 is a view of the healing of the wounds on the back of the mice in each group after drug treatment;
FIG. 10 is a comparison of the wound healing rates of groups of mice after drug treatment;
FIG. 11 is a comparison of the peripheral blood tumor necrosis factor alpha (TNF-. alpha.) content in the serum of mice in each group after drug treatment;
FIG. 12 is a comparison of the serum levels of the inflammatory factor interleukin-6 (IL-6) in groups of mice after drug treatment.
FIG. 13 shows the bacteriostatic and bactericidal results of the compound MW1810 for methicillin-resistant Staphylococcus aureus at different concentrations;
FIG. 14 is a comparison of the diffusion results of compound MW1810 to standard strains of Staphylococcus aureus and drug-resistant strains in paper.
Detailed Description
The species sources used in the following examples are as follows:
the staphylococcus aureus is from the microbiological laboratory of the life institute of transportation university in southwest;
methicillin-resistant staphylococcus aureus (MRSA) is obtained from the microbiological laboratory of the Life institute of south-West traffic university.
Example 1
A traditional Chinese medicine composition for resisting staphylococcus aureus and drug-resistant strains thereof comprises the following components in parts by volume: 10 parts of magnolia officinalis extract, 10 parts of Chinese gall extract, 20 parts of scutellaria baicalensis extract and 10 parts of benzoin extract.
The magnolia bark extract, the Chinese gall extract, the scutellaria baicalensis extract and the benzoin extract are prepared by the following methods: pulverizing cortex Magnolia officinalis, Galla chinensis, Scutellariae radix and Benzonum into 2mm granules, respectively, adding 70% ethanol, ultrasonic dispersing for 30min, extracting at 30 deg.C for 30 days, and filtering to obtain cortex Magnolia officinalis extract, Galla chinensis extract, Scutellariae radix extract and Benzonum extract; wherein the feed-liquid ratio is 1:4 g/mL.
The preparation method of the anti-staphylococcus aureus traditional Chinese medicine composition comprises the following steps: mixing cortex Magnolia officinalis extract, Galla chinensis extract, Scutellariae radix extract and Benzonum extract according to formula.
Example 2
A traditional Chinese medicine composition for resisting staphylococcus aureus and drug-resistant strains thereof comprises the following components in parts by volume: 10 parts of magnolia officinalis extract, 20 parts of Chinese gall extract, 20 parts of scutellaria baicalensis extract and 10 parts of benzoin extract.
The magnolia bark extract, the Chinese gall extract, the scutellaria baicalensis extract and the benzoin extract are prepared by the following methods: pulverizing cortex Magnolia officinalis, Galla chinensis, Scutellariae radix and Benzonum into 2mm particles, respectively, adding 70% ethanol, ultrasonic dispersing for 30min, extracting at 30 deg.C for 30 days, and filtering to obtain cortex Magnolia officinalis extract, Galla chinensis extract, Scutellariae radix extract and Benzonum extract; wherein the feed-liquid ratio is 1:4 g/mL.
The preparation method of the anti-staphylococcus aureus traditional Chinese medicine composition comprises the following steps: mixing cortex Magnolia officinalis extract, Galla chinensis extract, Scutellariae radix extract and Benzonum extract according to formula.
Example 3
A traditional Chinese medicine composition for resisting staphylococcus aureus and drug-resistant strains thereof comprises the following components in parts by volume: 10 parts of magnolia officinalis extract, 40 parts of Chinese gall extract, 40 parts of scutellaria baicalensis extract and 40 parts of benzoin extract.
The magnolia bark extract, the Chinese gall extract, the scutellaria baicalensis extract and the benzoin extract are prepared by the following methods: pulverizing cortex Magnolia officinalis, Galla chinensis, Scutellariae radix and Benzonum into 2mm granules, respectively, adding 70% ethanol, ultrasonic dispersing for 30min, extracting at 30 deg.C for 30 days, and filtering to obtain cortex Magnolia officinalis extract, Galla chinensis extract, Scutellariae radix extract and Benzonum extract; wherein the feed-liquid ratio is 1:4 g/mL.
The preparation method of the anti-staphylococcus aureus traditional Chinese medicine composition comprises the following steps: mixing cortex Magnolia officinalis extract, Galla chinensis extract, Scutellariae radix extract and Benzonum extract according to formula.
Example 4
A traditional Chinese medicine composition for resisting staphylococcus aureus and drug-resistant strains thereof comprises the following components in parts by volume: 20 parts of magnolia officinalis extract, 20 parts of Chinese gall extract, 40 parts of scutellaria baicalensis extract and 10 parts of benzoin extract.
The magnolia bark extract, the Chinese gall extract, the scutellaria baicalensis extract and the benzoin extract are prepared by the following methods: pulverizing cortex Magnolia officinalis, Galla chinensis, Scutellariae radix and Benzonum into 2mm granules, respectively, adding 70% ethanol, ultrasonic dispersing for 30min, extracting at 30 deg.C for 30 days, and filtering to obtain cortex Magnolia officinalis extract, Galla chinensis extract, Scutellariae radix extract and Benzonum extract; wherein the feed-liquid ratio is 1:4 g/mL.
The preparation method of the anti-staphylococcus aureus traditional Chinese medicine composition comprises the following steps: mixing cortex Magnolia officinalis extract, Galla chinensis extract, Scutellariae radix extract and Benzonum extract according to formula.
Example 5
A traditional Chinese medicine composition for resisting staphylococcus aureus and drug-resistant strains thereof comprises the following components in parts by volume: 20 parts of magnolia officinalis extract, 40 parts of Chinese gall extract, 10 parts of scutellaria baicalensis extract and 20 parts of benzoin extract.
The magnolia bark extract, the Chinese gall extract, the scutellaria baicalensis extract and the benzoin extract are prepared by the following methods: pulverizing cortex Magnolia officinalis, Galla chinensis, Scutellariae radix and Benzonum into 2mm granules, respectively, adding 70% ethanol, ultrasonic dispersing for 30min, extracting at 30 deg.C for 30 days, and filtering to obtain cortex Magnolia officinalis extract, Galla chinensis extract, Scutellariae radix extract and Benzonum extract; wherein the feed-liquid ratio is 1:4 g/mL.
The preparation method of the anti-staphylococcus aureus traditional Chinese medicine composition comprises the following steps: mixing cortex Magnolia officinalis extract, Galla chinensis extract, Scutellariae radix extract and Benzonum extract according to formula.
Example 6
A traditional Chinese medicine composition for resisting staphylococcus aureus and drug-resistant strains thereof comprises the following components in parts by volume: 20 parts of magnolia officinalis extract, 10 parts of Chinese gall extract, 20 parts of scutellaria baicalensis extract and 40 parts of benzoin extract.
The magnolia bark extract, the Chinese gall extract, the scutellaria baicalensis extract and the benzoin extract are prepared by the following methods: pulverizing cortex Magnolia officinalis, Galla chinensis, Scutellariae radix and Benzonum into 2mm granules, respectively, adding 70% ethanol, ultrasonic dispersing for 30min, extracting at 30 deg.C for 30 days, and filtering to obtain cortex Magnolia officinalis extract, Galla chinensis extract, Scutellariae radix extract and Benzonum extract; wherein the feed-liquid ratio is 1:4 g/mL.
The preparation method of the anti-staphylococcus aureus traditional Chinese medicine composition comprises the following steps: mixing cortex Magnolia officinalis extract, Galla chinensis extract, Scutellariae radix extract and Benzonum extract according to formula.
Example 7
A traditional Chinese medicine composition for resisting staphylococcus aureus and drug-resistant strains thereof comprises the following components in parts by volume: 40 parts of magnolia officinalis extract, 40 parts of Chinese gall extract, 20 parts of scutellaria baicalensis extract and 10 parts of benzoin extract.
The magnolia bark extract, the Chinese gall extract, the scutellaria baicalensis extract and the benzoin extract are prepared by the following methods: pulverizing cortex Magnolia officinalis, Galla chinensis, Scutellariae radix and Benzonum into 2mm granules, respectively, adding 70% ethanol, ultrasonic dispersing for 30min, extracting at 30 deg.C for 30 days, and filtering to obtain cortex Magnolia officinalis extract, Galla chinensis extract, Scutellariae radix extract and Benzonum extract; wherein the feed-liquid ratio is 1:4 g/mL.
The preparation method of the anti-staphylococcus aureus traditional Chinese medicine composition comprises the following steps: mixing cortex Magnolia officinalis extract, Galla chinensis extract, Scutellariae radix extract and Benzonum extract according to formula.
Example 8
A traditional Chinese medicine composition for resisting staphylococcus aureus and drug-resistant strains thereof comprises the following components in parts by volume: 40 parts of magnolia officinalis extract, 40 parts of Chinese gall extract, 10 parts of scutellaria baicalensis extract and 20 parts of benzoin extract.
The magnolia bark extract, the Chinese gall extract, the scutellaria baicalensis extract and the benzoin extract are prepared by the following methods: pulverizing cortex Magnolia officinalis, Galla chinensis, Scutellariae radix and Benzonum into 2mm granules, respectively, adding 70% ethanol, ultrasonic dispersing for 30min, extracting at 30 deg.C for 30 days, and filtering to obtain cortex Magnolia officinalis extract, Galla chinensis extract, Scutellariae radix extract and Benzonum extract; wherein the feed-liquid ratio is 1:4 g/mL.
The preparation method of the anti-staphylococcus aureus traditional Chinese medicine composition comprises the following steps: mixing cortex Magnolia officinalis extract, Galla chinensis extract, Scutellariae radix extract and Benzonum extract according to formula.
The components and contents (parts by volume) of the traditional Chinese medicine composition of the above examples 1-8 are shown in table 1:
TABLE 1 EXAMPLES 1-8 Components and amounts of Chinese medicinal compositions
Test example 1 bacteriostatic results of the respective Chinese medicinal compositions
The in vitro bacteriostasis tests of the traditional Chinese medicine compositions of the examples 1-8 are respectively carried out by adopting a test tube dilution method, and the bacteriostasis effect of each example on staphylococcus aureus is shown in figure 1 and table 2 under the condition of adding medicine concentration of 1:15 (6.25%).
TABLE 2 bacteriostatic results of the Chinese medicinal compositions of the examples
| Examples | Staphylococcus aureus colony count |
| Example 1 | 50 |
| Example 2 | 100+ |
| Example 3 | 200+ |
| Example 4 | 150+ |
| Example 5 | 25+ |
| Example 6 | -- |
| Example 7 | 0 |
| Example 8 | 50 |
Note: "- -" indicates that it is not calculable (in large quantities).
As shown in fig. 1 and table 2, the traditional Chinese medicine compositions of examples 1, 5, 7 and 8 have good effect when used for inhibiting staphylococcus aureus, the number of clones of staphylococcus aureus is less than or equal to 50, and especially, the traditional Chinese medicine composition of example 7 has the strongest killing effect on staphylococcus aureus and can play a role in completely inhibiting bacteria. The invention names the traditional Chinese medicine composition in the embodiment 7 as MW 1810.
Experimental example 2 antibacterial property study of MW1810
Referring to a test method of a dissolubility antibacterial product in GB 15979-2002 Standard design of Disposable sanitary articles, a specific test method is designed:
1. selecting staphylococcus aureus to be cultured in LB liquid medium until the concentration of the bacterial liquid is 1 multiplied by 107And taking out for standby at cfu/mL.
2. Experimental groups:
and (3) respectively adding 500 mu L of compound MW1810 liquid medicine and 10 mu L of bacterium liquid into 5 sterilized 1.5mL EP tubes, uniformly mixing until 4 action time points (2min, 5min, 10min and 20min), respectively adding 500 mu L of PBS, fully mixing, respectively taking 50 mu L of mixed liquid for plate coating, carrying out inverted culture at 37 ℃ for 24h, and carrying out colony counting.
3. Control group:
5 sterilized 1.5mL EP tubes were added with 500. mu.L of physiological saline and 10. mu.L of bacterial solution respectively and mixed until 4 action time points (2min, 5min, 10min, 20min), 500. mu.L of PBS was added respectively and mixed well, 50. mu.L of mixed solution was taken respectively and plated, inverted culture was carried out at 37 ℃ for 24h, and colony counting was carried out.
4. The bacteriostatic rate was calculated as follows:
X＝(a-b)/a×100％
in the formula: x is the bacteriostasis rate,%;
a is the average colony number of the control sample; b is the average colony number of the samples.
5. Experiments were performed in 3 parallel groups; the bacteriostasis rate is more than or equal to 50-90 percent, which indicates that the product has bacteriostasis; the bacteriostasis rate is more than or equal to 90 percent, which indicates that the product has stronger bacteriostasis.
The bacteriostatic results are shown in fig. 2 and table 3.
TABLE 3 antibacterial and bactericidal results of Staphylococcus aureus in experimental and control groups at different times
As can be seen from fig. 2 and table 3, no colonies are generated in the MW1810 experimental group in 2, 5, 10 and 20min, the bacteriostasis rate is 100%, and the control group cannot count too many colonies, so that the MW1810 has a strong bacteriostasis effect on staphylococcus aureus, and can be used as an anti-staphylococcus aureus medicament.
Test example 3 MIC and MBC determination of MW1810
The MIC and MBC determination process of MW1810 comprises the following steps:
1. adding a culture medium and 50 mu L of log-phase staphylococcus aureus liquid into a sterile test tube according to different dosing ratios of 1:15 (6.25%, volume concentration), 1:16 (5.88%), 1:17 (5.55%), 1:18 (5.26%), 1:18 (5.00%), and 1:20 (4.76%), shaking uniformly, then adding corresponding volumes of liquid medicine (experimental group) and absolute ethyl alcohol (control group) into the test tube according to the dosing ratios, and shaking uniformly, wherein the final volume of the test tube is 5.0 mL;
2. placing the test tube in an air bath oscillator at 37 ℃ and 210r/min for culturing for 7 h;
3. taking out 50 μ L of plate from each tube, spreading, standing for 10min, and performing inverted culture at 37 deg.C for 24 h;
4. plates were counted and photographed and data was recorded and the results are shown in figure 3.
As shown in fig. 3, the Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC) of the compound MW1810 of traditional Chinese medicine against staphylococcus aureus are respectively 1:18 (5.26%) and 1:17 (5.55%), wherein after the interference of the alcohol in the compound MW1810 is eliminated, the bacteriostatic effect of the traditional Chinese medicine compound MW1810 is obviously different from that of the alcohol under the same condition.
Test example 4 conductivity measurement
The conductivity measurement process comprises the following steps:
1. staphylococcus aureus growing to logarithmic phase was washed 3 times with 0.1mol/L phosphate buffer (pH7.4), centrifuged at 7000r/min for 2min, the supernatant was discarded, and washed with 5% glucose solution until the conductivity of the bacterial liquid was equivalent to that of the 5% glucose solution, and the bacterial liquid was used as isotonic bacteria.
2. Adding compound MW1810 medicinal liquid (control, 1MIC and 2MIC) with different concentrations into 5% glucose solution, mixing, measuring the electric conductivity, and recording as L1(ii) a Wherein the control group was added sterile water.
3. Adding isotonic bacteria solution into compound MW1810 medicinal liquid (0MIC, 1MIC and 2MIC) with different concentrations, culturing at 37 deg.C, taking out at 1, 3, 5, 7, 9 and 11 hr respectively, measuring conductivity, and recording as L2。
4. Cooling the bacterial liquid suspended in 5% glucose solution in boiling water bath for 5min, and measuring the conductivity as L0。
5. The relative conductivity of the bacterial membrane permeability is calculated according to the following formula:
relative conductivity (%) (L) of 100 × (L)2-L1)÷L0
The above 0MIC is no addition of MW1810 medicinal liquid, 1MIC is MW1810 medicinal liquid with 1-time MIC concentration, and 2MIC is MW1810 medicinal liquid with 2-time MIC concentration (the same below). The results of the conductivity measurements are shown in FIG. 4.
As can be seen from fig. 4, the relative conductivity of the bacterial liquid immediately increased after the addition of the relapse drug solution at a concentration of 1MIC or more, and rapidly increased with the increase in treatment time and the drug solution concentration 2MIC, as compared to the control. The increase in relative conductivity, which may be due to normal lysis and death of the bacteria, results in an increase in relative conductivity. This means that the compound MW1810 medicinal liquid can affect the permeability and cell membrane integrity of Staphylococcus aureus, causing its lysis, leakage of intracellular components, and especially the loss of electrolytes, including K+，Ca2+，Na+Etc., causing damage to metabolic pathways.
Test example 5 determination of alkaline phosphatase (AKP) Activity of bacterial liquid
The process for measuring the activity of alkaline phosphatase (AKP) of bacteria liquid comprises the following steps:
treatment of logarithmic phase Staphylococcus aureus (1 × 10) with compound MW1810 medicinal liquid7CFU/mL), final concentrations were 1MIC and 2 MIC. Bacterial suspensions without treatment with the combination MW1810 drug solution were used as controls. The mixture is incubated at 37 ℃ for 7 hours, samples are taken at five time points of 1h, 2h, 3h, 5h and 7h, then centrifugation is carried out at 7000r/min for 2min, the supernatant is taken, the content of extracellular alkaline phosphatase (AKP) in the supernatant is determined by using an AKP kit, and the change of the content of AKP in the bacterial liquid at different time points is recorded, and the result is shown in figure 5.
AKP is located between the cell wall and the cell membrane and therefore cannot be detected extracellularly through the intact cell wall. However, when the cell wall is damaged or damaged, AKP leaks out of the cell. Thus, AKP activity in the medium can be an indicator of cell wall integrity. As shown in fig. 5, AKP activity in the supernatant of the culture solution was significantly increased within 5 hours, compared to the control. AKP activity increased from 4.98 King's unit/100mL to 18.19 King's unit/100mL at 1MIC concentration and from 4.98 King's unit/100mL to 26.82 King's unit/100mL at 2MIC concentration after 5h of treatment with the combination MW1810 drug solution.
Thus, compound MW1810 treatment was more effective at increasing extracellular AKP levels at higher concentrations. Compound MW1810 was therefore postulated to be able to increase cell wall permeability, suggesting that damage to cell wall integrity may be the primary cause of AKP release into the supernatant.
Test example 6 Observation of bacterial surface by Electron microscope
1. And (3) treating and collecting thalli: taking Staphylococcus aureus (1 × 10) in logarithmic growth phase7CFU/mL), compound MW1810 liquid medicines (control, 1MIC and 2MIC) with different concentrations are respectively added into the bacterial suspension and cultured for 6h at 37 ℃ by a 210rpm gas bath oscillator.
2. Preparation of a thallus sample: centrifuging the suspension at 8000rpm for 3min, removing supernatant to obtain thallus, and performing the following operations: adding the thalli into 2.5 percent glutaraldehyde stationary liquid, and fixing the thalli in a refrigerator at 4 ℃ overnight; washing with PBS for 2-3 times; ③ gradient dehydrating with 30 percent, 50 percent, 70 percent, 85 percent and 95 percent of ethanol for 1 time (5-10 min/time) and 100 percent of ethanol for 2 times (15-20 min/time); (wherein, each step needs to be centrifuged at 8000rpm for 3-5min, the supernatant is discarded, and the next reagent is poured into the centrifuge, and the bacteria block is broken up by blowing and beating for 3-5 times with a pipette).
3. Naturally drying: and (4) sucking the bacterial liquid in the last step onto a silicon wafer by using a capillary, and naturally drying for 30 min.
4. Ion sputtering and gold spraying: after the ion sputtering gold spraying, the scanning electron microscope can be carried out to observe the morphological structure of the staphylococcus aureus, and the result is shown in figure 6.
After staphylococcus aureus is treated by the compound MW1810 liquid medicine with 1MIC and 2MIC for 6 hours respectively, the bacterial form and physical change are observed, as can be directly observed from figure 6, the thalli in an untreated control group are complete, the cell membrane is smooth, when the compound MW1810 liquid medicine with the concentration of 1MIC is added, the staphylococcus aureus loses the smooth spherical form, the rupture occurs, and the cell lysate is increased; when the concentration is increased to 2MIC, the staphylococcus aureus thalli is obviously wrinkled, the shape is seriously broken, obvious rupture occurs, and a large amount of cell lysates are obtained.
The observation result of an electron microscope shows that the compound MW1810 liquid medicine can cause the cell membrane of staphylococcus aureus to be cracked, and the more seriously the thallus surface is damaged along with the increase of the medicine adding concentration, the cell dissolved matter is increased, which is consistent with the measurement results of the conductivity and the AKP content.
Test example 7 detection of differential expression of specific Gene by real-time fluorescent quantitative PCR (qRT-PCR)
1. Alpha-toxin plays an essential role in the pathogenicity of staphylococcus aureus by causing tissue damage. Alpha-toxin is an extracellular secreted protein encoded by the hla gene, a pore-forming toxin that causes cell damage and death. This protein has been shown to be a virulence factor for many serious infections, including keratitis, mastitis, sinusitis, peritonitis, skin and soft tissue infections. The influence of compound MW1810 on the virulence function of bacterial cells is researched by detecting the differential expression of the specific gene hla, the auxiliary regulatory factors agr and RNAIII.
ATP-binding cassette (ABC) transport system proteins, which are ubiquitously distributed throughout an organism, are involved in the uptake or secretion of a variety of different biomolecules, nutrients, antimicrobials or ions across the plasma membrane of a cell. The system is characterized by the presence of ABC protein transporters in the cell membrane, molecules transported inside and outside the cell, which facilitate drug release and elimination of toxic substances, in such a way as to avoid the therapeutic effects of antibacterial agents, and it has also been reported that some of the proteins of the ABC system are associated with drug resistance. The influence of compound MW1810 on the ABC transport system protein of bacteria is researched by detecting the differential expression of ABC transport system related genes SAR0144, SAR1073, SAR1928 and SAR0306 (gene ID). The genes SAR0144, SAR1073 and SAR1928 have molecular functions as ATP binding protein of ABC type transporter, and SAR0306 is lipoprotein and ATP binding protein of release system.
The primers and sequences used for detecting the differential expression of specific genes by real-time fluorescent quantitative PCR (qRT-PCR) are as follows:
Gyrb
F：5'-GGTGGCGACTTTGATCTAGC-3'(SEQ ID NO：1)；
R：5'-TTATACAACGGTGGCTGTGC-3'(SEQ ID NO：2)；
hla
F：5'-GGTATATGGCAATCAAC-3'(SEQ ID NO：3)；
R：5'-CTCGTTCGTATATTACATCT-3'(SEQ ID NO：4)；
agr
F：5'-TCCAGCAGAATTAAGAACTCG-3'(SEQ ID NO：5)；
R：5'-ATATCATCATATTGAACATACACT-3'(SEQ ID NO：6)；
RNAIII
F：5'-GCACTGAGTCCAAGGAAACTAAC-3'(SEQ ID NO：7)；
R：5'-GCACTGAGTCCAAGGAAACTAAC-3'(SEQ ID NO：8)；
SAR0144
F：5'-AAGCCATCCCAACTTAATAACC-3'(SEQ ID NO：9)；
R：5'-TGGGTCTAATGAAGCAACTGG-3'(SEQ ID NO：10)；
SAR1073
F：5'-ATGTTGTTTAGAGGGGTCCAC-3'(SEQ ID NO：11)；
R：5'-CCAACTTCGCTGCCTACT-3'(SEQ ID NO：12)；
SAR1928
F：5'-TGAAGTCGTTGCATTTGGAG-3'(SEQ ID NO：13)；
R：5'-TCGCTTGGTTACGCATGT-3'(SEQ ID NO：14)；
SAR0306
F：5'-CGATTGGGTAGGAGGTGTA-3'(SEQ ID NO：15)；
R：5'-CCAGAAGGTCCAACTAATGC-3'(SEQ ID NO：16)。
the real-time fluorescent quantitative PCR reaction system is as follows: positive and negative primers are 0.6 mu L, ddH each2O6.3. mu. L, Mix 7.5.5. mu.L, template 1. mu.L.
The real-time fluorescent quantitative PCR reaction program is as follows: start (incubation at 95 ℃ for 30 s); two-step amplification, 40 cycles (5 s incubation at 95 ℃ C., 30s incubation at 60 ℃); melting one cycle (5 s incubation at 95 ℃, 60s incubation at 60 ℃, 1s incubation at 95 ℃); cooling (incubation at 50 ℃ for 30 s).
After the staphylococcus aureus is treated by the compound MW1810 under different concentrations, amplification curves and dissolution curves of virulence genes and ABC transporter corresponding genes are shown in figure 7, wherein 7-1 and 7-2 are respectively the amplification curve and the dissolution curve of the gene Gyrb; 7-3 and 7-4 are the amplification curve and the dissolution curve of the gene agr, respectively; 7-5 and 7-6 are the amplification curve and the lysis curve, respectively, for gene hla; 7-7 and 7-8 are the amplification curve and the dissolution curve of the gene RNAIII, respectively; 7-9 and 7-10 are the amplification curve and the lysis curve, respectively, of gene SAR 0144; 7-11 and 7-12 are respectively an amplification curve and a dissolution curve of the gene SAR 0306; 7-13 and 7-14 are respectively an amplification curve and a dissolution curve of the gene SAR 1073; 7-15 and 7-16 are the amplification curve and the dissolution curve of the gene SAR1928, respectively.
After staphylococcus aureus is treated by compound MW1810 under different concentrations, the expression levels of virulence genes and ABC transporter corresponding genes are shown in figure 8.
As can be seen from FIG. 7, the amplification curve of each gene primer is good, no specific amplification occurs, and the experimental result is reliable. As can be seen from FIG. 8, after 1h of treatment with 1/4MIC concentration of compound MW1810 drug, the genes agr, hla, RNAIII, SAR1073 and SAR1928 were significantly down-regulated compared to the control group: -76.5%, -41.35%, -46.9%, -52.5%, -52.7%, but SAR0144 and SAR0306 exhibit upregulation: + 35.1%, + 45.4%; after being treated by 1/4MIC compound MW1810 medicament for 4 hours, compared with a control group, the genes agr, hla, RNAIII, SAR0144, SAR1073, SAR1928 and SAR0306 are all in extremely obvious down-regulation: 98.7 percent, 96.5 percent, 99.2 percent, 99.8 percent, 98.9 percent, 98.6 percent and 98.5 percent. Therefore, the toxicity of the staphylococcus aureus can be effectively reduced by the drug treatment, the gene expression of an ABC transfer system can be well inhibited, the intake of nutrition and ions and the transfer of ATP, protein and other substances by the staphylococcus aureus can be prevented, and the staphylococcus aureus can be killed.
Experimental example 8 study on treatment effect and mechanism of skin wound surface staphylococcus aureus infection of mouse model
1. Establishment and experimental grouping of experimental animal models
A staphylococcus aureus infection mouse model is established by inducing BALB/C mice with staphylococcus aureus, and after the mice are anesthetized, 6 mu L of the anesthetized mice are injected into the median spine of the back to grow to the density of 1 multiplied by 10 in the logarithmic growth phase7The bacterial liquid of the staphylococcus aureus of CFU/mL is molded, and the grouping administration of the bacterial liquid is started twice a day after 24 hours according to the table 4.
Table 4 grouping and drug treatment
| Group of | Whether to make a mold | Drug treatment |
| Blank group | Whether or not | Is not processed at all |
| Model set | Is that | Is not processed at all |
| Positive group | Is that | Fusidic acid cream |
| MW1810 | Is that | MW1810 |
| MW1810+oil | Is that | MW1810+ tea seed oil (volume ratio 3:1) |
2. Index detection
2.1 wound healing Observation
The wound surface area is measured by a transparent film tracing and weighing method (Nagelschmidt method) in modeling 1d, 4d, 9d and 16d respectively, and the wound surface healing rate is calculated.
The wound healing rate is (original wound area-unhealed wound area)/original wound area × 100%
The observation result of wound healing is shown in 9, and the comparison result of the wound healing rate of each group of mice after drug treatment is shown in 10.
As can be seen from fig. 9 and 10, the anti-staphylococcus aureus composition has a healing effect on the wound infection of the mouse, and compared with the model composition, the wound areas of the positive composition, the MW1810 and the MW1810+ oil composition are all significantly reduced, and the infection conditions are all significantly improved; compared with the model group, the wound healing rates of the other groups are obviously increased (p <0.05), wherein the wound healing rate of the MW1810+ oil group is obviously increased (p <0.01) compared with the model group, and the comprehensive analysis on the wound infection healing conditions of the mice shows that: MW1810+ oil group > MW1810 group > positive group > model group.
2.2 peripheral blood tumor necrosis factor alpha (TNF-alpha), Interleukin-6 (IL-6) assays
After 16d, the changes of two inflammatory factors, TNF-alpha and IL-6, in each group of serum were measured by ELISA. After the mice are bled through eyeballs, blood samples are stood for 30min, centrifuged at 1000rpm for 10min, supernatants are taken for standby, and ELISA is carried out to detect relevant inflammatory factors, and the results are shown in figure 11 and figure 12.
The effect of the anti-Staphylococcus aureus formulation on the peripheral blood inflammatory factors TNF-. alpha.and IL-6 can be seen in FIGS. 11 and 12. Compared with the blank group, the contents of TNF-a and IL-6 in the other groups are increased to different degrees; compared with the model group, the contents of the two inflammatory factors in the positive group, the MW1810 group and the MW1810+ oil group show down-regulation trends (p <0.05), wherein the contents of the two inflammatory factors in the MW1810 group are extremely remarkably down-regulated. In conclusion, the formula shows remarkable inhibition effects on TNF-a and IL-6 inflammatory factors in serum to different degrees.
Experimental example 9 MIC and MBC determination of traditional Chinese medicine compound MW1810 to methicillin-resistant staphylococcus aureus
1. Activating methicillin-resistant staphylococcus aureus (MRSA) twice, and collecting logarithmic phase bacterial liquid (1 × 10)7CFU/mL) for use。
According to different dosing ratios of 1:15 (6.25%), 1:16 (5.88%), 1:17 (5.55%), 1:18 (5.26%), 1:19 (5.00%), and 1:20 (4.76%), a culture medium and 50 muL of log-phase methicillin-resistant staphylococcus aureus liquid are added into a sterile test tube according to the test tube dilution method, after shaking, the corresponding volume of the liquid medicine is added into the test tube according to the dosing ratio, and the shaking is carried out, wherein the final volume of the test tube is 5.0 mL.
2. Placing the test tube in an air bath oscillator at 37 ℃ and 210r/min for culturing for 7 h;
3. taking out 50 μ L of plate from each tube, spreading, standing for 10min, and performing inverted culture at 37 deg.C for 24 h;
4. plates were counted and photographed and data was recorded, the results are shown in figure 13.
As can be seen from fig. 13, the Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC) of the compound MW1810 of the traditional Chinese medicine against methicillin-resistant staphylococcus aureus are respectively 1:18 (5.26%) and 1:17 (5.56%). MIC and MBC of the combination against the standard strain Staphylococcus aureus were 1:18 (5.26%) and 1:17 (5.56%), respectively. The results of the test example 3 show that the traditional Chinese medicine compound MW1810 has the same effect on the in vitro bacteriostasis effect on the standard strain and the drug-resistant strain staphylococcus aureus.
Test example 10 paper diffusion method (K-B) drug sensitivity test
1. Respectively activating standard strains of staphylococcus aureus and methicillin-resistant staphylococcus aureus, and taking a logarithmic phase bacterial solution for later use;
2. respectively coating the two bacterial liquids in a solid culture medium, then placing two 6mm paper discs on the surface, wherein one disc is antibiotic oxacillin (10 ug/disc), the other disc is blank paper disc, and dripping 15 mu L of compound stock solution into the blank paper disc;
3. the solid medium plate was left at 37 ℃ overnight, and then the growth inhibition zone was measured, and the results are shown in FIG. 14.
As can be seen from FIG. 14, the SA discs on the left are standard strain Staphylococcus aureus plates, the SA2 on the right are MRSA methicillin-resistant Staphylococcus aureus plates, and one antibiotic oxacillin disc and one compound MW1810 stock solution disc are arranged in each of the two plates. The result clearly shows that although the antibiotic oxacillin has good inhibition effect on the standard strain staphylococcus aureus, the antibiotic oxacillin has no obvious inhibition effect on the drug-resistant staphylococcus aureus basically, and the traditional Chinese medicine compound MW1810 has good inhibition effect on the standard strain staphylococcus aureus and the drug-resistant staphylococcus aureus, thereby effectively solving the problem of drug resistance of bacteria.
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Claims (5)
1. The anti-staphylococcus aureus traditional Chinese medicine composition is characterized by comprising the following components in parts by volume: 10 parts of magnolia officinalis extract, 10 parts of gallnut extract, 20 parts of scutellaria baicalensis extract and 10 parts of benzoin extract; or
20 parts of magnolia officinalis extract, 40 parts of gallnut extract, 10 parts of scutellaria baicalensis extract and 20 parts of benzoin extract; or
40 parts of magnolia officinalis extract, 40 parts of gallnut extract, 20 parts of scutellaria baicalensis extract and 10 parts of benzoin extract; or
40 parts of magnolia officinalis extract, 40 parts of gallnut extract, 10 parts of scutellaria baicalensis extract and 20 parts of benzoin extract;
the magnolia bark extract is prepared by the following method: pulverizing cortex Magnolia officinalis, adding 70% ethanol, ultrasonic dispersing for 25-35min, extracting at 25-30 deg.C for 30-90 days, and filtering to obtain cortex Magnolia officinalis extract with a material-liquid ratio of 1:3-6 g/mL;
the gallnut extract, the scutellaria baicalensis extract and the benzoin extract are prepared by the following methods: pulverizing Galla chinensis, Scutellariae radix and Benzonum respectively, adding 70% ethanol, ultrasonic dispersing for 25-35min, extracting at 25-30 deg.C for 30-90 days, and filtering to obtain Galla chinensis extract, Scutellariae radix extract and Benzonum extract; wherein the feed-liquid ratio is 1:3-6 g/mL.
2. The anti-staphylococcus aureus traditional Chinese medicine composition of claim 1, further comprising pharmaceutically acceptable auxiliary materials.
3. The preparation method of the anti-staphylococcus aureus traditional Chinese medicine composition of any one of claims 1-2, which is characterized by comprising the following steps: mixing cortex Magnolia officinalis extract, Galla chinensis extract, Scutellariae radix extract and Benzonum extract according to formula.
4. The use of the anti-staphylococcus aureus traditional Chinese medicine composition of any one of claims 1-2 in the preparation of an anti-staphylococcus aureus medicine.
5. The application of the staphylococcus aureus resistant traditional Chinese medicine composition in the preparation of a staphylococcus aureus resistant strain medicine according to any one of claims 1-2 is characterized in that the traditional Chinese medicine composition is prepared from the following components in parts by volume: 40 parts of magnolia officinalis extract, 40 parts of Chinese gall extract, 20 parts of scutellaria baicalensis extract and 10 parts of benzoin extract.
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| CN104231875A (en) * | 2013-06-24 | 2014-12-24 | 江苏长松科技发展有限公司 | Antibacterial powder paint |
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