CN108853358A - A kind of antibacterial Chinese medicine composition and its preparation method and application - Google Patents
A kind of antibacterial Chinese medicine composition and its preparation method and application Download PDFInfo
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Abstract
The invention discloses a kind of antibacterial Chinese medicine composition and preparation method thereof and purposes, belong to antibacterials technical field.It includes：6-29 parts of 2-15 parts of 3-30 parts of 5-50 parts of 10-100 parts of Chinese rhubarb extract, dendrobium candidum extract, white balsamine extract, coptis extract, cinnamomum cassia extract based on parts by volume and 2-10 parts of Herba Blumeae Balsamiferae extract.The traditional chinese medicine composition of the invention can effectively kill candida albicans, and sterilizing rate is high, can kill clinical candida albicans, clinical Escherichia coli, clinical staphylococcus aureus and clinical P. aeruginosa.There is stronger synergic remove between the component of the traditional chinese medicine composition of the invention.
Description
Technical field
The present invention relates to antibacterials technical fields, and in particular to a kind of antibacterial Chinese medicine composition and preparation method thereof and uses
On the way.
Background technique
Bacterium/fungal infection is always the difficult medical problem of the puzzled mankind.Due to the abuse of antibiotic, most antibiotic are being answered
All occurs different degrees of reduction with rear drug effect, so that the speed that bacterium/Antifungal resistance occurs is far beyond the imagination.I
State's the traditional Chinese medicine medicinal herbs resource is extensive, and treatment bacterium/fungal infection disease is with toxic side effect is small, is not likely to produce the advantages such as drug resistance.Mesh
Preceding Chinese medicine antibacterial studies oneself through making great progress, with going deep into its bacteriostasis research, a variety of anti-infective Chinese medicines
It is found in succession, provides theoretical foundation for the clinical antibacterial therapy of drug.The diagnosis and treatment based on an overall analysis of the illness and the patient's condition of Chinese medicine and compound compatibility are theoretical,
New Research Thinking is provided for Bacterial resistant infections treatment.Different from the simple antibacterial mechanisms of Western medicine, Chinese medicine compound prescription compatibility is single absolutely not
Taste drug effect is simply added, while " eliminating evil " can " righting ", i.e., can pass through enhancing immunity of organisms while antibacterial, protect
Body tissue organ is protected, kills the anti-infectious purpose of pathogen to reach.
Candida albicans is widely present in nature, exists in normal human mouth, the upper respiratory tract, enteron aisle and vagina, is one
Kind opportunistic fungus.It can cause skin and mucosa shallow-layer or even whole body system fungal infection when immunity of organisms decline.Closely
Since 30 years, with the excessive use of broad-spectrum antibiotic, the extensive use of the treatment methods such as chemotherapy of tumors, organ transplant and exempt from
The increase of epidemic disease systemic disease patient, clinically there is the trend to grow in intensity in the infection as caused by fungi.Moreover, because clinical
Upper antifungal drug, especially antibacterial medicines are widely used, and Antifungal resistance is continuously increased and is on the rise, and this resistance to
Multidrug resistance usually occurs for the generation of pharmacological property, causes great difficulty, even treatment failure to the treatment of fungus Infection.
Mycotoruloides is clinically one of most common opportunistic fungus, and with candida albicans is most common in Mycotoruloides.
Staphylococcus aureus (Staphylococcus aureus) is also referred to as " S. aureus L-forms " a kind of important disease of the mankind
Opportunistic pathogen is under the jurisdiction of staphylococcus (Staphylococcus).Staphylococcus aureus is ubiquitous in nature, air,
It can all be found in the excreta of water, dust and humans and animals, be the most common pathogen in mankind's suppurative infection, office can be caused
Portion's suppurative infection can also cause pneumonia, pseudomembranous enteritis, pericarditis etc. or even the general infections such as septicemia, pyemia.In addition,
Staphylococcus aureus enterotoxin is that a worldwide health problem is eaten as caused by Staphylococcus aureus enterotoxin in the U.S.
Object poisoning, accounts for the 33% of entire food posioning, Canadian then more, accounts for 45%, Chinese staphylococcus aureus is drawn
The food poisoning risen also happens occasionally.
Escherichia coli (Escherichia coli), i.e. escherichia coli, are usually seen as opportunist, this be because
For under normal circumstances it be to colonize in a kind of prokaryotic micro-organisms harmless in human body large intestine and small intestine.In immunity of organism
In special circumstances, they can migrate the place other than enteron aisle for power reduction, the stimulation of enteron aisle long-term lacking, wound etc., such as gall-bladder,
The ground such as urethra, bladder, appendix cause the infection or whole body disseminated infections of corresponding site.
Pseudomonas aeruginosa (P.Aeruginosa), i.e. Pseudomonas aeruginosa, and a kind of common conditioned pathogen, are present in
The infection of the bacterium, which is common in, in the skin of normal person, respiratory tract and enteron aisle, in hospital suffers from metabolic disease, blood disease and pernicious swollen
In patient after the patient of tumor and postoperative or certain treatments.
To sum up, the treatment method majority of patient's encountered pathogenic bacteria infection is based on antibiotic usage, in view of the wide of antibiotic
It is general to use, even abuse, no matter bacterium or fungal infection, the drug resistance problems of pathogen are quite serious.Therefore, developmental research
It is high-efficiency low-toxicity, low or have no drug resistance while the antibacterials that use of hypoimmunity crowd being suitble to become compeling for medical personal
Cut task.Good safety, high-efficiency low-toxicity and the antibacterial Chinese medicine for being not likely to produce drug resistance are screened and developed from Chinese herbal medicine, effectively
Ground prevents and treats pathogenic bacterial infection, improves people's lives quality, has caused the concern and again of more and more medical scientific research workers
Depending on.
Summary of the invention
The purpose of the present invention is to provide a kind of antibacterial Chinese medicine composition and preparation method thereof and purposes, are provided in particular in
A kind of Chinese medicine composition and antimicrobial agent of anti-conditioned pathogen.Antibacterial Chinese medicine composition of the invention and antimicrobial agent sterilization
It is high-efficient, safety is good, is not likely to produce drug resistance, sterilization can be played the role of to a variety of conditioned pathogens simultaneously, had wide
Wealthy application prospect.
The technical solution that the present invention solves above-mentioned technical problem is as follows：
A kind of antibacterial Chinese medicine composition, including：10-100 parts of Chinese rhubarb extract, dendrobium candidum extract based on parts by volume
5-50 parts, 3-30 parts of white balsamine extract, 2-15 parts of coptis extract, 6-29 parts of cinnamomum cassia extract and Herba Blumeae Balsamiferae extract 2-
10 parts.
Rheum officinale：Hardship is trembled with fear.Can heat and toxic materials clearing away, clearing heat-fire, removing dampness through diuresis and removing jaundice, removing pattogenic heat from the blood and toxic material from the body, activating blood circulation method it is tired, by trace promoting menstruation.According to
Document report in recent years, effective component are mainly rheum emodin, Rhein and rheum officinale meal with wine etc., and rheum officinale has immunological regulation, removes
Oxygen radical bacteria-inhibiting anti-inflammation, leads to diarrhea, Hepatoprotective cholagogue, hemostasis, diuresis, adjusts also a variety of drug actions such as vascular system.
Dendrobium candidum：Have antibacterial, it is clearing heat and detoxicating, be beneficial to reinforce body immunity, have reinforcing stomach reg fluid, extend service life etc.
Effect.According to the literature, complex chemical composition in dendrobium nobile, polysaccharide rich in, phenolic constituent, sesquiterpenoids, alkaloid,
The compounds such as volatile oil, sterols.Its bioactivity also has diversity, mainly has：Immunization, antitumor, anti-oxidant, drop
The effects of blood glucose, anti-cataract, anti-aging.
White balsamine：Sweet, warm.Can blood circulation, wind-expelling pain-stopping, external application removing toxic substances.For amenorrhoea, traumatic injury, hemostasis
Swelling and pain, rheumatic arthritis, carbuncle boils boils, snake bite, the tinea manuum, tinea pedis.It acts on tinea bacterium：The flowers juice of balsamine, to red
Color table dermatophytes, trichophyton and epidermophyton inguinale, the special graceful golf Epidermophyton of husband have inhibiting effect.It is right simultaneously
Bacterium also inhibiting effect：This product decoction is to staphylococcus aureus, hemolytic streptococcus, Pseudomonas aeruginosa, typhoid bacillus and dysentery
Also there is different inhibiting effect.
The coptis：Hardship is trembled with fear.Have effects that heat-clearing and damp-drying drug, purging fire for removing toxin.It is main to contain Protoberberine Alkoloids, contain barberry
Blue alkali of alkali, Palmatine, coptisine, worenine, jateorrhizine, wood etc..The coptis is the traditional heat-clearing and detoxifying herb in China.It is modern
Medicine thinks that the coptis is broad spectrum antibiotic.
Cortex cinnamomi：It is pungent, sweet, big heat.According to《Compendium of Materia Medica》It records, " hundred medicines are preferably led in hard muscles and bones, promoting blood circulation ".With middle benefit gas
Kidney tonifying, eliminating cold to stop pain function play the role of antibacterial, calmness, decompression, prevention snail fever.To staphylococcus aureus, typhoid fever bar
Bacterium and Bacillus tuberculosis have significant antibacterial action.
Blumea balsamifera：Pungent, bitter, temperature has effects that timid wind dehumidifying, middle benefit gas stop gulf, promoting blood circulation and detoxication, is to extract natural borneol
Main source.Blumea balsamifera for a long time always for treat flu, rheumatic arthritis, puerperal exposure to wind pain, dysmenorrhea, traumatic injury,
Boil pain swollen, eczema, dermatitis etc..
The present invention with rheum officinale and dendrobium candidum for main antibacterials, in the association of white balsamine, the coptis, cortex cinnamomi and Blumea balsamifera
The more significant antifungal effect of curative effect is obtained under same-action.Contain a large amount of rheum emodin, aloe-emodin, big in rheum officinale
Effective antimicrobial component such as yellow acid, these ingredients can effectively inhibit pathogen growth, and lot of experimental data shows rheum officinale dialogue
Candida albicans, Escherichia coli, staphylococcus aureus and pseudomonas aeruginosa show superpower antibacterial effect, and white to drug resistance
Candida albicans also shows bactericidal bacteriostasis.Dendrobium candidum is rare Chinese medicine, in addition to have beneficial stomach raw leaching, nourishing Yin and clearing heat, resist it is tired
Other than labor, antitumor and other effects, the present invention has found that it also has stronger antibacterial action to candida albicans by many experiments.By
This, the present invention is selected using rheum officinale and dendrobium candidum as main antimicrobial component, and compatibility white balsamine, the coptis, cortex cinnamomi and Chinese mugwort
Fragrant wait of receiving has the Chinese medicine of anti-inflammatory, analgesia, antibacterial, guidance and other effects as auxiliary, cooperates with and plays excellent antibacterial effect,
A variety of germs are shown with strong bactericidal effect, and stronger bactericidal effect can also be generated to drug-fast bacteria.
Further, in preferred embodiments of the present invention, antibacterial Chinese medicine composition includes：Rheum officinale based on parts by volume mentions
10-80 parts of object, 5-45 parts of dendrobium candidum extract, 5-15 parts of white balsamine extract, 2-13 parts of coptis extract, cortex cinnamomi is taken to mention
Take 6-27 parts and Herba Blumeae Balsamiferae extract 2-6 parts of object.
Preferably, based on parts by volume, Chinese rhubarb extract is 10 parts, 20 parts, 30 parts, 35 parts, 40 parts, 45 parts, 50 parts, 55
Part, 60 parts, 70 parts, 80 parts, 90 parts or 100 parts.
Preferably, based on parts by volume, dendrobium candidum extract is 5 parts, 7 parts, 9 parts, 10 parts, 12 parts, 14 parts, 16 parts, 18
Part, 19 parts, 20 parts, 23 parts, 27 parts, 29 parts, 30 parts, 40 parts or 50 parts.
Preferably, based on parts by volume, Bai Fengxian extract flower for 5 parts, 6 parts, 7 parts, 8 parts, 9 parts, 10 parts, 11 parts, 12 parts,
13 parts or 15 parts.
Preferably, based on parts by volume, coptis extract is 2 parts, 3 parts, 4 parts, 5 parts, 6 parts, 7 parts, 8 parts, 9 parts, 10 parts, 11
Part, 13 parts, 14 parts or 15 parts.
Preferably, based on parts by volume, cinnamomum cassia extract is 6 parts, 8 parts, 9 parts, 10 parts, 12 parts, 14 parts, 15 parts, 17 parts, 19
Part, 20 parts, 21 parts, 23 parts, 25 parts, 26 parts, 27 parts, 28 parts or 29 parts.
Preferably, based on parts by volume, Herba Blumeae Balsamiferae extract is 2 parts, 3 parts, 4 parts, 5 parts, 6 parts, 8 parts or 10 parts.
Further, in preferred embodiments of the present invention, antibacterial Chinese medicine composition includes：Rheum officinale based on parts by volume mentions
Take 48 parts of object, 42 parts of dendrobium candidum extract, 12 parts of white balsamine extract, 6 parts of coptis extract, 6 parts of cinnamomum cassia extract and
6 parts of Herba Blumeae Balsamiferae extract.
Further, in preferred embodiments of the present invention, antibacterial Chinese medicine composition further includes pharmaceutically acceptable auxiliary
Material.
Above-mentioned auxiliary material includes but is not limited to solvent, propellant, solubilizer, cosolvent, emulsifier, colorant, binder, collapses
Solve agent, filler, lubricant, wetting agent, osmotic pressure regulator, stabilizer, glidant, corrigent, preservative, suspending agent, packet
Clothing material, aromatic, anti-binder, integrated agent, penetration enhancer, pH adjusting agent, buffer, plasticizer, surfactant,
Foaming agent, defoaming agent, thickener, inclusion agents, moisturizer, absorbent, diluent, flocculant and deflocculant, filter aid, release
Retarding agent etc..
Further, in preferred embodiments of the present invention, above-mentioned auxiliary material is solvent.Preferably, solvent is dehydrated alcohol
Or ethanol solution.
Preferably, the concentration of ethanol solution is 50%-95% (v/v), more preferably 60%-80% (v/v), such as 60%
(v/v), 65% (v/v), 70% (v/v), 72% (v/v), 75% (v/v), 77% (v/v) or 80% (v/v).
Preferably, the water in ethanol water is selected from least one of deionized water, pure water and distilled water.
Further, in preferred embodiments of the present invention, above-mentioned antibacterial Chinese medicine composition is vina, tablet, tincture, puts on the skin
Agent, pastille or pill.
Further, in preferred embodiments of the present invention, above-mentioned antibacterial Chinese medicine composition is sterilization (antibacterial) preparation.
Further, in preferred embodiments of the present invention, above-mentioned antibacterial Chinese medicine composition is external preparation, preferably skin
Skin external preparation, more preferably external preparation for skin sterilize (antibacterial) preparation.
Antibacterial Chinese medicine composition obtained by the present invention is external drug, in use, medicament is applied at infection.It is answering
Used time takes Chinese medicine composition of the invention to add auxiliary material (preferably solvent), uses after diluting 10-50 times.
The preparation method of above-mentioned antibacterial Chinese medicine composition, including：Chinese rhubarb extract, dendrobium candidum are mentioned according to said ratio
Take object, white balsamine extract, coptis extract, cinnamomum cassia extract and Herba Blumeae Balsamiferae extract mixing.
Further, in preferred embodiments of the present invention, above-mentioned Chinese rhubarb extract, dendrobium candidum extract, Bai Fengxian
Flower extract, coptis extract, cinnamomum cassia extract and Herba Blumeae Balsamiferae extract are made in accordance with the following methods：
Above-mentioned rheum officinale, dendrobium candidum, white balsamine, the coptis, cortex cinnamomi and Blumea balsamifera are broken into of 0.1-5mm respectively
Then grain, is 1 according to solid-liquid ratio respectively at ultrasonic vibration 15-30min in solvent：The ratio of (1-32) g/ml exists Chinese medicine
It is extracted 30-90 days in alcohol at 25-40 DEG C, filters, respectively obtain Chinese rhubarb extract, dendrobium candidum extract, white balsamine
Extract, coptis extract, cinnamomum cassia extract and Herba Blumeae Balsamiferae extract.
Preferably, the partial size of the particle after above-mentioned traditional Chinese medicine powder is broken be 0.1mm, 0.2mm, 0.3mm, 0.4mm, 0.5mm,
0.6mm, 0.7mm, 0.8mm, 0.9mm, 1mm, 2mm, 3mm or 4mm.
Preferably, the ultrasonic vibration time is 20min.The solvent used when ultrasonic vibration is preferably 75% alcoholic solution.
Preferably, solid-liquid ratio is (1:1)-(1:16) (g/ml), such as 1:1(g/ml),1:2(g/ml),1:3(g/ml),1:4(g/
ml),1:5(g/ml),1:6(g/ml),1:7(g/ml),1:8(g/ml),1:9(g/ml),1:10(g/ml)；1:11(g/ml),
1:12(g/ml),1:13(g/ml)1:14(g/ml),1:15 (g/ml) or 1:16(g/ml).It can also be 1:32(g/ml).
Preferably, extraction temperature is 25 DEG C, 30 DEG C, 27 DEG C, 35 DEG C or 40 DEG C.
Preferably, extraction time is 30-60 days, it is highly preferred that 30 days, 35 days, 40 days, 45 days, 50 days, 55 days or 60
It.
Above-mentioned antibacterial Chinese medicine composition is preparing antifungal drug, anti-Escherichia coli drug, anti-Staphylococcus aureus
Answering in bacterium drug, resisting pseudomonas aeruginosa drug, overriding resistance candida albicans drug or overriding resistance staphylococcus aureus drug
With.
The invention has the advantages that：
The traditional chinese medicine composition of the invention can effectively kill candida albicans, and sterilizing rate is high, can kill clinical candida albicans, face
Bed bacterium (clinical Escherichia coli, clinical staphylococcus aureus and clinical P. aeruginosa).The traditional chinese medicine composition of the invention
There is stronger synergic remove between component.
Detailed description of the invention
Fig. 1 is the compound Chinese medicinal preparation MZ1711B and control group of the embodiment of the present invention 5 under different time to candida albicans
Restraining and sterilizing bacteria result figure；
Fig. 2 is the compound Chinese medicinal preparation MZ1711B and control group of the embodiment of the present invention 5 under various concentration to candida albicans
Restraining and sterilizing bacteria result figure；
The compound Chinese medicinal preparation MZ1711B and control group that Fig. 3 is the embodiment of the present invention 5 are to candida albicans treated conductance
Rate variation diagram；
The compound Chinese medicinal preparation MZ1711B and control group that Fig. 4 is the embodiment of the present invention 5 are to candida albicans treated bacterium solution
The variation diagram of AKP amount；
Fig. 5 is after the compound Chinese medicinal preparation MZ1711B, control group and blank group of the embodiment of the present invention 5 handle candida albicans
The morphosis figure of candida albicans is observed under scanning electron microscope；
Fig. 6 is the compound Chinese medicinal preparation MZ1711B of the embodiment of the present invention 5 under various concentration to the antibacterial of clinical candida albicans
Sterilize result figure；
Fig. 7 (a) is the compound Chinese medicinal preparation MZ1711B of the embodiment of the present invention 5 under various concentration to clinical Escherichia coli
Restraining and sterilizing bacteria result figure；
Fig. 7 (b) is the compound Chinese medicinal preparation MZ1711B of the embodiment of the present invention 5 under various concentration to clinical golden yellow grape
The restraining and sterilizing bacteria result figure of ball group bacterium；
Fig. 7 (c) is the compound Chinese medicinal preparation MZ1711B of the embodiment of the present invention 5 under various concentration to clinical P vacation unit cell
The restraining and sterilizing bacteria result figure of bacterium；
Fig. 8 is the compound Chinese medicinal preparation MZ1711B of the embodiment of the present invention 5 under various concentration to being clinically separated the white beads of drug resistance
The restraining and sterilizing bacteria result figure of bacterium；
Fig. 9 is compound Chinese medicinal preparation MZ1711B, 5-flurocytosine positive controls and the blank group of the embodiment of the present invention 5
Mycelia state diagram after handling candida albicans；
Figure 10 is compound Chinese medicinal preparation MZ1711B, 5-flurocytosine positive controls and the blank group of the embodiment of the present invention 5
Phospholipase activity situation compares figure after processing candida albicans.
Specific embodiment
Principles and features of the present invention are described with reference to embodiments, the given examples are served only to explain the present invention,
It is not intended to limit the scope of the present invention.The person that is not specified actual conditions in embodiment, according to normal conditions or manufacturer suggests
Condition carries out.Reagents or instruments used without specified manufacturer is the conventional products that can be obtained by commercially available purchase.
Rheum officinale, dendrobium candidum, the coptis, cortex cinnamomi, Blumea balsamifera and the white balsamine that the following example uses are purchased from Chengdu
Chinese Medicinal Materials Markets, and identified through professional teacher.Candida albicans, clinical candida albicans, clinical Escherichia coli, clinical golden yellow grape
Coccus, clinical P. aeruginosa, clinical resistance to 5-flurocytosine candida albicans and clinical methicillin-resistant staphylococcus aureus
(MRSA) Microbiological Lab of Life Sciences of Southwest Jiaotong University is derived from, wherein clinical drug-resistant candida albicans is in Sharpe/LB
It is spare after at least being activated on culture medium twice.
Embodiment 1：
The antibacterial Chinese medicine composition of the present embodiment includes：10 parts of Chinese rhubarb extract, 5 parts of dendrobium candidum extract, Bai Fengxian
3 parts of flower extract, 2 parts of coptis extract, 6 parts of cinnamomum cassia extract and 2 parts of Herba Blumeae Balsamiferae extract.
The antibacterial Chinese medicine preparation method of composition of the present embodiment is：The Chinese medicine of said ratio is broken into 0.2-0.5mm's
Particle, respectively then ultrasonic vibration 20min in 80% (v/v) ethanol solution of 400mL is 1 according to solid-liquid ratio：The ratio of 4g/ml
Example extracts 30 days at 25 DEG C, filters, and mixes according to said ratio and obtains Chinese medicine composition, is added to the Chinese medicine composition auxiliary
Expect that antimicrobial agent is made.
Embodiment 2：
The antibacterial Chinese medicine composition of the present embodiment includes：100 parts of Chinese rhubarb extract, 50 parts of dendrobium candidum extract, Bai Feng
30 parts of celestial flower extract, 15 parts of coptis extract, 29 parts of cinnamomum cassia extract and 10 parts of Herba Blumeae Balsamiferae extract.
Preparation method is same as Example 1.
Embodiment 3：
The antibacterial Chinese medicine composition of the present embodiment includes：10 parts of Chinese rhubarb extract, 5 parts of dendrobium candidum extract, Bai Fengxian
5 parts of flower extract, 2 parts of coptis extract, 6 parts of cinnamomum cassia extract and 27 parts of Herba Blumeae Balsamiferae extract.
Preparation method is same as Example 1.
Embodiment 4：
The antibacterial Chinese medicine composition of the present embodiment includes：80 parts of Chinese rhubarb extract, 45 parts of dendrobium candidum extract, Bai Fengxian
15 parts of flower extract, 13 parts of coptis extract, 27 parts of cinnamomum cassia extract and 6 parts of Herba Blumeae Balsamiferae extract.
Preparation method is same as Example 1.
Embodiment 5：
The antibacterial Chinese medicine composition of the present embodiment includes：48 parts of Chinese rhubarb extract, dendrobium candidum based on parts by volume extract
42 parts of object, 12 parts of white balsamine extract, 6 parts of coptis extract, 6 parts of cinnamomum cassia extract and 6 parts of Herba Blumeae Balsamiferae extract.
Preparation method is same as Example 1.
The research of 1 bacteriostasis property of test example
With reference to GB 15979-2002《The design of Disposable Sanitary Accessory standard》Middle stripping property resists (suppression) bacterium product testing
Method designs specific experiment method：
1. choosing monoclonal to be incubated overnight
2. being diluted to respectively with sabouraud culture medium：10-1, 10-2, 10-3, 10-4, 10-5, 10-6It is spare.
3. taking be diluted to 10- respectively5, 10-6Bacterium solution 50ul carry out bed board counting, to estimate original bacteria liquid concentration.
4. experimental group：
4 sterilized 1.5ml EP pipes are taken, the medical fluid and 10 μ l for being separately added into 500 μ l embodiments 5 are diluted to 10-2
Bacterium solution, start timing, 2,5,10, after 20min, be separately added into 500 μ lPBS, mix well, 50 μ L mixed liquors is respectively taken to be applied
Plate, 30 DEG C of inversions cultivate 72h, carry out bacterium colony counting
Control group：
4 sterilized 1.5ml EP pipes are taken, 500 μ l sterile salines is separately added into and 10 μ l is diluted to 10-2's
Bacterium solution, starts timing, 2,5,10, after 20min, be separately added into 500 μ l 0PBS, mix well, 50 μ l mixed liquors is respectively taken to be applied
Plate, 30 DEG C of inversions cultivate 72h, carry out bacterium colony counting.
5. bacteriostasis rate is calculated as follows：
X=(a-b)/a × 100%
In formula：X- bacteriostasis rate, %；A- control sample average colony number；B- is tested sample average clump count.
6. 3 groups of experiment progress is parallel；Bacteriostasis rate >=50%-90%, product have bacteriostasis；Bacteriostasis rate >=90%, product
With stronger bacteriostasis.The result is shown in Figure 1 and table 1.
Table 1
By Fig. 1, table 1 as it can be seen that MZ1711B experimental group 2,5,10,20min do not have the generation of bacterium colony, and control group then has
Grow normal bacterium colony.Since bacterium colony can not be counted normally too much, the calculating of bacteriostasis rate, 2min are carried out to each group respectively
Completely inhibit, and 5-20min shows 100% fungistatic effect.Thus illustrate the Chinese medicine compound prescription system of the embodiment of the present invention
Agent MZ1711B has strong fungistatic effect, can be used as a kind of medicament of antifungal and uses.
Test example 2 MIC and MBC measurement
The sterile test tube for first taking 13 brace lids, with Sharpe fluid nutrient medium according to 1.875%, 3.75%, 5.625%,
7.5%, 9.375%, 11.25% and 2.5%, 5%, 7.5%, 10%, 12.5%, 15% different percentage concentration gradients difference
Prepare 5mL ethanol control group and MZ1711B experimental group each 6.Simultaneously using equivalent sterile distilled water as blank control group.Often
Pipe accesses 50ul candida albicans suspension, and vortex mixed is uniform, is incubated at 30 DEG C, 12h in the air bath oscillator of 180rpm, meat
Eye observation, takes pictures and records.
50 μ l are sucked out in the every pipe of bacteria suspension in above-mentioned each test tube and carry out culture dish coating, and are incubated at 30 DEG C of constant temperature training
In feeding case after for 24 hours~48h, the single colonie number grown on culture dish is observed and recorded.To there is the least maximum of single colonie number
Concentration is the minimal inhibitory concentration (MIC) of compound, and the Cmin grown with no single colonie is its minimum bactericidal concentration (MBC).
As a result see Fig. 2.
As shown in Figure 2, minimal inhibitory concentration (MIC) and minimum sterilization of the compound Chinese medicinal preparation MZ1711B to candida albicans
Concentration (MBC) is respectively 10% and 12.5%, wherein in excluding compound after the interference of middle alcohol bacteriostasis, equal conditions
The fungistatic effect of lower compound Chinese medicinal preparation MZ11711B is more obvious compared with alcohol.
3 conductance measurement of test example
Respectively by the compound of 1 times and 2 times of MBC concentration add to experiment bacterium solution in, control take respectively 1 times it is dense with 2 times 9.375%
The ethyl alcohol of degree is added in bacteria suspension, cultivates in 30 DEG C, 180rpm shaking table, the conductivity of culture solution is measured by sampling every 1h, really
The variation tendency of complexing metal ion exudation.As a result see Fig. 3.
From the figure 3, it may be seen that candida albicans is after MZ1711B experimental group compares with alcohol processing simultaneously, the conductivity of culture solution
The significantly raised trend of value；Wherein, 2 times 9.375% and 1 times 9.375% of control group processing after culture solution conductivity value highest
It respectively reaches(mean value), (mean value), and after 1MBC MZ1711B processing,
Conductivity value highest then reachesTreated that conductivity value is more up to by 2MBC MZ1711BIt is apparently higher than alcohol control group.And integrally moved towards from line chart trend analysis can be seen that with
The conductivity of the continuity of action time, bacterium solution is also increase accordingly, and the degree of leaking of thallus electrolyte significantly improves.
Be summarized as, compound Chinese medicinal preparation MZ1711B act on after in somatic cells environment and cell membrane stability by broken
Bad, the growth of candida albicans is suppressed, and inhibiting effect is apparently higher than alcohol control group.The compound Chinese medicinal preparation of 2MBC
MZ1711B is obvious to the function and effect of thallus, illustrates compound Chinese medicinal preparation MZ1711B concentration and its effect effect to somatic cells
Fruit is positively correlated.
Test example 4 bacterium solution alkaline phosphatase (AKP) determination of activity
The compound Chinese medicinal preparation MZ1711B of 1 times and 2 times of MBC concentration is added in bacteria suspension respectively, 30 DEG C, 180rpm
Shaking table culture, per timing sampling every other hour, 4000rpm is centrifuged 8min, and supernatant kit method is taken to measure alkaline phosphatase
The content of enzyme (AKP) detects the situation of change in different disposal time point bacterium solution AKP amount.As a result see Fig. 4.
As shown in Figure 4, after MZ1711B is handled, as action time lengthens, the content of the AKP outside somatic cells starts
Increase, and through 2MBC MZ1711B treated the 2nd hour, the seepage discharge of AKP reaches maximum value, isThough its amount is slightly reduced later, but still much higher than through 2MBC alcohol
(18.75%) control group handled.And each time point through 1MBC Compound B after effect, though its effect to candida albicans
Effect is weaker with respect to 2MBC MZ1711B, but its effect is much better than 1MBC alcohol (9.375%) control group, wherein after 1MBC processing
Phase AKP detected value peaks in the 4th hour, is
Passing through the comparison of different time seepage discharge, the results showed that compound MZ1711B enhances the permeability of somatic cells wall,
The integrality of somatic cells is destroyed, to inhibit the normal growth of candida albicans.
The observation of 5 phage surface Electronic Speculum of test example
(1) it handles and collects thallus：The candida albicans of logarithmic growth phase in centrifuge tube A, B, C of three 1.5mL, with
After 8000rpm is centrifuged 3-5min, supernatant is discarded, is washed 2 to 3 times using sterile phosphate buffer PBS, and be diluted to bacterium colony
Sum 105-106The bacteria suspension of cfu/mL is separately added into the compound Chinese medicinal preparation MZ1711B, 9.375% concentration of 1 times of MBC concentration
Alcohol, sterile distilled water in A, B, C, 30 DEG C, 180rpm gas bath oscillator culture 12h.
(2) thallus sample preparation：After above-mentioned bacterium solution is centrifuged 3-5min with 8000rpm, supernatant is discarded, after obtaining thallus
It performs the following operation respectively:1. above-mentioned thallus is added in 2.5% glutaraldehyde fixer fixed overnight in 4 DEG C of refrigerators；2. phosphorus
Acid buffer PBS clear Xian 2-3 times；3. Gradient elution using ethanol is followed successively by 30%, 50%, 70%, 85%, 95% each 1 (5-
10min/ times), 100% ethyl alcohol 2 times (15-20min/ times)；(wherein：Every step is both needed to discard after 3-5min centrifugation with 8000rpm
Supernatant, then it is poured into a kind of lower reagent, blown and beaten 3-5 times with liquid-transfering gun, break up fungus block).
(3) it spontaneously dries：Bacterium solution in previous step is drawn on silicon wafer with capillary, spontaneously dries 30min.
(4) ion sputtering metal spraying：After ion sputtering metal spraying.The form knot of Electronic Speculum observation candida albicans can be scanned
Structure.As a result see Fig. 5
As shown in Figure 5, after 1MBC compound Chinese medicinal preparation MZ1711B is handled 12 hours, thallus is severely deformed, phage surface
Start to become extremely coarse, cell wall brutal fracture, cytoplasm leaks exudation from thallus, and thalli morphology is seriously broken, can
See that the compound Chinese medicinal preparation MZ1711B of 1MBC has obviously destruction to candida albicans.And through 1 times of 9.375% wine
After precision processing 12 hours, thallus is slightly deformed, and cell wall starts to collapse, and only fewer parts cytoplasm leaks exudation from thallus；
It can be seen that the compound Chinese medicinal preparation MZ1711B of 1MBC is more significant compared with 1 times of 9.375% alcohol to the destruction of candida albicans.
Test example 6 measures compound Chinese medicinal preparation MZ1711B and sterilizes to minimal inhibitory concentration (MIC), the minimum of clinical strains
Concentration (MFC)
First take the sterile test tube with lid several, with Sharpe/LB liquid medium successively according to 2.5%, 5%,
7.5%, 10%, 12.5%, 15% different weight percentage concentration gradient prepares 5mL compound Chinese medicinal preparation MZ1711B experiment respectively
Totally 16 groups of group, each test tube is respectively connected to clinical candida albicans (10 kinds), the clinical Escherichia coli (2 that 50 μ L have been prepared
Kind), clinical staphylococcus aureus (3 kinds) and a kind of clinical P. aeruginosa suspension, vortex mixed is uniform, be incubated at 30 DEG C/
37 DEG C, after being incubated for 10-12h in the air bath oscillator of 180rpm/200rpm, by above-mentioned each invisible spectro every pipe of bacteria suspension
50 μ L are sucked out to carry out culture dish coating, and are incubated at 30 DEG C/37 DEG C, constant incubator in for 24 hours after -48h, observe and record
The single colonie number grown on culture dish.Criterion is to there is the least maximum concentration of single colonie number as compound Chinese medicinal preparation
The minimal inhibitory concentration (MIC) of MZ1711B, the maximum concentration grown with no single colonie is its minimum bactericidal concentration (MFC/MBC).
It the results are shown in Table 2 and Fig. 6, Fig. 7 (a)-(c).
Table 2
By table 2 and Fig. 6 it is found that the minimum for the candida albicans that compound Chinese medicinal preparation MZ1711B is clinically separated 10 plants is antibacterial
Respectively between 5%-10% and 7.5%-12.5%, fungistatic effect is good for concentration (MIC) and minimum bactericidal concentration (MFC).
By table 2 and Fig. 7 (a) it is found that the minimal inhibitory concentration (MIC) and minimum of the Escherichia coli being clinically separated to 2 plants are killed
Bacteria concentration (MBC) is then respectively 10% and 12.5%, and fungistatic effect is good.
By table 2 and Fig. 7 (b) it is found that the MIC and MBC of the staphylococcus aureus being clinically separated to 3 plants are then respectively
5% and 7.5% (wherein, 2. number being clinically separated staphylococcus aureus is methicillin-resistant staphylococcus aureus (MRSA)),
There is very strong inhibiting effect to the sensitivity and resistant Staphylococcus aureus that are clinically separated under low concentration.
By table 2 and Fig. 7 (c) it is found that being then 5% He respectively to the MIC and MBC of the pseudomonas aeruginosa being clinically separated
7.5%, equally there is extraordinary fungistatic effect.
Test example 7 measures compound Chinese medicinal preparation MZ1711B and presses down to the minimum of resistance to 5-flurocytosine candida albicans is clinically separated
Bacteria concentration (MIC), minimum bactericidal concentration (MBC/MFC) measurement
By the antibody-resistant bacterium being clinically separated on sabouraud's agar oneself at least passed on twice, first take 8 brace lids
Sterile test tube, with Sharpe fluid nutrient medium according to 2.5%, 5%, 7.5%, 10%, 12.5%, 15%, 17.5%, 20%,
Different percentage adding consistencies prepare.Every pipe accesses 50uL candida albicans suspension, and vortex mixed is uniform, is incubated at 30 DEG C,
12h in the air bath oscillator of 180rpm.50 μ l are sucked out in the every pipe of bacteria suspension in above-mentioned each test tube and carry out culture dish coating, and
It is incubated at the single colonie number for after 48h, observing and recording in 30 DEG C of constant incubator and growing on culture dish.To there is single colonie
The least maximum concentration of number is the minimal inhibitory concentration (MIC) of compound, and the Cmin grown with no single colonie is that its minimum is killed
Bacteria concentration (MFC).Testing result is as shown in Figure 8.
From figure 8, it is seen that Chinese medicine composition MZ1711B made from the embodiment of the present invention 5 is to being clinically separated the white thought of drug resistance
The minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MFC) of pearl bacterium are respectively 10% and 12.5%, and fungistatic effect is obvious.
8 MZ1711B of test example acts on the Mycelial growth for being clinically separated resistance to 5-flurocytosine candida albicans
1,5-flurocytosine mother liquor：Claim 5 Flucytosines in right amount in volumetric flask with balance precision, is added and adds appropriate nothing
Bacterium water is configured to 5 Flucytosines that concentration is 2560 μ g/mL, the mother liquid medicine that will be prepared after completely dissolution, 0.2 μm of membrane filtration
After being attached separately to the EP pipe to sterilize in advance, it is put into refrigerator and saves, it is spare.
2, by the antibody-resistant bacterium being clinically separated, oneself was at least passed on twice, using sterile on sabouraud's agar
Drug-resistant candida albicans bacterium solution is made into 1 × 10 by RPMI1640 (containing 20%FBS) fluid nutrient medium6CFU/mL is grouped as following to 6
Drug and above-mentioned bacterium solution are added in the different holes of orifice plate：
No. 1 hole：4000 μ L bacterium solutions；
No. 2 holes：3998.4+1.6 μ L of μ L bacterium solution, 5 Flucytosine (final concentration of 1.024 μ g/mL)；
No. 3 holes：+ 200 μ L MZ1711B (final concentration of 5%) of 3800 μ L bacterium solution
Sterile 6 orifice plates are put to 37 DEG C of constant incubator) in after constant temperature incubation 6h, washed away on coverslip not with sterile PBS
The drug-resistant candida albicans cell of adherency observes each group hole mycelia upgrowth situation under putting to microscope (10 × 10,20 × 10).Experiment
As a result see Fig. 9.
As shown in Figure 9：White thought in blank control group and 5-flurocytosine positive controls (final concentration of 1.024 μ g/mL)
Pearl bacterium cell is in apparent mycelia state growth, and the candida albicans cell in the MZ1711B experimental group of the embodiment of the present invention 5 is then
It is grown completely in yeast state.And mycelia state is the important symbol of candida albicans virulence, and to show that mycelia is grown to white for lot of documents
Candida albicans forms an important ring for biomembrane, related with the drug resistance of candida albicans, therefore, from this result it is found that 5% dosing
Under concentration, conversion of the bacterial strain to mycelia state is can be effectively suppressed in MZ1711B, reduces its virulence and drug resistance.
Influence of 10 MZ1711B of test example to the activity of phospholipase for being clinically separated drug-resistant candida albicans
1, the preparation of egg york medium：Weigh sodium chloride powder 1g, powdered beef 0.6g, peptone powder respectively with balance
3g, agar powder 3g, glucose powder 2g is into conical flask, sterile purified water 200mL, and it is close to adjust solution PH with pH test paper
7.5, basal medium is made in high pressure sterilization after glass bar stirring is substantially dissolved in all powder in water.Basis after sterilizing
50% yolk NaCl suspension (the 40mL suspension 20% is added in every 200mL basal medium) is added in culture medium when putting to 40-55 DEG C,
After glass bar stirs evenly, being poured into the glass culture dish to sterilize in advance while hot is egg yolk agar culture medium, is put into 4 DEG C of refrigerators, standby
With.
2, oneself was at least passed on twice the antibody-resistant bacterium being clinically separated on sabouraud's agar, used sterile Sharpe
Drug-resistant candida albicans bacterium solution is made into 1 × 105CFU/mL by fluid nutrient medium, by the following different Kong Zhongjia being grouped to 6 orifice plates
Enter drug and above-mentioned bacterium solution：
No. 1 hole：4000 μ L bacterium solutions；
No. 2 holes：3998.4+1.6 μ L of μ L bacterium solution, 5 Flucytosine (final concentration of 1.024 μ g/mL)；
No. 3 holes：+ 200 μ L MZ1711B (final concentration of 5%) of drug of 3800 μ L bacterium solution
After sterile 6 orifice plates are put into isothermal vibration incubator (75rpm, 35 DEG C) constant temperature incubation overnight, taken from each system
10 μ L liquid are vertically added dropwise on manufactured egg york medium, and scape is presented it round to the greatest extent, and egg york medium is placed in constant temperature
In incubator, the diameter of diameter and periphery of bacterial colonies the precipitating circle of the bacterium colony grown after 72h with vernier caliper measurement, the work of phosphatidase
Power is indicated with Pz：The diameter of Pz=bacterium colony/periphery of bacterial colonies phosphatidase precipitating circle diameter.
The physical relationship of Pz value and phospholipase activity is as follows：1 is negative for phospholipase activity, and 0.90-0.99 is phosphatide enzyme activity
Power is extremely low, and 0.80-0.89 is that phospholipase activity is low, and 0.70-0.79 is that phospholipase activity is high, and 0.69 and the following are phospholipase activities
It is high.
Experimental result is shown in Figure 10 and table 3.
Table 3
Phosphatidase is one of highly important virulence factor in Candida albicans, the enzyme can not only damage host cell and
Tissue and Candida albicans cause the signal of interest of death.In addition, the phosphatidase of hypersecretion and the height of pathogenic microorganism
Pathogenicity rate also has inseparable connection.
By the Pz value of above-mentioned experimental result it is found that blank control and 5-flurocytosine control group (final concentration of 1.024 μ g/
ML) phospholipase activity is high, and the MZ1711B under 5% concentration is acted on and is clinically separated Candida albicans and can make its phosphatidase
Vigor is negative, and greatly reduces its virulence and drug resistance.
Antibacterial Chinese medicine composition of the invention can be applied to by candida albicans, Escherichia coli, staphylococcus aureus, verdigris
The medicine preparation of the germ disease such as pseudomonad, such as scytitis, oral inflammation, reproduction and Urinary system inflammation,
Antibacterial Chinese medicine composition of the invention can be used as active constituent addition in the drug for treating corresponding disease.
The foregoing is merely presently preferred embodiments of the present invention, is not intended to limit the invention, it is all in spirit of the invention and
Within principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (8)
1. a kind of antibacterial Chinese medicine composition, which is characterized in that including：10-100 parts of Chinese rhubarb extract, iron sheet based on parts by volume
5-50 parts of Herba Dendrobii extract, 3-30 parts of white balsamine extract, 2-15 parts of coptis extract, 6-29 parts of cinnamomum cassia extract and Ai Na
Fragrant extract 2-10 parts.
2. antibacterial Chinese medicine composition according to claim 1, which is characterized in that including：Rheum officinale based on parts by volume extracts
10-80 parts of object, 5-45 parts of dendrobium candidum extract, 5-15 parts of white balsamine extract, 2-13 parts of coptis extract, cortex cinnamomi are extracted
6-27 parts and Herba Blumeae Balsamiferae extract 2-6 parts of object.
3. antibacterial Chinese medicine composition according to claim 2, which is characterized in that including：Rheum officinale based on parts by volume extracts
48 parts of object, 42 parts of dendrobium candidum extract, 12 parts of white balsamine extract, 6 parts of coptis extract, 6 parts of cinnamomum cassia extract and Chinese mugwort
Receive 6 parts of fragrant extract.
4. antibacterial Chinese medicine composition according to claim 1-3, which is characterized in that the antibacterial Chinese medicine composition
It further include pharmaceutically acceptable auxiliary material.
5. antibacterial Chinese medicine composition according to claim 1-3, which is characterized in that above-mentioned antibacterial Chinese medicine composition
For vina, tablet, tincture, liniment, pastille or pill.
6. the preparation method of the described in any item antibacterial Chinese medicine compositions of claim 1-3, which is characterized in that including：According to upper
Proportion is stated by Chinese rhubarb extract, dendrobium candidum extract, white balsamine extract, coptis extract, cinnamomum cassia extract and Ai Na
Fragrant extract mixing.
7. the preparation method of antibacterial Chinese medicine composition as claimed in claim 6, which is characterized in that the Chinese rhubarb extract, iron sheet
Herba Dendrobii extract, white balsamine extract, coptis extract, cinnamomum cassia extract and Herba Blumeae Balsamiferae extract are made in accordance with the following methods
?：
Above-mentioned rheum officinale, dendrobium candidum, white balsamine, the coptis, cortex cinnamomi and Blumea balsamifera are broken into the particle of 0.1-5mm respectively, point
Then the ultrasonic vibration 15-30min not in solvent is 1 according to solid-liquid ratio：The ratio of (1-32) g/ml is by Chinese medicine at 25-40 DEG C
Under extracted 30-90 days in alcohol, filter, respectively obtain Chinese rhubarb extract, dendrobium candidum extract, white balsamine extract,
Coptis extract, cinnamomum cassia extract and Herba Blumeae Balsamiferae extract.
8. any one of the claim 1-3 antibacterial Chinese medicine composition prepare antifungal drug, anti-Escherichia coli drug,
Anti-Staphylococcus aureus drug, resisting pseudomonas aeruginosa drug, overriding resistance candida albicans drug or overriding resistance golden yellow grape
Application in coccus drug.
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