CN1235585C - Use of berberine as anti fungus medicine synergist - Google Patents
Use of berberine as anti fungus medicine synergist Download PDFInfo
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- CN1235585C CN1235585C CN 200410052989 CN200410052989A CN1235585C CN 1235585 C CN1235585 C CN 1235585C CN 200410052989 CN200410052989 CN 200410052989 CN 200410052989 A CN200410052989 A CN 200410052989A CN 1235585 C CN1235585 C CN 1235585C
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Abstract
The present invention relates to the technical field of medicine, which relates to the new application of berberine as a synergistic agent for antifungal medicaments. In vivo and in vitro experiments prove that through the combination of berberine with antifungal medicaments comprising fluconazole, ketoconazole, sulconazole and the like, the therapeutic effects on shallow surficial fungal infection or deep fungal infection can be improved to different extents, and the action of the antifungal medicaments on medicine-resisting fungi can be restored. Therefore, berberine can be used as a synergistic agent for antifungal medicaments.
Description
Technical field
The present invention relates to medical technical field, is the new purposes that berberine strengthens the antifungal drug effect.
Background technology
Berberine, have another name called berberine, it is antimicrobial drug commonly used clinically, the clinical treatment action range, can be used for treating intestinal infection, bacillary dysentery, eye conjunctivitis, suppurative otitis media, arrhythmia, congestive heart failure etc., do not see the report that it has antifungic action, do not see more that itself and antifungal drug are united to use the mycotic report of treatment.
Summary of the invention
The invention provides the new purposes of a kind of berberine as antifungal medicine synergist.Confirm through cell in vitro experiment and interior animal experiment, it has clear and definite potentiation to antifungal compounds such as fluconazol, ketoconazole, sulconazole, miconazoles, endurance strain for clinical drug-resistant bacterial strain and laboratory-induced, when antifungal drug and berberine share, can obviously reduce the dosage of antifungal drug, strengthen inhibitory action the drug resistance fungus.Therefore the synergist of berberine as antifungal drug can be used for the treatment of different deeps and superficial fungal infection.
The present invention has opened up new purposes for berberine, use it for the synergist of antifungal drug, can not only improve the antifungic action of medicine, and it is general day by day at clinical pathomycete drug resistance, under the situation that drug-resistant intensity is on the rise, make the effect of antifungal drug recovery to the drug resistance fungus, its meaning is well imagined.
Description of drawings
Fig. 1 is 0.25 μ g/ml berberine and 10 μ g/ml fluconazol when share, survival number and the time-to-live broken line graph of treatment deep infection by Candida albicans mice.
Fig. 2 is 0.5 μ g/ml berberine and 20 μ g/ml fluconazol when share, survival number and the time-to-live broken line graph of treatment deep infection by Candida albicans mice.
The specific embodiment
Below in conjunction with specific embodiment, the present invention is described in detail.
Embodiment 1: berberine and fluconazol share the effect to different clinical fungus strains
Material and method
1. reagent:
Berberine hydrochloride: Shanghai Li Sheng pharmaceutical factory, lot number Y-001-001007.
Fluconazol: Changzhou second pharmaceutical factory, lot number 20000928.
Dimethyl sulfoxine: China Medicine (Group) Shanghai Chemical Reagent Co.,, lot number 20011022.
Berberine hydrochloride is made into 1600,100,50,10 μ g/ml variable concentrations respectively with dimethyl sulfoxine, and fluconazol is made into the concentration of 6.4mg/ml with tri-distilled water, is subjected to the reagent thing in-20 ℃ of preservations.Before the experiment, 35 ℃ of incubators are put in the taking-up of medicine storage liquid melted, fully mixing carries out pharmacodynamics test respectively.
2. bacterial strain:
Clinical strain: Candida albicans (930612,9703154,960468,950812,010213), Aspergillus fumigatus (102163), sporotrichum schenckii (9912063-23), provide by the Shanghai Changhai Hospital Mycology Lab, pick up from different section office of this hospital clinical sample respectively, and through morphology and biochemical evaluation.
All experiments are all drawn the plate activation in husky fort glucose agar medium (SDA) with bacterial strain, Candida albicans was cultivated 2 days in 30 ℃, Aspergillus fumigatus and sporotrichum schenckii are after 35 ℃ of 1 weeks of cultivation, the picking monoclonal is drawn the plate activation once more respectively, get for the second time the gained monoclonal and put the SDA inclined-plane, it is standby in 4 ℃ of preservations to cultivate the back with said method.
3. culture fluid:
RPMI RPMI-1640: RPMI1640 (Gibco BRL company) 10.0g, NaHCO3 2.0g, morphine quinoline propane sulfonic acid (Sigma) 34.5g adds tri-distilled water 900ml dissolving, and 1N NaOH transfers pH to 7.0, is settled to 1000ml, filters sterilization, 4 ℃ of preservations.
Husky fort agar glucose (SDA) culture medium: peptone 10g, glucose 40g, agar 18g adds tri-distilled water 900ml dissolving, adds 2mg/ml chloramphenicol solution 50ml, adjusts pH to 7.0, is settled to 1000ml, 4 ℃ of preservations behind the autoclaving.
The YEPD culture fluid: yeast extract 10g, peptone 20g, glucose 20g adds tri-distilled water 900ml dissolving, adds 2mg/ml chloramphenicol solution 50ml, is settled to 1000ml, 4 ℃ of preservations behind the autoclaving.
4. instrument:
Water isolation type electro-heating standing-temperature cultivator (Shanghai make a leapleap forward medical apparatus and instruments factory);
The desk-top constant temperature oscillator of THZ-82A (Shanghai make a leapleap forward medical apparatus and instruments factory);
511 type enzyme micro-plate readers (Shanghai the 3rd analytical tool factory);
5. bacterium liquid preparation:
(1) Candida albicans bacterium liquid
The picking Candida albicans is a small amount of on the SDA culture medium of 4 ℃ of preservations, is seeded to the 1mlYEPD culture fluid, in 30 ℃ with the activation of 200rpm shaken cultivation, make fungus be in later stage exponential phase of growth.Get this bacterium liquid to the 1mlYEPD culture fluid, activate 16 hours once more with said method after, with blood cell counting plate counting, adjust bacterial concentration to 3 * 10 with the RPMI1640 culture fluid
3～5 * 10
3The concentration of individual/ml.
The picking filamentous bacteria is seeded to the SDA inclined-plane on a small quantity on the SDA culture medium of 4 ℃ of preservations, in 35 ℃ cultivate a week after, picking is seeded to the SDA inclined-plane on a small quantity once more, cultivates weeks in 35 ℃.Add an amount of RPMI1640 culture fluid before the experiment in the SDA inclined-plane, blow and beat bacterium colony, fungal spore is free in the RPMI1640 culture fluid, filter through four layers of sterile gauze then with suction pipe.Culture fluid adds the RPMI1640 culture fluid and adjusts spore concentration to 3 * 10 behind the blood cell counting plate counting
3～5 * 10
3The concentration concentration of individual/ml.
6. drug sensitive plate preparation:
Get aseptic 96 orifice plates, add RPMI RPMI-1640 100 μ l in No. 1 hole of every row and make blank; 3～No. 12 the hole respectively adds freshly prepared bacterium liquid 99 μ l; No. 2 holes add the fluconazol solution 2 μ l of bacterium liquid 196 μ l and 6.4mg/ml respectively.The berberine 2 μ l that in No. 2 holes, add respective concentration, and 3～No. 11 holes add berberine 1 μ l.
10 grades of doubling dilutions are carried out in 2～No. 11 holes, make the final fluconazol concentration in 2～11 each hole be respectively 64,32,16,8,4,2,1,0.5,0.25 and 0.125 μ g/ml, and the maintenance of berberine concentration is constant; No. 1 hole is for not containing the RPMI RPMI-1640 of medicine, and as negative control, No. 12 holes are for not containing the bacterium liquid of medicine, as positive control.Each drug sensitive plate is in 30 ℃ of cultivations.
7.MIC
80Value is judged:
Candida albicans and filamentous bacteria were cultivated 24 hours respectively at 30 ℃ and after the week, are surveyed each hole OD value with enzyme micro-plate reader in 620nm.With the positive control boring ratio, be MIC with the drug level in the least concentration hole of OD value decline more than 80%
80(drug level when conk 80% is suppressed).
MIC when medicine
80Value surpasses when measuring concentration range, adds up by the following method: MIC
80When value is higher than maximum concentration 64 μ g/ml, count "＞64 μ g/ml "; MIC
80When value is lower than least concentration 0.125 μ g/ml, count "≤0.125 μ g/ml ".
The equal operation repetitive of above-mentioned experiment 2 to 3 times is worked as MIC
80Value just is accepted in the time of accurately repeating; Work as MIC
80Value differs a concentration when above, then needs experiment again, till meeting the requirements.
Experimental result sees Table 1, table 2
Table 1 berberine and fluconazol share the MIC to the clinical Candida albicans of 4 strains
80Value
| Numbering | Chemical compound (μ g/ml) | Bai Nian | Bai Nian | Bai Nian | Bai Nian |
| 930612 | 9703154 | 960468 | 950812 | ||
| 1 2 3 4 5 | Flu+Ber(1.0) Flu+Ber(0.5) Flu+Ber(0.1) Flu Ber(1) | 0.25 0.25 0.5 0.5 - | 0.5 0.25 2 2 - | 0.5 0.5 1 2 - | 0.5 0.5 1 1 - |
Annotate: Flu represents fluconazol, and Ber represents berberine (down together)
Table 2 berberine and fluconazol share the MIC to clinical sporothrix of 2 strains and Aspergillus fumigatus
80Value
| Numbering. | Chemical compound (μ g/ml) | Fibrillae of spores 9912063-23 | Cigarette song 102163 |
| 1 2 3 | Flu+Ber(16) Flu Ber(16) | ≤0.0625 ＞32 - | ≤0.0625 ＞32 - |
By table 1 and table 2 as seen, the berberine of 1 μ g/ml can make the MIC of 5 kinds of clinical Candida albicanss to fluconazol
802-4 times of value decline, the berberine of 16 μ g/ml can make the MIC of clinical sporothrix and Aspergillus fumigatus
80Value is reduced to 0.0625 μ g/ml from 32 μ g/ml, illustrates that berberine can obviously strengthen the antifungic action of fluconazol.
Embodiment 2: berberine and fluconazol share the effect to different clinical and laboratory-induced persisters
Material and method
1. reagent:
Berberine hydrochloride: Shanghai Li Sheng pharmaceutical factory, lot number Y-001-001007.
Fluconazol: Changzhou second pharmaceutical factory, lot number 20000928.
Dimethyl sulfoxine: China Medicine (Group) Shanghai Chemical Reagent Co.,, lot number 20011022.
Berberine hydrochloride is made into 1600,1280,200,50 μ g/ml variable concentrations respectively with dimethyl sulfoxine, and fluconazol is made into 6.4mg/ml with tri-distilled water, is subjected to the reagent thing in-20 ℃ of preservations.Before the experiment, 35 ℃ of incubators are put in the taking-up of medicine storage liquid melted, fully mixing carries out pharmacodynamics test respectively.
2. bacterial strain:
The clinical drug-resistant strain: Candida albicans (01,13,16,25,55) provides by the Shanghai Changhai Hospital Mycology Lab, picks up from different section office of this hospital clinical sample respectively, and through morphology and biochemical evaluation.
The laboratory-induced persister: Candida albicans (resis-Y01, resis-010213, resis-5314) is respectively Candida albicans type strain ATCC76625, Changhai clinical strain 01021, international strain SC5314 and induces the persister of formation through fluconazol, through morphology and biochemical evaluation.
Other experimental procedure and method are with embodiment 1.
Experimental result sees Table 3, table 4
Table 3 berberine and fluconazol share the MIC to 3 strain laboratory-induced Candida albicans persisters
80Value
| Numbering | Chemical compound (μ g/ml) | resis-Y01 | resis-01023 | resis-5314 |
| 1 2 3 4 5 6 | Flu+Ber(16) Flu+Ber(12.8) Flu+Ber(2) Flu+Ber(0.5) Flu Ber(16) | 0.5 0.5 0.5 1 ＞64 - | ≤0.125 ≤0.125 ≤0.125 ＞64 - | 0.25 ＞32 - |
Table 4 berberine and fluconazol share the MIC to the clinical Candida albicans persister of 5 strains
80Value
| Numbering. | Chemical compound (μ g/ml) | Bai Nian | Bai Nian | Bai Nian | Bai Nian | Bai Nian |
| 01 | 13 | 16 | 25 | 55 | ||
| 1 2 3 4 5 | Flu+Ber(2) Flu+Ber(1) Flu+Ber(0.5) Flu Ber(2) | 32 ＞64 ＞64 ＞64 - | 1 16 32 64 - | 2 16 ＞64 ＞64 - | 2 4 ＞64 ＞64 - | 1 ＞64 ＞64 ＞64 - |
By table 3, table 4 as can be seen, originally to the drug-fast clinical Candida albicans strain MIC of fluconazol
80Value＞64, the berberine that share 2 μ g/ml can make the MIC of fluconazol
80Value drops to 1-2 μ g/ml, and to the drug resistance Candida albicans of laboratory-induced, the berberine that share 2 μ g/ml can make the MIC of fluconazol
80Value drops to 0.5-0.125 μ g/ml, illustrates that berberine can make the sensitivity of bacterial strain recovery to medicine, and the antifungal that strengthens fluconazol is greatly renderd a service, and has dose dependent.
Embodiment 3: the drug combination of berberine and other triazole antifungal agents
Material and method
1. medicine
Sulconazole: organic teaching and research room of pharmaceutical college of The 2nd Army Medical College is synthetic, lot number 19991005.
Miconazole: organic teaching and research room of pharmaceutical college of The 2nd Army Medical College is synthetic, lot number 19990716.
Ketoconazole: organic teaching and research room of pharmaceutical college of The 2nd Army Medical College is synthetic, lot number 19970407.
Sulconazole, miconazole, ketoconazole are made into 6.4mg/ml concentration with dimethyl sulfoxine respectively, and berberine hydrochloride is made into 1600,800,400,200,100,50 and 0.25 μ g/ml variable concentrations respectively with dimethyl sulfoxine.Be subjected to the reagent thing in-20 ℃ of preservations.Before the experiment, 35 ℃ of incubators are put in the taking-up of medicine storage liquid melted, fully mixing carries out pharmacodynamics test respectively.
Other experimental procedure and method are with embodiment 1.
Experimental result sees Table 5,6,7
Table 5 berberine and sulconazole share the MIC to the clinical Candida albicans persister of 3 strains
80Value
| Numbering. | Chemical compound (μ g/ml) | White thought 55 | White thought 13 | White thought 16 |
| 1 2 3 4 5 6 7 8 | Sulcon+Ber(16) Sulcon+Ber(8) Sulcon+Ber(4) Sulcon+Ber(2) Sulcon+Ber(1) Sulcon+Ber(0.5) Sulcon+Ber(0.25) Sulcon(64) | ≤0.125 0.25 0.25 0.25 1 1 1 8 | ≤0.125 ≤0.125 ≤0.125 0.5 2 2 2 ＞64 | ≤0.125 1 2 |
Annotate: Sulcon represents sulconazole, and Ber represents berberine
Table 6 berberine and miconazole share the MIC to the clinical Candida albicans persister of 2 strains
80Value
| Numbering | Chemical compound (μ g/ml) | White thought 55 | White thought 13 |
| 1 2 3 4 5 6 7 8 | Micon+Ber(16) Micon+Ber(8) Micon+Ber(4) Micon+Ber(2) Micon+Ber(1) Micon+Ber(0.5) Micon+Ber(0.25) Micon(64) | 0.25 0.25 1 2 4 4 4 8 | ≤0.125 ≤0.125 0.5 1 1 2 2 32 |
Annotate: Micon represents miconazole, and Ber represents berberine
Table 7 berberine and ketoconazole share the MIC to the clinical Candida albicans persister of 3 strains
80Value
| Numbering | Chemical compound (μ g/ml) | White thought 55 | White thought 13 | White thought 16 |
| 1 2 3 4 | Ketocon+Ber(16) Ketocon+Ber(8) Ketocon+Ber(2) Ketocon | ≤0.125 ≤0.125 ≤0.125 4 | ≤0.125 ＞64 ＞64 ＞64 | ≤0.125 ≤0.125 ≤0.125 ≤0.125 |
Annotate: Ketocon represents ketoconazole, and Ber represents berberine
By table 5,6,7 as seen, berberine not only can share the generation synergism with fluconazol, and has antibiotic potentiation equally with antifungal agent such as sulconazole, miconazole, ketoconazoles.The berberine that share 16 μ g/ml can make sulconazole, miconazole, the ketoconazole MIC to the drug resistance Candida albicans
80Value drops to 0.25-0.125 μ g/ml from 8-64 μ g/ml, and increases the effectiveness enhancing with berberine dosage, has dose dependent.
Embodiment 4: berberine and fluconazol share the protective effect to the mice systemic fungal infection
Experimental technique:
The laboratory mice kind is the ICR mice, is provided by Chinese Academy of Sciences experimental animal feeding center.
Drug resistance Candida albicans (No. 25 clinical strains) after the virulence recovery, activates on the SDA culture medium in the mice body again, grows to exponential phase in the YEPD fluid medium, and adjusting concentration is 3.2 * 10
7Individual/ml, give mouse tail vein injection, cause mice deep fungal infection model.
48 mices experimentize in two batches.First 18 mices are divided into matched group, fluconazol (0.25mg/kg) group, fluconazol (0.25mg/kg) share 3 experimental grouies of berberine (10mg/kg) group, 6 every group, male and female half and half.After causing the mice deep fungal infection, the beginning gastric infusion, every day 1 time, continuous 7 days, to observe altogether 14 days, matched group gives the normal saline of equivalent.Second batch of 30 mice is divided into matched group, fluconazol (0.5mg/kg) group, fluconazol (0.5mg/kg) share 3 experimental grouies of berberine (20mg/kg) group, 10 every group, male and female half and half.Gastric infusion, every day 1 time, continuous 7 days, to observe altogether 21 days, matched group gives the normal saline of equivalent.Calculating the average survival natural law respectively organize mice, and be abscissa to infect natural law, is that vertical coordinate is made broken line graph with mice survival number of elements, and the emergency degree that broken line descends is represented the speed degree of dead mouse.
Experimental result sees Table 8 respectively, Fig. 1 and table 9, Fig. 2:
Table 8 berberine and fluconazol share the influence of mice time-to-live of Candida albicans deep infection
| The Mus group | Normal saline | Fluconazol (0.25mg/kg) | Share berberine (10mg/kg) |
| 123456 mean survival times (my god) standard deviation P | 3.5 they 3.5 days 3 days 3.5 days 3 days 3 days 3.25 0.27 | 4 days 3.5 days 5 days 2 days 5.5 days 9 days 4.83 2.38 0.137 | ＞14 days 4 days 4 days 12 days 11 days 13 days 8.80 4.44 0.013 |
Table 9 berberine and fluconazol share the influence of mice time-to-live of Candida albicans deep infection
| The Mus group | Normal saline | Fluconazol (0.5mg/kg) | Share berberine (20mg/kg) |
| 123456789 10 mean survival times (my god) standard deviation P | 7 days 3 days 3 days 7 days 3 days 3 days 5 days 7 days 3 days 3 days 4.40 1.90 | 6 days 4 days 9 days 6 days 6 days 12 days 14 days 12 days 13 days 11 days 9.30 3.56 0.001 | 6 days 9 days 6 days 3 days 9 days 12 days 13 days 15 days 19 days 20 days 11.20 5.65 0.002 |
By above-mentioned harmony in the exterior figure as seen, the fluconazol of the berberine of 0.25 μ g/ml and 10 μ g/ml share treatment deep infection by Candida albicans mice, compares with independent use fluconazol, can make the mean survival time of mice extend to 8.8 days from 4.8 days; 0.5 the fluconazol of the berberine of μ g/ml and 20 μ g/ml share treatment deep infection by Candida albicans mice, the mean survival time that can make mice of comparing with independent use fluconazol extended to 11.2 days from 9.3 days, illustrate that berberine and fluconazol share, all can prolong the time-to-live of Candida albicans deep infection mice, show the potentiation of berberine antimicrobial drug.
Claims (1)
1. berberine is characterized in that as the purposes of antifungal medicine synergist said antifungal is fluconazol, ketoconazole, sulconazole, miconazole, and berberine addition in antifungal valid density is 0.5～16 μ g/ml.
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