CN1313828C - Antifungal agent screening reagent kit - Google Patents
Antifungal agent screening reagent kit Download PDFInfo
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- CN1313828C CN1313828C CNB2004100529882A CN200410052988A CN1313828C CN 1313828 C CN1313828 C CN 1313828C CN B2004100529882 A CNB2004100529882 A CN B2004100529882A CN 200410052988 A CN200410052988 A CN 200410052988A CN 1313828 C CN1313828 C CN 1313828C
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Abstract
The present invention relates to the technical field of medicine, which discloses a reagent kit capable of sieving an antifungal agent and researching an antifungal mechanism and the toxicity to a human body. The reagent kit is composed of a medicine sensitive board and a reagent. According to the testing result of the reagent kit, whether a tested object has an antifungal function or not and is combined with ergosterol and cholesterol or not and the combined condition are used for judging whether the tested object is used as an antifungal agent and an antifungal action mechanism or not and whether the tested object is toxic to a human body or not. The reagent kit has the advantages of low cost and convenient operation. The antifungal agent can be rapidly and effectively sieved, and the action mechanism and the toxicity to a human body can be researched simultaneously.
Description
Technical field
The present invention relates to medical technical field, is a kind of kit that can be used for screening antifungal agent, and can study antimycotic mechanism and to human toxicity.
Background technology
In in the past nearly 100 years, antifungal drug research has obtained proud achievement, but the range of choice of clinical antifungal is still very narrow, and therefore kind seldom seeks new antifungal agent, and research antifungic action mechanism just becomes the task of top priority.
Ergosterol is a kind of special biotic component that contains on the fungal cell membrane, play a significant role for keeping fungal cell's membrane stability and flowability, existing antifungal agent spininess is to the biosynthesis pathway of ergosterol, thereby influenced the biosynthesizing of ergosterol, the performance antifungic action.Wherein polyenoid class antifungal drug mainly by combining with the direct of fungal cell membrane ergosterol, makes fungal cell membrane form the cavity, and content leaks and death; Though the cell membrane of human body cell does not contain ergosterol, but contain a large amount of cholesterol, some antifungal agent also can directly combine with cholesterol, can directly combine with cholesterol as polyenoid class medicine, so when simultaneously with polyenoid class medicine anti-fungal infection, to human body cell also possibility toxigenicity, just toxicity varies in size.Because this special mechanism of action, polyenoid class medicine antifungic action is strong, be difficult for producing drug resistance, and in the clinical critical role of occupying, be " goldstandard " of antifungal drug, the antifungal drug that therefore screens similar effect mechanism becomes an important research direction.
The conventional method of antifungal agent screening has micro-liquid base dilution method, test tube method, paper disk method etc., be to judge by the growing state of fungi in the pastille nutrient culture media, but can only carry out preliminary screening to antifungic action, whether the toxic important content that waits is difficult to determine for its mechanism of action and to human body, also needs other a large amount of experiments to be confirmed.Set up a kind of method, screening determines simultaneously that directly in conjunction with the antifungal agent of ergosterol its toxicity to human body is just extremely important for this reason.
Summary of the invention
The present invention contains ergosterol and does not contain cholesterol according to fungal cell membrane, human body cell contains cholesterol and does not contain the difference of ergosterol, a kind of kit that effectively screens antifungal compound fast is provided, testing result according to this kit, judge that this is tried thing and has or not antifungic action, whether produce antifungic action, and can or can not combine the toxicity that causes human body, thereby decide what to use with cholesterol by directly combining with ergosterol.
Kit of the present invention is by box body and be contained in drug sensitive plate in the box body and reagent A, B, C, D, E, each pipe of F are formed:
1. drug sensitive plate: one of porous plate, through cobalt 60 radiation sterilizations;
2. reagent A: 1 mol NaOH solution 5ml;
3. reagent B: peptone 10 grams, glucose 40 grams, agar 18 grams (facing the time spent adds 900 milliliters of dissolvings of tri-distilled water, adjusts pH to 7.0 with reagent A, is settled to 1000 milliliters, bevel behind the autoclaving, 4 ℃ of preservations);
4. reagent C: yeast extract 10 grams, peptone 20 grams, glucose 20 grams (face time spent add 900 milliliters of dissolvings of tri-distilled water, be settled to 1000 milliliters, 4 ℃ of preservations behind the autoclaving);
5. reagent D: RPMI 1,640 8.0 gram, sodium bicarbonate 2.0 grams, morphine quinoline propane sulfonic acid 34.5 grams [facing the time spent adds 900 milliliters of dissolvings of tri-distilled water, transfers pH to 7.0 (25 ℃) with reagent A, is settled to 1000 milliliters, filters sterilization, 4 ℃ of preservations];
6. reagent E: the DMSO solution 5ml (face the time spent, add 1000 milliliters of mentioned reagent D solution, filter sterilization, 4 ℃ of preservations) that contains 34.63 micromoles per liter ergosterols;
7. reagent F: the DMSO solution 5ml (face the time spent, add 1000 milliliters of mentioned reagent D solution, filter sterilization, 4 ℃ of preservations) that contains 34.63 micromoles per liter cholesterol.
RPMI 1640 is a U.S. Gibco company product, and ergosterol, cholesterol are U.S. Sigma company product, and all the other reagent are that homemade analysis is pure.
Kit using method of the present invention is as follows:
1. preparation is tried thing solution
Tried thing and be made into desired concn with dimethyl sulfoxide (DMSO) (DMSO), doubling dilution is the solution of 5～20 concentration gradients, and 4 ℃ of preservations are standby.
2. be mixed with as above corresponding inclined-plane and solution in the bracket with respectively managing reagent in the kit.
3. activate fungi
Preserve a small amount of fungi of picking the pipe with the inoculation circle from fungi, streak inoculation was cultivated 24 hours～48 hours for 35 ℃ on reagent B inclined-plane.A small amount of with the inoculation circle again from the bacterium colony picking fungi on the reagent B inclined-plane, be seeded in 1 milliliter of reagent C, in 37 ℃, 250 rev/mins shaken cultivation, activate 16 hours, make fungi be in the exponential growth later stage.
4. prepare fungi bacterium liquid
With above-mentioned activation fungi,, adjust bacterial concentration to (1～5) * 10 with reagent D respectively with the blood cell counting plate counting
5Individual/milliliter, generate a reagent D bacterium liquid; Be (1～5) * 10 with the reagent E dilution
5The bacterium liquid of individual/ml concn is made into the reagent E bacterium liquid that contains ergosterol; Be (1～5) * 10 with reagent F dilution
5The bacterium liquid of individual/ml concn is made into the reagent F bacterium liquid that contains cholesterol.
5. measure minimum inhibitory concentration value (MIC
80Value)
Get aseptic drug sensitive plate, No. 1 blank is made in the hole, adds 1 microlitre DMSO and 99 microlitre reagent D; What the 2nd～No. 11 hole added doubling dilution is tried thing solution 1 microlitre and 99 microlitre reagent D bacterium liquid; No. 12 hole do not contain is tried thing, adds 1 microlitre DMSO and 99 microlitre reagent D bacterium liquid are made positive control; Reagent E bacterium liquid and reagent F bacterium liquid are operated with method by mentioned reagent D bacterium liquid, are placed on the same sterilization drug sensitive plate each operation repetitive 2 times.
With this drug sensitive plate in 25～37 ℃ cultivate 12～168 hours after, survey each hole optical density value with enzyme micro-plate reader in 620 nanometers, each and corresponding positive control boring ratio are MIC with the substrate concentration that tried in the least concentration hole of optical density value decline more than 80%
80(the conk 80% repressed substrate concentration that tried).
The MIC that records with reagent D
80Value is as reference value, the MIC that reagent E, reagent F record
80Value and reference value compare, and the result is judged:
1. reference value 〉=64mgL of recording of reagent D
-1, show that being tried thing does not have antifungic action, need not compare that reagent E, reagent F record the result;
2. reference value＜64mgL of recording of reagent D
-1, reagent E result＞reference value, reagent F result＞reference value shows that being tried thing has antifungic action, and illustrates that itself and ergosterol directly combine the performance antifungic action, this is tried that thing can combine with cholesterol and toxic to human body;
3. reference value＜64mgL of recording of reagent D
-1, reagent E result＞reference value, reagent F result≤reference value shows that being tried thing has antifungic action, and it directly combines the performance antifungic action with ergosterol, and this is tried difficult the combination with cholesterol of thing and little to human toxicity, so can be used as antifungal agent;
4. reference value＜64mgL of recording of reagent D
-1, reagent E result≤reference value, reagent F result＞reference value shows that being tried thing has antifungic action, and its mechanism of action is still needed and is further studied, and this is tried thing and can be combined human body toxic with cholesterol;
5. reference value＜64mgL of recording of reagent D
-1, reagent E result≤reference value, reagent F result≤reference value shows that being tried thing has antifungic action, mechanism of action and toxicity all need further research.
This kit can be used for screening the thing that tried of separate sources, comprises natural products, chemosynthesis product, microbial fermentation product, marine extracts etc.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in detail.
Embodiment 1: the antifungic action that detects amphotericin B with kit of the present invention
One. obtain solution
1. tried the thing amphotericin B and be made into 6.4 mg/ml concentration with dimethyl sulfoxide (DMSO) (DMSO), and doubling dilution becomes the solution of 10 concentration gradients of 3.2,1.6,0.8,0.4,0.2,0.1,0.05,0.025,0.0125,0.00625 mg/ml, and 4 ℃ of preservations are standby.
2. reagent A: 1 mol NaOH solution 5ml;
3. reagent B: peptone 10 grams, glucose 40 grams, agar 18 grams add tri-distilled water 900ml dissolving, adjust pH to 7.0 with reagent A, are settled to 1000ml, bevel behind the autoclaving, 4 ℃ of preservations;
4. reagent C: yeast extract 10 grams, peptone 20 grams, glucose 20 grams add tri-distilled water 900ml dissolving, are settled to 1000ml, 4 ℃ of preservations behind the autoclaving;
5. reagent D: RPMI 16408.0 restrains, sodium bicarbonate 2.0 grams, and morphine quinoline propane sulfonic acid 34.5 grams add tri-distilled water 900ml dissolving, transfer pH to 7.0 (25 ℃) with reagent A, are settled to 1000ml, filtration sterilization, 4 ℃ of preservations;
6. reagent E: contain the DMSO solution 5ml of 34.63 micromoles per liter ergosterols, add 1000 milliliters of mentioned reagent D solution, filter sterilization, 4 ℃ of preservations;
7. reagent F: contain the DMSO solution 5ml of 34.63 micromoles per liter cholesterol, add 1000 milliliters of mentioned reagent D solution, filter sterilization, 4 ℃ of preservations.
Two. measure amphotericin B minimum inhibitory concentration value (MIC
80Value)
The inoculation circle is preserved a small amount of Candida albicans of picking the pipe from Candida albicans, and streak inoculation was cultivated 24 hours for 35 ℃ on reagent B inclined-plane.A small amount of with inoculation circle picking fungi from the reagent B inclined-plane again, be seeded to 1 milliliter of reagent C, in 37 ℃, 250 rev/mins shaken cultivation, activate 16 hours, make fungi be in the exponential growth later stage.
To activate fungi and count, adjust bacterial concentration to 3 * 10 with reagent D respectively with blood cell counting plate
5Individual/milliliter, generate a reagent D bacterium liquid; With the reagent E dilution is 3 * 10
5The bacterium liquid of individual/ml concn is made into the reagent E bacterium liquid that contains ergosterol; With reagent F dilution is 3 * 10
5The bacterium liquid of individual/ml concn is made into the reagent F bacterium liquid that contains cholesterol.
Aseptic 96 orifice plates, No. 1 blank is made in the hole, adds 1 microlitre DMSO and 99 microlitre reagent D; What the 2nd～No. 11 hole added doubling dilution is tried thing solution 1 microlitre and 99 microlitre reagent D bacterium liquid; No. 12 hole do not contain is tried thing, adds 1 microlitre DMSO and 99 microlitre reagent D bacterium liquid are made positive control, operation repetitive 2 times.Reagent E bacterium liquid and reagent F bacterium liquid are operated on same drug sensitive plate by the same method.
Drug sensitive plate after 24 hours, is surveyed each hole optical density value with enzyme micro-plate reader in 620 nanometers in 35 ℃ of cultivations.The amphotericin B that reagent D records is to Candida albicans MIC
80Value is 0.25 mcg/ml, and the prompting amphotericin B has stronger anti-candida albicans activity, is worth as reference with this; The MIC that reagent E records
80Value is 16 mcg/ml, obviously greater than above-mentioned MIC
80Reference value shows that amphotericin B brings into play antifungic action by combining with ergosterol; The MIC that reagent F records
80Value is 1 mcg/ml, is slightly larger than above-mentioned MIC
80Reference value shows that amphotericin B also can combine with cholesterol, has certain toxicity to human body.
Embodiment 2: the antifungic action that detects TTS-12 with kit of the present invention
One. obtain solution
1. being tried thing TTS-12 is the saponins natural products that extracts in the puncture vine, provide by phytochemistry teaching and research room of pharmaceutical college of The 2nd Army Medical College, be made into 6.4 mg/ml concentration with dimethyl sulfoxide (DMSO) (DMSO), and doubling dilution becomes the solution of 10 concentration gradients of 6.4,3.2,1.6,0.8,0.4,0.2,0.1,0.05,0.025,0.0125 mg/ml, and 4 ℃ of preservations are standby.
2. reagent A: 0.5 mol NaOH solution 5ml;
3. reagent B: peptone 10 grams, glucose 40 grams, agar 18 grams are joined method with embodiment 1;
4. reagent C: yeast extract 10 grams, peptone 20 grams, glucose 20 grams are joined method with embodiment 1;
5. reagent D: RPMI 1,640 8.0 restrains, sodium bicarbonate 2.0 grams, and morphine quinoline propane sulfonic acid 34.5 grams are joined method with embodiment 1;
6. reagent E: contain the DMSO solution 5ml of 34.63 micromoles per liter ergosterols, join method with embodiment 1;
7. reagent F: contain the DMSO solution 5ml of 34.63 micromoles per liter cholesterol, join method with embodiment 1.
Two. measure TTS-12 minimum inhibitory concentration value (MIC
80Value)
The inoculation circle is preserved a small amount of Candida albicans of picking the pipe from Candida albicans, and streak inoculation was cultivated 48 hours for 35 ℃ on reagent B inclined-plane.A small amount of with inoculation circle picking fungi from the reagent B inclined-plane again, be seeded to 1 milliliter of reagent C, in 37 ℃, 250 rev/mins shaken cultivation, activate 16 hours, make fungi be in the exponential growth later stage.
To activate fungi and count, adjust bacterial concentration to 2 * 10 with reagent D respectively with blood cell counting plate
5Individual/milliliter, generate a reagent D bacterium liquid; With the reagent E dilution is 2 * 10
5The bacterium liquid of individual/ml concn is made into the reagent E bacterium liquid that contains ergosterol; With reagent F dilution is 2 * 10
5The bacterium liquid of individual/ml concn is made into the reagent F bacterium liquid that contains cholesterol.
Aseptic 48 orifice plates, No. 1 blank is made in the hole, adds 1 microlitre DMSO and 99 microlitre reagent D; What the 2nd～No. 11 hole added doubling dilution is tried thing solution 1 microlitre and 99 microlitre reagent D bacterium liquid; No. 12 hole do not contain is tried thing, adds 1 microlitre DMSO and 99 microlitre reagent D bacterium liquid are made positive control, and reagent E bacterium liquid and reagent F bacterium liquid is operation by the same method on same drug sensitive plate, operation repetitive 2 times.
Again drug sensitive plate after 24 hours, is surveyed each hole optical density value with enzyme micro-plate reader in 620 nanometers in 25 ℃ of cultivations.The TTS-12 that reagent D records is to Candida albicans MIC
80Value is 2 mcg/ml, and prompting TTS-12 has stronger anti-candida albicans activity, is worth as the reference value with this; The MIC that reagent E records
80Value is 16 mcg/ml, greater than reference value, shows that TTS-12 can combine with ergosterol, the performance antifungic action; The MIC that reagent F records
80Value is 2 mcg/ml, equals reference value, shows that the TTS-12 difficulty combines with cholesterol, and is less to human toxicity.
Embodiment 3: the antifungic action that detects Philinopside A (I) with kit of the present invention
One. obtain solution
1. tried the lanostane-type triterpene saponin componds of thing Philinopside A (I) for intending from the sea life striped getting the cowfish, purity＞95%, through being accredited as 16 β-acetoxy-3-O-[3-O-β-D-methyl glucopyranose-(1 → 3)-β-D-xylopyranose-(1 → 4)-β-D-pyrans isorhodeose-(1 → 2)-4-O-sodium sulfonate-β-D-xylopyranose]-sea cucumber alkane-7,24-diene-3 β-alcohol, called after Philinopside A (I), provide by pharmaceutical college of The 2nd Army Medical College marine drug research centre, be made into 25.6 mg/ml concentration with dimethyl sulfoxide (DMSO) (DMSO), and doubling dilution becomes 25.6,12.8,6.4,3.2,1.6,0.8,0.4,0.2,0.1,0.05 the solution of 10 concentration gradients of mg/ml, 4 ℃ of preservations are standby.
2. reagent A: 0.5 mol NaOH solution 5ml;
3. reagent B: peptone 10 grams, glucose 40 grams, agar 18 grams are joined method with embodiment 1;
4. reagent C: yeast extract 10 grams, peptone 20 grams, glucose 20 grams are joined method with embodiment 1;
5. reagent D: RPMI 1,640 8.0 restrains, sodium bicarbonate 2.0 grams, and morphine quinoline propane sulfonic acid 34.5 grams are joined method with embodiment 1;
6. reagent E: contain the DMSO solution 5ml of 34.63 micromoles per liter ergosterols, join method with embodiment 1;
7. reagent F: contain the DMSO solution 5ml of 34.63 micromoles per liter cholesterol, join method with embodiment 1.
Two. measure Philinopside A (I) minimum inhibitory concentration value (MIC
80Value)
The inoculation circle is preserved a small amount of Candida albicans of picking the pipe from Candida albicans, and streak inoculation was cultivated 24 hours for 35 ℃ on reagent B inclined-plane.A small amount of with inoculation circle picking fungi from the reagent B inclined-plane again, be seeded to 1 milliliter of reagent C, in 37 ℃, 250 rev/mins shaken cultivation, activate 16 hours, make fungi be in the exponential growth later stage.
To activate fungi and count, adjust bacterial concentration to 2 * 10 with reagent D respectively with blood cell counting plate
5Individual/milliliter, generate a reagent D bacterium liquid; With the reagent E dilution is 3.2 * 10
5The bacterium liquid of individual/ml concn is made into the reagent E bacterium liquid that contains ergosterol; With reagent F dilution is 3.2 * 10
5The bacterium liquid of individual/ml concn is made into the reagent F bacterium liquid that contains cholesterol.
Aseptic 96 orifice plates, No. 1 blank is made in the hole, adds 1 microlitre DMSO and 99 microlitre reagent D; What the 2nd～No. 11 hole added doubling dilution is tried thing solution 1 microlitre and 99 microlitre reagent D bacterium liquid; No. 12 hole do not contain is tried thing, adds 1 microlitre DMSO and 99 microlitre reagent D bacterium liquid are made positive control, and reagent E bacterium liquid and reagent F bacterium liquid is operation by the same method on same drug sensitive plate, operation repetitive 2 times.
Again drug sensitive plate after 168 hours, is surveyed each hole optical density value with enzyme micro-plate reader in 620 nanometers in 37 ℃ of cultivations.The Philinopside A (I) that reagent D records is to Candida albicans MIC
80Value is 16 mcg/ml, and prompting Philinopside A (I) has the anti-candida albicans activity, is worth as the reference value with this; The MIC that reagent E records
80Value is 64 mcg/ml, greater than reference value, shows that TTS-12 can combine with ergosterol, the performance antifungic action; The MIC that reagent F records
80Value is 32 mcg/ml, greater than reference value, shows that TTS-12 and cholesterol have certain combination, and is toxic to human body.
More than experiment shows that kit of the present invention can be used for screening the thing that tried of separate sources, comprises natural products, chemosynthesis product, microbial fermentation product, marine extracts etc., and this kit is with low cost, and is easy and simple to handle, can effective and rapid screening antifungal agent.
Claims (1)
1. an antifungal agent screening reagent box comprises box body and reagent, it is characterized in that also comprising one of drug sensitive plate, and said reagent comprises reagent A, B, C, D, E, each pipe of F:
Reagent A: 1 mol NaOH solution 5ml;
Reagent B: peptone 10 grams, glucose 40 grams, agar 18 grams;
Reagent C: yeast extract 10 grams, peptone 20 grams, glucose 20 grams;
Reagent D: RPMI 1,640 8.0 grams, sodium bicarbonate 2.0 grams, morphine quinoline propane sulfonic acid 34.5 grams of 0.165 mol;
Reagent E: the DMSO solution 5ml that contains 34.63 micromoles per liter ergosterols;
Reagent F: the DMSO solution 5ml that contains 34.63 micromoles per liter cholesterol.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100529882A CN1313828C (en) | 2004-07-20 | 2004-07-20 | Antifungal agent screening reagent kit |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100529882A CN1313828C (en) | 2004-07-20 | 2004-07-20 | Antifungal agent screening reagent kit |
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| CN1588067A CN1588067A (en) | 2005-03-02 |
| CN1313828C true CN1313828C (en) | 2007-05-02 |
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| CNB2004100529882A Expired - Fee Related CN1313828C (en) | 2004-07-20 | 2004-07-20 | Antifungal agent screening reagent kit |
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH06245794A (en) * | 1993-02-19 | 1994-09-06 | Sekisui Chem Co Ltd | Method for testing antifungal performance |
| JPH10248565A (en) * | 1997-03-18 | 1998-09-22 | Kankyo Meneki Gijutsu Kenkyusho:Kk | Hapten compound of myclobutanil compound, antibody and measurement |
| CN1260482A (en) * | 1999-01-13 | 2000-07-19 | 罗姆和哈斯公司 | Use of spore-adhesion detection in screening fungicidal agent |
| WO2001071045A2 (en) * | 2000-03-23 | 2001-09-27 | Millennium Pharmaceuticals, Inc. | High throughput screening for inhibitors of fatty acid, ergosterol, sphingolipid, or phospholipid synthesis in fungi |
| JP2002335995A (en) * | 2001-05-16 | 2002-11-26 | Pola Chem Ind Inc | Method for evaluation of antimicrobial agent |
| WO2003058233A1 (en) * | 2001-12-28 | 2003-07-17 | Eisai Co., Ltd. | Method of screening compound having fungal cell wall synthesis inhibitory activity |
-
2004
- 2004-07-20 CN CNB2004100529882A patent/CN1313828C/en not_active Expired - Fee Related
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH06245794A (en) * | 1993-02-19 | 1994-09-06 | Sekisui Chem Co Ltd | Method for testing antifungal performance |
| JPH10248565A (en) * | 1997-03-18 | 1998-09-22 | Kankyo Meneki Gijutsu Kenkyusho:Kk | Hapten compound of myclobutanil compound, antibody and measurement |
| CN1260482A (en) * | 1999-01-13 | 2000-07-19 | 罗姆和哈斯公司 | Use of spore-adhesion detection in screening fungicidal agent |
| WO2001071045A2 (en) * | 2000-03-23 | 2001-09-27 | Millennium Pharmaceuticals, Inc. | High throughput screening for inhibitors of fatty acid, ergosterol, sphingolipid, or phospholipid synthesis in fungi |
| JP2002335995A (en) * | 2001-05-16 | 2002-11-26 | Pola Chem Ind Inc | Method for evaluation of antimicrobial agent |
| WO2003058233A1 (en) * | 2001-12-28 | 2003-07-17 | Eisai Co., Ltd. | Method of screening compound having fungal cell wall synthesis inhibitory activity |
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