CN102226806A - Compatibility method for acquiring optimal combined biological effect by testing binary mixture - Google Patents
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Abstract
The invention belongs to the field of environmental protection, and discloses a compatibility method for acquiring an optimal combined biological effect by testing a binary mixture. The method comprises the following steps: (1) the testing method is based on GB/T 15441-1995, and photobacterium phosphoreum is used as an indicator organism to test the single toxicity EC50-A and EC50-B of A and B compounds; (2) according to the EC50-A and the EC50-B, a mixed solution with an equivalent effect ratio is prepared by a testing method which is based on GB/T15441-1995, the biological effect concentration of the mixed solution is tested to obtain the concentration CA and CB of A and B in a mixed system when inhibition of the mixed system to the biological effect is 50 percent, and a combined biological effect index TU1:1 is calculated in the equivalent effect ratio; and (3) a combined biological effect regulation of the binary mixed system can be predicted according to the testing result TU1:1 of the combined biological effect, which is calculated in the equivalent effect ratio, and the optimal biological effect point can be acquired. The method can be applied in the aspects of environmental science, medication, pesticide and the like.
Description
Technical field
The invention belongs to field of environment protection, be specifically related to a kind of medicine compatibility method that two-component mixture obtains best associating biological effect of measuring.
Background technology
Along with the high speed development and the industrialization degree of modern science and technology improves constantly, chemicals is ubiquitous in the human lives, and the number of chemical product mix and exist often, and environment and human health have been produced profound influence.
Over nearly 40 years, people use quantitative structure activity relationship (QSAR) method, the environmental behaviour and the biological effect thereof of simplification compound have been carried out quite detailed research, and obtained many corresponding achievements.On this basis, many scholars study the biological effect of mixing cpd, and have proposed some Forecasting Methodologies.
Sprague (Sprague, J.B. " Lethal levels of mixed copper zinc solutions for juvenile salmon ", JFish Res Board Can 1965 22:425-432) waits the people to propose to estimate with toxic unit (TU) method of organic contaminant joint toxicity effect in the environment.Other scholar proposes to add and index (AI) and mix toxic unit (MTI) predicted method and study joint toxicity effect (Konemann H. " Quantitative structure-activity relationships in fish toxicity studies Part 1:Relationship for 50 industrial pollutants " .Toxicology 1981 in succession, 19 (3): 209-221) (Marking L " Method for assessing additive toxicity of chemical mixtures " .Aquatic toxicology and hazard evaluating, ASTM STP, 1977,634:99-108).Nirmalakhanda (Nirmalakhandan N, Arulgnanendran V, Mohsin M, Sun B, Cadena F. " Toxicity of mixtures of organic chemicals to microorganisms " .Water Res 1994,28 (3): 543-551) take the lead in proposing in the notion according to toxic unit in 1994, for the system that contains n simplification compound, under equitoxic hypothesis prerequisite, by the QSAR model of simplification compound, dope the MLC (median lethal concertration) LC of each simplification compound earlier
50, i, and then the toxicity of toxicity than the arbitrary organic compound in the mixed system of forming such as calculate.Xu (Xu S, Nirmalakhandan N. " Use of QSAR models in predicting joint effects in multi-component mixtures of organic chemicals " .Water Res 1998,32 (8): 2391-2399) inherited the theory of Nirmalakhanda, at the mixing cpd that contains a n-1 equitoxic organic compound and not equitoxic organic compound n, the method for a measurable compound n concentration has been proposed.Prakash split path (Prakash J in addition in 1996, Nirmalakhandan N, Sun B, Peace J. " Toxicity of binary mixtures of organic chemicals to microorganisms " .Water Res 1996.30 (6): 1459-1463), proposed a method that adopts arbitrary organic compound substrate concentration in the similar parameter λ prediction mixed system, these work make the associating biological effect of prediction potpourri become possibility.
But because existing method has following limitation, its application is restricted: on the one hand, existing method and research all fail the Changing Pattern that the associating biological effect of mixed system is formed with compound is explained, and cause the operability of its real world applications and feasibility relatively poor.On the other hand, the present mixed system of measuring such as mostly is at the toxicity ratio, and these methods toxicity such as are only applicable to than this perfect condition of mixed system of forming.Yet in actual life and production practices, almost be non-existent etc. toxicity than the mixed system of forming, mixed pollutants normally exists than form with toxicity such as non-.Therefore toxicity such as non-than mixed system in, the quantitative forecasting technique of biological effect is still a difficult problem, not seeing has the related data report.Therefore, press for a kind of conveniently method of seeking, compound is united when existing the mixed biologic effect under the different proportion condition predict, and then find suitable best biological effect point, instruct the economy input of people, produce best effect, improve the work effect with minimum.
Summary of the invention
The purpose of this invention is to provide a kind of medicine compatibility method that two-component mixture obtains best associating biological effect of measuring; this method is first according to mixed system biological effect Changing Pattern; solve binary mixture and how to have obtained best this challenge of uniting biological effect; the inventive method can be applicable to aspects such as environmental science, medicine and agricultural chemicals; provide scientific basis for seeking best associating biological effect point in pollution reduction in the environmental science, ecological environmental protection and medicine and the agricultural chemicals, have important scientific meaning and more practical value.
Technical scheme of the present invention is as follows:
The invention provides a kind of medicine compatibility method that two-component mixture obtains best associating biological effect of measuring, this method may further comprise the steps:
(1) mensuration of simplification compound biological effect concentration
Method of testing is indicator organism according to GB/T 15441-1995 with the photobacterium phosphoreum, measures the single toxicity of A, B two compounds, and simplification compound biological effect adopts EC50 to represent that these two kinds of simplification compound biological effect concentration are respectively EC
50-A, EC
50-B
(2) etc. effect than the time associating biological effect mensuration
The A that obtains according to mensuration and the single creature effective concentration EC of two kinds of compounds of B
50-AAnd EC
50-B, prepare the mixed solution of effect ratio such as, promptly two kinds of material concentrations of A and B are respectively EC in the mixed solution
50-AAnd EC
50-B, method of testing is measured the biological effect concentration of this mixed solution according to GB/T 15441-1995 " the mensuration photobacteria method of acute toxicity of water quality ", and can obtain that mixed system suppresses biological effect is 50% o'clock A and the concentration C of B in mixed system
AAnd C
B, calculate then etc. effect than the time associating biological effect index TU
1: 1
(3) mensuration of best biological effect point
According to step (2) calculate etc. effect than the measurement result TU of time associating biological effect
1: 1, the associating biological effect Changing Pattern of prediction binary mixture, and obtain best biological effect point.
The size of described associating biological effect index TU is used for estimating the associating biological effect:
TU
1: 1＜0.80 expression A and two kinds of compounds of B have produced cooperative effect;
TU
1: 1＞1.20 expression A and two kinds of compounds of B have produced picks up antiatherosclerotic effect;
TU
1: 1=1.00 ± 0.20 expression A and two kinds of compounds of B have produced additive effect, be also referred to as summation action (Broderius SJ, Kahl MD, Hoglund MD. " Use of joint toxic response to define the primary-mode of toxic action for diverse industrial organic chemicals " .Environ Toxicol Chem 1995,14 (9) 1591-1605).
Described TU
1: 1Adopt following formula to calculate:
If TU
1: 1＜0.80, show that this mixed system is waiting effect to present cooperative effect than the time, can predict the joint effect of mixed system when waiting effect point the strongest (cooperative effect is the strongest) that this two material is formed thus; Along with the concentration of mixed system from etc. effect concentration change such as effect specific concentration Xiang Fei, corresponding TU increases gradually and approaches 1.00 ± 0.20, that is to say from cooperative effect progressively to change to summation action; This just points out us in containing the discharge of wastewater of these compounds, the effect concentration discharge such as avoid, to reduce ecological hazard as far as possible; And agricultural chemicals unite use and medical drug combination aspect, two kinds of compounds with cooperative effect are waiting toxicity than putting an associating use, then can improve the medication effect greatly.
If TU
1: 1＞1.20, show that this mixed system is waiting effect to have the antiatherosclerotic effect of picking up than the time; Can predict the mixed system that these two kinds of compounds are formed, its associating biological effect the strongest (it is the strongest to pick up antiatherosclerotic effect) when waiting effect thus than point; Along with the concentration of mixed system from etc. effect concentration change such as effect specific concentration Xiang Fei, corresponding TU reduces gradually and approaches 1.00 ± 0.20.This just points out us when processing contains the waste water of this compounds, toxicity such as can adopt than the some discharging, utilizes it to pick up antiatherosclerotic effect, reduces processing cost and environmental hazard to greatest extent; As a same reason, for having two kinds of medicines picking up antiatherosclerotic effect, definitely avoid waiting toxicity, otherwise result of treatment will be had a greatly reduced quality than a drug combination.
If TU
1: 1=1.00 ± 0.20, show that this mixed system is waiting effect to have additive effect than the time; Can predict thus, for wait effect than the time be the binary system of additive effect, along with the concentration of these two kinds of materials from etc. effect concentration change such as effect specific concentration Xiang Fei, its associating biological effect size keeps constant (TU is between 0.80～1.20); That is to say, no matter these two kinds of materials with which kind of ratio mix, be additive effect all the time.For the mixed system that this two compounds is formed, when investigating its associating biological effect, can select arbitrary proportion to unite use according to economical and practical principle, reach economical and drop into---the purpose of output benefit doulbe-sides' victory.
Further, step (1) may further comprise the steps:
At first selecting photobacterium phosphoreum for use is indicator organism, according to " national standard that the photogen water quality toxicity detects " (GB/T15441-1995), measures the single creature effect of target compound (pollutant or compound), returns the back and calculates EC with interpolation method
50(being that luminous intensity inhibition ratio is 50% o'clock a compound concentration);
1, nutrient culture media preparation:
500ml water, 15g sodium chloride, 2.5g peptone, 1.5g yeast extract, 1.5g glycerine, 2.5g potassium dihydrogen phosphate, 2.5g sodium hydrogen phosphate.Each composition is mixed by proportioning, be heated to the solution clear, the NaOH adjustment pH with 1mol/L is 6.5-7.5 then, is sub-packed in the 100mL triangular flask, and every bottle of 40mL, kraft encase and frap.At 121 ℃ of high-temperature sterilization 20min, be stored in the refrigerator 4 ℃ of preservations then after the cooling.
2, bacterium liquid preparation:
(1) according to national standard method, photogen is selected photobacterium phosphoreum (Photobacterium phosphoreum) T3 mutation for use, purchases the Nanjing soil institute in the Chinese Academy of Sciences.Add the sterilized 3%NaCl solution of 1ml in the freeze-dried powder preparation of this photogen, fully mixing is placed 2min and is promptly recovered luminous under the room temperature.After the photogen recovery, be forwarded to the test tube slant with oese immediately, at 20 ℃ of following constant temperature culture 24h, and then the switching second generation, standby in 4 ℃ of preservations behind 20 ℃ of following constant temperature culture 24h, switching in every month is once.
(2) shake a bottle bacterium liquid: get three spoonfuls of inclined-plane photogens and be transferred in the 2ml conical flask that contains the 5ml nutrient solution, standby in 20 ℃ of shaken cultivation to exponential phase.Choosing the shaken cultivation time is 12h.
(3) work bacterium liquid preparation: draw the cultured bottle bacterium liquid (stir in advance 20min expose to the sun oxygen) in 20ml 3%NaCl solution that shakes of 0.2ml, use after stirring 40min.Dilute strength is advisable 1500,000 with the luminous intensity of control blank sample.
3, test system
(1) blank sample: blank sample is made up of the 3%NaCl solution of 0.8ml and the work bacterium liquid of 0.2ml.Blank luminous intensity is I
0
(2) specimen: get 0.8ml compound standard solution in the 1ml color comparison tube, add the work bacterium liquid of 0.2ml, leaving standstill 15min mensuration light value after the mixing is I
sKey takeaway: specimen will must be prepared with blank sample, measures simultaneously.Testing tool adopts fluorescence immunity analyzer (Beijing Analytical Instrument Factory, model BH9507).Sample is done at least six gradients, three replicate determinations of each concentration point by waiting logarithmic interval to set concentration gradient at every turn.The standard deviation of 3 groups of parallel samples must not be greater than 10%.Select proper concentration, selection principle is that correlativity is good, inhibiting rate is included in the linear extent (between 20-80%).
(3) calculate: inhibiting rate calculates as follows:
Inhibiting rate=(I
0-I
s)/I
0
Be x axle (unit is mol/L) then with the compound concentration, take the inhibiting rate as the mapping of y axle, obtaining inhibiting rate with interpolation method is 50% o'clock concentration, is the EC of simplification compound
50
Further, step (2) may further comprise the steps:
The A, the single creature effective concentration (EC of two kinds of compounds of B that obtain according to mensuration
50), prepare the mixed solution of effect ratio such as, promptly two kinds of material concentrations of A, B are respectively EC in the mixed solution
50-A, EC
50-B, method of testing is measured the biological effect concentration of this mixed solution according to GB/T15441-1995, and inhibition is 50% o'clock A, B concentration (C in mixed system to biological effect can to obtain mixed system
A, C
B).
1, mixed solution preparation
Measure A, the simplification compound biological effect concentration (EC of two kinds of materials of B that obtains according to step (1)
50-A, EC
50-B), the mixed solution of effect ratios such as preparation.
2, mixed solution test series
The degree of depth of supposing this mixed solution is 100%, according to waiting logarithmic interval to dilute this mixed solution, that is: and 100%, 80%, 56%, 32%, 18%, 10%, 8% ....As test series, be test organisms then, adopt fluorescence immunity analyzer to test this serial inhibiting rate with photobacterium phosphoreum (Photobacterium phosphoreum) T3 mutation.
3, calculate: with mixed system concentration (or extension rate) is the x axle, take the inhibiting rate as the mapping of y axle, and obtaining inhibiting rate with interpolation method is 50% o'clock concentration, and can obtain this mixed system inhibiting rate is 50% o'clock, and the concentration of A, two kinds of materials of B (is C in the mixed system
A, C
B).C
A, C
BAnd simplification compound biological effect concentration EC
50-A, EC
50-BEffects such as the substitution following formula can calculate than the time associating biological effect index TU
1: 1
Wherein, C
A, C
BThe binary mixture inhibiting rate that the effect ratio such as is illustrated in is two kinds of compounds of 50% o'clock A in mixed system, B concentration separately, EC
50-A, EC
50-BExpression simplification compound inhibiting rate is 50% o'clock a concentration, and unit is mol/L.
Further, step (3) may further comprise the steps:
For etc. toxicity than the time present the mixed system of cooperative effect (TU＜0.80).Can derive, this mixed system is the strongest at the joint effect that waits effect point, when etc. during effect concentration change such as effect concentration Xiang Fei, TU approaches 1.00, that is to say from cooperative effect progressively to change to summation action.This just points out us in containing the discharge of wastewater of these compounds, the effect concentration discharge such as avoid, to reduce ecological hazard as far as possible.And agricultural chemicals unite use and medical drug combination aspect, two kinds of compounds with cooperative effect are waiting toxicity than putting an associating use, then can improve the medication effect greatly.
For the system (TU＞0.80) of picking up antiatherosclerotic effect is arranged, its etc. toxicity more weak than some associating biological effect.This just points out us when processing contains the waste water of this compounds, toxicity such as can adopt than the some discharging, utilizes it to pick up antiatherosclerotic effect, reduces processing cost and environmental hazard to greatest extent.As a same reason, for having two kinds of medicines picking up antiatherosclerotic effect, definitely avoid waiting toxicity, otherwise result of treatment will be had a greatly reduced quality than a drug combination.
For the system (TU=1.00 ± 0.20) of additive effect, its associating biological effect size keeps constant (TU is between 0.80～1.20).For this compounds, can select arbitrary proportion to unite use according to economical and practical principle, reach economical and drop into---the purpose of output benefit doulbe-sides' victory.
The present invention compared with prior art has following advantage and beneficial effect:
Prior art can only be measured binary mixture associating biological effect under a certain ratio condition, still there is not correlative study about the associating biological effect with the concentration change rule, therefore, in the conventional method, if it is the strongest or the most weak to want to understand binary compositional system associating biological effect when what ratio, can only the ratio that might form test one by one, obtain a series of TU value, compare its size then, and then obtain best biological effect point.This method workload is very big, therefore needs to seek kind of the mixed system of a method prediction simply and easily associating biological effect.This method proposes a kind of shortcut of seeking best biological effect point at this problem.
1, the inventive method has been explained the Changing Pattern of binary mixture biological effect under the variable concentrations condition, point out best biological effect point with etc. the relation of effect point, solve binary mixture and how to have obtained best this challenge of uniting biological effect, this method has characteristics such as simple and convenient, workable, applied widely, is suitable for large-scale promotion.
2, the inventive method is united aspects such as use applicable to environmental protection, medicine and agricultural chemicals.
Description of drawings
Fig. 1 represents malononitrile and paranitrobenzaldehyde mixed system biological effect curve.
Fig. 2 represents benzene dinitrile and to dimethylamino benzaldehyde mixed system biological effect curve.
Fig. 3 represents acetonitrile and terephthalaldehyde mixed system associating biological effect curve.
Embodiment
The present invention is further illustrated below in conjunction with the accompanying drawing illustrated embodiment.
Used culture medium preparation method, the compound method of bacterium liquid and the compound method of experimental system among the embodiment
1, nutrient culture media preparation:
500ml water, 15g sodium chloride, 2.5g peptone, 1.5g yeast extract, 1.5g glycerine, 2.5g potassium dihydrogen phosphate, 2.5g sodium hydrogen phosphate.Each composition is mixed by proportioning, be heated to the solution clear, the NaOH adjustment pH with 1mol/L is 6.5-7.5 then, is sub-packed in the 100mL triangular flask, and every bottle of 40mL, kraft encase and frap.At 121 ℃ of high-temperature sterilization 20min, be stored in the refrigerator 4 ℃ of preservations then after the cooling.
2, bacterium liquid preparation:
(1) add the sterilized 3%NaCl solution of 1ml in the freeze-dried powder preparation of photogen, fully mixing is placed 2min and is promptly recovered luminous under the room temperature.After the photogen recovery, be forwarded to the test tube slant with oese immediately, at 20 ℃ of following constant temperature culture 24h, and then the switching second generation, standby in 4 ℃ of preservations behind 20 ℃ of following constant temperature culture 24h, switching in every month is once.
(2) shake a bottle bacterium liquid: get three spoonfuls of inclined-plane photogens and be transferred in the 2ml conical flask that contains the 5ml nutrient solution, standby in 20 ℃ of shaken cultivation to exponential phase.Choosing the shaken cultivation time is 12h.
(3) work bacterium liquid preparation: draw the cultured bottle bacterium liquid (stir in advance 20min expose to the sun oxygen) in 20ml 3%NaCl solution that shakes of 0.2ml, use after stirring 40min.Dilute strength is advisable 1500,000 with the luminous intensity of control blank sample.
3, experimental system:
(1) blank sample: blank sample is made up of the 3%NaCl solution of 0.8ml and the work bacterium liquid of 0.2ml.
(2) specimen: get 0.8ml compound standard solution in the 1ml color comparison tube, add the work bacterium liquid of 0.2ml, leave standstill 15min after the mixing and measure light value.Sample is done at least six gradients, three replicate determinations of each concentration point by waiting logarithmic interval to set concentration gradient at every turn.The standard deviation of 3 groups of parallel samples must not be greater than 10%.Select proper concentration, selection principle is that correlativity is good, inhibiting rate is included in the linear extent (between 20-80%).
(3) calculate: with compound concentration inhibiting rate is mapped, obtaining inhibiting rate with interpolation method is 50% o'clock concentration.
4, associating biological effect judgment criterion
That mixed system associating biological effect mainly comprises is collaborative, pick up anti-and add and three kinds, and the judgment criterion of these three kinds of effects is as follows:
TU＜two kinds of compounds of 0.80 expression A, B have produced cooperative effect, and antiatherosclerotic effect is picked up in TU＞1.20 expressions, and TU=1.00 ± 0.20 expression summation action is also referred to as additive effect.(Broderius?SJ，Kahl?MD，Hoglund?MD.“ Use?of?joint?toxic?response?to?define?the?primary?mode?of?toxic?action?for?diverse?industrial?organi
(1) determination step of simplification compound biological effect concentration
In the volumetric flask that contains the 5ml nutrient culture media, add photobacterium phosphoreum (available from Nanjing soil institute of the Chinese Academy of Sciences) liquid (T3) of 0.25ml, cultivated 12 hours, make and shake a bottle bacterium liquid; Get 0.2ml then and shake bottle bacterium liquid in 20ml 3% sodium chloride, aeration agitation 40min makes work bacterium liquid.Get an amount of malononitrile and paranitrobenzaldehyde, be made into the standard series of logarithm gradient, volume is 0.8ml, add 0.2ml work bacterium liquid, shake up contamination 15min, the reduction degree of system light value before and after the mensuration contamination, calculating inhibiting rate with interpolation method is 50% o'clock concentration, is the EC of this material (or system)
50Value.The EC that calculates
50Value is to be respectively 2.803 * 10
-3, 5.492 * 10
-5Mol/L.
(2) effect such as binary is than the mensuration of the associating biological effect of mixed system
The A, the single creature effective concentration (EC of two kinds of compounds of B that obtain according to mensuration
50), adopt and simplification compound (EC then
50) measure identical step, measure the biological effect concentration of this mixed solution, inhibition is 50% o'clock A, B concentration (C in mixed system to biological effect can to obtain mixed system
A, C
B).
Further, this step specifically comprises the steps (or crucial essential implementation):
1, mixed solution preparation
The A, the simplification compound biological effect concentration (EC of two kinds of materials of B that obtain according to mensuration
50-A, EC
50-B), the mixed solution of effect ratios such as preparation.Prepare the mixed solution of effect ratio such as, promptly two kinds of material concentrations of A, B are respectively 2.803 * 10 in the mixed solution
-3, 5.492 * 10
-5Mol/L.
2, mixed solution test series
The degree of depth of supposing this mixed solution is 100%, according to waiting logarithmic interval to dilute this mixed solution, that is: and 100%, 80%, 56%, 32%, 18%, 10%, 8%.As test series, be test organisms then, adopt fluorescence immunity analyzer to test this serial inhibiting rate with photobacterium phosphoreum (Photobacterium phosphoreum) T3 mutation.
3, calculate: with mixed system concentration (or extension rate) is the x axle, take the inhibiting rate as the mapping of y axle, and obtaining inhibiting rate with interpolation method is 50% o'clock concentration, and can obtain this mixed system inhibiting rate is 50% o'clock, the concentration C of A, two kinds of materials of B in the mixed system
A, C
BBe respectively 2.102 * 10
-4, 4.119 * 10
-6Mol/L
C
A, C
BAnd simplification compound biological effect concentration EC
50-A, EC
50-BEffects such as the substitution following formula can calculate than the time associating biological effect index TU
1: 1
(3) mensuration of best biological effect point
The result show malononitrile and paranitrobenzaldehyde mixed system wait effect than the time, TU
1: 1Value is 0.15, and according to the joint effect judgment principle, TU is a cooperative effect less than 0.80.Can predict that thus malononitrile and paranitrobenzaldehyde mixed system are the strongest at the joint effect that waits effect point (cooperative effect).Concentration when mixing along with these two kinds of materials from etc. effect than effects such as Xiang Fei than changing, joint effect (cooperative effect) will weaken gradually and be summation action.
This just points out us in containing the discharge of wastewater of these compounds, the effect concentration discharge such as avoid, to reduce ecological hazard as far as possible.
(4) checking of method reliability
In order to verify our this assay method, we have measured in the associating biological effect of effect such as non-than (promptly two kinds of materials mix with other concentration ratio) under the condition, their size relatively, result show the associating biological effect of mensuration be really act synergistically the strongest.
Effect such as non-than the associating biological effect method of testing of mixed solution under the condition with etc. effect than the time method of testing identical, comprising:
1) determination step of simplification compound biological effect concentration
In the volumetric flask that contains the 5ml nutrient culture media, add the photobacterium phosphoreum liquid (T3) of 0.25ml, cultivated 12 hours, make and shake a bottle bacterium liquid; Get 0.2ml then and shake bottle bacterium liquid in 20ml 3% sodium chloride, aeration agitation 40min makes work bacterium liquid.Get an amount of malononitrile and paranitrobenzaldehyde, be made into the standard series of logarithm gradient, volume is 0.8ml, add 0.2ml work bacterium liquid, shake up contamination 15min, the reduction degree of system light value before and after the mensuration contamination, calculating inhibiting rate with interpolation method is 50% o'clock concentration, is the EC of this material (or system)
50Value.The EC that calculates
50Value is to be respectively 2.803 * 10
-3, 5.492 * 10
-5Mol/L.
Effect such as 2) binary is non-is than the mensuration of the associating biological effect of mixed system
(1), effect such as non-is prepared than mixed solution
The A, the simplification compound biological effect concentration (EC of two kinds of materials of B that obtain according to mensuration
50-A, EC
50-B), the mixed solution of effect ratios such as preparation.Prepare the mixed solution of a series of effect ratios such as non-, suppose two material effects than being n: m, promptly two kinds of material degree of A, B are respectively n * EC in the mixed solution
50-A, m * EC
50-B(n * 2.803 * 10
-3, m * 5.492 * 10
-5Mol/L).
(2), mixed solution test series
The degree of depth of supposing this mixed solution is 100%, according to waiting logarithmic interval to dilute this mixed solution, that is: and 100%, 80%, 56%, 32%, 18%, 10%, 8%.......As test series, be test organisms then, adopt fluorescence immunity analyzer to test this serial inhibiting rate with photobacterium phosphoreum (Photobacterium phosphoreum) T3 mutation.
(3), calculate: with mixed system concentration (or extension rate) is the x axle, take the inhibiting rate as the mapping of y axle, and obtaining inhibiting rate with interpolation method is that 50% o'clock concentration is C '
A, C '
BC '
A, C '
BAnd simplification compound biological effect concentration EC
50-A, EC
50-BEffects such as the substitution following formula can calculate than the time associating biological effect index TU
1: 1
Table 1
With log n/m is horizontal ordinate, and the TU during the variable concentrations ratio is an ordinate, draws the binary mixture joint effect with concentration curve.
(n and m are illustrated in the concentration multiple of A in the mixed system, two kinds of materials of B to log n/m sign horizontal ordinate, and promptly two kinds of material degree of A, B are respectively n * EC in the mixed solution among Fig. 1
50-A, m * EC
50-B), the TU during the variable concentrations ratio is an ordinate, draws the binary mixture joint effect with concentration curve.
As seen from Figure 1, when waiting effect ratio point (logn/m=0), TU
1: 1Minimum, promptly synergy is the strongest.Along with concentration from etc. effect than skew such as effect point such as some Xiang Fei etc., TU value is increased to 1.00 ± 0.20 gradually, promptly synergy weakens gradually, finally weakens up to summation action.This result has shown that the used Forecasting Methodology of the present invention is accurate, reliable, can distich symphysis thing effect change rule predict well, and seeks best biological effect point.
(1) determination step of simplification compound biological effect concentration
In the volumetric flask that contains the 5ml nutrient culture media, add the photobacterium phosphoreum liquid (T3) of 0.25ml, cultivated 12 hours, make and shake a bottle bacterium liquid; Get 0.2ml then and shake bottle bacterium liquid in 20ml3% sodium chloride, aeration agitation 40min makes work bacterium liquid.Get an amount of benzene dinitrile (A compound) and paradime thylaminobenzaldehyde (B compound), be made into the standard series of logarithm gradient, volume is 0.8ml, add 0.2ml work bacterium liquid, shake up contamination 15min, the reduction degree of system light value before and after the mensuration contamination, calculating inhibiting rate with interpolation method is 50% o'clock concentration, is the EC of this material (or system)
50Value.Calculate the EC of benzene dinitrile (A compound) and paradime thylaminobenzaldehyde (B compound)
50Value is to be respectively 5.860 * 10
-4, 2.229 * 10
-6Mol/L.
(2) effect such as binary is than the mensuration of the associating biological effect of mixed system
The A, the single creature effective concentration (EC of two kinds of compounds of B that obtain according to mensuration
50), adopt and simplification compound (EC
50) measure identical step, measure the biological effect concentration of this mixed solution, inhibition is 50% o'clock A, B concentration (C in mixed system to biological effect can to obtain mixed system
A, C
B).
Further, this step specifically comprises the steps (or crucial essential implementation):
1, mixed solution preparation
The A, the simplification compound biological effect concentration (EC of two kinds of materials of B that obtain according to mensuration
50-A, EC
50-B), the mixed solution of effect ratios such as preparation.Prepare the mixed solution of effect ratio such as, promptly two kinds of material concentrations of A, B are respectively 5.860 * 10 in the mixed solution
-4, 2.229 * 10
-6Mol/L.
2, mixed solution test series
The degree of depth of supposing this mixed solution is 100%, according to waiting logarithmic interval to dilute this mixed solution, that is: and 100%, 80%, 56%, 32%, 18%, 10%, 8%.As test series, be test organisms then, adopt fluorescence immunity analyzer to test this serial inhibiting rate with photobacterium phosphoreum (Photobacterium phosphoreum) T3 mutation.
3, calculate: with mixed system concentration (or extension rate) is the x axle, take the inhibiting rate as the mapping of y axle, and obtaining inhibiting rate with interpolation method is 50% o'clock concentration, and can obtain this mixed system inhibiting rate is 50% o'clock, the concentration C of A, two kinds of materials of B in the mixed system
A, C
BBe respectively 4.952 * 10
-4, 1.884 * 10
-6Mol/L
C
A, C
BAnd simplification compound biological effect concentration EC
50-A, EC
50-BEffects such as the substitution following formula can calculate than the time associating biological effect index TU
1: 1
(3) mensuration of best biological effect point
The result show benzene dinitrile and paradime thylaminobenzaldehyde mixed system wait effect than the time, TU
1: 1Value is 1.69, according to the joint effect judgment principle, TU greater than 1.20 for picking up antiatherosclerotic effect.Can predict that thus benzene dinitrile and paradime thylaminobenzaldehyde mixed system are the strongest at the joint effect that waits effect point (picking up anti-).Concentration when mixing along with these two kinds of materials from etc. effect than effects such as Xiang Fei than changing, joint effect (picking up antiatherosclerotic effect) will weaken gradually and be summation action.
This just points out us when processing contains the waste water of this compounds, toxicity such as can adopt than the some discharging, utilizes it to pick up antiatherosclerotic effect, reduces processing cost and environmental hazard to greatest extent.As a same reason, for having two kinds of medicines picking up antiatherosclerotic effect, definitely avoid waiting toxicity, otherwise result of treatment will be had a greatly reduced quality than a drug combination.
(4) checking of method reliability
In order to verify our this assay method, we have measured in the associating biological effect of effect such as non-than (promptly two kinds of materials mix with other concentration ratio) under the condition, their size relatively, result show the associating biological effect of measuring be really act synergistically the strongest.The result as shown in Figure 2.
(n and m are illustrated in the concentration multiple of A in the mixed system, two kinds of materials of B to log n/m sign horizontal ordinate, and promptly two kinds of material degree of A, B are respectively n * EC in the mixed solution among Fig. 2
50-A, m * EC
50-B), the TU during the variable concentrations ratio is an ordinate, draws the binary mixture joint effect with concentration curve.
As seen from Figure 2, when waiting effect ratio point (logn/m=0), TU
1: 1Maximum, it is the strongest promptly to pick up anti-effect.Along with concentration from etc. effect than skew such as effect point such as some Xiang Fei etc., TU value is reduced to 1.00 ± 0.20 gradually, promptly picks up anti-effect to weaken gradually, finally weakens up to summation action.This result has shown that the used Forecasting Methodology of the present invention is accurate, reliable, can distich symphysis thing effect change rule predict well, and seeks best biological effect point.
(1) determination step of simplification compound biological effect concentration
In the volumetric flask that contains the 5ml nutrient culture media, add the photobacterium phosphoreum liquid (T3) of 0.25ml, cultivated 12 hours, make and shake a bottle bacterium liquid; Get 0.2ml then and shake bottle bacterium liquid in 20ml 3% sodium chloride, aeration agitation 40min makes work bacterium liquid.Get an amount of acetonitrile (A material) and terephthalaldehyde (B material) and be made into the standard series of logarithm gradient, volume is 0.8ml, add 0.2ml work bacterium liquid, shake up, contamination 15min, measure the reduction degree of system light value before and after the contamination, calculating inhibiting rate with interpolation method is 50% o'clock concentration, is the EC of this material (or system)
50Value.The EC that calculates
50Value is to be respectively 0.189,6.892 * 10
-5Mol/L.
(2) effect such as binary is than the mensuration of the associating biological effect of mixed system
The A, the single creature effective concentration (EC of two kinds of compounds of B that obtain according to mensuration
50), adopt and simplification compound (EC then
50) measure identical step, measure the biological effect concentration of this mixed solution, inhibition is 50% o'clock A, B concentration (C in mixed system to biological effect can to obtain mixed system
A, C
B).
Further, this step specifically comprises the steps (or crucial essential implementation):
1, mixed solution preparation
The A, the simplification compound biological effect concentration (EC of two kinds of materials of B that obtain according to mensuration
50-A, EC
50-B), the mixed solution of effect ratios such as preparation.Prepare the mixed solution of effect ratio such as, promptly two kinds of material concentrations of A, B are respectively 0.189,6.892 * 10 in the mixed solution
-5Mol/L.
2, mixed solution test series
The degree of depth of supposing this mixed solution is 100%, according to waiting logarithmic interval to dilute this mixed solution, that is: and 100%, 80%, 56%, 32%, 18%, 10%, 8%.As test series, be test organisms then, adopt fluorescence immunity analyzer to test this serial inhibiting rate with photobacterium phosphoreum (Photobacterium phosphoreum) T3 mutation.
3, calculate: with mixed system concentration (or extension rate) is the x axle, take the inhibiting rate as the mapping of y axle, and obtaining inhibiting rate with interpolation method is 50% o'clock concentration, and can obtain this mixed system inhibiting rate is 50% o'clock, the concentration C of A, two kinds of materials of B in the mixed system
A, C
BBe respectively 0.103,3.756 * 10
-5Mol/L
C
A, C
BAnd simplification compound biological effect concentration EC
50-A, EC
50-BEffects such as the substitution following formula can calculate than the time associating biological effect index TU
1: 1
(3) mensuration of best biological effect point
The result show acetonitrile and terephthalaldehyde mixed system wait effect than the time, TU
1: 1Value is 1.09, and according to the joint effect judgment principle, TU=1.00 ± 0.20 is an additive effect.Can predict thus, for wait effect than the time be the system (acetonitrile and terephthalaldehyde) of the mixture of additive effect, its joint toxicity effect size keeps constant (TU is between 0.8～1.2), does not change with two compound concentrations.For this compounds, can select proper ratio to unite use according to economical and practical principle, reach economical and drop into---the purpose of output benefit doulbe-sides' victory.
(4) checking of method reliability
In order to verify our this assay method, we have measured in the associating biological effect of effect such as non-than (promptly two kinds of materials mix with other concentration ratio) under the condition, their size relatively, and the result is as shown in Figure 3.
(n and m are illustrated in the concentration multiple of A in the mixed system, two kinds of materials of B to log n/m sign horizontal ordinate, and promptly two kinds of material degree of A, B are respectively n * EC in the mixed solution among Fig. 3
50-A, m * EC
50-B), the TU during the variable concentrations ratio is an ordinate, draws the binary mixture joint effect with concentration curve.
As seen from Figure 3, under the variable concentrations ratio, measure the associating biological effect be all to be additions really, do not change with concentration.This result has shown that the used Forecasting Methodology of the present invention is accurate, reliable, can distich symphysis thing effect change rule predict well, and seeks best biological effect point.
The above-mentioned description to embodiment is can understand and apply the invention for ease of those skilled in the art.The person skilled in the art obviously can easily make various modifications to these embodiment, and needn't pass through performing creative labour being applied in the General Principle of this explanation among other embodiment.Therefore, the invention is not restricted to the embodiment here, those skilled in the art are according to announcement of the present invention, and not breaking away from the improvement that category of the present invention makes and revise all should be within protection scope of the present invention.
Claims (3)
1. measure the medicine compatibility method that two-component mixture obtains best associating biological effect for one kind, it is characterized in that: this method may further comprise the steps,
(1) mensuration of simplification compound biological effect concentration
Method of testing is indicator organism according to GB/T 15441-1995 with the photobacterium phosphoreum, measures the single toxicity of A, B two compounds, and simplification compound biological effect adopts EC50 to represent that these two kinds of simplification compound biological effect concentration are respectively EC
50-A, EC
50-B
(2) etc. effect than the time associating biological effect mensuration
The A that obtains according to mensuration and the single creature effective concentration EC of two kinds of compounds of B
50-AAnd EC
50-B, prepare the mixed solution of effect ratio such as, promptly two kinds of material concentrations of A and B are respectively EC in the mixed solution
50-AAnd EC
50-B, method of testing is measured the biological effect concentration of this mixed solution according to GB/T 15441-1995, and can obtain that mixed system suppresses biological effect is 50% o'clock A and the concentration C of B in mixed system
AAnd C
B, calculate then etc. effect than the time associating biological effect index TU
1: 1
(3) mensuration of best biological effect point
According to step (2) calculate etc. effect than the measurement result TU of time associating biological effect
1: 1, measure the associating biological effect Changing Pattern of binary mixture, and obtain best biological effect point.
2. mensuration two-component mixture according to claim 1 obtains the medicine compatibility method of best associating biological effect, it is characterized in that: the size of described associating biological effect index TU is used for estimating the associating biological effect,
TU
1: 1＜0.80 expression A and two kinds of compounds of B have produced cooperative effect;
TU
1: 1＞1.20 expression A and two kinds of compounds of B have produced picks up antiatherosclerotic effect;
TU
1: 1=1.00 ± 0.20 expression A and two kinds of compounds of B have produced additive effect.
3. mensuration two-component mixture according to claim 1 obtains the medicine compatibility method of best associating biological effect, it is characterized in that: described TU
1: 1Adopt following formula to calculate
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