CN101717811A - Method for evaluating biological security of solubilizing auxiliary materials - Google Patents
Method for evaluating biological security of solubilizing auxiliary materials Download PDFInfo
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- CN101717811A CN101717811A CN200910237161A CN200910237161A CN101717811A CN 101717811 A CN101717811 A CN 101717811A CN 200910237161 A CN200910237161 A CN 200910237161A CN 200910237161 A CN200910237161 A CN 200910237161A CN 101717811 A CN101717811 A CN 101717811A
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Abstract
The invention relates to a method for evaluating the biological security of solubilizing auxiliary materials. In the method, tetrahymena thermophile is taken as a model, a microcalorimetry is combined, the influence on the growth metabolism of the tetrahymena thermophile from the solubilizing auxiliary materials is inspected, and the biological security of the solubilizing auxiliary materials is evaluated. By carrying out information analysis on a thermal activity spectrum with integral characteristics and adopting characteristic parameters that reflect different growth metabolism stages, the invention inspects the influence on the growth metabolism of the tetrahymena thermophile from the solubilizing auxiliary materials totally, objectively and quantitively, evaluates the biological security of the solubilizing auxiliary materials, is simple in operation and is beneficial to the detection normalization.
Description
Technical field
The present invention relates to a kind of method of evaluating biological security of solubilizing auxiliary materials, belong to field of pharmaceutical excipients.Particularly, the present invention is a living model with tetrahymena thermophila, in conjunction with microcalorimetry, investigates the influence of solubilizing auxiliary materials to tetrahymena thermophila's growth metabolism, thereby estimates the biological safety of solubilizing auxiliary materials.
Background technology
Solubilizing auxiliary materials is a class nonionogenic tenside mostly, and existing hydrophilic radical in its structure has lipophilic group again, can form micella or reverse micelle in solution, uses in the insoluble medicine liquid preparation as solubilizing agent, emulsifying agent.Along with improving of modern liquid preparation, and the appearance of modern novel form, it is important that the status of solubilizing auxiliary materials more shows.But existing toxicants such as oxyethane, dioxane, ethylene chlorhydrin, Diethylene Glycol residual in solubilizing auxiliary materials itself and the production storage process, is one of its reason that safety issue takes place frequently in application, especially in injection.Existing method for evaluating safety is mainly by conventional pharmacological evaluation such as red blood cell suspension haemolysis or aggegations, though the result is more directly perceived, that judges is subjective, and error is big, is difficult to realize the objective quantification evaluation.
Tetrahymena thermophila (Tetrahymena thermophila) is a unicellular eucaryon protozoon common in a kind of fresh water, not only have the typical subcellular structure of visible in most eukaryotic cells, and have good similarity with the higher animal cell.Its acellular wall directly contacts with environment by cytolemma, can react rapidly to environmental change, demonstrates the susceptibility higher than higher animal cell; And its growth meets typical microbial growth curve model.Can analyze the response situation of tetrahymena thermophila by the dynamic growth curve in view of the above to the poisonous substance character in the growing environment of living in.At present the tetrahymena thermophila has become the monitoring of biology and pharmaceutical research, environmental safety, and the ideal material of aquatic ecosystem pollutent safety toxicological evaluation, but it is used for pharmaceutical excipient, the especially evaluation of injection solubilizing auxiliary materials and yet there are no report.
Characteristics such as microcalorimetry has fast, sensitivity, good reproducibility can dynamically be followed the trail of the growth metabolism situation of organism under drug effect continuously, and this method independently carries out in the environment sealing, and are difficult for being affected by the external environment; Widespread use aspect micro-thermal distortion (neither endothermic nor exothermic) rules of organism in the growth metabolism process such as mensuration microorganism, cell, tissue and organ.
Know-why:
Be accompanied by metabolism in tetrahymena thermophila's growth, breeding, the decline process, heat release is arranged, the heat of release is with the variation relation in vegetative period, and promptly heat production power-time curve is called biological heat activity profile (claiming " thermography curve " or " biological effect spectrum " again).Under the effect of different pharmaceutical material, this biological effect spectrum meeting pests occurrence rule, characteristic change.
Tetrahymena thermophila's growth is similar to general microbial growth curve under set condition.Growth curve is divided into four phases, i.e. lag phase, exponential phase of growth, stationary phase and decline phase.Tetrahymena thermophila's growth rhythm index of coincidence growth mathematical model of exponential phase of growth.
P
t=P
0Exp (kt) or lnP
t=lnP
0+ kt (1)
In the formula, P
0Be tetrahymena thermophila's heat production power of exponential growth starting point, P
tBe t tetrahymena thermophila's heat production power constantly, k is a growth rate constant.Also can extract maximum heat production power (Pmax simultaneously
1, Pmax
2), peak time (T
1, T
2), the characteristic parameter of quantity of heat production hot activity profiles such as (Q).
Summary of the invention
The method that the purpose of this invention is to provide a kind of evaluating biological security of solubilizing auxiliary materials.
Method of the present invention is a model with tetrahymena thermophila, in conjunction with microcalorimetry, by investigating the influence of solubilizing auxiliary materials to tetrahymena thermophila's growth metabolism, estimates the biological safety of solubilizing auxiliary materials.
Said method comprising the steps of:
1) get solubilizing auxiliary materials 0.3-10.0mg respectively, add an amount of distilled water, be made into the solubilizing auxiliary materials solution of a series of different concns, under the aseptic condition, filter with 0.22 μ m filter membrane, 4 ℃ of preservations are standby;
2) under 25-37 ℃ of constant temperature, in each ampoule, tetrahymena thermophila's suspension 20-100 μ L is inoculated in the 1-5mL aseptic culture medium, the concentration of the preferred tetrahymena thermophila's suspension in inoculation back is 1000-2500 cell/mL, the solubilizing auxiliary materials solution 60-240 μ L that adds different concns respectively, place micro-calorimeter to measure the heat production power of tetrahymena thermophila's growth metabolism process, record heat production power-time curve;
3) relation of characteristic parameter or its parameter of deriving in the different concns of analysis solubilizing auxiliary materials and the heat production power-time curve, obtain promoter action, restraining effect or the toxic action result of solubilizing auxiliary materials, the biological safety of preliminary assessment solubilizing auxiliary materials to tetrahymena thermophila's growth metabolism;
Wherein, described characteristic parameter comprises growth rate constant, maximum heat production power, peak time and quantity of heat production, and the described parameter of deriving comprises inhibiting rate and 5% inhibition concentration (IC
5).
Aforesaid method can further include following steps:
4) characteristic parameter described in the step 3) is carried out principle component analysis, extraction can be represented the characteristic parameter of heat production power-time curve information more comprehensively;
5) by score coefficient and its contribution rate of each principal constituent, set up the composite evaluation function of solubilizing auxiliary materials, calculate the score of solubilizing auxiliary materials, the biological safety of comprehensive evaluation solubilizing auxiliary materials.
Wherein, the optimizing injection of solubilizing auxiliary materials described in step 1) solubilizing auxiliary materials, more preferably polysorbas20, tween 80, poly(oxyethylene glycol) 400, Polyethylene Glycol-600 or poloxamer 188; Typical case's representative that described preferred auxiliary material is a nonionogenic tenside, more extensive in the field of medicaments application, have solublization preferably; In addition, auxiliary materials such as tween 80, poloxamer 188 are existing at present to be used, but because still there is the potential potential safety hazard in its quality standard imperfection, therefore is necessary it is carried out the biological safety evaluation;
The concentration range of described solubilizing auxiliary materials solution is 0.3-10.0mg/mL, preferred series concentration 0.3mg/mL, 0.6mg/mL, 0.8mg/mL, 1.0mg/mL, 1.2mg/mL, more preferably series concentration 0.5mg/mL, 1.0mg/mL, 2.0mg/mL, 3.0mg/mL, 4.0mg/mL, more preferably series concentration 1.0mg/mL, 4.0mg/mL, 6.0mg/mL, 8.0mg/mL, 10.0mg/mL;
Step 2) preferred 28 ℃ of constant temperatures in, the concentration of the preferred tetrahymena thermophila's suspension in inoculation back is 1500 cell/mL, the volume that preferably adds solubilizing auxiliary materials is 120 μ L.
The present invention has measured the thermography curve of tetrahymena thermophila's growth metabolism under the different solubilizing auxiliary materials effects, and each characteristic parameter of use heat spectral curve is estimated, and investigates the biological safety of each solubilizing auxiliary materials.
The present invention adopts microcalorimetry to investigate the influence of different solubilizing auxiliary materials to tetrahymena thermophila's growth metabolism, with growth rate constant (k), maximum heat production power (Pmax
1, Pmax
2), peak time (T1, T2) and quantity of heat production biological heat dynamic characteristic parameters such as (Q) be index, and solubilizing auxiliary materials has been carried out objective quantitative evaluation to the influence of tetrahymena thermophila's growth metabolism.Having set up with tetrahymena thermophila is living model, adopts microcalorimetry that solubilizing auxiliary materials is carried out the method that biological safety is estimated.
The present invention is with the comprehensive mechanism of solubilizing auxiliary materials to tetrahymena thermophila's growth, by the thermography curve with globality feature is carried out information analysis, adopt the characteristic parameter of reflection different steps, comprehensively, objective quantification investigated the influence of solubilizing auxiliary materials, estimated the biological safety of solubilizing auxiliary materials tetrahymena thermophila's growth metabolism.This method is simple to operate, the standardization that helps detecting.
Technique effect of the present invention is:
1) is model with tetrahymena thermophila,, set up new biological security of solubilizing auxiliary materials evaluation method, estimate significant for the biological safety of injection solubilizing auxiliary materials in conjunction with microcalorimetry.
2) this method is carried out in the environment sealing independently, disturb little, truly dynamically reflection solubilizing auxiliary materials and tetrahymena thermophila's interaction comprehensively, the biological safety of objective evaluation solubilizing auxiliary materials.
3) selected living model can truly characterize the toxic effect of solubilizing auxiliary materials, thereby provides reference frame for its security.
4) this method is optimized conditions such as the concentration of tetrahymena thermophila's suspension, the volume that adds solubilizing auxiliary materials and growth temperatures, helps accurate mensuration.
5) parameter of Ping Jiaing is comprehensive, objective and can quantize, and automated operation is simple, the standardization that helps detecting.
6) set up the integrated evaluating method of solubilizing auxiliary materials to tetrahymena thermophila's growth effect.
Description of drawings
Fig. 1 is the heat production power-time curve of tetrahymena thermophila's growth metabolism under the normal condition.
Fig. 2 is the heat production power-time curve of tetrahymena thermophila's growth metabolism under the different solubilizing auxiliary materials effects.
Fig. 3 is the relation of tetrahymena thermophila's growth rate constant and concentration under the different solubilizing auxiliary materials effects.
Fig. 4 is the comprehensive evaluation shot chart of each sample.
Embodiment
Following examples are used to illustrate the present invention, but are not used for limiting the scope of the invention.
1. instrument: the heat-conducted isothermal calorimeter of TAM III 12 passage milliwatt levels (TA company, the U.S.), YXQ LS-B full-automatic vertical electric-heating steam Sterilizers (Shanghai falls and opens up plant and instrument company limited), 303AB-3 type water isolation type incubator (instrument company limited is established in Shanghai).
2. material and reagent: tetrahymena thermophila (Tetrahymena thermophila BF5) is provided by Wuhan University; Substratum (is got peptone 15g, yeast powder 5g, glucose 1g, is dissolved in the 1000mL distilled water; Get 5 parts, every part of 10mL is sub-packed in the test tube, and 121 ℃ of sterilization 20min cool off standby); Polysorbas20 (east, Beijing ring amalgamation factory, lot number: 960703); Tween 80 (Jiangsu Chenpai Pharmaceutical Co., Ltd, lot number: 9080220); Poly(oxyethylene glycol) 400 (Shantou Xilong Chemical Factory, Guangdong, lot number: 060908); Polyethylene Glycol-600 (Tianjin section close europeanized reagent development centre, lot number: 2041027); Poloxamer 188 (is gone up Taiwan Strait Exchange Association safe auxiliary material company limited, lot number: 090309); Sterile distilled water.
3. tetrahymena thermophila's cultivation of going down to posterity: get the substratum of sterilization, insert tetrahymena thermophila's suspension of 200 μ L exponentials phase of growth, in 28 ℃ of incubators, enter stationary phase behind the static cultivation 4d, standby.
1. trial-product preparation
Get 5 kinds of solubilizing auxiliary materials, every kind of auxiliary material is got 0.5mg, 1.0mg, 2.0mg, 3.0mg, 4.0mg respectively, the accurate title, decide, and adds the 1mL dissolved in distilled water, is mixed with concentration each solubilizing auxiliary materials solution of 0.5mg/mL, 1.0mg/mL, 2.0mg/mL, 3.0mg/mL, 4.0mg/mL respectively.Under the aseptic condition, filter with 0.22 μ m filter membrane, 4 ℃ of preservations are standby.
2. measuring method
Adopt the ampoule method, under 28 ℃ of constant temperatures, treat the instrument system baseline stability after, in each ampoule, tetrahymena thermophila's suspension inoculation that 50 μ L are grown stationary phase forms suspension in the 2mL aseptic culture medium, the concentration that makes tetrahymena thermophila's suspension is 1500 cell/mL.Add solubilizing auxiliary materials solution 120 μ L, mix, place micro-calorimeter to measure the heat production power of tetrahymena thermophila's growth metabolism process, record heat production power-time curve.Treat to finish experiment after curve is got back to baseline.
3. result
3.1 tetrahymena thermophila's mensuration of thermal power curve of growing under the normal condition
According to said determination method item, do not add solubilizing auxiliary materials (being blank sample), measure under the normal condition thermography curve of tetrahymena thermophila's growth metabolism and (seen Fig. 1, wherein A-B is that lag phase, B-C are that exponential phase of growth, C-D are that stationary phase, D-E are decline phase), it meets the microbial growth curve model.According to formula (1), lnPt, the t value of exponential phase of growth on the heat production power-time curve that obtains in the parallel laboratory test are carried out linear fit, obtain tetrahymena thermophila (BF
5) growth rate constant k.The match growth rate constant sees Table 1, and circulation ratio is good as a result.
Tetrahymena thermophila's growth rate constant measurement result under table 1 normal condition
| Experimental group | ??1 | ??2 | ??3 | ??4 | ??5 | ??6 |
| Growth rate constant k (h -1) | ??0.4335 | ??0.4302 | ??0.4347 | ??0.4365 | ??0.4352 | ??0.4325 |
| Correlation coefficient r | ??0.9853 | ??0.9854 | ??0.9918 | ??0.9878 | ??0.9913 | ??0.9886 |
3.2 the mensuration of tetrahymena thermophila's growth metabolism thermography curve has been measured the growth metabolism thermography curve of tetrahymena thermophila under different solubilizing auxiliary materials effects respectively, as shown in Figure 2 according to said determination method item under the different solubilizing auxiliary materials effects.
Parametric measurement the results are shown in Table 2.By analyzing as can be known, along with the increase of each solubilizing auxiliary materials concentration, tetrahymena thermophila's growth rate constant reduces.Wherein under the part concentration of tween 80 and poloxamer 188, big under the normal condition of its growth rate constant, and the linear relationship between the concentration better (P＜0.05).The tetrahymena thermophila constantly increases in generation time of growing.The first maximum heat production power reduces with the increase of solubilizing auxiliary materials concentration, except that the lower concentration of tween 80, its with normal condition under tetrahymena thermophila's heat production power compare all less; The second maximum heat production power also reduces with the increase of concentration, until disappearance.The existence that solubilizing auxiliary materials is described has produced restraining effect to tetrahymena thermophila's growth metabolism.
The different solubilizing auxiliary materials of table 2 are to the parametric measurement result of the thermography curve of tetrahymena thermophila's growth metabolism influence
Annotate: C represents solubilizing auxiliary materials concentration; K represents tetrahymena thermophila's growth rate constant; R represents the relation conefficient of k value match; T
1, T
2The expression peak time; Pmax
1, Pmax
2Represent maximum heat production power; I represents inhibiting rate.
3.3 inhibiting rate and 5% inhibition concentration (IC
5) mensuration
According to the parametric measurement result in the table 2 as can be known, different solubilizing auxiliary materials are to tetrahymena thermophila's growth-inhibiting difference, so be that index is calculated inhibiting rate (I) with the growth velocity:
I＝[(k
0-k)/k
0]×100％????????????????????????(2)
In the formula, k
0Be the growth rate constant of tetrahymena thermophila exponential phase of growth under the normal condition, k is the growth rate constant of tetrahymena thermophila exponential phase of growth under the solubilizing auxiliary materials effect.By calculating IC
5Be respectively: polysorbas20 is 0.0407mg/mL, and tween 80 is 1.202mg/mL, and poly(oxyethylene glycol) 400 is 0.668mg/mL, and Polyethylene Glycol-600 is 1.358mg/mL, and poloxamer 188 is 2.402mg/mL.Under the same concentrations, polysorbas20 suppresses stronger to tetrahymena thermophila's exponential growth metabolism stage as can be known, and toxicity is stronger.The restraining effect of poloxamer 188, Polyethylene Glycol-600 a little less than, tetrahymena thermophila's exponential growth metabolism stage is suppressed less, toxicity a little less than.
3.4 growth rate constant and solubilizing auxiliary materials concentration relationship
The growth rate constant k of exponential phase of growth sees Fig. 3 with the variation tendency of each solubilizing auxiliary materials different concns.Fitting of a straight line equation following (removing initial point), relation conefficient check P＜0.01, the concentration of each solubilizing auxiliary materials and growth rate constant k have the better linearity relation.
Polysorbas20: k=-0.0952c+0.3674 r=-0.9711
Tween 80: k=-0.0542c+0.4709 r=-0.9953
Poly(oxyethylene glycol) 400: k=-0.0380c+0.4332 r=-0.9801
Polyethylene Glycol-600: k=-0.0302c+0.4663 r=-0.9901
Poloxamer 188:k=-0.0216c+0.4678 r=-0.9918
Along with the increase of each solubilizing auxiliary materials concentration, tetrahymena thermophila's growth rate constant is all reducing, and shows that the increase of concentration can increase its toxic action to the tetrahymena thermophila.From the absolute value of solubilizing auxiliary materials concentration and growth rate constant relation curve slope as can be known, its toxic action is: the polysorbas20 maximum, and tween 80 and poly(oxyethylene glycol) 400 are taken second place, and Polyethylene Glycol-600 and poloxamer 188 are less.This shows the increase along with concentration, and polysorbas20 is very fast to toxic increase tetrahymena thermophila's exponential phase of growth, and the increase of Polyethylene Glycol-600 and poloxamer 188 is slower.
3.5 the principle component analysis of thermography parameter
By thermography curve and characteristic parameter as can be known, different solubilizing auxiliary materials there are differences the influence of tetrahymena thermophila's growth metabolism.With X1～X6 is variable, respectively representation parameter growth rate constant k, heat production power Q, peak time T
1, the first maximum heat production power P max
1, peak time T
2, the second maximum heat production power P max
2, adopt SPSS13.5 to carry out principle component analysis, extraction can be represented the characteristic parameter of thermography calibration curve information more comprehensively.
By the initial characteristics value (Initial Eigenvalues) of the characteristic parameter of principle component analysis, the summing value (Extraction Sums of Squared Loadings) of extracting load square and rotary load sum of squares (Rotation Sums of Squared loadings) as can be known, the eigenwert of preceding two principal constituents is all greater than 1, accumulation contribution rate (V) is 82.751%, and promptly preceding two principal constituents are represented 82.751% information of original parameter.See Table 3.
The comprehensive evaluation result of each sample of table 3
| Sample | | Polysorbas20 | Tween | 80 | Poly(oxyethylene glycol) 400 | Polyethylene Glycol-600 | Poloxamer 188 | Contribution rate (%) |
| The 1st principal component scores | ??-0.567 | ??-1.699 | ??0.877 | ??-0.025 | ??0.602 | ??0.812 | ??55.539 | |
| The 2nd principal component scores | ??0.971 | ??-0.477 | ??-1.385 | ??-0.220 | ??-0.233 | ??1.344 | ??27.212 | |
| The 3rd principal component scores | ??0.576 | ??0.306 | ??1.103 | ??-1.572 | ??-0.839 | ??0.426 | ??13.253 | |
| The 4th principal component scores | ??-0.380 | ??0.482 | ??-0.497 | ??-1.275 | ??1.658 | ??0.012 | ??3.084 | |
| The 5th principal component scores | ??-1.557 | ??0.852 | ??-0.119 | ??0.136 | ??-0.544 | ??1.233 | ??0.912 | |
| The 6th principal component scores | ??0 | ??0 | ??0 | ??0 | ??0 | ??0 | ??1*e-14 |
| Sample | | Polysorbas20 | Tween | 80 | Poly(oxyethylene glycol) 400 | Polyethylene Glycol-600 | Poloxamer 188 | Contribution rate (%) |
| Integrate score | ??-0.040 | ??-101.016 | ??24.008 | ??-32.010 | ??20.596 | ??88.463 | ??100 |
By the composition matrix of loadings as can be known, extracted two principal constituents, represented that with F its expression formula is:
F1＝0.943X1+0.969X2-0.510X3+0.989X4-0.342X5-0.384X6
F2＝0.080X1-0.038X2-0.816X3+0.030X4+0.228X5+0.522X6
The absolute value of coefficient is big more, illustrates that the relation of principal constituent and this parameter is close more.Therefore, first principal component and Q, k, Pmax
1In close relations, Second principal component, and T
1And Pmax
2In close relations.Two principal constituents have been represented 82.751% information of original 6 indexs.
By composition score matrix of coefficients as can be known, the expression formula of two principal constituents is as follows:
Z1＝0.283X1+0.291X2-0.0153X3+0.297X4-0.103X5-0.115X6
Z2＝0.049X1-0.024X2-0.500X3+0.019X4+0.507X5+0.320X6
3.6 solubilizing auxiliary materials is to the comprehensive evaluation of tetrahymena thermophila's growth effect
What the thermography curve was investigated is the Global Information of tetrahymena thermophila's growth metabolism, and the parameter of extraction also is the comprehensive parameters of its growth metabolism of reflection.Therefore, set up a kind of mathematical model evaluation method of science integration.By score coefficient and its contribution rate of each principal constituent, set up the composite evaluation function of each sample, calculate the score of each sample:
S＝V1*Z1+V2*Z2+V3*Z3+...+Vn*Zn
In the formula, S represents the sample integrate score; V represents each composition contribution rate; Z shows the score of each composition.Calculate the comprehensive mark of each sample.The results are shown in Table 3 and Fig. 4.As shown in Figure 4, compare with blank, poloxamer 188 scores are the highest, and tween 80 and Polyethylene Glycol-600 take second place, and polysorbas20 is minimum.Point out our poloxamer 188 and tween 80 less, the toxicity maximum of polysorbas20 to tetrahymena thermophila's toxicity.
4. conclusion
The result shows, poloxamer 188 (0～2.0mg/mL), Polyethylene Glycol-600 (0～1.0mg/mL), (growth rate constant under 0～0.5mg/mL) effect is big than the barren value, and tetrahymena thermophila's growth is had promoter action for tween 80.Polysorbas20, poly(oxyethylene glycol) 400 can suppress its growth to the toxic effect of tetrahymena thermophila's growth.Under the effect of 5 kinds of solubilizing auxiliary materials part lower concentrations, heat production power P max
1, Pmax
2Than the blank value height, under the effect of 5 kinds of solubilizing auxiliary materials part high densitys, heat production power P max
1, Pmax
2Low than blank value; No matter high density or lower concentration, quantity of heat production Q is low than normal value.By IC
5, principle component analysis and integrate score result as can be known, under the same concentrations, poloxamer 188, tween 80 and Polyethylene Glycol-600 are little than other to tetrahymena thermophila's toxicity, three's safety range is bigger.Analysis-by-synthesis as can be known, different solubilizing auxiliary materials are different to tetrahymena thermophila's growth metabolism influence.By the parameter analysis of growth thermography curve, can better estimate the biological safety of each solubilizing auxiliary materials.
Claims (9)
1. the method for an evaluating biological security of solubilizing auxiliary materials, it may further comprise the steps:
1) get solubilizing auxiliary materials 0.3-10.0mg respectively, add an amount of distilled water, be made into the solubilizing auxiliary materials solution of a series of different concns, under the aseptic condition, filter with 0.22 μ m filter membrane, 4 ℃ of preservations are standby;
2) under 25-37 ℃ of constant temperature, in each ampoule, tetrahymena thermophila's suspension 20-100 μ L is inoculated in the 1-5mL aseptic culture medium, the concentration of inoculation back tetrahymena thermophila's suspension is 1000-2500 cell/mL, the solubilizing auxiliary materials solution 60-240 μ L that adds different concns respectively, place micro-calorimeter to measure the heat production power of tetrahymena thermophila's growth metabolism process, record heat production power-time curve;
3) relation of characteristic parameter or its parameter of deriving in the different concns of analysis solubilizing auxiliary materials and the heat production power-time curve, obtain promoter action, restraining effect or the toxic action result of solubilizing auxiliary materials to tetrahymena thermophila's growth metabolism, wherein said characteristic parameter comprises growth rate constant, maximum heat production power, peak time and quantity of heat production, and the described parameter of deriving comprises inhibiting rate and 5% inhibition concentration;
Biological safety according to the evaluation of result solubilizing auxiliary materials.
2. the method for claim 1, it further comprises the steps:
4) characteristic parameter described in the step 3) is carried out principle component analysis, extraction can be represented the characteristic parameter of heat production power-time curve information more comprehensively;
5) by score coefficient and its contribution rate of each principal constituent, set up the composite evaluation function of solubilizing auxiliary materials, calculate the score of solubilizing auxiliary materials;
Biological safety according to the evaluation of result solubilizing auxiliary materials.
3. method as claimed in claim 1 or 2 is characterized in that, described solubilizing auxiliary materials is the injection solubilizing auxiliary materials.
4. method as claimed in claim 1 or 2 is characterized in that, described solubilizing auxiliary materials comprises polysorbas20, tween 80, poly(oxyethylene glycol) 400, Polyethylene Glycol-600 or poloxamer 188.
5. method as claimed in claim 1 or 2 is characterized in that, the concentration range of described solubilizing auxiliary materials solution is 0.3-10.0mg/mL.
6. method as claimed in claim 5 is characterized in that, the series concentration of described solubilizing auxiliary materials solution is 0.5mg/mL, 1.0mg/mL, 2.0mg/mL, 3.0mg/mL, 4.0mg/mL.
7. method as claimed in claim 1 or 2 is characterized in that step 2) described constant temperature is 28 ℃.
8. method as claimed in claim 1 or 2 is characterized in that step 2) concentration of described inoculation back tetrahymena thermophila's suspension is 1500 cell/mL.
9. method as claimed in claim 1 or 2 is characterized in that step 2) volume of described adding solubilizing auxiliary materials is 120 μ L.
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| CN111521631A (en) * | 2019-02-02 | 2020-08-11 | 中国人民解放军总医院第五医学中心 | Method for evaluating quality consistency of donkey-hide gelatin |
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