CN104293876A - Cigarette filter tip water extract cytotoxicity test positive control setting method - Google Patents
Cigarette filter tip water extract cytotoxicity test positive control setting method Download PDFInfo
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- CN104293876A CN104293876A CN201410489638.6A CN201410489638A CN104293876A CN 104293876 A CN104293876 A CN 104293876A CN 201410489638 A CN201410489638 A CN 201410489638A CN 104293876 A CN104293876 A CN 104293876A
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Abstract
The invention relates to a cigarette filter tip water extract cytotoxicity test positive control setting method which comprises the following steps: (1) selecting a to-be-detected sample group of cigarette filter tips and the same quantity of a positive control preparation group of cigarette filter tips; (2) preparing a 10-20 mg/ml lauryl sodium sulfate (SDS) water solution; (3) uniformly spraying the SDS water solution on the positive control preparation group of cigarette filter tips, and naturally drying for later use; (4) leaching the positive control group of cigarette filter tips and the same quantity of the to-be-detected sample group of cigarette filter tips by using the same process; (5) by using the leaching solution as a solvent, preparing a culture medium from a powder cell culture medium according to the culture medium specifications, and filtering and sterilizing the prepared solution for cytotoxicity test; and (6) carrying out cytotoxicity test on the treated extracting solution. The positive control set by the method can verify whether the cigarette filter tip leaching process and the cytotoxicity test on the cigarette filter tip water extract are reliable or not.
Description
Technical field
The invention belongs to technical field of tobacco, be specifically related to a kind of positive control method to set up when cell toxicity test carried out to cigarette filter water extract.
Background technology
Cell toxicity test be a class at ex vivo situation Imitating biology growing environment, detect the method for cell generation growth-inhibiting after tested material or its vat liquor exposing cell, changing function, dissolving, death or other toxic reaction.It is usually as one of the security Biological Detection means of food, medicine, dental material etc.
Because cigarette filter directly contacts with human oral, there is potential health risk, therefore, be necessary to adopt cell toxicity test means, the potential hazard that it may exist is investigated.When cell toxicity test is carried out to cigarette filter, first need to extract cigarette filter, and then extracting solution is detected.Therefore, need in process of the test to arrange positive control, with verify leaching process and follow-up cell toxicity test whether reliable.
Existing cell toxicity test positive control method to set up has two classes: first method, if tested material can be mixed with solution, then uses and certain densityly knownly has Cytotoxic reagent and be directly added in cell culture fluid and make positive control; Second method, test if tested material needs to prepare vat liquor, then positive control is preferably identical and close with tested material character and has Cytotoxic material, and after the method that this materials'use is identical with tested material is extracted, its vat liquor should be in test obvious cell-cytotoxic reaction.
But existing method be not suitable for the cell toxicity test of cigarette filter vat liquor, need to prepare cigarette filter vat liquor this is because carry out cell toxicity test to cigarette filter, therefore, use above-mentioned first method that positive control is set and then cannot verify that whether the process of extraction is reliable.And for use second method, because current existing cigarette filter kind does not find to have cytotoxicity, and not close with the cigarette filter character Cytotoxic material of tool, therefore for the leaching process of checking cigarette filter and the follow-up cell toxicity test that cigarette filter water extract is carried out whether reliable, be necessary to develop a kind of positive control method to set up for cigarette filter water extract cell toxicity test.
Through literature search, do not find the open report with content same document of the present invention.
Summary of the invention
The object of the present invention is to provide a kind of positive control method to set up when cell toxicity test carried out to cigarette filter water extract.By the positive control that the method is arranged, can verify the leaching process of cigarette filter and the follow-up cell toxicity test that cigarette filter water extract is carried out whether reliable.
The oral contact of filter tip and people during cigarette smoking, carries out extracting the solvent (water or artificial saliva) needing to select and people's saliva character is close to cigarette filter, therefore people is that the positive thing added on filter tip also should for water miscible.The present invention uses sodium lauryl sulphate (SDS) as the artificial lysis agent added.No. CAS of SDS is 151-21-3, is a kind of hydrophilic group tensio-active agent, can makes cell membrane lysis, is a kind of positive control agent conventional in cell toxicity test.Therefore, by SDS is added on filter tip as positive control sample, then prepare water extract and carry out cell toxicity test together with testing sample, the result if positive control is positive, then illustrate that the leaching process of cigarette filter and follow-up cell toxicity test of carrying out cigarette filter water extract are reliable.
The present invention realizes by following technical proposals:
Step (1) is chosen the testing sample group cigarette filter of equal amts and is met the cigarette filter (cigarette filter as the preparation of positive controls) preparing positive control conditions;
Step (2) compound concentration is the SDS aqueous solution of 10-20 mg/mL;
Step (3) is for the preparation of the SDS aqueous solution cigarette filter of positive controls evenly spraying step (2) and prepare, fountain height should make the complete stripping concentration of the theory of SDS in filter tip Leaching tests test system be more than or equal to 1 mg/mL, and make cigarette filter fully absorb SDS solution, for subsequent use as positive controls cigarette filter after its seasoning;
The positive controls cigarette filter that step (4) is prepared by the step (3) of identical method to equal amts and testing sample group cigarette filter carry out lixiviate;
The vat liquor that step (5) uses step (4) to obtain, as solvent, is prepared by the method that powder cell culture medium illustrates according to substratum, is then used for cell toxicity test by after the solution filtration sterilization after preparation;
Step (6) carries out cell toxicity test to the extracting solution processed through step (5), the concentration of Test extraction liquid in test system is 100%, 50%, 25% and 12.5% 4 dosage, the test fluid of lower concentration uses cell culture fluid dilution to obtain, and the cell open-assembly time of extracting solution is 24 hours.
The cigarette filter preparing positive control conditions that meets in described step (1) refers to: (1) can not react and affect its positive findings because there is comparatively extensive chemical with SDS for the preparation of the cigarette filter of positive control; (2) for the preparation of the cigarette filter of positive control, its positive findings can not be affected because producing stronger adsorption with SDS.
Theory complete stripping concentration described in step (3) refers to: the quantity of solvent according to the actual fountain height of prepared SDS solution actual concentrations, SDS and when extracting added by reality, the SDS supposing to be sprayed on filter tip in lixiviate time the concentration that can calculate under the condition of 100% stripping.
compared with prior art, its beneficial effect is in the present invention:operation is simple for cigarette filter water extract cell toxicity test positive control method to set up of the present invention, economical and practical.The positive thing SDS of the artificial interpolation that the present invention uses, it is a kind of water-soluble cell cracking agent, be easy to be dissolved in the Extraction solvent distilled water of filter tip, because the SDS aqueous solution is colourless, therefore can not the method such as centering RBC Toxicity method, MTT cytotoxicity method light absorption value measure cause interference.The concentration of the SDS aqueous solution set by the present invention and proof load can make cell toxicity test to be positive result, and present good dose-effect relationship.By the positive control that the method is arranged, can verify the leaching process of cigarette filter and the follow-up cell toxicity test that cigarette filter water extract is carried out whether reliable.
Accompanying drawing explanation
Fig. 1 prepares artificial positive filter tip with the inventive method as positive control, the water extract of the commercial cigarettes filter tip of 2 kinds of random chooses is carried out to the cell survival rate figure of the vat liquor of the different concns that cell toxicity test obtains.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail, but content of the present invention is not limited thereto.
Embodiment 1
Prepare artificial positive filter tip with the inventive method as positive control, cell toxicity test is carried out to the commercial cigarettes filter tip of 2 kinds of random chooses.
1, random choose 2 kinds of commercial cigaretteses, randomly draw 20 for the preparation of testing sample group cigarette filter for often kind.Randomly draw 20 meet prepare positive cigarette filter condition Kentucky 3R4F cigarette for the preparation of the cigarette filter of positive controls.Above-mentioned cigarette filter is cut down for subsequent use, the overlapping part switching to tipping paper and cigarette tissue during cutting, should be avoided, to prevent cigarette tissue and pipe tobacco to bring in filter tip sample.
2, compound concentration is the SDS aqueous solution of 10mg/mL.
3, spraying for the preparation of even on the 3R4F cigarette filter of positive control the SDS aqueous solution 10mL prepared in step 2, when making the complete stripping final concentration of the theory of SDS in cigarette filter Leaching tests test system be 1 mg/mL(extraction, amount of water is 100mL), cigarette filter is made fully to absorb the SDS solution of interpolation, for subsequent use as positive cigarette filter control group after cigarette filter seasoning.
4, the positive controls cigarette filter prepared step 3 with 100mL distilled water and 2 kinds of testing sample group cigarette filters (sample 1 and sample 2) leave standstill at 40 DEG C extracts 30min.
5, the water extract using step 4 to obtain is as solvent, prepare by the method that DMEM powder cell culture medium illustrates according to substratum, and add foetal calf serum, volume ratio shared by foetal calf serum body is made to be 10% of extracting solution cumulative volume, then by preparation after solution filtration sterilization, after for follow-up cell toxicity test.
6, use Chinese hamster ovary cell (CHO) cell to carry out MTT cell toxicity test to the extracting solution that processed through step 5, the concentration of Test extraction liquid in test system be 100%, 50%, 25% and 12.5% 4 dosage (test fluid of lower concentration uses cell culture fluid dilution to obtain).Chinese hamster ovary celI is planted in 96 orifice plates with the density in 1000 every holes, adds the extracting solution prepared and expose 24 hours.Then add after MTT solution continues to hatch 4 hours, extraction MTT is also determined at the light absorption value under 570 nm wavelength by microplate reader.The cell survival rate of each group different concns is calculated by light absorption value.
7, the cell survival rate under each sample different concns is shown in Fig. 1.From test-results, the extracting solution of the 2 kinds of filter tip samples tested all does not show obvious cytotoxicity and dose-response relationship under 4 tested concentration.The cell survival rate of artificial positive filter tip sample extracting solution increases with test concentrations and reduces, and has comparatively significantly cytotoxicity, and presents obvious dose-response relationship.Leaching process and follow-up cell toxicity test of carrying out cigarette filter water extract that cigarette filter in the present embodiment is described are reliable.
The compound concentration of the SDS aqueous solution can also be 15mg/mL, 20mg/mL etc.
More than show and describe ultimate principle of the present invention and principal character and advantage of the present invention.The technician of the industry should understand; the present invention is not restricted to the described embodiments; what describe in above-described embodiment and specification sheets just illustrates principle of the present invention; without departing from the spirit and scope of the present invention; the present invention also has various changes and modifications, and these changes and improvements all fall in the claimed scope of the invention.Application claims protection domain is defined by appending claims and equivalent thereof.
Claims (2)
1. a cigarette filter water extract cell toxicity test positive control method to set up, it is characterized in that, the method comprises the steps:
Step (1): choose the testing sample group cigarette filter of equal amts and meet the cigarette filter preparing positive control conditions;
Step (2): compound concentration is sodium lauryl sulphate (SDS) aqueous solution of 10-20 mg/mL;
Step (3): for the preparation of the SDS aqueous solution cigarette filter of positive controls evenly spraying step (2) and prepare, fountain height should make the complete stripping concentration of the theory of SDS in filter tip Leaching tests test system be more than or equal to 1 mg/mL, and make cigarette filter fully absorb SDS solution, for subsequent use as positive controls cigarette filter after its seasoning;
Step (4): the positive controls cigarette filter prepared by the step (3) of identical method to equal amts and testing sample group cigarette filter carry out lixiviate;
Step (5): the vat liquor that use step (4) obtains, as solvent, is prepared by the method that powder cell culture medium illustrates according to substratum, is then used for follow-up cell toxicity test by after the solution filtration sterilization after preparation;
Step (6): carry out cell toxicity test to the extracting solution processed through step (5), the concentration of Test extraction liquid in test system is 100%, 50%, 25% and 12.5% 4 dosage, and the cell open-assembly time of extracting solution is 24 hours.
2. cigarette filter water extract cell toxicity test positive control method to set up according to claim 1, is characterized in that: the lixiviate of the cigarette filter described in step (4) adopts distilled water as extracting solution.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104749353A (en) * | 2015-04-22 | 2015-07-01 | 云南中烟工业有限责任公司 | Method for detecting influence of buccal tobacco product on micronucleus rates of cells |
| CN104789633A (en) * | 2015-04-22 | 2015-07-22 | 云南中烟工业有限责任公司 | Test method for detecting cytotoxicity of buccal tobacco products |
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| CN102156188A (en) * | 2011-02-10 | 2011-08-17 | 云南烟草科学研究院 | Neutral erythrocyte toxicity testing method for biological evaluation of cigarette tipping paper |
| CN102495183A (en) * | 2011-12-16 | 2012-06-13 | 云南烟草科学研究院 | Detection method for cytotoxicity of gas-liquid contact type cigarettes under full-smoke exposure |
| CN103088105A (en) * | 2013-01-15 | 2013-05-08 | 中国烟草总公司郑州烟草研究院 | MTT (thiazolyl blue) cell toxicity test method of biological assessment of total particle matter in cigarette smoke |
| CN103604917A (en) * | 2013-11-28 | 2014-02-26 | 云南烟草科学研究院 | Screening method of positive substances for pyrolyzate trapping substance cytotoxicity test |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102156188A (en) * | 2011-02-10 | 2011-08-17 | 云南烟草科学研究院 | Neutral erythrocyte toxicity testing method for biological evaluation of cigarette tipping paper |
| CN102495183A (en) * | 2011-12-16 | 2012-06-13 | 云南烟草科学研究院 | Detection method for cytotoxicity of gas-liquid contact type cigarettes under full-smoke exposure |
| CN103088105A (en) * | 2013-01-15 | 2013-05-08 | 中国烟草总公司郑州烟草研究院 | MTT (thiazolyl blue) cell toxicity test method of biological assessment of total particle matter in cigarette smoke |
| CN103604917A (en) * | 2013-11-28 | 2014-02-26 | 云南烟草科学研究院 | Screening method of positive substances for pyrolyzate trapping substance cytotoxicity test |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104749353A (en) * | 2015-04-22 | 2015-07-01 | 云南中烟工业有限责任公司 | Method for detecting influence of buccal tobacco product on micronucleus rates of cells |
| CN104789633A (en) * | 2015-04-22 | 2015-07-22 | 云南中烟工业有限责任公司 | Test method for detecting cytotoxicity of buccal tobacco products |
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