CN111879598A - Method for trapping aerosol in smoke of heated non-combustible cigarette and method for testing cytotoxicity - Google Patents
Method for trapping aerosol in smoke of heated non-combustible cigarette and method for testing cytotoxicity Download PDFInfo
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Abstract
The invention belongs to the technical field of tobacco toxicity analysis, and particularly relates to a method for trapping aerosol in smoke of a heated non-combustible cigarette and a method for testing cytotoxicity. The method for trapping the aerosol in the smoke of the cigarette which is not combusted by heating comprises the following steps: (1) in the process of smoking and heating the non-burning cigarette, a Cambridge filter disc is adopted to capture total particulate matters of the smoke, and dimethyl sulfoxide is adopted to absorb smoke gaseous matters passing through the Cambridge filter disc; after the collection is finished, extracting the total particulate matters of the smoke gas collected on the Cambridge filter by using dimethyl sulfoxide, wherein the dimethyl sulfoxide extracted with the total particulate matters of the smoke gas and the dimethyl sulfoxide absorbed with the gas phase matters of the smoke gas in the step (1) jointly form the collection of the smoke gas aerosol. The invention establishes a novel method for trapping the smoke aerosol for the in vitro toxicity test of the heated non-burning cigarette, realizes the comprehensive trapping of the smoke aerosol of the heated non-burning cigarette, and provides a basis for comprehensively evaluating the toxicity test of the heated non-burning cigarette.
Description
Technical Field
The invention belongs to the technical field of tobacco toxicity analysis, and particularly relates to a method for trapping aerosol in smoke of a heated non-combustible cigarette and a method for testing cytotoxicity.
Background
The heating non-combustible cigarette has the characteristic of heating tobacco shreds or tobacco sheets instead of burning, compared with the traditional cigarette, the generation mode of smoke aerosol is different, the combustion cracking process is not carried out, the harmful components generated by the high-temperature combustion cracking of tobacco are reduced, and the release amount of the chemical components of main stream smoke is greatly reduced; compared with the electronic cigarette, the electronic cigarette can provide certain tobacco characteristic feeling for consumers. The cigarette is a novel cigarette with a very promising prospect because the cigarette has the advantages of low release of harmful ingredients and tobacco characteristic feeling based on heating and non-combustion. Currently, in vitro toxicity effect research of the cigarette which is not combusted by heating is one of indexes for evaluating the quality of the products.
The existing in vitro toxicity evaluation research of the cigarette which is not burnt by heating is mainly based on the in vitro toxicity evaluation method of the traditional cigarette to carry out in vitro toxicology test tests by collecting the total particulate matters of the smoke, for example, the Chinese patent application with the publication number of CN109593815A discloses a method for detecting the bacterial reversion experiment of the total particulate matters of the cigarette which is not burnt by heating. However, the smoke aerosol of the cigarette which is not burnt by heating comprises particulate matters and gas-phase matters, and the whole toxic effect of the smoke aerosol can not be truly and comprehensively reflected only by carrying out a toxicity test on the particulate matters.
In the prior art, when the toxicity test of the cigarette is carried out without combustion by heating, the capture mode of the smoke aerosol is to capture a particulate matter without simultaneously capturing a gas phase matter, the tested toxicity result is to analyze the particulate matter captured matter, the evaluation result is incomplete, and the toxicity effect of the gas phase matter is not considered.
In some existing technologies for capturing smoke gas-phase substances for cigarette smoke chemical analysis experiments, organic acid is used as a gas-phase substance leaching liquor, alkali liquor such as sodium hydroxide is used for adjusting the pH value, the organic acid and the alkali liquor can cause damage effects on cells, and in addition, the organic acid can react with partial chemical components in smoke, so that the chemical composition of the smoke is changed during subsequent toxicological tests, and the biological effect of actual smoke components cannot be truly reflected. In some existing technologies for capturing smoke gas-phase substances in cigarette smoke cell experiments, the steps are complex, for example, a multistage capturing mode that a phosphoric acid buffer solution (PBS) is firstly used for capturing the gas-phase substances and then an organic solvent is used for capturing is adopted, ice bath conditions are also needed when gas-phase components are captured, in addition, the capturing efficiency of the PBS and acetone or ethyl acetate on smoke components is different (the solvent properties are different), the smoke components captured by each solvent are also different, the test results of subsequent cell experiments can be influenced, and great differences exist; some existing trapping solvents such as acetone and ethyl acetate have the chemical characteristics of flammability, easiness in preparing toxin and the like, are low in boiling point, have the potential influence of volatilization at the environment temperature of 37 ℃ under the condition of cell culture, and cause large experimental error; in addition, when the cytotoxicity test is performed subsequently, the particulate phase component and the gas phase component are separately tested, the test of all fumes is not performed, and the cytotoxicity test results obtained by different solvents are also different, and the test results are not comparable.
Disclosure of Invention
The invention aims to provide a method for trapping smoke aerosol of a heated non-burning cigarette, which can comprehensively trap particulate matters and gas-phase matters in the smoke aerosol.
The invention also aims to provide a cytotoxicity test method for the cigarette which is not burnt and can comprehensively reflect the toxicity effect of the cigarette which is not burnt.
In order to achieve the purpose, the invention adopts the technical scheme that:
a method for collecting aerosol in smoke of a heated non-combustible cigarette comprises the following steps:
(1) in the process of smoking and heating the non-burning cigarette, a Cambridge filter disc is adopted to capture total particulate matters of the smoke, and dimethyl sulfoxide is adopted to absorb smoke gaseous matters passing through the Cambridge filter disc;
(2) after the collection is finished, extracting the total particulate matters of the smoke gas collected on the Cambridge filter by using dimethyl sulfoxide, wherein the dimethyl sulfoxide extracted with the total particulate matters of the smoke gas and the dimethyl sulfoxide absorbed with the gas phase matters of the smoke gas in the step (1) jointly form the collection of the smoke gas aerosol.
The invention establishes a novel method for trapping the smoke aerosol for the in vitro toxicity test of the heated non-combustible cigarette, can realize the comprehensive trapping of particulate matters and gas-phase matters in the smoke aerosol of the heated non-combustible cigarette, and provides a basis for comprehensively evaluating the toxicity of the heated non-combustible cigarette.
Only dimethyl sulfoxide (DMSO) is adopted in the trapping process, so that experimental errors caused by different solvents in the subsequent toxicity test process are avoided; meanwhile, DMSO has the advantages of high boiling point (189 ℃), good thermal stability and the like, is safe and nontoxic under a certain dosage, and has no influence on subsequent cytotoxicity tests; meanwhile, DMSO is a neutral solution and has high polarity, does not react with chemical components in the smoke, and has good absorption and extraction effects on the chemical components in the smoke.
After the collection in the step (2) is finished, extracting the total particulate matters of the smoke gas collected on the Cambridge filter by adopting pure DMSO (dimethyl sulfoxide), so as to obtain dimethyl sulfoxide with the total particulate matters of the smoke gas, and then mixing the dimethyl sulfoxide with the smoke gas phase matters absorbed in the step (1), so as to obtain the collection of the smoke gas aerosol; or directly extracting the total particulate matter of the smoke collected on the Cambridge filter by using the dimethyl sulfoxide absorbing the smoke gaseous matter to obtain the collected matter of the smoke aerosol, wherein the dimethyl sulfoxide for extraction is the dimethyl sulfoxide absorbing the smoke gaseous matter in the step (1).
When the total particulate matters of the flue gas captured on the Cambridge filter are directly extracted by dimethyl sulfoxide absorbed with the flue gas phase matters, the dimethyl sulfoxide needs to be supplemented for improving the extraction rate, and the addition amount is 60-70% of the dimethyl sulfoxide used in the step (1).
In the trapping method, the number of the Cambridge filter discs used in the step (1) is not less than 2, and all the Cambridge filter discs used in the step (1) are extracted during the extraction in the step (2). Preferably, the number of the cambridge filter sheets is 5.
Preferably, in order to improve the utilization rate of the cambridge filter and ensure that the amount of the total particulate matters in the captured smoke is suitable for the subsequent cytotoxicity analysis, the step (1) of capturing the total particulate matters in the smoke by the cambridge filter is specifically as follows: each Cambridge filter sheet captures 3-5 smoke total particulate matters of the smoke aerosol of the cigarette which is not burned after being heated.
Preferably, the dosage of the dimethyl sulfoxide in the step (1) is 10-30 mL, so that complete capture of the smoke gas phase substances is ensured.
In order to improve the extraction rate of the total particulate matters in the flue gas, the extraction in the step (2) is carried out under the oscillation condition, the oscillation speed is 120-180 rpm/min, and the time is 20-40 min.
The equipment used for suction is a linear type smoking machine, and the smoking parameters of the smoking machine can be set according to the experimental purpose. Preferably, each non-combustible heated cigarette is smoked for 8-10 mouths, the smoking capacity is 35 or 55mL per mouth, the smoking duration is 2s per mouth, and the smoking interval is 60 or 30s per mouth (according to the parameters of ISO or Canada deep smoking conditions).
Based on the trapping method for the smoke aerosol of the heating non-combustion cigarette, the invention also provides a cytotoxicity test method for the heating non-combustion cigarette, namely, the method for trapping the smoke aerosol of the heating non-combustion cigarette is adopted to obtain the extract of the smoke aerosol, and then the extract of the smoke aerosol is subjected to cytotoxicity test. The invention simultaneously carries out cytotoxicity test on the total particulate matter and the gas phase matter in the heated non-combustible cigarette aerosol, and the test result more comprehensively and truly reflects all the toxicity effects of the cigarette aerosol.
Drawings
FIG. 1 is a schematic flow chart in example 1 of the present invention;
FIG. 2 is the result of the cytotoxicity test of the heated non-burning cigarette in example 2 of the present invention;
FIG. 3 shows the results of the cytotoxicity test of DMSO in test example 1 of the present invention;
FIG. 4 is a result of cytotoxicity test of the conventional cigarette in Experimental example 2 of the present invention;
reference numerals:
1: heating the non-combustible cigarette; 2: a Cambridge filter; 3: a linear type smoking machine; 4: a DMSO solution; 5: an absorption bottle; 6: a conical flask; 7: shaking table.
Detailed Description
The invention is further described with reference to the following specific embodiments and the accompanying drawings.
Embodiment of method for collecting aerosol in smoke of heated non-combustible cigarette
Example 1
The flow of the method for trapping aerosol in smoke of a non-burning cigarette heated by the embodiment is shown in fig. 1, and the method comprises the following steps:
(1) a linear smoking machine 3 is adopted to carry out smoking on the heated non-burning cigarette test sample 1 in a Canada deep smoking mode (a bell-shaped smoking curve, the smoking interval time is 30s, the smoking capacity of each mouth is 55mL, and the smoking duration of each mouth is 2s), a Cambridge filter 2 with the diameter of 44mm is used for capturing total particulate matters of smoke in the smoking process, and gas phase matters of each mouth of smoking time are introduced into an absorption bottle 5 containing 15mL of DMSO solution 4 through the Cambridge filter;
during smoking, smoking 10 mouths of each heated non-combustible cigarette, capturing 4 total smoke particulate matters of the heated non-combustible cigarette by each Cambridge filter, and preparing a sample by 5 Cambridge filters;
(2) after the aggregation, 5 Cambridge filter discs 2 are transferred to a conical flask 6, then DMSO solution containing gas phase substances in an absorption bottle 5 is poured into the conical flask 6, 10mLDMSO is added, then the conical flask 6 (the total volume of DMSO is 25mL) is placed on a shaking table 7, shaking is carried out at room temperature at 150rpm/min for 40 minutes, then extract liquid is absorbed, and the extract of the flue gas aerosol is obtained and stored at-80 ℃ for standby. The flue gas trapping results are shown in table 1.
Table 1 flue gas trapping results
Note: in table 1, the mass of the total particulate matter in the smoke is the mass of the cambridge filter after suction-the mass of the cambridge filter before suction; smoke aerosol extract concentration (counts/mL) ═ number of cigarettes not burning by heating (4 x 5)/volume of DMSO in erlenmeyer flask; smoke aerosol extract concentration (mg/mL) is the sum of total smoke particulate matter captured on 5 cambridge filters/volume of DMSO in the flask.
Second, example of method for testing cytotoxicity of non-burning cigarette by heating
Example 2
In this example, cytotoxicity test was performed on the extract of the smoke aerosol obtained in example 1, the test cell was an a549 cell line, and the test was performed by using a CCK-8 kit, and the specific test method was: 1) inoculating the prepared cell suspension (15000 cells/well) into a 96-well plate, and culturing for 24 h; 2) removing the culture medium in the culture plate, adding a control culture medium and a culture medium (100 mu L/hole) containing smoke aerosol leaching liquor with different concentrations, and incubating for 24h at 37 ℃; 3) removing the solution in the culture plate, adding 100 μ L of the prepared CCK-8 solution into each well (adding 10 μ L of CCK-8 mother liquor into each 100 μ L of culture medium), and incubating at 37 deg.C for 0.5-2 h; 4) shaking at room temperature for 5min, and measuring light absorption value with microplate reader at 450 nm. Cell viability ═ (mean absorbance in experimental wells/mean absorbance in control wells) x 100%. The test results are shown in fig. 2.
Test example 1
The toxicity of the DMSO used in the test example is tested and analyzed, and the specific test method comprises the following steps: 1) inoculating the prepared cell suspension (15000 cells/well) into a 96-well plate, and culturing for 24 h; 2) removing the culture medium from the culture plate, adding a control culture medium and culture media (100 mu L/hole) containing DMSO at different concentrations, and incubating at 37 ℃ for 24 h; 3) removing the solution in the culture plate, adding 100 μ L of the prepared CCK-8 solution into each well (adding 10 μ L of CCK-8 mother liquor into each 100 μ L of culture medium), and incubating at 37 deg.C for 0.5-2 h; 4) shaking at room temperature for 5min, and measuring light absorption value with microplate reader at 450 nm. Cell viability ═ (mean absorbance in experimental wells/mean absorbance in control wells) x 100%. The test results are shown in fig. 3.
The test result shows that DMSO is non-toxic to cells under the concentration condition, and the influence of the solvent can be eliminated in the subsequent toxicity test of heating the non-burning cigarette.
Test example 2
In the test example, the cytotoxicity of all smoke of the conventional cigarette was tested by using the method of the present invention, and the results are shown in fig. 4. Comparing the cytotoxicity test results of fig. 2 and fig. 4, it can be seen that the cytotoxicity of the cigarette without burning is significantly less than that of the conventional cigarette. Therefore, if other solvents such as PBS are used for trapping, because some components in the gas phase substances cannot be completely trapped, the small difference between the toxicity of the smoke cannot be reflected, so that the toxicity of the cigarette which is not combusted by heating cannot be truly reflected.
Claims (9)
1. A method for trapping aerosol in smoke of a heating non-combustible cigarette is characterized by comprising the following steps:
(1) in the process of smoking and heating the non-burning cigarette, a Cambridge filter disc is adopted to capture total particulate matters of the smoke, and dimethyl sulfoxide is adopted to absorb smoke gaseous matters passing through the Cambridge filter disc;
(2) after the collection is finished, extracting the total particulate matters of the smoke gas collected on the Cambridge filter by using dimethyl sulfoxide, wherein the dimethyl sulfoxide extracted with the total particulate matters of the smoke gas and the dimethyl sulfoxide absorbed with the gas phase matters of the smoke gas in the step (1) jointly form the collection of the smoke gas aerosol.
2. The method for capturing the smoke aerosol of the non-burning cigarette as claimed in claim 1, wherein the dimethyl sulfoxide used for extraction in the step (2) is the dimethyl sulfoxide absorbed with the smoke gas phase substances in the step (1).
3. The method for trapping aerosol in smoke of heated non-combustible cigarettes according to claim 2, wherein dimethyl sulfoxide is supplemented during extraction, and the addition amount is 60-70% of the amount of dimethyl sulfoxide used in the step (1).
4. The method for capturing the smoke aerosol of the non-burning cigarette as claimed in claim 1, wherein the number of the Cambridge filter discs used in the step (1) is not less than 2, and the extraction in the step (2) is performed on all the Cambridge filter discs used in the step (1).
5. The method for capturing the smoke aerosol of the non-burning heated cigarettes according to any one of claims 1 to 4, wherein 3 to 5 total particulate matters of the smoke of the non-burning heated cigarettes are captured by each Cambridge filter.
6. The method for trapping aerosol in smoke of non-burning cigarettes according to any one of claims 1 to 4, wherein the amount of dimethyl sulfoxide used in step (1) is 10 to 30 mL.
7. The method for trapping aerosol in smoke of non-burning cigarette according to any one of claims 1 to 4, wherein the extraction in step (2) is carried out under the condition of oscillation, the speed of oscillation is 120-180 rpm/min, and the time is 20-40 min.
8. The method for trapping aerosol in smoke of heated non-combustible cigarettes according to any one of claims 1 to 4, wherein when the non-combustible cigarettes are heated by smoking, 8-10 cigarettes are smoked, the smoking capacity of each cigarette is 35 or 55mL, the duration of each smoking is 2s, and the time interval between each smoking is 60 or 30 s.
9. A method for testing cytotoxicity of a heated non-combustible cigarette, which is characterized in that the method for trapping the smoke aerosol of the heated non-combustible cigarette according to any one of claims 1-8 is adopted to trap the smoke aerosol to obtain an extract of the smoke aerosol, and then the extract of the smoke aerosol is subjected to cytotoxicity test.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113029710A (en) * | 2021-03-15 | 2021-06-25 | 中国烟草总公司郑州烟草研究院 | Method for extracting whole smoke of heated cigarette for in vitro toxicity test |
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Cited By (2)
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| CN113029710A (en) * | 2021-03-15 | 2021-06-25 | 中国烟草总公司郑州烟草研究院 | Method for extracting whole smoke of heated cigarette for in vitro toxicity test |
| CN113029710B (en) * | 2021-03-15 | 2023-12-15 | 中国烟草总公司郑州烟草研究院 | Extraction method of whole smoke of heated cigarettes for in-vitro toxicity test |
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