CN104730019B - Method for in-vitro evaluation on safety of cigarettes and tobaccos - Google Patents
Method for in-vitro evaluation on safety of cigarettes and tobaccos Download PDFInfo
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- flue gas
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Abstract
The invention discloses a method for in-vitro evaluation on safety of cigarettes and tobaccos. The method is characterized in that a biological reagent with TrxR (thioredoxin reductase) activity is adopted to detect TrxR activity inhibition ability of CSE (cigarette smoke extract) obtained after combustion of the cigarettes or the tobaccos so as to evaluate safety of the cigarettes and the tobaccos by taking an absorbance value of the CSE at 340nm as a quantification basis. By the method for in-vitro evaluation on safety of the cigarettes and the tobaccos, the problems of long evaluation period, complex evaluation mechanisms and the like in current cigarette harm evaluation are solved successfully to provide a reliable evaluation means for enterprises to improve tobacco quality and develop less harmful cigarettes.
Description
Technical field
The present invention relates to a kind of method of in-vitro evaluation Medicated cigarette and Nicotiana tabacum L. safety.Specifically use containing thioredoxin also
The solution of protoenzyme (TrxR) vigor, by detecting kinds of cigarettes and flue gas aqueous extract (CSE) made by Nicotiana tabacum L. to TrxR vigor
Rejection ability, the absorbance OD with CSE at 340nm340nmAs the side of quantitative basis, evaluating cigarette and Nicotiana tabacum L. safety
Method.
Background technology
Modern medicine study has enough evidences to prove that Smoking is harmful to your health, and flue gas aqueous extract CSE is also extensively used
In research flue gas toxicological mechanism (referring to document 1-5).Containing 4800 kinds of things having determined such as tar, nicotine and aldehydes in CSE
Matter, wherein aldehydes include acetaldehyde, formaldehyde and unsaturated aldehydes, and the dilute aldehyde of acrylic aldehyde and fourth of unsaturated aldehyde apoplexy due to endogenous wind is that hazardness is maximum
One of several materials.The unsaturation aldehydes such as acrylic aldehyde is high active electrophilic compound, nucleophilic occurs with nucleophile easily and adds
Into reaction.Research shows that acrylic aldehyde is the significant contributor that cigarette smoke causes cytotoxicity (referring to document 6)；In addition, propylene
Aldehyde can increase the risk (referring to document 7) of the Induced by Carcinogen cancer such as Benzpyrene in flue gas.
Thioredoxin reductase TrxR is a kind of dimer selenoenzyme, and the selenocystein (Sec) of TrxR is for TrxR
Catalysis activity is most important, and it is the analog of cysteine, its pKa (～5.2) far below cysteine pKa (～
8.3), thus Sec in physiological pH in non-protonated selenol salt state, this makes it have high nucleophilic vigor.TrxR
Pivotal role is played in the forming process of Deoxydization nucleotide, is requisite thing in DNA synthesis and proliferation process
Matter, while TrxR has inhibited apoptosis, improves various biological functions (referring to document 8) such as the activity of transcription factor.TrxR
Vigor is suppressed can cause the consequences such as tissue injury (referring to document 9 and 10).
Acrylic aldehyde in flue gas is also had been found to be combined with the TrxR containing Sec, is pressed down as active electrophilic compound
TrxR vigor processed (referring to document 11).
The current evaluation to Medicated cigarette harm relies primarily on component analyses and biological detection.Component analyses in cigarette industry
Extensively apply, but as smoke components are complicated, this disappears and other rises in the practice of reducing tar and reducing harm for different nuisances, depends on a few class things
The evaluation of matter has certain limitation.It is long using cell and the safe sexual cycle of animal experiment method evaluating cigarette, in Less harmful cigarette
In R＆D process, practicality is low.
Therefore, developing Less harmful cigarette needs a kind of instant, rapid, high flux to evaluate Medicated cigarette and Nicotiana tabacum L. safety
Means.
List of references：
1.Kosmider B,Messier EM,Chu HW,et.Epub 2011 Dec 7.Human alveolar
epithelial cell injury induced by cigarette smoke.PLoS One.2011；6(12).
2.Zhu L,Barrett EC,Xu Y,et.Regulation of Cigarette Smoke(CS)-Induced
Autophagy by Nrf2.PLoS One.2013 Apr 9；8(4).
3.Baglole CJ,Sime PJ,Phipps RP.Cigarette smoke-induced expression of
heme oxygenase-1 in human lung fibroblasts is regulated by intracellular
glutathione.Am J Physiol Lung Cell Mol Physiol.2008Oct；295(4).
4.Yageta Y,Ishii Y,Morishima Y,et.Mar 2.Role of Nrf2 in host defense
against influenza virus in cigarette smoke-exposed mice.J Virol.2011 May；85
(10).
5.Bertram KM,Baglole CJ,Phipps RP,rt.Molecular regulation of
cigarette smoke induced-oxidative stress in human retinal pigment epithelial
cells:implications for age-related macular degeneration.Am J Physiol Cell
Physiol.2009 Nov；297(5).
6.Noya Y,Seki K,Asano H,et.Epub 2013 Aug 24.Identification of stable
cytotoxic factors in the gas phase extract of cigarette smoke and
pharmacological characterization of their cytotoxicity.Toxicology.2013 Dec 6；
314(1):1-10.
7.Feng Z,Hu W,Hu Y,Tang MS.Acrolein is a major cigarette-related lung
cancer agent:Preferential binding at p53 mutational hotspots and inhibition
of DNA repair.Proc Natl Acad Sci USA.2006,103:15404-15409
8.Holmgren A,Lu J.Thioredoxin and thioredoxin reductase:current
research with special reference to human disease.Biochem Biophys Res
Commun.2010 May 21；396(1):120-4.
9.Wang X,Zhang J,Xu T.Thioredoxin reductase inactivation as a pivotal
mechanism of ifosfamide in cancer therapy.Eur J Pharmacol.2008Jan 28；579(1-
3):66-73.
10.Zhang J,Lu H.Ifosfamide induces acute renal failure via inhibition
of the thioredoxin reductase activity.Free Radic Biol Med.2007 Dec 15；43(12):
1574-83.
11.Zhang J1,Lu H.Randall MJ,Spiess PC,Hristova M.Acrolein-induced
activation of mitogen-activated protein kinase signaling is mediated by
alkylation of thioredoxin reductase and thioredoxin 1.Redox Biol 2013,1:265-
275.
12.A.D.Smith,O.A.Levander,High-throughput 96-well microplate assays
for determining specific activities of glutathione peroxidase and thioredoxin
reductase,Methods Enzymol.347 (2002)113–121.
13.E.S.Arnér,A.Holmgren,Measurement of thioredoxin and thioredoxin
reductase,in:M.Maines,L.Costa,D.Reed,S.Sassa(Eds.),Current Protocols in
Toxicology,John Wiley & Sons,Inc.,New York,2000,pp.7.4.1–7.4.14.
The content of the invention
It is an object of the invention to provide a kind of can instant, rapid, high-throughout in-vitro evaluation Medicated cigarette and Nicotiana tabacum L. safety
Method.
The present invention solves technical problem, adopts the following technical scheme that：
In-vitro evaluation Medicated cigarette of the present invention and the method for Nicotiana tabacum L. safety, its feature is：
Step 1：Each Medicated cigarette to be evaluated or Nicotiana tabacum L. are aspirated respectively, and are configured to flue gas aqueous extract respectively,
Detection absorbance OD of the flue gas aqueous extract at 340nm340nm, with OD340nmAs determining for evaluating cigarette or Nicotiana tabacum L. safety
Amount basis；
Step 2：The biological reagent containing thioredoxin reductase vigor is prepared, double (the 2- p-nitrophenyls of 5,5 '-two sulfur are used
Formic acid) (DTNB) reducing process determine the energy value of thioredoxin reductase in the biological reagent, be designated as H0；
Step 3：20min is carried out by different amounts of flue gas aqueous extract is added in the biological reagent to same volume
Incubation, make flue gas aqueous extract in the biological reagent thioredoxin reductase vigor produce suppression, determine suppress
Afterwards in biological reagent thioredoxin reductase energy value H1, and calculate acquisition flue gas aqueous extract to the sulfur in biological reagent
Suppression ratio T=(H0-H1)/H0 × 100% of oxygen also reductase proteins vigor, makes the dose effect curve of suppression ratio, from institute
State and remove
Suitable addition of the addition of any flue gas aqueous extract outside zero point as flue gas aqueous extract, is designated as m, and unit is micro-
Rise；It is determined that suppression of the flue gas aqueous extract to the thioredoxin reductase vigor in biological reagent under the suitable addition
Rate, is designated as T0；
Suitable addition m is corrected to 10 microlitres, the corrected value M=m/10 of suitable addition is obtained；
Step 4：Determine the flue gas aqueous extract of each Medicated cigarette or Nicotiana tabacum L. to the sulfur oxygen in biological reagent by the method for step 3
The also corrected value M of the suppression ratio T0 and suitable addition of reductase proteins vigor；The made flue gas water extraction of kinds of cigarettes or Nicotiana tabacum L.
Take liquid its suitable addition different, therefore the suitable addition collective of all kinds of cigarettes or Nicotiana tabacum L. made flue gas aqueous extract
10 microlitres are corrected to, more accurately to evaluate the safety of kinds of cigarettes or Nicotiana tabacum L..
Step 5：Calculate the safety evaluatio value S=T0/ (M × OD for obtaining each Medicated cigarette or Nicotiana tabacum L.340nm), with the safety
Property evaluation of estimate S compares the safety of each Medicated cigarette or Nicotiana tabacum L..
In-vitro evaluation Medicated cigarette of the present invention and Nicotiana tabacum L. security method menu, its feature lie also in：Flue gas water extraction described in step 1
Liquid is to prepare as follows：
One Medicated cigarette to be evaluated or Nicotiana tabacum L. are aspirated at the standard conditions by vacuum pump, produced main flume is successively
By being collected in the respectively GAS ABSORPTION bottle equipped with 5mL supplementary set liquid of two series connection, in a Medicated cigarette to be evaluated or Nicotiana tabacum L.
After suction is finished, suction 10s makes supplementary set liquid fully collect main flume, then will collect the supplementary set liquid Jing of main flume
0.22 μm of water film filtering, that is, obtain flue gas aqueous extract；The supplementary set liquid is the phosphate-buffered that concentration is 0.1M, pH=7.0
Liquid, containing the disodiumedetate (EDTA.Na that concentration is 10mM in the phosphate buffer2)。
Described in step 2, the biological reagent containing thioredoxin reductase vigor is the homogenate of animal tissue, plant group
The homogenate of homogenate, the homogenate of isolated cells or the microorganism knitted, or the pure enzyme preparation of thioredoxin reductase.
In step 2, the homogenate of the homogenate or plant tissue of animal tissue is to obtain as follows：Weigh 0.15g
In animal tissue or the plant tissue of -20 DEG C of low temperature cold storage, 1.5mL tissue homogenates are added, is then ground with high-flux tissue
Grinder grinds, 20min is centrifuged under the conditions of 15000rcf, 4 DEG C, takes supernatant, obtains final product the homogenate or plant group of animal tissue
The homogenate knitted；The tissue homogenate is concentration for 0.15M, pH=7.15 and containing the ethylenediaminetetraacetic acid that concentration is 1mM
The phosphate buffer of disodium.
In step 2, the homogenate of the homogenate or microbial body of isolated cells is to obtain as follows：By isolated cells
Or microbial body is suspended in cell pyrolysis liquid with the concentration of million/mL of 10-90, then with ultrasonic cleaner 4 DEG C,
Ultrasonic cell 20min under conditions of 40KHz, to reach broken purpose, is then centrifuged under conditions of 10000rpm, 4 DEG C
10min, takes supernatant, obtains final product the homogenate of the homogenate or microbial body of isolated cells；The cell pyrolysis liquid is that concentration is
The solution of the trishydroxymethylaminomethane (Tris-HCl) of 0.1M, pH7.4, includes in the solution of the trishydroxymethylaminomethane
Benzyl sulfonamide (PMSF) of the concentration for the dithiothreitol, DTT (DTT) and 1mM of 1mM.
In the present invention, with 5,5 '-two sulfur double (2- Nitrodracylic acids), (DTNB) reducing process is determined in the biological reagent
The method of the energy value of thioredoxin reductase is comprised the following steps that referring to document 12 and 13：
First prepare detection working solution (the detection working solution be concentration be 100mM, pH 7.0 phosphate buffer,
Comprising the EDTA-Na that concentration is 10mM in the detection working solution2, the DTNB of 5mM, the Sanguis Bovis seu Bubali of 240 μM of NADPH and 0.2mg/mL
Pure albumen), matching while using, 37 DEG C of temperature controls；
By biological reagent to be measured in killer tube and Auranofin by volume 9：1 mixing, biological reagent to be measured in non-inhibited pipe
With 5% ethanol by volume 9：1 mixing, acts on 20min respectively at 37 DEG C；Will be biological in killer tube after process and non-inhibited pipe
Reagent is added in 96 orifice plates respectively, treats to add 250 microlitres of detection working solutions to start reaction in gaging hole toward 96 orifice plates, and immediately 96
Orifice plate is detected in being placed into microplate reader.With 412nm wavelength detecting 6min in microplate reader under the conditions of 37 DEG C, per 12s readings
Once, enzymatic kinetic curve is read, reads the slope value (Vmax) of 120s～300s time periods, it is right with sample institute in non-inhibited pipe
The Vmax for answering deducts the Vmax in killer tube corresponding to sample, obtains biological examination calculating through specific formulation after obtaining the difference
In agent, (TrxR energy values unit definition is 1mg albumen oxidation μm ol NADPH per minute to the energy value of thioredoxin reductase
Ability), or be directly reduced to for the difference to be defined as the energy value of thioredoxin reductase in biological reagent with convenient
The calculating of data, this evaluation methodology is i.e. by the use of the difference as the energy value of thioredoxin reductase in biological reagent.
Industry standard be there is no because prepared by CSE, and CSE quantification when preparing, can not be realized, different product CSE live to TrxR
Power depression effect needs to be corrected based on a kind of accessible index, so as to realize the comparison of different products.The present invention is existed using CSE
Absorbance OD at 340nm340nmCSE is corrected, with OD340nmSuppress the quantitative base of TrxR vigor abilities as CSE
Plinth, namely the quantitative basis of evaluating cigarette or Nicotiana tabacum L. safety.The quantitative basis of evaluating cigarette or Nicotiana tabacum L. safety can also be
Tar content or nicotine content in flue gas aqueous extract.
Compared with the prior art, beneficial effects of the present invention are embodied in：
The invention provides a kind of instant, method of effectively evaluating cigarette or Nicotiana tabacum L. safety, successfully solves existing rank
The problems such as section Medicated cigarette or Nicotiana tabacum L. safety evaluatio mechanism are complicated, the cycle is long, operation sequence are simple, evaluation result accurately and reliably, and
With high pass flow characteristic, the research for developing reducing tar and reducing harm technology for tobacco enterprise and improving safety cigarette provides a reliability
Index.
Description of the drawings
Fig. 1 is CSE device for making figures；
Fig. 2 is that CSE is affected on several main enzymatic activitys in Hepar Mus homogenate；
Dose effect curves of the Fig. 3 for the suppression ratio in the embodiment of the present invention 1；
Fig. 4 is the comparison diagram of the safety evaluatio value of 18 kinds of Medicated cigarette in the embodiment of the present invention 1；
The time effect curve of suppression ratio of the Fig. 5 to suppress different biological reagents with CSE in the embodiment of the present invention 2；
The dose effect curve of suppression ratio of the Fig. 6 to suppress different biological reagents with CSE in the embodiment of the present invention 3；
Ratios pair of the Fig. 7 for the safety evaluatio value of Medicated cigarette A and Medicated cigarette R under different biological reagents in the embodiment of the present invention 3
Than figure.
Specific embodiment
Below in conjunction with the accompanying drawings, the present invention is further described by embodiment.
Embodiment 1
The present embodiment evaluates the safety of 18 kinds of Medicated cigarette (naming from A to R with letter respectively) as follows：
Step 1：18 kinds of Medicated cigarette to be evaluated are aspirated respectively, flue gas aqueous extract CSE is then configured to respectively, and is detected
Absorbance ODs of each CSE at 340nm340nm, with OD340nmAs evaluating cigarette or the quantitative basis of Nicotiana tabacum L. safety；
As shown in figure 1, CSE is to prepare as follows：By vacuum pump DP-01, (vacuum pressure is at the standard conditions
0.08Mpa) aspirate a Medicated cigarette to be evaluated, produced main flume pass sequentially through two series connection respectively be equipped with 5mL supplementary sets
It is collected in the GAS ABSORPTION bottle of liquid, after a Medicated cigarette to be evaluated or Nicotiana tabacum L. suction are finished, suction 10s makes supplementary set liquid
Main flume is fully collected, then 0.22 μm of water film filtering of supplementary set liquid Jing of main flume will have been collected, that is, obtained CSE；Supplementary set
Liquid is the phosphate buffer that concentration is 0.1M, pH=7.0, containing the second two that concentration is 10mM in the phosphate buffer
Amine tetraacethyl disodium (EDTA.Na2)。
Step 2：The biological reagent containing TrxR vigor is prepared, and thioredoxin in biological reagent is determined with DTNB reducing processs
The energy value of reductase, is designated as H0；
The compound method of the biological reagent containing TrxR vigor is：Hepar Mus of the 0.15g in -20 DEG C of low temperature cold storage are weighed, is added
1.5mL tissue homogenates, then grind with high-flux tissue dismembyator, 20min are centrifuged under the conditions of 15000rcf, 4 DEG C, take
Supernatant, obtains final product Hepar Mus homogenate, in this, as the biological reagent containing TrxR vigor；It is 0.15M, pH that tissue homogenate is concentration
=7.15 and containing concentration for 1mM disodiumedetate phosphate buffer.
By taking Hepar Mus homogenate as an example, the feelings of TrxR vigor (80%) in Hepar Mus homogenate are highly suppressed by detection in CSE
Other several relatively common and the enzyme with biological activity (glutathione s-transferase, gluathiones in Hepar Mus homogenate under condition
Fabk polypeptide, glutathion peroxidase, catalase, superoxide dismutase) activity change (Fig. 2), find this
The vigor of several enzymes is not changed in, and illustrates that CSE preferentially suppresses TrxR vigor in the biological reagent system containing TrxR vigor,
Other enzymes with biological activity suppress the ability of TrxR not affect CSE, therefore TrxR can be used as safety cigarette
The evaluation index of property.
Step 3：By a series of flue gas aqueous extract that different amounts of Medicated cigarette A are added in the biological reagent to same volume
Incubation 20min is carried out, is made flue gas aqueous extract produce the thioredoxin reductase vigor in biological reagent and is suppressed and determine
After suppression in biological reagent thioredoxin reductase energy value H1, calculate obtain flue gas aqueous extract in biological reagent
Suppression ratio T=(H0-H1)/H0 × 100% of thioredoxin reductase vigor, makes the dose effect curve of suppression ratio, such as
Shown in Fig. 3, suitable addition m of flue gas aqueous extract is determined from dose effect curve, and unit is microlitre (the present embodiment selection
20 μ L), suitable addition is the addition of flue gas aqueous extract and the linear model of suppression ratio in dose effect curve
The arbitrary value of the addition in enclosing；It is determined that under suitable addition flue gas aqueous extract to the thioredoxin in biological reagent also
The suppression ratio of protoenzyme vigor, is designated as T0；Suitable addition m is corrected to 10 microlitres, the corrected value M=m/ of suitable addition is obtained
10；
Step 4：Determine other 17 kinds flue gas aqueous extracts to the thioredoxin in biological reagent by the method for step 3
The corrected value M of the suppression ratio T0 and suitable addition of reductase vitality；
Step 5：Calculate the safety evaluatio value S=T0/ (M × OD for obtaining each Medicated cigarette or Nicotiana tabacum L.340nm), as shown in figure 4,
The safety of each Medicated cigarette is compared with safety evaluatio value S.As can be seen from the figure the S value highests of Medicated cigarette A, illustrate Medicated cigarette A's
Safety is poor, and the S values of Medicated cigarette R are minimum, illustrates that the safety of Medicated cigarette R is preferable；The gap of the S values of Medicated cigarette A and Medicated cigarette R reaches
2.59 again.
Embodiment 2
The present embodiment is used to prove the reason for incubation time selects 20min in the inventive method step 3：
20 μ L CSE by obtained in Medicated cigarette A are added in Hepar Mus homogenate obtained in embodiment 1, and when detecting difference
Between suppression ratio of the point CSE to TrxR vigor in Hepar Mus homogenate, draw suppression ratio time effect curve, as shown in Figure 5 a, from
It can be seen that in 20min, suppression ratio is tended towards stability in figure, therefore the time selected is 20min.
Additionally, biological reagent to be changed to Mus lung homogenate liquid and the pure enzyme preparation (production code members of TrxR respectively：T9698, sigma
Company produces), same test is carried out, as a result as illustrated in figures 5 b and 5 c, as can be seen from the figure CSE is suppressing different biological examinations
In the case of agent, 20min can all make suppression ratio stable.
Embodiment 3
The present embodiment is used to prove the inventive method reliable results, repeatable strong.
Use the biological reagent in embodiment 1 instead Mus lung homogenate liquid and the pure enzyme preparations of TrxR respectively, carry out same test,
The dose effect curve of the suppression ratio obtained in step 3 is respectively as shown in figure 6 a and 6b.
Three kinds of biological preparation evaluating cigarette safety acquired results are relatively used with the odds ratio of the S values of Medicated cigarette A and Medicated cigarette R,
As shown in Figure 7, it can be seen that though ratio obtained by when being evaluated from different biological preparation is differed, advise with identical
Rule, is all the S value of the S values more than Medicated cigarette R of Medicated cigarette A.Demonstrate the feasibility of the inventive method.
Embodiments of the invention are the foregoing is only, for a person skilled in the art, the present invention can have various
Change and change, obtain more preferable effect.All any modification/equivalents within the spirit and principles in the present invention, made, change
Enter, should be included within the scope of the present invention.
Claims (3)
1. a kind of method of in-vitro evaluation Medicated cigarette and Nicotiana tabacum L. safety, it is characterised in that：
Step 1：Each Medicated cigarette to be evaluated or Nicotiana tabacum L. are aspirated respectively, and is configured to flue gas aqueous extract respectively, detected
Absorbance OD of the flue gas aqueous extract at 340nm340nm, with OD340nmAs evaluating cigarette or the quantitative base of Nicotiana tabacum L. safety
Plinth；
Step 2：The biological reagent containing thioredoxin reductase vigor is prepared, double (the 2- p-nitrophenyl first of 5,5 '-two sulfur are used
Acid) reducing process determines the energy value of thioredoxin reductase in the biological reagent, is designated as H0；The biological reagent is Hepar Mus
Homogenate, Mus lung homogenate liquid or the pure enzyme preparations of TrxR；
Step 3：Incubating for 20min is carried out by different amounts of flue gas aqueous extract is added in the biological reagent to same volume
Educate, make flue gas aqueous extract suppression be produced to the thioredoxin reductase vigor in the biological reagent, determine raw after suppressing
The energy value H1 of thioredoxin reductase in thing reagent, and calculate acquisition flue gas aqueous extract to the sulfur oxygen in biological reagent also
Suppression ratio T=(H0-H1)/H0 × 100% of reductase proteins vigor, makes the dose effect curve of suppression ratio, from the suppression
Select except zero point in the addition of flue gas aqueous extract and the linear scope of suppression ratio in the dose effect curve of rate processed
Suitable addition of the addition of outer any flue gas aqueous extract as flue gas aqueous extract, is designated as m, unit for microlitre；Really
Suppression ratio of the flue gas aqueous extract to the thioredoxin reductase vigor in biological reagent under the suitable addition is scheduled on, is remembered
For T0；
Suitable addition m is corrected to 10 microlitres, the corrected value M=m/10 of suitable addition is obtained；
Step 4：Determine the flue gas aqueous extract of each Medicated cigarette or Nicotiana tabacum L. to the sulfur oxygen also egg in biological reagent by the method for step 3
The suppression ratio T0 and the corrected value M of suitable addition of white reductase vitality；
Step 5：Calculate the safety evaluatio value S=T0/ (M × OD for obtaining each Medicated cigarette or Nicotiana tabacum L.340nm), commented with the safety
Value S compares the safety of each Medicated cigarette or Nicotiana tabacum L..
2. in-vitro evaluation Medicated cigarette according to claim 1 and Nicotiana tabacum L. security method menu, it is characterised in that：Described in step 1
Flue gas aqueous extract is to prepare as follows：
One Medicated cigarette to be evaluated or Nicotiana tabacum L. are aspirated at the standard conditions by vacuum pump, produced main flume is passed sequentially through
It is collected in the respectively GAS ABSORPTION bottle equipped with 5mL supplementary set liquid of two series connection, aspirates in a Medicated cigarette to be evaluated or Nicotiana tabacum L.
After finishing, suction 10s makes supplementary set liquid fully collect main flume, then will collect 0.22 μm of the supplementary set liquid Jing of main flume
Water film filtering, that is, obtain flue gas aqueous extract；The supplementary set liquid is the phosphate buffer that concentration is 0.1M, pH=7.0, in institute
State in phosphate buffer containing concentration for 10mM disodiumedetate.
3. in-vitro evaluation Medicated cigarette according to claim 1 and Nicotiana tabacum L. security method menu, it is characterised in that：Evaluate in step 1
The quantitative basis of Medicated cigarette or Nicotiana tabacum L. safety can also be the tar content or nicotine content in flue gas aqueous extract.
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