CN104730019B - Method for in-vitro evaluation on safety of cigarettes and tobaccos - Google Patents

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

A method for evaluating the safety of cigarettes and tobacco in vitro

technical field

The invention relates to a method for evaluating the safety of cigarettes and tobacco in vitro. Specifically, the solution containing thioredoxin reductase (TrxR) activity is used to detect the inhibitory ability of different cigarette and tobacco smoke water extracts (CSE) to TrxR activity, and the absorbance value of CSE at 340nm is OD _340nm is used as a quantitative basis to evaluate the safety of cigarettes and tobacco.

Background technique

Modern medical research has enough evidence to prove that smoking is harmful to health, and smoke water extract CSE has also been widely used to study the toxicological mechanism of smoke (see literature 1-5). CSE contains 4,800 identified substances such as tar, nicotine, and aldehydes, among which aldehydes include acetaldehyde, formaldehyde, and unsaturated aldehydes. Among the unsaturated aldehydes, acrolein and butrolein are the most harmful ones. one of the substances. Unsaturated aldehydes such as acrolein are highly active electrophilic compounds, which are prone to nucleophilic addition reactions with nucleophiles. Studies have shown that acrolein is the main contributor to the cytotoxicity caused by cigarette smoke (see Document 6); in addition, acrolein can increase the risk of cancer induced by carcinogens such as benzopyrene in smoke (see Document 7).

Thioredoxin reductase TrxR is a dimeric selenium-containing enzyme. The selenocysteine (Sec) of TrxR is crucial for the catalytic activity of TrxR. It is an analog of cysteine, and its pKa (~ 5.2) is much lower than the pKa of cysteine (~8.3), so Sec is in the unprotonated selenolate state at physiological pH, which makes it highly nucleophilic. TrxR plays a key role in the formation of deoxynucleotides and is an essential substance in the process of DNA synthesis and cell proliferation. At the same time, TrxR has various biological functions such as inhibiting cell apoptosis and increasing the activity of transcription factors (see literature 8). Inhibition of TrxR activity can lead to consequences such as tissue damage (see literature 9 and 10).

As an active electrophilic compound, acrolein in smoke has also been confirmed to bind to Sec-containing TrxR and inhibit TrxR activity (see literature 11).

The current evaluation of the hazards of cigarettes mainly relies on component analysis and biological detection. Component analysis has been widely used in the cigarette industry. However, due to the complexity of smoke components, different harmful substances will rise and fall in the practice of reducing tar and harm, and the evaluation relying on several types of substances has certain limitations. It takes a long time to evaluate the safety of cigarettes by cell and animal experiments, and it has low practicability in the development of low-hazard cigarettes.

Therefore, the development of low-harm cigarettes requires an immediate, rapid and high-throughput method for evaluating the safety of cigarettes and tobacco.

references:

1. Kosmider B, Messier EM, Chu HW, et. Epub 2011 Dec 7. Human alveolarepithelial 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. 2008 Oct; 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 retina 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. 2008 Jan 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 byalkylation 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 thioredoxinreductase, 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., News York, 2000, pp.7.4.1–7.4.14.

Contents of the invention

The purpose of the present invention is to provide a method for evaluating the safety of cigarettes and tobacco in vitro which can be instant, rapid and high-throughput.

The present invention solves technical problem, adopts following technical scheme:

The method for evaluating the safety of cigarettes and tobacco in vitro of the present invention is characterized in that:

Step 1: Suck each cigarette or tobacco to be evaluated separately, and prepare the smoke water extract respectively, detect the absorbance value OD _340nm of the smoke water extract at 340nm, and use OD _340nm as the evaluation cigarette or tobacco safety Quantitative basis for sex;

Step 2: Prepare a biological reagent containing thioredoxin reductase activity, and use the 5,5'-dithiobis(2-p-nitrobenzoic acid) (DTNB) reduction method to determine the reduction of thioredoxin in the biological reagent Enzyme activity value, denoted as H0;

Step 3: adding different amounts of flue gas water extract to the same volume of the biological reagent and incubating for 20 minutes, so that the flue gas water extract can inhibit the activity of thioredoxin reductase in the biological reagent, Measure the activity value H1 of the thioredoxin reductase in the biological reagent after inhibition, and calculate and obtain the inhibition rate T=(H0-H1)/H0× 100%, make the dose-response curve of the inhibition rate, select any smoke water extract except the zero point from the range in which the amount of the smoke water extract in the dose-effect curve of the inhibition rate is linearly related to the inhibition rate The add-on of the flue gas water extract as the suitable add-on of flue gas, denoted as m, the unit is microliter; Determine the suppression of the thioredoxin reductase activity in the biological reagent by the flue gas water extract under the described suitable add-on rate, denoted as T0;

Correct the appropriate addition amount m to 10 microliters to obtain the corrected value M=m/10 of the appropriate addition amount;

Step 4: Determine the inhibition rate T0 and the correction value M of the appropriate addition amount of the smoke water extract of each cigarette or tobacco to the activity of thioredoxin reductase in the biological reagent according to the method of step 3; The appropriate addition amount of the smoke water extract is different, so the appropriate addition amount of the smoke water extract made by all different cigarettes or tobaccos is collectively corrected to 10 microliters, in order to more accurately evaluate the safety of different cigarettes or tobacco .

Step 5: Calculate and obtain the safety evaluation value S=T0/(M×OD _340nm ) of each cigarette or tobacco, and use the safety evaluation value S to compare the safety of each cigarette or tobacco.

The method for evaluating the safety of cigarettes and tobacco in vitro of the present invention is also characterized in that the smoke water extract described in step 1 is prepared as follows:

A cigarette or tobacco to be evaluated is sucked by a vacuum pump under standard conditions, and the mainstream smoke generated is sequentially collected through two series-connected gas absorption bottles each containing 5mL of replenishing liquid. After the cigarette or tobacco is smoked, air suck for 10 seconds, so that the replenishing liquid can fully collect the mainstream smoke, and then filter the replenishing liquid that has collected the mainstream smoke through a 0.22 μm water membrane to obtain the smoke water extract; The replenishing solution is a phosphate buffer solution with a concentration of 0.1M and pH=7.0, and the phosphate buffer solution contains disodium ethylenediaminetetraacetic acid (EDTA.Na ₂ ) at a concentration of 10 mM.

The biological reagent containing thioredoxin reductase activity described in step 2 is homogenate of animal tissue, homogenate of plant tissue, homogenate of isolated cells or homogenate of microorganism, or pure thioredoxin reductase Enzyme products.

The homogenate of animal tissue or plant tissue in step 2 is obtained as follows: Weigh 0.15 g of animal tissue or plant tissue frozen at -20°C, add 1.5 mL of tissue homogenate, and then use high-throughput Grind with a tissue grinder, centrifuge at 15,000 rcf and 4°C for 20 minutes, and take the supernatant to obtain a homogenate of animal tissue or a homogenate of plant tissue; the tissue homogenate has a concentration of 0.15M, pH=7.15 and contains Disodium edetate in phosphate buffer at a concentration of 1 mM.

The homogenate of isolated cells or microorganisms in step 2 is obtained as follows: suspend the isolated cells or microorganisms in the cell lysate at a concentration of 10-90 million/mL, and then use ultrasonic cleaning Sonicate the cells at 4°C and 40KHz for 20 minutes to achieve the purpose of crushing, then centrifuge at 10,000rpm and 4°C for 10 minutes, and take the supernatant to obtain the homogenate of isolated cells or homogenate of microorganisms ; The cell lysate is a solution of tris (Tris-HCl) with a concentration of 0.1M and pH7.4, and the solution of the tris contains dithiothreose with a concentration of 1 mM alcohol (DTT) and 1 mM phenylmethylsulfonamide (PMSF).

In the present invention, the method of using 5,5'-dithiobis(2-p-nitrobenzoic acid) (DTNB) reduction method to measure the activity value of thioredoxin reductase in the biological reagent is referred to literature 12 and 13, specifically Proceed as follows:

First prepare the detection working solution (the detection working solution is a phosphate buffer solution with a concentration of 100 mM and pH 7.0, which contains 10 mM EDTA-Na ₂ , 5 mM DTNB, 240 μM NADPH and 0.2 mg/ mL of bovine serum albumin), ready-to-use and ready-to-use, temperature controlled at 37°C;

Mix the biological reagent to be tested in the inhibition tube with auranofin at a volume ratio of 9:1, and the biological reagent to be tested in the non-inhibition tube and 5% ethanol at a volume ratio of 9:1, and act at 37°C for 20 minutes; Add the biological reagents in the post-inhibition tube and the non-inhibition tube to the 96-well plate respectively, add 250 microliters of detection working solution to the test well of the 96-well plate to start the reaction, and immediately place the 96-well plate in the microplate reader for detection . At 37°C, detect on a microplate reader at a wavelength of 412nm for 6 minutes, read the value every 12s, read the enzyme kinetics curve, and read the slope value (Vmax) of the time period from 120s to 300s, which corresponds to the sample in the non-inhibited tube The corresponding Vmax of the sample in the inhibition tube is subtracted from the Vmax, and after obtaining the difference, the activity value of thioredoxin reductase in the biological reagent is calculated through a specific formula (the unit of TrxR activity value is defined as 1 mg protein oxidized μmol NADPH per minute ability), or directly simplify to define the difference as the activity value of thioredoxin reductase in biological reagents to facilitate the calculation of data. This evaluation method uses this difference as the activity value of thioredoxin reductase in biological reagents vitality value.

Since there is no industry standard for the preparation of CSE, and CSE cannot be quantified during preparation, the inhibitory effect of CSE on TrxR activity of different products needs to be corrected based on a feasible index, so as to realize the comparison of different products. In the present invention, the absorbance value OD _340nm of CSE at 340nm is used to correct CSE, and OD _340nm is used as the quantitative basis for CSE's ability to inhibit TrxR activity, that is, the quantitative basis for evaluating the safety of cigarettes or tobacco. The quantitative basis for evaluating the safety of cigarettes or tobacco can also be the tar content or nicotine content in the smoke water extract.

Compared with the prior art, the beneficial effects of the present invention are reflected in:

The present invention provides an immediate and effective method for evaluating the safety of cigarettes or tobacco, which successfully solves the problems of complex mechanism and long period of safety evaluation of cigarettes or tobacco at the present stage, with simple operation procedures, accurate and reliable evaluation results, and high The flux characteristics provide a reliable indicator for tobacco companies to develop tar reduction and harm reduction technology and improve cigarette safety.

Description of drawings

Figure 1 is a diagram of the CSE preparation device;

Figure 2 is the effect of CSE on several main enzyme activities in rat liver homogenate;

Fig. 3 is the dose effect curve of the inhibition rate in the embodiment of the present invention 1;

Fig. 4 is a comparison chart of the safety evaluation values of 18 kinds of cigarettes in Example 1 of the present invention;

Fig. 5 is the time-effect curve of the inhibition rate of inhibiting different biological agents with CSE in Example 2 of the present invention;

Fig. 6 is the dose-effect curve of the inhibition rate of inhibiting different biological agents with CSE in Example 3 of the present invention;

Fig. 7 is a graph comparing the ratios of the safety evaluation values of cigarette A and cigarette R under different biological agents in Example 3 of the present invention.

detailed description

The present invention will be further described through the embodiments below in conjunction with the accompanying drawings.

Example 1

The present embodiment evaluates the safety of 18 kinds of cigarettes (named by letters from A to R respectively) by the following steps:

Step 1: Smoke the 18 kinds of cigarettes to be evaluated separately, and then prepare the smoke water extract CSE respectively, and detect the absorbance value OD _340nm of each CSE at 340nm, and use OD _340nm as the quantitative measure for evaluating the safety of cigarettes or tobacco Base;

As shown in Figure 1, CSE is formulated as follows: a cigarette to be evaluated is sucked under standard conditions (vacuum pressure is 0.08Mpa) by vacuum pump DP-01, and the mainstream smoke produced is sequentially passed through two series-connected Collect in gas absorption bottles each equipped with 5mL replenishment liquid. After smoking a cigarette or tobacco to be evaluated, suck for 10 seconds so that the replenishment liquid can fully collect the mainstream smoke, and then put the collected mainstream smoke CSE is obtained by filtering the replenishing solution through a 0.22 μm water membrane; the replenishing solution is a phosphate buffer solution with a concentration of 0.1M and pH=7.0, and the phosphate buffer solution contains 10 mM ethylenediaminetetra Disodium acetate ( _EDTA.Na2 ).

Step 2: Prepare a biological reagent containing TrxR activity, and use the DTNB reduction method to measure the activity value of thioredoxin reductase in the biological reagent, which is recorded as H0;

The preparation method of the biological reagent containing TrxR activity is as follows: Weigh 0.15g of mouse liver frozen at -20°C, add 1.5mL of tissue homogenate, and then use a high-throughput tissue grinder to grind it. Centrifuge for 20 minutes, and take the supernatant to obtain the rat liver homogenate, which is used as a biological reagent containing TrxR activity; the tissue homogenate has a concentration of 0.15M, pH=7.15, and contains disodium edetate at a concentration of 1mM of phosphate buffered saline.

Taking the rat liver homogenate as an example, by detecting several other common and biologically active enzymes (glutathione Peptide S transferase, glutathione reductase, glutathione peroxidase, catalase, superoxide dismutase) activity changes (Figure 2), found that the activity of these enzymes did not change , indicating that in the biological reagent system containing TrxR activity, CSE preferentially inhibits TrxR activity, and other biologically active enzymes have no effect on the ability of CSE to inhibit TrxR, so TrxR can be used as an evaluation index for cigarette safety.

Step 3: Add a series of different amounts of cigarette A smoke water extract to the same volume of biological reagent and incubate for 20 minutes, so that the smoke water extract can inhibit the activity of thioredoxin reductase in the biological reagent and Measure the activity value H1 of the thioredoxin reductase in the biological reagent after inhibition, and calculate the inhibition rate T=(H0-H1)/H0×100 of the flue gas water extract to the thioredoxin reductase activity in the biological reagent %, make the dose-response curve of the inhibition rate, as shown in Figure 3, determine the appropriate addition m of the smoke water extract from the dose-effect curve, the unit is microliter (the present embodiment selects 20 μ L for use), and the appropriate addition is In the dose-response curve, the addition of the flue gas water extract and the inhibition rate are any value of the addition within the scope of the linear relationship; determine the reduction of the flue gas water extract to the thioredoxin in the biological reagent under the appropriate addition The inhibition rate of the enzyme activity is recorded as T0; the appropriate addition amount m is corrected to 10 microliters, and the correction value M=m/10 of the appropriate addition amount is obtained;

Step 4: Determine the inhibition rate T0 of other 17 kinds of flue gas water extracts to the activity of thioredoxin reductase in biological reagents and the correction value M of the appropriate addition amount according to the method of step 3;

Step 5: Calculate and obtain the safety evaluation value S=T0/(M×OD _340nm ) of each cigarette or tobacco, as shown in FIG. 4 , compare the safety of each cigarette with the safety evaluation value S. It can be seen from the figure that the S value of cigarette A is the highest, indicating that the safety of cigarette A is poor, while the S value of cigarette R is the lowest, indicating that the safety of cigarette R is better; the difference between the S values of cigarette A and cigarette R is 2.59 times.

Example 2

This embodiment is used to prove that the incubation time in step 3 of the method of the present invention is selected for the reason of 20min:

Add 20 μL of CSE made from cigarette A to the rat liver homogenate obtained in Example 1, and detect the inhibitory rate of CSE to TrxR activity in the rat liver homogenate at different time points, and draw the time-effect curve of the inhibitory rate, As shown in Figure 5a, it can be seen from the figure that the inhibition rate has stabilized at 20 minutes, so the selected time is 20 minutes.

In addition, the biological reagents were changed to mouse lung homogenate and TrxR pure enzyme products (product number: T9698, produced by sigma company), and the same test was carried out. The results are shown in Figures 5b and 5c. It can be seen from the figure that CSE is inhibiting In the case of different biological reagents, the inhibition rate can be stabilized within 20 minutes.

Example 3

This example is used to prove that the results of the method of the present invention are reliable and repeatable.

The biological reagents in Example 1 were replaced by mouse lung homogenate and TrxR pure enzyme preparations, and the same test was carried out. The dose-effect curves of the inhibition rate obtained in step 3 are shown in Figures 6a and 6b, respectively.

Using the ratio of the S value of cigarette A and cigarette R to compare the results obtained by using three biological agents to evaluate the safety of cigarettes, as shown in Figure 7, it can be seen that although the ratios obtained when different biological agents are used for evaluation are different, they have the same The rule is that the S value of cigarette A is greater than that of cigarette R. The feasibility of the method of the present invention is proved.

The above descriptions are only embodiments of the present invention, and for those skilled in the art, various modifications and changes may be made to the present invention to obtain better effects. Any modification/equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection 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 340nm_340nm, with OD_340nmAs 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.