CN1133550A - Removal of noxious oxidants and carcinogenic volatile nitrosocompounds from cigarette smoke using biological substances - Google Patents

Abstract

This invention refers to a method of withholding noxious compound contained in cigarette smoke (NO, NOx, carcinogenic nitrosocompounds, free radicals, H2O2, CO, aldehydes, and trace elements) which were up to today insufficiently retained by conventional cigarette filters. The method described specifically refers to the enrichment of common convention filters with biological substances of the metal ions (Fe2+, Cu2+, Mg2+) complexed with prophirin ring as well as Fe2+ ions sterospecifically bound to protein molecules, either separately or in combination. The enrichment of these conventional filters with the above mentioned biological substances alters neither the physical properties of the cigarette smoke (odor, taste and appearance) nor the physical properties of the filter itself.

Description

Removal of harmful oxidizing agents and carcinogenic volatile nitroso compounds from cigarette smoke using biological substances

The present invention proposes a method for preventing the inhalation of harmful compounds during smoking, namely: nitric oxide, free radicals, aldehydes, hydrogen peroxide, carbon monoxide, trace elements and carcinogenic volatile nitroso compounds, which have not heretofore been effectively filtered out by conventional cigarette filters.

Many articles in international journals propose that cigarette smoke can be divided into two phases; a) solid phase (tar); and b) a gas phase. This separation occurs when using typical Cambridge glass fibers which retain 99.9% of the particles with a size greater than 0.1 μm. Cigarette tar contains very high concentrations of very stable free radicals which can be divided into at least four different types. Semiquinones in equilibrium with quinones and hydroxyquinones are considered to be free radicals with the most interesting chemical properties. The quinone system reduces molecular oxygen to superoxide (O)₂) The latter, via spontaneous disproportionation, produces hydrogen peroxide (H)₂O₂). In the gas phase to be inhaled, 10 are present in each puff¹⁵Organic groups with half-life less than 1 second. It seems paradoxical that, despite their short half-life, they maintain a higher activity in the gas phase for more than 10 minutes. In fact, the concentration of the above-mentioned radicals is significantly increased near the end of the cigarette filter. One explanation for this conflict is: a persistent steady state condition occurs; because free radicals are constantly generated (Pryor, W.A., stone, K., Ann.N.Y.Acad.Sci.686: 12-28, 1993).

Nitric Oxide (NO) is the most important free radical in the gas phase of the smoke during smoking, and it participates in a series of reactions that produce nitrogen dioxide, isoprenyl, hydrogen peroxide and alkoxy groups. Flue gasAlso contain considerable amounts of aldehydes which constitute their toxic effects. It has been shown that trace amounts of aldehydes extracted from smoke can lead to protein catabolism and oxidation of plasma protein sulfhydryl groups. These properties of aldehydes are due to the carbonyl group of aldehydes and the-SH groups and-NH groups of plasma proteins₂Resulting from reactions between radicals. For example, acrolein in flue gas rapidly reacts with-SH groups to form carbonyl compounds (Alving, K., Forhem, C., and Lundberg, J.M., Br.J.Pharmacol.110: 739-E746, 1993). The smoke tar contains trace elements such as iron, copper, manganese and cadmium, which are involved in many radical generating reactions and lead to the formation of very reactive secondary radicals (e.g., peroxy oxygen)Hydrogen radicals, alkoxy radicals, superoxides, cytotoxic aldehydes, etc.). The trace elements entering the lung during smoking result in a series of redox reactions in the lung fluid and alveolar macrophages, which lead to very reactive hydroxyl groups (OH)^-) Is performed. These hydroxyl groups are mainly formed by fenton reaction (Fento reaction) in the presence of iron. Copper also forms hydroxyl radicals in the lungs by reacting with hydrogen peroxide. Low concentration of manganese (10)^-7M) activates soluble guanylate cyclase of lung endothelial cells, generating nitric oxide and superoxide through a positive feedback mechanism (Youn, y.k.lalo-nde, c., and Demling, r., Free rad.biol.med.12: 409-415, 1992). Tobacco produces carbon monoxide on combustion, and even after smoke is given off, some amount of CO remains in the lungs, which activates it after reacting with the heme moiety of soluble guanylate cyclase in the endothelial cells and other cells of the lung tissue. Higher levels of cyclic GMP in cells associated with positive feedback mechanisms increase nitric oxide and superoxide production (Watson, A., Joyce, H., Hopper, L., and Pride, N.B., Thorax 48: 119-124, 1993). NO gas can be produced by a variety of cells, including vascular endothelial cells and reticuloendothelial cells, and can cause smooth muscle relaxation (Lowenstein, C.J., Din-erman, J.L., Snyder, S.H.Ann.Intern.Med.120: 227-. There is also exogenous NO that is believed to damage blood vessels and other tissues as well. It has been demonstrated that secondary aminesAnd tertiary amines can be reacted with nitrite and other nitrosating agents to form N-nitrosamines (Low-enstein, C.J., Dinerman, J.L., Snynder, S.H.Ann.Intern.Med.120: 227-. Since 1974, many studies have demonstrated that alkaloids are nitrosylated to tobacco specific N-nitrosamines (TSNAs) at harvest, tobacco processing and smoking. Among TSNAs identified in tobacco and/or its smoke, N-nitrosonornicotine (NNN), 4- (methylnitrosamino) -1- (3-pyridyl) -1-butanone (NNK) and 4- (methylnitrosamino) -1- (3-pyridyl) -1-butanol (NNAL) are strong animal carcinogens. NNN induces tumors in the rat lung, hamster trachea and nasal and esophageal tumors in rats. NNK induces lung cancer in rats, hamsters and rats, as well as liver cancer, nasal cancer and pancreas cancer in ratsCancer. Oral swabbing of the NNN and NNK mixture induced oral cancer and lung cancer in rats. Typical levels of NNK and NNN in raw stream smoke are 200 ng/cigarette (Hecht, S.S., Spr-att, T.E., and Trushin, N.Carcinogenesis, 9: 161-.

The present inventors' studies on the effects of smoke on lung tissue have revealed that NO reacts with superoxide to form the strong oxidizing group peroxynitrite (ONOO)^-) It can lead to secondary damage reactions of important biomolecules. The metabolic and destructive effects of NO in cells were studied in our laboratory by in vitro and in vivo experiments.

In the presence of oxygen, NO is oxidized to nitrogen dioxide (NO)₂). The oxidation rate depends on the oxygen concentration and the square of the NO concentration. It is clear that the cytotoxicity of nitrogen dioxide, when it enters into aqueous solution, is converted into nitrite and nitrate.Furthermore, NO can form complexes with trace elements and/or metalloproteins, such as hemoglobin (Wink, d.a., Darbyshire, j.f., Nims, r.w., Saavedra, j.e., and Ford, p.e., chem.res, toxicol.6: 23-27, 1993).

NO reacts with superoxide to produce the harmful compound ONOO^-Several superoxide toxicities could be demonstrated. For its strong oxidation potential (+1.4V), ONOO^-And (4) abnormal stability. In which it is divided intoDuring the decomposition, it generates strongly oxidized derivatives including hydroxyl, nitrogen dioxide and nitronium ions. Thus, any improvement in tissue production of NO and superoxide results in the generation of strong secondary oxidative groups (Deliconstaninos, G., Villiotou, V., Stavrid-es, J.C., Cancer mol. biol. 1: 77-86, 1994). Finally, ONOO^-And esters thereof (RO-ONO or RO-ONO)₂) Leads to the inactivation of α -1-protease inhibitor (α 1 PI). this is confirmed by the fact that a) α 1PI is not rapidly inactivated by hydrogen peroxide alone, but only in the presence of NO, to produce ONOO^-And rapid deactivation of α 1PI, b) tert-butyl peroxynitrite (RO-O-O-NO)₂) Or ONOO^-α 1PI is inactivated by the solution itself, and c) amines and amino acids prevent rapid inactivation of α 1PI protease (Mor-eno, J.J., and Pryor, W.A., chem.Res.Toxicol.5: 425-431, 1992). In addition to free radicals contained in smoke, activated alveolar macrophages are another important free radical generating source for smokers. Respiratory burst of alveolar macrophages activated by smoke results in more oxygen radicals (mainly O)₂ ^-NO and H₂O₂) Is generated. Smokers have a greater number of alveolar macrophages and circulating neutrophils. Oxygen free radicals in the smoke are also associated with the development of lung cancer. Inhaled smoke increases oxidative stress of lung cells, resulting in a decrease in intracellular antioxidantconcentration. H₂O₂By the production of hydroxyl groups, it reacts with DNA in the cell and causes double strand breaks. Since this cleavage can be avoided by addition of catalase, this indirectly confirms H₂O₂And hydroxyl group damage to cellular DNA (Leanderson, P., Ann.N.Y.Acad.Sci.686: 249-261, 1993). Furthermore, H₂O₂Can cause the change of lung airway epithelium, and is related to the attack of smoker bronchial cancer. Thus, H₂O₂The damaging effects on lung cells (contained in the smoke) and the onset of lung cancer have been strongly demonstrated. The smoke tar contains both semiquinone group and iron, thus forming a hydroxyl generating system. Various trace elements (Fe, Cu, Mn and Cd) contained in the smoke tar can act in cells and can also act among cells. Fe²⁺Can be used forThe known fenton reaction occurs:

various oxidation reactions can be induced by the hydroxyl groups. Similarly, Cd²⁺Hydroxyl radicals may also be generated. Mn²⁺Are representative activators of soluble guanylate cyclase. Cd contained in flue gas²⁺Especially, the lung is harmed. Smoker's lung Cd²⁺The content of (A) is 2 times of the normal concentration. This indicates that Cd²⁺Replaces Zn normally existing in the lung duct epithelium²⁺(Kostial, K., see "Trace elements in Human and Animal Nutrition (ed. W.Mertz) fifth edition, Vol.2: 319-345, Academic Press, Inc. Orlando, Fl., 1986). Aldehydes present in smoke and-SH and-NH in proteins₂Crotonaldehyde (α unsaturated aldehydes) contained in flue gas can reduce the concentration of-SH groups and increase the concentration of carbonyl proteins (Stadtman, E.R., Science 257: 1220-.

Filters for cigarettes are today highly appreciated. The ultimate goal of adding filters to cigarettes is to maximize the retention of harmful compounds in gas and solid phase smoke. Epidemiological studies of smokers have demonstrated that the onset of disease is positively correlated with the dose, whether smoke is ingested in the gas, solid or mixed phase (Surgeon General of the U.S. public Hea-lth service, the health consensus of using the small drugs tobaco, N.H. Publ.No. 862874, Bethesda, Md., 1986). Improvements to tobacco have proven themselves as a practical way to reduce the amount of harmful compounds in smoke. This was achieved initially; a conventional filter is used and the composition of the tobacco is then altered by chemical treatment. Cigarette production can also be improved by using porous paper or paper made from tobacco. Over the past 15 years, many attempts have been made to reduce the health hazard of smoking, with the following: reducing the smoke amount of each cigarette: changing the diameter of the cigarette; and porous filters are used. Porous filters are capable of diluting the smoke by 50% with air. Activated carbon is also used in conjunction with porous filters. This can significantly reduce tar and nicotine in the smoke. This technology is employed especially in developed countries like austria, canada, france, germany, sweden, uk and the united states. The tar and nicotine content of the American cigarette decreased from 38mg and 2.7mg in 1955 to 13mg and 1mg in 1991, respectively. In the european union, this trend to reduce tar and nicotine content in smoke is continuing. The allowable upper limit of tar at month 1 of 1993 was 15mg, and by the beginning of month 1 of 1998, this upper limit would be reduced to 12 mg. In other countries, however, the tar content in the smoke is 22mg (Mitacek, E.J., Brunne-man, K.D., Pollednak, A.P., Hoffman, D., and Suttajit, M., Prev.Med.20: 764-773, 1991). Improvements made in the manufacture of cigarettes have led to the specific removal of some of the toxic materials in the smoke; more specifically, cellulose acetate filters have been introduced, whereby semi-volatile phenols and volatile N-nitrosamines can be partially removed (Bru-nnemann, K.D., Hoffman, d., Recent.adv.Tobacco Res.17: 71-112, 1989). With porous filters, CO can be selectively reduced. The nitrite-rich tobacco is used to selectively reduce the concentration of carcinogenic Polynuclear Aromatics (PAHs). However, the reduction of Tobacco PAH by high concentrations of nitrite leads to an undesirable increase in carcinogenic N-nitrosamines, and therefore other approaches have to be taken to reduce PAH (Hoffman, D., Hoffman, I., Wynder, E.L., Lung Cancer and the Changing Cancer in Relevance to Human Cancer of N-Nitroso-com-ounds, Tobacco Smok and Mycotoxin (eds. O' Neil, I.K., Chen, J., and Bartsch, H.) Vol.105: 449-459, 1991).

As is clear from the above description, there is a need to produce a catalyst that retains harmful NO, free radicals, H₂O₂Aldehydes and carcinogenic nitroso compounds, which are responsible for the damaging effect of smoke on the respiratory and cardiovascular systems. In order to identify harmful compounds in smoke, chemical and biological experiments are carried out. The chemical experiments performed were:

a) a new chemical and biological method, which was created by our laboratory, was used to identify and quantify NO and NOx.

b) Free radicals are identified using a chemiluminescent process that relies on a lucigenin.

c) Aldehydes and quinones were identified by activating the luciferin-luciferase enzymatic system (this method was also created by our laboratory).

d) The trace elements were identified and quantified by the method of oxyluciferin, carried out by luciferase, in the presence of ATP (this method was created by our laboratory).

e) Identification and quantitative determination of H using isoluminol-dependent microperoxidase (isoluminol microperox-idase) chemiluminescence method₂O₂₀

f) Enhanced chemiluminescence of ONOO via luminol^-Spectrophotometric identification and quantitative determination were performed.

g) Carcinogenic nitroso compounds were identified by luminol-enhanced chemiluminescence.

The biological experiments performed were as follows:

a) identifying NO using the extracted soluble guanylate cyclase activity as a functional parameter.

b) By pairing ONOO^-Determination of induced oxidative stress (oxidative stress) of human erythrocytes to identify ONOO^-。

c) Identifying CO using the extracted soluble guanylate cyclase activity as a functional parameter.

In addition, we also performed the following in vitro experiments:

a) alveolar macrophages were extracted from rat lungs.

b) The oxidative stress of alveolar macrophages caused by t-butyl hydroperoxide (t-BHP) was measured.

c) Determination of NO/NO production by alveolar macrophages₂ ^-/ONOO^-。

d) Determination of H production by alveolar macrophages₂O₂₀

e) External source of H₂O₂Effect on NO production by alveolar macrophages.

In vivo experiments were performed with human volunteers in order to assay the following compounds:

a) the NO in the air exhaled by non-smokers was measured.

b) The NO in the air exhaled by the smoker is measured.

c) The NO in the discharged flue gas was measured.

d) Determination of ONOO in spit-out smoke^-。

e) The free radicals in the discharged flue gas were measured.

f) The aldehydes in the discharged flue gas were measured.

To determine the NO, NOx content in a) the smoke, b) released by alveolar macrophages after challenge with smoke and c) the smoke excreted by the volunteers, we designed and created a chamber with a transparent plexiglas solid rod 2.5cm in diameter, hollowed out one end of the solid plexiglas rod with a lathe, creating identical conical cavities on each plexiglas rod. The open ends are then further machined and polished to make a mating wedge bond, forming a very tight bond between the two tapered cavities. A thin teflon (te-flon) sheet (teflon. o.0015 inches thick) was sandwiched between the components and pressed together again by thumb screws. The two available tube access sections on both sides of the membrane allow for the injection, extraction or modification of the biologically active sample and active substance on either side of the membrane during the biological reaction.

A. Determination of NO by chemiluminescence

Standard NO solutions (Deliconstaninos, G., Villiotou, V., Fassitsas, C., (1992) J.Cardiovasc.Pharmacol.12, S63-S65) and (Deliconstaninos, G., Villiotou, V., Stavrides, J.C., (1994) see "Biology of Nitric Oxide", eds. Feelish, M.Busse, R.R., Moncanda, S., Portland Press, in print) were prepared according to the literature. The reaction solution was prepared from a Hank's Balanced Salt Solution (HBSS) pH 7.4; h₂O₂(500. mu.M); luminol (30. mu.M) in a total volume of 500. mu.l. The vial was stirred vigorously and then stirred in the Bedrthold Auto-Luminescence was recorded on a Lumat LB953 luminometer.

B、NO/NO₂ ^-Chemical assay of (4).

The chemical determination of NO is based on the diazotization of NO to the sulfonamide at acidic pH and the subsequent oxidation of the scopoletin. It can be determined by luminescence methods as described above (De-iconstantinos, G., Villiotou, V., Fassitsas, C., J.Cardi-ovasc.Pharmacol 12: S63-S65, 1992). Alveolar macrophages that will be present in HBSS (10)⁶Cells/ml) with 100. mu.l of a suspension of cells dissolved in 20% H₃PO₄20% of sulfanilamide and 25 μ M of scopoletin. The decay of fluorescence was monitored at room temperature (22 ℃) using an Amin-co SPF-500 fluorescence spectrophotometer. Fluorescence was monitored continuously until the slope of the line was measurable (approximately 8 minutes). The slope measurements were then converted to nmol concentrations of NO using a standard curve composed of various pure NO concentrations. Measurement of the most advanced NO synthesis by reaction with Grace reagent based on its accumulation in the supernatant of cultured cellsEnd product Nitrite (NO)₂ ^-)。

C. Peroxynitrite (ONOO)^-) Spectrophotometric determination of

ONOO^-Synthesized, titrated and stored as described previously (Deliconstaninos, G., Villiotou, V., Stavrides, J.C., see "Biology of nitric Oxi-de (eds. Feelish, M., Busse, R., and Moncada, S.) Portland Pre-ss (in print)). Due to ONOO^-Instability at pH7.4, in H₂O₂The UV spectrum was recorded immediately after mixing with the NO solution. According to 1670M^-1Cm^-1Determination of the value of ε 302nm of ONOO^-The concentration of (c). Minus H₂O₂The corresponding concentration of the basic UV spectrum, the UV spectrum is obtained.

D. Determination of free radicals

The determination of free radicals was carried out as described above using chemiluminescence induced by lucigenin/DAMCO (1, 4 diazabicyclo- [2, 2, 2]octane) (Deliconstatinos, G., Krueger, G.R.F., J.Viral Dis.1: 22-27, 1993). The reaction mixture was made up of HBSS, pH 7.4; lucigenin (30 μ M); DAMCO (100. mu.M). The reagent bottles were stirred vigorously and luminescence was recorded on a Bedrthold AutoLumat LB953 luminometer. Scavenger with oxygen free radical (SOD, mannitol, histidine, methionine) is used.

E. Determination of trace elements and aldehydes

This assay is based on the luciferase-catalysed oxidation of D-luciferin in the presence of ATP-magnesium salts, according to the following reaction:

trace element Cd²⁺，Cu²⁺，Fe²⁺The activity of luciferase can be improved, and the maximum chemiluminescence response is increased proportionally according to the concentration of trace elements and reaches 10 mug. The reaction was carried out in HBSS at pH7.4 in a total volume of 0.5 ml.

For the determination of aldehydes, the same enzymatic system luciferin/luciferase was used, but lacking ATP. The aldehyde reacts with the enzymatic system in the absence of ATP to produce chemiluminescence. Reagents used were taken from the ATP kit (Calbiochem-Novabiochem CA, u.s.a.).

F. Extraction of alveolar macrophages

Briefly, rats were killed by intravenous pentobarbital, the chest was dissected and treated with Ca-free²⁺The lungs were removed intact from the chest cavity by washing with cold (4 ℃) phosphate buffered saline (PBS, pH7.4) to remove blood from the lungs. Rat lung homogenate was obtained as follows: the lung tissue was repeatedly aspirated by syringe and sequentially passed through increasingly finer stainless steelscreens, 32, 62 and 68 holes per inch respectively, under a constant flow of Finkelstein balanced salt solution (FBSS; pH 7.4). The final suspension of alveolar macrophages was collected, filtered and centrifuged at 300 Xg for 10 min to pellet the cells. Cell pellets containing more than 98% macrophages were washed and resuspended in Ringer's solution. The above procedure was repeated two more times. About 10X 10 can be extracted from each rat⁸The macrophage cell of (1). Viability was assessed by trypan blue exclusion.

F. Identification of nitroso Compounds

The nitroso compound being H₂O₂Identified by the release of Nitric Oxide (NO) after treatment. The reaction solution is composed of dimethyl nitrosamine and/or diethyl nitrosamine (1 μ M); h₂O₂(500. mu.M); luminol (30 μ M), dissolved in HBSS, pH7.4, in a total volume of 0.5 ml. The reagent bottles were vigorously agitated and luminescence was recorded on a Bedrthold AutoLumatLB953 luminometer. Mannitol (100 mM); DMSO (100mM) and cysteine (3.0mM) were used to confirm the ONOO^-And (4) generating.

G. Isolation of alveolar macrophages

Briefly, rats were killed by intravenous pentobarbital, the chest was dissected and treated with Ca-free²⁺The lungs were removed intact from the chest cavity by washing with cold (4 ℃) phosphate buffered saline (PBS, pH7.4) to remove blood from the lungs. Rat lung homogenate was obtained as follows: the lung tissue was repeatedly aspirated by syringe and sequentially passed through increasingly finer stainless steel screens, each 32 per inch (mm), under a steady flow of Finkelstein balanced salt solution (FBSS; pH7.4)Screens of holes, 62 holes and 68 holes. The final suspension of alveolar macrophages was collected, filtered and centrifuged at 300 × g for 10 min; the cells were pelleted. Cell pellets containing more than 98% macrophages were washed and resuspended in Ringer's solution. The above procedure was repeated two more times. About 10X 10 can be extracted from each rat⁸And macrophages are formed. Viability was assessed by trypan blue exclusion.

H. Oxidative stress of alveolar macrophages induced by t-butyl peroxide (t-BHP)

The generation of oxygen free radicals in alveolar macrophages induced by t-BHP (2.5mM) was determined by luminol chemiluminescence. Chemiluminescent responses were recorded on a Bedrthold Autolumat LB953 luminometer as described above (Deliconstaninos, G., krue-ger, G.R.F., J.Viral Dis.1, 22-27, 1993).

I. Hydrogen peroxide (H)₂O₂) Measurement of (2)

An isoluminol/microperoxidase mix (100mM sodium borate, 1mM isoluminol, 0.01mM microperoxidase pH8 in 70% water and 30% methanol) was prepared. 50 μ l of the above reagent was mixed with extracted alveolar macrophages (10)⁶Individual cells) were mixed in HBSS in a total volume of 0.5 ml. By using a mixture of pure H₂O₂Concentration-configured calibration curves for conversion of chemiluminescence to H₂O₂The number of nmol (a).

J. Preparation and purification of soluble guanylate cyclase (sGC) for CO determination

sGC extracted from human endothelial cells was purified by GTP-agarose chromatography. The cytosol (10mg protein) was loaded onto a GTP agarose column (1.8X 9cm) containing 250mM sucrose and 10mM MnCl₂Pre-equilibrated with 25mM Tris&HCl buffer (pH 7.6). The sGC was then eluted from the column with 5ml of a running buffer to which 10mM GTP was added.

K. Determination of cyclic GMP

The concentration of cGMP was determined by radioimmunoassay after acetylation of the samples with acetic anhydride (Delikanstaninos, G., and Kopeikina, L., Anticancer Res.9: 753-760, 1989)). The reaction mixture contained triethanolamine/HCl (50 mM); creatine phosphate (5 mM); MgCl₂(3 mM); isobutylmethylxanthine (1 mM); creatine kinase (0.6 units); GTP (1 mM); soluble guanylate cyclase (1. mu.g protein) in a total volume of 150. mu.l. The reaction was initiated by addition of GTP and incubated at 37 ℃ for 10 min. The above incubation was aspirated and cGMP was extracted by addition of iced HCl (0.1M). After 10 minutes, the sample was transferred to a new dish, dried, and redissolved in 5mM sodium acetate (pH 4.75) for cGMP assay. The resulting cGMP was measured with cGMP kit (Amersham).

The object of the present invention is to propose and use a method using a biological substance capable of reacting with and eliminating the following substances inhaled during smoking:

a) the presence of NO and NOx,

b)CO，

c)H₂O₂，

d) the free radical(s) is (are),

e) (ii) an aldehyde-quinone compound, wherein,

f) a nitroso-compound that is carcinogenic,

g) and trace elements such as cadmium, copper, manganese, iron and the like are retained.

The present invention relies to a large extent on the concept of:

a) with optional suitable scavengers, e.g. haemoglobin or erythrocyte lysates or substances containing stereospecifically bound iron

b) Alternative scavengers contain porphyrin rings with iron (e.g. protoporphyrin)

c) Alternative scavengers containing porphyrin rings not necessarily with iron

d) Optionallycontaining other metals, e.g. Mg²⁺、Cu²⁺Scavengers of complex porphyrin rings

e) A biotechnological process will be devised for reinforcing the common conventional materials currently used in cigarette filter production to contain the above-mentioned biomass-scavenger.

The key to the present invention lies in the theory that: impregnating a conventional cigarette filter and/or a filter containing activated carbon,it can be reinforced by a biological substance, characterized by having Fe coordinated to the porphyrin ring²⁺、Cu²⁺、Mg²⁺And Fe stereospecifically binding to protein molecules²⁺In this way, the smoker is allowed to retain the harmful compounds contained in the cigarette before inhaling the smoke. This fact is a main feature of the present invention and constitutes a non-discriminatory innovation with great industrial application prospects. Method for industrial application

The present invention is prepared in the following way in view of its application to industrial production:

a1 mg/ml hemoglobin and/or red blood cell lysate dissolved in Phosphate Buffered Saline (PBS) (pH7.4) was prepared and 100mg activated charcoal was added to the solution. Incubate it at room temperature for 30 min and use S&S Carl Schleicher&Filter paper manufactured by Schuell Co u.s.a. The amount of hemoglobin in the filtrate that was not absorbed was measured spectrophotometrically. The charcoal fortified with hemoglobin was dried at room temperature. 200mg of hemoglobin-enhanced dry charcoal was sandwiched between two common filters so that all smoke drawn through contacted the active group (Fe) of the molecule²⁺，Fe³⁺、-SH，-NH₂) (FIG. 2). Such affinity materials will be used in the production of new cigarette filters, which will be referred to as biological filters from now on.

Alternatively, hemoglobin can be prepared by using a composition containing Fe as a metal ion to be coordinated with a porphyrin ring²⁺，Cu²⁺，Mg²⁺And Fe stereospecifically binding to protein molecules²⁺Such as transferrin, catalase, protoporphyrin, cytochrome C, chlorophyll.

In addition, a 5mg/ml solution of hemoglobin and/or red blood cell lysate in Phosphate Buffered Saline (PBS) (pH7.4) was prepared and scanned at 25 ℃ using an Acta Beck-man recording spectrophotometer. Absorption peaks were repeatedly observed at 540nm and 575nm (Smith, R.P., Kruszyma, H.J.Pharmacol. Exper. Ther.191, 557-. Ordinary conventional cigarette filters are soaked with the solution and air-dried at 25-35 ℃. Such affinity materials may be used to make new types of cigarette filters, which we will now refer to as biological filters. The new biological filter can ensure the full contact between the inhaled smoke and the active groups of hemoglobin molecules and/or dissolved products in the filter, and does not change the physical characteristics or taste of the smoke. For aesthetic reasons, a small portion (3mm) of a conventional filter may be used at the visible end of the biological filter. Other industrial processes include the following:

protoporphyrin was dissolved in buffer (PBS, pH7.4) to make a 5mg/ml solution and scanned at 25 ℃ with an Acta Backman recording spectrophotometer. Excitation of protoporphyrin with UV light (498-. The conventional filter is then impregnated (soaked) with the above solution and dried with hot air (25-35 ℃).

In addition, a 5mg/ml transferrin solution in PBS (pH7.4) was scanned using an Acta Beckman recording spectrophotometer. The transferrin exhibits a characteristic spectrum at 470 nm. The above-mentioned method of impregnating conventional filters currently used is employed.

In addition, a 5mg/ml catalase solution in PBS (pH7.4) was prepared.

The above-described method for producing a biological filter is then employed.

In addition, a 5mg/ml cytochrome C solution in PBS (pH7.4) was prepared. The method of making the biological filter described above is then employed.

In addition, a 5mg/ml chlorophyll solution in PBS (pH7.4) was prepared. The method for preparing the biological filter tip is adopted.

Alternatively, the biological substance is sandwiched in solid form between two conventional filters, so that all smoke drawn through the filters contacts the reactive group (Fe) of the molecule²⁺、Fe³⁺、-SH、-NH₂). Analysis of results

As shown in the following table, various biological substances used to reinforce conventional filters have been shown to retain toxic compounds (NO, CO, free radicals, H) in the smoke to varying degrees₂O₂Aldehydes, trace elementsPlain and nitroso compounds).Scavenger NO CO free radical H₂O₂Aldehyde nitroso compound trace elements

%%%%% of hemoglobin 90909080909095 transferrin 85906060607550 white peroxidized 85909090808080 hydroperoxide protoporphyrin 85907080707580 cell color 85807080606070 element C chlorophyll 15104015101080

The degree of retention of highly harmful substances in the smoke was obtained and the cigarette smoke (20ml) filtered from the biofilter was compared with the smoke (20ml) filtered from the conventional filter. Only 1ml of smoke filtered from a conventional filter was compared with 40ml of smoke filtered from a biofilter. The results show that the biological filter has a capacity of retaining trace elements 40 times that of the conventional filter.

Representative results are given in the following detailed experimental description to better understand the viability of this biological material.

a) Identifying NO contained in the smoke by using a chemiluminescence method:

NO was identified using the luminol-enhanced chemiluminescence method described in the experimental section. Figures 3 and 4 show a typical experiment of NO identification and determination, and NO removal after smoke has passed through a biofilter. The results show that more than 90% of NO is retained by hemoglobin. The action of the biological filter is remarkable in terms of retaining and neutralizing NO, which is associated with toxic reactions in the lung cells and lung fluids, especially when it is involved in the strong oxidant ONOO^-When (2) is formed.

b) Identifying the free radicals contained in the smoke by a chemiluminescence method:

free radicals in the smoke are identified by a chemiluminescent response. The luminescent response is caused after the reaction of the lucigenin/DAMCO system with free radicals. Figure 5 shows that the characteristic spectrum, which occurs within 2 seconds of the chemiluminescent response, is 100% suppressed after smoke has passed through the biofilter. The retention of free radicals by the biological filter means that oxidative stress in alveolar macrophages caused by conventional cigarette smoke is reduced.

c) Identification of H contained in flue gas by chemiluminescence₂O₂：

Determination of H by chemiluminescence response generated by isoluminol/microperoxidase System₂O₂₀FIG. 6 shows the result of H₂O₂The presence in the smoke results in a characteristic peak of chemiluminescence. In the presence of catalase (100 units/ml), the chemiluminescent responsewas inhibited by approximately 90%. After smoke passage through the biofilter, it was seen that the chemiluminescent response was suppressed by 80%. The isoluminol/microperoxidase system is specific for H₂O₂And (7) identifying. Free radicals contained in the smoke can cause weak chemiluminescence response after the action of isoluminol. Since catalase inhibits nearly 90% of the maximal luminescent response, this weak chemiluminescence appears to account only for H when free radicals are present₂O₂About 10% of the chemiluminescence induced. H₂O₂Is/are as followsRetention significantly reduces oxidative stress and NO produced by alveolar macrophages.

d) The trace elements and aldehydes contained in the smoke were identified using a luciferin/luciferase enzymatic system.

The trace elements contained in the smoke are identified by their ability to activate luciferase activity. FIG. 7 shows:

1) the chemiluminescent response caused by the oxidation of fluorescein in the presence of ATP,

2) presence of Cd²⁺Ion (0.5mg) was enhanced in the chemiluminescent response,

3) presence of Cu²⁺Enhanced chemiluminescent response of ions (0.5mg),

4) enhanced chemiluminescent response by smoke (1ml) and

5) chemiluminescence suppression (compared to chemiluminescence caused by smoke) resulted after 40ml of smoke had passed through the biological cigarette filter. It is clear that the chemiluminescent response caused by the trace elements contained in conventional smoke is 40 times higher than that caused by smoke passing through a biological filter. The biological filter has both short-term and long-term effects on the retention of trace elements. The short-term effect is manifested as inhibition of the redox reactions occurring in the lungs (Fe, Mn), and the long-term effect is manifested as inhibition of damage to components and substances in the blood (Cd).

The identification and determination of aldehydes contained in smoke was performed in the absence of ATP using the same luciferin/luciferase enzymatic system. Aldehydes can cause oxidation of fluorescein. FIG. 8 shows a characteristic chemiluminescent response that may last for more than 1 hour. The chemiluminescent response was inhibited by 100% after the smoke used passed through the biofilter, indicating that the effect of the biofilter on retaining toxic aldehydes was significant.

e) Identification of nitrosative chemicals in flue gas

The identification of nitroso compounds contained in smoke is carried out by using H₂O₂Obtained by measuring the slow release of NO from nitroso compounds after treatment. As shown in fig. 9, about 900 seconds worthTo a peak chemiluminescence response. After smoke passes through the biofilter, it is seen that its chemiluminescent response is suppressed by 90%, with a peak occurring at about 1200 seconds. Also shows that H is in use₂O₂Slow release of NO after treatment of Sodium Nitroprusside (SNP). FIG. 10 shows the NO slow release of the nitroso compounds diethylnitrosamine and dimethylnitrosamine; and with H₂O₂The NO of the treated hemoglobin enriched in nitroso compounds in the smoke is slowly released. It is clear that the NO release from the smoke nitroso compound, which forms an adduct with hemoglobin, follows the same pattern as the NO release from the nitroso compounds diethylnitrosamine and dimethylnitrosamine. FIG. 11 shows a graph obtained after irradiation with UVB (100 mJ/cm)²) The hemoglobin-nitroso compound adduct was added for 1 minute toThereafter, NO is released from the smoke nitroso compound which has formed an adduct with hemoglobin. In the presence of H₂O₂NO release was measured under conditions and gave a chemiluminescent response at 1 second. The gradual rise seen in FIG. 11 is due to H₂O₂Effect on hemoglobin (fenton reaction).

f) Production of NO by lung macrophages:

in vitro experiments were performed with the help of a special chamber of our laboratory invention as shown in figure 1. The teflon membrane separating the two parts of the chamber can pass NO, but not NO₂ ^-And ONOO^-. Unstimulated lung macrophages extracted as described in the experimental section were suspended in HBSS buffer (1X 10)⁶Cells/ml) and placed in part a of the chamber described above. In part B of the chamber 2.5ml of Grace reagent or sulfanilamide/scopoletin reagent was placed. NO released by macrophages in part a diffuses through the teflon membrane to part B and binds to the grits and/or sulfamoyltin reagent remaining in part B. This indicates that lung macrophages are capable of producing NO gas. The amount of NO present in fraction B is then determined spectrophotometrically or fluorometrically. ONOO contained in part A^-And NO₂ ^-The amounts were also determined using grice and/or sulfanilamide/scopoletin reagents. The above experiment was repeated with smoke stimulated macrophages before being placed in part a. The results shown in FIG. 12 indicate that smoke decreases NO production in lung macrophages and increases ONOO^-Indirectly evidence of the production of a large amount of NO and O₂ ^-Interaction ofGenerate ONOO^-。

Repetition of the above experiment with a biofilter (i.e.smoking of smoke through a biofilter) demonstrated that the same amount of NO was produced in fraction A as in macrophages not stimulated with smoke using the biomass used₂ ^-And ONOO^-And the same amount of NO is produced in part B. In the present application, the components of the grignard reaction are also used for the inhibition of NO/O in aqueous solutions at physiological pH₂The nitrosation kinetics of the intermediate formed in the reaction were examined. Smoke (50ml) was added to a solution containing 25mM sulfanilamideAnd 2.5mM N- (1-naphthylethylenediamine dihydrochloride (NEDD) in 100mM phosphate buffer (pH7.4) produced an absorption peak at λ max of 496mM, confirming the characteristic azo compound produced by nitration it is worth considering the implications of the above observations by comparing the expected reactivity of NO under relevant physiological conditions, it is estimated that the maximum concentration of NO in the cellular microenvironment is 0.5-10 μ M and that the NO concentration increases dramatically upon smoking, causing damage to lung cells.

g) Oxidative stress of lung macrophages:

the results of the effect of smoke on oxidative stress of lung macrophages are given in figure 13. The oxidative stress measured with t-BHP showed that smoke caused an oxidative stress 2 times higher than that of unstimulated macrophages. After smoke passes through the biofilter, the observed oxidative stress is similar to that of unstimulated lung macrophages. This clearly demonstrates its effect on the elimination of oxidative stress on macrophages caused by smoke. The smoke now contains no more substances that cause oxidative stress in lung macrophages.

h) H produced by pulmonary macrophages₂O₂：

H production by smoke stimulated macrophages₂O₂The yield is more than 10 times that of unstimulated macrophages. Compared with conventional filter, the biological filter can be used for filtering H₂O₂The production was reduced by 90% (fig. 14).It is clear that, as well as the oxidative stress induced by macrophages by smoke, it can increase the H-pairing of these cells₂O₂Is generated.

i) Reconstruction experiments:

the amount of cyclic GMP produced by NO released from alveolar macrophages was determined using the chamber shown in FIG. 1, where soluble guanylate cyclase was placed in part A and pulmonary macrophages were placed in part B. The amount of NO produced by macrophages was measured over a period of 50 minutes with and without cells stimulated with smoke. Macrophages treated with smoke (10ml) released approximately 10-fold less NO than untreated cells and therefore exhibited 10-fold less cyclic GMP production. By filters of biological originWherein the process is repeated for the passing flue gas. The results showed no statistically significant difference compared to unstimulated macrophages (control) (fig. 15). As shown in FIG. 16, when alveolar macrophages were depleted of H₂O₂The accumulated NO in part B increased more than 5-fold with treatment (5 mM). This indicates H₂O₂NO production is promoted by a positive feedback mechanism. The L-arginine/NO pathway in macrophages is responsible for NO/ONOO with smoke^-The theory of release is consistent.

k) Identification of carbon monoxide (CO) in flue gas:

the presence of CO in smoke is determined using a biological method based on the stimulation of soluble guanylate cyclase by CO.

Introducing HBSS saturated with smoke into section a of the chamber, in the presence of superoxide, to neutralize NO; and introducing soluble guanylate cyclase into part B, resulting in increased cyclic GMP production due to CO diffusion from part a to part B. Smoke, after passing through the biofilter, reduced cyclic GMP production by nearly 80% (figure 17). The above results show that the harmful substances NOx and CO in the smoke are retained and neutralized by the biofilter. In vivo experiments

a) We first confirmed NO and ONOO^-Presence in exhaled smoke. A cigarette with a conventional filter was smoked by the volunteer and the presence of NO in the exhaled smoke was identified after introducing the exhaled smoke into an acidic solution (50ml) (pH 4). Using what is described in the experimental partLuminol-enhanced chemiluminescence method measures NO concentration using a standard curve prepared from commercial NO. The resulting NO concentration was 0.045 mM. The experiment was repeated with a biofilter and the NO concentration in the inhaled smoke was reduced by nearly 70% compared to the conventional filter (fig. 18). Measurement of ONOO with 1.2M NaOH solution^-Concentration, showing an increase in absorption (. epsilon.) at 303nm (FIG. 19)_303nm＝1670 M^-1cm^-1). Our experiments show that the smoke discharged during smoking contains a large amount of ONOO^-(50ml of the expectorated Smoke was bubbled through 5ml of 1.2M NaOH to produce 0.9mM ONOO^-The solution of (a). Measuring NO/ONOO in the spitting smoke^-The ratio is 1: 20.

Thus, it appears that NOx in the lung is converted to ONOO after reaction with superoxide^-. Superoxide is released by macrophages and the redox reactions that occur in the lungs during smoking. The pumped smoke does not contain ONOO^-But with a certain amount of NOx reacted with superoxide or oxygen to form nitrite ions (NO)₂ ^-). Smoke only enters the lungs to form ONOO^-. Use of biological filters to deliver NO and ONOO^-The amount was reduced by 70%.

b)ONOO^-Reacts with bicarbonate in human erythrocytes according to the following reaction equation

Bicarbonate can oxidize luminol as well as aromatic and heterocyclic molecules. In addition, ONOO^-Bicarbonate can be peroxidized to form percarbonate, another strong oxidizing agent. On the other hand, superoxide dismutase (SOD) catalyzes ONOO^-And nitration of tyrosine in a variety of phenols, including proteins.

Thus, there are several potential mechanisms by which bicarbonate and SOD can affect ONOO^-Total reactivity in cells. Production of ONOO in the lungs due to inhalation of smoke^-This is manifested as a sharp increase in the oxidative stress of the erythrocytes, which is detected by a chemiluminescent response occurring within 5 seconds. The same experiment using the biological filter resulted in almost 100% inhibition of the oxidative stress of human erythrocytes (fig. 20). Contacting hemoglobin or red blood cell lysate with ONOO^-(contained in the spitting smoke) and then the normal condition of the cigarette is lostTwo absorption peaks of hemoglobin at 54Onm and 575nm are visible. A representative experiment similar to the one described above was performed in 12 volunteers, and the results are shown in fig. 21. When hemoglobin and/or lysate were exposed to a small amount of exhaled smoke (10ml), a shift in the absorption peaks from 540 and 575 to 525 and 555nm was seen, consistent with the production of nitrosylhemoglobin. The above experiment was repeated with a biological filter. The observed absorption peak maintains its characteristic wavelength.

d) Aldehydes in smoke gas spitted out by volunteers were identified by their characteristic chemiluminescence peaks. Repeating the above experiment with a biological filter, it was seen that the chemiluminescent response was reduced by 90% from the maximum chemiluminescent response observed with the conventional filter (figure 22). It is clear that biological filters are able to retain and neutralize aldehydes in smoke while retaining oxidizing agents, thereby significantly inhibiting redox reactions occurring in the lungs that result in the production of endogenous aldehydes.

e) Free radicals in smoke excreted by volunteers are identified by their characteristic chemiluminescence peaks. The cigarette used by the volunteer was provided with a conventional filter and a biological filter. Volunteers were instructed to spit smoke into acidic solution (0.01N HCl) (50ml, pH6) and to measure chemiluminescent responses after 5 and 60 minutes. When pH is 6, the discharged ONOO^-And (4) automatically decomposing. The chemiluminescent response of the smoke expectorated through the conventional filter increased 160% within 5 minutes compared to the smoke passing through the biological filter (figure 23). The difference in chemiluminescence response increased from 160% to 250% when placed in acid solution saturated with expectorated smoke for 1 hour (fig. 24). This is consistent with the theory that the oxidation-reduction reaction in smoke occurs continuously through quinones, and such reaction produces a series of active aerobic species that can cause biological damage. Discussion of the related Art

Our studies demonstrated that alveolar macrophages, like other cells, have endogenous NOSynthetase capable of releasing NO/ONOO for a long time after contacting with smoke^-. In addition, once these cells begin to release NO, NO production becomes self-supporting even after the excitation source is removed. Such a reaction would indicate that NO in the smoke stimulates alveolar macrophages to release NO and ONOO^-And can last for a period of several hours after the excitation source is removed. H production in the lung when alveolar macrophages are stimulated with smoke₂O₂Sucha reaction can also be initiated. H₂O₂Can stimulate the activity of NO-synthetase in lung cells, thereby removing NO and ONOO after excitation source^-The production of (2) lasts for more than 1 hour. I amThe experiments indeed demonstrated that smoke passage from the biofilter reduced the oxidative stress in rat macrophages by 90% (compared to conventional filters). ONOO produced in the lungs^-Statistical results show that smoking predisposes a person to emphysema (Southon, p.a., Pwis, g., Free radials in medicine, invasion in human disease, mayo clin.Proc.63: 390 cake 408, 1988.) in an in vivo test conducted by 12 volunteers, NO/ONOO is expectorated when inhaled smoke passes through a biofilter^-The reduction is 90%.

Oxygen radicals are also involved in the development of pouchitis caused by IgA antigen-antibody complexes. Pretreatment of animals with superoxide dismutase, catalase, iron chelator-desferrioxamine, or hydroxyl radical scavenger (DMSO) inhibits the development of lung damage. In contrast, the lungs of untreated positive control animals were characterized by the presence of more alveolar macrophages. Interstitial edema and hemorrhage may also occur. In addition, in this type of lung injury, L-arginine has a strong protective effect, which has been shown to reduce vascular permeability; bleeding of blood vessels; and damage to vascular endothelial cells and alveolar epithelial cells. The above findings indicate that macrophages are made of NO, O₂ ^-，H₂O₂And the source of damage caused by OH compounds (mullignan, m.s., Johnson, k.j., Ward, p.a., see "Biological Oxidants:generation and Injurious sequences "(eds. Cochrane, C.G., and Gilbron, M.A., Jr. academic Press 157-.

The biological filter has obvious effects on the retention and neutralization of oxidants in smoke and the activity of oxidoreductase which is directlyrelated to the oxidative stress of lung cells. Biological filters can greatly reduce the oxidative stress caused by inhaled smoke. The NO, NOx, oxygen-containing free radicals and/or aldehydes contained in the smoke can induce oxidative stress in lung macrophages and lung vascular epithelial cells. In addition, the biological filter has no aldehyde and trace amountThe retention of elements, especially Cd, has a considerable time-consuming role in preserving cytoplasmic antioxidants and inhibiting the development of arthrosclerosis haemoglobin has several neutrophilic centres which react covalently with electrophiles, said centres attracting the N-terminal valine residues of the α -and β -chains, the N of the histidine residues₁And N₃Atoms, and the sulfhydryl group of a cysteine residue. During the combustion of the cigarette, the carcinogenic nitroso compound 4- (methylnitrosamino) -1- (3-pyridyl) -1-butanone (NNK) in tobacco is transferred to the smoke, the content of which in the mainstream smoke of each cigarette fluctuates in the range of 4-1700 ng. NNK can form adducts with hemoglobin (Hecht, s.s., Karan, s., and Carmella, s.g., see "Humam cardiogen expose" (eds. garmer, r.c., Farmer, p.b., steel, g.i., and Wricht, A.S.) IRL Press pp267-274, 1991). Clearly, the only way to avoid tobacco-related diseases is to abstain from chewing and smoking tobacco. However, statistics on existing smokers suggest that a significant step can be taken to reduce exposure to tobacco carcinogens and improve their mode of action. The main approaches to achieve this goal are: 1) improvement of tobacco products, 2) inhibition of metabolic activation and endogenous production of tobacco carcinogens by certain small and large nutrients and chemical inhibitors, 3) use of special filters applicable to tobacco, which retain tobacco carcinogens. Our invention, using biomass to produce biofilters, ultimately relates to the discovery that: nitrous oxides in the inhaled smokeThe base compoundis retained by the biological substance, which not only protects the health of smokers, but also protects the health of non-smokers.

Claims

Modification according to article 19 of the treaty

1. A filter for filtering cigarette smoke, characterized in that it comprises a fibrous matrix reinforced with biological material. The biological substance is selected from one or more substances containing iron, copper and/or magnesium complexed to a porphyrin ring and containing iron stereospecifically bound to a protein molecule.

2. A filter according to claim 1, characterised in that it comprises activated carbon reinforced with the biomass.

3. A filter according to claim 1 or 2, characterised in that the reinforced fibrous matrix is preceded and followed by a fibrous matrix which is not reinforced with the biological substance.

4. A filter as claimed in any one of claims 1 to 3 wherein the biological substance comprises haemoglobin and/or a lysate of red blood cells.

5. A filter as claimed in any one of claims 1 to 3, characterised in that the biological substance is selected from one or more of transferrin, catalase, protoporphyrin, cytochrome C and chlorophyll, which bind stereospecifically Fe2 +.

6. A filter according to any of claims 1 to 5, wherein the biological material is in solid form.

7. A cigarette, characterised in that it is provided with a filter according to any one of claims 1 to 6.

8. A method of making a cigarette filter according to any of claims 1 to 6 comprising impregnating a conventional cigarette filter with one or more of said biological substances.

9. The method of claim 8, wherein the filter comprises activated carbon.

10. A method according to claim 8 or 9, wherein the biological substance comprises haemoglobin and/or a lysate of red blood cells.

11. A method according to any one of claims 8 to 10, wherein the biological material is provided as a solution of 1 to 10mg/ml in a phosphate buffered saline solution at pH 7.4.

12. A method of filtering tobacco smoke comprising providing a filter according to any one of claims 1 to 6 and passing tobacco smoke therethrough.

13. A method according to claim 12, wherein the filter is capable of retaining 15-90% of the NO present in the tobacco smoke prior to passage through the filter; 10-90% CO; 40-90% free radicals; 10-90% aldehydes; 10 to 90 percentA carcinogenic nitroso compound; 15-90% H₂O₂And 50-95% of trace elements.

14. A method according to claim 13, wherein the filter is capable of retaining 85-90% of the NO present in the tobacco smoke prior to passage through the filter; 80-90% CO; 60-90% free radicals; 60-90% H₂O₂(ii) a 60-90% aldehydes; 60-90% carcinogenic nitroso compound; and 70-95% of trace elements.

Claims (22)

1. A method for retaining and neutralizing harmful compounds (NO, NOx, free radicals, aldehydes and H) in cigarette smoke is developed₂O₂CO, trace elements and carcinogenic nitroso compounds) conventional cigarette filters do not adequately retain these harmful compounds. The method is characterized in that a conventional cigarette filter made of a fibrous matrix or activated carbon is reinforced with a biological substance containing iron, copper and/or magnesium complexed with a porphyrin ring, and stereospecifically bound iron in protein molecules, alone or in combination. The reinforcement of conventionalfilters or activated carbon with the above substances does not alter either the physical characteristics of the smoke (smell, taste and appearance) or the physical characteristics of the filter itself.

2. The method of claim 1, wherein the hemoglobin and/or lysate solution is prepared in Phosphate Buffered Saline (PBS), 5-10mg/ml (this amount depends on tobacco and cigarette quality), ph 7.4. A conventional cigarette filter is dipped in the above biological solution. The soaked biofilter was then air dried at 25-35 ℃. The method can retain at least 90% of NO, 90% of CO, 90% of free radicals, 90% of aldehydes, 90% of carcinogenic nitroso compounds, and 80% of H₂O₂And 95% trace elements.

3. The method according to claims 1 and 2, characterized in that hemoglobin and/or lysate is used in solid form. 5-10mg (this amount depends on the tobacco mass and the cigarette mass) of hemoglobin and/or lysate are sandwiched between two parts of a common conventional cigarette filter. This method ensures the effect described in claim 2 when smoking.

4. Method according to claims 1 and 2, characterized in that a 1mg/ml (only indicative amount) solution of hemoglobin and/or erythrocyte lysate is prepared with Phosphate Buffered Saline (PBS), ph 7.4. 100mg (indicative only) of activated carbon was added to the solutionAnd the mixture was incubated at room temperature for about 30 minutes. 200mg (indicative only) of dry activated carbon fortified with haemoglobin were sandwiched between two parts of a common conventional filter so that all the smoke drawn through the filter could contact the active group (Fe) of the molecule²⁺，Fe³⁺，-SH，-NH₂). This method also ensures the effect described in claim 2 when smoking.

5. The method according to claim 1, characterized in that a transferrin solution is prepared in 5-10mg/ml (the amount depends on tobacco mass and cigarette mass) in Phosphate Buffered Saline (PBS), ph 7.4. A common conventional filter is immersed in the above-mentioned biological solution. And then drying the soaked biological filter tip by hot air at 25-35 ℃. The method can retain at least 85% of NO, 90% of CO, 60% of free radicals, 60% of aldehydes, 75% of carcinogenic nitroso compounds, and 60% of H₂O₂And 50% trace elements.

6. The method according to claims 1 and 5, characterized in that transferrin is used in solid form. Between two parts of a conventional cigarette filter, 5-10mg (the amount depends on the tobacco mass and the cigarette mass) of transferrin is sandwiched. The method can ensure the effect of claim 5 when smoking.

7. Method according to claims 1 and 5, characterized in that a 1mg/ml (indicative only) transferrin solution is prepared with phosphate buffered saline solution (PBS), pH 7.4. Will be provided with100mg (indicative only) of activated charcoal was added to the solution and incubated at room temperature for about 30 minutes. 200mg (indicative only) of transferrin-reinforced dry carbon are sandwiched between two ordinary conventional filter parts so that all smoke drawn through the filter is in contact with the active group (Fe) of the molecule²⁺，Fe³⁺，-SH，-NH₂). The method can ensure the effect of claim 5 when smoking.

8. The method according to claim 1, wherein 5-10mg/ml (amount depending on tobacco quality and cigarette quality) of catalase is prepared using Phosphate Buffered Saline (PBS)Solution, pH 7.4. A common conventional filter is immersed in the above-mentioned biological solution. The soaked biofilter is then air dried at 25-35 ℃. The method can retain at least 85% of NO, 90% of CO, 90% of free radicals, 80% of aldehydes, 80% of carcinogenic nitroso compounds, and 90% of H₂O₂And 80% trace elements.

9. The method as claimed in claims 1 and 8, characterized in that catalase is used in solid form. 5-10mg (amount depending on tobacco mass and cigarette mass) of catalase was sandwiched between two sections of a common conventional filter. The method ensures the effect described in claim 8 when smoking.

10. The method according to claims 1 and 8, characterized in that a 1mg/ml (indicative only) catalase solution is prepared with phosphate buffered saline solution (PBS), ph 7.4. 100mg (indicative only) of activated carbon was added to the solution and incubated at room temperature for about 30 minutes. 200mg (indicative only) of dry charcoal fortified with catalase were sandwiched between two sections of a common conventional filter so that all smoke drawn through the filter could contact the active groups (Fe) of the molecule²⁺，Fe³⁺，-SH，-NH₂). The method can ensure the effect of claim 8 when smoking.

11. The method according to claim 1, wherein a solution of 5-10mg/ml (depending on tobacco and cigarette quality) of protoporphyrin is prepared in Phosphate Buffered Saline (PBS), ph 7.4. A common conventional filter is immersed in the above-mentioned biological solution. The soaked biofilter was then air dried at 25-35 ℃. The method can retain at least 85% of NO, 90% of CO, 70% of free radicals, 70% of aldehydes, 75% of carcinogenic nitroso compounds, and 80% of H₂O₂And 80% trace elements.

12. The method according to claims 1 and 11, characterized in that protoporphyrin is used in solid form. 5-10mg (amount depending on tobacco mass and cigarette mass) of protoporphyrin was sandwiched between two sections of a common conventional filter. The method ensures the effect described in claim 11 when smoking.

13. Method according to claims 1 and 11, characterized in that a 1mg/ml (indicative only) solution of protoporphyrin is prepared with Phosphate Buffered Saline (PBS), ph 7.4. 100mg (indicative only) of activated carbon was added to the solution and the mixture was incubated at room temperature for about 30 minutes. 200mg (indicative only) of protoporphyrin-reinforced dry carbon was sandwiched between two parts of a common conventional filter so that all smoke drawn through the filter could contact the active group (Fe) of the molecule²⁺，Fe³⁺，-SH，-NH₂). The method ensures the effect described in claim 11 when smoking.

14. The method according to claim 1, wherein 5-10mg/ml (amount depending on tobacco quality and cigarette quality) of cytochrome solution is prepared using Phosphate Buffered Saline (PBS), ph 7.4. A common conventional filter is immersed in the above-mentioned biological solution. The soaked biofilter was then air dried at 25-35 ℃. The method can retain at least 85% of NO, 80% of CO, 70% of free radicals, 60% of aldehydes, 60% of carcinogenic nitroso compounds, and 80% of H₂O₂And 70% trace elements.

15. The method according to claims 1 and 14, characterized in that cytochrome C is used in solid form. The method of sandwiching between two conventional filter portions between 5-10mg (amount depending on tobacco quality and cigarette quality) of cytochrome C ensures the effect of claim 14 when smoking.

16. Method according to claims 1 and 14, characterized in that a 1mg/ml (indicative only) cytochrome C solution is prepared with Phosphate Buffered Saline (PBS), pH 7.4. 100mg (indicative only) of activated carbon was added to the solution, and the mixture was incubated at room temperature for about 30 minutes. 200mg (indicative only) of dry charcoal fortified with cytochrome C were sandwiched between two parts of a common conventional filter so that all smoke drawn through the filter could contact the active group (Fe) of the molecule²⁺，Fe³⁺，-SH，-NH₂)。The method ensures the effect described in claim 14 when smoking.

17. The method according to claim 1, wherein 5-10mg/ml (amount depending on tobacco quality and cigarette quality) of chlorophyll solution is prepared with Phosphate Buffered Saline (PBS), ph 7.4. A common conventional filter is immersed in the above-mentioned biological solution. And then drying the soaked biological filter tip with hot air at 25-35 ℃. The method can retain at least 15% of NO, 10% of CO, 40% of free radicals, 10% of aldehydes, 10% of carcinogenic nitroso compounds, and 15% of H₂O₂And 80% trace elements.

18. A method according to claims 1 and 17, characterized in that chlorophyll is used in solid form. 5-10mg (the amount depends on the tobacco quality and cigarette quality) of chlorophyll is sandwiched between two parts of a common conventional filter. The method ensures the effect described in claim 17 when smoking.

19. Method according to claims 1 and 17, characterized in that a 1mg/ml (indicative only) chlorophyll solution is prepared with a phosphate buffered saline solution, ph 7.4. 100mg (indicative only) of activated carbon was added to the solution and the mixture was incubated at room temperature for about 30 minutes. 200mg (indicative only) of chlorophyll-fortified dry carbon was sandwiched between two parts of a common conventional filter so that all smoke drawn through the filter could contact the active group (Fe) of the molecule²⁺，Fe³⁺，-SH，-NH₂). The method ensures the effect described in claim 17 when smoking.

20. The method according to claim 1, characterized in that a solution and/or a solid biological substance is prepared, which have the common feature that:

a) any molecule containing heme iron (heme, hemin, etc.).

b) Any macromolecule containing stereospecifically bound iron or copper (ferritin, ceruloplasmin, etc.).

c) Any macromolecule containing a porphyrin ring, which does not necessarily contain iron.

d) Any macromolecule containing a porphyrin ring coordinated to a metal other than iron (Mg, Cu).

21. The invention is directed to biochemical-pathophysiological mechanisms and technical countermeasures associated with lung and blood (constituent and substance) damage. These mechanisms are particularly related to pulmonary alveolar macrophages and endothelial cells which, when stimulated by cigarette smoke, produce oxidative stress leading to prolonged production of large amounts of NO/ONOO^-And H₂O₂₀H₂O₂Further stimulating NO/ONOO^-The generation of (2) to form a vicious circle. ONOO^-Group inhibiting important protective mechanisms α 1-protease inhibitor (α 1PI) in lung tissue in addition, the production method of claim 1 is capable of retaining and neutralizing harmful compounds contained in smoke, i.e. NO, CO, aldehydes, free radicals, H₂O₂Trace elements, carcinogenic nitroso compounds, and thus protect active and passive smokers to a large extent from diseases such as emphysema, lung cancer, chronic bronchitis, and chronic diseases of the cardiovascular system.

22. A new cigarette filter, characterized in that either the filter is reinforced with the biological substance according to claims 1 and 20 or activated carbon reinforced with the biological substance according to claims 1 and 20 is placed at both ends or at one end of the new filter. Both ends or one end of such a filter are made of a fibrous substrate used to make conventional filters.

Images