Use of polyphenol compounds or derivatives thereof in the preparation of additives having inhibitory effect on carcinogenicity of electronic cigarettes

Abstract

This invention relates to the application of polyphenolic compounds or their derivatives in the preparation of additives with inhibitory effects on the carcinogenicity of e-cigarettes, and provides an additive with inhibitory effects on the carcinogenicity of e-cigarettes; the polyphenolic compounds or their derivatives can be obtained by chemical synthesis, biotechnology or extraction from plants; the plants are preferably plants of the Lamiaceae family such as rosemary.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, specifically to the application of polyphenolic compounds or their derivatives in the preparation of additives with inhibitory effects on the carcinogenicity of e-cigarettes, and particularly in the prevention of malignant transformation of normal bronchial epithelial cells caused by e-cigarettes. Background Technology

[0002] With the gradual implementation of the Framework Convention on Tobacco Control and the increasing health awareness of the public, people's lifestyles and consumption concepts are changing, and the traditional cigarette market is facing a strong impact. The development of new tobacco products has become inevitable. In recent years, e-cigarettes, especially in developed countries in Europe and America, have shown a strong development trend.

[0003] E-cigarettes are electronic products that mimic traditional cigarettes, possessing the same appearance, smoke, taste, and feel. They are nicotine-containing aerosols, free from carcinogenic byproducts of incomplete combustion, such as nitrosamines, nicotine nitrosamines, and tar, and were once considered a safe smoking method. However, recent research indicates that both e-cigarette and traditional tobacco users exhibit reduced expression of immune genes in respiratory epithelial cells. Furthermore, e-cigarette users also show suppressed expression of immune genes. This suppression of immune gene expression in respiratory epithelial cells can increase the likelihood of bacterial and viral infections and inflammation. Another study found that vaporized e-cigarette liquids are cytotoxic, cause inflammation, and inhibit phagocytosis by alveolar macrophages. E-cigarettes have also been found to be carcinogenic to lung and bladder cancer in mice, and can damage the heart in mice. Current laboratory evidence is sufficient to prove that e-cigarettes can increase the incidence of lung and bladder cancer in users. Because nicotine, the main component of e-cigarettes, and its metabolite NNK induce the formation of DNA adducts, causing DNA damage and weakening DNA repair activity, leading to genomic instability and increased susceptibility to gene mutations, while also downregulating immune-related genes and inhibiting immune cell activity, they exert a tumorigenic effect. Currently, no research has reported compounds that inhibit the carcinogenicity of e-cigarettes. Therefore, although the harm of e-cigarettes is less than that of traditional cigarettes, there is still an urgent need to find compounds that inhibit the carcinogenicity of e-cigarettes to be used as additives to reduce nicotine toxicity in the e-cigarette industry. Summary of the Invention

[0004] In their research on polyphenolic compounds and their derivatives, the inventors discovered that these compounds exhibit excellent anti-nicotine-induced bronchial epithelial cell carcinogenesis effects and effectively inhibit the carcinogenic effects of e-cigarettes. Therefore, this invention aims to provide the e-cigarette industry with an application of polyphenolic compounds or their derivatives in the preparation of additives with anti-carcinogenic effects from e-cigarettes.

[0005] The polyphenolic compounds used in this invention are selected from caryophyllene, rosmarinic acid, or caryophyllic acid and their derivatives. The derivatives refer to compounds derived from polyphenolic compounds by substituting a hydrogen atom of at least one hydroxyl group in the polyphenolic compound with a C1-C6 alkyl or (C1-C4 alkyl) carbonyl group. Preferred are acetates such as caryophyllic acid acetate and rosmarinic acid acetate.

[0006] The polyphenolic compounds mentioned in this invention, such as caryophyllene, rosmarinic acid, and derivatives of caryophyllene, also refer to isomers of the polyphenolic compounds, such as epirosmarinic acid or isorrosmarinic acid.

[0007] These compounds can be obtained through common chemical synthesis or through biotechnological methods as known to those skilled in the art, or they can be isolated from plant extracts.

[0008] The polyphenolic compounds can also be used in the present invention in the form of plant extracts, preferably extracts from plants of the Lamiaceae family, especially rosemary extract.

[0009] These plant extracts can be obtained by extraction with polar solvents such as alcohols or aqueous alcohols. Ethanol is preferred as the solvent.

[0010] The polyphenolic compounds or their derivatives described in this invention can be obtained by chemical synthesis or biotechnology or from plants in accordance with methods known to those skilled in the art.

[0011] The polyphenolic compound or its derivatives may also be plant extracts, preferably extracts from plants of the Lamiaceae family, and most preferably rosemary extract.

[0012] The rosemary extract was obtained by extraction from rosemary, a plant of the Lamiaceae family, in an alcohol or aqueous alcohol solvent.

[0013] The polyphenolic compounds or their derivatives described in this invention can be used alone or in combination. Preferably, they are mixtures of caryophyllophenol, rosmarinic acid, or caryophyllic acid and their derivatives; more preferably, they are mixtures of rosmarinic acid and caryophyllic acid; and most preferably, they are rosmarinic acid.

[0014] Another object of the present invention is to provide an additive that has the effect of inhibiting the carcinogenicity of e-cigarettes, wherein the additive contains the above-mentioned polyphenolic compound or its derivative, preferably one or more of caryophyllophenol, rosmarinic acid or caryophylloic acid and its derivatives.

[0015] The present invention also provides an electronic cigarette filter, the filter comprising a shell and an inner core disposed within the shell, the inner core being filled with the aforementioned additive having an inhibitory effect on the carcinogenicity of electronic cigarettes. The polyphenol compound or its derivative thereof is added to the electronic cigarette filter in an amount of 20 mg to 50 mg, preferably, the polyphenol compound or its derivative thereof is added to the additive having an inhibitory effect on the carcinogenicity of electronic cigarettes in an amount of 30 mg. Attached Figure Description

[0016] Figure 1 Figure 1 shows the effect of rosmarinic acid and sage acid on nicotine-induced Beas-2B soft agar sphere formation in Experiment Example 1. Figure 2 Figure 5 shows the results of rosmarinic acid inhibiting the tumorigenic effect of nicotine-induced BEAS-2A in nude mice. Detailed Implementation

[0017] The inventors discovered in their research that nicotine produced by e-cigarettes can induce malignant transformation of normal human bronchial epithelial cells, while the additive described in this invention can inhibit the ability of nicotine to induce malignant transformation of normal human bronchopulmonary epithelial cells. This is because the additive has strong antioxidant activity, can enhance the regulatory capacity of immune cells, can maintain genomic stability, and helps prevent cell damage caused by free radicals, thus reducing the risk of cancer and arteriosclerosis. This invention has significant social implications and can also generate substantial economic benefits.

[0018] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0019] To explain in detail the technical content, objectives, and effects of the present invention, the following description is provided in conjunction with the embodiments and accompanying drawings.

[0020] Example 1: Effects of rosmarinic acid and sage acid on nicotine-induced sphere formation on Beas-2B soft agar

[0021] 1. Source of materials

[0022] Rosmarinic acid (RA, commercially available), sarsaparilla acid (CA, commercially available); Nicotine (purchase application requested); Human intestinal epithelial cells Beas-2B (preserved at the Department of Pharmaceutical Chemistry, Xiangya School of Pharmacy, Central South University); Spanish soft agar.

[0023] 2. Soft agar sphere formation experiment

[0024] Take Beas-2B in the logarithmic growth phase and seed it at a density of 1*10⁵ / ml. Incubate Beas-2B with DMSO, nicotine (200 μM), or rosmarinic acid (1, 5, 20 μM) or carrageenan (1, 5, 20 μM) for 2 hours. Mix 1.2% soft agar and 2* complete medium (2*DMEM + 20% FBS + 2% antibiotics) and take 2 ml to quickly spread it on a six-well plate. Mix 0.7% soft agar and 2* complete medium evenly to prepare the top layer gel. Incubate at 42℃ to keep it in a liquid state. Add the drug-treated Beas-2B at a density of 1*10⁵ / ml to the top layer gel, mix evenly, and quickly spread it on the solidified bottom layer gel. Add 100 μl of complete medium every 2 days to prevent the top gel from drying out.

[0025] 3. Experimental Results

[0026] like Figure 1 As shown, nicotine significantly promoted the formation of spheroids (malignant proliferation) of Beas-2B cells in soft agar, while RA and CA inhibited nicotine-induced spheroidization of Beas-2B cells in a concentration gradient. These results indicate that rosmarinic acid and sarsaparilla acid can inhibit nicotine-induced spheroidization of Beas-2B cells. Normally derived cells do not exhibit excessive proliferation in liquid or semi-solid culture media; only malignantly transformed cells can proliferate in clusters in semi-solid media. Therefore, our experimental results demonstrate that nicotine induces malignant transformation of normal human bronchopulmonary epithelial cells, while rosmarinic acid and sarsaparilla acid have the ability to inhibit nicotine-induced malignant transformation of normal human bronchopulmonary epithelial cells.

[0027] Experiment Example 2: Effect of Rosmarinic Acid on Nicotine-Induced DNA Damage in Mice

[0028] 1. Source of materials

[0029] Smoke extraction machine (commercial purchase); Aerosol generator tower (commercial purchase); C57BL / 6j (Hunan Silek Jingda)

[0030] 2. Test methods

[0031] E-cigarette cartridges containing different concentrations (10, 40 mg / ml) of rosmarinic acid were prepared. Mice were exposed daily to solvent aerosols, 10 mg / ml nicotine, 10 mg / ml nicotine + 10 mg / ml rosmarinic acid, and 10 mg / ml nicotine + 40 mg / ml rosmarinic acid, respectively, for 3 hours daily, 5 days per week, for 12 consecutive weeks. Circular PdG and O6-medG adducts formed in genomic DNA were measured using Western blotting. The expression levels of DNA repair proteins XPC and OGG1 / 2 were determined using Western blotting.

[0032] 3. Test Results

[0033] The experimental results show that, compared with the control group, the e-cigarette model containing only nicotine significantly promoted the formation of DNA adducts PDG and O6-medG, and inhibited the expression levels of DNA repair proteins XPC and OGG1 / 2. In contrast, the e-cigarette model containing both rosmarinic acid and nicotine significantly inhibited the formation of DNA adducts PDG and O6-medG, and upregulated the expression levels of DNA repair proteins XPC and OGG1 / 2, compared with the nicotine-only model. Therefore, rosmarinic acid inhibits nicotine-induced DNA damage and maintains genome stability by reducing the formation of DNA adducts and the expression of DNA repair proteins.

[0034] Experiment Example 3: Effect of Rosmarinic Acid on Nicotine-Induced Mutation Sensitivity

[0035] The shuttle vector pSP189 plasmid (1500 J / m2) carrying the tyrosine repressor gene tRNA encoding the supF gene was modified by UV irradiation or H2O2 (100 mM, 37℃ for 1 h) and then transfected into BEAS-2B cells pretreated with nicotine, cells without nicotine, and cells pretreated with different concentrations of rosmarinic acid (0.1, 1, 5, 10 μM), and incubated at 37℃ for 1 h. Mutations were detected in supF mutations.

[0036] Compared with the control group, the nicotine-treated model group showed a significant increase in the supF mutation rate induced by ultraviolet irradiation and H2O2, while rosmarinic acid inhibited the nicotine-induced supF mutation rate increase in a concentration gradient. This means that nicotine increases susceptibility to gene mutations, while rosmarinic acid inhibits nicotine-induced susceptibility to gene mutations and maintains genome stability.

[0037] Experiment Example 4: Effect of rosmarinic acid on nicotine-induced malignant transformation of bronchial epithelial cells in pulmonary cells of FVB / N mice

[0038] After euthanizing mice by cervical dislocation, bronchial epithelial tissue was dissected in a clean bench. The tissue was washed with cold, sterile PBS and dissociated into single-cell suspensions in mouse bronchial dissociation medium. These suspensions were then mixed with matrix gel in mouse bronchial epithelial organoid culture medium and seeded into 96-well plates. Initial organoid formation was observed after 7 days. The organoids were incubated for 7 days with nicotine at varying concentrations (100, 200, 400, 800 μM). Immunohistochemistry was used to determine the optimal concentration for nicotine-induced carcinogenesis of bronchial epithelial cells. After pre-incubating the organoids for 1 day with different concentrations of rosmarinic acid (1, 5, 10 μM), they were then incubated for 7 days in organoid culture media with or without nicotine. Immunohistochemistry was used to observe the effect of rosmarinic acid on nicotine-induced carcinogenesis of bronchial epithelial cell organoids.

[0039] HE staining revealed that nicotine induced organoid transformation of mouse bronchial epithelial cells into lung adenocarcinoma, while rosmarinic acid inhibited the occurrence of nicotine-induced lung adenocarcinoma. This indicates that rosmarinic acid plays a role in maintaining gene stability and inhibiting nicotine-induced bronchial carcinogenesis.

[0040] Experimental Example 5: Rosmarinic acid inhibits nicotine-induced tumorigenicity of BEAS-2A in nude mice.

[0041] 1. Source of materials

[0042] Nude mice (Hunan Sleike Jingda)

[0043] 2. Test methods

[0044] Beas-2B mice in the logarithmic growth phase were seeded at a density of 1*10⁵ / ml. The model group was induced with 20 μM nicotine for 30 days, followed by approximately 30 passages. The experimental group was co-incubated with rosmarinic acid (20 μM, 10 μM) and nicotine (20 μM) for 30 days, followed by 30 passages. Five-week-old male nude mice were seeded at a density of 5*10⁵ / ml. 6 Cells were resuspended in 200 ml of ice-cold PBS and inoculated into the upper inguinal region of nude mice within 1 hour. The largest tumor reached 1000 mm. 3 The experiment ends when the time is right.

[0045] 3. Test Results

[0046] Compared with the control group, the nicotine model group showed a significant increase in the tumorigenicity of BEAS-2A induced by long-term low-dose nicotine exposure in vitro, indicating that nicotine induces malignant transformation of normal cells and promotes the carcinogenesis of normal cells. In contrast, the experimental groups, with high concentrations (20 μM) and low concentrations (1 μM) of rosmarinic acid, showed significantly smaller tumor volume compared to the model group, demonstrating that rosmarinic acid can inhibit nicotine-induced malignant transformation of BEAS-2A and thus inhibit nicotine-promoted tumorigenesis.

Claims

1. The use of polyphenolic compounds or their derivatives in the preparation of additives for inhibiting the carcinogenic effects of nicotine in electronic cigarette filters, wherein the polyphenolic compounds and their derivatives are present in the form of rosemary extract, wherein the rosemary extract contains at least one of rosmarinic acid and sauropic acid.

2. The application according to claim 1, wherein the rosemary extract is obtained by extraction in an alcohol or an aqueous alcohol solvent.

3. The application according to claim 1, wherein the additive for inhibiting the carcinogenic effect of nicotine in electronic cigarette filters is present in the form of a mixture consisting entirely or partially of rosmarinic acid.

4. The application according to claim 1, wherein the additive for inhibiting the carcinogenic effect of nicotine in electronic cigarette filters is present in the form of a mixture consisting entirely or partially of oxalic acid.

5. The application according to claim 1, wherein the additive for inhibiting the carcinogenic effect of nicotine in electronic cigarette filters is added to the electronic cigarette filters in an amount of 20 mg to 50 mg / g.

6. The application according to claim 1, wherein the additive for inhibiting the carcinogenic effect of nicotine in electronic cigarette filters is added to the electronic cigarette filter in an amount of 30 mg.

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