Composition and application thereof in preparation of tobacco additive

Abstract

The invention provides a tobacco additive composition for preventing and treating sphagitis. The tobacco additive composition comprises isorhamnetin and naringenin. The tobacco additive has unique properties, when tobacco is ignited, isorhamnetin and naringenin are not changed through a combustion mode but are converted into a gaseous state, and the isorhamnetin and the naringenin enter the respiratory tract of the human body along with gas generated by tobacco combustion. Due to the characteristic, the traditional Chinese medicine composition can play a positive role in chronic pharyngitis and pharyngolaryngitis, effectively relieve symptoms, prevent diseases and assist the treatment process, does not cause any complications after being used for a long time, and greatly guarantees the health and safety of a user. From the perspective of use experience, the tobacco product is endowed with good taste by the additive, and the additive can be appreciated and accepted by the majority of smokers.

Description

Technical Field

[0001] This invention relates to the field of tobacco technology, and in particular to a composition and its application in the preparation of tobacco additives. Background Technology

[0002] Cigarette smoke contains various harmful components such as nicotine, tar, and aldehydes. When people smoke, these substances directly irritate the mucous membranes of the throat, causing congestion and swelling, and lowering their resistance, making them more susceptible to bacterial and viral infections, ultimately leading to pharyngitis. Furthermore, long-term smoking can worsen pharyngitis, making treatment more difficult and significantly increasing the recurrence rate.

[0003] Currently, some synthetic small-molecule compounds have also entered the research field of tobacco anti-inflammatory effects. For example, certain metal complexes can mimic the activity of natural antioxidant enzymes, decomposing harmful substances such as peroxides generated during tobacco combustion, thereby blocking the oxidative stress phase in the inflammatory response. In addition, some artificially synthesized peptide compounds can specifically bind to inflammatory factors, rendering them inactive and thus reducing inflammation. However, the safety and stability of synthetic compounds in the complex combustion environment of tobacco still require more in-depth and comprehensive research and verification.

[0004] It is important to note that tobacco itself has an extremely complex composition, and the anti-inflammatory effects of additives can be significantly affected by various factors. For example, different tobacco varieties, processing techniques, and smoking methods can all influence the interaction between additives and harmful substances in tobacco, thereby greatly reducing their actual anti-inflammatory efficacy.

[0005] Therefore, it is essential to address the problems caused by cigarette-related pharyngitis in existing technologies and reduce the prevalence of pharyngitis among long-term smokers. Summary of the Invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide a tobacco additive that can prevent and treat pharyngitis.

[0007] The present invention provides a tobacco additive composition for preventing and treating pharyngitis, comprising isorhamnetin of formula (I) and naringenin of formula (II).

[0008]

[0009] Formula I Formula II

[0010] According to the present invention, the mass ratio of isorhamnetin and naringenin is 1:1.

[0011] The compound of Formula I of this invention, chemically named isorhamnetin, is a flavonoid compound believed to exert anti-inflammatory effects through multiple mechanisms. Studies have shown that it can inhibit the activation of inflammatory cells and reduce the release of pro-inflammatory and chemokine factors, thereby alleviating inflammatory responses. Isorhamnetin has also been shown to inhibit the activation of the NF-κB (nuclear factor κB) signaling pathway, a key transcription factor in inflammatory responses that regulates the expression of various inflammatory genes.

[0012] The compound of Formula II of this invention, chemically named naringenin, has been shown to possess significant antioxidant properties. In vitro and animal studies have demonstrated its ability to reduce oxidative damage to DNA.

[0013] Isorhamnetin and naringenin belong to the flavonoid family and possess relatively stable chemical structures and a certain degree of thermal stability. Even at high temperatures, their molecular structures do not easily decompose. This stability allows them to retain their chemical properties during tobacco combustion without undergoing rapid degradation. Furthermore, these two compounds convert to gases upon heating and enter the flue gas through gas-phase diffusion and volatilization, maintaining their activity and preventing complete decomposition and inactivation due to the high temperatures of combustion. Literature also reports the detection of these two compounds in flue gas using HPLC analysis.

[0014] The tobacco additives described in this invention preferably also include one or more of the following: flavoring agents, combustion regulators, preservatives, pH adjusters, fillers, humectants, or flavoring agents; this invention does not limit the specific components of the above-mentioned additives, as those well known to those skilled in the art are acceptable.

[0015] The flavoring agent preferably includes one or more of the following: lemon oil, orange peel oil, cinnamon oil, lavender oil, fennel oil, menthol, vanillin, clove oil, ylang-ylang oil, or brandy essential oil.

[0016] In some specific embodiments, the smoking material may also include additives, such as wetting agents. Wetting agents may include at least one of glycerin, propylene glycol, ethylene glycol, dipropylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, and oleyl alcohol.

[0017] This invention provides a method for preparing a tobacco additive composition, comprising:

[0018] Isorhamnetin and naringenin are dissolved in a solvent to obtain a tobacco additive solution; the solvent is preferably anhydrous ethanol.

[0019] In this invention, the mass-to-volume ratio of isorhamnetin to anhydrous ethanol is 1 mg: 1.5 mL;

[0020] Specifically, the mass-to-volume ratio of naringenin to anhydrous ethanol is 1 mg: 1.5 mL;

[0021] The mixing temperature described in this invention is 15~25℃; specifically, it can be 15℃, 16℃, 17℃, 18℃, 19℃, 20℃, 21℃, 22℃, 23℃, 24℃, 25℃; or any value between the above two.

[0022] This invention provides a tobacco material, including the tobacco additives described in any one of the above technical solutions.

[0023] The tobacco materials of this invention may include, for example, tobacco leaves, shredded tobacco, tobacco stems, or materials processed therefrom. More specifically, the tobacco materials may include pulverized tobacco leaves, pulverized reconstituted tobacco, expanded shredded filler, expanded stems and leaflets, etc. However, this invention is not limited thereto.

[0024] This invention provides a method for preparing tobacco material, comprising:

[0025] The tobacco material is impregnated with the tobacco additive solution described in any of the above technical solutions and then dried to obtain the final product.

[0026] The immersion temperature described in this invention is 15~30℃, and the immersion time is 30min. This invention does not limit the specific immersion method, but any method known to those skilled in the art is acceptable.

[0027] In some specific solutions, the immersion temperature can be 15℃, 17℃, 19℃, 20℃, 21℃, 23℃, 25℃, 27℃, 28℃, 29℃, 30℃; or any value between the above two.

[0028] The tobacco additive solution of the present invention is an ethanol solution of isorhamnetin and naringenin;

[0029] The drying temperature described in this invention is 50~80℃; the drying time is 15~30min;

[0030] In some specific embodiments, the drying temperature can be 50℃, 55℃, 60℃, 65℃, 70℃, 75℃, or 80℃; or any value within a range of the above. The drying process of this invention is preferably performed under pressure, atmospheric pressure, or reduced pressure, preferably under reduced pressure, and more preferably under vacuum conditions. The drying time can be 15 min, 16 min, 17 min, 18 min, 20 min, 22 min, 24 min, 26 min, 28 min, or 30 min; or any value within a range of the above.

[0031] In some specific formulations, the total mass ratio of isorhamnetin and naringenin in the tobacco additive to the mass of tobacco shreds is 1 mg: 50 g.

[0032] According to the present invention, the mass ratio of isorhamnetin and naringenin is 1:1.

[0033] This invention provides the use of any of the above-described tobacco additives in the preparation of tobacco products for the prevention or treatment of pharyngitis.

[0034] According to the present invention, the prevention and treatment of pharyngitis includes reducing the levels of inflammatory factors IL-6, TNF-α, or nitric oxide.

[0035] In this embodiment of the invention, a mixture of isorhamnetin and naringenin was added to DMEM high-glucose complete culture medium containing 0.5% fetal bovine serum. The final concentration of the mixed solution of isorhamnetin and naringenin was 50 μg / mL or 100 μg / mL.

[0036] Experimental results showed that treatment with TPM at concentrations of 100 μg / ml and 50 μg / ml significantly reduced the production of nitric oxide (NO). Treatment with TPM at a concentration of 100 μg / ml significantly reduced the production of inflammatory factors IL-6 and TNF-α.

[0037] The present invention provides a tobacco product comprising the tobacco additives described in any one of the above technical solutions or the tobacco shreds described in the above technical solutions.

[0038] "Tobacco products" refers to any product that enables smoking, whether it is based on tobacco, tobacco derivatives, expanded tobacco, reconstituted tobacco, or tobacco substitutes, or any product that provides the smoking experience. Examples include cigarettes, cigars, and mini cigars.

[0039] The tobacco products described in this invention may include, for example, tobacco materials. Tobacco materials may include, for example, tobacco leaves, tobacco stems, or materials processed therefrom. Tobacco materials may include pulverized tobacco leaves, pulverized reconstituted tobacco, expanded cornflower, expanded stems and leaflets, etc. Furthermore, tobacco materials include, but are not limited to, the form of tobacco shreds, tobacco pellets, tobacco sheets, tobacco granules, or tobacco extracts.

[0040] This invention provides a tobacco additive comprising isorhamnetin and naringenin of formula (I). The tobacco additive of this invention possesses unique properties: when tobacco is ignited, the isorhamnetin and naringenin are not transformed through combustion, but rather converted into a gaseous form, entering the human respiratory tract along with the gases produced by tobacco combustion. This characteristic allows it to play a positive role in chronic pharyngitis and laryngitis, effectively alleviating symptoms, preventing disease occurrence, and assisting in the treatment process. Furthermore, long-term use will not cause any complications, greatly ensuring the health and safety of users. From a user experience perspective, this additive imparts a pleasant taste to tobacco products, making it readily accepted by a wide range of smokers. In terms of production process, the preparation process of cigarettes containing this additive is relatively simple, requiring no complex technical means or high equipment investment, thereby effectively reducing production costs, improving production efficiency, and demonstrating significant economic benefits. Rigorous cell experiments have verified that this tobacco additive exhibits excellent anti-inflammatory properties, precisely intervening in pharyngeal inflammation caused by long-term smoking, significantly alleviating inflammatory responses in the throat, and effectively preventing the recurrence of inflammation. This invention undoubtedly provides innovative ideas and highly promising solutions for the throat health problems of smokers, marking an important step in the product improvement process of the tobacco industry and opening up new avenues for protecting the health of smokers. It has extremely broad application prospects and far-reaching positive significance. Attached Figure Description

[0041] Figure 1 Schematic diagram of nitric oxide level testing in this invention;

[0042] Figure 2 Schematic diagram of ELISA enzyme-linked immunosorbent assay for IL-6 levels in this invention;

[0043] Figure 3 Schematic diagram of the ELISA enzyme-linked immunosorbent assay for TNF-α levels in this invention;

[0044] Figure 4 Effects of TPM solution intervention with isorhamnetin and naringenin for 24 hours on the proliferative activity of macrophages in RAW264.7 mice;

[0045] Figure 5Effects of TPM solutions of isorhamnetin and naringenin on LPS-induced NO production in RAW264.7 mouse macrophages (n=3);

[0046] Figure 6 Effects of TPM solutions of isorhamnetin and naringenin on LPS-induced IL-6 production in RAW264.7 mouse macrophages (n=3);

[0047] Figure 7 Effects of TPM solutions of isorhamnetin and naringenin on LPS-induced TNF-α production in RAW264.7 mouse macrophages (n=3). Detailed Implementation

[0048] This invention provides a tobacco additive, its preparation method, and its application. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and fall within the scope of protection of this invention. The method and application of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the method and application described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0049] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0050] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0051] In this application, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural.

[0052] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0053] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and is not intended to limit the scope of the invention unless the claims are made. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0054] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately". Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0055] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0056] The embodiments and comparative examples of this invention describe some examples, in which the embodiments illustrate certain implementations of the invention. However, this does not mean that the effects of the invention can only be achieved in these examples.

[0057] To further illustrate the present invention, the following detailed description of a tobacco additive, its preparation method, and its application is provided in conjunction with embodiments.

[0058] The isorhamnetin and naringenin used in the examples were commercially available, such as those from Chengdu Dester Biotechnology Co., Ltd., and the purity of the samples used all met pharmaceutical standards.

[0059] Example 1

[0060] 1. Experimental Materials

[0061] 1.1 Medicinal Materials

[0062] Isorhamnetin (CAS: 480-19-3, molecular formula: C) 16 H 12 O7, molecular weight 316.26, HPLC purity ≥98%, Chengdu Manster Biotechnology Co., Ltd. Naringenin (CAS: 67604-48-2, molecular formula: C 15 H 12 O5, molecular weight 272.25, HPLC purity ≥98%, Chengdu Manster Biotechnology Co., Ltd.

[0063] 1.2 Cell Culture

[0064] Mouse mononuclear macrophage leukemia cells RAW264.7 (purchased from Shanghai Cell Bank, Chinese Academy of Sciences, TCM13) were cultured in high-glucose DMEM (Shanghai Datashire Biotechnology Co., Ltd.) medium containing 15% fetal bovine serum (Cellmax, FBS) and 1% penicillin-streptomycin mixture (Boster Biological Engineering Co., Ltd.) at 37°C and 5% CO2 saturated humidity.

[0065] 1.3 Cell resuscitation

[0066] Preheat the water bath to 37°C. Remove the RAW264.7 cryovials of mouse macrophages from liquid nitrogen and quickly thaw them in the preheated water bath, shaking within 2 minutes. Transfer the thawed cells to 10ml centrifuge tubes containing 7ml of DMEM complete medium and centrifuge at 1000rpm for 5 minutes. After centrifugation, discard the supernatant, add 1ml of complete medium, and resuspend the cells thoroughly by pipetting. Transfer the cell suspension to T25 cell culture flasks containing 4ml of complete medium. After thorough mixing, incubate in a CO2 cell culture incubator at 7°C and 5% CO2 saturated humidity. After overnight incubation, change the medium after cell adhesion.

[0067] 1.4 Cell passage

[0068] RAW264.7 macrophages were cultured in a CO2 cell incubator and observed under an inverted biological microscope. When the cells reached the logarithmic growth phase (i.e., confluence of 80% or higher), the original culture medium was discarded, and 2 ml of pre-chilled neutral PBS (Wuhan Sewell Biotechnology Co., Ltd.) buffer was added. After gently washing the bottom of the cell culture flask, the PBS buffer was discarded, and 2 ml of fresh DMEM complete culture medium was added. The cells were gently pipetted to detach the adherent cells and mix well. The cell suspension was then added to new culture flasks according to the required passage ratio (1:2~1:6), and an appropriate amount of DMEM complete culture medium was added before culturing in a cell incubator.

[0069] 1.5 Cell Count

[0070] Wipe the hemocytometer and coverslip with 75% alcohol and let them air dry. Discard the original culture medium, add 2 ml of pre-chilled PBS buffer, gently wash the bottom of the cell culture flask, discard the PBS buffer, add 2 ml of fresh DMEM complete culture medium, and gently pipette to detach the adherent cells and mix well. Transfer the cell suspension to a 10 ml centrifuge tube and centrifuge at 1000 rpm for 5 min. After centrifugation, discard the supernatant, add 1 ml of complete culture medium, and gently pipette to mix thoroughly. Use a pipette to draw 10 μl of the mixed cell suspension and add it to the gap between the sterilized hemocytometer and coverslip, ensuring the area under the coverslip is filled with cell suspension without overflowing. Under an inverted biological microscope, calculate the number of cells in the suspension using the following formula:

[0071] Total cell count (cells / ml suspension) = (Total number of cells in four large squares / 4) × 10 4

[0072] 1.6 Data Processing and Statistical Analysis

[0073] Quantitative data that follow a normal distribution are expressed as mean ± standard deviation (±SD). One-way ANOVA is used to compare the significance between groups. p < 0.05 is considered statistically significant.

[0074] 2. Experimental Methods

[0075] 2.1 Preparation of DMEM complete culture medium

[0076] Prepare a mixture of DMEM high-glucose medium, fetal bovine serum (inactivated), and antibiotics at a ratio of 100:15:1. Store at 4°C for later use.

[0077] 2.2 TPM capture in cigarettes

[0078] The Canadian deep aspiration method (HCI, aspiration volume of 55 mL, aspiration time of 2 s, and aspiration interval of 30 s) was used to aspirate samples (four cigarettes each) from the control group and the treatment group, and the mainstream smoke TPM was collected using a 44 mm Cambridge filter.

[0079] 2.3 Dissolve DMSO overnight

[0080] Add 400 μL of dimethyl sulfoxide (DMSO, Bosch Biotech Co., Ltd.) solution to each sterilized and dried EP tube for dissolution, and place in a refrigerator at 4 s ℃ overnight.

[0081] 2.4 Ultrasound and Filtration

[0082] The overnight DMSO solution was removed and ultrasonically cleaned for 15 minutes in an ultrasonic cleaner (KQ-700E model) to ensure thorough agitation. Each compound was filtered through a 0.22 μM microporous membrane. The filtered solution was the stock solution of the compound with a concentration of 100 mg / mL, and stored at -20°C for later use. For drug administration, the solution was diluted with high-glucose DMEM containing 0.5% serum to the appropriate concentration, ensuring that the final concentration of DMSO in the working solution was below 0.1% during dilution.

[0083] 2.5 Drug dilution

[0084] Take 10 μL of the stock solutions of the compounds (isorhamnoside and naringenin) with a concentration of 100 mg / mL, and prepare 10 mL solutions with DMEM high-glucose complete medium containing 0.5% fetal bovine serum, to a final concentration of 100 μg / mL. Using the 100 μg / mL stock solution, further dilute downwards to prepare solutions of 50 μg / mL, 25 μg / mL, and 12.5 μg / mL, and use immediately after preparation.

[0085] 2.6 Preparation of Modeling Drugs

[0086] Accurately weigh 1 mg of cell-grade lipopolysaccharide (LPS) powder (purchased from Sigma-Aldrich, L2630-100 mg) and dissolve it in 1 ml of PBS (purchased from Seville Biosciences) until completely dissolved, preparing a 1 mg / mL LPS stock solution. Store at -20°C for later use. For modeling, dilute with high-glucose DMEM containing 0.5% serum to a final concentration of 1 μg / mL LPS solution, preparing fresh before use.

[0087] 2.7 Cell viability assay

[0088] Logarithmically growing RAW264.7 cells were seeded in 96-well plates (all plates used in this experiment were purchased from CEPREI) at a density of 1.5 × 10⁵ cells / mL, with a volume of 100 μL per well. The cells were incubated overnight at 37 ℃, 5% CO₂, and 95% humidity. Cells were then cultured for 24 h with compounds at concentrations of 100 μg / mL, 50 μg / mL, 25 μg / mL, and 12.5 μg / mL, with five replicates for each concentration. Cell viability was measured using a CCK8 assay (purchased from Biolight Biotech, Lot. #X2544481X), and absorbance (OD value) was measured at 450 nm using a thermoelectron corporation microplate reader. The OD value indicates the number of viable cells. The effect of each compound on the proliferative activity of mouse macrophages RAW264.7 was investigated to determine the safe concentration range of each compound in this cell line.

[0089] 2.8 Determination of Nitric Oxide (NO) Levels

[0090] RAW264.7 cells in logarithmic growth phase were seeded at a density of 1 × 10⁶ cells / mL in 24-well plates, with a volume of 500 μL per well. The cells were incubated overnight at 37 ℃, 5% CO₂, and 95% humidity. Stimulation with 1 μg / mL LPS was performed for 0.5 h. After 0.5 h, half of the supernatant from the model group was discarded, and the medium was replenished to 500 μL with DMEM high-glucose complete medium containing 0.5% fetal bovine serum. Similarly, half of the supernatant from the drug-treated group was discarded, and the medium was replenished to 500 μL with two concentrations of the respective compound (100 μg / mL and 50 μg / mL) for another 24 h. After stimulation, the cell culture medium was collected, and the supernatant was used to detect NO levels using the Griess method (NO kit purchased from Shanghai Beyotime Biotechnology Co., Ltd., S0021S, Lot No. 052223231108).

[0091] Dilute the NO kit standards with high-glucose medium containing 0.5% fetal bovine serum. Standard concentrations of 0, 1, 2, 5, 10, 20, 40, 60, and 100 μM were used. Add 50 μL of the standard and cell supernatant to each well of a 96-well plate, followed by adding pre-warmed Griess Reagent I and II to each well. Measure the absorbance at 560 nm using a microplate reader. Calculate the nitric oxide concentration in the cell supernatant based on the standard curve.

[0092] 2.9 ELISA enzyme-linked immunosorbent assay

[0093] RAW264.7 cells in logarithmic growth phase were seeded at a density of 1 × 10⁶ cells / mL in 24-well plates, with a volume of 500 μL per well. The cells were incubated overnight at 37 ℃, 5% CO₂, and 95% humidity. Stimulation with 1 μg / mL LPS was performed for 0.5 h. After 0.5 h, half of the supernatant from the model group was discarded, and the medium was replenished to 500 μL with DMEM high-glucose complete medium containing 0.5% fetal bovine serum. Similarly, half of the supernatant from the drug-treated group was discarded, and the medium was replenished to 500 μL with two concentrations of the respective compound (100 μg / mL and 50 μg / mL) for another 24 h. After stimulation, the cell culture medium was collected, and the supernatant was used for mouse interleukin-6 (IL-6) enzyme-linked immunosorbent assay (ELISA kit purchased from Wuhan Elite Biotechnology Co., Ltd., Lot, WA02060J0794).

[0094] RAW264.7 cells in logarithmic growth phase were seeded at a density of 1 × 10⁶ cells / mL in 24-well plates, with a volume of 500 μL per well. The cells were incubated overnight at 37 ℃, 5% CO₂, and 95% humidity. Stimulation with 1 μg / mL LPS was performed for 0.5 h. After 0.5 h, half of the supernatant from the model group was discarded, and the medium was replenished to 500 μL with DMEM high-glucose complete medium containing 0.5% fetal bovine serum. Similarly, half of the supernatant from the drug-treated group was discarded, and the medium was replenished to 500 μL with two concentrations of the respective compound (100 μg / mL and 50 μg / mL) for another 2 h. After stimulation, the cell culture medium was collected, and the supernatant was used for enzyme-linked immunosorbent assay (ELISA) of tumor necrosis factor-α (TNF-α) (ELISA kit purchased from Wuhan Elite Biotechnology Co., Ltd., Lot, WA0742L23345).

[0095] Centrifuge the collected cell supernatant at 1000×g for 20 minutes to remove impurities and cell debris. Use the supernatant for detection. Remove the kit from the refrigerator 20 minutes in advance and allow it to equilibrate to room temperature (18-25℃). Dilute the concentrated wash buffer from the IL-6 and TNF-α kits with double-distilled water (1:24). In this experiment, take 15 mL of the concentrated wash buffer from each kit, add 375 mL of double-distilled water to dilute, and mix well before use.

[0096] Centrifuge the standard at 10000×g for 1 minute. Add 1 mL of standard & sample diluent to the lyophilized standard, tighten the cap, and let stand for 10 minutes. Invert the tube several times to ensure complete dissolution. Gently mix, avoiding foaming. Prepare a 2000 pg / mL working solution of the standard powder from the IL-6 kit, and then serially dilute as needed. Prepare the following concentrations: 2000, 1000, 500, 250, 125, 62.5, 31.25, and 0 pg / mL. Prepare a 500 pg / mL working solution of the standard powder from the TNF-α kit, and then serially dilute as needed. Prepare the following concentrations: 500, 250, 125, 62.5, 31.25, 15.625, 7.8125, and 0 pg / mL.

[0097] Fifteen minutes before use, centrifuge the concentrated biotinylated antibody at 800×g for 1 minute. Dilute the 100× concentrated biotinylated antibody to a working concentration using biotinylated antibody diluent. In this experiment, take 100 μL of the concentrated biotinylated antibody from each IL-6 and TNF-α kit and add it to 9900 μL of biotinylated antibody diluent for proportional dilution. Prepare and use immediately. Fifteen minutes before use, centrifuge the concentrated HRP enzyme conjugate at 800×g for 1 minute. Dilute the 100× concentrated HRP enzyme conjugate to a working concentration using enzyme conjugate diluent. In this experiment, take 100 μL of the concentrated HRP enzyme conjugate from each IL-6 and TNF-α kit and add it to 9900 μL of enzyme conjugate diluent for proportional dilution. Prepare and use immediately.

[0098] Set up standard wells, blank wells, and sample wells separately. Add 100 μL of serially diluted standard to the standard wells, 100 μL of standard and sample diluent to the blank wells, and 100 μL of the sample to be tested to the remaining wells. After adding the samples, cover the microplate with a membrane and incubate at 37°C for 90 minutes. After incubation, discard the liquid from the wells without washing. Add 100 μL of biotinylated antibody working solution to each well, cover the microplate with a membrane, and incubate at 37°C for 1 hour. Discard the liquid from the wells and pat dry on clean absorbent paper. Add 350 μL of washing buffer to each well, soak for 1 minute, aspirate or discard the liquid from the microplate, and pat dry. Repeat this washing step 3 times. Add 100 μL of HRP enzyme conjugate working solution to each well, cover the microplate with a membrane, and incubate at 37°C for 30 minutes. Discard the liquid from the wells, wash the plate 5 times, add 90 μL of substrate solution (TMB) to each well, cover the plate with a membrane, and incubate at 37°C in the dark for about 15 minutes. Add 50 μL of stop solution to each well to stop the reaction. Immediately measure the optical density (OD value) of each well at 450 nm using a microplate reader.

[0099] 2.10 Statistical processing of data

[0100] Data were processed using GraphPad Prism 9.3.0 software, and the data are expressed as mean ± SEM. Analysis of variance was used to compare means among multiple groups, and t-tests were used for pairwise comparisons between multiple groups. P < 0.05 was considered statistically significant.

[0101] 3 Results

[0102] 3.1 Cytotoxicity of isorhamnetin and naringenin

[0103] according to Figure 4It was found that treatment with TPM at concentrations of 100 μg / ml, 50 μg / ml, 25 μg / ml, and 12.5 μg / ml for 24 h did not significantly affect the proliferation activity of RAW264.7 mouse macrophages.

[0104] Figure 4 Effects of TPM solution treatment with isorhamnetin and naringenin on macrophage proliferation activity in RAW264.7 mice for 24 hours

[0105] 3.2 Inhibitory effects of isorhamnetin and naringenin on LPS-induced inflammation

[0106] according to Figure 5 It was found that treatment with TPM concentrations of 100 μg / ml and 50 μg / ml significantly reduced the production of nitric oxide (NO). Based on... Figure 6 , Figure 7 It can be seen that treatment at TPM concentrations of 100 μg / ml and 50 μg / ml can significantly reduce the production of inflammatory factors IL-6 and TNF-α.

[0107] Figure 5 Effects of TPM solutions of isorhamnetin and naringenin on LPS-induced NO production in RAW264.7 mouse macrophages. (n=3)

[0108] Figure 6 Effects of TPM solutions of isorhamnetin and naringenin on LPS-induced IL-6 production in RAW 264.7 macrophages. (n=3)

[0109] Figure 7 Effects of TPM solutions of isorhamnetin and naringenin on LPS-induced TNF-α production in RAW 264.7 macrophages. (n=3)

[0110] The above results indicate that the use of isorhamnetin and naringenin can inhibit LPS-induced inflammation.

[0111] The addition of isorhamnetin and naringenin to tobacco does not affect cell proliferation and can significantly inhibit LPS-induced inflammation of RAW264.7 mouse macrophages.

[0112] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A tobacco additive composition for preventing and treating pharyngitis, characterized in that, Including isorhamnetin and naringenin.

2. The tobacco additive composition according to claim 1, characterized in that, The mass ratio of isorhamnetin to naringenin is 1:

1.

3. A method for preparing a tobacco additive composition, characterized in that, include: Isorhamnetin and naringenin were dissolved in a solvent to obtain a tobacco additive solution; The solvent is anhydrous ethanol.

4. The preparation method according to claim 3, characterized in that, The mass-to-volume ratio of isorhamnetin to anhydrous ethanol is 1 mg: 1.5 mL; The mass-to-volume ratio of naringin to anhydrous ethanol was 1 mg: 1.5 ml. The dissolution temperature is 15~25℃.

5. A tobacco material, characterized in that, Includes the tobacco additives described in any one of claims 1 to 2.

6. A method for preparing a tobacco material, characterized in that, include: The tobacco material is impregnated with a solution of the tobacco additive composition according to any one of claims 1 to 2 and then dried to obtain the final product.

7. The preparation method according to claim 6, characterized in that, The solution of the tobacco additive composition is an ethanol solution of isorhamnetin and naringenin; the total mass ratio of isorhamnetin and naringenin in the tobacco additive to the mass of tobacco shreds is 1 mg: 50 g. The immersion temperature is 15~30℃ and the immersion time is 30min; the drying temperature is 50~80℃ and the drying time is 15~30min.

8. The use of the tobacco additive composition according to any one of claims 1 to 2 in the preparation of tobacco products for the prevention and / or treatment of pharyngitis.

9. The application according to claim 8, characterized in that, The prevention and / or treatment of pharyngitis includes reducing the levels of inflammatory factors IL-6, TNF-α, or nitric oxide.

10. A tobacco product, characterized in that, It includes the tobacco additive composition according to any one of claims 1 to 2 or the tobacco material according to claim 5.

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