Composition for alleviating, preventing, and treating oral soft tissue inflammation using helium plasma treatment solution and preparation method therefor

Abstract

The present invention relates to a technique for preventing and treating oral soft tissue inflammation and, more specifically, to a novel use of a helium plasma treatment liquid effective for preventing and treating oral soft tissue inflammation and to a composition for alleviating, preventing, and treating oral soft tissue inflammation using same.

Description

Composition for preventing and treating oral soft tissue inflammation using helium plasma treatment solution and method for producing the same

[0001] The present invention relates to a technology for preventing and treating oral soft tissue inflammation, and more specifically, to a new use of a helium plasma treatment solution effective in preventing and treating oral soft tissue inflammation, and a composition for preventing and treating oral soft tissue inflammation using the same.

[0002] The manifestation of inflammation in oral soft tissue cells varies depending on the type and cause of the oral ulcer. Oral ulcers can be caused by infections, systemic diseases, physical or chemical irritants, and allergic reactions. However, in many cases, the cause is difficult to determine. Furthermore, since normal saliva secretion helps protect the oral lining, decreased saliva production increases the risk of oral ulcers. The most common known causes of oral ulcers include recurrent aphthous stomatitis (cancers), viral infections (such as herpes simplex and shingles), other infections (such as fungal or bacterial infections), injury, irritating foods, chemicals, smoking, medications (such as chemotherapy drugs), and radiation therapy.

[0003] Iron imbalance is also a contributing factor to oral inflammation, as it can alter the mucous membrane, increase the risk of developing periodontal disease, and affect the body's oxygen supply. Oral tissues are sensitive to oxygen supply, and an inadequate oxygen supply can damage oral tissues and increase the risk of developing periodontal disease or oral inflammation. Iron can play a significant role in the growth and control of immune cells and bacteria. An imbalance due to iron deficiency or excess can suppress immune cell activation and promote bacterial growth, thereby lowering immunity. A weakened immune system can lower resistance to oral infections. This can increase the likelihood of oral bacterial growth and inflammation. Therefore, iron imbalance can negatively impact oral health, which can increase the likelihood of developing oral inflammation and disease. The iron-induced lipid peroxidation mechanism involves a series of metabolic modifications, including the production of reduced glutathione (GSH) via the cysteine / glutamate xc- exchanger. An imbalance in glutathione (GSH) production disrupts cellular redox reactions, causing damage to cell membranes due to lipid peroxidation, leading to cell death via the ferroptosis mechanism. This cell death then causes inflammation in oral soft tissues.

[0004] Diseases caused by inflammation of oral soft tissues are medically termed as periodontitis, periodontal disease, oral mucositis, oral lichen planus, oral throbbing, etc. This inflammation can occur in various parts of the oral cavity and is related to the immune response.

[0005] Inflammation of the oral soft tissues can cause swelling and redness of the lining, and painful ulcers can form, making eating difficult until they heal, which can lead to dehydration and malnutrition. Some ulcers often recur after disappearing. Current treatments for stomatitis include oral antibiotics or antivirals, topical anti-inflammatory analgesics applied directly to the mucosa, topical steroids, and strong acidic drugs, which can cause strong irritation when applied to the oral mucosa. Other treatments include lasers. Depending on the type of these agents (liquid, ointment, gargle, patch, etc.), there are contraindications for treatment, including pregnancy and lactation, children, the elderly, patients with oral viruses, patients with fungal infections, and patients allergic to nonsteroidal anti-inflammatory drugs.

[0006] Inflammatory oral tissues are known to have high expression of nitric oxide synthase (NOS). This enzyme is involved in the production of nitric oxide (NO), which is produced intracellularly using L-arginine and oxygen. There are three main types of NOS, each activated in different tissues and physiological processes. nNOS, expressed in neurons (nerve cells), acts as a neurotransmitter; eNOS, expressed in vascular endothelial cells, dilates blood vessels and regulates blood circulation; and iNOS, activated during inflammation and immune responses, is involved in the production of inflammatory chemicals and immune responses. iNOS is known to be involved in the overproduction of NO, promoting inflammatory responses such as vascular permeability and edema, and promoting the biosynthesis of inflammatory mediators, which exacerbates inflammation.

[0007] Overexpression of inducible nitric oxide synthase (iNOS) is primarily activated in inflammatory situations. Excessive iNOS activation leads to the production of high levels of nitric oxide (NO), which can enhance the inflammatory response. Unregulated overactivation of iNOS can lead to persistent inflammation. This leads to oxidative stress by reacting with radicals, which can lead to cell damage and increase the risk of diseases associated with autoimmune attacks or compromised immune function, as well as tissue damage and dysfunction. Furthermore, studies related to autoimmune diseases have shown that overexpression of iNOS can influence the development and progression of immune-mediated diseases. For these reasons, regulation of iNOS activation is crucial, and overactivation must be prevented.

[0008] To date, there is no adequate treatment for reducing pain and preventing recurrence due to oral soft tissue inflammation related to oral soft tissue cells. Therefore, there is a need to develop an effective technique that reduces drug-induced side effects, alleviates pain as much as possible, and eliminates inflammation recurrence.

[0009] Accordingly, the purpose of the present invention is to confirm a new use of a helium plasma treatment liquid, in which a liquid treated with low-temperature atmospheric pressure plasma using helium (He) gas using a low-temperature atmospheric pressure plasma device is effective in preventing and treating inflammation of oral soft tissue, and to provide a composition for preventing and treating inflammation of oral soft tissue containing a helium plasma treatment liquid and a method for producing the same.

[0010] Another object of the present invention is to provide an oral cleaning composition containing a helium plasma treatment solution and a method for manufacturing the same.

[0011] The purposes of the present invention are not limited to those mentioned above, and other purposes not mentioned will be clearly understood by those skilled in the art from the description below.

[0012] In order to achieve the above-described object of the present invention, the present invention provides a composition for preventing and treating oral soft tissue inflammation, which comprises a helium plasma treatment solution as an active ingredient.

[0013] In a preferred embodiment, the helium plasma treatment solution is obtained by generating power by setting the frequency and voltage to 30 kHz or higher and 5 kV or higher in a cold atmospheric plasma device, and then passing He (helium) gas at 3 to 5 L / min and O2 (oxygen) at 40 to 60 SCCM through water, an aqueous solution, or an aqueous dispersion.

[0014] In a preferred embodiment, when the water or aqueous solution is 5 ml, the plasma treatment by the low-temperature atmospheric pressure plasma device is performed for 10 to 60 seconds.

[0015] In a preferred embodiment, when the water, aqueous solution or aqueous dispersion is 5 ml, the frequency and voltage of the low-temperature atmospheric pressure plasma device are 40 kHz and 6 kV, the He (helium) gas is 4 L / min, the O2 (oxygen) is 50 SCCM, and the plasma treatment is performed for 15 seconds.

[0016] In addition, the present invention provides an oral hygiene composition comprising a helium plasma treatment solution.

[0017] In a preferred embodiment, the helium plasma treatment solution is obtained by generating power by setting the frequency and voltage to 30 kHz or higher and 5 kV or higher in a cold atmospheric plasma device, and then passing He (helium) gas at 3 to 5 L / min and O2 (oxygen) at 40 to 60 SCCM through water, an aqueous solution, or an aqueous dispersion.

[0018] In a preferred embodiment, when the water, aqueous solution or aqueous dispersion is 5 ml, the plasma treatment by the low-temperature atmospheric pressure plasma device is performed for 10 to 60 seconds.

[0019] In a preferred embodiment, when the water, aqueous solution or aqueous dispersion is 5 ml, the frequency and voltage of the low-temperature atmospheric pressure plasma device are 40 kHz and 6 kV, the He (helium) gas is 4 L / min, the O2 (oxygen) is 50 SCCM, and the plasma treatment is performed for 15 seconds.

[0020] In a preferred embodiment, the helium plasma treatment solution is included in an amount of 50% or less of the total composition volume.

[0021] In addition, the present invention provides a method for preparing a composition for alleviating, preventing, and treating oral soft tissue inflammation, comprising the steps of: preparing any one liquid precursor selected from the group consisting of water, an aqueous solution, and an aqueous dispersion; generating power by setting a frequency and voltage of 30 kHz or more and 5 kV or more in a cold atmospheric plasma device; and performing plasma treatment with the cold atmospheric plasma device in which the power is generated while passing He (helium) gas of 3 to 5 L / min and O2 (oxygen) of 40 to 60 SCCM through the prepared liquid precursor to obtain a helium plasma treatment liquid.

[0022] In a preferred embodiment, when the liquid precursor is 5 ml, plasma treatment by the low-temperature atmospheric pressure plasma device is performed for 10 to 60 seconds.

[0023] In a preferred embodiment, when the liquid precursor is 5 ml, the frequency and voltage of the low-temperature atmospheric pressure plasma device are 40 kHz and 6 kV, the He (helium) gas is 4 L / min, the O2 (oxygen) is 50 SCCM, and the plasma treatment is performed for 15 seconds.

[0024] In addition, the present invention provides a method for manufacturing an oral hygiene composition, comprising: a step of preparing any one liquid precursor selected from the group consisting of water, an aqueous solution, and an aqueous dispersion; a step of generating power by setting a frequency and voltage of 30 kHz or more and 5 kV or more in a cold atmospheric plasma device; a step of passing He (helium) gas of 3 to 5 L / min and O2 (oxygen) of 40 to 60 SCCM through the prepared liquid precursor while performing plasma treatment with the cold atmospheric plasma device in which the power is generated to obtain a helium plasma treatment liquid; and a step of diluting the helium plasma treatment liquid so that it is comprised of 50% or less of the total volume of the composition.

[0025] In a preferred embodiment, when the liquid precursor is 5 ml, plasma treatment by the low-temperature atmospheric pressure plasma device is performed for 10 to 60 seconds.

[0026] In a preferred embodiment, when the liquid precursor is 5 ml, the frequency and voltage of the low-temperature atmospheric pressure plasma device are 40 kHz and 6 kV, the He (helium) gas is 4 L / min, the O2 (oxygen) is 50 SCCM, and the plasma treatment is performed for 15 seconds.

[0027] According to the present invention described above, by using atmospheric plasma having an anti-inflammatory effect, the expression of iNOS is lowered, thereby inducing a normal immune response, thereby reducing cytotoxicity by nitrogen oxides, weakening excessive inflammation and immune response, thereby inducing normal intercellular signal transmission, thereby being effective in preventing and treating inflammation of oral soft tissues.

[0028] Therefore, according to the present invention, an effective technique can be secured to reduce side effects caused by drugs, alleviate pain as much as possible, and eliminate inflammation recurrence.

[0029] The effects of the present invention are not limited to the effects mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the description below.

[0030] Figure 1a is a photograph of plasma generation through eight nozzles, and Figure 1b is a graph showing the results of observing lactate dehydrogenase (LDH) secreted from dead cells after exposing human gingival fibroblasts to He plasma in a time-dependent manner.

[0031] Figure 2a shows the electrical characteristics of the current-voltage waveform of a cold atmospheric plasma (CAP) device generating helium plasma using an oscilloscope at 6 kV / 40 kHz, and Figures 2b and 2c show the He, O2 in the range of 280 to 860 nm. + , O + , optical emission spectra (OES) for helium and oxygen showing emission lines of O2 and O, and Fig. 2d shows the image sensor electron temperature measurements according to the He CAP treatment time.

[0032] Figures 3a to 3c illustrate the results of establishing conditions for time and concentration by treating oral fibroblasts with LPS and Erastin for cell viability, Figure 3d illustrates the results of confirming the results of mRNA expression of IL-1β, TNFα, and IL-6 after treating with LPS and Erastin, and Figures 3e and 3f illustrate the results of observing the results of mRNA and protein expression of iNOS and COX-2 expressed when inflammation of oral cells increases.

[0033] Figures 4a and 4b show the results of mRNA expression and protein expression of Bax, Atg5, and P62 after treatment with LPS and Erastin, Figures 4c and 4d show the results of confirming the expression of GPX4 related to ferroptosis by Atg5, which was significantly increased by Erastin treatment, Figure 4e shows the results of confirming the results of decreasing the mRNA expression and protein expression of GPX4 by Erastin treatment, and Figures 4f and 4g show the results of confirming the expression of NCOA4, a protein related to iron degradation by binding to intracellular iron, after treatment with Erastin in a time- and concentration-dependent manner.

[0034] FIG. 5a and FIG. 5b illustrate the results of observing the mRNA expression of iNOS, IL-1β, TNFα, and IL-6 cytokines after treating with a helium plasma treatment solution obtained by controlling the treatment time of helium CAP and the flow concentration of helium gas to find the conditions for suppressing inflammation in oral cells, FIG. 5c illustrates the results of observing the mRNA expression of Bax, Atg5, and P62 after treating with a helium plasma treatment solution obtained by controlling the treatment time of helium CAP and the flow concentration of helium gas, and FIG. 5d illustrates the results of observing the expression of proteins related to inflammation and increased cell death using a helium plasma treatment solution obtained by treating with a helium CAP for 15 seconds in a helium gas flow of 4 L / min.

[0035] Figures 6a and 6b illustrate the results of confirming the reason for the reduction in inflammation and cell death by helium plasma treatment solution in relation to ferroptosis, and Figure 6c illustrates the results of observing the cell membrane potential of mitochondria using a helium plasma treatment solution obtained by time-dependent treatment under 4 L / min conditions with helium CAP.

[0036] Figures 7a to 7e illustrate the results showing that the mRNA expression of iNOS, IL-1β, TNFα, IL-6, and COX-2, which was increased by Erastin treatment in human gingival fibroblast-1 cells, was reduced by helium plasma treatment. Figure 7e illustrates the results showing that Atg5 and P62, which were increased by Erastin treatment, were reduced by helium plasma treatment. Figures 7f and 7g illustrate the results showing that the ferroptosis, which was increased by Erastin treatment, was restored by helium plasma treatment.

[0037] Figure 8a shows the results of observing the recovery of the mitochondrial membrane potential decreased by Erastin treatment by helium plasma treatment solution through JC-1 staining (red staining: healthy cells have high mitochondrial membrane potential, so they form J-aggregates when stained with JC-1 and exhibit red fluorescence), and Figures 8b to 8d show the results of observing the effect of helium plasma treatment solution on the expression of VDAC1, which is located in the mitochondrial outer membrane and plays an important role in regulating the exchange of ions and metabolites between mitochondria and the cytoplasm.

[0038] Figure 9a shows the results of measuring cytosolic and mitochondrial ROS, respectively, to observe the intracellular reaction by helium plasma treatment solution, Figures 9b and 9c show the results of observing the mRNA and protein expression of VDAC1 in response to helium plasma treatment solution, Figure 9d shows the results of confirming the state of mitochondria recovered by helium plasma treatment solution, and Figure 9e shows the results of confirming lipid peroxidation for the reduction of ferroptosis cell death by helium plasma treatment.

[0039] The terms used in this invention have been selected from widely used, common terms, taking into account the functions of the invention. However, these terms may vary depending on the intentions of those skilled in the art, precedents, the emergence of new technologies, etc. Furthermore, in certain cases, the applicant may arbitrarily select terms, and in such cases, their meanings will be described in detail in the description of the relevant invention.

[0040] In the present invention, when the terms "includes," "has," and "consists of," etc. are used, other parts may be added unless "only" is used. When a component is expressed in the singular, it includes cases where the plural is included unless there is a specifically stated description.

[0041] When interpreting a component, it is interpreted as including the error range even if there is no separate explicit description.

[0042] The characteristic parts of each of the various implementation examples of the present invention can be partially or wholly combined or combined with each other, and can be technically linked and driven in various ways, and each implementation example can be implemented independently of each other or can be implemented together in a related relationship.

[0043] Hereinafter, the technical configuration of the present invention will be described in detail with reference to the attached drawings and preferred embodiments.

[0044] However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. The same reference numbers used to describe the present invention throughout the specification represent the same components.

[0045] The technical feature of the present invention is that it is confirmed that a liquid treated with low-temperature atmospheric pressure plasma using helium (He) gas is effective in preventing and treating inflammation of oral soft tissues by lowering the expression of iNOS, thereby inducing a normal immune response, thereby lowering cytotoxicity by nitrogen oxides, weakening excessive inflammation and immune response, and inducing normal intercellular signal transmission, and by using this, a composition for alleviating and treating inflammation of oral soft tissue cells is developed, which contains a helium plasma treatment liquid generated by atmospheric pressure plasma using helium (He) gas as an active ingredient.

[0046] That is, although the role of low-temperature atmospheric pressure plasma as an inflammatory response factor is known from several reports, the plasma treatment solution itself has not been studied, and in particular, it has not yet been discovered that the helium plasma solution generated by low-temperature atmospheric pressure plasma using helium (He) gas has an effect related to relieving inflammation of oral soft tissue cells in relation to nitrogen oxides.

[0047] Therefore, the composition for preventing and treating oral soft tissue inflammation of the present invention contains a helium plasma treatment solution as an active ingredient.

[0048] The ions produced in helium plasma are helium ions (He +) is one of the most commonly produced ions, and ionized helium is electrically active and can participate in chemical reactions in the ionized state. In addition, some helium atoms (He) may be created and remain in an un-ionized state, and can participate in chemical reactions and physical interactions. In addition, free electrons are created in the electrically active state, and these are important for transferring electrical energy or interacting with other substances. In particular, low-temperature atmospheric pressure plasma can discharge in open air, and in addition to the inherent excited species of the discharge gas, it can react with oxygen, water, and nitrogen to generate various reactive oxygen species or chemical species with metastable excited state energies. Therefore, helium plasma generates various ion and atomic forming substances through collisions and chemical interactions with substances, and these substances can cause various chemical reactions through interactions with the surrounding environment.

[0049] The helium plasma treatment solution of the present invention was obtained by generating power by setting the frequency and voltage to 30 kHz or more and 5 kV or more in a cold atmospheric plasma device, and then passing He (helium) gas at 3 to 5 L / min and O2 (oxygen) at 40 to 60 SCCM through water or an aqueous solution. When the frequency and voltage were set to less than 30 kHz and less than 5 kV, the decrease in the expression of cytokines related to inflammation suppression was reduced, and when He (helium) gas was treated at less than 3 L / min, the amount of helium gas discharged was reduced, weakening plasma generation. When the treatment exceeded 5 L / min, the plasma discharge was unstable. Here, the helium plasma treatment liquid obtained by setting the frequency and voltage to 60 kHz and 8 kV did not show better characteristics than the helium plasma treatment liquid obtained by setting the frequency and voltage to 40 kHz and 6 kV to generate power of 7.5 W. Therefore, a helium plasma treatment liquid having desired characteristics can be obtained as long as the frequency and voltage are 30 kHz or higher and 5 kV or higher, but for energy saving, the frequency and voltage can be set to the range of 30 to 50 kHz and 5 to 7 kV.

[0050] The helium plasma treatment solution of the present invention can be obtained by performing plasma treatment using a low-temperature atmospheric pressure plasma device for 10 to 60 seconds when the amount of water, aqueous solution or aqueous dispersion to be treated is 5 ml. However, if the treatment time is less than 10 seconds, there is a problem that the reduction in inflammation suppression is weakened, and if it exceeds 60 seconds, there is a problem that cell damage to oral cells is increased.

[0051] In particular, the helium plasma treatment solution of the present invention can be obtained when the frequency and voltage of the low-temperature atmospheric pressure plasma device are 40 kHz and 6 kV, the He (helium) gas is 4 L / min, the O2 (oxygen) is 50 SCCM, and the plasma treatment is performed for 15 seconds when the water, aqueous solution, or aqueous dispersion is 5 ml.

[0052] Next, the oral hygiene composition of the present invention comprises a helium plasma treatment solution as an active ingredient. Here, since the helium plasma treatment solution used has the same characteristics as described above, a detailed description thereof will be omitted. However, since the oral hygiene composition of the present invention is used for preventive purposes on a regular basis, it is of course possible to use the helium plasma treatment solution diluted rather than directly, that is, to include the helium plasma treatment solution in an amount of 50% or less of the total composition volume.

[0053] Next, the method for manufacturing a composition for alleviating and preventing oral soft tissue inflammation of the present invention may include the steps of preparing any one liquid precursor selected from the group consisting of water, an aqueous solution, and an aqueous dispersion; generating power by setting the frequency and voltage to 30 kHz or more and 5 kV or more in a cold atmospheric plasma device; and performing plasma treatment with the cold atmospheric plasma device in which the power is generated while passing 3 to 5 L / min of He (helium) gas and 40 to 60 SCCM of O2 (oxygen) through the prepared liquid precursor to obtain a helium plasma treatment liquid. Here, when the liquid precursor is 5 ml, the plasma treatment by the low-temperature atmospheric pressure plasma device can be performed for 10 to 60 seconds. In particular, when the liquid precursor is 5 ml, the frequency and voltage of the low-temperature atmospheric pressure plasma device are 40 kHz and 6 kV, the He (helium) gas is 4 L / min, and the O2 (oxygen) is 50 SCCM, and the plasma treatment can be performed for 15 seconds.

[0054] Next, the method for manufacturing an oral hygiene composition of the present invention includes the steps of: preparing any one liquid precursor selected from the group consisting of water, an aqueous solution, and an aqueous dispersion; generating power by setting the frequency and voltage to 30 kHz or more and 5 kV or more in a cold atmospheric plasma device; performing plasma treatment with the cold atmospheric plasma device in which the power is generated while passing He (helium) gas at 3 to 5 L / min and O2 (oxygen) at 40 to 60 SCCM through the prepared liquid precursor to obtain a helium plasma treatment solution; and diluting the helium plasma treatment solution so that it is comprised of 50% or less of the total volume of the composition. Here, the method for manufacturing the helium plasma treatment solution is the same as described above, so a detailed description thereof is omitted.

[0055] Example

[0056] 5 ml of a medium composition consisting of DMEM (high glucose) + 10% FBS was prepared, and the frequency and voltage were set to 40 kHz and 6 kV in a cold atmospheric plasma device to generate 7.5 W of power, and then 4 L / min of He (helium) gas and 50 SCCM of O2 (oxygen) were passed through the medium composition for 15 seconds to obtain a helium plasma treatment solution (He CAP).

[0057] Experimental Example 1

[0058] Cell viability was analyzed by helium low-temperature plasma (He CAP) treatment in human gingival fibroblasts (HGF), and the results are shown in Fig. 1a and Fig. 1b.

[0059] As shown in Fig. 1a, helium plasma was generated through eight nozzles at a frequency of 40 kHz, a voltage of 6 kV, and a power of 7.5 W. The flow of He gas was determined to be 4 L / min and O2 to be 50 sccm.

[0060] As shown in Figure 1b, after time-dependent exposure of human gingival fibroblasts to helium plasma, the secretion of lactate dehydrogenase (LDH) from dead cells was observed to increase in a time-dependent manner. LDH release was analyzed using the LDH-Glo cytotoxicity assay, which showed an increase in luminescence signal with treatment time. Statistical significance was determined using the Mann-Whitney test. All values ​​are expressed as mean ± SD, and * indicates P < 0.05, compared to the control group.

[0061] A bioluminescence-based LDH-Glo™ Cytotoxicity Assay was performed, and it was confirmed that cell viability decreased after 30 seconds of helium plasma exposure.

[0062] As shown in Fig. 1b, it can be seen that cytotoxicity is significantly exhibited when plasma treatment is performed for more than 60 seconds, so the treatment time when preparing a helium plasma treatment solution may be 60 seconds or less.

[0063] Experimental Example 2

[0064] The electrical characteristics of the low-temperature atmospheric pressure plasma device under helium CAP treatment conditions were analyzed using an oscilloscope and optical emission spectrum (OES), and the results are shown in FIGS. 2a to 2d.

[0065] In Fig. 2a, the electrical characteristics of the current-voltage waveform of the low-temperature atmospheric pressure plasma device are displayed using an oscilloscope at 6 kV / 40 kHz. Fig. 2b and Fig. 2c show the He, O2 in the range of 280 to 860 nm. + , O + , optical emission spectra (OES) for helium and oxygen showing emission lines of O2 and O. Figure 2d shows the image sensor electron temperature measurements according to He CAP treatment time, where He CAP was measured for 0, 10, 30 and 180 seconds, and it can be seen that the temperature increases over time.

[0066] Experimental Example 3

[0067] The response of inflammatory cytokines to LPS and Erastin treatment in human gingival fibroblast-1 cells (HGF-1, Human Gingival Fibroblast-1) was observed, and the results are shown in Figures 3a to 3f.

[0068] In order to establish the conditions for time and concentration by treating oral fibroblasts with LPS and Erastin, the cells were treated with 200 ng / ml of LPS and 10 μM Erastin for 0, 0.5, 1, 3, and 6 hours, and the results of mRNA expression of IL-1β, TNFα, and IL-6 are shown in Figs. 3a to 3c. The results in Fig. 3d are for finding the conditions for increasing inflammation while maintaining cell viability, and Figs. 3e to 3f are for observing the results of mRNA and protein expression of iNOS and COX-2, which are expressed when inflammation in oral cells increases.

[0069] Experimental Example 4

[0070] The expression of apoptosis-related genes by LPS and Erastin treatment in human gingival fibroblast-1 cells was observed, and the results are shown in Figs. 4a to 4g.

[0071] Figures 4a and 4b show the results of mRNA expression and protein expression of Bax, Atg5, and P62 after treatment with 200 ng / ml of LPS and 10 μM Erastin for 0, 0.5, 1, 3, and 6 hours to find cell death conditions related to the cause of increased inflammation. It can be confirmed that Atg5 is significantly increased by Erastin treatment.

[0072] Figures 4c to 4e are the results of confirming the expression of GPX4 in relation to ferroptosis by Atg5 increased by Erastin treatment, which is a cause of cell death due to increased inflammation and cell death, and it can be confirmed that the mRNA expression and protein expression of GPX4 were reduced by Erastin treatment.

[0073] Figures 4f and 4g show the results of confirming the expression of NCOA4, a protein involved in iron degradation by binding to intracellular iron after treatment with Erastin in a time- and concentration-dependent manner. Through the mRNA and protein expression of NCOA4, which regulates intracellular iron utilization through stimulation with iron ions, the mechanism related to ferroptosis of oral cells was confirmed.

[0074] Experimental Example 5

[0075] The expression of inflammatory cytokines and apoptosis-related genes by He CAP treatment in human gingival fibroblast-1 cells was observed, and the results are shown in Figures 5a to 5d.

[0076] In order to establish the manufacturing conditions of the helium plasma treatment solution, the mRNA expression of iNOS was observed by controlling the treatment time per second of the helium plasma and the flow rate of the helium gas that generates the plasma (standard liters per minute (slm) flow rate). Figure 5a shows the mRNA expression of iNOS measured for 0, 15, 30, 60, 120, and 180 seconds under the condition of a helium gas flow of 4 L per minute (4 slm, 4 L / min). In addition, the mRNA expression of iNOS was observed by measuring for 15 seconds each under the conditions of 0, 1, 2, 3, 4, and 5 slm of helium gas flow. From these results, the condition that minimizes the mRNA expression of iNOS by helium plasma is exposure to plasma for 15 seconds under the condition of a 4 slm helium gas flow. In Fig. 5b, in order to find the conditions for minimizing the mRNA expression of IL-1β, TNFα, and IL-6 cytokines, measurements were made for 0, 15, 30, 60, 120, and 180 seconds under the condition of 4 slm helium gas flow. As a result, the times when the expression of IL-1β, TNFα, and IL-6 mRNA decreased the most were 15 and 30 seconds, and when the helium gas flow was fixed at 15 seconds and the conditions of 0, 1, 2, 3, 4, and 5 slm were applied, the mRNA expression of cytokines was minimized under the 4 slm condition. Therefore, in order to find the conditions for suppressing inflammation of oral cells, the treatment time of helium CAP and the flow concentration of helium gas were controlled to observe the mRNA expression results of iNOS and IL-1β, TNFα, and IL-6 cytokines. From Fig. 5a and Fig. 5b, it can be confirmed that the expression of iNOS, IL-1β, TNFα, and IL-6 is significantly reduced by the helium plasma treatment solution obtained by treating for 15 seconds in a helium gas flow of 4 L / min.

[0077] Figure 5c shows the results of observing the expression of Bax, Atg5, and P62 mRNA by controlling the treatment time of the helium CAP and the flow concentration of helium gas under the same conditions as Figures 5a and 5b, and it was observed that P62 expression increased.

[0078] Figure 5d shows the results of observing protein expression related to increased inflammation and cell death by helium CAP treated for 15 seconds in a 4 L / min helium gas flow, i.e., the helium plasma treatment solution obtained in the example, and confirming the expression of P62, and confirming that the mRNA expression and protein expression of GPX4, which were reduced by Erastin treatment, were increased.

[0079] Experimental Example 6

[0080] The effects of He CAP treatment on GPX4 expression and mitochondrial membrane potential in human gingival fibroblast-1 cells were determined, and the results are shown in Figures 6a to 6c.

[0081] Figure 6a observed the mRNA expression of GPX4 as a major factor in antioxidant regulation. In Figures 5a to 5d, it was observed whether the helium plasma conditions performed to find conditions that reduce the expression of inflammatory cytokines affected the mRNA expression of GPX4. The mRNA expression of GPX4 measured for 0, 15, 30, 60, 120, and 180 seconds under the conditions of a helium gas flow of 4 L per minute (4 slm, 4 L / min) showed the highest expression at 15 seconds of exposure, and when the helium gas flow conditions of 0, 1, 2, 3, 4, and 5 slm were measured for 15 seconds each, the highest mRNA expression of GPX4 was observed at 3 L and 4 L. From these results, it can be seen that the condition that increases the mRNA expression of GPX4 is treatment with a helium plasma treatment solution obtained by treating for 15 seconds under a 4 slm helium gas flow. Figure 6b shows the results of confirming the reason for the reduction in inflammation and cell death by helium CAP treatment in relation to ferroptosis. It shows that the expression of GPX4 according to the progression of ferroptosis increased when treated for 15 seconds under the same conditions as the helium plasma treatment solution obtained in the example, that is, at a helium gas flow of 4 L / min. However, it can be seen that the expression of GPX4 in the cells disappears after 60 seconds.

[0082] Figure 6c shows the results of observing the mitochondrial membrane potential after time-dependent treatment with helium CAP at 4 L / min conditions. From 30 seconds onwards, the mitochondrial membrane potential decreases and apoptosis is confirmed (green fluorescence: JC-1 staining stains cells existing in a monomeric state in mitochondria with low membrane potential, indicating cell death). The graph on the right is a bar graph showing the intensity of green fluorescence, which indicates cell death.

[0083] Experimental Example 7

[0084] The effect of Helium CAP treatment on the expression of Erastin-induced inflammatory cytokines and apoptosis-related genes in human gingival fibroblast-1 cells was confirmed, and the results are shown in Figures 7a to 7g.

[0085] Figures 7a to 7e are the results confirming the inhibition of inflammation of oral cells by treatment with helium CAP, and it can be observed that the mRNA expression of iNOS, IL-1β, TNFα, IL-6, and COX-2 increased by treatment with erastin is reduced by the helium plasma treatment solution obtained by treatment with 4 L / min and 15 seconds of helium CAP. In particular, Figure 7e shows that Atg5 and P62 increased by treatment with erastin are reduced by helium CAP.

[0086] Figures 7f and 7g show that the increased ferroptosis by Erastin treatment is recovered by helium CAP, as observed through increased protein expression of GPX4 and increased ratio of GSH / GSSG, and this is observed as a result of decreased protein expression of NCOA4 resulting in decreased decomposition of iron ions.

[0087] Experimental Example 8

[0088] The effects of He CAP treatment on mitochondrial membrane potential and VDAC1 expression in human gingival fibroblast-1 cells were confirmed, and the results are shown in Figures 8a to 8d.

[0089] Figure 8a shows the results of JC-1 staining, which shows that the mitochondrial membrane potential decreased by Erastin treatment is restored by helium CAP treatment. In the figure, red indicates healthy cells. This is because healthy cells have high mitochondrial membrane potential, forming J-aggregates when stained with JC-1, resulting in red fluorescence.

[0090] Figures 8b to 8d show the results of observing the effect of helium CAP on the expression of VDAC1, which is located in the mitochondrial outer membrane and plays an important role in regulating the exchange of ions and metabolites between mitochondria and the cytoplasm. It can be seen through the expression of red fluorescence that the protein expression of VDAC1 increases when treated with helium CAP and increases significantly after 20 seconds. Figure 8c shows the protein expression of VDAC1 after treating the helium plasma treatment solution obtained after plasma exposure for 0, 15, 30, 60, and 120 seconds under a helium plasma 4 slm condition. It was found that the protein expression of VDAC1 increased significantly at 15 seconds and gradually decreased after 30 seconds. In addition, it can be seen that VDAC1 is reduced by erastin treatment, which is related to ferroptosis cell death and that helium CAP restores it.

[0091] Experimental Example 9

[0092] The response of VDAC1 to He CAP treatment in human gingival fibroblast-1 cells is shown in Figures 9a to 9e.

[0093] As shown in Figure 9a, cytosolic and mitochondrial ROS were measured separately to observe the intracellular response to helium CAP. The results showed that the rate of mitochondrial ROS reduction was higher when cytosolic ROS was increased by Erastin compared to when helium CAP was reduced. This confirms that helium CAP has a greater effect on mitochondrial recovery.

[0094] Figures 9b and 9c show the results of observing the mRNA and protein expression of VDAC1 in response to helium CAP treatment, showing that the expression of VDAC1 reduced by Erastin is restored by the helium plasma treatment solution.

[0095] Figure 9d confirms the status of mitochondria recovered by helium CAP treatment. That is, Rhod-2 is used to visualize calcium concentration within mitochondria by specifically binding calcium, and the recovery of mitochondrial function can be confirmed by an increase in red fluorescence.

[0096] Figure 9e confirms lipid peroxidation for the reduction of ferroptosis cell death by helium CAP treatment, and it can be seen that lipid peroxidation increased by Erastin is reduced by helium CAP treatment.

[0097] Although the present invention has been illustrated and described with reference to preferred embodiments as described above, it is not limited to the above-described embodiments, and various changes and modifications may be made by a person having ordinary skill in the art to which the invention pertains within a scope that does not depart from the spirit of the present invention.

Claims

1. A composition for preventing and treating oral soft tissue inflammation, comprising a helium plasma treatment solution as an active ingredient.

2. In paragraph 1, The above helium plasma treatment solution is obtained by setting the frequency and voltage to 30 kHz or more and 5 kV or more in a cold atmospheric plasma device to generate power, and then passing the helium plasma treatment solution through at least one of water, an aqueous solution, or an aqueous dispersion with 3 to 5 L / min of He (helium) gas and 40 to 60 SCCM of O2 (oxygen). A composition for preventing and treating inflammation of oral soft tissues.

3. In paragraph 2, A composition for preventing and treating oral soft tissue inflammation, characterized in that the plasma treatment by the low-temperature atmospheric pressure plasma device is performed for 10 to 60 seconds when the water, aqueous solution or aqueous dispersion is 5 ml.

4. In paragraph 3, A composition for preventing and treating oral soft tissue inflammation, characterized in that when the water, aqueous solution or aqueous dispersion is 5 ml, the frequency and voltage of the low-temperature atmospheric pressure plasma device are 40 kHz and 6 kV, the He (helium) gas is 4 L / min, the O2 (oxygen) is 50 SCCM, and the plasma treatment is performed for 15 seconds.

5. Oral cleaning composition containing a helium plasma treatment solution.

6. In paragraph 5, The above helium plasma treatment solution is characterized in that it is obtained by passing He (helium) gas at 3 to 5 L / min and O2 (oxygen) at 40 to 60 SCCM through at least one of water, an aqueous solution, or an aqueous dispersion after generating power by setting the frequency and voltage to 30 kHz or more and 5 kV or more in a cold atmospheric plasma device.

7. In paragraph 6, An oral hygiene composition characterized in that the plasma treatment by the low-temperature atmospheric pressure plasma device is performed for 10 to 60 seconds when the water, aqueous solution or aqueous dispersion is 5 ml.

8. In paragraph 7, An oral hygiene composition characterized in that when the water, aqueous solution or aqueous dispersion is 5 ml, the frequency and voltage of the low-temperature atmospheric pressure plasma device are 40 kHz and 6 kV, the He (helium) gas is 4 L / min, the O2 (oxygen) is 50 SCCM, and the plasma treatment is performed for 15 seconds.

9. In any one of paragraphs 5 to 8, An oral hygiene composition characterized in that the helium plasma treatment liquid is contained in an amount of 50% or less of the total composition volume.

10. A step of preparing any one liquid precursor selected from the group consisting of water, aqueous solution or aqueous dispersion; A step of generating power by setting the frequency and voltage to 30 kHz or higher and 5 kV or higher in a cold atmospheric plasma device; and A method for producing a composition for preventing and treating inflammation of oral soft tissue, comprising: a step of obtaining a helium plasma treatment solution by passing 3 to 5 L / min of He (helium) gas and 40 to 60 SCCM of O2 (oxygen) through the prepared liquid precursor, and performing plasma treatment using a low-temperature atmospheric pressure plasma device in which the power is generated; 11. In paragraph 10, A method for producing a composition for preventing and treating oral soft tissue inflammation, characterized in that plasma treatment using a low-temperature atmospheric pressure plasma device is performed for 10 to 60 seconds when the liquid precursor is 5 ml.

12. In paragraph 10, A method for producing a composition for preventing and treating oral soft tissue inflammation, characterized in that when the liquid precursor is 5 ml, the frequency and voltage of the low-temperature atmospheric pressure plasma device are 40 kHz and 6 kV, the He (helium) gas is 4 L / min, the O2 (oxygen) is 50 SCCM, and the plasma treatment is performed for 15 seconds.

13. A step of preparing any one liquid precursor selected from the group consisting of water, aqueous solution or aqueous dispersion; A step of generating power by setting the frequency and voltage to 30 kHz or higher and 5 kV or higher in a cold atmospheric plasma device; A step of obtaining a helium plasma treatment solution by passing He(helium) gas 3~5L / min and O2(oxygen) 40~60SCCM through the prepared liquid precursor and performing plasma treatment using a low-temperature atmospheric pressure plasma device in which the power is generated; and A method for manufacturing an oral hygiene composition, comprising the step of diluting the helium plasma treatment solution to less than 50% of the total composition volume.

14. In paragraph 13, A method for manufacturing an oral hygiene composition, characterized in that when the liquid precursor is 5 ml, plasma treatment using the low-temperature atmospheric pressure plasma device is performed for 10 to 60 seconds.

15. In paragraph 13, A method for manufacturing an oral hygiene composition, characterized in that when the liquid precursor is 5 ml, the frequency and voltage of the low-temperature atmospheric pressure plasma device are 40 kHz and 6 kV, the He (helium) gas is 4 L / min, the O2 (oxygen) is 50 SCCM, and the plasma treatment is performed for 15 seconds.

Images