WO2024085659A1 - Composition for preventing or treating oral disease, containing steamed ginseng berry extract as active ingredient - Google Patents

Abstract

The present invention relates to a composition for preventing or treating oral disease, containing a steamed ginseng berry extract as an active ingredient. Particularly, the present invention inhibits the expression of inflammatory cytokines of IL-6, IL-1β or TNF-α in periodontal ligament cells, induces differentiation of osteoblasts and exhibits antioxidant activity, and thus can be effectively used in the prevention and treatment of oral diseases.

Description

Composition for preventing or treating oral diseases containing steamed ginseng fruit extract as an active ingredient

The present invention relates to a composition for preventing or treating oral diseases containing steamed ginseng fruit extract as an active ingredient.

Oral diseases are diseases that occur within the oral cavity, and can affect anyone, including periodontal disease (periodontal disease) and dental caries (cavities), and can cause pain, masticatory dysfunction, destruction of periodontal tissue, bad breath, and coldness. It is known to be the main factor that causes various symptoms and causes tooth loss.

Periodontal disease is a chronic disease with a very high prevalence, affecting more than 7 out of 10 adults. Periodontal disease is largely divided into gingivitis and periodontitis. When the inflammation is limited to the gums, or soft tissue, it is called gingivitis, and when this inflammation progresses to the gums and gum bones, it is called periodontitis.

Periodontitis, which is caused by complex causes caused by periodontitis bacteria such as gingivalis, causes inflammation of the tooth-supporting structure and simultaneously causes destruction of the periodontal ligament and loss of alveolar bone, so it has complex antibacterial and anti-inflammatory properties, and promotion of osteoblast differentiation. A treatment method is needed. Currently, the most effective treatment for periodontitis is a combination of drug treatment and surgery, but this treatment method has a poor treatment prognosis and can cause pain and side effects in patients. In addition, drugs used to treat oral diseases, such as antibacterial and anti-inflammatory drugs, are not treatments for fundamental periodontal disease and are accompanied by side effects due to long-term use. Accordingly, there is a need to secure natural materials and discover new herbal medicine/natural materials for the prevention and treatment of periodontitis.

Meanwhile, ginseng fruit is a rare fruit that grows from May to July in 4-year-old ginseng. Previously, it was simply recognized as a ginseng seed, but through clinical trials and research results in the medical community in the United States, Japan, and Korea, it was revealed that it contains a large amount of ginsenoside, a saponin of ginseng. However, due to the nature of ginseng fruits, which wither within a day after harvesting, they require intensive harvesting in a short period of time, making cultivation and management difficult.

As a prior art, Korean Patent No. 10-1394832 describes a composition for suppressing oral bacteria containing ginseng fruit extract as an active ingredient, and Korean Patent Publication No. 10-2016-00065472 describes the pulp and skin of ginseng fruit. A seed processing method of ginseng fruit is disclosed in which the seed is removed and only the seeds are roasted to produce a fine powder. However, research is needed to increase the utilization rate of ginseng fruits and enhance their medicinal properties through appropriate processing of ginseng fruits to discover new uses.

The purpose of the present invention is to provide a composition for preventing or treating oral diseases containing steamed ginseng fruit extract as an active ingredient.

In order to solve the above problems, the present invention provides a composition for preventing or treating oral diseases containing steamed ginseng fruit extract as an active ingredient.

The steamed ginseng fruit extract can be used in a concentrated form, and the steamed ginseng fruit concentrate can be used in the form of a powder obtained by freeze-drying the steamed ginseng fruit concentrate or the steamed ginseng fruit concentrate.

The steamed ginseng fruit extract is an extraction concentrate of ginseng fruit water, lower alcohol (methanol, ethanol, propanol, butanol), lower alcohol aqueous solution, or organic solvent (chloroform, ethyl acetate, dichloromethane, dimethyl sulfoxide, hexane, etc.) It contains a steamed ginseng fruit concentrate steamed at 100-130°C and 0.11-15 MPa for 4-12 hours, and the extraction solvent for the ginseng fruit is preferably water or lower alcohol (methanol, ethanol, propanol, butanol). , an aqueous solution of lower alcohol, more preferably 30 to 99% ethanol.

The steamed ginseng fruit extract may be obtained by steaming 50% alcohol extract of ginseng fruit at a temperature of 100 to 130°C and a pressure of 0.11 to 15 MPa for 4 to 12 hours.

In the present invention, the steamed ginseng fruit extract may be a steamed ginseng fruit concentrate steamed by microwave treatment at high temperature and high pressure. The steamed ginseng fruit concentrate steamed by microwave treatment at high temperature and high pressure is extracted from ginseng fruit water, lower alcohol (methanol, ethanol, propanol, butanol), or lower alcohol aqueous solution at 300 to 400 W, 2.5 to 3.5 MPa. , It is a steamed ginseng fruit concentrate steamed by microwave treatment at 110~130℃, and high temperature and pressure for 30~90 minutes.

In the present invention, the steamed ginseng fruit concentrate steamed at high temperature and pressure or by microwave treatment at high temperature and pressure contains the active ingredients 0.5 to 2% by weight of ginsenoside Rg6, 0.5 to 2% by weight of ginsenoside Rg4, and ginsenoside. It may contain 0.5 to 5% by weight of side Rh4.

In the present invention, the oral disease may be dental caries, periodontitis, or gingivitis.

In the present invention, “dental cavities” refers to an infectious disease caused by bacteria living on the surface of teeth. Also called cavities, it is specifically a milky-white, translucent, hard substance that covers the surface of the head of the tooth and protects the dentin of the tooth. It occurs when tooth enamel or enamel is damaged by acids produced during the decomposition of sugar, starch, etc. by oral pathogens that live in the mouth.

In the present invention, “periodontitis” is a disease in which inflammation occurs in the gingiva, periodontal ligament, and bone tissue that support the teeth.

In the present invention, “gingivitis” is a form of periodontal disease limited to the gums, i.e., soft tissue.

Bacteria that cause periodontal diseases such as periodontitis or gingivitis, specifically Actinobacillus actinomycetemcomitans , Porphyromonas gingivalis , and Tannerella forsythia. , Treponema denticola and Fusobacterium nucleatum may be one or more species selected from the group consisting of, and more specifically, Porphyromonas gingivalis.

The Porphyromonas gingivalis is a type of Bacteroide soft bacteria, a Gram-negative bacterium, an anaerobic bacterium, and is found in the oral cavity where periodontal disease occurs, and is also found in the upper gastrointestinal tract, respiratory tract, and colon. In patients with chronic periodontal disease, collagen is decomposed by the collagenase enzyme of the bacteria, and the bacteria can invade gingival fibroblasts and survive even under high concentrations of antibiotics. Additionally, the bacteria can invade gingival epithelial cells and survive for a long time.

The composition for preventing or treating oral diseases containing the steamed ginseng fruit extract of the present invention as an active ingredient has an antibacterial effect against the causative bacteria that cause periodontal disease, and in particular has an antibacterial effect against the Porphyromonas gingivalis strain. It is characterized by having

The ginseng fruit of the present invention is Korean ginseng ( Panax ginseng ), American ginseng ( Panax quinquefolius ), Panax notoginseng ( Panax notoginseng ), Panax japonicus , trifoliate ginseng (American ginseng; Panax trifolius ), and Himalayan ginseng ( Panax ). pseudoginseng ) or Vietnamese ginseng ( Panax vietnamensis ) fruits can be used, but are not limited to these.

The composition for preventing or treating oral diseases containing the steamed ginseng fruit extract of the present invention as an active ingredient can inhibit the expression of inflammatory cytokines such as IL-6, IL-1β or TNF-α in periodontal ligament cells. In addition, the composition can induce differentiation of osteoblasts from periodontal ligament cells, and the induction of differentiation of osteoblasts increases the expression of alkaline phosphatase (ALP) or osteocalcin (OCN) or matrix metalloproteinase. It may be inhibiting the expression of matrix metalloproteinase (MMP).

The composition for preventing or treating oral diseases containing the steamed ginseng fruit extract of the present invention as an active ingredient can prevent or treat oral diseases through antioxidant activity in periodontal ligament cells.

The antioxidant activity can be shown by increasing the expression of superoxide dismutase (SOD), catalase (CAT), or glutathione peroxidase (GPx) in periodontal ligament cells, etc., and reactive oxygen species (ROS) ) inhibits the production of

The composition according to the present invention can be provided as a pharmaceutical composition and can be formulated in a suitable form with a commonly used pharmaceutically acceptable carrier. “Pharmaceutically acceptable” refers to a composition that is physiologically acceptable and does not typically cause allergic or similar reactions such as gastrointestinal upset, dizziness, etc. when administered to humans. In addition, the composition can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injection solutions according to conventional methods.

Preferably, the composition of the present invention is in the form of one or more selected from the group consisting of toothpaste, mouthwash, mouthwash, gum, candy, oral spray, oral gel, oral ointment, oral varnish, gum massage cream, and oral patch. It can be formulated as:

Carriers, excipients, and diluents that may be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, and methyl. It may include, but is not limited to, cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl paraoxybenzoate, propyl paraoxybenzoate, talc, magnesium stearate, and mineral oil. When formulated, it is prepared using diluents or excipients such as commonly used fillers, stabilizers, binders, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations include the ginsenoside of the present invention with at least one excipient, such as starch, microcrystalline cellulose, sucrose or It is prepared by mixing lactose, low-substituted hydroxypropyl cellulose, and hypromellose. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. . Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurel, glycerol, gelatin, etc. can be used. In order to formulate a formulation for parenteral administration, the ginsenoside or a pharmaceutically acceptable salt thereof is sterilized or added with preservatives, stabilizers, wetting agents or emulsification accelerators, auxiliaries such as salts or buffers for osmotic pressure adjustment, and other therapeutic agents. It can be prepared as a solution or suspension by mixing it in water with useful substances, and it can be prepared in ampoule or vial unit dosage form.

The pharmaceutical composition can be administered to mammals such as rats, livestock, and humans through various routes. All modes of administration are contemplated, for example, oral, rectal or by intravenous, intramuscular, subcutaneous, intrathecal or intracerebrovascular injection. The dosage is determined by the age, gender, and weight of the subject to be treated, the specific disease or pathological state to be treated, the severity of the disease or pathological state, administration time, route of administration, absorption, distribution and excretion rate of the drug, types of other drugs used, and the prescriber's It will vary depending on judgment, etc. Dosage determinations based on these factors are within the level of one skilled in the art, and dosages generally range from 0.01 mg/kg/day to approximately 5000 mg/kg/day. A more preferred dosage is 1 mg/kg/day to 500 mg/kg/day. Administration may be administered once a day, or may be administered in several divided doses. The above dosage does not limit the scope of the present invention in any way.

In another aspect, the present invention provides a health functional food for preventing or improving oral diseases containing a steamed ginseng fruit concentrate.

The health functional food refers to food manufactured or processed using raw materials or ingredients with useful functionality, and includes, for example, health supplements, functional foods, nutrients, supplements, etc.

The steamed ginseng fruit concentrate is preferably 0.001% by weight to 50% by weight, more preferably 0.001% by weight to 40% by weight, and most preferably 0.001% by weight to 30% by weight, based on the total weight of the entire health functional food. It can be added. The above addition amount does not limit the scope of the present invention in any way.

The health functional food of the present invention includes forms such as tablets, capsules, pills, and liquids. Foods to which the ginsenoside of the present invention can be added include, for example, various foods, beverages, gum, tea, and vitamins. There are combination drugs, etc.

The present invention relates to a composition for preventing or treating oral diseases containing steamed ginseng fruit extract as an active ingredient, and specifically, inhibits the expression of inflammatory cytokines such as IL-6, IL-1β or TNF-α in periodontal ligament cells. It induces the differentiation of osteoblasts and exhibits antioxidant activity, making it useful for the prevention and treatment of oral diseases.

Figure 1 is a graph showing the effect of steamed ginseng fruit concentrate and steamed ginseng fruit concentrate powder according to the present invention on the expression of inflammatory cytokines in human periodontal ligament cells treated with PS-LPS.

Figure 2 is a graph showing the effect of the steamed ginseng fruit concentrate according to the present invention on osteoblast differentiation in human periodontal ligament cells treated with PS-LPS.

Figure 3 is a graph showing the effect of the steamed ginseng fruit concentrate powder according to the present invention on osteoblast differentiation in human periodontal ligament cells treated with PS-LPS.

Figure 4 is a graph showing the effect on the antioxidant activity of the steamed ginseng fruit concentrate according to the present invention in human periodontal ligament cells treated with PS-LPS.

Figure 5 is a graph showing the effect on the antioxidant activity of the steamed ginseng fruit concentrate powder according to the present invention in human periodontal ligament cells treated with PS-LPS.

Figure 6 is a result showing the MIC test against Porphyromonas gingivalis of the steamed ginseng fruit concentrate according to the present invention.

Figure 7 shows the results of an MIC test against Porphyromonas gingivalis of the steamed ginseng fruit concentrate powder according to the present invention.

Figure 8 shows the results of the MBC test for Porphyromonas gingivalis of the steamed ginseng fruit concentrate according to the present invention.

Figure 9 is a result showing the MBC test for Porphyromonas gingivalis of the steamed ginseng fruit concentrate powder according to the present invention.

Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms, and the content introduced here is provided to sufficiently convey the idea of the present invention.

<Example 1. Preparation of steamed ginseng fruit concentrate steamed at high temperature and high pressure>

600kg of 50% alcohol was added to 50kg of ginseng fruit and extracted twice for 6 hours at 85°C. The extract was then filtered and concentrated to 70 brix. This was steamed at a temperature of 121°C and a pressure of 0.13 MPa for 4 hours to obtain a steamed ginseng fruit concentrate. Table 1 below summarizes the types and contents of ginsenoside components in the steamed ginseng fruit concentrate concentrated to 70 brix.

<Example 2. Preparation of steamed ginseng fruit concentrate by microwave steaming at high temperature and high pressure>

600kg of 50% alcohol was added to 50kg of ginseng fruit and extracted twice for 6 hours at 85°C. The extract was then filtered and concentrated to 70 brix. This was irradiated with a microwave at 130°C for 60 minutes under conditions of 400 W and 3.0 MPa to obtain a steamed ginseng fruit concentrate subjected to microwave steaming. Table 1 below summarizes the types and contents of ginsenoside components in the steamed ginseng fruit concentrate that was medium-ripened by irradiating microwaves at high temperature and pressure.

<Comparative Example 1. Preparation of non-steamed ginseng fruit concentrate>

600kg of 50% alcohol was added to 50kg of ginseng fruit and extracted twice for 6 hours at 85°C. Then, the extract was filtered and concentrated to 70 brix to obtain ginseng fruit concentrate. Table 1 below summarizes the types and contents of ginsenoside components in ginseng fruit concentrate concentrated to 70 brix without undergoing a steaming process.

Ginsenoside	Comparative Example 1. Ginseng fruit concentrate before steaming Ginsenoside content (mg/g)	Example 1. Ginsenoside content in ginseng fruit concentrate steamed at high temperature and pressure (mg/g)	Example 2. Ginseng fruit concentrate processed by microwave steaming at high temperature and pressure Ginsenoside content (mg/g)
Re+Rg1	64.93	5.61	0
Rb1	21.97	4.88	0
Rc	17.64	5.71	0
Rb2	15.68	6.71	0
Rd	16.23	13.21	0
Rg2-S	2.87	14.19	7.37
Rh1-S	0	1.37	3.04
Rg2-R	0	5.01	3.98
Rh1-R	0	1.03	2.35
Rg6	0	10.38	10.15
Rg4	0	11.31	10.99
Rh4	0	11.98	30.08
Rg3-S	0	7.78	9.18
Rg3-R	0	7.18	9.33
Rk1	0	6.03	14.42
Rg5	0	8.66	19.44
total amount	139.32	121.04	120.33

Table 1 above summarizes the types of ginsenoside components and their contents according to the treatment after concentrating ginseng fruit. While the ginseng fruit concentrate before steaming (Comparative Example 1) contains a lot of ginsenoside Re+Rg1, it is concentrated at high temperature and pressure. In the steamed ginseng fruit concentrate (Example 1), the ginsenoside components (Re+Rg1, Rb1, Rc, Rb2, Rd) present before steaming were reduced, and low molecular weight ginsenosides Rg6, Rg4, Rh4, Rg2, Rh1, Rg3, Rk1, Rg5, etc. were produced, and in particular, it was confirmed that ginsenoside Rg6, Rg4, and Rh4 components were significantly increased. In addition, in the ginseng fruit concentrate (Example 2) that was microwave-steamed at high temperature and pressure, the ginsenoside components that existed before steaming were completely destroyed, and the steamed ginseng fruit concentrate with significantly enhanced ginsenoside Rg6, Rg4, and Rh4 components was obtained. I was able to make it.

Accordingly, in the subsequent experiments, the ginseng fruit concentrate steamed at high temperature and high pressure according to Example 2 was used, and the steamed ginseng fruit concentrate steamed at high temperature and high pressure was frozen to facilitate storage and distribution. This was conducted using dried steamed ginseng fruit concentrate powder.

<Experimental Example 1. Confirmation of anti-inflammatory effect of steamed ginseng fruit concentrate>

Representative periodontal pathogens among oral bacteria include Porphyromonas gingivalis, Treponema denticola, Prevotella intermedia , and Aggregatibacter actinomycetemcomitans . The lipopolysaccharides (LPS) endotoxins or metabolites formed by these microorganisms increase the secretion of proinflammatory cytokines from tissues and immune cells. These pro-inflammatory cytokines damage periodontal tissue and destroy connective tissue by inducing enzyme activity such as matrix metalloproteinase (MMP). Therefore, inhibition of these pro-inflammatory cytokines in periodontitis is an important treatment strategy for suppressing periodontitis. Accordingly, the effect of steamed ginseng fruit concentrate on regulating representative inflammatory cytokines of periodontal inflammation in human periodontal ligament cells stimulated with Porphyromonas gingivalis -LPS (PG-LPS) was evaluated.

Human periodontal ligament cells were donated and used by Kyungpook National University College of Dentistry. Human preiodontal ligament (HPDL) cells were cultured in a 96-well plate at a concentration of 5×10 ³ cell/mL at a temperature of 37°C and 5% CO _{2 .} Cultured for 24 hours under culture conditions. Afterwards, the steamed ginseng fruit concentrate or steamed ginseng fruit concentrate powder was treated at concentrations of 0, 50, 100, 150, and 200 μg/mL and incubated at a temperature of 37°C and 5% CO _2. Cultured for 6 hours under culture conditions. After stimulation with PG-LPS for 6 hours, the total cDNA extracted using TRIzol/chloroform reagent was quantified using nanodrop, and the inflammatory cytokine IL- was extracted using PrimeScript-RT reagent kit (SYBR Premix Ex Taq). 6, IL-1β and TNFα genes were amplified and analyzed. The threshold (Ct) value of each target gene was normalized to the level of GAPDH, a housekeeping gene, and the results are shown in Figure 1.

As shown in Figure 1, both steamed ginseng fruit concentrate and steamed ginseng fruit concentrate powder suppressed the expression of IL-6, IL-1β, and TNFα in a concentration-dependent manner. In particular, steamed ginseng fruit concentrate powder significantly down-regulated the expression of IL-6 at a lower concentration than steamed ginseng fruit concentrate. In this way, it was confirmed that steamed ginseng fruit concentrate or steamed ginseng fruit concentrate powder can effectively downregulate inflammatory mediators and cytokines increased by PG-LPS in human periodontal ligament cells, thereby exhibiting an inhibitory effect on periodontal inflammation.

<Experimental Example 2. Confirmation of osteoblast differentiation inducing effect of steamed ginseng fruit concentrate>

If initial gingivitis becomes chronic inflammation and becomes prolonged, not only the gums but also the alveolar bone below the gums erode, which is called periodontitis. Once alveolar bone is lost, it is difficult to restore it to its original state, and as periodontitis progresses, alveolar bone loss increases. The expression of alkaline phosphatase (ALP) and osteocalcin (OCN), which are used as indicators of the activity of osteocytes, are known to be important factors in the formation of alveolar bone in human periodontal ligament cells, and thus the differentiation of these osteocytes lost due to periodontitis. Restoration of activity indicators has become another important treatment strategy in the treatment of periodontitis. Therefore, the osteoblastic differentiation capacity of steamed ginseng fruit concentrate was evaluated in human periodontal ligament cells stimulated with PG-LPS, and its effect on the regeneration of human periodontal ligament cells was evaluated.

2.1 Evaluation of osteoblast differentiation induction effect of human periodontal ligament cells

Human periodontal ligament (HPDL) cells were grown in a 24-well plate at a concentration of 5×10 ² cell/mL at a temperature of 37°C and 5% CO ₂ Cultured for 24 hours under culture conditions. After removing the culture medium, the culture medium was replaced with α-MEM containing 10% fetal bovine serum, 100U/ml penicilline, 100 μg/mL streptomycin, 50 μg/mL ascorbic acid, and 10 mM β-glycerophosphate to promote osteoblast differentiation. induced. After inducing osteoblastic differentiation and treating steamed ginseng fruit concentrate (EX) or steamed ginseng fruit concentrate powder (EX powder) at concentrations of 0, 50, 100, 150, and 200 μg/mL, PG-LPS (1 μg/mL ) After treatment, temperature of 37 ℃, 5% CO ₂ Cultured for 14 days under culture conditions. After completion of incubation, cells were fixed with 4% formaldehyde for 30 minutes, stained with 0.1% Alizarin Red S (pH 4.3) for 1 hour, washed with deionized water for 30 minutes at room temperature, and then observed as mineralized nodules under a microscope. ) After confirming the formation, it was dissolved in 400 uL of 10% cetylpyridinium chloride and the absorption (570 nm) was measured and shown in Figures 2 and 3.

As shown in Figures 2 and 3, both steamed ginseng fruit concentrate (EX) and steamed ginseng fruit concentrate powder (EX powder) increased the osteoblast differentiation ability of human periodontal ligament cells, whose osteoblast differentiation was suppressed by PG-LPS. It effectively induced mineralization of human periodontal ligament cells.

2.2 Evaluation of the effect of inducing osteoblastic differentiation-specific gene expression in human periodontal ligament cells

Human preiodontal ligament (HPDL) cells were cultured in a 6-well plate at a concentration of 5×10 ⁵ cells/mL at a temperature of 37°C and 5% CO _{2 .} Cultured for 24 hours under culture conditions. Afterwards, steamed ginseng fruit concentrate (EX) or steamed ginseng fruit concentrate powder (EX powder) was treated for 6 hours at concentrations of 0, 50, 100, 150, and 200 μg/mL, followed by PG-LPS (1 μg/mL). was stimulated for 12 hours. Afterwards, the total cDNA extracted using TRIzol/chloroform reagent was quantified using nanodrop, and the genes of alp, ocn, and mmp9 were amplified and analyzed using PrimeScript-RT reagent kit (SYBR Premix Ex Taq). Afterwards, the threshold (Ct) value of each gene was normalized to the GAPDH level and shown in Figures 2 and 3.

As shown in Figures 2 and 3, both steamed ginseng fruit concentrate (EX) and steamed ginseng fruit concentrate powder (EX powder) concentration-dependently expressed the expression of alp and ocn genes, which are indicators of bone activity lost due to PG-LPS. increased. In addition, it increased the osteoblastic differentiation ability of human periodontal ligament cells whose osteoblast differentiation was suppressed and effectively induced mineralization of human periodontal ligament cells. In addition, the expression of MMP9 (Matrix metalloproteinase 9) enzyme, which destroys gum connective tissue, was suppressed in a concentration-dependent manner.

<Experimental Example 3. Confirmation of antioxidant effect of steamed ginseng fruit concentrate>

Active oxygen generated during the cause or progression of periodontitis damages periodontal tissue, causing periodontal disease or worsening ongoing periodontal disease. Free radicals, known as harmful oxygen, form lipid peroxides, so when the lipid peroxides of these free radicals are not properly eliminated, they cause various inflammatory reactions, including periodontitis. Therefore, suppressing the production of oxidative stress through antioxidant action is known to be an important mechanism for improving periodontitis. Accordingly, in human periodontal ligament cells stimulated with PG-LPS, steamed ginseng fruit concentrate (EX) or steamed ginseng fruit concentrate powder (EX powder) removed reactive oxygen species (ROS) and produced antioxidant enzymes (Superoxide Dismutase, SOD). , the effect on the level of catalase (CAT) production was evaluated.

3.1 Antioxidant activity evaluation

Human preiodontal ligament (HPDL) cells were cultured in a 6-well plate at a concentration of 5×10 ³ cell/mL at a temperature of 37°C and 5% CO _{2 .} Cultured for 24 hours under culture conditions. Afterwards, 0, 50, 100, 150, and 200 μg/mL of steamed ginseng fruit concentrate (EX) or steamed ginseng fruit concentrate powder (EX powder) and PG-LPS (1 μg/mL) were treated at a temperature of 37°C, 5% _CO2 Cultured for 2 hours under culture conditions. After completion of incubation, cells were treated with medium supplemented with 10 μM dichlorofluorescin diacetate (DCF-DA) for 30 min at a temperature of 37°C and 5% CO _2. Cultured under culture conditions, the cultured cells were washed with PBS, treated with 1% trypsin-EDTA solution, harvested, washed again with PBS, incubated for 30 min, and incubated with the Incucyte® Live-Cell analysis system. The amount of change in ROS was measured and shown in Figures 4 and 5.

As shown in Figures 4 and 5, both steamed ginseng fruit concentrate (EX) and steamed ginseng fruit concentrate powder (EX powder) reduced the increased production of reactive oxygen species (ROS) due to PG-LPS in a concentration-dependent manner. These active oxygen species cause periodontitis by causing non-selective and irreversible destruction of cellular components such as phospholipids, proteins, sugars, and DNA. Therefore, it can be seen that steamed ginseng fruit concentrate (EX) or steamed ginseng fruit concentrate powder (EX powder) improves periodontitis by measuring the effect of suppressing oxidative stress through antioxidant action.

3.2 Evaluation of antioxidant enzyme protection effect

Superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) are enzymes that catalyze antioxidant efficacy and form the first line of defense against cell damage caused by free radicals. Compose. In the present invention, it was determined whether steamed ginseng fruit concentrate (EX) or steamed ginseng fruit concentrate powder (EX powder) affects the protein expression of these antioxidant enzymes in human periodontal ligament cells stimulated with PG-LPS.

Human periodontal ligament (HPDL) cells were grown in a 6-well plate at a concentration of 5×10 ⁵ cells/mL at a temperature of 37°C and 5% CO _{2 .} After culturing for 24 hours under culture conditions, treatment with steamed ginseng fruit concentrate (EX) or steamed ginseng fruit concentrate powder (EX powder) at concentrations of 0, 50, 100, 150, and 200 μg/mL for 6 hours and then treated with PG-LPS. Stimulation was performed for 12 hours. Then, after lysis with cell lysis buffer (RIPA buffer), centrifugation at 23,000 20 μg was loaded into each well and transferred to a PVDF membrane. Afterwards, after blocking with PVDF membrane skim milk at room temperature for 1 hour, the primary antibodies of SOD, CAT, and GPx were each reacted at 4°C for 24 hours, and then the secondary antibodies for each corresponding primary antibody were sequentially reacted. Proteins activated by binding to each antibody were measured using an ECL Western blotting detection imaging system, and the results are shown in Figures 4 and 5.

As shown in Figures 4 and 5, steamed ginseng fruit concentrate (EX) or steamed ginseng fruit concentrate powder (EX powder) contains the antioxidant enzymes SOD, CAT and GPx lost from human periodontal ligament cells stimulated with PG-LPS. Protein expression was upregulated in a concentration-dependent manner. These research results confirmed that steamed ginseng fruit concentrate (EX) or steamed ginseng fruit concentrate powder (EX powder) can suppress oxidative stress through antioxidant activity.

<Experimental Example 4. Confirmation of antibacterial effect of steamed ginseng fruit concentrate>

The main cause of periodontitis is bacteria present in the oral cavity, and periodontal disease occurs due to inflammatory progression due to the reaction of periodontal tissue due to mixed infection caused by several types of bacteria present in plaque. Among these bacteria, Porphyromonas gingivalis, which belongs to the red complex, is known to be the most important cause of periodontal disease. P. gingivalis is a Gram-negative anaerobic bacterium that increases in number in the lesions of patients with periodontal disease, and is often found in the crevicular epithelium as periodontal disease progresses. P. gingivalis produces metabolites or toxins in the process of attaching to or infiltrating host tissues and cells, causing direct harm to periodontal tissue, but is also caused by cytokines produced in cells that react with P. gingivalis , and cytokines. Substances produced by reacting host cells further destroy periodontal tissue, thereby accelerating periodontitis. Therefore, we investigated the antibacterial effect of steamed ginseng fruit concentrate against P. gingivalis, a representative bacteria causing periodontitis.

4.1 Evaluation of minimum inhibitory concentration of steamed ginseng fruit concentrate

Minimum Inhibitory Concentration (MIC) is the most basic indicator for measuring antibacterial activity and is the minimum test substance concentration that can inhibit the growth of microorganisms in an in vitro bacterial susceptibility test.

P. gingivalis (KCTC 5352) The strain was distributed from the Biological Resources Center of the Jeonbuk Branch of the Korea Research Institute of Bioscience and Biotechnology. After injecting into TSA (Tryptic Soy Agar) hemin menadione medium at a concentration of 1 × 10 ⁵ [CFU]/mL, steamed ginseng fruit concentrate (EX) or steamed ginseng fruit concentrate powder (EX powder) was added to 0, Treated at concentrations of 2.5, 5, 10, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, and 220 μg/mL and cultured under anaerobic conditions at 37°C. After completion of the culture, the MIC was analyzed by analyzing the absorbance at a wavelength of 600 nm using a microplate analyzer, and the results are shown in Figures 6 and 7.

As shown in Figures 6 and 7, the growth of P. gingivalis was not observed from 160 μg/mL for steamed ginseng fruit concentrate (EX) and 160 μg/mL for steamed ginseng fruit concentrate powder (EX powder), so that the steamed ginseng fruit The minimum inhibitory concentration (MIC) of concentrate (EX) and steamed ginseng fruit concentrate powder (EX powder) against P. gingivalis was 160 μg/mL and 180 μg/mL, respectively.

4.2 Evaluation of minimum sterilization concentration of steamed ginseng fruit concentrate

If the Minimum Inhibitory Concentration (MIC) refers to the lowest concentration of an antibacterial agent necessary to inhibit the visible growth of bacteria, the Minimum Bicteriocidal Concentration (MBC) is the minimum concentration of an antibacterial agent that kills bacteria.

In Example 4.1, samples from the 160 to 220 μg/mL treatment group were re-cultivated to confirm the minimum bactericidal concentration (MBC) of the steamed ginseng fruit concentrate (EX) and the steamed ginseng fruit concentrate powder (EX powder) against P. gingivalis. And the results are shown in Figures 8 and 9. As shown in Figures 8 and 9, it was confirmed that the strain did not grow from 180 μg/mL in both steamed ginseng fruit concentrate (EX) and steamed ginseng fruit concentrate powder (EX powder). Accordingly, it was confirmed that the minimum bactericidal concentration (MBC) of steamed ginseng fruit concentrate (EX) and steamed ginseng fruit concentrate powder (EX powder) against P. gingivalis was the same at 180 μg/mL.

As described above, steamed ginseng fruit concentrate (EX) and steamed ginseng fruit concentrate powder (EX powder) were confirmed to exhibit antibacterial and sterilizing effects against P. gingivalis strains, the main causative bacteria causing periodontal disease, and these steamed ginseng fruits It is believed that the antibacterial and sterilizing effects of concentrate (EX) and steamed ginseng fruit concentrate powder (EX powder) will play an important role in controlling periodontitis along with the anti-inflammatory, osteoblast differentiation inducing, and antioxidant effects identified above.

<Formulation Example 1. Preparation of tablets>

20 g of the steamed ginseng fruit concentrate of the present invention was mixed with 175.9 g of lactose, 180 g of potato starch, and 32 g of colloidal silicic acid. A 10% gelatin solution was added to this mixture, then pulverized and passed through a 14 mesh sieve. This was dried and 160g of potato starch, 50g of talc, and 5g of magnesium stearate were added to make the resulting mixture into tablets.

<Formulation Example 2. Preparation of powder>

20 g of the steamed ginseng fruit concentrate of the present invention was mixed with 50 g of dextrin, then the ingredients were mixed and spray dried according to a conventional powder manufacturing method to prepare a powder.

<Formulation example 3. Preparation of capsule>

According to the present invention, 100 mg of steamed ginseng fruit concentrate, 100 mg of corn starch, 100 mg of lactose, and 2 mg of magnesium stearate are mixed, then the above ingredients are mixed according to a typical capsule manufacturing method and filled into a gelatin capsule to prepare a capsule. did.

<Formulation example 4. Manufacturing of health functional food>

20g of steamed ginseng fruit concentrate of the present invention, appropriate amount of vitamin mixture, 70㎍ of vitamin A acetate, 1.0㎎ of vitamin E, 0.13㎎ of vitamin B1, 0.15㎎ of vitamin B2, 0.5㎎ of vitamin B6, 0.2㎎ of vitamin B12, 10㎎ of vitamin C, biotin 10㎍, nicotinic acid amide 1.7㎎, folic acid 50㎍, calcium pantothenate 0.5㎎, appropriate amount of mineral mixture, ferrous sulfate 1.75㎎, zinc oxide 0.82㎎, magnesium carbonate 25.3㎎, potassium phosphate monobasic 15㎎, calcium phosphate 255 ㎎, 90㎎ potassium citrate, 100㎎ calcium carbonate, and 24.8㎎ magnesium chloride were mixed to form granules, but it can be manufactured by modifying it into various dosage forms depending on the purpose. In addition, the composition ratio of the above vitamin and mineral mixture may be modified arbitrarily, and the above ingredients may be mixed according to a typical health functional food manufacturing method.

<Formulation example 5. Manufacturing of health functional beverage>

A beverage was prepared by mixing 1 g of the steamed ginseng fruit concentrate of the present invention, 0.1 g of citric acid, 100 g of fructooligosaccharide, and 900 g of purified water and stirring, heating, filtering, sterilizing, and refrigerating according to a typical beverage production method.

Claims (12)

1. A composition for preventing or treating oral diseases containing steamed ginseng fruit extract as an active ingredient.
2. According to paragraph 1,

   The steamed ginseng fruit extract is a composition for preventing or treating oral diseases, characterized in that 50% alcohol extract of ginseng fruit is steamed for 4 to 12 hours at a temperature of 100 to 130 ℃ and a pressure of 0.11 to 15 MPa.
3. According to paragraph 1,

   Oral disease, characterized in that the steamed ginseng fruit extract is microwave-steamed by irradiating 50% alcohol extract of ginseng fruit with a microwave at 110-130°C for 30-90 minutes under conditions of 300-400 W and 2.5-3.5 MPa. Composition for prevention or treatment.
4. According to paragraph 1,

   The steamed ginseng fruit extract is a composition for preventing or treating oral diseases, characterized in that it contains 0.5 to 2% by weight of ginsenoside Rg6, 0.5 to 2% by weight of ginsenoside Rg4, and 0.5 to 5% by weight of ginsenoside Rh4. .
5. According to any one of claims 1 to 4,

   A composition for preventing or treating oral diseases, wherein the oral diseases are dental caries, periodontitis, or gingivitis.
6. According to any one of claims 1 to 4,

   The composition is a composition for preventing or treating oral diseases, characterized in that it has an antibacterial effect against Porphyromonas gingivalis strains.
7. According to any one of claims 1 to 4,

   The composition is a composition for preventing or treating periodontitis, characterized in that it inhibits the expression of inflammatory cytokines IL-6, IL-1β or TNF-α in periodontal ligament cells.
8. According to any one of claims 1 to 4,

   The composition is a composition for preventing or treating periodontitis, characterized in that it induces differentiation of osteoblasts in periodontal ligament cells.
9. According to clause 8,

   Periodontitis, characterized in that the induction of differentiation of osteoblasts involves increasing the expression of alkaline phosphatase (ALP) or osteocalcin (OCN) or suppressing the expression of matrix metalloproteinase (MMP) Composition for prevention or treatment.
10. According to any one of claims 1 to 4,

    The composition is a composition for preventing or treating periodontitis, characterized in that it increases the expression of superoxide dismutase (SOD), catalase (CAT), or glutathione peroxidase (GPx) in periodontal ligament cells. .
11. According to any one of claims 1 to 4,

    The composition is an oral application characterized by one or more selected from the group consisting of toothpaste, mouthwash, mouthwash, gum, candy, oral spray, oral gel, oral ointment, oral varnish, gum massage cream, and oral patch. Composition for preventing or treating disease.
12. A health functional food comprising a composition for preventing or treating periodontitis comprising the steamed ginseng fruit extract of any one of claims 1 to 4 as an active ingredient.

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