Method for preparing rosa canina fruit extract and composition for preventing or treating oral diseases comprising same as active ingredient

Abstract

The present invention relates to a method for preparing a Rosa canina fruit extract and a composition for preventing or treating oral diseases, the composition comprising same as an active ingredient. The extract, which is obtained by mixing the peels and seeds of Rosa canina fruits at various ratios and subjecting the mixture to supercritical fluid extraction (SFE), is clearly distinguished in terms of component composition from conventional hot water extracts, organic solvent extracts, and laboratory-scale supercritical extracts, and thus finds specific applications in oral diseases.

Description

Method for preparing rosehip fruit extract and a composition for preventing or treating oral diseases containing the same as an active ingredient

[0001] The present invention relates to a composition for preventing or treating oral diseases by controlling causative bacteria and other bacteria, using the resulting product obtained by mixing the peel and seeds of rosehip fruit in proportions and performing Supercritical Fluid Extraction (SFE).

[0002] Rose hips are fruits formed after wild roses bloom, and the plant is a shrubby native primarily to the southern Andes Mountains, Central Asia, and Turkey. It belongs to the Rosaceae family, and its scientific name is Rosa canina L. The plant body features lower branches that are relatively straight and upper branches that bend and droop downwards, along with numerous side branches and thorns. The leaves are oval-shaped and consist of 5 to 7 leaflets, while the calyx is long and sharp, with some containing lateral projections. The petals vary in color from white to red, and the fruit is a red, oval shape with a diameter of approximately 1 to 2 cm.

[0003] Rosehip fruit consists of a peel and seeds, and it is known that each part contains a large amount of distinct pharmacologically active ingredients. However, most rosehip extracts used in conventional technology utilize strong polar organic solvents such as hexane, which inherently raises safety concerns regarding residual solvents. In particular, hexane is classified as a carcinogen, restricting its use in products intended for human application; furthermore, the processing of extracts and extract residues produced using such solvents is subject to strict regulations. Nevertheless, existing patents and literature merely describe cases where rosehip extracts were applied as raw materials for oral compositions without specific analysis of their active ingredients or verification of their mechanisms of action; consequently, it is difficult to clearly attribute these effects to the inherent pharmacological action of rosehip.

[0004] Meanwhile, oral diseases are caused by local and systemic factors, with local factors, such as dental plaque, being the primary cause for the majority. Dental plaque consists of numerous bacteria bound to microscopic food residues and glycoproteins attached to the tooth surface; the acids and toxins produced by these bacteria induce tooth decay and periodontal disease. If dental plaque is not removed for a long period, it combines with calcium components in saliva to harden into tartar, which leads to gum inflammation, periodontitis, and, in severe cases, tooth loosening and loss.

[0005] In particular, as we enter an aging society, the importance of oral health is increasing, and the number of patients undergoing implant procedures is also continuously rising. Due to the nature of the procedure, which involves eating, there is a high risk of bacterial infection, with an infection rate reported to reach approximately 40%; in severe cases, serious instances leading to jawbone loss occur. Accordingly, there is a continuously being raised need for the development of safe and effective materials for the prevention and treatment of oral diseases, including peri-implantitis.

[0006] To date, no invention has been reported that fractionates and isolates active ingredients from rosehip extract and verifies their specific effects against bacteria causing oral diseases. In particular, there are no cases of selectively maximizing medicinal components by separating the peel and seeds of rosehip fruit and varying their mixing ratios, and applying this as a composition for oral prevention and treatment.

[0007] The present invention relates to a technology for selectively extracting, separating, and purifying medicinal components by mixing the peels and seeds of rosehip fruits in different ratios, by applying a supercritical fluid extraction (SFE) method that utilizes the physical property changes of gaseous carbon dioxide (CO₂) with controllable temperature and pressure, without using toxic organic solvents.

[0008] The objective of the present invention is to provide a product with maximized pharmacological effects and a method for producing the same by utilizing only rosehip fruits rich in nutritional and pharmacological effects among shrubby plants, separating the husks and seeds, mixing them in proportions, and applying a supercritical fluid extraction (SFE) process.

[0009] Furthermore, another objective of the present invention is to provide an oral composition based on raw materials with clear efficacy and excellent reproducibility, while excluding the use of herbal raw materials with insufficient efficacy verification in oral prevention and hygiene products and minimizing the use of chemical additives.

[0010] To achieve the above objective, the present invention provides a method for preparing a rosehip fruit extract comprising: a first step of low-temperature drying of rosehip (Rosa canina) fruit; a second step of separating the peel and seeds of the dried rosehip fruit; a third step of grinding the separated peel and seeds of the rosehip fruit; a fourth step of mixing the separated peel and seeds of the rosehip fruit; and a fifth step of Supercritical Fluid Extraction (SFE) of the mixture.

[0011] Alternatively, the present invention provides a pharmaceutical composition for the prevention or treatment of oral diseases comprising rosehip (Rosa canina) fruit extract as an active ingredient.

[0012] In addition, the present invention provides a quasi-drug composition for preventing or improving oral diseases, comprising rosehip (Rosa canina) fruit extract as an active ingredient.

[0013] In addition, the present invention provides a food composition for preventing or improving oral diseases comprising rosehip (Rosa canina) fruit extract as an active ingredient.

[0014] In addition, the present invention provides an oral composition for pets comprising rosehip (Rosa canina) fruit extract as an active ingredient.

[0015] In addition, the present invention provides a feed composition for preventing or improving oral diseases comprising rosehip (Rosa canina) fruit extract as an active ingredient.

[0016] According to the present invention, an extract obtained by mixing the peel and seeds of rosehip (Rosa canina) fruit in specific proportions and using a supercritical fluid extraction (SFE) process exhibits clear differentiation in terms of component composition compared to conventional hot water extracts, organic solvent extracts, and laboratory-scale supercritical extracts, thereby enabling specific applications for oral diseases.

[0017] Furthermore, the product of the present invention has an expanded compositional range of active ingredients, allowing it to exhibit excellent efficacy with only a small amount of addition compared to existing oral preventive or therapeutic products, thereby enabling the manufacture of high-efficiency products.

[0018] Furthermore, conventional oral preventive products had the problem of complex compositions due to the mixing of multiple Eastern and Western herbal ingredients without sufficient verification to compensate for the limitations of the efficacy of a single ingredient; however, the product obtained according to the present invention exhibits a direct and effective action in preventing oral diseases despite being based on a single raw material, thereby enabling the provision of a simpler and more reliable oral composition.

[0019] Figure 1 shows the antibacterial effect of the SEF product (RH-M) and cold-pressed extract (RH-cold) of rosehip fruit peel and seeds on P. intermedia.

[0020] Figure 2 shows the antibacterial effect of the SEF product (RH-M) and cold-pressed extract (RH-cold) of rosehip fruit peel and seeds against P. gingivalis.

[0021] Figure 3 shows the antibacterial effects of the SEF product (RH-M) and cold-pressed extract (RH-cold) of rosehip fruit peel and seeds against S. mutans.

[0022] Figure 4 shows the SEF results of rosehip fruit peel and seeds and the effect of cold-pressed extract on the biofilm test.

[0023] Figure 5 shows the cytotoxicity of rosehip extract.

[0024] Figure 6 shows cell viability in THP-1 cells according to rosehip extract concentration. (**P<0.01, ***P<0.001, ****P<0.0001 is compared with 0㎍ / ㎖Rosehip)

[0025] Figure 7 shows cell viability in THP-1 cells at different ibuprofen concentrations. (*P<0.05, **P<0.01, ***P<0.001 is compared with 0 μg / ml ibuprofen)

[0026] Figure 8 shows the effects of rosehip extract and ibuprofen on cytokine IL-6 production in LPS-induced THP-1 cells. (***P<0.001, ****P<0.0001 is compared with LPS)

[0027] Figure 9 shows the effects of rosehip extract and ibuprofen on cytokine TNF-α production in LPS-induced THP-1 cells. (*P<0.05, ****P<0.0001 is compared with LPS)

[0028] Figure 10 shows the effects of rosehip extract and ibuprofen on cytokine IL-6 mRNA expression in LPS-induced THP-1 cells. (***P<0.001, ****P<0.0001 is compared with LPS)

[0029] Figure 11 shows the effects of rosehip extract and ibuprofen on cytokine TNF-α mRNA expression in LPS-induced THP-1 cells. (****P<0.0001 is compared with LPS)

[0030] The present invention will be described in more detail below.

[0031] The present invention provides a method for producing a rosehip fruit extract comprising: a first step of low-temperature drying of rosehip (Rosa canina) fruit; a second step of separating the peel and seeds of the dried rosehip fruit; a third step of grinding the separated peel and seeds of the rosehip fruit; a fourth step of mixing the separated peel and seeds of the rosehip fruit; and a fifth step of Supercritical Fluid Extraction (SFE) of the mixture.

[0032] Preferably, the first step can be performed at 35 to 60 ℃ for 5 to 48 hours.

[0033] Preferably, after the first step, the moisture content may be 4 to 10 parts by weight per 100 parts by weight of the whole rosehip fruit.

[0034] Preferably, the powder ground after the third step may have a particle size of 20 to 200 mesh.

[0035] Preferably, the fourth step may consist of 0 to 10 parts by weight of rosehip fruit peel per 100 parts by weight of rosehip fruit extract.

[0036] Preferably, the fourth step may consist of 0 to 10 parts by weight of rosehip fruit seeds per 100 parts by weight of rosehip fruit extract.

[0037] Preferably, the fifth step may be performed at 35 to 50°C at 200 to 400 bar for 150 to 350 minutes.

[0038] Preferably, the solvent for the extraction may be one or more selected from the group consisting of carbon dioxide (CO2), water (H2O), ethanol (C2H5OH), propane (C3H8), normal butane (n-Butane), methanol (CH3OH), and a co-solvent thereof.

[0039]

[0040] Alternatively, the present invention provides a pharmaceutical composition for the prevention or treatment of oral diseases comprising rosehip (Rosa canina) fruit extract as an active ingredient.

[0041] Preferably, the oral disease may be one or more selected from the group consisting of dental caries, gum disease, malocclusion, abrasion, other bruxism, weak joint disorder, oral cancer, periodontitis, peri-implantitis, periodontal pocket, periodontal ligament disease, and gingivitis.

[0042] Preferably, the oral disease is Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, Prevotella nigrescens, Eubacterium nodatum, Parvimonas micra, Campylobacter rectus, Prevotella intermedia, Fusobacterium nucleatum, Streptococcus mutans, and Streptococcus sobrinus. It can be caused by one or more selected from the formed group.

[0043] Preferably, the rosehip (Rosa canina) fruit extract may have antibacterial, bactericidal, or anti-inflammatory effects.

[0044] Preferably, the rosehip (Rosa canina) fruit extract may have an antiviral effect.

[0045] Preferably, the pharmaceutical composition may be a liposomalized rosehip fruit extract.

[0046]

[0047] In this specification, “antimicrobial” means an action that inhibits the proliferation, survival, or metabolic activity of microorganisms, particularly bacteria, and is not limited to cases where the microorganisms are killed, but includes actions that reduce the growth rate or stop the proliferation of microorganisms. The antimicrobial effect may be confirmed, for example, through inhibition of the increase in the number of bacteria, a decrease in colony-forming units (CFU), or changes in the bacterial growth curve, and in this specification, “antimicrobial” may or may not include a bactericidal action.

[0048] In this specification, “sterilization” means an action that kills microorganisms, particularly bacteria, or renders them irreversibly unviable. The sterilization effect may be confirmed, for example, through indicators such as a decrease in bacterial survival rate before and after treatment, loss of colony-forming ability, or damage to bacterial cell membranes and degradation of nucleic acids. In this specification, “sterilization” is a form of antimicrobial action that includes, but is not limited to, cases involving substantial bacterial death beyond the level of inhibiting microbial growth.

[0049] In this specification, the “pharmaceutical composition” may be prepared in a unit dose form or contained in a multi-dose container by formulation using a pharmaceutically acceptable carrier according to a method that can be easily carried out by a person skilled in the art to which the invention pertains.

[0050] The above-mentioned pharmaceutically acceptable carriers are those commonly used in formulations and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above components, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc.

[0051] In the present invention, the content of the additive included in the pharmaceutical composition is not particularly limited and can be appropriately adjusted within the content range used in conventional formulations.

[0052] The above pharmaceutical composition may be formulated into one or more external forms selected from the group consisting of injectable formulations such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, tablets, creams, gels, patches, sprays, ointments, warning agents, lotions, liniments, pastes, and cataplasms.

[0053] The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier and a diluent for formulation. The pharmaceutically acceptable carrier and diluent include, but are not limited to, excipients such as starch, sugar, and mannitol; fillers and extenders such as calcium phosphate; cellulose derivatives such as carboxymethylcellulose and hydroxypropylcellulose; binders such as gelatin, alginates, and polyvinylpyrrolidone; lubricants such as talc, calcium stearate, hydrogenated castor oil, and polyethylene glycol; disintegrants such as povidone and crospovidone; and surfactants such as polysorbate, cetyl alcohol, and glycerol. The pharmaceutically acceptable carrier and diluent may be biologically and physiologically affinity to the target. Examples of diluents include, but are not limited to, saline solution, aqueous buffer solution, solvent, and / or dispersion media.

[0054] The pharmaceutical composition of the present invention may be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) depending on the intended method. For oral administration, it may be formulated into tablets, troches, lozenges, water-soluble suspensions, oily suspensions, prepared powders, granules, emulsions, hard capsules, soft capsules, syrups, or elixirs. For parenteral administration, it may be formulated into injectable solutions, suppositories, powders for respiratory inhalation, aerosols for sprays, ointments, powders for topical application, oils, creams, etc.

[0055] The dosage of the pharmaceutical composition of the present invention may vary depending on the patient's condition and weight, age, gender, health status, dietary constitutional specificity, properties of the formulation, degree of disease, time of administration of the composition, method of administration, duration or interval of administration, excretion rate, and form of the drug, and may be appropriately selected by a person skilled in the art. For example, it may be in the range of about 0.1 to 10,000 mg / kg, but is not limited thereto, and may be administered once or several times a day.

[0056]

[0057] In addition, the present invention provides a quasi-drug composition for preventing or improving oral diseases, comprising rosehip (Rosa canina) fruit extract as an active ingredient.

[0058] Preferably, the formulation of the above quasi-drug composition may be one or more formulations selected from the group consisting of toothpaste (paste, bubble, or solid type), mouthwash, mouthwash, gum, candy, oral spray, oral ointment, oral varnish, mouthwash, and gum massage cream.

[0059] In this specification, the term “quasi-drug” refers to a fiber or rubber product used for the purpose of treating, alleviating, managing, or preventing diseases in humans or animals, which has a weak effect on the human body or does not act directly on the human body.

[0060] Examples of the quasi-drug compositions of the present invention include external preparations, powders, disinfectants, toothpastes, ointments, lotions, internal preparations (vitamin-mineral preparations, nutritional tonics), wet wipes, spray patches, bandages, or patches, but are not particularly limited thereto. The formulation method, dosage, method of use, and components of the quasi-drug may be appropriately selected by a person skilled in the art from the ordinary technology known in the relevant technical field.

[0061]

[0062] In addition, the present invention provides a food composition for preventing or improving oral diseases comprising rosehip (Rosa canina) fruit extract as an active ingredient.

[0063]

[0064] In addition, the present invention provides an oral composition for pets comprising rosehip (Rosa canina) fruit extract as an active ingredient.

[0065]

[0066] In addition, the present invention provides a feed composition for preventing or improving oral diseases comprising rosehip (Rosa canina) fruit extract as an active ingredient.

[0067] In this specification, the term “feed” refers to a substance that supplies organic or inorganic nutrients necessary to sustain the life and rear an individual. The feed may include nutrients such as energy, protein, lipids, vitamins, and minerals required by the individual consuming the feed, but is not specifically limited thereto. The individual refers to the subject of rearing and includes any living organism capable of consuming the feed of the present invention without limitation.

[0068] The feed composition of the present invention can be produced in various forms such as dry feed, semi-moist feed, and wet feed according to methods known in the art, and can be made in any one of the following forms, for example, powder, granules, pills, pellets, jelly, canned feed, biscuits, croquettes, nuggets, flakes, snacks, etc., but is not limited thereto.

[0069] The feed composition of the present invention may include one or more carriers, excipients, or diluents, and examples of said carriers, excipients, and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. Additionally, it may further include fillers, anticoagulants, lubricants, wetting agents, flavorings, emulsifiers, and preservatives.

[0070] In addition, the feed composition of the present invention may include components commonly added to feed, such as carbohydrates, proteins, fats, minerals, vitamins, minerals, and water. There are no particular limitations on the types of each of these components, and any commonly used in the field may be used.

[0071]

[0072] Hereinafter, the present invention will be described in detail with reference to examples to aid in understanding. However, the following examples are merely illustrative of the content of the present invention and the scope of the present invention is not limited to the following examples. The examples of the present invention are provided to more completely explain the present invention to those with average knowledge in the art.

[0073] <Example>

[0074] Example 1. Process for separating rosehip fruit husks and seeds

[0075] Rosehips are Rosa canina L. or Rosa rubiginosa species, which grow wild or are cultivated in the Andes Mountains of Chile. They are distributed from the west-central to the south, with a harvest season from March to August. The harvested rosehip fruits were dried at 45–70°C, and the fruit remnants and impurities were removed after drying. The first grinding process is designed to effectively separate the seeds from the husks; the pulp, which resembles fine downy hairs, was carefully separated to remove impurities. The rosehip husks, from which impurities had been removed, were ground to a particle size of 200 mesh. The rosehip seeds separated after the first grinding were also cleaned of impurities. The rosehip seeds, free of impurities, were ground to a particle size of 200 mesh.

[0076]

[0077] Example 2. Process for extracting pharmacological components from a mixed formulation of rosehip fruit peel and seeds using an SFE (Supercritical Fluid Extraction) system

[0078] The peels and seeds of rosehip fruits were separated and ground separately to prepare raw materials with a uniform particle size distribution. To perform SFE extraction using the rosehip fruit peel powder and seed powder, they were mixed in the proportions shown in Table 1.

[0079] Mixing ratio of rosehip husks and seeds Order Husks (Weight%) Seeds (Weight%) 10 10 22 83 46 46 45 82 6 100

[0080] Example 3. Extraction of rosehip fruit oil using the cold-pressing method

[0081] To use a cold-pressed rosehip extract as a control group, rosehip fruits were dried under natural drying conditions (45–70℃) and then cold-pressed at 40–50℃.

[0082]

[0083] Example 4. Culture of causative bacteria of oral diseases

[0084] For the antibacterial activity test of rosehip fruit extract, Streptococcus mutans ATCC 25175, a bacterium associated with dental caries, was used as the Gram-positive bacterium, and Porphyromonas gingivalis ATCC 33277 and Prevotella intermedia ATCC 25611, bacteria associated with periodontitis and peri-implantitis, were used as the Gram-negative bacteria. S. mutans was cultured aerobically in Typticase soy broth (TSB) (BD bioscience, San Jose, CA, USA), while P. gingivalis and P. intermedia were cultured anaerobically at 37°C using brain heart infusion (BHI) (BD bioscience, San Jose, CA, USA) supplemented with hemin (1 μg / ml) and vitamin K (0.2 μg / ml).

[0085]

[0086] Example 5. Antimicrobial activity test

[0087] The above bacteria cultured in liquid media were centrifuged at 4,000 × g for 5 minutes, and each liquid medium was added to measure the bacterial counting chamber. Using clean medium on the measured bacterial suspension, S. mutans was 1.0 × 10⁶ 6 Adjust to cells / mL, and P. gingivalis and P. intermeida at 1.5 × 10 6 The concentration was adjusted to cell / mL and used for the test. 180 µl of each medium was added to a 96-well plate, and two prepared rosehip fruit extracts (diluted 10-fold in DMSO) were added to the first column and serially diluted twofold. Subsequently, 20 µl of bacterial suspension was added to each well, and the plates were incubated in an anaerobic incubator for 24 or 48 hours. Afterward, bacterial growth was analyzed by measuring absorbance at 660 nm using a spectrophotometer.

[0088]

[0089] 5.1. Antimicrobial test on P. intermedia

[0090] Rosehip SFE extract completely inhibited the growth of P. intermedia in samples diluted 5,120 times. On the other hand, rosehip cold-press extract inhibited the growth of P. intermedia in samples diluted 40 times, and completely inhibited the growth of P. intermedia in samples diluted 20 times.

[0091]

[0092] 5.2. Antimicrobial test against P. gingivalis

[0093] Rosehip SFE extract inhibited the growth of P. gingivalis in samples diluted 204,800 times and completely inhibited growth in samples diluted 10,240 times. On the other hand, rosehip cold-press extract inhibited the growth of P. gingivalis in samples diluted 80 times and completely inhibited growth in samples diluted 20 times.

[0094]

[0095] 5.3. Antimicrobial test against S. mutans

[0096] Rosehip SFE extract completely inhibited the growth of S. mutans in a sample diluted 204,800 times. On the other hand, rosehip cold-press extract inhibited the growth of S. mutans in a sample diluted 80 times, and completely inhibited the growth of S. mutans in a sample diluted 40 times.

[0097]

[0098] Example 6. Sterilization power test

[0099] Bacteria cultured in liquid medium were centrifuged at 4,000 × g for 5 minutes, clean liquid medium was added, and the bacterial count was measured using a bacterial counting chamber; using clean medium, S. mutans was 1.0 × 10⁶6 Adjust to cells / mL, and P. gingivalis and P. intermeida at 1.5 × 10 6 The concentration was adjusted to cell / mL and used in the test. Two rosehip extracts were diluted 100-fold with DMSO, and the rosehip extracts were mixed with each bacterial suspension at a ratio of 9:1 and left at room temperature for 3 minutes. Immediately thereafter, they were placed in 40 mL of fresh medium to inhibit antimicrobial activity as much as possible. Then, 50 µL aliquots were dispensed onto TSA solid medium, and 50 µL aliquots were dispensed onto blood agar medium containing hemin and vitamin K. The samples were spread evenly using a spreader (SPL lifescience). The control group was left at room temperature and treated for the same amount of time. Subsequently, the solid media were incubated anaerobically at 37°C for 36 hours, after which the colony count was measured. The viable cell count and bactericidal reduction rate were determined according to the following criteria.

[0100] Sterilization reduction rate (%)

[0101] R(%) = [(AB) / A] ×100

[0102] R: Sterilization reduction rate

[0103] A: Initial bacterial count

[0104] B: Number of bacteria after a certain period of time

[0105]

[0106] 6.1.P. Sterilizing power against intermedia

[0107] Results of the sterilization test for P. intermedia, initial inoculation count [1.34 × 10⁶ 6 ±3.92 × 10⁻⁶ 4 In the group treated with rosehip SFE extract, the level was 194 ± 52 CFU / mL (≥99.99%), and the rosehip cold-pressed extract treatment group was 1.12 × 10⁶ 6 ±4.11 × 10⁻⁶ 4 CFU / ㎖ (6.03%) was found.

[0108]

[0109] 6.2. Bactericidal activity against P. gingivalis

[0110] Results of the bactericidal test against P. gingivalis, initial inoculation count [1.16 × 10⁶ 6 ±4.51 ×10 4 In the group treated with rosehip SFE extract, the level was 156 ±34 CFU / mL (≥99.99%), and the rosehip cold-pressed extract treatment group was 1.09 × 10⁻⁶ 6 ±6.81 × 10⁻⁶ 4 CFU / ㎖ (6.03%) was found.

[0111]

[0112] 6.3.S. Sterilizing power against mutans

[0113] Results of the bactericidal test against S. mutans, initial inoculation count [9.72 × 10⁶ 5 ±6.02 ×10 3 In the group treated with rosehip SFE extract, the level was 124 ± 29 CFU / mL (≥99.99%), and the rosehip cold-pressed extract treatment group was 9.64 × 10⁶ 5 ±5.97 ×10 3 CFU / ㎖ (6.03%) was found.

[0114]

[0115] Example 7. Test on biofilm

[0116] S. mutans(1 × 10 6The cells (cells / mL) were suspended in TSB supplemented with 2% sucrose and 400 µL was dispensed into each well of an 8-well glass plate (BD Bioscience, San Jose, CA, USA). The medium was changed daily, and a biofilm was formed for 4 days. Subsequently, the control group was left at room temperature and treated with two types of rosehip extracts (diluted 1000-fold with DMSO) for 3 minutes. The biofilms were washed with phosphate buffer solution, fluorescent staining was performed using the LIVE / DEAD bacterial viability kit (Invitrogen, Eugene, OR, USA), and 3D images were obtained using a confocal laser microscope (CLSM; Carl Zeiss Co., Baden-Wurttemberg, Germany).

[0117] As a result, the effects of rosehip fruit SEF extract and cold-pressed extract on biofilms were found to be very desirablely high. The change to yellow indicates that the rosehip SEF extract has a strong bactericidal effect against oral bacteria in biofilms.

[0118]

[0119] Example 8. Cytotoxicity

[0120] 8.1. Cytotoxicity of Rosehip Extract on Gingival Fibroblasts

[0121] Gingival fibroblasts were cultured in 12-well plates until full, and to investigate the cytotoxicity of rosehip extract, they were treated with rosehip extract at various concentrations and cultured for 12 hours. Subsequently, cell viability was assessed using the MTT reagent. To examine the effect of DMSO, an equal amount of DMSO as the rosehip extract treatment was administered as a control. Cell viability decreased in groups diluted 80-fold or less with DMSO, while it decreased in groups diluted 160-fold or less with rosehip extract. Regarding the bactericidal activity at 5 minutes, neither rosehip extract nor DMSO had any effect on cell viability.

[0122]

[0123] Example 9. Inflammation experiment

[0124] 9.1. Cell Culture

[0125] THP-1 cells were obtained from the Korean Cell Line Bank and used. They were subcultured every 5 days in RPMI 1,640 (CORNING, USA) medium with 10% Fetal bovine serum (FBS, CORNING, USA) and 1% Penicillin / sterptomycin (CORNING, USA) in a 5% CO2 incubator at 37℃.

[0126] To differentiate human monocyte cells into macrophages, THP-1 cells were cultured for 24 hours in RPMI 1,640 medium containing 10 nM PMA (phorbol 12-myristate 13-acetate, Sigma-Aldrich, USA).

[0127] 9.2. Cell Cytotoxicity Assay

[0128] The cytotoxicity of THP-1 cells according to different concentrations of the experimental group (rosehip SFE extract) and the positive control (ibuprofen) was measured using a CCK-8 assay. THP-1 cells were seeded into a 96-well plate at a density of 5,000 cells / well. The following day, rosehip extract and ibuprofen were treated at various concentrations, and the cells were incubated for 24 hours in a 5% CO2 incubator at 37°C. Subsequently, 10 µl of CCK-8 reagent was added to each well, and the cells were incubated for an additional 2 hours. After 2 hours, the absorbance was measured at 450 nm using an ELISA reader.

[0129] 9.3. Enzyme-linked immunosorbent assay (ELISA)

[0130] 1 × 10⁶ THP-1 cells in a 6-well plate 6 Cells were seeded according to the number of cells per well and treated with PMA, then cultured for 24 hours in a 5% CO2 incubator at 37°C. Differentiated cells were treated with rosehip extract and ibuprofen at various concentrations for 1 hour, followed by the addition of 100 ng / ml of LPS. After 24 hours, the cell culture supernatant was collected, and the production of cytokines IL-6 and TNF-α was measured using an ELISA kit. Cytokine levels were measured at 450 and 570 nm using an ELISA reader.

[0131] 9.4. Real Time-PCR

[0132] 1 × 10⁶ THP-1 cells in a 6-well plate 6Cells were seeded according to the number of cells per well and treated with PMA, then cultured for 24 hours in a 5% CO2 incubator at 37°C. Differentiated cells were treated with rosehip extract and ibuprofen at various concentrations for 1 hour, followed by the addition of 100 ng / ml LPS. After 24 hours, the medium was removed, and total RNA was separated using TRlzol. RNA quantification was performed using Nano Dorp, and 1 µg of total RNA was analyzed using Oligo-dT primer and AccuPower ⓡ RocketScript TM cDNA was synthesized by mixing RT premix. AccuPower® 2xGreensStar in a Real Time PCR system. TM Cytokine expression was analyzed using qPCR Master Mix. Subsequently, relative mRNA expression levels were calculated using the 2-△△Ct method and normalized to GAPDH.

[0133] Primers used in real-time PCRForward (5'-3')Reverse (5'-3')IL-6AAC CTG TCC ACT GGG CAC ATCT GGC TCT GAA ACA AAG GATTNF-αCAG GGA CCT CTC TCT AAT CAAGC TGG TTA TCT CTC AGC TCGAPDHGTG GTG GAC CTG ACC TGCTGA GCT TGA CAA AGT GGT CG

[0134] 9.5. Statistical Processing

[0135] All experimental results were analyzed using Graphpad Prism (GraphPad Software, USA). Data were expressed as mean ± standard deviation. Ordinary one-way analysis of variance (ANOVA) was performed to verify the significance between experimental groups and the differences between each treatment sample (n=3). P<0.05 was considered to reflect statistical significance. In the plot, P-values ​​are indicated as follows: *P<0.05, **P<0.01, **P<0.001, and****P<0.0001.

[0136] 9.6. Results

[0137] To measure the cell viability of rosehip extract, THP-1 cells were treated with rosehip extract and ibuprofen at various concentrations. As a result, the viability of THP-1 cells decreased starting from a rosehip extract concentration of 10 µg / ml. On the other hand, the viability of THP-1 cells was inhibited by the positive control, ibuprofen, starting from a concentration of 20 µg / ml.

[0138] To determine the effect of rosehip extract on the production of cytokines IL-6 and TNF-α induced by LPS-induced inflammation, THP-1 cells were treated with rosehip extract and ibuprofen at various concentrations, followed by the addition of 100 μg / ml of LPS to check cytokine production. As a result, cytokines IL-6 and TNF-α were significantly reduced when rosehip extract was treated at concentrations of 5 and 10 μg / ml compared to the LPS group, and the mRNA levels of IL-6 and TNF-α were also significantly reduced. Based on these results, rosehip extract inhibited the LPS-induced inflammation in THP-1 cell experiments and suppressed the expression of cytokines IL-6 and TNF-α, indicating that rosehip extract (supercritical) regulates the inflammation response.

[0139]

[0140] Foregoing, specific parts of the present invention have been described in detail. It will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

Claims

1. First step of low-temperature drying of rosehip (Rosa canina) fruits; A second step of separating the peel and seeds of the dried rosehip fruit; A third step of crushing the peels and seeds of the separated rosehip fruit; A fourth step of mixing the peels and seeds of the separated rosehip fruit; and A method for preparing rosehip fruit extract comprising a fifth step of performing SFE (Supercritical Fluid Extraction) on the above mixture.

2. A method for preparing rosehip fruit extract according to claim 1, characterized in that the first step is performed at 35 to 60 ℃ for 5 to 48 hours.

3. A method for preparing rosehip fruit extract according to claim 1, characterized in that, after the first step, the moisture content is 4 to 10 parts by weight per 100 parts by weight of the total rosehip fruit.

4. A method for preparing rosehip fruit extract according to claim 1, characterized in that the powder ground after the third step has a particle size of 20 to 200 mesh.

5. A method for preparing rosehip fruit extract according to claim 1, wherein the fourth step comprises 0 to 10 parts by weight of rosehip fruit peel per 100 parts by weight of total rosehip fruit extract.

6. A method for preparing rosehip fruit extract according to claim 1, wherein the fourth step comprises 0 to 10 parts by weight of rosehip fruit seeds per 100 parts by weight of total rosehip fruit extract.

7. A method for preparing rosehip fruit extract according to claim 1, wherein the fifth step is performed at 35 to 50℃ at 200 to 400 bar for 150 to 350 minutes.

8. A method for preparing rosehip fruit extract according to claim 7, wherein the solvent for extraction is one or more selected from the group consisting of carbon dioxide (CO2), water (H2O), ethanol (C2H5OH), propane (C3H8), normal butane (n-Butane), methanol (CH3OH), and a mixed solvent (Co-solvent) thereof.

9. A pharmaceutical composition for the prevention or treatment of oral diseases comprising rosehip (Rosa canina) fruit extract as an active ingredient.

10. A pharmaceutical composition according to claim 9, characterized in that the oral disease is one or more selected from the group consisting of dental caries, gum disease, malocclusion, abrasion, other bruxism, weak joint disorder, oral cancer, periodontitis, peri-implantitis, periodontal pocket, periodontal ligament disease, and gingivitis.

11. In paragraph 9, the above oral disease is Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, Prevotella nigrescens, Eubacterium nodatum, Parvimonas micra, Campylobacter rectus, Prevotella intermedia, Fusobacterium nucleatum, Streptococcus mutans and Streptococcus A pharmaceutical composition characterized by being induced by one or more selected from the group consisting of Streptococcus sobrinus.

12. A pharmaceutical composition according to claim 9, characterized in that the rosehip (Rosa canina) fruit extract has antibacterial, bactericidal, or anti-inflammatory effects.

13. A pharmaceutical composition according to claim 9, characterized in that the rosehip (Rosa canina) fruit extract has an antiviral effect.

14. A pharmaceutical composition according to either claim 12 or 13, wherein the pharmaceutical composition is characterized by liposomalizing a rosehip fruit extract.

15. A quasi-drug composition for preventing or improving oral diseases, comprising rosehip (Rosa canina) fruit extract as an active ingredient.

16. A quasi-drug composition according to claim 14, characterized in that the formulation of the above quasi-drug composition is one or more formulations selected from the group consisting of toothpaste (paste, bubble, or solid type), mouthwash, mouthwash, gum, candy, oral spray, oral ointment, oral varnish, mouthwash, and gum massage cream.

17. A food composition for preventing or improving oral diseases containing rosehip (Rosa canina) fruit extract as an active ingredient.

18. A composition for the oral use of pets containing rosehip (Rosa canina) fruit extract as an active ingredient.

19. A feed composition for preventing or improving oral diseases containing rosehip (Rosa canina) fruit extract as an active ingredient.

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