Composition comprising pyrrosia hastata (thunb. ex houtt.) ching extract thereof for preventing and improving of periodontitis

Abstract

The present invention relates to a composition for preventing and improving periodontal disease comprising an extract of *Sedum trichomanes* as an active ingredient. By inhibiting the release of tumor necrosis factor-alpha (TNF-α), the composition can prevent inflammation, promote the proliferation and differentiation of osteoblasts, inhibit the destruction of alveolar bone by reducing the formation and activity of osteoclasts, and increase the proliferation and cellular activity of periodontal ligament cells.

Description

Technology Field

[0001] The present invention relates to a composition for preventing and improving periodontal disease comprising an extract of *Sedum trichomanes* as an active ingredient, and more specifically, to a composition that exhibits the effect of preventing and improving periodontal disease by including the extract of *Sedum trichomanes* as an active ingredient and has excellent functionality. Background Technology

[0003] Periodontal disease is reported to be directly or indirectly correlated with diseases such as gastrointestinal disorders, lipid metabolism disorders, and stroke; furthermore, periodontal disease cannot be explained by a single cause and is generally considered a chronic disease resulting from complex causes.

[0004] Periodontal tissue consists of the alveolar bone, gingiva (gums), and periodontal ligaments. The gums are part of the tooth-supporting tissue visible in the mouth, and it is here that diseases (gingivitis) begin. As the disease progresses and spreads deeper into the supporting tissues, the periodontal ligaments—which are connected to the tooth root surface and the surrounding bone tissue—are destroyed, and the alveolar bone surrounding the tooth is also destroyed, leading to the development of periodontal disease.

[0005] Periodontal disease, including gingivitis and periodontitis, is an inflammatory condition of the tooth-supporting tissues caused by bacteria. It leads to tooth loss due to bleeding, the formation of periodontal pockets, and the destruction of alveolar bone. This disease progresses through a process involving bacterial colonization, bacterial infiltration of periodontal tissues, and the destruction of these tissues. Specifically, when oral bacteria thrive due to poor oral hygiene, they form a bacterial film, causing inflammation, occasional gum bleeding, and bad breath. If this condition progresses further, the gap between the teeth and gums deepens, forming periodontal pockets where disease-causing bacteria proliferate, leading to periodontitis. As periodontitis progresses, even mild stimulation such as brushing can cause the gums to bleed and swell; often, this escalates into acute inflammation, causing pain. This inflammation impairs bone formation and increases bone resorption, resulting in the gradual loss and destruction of alveolar bone, ultimately leading to tooth loss.

[0006] Although there are various causes of periodontal disease, when plaque accumulates within the periodontal pocket due to local factors, it becomes a habitat for surrounding anaerobic Gram-negative bacteria, causing them to proliferate deep within the pocket. At this stage, the toxins and all products of these proliferated anaerobic Gram-negative bacteria directly destroy tissues or stimulate the immune system; through various actions of this stimulated immune system, they induce inflammation along with the destruction of periodontal tissues. As a defense mechanism against this, the function of polymorphonuclear leukocytes and immune responses act as systemic factors.

[0007] In other words, the destruction of periodontal tissue and inflammation are caused by direct tissue destruction resulting from cytotoxins such as hydrogen sulfide, ammonia, and toxic amines—metabolic products of anaerobic Gram-negative bacteria—and endotoxins such as lipopolysaccharides, which are components of the cell walls of anaerobic Gram-negative bacteria; or by various cytokines, such as reactive oxygen species, prostaglandins, leukotrienes, histamine, and interleukin, secreted extracellularly through the action of the stimulated humoral and cellular immune systems that trigger the body's immune system. Furthermore, collagen, the matrix of periodontal tissue, is broken down by enzymes such as collagenase secreted by bacteria and leukocytes, leading to gum recession; if left untreated, this progresses to periodontal disease.

[0008] Therefore, antibacterial and bacteriostatic actions against anaerobic Gram-negative bacteria, which are the root cause of periodontal disease, as well as the removal of bacterial toxic products and the restoration of lost periodontal tissues, are important factors in the prevention and treatment of periodontal disease.

[0009] Currently, the treatment of periodontal disease utilizes methods such as improving oral hygiene, non-surgical treatments (non-steroidal anti-inflammatory drugs), and surgical treatments (scaling, root planing, gingival curettage, and periodontal tissue regeneration using new attachments). Although surgical treatment is the most effective method, it is often performed only after the disease has progressed to a certain extent rather than for prevention, which limits effective treatment and causes the disease to mostly progress into a chronic condition. As a result, systemic antibiotics and topical sustained-release agents have been used as supplementary treatments; however, these methods present serious problems, including the delivery of excessive amounts of medication to unnecessary areas leading to side effects, and reports of the isolation of periodontal disease bacteria resistant to recently used antibiotics.

[0010] To complement the limitations of such surgical treatments and the problems associated with antibiotic use, and to improve the prevention and treatment effects of periodontal disease, there is an urgent need for the development of agents capable of restoring destroyed and lost periodontal tissues to their original state.

[0011] Against this backdrop, research on periodontal disease treatments utilizing natural products is actively underway to address these conventional problems; however, most of these agents exhibit poor antibacterial effects or merely demonstrate the ability to prevent the onset of periodontal disease through abstract efficacy such as anti-inflammatory, astringent, hemostatic, and blood circulation promotion, making it difficult to utilize them as actual treatments for periodontal disease. Prior art literature

[0012] (Patent Document 0001) KR 10-2474229 B (Patent Document 0002) KR 10-1978355 B The problem to be solved

[0013] The objective of the present invention is to provide a composition for preventing and improving periodontal disease comprising an extract of *Sedum trichomanes* as an active ingredient, which prevents inflammation by inhibiting the release of tumor necrosis factor-alpha (TNF-α), promotes the proliferation and differentiation of osteoblasts, inhibits the destruction of alveolar bone by reducing the formation and activity of osteoclasts, and increases the proliferation and cellular activity of periodontal ligament cells. means of solving the problem

[0015] A composition for preventing and improving periodontal disease according to one embodiment of the present invention comprises an extract of *Syneilesis aconitifolia (Bunge) Maxim* as an active ingredient.

[0016] The above extract is extracted using an extraction solvent selected from the group consisting of water, C1 to C6 lower alcohols, and mixtures thereof.

[0017] The above periodontal disease includes a disease selected from the group consisting of gingivitis, periodontitis, and mixtures thereof.

[0018] The above composition exhibits an effect of preventing and improving inflammation by inhibiting the release of tumor necrosis factor-alpha (TNF-α).

[0019] A toothpaste for preventing periodontal disease according to another embodiment of the present invention comprises the above-mentioned composition for preventing and improving periodontal disease.

[0020] A mouthwash for preventing periodontal disease according to another embodiment of the present invention comprises the above-mentioned composition for preventing and improving periodontal disease.

[0021] An oral spray for preventing periodontal disease according to another embodiment of the present invention comprises the composition for preventing and improving periodontal disease.

[0023] The present invention will be described in more detail below.

[0025] All technical terms used in this invention, unless otherwise defined, have the following definitions and correspond to the meaning generally understood by those skilled in the art in the relevant field of this invention. Additionally, while preferred methods or samples are described herein, similar or equivalents are also included within the scope of this invention.

[0027] In the specification of the present invention, plants are not limited to any type and can be used without restriction on their source, such as using cultivated ones or commercially purchased ones.

[0028] In the specification of the present invention, the extract may be extracted by a method known in the art, and the method is not particularly limited. Alternatively, a commercially available extract may be used.

[0029] The above plant extract may utilize any part of the plant and is not limited to the extraction site. The preparation of the above plant extract is not limited by the form of the plant body, and includes all plant bodies that have undergone processing such as drying. For example, the plant may be the whole plant, roots, stems, leaves, fruits, flowers, shoots, branches, bark, sap, bulbils, and / or seeds of the aforementioned plant.

[0030] The natural extract used in the present invention can be prepared in a powder state by additional processes such as vacuum distillation, freeze-drying, or spray-drying.

[0032] In the present invention, 'prevention' means any act of suppressing or delaying the progression of a disease or illness to which the term applies, or one or more symptoms of said disease or illness, by administering a composition of the present invention.

[0033] In the present invention, "improvement" refers to any act in which the disease or illness to which the term applies, or one or more symptoms of said disease or illness, are improved or beneficially altered by the administration of the composition of the present invention.

[0034] In the present invention, "treatment" means reversing, alleviating, inhibiting the progression of, or preventing a disease or illness to which the term applies, or one or more symptoms of said disease or illness, and as used herein, the term "treatment" refers to the act of treating when "treating" is defined as above.

[0035] As used in this specification, the term "active ingredient" means a component that exhibits the intended activity alone or can exhibit activity in combination with a carrier that is inactive itself.

[0036] As used in this specification, the term 'extract' has the meaning commonly used in the art as a crude extract, as described above, but in a broader sense, it also includes fractions obtained by further fractionating the extract. That is, the plant extract includes not only that which is obtained using the extraction solvent described above, but also that which is obtained by additionally applying a purification process thereto. For example, fractions obtained through various additional purification methods, such as a fraction obtained by passing the extract through an ultrafiltration membrane having a certain molecular weight cut-off value, or separation by various chromatographs (designed for separation based on size, charge, hydrophobicity, or affinity), are also included in the plant extract of the present invention.

[0037] In addition, it includes the extract itself and extracts of all formulations that can be formed using the extract, such as diluted or concentrated extracts of the extract, dried products obtained by drying the extract, adjusted or purified products of the extract, fermented products of the extract, or mixtures thereof. It also includes juices obtained by directly pressing or crushing plants and then filtering. The plants may be extracted as they are or after processing.

[0038] The above "Poje" refers to a pharmaceutical technology that alters the original properties of medicinal materials by processing them based on traditional Korean medicine theory. Examples include the stir-frying method, which involves roasting the medicinal materials; the roasting method, which involves roasting the medicinal materials together with a certain amount of liquid auxiliary material to allow the auxiliary material to permeate into the medicinal tissue; and the steaming method, which involves adding and mixing liquid auxiliary material according to the Poje regulations for each medicinal item, heating and steaming it in a suitable container, or steaming and drying it until it reaches a certain level. Additionally, it may refer to the strain itself, the crushed material of the strain, its culture, its dead body, or a mixture thereof, extracted using an extraction solvent.

[0039] The term "fraction" as used in this invention refers to a result obtained by performing fractionation to separate a specific component or a specific group of components from a mixture containing various constituent components.

[0040] The terms used herein are for describing embodiments and are therefore not intended to limit the invention. In this specification, the singular form includes the plural form unless specifically stated otherwise in the text. As used herein, 'comprise' and / or 'comprising' do not exclude the presence or addition of one or more other components, steps, actions, and / or elements to the mentioned components, steps, actions, and / or elements.

[0041] Specifically, among the terms used in this specification, "containing as an active ingredient" means containing an amount sufficient to achieve the efficacy or activity of the following natural extracts.

[0042] As used herein, the term “about” means an amount, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length that varies by about 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% with respect to a reference amount, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length.

[0043] In this specification, "health functional food" refers to a group of foods to which added value has been imparted by utilizing physical, biochemical, or biotechnological methods to enable the function of the food to act or manifest for a specific purpose, or to foods designed and processed to fully express the biological regulatory functions related to the regulation of biological defense rhythms, disease prevention, and recovery inherent in the food composition. Compared to general foods, they possess active effects for maintaining or promoting health, and "health supplement food" refers to foods intended for the purpose of health support. In some cases, the terms functional food, health food, and health supplement food are used interchangeably.

[0045] A composition for preventing and improving periodontal disease according to one embodiment of the present invention comprises an extract of *Pyrrosia hastata* (Thunb. ex Houtt.) Ching as an active ingredient.

[0046] The aforementioned *Pyrrosia hastata* (Thunb. ex Houtt.) Ching is an evergreen perennial herb that grows on rock surfaces, reaching a height of 15 to 20 cm. Its rhizomes spread laterally, and the scales are dark brown, lanceolate, or ovate-lanceolate. The leaves are spear-shaped with margins divided into 3 to 5 lobes, with the central lobe being the longest. The sporangia are arranged in an orderly fashion in 3 to 6 rows between the main veins. It is distributed in southern Korea, China, and Japan.

[0048] The above-mentioned three-horned sedge extract is extracted using an extraction solvent selected from the group consisting of water, C1 to C6 lower alcohols, and mixtures thereof.

[0049] Specifically, to prepare a three-horned sedum extract, the method comprises the steps of: washing the three-horned sedum; drying it after washing; grinding the three-horned sedum after drying; leaching the ground material using an organic solvent; drying the sample after leaching; leaching it using water; and leaching, thereby obtaining the three-horned sedum extract.

[0050] The method for preparing the above extract may be a conventional extraction method in the art, such as solvent extraction, hot water extraction, filtration, ultrasonic extraction, leaching, and reflux extraction, and these may be performed alone or in combination of two or more methods. In the present invention, the type of extraction solvent used for extraction is not particularly limited, and any solvent known in the art may be used.

[0051] Non-limiting examples of extraction solvents include water; lower alcohols having 1 to 6 carbon atoms, such as methanol, ethanol, propyl alcohol, and butyl alcohol; polyhydric alcohols, such as glycerin, butylene glycol, and propylene glycol; hydrocarbon solvents, such as methyl acetate, ethyl acetate, acetone, benzene, hexane, diethyl ether, and dichloromethane; or mixtures thereof. Specifically, water, lower alcohols, 1,3-butylene glycol, and ethyl acetate may be used individually or in a mixture of two or more. In this case, when two or more solvents are mixed, the mixing ratio between the solvents is not particularly limited.

[0052] Specifically, the extract may be obtained by extracting the extract of *Sedum trichomanes*, from which foreign substances have been removed by washing and drying, with water, an alcohol having 1 to 6 carbon atoms, or a mixture of these solvents, and may be an extract obtained by sequentially applying the solvents to a sample.

[0053] For extraction, the sample may be left in an extraction solvent for leaching for 1 to 72 hours, and more specifically, for 24 to 48 hours.

[0054] In the present invention, the extraction method may be applied using a solvent with a weight of 1 to 100 times, specifically 1 to 50 times, more specifically 2 to 20 times, based on the weight of the dried plant material, at an extraction temperature of 10 to 80°C, specifically 15 to 50°C, for an extraction time of 2 hours to 30 days, specifically 12 hours to 18 days, and may include a process of obtaining a liquid crude extract by performing continuous extraction 1 to 5 times including the dried and crushed material.

[0055] The extract obtained using the above organic solvent may further include a step of fractionating using an organic solvent, and the method for preparing the extract may be a conventional extraction method in the art, such as ultrasonic extraction, leaching, and reflux extraction.

[0056] The above reflux extraction method is based on 10 to 30 g of crushed natural product, 1 to 3 hours of reflux, and 50 to 100% of alcohol or water with 1 to 6 carbon atoms, based on 100 mL of alcohol with 1 to 6 carbon atoms. More specifically, based on 100 mL of alcohol or water with 1 to 6 carbon atoms, 10 to 20 g of crushed natural product, 1 to 2 hours of reflux, and 70 to 90% of alcohol or water with 1 to 4 carbon atoms.

[0057] The above leaching method is carried out at 15 to 30°C for 24 to 72 hours, and water or 50 to 100% alcohol having 1 to 6 carbon atoms is used as the extraction solvent. More specifically, it is carried out at 20 to 25°C for 30 to 54 hours, and the extraction solvent is water or 70 to 80% alcohol having 1 to 6 carbon atoms.

[0058] The above ultrasonic extraction method is carried out at 30 to 50°C for 0.5 to 2.5 hours, and the extraction solvent is water or 50 to 100% alcohol having 1 to 6 carbon atoms. Specifically, the extraction is carried out at 40 to 50°C for 1 to 2.5 hours, and the extraction solvent is water or 70 to 80% alcohol having 1 to 6 carbon atoms.

[0059] The above extraction solvent may be used in an amount of 2 to 50 times the weight of the sample, more specifically 2 to 20 times. For extraction, the sample may be left in the extraction solvent for 1 to 72 hours, more specifically 24 to 48 hours.

[0060] After extraction, the extract can be fractionated by sequentially applying a new fractionation solvent. The fractionation solvent used for fractionation is one or more selected from the group consisting of water, hexane, butanol, ethyl acetic acid, ethyl acetate, methylene chloride, and mixtures thereof, preferably ethyl acetate or methylene chloride. After obtaining the extract or fraction, additional methods such as concentration or freeze-drying may be used.

[0062] The above periodontal disease includes a disease selected from the group consisting of gingivitis, periodontitis, and mixtures thereof.

[0063] Periodontal disease is classified into gingivitis and periodontitis depending on the severity of the condition. Gingivitis refers to a relatively mild and fast-recovering form of periodontal disease confined to the gums, or soft tissues, while periodontitis refers to cases where this inflammation has progressed to the gums and surrounding alveolar bone. Diagnosis of gingivitis and periodontitis is made by checking the condition of the teeth and gums through dental and periodontal examinations, and the extent of alveolar bone destruction can be assessed through radiographic examinations.

[0064] Gingivitis is a stage where bacterial inflammatory response is confined to the superficial soft tissues, whereas periodontitis is when the bacteria attack the area below the gum line within the sulcus, damaging the periodontal ligament and surrounding bone tissue. As inflammation progresses and more periodontal ligament tissue is damaged, the narrow, shallow sulcus deepens and develops into a periodontal pocket. The more severe the periodontitis, the deeper the periodontal pocket becomes, leading to the loss of the alveolar bone surrounding the tooth roots. As inflammation progresses, the gap between the gums and teeth widens, eventually causing the teeth to loosen; if left untreated, the teeth may fall out spontaneously.

[0065] The most fundamental aspect of treating periodontal disease is thoroughly removing bacterial plaque and tartar to prevent the creation of an environment favorable for bacterial proliferation. Oral gum medications are not very helpful for treatment. Treatment may involve using a mouthwash containing chlorhexidine, placing special agents between the gums and teeth, or using antibiotics to eradicate specific bacteria present in the gums.

[0066] Interleukin-1 (IL-1) and tumor necrosis factor are known to be the major cytokines associated with bone loss, inflammation, and connective tissue destruction associated with periodontal disease.

[0068] The above composition exhibits effects of preventing and improving dry mouth.

[0069] Healthy adults secrete 1 to 1.5 liters of saliva per day, and if less saliva is produced, they feel that their mouth is dry. Alternatively, they may subjectively feel dry mouth as moisture evaporates from the mouth while breathing through it.

[0070] Severe dry mouth can make it difficult to chew and swallow food, and may also make speaking difficult. Furthermore, since saliva plays a role in digestion, digestive problems may arise, and eating spicy or irritating foods may cause pain in the mouth. It also increases the risk of developing gingivitis or cavities.

[0071] The above dry mouth includes a disease selected from the group consisting of acid erosion, oral candidiasis, dysgeusia, dysphagia, oral dysesthesia, intraoral halitosis, and mixtures thereof.

[0072] Xerostomia is the most common clinical symptom associated with the salivary glands. It is diagnosed when the volume of saliva secreted without stimulation is less than 0.1 mL per minute. The primary causes of xerostomia are side effects of various medications, such as antidepressants, antidiabetics, and anti-allergics. Xerostomia caused by natural aging occurs when the parenchymal tissue of the salivary glands decreases and is replaced by fatty tissue, or when the volume of the salivary sacs decreases. Additionally, it may occur due to the destruction of salivary glands or blockage of blood flow caused by autoimmune diseases such as Sjögren's syndrome or radiation therapy. A decrease in the volume of saliva in the oral cavity is accompanied by dental caries, acid erosion, oral candidiasis, dysgeusia, dysphagia, oral dysesthesia, and intraoral halitosis.

[0074] Preferably, the composition for preventing and improving periodontal disease according to the present invention may additionally include a natural extract in addition to the three-horned stonecrop extract.

[0075] The above natural extract is selected from the group consisting of Lithospermum root extract, Lithospermum root extract, Perilla leaf extract, and mixtures thereof.

[0076] The aforementioned *Lithospermum erythrorhizon* is a perennial herb belonging to the family *Lithospermum* in the order Lamiales of dicotyledonous plants, growing in grasslands in mountains and fields. Its roots are thick and purple, penetrating deep into the ground. The stems stand upright and branch out, reaching a height of 30–70 cm, and are covered with numerous upward-pointing fine hairs. The leaves are alternate, lanceolate or oblong-lanceolate in shape, measuring 3–7 cm in length; they are pointed at the tip and narrow at the base, resembling a petiole, and are densely covered with coarse hairs. The upper surface of the leaves is deeply wrinkled along the veins, the margins are smooth, and there are no petioles. White flowers bloom from May to June, forming spike inflorescences at the tips of stems and branches. The bracts are leaf-shaped, and the calyx is green, deeply divided into five lobes; the lobes are broadly linear with blunt tips and are longer than the tubular part of the corolla. The corolla is 6–7 mm long and splits into five lobes at the tip; the lobes are round and spread horizontally. There are five stamens and one pistil. The fruit is a schizocarp (a fruit that splits into separate segments), grayish-white, smooth, and glossy. In traditional Korean medicine, the root is used as a medicinal herb called *Jacho*, which is effective for hemoptysis, nosebleeds, urinary hemorrhage, and measles. It is also used externally as a disinfectant for burns, frostbite, eczema, rashes, and skin ulcers. In the past, it was used as a purple dye. It is distributed in Korea, Japan, China, and Amur. It is preferable to use the root of *Jichi* to be used as the extract of the present invention.

[0077] The above-mentioned *Echinops setifer Iljin* is a perennial herb belonging to the Asteraceae family, order Campanulales, and class Dicotyledonous plants; it is also known as *Gae-seurichwi* or *Jeolgudae*. It grows in sunny grasslands. It reaches a height of approximately 1 meter, has slightly branched stems, and is covered with cottony hairs, giving the entire plant a whitish appearance. Leaves emerging from the roots have long petioles; the upper surface is green and the lower surface is white. Additionally, the margins are split like thistles and have thorns. Leaves emerging from the stem are sessile, long elliptical, and divided into 5 to 6 pairs. Flowers bloom in July and August, are purplish in color, and are tubular flowers with a diameter of about 5 cm. The corolla is split into five lobes at the tip and curls backward, while the involucre has a thorn-like tip. The fruit is an achene with dense hairs, and the pappus resembles scales. Young leaves are edible, and the roots are used for boils. It is distributed in Korea and Japan. It is preferable to use the roots or leaves of *Echinops setifer* to serve as the extract for the present invention.

[0078] The aforementioned *Perilla frutescens var. acuta* is an annual herb belonging to the Lamiaceae family in the order Lamiales of dicotyledonous plants. Also known as *Chajogi* or *Chazugi*, it is native to China. The stem stands upright to a height of 20–80 cm, has a square cross-section, is purplish in color, and is fragrant. The leaves are opposite, broadly ovate with a pointed tip and a rounded base, and have serrated margins. Both sides of the leaves are hairy, with long hairs on the veins of the underside, and the petioles are long. Flowers bloom in August and September in a pale purple color, forming racemes at the tips of stems and branches. The calyx is hairy and splits into two; the upper segment splits into three, while the lower segment splits into two. The corolla is short and tubular, with a lip-like tip, and the lower lip is slightly longer than the upper lip. There are four stamens, two of which are longer. The fruit is a schizocarp, round, 1.5 mm in diameter, and enclosed within the calyx. In traditional Korean medicine, the leaves are called Soyeop and the seeds are called Jasoja; they are used as diaphoretics, antitussives, stomachics, diuretics, sedatives, and analgesics. When suffering from food poisoning after eating fish or crab, the fresh juice of the leaves is drunk or the leaves are boiled and eaten.

[0079] More preferably, the composition for preventing and improving periodontal disease according to the present invention comprises, with respect to 100 parts by weight of *Sedum trichomanes* extract, 20 to 300 parts by weight of *Ligusticum chuanxiong* extract, 20 to 300 parts by weight of *Lysimachia japonica* extract, and 20 to 300 parts by weight of *Perilla frutescens* extract, and preferably may comprise 20 to 200 parts by weight of *Ligusticum chuanxiong* extract, 20 to 200 parts by weight of *Lysimachia japonica* extract, and 20 to 200 parts by weight of *Perilla frutescens* extract. Even more preferably, it comprises 50 to 200 parts by weight of *Ligusticum chuanxiong* extract, 50 to 200 parts by weight of *Lysimachia japonica* extract, and 50 to 200 parts by weight of *Perilla frutescens* extract.

[0080] When the composition for preventing and improving periodontal disease according to the present invention further includes a natural extract selected from the group consisting of Lithospermum erythrorhizon extract, Lithospermum erythrorhizon extract, Perilla frutescens extract, and mixtures thereof in addition to the above-mentioned *Sedum trichomanes* extract, it can superiorly inhibit the release of tumor necrosis factor-alpha (TNF-α) compared to the case where only *Sedum trichomanes* extract is included, and can induce an increased activity capable of inhibiting alveolar bone destruction by promoting the proliferation and differentiation of osteoblasts and reducing the formation and activity of osteoclasts.

[0082] A toothpaste for preventing periodontal disease according to another embodiment of the present invention comprises the above-mentioned composition for preventing and improving periodontal disease.

[0083] When the above composition for the prevention and improvement of periodontal disease is used as toothpaste, the composition may be added directly to the toothpaste, or mixed or added with functional foods or functional ingredients, and may be used appropriately according to conventional methods. The mixing amount of the active ingredient may be appropriately determined according to the purpose of use.

[0085] A mouthwash for preventing periodontal disease according to another embodiment of the present invention comprises the above-mentioned composition for preventing and improving periodontal disease.

[0086] The above-mentioned gargle solution is defined as comprising all compositions capable of gargling the oral cavity and other agents for cleaning the oral cavity. Meanwhile, the above-mentioned gargle solution for achieving the purpose of the present invention must have an oil-based composition, and it is preferable not to mix surfactants or allols. The above-mentioned specific formulation is defined as including all compositions and mixing ranges that can be selected and mixed by a person with ordinary knowledge in the relevant field.

[0088] The toothpaste composition, mouthwash (gargle), and oral spray of the present invention may additionally contain one or more ingredients selected from the group consisting of: sodium fluoride, sodium fluoride phosphate, amine fluoride, tin fluoride, or xylitol as active ingredients that are caries preventive agents; chlorhexidine, cetylpyridium chloride, or triclosan as antiseptics; bamboo salt or propolis as herbal medicines; allantoin, aminocaproic acid, tranexamic acid, tocopherol acetate, or methylsulfonylmethane as anti-inflammatory agents; and dextranase, glucose oxidase, glucoamylase, α, β-amylase, lactoperoxidase, and lysozyme as enzyme agents.

[0089] In addition, the toothpaste composition, mouthwash, and oral spray of the present invention may further include one or more substances selected from the group consisting of abrasives, humectants, binders, foaming agents, sweeteners, anionic surfactants, fragrances, and coloring agents. The composition may be in the form of a paste, powder, liquid (gargle form), or powder. Effects of the invention

[0091] The present invention relates to a composition for preventing and improving periodontal disease comprising an extract of *Sedum trichomanes* as an active ingredient. By inhibiting the release of tumor necrosis factor-alpha (TNF-α), the composition can prevent inflammation, promote the proliferation and differentiation of osteoblasts, inhibit the destruction of alveolar bone by reducing the formation and activity of osteoclasts, and increase the proliferation and cellular activity of periodontal ligament cells. Specific details for implementing the invention

[0093] Hereinafter, embodiments of the present invention are described in detail so that those skilled in the art can easily implement the invention. However, the present invention may be embodied in various different forms and is not limited to the embodiments described herein.

[0094] [Preparation Example 1: Preparation of Natural Extract]

[0095] 1. Preparation of Trichosanthes extract

[0096] The three-horned sedum was washed and dried, then ground. The ground material was mixed with ethanol and maintained at 98 to 100°C for 2 hours, then cooled and filtered through Whatman filter paper to obtain the filtrate. This was prepared as a three-horned sedum extract (PHE).

[0097] 2. Preparation of other natural extracts

[0098] Using the same method as the above-mentioned method for preparing the *Sedum trichomanes* extract (PHE), *Lithospermum erythrorhizon* extract (LE), *Euryale ferox* extract (EE), and *Perilla frutescens* extract (PFE) were prepared.

[0099] 3. Preparation of mixed extract

[0100] A mixed extract was prepared by mixing the above-prepared *Sedum trichomanes* extract (PHE), *Lithospermum erythrorhizon* extract (LE), *Euonymus japonicus* extract (EE), and *Perilla frutescens* extract (PFE) within the weight ranges shown in Table 1 below.

[0101] M1 M2 M3 M4 M5 M6 M7 M8 M9 PHE 100 100 100 100 100 100 100 100 100 LE - 100 - - 20 50 100 200 300 EE - - 100 - 20 50 100 200 300 PFE - - - 100 20 50 100 200 300

[0102] (Unit: parts by weight)

[0104] [Example 1: Experiment on Inhibition of TNF-α Secretion]

[0105] The following experiment was conducted to determine the effect of the three-horned sedge extract (PHE, M1) and mixed extracts M2 to M9 prepared in Preparation Example 1 above on inhibiting the release of cytokine TNF-α in THP-1 cells, a human monocytic cell line.

[0106] Human-derived THP-1 cells (ATCC No. TIB-202) were purchased from ATCC (Rockville, USA) and cultured in RPMI 1640 (Gibco, BRL, USA) supplemented with 10% FBS (fetal bovine serum) before being used in the experiment. The cell line was cultured in 96-well plates at a density of 5 × 10⁶ cells per ml. 5 Cells were divided into groups and lipopolysaccharide (LPS) was added to activate the cells to secrete TNF-α.

[0107] Simultaneously with LPS, the experimental group was treated with three-horned sedge extract (PHE) and mixed extracts M2 to M9, and after treatment, the amount of TNF-α in the cell culture medium was measured by enzyme immunoassay (ELISA), and the results are shown in Table 2 below.

[0108] M1 M2 M3 M4 M5 M6 M7 M8 M9 TNF-α secretion amount 94.16 ± 2.32 90.27 ± 1.73 84.89 ± 1.76 78.49 ± 1.32 71.21 ± 0.96 57.98 ± 5.63 43.27 ± 0.88 49.45 ± 2.07 65.61 ± 2.86

[0109] (Unit: mg / ml)

[0110] As shown in Table 2 above, when the three-horned sedge extract (PHE, M1) and mixed extracts M2 to M9 of the present invention were treated, it was confirmed that the secretion of TNF-α was significantly inhibited in a concentration-dependent manner in M2 to M9, which further contained Lithospermum erythrorhizon extract (LE), Lithospermum erythrorhizon extract (EE), and Perilla frutescens extract (PFE), compared to M1, which contained only the three-horned sedge extract (PHE).

[0111] Specifically, in the case of M2 to M4, which additionally include the extracts of *Sedum trichomanes*, *Sedum sarmentosum*, and *Perilla frutescens* respectively in addition to the *Sedum trichomanes* extract, the inhibitory effect on TNF-α secretion was slightly enhanced; meanwhile, in the case of complex extracts M5 to M9, which include all of the aforementioned natural extracts, it was confirmed that the inhibitory effect on TNF-α secretion was further enhanced. Preferably, in the case of complex extracts M6 to M8, it was confirmed that TNF-α secretion was excellently inhibited, and furthermore, it was confirmed that the effect was maximized in M7.

[0112] Therefore, it was found that the composition according to the present invention effectively inhibits the secretion of TNF-α.

[0114] [Example 2: Osteoblast Proliferation Experiment]

[0115] In order to determine the effects of the three-horned stonecrop extract (PHE, M1) and mixed extracts M2 to M9 prepared in Preparation Example 1 above on the proliferation of osteoblasts, the following experiments were conducted.

[0116] Mouse osteoblasts were obtained by purchasing mouse cranial cell line MC3T3-E1 cells (ATCC No. CRL-2593) from ATCC (Rockville, USA) and culturing them in DMEM (Gibco, BRL, USA) supplemented with 10% FBS (Fetal bovine serum) before use in the experiment.

[0117] The above osteoblasts were placed in a 24-well plate at a density of 20,000 cells per well and cultured in a 5% CO2 incubator for 48 hours. Then, the culture medium was replaced with a mixture of the *Sedum trichomonas* extract (PHE, M1) and mixed extracts M2 to M9 according to the present invention at various mixing ratios, and the mixture was cultured for another 48 hours. After removing the culture medium, trypsin-EDTA was added, and the number of cells was counted using a hemacytometer and shown in % units in Table 3 below.

[0118] M1 M2 M3 M4 M5 M6 M7 M8 M9 Osteoblast proliferation 64 67 71 75 81 89 95 91 84

[0119] (unit: %)

[0120] As shown in Table 3 above, when treated with the three-horned sedge extract (PHE, M1) and mixed extracts M2 to M9 of the present invention, it was confirmed that cell proliferation was promoted by up to 30% in M7, which further includes Lithospermum erythrorhizon extract (LE), Lithospermum erythrorhizon extract (EE), and Perilla frutescens extract (PFE), compared to the case containing only the three-horned sedge extract (PHE, M1).

[0121] Specifically, in the case of M2 to M4, which additionally included the extracts of *Sedum trichomanes*, *Sedum sarmentosum*, and *Perilla frutescens*, respectively, to the *Sedum trichomanes* extract, osteoblast proliferation was slightly enhanced; meanwhile, it was confirmed that the osteoblast proliferation effect was further enhanced in the case of the complex extracts M5 to M9, which included all of the aforementioned natural extracts. Preferably, it was confirmed that osteoblast proliferation was excellently enhanced in the case of the complex extracts M6 to M8, and furthermore, that the effect was maximized in M7.

[0122] Therefore, it was found that the composition according to the present invention effectively induces osteoblast proliferation.

[0124] [Example 3: Inhibition of Osteoclast Activation]

[0125] In order to confirm the osteoclast growth and activity of the three-horned sedum extract (PHE, M1) and mixed extracts M2 to M9 prepared in Preparation Example 1 above, osteoclast progenitor cells were cultured on a plate coated with hydroxyapatite (OAAS, OCT Inc.) and the activity and resorption of TRAP, a marker enzyme of osteoclasts, were observed.

[0126] First, to isolate mouse bone marrow cells, 7 to 9-week-old male mice were sacrificed by cervical torsion. The femur and tibia were aseptically excised, soft tissues were removed, and both ends of the long bone were desecrated. Then, using a 26G needle, 1 mL of an enzyme solution containing 0.1% collagenase (Gibco), 0.05% trypsin, and 0.5 mM EDTA (Gibco) was injected into the bone marrow cavity of one end to extract the bone marrow. The bone marrow cells were collected by stirring for 30 minutes and pre-cultured for 24 hours in α-MEM containing 10% FBS to obtain non-adherent cells. Non-adherent cells, which serve as precursors to osteoclasts, were collected at a rate of 2 × 10⁶ per well. 5 The cells were divided and cultured until they became dog cells.

[0127] Among the cultured cells above, adherent cells were washed with PBS, fixed with citrate-acetate-formaldehyde for 5 minutes, and incubated for 1 hour in 37°C acetate buffer (pH 5.0) containing naphthol AS-BI phosphate, Fast Garnet GBC solution, and 7 mM tartrate buffer (pH 5) to perform TRAP staining. At this time, TRAP(+) multinucleated cells having three or more nuclei were considered as osteoclasts.

[0128] To induce the differentiation of osteoclasts, bone marrow known to contain osteoclast progenitor cells was used; cells that were TRAP-positive (+) and multinucleated were considered osteoclasts, and osteoclast progenitor cells were cultured.

[0129] The culture medium was removed from the cultured osteoclast progenitor cells. After cell culture, to remove attached cells, the OAAS plate was washed with distilled water, then a 5% sodium hypochlorite solution was added and left for 5 minutes, then washed thoroughly with distilled water again and dried, after which the absorbed area was observed using Image pro plus. That is, to examine the activity of osteoclasts, which are primarily responsible for bone resorption in bone tissue, osteoclast progenitor cells were cultured using a calcium and phosphate-coated plate made to resemble the mineral part of bone tissue, and when the cerebral sedum extract (PHE, M1) and mixed extracts M2 to M9 according to the present invention were treated, changes in the absorption area were observed.

[0130] The results are shown in Table 4 below.

[0131] M1 M2 M3 M4 M5 M6 M7 M8 M9 Osteoclast activity inhibition rate 28 32 37 43 49 63 73 69 56

[0132] (unit: %)

[0133] As shown in Table 4 above, when osteoclast precursor cells were treated with the other three-horned sedge extract (PHE, M1) and mixed extracts M2 to M9 of the present invention, it was confirmed that the activity and absorption by osteoclasts were reduced by culturing them on a calcium-phosphate-coated plate.

[0134] In particular, when using the mixed extracts M6 to M8 according to the present invention, an excellent inhibition rate of osteoclast activity was confirmed.

[0135] Specifically, compared to M1, which contains only *Pterocarya trichomanes* extract (PHE), M2 to M4, which additionally contain *Pterocarya trichomanes* extract, *Pterocarya persica* extract, and *Perilla frutescens* extract respectively in addition to *Pterocarya trichomanes* extract, showed a slight improvement in the inhibition rate of osteoclast activity; furthermore, it was confirmed that the inhibitory effect on osteoclast activity was further enhanced in the complex extracts M5 to M9, which contain all of the aforementioned natural extracts. Preferably, it was confirmed that the complex extracts M6 to M8 not only excellently inhibited activity and absorption by osteoclasts, but also that the effect was maximized in M7.

[0136] Therefore, it was found that the composition according to the present invention effectively inhibits activity and absorption by osteoclasts.

[0138] [Example 4: Experiment on the proliferation of gingival fibroblasts and periodontal ligament cells]

[0139] In order to investigate the effects of the three-horned sedum extract (PHE, M1) and mixed extracts M2 to M9 prepared in Preparation Example 1 above on the proliferation and increase in cell activity of human periodontal ligament cells and gingival fibroblasts, the following experiments were conducted.

[0140] To confirm osteoclast growth and activity, osteoclast progenitor cells were cultured on hydroxyapatite-coated plates (OAAS, OCT Inc.), and the activity and resorption of TRAP, a marker enzyme of osteoclasts, were observed.

[0141] The periodontal ligament portion of a premolar extracted from a healthy person for orthodontic treatment was aseptically scraped using a curette and placed on a culture dish. A small amount of culture medium containing 10% FBS (fetal bovine serum) was added to DMEM (Gibco, BRL, USA) for culture. Gingival fibroblasts were cultured in the same manner using a portion of the gingiva removed from the premolar after extraction. During culture, microscopic observation was performed to confirm the emergence of cells as explants from the tissue. After the cells grew in colonies, individual tissue specimens were removed, and the cells were allowed to grow in a single layer. Subculture was then performed for use in experiments. Human periodontal ligament cells and gingival fibroblasts were placed in a 24-well plate with 20,000 cells per well and cultured in a 5% CO2 incubator for 48 hours. Subsequently, the extract of *Sedum trichomonas* (PHE, M1) and mixed extracts M2 to M9 of the present invention were added, and the samples were cultured for another 48 hours.

[0142] After removing the culture medium, trypsin-EDTA was added, and the number of cells was counted using a hemacytometer and expressed as % in Table 5 below.

[0143] M1 M2 M3 M4 M5 M6 M7 M8 M9 Gingival fibroblast proliferation rate 88 87 85 82 80 76 73 74 78 Periodontal ligament cell proliferation rate 41 45 52 59 63 70 78 75 67

[0144] (unit: %)

[0145] As shown in Table 5 above, it was confirmed that the proliferation of gingival fibroblasts by the extract according to the present invention was maintained at a normal or lower level compared to when the extract of *Pterygota cervico* (PHE, M1) was treated alone.

[0146] In addition, it was confirmed that cell proliferation was promoted by up to 40% when the mixed extract M7 was treated to periodontal ligament cells compared to when the extract (PHE, M1) was treated alone.

[0147] Therefore, it was confirmed that the extract according to the present invention can effectively proliferate periodontal ligament cells, which play the most important role in the regeneration of periodontal tissue, without significantly affecting the proliferation of human gingival fibroblasts.

[0149] [Example 5: Sensory Test]

[0150] A mouthwash containing each of the above-mentioned complex extracts was prepared, and 30 adult male and female test subjects aged 20 to 60 were asked to taste it and evaluate its aroma and flavor, with the preference being rated on an index of 1 to 10. For relative evaluation, the *Pterocarya trichomanes* extract (PHE) of Preparation Example 1 of the present invention was fixed at an index of 5 to evaluate relative index values, and the results were aggregated and averaged, and are shown in Table 6 below. The higher the index below, the better the preference, and each index is expressed by applying the average value.

[0151] M1 M2 M3 M4 M5 M6 M7 M8 M9 taste 5 5 5.7 5.3 6 6.5 7.5 7 6.5 incense 5 5.5 6 5 6.5 7 8.1 8 6.8 Overall preference 5 5.3 5.9 5.2 6.3 6.8 7.8 7.5 6.7

[0152] (Unit: exponent)

[0153] Referring to Table 6 above, it was confirmed that M2 to M4, each containing a natural extract, showed slightly improved preference compared to M1, while M5 to M9, each containing a natural extract, showed superior preference.

[0154] In particular, in the case of M5 to M9, which further include Lithospermum erythrorhizon extract, Ligusticum chuanxiong extract, and Perilla frutescens extract with respect to 100 parts by weight of the preferred weight part of Sophora trichosanthes extract (PHE), and more preferably M6 to M8, it was confirmed that the preference for taste and aroma is superior compared to M1, which includes only Sophora trichosanthes extract, thus confirming that it is possible to provide a composition with high preference by including the complex extract of the present invention. Furthermore, it was confirmed that the effect is maximized in M7.

[0155] In other words, it can be seen that when the composition of the present invention is processed, the overall preference for taste and aroma is the best, thereby providing a composition with high preference.

[0157] Although preferred embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and improvements by those skilled in the art using the basic concept of the present invention as defined in the following claims also fall within the scope of the present invention.

Claims

Claim 1 A composition for preventing and improving periodontal disease comprising an extract of *Sedum trichomanes* as an active ingredient, wherein the periodontal disease comprises a disease selected from the group consisting of gingivitis, periodontitis, and mixtures thereof. Claim 2 delete Claim 3 In claim 1, the composition is a composition for preventing and improving periodontal disease, which exhibits an effect of preventing and improving inflammation by inhibiting the release of tumor necrosis factor-alpha (TNF-α). Claim 4 A composition for preventing and improving tooth disease, wherein the extract is extracted using an extraction solvent selected from the group consisting of water, C1 to C6 lower alcohols, and mixtures thereof. Claim 5 Toothpaste for preventing periodontal disease comprising a composition for preventing and improving periodontal disease according to any one of paragraphs 1, 3, and 4. Claim 6 A mouthwash for preventing periodontal disease comprising a composition for preventing and improving periodontal disease according to any one of paragraphs 1, 3, and 4. Claim 7 An oral spray for preventing periodontal disease comprising a composition for preventing and improving periodontal disease according to any one of paragraphs 1, 3, and 4.

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