KR101879398B1 - Pharmaceutical composition for enhancing osteogenesis comprising garcinia mangostana extract and propolis extract - Google Patents

Abstract

The present invention provides a composition for promoting osteogenesis, comprising mangosteen extract and propolis extract, and a preparation or food for preventing, improving or treating osteogenesis-related diseases comprising the same.
The composition of the present invention shows excellent synergistic effect on osteogenesis by containing mangosteen extract and propolis and thus can be widely used in medicines and foods for the prevention, improvement or treatment of various diseases requiring bone formation promotion have.

Description

[0001] The present invention relates to a composition for promoting osteogenesis, comprising mangosteen extract and propolis extract, and a method for preparing the same,

The present invention relates to a composition for promoting bone formation. More specifically, the present invention relates to a pharmaceutical composition or food composition having excellent bone formation promoting activity using mangosteen extract, alpha-mangosteen or gamma-mangosteen and propolis extract as active ingredients.

The bone of the human body maintains its homeostasis through bone modeling and bone remodeling by the action of osteoblasts and osteoclasts. However, as the age increases, an increase in bone resorption due to various causes weakens the bone tissue, resulting in various bone diseases such as osteoporosis and fracture. Therefore, development of an osteogenesis promoter for maintaining homeostasis has been demanded.

Bone related diseases include osteoporosis, fracture, osteoarthritis, periodontal disease, malocclusion, and alveolar bone destruction in periodontal disease.

Osteoporosis is a skeletal disease that increases the risk of fracture and vulnerability of the bone due to low bone mass and destruction of bone matrix. It causes bone mass to decrease due to various causes and the risk of fracture due to microstructure degeneration of bone tissue is continuously increased do. The osteoporosis drugs currently being used can be classified as bone resorption inhibitors and bone formation promoters.

Bone resorption inhibitors basically prevent excessive bone resorption by osteoclasts, increase bone density to reduce fractures, and osteogenic stimulants decrease the number and differentiation of osteoblasts that lead to bone formation.

A commonly used therapeutic agent for osteoporosis is a bone resorption inhibitor series which weakens the osteoclast function and induces the death, and is mainly composed of bisphosphonate series such as Fosamax (alendronate) and Actonel (ingredient name: risedronate) Has been used as a therapeutic agent. In addition, parathyroid hormone is used as an accelerator for osteogenesis, but it is prohibited to use for more than 18 months because of the increased incidence of osteosarcoma in long term use.

Fracture is a condition in which the continuity of bone, bone plate, or joint surface is abnormally broken, and it is known to occur due to external impact such as trauma, osteoporosis, and metabolic abnormalities such as bone cancer. In general, fracture may not be a big problem in itself, but it is known that secondary damage due to fracture, or complication, becomes a problem.

Osteodystrophy is also called renal osteodystrophy and is mainly caused by kidney disease. Dysphagia is not a disease but a disease that binds several kinds of diseases (hyperparathyroidism, osteomalacia, bone disease caused by aluminum, amorphous sacrocolic dystrophy), and it is known that it is caused by a combination of one or several .

On the other hand, orthodontics is performed to treat or prevent irregular dentition such as malocclusion. It is known that orthodontic tooth movement for orthodontics is accompanied by alveolar bone turnover and that physiological alveolar bone replacement is changed by orthodontic force. During the orthodontic tooth movement, the alveolar bone replacement process involves repeated osteogenesis on the tension side and bone resorption on the compression side, and bone resorption occurs early (3-5 days) It is known that bone formation occurs not only in the alveolar bone but also throughout the periodontal tissue. Bone resorption induced by orthodontic force lasts for 1-14 days, and osteoclasts derived from bone marrow cells are present for 12 days at the compression side during tooth movement for 7 days, and acid phosphatase (ACP) and tartrate - tartrate-resistant acid phosphatase (TRAP) activity. Highly alkaline phosphatase (LP) activity is observed in the cells involved in osteogenesis. It is known to last. In such an orthodontic treatment, it is necessary to develop a drug or a food that is capable of orthodontic treatment by promoting osteogenesis because it enables quick and effective orthodontic treatment by promoting osteogenesis by the orthodontic force.

Furthermore, periodontal disease is an inflammation of gingiva and alveolar bone, such as gingivitis and periodontitis. In particular, periodontal disease is destroyed and destroyed in case of periodontitis. In severe cases, periodontal surgery or artificial bone grafting may occur. It is necessary to develop a drug or a food having a bony characteristic capable of alleviating the symptoms and restoring the lost alveolar bone.

In addition, in recent years, implant treatment has become popular. In this regard, when the alveolar bone is so poor that it can not support the natural teeth, the implant may not be supported normally if the tooth is lost. It is an essential condition. Therefore, in recent years, besides bone grafting, bone formation is performed so that the amount and shape of the alveolar bone can be reconstructed so as to be closer to the normal, and further development of drug or food is needed.

The present invention aims at providing a pharmaceutical composition for promoting bone formation and a food composition for promoting bone formation.

In one aspect, the present invention relates to a pharmaceutical composition for promoting bone formation comprising mangosteen extract and propolis extract.

In the present invention, the mixture of the mangosteen extract and the propolis extract is also defined as "mangosteen combination extract ".

The composition according to the present invention comprises, as one active ingredient, a mangosteen extract.

Mangosteen (Garcinia mangostana) is a fruit of a dicotyledonous plant belonging to the rootstock pepper tree of the origin of Malaysia. It forms a fruit of a size slightly larger than a flat ball-shaped table tennis ball. Mangosteen is known to have excellent antioxidant ability because it contains a typical antioxidant component called xanthone which is a type of polyphenol. In Thailand and other Southeast Asian countries, it has been used for a long time as a civilian medicine for treating wounds such as inflammation and wound. .

The above-mentioned mangosteen extract can be obtained by purchasing a commercially available product or by appropriately selecting it without limitation of an extraction method and an extraction solvent generally known in the pharmaceutical or food industry. Typical extraction methods include, but are not limited to, ultrasonic extraction, filtration, and reflux extraction. Preferably, mangosteen can be extracted using one or more extraction solvents selected from the group consisting of water and lower alcohols having 1 to 6 carbon atoms. More preferably, alpha-mangosteen and gamma-mangosteen, which are effective ingredients for accelerating osteogenesis in the above mangosteen extract, are lipophilic substances. Therefore, for example, water such as an aqueous ethanol solution of less than 50 to 100 (v / v) And a functional organic solvent of ethanol or an ethanol solvent can be used. In one specific embodiment, the mangosteen extract used in the present invention is prepared by mixing 3 to 5 parts by weight of an aqueous ethanol solution of 50 to less than 100% in 1 part by weight of mangosteen, and heating the mixture at a temperature of 60 to 80 for 8 to 10 hours After the extraction, the extracted liquid is filtered by a suitable filtration method such as filtration under reduced pressure using a filter or the like, and the extraction solvent is removed from the filtrate under the conditions of 40 to 60 temperature range and vacuum degree of 0.2 to 0.5 atm, Stin extract can be obtained. These mangosteen extracts may be in various forms such as liquid or powder, but are preferably in powder form.

In addition, the mangosteen extract of the present invention is characterized by containing alpha-mangosteen, gamma-mangosteen, and a mixture thereof as an active ingredient.

Alpha-mangosteen is a minor tonometric compound having the structure of the following formula (1).

 [Chemical Formula 1]

In addition, gamma-mangosteen is a minor tonic compound having a structure represented by the following formula (2).

(2)

As a specific embodiment, the composition according to the present invention contains the above-mentioned mangosteen extract in an amount of 5 to 10 mg / 1 dose unit, more preferably 7.5 to 9 mg / 1 dose unit, in terms of alpha-mangosteen, .

In a preferred embodiment, the mangosteen extract has lipophilicity and is formulated in the form of a water soluble mangosteen molecular encapsulation in an inclusion compound such as chitosan or gamma-dextrin for effective formulation and absorption into the body. .

The composition according to the present invention may further comprise propolis extract as one active ingredient. "Propolis" is a substance made by mixing bees with their own needles and enzymes in a material such as resin (resin) extracted from various plants for their survival and propagation, and flavon (for example, Such as benzoic acid and coumarin benzyl ester, aromatic acids such as coumarin benzyl ester, sialic acid, pinovansine, pinosembrine, and the like. Antifungal, antioxidant, and tooth protecting effect. The propolis extract is prepared by processing the extract of propolis powder through a concentration process. Concretely, it may be produced by pulverizing the propolis raw material, adding thereto a solvent (for example, ethanol) at an appropriate concentration, and extracting, concentrating and drying, or a commercially available product may be used.

In a preferred embodiment, the ratio of the propolis extract to the mangosteen extract in the composition according to the present invention is in the range of 5 to 40: 0.2 to 2, preferably 7 to 36: 0.3 to 0.7 , More preferably 34: 0.5 or 34: 1. Most preferably 34: 1. In addition, the propolis extract in the composition according to the present invention may be included so that the daily dose may be 150-200 mg, preferably 180 mg (16.2 mg as flavonoid).

In one specific embodiment, it is possible to obtain an excellent effect of an alkaline phosphatase (ALP) activity of a mixture of mangosteen extract and propolis effective ingredient according to the present invention, mineralization activity performance of human osteosarcoma cell line MG63 cells and osteoblast gene expression ability Activity. ≪ / RTI > In addition, the use of the mangosteen extract and the propolis extract in combination with the mangosteen extracts showed a synergistic superiority in overall bone morphology.

In a further embodiment of the present invention, the composition according to the present invention may further comprise at least one member selected from the group consisting of soybean insolubles, natto cultures, calcium, vitamin C, xylitol, vitamin E and chamomile extract.

The "natto culture" is a culture obtained by culturing Bacillus natto, which is a substance including natto kinase and vitamin K2. Vitamin K2 (Vit K2) is a compound that inhibits arteriosclerosis, increases bone formation, accelerates the differentiation of dendritic cells by carboxylation of matrix Gla-protein (MGP) with bone formation promoting effect Indicating alveolar bone regeneration. In general, the form of vitamin K2 contained in the natto culture is menadizinone-7 (MK-7).

The above-mentioned "chamomile extract" is an extract of Camomile (Matricaria recutita L.) which is a plant of the family Asteraceae, and is widely used as a herbal tea. For many years, chamomile has been used to relieve intestinal gas, headache, insomnia, back pain, neuralgia, rheumatism, skin diseases, indigestion and gout diseases. Recent studies have shown that chamomile essential oils are highly effective in antimicrobial and antioxidant (Natarajan et al., Phytotherapy Research, 27 (1) 118-125, 2013). Chamomile essential oils contain a variety of phytochemicals including flavonoids, some of which exhibit anti-inflammatory and antiallergic activity. The above-mentioned chamomile extract may be obtained by extracting chamomile according to a known extraction method by cutting the camomile into a fresh or dried state, without limitation.

Preferably, the chamomile extract is added to the propolis extract and the mangosteen extract in an amount of 5 to 40: 0.2 to 2: 3 to 15, preferably 7 to 36: 0.3 as propolis extract: mangosteen extract: To 0.7: 5-13.

The term "soybean unsaponifiable" refers to a pure compound that is hydrophobic in soybean oil and does not react with strong base components such as potassium hydroxide to produce soluble soap, and includes phytosterol. Specifically, the soybean insolubles include components such as beta-sitosterol, campesterol, stigmasterol, brassicasterol, resveratrol, lignan, genistein isoflavone, lecithin, phosphatidylcholine, sphingolipid, phosphatidylserine.

Preferably, the soybean unsaponifiable concentrate may be present in an amount of 7 to 13% by weight based on the total weight of the composition according to the present invention.

The calcium may preferably be water-soluble calcium. In the present invention, "water-soluble calcium" means calcium which has been solubilized through a process of increasing the solubility in water for effective formulation of calcium and absorption into the body. Such a solubilizing method may be a known solubilizing method without limitation, Quot; means that it is prepared in the form of a water-soluble molecular encapsulation, using an inclusion compound such as chitosan, gamma-dextrin and the like.

"Vitamin C" is an antioxidant that eliminates free radicals in the body to prevent cell damage, promotes the synthesis of collagen to rapidly regenerate wound tissue, strengthens blood vessel walls, revitalizes thrombin, It acts to prevent. Preferably, the vitamin C may be included in an amount of 3 to 10% by weight based on the total weight of the composition according to the present invention.

The above "vitamin E " prevents damage to tissues through antioxidation, microcirculation improvement, and the like through cell membrane stabilization, and may be included in an amount of 0.5 to 2% by weight based on the total weight of the composition of the present invention

The above-mentioned "xylitol" is a sugar alcohol-based natural sweetener extracted from birch or oak tree and is capable of preventing the propagation of the insecticidal bacteria due to its pentagonal structure. It is contained in an amount of 1 to 7% by weight based on the total weight of the composition according to the present invention .

When such additional components are further included, the composition according to the present invention exhibits an excellent effect on bone formation and related diseases, in particular, restoration and alleviation of alveolar bone destruction in periodontal disease.

More preferably, the composition according to the present invention may further comprise at least one ingredient selected from the group consisting of seaweed powder, brewer's yeast, dry yeast and chromium yeast in addition to the above-mentioned additional ingredients.

In one specific embodiment, the pharmaceutical composition for promoting bone formation of the present invention comprises 7 to 13% by weight of a soybean insoluble material, 5 to 10% by weight of a natto culture, 2 to 10% by weight of a water- 3 to 10% by weight of vitamin C, 1 to 7% by weight of xylitol, 0.5 to 2% by weight of vitamin E, 10 to 15% by weight of chamomile extract and 1 to 5% by weight of brewer's yeast.

The composition of the present invention includes, in addition to the above-described effective ingredients for administration, at least one pharmaceutically acceptable carrier. In the present invention, the term "pharmaceutically acceptable carrier" means a known pharmaceutical excipient that is useful when formulating a pharmaceutically active compound for administration and is substantially non-toxic and non-sensitive under the conditions of use. The exact ratio of such excipients is determined by standard pharmaceutical practice, as well as the solubility and chemical properties of the active compound, the route of administration chosen.

The pharmaceutical composition of the present invention may be formulated into a form suitable for a desired administration method using suitable and physiologically acceptable excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, lubricants, .

The pharmaceutical composition may be formulated in the form of tablets, capsules, pills, granules, powders, injections or solutions, though it is not limited thereto.

Formulations of pharmaceutical compositions and pharmaceutically acceptable carriers may be appropriately selected according to techniques known in the art.

The composition of the present invention can be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) according to the intended method If you can not provide evidence, I think it might work against the patent examination),

The dosage ranges vary depending on the individual's body weight, age, sex, health condition, diet, time of administration, administration method, excretion rate, and severity of disease. The daily dose of the mangosteen extract is about 60 to 90 mg, the daily dose of alpha-mangosteen contained in the mangosteen extract is about 7.5 to 9.0 mg on an adult basis, and the gamma-mangosteen is 0.9 to 1.2 mg . It is preferable to administer them once or several times a day, and when a composition is prepared in a daily dosage form so as to be administered in this way, it exhibits excellent pharmacological effects.

When the composition according to the present invention is used for food, the composition for food may be used as a health functional food itself through formulation, or as an additive for health functional food. A health functional food refers to a food having a biological control function such as prevention or improvement of disease, bio-defense, immunity, recovery after disease, suppression of aging, and should be harmless to human body when taken over a long period of time. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment).

There is no particular limitation on the kind of the food. Examples of the foods to which the above substances can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen noodles, gums, ice cream, various soups, drinks, tea, Alcoholic beverages, and vitamin complexes, all of which include health functional foods in a conventional sense.

The food composition of the present invention may contain conventional ingredients used for the production of foods or food additives, in particular, flavorings; Natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, natural sweeteners such as dextrin and cyclodextrin, synthetic sweeteners such as saccharin and aspartame; Nutrients; vitamin; Electrolyte; coloring agent; Organic acids; Protective colloid thickener; pH adjusting agents; Stabilizers; antiseptic; glycerin; Alcohol; A carbonating agent used in a carbonated beverage, and the like.

The composition according to the present invention may also be used as an oral composition for oral hygiene. In this case, formulations such as toothpaste, mouthwash, mouthwash, oral massage cream, and the like may be used. In addition, the compounding ingredients of the oral composition provided in the present invention may be used in various formulations in addition to the above-described ingredients, depending on the use and type of the composition.

As described above, the present invention has excellent bone properties when the mangosteen extract, that is, the mangosteen extract and the propolis mixture, are included as the main components, so that the present invention can provide various diseases related to bone formation, For example, it exhibits a potent effect on the treatment of osteoporosis, fracture, bone formation disorder, periodontal disease, malocclusion, alveolar bone destruction upon periodontal disease, and the like. It can also be used as an adjunct to facilitate the implant by repairing the alveolar bone that is destroyed at the time of implantation.

In particular, as can be seen from the specific examples, the present invention has low cytotoxicity, exhibits a strong anti-inflammatory and antimicrobial effect as well as a bone morphology, and, among the diseases associated with osteogenesis, Can be preferably used for alleviating alveolar bone destruction and inflammation at a high disease, in particular, periodontal disease.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

The composition according to the present invention shows excellent synergistic effect on osteogenesis by containing mangosteen extract and propolis and can be widely used in medicines and foods for prevention, .

FIG. 1 is a graph showing the toxicity test results of hTERT-hNOF cells in each experimental group. * Means a significance of 0.05 (5%) or less (p <0.05) with negative control at 95% confidence level.
FIG. 2 is a graph showing the Porphyromonas gingivalis 6 is a graph showing the results of inhibiting IL-6 expression in hTERT-hNOF cells treated with KCOM 2804 LPS. * Showed a significance of 0.05 (5%) or less (p <0.05) with the positive control group at the 95% confidence level.
Fig. 3 shows the results of Porphyromonas gingivalis 8 shows the results of inhibiting IL-8 expression in hTERT-hNOF cells treated with KCOM 2804 LPS. * Means a significance of 0.05 (5%) or less (p <0.05) with the positive control at 95% confidence level.
Fig. 4 shows the results of Porphyromonas gingivalis The results of inhibition of PEG ₂ expression in hTERT-hNOF cells treated with KCOM 2804 LPS are shown. * Means a significance of 0.05 (5%) or less (p <0.05) with the positive control at 95% confidence level.
FIG. 5 is a graph showing the effect of Pipipropolis extract and Mangosteen extract on ALP activity on human osteosarcoma cell line MG63 cell line. * Means a significance of 0.05 (5%) or less (p <0.05) with negative control at 95% confidence level.
FIG. 6 shows a photograph of the synthesis of calcified hard tissues of Pipipropolis extract and mangosteen extract on human osteosarcoma cell line MG63 cell line by Alizarin red S staining.
FIG. 7 is a graph showing Alizarin red S staining for the synthesis of calcified hard tissues of Pipipropolis extract and mangosteen extract on human osteosarcoma cell line MG63 cell line. * Was less than 0.05% (5%) with negative control at 95% confidence level (p <0.05).
FIG. 8 is a graph showing the results on the RUNX2 expression of the Pipipropolis extract and the mangosteen extract on human osteosarcoma cell line MG63 cells. * Was less than 0.05% (5%) with negative control at 95% confidence level (p <0.05).
FIG. 9 is a graph showing the results on the expression of OSPH on MG63 cells, a human osteosarcoma cell line, and the extract of Pifipropolis and Mangosteen. * Was less than 0.05% (5%) with negative control at 95% confidence level (p <0.05).
FIG. 10 is a graph showing the results on the type I collagen? Expression of the Pipipropolis extract and the mangosteen extract on human osteosarcoma cell line MG63 cells. * Was less than 0.05% (5%) with negative control at 95% confidence level (p <0.05).
FIG. 11 is a graph showing the results on the expression of oepocalcin (OCN) in the human osteosarcoma cell line, MG63 cells, of the extract of Pifipropolis and the extract of mangosteen. * Was less than 0.05% (5%) with negative control at 95% confidence level (p <0.05).

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

I. Purpose of Testing

In order to confirm the bone structure performance, antibacterial effect and anti-inflammatory effect according to the combination of the mangosteen extract and the propolis extract according to the present invention, PEP-PROPOLIS EXTRACT POWDER, Australian propolis, Threespears Nutritional Pty LTd, Australia) and mangosteen (scientific name: Garcinia Mangostana L.) In the case of single or complex treatment of extract powder (containing 40% of mangosteen, Maypro Industries, LLC, USA, hereinafter referred to as mangosteen), Porphyromonas gingivalis , Prevotella intermedia , Fusobacterium nucleatum , Aggregatibacter actinomycetemcomitans , Tannerella forsythia ATCC 43037 ^T and Treponema denticolla ATCC 35405 ^T ). In the second, immortalized human gingival fibroblasts (hTERT-hNOF) were treated with LPS extracted from P. gingivalis KCOM 2804 strain to induce inflammatory response, followed by the addition of a mixture of Pepipopropis extract powder and Mangosteen extract powder Anti - inflammatory effect. Finally, osteosarcoma cells (human osteosarcoma, human osteoblast-like cells, MG-63) were treated with a powder of Pifipropolis extract and a powder of mangosteen extract.

II. Test Methods

II-1. Antibacterial experiment

1) Bacteria and culture method

The strains used in this study were Porphyromonas gingivalis KCOM 2804, Prevotella intermedia KCOM 1107, Fusbacterium nucleatum subsp. polymorphum KCOM 1232 and Aggregatibacter actinomycetemcomitans KCOM 1306, Tannerella forsythia ATCC 43037 ^T and Treponema denticolla was ATCC 35405 ^T. These strains were purchased from the Korean Institute of Oral Microbiology, Gwangju, Korea and the American Type Culture Collection (ATCC, Manassas, VA, USA).

T. denticola was added to the heart infusion broth with 1.0% trypticase peptone, 0.25% yeast extract, 0.05% sodium thioglycollate, 0.1% L-cysteine HCl-H ₂ O, 0.025% L-asparagine 0.2% D- glucose, 0.0001% resazurin, 0.20025 % NaHCO ₃ , 2% rabbit serum and 2% TPP / VFA solution (0.03% thiamine pyrophosphate, 0.05% isobutyric acid, 0.05% 2-methylbutyric acid, 0.05% isovaleric acid, 0.05% valeric acid and 0.098N NaOH) (ATCC Medium: 1494 Modified NOS Medium, https://www.atcc.org/~/media/C14CA5B6B5D246A6B8073D6894BFE5EB.ashx). P. gingivalis , P. intermedia , F. nucleatum And A. actinomycetemcomitans were cultured in Tryptic Soy broth containing 0.5% yeast extract, 0.05% cysteine HCl-H ₂ O, 0.5 mg / ml hemin and 2 μg / ml vitamin K ₁ , and T. forsythia In the culture medium 10 μg N-acetylmuraminic acid ml1; by the addition of (NAM Sigma) 37 anaerobic chamber ( Bactron I, Sheldon Manufacturing Inc., Cornelius, OR, USA) and anaerobic conditions _{(10% H 2, 5%} CO 2 , 85% N ₂ ).

2) Determination of minimum bactericidal concentration (MBC) value

MBC measurements were made using the micro-dilution method proposed by the Clinical and Laboratory Standards Institute (CLSI). Briefly, each bacterium was inoculated on the previously described medium, cultured in a bacterial incubator for 37-48 hours, diluted to 1 × 10 ⁵ CFU / ml, and dispensed into a 96-well plate. Then, either the Pifipropolis extract powder or the Mangosteen extract powder was added to the bacterial culture solution so as to have an appropriate concentration. As a negative control, DMSO (1% DMSO bacterial culture solution) was added to the bacterial culture solution as the solvent of the Pipipropolis extract powder and the mangosteen extract powder, and the positive control was a culture medium containing 100 μg / ml of ampicillin (Table 1). The cells were cultured in 96-well plates at 37 ° C for 24 hours, and then cultured in bacterial culture. The cells were plated on agar medium and cultured for 48 hours at 37 ° C. The minimum concentration was determined as the MBC value. Each reaction was repeated three times.

group Pifipropolis extract powder (A) (μg / ml) Mangosteen extract powder (B) (μg / ml) (A) :( B) Negative control (1% DMSO) - - - Positive control (Ampicillin, 100 μg / ml) - - - Experiment 1 34 - 34: 0 Experiment 2 17 - 17: 0 Experiment group 3 8.5 - 8.5: 0 Experiment group 4 - One 0: 1 Experiment group 5 - 0.5 0: 0.5 Experiment group 6 34 One 34: 1 Experiment group 7 17 One 17: 1 Experiment group 8 8.5 One 8.5: 1 Experiment group 9 34 0.5 34: 0.5 Experiment group 10 17 0.5 17: 0.5 Experiment group 11 8.5 0.5 8.5: 0.5

II-2. Cytotoxicity experiment

Immortalized human gingival fibroblasts (hTERT-hNOF) used in this study were purchased from Professor Kim Jin, Department of Oral Pathology, Yonsei University. hTERT-hNOF cells were cultured in F medium (composed of Dulbecco's Modified Eagles Medium [DMEM] (Gibco, BRL, USA) and Ham's Nutrient Mixture F-12 (Gibco, 1 supplemented with 10% fetal bovine serum [FBS] (Hyclone, UT, USA) and 1% penicillin / streptomycin (Invitrogen, NY, USA] for the 37, 5% CO _2, 100% cell incubator which humidity is maintained using HTERT-hNOF cells were seeded at 5 × 10 ⁴ cells in a 24-well plate and cultured overnight. After 80% confluence, the haplotypic extract powder and the mangosteen extract powder mixture were used alone or in combination (MTT, Sigma) was added to each well and incubated for 4 hours.The resulting formazan crystals were dissolved in DMSO (50 mM Tris-HCl, , And the absorbance was measured at 570 nm using an ELISA reader, and the results were analyzed. Three times was performed.

group Pifipropolis extract powder (A) (μg / ml) Mangosteen extract
Powder (B) (占 퐂 / ml) LPS (A) :( B) Negative control (1% DMSO) - - (-) or (+) - Experiment 1 34 - (-) or (+) 34: 0 Experiment 2 17 - (-) or (+) 17: 0 Experiment group 3 8.5 - (-) or (+) 8.5: 0 Experiment group 4 - One (-) or (+) 0: 1 Experiment group 5 - 0.5 (-) or (+) 0: 0.5 Experiment group 6 34 One (-) or (+) 34: 1 Experiment group 7 17 One (-) or (+) 17: 1 Experiment group 8 8.5 One (-) or (+) 8.5: 1 Experiment group 9 34 0.5 (-) or (+) 34: 0.5 Experiment group 10 17 0.5 (-) or (+) 17: 0.5 Experiment group 11 8.5 0.5 (-) or (+) 8.5: 0.5

II-3. Anti-inflammatory experiment

One) P. gingivalis KCOM  2804 LPS  extraction

P. gingivalis causing inflammatory response to hTERT-hNOF cells The LPS of KCOM 2804 was extracted using the LPS extraction kit from Intron Company according to the enclosed protocol. The extracted LPS was lyophilized and weighed and used in the following experiment.

group Pifipropolis extract powder (A) (μg / ml) Mangosteen extract
Powder (B) (占 퐂 / ml) (A) :( B) Negative control [1% DMSO, LPS (-)] - - - Positive control [1% DMSO, LPS (+)] - - - Experimental group 1 [LPS (+)] 34 - 34: 0 Experimental group 2 [LPS (+)] 17 - 17: 0 Experimental group 3 [LPS (+)] 8.5 - 8.5: 0 Experimental group 4 [LPS (+)] - One 0: 1 Experimental group 5 [LPS (+)] - 0.5 0: 0.5 Experimental group 6 [LPS (+)] 34 One 34: 1 Experimental group 7 [LPS (+)] 17 One 17: 1 Experimental group 8 [LPS (+)] 8.5 One 8.5: 1 Experimental group 9 [LPS (+)] 34 0.5 34: 0.5 Experimental group 10 [LPS (+)] 17 0.5 17: 0.5 Experimental group 11 [LPS (+)] 8.5 0.5 8.5: 0.5

2) Inflammation related Cytokine  Measurement of gene expression level

hTERT-hNOF cells were seeded at 3 × 10 ⁵ cells in a 6-well plate overnight. After incubation at 80% confluence, the samples were incubated with P. gingivalis LPS (100 ng / ml) of KCOM 2804 was added. In this case, 200 ng / ml CD14 (R & D systems) and 50 ng / ml LPS-binding protein (LBP, R & D systems) were added together to increase the reactivity of HGF to LPS. IL-8 and PGE ₂ gene expression was measured by ELISA (human IL-6 ELISA kit, IL-8 ELISA kit, Biolegent, human PGE 2, ₂ ELISA kit (Enzo) according to the enclosed protocol and measured]. Each reaction was repeated three times. All experimental results were calculated as a percentage of each control. The negative control group was grown with medium supplemented with 1% of Pipipropolis extract powder and DMSO as a powdered mangosteen extract powder. In the positive control group, LPS, CD14 and LBP were added to the medium supplemented with 1% of DMSO Respectively. Each reaction was repeated three times. The experimental group is shown in Table 3.

II-4. goal Formability  ( in vitro  mineral formation experiments

1) Cell and cell culture

Human osteosarcoma (human osteoblast-like cells, MG-63) used in this experiment was purchased from Korean Cell Line Bank. 5% CO ₂ and 100% humidity were maintained in a cell culture medium containing 10% fetal bovine serum (FBS, PAA) and 1% penicillin / streptomycin in Dulbecco's Modified Eagles Medium (DMEM, Sigma) Lt; / RTI &gt; Table 4 shows the control and experimental groups used in the bone performance test.

2) Alkaline 포스화제  (ALP) activity measurement

The MG63 cell line in the 6 well plate 3 10 ⁵ (SIGMA A-4034) and 10 mM β-glycerophosphate (SIGMA G-9891) were added to the samples for 1, 4, 7, and 14 days after 80% confluence. (Dolder Jvd, Bancroft GN, Sikavitsas VI, Spauwen PHM, Flow perfusion culture of marrow stromal osteoblasts in titanium fiber mesh, J Biomed Mater Res 2002; 64A : 235-41). &Lt; / RTI &gt;

ALP activity is measured by the principle that colorless p-nitrophenyl phosphate becomes yellow reaction product p-nitrophenol at pH 10.4. First, each sample was lysed with cold Triton X-100, centrifuged, and the supernatant was mixed with 0.1 M glycine-NaOH buffer (pH 10.4) and 15 mM p-nitrophenyl phosphate (Sigma) Respectively. After that, 1.2N NaOH was mixed in the same amount to stop the reaction, transferred to an ELISA reader, and absorbance was measured at 405 nm. Protein concentration was measured using the BCA Protein Assay kit (Pierce, USA). Enzyme activity was calculated as μmole p-nitrophenol / min / μg protein.

3) Alizarin red staining

MG63 cell lines were seeded at 1 × 10 ⁵ cells / well in a 12-well plate and incubated overnight. After incubation at 80% confluence, samples were incubated with 50 μg / ml ascorbic acid (SIGMA A-4034) and 10 mM β-glycerophosphate (SIGMA G- And 28 days later, Reinholz et al. (Reinholz GG, Getz B, Pederson L, Sanders ES, Subramaniam M, Ingle JN, Spelsberg TC. Bisphosphonates directly regulate cell proliferation, differentiation, and gene expression in human osteoblasts. : 6001-7). Alizarin red staining was performed to observe the minerals produced in the extracellular matrix.

First, the medium was removed, washed with PBS, and fixed with 2.5% glutaraldehyde for 15 minutes at room temperature. After washing with PBS, the cells were stained with 2% Alizarin red (pH 4.2) solution for 10 minutes, washed with distilled water, and then calcified to a degree of red staining. After drying, it was dissolved in a resolving solution (20% methanol, 10% acetic acid), absorbance was measured at 450 nm with an ELISA reader, and the results were analyzed.

4) Osteoblast  Measurement of differentiated gene expression

(RUNX2), osterix (OSX = Sp7 transcription factor [SP7]), collagen type I alpha 1 chain (COL1A1) and osteocalcin (OCN), which are expressed specifically in osteoblast differentiation ELISA assay. MG63 cell line was seeded at 3 × 10 ⁵ cells in a 6-well plate overnight. After overnight incubation, samples were incubated with 50 μg / ml ascorbic acid (SIGMA A-4034) and 10 mM β-glycerophosphate (SIGMA G-9891) And cell culture supernatant and cell lysate were extracted 1, 2, 4, and 8 days later and ELISA was performed. ELISA was performed using the human runx2 ELISA kit (Biomatik), human osterix ELISA kit (Biomatik), human type I collagen ELISA kit (Biomatik) and human osteocalcin ELISA kit (Biomatik) Each reaction was repeated three times. All experimental results were calculated as a percentage of each control.

group Pifipropolis extract powder (A) (μg / ml) Mangosteen extract
Powder (B) (占 퐂 / ml) (A) :( B) Negative control (1% DMSO) - - - The positive control (BMP2, 10 ng / ml) - - - Experiment 1 34 - 34: 0 Experiment 2 17 - 17: 0 Experiment group 3 8.5 - 8.5: 0 Experiment group 4 - One 0: 1 Experiment group 5 - 0.5 0: 0.5 Experiment group 6 34 One 34: 1 Experiment group 7 17 One 17: 1 Experiment group 8 8.5 One 8.5: 1 Experiment group 9 34 0.5 34: 0.5 Experiment group 10 17 0.5 17: 0.5 Experiment group 11 8.5 0.5 8.5: 0.5

II-5. Statistical analysis

A comparative sample T test was conducted using the SPSS statistical program (version 12, SPSS Inc., Chicago, IL, USA) to compare the differences between the control and experimental groups. Respectively. Analysis showed that there was a significant difference between the two groups when the significance was less than 0.05 (significance level 5%) at 95% confidence level (p <0.05).

III. Test result

III-1. Results of antibacterial experiments

In the experimental group, P. gingivalis KCOM 2804, P. intermedia KCOM 1107, F. nucleatum subsp. polymorphum KCOM 1232 and A. actinomycetemcomitans KCOM 1306, T. forsythia ATCC 43037 ^T and T. denticolla ATCC 35405 ^T (Table 5). In Table 5, + indicates 99.9% bacterial killing, indicating that there is no antimicrobial activity.

Experimental group Aa
KCOM 1306 Pi
KCOM 1107 Pg
KCOM 2804 Fn
KCOM 1232 Td
ATCC 35405 ^T Tf
ATCC 43037 ^T Negative control group
(1% DMSO) - - - - - - The positive control (Ampicillin,
100 [mu] g / ml) + + + + + + Experiment 1 - - - - - - Experiment 2 - - - - - - Experiment group 3 - - - - - - Experiment group 4 - - - - - - Experiment group 5 - - - - - - Experiment group 6 - - - - - - Experiment group 7 - - - - - - Experiment group 8 - - - - - - Experiment group 9 - - - - - - Experiment group 10 - - - - - - Experiment group 11 - - - - - -

(Aa, Aggregatibacter actinomycetemcomitans ; Pi, Prevotella intermedia ; Pg, Porphyromonas gingivalis ; Fn, Fusbacterium nucleatum subsp. polymorphum ; Td, Treponema denticolla ; Tf, Tannerella forsythia )

III-2. Cytotoxicity test results

The cell survival rate for immortalized human gingival fibroblasts (hTERT-hNOF) in all the experimental groups used in this experiment was more than 80% (Table 6 and Fig. 1). In particular, the survival rate of the test group 9 (34: 0.5) was 23.3% higher than that of the negative control group (p <0.05). The P. gingivalis used in this experiment In order to investigate the cytotoxicity of human gingival fibroblasts (hTERT-hNOF) by KCOM 2804 LPS (100 ng / ml), it was found that the hTERT-hNOFDP cells There was no difference in cell viability, and P. gingivalis KCOM 2804 LPS (100 ng / ml) had no effect on cell viability (or cytotoxicity) on hTERT-hNOFDP cells (Table 6 and Fig. 1).

group Pifipropolis extract powder (A) (μg / ml) Mangosteen extract
Powder (B) (占 퐂 / ml) Relative cell viability (mean ± deviation [%]) Probability of significance
(Both sides) LPS (-) LPS (+) LPS (-) LPS (+) Negative control (1% DMSO) - - 100.0 + - 3.4 103.9 ± 8.1 - 0.301 Experiment 1 34 - 109.4 ± 9.0 102.6 + - 5.4 0.107 0.479 Experiment 2 17 - 103.3 ± 4.5 106.3 ± 6.0 0.317 0.158 Experiment group 3 8.5 - 92.2 ± 5.7 86.9 ± 1.4 0.037 0.012 Experiment group 4 - One 96.8 ± 0.9 93.8 ± 9.9 0.181 0.311 Experiment group 5 - 0.5 93.0 ± 6.9 95.1 ± 3.3 0.136 0.148 Experiment group 6 34 One 99.4 ± 5.3 102.4 ± 5.8 0.715 0.242 Experiment group 7 17 One 92.0 ± 5.5 87.8 ± 3.9 0.026 * 0.065 Experiment group 8 8.5 One 85.3 ± 7.7 87.2 ± 6.9 0.042 * 0.058 Experiment group 9 34 0.5 123.3 ± 1.3 124.1 ± 6.8 0.012 * 0.027 * Experiment group 10 17 0.5 112.7 ± 6.7 128.8 ± 9.0 0.110 0.014 * Experiment group 11 8.5 0.5 91.7 ± 2.8 93.5 ± 1.9 0.008 * 0.149

*: Significant difference between negative control and 95% confidence level was 0.05 (5%) or less (p <0.05).

III-3. Anti-inflammatory test result

P. gingivalis IL-6 of the extract of Pifipropolis and mangosteen extract on human gingival fibroblasts (hTERT-hNOF) treated with LPS from KCOM 2804 The expression levels of IL-1 and IL-6 in the experimental group 1 (34: 0), experimental group 2 (17: 0), experimental group 3 (8.5: 0), experimental group 6 (34: 6 Expression decreased by 11-23% (p <0.05) (Table 7 and Fig. 2).

group Pifipropolis extract powder (A) (μg / ml) Mangosteen extract
Powder (B) (占 퐂 / ml) Relative IL-6 expression level
(Mean ± deviation [%]) Probability of significance
(Both sides) Negative control group
[1% DMSO, LPS (-)] - - 5 ± 1 0.000 Positive control group
[1% DMSO, LPS (+)] - - 100 ± 1 - Experiment 1 34 - 83 ± 1 0.006 * Experiment 2 17 - 87 ± 3 0.006 * Experiment group 3 8.5 - 81 ± 1 0.000 * Experiment group 4 - One 91 ± 4 0.063 Experiment group 5 - 0.5 79 ± 2 0.006 * Experiment group 6 34 One 89 ± 5 0.046 * Experiment group 7 17 One 106 ± 2 0.047 * Experiment group 8 8.5 One 88 ± 2 0.004 * Experiment group 9 34 0.5 77 ± 2 0.004 * Experiment group 10 17 0.5 102 ± 3 0.414 Experiment group 11 8.5 0.5 83 ± 4 0.031 *

*: Significant difference between the positive control group and the 95% confidence level was 0.05 (5%) or less (p <0.05).

P. gingivalis IL-8 of the extract of Pifipropolis and mangosteen extract on human gingival fibroblast (hTERT-hNOF) treated with LPS from KCOM 2804 Expression of IL-8 in the experimental group 1 (34: 0), experimental group 3 (8.5: 0), experimental group 5 (0: 0.5) Expression decreased by 11-35% (p <0.05) (Table 8 and Fig. 3).

group Pifipropolis extract powder (A) (μg / ml) Mangosteen extract
Powder (B) (占 퐂 / ml) Relative IL-8 expression level
(Mean ± deviation [%]) Probability of significance
(Both sides) Negative control group
[1% DMSO, LPS (-)] - - 4 ± 0 0.000 Positive control group
[1% DMSO, LPS (+)] - - 100 ± 3 - Experiment 1 34 - 89 ± 2 0.016 * Experiment 2 17 - 91 ± 4 0.147 Experiment group 3 8.5 - 71 ± 4 0.014 * Experiment group 4 - One 83 ± 6 0.075 Experiment group 5 - 0.5 65 ± 2 0.006 * Experiment group 6 34 One 96 ± 5 0.482 Experiment group 7 17 One 119 ± 7 0.077 Experiment group 8 8.5 One 90 ± 4 0.113 Experiment group 9 34 0.5 93 ± 4 0.179 Experiment group 10 17 0.5 122 ± 6 0.050 * Experiment group 11 8.5 0.5 77 ± 4 0.004 *

*: Significant difference between the positive control group and the 95% confidence level was 0.05 (5%) or less (p <0.05).

P. gingivalis Pipipropolis extract of human gingival fibroblast (hTERT-hNOF) and PEG ₂ of mangosteen extract treated with LPS from KCOM 2804 Expression experiments showed a statistically significant decrease (p <0.05) in all the experimental groups compared to the positive control (Table 9 and FIG. 4). In particular, PEG ₂ (34: 0), experimental group 6 (34: 1) and experimental group 9 Expression decreased by 78-82% (p <0.05) (Table 9 and Fig. 4).

group Pifipropolis extract powder (A) (μg / ml) Mangosteen extract
Powder (B) (占 퐂 / ml) Relative PEG ₂ Expression level
(Mean ± deviation [%]) Probability of significance
(Both sides) Negative control group
[1% DMSO, LPS (-)] - - 22 ± 0 0.000 Positive control group
[1% DMSO, LPS (+)] - - 100 ± 1 - Experiment 1 34 - 18 ± 0 0.000 * Experiment 2 17 - 39 ± 1 0.000 * Experiment group 3 8.5 - 59 ± 0 0.000 * Experiment group 4 - One 80 ± 2 0.005 * Experiment group 5 - 0.5 74 ± 3 0.002 * Experiment group 6 34 One 18 ± 0 0.000 * Experiment group 7 17 One 42 ± 2 0.000 * Experiment group 8 8.5 One 65 ± 1 0.001 * Experiment group 9 34 0.5 22 ± 0 0.000 * Experiment group 10 17 0.5 40 ± 3 0.001 * Experiment group 11 8.5 0.5 53 ± 3 0.001 *

*: Significant difference between the positive control group and the 95% confidence level was 0.05 (5%) or less (p <0.05).

P. gingivalis IL-12, IL-10, and IL-12 in the human gingival fibroblast (hTERT-hNOF) treated with LPS from KCOM 2804 except the three inflammatory cytokines (IL-6, IL-8 and PGE ₂ ) -1β, TNF-α, COX-1, and COX-2 (Table 10). As a result, IL-12, IL-10, IL-1β and TNF-α were not expressed in hTERT-hNOF cells and P. gingivalis But not at 1 μg / ml LPS extracted from KCOM 2804 (Table 10). In addition, IL-12, IL-10, IL-1β and TNF-α were also expressed in hTERT-hNOF cells against LPS extracted from P. intermedia (KCOM 1107) and F. nucletatum (KCOM 1232) It was not.

Expression of iNOS, COX-1 and COX-2 was also observed in hTERT-hNOF cells but there was almost no induction of LPS from P. gingivalis KCOM 2804, P. intermedia KCOM 1107 and F. nucletatum KCOM 1232 (Table 10) .

For this reason, no further experiments were performed with iNOS, IL-12, IL-10, IIL-1β, TNF-α, COX-1 and COX-2.

Experimental group The relative expression level (fold) with the negative control group IL-6 IL-8 PGE ₂ iNOS COX-1 COX-2 IL-12 IL-10 IL-1b TNF-a Medium (negative control) 1.0 1.0 1.0 1.0 1.0 1.0 ND * ND ND ND P. gingivalis KCOM 2804
LPS (0.1 μg / ml) 20.0 24.0 4.5 1.0 1.2 1.1 ND ND ND ND P. gingivalis KCOM 2804
LPS (1 [mu] g / ml) 27.8 16.8 6.2 1.1 1.3 1.3 ND ND ND ND P. intermedia KCOM 1107
LPS (0.1 μg / ml) 4.3 2.4 3.6 1.1 1.1 1.0 ND ND ND ND P. intermedia KCOM 1107
LPS (1 [mu] g / ml) 3.7 2.2 2.2 1.1 1.3 1.3 ND ND ND ND F. nucletatum KCOM 1232
LPS (0.1 μg / ml) 6.5 3.7 4.5 1.1 0.9 1.1 ND ND ND ND F. nucletatum KCOM 1232
LPS (1 [mu] g / ml) 15.6 8.2 5.9 1.1 0.9 1.1 ND ND ND ND

* ND: not detected

III-4. goal Formability  ( in vitro  mineral formation)

1) Alkaline 포스화제  (ALP) activity measurement results

The results of the experiment on the ALP activity of the extract of Pifipropolis and Mangosteen on the human osteosarcoma cell line MG63 cell line showed that the mangosteen extract showed a statistically significant increase from the 7th day when compared with the negative control group (P <0.05) (Table 11 and Fig. 5). From the 14th day, ALP activity was significantly increased in all experimental groups except the experimental group 3 (8.5: 0) and the experimental group 5 (0: 0.5) compared to the negative control group (Table 11 and FIG. 5). In MG63 cells treated with rhBMP2 as a positive control, ALP activity was increased compared with the negative control group except for 14 days (p <0.05, Table 11 and Fig. 5).

Groups ALP activity mean ± deviation (%) Probability of significance (both sides) 1 day 4 days 7 days 14 days 1 day 4 days 7 days 14 days (-) Control 100.0 + - 12.0 100.0 + - 6.8 100.0 ± 2.6 100.0 9.7 - - - - (+) Control 166.7 ± 0.0 168.9 ± 2.2 128.5 ± 11.5 89.5 ± 3.8 0.011 * 0.005 * 0.052 * 0.170 Experiment 1 105.6 ± 4.4 111.5 ± 4.4 96.2 ± 6.5 146.3 ± 5.3 0.614 0.115 0.452 0.027 * Experiment 2 95.1 ± 18.0 138.9 ± 16.2 103.9 ± 3.8 131.1 ± 2.7 0.594 0.091 0.081 0.018 * Experiment group 3 91.2 ± 8.7 115.7 ± 17.8 115.4 ± 24.2 106.9 ± 5.5 0.532 0.162 0.343 0.502 Experiment group 4 92.2 ± 2.8 118.6 ± 12.2 132.0 ± 5.1 152.9 ± 5.2 0.457 0.179 0.002 * 0.020 * Experiment group 5 88.1 ± 4.6 105.8 ± 8.9 134.7 ± 2.6 144.3 ± 10.1 0.230 0.497 0.005 * 0.060 Experiment group 6 104.8 ± 4.0 96.2 ± 5.0 122.8 ± 3.4 162.4 ± 3.2 0.652 0.629 0.004 * 0.014 * Experiment group 7 96.4 ± 12.7 109.1 ± 5.3 135.6 ± 17.0 190.3 ± 17.3 0.824 0.127 0.063 0.021 * Experiment group 8 97.5 ± 5.9 97.1 + - 10.3 117.6 ± 6.5 180.8 ± 3.0 0.776 0.770 0.038 * 0.003 * Experiment group 9 112.9 ± 10.0 102.2 ± 5.3 124.6 ± 4.7 196.2 ± 2.1 0.133 0.784 0.009 * 0.003 * Experiment group 10 106.5 ± 3.8 101.7 ± 8.3 140.7 ± 3.9 180.8 ± 4.3 0.315 0.857 0.006 * 0.009 * Experiment group 11 100.2 ± 2.4 146.3 ± 8.8 112.3 ± 8.1 148.0 + - 9.3 0.977 0.001 * 0.128 0.048 *

*: Significant difference between negative control and 95% confidence level was 0.05 (5%) or less (p <0.05).

2) Results of Alizarin red S staining

In order to investigate the mineralization activity of MG63 cells, a human osteosarcoma cell line, propolis extract and mangosteen extract were tested by Alizarin red S staining. The positive control group (10 ng / ml rhBMP2 treated group) (17: 1), experimental group 8 (8.5: 1), experimental group 9 (34: 0.5), experimental group 10 (17: (P <0.05) (Figs. 6 and 7 and Table 12) in the experimental group 11 (8.5: 0.5) and the negative control group (9.3-37.4%).

Murphy et al. (Murphy MG, Mailhot J, Borke J, Wataha J, Sharawy M, Smith A. Effects of rhBMP-2 on human osteosarcoma cells and human gingival fibroblasts in vitro. J Oral Implantol 2001; 27: 16-24) Reported a decrease in bone mineral formation (analyzed by von Kosa staining) in MG63 cells treated with 10 ng / ml rhBMP2 for 3 days (after 18 days of culture), and 10 ng / ml rhBMP2 inhibited the mineralization of MG63 cells . However, in this experiment, the MG63 cell group treated with 10 ng / ml rhBMP2 showed about twice as much inorganic material as the negative control group (Table 12 and FIGS. 6 and 7). This may be due to the difference between the two experimental methods. In other words, in Murphy's experiment, rhBMP2 was treated after culturing for 18 days. In this experiment, it is thought that when cells were cultured, rhBMP2 was continuously treated every time the medium was changed for 4 weeks. Recent studies have shown that osteoblasts are more likely to undergo apoptosis in BMP2 treatment than in mature osteoblasts (human normal osteoblasts> MG63 cells> undifferentiated mesenchymal stem cells) (Hyzy SL, Olivares-Navarrete R, Schwartz Z, Boyan BD. BMP2 induces osteoblast apoptosis in a maturation state and noggin-dependent manner. J Cell Biochem. 2012 Oct; 113 (10): 3236-45).

group Pifipropolis extract powder (A) (μg / ml) Mangosteen extract
Powder (B) (占 퐂 / ml) Alizarin Red S stain (% of control) Average (%) Deviation (%) Probability of significance (both sides) Negative control group
(1% DMSO) - - 100.0 0.0 - Positive control group
(rhBMP2, 10 ng / ml) - - 201.0 ± 19.2 0.06 * Experiment 1 34 - 137.4 ± 15.1 0.026 * Experiment 2 17 - 104.8 ± 5.2 0.153 Experiment group 3 8.5 - 97.0 ± 6.2 0.356 Experiment group 4 - One 109.3 ± 4.3 0.033 * Experiment group 5 - 0.5 100.0 ± 3.1 0.991 Experiment group 6 34 One 137.8 ± 18.8 0.039 * Experiment group 7 17 One 123.0 + - 8.3 0.021 * Experiment group 8 8.5 One 120.3 ± 0.3 0.000 * Experiment group 9 34 0.5 129.1 ± 11.7 0.026 * Experiment group 10 17 0.5 120.1 ± 11.1 0.047 * Experiment group 11 8.5 0.5 120.0 ± 0.9 0.000 *

*: Significant difference between negative control and 95% confidence level was 0.05 (5%) or less (p <0.05).

3) Osteoblast  Results of differentiation gene expression measurement

RUNX2 is a transcription factor involved in the expression of collagen type I alpha 1 chain (COL1A1), and RUNX2 and OSX is a transcription factor involved in osteocalcin (OCN) expression. RUNX2 has been reported to induce differentiation into mesenchymal stem cells into preosteoblasts and to block differentiation into adipocytes and OSX plays a role in promoting the differentiation of preosteoblasts into mature osteoblasts (Komori T, Yagi H, Nomura S, Yamaguchiya, Sasaki K, Deguchi K, Shimizu Y, Bronson RT, Gao YH, Inada M, Sato M, Okamoto R, Kitamura Y, Yoshiki S, Kishimoto T. Targeted disruption of Cbfa 1 results in a complete lack of bone Formation owing to maturational arrest of osteoblasts. Cell 1997; 89: 755-764; and Nakashima K, Zhou X, Kunkel G, Zhang Z, Deng JM, Behringer RR, de Crombrugghe B. The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation. Cell 2002; 108: 17-29). Type I collagen is a major substrate protein of bone tissue, and osteocalcin is the most abundant of type I collagen in bone tissue proteins and is closely related to bone mineralization (calcification). For this reason, RUNX2, OSX, COL1A1, and OCN are known to be the most important osteoblast differentiation marker genes.

RUNX2 tended to increase on days 4 and 8 (Table 13 and Figure 8) in the experimental group to which the Pifipropolis extract and Mangosteen extract were added. RUNX2 expression was significantly increased (P <0.05) in the experimental group 5, the experimental group 6, the experimental group 8, and the experimental group 9 on days 4 and 8 compared with the negative control group. In contrast, BMP2-treated MG63 cells showed maximal RUNX2 expression on day 2 and then decreased in proportion to time (P <0.05).

OSX expression was higher in the experimental groups 1, 2, 3, 4 and 5 than in the negative control group on day 4 (P <0.005), but the expression levels were similar or decreased in the other experimental groups (Table 14 and FIG. In the MG63 cells treated with BMP2 used as a control, OSX expression was decreased in proportion to time from day 2 compared to negative control (P <0.05).

COL1A1 showed a similar expression level to negative control group in the experimental group 5 (0: 0.5) and the experimental group 8 (8.5: 1) on the first day after the treatment with the pepropropolis extract and the mangosteen extract, (Table 15 and Figure 10). In particular, the experimental group 1 (34: 0), the experimental group 2 (17: 0), the experimental group 3 (8.5: 0), the experimental group 4 (0: The expression level of COL1A1 was similar to or greater than that of the positive control (BMP2 10 ng / ml) in the experimental group 9 (34: 0.5), the experimental group 10 (17: 0.5) and the experimental group 11 (8.5: 0.5). On the second day after treatment with the extracts of Pifipropolis and Mangosteen, the expression level of COL1AI in the positive control group was similar to that of the negative control group, but decreased in most experimental groups (Table 15 and FIG. 10). On the 4th and 8th day after the treatment with the extract of Pifipropolis and Mangosteen, the expression of COL1A1 was significantly increased in the positive control (p <0.05). However, the expression of COL1A1 in the negative control group was decreased by 15.4-5.2% in most experimental groups.

On the first day of treatment with Pepipropolis extract and Mangosteen extract, OCN was observed in the experimental group 1 (34: 0), experimental group 2 (17: 0), experimental group 6 (34: 1), experimental group 7 (P <0.05) (Table 16 and FIG. 11), whereas the expression levels were decreased in the other experimental groups (p <0.05) . However, from day 2, the expression level of the control group was similar or increased. In particular, in the experimental group 2 (17: 0), the amount of OCN expression increased from day 2 (p <0.05). On the 8th day after treatment with the extract of Pifipropolis and Mangosteen, the experimental group 3 (8.5: 0), the experimental group 4 (0: 1), the experimental group 5 (0: 0.5) OCN expression was increased by 17.3-64.9% (p <0.05) in the control group (8: 8.5: 1) and the experimental group (10: 17: 0.5)

Groups RUNX2 Relative expression level [mean ± deviation (%)] Probability of significance (both sides) 1 day 2 days 4 days 8 days 1 day 2 days 4 days 8 days (-) Control 100.0 ± 3.1 100.0 ± 7.1 100.0 + - 8.8 100.0 7.7 - - - - (+) Control 78.8 ± 3.3 141.7 ± 2.0 101.3 ± 3.9 66.8 ± 3.2 0.005 * 0.010 * 0.841 0.022 * Experiment 1 109.3 ± 8.8 52.0 ± 9.3 148.9 ± 15.1 168.3 ± 13.8 0.123 0.013 * 0.053 0.017 * Experiment 2 60.4 ± 5.0 86.1 ± 9.5 141.9 ± 22.0 166.4 ± 16.2 0.002 * 0.122 0.085 0.034 * Experiment group 3 75.9 ± 8.2 48.9 ± 12.1 115.4 ± 7.8 125.0 + - 11.3 0.058 0.036 * 0.197 0.055 Experiment group 4 77.4 ± 11.7 47.3 ± 5.7 121.8 ± 4.1 143.9 ± 15.9 0.082 0.007 * 0.053 0.016 * Experiment group 5 71.6 13.8 46.7 ± 9.0 145.1 ± 20.9 191.1 ± 20.1 0.086 0.006 * 0.040 * 0.021 * Experiment group 6 70.4 ± 3.5 86.5 ± 4.4 146.5 ± 7.4 148.9 ± 16.4 0.011 * 0.115 0.014 * 0.039 * Experiment group 7 13.2 ± 1.0 45.6 ± 4.0 130.6 ± 7.4 139.7 ± 14.5 0.000 * 0.004 * 0.058 0.054 Experiment group 8 70.7 ± 9.7 42.1 ± 8.5 169.1 ± 17.0 128.9 ± 3.4 0.035 * 0.003 * 0.015 * 0.016 * Experiment group 9 120.2 ± 4.8 63.7 ± 3.6 145.9 ± 3.5 110.4 ± 6.9 0.045 * 0.004 * 0.008 * 0.008 * Experiment group 10 70.2 ± 7.7 33.0 ± 5.8 141.7 ± 13.2 115.2 ± 7.7 0.014 * 0.000 * 0.053 0.008 * Experiment group 11 71.9 ± 3.4 54.5 ± 6.3 128.7 ± 5.1 124.7 ± 5.8 0.008 * 0.001 * 0.019 * 0.003 *

*: Significant difference between negative control and 95% confidence level was 0.05 (5%) or less (p <0.05).

Groups Osterix Relative expression level [mean ± deviation (%)] Probability of significance (both sides) 1 day 2 days 4 days 8 days 1 day 2 days 4 days 8 days (-) Control 100.0 + - 11.3 100.0 + - 12.4 100.0 0.0 100.0 8.3 - - - - (+) Control 73.8 ± 6.3 73.0 ± 9.3 30.4 ± 1.1 16.4 ± 1.3 0.112 0.013 * 0.000 * 0.002 * Experiment 1 113.3 ± 0.2 96.8 ± 6.8 181.8 + - 16.4 76.7 ± 12.9 0.178 0.719 0.013 * 0.040 * Experiment 2 115.4 ± 5.6 104.0 ± 10.1 199.4 ± 23.9 94.0 ± 6.1 0.082 0.650 0.019 * 0.445 Experiment group 3 107.4 ± 3.8 89.6 ± 11.7 177.1 ± 19.7 80.1 ± 13.7 0.263 0.073 0.021 * 0.122 Experiment group 4 99.6 ± 5.7 89.5 ± 1.7 159.3 ± 17.5 96.9 ± 3.3 0.944 0.239 0.028 * 0.414 Experiment group 5 158.3 ± 3.5 93.1 ± 9.4 146.9 ± 15.9 118.7 ± 4.9 0.007 * 0.627 0.036 * 0.057 Experiment group 6 84.2 ± 7.1 86.2 ± 8.6 83.6 ± 6.4 64.8 ± 9.5 0.037 * 0.370 0.047 * 0.011 * Experiment group 7 60.7 ± 9.0 87.2 ± 1.9 93.5 ± 10.9 88.5 ± 12.3 0.073 0.231 0.411 0.122 Experiment group 8 78.2 + - 11.4 80.5 ± 0.7 97.8 ± 12.8 91.2 ± 2.9 0.010 * 0.119 0.799 0.099 Experiment group 9 94.3 ± 8.9 80.2 ± 8.2 95.1 ± 4.0 74.6 ± 5.3 0.611 0.127 0.170 0.022 * Experiment group 10 104.4 ± 2.7 85.7 ± 3.9 96.1 ± 8.4 114.5 ± 3.4 0.512 0.245 0.508 0.112 Experiment group 11 104.5 ± 15.2 81.3 ± 5.2 93.4 ± 5.0 89.3 ± 1.3 0.535 0.052 0.153 0.078

*: Significant difference between negative control and 95% confidence level was 0.05 (5%) or less (p <0.05).

Groups Type I collagen α Relative expression level [Mean ± deviation (%)] Probability of significance (both sides) 1 day 2 days 4 days 8 days 1 day 2 days 4 days 8 days (-) Control 100.0 ± 0.5 100.0 ± 2.3 100.0 ± 8.1 100.0 + - 5.0 - - - - (+) Control 130.4 ± 17.9 116.1 ± 19.0 168.2 ± 15.7 679.4 ± 55.3 0.095 0.259 0.023 * 0.003 * Experiment 1 192.7 ± 8.8 46.2 ± 12.8 108.9 ± 10.6 84.6 ± 6.1 0.001 * 0.014 * 0.405 0.129 Experiment 2 134.3 ± 13.7 53.5 ± 5.8 106.3 ± 5.0 79.2 ± 2.0 0.050 * 0.009 * 0.273 0.031 * Experiment group 3 170.2 + 21.3 78.7 ± 10.1 112.9 ± 2.2 92.9 ± 7.1 0.028 * 0.067 0.061 0.103 Experiment group 4 142.3 ± 14.9 82.0 ± 3.0 99.6 ± 2.2 87.2 ± 4.2 0.039 * 0.027 * 0.897 0.101 Experiment group 5 109.4 ± 15.9 62.0 + - 6.7 103.6 ± 5.8 85.5 ± 3.4 0.418 0.006 * 0.623 0.004 * Experiment group 6 110.6 ± 2.8 81.4 ± 8.7 97.6 ± 6.0 91.4 ± 1.8 0.014 * 0.037 * 0.003 * 0.049 * Experiment group 7 157.7 ± 13.6 92.1 ± 10.8 100.7 ± 7.7 90.2 ± 3.4 0.009 * 0.242 0.930 0.096 Experiment group 8 96.4 ± 14.7 77.9 ± 6.2 84.3 ± 9.2 93.5 ± 5.3 0.705 0.028 * 0.036 * 0.242 Experiment group 9 133.4 ± 2.0 76.0 7.7 112.7 ± 6.0 92.0 + - 6.4 0.000 * 0.022 * 0.118 0.345 Experiment group 10 136.5 ± 16.6 68.4 ± 13.4 104.6 ± 10.8 89.9 ± 2.6 0.065 0.063 0.599 0.128 Experiment group 11 142.8 ± 1.5 81.4 ± 3.3 92.9 ± 3.1 94.8 ± 3.1 0.000 * 0.010 * 0.110 0.150

*: Significant difference between negative control and 95% confidence level was 0.05 (5%) or less (p <0.05).

Groups Relative expression level of osteocalcin [mean ± deviation (%)] Probability of significance (both sides) 1 day 2 days 4 days 8 days 1 day 2 days 4 days 8 days (-) Control 100.0 + - 0.4 100.0 ± 3.3 100.0 + - 8.8 100.0 ± 3.5 - - - - (+) Control 83.8 ± 4.2 74.8 ± 3.9 64.6 ± 0.7 37.2 ± 5.6 0.011 * 0.022 * 0.022 * 0.001 * Experiment 1 106.7 ± 14.5 106.6 ± 1.2 117.1 ± 11.9 112.7 ± 7.5 0.498 0.124 0.288 0.117 Experiment 2 108.8 ± 13.6 127.5 ± 2.8 134.7 ± 11.5 141.3 ± 12.8 0.388 0.002 * 0.011 * 0.047 * Experiment group 3 80.5 ± 4.3 115.7 ± 6.8 108.9 ± 10.4 117.3 ± 2.7 0.014 * 0.067 0.505 0.039 * Experiment group 4 85.6 ± 11.0 112.5 ± 6.1 106.7 ± 7.0 128.3 ± 6.5 0.147 0.045 * 0.288 0.009 * Experiment group 5 77.0 ± 8.5 122.3 + - 11.8 121.9 ± 12.0 164.9 ± 8.3 0.040 * 0.123 0.082 0.010 * Experiment group 6 108.0 ± 10.5 107.4 ± 9.6 111.1 ± 3.1 104.7 ± 5.6 0.319 0.390 0.184 0.291 Experiment group 7 62.6 ± 7.3 118.4 ± 3.2 95.0 ± 6.0 110.4 ± 9.4 0.012 * 0.010 * 0.239 0.241 Experiment group 8 81.4 ± 4.8 116.4 ± 9.9 110.4 ± 13.9 124.2 ± 2.8 0.018 * 0.084 0.426 0.021 * Experiment group 9 83.1 + - 13.4 98.5 ± 4.6 90.5 ± 6.7 102.7 + 1.7 0.157 0.712 0.231 0.431 Experiment group 10 97.6 ± 7.9 107.7 ± 4.7 92.7 ± 8.8 124.2 ± 8.0 0.632 0.200 0.240 0.049 * Experiment group 11 78.3 ± 3.3 100.7 ± 3.4 98.0 ± 7.5 102.7 ± 10.8 0.008 * 0.819 0.758 0.712

*: Significant difference between negative control and 95% confidence level was 0.05 (5%) or less (p <0.05).

IV. Summary of test results

Combining anti-inflammatory and osseointegration performance experiments, the efficacy of Experiment 9 (34 μg / ml of propolis extract and 0.5 μg / ml of mangosteen extract) was the highest (Table 17).

In the case of propolis extract (34 μg / ml), P. gingivalis The highest inhibition of PEG ₂ expression was observed in human gingival fibroblasts (hTERT-hNOF) treated with KCOM 2804 and mangosteen extract (0.5 μg / ml) inhibited the expression of IL-6 and IL-8 (Table 17).

In the evaluation of bone morphology, the results of ALP activity and Alizarin Red S staining showed that the combination of Pifipropolis extract and Mangosteen extract had better bone performance than those used alone (Table 17).

group Pifipropolis extract powder (A) (μg / ml) Mangosteen extract
Powder (B) (占 퐂 / ml) Anti-inflammatory properties * Bone Performance Rating ** All rankings
Sum IL-6 IL-8 PEG ₂ Ranking subtotal ALP
activation
(14 days) Alizarin Red S staining Ranking subtotal Experiment 1 34 - 4 5 One 10 8 2 10 20 Experiment 2 17 - 5 7 4 16 10 9 19 35 Experiment group 3 8.5 - 3 2 8 13 11 11 22 35 Experiment group 4 - One 8 4 11 23 6 8 14 37 Experiment group 5 - 0.5 2 One 10 13 9 10 19 32 Experiment group 6 34 One 7 9 One 17 5 One 6 23 Experiment group 7 17 One 10 10 6 26 2 4 6 32 Experiment group 8 8.5 One 6 6 9 21 3 5 8 29 Experimental group  9 34 0.5 One 8 2 11 One 3 4 15 Experiment group 10 17 0.5 9 11 5 25 3 6 9 34 Experiment group 11 8.5 0.5 4 3 7 14 7 7 14 28

*: The lower the ranking (number), the greater the anti-inflammatory activity.

**: The lower the ranking (numerical value), the greater the bone performance.

Claims (11)

Wherein the weight ratio of the mangosteen extract to the propolis extract is 0.2 to 2: 5 to 40, wherein the weight ratio of the mangosteen extract to the propolis extract is 0.2: 2: 5 to 40: delete The pharmaceutical composition for accelerating bone formation according to claim 1, wherein the weight ratio of the mangosteen extract: propolis extract is 0.5: 34. 2. The method according to claim 1, further comprising at least one member selected from the group consisting of soybean unsaponifiable matter, natto culture, water soluble calcium, vitamin C, xylitol, vitamin E, chamomile extract and brewer's yeast. A pharmaceutical composition. 5. The composition of claim 4, wherein said composition comprises 7 to 13% by weight of soybean insolubles, 5 to 10% by weight of natto culture, 2 to 10% by weight of water-soluble calcium, 3 to 10% by weight of vitamin C, 7 to 7 wt%, vitamin E 0.5 to 2 wt%, chamomile extract 10 to 15 wt%, and brewer's yeast 1 to 5 wt%. The pharmaceutical composition for promoting bone formation according to claim 1, wherein the mangosteen extract contains alpha-mangosteen in an amount of 7.5 to 9.0 mg / 1 dose unit. A pharmaceutical preparation for preventing or treating alveolar bone destruction at the time of periodontal disease by the formation of a destroyed alveolar bone comprising the composition according to any one of claims 1 and 3 to 6. delete 7. An implant assistant preparation, comprising a composition according to any one of claims 1 and 6 to form a fractured alveolar bone. Wherein the weight ratio of the mangosteen extract to the propolis extract is 0.2 to 2: 5 to 40, wherein the weight ratio of the mangosteen extract to the propolis extract is 0.2 to 2: 5 to 40. A health functional food for preventing or alleviating alveolar bone destruction in periodontal disease, comprising the composition according to claim 10.

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