KR102344959B1 - Composition for improving osteointegration of implants comprising rosmarinus officinalis extract - Google Patents

Abstract

The present invention relates to a composition for improving osseointegration of an implant comprising a rosemary extract, and the effect of MC3T3-E1 preosteoblasts on the surface of the implant body and the effect of focal adhesion on the surface of the implant body It is possible to improve the osseointegration between the jawbone and the implanted body surface by improving the cell proliferation effect.
Furthermore, it is possible to provide a pharmaceutical composition and a functional food composition for improving osseointegration of implants comprising a rosemary extract as an active ingredient.

Description

Composition for improving osseointegration of implants comprising rosemary extract

The present invention relates to a composition for improving osseointegration of an implant comprising a rosemary extract. More particularly, it relates to a composition capable of improving osseointegration between the jawbone and an implanted implant body surface.

A dental implant is a structure that is placed in the jawbone to serve as a root when a tooth is damaged or lost. . The implant body (fixture) is a portion serving as a root of a tooth, and the abutment (abutment) is a portion serving to connect the crown and the implant body and corresponds to the neck of a natural tooth. The upper prosthesis of the implant corresponds to the head of the tooth in natural teeth.

By using this dental implant treatment, the implant body can be placed in the upper and lower jaw bones instead of the natural teeth in the area where the natural teeth are lost, and the oral function and the function of the teeth can be stably restored. Loss of dental function not only causes great discomfort in food intake, but also causes additional problems such as digestive disorders and deformities, so implants are widely recognized as a universal treatment method in recent years.

A close osseointegration is required between the surface of the implant body and the jawbone in order to restore the original function and aesthetic function of teeth through surgery or treatment using implants.

As factors influencing the functional recovery and stable osseointegration of the implant, the material, design, surface characteristics, bone mass and quality of the implant, surgical procedure, load conditions, and the oral environment of the patient can be exemplified. Therefore, when implants are implanted in the alveolar bone in the oral cavity, it is necessary to select a material with good biocompatibility to the living tissue and select a material that has no biochemical side effects with the living tissue, specifically, the material of the implant. Metal materials such as titanium (Ti) or titanium (Ti) alloy, cobalt alloy, stainless steel, platinum, iridium, niobium, and tantalum may be used as the example.

Among the metals exemplified above, titanium (Ti) or a titanium (Ti) alloy has good biocompatibility, corrosion resistance, strength, toughness and durability, and in particular, when used as an implant material, dissolution rate Since the fusion is slow and the lysate is chemically inert, it allows bone growth and effectively induces an osseointegration reaction at the interface with the bone. However, titanium (Ti) or titanium (Ti) alloy releases cytotoxic metal ions to inhibit cell proliferation of osteoblasts on the surface of the implant body, and thus successful osseointegration is not achieved. However, there is a problem that diseases such as peri-implantitis and periodontitis may occur.

In order to overcome the above-mentioned problems, various attempts have been made to increase the success rate of implants and shorten the healing period by giving bioactivity to the implant through surface treatment such as coating the surface of the metallic implant body with a bioactive material. has been made As such, surface treatment that imparts activity to the surface of titanium (Ti) or titanium (Ti) alloy can induce rapid osseointegration without inducing inflammation in the alveolar bone. can reduce

Recently, due to the rapid development of nanotechnology, a method of forming a nanostructured oxide layer on a metal surface such as titanium (Ti) has been studied in the field of implant materials. For example, _{it is reported that a uniform nanotube TiO 2} layer expands the surface area in contact with the bone and thus acts favorably on osseointegration, and osseointegration reaction by the hydroxyapatite coating method It is known that silver exhibits a tendency to peel from a thick film layer because a strong bond between the titanium (Ti) surface and the coating layer is difficult.

On the other hand, a calcification cycle treatment method that has a similar effect to the hydroxyapatite coating method and can form a thin film layer is used, and the surface of chemical acid treatment, spray treatment using fine particles, anodization treatment, plasma spray treatment, etc. Treatment methods are also being used. Through this, a technique for stabilizing the formation of a blood clot at the interface between the implant and the bone, promoting the differentiation of osteoblast cells, and also improving the bone formation around the implant in the early stage after the procedure has been reported. .

However, in this method, the hydrophilicity is reduced by the growth of the oxide layer and the adsorption of various contaminants after surface treatment, thereby inevitably lowering the osseointegration. In order to alleviate this problem, a method of surface treatment with a mixed solution containing a metal salt and an organic compound having a hydrophilic group such as sugars and proteins after performing a pretreatment such as sand blasting (Korean Patent No. 1404632) ), a method of surface treatment with a solution containing a sugar alcohol such as glycerol, sorbitol, and xylitol (Korean Patent No. 1304217) has been known. In addition, a cyclic molecule is used to form a polyrotaxane-like polyrotaxane on a biocompatible linear polymer having a first functional group at both ends, such as polyethylene glycol (PEG), and capping ( A technology of chemically bonding with a capping molecule to convert it into polyotaxane, then combining it with an implant base into which a third functional group that reacts with a second functional group in the capping molecule is introduced and hydrolyzing it (Korean Patent Publication) No. 2012-133588) was also developed.

However, even with the implant according to the method reported in the prior art, osseointegration that forms bone at the interface between the implant body and the surrounding tissue cannot be formed to a satisfactory level, and the surface-treated coating layer may be removed. There is a problem.

Therefore, as a fundamental method to improve osseointegration, cell adhesion of preosteoblasts to the implant surface and osteoblasts ( In order to improve the process of osseointegration between the jawbone and the surface of the implant body by promoting differentiation and proliferation into osteoblast), the present applicant provides a composition for improving osseointegration of an implant comprising a natural extract, rosemary extract, as an active ingredient came to invent

KR 10-1404632 B1 KR 10-1304217 B1

It is an object of the present invention to provide a composition for improving osseointegration of an implant comprising a rosemary extract.

Another object of the present invention is to improve the effect of MC3T3-E1 preosteoblasts on the surface of the implant body and the cell proliferation effect on the surface of the implant body, so that the surface between the jawbone and the implanted body surface is excellent. To provide a composition for improving osseointegration of an implant capable of improving osseointegration.

Another object of the present invention is to provide a pharmaceutical composition for improving osseointegration of implants comprising a rosemary extract as an active ingredient.

Another object of the present invention is to provide a functional food composition for improving osseointegration of implants comprising a rosemary extract as an active ingredient.

In order to achieve the above object, the composition for improving osseointegration of an implant according to an embodiment of the present invention contains a rosemary extract as an active ingredient, and an osseointegration effect between the jawbone and the implanted body surface. is excellent

The composition modulates the FAK/Paxillin signaling pathway, and thus has excellent focal adhesion effect of MC3T3-E1 preosteoblasts on the surface of the implant body.

The composition modulates the FAK/Grab2/Ras/ERK1,2 signaling pathway, and thus has an excellent effect on the proliferation of MC3T3-E1 preosteoblasts on the surface of the implant body.

The rosemary extract is rosmarinic acid.

The extract is extracted using an extraction solvent selected from the group consisting of water, alcohols having 1 to 4 carbon atoms, ether, chloroform, ethyl acetate, dimethyl sulfoxide, and mixtures thereof.

A pharmaceutical composition for improving osseointegration of an implant according to another embodiment of the present invention includes the composition.

A functional food composition for improving osseointegration of implants according to another embodiment of the present invention includes the composition.

Hereinafter, the present invention will be described in more detail.

As used herein, the term 'extract' has a meaning commonly used as a crude extract in the art, but in a broad sense also includes a fraction obtained by additionally fractionating the extract. That is, the plant extract includes not only those obtained by using the above-described extraction solvent, but also those obtained by additionally applying a purification process thereto. For example, a fraction obtained by passing the extract through an ultrafiltration membrane having a constant molecular weight cut-off value, separation by various chromatography (prepared for separation according to size, charge, hydrophobicity or affinity), etc. Fractions obtained through various purification methods are also included in the extract of the present invention.

The extract used in the present invention may be prepared in a powder state by an additional process such as distillation under reduced pressure and freeze-drying or spray-drying.

As used herein, the term "comprising as an active ingredient" refers to an amount sufficient to affect a desired biological effect, such as, but not limited to, beneficial results, including, but not limited to, prevention, reduction, alleviation or elimination of signs or symptoms of a disease or disorder. is meant to include, and this amount is referred to as an “effective amount”. Accordingly, the total amount of each active ingredient of a composition or method is sufficient to exhibit a significant individual benefit. Accordingly, the effective amount will vary depending on the circumstances in which it is administered. An effective amount may be administered in one or more prophylactic or therapeutic administrations.

The term 'implant body' used in the specification of the present invention refers to a site serving as a root of a tooth, including zirconium, an alloy and/or oxide thereof, tantalum, an alloy and/or oxide thereof, hafnium, an alloy and/or oxide thereof, niobium, alloys and/or oxides thereof, chromium-vanadium alloys and/or bonded oxides, and other metal(s) such as stainless steel, other metal alloy(s) and/or metal oxide(s), for example titanium, One or more metals, such as, but not limited to, titanium alloys and/or titanium oxides, metal alloys and/or metal oxides.

The composition for improving osseointegration of an implant according to an embodiment of the present invention contains a rosemary extract as an active ingredient, and has an excellent osseointegration effect between the jawbone and the implanted body surface.

The rosemary (Rosmarinus officinalis L.), also referred to as rosemary or bitter gourd, is an evergreen small shrub of the Lamiaceae family (Labiatae) whose leaves, shoots and flowers are edible. It is a perennial plant with needle-like leaves and is a herb native to the Mediterranean coast. It has green leaves and a characteristic fragrance. Flowers bloom in winter and spring, and the colors are white, pink, purple, blue, etc. It has been widely used in Western traditional cuisine since ancient times, and is still used in almost all cuisines in Italy. In particular, it is often used for meat dishes along with sage or thyme because the flavor is preserved even when heat is applied.

Main ingredients include beta-carotene, beta-sitosterol, bedulin acid, borneol, coffee acid, caper, capnosol, carvacrol, carbon, caryophyllene, chlorogenic acid, diosmin, genkwanin, geraniol, hespei Dean, limonene, linalool, olenic acid, 1,8-cineyl, rosemarinol, rosmaric acid, salicylic acid, squalene, tannin, thymol, assolic acid, calcium, magnesium, manganese, phosphorus, potassium, zinc, vitamin B1 , vitamin B3 and vitamin C, and a wide variety of active ingredients exist.

In addition, rosemary is known to have various effects such as antibacterial activity, antioxidant activity, whitening activity, and antiviral activity due to the above components. In particular, the most widely used function currently industrially is the antioxidant effect of oils and fats, and oil-soluble antioxidant products using rosemary extract are being sold.

The osseointegration is an important factor determining the success of the implant procedure, and after the implant body made of titanium (Ti) or titanium (Ti) alloy is surgically implanted in the jawbone, osteoblasts are implanted It refers to the process of forming a morphological, physiological, and direct connection between the jawbone and the implanted body surface by proliferating on the surface of the implanted body. By this osseointegration, the implant body is firmly fixed to the jawbone.

The composition containing the rosemary extract as an active ingredient is an essential process in the initial stage of osseointegration between the jawbone and the implanted body surface. Local adhesion of MC3T3-E1 preosteoblasts to the implant body surface Focal adhesion) and proliferative action of MC3T3-E1 preosteoblasts on the surface of the implant body can be improved to improve osseointegration between the jawbone and the surface of the implant body.

The composition modulates the FAK/Paxillin signaling pathway, and thus has excellent focal adhesion effect of MC3T3-E1 preosteoblasts on the surface of the implant body.

Osteoblasts refer to cells that have the ability to mineralize bone tissue by synthesizing and secreting bone matrix and depositing inorganic salts such as calcium and magnesium ions in the matrix. These are the cells that can be seen at the site of neogenesis.

On the other hand, preosteoblasts are cells that are differentiated into osteoblasts through a differentiation process into precursor cells of osteoblasts, and in the present invention, the preosteoblasts are MC3T3-E1 precursor osteoblasts. (preosteoblasts), but is not limited thereto, and may be used without limitation as long as it corresponds to the type of preosteoblasts that can act in the osseointegration process.

The focal adhesion is an interaction between titanium (Ti) or titanium (Ti) alloy and MC3T3-E1 preosteoblasts constituting the implant body, osseointegration between the jawbone and the implant body surface. For MC3T3-E1, it plays a role in regulating cell proliferation, cell migration, and differentiation into osteoblasts of preosteoblasts.

This focal adhesion is through a complex of extracellular matrix (ECM) proteins, integrins, cytoskeletal proteins and focal adhesion kinase (FAK)). It is regulated by signal transduction.

The focal adhesion of the MC3T3-E1 preosteoblasts and the subsequent cell proliferation of MC3T3-E1 preosteoblasts on the implant body surface are osseointegration between the jawbone and the implant body surface. (osseointegration) inducing influence.

The integrin is a protein located in the cell membrane of preosteoblasts, and acts as an anchor capable of forming a cytoskeleton by directly binding with actin, a cytoskeletal protein, to form a precursor bone. It plays an important role in focal adhesion and differentiation of cells (preosteoblasts).

Focal adhesion of MC3T3-E1 preosteoblasts interacts with the extracellular matrix (ECM) through integrin receptors, and processes such as activation and phosphorylation of local adhesion kinase (FAK) is conducted by

In addition, the focal adhesion is the F-actin bundle (F-actin bundle) and the interaction with the signaling molecules that affect the stability of focal adhesion (FAK) and paxillin proceeds through

The paxillin is a protein encoded by the human PXN gene, and functions to attach cells to the extracellular matrix (ECM). Specifically, it interacts directly with local adhesion kinase (FAK).

That is, according to the composition comprising the rosemary extract as an active ingredient, phosphorylation of the local adhesion kinase (FAK), which is a biomarker of the focal adhesion, is induced, as well as the precursor of MC3T3-E1. It is possible to increase the expression of paxillin and the formation of F-actin in osteoblasts (preosteoblasts), and through the regulation of this FAK/Paxillin signaling pathway, the implant body of MC3T3-E1 preosteoblasts It is possible to improve the effect of local adhesion to the surface.

The composition modulates the FAK/Grab 2/Ras/ERK 1,2 signaling pathway, and thus has an excellent effect on the proliferation of MC3T3-E1 preosteoblasts on the surface of the implant body.

Phosphorylated FAK (pFAK) binds directly to Grab 2 during focal adhesion and cytoskeletal formation of MC3T3-E1 preosteoblasts to the implant body surface.

The Grab2 is a protein expressed for regulating various functions of cells, and interacts with growth factor receptors to regulate the activities of Ras and ERK 1 and 2.

Specifically, growth factor receptors induce Grab 2 to the plasma membrane and then activate Ras and ERK 1 and 2.

Then, activated ERK 2 induces cell proliferation, cell migration, and differentiation into osteoblasts of MC3T3-E1 preosteoblasts through a series of FAK/Grab 2/Ras/ERK 1 and 2 signaling pathways. do.

As a result, according to the composition containing the rosemary extract as an active ingredient, the expression of Grab 2, Ras, and pERK 1 and 2 in MC3T3-E1 preosteoblasts was induced, and thus the expression of MC3T3-E1 preosteoblasts was induced. It can increase cell proliferation and mitosis. That is, it is possible to increase cell proliferation and mitosis on the surface of the implant body of MC3T3-E1 preosteoblasts through regulation of the FAK/Grab 2/Ras/ERK 1 and 2 signaling pathways.

Furthermore, the composition can increase the expression of SLPI and Tβ4 in MC3T3-E1 preosteoblasts on the surface of the implant body.

The SLPI is a protein present in saliva and seminal plasma, and protects tissues from proteolytic enzymes, improves cell proliferation, and is related to the immune function of the wounded site.

The Tβ4 is involved in cell proliferation, cell differentiation and motility through regulation of the actin cytoskeleton, and is a protein that plays an important role in the differentiation and mineralization of dentin of bones and teeth.

The SLPI and Tβ4 enhance the effect of focal adhesion of MC3T3-E1 preosteoblasts on the surface of the implant body through regulation of the FAK/Paxillin signaling pathway, and FAK/Grab 2/Ras/ERK It is possible to increase the cell proliferation of MC3T3-E1 preosteoblasts, as well as enhance the interaction between the implant body surface and MC3T3-E1 preosteoblasts through the regulation of 1 and 2 signaling pathways. known as nanomolecules.

That is, according to the composition containing the rosemary extract as an active ingredient, the expression of SLPI and Tβ4 in MC3T3-E1 preosteoblasts is increased, and the local adhesion of MC3T3-E1 preosteoblasts to the surface of the implant body. (focal adhesion) and may increase cell proliferation.

Preferably, the rosemary extract may further include a natural extract selected from the group consisting of a mulberry leaf extract, a gulpi tree leaf extract, a wisteria leaf extract, an elm leaf extract, and mixtures thereof.

The Cudrania tricuspidata (Carr.) Bureau ex Lavallee) is a small tree belonging to the dicotyledonous nettle family, and contains vitamins A, B1, B2, C, dietary fiber, flavonoids, gamma aminobutyric acid, rutin, etc. , hypertension, diabetes, cholesterol improvement, bowel activity improvement, antioxidant action, anticancer action, etc. The root bark, tree bark, bark and leaves of Cudrania biloba contain various ingredients effective for the human body. It has been used as a medicinal agent, such as a treatment, antipyretic, anti-dry, expectorant, diuretic, hemostatic, antipyretic, etc., and is used as an antifungal agent for athlete's foot and for chronic indigestion caused by weakness of the digestive system.

The cornus kousa (Cornus kousa) is a deciduous small tree of the Astragalus family, and mainly grows in the Yangji or seaside Suseongam area at the foot of the mountain in Korea (South Chungcheong Province), Japan, Taiwan, and China. The leaves are odd pinnate leaves, and consist of 7 to 19 small leaves without petioles. The small leaves are oval lancet-shaped or egg-shaped lancet-shaped with a sharp tip and deep serrations on the edge. There are white hairs on both sides of the leaves, but they gradually disappear. There is also hair on the peduncle, but it gradually disappears. The flowers are yellowish green flowers. The male ear is 5 to 8 cm and the female ear is 2 to 4 cm, and the mature female ear is in the shape of a pine cone. The ears of the fruit are long oval, blackish-brown, and hairless. The bracts do not fall off and are lanceolate, and the fruit is a winged nut and ripens in September to October.

The fruit bark (peel) of the gulpi tree contains eugloon and the fruit contains tannins, so it is used in folklore as a phlegm, insecticide, and pregnancy emetic.

The Eupatorium japonicum Thunb. ex Murray is a perennial herb that grows in mountains and fields in Korea. The growing environment is independent of the fertility of the soil, and it grows in semi-shaded places and sunny places. The height is 70 to 150 cm, the leaves are opposite to each other in an egg-shaped long oval or oval shape, and the pointed end is 10 to 18 cm long and 3 to 8 cm wide. disappears Small flowers are formed in flat clusters at the end of the main stem. The fruits ripen in October or November, and the seeds have white feathers. It is used for ornamental purposes, the young shoots are edible, and the outpost including the roots is used for medicinal purposes.

Ayapin, which is contained in stamens, is used in oriental medicine for jaundice, colic, stroke, hypertension, postpartum abdominal pain, hematemesis, and pneumonia in oriental medicine. In addition, it is known that the leaf stalk is a diuretic to remove edema and is good for fatigue recovery as a bathing agent as a known efficacy of stamens. Furthermore, it is known that it has an antiviral action, a direct inhibitory action against an epidemic virus, indigestion, abdominal pain, and the like.

The elm tree (Ulmus davidiana var. Japonica) belongs to a deciduous broad-leaved tree of the dicotyledonous plant Nettle family Elmaceae. It is distributed in Korea, Japan, Sakhalin, the Kuril Islands, northern China and Siberia, and grows to a height of 20m and a diameter of 60cm near a mountain stream or valley. There are several types of elm, but the shape of the leaves and the use of medicines are all the same. These elm trees have been used since ancient times as a diuretic, as a remedy for sore throats or boils. As medicine, the root bark of elm is used, which is known to contain flavonoids, saponins, tannins, or a large amount of mucilage.

When the rosemary extract further includes a natural extract selected from the group consisting of a mulberry leaf extract, a gulberry leaf extract, a staphylococcus leaf extract, an elm leaf extract, and a mixture thereof, the FAK/Paxillin signaling pathway is regulated Thus, it is possible to maximize the effect of focal adhesion of MC3T3-E1 preosteoblasts on the surface of the implant body, and the formation of F-actin in MC3T3-E1 preosteoblasts and Maximize the number of Paxillin in contact with MC3T3-E1 preosteoblasts.

Furthermore, by regulating the FAK/Grab 2/Ras/ERK 1 and 2 signaling pathway, cell proliferation and mitosis of MC3T3-E1 preosteoblasts can be maximized, and MC3T3-E1 precursor bone It is possible to maximize the expression level of SLPI and Tβ4 proteins in cells (preosteoblasts).

Due to the synergistic effect, it is possible to maximize the effect of osseointegration between the jawbone and the surface of the implant body.

More preferably, the rosemary extract, mulberry leaf extract, gulpi tree leaf extract, staphylococcus leaf extract and elm leaf extract are in a weight ratio of 1:0.4:0.4:0.5:0.5 to 1:1:1:1:1 By mixing with MC3T3-E1 preosteoblasts, the effect of local adhesion on the surface of the implant body was improved by better controlling the FAK/Paxillin signaling pathway within the above weight range. can be maximized, and the formation of F-actin in MC3T3-E1 preosteoblasts and the number of Paxillin in contact with MC3T3-E1 preosteoblasts can be maximized.

In addition, within the above weight range, the cell proliferation and mitosis of MC3T3-E1 preosteoblasts can be maximized through the excellent FAK/Grab 2/Ras/ERK 1 and 2 signaling pathway regulation mechanism. , Maximize the expression level of SLPI and Tβ4 proteins in MC3T3-E1 preosteoblasts.

However, when it is less than or exceeding the range value, there is a problem in that the effect is significantly reduced.

In the present invention, the rosemary extract is rosmarinic acid.

Rosmarinic acid is a compound that is a biochemical composition found in various plants such as rosemary, sage, basil, and mint. It is known as a substance that prevents spoilage caused by oxidation of food. It is known that the main action has antioxidant action, anti-inflammatory action, anti-mutagenic action, anti-microbial action, anti-viral action, and anti-cancer action. In addition, after a meal, the blood condition temporarily thickens for nutrients to enter the blood, and rosmarinic acid decomposes maltose into glucose, thereby discharging the sugar dispersed in the blood to the outside of the body. It has been reported that it suppresses the rise in blood sugar levels and prevents hypertension, diabetes, and hyperlipidemia.

In addition, rosmarinic acid is effective for headache and is known as a good substance for the circulatory system. Furthermore, rosmarinic acid has a strong antiseptic action, is effective in preventing hair loss, helps in hair management, has a function of restoring dry and aged skin to normal, and promotes cell regeneration to reduce wrinkles serves to reduce Because of these functions, it is a material that is currently attracting attention as a very important ingredient in the cosmetic field.

The rosmarinic acid is represented by the following Chemical Formula 1:

[Formula 1]

In the present invention, the extract is extracted using an extraction solvent selected from the group consisting of water, alcohols having 1 to 4 carbon atoms, ether, chloroform, ethyl acetate, dimethyl sulfoxide, and mixtures thereof. The 'rosemary extract' refers to an extract obtained by extraction from rosemary.

The rosemary extract is a pulverized product of dried rosemary having a mixing ratio of 1:0.1 to 1:10 of an alcohol having 1 to 4 carbon atoms, such as water, ethanol, methanol, etc., a polar solvent such as dimethyl sulfoxide, or alcohol and water. Elution may be performed with a mixed solvent, and in more detail, ethanol may be used as an extraction solvent. At this time, the extraction temperature may be 10 ℃ to 100 ℃, preferably the extract extracted at 25 ℃.

The extraction solvent may be used in an amount of 2 to 50 times, preferably 2 to 20 times, based on the weight of the sample. For extraction, the sample may be left for 1 to 72 hours for leaching in the extraction solvent, and in particular, from 1 to 24 hours.

It may be a result obtained by concentrating the filtered extract under reduced pressure with a vacuum rotary concentrator, but as long as it is a rosemary extract that can exhibit an excellent effect of osseointegration between the jawbone and the implanted body surface of the present invention, it is not limited thereto. It includes all extracts, diluted or concentrated solutions of extracts, dried products obtained by drying the extracts, or prepared or purified products thereof.

The Cuji mulberry leaf extract, Gulpi tree leaf extract, Staple tree leaf extract, and Elm leaf extract can also be prepared using the same method as the rosemary extract.

A pharmaceutical composition for improving osseointegration of an implant according to another embodiment of the present invention includes the composition.

The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. The composition comprising a pharmaceutically acceptable carrier may be in various oral or parenteral formulations. In the case of formulation, it can be prepared using a diluent or excipient such as a filler, extender, binder, wetting agent, disintegrant, surfactant, etc. commonly used. Solid preparations for oral administration may include tablet pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in one or more compounds, for example, starch, calcium carbonate, sucrose or lactose ( lactose), gelatin, etc. can be mixed to prepare it. In addition to simple excipients, lubricants such as magnesium stearate, talc and the like may also be used. Liquid preparations for oral administration include suspensions, internal solutions, emulsions, syrups, etc. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have. Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like can be used.

In addition, the pharmaceutical composition of the present invention is from the group consisting of tablets, pills, powders, granules, capsules, suspensions, internal solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations and suppositories It may have any one formulation selected.

The rosemary extract of the present invention has been used for food and medicine since ancient times, and there is no particular restriction on its dosage, body absorption, body weight, age, sex, health status, diet, administration time, administration method, excretion rate of the patient. , and the severity of the disease. In general, the rosemary extract may be administered in an amount of about 10 to 1000 mg per 1 kg of body weight, and more specifically, about 50 to 500 mg per kg of body weight.

The pharmaceutical composition comprising the rosemary extract of the present invention as an active ingredient is to be prepared in consideration of the effective amount range, and the unit dosage form formulated in this way is the judgment of an expert who monitors or observes the administration of the drug as needed and the needs of the individual. Depending on the patient, a specialized dosing method may be used, or several doses may be administered at regular time intervals.

A functional food composition for improving osseointegration of implants according to another embodiment of the present invention includes the composition.

There is no particular limitation on the type of the functional food of the present invention. Examples of health functional foods to which the extract mixture can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, tea , drinks, alcoholic beverages, and vitamin complexes, and may include all health functional foods in a conventional sense, and may include foods used as feed for animals. In addition to the above, the health functional food composition of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin , alcohol, a carbonation agent used in carbonated beverages, and the like. In addition, it may contain the pulp for the production of natural fruit juice, fruit juice beverage and vegetable beverage.

According to the composition for improving osseointegration of implants comprising the rosemary extract of the present invention, the effect of MC3T3-E1 preosteoblasts on the surface of the implant body and cell proliferation on the surface of the implant body By improving the effect, it is possible to improve the osseointegration between the jawbone and the surface of the implanted implant body.

Furthermore, it is possible to provide a pharmaceutical composition and a functional food composition for improving osseointegration of implants comprising a rosemary extract as an active ingredient.

1 is an experimental result showing the effect of rosmarinic acid on the focal adhesion of MC3T3-E1 preosteoblasts on the surface of a titanium plate (Ti discs) according to an embodiment of the present invention.
Figure 2 is a rosmarinic acid (rosmarinic acid) according to an embodiment of the present invention through the regulation of the FAK / Paxillin signaling pathway on the titanium plate (Ti discs) surface MC3T3-E1 precursor osteoblasts (focal adhesion) (focal adhesion) ) is the experimental result showing the effect on
Figure 3 shows the formation of F-actin in MC3T3-E1 preosteoblasts and MC3T3-E1 preosteoblasts on the surface of titanium plates (Ti discs) according to an embodiment of the present invention; Experimental results showing the effect of rosmarinic acid on the number of contacting Paxillin.
4 is an experimental result showing the effect of rosmarinic acid on the cell proliferation of MC3T3-E1 preosteoblasts on the surface of titanium plates (Ti discs) according to an embodiment of the present invention.
5 is rosmarinic acid according to an embodiment of the present invention through the regulation of the FAK/Grab 2/Ras/ERK 1,2 signal transduction pathway on the titanium plate (Ti discs) surface MC3T3-E1 precursor osteoblasts ( These are the experimental results showing the effect on cell proliferation and mitosis of preosteoblasts).
6 is an experimental result showing the effect of rosmarinic acid on the expression of SLPI and Tβ4 protein in MC3T3-E1 preosteoblasts on the surface of titanium plates (Ti discs) according to an embodiment of the present invention.
7 is a view showing the osseointegration of the jawbone and the implant body surface by inducing focal adhesion and cell proliferation of MC3T3-E1 preosteoblasts on the surface of the titanium plate (Ti discs) according to an embodiment of the present invention. It is a block diagram showing the effect of rosmarinic acid on the process of forming osseointegration.

Hereinafter, embodiments of the present invention will be described in detail so that those of ordinary skill in the art can easily carry out the present invention. However, the present invention may be embodied in several different forms and is not limited to the embodiments described herein.

[Preparation Example 1: Manufacture of titanium plates (Ti discs)]

The polished titanium plates (Ti discs) were cut to have a thickness of 2 mm and diameters of 20 mm and 48 mm.

[Preparation Example 2: Preparation of rosemary extract (rosmarinic acid)]

After dissolving rosmarinic acid (Sigma-Aldrich, MO, USA) in DMSO (dimethyl sulfoxide) as a solvent, it was stored at -20 °C to prepare a rosemary extract (rosmarinic acid).

Thereafter, the stock solution was diluted to the treatment concentration applied in the experiment before use.

[Preparation Example 3: Preparation of other extracts]

Cuji mulberry leaves were pulverized with a blender and then prepared into powder. After adding 50% ethanol as an extraction solvent to the powder sample at a ratio of 1:10 (w; v), and then completely immersing the powder sample, extraction was repeated 3 times for 3 hours at 80°C while refluxing. The extract is Whatman No. 2 filtered with filter paper. The filtrate was concentrated under reduced pressure at 60° C. to prepare a Cuji mulberry leaf extract.

In the same manner as in the preparation of the Cuji mulberry leaf extract, a gulpi tree leaf extract, a stasis leaf extract and an elm leaf extract were prepared.

[Preparation Example 4: Preparation of mixed extract]

The rosemary extracts of rosmarinic acid (RA), mulberry leaf extract (BE), gulberry leaf extract (GE), staphylococcus leaf extract (DE) and elm leaf extract (NE) are shown in Table 1 below. Mixed extracts were prepared by mixing within the weight range.

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 T13 T14 T15 T16 RA 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 BE - 70 70 70 70 70 30 40 70 100 120 GE - 70 70 70 70 70 30 40 70 100 120 DE - 75 75 75 75 75 40 50 75 100 130 NE - 75 75 75 75 75 40 50 75 100 130

(Unit: parts by weight)

[Experimental Example 1: Experiment on the improvement effect of osseointegration with the jawbone and the implanted body surface]

1. Experimental method

cell culture

MC3T3-E1 cells, a cell line of preosteoblasts collected from the cranial tube of a mouse, were smeared on the surface of Ti discs, and 10% fetal bovine serum (FBS, WelGENE) and 1 % Antibiotic and antifungal solution (WelGene, Daegu, Korea) containing Alpha modified eagle's medium (α-MEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), at 37 ℃ 5% CO ₂ of The state was maintained and cultured.

Cell adhesion and Proliferation assay

MC3T3-E1 preosteoblasts on the surface of Ti discs cultured for 12 hours in serum-free medium were treated with 14 μg/ml rosmarinic acid and not treated. They were incubated for 10 minutes, 30 minutes and 60 minutes, respectively, under non-renewable conditions.

3-(4,5-dimethylthiazol-2-yl)- An MTT assay using 2,5-diphenyltetrazolium bromide was used.

Western Blot analysis

^{1×10 6} cells/ml of MC3T3-E1 preosteoblasts on the surface of Ti discs cultured for 12 hours in serum-free medium were treated with 14 μg/ml of rosmarinic acid. After incubation for 10 minutes, 30 minutes, and 60 minutes, respectively, under the treated and untreated conditions, the total protein of the cells was extracted.

30 μg of protein was used to determine the extent of protein expression.

After electrophoresis, the protein was transferred to a polyvinylidene difluoride (PVDF) membrane (Merck Millipore, Darmstadt, Germany).

Thereafter, the membrane was blotted using a primary antibody at 4° C. for 16 hours. Examples of the primary antibody used in the experiment include 1:1000 anti-rabbit phosphorylated FAK (pFAK, Cell signaling, USA), 1:1,000 anti-rabbit FAK (Upstate, USA), and 1:2,500 anti- rabbit phosphorylated ERK 1, 2 (pERK 1, 2, Cell signaling), 1:2,500 anti-rabbit ERK 1, 2 (Upsate), 1:40,000 anti-mouse paxillin (Transduction laboratories, USA), 1:1,000 anti-mouse Grb 2 (Upstate), 1:1,000 of anti-rabbit Ras (Upstate), 1:1,000 of anti-rabbit SLPI, 1:1,000 of anti-rabbit thymosin β4 (SantaCruz Biotechnology, USA), and 1:2,500 of anti-mouse β-actin (Santa Cruz Biotechnology) was used.

Thereafter, after washing the membrane, blotting was performed with either HRP-conjugated goat anti-rabbit or mouse-IgG (Enzo Life Sciences, USA) at 1:5,000.

Development was performed using an X-ray film (Fuji Film, Japan) after detection using an ECL solution (Merck Millipore). The density of the expressed band was measured using Science Lab Image Gauge (Fuji Film).

Immunofluorescence Staining

^{1×10 5} cells/ml of MC3T3-E1 preosteoblasts on the surface of titanium discs were treated with and without 14 μg/ml of rosmarinic acid on a serum-free medium. Incubated for 30 minutes, 60 minutes and 3 hours, respectively.

Thereafter, the cells were fixed in 4% paraformaldehyde for 10 minutes, and treated with 0.15% triton X-100 for 10 minutes. Cells were blocked with 1% BSA for 1 hour, and reacted with anti-mouse paxillin (1 μg/ml, Transduction Laboratories) at 4° C. for 16 hours.

Thereafter, washing was performed, and the cells were cultured for 1 hour at 25°C under goat anti-mouse IgG (ThermoFisher, USA) conjugated with FITC.

For F-actin staining, cells were incubated for 40 minutes at 25° C. under phalloidin (Sigma, USA) conjugated with 50 μg/ml FITC. A mounting medium (Vector Lab, USA) containing DAPI was used to mount the cells on the surface of the titanium plate (Ti discs).

Contact between actin-labeled cells and paxillin was observed and photographed by fluorescence microscopy (Carl Zeiss, GER). During the experiment, the number of contacts of paxillin per cell was calculated in real time, and the experimental result means the average value of 40 to 60 cells calculated each time.

Statistical Analysis

All experiments were repeated three times, and all data were calculated as mean and standard deviation determined using SPSS 12.0 (SPSS, USA). Statistical analysis was performed with independent samples t-test, and differences at p < 0.05 were considered statistically significant.

2. Experimental results

Experiments on the effect of rosmarinic acid on focal adhesion of MC3T3-E1 preosteoblasts

Local adhesion to the surface of Ti discs of MC3T3-E1 preosteoblasts when treated with 14 μg/ml of rosmarinic acid compared to the case without treatment with rosmarinic acid (focal adhesion) effect was confirmed by MTT assay.

The focal adhesion rates of MC3T3-E1 preosteoblasts were 10 min, 30 min, and 60 min in the case of treatment with rosmarinic acid compared to the case without treatment with rosmarinic acid. each increased 1.2-fold during the period (Fig. 1).

The above results demonstrate that rosmarinic acid enhances the effect of focal adhesion of MC3T3-E1 preosteoblasts.

Experiments on the effect of rosmarinic acid on focal adhesion of MC3T3-E1 preosteoblasts by FAK/Paxillin signaling pathway

In order to confirm the effect of rosmarinic acid on focal adhesion of MC3T3-E1 preosteoblasts by the FAK/Paxillin signaling pathway, rosmarinic acid was not treated and 14 When μg/ml of rosmarinic acid was treated for 10, 30, and 60 minutes, respectively, the entire protein of MC3T3-E1 preosteoblasts was extracted, and local adhesion phosphorylation was performed through western blot analysis. The expression of enzyme (FAK), phosphorylated localized kinase (pFAK) and paxillin was confirmed (FIG. 2A).

In the case of MC3T3-E1 preosteoblasts treated with rosmarinic acid, the expression of phosphorylated localized kinase (pFAK) was reduced at 10 minutes and 30 minutes compared to the case of not treated with rosmarinic acid. Treatment for minutes increased 1.6-fold and 1.1-fold, respectively. However, when treated for 60 minutes, it decreased by 1.1 times ( FIGS. 2A and 2B ).

The expression of paxillin was also increased when treated with rosmarinic acid compared to the case without treatment with rosmarinic acid.

Specifically, in the case of treatment with rosmarinic acid, the expression rate of paxillin increased by 2.8 times when treated with rosmarinic acid for 10 minutes compared to the case without treatment with rosmarinic acid, and increased by 1.2 times when treated for 30 minutes, and increased for 60 minutes. Treatment increased by 1.3-fold ( FIGS. 2A and 2B ).

The above results demonstrate that rosmarinic acid induces focal adhesion of MC3T3-E1 preosteoblasts by regulating the FAK/Paxillin signaling pathway.

Experiments on the effect of rosmarinic acid on the formation of F-actin and the number of Paxillin in contact with MC3T3-E1 preosteoblasts

In order to observe the contact of F-actin and paxillin through a fluorescence microscope, MC3T3-E1 preosteoblasts treated with rosmarinic acid and without rosmarinic acid were treated with rosmarinic acid. It was fixed and fluorescently stained.

The formation of F-actin, an actin fiber, was increased in MC3T3-E1 preosteoblasts treated with rosmarinic acid compared to the case not treated with rosmarinic acid (FIG. 3A) ).

Contact with paxillin also increased by 1.3 times when treated with rosmarinic acid and 1.2 times when treated for 30 minutes in MC3T3-E1 preosteoblasts treated with rosmarinic acid compared to the case without treatment with rosmarinic acid. It increased 1.1-fold after 3 h treatment ( FIGS. 3B and 3C ).

Experiments on the effect of rosmarinic acid on cell proliferation of MC3T3-E1 preosteoblasts

Cell proliferation on the surface of Ti discs of MC3T3-E1 preosteoblasts when treated with 14 μg/ml of rosmarinic acid compared to the case without treatment with rosmarinic acid The effect was confirmed by MTT assay.

Cell proliferation was further increased in the case of MC3T3-E1 preosteoblasts treated with rosmarinic acid compared to the case of not treated with rosmarinic acid. Specifically, the cell proliferation rate was 12 hours and 24 hours. and in culture for 48 hours, all increased 1.1-fold ( FIG. 4 ).

The above results prove that rosmarinic acid effectively induces cell proliferation of MC3T3-E1 preosteoblasts.

Experiments on the effect of rosmarinic acid on proliferation and mitosis of MC3T3-E1 preosteoblasts by FAK/Grab 2/Ras/ERK 1, 2 signaling pathway

The expression of Grab 2, Ras, ERK 1, 2 and pERK 1 and 2 proteins in MC3T3-E1 preosteoblasts treated with rosmarinic acid and without rosmarinic acid was analyzed by western blot. It was confirmed through analysis (FIG. 5A).

The expression of Grab 2 was increased by 1.7 times when treated with rosmarinic acid for 10 minutes in MC3T3-E1 preosteoblasts treated with rosmarinic acid compared to the case without treatment with rosmarinic acid, and was increased by 1.7 times after treatment for 30 minutes. It increased by 1.1 times at the time of treatment, and increased by 1.2 times when treated for 60 minutes (FIG. 5B).

The expression of Ras was increased by 1.3 times when treated with rosmarinic acid for 10 minutes in MC3T3-E1 preosteoblasts treated with rosmarinic acid compared to the case without treatment with rosmarinic acid, 30 minutes and 60 minutes There was a 1.2-fold increase in the minute treatment (FIG. 5B).

The expression of pERK 1 and 2 was increased 1.5-fold when treated for 10 minutes in MC3T3-E1 preosteoblasts treated with rosmarinic acid compared to the case without treatment with rosmarinic acid, 30 It increased by 1.3 times when treated for minutes, and increased by 1.1 times when treated for 60 minutes (FIG. 5B).

The above results prove that rosmarinic acid effectively induces proliferation and mitosis of MC3T3-E1 preosteoblasts by regulating FAK/Grab 2/Ras/ERK 1 and 2 signaling pathways.

Experiments on the effect of rosmarinic acid on the expression of SLPI and Tβ4 in MC3T3-E1 preosteoblasts

Western blot analysis was performed to examine the effect of rosmarinic acid on the expression of SLPI and Tβ4 in MC3T3-E1 preosteoblasts ( FIG. 6A ).

The expression of secreted SLPI protein (Secreted protein) increased by 1.2 times when treated with rosmarinic acid for 30 minutes in MC3T3-E1 preosteoblasts compared to the case without rosmarinic acid treatment However, it decreased during treatment for 10 minutes and 60 minutes, and was at a level similar to the case where rosmarinic acid was not treated (FIG. 6B).

The expression of cytoplasmic protein (SLPI) was observed in MC3T3-E1 preosteoblasts treated with rosmarinic acid for 10 minutes and 60 minutes after treatment with rosmarinic acid compared to the case without treatment with rosmarinic acid. It increased by 1.4 and 1.1 times, respectively, but decreased by 1.1 times when treated for 30 minutes (FIG. 6B).

The expression of Tβ4 was 1.4 times when treated with rosmarinic acid for 10 minutes, 30 minutes, and 60 minutes, respectively, in MC3T3-E1 preosteoblasts treated with rosmarinic acid compared to the case without treatment with rosmarinic acid. , increased by 1.2-fold and 1.1-fold (Fig. 6B).

The above results show that rosmarinic acid can increase the expression of SLPI and Tβ4 in MC3T3-E1 preosteoblasts, thereby increasing focal adhesion and cell proliferation of MC3T3-E1 preosteoblasts. show that there is

Synergistic effect by combined treatment of rosemary extract (rosmarinic acid), mulberry leaf extract, gulberry leaf extract, stalagmite leaf extract and elm leaf extract

In order to test whether the effect of improving osseointegration between the jawbone and the surface of the implant body by the combined treatment of the extracts appears,

MC3T3 according to the treatment of single treatment of rosemary extract, rosmarinic acid, and the mixed extract mixed with rosmarinic acid, mulberry leaf extract, gulberry leaf extract, stamen leaf extract and elm leaf extract in a certain weight ratio -Effect on focal adhesion of E1 preosteoblasts, modulating effect of FAK/Paxillin signaling pathway related to focal adhesion of MC3T3-E1 preosteoblasts, F-actin Effect on the formation of (F-actin) and the number of Paxillin in contact with MC3T3-E1 preosteoblasts, on the cell proliferation of MC3T3-E1 preosteoblasts, MC3T3-E1 precursor osteoblasts ( Preosteoblasts) proliferation and mitosis-related modulating effects of FAK/Grab 2/Ras/ERK 1 and 2 signaling pathways and the expression effects of SLPI and Tβ4 in MC3T3-E1 preosteoblasts etc. were used for experiments.

For relative comparison, the degree of focal adhesion of MC3T3-E1 preosteoblasts in the case of single treatment (T1) with rosemary extract, rosmarinic acid, was set to index 5, and The degree of focal adhesion of MC3T3-E1 preosteoblasts was evaluated as an index.

The index is evaluated on a scale from 1 to 10, and a higher value means better effect.

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 T13 T14 T15 T16 Degree of focal adhesion of MC3T3-E1 preosteoblasts 5 5.1 4.8 4.9 3.9 4.5 5.5 4.8 6.5 4.8 6.2 4.5 8.5 9 8.6 4.8

According to Table 2, the rosemary extract rosmarinic acid, mulberry leaf extract, gulpi tree leaf extract, stamen leaf extract and elm leaf extract 1:0.4:0.4:0.5:0.5 to 1:1:1 It has been demonstrated that MC3T3-E1 preosteoblasts have a better effect on focal adhesion to the implant body surface when mixed in a weight ratio of 1:1:1.

On the other hand, when the mixing ratio of rosemary extract, rosmarinic acid, Cudrania mulberry leaf extract, gulberry leaf extract, staphylococcus leaf extract, and elm leaf extract, is less than or greater than the above range value, MC3T3-E1 preosteoblasts ) has been proven to have a lower effect of focal adhesion on the surface of the implant body.

Furthermore, the regulatory effect of the FAK/Paxillin signaling pathway related to focal adhesion of MC3T3-E1 preosteoblasts was confirmed through an experiment. For relative comparison, rosmarinic acid, a rosemary extract, was used alone. In the case of treatment (T1), the modulating effect of the FAK/Paxillin signaling pathway was set to 5, and the modulating effect of the mixed extract was evaluated as an index.

The index is rated on a scale of 1 to 10, with higher values indicating superior effects.

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 T13 T14 T15 T16 Modulating effect of FAK/Paxillin signaling pathway 5 4.8 5.2 3.2 4.7 5 6 5.5 5.1 3.9 4 3.9 8.8 9.2 8.7 4.8

According to Table 3, the rosemary extract rosmarinic acid, mulberry leaf extract, gulpi tree leaf extract, stamen leaf extract and elm leaf extract 1:0.4:0.4:0.5:0.5 to 1:1:1 It can be seen that the control effect of the FAK/Paxillin signaling pathway is more excellent when mixed and used in a weight ratio of 1:1:1.

On the other hand, when the mixing ratio of the rosemary extract rosmarinic acid, Cudrania mulberry leaf extract, Gyulphus leaf extract, staphylococcus leaf extract and elm leaf extract is less than or greater than the above range value, the FAK / Paxillin signaling pathway It can be seen that the control effect decreases.

On the other hand, the difference between the formation of F-actin and the number of Paxillin in contact with MC3T3-E1 preosteoblasts was confirmed through experiments.

For relative comparison, the formation of F-actin and the degree of Paxillin in contact with MC3T3-E1 preosteoblasts in the case of treatment with rosmarinic acid, a rosemary extract alone (T1), was index 5 and the synergistic effect of the mixed extract was evaluated as an index.

The index is rated on a scale of 1 to 10, with higher values indicating superior effects.

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 T13 T14 T15 T16 Formation of F-actin and the level of Paxillin in contact with MC3T3-E1 preosteoblasts 5 4.8 3.8 5.2 5 4.7 5.5 6 3.9 5.1 3.9 4 8 8.9 8.6 4.3

According to Table 4, the rosemary extract, rosmarinic acid, mulberry leaf extract, gulpi tree leaf extract, stamen leaf extract and elm leaf extract 1:0.4:0.4:0.5:0.5 to 1:1:1 It was confirmed that the formation of F-actin and the degree of Paxillin in contact with MC3T3-E1 preosteoblasts were further increased when mixed in a weight ratio of 1:1:1.

On the other hand, it can be seen that when the mixing ratio of rosemary extract, rosmarinic acid, Cudrania mulberry leaf extract, gulberry leaf extract, staphylococcus leaf extract, and elm leaf extract, is less than or greater than the above range value, the effect is lowered. have.

On the other hand, the synergistic effect on cell proliferation of MC3T3-E1 preosteoblasts was confirmed through an experiment.

For a relative comparison, the effect on cell proliferation of MC3T3-E1 preosteoblasts in the case of single treatment (T1) with rosemary extract, rosmarinic acid, was set to index 5, and the synergistic effect of the mixed extract was The index was evaluated.

The index is rated on a scale of 1 to 10, with higher values indicating superior effects.

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 T13 T14 T15 T16 Effect on cell proliferation of MC3T3-E1 preosteoblasts 5 4.8 4.9 5.1 5.5 4.5 3.9 6.5 5.5 4.8 3.8 6.2 8.8 9 8 4.3

According to Table 5, rosemary extract rosmarinic acid (rosmarinic acid), Cudrania mulberry leaf extract, gulpi tree leaf extract, stamen leaf extract and elm leaf extract 1:0.4:0.4:0.5:0.5 to 1:1:1 It was confirmed that the effect on cell proliferation of MC3T3-E1 preosteoblasts was more excellent when mixed in a weight ratio of 1:1:1.

On the other hand, it can be seen that when the mixing ratio of rosemary extract, rosmarinic acid, Cudrania mulberry leaf extract, Gyulpi tree leaf extract, Staple tree leaf extract, and Elm leaf extract is less than or greater than the above range value, the effect is lowered. have.

On the other hand, the synergistic effect of regulation of FAK/Grab 2/Ras/ERK 1 and 2 signaling pathways related to proliferation and mitosis of MC3T3-E1 preosteoblasts was confirmed through an experiment.

For relative comparison, the modulating effect of FAK/Grab 2/Ras/ERK 1 and 2 signaling pathways in the case of single treatment (T1) of rosemary extract, rosmarinic acid, was set to index 5, and the increase by the mixed extract The effect was evaluated exponentially.

The index is evaluated on a scale from 1 to 10, and a higher value means better effect.

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 T13 T14 T15 T16 Modulating effect of FAK/Grab 2/Ras/ERK 1, 2 signaling pathway 5 4.3 4.5 5 4.1 3.8 5.1 6.5 5.5 4.2 6 4.6 8.8 9.1 8.7 4

According to Table 6, rosmarinic acid, which is a rosemary extract, a mulberry leaf extract, a gulpi tree leaf extract, a stamen leaf extract and an elm leaf extract were 1:0.4:0.4:0.5:0.5 to 1:1:1. When mixed and used in a weight ratio of 1:1:1, it was confirmed that the regulatory effect of FAK/Grab 2/Ras/ERK 1 and 2 signaling pathways was more excellent.

On the other hand, when the mixing ratio of rosemary extract, rosmarinic acid, Cudrania mulberry leaf extract, Ginger biloba leaf extract, Staple tree leaf extract, and Elm leaf extract is less than or greater than the above range value, FAK/Grab 2/Ras/ERK It can be seen that the regulatory effect of the 1 and 2 signal transduction pathways is lowered.

On the other hand, the difference in the expression effect of SLPI and Tβ4 in MC3T3-E1 preosteoblasts was confirmed through an experiment.

For relative comparison, the expression effect of SLPI and Tβ4 in MC3T3-E1 preosteoblasts when treated with rosmarinic acid, a rosemary extract alone (T1), was set to index 5, and the synergistic effect by the mixed extract was evaluated as an index.

The index is evaluated on a scale from 1 to 10, and a higher value means better effect.

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 T13 T14 T15 T16 Effect of SLPI and Tβ4 Expression in MC3T3-E1 Progenitor Osteoblasts 5 4.5 3.5 5 5.2 4.7 5.1 3.9 3.9 4 4.1 4 8.9 9.3 8.4 4.2

According to Table 7, rosmarinic acid, which is a rosemary extract, a mulberry leaf extract, a gulpi tree leaf extract, a stamen leaf extract and an elm leaf extract 1:0.4:0.4:0.5:0.5 to 1:1:1 It was confirmed that the expression effect of SLPI and Tβ4 was maximized in MC3T3-E1 preosteoblasts when mixed in a weight ratio of 1:1:1.

On the other hand, when the mixing ratio of rosemary extract, rosmarinic acid, Cudrania mulberry leaf extract, gulberry leaf extract, staphylococcus leaf extract, and elm leaf extract, is less than or greater than the above range value, MC3T3-E1 preosteoblasts ), it can be seen that the expression effect of SLPI and Tβ4 is lowered.

Looking at the results of the above experiments, compared to the case of treatment with rosmarinic acid, which is a rosemary extract, alone, rosmarinic acid, which is a rosemary extract, a currant mulberry leaf extract, a gulpi tree leaf extract, a stamen leaf extract and elm When the tree leaf extract is mixed in a weight ratio of 1:0.4:0.4:0.5:0.5 to 1:1:1:1:1, the effect of focal adhesion of MC3T3-E1 preosteoblasts, the above Modulating effect of FAK/Paxillin signaling pathway, formation of F-actin and level of Paxillin in contact with MC3T3-E1 preosteoblasts, cell proliferation effect of MC3T3-E1 preosteoblasts , Maximizes the regulatory effect of the FAK/Grab 2/Ras/ERK 1 and 2 signaling pathway and the expression effect of SLPI and Tβ4 in MC3T3-E1 preosteoblasts, resulting in osseointegration between the jawbone and the surface of the implant body It can be seen that (osseointegration) improves the action.

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 are also provided. is within the scope of the

Claims (7)

It contains rosemary extract, mulberry leaf extract, gulberry leaf extract, stamen leaf extract and elm leaf extract as active ingredients,
Excellent osseointegration effect between the jawbone and the implant body surface
A composition for improving osseointegration of implants. The method of claim 1,
The composition modulates the FAK / Paxillin signaling pathway,
Excellent focal adhesion effect of MC3T3-E1 preosteoblasts on the surface of the implant body
A composition for improving osseointegration of implants. The method of claim 1,
The composition modulates the FAK/Grab2/Ras/ERK1,2 signaling pathway,
Excellent proliferation effect of MC3T3-E1 preosteoblasts on the surface of the implant body
A composition for improving osseointegration of implants. The method of claim 1,
The rosemary extract is rosmarinic acid
A composition for improving osseointegration of implants. The method of claim 1,
The extract is extracted using an extraction solvent selected from the group consisting of water, alcohols having 1 to 4 carbon atoms, ether, chloroform, ethyl acetate, dimethyl sulfoxide, and mixtures thereof.
A composition for improving osseointegration of implants. A composition comprising the composition according to any one of claims 1 to 5
A pharmaceutical composition for improving osseointegration of implants. A composition comprising the composition according to any one of claims 1 to 5
A functional food composition for improving osseointegration of implants.

Images