KR101882575B1 - Composition for preventing or improving periodontal disease comprising natural extract using microorganism culture - Google Patents

Abstract

The present invention relates to a health functional food and composition for preventing or ameliorating periodontal disease containing natural extracts through microbial cell culture, and more particularly, to a health functional food and composition for prevention or improvement of periodontal disease by inhibiting NF- The present invention relates to a health functional food and a pharmaceutical composition for preventing or ameliorating periodontal disease, which comprises a fermented product obtained by cultivating seaweed and olive leaves which are useful for the treatment and prevention of diseases. The health functional food and pharmaceutical composition for prevention or improvement of periodontal disease including the fermented product of the sea tangle and the olive leaf leaf obtained by the present invention have excellent anti-inflammatory effect while minimizing side effects as a natural material, And it can be utilized effectively. Also, the seaweed seaweed, which is rich in seaweed, can be used to provide a great economic effect.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for preventing or improving periodontal disease, which comprises a natural fermented extract through microbial cell culture,

The present invention relates to a composition for preventing or ameliorating periodontal disease, which contains a natural extract through microbial cell culture, and more particularly to a composition for preventing or treating periodontal disease by significantly inhibiting NF-κB transcription activation, The present invention relates to a health functional food and a pharmaceutical composition for preventing or ameliorating periodontal disease, which comprises a fermented product obtained by cultivating kelp and olive leaves useful for prevention.

Inflammatory reactions are caused by various inflammatory mediators and immune cells in the local blood vessels and body fluids when they are infected with external infections such as cell or tissue damage, bacteria, fungi, viruses, various allergens, Secretion, fluid infiltration, cell migration, tissue destruction, and other external symptoms such as erythema, edema, fever, and pain. In normal cases, the inflammatory reaction removes the external infectious agent and regenerates the damaged tissue to regenerate the organism's function. However, if the antigen is not removed or the internal substance causes the inflammatory reaction to occur excessively or continuously, Some lead to diseases such as cancer. Various biological and biochemical phenomena are involved in the cause of inflammation in living body. In particular, nitric oxide synthase (NOS), which is an enzyme that generates nitric oxide (NO), and enzymes related to prostaglandin biosynthesis Are known to play an important role in mediating the inflammatory response. Therefore, blocking inflammation by inhibiting the activity of nitric oxide synthase (NOS), an enzyme that produces NO from L-arginine, and cyclooxygenase (COX), an enzyme involved in the synthesis of prostaglandins from arachidonic acid, It is becoming a target for the development of therapeutic drugs. In addition, it is known that siccloxigenin-2 (COX-2) plays an important role in the inflammatory response by rapidly increasing in the cell during the inflammatory reaction. Transcriptional inflammatory factors, including iNOS and COX-2, that improve the degree of NO and PGs are involved in a variety of sclerosis and chronic diseases.

Nuclear transcription factor NF-κB is a nuclear protein that binds to I-κB in the cytoplasm and is inactive. However, reactive oxygen species, TNF-α and interleukin-1 (IL) I-kappaB kinase is activated by various stimuli such as chemokines or lipopolysaccharides (LPS) such as IL-1, and I-kappaB is released through the phosphorylation process. It is known that NF-κB, composed of p50 and p65 heterodimers, is activated and promotes the expression of genes that migrate to the nucleus and induce an inflammatory response.

On the other hand, periodontal tissue consists of cementum, periodontal ligament, alveolar bone and gingiva. Periodontal disease, including gingivitis and periodontitis, is an inflammatory condition of the tooth supporting tissues caused by bacteria, which can lead to loss of teeth due to bleeding, formation of the periodontal pouch and destruction of the alveolar bone. The inflammation of periodontal tissues is directly induced by endotoxins such as cytotoxins such as hydrogen sulfide, ammonia, and toxic amines, metabolites of anaerobic gram-negative bacteria, and lipopolysaccharide, a cell wall component of anaerobic gram-negative bacteria It is induced by various cytokines such as active oxygen, prostaglandins, rucotrienes, histamines, interleukins, etc., which are destroyed or secreted outside the cells by the action of the humoral and cellular immune system stimulated by stimulating the vital immune system.

In recent years, in accordance with the natural and environmentally friendly consumption tendency, the effective ingredients for oral products have been used in various forms based on their usefulness as well as chemical substances as well as natural materials. In the vegetable raw materials containing herbal medicines, The development of products using natural materials ranging from raw materials to raw materials is coming to an era of natural products. The natural products that have been used as additive in the past are expanding their usability to functional oral products based on their pharmacological properties.

On the other hand, several seaweeds have been reported to have blood pressure and blood cholesterol regulation, anti-inflammation, immunostimulatory effect, and bioactivity such as antibiotic, antioxidant, and antivirus through previous studies, but the yield of the extract is low, The active ingredient is abandoned and polysaccharides such as alginic acid and fucoidan, which constitute most of the ingredients, are disadvantageous in that they are not easily absorbed by the human body.

Korean Patent Publication No. 10-2011-0103267

Therefore, the inventors of the present invention have found that a seed culture fermented product obtained by using a functional strain of sea tangle and olive leaf extract, which has not yet been disclosed in relation to the anti-inflammatory effect, can be used as a nuclear transcription factor of NF- lt; / RTI > transcriptional activation, thereby inhibiting the inflammatory activity.

It is an object of the present invention to provide a composition for health functional food for preventing or improving periodontal disease containing a fermented tuna starch and an olive leaf fermented product.

Another object of the present invention is to provide a medicament for the prevention or treatment of periodontal disease containing a fermented tuna starch and an olive leaf fermented product.

Therefore, the present invention relates to a method for producing kelp extract, which comprises adding to a fermentation product obtained by firstly fermenting seaweed extract using a strain of Saccharomyces, a strain of Lactobacillus, Streptococcus, Bacillus and Rhodopseudomonas A fermented product obtained by inoculating a selected one or more seeds to produce a fermented product which is secondarily fermented by public cultivation, adding an olive leaf extract to the second fermented fermented product, and fermenting the fermented product by tertiary fermentation A composition for health functional food for prevention or improvement.

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In an embodiment of the present invention, the sea tangle extract may be extracted with water, methanol, ethanol, butanol or a mixture thereof.

In one embodiment of the present invention, the kelp extract may be room temperature extraction, hot water extraction, cold extraction, reflux extraction, ultrasonic extraction, or steam extraction.

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In one embodiment of the present invention, the olive leaf extract may be extracted with water, methanol, ethanol, butanol or a mixture thereof.

In one embodiment of the present invention, the olive leaf extract may be room temperature extraction, hot water extraction, cold extraction, reflux extraction, ultrasonic extraction, or steam extraction.

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In one embodiment of the present invention, the olive leaf fermentation product may be obtained by adding a brown rice germ powder to ferment.

The present invention also provides a method for producing a fermented product, comprising: obtaining a fermented product by firstly fermenting kelp extract with a strain of Saccharomyces; The fermentation product obtained by secondary fermentation by co-cultivation by adding at least one selected from the group consisting of Lactobacillus, Streptococcus, Bacillus and Rhodopseudomonas to the primary fermented product, ; A method for preparing a composition for health functional foods for prevention or improvement of periodontal disease containing kelp and olive leaf fermented product, which comprises the step of thirdly fermenting the fermented fermented product with an olive leaf extract, .

In one embodiment of the present invention, in the step (a), the olive leaf fermented product may be cultured by adding a brown rice germ powder at the time of culturing.

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Furthermore, the present invention relates to a method for producing a fermented seaweed extract, which comprises fermenting a fermentation product firstly fermented with Saccharomyces strains, into a fermentation product of Lactobacillus, Streptococcus, Bacillus and Rhodopseudomonas A fermented product obtained by fermenting the fermented product in a secondary culture by adding at least one selected fermented product to a fermented product by secondary culture, adding an olive leaf extract to the fermented fermented product, Thereby providing an agent for prevention or treatment.

 The health functional food and pharmaceutical composition for prevention or improvement of periodontal disease including the fermented product of the sea tangle and the olive leaf obtained by the invention have excellent anti-inflammatory effects while minimizing side effects as natural materials, and thus are useful for the development of periodontal disease therapeutic agents And it is possible to use the seaweed, which is rich in seaweed, to have a great economic effect on industry.

FIG. 1 is a graph showing the expression level of inflammatory cytokine IL-1β in periodontal ligament cells in which an alginate fermented product, an olive leaf fermented product and an extract mixture are subjected to an inflammatory reaction.
FIG. 2 is a graph showing the expression level of inflammatory cytokine IL-6 in periodontal ligament cells in which the tangle of tuna fermented product, the fermented olive leaf, and the extracted mixture are subjected to inflammatory reaction.
FIG. 3 shows a process for preparing a composition containing a co-cultured fermented product of sea tangle and olive leaves according to the present invention.
Fig. 4 shows the tissue photographs.
Figure 5 shows the results of periodontal biopsy evaluation through immunostaining.
6 shows the result of immunocyte measurement in blood.
FIG. 7 shows the results of measurement of pro-inflammatory cytokines in serum.
Fig. 8 shows the results of measurement of expression of NF-kB from periodontal tissue.
FIG. 9 shows the measurement results of iNOS and COX2 expression from periodontal tissue.
Fig. 10 shows the result of measurement of periodontal bone density by Micro CT.

The present invention is characterized by providing a health functional food and a pharmaceutical composition for prevention or improvement of periodontal disease containing a fermented tuna starch and an olive leaf fermented product.

In the present invention, the periodontal disease is a disease caused by inflammation of periodontal tissue, and may include periodontitis, gingivitis, or dental caries.

In the present invention, the ternaceae fermented product may be a fermented product of seaweed extract.

In the present invention, the kelp extract may be extracted with water, methanol, ethanol, butanol or a mixture thereof.

In the present invention, the seaweed extract may be at room temperature extraction, hot water extraction, cold extraction, reflux extraction, ultrasonic extraction, or steam extraction.

In the present invention, the tangle of tangle may be fermented into at least one strain selected from the group consisting of Saccharomyces, Lactobacillus, Streptococcus, Bacillus and Rhododius monas, And fermenting by fermentation using ii) Lactobacillus, Streptococcus, Bacillus and Rhododius Mounus strains, and fermenting.

In the present invention, the Saccharomyces strain includes, but is not limited to, Saccharomyces cerevisiae , Saccharomyces ellipsoideus Or Saccharomyces coreanus may be used. Streptococcus thermophilus may be used as the Streptococcus sp. Strain, and the Rhodo- Rhodopseudomonas capsulate can be used.

Bacillus licheniformis , Bacillus subtilis, and Bacillus amyloliquefaciens may be used as the Bacillus sp. Strain. Lactobacillus acidophilus, Lactobacillus bulgaricus, and Lactobacillus casei may be used as the Lactobacillus sp. Strain.

Here, the stepwise progressive 'public incubation' means culturing various strains together with each natural product step by step according to each condition.

In the present invention, the olive leaf fermented product may be a fermented product of an olive leaf extract.

In the present invention, the olive leaf extract may be extracted with water, methanol, ethanol, butanol or a mixture thereof.

 In the present invention, the olive leaf extract may be at room temperature extraction, hot water extraction, cold extraction, reflux extraction, ultrasonic extraction, or steam extraction.

  In the present invention, the olive leaf fermented product may be fermented into one or more strains selected from the group consisting of Lactobacillus, Streptococcus, Bacillus,

In particular, it may be fermented by co-culturing with Lactobacillus, Streptococcus, Bacillus and Rhododius Mounus strains.

In the present invention, the olive leaf fermented product may be characterized by adding a brown rice germ powder to ferment.

The present invention provides a method for preparing a health functional food and a pharmaceutical composition for preventing or ameliorating periodontal disease containing a fermented tuna extract and an olive leaf fermented product comprising the steps of:

(a) culturing the kelp extract and the olive leaf extract with at least one strain selected from the group consisting of Saccharomyces, Lactobacillus, Streptococcus, Bacillus and Rhododius monas, respectively, to obtain a culture;

(b) mixing the kelp culture and the olive leaf culture.

In the step (a) of the present invention, the olive leaf fermented product may be cultured by adding a brown rice germ powder during the culture.

In the step (a) of the present invention, the tangle of tangle may be prepared by i) culturing the strain using Saccharomyces strain, followed by fermentation, and ii) using Lactobacillus, Streptococcus, Bacillus, And then cultivating it by co-culture.

In the present invention, the mixing ratio of the kelp-extracted fermented product to the olive leaf-extracted fermented product may be 1: 3 to 3: 1, preferably 1: 1.

In the present invention, the manufacturing process will be described in detail as follows.

(1) Preparation of fermented sea tangle

* Seaweeds are cut and rinsed in seaweed to an appropriate size, then immersed in purified water to dissolve and remove alginate. Purified water or alcohol is extracted as an extraction solvent, and then subjected to filtration and vacuum concentration. That is, 50 g of kelp was extracted with 1500 ml of extraction solvent, and then the extraction solvent was filtered and concentrated in vacuo. After the primary extraction, the kelp is further extracted with purified water, filtered and concentrated in vacuo to obtain a secondary extract.

(2) Preparation of fermented olive leaves

An olive leaf which is easy to obtain polyphenol among natural products is cut and extracted with an alcohol solvent, and then concentrated in vacuo to obtain a first extract, followed by second extraction using purified water. These extracts are concentrated in vacuo and filtered to give a crude extract.

(3) Natural product cultivation

The seaweed extract of (1) above is mixed with purified water, alcohol sugar, fructose and the like, sterilized, and then yeast Saccharomyces ( Lactobacillus spp.), Streptococcus sp., Bacillus spp., and Bacillus spp.) were inoculated and cultured at 25 ° C to 37 ° C for 2 to 3 days. Secondary incubation is carried out at 15 ° C to 30 ° C for 10 to 30 days using monos ( Rhodopseudomonas sp.).

The extract of (2) and the brown rice germ powder are further added and incubated for 10 to 30 days at 15 ° C to 25 ° C.

In this case, it can be mixed with 40-50% of tangle of tangle, 40-50% of olive leaf fermented product, and 5-20% of brown rice embryo powder. Preferably, the fermented sea tangle: olive leaf fermented product: %: 45%: 10%.

(4) Filtration and molding process and composition manufacturing

In the above (3), 300 ml of the extract, 1000 ml of the extracts of the above (1) and (2), 200 g of the brown rice germ powder, 400 ml of purified water, 15 g of alcohol and 5 g of fructose were mixed to prepare 2000 ml, Lt; RTI ID = 0.0 > 30 C < / RTI > After sterilization, the mixture was filtered on a filter press Kelp and olive leaf blend fermented.

As described above, the present invention provides a composition for health functional food for preventing or improving periodontal disease, which comprises fermented tuna and fermented olive leaf as an active ingredient.

The health functional food of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, and rings for the purpose of preventing or improving periodontal disease.

In the present invention, the term " health functional food " refers to foods manufactured and processed using raw materials or ingredients having useful functions in accordance with Law No. 6727 on Health Functional Foods. Or to obtain a beneficial effect in health use such as physiological action.

The health functional foods of the present invention may contain conventional food additives and, unless otherwise specified, whether or not they are suitable as food additives are classified according to the General Rules for Food Additives approved by the Food and Drug Administration, Standards and standards.

Examples of the items listed in the above-mentioned "food additives" include chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; Natural additives such as persimmon extract, licorice extract, crystalline cellulose, high color pigment and guar gum; L-glutamic acid sodium preparations, noodle-added alkalis, preservative preparations, tar coloring preparations and the like.

For example, the health functional food in the form of tablets may be prepared by granulating a mixture obtained by mixing the melon seed extract, which is an active ingredient of the present invention, with an excipient, a binder, a disintegrant and other additives in a usual manner, Or the mixture can be directly compression molded. In addition, the health functional food of the tablet form may contain a mating agent or the like if necessary.

The hard capsule of the capsule type health functional food can be prepared by filling a normal hard capsule with a mixture of an active ingredient of the present invention and an additive such as an excipient and the soft capsule is prepared by mixing the active ingredient with an additive such as an excipient And filling the mixture into a capsule base such as gelatin. The soft capsule may contain a plasticizer such as glycerin or sorbitol, a coloring agent, a preservative and the like, if necessary.

The ring-shaped health functional food can be prepared by molding a mixture of the active ingredient of the present invention with an excipient, a binder, a disintegrant, etc. by a conventionally known method and, if necessary, , Or a surface such as starch, talc, or the like.

The granular health functional food may be prepared by granulating the active ingredient of the present invention with an excipient, a binder, a disintegrant or the like by a conventional method, and may contain a flavoring agent, a mating agent or the like if necessary have.

The health functional food may be a beverage, a meat, a chocolate, a food, a confectionery, a pizza, a ramen, a noodle, a gum, a candy, an ice cream, an alcoholic beverage, a vitamin complex and a health supplement food.

The present invention also provides a pharmaceutical composition for preventing or treating periodontal disease, which comprises a fermented tuna extract and fermented olive leaf as an active ingredient.

The pharmaceutical composition according to the present invention can be formulated into various forms of external preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, and sterilized injection solutions according to conventional methods, Examples of carriers, excipients and diluents which may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used.

Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose ), Lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As a suppository base, witepsol, macrogol, tween 60, cacao paper, laurin, glycerogelatin and the like can be used.

The preferred dosage of the fermented product of the present invention varies depending on the condition and body weight of the patient, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect,

It is preferably administered at 0.0001 to 100 mg / kg, preferably 0.001 to 100 mg / kg. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

When the fermented product is used as a food additive, the fermented product may be directly added or used together with other food or food ingredients, and may be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to its intended use (prevention, health or therapeutic treatment). Generally, the fermentation product of the present invention is added in an amount of not more than 15 parts by weight, preferably not more than 10 parts by weight, based on the raw material, in the production of food or beverage. However, in the case of long-term consumption intended for health and hygiene purposes or for health control purposes, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range .

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

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in detail with reference to the following examples and experiments.

[Example 1]

Preparation of containing kelp and olive leaves public order fermentation culture according to the invention the composition

1-1. Extraction and recovery of seaweed

50 g of kelp in seaweed is cut and rinsed to an appropriate size, and then immersed in 20-fold purified water for 10 hours or more to elute the alginate. Purified water or 1500 ml of 80% alcohol is used as an extraction solvent and extracted at 80 ° C for 2 to 10 hours. The extract is filtered, and purified water or alcohol is separated and recovered through vacuum concentration. 300 ml of sea tangle extract is obtained through primary extraction and solvent recovery. In the second extraction step, 700 ml of purified water is added, and the mixture is extracted at 90 ° C for 3 hours, filtered and concentrated in vacuo to obtain 300 ml of a second kelp extract.

1-2. Extraction and recovery of olive leaf

Olive leaves, which are easy to obtain polyphenol among natural products, were used. 50 g of dried olive leaves are cut and 2000 ml of 50 ~ 80% alcohol is added, followed by extraction at 70 ~ 100 ℃ for 3 ~ 12 hours. The extract was concentrated in vacuo to remove the solvent, and 450 ml of the primary extract was obtained.

1000 ml of purified water is added in the second extraction step and extracted at 90 ° C for 3 hours. The extract is concentrated in vacuo to a volume of 550 ml, filtered, and the remaining Alcohol is recovered and removed and mixed with the first extract to make 1000 ml.

1-3. Fermentation process of seaweed and olive leaf extract

300 ml of each of the first and second extracts of Example 1-1, 400 ml of purified water, 15 g of alcohol, and 5 g of fructose are mixed to prepare 1000 ml, and sterilized at 121 ° C for 30 minutes or more. Yeast Saccharomyces After incubation for 2 ~ 3 days at 25 ℃ ~ 37 ℃, Lactobacillus spp., Stretococcus sp., Bacillus spp. and Rhodopseudomonas spp. sp.) is secondarily incubated for 10 to 30 days at 15 ° C to 30 ° C. To 1000 ml of the extract of Example 1-2 and 200 g of brown rice germ powder are added to the above culture, After incubation, filter on a filter press to produce a fermented product.

As the strains used for the above-mentioned public cultivation, specifically Saccharomyces, Saccharomyces cerevisiae , Saccharomyces ellipsoideus Or Saccharomyces coreanus was used. Streptococcus thermophilus was used as the Streptococcus sp. Strain, and Rhodopseudomonas capsulase was used as the Rhodotaxin sp. Strain. Bacillus licheniformis , Bacillus subtilis and Bacillus amyloliquefaciens were used as Bacillus subtilis strains. Lactobacillus acidophilus, Lactobacillus bulgaricus and Lactobacillus casei were used as Lactobacillus spp. Strains.

1-4. Preparation of extraction mixture

300 ml of each of the first and second extracts of Example 1-1, 1000 ml of the extract of Example 1-2, 200 g of brown rice germ powder, 400 ml of purified water, 15 g of alcohol, 5 g of fructose, And then sterilized at 121 캜 for 30 minutes or more. After sterilization, the mixture was filtered on a filter press Kelp and olive leaf blend extract.

[Example 2]

Analysis of inflammatory cytokine expression in periodontal ligament cells (IL-1β, IL-6)

The tannin and olive leaf mixed fermented product obtained through the stepwise co-cultivation described in Example 1 above and each of the extract mixtures not subjected to fermentation in the above <Example 1-4> inhibited the expression of inflammatory cytokines in periodontal ligament cells To confirm the effect, periodontal ligament cells were treated with LPS to induce inflammation.

Specifically, periodontal ligament cells (PDL) were cultured in DMEM containing 1% antibiotics and 10% FBS at 37 ° C and 5% CO ₂ . Periodontal ligament cells were seeded at a density of 5 × 10 ⁵ cells / ml per well in a 96-well plate and cultured at 37 ° C. for 24 hours. To induce the inflammatory reaction, 1 ㎍ / ml of LPS was treated, and then the fermented product and the extracted mixture were treated at a concentration of 100 ㎍ / ml and cultured at 37 ° C and 5% CO ₂ for 48 hours. Expression of IL-1β and IL-6 from cell suspensions treated with LPS, fermentation products and extract mixtures was measured using an ELISA kit (R & D system). The cell suspension was diluted stepwise and the samples were pretreated according to the reagents and methods provided in the ELISA kit. After pre-treatment, the absorbance at 450 nm was measured by an ELISA reader to measure the expression of inflammatory cytokines.

LPS acting as a typical endotoxin of periodontal disease is present in the extracellular membrane of Gram-negative bacteria, and pro-inflammatory cytokines such as TNF-α, IL-6 and IL-1β in macrophages or monocytes . IL-1β, and IL-6, respectively, were induced by stimulation of LPS with periodontal ligament cells and treatment of extracts from sea tangle and olive leaves.

The expression of IL-1β and IL-6, which are pro-inflammatory cytokines, was significantly lower in the untreated group than in the control group. In addition, the expression levels of IL-1β and IL-6 in the treated group were lower than those of the LPS-treated control group when the extract mixture was treated. 1 and 2) in the fermented water-treated group treated with kelp and olive leaf extracts compared to the LPS treated control group and the extract mixture treated group (see FIGS. 1 and 2).

  Therefore, it was confirmed that the inhibitory effect of the inflammatory factor when the inflammation was induced in the periodontal ligament cells was inhibited by the extract of the kelp and olive leaf extract and the fermented fermented product from the sea tangle and olive leaf, compared to the LPS-treated control group , Kelp, and olive leaf, the fermented product fermented was found to be more effective in inhibiting the expression of inflammatory factors (see FIGS. 1 and 2).

Experimental Example 1: Results of administration of periodontal tissue of the composition according to the present invention

To induce periodontitis, 10 μg of LPS was dissolved in PBS at 6 weeks of age in C57BL / 6 mice. Normal mice were injected with PBS only. In the control group (LPS), LPS was dissolved in PBS and injected into the first molar at the lower left for 48 weeks for 13 weeks. In the treated group, LPS was dissolved in PBS and injected into the first molar at the lower left for 48 weeks for 13 weeks. The test substance kelp and the olive leaf fermented product were orally administered. As a method of preparing the test materials, the sea tangle and the olive leaf fermented product were finely ground with a mortar as a cyclic material and then dissolved in PBS.

As shown in FIG. 4, the inflammation due to periodontitis progressed in the soft tissue and epithelial tissue surrounding the left tooth, and the color of the tissue was pink It seemed to be a dark red color. In the same way, inflammation of the red gingiva in the frontal gingival region was also confirmed by photographing. That is, the oral administration of the sea tangle and olive leaf fermented product prepared in Example 1 at a concentration of 5, 10 and 20 mg / kg of mouse for one week for 13 weeks resulted in lowering of periodontal inflammation compared to the group administered with LPS alone Confirmed.

Experimental Example 2: H & E Histological Analysis

After 13 weeks of induction of periodontal disease in the mouse model as in Experimental Example 1, the skull fragment including the surgical site was taken for histological examination, fixed in 10% neutral formalin for 2 weeks, and then placed in 5% nitric acid for 5 days Demineralized and embedded in paraffin. Cut the gums and teeth into 5-mm thick specimens and stain them with hematoxylin and eosin (HE). Then, the osteogenic findings are observed by taking a tissue microscope. For COX-2 and iNOS immunostaining, the digested samples were placed in 0.1 M citrate buffer (pH 6.0), heated in a microwave oven for 3 minutes without boiling, cooled for 1 hour at room temperature, and washed three times for 10 minutes with PBS. To block non-specific endogenous peroxidase, the cells were incubated with 0.3% hydrogen peroxide solution for 15 minutes at room temperature. Nonspecific endogenous peroxidase was blocked and reacted with the primary antibody, COX-2, iNOS (1: 500 dilution, Cayman Chemical, Ann Arbor, Mich., USA) overnight at 4 ° C. The primary antibody was washed three times with PBS and reacted with Dako Envision + System-HRP Labeled Polymer Anti-Mouse and anti-rabbit (DAKO, USA) for 30 minutes at room temperature. After washing three times with PBS, diaminobenzidine (DAB) . After color development, the cells were washed three times with distilled water and counterstained with Mayer's hematoxylin. We then examined the expression of COX-2 and iNOS by histological microscopy.

As a result, as shown in FIG. 5, when H & E staining was performed, compared with the normal group, the LPS administration group showed a large amount of alveolar bone damaged and a large number of gums were observed. In addition, it was confirmed that the cementum was worn. The expression of iNOS and COX-2 was confirmed by immunohistochemical staining. As a result, the expression of iNOS and COX2 was observed in the alveolar bone, (5, 10, and 20 mg / kg) at concentrations comparable to the clinical concentrations, the concentrations of iNOS and COX2 were decreased in a concentration-dependent manner compared to the LPS-treated group.

Experimental Example 3: Expression test of NF-kB

After 13 weeks of induction of periodontitis in the mouse model as in Experimental Example 1, the CBC test was performed to confirm the increase of inflammation and the level of WBC, which is an immune cell, due to the expression of NF-κB. That is, after 13 weeks from the induction of periodontitis in the mouse model as in Experimental Example 1, blood was collected and the blood was coagulated upside down in an EDTA tube to prevent blood coagulation, 4 &lt; 0 &gt; C. After that, white blood cells were measured using an automatic hemocytometer (Technicon Instruments, Miles Laboratories, Tarrytown, NY) at Chungbuk National University Experimental Animal Research Support Center.

As a result, as shown in FIG. 6, the control group treated with LPS showed a high WBC level, but the animals fed with the tangle and olive leaf fermentation obtained by the stepwise co-culture of the microorganisms decreased in a concentration-dependent manner.

Experimental Example 4 Expression Analysis of TNF-alpha, IL-6 and IL-1 Beta

We measured levels of TNF-alpha, IL-6 and IL-1 beta with serum collected at the time of autopsy in order to confirm the increase of inflammation and the level of pro-inflammatory cytokines due to the expression of NF-κB.

For the cytokine analysis, ELISA kit (R & D system) was used. For the quasi-solution, 500ul of sample diluents was added to the antigen standard powder and diluted stepwise. Experimental procedure was as follows according to the reagents and methods provided. Serum was added to each well of a microplate (TNF-alpha, IL-6, IL-1 beta) in a volume of 50 ul and reacted at room temperature with 500 rpm intensity for 2 hours on a plate shaker. After washing 3 times, 50μl of Detection mix solution was added to each well, and reacted in a plate shaker for 1 hour at room temperature and 500rpm strength. After washing three times, 200 μl of the conjugate solution was dispensed and reacted in a plate shaker for 2 hours at a temperature of 500 rpm at room temperature. After washing again 6 times, Substance A and Substance B solutions were mixed 1: 1 and the mixed substrate was dispensed in 50 μl of each well and reacted in a plate shaker at room temperature and 500 rpm for 20 minutes. Then, 50 μl of the reaction stop solution was dispensed and 450 nm was measured within 30 minutes.

As a result, as shown in FIG. 7, although the levels of pro-inflammatory cytokines in the control group treated with LPS alone were high, in the animal model in which kelp and olive leaf fermented by the stepwise co-cultivation of microorganisms were administered The levels of cytokines decreased in a dose-dependent manner.

Experimental Example 5: iNOS and COX2 expression analysis

Western blot assay was performed to measure the expression of iNOS and COX2 proteins, which are inflammatory markers in periodontal tissue.

For this purpose, periodontal model tissues were lysed in lysis buffer (50 mM Tris pH 8.0, 150 mM NaCl, 0.02% sodium azide, 0.2% SDS, 1 mM PMFS, 10 μl / ml aprotinin, 1% igapel 630 , MO, USA), 10 mM NaF and 0.5 mM EDTA] and centrifuged at 23,000 × g for 15 minutes. Protein concentrations were measured and then loaded on SDS / 12% -polyacrylamide gels to 20 μg, and then transferred to Hybond ECL Nitrocellulose membrane (Amersham Pharmacia Biotech Inc., Piscataway, NJ, USA). The membrane is incubated at room temperature for 2 hours with 5% (w / v) non-fat dried milk dissolved in tris buffer [10 mM Tris (pH 8.0) and 150 mM NaCl] containing 0.05% . Each membrane is incubated with primary antibody monoclonal antibodies iNOS and COX-2 (1: 1000 dilution, Cayman Chemical, Ann Arbor, Mich., USA) for 5 h at room temperature and washed with tris buffer. After this, anti-rabbit and anti mouse (1: 2,000 dilution, Santa Cruz Biotechnology Inc.) are incubated for 2 hours with secondary antibody and washed with tris buffer. Proteins activated by binding to the antibody were measured using an ECL Western blotting detection system. In this case, β-actin protein was used as a loading control.

As a result, as shown in FIG. 8, the expression of iNOS and COX2 protein was high in the control group to which LPS was administered, but the tuna and olive leaf fermentation products obtained by the stepwise co-culture of LPS and microorganisms were also administered One animal decreased in concentration-dependent manner.

Experimental Example 6: Analysis of NF-kB expression change

Binding activity of 32P-labeled oligonucleotides specific for NF-κB was determined by electrophoretic mobility shift assays (EMSA) in tissue nuclear extracts of periodontal models treated with kelp and olive leaf fermented products.

To this end, the periodontal model was treated with a solution A (50 mM HEPES, pH 7.4, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 0.1 μg / ml phenylmethylsulfonyl, 1 μg / ml pepstatin A) (v / v) and incubated in an ice bath for 10 minutes. Solution C (Solution A and 10% glycellol and 400 mM KCl) is added to the pellet in a 2: 1 ratio (v / v) and stirred for 20 minutes in an ice bath. The cells were centrifuged at 15,000 g for 7 min and the nuclear extract supernatant was collected in tubes. The oligonucleotide [γ-32P] and ATP were positively reacted with T4 polynucleotide kinase at 37 ℃ for 10 min. After the gel-transfer reaction, the mixture was incubated at room temperature for 10 minutes, 1 olig of oligonucleotide of 50,000 to 200,000 cpm of 32P end-label was added, and the mixture was incubated at room temperature for 20 minutes. Subsequently, 1 μl of gel loading buffer was added and loaded with 4% denaturation gel, and electrophoresis was performed until 3/4 of the dye had penetrated the gel. The gel was dried at 80 ° C for 1 hour, exposed to film overnight at 70 ° C, and then developed.

As a result, as shown in FIG. 9, the expression of NF-kB was very high in the control group administered with LPS alone, but in the animals treated with the sea tangle and olive leaf fermentation obtained by the stepwise co-culture of LPS and microorganisms Expression of NF-kB decreased in a concentration-dependent manner.

Experimental Example 7: Dental bone mineral density analysis

Inflammation caused by periodontitis occurs around the gums, and inflammation penetrates deeply into the gums and spreads widely around. The purpose of this study was to evaluate the bone density of the left alveolar bone, which can be lost by periodontitis, compared with the normal alveolar bone density.

For this purpose, 13 weeks after the induction of periodontitis in the mouse model as in Experimental Example 1, a specimen was taken and micro CT (SkyScan 1072, SkyScan, Aartselaar, Belgium) was taken at Yonsei University College of Medicine . Shooting method Alveolar bone loss stereomicroscopic analysis was evaluated using tomography. The tissue specimens were then scanned at a resolution of 35 ㎛ (100 kV, 100 μA) and the data were digitally imaged for reconstruction of the 3D image to evaluate bone loss in the teeth.

As a result, as shown in FIG. 10, in the case of administration of the kelp and olive leaf fermented product obtained by the stepwise co-cultivation of microorganisms (5, 10, 20 mg / kg) , The damage of alveolar bone density was significantly reduced.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (16)

One or more selected from among Lactobacillus, Streptococcus, Bacillus and Rhodopseudomonas strains were added to the fermentation product obtained by firstly fermenting seaweed extract with Saccharomyces strain The fermented product was inoculated and subjected to secondary fermentation by public incubation,
A composition for health functional foods for prevention or improvement of periodontal disease containing fermented product which is fermented by tertiary fermentation in a public incubation by adding olive leaf extract to a fermented fermented product. delete The method according to claim 1,
Wherein the sea tangle extract is extracted with water, methanol, ethanol, butanol or a mixture thereof. The method of claim 3,
Wherein the sea tangle extract is at room temperature extraction, hot water extraction, cold extraction, reflux extraction, ultrasonic extraction or steam extraction. delete delete delete The method according to claim 1,
Wherein the olive leaf extract is extracted with water, methanol, ethanol, butanol or a mixture thereof. 9. The method of claim 8,
Wherein the olive leaf extract is at room temperature extraction, hot water extraction, cold extraction, reflux extraction, ultrasonic extraction or steam extraction. delete delete The method according to claim 1,
Wherein the fermented product obtained by the third fermentation is fermented by adding a brown rice germ powder. Obtaining a fermented product obtained by firstly fermenting kelp extract with a strain of Saccharomyces;
The fermentation product obtained by secondary fermentation by co-cultivation by adding at least one selected from the group consisting of Lactobacillus, Streptococcus, Bacillus and Rhodopseudomonas to the primary fermented product, ;
A method for preparing a composition for health functional foods for prevention or improvement of periodontal disease containing kelp and olive leaf fermented product, which comprises the step of thirdly fermenting the fermented fermented product with an olive leaf extract, . 14. The method of claim 13,
Wherein the third fermented product is cultured by adding a brown rice germ powder during the culture. delete One or more selected from among Lactobacillus, Streptococcus, Bacillus and Rhodopseudomonas strains were added to the fermentation product obtained by firstly fermenting seaweed extract with Saccharomyces strain To prepare a fermented product which is secondarily fermented by a public incubation,
A medicament for the prevention or treatment of periodontal disease containing fermented product fermented by co-culture with olive leaf extract added to the fermented product of the second fermentation as an active ingredient.

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