KR102007144B1 - A novel strain of Lactobacillus plantarum SKB1234 having an improving effect on arthritis and a method for producing a mixture of the fermented Achyranthes japonica Nakai extract using the Lactobacillus plantarum SKB1234, Angelica gigas Nakai extract and Eucommia ulmoides Oliver extract - Google Patents

Abstract

The present invention relates to a novel strain of Lactobacillus plantarum SKB1234, and a method for producing a mixture of fermented Achyranthes japonica extract using the strain, Angelica gigas extract and Eucommia ulmoides extract. The fermented Achyranthes japonica extract obtained by fermenting an Achyranthes japonica extract with the Lactobacillus plantarum SKB1234 strain, or a mixture of the Angelica gigas extract and the Eucommia ulmoides extract added herein is excellent in cartilage production and inflammation inhibition efficacy in articular cartilage or arthritis induced animal model, thereby capable of being easily applied to various pharmaceutical compositions or functional health foods having the efficacy of preventing, ameliorating or treating arthritis.

Description

A novel strain of Lactobacillus plantarum SKB1234 having an improving effect on arthritis and a method for producing a mixture of the fermented Achyranthes japonica Nakai extract using the Lactobacillus plantarum SKB1234, Angelica gigas Nakai extract and Eucommia ulmoides Oliver extract}

The present invention relates to a method for preparing a mixture of Lactobacillus plantarum SKB1234 ( Lactobacillus plantarum SKB1234, Accession No .: KCCM12450P) novel strain, fermented dew extract, Angelica extract and tofu extract of the arthritis.

Osteoarthritis is a general term for several diseases involving joint damage and can be broadly divided into osteoarthritis (Osteoarthritis) and rheumatoid arthritis (Rheumatoid Arthritis).

Osteoarthritis is a disease in which the cartilage layer developed in the joints of the body is damaged to alleviate the impact and friction of the bones that are directly hit by the bones. May occur mainly in one joint, such as the knee, hip, hand, foot, and spine, and may occur simultaneously in multiple joints. Rheumatoid arthritis is an autoimmune disease that causes inflammation by attacking a part of one's joints or body due to an abnormality of the immune system and causes pain and swelling of joints such as fingers, hands, feet, wrists, ankles, knees, muscles, skin, It is a systemic chronic inflammatory disease that can cause abnormalities in many other organs such as the lungs and eyes.

Arthritis patients cause severe pain, so be sure to take anti-inflammatory drugs, etc. These chemotherapy drugs have drawbacks such as side effects that prevent long-term use of the drug, lack of anti-inflammatory effects, and lack of efficacy against arthritis that has already occurred. As a solution to this problem, there is a continuing need for the development of effective arthritis therapeutics.

Most of the currently used arthritis medications have a difference in degree and individual differences, but all have side effects. Especially, in order to treat rheumatoid arthritis, it is necessary to take medicine for a long time. very important. Osteoarthritis is a senile disease in general, and it is a disease caused by loss of research and aging, excessive use of joints, obesity, etc. Rheumatoid arthritis (inflammatory arthritis) is an autoimmune disease suffered by about 1% of the Korean population. Rheumatoid arthritis usually occurs in the 20s and 40s and is three times more common in females than in males. It is a disease involving joint swelling and pain caused by chronic inflammation caused by the release of many inflammatory mediators and cytokines. .

Ussul is the name of the root of the Anchyranthes japonica Nakai, a herb that is distributed in all regions of Korea, Japan, and sub- Hwangha in Korea. It is named after a bark that swells like a cow's knee. The taste of hyssop is bitter and sour, non-toxic and works on the liver and kidney. 'Is introduced. In particular, hyssop is effective in diuresis, granulation, arthritis, etc., and major ingredients include saponin, 20-Hydroxyecdysone, oleanolic glycoside, and succinic acid.

Angelica is the root of perennial aromatic herbaceous genus belonging to the Apiaceae family, and the herbaceous tree has a "cough, coughing up, severe, coughing up of skin, miscarriage, miscarriage, all boils, swells, gold swelling and drinking juice." . "," Warms the inside, relieves pain and removes clumps of blood. Also, it prevents sweating due to invasion of fraud, symptoms caused by wet hygiene, invasion of severe fraud, and cold and loose symptoms. And the five chapters and to improve the house. " The main components of Angelica are vitamin B12, folic acid, vitamin E, Decrucin, and Decruxianzerate.

The larvae are stem bark of E. ulmoides Oliv. Belonging to the genus Eucommia of the family Eucommiaceae. Tofu is a herb listed in the middle part of the 'new agricultural herbaceous', and is one of the oldest herbal medicines used in medicine. Stems and bark are reported to be used for high blood pressure, erectile dysfunction, back pain and sciatica. In Korea and Japan, the leaves of the larvae are used as medicinal and beverages. Recently, the larvae of larvae are drinking tea blossoms. The main active ingredients of larvae are lignans and iridoids. Phenolics, steroids, terpenoids and flavonoids are also included in the larvae.

On the other hand, to date, most health foods for the treatment of arthritis have cartilage components as a main ingredient. Among them, the method of applying the therapeutic agent using lactic acid bacteria has been actively studied recently. Therefore, while the present inventors have studied various physiological activities of these lactic acid bacteria, fermented dew extract fermented by Lactobacillus plantarum SKB1234 new strain, or a mixture (composite) of the mixture of the Angelica extract and the worm extract in this fermented dew extract arthritis Confirmed that the therapeutic efficacy of the superior to complete the present invention.

Republic of Korea Patent Registration No. 10-1720051 (Invention: Pharmaceutical composition for preventing or treating arthritis, including as an active ingredient as an active ingredient, Applicant: Kyungwoon University Industry-Academic Cooperation Group and one other person, registered date: March 21, 2017) Republic of Korea Patent No. 10-1338941 (Invention name: Fermented dew extract for the prevention and treatment of osteoarthritis or rheumatoid arthritis, and a method for producing the same, Applicant: NYU Co., Ltd., Life and Health and others, registered date: December 2013 Work) Republic of Korea Registered Patent No. 10-0415815 (Invention name: pharmaceutical composition containing the main ingredient, such as hyssop, five holes, two worms, ogapi, windproof, and pharmaceutical preparations containing it as a main ingredient, Applicant: Shin Junsik, registered date: January 2004 Work) Republic of Korea Patent Publication No. 10-2008-0105825 (Name of the invention: herbal composition for the treatment and prevention of arthritis and a method of manufacturing the same, Applicant: Kim Sun-jeong, Publication Date: December 04, 2008)

It is an object of the present invention to provide a method for producing a mixture of Lactobacillus plantarum SKB1234 ( Lactobacillus plantarum SKB1234, Accession No .: KCCM12450P) novel strain, fermented dew extract, Angelica extract, and tofu extracts having an effect of improving arthritis. .

The present invention relates to a new strain of Lactobacillus plantarum SKB1234 (Accession No .: KCCM12450P) having an improvement in arthritis.

The arthritis may be a disease selected from osteoarthritis (Osteoarthritis) or rheumatoid arthritis (Rheumatoid Arthritis).

The present invention provides a method for producing a fermented dew extract complex using the strain.

Preferably the fermented dew extract complex,

(First step) preparing the dew extract, Angelica extract and worm extract, respectively;

(Second step) inoculating the Lactobacillus plantarum SKB1234 strain to the dew extract obtained in the first step to obtain a fermented dew extract; And

(Third step) mixing the fermented dew extract obtained in the second step, the Angelica extract of the first step and the worm extract of the first step;

. ≪ / RTI >

In the first step, the extract of dew drops, Angelica extract or larvae extract 10 to 40 times the weight of water to each raw medicinal herb, Angelica or larvae and extract for 2 to 10 hours under conditions of 90 to 100 ℃, The extract obtained by filtration may be concentrated and prepared as an extract containing 10 to 30% by weight of solids.

In addition, in the first step in the preparation of the dew extract, Angelica extract and the worm extract, after extracting the primary by adding water to each of the dew, Angelica, worms, take the extract, and then again to the remaining dew, donggi or worms (in the same way) Additional extraction can be carried out by adding water, and the second or third extraction can be carried out consecutively.

On the other hand, the Angelica extract and the worm extract may be carried out only the first extraction, but in the preparation of the extract of the dew extract in the first step, after the first extraction by adding water to the dew, after taking the extract and adding water to the remaining dew It is better to proceed with the second extraction.

The fermented dew drop extract of the second step is obtained by inoculating Lactobacillus plantarum SKB1234 strain to the dew drop extract and concentrating the liquid obtained by heat treatment and filtration to a concentration of 10 to 30% by weight of solids. have.

In more detail, the fermented dew extract is inoculated with Lactobacillus plantarum SKB1234 strain to the extract, and the liquid obtained by heat treatment and filtration to the culture obtained by incubating at 30 to 37 ℃ for 15 to 24 hours, the solid content 10 again Can be obtained by concentration to ˜30% by weight.

In the preparation of the fermented dew extract, Lactobacillus plantarum SKB1234 strain is preferably inoculated so that the strain is 1 to 10% by weight when the extract containing the strain is 100% by weight of the culture raw material. The strain inoculated into the dew extract may be a cell or a preculture of the cell.

In the third step, dextrin or beta-cyclodextrin may be mixed or added as an excipient when the fermented dew extract, the Angelica extract and the worm extract are mixed.

In the third step, the mixing ratio of the fermented dew extract, Angelica extract and tofu extract, based on 30 parts by weight of fermented dew extract, 15-30 parts by weight of Angelica extract, 10-22 parts by weight of Tofu extract, For powder formulation, further 25-50 parts by weight of beta-cyclodextrin may be added.

At this time, when the Angelica extract is mixed by less than 15 parts by weight, or when the Angelica extract is mixed by more than 30 parts by weight of fermented dew extract, or when the larvae extract is mixed by less than 10 parts by weight, When mixed in excess of parts, the stability of the formulation may not be guaranteed, which is undesirable.

In addition, the fermented dew extract, Angelica extract, and Tobacco extract may be mixed in such a mixed ratio to prepare an extract complex having excellent pharmacological activity. In addition, in the preparation of extract composites, the addition of excipients enhances formulation stability during drying and stability during storage.

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

Arthritis in the present invention may be selected from osteoarthritis (Osteoarthritis) or rheumatoid arthritis (Inflammatory arthritis, Rheumatoid Arthritis). Even more preferably, it may be osteoarthritis.

Lactobacillus plantarum SKB1234 strain in the present invention is a cell of the strain; Lysate of the cells; Culture of said strain; Culture medium from which the cells are removed from the culture of the strain; Cell extract of the strain; Extracts of cultures of said strains; And, the extract of the culture solution to remove the cells from the culture of the strain; may be replaced or included in the selected from the group consisting of.

Lactobacillus plantarum SKB1234 strain of the present invention can be cultured in a liquid or solid medium (broth or agar) of MRS, it can be cultured to a concentration of 3.0 × 10 ⁸ ~ 3.0 × 10 ⁹ CFU / ㎖.

The strain is preferably incubated for 16 to 24 hours at 30 to 37 ℃, the optimum temperature for cultivation is 37 ℃, the minimum temperature is 24 ℃, the maximum temperature is 40 ℃, the optimum pH is 6.0 ~ 6.5, the minimum cultivable pH 4.0 maximum pH is 6.5. The optimum incubation time is 18 hours and the minimum incubation time is 16 hours and can be incubated for up to 2 days.

Each composition derived from the strain can be used without additional processing, and may be purified or dried through various methods and added to the pharmaceutical composition or health functional food. The strain-derived composition may be dried by performing a lyophilization method, and may include various excipients included when drying a conventional strain or a composition derived therefrom. The lyophilized excipients are gluconic acid, alginic acid, sodium alginate, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, sodium carboxymethyl cellulose, methyl cellulose, carbomer, hyaluronic acid, tragacanth, karaya gum, water soluble starch, pectin, Gelatin, polyvinyl alcohol, polyvinyl pyrrolidone, oligosaccharides, sugar alcohols, calcium gluconate, calcium lactate, polymethyl methacrylate, wheat protein, soy protein, methyl cellulose, aqua coat, guar gum, locust bean gum, Xanthan gum, gellan gum, gum arabic, trehalose and the like.

The present invention provides a fermented dew extract which fermented the dew extract as a culture of Lactobacillus plantarum SKB1234 strain. The fermented dew extract may be used as a fermented dew extract complex by mixing with the extract of Angelica sinensis and tofu extract, and the fermented dew extract complex of the fermented dew extract or mixed with Angelica extract and soybean extract is also applied as a composition for the prevention or treatment of arthritis It is possible.

In another aspect, the present invention can provide a dietary supplement for the prevention or improvement of arthritis comprising the strain.

Thus, in the present invention, it is possible to prepare a health functional food for the prevention or improvement of arthritis, which contains the fermented dew extract extract complex fermented dew extract, Angelica extract and worm extract.

In the preparation of the fermentation dew extract extract of each of the herbal extracts mixed with the fermented dew extract derived from the strain, as an excipient in the drying or mixing process, a commonly used excipient such as dextrin or beta-cyclodextrin may be used, more preferably. It is recommended to use beta-cyclodextrin. If no excipient is added, drying is not good, so it is better to proceed with drying at the minimum possible condition.When the total weight immediately after drying is 100% by weight, the excipient ratio is 10 to 50% by weight. Better but more preferably 33% by weight.

In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of arthritis, containing various compositions derived from the strain, fermented dew extract or fermented dew extract complex prepared using the same. The strain-derived composition, fermented dew extract, fermented dew extract complex may be added to the pharmaceutical composition as 0.001 to 30% by weight.

The pharmaceutical compositions may be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories, etc., according to conventional methods. Examples of carriers, excipients and diluents that can be contained in the pharmaceutical composition 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. 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 preparations for oral administration include tablets, pills, powders, granules, capsules and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate, sucrose or lactose, It is prepared by mixing gelatin. 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 sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, suppositories, 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. Examples of suppository bases include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like.

The dosage of the pharmaceutical composition of the present invention will vary depending on the age, sex, weight of the subject to be treated, the specific disease or pathology to be treated, the severity of the disease or pathology, the route of administration and the judgment of the prescriber. Dosage determinations based on these factors are within the level of ordinary skill in the art and generally the dosage ranges from 0.01 mg / kg / day to approximately 2000 mg / kg / day. A more preferable dosage is 1 mg / kg / day to 500 mg / kg / day. 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.

The pharmaceutical composition of the present invention can be administered to mammals such as rats, livestock, humans, etc. by various routes. Since the strain of the present invention has almost no toxicity and side effects, it is a drug that can be used safely even for long-term administration for the purpose of prevention.

In addition, the present invention provides a health functional food having an effect of preventing or improving arthritis containing the strain-derived composition, fermented dew extract, and fermented dew extract complex. The strain-derived composition, fermented dew extract, fermented dew extract complex may be added as 0.001 to 50% by weight to the health functional food of the present invention.

The health functional food of the present invention includes the form of tablets, capsules, pills, or liquids, and foods to which the strain-derived composition of the present invention can be added include, for example, meat, sausage, bread, candy, and snacks. , Noodles, ice cream, dairy products, fermented milk, soups, ionic drinks, beverages, alcoholic beverages, gums, teas and vitamin complexes.

The present invention relates to a method for preparing a new strain of Lactobacillus plantarum SKB1234, its fermented dew extract, Angelica extract and tofu extract, fermented dew extract obtained by fermenting the dew extract with the Lactobacillus plantarum SKB1234 strain or The complex mixture of Angelica extract and S. larvae extract can be easily applied to various pharmaceutical compositions or health functional foods that are effective in preventing, improving or treating arthritis due to excellent cartilage production and inflammation suppression effect in articular cells or arthritis-inducing animal models. .

Figure 1 is a phylogenetic tree showing the genetic location of the Lactobacillus plantarum SKB1234 strain of the present invention.
Figure 2 is a sequence showing the 16S rRNA gene of Lactobacillus plantarum SKB1234 strain of the present invention.
Figure 3 is a schematic process diagram showing the manufacturing process of the fermented dew extract extract of Example 4 using the Lactobacillus plantarum SKB1234 strain of the present invention.
Figure 4 is the result of confirming the change in the production amount of PGE ₂ (Prostaglandin E ₂ ) appearing in the animal model arthritis due to the treatment of fermented dew extract complex of the present invention.
Figure 5 is a result confirming the change in the amount of IL-1β produced in the arthritis animal model due to the treatment of the fermented dew extract extract of the present invention.
Figure 6 is a result confirming the change in the amount of IL-6 produced in the animal model arthritis due to the treatment of the fermented dew extract extract of the present invention.
Figure 7 is a result confirming the change in the amount of production of TNF-α appear in arthritis animal model due to the treatment of the fermented dew extract complex of the present invention.
Figure 8 is a result confirming the mRNA changes of Collagen type I appear in arthritis animal model due to the treatment of fermented dew extract extract of the present invention.
Figure 9 is a result confirming the mRNA changes of Collagen type II in the animal model of arthritis due to the treatment of fermented dew extract extract of the present invention.
10 is a result confirming the mRNA changes of Aggrecan appear in the arthritis animal model due to the treatment of the fermented dew extract complex of the present invention.
Figure 11 is the result of confirming the mRNA changes of MMP-3 appear in the arthritis animal model due to the treatment of the fermented dew extract extract of the present invention.
12 is a result confirming the mRNA change of MMP-7 in the animal model of arthritis due to the treatment of fermented dew extract complex of the present invention.
Figure 13 shows the results of confirming the mRNA changes of NF-κB appear in the arthritis animal model due to the treatment of the fermented dew extract complex of the present invention.
Figure 14 is a result confirming the mRNA changes of IL-1β appearing in the arthritis animal model due to the treatment of the fermented dew extract complex of the present invention.
Figure 15 shows the results of confirming the mRNA changes of TNF-α appear in the arthritis animal model due to the treatment of the fermented dew extract complex of the present invention.
Figure 16 shows the results of confirming the mRNA changes of COX-2 appearing in the arthritis animal model due to the treatment of the fermented dew extract extract of the present invention.

Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein but may be embodied in other forms. Rather, it is provided to fully convey the spirit of the present invention to those skilled in the art so that the contents introduced herein are thoroughly and completely.

<Example 1. Genetic / physiological characterization of strains>

In order to find lactic acid strains and cultures thereof having an effect on arthritis, the inventors selected SKB strains having the highest relative activity among various lactic acid bacteria (described in the following examples).

First, phylogenetic analysis was performed through 16S rRNA gene sequence comparison to identify selected SKB strains. The 16S rRNA (SEQ ID NO: 1) and the Lactobacillus plantarum standard strain ATCC 14917 (JCM 1149) and the 16S rRNA were compared using the Blast similarity search program (National Institute of Biotechnology Information). .

As a result, it showed a high homology of 99.8% between the two strains. In addition, the multisequence alignment was performed with 16S rRNA gene sequences of strains of the same series registered in GenBank, and then the positions in the phylogenetic tree were determined (FIG. 1). On the phylogenetic tree, the 16S rRNA gene of the SKB strain was located closest to Lactobacillus plantarum . As a result of molecular molecular identification of the SKB1234 strain as described above, this strain was classified as Lactobacillus plantarum. Therefore, the strain having an effect on arthritis was named 'Lactobacillus plantarum SKB1234' and was entrusted to the Korean Culture Center of Microorganisms (KCCM, http://www.kccm.or.kr/). Accession number: KCCM12450P).

SEQ ID NO: 1: Lactobacillus plantarum SKB1234 16S rRNA patial sequence (see FIG. 2)

In order to confirm the morphological characteristics of the strain, when cultured in MRS agar plate medium for 1 day at 37 ℃ conditions, the cell morphology is bacilli (chain), it is confirmed that there is no motility. The sporulation ability of the strain was not confirmed and is a Gram positive strain. The form of the strain colony is confirmed to form a smooth smooth colony of translucent grayish color. When confirming the physiological characteristics of the strain, can be cultured at 25 ~ 40 ℃, the optimum growth temperature is 37 ℃. The viable pH was 4.0-6.5 and the optimal growth pH was 6.0-6.5. The optimal growth time is 18 hours and can be cultured for a minimum of 16 hours and a maximum of 2 days. It was confirmed that no gas was generated as the anaerobic strain.

<Example 2: Excellent strain screening suitable for fermentation of dew extract through fermentation>

Fermentation was carried out using various strains to select strains suitable for fermentation of hyssop extract. First, the hot water extract was filtered, concentrated to about 10% by weight of solids, inoculated with 5% by weight of each strain (100% by weight of total strain + concentrate), and incubated for 18 hours at 37 ° C. Comparison was made to confirm the increase and decrease rate. Thereafter, the samples used in the examples were also prepared by fermentation in the same manner as above.

As the strain of Table 1, the Lactobacillus plantarum SKB1234 KCCM12450P strain, L. plantarum KCCM12116, L. acidophilus KCCM32820, L. fermentum KCCM35469, L. casei KCCM12452, and S. cerevisia (commercially available) identified in Example 1 were used. * L .: Lactobacillus / S .: Saccharomyces

The comparison result of the bacterial growth rate for each strain is shown in Table 1 below, it is confirmed that the strain of the present invention is the best strain.

Culture strain % Increase compared to the beginning of culture L. acidophilus KCCM32820 147 Inventive strain L. plantarum SKB1234 KCCM12450P 429 L. plantarum KCCM12116 243 L. fermentum KCCM35469 184 L. casei KCCM12452 261 S. cerevisiae (commercially available) 221

<Example 3: Comparison of metabolites of dew drop extract and fermented dew drop extract through selected lactic acid strains>

For LC-MS analysis of metabolite between extract of fermented dew and fermented extract, after methanol was added for 10 minutes for the extraction of metabolite contained in each extract, methanol was removed and the remaining precipitate was removed. It was dissolved in% (v / v) acetonitrile and used for analysis. As a column, a CSH C18 column was used, mobile phase solvent A used 0.1% (w / v) formic acid (in D.W), and mobile phase solvent B used 0.1% (w / v) formic acid (in ACN). The metabolite increased in fermented dew extract as compared to the dew extract was as follows.

Content of Metabolite Enhanced by Fermentation (fold) Metabolite Fold change Taurohyocholate 16.200 L-Tyrosine 15.580 Cortisol 4.938 Lactosamine 3.296 L-Lysine 3.227 L-prolyl-L-proline 2.845 LysoPC (18: 3) 2.166 Phenylglyoxylic acid 2.112 sn-Glycero-3-phosphocholine 2.081 Phenylalanine 1.986 Isonicotinic acid 1.935 Isoleucine 1.750 Aminoadipic acid 1.711 1,25-Dihydroxyvitamin D3-26,23-lactone 1.558 20-hydroxyecdysone 1.330 25s-inokosterone 1.291 Corticosterone 1.278 Glycine-betaine 1.128 Choline 1.061

As a result of Table 2, it is confirmed that a large amount of Metabolite is greatly increased through fermentation.

Example 4 Preparation of Fermented Wax Extract Complex

Fermented dew extract was prepared using a novel Lactobacillus plantarum SKB1234 strain having an effect on arthritis through the above example.

The fermented dew extract was prepared by primary extraction for 4 hours under conditions of 95 ° C. using 15 times the weight of water and then secondary extraction for 2 hours using 10 times the weight of water. After extraction, the filter press was filtered and concentrated to a solid content of 5 to 15% by weight to prepare a dew extract.

When the extract containing Lactobacillus plantarum SKB1234 strain was used as a whole culture raw material, the extract was inoculated so that the strain became 5% (v / v) (culture solution, 3.0 × 10 ⁸ to 3.0 × 10 ⁹ CFU). / Ml), and incubated at 37 ℃ for 24 hours. The culture liquid obtained after the cultivation was subjected to heat treatment and filtration to concentrate the liquid obtained again to a solid content of 15% by weight to obtain a fermented dew extract.

In addition, after investigating the efficacy of various herbal extracts as raw materials that can increase the efficacy of fermented dew extract, after confirming the efficacy of the Angelica extract and the worm extract good through a preliminary experiment, and confirmed the effect when prepared as a composite raw material In order to prepare a Angelica extract and a worm extract as follows.

In order to obtain a Angelica extract, the extract was extracted for 6 hours under conditions of 95 ° C using 15 times the weight of Angelica. After extraction, the filter press was filtered and concentrated to 15% by weight of solids.

Tobacco extract was also extracted, filtered and concentrated under the same conditions as Angelica extract.

The fermented dew extract complex was prepared by mixing evenly the fermented dew extract of the concentrate state: Angelica extract: Tofu extract: excipient (beta-cyclodextrin) in a weight ratio of 30: 22: 15: 33.

Each of these manufacturing steps is summarized and shown schematically in FIG. 3.

Thus, while preparing the fermented dew extract complex, at each step, the dew extract, the fermented dew extract, the larvae extract, the Angelica extract was also prepared in the same manner for further experiments.

Comparative Example 1: Preparation of Nonfermented Composites

In Example 1, the composite was prepared according to the method of preparing the fermented dew extract extract, but instead of the fermented dew extract, the extract of the dew extract before fermentation was prepared by extracting the larvae extract and the donkey extract.

<Experimental Example 1: Confirming the effect of cell repair (regeneration) in the joint cavity>

Fermented dew extract and mixed with other herbal extracts were confirmed whether the intraarticular cell regeneration function.

Lactobacillus plantarum SKB1234 KCCM12450P strain, L. plantarum KCCM12116, L. acidophilus KCCM32820, L. fermentum KCCM35469, L. casei KCCM12452, S.cerevisia (Commercially available product) Was inoculated and used for strain selection experiments after incubation. * L .: Lactobacillus / S .: Saccharomyces .

Each efficacy experiments MH7A cells (human rheumatoid arthritis synovial cells) to MTT to determine the effect on cell damage after treatment for 2 hours H ₂ O ₂ at a concentration of 300 μM assay (3- (4,5- Dimethylthiazol -2-yl) -2,5-diphenyltetrazolium bromide assay was used to confirm cell viability. For this purpose, MH7A cells were diluted in a 96-well plate at a concentration of 1.0 × 10 ⁴ cells / well for 10% ( v / v) After dispensing in DMEM (Dulbecco's Modified Eagle Medium, Welgene, Korea) culture solution added with FBS and stabilizing for 12 hours, each sample was incubated with 100 µg / ml simultaneously with H ₂ O ₂ for 2 hours. It was.

At this time, each extract or fermented product used in the cell experiment is the same as the method applied in the process of preparing the raw material for producing the fermented dew extract complex in Example 4, each content is based on the solid content of dry weight It is calculated by conversion.

In addition, the fermented dew extract extract complex was prepared by the method of Example 4 using the fermented dew extract, larvae extract, Angelica extract as a raw material, was applied to calculate the content value except beta-cyclodextrin (in the following animal experiments Provided at the time of feeding, calculated as a dose including beta-cyclodextrin).

Thereafter, 20 μl of MTT reagent was treated and incubated at 37 ° C. for up to 3 hours, and then the medium and the MTT reagent were removed, and the absorbance was measured at 560 nm by adding DMSO.

The results are shown in Tables 3 and 4 below.

Condition Cell viability (%) Control group 100 H ₂ O ₂ alone 45 H ₂ O ₂ + Hyssop Extract 61 Fermented Emulsion Extract of H ₂ O ₂ + L. acidophilus KCCM32820 70 Fermented Emulsion Extract of H ₂ O ₂ + L. plantarum SKB1234 KCCM12450P 79 Fermented Emulsion Extract of H ₂ O ₂ + L. plantarum KCCM12116 68 Fermented Emulsion Extract of H ₂ O ₂ + L. fermentum KCCM35469 69 Fermented Emulsion Extract of H ₂ O ₂ + L. casei KCCM12452 68 Fermented Hyssop Extract of H ₂ O ₂ + S.cerevisiae (commercially available) 70

Condition Cell viability (%) Control group 100 H ₂ O ₂ alone 45 H ₂ O ₂ + Angelica Extract 71 H ₂ O ₂ + Tobacco Extract 70 H ₂ O ₂ + Fermented Wax Extract Complex (Example 4) 106 H ₂ O ₂ + Harrow Extract Complex (Comparative Example 1) 73

Referring to Table 3, the fermented dew extract was confirmed that the fermentation of Lactobacillus plantarum SKB1234 strain to recover the most cytotoxicity from H ₂ O ₂ , Table 4 shows each herbal extract of Angelica extract or Tofu extract The effect was also good, but the most noticeably high cell viability was confirmed and the best effect was found in the composite of Example 4, which had some proliferative effect despite the toxicity of H ₂ O ₂ . On the other hand, in Comparative Example 1 complex prepared with the extract of dew drops instead of the fermented dew extract had almost no significant enhancement effect.

Thus, in the present invention, the reason why the extract was fermented with the strain Lactobacillus plantarum SKB1234 because the extracts of Angelica ulna and S. larvae have similar enhancement effect as the fermented dew extract without fermentation. Because arthritis improvement function is the best.

Experimental efficacy of arthritis treatment with fermented dew extract through this result was tested using only fermented with Lactobacillus plantarum SKB1234 strain from now on. That is, the fermented dew extract from the following experiment refers only to the fermented with the strain.

Experimental Example 2: Confirming the Efficacy of Arthritis Treatment of Fermented Beetle Extract and Its Complex- in vitro >

Experimental Example 2-1. mRNA expression inhibition effect

IL-1β, IL-6 and TNF-α, which are known as pro-inflammatory cytokine in arthritis, are known as cytokine that affects the catabolism of chondrocyte tissues, among which TNF-α is chondrocyte It stimulates osteoclasts, promotes the production of inflammatory cytokines such as IL-6, and is a major factor in cartilage destruction in arthritis. IL-1β induces the expression of MMPs (metalloproteinases) in the catabolism of chondrocytes and prevents TGF-α from proliferating chondrocytes. In degenerative arthritis, chondrocytes express more than twice the IL-1β receptor and inhibit the synthesis of genetic factors involved in chondrogenesis. Collagen is also a representative component of cartilage that protects joints through its buffering role.

To confirm the effects on each of these cytokines or cartilage components, MH7A cells were dispensed in DMEM medium containing 10% (v / v) FBS at a concentration of 5 × 10 ⁶ cells / well in a 6-well plate. After stabilization for 12 hours, each sample was treated with H ₂ O ₂ for 2 hours in stabilized MH7A cells, and the cells were collected with 0.25% (w / v) trypsin-EDTA to collect RNeasy extraction kit (QIAGEN, Germantown). , MD, USA) to extract total RNA according to the manufacturer's manual and 5X iScript select reaction mix to 5 ㎍ total RNA using iScript Select cDNA Synthesis kit (BIO-RAD, Singapore, Singapore) for the synthesis of cDNA. 4 μl, Oligo (dT) Primer set 2 μl (Table 5), 5 μl RNA sample, 8 μl Nuclease-Free Water were added, and 1 μl iScript Reverse Transcriptase was added and mixed. After 60 minutes at 42 ° C and 5 minutes at 85 ° C, the synthesized cDNA was used for the PCR reaction. The machine used in the Realtime PCR experiment was a Step One Real-Time PCR system (Applied Biosystems, Foster, CA, USA) and was performed according to the protocol of iQ SYBR Green Supermix (BIO-RAD). Final concentration of the mixed solution for PCR was 2 μl of cDNA (10-100 ng), 10 μl of 2X iQ SYBR Green supermix, 1 μl of each forward and reverse primer (250 nM), and 7 μl of H ₂ O. After 10 minutes hot start at 95 ℃, PCR was performed by 40 cycling for 15 seconds at 95 ℃, 15 seconds at 55 ℃, 30 seconds at 72 ℃, and finally 15 seconds at 95 ℃, 1 minute at 60 ℃, 95 ℃ PCR analysis was performed after 15 seconds of polishing step.

Primer set sequence used for Real-Time PCR Sequence Name Forward Sequence Reverse sequence TNF-α AGCCTCTTCTCCTTCCTGATCGTG GGCTGATTAGAGAGAGGTCCCTGG IL-1β ACAGATGAAGTGCTCCTTCCA GTCGGAGATTCGTAGCTGGAT collagen type I ATGCCTGGTGAACGTGGT AGGAGAGCCATCAGCACCT

The results are shown in Table 6 below.

object
gene Result (fold) Control group H ₂ O ₂
Treated group H ₂ O ₂ +
Dew
extract H ₂ O ₂ +
Fermented dew
extract H ₂ O ₂ +
Angelica
extract H ₂ O ₂ +
Mushrooms
extract H ₂ O ₂ +
Fermented dew
Extraction complex TNF-α 1.0 1.95 1.67 1.43 1.72 1.55 1.10 IL-1β 1.0 1.62 1.55 1.30 1.42 1.43 1.05 collagen type I 1.0 0.55 0.64 0.82 0.72 0.75 1.20

Analyzing Table 6 above, TNF-α and IL-1β produced from H ₂ O ₂ stimulation are most strongly inhibited in fermented dew extract and its extract complex, and expression of collagen type 1 is inhibited from H ₂ O ₂ stimulation. Its expression was markedly enhanced in fermented dew extract and its extract complex.

Experimental Example 2-2. Protein expression inhibition effect

Among the results of confirming mRNA expression, For IL-1β, the effect of inhibiting protein expression was further confirmed on the cells. The cell treatment conditions were the same as the experimental conditions for identifying mRNA, and 50 ul / ml of the cell culture supernatant was followed by enzyme-linked immunosorbent assay (ELISA) kit (PeproTech, Rocky Hill, NJ, USA; and R & D Systems, Minneapolis, MN, USA) was measured according to the manual.

The results are shown in Table 7.

object
protein Result value (pg / mL) Control group H ₂ O ₂
Treated group H ₂ O ₂ +
Dew
extract H ₂ O ₂ +
Fermented dew
extract H ₂ O ₂ +
Angelica
extract H ₂ O ₂ +
Mushrooms
extract H ₂ O ₂ +
Fermented dew
Extraction complex IL-1β 3.0 5.2 4.6 3.9 4.6 4.2 2.9

Referring to the results of Table 7, it is confirmed that the expression of IL-1β protein in the pattern similar to the expression of mRNA is most strongly inhibited in the fermented dew extract and its extract complex.

Therefore, in the arthritis-induced cell model as described above, it was confirmed that the hyssop extract, the Angelica extract and the worm extract have a slight arthritis-improving effect, and the fermented sesame extract fermented with Lactobacillus plantarum SKB1234 strain has an improvement of about 50% or more. In the case of fermented dew extract extracts prepared by mixing them, the effect of restoring the function of the cells to the state before inducing arthritis is considered to be very excellent.

Therefore, the effect of the fermented dew extract extract complex is applied to the arthritis-induced animal model is the same to determine whether it is suitable for use as a clinical material through the following experiment.

Experimental Example 3: Confirmation of the Arthritis Treatment Efficacy of Fermented Beetle Extract Complex- in vivo >

Experimental Example 3-1. Preparation of experimental animals and derivation of arthritis models

As an animal used to induce arthritis, Saronbio Co., Ltd. received a male SD rat of 5 weeks old at around 250 g. Each animal was used for the experiment after 1 week under conditions of 12 hours (light / dark cycle) of light and dark, temperature of 23 ± 2 ° C., and relative humidity of 50 ± 5%. During the acclimation period, AIN-93G diet was supplied, drinking water was freely ingested, and weights were measured. This study was approved by the Animal Experimentation Ethics Committee of Kyung Hee University.

In order to observe the arthritis prevention and cartilage protection effect by providing the fermented dew extract complex, 100 mg / kg bw (S100) and 300 mg / kg bw (S300) Was supplied.

Arthritis was induced by MIA (Monosodium Iodoacetate, Sigma-aldrich, St Louis, MO, USA). To explain this in more detail, a 6-week-old male SD Rat was anesthetized using a respiratory apparatus with isoflurane. Hair was removed around the knee of the anesthetized rat. Inject μl (60 mg / ml). At this time, when diluting MIA, saline solution was used, and 7 days after drug injection, the degree of swelling of the joint area and the walking state of the experimental animal were observed to confirm the induction of arthritis.

In this way, only the MIA-treated group was indicated as a control (Control). In positive control (PC), MSM (Methyl Sulfonyl Methane) was added 300 mg / kg b.w. It was provided by the comparison. AIN93G was provided for the basic diet of all experimental groups, including the normal control (NC).

These experimental conditions are summarized in Table 8 below.

Experimental design animals (n = 8 / group) Groups Induced arthritis Dietary administration Orally administration Normal control
(Normal control, NC) - AIN 93G diet Control group
(Control, C) + AIN 93G diet Positive control group
(Positive control, PC) + AIN 93G diet MSM 300 mg / kg S100 + AIN 93G diet Fermented Beetle Extract Complex
100 mg / kg S300 + AIN 93G diet Fermented Beetle Extract Complex
300 mg / kg

Experimental Example 3-3. Identify the relationship between blood collection, organ harvesting, weight change, and dietary efficiency

After the induction of arthritis and the feeding of the sample, the animals were fasted for 12 hours, anesthetized, opened, and blood was collected through the abdominal aorta. Before blood collection, 10% (w / v) EDTA solution was soaked in syringe and tube to prevent blood coagulation. Next, the organs (liver, kidney, spleen) were extracted and weighed, and the results of measuring the effects on weight change, dietary efficiency and organ weight of each animal group are shown in Table 9.

Body weight gain, food efficiency ratio, and organ weight of experimental animals for 4 weeks Measurements NC Induced Arthritis C PC S100 S300 Weight gain (g) 278.40 ± 44.08 239.36 ± 16.12 264.16 ± 15.55 252.29 ± 20.14 272.19 ± 37.12 Food consumption (g / day) 22.67 ± 3.08 20.11 ± 4.23 22.59 ± 3.45 21.54 ± 3.80 22.66 ± 3.04 Food efficiency ratio 34.02 ± 5.39 32.84 ± 2.21 32.38 ± 1.91 32.39 ± 2.58 33.27 ± 4.54 Liver (g) 2.75 ± 0.30 2.59 ± 0.13 2.67 ± 0.06 2.62 ± 0.16 2.57 ± 0.10 Kidney (g) 0.65 ± 0.06 0.67 ± 0.04 0.65 ± 0.03 0.66 ± 0.04 0.63 ± 0.05 Spleen (g) 0.17 + 0.03 0.20 ± 0.02 0.18 ± 0.03 0.18 ± 0.02 0.17 ± 0.03

Referring to Table 9, the control (C) group administered with MIA significantly reduced body weight compared to the normal control (NC), and the PC group and the fermented dew extract extract administration group (S100, S300) administered MSM. ) Tended to increase compared to group C, but there was no significant difference. It was confirmed that there was no significant difference in dietary intake, dietary efficiency and long-term weight among the experimental groups.

Experimental Example 3-5. PGE ₂  Secretion measurement

PGE ₂ (Prostaglandin E ₂ ) is produced under the influence of TNF-α in inflammatory reactions and is a factor involved in articular cartilage degradation during the onset of arthritis. It also forms oetophyte, resulting in inconsistent bone growth and pain. These symptoms are known to be common in degenerative arthritis. Thus, the blood obtained after the sacrifice of arthritis-induced experimental animals was centrifuged to obtain serum, and experimented according to R & D Systems' protocol using the Prostaglandin E ₂ Assay kit of R & D Systems (Minneapolis, MN, USA). -rad, California, USA) was used to measure the absorbance at 655 nm.

The results are shown in Figure 4, the serum PGE ₂ level of the C group was confirmed that the inflammation increased by 318.49% significantly compared to the normal control group, MSM administration group PC group and fermented dew extract extract administration group S100 The serum PGE ₂ levels in the S300 group were reduced by 46.51%, 23.13%, and 42.19% compared to the C group. In particular, the PC group and the S300 group was significantly different from the C group. Through this, it can be confirmed that the fermented dew extract extract composite prepared by the method of the present invention is a very excellent composition to be applied as a therapeutic agent for arthritis.

Experimental Example 3-5. Serum pro-inflammatory cytokine (IL-1β, IL-6, TNF-α) measurement

Cytokines are water-soluble proteins secreted by cells, expressed by specific signaling substances, and are influenced by surrounding cells, distinct from enzymes, and their half-lives are so short that their range of action is limited to adjacent cells that secrete cytokines. do.

Among them, interleukin (IL) -1β, IL-6, and tumor necrosis factor (TNF) -α are cytokines that affect the catabolism of chondrocytes. Inhibiting the synthesis of collagen II and proteoglycan related to IL-1β cartilage production, and in chondrocytes associated with degenerative arthritis, IL-1β has twice as many IL-1β receptors as normal chondrocytes. Plays a similar role.

After the administration of the feed during the experiment the effect on the serum cytokines (IL-1β, IL-6, TNF-α) measured using the kit of R & D Systems as in Experiment 3-4 is shown in Figures 5-7 Shown in

Referring to FIG. 5, the serum IL-1β level of the arthritis-induced group (C) was significantly increased by 213.66% compared to that of the normal control group (NC). Serum IL-1β levels were decreased by 9.46%, 22.19%, and 23.79%, respectively, compared to group C. Especially, S300 group was found to be significantly different from group C.

According to the data disclosed in FIG. 6, the serum IL-6 level of the arthritis-induced group (C) was significantly increased by 252.83% compared to that of the normal control group (NC). Serum IL-6 levels in the S300 group were significantly decreased by 73.39%, 27.10% and 43.43%, respectively, compared to the C group.

7, the serum TNF-α level in the arthritis-induced group (C) was significantly increased by 153.83% compared to the NC group. Serum TNF-α levels were significantly reduced by 25.96%, 17.79%, and 32.04%, respectively, compared to the C group, and the PC and S300 groups were decreased to similar levels as the NC group.

These results demonstrate that ingestion of fermented dew extract extracts has an effect on reducing serum pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) due to arthritis.

Experimental Example 3-6. Gene Expression Measurement by Real-Time Polymerase Chain Reaction

Maintaining the physiological and biological action of chondrocyte tissue is known to be due to the balance of anabolism and catabolism of tissue. Involvement in assimilation includes collagen, proteoglycan including aggrecan, and catabolism includes metalloproteinase (MMP), nitric oxide (NO), TNF-a, collagenase, and aggrecanase.

In addition, collagen type I is the most widely distributed in human cells as well as chondrocytes. Collagen type II accounts for 50% of cartilage protein and about 85-90% of collagen types in cartilage. Aggrecan, along with the formation of collagen type I and II, is an important factor in determining the state of cartilage tissue. Aggrecan is one of the proteoglycan core proteins that synthesize cartilage tissue. Aggrecan is composed of G1, G2 and G3, globular proteins involved in aggregation, hyaluronan binding, cell adhesion and chondrocyte apoptosis. It is known to exhibit resistance and maintain a joint.

MMP-3 (Stromelysin) is a metalloproteinase that plays an important role in the degeneration of proteoglycan. The increase in MMP-3 in the joint cavity is also associated with severe cartilage damage, and MMP-7 is particularly expressed in arthritis compared to rheumatoid arthritis. It is an increasing factor.

Nuclear transcription factor κB (NF-κB) is a transcription factor of several genes involved in immune and inflammatory responses. It is a factor that regulates the transcription of inflammation-related cytokines such as TNF-α and IL-6. Most inflammatory reactions are triggered by inflammatory cytokines such as COX-2, IL-6, TNF-α, and activated NF-κB transfers to the nucleus to produce TNF-α. It is used to reactivate NF-κB and is known to accelerate the inflammatory response by genetically synthesizing Cox-2. Therefore, proper production regulation of TNF-α is an important factor in controlling the inflammatory response.

Therefore, later experiments confirmed the expression of these factors associated with arthritis.

Therefore, after sacrifice of arthritis-induced experimental animals, chondrocytes isolated from each animal were stored in 1 mL Trizol (QIAZEN), and 200 μl Chloroform was collected to collect supernatant, followed by RNA extraction and cDNA synthesis. Was measured.

To this end, after 10 minutes hot start at 95 ℃ for 15 seconds at 95 ℃, 15 seconds at 55 ℃, 40 cycles for 30 seconds at 72 ℃ after PCR was performed for 15 seconds at 95 ℃, 1 minute at 60 ℃, PCR analysis was performed through a polishing step at 95 ° C for 15 seconds. Primer used in the reaction is shown in Table 10.

Primer set sequence used for Real-Time PCR Sequence
Name Forward Sequence Reverse sequence GAPDH TGG CCT CCA AGG AGT AAG AAA C CAG CAA CTG AGG GCC TCT CT Aggrecan GAA GTG GCG TCC AAA CCAA CGT TCC ATT CAC CCC TCTCA Collagen type i GAG CGG AGA GTA CTG GAT CGA CTG ACC TGT CTC CAT GTT GCA Collagen type ii GCA ACA GCA GGT TCA CGT ACA TCG GTA CTC GAT GAT GGT CTT G MMP-3 GAG TGT GGA TTC TGC CAT TGA G TTA TGT CAG CCT CTC CTT CAG AGA MMP-7 ACT CTA GGC CAT GCC TTT GC CCA TCC GTC CAG TAC TCA TCC T NF-κB GCA CCA AGA CCG AAG CAA TT GAA ACC CCA CAT CCT CTT CCT T IL-1β GGC TTC GAG ATG AAC AAC AAA AA GTC CAT TGA GGT GGA GAG CTT T TNF-α ACA AGG CTG CCC CGA CTA T CTC CTG GTA TGA AGT GGC AAA TC COX2 AGA GAA AGA AAT GGC TGC AGA GTT AGC AGG GCG GGA TAC AGT

As a result of measuring the expression of collagen type I & II, Aggrecan gene is shown in Figs. 8 to 10, as a result of measuring the expression of the collagen type I & II and Aggrecan gene, arthritis-induced group (C) significantly reduced compared to the normal control group It was confirmed that.

The results of measuring the expression of MMP-3 and MMP-7 genes are shown in FIGS. 11 and 12.

According to FIG. 11, MMP3 gene expression of arthritis-induced group (C) was significantly increased compared to normal control group (NC), and MMP3 gene expression of PC group and MS100-administered fermented dew extract group S100 and S300 groups Compared with the arthritis-induced group (C), the rate was reduced by 29.54%, 38.01% and 42.87%, respectively. In particular, S100 and S300 groups treated with fermented dew extract extract showed significant differences from arthritis-induced group (C).

Similarly, as shown in Figure 12, MMP7 gene expression was also measured, arthritis induced group (C) significantly increased compared to the normal control group (NC), MSM administration group PC group and fermented wort complex extract administration group S100, MMP7 gene expression in the S300 group was reduced by 36.28%, 14.00% and 78.33%, respectively, compared to the arthritis-induced group (C). In particular, the PC group and the S300 group showed a significant difference from the arthritis-induced group (C). Therefore, it can be seen that the intake of fermented dew extract reduced MMP3 and MMP7 increased due to arthritis.

The results of measuring NF-κB, IL-1β, TNF-α, and COX-2 gene expression are shown in FIGS. 13 to 16.

Referring to FIG. 13, the NF-κB gene expression in the arthritis-induced group (C) was increased by 158.28% compared to the normal control group, but there was no significant difference. The expression of NF-κB in the PC group and the fermented dew extract complex group S100 and S300 group decreased by 33.13%, 7.03% and 51.41%, respectively, compared to the arthritis-induced group (C). There was a significant difference.

According to FIG. 14, as a result of measuring IL-1β gene expression, the gene expression of the arthritis-induced group (C) was significantly increased by 1003.53% compared to the normal control group (NC). The S100 and S300 groups were significantly decreased by 38.65% and 47.52% and 91.86%, respectively, compared to the arthritis-induced group (C). In particular, the S300 group was confirmed to decrease to the normal control level.

15, the TNF-α gene expression of arthritis-induced group (C) was significantly increased by 288.49% compared to the normal control group (NC). It can be seen that the reduction was 57.71%, 0.45%, 67.60% compared to the arthritis group (C). The PC group and the S300 group were significantly different from the arthritis-induced group (C).

As shown in Figure 16, as a result of measuring the COX-2 gene expression, the gene expression of the arthritis-induced group (C) significantly increased 398.13% compared to the normal control group (NC), MSM administration group PC and fermented dew extract extract complex S100 and S300 groups were 48.79%, 20.75% and 72.52% less than the arthritis-induced group (C), respectively. In particular, the PC group and the S300 group showed a significant difference from the arthritis-induced group (C) and decreased to the normal control group (NC) level.

Therefore, the above results, it was comprehensively proved that the level of inflammatory biomarker due to arthritis induced by the intake of fermented dew extract complex to the normal control level.

Experimental Example 4 Experiment for Manufacturing Conditions of Fermented Wax Extract Complex

In order to confirm the preferred production conditions of the fermented dew extract extract composite prepared in Example 4 in more detail, after preparing the extract composite under the following Table 11 conditions, the state was confirmed while spray drying.

Condition Fermented Dew Extract Extract Composite Raw Material Weight (g) Fermented dew extract Angelica Extract Tofu extract beta-
Cyclo
dextrin sum Preparation Example 1-1
(Example 4 conditions) 30 22 15 33 100 Production Example 1-2 30 15 22 33 100 Production Example 1-3 30 25 20 25 100 Production Example 1-4 30 30 15 25 100 Production Example 1-5 30 27 10 33 100 Comparative Production Example 1-1 30 5 32 33 100 Comparative Production Example 1-2 30 32 5 33 100 Comparative Production Example 1-3 70 15 15 0 100 Comparative Production Example 1-4 30 40 30 0 100 Comparative Production Example 1-5 30 25 25 20 100

Condition aridity Preparation Example 1-1 (Example 4 Conditions) Good Production Example 1-2 Good Production Example 1-3 Good Production Example 1-4 Good Production Example 1-5 Good Comparative Production Example 1-3 Not dry Comparative Production Example 1-4 Not dry Comparative Production Example 1-5 Not dry

In addition, in addition, each complex was converted to a concentration except for beta-cyclodextrin, and cell viability (%) was measured under the conditions of Experimental Example 1.

Condition Cell viability (%) Control group 100 H ₂ O ₂ alone 46 H ₂ O ₂ + Preparation Example 1-1 126 H ₂ O ₂ + Preparation 1-2 121 H ₂ O ₂ + Preparation 1-3 113 H ₂ O ₂ + Preparation Example 1-4 131 H ₂ O ₂ + Preparation Example 1-5 115 H ₂ O ₂ + Comparative Production Example 1-1 79 H ₂ O ₂ + Comparative Production 1-2 75

The conditions of Table 12 and Table 13 above support a preferred embodiment of the fermented dew extract complex preparation conditions of the present invention.

Preparation Example 1. Pharmaceutical Formulations

Formulation Example 1-1. Manufacture of tablets

200 g of the lyophilized extract of the fermented dew drop of the present invention was mixed with 175.9 g of lactose, 180 g of potato starch, and 32 g of colloidal silicic acid. 10% (w / v) gelatin solution was added to the mixture, which was then ground and passed through a 14 mesh sieve. It was dried and the mixture obtained by adding 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate was made into a tablet for treating arthritis.

Formulation Example 1-2. Preparation of Ointment

1 g of the lyophilized extract of the fermented dew drop extract, Angelica extract and the worm extract of the present invention were mixed with petrolatum 99g to prepare an ointment for treating arthritis.

<Formulation Example 2: Food Preparation>

Formulation Example 2-1. Manufacture of cooking seasonings

The lyophilized extract of the fermented dew drop extract, Angelica extract and the worm extract extract of the present invention was added to the cooking seasoning by 1% by weight to prepare a cooking seasoning for health promotion.

Formulation Example 2-2. Manufacture of dairy products

The freeze-dried product of the extract complex of the fermented dew drop extract, Angelica extract and the worm extract of the present invention was added to the milk at 0.1% by weight, and various dairy products such as butter and ice cream were prepared using the milk.

Preparation Example 2-3. Vegetable juice manufacturing

The lyophilized extract of the fermented dew drop extract, Angelica extract and the worm extract extract of the present invention was added to 1,000 ml of tomato juice or carrot juice to prepare vegetable juice for health promotion.

Formulation Example 2-4. Manufacture of fruit juice

The lyophilized extract of the fermented dew drop extract, Angelica extract and the worm extract extract of the present invention was added to 1,000 ml of apple juice or grape juice to prepare fruit juice for health promotion.

[Trusted agency]

Depositary Name: Korea Microorganism Conservation Center

Accession number: KCCM12450P

Deposit Date: 20190305

<110> SK bioland Co., Ltd. <120> A novel strain of Lactobacillus plantarum SKB1234 having an          improving effect on arthritis and a method for producing a          mixture of the fermented Achyranthes japonica Nakai extract using          the Lactobacillus plantarum SKB1234, Angelica gigas Nakai extract          and Eucommia ulmoides Oliver extract <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1502 <212> DNA <213> Unknown <220> <223> Lactobacillus plantarum <400> 1 gacgaacgct ggcggcgtgc ctaatacatg caagtcgaac gaactctggt attgattggt 60 gcttgcatca tgatttacat ttgagtgagt ggcgaactgg tgagtaacac gtgggaaacc 120 tgcccagaag cgggggataa cacctggaaa cagatgctaa taccgcataa caacttggac 180 cgcatggtcc gagcttgaaa gatggcttcg gctatcactt ttggatggtc ccgcggcgta 240 ttagctagat ggtggggtaa cggctcacca tggcaatgat acgtagccga cctgagaggg 300 taatcggcca cattgggact gagacacggc ccaaactcct acgggaggca gcagtaggga 360 atcttccaca atggacgaaa gtctgatgga gcaacgccgc gtgagtgaag aagggtttcg 420 gctcgtaaaa ctctgttgtt aaagaagaac atatctgaga gtaactgttc aggtattgac 480 ggtatttaac cagaaagcca cggctaacta cgtgccagca gccgcggtaa tacgtaggtg 540 gcaagcgttg tccggattta ttgggcgtaa agcgagcgca ggcggttttt taagtctgat 600 gtgaaagcct tcggctcaac cgaagaagtg catcggaaac tgggaaactt gagtgcagaa 660 gaggacagtg gaactccatg tgtagcggtg aaatgcgtag atatatggaa gaacaccagt 720 ggcgaaggcg gctgtctggt ctgtaactga cgctgaggct cgaaagtatg ggtagcaaac 780 aggattagat accctggtag tccataccgt aaacgatgaa tgctaagtgt tggagggttt 840 ccgcccttca gtgctgcagc taacgcatta agcattccgc ctggggagta cggccgcaag 900 gctgaaactc aaaggaattg acgggggccc gcacaagcgg tggagcatgt ggtttaattc 960 gaagctacgc gaagaacctt accaggtctt gacatactat gcaaatctaa gagattagac 1020 gttcccttcg gggacatgga tacaggtggt gcatggttgt cgtcagctcg tgtcgtgaga 1080 tgttgggtta agtcccgcaa cgagcgcaac ccttattatc agttgccagc attaagttgg 1140 gcactctggt gagactgccg gtgacaaacc ggaggaaggt ggggatgacg tcaaatcatc 1200 atgcccctta tgacctgggc tacacacgtg ctacaatgga tggtacaacg agttgcgaac 1260 tcgcgagagt aagctaatct cttaaagcca ttctcagttc ggattgtagg ctgcaactcg 1320 cctacatgaa gtcggaatcg ctagtaatcg cggatcagca tgccgcggtg aatacgttcc 1380 cgggccttgt acacaccgcc cgtcacacca tgagagtttg taacacccaa agtcggtggg 1440 gtaacctttt aggaaccagc cgcctaaggt gggacagatg attagggtga agtcgtccaa 1500 gg 1502

Claims (7)

Lactobacillus plantarum SKB1234 strain (Accession No .: KCCM12450P), characterized in that it has an improving effect of osteoarthritis or rheumatoid arthritis. delete (First step) preparing the dew extract, Angelica extract and worm extract, respectively;
(Second step) inoculating the Lactobacillus plantarum SKB1234 strain of claim 1 to the dew extract obtained in the first step to obtain a fermented dew extract; And
(Third step) mixing the fermented dew extract obtained in the second step, the Angelica extract of the first step and the worm extract of the first step;
Method for producing a fermented dew extract extract having an effect of improving or treating osteoarthritis or rheumatoid arthritis comprising a. The method of claim 3,
In the first step, the extract of dew drops, Angelica extract or larvae extract 10 to 40 times the weight of water to each raw medicinal herb, Angelica or larvae and extract for 2 to 10 hours under conditions of 90 to 100 ℃, Method for producing a fermented dew extract extract complex having an improved or therapeutic efficacy of osteoarthritis or rheumatoid arthritis, characterized in that the extract obtained by filtration is concentrated to an extract containing 10 to 30% by weight solids. The method of claim 3,
In the preparation of the extract of the dew drop extract in the first step, after extracting the primary by adding water to the dew, and then taking the extract and the second extraction by adding water to the remaining dew to improve the osteoarthritis or rheumatoid arthritis A method for preparing a fermented dew extract extract having therapeutic effect. The method of claim 3,
The fermented dew drop extract of the second step is obtained by inoculating Lactobacillus plantarum SKB1234 strain to the dew drop extract and concentrating the liquid obtained by heat treatment and filtration to a solid content of 10-30% by weight. A method for producing a fermented wadded extract complex having the effect of improving or treating osteoarthritis or rheumatoid arthritis. The method of claim 3,
Preparation of fermented dew extract extract having improved or therapeutic efficacy of osteoarthritis or rheumatoid arthritis, characterized in that dextrin or beta-cyclodextrin is added as an excipient when mixing the fermented dew extract, Angelica extract and worm extract in the third step Way.

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