KR20230104514A - Bioconverted Polysaccharide Extract of Gloiopeltis furcata with High Anti-inflammation Activity, and Use thereof - Google Patents

Abstract

The present invention relates to a fermented product obtained by fermenting the polysaccharide of a starfish with anti-inflammatory properties by fermenting it with ganoderma lucidum mycelium. The fermented starfish polysaccharide extract of the present invention is a natural product having high anti-inflammatory activity against intracellular inflammation caused by oxidative stress, etc., and can be usefully used as a natural anti-inflammatory material in products such as quasi-drugs, foods, and cosmetics.

Description

Fermented Polysaccharide Extract of Gloiopeltis furcata with High Anti-inflammation Activity, and Use It}

The present invention relates to a fermented product obtained by extracting and separating polysaccharides from Seaweed furcata and Gloiopeltis Furcata , and then bioconverting them into mushroom mycelium to have anti-inflammatory activity. More specifically, it relates to a fermented product obtained by bioconversion of the polysaccharide extract of Ganoderma lucidum Karstena using Ganoderma lucidum Karstena to have excellent anti-inflammatory activity.

The present invention is to provide a polysaccharide-extracted fermented product of L. ligament that has a completely new anti-inflammatory activity that has not been previously possessed by isolating and bioconverting polysaccharide from L.

Excessive oxidative stress in vivo is known to cause various diseases such as decreased immune function, induction of inflammation, cancer, arteriosclerosis, and cardiovascular disease, and promotes aging of all organs.

All living organisms, including humans, that undergo aerobic respiration require oxygen to produce energy necessary for sustaining life, and superoxide radicals (ㆍO ₂ ^- ), It generates various reactive oxygen species (ROS) such as hydrogen peroxide (H ₂ O ₂ ), singlet oxygen (ㆍO ₂ ), and hydroxyl radical (ㆍOH) (Kang KA, Chae SC, Kang DG, Kim JS, Jin WH. 2005. Screening of antioxidative effect of herbal extracts on oxidative stress. Kor. J. Pharmacogn. 36: 159-163).

Living organisms maintain homeostasis through the production and removal of these oxygen species to perform cellular functions. However, when this balance is imbalanced, cells and the like are subjected to harmful effects due to abnormally increased ROS, which is called oxidative stress.

Oxidative stress causes various diseases, and it is known that cancer, arteriosclerosis, or cardiovascular disease damages cells or tissues, is involved in aging, and causes progressive memory loss and cognitive impairment (Kamat PK, Kalani A, Rai S, Swarnkar S, Tota S, Nath C, Tyagi N. 2016. Mechanism of Oxidative Stress and Synapse Dysfunction in the Pathogenesis of Alzheimer's Disease: Understanding the Therapeutics Strategies. Mol Neurobiol. 53: 648-661). That is, oxidative stress occurs due to excessive generation of reactive oxygen species, and various diseases appear through oxidative damage of biological molecules such as proteins, lipids, and DNA.

To counteract this, there exists a mechanism to prevent the accumulation of reactive oxygen species, which can be largely classified into two types. Antioxidant enzymes such as superoxide dismutase (SOD), catalse (CAT), and glutathione (GSH), and non-enzymatic free radical scavengers such as vitamin C, vitamin E, and flavonoid prevent accumulation of reactive oxygen species, resulting in to defend against oxidative stress ((Rhee, S.-J. and Choi, J.-H. 2001. Effects of green tea catechin on the superoxide dismutase, glutathione peroxidase and xanthine oxidase activities of kidney in diabetic rats. Kor (Nutr. Soc. 34, 734-740).

In order to alleviate oxidative stress, research is being conducted on substances that inhibit active oxygen and lipid peroxidation reactions and substances that improve inflammatory responses. Representative antioxidants currently reported include vitamins, phenolic acids such as caffeic acid, and catechin. There are tannins and flavonoids such as In addition, in Korea, studies on the antioxidant effect through natural substances and food materials such as red ginseng and green tea are being actively conducted.

Inflammation caused by oxidative stress is the expression of a normal and protective in vivo defense mechanism that appears locally against tissue damage caused by harmful chemicals or irritants among metabolites in vivo. Such inflammation is triggered by various chemical mediators produced from damaged tissue and migrating cells, and these chemical mediators are known to vary according to the type of inflammatory process.

In normal cases, the living body neutralizes or removes disease factors through an inflammatory response and regenerates damaged tissues to restore normal structures and functions, but in other cases, it may progress to a disease state such as chronic inflammation.

The most common inflammatory disease prevention or treatment solvents for treating inflammatory diseases are largely classified into steroidal and non-steroidal inflammatory disease prevention or treatment solvents, and most of the synthetic inflammatory disease prevention or treatment solvents have various It has many disadvantages, often accompanied by side effects. In other words, in order to treat inflammatory diseases, appropriate non-steroidal inflammatory disease prevention or treatment drugs (NSAIDs) are used to relieve inflammation, and if inflammation is severe or NSAIDs do not have the efficacy, steroid preparations are used, and in intractable cases, immunosuppressants are used. I will perform surgery.

NSAIDs used in this case mainly inhibit cyclooxygenase (COX) to inhibit the production of prostaglandin involved in inflammatory reactions, thereby preventing or treating inflammatory diseases, but gastrointestinal disorders and liver dysfunction. There is a problem that it is difficult to use for a long period of time because it causes side effects such as kidney dysfunction and the like.

On the other hand, due to the increased interest in health, beauty and the environment, research to obtain biomaterials from natural resources has been extensively conducted. In particular, various studies are being conducted on the judgment that the diversity and abundance of marine life has potential and value as a good material. Among them, as various bioactive substances of seaweed are known, potential as a new development resource is being recognized (Yoon SJ, Cho YS, Nam JH, Lee HH, Kim E and Hong YK. 2008. Effects of several seaweed extracts on the viability of human kerationoyte HaCaT cells.J Kor Fish Soc 41(1), 68-72.).

nguez H. 2013. In vitro antioxidant properties of crude extracts and compounds from brown algae. Food Chem 138, 1764-1785.). 국내 연구에서도 대황, 감태, 지충이 등에서 항산화 활성 및 α-glucosidase 저해 활성을 보고하였고(Kim JH, Kang HM, Lee SH, Lee JY, Park LY. 2015. Antioxidant and α-glucosidase inhibition activity of seaweed extracts. Kor J Food Preserv 22(2), 290-296), 김, 미역, 다시마와 톳 추출물들에서 항산화 효과, 지질 산화 억제 능력 및 tyrosinase 억제 활성을 발견하였다(Lee NY. 2013. Antioxidant effect and tyrosinase inhibition activity of seaweeds ethanol extracts. J Korean Soc Food Sci Nutr 42(12), 1893-1898.). Seaweeds have various types of polysaccharides, fatty acids, proteins, phenolic compounds, terpenes, etc., and exhibit anticancer, antimicrobial and antiviral effects and antioxidant activity, so they are highly valuable as biomaterials (Balboa EM, Conde E, Moure A, Falqu*?* E, Dom

nguez H. 2013. In vitro antioxidant properties of crude extracts and compounds from brown algae. Food Chem 138, 1764-1785.). A domestic study also reported antioxidant activity and α-glucosidase inhibitory activity in rhubarb, Ecklonia cava, and streptococci (Kim JH, Kang HM, Lee SH, Lee JY, Park LY. 2015. Antioxidant and α-glucosidase inhibition activity of seaweed extracts. Kor J Food Preserv 22(2), 290-296), antioxidant effect, lipid oxidation inhibitory ability and tyrosinase inhibitory activity were found in seaweed, seaweed, kelp and fusiformis extracts (Lee NY. 2013. Antioxidant effect and tyrosinase inhibition activity of seaweeds ethanol extracts.J Korean Soc Food Sci Nutr 42(12), 1893-1898.).

In addition, research to secure high value-added materials from seaweed, such as application of processing technology such as fermentation, is actively underway (Lee CW, Kim HA, Yoon HR, and Jeon TY. 2019. Establishment of seaweed fermentation process for cosmetic material research. J Korea Aca Ind Coop Soc 20(9), 14-19.).

Among various seaweeds, Gloiopeltis Furcata is a seaweed of the red algae plant Pulgasariaceae, which lives in colonies on the upper part of rocky shores, and is used as a thickener, an additive added to increase the viscosity of foods such as gum and starch. The distribution area is known as Korea, Japan, Sakhalin, the Kuril Islands, and the west coast of the American continent.

It has a height of 5 to 10 cm and a branch diameter of 2 to 5 mm, and the stem is cylindrical. Branches are irregularly branched in a Y shape, and the body is constricted to look like a joint. The stem is short and the shape variation is severe. It is thin at the divergence point and the top is round or pointed, so it is also called triangular gasari. Strictly speaking, it is a different kind. Jill is tough like thin leather, rich in mucilage, and adheres well to the ground when dry. In Korea, it is especially abundant in the southern coast. It is similar in appearance to Pulgasari, but it can be distinguished because the stem is hollow. It grows attached to rocks or stones in the upper intertidal zone. Several individuals occur in one place and live in colonies. From the end of March to April, spores appear, and after the primary proliferation until early summer, the erect body that released the spores dies. The released spores soon attach to others and germinate, repeating the growth activity of creating new erect bodies in the fall and spring of the following year. It is used as a raw material for starch feeding on fabrics or eaten as bibimbap, seasoning, etc. (Ryo T, Hiroko IS, Kaeko H, Saburo H, Susumu H. 1998. Concurrence of agaroid and carrageenan chains in funoran from the red seaweed Gloiopeltis furcata post. et ruprecht. Carborate Polymers 35, 81-87).

Although not many studies have been reported on the physiological activity of Pulgasari Pulgasari, the physiological activity effect of ethanol extract of Pulgasari Pulgasari (Kwon Da-hye. 2016. Anti-inflammatory function and physiological activity of Ethanol Extract of Pulgasari Pulgasari. Master's thesis, Dong-eui University), on the bacteria causing tooth decay Antibacterial activity against cariogenic bacteria and inhibition of insoluble glucan production by free fatty acids obtained from dried Gloiopeltis furcata . Fish Sci 65, 129-132. ), etc. As a study on the polysaccharide of Pulgasari Pulgasari, the physiological activity was improved when the polysaccharide of Pulgasari Pulgasari was treated through enzyme treatment (Lee Dae-hoon, Hong Joo-heon. 2017. Physicochemical characteristics and physiological activity of polysaccharide derived from Pulgasi Pulgasari according to the enzyme type. Korea Food Journal of Storage Distribution Society 24(3), 455-463.) and development of cosmetic materials that improve skin wrinkles (Lee Dae-hoon, Hong Joo-heon. 2017. Physicochemical quality characteristics and skin wrinkle improvement effect according to molecular weight of Undeungpulgasari polysaccharide. Korea Food Storage & Distribution Journal of Korean Society of Food Storage and Distribution 24(5), 688-696.) and immune-enhancing effect (Lee Dae-hoon, Hong Joo-heon. 2018. Immune-enhancing effect according to the molecular weight of the polysaccharide of Undeungpulgasari. Journal of the Korean Society of Food Storage and Distribution 25(1), 1-6.) reported.

In addition, the anti-cancer effect of Pulgasari (Park Seong-young, Bok-mi Jeong, Young-hyun Choi, Bae Seong-ja. 2005. Cancer cell growth inhibitory effect of the fraction of Bul-deung-Pulgasari. Korean Journal of Food and Nutritional Science 34(6), 771-775.; Woo-Young Choi, Ki-Young Kim, Won-Ho Lee, Bae Sang Here, Choi, Young - Hyun.

Prior art related to the present invention is a composition for decomposing alcohol and relieving hangover containing seaweed extract as an active ingredient (Korean Registered Patent Publication No. 10-1725261) in the group consisting of Pulgasari, Undeunggasari, Jindubal, and Single seaweed. Alcohol decomposition effect and hangover relief containing seaweed extract containing at least one selected seaweed and at least one oriental medicinal herb selected from the group consisting of raisin root, brown root, cornelian cornus, Schisandra chinensis, licorice, raspberry and jujube as active ingredients It's about the composition.

In addition, a composition for whitening containing a Pulgasari Pulgasari extract and a manufacturing method thereof (Korean Patent Registration No. 10-1821024) fermented Pulgasari Pulgasari extract with Lactobacillus casei and fermented with rice water. It relates to a manufacturing method capable of improving skin whitening activity by mixing a juniper hot-water extract and a hot-water extract of B. chinensis fermented with rice water at a certain ratio to inhibit melanin production without causing side effects due to non-cytotoxicity.

On the other hand, a seaweed mixture containing kelp and kelp as a fermented seaweed mixture containing kelp and a cosmetic composition containing it as an active ingredient (Registration Patent Publication No. 10-2099251) is Lactobacillus sakei , KCTC3603), it relates to a cosmetic composition containing fermented product obtained by fermentation as an active ingredient, it relates to a technology capable of providing antioxidant function, skin moisturizing function and anti-aging function, and antioxidant function containing complex fermented seaweed extract. and a whitening active composition (Korean Patent Publication No. 10-2123588) containing, as an active ingredient, a fermented product obtained by fermenting a hot-water extract of a mixture of wakame, kelp, and Lactobacillus sakei (ATCC15521). and compositions having a moisturizing effect.

However, it is different from the present invention, which separates and purifies the polysaccharide of Euphorbia presented in this specification and then bioconverts it through mycelium fermentation to have a completely new anti-inflammatory activity that has not been previously reported.

On the other hand, bioconversion is a kind of fermentation process using microorganisms or enzymes, and is a generic term for a technology for producing and manufacturing a desired product from a precursor. While the conventional fermentation process starts with relatively simple raw materials, the bioconversion process uses the selectivity of microorganisms or enzymes for substrates to change the structure of existing materials to increase the content of active ingredients, improve efficacy, and increase absorption rate. It is being used as a technology to induce change.

Accordingly, the inventors of the present invention completed the present invention by finding that, when polysaccharide was separated and purified from L. ganoderma, and then bioconversion was performed into ganoderma lucidum mycelium, a high anti-inflammatory effect not found in the polysaccharide extracted from L. ganoderma prepared by a general method appeared. did

Republic of Korea Patent Registration No. 10-1725261 Republic of Korea Patent Registration No. 10-1821024 Republic of Korea Patent Registration No. 10-2099251 Republic of Korea Patent Registration No. 10-2123588

The present inventors have intensively tried to develop an anti-inflammatory agent that can be administered for a long period of time from a natural extract without causing side effects or drug resistance and having low toxicity. As a result, the present invention was completed by confirming that the fermented product obtained by bioconversion of the polysaccharide extract of Ganoderma luciferus by using the mycelium of Ganoderma luciferus showed high anti-inflammatory activity.

Accordingly, an object of the present invention is to provide a fermented product obtained by bioconversion of ganoderma lucidum mycelium to extract polysaccharide of L. elegans having anti-inflammatory activity.

Another object of the present invention is to provide a method for producing a fermented product obtained by bioconversion of ganoderma lucidum mycelium to extract polysaccharide of L. elegans having anti-inflammatory activity.

Another object of the present invention is to provide a natural anti-inflammatory agent containing the fermented product of the polysaccharide extract of Ungulgasari as an active ingredient.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

In order to achieve the object of the present invention, the present invention provides a fermented product extracted from the polysaccharide of L. ligamentum having high anti-inflammatory activity.

More specifically, the present invention provides a fermented product of the polysaccharide extract of the Lungium gashi having high anti-inflammatory activity by bioconversion of the polysaccharide extract of the Lungji mushroom using the mycelium of Ganoderma lucidum.

The present invention also includes the steps of (a) extracting polysaccharides from turmeric by using a low-carbon alcohol solvent; and (b) bioconverting the Ganoderma luciferase polysaccharide extract obtained in step (a) into ganoderma lucidum mycelium, and providing a method for producing a fermented product extracted from Ganoderma lucidum polysaccharide having a new anti-inflammatory effect.

In the following, the present invention is described in more detail.

In a previous study, the present inventors confirmed the antioxidant activity of polysaccharide extracts from various seaweed polysaccharide extracts. When prepared as an aqueous solution, the polysaccharide extract of Undungulgasari was judged to have a high molecular weight of about 840,000 daltons and high viscosity, limiting its use as an additive for pharmaceuticals, foods, and cosmetics. During the test by introducing the chemical method, it was found that bioconversion proceeded in the mycelium of Ganoderma lucidum when fermented the polysaccharide extract of Iridescent spp. Therefore, the optimum time point for fermentation of Ganoderma lucidum mycelium for bioconversion of the polysaccharide extract of Undung Pulgasi was set, and in the process of measuring the physiological activity of the bioconverted fermented Pulgasi polysaccharide extract, an anti-inflammatory activity that was not previously found was found, and fermentation 7 It was confirmed that it has the highest anti-inflammatory activity in the first day.

In addition, according to the following examples of the present invention, the fermented product extracted from the polysaccharide of Ganoderma lucidum, which was bioconverted into the mycelium of the ganoderma lucidum of the present invention, inhibited the inflammatory response of cells, alleviated oxidative stress, and induced atopic dermatitis. When fed to animals, it was confirmed that not only inflammatory diseases were alleviated, but also the damaged skin barrier was improved, and it could be usefully used as a natural anti-inflammatory agent.

Accordingly, the present invention provides an anti-inflammatory agent containing, as an active ingredient, a fermented polysaccharide extract obtained after bioconversion of the polysaccharide extract of Ganoderma lucifera lucidum using ganoderma lucidum mycelium.

Of course, "Seaweed furcata, Gloiopeltis Furcata " of the present invention belongs to the red algae family and includes its subspecies and variants. In addition, the bulbous plants of the present invention include cognate herbal medicines that are obvious in the art and can be used for the same or similar purposes as those of the present invention.

In the present invention, the "fermented fermented product of polysaccharide extraction of L. L. L." means a fermented extract obtained after extracting and separating polysaccharide from L. L. ganoderma and bioconversion using Ganoderma lucidum Karstena mycelium for 7 days.

The polysaccharide extract of Pulgasii puerperium according to the present invention can be extracted using a low-carbon alcohol solvent. The bulgeungasari can be used for drying or its powder, which is generally sold on the market. In addition, it can also be prepared directly by cutting the bulgeungpulgasari and then drying it.

Methanol, ethanol, propanediol, and mixed solvents thereof may be used as the low-carbon alcohol solvent, and ethanol is preferably used. Obtaining the polysaccharide extract of Undulgasari by using ethanol as an extraction solvent has the advantage of a high recovery rate of the polysaccharide extract.

Accordingly, in the present invention, the term "extract of polysaccharide of Ungulgasari" is a concept including all extracts, fractions, and purified products obtained in each step of extraction, fractionation, or purification, as well as dilutions, concentrates, or dried products thereof. The polysaccharide extract of the present invention can be prepared in a powder state by additional processes such as distillation under reduced pressure and lyophilization or spray drying.

Ganoderma lucidum used in the present invention is a year-old mushroom that sprouts from broad-leaved trees in summer, and is also called Ganoderma lucidum, Ganoderma lucidum, and Bulrocho. The stem is about 10 cm high, and the hat is heart-shaped or circular. The lampshade and the surface of the handle have a luster like lacquer. At first, it turns into egg yolk white, yellowish brown, or reddish brown, and when it is aged, the whole is leathery cork, hard and reddish brown or purple brown. There are two layers above and below, the upper layer is almost white, and the lower layer of the tube part is pale yellow. The shade is semi-circular, kidney-shaped or fan-shaped, and the surface is flat and has concentric rings. According to the color, it is divided into purple, black, blue, white, and yellow, but there is also a view that the cockerel mushroom is a close relative. Distributed in Korea, Japan and northern temperate regions of the Northern Hemisphere. In oriental medicine, it is used for nervous breakdown, heart disease, high blood pressure, and various cancers because it has effects such as tonic, cough, and small tumor. In the present invention, the ganoderma lucidum belongs to the mushroom family Bulloaceae, including subspecies and variants thereof.

In the present invention, when using Ganoderma lucidum or mycelium of Ganoderma lucidum to ferment the polysaccharide extract of Ganoderma lucidum, preferably, but not limited to, fermented using Ganoderma lucidum mycelium: Ganoderma lucidum polysaccharide extract in a weight ratio of 10: 1 to 20: 1 let it If it is less than the above range, the polysaccharide extract of Amaryllidaceae may not be properly bioconverted, and if it exceeds the above range, active ingredients may be destroyed due to over-fermentation.

According to a preferred embodiment of the present invention, the present invention provides an anti-inflammatory agent containing, as an active ingredient, a fermented product of polysaccharide extraction of Ganoderma luciferus bioconverted into Ganoderma lucidum mycelium. According to the present invention, the fermentation product of the Ganoderma lucidum polysaccharide extract bioconverted with the Ganoderma lucidum mycelium having anti-inflammatory activity of the present invention shows a lower viscosity of about 75% compared to the non-bioconverted Ganoderma lucidum polysaccharide extract (Table 3). , The active radical scavenging ability is very high, and the cell inflammation inhibitory effect is excellent (FIG. 2, FIG. 3, FIG. 4, FIG. 5), promoting collagen production in skin cells (FIG. 6) and suppressing melanin production (FIG. 7, FIG. 8), and it was shown to rapidly recover the skin condition of the experimental animals induced by oxidative stress through dietary feeding experiments (FIGS. 9 and 10). The fermented extract of the present invention may be contained in an amount of 0.1% by weight or more based on the total weight. If it is less than 0.1% by weight, it is difficult to expect a substantial anti-inflammatory effect of the fermented product extracted from the polysaccharide extract of Undulgasari. The fermented extract of the present invention has very high solubility in water, is odorless and tasteless, and has high selectivity as an additive. The anti-inflammatory agent according to the present invention may be prepared as a solution, powder, suspension, dispersion, emulsion, oil dispersion, paste, dust, dust, or granules, but is not limited thereto.

In addition, the present invention provides a pharmaceutical composition for preventing and improving inflammation comprising a fermented product of polysaccharide extraction of Ganoderma luciferus bioconverted into Ganoderma lucidum mycelium.

The pharmaceutical composition for preventing and improving inflammation according to the present invention may further include a pharmaceutically acceptable carrier, and is formulated with the carrier for pharmaceuticals, foods (health functional food), feed additives, drinking water additives, cosmetic compositions, etc. can be provided as

As used herein, the term 'pharmaceutically acceptable carrier' may refer to a carrier or diluent that does not inhibit the biological activity and properties of the administered compound while not stimulating living organisms.

The type of the carrier that can be used in the present invention is not particularly limited, and any carrier commonly used in the art and pharmaceutically acceptable can be used. Non-limiting examples of the carrier include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and the like. These may be used alone or in combination of two or more.

In addition, if necessary, other general additives such as other anti-inflammatory agents, buffers and/or bacteriostatic agents may be added and used, and diluents, dispersants, surfactants, binders, lubricants, etc. may be additionally added to form solutions such as aqueous solutions, suspensions, emulsions, etc. It may be formulated into an injection formulation, pill, capsule, granule or tablet and the like.

The administration method of the pharmaceutical composition for preventing and improving inflammation according to the present invention is not particularly limited, and may follow a method commonly used in the art. As a non-limiting example of the administration method, the composition may be administered by oral administration or parenteral administration. The pharmaceutical composition for preventing and improving inflammation of the present invention may be prepared in various formulations depending on the desired administration method.

The pharmaceutical composition of the present invention can be used as a single formulation, and can be prepared and used as a combination formulation by further including a drug known to have a recognized anti-inflammatory effect, and can be formulated using a pharmaceutically acceptable carrier or excipient to unite It can be prepared in dosage form or introduced into a multi-dose container.

As another aspect, the present invention provides a method for preventing and improving inflammation comprising administering the pharmaceutical composition for preventing and improving inflammation to a subject.

As used herein, the term "subject" may refer to all animals, including humans, that require prevention and improvement of inflammation.

Specifically, the treatment method of the present invention may include the step of administering a pharmaceutically effective amount of a fermented L. ganoderma polysaccharide extract fermented with the ganoderma lucidum mycelium to a subject in need of prevention and improvement of inflammation.

As used herein, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and is generally administered in an amount of 100 mg to 10 g once a day to several times. It can be administered in divided doses. However, for the purpose of the present invention, a suitable total daily amount of the composition containing the fermented extract can be determined by a treating physician within the scope of sound medical judgment, and can be administered once or divided into several times. However, for the purposes of the present invention, a specific therapeutically effective amount for a specific patient is determined by the type and extent of the response to be achieved, the specific composition, including whether other agents are used as the case may be, the patient's age, weight, general health condition, It is preferable to apply differently according to various factors including gender and diet, administration time, administration route and secretion rate of the composition, treatment period, drugs used together with or concurrently used with a specific composition, and similar factors well known in the medical field.

The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. And it can be single or multiple administrations. It is important to administer the amount that can obtain the maximum effect with the minimum amount without causing side effects in consideration of all of the above factors, and can be easily determined by those skilled in the art.

As used herein, the term "administration" means introducing the pharmaceutical composition of the present invention to a patient by any suitable method, and the route of administration of the composition of the present invention is oral or parenteral as long as it can reach the target tissue. It can be administered through various routes.

The administration method of the pharmaceutical composition according to the present invention is not particularly limited, and may follow a method commonly used in the art. As a non-limiting example of the administration method, the composition may be administered by oral administration or parenteral administration. The pharmaceutical composition according to the present invention may be prepared in various formulations depending on the desired administration method.

The frequency of administration of the composition of the present invention is not particularly limited thereto, but may be administered once a day or administered several times by dividing the dose.

In addition, when the fermented product of fermented Ganoderma lucidum polysaccharide extract fermented with mycelium of Ganoderma lucidum of the present invention is used as a food additive, the fermented product can be added as it is or used together with other foods or food ingredients, and can be used appropriately according to a conventional method. can The mixing amount of the active ingredient may be appropriately determined depending on the purpose of use (prevention, health or therapeutic treatment).

There is no particular limitation on the type of food. Examples of foods to which the substance can be added include meat, sausage, bread, chocolate, candy, snacks, baked goods, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea , drinks, alcoholic beverages, and vitamin complexes.

The food composition may include a food chemically acceptable carrier.

The food composition may be a health functional food. Functional food is the same term as food for special health use (FoSHU), and refers to food with high medical and medical effects processed to efficiently display bioregulatory functions in addition to nutrient supply. , The food may be prepared in various forms such as tablets, capsules, powders, granules, liquids, and pills in order to obtain useful effects for preventing or improving inflammation.

At this time, the content of the fermented polysaccharide extract of Ganoderma lucidum fermented with Ganoderma lucidum mycelium contained in the food is not particularly limited thereto, but is 0.1 to 100% by weight, more preferably 1 to 80% by weight, based on the total weight of the food composition can be included If the food is a beverage, it may be included in a ratio of 1 to 30 g, preferably 3 to 10 g, based on 100 mL.

The health functional food can be prepared by a method commonly used in the art, and can be prepared by adding raw materials and components commonly added in the art during the manufacture. In addition, unlike general drugs, there is an advantage in that there is no side effect that may occur when taking a drug for a long time by using food as a raw material, and it can be excellent in portability.

According to the present invention, the polysaccharide-extracted fermented product of Ganoderma lucida bioconverted with the ganoderma lucidum mycelium of the present invention has an inhibitory effect on melanin production (FIGS. 7 and 8). The extract of the present invention may be contained in the range of 0.1 to 30% by weight based on the total weight. If it is less than 0.1% by weight, it is difficult to expect a practical whitening effect of the fermented fermented sage extract, and if it exceeds 30% by weight, the synergistic effect due to the excess input is small and it is not economical.

In addition, the present invention provides a composition for food (health functional food) containing, as an active ingredient, a fermented polysaccharide-extracted product of Ganoderma lucidum fermented with ganoderma lucidum mycelium, which can prevent or improve inflammation. Ingestion of the fermented polysaccharide-extracted product of the present invention keeps the skin moisturized and quickly recovers the skin connective tissue damage caused by inflammation, and can be provided as a food for preventing or improving skin damage using it as an active ingredient. These health functional foods for skin moisturizing and tissue damage recovery can be consumed as edible cosmetics (Inner beauty).

According to another embodiment of the present invention, the present invention provides a quasi-drug composition for preventing, ameliorating, or treating inflammation, including a fermented product of polysaccharide extract of L. ganoderma fermented with ganoderma lucidum mycelium. The fermented product of polysaccharide extract of Ganoderma lucidum fermented with mycelium of Ganoderma Lucidum of the present invention can be added to a quasi-drug composition for the purpose of preventing, improving or treating inflammation.

In the present invention, the term "quasi-drugs" refers to textiles, rubber products or similar products used for the purpose of treating, mitigating, treating or preventing diseases of humans or animals, products that have weak effects on the human body or do not directly act on the human body, and devices or non-machines and similar items, products falling under one of the agents used for sterilization, insecticidal, and similar purposes to prevent infectious diseases, for the purpose of diagnosing, treating, mitigating, treating, or preventing human or animal diseases It may refer to items other than instruments, machines or devices among items used and items other than instruments, machines or devices among items used for the purpose of pharmacologically affecting the structure and function of humans or animals.

In addition, the quasi-drugs may include external skin preparations and personal hygiene products. Preferably, it may be a disinfectant cleaner, shower foam, gargreen, wet tissue, detergent soap, hand wash, or ointment, but is not limited thereto.

When the composition according to the present invention is used as a quasi-drug additive, the fermented product of fermented Ganoderma luciferase polysaccharide extract fermented with the ganoderma lucidum mycelium can be added as it is or used together with other quasi-drugs or quasi-drug ingredients, and can be appropriately used according to a conventional method there is. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of use.

According to another embodiment of the present invention, the present invention provides a drinking water additive for preventing or ameliorating inflammation, including fermented Ganoderma lucidum polysaccharide extract fermented with the Ganoderma lucidum mycelium. The drinking water additive of the present invention can be used in a manner in which the fermentation product fermented with the ganoderma lucidum mycelium extract polysaccharide is separately prepared in the form of a drinking water additive and mixed with drinking water, or directly added during preparation of drinking water. The drinking water additive of the present invention may be in a liquid or dry state, preferably in a dried powder form.

A drying method for preparing the drinking water additive of the present invention in a dried powder form is not particularly limited, and a method commonly used in the art may be used.

The drinking water additive of the present invention may further include other additives if necessary. Non-limiting examples of the usable additives include binders, emulsifiers, and preservatives added to prevent deterioration of drinking water quality; There are amino acids, vitamins, enzymes, probiotics, flavors, non-protein nitrogen compounds, silicates, buffers, coloring agents, extractants or oligosaccharides added to increase the effectiveness of feed or drinking water, and other feed additives are added. can be included with These may be used singly or two or more may be added together.

The fermented product of fermented Ganoderma lucidum polysaccharide extract fermented with the mycelium of Ganoderma lucidum of the present invention exhibits an anti-inflammatory effect that general extracts of Ganoderma lucidum do not have, and can be used as a natural anti-inflammatory agent, and can be used as a pharmaceutical composition, quasi-drug composition, food (health function) It can be used as a composition for food), an additive for drinking water, and an additive for feed. It is effective in improving skin atopy when applied to cosmetics because of its excellent anti-inflammatory effect in skin cells.

In addition, the fermented product of fermented Ganoderma lucidum polysaccharide extract fermented with Ganoderma lucidum mycelium of the present invention does not show hepatotoxicity and renal toxicity as a result of animal experiments, and is derived from a natural product, so it is safe and continuously without applying serious stimulation to the body or causing harmful effects. can be used

Figure 1 shows the effect of the polysaccharide extract (Example 4) fermented for 7 days with Ganoderma lucidum mycelium on the cell viability of HaCat cells.
Figure 2 shows the effect of the polysaccharide extract (Example 4) fermented for 7 days with Ganoderma lucidum mycelium in HaCat cells on the expression of NFAT5 and NF-κB, which are inflammation-related transcription factors.
Figure 3 shows the effect of the polysaccharide extract (Example 4) fermented for 7 days with Ganoderma lucidum mycelium in HaCat cells on the expression of IL-6, IL-1β and IL-4, which are cytokine mRNAs.
FIG. 4 shows the effect of the H ₂ O ₂ -induced HaCat cell toxicity inhibition and intracellular ROS generation inhibitory effect of the polysaccharide extract (Example 4) fermented with Ganoderma lucidum mycelium for 7 days.
Figure 5 shows the cytoprotective action mechanism against oxidative stress of the polysaccharide extract (Example 4) fermented for 7 days with Ganoderma lucidum mycelium in HaCat cells.
Figure 6 shows the effect of the polysaccharide extract (Example 4) fermented for 7 days with Ganoderma lucidum mycelium in HaCat cells on MMP-1 and collagen production and secretion.
Figure 7 shows the tyrosinase activity and melanin production inhibitory effect of the polysaccharide extract (Example 4) fermented for 7 days with Ganoderma lucidum mycelium.
8 shows the effect of the polysaccharide extract (Example 4) fermented for 7 days with Ganoderma lucidum mycelium on the expression of MITF and target factors involved in melanin production.
Figure 9 shows the in vivo skin moisturizing effect after oral administration of the polysaccharide extract (Example 4) fermented for 7 days with Ganoderma lucidum mycelium to experimental animals for 4 weeks.
Figure 10 shows collagen expression in tissues and expression of skin inflammatory factors (TNF-α and IL-6) after oral administration of the polysaccharide extract (Example 4) fermented for 7 days with Ganoderma lucidum mycelium to experimental animals for 4 weeks. It shows the result of observing the change in .

Hereinafter, the present invention will be described in more detail through examples. These examples are only for explaining the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited thereto.

[Comparative example]

<Preparation of polysaccharide from sagebrush>

After immersing 20 g of dried Pulgasari in distilled water for about 1 hour in 400 mL of distilled water (v/v, 1:20), it was pulverized with a grinder, left to stand for another 1 hour, and then water-soluble substances were extracted using a vacuum extractor.

The extraction solution was centrifuged (5,000 rpm, 4°C, 20 minutes) to collect the supernatant, and the volume was measured. After adding about twice the amount of ethanol, the polysaccharide was precipitated while standing in a refrigerator at 4°C for 24 hours.

The precipitated polysaccharide was filtered using filter paper and then dried at room temperature. At this time, about 10.4 g of polysaccharide was obtained, and the recovery rate was measured as 48-52%. Dried turmeric powder was used as an untreated group (comparative example).

[Examples 1 to 4]

<Manufacture of bioconverted polysaccharide from Ganoderma lucidum mycelium using mycelium>

After preparing by culturing the mycelium of Ganoderma lucidum in a liquid phase, 0.2% by weight of polysaccharide powder (Comparative Example) was added based on the culture medium, and then cultured at 37 ° C. During the culture, the culture medium was collected on the first day (Example 1), the third day (Example 2), the fifth day (Example 3), the seventh day (Example 4), and the 9th day (Example 5), respectively, and a part of the culture medium. was separated and freeze-dried without any treatment to prevent inactivation of the enzyme by heat treatment, etc., when observing changes in α-glucosidase activity as part of an experiment to set the optimal time point for biotransformation. After filtration using 2), the filtrate was put into an Erlenmeyer flask, the inlet was sealed with cotton, and then fixed in a stirred water bath fixed at 90 ° C., and then heated in water bath for 30 minutes to stop bioconversion. After heat treatment, the Erlenmeyer flask was allowed to cool, concentrated with nitrogen gas, and freeze-dried.

Table 1 below shows the fermentation time of Ganoderma lucidum mycelium for each sample of the polysaccharide extract of Undeungpulgasari.

sample name Fermentation time (days) comparative example 0 Example 1 One Example 2 3 Example 3 5 Example 4 7 Example 5 9

[Experimental Example 1]

<Setting the optimal bioconversion period>

<1-1> Measurement of α-glucosidase activity change according to culture period

In order to set the optimal bioconversion period, α-glucosidase activity, that is, Ganoderma lucidum mycelium secreted to obtain energy source from the polysaccharide extract and grow The activity of the enzyme to be measured.

In the α-Glucosidase activity test, substrate solution and test solution (sample solution) were mixed in a test tube, incubated at 40 ° C for 10 minutes, 10% sodium carbonate solution was added, cooled for 5 minutes, and absorbance (A ₁ ) was measured at 420 nm. .

For the enzyme blank test, the absorbance (A ₂ ) was measured at 420 nm in the same manner as in the experimental group without adding the substrate solution. At this time, a calibration curve of enzyme titer was prepared through p-nitrophenol.

Potency (units/g) = (AS-A2)×F×(3/0.5)×(1/10)×(1/W)

A1: absorbance of enzyme reaction solution

A2: absorbance of enzyme blank test solution

F: When the absorbance difference is 1 in the calibration curve, the amount of p-nitrophenol

10: reaction time (minutes)

W: Amount of sample contained in 1 ml of test solution (g)

Table 2 below shows the change in activity of α-glucosidase according to the culture period.

sample name α-glucosidase activity (U/g) comparative example 0 Example 1 2.72 ^a Example 2 ^3.21b Example 3 ^4.58c Example 4 4.65 ^c Example 5 ^4.64c

Different superscripts ( ^ac ) of α-glucosidase activity result values according to the culture period have statistical significance.

As a result, it was confirmed that α-glucosidase enzyme activity increased until the 5th day of culture, but remained unchanged after the 7th day.

<1-2> Measurement of change in viscosity during biotransformation period

Viscosity of the polysaccharide of fermented Pulgasi fermented by another method was measured to confirm the completion point of bioconversion. After fermenting, filtering, and heat-treating Ganoderma lucidum mycelium, freeze-dried lyophilized polysaccharide extract fermented powder was prepared into a 1.0% by weight solution using distilled water, and then the viscosity was measured using a viscometer.

Table 3 below shows changes in the activity of α-glucosidase according to the culture period.

sample name Viscosity (cP) comparative example 45.7 ^a Example 1 44.3 ^ab Example 2 ^42.3b Example 3 37.6 ^c Example 4 ^34.2d Example 5 ^34.1d

The other superscripts ( ^ad ) of the viscosity measurement result of polysaccharide of Idyllus spp. according to the culture period have statistical significance.

When the viscosity of the polysaccharide fermented with ganoderma lucidum mycelium was measured, the viscosity of the control group was 42.7 cP, and as the culture progressed, the viscosity of the test groups gradually decreased to 37.6 cP on the 5th day (comparison group 3) and 37.6 cP on the 7th day (comparison group 3). In group 4), it was measured at 34.2 cP, and on day 9 (comparative group 5), there was no statistical difference at 34.1 cP, indicating that fermentation was completed on day 7 of culture.

<1-3> Measurement of changes in polysaccharide molecular weight during bioconversion

As another method for determining the completion point of bioconversion, the change in the molecular weight of the polysaccharide of Idyllus phlegmon was measured during the bioconversion period. After bioconversion, filtration, and heat treatment, the powder of the fermented product extracted from the polysaccharide extract of lyophilus lyophilus was prepared as a 1.0% by weight solution using distilled water, and then the molecular weight was measured using GPC-HPLC. As a result, as the number of biotransformation days increased, low molecular weight progressed, and on the 9th day, it was found that the number was reduced by 3 to 560 times to 1,404 to 255,578 Da.

Table 4 below shows the change in the molecular weight of the polysaccharide of Undysphorus spp. according to the culture period.

sample name Molecular Weight (Dalton) comparative example 800,918 ^a Example 1 727,578 ^AB Example 2 8,559 to 655,078 ^b Example 3 5,256 to 389,000 ^c Example 4 3,526 to 298,265 ^d Example 5 1,404 to 255,578 ^d.

<1-4> Measurement of antioxidant change during biotransformation period

As the activity of the mycelium increased, it was confirmed that the molecular weight of the polysaccharide was decreased, and the change in antioxidant activity was measured. As a result, the DPPH and ABTS radical scavenging abilities of the polysaccharide of L. japonica before bioconversion were improved from 12.4 ± 1.2% and 7.3 ± 0.9%, respectively, to 28.4 ± 0.8% and 25.4 ± 1.2% on the 9th day of bioconversion.

Table 5 below shows the change in antioxidant activity of the fermented product extracted from the polysaccharide extract of Undulgasari according to the culture period.

sample name DPPH radical scavenging activity (%) ABTS radical scavenging activity (%) comparative example 12.4 ± 1.2 ^a 7.3 ± 0.9 ^a Example 1 13.4 ± ^1.4ab 14.8 ± ^1.2ab Example 2 19.4 ± ^1.1b 15.6 ± ^1.1b Example 3 22.6± ^1.2c 19.8± ^0.9c Example 4 25.3 ± 1.1 ^d 23.1 ± 1.1 ^d Example 5 28.4 ± 0.8 ^d 25.4 ± 1.2 ^d

[Experimental Example 2]

<Inflammation-inhibiting activity measurement test of polysaccharide of Undulgasari>

In Experimental Example 1, as the fermentation of the polysaccharide extract of Undeungpulgasari was completed on the 7th day of fermentation, subsequent anti-inflammatory tests were conducted using Example 4, which is a sample on the 7th day of fermentation, as a test group.

<2-1> Measurement of anti-inflammatory effect in HaCat cells

When TNF-α and INF-γ were treated with 20 ng/ml of keratinocyte HaCat cells, they showed a pattern similar to atopy, a representative symptom of dermatitis. Accordingly, an atopic dermatitis cell model was constructed to measure the anti-inflammatory effect of the bioconverted polysaccharide of Phlegans.

First, in order to measure cytotoxicity, HaCat cells were cultured in a 96-well plate at 1×10 ⁴ /well, and then samples or samples were treated with TNF-α and INF-γ, and cell viability was measured by MTT assay after 24 hours. measured (FIG. 1). The head mark (*, 0.05; #, 0.01) of the cell viability result value for HaCat cells of the polysaccharide euphorbiae is statistically significant.

When the HaCat cells were treated with the polysaccharide cultured for 7 days up to a concentration of 200 μg/ml, no change in cell viability was observed, confirming that the fermented product fermented with the mycelium of the Ganoderma lucidum mycelium had no cytotoxicity. When treated with TNF-α and INF-γ for 7 days of bioconverted polysaccharide, cell viability decreased by about 29.3% when treated with TNF-α and INF-γ alone, whereas Example 4 (fermentation Day 7), the cell viability tended to increase slightly in a concentration-dependent manner, and the cell viability increased significantly at a concentration of 100-200 μg/ml.

This result showed that the bioconverted euphorbia polysaccharide significantly protected HaCat cells from cell damage caused by TNF-α and INF-γ without cytotoxicity.

<2-2> Measurement of inflammatory factor expression regulation effect

In order to measure the anti-inflammatory effect of the biotransformed polysaccharide, the expression of NFAT5 and NF-κB, which are transcription factors, was confirmed by Western blot. HaCaT cells were cultured in a 6-well plate (2×10 ⁴ cells/mL), and pre-treated with biotransformed euphorbia for 1 hour at each concentration (6.25-100 μg/mL), followed by TNF-*?*/INF-γ ( 20 ng/mL) and cultured in a CO ₂ incubator for 18 hours. Thereafter, intracellular proteins were extracted with a passive lysis buffer, and the protein concentration of the cell lysate was measured using a BCA protein assay kit (Thermo scientific, Waltham, MA, USA).

After separating proteins through 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the separated proteins on a nitrocellulose membrane (0.45 μm) were blocked with 5% skim milk for 1 hour. Thereafter, the membrane was reacted with the primary antibody at 4° C. for 12 hours, washed, and then reacted with the HRP-conjugated secondary antibody for 1 hour at room temperature. At this time, protein expression was analyzed using an enhanced chemiluminescence (ECL) detection system (AI680, GE healthcare, Uppsala, Sweden).

As a result of examining the effect on the expression of NFAT5 and NF-κB, known as major inflammatory regulators, the expression of NFAT5 and NF-κB increased by TNF-α / INF-γ compared to Comparative Example 1. Fermented with Ganoderma lucidum mycelium In Example 4, it was confirmed that the concentration-dependent decrease was observed (FIG. 2).

In particular, NFAT5, known as an upstream regulator of NF-κB in diabetic nephropathy or inflammatory diseases, was reduced at all concentrations regardless of the B-GFPS treatment concentration, and NF-κB p65 phosphorylation decreased in a concentration-dependent manner in Example 4, then 200 As the protein expression was also reduced at the concentration of μg/ml, this is thought to be a decrease in phosphorylation due to a decrease in protein expression.

In order to measure the reduced transcriptional activity of NFAT5 at all concentrations regardless of the treatment concentration of the bioconverted luciferase polysaccharide, HaCat cells were transfected with the luciferase promoter having the NFAT5 promoter and measured. As a result, NFAT5 promoter activity showed a tendency to decrease significantly like protein expression when treated with B-GFPS.

<2-3> Measurement of effect on inflammatory factor expression and cytokine expression

Since the transcriptional activity of NF-κB was also found to decrease in the same pattern as protein expression, the effect of these transcription factors on cytokine expression was measured (FIG. 3).

Quantitative real-time PCR (qRT-PCR) was performed to measure the effect on the transcription of inflammatory cytokines in biotransformed euphorbia. HaCaT cells were cultured in a 6-well plate (2×10 ⁴ cells/mL), pretreated with black ginseng extract at each concentration (6.25-100 μg/mL) for 1 hour, and then treated with TNF-α/INF-γ (20 ng/mL). ) and incubated for 18 hours in a CO ₂ incubator.

After washing the cells three times with PBS, total RNA was isolated from the cells using TRIzol reagent. Synthesize cDNA using a PCR thermal cycler device (Takara Bio Inc., Shiga, Japan), and use SYBR green on a CFX connect Real-Time PCR detection system (Bio-Rad ***Laboratories Inc., Hercules, CA, USA). Quantitative real-time PCR was performed using mastermix.

As a result, it was confirmed that IL-6, IL-1β, and IL-4 mRNA expressions were all decreased according to the decrease in activity and expression of transcription factors. In other words, as the expression and activity of inflammation-related transcription factors in the biotransformed spp.

<2-4> Measurement of oxidative stress relieving effect in HaCat cells

Nrf2 is known to regulate the expression of various antioxidant enzymes as a redox-sensitive transcription factor. Nrf2 usually exists in an inactive complex with Keap1 in the cytoplasm, but when activated, it moves into the nucleus and binds to the Antioxidant Response Element (ARE), increasing the expression of antioxidant enzymes such as HO-1 and Catalase, and generating these genes. It plays a central role in the biological defense mechanism against intracellular oxidative stress by regulating the expression of and enhancing protein expression.

Therefore, in this experiment, the cytoprotective effect of fermented Ganoderma lucidum polysaccharide fermented with Ganoderma lucidum mycelium against oxidative stress induced by hydrogen peroxide (H ₂ O ₂ ) in HaCaT cells was confirmed, and Nrf-2 as its mechanism of action An experiment was conducted on the correlation with /HO-1 protein expression (FIG. 4).

First _, as a _result of examining the protective effect of HaCaT cells of the biotransformed _{spp .} In addition, it was determined that the ROS induced by H ₂ O ₂ was reduced by the bioconverted euphorbia polysaccharide in the same way, and thus, there was a cytoprotective effect according to the reduction of intracellular ROS (FIG. 4).

As a result of examining the expression of Nrf2, a representative transcription factor that protects against damage caused by oxidative stress in cells, and its target protein, oxidase, Nrf2, which was reduced by H ₂ O ₂ , was recovered by fermented Ganoderma lucidum polysaccharide. HO-1, Catalase, and SOD showed the same tendency according to Nrf2 recovery. In addition, as the mRNA expression of HO-1, Catalase, and SOD also increased in the same way as protein, the cytoprotective effect of the polysaccharide extract of Ganoderma lucidum fermented with Ganoderma lucidum mycelium against oxidative stress was related to Nrf2/HO-1 expression. there will be (Fig. 5).

[Experimental Example 3]

<Skin improvement effect test due to anti-inflammatory effect>

<3-1> Measurement of effect on collagen production

MMP-1 is a connective tissue proteolytic factor responsible for tissue remodeling and disassembly of the extracellular matrix (ECM) including collagen, elastin, gelatin and matrix glycoproteins, and is generally minimally expressed under normal physiological conditions, thus maintaining homeostasis. maintain.

However, when oxidative stress such as UV irradiation is induced, it not only causes cell damage but also promotes the activity of MMP-1, which accelerates skin aging as connective tissue proteins such as collagen are decomposed and reduced.

As it was confirmed that the polysaccharide extract of Ganoderma lucidum fermented with Ganoderma lucidum mycelium had a cytoprotective action against oxidative stress, the effect on the production and secretion of MMP-1 and collagen was measured.

As a result, the polysaccharide fermented with Ganoderma lucidum mycelium reduced the expression of MMP-1, which was increased by H ₂ O ₂ , and increased the expression of collagen. It was found to be secreted in the same way (FIG. 6).

<Melanin formation inhibitory effect>

As tyrosine is oxidized to dopaquinone by tyrosinase, pigmentation occurs in the skin. The tyrosinase inhibitory activity was measured in order to confirm whether the polysaccharide fermented with Ganoderma lucidum mycelium exhibits an inhibitory effect on tyrosinase, which makes the skin black.

As a result, when the polysaccharide extract (Example 4) fermented for 7 days with Ganoderma lucidum mycelium was treated at a concentration of 100 to 200 μg/ml, the activity of tyrosinase e was inhibited by 81.3 to 84.5%. When used as an unfermented Pulgasi polysaccharide extract (Comparative Example), 85.7% was inhibited at a concentration of 200 μg/ml, and the polysaccharide fermented Pseudomonas lucidum fermented with Ganoderma lucidum mycelium showed a similar inhibitory effect on tyrosinase activity as that of arbutin, a positive control. (FIG. 7).

When the skin is exposed to ultraviolet rays, melanin produced through a series of oxidative polymerizations from tyrosine causes melanin, erythema, etc., and accelerates skin aging. It is known that inhibition of tyrosinase enzyme can inhibit melanin synthesis. there is. Therefore, in order to directly confirm whether the Ganoderma lucidum polysaccharide fermented with Ganoderma lucidum mycelium inhibits melanin synthesis, melanin was quantitatively analyzed by treating Melan A cells with Ganoderma lucidum polysaccharide fermented with Ganoderma lucidum mycelium.

As a result, the melanin increased by α-MSH was reduced in a concentration-dependent manner in the polysaccharide extract (Example 4) fermented with ganoderma lucidum mycelium for 7 days (FIG. 7). MITF is involved in the proliferation and differentiation of melanocytes, and acts as an important regulator of the melanin synthesis mechanism by increasing or decreasing the expression of tyrosinase, TRP-1, and TRP-2, which are melanin synthesis-related enzymes.

Since the polysaccharide extract (Example 4) fermented with ganoderma lucidum mycelium for 7 days was found to inhibit melanin synthesis, changes in the expression of proteins related to the melanin synthesis mechanism were confirmed. As a result, the polysaccharide extract (Example 4) fermented for 7 days with α-MSH or α-MSH and ganoderma lucidum mycelium did not show a significant difference in MITF expression, whereas TRP-1 and -2, target factors of MITF , and tyrosinase was increased by α-MSH.

However, when treated with α-MSH and B-GFPS, MITF expression was decreased at all concentrations, unlike MITF expression (FIG. 8). This was the same as the mRNA expression level and was consistent with the results of arbutin, which is well known as a tyrosinase inhibitor.

<Experimental Example 4>

<Anti-inflammatory effect test according to the feeding of the polysaccharide extract fermented with ganoderma lucidum mycelium in laboratory animals>

In an in vitro model, the polysaccharide extract (Example 4) fermented with ganoderma lucidum mycelium for 7 days showed anti-inflammatory, oxidative stress inhibitory, and melanin production inhibitory effects. In other words, it showed potential as a functional inner beauty material.

Accordingly, NC/Nga Tnd mice, which are suitable as animal models for dry skin, were fed 50 mg/Kg and 100 mg/Kg of polysaccharide fermented with Ganoderma lucidum mycelium for 4 weeks, followed by acetone and ether 1 week before sacrifice. After soaking a cotton pad (2 × 2 cm) in the solution mixed with No. 1, placing it on the back for 15 seconds, wiping the same area with distilled water for 30 seconds to build a skin dry model.

During the construction of the dry skin model, the amount of skin moisture and the amount of transepidermal water loss were measured every morning and evening, and after one week of dry skin, the mice were sacrificed and blood and skin tissue were cut.

<4-1> In vivo skin moisturizing effect measurement

Transepidermal water loss (TEWL) is a measure of the amount of water evaporated from the skin surface, and the lower the measured value, the smaller the water loss from the skin surface.

As shown in FIG. 9, in the case of the control group in which dry skin was induced, compared to the normal group, TEWL was significantly increased, which indicates that moisture on the skin surface was lost. However, it was observed that the TEWL value was significantly lowered in the group ingesting 100 mg/Kg of polysaccharide fermented with B ganoderma lucidum mycelium for 4 weeks.

As a result of measuring the moisture content of the stratum corneum of each experimental group, there was no change in the moisture content of the epidermal layer in the Normal group, but in the fermented Ganoderma lucidum mycelium (Example 4) group, the moisture content was significant compared to the control group. It increased in a concentration dependent manner (FIG. 9).

<4-2> Changes in elastic fibers

10 shows changes in elastic fibers in the skin dermis through Masson's trichrome staining, and in the control group (comparative example), elastic fibers denatured in the dermis due to dryness induction were easily identified, and collagen was stained blue. The amount of was significantly reduced compared to the normal group.

However, in the group (Example 4) ingesting the polysaccharide extract fermented for 7 days with ganoderma lucidum mycelium for 4 weeks, compared to the control group, the amount of collagen was relatively increased and dyed blue more intensely, It was confirmed that oral administration of the polysaccharide extract of Ganoderma lactobacillus fermented for 7 days with Ganoderma lucidum mycelium maintained the collagen layer in the skin tissue in NC/Nga Tnd mice despite skin dryness induced by acetone and ether (FIG. 10).

In the skin damaged by dryness, external antigens easily penetrate into the skin and secrete various inflammation-inducing cytokines in keratinocytes, causing skin inflammation. TNF-α and IL- As a result of confirming the mRNA expression of 6, in the group fed the polysaccharide fermented with Ganoderma lucidum mycelium, the mRNA expression of the cytokine was significantly decreased, which also increased the reduced collagen expression. appeared (FIG. 10). In other words, it was found that intake of fermented luciferase polysaccharide extract (Example 4) fermented with ganoderma lucidum mycelium for 7 days not only maintains and restores skin moisture, but also regulates factors involved in skin inflammation as a whole.

[Statistical analysis]

Statistical analysis of the experimental results was performed using the 'Independent-Sample-T-Test method', an SPSS 13.0 statistical software, and expressed as the average value ± standard difference. In addition, multiple comparisons were indicated by the LSD method, P>0.05: the difference is not clear, P<0.05: it means the difference is clear.

<Preparation Example 1> Preparation of powder formulation

An anti-inflammatory preparation containing the fermented product obtained from the Ganoderma lucidum polysaccharide extract bioconverted with Ganoderma lucidum mycelium obtained in Examples 1 to 5 was prepared by mixing with an excipient after drying by a conventional freeze-drying method or a hot air drying method.

<Preparation Example 2> Preparation of spray for nasal cavity (nose)

<2-1> Manufacture of nasal decongestant (nasal decongestant) spray

According to a method commonly performed in the art, the nasal decongestant spray for decongestion is 10.00% by weight of powder of a fermented polysaccharide extract of L. ganoderma, bioconverted for 7 days with the ganoderma lucidum mycelium obtained in Example 4, oxymetazoline or xylo It was prepared by mixing 0.10% by weight of metazoline, 0.01% by weight of chlorophenylamine, 0.10% by weight of sorbitol, and the remaining amount of physiological saline.

<2-2> Preparation of nasal spray for moisturizing

According to a method commonly performed in the art, the nasal spray for moisturizing the nasal cavity is 10.00% by weight of powder of fermented polysaccharide extract of Ganoderma lucidum, 0.10% by weight of sorbitol, and physiological saline solution obtained in Example 4. It was prepared by mixing the remaining weight %.

<Preparation Example 3> Preparation of skin external preparation (ointment)

The ointment contains 10.00% by weight of a fermented product powder extracted from the polysaccharide extracted from L. ganoderma, 10.00% by weight of caprine/capryl triglyceride, and liquid paraffin 10.00 wt%, sorbitan sesquinoleate 6.00 wt%, octyldodeceth-25 9.00 wt%, cetylethylhexanoate 10.00 wt%, squalane 1.00 wt%, salicylic acid 1.00 wt%, glycerin 15.00 wt%, sorbitol 10.00 wt% %, was prepared by mixing the remaining amount by weight of purified water.

<Preparation Example 4> Preparation of food and beverage (health functional food) composition

<4-1> Manufacture of beverages

The beverage was prepared by mixing the ingredients of the formulations presented below according to a method commonly practiced in the art.

Honey 522 mg, thioctic acid amide 5 mg, nicotinic acid amide 10 mg, riboflavin sodium hydrochloride 3 mg, pyridoxine hydrochloride 2 mg, inositol 30 mg, ortic acid 50 mg, biotransformation with ganoderma lucidum mycelium obtained in Example 4 for 7 days 10 g of the fermented product powder of the polysaccharide extract of one Ungulgasari, 200 mL of water.

<2-2> Preparation of powder

The powder is 2 g of powder of fermented polysaccharide extract of L. lily, bioconverted for 7 days with Ganoderma lucidum mycelium obtained in Example 4 according to a method commonly performed in the art, complex probiotic (1 × 10 ⁹ CFU / g) 2 g, 5 g of lactose, and 1 g of talc were mixed and filled in an airtight bag to prepare a powder.

<2-3> Manufacture of tablets

The tablet was bioconverted for 7 days with the mycelium of Ganoderma lucidum obtained in Example 4 according to a method commonly performed in the art. 250 mg of fermented product powder extracted from polysaccharide, 150 mg of corn starch, 100 mg of lactose, stearic acid Tablets were prepared by mixing 20 mg of magnesium and then tableting according to a conventional tablet manufacturing method.

<2-4> Preparation of capsules

The capsule was bioconverted for 7 days with the Ganoderma lucidum mycelium obtained in Example 4 according to a method commonly performed in the art. 50 mg of powder of fermented polysaccharide extract, crystalline cellulose 3 mg, lactose 14.8 mg, magnesium stearate Capsules were prepared by mixing 0.2 mg of latex according to a conventional capsule preparation method and filling gelatin capsules.

<2-5> Preparation of mixed powder formulation

The mixed powder is 5 g of fermented fermented polysaccharide powder obtained by biotransformation for 7 days with Ganoderma lucidum mycelium obtained in Example 4 according to a method commonly performed in the art, appropriate amount of vitamin mixture (vitamin A acetate 70 μg, vitamin E 1.0 mg, vitamin B1 0.13 mg, vitamin B2 0.15 mg, vitamin B6 0.5 mg, vitamin B12 0.2 μg, vitamin C 10 mg, biotin 10 μg), nicotinamide 1.7 mg, folic acid 50 μg, calcium pantothenate 0.5 mg, mineral mixture Appropriate amounts (ferrous sulfate 1.75 mg, zinc oxide 0.82 mg, magnesium carbonate 25.3 mg, monobasic potassium phosphate 15 mg, dibasic calcium phosphate 55 mg, potassium citrate 90 mg, calcium carbonate 100 mg, magnesium chloride 24.8 mg) After mixing according to the health functional food manufacturing method of, to prepare the granules, it can be used to prepare a health functional food composition according to a conventional method.

The composition ratio of the above vitamin and mineral mixture was prepared by mixing ingredients suitable for a relatively health functional food in a preferred manufacturing example, but the mixing ratio may be arbitrarily modified.

<Preparation Example 5> Preparation of feed composition

According to a method commonly performed in the art, the composition for feed was 45 g of corn, 30 g of soybean meal, 15 g of wheat, 4 g of beef tallow, 3 g of molasses, 2 g of calcium phosphate, 0.4 g of limestone, 0.2 g of salt, in Example 4. It was prepared by mixing 10 g of the fermented product powder extracted from the polysaccharide extracted from the Ganoderma lucidum which was bioconverted for 7 days with the obtained mycelium of Ganoderma lucidum.

<Production Example 6> Manufacturing of cosmetics

<6-1> Preparation of lotion

According to a method commonly performed in the field, the lotion was prepared by adding 0.05 g of polypyrrolidone, 0.1 g of polyol alcohol, 0.2 g of polyoxyethylene monooleate, 0.2 g of perfume, 0.1 g of paraoxybenzoic acid methyl ester, and a small amount of 8 g of 95% ethanol. of antioxidants and a small amount of pigment were mixed and dissolved.

1 g of polysaccharide-extracted fermented powder powder of Ganoderma lucidum obtained in Example 4 bioconverted for 7 days with mycelium of Ganoderma lucidum obtained in Example 4 and 5 g of glycerin were dissolved in the remaining weight % of purified water. After adding the mixture, the mixture was stirred to have a soothing effect on the skin caused by inflammation I got the lotion in there.

<6-2> Manufacture of Lotion

The lotion was 1.00% by weight of powder of fermented polysaccharide extract of L. ginseng, 3.00% by weight of glycerin, 0.10% by weight of carbomer, xanthan 0.05% by weight of gum, 3.00% by weight of 1,3-butylene glycol, 1.50% by weight of polyglyceryl-3 methylglucose distearate, 0.50% by weight of glyceryl stearate, 0.30% by weight of cetylaryl alcohol, 3.00% by weight of jojoba oil , 2.00% by weight of liquid paraffin, 3.00% by weight of squalane, 0.50% by weight of dimethicone, 0.20% by weight of tocopheryl acetate, 0.10% by weight of triethanolamine, preservatives, fragrances, trace amounts of pigments, and the remaining amount of purified water.

<6-3> Manufacture of emulsion

The emulsion was prepared according to a method commonly performed in the art, including 1.2 g of cetyl alcohol, 10 g of squalane, 2 g of petrolatum, 0.2 g of p-oxybenzoic acid ethyl ester, 1 g of glycerin monoester, and polyoxyethylene (20 moles added). 1 g of monooleate and 0.1 g of perfume were mixed and dissolved by heating at 70 ° C, and bioconverted for 7 days with Ganoderma lucidum mycelium obtained in Example 4. 1.00 g of powder of fermented polysaccharide extract, 5 g of dipropylene glycol, polyethylene glycol 1500 2 g, 0.2 g of triethanolamine, and the remaining weight % of purified water were heated to 75 ° C and dissolved by heating.

The mixture was emulsified and then cooled to obtain an emulsion having an effect of improving skin caused by water/oil-based inflammation.

<6-4> Preparation of serum

According to a method commonly performed in the art, the essence is 95% ethyl alcohol 5 g, polyoxyethylene sorbitan monooleate 1.2 g, kitulose 0.3 g, sodium hyaluronate 0.2 g, vitamin E-acetate 0.2 g, licorice acetic acid 0.2 g of sodium, 0.1 g of p-oxybenzoic acid ester ester, 1 g of powder of fermented polysaccharide extract of L. aurigasi biotransformed for 7 days with Ganoderma lucidum mycelium obtained in Example 4, and an appropriate amount of pigment are mixed to improve the skin caused by inflammation I got a beauty essence.

From the above description, those skilled in the art to which the present invention pertains will be able to understand that the present invention may be embodied in other specific forms without changing its technical spirit or essential features. In this regard, the manufacturing examples described above should be understood as illustrative in all respects and not limiting.

The scope of the present invention should be construed as including all changes or modifications derived from the meaning and scope of the claims to be described later and equivalent concepts rather than the detailed description above are included in the scope of the present invention.

As described above, the polysaccharide-extracted fermented product of Lingji mushroom bioconverted for 7 days with the Ganoderma lucidum mycelium of the present invention has an excellent effect on anti-inflammatory activity, and in particular, when ingested as a diet, the skin caused by skin inflammation due to oxidative stress It can be usefully used in the development and production of natural anti-inflammatory drugs, health functional foods, and whitening functional cosmetics by effectively improving the condition of the skin.

Claims (10)

Fermented product obtained by bioconversion of Seaweed furcata ( Gloiopeltis Furcata ) extracted polysaccharide using Ganoderma lucidum (Leyss. ex. Fr) Karst mycelium. The fermented product according to claim 1, wherein the Ganoderma lucidum or its mycelium is bioconverted using Ganoderma lucidum: Polysaccharide of Ganoderma lucidum in a weight ratio of 10:1 to 20:1. The fermented product of claim 1, characterized by bioconversion of the polysaccharide of the luciferase lucidum to ganoderma lucidum mycelium for 7 days. The fermented product extracted from Irregular Starfish polysaccharide according to claim 1, characterized in that the fermented extract is used for inhibiting inflammation. A pharmaceutical composition for the prevention, improvement or treatment of inflammation comprising the fermented product of extracting the polysaccharide extracted from any one of claims 1 to 4 as an active ingredient. A quasi-drug composition for the prevention, improvement or treatment of inflammation, comprising the fermented product extracted from the polysaccharide extracted from any one of claims 1 to 4 as an active ingredient. A food composition for preventing or improving inflammation, comprising the fermented product of extracting the polysaccharide extracted from any one of claims 1 to 4 as an active ingredient. A drinking water additive comprising the fermented product of extracting the polysaccharide extracted from Irregular Starfish according to any one of claims 1 to 4 as an active ingredient. A feed additive comprising the fermented product of extracting the polysaccharide extracted from Irregular Starfish according to any one of claims 1 to 4 as an active ingredient. A cosmetic composition for preventing or improving skin inflammation, comprising the fermented product of extracting the polysaccharide extracted from any one of claims 1 to 4 as an active ingredient.

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