KR102515226B1 - Pharmaceutical composition comprising mixing fermentation product of Citrus onshiu and Raphanus sativus - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating inflammatory diseases or obesity comprising fermented lactic acid bacteria of green tangerine or a mixture of green tangerine and black radish, a method for preventing or treating inflammatory disease or obesity using the pharmaceutical composition, green tangerine or green tangerine and It relates to a food composition for improving inflammatory diseases or obesity containing a lactic acid bacteria fermentation product of a black radish mixture and a feed composition for improving inflammatory diseases or obesity containing a green tangerine or a lactic acid bacteria fermentation product of a green tangerine and black radish mixture. The composition of the present invention is effective in improving anti-obesity function because it has an anti-inflammatory effect and an effect of inhibiting fat formation in adipocytes, which is one of the indicators of fat accumulation inhibition.

Description

Pharmaceutical composition comprising a mixed fermentation product of Citrus onshiu and Raphanus sativus}

The present invention relates to a pharmaceutical composition comprising a mixed fermented product of green tangerine and black radish, and more specifically, the present invention relates to a pharmaceutical composition for preventing or treating inflammatory diseases or obesity, including a fermented product of lactic acid bacteria of green tangerine or a mixture of green tangerine and black radish A composition, a method for preventing or treating inflammatory diseases or obesity using the pharmaceutical composition, a food composition for improving inflammatory diseases or obesity containing a fermented product of lactic acid bacteria of green tangerine or a mixture of green tangerine and black radish, and a mixture of green tangerine or green tangerine and black radish It relates to a feed composition for improving inflammatory diseases or obesity containing a lactic acid bacteria fermentation product.

Obesity plays the most important role in relation to fat synthesis and metabolism. In adipose tissue, preadipocytes differentiate into adipocytes through the adipogenesis stage to accumulate triglyceride (TG) (Foretz et al., 1999). Adipocyte differentiation is closely related to endocrine organs that secrete various hormones such as adiponectin, leptin, and adipsin, and glucose and lipid metabolism (Rosen et al., 2000; Visscher and Seidell, 2001). Today, the obese population is rapidly increasing in modern society. According to the 2010 National Health and Nutrition Examination, the adult obesity rate is 30.8%, which means that one out of three people is obese. Obesity is a phenomenon in which excessive body fat is accumulated when intake exceeds consumption due to an imbalance between energy intake and consumption, and is a factor that increases the incidence of various diseases such as hyperlipidemia, hypertension, diabetes, and arteriosclerosis.

Among human intestinal bacteria, lactic acid bacteria are the most commonly used probiotics, and they improve nutritional and physiological health by regulating intestinal movement, inhibiting pathogenic bacteria, promoting digestion and absorption, and preventing constipation and diarrhea, as well as fermented milk products and naturally fermented foods. , it is widely used in livestock feed and pharmaceutical fields. Probiotics have been reported to show physiological activity in various diseases such as intestinal function, immune enhancement and regulation, anti-cholesterol, lipid metabolism balance and inflammatory bowel disease, alcoholic and non-alcoholic liver disease. For example, L. plantarum lactobacilli are effective in preventing chronic and acute alcohol-induced liver damage by improving intestinal barrier function, restoring gut microbiota, regulating pro-inflammatory molecules, inhibiting oxidative stress, and disrupting endotoxinemia. According to reports by Li et al (2014) and Zhang et al (2017), L. plantarum alleviates non-alcoholic fatty liver disease by inducing adipose leptin, increasing antioxidant capacity, and reducing lipid peroxidation. And L. plantarum alleviates liver damage induced by endotoxin, D-galactosamine, lipopolysaccharide and cadmium. Among various health-promoting actions of lactic acid bacteria and fermented foods, if attention is paid to the lipid metabolism improving effect, cholesterol-lowering effects of Lactobacillus acidophilus, Streptococcus faecalis, Lactobacillus plantarum, and Enterococcus faecium have been revealed. In addition, in the case of Gochujang, a representative fermented food, it inhibits fat accumulation in 3T3-L1 fat cells and lowers sterol regulatory element-binding protein 1-c (SREBP-1c) and peroxisome proliferator-activated receptor γ (PPAR-γ) to reduce fat It has been shown to inhibit the production, and the anti-obesity effect of kimchi lactobacillus powder has also been found. As described above, research on the anti-obesity effect of lactic acid bacteria and fermented foods is being actively conducted, but there is no research on green tangerine fermentation using lactic acid bacteria or development of green tangerine-black radish mixed fermentation.

Recently, research on fermented herbal medicines has been actively conducted. Fermented herbal medicines refer to fermented herbal medicines into beneficial seed germs to increase the efficacy of the original herbal medicine, reduce side effects, and add new functions. Fermented herbal medicines are attracting attention recently because they maximize the absorption rate and bioavailability of active ingredients in the body, minimize side effects such as digestive intolerance, enhance the effectiveness of existing herbal medicines, and help balance the intestinal microflora. Studies on the effect of obesity are still lacking. When ephedra, a drug widely used in herbal medicine prescribed for obesity treatment, was fermented with Lactococcus confusus, the expression level of PPAR-γ was decreased and tumor necrosis factor-α (TNF-α) was increased in 3T3-L1 cells. , There have been studies showing that fermented ephedra reduced autonomic nervous system side effects such as anxiety in animal experiments, but studies on the anti-obesity and anti-inflammatory effects of fermented green tangerine lactobacillus have not been reported. When an imbalance in fat synthesis and metabolism is induced, adipose tissue accumulates in blood vessels, muscle cells, and hepatocytes, resulting in various metabolic syndrome diseases such as arteriosclerosis, type 2 diabetes, fatty liver, and liver cirrhosis. Reactive oxygen species (ROS) are generated during metabolism and cause oxidative damage to proteins, lipids, and DNA. This damage further promotes fat accumulation and fibrosis in hepatocytes, further progressing fatty liver or liver cirrhosis.

Green tangerine ( Citrus onshiu ) refers to an immature mandarin orange harvested between July and September. In general, Onju tangerines are divided into very early and early ripening, and to be used as raw fruits, extremely early ripening is harvested in October and early ripening is harvested between November and December. In general, green tangerines are reported to be very rich in vitamins, flavonoids, chlorophyll, dietary fiber, limonoids, etc. compared to mature fruits. Fermented tea containing lactic acid bacteria improves flavor and bioavailability of phenolic compounds through reduction of epigallocatechin gallate epigallocatechin and epicatechin. Microbial-derived fermented tea is actively studied for promoting digestion, anti-cancer effects, and treatment and prevention of cardiovascular diseases.

The β-glucosidase enzyme secreted by lactic acid bacteria hydrolyzes various glycosides, increasing the bioavailability of glucosides. In addition, it shows effects on metabolite detoxification, xenobiotic, and mycotoxin defense by increasing water solubility and chemical stability. Lactic acid bacteria that secrete β-glucosidase include L. acidophilus, bulgaricus, L. casei, L. plantarum, L. fermentum, and Bifidobacterium sp. etc. have been reported. These lactic acid bacteria can hydrolyze a wide range of substrates through deglycosylation, dihydroxylation, or O-methylation, such as β-glucosidase and rhanosidase.

Flavonoid compounds reported in green tangerines include hesperidin, naringin, and narirutin, and polymethoxy flavoniods include tangeritin, limocitrin-3-glucose, 8-methoxy quercetin 3-O-[ 6′-(3′-hydroxy-3′-methyl-glutaroyl)-beta-D-glucopyranoside](8-methoxy quercetin 3-O-[6′-(3′-hydroxy-3- methyl-glutaroyl)-β-D-glucopyranoside], quercetin3-O-[6′-(3′-hydroxy-3′-methyl-glucopyranoyl)-beta-di-glucopyranoside] (quercetin3- O-[6′-(3′-hydroxy-3′-methyl-glutaroyl)-β-D-glucopyranoside]) and 5,6,7,8,3’,4’-hexamethoxyquercetin 3-O- [6”-(3”’-hydroxy-glutaroyl)-beta-D-glucopyranoside](5,6,7,8,3’,4’-HexaMethoxyQuercetin3-O-[6”-( 3”'-hydroxy-3”’-methyl-glutaroyl)-β-D-glucopyranoside]), etc., and various physiological activities such as antihyperlipidemia, skin moisturizing, and anti-inflammation have been reported as major functions. Despite this, green tangerines are not in the limelight from the public due to their low palatability, which is becoming an obstacle to fostering the green tangerine industry.

Accordingly, the inventors of the present invention made diligent research efforts to develop new ways to utilize green tangerine, and as a result, it was confirmed that the lactic acid bacteria fermented product of green tangerine exhibited superior anti-inflammatory activity and anti-obesity activity compared to green tangerine itself, and completed the present invention.

A main object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of inflammatory diseases or obesity, comprising a fermented product of lactic acid bacteria of green tangerine.

Another object of the present invention is to provide a method for preventing or treating inflammatory diseases or obesity using the pharmaceutical composition.

Another object of the present invention is to provide a food composition for improving inflammatory diseases or obesity containing a fermented product of lactic acid bacteria of green tangerine.

Another object of the present invention is to provide a feed composition for inflammatory disease or obesity improvement comprising a fermented product of lactic acid bacteria of green tangerine.

Another object of the present invention is to provide a use of a fermented product of lactic acid bacteria of green tangerine for the preparation of a pharmaceutical composition for preventing or treating inflammatory diseases or obesity.

One embodiment of the present invention for achieving the above object provides a pharmaceutical composition for preventing or treating inflammatory diseases or obesity, comprising a fermented product of lactic acid bacteria of green tangerine.

The term "green tangerine" of the present invention refers to an immature citrus fruit ( Citrus unshiu ), and generally the entire surface of the fruit shows a green state.

In the present invention, the green tangerine may be all, a part, or a by-product of green tangerine, which is an immature fruit of all citrus fruits that can be used for lactic acid fermentation. The by-product of the immature fruit may comprehensively include an extract of the immature fruit, a fraction of the extract, and the like.

The term "lactic acid bacteria fermented product" of the present invention means a material fermented using lactic acid bacteria (lactic acid bacteria) regardless of whether dairy products are included.

In the present invention, it can be understood that the lactic acid bacteria fermented product is a product obtained by inoculating and fermenting lactic acid bacteria in raw materials including green mandarins.

The method for obtaining the fermented product of lactic acid bacteria is not particularly limited thereto, but includes inoculating lactic acid bacteria into a pulverized product obtained by grinding a raw material including all or part of a green tangerine and fermenting it.

In the above method, the pulverized material may be used when the pH is titrated to 6.0 to 8.0.

In addition, the lactic acid bacteria are not particularly limited thereto, but as an example, Lactobacillus sp. strain, Lactococcus sp. strain, Enterococcus sp. strain, Streptococcus sp . sp.) strains, Pediococcus sp. strains, Leuconostoc sp. strains, and Bifidobacterium sp. strains, etc. may be used alone or in combination, and , As another example, Lactobacillus plantarum ( Lactobacillus plantarum ), Lactobacillus casei ( Lactobacillus casei ), Lactobacillus helveticus ( Lactobacillus helveticus ), Lactobacillus reuteri ( Lactobacillus sp.) Microorganisms can be used.

In addition, the temperature condition for performing the fermentation is not particularly limited thereto, but as an example, it may be 30 to 37 ° C, as another example, it may be 32 to 35 ° C, and as another example, 34 ° C It can be.

Finally, the time condition for carrying out the fermentation is not particularly limited thereto, but as an example, it may be 20 to 50 hours, as another example, it may be 30 to 40 hours, and as another example, 36 hours This can be.

On the other hand, the raw material may include not only green tangerines but also various additional materials. The additional materials are not particularly limited thereto, but may be, for example, black radish.

The term "black radish ( Raphanus sativus L. var. niger)" of the present invention means one of the colored radish varieties showing a black outer skin, which is known as a crop that has been widely used for medicinal purposes in Europe.

In the present invention, the black radish can be used as an additive material for the lactic acid bacteria fermentation of green tangerine provided in the present invention. It may include leaves, stems, roots, flowers, etc. of black radish, and the by-products may comprehensively include the extract of black radish, fractions of the extract, and the like.

The term "inflammatory disease" of the present invention refers to an inflammation-inducing factor or TNF-α (tumor necrosis factor- α), IL-1 (interleukin-1), IL-6, prostaglandin (prostagladin), leucotriene (luecotriene) or nitric oxide (nitric oxide (NO)) caused by proinflammatory substances (inflammatory cytokines) means disease.

In the present invention, the inflammatory disease is not particularly limited thereto, but as an example, it may be an allergic inflammatory disease, an inflammatory skin disease, an inflammatory eye disease, an inflammatory osteoarthritis, an inflammatory muscle disease, an inflammatory bowel disease, and the like, and other For example, allergic inflammatory diseases such as allergic rhinitis, angioedema, and allergic conjunctivitis; inflammatory skin diseases such as atopic dermatitis, psoriasis, contact dermatitis, eczematous dermatitis, actinic dermatitis, seborrheic dermatitis, dermatitis herpetiformis, lichen planus, lichen sclerosus, pyoderma gangrenosum, pemphigus, epidermolysis bullosa; inflammatory eye diseases such as blepharitis, degenerative or inflammatory blepharitis; inflammatory bone and joint diseases such as arthritis, rheumatoid arthritis, and spondylitis; inflammatory muscle diseases such as systemic sclerosis, dermatomyositis, polymyositis, and inflammatory myopathy; Or it may be an inflammatory bowel disease such as ulcerative colitis or Crohn's disease.

The term "obesity" of the present invention refers to a condition in which adipose tissue is excessive in the body in general or a body mass index (Body mass index: weight (kg) divided by the square of height (m)) is 25 or more means It is known that in individuals with symptoms of obesity, fatty acids and glucose introduced from blood plasma into adipocytes are esterified and mainly accumulated in the form of neutral fat.

The term "prevention" as used herein refers to all activities that suppress or delay the onset of inflammatory diseases or obesity by administering a pharmaceutical composition containing a fermented product of lactic acid bacteria of green tangerine provided in the present invention.

As used herein, the term "treatment" refers to all activities that improve or beneficially change inflammatory diseases or obesity by the administration of the pharmaceutical composition.

According to one embodiment of the present invention, compared to unfermented green tangerine, the fermented product of green tangerine with lactic acid bacteria not only exhibits excellent anti-inflammatory effects such as NO production inhibition, Cox-2 expression inhibition, and iNOS expression inhibition effect, but also fat globules formation It was confirmed that it exhibits excellent anti-obesity effects such as inhibition of PPARγ expression, inhibition of aP2 expression, and inhibition of C/EBP-a expression.

In addition, it was confirmed that the lactic acid bacteria fermented product using a mixture of green tangerine and black radish exhibited relatively excellent anti-inflammatory and anti-obesity effects compared to the lactic acid bacteria fermented product of green tangerine.

Therefore, it was analyzed that the lactic acid bacteria fermented green tangerine provided in the present invention has a therapeutic effect on inflammatory diseases or obesity.

The pharmaceutical composition of the present invention may include 0.0001 to 50% by weight based on the weight of the total composition, specifically 0.01% to 10% by weight, but is not limited thereto.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier, excipient or diluent commonly used in the preparation of pharmaceutical compositions, and the carrier may include a non-naturally occurring carrier. can

The term of the present invention, "pharmaceutically acceptable" means exhibiting characteristics that are not toxic to cells or humans exposed to the composition.

Specifically, the pharmaceutical composition is formulated in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories and sterile injection solutions according to conventional methods, respectively. can In the present invention, carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations contain at least one excipient such as starch, calcium carbonate, sucrose or lactose. It is prepared by mixing lactose and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid formulations for oral use include suspensions, solutions for internal use, emulsions, syrups, etc., and various excipients such as wetting agents, sweeteners, aromatics, preservatives, etc. may be included in addition to water and liquid paraffin, which are commonly used simple diluents. there is. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for the suppository, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogeratin and the like may be used.

Another aspect of the present invention provides a method for preventing or treating an inflammatory disease or obesity comprising administering the pharmaceutical composition to a subject other than a human who has or is likely to develop an inflammatory disease or obesity.

At this time, the definitions of green tangerine, lactic acid bacteria fermented product, inflammatory disease, obesity, prevention and treatment are as described above.

As used herein, the term "administration" means introducing a predetermined substance into a subject by an appropriate method.

As used herein, the term "individual" refers to all animals, such as rats, mice, livestock, and the like, including humans who have or may develop inflammatory diseases or obesity. As a specific example, it may be mammals including humans.

Specifically, the method for preventing or treating inflammatory diseases or obesity of the present invention comprises the steps of administering a pharmaceutical composition for preventing or treating inflammatory diseases or obesity containing a lactic acid bacteria fermented product of green tangerine in a pharmaceutically effective amount to a non-human subject can include

As used herein, the term "pharmaceutically effective amount" means an amount that is sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment and does not cause side effects, and the effective dose level is dependent on the gender of the patient. , age, weight, health condition, type of disease, severity, activity of drug, sensitivity to drug, method of administration, time of administration, route of administration, and excretion rate, duration of treatment, factors including drugs used in combination or concurrently, and others It can be readily determined by a person skilled in the art according to factors well known in the medical arts.

Specifically, the composition of the present invention may be administered at 0.0001 to 100 mg/kg of body weight per day, more specifically at 0.001 to 100 mg/kg of body weight, based on solid content. As for administration, the recommended dose may be administered once a day, or may be administered in several divided doses.

In the method for preventing or treating inflammatory diseases or obesity of the present invention, the administration route and administration method for administering the composition are not particularly limited, and any administration as long as the composition containing the composition can reach the target site. It may depend on the route and mode of administration. Specifically, the composition may be administered through various oral or parenteral routes, and non-limiting examples of the route of administration include oral, rectal, topical, intravenous, intraperitoneal, intramuscular, intraarterial, transdermal, nasal It may be administered intralaterally or through inhalation or the like.

Another aspect of the present invention provides a health food composition for preventing or alleviating inflammatory diseases or obesity, including the lactic acid bacteria fermented product of green tangerine.

At this time, the definitions of green tangerine, lactic acid bacteria fermented product, inflammatory disease, obesity and prevention are as described above.

The term "improvement" as used herein refers to all actions that at least reduce the parameters related to the condition to be treated, for example, the severity of symptoms, by administering the composition containing the lactic acid bacteria fermented green tangerine provided in the present invention.

The term "food" in the present invention means meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages , vitamin complexes, health functional foods and health foods, etc., and include all foods in the conventional sense.

Since the food composition of the present invention can be consumed on a daily basis, an excellent inflammatory disease or obesity improvement effect can be expected, so it can be used very usefully for health promotion purposes.

The health functional food (functional food) is the same term as food for special health use (FoSHU), and is a medicine processed to efficiently display bioregulatory functions in addition to nutritional supply, and has high medical effect. means food. Here, 'function (sex)' means obtaining useful effects for health purposes such as regulating nutrients for the structure and function of the human body or physiological functions. The food of the present invention can be prepared by a method commonly used in the art, and can be prepared by adding raw materials and ingredients commonly added in the art during the preparation. In addition, the formulation of the food may also be prepared without limitation as long as the formulation is recognized as a food. The composition for food of the present invention can be prepared in various types of formulations, and unlike general drugs, it has the advantage of not having side effects that may occur when taking drugs for a long time by using food as a raw material, and has excellent portability. The food of the invention can be consumed as an adjuvant to enhance the effect of improving inflammatory diseases or obesity.

The health food (health food) means a food having an active health maintenance or promotion effect compared to general food, health supplement food (health supplement food) means a food for the purpose of health supplement. In some cases, the terms health functional food, health food, and health supplement food are used interchangeably.

Specifically, the health functional food is a food prepared by adding the compound of the present invention to food materials such as beverages, teas, spices, chewing gum, confectionery, etc., or encapsulated, powdered, suspended, etc. It means to bring about an effect, but unlike general drugs, it has the advantage of not having side effects that can occur when taking drugs for a long time by using food as a raw material.

The food composition may further include a physiologically acceptable carrier. The type of carrier is not particularly limited, and any carrier commonly used in the art may be used.

In addition, the food composition may include additional ingredients that are commonly used in food compositions and can improve smell, taste, and vision. For example, vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, panthotenic acid, and the like may be included. In addition, minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), and chrome (Cr); and amino acids such as lysine, tryptophan, cysteine, and valine.

In addition, the food composition may include preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), bactericides (bleaching powder, high bleaching powder, sodium hypochlorite, etc.), antioxidants (butylhydroxyanisole (BHA), butyl hydroxy Loxytoluene (BHT), etc.), coloring agents (tar color, etc.), coloring agents (sodium nitrite, sodium nitrite, etc.), bleaching agents (sodium sulfite, etc.), seasonings (MSG sodium glutamate, etc.), sweeteners (dulcin, cyclemate, saccharin) , sodium, etc.), flavoring (vanillin, lactones, etc.), expanding agent (alum, D-potassium hydrogen stannate, etc.), strengthening agent, emulsifier, thickener (thickener), coating agent, gum base agent, foam inhibitor, solvent, improver, etc. food May contain food additives. The additive may be selected according to the type of food and used in an appropriate amount.

An example of the food composition of the present invention may be used as a health beverage composition, and in this case, it may contain various flavoring agents or natural carbohydrates as additional components, like conventional beverages. The aforementioned natural carbohydrates include monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; polysaccharides such as dextrins and cyclodextrins; It may be a sugar alcohol such as xylitol, sorbitol, or erythritol. Sweeteners include natural sweeteners such as thaumatin and stevia extract; Synthetic sweeteners such as saccharin and aspartame may be used. The ratio of the natural carbohydrates may be generally about 0.01 to 0.04 g, specifically about 0.02 to 0.03 g per 100 mL of the health drink composition of the present invention.

In addition to the above, the health beverage composition includes various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid, salts of pectic acid, alginic acid, salts of alginic acid, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, It may contain alcohol or a carbonating agent, and the like. In addition, it may contain fruit flesh for the manufacture of natural fruit juice, fruit juice beverages, or vegetable beverages. These components may be used independently or in combination. The ratio of these additives is not very important, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the health beverage composition of the present invention.

Another aspect of the present invention provides a feed composition for preventing or improving inflammatory diseases or obesity, comprising the lactic acid bacteria fermented product of the green tangerine.

At this time, the definitions of green tangerine, lactic acid bacteria fermented product, inflammatory disease, obesity, prevention and treatment are as described above.

The term "feed" as used herein refers to any natural or artificial diet, meal, etc. or component of said meal, intended for or suitable for consumption by livestock.

The feed may include feed additives or feed supplements.

The type of feed is not particularly limited, and feeds commonly used in the art may be used. Non-limiting examples of the feed include vegetable feeds such as grains, root fruits, food processing by-products, algae, fibers, pharmaceutical by-products, oils and fats, starches, meal or grain by-products; Animal feed such as proteins, inorganic materials, oils, mineral oils, oils, single cell proteins, zooplankton, or food may be mentioned. These may be used alone or in combination of two or more.

Another aspect of the present invention provides the use of a fermented product of lactic acid bacteria of green tangerine for the preparation of a pharmaceutical composition for preventing or treating inflammatory diseases or obesity.

Since the lactic acid bacteria fermented product of green tangerine provided in the present invention can exhibit anti-inflammatory and anti-obesity effects, it will be widely used in the development of safe anti-inflammatory and anti-obesity drugs using natural products.

1 is a graph showing the results of comparing the total polyphenol content and total flavonoid content measured in green tangerine, black radish, or each fermented product of these mixtures.
Figure 2 is a graph showing the results of comparing the cytotoxicity level of each fermented product of green tangerines, black radishes, or mixtures thereof.
Figure 3 is a photomicrograph and graph showing the results of comparing the fat globule formation inhibitory ability of each fermented green tangerine or a mixture of green tangerine and black radish, Figure 3a shows the fermented green tangerine, Figure 3b shows the green tangerine extract, It shows the mixed fermented product of green tangerine and black radish, and FIG. 3d shows the results of quantitative analysis thereof.
4 is a Western blot analysis result showing the results of comparing the expression levels of aP2, PPAR-r, and C/EBP-a in adipocytes treated with green tangerine or a mixture of green tangerine and black radish.
Figure 5 is a quantitative analysis graph of the results of Western blot analysis showing the results of comparing the expression levels of aP2, PPAR-r, and C/EBP-a in adipocytes treated with green tangerine or a mixture of green tangerine and black radish. , Fig. 5a shows aP2, Fig. 5b shows PPAR-r, and Fig. 5c shows C/EBP-a.
Figure 6 is a graph showing the results of comparing the NO production inhibitory effect of each fermented product of green tangerine or a mixture of green tangerine and black radish.
FIG. 7 is a Western blot analysis result showing the results of comparing the expression levels of Cox-2 and iNOS in RAW264.7 cells treated with green tangerine or each fermented product of green tangerine and black radish mixture.
8 is a quantitative analysis graph of the results of Western blot analysis showing the result of comparing the expression level of Cox-2 in RAW264.7 cells treated with green tangerine or each fermented product of green tangerine and black radish mixture.
9 is a graph of quantitative analysis of the results of Western blot analysis showing the results of comparison of iNOS expression levels in RAW264.7 cells treated with green tangerine or each fermented product of green tangerine and black radish mixture.
10 is an HPLC analysis result showing the result of measuring the content change of flavonoids before and after fermentation of green tangerine or a mixture of green tangerine and black radish.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are intended to illustrate the present invention by way of example, and the scope of the present invention is not limited to these examples.

Example 1: Preparation of green tangerine nutrient source

Green tangerines were washed with purified water, washed once with sterilized distilled water, wet-ground, and mixed with the same amount of purified water.

Example 2: Preparation of green tangerine lactic acid bacteria fermented product

Example 2-1: Distribution of lactic acid bacteria

Lactobacillus plantarum was purchased from the Korea Culture Center of Microorganisms (KCCM) and used in the experiment.

Example 2-2: Seed culture

The strain received in Example 2-1 was subcultured to slope medium and liquid medium and used in the experiment. Specifically, lactic acid bacteria were inoculated into a slanted medium and cultured in a 37 ° C incubator for 24 hours, and when a lactic acid bacteria colony was formed, it was inoculated into a liquid medium and cultured in a 37 ° C incubator for 48 hours. MRS medium (Difco, USA) was used for seed culture.

Example 2-3: Preparation of green tangerine lactic acid bacteria fermented product

To prepare green tangerine lactobacillus fermented product, green tangerines washed with purified water were wet-pulverized, mixed with the same amount of purified water, adjusted to pH 7.0, and then inoculated with a pre-cultured seed culture medium at 0.7% to 1.0%. Liquid fermentation was performed by shaking culture for 36 hours in a 37 ° C incubator. After fermentation, it was lyophilized and powdered.

Example 3: Preparation of green tangerine extract

A 15-fold mixture of 80% alcohol was added to the freeze-dried green tangerine fermentation product, and primary extraction was performed at room temperature for 24 hours. After the first extraction, the same extraction solvent as in the first extraction was added 10 times to the residue remaining after filtration, and the second extraction was performed at the same temperature for 24 hours, followed by filtration. The primary extract and the secondary extract were mixed, concentrated, and lyophilized to powder.

Example 4: Total polyphenol compound and total flavonoid compound content

The contents of total polyphenolic compounds and total flavonoid compounds, which can be seen as indirect indicators of antioxidant efficacy, were measured. The total polyphenol content was determined by mixing 100 ul of Folin-denis reagent (Sigma, USA) with 20 ul of the sample according to the Folin-denis method (9), reacting at 40 ° C for 1 minute, and then adding 80 ul of 7.5% Na ₂ CO ₃ solution. was added and reacted at 40 °C for 15 minutes. After completion of the reaction, absorbance was measured at 765 nm. The total polyphenol content was expressed as gallic acid equivalent (GAE) by substituting the absorbance of the sample into the standard curve prepared in the same way using the gallic acid standard in advance. The experiment was repeated three times, and significance was confirmed at the p<0.05 level through Duncan analysis.

The content of total flavonoid compounds was measured according to the method of Davis (10). After mixing 200 ul of diethylene glycol and 20 ul of 1N NaOH in 20 ul of the sample, reacting at 37 ° C for 1 hour, the absorbance was measured at 420 nm. The total flavonoid content was expressed as quercertin equivalent (QE) by substituting the absorbance of the sample into the standard curve prepared in the same way using the quercertin standard in advance. The experiment was repeated three times, and significance was confirmed at the p<0.05 level through Duncan analysis.

Total polyphenol content and total flavonoid content are shown in Figure 1. As a result of measuring the total polyphenolic compound content, the green tangerine fermented extract showed the highest content, followed by the green tangerine-black radish mixed fermented extract. In addition, the total flavonoid content was higher in fermented green tangerine and fermented green tangerine-black radish mixture than in unfermented material (Fig. 1).

Example 5: Efficacy evaluation of inhibiting fat formation

In this experiment, cytotoxicity was measured to set an appropriate concentration of the sample to evaluate liver function improvement efficacy of fermented black radish lactic acid bacteria, and fat formation inhibitory efficacy of 3T3-L1 cells was evaluated.

Example 5-1: Measurement of cytotoxicity and cell proliferation effect of mixed fermented extract of green tangerine-black radish

Before the experiment, the MTT reagent was used after dissolving Thizolyl blue tetrazolium bromide (Sigma, USA) in 1xPBS to a final concentration of 5 mg/ml and blocking light. To measure cytotoxicity, cells were dispensed in a 96-well plate at 1x10 ⁵ cells/ml, cultured for 24 hours, and each sample dissolved in the medium was treated by concentration (100 ug/ml, 500 ug/ml) and cultured for 24 hours. . After incubation, MTT reagent was added at a ratio of about 10:1 and allowed to react for 3 hours. After removing the culture medium, 100 μl of DMSO was dispensed to dissolve MTT formazan, and absorbance was measured at 540 nm, and cell viability was measured by substituting the absorbance into the following formula. The experiment was repeated three times, and significance was confirmed at the p<0.05 level through Duncan analysis. The measurement results are shown in FIG. 2 .

As a result of the measurement, no significant toxicity was observed for 3T3-L1 cells at each sample concentration of 1000, 500, 250, and 125ug/ml.

Cell viability (%) = absorbance of sample/absorbance of control x 100

In addition, 1000, 500, 250,125ug/ml of green tangerine-black radish mixed extract was treated and cell viability was analyzed at 24 and 48 hours. As a result, 141% (1000ug/ml), 103% (500ug/ml), 111% (250ug/ml), and 105% (125ug/ml) of cell proliferation effect were shown compared to the control at 48 hours. In addition, green tangerine lactobacillus fermented products also increased by 104% (1000ug/ml), 114% (500ug/ml), 123% (250ug/ml), and 105% (125ug/ml) compared to the control. These results suggest that lactic acid fermentation removes cytotoxicity and induces a proliferative effect.

Example 5-2: Efficacy evaluation of inhibition of fat formation

First, 3T3-L1 cells were dispensed in a 24-well plate at 2x10 ⁵ cells/ml and cultured for 2 days. Differentiation into adipocytes was induced by treatment with MDI (3-isobutyl-1-methylxanthine, Dexamethasone, Insulin). After 2 days, the existing medium was removed and replaced with a DMEM/10% FBS culture medium containing 10 μg/ml insulin and cultured for 2 days, and then cultured with DMEM/10% FBS culture medium. In addition, each sample was treated at 1000, 500, and 250 ug/ml concentrations at intervals of 2 days and cultured for 6 days. For staining, after removing the culture medium, washing twice with 1xPBS, treating with 10% formalin diluted in formaldehyde solution (Sigma, USA) at room temperature for more than 1 hour, removing formalin, washing twice with distilled water, and then using Oil-Red O ( ORO) (Sigma, USA) solution at room temperature for 30 minutes. After removing the ORO solution, it was washed 4 times with distilled water, and the dye stained in fat globules was dissolved with isopropanol (Sigma, USA), transferred to a 96-well plate, and absorbance was measured at 490 nm. The fat globule formation rate was calculated by substituting the absorbance into the following formula. The experiment was repeated three times, and significance was confirmed at the p<0.05 level through Duncan analysis. The measurement results are shown in Figure 3.

As a positive control group, fat globules were formed in the MDI-treated group. Fermented green tangerine and black radish fermented extract showed 10% inhibitory effect. The green tangerine lactobacillus fermented product had a significantly lower rate of formation of fat globules compared to the MDI-treated group and was equivalent to that of the non-treated group. In addition, the inhibition rate of fat globules was 70% and 40% in 1000 and 500ug/ml treatment of green tangerine-black radish mixed lactobacillus fermented extract, respectively. Therefore, the green tangerine fermented extract and the green tangerine-black radish mixed fermented extract showed significant inhibitory effects at 1000 and 500ug/ml of fat globule formation, but showed a low inhibition rate at 250ug/ml treatment.

Fat globules formation rate (%) = absorbance of sample/absorbance of control x 100

Example 5-3: Inhibition of aP2, PPAR-r, and C/EBP-a protein expression in adipocyte model

The results of analyzing the expression levels of aP2, PPAR-r, and C/EBPa using western blot after protein extraction from the cells of Example 5 are shown in FIG. 4 . Expressions of aP2, PPAR-r, and C/EBPa were inhibited in the treatment group of black radish and green tangerine, but there was no significant difference. The aP2 expression inhibition rate was suppressed by 90~40% in the 1000, 500, and 250ug/ml treatment groups of green tangerine lactobacillus fermentation and green tangerine-black radish mixed lactobacillus fermentation. Inhibition of PPAR-r expression was suppressed by nearly 100 and 50% in the treatment of 1000 and 500ug/ml of green tangerine lactobacillus fermented product. However, it was not suppressed in the 250ug/ml treatment. In the green tangerine-black radish mixed fermented product treatment, 40% (1000ug/ml), 30% (500ug/ml), and 20% (250ug/ml) were inhibited. This result suggests that lactic acid bacteria fermentation inhibits adipogenesis factors by the synergistic effect of black radish and green tangerine. In the C/EBP-a expression inhibition assay, it was strongly inhibited in the fermentation of green tangerine lactic acid bacteria and in the fermentation of mixed lactobacillus of green tangerine-black radish. However, green tangerine-black radish mixed lactic acid bacteria fermentation 250ug/ml treatment did not inhibit it. Therefore, the result of suppressing the expression of the adipogenesis transcription factor and the inhibition of the formation of fat globule (lipid content) were consistent with the fermented product of green tangerine lactobacillus and the mixed fermented product of green tangerine-black radish lactic acid bacteria. Therefore, it was proved that it is an anti-agonist of PPAR-r and C/EBPa transcription factors. The above fermented product can be seen to be involved in anti-obesity and inhibition of differentiation from preadipocytes to adipocytes.

Example 6: Confirmation of inhibition of NO secretion in inflammatory model cells

RAW264.7 cells (mouse macrophages), an inflammatory cell model, were treated with LPS (500ng/ml), green tangerine extract, black radish lactobacillus fermented extract, green radish lactobacillus fermented extract, and black radish-green tangerine mixed lactobacillus fermented extract at various concentrations. Formation of nitric oxide (NO) was confirmed using Griess reagent and is shown in FIG. 8 . Inhibition of NO production was 17.2% and 4.4%, respectively, in the treatment of green tangerine extract and fermented black radish extract 1000ug/ml. However, 51, 451.%, 65.3, and 51.4% of inhibitory effects were shown in the treatment of green tangerine lactobacillus fermentation product and green tangerine-black radish lactobacillus fermentation product 1000, 500ug/ml. The green tangerine fermented product showed an inhibitory effect of 300% compared to the non-fermented product, and the mixed lactic acid bacteria fermented product (black radish-green tangerine) showed an inhibitory effect of more than 400% (FIG. 6).

Example 6-1: Inhibition of Cox-2 and iNOS expression of green tangerine lactobacillus fermented product and green tangerine-black unmixed lactobacillus fermentation in inflammatory model cells

Recently, it has been reported that inflammatory factors rapidly increase when RAW264.7 cells are stimulated with LPS (lipopolysaccharide) as an inflammatory cell model. Cox-2 (cyclooxygenase-2) and iNOS (nitrix oxide synthesis) are representative proteins that form inflammatory factors. When inflammatory factors LPS, interferon-r, and various pathogenic microorganisms are infected, cox-2, iNOS, PGE2, and inflammatory cytokines (IL1b, TNF-a, IL6, etc.) rise rapidly.

As shown in the graph of FIG. 9, compared to the group treated with only LPS, COX-2 protein expression was very specifically inhibited by the treatment with 1000 and 500 ug/ml of green tangerine extract. However, there was no inhibition in the black radish fermented extract treatment. In addition, the fermented extract of green tangerine lactobacillus showed an inhibitory effect in a concentration-dependent manner. In the treatment of 1000, 500, and 250ug/ml of green tangerine fermented extract, 80, 70, and 40% inhibitory effects were shown. In addition, 70, 50, and 30% of inhibitory effects were shown in 1000, 500, and 250ug/ml of green tangerine-black radish mixed lactobacillus fermented product treatment group. In particular, the black radish lactic acid bacteria fermented product showed no inhibitory effect, but the mixed fermented product showed an inhibitory effect of more than 80%, showing a synergistic effect with the mixed fermented product (Figs. 7 and 8).

In the iNOS protein effect study, as shown in FIG. 10, 18% and 0% inhibition rates were shown in the treatment of 1000 and 50ug/ml green mandarin extract, and almost no inhibitory effect was found in the fermented black radish. However, 1000, 500, and 250ug/ml of green tangerine lactic acid bacteria fermentation showed 80, 60, and 40% inhibitory effects. In addition, green tangerine-black radish mixed lactobacillus fermented product showed the same inhibitory effect as green tangerine lactobacillus fermented product. This result was consistent with the effect of inhibiting NO production. This result shows that the green tangerine lactobacillus fermented product and the green tangerine-black radish mixed lactobacillus fermented extract in Examples 8 and 9 have a strong anti-inflammatory effect, and are therefore worthy of development as food and anti-inflammatory drugs. In particular, in the fermented product of the mixed extract, it is the first composition that has a synergistic effect of fermentation of black radish and green tangerine by lactic acid bacteria, which strongly suppresses inflammation despite having about 1/2 the content of green tangerine component (FIG. 9).

Example 7: flavonoid glycoside change by bioconversion

We analyzed HPLC to confirm the flavonoid changes before and after fermentation in three extracts (green tangerine, green tangerine lactobacillus fermented extract, and green tangerine-black radish mixed fermented extract). HPLC analysis was performed on each extract. For analysis preparation, the extract was dissolved at 100 mg/mL in DMSO, diluted to 10 mg/mL with ethanol, and then used for analysis. For HPLC (High performance liquid chromatography), Alliance 2695-2998 system (Waters Co., Massachusetts, USA) was used.

Nine peaks were selected through HPLC analysis and the area of each peak before and after fermentation was confirmed.

Except for peaks 7 and 8, it decreased between 25 and 51%. In particular, peaks 2 and 3 were reduced by 34% and 51%. This result shows that hydrophilic flavonoid compounds, that is, flavonoids with glycosides, peak at an early stage, and flavonoid-type compounds with glycosides removed appear in the latter half. Therefore, peaks 2 and 3 in the unfermented green tangerine decreased by 37 and 57%, and peaks 7 and 8 increased by 134% and 214%, respectively. This result suggests that peak 2 and 3 are changed to peka 7 and 8 by deglycosylation, dihydroxylation, and O-methylation, such as glucosidase and rhanosidase secreted by lactic acid bacteria, in flavonoids attached with aglycones. These peaks could be expected as the main component of green tangerines, and four lactobacilli (Lactobacillus plantarum, Lactobacillus casei, Lactobacillus helveticus, Lactobacillus reuteri) ) and the main peakd that appears during fermentation. In addition, it was confirmed that peaks 7 and 8 were significantly increased in the green tangerine-black radish mixed fermented product, and peka 8 in particular increased by more than 250%. Therefore, it was confirmed that the above peak was analyzed in common with lactic acid bacteria fermentation (Fig. 11). Although peaks 7 and 8 compounds have not yet been analyzed for their definite structure, they can be represented as off-label components in this fermentation study (FIG. 10).

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 embodiments 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.

Claims (12)

A food composition for improving obesity comprising fermented green tangerine with lactic acid bacteria.
According to claim 1,
The green tangerine is a food composition that is all, part or a by-product of green tangerine.
According to claim 1,
The lactic acid bacteria fermented product is a food composition obtained by inoculating lactic acid bacteria into raw materials containing green tangerines and fermenting them.
According to claim 3,
The lactic acid bacteria are Lactobacillus sp. strains, Lactococcus sp. strains, Enterococcus sp. strains, Streptococcus sp. strains, and Pediococcus sp. .) strains, Leuconostoc sp. strains, Bifidobacterium sp. strains, and strains selected from the group consisting of combinations thereof, a food composition.
According to claim 3,
The temperature at which the fermentation is carried out is 30 to 37 ℃, the food composition.
According to claim 3,
The fermentation time is 20 to 50 hours, the food composition.
According to claim 3,
The raw material is a food composition that further comprises black radish.
According to claim 7,
The black radish is a food composition that is the whole of black radish, a part or a by-product thereof.
According to claim 1,
Wherein the food composition further comprises a physiologically acceptable carrier.
delete delete A feed composition for improving obesity, comprising a fermented product of lactic acid bacteria of green tangerines.

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