Composition for preventing, ameliorating or treating sepsis comprising black ginseng and black Lycium chinense mixed fermented product as effective component

Abstract

The present invention relates to a composition for the prevention, improvement, or treatment of sepsis comprising a mixed fermented product of black ginseng and black goji berries as an active ingredient. The mixed fermented product of black ginseng and black goji berries has almost no cytotoxicity, reduces the production of inflammatory factors in macrophages, lowers body temperature and increases survival rates in animal models of sepsis, and has the effect of reducing the content of inflammatory factors in the serum; therefore, it can be usefully used for the prevention, improvement, or treatment of sepsis.

Description

Technology Field

[0001] The present invention relates to a composition for the prevention, improvement, or treatment of sepsis comprising a mixed fermented product of black ginseng and black goji berries as an active ingredient. Background Technology

[0002] Sepsis refers to a condition in which a severe systemic inflammatory response occurs due to microbial infection. The primary cause of sepsis is microbial infection. Pathogens that cause sepsis include Streptococcus, Staphylococcus, Escherichia coli, Pneumococcus, Pseudomonas aeruginosa, fungi, and Klebsiella pneumoniae. The site of infection can be any organ in the body, and conditions such as pneumonia, pyelonephritis, meningitis, cellulitis, infectious endocarditis, peritonitis, pressure ulcers, cholecystitis, and cholangitis can lead to sepsis. When these infections occur, the causative microorganisms can invade the bloodstream and cause sepsis. However, systemic sepsis can also occur even without microorganisms penetrating the bloodstream, driven by inflammatory responses and the production of inflammatory substances in specific parts of the body.

[0003] Currently, antibiotics and steroids are administered in combination or as adjuvants to treat sepsis; however, the development of treatments that demonstrate a significant clinical effect on mortality remains insufficient. Since one of the clinical characteristics of sepsis progression is the overactivation of the systemic inflammatory system, numerous attempts have been made to treat sepsis by focusing on inhibitors or suppressors capable of inhibiting inflammatory mediators such as TNF-α, Interleukin-1 (IL-1), Interleukin-6 (IL-6), and HMGB1, which involve or promote such inflammatory responses. However, these efforts have failed to improve patient survival rates, and to date, there are almost no cases of drugs successfully passing clinical trials and being developed as sepsis treatments. Therefore, to prevent or treat sepsis, it is crucial not only to suppress the concentration of inflammatory cytokines but also to improve the mortality rate—that is, the survival rate—caused by sepsis. Furthermore, while the inflammatory process generally serves as a defense mechanism against pathogenic factors, it can induce pain even in the absence of stimulation by toxic substances. Local inflammatory responses release pro-inflammatory cytokines and upregulate cyclooxygenase-2 to synthesize prostaglandins. This sensitizes primary neurons, activating neuroimmunity by sensitizing surrounding neurons. Consequently, thermal or mechanical hypersensitivity ultimately occurs.When the aforementioned inflammatory response occurs, it is accompanied by pain. This pain can be distinguished into nociceptive pain, which is a type of warning sensation caused by harmful stimuli that damage or potentially damage tissues; inflammatory pain, which occurs when inflammatory mediators such as IL-1β, activated and secreted from damaged tissues and surrounding tissues, act on nociceptors; and neuropathic pain, which persists even in response to non-harmful stimuli due to self-damage to the peripheral and central nervous systems.

[0004] Meanwhile, fresh ginseng undergoes a red ginseng stage through multiple steaming and drying processes to acquire a red color, and red ginseng is transformed into black ginseng by steaming and drying it multiple times again. It is known that red and black ginseng contain superior active ingredient content compared to fresh ginseng due to changes in its pharmacological effects resulting from the enrichment of functional ginsenosides through these repeated steaming and drying processes. Similarly, in the case of black ginseng, it is known that the process of steaming and drying red ginseng multiple times further enhances trace ginsenoside components, including ginsenoside Rg3, which are present in small amounts in red ginseng. Ginsenosides are saponins found in ginseng that have recently begun to receive attention as physiologically active substances following the discovery of their anticancer, antioxidant, and cholesterol-lowering effects. Ginseng saponins have a unique chemical structure distinct from saponins found in other plants and possess unique pharmacological effects, so they are called 'ginsenosides,' meaning ginseng glycosides.

[0005] Goji berries are the name of the fruit, while the tree is called the goji berry tree. In pure Korean, it was formerly known as the "Gwaejotnamu" or "Mulgochunamu," but it is now commonly referred to as the goji berry tree, named after the fruit, due to the sound of the names. It is widely distributed throughout Asia, including Korea. It has been used for various purposes across Asia, ranging from a snack to a precious medicinal herb, and is one of the plants believed to be the "elixir of immortality" mentioned in the Ten Symbols of Longevity, alongside Reishi mushrooms. Goji berry fruits are said to be very beneficial for eyesight, and are particularly recommended when added to Samgyetang (chicken soup). The fruit is famous as a tonic, and in Korea, it has been used as an aphrodisiac since ancient times. However, due to its cold nature, excessive consumption can cause severe diarrhea; therefore, it is advised not to use it alone but to combine it with complementary herbs. Even when consuming dried goji berries, excessive amounts should be avoided, and it is recommended to eat them with warm tea. Every part—leaves, fruit, and root bark—is used as both a medicinal and culinary ingredient. The fruit is known as goji berry, and the root bark is called Jigolpi, both of which are traditional Korean medicinal herbs. Key benefits include improved immune function, prevention of brain cell damage, skin health, eye health, blood sugar levels, and liver function.

[0006] Regarding technologies related to sepsis, Korean Registered Patent No. 1363933 discloses a composition for improving Vibrio sepsis containing Terminalia chebula extract and Ligusticum chuanxiong extract as active ingredients, and Korean Registered Patent No. 2368983 discloses a polypeptide having antibacterial activity, a composition for preventing or treating sepsis containing the same, and an antibacterial composition; however, there has not yet been any disclosure of a composition for preventing, improving, or treating sepsis containing a mixed fermented product of black ginseng and black Lycium barbarum of the present invention as an active ingredient. The problem to be solved

[0007] The present invention was derived from the above-mentioned requirements. The present invention provides a composition for the prevention, improvement, or treatment of sepsis comprising a mixed fermented product of black ginseng and black goji berries as an active ingredient. The invention was completed by confirming that the mixed fermented product of black ginseng and black goji berries has almost no cytotoxicity, reduces the production of inflammatory factors in macrophages, lowers body temperature and increases survival rates in a sepsis animal model, and has the effect of reducing the content of inflammatory factors in the serum. means of solving the problem

[0008] To achieve the above objective, the present invention provides a health functional food composition for the prevention or improvement of sepsis comprising a mixed fermented product of black ginseng and black goji berries as an active ingredient.

[0009] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of sepsis comprising a mixed fermented product of black ginseng and black goji berries as an active ingredient.

[0010] In addition, the present invention provides a feed additive for the prevention or improvement of sepsis comprising a mixed fermented product of black ginseng and black goji berries as an active ingredient.

[0011] In addition, the present invention provides a veterinary composition for the prevention or treatment of sepsis comprising a mixed fermented product of black ginseng and black goji berries as an active ingredient.

[0012] In addition, the present invention provides a herbal medicine composition for the prevention or treatment of sepsis comprising a mixed fermented product of black ginseng and black goji berries as an active ingredient. Effects of the invention

[0013] The present invention was derived from the above-mentioned needs, and the present invention relates to a composition for the prevention, improvement, or treatment of sepsis comprising a mixed fermented product of black ginseng and black goji berries as an active ingredient. The mixed fermented product of black ginseng and black goji berries has almost no cytotoxicity, reduces the production of inflammatory factors in macrophages, lowers body temperature and increases survival rate in a sepsis animal model, and has the effect of reducing the content of inflammatory factors in the serum. Brief explanation of the drawing

[0014] Figure 1 shows 1×10⁶ RAW 264.7 macrophages. 9 (A), 2×10 9 (B) and 5×10 9 (C) This is the result of an MTT assay confirming the cell viability of a mixed fermented product of black ginseng and black goji berries prepared by treatment with CFU / g of complex lactic acid bacteria. Figure 2 shows 1×10⁶ RAW 264.7 macrophages. 9 (A), 2×10 9 (B) and 5×10 9 (C) This is the result of confirming the change in NO production amount following treatment with a mixed fermented product of black ginseng and black goji berries prepared by treating with a complex of lactic acid bacteria of CFU / g. *, **, and *** indicate that the NO production amount of the experimental group treated with the mixed fermented product of black ginseng and black goji berries of the present invention was statistically significantly reduced compared to the control group (0) in which inflammation was induced by treatment with LPS, where * is p<0.05, ** is p<0.01, and *** is p<0.001. Figure 3 shows 1×10⁶ RAW 264.7 macrophages. 9 (A), 2×10 9 (B) and 5×10 9(C) These are the results of confirming the changes in TNF-α(A) and IL-6(B) content following treatment with a mixed fermented product of black ginseng and black goji berries prepared by treating with CFU / g of complex lactic acid bacteria. *, **, and *** indicate that the content of TNF-α or IL-6 in the experimental group treated with the mixed fermented product of black ginseng and black goji berries of the present invention was statistically significantly reduced compared to the control group (0) in which inflammation was induced by treatment with LPS, where * is p<0.05, ** is p<0.01, and *** is p<0.001. Figure 4 is a Western blot result confirming the change in expression levels of APK signaling pathway activators (p38, ERK, and JNK) following treatment of RAW 264.7 macrophages with a mixed fermented product of black ginseng and black goji berries. Figure 5 is an ELISA result confirming the change in IL-1β expression levels in bone marrow-derived macrophages (BMDM) after treatment with a mixed fermented product of black ginseng and black goji berries. ** and *** indicate that the IL-1β expression levels of the experimental group treated with the mixed fermented product of black ginseng and black goji berries of the present invention were statistically significantly reduced compared to the control group (0) in which inflammation was induced by treatment with LPS, where ** is p<0.01 and *** is p<0.001. Figure 6 shows the change in body temperature following the administration of a mixed fermented product of black ginseng and black goji berries to an animal model of LPS-induced sepsis. # indicates that the body temperature of the sepsis-induced group (LPS) increased statistically significantly compared to the normal group (N), with p<0.05. * and ** indicate that the body temperature of the experimental group administered the mixed fermented product of black ginseng and black goji berries of the present invention decreased statistically significantly compared to the sepsis-induced group (LPS), with * being p<0.05 and ** being p<0.01. Figure 7 shows the change in survival rate following the administration of a mixed fermented product of black ginseng and black goji berries to an animal model of LPS-induced sepsis. ### indicates that the survival rate of the sepsis-induced group (LPS) was statistically significantly reduced compared to the normal group (N), with p<0.001. * and ** indicate that the survival rate of the experimental group administered the mixed fermented product of black ginseng and black goji berries of the present invention was statistically significantly increased compared to the sepsis-induced group (LPS), with * being p<0.05 and ** being p<0.01. Figure 8 shows the results of confirming changes in the levels of serum inflammatory factors IL-6 (A), TNF-α (B), and IL-1β (C) following the administration of a mixed fermented product of black ginseng and black goji berries to an animal model of LPS-induced sepsis. ### indicates that the levels of IL-6, TNF-α, or IL-1β in the sepsis-induced group (LPS) increased statistically significantly compared to the normal group (N), with p<0.001. * and ** indicate that the levels of IL-6, TNF-α, or IL-1β in the experimental group administered the mixed fermented product of black ginseng and black goji berries of the present invention decreased statistically significantly compared to the sepsis-induced group (LPS), with * being p<0.05 and ** being p<0.01. Specific details for implementing the invention

[0015] The present invention relates to a health functional food composition for the prevention or improvement of sepsis comprising a mixed fermented product of black ginseng and black goji berries as an active ingredient.

[0016] The above-mentioned mixed fermented product of black ginseng and black goji berries is characterized by mixing black ginseng extract and black goji berry extract and then fermenting it with lactic acid bacteria. The above-mentioned black ginseng extract or black goji berry extract comprises: (1) a step of preparing black ginseng or black goji berries by repeating a steaming process 3 to 9 times in which ginseng or goji berry fruits are steamed at 85 to 95°C for 4 to 6 hours and dried at 45 to 55°C for 4 to 6 hours, and then pulverizing them; and

[0017] (2) A step of extracting by adding 8 to 12 parts by weight of an extraction solvent to 1 part by weight of black ginseng or black goji berry powder prepared in step (1) above; is preferred, but not limited thereto.

[0018] In step (2) above, the extraction solvent is preferably water, a C1-C4 lower alcohol, or a mixture thereof, and more preferably water, but is not limited thereto.

[0019] In the above manufacturing method, the extraction method may utilize any conventional method known in the art, such as filtration, hot water extraction, immersion extraction, reflux cooling extraction, and ultrasonic extraction. The extraction temperature is preferably 20 to 110°C, more preferably 80 to 100°C, but is not limited thereto. Additionally, the extraction time is preferably 1 to 48 hours, more preferably 1 to 24 hours, and most preferably 8 hours, but is not limited thereto. Furthermore, drying is preferably performed by reduced pressure drying, vacuum drying, boiling drying, spray drying, or freeze-drying, but is not limited thereto.

[0020] The above lactic acid bacteria is Lactobacillus plantarum ( L. plantarum ), Lactobacillus acidophilus ( L. acidophilus ), Bifidobacterium animalis lactis ( Bi. animalis ssp. lactis ), Lactococcus lactis( Lc.lactis ), Lactobacillus reuteri( L. reuteri ), Streptococcus thermophilus( S. thermophilus ), Lactobacillus rhamnosus ( L. rhamnosus ), Lactobacillus paracasei( L. paracasei ), Bifidobacterium longum( Bi. longum ), Lactobacillus casei( L. casei ), Lactobacillus bulgaricus( L. bulgaricus ), Lactobacillus fermentum( L. fermentum ), Bifidobacterium bifidum ( Bi. bifidum ), Bifidobacterium breve ( Bi. breve ), Lactobacillus gasseri( L. gasseri), Lactobacillus salivarius( L. salivarius ) and Lactobacillus helveticus ( L. helveticus It is preferable that one or more selected from ) are used, and most preferably, the above 17 types of complex lactic acid bacteria are used, but are not limited thereto.

[0021] The above-mentioned fermented mixture of black ginseng and black goji berries increases the survival rate reduced due to sepsis and reduces the production or expression of inflammatory cytokines increased due to sepsis, and the sepsis is characterized as bacterial sepsis caused by lipopolysaccharide present in the outer membrane of Gram-negative bacteria.

[0022] The above composition is preferably prepared in any one formulation selected from powder, granules, pills, tablets, capsules, candy, syrup, and beverage, but is not limited thereto.

[0023] The health functional food composition of the present invention may be prepared by adding the mixed fermented product of black ginseng and black goji berries as is, or by mixing it with other foods or food ingredients, and may be appropriately prepared according to conventional methods. Examples of foods to which the mixed fermented product of black ginseng and black goji berries may be any one form selected from caramel, meat, sausage, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and include all health functional foods in the conventional sense. That is, there are no special restrictions on the types of foods. The above health functional food composition may contain various nutritional supplements, vitamins, minerals (electrolytes), synthetic and natural flavoring agents, coloring agents and promoters (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. Additionally, it may contain fruit pulp for the production of natural fruit juices and vegetable beverages. The above ingredients may be used independently or in combination. Furthermore, the health functional food composition of the present invention may contain various flavoring agents or natural carbohydrates, etc. as additional ingredients, and the natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. The proportion of the above natural carbohydrates is not of great importance, but it is preferable that it be 0.01 to 0.04 g per 100 g of the composition of the present invention, and more preferably 0.02 to 0.03 g, but is not limited thereto. As a sweetener, natural sweeteners such as taumatin and stevia extract, synthetic sweeteners such as saccharin and aspartame may be used.

[0024] In addition, the present invention relates to a pharmaceutical composition for the prevention or treatment of sepsis comprising a mixed fermented product of black ginseng and black goji berries as an active ingredient.

[0025] In addition to the above-mentioned fermented mixture of black ginseng and black goji berries, pharmaceutically acceptable carriers, excipients, or diluents may be further included. Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are those commonly used in formulations and include, but are not limited to, saline solution, sterile water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0026] In addition, in addition to the above components, the composition may further include antioxidants, buffers, bacteriostatic agents, diluents, surfactants, binders, lubricants, humectants, sweeteners, flavorings, emulsifiers, suspending agents, or preservatives. Suitable dosages of the pharmaceutical composition of the present invention may be prescribed in various ways depending on factors such as the formulation method, mode of administration, age, body weight, sex, pathological condition of the patient, food, time of administration, route of administration, excretion rate, and response sensitivity.

[0027] The pharmaceutical composition for the prevention or treatment of retinal disease of the present invention may be administered via any general route as long as it can reach the target tissue. The pharmaceutical composition of the present invention is not particularly limited thereto, but may be administered via routes such as oral administration, ophthalmic administration, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, or transdermal patch administration, depending on the purpose, and preferably orally or ophthalmicly.

[0028] In addition, the present invention relates to a feed additive for the prevention or improvement of sepsis comprising a mixed fermented product of black ginseng and black goji berries as an active ingredient.

[0029] The feed additive of the present invention corresponds to an auxiliary feed under the Feed Management Act. In the present invention, the term "feed" may refer to any natural or artificial prescribed food, single meal, etc., or the components of said single meal, intended for or suitable for animals to eat, consume, and digest. The types of said feed are not particularly limited, and feeds commonly used in the relevant technical field may be used. Non-limiting examples of said feed include plant-based feeds such as grains, root vegetables, food processing by-products, algae, fibers, pharmaceutical by-products, oils and fats, starches, meal, or grain by-products; and animal-based feeds such as proteins, inorganic substances, oils and fats, minerals, single-cell proteins, zooplankton, or food waste. These may be used individually or in a mixture of two or more types.

[0030] In addition, the present invention relates to a veterinary composition for the prevention or treatment of sepsis comprising a mixed fermented product of black ginseng and black goji berries as an active ingredient. The veterinary composition of the present invention may further include appropriate excipients and diluents according to conventional methods. Examples of excipients and diluents that may be included in the veterinary composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, ethanol, stearyl alcohol, liquid paraffin, sorbitan monostearate, polysorbate 60, methylparaben, propylparaben, and mineral oil. The veterinary composition according to the present invention may further include fillers, anticoagulants, lubricants, humectants, spices, emulsifiers, preservatives, etc. The veterinary composition according to the present invention may be formulated using methods well known in the art to provide rapid, sustained, or delayed release of the active ingredient after administration to an animal, and the formulation may be in the form of powders, granules, tablets, capsules, suspensions, emulsions, solutions, syrups, aerosols, soft or hard gelatin capsules, suppositories, sterile injectable solutions, sterile topical preparations, etc. The effective amount of the veterinary composition according to the present invention may be appropriately selected according to the individual animal. It may be determined according to the severity of the disease or condition, the age, weight, health status or sex of the individual, sensitivity to the active ingredient of the present invention, the route of administration, the duration of administration, factors including other compositions combined with or used simultaneously with the composition, and other factors well known in the physiological or veterinary field.

[0031] In addition, the present invention relates to a herbal medicine composition for the prevention or treatment of sepsis comprising a mixed fermented product of black ginseng and black goji berries as an active ingredient.

[0032] The herbal medicine composition according to the present invention refers to a conventional dried medicinal material used in the manufacture of herbal medicine. The herbal medicine composition of the present invention may be in the form of a powder obtained by fermenting and drying an extract obtained by extracting water, C1-C4 lower alcohols, or mixtures thereof using an extraction solvent selected from water, C1-C4 lower alcohols, or mixtures thereof according to conventional methods, and there are no particular restrictions on the formulation. For example, it may be manufactured in the form of pills, tablets, capsules, or liquids.

[0033] The present invention will be described in more detail below using examples. These examples are solely for the purpose of more specifically explaining the present invention, and it is obvious to those skilled in the art that the scope of the present invention is not limited by them.

[0034] Example 1. Preparation of steamed black ginseng and black goji berries and extracts

[0035] 1) Preparation of black ginseng extract

[0036] Black ginseng was prepared by repeating the steaming process 5 times, in which 5-year-old ginseng was steamed at 90±5℃ for 5 hours and then dried at 50±5℃ for 5 hours. Subsequently, 50g of black ginseng was finely ground, placed in 10 times the amount of water, and heated to extract at 80~100℃ for 8 hours to prepare black ginseng extract.

[0037] 2) Preparation of black goji berry extract

[0038] Similar to the case of black ginseng, black goji berries were prepared by repeating the steaming process three times, in which goji berries were steamed at 90±5℃ for 5 hours and then dried at 50±5℃ for 5 hours. Subsequently, 50g of black goji berries were finely ground, placed in 10 times the amount of water, and heated to extract at 80~100℃ for 8 hours to prepare black goji berry extract.

[0039] Example 2. Preparation of a mixed fermented product of black ginseng and black goji berries using complex lactic acid bacteria

[0040] The mixed fermented product of black ginseng and black goji berries is a lactic acid bacteria mixed powder containing 17 types of lactic acid bacteria (1×10 9It was prepared using CFU / g). The 17 types of lactic acid bacteria used in the present invention were purchased from Mediogen Co., Ltd. and are complex lactic acid bacteria composed of the composition as described in Table 1 below.

[0041] Composition of lactic acid bacteria mixed powder Types of lactic acid bacteria 1 L. plantarum 2 L. acidophilus 3 Bi. animalis ssp. lactis 4 Lc.lactis 5 L. reuteri 6 S. thermophilus 7 L. rhamnosus 8 L. paracasei 9 Bi. longum 10 L. casei 11 L. bulgaricus 12 L. fermentum 13 Bi. bifidum 14 Bi. breve 15 L.gasseri 16 L. salivarius 17 L. helveticus

[0042] After mixing the black ginseng extract and black goji berry extract prepared in Example 1 above in a weight ratio of 1:1, for every 100 ml of black ginseng and black goji berry extract, 1×10 9 , 2×10 9 and 5×10 9 Each CFU / g of complex lactic acid bacteria was added and cultured with shaking at 37°C for 24 hours. These lactic acid bacteria were sterilized to prepare a mixed fermented product (original extract) of black ginseng and black goji berries.

[0043] Example 3. Evaluation of cytotoxicity of a mixed fermented product of black ginseng and black goji berries

[0044] 5×10⁶ RAW 264.7 macrophages in a 96-well plate 4 After placing in a well and stabilizing for 6 hours, 1×10 from Example 1 above 9 , 2×10 9 and 5×10 9 The undiluted extract of the mixed fermentation product of black ginseng and black goji berries, prepared by treating with CFU / g of complex lactic acid bacteria, was diluted in cell culture medium to a ratio of 400 to 6,400 and treated with RAW 264.7 macrophages. After 24 hours, the cell viability of the mixed fermentation product of black ginseng and black goji berries in RAW 264.6 macrophages was analyzed using the MTT method.

[0045] As a result, as disclosed in Fig. 1, the cell viability of the mixed fermented product of black ginseng and black goji berries of the present invention was found to be at a level similar to that of the untreated group, and it was determined that there was almost no cytotoxicity.

[0046] Example 4. Evaluation of the anti-inflammatory activity of a mixed fermented product of black ginseng and black goji berries

[0047] 5 × 10⁶ RAW 264.7 macrophages per well in a 96-well plate4 After inoculating cells and stabilizing for 6 hours, the undiluted extract of the mixed fermentation of black ginseng and black goji berries prepared in Example 1 was diluted in cell medium to a ratio of 400 to 6,400 and treated on RAW 264.7 macrophages. After 12 hours, 100 ng / ml of LPS was administered, and inflammation was induced for 24 hours. Subsequently, the cell culture medium was collected, and the content of inflammatory mediators NO, TNF-α, and IL-6 was checked.

[0048] Nitric Oxide (NO) was quantitatively analyzed at 540 nm after treatment with Griess reagent. As a result, as disclosed in Fig. 2, the amount of Nitric Oxide (NO) produced significantly increased due to LPS treatment; in contrast, it was found that treatment with the mixed fermented product of black ginseng and black goji berries of the present invention had the effect of reducing NO production. In particular, 1×10 9 or 5×10 9 Compared to the mixed fermented product of black ginseng and black goji berries prepared by treatment with CFU / g of complex lactic acid bacteria, 2×10 9 A significant reduction in NO production was observed in the mixed fermented product of black ginseng and black goji berries prepared by treatment with complex lactic acid bacteria at CFU / g.

[0049] In addition, the content of TNF-α and IL-6 was quantified using an ELISA kit. As a result, as disclosed in Fig. 3, the content of TNF-α and IL-6 significantly increased due to LPS treatment. In contrast, it was found that treatment with the mixed fermented product of black ginseng and black goji berries of the present invention had the effect of reducing the content of TNF-α and IL-6. In particular, 1×10 9 or 5×10 9 Compared to the mixed fermented product of black ginseng and black goji berries prepared by treatment with CFU / g of complex lactic acid bacteria, 2×10 9A significant reduction in the content of TNF-α and IL-6 was observed in the mixed fermented product of black ginseng and black goji berries prepared by treatment with CFU / g of complex lactic acid bacteria.

[0050] Example 5. Analysis of expression levels of MAPK signaling pathway activators related to the anti-inflammatory activity of a mixed fermented product of black ginseng and black goji berries

[0051] Western blot was performed to analyze the expression levels of MAPK signaling pathway activators related to the anti-inflammatory activity of the mixed fermented extract of black ginseng and black goji berries. Specifically, RAW 264.7 cells (1×10⁶ 6 In cells / well, 2×10 9 A mixed fermented product of black ginseng and black goji berries, fermented with a complex of lactic acid bacteria in CFUs, was treated to cells at dilutions of 500-fold or 1,000-fold for 15 minutes. Afterward, the cells were washed three times with cold PBS, lysed with RIPA buffer, a protease and phosphatase inhibitor cocktail, and protein concentration was quantified using a BCA protein assay kit. 30 µg of protein was developed on 10% Gel SDS-PAGE and transferred to a PVDF (Polyvinylidene difluoride) membrane for 2 hours. Subsequently, the cells were blocked in 5% skim milk for 2 hours, and antibodies against p-JNK, JNK, p-ERK, ERK, p-p38, p38, and β-actin were reacted, respectively. The anti-Rabbit secondary antibody conjugated with HRP (Horseradish peroxidase) was diluted 1:5,000 and reacted for 2 hours, and washed a total of 8 times for 5 minutes each with TBST buffer containing 0.05% Tween-20. Subsequently, the antibodies were reacted with an ECL substrate, and each band was analyzed using a chemiluminescence imaging system (BD Bioscience, Milpitas, CA, USA).

[0052] As a result, as disclosed in Fig. 4, compared to the control group (-, -) that received no treatment, the expression levels of p-JNK, p-ERK, and p-p38 in the inflammation-induced group (+, -) were significantly increased, and compared to the inflammation-induced group, the expression levels of p-JNK, p-ERK, and p-p38 in the treatment group (+, X1000 / +, X500) of the black ginseng and black goji berry mixed fermented product of the present invention were significantly decreased. Meanwhile, it was found that there was almost no change in the expression levels of JNK, ERK, and p38. From these results, it was determined that the black ginseng and black goji berry mixed fermented product of the present invention does not affect the expression levels of inflammation-related factors (JNK, ERK, and p38), but does affect their activation.

[0053] Example 6. Confirmation of changes in IL-1β expression levels in bone marrow-derived macrophages (BMDM) following treatment with a mixed fermented product of black ginseng and black goji berries.

[0054] Inflammasomes, a type of NLR induced by external stimuli such as cellular stress and infection, are composed of a complex of several proteins. Among the known types of inflammasomes, such as NAIP / NLRC4, NLRP3 / 6 / 7, or AIM2 / IHI1, NLRP3 activates caspase-1 to convert pro-IL-1β and pro-IL-18 into active IL-1β and IL-18, thereby causing inflammation, and it is known that the activation of NF-κB increases the expression of NLPR3, caspase-1, and pro-IL-1β in inflammasomes.

[0055] In Example 6, it was confirmed whether the anti-inflammatory activity of the mixed fermented product of black ginseng and black goji berries has an inhibitory effect on NLRP3 inflammasome-related inflammatory responses.

[0056] Bone marrow-derived macrophages (BMDMs) were generated by isolating the femur and pelvic bones of C57BL / 6 mice, collecting bone marrow cells, and differentiating them for 6–7 days in IMDM medium containing L929 conditioned medium. The L929 conditioned medium used was the supernatant obtained by culturing L929 cells for 7 days. The inflammatory response of BMDM cells was 2×10⁶ 6 Cells in cells / well were induced by treating them with LPS (100 ng / ml) for 2 hours and reacting with ATP (5 mM) for 40 minutes, and the mixed fermented product of black ginseng and black goji berries was treated at each concentration 5 hours before LPS treatment. After the end of culture, the cell culture medium was taken and the concentration of IL-1β was quantified using an ELISA kit.

[0057] When BMDM differentiated from mouse bone marrow cells was stimulated with LPS and ATP and then treated with a mixed fermented product of black ginseng and black goji berries, it was confirmed that the expression of IL-1β in the cell culture medium was significantly reduced (Fig. 5).

[0058] Example 7. Confirmation of sepsis induction and sepsis inhibition effects

[0059] Sepsis was induced in C57BL / 6 mice by intravenously injecting LPS at a dose of 500 µg / mouse. After 12 hours, changes in the mice's body temperature were measured, and blood was collected to separate the serum, after which the levels of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) were quantified using an ELISA kit.

[0060] As a result, body temperature increased in the sepsis-induced group (LPS) compared to the normal group (N), and in contrast, the group administered the mixed fermented product of black ginseng and black goji berries showed a decrease in body temperature (Fig. 6), and the survival rate reduced by sepsis was significantly increased in the group treated with the mixed fermented product of black ginseng and black goji berries of the present invention (Fig. 7).

[0061] In addition, as a result of examining changes in the expression levels of pro-inflammatory cytokines in the blood, it was confirmed that the expression levels of inflammatory cytokines (IL-1β, IL-6, and TNF-α) increased by sepsis induction were reduced by the administration of the mixed fermented product of black ginseng and black goji berries of the present invention (Fig. 8).

Claims

Claim 1 A health functional food composition for the prevention or improvement of sepsis comprising a mixed fermented product of black ginseng and black goji berries as an active ingredient. Claim 2 A health functional food composition for the prevention or improvement of sepsis according to claim 1, characterized in that the mixed fermented product of black ginseng and black goji berries is prepared by mixing black ginseng extract and black goji berry extract and then fermenting it with lactic acid bacteria. Claim 3 A health functional food composition for the prevention or improvement of sepsis according to claim 2, wherein the black ginseng extract or black goji berry extract is prepared by a method comprising: (1) a step of preparing black ginseng or black goji berries by repeating a steaming process 3 to 9 times in which ginseng or goji berry fruits are steamed at 85 to 95°C for 4 to 6 hours and dried at 45 to 55°C for 4 to 6 hours, and then powdering them; and (2) a step of extracting by adding 8 to 12 parts by weight of an extraction solvent to 1 part by weight of the black ginseng or black goji berry powder prepared in step (1). Claim 4 A health functional food composition for the prevention or improvement of sepsis, characterized in that, in step (2) above, the extraction solvent is water, a C1-C4 lower alcohol, or a mixture thereof. Claim 5 In paragraph 2, the above lactic acid bacteria is Lactobacillus plantarum ( L. plantarum ), Lactobacillus acidophilus ( L. acidophilus ), Bifidobacterium animalis lactis ( Bi. animalis ssp. lactis ), Lactococcus lactis( Lc.lactis ), Lactobacillus reuteri( L. reuteri ), Streptococcus thermophilus( S. thermophilus ), Lactobacillus rhamnosus ( L. rhamnosus ), Lactobacillus paracasei( L. paracasei ), Bifidobacterium longum( Bi. longum ), Lactobacillus casei( L. casei ), Lactobacillus bulgaricus( L. bulgaricus ), Lactobacillus fermentum( L. fermentum ), Bifidobacterium bifidum ( Bi. bifidum ), Bifidobacterium breve ( Bi. breve ), Lactobacillus gasseri( L. gasseri ), Lactobacillus salivarius( L. salivarius ) and Lactobacillus helveticus ( L. helveticus A health functional food composition for the prevention or improvement of sepsis, characterized by having one or more selected from ). Claim 6 A health functional food composition for the prevention or improvement of sepsis, characterized in that, in claim 1, the mixed fermented product of black ginseng and black goji berries increases the survival rate reduced due to sepsis. Claim 7 A health functional food composition for the prevention or improvement of sepsis, characterized in that, in claim 1, the mixed fermented product of black ginseng and black goji berries reduces the production or expression of inflammatory cytokines increased due to sepsis. Claim 8 A health functional food composition for the prevention or improvement of sepsis, characterized in that, in claim 1, the sepsis is bacterial sepsis caused by lipopolysaccharide present in the outer membrane of Gram-negative bacteria. Claim 9 A health functional food composition for the prevention or improvement of sepsis according to claim 1, characterized in that the composition is manufactured in any one formulation selected from powder, granules, pills, tablets, capsules, candies, syrups, and beverages. Claim 10 A pharmaceutical composition for the prevention or treatment of sepsis comprising a mixed fermented product of black ginseng and black goji berries as an active ingredient. Claim 11 A pharmaceutical composition for the prevention or treatment of sepsis according to claim 10, characterized by further comprising, in addition to the active ingredient, a pharmaceutically acceptable carrier, excipient, or diluent. Claim 12 A feed additive for the prevention or improvement of sepsis containing a mixed fermented product of black ginseng and black goji berries as an active ingredient. Claim 13 A veterinary composition for the prevention or treatment of sepsis comprising a mixed fermented product of black ginseng and black goji berries as an active ingredient. Claim 14 A herbal medicine composition for the prevention or treatment of sepsis containing a mixed fermented product of black ginseng and black goji berries as an active ingredient.

Images