WO2022055012A1 - Use of choerospondias axillaris extract - Google Patents

Use of choerospondias axillaris extract Download PDF

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WO2022055012A1
WO2022055012A1 PCT/KR2020/013055 KR2020013055W WO2022055012A1 WO 2022055012 A1 WO2022055012 A1 WO 2022055012A1 KR 2020013055 W KR2020013055 W KR 2020013055W WO 2022055012 A1 WO2022055012 A1 WO 2022055012A1
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extract
composition
cells
administration
control group
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French (fr)
Korean (ko)
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가우탐라비
허용
김창열
조지훈
양수정
이재희
김형아
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주식회사 드래곤이뮤노
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/22—Anacardiaceae (Sumac family), e.g. smoketree, sumac or poison oak
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders

Definitions

  • the present invention is Cherospondias axillaris ( Choerospondias axillaris ) It relates to a composition for enhancing immunity comprising an extract as an active ingredient.
  • Choerospondias axillaris commonly called Nepali hog plum or Lapsi, is native to Nepal and is a large edible fruit tree belonging to the Anacardiaceae family.
  • Lush fruit is composed of various components such as amino acids, vitamin C, reducing sugars, non-reducing sugars, pectins, organic acids and trace elements, chemically dihydroquercetin (quercetin), quercetin, protocatechuic acid (protocatechuic) acid), gallic acid, 3,3-di-o-methylellagic acid, B-sitosterol, docosterol (daucosterol), stearic acid (steric acid) is composed of triacontanoic acid, octacosanol, syringaldehyde, vanillic acid and citric acid.
  • TFC Toxicol. Environ. Heal.-Part A Curr. Issues., 2016, 79:878-883.
  • the functions of TFC include hypoxic tolerance, myocardial ischemia protection, platelet congregation inhibition, hemorheology improvement and mouse immune function enhancement. (Lijuan, DGJL Sport. Sci., 2002, 5).
  • TFC inhibits dexamethasone-induced thymocyte apoptosis in rats and creatine kinase (CK), creatine kinase-MB (Creatine kinase-MB) in isoproterenol-induced damage.
  • LDH lactate dehydrogenase
  • Plant extract studies on immune modulation mainly focus on their effects on T lymphocytes, cytokine production, antibody production, autoimmune disorders, apoptosis, antimicrobial, anticancer or antioxidant effects (Huang, CF et al. Cell Mol. Immunol). ., 2008, 5:23).
  • inflammatory diseases are mainly treated by administering pharmaceutical agents that reduce the physical discomfort of the inflammatory response, but general anti-inflammatory medicines are used for the treatment of a wide range of diseases, and the same medicine is often used to treat different diseases. Because it is used for treatment, it shares therapeutic action and side effects. As side effects, symptoms such as respiratory stimulation, circulatory collapse, epigastric pain, vomiting, gastrointestinal bleeding, liver damage, and platelet inhibition have been known. Because of these side effects, it is difficult to use the existing anti-inflammatory drugs in the long term, and the serious side effects of the current treatment methods are large, so the development of new or improved therapeutic agents is essential and urgent. The need for safe anti-inflammatory drugs is emerging.
  • An object of the present invention is Cherospondias axillaris for enhancing the immune system of a subject ( Choerospondias axillaris ) To provide a composition comprising an extract as an active ingredient.
  • an object of the present invention is Cherospondias axillaris for use as an anti-inflammatory medicament ( Choerospondias axillaris ) To provide a composition comprising an extract as an active ingredient.
  • Cherospondias axillaris Choerospondias axillaris ) Provides a composition for enhancing immunity comprising an extract as an active ingredient.
  • the Cherospondias axillaris The extract may be an extract of a fruit part.
  • the Cherospondias axillaris The extract may be extracted using ethanol.
  • composition for enhancing immunity of the present invention may increase the level of IgG2a/IgG1 in the subject after administration as compared to before administration.
  • composition for enhancing immunity of the present invention can promote an immune response by type 1 helper T lymphocytes (Th1).
  • composition for enhancing immunity of the present invention can reduce the level of one or more selected from IgE antibody and IgG1 antibody in the subject after the administration compared to before the administration.
  • composition for enhancing immunity of the present invention may increase the IgA antibody level in the subject after the administration compared to before the administration.
  • composition for enhancing immunity of the present invention is nitric oxide (NO), prostaglandin E 2 (PGE 2 ), COX-2 and reactive oxygen species (ROS) in the subject after the administration compared to before the administration.
  • NO nitric oxide
  • PGE 2 prostaglandin E 2
  • COX-2 reactive oxygen species
  • ROS reactive oxygen species
  • composition for enhancing immunity of the present invention can reduce the level of pro-inflammatory cytokines in the subject after the administration compared to before the administration.
  • the pro-inflammatory cytokine may be one or more selected from TNF- ⁇ , IL-1 ⁇ , IL-6, IL-8 and IL-17.
  • composition for enhancing immunity of the present invention may be a functional food.
  • composition for enhancing immunity of the present invention may be an anti-inflammatory drug.
  • the present invention is Cherospondias axillaris ( Choerospondias axillaris ) Extract, that is, provides a composition for enhancing immunity comprising the ruby extract as an active ingredient.
  • the ruby The extract may be an extract of a fruit part.
  • the ruby The extract may be extracted using ethanol.
  • the ruby extract of the present invention means a solvent extract of ruby or a fraction fractionated therefrom.
  • the solvent is water or a low-cost alcohol having 5 or less carbon atoms, preferably water or a low-cost alcohol having 3 or less carbon atoms, more preferably water or ethanol, and most preferably ethanol.
  • the ruby extract may be a fraction prepared by lyophilizing the concentrated concentrate after filtration by adding ethanol to ruby fruit, for example, peel and pulp powder.
  • T lymphocytes are considered more important because activation of T lymphocytes requires antigen specificity and the production of multiple cytokines from T lymphocytes (Fox, DA Off. J. AM. Coll. Rheumatol., 1997, 40: 598-609).
  • T lymphocytes can be classified into two different types according to their cytokine production patterns: IFN- ⁇ and IL-2 are typically induced by Type-1 helper T lymphocytes (Th1) cells.
  • Type 2 helper T lymphocytes (Th2) mainly produce IL-4, IL-5, IL-13 and IL-10.
  • antibodies are used by the immune system to detect and neutralize foreign substances, such as various bacteria.
  • antibodies, cytokines, etc. can destroy host tissues in autoimmune diseases. Therefore, herbal extracts that not only stimulate the immune system but also have inhibitory effects may be of special interest.
  • Immunosuppression refers to temporary or permanent immune damage that can make the host more susceptible to pathogens (Liang, M. et al. Microb. Pathog., 2013, 54: 40-45).
  • Previous studies have shown that a single high-dose injection of cyclophosphamide or repeated low-dose injections mainly affects B lymphocytes and suppressive T lymphocytes (Graziano, F. et al., J. Immunol., 1981, 127: 1067-1070; Shukla, ML and Chaturvedi, UC, Br. J. Exp. Pathol., 1984, 65:397).
  • Cyclophosphamide also affects CD4+ and CD8+ memory T lymphocyte subpopulations located in lymphoid and non-lymphatic tissues (Siracusa, F. et al., Eur. J. Immunol., 2017, 47:1900-1905; Sheeja , K and Kuttan, G., Asian Pacific J. Cancer Prev., 2006, 7:609-614). Natural products have been used in various experiments on the antagonism of cyclophosphamide-induced immunosuppression in mice.
  • Ruby extract can activate both humoral and cellular immunity of immunosuppressed mice injected with CYP.
  • Rubsy extract can significantly control changes in spleen and thymus weight following CYP injection, and also control quantitative changes in hematological parameters.
  • Rubsy extract can also up-regulate the quantitative level of peripheral immune cells that are reduced by immunosuppression, in particular, it can significantly increase the number of natural killer cells.
  • the composition for enhancing immunity of the present invention may increase the IgG2a/IgG1 level in the subject after the administration compared to before the administration.
  • composition for enhancing immunity of the present invention can promote an immune response by type 1 helper T lymphocytes (Th1).
  • Th1 type 1 helper T lymphocytes
  • T lymphocytes are broadly classified into apoptotic T lymphocytes (CD8+ T lymphocytes) that act directly on cancer cells or virus-infected cells, and helper T lymphocytes that support the functions of other immune cells through mediators such as cytokines (Globerson, A. , Int. Arch. Allergy Immunol, 1995, 107:491-497).
  • cytokines Globerson, A. , Int. Arch. Allergy Immunol, 1995, 107:491-497.
  • NK cells play an important role in killing cancer cells or virus-infected cells, which are important in innate immunity.
  • IgG1 antibody is isotype switched by IL-4 produced by type 2 helper T lymphocytes and IgG2a is isotype switched by IFN- ⁇ produced by type I helper T lymphocytes. Accordingly, as the IgG2a/IgG1 ratio is relatively high, the type 1 helper T lymphocyte response (type-1 resposne), which is the key to defense against cancer and viral infection, for example, is enhanced, and the type 2 helper T lymphocyte response ( type-2 resposne) is inhibited.
  • type-1 resposne the type 1 helper T lymphocyte response
  • type-2 resposne type-2 resposne
  • composition for enhancing immunity of the present invention may reduce the level of one or more selected from an IgE antibody and an IgG1 antibody in a subject after the administration compared to before the administration.
  • composition for enhancing immunity of the present invention may increase the IgA antibody level in the subject after the administration compared to before the administration.
  • IL-4 induces isotype switching to IgE, a representative antibody marker for allergy development (Snapper, CM et al., Immunol. Rev., 1988, 102:51-75, Bosie, A. and Vietta, ES, Cell Immunol., 1991, 135:95-104).
  • Administration of the ruby extract in the present invention can reduce the IgE level in the subject in a concentration-dependent manner.
  • an extract containing a mixture of polyphenols and anthocyanidins reduced the levels of IgE and IgG1 in DNCB-induced atopic dermatitis mice
  • the reduction in the IgE antibody level was related to the polyphenols and It may be inferred that the anthocyanidins may be an enhancement of the type 1 response.
  • administration of the ruby extract in the present invention can increase the level of IgA, an important antibody in mucus immunity, in a concentration-dependent manner.
  • the composition for enhancing immunity of the present invention can reduce one or more levels selected from NO, PGE 2 , COX-2 and ROS in the subject after the administration compared to before the administration.
  • the ruby extract can significantly reduce the production of NO, PGE 2 and intracellular ROS in LPS-activated phagocytes.
  • the ruby extract of the present invention can suppress the protein expression of COX-2 involved in the production of PGE 2 and also inhibit the production of the related PGE 2 .
  • the composition for enhancing immunity of the present invention can reduce the level of pro-inflammatory cytokines in the subject after the administration compared to before the administration.
  • the pro-inflammatory cytokine may be one or more selected from TNF- ⁇ , IL-1 ⁇ , IL-6, IL-8 and IL-17.
  • pro-inflammatory cytokine production such as TNF- ⁇ , IL-1 ⁇ , and IL-6
  • the ruby extract significantly lowered the levels of IL-6, TNF ⁇ , and IL-1 ⁇ produced from RAW 264.7 cells according to LPS activation in a wide range of extract concentrations in a dose-dependent manner, and the high concentration of the ruby extract significantly reduced TNF- ⁇ It was confirmed that it can be significantly reduced.
  • IL-6, TNF- ⁇ , IL-1 ⁇ , and IL-8 levels could be decreased in a dose-dependent manner according to the concentration of the Rubsy extract. Controlling the production of pro-inflammatory cytokines is a target of anti-inflammatory therapy. It is thought to suggest
  • Th1-cell-mediated immunity protects individuals from cancer progression, viral infection, and intracellular infectious disease
  • Th2-cell-mediated immunity is mainly associated with respiratory and skin allergic reactions
  • IFN- ⁇ produced by Th1 cells and IL-4 produced by Th2 cells play an important role in homeostasis of immune responses in the body. It is a measure of monitoring Th2 balance (Paludan, SR, Scand. J. Immunol., 1998, 48:459-468).
  • cytokines secreted by Th1 and Th2 cells play a role in determining the direction and outcome of the immune response.
  • the statistically significantly changed cytokines that would have activated spleen-derived T lymphocytes in vitro were significantly lower in the 5 and 50 mg/kg body weight administration groups than in the control group with the pro-inflammatory cytokine TNF- ⁇ . This suggests that ruby extract may be involved in the inflammatory response by inhibiting the production of this cytokine at certain concentrations.
  • ruby extract can lower IL-17, a representative cytokine that contributes to the promotion of inflammatory responses in the intestinal tract.
  • CYP-induced immunosuppressive mice are commonly used to evaluate the recovery or activation of immunosuppression of various drugs and medicinal plant preparations (Huang, Y. and Li, L., Transl. Cancer Res., 2013, 2:144). Therefore, in the present invention, CYP-induced immunosuppression mice were used to evaluate the recovery or activation potential of Rubsy fruit extract.
  • the thymus and spleen play important roles in the body's immune response. The weight of these organs changes in response to various stimuli that directly affect the immune system (Cui, H. et al., J. Sci. Food Agric, 2011, 91:2180-2185). Immune suppression by such cyclophosphamide can be restored by administration of the rubsy extract of the present invention.
  • CYP has been reported to increase neutrophils in the bronchoalveolar lavage fluid of mice and decrease the level of hematological parameters such as red blood cell count, hemoglobin and % lymphocytes in Wistar rats (Said Abd-Elkhalek, E. et al., Can. J. Physiol. Pharmacol., 94: 347-358, Muruganandan, S. et al., Toxicology, 2005, 215: 57-68).
  • TGF- ⁇ 1 has the ability to induce Th17 cell differentiation and promote IL-17 secretion, which has the property of prolonging the acute inflammatory process (McDonald, DM, Am. J. Respir. Crit. Care Med, 2016). , 164: S39-S45).
  • CYP CYP promotes Th17 cell differentiation to enhance IL-17 production (Viaud, S. et al., Cancer Res., 2011, 71: 661-665). Accordingly, the increased levels of IL-17 and TGF- ⁇ 1 in mice administered with CYP can be explained.
  • the rubsy extract can reduce the level of the corresponding cytokine production at some concentrations.
  • IL-17 similar results were observed in mesenteric lymph nodes.
  • CYP-induced immune dysfunction affects the intestinal mucosal immune system more and disrupts the balance of the intestinal microflora (Matsuoka, K. and Kanai, T., Semin. Immunopathol., 2015, 37:47- 55; Xu, X. and Zhang, X., Microbiol. Res., 2015, 171: 97-106)
  • the decrease in IL-17 level by Ruby extract suggests that Ruby extract is effective in protecting intestinal mucosal immunity. .
  • the composition of the present invention can prevent or treat autoimmune diseases, preferably autoimmune diseases mediated by T cells.
  • the autoimmune disease includes, for example, rheumatoid arthritis, psoriasis, systemic lupus erythematosis, E-hyperimmunoglobulin E, Hashimoto's thyroiditis, Greves Grave's Disease, multiple sclerosis, Scleroderma, progressive systemic sclerosis, myasthenia gravis, type I diabetes, uveitis, Allergic encephalomyelitis, glomerulonephritis, Vitilligo, Goodpasture syndrome, Becet's Disease, Crohn's Disease, Ankylosing Spondylitis Thrombocytopenic purpura, Pemphigus vulgaris, Autoimmune Anemia, Cryoglobulinemia, ALD, Systemic Lupus Erythematosus
  • the number of helper T lymphocytes, apoptotic T lymphocytes, B lymphocytes, and natural killer cells were all lower than that of the excipient control group, as expected in the model control group mice injected with CYP.
  • the ratio of these cells was significantly increased compared to the model control group, and the level of natural killer cells was significantly higher than that of the excipient control group.
  • Natural killer cells are known to produce an excess of IFN- ⁇ (Arase, H. at al., J. Exp. Med., 1996, 183: 2391-2396). The effect of inducing an increase in the number of killer cells, ultimately leading to an increase in the production of IFN- ⁇ , which plays a key role in the cellular immune response, suggests that ruby extract may contribute to the increase in immunity.
  • the composition for enhancing immunity of the present invention may be a functional food.
  • the functional food includes, but is not limited to, adding the ruby extract of the present invention to natural food, processed food, patient food, and general food materials.
  • Food in the present invention the composition for enhancing immunity of the present invention may be used as it is or may be used together with other food or food compositions, and may be appropriately used according to a conventional method.
  • the mixing amount of the active ingredient may be appropriately determined depending on the purpose of its use (prophylaxis, improvement or therapeutic treatment).
  • the ruby extract of the present invention may be added in 0.1 to 99.9 parts by weight based on 100 parts by weight of the food raw material during food production.
  • the effective dose of the ruby extract in the food can be used according to the effective dose of the pharmaceutical composition.
  • the functional food may be used in the form of formulations for oral administration such as tablets, hard or soft capsules, solutions, suspensions, etc., and these formulations are acceptable conventional carriers, for example, excipients in the case of formulations for oral administration, It may be prepared using a binder, a disintegrant, a lubricant, a solubilizer, a suspending agent, a preservative or an extender, and the like.
  • a food to which the ruby extract can be added there is no particular limitation as an example of a food to which the ruby extract can be added.
  • foods to which the ruby extract can be added include drinks, meat, sausage, bread, biscuits, rice cakes, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, and various soups. , beverages, alcoholic beverages and vitamin complexes, dairy products, and dairy products, and includes all health functional foods in the ordinary sense.
  • the functional food of the present invention is not particularly limited in other ingredients except for containing a ruby extract as an essential ingredient, and may contain various flavoring agents or natural carbohydrates as additional ingredients like conventional food and beverages.
  • natural carbohydrates include monosaccharides such as glucose, fructose and the like; disaccharides such as maltose, sucrose and the like; and polysaccharides such as conventional sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol.
  • flavoring agents other than those described above, natural flavoring agents and synthetic flavoring agents may be used.
  • the ratio of the natural carbohydrate may be appropriately determined by the selection of those skilled in the art.
  • the functional food composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), synthetic flavoring agents and natural flavoring agents, coloring agents and thickeners (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonates used in carbonated beverages, and the like. These components may be used independently or in combination. The proportion of these additives may also be appropriately selected by those skilled in the art.
  • composition for enhancing immunity of the present invention may be an anti-inflammatory drug.
  • Inflammation is the body's defense mechanism against harmful effects such as tissue damage and infection. Inflammation is one of the defense responses of biological tissues to certain stimuli, and refers to a complex lesion that combines three types: tissue deterioration, circulatory disorder and exudation, and tissue proliferation. In addition, it can be said that it is the expression of a defense mechanism in vivo against various types of infection or irritants in in vivo metabolites, and various chemical mediators are involved in the expression mechanism of inflammation, and the pathogenesis is very complicated. . It is a local protective reaction induced by tissue injury or destruction, and acts to destroy, weaken, or mask both the injury-causing agent and the injured tissue.
  • microvessels are perforated, blood components leak into the interstitial space, and leukocytes move to the inflamed tissue, which is usually accompanied by clinical symptoms such as erythema, edema, hyperalgesia and pain.
  • a variety of diseases are associated with chronic inflammation, including atherosclerosis, asthma, psoriasis, rheumatoid atthritis, inflammatory bowel disease or cancer (Lawrence, T., Cold Spring Harb. Perspect. Biol., 2009, 1: a001651).
  • NOS an enzyme that produces NO from L-arginine
  • COX cyclooxygenase
  • NOS is always expressed at a certain level in the body, and the small amount of NO produced by them plays an important role in maintaining normal body improvement, such as inducing neurotransmission or vasodilation.
  • NO which is rapidly excessively generated by iNOS induced by various cytokines or external stimulants, is known to cause cytotoxicity or various inflammatory reactions, and there is a study that chronic inflammation is related to an increase in iNOS activity (Murakami, A. and Ohigashi, H., Int. J. Cancer, 2007, 121: 2357-2363; Rehman, MU et al., Inflamm. Res., 2012, 61:1177-1185).
  • Macrophages are cytokines / chemokines and tumor necrosis factor-alpha (TNF- ⁇ ), interleukin (interleukin-6, IL-6), IL-1 ⁇ , cyclooxygenase-2 (cyclooxygenase) -2, COX-2), nitric oxide (NO) and prostaglandins are major inflammatory cells that produce many inflammatory mediators (Erwig, LP, and Rees, AJ, Kidney Blood Press. Res., 1999, 22:21-25; Zhang, X. and Mosser, DM, J. Pathol. A J. Pathol. Soc. Gt. England Irel., 2008, 214:161-178).
  • iNOS synthesizes NO from L-arginine using NADPH and oxygen, and COX-2 converts arachidonic acid to prostaglandin E 2 (PGE 2 ) (Murakami, A. and Ohigashi, H., Int. J. Cancer, 2007, 121:2357-2363; Aoki, T. and Narumiya, S., Trends Pharmacol. Sci., 2012, 33:304-311).
  • Nuclear factor ⁇ B (NF- ⁇ B) transcription factor plays an important role in the regulation of transcription of various inflammatory factors and cytokines (Ghosh, S. et al., Annu. Rev. Immunol, 1998, 16:225-260, Baeuerle, PA and Henkel, T., Annu. Rev. Immunol., 1994, 12:141-179).
  • NF- ⁇ B nuclear factor ⁇ B
  • I ⁇ B ⁇ I ⁇ B ⁇ that inhibits translocation to the nucleus
  • NF- ⁇ B When NF- ⁇ B is activated by lipopolysaccharide (LPS), it is activated through I ⁇ B-kinase (IKK) complex activation. Activated IKK phosphorylates I ⁇ B ⁇ through ubiquitination and subsequent proteasomal degradation (Gloire, G. et al., Biochem. Pharmacol., 2006, 72:1493-1505), Accordingly, NF- ⁇ B is released from the complex with I ⁇ B ⁇ .
  • LPS lipopolysaccharide
  • IKK I ⁇ B-kinase
  • NF- ⁇ B moves to the nucleus, and pro-inflammatory cytokines (IL-1 ⁇ , IL-2, IL-6, TNF- ⁇ ), chemokines [IL-8, macrophage chemotactic protein-1, MCP- 1)], inflammatory enzymes (iNOS, inducible COX-2), adhesion molecules [intracellular adhesion molecule-1, ICAM-1, vascular adhesion molecule-1, VCAM- 1), E-selectin] and promotes the transcription of various inflammatory mediators (Barnes, PJ and Karin, M.).
  • pro-inflammatory cytokines IL-1 ⁇ , IL-2, IL-6, TNF- ⁇
  • chemokines IL-8, macrophage chemotactic protein-1, MCP- 1
  • iNOS inducible COX-2
  • adhesion molecules Intracellular adhesion molecule-1, ICAM-1, vascular adhesion molecule-1, VCAM- 1), E-selectin
  • MAPK Mitogen-activated protein kinase
  • JNK c-Jun NH2-terminal kinase
  • ERK extracellular signal-regulated kinase
  • MAPK is phosphorylated to induce a signaling cascade of NF- ⁇ B and/or AP-1 activation (Kaminska, B., Biochimica et Biophysica Acta - Proteins and Proteomics, 2005, 1754:253-262; Guha, M. and Mackman, N., Cell. Signal., 2001, 13:85-94).
  • IL-1 ⁇ is a cytokine that plays a critical role in modulating innate and adaptive immune responses (Dinarello, C.A., Annu. Rev. Immunol., 2009, 27:519-550).
  • IL-1 ⁇ activates T-lymphocytes to enhance production of proinflammatory cytokines such as TNF- ⁇ and IL-6 (Ciraci, C. et al., Microbes Infect., 2012, 14:1263-1270, Dinarello, CA, Eur. J. Immunol., 2011, 41:1203-1217).
  • IL-1 ⁇ Activation of IL-1 ⁇ from pro-IL-1 ⁇ , a pro-cytokine, is dependent on caspase-1, which is activated by the inflammasome (Lamkanfi, M. and Kanneganti, TD, Int. J. Biochem). (Cell Biol., 2010, 42:792-795).
  • NLRP3 is the most intensively studied inflammasome and is implicated in a wide range of diseases including inflammation, autoimmunity and infection (Stutz, A. et al., J. Clin. Invest. 2009, 119:3502-3511; Menu, P. and Vince, JE, Clin. Exp. Immunol., 2011, 166:1-15).
  • Activation of the NLRP3 inflammasome requires two steps: a priming step and an activation step.
  • the priming step the expression of NF- ⁇ B-linked NLRP3, pro-IL-1 ⁇ or pro IL-18 is induced by a Toll-like receptor-4 agonist (eg, LPS).
  • the activation step removes pore-forming toxins, extracellular ATP, microbial DNA and RNA, inhaled particulates, uric acid and cholesterol crystals. by a wide range of substances including (Bauernfeind, F. et al., J. Immunol., 2010, 183:787-791; Rajamaki, K. et al., PLoS One, 2010, 5:e11765).
  • Activation of NLRP3 is achieved through caspase-1 attraction through ASC adapter protein (apoptosis-associated speck-like protein containing a caspase-recruitment domain).
  • ASC adapter protein apoptosis-associated speck-like protein containing a caspase-recruitment domain.
  • the activated NLRP3 inflammasome complex activates caspase-1 and 1 converts pro-IL-1 ⁇ and pro-IL-18 to IL-1 ⁇ and IL-18 (Gross, O. et al., Immunol. Rev., 2011, 243:136-151; Davis, BK et al. al., Annu. Rev. Immunol., 2011, 29:707-735).
  • NLRP3 Since the macrophage cell line of RAW 264.7 mice lacks the ASC adapter, NLRP3 has been successfully studied in PMA (phorbol 12-myristate 13-acetate) stimulated THP-1-derived macrophages (Kong, F. et al., Biomed. Pharmacother., 2016, 82:167-172; Pelegrin, P.. et al., J. Immunol., 2008, 180:7147-7157). THP-1 macrophages activated with PMA (10-400 ng/ml) exhibit metabolic and morphological similarities to human macrophages, and are therefore widely used as in vitro test models for human macrophages (Park, Ek et al., Inflamm. Res., 2007, 56:45-50).
  • the intracellular signaling pathway NF- ⁇ B or MAPK signaling pathway
  • LPS induces activation of I ⁇ B ⁇ , NF- ⁇ B, p-38, JNK, and ERK.
  • the rubsy extract shows that NF- ⁇ B translocation into the nucleus is inhibited.
  • the ruby extract of the present invention can also control phosphorylation of p-38, JNK, and ERK.
  • Ruby extract inhibits the production and release of pro-inflammatory mediators by controlling the activities of I ⁇ B ⁇ , NF- ⁇ B, p-38, JNK, and ERK in LPS-activated RAW 264.7 cells, and relieves the level of inflammatory response induced by LPS. suggest that it can be done.
  • the level of NLRP3 protein one of the intracellular inflammasome protein complexes, was enhanced.
  • the ruby extract of the present invention can significantly reduce the NLRP3 protein expression level, which shows that the ruby extract can control the inflammasome-related inflammatory response.
  • the ruby extract of the present invention can control the production of proinflammatory cytokines in mouse and human macrophages and modulate the mechanistically related intracellular signaling system.
  • the pro-inflammatory cytokines TNF- ⁇ and IL-1 ⁇ can cause cell degeneration and death and can cause dysfunction of several organs, and also stimulate the production of pro-inflammatory cytokines such as IL-6 and IL-8 (Tracey). , KJ et al., Nature, 1987,330:662).
  • the ruby extract of the present invention can prevent the aggravation of diseases associated with abnormal inflammatory responses by inhibiting excessive production of these cytokines.
  • the anti-inflammatory effect of rubsy extract will be related to the constituents present in the extract.
  • quercetin inhibits NLRP3 protein-related reactions
  • dihydroquercetin inhibits ROS production and NLRP3 complex production
  • procyanidin inhibits ROS production
  • catechin also inhibits ROS production. It has been reported to inhibit NLRP3 protein-related responses (Wang, W. et al., Br. J. Pharmacol., 169:1352-1371; Ding, T. et al., Phytomedicine, 2018, 41:45- 53; Liu, HJ et al., J. Neuroinflammation, 2017, 14:74; Jhang, J. et al., Mol. Nutr. Food Res., 2016, 60:2297-2303).
  • flavonoids other components of the extract, decreased the expression of other proinflammatory cytokines including TNF- ⁇ , IL-1 ⁇ , IL-6, and IL-8 in the RAW 264.7 cell line, and this action was , inferred to be due to inhibition of AP-1, MAPK activity (Santangelo, C. et al., Ann. Ist. Super. Sanita, 2007, 43:394; Bode, AM and Dong, Z., Mutat. Res- Fundam. Mol. Mech. Mutagen, 2004, 555: 33-51).
  • the present invention further proves the anti-inflammatory efficacy of the rubsy extract from the fragmentary efficacy confirmation.
  • the composition of the present invention can prevent or treat inflammatory diseases.
  • inflammatory disease refers to a disease resulting from, arising from, or inducing inflammation.
  • the term “inflammatory disease” may also refer to a dysregulated inflammatory response caused by an excessive response by macrophages, granulocytes, and/or T lymphocytes resulting in abnormal tissue damage and cell death.
  • the inflammatory disease comprises an antibody mediated inflammatory process.
  • An “inflammatory disease” may be an acute or chronic inflammatory condition and may arise from an infectious or non-infectious cause.
  • the inflammatory diseases include, for example, atherosclerosis, arteriosclerosis, autoimmune disorders, multiple sclerosis, systemic lupus erythematosus, polymyalgia rheumatism (PMR), gouty arthritis, degenerative arthritis, tendinitis, bursitis, psoriasis, cystic fibrosis, joint Osteoarthritis, rheumatoid arthritis, inflammatory arthritis, Sjogren's syndrome, giant cell arteritis, progressive systemic sclerosis (scleroderma), ankylosing spondylitis, polymyositis, dermatomyositis, pemphigus, pemphigus, diabetes (eg type 1), myasthenia gravis, Hashimoto Thyroiditis, Graves' disease, Good
  • the medicament comprising the ruby extract of the present invention may be administered to patients with reduced immune function, cancer patients, and the like, and may be administered to subjects with a high risk of disease due to decreased immune function.
  • the medicament of the present invention may contain 0.1 to 99.9 parts by weight of the ruby extract of the present invention based on 100 parts by weight of the composition as a medicament.
  • this can be increased or decreased according to the needs of the user, and it can be appropriately increased or decreased according to circumstances such as diet, nutritional status, disease progression, and brain dysfunction.
  • the drug of the present invention can be administered orally or parenterally, and can be used in the form of a general pharmaceutical formulation.
  • Preferred pharmaceutical formulations include formulations for oral administration such as tablets, hard or soft capsules, solutions, suspensions, and the like, and these pharmaceutical formulations include conventional pharmaceutically acceptable carriers, for example, excipients, binders, and binders for oral formulations; It may be prepared by further including a disintegrant, a lubricant, a solubilizer, a suspending agent, a preservative or an extender, and the like.
  • Another preferred pharmaceutical preparation is a skin external preparation (skin application preparation) such as a patch, gel, ointment, cream, etc.
  • These pharmaceutical preparations are also commonly used in external preparations for skin to the extent that the effect of the ruby extract is not inhibited. It can be prepared by including preservatives, disinfectants and/or various additives as optional ingredients.
  • additives include surfactants, wetting agents, silicone compounds, high molecular substances (polymer compounds), alcohols, ultraviolet absorbers, pigments, pigments, vitamins, antioxidants, sequestering agents, anti-inflammatory agents, pH adjusters, pearlescent agents, nucleic acids, enzymes , and natural extracts.
  • Pharmaceutically acceptable carriers that may be included in the medicament of the present invention are those commonly used in formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, silicic acid. calcium, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like. it is not Suitable pharmaceutically acceptable carriers and agents are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).
  • the dosage of the medicament containing the ruby extract of the present invention can be determined by a person skilled in the art according to various factors such as the patient's condition, age, sex, and complications, but generally 0.1 mg to 10 g per 1 kg for adults, Preferably, it may be administered in a dose of 10 mg to 5 g.
  • the daily dose or 1/2, 1/3 or 1/4 of the dose of the drug per unit dosage form is contained, and may be administered 1 to 6 times a day.
  • the above amount may be less than the above range, and an amount above the above range may be used under the judgment of an expert that there is no problem in terms of safety.
  • the in vivo administration results confirmed in the Examples of the present invention show that the Rubsy extract does not induce specific organ toxicity even at a concentration of up to 500 mg/kg body weight.
  • the medicament of the present invention may be prepared in a unit dose form by formulation using a pharmaceutically acceptable carrier and/or excipient according to a method readily practiced by those skilled in the art, or may be prepared by internalizing in a multi-dose container.
  • the formulation may be in the form of a solution, suspension, or emulsion in oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet or capsule, and may additionally include a dispersant or stabilizer.
  • composition for enhancing immunity of the present invention comprising an extract is effective in anti-inflammatory and immune enhancement, and has an effect that can be usefully used in inflammatory diseases and diseases related to inflammatory reactions.
  • Ruby extract also has the property of promoting a Th1 response versus a Th2 response in humoral immunity, lowering serum antibody levels related to allergy and increasing antibody levels related to mucosal and gut immunity. It was confirmed that the extract could inhibit the production of some proinflammatory cytokines from splenic T lymphocytes by the extract.
  • 1A-1B Effect of extracts on cell viability of macrophages.
  • Figures 2a-2b Effect of extracts on NO and PGE 2 production following LPS activation in RAW 264.7 cells.
  • NO production level NO production level.
  • PGE 2 levels #, *: Significant difference (p ⁇ 0.05) compared to LPS inactive control group (inactive control group) or excipient control group without extract (excipient control group), respectively.
  • Figures 3a-3d Effect of extracts on the level of reactive oxygen species produced in RAW 264.7 cells following LPS activation.
  • (b) macrophage gating during flow cytometry analysis of whole cells when LPS is activated
  • (c) fluorescence staining intensity graph for representative reactive oxygen species generation by activation and extract addition conditions
  • Figures 4a-4c Effect of extracts on the level of pro-inflammatory cytokines produced in RAW 264.7 cells following LPS activation.
  • #, * Significant difference (p ⁇ 0.05) compared to LPS inactive control group (inactive control group) or excipient control group without extract (excipient control group), respectively.
  • Figure 5a-5d Effect of extracts on the level of pro-inflammatory cytokines produced in THP-1 cell-derived macrophages (TDM) following LPS activation.
  • #, * Significant difference (p ⁇ 0.05) compared to LPS inactive control group (inactive control group) or excipient control group without extract (excipient control group), respectively.
  • FIG. 6 Effect of extracts on the expression of COX-2 in RAW 264.7 cells.
  • Figure 7 Effect of extracts on the expression of NF- ⁇ B in RAW 264.7 cells.
  • Figure 8 Effect of extracts on MAPK phosphorylation signaling protein expression in RAW 264.7 cells.
  • FIG. 9 Effect of LPS/ATP activation by extracts on NLRP3 inflammasome expression in THP-1-derived macrophages (TDM).
  • 10A-10E Effect of extract administration on IgG subtypes (IgG1 and IgG2a), IgA and IgE immunoglobulin levels. * Significant difference compared to control (p-value ⁇ 0.05).
  • 11A-11F Effect of extract administration on cytokine levels produced in splenic T lymphocyte culture supernatant. * Significant difference compared to control (p-value ⁇ 0.05).
  • 13A-13D Effect of extracts on hematologic index levels in mice administered CYP or extracts.
  • RBCs red blood cells
  • HGB haemoglobin (hemoglobin); NEUT, neutrophils (neutrophils); LYM, lymphocytes (lymphocytes).
  • # Significance was the difference (p-value ⁇ 0.05) compared to the excipient control group and the CYP administration control group (extract non-administration model control group), respectively.
  • 15A-15B Antibody levels in culture supernatants generated from splenic B lymphocytes of mice administered with CYP or extracts. # and *: the difference (p-value ⁇ 0.05) compared to the excipient control group and the CYP administration control group (extract non-administration model control group), respectively.
  • 16A-16F Effect of extracts on cytokine levels produced in splenic T lymphocyte culture supernatants of CYP-injected mice. # and *: the difference (p-value ⁇ 0.05) compared to the excipient control group and the CYP administration control group (extract non-administration model control group), respectively.
  • 17A-17B Effect of extracts on cytokine levels produced in mesenteric lymph node T lymphocyte culture supernatants of CYP-injected mice. # and *: the difference (p-value ⁇ 0.05) compared to the excipient control group and the CYP administration control group (extract non-administration model control group), respectively.
  • 18A-18F Effect of extracts on the proportion of each spleen immune cell in mice injected with CYP. # and *: the difference (p-value ⁇ 0.05) compared to the excipient control group and the CYP administration control group (extract non-administration model control group), respectively.
  • Plant collection and extraction preparation Plant collection and extraction preparation:
  • TDM macrophages
  • Mouse macrophage RAW 264.7 cells were cultured in DMEM (Dulbecco's modified eagle's medium) medium supplemented with 1% penicillin-streptomycin-neomycin and 10% heat-inactivated fetal bovine serum (FBS). cultured. For subculture, the cells were seeded at a density of 1.5 ⁇ 10 6 cells per T75 culture flask. Subculture was performed at a cell culture density of about 70-80%.
  • DMEM Dynabecco's modified eagle's medium
  • FBS heat-inactivated fetal bovine serum
  • the THP-1 cell line was cultured in RPMI-1640 medium supplemented with 0.05 mM 2-mercaptoethanol, 1% penicillin-streptomycin-neomycin mixture and 10% FBS, and subcultured every 2-3 days.
  • PMA phorbol 12-myristate-13-acetate
  • Differentiated THP-1 cell line-derived macrophages were stabilized in PMA-free medium for 48 h before treatment with extract and LPS.
  • RAW 264.7 macrophages (1 ⁇ 10 4 cells/100 ⁇ l) and TDM (4 ⁇ 10 4 cells/100 ⁇ l) were spread on a 96-well plate and cultured at 37° C., 5% CO 2 in an incubator for 24 hours.
  • the cells were treated with extracts of various concentrations, incubated for 2 hours, and then cultured for 24 hours after addition of 1 ⁇ g/ml LPS. 10 ⁇ l of CCK-8 reagent was added to each well and incubated for another 3 hours.
  • the absorbance was read at 450 nm under a calibration wavelength of 650 nm.
  • nitrite a stable oxidizing substance of NO
  • the production level of nitrite, a stable oxidizing substance of NO, in the cell culture medium was measured using the Griess reagent.
  • the cells were laid out in a 24-well culture plate at a density of 5 ⁇ 10 4 cells/ml and cultured at 37° C., 5% CO 2 in an incubator for 24 hours. Thereafter, the cells were treated with extracts of various concentrations, and after incubation for 2 hours, LPS (1 ⁇ g/ml) was added to all wells except for the LPS inactive control group (inactive control group). After incubation for 24 hours, the supernatant was collected by centrifugation.
  • ROS Intracellular Reactive Oxygen Species
  • Non-fluorescent 2', 7'-dichlorodihydrofluorescein-diacetate (2', 7'-dichlorodihydrofluorescein diacetate, H2DCF-DA) is a method for converting DCF, a highly fluorescent derivative, in the presence of ROS. Put 5 ⁇ 10 5 cells/ml per well in a 24-well culture plate, and incubate at 37° C., 5% CO 2 in an incubator for 24 hours.
  • the cells were laid out in a 24-well culture plate at a density of 5 ⁇ 10 4 cells/ml and cultured at 37° C., 5% CO 2 in an incubator for 24 hours. Thereafter, the cells were treated with extracts of various concentrations, and after incubation for 2 hours, LPS (1 ⁇ g/ml) was added to all wells except for the LPS inactive control group (inactive control group). After incubation for 24 hours, the supernatant was collected by centrifugation. Using the obtained supernatant, the production levels of prostaglandin E 2 , IL-6, IL-1 ⁇ and TNF- ⁇ were analyzed by ELISA method.
  • the cells were laid out in a 24-well culture plate at a density of 5 ⁇ 10 4 cells/ml and cultured at 37° C., 5% CO 2 in an incubator for 24 hours. Thereafter, the cells were treated with extracts of various concentrations, and after incubation for 2 hours, LPS (1 ⁇ g/ml) was added to all wells except for the LPS inactive control group (inactive control group). After incubation for 24 hours, the supernatant was collected by centrifugation. Using the obtained supernatant, TNF ⁇ , IL-8, and IL-6 were analyzed by ELISA method. In the case of IL-1 ⁇ , 3 mM ATP was added to the supernatant 45 minutes before supernatant collection and quantified by ELISA method.
  • RAW 264.7 cells (2 ⁇ 10 6 ) were cultured in a 90 x 20mm cell culture plate for 24 hours, treated with extracts of various concentrations, incubated for 3 hours, and then treated with LPS (1 ⁇ g/ml).
  • LPS 1 ⁇ g/ml
  • DPBS addition after 15 min (for phospho-ERK, total ERK, phospho-JNK, JNK, phospho-p38, p38), 30 min (for phospho-I ⁇ B alpha and NF- ⁇ B) and 24 h (for COX2) After that, the cells were collected by scraping.
  • Cells were collected in 1.5 ml Eppendorf tubes by centrifugation at 4° C., 1500 rpm for 5 minutes, and then washed twice with 1 ml of DPBS at 4° C., 4000 rpm for 3 minutes.
  • the collected cells were lysed in RIPA buffer containing 1% protease inhibitor cocktail, 1 mM sodium fluoride (NaF) and 1 mM phenylmethylsulfonyl fluoride (PMSF).
  • RIPA buffer was added while the cells were refrigerated and then vortexed every 10 minutes for 30 minutes. Then, the tube was centrifuged at 4 °C, 1600 ⁇ g for 30 minutes, and the supernatant was collected.
  • Nuclear lysates were extracted using NE-PER nuclear and cytoplasmic extraction reagent to analyze nuclear NF- ⁇ B (nuclear NF- ⁇ B) levels.
  • PVDF membrane polyvinylidene difluoride membrane moved to Non-specific binding was blocked by placing the membrane in tris-buffered saline (TBS) containing 0.1% Tween 20 (TBST) and 5% DifcoTM skim milk or 5% BSA and rotational stirring at room temperature for 2 hours.
  • TDM 8.8 ⁇ 10 6 cells
  • TDM 8.8 ⁇ 10 6 cells
  • LPS 1 ⁇ g/ml
  • 3 mM ATP was added to the cells and further incubated for 45 minutes.
  • the following procedure was the same as that of RAW 264.7 cells.
  • NLRP3 (D2P5E) rabbit monoclonal antidody was added to 5% BSA-TBST buffer, and anti rabbit IgG HRP antibody was added to 5% skim milk, and used as primary and secondary antibodies, respectively. .
  • mice Specific pathogen-free 4-week-old male BALB/c mice were used for the study. Animals were housed in sterile cages with ventilation chambers in a specific pathogen-free facility maintained at 22 ⁇ 2 °C with 50 ⁇ 5% relative humidity and a 12 hour light-dark cycle. All mice had free access to standard rodent diet and autoclaved filtered water. All management and experimental procedures were performed in accordance with the approval of the Daegu Catholic University Animal Ethics Committee (IACUC-2018-015).
  • mice were divided into 4 groups, 6 mice per group, for screening for the immunomodulatory ability test of the extract.
  • Excipients were administered to 1 group out of 4 groups, and extracts at different concentrations (5, 50, 500 mg/kg body weight/day/100 ⁇ l) were administered to the other 3 groups for 21 days (4 weeks excluding weekends). Administered through the inner tube.
  • mice were divided into 6 groups. Of these groups, two groups (the excipient control group in which only excipients were administered without CYP injection, and the model control group in which CYP injections and excipients were administered without administration of excipients) were administered intragastrically, and the remaining groups were administered with different concentrations of the excipients suspended in the excipients.
  • the extract was administered intragastrically.
  • As an excipient DMSO and physiological saline containing 2% of Tween 80, respectively, were used.
  • Vehicle control mice were injected with 100 ⁇ l of vehicle, and all other mice were intraperitoneally injected with 100 mg/kg body weight/100 ⁇ l of CYP on days 1, 7, and 14 to induce immunosuppression.
  • DGMIF Daegu Gyeongbuk Medical Innovation Foundation
  • Serum isolated from cardiac blood was used to evaluate the level of immunoglobulin in the serum.
  • serum Serum isolated from cardiac blood was used to evaluate the level of immunoglobulin in the serum.
  • spleen single cells were treated with 1 ⁇ g of LPS, recombinant mouse IL-4 (50 ng) and recombinant human APRIL (10 ng) added to 37° C., 5% CO 2 incubator for 96 hours. during incubation in complete RPMI medium.
  • Antibody levels were determined using a sandwich ELISA.
  • Splenic and mesenteric lymph node T lymphocytes were activated with 5-unit recombinant human IL-2 and immobilized anti-CD3e mAb (5 ⁇ g/5 ⁇ 10 5 cells) in an incubator at 37° C., 5% CO 2 for 48 hours.
  • cytokines interferon-gamma (IFN- ⁇ ), tumor necrosis factor-alpha (TNF- ⁇ ), interleukin-4 (interleukin-4, IL-4), interleukin-17 (IL-17) and transforming growth factor-beta 1 (TGF- ⁇ 1) measured in spleen cell T lymphocyte culture supernatant and sandwiched levels of IFN- ⁇ and IL-17 in mesenteric lymph node T lymphocyte culture supernatant It was measured using ELISA.
  • IFN- ⁇ interferon-gamma
  • TNF- ⁇ tumor necrosis factor-alpha
  • TNF- ⁇ tumor necrosis factor-alpha
  • IL-4 interleukin-4
  • IL-17 interleukin-17
  • TGF- ⁇ 1 transforming growth factor-beta 1
  • Splenocytes and thymocytes were processed for flow cytometry analysis.
  • Splenocytes are screened for CD4+ CD4+ helper T lymphocytes), CD8+ apoptotic T lymphocytes), B220 B lymphocytes and natural killer cell (NK cell) phenotypes, and thymocytes are CD4+, CD8+, and CD4+CD8+ double-positive T Lymphocytes were analyzed. 10 6 cells were washed with sodium azide-containing phosphate buffer solution (PBS).
  • PBS sodium azide-containing phosphate buffer solution
  • Anti-CD3 PE, anti-CD3 FITC, anti-CD4 FITC, anti-CD8 PE, anti-CD45RB/B220 PE, anti-CD335 (NKp46) PE in the state of preventing non-specific binding with FcBlock (1 ⁇ g/10 6 cells) and anti-CD49b APC were used to sort specific cell populations.
  • FITC-conjugated, PE-conjugated, and APC-conjugated isotype controls were also used.
  • Data were expressed as mean ⁇ standard deviation (SD) or mean ⁇ standard error of mean (SEM). Data were screened for normal distribution and equal variance. Significant differences between groups were investigated using one-way analysis of variance (ANOVA) or Kruskal-Wallis rank test according to data normality. If there was a significant difference between groups, the Student-Newman-Keuls (SNK) post-hoc method was additionally used. When it was necessary to perform a comparison between two groups, either the Student's t-test or the non-distributed nonparametric test was used depending on the data distribution. A p-value of 0.05 or less was considered statistically significant.
  • SD standard deviation
  • SEM mean ⁇ standard error of mean
  • Example 1 Effect of extract on viability of LPS-activated macrophages:
  • Example 2 Inhibition of NO, PGE 2 and ROS production by extracts in LPS-activated RAW 264.7 macrophages:
  • Example 3 Efficacy of inhibiting proinflammatory cytokine production by extracts in LPS-activated RAW 264.7 cell line and THP-1 derived macrophage cell line:
  • TDM THP-1 cell-derived macrophages
  • the extract was added to the TDM-differentiated cells, incubated for 2 hours, LPS (1 ⁇ g/ml) was added, and the culture supernatant was collected after 24 hours of incubation.
  • IL-6, TNF- ⁇ , IL-1 ⁇ , and IL-8 levels in culture were analyzed using ELISA. Values are presented as the mean ⁇ SEM of three independent experiments.
  • IL-6, TNF ⁇ , and IL-1 ⁇ significantly produced from RAW 264.7 cells following LPS activation were decreased by the addition of the extract.
  • IL-6 and IL-1 ⁇ were dose-dependently lowered by extract concentrations ranging from 30 to 125 ⁇ g/ml (Figs. 4a, 4c), and for TNF- ⁇ production at concentrations of 100 ⁇ g/ml and 125 ⁇ g/ml was significantly reduced (Fig. 4b).
  • IL-6, TNF- ⁇ , IL-1 ⁇ , and IL-8 levels were dose-dependently decreased according to the concentration of the extract ( FIG. 5 ).
  • Example 4 Effect of extracts on COX-2 protein expression in LPS-activated RAW 264.7 cells:
  • LPS significantly increased COX-2 protein expression by about 3 times compared to the inactive control group, and the extract inhibited COX-2 expression in a dose-dependent manner.
  • COX-2 inhibitors inhibit inflammation by blocking the synthesis of PGE 2 (Eliopoulos, AG, EMBO. J., 2002, 21: 4831-4840). Accordingly, it can be inferred that the extract of the present invention is accompanied by a decrease in PGE 2 production according to the inhibition of COX-2 expression.
  • Example 5 Effect of extracts on I ⁇ B ⁇ and NF- ⁇ B activation in LPS-activated RAW 264.7 cells:
  • Example 6 Effect of extracts on MAPK activation in LPS-activated RAW 264.7 cells:
  • the MAPK activation pathway consists of c-Jun N-terminal kinase (JNK), extracellular signal-regulated protein kinases (ERK1/2), and p38 MAPK, leading to an inflammatory response. It has been reported to play a substantial major role in product regulation.
  • JNK c-Jun N-terminal kinase
  • ERK1/2 extracellular signal-regulated protein kinases
  • p38 MAPK extracellular signal-regulated protein kinases
  • Example 7 Effect of extracts on NLRP3 expression in LPS-activated TDM cell lines:
  • TDM THP-1-derived macrophages
  • NLRP3 enhancement of THP-1-derived macrophages was observed following the addition of LPS and ATP, and the extract inhibited NLRP3 expression in a dose-dependent manner ( FIG. 9 ).
  • Example 8 Analysis of histopathological characteristics according to the administration of the extract:
  • the % of neutrophils in the 50 mg/kg extract administration group was higher than that of the excipient control group, and the % of monocytes was lower than the control group at various administration concentrations.
  • the lymphocyte % was higher in the 50 mg/kg and 500 mg/kg extract administration groups (Table 2).
  • WBCs white blood cells (white blood cells); RBCs, red blood cells; HGB, hemoglobin (hemoglobin); NEUT, neutrophils (neutrophils); LYM, lymphocytes (lymphocytes); MONO, monocytes (monocytes); EOS, eosinophils (eosinophils); LUC, large unstained cells; BASO, basophils (basophils).
  • Data are presented as mean ⁇ SD . *, #, $ A significant difference compared with the control group, 50 mg/kg and 500 mg/kg ruby administration groups, respectively ( p -value ⁇ 0.05).
  • IgG1 and IgG2a The effect of extract administration on IgG subtypes (IgG1 and IgG2a), IgA and IgE serum levels was evaluated. Immunoglobulin levels were analyzed in serum isolated from mouse heart blood collection. Data are expressed as mean ⁇ SEM.
  • the level of IgE which is an indicator of allergic reaction, was significantly lower in the 500 mg/kg extract-administered group than in the control group ( FIG. 10d ).
  • the level of IgA an important index for the mucin immune response, was high in a concentration-dependent manner in the extract ( FIG. 10e ).
  • Example 11 Effect of extract on splenic T lymphocyte cytokine production:
  • cytokine levels produced in splenic T lymphocyte culture supernatants was evaluated. Data are expressed as mean ⁇ SEM. After activation with immolized anti-CD3e mAb (5 ⁇ g/5x10 5 cells) for 48 hours at 37°C in a 5% CO 2 incubator, the culture supernatant was collected and cytokines (IFN- ⁇ , IL-4, IL-17, TNF- ⁇ and TGF- ⁇ 1) were measured. The final concentration was obtained by subtracting the cytokine concentration from the inactivated cells as a control from the cytokine concentration in the immolized anti-CD3e mAb activated cells.
  • immolized anti-CD3e mAb 5 ⁇ g/5x10 5 cells
  • cytokines IFN- ⁇ , IL-4, IL-17, TNF- ⁇ and TGF- ⁇ 1
  • IFN- ⁇ level was lower than that of the control group in the 5 and 50 mg/kg extract administration group (FIG. 11a), but there was no difference in the 500 mg/kg administration group and the control group.
  • IL-4 production levels did not differ significantly between groups (Fig. 11b). Accordingly, the ratio of IFN- ⁇ :IL-4 was lower in the 5 and 50 mg/kg extract-administered groups than in the control group, and was high in the 500 mg/kg-administered group ( FIG. 11c ).
  • the pro-inflammatory cytokine IL-17 was lower in the 500 mg/kg administration group than in the control group, but there was no significant difference ( FIG. 11d ).
  • TNF- ⁇ another proinflammatory cytokine
  • TGF- ⁇ 1 was significantly lower in the 5 and 50 mg/kg administration groups than in the control group, and the inflammatory response promoting cytokine TGF- ⁇ 1 did not show a significant difference from the control group ( FIGS. 11e and 11f ).
  • Example 12 Quantitative analysis of splenic and thymic lymphocyte subpopulation rates:
  • Example 13 Effect of extracts on spleen and thymus indices of immunosuppressed mice injected with CYP:
  • the effect of the extract on the spleen and thymus indices of mice injected with cyclophosphamide (CYP) was evaluated. Data are expressed as mean ⁇ SEM.
  • the spleen index and thymus index are calculated by dividing the spleen or thymus weight of the mouse by the body weight.
  • the spleen index was significantly higher in the mice injected with CYP, and the spleen index was lower in the extract-administered group than in the model control group administered only with CYP.
  • the thymus index was significantly lower in the CYP-administered group than in the excipient control group, but the thymus index was higher in the extract-administered group in a dose-dependent manner, and in particular, the extract at the highest concentration showed a statistically significant difference (FIG. 12) .
  • Example 14 Effect of extracts on hematological parameters of immunosuppressed mice injected with CYP:
  • Example 15 Effect of extracts on immunoglobulin levels in serum and B lymphocyte cultures of immunosuppressed mice injected with CYP:
  • Serum immunoglobulin levels were evaluated in mice administered CYP or extract. Data are expressed as mean ⁇ SEM.
  • the level of immunoglobulin in the culture supernatant generated from splenic B lymphocytes of mice administered with CYP or the extract was evaluated. Data are expressed as mean ⁇ SEM. Splenocytes (1x10 6 ) were activated and cultured with LPS, recombinant mouse IL-4 and recombinant human APRIL for 96 h. The final concentration was obtained by subtracting the level of immunoglobulin generated from non-activated cells as a control from the level of immunoglobulin in activated cells.
  • the levels of all immunoglobulins (IgG1, IgG2a, IgE and IgA) measured in the serum were significantly lower in the CYP-injected group compared to the vehicle control group ( FIG. 14 ).
  • all immunoglobulin levels were enhanced compared to the model control group injected with only CYP.
  • the levels of IgG2a and IgA were significantly higher in the extract-administered group compared to the model control group ( FIGS. 14b and 14e ).
  • the levels of IgG1 and IgE were still lower than that of the excipient control group ( FIGS. 14a and 14d ).
  • the IgG2a/IgG1 ratio was significantly higher in the extract-administered group than in the excipient control group and the model control group (FIG. 14c).
  • the CYP immunosuppressed model control group showed decreased IgG1 and IgG2a levels compared to the excipient control group, and the extract-administered group showed decreased IgG1, The results of restoring the IgG2a level were shown (FIGS. 15a, 15b).
  • Example 16 Effect of extracts on splenic T lymphocyte-producing cytokine levels in immunosuppressed mice injected with CYP:
  • the effect of the extract on the cytokine levels produced in the splenic T lymphocyte culture supernatant of mice injected with CYP was evaluated. Data are expressed as mean ⁇ SEM. After activation with immolized anti-CD3e mAb (5 ⁇ g/5x10 5 cells) for 48 hours in a 37° C., 5% CO 2 incubator, the culture supernatant was collected to measure cytokines. The final concentration was obtained by subtracting the cytokine concentration from the non-activated cells as a control from the cytokine concentration in the immolized anti-CD3e mAb activated cells.
  • mice When immunosuppression was induced by CYP, the spleens of these mice were harvested and activated in vitro. Compared to the excipient control group, IFN- ⁇ and IL-4 production levels decreased, while IL-17 and TGF- ⁇ 1 levels increased, and TNF ⁇ was no difference (Fig. 16). In particular, IFN- ⁇ , IL-4, and TNF- ⁇ production levels were higher in the extract-administered group than in the model control group, and were most pronounced in the low-concentration extract-administered group (5 mg/kg body weight).
  • Example 17 Effect of extracts on mesenteric lymph node T-lymphocyte-producing cytokine levels in immunosuppressed mice injected with CYP:
  • the effect of the extract on the cytokine levels produced in the mesenteric lymph node T lymphocyte culture supernatant of mice injected with CYP was evaluated. Data are expressed as mean ⁇ SEM.
  • Mesenteric lymph node single cells were activated with immolized anti-CD3e mAb (5 ⁇ g/5x10 5 cells) in a 5% CO 2 incubator at 37° C. for 48 hours, and then the culture supernatant was collected to measure cytokines. The final concentration was obtained by subtracting the cytokine concentration from the non-activated cells as a control from the cytokine concentration in the immolized anti-CD3e mAb activated cells.
  • IFN- ⁇ and IL-17 As a result of evaluating the effect of the extract on intestinal immunity through cytokine level analysis produced by activating mesenteric lymph node T lymphocytes in vitro, significant differences were observed in IFN- ⁇ and IL-17 ( FIG. 17 ).
  • the level of IFN- ⁇ was significantly higher in the extract-administered group compared to both the excipient control group and the model control group, and the inflammatory cytokine IL-17 level, which has been proven to be involved in the pathogenesis of inflammatory bowel disease (IBD), was higher in the model control group than in the model control group. was significantly lower than
  • Example 18 Effect of extracts on the proportion of splenic lymphocyte subpopulations in mice administered CYP:
  • the number of helper T lymphocytes, apoptotic T lymphocytes, B lymphocytes, and natural killer cells were all lower than those of the vehicle control group ( FIG. 18 ).
  • the proportion of these cells was significantly higher than that of the model control group, but it was still lower than the level of the excipient control group.
  • the level of natural killer cells was significantly higher than that of the excipient control group.

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Abstract

The present invention provides a composition for boosting the immunity of a subject, the composition comprising a <i />(Choerospondias axillaris) extract as an active component. The present invention also provides a composition for use as an anti-inflammatory medicine, the composition comprising a <i />(Choerospondias axillaris) extract as an active component.

Description

체로스폰디아스 액실라리스 추출물의 이용 Use of Cherospondias axillaris extract
본 발명은 체로스폰디아스 액실라리스 ( Choerospondias axillaris) 추출물을 유효 성분으로 포함하는 면역 증강용 조성물에 관한 것이다.The present invention is Cherospondias axillaris ( Choerospondias axillaris ) It relates to a composition for enhancing immunity comprising an extract as an active ingredient.
최근 기능성 식품의 개념 및 건강 증진과 질병 예방에서의 그 역할에 대한 관심이 늘어나고 있다. 이전의 연구에서 과일 및 채소의 섭취가 심혈관 질환, 노화 관련 문제, 암 또는 염증 위험성 감소와 관련이 있다고 제안되었다 (Hasler, C.M. Food Technol., 1998, 52: 63-147; Lu, C.C. and Yen, G.C. Curr. Opin. Food Sci., 2015, 2:1-8). 이러한 효과는 카로티노이드 (carotenoids), 비타민 C, 페놀릭 (phenolics) 성분과 같은 과일에 함유되어 있는 성분에 기인한다. Recently, interest in the concept of functional food and its role in health promotion and disease prevention is increasing. Previous studies have suggested that consumption of fruits and vegetables is associated with a reduced risk of cardiovascular disease, age-related problems, cancer, or inflammation (Hasler, CM Food Technol., 1998, 52: 63-147; Lu, CC and Yen, GC Curr. Opin. Food Sci., 2015, 2:1-8). This effect is due to the ingredients in the fruit, such as carotenoids, vitamin C, and phenolics.
일반적으로 네팔 돼지 감자 (네팔 호그 플럼, Nepali hog plum) 또는 럽시 (Lapsi)라고도 불리는 체로스폰디아스 액실라리스 ( Choerospondias axillaris)는 원산지가 네팔이며, 옻나무과 ( Anacardiaceae)에 속하는 큰 식용 과수이다. Choerospondias axillaris , commonly called Nepali hog plum or Lapsi, is native to Nepal and is a large edible fruit tree belonging to the Anacardiaceae family.
럽시 과실은 아미노산, 비타민 C, 환원성 당류, 비 환원성 당류, 펙틴, 유기산 및 미량 원소와 같은 다양한 성분으로 구성되며, 화학적으로 디히드로퀘르세틴 (dihydroquercetin, quercetin), 퀘르세틴 (quercetin), 프로토카테산 (protocatechuic acid), 갈산 (gallic acid), 3,3-디-o-메틸엘라그산 (3,3-di-o-methylellagic acid), B-시토스테롤 (B-sitosterol), 도코스테롤 (daucosterol), 스테아르산 (steric acid,) 트리아코탄산 (triacontanoic acid), 옥타코사놀 (octacosanol), 시린알데히드 (syringaldehyde), 바닐산 (vanillic acid) 및 시트르산 (citric acid)으로 구성된다.Lush fruit is composed of various components such as amino acids, vitamin C, reducing sugars, non-reducing sugars, pectins, organic acids and trace elements, chemically dihydroquercetin (quercetin), quercetin, protocatechuic acid (protocatechuic) acid), gallic acid, 3,3-di-o-methylellagic acid, B-sitosterol, docosterol (daucosterol), stearic acid (steric acid) is composed of triacontanoic acid, octacosanol, syringaldehyde, vanillic acid and citric acid.
"생약(crude drug)"에는 다양한 혼합물이 내재되어 단일 물질에 대한 효과를 규명하지 못한다는 비판은 있지만 많은 연구에서 약용 식물의 효과는 단일 물질의 효과라기 보다는 여러 화합물의 복합, 상승 작용에 기인한다고 많은 연구자들을 적시하고 있다 (Spleman, K. et al., Nat. Prod. From Plants., 2006, 475-501; Wagner, H., Environ. Health Perspect., 1999, 107:779-781; Williamson, E.M., Phytomedicine, 2001, 8:401-409).Although there are criticisms that "crude drugs" contain various mixtures and cannot determine the effect on a single substance, many studies say that the effect of medicinal plants is due to the complex and synergistic action of several compounds rather than the effect of a single substance. Numerous researchers are pointed out (Spleman, K. et al., Nat. Prod. From Plants., 2006, 475-501; Wagner, H., Environ. Health Perspect., 1999, 107:779-781; Williamson, EM, Phytomedicine, 2001, 8:401-409).
건조하고 잘 익은 럽시 과실은 일반적으로 중국 및 몽골의 전통의학에서 진정제 (sedative) 및 강심제 (cardiotonic)로 사용된다 (Tang, W. and Eisenbrand. G. Chinese drugs of plant origin, 1992, 710-737; Li, C. et al. Cardiovasc.Toxicol., 2014, 14:145-152). 럽시 과실의 총 플라본 (total flavones of Choerospondias axillaris Fructus, TFC)은 랫드에서 아드리아마이신 (Adriamycin)으로 유도된 심근과산화손상 (myocardial peroxidation injury)에 대한 보호 효과를 갖는 것으로 밝혀졌으며, 잎에서 추출한 총 플라본 (TFC folium, TFCF)은 아코니틴 (aconitine)으로 생성되는 부정맥에서 항부정맥 작용을 나타낸다 (Xinyuan, Z. et al. J. Chinese Med. Mater., 2001, 3; Qiu, M. et al. J. Toxicol. Environ. Heal.-Part A Curr. Issues., 2016, 79:878-883). 또한, TFC의 기능에는 저산소 내성 (hypoxic tolerance), 심근 허혈 보호 (myocardial ischemia protection), 혈소판 응집 억제 (platelet congregation inhibition), 혈류 개선 (hemorheology improvement) 및 마우스 면역 기능 향상 (immune function enhancement)이 포함된다 (Lijuan, D.G.J.L Sport. Sci.,2002, 5). 또한, TFC는 랫드에서 덱사메타손 (dexamethasone)으로 유발된 흉선세포 자멸사를 억제하고 이소프로테레놀 (isoproterenol)로 유발된 손상에서 크레아틴 키나아제 (Creatine kinase, CK), 크레아틴 키나아제-MB (Creatine kinase-MB) 및 락트산 탈수소효소 (Lactic Acid Dehydrogenase, LDH)의 혈청 수준을 감소시켰다 (Li, B. et al. Chinese J. Microbiol. Immunol., 1998, 18 : 235-241; Ao, J. et al. Cardiovasc. Drugs Ther., 2007, 21 : 235-241).Dried and ripe ruby fruits are commonly used as sedative and cardiotonic in traditional Chinese and Mongolian medicine (Tang, W. and Eisenbrand. G. Chinese drugs of plant origin, 1992, 710-737; Li, C. et al. Cardiovasc. Toxicol., 2014, 14:145-152). Total flavones of Choerospondias axillaris Fructus (TFC) from the Ruby fruit were found to have a protective effect against adriamycin-induced myocardial peroxidation injury in rats, and total flavones ( TFC folium, TFCF) exhibits antiarrhythmic action in arrhythmias produced by aconitine (Xinyuan, Z. et al. J. Chinese Med. Mater., 2001, 3; Qiu, M. et al. J (Toxicol. Environ. Heal.-Part A Curr. Issues., 2016, 79:878-883). In addition, the functions of TFC include hypoxic tolerance, myocardial ischemia protection, platelet congregation inhibition, hemorheology improvement and mouse immune function enhancement. (Lijuan, DGJL Sport. Sci., 2002, 5). In addition, TFC inhibits dexamethasone-induced thymocyte apoptosis in rats and creatine kinase (CK), creatine kinase-MB (Creatine kinase-MB) in isoproterenol-induced damage. and lactate dehydrogenase (LDH) (Li, B. et al. Chinese J. Microbiol. Immunol., 1998, 18:235-241; Ao, J. et al. Cardiovasc. Drugs Ther., 2007, 21:235-241).
이전 연구에서는 대부분 럽시의 과실, 껍질 또는 잎으로부터의 얻어진 총 플라본을 사용했으며 심장 관련 장애 또는 산화 스트레스에서의 그 유효성에 대해 시험하였다 (Li, C. al., Cardiovasc. Toxicol., 2014, 14 : 145- 152). 물 또는 에탄올 추출물 전체를 이용한 연구는 이루어지지 않았다. 총 과실 추출물에 대한 한 연구에서 럽시 추출물을 DPPH 라디칼 분석으로 정량한 바, 아스코르브산 (ascorbic acid )과 유사한 항산화 기능 가능성이 있다고 발표되었다 (Labh, S.N. et al., J. Pharmacogn. Phytochem., 2015, 4 : 194). 또 다른 실험에서, 럽시 추출물이 포함된 사료를 매일 급여한 어류에서 혈액학적 지표치들의 항진이 보고되었다 (Labh, S.N. and Shakya, S.R., 2016, 4:127-131). Previous studies have mostly used total flavones obtained from the fruits, skins or leaves of rupsi and tested their effectiveness in heart-related disorders or oxidative stress (Li, C. al., Cardiovasc. Toxicol., 2014, 14: 145- 152). No studies were conducted with water or ethanol extracts as a whole. In one study on total fruit extract, it was reported that Rubsy extract had a potential for antioxidant function similar to that of ascorbic acid as quantified by DPPH radical analysis (Labh, SN et al., J. Pharmacogn. Phytochem., 2015 , 4:194). In another experiment, enhancement of hematological parameters was reported in fish fed daily diets containing ruby extract (Labh, S.N. and Shakya, S.R., 2016, 4:127-131).
면역 조절에 대한 식물 추출물 연구는 주로 T 림프구, 싸이토카인 생성, 항체 생성, 자가 면역 장애, 세포자멸사, 항 미생물, 항암 또는 항산화 효과에 대한 영향을 중심으로 한다 (Huang, CF et al. Cell Mol. Immunol., 2008, 5:23). Plant extract studies on immune modulation mainly focus on their effects on T lymphocytes, cytokine production, antibody production, autoimmune disorders, apoptosis, antimicrobial, anticancer or antioxidant effects (Huang, CF et al. Cell Mol. Immunol). ., 2008, 5:23).
이때, 자가 면역 질환에서는 항체, 싸이토카인등이 숙주 조직을 파괴할 수도 있기 때문에, 면역 조절과 관련한 연구에 있어서는 면역 체계를 자극하는 것 뿐만 아니라 면역 체계를 억제하는 것 역시 특별한 관심 대상이 될 수 있다. At this time, since antibodies, cytokines, etc. may destroy host tissues in autoimmune diseases, suppressing the immune system as well as stimulating the immune system may be of special interest in research related to immune regulation.
한편, 염증 질환과 관련하여 현재는 주로 염증 반응의 물리적 불편함을 감소시키는 의약 제제를 투여하여 염증 질환을 치료하고 있으나 일반적인 항염증성 의약은 광범위한 질병의 치료에 사용되고, 동일한 의약이 종종 상이한 질병의 치료에 이용되기 때문에 치료 작용 및 부작용을 공유하게 된다. 부작용으로는 호흡 촉진 현상, 순환계 붕괴, 상복부 통증, 구토, 위장 출혈, 간 손상 및 혈소판 저해 등의 증상이 알려진 바 있다. 이러한 부작용 때문에 기존의 항염증용 의약은 장기적으로 사용하기가 곤란하고, 현재까지의 치료법에 부작용의 심각성이 크기 때문에 새롭거나 개선된 치료제 개발은 필수적이고 시급한 실정이며, 부작용에 대한 우려가 없는 인체에 안전한 항염증용 의약의 필요성이 대두되고 있다.On the other hand, in relation to inflammatory diseases, currently, inflammatory diseases are mainly treated by administering pharmaceutical agents that reduce the physical discomfort of the inflammatory response, but general anti-inflammatory medicines are used for the treatment of a wide range of diseases, and the same medicine is often used to treat different diseases. Because it is used for treatment, it shares therapeutic action and side effects. As side effects, symptoms such as respiratory stimulation, circulatory collapse, epigastric pain, vomiting, gastrointestinal bleeding, liver damage, and platelet inhibition have been known. Because of these side effects, it is difficult to use the existing anti-inflammatory drugs in the long term, and the serious side effects of the current treatment methods are large, so the development of new or improved therapeutic agents is essential and urgent. The need for safe anti-inflammatory drugs is emerging.
이에, 면역 기능을 증강시키고 염증에 대항하기 위해 합성 약물과 화학 물질을 대체할 천연 유래 제품에 대한 요구는 계속되고 있다. Accordingly, there is a continuing demand for natural products to replace synthetic drugs and chemicals to enhance immune function and fight inflammation.
본 발명의 목적은 대상체의 면역 증강을 위한 체로스폰디아스 액실라리스 ( Choerospondias axillaris) 추출물을 유효 성분으로 포함하는 조성물을 제공하는 것이다. 또한, 본 발명의 목적은 항염증용 의약으로 사용하기 위한 체로스폰디아스 액실라리스 ( Choerospondias axillaris) 추출물을 유효 성분으로 포함하는 조성물을 제공하는 것이다.An object of the present invention is Cherospondias axillaris for enhancing the immune system of a subject ( Choerospondias axillaris ) To provide a composition comprising an extract as an active ingredient. In addition, an object of the present invention is Cherospondias axillaris for use as an anti-inflammatory medicament ( Choerospondias axillaris ) To provide a composition comprising an extract as an active ingredient.
상기 목적을 달성하기 위하여, 본 발명의 일 측면은 체로스폰디아스 액실라리스 ( Choerospondias axillaris) 추출물을 유효 성분으로 포함하는 면역 증강용 조성물을 제공한다. In order to achieve the above object, one aspect of the present invention is Cherospondias axillaris ( Choerospondias axillaris ) Provides a composition for enhancing immunity comprising an extract as an active ingredient.
상기 체로스폰디아스 액실라리스 추출물은 과실부의 추출물인 것일 수 있다.The Cherospondias axillaris The extract may be an extract of a fruit part.
상기 체로스폰디아스 액실라리스 추출물은 에탄올을 이용하여 추출된 것일 수 있다.The Cherospondias axillaris The extract may be extracted using ethanol.
상기 본 발명의 면역 증강용 조성물은 그 투여 전과 비교하여 그 투여 후에 대상체에서 IgG2a/IgG1 수준을 증가시킬 수 있다.The composition for enhancing immunity of the present invention may increase the level of IgG2a/IgG1 in the subject after administration as compared to before administration.
상기 본 발명의 면역 증강용 조성물은 1형 보조 T 림프구 (Type-1 helper T lymphocyte, Th1)에 의한 면역 반응을 촉진시킬 수 있다.The composition for enhancing immunity of the present invention can promote an immune response by type 1 helper T lymphocytes (Th1).
상기 본 발명의 면역 증강용 조성물은 그 투여 전과 비교하여 그 투여 후에 대상체에서 IgE 항체 및 IgG1 항체 중에서 선택되는 하나 이상의 수준을 감소시킬 수 있다.The composition for enhancing immunity of the present invention can reduce the level of one or more selected from IgE antibody and IgG1 antibody in the subject after the administration compared to before the administration.
상기 본 발명의 면역 증강용 조성물은 그 투여 전과 비교하여 그 투여 후에 대상체에서 IgA 항체 수준을 증가시킬 수 있다.The composition for enhancing immunity of the present invention may increase the IgA antibody level in the subject after the administration compared to before the administration.
상기 본 발명의 면역 증강용 조성물은 그 투여 전과 비교하여 그 투여 후에 대상체에서 산화질소 (nitric oxide, NO), 프로스타글란딘 E 2 (PGE 2), COX-2 및 활성 산소종 (reactive oxygen species, ROS) 중에서 선택되는 하나 이상의 수준을 감소시킬 수 있다.The composition for enhancing immunity of the present invention is nitric oxide (NO), prostaglandin E 2 (PGE 2 ), COX-2 and reactive oxygen species (ROS) in the subject after the administration compared to before the administration. One or more levels selected from among may be reduced.
상기 본 발명의 면역 증강용 조성물은 그 투여 전과 비교하여 그 투여 후에 대상체에서 전염증성 싸이토카인 (pro-inflammatory cytokine)의 수준을 감소시킬 수 있다.The composition for enhancing immunity of the present invention can reduce the level of pro-inflammatory cytokines in the subject after the administration compared to before the administration.
상기 전염증성 싸이토카인은 TNF-α, IL-1β, IL-6, IL-8 및 IL-17 중에서 선택되는 하나 이상인 것일 수 있다.The pro-inflammatory cytokine may be one or more selected from TNF-α, IL-1β, IL-6, IL-8 and IL-17.
상기 본 발명의 면역 증강용 조성물은 기능성 식품일 수 있다.The composition for enhancing immunity of the present invention may be a functional food.
상기 본 발명의 면역 증강용 조성물은 항염증용 의약일 수 있다.The composition for enhancing immunity of the present invention may be an anti-inflammatory drug.
이하, 본 발명을 보다 상세하게 설명한다.Hereinafter, the present invention will be described in more detail.
본 발명은 체로스폰디아스 액실라리스 ( Choerospondias axillaris) 추출물, 즉 럽시 추출물을 유효 성분으로 포함하는 면역 증강용 조성물을 제공한다. The present invention is Cherospondias axillaris ( Choerospondias axillaris ) Extract, that is, provides a composition for enhancing immunity comprising the ruby extract as an active ingredient.
일 구체예에 있어서, 상기 럽시 추출물은 과실부의 추출물인 것일 수 있다.In one embodiment, the ruby The extract may be an extract of a fruit part.
일 구체예에 있어서, 상기 럽시 추출물은 에탄올을 이용하여 추출된 것일 수 있다.In one embodiment, the ruby The extract may be extracted using ethanol.
상기 본 발명의 럽시 추출물은 럽시의 용매 추출물 또는 이로부터 분획한 획분을 의미한다. 상기 용매는 물 또는 탄소수 5 이하의 저가 알코올이며, 바람직하게는 물 또는 탄소수 3 이하의 저가 알코올이고, 더욱 바람직하게는 물 또는 에탄올이고, 가장 바람직하게는 에탄올이다. 예컨대, 상기 럽시 추출물은 럽시 과실, 예컨대 껍질과 과육분말에 에탄올을 첨가하고 여과 후 농축한 농축물을 동결건조하여 조제한 획분일 수 있다. The ruby extract of the present invention means a solvent extract of ruby or a fraction fractionated therefrom. The solvent is water or a low-cost alcohol having 5 or less carbon atoms, preferably water or a low-cost alcohol having 3 or less carbon atoms, more preferably water or ethanol, and most preferably ethanol. For example, the ruby extract may be a fraction prepared by lyophilizing the concentrated concentrate after filtration by adding ethanol to ruby fruit, for example, peel and pulp powder.
모든 면역 세포 중에서, T 림프구의 활성화에는 항원 특이성이 필요하며 T 림프구로부터 다수의 싸이토카인이 생성되기 때문에 T 림프구는 더욱 중요한 것으로 간주된다 (Fox, D.A Off. J. AM. Coll. Rheumatol., 1997, 40: 598-609). T 림프구는 싸이토카인 생성 패턴에 따라 다음과 같은 두 가지 서로 다른 유형으로 분류할 수 있다: IFN-γ 및 IL-2는 전형적으로 1형 보조 T 림프구 (Type-1 helper T lymphocyte, Th1) 세포에 의해 생성되며 2형 보조 T 림프구 (Type-2 helper T lymphocyte, Th2)는 주로 IL-4, IL-5, IL-13 및 IL-10을 생성한다. 이와 유사하게, 항체들은 면역 체계에서 다양한 세균 등의 이물질을 찾아 중화하는데 사용된다. 그러나, 자가 면역 질환에서는 항체, 싸이토카인 등이 숙주 조직을 파괴할 수 있음도 보고되었다. 따라서, 면역체계를 자극할 뿐만 아니라 억제작용을 보이는 본초 추출물도 특별한 관심 대상이 될 수 있다. Of all immune cells, T lymphocytes are considered more important because activation of T lymphocytes requires antigen specificity and the production of multiple cytokines from T lymphocytes (Fox, DA Off. J. AM. Coll. Rheumatol., 1997, 40: 598-609). T lymphocytes can be classified into two different types according to their cytokine production patterns: IFN-γ and IL-2 are typically induced by Type-1 helper T lymphocytes (Th1) cells. Type 2 helper T lymphocytes (Th2) mainly produce IL-4, IL-5, IL-13 and IL-10. Similarly, antibodies are used by the immune system to detect and neutralize foreign substances, such as various bacteria. However, it has also been reported that antibodies, cytokines, etc. can destroy host tissues in autoimmune diseases. Therefore, herbal extracts that not only stimulate the immune system but also have inhibitory effects may be of special interest.
면역 억제란 숙주를 병원체에 보다 취약하게 할 수 있는 일시적 또는 영구적인 면역 손상을 말한다 (Liang, M. et al. Microb. Pathog., 2013, 54 : 40-45). 기존 연구에 따르면 고용량의 단일 싸이클로포스파미드 주사 또는 저용량 반복 주사는 주로 B 림프구 및 억제 T 림프구에 영향을 미친다 (Graziano, F. et al., J. Immunol., 1981, 127 : 1067-1070; Shukla, ML 및 Chaturvedi, UC, Br. J. Exp. Pathol., 1984, 65 : 397). 또한, 싸이클로포스파미드는 림프 및 비림프 조직에 위치한 CD4+ 및 CD8+ 기억 T림프구 아집단에 영향을 미친다 (Siracusa, F. et al., Eur. J. Immunol., 2017, 47:1900-1905; Sheeja, K and Kuttan, G., Asian Pacific J. Cancer Prev., 2006, 7:609-614). 천연물 제품은 마우스에서 싸이클로포스파미드로 유발된 면역억제의 길항작용에 대한 다양한 실험에서 사용되고 있다. Immunosuppression refers to temporary or permanent immune damage that can make the host more susceptible to pathogens (Liang, M. et al. Microb. Pathog., 2013, 54: 40-45). Previous studies have shown that a single high-dose injection of cyclophosphamide or repeated low-dose injections mainly affects B lymphocytes and suppressive T lymphocytes (Graziano, F. et al., J. Immunol., 1981, 127: 1067-1070; Shukla, ML and Chaturvedi, UC, Br. J. Exp. Pathol., 1984, 65:397). Cyclophosphamide also affects CD4+ and CD8+ memory T lymphocyte subpopulations located in lymphoid and non-lymphatic tissues (Siracusa, F. et al., Eur. J. Immunol., 2017, 47:1900-1905; Sheeja , K and Kuttan, G., Asian Pacific J. Cancer Prev., 2006, 7:609-614). Natural products have been used in various experiments on the antagonism of cyclophosphamide-induced immunosuppression in mice.
본 발명에 따르면, 럽시 추출물은 CYP를 주사한 면역억제 마우스의 체액 및 세포성 면역 (humoral and cellular immunity)을 모두 활성화시킬 수 있다. 럽시 추출물은 CYP 주사에 따른 비장 및 흉선의 무게 변화를 유의하게 제어하고, 혈액학적 지표치들의 정량적 변화도 제어할 수 있다. 또한 알레르기와 관련된 혈청 중 항체 수준 변동에 대한 영향은 최소화하면서 면역 억제 마우스의 전반적 항체 생성을 항진시킬 수 있다. 럽시 추출물은 또한 면역 억제로 저하된 말초 면역 세포 (peripheral immune cell)들의 양적 수준도 상향 조절할 수 있는데 특히 자연 살해 세포의 수를 유의하게 증가시킬 수 있다.According to the present invention, Ruby extract can activate both humoral and cellular immunity of immunosuppressed mice injected with CYP. Rubsy extract can significantly control changes in spleen and thymus weight following CYP injection, and also control quantitative changes in hematological parameters. In addition, it is possible to enhance overall antibody production in immunosuppressed mice while minimizing the effect on allergy-related fluctuations in serum antibody levels. Rubsy extract can also up-regulate the quantitative level of peripheral immune cells that are reduced by immunosuppression, in particular, it can significantly increase the number of natural killer cells.
일 구체예에 있어서, 상기 본 발명의 면역 증강용 조성물은 그 투여 전과 비교하여 그 투여 후에 대상체에서 IgG2a/IgG1 수준을 증가시킬 수 있다.In one embodiment, the composition for enhancing immunity of the present invention may increase the IgG2a/IgG1 level in the subject after the administration compared to before the administration.
또 다른 일 구체예에 있어서, 상기 본 발명의 면역 증강용 조성물은 1형 보조 T 림프구 (Type-1 helper T lymphocyte, Th1)에 의한 면역 반응을 촉진시킬 수 있다.In another embodiment, the composition for enhancing immunity of the present invention can promote an immune response by type 1 helper T lymphocytes (Th1).
T 림프구는 암 세포 또는 바이러스에 감염된 세포에 직접 작용하는 세포파괴 T 림프구 (CD8+ T림프구)와 싸이토카인 등 매개 물질을 통하여 다른 면역 세포의 기능을 지원하는 보조 T 림프구로 크게 분류된다 (Globerson, A., Int. Arch. Allergy Immunol, 1995, 107:491-497). 또한, NK 세포는 선척 면역에서 중요한 암세포 혹은 바이러스 감염세포를 사멸시키는데 중요한 역할을 수행한다. T lymphocytes are broadly classified into apoptotic T lymphocytes (CD8+ T lymphocytes) that act directly on cancer cells or virus-infected cells, and helper T lymphocytes that support the functions of other immune cells through mediators such as cytokines (Globerson, A. , Int. Arch. Allergy Immunol, 1995, 107:491-497). In addition, NK cells play an important role in killing cancer cells or virus-infected cells, which are important in innate immunity.
IgG1 항체는 2형 보조 T 림프구에서 생성되는 IL-4에 의해 이소형 스위칭되고 IgG2a는 1형 보조 T 림프구에서 생성되는 IFN-γ에 의해 이소형 스위칭된다. 이에 IgG2a/IgG1 비가 상대적으로 높을수록 예컨대 암 발생, 바이러스 감염에 대한 방어 능력의 핵심인 1형 보조 T 림프구 반응 (type-1 resposne)이 항진되고 알레르기 발생과 연계되어 있는 2형 보조 T 림프구 반응 (type-2 resposne)이 억제되는 것으로 해석된다. 럽시 추출물 투여군에서 IgG2a/IgG1 비가 대조군에 비해 유의하게 높다는 점은 럽시 추출물이 1형 반응을 상대적으로 촉진시킬 수 있는 기능이 있음을 암시한다.IgG1 antibody is isotype switched by IL-4 produced by type 2 helper T lymphocytes and IgG2a is isotype switched by IFN-γ produced by type I helper T lymphocytes. Accordingly, as the IgG2a/IgG1 ratio is relatively high, the type 1 helper T lymphocyte response (type-1 resposne), which is the key to defense against cancer and viral infection, for example, is enhanced, and the type 2 helper T lymphocyte response ( type-2 resposne) is inhibited. The fact that the IgG2a/IgG1 ratio was significantly higher in the Rubsy extract-administered group than in the control group suggests that the Rubsy extract has the ability to relatively promote the type 1 response.
또 다른 구체예에 있어서, 상기 본 발명의 면역 증강용 조성물은 그 투여 전과 비교하여 그 투여 후에 대상체에서 IgE 항체 및 IgG1 항체 중에서 선택되는 하나 이상의 수준을 감소시킬 수 있다.In another embodiment, the composition for enhancing immunity of the present invention may reduce the level of one or more selected from an IgE antibody and an IgG1 antibody in a subject after the administration compared to before the administration.
또 다른 구체예에 있어서, 상기 본 발명의 면역 증강용 조성물은 그 투여 전과 비교하여 그 투여 후에 대상체에서 IgA 항체 수준을 증가시킬 수 있다.In another embodiment, the composition for enhancing immunity of the present invention may increase the IgA antibody level in the subject after the administration compared to before the administration.
IL-4는 알레르기 발생의 대표적 지표 항체인 IgE로의 이소형 스위칭을 유도한다 (Snapper,C.M. et al., Immunol. Rev., 1988, 102:51-75, Bosie, A. and Vietta, E.S., Cell Immunol., 1991, 135:95-104). 본 발명에서 럽시 추출물의 투여는 농도 의존적으로 대상체 내 IgE 수준을 감소시킬 수 있다. 폴리페놀과 안토시아니딘 혼합물을 함유하는 추출물이 DNCB로 유발된 아토피피부염 마우스에서 IgE 및 IgG1의 수준을 감소시켰다는 기존의 보고를 참고한다면 상기 IgE 항체 수준의 감소가 럽시 추출물에 존재하는 폴리페놀 및 안토시아니딘에 의한 1형 반응의 항진일 수 있다는 추론이 가능할 것이다. 또한, 본 발명에서 럽시 추출물의 투여는 점액 면역에서 중요한 항체인 IgA 수준을 농도 의존적으로 증가시킬 수 있다.IL-4 induces isotype switching to IgE, a representative antibody marker for allergy development (Snapper, CM et al., Immunol. Rev., 1988, 102:51-75, Bosie, A. and Vietta, ES, Cell Immunol., 1991, 135:95-104). Administration of the ruby extract in the present invention can reduce the IgE level in the subject in a concentration-dependent manner. Referring to the previous report that an extract containing a mixture of polyphenols and anthocyanidins reduced the levels of IgE and IgG1 in DNCB-induced atopic dermatitis mice, the reduction in the IgE antibody level was related to the polyphenols and It may be inferred that the anthocyanidins may be an enhancement of the type 1 response. In addition, administration of the ruby extract in the present invention can increase the level of IgA, an important antibody in mucus immunity, in a concentration-dependent manner.
일 구체예에 있어서, 상기 본 발명의 면역 증강용 조성물은 그 투여 전과 비교하여 그 투여 후에 대상체에서 NO, PGE 2, COX-2 및 ROS 중에서 선택되는 하나 이상의 수준을 감소시킬 수 있다.In one embodiment, the composition for enhancing immunity of the present invention can reduce one or more levels selected from NO, PGE 2 , COX-2 and ROS in the subject after the administration compared to before the administration.
ROS, NO, 유도성 NO 합성효소 (inducible NO synthase, iNOS) 및 COX-2와 같은 다양한 전염증성 매개 물질 생성에 있어서 장기간의 조절 이상이 유도되면 암을 포함한 다양한 질병 진행과 연계된 만성 염증을 유발하기도 한다 (Murakami, A. and Ohigashi, H., Int. J. Cancer, 2007, 121: 2357-2363; Rehman, M.U. et al., Inflamm. Res., 2012, 61:1177-1185). ROS는 싸이토카인 분비부터 세포 증식까지의 세포 활성을 자극하고 필요 이상 높은 농도에서는 세포 손상 및 사망을 유발하는 신호 전달 분자로서 기능을 수행하는 것으로 보고되고 있다. COX-2 및 iNOS는 ROS 생성 유도에 관여한다 (Lum, H. and Roebuck, K.A., Am. J. Physiol. Cell Physiol., 2001, 280:C719-C741; Liu, M.Y., Shock, 2005, 23:360-364). iNOS의 과다 발현으로 인한 NO 생성의 증가는 염증, 패혈성 쇼크 또는 암 발생 병리 기전에도 관여한다 (Ohshuma, H. and Bartsch, H., Fundam. Mol. Mech. Mutagen, 1994, 305:253-264). iNOS 및 COX-2 유전자 발현을 억제하여 NO 및 PGE 2 생성 수준을 감소시킬 수 있는 제제는 염증의 강도를 감소시킬 수 있다. 본 발명에서 럽시 추출물은 LPS로 활성화된 탐식세포에서 NO, PGE 2 및 세포내 ROS 생성을 유의하게 감소시킬 수 있다. 또한, 본 발명의 럽시 추출물은 PGE 2의 생성에 관여하는 COX-2의 단백질 발현을 억제하고 관련된 PGE 2의 생성도 억제할 수 있다.Long-term dysregulation in the production of various proinflammatory mediators, such as ROS, NO, inducible NO synthase (iNOS) and COX-2, induces chronic inflammation associated with the progression of various diseases, including cancer. (Murakami, A. and Ohigashi, H., Int. J. Cancer, 2007, 121: 2357-2363; Rehman, MU et al., Inflamm. Res., 2012, 61:1177-1185). It has been reported that ROS functions as a signal transduction molecule that stimulates cellular activity from cytokine secretion to cell proliferation, and causes cell damage and death at concentrations higher than necessary. COX-2 and iNOS are involved in the induction of ROS production (Lum, H. and Roebuck, KA, Am. J. Physiol. Cell Physiol., 2001, 280: C719-C741; Liu, MY, Shock, 2005, 23: 360-364). Increased NO production due to overexpression of iNOS is also involved in the pathogenesis of inflammation, septic shock or cancer (Ohshuma, H. and Bartsch, H., Fundam. Mol. Mech. Mutagen, 1994, 305:253-264) ). Agents capable of reducing the levels of NO and PGE 2 production by inhibiting iNOS and COX-2 gene expression can reduce the intensity of inflammation. In the present invention, the ruby extract can significantly reduce the production of NO, PGE 2 and intracellular ROS in LPS-activated phagocytes. In addition, the ruby extract of the present invention can suppress the protein expression of COX-2 involved in the production of PGE 2 and also inhibit the production of the related PGE 2 .
일 구체예에서, 상기 본 발명의 면역 증강용 조성물은 그 투여 전과 비교하여 그 투여 후에 대상체에서 전염증성 싸이토카인의 수준을 감소시킬 수 있다.In one embodiment, the composition for enhancing immunity of the present invention can reduce the level of pro-inflammatory cytokines in the subject after the administration compared to before the administration.
또 다른 구체예에서, 상기 전염증성 싸이토카인은 TNF-α, IL-1β, IL-6, IL-8 및 IL-17 중에서 선택되는 하나 이상인 것일 수 있다.In another embodiment, the pro-inflammatory cytokine may be one or more selected from TNF-α, IL-1β, IL-6, IL-8 and IL-17.
염증성 질환에서는 TNF-α, IL-1β 및 IL-6 등 전염증성 싸이토카인 생성 항진이 두드러진다. 본 발명에서 럽시 추출물은 LPS 활성화에 따른 RAW 264.7 세포로부터 유의하게 생성된 IL-6, TNFα, IL-1β 수준을 광범위한 추출물 농도에서 용량-의존적으로 저하시키고 고농도의 상기 럽시 추출물은 TNF-α를 유의하게 감소시킬 수 있음이 확인되었다. 이와 유사하게, TDM 세포의 경우 IL-6, TNF-α, IL-1β, IL-8 수준은 럽시 추출물 농도에 따라 용량-의존적으로 저하될 수 있었다. 전염증성 싸이토카인 생성 제어는 항염증 요법의 표적인데 마우스 대식세포 및 인간 대식세포 모두에서 전염증성 싸이토카인을 성공적으로 억제하는 것으로 보여지는 본 발명의 럽시 추출물은 항염증용 의약의 후보 물질이 될 수 있음을 시사한다고 여겨진다.In inflammatory diseases, pro-inflammatory cytokine production, such as TNF-α, IL-1β, and IL-6, is prominent. In the present invention, the ruby extract significantly lowered the levels of IL-6, TNFα, and IL-1β produced from RAW 264.7 cells according to LPS activation in a wide range of extract concentrations in a dose-dependent manner, and the high concentration of the ruby extract significantly reduced TNF-α It was confirmed that it can be significantly reduced. Similarly, in the case of TDM cells, IL-6, TNF-α, IL-1β, and IL-8 levels could be decreased in a dose-dependent manner according to the concentration of the Rubsy extract. Controlling the production of pro-inflammatory cytokines is a target of anti-inflammatory therapy. It is thought to suggest
T 림프구는 기능적으로 크게 Th1 및 Th2 세포로 나뉜다. Th1 세포 매개 면역은 암의 진행, 바이러스 감염, 세포 내 감염성 질환으로부터 개체를 보호하는 반면, Th2 세포 매개 면역은 주로 호흡기, 피부 알레르기 반응 발생과 관련이 있다 (Kidd, P., Alternative medicine review., 2003, 8: 223-246, Heo, Y. et al., Ind. Health, 2010, 48:171-177). Th1 세포에서 생성되는 IFN-γ와 Th2 세포에서 생성되는 IL-4는 체내 면역반응의 항상성에 중요한 역할을 수행하는데, 이에 T 림프구에서 생성되는 IFN-γ:IL-4 비는 세포 면역의 Th1 및 Th2 균형을 모니터링하는 척도이다 (Paludan, S.R., Scand. J. Immunol., 1998, 48:459-468). 관련하여 Th1 및 Th2 세포에서 분비되는 싸이토카인들은 면역 반응의 방향성 및 결과에 결정하는 역할을 한다. 본 발명에서 비장 유래 T 림프구를 시험관내에서 활성화시켰을 통계적으로 유의하게 변화된 싸이토카인은 전염증성 싸이토카인인 TNF-α로 5, 50 mg/kg체중 투여군에서 대조군에 비해 유의하게 낮았다. 이는 럽시 추출물이 특정 농도에서 이 싸이토카인 생성을 억제하여 염증 반응에 관여할 수 있음을 시사한다. T lymphocytes are functionally largely divided into Th1 and Th2 cells. Th1-cell-mediated immunity protects individuals from cancer progression, viral infection, and intracellular infectious disease, whereas Th2-cell-mediated immunity is mainly associated with respiratory and skin allergic reactions (Kidd, P., Alternative medicine review., 2003, 8: 223-246, Heo, Y. et al., Ind. Health, 2010, 48:171-177). IFN-γ produced by Th1 cells and IL-4 produced by Th2 cells play an important role in homeostasis of immune responses in the body. It is a measure of monitoring Th2 balance (Paludan, SR, Scand. J. Immunol., 1998, 48:459-468). In this regard, cytokines secreted by Th1 and Th2 cells play a role in determining the direction and outcome of the immune response. In the present invention, the statistically significantly changed cytokines that would have activated spleen-derived T lymphocytes in vitro were significantly lower in the 5 and 50 mg/kg body weight administration groups than in the control group with the pro-inflammatory cytokine TNF-α. This suggests that ruby extract may be involved in the inflammatory response by inhibiting the production of this cytokine at certain concentrations.
또한, 럽시 추출물은 장관에서 염증성 반응 촉진에 기여하는 대표적 싸이토카인인 IL-17을 저하시킬 수 있다.In addition, ruby extract can lower IL-17, a representative cytokine that contributes to the promotion of inflammatory responses in the intestinal tract.
CYP로 유도된 면역억제 마우스는 일반적으로 다양한 약물 및 약용 식물 제제의 면역 억제 회복 또는 활성화 평가에 사용된다 (Huang, Y. and Li, L., Transl. Cancer Res., 2013, 2:144). 따라서 본 발명에서도 CYP로 유도된 면역억제 마우스를 럽시 과실 추출물의 면역억제 회복 또는 활성화 가능성 평가에 사용하였다. 흉선과 비장은 신체의 면역 반응에서 중요한 역할을 한다. 이들 기관의 무게는 면역 체계에 직접적으로 영향을 미치는 다양한 자극에 반응하여 변화한다 (Cui, H. et al., J. Sci. Food Agric, 2011, 91:2180-2185). 이러한 싸이클로포스파미드에 의한 면역 억제는 본 발명의 럽시 추출물 투여로 회복될 수 있다. CYP-induced immunosuppressive mice are commonly used to evaluate the recovery or activation of immunosuppression of various drugs and medicinal plant preparations (Huang, Y. and Li, L., Transl. Cancer Res., 2013, 2:144). Therefore, in the present invention, CYP-induced immunosuppression mice were used to evaluate the recovery or activation potential of Rubsy fruit extract. The thymus and spleen play important roles in the body's immune response. The weight of these organs changes in response to various stimuli that directly affect the immune system (Cui, H. et al., J. Sci. Food Agric, 2011, 91:2180-2185). Immune suppression by such cyclophosphamide can be restored by administration of the rubsy extract of the present invention.
혈액 내 각 세포들의 경우에도 양적인 변화가 CYP 주사에 따라 나타나는 바, 본 발명의 럽시 추출물 투여로 그 변화가 회복될 수 있다. CYP는 마우스 기관지 폐포 세척액에서 호중구를 증가시키고 Wistar 랫드에서 적혈구 수, 헤모글로빈 및 림프구 %와 같은 혈액학적 지표치 수준을 저하시키는 것으로 보고되었다 (Said Abd-Elkhalek, E. et al., Can. J. Physiol. Pharmacol., 94 : 347-358, Muruganandan, S. et al., Toxicology, 2005, 215 : 57-68). 천식 환자에서 IL-17 분비 호중구의 증가에 대한 연구도 보고된 바 있다 (Ramirez-Velazquez, C. al., Allergy, Asthma, Clin. Immunol., 2013, 9:23). 이에 본 발명에서 럽시 추출물 투여에 따른 호중구 % 증가 억제는 호중구와 관련된 알레르기 발생 기전에 억제력을 부여할 수 있을 것이다. 또한 럽시 추출물 투여에 따른 적혈구 수 및 헤모글로빈 수치 증가는 럽시 추출물 내 존재하는 플라보노이드가 기여하는 것으로 판단된다. In the case of each cell in the blood, a quantitative change appears according to CYP injection, and the change can be restored by administration of the rubsy extract of the present invention. CYP has been reported to increase neutrophils in the bronchoalveolar lavage fluid of mice and decrease the level of hematological parameters such as red blood cell count, hemoglobin and % lymphocytes in Wistar rats (Said Abd-Elkhalek, E. et al., Can. J. Physiol. Pharmacol., 94: 347-358, Muruganandan, S. et al., Toxicology, 2005, 215: 57-68). An increase in IL-17-secreting neutrophils in asthmatic patients has also been reported (Ramirez-Velazquez, C. al., Allergy, Asthma, Clin. Immunol., 2013, 9:23). Accordingly, in the present invention, inhibition of the increase in neutrophil % according to the administration of the Rubsy extract may impart inhibitory power to the neutrophil-related allergy development mechanism. In addition, it is judged that the increase in the number of red blood cells and hemoglobin levels according to the administration of the rubsy extract is attributed to the flavonoids present in the ruby extract.
본 발명에서, 럽시 추출물을 투여한 결과 CYP만 주사한 모델대조군에 비해 모든 항체 수준이 항진되었다. 특히 IgG2a 및 IgA의 수준은 모델대조군과 비교하여 추출물 투여군들에서 유의하게 높았던 반면, 알레르기 발생과 연관이 있는 IgG1 및 IgE의 수준은 여전히 부형제대조군에 비해서는 낮았다. 이러한 결과는 럽시 추출물 투여시 1형 보조 T 림프구 반응이 2형 보조 T 림프구 반응에 비해 우세한 체액 면역 반응을 촉진할 수 있고, 이에 따라 알레르기 반응을 제어할 기능이 있음을 시사하는 것으로 판단된다. 이러한 결과는 시험관내에서 비장 B 림프구를 활성화시켜 생성된 항체 수준을 평가한 결과에서도 비슷하게 나타났다. In the present invention, as a result of administering the Rubsy extract, all antibody levels were enhanced compared to the model control group injected with only CYP. In particular, the levels of IgG2a and IgA were significantly higher in the extract-treated group compared to the model control group, whereas the levels of IgG1 and IgE, which are associated with allergy development, were still lower than that of the excipient control group. These results are considered to suggest that the type 1 auxiliary T lymphocyte response can promote a predominant humoral immune response compared to the type 2 auxiliary T lymphocyte response when the rubsy extract is administered, and thus has a function to control the allergic reaction. These results were similar to the results of evaluating the level of antibodies produced by activating splenic B lymphocytes in vitro.
본 발명에서, CYP에 의해 면역 억제가 유도된 경우 이들 마우스 비장을 채취하여 시험관내에서 활성화시킨 결과 부형제대조군에 비해 IFN-γ 및 IL-4 생성 수준이 감소한 점은 다른 연구 결과들과 비슷하였고 (Xu,X. and Zhang,X., Microbiol. Res., 2015, 171:97-106), IL-17, TGF-β1 수준은 증가하였고 TNFα는 차이가 없었다. TGF-β1은 염증 부위에서 대식세포, 과립구 및 기타 세포의 빠른 축적을 유인하는 특성을 갖고 있는데, CYP로 유도된 방광염에 동반되는 방광 기능 장애시 증가됨이 보고된 바 있다 (Massague, J. et al., Cancer Surv, 1992, 12:81-103; Gonzalez, E.J.,2016). 또한 TGF-β1은 Th17 세포 분화를 유도하여 급성 염증 과정을 지속시키는 특성을 갖고 있는 IL-17 분비를 촉진할 수 있는 특성도 있다 (McDonald, D.M., Am. J. Respir. Crit. Care Med, 2016, 164: S39-S45). 기존 암 환자 대상 연구에서, CYP는 Th17 세포 분화를 촉진하여 IL-17 생성이 항진되는 점이 보고된 바 있다 (Viaud, S. et al., Cancer Res., 2011, 71: 661-665). 그에 따라 CYP를 투여한 마우스에서의 상기 IL-17 및 TGF-β1의 수준 증가는 설명될 수 있다. 본 발명에서, 럽시 추출물은, 일부 농도에서 해당 싸이토카인 생성 수준을 감소시킬 수 있다. IL-17의 경우 장간막 림프절에서도 비슷한 결과가 관찰되었다. CYP에 의한 면역 기능 저하는 장점막 면역 체계에 더 영향이 있고 장내 미생물총의 균형을 깨트린다는 보고에 비추어 보면 (Matsuoka, K. and Kanai, T., Semin. Immunopathol., 2015, 37:47-55; Xu, X. and Zhang, X., Microbiol. Res., 2015, 171: 97-106), 럽시 추출물에 의한 IL-17 수준 저하는 럽시 추출물이 장점막 면역력을 보호하는 효과가 있음을 시사한다. In the present invention, when immunosuppression was induced by CYP, the spleens of these mice were collected and activated in vitro. As a result, the IFN-γ and IL-4 production levels decreased compared to the excipient control group, similar to the results of other studies ( Xu, X. and Zhang, X., Microbiol. Res., 2015, 171:97-106), IL-17, and TGF-β1 levels were increased, but there was no difference in TNFα. It has been reported that TGF-β1 induces rapid accumulation of macrophages, granulocytes and other cells at the site of inflammation, and that it is increased during bladder dysfunction accompanying CYP-induced cystitis (Massague, J. et al. ., Cancer Surv, 1992, 12:81-103; Gonzalez, EJ, 2016). In addition, TGF-β1 has the ability to induce Th17 cell differentiation and promote IL-17 secretion, which has the property of prolonging the acute inflammatory process (McDonald, DM, Am. J. Respir. Crit. Care Med, 2016). , 164: S39-S45). In a previous study on cancer patients, it has been reported that CYP promotes Th17 cell differentiation to enhance IL-17 production (Viaud, S. et al., Cancer Res., 2011, 71: 661-665). Accordingly, the increased levels of IL-17 and TGF-β1 in mice administered with CYP can be explained. In the present invention, the rubsy extract can reduce the level of the corresponding cytokine production at some concentrations. In the case of IL-17, similar results were observed in mesenteric lymph nodes. In light of reports that CYP-induced immune dysfunction affects the intestinal mucosal immune system more and disrupts the balance of the intestinal microflora (Matsuoka, K. and Kanai, T., Semin. Immunopathol., 2015, 37:47- 55; Xu, X. and Zhang, X., Microbiol. Res., 2015, 171: 97-106), the decrease in IL-17 level by Ruby extract suggests that Ruby extract is effective in protecting intestinal mucosal immunity. .
본 발명의 럽시 추출물의 투여에 따른 상기 싸이토카인 및 항체 수준의 변화로부터, 본 발명의 조성물은 자가면역 질환, 바람직하게는 T 세포에 의해 매개되는 자가면역질환을 예방 또는 치료할 수 있다. 상기 자가면역질환으로는 예컨대, 류머티스 관절염 (rheumatoid arthritis), 건선, 전신성홍반성낭창 (systemic lupus erythematosis), E-과면역글로불린혈증증후군 (hyperimmunoglobulin E), 하시모토씨 갑상선염 (Hashimoto's thyroiditis), 그레브스 병 (Grave' s Disease), 다발성경화증 (multiple sclerosis), 경피증 (Scleroderma), 전신성 경피증 (progressive systemic sclerosis), 중증근무력증 (myasthenia gravis), 제1형 당뇨 (type I diabetes), 포도막염 (uveitis), 알레르기성 뇌척수염 (allergic encephalomyelitis), 사구체신염 (glomerulonephritis), 백반증 (Vitilligo), 굿패스쳐 신드롬 (Goodpasture syndrome), 베제트병 (Becet's Disease), 크론병 (Crohn's Disease), 강직성 척추염 (Ankylosing Spondylitis), 혈소판 감소성 자반증 (Thrombocytopenic purpura), 심상성 천포창 (Pemphigus vulgaris), 자가면역성 용혈성 빈혈 (Autoimmune Anemia), 크라일로글로불린증 (Cryoglobulinemia), 부신백질이영양증 (ALD), 전신성 홍반성 낭창 (Systemic Lupus Erythematosus,SLE) 등을 들 수 있으나, 이에 국한되는 것은 아니다. From the changes in the cytokine and antibody levels according to the administration of the ruby extract of the present invention, the composition of the present invention can prevent or treat autoimmune diseases, preferably autoimmune diseases mediated by T cells. The autoimmune disease includes, for example, rheumatoid arthritis, psoriasis, systemic lupus erythematosis, E-hyperimmunoglobulin E, Hashimoto's thyroiditis, Greves Grave's Disease, multiple sclerosis, Scleroderma, progressive systemic sclerosis, myasthenia gravis, type I diabetes, uveitis, Allergic encephalomyelitis, glomerulonephritis, Vitilligo, Goodpasture syndrome, Becet's Disease, Crohn's Disease, Ankylosing Spondylitis Thrombocytopenic purpura, Pemphigus vulgaris, Autoimmune Anemia, Cryoglobulinemia, ALD, Systemic Lupus Erythematosus ), but is not limited thereto.
본 발명에서, CYP를 주사한 모델대조군 마우스의 경우 예상된 바와 같이 보조 T 림프구, 세포파괴 T 림프구, B 림프구, 자연 살해 세포 수가 모두 부형제대조군에 비해 낮았다. 그러나, 럽시 추출물을 투여하였을 때 이들 세포들의 비율은 모델대조군에 비해 유의하게 높아졌고 특히 자연 살해 세포 수준은 부형제대조군보다도 유의하게 높았다. 자연 살해 세포는 과량의 IFN-γ을 생성하는 것으로 알려져 있는데 (Arase, H. at al., J. Exp. Med., 1996, 183: 2391-2396), 상기 본 발명 럽시 추출물의 투여 결과는 자연 살해 세포 수 증가를 유도하여 궁극적으로 세포 면역 반응에서 핵심적인 역할을 수행하는 IFN-γ 생성 증가에 이르는 효과를 통해 럽시 추출물이 면역력 증가에 기여할 수 있음을 시사한다. In the present invention, the number of helper T lymphocytes, apoptotic T lymphocytes, B lymphocytes, and natural killer cells were all lower than that of the excipient control group, as expected in the model control group mice injected with CYP. However, when the rubsy extract was administered, the ratio of these cells was significantly increased compared to the model control group, and the level of natural killer cells was significantly higher than that of the excipient control group. Natural killer cells are known to produce an excess of IFN-γ (Arase, H. at al., J. Exp. Med., 1996, 183: 2391-2396). The effect of inducing an increase in the number of killer cells, ultimately leading to an increase in the production of IFN-γ, which plays a key role in the cellular immune response, suggests that ruby extract may contribute to the increase in immunity.
일 구체예에 있어서, 상기 본 발명의 면역 증강용 조성물은 기능성 식품일 수 있다. 본 발명에서 상기 기능성 식품은 천연식품, 가공식품, 환자식품, 일반적인 식자재 등에 본 발명의 럽시 추출물을 첨가하는 것을 비제한적으로 포함한다. 본 발명에서 식품은, 상기 본 발명의 면역 증강용 조성물을 그대로 사용하거나 다른 식품 또는 식품 조성물과 함께 사용될 수 있으며, 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효 성분의 혼합양은 그의 사용 목적 (예방, 개선 또는 치료적 처치)에 따라 적합하게 결정될 수 있다. 일반적으로, 본 발명의 럽시 추출물은, 식품 제조 시에 식품 원료 100 중량부에 대하여 0.1 내지 99.9 중량부 첨가될 수 있다. 상기 식품 내에서 럽시 추출물의 유효 용량은 의약 조성물의 유효 용량에 준해서 사용할 수 있다. In one embodiment, the composition for enhancing immunity of the present invention may be a functional food. In the present invention, the functional food includes, but is not limited to, adding the ruby extract of the present invention to natural food, processed food, patient food, and general food materials. Food in the present invention, the composition for enhancing immunity of the present invention may be used as it is or may be used together with other food or food compositions, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of its use (prophylaxis, improvement or therapeutic treatment). In general, the ruby extract of the present invention may be added in 0.1 to 99.9 parts by weight based on 100 parts by weight of the food raw material during food production. The effective dose of the ruby extract in the food can be used according to the effective dose of the pharmaceutical composition.
상기 기능성 식품의 종류에는 특별한 제한은 없다. 상기 기능성 식품은 정제, 경질 또는 연질 캅셀제, 액제, 현탁제 등과 같은 경구 투여용 제제의 형태로 이용될 수 있으며, 이들 제제는 허용 가능한 통상의 담체, 예를 들어 경구 투여용 제제의 경우에는 부형제, 결합제, 붕해제, 활택제, 가용화제, 현탁화제, 보존제 또는 증량제 등을 사용하여 조제될 수 있다.There is no particular limitation on the type of the functional food. The functional food may be used in the form of formulations for oral administration such as tablets, hard or soft capsules, solutions, suspensions, etc., and these formulations are acceptable conventional carriers, for example, excipients in the case of formulations for oral administration, It may be prepared using a binder, a disintegrant, a lubricant, a solubilizer, a suspending agent, a preservative or an extender, and the like.
또한, 상기 럽시 추출물을 첨가할 수 있는 식품의 예로 특별한 제한은 없다. 상기 럽시 추출물을 첨가할 수 있는 식품의 예로는 드링크제, 육류, 소시지, 빵, 비스킷, 떡, 초콜릿, 캔디류, 스낵류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 알코올 음료 및 비타민 복합제, 유제품 및 유가공 제품 등이 있으며, 통상적인 의미에서의 건강 기능성 식품을 모두 포함한다.In addition, there is no particular limitation as an example of a food to which the ruby extract can be added. Examples of foods to which the ruby extract can be added include drinks, meat, sausage, bread, biscuits, rice cakes, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, and various soups. , beverages, alcoholic beverages and vitamin complexes, dairy products, and dairy products, and includes all health functional foods in the ordinary sense.
본 발명의 기능성 식품은 필수 성분으로서 럽시 추출물을 함유하는 것 외에는 다른 성분에는 특별한 제한이 없으며 통상의 식음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 상술한 천연 탄수화물의 예는 모노사카라이드, 예를 들어, 포도당, 과당 등; 디사카라이드, 예를 들어 말토스, 슈크로스 등; 및 폴리사카라이드, 예를 들어 덱스트린, 시클로덱스트린 등과 같은 통상적인 당, 및 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이다. 상술한 것 이외의 향미제로서 천연 향미제 및 합성 향미제를 사용할 수 있다. 상기 천연 탄수화물의 비율은 당업자의 선택에 의해 적절하게 결정될 수 있다.The functional food of the present invention is not particularly limited in other ingredients except for containing a ruby extract as an essential ingredient, and may contain various flavoring agents or natural carbohydrates as additional ingredients like conventional food and beverages. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; disaccharides such as maltose, sucrose and the like; and polysaccharides such as conventional sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents and synthetic flavoring agents may be used. The ratio of the natural carbohydrate may be appropriately determined by the selection of those skilled in the art.
본 발명의 기능성 식품 조성물은 상기 외에 여러 가지 영양제, 비타민, 광물 (전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 중진제 (치즈, 초콜릿 등), 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산음료에 사용되는 탄산화제 등을 함유할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. 이러한 첨가제의 비율 또한 당업자에 의해 적절히 선택될 수 있다. In addition to the above, the functional food composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), synthetic flavoring agents and natural flavoring agents, coloring agents and thickeners (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonates used in carbonated beverages, and the like. These components may be used independently or in combination. The proportion of these additives may also be appropriately selected by those skilled in the art.
일 구체예에 있어서, 상기 본 발명의 면역 증강용 조성물은 항염증용 의약일 수 있다.In one embodiment, the composition for enhancing immunity of the present invention may be an anti-inflammatory drug.
염증은 조직 손상 및 감염과 같은 위해 영향에 대한 신체의 방어 기전이다. 염증이란 어떤 자극에 대한 생체조직의 방어반응의 하나로, 조직 변질, 순환장애와 삼출, 조직 증식의 세 가지를 병발하는 복잡한 병변을 일컫는다. 또한, 여러 가지 형태의 감염 (infection)이나 생체 내 대사산물 중의 자극성 물질에 대한 생체 내 방어 기전의 발현이라 할 수 있고, 다양한 화학적 매개체가 염증의 발현 기전에 관여하고 있으며, 그 병인도 매우 복잡하다. 이는 조직의 상해 또는 파괴에 의해 유발되는 국소 보호 반응으로, 상해 유발 물질과 상해된 조직 모두를 파괴, 약화시키거나 차폐하는 작용을 한다. 이러한 염증의 특징은 미세혈관이 천공되고, 혈액 성분이 틈새 공간으로 누출되며, 백혈구가 염증 조직으로 이동한다는 것으로, 통상적으로 홍반, 부종, 통각과민 및 통증 등의 임상적 증상들을 동반한다.Inflammation is the body's defense mechanism against harmful effects such as tissue damage and infection. Inflammation is one of the defense responses of biological tissues to certain stimuli, and refers to a complex lesion that combines three types: tissue deterioration, circulatory disorder and exudation, and tissue proliferation. In addition, it can be said that it is the expression of a defense mechanism in vivo against various types of infection or irritants in in vivo metabolites, and various chemical mediators are involved in the expression mechanism of inflammation, and the pathogenesis is very complicated. . It is a local protective reaction induced by tissue injury or destruction, and acts to destroy, weaken, or mask both the injury-causing agent and the injured tissue. The hallmark of such inflammation is that microvessels are perforated, blood components leak into the interstitial space, and leukocytes move to the inflamed tissue, which is usually accompanied by clinical symptoms such as erythema, edema, hyperalgesia and pain.
죽상 경화증 (atherosclerosis), 천식 (asthma), 건선 (psoriasis), 류마티스 관절염 (rheumatoid atthritis), 염증성 장 질환 (inflammatory bowel disease) 또는 암 (cancer)을 포함한 다양한 질병이 만성 염증과 관련이 있다 (Lawrence, T., Cold Spring Harb. Perspect. Biol., 2009, 1: a001651). A variety of diseases are associated with chronic inflammation, including atherosclerosis, asthma, psoriasis, rheumatoid atthritis, inflammatory bowel disease or cancer (Lawrence, T., Cold Spring Harb. Perspect. Biol., 2009, 1: a001651).
생체에 있어서 염증의 발생 원인으로는 다양한 생화학적인 현상이 관여하고 있으며, 특히 NO를 발생시키는 효소인 NOS와 프로스타글란딘 (prostaglandin)의 생합성에 관련된 효소들이 염증 반응을 매개하는데 있어서 중요한 역할을 하고 있는 것으로 알려진 바 있다. 이에 L-아르기닌으로부터 NO를 생성시키는 효소인 NOS나, 아라키돈산으로부터 프로스타글란딘류를 합성하는데 관련된 효소인 싸이클로옥시게나제 (cyclooxygenase, COX)는 염증을 차단하는데 있어서 주된 작용을 하고 있다.Various biochemical phenomena are involved as the cause of inflammation in the living body, and in particular, enzymes involved in the biosynthesis of NOS and prostaglandin, which are enzymes that generate NO, are known to play an important role in mediating the inflammatory reaction. there is a bar Accordingly, NOS, an enzyme that produces NO from L-arginine, and cyclooxygenase (COX), an enzyme involved in synthesizing prostaglandins from arachidonic acid, play a major role in blocking inflammation.
연구 결과에 따르면, NOS는 체내에 항상 일정수준으로 발현되고 있고, 이들에 의해 소량 생성되는 NO는 신경전달이나 혈관확장을 유도하는 등 정상적인 신체의 향상성 유지에 중요한 역할을 한다. 이에 반하여, 각종 싸이토카인이나 외부 자극물질에 의해 유도되는 iNOS에 의해 급격히 과량 발생되는 NO는 세포독성이나 각종 염증반응을 일으키는 것으로 알려져 있으며, 만성 염증은 iNOS 활성의 증가와 관련 있다는 연구가 있다 (Murakami, A. and Ohigashi, H., Int. J. Cancer, 2007, 121: 2357-2363; Rehman, M.U. et al., Inflamm. Res., 2012, 61:1177-1185).According to the research results, NOS is always expressed at a certain level in the body, and the small amount of NO produced by them plays an important role in maintaining normal body improvement, such as inducing neurotransmission or vasodilation. On the other hand, NO, which is rapidly excessively generated by iNOS induced by various cytokines or external stimulants, is known to cause cytotoxicity or various inflammatory reactions, and there is a study that chronic inflammation is related to an increase in iNOS activity (Murakami, A. and Ohigashi, H., Int. J. Cancer, 2007, 121: 2357-2363; Rehman, MU et al., Inflamm. Res., 2012, 61:1177-1185).
대식세포 (macrophages)는 싸이토카인/케모카인 및 종양 괴사 인자-알파 (tumor necrosis factor-alpha, TNF-α), 인터루킨 (interleukin-6, IL-6), IL-1β, 싸이클로옥시게나제-2 (cyclooxygenase-2, COX-2), 산화질소 (nitric oxide, NO) 및 프로스타글란딘 (prostaglandin)과 같은 많은 염증 매개체를 생성하는 주요 염증 세포이다 (Erwig, L.P., and Rees, A.J., Kidney Blood Press. Res.,1999, 22:21-25; Zhang, X. and Mosser, D.M., J. Pathol. A J. Pathol. Soc. Gt. Britain Irel., 2008, 214:161-178). iNOS는 NADPH와 산소를 사용하여 L-아르기닌 (L-arginine)으로부터 NO를 합성하며 COX-2는 아라키돈산 (arachidonic acid)을 프로스타글란딘 E 2 (PGE 2)로 전환시킨다 (Murakami, A. and Ohigashi, H., Int. J. Cancer, 2007, 121:2357-2363; Aoki, T. and Narumiya, S., Trends Pharmacol. Sci.,2012, 33:304-311). Macrophages are cytokines / chemokines and tumor necrosis factor-alpha (TNF-α), interleukin (interleukin-6, IL-6), IL-1β, cyclooxygenase-2 (cyclooxygenase) -2, COX-2), nitric oxide (NO) and prostaglandins are major inflammatory cells that produce many inflammatory mediators (Erwig, LP, and Rees, AJ, Kidney Blood Press. Res., 1999, 22:21-25; Zhang, X. and Mosser, DM, J. Pathol. A J. Pathol. Soc. Gt. Britain Irel., 2008, 214:161-178). iNOS synthesizes NO from L-arginine using NADPH and oxygen, and COX-2 converts arachidonic acid to prostaglandin E 2 (PGE 2 ) (Murakami, A. and Ohigashi, H., Int. J. Cancer, 2007, 121:2357-2363; Aoki, T. and Narumiya, S., Trends Pharmacol. Sci., 2012, 33:304-311).
대식세포에서 전염증성 유전자의 발현에는 여러 가지 전사인자 (transcription factor) 및 세포 신호 전달 경로 (cell signaling pathway)가 관여한다 (Guha, M. and Mackman, N., Cell. Signal., 2001, 13:85-94). 핵 인자 κB (NF-κB) 전사인자는 다양한 염증 인자 및 싸이토카인 전사 조절에서 중요한 역할을 한다 (Ghosh, S. et al., Annu. Rev. Immunol, 1998, 16:225-260, Baeuerle, P.A. and Henkel, T., Annu. Rev. Immunol., 1994, 12:141-179). 비활성화 상태에서, NF-κB는 핵으로의 전위를 억제하는 IκBα와의 복합체로서 세포질에 위치한다 (Zhang, T. et al., Mol. Pharm., 2012, 9:671-677). NF-κB는 지질 다당류 (LPS)에 의해 활성화되면 IκB-키나제 (IκB-kinase, IKK) 복합체 활성화를 통한 활성화가 이루어진다. 활성화된 IKK는 유비퀴틴화 (ubiquitination)과정과 이어지는 프로테아좀 분해 (proteasomal degradation) 과정을 거쳐 IκBα를 인산화시키고 (Gloire, G. et al., Biochem. Pharmacol., 2006, 72:1493-1505), 이에 따라 IκBα와의 복합체로에서 NF-κB가 유리된다. 이후 NF-κB는 핵으로 이동하여 전염증성 싸이토카인 (IL-1β, IL-2, IL-6, TNF-α), 케모카인 [IL-8, 대식세포 화학주성 단백질 (macrophage chemotactic protein-1, MCP-1)], 염증성 효소 (iNOS, inducible COX-2), 부착 분자 (adhension molecules)[세포내 부착 분자(intracellular adhesion molecule-1, ICAM-1), 혈관부착분자 (vascular adhesion molecule-1, VCAM-1), E-셀렉틴 (selectin)] 등의 다양한 염증 매개체의 전사를 촉진한다 (Barnes, P.J. and Karin, M.). Various transcription factors and cell signaling pathways are involved in the expression of proinflammatory genes in macrophages (Guha, M. and Mackman, N., Cell. Signal., 2001, 13: 85-94). Nuclear factor κB (NF-κB) transcription factor plays an important role in the regulation of transcription of various inflammatory factors and cytokines (Ghosh, S. et al., Annu. Rev. Immunol, 1998, 16:225-260, Baeuerle, PA and Henkel, T., Annu. Rev. Immunol., 1994, 12:141-179). In the inactive state, NF-κB is localized in the cytoplasm as a complex with IκBα that inhibits translocation to the nucleus (Zhang, T. et al., Mol. Pharm., 2012, 9:671-677). When NF-κB is activated by lipopolysaccharide (LPS), it is activated through IκB-kinase (IKK) complex activation. Activated IKK phosphorylates IκBα through ubiquitination and subsequent proteasomal degradation (Gloire, G. et al., Biochem. Pharmacol., 2006, 72:1493-1505), Accordingly, NF-κB is released from the complex with IκBα. Thereafter, NF-κB moves to the nucleus, and pro-inflammatory cytokines (IL-1β, IL-2, IL-6, TNF-α), chemokines [IL-8, macrophage chemotactic protein-1, MCP- 1)], inflammatory enzymes (iNOS, inducible COX-2), adhesion molecules [intracellular adhesion molecule-1, ICAM-1, vascular adhesion molecule-1, VCAM- 1), E-selectin] and promotes the transcription of various inflammatory mediators (Barnes, PJ and Karin, M.).
마이토젠 활성화 단백질 키나제 (MAPK)[c-Jun NH2-terminal kinase (JNK), p38, 세포 외 신호 조절 키나제 (extracellular signal-regulated kinase, ERK)] 역시 염증 반응시 전염증성 유전자의 발현 기능을 수행한다. LPS 활성화시 MAPK는 인산화되어 NF-κB 및/또는 AP-1 활성화의 신호전달 연쇄반응을 유도한다 (Kaminska, B., Biochimica et Biophysica Acta - Proteins and Proteomics, 2005, 1754:253-262; Guha, M. and Mackman, N., Cell. Signal., 2001, 13:85-94). Mitogen-activated protein kinase (MAPK) [c-Jun NH2-terminal kinase (JNK), p38, extracellular signal-regulated kinase (ERK)] also plays a role in the expression of pro-inflammatory genes during inflammatory responses. . Upon LPS activation, MAPK is phosphorylated to induce a signaling cascade of NF-κB and/or AP-1 activation (Kaminska, B., Biochimica et Biophysica Acta - Proteins and Proteomics, 2005, 1754:253-262; Guha, M. and Mackman, N., Cell. Signal., 2001, 13:85-94).
전염증성 싸이토카인 중에서, IL-1β는 선천면역 및 적응면역 반응을 조정하는데 결정적인 역할을 하는 싸이토카인이다 (Dinarello, C.A., Annu. Rev. Immunol., 2009, 27:519-550). IL-1β는 T-림프구를 활성화시켜 TNF-α 및 IL-6과 같은 전염증성 싸이토카인의 생성을 항진시킨다 (Ciraci, C. et al., Microbes Infect., 2012, 14:1263-1270, Dinarello, C.A., Eur. J. Immunol., 2011, 41:1203-1217). 전구 싸이토카인인 pro-IL-1β로부터 IL-1β의 활성화는 인플라마좀 (inflammasome)에 의해 활성화되는 카스파제 (caspase-1)에 의존한다 (Lamkanfi, M. and Kanneganti, T.D., Int. J. Biochem. Cell Biol., 2010, 42:792-795). NLRP3는 가장 집중적으로 연구된 인플라마좀으로 염증, 자가면역 및 감염을 포함한 광범위한 질병에 관여되어 있다 (Stutz, A. et al., J. Clin. Invest. 2009, 119:3502-3511; Menu, P. and Vince, J.E., Clin. Exp. Immunol., 2011, 166:1-15). NLRP3 인플라마좀의 활성화에는 프라이밍 (priming) 단계와 활성화 (activation) 단계의 두 단계가 필요하다. 프라이밍 단계에서는 톨-유사 수용체 (Toll-like receptor)-4 작용제에 의해 (예컨대, LPS) NF-κB 연계 NLRP3, pro-IL-1β 또는 pro IL-18 발현이 유도되는 단계이다. 활성화 단계는 기공 형성 독소 (pore-forming toxins), 세포 외 ATP (extracellular ATP), 미생물 DNA (microbial DNA) 및 RNA, 흡입 미립자 (inhaled particulates), 요산 (uric acid) 및 콜레스테롤 결정 (cholesterol crystals)을 포함한 광범위한 물질에 의해 이루어진다 (Bauernfeind, F. et al., J. Immunol., 2010, 183:787-791; Rajamaki, K. et al., PLoS One, 2010, 5:e11765). NLRP3의 활성화는 ASC 어댑터 단백질 (apoptosis-associated speck-like protein containing a caspase-recruitment domain)을 통한 caspase-1 유인을 통해 이루어지는데, 활성화된 NLRP3 인플라마좀 복합체는 caspase-1을 활성화시키고 이 caspase-1은 pro-IL-1β 및 pro-IL-18을 IL-1β 및 IL-18로 전환시킨다 (Gross, O. et al., Immunol. Rev., 2011, 243:136-151; Davis, B.K et al., Annu. Rev. Immunol., 2011, 29:707-735). RAW 264.7 마우스의 대식세포 세포주에는 ASC 어댑터가 결여되어 있으므로 NLRP3는 PMA (phorbol 12-myristate 13-acetate) 자극 THP-1 유래 대식세포에서 성공적으로 연구되어 왔다 (Kong, F. et al., Biomed. Pharmacother., 2016, 82:167-172; Pelegrin, P.. et al., J. Immunol., 2008, 180:7147-7157). PMA(10~400 ng/ml)로 활성화시킨 THP-1 대식세포는 인간 대식세포와 대사 및 형태학적 유사성을 나타내므로 인간 대식세포의 시험관내 시험모델로 널리 사용된다 (Park, E.k. et al., Inflamm. Res., 2007, 56:45-50). Among proinflammatory cytokines, IL-1β is a cytokine that plays a critical role in modulating innate and adaptive immune responses (Dinarello, C.A., Annu. Rev. Immunol., 2009, 27:519-550). IL-1β activates T-lymphocytes to enhance production of proinflammatory cytokines such as TNF-α and IL-6 (Ciraci, C. et al., Microbes Infect., 2012, 14:1263-1270, Dinarello, CA, Eur. J. Immunol., 2011, 41:1203-1217). Activation of IL-1β from pro-IL-1β, a pro-cytokine, is dependent on caspase-1, which is activated by the inflammasome (Lamkanfi, M. and Kanneganti, TD, Int. J. Biochem). (Cell Biol., 2010, 42:792-795). NLRP3 is the most intensively studied inflammasome and is implicated in a wide range of diseases including inflammation, autoimmunity and infection (Stutz, A. et al., J. Clin. Invest. 2009, 119:3502-3511; Menu, P. and Vince, JE, Clin. Exp. Immunol., 2011, 166:1-15). Activation of the NLRP3 inflammasome requires two steps: a priming step and an activation step. In the priming step, the expression of NF-κB-linked NLRP3, pro-IL-1β or pro IL-18 is induced by a Toll-like receptor-4 agonist (eg, LPS). The activation step removes pore-forming toxins, extracellular ATP, microbial DNA and RNA, inhaled particulates, uric acid and cholesterol crystals. by a wide range of substances including (Bauernfeind, F. et al., J. Immunol., 2010, 183:787-791; Rajamaki, K. et al., PLoS One, 2010, 5:e11765). Activation of NLRP3 is achieved through caspase-1 attraction through ASC adapter protein (apoptosis-associated speck-like protein containing a caspase-recruitment domain). The activated NLRP3 inflammasome complex activates caspase-1 and 1 converts pro-IL-1β and pro-IL-18 to IL-1β and IL-18 (Gross, O. et al., Immunol. Rev., 2011, 243:136-151; Davis, BK et al. al., Annu. Rev. Immunol., 2011, 29:707-735). Since the macrophage cell line of RAW 264.7 mice lacks the ASC adapter, NLRP3 has been successfully studied in PMA (phorbol 12-myristate 13-acetate) stimulated THP-1-derived macrophages (Kong, F. et al., Biomed. Pharmacother., 2016, 82:167-172; Pelegrin, P.. et al., J. Immunol., 2008, 180:7147-7157). THP-1 macrophages activated with PMA (10-400 ng/ml) exhibit metabolic and morphological similarities to human macrophages, and are therefore widely used as in vitro test models for human macrophages (Park, Ek et al., Inflamm. Res., 2007, 56:45-50).
대식세포 표면 발현 TLR4가 LPS와 결합되면 이후 세포내 신호전달 pathway인 NF-κB 또는 MAPK 신호전달 경로가 작동한다. LPS는 IκBα, NF-κB, p-38, JNK, ERK의 활성화를 유도한다. 본 발명에서 럽시 추출물은 IκBα의 인산화를 제어한 결과 NF-κB의 핵 내 전이가 억제됨을 보여준다. 본 발명의 럽시 추출물은 p-38, JNK, ERK의 인산화 또한 제어할 수 있다. 종합하면 럽시 추출물은 LPS 활성 RAW 264.7 세포에서 IκBα, NF-κB, p-38, JNK, ERK의 활성도를 제어함으로써 전염증성 매개물질의 생성, 배출을 억제하고 LPS로 인해 유발된 염증반응 수준을 완화시킬 수 있는 것임을 시사한다. When macrophage surface-expressed TLR4 binds to LPS, the intracellular signaling pathway, NF-κB or MAPK signaling pathway, is then activated. LPS induces activation of IκBα, NF-κB, p-38, JNK, and ERK. In the present invention, as a result of controlling phosphorylation of IκBα, the rubsy extract shows that NF-κB translocation into the nucleus is inhibited. The ruby extract of the present invention can also control phosphorylation of p-38, JNK, and ERK. In summary, Ruby extract inhibits the production and release of pro-inflammatory mediators by controlling the activities of IκBα, NF-κB, p-38, JNK, and ERK in LPS-activated RAW 264.7 cells, and relieves the level of inflammatory response induced by LPS. suggest that it can be done.
본 발명에서는 한편, LPS와 ATP 복합처리를 통하여 THP-1 유래 대식세포를 활성화시킨 결과, 세포내 인플라마좀 단백질 복합체 중 하나인 NLRP3 단백질 수준이 항진됨을 확인하였다. 본 발명의 럽시 추출물은 NLRP3 단백질 발현 수준을 유의하게 감소시킬 수 있는데, 이는 럽시 추출물이 인플라마좀 관여 염증 반응을 제어할 수 있음을 보여주는 것이다. Meanwhile, in the present invention, as a result of activating THP-1-derived macrophages through complex treatment with LPS and ATP, it was confirmed that the level of NLRP3 protein, one of the intracellular inflammasome protein complexes, was enhanced. The ruby extract of the present invention can significantly reduce the NLRP3 protein expression level, which shows that the ruby extract can control the inflammasome-related inflammatory response.
결국, 본 발명의 럽시 추출물은 마우스 및 인간 대식세포에서 전염증성 싸이토카인의 생성을 제어하고 기전상 관련된 세포 내 신호 전달 체계를 조절할 수 있는 것으로 보인다. 전염증성 싸이토카인인 TNF-α 및 IL-1β는 세포 변성 및 사망을 유발할 수 있고 여러 기관의 기능 장애를 유발할 수 있으며, 또한 IL-6 및 IL-8과 같은 전염증성 싸이토카인의 생성도 자극한다 (Tracey, K.J et al., Nature, 1987,330:662). 본 발명의 럽시 추출물은 이러한 싸이토카인들의 과다 생성을 억제함으로써 비정상적 염증 반응과 연관된 질병의 악화를 방지할 수 있다. 럽시 추출물의 항염증 효능은 추출물에 존재하는 구성 성분과 관련이 있을 것이다. 성분 중 하나인 퀘르세틴 (quercetin)은 NLRP3 단백질 관련 반응을 억제하고 디하이드로퀘르세틴 (dihydroquercetin)은 ROS 생성을 억제하고 NLRP3 복합체 생성을 억제하며, 프로시아니딘 (procyanidin)은 ROS 생성을 억제하고 카테킨 (catechin) 역시 NLRP3 단백질 관련 반응을 억제하는 것으로 보고된 바 있다 (Wang, W. et al., Br. J. Pharmacol., 169:1352-1371; Ding, T. et al., Phytomedicine, 2018, 41:45-53; Liu, H.J et al., J. Neuroinflammation, 2017, 14:74; Jhang, J. et al., Mol. Nutr. Food Res., 2016, 60:2297-2303). 또한 추출물 다른 구성성분인 플라보노이드는 RAW 264.7 세포주에서 TNF-α, IL-1β, IL-6, IL-8을 포함한 다른 전염증성 싸이토카인의 발현을 감소시켰으며, 이러한 작용은 폴리페놀에 의한 NF-κB, AP-1, MAPK 활성 억제에 기인한 것으로 추론되었다 (Santangelo, C. et al., Ann. Ist. Super. Sanita, 2007, 43:394; Bode, A.M. and Dong, Z., Mutat. Res-Fundam. Mol. Mech. Mutagen, 2004, 555: 33-51). 본 발명은 상기 단편적 효능 확인에서 더 나아가 럽시 추출물의 항염증 효능을 증명한 것이다. In conclusion, it appears that the ruby extract of the present invention can control the production of proinflammatory cytokines in mouse and human macrophages and modulate the mechanistically related intracellular signaling system. The pro-inflammatory cytokines TNF-α and IL-1β can cause cell degeneration and death and can cause dysfunction of several organs, and also stimulate the production of pro-inflammatory cytokines such as IL-6 and IL-8 (Tracey). , KJ et al., Nature, 1987,330:662). The ruby extract of the present invention can prevent the aggravation of diseases associated with abnormal inflammatory responses by inhibiting excessive production of these cytokines. The anti-inflammatory effect of rubsy extract will be related to the constituents present in the extract. One of the components, quercetin, inhibits NLRP3 protein-related reactions, dihydroquercetin inhibits ROS production and NLRP3 complex production, procyanidin inhibits ROS production, and catechin also inhibits ROS production. It has been reported to inhibit NLRP3 protein-related responses (Wang, W. et al., Br. J. Pharmacol., 169:1352-1371; Ding, T. et al., Phytomedicine, 2018, 41:45- 53; Liu, HJ et al., J. Neuroinflammation, 2017, 14:74; Jhang, J. et al., Mol. Nutr. Food Res., 2016, 60:2297-2303). In addition, flavonoids, other components of the extract, decreased the expression of other proinflammatory cytokines including TNF-α, IL-1β, IL-6, and IL-8 in the RAW 264.7 cell line, and this action was , inferred to be due to inhibition of AP-1, MAPK activity (Santangelo, C. et al., Ann. Ist. Super. Sanita, 2007, 43:394; Bode, AM and Dong, Z., Mutat. Res- Fundam. Mol. Mech. Mutagen, 2004, 555: 33-51). The present invention further proves the anti-inflammatory efficacy of the rubsy extract from the fragmentary efficacy confirmation.
본 발명 럽시 추출물의 항염증 효능에 비추어 볼 때, 본 발명의 조성물은 염증성 질환을 예방 또는 치료할 수 있다. In view of the anti-inflammatory effect of the ruby extract of the present invention, the composition of the present invention can prevent or treat inflammatory diseases.
본원에서 사용되는 용어 "염증성 질환"이란 염증에서 기인하거나 염증에서 발생하거나 염증을 유도하는 질환을 지칭한다. 용어 "염증성 질환"은 또한 비정상적인 조직 손상 및 세포사를 초래하는 대식세포, 과립구, 및/또는 T 림프구에 의한 과도한 반응에 의해 유발되는 조절이상 (dysregulated) 염증성 반응을 지칭할 수도 있다. As used herein, the term “inflammatory disease” refers to a disease resulting from, arising from, or inducing inflammation. The term “inflammatory disease” may also refer to a dysregulated inflammatory response caused by an excessive response by macrophages, granulocytes, and/or T lymphocytes resulting in abnormal tissue damage and cell death.
특정 구체예에서, 염증성 질환은 항체 매개 염증성 과정을 포함한다. "염증성 질환"은 급성 또는 만성 염증성 병태일 수 있고 감염 또는 비감염성 원인에서 발생할 수 있다. 상기 염증성 질환으로는 예컨대, 죽상동맥경화증, 동맥경화증, 자가면역 장애, 다발성 경화증, 전신 홍반성 루프스, 다발성근육통 류마티즘(PMR), 통풍 관절염, 퇴행성 관절염, 건염, 활액낭염, 건선, 낭포성 섬유증, 관절골염, 류마티스 관절염, 염증성 관절염, 쇼그렌 증후군, 거대 세포 동맥염, 진행성전신성경화증 (강피증), 강직성 척추염, 다발성근염, 피부근염, 천포창, 유천포창, 당뇨병 (예, 제1형), 중증 근무력증, 하시모토 갑상선염, 그레이브스병, 굿패스쳐 질환, 혼합 결합 조직병, 경화성담관염, 염증성 장 질환, 크론병, 궤양성 대장염, 악성 빈혈, 염증성 피부병, 통상성 간질성 폐렴 (UIP), 석면병, 규폐증, 기관지 확장증, 베릴륨중독, 활석증, 진폐증, 유육종증, 박리성간질성폐렴, 임파구성 간질성 폐렴, 거대 세포 간질성 폐렴, 세포 간질성 폐렴, 외인성 알레르기성 폐포염, 베게너 육아종증 및 맥관염 관련 형태 (측두동맥염 및 결절성다발성동맥염), 염증성 피부병, 간염, 지연형 과민 반응 (예, 옻중독), 폐렴, 기도 염증, 성인 호흡 장애 증후군 (ARDS), 뇌염, 즉시성 과민 반응, 천식, 건초열, 알레르기, 급성 아나필락시스, 류마티스성 열, 사구체신염, 신우신염, 봉와직염, 방광염, 만성 담낭염, 국소 빈혈 (허혈성 손상), 동종이식 거부반응, 숙주대이식편 거부반응, 맹장염, 동맥염, 안검염, 세기관지염, 기관지염, 자궁경관염, 담관염, 융모양막염, 결막염, 누선염, 피부근염, 심장내막염, 자궁내막염, 장염, 전장염, 상과염, 부고환염, 근막염, 결합조직염, 위염, 위장염, 치은염, 회장염, 홍채염, 후두염, 척수염, 심근염, 신염, 제염, 난소염, 고환염, 골염, 이염, 췌장염, 이하선염, 심낭염, 인두염, 능막염, 정맥염, 간질성폐렴, 직장항문염, 전립선염, 비염, 난관염, 부비강염, 구내염, 활액막염, 고환염, 편도염, 요도염, 방광 감염 (urocystitis), 포도막염, 질염, 맥관염, 음문염, 및 외음질염, 혈관염, 만성 기관지염, 골수염, 시신경염, 측두동맥염, 횡단척수염, 뇌사성 근막염, 및 뇌사성 전장염을 들 수 있으나, 이에 국한되는 것은 아니다. In certain embodiments, the inflammatory disease comprises an antibody mediated inflammatory process. An “inflammatory disease” may be an acute or chronic inflammatory condition and may arise from an infectious or non-infectious cause. The inflammatory diseases include, for example, atherosclerosis, arteriosclerosis, autoimmune disorders, multiple sclerosis, systemic lupus erythematosus, polymyalgia rheumatism (PMR), gouty arthritis, degenerative arthritis, tendinitis, bursitis, psoriasis, cystic fibrosis, joint Osteoarthritis, rheumatoid arthritis, inflammatory arthritis, Sjogren's syndrome, giant cell arteritis, progressive systemic sclerosis (scleroderma), ankylosing spondylitis, polymyositis, dermatomyositis, pemphigus, pemphigus, diabetes (eg type 1), myasthenia gravis, Hashimoto Thyroiditis, Graves' disease, Goodpasture's disease, mixed connective tissue disease, sclerosing cholangitis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, pernicious anemia, inflammatory dermatosis, common interstitial pneumonia (UIP), asbestos disease, silicosis, bronchiectasis , beryllium poisoning, talcosis, pneumoconiosis, sarcoidosis, dissociative interstitial pneumonia, lymphocytic interstitial pneumonia, giant cell interstitial pneumonia, cell interstitial pneumonia, exogenous allergic alveolitis, Wegener's granulomatosis and vasculitis-associated forms (temporal Arteritis and polyarteritis nodosa), inflammatory dermatosis, hepatitis, delayed-type hypersensitivity reactions (eg poison poisoning), pneumonia, airway inflammation, adult respiratory distress syndrome (ARDS), encephalitis, immediate hypersensitivity reaction, asthma, hay fever, allergy, acute Anaphylaxis, rheumatic fever, glomerulonephritis, pyelonephritis, cellulitis, cystitis, chronic cholecystitis, ischemia (ischemic injury), allograft rejection, host-to-graft rejection, appendicitis, arteritis, blepharitis, bronchiolitis, bronchitis, cervicitis , cholangitis, chorioamnionitis, conjunctivitis, laryngitis, dermatomyositis, endocarditis, endometritis, enteritis, total colitis, epididymitis, epididymitis, fasciitis, connective tissueitis, gastritis, gastroenteritis, gingivitis, ileitis, iritis, laryngitis, myelitis, Myocarditis, nephritis, decontamination, oophoritis, orchitis, osteitis, otitis, pancreatitis, parotitis, pericarditis, pharyngitis, synovitis, phlebitis, interstitial pneumonia, proctitis, prostatitis, rhinitis, salpingitis, sinusitis, stomatitis, synovitis, orchitis, Tonsillitis, urethritis, bladder infection (urocystitis), uveitis, vaginitis, vasculitis, vulvitis, and vulvovaginitis, vasculitis, bay sexual bronchitis, osteomyelitis, optic neuritis, temporal arteritis, transverse myelitis, cerebrospinal fasciitis, and cerebrospinalitis.
본 발명의 럽시 추출물을 포함하는 의약은 면역 기능이 저하된 환자, 암 환자 등에게 투여할 수 있으며, 면역능이 저하되어 질병에 걸릴 위험이 높은 대상체에게도 투여할 수 있다.The medicament comprising the ruby extract of the present invention may be administered to patients with reduced immune function, cancer patients, and the like, and may be administered to subjects with a high risk of disease due to decreased immune function.
본 발명의 의약은 의약으로서의 조성물 100 중량부에 대하여 본 발명의 럽시 추출물을 0.1 내지 99.9 중량부 포함할 수 있다. 그러나 이는 투약자의 필요에 따라 증감할 수 있으며, 식생활, 영양 상태, 병의 진행 정도, 뇌 기능 장애 정도 등 상황에 따라 적절히 증감할 수 있다. The medicament of the present invention may contain 0.1 to 99.9 parts by weight of the ruby extract of the present invention based on 100 parts by weight of the composition as a medicament. However, this can be increased or decreased according to the needs of the user, and it can be appropriately increased or decreased according to circumstances such as diet, nutritional status, disease progression, and brain dysfunction.
본 발명의 의약은 경구 또는 비경구로 투여가 가능하며 일반적인 의약품 제제의 형태로 사용될 수 있다. 바람직한 의약 제제는 정제, 경질 또는 연질 캅셀제, 액제, 현탁제 등과 같은 경구 투여용 제제가 있으며 이들 의약 제제는 약학적으로 허용 가능한 통상의 담체, 예를 들어 경구 투여용 제제의 경우에는 부형제, 결합제, 붕해제, 활택제, 가용화제, 현탁화제, 보존제 또는 증량제 등을 더 포함하여 조제될 수 있다. 또 다른 바람직한 의약 제제는 패치제, 겔제, 연고제, 크림제 등의 피부 외용 제제 (피부 도포용 제제)가 있으며 이러한 의약 제제 역시 럽시 추출물에 의한 효과를 억제하지 않는 범위에서 피부 외용 제제에 통상적으로 사용되는 방부제, 살균제 및/또는 각종 첨가물을 임의 성분으로 포함하여 조제될 수 있다. 첨가물의 예로는 계면활성제, 습윤제, 실리콘 화합물, 고분자 물질 (고분자 화합물), 알코올류, 자외선 흡수제, 색소, 안료, 비타민, 산화방지제, 금속 이온 봉쇄제, 소염제, pH 조정제, 펄광택제, 핵산, 효소, 천연 추출물 등을 들 수 있다. The drug of the present invention can be administered orally or parenterally, and can be used in the form of a general pharmaceutical formulation. Preferred pharmaceutical formulations include formulations for oral administration such as tablets, hard or soft capsules, solutions, suspensions, and the like, and these pharmaceutical formulations include conventional pharmaceutically acceptable carriers, for example, excipients, binders, and binders for oral formulations; It may be prepared by further including a disintegrant, a lubricant, a solubilizer, a suspending agent, a preservative or an extender, and the like. Another preferred pharmaceutical preparation is a skin external preparation (skin application preparation) such as a patch, gel, ointment, cream, etc. These pharmaceutical preparations are also commonly used in external preparations for skin to the extent that the effect of the ruby extract is not inhibited. It can be prepared by including preservatives, disinfectants and/or various additives as optional ingredients. Examples of additives include surfactants, wetting agents, silicone compounds, high molecular substances (polymer compounds), alcohols, ultraviolet absorbers, pigments, pigments, vitamins, antioxidants, sequestering agents, anti-inflammatory agents, pH adjusters, pearlescent agents, nucleic acids, enzymes , and natural extracts.
본 발명의 의약에 포함될 수 있는 약학적으로 허용 가능한 담체는 제제 시에 통상적으로 이용되는 것으로서, 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 전분, 아카시아 고무, 인산 칼슘, 알기네이트, 젤라틴, 규산칼슘, 미세 결정성셀룰로스, 폴리비닐피롤리돈, 셀룰로스, 물, 시럽, 메틸 셀룰로스, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 활석, 스테아르산 마그네슘 및 미네랄 오일 등을 포함하나, 이에 국한되는 것은 아니다. 적합한 약학적으로 허용되는 담체 및 제제는 Remington's Pharmaceutical Sciences (19th ed., 1995)에 상세히 기재되어 있다.Pharmaceutically acceptable carriers that may be included in the medicament of the present invention are those commonly used in formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, silicic acid. calcium, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like. it is not Suitable pharmaceutically acceptable carriers and agents are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).
본 발명의 럽시 추출물을 포함하는 의약의 투여 용량은, 환자의 상태, 연령, 성별 및 합병증 등의 다양한 요인에 따라 당업자인 전문가에 의해 결정될 수 있지만 일반적으로는 성인 1 kg 당 0.1 ㎎ 내지 10 g, 바람직하게는 10 mg 내지 5 g의 용량으로 투여될 수 있다. 또, 단위 제형당 상기 의약의 1일 용량 또는 이의 1/2, 1/3 또는 1/4의 용량이 함유되도록 하며, 하루 1 내지 6 회 투여될 수 있다. 그러나 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취의 경우에는 상기 양은 상기 범위 이하일 수 있으며, 안전성 면에서 아무런 문제가 없다는 전문가의 판단 아래 상기 범위 이상의 양으로도 사용될 수 있다. 본 발명의 실시예에서 확인한 생체내 투여 결과는 럽시 추출물이 최대 500 mg/kg체중 투여 농도에서도 특정 장기 독성을 유발하지 않음을 보여준다.The dosage of the medicament containing the ruby extract of the present invention can be determined by a person skilled in the art according to various factors such as the patient's condition, age, sex, and complications, but generally 0.1 mg to 10 g per 1 kg for adults, Preferably, it may be administered in a dose of 10 mg to 5 g. In addition, the daily dose or 1/2, 1/3 or 1/4 of the dose of the drug per unit dosage form is contained, and may be administered 1 to 6 times a day. However, in the case of long-term intake for health and hygiene or health control, the above amount may be less than the above range, and an amount above the above range may be used under the judgment of an expert that there is no problem in terms of safety. The in vivo administration results confirmed in the Examples of the present invention show that the Rubsy extract does not induce specific organ toxicity even at a concentration of up to 500 mg/kg body weight.
본 발명의 의약은 당업자가 용이하게 실시할 수 있는 방법에 따라, 약학적으로 허용되는 담체 및/또는 부형제를 이용하여 제제화함으로써 단위 용량 형태로 제조되거나 또는 다용량 용기 내에 내입시켜 제조될 수 있다. 이때 제형은 오일 또는 수성 매질중의 용액, 현탁액 또는 유화액 형태이거나 엑스제, 분말제, 과립제, 정제 또는 캅셀제 형태일 수도 있으며, 분산제 또는 안정화제를 추가적으로 포함할 수 있다.The medicament of the present invention may be prepared in a unit dose form by formulation using a pharmaceutically acceptable carrier and/or excipient according to a method readily practiced by those skilled in the art, or may be prepared by internalizing in a multi-dose container. At this time, the formulation may be in the form of a solution, suspension, or emulsion in oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet or capsule, and may additionally include a dispersant or stabilizer.
체로스폰디아스 액실라리스 ( Choerospondias axillaris) 추출물을 포함하는 본 발명의 면역 증강용 조성물은 항염증 및 면역 증강에 효과가 있으며, 염증 질환 및 염증 반응과 관련된 질환에 유용하게 사용할 수 있는 효과가 있다.cherospondias axillaris ( Choerospondias axillaris ) The composition for enhancing immunity of the present invention comprising an extract is effective in anti-inflammatory and immune enhancement, and has an effect that can be usefully used in inflammatory diseases and diseases related to inflammatory reactions.
체로스폰디아스 액실라리스 ( Choerospondias axillaris) 추출물, 즉 럽시 추출물이 마우스 모델 및 인체 대식세포 시험관내 실험 모델에서 항염증 효능을 가짐이 확인되었다. 럽시 추출물이 LPS로 자극된 염증 표지자를 억제한다는 것이 확인되었고 또한, 마우스 및 인체 대식세포 모두에서 전염증성 싸이토카인 생성을 억제할 수 있음이 확인되었다. 이로부터, 럽시 추출물의 항염증 활성이 NF-κB, MAPK, 인플라마좀 경로 모두에 작용한다는 것을 알 수 있다.cherospondias axillaris It was confirmed that ( Choerospondias axillaris ) extract, ie, Ruby extract, had anti-inflammatory effects in mouse model and human macrophage in vitro experimental model. It was confirmed that ruby extract inhibited LPS-stimulated inflammatory markers, and it was also confirmed that it could inhibit pro-inflammatory cytokine production in both mouse and human macrophages. From this, it can be seen that the anti-inflammatory activity of the rubsy extract acts on all of the NF-κB, MAPK, and inflammasome pathways.
럽시 추출물은 또한, 체액 면역 (humoral immunity)에서 Th2 반응 대비 Th1 반응을 촉진하는 특성이 있어, 알레르기와 관련된 혈청 항체 수준은 낮추고 점막 (mucosal) 및 장 면역 (gut immunity)과 관련된 항체 수준은 증가시킬 수 있음이 확인되었고 럽시 추출물에 의하여 비장 T 림프구로부터 일부 전염증성 싸이토카인의 생성을 억제할 수도 있음이 확인되었다.Ruby extract also has the property of promoting a Th1 response versus a Th2 response in humoral immunity, lowering serum antibody levels related to allergy and increasing antibody levels related to mucosal and gut immunity. It was confirmed that the extract could inhibit the production of some proinflammatory cytokines from splenic T lymphocytes by the extract.
도 1a-1b: 대식세포의 세포 생존력에 대한 추출물의 영향. (a) RAW 264.7 대식세포. (b) THP-1 유래 대식세포 (THP-1 derived macrophage, TDM).1A-1B: Effect of extracts on cell viability of macrophages. (a) RAW 264.7 macrophages. (b) THP-1 derived macrophage (TDM).
도 2a-2b: RAW 264.7 세포에서 LPS 활성화에 따른 NO 및 PGE 2 생성에 대한 추출물의 영향. (a) NO 생성 수준. (b) PGE 2 수준. #, *: 각각 LPS 비활성대조군 (비활성대조군) 또는 추출물 첨가하지 않은 부형제대조군 (부형제대조군) 대비 유의한 차이 (p ≤ 0.05).Figures 2a-2b: Effect of extracts on NO and PGE 2 production following LPS activation in RAW 264.7 cells. (a) NO production level. (b) PGE 2 levels. #, *: Significant difference (p ≤ 0.05) compared to LPS inactive control group (inactive control group) or excipient control group without extract (excipient control group), respectively.
도 3a-3d: LPS 활성화에 따른 RAW 264.7 세포에서 생성되는 활성 산소종 수준에 대한 추출물의 영향. (a) 비활성대조군 유세포 분석시 대식세포 게이팅, (b) LPS 활성된 경우 전체 세포에 대한 유세포 분석시 대식세포 게이팅, (c) 활성화 및 추출물 첨가 조건별 대표적인 활성 산소종 생성에 대한 형광 염색 강도 그래프, (d) 비활성대조군 대비 형광 강도에 대한 3 회 반복 시험 평균 ± SEM. #, *: 각각 LPS 비활성대조군 (비활성대조군) 또는 추출물 첨가하지 않은 부형제대조군 (부형제대조군) 대비 유의한 차이 (p ≤ 0.05).Figures 3a-3d: Effect of extracts on the level of reactive oxygen species produced in RAW 264.7 cells following LPS activation. (a) macrophage gating during flow cytometry analysis of inactive control group, (b) macrophage gating during flow cytometry analysis of whole cells when LPS is activated, (c) fluorescence staining intensity graph for representative reactive oxygen species generation by activation and extract addition conditions , (d) Mean ± SEM of three replicates of fluorescence intensity versus inactive control. #, *: Significant difference (p ≤ 0.05) compared to LPS inactive control group (inactive control group) or excipient control group without extract (excipient control group), respectively.
도 4a-4c: LPS 활성화에 따른 RAW 264.7 세포에서 생성되는 전염증성 싸이토카인 수준에 대한 추출물의 영향. 배양액 내 수준 IL-6 (a), TNF-α (b), IL-1β (c). #, *: 각각 LPS 비활성대조군 (비활성대조군) 또는 추출물 첨가하지 않은 부형제대조군 (부형제대조군) 대비 유의한 차이 (p ≤ 0.05).Figures 4a-4c: Effect of extracts on the level of pro-inflammatory cytokines produced in RAW 264.7 cells following LPS activation. Levels in culture medium IL-6 (a), TNF-α (b), IL-1β (c). #, *: Significant difference (p ≤ 0.05) compared to LPS inactive control group (inactive control group) or excipient control group without extract (excipient control group), respectively.
도 5a-5d: LPS 활성화에 따른 THP-1세포 유래 대식세포 (TDM)에서 생성되는 전염증성 싸이토카인 수준에 대한 추출물의 영향. 배양액 내 수준 IL-6 (a), TNF-α (b), IL-1β (c), IL-8 (d). #, *: 각각 LPS 비활성대조군 (비활성대조군) 또는 추출물 첨가하지 않은 부형제대조군 (부형제대조군) 대비 유의한 차이 (p ≤ 0.05).Figure 5a-5d: Effect of extracts on the level of pro-inflammatory cytokines produced in THP-1 cell-derived macrophages (TDM) following LPS activation. Levels in culture IL-6 (a), TNF-α (b), IL-1β (c), IL-8 (d). #, *: Significant difference (p ≤ 0.05) compared to LPS inactive control group (inactive control group) or excipient control group without extract (excipient control group), respectively.
도 6: RAW 264.7 세포에서 COX-2의 발현에 대한 추출물의 영향. Figure 6: Effect of extracts on the expression of COX-2 in RAW 264.7 cells.
도 7: RAW 264.7 세포에서 NF-κB의 발현에 대한 추출물의 영향.Figure 7: Effect of extracts on the expression of NF-κB in RAW 264.7 cells.
도 8: RAW 264.7 세포에서 MAPK 인산화 신호전달 단백질 발현에 대한 추출물의 영향.Figure 8: Effect of extracts on MAPK phosphorylation signaling protein expression in RAW 264.7 cells.
도 9: THP-1 유래 대식세포 (TDM)에서 추출물에 의한 LPS/ATP 활성화에 따른 NLRP3 인플라마좀 발현에 대한 영향.Figure 9: Effect of LPS/ATP activation by extracts on NLRP3 inflammasome expression in THP-1-derived macrophages (TDM).
도 10a-10e: IgG 아형 (IgG1 및 IgG2a), IgA 및 IgE 면역글로불린 수준에 대한 추출물 투여의 영향. * 대조군 대비 유의한 차이 (p-값 ≤ 0.05).10A-10E: Effect of extract administration on IgG subtypes (IgG1 and IgG2a), IgA and IgE immunoglobulin levels. * Significant difference compared to control (p-value ≤ 0.05).
도 11a-11f: 비장 T림프구 배양 상층액에서 생성된 싸이토카인 수준에 대한 추출물 투여의 영향. * 대조군 대비 유의한 차이 (p-값 ≤ 0.05).11A-11F: Effect of extract administration on cytokine levels produced in splenic T lymphocyte culture supernatant. * Significant difference compared to control (p-value ≤ 0.05).
도 12a-12b: 싸이클로포스파미드 (CYP)를 주사한 마우스의 비장 및 흉선 지수에 대한 추출물의 영향. #, * 유의성은 각각 부형제대조군 및 CYP 투여 대조군 (추출물 비투여 모델대조군)과 비교한 차이 (p-값 ≤ 0.05). 12A-12B: Effect of extracts on spleen and thymus indices of mice injected with cyclophosphamide (CYP). #, * Significance was the difference (p-value ≤ 0.05) compared to the excipient control group and the CYP administration control group (extract non-administration model control group), respectively.
도 13a-13d: CYP 또는 추출물을 투여한 마우스에서 혈액학적 지표치 수준에 대한 추출물의 영향. RBC, red blood cells (적혈구); HGB, haemoglobin (헤모글로빈); NEUT, neutrophils (호중구); LYM, lymphocytes (림프구). #, * 유의성은 각각 부형제대조군 및 CYP 투여 대조군 (추출물 비투여 모델대조군)과 비교한 차이 (p-값 ≤ 0.05).13A-13D: Effect of extracts on hematologic index levels in mice administered CYP or extracts. RBCs, red blood cells; HGB, haemoglobin (hemoglobin); NEUT, neutrophils (neutrophils); LYM, lymphocytes (lymphocytes). #, * Significance was the difference (p-value ≤ 0.05) compared to the excipient control group and the CYP administration control group (extract non-administration model control group), respectively.
도 14a-14e: CYP 또는 추출물을 투여한 마우스에서 혈청 면역글로불린 수준. # 및 *: 각각 부형제대조군 및 CYP 투여 대조군 (추출물 비투여 모델대조군)과 비교한 차이 (p-값 ≤ 0.05).14A-14E: Serum immunoglobulin levels in mice administered CYP or extracts. # and *: the difference (p-value ≤ 0.05) compared to the excipient control group and the CYP administration control group (extract non-administration model control group), respectively.
도 15a-15b: CYP 또는 추출물을 투여한 마우스 비장 B림프구에서 생성된 배양 상층액 중 항체 수준. # 및 *: 각각 부형제대조군 및 CYP 투여 대조군 (추출물 비투여 모델대조군)과 비교한 차이 (p-값 ≤ 0.05).15A-15B: Antibody levels in culture supernatants generated from splenic B lymphocytes of mice administered with CYP or extracts. # and *: the difference (p-value ≤ 0.05) compared to the excipient control group and the CYP administration control group (extract non-administration model control group), respectively.
도 16a-16f: CYP를 주사한 마우스의 비장 T림프구 배양 상층액에서 생성된 싸이토카인 수준에 대한 추출물의 영향. # 및 *: 각각 부형제대조군 및 CYP 투여 대조군 (추출물 비투여 모델대조군)과 비교한 차이 (p-값 ≤ 0.05).16A-16F: Effect of extracts on cytokine levels produced in splenic T lymphocyte culture supernatants of CYP-injected mice. # and *: the difference (p-value ≤ 0.05) compared to the excipient control group and the CYP administration control group (extract non-administration model control group), respectively.
도 17a-17b: CYP를 주사한 마우스의 장간막 림프절 T림프구 배양 상층액에서 생성된 싸이토카인 수준에 대한 추출물의 영향. # 및 *: 각각 부형제대조군 및 CYP 투여 대조군 (추출물 비투여 모델대조군)과 비교한 차이 (p-값 ≤ 0.05).17A-17B: Effect of extracts on cytokine levels produced in mesenteric lymph node T lymphocyte culture supernatants of CYP-injected mice. # and *: the difference (p-value ≤ 0.05) compared to the excipient control group and the CYP administration control group (extract non-administration model control group), respectively.
도 18a-18f: CYP를 주사한 마우스의 각 비장 면역 세포의 비율에 대한 추출물의 영향. # 및 *: 각각 부형제대조군 및 CYP 투여 대조군 (추출물 비투여 모델대조군)과 비교한 차이 (p-값 ≤ 0.05).18A-18F: Effect of extracts on the proportion of each spleen immune cell in mice injected with CYP. # and *: the difference (p-value ≤ 0.05) compared to the excipient control group and the CYP administration control group (extract non-administration model control group), respectively.
이하, 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있도록 본 발명의 실시예에 대하여 첨부한 도면을 참고로 하여 상세히 설명한다. 그러나 본 발명은 여러 가지 상이한 형태로 구현될 수 있으며 여기에서 설명하는 실시예에 한정되지 않는다. Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings so that those of ordinary skill in the art can easily carry out the present invention. However, the present invention may be embodied in several different forms and is not limited to the embodiments described herein.
실험 재료 및 방법:Experimental Materials and Methods:
식물 수집 및 추출 준비:Plant collection and extraction preparation:
건강하고 신선한 체로스폰디아스 액실라리스 ( Choerospondias axillaris, Lapsi, 럽시) 과실을 네팔 카트만두의 현지 시장에서 구매하였다. 과실을 증류수로 세척하고 껍질과 과육을 씨와 분리하였다. 그늘에서 말린 과실의 껍질과 과육 분말 (10 g)을 300 ml의 70% 에탄올에 첨가하였다. 혼합물을 30분 동안 초음파 처리하고 공정을 2 회 반복하였다. 여과액을 혼합하고 Whatman 여과지를 사용하여 여과하였다. 여과액을 45℃에서 진공 하에서 회전 증발을 통해 농축했다. 마지막으로, 얻어진 추출물을 동결 건조하여 동결 건조 분말을 얻었다.Healthy and fresh fruits of Choerospondias axillaris (Lapsi) were purchased from a local market in Kathmandu, Nepal. The fruit was washed with distilled water, and the skin and pulp were separated from the seeds. Shade dried fruit peel and pulp powder (10 g) was added to 300 ml of 70% ethanol. The mixture was sonicated for 30 minutes and the process repeated twice. The filtrate was mixed and filtered using Whatman filter paper. The filtrate was concentrated via rotary evaporation at 45° C. under vacuum. Finally, the obtained extract was freeze-dried to obtain a freeze-dried powder.
항염증 효능 평가를 위한 in vitro 시험 사용 세포주:Cell lines used in vitro to evaluate anti-inflammatory efficacy:
추출물의 항염증 효능 및 관련 기전을 연구하기 위해, 한국 세포주 은행에서 구매한 마우스 대식세포 RAW 264.7 세포주와 인간 THP-1 세포주 유래 대식세포 (TDM)의 두 가지를 사용하였다. THP-1 세포는 American type culture collection (ATCC) 에서 구입하였다.To study the anti-inflammatory efficacy and related mechanisms of the extract, two types of macrophages (TDM) derived from a mouse macrophage RAW 264.7 cell line and a human THP-1 cell line were used. THP-1 cells were purchased from the American type culture collection (ATCC).
세포 배양:Cell culture:
마우스 대식세포 RAW 264.7 세포를 1% 페니실린 (penicilline)-스트렙토마이신 (streptomycin)-네오마이신 (neomycin) 및 10% 열-불활성화 FBS (fetal bovine serum)가 첨가된 DMEM (Dulbecco's modified eagle's medium) 배지에 배양하였다. 계대 배양을 위해서 T75 배양 플라스크당 1.5 × 10 6 세포 밀도로 접종하였다. 세포 배양 밀도 70~80% 정도에서 계대 배양을 실시하였다.Mouse macrophage RAW 264.7 cells were cultured in DMEM (Dulbecco's modified eagle's medium) medium supplemented with 1% penicillin-streptomycin-neomycin and 10% heat-inactivated fetal bovine serum (FBS). cultured. For subculture, the cells were seeded at a density of 1.5 × 10 6 cells per T75 culture flask. Subculture was performed at a cell culture density of about 70-80%.
THP-1 세포주는 0.05 mM 2-메르캅토에탄올 (mercaptoethanol), 1% 페니실린-스트렙토마이신-네오마이신 혼합물 및 10% FBS가 추가된 RPMI-1640 배지에서 배양하고 2~3일마다 계대 배양하였다. THP-1 세포를 대식세포로 분화시키기 위해 100 ng/ml의 PMA (phorbol 12-myristate-13-acetate)를 첨가하여 48 시간 동안 배양하였다. 분화된 THP-1 세포주 유래 대식세포는 추출물 및 LPS로 처리하기 전 48 시간 동안 PMA가 없는 배지에서 안정화시켰다.The THP-1 cell line was cultured in RPMI-1640 medium supplemented with 0.05 mM 2-mercaptoethanol, 1% penicillin-streptomycin-neomycin mixture and 10% FBS, and subcultured every 2-3 days. To differentiate THP-1 cells into macrophages, 100 ng/ml of PMA (phorbol 12-myristate-13-acetate) was added and cultured for 48 hours. Differentiated THP-1 cell line-derived macrophages were stabilized in PMA-free medium for 48 h before treatment with extract and LPS.
대식세포의 세포 생존력 평가:Assessing cell viability of macrophages:
RAW 264.7 대식세포 (1×10 4 cells/100 μl) 및 TDM (4×10 4 cells/100μl)을 96-well 플레이트에 세포를 깔고 37℃, 5% CO 2 배양기에서 24 시간 동안 배양하였다. 세포를 다양한 농도의 추출물로 처리한 후 2 시간 배양하고 LPS 1μg/ml를 첨가한 뒤 24 시간 배양하였다. 10 μl의 CCK-8 시약을 각 well에 첨가하고 3 시간 더 배양하였다. 650 nm의 보정 파장 하에 450 nm 에서 흡광도를 판독하였다.RAW 264.7 macrophages (1×10 4 cells/100 μl) and TDM (4×10 4 cells/100 μl) were spread on a 96-well plate and cultured at 37° C., 5% CO 2 in an incubator for 24 hours. The cells were treated with extracts of various concentrations, incubated for 2 hours, and then cultured for 24 hours after addition of 1 μg/ml LPS. 10 μl of CCK-8 reagent was added to each well and incubated for another 3 hours. The absorbance was read at 450 nm under a calibration wavelength of 650 nm.
RAW 264.7 세포의 산화 질소 (NO) 생성 분석:Analysis of Nitric Oxide (NO) Production in RAW 264.7 Cells:
세포 배양 배지 내 NO의 안정한 산화물질인 아질산염 (nitrite)의 생성 수준을 Griess 시약을 사용하여 측정하였다. 이를 위해, 세포를 24-well 배양 플레이트에 5×10 4 cells/ml 밀도로 세포를 깔고 37℃, 5% CO 2 배양기에서 24 시간 동안 배양하였다. 이후 세포를 다양한 농도의 추출물로 처리하고, 2 시간 동안 배양한 후 LPS 비활성대조군 (비활성대조군)을 제외한 모든 well에 LPS (1 μg/ml)를 첨가하였다. 24 시간 동안 배양한 후, 원심분리하여 상등액을 수집하였다. 이를 통해 얻은 상등액 50 μl를 96-well 플레이트에 첨가한 후, Griess 시약 50 μl 를 첨가하였다. Griess 시약은 Verdon et al. (1995)에 기술된 대로 제조하였다. 5% 인산 (phosphoric acid) 내 최종 농도가 1% 설파닐아미드 (sulfanilamide), 0.5% 나프틸 에틸렌디아민 디히드로클로라이드 (naphthyl ehtylenediamine dihydrochloride) 되도록 제조하였다 (Verdon, C.P. et al., Anal. Biochem., 1995, 224:502-508). 시약을 첨가한 후, 플레이트를 실온에서 15 분 동안 배양하고 540 nm에서 흡광도를 측정하고 아질산나트륨 표준 곡선을 통해 정량하였다.The production level of nitrite, a stable oxidizing substance of NO, in the cell culture medium was measured using the Griess reagent. To this end, the cells were laid out in a 24-well culture plate at a density of 5×10 4 cells/ml and cultured at 37° C., 5% CO 2 in an incubator for 24 hours. Thereafter, the cells were treated with extracts of various concentrations, and after incubation for 2 hours, LPS (1 μg/ml) was added to all wells except for the LPS inactive control group (inactive control group). After incubation for 24 hours, the supernatant was collected by centrifugation. 50 μl of the obtained supernatant was added to a 96-well plate, and then 50 μl of Griess reagent was added. Griess reagent is described in Verdon et al. (1995) as described. It was prepared so that the final concentration in 5% phosphoric acid was 1% sulfanilamide, 0.5% naphthyl ethylenediamine dihydrochloride (Verdon, CP et al., Anal. Biochem., 1995, 224:502-508). After addition of the reagents, the plates were incubated at room temperature for 15 minutes and the absorbance was measured at 540 nm and quantified via a sodium nitrite standard curve.
RAW 264.7 세포내 활성 산소종 (ROS) 측정:RAW 264.7 Intracellular Reactive Oxygen Species (ROS) Measurement:
LPS 및 추출물로 처리한 RAW 264.7 세포내 ROS의 생성을 측정하기 위해 DCF-DA 염색 분석을 이용하였다. 비형광성인 2', 7'-디클로로디하이드로플루오레세인-디아세테이트 (2', 7'-dichlorodihydrofluorescein diacetate, H2DCF-DA)는 ROS 존재시 고형광성 유도체인 DCF로 전환되는 방법이다. 24-well 배양 플레이트에 well당 5×10 5 cells/ml를 넣고 37℃, 5% CO 2 배양기에서 24 시간 동안 배양하였다. 이후 세포를 다양한 농도의 추출물로 처리하고, 2 시간 동안 배양한 후 LPS 비활성대조군 (비활성대조군)을 제외한 모든 well에 LPS (1 μg/ml)를 첨가하여 24 시간 동안 배양한 후 세포를 수집하였다. 2 ml의 DPBS를 첨가하여 2 회 세척후, 10 μM H2DCF-DA를 튜브에 첨가하고 37℃에서 20 분 동안 정치시켰다. 형광 강도는 유세포측정기를 사용하여 분석하였다.DCF-DA staining analysis was used to measure the production of ROS in RAW 264.7 cells treated with LPS and extract. Non-fluorescent 2', 7'-dichlorodihydrofluorescein-diacetate (2', 7'-dichlorodihydrofluorescein diacetate, H2DCF-DA) is a method for converting DCF, a highly fluorescent derivative, in the presence of ROS. Put 5×10 5 cells/ml per well in a 24-well culture plate, and incubate at 37° C., 5% CO 2 in an incubator for 24 hours. Thereafter, the cells were treated with extracts of various concentrations, and after incubation for 2 hours, LPS (1 μg/ml) was added to all wells except for the LPS inactive control group (inactive control group) and cultured for 24 hours, and then the cells were collected. After washing twice with the addition of 2 ml of DPBS, 10 μM H2DCF-DA was added to the tube and allowed to stand at 37°C for 20 minutes. Fluorescence intensity was analyzed using a flow cytometer.
RAW 264.7 세포에 의해 생성된 전염증성 싸이토카인 및 염증 표지자의 평가:Assessment of proinflammatory cytokines and inflammatory markers produced by RAW 264.7 cells:
세포를 24-well 배양 플레이트에 5×10 4 cells/ml 밀도로 세포를 깔고 37℃, 5% CO 2 배양기에서 24 시간 동안 배양하였다. 이후 세포를 다양한 농도의 추출물로 처리하고, 2 시간 동안 배양한 후 LPS 비활성대조군 (비활성대조군)을 제외한 모든 well에 LPS (1 μg/ml)를 첨가하였다. 24 시간 동안 배양한 후, 원심분리하여 상등액을 수집하였다. 이를 통해 얻은 상등액을 이용하여 프로스타글란딘 E 2, IL-6, IL-1β 및 TNF-α의 생성 수준을 ELISA방법으로 분석하였다.The cells were laid out in a 24-well culture plate at a density of 5×10 4 cells/ml and cultured at 37° C., 5% CO 2 in an incubator for 24 hours. Thereafter, the cells were treated with extracts of various concentrations, and after incubation for 2 hours, LPS (1 μg/ml) was added to all wells except for the LPS inactive control group (inactive control group). After incubation for 24 hours, the supernatant was collected by centrifugation. Using the obtained supernatant, the production levels of prostaglandin E 2 , IL-6, IL-1β and TNF-α were analyzed by ELISA method.
TDM 생성 싸이토카인 수준 분석:Analysis of TDM-producing cytokine levels:
세포를 24-well 배양 플레이트에 5×10 4 cells/ml 밀도로 세포를 깔고 37℃, 5% CO 2 배양기에서 24 시간 동안 배양하였다. 이후 세포를 다양한 농도의 추출물로 처리하고, 2시간 동안 배양한 후 LPS 비활성대조군 (비활성대조군)을 제외한 모든 well에 LPS (1 μg/ml)를 첨가하였다. 24 시간 동안 배양한 후, 원심분리하여 상등액을 수집하였다. 이를 통해 얻은 상등액을 이용하여 TNFα, IL-8, IL-6을 ELISA 방법으로 분석하였다. IL-1β의 경우, 상등액 수집 45 분 전 3 mM ATP를 상등액에 첨가하고 ELISA 방법으로 정량하였다.The cells were laid out in a 24-well culture plate at a density of 5×10 4 cells/ml and cultured at 37° C., 5% CO 2 in an incubator for 24 hours. Thereafter, the cells were treated with extracts of various concentrations, and after incubation for 2 hours, LPS (1 μg/ml) was added to all wells except for the LPS inactive control group (inactive control group). After incubation for 24 hours, the supernatant was collected by centrifugation. Using the obtained supernatant, TNFα, IL-8, and IL-6 were analyzed by ELISA method. In the case of IL-1β, 3 mM ATP was added to the supernatant 45 minutes before supernatant collection and quantified by ELISA method.
Western blot 방법에 의한 추출물의 RAW 264.7 세포에서 항염증기전 분석:Analysis of anti-inflammatory mechanisms in RAW 264.7 cells of extracts by Western blot method:
항염증 활성과 관련된 기전은 염증 매개 단백질 발현을 정량하여 확인하였다. RAW 264.7 세포 (2×10 6)를 90 x 20mm cell 배양 플레이트에서 24 시간 동안 배양하고 다양한 농도의 추출물로 처리한 후 3 시간 동안 배양한 후 LPS (1 μg/ml)로 처리하였다. 15 분 (phospho-ERK, total ERK, phospho-JNK, JNK, phospho-p38, p38의 경우), 30 분 (phospho-IκB alpha 및 NF-κB의 경우) 및 24 시간 (COX2의 경우) 후 DPBS 첨가한 뒤 스크래핑하여 세포를 수집하였다. 4℃, 1500 rpm에서 5 분간 원심 분리하여 세포만을 1.5 ml 에펜도르프 (Eppendorf) 튜브에 수집한 뒤, 4℃, 4000 rpm에서 3 분 동안 1 ml의 DPBS로 2 회 세척하였다. 수집된 세포는 1% 프로테아제 억제제 칵테일 (protease inhibitor cocktail), 1 mM 플루오르화 나트륨 (sodium fluoride, NaF) 및 1 mM 페닐메틸 술포닐플루오라이드 (phenylmethylsulfonyl fluoride, PMSF)를 첨가한 RIPA 완충액으로 용해시켰다. RIPA 완충액은 세포를 냉장상태에 위치시키면서 첨가한 뒤 30 분 동안 10 분마다 볼텍싱 교반하였다. 이후 튜브를 4℃, 1600 Х g에서 30 분 동안 원심분리한 뒤 상등액을 채취하였다. NE-PER 핵 및 세포질 추출 시약을 사용하여 핵 용해물을 추출하여 핵 NF-κB (nuclear NF-κB) 수준을 분석하였다. Mechanisms related to anti-inflammatory activity were confirmed by quantifying the expression of inflammation-mediated proteins. RAW 264.7 cells (2×10 6 ) were cultured in a 90 x 20mm cell culture plate for 24 hours, treated with extracts of various concentrations, incubated for 3 hours, and then treated with LPS (1 μg/ml). DPBS addition after 15 min (for phospho-ERK, total ERK, phospho-JNK, JNK, phospho-p38, p38), 30 min (for phospho-IκB alpha and NF-κB) and 24 h (for COX2) After that, the cells were collected by scraping. Cells were collected in 1.5 ml Eppendorf tubes by centrifugation at 4° C., 1500 rpm for 5 minutes, and then washed twice with 1 ml of DPBS at 4° C., 4000 rpm for 3 minutes. The collected cells were lysed in RIPA buffer containing 1% protease inhibitor cocktail, 1 mM sodium fluoride (NaF) and 1 mM phenylmethylsulfonyl fluoride (PMSF). RIPA buffer was added while the cells were refrigerated and then vortexed every 10 minutes for 30 minutes. Then, the tube was centrifuged at 4 ℃, 1600 Х g for 30 minutes, and the supernatant was collected. Nuclear lysates were extracted using NE-PER nuclear and cytoplasmic extraction reagent to analyze nuclear NF-κB (nuclear NF-κB) levels.
용해액 내 BCA 단백질 분석 키트를 사용하여 총단백질 정량을 시행한 뒤, 30 μg을 10~12%의 폴리아크릴아미드 (polyacrylamide) SDS 겔을 사용하여 분리하고 폴리비닐리덴 디플루오라이드 멤브레인 (PVDF membrane)으로 옮겼다. 막을 0.1% Tween 20 (TBST) 및 5% DifcoTM skim milk 또는 5% BSA를 함유하는 tris-bufferd saline(TBS)에 넣고 실온에서 2 시간 동안 회전교반함으로써 비특이적 결합을 차단시켰다. TBS-0.1% Tween 20으로 세척한 후, 5% BSA-TBST에 phospo-p38, phospho-JNK, p38, JNK, phospho-ERK1/2, ERK1/2, NF-κB p65, phospho-IκBα, COX-2 및 β-actin 항체들을 넣고 4℃ 에서 회전교반을 밤새 실시하였다. 멤브레인을 각각 5 분 동안 TBS로 2 회 및 TBST로 3 회 세척하고, 5% skim milk-TBST 또는 3% BSA-TBST에 m-IgGκ BP-HRP 또는 anti-rabbit IgG HRP 항체를 첨가하여 1 시간 동안 실온에서 회전교반하였다. TBST로 각각 10 분 동안 3 회 세척한 후 TBS로 각각 5 분 동안 3 회 헹구고, Western Chemiluminescent HRP substrate을 사용하여 화학 발광 검출을 수행하였다. 필요시 스트리핑을 실시한 뒤 재프루빙하였다.After performing total protein quantification using the BCA protein analysis kit in the lysate, 30 μg was separated using a 10-12% polyacrylamide SDS gel and polyvinylidene difluoride membrane (PVDF membrane) moved to Non-specific binding was blocked by placing the membrane in tris-buffered saline (TBS) containing 0.1% Tween 20 (TBST) and 5% Difco™ skim milk or 5% BSA and rotational stirring at room temperature for 2 hours. After washing with TBS-0.1% Tween 20, phospo-p38, phospho-JNK, p38, JNK, phospho-ERK1/2, ERK1/2, NF-κB p65, phospho-IκBα, COX- 2 and β-actin antibodies were added, and rotational stirring was performed at 4°C overnight. The membrane was washed twice with TBS and three times with TBST for 5 min each, and m-IgGκ BP-HRP or anti-rabbit IgG HRP antibody was added to 5% skim milk-TBST or 3% BSA-TBST for 1 hour. Rotational stirring was carried out at room temperature. After washing 3 times with TBST for 10 min each, rinse 3 times with TBS for 5 min each, chemiluminescent detection was performed using Western Chemiluminescent HRP substrate. If necessary, stripping was performed and then reproving was performed.
Western blot 방법에 의한 추출물의 TDM 세포에서 항염증 기전 분석:Analysis of anti-inflammatory mechanisms in TDM cells of extracts by Western blot method:
TDM (8.8×10 6 cells) 세포를 90 x 20mm cell 배양 플레이트에서 24 시간 동안 배양하고 다양한 농도의 추출물로 처리한 후 3 시간 동안 배양한 후 LPS (1 μg/ml)를 세포에 첨가하고 3 시간 더 배양하였다. 이후 3mM ATP를 세포에 첨가하고 45분 동안 추가 배양하였다. 그 다음 과정은 RAW 264.7 세포의 경우와 같았는데, 5% BSA-TBST 완충액에 NLRP3 (D2P5E) rabbit monoclonal antidody를 넣고 5% skim milk에 anti rabbit IgG HRP 항체를 넣어 각각 1 차 및 2 차 항체로 사용하였다.TDM (8.8×10 6 cells) cells were cultured in a 90 x 20 mm cell culture plate for 24 hours, treated with extracts of various concentrations, incubated for 3 hours, and then LPS (1 μg/ml) was added to the cells for 3 hours. further cultured. Then, 3 mM ATP was added to the cells and further incubated for 45 minutes. The following procedure was the same as that of RAW 264.7 cells. NLRP3 (D2P5E) rabbit monoclonal antidody was added to 5% BSA-TBST buffer, and anti rabbit IgG HRP antibody was added to 5% skim milk, and used as primary and secondary antibodies, respectively. .
추출물의 면역 조절/항진 기능을 평가하기 위한 in vivo 실험:In vivo experiments to evaluate the immune modulating/stimulating function of extracts:
특정 병원체 부재 4 주령의 수컷 BALB/c 마우스를 연구에 사용하였다. 실험동물을 50 ± 5% 상대 습도 및 12 시간 명암 주기로 22 ± 2℃에서 유지되는 특정 병원체가 없는 시설에서 환기 챔버를 갖춘 멸균 케이지에 넣었다. 모든 마우스는 표준 설치류 사료와 가압멸균 여과수에 자유로이 접근할 수 있었다. 모든 관리 및 실험 절차는 대구가톨릭대학교 동물윤리위원회 (IACUC-2018-015)의 승인에 따라 이루어졌다.Specific pathogen-free 4-week-old male BALB/c mice were used for the study. Animals were housed in sterile cages with ventilation chambers in a specific pathogen-free facility maintained at 22 ± 2 °C with 50 ± 5% relative humidity and a 12 hour light-dark cycle. All mice had free access to standard rodent diet and autoclaved filtered water. All management and experimental procedures were performed in accordance with the approval of the Daegu Catholic University Animal Ethics Committee (IACUC-2018-015).
적응 1주 후, 마우스들을 추출물의 면역조절력 시험에 대한 스크리닝을 위해 군당 6 마리, 4 개 군으로 나누었다. 4 개 군 중에서 1 개 군에는 부형제를 투여하고, 나머지 3 개 군에는 상이한 농도 (5, 50, 500 mg/kg 체중/day/100 μl)의 추출물을 21 일(주말을 제외한 4 주) 동안 위내관을 통해 투여하였다.After 1 week of adaptation, the mice were divided into 4 groups, 6 mice per group, for screening for the immunomodulatory ability test of the extract. Excipients were administered to 1 group out of 4 groups, and extracts at different concentrations (5, 50, 500 mg/kg body weight/day/100 μl) were administered to the other 3 groups for 21 days (4 weeks excluding weekends). Administered through the inner tube.
추출물의 면역 항진 특성을 조사하기 위해, 마우스를 6 개의 군으로 나누었다. 이 군들 중 2 개 군 (CYP 주사 하지 않고 부형제만 투여한 부형제대조군, CYP 주사하고 추출물을 투여하지 않고 부형제 투여한 모델대조군)에는 부형제를 위 내로 투여하고 나머지 군에는 부형제에 현탁시킨 서로 다른 농도의 추출물을 위 내로 투여하였다. 부형제로는 DMSO와 Tween 80가 각각 2% 함유된 생리식염수를 사용하였다. 부형제대조군 마우스에는 부형제 100 μl를 주사하고 다른 모든 마우스에는 면역억제를 유도하기 위해 제 1, 7, 14 일에 복강 내로 CYP 100 mg/kg 체중/100 μl를 주사하였다.To investigate the immunostimulating properties of the extract, mice were divided into 6 groups. Of these groups, two groups (the excipient control group in which only excipients were administered without CYP injection, and the model control group in which CYP injections and excipients were administered without administration of excipients) were administered intragastrically, and the remaining groups were administered with different concentrations of the excipients suspended in the excipients. The extract was administered intragastrically. As an excipient, DMSO and physiological saline containing 2% of Tween 80, respectively, were used. Vehicle control mice were injected with 100 μl of vehicle, and all other mice were intraperitoneally injected with 100 mg/kg body weight/100 μl of CYP on days 1, 7, and 14 to induce immunosuppression.
혈액 및 조직병리학적 평가:Hematological and histopathological evaluation:
K 2EDTA 채혈관에 혈액샘플을 채취한 후 혈액학적 분석을 실시하였다. 군당 마우스 1 마리로부터 비장, 흉선, 장간막 림프절을 채취하여 10% 완충 포르말린에 저장하였다. 대구경북 첨단 의료산업진흥재단 (Daegu Gyeongbuk Medical Innovation Foundation, DGMIF)의 실험 동물 센터에서 헤마톡실린 및 에오신 염색 (hematoxylin and eosin staining)을 통해 조직병리학적 평가를 진행하였다.After collecting blood samples from K 2 EDTA blood collection vessels, hematological analysis was performed. Spleen, thymus, and mesenteric lymph nodes were collected from one mouse per group and stored in 10% buffered formalin. Histopathological evaluation was performed through hematoxylin and eosin staining at the experimental animal center of the Daegu Gyeongbuk Medical Innovation Foundation (DGMIF).
혈청 면역글로불린 수준 및 비장 B 림프구 배양 후 면역글로불린 생성에 대한 추출물의 영향:Effect of extracts on serum immunoglobulin levels and immunoglobulin production after splenic B lymphocyte culture:
심장 혈액에서 분리한 혈청을 사용하여 혈청 내 면역글로불린 수준을 평가하였다. B 림프구 생성 면역글로불린 수준을 평가하기 위해, 비장 단일 세포를 LPS 1 μg, 재조합 마우스 IL-4 (50 ng) 및 재조합 인간 APRIL (10 ng)을 첨가하여 37℃, 5% CO 2 배양기에서 96 시간 동안 complete RPMI 배지에서 배양하였다. 항체 수준은 샌드위치 ELISA를 사용하여 측정하였다.Serum isolated from cardiac blood was used to evaluate the level of immunoglobulin in the serum. To assess B lymphocyte-producing immunoglobulin levels, spleen single cells were treated with 1 μg of LPS, recombinant mouse IL-4 (50 ng) and recombinant human APRIL (10 ng) added to 37° C., 5% CO 2 incubator for 96 hours. during incubation in complete RPMI medium. Antibody levels were determined using a sandwich ELISA.
비장 및 장간막 림프절 T림프구 배양 수준에 대한 추출물의 영향:Effect of extract on spleen and mesenteric lymph node T lymphocyte culture levels:
비장 및 장간막 림프절 T림프구를 37℃, 5% CO 2 배양기에서 48 시간 동안 동안 5-unit 재조합 인간 IL-2 및 immobilized anti-CD3e mAb(5 μg/5x10 5 cell)로 활성화시켰다. 다양한 싸이토카인, 인터페론-감마 (IFN-γ), 종양 괴사 인자-알파 (tumor necrosis factor-alpha, TNF-α), 인터루킨-4 (interleukin-4, IL-4), 인터루킨-17 (IL-17) 및 형질전환 성장 인자-베타 1 (transforming growth factor-beta 1, TGF-β1)을 비장 세포 T 림프구 배양 상층액에서 측정하고 장간막 림프절 T 림프구 배양 상층액에서는 IFN-γ 및 IL-17의 수준을 샌드위치 ELISA를 사용하여 측정하였다.Splenic and mesenteric lymph node T lymphocytes were activated with 5-unit recombinant human IL-2 and immobilized anti-CD3e mAb (5 μg/5×10 5 cells) in an incubator at 37° C., 5% CO 2 for 48 hours. Various cytokines, interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), interleukin-4 (interleukin-4, IL-4), interleukin-17 (IL-17) and transforming growth factor-beta 1 (TGF-β1) measured in spleen cell T lymphocyte culture supernatant and sandwiched levels of IFN-γ and IL-17 in mesenteric lymph node T lymphocyte culture supernatant It was measured using ELISA.
비장 및 흉선 면역세포 구성비율에 대한 추출물의 영향:Effect of extract on spleen and thymic immune cell composition:
비장세포 및 흉선세포를 유세포 분석을 위해 처리하였다. 비장세포는 CD4+ CD4+ 보조 T 림프구), CD8+ 세포파괴 T 림프구), B220 B 림프구 및 NK 세포 (natural killer cell) 표현형에 대해 스크리닝하고, 흉선세포는 CD4+, CD8+, 그리고 CD4+CD8+ 2중-양성 T 림프구에 대해 분석하였다. 10 6 개의 세포를 소듐 아자이드가 함유된 인산염 완충액 (phosphate buffer solution, PBS)으로 세척하였다. FcBlock (1μg/10 6 세포)으로 비특이적 결합을 방지한 상태에서 anti-CD3 PE, anti-CD3 FITC, anti-CD4 FITC, anti-CD8 PE, anti-CD45RB/B220 PE, anti-CD335 (NKp46) PE 및 anti-CD49b APC를 사용하여 특정 세포 집단을 분류하였다. 또한 FITC-컨쥬게이션, PE-컨쥬게이션, 및 APC-컨쥬게이션 이소형 대조군도 사용하였다.Splenocytes and thymocytes were processed for flow cytometry analysis. Splenocytes are screened for CD4+ CD4+ helper T lymphocytes), CD8+ apoptotic T lymphocytes), B220 B lymphocytes and natural killer cell (NK cell) phenotypes, and thymocytes are CD4+, CD8+, and CD4+CD8+ double-positive T Lymphocytes were analyzed. 10 6 cells were washed with sodium azide-containing phosphate buffer solution (PBS). Anti-CD3 PE, anti-CD3 FITC, anti-CD4 FITC, anti-CD8 PE, anti-CD45RB/B220 PE, anti-CD335 (NKp46) PE in the state of preventing non-specific binding with FcBlock (1μg/10 6 cells) and anti-CD49b APC were used to sort specific cell populations. FITC-conjugated, PE-conjugated, and APC-conjugated isotype controls were also used.
통계 분석:Statistical analysis:
데이터는 평균±표준 편차 (standard deviation, SD) 또는 평균±평균의 표준오차 (standard error of mean, SEM)로 표시하였다. 데이터를 정규 분포와 등분산에 대해 스크리닝하였다. 데이터의 정규성에 따라 일원분산분석 (one-way analysis of variance, ANOVA) 또는 Kruskal-Wallis 순위 검정을 이용하여 군 간의 유의한 차이를 조사하였다. 군 간에 유의한 차이가 존재하는 경우 Student-Newman-Keuls (SNK) 사후 방법을 추가로 사용하였다. 두 군간 비교를 시행해야 하는 경우, 데이터 분포에 따라 Student t-검정 또는 비분포 비모수 검정을 사용하였다. 0.05 이하의 p-값을 통계적으로 유의한 것으로 간주하였다.Data were expressed as mean±standard deviation (SD) or mean±standard error of mean (SEM). Data were screened for normal distribution and equal variance. Significant differences between groups were investigated using one-way analysis of variance (ANOVA) or Kruskal-Wallis rank test according to data normality. If there was a significant difference between groups, the Student-Newman-Keuls (SNK) post-hoc method was additionally used. When it was necessary to perform a comparison between two groups, either the Student's t-test or the non-distributed nonparametric test was used depending on the data distribution. A p-value of 0.05 or less was considered statistically significant.
실시예:Example:
실시예 1: LPS 활성 대식세포의 생존력에 대한 추출물의 영향:Example 1: Effect of extract on viability of LPS-activated macrophages:
대식세포의 세포 생존력에 대한 추출물의 영향을 평가하였다. RAW 264.7 대식세포 및 THP-1 유래 대식세포 (THP-1 derived macrophage, TDM) 각각을 24 시간 동안 추출물 및 LPS (1 μg/ml)와 함께 배양한 뒤 생존력을 CCK-8 세포 계수 키트 (CCK-8 cell counting kit)를 사용하여 결정하였다. 결과는 3 번의 독립적 실험의 평균 ± SD로 나타내었다.The effect of the extract on the cell viability of macrophages was evaluated. RAW 264.7 macrophages and THP-1 derived macrophage (TDM) each were incubated with extract and LPS (1 μg/ml) for 24 h and then viability was assessed with CCK-8 cell counting kit (CCK- 8 cell counting kit) was used. Results are presented as mean±SD of three independent experiments.
시험한 추출물 농도 (0, 8, 16, 31, 63, 125, 250 및 500 μg/ml) 중에서 250 μg/ml 및 500 μg/ml 는 세포 생존률을 현저히 저하시켰다. 125 μg/ml 농도에서는 RAW 264.7 세포주 및 TDM에서 각각 세포사멸이 없거나 75% 정도의 세포 생존율을 유지하였다 (도 1). 추출물의 75% 세포 생존율 (CV 75%)은 RAW 264.7 세포주는 149 μg/ml 및 TDM 세포주는 125 μg/ml로 계산되었다. Among the extract concentrations tested (0, 8, 16, 31, 63, 125, 250 and 500 μg/ml), 250 μg/ml and 500 μg/ml significantly reduced cell viability. At a concentration of 125 μg/ml, there was no apoptosis or cell viability was maintained at about 75% in RAW 264.7 cell line and TDM, respectively ( FIG. 1 ). The 75% cell viability (CV 75%) of the extract was calculated to be 149 μg/ml for RAW 264.7 cell line and 125 μg/ml for TDM cell line.
실시예 2: LPS 활성 RAW 264.7 대식세포에서 추출물에 의한 NO, PGE 2 및 ROS 생성 억제:Example 2: Inhibition of NO, PGE 2 and ROS production by extracts in LPS-activated RAW 264.7 macrophages:
RAW 264.7 세포에서 LPS 활성화에 따른 NO, PGE 2 및 ROS 생성에 대한 추출물의 영향을 평가하였다. Effects of extracts on NO, PGE 2 and ROS production according to LPS activation in RAW 264.7 cells were evaluated.
RAW 264.7 세포 배양 직후 추출물을 넣고 2 시간 배양한 뒤 LPS (1 μg/ml)을 넣고 24 시간 배양한 뒤 배양 상등액을 수집하였고, Griess 시험으로 NO 생성 수준을 분석하였고, PGE 2 수준은 ELISA를 사용하여 분석하였다. 값은 3번의 독립적 실험의 평균 ± SEM으로 나타내었다.Immediately after culturing RAW 264.7 cells, extract was added and incubated for 2 hours, then LPS (1 μg/ml) was added and cultured for 24 hours, the culture supernatant was collected, NO production level was analyzed by Griess test, and PGE 2 level was ELISA. and analyzed. Values are presented as the mean ± SEM of three independent experiments.
RAW 264.7 세포 배양 직후 추출물을 넣고 2시간 배양한 뒤 LPS (1 μg/ml)을 넣고 24 시간 배양한 뒤 세포 내 활성 산소종 수준을 DCFH-DA 형광 염료를 사용하여 측정하였다. 유세포 분석에 총 10000 개의 세포가 사용되었다.Immediately after culturing RAW 264.7 cells, the extract was added and incubated for 2 hours, then LPS (1 μg/ml) was added and incubated for 24 hours. The intracellular reactive oxygen species level was measured using DCFH-DA fluorescent dye. A total of 10000 cells were used for flow cytometry.
본 실험을 위하여 추출물 5가지 농도를 사용하였다. LPS 비활성대조군 (비활성대조군)에 비해 부형제대조군 (LPS 활성, 추출물에 대한 부형제만 첨가)에서 유의하게 증가된 NO, PGE 2 및 ROS를 생성하였고 추출물은 용량-의존적으로 (도 2) 또는 처리 용량에서 비숫하게 (도 3) 이들 지표치 생성을 감소시켰다. Five concentrations of extracts were used for this experiment. Compared to the LPS inactive control group (inactive control group), the excipient control group (LPS activity, only adding excipients to the extract) produced significantly increased NO, PGE 2 and ROS, and the extract was dose-dependently ( FIG. 2 ) or at the treatment dose. Equally ( FIG. 3 ) the production of these indicators was reduced.
실시예 3: LPS 활성 RAW 264.7 세포주 및 THP-1 유래 대식세포주에서 추출물에 의한 전염증성 싸이토카인 생성 억제 효능:Example 3: Efficacy of inhibiting proinflammatory cytokine production by extracts in LPS-activated RAW 264.7 cell line and THP-1 derived macrophage cell line:
LPS 활성화에 따른, RAW 264.7 세포 및 THP-1세포 유래 대식세포 (TDM)에서 생성되는 전염증성 싸이토카인 수준에 대한 추출물의 영향을 평가하였다.The effect of the extract on the level of pro-inflammatory cytokines produced in RAW 264.7 cells and THP-1 cell-derived macrophages (TDM) according to LPS activation was evaluated.
RAW 264.7 세포 배양 직후 추출물을 넣고 2시간 배양한 뒤 LPS (1 μg/ml)을 넣고 24 시간 배양한 뒤 배양 상등액을 수집하였다. 배양액 내 IL-6, TNF-α, IL-1β 수준은 ELISA를 사용하여 분석하였다. 값은 3번의 독립적 실험의 평균 ± SEM으로 나타내었다.Immediately after culturing RAW 264.7 cells, the extract was added and incubated for 2 hours, then LPS (1 μg/ml) was added and cultured for 24 hours, and the culture supernatant was collected. IL-6, TNF-α, IL-1β levels in culture were analyzed using ELISA. Values are presented as the mean ± SEM of three independent experiments.
TDM으로 분화가 완료된 세포에 세포 배양 직후 추출물을 넣고 2 시간 배양한 뒤 LPS (1 μg/ml)을 넣고 24 시간 배양한 뒤 배양 상등액을 수집하였다. 배양액 내 IL-6, TNF-α, IL-1β, IL-8 수준은 ELISA를 사용하여 분석하였다. 값은 3 번의 독립적 실험의 평균 ± SEM으로 나타내었다.Immediately after cell culture, the extract was added to the TDM-differentiated cells, incubated for 2 hours, LPS (1 μg/ml) was added, and the culture supernatant was collected after 24 hours of incubation. IL-6, TNF-α, IL-1β, and IL-8 levels in culture were analyzed using ELISA. Values are presented as the mean ± SEM of three independent experiments.
LPS 활성화에 따른 RAW 264.7 세포로부터 유의하게 생성된 IL-6, TNFα, IL-1β 수준은 추출물 첨가로 저하되었다. IL-6 및 IL-1β는 30~125 μg/ml 범위의 추출물 농도에 의해 용량-의존적으로 저하되었으며(도 4a, 4c), TNF-α 생성의 경우 100 μg/ml 및 125 μg/ml 농도에서 유의하게 감소되었다(도 4b). TDM의 경우 IL-6, TNF-α, IL-1β, IL-8 수준은 추출물 농도에 따라 용량-의존적으로 저하되었다 (도 5). The levels of IL-6, TNFα, and IL-1β significantly produced from RAW 264.7 cells following LPS activation were decreased by the addition of the extract. IL-6 and IL-1β were dose-dependently lowered by extract concentrations ranging from 30 to 125 μg/ml (Figs. 4a, 4c), and for TNF-α production at concentrations of 100 μg/ml and 125 μg/ml was significantly reduced (Fig. 4b). In the case of TDM, IL-6, TNF-α, IL-1β, and IL-8 levels were dose-dependently decreased according to the concentration of the extract ( FIG. 5 ).
실시예 4: LPS 활성 RAW 264.7 세포에서 COX-2 단백질 발현에 대한 추출물의 영향:Example 4: Effect of extracts on COX-2 protein expression in LPS-activated RAW 264.7 cells:
RAW 264.7 세포에서 COX-2의 발현에 대한 추출물의 영향을 평가하였다. RAW 264.7 세포에 추출물 75, 100 μg/ml을 첨가한 뒤 3 시간 후 LPS (1 μg/ml)를 첨가하여 24 시간 배양하였다. 단백질 발현은 western blot 방법으로 분석하고, β-액틴을 internal control로 사용하여 발현 정도를 표준화하였다. 발현 단백질의 상대 수치는 비활성대조군 대비 %로 나타내었고, 2회의 독립적 반복 실험의 평균이다.The effect of the extract on the expression of COX-2 in RAW 264.7 cells was evaluated. After adding 75 and 100 μg/ml of extract to RAW 264.7 cells, 3 hours later, LPS (1 μg/ml) was added and incubated for 24 hours. Protein expression was analyzed by western blot method, and expression level was standardized using β-actin as an internal control. The relative level of the expressed protein was expressed as a percentage compared to the inactive control group, and is the average of two independent replicates.
도 6에서 보여지듯이 LPS는 COX-2 단백질 발현을 유의하게 비활성대조군에 비해 3배정도 상승시켰으며, 추출물은 용량 의존적으로 COX-2 발현을 억제하였다. 기존 연구에서 COX-2 억제제는 PGE 2의 합성을 차단하여 염증을 억제하는 것으로 보고된 바 있다 (Eliopoulos, A.G., EMBO. J., 2002, 21: 4831-4840). 이에 본 발명의 추출물은 COX-2 발현 억제에 따라 PGE 2 생성 감소를 수반한 것으로 추론할 수 있다.As shown in FIG. 6 , LPS significantly increased COX-2 protein expression by about 3 times compared to the inactive control group, and the extract inhibited COX-2 expression in a dose-dependent manner. In previous studies, it has been reported that COX-2 inhibitors inhibit inflammation by blocking the synthesis of PGE 2 (Eliopoulos, AG, EMBO. J., 2002, 21: 4831-4840). Accordingly, it can be inferred that the extract of the present invention is accompanied by a decrease in PGE 2 production according to the inhibition of COX-2 expression.
실시예 5: LPS 활성 RAW 264.7 세포에서 IκBα 및 NF-κB 활성화에 대한 추출물의 영향:Example 5: Effect of extracts on IκBα and NF-κB activation in LPS-activated RAW 264.7 cells:
RAW 264.7 세포에서 NF-κB의 발현에 대한 추출물의 영향을 평가하였다. RAW 264.7 세포에 추출물 75, 100 μg/ml을 첨가한 뒤 3 시간 후 LPS (1 μg/ml)를 첨가하여 30분 배양하였다. 단백질 발현은 western blot방법으로 분석하고, β-액틴을 internal control로 사용하여 발현 정도를 표준화하였다. 발현 단백질의 상대 수치는 비활성대조군 대비 %로 나타내었고, 2 회의 독립적 반복 실험의 평균이다.The effect of the extract on the expression of NF-κB in RAW 264.7 cells was evaluated. After adding 75 and 100 μg/ml of extract to RAW 264.7 cells, 3 hours later, LPS (1 μg/ml) was added and incubated for 30 minutes. Protein expression was analyzed by western blot method, and expression level was standardized using β-actin as an internal control. The relative level of the expressed protein was expressed as a percentage compared to the inactive control group, and it is the average of two independent replicates.
RAW 264.7 세포에서 염증 반응에 따른 싸이토카인/케모카인 생성과 관련된 세포 내 신호전달 pathway 중 주요한 NF-κB pathway를 분석하였다. LPS 비활성대조군에 비해 부형제대조군의 경우 NF-κB 핵 내 전이가 2 배 정도 더 이루어졌으며, 추출물은 이러한 핵 내 전이를 억제하는 것으로 나타났다 (도 7). 이러한 현상은 추출물에 의한 IκBα의 인산화 억제에 기인한 것으로 판단된다.In RAW 264.7 cells, the major NF-κB pathway among intracellular signaling pathways related to cytokine/chemokine production following an inflammatory response was analyzed. In the case of the excipient control group compared to the LPS inactive control group, NF-κB nuclear metastasis was made about 2 times more, and the extract was found to inhibit this nuclear metastasis ( FIG. 7 ). This phenomenon is considered to be due to the inhibition of phosphorylation of IκBα by the extract.
실시예 6: LPS 활성 RAW 264.7 세포에서 MAPK 활성화에 대한 추출물의 영향:Example 6: Effect of extracts on MAPK activation in LPS-activated RAW 264.7 cells:
RAW 264.7 세포에서 MAPK 인산화 신호전달 단백질 발현에 대한 추출물의 영향을 평가하였다. RAW 264.7 세포에 추출물 75, 100 μg/ml을 첨가한 뒤 3 시간 후 LPS (1 μg/ml)를 첨가하여 15 분 배양하였다. 단백질 발현은 western blot 방법으로 분석하고, β-액틴을 internal control로 사용하여 발현 정도를 표준화하였다. 발현 단백질의 상대 수치는 비활성대조군 대비 %로 나타내었고, 2 회의 독립적 반복 실험의 평균이다.The effect of extracts on MAPK phosphorylation signaling protein expression in RAW 264.7 cells was evaluated. After adding 75 and 100 μg/ml of extract to RAW 264.7 cells, 3 hours later, LPS (1 μg/ml) was added and incubated for 15 minutes. Protein expression was analyzed by western blot method, and expression level was standardized using β-actin as an internal control. The relative level of the expressed protein was expressed as a percentage compared to the inactive control group, and it is the average of two independent replicates.
MAPK 활성화 경로는 c-Jun N-말단 키나제 (c-Jun N-terminal kinase, JNK), 세포 외 신호-조절 단백질 키나제 (extracellular signal-regulated protein kinases, ERK1/2) 및 p38 MAPK로 구성되어 염증 반응 생성 물질 조절에 실질적인 주요한 역할을 하는 것으로 보고되어 왔다. 본 발명에서도 LPS 활성화에 따라 인산화된 p38, ERK1/2 및 JNK의 발현 수준이 증가하였으며, 추출물은 이들 단백질들의 인산화를 억제하였다 (도 8). 이러한 결과는 염증 반응에 따른 싸이토카인/케모카인 생성과 관련된 세포 내 신호전달 pathway 중 MAPK 신호전달 pathway 경로도 추출물이 제어함을 보여주는 것이다.The MAPK activation pathway consists of c-Jun N-terminal kinase (JNK), extracellular signal-regulated protein kinases (ERK1/2), and p38 MAPK, leading to an inflammatory response. It has been reported to play a substantial major role in product regulation. In the present invention, the expression levels of phosphorylated p38, ERK1/2 and JNK increased according to LPS activation, and the extract inhibited phosphorylation of these proteins (FIG. 8). These results show that the extract also controls the MAPK signaling pathway among the intracellular signaling pathways related to cytokine/chemokine production according to the inflammatory response.
실시예 7: LPS 활성 TDM 세포주에서 NLRP3 발현에 대한 추출물의 영향:Example 7: Effect of extracts on NLRP3 expression in LPS-activated TDM cell lines:
THP-1 유래 대식세포 (TDM)에서 추출물에 의한 LPS/ATP 활성화에 따른 NLRP3 인플라마좀 발현에 대한 영향을 평가하였다. TDM 세포에 추출물 75, 100 μg/ml을 첨가한 뒤 3 시간 후 LPS (1 μg/ml)를 첨가하여 3 시간 배양하였다. 이후 3 mM ATP를 첨가한 뒤 45 분간 배양하였다. 단백질 발현은 western blot 방법으로 분석하고, β-액틴을 internal control로 사용하여 발현 정도를 표준화하였다. 발현 단백질의 상대 수치는 비활성대조군 (이 경우에는 ATP는 첨가하였음) 대비 %로 나타내었고, 2 회의 독립적 반복 실험의 평균이다.The effect on NLRP3 inflammasome expression according to LPS/ATP activation by the extract in THP-1-derived macrophages (TDM) was evaluated. After adding 75 and 100 μg/ml of extract to TDM cells, 3 hours later, LPS (1 μg/ml) was added and incubated for 3 hours. Then, 3 mM ATP was added and incubated for 45 minutes. Protein expression was analyzed by western blot method, and expression level was standardized using β-actin as an internal control. The relative level of the expressed protein was expressed as a percentage compared to the inactive control group (in this case, ATP was added), and is the average of two independent replicates.
LPS와 ATP 첨가에 따른 THP-1 유래 대식세포의 NLRP3 항진이 관찰되었으며, 추출물은 용량 의존적으로 NLRP3 발현을 억제하였다 (도 9). NLRP3 enhancement of THP-1-derived macrophages was observed following the addition of LPS and ATP, and the extract inhibited NLRP3 expression in a dose-dependent manner ( FIG. 9 ).
실시예 8: 추출물 투여에 따른 조직병리학적 특성 분석:Example 8: Analysis of histopathological characteristics according to the administration of the extract:
흉선, 비장 및 장간막 림프절의 조직병리학적 분석에서 럽시 추출물로 인한 비정상적 변화는 나타나지 않았던 반면, 싸이클로포스파미드 주사와 관련된 조직병리학적 변화는 명확하게 나타났다. CYP 투여 마우스에서, 흉선에서의 피질 대 수질 비가 증가했고, 비장 백색속질의 변연부에서 세포충실성에 변화가 있었으며, 림프절 피질의 세포충실성이 감소하였다. 추출물 500 mg/kg 체중 투여군에서는 흉선에서 관찰된 CYP의 영향이 관찰되지 않았다 (표 1).Histopathological analysis of thymus, spleen, and mesenteric lymph nodes showed no abnormal changes due to Rubsy extract, whereas histopathological changes related to cyclophosphamide injection were clearly identified. In CYP-administered mice, the cortex to medulla ratio in the thymus was increased, there was a change in cellularity in the marginal area of the splenic white medullary, and the cellularity of the lymph node cortex was decreased. The effect of CYP observed in the thymus was not observed in the extract 500 mg/kg body weight group (Table 1).
흉선, 비장 및 장간막 림프절에서의 조직병리학적 소견Histopathological findings in thymus, spleen, and mesenteric lymph nodes
군 / 용량(mg/kg)group/dose (mg/kg) 조직병리 소견histopathological findings
부형제대조군excipient control group 주목할 만한 소견 없음no noteworthy findings
CYPCYP 흉선(thymus) - 피질(cortex):수질(medulla) 비 증가비장 - 백색속질(white pulp) 변연부(marzinal zone)에서 세포 수(cellularity) 변화 림프절 - 피질에서 세포 수 감소thymus - cortex: medulla ratio increased spleen - white pulp cellularity change in marginal zone lymph node - decreased cell number in cortex
Lapsi (5 mg/kg)Lapsi (5 mg/kg) 주목할 만한 소견 없음no noteworthy findings
Lapsi (50 mg/kg)Lapsi (50 mg/kg) 주목할 만한 소견 없음no noteworthy findings
Lapsi (500 mg/kg)Lapsi (500 mg/kg) 주목할 만한 소견 없음no noteworthy findings
CYP + Lapsi (5 mg/kg)CYP + Lapsi (5 mg/kg) 흉선 - 피질:수질 비 증가비장 - 백색속질 변연부 세포 수 변화, 적색속질(red pulp)에서 다핵거대세포(multinucleated giant cell) 증가 림프절 - 피질에서 세포 수 감소Thymus - Cortex:Medulum ratio increase Spleen - White medullary marginal cell number change, multinucleated giant cells increase in red pulp Lymph node - Cell number decrease in cortex
CYP + Lapsi (50 mg/kg)CYP + Lapsi (50 mg/kg) 흉선 - 피질에서 follicle 관찰비장 - 백색속질 변연부 세포 수 변화림프절 - 피질에서 세포 수 감소Thymus - follicle observed in cortex Spleen - white medullary marginal cell number change Lymph node - reduced cell number in cortex
CYP + Lapsi (500 mg/kg)CYP + Lapsi (500 mg/kg) 비장 - 백색속질 변연부 세포 수 변화림프절 - 피질에서 세포 수 감소Spleen - white medullary marginal area change in cell number Lymph node - decrease in cell number in cortex
실시예 9: 말초혈액 세포 수 정량 분석:Example 9: Quantitative analysis of peripheral blood cell count:
통계적으로 유의하지는 않다 하더라도, 호중구 %가 50 mg/kg 추출물 투여군에서 부형제대조군에 비해 높았고 단핵구 %는 여러 투여 농도에서 대조군에 비해 낮았다. 그러나 림프구 %는 50 mg/kg 및 500 mg/kg 추출물 투여군에서 높았다 (표 2).Although not statistically significant, the % of neutrophils in the 50 mg/kg extract administration group was higher than that of the excipient control group, and the % of monocytes was lower than the control group at various administration concentrations. However, the lymphocyte % was higher in the 50 mg/kg and 500 mg/kg extract administration groups (Table 2).
군별 말초혈액 세포 평균 (각군 6수)Average of peripheral blood cells by group (6 numbers in each group)
매개변수 parameter 대조군control Lapsi 5 Lapsi 5 Lapsi 50 Lapsi 50 Lapsi 500 Lapsi 500 p-값ANOVA 또는K-Wallis p -value ANOVA or K-Wallis
WBCs (10 3/μl)WBCs (10 3 /μl) 5.780 ± 1.2665.780 ± 1.266 5.163 ± 1.0365.163 ± 1.036 4.953 ± 0.9084.953 ± 0.908 4.908 ± 1.4244.908 ± 1.424 0.9490.949
RBCs (10 6/μl)RBCs (10 6 /μl) 11.01 ± 0.20911.01 ± 0.209 10.81 ± 0.30510.81 ± 0.305 10.74 ± 0.10410.74 ± 0.104 10.40 ± 0.26510.40 ± 0.265 0.3480.348
HGB (g/dL)HGB (g/dL) 16.58 ± 0.26516.58 ± 0.265 16.05 ± 0.38216.05 ± 0.382 16.02 ± 0.17216.02 ± 0.172 15.40 ± 0.30415.40 ± 0.304 0.0680.068
Platelets (10 3/μl)Platelets (10 3 /μl) 894.7 ± 89.60894.7 ± 89.60 1027 ± 56.891027 ± 56.89 892.5 ± 64.53892.5 ± 64.53 903.3 ± 184.2903.3 ± 184.2 0.6820.682
%NEUT (%)%NEUT (%) 24.87 ± 3.78024.87 ± 3.780 25.08 ± 2.55625.08 ± 2.556 15.88 ± 1.46315.88 ± 1.463 22.20 ± 1.63822.20 ± 1.638 0.0620.062
%LYM (%)%LYM (%) 67.72 ± 3.31067.72 ± 3.310 63.77 ± 6.59763.77 ± 6.597 70.57 ± 2.11170.57 ± 2.111 68.23 ± 1.25168.23 ± 1.251 0.7140.714
%MONO (%)%MONO (%) 1.983 ± 0.4191.983 ± 0.419 1.650 ± 0.2141.650 ± 0.214 1.250 ± 0.0921.250 ± 0.092 1.333 ± 0.1361.333 ± 0.136 0.1810.181
%EOS (%)%EOS (%) 3.200 ± 0.2663.200 ± 0.266 6.600 ± 4.2796.600 ± 4.279 4.833 ± 2.3804.833 ± 2.380 5.467 ± 1.3195.467 ± 1.319 0.3320.332
%LUC (%)%LUC (%) 1.850 ± 1.0661.850 ± 1.066 2.567 ± 0.5592.567 ± 0.559 6.817 ± 1.304 *# 6.817 ± 1.304 *# 2.383 ± 1.204 $ 2.383 ± 1.204 $ 0.0260.026
%BASO (%)%BASO (%) 0.700 ± 0.2480.700 ± 0.248 0.800 ± 0.0810.800 ± 0.081 1.033 ± 0.2741.033 ± 0.274 0.500 ± 0.1030.500 ± 0.103 0.1230.123
WBCs, white blood cells (백혈구); RBCs, red blood cells (적혈구); HGB, hemoglobin (헤모글로빈); NEUT, neutrophils (호중구); LYM, lymphocytes (림프구); MONO, monocytes (단핵구); EOS, eosinophils (호산구); LUC, large unstained cells (거대 비염색 세포); BASO, basophils (호염구). 데이터는 평균 ± SD로 표시 . *, #, $ 대조군, 50 mg/kg 및 500 mg/kg 럽시 투여군과 비교하여 각각 유의한 차이 ( p-값 ≤ 0.05). WBCs, white blood cells (white blood cells); RBCs, red blood cells; HGB, hemoglobin (hemoglobin); NEUT, neutrophils (neutrophils); LYM, lymphocytes (lymphocytes); MONO, monocytes (monocytes); EOS, eosinophils (eosinophils); LUC, large unstained cells; BASO, basophils (basophils). Data are presented as mean ± SD . *, #, $ A significant difference compared with the control group, 50 mg/kg and 500 mg/kg ruby administration groups, respectively ( p -value ≤ 0.05).
실시예 10: 혈청 내 면역글로불린 수준에 대한 추출물의 영향:Example 10: Effect of Extracts on Immunoglobulin Levels in Serum:
IgG 아형 (IgG1 및 IgG2a), IgA 및 IgE 혈청 수준에 대한 추출물 투여의 영향을 평가하였다. 면역글로불린 수준은 마우스 심장 혈액 채취 후 분리한 혈청에서 분석하였다. 데이터는 평균 ± SEM으로 표시하였다. The effect of extract administration on IgG subtypes (IgG1 and IgG2a), IgA and IgE serum levels was evaluated. Immunoglobulin levels were analyzed in serum isolated from mouse heart blood collection. Data are expressed as mean ± SEM.
알레르기 반응 지표 면역글로불린인 IgE의 수준은 500 mg/kg 추출물 투여군에서 유의하게 대조군에 비해 낮았다 (도 10d). IgG1 수준은 추출물 투여 용량 의존적으로 낮았으며 추출물 500 mg/kg 투여군에서 대조군에 비해 유의하게 낮았다 (p-값 = 0.015) (도 10a). 혈청 IgG2a의 경우에는 그러한 변화가 보이지는 않았지만, 모든 추출물 농도 투여군에서 IgG2a/IgG1 비 (ratio)가 대조군에 비해 유의하게 높았다 (도 10b, 10c). 또한 점액면역 반응에 대한 중요한 지표치인 IgA의 수준은 추출물 농도 의존적으로 높았다 (도 10e).The level of IgE, which is an indicator of allergic reaction, was significantly lower in the 500 mg/kg extract-administered group than in the control group ( FIG. 10d ). The level of IgG1 was low in a dose-dependent manner in the extract administration and was significantly lower in the extract 500 mg/kg administration group than in the control group (p-value = 0.015) ( FIG. 10A ). In the case of serum IgG2a, no such change was observed, but the IgG2a/IgG1 ratio was significantly higher in all extract concentration-administered groups than in the control group ( FIGS. 10b and 10c ). In addition, the level of IgA, an important index for the mucin immune response, was high in a concentration-dependent manner in the extract ( FIG. 10e ).
실시예 11: 비장 T 림프구 싸이토카인 생성에 대한 추출물의 영향:Example 11: Effect of extract on splenic T lymphocyte cytokine production:
비장 T 림프구 배양 상층액에서 생성된 싸이토카인 수준에 대한 추출물 투여의 영향을 평가하였다. 데이터는 평균±SEM으로 표시하였다. 5% CO 2 배양기에서 37℃에서 48 시간 동안 immolized anti-CD3e mAb (5μg/5x10 5 세포)로 활성화시킨 뒤 배양 상층액을 수집하여 싸이토카인 (IFN-γ, IL-4, IL-17, TNF-α 및 TGF-β1)을 측정하였다. immolized anti-CD3e mAb 활성화 세포에서의 싸이토카인 농도로부터 대조군으로 비활성화 세포로부터 생성된 싸이토카인 농도를 빼서 최종 농도로 하였다. The effect of extract administration on cytokine levels produced in splenic T lymphocyte culture supernatants was evaluated. Data are expressed as mean±SEM. After activation with immolized anti-CD3e mAb (5μg/5x10 5 cells) for 48 hours at 37°C in a 5% CO 2 incubator, the culture supernatant was collected and cytokines (IFN-γ, IL-4, IL-17, TNF- α and TGF-β1) were measured. The final concentration was obtained by subtracting the cytokine concentration from the inactivated cells as a control from the cytokine concentration in the immolized anti-CD3e mAb activated cells.
IFN-γ 수준은 5 및 50 mg/kg 추출물 투여군에서 대조군에 비해 낮았지만 (도 11a), 500 mg/kg 투여군에서는 대조군과 차이가 없었다. IL-4 생성 수준은 군간 뚜렷한 차이가 없었다 (도 11b). 이에 따라 IFN-γ:IL-4의 비는 5 및 50 mg/kg 추출물 투여군에서 대조군에 비해 낮았고 500 mg/kg 투여군에서는 높았다 (도 11c). 전염증성 싸이토카인 IL-17은 500 mg/kg 투여군에서 대조군에 비해 낮았지만, 유의한 차이는 아니었다 (도 11d). 반면, 또 다른 전염증성 싸이토카인인 TNF-α은 5 및 50 mg/kg 투여군에서 대조군에 비해 유의하게 낮았으며 염증반응 촉진 싸이토카인 TGF-β1은 대조군과 유의한 차이를 나타내지 않았다 (도 11e, 11f).IFN-γ level was lower than that of the control group in the 5 and 50 mg/kg extract administration group (FIG. 11a), but there was no difference in the 500 mg/kg administration group and the control group. IL-4 production levels did not differ significantly between groups (Fig. 11b). Accordingly, the ratio of IFN-γ:IL-4 was lower in the 5 and 50 mg/kg extract-administered groups than in the control group, and was high in the 500 mg/kg-administered group ( FIG. 11c ). The pro-inflammatory cytokine IL-17 was lower in the 500 mg/kg administration group than in the control group, but there was no significant difference ( FIG. 11d ). On the other hand, TNF-α, another proinflammatory cytokine, was significantly lower in the 5 and 50 mg/kg administration groups than in the control group, and the inflammatory response promoting cytokine TGF-β1 did not show a significant difference from the control group ( FIGS. 11e and 11f ).
실시예 12: 비장 및 흉선 림프구 아집단 (subpopulation) 비율에 대한 정량 분석:Example 12: Quantitative analysis of splenic and thymic lymphocyte subpopulation rates:
비장 및 흉선의 모든 면역세포에 대해 추출물 투여군과 대조군에서 유의한 차이가 관찰되지 않았다. 유의한 차이는 없었지만, 대조군과 비교했을 때 비장 B 림프구 %는 추출물 투여군에서 높았다. 비장에서 보조 T 림프구/세포파괴 T 림프구 비는 5 mg/kg 및 50 mg/kg를 투여한 마우스에서 대조군에 비해 높았다. 흉선 내 보조 T 림프구 및 세포파괴 T 림프구의 %는 추출물 투여군과 대조군 간 유의한 차이는 보이지 않았지만, 보조 T 림프구/세포파괴 T 림프구 비는 500 mg/kg 투여군에서 대조군에 비해 높았다 (표 3).No significant difference was observed between the extract-administered group and the control group for all immune cells in the spleen and thymus. Although there was no significant difference, the percentage of splenic B lymphocytes was higher in the extract-treated group compared with the control group. The helper T lymphocyte/cytotoxic T lymphocyte ratio in the spleen was higher in mice administered 5 mg/kg and 50 mg/kg compared to the control group. The percentage of auxiliary T lymphocytes and apoptotic T lymphocytes in the thymus did not show a significant difference between the extract-treated group and the control group, but the auxiliary T lymphocyte/cytotoxic T lymphocyte ratio was higher in the 500 mg/kg group than in the control group (Table 3).
비장 및 흉선 면역세포 분포(%)에 대한 추출물의 영향 (평균±SD)Effect of extract on spleen and thymus immune cell distribution (%) (mean±SD)
장기long time 면역세포immune cells 대조군control Lapsi 5 Lapsi 5 Lapsi 50 Lapsi 50 Lapsi 500Lapsi 500 p-값ANOVA 또는K-Wallis p -value ANOVA or K-Wallis
비장spleen B림프구 (%)B lymphocytes (%) 22.76 ± 8.29122.76 ± 8.291 30.06 ± 3.96630.06 ± 3.966 29.70 ± 2.03529.70 ± 2.035 27.48 ± 8.70327.48 ± 8.703 0.2870.287
CD3+ T림프구 (%)CD3+ T lymphocytes (%) 60.61 ± 10.0660.61 ± 10.06 53.41 ± 2.98353.41 ± 2.983 52.93 ± 2.29952.93 ± 2.299 57.49 ± 10.7657.49 ± 10.76 0.5830.583
CD4+ T림프구 (%)CD4+ T lymphocytes (%) 30.81 ± 7.13830.81 ± 7.138 30.71 ± 2.45030.71 ± 2.450 29.06 ± 3.64129.06 ± 3.641 26.97 ± 3.61426.97 ± 3.614 0.5880.588
CD8+ T림프구 (%)CD8+ T lymphocytes (%) 20.30 ± 6.56720.30 ± 6.567 16.38 ± 3.04216.38 ± 3.042 15.14 ± 2.01615.14 ± 2.016 20.02 ± 5.17220.02 ± 5.172 0.2250.225
CD4+/CD8+ (비)CD4+/CD8+ (non) 1.598 ± 0.4791.598 ± 0.479 1.937 ± 0.4671.937 ± 0.467 1.876 ± 0.3971.876 ± 0.397 1.391 ± 0.2441.391 ± 0.244 0.1790.179
NK 세포 (%)NK cells (%) 5.389 ± 0.5045.389 ± 0.504 4.447 ± 1.4904.447 ± 1.490 4.577 ± 2.4204.577 ± 2.420 4.791 ± 0.7904.791 ± 0.790 0.7630.763
흉선thymus CD4+ T림프구 (%)CD4+ T lymphocytes (%) 29.30 ± 1.36529.30 ± 1.365 28.42 ± 8.45928.42 ± 8.459 25.10 ± 3.41425.10 ± 3.414 28.96 ± 7.74428.96 ± 7.744 0.7940.794
CD8+ T림프구 (%)CD8+ T lymphocytes (%) 18.38 ± 2.60618.38 ± 2.606 18.06 ± 1.84318.06 ± 1.843 18.06 ± 2.00418.06 ± 2.004 14.56 ± 2.51714.56 ± 2.517 0.0590.059
CD4+CD8+ T림프구 (%)CD4+CD8+ T lymphocytes (%) 35.54 ± 8.47635.54 ± 8.476 35.56 ± 4.08235.56 ± 4.082 34.06 ± 5.35834.06 ± 5.358 41.30 ± 7.56941.30 ± 7.569 0.3810.381
CD4+/CD8+ (비)CD4+/CD8+ (non) 1.570 ± 0.2771.570 ± 0.277 1.618 ± 0.6401.618 ± 0.640 1.412 ± 0.3021.412 ± 0.302 2.032 ± 0.6072.032 ± 0.607 0.3740.374
실시예 13: CYP를 주사한 면역억제 마우스의 비장 및 흉선 지수에 대한 추출물의 영향:Example 13: Effect of extracts on spleen and thymus indices of immunosuppressed mice injected with CYP:
싸이클로포스파미드 (CYP)를 주사한 마우스의 비장 및 흉선 지수에 대한 추출물의 영향을 평가하였다. 데이터는 평균±SEM으로 표시하였다. 비장 지수 및 흉선 지수는 마우스의 비장 또는 흉선 무게를 체중으로 나누어 계산한다.The effect of the extract on the spleen and thymus indices of mice injected with cyclophosphamide (CYP) was evaluated. Data are expressed as mean±SEM. The spleen index and thymus index are calculated by dividing the spleen or thymus weight of the mouse by the body weight.
부형제대조군에 비해 CYP를 주사한 마우스에서 비장 지수가 유의하게 높았으며 추출물 투여군에서는 비장 지수가 CYP만을 투여한 모델대조군에 비해 낮아졌다. 흉선의 경우 부형제대조군에 비해 CYP 투여군에서 흉선 지수가 유의하게 낮았으나 추출물 투여군에서는 용량 의존적으로 방식으로 흉선 지수가 높았졌으며, 특히 가장 높은 농도의 추출물에서는 통계적으로도 유의한 차이를 보였다 (도 12).Compared to the excipient control group, the spleen index was significantly higher in the mice injected with CYP, and the spleen index was lower in the extract-administered group than in the model control group administered only with CYP. In the case of thymus, the thymus index was significantly lower in the CYP-administered group than in the excipient control group, but the thymus index was higher in the extract-administered group in a dose-dependent manner, and in particular, the extract at the highest concentration showed a statistically significant difference (FIG. 12) .
실시예 14: CYP를 주사한 면역억제 마우스의 혈액학적 지표치에 대한 추출물의 영향:Example 14: Effect of extracts on hematological parameters of immunosuppressed mice injected with CYP:
CYP 또는 추출물을 투여한 마우스에서 혈액학적 지표치 수준에 대한 추출물의 영향을 평가하였다. The effect of the extract on the level of hematological parameters in mice administered with CYP or the extract was evaluated.
추출물이 CYP에 의해 변동된 혈액학적 지표치들을 정상 상태로 환원시킬 수 있는지 평가하였다. CYP에 의해 유의하게 저하되었던 적혈구 수 및 림프구 백분율은 추출물 투여로 회복되는 추세를 보였다 (도 13a, 13d). 이와 유사하게, 헤모글로빈의 평균 수준이 CYP에 의해 감소되었는데 추출물 50 및 500 mg/kg 투여군에서 그 수준이 증가되었다 (도 13b). 반면, CYP를 주사한 마우스에서 유의하게 높았던 호중구 백분율은 추출물 투여군에서 낮아지는 경향을 보였다 (도 13c).It was evaluated whether the extract could reduce the hematological parameters changed by CYP to a normal state. The number of red blood cells and the percentage of lymphocytes, which were significantly lowered by CYP, showed a trend of recovery with the administration of the extract ( FIGS. 13A and 13D ). Similarly, the mean level of hemoglobin was decreased by CYP, but the level was increased in the extract 50 and 500 mg/kg administration groups ( FIG. 13b ). On the other hand, the significantly higher percentage of neutrophils in the mice injected with CYP showed a tendency to decrease in the extract-administered group (FIG. 13c).
실시예 15: CYP를 주사한 면역억제 마우스의 혈청 및 B림프구 배양액 중 면역글로불린 수준에 대한 추출물의 영향:Example 15: Effect of extracts on immunoglobulin levels in serum and B lymphocyte cultures of immunosuppressed mice injected with CYP:
CYP 또는 추출물을 투여한 마우스에서 혈청 면역글로불린 수준을 평가하였다. 데이터는 평균 ± SEM으로 표시하였다.Serum immunoglobulin levels were evaluated in mice administered CYP or extract. Data are expressed as mean ± SEM.
CYP 또는 추출물을 투여한 마우스 비장 B림프구에서 생성된 배양 상층액 중 면역글로불린 수준을 평가하였다. 데이터는 평균±SEM으로 표시하였다. 비장세포 (1x10 6)를 96 시간 동안 LPS, 재조합 마우스 IL-4 및 재조합 인간 APRIL로 활성화 배양했다. 활성화 세포에서의 면역글로불린 수준으로부터 대조군으로 비 활성화 세포로부터 생성된 면역글로불린 수준을 빼서 최종 농도로 하였다.The level of immunoglobulin in the culture supernatant generated from splenic B lymphocytes of mice administered with CYP or the extract was evaluated. Data are expressed as mean±SEM. Splenocytes (1x10 6 ) were activated and cultured with LPS, recombinant mouse IL-4 and recombinant human APRIL for 96 h. The final concentration was obtained by subtracting the level of immunoglobulin generated from non-activated cells as a control from the level of immunoglobulin in activated cells.
혈청 중 측정한 모든 면역글로불린 (IgG1, IgG2a, IgE 및 IgA)의 수준은 부형제대조군에 비해 CYP를 주사한 군에서 유의하게 낮았다 (도 14). 추출물을 투여한 군들에서는 CYP만 주사한 모델대조군에 비해 모든 면역글로불린 수준이 항진되었다. 특히 IgG2a 및 IgA의 수준은 모델대조군과 비교하여 추출물 투여군들에서 유의하게 높았다 (도 14b, 14e). 반면, IgG1 및 IgE의 수준은 여전히 부형제대조군에 비해서는 낮았다 (도 14a, 14d). 이에 따라 IgG2a/IgG1 비가 추출물 투여군에서 부형제대조군과 모델대조군에 비해 유의하게 높았다 (도 14c). 이러한 결과는 추출물 투여가 1형 보조 T 림프구 반응이 2형 보조 T 림프구 반응에 비해 우세한 체액 면역 반응을 촉진할 수 있음을 시사하는 것으로 판단된다.The levels of all immunoglobulins (IgG1, IgG2a, IgE and IgA) measured in the serum were significantly lower in the CYP-injected group compared to the vehicle control group ( FIG. 14 ). In the group to which the extract was administered, all immunoglobulin levels were enhanced compared to the model control group injected with only CYP. In particular, the levels of IgG2a and IgA were significantly higher in the extract-administered group compared to the model control group ( FIGS. 14b and 14e ). On the other hand, the levels of IgG1 and IgE were still lower than that of the excipient control group ( FIGS. 14a and 14d ). Accordingly, the IgG2a/IgG1 ratio was significantly higher in the extract-administered group than in the excipient control group and the model control group (FIG. 14c). These results are considered to suggest that the administration of the extract can promote the humoral immune response in which the type 1 helper T lymphocyte response is dominant compared to the type 2 helper T lymphocyte response.
시험관내에서 비장 B림프구를 활성화시켜 생성된 항체 수준을 평가한 결과, CYP에 의해 면역억제된 모델대조군 경우 부형제대조군에 비해 저하된 IgG1 및 IgG2a 수준을 보였으며, 추출물 투여군의 경우에는 저하된 IgG1, IgG2a 수준을 회복시키는 결과를 나타내었다 (도 15a, 15b)As a result of evaluating the level of antibodies generated by activating splenic B lymphocytes in vitro, the CYP immunosuppressed model control group showed decreased IgG1 and IgG2a levels compared to the excipient control group, and the extract-administered group showed decreased IgG1, The results of restoring the IgG2a level were shown (FIGS. 15a, 15b).
실시예 16: CYP를 주사한 면역억제 마우스의 비장 T 림프구 생성 싸이토카인 수준에 대한 추출물의 영향:Example 16: Effect of extracts on splenic T lymphocyte-producing cytokine levels in immunosuppressed mice injected with CYP:
CYP를 주사한 마우스의 비장 T림프구 배양 상층액에서 생성된 싸이토카인 수준에 대한 추출물의 영향을 평가하였다. 데이터는 평균 ± SEM으로 표시하였다. 37℃, 5% CO 2 배양기에서 48 시간 동안 immolized anti-CD3e mAb (5μg/5x10 5 세포)로 활성화시킨 뒤 배양 상층액을 수집하여 싸이토카인을 측정하였다. immolized anti-CD3e mAb 활성화 세포에서의 싸이토카인 농도로부터 대조군으로 비 활성화 세포로부터 생성된 싸이토카인 농도를 빼서 최종 농도로 하였다.The effect of the extract on the cytokine levels produced in the splenic T lymphocyte culture supernatant of mice injected with CYP was evaluated. Data are expressed as mean ± SEM. After activation with immolized anti-CD3e mAb (5μg/5x10 5 cells) for 48 hours in a 37° C., 5% CO 2 incubator, the culture supernatant was collected to measure cytokines. The final concentration was obtained by subtracting the cytokine concentration from the non-activated cells as a control from the cytokine concentration in the immolized anti-CD3e mAb activated cells.
CYP에 의해 면역억제가 유도된 경우 이들 마우스 비장을 채취하여 시험관내에서 활성화시킨 결과 부형제대조군에 비해 IFN-γ 및 IL-4 생성 수준은 감소한 반면, IL-17, TGF-β1 수준은 증가하였고 TNFα는 차이가 없었다 (도 16). 특히 IFN-γ, IL-4, TNF-α 생성 수준은 추출물을 투여한 군에서 모델대조군 수준보다 높았는데 저농도 추출물 투여군 (5 mg/kg 체중)에서 가장 현저하였다. When immunosuppression was induced by CYP, the spleens of these mice were harvested and activated in vitro. Compared to the excipient control group, IFN-γ and IL-4 production levels decreased, while IL-17 and TGF-β1 levels increased, and TNFα was no difference (Fig. 16). In particular, IFN-γ, IL-4, and TNF-α production levels were higher in the extract-administered group than in the model control group, and were most pronounced in the low-concentration extract-administered group (5 mg/kg body weight).
실시예 17: CYP를 주사한 면역 억제 마우스의 장간막 림프절 T림프구 생성 싸이토카인 수준에 대한 추출물의 영향:Example 17: Effect of extracts on mesenteric lymph node T-lymphocyte-producing cytokine levels in immunosuppressed mice injected with CYP:
CYP를 주사한 마우스의 장간막 림프절 T 림프구 배양 상층액에서 생성된 싸이토카인 수준에 대한 추출물의 영향을 평가하였다. 데이터는 평균±SEM으로 표시하였다. 장간막 림프절 단일 세포를 5% CO 2 배양기에서 37℃ 에서 48 시간 동안 immolized anti-CD3e mAb (5μg/5x10 5 세포)로 활성화시킨 뒤 배양 상층액을 수집하여 싸이토카인을 측정하였다. immolized anti-CD3e mAb 활성화 세포에서의 싸이토카인 농도로부터 대조군으로 비 활성화 세포로부터 생성된 싸이토카인 농도를 빼서 최종 농도로 하였다. The effect of the extract on the cytokine levels produced in the mesenteric lymph node T lymphocyte culture supernatant of mice injected with CYP was evaluated. Data are expressed as mean±SEM. Mesenteric lymph node single cells were activated with immolized anti-CD3e mAb (5μg/5x10 5 cells) in a 5% CO 2 incubator at 37° C. for 48 hours, and then the culture supernatant was collected to measure cytokines. The final concentration was obtained by subtracting the cytokine concentration from the non-activated cells as a control from the cytokine concentration in the immolized anti-CD3e mAb activated cells.
장간막 림프절 T 림프구를 시험관내에서 활성화시켜 생성되는 싸이토카인 수준 분석을 통한 장 면역력에 대한 추출물의 영향을 평가한 결과, IFN-γ와 IL-17에 있어서 유의한 차이가 관찰되었다 (도 17). IFN-γ 수준은 부형제대조군, 모델대조군 모두에 비해 추출물 투여군에서 유의하게 높았으며, 염증성 장 질환 (inflammatory bowel disease, IBD)의 발병기전에 관여하는 것으로 입증된 염증성 싸이토카인 IL-17 수준은 모델대조군에 비해 유의하게 낮았다.As a result of evaluating the effect of the extract on intestinal immunity through cytokine level analysis produced by activating mesenteric lymph node T lymphocytes in vitro, significant differences were observed in IFN-γ and IL-17 ( FIG. 17 ). The level of IFN-γ was significantly higher in the extract-administered group compared to both the excipient control group and the model control group, and the inflammatory cytokine IL-17 level, which has been proven to be involved in the pathogenesis of inflammatory bowel disease (IBD), was higher in the model control group than in the model control group. was significantly lower than
실시예 18: CYP를 투여한 마우스에서 비장 림프구 아집단의 비율에 대한 추출물의 영향:Example 18: Effect of extracts on the proportion of splenic lymphocyte subpopulations in mice administered CYP:
CYP를 주사한 마우스의 각 비장 면역 세포의 비율에 대한 추출물의 영향을 평가하였다. 데이터는 평균 ± SEM으로 표시하였다.The effect of the extract on the proportion of each spleen immune cell in mice injected with CYP was evaluated. Data are expressed as mean ± SEM.
CYP를 주사한 모델대조군 마우스의 경우 보조 T 림프구, 세포파괴 T 림프구, B 림프구, 자연살해세포 수가 모두 부형제대조군에 비해 낮았다 (도 18). 추출물을 투여하였을 때 이들 세포들의 비율은 모델대조군에 비해 유의하게 높아졌으나 아직 부형제대조군 수준보다는 낮았다. 반면 자연살해세포 수준은 부형제대조군보다도 유의하게 높았다. In the case of model control mice injected with CYP, the number of helper T lymphocytes, apoptotic T lymphocytes, B lymphocytes, and natural killer cells were all lower than those of the vehicle control group ( FIG. 18 ). When the extract was administered, the proportion of these cells was significantly higher than that of the model control group, but it was still lower than the level of the excipient control group. On the other hand, the level of natural killer cells was significantly higher than that of the excipient control group.
이상에서 본 발명의 바람직한 실시예에 대하여 상세하게 설명하였지만 본 발명의 권리범위는 이에 한정되는 것은 아니고 다음의 청구범위에서 정의하고 있는 본 발명의 기본 개념을 이용한 당업자의 여러 변형 및 개량 형태 또한 본 발명의 권리범위에 속하는 것이다.Although preferred embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and improvements by those skilled in the art using the basic concept of the present invention as defined in the following claims are also provided. is within the scope of the

Claims (12)

  1. 체로스폰디아스 액실라리스 ( Choerospondias axillaris) 추출물을 유효 성분으로 포함하는 면역 증강용 조성물.cherospondias axillaris ( Choerospondias axillaris ) A composition for enhancing immunity comprising an extract as an active ingredient.
  2. 제1항에 있어서, 상기 체로스폰디아스 액실라리스 추출물은 과실부의 추출물인 것인 조성물.The method of claim 1, wherein the Cherospondias axillaris The extract is an extract of the fruit part composition.
  3. 제1항에 있어서, 상기 체로스폰디아스 액실라리스 추출물은 에탄올을 이용하여 추출된 것인 조성물.The method of claim 1, wherein the Cherospondias axillaris The extract is a composition that is extracted using ethanol.
  4. 제1항에 있어서, 상기 조성물의 투여 전과 비교하여 그 투여 후에 대상체에서 IgG2a/IgG1 수준을 증가시키는 것인 조성물.The composition of claim 1 , wherein the composition increases IgG2a/IgG1 levels in the subject after administration of the composition as compared to before administration.
  5. 제1항에 있어서, 상기 조성물은 1형 보조 T 림프구 (Type-1 helper T lymphocyte, Th1)에 의한 면역 반응을 촉진시키는 것인 조성물.The composition of claim 1, wherein the composition promotes an immune response by type 1 helper T lymphocytes (Th1).
  6. 제1항에 있어서, 상기 조성물의 투여 전과 비교하여 그 투여 후에 대상체에서 IgE 항체 및 IgG1 항체 중에서 선택되는 하나 이상의 수준을 감소시키는 것인 조성물.The composition of claim 1 , wherein the level of at least one selected from an IgE antibody and an IgG1 antibody is reduced in the subject after administration of the composition as compared to before administration of the composition.
  7. 제1항에 있어서, 상기 조성물의 투여 전과 비교하여 그 투여 후에 대상체에서 IgA 항체 수준을 증가시키는 것인 조성물.The composition of claim 1 , wherein the composition increases IgA antibody levels in the subject after administration of the composition as compared to before administration.
  8. 제1항에 있어서, 상기 조성물의 투여 전과 비교하여 그 투여 후에 대상체에서 산화질소 (NO), PGE 2, COX-2 및 활성 산소종 (ROS) 중에서 선택되는 하나 이상의 수준을 감소시키는 것인 조성물.The composition of claim 1 , wherein the level of one or more selected from nitric oxide (NO), PGE 2 , COX-2 and reactive oxygen species (ROS) is reduced in the subject after administration of the composition as compared to before administration of the composition.
  9. 제1항에 있어서, 상기 조성물의 투여 전과 비교하여 그 투여 후에 대상체에서 전염증성 싸이토카인 (pro-inflammatory cytokine)의 수준을 감소시키는 것인 조성물.The composition of claim 1 , wherein the composition reduces the level of pro-inflammatory cytokines in the subject after administration of the composition as compared to before administration.
  10. 제9항에 있어서, 상기 전염증성 싸이토카인은 TNF-α, IL-1β, IL-6, IL-8 및 IL-17 중에서 선택되는 하나 이상인 것인 조성물.The composition of claim 9, wherein the pro-inflammatory cytokine is one or more selected from TNF-α, IL-1β, IL-6, IL-8 and IL-17.
  11. 제1항 내지 제10항 중 어느 하나의 항에 있어서, 기능성 식품인 것인 조성물.The composition according to any one of claims 1 to 10, which is a functional food.
  12. 제1항 내지 제10항 중 어느 하나의 항에 있어서, 항염증용 의약인 것인 조성물.The composition according to any one of claims 1 to 10, which is an anti-inflammatory medicament.
PCT/KR2020/013055 2020-09-11 2020-09-25 Use of choerospondias axillaris extract WO2022055012A1 (en)

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Citations (3)

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