WO2022055012A1 - Utilisation d'extrait de choerospondias axillaris - Google Patents

Utilisation d'extrait de choerospondias axillaris Download PDF

Info

Publication number
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
Authority
WO
WIPO (PCT)
Prior art keywords
extract
composition
cells
administration
control group
Prior art date
Application number
PCT/KR2020/013055
Other languages
English (en)
Korean (ko)
Inventor
가우탐라비
허용
김창열
조지훈
양수정
이재희
김형아
Original Assignee
주식회사 드래곤이뮤노
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 주식회사 드래곤이뮤노 filed Critical 주식회사 드래곤이뮤노
Publication of WO2022055012A1 publication Critical patent/WO2022055012A1/fr

Links

Images

Classifications

    • 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.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Natural Medicines & Medicinal Plants (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Mycology (AREA)
  • Botany (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Medical Informatics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Alternative & Traditional Medicine (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Epidemiology (AREA)
  • Nutrition Science (AREA)
  • Food Science & Technology (AREA)
  • Polymers & Plastics (AREA)
  • Medicines Containing Plant Substances (AREA)

Abstract

La présente invention concerne une composition destinée à renforcer l'immunité d'un sujet, la composition comprenant un extrait de <i />(Choerospondias axillaris) en tant que principe actif. La présente invention concerne également une composition destinée à être utilisée en tant que médicament anti-inflammatoire, la composition comprenant un extrait de <i />(Choerospondias axillaris) en tant que principe actif.
PCT/KR2020/013055 2020-09-11 2020-09-25 Utilisation d'extrait de choerospondias axillaris WO2022055012A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR10-2020-0116767 2020-09-11
KR20200116767 2020-09-11

Publications (1)

Publication Number Publication Date
WO2022055012A1 true WO2022055012A1 (fr) 2022-03-17

Family

ID=80630298

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2020/013055 WO2022055012A1 (fr) 2020-09-11 2020-09-25 Utilisation d'extrait de choerospondias axillaris

Country Status (1)

Country Link
WO (1) WO2022055012A1 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2003055245A (ja) * 2001-08-07 2003-02-26 Maruzen Pharmaceut Co Ltd 皮膚化粧料および飲食品
CN102579527A (zh) * 2012-02-24 2012-07-18 杨玉梅 一种广枣叶总黄酮及其提取方法和用途
KR20130068307A (ko) * 2011-12-15 2013-06-26 조선대학교산학협력단 식물 추출물을 유효성분으로 함유하는 15-하이드록시프로스타글란딘 탈수소효소（15－ｐｇｄｈ） 억제용 조성물

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2003055245A (ja) * 2001-08-07 2003-02-26 Maruzen Pharmaceut Co Ltd 皮膚化粧料および飲食品
KR20130068307A (ko) * 2011-12-15 2013-06-26 조선대학교산학협력단 식물 추출물을 유효성분으로 함유하는 15-하이드록시프로스타글란딘 탈수소효소（15－ｐｇｄｈ） 억제용 조성물
CN102579527A (zh) * 2012-02-24 2012-07-18 杨玉梅 一种广枣叶总黄酮及其提取方法和用途

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
JUMAHAT N M, ZAIPUL-ANUAR N F, MOHD-ZAIN Z, KUMARI P N, CHANDRA M A, HUSSAINI J: "Abstracts of the 4th International Interscience Conference of Infection and Chemotherapy and 12th International Symposium on Antimicrobial Agents and Resistance (ICIC & ISAAR 2019", INFECTION & CHEMOTHERAPY INFECTION & CHEMOTHERAPY S83, 28 September 2019 (2019-09-28), pages S83 - S190, XP055911552, Retrieved from the Internet <URL:https://icjournal.org/src/sm/ic-51-S1-s005.pdf> [retrieved on 20220411] *
LABH SHYAM NARAYAN: "Effects of Lapsi Choerospondias Axillaris on Growth and Immune-Related Genes in Silver Carp (Hypophthalmichthys Molitrix)", SCIMEDICINE JOURNAL, vol. 2, no. 2, 30 June 2020 (2020-06-30), pages 86 - 99, XP055911557, DOI: 10.28991/SciMedJ-2020-0202-6 *
MANN SONIA, SHARMA ANKITA, SARKAR ASHISH, KHARB RUPSI, MALHOTRA RAJESH, DATTA BARUN, GUPTA RAJINDER, BISWAS SAGARIKA: "Evaluation of Anti-inflammatory Effects of Choerospondias axillaris Fruit`s Methanolic Extract in Synoviocytes and CIA Rat Model", CURRENT PHARMACEUTICAL BIOTECHNOLOGY, vol. 21, no. 7, 1 June 2020 (2020-06-01), NL , pages 596 - 604, XP009535248, ISSN: 1389-2010, DOI: 10.2174/1389201021666191210114127 *

Similar Documents

Publication Publication Date Title
WO2012074183A1 (fr) Composition pharmaceutique pour prévenir ou traiter des maladies inflammatoires comprenant un extrait de trachelospermi caulis et un extrait de paeonia suffruticosa andrews, et son procédé de préparation
WO2020040462A1 (fr) Composition comprenant un extrait de plante en tant que substance active pour la prévention ou le traitement d&#39;une maladie respiratoire induite par une matière
WO2016032249A1 (fr) Composition pharmaceutique contenant un extrait de vaccinium bracteatum thunb. ou une fraction de celui-ci comme principe actif pour prévenir ou traiter une inflammation des nerfs ou des maladies neurodégénératives
KR20110087448A (ko) 선복화 추출물로부터 분리된 화합물을 유효성분으로 함유하는 염증 또는 알러지 질환의 예방 및 치료용 조성물
WO2019177428A1 (fr) Composition de matière première de médicament pour prévenir ou traiter des maladies respiratoires
WO2022055012A1 (fr) Utilisation d&#39;extrait de choerospondias axillaris
WO2015064975A1 (fr) Composition comprenant un extrait de viola herba, ou un extrait de viola herba, persicae semen, cinnamomi ramulus, et glycyrrhiza spp. pour la prévention ou le traitement de maladies cardiovasculaires liées aux lipides et de l&#39;obésité
WO2014098306A1 (fr) Composition pharmaceutique pour prévenir ou traiter la démence
WO2014065640A2 (fr) Composition pour le traitement ou la prévention de maladies inflammatoires contenant un extrait de myagropsis myagroides ou fraction de celui-ci comme principe actif
KR101113202B1 (ko) 트리테르페노이드의 자가 면역 질환의 예방 및 치료 용도
WO2013012117A1 (fr) Utilisation de compositions pharmaceutiques comprenant un phytostérol pour prévenir ou traiter les maladies inflammatoires
KR102002962B1 (ko) 멍게로부터 정제된 바나듐 결합 단백질을 이용한 항비만용 조성물 및 이를 포함한 기능성 식품 조성물
WO2015002430A1 (fr) Composition pharmaceutique pour la prévention ou le traitement de l&#39;asthme comprenant un extrait de pistacia weinmannifolia j. poiss. ex franch ou une fraction de celui-ci
WO2018221922A1 (fr) Composition pour la prévention et le traitement de maladies musculaires, contenant un extrait de coptidis rhizoma, et son utilisation
KR20120025826A (ko) 곰취 추출물을 포함하는 뇌신경세포 보호용 조성물
WO2014157803A1 (fr) Composition comprenant un extrait de mollusques et crustacés comme principe actif qui est destinée au soulagement de l&#39;anxiété, à l&#39;amélioration de convulsant, à une action sédative ou à l&#39;induction ou à l&#39;amélioration du sommeil
WO2009151236A2 (fr) Composition comprenant des extraits ou fractions de magnolia obovata thunb. utilisable pour le traitement et la prévention des affections inflammatoires
WO2015105373A1 (fr) Composition pour la prévention ou le traitement de l&#39;asthme, comprenant un extrait de l&#39;e uonymus alatus ou une fraction de ce dernier
WO2020204479A1 (fr) Composition pour soulager des troubles de la circulation veineuse comprenant un extrait de feuille de raisin et un extrait de centella asiatica
Sarker et al. Dietary coumarins
EP3310799A1 (fr) Nouveau composé (ks 513) isolé de pseudolysimachion rotundum var. subintegrum, la composition le comprenant comme ingrédient actif pour la prévention ou le traitement de l&#39;allergie, d&#39;une maladie inflammatoire, de l&#39;asthme ou d&#39;une maladie pulmonaire obstructive chronique et son utilisation
WO2019231198A1 (fr) Composition comprenant un extrait de cimicifuga dahurica, une fraction de ce dernier ou un composé dérivé de cimicifuga dahurica en tant que principe actif permettant la prévention ou le traitement de maladies dégénératives du cerveau
WO2019198982A1 (fr) Composition pour prévenir ou améliorer l&#39;atrophie musculaire squelettique contenant de la kukoamine a et de la kukoamine b en tant que principes actifs
WO2019151547A1 (fr) Composition pour préven, améliorer ou traiter une maladie neurologique dégénérative comprenant une fraction d&#39;un extrait d&#39;a quilaria agallocha roxburgh à titre de principe actif
WO2019078386A1 (fr) Fraction d&#39;extrait de feuille de zanthoxylum piperitum, efficace pour soulager la douleur

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20953414

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20953414

Country of ref document: EP

Kind code of ref document: A1