KR101507221B1 - Composition for induction of Heat Shock Protein comprising the compounds isolated from the extract of Eucommia ulmoides as an active ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition comprising a compound isolated from bivalves with heat shock protein inducing activity. The compounds isolated from the bugs according to the present invention have a heat shock protein inducing activity and can particularly control the expression of HSF 1, HSP 27 and / or HSP 70. Accordingly, it has a protecting and regenerating effect on cells through the induction of heat shock protein and can be useful for the treatment or prevention of neurological diseases, digestive diseases, intestinal diseases, renal diseases, eye diseases, cardiovascular diseases or skin diseases. Further, it can be usefully used as a cosmetic composition for regenerating damaged skin cells.

Description

[0001] The present invention relates to a composition having a heat shock protein inducing activity, which comprises a compound isolated from a bacterium,

The present invention relates to a composition having a thermal shock protein-inducing activity comprising a compound isolated from bugs.

Eucommia ulmoides Oliv (Eucommiaceae) is a plant belonging to the genus 1st genus. The bark of Eucommia ulmoides, which is a bivalve plant, is used as a tonic in Korea, China and Japan. It is known that diarrhea is effective for hypotension and has a diuretic effect. (Tomada, Gonda, Shimizu, & Kanari, 1990). (Ho et al., 2005; Hua, Yin, Li, Hu, & Pei, 2002), Kim, Moon and others have reported that various lignans, flavonoids, iridoids and terpenoids , Lee, & Choi, 2004; Tanaka, Nakamura, Nakazawa, & Nohara, 1997). Some of the compounds isolated from the bugs are reported to exhibit the effects of antioxidants, hypotensives, anti-inflammatory agents, osteoblast proliferation and anti-thrombosis (Hoet al., 2005; Hua, Yin, Pei, 2002; Kim, Moon, Lee, & Choi, 2004; Tanaka, Nakamura, Nakazawa, & Nohara, 1997).

Heat shock factor 1 (HSF 1) is a major regulator of heat shock protein (HSP) gene expression in stressful conditions such as inflammation, ischemia, oxidative stress, starvation and viral infection (Lee et al., 2006). Under stressful conditions, HSF 1 is released from the chaperone complex and induces the expression of heat shock proteins such as HSP 25, HSP 27, HSP 70, HSP 72 and HSP 90. Increased expression of heat shock protein (HSP) has been reported to inhibit cell damage and promote cell regeneration.

In particular, Parkinson's disease has been reported to have a therapeutic effect (Luo, G.-R., et al., Is heat shock proteins for Parkinson's disease? Int J Biol Sci 3 (2007) 20-26) , HSP25 expression has been reported to protect damaged submandibular gland cells (Lee, H.-J. et al., (2006).) Radioprotective effect of heat shock protein 25 on submandibular glands of rats. Am. J. Pathol, 169 (5), 1601-1611). In addition, an increase in expression of heat shock protein (HSP 70) can inhibit gastric mucosal cell damage by monocloramine (NH ₂ Cl) generated by neutrophil-induced hypochlorous acid or Helicobacter pylori-induced ammonia, (Oyake, J. et al., Over-expression of 70-kDa heat shock protein confers protection against monochloramine-induced gastric mucosal cell injury. Life Sciences 79 (2006) 300-305). In addition, the molecular chaperone function of HSP90 due to increased expression of heat shock protein (HSP90) can inhibit the damage of small intestinal mucosa cells, and it can be used to treat intestinal mucosal damage and inflammatory bowel disease by NSAID (Tamaki, K. et al., Evidence for Enhanced Cytoprotective Function of HSP 90-Overexpressing Small Intestinal Epithelial Cells. Dig Dis Sci 56 (2011) 1954-1961). In addition, it has been reported that induction of heat shock protein (HSP 70) can prevent ischemic renal injury due to acute renal injury (Wang, Z. et al., Induction of heat shock protein 70 inhibits ischemic renal injury. Kidney International 79 (2011), 861-870). In addition, it has been reported that DNA damage of human lens epithelial cells (hLEC) can be prevented by increasing the expression of heat shock protein (HSP 70) (Lixia, S. et al., Effects of 1.8 GHz radiofrequency field on DNA damage and expression of heat shock protein 70 in human lens epithelial cells. Mutation Research 602 (2006) 135-142). In addition, it has been reported that HSP 27 and HSP 72 can be used as therapeutic agents for intestinal diseases such as colitis (Ohkawara, T. et al., Polaprezinc protects human colon cells from oxidative injury induced by hydrogen peroxide, Relevant to cytoprotective heat shock proteins. World J Gastroenterol 12 (38), (2006) 6178-6181). In addition, it has been reported that HSP 27 has a protective effect against damaged neurons (Toth, ME et al., Neuroprotective effect of small heat shock protein, HSP 27, after acute and chronic alcohol administration. and Chaperones 15 (2010) 807-817). In addition, it has been reported that HSP 70 induces protective effects on damaged myocardial cells (Morris, SD et al, Specic Induction of the 70-kD Heat Stress Proteins by the Tyrosine Kinase Inhibitor Herbimycin-A Protects Rat Neonatal Cardiomyocytes, J. Clin. Invest., 97 (3), (1996) 706-712). In addition, it has been reported that it is effective in regenerating damaged skin cells (Zhou, X. et al., Heat Shock Transcription Factor-1 Regulates Heat Shock Protein-72 Expression in Human Keratinocytes Exposed to Ultraviolet B Light. , 111 (2), (1998) 194-198). (A) Dillmann, WH, Small heat shock proteins and protection against injuries. (A) Dillmann, WH; Ann. NY Acad. Sci., 874 (Heart in Stress), 66-68, 1999; b) Bluhm, WF et al., Specific heat shock proteins protect microtubules during simulated ischemia in cardiac myocytes. Am. J. Physiol. 275 (Heart Circ. Physiol.): H2243-H2249, 1998).

Accordingly, the inventors of the present invention confirmed that a compound isolated from the shell of Eucommia ulmoides had heat-shock protein inducing activity while studying to find an HSP-expression regulator from a natural substance, thereby completing the present invention.

 Bava, S. V., Puliappadamba, V. T., Deepti, A., Nair, A., Karunagaran, D., & Anto, R. J. (2005). Sensitization of taxol-induced apoptosis by curcumin-down-regulation of nuclear factor-kB and the serine / threonine kinase Akt and is independent of tubulin polymerization. Journal of Biological Chemistry, 280 (8), 6301-6308.  Ha, H., Ho, J., Shin, S., Kim, H., Koo, S., Kim, I., & Kim, C. (2003). Effects of eucommiae cortex on osteoblast-like cell proliferation and osteoclast inhibition. Archives of Pharmacal Research, 26 (11), 929-936.  Ho, J. N., Cho, H. Y., Lim, E. J., & Kim, H. K. (2009). Effects of aucubin isolated from Eucommia ulmoides on UVB-induced oxidative stress in human keratinocytes HaCaT. Food Science and Biotechnology, 18 (2), 475-480.  Ho, J. N., Lee, Y. H., Park, J. S., Jun, W. J., Kim, H. K., Hong, B. S., Shin, D. H., & Cho, H. Y. (2005). Protective effects of aucubin were isolated from Eucommia ulmoides against UVB-induced oxidative stress in human skin fibroblasts. Biological & Pharmaceutical Bulletin, 28 (7), 1244-1248.  Hua, H.-M., Yin, H.-Q., Li, B.-Q., Hu, B., & Pei, Y.-H. (2002). A new monoterpene from the bark of Eucommia ulmoides. Journal of Asian Natural Products Research, 4 (3), 201-204.  Kim, H. Y., Moon, B. H., Lee, H. J., & Choi, D. H. (2004). Flavonol glycosides from the leaves of Eucommia ulmoides O. with glycation inhibitory activity. Journal of Ethnopharmacology, 93 (2-3), 227-230.  Kobayashi, H., Karasawa, H., Miyase, T., & Fukushima, S. (1985). Studies on the constituents of Cistanchis Herba. VI. Isolation and structure of a new iridoid glycoside, 6-deoxycatalpol. Chemical & Pharmaceutical Bulletin, 33 (9), 3645-3650.  Lami, N., Kadota, S., Kikuchi, T., & Momose, Y. (1991). Constituents of the roots of Boerhaavia diffusa L. III. Identification of calcium channel antagonistic compound from the methanol extract. Chemical & Pharmaceutical Bulletin, 39 (6), 1551-1555.  Lee, Y.-J., Kwon, H.-C., Bae, S., Kim, S.-H., Min, J.-J., Cho, C.- K., & Lee, Y.-S. (2006). Radioprotective effect of heat shock protein 25 on submandibular glands of rats. American Journal of Pathology, 169 (5), 1601-1611.  Lee, Y.-J., Kim, E.-H., Lee, J. S., Jeoung, D., Bae, S., Kwon, S.H., & Lee, Y.-S. (2008). HSF 1 as a mitotic regulator: phosphorylation of HSF 1 by Plk1 is essential for mitotic progression. Cancer Research, 68 (18), 7550-7560.  Miyagoshi, M., Amagaya, S., & Ogihara, Y. (1987). The structural transformation of the gardenoside and its related iridoid compounds by acid and glucosidase. Planta Medica, 53 (5), 462-464.  Okada, N., Shirata, K., Niwano, M., Koshino, H., & Uramoto, M. (1994). Immunosuppressive activity of a monoterpene from Eucommia ulmoides. Phytochemistry, 37 (1), 281-282.  Ono, M., Ueno, M., Masuoka, C., Ikeda, T., & Nohara, T. (2005). Iridoid glucosides from the fruit of Genipa americana. Chemical & Pharmaceutical Bulletin, 53 (10), 1342-1344.  Schumacher, B., Scholle, S., Hoelzl, J., Khudeir, N., Hess, S., & Mueller, C. E. (2002). Lignans isolated from Valerian: Identification and characterization of a new adrenaline derivative with partial adrenergic activity at A1 adenosine receptors. Journal of Natural Products, 65 (10), 1479-1485.  Seo, H. R., Chung, D. Y., Lee, Y. J., Lee, D. H., Kim, J. I., Bae, S., Chung, H. Y., Lee, S. J., Jeoung, D., & Lee, Y. S. (2006). Heat shock protein 25 or inducible heat shock protein 70 activates heat shock factor 1: dephosphorylation on serine 307 through inhibition of ERK1 / 2 phosphorylation. J Biol Chem, 281 (25), 17220-17227.  Steeves, V., Forster, H., Pommer, U., & Savidge, R. (2001). Coniferyl alcohol metabolism in conifers - I. Glucosidic turnover of cinnamyl aldehydes by UDPG: Coniferyl alcohol glucosyltransferase from pine cambium. Phytochemistry, 57 (7), 1085-1093.  Takeda, T., Narukawa, Y., & Hada, N. (1999). Studies on the constituents of Leonotis nepetaefolia. Chemical & Pharmaceutical Bulletin, 47 (2), 284-286.  Tanaka, C., Nakamura, T., Nakazawa, Y., & Nohara, T. (1997). A new triterpenoid from the leaves of Eucommia ulmoides Oliv. Chemical & Pharmaceutical Bulletin, 45 (8), 1379-1380.  Tomoda, M., Gonda, R., Shimizu, N., & Kanari, M. (1990). A reticuloendothelial system-activating glycan from the barks of Eucommia ulmoides. Phytochemistry, 29 (10), 3091-3094.  Walcott, S. E., & Heikkila, J. J. (2010). Celastrol can inhibit proteasome activity and upregulate the expression of heat shock protein genes, hsp 30 and HSP 70, in Xenopus laevis A6 cells. Comparative Biochemistry and Physiology, Part A: Molecular & Integrative Physiology, 156A (2), 285-293.  Oyake, J. et al., Over-expression of 70-kDa heat shock protein confers protection against monochloramine-induced gastric mucosal cell injury. Life Sciences 79 (2006) 300-305.  Tamaki. K. et al., Evidence for Enhanced Cytoprotective Function of HSP 90-Overexpressing Small Intestinal Epithelial Cells. Dig Dis Sci 56 (2011) 1954-1961.  Wang, Z. et al., Induction of heat shock protein 70 inhibits ischemic renal injury. Kidney International 79 (2011), 861-870.  Lixia, S. et al., Effects of 1.8 GHz radiofrequency eld on DNA damage and expression of heat shock protein 70 in human lens epithelial cells. Mutation Research 602 (2006) 135-142.  Ohkawara, T. et al., Polaprezinc protects human colon cells from oxidative injury induced by hydrogen peroxide: Relevant to cytoprotective heat shock proteins. World J Gastroenterol 12 (38), (2006) 6178-6181.  Toth, M. E. et al., Neuroprotective effect of small heat shock protein, HSP 27, after acute and chronic alcohol administration. Cell Stress and Chaperones 15 (2010) 807-817.  Morris, S. D. et al., Specic Induction of the 70-kD Heat Stress Proteins by Tyrosine Kinase Inhibitor Herbimycin-A Protects Rat Neonatal Cardiomyocytes. J. Clin. Invest. 97 (3), (1996) 706-712.  Luo, G.-R. et al., Are heat shock proteins therapeutic target for Parkinson's disease? Int J Biol Sci 3 (2007) 20-26.  Zhou, X. et al., Heat Shock Transcription Factor-1 Regulates Heat Shock Protein-72 Expression in Human Keratinocytes Exposed to Ultraviolet B Light. THE JOURNAL OF INVESTIGATIVE DERMATOLOGY, 111 (2), (1998) 194-198.

The present invention is to provide a composition having a heat shock protein-inducing activity containing a compound isolated from mites as an active ingredient.

The present invention also provides a pharmaceutical composition for preventing or treating neurological diseases, gastrointestinal diseases, intestinal diseases, renal diseases, eye diseases, cardiovascular diseases or skin diseases, which comprises a compound isolated from bivalves having heat shock protein inducing activity as an active ingredient .

The present invention also provides a cosmetic composition for regenerating damaged skin cells, which comprises, as an active ingredient, a compound isolated from bivalves having heat shock protein-inducing activity.

In order to solve the above problems, the present invention provides a composition having a heat shock protein-inducing activity comprising at least one compound selected from the group consisting of compounds represented by the following formulas (1) to (7) as an active ingredient.

[Chemical Formula 1]

(2)

(3)

[Chemical Formula 4]

[Chemical Formula 5]

[Chemical Formula 6]

(7)

In the above formula, Glc is a D-glucopyranosyloxy group.

The term " Eucommia ulmoides "as used in the present invention is a deciduous arboreous tree and is a plant mainly distributed in China. It is mainly used as dried bark of tree sap, and it is mainly used as tonic, arthritis and rheumatic analgesic.

The term "extraction" used in the present invention refers to separation of Eucommia ulmoides by using a liquid solvent. Examples of the solvent include C ₁ -C ₆ alcohols such as water, methanol, ethanol and butanol, ethyl acetate, Mixed solvents of these may be used. In the present invention, it is preferable to extract the extract from methanol using bark beetle. In the present invention, the methanol extracts of the bivalves may be fractionated with butanol to separate the compounds 1 to 7.

Also, the present invention provides a method for producing a mucilage comprising the steps of: extracting Eucommia ulmoides with methanol to obtain a mugwort extract; Fractionating the mulberry extract with butanol to obtain mulberry fraction; And a step of subjecting the bifloride fraction to column chromatography to obtain a compound represented by the following formula (1), (2) or (5).

[Chemical Formula 1]

(2)

[Chemical Formula 5]

In the above formula, Glc is a D-glucopyranosyloxy group.

The fact that the compound represented by the above 1, 2 or 5 can be separated from the two species was not known in the prior art, and the present invention can separate the above-mentioned compounds represented by 1, 2 or 5 from the two- I said for the first time. According to an embodiment of the present invention, the structure of each compound was analyzed using 1D and 2D NMR such as ¹ H, ¹³ C, DEPT, COZY, HSQC, HMBC and NOESY.

The compounds of Formulas 1 to 7 will be briefly described as Compounds 1 to 7, respectively. Seven compounds were identified as coniferaldehydeglucoside (Steeves, Forster, Pommer, & Savidge, 2001), bartsioside (Kobayashi, Karasawa, Miyase, & Fukushima, 1985), geniposidic acid (Takeda, (Oncoprotein) (Narukawa, & Hada, 1999), compound 4 (geno), Ono, Ueno, Masuoka, Ikeda, and Nohara, 2005, compound 5 ferredoside (Miyagoshi, Amagaya, & Ogihara, 1987), compound 6 (pinoresinoldiglucoside Schumacher, Scholle, Hoelzl, Khudeir, Hess, & Mueller, 2002) and liriodendrin (Lami, Kadota, Kikuchi, & Momose, 1991).

The fraction obtained by fractionating the mulberry extract with the butanol solution may have a thermal shock protein inducing activity as it contains a large amount of the above compounds 1 to 7.

As used herein, the term "heat shock protein inducing activity" means activating a regulatory factor (HSF 1) that induces the expression of a heat shock protein and activating the heat shock protein to induce it or increasing the expression of a heat shock protein (HSP) . Heat shock factor 1 (HSF 1) is a transcription factor that regulates the heat-shock response of a cell, and the heat shock protein is an enzyme that induces synthesis when the cell, tissue or individual is at a temperature 5 to 10 ° C higher than the physiological temperature Lt; / RTI >

Exposure of cells to various environmental disorders, including heat shock, alcohol, heavy metals and oxidative disorders, typically results in cell accumulation of many chaperones known as heat shock proteins (HSPs). Induction of HSP protects cells against early impairment impairment and enhances maintenance recovery and induction of impaired resistance status. In addition, any HSP can act as a major molecular chaperone under normal disorder-free conditions by regulating the correct folding, degradation, integration and function of growth lists of important cellular proteins.

They are classified according to molecular weight, for example HSP 25, HSP 27, HSP 70, HSP 72 and HSP 90. Some diseases of humans can be obtained according to the results of incorrectly folded proteins. Since HSP is involved in preventing protein denaturation in cells or regenerating denatured proteins, it can be useful for the treatment of inflammatory diseases of the organs such as tissue damage.

Thus, by inducing the expression of HSP, it is possible to treat diseases particularly related to cell regeneration. In one embodiment of the invention, the effects on HSF 1 protein expression and its transcription target, HSP 27 and HSP 70 were measured, and compounds 1 and 7 increased HSF 1 expression by 1.214 and 1.167 fold at 3 uM, respectively. The expression of HSP 27 was 1.316, 1.357 and 1.333 times, respectively, by compounds 1, 5 and 7, respectively. Compound 2 showed an effect of inducing an increase in expression of HSP70 at 1.162-fold. Compound 1 also activated the luciferase promoter of HSP 25 (1.47-fold) and HSP 70 (1.09-fold) at 50 uM. As a result, it was found that the compounds 1 to 7 extracted from the bark bark had an inducing activity of HSF 1, HSP 27 and HSP 70.

That is, the compounds 1 to 7 of the present invention can regulate the expression of HSF 1 and induce the expression of HSP 27 and / or HSP 70. In particular, the compounds 1 to 7 of the present invention are capable of inducing both HSF 1, HSP 27 and HSP 70, and thus have excellent HSP inducing ability.

The present invention relates to a pharmaceutical composition for the prevention and treatment of neurological diseases, gastrointestinal diseases, intestinal diseases, renal diseases, eye diseases, cardiovascular diseases and the like, which comprises a composition having at least one compound selected from the group consisting of the above- Or a pharmaceutical composition for preventing or treating skin diseases. In the present invention, the neurological disease is preferably Parkinson's disease as a degenerative brain disease. The gastrointestinal disease is preferably a gastric ulcer or a duodenal ulcer as a peptic ulcer. The intestinal disease is preferably inflammatory bowel disease as a colitis. The renal disease is preferably an ischemic renal disease. It is preferable that the ocular disease is glaucoma, cataract or conjunctivitis. The cardiovascular disease is preferably an ischemic heart disease or myocardial infarction.

It has been reported that the neurological disorder such as Parkinson's disease is induced by the expression induction of HSP prior to the invention of the present invention, and the increase of expression of HSP25 has been reported to protect injured submandibular gland cells. In addition, an increase in expression of heat shock protein (HSP 70) can inhibit gastric mucosal cell damage by monocloramine (NH ₂ Cl) generated by neutrophil-induced hypochlorous acid or Helicobacter pylori-induced ammonia, It has been reported that it can be applied to therapy. In addition, the molecular chaperone function of HSP90 due to increased expression of heat shock protein (HSP90) can inhibit the damage of small intestinal mucosa cells, and it can be used to treat intestinal mucosal damage and inflammatory bowel disease by NSAID It has been reported that it can be applied. In addition, it has been reported that induction of heat shock protein (HSP 70) can prevent ischemic renal damage due to acute renal injury. In addition, it has been reported that DNA damage of human lens epithelial cells (hLEC) can be prevented by increasing expression of heat shock protein (HSP 70). In addition, it has been reported that the expression of HSP 27 and HSP 72 can be applied as a therapeutic agent for intestinal diseases such as colitis. In addition, it has been reported that a protective effect against damaged neurons by the increase of HSP 27 expression has been reported. In addition, it has been reported that HSP 70 has protective effect on damaged myocardial cells by the induction effect. It has also been reported to be effective in regenerating damaged skin cells. It has also been reported that induction of the expression of HSF 1, HSP 27 and HSP 70 is effective in the prevention and treatment of ischemic heart disease.

As used herein, the term "treatment" refers to the administration of a composition comprising at least one compound selected from the group consisting of the compounds 1 to 7 as an active ingredient to treat neurological diseases, gastrointestinal diseases, intestinal diseases, A disease, a cardiovascular disease, or a skin disease is improved or cured. The term "prevention ", as used herein, refers to the administration of a composition comprising at least one compound selected from the group consisting of the compounds 1 to 7 as an active ingredient, to inhibit or delay the onset of the disease Acts.

The composition of the present invention may contain, for administration, a pharmaceutically acceptable carrier, excipient or diluent in addition to the above-described effective ingredients. Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The composition of the present invention may be formulated in the form of oral, granule, tablet, capsule, suspension, emulsion, syrup, aerosol or the like oral preparation, external preparation, suppository or sterilized injection solution according to a conventional method. In detail, when formulating, it can be prepared by using diluents or excipients such as fillers, weighing agents, binders, humectants, disintegrants, surfactants and the like which are generally used. Solid formulations for oral administration include, but are not limited to, tablets, pills, powders, granules, capsules, and the like. Such a solid preparation can be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose, lactose, gelatin and the like in the composition. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration, liquid paraffin, and various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like. Examples of the suppository base include withexol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The composition of the present invention may be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) depending on the desired method, and the dose may be determined depending on the condition and weight of the patient, The mode of administration, the route of administration, and the time, but may be suitably selected by those skilled in the art. The daily dose of the composition is preferably 1 mg / kg to 500 mg / kg, and may be administered once or several times a day, if necessary.

The present invention also provides a cosmetic composition for regenerating damaged skin cells, which comprises a composition having at least one compound selected from the group consisting of the compounds represented by formulas (1) to (7) as an active ingredient and having a heat shock protein inducing activity. The cosmetic composition according to the present invention may be formulated into a cosmetic lotion, a gel, a water-soluble powder, a fat-soluble powder, a water-soluble liquid, a cream or an essence by a conventional method for producing a cosmetic by adding a pH adjusting agent, a flavoring agent, an emulsifier, .

The compounds isolated from the bugs according to the present invention have a heat shock protein inducing activity and can particularly control the expression of HSF 1, HSP 27 and / or HSP 70. Accordingly, it has a protecting and regenerating effect on cells through the induction of heat shock protein and can be useful for the treatment or prevention of neurological diseases, digestive diseases, intestinal diseases, renal diseases, eye diseases, cardiovascular diseases or skin diseases. Further, it can be usefully used as a cosmetic composition for regenerating damaged skin cells.

FIG. 1 shows the results of Western blotting the expression of HSF 1, HSP 27 and HSP 70 after treating L132 cells with a compound isolated from Eucommia ulmoides bark according to an embodiment of the present invention. β-Actin was used as an internal control, and L132 cells were treated with 3 μM compound for 12 h.
FIG. 2 shows the activity of HSP 25 or HSP 70 promoter after treating Compound 1 isolated from bivalve shells according to an embodiment of the present invention to NCI-H460 cells. Where each value is the mean ± SD of three independent test groups. * P < CON group, ** p < CON group.
Figure 3, shows the result of measuring a cell viability of Taxol ^® in a MTT viability of L132 cells and the control group was treated from the bark of the two layers in the extracted compounds 1 to 7 according to an embodiment of the present invention.
A of Figure 4 shows the result after induced cell death in Western blotting, by observing the cut caspase 3 and PARP cleavage-1 sikindaneun the compound 1 reduced the cell death induced by radiation and Taxol ^®. B represents the cell viability (%) after IR treatment for Compound 1. [ C represents the cell viability (%) after treatment with Taxol ( ^R) for Compound (1).
5 is a graph showing that the mortality rate by radiation by the compound 1 after radioactivity irradiation of the mouse to the lethal dose is reduced.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided to further understand the present invention, and the present invention is not limited by the examples.

Example  1: Materials and Methods

Optical rotation at 25 캜 was measured with a P-1010 polarimeter (JASCO, Japan). UV and IR spectra were recorded on a U-3000 spectrophotometer (Hitachi, Japan) and an FTS 135 FT-IR spectrometer (Bio-Rad, CA). 1D and 2D NMR experiments were performed with a UNITY INOVA 400 MHz FT-NMR instrument (Varian, CA) with tetramethylsilane (TMS) as internal standard. Mass spectrometry was performed with a Waters ACQUITY UPLC system coupled with a Micromass Q-Tof micro mass spectrometer and an Agilent 6220 Accurate-Mass TOF LC / MS system. Column chromatography was performed on silica gel (230-400 mesh, Merck, Germany), RP-18 (YMC gel ODS-A, 12 nm, S-150 urn), Sephadex LH-20 (Amersham Pharmacia Biotech) 20 (Mitsubishi Chemical Co.). The TLC was performed with Kieselgel 60 F 254 (silica gel, 0.25-mm layer thickness, Merck, Germany) and RP-18 F 254s (Merck, Germany) plates and exposed to UV light (254 nm and 365 nm) v) H ₂ SO ₄ And visualized with a spray and heated at 120 ° C for 5 minutes.

Example  2: Eucommia ulmoides ) Preparation of methanol extract

Twenty kilograms of mites were placed in 18 L of 100% methanol, extracted three times at room temperature for 24 hours, and concentrated under reduced pressure to obtain 1.4 kg of methanol extract.

Example  3: Eucommia ulmoides ) &Lt; / RTI & Fraction  Produce

After 2 L of distilled water was added to 1.4 kg of the methanol extract of Example 2 and suspended, hexane (2 L x 8 times) was added to dissolve it. Only the components dissolved in the hexane layer were separated and vacuum dried to obtain 210 g of hexane fraction Respectively. Ethyl acetate (2 L x 4 times) was added to the remaining aqueous phase to dissolve, and the components dissolved in the ethyl acetate layer were separated and vacuum dried to obtain 200 g of the ethyl acetate fraction. The same procedure as above was conducted to obtain 450 g (2 L x 5 times) of n-butanol fraction and 474 g of an aqueous layer.

Example  4: Eucommia ulmoides ) Butanol  Separation of compounds from fractions

450 g of the butanol fraction of Example 3 was divided into 6 fractions (F01-F06) by performing a Diaion HP-20 column with a methanol-water mixed solvent concentration gradient (100% water → methanol 100%). Compound 3 (2.3 g, 0.0115% w / w) was obtained from the F05 fraction by precipitation. 3.2 g of the F02 fraction was subjected to silica gel column chromatography using a chloroform: methanol mixed solvent (24: 1 to 19: 1) to obtain Compound 4 (200 mg, 0.001% w / w). Further, silica gel column chromatography was performed on 124.0 g of the F03 fraction with a solvent mixture of chloroform: methanol: water (9: 1: 0.1 → 7: 3: 0.5) to obtain 13 fractions (F03.01-F03.13) . Compound 7 (36 mg, 0.00018% w / w) and compound 6 (270 mg, 0.00135% w / w) were obtained from three fractions F03.06 and F03.07, respectively, by recrystallization using methanol. Next, 2.4 g of the three fractions F03.08 were subjected to reverse phase column chromatography (YMC gel ODS-A, 12 nm, S-150 μm) in a methanol / water mixture (6: 4 → 9: Fraction (F03.08.01-F03.08.04) was obtained. (5 mg, 0.000025% w / w) and Compound 5 (5 mg, 0.000025% w / w) were obtained from fractions F 03.08.02 and F03.08.03 by proceeding with a Sephadex LH-20 column (Amersham Pharmacia Biotech) w) respectively. Fractional F05 (35 g) was subjected to silica gel column chromatography using chloroform: methanol: water mixed solvent (9: 1: 0.1 → 7: 3: 0.5) to obtain 10 fractions (F05.01-F05.10) &Lt; / RTI &gt; Reversed-phase column chromatography (YMC gel ODS-A, 12 nm, S-150 μm) was performed on 12 g of F05.01 with a methanol / water mixed solvent (9: 1) to obtain four fractions (F05.01.01- F05.01.04), and the second fraction (F05.01.02) was subjected to a Sephadex LH-20 column (Amersham Pharmacia Biotech) with a solvent of 100% methanol to obtain Compound 1 (10 mg, 0.00005% w / w) .

It has been shown for the first time that the compound 1, compound 2, and compound 5 can be separated from the bivalves by the above separation method. Therefore, the structures of Compound 1, Compound 2 and Compound 5 were analyzed using 1D and 2D NMR such as ¹ H, ¹³ C, DEPT, COZY, HSQC, HMBC and NOESY as follows.

Compound 1 ( Coniferaldehydeglucoside )of NMR Data

White solid; ^{_{[a] 25 D -48.0 (c}} 0.05, MeOH); UV (MeOH) _max (log e) 300 (4.2), 327 (4.7) nm; _{IR (KBr) v max 3385,} 1717, 1650, 1541 cm -1; _{^{1 H NMR (CD 3 OD,}} 400 MHz) d9.61 (1H, d, J = 7.6 Hz, H-9), 7.62 (1H, d, J = 15.6 Hz, H-7), 7.32 (1H, d , J = 1.8 Hz, H- 2), 7.26 (1H, dd, J = 8.4, 1.8 Hz, H-6), 7.21 (1H, d, J = 8.4 Hz, H-5), 6.72 (1H, dd , J = 15.6, 7.6 Hz, H-8), 5.00 (1H, d, J = 7.6 Hz, Glc-1 '), 3.91 (3H, s, OCH 3 -3), 3.88 (1H, dd, J = 11.8, 2 Hz, Glc-6'b ), 3,69 (1H, dd, J = 11.8, 5.2 Hz, Glc-6'a), 3.48 (4H, m, Glc-2 ', 3', 4 ' , 5 '); _{^{13 C NMR (CD 3 OD,}} 100 MHz) d130.5 (C, C-1), 128.4 (CH, C-8), 124.5 (CH, C-6), 117.4 (CH, C-5), 112.9 (CH, CI-2 '), 102.2 (CH, Glc-1'), 78.5 CH, Glc-4 '), 62.6 (CH 2, Glc-6'), 56.9 (CH 3, OCH 3 -3); HR-ESIMS m / z 363.1056 [ M + Na] + (calcd for C 16 H 20 O 8 Na, 363.1050).

Compound 2 ( Bartsioside )of NMR Data

Yellow oil; [a] ²⁵ _D -94.5 ( c 0.1, MeOH); UV (MeOH)? _Max (log?) 218 (3.2) nm; _{IR (KBr) v max 3318,} 1710 cm -1; _{^{1 H NMR (CD 3 OD,}} 400 MHz) δ6.26 (1H, dd, J = 6, 2 Hz, H-3), 5.72 (1H, brs, H-7), 5.13 (1H, d, J = 6 Hz, H-1), 4.88 (1H, dd, J = 6, 3.6 Hz, H-4), 4.68 (1H, d, J = 7.6 Hz, Glc-1 '), 4.28 (1H, brd, J = 14.2 Hz, H-10a) , 4.16 (1H, brd, J = 14.2 Hz, H-10b), 3.84 (1H, m, Glc-6'a), 3.66 (1H, m, Glc-6'b) , 3.19 - 3.45 (4H, m , Glc-2 ', 3', 4 ', 5'), 2.91 (1H, m, H-5), 2.77 (1H, t, J = 6.8 Hz, H-9) , 2.61 (1H, m, H-6b), 2.06 (1H, m, H-6a); _{^{13 C NMR (CD 3 OD,}} 100 MHz) δ145.1 (C, C-8), 141.0 (CH, C-3), 127.9 (CH, C-7), 108.4 (CH, C-4), 100.0 (CH, Glc-1 '), 96.5 (CH, C-1), 78.4 (CH, Glc-3'), 78.1 CH, Glc-4 '), 62.9 (CH 2, Glc-6'), 61.6 (CH 2, C-10), 48.9 (CH, C-9), 40.0 (CH 2, C-6), 35.8 ( CH, C-5); HR-ESIMS m / z 353.1212 [ M + Na] + (calcd for C 15 H 22 O 8 Na, 353.1207).

Compound 5 ( Feretoside )of NMR Data

Pale yellow solid; ^{_{[a] 25 D -62.0 (c}} 0.2, MeOH); _{UV (MeOH) λ max (log} ε 232 (3.6) nm; IR (KBr) v max 3316, 1698 cm -1; 1 H NMR (CD 3 OD, 400 MHz) δ7.51 (1H, d, J = 1.2 Hz, H-3), 5.80 (1H, t, J = 1.4 Hz, H-7), 5.19 (1H, d, J = 6 Hz, H-1), 4.67 (1H, d, J = 8 Hz, Glc-1 '), 4.54 ( 1H, m, H-6), 4.34 (1H, brd, J = 16 Hz, H-10b), 4.18 (1H, brd, J = 16 Hz, H-10a), 3.86 (1H, dd, J = 12 , 2 Hz, Glc-6'b), 3.75 (3H, s COOCH 3 -4), 3,63 (1H, m, Glc-6'a), 3.28 (4H, m , Glc-2 ', 3' , 4 ', 5'), 3.03 (1H, m, H-9), 2.99 (1H ddd, J = 7.6, 4.4, 1.2 Hz, H-5);. 13 C NMR (CD ₃ OD, 100 MHz)? 170.8 (C, C-11), 154.4 (CH, C-3), 148.1 C-4), 100.8 (CH, Glc-1 '), 98.8 (CH, C-1), 82.8 '), 75.3 (CH, Glc -2'), 72.0 (CH, Glc-4 '), 63.2 (CH 2, Glc-6'), 61.5 (CH 2, C-10), 52.6 (CH 3, COOCH _{3 -4), 47.6 (CH,} C-9), 46.1 (CH, C-5); HR-ESIMS m / z 427.1204 [m + Na] + (calcd for C 17 H 24 O 11 Na, 427.1211).

Example  5: Western blot  analysis

(Lee et al., 2008) for the measurement of HSF 1 and HSPs expression of compounds isolated from B. tuberculosis. Protein in the lysates was separated by SDS-PAGE, electrotransferred to nitrocellulose membranes (GE Healthcare, UK) and blotted with specific antibodies. And visualized with an ECL detection system (Thermo Scientific, USA). Anti-HSF 1, anti-HSP 27, anti-HSP 70 and anti-b-actin antibodies were purchased from Santa Cruz Biotechnology (USA).

To determine whether the extract had an inducing effect on HSF 1 and its transcriptional targets, HSP 27 and HSP 70, normal lung fibroblasts, L132 cells were treated with the compound and then subjected to Western blot for 12 hours. HSF 1 expression was increased by treatment with the compounds 1 to 7. In particular, statistically high expression increases were observed in Compound 1 and Compound 7. In the case of HSP 27 and HSP 70, all compounds induced their protein expression enhancement. In particular, when compared statistically with the untreated control, high expression enhancement was observed for compounds 1, 5 and 7 for HSP 27 and high expression was observed for compound 2 for HSP 70 (FIG. 1 and Table 1) .

Celastrol, a positive control for measuring HSP expression (Walcott & Heikkila, 2010), increased the expression of HSP 27 and HSP 70 without increasing the expression of HSF 1 protein. This shows that celastrol and bidentate compounds act as different mechanisms. The most effective increase of HSF 1 occurred in Compound 1.

Example   6: MTT assay

Cells were treated with 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (prepared according to Bava, Puliappadamba, Deepti, Nair, Karunagaran, & Anto, 2005) MTT; Sigma) test to determine their cytotoxicity.

Compounds isolated from the bark peptides did not show any cytotoxicity in L132 cells (IC ₅₀ value > 5 uM), whereas the anticancer drug Taxol ^® had an IC ₅₀ value of 12.3 uM and showed cytotoxicity (Fig. 3).

Example   7: HSP  Promoter analysis

The promoter activity of HSP 25 or HSP 70 was measured using the luciferase reporter construct according to the established protocol (Seo et al., 2006). Human lung cancer NCI-H460 cells were plated at 1 × 10 ⁵ cells in 35-mm dishes and co-transfected with 400 ng luciferase reporter construct and 400 ng β-galactosidase expression vector . After 24 hour incubation, the cells were chemically treated and harvested after 12 h. Luciferase activity was measured and normalized to? -Galactosidase expression (Promega). All results were expressed as mean ± SD of three independent test groups. Statistical significance ( p- value) was determined by Student's t-test with untreated controls.

Analysis of HSP 25 (murine form of HSP 27) and HSP 70 was performed using Compound 1 showing the most effective expression increase of HSF 1 in Example 8 above. Compound 1 activated the luciferase promoter activity of HSP 25 and HSP 70 depending on the dose. In particular, as shown in FIG. 2, Compound 1 increased HSP 25 1.47-fold and HSP 70 1.09-fold promoter activity at 50 uM.

The quantitative immunoblot results of HSF 1, HSP 27 and HSP 70 in L132 (human embryonic lung tissue cells) after normalization with β-Actin signal are summarized in Table 1 below.

Degree of improvement (multiples) compound HSF1 HSP27 HSP70 IC ₅₀ (uM) One 1.214 + 0.013 * 1.316 + 0.067 * 1.102 + - 0.057 > 5 uM 2 1.144 + 0.066 1.270 + 0.085 1.162 + 0.048 * > 5 uM 3 1.159 + 0.108 1.331 + 0.115 1.205 + 0.063 > 5 uM 4 1.114 ± 0.054 1.295 + 0.109 1.157 ± 0.077 > 5 uM 5 1.153 + - 0.052 1.357 + 0.082 * 1.129 + 0.043 > 5 uM 6 1.041 + 0.086 1.179 + 0.073 1.158 ± 0.057 > 5 uM 7 1.167 + - 0.046 * 1.333 + 0.093 * 1.152 + 0.091 > 5 uM Celastrol 1.019 ± 0.003 1.193 + 0.038 * 1.319 0.040 * > 5 uM Taxol ^® ND ND ND 12.3

Data values are mean SD of the three experimental groups 12 hours after treatment. Significant statistical differences by comparison of HSP 70, HSP 27 or HSF 1 level amounts between the treated and untreated groups were expressed as * p < 0.05. The IC ₅₀ value is the concentration (uM) required to inhibit the growth of L132 cells by 50%. Celastrol was used as a control for HSP expression. And Taxol ^® was used as a control for cytotoxicity.

In conclusion, compounds 1 and 7 increased the expression of HSFl by 1.214 and 1.167 times, respectively, at 3 uM. The expression of HSP 27 was 1.316, 1.357 and 1.333 times, respectively, by compounds 1, 5 and 7, respectively. Compound 2 showed an effect of inducing an increase in expression of HSP70 at 1.162-fold. Compound 1 also activated the luciferase promoter of HSP 25 (1.47-fold) and HSP 70 (1.09-fold) at 50 uM. This fact indicates that Compound 1 is the most effective compound on the present system. This is the first report of the induction activity of HSF 1, HSP 27 and HSP 70 of Compounds 1 to 7 extracted from the bark of the bark.

Example  8: Cell viability assay

(1) (coniferalaldehyde glucoside and coniferalaldehyde have the same HSF1 and HSP production ability under the condition that the cells are treated with toxic compounds that can damage cell viability such as Taxol and IR (radiation), or are exposed to ionizing radiation and the survival rate is about 50% And the coniferalaldehyde compound was purchased instead of the glucoside form of the compound of formula (I) of the present invention in order to treat with a certain amount of the compound. The cells were treated with trypan blue at the same time, . Cell viability was determined by Western blot method using a hematocytometer.

Expression of cleaved caspase-3 and truncated PARP1 protein was measured to determine the effect of Compound 1 on cell death. In the lysate the proteins were separated by SDS-PAGE, electro-transferred to nitrocellulose membranes (GE Healthcare, UK) and blotted with specific antibodies. And visualized with an ECL detection system (Thermo Scientific, USA). Anti-cleaved caspase 3, anti-cleaved PARP1, and anti-β-actin antibodies were purchased from Santa Cruz Biotechnology (USA).

To confirm the protective effect of L132 cells, which are human embryonic lung cells, on IR and Taxol ^® damage, Cleaved PARP and Cleaved caspase 3, apoptosis markers, were examined. As a result, Compound 1 (3 μM) In the treated group, the protective effect was shown and the survival rate of the cells was examined. As a result, it was confirmed that the survival capacity was increased due to the damage (FIG. 4).

Example  9: Animal viability analysis

Mice were exposed to radiation of 7 Gy to confirm the viability of Compound 1. Radiation treatment One day before coniferalaldehyde was administered intraperitoneally (5 mg / kg, 10 mg / kg), the animals were exposed to the whole body exposure the next day, followed by intraperitoneal administration of additional drug for 3 days. After observing the survival days until the death of all of the animals, the radiation protection effect was confirmed. As a result, it was confirmed that the survival ability was increased to 3.7 days in the 5 mg / kg group and 3.9 days in the 10 mg / kg group 5).

Claims (12)

A pharmaceutical composition for preventing or treating cytotoxicity by radiation or a toxic drug containing the compound of formula (1) as an active ingredient:
[Chemical Formula 1]

The pharmaceutical composition according to claim 1, wherein the compound of formula (1) is isolated from a butanol fraction of a methanol extract of Eucommia ulmoides .
The method according to claim 1, wherein the compound of formula (1) is used to increase the expression of Heat Shock Factor 1 (HSF 1), Heat Shock Protein 27 (HSP 27) and Heat Shock Protein 70 &Lt; / RTI &gt;
The pharmaceutical composition according to claim 1, wherein the toxic drug is paclitaxel anticancer agent.
The pharmaceutical composition according to claim 1, wherein the radiation is ionized radiation (IR).
delete delete delete delete delete delete Extracting Eucommia ulmoides with methanol to obtain a mulberry extract; Fractionating the mulberry extract with butanol to obtain mulberry fraction; And subjecting the bifloride fraction to column chromatography to obtain a compound represented by the following formula (1): &lt; EMI ID =
[Chemical Formula 1]

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