WO2013172640A1 - Composition having heat shock protein induction activity and comprising compound isolated from eucommia ulmoides - Google Patents

Abstract

The present invention relates to a pharmaceutical composition having a heat shock protein induction activity and comprising a compound isolated from Eucommia ulmoides. The compound isolated from Eucommia ulmoides according to the present invention has a heat shock protein induction activity, and may particularly control the expression of HSF 1, HSP 27 and/or HSP 70. Thus, the composition of the present invention may provide the effects of protecting and regenerating cells through the induction of heat shock proteins and can be effectively used in treating or preventing nerve disorders, digestive diseases, enteropathy, renal diseases, eye diseases, cardiovascular diseases, or skin diseases. Further, the composition of the present invention can be effectively used as a cosmetic composition for regenerating damaged skin cells.

Description

A composition having a thermal shock protein inducing activity comprising a compound isolated from the worms

The present invention relates to a composition having a thermal shock protein inducing activity comprising a compound isolated from the two worms.

Toothworm Eucommia ulmoides Oliv (Toothworm Eucommiaceae) is a plant of the first genus. The skin of the toothworm, Eucommia ulmoides, is used as a tonic in Korea, China and Japan. In addition, it is known that drinking the leaves of the larvae with tea has a therapeutic effect on hypotension and a diuretic effect (Okada, Shirata, Niwano, Koshino, & Uramoto, 1994; Tomoda, Gonda, Shimizu, & Kanari, 1990). Previous studies have reported that various lignans, flavonoids, iridoids and terpenoids can be isolated from larvae (Ho et al., 2005; Hua, Yin, Li, Hu, & Pei, 2002; Kim, Moon). , Lee, & Choi, 2004; Tanaka, Nakamura, Nakazawa, & Nohara, 1997). Some of the compounds isolated from the worms have been reported to exhibit the effects of antioxidants, hypotensive agents, anti-inflammatory agents, osteoblast proliferation and antithrombosis (Ho et al., 2005; Hua, Yin, Li, Hu, & 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 infections (Lee et al., 2006). Under stressful conditions, HSF 1 is released from chaperone complexes and induces expression of thermal 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.

Specifically, it has been reported to have a therapeutic effect on neurological disorders such as Parkinson's disease (Luo, G.-R. et al., Are heat shock proteins therapeutic target for Parkinson's disease? Int J Biol Sci 3 (2007) 20-26) Increasing expression of HSP 25 has been reported to protect damaged submandibular 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 the expression of heat shock protein (HSP 70) is a neutrophil-derived hypo chlorite or Helicobacter pylori urease - it is possible to suppress the above damage to the mucosal cells of the mono-chloramine (NH ₂ Cl) generated by the induction ammonia PUD It has been reported to be applicable to treatment (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 HSP 90 due to increased expression of heat shock protein (HSP 90) can inhibit the damage of small intestinal mucosa cells, thereby preventing the intestinal mucosal damage and inflammatory bowel disease caused by nonsteroidal anti-inflammatory drugs (NSAIDs). Application has been reported (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 ischemic renal injury due to acute kidney injury can be prevented by the induction effect of heat shock protein (HSP 70) (Wang, Z. et al., Induction of heat shock protein 70 inhibits ischemic renal injury. Kidney International 79 (2011), 861-870). In addition, increased expression of heat shock protein (HSP 70) has been reported to prevent DNA damage in human lens epithelial cells (hLEC) (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 the increased expression of HSP 27 and HSP 72 can be applied as a therapeutic agent 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 there is a protective effect against damaged neurons by increased expression of HSP 27 (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 protects against damaged cardiomyocytes (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). It has also been reported to be effective for damaged skin cell regeneration (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). In addition, induction of the expression of HSF 1, HSP 27 and HSP 70 has been reported to be effective in the prevention and treatment of ischemic heart disease (a) Dillmann, WH, Small heat shock proteins and protection against injury. 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).

Thus, the present inventors completed the present invention by confirming that the compound isolated from the shell of the eucommia ulmoides has thermal shock protein inducing activity while studying to find HSP-expressing regulator from natural substances.

(Non-Patent Document 0001) Bava, S. V., Puliappadamba, V. T., Deepti, A., Nair, A., Karunagaran, D., & Anto, R. J. (2005). Sensitization of taxol-induced apoptosis by curcumin involves 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.

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(Non-Patent Document 0009) Lee, H.-J., 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.

(Non-Patent Document 0010) 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.

(Non-Patent Document 0011) Miyagoshi, M., Amagaya, S., & Ogihara, Y. (1987). The structural transformation of gardenoside and its related iridoid compounds by acid and glucosidase. Planta Medica, 53 (5), 462-464.

(Non-Patent Document 0012) Okada, N., Shirata, K., Niwano, M., Koshino, H., & Uramoto, M. (1994). Immunosuppressive activity of a monoterpene from Eucommia ulmoides. Phytochemistry, 37 (1), 281-282.

(Non-Patent Document 0013) 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.

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(Non-Patent Document 0015) Seo, HR, Chung, DY, Lee, YJ, Lee, DH, Kim, JI, Bae, S., Chung, HY, Lee, SJ, Jeoung, D., & Lee, YS (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.

(Non-Patent Document 0016) 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.

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constituents of Leonotis nepetaefolia. Chemical & Pharmaceutical Bulletin, 47 (2), 284-286.

(Non-Patent Document 0018) 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.

(Non-Patent Document 0019) 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.

(Non-Patent Document 0020) 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.

(Non-Patent Document 0021) 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.

(Non-Patent Document 0022) Tamaki. K. et al., Evidence for Enhanced Cytoprotective Function of HSP 90-Overexpressing Small Intestinal Epithelial Cells. Dig Dis Sci 56 (2011) 1954-1961.

(Non-Patent Document 0023) Wang, Z. et al., Induction of heat shock protein 70 inhibits ischemic renal injury. Kidney International 79 (2011), 861-870.

(Non-Patent Document 0024) 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.

(Non-Patent Document 0025) 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.

(Non-Patent Document 0026) 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.

(Non-Patent Document 0027) Morris, S. D. 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.

(Non-Patent Document 0028) Luo, G.-R. et al., Are heat shock proteins therapeutic target for Parkinson's disease? Int J Biol Sci 3 (2007) 20-26.

(Non-Patent Document 0029) 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 thermal shock protein inducing activity containing a compound isolated from the two insects as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of neurological diseases, digestive diseases, intestinal diseases, kidney diseases, eye diseases, cardiovascular diseases or skin diseases comprising a compound isolated from the worms having heat shock protein inducing activity as an active ingredient It is to provide.

In addition, the present invention is to provide a cosmetic composition for rejuvenating damaged skin cells comprising a compound isolated from the worms having heat shock protein inducing activity as an active ingredient.

In order to solve the above problems, the present invention provides a composition having a thermal shock protein inducing activity containing at least one compound selected from the group consisting of compounds of the general formula (1) as an active ingredient.

[Formula 1]

[Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

[Formula 6]

[Formula 7]

Wherein Glc is a D-glucopyranosyloxy group.

The term "Eucommia ulmoides" used in the present invention is a deciduous arborescent, a plant mainly distributed in China. It is mainly used to dry the bark of the larvae and is mainly used as a tonic, arthritis, and rheumatism painkiller.

The term "extraction" used in the present invention is to separate the eucommia ulmoides using a liquid solvent, the solvent is water, methanol, ethanol, C ₁ -C ₆ alcohols such as butanol, ethyl acetate, or These mixed solvents can be used. In the present invention, it is preferable to extract with methanol using the shell portion of the larvae. In the present invention, the methanol extract of the two insects may be fractionated with butanol to separate the compounds of 1 to 7.

In addition, the present invention comprises the steps of extracting the larvae extract (Eucommia ulmoides) with methanol; Fractionating the larvae extract with butanol to obtain a larvae fraction; And performing the column chromatography of the larvae fraction to obtain a compound represented by 1, 2 or 5 below.

[Formula 1]

[Formula 2]

[Formula 5]

Wherein Glc is a D-glucopyranosyloxy group.

The fact that the compound represented by 1, 2 or 5 can be separated from the larvae is not known in the prior art, and in the present invention, the compound represented by the 1, 2 or 5 can be separated from the larvae-based plant. 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, NOESY.

The compounds of Formulas 1 to 7 will be briefly described as compounds 1 to 7, respectively. The seven compounds are compound 1 (coniferaldehydeglucoside) (Steeves, Forster, Pommer, & Savidge, 2001), compound 2 (bartsioside) (Kobayashi, Karasawa, Miyase, & Fukushima, 1985), compound 3 (geniposidic acid) (Takeda, Narukawa, & Hada, 1999), compound 4 (geniposide) (Ono, Ueno, Masuoka, Ikeda, & Nohara, 2005), compound 5 (feretoside) (Miyagoshi, Amagaya, & Ogihara, 1987), compound 6 (pinoresinoldiglucoside) ( Schumacher, Scholle, Hoelzl, Khudeir, Hess, & Mueller, 2002), compound 7 (liriodendrin) (Lami, Kadota, Kikuchi, & Momose, 1991).

The fraction obtained by dividing the tofu extract with a butanol solution includes a large amount of Compounds 1 to 7, and may have thermal shock protein inducing activity.

As used herein, the term “heat shock protein inducing activity” refers to activating a regulatory factor (HSF 1) that induces the expression of a heat shock protein to activate a heat shock protein or to increase the expression of a heat shock protein (HSP). Means to induce. Heat shock factor 1 (HSF 1) is a transcription factor that regulates the heat-shock response of cells, and heat shock proteins induce synthesis when cells, tissues or individuals are brought to a temperature between 5 ° C and 10 ° C above physiological temperature. It is a protein.

Cell exposure to various environmental disorders, including thermal shock, alcohol, heavy metals, and oxidative disorders, results in cellular accumulation of many chaperones, commonly known as heat shock proteins (HSPs). Induction of HSP protects cells against early disorder damage and enhances maintenance recovery and induction of disorder tolerance. In addition, any HSP can also serve as a major molecular chaperone under normal disorder-free conditions by regulating the precise folding, degradation, accumulation and function of the growth list of important cellular proteins.

These are classified according to molecular weight, for example HSP 25, HSP 27, HSP 70, HSP 72 and HSP 90. Some diseases in humans can be obtained as a result of misfolded proteins. Since HSP is involved in preventing degeneration of proteins in cells or regenerating denatured proteins, HSP may be particularly useful for the treatment of inflammatory diseases of body 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 present invention was measured for HSF 1 protein expression and its impact on its transcription target, HSP 27 and HSP 70, compounds 1 and 7 increased the expression of HSF 1 at 3 uM by 1.214 and 1.167 fold, respectively. The expression of HSP 27 was increased by 1.316, 1.357 and 1.333 fold by compounds 1, 5 and 7, respectively. Compound 2 showed an effect of inducing an improvement in expression of HSP 70 by 1.162 fold. Compound 1 also activated the luciferase promoters of HSP 25 (1.47 fold) and HSP 70 (1.09 fold) at 50 uM. Accordingly, it can be seen that Compounds 1 to 7 extracted from the shell of the larvae have inducible activities of HSF 1, HSP 27 and HSP 70.

That is, the compounds 1 to 7 of the present invention may regulate the expression of HSF 1 and may induce the expression of HSP 27 and / or HSP 70. In particular, the compounds 1 to 7 of the present invention can induce all of HSF 1, HSP 27 and HSP 70, and excellent in HSP inducing ability.

The present invention comprises a neurological disease, digestive disease, intestinal disease, kidney disease, eye disease, cardiovascular disease comprising a composition having a thermal shock protein inducing activity containing at least one compound selected from the group consisting of compounds 1 to 7 as an active ingredient Or it provides a pharmaceutical composition for the prevention or treatment of skin diseases. In the present invention, the neurological disease is a degenerative brain disease, which is preferably Parkinson's disease. In addition, the gastrointestinal disease is preferably a peptic ulcer or duodenal ulcer as a peptic ulcer. In addition, the bowel disease is preferably inflammatory bowel disease, colitis. In addition, the renal disease is preferably ischemic renal disease. In addition, the eye disease is preferably glaucoma, cataracts or conjunctivitis. In addition, the cardiovascular disease is preferably ischemic heart disease or myocardial infarction.

Prior to the present invention, the expression of HSP has been reported to be effective in the treatment of neurological disorders such as Parkinson's disease and increased expression of HSP 25 has been reported to protect the damage of damaged submandibular cells. In addition, increased expression of the heat shock protein (HSP 70) may inhibit gastric mucosal cell damage caused by neutrophil-induced hypochloric acid or Helicobacter pylori urease-induced ammonia, which is caused by monochloramine (NH ₂ Cl). It has been reported that it can be applied to treatment. In addition, the molecular chaperone function of HSP 90 due to increased expression of heat shock protein (HSP 90) can inhibit the damage of small intestinal mucosa cells, thereby preventing the intestinal mucosal damage and inflammatory bowel disease caused by nonsteroidal anti-inflammatory drugs (NSAIDs). It has been reported to be applicable. In addition, it has been reported that the ischemic kidney injury caused by acute kidney injury can be prevented by the induction effect of the heat shock protein (HSP 70). In addition, increased expression of heat shock protein (HSP 70) has been reported to prevent DNA damage of human lens epithelial cells (hLEC). In addition, it has been reported that the increased 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 there is a protective effect on damaged neurons by increased expression of HSP 27. In addition, it has been reported that there is a protective effect on cardiomyocytes damaged by the induction effect of HSP 70. It has also been reported to be effective in repairing damaged skin cells. In addition, induction of the expression of HSF 1, HSP 27 and HSP 70 has been reported to be effective in the prevention and treatment of ischemic heart disease.

The term "treatment" used in the present invention, the administration of a composition comprising at least one compound selected from the group consisting of compounds of compounds 1 to 7 as an active ingredient neurological diseases, digestive diseases, intestinal diseases, kidney diseases, eye Means any behavior that improves or cures a condition of a disease, cardiovascular disease or skin disease. In addition, the term "prevention" as used in the present invention, any one that inhibits or delays the onset of the disease by administration of a composition comprising as an active ingredient at least one compound selected from the group consisting of compounds of compounds 1-7. Say an act.

The composition of the present invention may include a pharmaceutically acceptable carrier, excipient or diluent in addition to the above-described active ingredient for administration. The carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The compositions of the present invention can be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral formulations, suppositories, or sterile injectable solutions, respectively, according to conventional methods. Specifically, when formulated, it may be prepared by using diluents or excipients such as fillers, weighting agents, binders, wetting agents, disintegrating agents, and surfactants which are commonly used. Solid preparations for oral administration include, but are not limited to, tablets, pills, powders, granules, capsules, and the like. Such solid preparations may be prepared by mixing at least one excipient such as starch, calcium carbonate, sucrose, lactose, gelatin and the like in the composition. In addition to simple excipients, lubricants such as magnesium stearate, talc can also be used. It may be prepared by adding various excipients such as humectants, sweeteners, fragrances, preservatives and the like in addition to liquid oral liquids or liquid paraffin for oral use. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate and the like can be used. As the base of the suppository, utopsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The compositions of the present invention can be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally or topically) according to the desired method, and the dosage is based on the condition and weight of the patient, the extent of the disease, Depending on the drug form, route of administration, and time, it may be appropriately selected by those skilled in the art. The daily dosage of the composition is preferably 1 mg / kg to 500 mg / kg, may be administered once to several times daily if necessary.

In another aspect, the present invention provides a cosmetic composition for damaged skin cell regeneration comprising a composition having a thermal shock protein inducing activity containing at least one compound selected from the group consisting of compounds of Formulas 1 to 7 as an active ingredient. The cosmetic composition according to the present invention may be formulated with a lotion, gel, water-soluble powder, fat-soluble powder, water-soluble liquid, cream or essence by adding a pH adjuster, flavoring, emulsifier, preservative, etc. as necessary in a conventional cosmetic preparation method. .

Compounds isolated from the worms according to the invention have thermal shock protein inducing activity and in particular can modulate the expression of HSF 1, HSP 27 and / or HSP 70. Accordingly, there is a protective and regenerative effect of cells through the induction of thermal shock proteins, and can be useful for the treatment or prevention of neurological diseases, digestive diseases, intestinal diseases, kidney diseases, eye diseases, cardiovascular diseases or skin diseases. In addition, it can be usefully used as a cosmetic composition for rejuvenating damaged skin cells.

Figure 1 shows the results of measuring the expression of HSF 1, HSP 27 and HSP 70 by Western blotting after treatment with L132 cells of a compound isolated from the Eucommia ulmoides shell according to an embodiment of the present invention. β-Actin was used as internal control and L132 cells were treated with 3 μM compound for 12 h.

Figure 2 shows the HSP 25 or HSP 70 promoter activity after treatment with NCI-H460 cells Compound 1 isolated from the shell of the worms according to an embodiment of the present invention. Each value is the mean ± SD of three independent experimental groups. And * p <0.05 vs. CON group, ** p <0.01 vs. CON group.

Figure 3 shows the results of measuring the viability of L132 cells treated with Compounds 1 to 7 extracted from the shell of the two layers according to an embodiment of the present invention and the cell viability of Taxol® as a control by MTT.

4A shows that after induction of cell death by Western blotting, cleavage caspase 3 and cleavage PARP-1 observed that Compound 1 reduces cell death by radiation and Taxol ^® . B shows cell viability (%) after IR treatment for Compound 1. C shows the cell viability (%) after Taxol ^® treatment for Compound 1.

5 is a graph showing that the mortality caused by radiation is reduced by Compound 1 after the mouse is irradiated with a lethal amount of radiation systemically.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the examples.

Example 1: Materials and Methods

Optical rotation at 25 ° C. was measured with a P-1010 polarimeter (JASCO, Japan). UV and IR spectra were recorded with a U-3000 spectrophotometer (Hitachi, Japan) and a FTS 135 FT-IR spectrometer (Bio-Rad, CA), respectively. 1D and 2D NMR experiments were performed with a UNITY INOVA 400 MHz FT-NMR instrument (Varian, CA) using tetramethylsilane (TMS) as an internal standard. Mass spectrometry was performed with a Micromass Q-Tof micromass spectrometer and a Waters ACQUITY UPLC system combined with an Agilent 6220 Accurate-Mass TOF LC / MS system. Column chromatography includes silica gel (230-400 mesh, Merck, Germany), RP-18 (YMC gel ODS-A, 12 nm, S-150 μm), Sephadex LH-20 (Amersham Pharmacia Biotech), and Diaion HP- 20 (Mitsubishi Chemical Co.). 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, UV light (254 nm and 365 nm) and 10% (v / v) Visualized with H ₂ SO ₄ spray and heated at 120 ° C. for 5 minutes.

Example 2: Tofu Eucommia ulmoides A) methanol extract manufacturing

20 kg of worms were added to 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: worms Eucommia ulmoides Solvent fractions)

2 L of distilled water was added to 1.4 kg of the methanol extract of Example 2 and suspended. Then, hexane (2 L x 8 times) was added thereto to dissolve, and only the components dissolved in the hexane layer were separated and dried in vacuo to obtain 210 g of a hexane fraction. It was. Ethyl acetate (2 L x 4 times) was added to the remaining aqueous layer to dissolve, and only the components dissolved in the ethyl acetate layer were separated and dried in vacuo to yield 200 g of an ethyl acetate fraction. In the same manner as above, 450 g (2 L × 5 times) of n-butanol fraction and 474 g of aqueous layer were obtained.

Example 4: Toothworm Eucommia ulmoides Separation of Compounds from Butanol Fraction

450 g of butanol fraction of Example 3 was divided into six fractions (F01-F06) by a Diaion HP-20 column with a methanol-water mixed solvent concentration gradient (from 100% to 100% methanol). Compound 3 (2.3 g, 0.0115% w / w) was obtained by precipitation from the F05 fraction. 3.2 g of the F02 fractions were purified by silica gel column chromatography with a chloroform: methanol mixed solvent (24: 1 → 19: 1) to obtain compound 4 (200 mg, 0.001% w / w). In addition, 13 fractions (F03.01-F03.13) were subjected to silica gel column chromatography on 124.0 g of the F03 fraction using a chloroform: methanol: water mixed solvent (9: 1: 0.1 → 7: 3: 0.5). Got it. From subfractions F03.06 and F03.07, compound 7 (36 mg, 0.00018% w / w) and compound 6 (270 mg, 0.00135% w / w) were obtained by recrystallization with methanol. Next, 2.4 g of subfraction F03.08 was subjected to reverse phase column chromatography (YMC gel ODS-A, 12 nm, S-150 μm) using a methanol: water mixed solvent (6: 4 → 9: 1). Fraction (F03.08.01- F03.08.04) was obtained. Compound 2 (5 mg, 0.000025% w / w) and Compound 5 (5 mg, 0.000025% w / w) from fractions F 03.08.02 and F03.08.03 were run on a Sephadex LH-20 column (Amersham Pharmacia Biotech) with 100% methanol. w) was obtained respectively. In addition, fraction F05 (35 g) was subjected to silica gel column chromatography using a chloroform: methanol: water mixed solvent (9: 1: 0.1 → 7: 3: 0.5) to obtain 10 subfractions (F05.01-F05.10). Got. Among them, four fractions (F05.01.01-) were subjected to reversed phase column chromatography (YMC gel ODS-A, 12 nm, S-150 μm) using a methanol: water mixed solvent (9: 1). F05.01.04), and the second fraction (F05.01.02) was subjected to a Sephadex LH-20 column (Amersham Pharmacia Biotech) with 100% methanol to obtain Compound 1 (10mg, 0.00005% w / w). .

It was found for the first time that Compound 1, Compound 2, and Compound 5 can be separated from the worm family plants 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.

NMR Data of Compound 1 (Coniferaldehydeglucoside)

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, C-2), 102.2 (CH, Glc-1 '), 78.5 (CH, Glc-3'), 78.0 (CH, Glc-5 '), 74.9 (CH, Glc-2'), 71.4 ( 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).

NMR Data of Compound 2 (Bartsioside)

Yellow oil; [a] ²⁵ _D -94.5 ( c 0.1, MeOH); UV (MeOH) λ _max (log ε) 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) δ 145.1 (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-5 '), 75.1 (CH, Glc-2'), 71.8 ( 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).

NMR Data of Compound 5 (Feretoside)

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) δ 170.8 (C, C-11), 154.4 (CH, C-3), 148.1 (C, C-8), 130.6 (CH, C-7), 111.3 (C, C-4), 100.8 (CH, Glc-1 '), 98.8 (CH, C-1), 82.8 (CH, C-6), 78.9 (CH, Glc-3'), 78.4 (CH, Glc-5 '), 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

Measurements were made by established protocols (Lee et al., 2008) to measure HSF 1 and HSPs expression of compounds isolated from worms. Proteins in the lysates were separated by SDS-PAGE, electro-transferred to nitrocellulose mucosa (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).

In order to determine whether the extract has an inducing effect on HSF 1 and its transcription targets, HSP 27 and HSP 70, Western blot was performed for 12 hours after treatment with the normal lung fibroblasts and L132 cells. HSF 1 expression was shown to increase by treatment of compounds 1-7. In particular, high expression increase was observed in compound 1 and compound 7 statistically. In the case of HSP 27 and HSP 70 all compounds induced an improvement in their protein expression. In particular, a statistically significant improvement in expression of compounds 1, 5 and 7 was observed for HSP 27 and a higher expression of compound 2 was observed for HSP 70 compared to untreated controls (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 the locust isolate compound act by different mechanisms. The most effective increase in HSF 1 was found in compound 1.

Example 6: MTT assay

Cells were treated with 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (Bava, Puliappadamba, Deepti, Nair, Karunagaran, & Anto, 2005) according to established protocols. Their cytotoxicity was measured by MTT; Sigma) test.

The compound isolated from the bark of Eucommia while not show any cytotoxicity in L132 cells (IC ₅₀ values> 5 uM), anticancer agent Taxol ^® was has an IC ₅₀ value of 12.3 uM indicate the cytotoxicity (FIG. 3).

Example 7: HSP promoter analysis

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

Assay 1 was used to analyze HSP 25 (a murine form of HSP 27) and HSP 70 using Compound 1, which showed the most effective increase in expression of HSF 1. 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 the promoter activity of HSP 25 1.47 fold and HSP 70 1.09 fold at 50 uM.

The results of quantitative immunoblots 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.

Table 1

Improvement degree (multiple)
compound	HSF	1	HSP 27	HSP 70	IC 50 (uM)
One	1.214 ± 0.013 *	1.316 ± 0.067 *	1.102 ± 0.057	> 5uM
2	1.144 ± 0.066	1.270 ± 0.085	1.162 ± 0.048 *	> 5uM
3	1.159 ± 0.108	1.331 ± 0.115	1.205 ± 0.063	> 5uM
4	1.114 ± 0.054	1.295 ± 0.109	1.157 ± 0.077	> 5uM
5	1.153 ± 0.052	1.357 ± 0.082 *	1.129 ± 0.043	> 5uM
6	1.041 ± 0.086	1.179 ± 0.073	1.158 ± 0.057	> 5uM
7	1.167 ± 0.046 *	1.333 ± 0.093 *	1.152 ± 0.091	> 5uM
Celastrol	1.019 ± 0.003	1.193 ± 0.038 *	1.319 ± 0.040 *	> 5uM
Taxol ®	ND	ND	ND	12.3

Data values are mean ± SD of three experimental groups 12 hours after treatment. Statistically significant differences through comparison of HSP 70, HSP 27 or HSF 1 level amounts between treated and untreated groups are indicated by * 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. Taxol ^® was used as a control for cytotoxicity.

In conclusion, compounds 1 and 7 increased the expression of HSF 1 at 3 uM by 1.214 and 1.167 fold, respectively. The expression of HSP 27 was increased by 1.316, 1.357 and 1.333 fold by compounds 1, 5 and 7, respectively. Compound 2 showed an effect of inducing an improvement in expression of HSP 70 by 1.162 fold. Compound 1 also activated the luciferase promoters 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 current system. This is the first report of the inducible activity of HSF 1, HSP 27 and HSP 70 of Compounds 1 to 7 extracted from the shells of larvae.

Example 8: Cell Viability Assay

Treatment of toxic compounds that may damage cell survival, such as Taxol and IR (radiation), or exposure to ionizing radiation results in the same HSF1 and HSP production of compound 1 (coniferalaldehyde glucoside and coniferalaldehyde) under conditions of approximately 50% survival. In order to confirm that, in order to treat with a certain amount of the compound instead of the glucoside form of the compound of formula 1 of the present invention was purchased by coniferalaldehyde compound experiment was carried out simultaneously) and after a certain time the cells trypan blue (trypan blue) After staining with the hematocytometer, the cell number was measured and viability was analyzed by Western blot method.

Cleavage caspase-3 and cleavage PARP1 protein expression were measured to determine the effect on compound cell death. Proteins in the lysates were separated by SDS-PAGE, electro-transferred to nitrocellulose mucosa (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).

In addition, a combination of Compound 1 (3 μM) was used to identify the apoptosis markers Cleaved PARP and Cleaved caspage3 to determine the protective effect of IR and Taxol ^® damage on L132 cells, which are human embryonic lung cells. In the treated group showed a protective effect against damage and confirmed the survival rate of the cell was able to confirm the increase in viability according to the damage (Fig. 4).

Example 9: Animal Viability Assay

The mice were exposed to radiation 7Gy to confirm viability for compound 1. Compound 1 (coniferalaldehyde) was given intraperitoneally one day before the radiation treatment (5 mg / kg, 10 mg / kg) and the next day radiation (whole body exposure) was exposed to the animals followed by intraperitoneal administration of additional drugs for three days. After that, the survival date was observed until the day when all animals died, and the results showed that the survival ability increased by 3.7 days in the average 5mg / kg group and 3.9 days in the average 10mg / kg group. 5).

Claims (12)

1. A composition having a thermal shock protein inducing activity containing as an active ingredient at least one compound selected from the group consisting of compounds of the formula

   [Formula 1]

   

   [Formula 2]

   

   [Formula 3]

   

   [Formula 4]

   [Formula 5]

   

   [Formula 6]

   

   [Formula 7]

   

   Wherein Glc is a D-glucopyranosyloxy group.
2. According to claim 1, wherein the compound of Formula 1 to 7 has a thermal shock protein inducing activity, characterized in that separated from the butanol fraction of the methanol extract Eucommia ulmoides .
3. According to claim 1, wherein the heat shock protein inducing activity is a composition having a heat shock protein inducing activity, characterized in that by increasing the expression of HSF 1, HSP 27 and HSP 70.
4. Pharmaceutical composition for the prevention or treatment of neurological diseases, digestive diseases, intestinal diseases, kidney diseases, eye diseases, cardiovascular diseases or skin diseases containing the composition according to claim 1.
5. The pharmaceutical composition according to claim 4, wherein the neurological disease is Parkinson's disease as a degenerative brain disease.
6. The pharmaceutical composition of claim 4, wherein the digestive disease is a gastric ulcer or duodenal ulcer as a peptic ulcer.
7. The pharmaceutical composition according to claim 4, wherein the enteric disease is colitis as an inflammatory bowel disease.
8. The pharmaceutical composition according to claim 4, wherein the renal disease is ischemic renal disease.
9. The pharmaceutical composition of claim 4, wherein the ocular disease is glaucoma, cataract or conjunctivitis.
10. The pharmaceutical composition of claim 4, wherein the cardiovascular disease is ischemic heart disease or myocardial infarction.
11. A cosmetic composition for damaged skin cell regeneration comprising the composition according to claim 1.
12. Extracting worms (Eucommia ulmoides) with methanol to obtain a worms extract; Fractionating the larvae extract with butanol to obtain a larvae fraction; And performing column chromatography on the larvae fraction to obtain a compound represented by 1, 2 or 5 below.

    [Formula 1]

    

    [Formula 2]

    

    [Formula 5]

    

    Wherein Glc is a D-glucopyranosyloxy group.

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