KR100965305B1 - Composition for preventing or treating a disease mediated by overexpression of heat shock protein 27 - Google Patents

Abstract

The present invention is a seaweed ( Trunterygium) regelii ) or a composition for preventing or treating a disease mediated by overexpression of HSP 27 (Heat shock protein 27) comprising an active compound extracted from wakame tree, a method for extracting the active compound from wakame tree, and the It provides a pharmaceutical and dietary supplement comprising the composition.

Description

Composition for preventing or treating a disease mediated by overexpression of heat shock protein 27}

The present invention relates to a composition for inhibiting expression of HSP 27 (Heat Shock Protein 27) including Tripterygium regelii , more specifically, a compound isolated from Wakame tree, its extract, or Wakame tree extract A composition for the prevention or treatment of diseases mediated by overexpression of HSP 27 comprising as an active ingredient.

The low-molecular heat shock protein HSP 27 has a chaperon function that protects against stress by forming a cluster of itself against external environmental factors such as free radicals, heat and toxins. It is a protein having a molecular weight. Problems of currently used anticancer drugs include side effects due to unintended death of normal cells during treatment and development of resistance of cancer cells to anticancer drugs. According to the present inventors and several other studies, one of the main causes of anticancer drug resistance of various cancer cells is due to the low-molecular shock protein that is expressed in cancer cells, although the amount is small in normal cells (Yun-Sil Lee, et al., 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, 2006, 281 (25), 17220-17227; Kroemer G, et al. Heat shock proteins 27 and 70: anti-apoptotic proteins with tumorigenic properties.Cell cycle, 2006, 5 (22), 2592-2601). Lung cancer, osteosarcoma, uterine cancer and breast cancer have been reported as resistance to cancer induced by HSP 27 (Yun-Sil Lee, et al. Inhibition of heat shock protein 27-mediated resistance to DNA damaging agents by a novel PKC delta-V5 heptapeptide.Cancer Res. 2007, 67 (13), 6333-6341; Kopecek J. et al., Free and N- (2-hydroxypropyl) methacrylamide copolymer-bound geldanamycin derivative induce different stress responses in A2780 human ovarian carcinoma cells, Cancer Res, 2003, 63 (22), 7876-7882; Head JF, et al. Gene targets of antisense therapies in breast cancer, Expert Opin Ther Targets, 2002, 6 (3), 375-385) . Therefore, it is known that inhibiting the expression of low molecular heat shock protein HSP 27 is a novel prophylaxis or treatment of HSP 27 resistant cancer disease.

In addition, it has been reported that the expression of low molecular heat shock protein HSP 27 is abnormally common in Alzheimer's, Parkinson's, and Huntington's disease, which are neurodegenerative diseases. Therefore, the method of inhibiting the expression of HSP 27 is reported to be effective in the prevention and treatment of neurological diseases such as Alzheimer's, Parkinson's disease, and Huntington's disease in addition to cancer (MMM Wilhelmus et al., Sepcific association of small heat). shock proteins with the pathological hallmarks of Alzheimer's disease brains, Neuropathology and Applied Neurobiology (2006), 32, 119-130; Bobby Thomas and M. Flint Beal, Parkinson's disease, Journal of neuroscience Research (2007), 85, 1288-1294; Valerie Perrin et al., Meuroprotection by Hsp104 and HSP 27 in lentiviral-based rat models of huntington's disease, Molecular Therapy (2007), 15 (5), 903-911).

At present, there is an active research on heat shock proteins having different molecular weight from HSP 27. For example, 17 allyl amino geldanamycin (17AAG), which has inhibitory activity against heat shock protein (HSP90) with a molecular weight of 90,000, is already in phase 2 clinical trials and developed an inhibitor for heat shock protein HSPA (HSP70) with a molecular weight of 70,000. It is also actively underway. However, little inhibitors for HSP 27 (eg HSPB1) are currently being developed.

On the other hand, Wakame tree is a deciduous broad-leaved mandarin tree belonging to the family of Celastraceae and is distributed in Korea, Japan, and northern China. Wakame tree is used for ornamental and medicinal purposes. In oriental medicine and folk medicine, roots and stems are used for inflammation, anti-inflammatory and detoxification. Separated and reported components from the roots and stems of seaweed tree include sesquiterpenoids, diternoids, triterpenoids, and quinonemethides (Anita M. Brinker et al., Medicinal chemistry and pharmacology) of genus Tripterygium (Celastaceae), Phytochemistry 68 (2007) 732-766; The triterpenoid quinonemethide pristimerine inhibits induction of inducible nitric oxide synthase in murine macrophages, European Journal of Pharmacology 336 (1997) 211-217; Alex Haroldo Jeller et al., Antioxidant phenolic and quinonemethide triterpenes from Cheiloclinium cognatum, Phytochemistry 65 (2004) 1977-1982). However, there is no published report on the effects of the compounds contained in the wakame tree on the expression of the low molecular heat shock protein HSP 27.

Therefore, the present inventors have searched for inhibitors that can inhibit the expression of low molecular shock protein HSP 27 in order to develop a drug capable of preventing or treating a disease mediated by overexpression of low molecular shock protein HSP 27, resulting in plant herbal medicines. It was confirmed that the compound of inhibits the expression of HSP 27, and completed the present invention.

Accordingly, it is an object of the present invention to provide a composition derived from a plant herb that can prevent or treat a disease mediated by overexpression of HSP 27.

Another object of the present invention is to provide a composition capable of preventing or treating a disease mediated by overexpression of HSP 27 comprising a compound derived from a plant herb.

Another object of the present invention is to provide a method for separating a compound having HSP 27 expression inhibiting activity from a plant herb.

Still another object of the present invention is to provide a medicine comprising the composition.

Another object of the present invention to provide a dietary supplement comprising the composition.

In order to achieve the above object, the present invention provides a composition for the prevention or treatment of diseases mediated by overexpression of HSP 27 comprising wakame bark as an active ingredient.

The present invention also provides a composition for the prophylaxis or treatment of a disease mediated by HSP 27 overexpression comprising, as an active ingredient, at least one compound selected from the group consisting of compounds of Formulas 1 to 5 or a pharmaceutically acceptable salt thereof. do:

The present invention also provides a method for separating one or more compounds selected from the group consisting of the compounds of Formulas 1 to 5 from Wakame tree.

The present invention also provides a medicine comprising the composition.

In addition, the present invention provides a health supplement comprising the composition.

Hereinafter, the present invention will be described in more detail.

The present inventors have conducted research on various plant herbals to find plant herbals having the activity of inhibiting the expression of HSP 27, and found that Wakame tree extract has excellent HSP 27 expression inhibiting activity.

Accordingly, in one aspect, the present invention provides a composition for the prevention or treatment of diseases mediated by overexpression of HSP 27 comprising Wakame tree as an active ingredient.

Seaweed willow, which is an active ingredient of the composition, may be included in the form of an extract, or may be included as a pulverized powder of the herbal medicine itself or as a dry pulverized powder of the herbal medicine.

The extract of seaweed tree may be a crude extract of a C ₁ -C ₄ alcohol solvent, preferably an extract of ethanol, and more preferably 70 to 100% ethanol extract. When extracting with a solvent, the extraction may be heat extraction, cold extraction, reflux extraction, or ultrasonic extraction. Such extraction may be carried out at room temperature or under warm conditions. Preferably, ethanol is added to the dry bark of wakame jawwood and stored at room temperature for at least 5 days and then filtered through a filter paper. The extraction may be extracted one or more times several times in order to obtain a larger amount of the active ingredient, preferably three times the continuous extraction can be used combined extract.

The composition comprising wakame sapling according to the present invention may include wakame sapling as a crude extract as described above, but may also include the crude extract as a soluble extract of a nonpolar solvent obtained by further extracting with a nonpolar organic solvent. As the soluble extract of the non-polar solvent, it is preferable to use the chloroform soluble fraction obtained by suspending the crude extract of the C ₁ -C ₄ alcohol solvent with water and then fractionally extracting in the order of hexane, ethyl acetate, and chloroform. The fractional extraction may use a conventional fractional extraction method, preferably a fractional funnel.

The extract extracted by the method as described above may be used as it is, but may be used in the form of concentrated X, or may be concentrated and then lyophilized to be used as a lyophilized form. Preferably, it is used in concentrated form so that it can be used at a sufficient concentration.

The chloroform soluble fraction may be further purified by chromatography to separate specific active ingredients having HSP 27 expression inhibiting activity from Wakame tree.

The chromatography may use silica gel chromatography, and purification may be performed in the order of chloroform, chloroform and acetone mixed solvent, and methanol as a mobile phase. Thus, a fraction of the crude fraction obtained as a mobile phase was further subjected to silica gel chromatography using a mixed solvent of n-hexane and acetone to obtain a fraction containing the compound represented by the following formulas (1) and (2), and mixed with n-hexane / acetone as a mobile phase. By further performing silica gel chromatography using a solvent, the compounds of Formulas 1 and 2 may be separated, respectively.

Further fractionation of the crude fraction obtained above was carried out by silica gel chromatography using a mixed solvent of chloroform and acetone as a mobile phase to obtain a fraction containing the compound represented by the following Formulas 3 to 5, and mixed with n-hexane and acetone as a mobile phase. Compounds of the following Chemical Formulas 3 to 5 may be separated by further performing silica gel chromatography using a solvent, a chloroform acetone mixed solvent, or an n-hexane / ethyl acetate mixed solvent.

The n-hexane / acetone mixed solvent used as the mobile phase is preferably in a volume ratio of 50: 1 to 1: 2, the chloroform-acetone mixed solvent is preferably (volume ratio range), and the n-hexane / acetate mixed solvent is preferred. Preference is given to a volume ratio of 60: 1 to 1: 2. The chromatography may be performed once or several times until a single compound is purified, and may be concentrated or recrystallized as necessary. Thus, the separated compound may be analyzed by molecular weight measurement, NMR measurement, and the like, and the following Formula 1 (celastrol), Formula 2 (pristimerine), Formula 3 (iguestrein), Formula 4 (tingenone), and Formula 5 (22-β) -hydroxy tingenone) was isolated:

[Formula 1]

[Formula 2]

(3)

[Formula 4]

[Chemical Formula 5]

Thus, in another aspect of the invention, the invention

Extracting brown seaweed from C ₁ -C ₄ alcohol solvent to obtain a crude extract;

Suspending the crude extract with water and then fractionally extracting in the order of hexane, ethyl acetate and chloroform to obtain a chloroform soluble fraction; And

Provided is a method for separating a compound selected from the group consisting of the compounds of Formulas 1 to 5 from the wakaken tree performing the step of purifying the chloroform soluble fraction by chromatography.

Each of the isolated compounds was tested for HSP 27 inhibitory activity, and each was found to have excellent HSP 27 expression inhibitory activity.

Therefore, in another aspect of the present invention, the present invention is mediated by the overexpression of HSP 27 comprising as an active ingredient at least one compound selected from the group consisting of compounds of Formulas 1 to 5 or a pharmaceutically acceptable salt thereof It provides a composition for the prevention or treatment of diseases.

The composition of the present invention comprising as an active ingredient a compound or a pharmaceutically acceptable salt thereof selected from the group consisting of Wakame tree or the compound of Formulas 1 to 5 is used for the prevention or treatment of diseases mediated by overexpression of HSP 27 Can be. The disease mediated by the overexpression of HSP 27 refers to any disease in which the inhibitory activity of HSP 27 expression may be beneficial for the treatment or prevention of the disease. Such diseases include, but are not limited to, HSP 27 resistant cancer disease, Alzheimer's, Parkinson's disease, and Huntington's disease. The HSP 27 resistant cancer disease refers to any cancer disease that exhibits resistance by expression of HSP 27, and examples thereof include lung cancer, osteosarcoma, uterine cancer, and breast cancer, but are not limited thereto.

It is demonstrated in the following examples that the composition according to the present invention has HSP 27 expression inhibiting activity. Specifically, to the extent that the expression of HSP 27 is inhibited by treating each of the compounds of Formulas 1 to 5 isolated from the extracts of wakame tree according to one embodiment of the present invention on cancer cells overexpressed HSP 27 It was confirmed. In addition, lung cancer cells known to be resistant to HSP 27 were transplanted into the femoral thighs of nude mice and then administered in vivo with the pristinerin compound of Formula 2, compared with the control group and the cisplatin group, and compared with the control group. It slowly increased in size and appeared to have a similar effect to the cisplatin-treated group.

A composition for the prevention or treatment of diseases mediated by overexpression of HSP 27 comprising as an active ingredient a compound selected from the group consisting of wakame tree or a compound of formulas 1 to 5 according to the present invention or a pharmaceutically acceptable salt thereof Can be used as a raw material for pharmaceuticals for the treatment or prevention of diseases mediated by overexpression of HSP 27.

Accordingly, the present invention in another aspect, the present invention is a composition comprising a compound selected from the group consisting of Wakame tree or the compound of Formula 1 to 5 or a pharmaceutically acceptable salt thereof, and pharmaceutically acceptable Provided is a medicament comprising a possible carrier or additive.

The medicament may be formulated in conventional pharmaceutical formulations known in the art. The medicament may be formulated and administered in any dosage form including, but not limited to, oral, injectable, suppository, transdermal, and non-administrative agents, but preferably liquids, suspensions, powders, granules, It may be formulated into a formulation for oral administration such as tablets, capsules, pills, or excipients.

When formulated into each of the above formulations, it may be prepared by the addition of a pharmaceutically acceptable carrier or additive necessary for the preparation of each formulation. Representatively, when formulated into a dosage form for oral administration, one or more of a diluent, a lubricant, a binder, a disintegrant, a sweetener, a stabilizer, and a preservative may be used as the carrier, and as an additive, flavors, vitamins, and antioxidants may be used. One or more types can be selected and used out of them.

The carrier and the additive may be any pharmaceutically acceptable, and specifically, lactose, corn starch, soybean oil, microcrystalline cellulose, or mannitol as a diluent, magnesium stearate or talc as a lubricant, and polyvinylpyrrolidone as a binder. Or hydroxypropyl cellulose is preferred. In addition, as a disintegrant, calcium carboxymethyl cellulose, sodium starch glycolate, potassium polyacrylic acid, or crospovidone, sweetener as white sugar, fructose, sorbitol, or aspartame, stabilizer as carboxymethyl cellulose sodium, beta-cyclodextrin, As lead, or xanthan gum, and preservative, methyl paraoxybenzoate, propyl paraoxybenzoate, or potassium sorbate is preferable.

In addition to the above ingredients, in order to enhance the taste as a known additive, natural flavors such as plum flavor, lemon flavor, pineapple flavor, herbal flavor, natural pigments such as natural fruit juice, chlorophyllin, flavonoid, fructose, honey, sugar alcohol, sugar Sweetening ingredients such as, or may be used by mixing an acidulant such as citric acid, sodium citrate.

In order to obtain a prophylactic or therapeutic effect of HSP 27-resistant cancer disease, Alzheimer's disease, Parkinson's disease, and HSP 27 expression inhibiting activity including Huntington's disease, the total daily dose based on an adult as an active ingredient It may be administered in several divided doses so as to be 0.5 mg / kg-3 mg / kg as an extract or compound, and 10 mg / kg-50 mg / kg as a crude drug. The dosage may be appropriately increased or decreased depending on the route of administration, disease progression, sex, age, weight, and the like.

In addition, the composition for the prevention or treatment of diseases mediated by the overexpression of HSP 27 as an active ingredient in the seaweed tree according to the present invention or a compound selected from the group consisting of compounds of Formulas 1 to 5 by overexpression of the HSP 27 It may be used as a raw material for dietary supplements that is effective in preventing or treating a mediated disease.

Accordingly, in another aspect of the present invention, the present invention is a composition comprising a compound selected from the group consisting of wakame tree or the compound of formula 1 to 5 or a pharmaceutically acceptable salt thereof, and food It provides a dietary supplement comprising a possible carrier or additive.

Such dietary supplements may be formulated in the form of conventional dietary supplements known in the art. The dietary supplement may be prepared, for example, as a powder, granules, tablets, capsules, suspensions, emulsions, syrups, liquids, extracts, teas, jelly, extracts, or beverages. As the food acceptable carrier or additive, any carrier or additive known to be usable in the art may be used to prepare a formulation to be prepared. As such, the health supplement of the present invention processed in various forms can be used not only for the prevention of various diseases mediated by HSP 27 overexpression, but also for ease of use.

In addition, the young sprouts of seaweeds are currently edible as herbs, so the seaweeds extract or the compounds of formula 1 to 5 derived from it can be seen that there is no problem of toxicity or side effects, the medicines and health according to the present invention Supplements have the advantage that can be used with confidence even for prolonged use for the prevention or treatment of diseases.

As described above, the composition comprising a seaweed tree according to the present invention or a compound selected from the group consisting of compounds of Formulas 1 to 5 or a pharmaceutically acceptable salt thereof as an active ingredient is excellent in inhibiting activity of HSP 27 expression. It can be used for the prevention or treatment of various diseases mediated by overexpression of HSP 27, such as cancer, Alzheimer's, Parkinson's, Huntington's disease. In addition, there is an advantage that can eliminate various side effects that can be exhibited by conventional anticancer drugs and neurological diseases drugs by using a plant herbal medicine proven safety.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are only for the understanding of the present invention, and the scope of the present invention is not limited by them in any sense.

Example 1

Extraction, Separation and Purification of Compounds from Seaweed

4.0 kg of dried Wakame tree roots and stems (Jiri, Gyeongsangnam-do) were put in 20 l of 95% ethanol, stored at room temperature for 10 days, filtered through filter paper, and concentrated under reduced pressure to obtain 780 g of a brownish brown extract. 4 L of distilled water was added and suspended, and the extract was separated and extracted in the order of hexane, ethyl acetate, chloroform and butanol to obtain 210 g of a chloroform fraction. The obtained chloroform fraction was subjected to silica gel column chromatography (3 kg, 240 to 400 mesh, Merk), and chloroform, chloroform: acetone (200: 1 to 1: 7 mixed ratio) mixed solvent, and methanol were sequentially used as a mobile phase solvent. To obtain 24 fractions (fractions 1-1 to 24). Among them, fractions 2-10 (5.2 g) were adsorbed onto silica gel (80 g, 70-230 mesh), and silica gel column chromatography (1 kg, 230-400 mesh) was performed, wherein n -hexane: acetone was used as the mobile phase solvent. Fractions 1-2-1 to 18 were obtained using a mixed solvent of. Fr.1-2-1 to 12, which are small fractions, are second column chromatography (20 g, 230 to 400 mesh) using a mixed solvent of n -hexane: acetone (15: 1 volume ratio) as the mobile phase developing solvent. To obtain the compound of Formula 1 (240 mg), Compound 2 of the compound (300 mg).

In addition, fractions 13-17 (6.2 g) in 24 fractions were adsorbed onto silica gel (100 g, 70-230 mesh), and silica gel column chromatography (1.4 kg, 230-400 mesh) was performed, wherein chloroform was used as the mobile phase solvent. : Fractions 2-1-1 to 24 were obtained using acetone mixed solvent. Fr.2-1-1 to 11, which is a small fraction, of the second column chromatography (30 g, 230 to 400 mesh) using a mixed solvent of n -hexane: acetone (10: 1 volume ratio) as the mobile phase developing solvent. The compound of formula 3 (105 mg) was obtained, and the small fraction Fr.2-1-12-Fr 2-1-19 was prepared by using a mixed solvent of chloroform: acetone (5: 1 volume ratio) as a mobile phase developing solvent. Second column chromatography (10 g, 230-400 mesh) was performed to obtain the compound of formula 4 (90 mg), respectively. Finally, Fr. 2-1-20-Fr. 2-1-23 was prepared by performing a second column chromatography (23 g, 230-400 mesh) using a mixed solvent of n-hexane: ethyl acetate (8: 1, by volume) as a mobile phase developing solvent. (30 mg) was obtained.

Example 2

Structural Analysis of Compounds Isolated from Natural Products

For each material obtained in Example 1, the molecular weight and molecular formula were determined using a VG high resolution GC / MS spectrometer (Election Ionization MS, JEOLJMS-700). In addition, the photoluminescence was measured using a polarizer (Perkin-Elmer polarimeter 343), ¹ H NMR, ¹³ C NMR, HOMO-COSY, HMQC (Bruker Bruker IFS 66) ¹ H-Detected heteronuclear Multiple-Quantum Coherence (HMBC), Heteronuclear Multiple-Bond Coherence (HMBC), and DPT (Distortionless Enhancement by Polarization) spectra were obtained and the molecular structure was determined.

The physical properties, molecular weight, molecular formula, NMR analysis, and mass spectrometry of each compound obtained as a result are shown below.

[Formula 1]

Celastrol

1) Physical property: Red crystal (melting point: 203 ~ 205 ℃)

2) Molecular Weight: 450

3) Molecular formula: C ₂₉ H ₃₈ O ₄

4) ¹ H NMR (CDCl ₃ , 500 MHz) d 0.57 (3H, s, H-27), 0.91 (1H, m, H-22a), 1.09 (3H, s, H-28), 1.24 (3H, s, H-26), 1.27 (3H, s, H-29), 1.35 (1H, m, H-21a), 1.42 (3H, s, H-25), 1.48 (1H, m, H-16a) , 1.55 (2H, m, H-15), 1.57 (1H, m, H-18), 1.63 (1H, m, H-12a), 1.75 (1H, m, H-19a), 1.76 (1H, m , H-22b), 1.79 (1H, m, H-11a), 1.87 (1H, m, H-12b), 1.88 (1H, m, H-16b), 2.20 (3H, s, H-23), 2.13 (1H, m, H-11b), 2.26 (1H, d, J = 13.7 Hz, H-21b), 2.51 (1H, d, J = 15.9 Hz to H-19b), 6.31 (1H, d, J = 7.1 Hz, H-7), 6.49 (1H, s, H-1), 7.05 (1H, d, J = 7.0 Hz, H-6)

5) EIMS m / z 450 [M] ⁺ (100), 390 (100), 376 (80).

[Formula 2]

Pristimerine

1) Physical property: Orange crystal (melting point: 212 ~ 213 ℃)

2) Molecular Weight: 464

3) Molecular formula: C ₃₀ H ₄₀ O ₄

4) ¹ H-NMR (500 MHz, CDCl ₃ ) δ 0.53 (3H, s, H-27), 0.97 (1 H, m, H-22a), 1.10 (3H, s, H-28), I. 17 (3H, s, H-29), 1.24 (3H, s, H-26), 1.37 (IH, m, H-21 a), 1.45 (3H, s, H-25), 1.48 (IH, m , H-16a), 1.53 (lH, m, H-15a), 1.58 (lH, m, H-18), 1.64 (lH, m, H-15b), 1.67 (lH, m, H-12a), 1.69 (lH, m, H-19a), 1.78 (lH, m, H-12b), 1.82 (lH, m, H-lla), 1.86 (IH, m, H-16b), 2.05 (lH, ddd, J = 10.0, 10.0,4.0 Hz, H-22b), 2.17 (lH, m, H-llb), 2.20 (3H, s, H-23), 2.21 (lH, m, H-21b), 2.43 (lH , d, J = 15.8 Hz, H-19b), 3.55 (3H, s, -OCH,), 6.34 (IH, d, J = 7.2 Hz, H-7), 6.52 (lH, s, H-1) , 7.01 (lH, d, J = 7.1 Hz, H-6)

5) EIMS m / z 464 [M] < + > (100), 406 (100), 376 (80), 308 (70).

(3)

Iguesterin

1) Physical property: Orange crystal (melting point: 231 ~ 233 ℃)

2) Molecular Weight: 404

3) Molecular formula: C ₂₈ H ₃₆ O ₂

4) ¹ H-NMR (500 MHz, CDCl ₃ ) δ 0.94 (3H, s, H-27), 1.05 (3H, s, H-30), 1.15 (3H, S, H-28), 1.27 (3H , s, H-26), 1.32 (1H, m, H-16a), 1.57 (3H, s, H-25), 1.69 (1H, m, H-15a), 1.87 (1H, d, J = 8.2 Hz, H-18), 1.89 (1H, m, H-19), 1.91 (1H, d, J = 14.0 Hz, H-15b), 1.95 (1H, m, H-12), 1.98 (1H, d , J = 16.3 Hz, H-22b), 2.00 (1H, m, H-12), 2.10 (1H, d, J = 15.0 Hz, H-15b), 2.12 (1H, td, J = 14.7, 6.2 Hz , 11a), 2.20 (1H, d, J = 15.0 Hz, H-16a), 2.34 (3H, s, H-23), 2.46 (1H, m, 11b), 2.50 (1H, m, H-20) , 2.82 (1H, d, J = 14.1 Hz, H-22a), 6.48 (1H, d, J = 7.8 Hz, H-7), 6.51 (1H, d, J = 1.7 Hz, H-1), 6.89 (1H, s, 3-OH), 7.12 (1H, doublet of doublets, J = 6.8, 2.5 Hz, H-6).

5) EIMS m / z 464 [M] < + > (100), 406 (100), 376 (80), 308 (70).

[Formula 4]

Tingenone

1) Physical property: Orange crystal (melting point: 203 ~ 204 ℃)

2) Molecular Weight: 420

3) Molecular formula: C ₂₈ H ₃₆ O ₃

4) ¹ H NMR (CDCl ₃ , 500 MHz) d 0.98 (3H, s, H-27), 1.00 (3H, s, H-30), 1.02 (3H, S, H-28), 1.35 (3H, s, H-26), 1.46 (1H, m, H-16a), 1.51 (3H, s, H-25), 1.65 (1H, m, H-15a), 1.67 (1H, d, J = 7.2 Hz , H-18), 1.76 (1H, m, H-19), 1.81 (1H, d, J = 14. 0 Hz, H-15b), 1.84 (1H, m, H-12), 1.86 (1H, d, J = 15.1 Hz, H-22b), 1.87 (1H, m, H-12), 1.91 (1H, d, J = 14.0 Hz, H-15b), 2.02 (1H, td, J = 13.7, 5.8 Hz, 11a), 2.20 (1H, d, J = 15.0 Hz, H-16a), 2.23 (3H, s, H-23), 2.26 (1H, m, 11b), 2.50 (1H, m, H-20 ), 2.92 (1H, d, J = 15.1 Hz, H-22a), 6.38 (1H, d, J = 7.0 Hz, H-7), 6.52 (1H, d, J = 1.5 Hz, H-1), 6.98 (1H, s, 3-OH), 7.01 (1H, doublet of doublets, J = 7.0, 1.5 Hz, H-6).

5) EIMS m / z 420 [M] < + > (100), 404 (80), 376 (60).

[Chemical Formula 5]

22β-hydroxy-tingenone

1) Physical property: Red color crystal (melting point: 240 ~ 242 ℃)

2) Molecular Weight: 436

3) Molecular formula: C ₂₈ H ₃₆ O ₄

4) ¹ H NMR (CDCl ₃ , 500 MHz) d 0.87 (3H, s, H-28), 0.99 (3H, s, H-27), 1.07 (3H, d, J = 6.3 Hz, H-29) , 1.27 (lH, m, H-16a), 1.37 (3H, s, H-26), 1.51 (3H, s, H-25), 1.60 (lH, m, H-16b), 1.66 (IH, m , H-15a), 1.77 (IH, m, H-19a), 1.79 (lH, m, H-18), 1.85 (lH, m, H-15b), 1.88 (2H, m, H-12), 2.01 (lH, m, H-lla), 2.21 (IH, m, H-19b), 2.23 (3H, s, H-23), 2.26 (lH, m, H-llb), 2.67 (IH, m, H-20), 4.54 (lH, s, H-22), 6.37 (lH, d, J = 7.1 Hz, H-7), 6.53 (IH, s, H-1), 7.04 (lH, d, J = 7.0 Hz, H-6);

5) EIMS m / z 436 [M] < + > (100), 420 (78), 390 (50).

Example 3

HSP 27 expression inhibitory activity test

Five dishes were used to express human lung cancer cells (NCI-H1299, NCI-H460), osteosarcoma (Hos), and uterine cancer cells (HeLa), which express more HSP 27 than normal cells. bovine serum) and RPMI 1640 medium (GIBCO BRL) containing penicillin and streptomycin. Cultivation was carried out for one day in 37 ° C CO ₂ incubator divided into five 60 mm dishes. Each of the compounds of Chemical Formulas 1 to 5 obtained in Example 1 was administered and subjected to Western blotting tests by concentration and time. Human lung cancer cells were lysed with lysis buffer (lysis buffer: 20 mM Tris / HCl, pH 8.0, 0.1 mM EDTA, 1% SDS and 0.5 mg / ml proteinase K), followed by centrifugation to extract proteins, and SDS. Proteins were separated on PAGE and transferred to nitrocellulose membranes. After HSP 27 antibody was attached to the membrane and the secondary antibody (HRP) was attached to the secondary antibody (horseradish peroxide), the expression of HSP 27 was confirmed using ECL reagent (PerkinElmer Life Sciencec, Inc.). The results are shown in FIGS. 1 to 3.

1 is a diagram showing the results of Western blotting of HSP 27 of human lung cancer cells (NCI-H1299, NCI-H460), osteosarcoma (Hos), and uterine cancer cells (HeLa), and FIG. Each of the compounds of Chemical Formulas 1 to 5 obtained in 1 was treated to human lung cancer cells (H1299) (based on the concentration and time of treatment) and then the results of Western blotting of the expression amount of HSP 27. Figure 3 is a view showing the results of Western blotting of the HSP 27 expression of the compound of Formula 2 by the concentration, time for the human lung cancer cell line (H1299).

As can be seen in the results of Figures 1, 2, and 3, each of the compounds of Formulas 1 to 5, which is a quinonemethide compound isolated from the wakame tree, apoptosis, Alzheimer's, Parkinson's disease, And significantly reduce the expression of HSP 27, a major protein that controls Huntington's disease.

Example 4

Cytotoxicity test

In order to confirm the cytotoxic effects of the compounds of Formulas 1 to 5 obtained in Example 1, human lung cancer cells (NCI-H1299, NCI-H460), osteosarcoma (Hos), uterine cancer cells (HeLa), respectively MTT assays were performed on the subjects. Each chemical was added to a 6 well plate at 0, 0.5, 1, 1.5, 3, 5, 10 μM to confirm the viability of the cells after 2 days. The results are shown in FIG. According to the results of FIG. 4, it was shown that the compound of formula 2, pristinerin, had the best cytotoxic effect.

Example 5

In vivo animal testing

15 g of nude mice were divided into three groups, and human lung cancer cells (NCI-H1299 cells) were transplanted to the thighs of nude mice, respectively, so that the tumor size reached about 120-150 cm ³ . The first group was a control group, and the second group was dissolved in DMSO with prestimerin daily for 7 days by intratumor injection or intraperitoneal injection at a dose of 1 mg / kg, and the third group was also dissolved in cisplatin and DMSO. Daily administration at a dose of 1 mg / kg for 7 days by intratumoral injection. Each control and administration group was then monitored for tumor size without drug administration for 3 weeks. In each treatment group, intratumoral injection was measured at 7 day intervals, and intraperitoneal injection was measured at 3 day intervals. The results are shown in FIGS. 5 and 6, respectively. In addition, photographs 3 weeks after intratumoral injection of control and pristinerin are shown in FIG. 7.

As can be seen in Figures 5 to 7, it can be seen that the size of the cancer tissue is significantly reduced in the group administered with the pre-stimerin. In addition, as the number of days of culture of nude mice increased, the tumor size increased rapidly in the group transplanted with lung cancer cells only. The size of tumor in the group treated with pristine in the nude mouse transplanted with lung cancer cells increased. Increased slowly. In addition, according to the results shown in FIG. 5, the pristinerin has a tumor therapeutic effect similar to that of cisplatin used as a conventional anticancer agent. Thus, pristinerins have been observed to effectively treat tumors at the cultured cell level as well as in animal studies using human cancer cell transplanted mice.

1 is a diagram showing the results of Western blotting on the degree of expression of HSP 27 in each of various cancer cells.

2 is a view showing the results of Western blotting the expression level of HSP 27 by treating each of the quinonemethide-based compounds isolated from wakaken bark in accordance with the method of the present invention in human lung cancer cell line (H1299).

Figure 3 is a view showing the results confirmed by Western blotting the degree of expression of HSP by treatment with concentration, time by human lung cancer cell line (H1299) of the pristine rinse isolated from Wakame tree according to the method of the present invention.

4 is a table showing the results of the cytotoxic effects of various quinone-methoxide-based compounds isolated from wakaken tree according to the method of the present invention measured by MTT assay.

Figure 5 is implanted human lung cancer cell line (H1299) into the thigh of the mouse already tumor-forming mice, prestimulant isolated from Wakame tree in accordance with the method of the present invention for 7 days after intratumoral injection, the tumor size The change observed every 7 days is a graph showing the results of the negative control group and the positive control group (cisplatin administration group).

Figure 6 is implanted human lung cancer cell line (H1299) into the thigh of the mouse already tumor-formed mice, intraperitoneal injection of pristinerin isolated from Wakame tree according to the method of the present invention for 7 days, and then the size of the tumor The result of observing every 3 days is a graph with a negative control.

Figure 7 is implanted human lung cancer cell line (H1299) into the thigh of the mouse already tumor-forming mice, intratumoral injection of pristinerin isolated from Wakame tree in accordance with the method of the present invention at 1 mg / Kg daily for 7 days Three weeks after the change in tumor size was taken.

Claims (12)

A composition for the prevention or treatment of diseases selected from the group consisting of Alzheimer's, Parkinson's disease, and Huntington's disease, including Wakame tree ( Tripterygium regelii ) as an active ingredient. delete The composition of claim 1, wherein the wakame tree is contained as a pulverized product of the herbal medicine itself, a dry pulverized product of the herbal medicine, or a crude extract of a C ₁ -C ₄ alcohol solvent. 4. The composition according to claim 3, wherein the wakame tree is contained as an chloroform extract of the crude extract of the C ₁ -C ₄ alcohol solvent. A composition for the prophylaxis or treatment of a disease selected from the group consisting of Alzheimer's, Parkinson's disease, and Huntington's disease comprising as an active ingredient at least one compound selected from the group consisting of compounds of Formulas 1 to 5 or a pharmaceutically acceptable salt thereof: [Formula 1]

[Formula 2]

(3)

[Formula 4]

[Chemical Formula 5]

delete The composition of claim 5, wherein the compound of Chemical Formulas 1 to 5 is extracted from Wakame tree. delete A disease selected from the group consisting of Alzheimer's, Parkinson's disease, and Huntington's disease, comprising the composition according to any one of claims 1, 3 to 5, and 7 and a pharmaceutically acceptable carrier or additive. Prophylactic or therapeutic medicines. The pharmaceutical product according to claim 9, which is in the form of powder, granule, tablet, capsule, suspension, emulsion, syrup, liquid, aerosol, extract, injection, transdermal or suppository. A dietary supplement comprising the composition according to any one of claims 1, 3 to 5, and 7, and a food acceptable carrier or additive. The dietary supplement according to claim 11, which is in the form of powder, granule, tablet, capsule, suspension, emulsion, syrup, liquid, aerosol, extract, tea, jelly, or beverage.

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