KR20090087684A - Composition for preventing or treating a disease mediated by overexpression of heat shock protein 27 - Google Patents
Composition for preventing or treating a disease mediated by overexpression of heat shock protein 27 Download PDFInfo
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- KR20090087684A KR20090087684A KR1020080013085A KR20080013085A KR20090087684A KR 20090087684 A KR20090087684 A KR 20090087684A KR 1020080013085 A KR1020080013085 A KR 1020080013085A KR 20080013085 A KR20080013085 A KR 20080013085A KR 20090087684 A KR20090087684 A KR 20090087684A
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Abstract
Description
본 발명은 미역줄나무(Tripterygium regelii)를 포함하는 HSP 27(Heat Shock Protein 27) 발현 억제용 조성물에 관한 것으로, 보다 구체적으로는 미역줄나무, 그 추출물, 또는 미역줄나무 추출물로부터 분리된 화합물을 활성성분으로서 포함하는 HSP 27의 과발현에 의해 매개되는 질병의 의 예방 또는 치료용 조성물에 관한 것이다.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.
저분자 열충격 단백질 HSP 27은 활성산소(free radical), 열(heat), 독소(toxins)와 같은 외부환경적인 요인에 대하여 스스로 군집을 형성하여 이러한 스트레스에 방어 능력을 가지는 샤페론(Charperon) 기능을 가지는 저분자량을 가지는 단백질이다. 현재 사용되고 있는 항암제의 문제점은 치료시 의도하지 않은 정상세포의 사멸로 인한 부작용과 암세포의 항암제에 대한 내성의 발생이다. 본 발명자들의 연구 및 다른 여러 연구에 따르면, 여러 암세포의 항암제 내성의 주된 원인 중에 하나가 정상세포에 그 양이 매우 적으나 암세포에서 많이 발현되고 있는 저분 자열충격단백질 때문인 것으로 나타났다(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). 이러한 HSP 27에 의해 내성이 유도되는 암 질환으로는 폐암, 골육종, 자궁암, 유방암등이 보고되고 있다(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). 따라서, 저분자 열충격단백질 HSP 27의 발현을 저해하는 것이 HSP 27 저항성 암 질환의 새로운 예방 또는 치료법인 것으로 알려져 있다. 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
또한, 신경조직의 퇴행성질환인 알츠하이머, 파킨슨병, 및 헌팅톤병에서 공통적으로 저분자열충격단백질 HSP 27의 발현이 비정상적으로 높게 나타나는 것으로 보고되었다. 따라서, 이러한 HSP 27의 발현을 억제하는 방법이 암 이외에도 알츠하이머, 파킨슨병, 및 헌팅톤병과 같은 신경질환의 예방 및 치료에도 효과가 있는 것으로 보고되고 있다(M. M. M. 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). 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).
현재, HSP 27과 다른 분자량의 열충격단백질에 대해서는 연구가 활발이 이루어지고 있다. 예를 들어, 분자량이 90,000인 열충격단백질(HSP90)에 대한 저해활성을 가지는 17AAG(17 allyl amino geldanamycin)은 이미 2상 임상시험에 들어가 있고, 분자량이 70,000인 열충격단백질 HSPA (HSP70)에 대한 저해제 개발도 활발히 진행되고 있다. 그러나, HSP 27(예를 들면, HSPB1)에 대한 저해제는 현재 개발되고 있는 것이 거의 없는 실정이다. 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
한편, 미역줄나무는 노박덩쿨과(Celastraceae)에 속하는 낙엽활엽 만경목으로 전 세계적으로 우리나라와 일본, 중국북부에 분포한다. 미역줄나무는 관상용, 약용으로 쓰이고, 한방과 민간에서는 뿌리와 줄기를 염증, 소염, 해독 등에 약재로 이용하고 있다. 미역줄나무의 뿌리와 줄기에서 분리, 보고된 성분으로는 세스퀴테르페노이드, 디테르노이드, 트리테르페노이드, 퀴논메차이드 등이 있다(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). 그러나, 아직까지 상기 미역줄나무에 함유된 화합물들이 저분자 열충격단백질 HSP 27의 발현에 미치는 영향에 대해서는 발표된 바는 없다.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.
이에 본 발명자들은 저분자열충격단백질 HSP 27의 과발현에 의해 매개되는 질병을 예방 또는 치료할 수 있는 약물을 개발하기 위해, 저분자열충격단백질 HSP 27의 발현을 저해할 수 있는 저해제에 대해 탐색한 결과, 식물 생약유래의 화합물이 HSP 27의 발현 억제효과가 있음을 확인하여, 본 발명을 완성하게 되었다.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.
따라서, 본 발명의 목적은 HSP 27의 과발현에 의해 매개되는 질병을 예방 또는 치료할 수 있는 식물 생약 유래의 조성물을 제공하는 것이다.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.
본 발명의 다른 목적은 식물 생약 유래의 화합물을 포함하는 HSP 27의 과발현에 의해 매개되는 질병을 예방 또는 치료할 수 있는 조성물을 제공하는 것이다.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.
본 발명의 또 다른 목적은 식물 생약으로부터 HSP 27의 발현 억제 활성을 갖는 화합물을 분리하는 방법을 제공하는 것이다.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.
본 발명의 또 다른 목적은 상기 조성물을 포함하는 의약품을 제공하는 것이다.Still another object of the present invention is to provide a medicine comprising the composition.
본 발명의 또 다른 목적은 상기 조성물을 포함하는 건강보조식품을 제공하는 것이다. Another object of the present invention to provide a dietary supplement comprising the composition.
상기 목적을 달성하기 위하여, 본 발명은 미역줄나무를 활성성분으로서 포함하는 HSP 27 과발현에 의해 매개되는 질병의 예방 또는 치료용 조성물을 제공한다.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.
또한, 본 발명은 하기 화학식 1 내지 5의 화합물로 구성된 그룹에서선택된 하나 이상의 화합물 또는 약제학적으로 허용 가능한 그의 염을 활성성분으로서 포함하는 HSP 27 과발현에 의해 매개되는 질병의 예방 또는 치료용 조성물을 제공한다: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
또한, 본 발명은 상기 화학식 1 내지 5의 화합물로 구성된 그룹에서선택된 하나 이상의 화합물을 미역줄나무로부터 분리하는 방법을 제공한다.The present invention also provides a method for separating one or more compounds selected from the group consisting of the compounds of
또한, 본 발명은 상기 조성물을 포함하는 의약품을 제공한다. The present invention also provides a medicine comprising the composition.
또한, 본 발명은 상기 조성물을 포함하는 건강보조식품을 제공한다.In addition, the present invention provides a health supplement comprising the composition.
이하, 본 발명을 보다 상세하게 설명한다. Hereinafter, the present invention will be described in more detail.
본 발명자들은 HSP 27의 발현을 억제하는 활성을 갖는 식물 생약을 발굴하기 위해 다양한 식물 생약에 대한 연구를 수행한 결과, 미역줄나무 추출물이 탁월한 HSP 27 발현 억제활성을 갖는다는 것을 발견하게 되었다. 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.
따라서, 본 발명은 일 측면에 있어서, 미역줄나무를 활성성분으로서 포함하는 HSP 27의 과발현에 의해 매개되는 질병의 예방 또는 치료용 조성물을 제공한다.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.
상기 조성물의 활성성분인 미역줄나무는 추출물의 형태로 포함되거나, 생약 자체의 분쇄물 또는 생약의 건조 분쇄물로서 포함될 수도 있다.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.
상기 미역줄나무의 추출물은 C1-C4 알콜 용매의 조추출물일 수 있으며, 바람직하게는 에탄올의 추출물이며, 더욱 바람직하게는 70 내지 100%의 에탄올 추출물 일 수 있다. 용매로 추출 시, 추출은 가열 추출, 냉침 추출, 환류냉각 추출, 또는 초음파 추출 등이 이용될 수 있다. 이러한 추출은 실온에서 수행할 수도 있으며, 가온 조건 하에서 수행할 수도 있다. 바람직하게는, 미역줄나무의 음건 세절물에 에탄올을 넣고 5일 이상 실온에서 보관한 다음 거름종이로 여과하여 얻을 수 있다. 상기 추출은 활성성분을 보다 다량 수득하기 위해 1 회 이상 여러 번 추출할 수 있으며, 바람직하게는 3 회 연속추출하여 합한 추출액을 이용할 수 있다. 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.
본 발명에 따른 미역줄나무을 포함하는 조성물은 미역줄나무을 상기와 같이 조추출물로서 포함할 수도 있으나, 상기 조추출물을 비극성 유기 용매로 더욱 추출하여 얻어진 비극성 용매의 가용성 추출물로서 포함할 수도 있다. 상기 비극성 용매의 가용성 추출물은 상기 C1-C4 알콜 용매의 조추출물을 물로 현탁시킨 다음, 헥산, 에틸 아세테이트, 클로로포름의 순서로 분별추출하여 얻어진 클로로포름 가용성 분획을 이용하는 것이 바람직하다. 상기 분별추출은 통상의 분별 추출 방법을 이용할 수 있으며, 바람직하게는 분별 깔대기를 사용할 수 있다. 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.
상기한 바와 같은 방법에 의해 추출한 추출물은 그대로 사용할 수도 있으나, 농축한 엑스 형태로 사용될 수도 있고, 농축한 다음 동결 건조하여 동결건조물의 형태로서 사용될 수도 있다. 바람직하게는, 충분한 농도로 사용될 수 있도록 농축된 형태로서 사용한다. 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.
상기 클로로포름 가용성 분획을 크로마토그래피에 의한 정제를 더욱 수행하여 미역줄나무로부터 HSP 27의 발현 억제활성을 갖는 구체적인 활성성분을 분리해 낼 수 있다. The chloroform soluble fraction may be further purified by chromatography to separate specific active ingredients having HSP 27 expression inhibiting activity from Wakame tree.
상기 크로마토그래피는 실리카겔 크로마토그래피를 사용할 수 있으며, 이동상으로서 클로로포름, 클로로포름·아세톤 혼합용매, 및 메탄올의 순서로 하여 정제를 수행할 수 있다. 그리하여 얻어진 조분획물 중의 일부를 이동상으로서 n-헥산·아세톤 혼합용매를 이용하여 실리카겔 크로마토그래피를 추가적으로 수행함으로써 하기 화학식 1 및 2의 화합물을 포함하는 분획물을 얻을 수 있으며, 이동상으로서 n-헥산·아세톤 혼합용매를 이용하여 실리카겔 크로마토그래피를 더욱 수행함으로써 하기 화학식 1 및 2의 화합물을 각각 분리할 수 있다. 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
상기 얻어진 조분획물 중의 또 다른 일부를 이동상으로서 클로로포름·아세톤 혼합용매를 이용하여 실리카겔 크로마토그래피를 추가적으로 수행함으로써 하기 화학식 3 내지 5의 화합물을 포함하는 분획물을 얻을 수 있으며, 이동상으로서 n-헥산·아세톤 혼합용매, 클로로포름·아세톤 혼합용매, 또는 n-헥산·에틸아세테이트 혼합용매를 이용하여 실리카겔 크로마토그래피를 더욱 수행함으로써 하기 화학식 3 내지 5의 화합물을 각각 분리해 낼 수 있다.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
상기 이동상으로서 사용되는 n-헥산·아세톤 혼합용매는 50:1 내지 1:2 부피비가 바람직하며, 클로로포름·아세톤 혼합용매는 (부피비 범위 기재)가 바람직하 고, n-헥산·에틸아세테이트 혼합용매는 60:1 내지 1:2의 부피비가 바람직하다. 상기 크로마토그래피는 단일 화합물이 정제될 때까지 1 회 내지 수회에 걸쳐 수행할 수 있으며, 필요에 따라 농축, 재결정을 수행할 수도 있다. 그리하여 분리된 화합물은 분자량 측정, NMR 측정 등의 방법으로 분석할 수 있으며, 하기 화학식 1(celastrol), 화학식2(pristimerine), 화학식 3(iguestrein), 화학식 4(tingenone), 화학식 5(22-β-hydroxy tingenone)의 화합물이 분리되었다: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:
[화학식 1][Formula 1]
[화학식 2][Formula 2]
[화학식 3][Formula 3]
[화학식 4][Formula 4]
[화학식 5][Formula 5]
따라서, 본 발명의 다른 측면에서 있어서, 본 발명은 Thus, in another aspect of the invention, the invention
미역줄나무를 C1-C4 알콜 용매로 추출하여 조추출물을 획득하는 단계; Extracting brown seaweed from C 1 -C 4 alcohol solvent to obtain a crude extract;
상기 조추출물을 물로 현탁시킨 다음, 헥산, 에틸아세테이트, 클로로포름의 순서로 분별추출하여 클로로포름 가용성 분획을 획득하는 단계; 및Suspending 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
상기 클로로포름 가용성 분획을 크로마토그래피를 수행하여 정제하는 단계를 수행하는 미역줄나무로부터 상기 화학식 1 내지 5의 화합물로 구성된 그룹에서 선택된 화합물을 분리하는 방법을 제공한다. Provided is a method for separating a compound selected from the group consisting of the compounds of
상기 각각 분리된 화합물은 HSP 27 억제 활성에 대해 시험한 결과, 각각은 우수한 HSP 27 발현 억제 활성을 갖는 것으로 확인되었다. Each of the isolated compounds was tested for HSP 27 inhibitory activity, and each was found to have excellent HSP 27 expression inhibitory activity.
따라서, 본 발명의 또 다른 측면에서 있어서, 본 발명은 상기 화학식 1 내지 5의 화합물로 구성된 그룹에서 선택된 하나 이상의 화합물 또는 약제학적으로 허용 가능한 그의 염을 활성성분으로서 포함하는 HSP 27의 과발현에 의해 매개되는 질병의 예방 또는 치료용 조성물을 제공한다. 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
상기 미역줄나무 또는 화학식 1 내지 5의 화합물로 구성된 그룹에서 선택된 화합물이나 약제학적으로 허용 가능한 그의 염을 활성성분으로 하는 본 발명의 조성물은 HSP 27의 과발현에 의해 매개되는 질병의 예방 또는 치료에 이용될 수 있다. 상기 HSP 27의 과발현에 의해 매개되는 질병이란 HSP 27 발현의 억제 활성이 질병의 치료 또는 예방에 유익할 수 있는 임의의 질병을 말한다. 이러한 질병으로는 HSP 27 저항성 암질환, 알츠하이머, 파킨슨병, 및 헌팅톤병 등이 있으나, 이에 한정되는 것은 아니다. 상기 HSP 27 저항성 암질환이란 HSP 27의 발현에 의해 내성을 나타내는 임의의 암질환을 말하며, 예를 들어 폐암, 골육종, 자궁암, 유방암 등이 있으나, 이에 한정되는 것은 아니다. 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
본 발명에 따른 조성물이 HSP 27 발현 억제 활성이 있다는 것은 하기 실시예에서 입증하였다. 구체적으로는, 본 발명의 분리방법의 일 구현예에 따라 미역줄 나무의 추출물에서 분리된 상기 화학식 1 내지 5의 화합물 각각을 HSP 27이 과발현된 암세포에 대해 처리하여 HSP 27의 발현이 억제되는 정도를 확인하였다. 또한 HSP 27에 대한 내성을 갖는 것으로 알려진 폐암세포를 누드 마우스이 대퇴부에 이식한 다음 상기 화학식 2의 프리스티머린 화합물을 생체내로 투여하여, 대조군 및 시스플라틴 투여 그룹과 비교한 결과 대조군에 비해 현저한 정도로 암 조직의 크기가 느리게 증가하였으며, 시스플라틴 투여군과 거의 유사한 정도의 효과를 갖는 것으로 나타났다. 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
상기 본 발명에 따른 미역줄나무 또는 화학식 1 내지 5의 화합물로 구성된 그룹에서 선택된 화합물이나 약제학적으로 허용 가능한 그의 염을 활성성분으로서 포함하는 HSP 27의 과발현에 의해 매개되는 질병의 예방 또는 치료용 조성물은 HSP 27의 과발현에 의해 매개되는 질병의 치료 또는 예방을 위한 의약품의 원료로서 사용될 수 있다. 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
따라서, 본 발명은 또 다른 일 측면에 있어서, 본 발명은 상기 미역줄나무 또는 화학식 1 내지 5의 화합물로 구성된 그룹에서 선택된 화합물이나 약제학적으로 허용 가능한 그의 염을 포함하는 조성물, 및 약제학적으로 허용 가능한 담체 또는 첨가제를 포함하는 의약품을 제공한다.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
상기 의약품은 당해 기술분야에 공지되어 있는 통상적인 약제학적 제형으로 제제화될 수 있다. 상기 의약품은 경구투여제제, 주사제, 좌제, 경피투여제제, 및 경비투여제제를 포함하지만, 이에 한정되지 않는 임의의 제형으로 제제화되어 투여될 수도 있으나, 바람직하게는 액제, 현탁제, 산제, 과립제, 정제, 캡슐제, 환제, 또는 엑스제와 같은 경구 투여용 제형으로 제제화될 수 있다. 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.
상기 각각의 제형으로 제제화 시, 각각의 제형의 제조에 필요한 약제학적으로 허용 가능한 담체 또는 첨가제를 부가하여 제조할 수 있다. 대표적으로 경구 투여용 제형으로 제제화 시 상기 담체로서 희석제, 활택제, 결합제, 붕해제, 감미제, 안정제, 및 방부제 중에서 1 종 이상을 선택하여 사용할 수 있으며, 첨가제로는 향료, 비타민류, 및 항산화제 중에서 1 종 이상을 선택하여 사용할 수 있다.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.
상기 담체 및 첨가제는 약제학적으로 허용 가능한 것은 모두 가능하며, 구체적으로 희석제로는 유당, 옥수수 전분, 대두유, 미정질 셀룰로오스, 또는 만니톨, 활택제로는 스테아린산 마그네슘 또는 탈크, 결합제로는 폴리비닐피롤리돈 또는 히드록시프로필셀룰로오스가 바람직하다. 또한, 붕해제로는 카르복시메틸셀룰로오스 칼슘, 전분글리콜산나트륨, 폴라크릴린칼륨, 또는 크로스포비돈, 감미제로는 백당, 과당, 솔비톨, 또는 아스파탐, 안정제로는 카르복시메틸셀룰로오스나트륨, 베타-사이클로덱스트린, 백납, 또는 잔탄검, 방부제로는 파라옥시안식향산메틸, 파라옥시안식향산프로필, 또는 솔빈산칼륨이 바람직하다. The carrier and the additive may be any pharmaceutically acceptable, and specifically, a diluent may include lactose, corn starch, soybean oil, microcrystalline cellulose, or mannitol, and a lubricant may include magnesium stearate or talc, and a binder may be polyvinylpyrrolidone. 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.
또한, 상기 성분 이외에도 공지의 첨가제로서 미각을 돋구기 위하여, 매실향, 레몬향, 파인애플향, 허브향 등의 천연향료, 천연과즙, 클로로필린, 플라보노이드 등의 천연색소, 과당, 벌꿀, 당알코올, 설탕과 같은 감미성분, 또는 구연산, 구연산 나트륨과 같은 산미제를 혼합하여 사용할 수도 있다. 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.
상기 의약품은 HSP 27 저항성 암질환, 알츠하이머, 파킨슨병, 및 헌팅톤병을 포함한 HSP 27 발현 억제활성이 효과가 있는 질병의 예방 또는 치료 효과를 얻기 위하여, 유효성분으로서 성인을 기준으로 1 일 총 투여량이 추출물 또는 화합물로서 0.5mg/kg - 3 mg/kg, 생약으로서 10mg/kg - 50 mg/kg이 되도록 임의로 수회 나누어서 투여할 수 있다. 상기 투여량은 투여 경로, 질병의 진행 정도, 성별, 나이, 체중 등에 따라 적절히 증감될 수 있다.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.
또한, 본 발명에 따른 미역줄나무 또는 화학식 1 내지 5의 화합물로 구성된 그룹에서 선택된 화합물을 활성성분으로 하는 HSP 27의 과발현에 의해 매개되는 질병의 예방 또는 치료용 조성물은 상기 HSP 27의 과발현에 의해 매개되는 질병의 예방 또는 치료에 효과가 있는 건강보조식품의 원료서 사용될 수 있다. 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
따라서, 본 발명의 또 다른 일 측면에 있어서, 본 발명은 상기 미역줄나무 또는 화학식 1 내지 5의 화합물로 구성된 그룹에서 선택된 화합물이나 약제학적으로 허용 가능한 그의 염을 포함하는 조성물, 및 식품학적으로 허용 가능한 담체 또는 첨가제를 포함하는 건강보조식품을 제공한다.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
이러한 건강보조식품은 당해 기술분야에 공지되어 있는 통상적인 건강보조식품의 제형으로 제제화될 수 있다. 상기 건강보조식품은 예를 들어 산제, 과립제, 정제, 캅셀제, 현탁액, 에멀젼, 시럽제, 액제, 엑스제, 차, 젤리, 엑스, 또는 음료 등으로 제조될 수 있다. 상기 식품학적으로 허용 가능한 담체 또는 첨가제로는 제조하고자 하는 제형의 제조에 당해 기술분야에서 사용 가능한 것으로 공지되어 있는 임의의 담체 또는 첨가제가 이용될 수 있다. 이와 같이 다양한 형태로 가공된 본 발명의 건강보조식품은 HSP 27 과발현에 의해 매개되는 각종 질환의 예방에 사용될 수 있을 뿐만 아니라, 복용이 용이하여 편리하게 사용될 수 있다. 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.
또한, 현재 미역줄나무의 어린 순은 나물로서 식용되고 있는 것이어서, 이로부터 유래된 미역줄나무 추출물 또는 화학식 1 내지 5의 화합물 역시 독성 또는 부작용의 문제가 없다고 볼 수 있으므로, 본 발명에 따른 의약품 및 건강보조식품은 질병의 예방 또는 치료 목적으로 장시간 사용 시에도 안심하고 사용할 수 있는 장점이 있다. In addition, the young sprouts of seaweeds are currently edible as herbs, so the seaweeds extract or the compounds of
앞서 설명한 바와 같이, 본 발명에 따른 미역줄나무 또는 상기 화학식 1 내지 5의 화합물로 구성된 그룹에서 선택된 화합물이나 약제학적으로 허용 가능한 그의 염을 활성성분으로서 포함하는 조성물은 HSP 27 발현의 억제 활성이 탁월하여 HSP 27의 과발현에 의해 매개되는 각종 질환, 즉 암, 알츠하이머, 파킨슨, 헌팅톤병과 같의 질환의 예방 또는 치료에 이용될 수 있다. 또한, 안전성이 입증된 식물 생약을 원료로 함으로써 종래 항암제나 신경질환 약물이 나타낼 수 있는 각종 부작용을 해소할 수 있는 장점이 있다. 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
이하, 본 발명을 하기 실시예에 의해 더욱 구체적으로 설명한다. 그러나, 이들 실시예는 본 발명에 대한 이해를 돕기 위한 것일 뿐, 어떤 의미로든 본 발명의 범위가 이들에 의해 제한되는 것은 아니다. 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.
실시예 1Example 1
미역줄나무로부터 화합물의 추출, 분리 및 정제Extraction, Separation and Purification of Compounds from Seaweed
건조된 미역줄나무의 뿌리 및 줄기(경상남도 지리산) 4.0 kg을 95% 에탄올 20ℓ에 넣어 실온에서 10 일 동안 보관한 후 여과지로 여과한 다음, 감압농축시켜 흑갈색의 추출물 780 g을 얻었다. 여기에 증류수 4 ℓ를 넣어 현탁시키고, 헥산, 에틸아세테이트, 클로로포름, 부탄올의 순으로 분별 추출하여 그중 클로로포름 분획물 210 g을 얻었다. 얻어진 클로로포름 분획물은 실리카겔 컬럼크로마토그래피(3 kg, 240 ~ 400 메쉬, Merk)를 수행하였으며, 이동상 용매로서 클로로포름, 클로로포름:아세톤(200:1 ~ 1:7 혼합비) 혼합용매, 및 메탄올을 순차적으로 사용하여 24개의 분획(분획물 1-1 ~ 24)을 얻었다. 그중 분획물 2 - 10(5.2 g)을 실리카겔(80 g, 70 ~ 230 메쉬)에 흡착시켜, 실리카겔 컬럼크로마토그래피(1 kg, 230 ~ 400 메쉬)를 수행하였으며, 이때 이동상 용매로서 n-헥산:아세톤의 혼합용매를 사용하여 분획물 1-2-1 ~ 18을 얻었다. 이중에서 소분획인 Fr.1-2-1 ~ 12은 n-헥산:아세톤(15:1 부피비)의 혼합용매를 이동상 전개용매로 사용하여 두 번째 컬럼크로마토그래피(20 g, 230 ~ 400 메쉬)를 수행하여 화학식 1의 화합물(240 mg), 화합물 2의 화합물(300 mg)을 각각 획득하였다. 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 resulting chloroform fractions were 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. Was carried out to obtain a compound of formula 1 (240 mg), and a compound of compound 2 (300 mg), respectively.
또한, 분획물 24 개중 분획물 13-17(6.2 g)을 실리카겔(100 g, 70 - 230 메쉬)에 흡착시키고, 실리카겔 컬럼크로마토그래피(1.4 kg, 230 ~ 400 메쉬)를 수행하였으며, 이때 이동상 용매로서는 클로로포름:아세톤의 혼합용매를 사용하여 분획물 2-1-1 ~ 24를 얻었다. 이중에서 소분획인 Fr.2-1-1 ~ 11은 n-헥산:아세톤(10:1 부피비)의 혼합용매를 이동상 전개용매로 사용하여 두 번째 컬럼크로마토그래피(30 g, 230 ~ 400 메쉬)를 수행하여 화학식 3의 화합물(105 mg)을 얻었고, 소분획물 Fr.2-1-12 - Fr 2-1-19 은 클로로포름: 아세톤(5:1 부피비)의 혼합용매를 이동상 전개용매로 사용하여 두 번째 컬럼크로마토그래피(10 g, 230 - 400 메쉬)를 수행하여 화학식 4의 화합물(90 mg)을 각각 얻었다. 마지막으로 Fr. 2-1-20 - Fr. 2-1-23은 n-헥산:에틸아세테이트(8:1, 부피비)의 혼합용매를 이동상 전개용매로 사용하여 두 번째 컬럼크로마토그래피(23 g, 230 - 400 메쉬)를 수행하여 화학식 5의 화합물(30 mg)을 얻었다. 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.
실시예 2Example 2
천연물로부터 분리한 화합물의 구조 분석Structural Analysis of Compounds Isolated from Natural Products
상기 실시예 1에서 얻어진 각각의 물질에 대해 VG 고분해능 GC/MS 분광기(VG high resolution GC/MS spectrometer, Election Ionization MS, JEOLJMS-700 )를 사용하여 분자량 및 분자식을 결정하였다. 또한, 편광기(Perkin-Elmer polarimeter 343 )를 사용하여 선광도를 측정하였으며, 핵자기공명 분석(Bruker Bruker IFS 66)을 통하여 1H NMR, 13C NMR, 호모-코지(HOMO-COSY), HMQC(1H-Detected heteronuclear Multiple-Quantum Coherence), HMBC(Heteronuclear Multiple-Bond Coherence), DEPT(Distortionless Enhancement by Polarization) 스펙트럼을 얻고, 분자구조를 결정하였다. 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.
그 결과 얻어진, 각각의 화합물의 물성, 분자량, 분자식, NMR 분석, 및 질량분광분석 결과를 아래에 나타내었다.The physical properties, molecular weight, molecular formula, NMR analysis, and mass spectrometry of each compound obtained as a result are shown below.
[화학식 1][Formula 1]
CelastrolCelastrol
1) 물성 : 붉은색 결정체(녹는점 : 203 ~ 205 ℃)1) Physical property: Red crystal (melting point: 203 ~ 205 ℃)
2) 분자량 : 4502) Molecular Weight: 450
3) 분자식 : C29H38O4 3) Molecular formula: C 29 H 38 O 4
4) 1H NMR (CDCl3, 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 ~ 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)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).5) EIMS m / z 450 [M] + (100), 390 (100), 376 (80).
[화학식 2][Formula 2]
Pristimerine Pristimerine
1) 물성 : 오렌지색 결정체(녹는점 : 212 ~ 213 ℃)1) Physical property: Orange crystal (melting point: 212 ~ 213 ℃)
2) 분자량 : 4642) Molecular Weight: 464
3) 분자식 : C30H40O4 3) Molecular formula: C 30 H 40 O 4
4) 1H-NMR(500 MHz, CDCl3) 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)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).5) EIMS m / z 464 [M] < + > (100), 406 (100), 376 (80), 308 (70).
[화학식 3][Formula 3]
IguesterinIguesterin
1) 물성 : 오렌지색 결정체(녹는점 : 231 ~ 233 ℃)1) Physical property: Orange crystal (melting point: 231 ~ 233 ℃)
2) 분자량 : 4042) Molecular Weight: 404
3) 분자식 : C28H36O2 3) Molecular formula: C 28 H 36 O 2
4) 1H-NMR(500 MHz, CDCl3) 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, dd, J = 6.8, 2.5 Hz, H-6).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).5) EIMS m / z 464 [M] < + > (100), 406 (100), 376 (80), 308 (70).
[화학식 4][Formula 4]
TingenoneTingenone
1) 물성 : 오렌지색 결정체(녹는점 : 203 ~ 204 ℃)1) Physical property: Orange crystal (melting point: 203 ~ 204 ℃)
2) 분자량 : 4202) Molecular Weight: 420
3) 분자식 : C28H36O3 3) Molecular formula: C 28 H 36 O 3
4) 1H NMR (CDCl3, 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, dd, J = 7.0, 1.5 Hz, H-6).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).5) EIMS m / z 420 [M] < + > (100), 404 (80), 376 (60).
[화학식 5][Formula 5]
22β-hydroxy-tingenone22β-hydroxy-tingenone
1) 물성 : 붉은색 결정체(녹는점 : 240 ~ 242 ℃)1) Physical property: Red color crystal (melting point: 240 ~ 242 ℃)
2) 분자량 : 4362) Molecular Weight: 436
3) 분자식 : C28H36O4 3) Molecular formula: C 28 H 36 O 4
4) 1H NMR (CDCl3, 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=7.1Hz, H-7), 6.53 (IH, s, H-1), 7.04 (lH, d, J= 7.0 Hz, H-6);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).5) EIMS m / z 436 [M] < + > (100), 420 (78), 390 (50).
실시예 3Example 3
HSP 27 발현 억제활성 시험 HSP 27 expression inhibitory activity test
5개의 디쉬를 사용하여 정상세포에 비해 HSP 27의 발현이 많은 인체 폐암세포(NCI-H1299, NCI-H460), 골육종세포(Osteosarcoma)(Hos), 자궁암세포(HeLa)를 10%의 FBS(fetal bovine serum)와 페니실린 및 스트렙토마이신이 함유된 RPMI 1640 배지(GIBCO BRL) 중에서 배양하였다. 배양은 5개의 60 mm 디쉬(dish)에 나누어 37℃의 CO2 배양기에서 하루동안 실시하였다. 상기 실시예 1에서 얻어진 화학식 1 내지 5의 화합물 각각을 투여하고 농도별, 시간별 웨스턴 블럿팅(Western Blotting) 시험을 하였다. 인체 폐암세포를 세포용해용 완충액(lysis buffer: 20mM Tris/HCl, pH 8.0, 0.1 mM EDTA, 1% SDS 및 0.5 mg/ml 프로티나아제 K)으로 용해시킨 후 원심분리하여 단백질을 추출하고, SDS PAGE에서 단백질을 분리하여 니트로셀룰로오스막으로 전이시켰다. HSP 27 항체를 막에 부착시키고 HRP(horseradish peroxide)가 연결된 2차 항체(secondary antibody)를 결합시킨 후 ECL시 약(PerkinElmer Life Sciencec, Inc.)을 사용하여 HSP 27의 발현을 확인하였다. 그 결과를 도 1 내지 도 3에 나타내었다. 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
도 1은 인체폐암세포(NCI-H1299, NCI-H460), 골육종세포(Osteosarcoma)(Hos), 자궁암세포(HeLa) 각각의 HSP 27의 웨스턴 블랏팅 결과를 나타낸 도면이고, 도 2는 상기 실시예 1에서 획득한 화학식 1 내지 5의 화합물 각각을 인체폐암세포(H1299)에 처리한 다음(처리농도 및 시간 기재) HSP 27의 발현양을 웨스턴 블랏팅한 결과를 나타낸 도면이다. 도 3은 상기 화학식 2의 화합물의 HSP 27의 발현 억제활성을 인체폐암세포주(H1299)에 대해 농도별, 시간별로 웨스턴 블럿팅한 결과를 나타낸 도면이다. 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
도 1, 도 2, 및 도 3의 결과에서 확인할 수 있는 바와 같이, 상기 미역줄나무로부터 분리된 퀴논메차이드계 화합물인 화학식 1 내지 5의 화합물 각각은 세포사멸(apoptosis), 알츠하이머, 파킨슨병, 및 헌팅톤 질환을 조절하는 주요 단백질인 HSP 27의 발현을 현저히 감소시킨다는 것을 알 수 있다.As can be seen in the results of Figures 1, 2, and 3, each of the compounds of
실시예 4Example 4
세포 독성 시험Cytotoxicity test
상기 실시예 1에서 얻어진 화학식 1 내지 5의 화합물의 세포 독성 효과를 확인하기 위해, 인체 폐암세포(NCI-H1299, NCI-H460), 골육종세포(Osteosarcoma)(Hos), 자궁암세포(HeLa) 각각을 대상으로 MTT 어세이를 수행하였다. 6 웰 플레이트에 각각의 화학물을 0, 0.5, 1, 1.5, 3, 5, 10 μM 씩 첨가하여 2 일후 세포의 생존율을 확인하였다. 그 결과를 도 4에 나타내었다. 도 4의 결과에 따르면, 화학식 2의 화합물인 프리스티머린이 가장 우수한 세포독성 효과를 갖는 것으로 나타났다. In order to confirm the cytotoxic effects of the compounds of
실시예 5Example 5
생체내 (in vivo) 동물실험In vivo animal testing
15 g의 누드 마우스를 3 개의 군으로 나누어, 각각 인체 폐암세포(NCI-H1299 세포)를 누드 마우스의 대퇴부에 이식하여 종양 크기가 약 120-150 cm3에 이르도록 하였다. 제 1 군은 대조군으로 하고, 제 2 군은 프리스티머린을 DMSO에 용해시켜 1 mg/kg의 용량으로 종양내 주사 또는 복강내 주사로 7 일동안 매일 수행하였고, 제 3 군은 시스플라틴 또한 DMSO에 용해시켜 종양내 주사로 7 일동안 1 mg/kg의 용량으로 매일 투여하였다. 그런후 각각의 대조군과 투여군을 3 주동안 약물투여 없이 종양의 크기를 관찰하였다. 각각의 처리군에서 종양내주사는 7 일 간격으로, 복강내주사는 3 일 간격으로 종양의 부피를 측정하였으며, 그 결과를 각각 도 5 및 도 6에 나타내었다. 또한, 대조군 및 프리스티머린을 종양내 주사한 다음 3 주 후의 사진을 도 7에 나타내었다. 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 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.
도 5 내지 도 7에서 확인할 수 있는 바와 같이, 프리스티머린을 투여한 군에서 암 조직의 크기가 현저히 감소함을 알 수 있다. 또한, 누드 마우스의 배양 일수가 증가함에 따라 폐암 세포만 이식한 군은 배양 일수에 의존적으로 종양의 크기 가 빠르게 증가하였고, 폐암세포를 이식한 누드 마우스에 프리스티머린을 처리한 군은 종양의 크기가 느리게 증가하였다. 또한, 도 5에 나타난 결과에 따르면, 프리스티머린은 종래 항암제로서 사용되는 시스플라틴의 종양 치료 효과와 유사한 정도의 종양 치료효과를 갖는 것으로 나타났다. 따라서, 프리스티머린은 배양세포 수준에서 뿐만 아니라 인체 암세포 이식 마우스를 이용한 동물시험에서도 종양을 효과적으로 치료하는 것으로 관찰되었다. 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.
도 1은 각종 암세포를 배양하여 각각을 대상으로 HSP 27의 발현 정도를 웨스턴 블랏팅 결과를 나타낸 도면이다. 1 is a diagram showing the results of Western blotting on the degree of expression of HSP 27 in each of various cancer cells.
도 2는 본 발명의 방법에 따라 미역줄나무에서 분리된 퀴논메차이드계 화합물 각각을 인체폐암세포주(H1299)에 처리하여 HSP 27의 발현 정도를 웨스턴 블랏팅으로 확인한 결과를 나타낸 도면이다. 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).
도 3는 본 발명의 방법에 따라 미역줄나무에서 분리된 프리스티머린의 인체폐암세포주(H1299)에 농도별, 시간별로 처리하여 HSP의 발현 정도를 웨스턴 블랏팅으로 확인한 결과를 나타낸 도면이다. 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.
도 4는 본 발명의 방법에 따라 미역줄나무에서 분리된 퀴논메차이드계 화합물 각각의 각종 암세포에 대한 세포독성의 효과를 MTT 어세이에 의해 측정한 결과를 정리한 표이다. 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.
도 5는 인체폐암세포주(H1299)를 마우스의 대퇴부에 이식하여 이미 종양이 형성된 마우스에, 본 발명의 방법에 따라 미역줄나무에서 분리된 프리스티머린을 7 일간 매일 종양내주사한 다음, 종양의 크기 변화를 7일마다 관찰한 결과를 음의 대조군 및 양의 대조군(시스플라틴 투여군)의 결과와 함께 나타낸 그래프이다. 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).
도 6은 인체폐암세포주(H1299)를 마우스의 대퇴부에 이식하여 이미 종양이 형성된 마우스에, 본 발명의 방법에 따라 미역줄나무에서 분리된 프리스티머린을 7 일간 매일 복강주사한 다음, 종양의 크기 변화를 3일마다 관찰한 결과를 음의 대조군과 함께 나타낸 그래프이다.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.
도 7은 인체폐암세포주(H1299)를 마우스의 대퇴부에 이식하여 이미 종양이 형성된 마우스에, 본 발명의 방법에 따라 미역줄나무에서 분리된 프리스티머린을 7 일동안 매일 1 mg/Kg으로 종양내 주사한 후 3 주후에 종양 크기의 변화를 촬영한 사진이다.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.
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