TWI430800B - A triterpenoid compound, its preparation method and uses - Google Patents
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本揭示內容大致是有關於一種自靈芝中分離出來的新穎化合物，製備此新穎化合物的方法及其用途。詳言之，本揭示內容係有關自黃芝(Ganoderma colossum (Fr.)Baker)中分離出來的一種包含有氧雜-A,B-升二-類固醇(oxa-A,B-dihomo-steroidal)骨架的新穎三萜化合物；此新穎三萜化合物的分離方法；以及以此新穎三萜化合物來製備抗癌或抗發炎藥物的用途。The present disclosure is broadly related to a novel compound isolated from Ganoderma lucidum, a process for preparing the novel compound, and uses thereof. In particular, this disclosure relates to an oxa-A, B-dihomo-steroidal, isolated from Ganoderma colossum (Fr.) Baker. a novel triterpenoid compound of the skeleton; a method for isolating the novel triterpenoid compound; and the use of the novel triterpenoid compound for the preparation of an anticancer or anti-inflammatory drug.
食藥用真菌被廣泛做為保健食品，其中又以靈芝為大宗。在東南亞地區，更常以靈芝子實體入藥。由於靈芝可在生體內啟動諸多正面的反應，相關活性成分的研究很多，其活性成分大致被區分成五大類：靈芝三萜類(triterpenoids)、多醣體(polysaccharides)、核苷(nucleotides)、植物固醇類(phytosterols)及多肽類(polypeptides)。Food and medicinal fungi are widely used as health foods, among which Ganoderma lucidum is a large one. In Southeast Asia, Ganoderma lucidum fruiting bodies are more commonly used. Since Ganoderma lucidum can initiate many positive reactions in the living body, there are many studies on related active ingredients, and the active ingredients are roughly divided into five categories: triterpenoids, polysaccharides, nucleotides, plants. Phytosterols and polypeptides.
黃芝(G .colossum )是靈芝科靈芝屬中的一屬，因黃芝較為稀少，因此相關活性成分的研究並不多。本申請案發明人即是在進行黃芝研究過程中，意外發現一種新穎的靈芝三萜化合物，且於離體試驗中證實此一新發現的靈芝三萜化合物具有可抑制癌細胞生長、轉移以及發炎基因表現的功效，因此可做為未來開發成抗腫瘤生長藥物或抗發炎藥物的先導化合物。 G. colossum is a genus of Ganoderma lucidum. Because of the scarcity of yellow zhizhi , there are not many studies on related active ingredients. The inventor of the present application accidentally discovered a novel Ganoderma lucidum triterpenoid compound during the research of Huangzhi, and confirmed in an in vitro test that the newly discovered Ganoderma lucidum triterpenoid compound can inhibit the growth and metastasis of cancer cells. The efficacy of inflammatory gene expression can be used as a lead compound for future development of anti-tumor growth drugs or anti-inflammatory drugs.
如本揭示內容所廣泛描述與例示，本揭示內容目標為提供一種自黃芝中分離出來的新穎化合物，以及此新穎化合物的用途。As broadly described and exemplified in the present disclosure, the present disclosure aims to provide a novel compound isolated from yellow cherries and the use of such novel compounds.
因此，本揭示內容第一目的在於提供一種自黃芝中分離出來之具備有氧雜-A,B-升二-類固醇(oxa-A,B-dihomo-steroidal)骨架的新穎三萜化合物，具有如下式(I)之結構：Accordingly, a first object of the present disclosure is to provide a novel triterpenoid compound having an oxa-A, B-dihomo-steroidal skeleton separated from yellow lucidum, having the following formula (I) Structure:
依據本發明一實施方式，此新穎之式(I)化合物具有抑制癌細胞生長、轉移或抑制發炎基因表現的功效，故未來可開發成抗癌或抗發炎藥物。According to one embodiment of the present invention, the novel compound of the formula (I) has an effect of inhibiting the growth, metastasis or inhibition of the expression of an inflammatory gene, and thus can be developed into an anticancer or anti-inflammatory drug in the future.
據此，本揭示內容第二目的在於提供一種分離上述新穎式(I)化合物之方法，包含以下步驟：Accordingly, a second object of the present disclosure is to provide a method of isolating the above novel compound of formula (I) comprising the steps of:
(a) 以一第一溶液萃取一黃芝(G. colossum )子實體，以獲得一第一萃出物，其中該黃芝子實體與該第一溶液係以重量：體積約為1：10至1:50之比例混合；(a) extracting a G. colossum fruiting body with a first solution to obtain a first extract, wherein the yellowing fruit body and the first solution are weighted: a volume of about 1:10 to 1 a ratio of 50:
(b) 以一由水及有機溶劑組成之第二溶液萃取該第一萃出物，以獲得一水層與一有機層萃出物，其中水及有機溶劑是以體積比約1:1至2:3之比例混合；(b) extracting the first extract with a second solution consisting of water and an organic solvent to obtain an aqueous layer and an organic layer extract, wherein the water and the organic solvent are in a volume ratio of about 1:1 to a ratio of 2:3;
(c) 在一矽膠管柱中，以一第三溶液沖提該有機層萃出物，並收集一第一沖提液；(c) extracting the organic layer extract with a third solution in a cartridge column and collecting a first extract;
(d) 在一逆相高效液相層析管柱(reverse-phase high performance liquid chromatography)中，以一濃度介於20%至95%(體積%)間的第四溶液沖提該第一沖提液，以獲得一第二沖提液；以及(d) in a reverse-phase high performance liquid chromatography, the first rush is flushed with a fourth solution having a concentration between 20% and 95% by volume. Lifting liquid to obtain a second extract;
(e) 濃縮乾燥該第二沖提液以獲得該式(I)化合物。(e) The second extract is concentrated and dried to obtain the compound of the formula (I).
依據本揭示內容一實施方式，所述步驟(a)中該第一溶液為70%-95%(體積%)之乙醇；所述步驟(b)中該有機溶劑是乙酸乙酯，且該第二溶液是由等體積之水與乙酸乙酯混合而成；所述步驟(c)中該第三溶液是由體積比介於1:0至0:1之己烷與乙酸乙酯所組成；所述步驟(d)中該第四溶液為濃度介於60%-80%(體積%)之甲醇。According to an embodiment of the present disclosure, in the step (a), the first solution is 70%-95% (% by volume) of ethanol; in the step (b), the organic solvent is ethyl acetate, and the first The second solution is prepared by mixing an equal volume of water and ethyl acetate; in the step (c), the third solution is composed of hexane and ethyl acetate in a volume ratio of 1:0 to 0:1; In the step (d), the fourth solution is methanol having a concentration of between 60% and 80% by volume.
本揭示內容第三目的在於提供一種可抑制癌細胞增生、轉移或抑制發炎基因表現之藥學組合物，包含一有效量之式(I)化合物；及其藥學上可接受的載劑(excipient)。A third object of the present disclosure is to provide a pharmaceutical composition which inhibits the proliferation, metastasis or inhibition of the expression of an inflammatory gene, comprising an effective amount of a compound of formula (I); and a pharmaceutically acceptable excipient thereof.
依據本發明一實施方式，適合以所述藥學組合物來抑制其生長或遷移的癌細胞為肝癌或肺癌細胞。According to an embodiment of the present invention, the cancer cells suitable for inhibiting growth or migration by the pharmaceutical composition are liver cancer or lung cancer cells.
據此，本揭示內容第四目的在於提供一種可延緩一個體體內之癌細胞生長、轉移或抑制該個體之發炎狀況的方法，包含對該個體施用上述之藥學組合物。Accordingly, a fourth object of the present disclosure is to provide a method of delaying the growth, metastasis or inhibition of inflammatory conditions in a subject, comprising administering to the individual a pharmaceutical composition as described above.
依據本發明一實施方式，適合以所述方法來治療(亦即，延緩癌細胞生長或遷移)的癌症可為肝癌、肺癌或乳癌；特別是具有轉移能力的肺癌或乳癌。適合接受所述方法治療的個體為哺乳動物，例如，人類。According to an embodiment of the invention, the cancer suitable for treatment by the method (i.e., delaying the growth or migration of cancer cells) may be liver cancer, lung cancer or breast cancer; in particular, lung cancer or breast cancer having metastatic ability. An individual suitable for treatment by the methods is a mammal, for example, a human.
以下所提供之發明詳細說明與附隨圖示僅在解釋本發明實施方式之用，並非本發明多種實施方式的唯一代表實施方式。以下文字將說明實施例的功能以及如何建構與操作這些實施例的步驟和順序。但是，本發明請求範圍並不僅限於所揭示的實施態樣。The detailed description of the invention, which is set forth below, and the accompanying drawings are merely illustrative of the embodiments of the invention The following text will explain the functions of the embodiments and the steps and sequences of how to construct and operate the embodiments. However, the scope of the present invention is not limited to the disclosed embodiments.
黃芝(G. colossum )是靈芝科真菌中較為罕見的一個品種，由於黃芝非常稀有，因此有關其活性成分的研究並不多。有鑑於此，本案發明人利用溶劑萃取以及層析技術，包括矽膠管柱層析與逆相高效液相層析(reverse-phase high performance liquid chromatography)，而自乾燥的黃芝子實體中分離出一種新穎化合物，經過結構鑑定，確認其為含有氧雜-A,B-升二-類固醇(oxa-A,B-dihomo-steroidal)骨架的靈芝三萜類化合物。此新穎化合物在體外試驗(in vitro study)中被證實可抑制癌細胞生長及發炎基因表現，故未來可開發成抗癌或抗發炎藥物。 G. colossum is a rare species in Ganoderma lucidum fungi. Because of its rareness, Huangzhi is not a research on its active ingredients. In view of this, the inventors of the present invention used solvent extraction and chromatography techniques, including tantalum column chromatography and reverse-phase high performance liquid chromatography, to isolate a novel from the dried yellow lucidum fruit body. The compound, which was structurally identified, was confirmed to be a Ganoderma lucidum triterpenoid containing an oxa-A, B-dihomo-steroidal skeleton. This novel compound has been shown to inhibit cancer cell growth and inflammatory gene expression in in vitro studies, and may be developed into an anti-cancer or anti-inflammatory drug in the future.
因此，本發明一方面是提供一種自黃芝子實體中分離出來的新穎靈芝三萜化合物，即(22S ,23R )-A,B-升二-19-降-15-β-乙醯氧-5,23-二羥-4-氧雜-3-氧代羊毛固醇-1,8,19,24-四烯-26,22-內酯[(22S ,23R )-A,B-dihomo-19-nor-15-β-acetoxy-5,23-dihydroxy-4-oxa-3-oxolanosta-1,8,19,24-tetraen-26,22-olide)]或稱羊毛固醇三萜H(Colossolactone H)，其具有以下式(I)之結構：Accordingly, in one aspect of the invention, there is provided a novel Ganoderma lucidum triterpenoid compound isolated from a fruiting body of the yellow lucidum, namely (22 S , 23 R )-A, B-L 2-19--15-β-acetyloxy- 5,23-dihydroxy-4-oxa-3-oxo lanosterol-1,8,19,24-tetraene-26,22-lactone [(22 S ,23 R )-A,B- Dihomo-19-nor-15-β-acetoxy-5,23-dihydroxy-4-oxa-3-oxolanosta-1,8,19,24-tetraen-26,22-olide)] or lanosterol H (Colossolactone H) having the structure of the following formula (I):
本發明另一方面是要提供一種自黃芝子實體中分離出上述式(I)化合物的方法，包含以下步驟：Another aspect of the present invention is to provide a method for isolating a compound of the above formula (I) from a fruiting body of a yellow gum, comprising the steps of:
(a) 以一第一溶液萃取黃芝子實體，以獲得一第一萃出物，其中該黃芝子實體與該第一溶液係以重量：體積約為1：10至1:50之比例混合；(a) extracting a yellow fruiting body with a first solution to obtain a first extract, wherein the yellow fruiting body and the first solution are mixed in a weight: a volume of about 1:10 to 1:50;
(b) 以一由水及有機溶劑組成之第二溶液萃取該第一萃出物，以獲得一水層與一有機層萃出物，其中水及有機溶劑是以體積比約1:1至2:3之比例混合；(b) extracting the first extract with a second solution consisting of water and an organic solvent to obtain an aqueous layer and an organic layer extract, wherein the water and the organic solvent are in a volume ratio of about 1:1 to a ratio of 2:3;
(c) 在一矽膠管柱中，以一第三溶液沖提該有機層萃出物，並收集一第一沖提液；(c) extracting the organic layer extract with a third solution in a cartridge column and collecting a first extract;
(d) 在一逆相高效液相層析管柱中，以一濃度介於20%至95%(體積%)間的第四溶液沖提該第一沖提液，以獲得一第二沖提液；以及(d) in a reverse phase high performance liquid chromatography column, the first extract is flushed with a fourth solution having a concentration between 20% and 95% by volume to obtain a second flush Lifting liquid;
(e) 濃縮乾燥該第二沖提液以獲得該式(I)化合物。(e) The second extract is concentrated and dried to obtain the compound of the formula (I).
依據本揭示內容一實施方式，在上述步驟(a)中，黃芝子實體較佳是先經過乾燥處理使其含水量下降，以避免新鮮的黃芝子實體腐敗而使其中活性化合物改變或含量下降。接著，將乾燥的子實體粉碎成大小均勻的碎屑或粉末，才與第一溶液混合並進行萃取，以獲得第一萃出物。適合的第一溶液為醇類，包括(但不限於)乙醇、丙醇、異丙醇、正丁醇及異丁醇。在一較佳實施方式中，第一溶液為70%-95%(體積%)之乙醇；較佳是95%(體積%)之乙醇。乾燥的黃芝子實體粉末與第一溶液(如，乙醇)較佳是以乾燥粉末重量：第一溶液體積為1：10至1:50之比例混合，更佳是以1:20至1:40的比例混合；最佳是以約1:25至約1:35比例混合。舉例來說，1公斤的黃芝子實體乾燥粉末至少需添加10公升之95%(體積%)乙醇。混合物可以加熱震盪、廻流浸煮或室溫浸泡等方式進行萃取。在一較佳實施方式中，黃芝子實體乾燥粉末與第一溶液是以1：24的比例混合，然後在37℃下浸煮混合物約24小時後，即可得一粗萃液。接著，將此粗萃液過濾、濃縮並乾燥(可以任何習知方式來執行此過濾、濃縮及乾燥的步驟)成粗萃物。According to an embodiment of the present disclosure, in the above step (a), the yellow berry fruit body is preferably subjected to a drying treatment to reduce the water content to prevent the fresh yellow lucidum fruit body from being spoiled and the active compound therein to be changed or decreased. Next, the dried fruit body is pulverized into chips or powder of uniform size before being mixed with the first solution and subjected to extraction to obtain a first extract. Suitable first solutions are alcohols including, but not limited to, ethanol, propanol, isopropanol, n-butanol, and isobutanol. In a preferred embodiment, the first solution is 70% to 95% by volume of ethanol; preferably 95% by volume of ethanol. The dried yellow lucidum fruit body powder and the first solution (eg, ethanol) are preferably mixed in a dry powder weight: first solution volume in a ratio of 1:10 to 1:50, more preferably 1:20 to 1:40. Proportional mixing; optimally mixed in a ratio of from about 1:25 to about 1:35. For example, at least 10 liters of 95% (% by volume) ethanol is added to 1 kg of dried powder of the yellow body fruit. The mixture can be extracted by heating, turbulent dip or room temperature soaking. In a preferred embodiment, the dried powder of the yellow body fruit body and the first solution are mixed at a ratio of 1:24, and then the mixture is immersed at 37 ° C for about 24 hours to obtain a crude extract. Next, the crude extract is filtered, concentrated and dried (the filtration, concentration and drying steps can be carried out in any conventional manner) to form a crude extract.
接著，在步驟(b)中，以一種由水與有機溶劑組成的第二溶液來萃取步驟(a)所獲得的第一萃出物。由於第二溶液是由彼此不互溶或極性不同的兩種溶劑以約1:1至約2:3之比例組成，因此萃取後會分成兩層，分別為水層以及有機層。適合用於此步驟中的有機溶劑為低碳數的酯類，包括，但不限於甲酸乙酯、乙酸甲酯、乙酸乙酯、丙酸甲酯、丙酸乙酯及丙酸丙酯；較佳是乙酸乙酯。在一較佳實施方式中，此第二溶液是由等體積之水與乙酸乙酯混合而成，但需知水與乙酸乙酯亦可以其他適合的比例混合，此領域中習知技藝人士可依據實際操作狀況，透過簡單的試驗找到最適合當時狀況的混合比例。在以第二溶液萃取上述步驟(a)之第一萃出物後，保留有機層之萃出物，用於接續的分離步驟(c)至(e)中。Next, in the step (b), the first extract obtained in the step (a) is extracted with a second solution composed of water and an organic solvent. Since the second solution is composed of two solvents which are mutually incompatible or different in polarity, in a ratio of about 1:1 to about 2:3, the extraction is divided into two layers, an aqueous layer and an organic layer, respectively. The organic solvent suitable for use in this step is a low carbon number ester, including, but not limited to, ethyl formate, methyl acetate, ethyl acetate, methyl propionate, ethyl propionate and propyl propionate; It is preferably ethyl acetate. In a preferred embodiment, the second solution is formed by mixing an equal volume of water with ethyl acetate, but it is understood that water and ethyl acetate may also be mixed in other suitable ratios, as those skilled in the art may According to the actual operating conditions, through simple tests, find the mixing ratio that is most suitable for the current situation. After extracting the first extract of the above step (a) with the second solution, the extract of the organic layer is retained for successive separation steps (c) to (e).
可重複進行上述步驟(b)數次，例如，1、2或3次，將每一次獲得的有機層萃出物合併，用於接續的分離步驟(c)至(e)中。The above step (b) may be repeated several times, for example, 1, 2 or 3 times, and the organic layer extracts obtained each time are combined for successive separation steps (c) to (e).
在步驟(c)中，將上述步驟(b)之乙酸乙酯層萃出物載入至矽膠管柱中，接著以由體積比介於1:0至0：1之己烷與乙酸乙酯所組成的該第三溶液沖提，並收集所得第一沖提液。適合用來沖提矽膠管柱的第三溶液是由體積比介於1:0至0：1之己烷與乙酸乙酯所組成。In the step (c), the ethyl acetate layer extract of the above step (b) is loaded into a ruthenium tube column, followed by hexane and ethyl acetate in a volume ratio of 1:0 to 0:1. The third solution composed is washed and the resulting first extract is collected. A third solution suitable for use in the stripping of the gum column is composed of hexane and ethyl acetate in a volume ratio of 1:0 to 0:1.
在步驟(d)中，進一步地將步驟(c)所獲得的第一沖提液載入至一逆相高效液相層析管柱中，在約0.5~20毫升/分鐘的流速下(較佳是約1、2、3、4、5、6、7、8、9、10、11、12、13或14毫升/分鐘，更佳是約2、3、4、5、6、7、8或9毫升/分鐘)，以濃度在20%至95%(體積%)間，例如約20%、30%、35%、40%、45%、50%、55%、60%、65%、70%、75%、80%、85%、90%或95%；更佳是約50%至90%(體積%)間，例如約50%、55%、60%、65%、70%、75%、80%、85%或90%；最佳是60%至80%(體積%)間，例如約60%、65%、70%、75%或80%之第四溶液進行沖提，並收集所得沖提液。適合用來沖提管柱的第四溶液包括，但不限於水或醇類，包括水、甲醇、乙醇、丙醇、異丙醇、正-丁醇、異-丁醇等。在一特定實例中，此第四溶液是濃度介於60-80%(體積%)的甲醇溶液。在一特定實例中，此第四溶液是濃度介於60-80%(體積%)的甲醇溶液。In the step (d), the first extract obtained in the step (c) is further loaded into a reverse phase high performance liquid chromatography column at a flow rate of about 0.5 to 20 ml/min (compared Preferably, it is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 ml/min, more preferably about 2, 3, 4, 5, 6, 7, 8 or 9 ml/min), at a concentration between 20% and 95% (% by volume), for example about 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% 70%, 75%, 80%, 85%, 90% or 95%; more preferably between about 50% and 90% (% by volume), for example about 50%, 55%, 60%, 65%, 70% , 75%, 80%, 85% or 90%; optimally between 60% and 80% (% by volume), for example about 60%, 65%, 70%, 75% or 80% of the fourth solution is stripped And collect the resulting extract. A fourth solution suitable for use in flushing the column includes, but is not limited to, water or alcohols including water, methanol, ethanol, propanol, isopropanol, n-butanol, iso-butanol, and the like. In a specific example, the fourth solution is a methanol solution having a concentration between 60 and 80% by volume. In a specific example, the fourth solution is a methanol solution having a concentration between 60 and 80% by volume.
在此一實例中，可將沖提後的沖提液收集在一起，並經減壓濃縮及冷凍乾燥後，獲得欲求的黃芝化合物。在一特定實例中，所沖提出來的新穎化合物經NMR、UV及高解析度電噴霧電離質譜(high resolution electrospray ionization mass spectroscopy,HREIMS)等儀器分析鑑定後，確認其為(22S ,23R )-A,B-升二-19-降-15-β-乙醯氧-5,23-二羥-4-氧雜-3-氧代羊毛固醇-1,8,19,24-四烯-26,22-內酯，又稱羊毛固醇三萜H。經由本發明方法分離出來的新穎化合物產率約為0.04%(此百分比係為式(I)化合物重量/子實體乾重)。此外，可以任何習知的方式來將產物濃縮、乾燥。在一實例中，係在一減壓環境下，例如壓力約0.1 torr的環境下，將沖提液濃縮，待沖提液中的固形物含量達20%時，即停止此濃縮處理。接著，將濃縮物乾燥，例如，在-30℃~-50℃下冷凍乾燥。In this example, the rinsed extract can be collected together, concentrated under reduced pressure, and lyophilized to obtain the desired yellow gum compound. In a specific example, the proposed novel compound was identified by NMR, UV, and high resolution electrospray ionization mass spectroscopy (HREIMS) and identified as (22 S , 23 R )-A, B-Like II-19-Lower-15-β-Ethyloxy-5,23-dihydroxy-4-oxa-3-oxo lanosterol-1,8,19,24-four Alkene-26,22-lactone, also known as lanosterol triterpene H. The yield of the novel compound isolated by the process of the invention is about 0.04% (this percentage is the weight of the compound of formula (I) / dry weight of the fruit body). Furthermore, the product can be concentrated and dried in any conventional manner. In one example, the extract is concentrated under a reduced pressure environment, for example, at a pressure of about 0.1 torr, and the concentration treatment is stopped when the solids content in the rinse is 20%. Next, the concentrate is dried, for example, freeze-dried at -30 ° C to -50 ° C.
據此，本發明另一發明目的是提供一種可抑制癌細胞增生或抑制發炎的藥學組合物，包含一有效量之式(I)化合物；及其藥學上可接受的載體。Accordingly, another object of the present invention is to provide a pharmaceutical composition which inhibits proliferation of cancer cells or inhibits inflammation, comprising an effective amount of a compound of formula (I); and a pharmaceutically acceptable carrier therefor.
「一有效量(an effective amount)」一詞意指對治療對象施用一具有療效之化合物或組合物的劑量。療效之衡量可為客觀的(利用一些試驗或標誌來衡量)或是主觀的(治療對象給予其對效果與感覺之敘述)。本發明藥學組合物的有效劑量範圍可自約1毫克/公斤至約1000毫克/公斤，或是自10毫克/公斤至約100毫克/公斤。有效劑量亦會隨著施用途徑之不同以及可能並用的其他藥劑之不同而有所改變。熟知此技藝之人會知道有可能會使用比上述多或少之劑量。對特定病人所使用之特定劑量與治療方法將會受許多變因的影響，包含使用藥物之活性、年齡、體重、整體健康狀況、性別、飲食、施用時間、施用途徑、施用頻率、***速率、合併用藥、疾病的進程與嚴重度、患者疾病位置、症狀狀況、醫生對疾病之處理與判斷。By "an effective amount" is meant a dose of a therapeutically effective compound or composition administered to a subject. The measurement of efficacy can be objective (measured by some test or marker) or subjective (the subject gives a narrative of its effects and feelings). The effective dosage of the pharmaceutical compositions of the present invention can range from about 1 mg/kg to about 1000 mg/kg, or from 10 mg/kg to about 100 mg/kg. The effective dose will also vary with the route of administration and the other agents that may be used in combination. Those skilled in the art will recognize that it is possible to use more or less than the above. The particular dose and method of treatment used for a particular patient will be affected by a number of variables, including the activity of the drug used, age, weight, overall health, sex, diet, time of administration, route of administration, frequency of administration, rate of excretion, Combined medication, the course and severity of the disease, the location of the patient's disease, the state of the symptoms, and the treatment and judgment of the doctor.
因此，本發明一態樣為提供一種延緩或抑制一個體身上多數癌細胞生長或轉移的方法，其至少包含對該個體施用一有效量之所述藥學組合物，使得該個體身上約90%的該些癌細胞的生長停滯或是不會轉移，因而能達到延緩腫瘤或癌生長或擴散的目的。適合以本發明方法或藥學組合物來治療(亦即，延緩癌細胞生長或轉移)的癌症為肝癌、肺癌或乳癌；特別是具有轉移能力的肺癌或乳癌。Accordingly, it is an aspect of the present invention to provide a method of delaying or inhibiting the growth or metastasis of a majority of cancer cells in a subject, comprising at least administering to the individual an effective amount of the pharmaceutical composition such that about 90% of the individual is The growth of these cancer cells is stagnant or does not metastasize, and thus can achieve the purpose of delaying the growth or spread of tumors or cancers. A cancer suitable for treatment with the method or pharmaceutical composition of the invention (i.e., delaying the growth or metastasis of cancer cells) is liver cancer, lung cancer or breast cancer; in particular, lung cancer or breast cancer with metastatic ability.
本發明另一態樣則是提供一種可抑制或減緩一個體之發炎狀況的方法，其至少包含對該個體施用一有效量之所述藥學組合物，使得該個體之發炎基因表現受到抑制，因而能達成減緩發炎的效果。Another aspect of the present invention is to provide a method for inhibiting or slowing the inflammatory condition of a subject comprising administering to the individual an effective amount of the pharmaceutical composition such that the inflammatory gene expression of the individual is inhibited, thereby Can achieve the effect of slowing down inflammation.
「個體(subject)」一詞在本文中代表可接受本發明藥學組合物之任何一種動物(例如，人類)，包括，但不限於，人類、非人類的靈長類，如哺乳動物、狗、貓、馬、羊、豬、牛等。一般來說，「患者(patient)」一詞及「個體(subject)」一詞在本文中可彼此交替使用。The term "subject" as used herein refers to any animal (eg, human) that can accept a pharmaceutical composition of the invention, including, but not limited to, human, non-human primates, such as mammals, dogs, Cats, horses, sheep, pigs, cattle, etc. In general, the terms "patient" and "subject" are used interchangeably herein.
除非另行定義，文中所使用之所有專業與科學用語與習知技藝者所熟悉之意義相同。此外，任何與所記載內容相似或均等之方法及材料皆可應用於本發明方法中。文中所述之較佳實施方法與材料僅做示範之用。於本申請書中所提到之所有參考文獻均全體納入參考，以揭露並敘述該文獻所記載之相關方法及/或材料。此外，文中所討論之文獻僅揭露本發明申請日前之習知技術。並且無任何文獻顯示本發明內容曾為習知技術所揭露。本發明內容所得到之實際數據會因個別的實施條件而與本發明揭露於說明書內容中之數據有所不同。Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those skilled in the art. In addition, any methods and materials similar or equivalent to those described may be employed in the methods of the invention. The preferred embodiments and materials described herein are for illustrative purposes only. All references cited in this application are hereby incorporated by reference in their entirety to the extent of the disclosure of the disclosure of the disclosure. Moreover, the documents discussed herein merely disclose prior art techniques of the present application. And there is no literature showing that the present invention has been disclosed in the prior art. The actual data obtained in the context of the present invention may differ from the data disclosed in the present disclosure by the present invention.
除非另外指明，否則在本文及附隨之請求項範圍中所述及之單數型式詞，「一(“a“,“an”)或該(“the”)」均涵蓋其複數形式。Unless otherwise indicated, the singular "a", "an", "the" or "the"
以下，將透過實施例來闡述本發明的較佳實施方式。Hereinafter, preferred embodiments of the present invention will be described by way of examples.
羊毛固醇三萜H(Colossolactone H)Wool sterol H (Colossolactone H)
(22S ,23R )-A,B-升二-19-降-15-β-乙醯氧-5,23-二羥-4-氧雜-3-氧代羊毛固醇-1,8,19,24-四烯-26,22-內酯(22 S ,23 R )-A,B-Like II-19-Lower-15-β-Ethyloxy-5,23-dihydroxy-4-oxa-3-oxo lanosterol-1,8 ,19,24-tetraene-26,22-lactone
(22S ,23R )-A,B-dihomo-19-nor-15-β-acetoxy-5,23-dihydroxy-4-oxa-3-oxolanosta-1,8,19,24-tetraen-26,22-olide(22 S ,23 R )-A,B-dihomo-19-nor-15-β-acetoxy-5,23-dihydroxy-4-oxa-3-oxolanosta-1,8,19,24-tetraen-26, 22-olide
將乾重為50克的黃芝子實體以粉粹機碎裂成粉末後，置於震盪萃取裝置內。接著以黃芝子實體粉末乾重量：溶劑體積=1：24之比例，加入1,200毫升的酒精溶劑，在37℃下浸煮一天後，將浸煮液濃縮。重複此萃取、濃縮步驟數次，合併所得的萃取液並經過濾與減壓濃縮抽乾後，可得13克(產率：26%)的黃芝子實體醇溶粗萃物。接著，在40℃恆溫真空乾燥系統內隔夜抽濾去除所得黃芝子實體醇溶粗萃物中的殘餘溶劑。The 50 g dry weight of the yellow lucidum fruit body was broken into powder by a powder machine and placed in an oscillating extraction device. Next, the dilute liquid was concentrated by adding 1,200 ml of an alcohol solvent to the dry weight of the powder of the yellow berry fruit: solvent volume = 1:24, and digesting at 37 ° C for one day. This extraction and concentration steps were repeated several times, and the resulting extracts were combined and filtered and concentrated under reduced pressure to give 13 g (yield: 26%). Next, the residual solvent in the alcoholic crude extract of the resulting yellow body fruit body was removed by suction filtration overnight in a 40 ° C constant temperature vacuum drying system.
於上述實施例1.1所得之乾重為13.0克的黃芝子實體醇溶粗萃物中，加入以等體積比例混合之水與乙酸乙酯(H2 O: ethyl acetate=1：1)的混合溶液，以進行萃取，可得一水層溶液以及乙酸乙酯萃取層溶液，將此乙酸乙酯萃取層溶液濃縮乾燥後，以40℃恆溫真空乾燥系統隔夜抽濾去除殘餘溶劑，可得乾重為9.5克的黃芝子實體乙酸乙酯萃取物(GCAE)，相對於總黃芝子實體醇溶粗萃物而言，此GCAE產率約為19%。A mixed solution of water and ethyl acetate (H 2 O: ethyl acetate = 1:1) mixed in an equal volume ratio was added to the crude extract of the yellow lucidum fruit body obtained by the above Example 1.1 in a dry weight of 13.0 g. For extraction, an aqueous layer solution and an ethyl acetate extraction layer solution are obtained, and the ethyl acetate extraction layer solution is concentrated and dried, and then the residual solvent is removed by suction filtration at 40 ° C under a vacuum drying system overnight to obtain a dry weight of 9.5. The acetic acid extract (GCAE) of the yellow lucidum fruiting body has a GCAE yield of about 19% relative to the total alcoholic crude extract of the yellow gum fruit body.
將上述實施例1.2所得乾燥的黃芝子實體乙酸乙酯萃取物(GCAE)，以開放式矽膠管柱(silica gel 60,25×3.5 cm)進行層析，利用己烷：乙酸乙酯=1:1之溶劑系統進行梯度沖提後，將其劃分為12個區分層，分別稱為第1至12區分層。進一步地，利用逆向高壓液相層析系統(HPLC/Diode array)以60%-80%甲醇溶液系統對第11個區分層(247.7毫克)進行分離純化，可得18毫克之式(I)化合物的白色粉末狀固體，相對於總黃芝子實體醇溶粗萃物而言，此式(I)化合物的產率為0.04%。The dried ethyl acetate extract (GCAE) obtained from the above Example 1.2 was subjected to chromatography on an open gel column (silica gel 60, 25×3.5 cm) using hexane:ethyl acetate=1:1. After the solvent system is subjected to gradient elution, it is divided into 12 distinguishing layers, which are referred to as layer 1 to layer 12, respectively. Further, the 11th discrimination layer (247.7 mg) was separated and purified by a reversed-pressure high pressure liquid chromatography system (HPLC/Diode array) in a 60%-80% methanol solution system to obtain 18 mg of the compound of the formula (I). The white powdery solid has a yield of the compound of the formula (I) of 0.04% relative to the total alcoholic crude extract of the yellow gum fruit body.
進一步以IR、UV、1 HNMR、13 CNMR、HREIMS及CD對此式(I)化合物進行鑑定，相關數據如下示：mp 149-156℃;+48.5(c 0.40,MeOH);IR(KBr)νmax 3418,2930,1705,1576,1446,1372,1248,1142,1021 cm-1 ;UV(MeOH)λ max 250(sh),340 nm;CD Δε251 -5.446,Δε353 ,+0.936(c 1.44×10-4 mM,MeOH);1 H NMR(CD3 OD,500 MHz)δ7.08(1H,d,J =10.0 Hz,H-1),6.62(1H,m,H-24),6.38(1H,s,H-19),5.80(1H,d,J =10.0 Hz,H-2),4.91(1H,d,brs,J =7.0 Hz,H-15),4.41(1H,dd,J =10.5,2.0 Hz,H-23),2.67(1H,m,H-16a),2.34(1H,m,H-6a),2.33(2H,m,H-11),2.24(1H,m,H-6b),2.12(1H,m,H-17),2.08(1H,m,H-20),2.05(3H,s,H-2' ),1.88(3H,s,H-27),1.83(2H,m,H-12),1.51(1H,m,H-16b),1.24(3H,s,H-29),1.17(3H,s,H-18),1.14(3H,s,H-28),1.06(3H,d,J =5.5 Hz,H-21),1.03(3H,s,H-30);13 C NMR(CD3 OD,125 MHz)δ172.0(s,C-1' ),167.5(s,C-3),167.4(s,C-26),150.6(d,C-1),150.2(s,C-8),147.5(d,C-24),141.2(d,C-19),134.8(s,C-10),129.7(s,C-25),127.7(s,C-9),117.0(d,C-2),94.9(s,C-5),85.8(d,C-22),79.9(s,C-15),77.9(s,C-4),64.1(d,C-23),56.6(s,C-14),47.2(d,C-17),45.9(t,C-6),45.7(s,C-13),39.2(t,C-16),36.9(d,C-20),32.6(t,C-12),28.9(t,C-11),27.6(t,C-7),25.1(q,C-28),24.7(q,C-29),24.7(q,C-30),21.6(q,C-2' ),17.7(q,C-18),16.9(q,C-27),13.3(q,C-21);HRESIMSm/z 577.2767[M+Na]+ (Calc. for C32 H42 O8 Na,577.2772).Further, the compound of the formula (I) was identified by IR, UV, 1 H NMR, 13 C NMR, HREIMS and CD, and the relevant data are as follows: mp 149-156 ° C; +48.5 ( c 0.40, MeOH); IR (KBr) ν max 3418, 2930, 1705, 1576, 1446, 1372, 1248, 1142, 1021 cm -1 ; UV (MeOH) λ max 250 (sh), 340 nm; CD Δε 251 -5.446, Δε 353 , +0.936 ( c 1.44 × 10 -4 mM, MeOH); 1 H NMR (CD 3 OD, 500 MHz) δ 7.08 (1H, d, J =10.0 Hz, H-1 ), 6.62 (1H, m, H-24), 6.38 (1H, s, H-19), 5.80 (1H, d, J = 10.0 Hz, H-2), 4.91 (1H, d, brs, J = 7.0 Hz, H-15), 4.41 (1H, dd, J = 10.5, 2.0 Hz, H-23), 2.67 (1H, m, H-16a), 2.34 (1H, m, H-6a), 2.33 ( 2H, m, H-11), 2.24 (1H, m, H-6b), 2.12 (1H, m, H-17), 2.08 (1H, m, H-20), 2.05 (3H, s, H-) 2 ' ), 1.88 (3H, s, H-27), 1.83 (2H, m, H-12), 1.51 (1H, m, H-16b), 1.24 (3H, s, H-29), 1.17 ( 3H, s, H-18), 1.14 (3H, s, H-28), 1.06 (3H, d, J = 5.5 Hz, H-21), 1.03 (3H, s, H-30); 13 C NMR (CD 3 OD, 125 MHz) δ 172.0 (s, C-1 ' ), 167.5 (s, C-3), 167.4 (s, C-26), 150.6 (d, C-1), 150.2 (s , C-8), 147.5 (d, C-24), 141.2 (d, C-19), 134.8 (s, C-10), 129.7 (s, C-25), 127.7 (s, C-9) , 117.0 (d, C-2), 94.9 (s, C-5), 85.8 (d, C-22), 79.9 (s, C-15), 77.9 (s, C-4), 64.1 (d, C-23), 56.6 (s, C-14), 47.2 (d, C-17), 45.9 (t, C-6), 45.7 (s, C-13), 39.2 (t, C-16), 36.9 (d, C-20), 32.6 (t, C-12), 28.9 (t, C-11), 27.6 (t, C-7), 25.1 (q, C-28), 24.7 (q, C) -29), 24.7 (q, C -30), 21.6 (q, C-2 ' ), 17.7 (q, C-18), 16.9 (q, C-27), 13.3 (q, C-21); HRESIMS m/z 577.2767 [M+Na ] + (Calc. for C 32 H 42 O 8 Na, 577.2772).
在本試驗中，係以人類肝癌細胞株HepG2以及人類肺癌細胞株A549進行活體外抗癌分析；將細胞以不同濃度的式(I)化合物處理後，測量細胞內酸性磷酸酵素(Acid phosphatase,ACP)的活性，作為細胞活性的指標。In this experiment, the human liver cancer cell line HepG2 and the human lung cancer cell line A549 were used for in vitro anticancer analysis; after treating the cells with different concentrations of the compound of formula (I), the intracellular acid phosphatase (ACP) was measured. Activity as an indicator of cellular activity.
以DMEM(Dulbecco's Modified Eagle Medium)培養液當作HepG2或A549細胞株的基礎培養液，並在其中添加盤尼西林G(100 units/ml)、鏈黴素硫酸鹽(100 ng/ml)、L-麩醯胺、非必要胺基酸、胎牛血清(10%)及丙酮酸鈉(1%)。平時將細胞培養於10公分培養盤內，當細胞長到八分滿時進行繼代培養。繼代培養方式係先吸去舊的培養液後，以3毫升的磷酸緩衝液(PBS)清洗培養細胞一次，接著，在37℃下以1毫升之胰蛋白酶/EDTA(0.05%/0.025%)溶液處理細胞5分鐘，使細胞脫離培養盤成懸浮狀態；接著，在懸浮細胞中加入2毫升新鮮的培養液，以中和胰蛋白酶的活性。然後，將細胞打散使平均分布於細胞懸浮液中，留下適當比例的細胞數後，加入新培養液補至適當體積並混勻後，放入37℃培養箱內培養。DMEM (Dulbecco's Modified Eagle Medium) culture medium was used as the base medium of HepG2 or A549 cell line, and penicillin G (100 units/ml), streptomycin sulfate (100 ng/ml), L-brate was added thereto. Indoleamine, non-essential amino acid, fetal bovine serum (10%) and sodium pyruvate (1%). Cells are usually cultured in a 10 cm culture dish and subcultured as the cells grow to eight minutes. The subculture method was to first aspirate the old culture medium, and then wash the culture cells once with 3 ml of phosphate buffer (PBS), followed by 1 ml of trypsin/EDTA (0.05%/0.025%) at 37 °C. The cells were treated with the solution for 5 minutes, and the cells were detached from the culture plate to be in a suspended state; then, 2 ml of fresh culture solution was added to the suspension cells to neutralize the activity of trypsin. Then, the cells were dispersed to be evenly distributed in the cell suspension, and after a proper ratio of the number of cells was left, the new culture solution was added to an appropriate volume and mixed, and then cultured in a 37 ° C incubator.
取約3,000個HepG2細胞，將之接種在平底96孔盤中，以培養液補充每一培養孔內的培養液，使體積達180 μL，接著將培養盤放入37℃之培養箱培養過夜。次日加入以不含胎牛血清(fetal bovine serum,FBS)的上述基礎培養液稀釋成不同濃度的式(I)化合物，各培養孔內最終體積為200 μL，式(I)化合物濃度分別為0、5、10、20及40 μg/ml，每個濃度進行三重覆，放入37℃培養箱培養，於72小時測定細胞活性。Approximately 3,000 HepG2 cells were inoculated into a flat-bottom 96-well plate, and the culture solution in each well was supplemented with a culture medium to a volume of 180 μL, and then the plate was incubated overnight in a 37 ° C incubator. The next day, the above basic medium containing no fetal bovine serum (FBS) was added to dilute to different concentrations of the compound of formula (I), and the final volume in each culture well was 200 μL. The concentration of the compound of formula (I) was 0, 5, 10, 20 and 40 μg/ml, each concentration was triple-coated, placed in a 37 ° C incubator, and assayed for cell activity at 72 hours.
細胞活性測定：活細胞中有大量酸性磷酸酶(ACP)可將其受質磷酸對硝基苯酯(p -nitrophenyl phosphate,p -NPP)轉換成對-硝基苯酚(p -nitrophenol,p -NP)，p -NP在405 nm波長下有最大吸光值，故可藉測定吸光值可定量出細胞中ACP活性，進而推算出細胞的活性。Cell viability assay: A large number of acid phosphatase (ACP) in living cells can convert p -nitrophenyl phosphate ( p- NPP) into p-nitrophenol ( p- nitrophenol, p- NP), p- NP has the maximum absorbance at 405 nm, so the absorbance can be used to quantify the ACP activity in the cell, and then the cell activity can be deduced.
測定細胞活性時，先吸去培養液，以200 μL PBS清洗一次，加入100 μl的分析用緩衝液(內含10 mMp -NPP、0.1 M醋酸鈉、0.1% Triton X-100，且pH約為5.5)，於37℃反應30分鐘後加入10 μl的氫氧化鈉(0.1 N)終止反應，以MRX II ELISA Reader(DYN-EX Tech.,USA)於波長410 nm測定吸光值，進而換算出不同條件處理下的細胞活性。結果示於第1及2圖。To measure cell viability, first aspirate the culture solution, wash once with 200 μL PBS, and add 100 μl of assay buffer (containing 10 mM p- NPP, 0.1 M sodium acetate, 0.1% Triton X-100, and pH approx. 5.5), after reacting at 37 ° C for 30 minutes, 10 μl of sodium hydroxide (0.1 N) was added to terminate the reaction, and the absorbance was measured by MRX II ELISA Reader (DYN-EX Tech., USA) at a wavelength of 410 nm, and then converted. Cell viability under different conditions. The results are shown in Figures 1 and 2.
如第1圖所示，當細胞以式(I)化合物處理後，活性下降，且隨著式(I)化合物的濃度上升以及處理時間增加，細胞活性下降越明顯，式(I)化合物對於HepG2的IC50 (抑制50%細胞活性之濃度)約為25 μM。類似的趨勢也可在A549細胞株中觀察到，參見第2圖，處理72小時後，式(I)化合物對於A549細胞的IC50 約為38 μM。As shown in Fig. 1, when the cells are treated with the compound of formula (I), the activity decreases, and as the concentration of the compound of formula (I) increases and the treatment time increases, the decrease in cell activity becomes more pronounced, and the compound of formula (I) is for HepG2. the IC 50 (50% inhibitory concentration of cell viability) is approximately 25 μM. Similar trends were also observed in the A549 cell line, see Fig. 2, 72 hours after treatment, the compound of formula (I) for the A549 cells IC 50 of about 38 μM.
當細胞受到外來刺激時，會活化環氧合酶(cyclooxygenase,COX)，使花生四烯酸(arachidonic acid)轉變成***素(prostaglandin G2,PG)，再轉變成不同的代謝物，如***素E2(PGE2)、***素D2(PGD2)等等，進而引起血管擴張與紅、腫、熱、痛等發炎反應。在細胞中有兩種COX酵素存在，分別為COX-1與COX-2，COX-1平時即存在於細胞中，於各組織中表現並合成***素以調節正常的生理功能，如調控腎血管阻力與維持胃腸道微血管的完整性。COX-2在正常細胞中的表現量非常低，只有在受到發炎性刺激或細胞素(cytokine)刺激時，才會被誘導生成，進而大量合成***素。COX-2能夠被選擇性的抑制，因其與COX-1的活化位置不同，若一待測物能抑制COX-2的表現量，即表示此待測物具有抑制發炎反應的活性。When the cells are stimulated by external stimuli, they activate cyclooxygenase (COX), which converts arachidonic acid into prostaglandin G2 (PG) and then transforms into different metabolites such as prostaglandins. E2 (PGE2), prostaglandin D2 (PGD2), etc., and then cause vasodilation and red, swollen, heat, pain and other inflammatory reactions. There are two kinds of COX enzymes in the cells, COX-1 and COX-2. COX-1 is usually present in cells, and it expresses and synthesizes prostaglandins in various tissues to regulate normal physiological functions, such as regulating renal blood vessels. Resistance and maintenance of microvascular integrity of the gastrointestinal tract. The amount of COX-2 in normal cells is very low, and it is only induced when it is stimulated by inflammatory stimuli or cytokine, and a large amount of prostaglandin is synthesized. COX-2 can be selectively inhibited because it is different from the activation position of COX-1. If a test substance can inhibit the expression of COX-2, it means that the analyte has an activity of inhibiting the inflammatory reaction.
在本實驗例中，以老鼠巨噬細胞RAW264.7為細胞模式，施以脂多醣(lipopolysaccharide,LPS)處理促使細胞內COX-2的表現量上升，再觀察式(I)化合物是否可抑制COX-2的表現，以評估式(I)化合物是否可做為抗發炎藥物。In this experimental example, the mouse macrophage RAW264.7 was used as the cell model, and lipopolysaccharide (LPS) treatment was used to increase the expression of COX-2 in the cells. Then, whether the compound of formula (I) can inhibit COX The performance of -2 to assess whether the compound of formula (I) can be used as an anti-inflammatory drug.
將老鼠巨噬細胞株RAW264.7培養在含有10%胎牛血清(FBS)的DMEM培養液中，並大致依實施例2.1之操作方式進行培育。The mouse macrophage cell line RAW264.7 was cultured in DMEM medium containing 10% fetal calf serum (FBS) and cultured in the same manner as in Example 2.1.
測定細胞內mRNA表現量 在直徑6公分的細胞培養盤中，放入3×105 個細胞，待其貼附至培養盤底部後，分別加入1、5、10、20μg/ml之式(I)化合物(相當於1.8、9、18、36μM之式(I)化合物)，於37℃下培養1小時後，再以脂多醣(1μg/ml)處理7小時，接著萃取細胞內的RNA。 Determination of intracellular mRNA expression In a 6 cm diameter cell culture dish, 3 × 10 5 cells were placed, and after attaching to the bottom of the culture plate, 1, 5, 10, 20 μg/ml were added, respectively. The compound (corresponding to 1.8, 9, 18, 36 μM of the compound of the formula (I)) was cultured at 37 ° C for 1 hour, and then treated with lipopolysaccharide (1 μg / ml) for 7 hours, followed by extraction of RNA in the cells.
取2μg RNA進行反轉錄反應(此反轉錄反應溶液中含有0.5 mM dNTPs、10 mM DTT、50 mM Tris-HCl(pH 8.3)、75 mM KCl,3 mM MgCl2 以及200 unit SuperScriptTM III RNase H-反轉錄酶)，於50℃下反應2小時，使mRNA完全反轉錄成cDNA。接著，進行即時聚合酶連鎖反應，取200 ng cDNA，與最終濃度為400 nM的一對引子，即5’-CCT CTG CGA TGC TCT TCC GA-3’(序列編號：1)及5’-CAC TTA TAC TGG TCA AAT CC-3’(序列編號：2)，混合均勻後，加入10 μl的反應緩衝液(含有dNTP混合物、緩衝液、Mg2+ 、Tag聚合酶以及SYBR綠I與參考顏料)，加水至終體積為20μL。以GADPH基因表現量為校正基準，並以未加藥處理之細胞組別為控制組，以應用生物系統7500快速即時PCR分析儀(Applied Biosystems 7500 Fast Real Time PCR)，測定PCR產物之螢光變化，並據此回推出各基因的表現量。依據製造商所提供的軟體計算出各表現基因的變化量。結果示於第3圖中。2 μg of RNA was subjected to reverse transcription reaction (this reverse transcription reaction solution contained 0.5 mM dNTPs, 10 mM DTT, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl 2 and 200 unit SuperScript TM III RNase H- Reverse transcriptase), reacted at 50 ° C for 2 hours to completely reverse transcribe mRNA into cDNA. Next, an instant polymerase chain reaction was performed to obtain 200 ng of cDNA with a pair of primers with a final concentration of 400 nM, namely 5'-CCT CTG CGA TGC TCT TCC GA-3' (SEQ ID NO: 1) and 5'-CAC TTA TAC TGG TCA AAT CC-3' (sequence number: 2), mix well, add 10 μl of reaction buffer (containing dNTP mixture, buffer, Mg 2+ , Tag polymerase and SYBR Green I and reference pigment) Add water to a final volume of 20 μL. The GADPH gene expression was used as the calibration standard, and the untreated drug cell group was used as the control group, and the fluorescence change of the PCR product was determined by using the Applied Biosystems 7500 Fast Real Time PCR (Applied Biosystems 7500 Fast Real Time PCR). And based on this, the performance of each gene is released. The amount of change in each gene was calculated based on the software provided by the manufacturer. The results are shown in Figure 3.
如第3圖所示，相較於控制組(即，只施以LPS處理的細胞)，隨著式(I)化合物濃度增加，其抑制COX-2基因表現的能力也隨之上升。根據本實驗結果初步顯示，式(I)化合物具有抑制發炎反應的活性，因此未來可開發成為抗發炎藥物。As shown in Fig. 3, as the concentration of the compound of formula (I) increases, the ability to inhibit the expression of the COX-2 gene increases as compared to the control group (i.e., cells treated with LPS only). According to the results of the present experiment, the compound of the formula (I) has an activity of inhibiting an inflammatory reaction, and thus it can be developed into an anti-inflammatory drug in the future.
惡性腫瘤轉移是癌症難以痊癒的原因之一，因此化合物能抑制癌轉移的能力極有醫藥價值。我們利用三株具有高轉移能力的癌細胞株，包括肺癌細胞株PC9和CL1-5，以及乳癌細胞株MDA-MB231,在體外以傷口癒合試驗(wound-healing assay)來分析癌細胞轉移的能力。Malignant tumor metastasis is one of the reasons why cancer is difficult to heal, so the ability of compounds to inhibit cancer metastasis is of great medical value. We used three cancer cell lines with high metastatic potential, including lung cancer cell lines PC9 and CL1-5, and breast cancer cell line MDA-MB231, to analyze the ability of cancer cell metastasis in vitro by wound-healing assay. .
在24孔培養皿中置入細胞培養間隔物(Culture-Insert，購自ibidi,Gmbh,Germany)，在每個有***細胞培養間隔物的培養孔中加入70 μl細胞懸浮液(含3x104 個細胞)，搖晃使細胞分布均勻，將培養皿置入37℃的二氧化碳培養箱中培養，待細胞貼附後移除細胞培養間隔物，可以看到細胞被培養間隔物造成的分隔(稱為『傷口』)，將培養基置換成含1% FBS的DMEM培養基，並加入式(I)化合物，分別於0小時、12小時、24小時及48小時以顯微鏡(10X物鏡)觀察『傷口』被細胞覆蓋的的狀況並拍照，再以Image-J軟體定量分析『傷口』的癒合。結果顯示於第4及5圖中。Cell culture spacers (Culture-Insert, purchased from ibidi, Gmbh, Germany) were placed in 24-well culture dishes, and 70 μl of cell suspension (containing 3×10 4 cells) was added to each well with the cell culture spacer inserted. Cells), shake the cells to make the cells evenly distributed, and place the culture dish in a carbon dioxide incubator at 37 ° C. After the cells are attached, remove the cell culture spacers, and you can see the separation of the cells by the culture spacers (called " Wounds), the medium was replaced with DMEM medium containing 1% FBS, and the compound of formula (I) was added, and the "wound" was observed by the microscope at 10 hours, 12 hours, 24 hours, and 48 hours, respectively. The situation and taking pictures, and then quantitative analysis of the "wound" healing with Image-J software. The results are shown in Figures 4 and 5.
如第4圖所示，相較於控制組(即，未經式(I)化合物處理的細胞)，隨著式(I)化合物濃度增加，其抑制PC9肺癌細胞爬行的能力也隨之上升。根據本實驗結果顯示，如第5圖所示，式(I)化合物具有抑制PC9和CL1-5兩種肺癌細胞以及MDA-MB231乳癌細胞爬行的活性，因此未來可開發成為抗癌轉移的藥物。As shown in Fig. 4, as the concentration of the compound of formula (I) increases, its ability to inhibit the crawling of PC9 lung cancer cells increases as compared to the control group (i.e., cells that have not been treated with the compound of formula (I)). According to the results of the present experiment, as shown in Fig. 5, the compound of the formula (I) has an activity of inhibiting the crawling of two lung cancer cells, PC9 and CL1-5, and MDA-MB231 breast cancer cells, and thus can be developed into a drug for anti-cancer metastasis in the future.
雖然本揭示內容已以實施方式揭露如上，然其並非用以限定本揭示內容，任何熟習此技藝者，在不脫離本揭示內容之精神和範圍內，當可作各種之更動與潤飾。舉例來說，上述的說明書、實施例與數據已提供了與本發明相關之結構與實施例之完整說明，任何習知技藝人士將可在不背離本發明精神與範疇下，參照上述一或多個特定實施方式進行各種改良或變化，這些改良或變化應該被視為本發明下附請求範圍之範疇。The present disclosure has been disclosed in the above embodiments, and is not intended to limit the scope of the disclosure, and various modifications and changes may be made without departing from the spirit and scope of the disclosure. For example, the above description, the embodiments and the data are provided to provide a complete description of the structure and the embodiments of the present invention. Any one skilled in the art can refer to the above one or more without departing from the spirit and scope of the present invention. Various modifications and changes may be made to the specific embodiments, and such modifications or variations are considered to be within the scope of the appended claims.
以下提供本揭示內溶附隨圖式之簡要說明，其中：A brief description of the dissolution patterns in this disclosure is provided below, where:
第1圖係依據本發明一實施方式以式(I)化合物來抑制HepG2肝癌細胞生長的示意圖；1 is a schematic diagram of inhibiting the growth of HepG2 liver cancer cells with a compound of formula (I) according to an embodiment of the present invention;
第2圖係依據本發明一實施方式以式(I)化合物來抑制A549肺癌細胞生長的示意圖；2 is a schematic diagram of inhibiting the growth of A549 lung cancer cells with a compound of formula (I) according to an embodiment of the present invention;
第3圖係依據本發明一實施方式以式(I)化合物來抑制發炎指標COX-2基因表現量的示意圖；Figure 3 is a schematic illustration of inhibition of the inflammatory index COX-2 gene expression by a compound of formula (I) in accordance with one embodiment of the present invention;
第4圖係依據本發明一實施方式以式(I)化合物來抑制PC9肺癌細胞體外癌轉移的示意圖；以及Figure 4 is a schematic diagram of inhibiting cancer metastasis of PC9 lung cancer cells in vitro by a compound of formula (I) according to an embodiment of the present invention;
第5圖係依據本發明一實施方式以式(I)化合物抑制PC9和CL1-5兩種肺癌細胞以及MDA-MB231乳癌細胞之體外癌轉移活性量化的示意圖。Figure 5 is a graph showing quantification of in vitro cancer metastasis activity of PC9 and CL1-5 lung cancer cells and MDA-MB231 breast cancer cells by a compound of formula (I) according to an embodiment of the present invention.
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