201223526 六、發明說明： 【發明所屬之技術領域】 本發明係關於一種抑制癌細胞基因表現的橘皮化合 物’特別是一種抑制癌細胞乙二醛酶(Glyoxaiase I )基因表 現的橘皮化合物及其在醫療上之用途。 【先前技術】 癌症的發生係由於細胞不正常增生所形成的異常腫 籲 塊，並且該腫塊會壓迫到其他組織或器官，如該腫塊發峰 轉移，不正常增生的細胞藉由血管轉移到人體的其他部位 - 即為惡性腫瘤’也就是俗稱的癌症。 - 事實上，癌細胞是正常細胞基因發生改變，引起細胞 不正常增生，一般來說，可將與引發癌症相關的基因歸類 為二：一類為致癌基因（Oncogene)，即該致癌基因發生活 化表現時，會使正常細胞發生異常的增生現象；另一類為 抑癌基因（Tumor Suppressor Gene) ’即該抑癌基因係於正常 鲁 細胞中表現’當該抑癌基因發生缺失或失活，使得正常細 胞發生異常的增生現象。舉例而言，根據學者專家們投入 癌症基因的研究發現，血癌、淋巴癌是跟致癌基因的活化 有關，而肺癌、乳癌、大腸直腸癌、肝癌、腦癌或膀胱癌 則與抑癌基因的缺失有關。正常細胞的癌化過程大致上是 人體與致癌因子接觸，造成正常細胞中致癌基因啟動或抑 癌基因缺失，使得正常細胞的代謝發生異常而發生不正常 的增生現象，正常細胞則演變成癌細胞，經過數十年後癌 細胞數量的累積，而形成腫瘤。 201223526 通常發現癌細胞之後會依據狀況進行手術切除癌細 胞，亚且辅以其他治療法如放射線治療、化學治療、藥物 治療等抑制癌細胞的再發，然而用以抑制癌細胞的習知藥 物係以針對細胞毒殺性(cy t〇i〇xic)抑制癌細胞，因此對正常 細胞亦有毒殺作用，當癌症病人服用該習知藥物時，雖然 使癌㈣1的％生現象得以控制，但癌症病人的正常細胞也 會受到抑㈣產生副作帛，如織賴癌細胞具有更專一 性的抑制途徑就能使癌症病人在服用藥物的過程中降低其 副作用，對癌症病人會是一大福音。 因此201223526 VI. Description of the Invention: [Technical Field] The present invention relates to an orange peel compound which inhibits the expression of a cancer cell gene, particularly an orange peel compound which inhibits the expression of a cancer cell Glyoxaiase I gene and For medical purposes. [Prior Art] The occurrence of cancer is caused by an abnormal swelling of the cells, and the tumor is pressed to other tissues or organs, such as the peak of the tumor, and the abnormally proliferating cells are transferred to the human body by blood vessels. The other part - the malignant tumor - is also known as cancer. - In fact, cancer cells are genetically altered in normal cells, causing abnormal proliferation of cells. In general, genes associated with cancer can be classified as two: one is an oncogene, that is, the oncogene is activated. In the performance, normal cells will undergo abnormal proliferation; the other is Tumor Suppressor Gene, which means that the tumor suppressor gene is expressed in normal Lu cells. When the tumor suppressor gene is deleted or inactivated, Abnormal proliferation of normal cells. For example, according to research conducted by scholars and experts on cancer genes, blood cancer and lymphoma are related to the activation of oncogenes, while lung cancer, breast cancer, colorectal cancer, liver cancer, brain cancer or bladder cancer are associated with the absence of tumor suppressor genes. related. The process of cancerization of normal cells is generally in contact with human carcinogenic factors, causing the initiation of oncogenes or the deletion of tumor suppressor genes in normal cells, causing abnormal metabolism of normal cells and abnormal proliferation. Normal cells evolve into cancer cells. After a few decades, the number of cancer cells accumulates to form a tumor. 201223526 It is usually found that cancer cells are surgically resected according to the condition, and other treatments such as radiation therapy, chemotherapy, drug therapy, etc., inhibit the recurrence of cancer cells, but the conventional drug system for inhibiting cancer cells It inhibits cancer cells against cytotoxicity (cy t〇i〇xic), and therefore has a toxic effect on normal cells. When a cancer patient takes the conventional drug, although the cancer of the cancer (4) 1 is controlled, the cancer patient The normal cells will also be inhibited. (4) The side effects of paralysis, such as the more specific inhibition of cancer cells, can make cancer patients reduce their side effects during the course of taking drugs, which will be a great boon for cancer patients. therefore
旰夕寻豕學者們針對癌細胞中有表現，而正常 細胞中無表現/或表現量較少的蛋白質（酶)進行研究，並針 對該蛋白質(酶)表現差異進行抑制癌細胞的研究，例如： 細胞核醣轉位酶（T〇p〇isomerase 1}、胸腺核合成酶 (Thymidilate Synthase)及乙二醛酶(吻咖⑻ι )。專家學旰 豕 豕 们 针对 豕 豕 豕 豕 豕 豕 豕 豕 豕 豕 针对 针对 针对 针对 针对 针对 针对 针对 针对 针对 针对 针对 针对 针对 针对 针对 针对 针对 针对 针对 针对 针对 针对 针对 针对 针对 针对 针对 针对 针对 针对 针对 针对: Cell ribose transposase (T〇p〇isomerase 1}, Thymidilate Synthase and Glyoxalase (Kiss coffee (8) ι). Experts
者們再藉由尋找能夠抑制這些蛋白質（酶)的藥物，進而= 癌細胞的增生得到控制，例如Campt(^是用來抑制癌細 胞中細胞核轉_㈣物，但癌症紅會有㈣的副作 用；Thymitage與職rexed是用來抑制癌細胞中胸腺核合 成酶’但癌症病人會有㈣、腎魏下降、自 等副作用。 Γ Τ 目前許多研究已證實乙 一醛酶與癌細胞之間且有關 聯性。由於癌細胞的快速增生伴隨著癌細胞中♦生/大 醣解個，㈣㈣用的反齡伴隨著細胞料 ^，、 因此癌細胞需要足夠的乙二搭酶清降置拉—+、 的主喜—〜山 月除累積在癌細胞中的細 胞毋素’-―癌細胞中的乙二輕酶基因被抑制，使癌細胞 1 4 丨丨 201223526 中乙二_的含量降低使得細胞毒素的清除率降低，癌細 胞會累積大量的細胞毒素，進而造成癌細胞〉周亡 (Apoptosis)。 目前，已知在大腸癌、胰臟癌、乳癌及腎臟癌等均可 觀察到大量的乙二_，顯示癌細胞需大量表現乙二藤酶 基因，即時清除癌細胞大量增生_產生的細胞毒素，以 維持癌細胞的正常生長’目此添加乙謂酶抑制劑， 使癌細胞的乙二_基因表現受限，可使癌細胞中細胞毒 素的累積，造成癌細胞凋亡。 至今為止尚未確立關於具有抑制癌細胞中乙二醛酶 基因表現的化合物相關報告，本發明係提供針對具有抑制 癌細胞中乙二醛酶基因表現的橘皮化合物。 【發明内容】 本發明之主要目的係提供一種橘皮化合物係具有抑 制癌細胞中的乙二搭酶(Glyoxalase I )基因表現。 本發明之次一目的係提供一種橘皮化合物係具有使 癌細胞發生細胞凋亡(Apoptosis)，進而抑制癌細胞之增生。 本發明再一目的係提供一種橘皮化合物，可有效抑制 癌細胞中乙二醛酶基因的表現，可應用於開發用以治療或 預防癌症之相關保健食品。 為達到前述發明目的，本發明所運用之技術手段包含 有： 一種抑制癌細胞基因表現的橘皮化合物，係用以抑^制 癌細胞乙二醛酶基因之表現。 201223526 種抑制癌細胞基因表現的橘皮化合物之用途，該橘 皮匕口物係製備用以治療或預防癌.症疾病的保健食品。 【實施方式】 λ為讓本發明之上述及其他目的、特徵及優雜更明顯 易懂，下文脾本發明之較佳實闕，並配合所附圖式， 作詳細說明如下： 本《月係提供針對具有抑制癌細胞中乙二盤酶基因 表現的橘皮化合物，該橘皮化合物包含有如第la圖所示之 橘紅素(Tangeritin)結構，其IupAC化學名稱為ms她 也础呵冬⑷刪㈣咖崎犯小⑻哪澤本·;以及 如第1b圖所不之川陳皮素(N〇biletin)結構，其KJPAC化學. 名稱為 2-(3,4-dimethoxyphenyl)-5?6,758-tetramethoxychromen- 4-one。該橘皮化合物屬於黃嗔Flav〇n〇id❼化合物係可專 -性抑制癌細胞中乙二酸酶基因表現，使癌細胞中之細胞 毒素無法清除，使癌細胞發生壯而無法增生，可有效抑 制癌細胞之擴散，因此，該橘皮化合物係可應用於製備治 療或預防癌症之保健食品，以改善癌症之治療現況。 本發明之—種抑制癌細胞中乙二㈣基因表現的橘 皮化合物’係可由各種人工合成方式獲得，或者由天然植 物(如柑、橘、橙等柑橘類之果皮）中萃取而得。 為證實本發明所提供橘皮化合物具有抑織細胞中 乙二酸酶基因表現之功能，將本發明之橘皮化合物與一癌 細胞株共同培養一段時間，再分別針對以下各試驗(a) 201223526 5亥癌細胞中乙一搭轉基因之表現量、（B)癌細胞之存活率、 (C)流式細胞儀偵測癌細胞之細胞週期以及(D)癌細胞致痒 因子NFkB之位置分布等分析試驗，紀錄該癌細胞株之二 胞生理變化。 本實施例係選用人類前骨髓性白血病癌細胞株 HL-60，該癌細胞株係購自台灣新竹食品工業科學研究 所，編號為BCRC 60027。本實施例將該癌細胞培養於_ 適當培養基中進行繼代培養，待該癌細胞增殖至培養容器 • 之7〜8分滿，取一緩衝液，將增殖培養之人類癌細胞株由 該〃培養容器之器壁沖刷至該緩衝液中，以方便進行該人類' - 癌細胞株之細胞計數，以及後續細胞存活率、細胞蛋白質 表現以及流式細胞儀之分析。 更δ羊5之’本賞施例係將該癌細胞增殖培養於含有 10%胎牛血清蛋白之RPMI培養基[10% fetal bovine serum(FBS)/R〇swell Park Memorial Institute medium]，待該 癌細胞長至培養容器之7成滿，以一商用EDTA緩衝液 鲁 （Ethylenediaminetetraacetic acid ; MERCK)重複沖洗該培養 容器’以便將貼壁生長的癌細胞沖刷至該EDTA緩衝液， 進行細胞計數。本發明l〇%FBS/RPMI培養基之配方如第1 表所示： 第1表：10%FBS/RPMI培養基(1升） 配方 劑量 (毫升；ml) PRMI 1640 900 201223526 胎牛血清蛋白（FBS) 100 抗生素(antibiotic) 10 穀胺醒酸(L-Glutamine) 10 ------------------------------ (A)癌細胞中乙二醛酶基因之表現量 癌細胞為了能夠大量增生’癌細胞會表現乙二酸酶基 因’較正常細胞產生較多的乙二醛酶。為證實添加本發明 之橘皮化合物可使癌細胞中乙二醛酶基因之表現被抑制， 以西方墨點法(Western Blotting)偵測該癌細胞中乙二齡酶 基因之表現量。 本實施例係將6組癌細胞（各組均含有一定細胞數量 之癌細胞’如 lxl〇4〜7 cells/ml)分別與 0、1〇、20、3〇、 及50微莫耳濃度(μΜ)之橘皮化合物共同培養24小時，係 為未添加之對照組AO、Α卜Α2、A3、Α4及Α5組，進行 西方墨點法之試驗，確認各組癌細胞中所含乙二醛酶之量。 更详言之’本實施例係自上述A0〜A5組中分別取出適 量癌細胞，以一定比例(較佳係1:1〇)稀釋於一商用蛋白質 抽取溶劑(lysis buffer ; Sigma)，進行均質處理，造成各組 細胞溶解、破裂，以獲得細胞内的蛋白質；再由一商用蛋 白質定量套組(Protein Assay Kit ; Invitrogen)分析由各組 (A0〜A5組)細胞所抽取的蛋白質總量’取等量蛋白質，利 用取自兔子之商用抗乙二醛酶多株抗體(Sigma)進行西方 墨點法分析。 ^ 晴參照第2圖西方墨點法之蛋白質染色結果所示，本 貝知例係以癌細胞中正常表現之β-actin的表現量為定量標 201223526 2，分別比較各組細胞中乙二醛酶的表現情況；由第2圖 結果，第AO、A1及A2組的細胞中具有較多量的乙二搭 • 酶表現(其染色訊號較明顯），而其他A3、A4及A5組細胞 • 中，乙二醛酶的表現量則相對較少（訊號較不明顯），並且^ 各組在乙二醛酶表現量之差異係與本發明橘皮化合物之劑 量呈現反向趨勢關係。由此可證實，本發明之橘皮化合^ 係可以抑制人類癌細胞中乙二醛酶的表現，而較高劑量(如 30〜50微莫耳濃度)之該橘皮化合物則可呈現較佳之抑制效 _ 果。 夕 (Β)癌細胞之存活率 ~、 進一步確認由於添加本發明之橘皮化合物使癌細胞 - 的乙一基因表現量減少，進而使癌細胞之增生現象被 抑制。ΜΤΤ鈿胞活性染色法(MTT assay)係一般用以測定細 胞活性的測試方法，利用活細胞的粒線體中所含有之去氣 酶(dehydrogenase)可代謝溶於癌細胞培養液中的黃色Μττ (3- (4,5-cimethylthiazol- 2-yl)- 2,5-diphenyl tetrazolium 鲁 bromide)，將MTT中的tetrazolium轉為一藍色產物 formazan，且堆積在細胞中，再添加DMSO將formazan溶 解測得細胞中formazan之含量’formazan於波長570nm之 處有吸收值，當活細胞數越多則57〇nm之吸收值越高，故 測量570nm之吸收值以估計細胞之存活率。 請參照第3圖，本實施例係將6組癌細胞（各組均含 有一定細胞數量之HL-60，如lxlO4〜7 cells/ml)分別與〇、 10、20、30、40及50微莫耳濃度(μΜ)之橘皮化合物共同 培養24小時，係為未添加之對照組Β0、Bl、Β2、Β3、Β4 201223526 及B5組，進行細胞活性染色法之試驗，確認各組癌細胞 之存活率。以B0組所測得之吸光值為基準，（癌細胞存活 率為100%)，其餘組別之癌細胞存活率分別依序為99%、 75%、35%、2G%及12%’由細胞活性染色法顯示當添加橘 皮化合物之濃度增加而該癌細胞之存活率會隨著減少，並 且選用濃度為30〜之劑量可有效地抑制癌細胞之細 胞活性’造成癌細胞之死亡；此外，該橘皮化合物對於癌 細胞的抑制作用係呈現一正向趨勢；因此，證實本發明之 橘农化合物確實具有影響癌細胞正常細胞表現之作用性， 以致癌細胞之生長、增生受阻。 (C)癌細胞的細胞凋亡 為證實癌細胞之存活率下降係與細胞凋亡(Ap〇pt〇sis) 或細胞的增生作用停滯(Rest)相關，以流式細胞儀進行癌細 胞之細胞週期測定。請參照第4a〜4d圖，以30μΜ橘皮化 合物與癌細胞共同培養’分別在培養第0(紀錄於第4a圖）、 3(纪錄於第4b圖）、6(紀錄於第4c圖）及9小時(紀錄於第 4d圖）進行流式細胞儀之分析結果。 本實施例係將分別在培養第〇、3、6及9小時，自選 取適夏之癌細胞’利用一商用螢光染劑[如pr〇pidium iodine (PI)、acridine organe (AO)及 fluorescein diacetate (FDA);They are also controlled by finding drugs that inhibit these proteins (enzymes), and then the proliferation of cancer cells, such as Campt (^ is used to inhibit the nucleus of cancer cells, but the side effects of cancer redness (4) Thymitage and rexed are used to inhibit thymocyte synthase in cancer cells, but cancer patients may have (four), decreased kidney and self-reporting side effects. Γ Τ Many studies have confirmed the association between beta-aldase and cancer cells. Because the rapid proliferation of cancer cells is accompanied by the ♦ raw/large sugar solution in cancer cells, (4) (4) the relative age is accompanied by the cell material ^, so the cancer cells need enough B2 enzyme to clear the pull-+, The main joy - ~ Shanyue in addition to the accumulation of cellulite in cancer cells - the cancer cell in the E2 light enzyme gene is inhibited, so that the cancer cell 14 4 丨丨 201223526 in the content of ethylene _ reduced cytotoxin The clearance rate is reduced, and cancer cells accumulate a large amount of cytotoxins, which in turn cause cancer cells to become "Apoptosis." Currently, it is known that a large amount of ethylene is observed in colorectal cancer, pancreatic cancer, breast cancer, and kidney cancer. _ It shows that cancer cells need to display a large number of genes, and instantly eliminate the cytotoxin produced by the proliferation of cancer cells to maintain the normal growth of cancer cells. This is to add an enzyme inhibitor to the B cells. Restricted, can cause accumulation of cytotoxins in cancer cells, causing apoptosis of cancer cells. So far, there has not been established a report on compounds having inhibitory expression of glyoxalase genes in cancer cells, and the present invention provides for targeting cancer cells. The present invention aims to provide an orange peel compound which inhibits the expression of the Glyoxalase I gene in cancer cells. The invention provides an orange peel compound which has apoptosis (Apoptosis) and inhibits proliferation of cancer cells. A further object of the present invention is to provide an orange peel compound which can effectively inhibit the glyoxalase gene in cancer cells. Performance can be applied to the development of health foods for treating or preventing cancer. To achieve the aforementioned objectives, this The technical means used in the invention include: an orange peel compound which inhibits the expression of cancer cell genes, and is used for suppressing the expression of the cancer cell glyoxalase gene. 201223526 The use of orange peel compounds for inhibiting the expression of cancer cell genes, The cellulite mouthwash is a health food for treating or preventing cancer diseases. [Embodiment] λ is to make the above and other objects, features and advantages of the present invention more obvious and easy to understand. Preferably, and in conjunction with the accompanying drawings, the detailed description is as follows: The "Monthlet" provides an orange peel compound having the performance of inhibiting the expression of the E2CD enzyme gene in cancer cells, and the orange peel compound comprises as shown in FIG. The structure of the Tangeritin, the chemical name of the IupAC is ms, she is also the foundation of the winter (4), and the (4) Kawasaki is a small (8) Nakamoto, and the structure of N〇biletin, as shown in Figure 1b. KJPAC chemistry. The name is 2-(3,4-dimethoxyphenyl)-5?6,758-tetramethoxychromen- 4-one. The orange peel compound belongs to the Astragalus Flav〇n〇id❼ compound system, which can specifically inhibit the expression of the oxalic acid gene in cancer cells, so that the cytotoxin in the cancer cells cannot be cleared, and the cancer cells can grow strong and cannot proliferate, and can be effective. Inhibition of the spread of cancer cells, therefore, the orange peel compound can be applied to the preparation of health foods for treating or preventing cancer to improve the treatment status of cancer. The orange peel compound of the present invention which inhibits the expression of the ethylene (tetra) gene in cancer cells can be obtained by various artificial synthesis methods or by extraction from natural plants (such as citrus peels such as mandarin, orange, and orange). In order to confirm that the orange peel compound provided by the present invention has the function of expressing the oxalic acid gene in the weaving cell, the orange peel compound of the present invention is cultured together with a cancer cell strain for a period of time, and respectively for the following tests (a) 201223526 The expression of transgenic genes in B-cell cancer cells, (B) the survival rate of cancer cells, (C) the cell cycle of cancer cells detected by flow cytometry, and (D) the location distribution of NFkB in cancer cells Test to record the physiological changes of the two cells of the cancer cell line. In this example, a human promyelocytic leukemia cancer cell line HL-60 was selected, which was purchased from the Hsinchu Food Industry Research Institute of Taiwan under the number BCRC 60027. In this embodiment, the cancer cells are cultured in a suitable medium for subculture, and the cancer cells are propagated to the culture container for 7 to 8 minutes, and a buffer is taken to proliferate the cultured human cancer cell line from the sputum. The walls of the culture vessel are flushed into the buffer to facilitate cell counting of the human '-cancer cell line, as well as subsequent cell viability, cellular protein performance, and flow cytometry analysis. The more δ sheep 5's present application is to culture the cancer cells in RPMI medium containing 10% fetal bovine serum albumin [10% fetal bovine serum (FBS) / R〇swell Park Memorial Institute medium] The cells were grown to 7% of the culture vessel, and the culture vessel was repeatedly washed with a commercial EDTA buffer (Ethylenediaminetetraacetic acid; MERCK) to wash the adherently grown cancer cells into the EDTA buffer for cell counting. The formulation of the present invention 1% FBS/RPMI medium is shown in Table 1: Table 1: 10% FBS/RPMI medium (1 liter) Formulation dose (ml; ml) PRMI 1640 900 201223526 Fetal bovine serum albumin (FBS) 100 antibiotics (antibiotic) 10 glutamine citrate (L-Glutamine) 10 ------------------------------ (A) cancer The expression of the glyoxalase gene in the cells in order to be able to proliferate in a large amount, 'the cancer cells will exhibit the oxalate gene', which produces more glyoxalase than the normal cells. To confirm that the orange peel compound of the present invention was added, the expression of the glyoxalase gene in the cancer cells was inhibited, and the expression amount of the second instar enzyme gene in the cancer cells was detected by Western Blotting. In this embodiment, 6 groups of cancer cells (each group containing a certain number of cells of cancer cells such as lxl〇4~7 cells/ml) and 0, 1〇, 20, 3〇, and 50 micromolar concentrations, respectively ( The orange peel compound of μΜ) was co-cultured for 24 hours, and the untreated AO, Α卜Α2, A3, Α4, and Α5 groups were tested in the Western blotting method to confirm the glyoxal contained in each group of cancer cells. The amount of enzyme. More specifically, the present embodiment extracts an appropriate amount of cancer cells from the above A0~A5 groups, and dilutes them in a commercial protein extraction solvent (Sigma buffer) in a certain ratio (preferably 1:1 ,) for homogenization. Treatment, causing each group of cells to dissolve and rupture to obtain intracellular proteins; and then analyzing the total amount of protein extracted from each group (A0~A5 group) by a commercial protein quantitative kit (Protein Assay Kit; Invitrogen) Equal amounts of protein were taken and Western blot analysis was performed using a commercial anti-glyoxalase antibody (Sigma) from rabbits. ^ Clear reference Figure 2 shows the protein staining results of the Western blot method. The results of this method are based on the normal expression of β-actin in cancer cells as the quantitative standard 201223526 2, respectively, comparing the glyoxal in each group of cells. The performance of the enzyme; from the results of Figure 2, the cells in the AO, A1 and A2 groups have a greater amount of Ethylene-enzyme (the staining signal is more obvious), while the other cells in the A3, A4 and A5 groups The performance of glyoxalase is relatively small (signal is less obvious), and the difference in the amount of glyoxalase exhibited by each group is inversely related to the dose of the orange peel compound of the present invention. It can be confirmed that the orange peel compound of the present invention can inhibit the performance of glyoxalase in human cancer cells, and the orange peel compound of a higher dose (e.g., 30 to 50 micromolar concentration) can be better. Inhibitory effect. Survival rate of cancer cells in the evening ( ~) Further, it was confirmed that the expression of the B-gene of the cancer cells was reduced by the addition of the orange peel compound of the present invention, and the proliferation of cancer cells was suppressed. The cell viability staining method (MTT assay) is a test method generally used for measuring cell activity, and the dehydrogenase contained in the mitochondria of living cells can be metabolized and metabolized in the yellow Μττ of the cancer cell culture solution. (3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide), converts tetrazolium from MTT into a blue product formazan, and accumulates in cells, then adds DMSO to dissolve formazan The amount of formazan in the cells was measured. The amount of formazan had an absorption value at a wavelength of 570 nm. When the number of living cells increased, the absorption value at 57 〇 nm was higher. Therefore, the absorption value at 570 nm was measured to estimate the survival rate of the cells. Please refer to Fig. 3, in this embodiment, 6 groups of cancer cells (each group contains a certain number of cells of HL-60, such as lxlO4~7 cells/ml) and 〇, 10, 20, 30, 40 and 50 micro respectively. The cellophane compound of the molar concentration (μΜ) was co-cultured for 24 hours, and the untreated control group Β0, B1, Β2, Β3, Β4 201223526 and B5 groups were tested for cell activity staining, and the cancer cells of each group were confirmed. Survival rate. Based on the absorbance measured by the B0 group (the cancer cell survival rate was 100%), the survival rates of the remaining groups of cancer cells were 99%, 75%, 35%, 2G%, and 12%, respectively. The cell-active staining method shows that when the concentration of the added orange peel compound is increased, the survival rate of the cancer cell is decreased, and the concentration of 30~ is selected to effectively inhibit the cell activity of the cancer cells to cause cancer cell death; The inhibitory effect of the orange peel compound on cancer cells is a positive trend; therefore, it has been confirmed that the orange farm compound of the present invention does have an effect of affecting the normal cell expression of cancer cells, so that the growth and proliferation of cancer cells are hindered. (C) Apoptosis of cancer cells To confirm that the decrease in the survival rate of cancer cells is related to apoptosis (Ap〇pt〇sis) or the proliferation of cells (Rest), and the cells of cancer cells are flow cytometry. Cycle determination. Please refer to pictures 4a to 4d, and co-culture with 30μΜ orange peel compound and cancer cells' in culture 0 (recorded in Figure 4a), 3 (recorded in Figure 4b), and 6 (recorded in Figure 4c). The results of the analysis by flow cytometry were performed for 9 hours (recorded in Figure 4d). In this example, a commercial fluorescent dye [such as pr〇pidium iodine (PI), acridine organe (AO), and fluorescein will be utilized in the selection of summer cancer cells at the third, third, sixth, and ninth hours of culture. Diacetate (FDA);
Betcon Dickinson]進行細胞染色’再通入一流式細胞儀 (FACScan laser flow cytometer analysis system ； Becton Dickenson) ’分析該癌細胞在波長488 nm之雷射光激發下 所產生之散射光’以推得該癌細胞在各培養階段所呈現之 細胞凋亡狀態。 201223526 ’添加本發明之橘皮化合物於癌細Betcon Dickinson] performs cell staining 'FACScan laser flow cytometer analysis system; Becton Dickenson' 'analyzes the scattered light generated by the cancer cell under excitation of 488 nm laser light to extract the cancer The state of apoptosis exhibited by the cells at each stage of culture. 201223526 'Adding the orange peel compound of the present invention to cancer
卜(’ 4c圖）’ 9個小時後增加為4〇 %%的癌細胞發生細 胞〉周亡(第4d圖）’表示添加本發明之橘皮化合物使癌細胞 Ik著間增加發生細胞;周亡，造成癌細胞之存活率下降， 而非細胞增生作崎滯之朗，本發日狀化合物係可造成 癌細胞之細胞;壯，並且隨料科_增加，該癌細胞 晴翏照第4a〜4d圖，添加本發 胞之培養液後第〇小時之癌細胞僅 一對照組（第4a圖），經過3個小陆 凋亡的狀態則逐漸加劇。 (Ρ)癌細胞之致癌基因轉錄因子_邪也 為證實癌細胞發生細胞凋亡係由於添加本發明之橘 皮化合物相關’以共軛焦螢光顯微鐃觀察與調控致癌基因 轉錄相關的§周控因子(Nuciear Factor κ-light-chain-enhancer of activated B cells)於癌細胞中分佈 的情形’其中NFkB以螢光標定追蹤，當NFkB活化會進 入細胞核中’使細胞發生增生作用，因此可利用NFkB做 為標定癌細胞增生之現象。 以0和30μΜ橘皮化合物與癌細胞共同培養24小時， 利用免疫染色的方式標定一特定轉錄因子在兩組癌細胞中 之分布知況’進一步推定兩組癌細胞的細胞狀態。更詳言 之，本發明係於培養第24小時分別自添加〇和30μΜ橘皮 化合物共同培養之癌細胞組別中挑取適量細胞，利用取自 於兔子之商用抗NFkB抗體(Sigma)進行免疫染色反應，再 以一共輛焦顯微鏡(Leica TCS SP2 confocal microscope)觀 201223526 祭，以測定兩組癌細胞中NFkB之分布狀況與細胞狀態。 D月參如、弟5a及5b圖，將癌細胞添加本發明之橘皮化 δ物培養經過24小時，以螢光標定癌細胞2NFkB，未添 加本發明之橘皮化合物之對照組(第5a圖），癌細胞之細胞 核中存在有NFkB，而相較於添加3〇μΜ本發明之橘皮化合 物之組別（第5b圖），NFkB無法進入癌細胞之細胞核中， 使得癌細胞無法進行細胞增生。由試驗(D)可證實本發明之 橘皮化合物可使癌細胞中調控細胞增生之轉錄因子(NFkB) 無法進入癌細胞的細胞核中，使癌細胞無法進行細胞增生。 承上所述，本發明一種抑制癌細胞基因表現的橘皮化 合物，包含有橘紅素及川陳皮素，係可以阻斷的作 用路徑’抑制人類癌細胞之乙二醛酶基因表現，因此，癌 細胞無法有效排除由醣類代謝所產生的細胞毒素，使大量 毒素堆積於癌細胞内，影響其細胞活性，最後引發癌細胞 之自發性的死亡(apoptosis)，進而抑制癌細胞的增生與擴 散，同理可知，由二烯丙基硫醚類相關之化合物，包含 2，l，2-Dithiol-3-thione、Oltipraz、ADT 及其衍生物所組成之 族群皆具有阻斷NFkB的作用路徑，抑制人類癌細胞之乙 一酸r I#基因表現’進而達到抑制癌細胞增生與擴散的功 效；據此，本發明之橘皮化合物確實具有抑制癌細胞乙二 醛酶基因表現之功能’可應用於開發用以預防或治療癌症 之藥物化合物、食品添加成分以及相關保健食品，具有提 升臨床醫療品質之功效。 本發明一種抑制癌細胞基因表現之橘皮化合物，係可 以做為一藥物活性成分(active substrate)，以各種方式單 —12 — 201223526 獨’或者結合至少-種藥物佐劑、藥物載劑、其他副成分、 營養成分或錄藥物雜絲共同㈣各齡物個體，較 佳係經由口服方式定期給予各種生物個體—適#劑量，如 每天給予！〜2次，每次給予3〇〜4〇微莫耳濃度(μΜ)/每單位 重量公斤(kg);再者，本發明之橘皮化合物可以藉由任何 食品加工方法製成各種習知造型的口験劑(城、膠囊、 粉末或義)或相關食品(飲品、乳品或精力料），以符合 各種生物個體之使用。 σ 因此’本發明之橘皮化合物具有抑制癌細胞中的乙二 酸酶基因表現’使癌細胞中乙二_之含量不^以清除癌 細胞内的毒素累積’進而達到抑制癌細胞增生之功效/ 本發明之橘皮化合物具有使癌細胞發生細胞祠亡，進 而達到抑制癌細胞增生之功效。 本發明之橘皮化合物，具有抑制癌細胞生長及擴散之 特性’因此’可以應用於開發用以治療或預防癌症之相關 保健食品，以提升臨床醫療的水準，為本發明之功效。 雖然本發明已利用上述較佳實施例揭示，然其並非用 以，定本發明’任何熟習此技藝者在不脫離本發明之精神 和範圍之内，相對上述實關進行各觀動鄕改仍屬本 發明所保叙肋射，因此本發明之職 之申請專利範圍所界定者為準。 · 【圖式簡單說明】 第la圖：本發明一種抑制癌細胞乙二醛酶基因表現之 —13 — 201223526 橘皮化合物化學結構示意圖。 第lb圖：本發明一種抑制癌細胞乙二醛酶基因表現之 橘皮化合物化學結構示意圖。 第2圖：本發明一種抑制癌細胞乙二醛酶基因表現之橘 皮化合物之西方墨點法分析結果。 第3圖：本發明一種抑制癌細胞乙二醛酶基因表現之橘 皮化合物之細胞活性染色分析結果。 第4a圖：本發明一種抑制癌細胞乙二醛酶基因表現之 橘皮化合物之流式細胞儀分析結果之一。 第4b圖：本發明一種抑制癌細胞乙二醛酶基因表現之 橘皮化合物之流式細胞儀分析結果之二。 第4c圖：本發明一種抑制癌細胞乙二醛酶基因表現之 橘皮化合物之流式細胞儀分析結果之三。 第4d圖：本發明一種抑制癌細胞乙二醛酶基因表現之 橘皮化合物之流式細胞儀分析結果之四。 第5a圖：本發明一種抑制癌細胞乙二醛酶基因表現之 橘皮化合物之免疫染色結果之一。 第5b圖：本發明一種抑制癌細胞乙二醛酶基因表現之 橘皮化合物之免疫染色結果之二。 【主要元件符號說明】 〔無〕Bu ('4c map)' increased to 4%% of cancer cells in 9 hours after the occurrence of cells> Weekly death (Fig. 4d)' indicates the addition of the orange peel compound of the present invention to increase the growth of cancer cells Ik; Death, resulting in a decline in the survival rate of cancer cells, rather than cell proliferation, the local compound can cause cancer cells; strong, and with the increase in the number of cells, the cancer is sunny 4a ~4d, after adding the cell culture medium, the cancer cells in the third hour were only in the control group (Fig. 4a), and the state of apoptosis after 3 small colonies gradually increased. (Ρ) Oncogene transcription factor of cancer cells _ evil also confirms the apoptosis of cancer cells due to the addition of the orange peel compound of the present invention. § Peripheral control related to the regulation of oncogene transcription by conjugated focal fluorescence microscopy The distribution of factors (Nuciear Factor κ-light-chain-enhancer of activated B cells) in cancer cells 'where NFkB is tracked by the fluorescing cursor, and when NFkB is activated, it enters the nucleus', causing the cells to proliferate, so NFkB can be utilized. As a phenomenon to calibrate cancer cell proliferation. The 0 and 30 μΜ orange peel compounds were co-cultured with cancer cells for 24 hours, and the distribution of a specific transcription factor in the two groups of cancer cells was calibrated by immunostaining to further estimate the cell state of the two groups of cancer cells. More specifically, the present invention selects an appropriate amount of cells from the cancer cell group co-cultured with the addition of sputum and 30 μ Μ orange peel compound at the 24th hour of culture, and is immunized with a commercial anti-NFkB antibody (Sigma) obtained from rabbits. The staining reaction was carried out on a Leica TCS SP2 confocal microscope to observe the 201223526 sacrifice to determine the distribution and cell status of NFkB in the two groups of cancer cells. In the D-month reference, the brothers 5a and 5b, the cancer cells were added to the orange peeling delta of the present invention for 24 hours, and the cancer cells 2NFkB were set by the cursor, and the control group of the orange peel compound of the present invention was not added (the 5a). Figure VIII, NFkB is present in the nucleus of cancer cells, and NFkB cannot enter the nucleus of cancer cells, making it impossible for cancer cells to perform cells, compared to the group of 3 〇μΜ of the orange peel compound of the present invention (Fig. 5b). Hyperplasia. From the test (D), it was confirmed that the cellulite compound of the present invention can prevent the transcription factor (NFkB) which regulates cell proliferation in cancer cells from entering the nucleus of cancer cells, thereby preventing cancer cells from undergoing cell proliferation. According to the above, an orange peel compound which inhibits the expression of a cancer cell gene, comprising an orange pigment and a nobiletin, can block the action pathway of 'suppressing the glyoxal enzyme gene expression of a human cancer cell, and therefore, the cancer cell It is impossible to effectively eliminate the cytotoxin produced by carbohydrate metabolism, causing a large amount of toxin to accumulate in cancer cells, affecting its cell activity, and finally triggering spontaneous apoptosis of cancer cells, thereby inhibiting the proliferation and spread of cancer cells. It is known that the compounds composed of diallyl sulfide-related compounds, including 2,1,2-Dithiol-3-thione, Oltipraz, ADT and their derivatives all have a pathway to block NFkB and inhibit humans. The effect of the ethyl acetate-I I gene expression of cancer cells in order to inhibit the proliferation and spread of cancer cells; accordingly, the orange peel compound of the present invention does have a function of inhibiting the expression of the glyoxalase gene of cancer cells', and can be applied for development. The pharmaceutical compounds for preventing or treating cancer, food-added ingredients, and related health foods have the effect of improving clinical medical quality. The orange peel compound for inhibiting the expression of cancer cell genes can be used as an active substrate, in various ways, or in combination with at least one drug adjuvant, drug carrier, other Sub-components, nutrients, or drug-mixed silks (4) Individuals of all ages, preferably by regular oral administration to various biological individuals - appropriate doses, such as daily! ~2 times, each time giving 3〇~4〇 micromolar concentration (μΜ) / kilogram (kg) per unit weight; further, the orange peel compound of the present invention can be made into various conventional shapes by any food processing method Oral sputum (city, capsule, powder or meaning) or related foods (drinks, dairy or energy) to meet the use of various biological individuals. σ Therefore, the 'orange peel compound of the present invention has the effect of inhibiting the expression of the oxalic acid gene in the cancer cells, so that the content of the ethylene glycol in the cancer cells is not sufficient to eliminate the accumulation of toxins in the cancer cells, thereby achieving the effect of inhibiting the proliferation of cancer cells. / The orange peel compound of the present invention has the effect of causing cell death of cancer cells, thereby inhibiting the proliferation of cancer cells. The orange peel compound of the present invention has a property of inhibiting the growth and spread of cancer cells. Therefore, it can be applied to the development of a health food for treating or preventing cancer, so as to improve the level of clinical care, and is an effect of the present invention. The present invention has been disclosed in the above-described preferred embodiments, and is not intended to be used in any way. The invention is described in terms of the ribs and is therefore defined by the scope of the patent application of the present invention. · [Simplified description of the schema] Figure la: A schematic diagram of the chemical structure of the orange peel compound of the present invention, which inhibits the expression of the glyoxalase gene of the cancer cell. Figure lb is a schematic diagram showing the chemical structure of an orange peel compound which inhibits the expression of the glyoxalase gene of cancer cells. Fig. 2 is a view showing the results of Western blot analysis of an orange peel compound which inhibits the expression of the cell glyoxalase gene of the present invention. Fig. 3 is a graph showing the results of cell staining analysis of an orange peel compound which inhibits the expression of the cell glyoxalase gene of the present invention. Fig. 4a is a graph showing the results of flow cytometry analysis of an orange peel compound which inhibits the expression of the glyoxalase gene of cancer cells. Fig. 4b is a diagram showing the results of flow cytometry analysis of an orange peel compound which inhibits the expression of the glyoxalase gene of the cancer cell of the present invention. Fig. 4c is a graph showing the results of flow cytometry analysis of an orange peel compound which inhibits the expression of the glyoxalase gene of the cancer cell of the present invention. Fig. 4d is a graph showing the results of flow cytometry analysis of an orange peel compound which inhibits the expression of the glyoxalase gene of the cancer cell of the present invention. Fig. 5a: One of the results of immunostaining of an orange peel compound which inhibits the expression of the glyoxalase gene of the cancer cell of the present invention. Fig. 5b is a graph showing the results of immunostaining of an orange peel compound which inhibits the expression of the cell glyoxalase gene of the present invention. [Main component symbol description] [None]