201233384 四、指定代表圖： (一) 本案指定代表圖為：無。 (二) 本代表圖之元件符號簡單說明： 五、本案若有化學式時，請揭示最能顯示發明特徵的化學式： 無。 六、發明說明： 201233384 【發明所屬之技術領域】 本發明係—種龍眼籽多酚提取物之生產方法及應用， 特別係私一種可以提升由龍眼籽多酚提取物中多酚類物質 含量的生產方法和利用其抑制癌細胞增生的應用。 【先前技術】 植物袍果與種子的多酚物質已經被證實與癌症發生率 降低有密切的關係。龍眼是是台灣常見的夏季水果之一， 也节見於中國以及東南亞一帶，其係屬於無患子科的常綠 Φ 阿木，其果實即稱龍眼，又稱桂圓，龍眼籽常常被當作廢 棄物丟棄，然而近年的許多研究卻發現其含有高量的多酚 物質且具有抗氧化及自由基清除的能力，本草綱目中說： 食品以荔枝為貴，而資益以龍眼為良。」，蘇頌的圖經 本草也說：『龍眼味甘平無毒，主治五臟邪氣，安志壓食， 久服強魄聰明，輕身不老，通神明。』，龍眼果肉主治虛 勞赢弱’失眠健忘，虛瀉濁，產後浮腫等症。果殼、花、 及根等也可以作為配伍的中藥（黃鎖義等；2006)。 φ 龍眼核佔龍眼果約20%的重量，古籍記載主要用在外傷 的定疼止血與生肌，近年來在龍眼籽的成分研究上發現其 主要含有 gallic acid、corilagin、ellagic acid(Rangladil〇k et al.，2005; Soong andBarlow，2005)，然關於龍眼籽萃取物的 研九多者眼於自由基清除與抑制tyrosinase活性的能力 (Rangladilok et al.，2007)，然而，其富含多酚物質的特性， 卻還沒有對防癌或抗癌的功能方面有任何的研究與開發。 【發明内容】 本發明之一目的在於提供一種龍眼籽多酚提取物之生 201233384201233384 IV. Designated representative map: (1) The representative representative of the case is: None. (2) A brief description of the symbol of the representative figure: 5. If there is a chemical formula in this case, please disclose the chemical formula that best shows the characteristics of the invention: None. VI. Description of the invention: 201233384 [Technical field to which the invention belongs] The present invention relates to a method and a method for producing a longan seed polyphenol extract, in particular, a private one can enhance the content of polyphenols in the polyphenol extract of longan seeds. Production methods and applications for inhibiting proliferation of cancer cells. [Prior Art] Polyphenols in vegetal and seed have been shown to be closely related to the reduction in cancer incidence. Longan is one of the common summer fruits in Taiwan. It is also found in China and Southeast Asia. It belongs to the evergreen Φ Amu of the Sapindaceae family. Its fruit is called longan, also known as longan. Longan seeds are often discarded as waste. However, many studies in recent years have found that it contains high amounts of polyphenols and has the ability to resist oxidation and free radical scavenging. The Compendium of Materia Medica says: Food is lychee expensive, and the benefits are good for longan. Su Shi's picture of the scriptures also said: "Longan taste Ganping is non-toxic, attending the five evil spirits, Anzhi pressure food, long-wearing strong and clever, light and not old, through the gods. 』, Longan pulp is inherited from the virtual work to win the weak 'insomnia, forgetfulness, vaginal turbidity, postpartum edema embolism. Fruit shells, flowers, and roots can also be used as compatible Chinese medicines (Huang Suoyi et al.; 2006). φ Longan nucleus accounts for about 20% of the weight of longan fruit. The ancient records are mainly used for painful hemostasis and myogenic muscles in trauma. In recent years, it has been found in the composition of longan seeds mainly containing gallic acid, corilagin, ellagic acid (Rangladil〇k Et al., 2005; Soong and Barlow, 2005), but the research on longan seed extracts has the ability to scavenge free radicals and inhibit tyrosinase activity (Rangladilok et al., 2007), however, it is rich in polyphenols. The nature of the substance, but there is no research and development on the function of anti-cancer or anti-cancer. SUMMARY OF THE INVENTION One object of the present invention is to provide a longan seed polyphenol extract for life 201233384
令的多齡 眼籽乾粉； 下列步驟： 後以研磨機將龍眼籽粉碎為龍 萃取.將此龍眼籽乾粉浸泡於一醋酸丙酮 生一粗萃液； 一醋酸丙酮溶液，以產 蒸餾：將此粗萃液進行分離並收集溶液離分，The multi-aged eye seed dry powder; the following steps: After the longan seed is crushed into a dragon extract by a grinder, the longan seed dry powder is soaked in a crude acetic acid acetone solution; an acetic acid acetone solution to produce distillation: The crude extract is separated and the solution is separated.
劑進行提取，以產生—提取物；及 乾燥：除去提取液中的乙酸乙酯溶劑，並進行冷凍乾 燥產生粉末狀的龍眼籽多酚提取物。 本發明之另一目的在於提供一種龍眼籽多酚提取物的 應用，其係可以有效抑制癌症細胞之聚落形成，亦可抑制 癌症細胞的生長週期’進而有效控制癌症細胞的發展進 程’本發明之龍眼籽多酚提取物係可應用於大腸直腸癌細 胞、乳癌細胞、肺癌細胞、子宮頸癌細胞和肝癌細胞的控 制。 【實施方式】 以下僅以實施例對於龍眼籽多酚提取物之生產方法及 其應用進行詳細之說明。 實施例一 本實施例係說明龍眼籽多酚提取物之生產方法，其係 在收集龍眼籽後，以清水洗滌龍眼籽兩次並且使其乾燥’ 乾燥後的龍眼籽利用研磨機加以研磨成龍眼籽粉末’取100 201233384 公克龍眼籽粉末浸泡於800毫升醋酸丙，I溶液（1 〇〇mM醋 酸溶解於30%體積百分比之丙酮溶液，pH值4.8 )中，於 室溫下浸置12小時以形成一粗萃物，將此粗萃物利用Ν〇ι 濾紙過濾’並離心以收集溶液離分，將溶液離分利用轉動 式蒸餾機在減壓及水溫3 5 °C以下的水浴槽中進行蒸館以除 去丙酮並形成一蒸餾液，收集此蒸餾液以2〇〇毫升乙酸乙 酯（ethyl acetate)為溶劑提取四次並收集提取後的提取液， 在以蒸餾方式將此提取液中的乙酸乙酯去除並進行冷康乾 燥產生粉末狀的龍眼籽多酚提取物。 總酚類化合物之含量測定：係將〇· 1毫升濃度為每毫升 2毫克的龍眼軒多紛提取物與1毫升鱗翻酸紛試劑 (Folin-Ciocalteu reagent)及2毫升混合後均質震盪，再加 入5毫升濃度為20%的碳酸納水溶液進行2〇分鐘避光反 應，測定波長735nm的吸光值，本檢測係以濃度為〇、 15.625、31.25、62.5、125、250、500 和 1〇〇〇 (微克 / 毫升） 的沒食子酸建立標準曲線。 總黃綱類化合物之含量測定：係將2 5毫克的龍眼籽多 酚提取物溶解於80%的乙醇中以形成一龍眼籽多酚提取物 溶液’取20微升的龍眼籽多齡提取物溶液與122微升的二 次水和6微升濃度為5°/。（克/毫升）的亞硝酸納（NaN〇2 ) 進行反應六分鐘後’加入12微升濃度為10%的氣化鋁 (AlCl3(6H2〇))在反應五分鐘後，再加入40微升濃度為ιΜ 的氫氧化鈉進行反應’並檢測波長490nm的吸光值，本檢 測係以濃度為0、125、250、500、1〇〇〇、和2000 (微克/ 毫升）的兒茶素（Catechin)建立標準曲線。 201233384 縮合單寧（condensed tannins )之含量測定：係將25 毫克的龍眼籽多酚提取物粉末溶解於80%的乙醇中以形成 一龍眼籽多酚提取物溶液，取5微升的龍眼籽多酚提取物 /谷液與150微升漠度為4%(克/毫升）的香草路（vanillin，溶 解於甲醇中）進行均質混合後，加入75毫升濃度為12N的 鹽酸混合後在25°C的溫度下進行20分鐘的避光反應，並測 定波長490nm的吸光值，本檢測係以濃度為〇、125、25〇、 500、1〇〇〇、和2000 (微克/毫升）的兒茶素（Catechin)建立 標準曲線》 前花青素（Proanthocyanidin)含量測定：取50微升濃度 為每毫升1毫克之龍眼籽多酚提取物樣品加入6〇〇微升丁 醇/鹽酸混合液（n-butanol/HCl，95:5 v/v)，在避光環境下 以震盪器均勻混合，再加入6.8微升濃度為1 〇〇/0之硫酸鐵銨 (NH4Fe(S〇4)2 . 12仏0 )均勻混合，以95〇c水浴槽中反應 4〇分鐘，混合均勻後取出100微升混合液檢測吸光波長 55〇nm下之吸光值，並以氣化矢車菊素（cyanidin 心） 建立標準曲線（沒有數據？）。 请參見表1所示，若是將此龍眼籽多酚提取物進行比 色以進行其中多酚類物質含量分析可以發現，利用乙醇乙 酯為溶劑進行提取時，所產生的龍眼籽多酚提取物中含有 最高量的總多酚類、總黃酮類和濃縮丹寧酸等物質。 表1、利用不同種類溶劑提取之龍眼籽多酚提取物中總 多酚類、總黃酮類和濃縮丹寧各量比較表。 提取溶劑 ——總多酚類含量 -峨含f 縮合單寧酸含量 -----每克龍眼籽多酚提取物乾重 201233384 43.08+1.19 30.18+2.44 48.01+3.99 水 268.94±44.68 38.02+2.39 甲醇 301.81±22.77 84.44+5.23 乙酸乙酯 695.32±23.02 150.45+4.36 實施例二 本實施例係說明龍眼籽多酚提取物對於癌細胞的影 響’其中所使用的癌細胞株包括有大腸直腸癌細胞株C〇l〇 320DM、SW480、HT-29 和 LoVo ’ 肺癌細胞株 A549，肝癌 細胞株HepG2 ’子宮頸癌細胞株C_33a以及乳癌細胞株 ]\40八-148-231(前述細胞皆購自新竹生物資源保存中心），並 以老鼠小腸表皮細胞IEC-6做為對照使用，其中細胞株c〇1〇 320DM和HT-29係培養於添加有10%體積百分比熱不活化 (heat-inactive)胎牛血清的RPMI培養基164〇,細胞株SW48〇 係培養於添加有10%體積百分比熱不活化胎牛血清的L_15 (Leibovitz)培養基，細胞株Lov〇培養於添加有2〇%體積百 分比熱不活化胎牛血清的漢氏！?_12培養基（Ham，s F12 medium) ’而老鼠小腸表皮細胞IEC_6則培養於添加有每毫 升0.1U和1〇%胎牛血清的DEME培養基中，所有培養基都 添加有青黴素（25U/毫升）和鏈黴素（25毫克/毫升）。 龍眼籽多酚提取物對癌細胞存活率的影響： 在直控6公分的培養盤上分別接種1 〇,〇〇〇個上述細胞 株，經過18小時培養後，於培養盤中添加濃度分別為〇、 25、50、1〇〇和2〇〇微克/毫升之龍眼籽多酚提取物（溶解於 DMSO溶劑）繼續進行培養48小時，將細胞由培養盤洗下並 以錐蟲藍（Trypan blue)進行染色，計算存活之細胞數。 凊參見第1A和1B圖所示，對於不同的大腸直腸癌細 201233384 胞株來說’當龍眼籽多酚提取物的添加量增加時，各細胞 株的細胞存活率都有明顯下降的趨勢，且當龍眼籽多齡提 取物的添加量為1〇〇微克/毫升時，大腸直腸癌細胞株c〇l〇 320DM和SW480的存活率都可以下降至40%以下，且細胞 株HT-29的存活率也可以下降至6〇%以下，顯見本發明之 龍眼籽多酚提取物有助於抑制大多數大腸直腸癌細胞株的 存活；又相同的效果也可見於龍眼籽多酚提取物對於肺癌 細胞株、肝癌細胞株、子宮頸癌細胞株和乳癌細胞株影響， 尤其是在添加100微克/毫升龍眼籽多酚提取物時，各種類 的癌細胞活率都可以有效被降低至4〇%以下。 龍眼籽多酚提取物對於癌細胞聚落生成的影響： 在直徑6公分的培養盤上分別接種2〇〇個大腸直腸癌 細胞株SW480、HT-29和LoVo，而Colo 320DM細胞株則 接種於添加有0.6%壤脂（agarose)和20%血清的培養基中， 培養液中皆添加有濃度分別為〇、1、5、1〇、25和5〇微克 /毫升之龍眼軒多紛提取物’經過18小時培養後利用結晶紫 染劑染色並估算細胞聚落生成的情形。 請參見第2A和2B圖所示，對於大腸直腸癌細胞株c〇1〇 320DM、SW480、HT-29來說，只要微量（5微克/毫升）的龍 眼籽多酚提取物，便可以大幅抑制癌細胞的聚落性，甚至 龍眼籽多盼提取物添加量達到25微克/毫升時，該些細胞株 幾乎無法形成群落’又龍眼籽多紛提取物對於癌細胞群落 形成能力的影響也可以作用於肺癌細胞株、肝癌細胞株、 子宮頸癌細胞株和乳癌細胞株上，尤其對乳癌細胞株來說 其抑制癌細胞群落形成的功效更甚明顯，只需添加丨微克/ 201233384 毫升的龍眼籽多酚提取物，可以產生群落的細胞比例便會 降低約70%，當龍眼籽多酚提取物的添加量增加至10微克 /毫升時，乳癌細胞則完全無法形成群落，用來減少肺炎細 胞、肝癌細胞和子宮頸癌細胞產生群落所需添加的龍眼籽 多酚提取物含量也各有所不同，但是只要一提升龍眼籽多 酚提取物的添加量，癌細胞的群落形成比例便會有所降低。 龍眼籽多酚提取物對於癌細胞生長周期的影響： 將利用不同添加量龍眼籽多酚提取物共培養之各種癌 φ 細胞由培養盤上洗下並去除多餘培養液後，浸泡於70%酒 精中在-20°C下反應至少30分鐘，利用離心方式收集細胞， 以碘化丙啶染劑（其中含有 20微克/毫升碘化丙啶 (Propidium Iodide)和10微克/毫升核醣核酸酵素A(RNaseA)) 在暗室中進行細胞染色，以流式細胞儀觀察細胞之染色狀 態並利用MODFIT軟體計算不同癌細胞株的不同生長周期 的細胞比例。 請參考第3A至3H及第4A至4H所示，由圖中可看出 % 龍眼籽多酚提取物確實可以抑制不同癌症細胞株的生長， 且各個細胞株對於龍眼籽多盼提取物的敏感度並不盡相 同，舉例來說’大腸直腸癌細胞株HT-29和Lovo便需要較 高濃度的龍眼籽多酚提取物才能較為有效的抑制癌細胞的 生長’且一般來說，對於龍眼籽多酚提取物反應較為靈敏 的癌症細胞株來說（大腸直腸癌細胞例如搭C〇1〇320DM和 S W480 ) ’其細胞生長大多會停滯在s期，且周期素a蛋 白質的表現直也會隨著龍眼軒多紛提取物的濃度增加而減 少。 201233384 龍眼籽多酚提取物對於癌症細胞调亡的影響： 係利用連結有螢光指示劑的墙脂結合蛋白（Annexin V ) 對各種癌症細胞株進行染色，再利用流式細胞儀觀察細胞 的狀態。 凊參考第5A至5E所示，一般而言，當龍眼籽多酚提 取物的添加量增加時，各種癌細胞的凋亡比例也會有所增 加’若是進一步以免疫分析法觀察蛋白質表現狀況可以發 現’龍眼杆多驗提取物可能會引發硫胱氨酸蛋白酶 (caspases)的活性，並因為Bcl_2 (即B-細胞淋巴瘤基因 2 )蛋白質家族（的作用和Bax癌細胞粒腺體蛋白質表現產 生改變並進而造成癌症細胞的凋亡。 綜上所述，本發明提供了 一種龍眼籽多酚提取物之生 產方法’其可以有效提升龍眼籽多酚提取物中多酚類物質 的含量，且本發明所提供的龍眼籽多酚提取物可以有效降 低癌症細胞的群落形成現象，亦可抑制癌症細胞的細胞生 長週期8 【圖式簡單說明】 第1A圖為本發明龍眼籽多酚提取物對於不同大腸直腸 癌細胞株之細胞存活率的影響。 第1B圖為本發明龍眼籽多酚提取物對於肺癌細胞、肝 癌細胞、子宮頸癌細胞、以及乳癌細胞之細胞存活率的影 響。 第2 A圖為本發明龍眼籽多酚提取物對於不同大腸直腸 癌細胞株之細胞聚落形成比率的影響。 第2B圖為本發明龍眼籽多酚提取物對於肺癌細胞、肝 201233384 癌細胞、子宮頸癌細胞、以及乳癌細胞之細胞聚落形成比 率的影響。 第3A圖為以不同濃度龍眼籽多酚提取物共培養後各個 不同細胞生長階段大腸直腸癌細胞株Colo 320DM的比例。 第3B圖為以不同濃度龍眼籽多酚提取物共培養後各個 不同細胞生長階段大腸直腸癌細胞株SW480的比例。 第3C圖為以不同濃度龍眼籽多酚提取物共培養後各個 不同細胞生長階段大腸直腸癌細胞株HT-29的比例。 φ 第3D圖為以不同濃度龍眼籽多酚提取物共培養後各個 不同細胞生長階段大腸直腸癌細胞株LoVo的比例。 第3E圖為以不同濃度龍眼籽多酚提取物共培養後各個 不同細胞生長階段肺癌細胞株A549的比例。 第3F圖為以不同濃度龍眼籽多酚提取物共培養後各個 不同細胞生長階段肝癌細胞株HepG2的比例。 第3G圖為以不同濃度龍眼籽多酚提取物共培養後各個 不同細胞生長階段子宮頸癌細胞株C-33 A的比例。 • 第3H圖為以不同濃度龍眼籽多酚提取物共培養後各個 不同細胞生長階段乳癌細胞株MDA-MB-23 1的比例。 第4A圖為以不同濃度龍眼籽多酚提取物共培養後大腸 直腸癌細胞株Colo 320DM所生產的各種蛋白質比例。 第4B圖為以不同濃度龍眼籽多酚提取物共培養後大腸 直腸癌細胞株S W 4 8 0所生產的各種蛋白質比例。 第4C圖為以不同濃度龍眼籽多酚提取物共培養後大腸 直腸癌細胞株HT-29所生產的各種蛋白質比例。 第4D圖為以不同濃度龍眼軒多齡提取物共培養後大腸 201233384 直腸癌細胞株LoVo所生產的各種蛋白質比例β 第4Ε圖為以不同濃度龍眼籽多酚提取物共培養後肺癌 細胞株A549所生產的各種蛋白質比例。 第4F圖為以不同濃度龍眼軒多齡提取物共培養後肝癌 細胞株HepG2所生產的各種蛋白質比例。 第4G圖為以不同濃度龍眼籽多酚提取物共培養後子宮 頸癌細胞株C-33A所生產的各種蛋白質比例。 第4H圖為以不同激度龍眼籽多齡提取物共培養後乳癌 細胞株MDA-MB-23 1所生產的各種蛋白質比例。 第5A圖為以不同濃度龍眼籽多酚提取物共培養後大腸 直腸癌細胞株Colo 320DM的細胞凋亡率。 第5B圖為以不同濃度龍眼籽多酚提取物共培養後大腸 直腸癌細胞株SW480的細胞凋亡率。 第5C圖為以不同濃度龍眼籽多酚提取物共培養後大腸 直腸癌細胞株HT-29的細胞凋亡率。 第5D圖為以不同濃度龍眼籽多酚提取物共培養後大腸 直腸癌細胞株LoVo的細胞凋亡率。 第5E圖為以不同濃度龍眼籽多酚提取物共培養後不同 癌症細胞株的細胞凋亡率。 【主要元件符號說明】 1大腸直腸癌細胞株Colo 320DM 2大腸直腸癌細胞株SW480 3大腸直腸癌細胞株HT-29 4大腸直腸癌細胞株LoVo 5老鼠小腸表皮細胞ICE-6 12 201233384 6無添加龍眼籽多酚提取物 7龍眼籽多酚提取物1微克/毫升 8龍眼籽多酚提取物5微克/毫升 9龍眼籽多酚提取物10微克/毫升 10龍眼籽多酚提取物25微克/毫升 11龍眼籽多酚提取物50微克/毫升 12龍眼籽多酚提取物100微克/毫升 13龍眼籽多酚提取物200微克/毫升 A周期素D1蛋白質 B周期素E蛋白質 C周期素A蛋白質 D肌動蛋白The extract is extracted to produce an extract; and dried: the ethyl acetate solvent in the extract is removed, and freeze-dried to produce a powdered longan seed polyphenol extract. Another object of the present invention is to provide an application of a longan seed polyphenol extract, which can effectively inhibit the colony formation of cancer cells, and can also inhibit the growth cycle of cancer cells, thereby effectively controlling the progression of cancer cells. Longan seed polyphenol extract can be applied to the control of colorectal cancer cells, breast cancer cells, lung cancer cells, cervical cancer cells and liver cancer cells. [Embodiment] Hereinafter, the production method of the longan seed polyphenol extract and its application will be described in detail by way of examples only. EXAMPLE 1 This example illustrates the production method of longan seed polyphenol extract, which is to wash the longan seed twice with water and dry it after collecting the longan seed. The dried longan seed is ground into a longan by a grinder. Seed powder 'take 100 201233384 grams of longan seed powder soaked in 800 ml of acetic acid, I solution (1 〇〇 mM acetic acid dissolved in 30% by volume of acetone solution, pH 4.8), immersed at room temperature for 12 hours A crude extract is formed, and the crude extract is filtered by Ν〇ι filter paper and centrifuged to collect the solution, and the solution is separated by using a rotary distillation machine in a water bath under reduced pressure and a water temperature of 3 5 ° C or lower. Steaming to remove acetone and forming a distillate, collecting the distillate with 2 ml of ethyl acetate as solvent to extract four times and collecting the extracted extract, and extracting the extract by distillation The ethyl acetate was removed and subjected to cold drying to produce a powdery longan seed polyphenol extract. Determination of total phenolic compounds: The concentration of 龙·1 ml of 2 mg of Longyanxuan extract and 1 ml of Folin-Ciocalteu reagent and 2 ml were mixed and homogenized. Add 5 ml of 20% aqueous sodium carbonate solution for 2 〇 minutes to avoid light absorption, and measure the absorbance at 735 nm. The concentration is 〇, 15.625, 31.25, 62.5, 125, 250, 500 and 1〇〇〇. A standard curve was established for gallic acid (micrograms per milliliter). Determination of the total yellow compound: the 25 mg of longan seed polyphenol extract was dissolved in 80% ethanol to form a longan seed polyphenol extract solution. Take 20 microliters of longan seed multi-age extract. The solution was mixed with 122 μl of secondary water and 6 μl at a concentration of 5 °/. (g/ml) of sodium nitrite (NaN〇2) After reacting for six minutes, 'add 12 μl of 10% aluminum oxide (AlCl3(6H2〇)) for five minutes, then add 40 μl. The reaction was carried out with sodium hydroxide at a concentration of ιΜ and the absorbance at a wavelength of 490 nm was detected. The test was performed at concentrations of 0, 125, 250, 500, 1 〇〇〇, and 2000 (μg/ml) of catechin (Catechin). ) Establish a standard curve. 201233384 Condensed tannins content determination: 25 mg of longan seed polyphenol extract powder is dissolved in 80% ethanol to form a longan seed polyphenol extract solution, taking 5 microliters of longan seeds Phenol extract / gluten solution and 150 μl of vanilla (vanillin, dissolved in methanol) with a gradient of 4% (g / ml) for homogeneous mixing, add 75 ml of 12N hydrochloric acid and mix at 25 ° C The light-proof reaction was carried out for 20 minutes at a temperature, and the absorbance at a wavelength of 490 nm was measured. The test was carried out with catechins at concentrations of 〇, 125, 25 〇, 500, 1 〇〇〇, and 2000 (μg/ml). (Catechin) establishes a standard curve. Preanthocyanidin content determination: Take 50 μl of a sample of 1 mg of longan seed polyphenol extract per ml and add 6 μl of microliter of butanol/hydrochloric acid mixture (n- Butanol/HCl, 95:5 v/v), uniformly mixed with an oscillator in a dark environment, and then add 6.8 μl of ammonium iron sulfate (NH4Fe(S〇4)2. 12仏 with a concentration of 1 〇〇/0. 0) Mix evenly, react in a 95〇c water bath for 4 minutes, mix well and take out 100 micro The liter mixture was tested for absorbance at an absorbance wavelength of 55 〇 nm and a standard curve was established with a gasified cyanidin (no data?). Please refer to Table 1. If the longan seed polyphenol extract is colorimetrically analyzed for the content of polyphenols, it can be found that the longan seed polyphenol extract is produced by extracting with ethyl alcohol as solvent. It contains the highest amount of total polyphenols, total flavonoids and concentrated tannins. Table 1. Comparison table of total polyphenols, total flavonoids and concentrated tannins in longan seed polyphenol extracts extracted with different kinds of solvents. Extraction solvent - total polyphenol content - 峨 f content of condensed tannic acid ---- dry weight per gram of longan seed polyphenol extract 201233384 43.08+1.19 30.18+2.44 48.01+3.99 water 268.94±44.68 38.02+2.39 Methanol 301.81±22.77 84.44+5.23 Ethyl acetate 695.32±23.02 150.45+4.36 Example 2 This example illustrates the effect of longan seed polyphenol extract on cancer cells. The cancer cell line used includes colorectal cancer cell lines. C〇l〇320DM, SW480, HT-29 and LoVo 'Lung cancer cell line A549, liver cancer cell line HepG2 'cervical cancer cell line C_33a and breast cancer cell line>\40 八-148-231 (the aforementioned cells are all purchased from Hsinchu Biotechnology) Resource Conservation Center), and used mouse small intestinal epithelial cells IEC-6 as a control, in which cell lines c〇1〇320DM and HT-29 were cultured with 10% by volume of heat-inactive fetal calf Serum RPMI medium was 164 〇, cell line SW48 〇 was cultured in L_15 (Leibovitz) medium supplemented with 10% by volume of heat-inactivated fetal bovine serum, and cell line Lov 〇 was cultured with 2% by volume of heat-inactivated fetus. Bovine blood Han's! ?_12 medium (Ham, s F12 medium)' and mouse intestinal epithelial cells IEC_6 were cultured in DEME medium supplemented with 0.1 U and 1% fetal calf serum per ml, all medium was supplemented with penicillin (25 U/ml) and Streptomycin (25 mg/ml). Effect of longan seed polyphenol extract on cancer cell survival rate: 1 〇 was inoculated on a 6 cm culture plate, and the above cell lines were cultured. After 18 hours of culture, the concentration was added to the culture plate. 〇, 25, 50, 1 〇〇 and 2 〇〇 micrograms/ml of longan seed polyphenol extract (dissolved in DMSO solvent) were further cultured for 48 hours, the cells were washed from the culture dish and trypan blue (Trypan blue) ) staining was performed to calculate the number of viable cells.凊 See Figures 1A and 1B. For different colorectal cancer 201233384 cell lines, when the amount of longan seed polyphenol extract increased, the cell viability of each cell line decreased significantly. And when the longan seed multi-age extract is added in an amount of 1 μg/ml, the survival rate of the colorectal cancer cell lines c〇l〇320DM and SW480 can be reduced to below 40%, and the cell line HT-29 The survival rate can also be reduced to less than 6%. It is obvious that the longan seed polyphenol extract of the present invention can inhibit the survival of most colorectal cancer cell lines; the same effect can also be seen in the longan seed polyphenol extract for lung cancer. Cell line, liver cancer cell line, cervical cancer cell line and breast cancer cell line, especially when 100 μg/ml longan seed polyphenol extract is added, the activity rate of various types of cancer cells can be effectively reduced to 4%. the following. Effects of longan seed polyphenol extract on colony formation of cancer cells: 2 colonic rectal cancer cell lines SW480, HT-29 and LoVo were inoculated on a 6 cm diameter plate, while Colo 320DM cell line was inoculated. In the medium with 0.6% agarose and 20% serum, the culture medium was added with longan extracts of 〇, 1, 5, 1 〇, 25 and 5 〇 micrograms/ml, respectively. After 18 hours of incubation, the crystal violet stain was used to stain and estimate the formation of cell colonies. Please refer to Figures 2A and 2B. For colorectal cancer cell lines c〇1〇320DM, SW480, HT-29, as long as a small amount (5 μg/ml) of longan seed polyphenol extract can be greatly inhibited The colony of cancer cells, even when the amount of longan seeds is more than 25 μg/ml, the cell lines can hardly form a community. The effect of extracts of longan seeds on the formation of cancer cell communities can also be applied to The lung cancer cell line, the liver cancer cell line, the cervical cancer cell line and the breast cancer cell line, especially for the breast cancer cell line, the effect of inhibiting the formation of the cancer cell community is more obvious, and only need to add 丨 microgram / 201233384 ml of longan seeds. Phenol extract can reduce the proportion of cells that can produce colonies by about 70%. When the amount of longan seed polyphenol extract is increased to 10 μg/ml, breast cancer cells can not form a community at all, which is used to reduce pneumonia cells and liver cancer. The content of polyphenol extract in longan seeds to be added to cells and cervical cancer cells is also different, but as long as the polyphenol extract of longan seeds is raised Increase the amount of formation ratio will be decreased cancer community. Effect of longan seed polyphenol extract on cancer cell growth cycle: Various cancer φ cells co-cultured with different added amount of longan seed polyphenol extract were washed from the culture plate and the excess culture solution was removed, and then immersed in 70% alcohol. The reaction was carried out at -20 ° C for at least 30 minutes, and the cells were collected by centrifugation with a propidium iodide dye containing 20 μg/ml propidium Iodide and 10 μg/ml ribonuclease A ( RNaseA)) Cell staining was performed in a dark room, the staining state of the cells was observed by flow cytometry, and the proportion of cells of different growth cycles of different cancer cell lines was calculated using MODFIT software. Please refer to Figures 3A to 3H and 4A to 4H. It can be seen from the figure that % longan seed polyphenol extract can indeed inhibit the growth of different cancer cell lines, and the sensitivity of each cell line to longan seeds The degrees are not the same. For example, 'colorectal cancer cell lines HT-29 and Lovo require higher concentrations of longan seed polyphenol extract to inhibit cancer cell growth more effectively' and, in general, for longan seeds For cancer cell lines with more sensitive polyphenol extract reaction (colorectal cancer cells such as C〇1〇320DM and S W480), most of their cell growth will be arrested in s phase, and the performance of cyclin a protein will also follow The concentration of Longyanxuan extract increased and decreased. 201233384 Effects of polyphenol extract from longan seed on apoptosis of cancer cells: Various cancer cell lines were stained with wall-binding protein (Annexin V) linked with a fluorescent indicator, and the state of the cells was observed by flow cytometry. .凊 Referring to Figures 5A to 5E, in general, when the amount of longan seed polyphenol extract is increased, the percentage of apoptosis of various cancer cells is also increased. 'If the protein expression is further observed by immunoassay, It was found that 'longan rod multiple extracts may trigger the activity of thiocysteine protease (caspases), and because of the Bcl_2 (ie B-cell lymphoma gene 2) protein family (the role of Bax cancer cell granule gland protein expression) Altering and causing apoptosis of cancer cells. In summary, the present invention provides a method for producing longan seed polyphenol extract, which can effectively increase the content of polyphenols in longan seed polyphenol extract, and The longan seed polyphenol extract provided by the invention can effectively reduce the colony formation phenomenon of cancer cells, and can also inhibit the cell growth cycle of cancer cells. 8 [Simple description of the figure] FIG. 1A is a view of different longan seed polyphenol extracts of the present invention. Effect of cell survival rate of colorectal cancer cell line. Fig. 1B is a view of the longan seed polyphenol extract of the present invention for lung cancer cells, liver cancer cells, Effect of cell survival rate of cervical cancer cells and breast cancer cells. Figure 2A shows the effect of longan seed polyphenol extracts on the cell colony formation ratio of different colorectal cancer cell lines. Figure 2B is the longan seed of the present invention. The effect of polyphenol extract on the cell colony formation ratio of lung cancer cells, liver 201233384 cancer cells, cervical cancer cells, and breast cancer cells. Figure 3A shows the growth stages of different cells after co-culture with different concentrations of longan seed polyphenol extracts. The proportion of colorectal cancer cell line Colo 320DM. Figure 3B shows the proportion of colorectal cancer cell line SW480 at different growth stages after co-cultivation of polyphenol extracts of different longan seeds. Figure 3C shows the concentration of longan seeds at different concentrations. The proportion of colorectal cancer cell line HT-29 in different cell growth stages after co-culture of phenol extract. φ Figure 3D shows the colorectal cancer cell line LoVo in different cell growth stages after co-culture with different concentrations of longan seed polyphenol extract. The ratio of Figure 3E shows the growth order of different cells after co-culture with different concentrations of longan seed polyphenol extracts. The proportion of lung cancer cell line A549. Figure 3F shows the proportion of HepG2 cells in different cell growth stages after co-culture of polyphenol extracts from different concentrations of longan seeds. Figure 3G shows the total concentration of polyphenol extracts from longan seeds at different concentrations. The proportion of cervical cancer cell line C-33 A at different growth stages after culture. • Figure 3H shows the growth of breast cancer cell line MDA-MB-23 1 at different cell growth stages after co-culture with different concentrations of longan seed polyphenol extract. The ratio of the various proteins produced by the colorectal cancer cell line Colo 320DM co-cultured with different concentrations of longan seed polyphenol extracts. Figure 4B shows the co-culture of the large intestine with different concentrations of longan seed polyphenol extract. The proportion of various proteins produced by the rectal cancer cell line SW 4 80 . Fig. 4C is a graph showing the proportions of various proteins produced by the colorectal cancer cell line HT-29 after co-cultivation of polyphenol extracts of different longan seeds. The 4D is the ratio of various proteins produced by the rectal cancer cell line LoVo in the large intestine 201233384 after co-culture with different concentrations of Longyanxuan age extract. The fourth picture shows the lung cancer cell line A549 co-cultured with different concentrations of longan seed polyphenol extract. The proportion of various proteins produced. Figure 4F shows the proportions of various proteins produced by HepG2 cells co-cultured with different concentrations of Longyanxuan. Figure 4G shows the proportions of various proteins produced by cervical cancer cell line C-33A after co-culture of polyphenol extracts from different concentrations of longan seeds. Figure 4H shows the proportions of various proteins produced by breast cancer cell line MDA-MB-23 1 co-cultured with different concentrations of longan seed multi-age extracts. Figure 5A shows the apoptosis rate of colorectal cancer cell line Colo 320DM after co-culture of polyphenol extracts from different concentrations of longan seeds. Figure 5B shows the apoptosis rate of colorectal cancer cell line SW480 after co-culture with different concentrations of longan seed polyphenol extract. Figure 5C shows the apoptosis rate of colorectal cancer cell line HT-29 after co-culture with different concentrations of longan seed polyphenol extract. Figure 5D shows the apoptosis rate of colorectal cancer cell line LoVo after co-culture with different concentrations of longan seed polyphenol extract. Figure 5E shows the apoptosis rate of different cancer cell lines after co-culture with different concentrations of longan seed polyphenol extract. [Key device symbol description] 1 colorectal cancer cell line Colo 320DM 2 colorectal cancer cell line SW480 3 colorectal cancer cell line HT-29 4 colorectal cancer cell line LoVo 5 mouse small intestine epithelial cell ICE-6 12 201233384 6 no added Longan seed polyphenol extract 7 longan seed polyphenol extract 1 μg / ml 8 longan seed polyphenol extract 5 μg / ml 9 longan seed polyphenol extract 10 μg / ml 10 longan seed polyphenol extract 25 μg / ml 11 longan seed polyphenol extract 50 μg / ml 12 longan seed polyphenol extract 100 μg / ml 13 longan seed polyphenol extract 200 μg / ml A cycle D1 protein B cyclin E protein C cyclin A protein D muscle Kinesin
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