201023872 六、發明說明： 【發明所屬之技術領域】 本發明係關於一種龍眼籽萃取物的製備方法及其應 用，所得之龍眼籽萃取物應用於生物體，可具有抗發炎、 降低血液中尿酸的濃度、促進皮膚角質細胞的生長、增強 傷口癒合機轉及抵制細菌活性等多種功效。 【先前技術1 一、發炎（Inflammation ): 發炎在藥物治療上是一種不可忽視的現象，對人體而 言，它是生成疾病前的警告訊息。當人體組織受傷時，不 論是由細菌、外傷、化學物品還是其他原因導致的，受傷 組織附近的巨噬細胞（Macrophage)會被活化而進行吞噬 外來物質的任務，同時也會釋放一些因數來啓動更深一層 的防禦反應；倘若發炎組織持續受外來物質刺激，其防禦 機轉可達數月甚至數年，故處於發炎狀態時，所釋放的因 數含量往往較多。經硏究證實：所述因數包含有：一氧化 氮（Nitric oxide, NO)、腫瘤壞死因數（Tumor necrosis factor, TNF )、細胞界白素（Interleukin, IL )、顆粒白血球群落刺 激因數（Granulocyte colony stimulating factor, G-CSF)、單 核球群落刺激因數（Monocyte colony stimulating factor, M-CSF )、顆粒白血球及單核球群落刺激因數 (Granulocyte-monocyte colony stimulating factor, GM-CSF)， 學者將由單核細胞與巨噬細胞產生的TNF命名爲TNF- a， 將T淋巴細胞產生的淋巴毒素（Lymphotoxin, LT)命名爲 201023872 - TNFj來加以區別。 . 脂多糖（Lipopolysaccharides, LPS)是內毒素的主要成 份，經由動物實驗證明：LPS能夠延緩胃部排空，此作用 ' 與LPS誘導下前炎細胞因數及一氧化氮的升高有關。機體 受到LPS的刺激後，會觸發單核吞噬細胞系統合成TNF-α、IL-1^、IL-6等多種細胞因數，參與機體防禦反應和 修復。TNF-α、IL-lyS、IL-6這三種細胞因數的合成是有 • 序的級聯，即LPS誘導TNF-α的合成、TNF-0：誘導IL-1 φ 冷的合成、而IL-1/5又誘導IL-6的合成。但過量的細胞因 數也會對機體產生不良作用，例如過量的TNF-α會引起多 種臟器功能衰竭、彌漫性血管內凝血及中毒性休克，甚至 引起死亡，而應用TNF抗體的動物則能有效地阻止致死性 內毒素休克的發生。 現今醫藥界所使用的抗發炎藥物種類繁多，例如抗生 素（Antibiotics )、非類固醇抗發炎藥（Non-steroidal anti-inflammation drugs ; NSAIDs )、抗組織氨藥 ® ( Anti-histamine drugs )等等，雖具有相當良好的消炎作用， 卻有抗藥性及損傷腸胃等副作用。 二、痛風： 痛風是嘌呤代謝異常或尿酸***減少所引起的一種疾 • 病，其臨床特點爲高尿酸血症（Hyperuricemia)、反復發 - 作的急性單一關節炎（Recurrent acute monoarthritis )、尿 酸鈉鹽形成痛風石（Tophi)沉積、慢性痛風石關節炎等， 若未經適當治療，通常最終會發展爲痛風性腎病（Gouty 201023872 nephropathy)。本病主要分爲原發性和繼發性兩大類，原 發性痛風患者中有近1%患者爲酶缺陷所致，而大多數病 因不明，臨床以痛風性關節炎爲主要表現，且常伴有高血 脂病、高血壓病、糖尿病、動脈硬化及冠心病等；繼發性 痛風常由腎臟病、血液病及藥物等原因引起，痛風爲其倂 發症。 高尿酸血症是痛風最重要的生化基礎，但並不是痛風 的同義詞，硏究指出：約有5〜18.8%的高尿酸血症患者最 終會發展爲痛風，但痛風患者在其病程中的某一階段必將 有高尿酸血症的存在。 實驗室可利用尿酸酶法（Uricase differential spectrophotometric method)精確測定血尿酸値。高尿酸血 症可分爲絕對性和相對性兩大類。當血中尿酸濃度超過可 溶性的上限時，稱爲絕對性高尿酸血症，在37°C時血中尿 酸飽和値是7mg/dl，超過這個飽和點，逐漸有針狀晶體析 出。一般流行病學硏究則以正常人血尿酸平均値加上二個 標準差爲上限，認爲男性血中的尿酸値超過7mg/dl，女性 超過6mg/dl時，稱爲相對性高尿酸症。若血尿酸値超過 7mg/dl，則痛風或腎結石的發生率將會增加。 痛風的臨床表現分爲四個階段：無症狀高尿酸血症 (Asymptomatic hyperuricemia )、急性痛風關節炎（Acute gouty arthritis)、間歇期（Inter-critical gout)、慢性痛風 石關節炎（Chronic tophaceous gout)。 診斷痛風較正確的方法爲：在急性痛風關節炎發作時 201023872 - 抽取關節液，如發現有被嗜中性白血球吞噬的針狀尿酸鹽 . 結晶（Monosodium urate crystal )，在偏光顯微鏡下呈現負 性雙折光（Negative birefringent)，即爲痛風。其他常見的 臨床表徵或實驗室檢查：突然發作第一大腳趾、足背、踝 等單一關節紅腫劇痛、秋水仙鹼治療有特效者或高尿酸血 症者，僅可作爲診斷痛風的參考。 由於原發性痛風缺乏病因治療，因此不能根治。臨床 治療的目的在於：1、及時控制痛風性關節炎的急性發作； • 2、長期治療高尿酸血症，以預防尿酸鈉鹽沉積造成的關 節破壞及腎臟損害。 至於降尿酸藥物的選擇：在腎功能正常或輕度損害， 24小時尿酸排出量低於600mg時，可使用促進尿酸*** 藥；在腎功能爲中等損害（肌酐廓清率< 35ml/分鐘）或24 小時尿液尿酸明顯升高時，應使用抑制尿酸生成物。血尿 酸明顯升高及痛風石大量沉積時，可合用以上兩種藥物， 以防止漸進性痛風性倂發症。 • 一、 、 、 HU述的促進尿酸***藥（Uricosuric agent)主要通過抑 制近端腎小管對尿酸的重吸收而促進尿酸***。爲防止尿 酸在腎臟大量排出時引起腎臟損害及腎結石的副作用，均 應從小劑量開始，於7〜10天內逐漸加量，並考慮鹹化尿 * 液。此類藥品有丙磺殊（Probenecid )、苯溴馬隆 * ( Benzbromarone )等。 前述的抑制尿酸生成藥（Xanthine oxidase inhibitor)， 僅有別嘌呤醇（Allopurinol )，其結構類似次黃嘌呤 201023872 (hypoxanthine )，有較強的抑制黃嘿玲氧化酶（xanthine oxidase)的作用，可抑制痛風石和腎結石的形成，並促進 痛風石的溶解。同時使用抗癌藥如疏嘌昤（Mercaptopurine ) 或硫唑嘌暗（Azathioprine )時，會提高抗癌藥的血中濃度， 此時需酌量或留心臨床副作用。 三、傷 口癒合（Wound healing): 傷口癒合是一種動態的（Dynamic )、許多細胞交互作 用（Interactive)且爲多重步驟的（Multiple steps)過程， 包括細胞移動、細胞增生、細胞分化、細胞外基質的合成 與組織再造（Tissue remodeling)等。這個過程與傷口處表 皮的再生以及皮下結締組織的修復有關。在皮膚傷口癒合 的過程中，傷口兩邊表皮邊緣的角質細胞會增生，並且向 傷口中間移動而形成新的表皮層，這些過程需幾天甚至數 星期才能完成。 與傷口癒合有密切關係的生長因數（Growth factors )， 包括有成纖維細胞生長因數2 ( Hbroblast growth factor 2， FGF2 )、血小板衍化生長因數（PI at el et-derive growth factor， PDGF )、表皮生長因數（Epidermal growth factor，EGF )、 角質細胞生長因數（Keratinocyte growth factor，KGF )、轉 化生長因數-a (Transforming growth factor-a，TGF-α )、 轉化生長因數-/5 (Transforming growth factor-/S，TGF-/3 ) 以及血管內皮生長因數（Vascular endothelial growth factor，VEGF )。這些生長因數（Growth factors )中，PDGF、 EGF、TGF- /3與VEGF由角質細胞分泌。其中，PDGF能夠 201023872 - 吸引巨啦細胞（Macrophages )與成纖維細胞（Fibroblasts)， . 並促進基質蛋白（Matrix protein )的製造；EGF能夠以自 分泌（Autocrine)的形式促進自身的移動與增生；TGF-/3 能夠促進成纖維細胞（Fibroblasts)的增生、細胞移動及血 管新生，而且在傷口癒合的初期就會大量釋出；VEGF則能 夠促進血管通透性、促血管再生基質（Proangiogenic matrix ) 的沉積及血管新生，並且能夠刺激單核細胞（Monocyte) 的移動。由以上硏究結果顯示：這些角質細胞所釋放的生 〇 長因數（Growth factors)均與傷口癒合過程密切相關。 另一方面，根據《全國中草藥彙編》記載：龍眼籽可 用於治療胃痛、燒烫傷、刀傷出血、疳氣痛、外傷出血、 疥癖、濕瘡等。古人將龍眼籽用於治療外傷，有良好的止 血、定痛、生肌之效。《黃氏醫抄方》記載：「治刀斧傷， 桂圓核不拘多少，用火燒枯存性，硏末，摻患處即愈」。 古文獻記載：「龍眼籽末，敷金刀傷，昔在西秦及巴理坤 軍營救愈多人」。《殷紅趾傳方》亦雲：「治刀傷出血， ^ 以龍眼籽炒搗細磨，敷之」。由以上古書及藥典的記載可 以知道：龍眼籽對皮膚刀傷及相關疾病具有治療效果，對 促進傷口的癒合有效果，但是其作用機轉並不清楚。 【發明内容】 _ 本案發明人從事相關產業的硏究與發展數載，據由前 ' 述試驗報告得知悉，治療痛風最重要地即是抑制體內的 xanthine oxidase活性，從而減少尿酸生成，同時阻止或減 低尿酸鹽的沉著；據此，本案發明人積極尋求解決之道， 9 201023872 在經過長期努力之硏究與測試之後，終於完成本發明。 . 緣此，本發明之主要目的，在於提供一種龍眼籽萃取 物的製備方法及其在生物體中的應用。 ’ 依據上述之目的，本發明首先提供一種龍眼籽萃取物 的製備方法，包含下列步驟： 一、 配製特定濃度的萃取溶液；該萃取溶液可爲水溶 液或濃度爲20%〜95%的乙醇溶液； 二、 將萃取溶液溫度加熱至70°C〜90°C ; 三、 於萃取溶液中放入打碎的龍眼籽顆粒，進行萃取，❹ 萃取溫度爲70°C〜90°C，萃取時間爲1〜3小時； 四、 將萃取後的溶液過濾、濃縮； 五、 再將濃縮後的溶液進行冷凍及乾燥； 六、 製備出龍眼籽萃取物。 本發明進一步揭示，由上述製備方法制得的龍眼籽萃 取物，主要可應用於生物體抗發炎；降低生物體的尿酸； 促進生物體傷口癒合；抑制細菌活性。 據由前述，本發明提供的龍眼籽萃取物，可以應用於 ® 生物體，能夠產生抗發炎反應、降低尿酸生成、抑制細菌 活性，並可促進皮膚角質細胞生長、增加傷口愈合速度， 同時不會造成生物體內臟器受損或增加臟器負擔。 爲期使對於本發明之目的、功效以及構造特徵能有更 詳細明確的瞭解，茲舉出如下述之較佳實施例並配合圖式 說明如後。 【實施方式】 10 201023872 - 本發明所述之龍眼籽萃取物，係透過以下步驟逐步進 . 行製備： 一、 以親水性溶液（例如水）或親脂性溶液作爲萃取 溶液；本實施例係配製濃度爲20〜95%的乙醇溶液作爲萃 取溶液； 二、 將萃取溶液溫度加熱至70〜90°C ; 三、 將打碎後的龍眼籽放入萃取溶液中，使萃取溫度 維持在70〜90°C，萃取時間約爲1〜3小時，進行萃取； Φ 四、將萃取後的溶液經過濾、濃縮； 五、 再進行低溫低壓的冷凍乾燥； 六、 製備出龍眼籽萃取物。 利用高效液相層析儀（High Performance liquid Chromatography，HPLC)分析製備的龍眼籽萃取物，可得出 其主要組成成份包含沒食子酸（Gallic acid )、鞣料雲實素 (Corilagin )、隸花酸（Ellagic acid )，結構式分別如下， 高效液相層析儀的分析條件則如表格一所示： 沒食子酸（Gallic acid)201023872 VI. Description of the Invention: [Technical Field] The present invention relates to a method for preparing a longan seed extract and an application thereof, and the obtained longan seed extract is applied to a living body, which has anti-inflammatory and lowering uric acid in blood. Concentration, promote the growth of skin keratinocytes, enhance the wound healing machine and resist bacterial activity. [Prior Art 1] Inflammation: Inflammation is a phenomenon that cannot be ignored in drug therapy. For the human body, it is a warning message before the disease is generated. When human tissue is injured, whether caused by bacteria, trauma, chemicals, or other causes, macrophage near the injured tissue is activated to devour foreign matter, and some factors are released to activate A deeper defense response; if the inflamed tissue continues to be stimulated by foreign substances, its defense machine can be transferred for several months or even years, so when it is in an inflammatory state, the factor is often released. It has been confirmed by research that the factors include: Nitric oxide (NO), Tumor necrosis factor (TNF), Interleukin (IL), Granulocyte colony. Stimulating factor (G-CSF), Monocyte colony stimulating factor (M-CSF), Granulocyte-monocyte colony stimulating factor (GM-CSF), scholars will The TNF produced by nuclear cells and macrophages is named TNF-a, and the lymphotoxin (Lymphotoxin, LT) produced by T lymphocytes is named 201023872 - TNFj to distinguish them. Lipopolysaccharides (LPS) are the main components of endotoxin. It has been shown in animal experiments that LPS can delay gastric emptying, which is related to the increase of proinflammatory cytokines and nitric oxide induced by LPS. After being stimulated by LPS, the body triggers the synthesis of TNF-α, IL-1^, IL-6 and other cytokines by the mononuclear phagocytic system, and participates in the body's defense response and repair. The synthesis of three cytokines of TNF-α, IL-lyS, and IL-6 is a sequence of sequences, that is, LPS induces the synthesis of TNF-α, TNF-0: induces the synthesis of IL-1 φ cold, and IL- 1/5 induced the synthesis of IL-6. However, excessive cytokines may also have adverse effects on the body. For example, excessive TNF-α may cause various organ failure, diffuse intravascular coagulation and toxic shock, and even cause death, while animals using TNF antibody can be effective. Prevent the occurrence of lethal endotoxin shock. Today's pharmaceutical industry uses a wide variety of anti-inflammatory drugs, such as antibiotics, non-steroidal anti-inflammation drugs (NSAIDs), anti-histamine drugs, etc. Has a very good anti-inflammatory effect, but has drug resistance and damage to the stomach and other side effects. Second, gout: Gout is a disease caused by abnormal metabolism of sputum or decreased uric acid excretion. Its clinical features are hyperuricemia, recurrent acute monoarthritis, sodium urate. Salt formation of tophi (Tophi) deposition, chronic tophus arthritis, etc., if not properly treated, usually eventually develops into gouty nephropathy (Gouty 201023872 nephropathy). The disease is mainly divided into two major categories: primary and secondary. Nearly 1% of patients with primary gout are caused by enzyme deficiency, and most of the causes are unknown. Clinically, gouty arthritis is the main manifestation, and often Accompanied by hyperlipidemia, hypertension, diabetes, arteriosclerosis and coronary heart disease; secondary gout is often caused by kidney disease, blood diseases and drugs, and gout is a symptom of it. Hyperuricemia is the most important biochemical basis of gout, but it is not synonymous with gout. The study pointed out that about 5 to 18.8% of patients with hyperuricemia will eventually develop gout, but gout patients in the course of their disease There must be hyperuricemia in one stage. The laboratory can accurately measure blood urate by the Uricase differential spectrophotometric method. Hyperuricemia can be divided into two categories: absolute and relative. When the concentration of uric acid in the blood exceeds the upper limit of solubility, it is called absolute hyperuricemia, and the uric acid saturation in blood is 7 mg/dl at 37 ° C. Above this saturation point, needle crystals gradually precipitate. The general epidemiological study is based on the average person's blood uric acid average 値 plus two standard deviations. It is considered that the uric acid sputum in male blood exceeds 7 mg/dl, and when the female exceeds 6 mg/dl, it is called relative high uric acid. . If the blood uric acid sputum exceeds 7 mg/dl, the incidence of gout or kidney stones will increase. The clinical manifestations of gout are divided into four stages: Asymptomatic hyperuricemia, Acute gouty arthritis, Inter-critical gout, Chronic tophaceous gout . The correct way to diagnose gout is: in the onset of acute gout arthritis 201023872 - extraction of joint fluid, if found acicular urate phagocytized by neutrophils. Crystal (Monosodium urate crystal), negative under polarized light microscope Negative birefringent, which is gout. Other common clinical characterizations or laboratory tests: sudden onset of the first major toe, foot, sputum and other single joint redness and severe pain, colchicine treatment with special effects or hyperuricemia, can only be used as a reference for the diagnosis of gout. Because primary gout lacks the cause of treatment, it cannot be cured. The purpose of clinical treatment is: 1. To timely control the acute onset of gouty arthritis; 2. Long-term treatment of hyperuricemia to prevent joint damage and kidney damage caused by sodium urate deposition. As for the choice of uric acid-lowering drugs: in normal or mild renal damage, 24 hours of uric acid excretion below 600mg, can be used to promote uric acid excretion; moderate damage in renal function (creatinine clearance rate < 35ml / min) or When 24-hour urine uric acid is significantly elevated, uric acid production should be inhibited. When the blood uric acid is significantly elevated and the tophi is deposited in large amounts, the above two drugs can be used together to prevent progressive gout. • The Uricosuric agent promoted by I, HU, and HU promotes uric acid excretion primarily by inhibiting the reabsorption of uric acid by proximal tubules. In order to prevent kidney damage and kidney stones from causing side effects when uric acid is excreted in large amounts in the kidneys, it should be started from a small dose and gradually added in 7 to 10 days, taking into account the salted urine. Such drugs include Probenecid, Benzbromarone, and the like. The aforementioned Xanthine oxidase inhibitor, only allopurinol, has a structure similar to that of hypoxanthine 201023872 (hypoxanthine), and has a strong inhibitory effect on xanthine oxidase. Inhibits the formation of tophi and kidney stones and promotes the dissolution of tophi. When using an anticancer drug such as Mercaptopurine or Azathioprine, it will increase the blood concentration of the anticancer drug. At this time, it is necessary to discretion or pay attention to clinical side effects. Wound healing: Wound healing is a dynamic, multi-cell interaction and multiple steps process, including cell migration, cell proliferation, cell differentiation, extracellular matrix. Synthesis and tissue remodeling (Tissue remodeling). This process is associated with the regeneration of the epidermis at the wound site and the repair of subcutaneous connective tissue. During the healing of the skin wound, the keratinocytes at the edge of the epidermis on both sides of the wound proliferate and move toward the middle of the wound to form a new epidermal layer. These processes take days or even weeks to complete. Growth factors closely related to wound healing, including fibroblast growth factor 2 (FGF2), platelet-derived growth factor (PDGF), epidermal growth Epidermal growth factor (EGF), Keratinocyte growth factor (KGF), Transforming growth factor-a (TGF-α), Transforming growth factor-/5 (Transforming growth factor-/) S, TGF-/3) and Vascular endothelial growth factor (VEGF). Among these growth factors, PDGF, EGF, TGF-/3 and VEGF are secreted by keratinocytes. Among them, PDGF can 201023872 - attracts macrophages and fibroblasts, and promotes the production of matrix proteins; EGF can promote its own movement and proliferation in the form of autocrine; TGF-/3 can promote the proliferation, cell migration and angiogenesis of fibroblasts, and it will be released in the early stage of wound healing. VEGF can promote vascular permeability and proangiogenic matrix. Deposition and angiogenesis, and can stimulate the movement of monocytes. The results of the above studies show that the growth factors released by these keratinocytes are closely related to the wound healing process. On the other hand, according to the “National Chinese Herbal Medicine Compilation”, longan seeds can be used to treat stomach pain, burns, burns, stab wounds, hernias, traumatic bleeding, phlegm, and sores. The ancients used longan seeds to treat trauma, and had good hemostasis, pain, and muscle effects. "Huang's Medical Copying" records: "The knife is injured, the longan core is not limited, and the fire is burned, and the end of the war is mixed." The ancient literature records: "At the end of the longan seed, there is a gold knife wound. In the West Qin and Ballykun, the army rescued more people." "Yin Hong Toe Chuan Fang" also said: "The knife is wounded and bleeding, ^ It is finely ground with longan seeds, and it is applied." It can be known from the above ancient books and pharmacopoeia that longan seeds have a therapeutic effect on skin wounds and related diseases, and have an effect on promoting wound healing, but the action is not clear. [Summary of the Invention] _ The inventor of the case engaged in the research and development of related industries. According to the previous test report, the most important thing in the treatment of gout is to inhibit the activity of xanthine oxidase in the body, thereby reducing uric acid production and preventing Or reduce the uric acid deposition; accordingly, the inventor of the present invention actively seeks solutions, 9 201023872 After a long period of hard work and testing, the invention has finally been completed. Accordingly, it is a primary object of the present invention to provide a method for preparing a longan seed extract and its use in a living organism. According to the above purpose, the present invention firstly provides a method for preparing a longan seed extract, comprising the following steps: 1. preparing a specific concentration of the extraction solution; the extraction solution may be an aqueous solution or a concentration of 20% to 95% ethanol solution; Second, the temperature of the extraction solution is heated to 70 ° C ~ 90 ° C; Third, the crushed longan seed particles are added to the extraction solution for extraction, ❹ extraction temperature is 70 ° C ~ 90 ° C, extraction time is 1 ~3 hours; Fourth, the extracted solution is filtered and concentrated; 5. The concentrated solution is then frozen and dried; 6. The longan seed extract is prepared. The invention further discloses that the longan seed extract obtained by the above preparation method can be mainly applied to anti-inflammatory of the organism; reducing uric acid of the organism; promoting wound healing of the organism; and inhibiting bacterial activity. According to the foregoing, the longan seed extract provided by the present invention can be applied to a living organism, which can produce an anti-inflammatory reaction, reduce uric acid production, inhibit bacterial activity, and promote skin keratinocyte growth and increase wound healing speed without Causes damage to organs in the living organism or increases the burden on organs. For a more detailed and detailed understanding of the purpose, the <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; [Embodiment] 10 201023872 - The longan seed extract of the present invention is prepared by the following steps: 1. Using a hydrophilic solution (such as water) or a lipophilic solution as an extraction solution; this embodiment is formulated. The ethanol solution having a concentration of 20 to 95% is used as an extraction solution; 2. The temperature of the extraction solution is heated to 70 to 90 ° C; 3. The broken longan seeds are placed in the extraction solution to maintain the extraction temperature at 70 to 90 ° °C, extraction time is about 1~3 hours, extraction; Φ Fourth, the extracted solution is filtered and concentrated; Fifth, low temperature and low pressure freeze-drying; 6. Preparation of longan seed extract. The high-temperature liquid Chromatography (HPLC) analysis of the prepared longan seed extract can be used to determine that the main components include gallic acid, corilagin, and Ellagic acid, the structural formula is as follows, the analytical conditions of high performance liquid chromatography are shown in Table 1: Gallic acid
OHOH
HO、JL 鞣花酸（Ellagic acid)HO, JL Ellagic acid
OH 11 201023872 驟料雲實素（Corilagin)OH 11 201023872 Suddenly Clouds (Corilagin)
表格一、尚效液相層析儀的分析條件 OH Θ 儀器設備 Agilent 1100 層析管 Atlantis®T3 5 // m 4.6 X 10 mm, Waters 前置管柱 Cosmosil 5C18-AR-II 4.6 X 10 mm, Nacalai Tesque 管柱溫度 25 °C 流速 1.0 ml/min 檢測波長 UV 270 nm 注射體積 10 μ\ 時間（分） 0 5 15 30 45 60 75 85 0.1% Η3Ρ04 ( % ) 98 97 97 87 86 81 79 0 Acetonitrile ( % ) 2 3 3 13 14 19 21 100 以包含沒食子酸、鞣料雲實素、鞣花酸的標準溶液爲 參考溶液，利用上述高效液相層析儀的分析條件進行分 析，得到第一圖所示結果，該結果表明：沒食子酸（42.42 12 201023872 - // g/ml)、鞣料雲實素（52.72/z g/ml)、鞣花酸（22.4// g/ml) . 的保留時間（Retention Time)分別爲 14.409、43.304、63.489 分鐘；將龍眼籽萃取物利用高效液相層析儀分析後’得到 第二圖所示結果，顯示龍眼籽萃取物中峰値成分的保留時 間分別爲14.461、43.302、63.476分鐘，因此’該龍眼籽萃 取物的主要成分與標準溶液相同，即該龍眼籽萃取物包含 有沒食子酸、鞣料雲實素和鞣花酸。 再者，爲了揭示前述製備的龍眼籽萃取物的療效，茲 〇 利用前述製備之龍眼籽萃取物進行各類體內試驗與體外試 驗· 一、抗發炎的體內試驗： (一） 準備材料： 龍眼籽萃取物（萃取溶液爲50%乙醇溶液）、龍眼籽 萃取物Α (萃取溶液爲純水）、龍眼籽萃取物Β (萃取溶 液爲20%乙醇溶液）。 (二） 體內試驗（餵食試驗）： 參 1、取雄性 S D 鼠（Sprague-dawley rats) 24 隻，每隻 體重約爲200g〜250g，控制飼養室室內溫度23t，光線明、 暗各12小時，使用S D鼠專用飼料，飲用水經過逆滲透處 理。 . 2、隨機分成九組，每組6隻，分別餵食： ' (1 )控制組，僅口投逆滲透水； (2 )口投龍眼籽萃取物（0.5g/Kg，每Kg鼠重口投0.5g 龍眼籽萃取物）一星期後，腹腔注射大腸桿菌脂 13 201023872 多糖（Lipopolysaccharides, LPS，2.5 mg/Kg，每Table 1. Analytical conditions for the liquid chromatograph OH Θ Instrumentation Agilent 1100 Chromatography Atlantis® T3 5 // m 4.6 X 10 mm, Waters Front column Cosmosil 5C18-AR-II 4.6 X 10 mm, Nacalai Tesque column temperature 25 °C flow rate 1.0 ml/min detection wavelength UV 270 nm injection volume 10 μ\ time (minutes) 0 5 15 30 45 60 75 85 0.1% Η3Ρ04 ( % ) 98 97 97 87 86 81 79 0 Acetonitrile ( % ) 2 3 3 13 14 19 21 100 Using a standard solution containing gallic acid, sputum, and ellagic acid as a reference solution, the analysis conditions of the above high performance liquid chromatography are used to analyze The results shown in the figure show that gallic acid (42.42 12 201023872 - // g/ml), sputum protein (52.72/zg/ml), ellagic acid (22.4//g/ml) The retention times are 14.409, 43.304, and 63.489 minutes, respectively. After the longan seed extract is analyzed by high performance liquid chromatography, the results shown in the second graph are obtained, showing the peak 値 composition in the longan seed extract. The retention time is 14.461, 43.302, 63.476 minutes, so 'the longan seed extract The main component of the standard solution was the same, i.e., the seed longan extract with gallic acid, tanning Caesalpinia hormone and ellagic acid. Furthermore, in order to reveal the curative effect of the longan seed extract prepared as described above, the above-prepared longan seed extract is used for various in vivo tests and in vitro tests. 1. Inflammatory in vivo test: (1) Preparation materials: Longan seeds The extract (the extraction solution is a 50% ethanol solution), the longan seed extract Α (the extraction solution is pure water), the longan seed extract Β (the extraction solution is a 20% ethanol solution). (2) In vivo test (feeding test): References 1, 24 male Sprague-dawley rats, each weighing about 200g~250g, controlling the indoor temperature of the breeding room 23t, light and dark for 12 hours. The drinking water is subjected to reverse osmosis treatment using a special feed for SD rats. 2, randomly divided into nine groups, each group of 6, respectively fed: ' (1) control group, only oral injection of reverse osmosis water; (2) oral longan seed extract (0.5g / Kg, per Kg rat heavy mouth After 0.5 mL of longan seed extract, a week after intraperitoneal injection of E. coli lipid 13 201023872 polysaccharide (Lipopolysaccharides, LPS, 2.5 mg/Kg, per
Kg鼠重注射2.5mg大腸桿菌脂多糖）後’待24 . 小時； (3 ) 口投龍眼籽萃取物（0.5g/Kg) —星期後’腹腔注 射 LPS ( 2.5mg/Kg)待 48 小時； (4 )腹腔注射LPS ( 2.5mg/Kg)後待24小時，口投龍 眼籽萃取物A (0.5g/Kg); (5 )腹腔注射LPS ( 2_5mg/Kg)後待24小時，口投龍 眼籽萃取物B ( 0.5g/Kg) ; © (6 )腹腔注射LPS ( 2.5mg/Kg)後待24小時，口投龍 眼籽萃取物（〇.5g/Kg); (7 )腹腔注射LPS ( 2.5mg/Kg)後待48小時，口投龍 眼籽萃取物（〇.5g/Kg); (8 )單獨腹腔注射LPS ( 2.5mg/Kg)後待24小時； (9 )單獨口投龍眼籽萃取物（〇.5g/Kg);Kg mice were reinjected with 2.5 mg of E. coli lipopolysaccharide) after '24 hours; (3) Longan seed extract (0.5 g/Kg) - after week, 'intraperitoneal injection of LPS (2.5 mg/Kg) for 48 hours; (4) After intraperitoneal injection of LPS (2.5mg/Kg) for 24 hours, oral longan seed extract A (0.5g/Kg); (5) after intraperitoneal injection of LPS (2_5mg/Kg) for 24 hours, mouth longan Seed extract B (0.5g/Kg); © (6) 24 hours after intraperitoneal injection of LPS (2.5mg/Kg), longan seed extract (〇.5g/Kg); (7) intraperitoneal injection of LPS ( 2.5mg/Kg) for 48 hours, oral longan seed extract (〇.5g/Kg); (8) after intraperitoneal injection of LPS (2.5mg/Kg) for 24 hours; (9) single oral longan seed Extract (〇.5g/Kg);
投藥結束，經過一晚絕食後，在***麻醉下，由S D ❹ 鼠腹腔動脈採血，提供血清免疫學檢查。 3、 血清免疫學檢查：使用酶聯免疫分析法（ELISA) 檢測腫瘤壞死因數（TNF-α) '細胞白介素（IL-ie)。 4、 統計方法：本實驗所得資料，均以單因數變異數 分析（One-way analysis of variance, AN0VA)。 5、 實驗結果：由表格二可以看出：在IL-Ιβ的部分， 口投龍眼籽萃取物與腹腔注射LPS均可對S D鼠造成免疫 反應；由表格三與第三圖可看出：在TNF-α的部分，預先 14 201023872 - 口投龍眼籽萃取物再腹腔注射LPS及單獨口投龍眼籽萃取 . 物均有抗發炎的作用，而LPS逆境處理後再口投龍眼籽萃 取物，則對SD鼠不具有抗發炎的作用。 表格二、抗發炎體內實驗的IL-1/S値對比 IL-Ιβ (ng/L) 開始（original) IL-l/S (ng/L) 給藥後（after stress) 控制組 119±12 117±22 龍眼籽萃取物（7d) +LPS (24h) 131±20 212±15 龍眼籽萃取物（7d) +LPS (48h) 102±16 213+19 LPS (24h) +龍眼籽萃取物A 116±14 212+18 LPS (24h) +龍眼籽萃取物B 104±17 190±20 LPS (24h) +龍眼籽萃取物 125±11 207+13 LPS (48h) +龍眼籽萃取物 110±16 211±10 LPS (24h) 111±20 209±9 龍眼籽萃取物 124±8 198±9 表格三、抗發炎體內實驗的TNF- α値對比 TNF-α (ng/L) 開始（original) TNF-α (ng/L) 給藥後（after stress) 控制組 549±37 527±22 龍眼籽萃取物（7d) +LPS(24h) 551±25 581±34 龍眼籽萃取物（7d) +LPS(48h) 547±30 633±28 LPS (24h) +龍眼籽萃取物A 586±22 744±41 LPS (24h) +龍眼籽萃取物B 564±31 753+33 LPS (24h) +龍眼籽萃取物 535±28 737±34 LPS (48h) +龍眼籽萃取物 599±33 741±35 LPS (24h) 531±31 769±35 龍眼籽萃取物 554±27 687±31 15 201023872 二、抗痛風的體內試驗與體外試驗： (一） 準備材料：龍眼籽萃取物（萃取溶液爲50%乙 醇溶液）。 (二） 體內試驗（餵食試驗）： 1、 取雄性 S D 鼠（Sprague-Dawley rats) 24 隻，每隻 體重約爲200g〜250g，控制飼養室室內溫度約23°C，光線 明、暗各12小時，使用S D鼠專用飼料，飲用水經過逆滲 透處理。 2、 隨機分成三組，每組各8隻，其中一組爲對照組， 該組經口投逆滲透水；一組爲實驗組，該組投300 mg/Kg 鼠重的6-羥基嚷卩令（Hypoxathine)加上250 mg/Kg鼠重的 氧嗪酸（Oxonic acid，一種尿酸酶抑制劑）；另一組爲龍 眼籽實驗組，該組口投300 mg/Kg鼠重的6-羥基嘌呤 (Hypoxathine) 、250 mg/Kg 鼠重的氧嗪酸（Oxonic acid) 及龍眼籽0.1% ( wt% );添加氧嗪酸（Oxonic acid)的主 要目的，在於誘導SD老鼠體內形成高度尿酸者；SD鼠 可任意飮水，投藥期間，每天觀察動物兩次，每天稱體重 一次。投藥結束，經過一晚絕食後，在***麻醉下，由S D鼠尾部採血，提供血清生化學檢查。 (1 )對照組：口投逆滲透水。 (2 )實驗組：口投300 mg/Kg鼠重的6-羥基嘌呤 (Hypoxathine )，加上250 mg /Kg鼠重的氧嗪酸 (Oxonic acid ) ° (3 )龍眼籽組：投予300mg/Kg鼠重的6-羥基嘌呤 201023872 . (Hypoxanthine )，加上250mg/Kg鼠重的氧嗪酸 (Oxonic acid )，再加上 〇· 1 % ( Wt% )龍眼好。 3、 血清生化學檢查：使用生化自動分析儀 (Ciba-cornint 550)測定，檢測S D鼠血清尿酸濃度》 4、 統計方法：本實驗所得資料，均以單因數變異數 分析（One-way analysis of variance，ANOVA)。 5、 實驗結果：由表格四以及第四圖可以看出：龍眼 籽萃取物（萃取溶液爲50%乙醇溶液）能有效降低SD鼠 0 血清尿酸，降低效果高達32%。 表格四、抗痛風體內實驗的尿酸濃度對比 時間 對照組尿酸濃度 實驗組尿酸濃度 龍眼籽組尿酸濃度 0小時 7.3±1.5 mg/dl 8·8±1.2 mg/dl 8.2±1.7 mg/dl 1小時 7.6±1.6 mg/dl 40.6±12.1 mg/dl 27.717.3 mg/dl (三）體外實驗（黃嘌呤氧化酶（Xanthine oxidase)抑制 實驗）： 1、 以磷酸緩衝溶液（PBS)配製出50mmol/L的黃嘌 呤（Xanthine)。 2、 將黃嘌呤氧化酶（Xanthine oxidase)以磷酸緩衝溶液 (PBS)配製成 〇.1 〜〇·2 unit/ml。 3、 配製各式龍眼籽萃取物的樣本： (1 )配製純水，萃取龍眼籽。 (2)配製20%乙醇溶液，萃取龍眼籽。 17 201023872 (3 )配製50%乙醇溶液，萃取龍眼籽。 (4)配製95%乙醇溶液，萃取龍眼籽。 4、 取一陽性對照組——臨床用藥別嘌呤醇 (Allopurinol )，於陽性對照組中加入黃嘌呤氧化酶（Xanthine oxidase)，反應5分鐘後，加入黃嘌呤（Xanthine)。 5、 取一空白對照組，該組僅添加純水。 6、 於各種龍眼籽萃取物樣本中，加入黃嘌呤氧化酶 (Xanthine oxidase)，反應5分鐘，再加入黃嚷卩令（Xanthine)。 7、 利用分光光度儀，選取波長290nm，偵測各種龍 眼籽萃取物樣本及對照組的吸光値變化，每20秒偵測一 次，共偵測5分鐘，再經儀器計算其酵素活性。 8、 活性抑制率=〔1-（試驗組活性/對照組活性）〕 9、 實驗結果：由表格五可看出：各種龍眼籽萃取物 均能抑制黃嘌玲氧化酶（Xanthine oxidase)活性，活性抑制率 最高可達60%。 表格五、抗痛風體外實驗（黃嘌呤氧化酶抑制實驗） 的結果對比 樣本組 嘌呤醇 純水萃取龍 眼籽的樣本 20%乙醇萃取 龍眼籽的樣本 50%乙醇萃取 龍眼籽的樣本 95%乙醇萃取 龍眼籽的樣本 濃度（ppm) 5 50 50 50 50 活性抑制率（％ ) 83 12 36 60 44 三、抗痛風毒性試驗： (一）準備材料：龍眼籽萃取物。 201023872 (二）急性毒性試驗：S D鼠分成2組，每組8〜10隻， . 實驗前絕食一晚，但不絕水，經口投龍眼籽萃取物（450 mg/ml)，觀察中毒症狀並記錄14天內體重變化和死亡情 " 形；一半致死劑量（LD50)和95%可信賴限依照Litctchfield 及Wilcoxon二氏的方法計算。 (三） 28日餵食急性毒性試驗：分成2組，每組各10 隻，分別口投龍眼籽萃取物lg/kg、3 g/kg及去離子水（1 ml/100g)，連續28天。投藥期間，每天觀察動物兩次，每 ❿ 週稱重一次。投藥結束，經過一晚絕食，再***麻醉下由 S D鼠腹腔動脈採血，提供血液學及血清生化學檢查。 (四） 血清生化學檢查：用生化自動分析儀（Ciba-cornint 550)測定，檢測項目包含麩氨酸草乙酸轉氨酶（GOT)、 麩氨酸丙氨基轉氨酶（GPT)、白蛋白（Albumin)、球蛋 白（Globulin)、肌酸酐（Greatinine)。 (五） 統計方法：本實驗所得資料，均以單因數變異數 分析（One-way analysis of variance, ANOVA )，並進行 Dunnett ® 檢驗，以P値小於0.01認爲有顯著差異。 (六） 實驗結果： 1、急性毒性： 急性毒性試驗主要目的是尋求一次投予高劑量的藥物 ’ 造成試驗動物一半死亡的劑量，SD鼠空腹最大量每100 g • 重量可投予1.0 ml的劑量，藥物濃度爲450 mg/ml的濃度， 因此急性毒性試驗的劑量以15 g/kg爲基準，若SD鼠完全 沒有死亡，則不再進行。 19 201023872 S D鼠10隻，經口一次投予龍眼籽萃取物15 g/kg爲 基準，觀察14天，沒有死亡情形’即S D鼠的一半致死劑 . 量（LD50)大於15 g/kg。投予龍眼籽萃取物後至第14天 S D鼠體重與控制組比較沒有差異。 2、 28日連續餵食毒性試驗·’ 使用最高劑量以一半致死劑量的1/5爲原則，因此選 用最高劑量爲3 g/kg，和1 g/kg。 (1 )每組SD鼠8〜10隻，連續28天口投龍眼籽萃 .取物1 g/kg和3 g/kg，無死亡情形，與控制組比 © 較體重也沒有明顯變化。 (2 )血清生化學檢查：如表格六所示，連續28天經 口投龍眼籽萃取物，S D鼠1 g/kg和3 g/kg萃取 物組和正常對照組的血清生化指標沒有差異。 (3 )主要臟器重量：連續28天經口投龍眼籽萃取物’ S D鼠1 g/kg和3 g/kg組的肝臟及腎臟的重量和 控制組的沒有差異，表明：龍眼籽萃取物對主要 臟器重量沒有影響。 ® (4 )病理檢驗：連續28日經口投龍眼籽萃取物1 g/kg 和3 g/kg組進行病理切片檢查，S D鼠的心臟、 肝臟、腎臟、脾臟、腎上腺、精囊、睾九等皆無 變化。 3、 實驗結果：龍眼籽萃取物不會造成生物體內臟器 受損或增加臟器負擔。 20 201023872 - 表格六、連續餵食毒性試驗結果 ^ 控制組 14 Days 28 Days 28 Days SD 鼠數量（Number of mice) 8 10 10 10 實驗前體重（Originalweight) 19.2±1.8 19.6±1.1 19.1+1.5 19.5+0.8 實驗後體重（Final weight) 19.2±1.8 19.2±1.8 19.2+1.8 25.9±1.07 口投藥量（OralSample) 0 15 g/kg 1 g/kg 3 g/kg 肌酸酐(Creatinine) 0.395±0.15 0.435±0.07 0.51±0.084 29·9±14·8 麩氨酸草乙酸轉氨酶（GOT) 30.2±15.1 32.88±15.6 38.9+6.2 29.9±14.8 麩氨酸丙氨基轉氨酶（GPT) 8.27±5.54 14.95±10.1 7.8±3.8 9.4413.29 白蛋白（Albumin) 2.肚 1.37 2.75+1.08 2.63±1.64 2.5710.8 球蛋白（Globulin) 2.5±1.12 2.55±1.5 3.3±1.79 2.84±0.68 四、抑菌試驗： (一） 準備材料： 取以龍眼籽萃取物爲核心成分2.5 mg/ml (每ml” P豆 凝膠”中含有2.5 mg龍眼籽萃取物）的” P豆凝膠（含龍眼 籽萃取物之產品名）”，以逆滲透水配製出一倍的等張磷 酸液（lxPBS，等張溶液是指不引起紅細胞膜變形的溶液）， 再將配製的等張磷酸液利用無菌濾膜（Mini pore)過濾， 製備出無菌狀態的” P豆凝膠”等張磷酸液。 (二） 試驗菌種： 大腸桿菌（Escherichia coli )與金黃色葡萄球菌 (Staphylococcus aureus):At the end of the administration, after one night of hunger strike, blood was collected from the celiac artery of S D sputum under ether anesthesia to provide serum immunological examination. 3. Serum immunological examination: Tumor necrosis factor (TNF-α) 'cell interleukin (IL-ie) was detected by enzyme-linked immunosorbent assay (ELISA). 4. Statistical methods: The data obtained in this experiment were analyzed by One-way analysis of variance (AN0VA). 5, the experimental results: from Table 2 can be seen: in the IL-Ιβ part, oral longan seed extract and intraperitoneal injection of LPS can cause immune response to SD rats; from Table 3 and Figure 3 can be seen: Part of TNF-α, pre-14 201023872 - Longan seed extract and then intraperitoneal injection of LPS and oral longan seed extraction. The anti-inflammatory effect of the substance, and LPS adverse treatment after the longan seed extract, then It does not have an anti-inflammatory effect on SD rats. Table 2. IL-1/S値 vs. IL-Ιβ (ng/L) in anti-inflammatory in vivo experiments. Original IL-l/S (ng/L) After stress control group 119±12 117 ±22 Longan Seed Extract (7d) +LPS (24h) 131±20 212±15 Longan Seed Extract (7d) +LPS (48h) 102±16 213+19 LPS (24h) + Longan Seed Extract A 116± 14 212+18 LPS (24h) + longan seed extract B 104±17 190±20 LPS (24h) + longan seed extract 125±11 207+13 LPS (48h) + longan seed extract 110±16 211±10 LPS (24h) 111±20 209±9 Longan seed extract 124±8 198±9 Table 3. Anti-inflammatory in vivo TNF-α値 comparison TNF-α (ng/L) initial TNF-α (ng /L) After stress control group 549±37 527±22 Longan seed extract (7d) +LPS(24h) 551±25 581±34 Longan seed extract (7d) +LPS(48h) 547± 30 633±28 LPS (24h) + longan seed extract A 586±22 744±41 LPS (24h) + longan seed extract B 564±31 753+33 LPS (24h) + longan seed extract 535±28 737± 34 LPS (48h) + longan seed extract 599±33 741±35 LPS (24h) 531±31 769±35 Longan seed extract 5 54±27 687±31 15 201023872 2. In vivo and in vitro tests for gout resistance: (1) Preparation materials: longan seed extract (extraction solution is 50% ethanol solution). (2) In vivo test (feeding test): 1. Take 24 male Sprague-Dawley rats, each weighing about 200g~250g, controlling the indoor temperature of the breeding room to about 23°C, and the light is bright and dark. Hours, using special feed for SD rats, drinking water is subjected to reverse osmosis treatment. 2, randomly divided into three groups, each group of 8 each, one group is the control group, the group is orally administered reverse osmotic water; one group is the experimental group, the group is administered 300 mg / Kg rat heavy 6-hydroxy oxime (Hypoxathine) plus 250 mg/Kg murine oxoxine (Oxonic acid, a uric acid enzyme inhibitor); the other group is the longan seed experimental group, which is administered with a 300 mg/kg rat heavy 6-hydroxyl group. Hypoxathine, 250 mg/Kg Oxonic acid and 0.1% (wt%) of longan seeds; the main purpose of adding Oxonic acid is to induce the formation of high uric acid in SD rats. SD rats can be arbitrarily drowned. During the administration, the animals are observed twice a day and weighed once a day. At the end of the drug administration, after one night of hunger strike, blood was collected from the tail of the S D mouse under ether anesthesia to provide a serum biochemical examination. (1) Control group: Oral administration of reverse osmosis water. (2) Experimental group: Oral administration of 300 mg/Kg rat heavy 6-hydroxypurine (Hypoxathine) plus 250 mg / Kg rat weight of Oxonic acid ° (3) Longan seed group: 300 mg administered /Kg rat heavy 6-hydroxy 嘌呤 201023872 . (Hypoxanthine), plus 250mg / Kg rat weight of Oxonic acid, plus 〇 · 1% (Wt%) longan is good. 3. Serum biochemical examination: detection of serum uric acid concentration in SD rats by biochemical automatic analyzer (Ciba-cornint 550) 4. Statistical methods: The data obtained in this experiment are analyzed by single factor analysis. Variance, ANOVA). 5. Experimental results: It can be seen from Table 4 and Figure 4 that longan seed extract (50% ethanol solution in extraction solution) can effectively reduce serum uric acid in SD rats, and the effect is as high as 32%. Table 4, uric acid concentration in anti-gout in vivo experiment vs. time control uric acid concentration experimental group uric acid concentration longan seed group uric acid concentration 0 hours 7.3 ± 1.5 mg / dl 8 · 8 ± 1.2 mg / dl 8.2 ± 1.7 mg / dl 1 hour 7.6 ±1.6 mg/dl 40.6±12.1 mg/dl 27.717.3 mg/dl (III) In vitro experiment (Xanthine oxidase inhibition experiment): 1. Prepare 50mmol/L with phosphate buffer solution (PBS). Xanthine. 2. Xanthine oxidase was prepared as a 〇.1 ~〇·2 unit/ml in phosphate buffered saline (PBS). 3. Prepare samples of various longan seed extracts: (1) Prepare pure water and extract longan seeds. (2) Prepare 20% ethanol solution and extract longan seeds. 17 201023872 (3) Prepare 50% ethanol solution and extract longan seeds. (4) Prepare a 95% ethanol solution to extract longan seeds. 4. Take a positive control group - clinical use of allopurinol, add Xanthine oxidase to the positive control group, and add Xanthine after 5 minutes. 5. Take a blank control group, which only adds pure water. 6. Add Xanthine oxidase to various longan seed extract samples and react for 5 minutes, then add Xanthine. 7. Using a spectrophotometer, select a wavelength of 290 nm to detect changes in the absorbance of various samples of the longan seed extract and the control group, and detect it every 20 seconds for 5 minutes, and then calculate the enzyme activity by the instrument. 8. Activity inhibition rate = [1 - (test group activity / control group activity)] 9. Experimental results: It can be seen from Table 5 that various longan seed extracts can inhibit Xanthine oxidase activity and inhibit activity. The rate can be up to 60%. Table 5. Anti-gout in vitro test (xanthine oxidase inhibition experiment) Results comparison sample group sterol pure water extraction longan seed sample 20% ethanol extraction longan seed sample 50% ethanol extraction longan seed sample 95% ethanol extraction longan Seed sample concentration (ppm) 5 50 50 50 50 Activity inhibition rate (%) 83 12 36 60 44 III. Anti-gout toxicity test: (1) Preparation materials: longan seed extract. 201023872 (2) Acute toxicity test: SD rats were divided into 2 groups, 8~10 per group. Before the experiment, hunger strike for one night, but not water-tight, oral longan seed extract (450 mg/ml), observe the symptoms of poisoning The changes in body weight and death were recorded within 14 days; the half lethal dose (LD50) and the 95% confidence limit were calculated according to the methods of Litctchfield and Wilcoxon. (3) Acute toxicity test on the 28th: divided into 2 groups, 10 in each group, and the longan seed extracts were lg/kg, 3 g/kg and deionized water (1 ml/100g) for 28 consecutive days. During the administration, the animals were observed twice a day and weighed once every week. At the end of the drug administration, after one night of hunger strike, blood was collected from the abdominal artery of S D mice under ether anesthesia to provide hematology and serum biochemical examination. (iv) Serum biochemical examination: measured by biochemical automatic analyzer (Ciba-cornint 550), including glutamic acid oxaloacetate transaminase (GOT), glutamic acid aminotransferase (GPT), albumin (Albumin), Globulin, creatinine (Greatinine). (V) Statistical methods: The data obtained in this experiment were analyzed by One-way analysis of variance (ANOVA) and Dunnett ® test. P値 was less than 0.01 and considered to be significantly different. (VI) Experimental results: 1. Acute toxicity: The main purpose of the acute toxicity test is to seek a high dose of the drug once, which causes half of the death of the test animals. The maximum amount of SD rats per 100 g • weight can be administered to 1.0 ml. The dose, the concentration of the drug is 450 mg / ml, so the dose of the acute toxicity test is based on 15 g / kg, and if the SD mouse does not die at all, it will not be carried out. 19 201023872 S 10 rats were administered with 15 g/kg of longan seed extract once orally. After 14 days of observation, there was no death condition, ie, half of the lethal agent of S D rats. The amount (LD50) was greater than 15 g/kg. There was no difference between the body weight of the S D mice and the control group after the longan seed extract was administered. 2, 28th continuous feeding toxicity test ·' The highest dose is based on 1/5 of the half lethal dose, so the highest dose is 3 g/kg, and 1 g/kg. (1) 8 to 10 SD rats in each group, and the longan seed extract was taken for 28 consecutive days. The samples were taken at 1 g/kg and 3 g/kg, and there was no death. Compared with the control group, there was no significant change in body weight. (2) Serum biochemical examination: As shown in Table 6, there was no difference in the serum biochemical parameters of the S d rats 1 g/kg and 3 g/kg extract group and the normal control group for 28 consecutive days of oral longan seed extract. (3) Main organ weight: No significant difference in liver and kidney weight and control group of the SD rats 1 g/kg and 3 g/kg group for 28 consecutive days, indicating: longan seed extract No effect on the weight of major organs. ® (4) Pathological examination: pathological biopsy was performed on the 28th day of oral longan seed extract 1 g/kg and 3 g/kg. The heart, liver, kidney, spleen, adrenal gland, seminal vesicle, testis, etc. of SD rats No change. 3. Experimental results: Longan seed extract does not cause damage to organs in the living body or increase the burden on organs. 20 201023872 - Form VI. Results of continuous feeding toxicity test^ Control group 14 Days 28 Days 28 Days SD Number of mice 8 10 10 10 Pre-test weight (Originalweight) 19.2±1.8 19.6±1.1 19.1+1.5 19.5+0.8 Final weight after surgery 19.2±1.8 19.2±1.8 19.2+1.8 25.9±1.07 Oral dose (OralSample) 0 15 g/kg 1 g/kg 3 g/kg Creatinine (Creatinine) 0.395±0.15 0.435±0.07 0.51 ±0.084 29·9±14·8 glutamic acid oxaloacetate transaminase (GOT) 30.2±15.1 32.88±15.6 38.9+6.2 29.9±14.8 glutamic acid aminotransferase (GPT) 8.27±5.54 14.95±10.1 7.8±3.8 9.4413. 29 Albumin (Albumin) 2. Belly 1.37 2.75+1.08 2.63±1.64 2.5710.8 Globulin 2.5±1.12 2.55±1.5 3.3±1.79 2.84±0.68 IV. Bacteriostatic test: (1) Preparation materials: Longan seed extract is a core ingredient of 2.5 mg/ml (2.5 ml of longan seed extract per ml of P bean gel) containing "P bean gel (product name containing longan seed extract)" to reverse osmosis Water to prepare twice the isotonic phosphate solution (lxPBS, isotonic solution means not caused Membrane deformable solution), and then formulated using isotonic phosphate solution sterile filtration membranes (Mini pore) was filtered, prepared sterility "P beans gel" isotonic phosphate solution. (ii) Test strains: Escherichia coli and Staphylococcus aureus:
1、將大腸桿菌、金黃色葡萄球菌等細菌，分別用LB 21 201023872 broth液體培養基、37°C連續培養16小時，將培養好的菌 -液以一倍的等張磷酸液（lxPBS)清洗三次’每次清洗後以 -3000 rpm離心10分鐘，去除上清液。將清洗後的菌液利用 光譜儀測試其吸光度（〇D value)，最後再以一倍的等張 磷酸液稀釋，製備出吸光度爲0.3 ( 0D = 0.3)的菌液，以 進行實驗。 2、 將配製好的一倍的等張磷酸液（lxPBS)分別加入 10倍連續稀釋的大腸桿菌、金黃色葡萄球菌等菌液，以37 °c下連續處理1小時。 © 3、 將處理1小時後的菌液取5、10、20、50、100 μΐ 塗於LB培養基上，37t下連續培養18小時後，計數每盤 培養基的菌數，菌液以P豆凝膠等張磷酸液處理者爲實驗 組’菌液以逆滲透水等張磷酸液處理者爲對照組。 4、 以上步驟分別進行三次，最後統計結果。 5、 實驗結果：如表格七與第五圖所示，與對照組相 比’實驗組明顯降低了大腸桿菌與金黃色葡萄球菌的細菌 數目，從而證實：以龍眼籽萃取物爲核心成分的” P豆凝 ® 膠確實具有抑制大腸桿菌與金黃色葡萄球菌的功能，可 以應用於抑制青春痘的生成。 七、抑菌（大腸桿菌和金黃色葡萄球菌）試驗結果 i腸桿菌 (colonies/plate) 金黃色葡萄球菌 (colonies/plate) 實驗次序 對照組 實驗組~ 對照組 實驗組 22 201023872 第一次 232 152 218 149 第二次 121 82 239 134 第三次 169 94 153 99 平均値±誤差 174+38 109±28 203±45 127±26 (三）試驗菌種：痤瘡桿菌（Propionibacterium acne): 1、 將痤瘡桿菌利用anBAP broth液體培養基、37°C 下連續培養48小時，將培養好的菌液以一倍的等張磷酸液 參 （lxPBS)清洗三次，每次清洗後以3000 rpm離心10分鐘， 去除上清液。將清洗後的菌液利用光譜儀測試其吸光度 (OD value)，最後再以一倍的等張磷酸液稀釋，制得吸光 度爲0.3 ( 0D = 0.3)的菌液，以進行實驗。 2、 將配製好的一倍的等張磷酸液（lxPBS)分別加入 10倍連續稀釋的痤瘡桿菌，3(TC下連續處理1小時。 3、 將處理1小時後的菌液取5、10、20、50、ΙΟΟμΙ 塗於LB培養基上，30°C下連續培養48小時後，計數每盤 ® 培養基的菌數，菌液以” P豆凝膠”等張磷酸液處理者爲 實驗組，菌液以逆滲透水等張磷酸液處理者爲對照組。 4、 以上步驟分別進行三次，最後統計結果。 5、 實驗結果：如表格八與第六圖所示’與對照組相 '較，實驗組明顯降低了痤瘡桿菌的數目，從而證實：以龍 眼籽萃取物爲核心成分的” P豆凝膠”確實具有抑制痤瘡 桿菌的功能，可以應用于抑制青春痘的生成。 23 201023872 表格八、抑菌（痤瘡桿菌）試驗結果 痤瘡桿菌（colonies/plate) 實驗次序 對照組 實驗組 第一次 76 44 第二次 47 19 第三次 65 22 平均値±誤差 63±15 28±14 (四）試驗菌種：紅色毛癬菌（Trichophyton rubrum) ·· 1、 將紅色毛癖菌利用 IMA plate ( Inhibit mold Agar ) Q 培養基、30°C下連續培養96小時，將培養好的菌液以一倍 的等張磷酸液（lxPBS)清洗三次，每次清洗後以3000 rpm 離心10分鐘，去除上清液。將清洗後的菌液利用光譜儀測 試其吸光度（〇D value)，最後以一倍的等張磷酸液稀釋 製備出吸光度爲0.1 ( 〇D = 0.1)的菌液，以進行實驗。 2、 將配製好的一倍的等張磷酸液（lxPBS)分別加入 10倍連續稀釋的紅色毛癬菌，30°C下連續處理1小時。 3、 處理1小時後的菌液取5、10、20、50、100 μΐ塗 ® 於LB培養基，130°C下連續培養96小時後，計數每盤培養 基的菌數，菌液以” P豆凝膠”等張磷酸液處理者爲實驗 組，菌液以逆滲透水等張磷酸液處理者爲對照組。 4、 以上步驟分別進行三次，最後統計結果。 5、 實驗結果：如表格九與第七圖所示，與對照組相 較，實驗組明顯降低了紅色毛癬菌的細菌數目（約30%)， 表明··以龍眼籽萃取物爲核心成分的” P豆凝膠”確實具 24 201023872 - 有抑制紅色毛癖菌等黴菌的功能，可以應用於抑制香港腳 的生成。 表格九、抑菌（紅色毛癬菌）試驗結果 糸工色毛癣菌（colonies/plate) 實驗次序 對照組 實驗組 第一次 176 128 第二次 136 93 第三次 182 133 平均値±誤差 165±25 118±22 五、促進傷口癒合機轉試驗： (一） 準備材料： 1、 取龍眼籽萃取物5 g溶解於250 ml二次水中，以 2號濾紙過濾，再以0.45 μιη、0.22 μιη過濾膜過濾後備用。 2、 配製 0%、0.25%、2.5%、5.0% 與 10.0% ( Wt% ) 龍眼籽萃取物，作爲角質細胞培養液。 3、 人類角質細胞（Human epidermal keratinocytes ; HEKa-C005-5C)。 (二） 細胞培養：取1 x 104 cells/ml人類角質細胞 (HEKa-C005-5C )，用添加有角質細胞生長因數（KC supplements)及青黴素一鏈黴素（Penicillin — Streptomycin ) 的角質細胞培養基（Cascade biologies，USA)，於 37°C、 含5% C02的培養箱中進行培養，此批細胞爲第一代；每隔 兩天換一次培養基，直到第一代細胞八分滿後，再作繼代 25 201023872 培養。加入0.25%的胰酶細胞消化液（Trypsin-EDTA solution, · 含0.25%的胰酶和0.02%的EDTA) ’於37°C作用5分鐘 . 後，將懸浮的角質細胞以10%上清液沖洗中和後，置入離 心管中，15OOrpm離心10分鐘，去除上清液，再以角質細 胞培養基重新懸浮細胞，以1 : 3稀釋，並繼續於含濃度5 % CCh的培養箱中培養；取其第三代進行實驗。 (三） 細胞增生能力測試：以結晶紫（Crystal violet) 染色法進行；人類角質細胞（HEKa-C005-5C)經24、48、 72小時培養後，先以倒立式顯微鏡觀察細胞生長的實際狀 © 況，用200μ1上清液清洗細胞兩次，以細胞固定液固定細 胞20分鐘，再用200μ1 PBST清洗細胞兩次，以100 μΐ結 晶紫（Crystal violet)溶液於室溫下染色30分鐘，再用200μ1 上清液清洗細胞三次，以1%SDS溶解細胞，於室溫下迴旋 震盪培養1小時，將染上細胞核的結晶紫染料溶出，測定 波長595 nm下的吸光度（OD値），同時以650 nm的參考 波長修正吸光度（OD値）。 以未添加龍眼籽萃取物的組別作爲控制組，求得生長 ® 促進率。 (四） 以ELISA測定人類角質細胞所分泌生長因數 (growth factors)的釋出量： 1、 人類角質細胞經刺激後收集其上清液，以 Commercial kits測定培養液中生長因數（growth factors )的 量。 2、 首先取96孔板（96 well plates)的微量滴定板 26 201023872 - (Microtitration plate)，以牛血清蛋白（Bovine serum albumin) . 阻抑未結合位置，再以PBS-Tween洗淨，加入經刺激作用 後的上清液各ΙΟΟμΙ，於37°C反應2小時後，以PBS—Tween 洗淨，然後加入兔抗生長因數（Rabbit-anti-growth factor Ab-HRP Chemicon，Temecula，CA)，於 37〇C 反應 2 小時後 洗淨，加入呈色的底物（Substrate，內含鄰苯二胺 O-phenyldiamine)，呈色後加入50μ1的2NH2S04終止反應， 並測定波長450 nm下的吸光度，從而測出生長因數（growth Φ factors)中的血管內皮細胞生長因數（VEGF)的濃度。 (五）實驗結果： 1、如表格十與第八圖所示，以結晶紫（Crystal violet) 染色法測定增生能力與顯微鏡下的結果相符，顯示2.5%、 5.0 %與10 %的組別其促進角質細胞生長的倍數分別爲控 制組的1.25、1.26與1.50倍，其中僅10%組別具有統計上 的顯著意義（其P値小於0.05)，表明：僅須10%龍眼籽 萃取物，即可有效促進人類角質細胞的生長。 ® 2、如表格十一與第九圖所示，經由酶聯免疫吸附 (ELISA)測定得到的結果，在塗覆膠原I ( Collagen I，CI) 和纖維連接蛋白（FibronectU，FN )的組別，加入5 %及 10%龍眼籽萃取物培養後，上清液中血管內皮細胞生長因 * 數（VEGF)隨劑量增加而顯著上升（p<0.05)，表明：龍 • 眼籽萃取物在促進傷口癒合過程中包含有促進血管增生的 機制，能夠促進生物體傷口癒合。 27 201023872 表格十、結晶紫染色法測定增生能力結果 劑量（V/V %) 生長倍數 0 1±0.060 1.25 0.9810.075394 2.5 1.2610.059782 5 1.2710.090245 10 1.5010.119184* 表格十一、酶聯免疫吸附測定結果 劑量（Dose，v/v %) Cl ( pg/ml) CVI ( pg/ml) FN ( pg/ml) 0 144.8114.61 150.1133.47 53.518.96 5 84.9±7.54 73.6±23.57 80.9+11.31 10 269.8±36.77* 171.1+3.77 141.1±1.89* 由以上列舉的體內試驗、體外試驗與毒性試驗可知： 本發明提供的龍眼籽萃取物，可用於生物體並產生抗發炎 反應、降低尿酸生成、抑制細菌活性，還可促進皮膚角質 細胞的生長，增加傷口癒合的速度，同時該龍眼籽萃取物 @ 並不會造成生物體內的臟器受損或增加其負擔，故能安心 使用。 本發明在同類領域中，具有極佳之進步性及實用性， 同時查遍國內外關於此類架構之技術資訊文獻，亦未發現 有相同近似之構造存在於先，應已符合『創作性』、『合 於產業利用性』、『新穎性』以及『進步性』的專利要件， 爰依法提出申請。 28 201023872 . 惟，以上所述者僅係本發明之較佳實施例而已，故舉 . 凡應用本專利說明書以及申請專利範圍所爲之其他等效方 法結構變化者，均屬可行，理應包含在本發明之申請專利 ' 範圍內。1. Bacteria such as Escherichia coli and Staphylococcus aureus were continuously cultured for 16 hours at 37 ° C with LB 21 201023872 broth liquid medium, and the cultured bacteria-liquid was washed three times with one-fold isotonic phosphate solution (lxPBS). 'After each wash, centrifuge at -3000 rpm for 10 minutes to remove the supernatant. The washed bacteria solution was tested for absorbance (〇D value) by spectrometer, and finally diluted with one-fold isotonic phosphoric acid to prepare a bacterial solution having an absorbance of 0.3 (0D = 0.3) for the experiment. 2. The prepared isotonic phosphate solution (lxPBS) was separately added to a 10-fold serial dilution of Escherichia coli, Staphylococcus aureus, and the like, and continuously treated at 37 ° C for 1 hour. © 3, the bacterial solution after 1 hour of treatment was taken 5, 10, 20, 50, 100 μΐ on LB medium, and cultured continuously for 37 hours at 37t, the number of bacteria in each medium was counted, and the bacterial solution was condensed with P bean. The gel isotonic phosphate solution was treated by the experimental group 'bacterial solution with the reverse osmosis water isotonic phosphate solution as the control group. 4. The above steps are performed three times separately, and the final statistical results. 5. Experimental results: As shown in Tables 7 and 5, compared with the control group, the experimental group significantly reduced the number of bacteria in Escherichia coli and Staphylococcus aureus, thus confirming that the longan seed extract was the core component. P Bean Gelatin does have the function of inhibiting Escherichia coli and Staphylococcus aureus and can be used to inhibit the formation of acne. 7. Antibacterial (E. coli and Staphylococcus aureus) test results I enterobacteria (colonies/plate) Staphylococcus aureus (colonies/plate) experimental order control group experimental group ~ control group experimental group 22 201023872 first 232 152 218 149 second 121 82 239 134 third 169 94 153 99 average 値 ± error 174 + 38 109±28 203±45 127±26 (III) Test species: Propionibacterium acne: 1. The acne bacillus was cultured in anBAP broth liquid medium at 37 ° C for 48 hours. Double the isotonic phosphate ginseng (lxPBS) three times, and after each washing, centrifuge at 3000 rpm for 10 minutes to remove the supernatant. The washed bacteria solution was tested with a spectrometer. The OD value was finally diluted with one-fold isotonic phosphoric acid to prepare a bacterial solution with an absorbance of 0.3 (0D = 0.3) for the experiment. 2. The prepared isotonic phosphoric acid solution ( lxPBS) were added 10 times serial dilution of acne bacillus, 3 (continuous treatment for 1 hour under TC. 3. Apply 5, 10, 20, 50, ΙΟΟμΙ of the bacterial solution after 1 hour of treatment to LB medium, 30 °C After 48 hours of continuous culture, the number of bacteria in each plate of the medium was counted. The bacteria liquid was treated with the "P bean gel" isotonic phosphate solution as the experimental group, and the bacterial liquid was treated with the reverse osmosis water isotonic phosphate solution as the control group. 4. The above steps were performed three times and finally the statistical results. 5. Experimental results: As shown in Table 8 and Figure 6 'Compared with the control group', the experimental group significantly reduced the number of acne bacteria, thus confirming: longan The "P bean gel" with the core extract as the core ingredient does have the function of inhibiting the acne bacillus and can be used to inhibit the formation of acne. 23 201023872 Table 8. Antibacterial (Acne bacillus) test results Coronide (plate) Experimental order Photo group experimental group first 76 44 second 47 19 third 65 22 average 値 ± error 63 ± 15 28 ± 14 (four) test strain: Trichophyton rubrum · · 1, will be red Trichophyton spp. was continuously cultured for 96 hours at 30 °C using IMA plate (Inhibit mold Agar) Q medium, and the cultured bacterial solution was washed three times with one-fold isotonic phosphate solution (lxPBS) at 3000 rpm after each washing. Centrifuge for 10 minutes and remove the supernatant. The washed bacteria solution was measured for absorbance (〇D value) by a spectrometer, and finally diluted with one-fold isotonic phosphoric acid to prepare a bacterial solution having an absorbance of 0.1 (〇D = 0.1) for the experiment. 2. The prepared isotonic phosphoric acid solution (lxPBS) was separately added to 10-fold serially diluted Trichophyton rubrum, and continuously treated at 30 ° C for 1 hour. 3. After 1 hour of treatment, take 5, 10, 20, 50, 100 μΐ of the bacterial solution in LB medium, and continuously culture at 130 °C for 96 hours. Count the number of bacteria in each medium, and the bacteria solution is “P bean”. The "gel" isotonic phosphate solution was treated in the experimental group, and the bacterial solution was treated with reverse osmosis water isotonic phosphate solution as a control group. 4. The above steps are performed three times separately, and the final statistical results. 5. Experimental results: As shown in Tables 9 and 7, compared with the control group, the experimental group significantly reduced the number of bacteria (about 30%) of Trichophyton rubrum, indicating that the longan seed extract was the core component. "P bean gel" does have 24 201023872 - It has the function of inhibiting molds such as Trichophyton rubrum and can be used to suppress the formation of Hong Kong feet. Table IX, Antibacterial (Red Trichophyton) test results for the work of Coriolis versicolor (colonies/plate) Experimental sequence Control group Experimental group first 176 128 Second 136 93 Third 182 133 Average 値 ± Error 165 ±25 118±22 V. Promote wound healing machine test: (1) Prepare materials: 1. Take 5 g of longan seed extract dissolved in 250 ml of secondary water, filter with No. 2 filter paper, and then 0.45 μηη, 0.22 μηη The filter membrane was filtered and used. 2. Prepare 0%, 0.25%, 2.5%, 5.0%, and 10.0% (Wt%) longan seed extracts as keratinocyte culture fluid. 3. Human epidermal keratinocytes (HEKa-C005-5C). (ii) Cell culture: Take 1 x 104 cells/ml human keratinocytes (HEKa-C005-5C) with keratinocyte medium supplemented with keratinocyte growth factor (KC supplements) and penicillin-streptomycin (Penicillin-Streptomycin) (Cascade biologies, USA), cultured in a 5% CO 2 incubator at 37 ° C. The batch of cells is the first generation; the medium is changed every two days until the first generation of cells is eight points full, then Cultivated as a successor 25 201023872. Add 0.25% trypsin-EDTA solution (containing 0.25% trypsin and 0.02% EDTA) to action at 37 ° C for 5 minutes. After the suspension of keratinocytes with 10% supernatant After washing and neutralizing, the cells were placed in a centrifuge tube, centrifuged at 15 rpm for 10 minutes, the supernatant was removed, and the cells were resuspended in keratinocyte medium, diluted 1:3, and continuously cultured in an incubator containing 5% CCh; Take the third generation to conduct experiments. (C) Cell proliferation test: by crystal violet staining method; human keratinocytes (HEKa-C005-5C) were cultured at 24, 48, 72 hours, and then observed the actual growth of cells by inverted microscope. © condition, wash the cells twice with 200 μl supernatant, fix the cells with cell fixative for 20 minutes, wash the cells twice with 200 μl PBST, and stain with 100 μM Crystal violet solution for 30 minutes at room temperature. The cells were washed three times with 200 μl of the supernatant, and the cells were lysed with 1% SDS, and shaken at room temperature for 1 hour, and the crystal violet dye stained with the nuclei was eluted, and the absorbance at a wavelength of 595 nm (OD値) was measured. The reference wavelength at 650 nm corrects the absorbance (OD値). The growth ® promotion rate was determined by using the group without the addition of longan seed extract as the control group. (4) The release amount of growth factors secreted by human keratinocytes was determined by ELISA: 1. The human keratinocytes were collected after stimulation, and the growth factors in the culture medium were determined by Commercial kits. the amount. 2. First, take a 96-well plate of 96 well plates. The microtiter plate 26 201023872 - (Microtitration plate), bovine serum albumin. Reinforce the unbound position, then wash with PBS-Tween, add the After stimulating the supernatant, each reaction was incubated at 37 ° C for 2 hours, washed with PBS-Tween, and then added rabbit anti-growth factor Ab-HRP Chemicon (Temecula, CA). After 37 hrs of reaction, wash for 2 hours, add a colored substrate (Substrate containing o-phenyldiamine), and then add 50 μl of 2NH2S04 to terminate the reaction, and measure the absorbance at a wavelength of 450 nm. The concentration of vascular endothelial cell growth factor (VEGF) in growth Φ factors was measured. (V) Experimental results: 1. As shown in Tables 10 and 8, the results of the method of crystal violet staining were consistent with the results under the microscope, showing 2.5%, 5.0% and 10% of the group. The folds that promote keratinocyte growth were 1.25, 1.26, and 1.50 times of the control group, respectively, of which only 10% were statistically significant (its P値 was less than 0.05), indicating that only 10% of longan seed extract was required, ie It can effectively promote the growth of human keratinocytes. ® 2. As shown in Tables 11 and 9, the results obtained by enzyme-linked immunosorbent assay (ELISA) were applied to the group of collagen I ( Collagen I, CI) and fibronectin (FN). After adding 5% and 10% longan seed extract, the growth factor (VEGF) of vascular endothelial cells in the supernatant increased significantly with the increase of dose (p<0.05), indicating that: Long• eye seed extract is promoting The wound healing process contains mechanisms to promote vascular proliferation and promote wound healing in living organisms. 27 201023872 Table X. Determination of proliferative capacity by crystal violet staining Results dose (V/V %) Growth multiples 0 1±0.060 1.25 0.9810.075394 2.5 1.2610.059782 5 1.2710.090245 10 1.5010.119184* Table XI, enzyme-linked immunosorbent assay Adsorption assay dose (Dose, v/v %) Cl (pg/ml) CVI (pg/ml) FN (pg/ml) 0 144.8114.61 150.1133.47 53.518.96 5 84.9±7.54 73.6±23.57 80.9+11.31 10 269.8±36.77* 171.1+3.77 141.1±1.89* It can be seen from the above-mentioned in vivo test, in vitro test and toxicity test that the longan seed extract provided by the present invention can be used for living body and produces anti-inflammatory reaction, reducing uric acid production and inhibiting Bacterial activity can also promote the growth of skin keratinocytes and increase the speed of wound healing. At the same time, the longan seed extract@ does not cause damage or increase the burden on organs in the living body, so it can be used with peace of mind. The invention has excellent advancement and practicability in the same field, and at the same time, it has searched the domestic and foreign technical information literatures on such structures, and has not found that the structure with the same approximation exists first, and should have conformed to the "creative". , "Combined with industrial use", "novelty" and "progressive" patent requirements, 提出 apply in accordance with the law. 28 201023872. However, the above description is only for the preferred embodiment of the present invention, and all other equivalent method structural changes that apply to this patent specification and the scope of the patent application are feasible and should be included in Within the scope of the patent application of the present invention.
29 201023872 【圖式簡單說明】 . 第一圖係爲包含沒食子酸、鞣料雲實酸、鞣花酸之標 . 準品溶液的質譜分析圖。 ’ 第二圖係爲本發明龍眼籽萃取物的質譜分析圖。 ’ 第三圖係爲表格三所建構之折線圖。 第四圖係爲表格四所建構之折線圖。 第五圖係爲表格七之平均値所建構的長條圖。 第六圖係爲表格八之平均値所建構的長條圖。 第七圖係爲表格九之平均値所建構的長條圖。 © 第八圖係爲表格十所建構的折線圖。 第九圖係爲表格十一所建構的折線圖。 【主要元件符號說明】 無。 3029 201023872 [Simple description of the diagram] The first diagram is the mass spectrometry diagram of the standard solution containing gallic acid, sputum acid and ellagic acid. The second figure is a mass spectrogram of the longan seed extract of the present invention. The third picture is a line chart constructed in Table 3. The fourth figure is a line drawing constructed in Table 4. The fifth picture is the bar chart constructed by the average of Table 7. The sixth picture is the bar chart constructed by the average of Table 8. The seventh picture is the bar chart constructed by the average of Table 9. © Figure 8 is a line drawing constructed in Table 10. The ninth figure is a line drawing constructed in Table 11. [Main component symbol description] None. 30