200916110 九、發明說明： 【發明所屬之技術領域】 本發明係有關一種由桂花中萃取抗氣喘物質的方法，乃 以此方法在桂花中萃取一抗氣喘物質之桂花萃取物，該桂花萃 取物具有抗氣喘效果，對於生物體具明顯之保健作用，包含較 佳之抗氣喘的生理功效，減缓呼吸道以及肺部慢性發炎症狀， 更可舒緩氣喘症狀，及預防與減少氣喘之發作者。 【先前技術】 依據世界衛生組織(WHO)估計，全球有約有3億人患有氣 喘’其t氣喘重症患者約佔ions%。歐美地區氣喘患者每 10年約增加2倍，每年因氣喘而死亡的人數約萬人，根據 統計，氣喘發病率在世界各地都有逐年攀升的趨勢，並隨著社 會現代化^逐年升高。細_綱有萬氣喘患者，盛行 率高達8.5 %’罹患率及致死率在二十年中持續增加，目前台 灣每三個小孩中有—個是過敏兒，台北地區7、12歲兒童氣喘 發生率南達12-13%，顯示氣喘確實在台灣是高發性疾病之一。 引起氣喘的機轉至今未明，但近年來的研究已經證實，氣 喘事實上f種呼吸道的慢性發炎性疾病，其主要是經由呼吸 道吸入過敏原後’不钊起支氣管平滑肌收縮，而且有許多炎 ，介質(如Hista聰e、LTC4、驗等)的參與，同時呼吸道發 火能誘發婦續錢#的過度反應和獨 臨床上病人的主要特徵是在血清中有高於正常二^ 200916110 度，尤其#可以測到針對某種環境過敏原有特異性的每β抗 體另-個重要特徵是在病人的呼吸道中，可以看到多種免疫 細胞參與整個發病過程，其中又以嗜酸性白血球 、:mast cells (肥 胖細胞）及T細胞較為重要。而在氣喘過針 ，炎症細胞（嘻 中性白血球、倾性自树、淋巴球）、炎症介f、呼吸道平 滑肌及其神經性觸之相互_，被人認為是氣喘致病機轉的 ，基礎。因此’氣喘是—種慢性發炎反應所引触疾病，目 前對=抑制氣喘疾病的藥物，主要以支氣管擴張劑，及長期預 防的藥物，或稱抗發炎藥物，包括類_ (吸入型，口服型）、 抗白三埽藥物、咽達永樂等。因此目前使用的氣喘藥物大多含 有类員固醇及荷·等成分，或是其触絲的化合物 ，這些化 學藥品對生物體之免疫系統容易造成傷害，長麻用下除了產 生抗藥性之外，也料導致生長不正常_鮮病變的產生。 因此國内各研究單位針對氣喘用藥進行研究開發，希望能研發 出更，、功效蚊細伽的絲性產品。如今年年初工業技術 研究院所研發的植物萃取複方「抗氣喘藥物 BMEC-1217B」， 便獲美國FDA臨床測試許可，並且成功技術轉移給國内生物 科技A司廠商’現在更進行臨床測試。工業技術研究院產業經 濟與趨勢研究巾心職，今年全球氣喘藥品料難額，將超 過105億美元，商機無窮。 【發明内容】 本發明之目的，為提供一種由乾燥桂花(Osnianthiis fragrans fiower ; kwe卜_中萃取抗氣喘成份之方法，此方 200916110 法之程序簡單，可獲得一具有錢瑞物質的桂花萃取物 本發明之另-目的，該桂花萃取物具魏氣喘功效 =下將以實施舰-步說明本伽之實施方式， 下述所列 圍’任 ^=^_本發明，並麵戰她之範〇 林麟树和範_，當可辦 =更U飾’因財卿之㈣朗#視 圍所界定者為準。 Τ 【實施方式】 為使能進-步了解本發明之内容，及所能達成之功效，兹 配合圖示列舉較具體之實施例，詳細說明如下： 實施例1·桂花萃取物之製備 首先，請參閱第一圖所示，係將乾燥桂花以磨粉機研磨成 粉，以五倍體積75 %乙醇萃取桂花粉末2_3次，萃取方式為 浸泡萃取72，i、時。之後過齡除桂花粉末，轉其桂花^取 液，先經賴濃縮絲乙醇，接著_餘桂辟取朗冷滚乾 燥機冷束乾燥，以確保桂花萃取物之剩餘水分完全去除。、人、東 乾燥完成之桂花萃取物收集於樣品瓶，儲存於_2〇它備用。 實施例2.抗氣喘模式試驗 本試驗依氣喘生理下的反應，測定血清中特異性抗體 IgE、IgGl、IgG2a，肺沖洗液中炎症細胞嗜酸性白血球 (Eosinophil)組成比例，及呼吸道阻力測定作為評估氣喘反應之 200916110 條件。 實施例2-1. BALB/c小鼠餵食桂花萃取物28天及誘導氣喘反應 之條件 動物來源是購自財團法人國家實驗研究院國家實驗動物 繁殖及研究中心六週大BALB/c小鼠，將小鼠隨機分成六 組’每組六隻小鼠，分別依體重餵食，分成桂花萃取物組（低 劑量10〇!1^灯丑/]^及高劑量1_„^5^/]^)、正控制組及200916110 IX. Description of the Invention: [Technical Field] The present invention relates to a method for extracting anti-asthmatic substances from sweet-scented osmanthus, and extracting an anti-asthmatic substance of osmanthus extract in sweet-scented osmanthus by using the method, the sweet-scented osmanthus extract has Anti-asthmatic effect, has obvious health effects for living organisms, contains better physiological effects against asthma, slows down the symptoms of chronic inflammation of the respiratory tract and lungs, relieves asthma symptoms, and prevents and reduces asthma. [Prior Art] According to estimates by the World Health Organization (WHO), there are approximately 300 million people worldwide suffering from asthma, and their patients with severe asthma account for about %%. The number of asthmatic patients in Europe and the United States is about doubled every 10 years. The number of people who die from asthma every year is about 10,000. According to statistics, the incidence of asthma has been increasing year by year in the world, and it has increased year by year with the modernization of society. _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ The rate is 12-13% for Nanda, indicating that asthma is indeed one of the most common diseases in Taiwan. The mechanism of causing asthma has not been known so far, but recent studies have confirmed that asthma is a chronic inflammatory disease of the respiratory tract, which is mainly caused by the inhalation of allergens through the respiratory tract, which does not cause bronchial smooth muscle contraction, and there are many inflammations. The participation of media (such as Hista Cong e, LTC4, test, etc.), while the respiratory tract igniting can induce over-reaction of the woman's continued money and the main characteristic of the patient is that the serum is higher than the normal two ^ 200916110 degrees, especially # Another important feature that can be detected for each beta antibody specific to an environmental allergy is that in the patient's respiratory tract, a variety of immune cells can be seen to participate in the entire pathogenesis, including eosinophils, :mast cells ( Obese cells) and T cells are more important. In asthmatic needles, inflammatory cells (neutrophils, sputum, and lymphocytes), inflammation, respiratory muscles, and neurological touches are considered to be asthmatic pathogens. . Therefore, 'asthma is a disease caused by chronic inflammatory reaction, and currently drugs for suppressing asthma, mainly bronchodilators, and long-term preventive drugs, or anti-inflammatory drugs, including class _ (inhalation type, oral type) ), anti-white Sancha drugs, pharyngeal Yongle and so on. Therefore, most of the asthma medications currently used contain components such as steroids, ketones, and the like, or compounds that touch the filaments. These chemicals are likely to cause damage to the immune system of the organism, and in addition to producing resistance to marijuana, It is also expected to cause abnormal growth - the production of fresh lesions. Therefore, various research units in China have researched and developed anti-asthmatic drugs, and hope to develop more silky products with improved efficacy. For example, the plant extract compound “Anti-asthmatic drug BMEC-1217B” developed by the Industrial Technology Research Institute at the beginning of this year was approved by the US FDA clinical test, and the successful transfer of technology to the domestic biotechnology A division manufacturer is now undergoing clinical testing. Industrial Technology Research Institute's industrial economy and trend research towel, this year's global asthma medicine material is difficult, will exceed $10.5 billion, business opportunities are endless. SUMMARY OF THE INVENTION The object of the present invention is to provide a method for extracting anti-asthmatic components from dried osmanthus (Osnianthiis fragrans fiower; kwe), the procedure of the method 200916110 is simple, and an osmanthus extract having a medicinal substance can be obtained. According to another aspect of the present invention, the osmanthus extract has a Wei asthma effect = the following will be implemented in a ship-step description of the embodiment of the gamma, the following list of the 'Yong ^ = ^ _ the invention, and face her 〇林麟树和范_,When it can be done = more U ornaments 'Because of the definition of the four sides of the 财 卿 四 四 视 视 视 实施 实施 实施 实施 实施 实施 实施 实施 实施 实施 实施 实施 实施 实施 实施 实施 实施 实施 实施 实施 实施 实施 实施 实施 实施 实施 实施 实施 实施 实施 实施 实施The effect achieved is described in detail with reference to the specific examples, which are described as follows: Example 1 Preparation of Osmanthus fragrans Extract First, please refer to the first figure, which is to grind dry osmanthus to a powder by a mill. The osmanthus powder is extracted 2 times 3 times with 5 times volume of 75% ethanol, and the extraction method is soaking extraction 72, i, hour. After the mulberry flower powder is removed, the osmanthus extract liquid is taken, and the silk ethanol is firstly concentrated, then _ Yugui Cold-drying dryer To ensure that the residual moisture of the sweet-scented osmanthus extract is completely removed. The human and East dried osmanthus extract is collected in a sample vial and stored in _2 〇 for use. Example 2. Anti-asthmatic mode test This test is based on asthmatic physiology The reaction measures the specific antibodies IgE, IgG1, IgG2a in serum, the proportion of inflammatory cells eosinophil in the lung lavage fluid, and the airway resistance measurement as a condition for evaluating the asthma response 200916110. Example 2-1. BALB/c Conditions for feeding the sweet-scented osmanthus extract for 28 days in mice and inducing asthma response The source of the animal was purchased from the National Experimental Animal Breeding and Research Center of the National Experimental Animal Breeding and Research Center of the National Experimental Research Institute. The BALB/c mice were randomly divided into six groups. Six mice in each group were fed with body weight and divided into osmanthus extract group (low dose 10 〇! 1^灯 ugly /] ^ and high dose 1_„^5^/]^), positive control group and
負控制組（0.9 % normal saline)等方式餵食，投與體積為！ mL/100g小鼠體重’以塑膠針管套附傲食管（直徑〇9 mm，長度50mm)，以強迫方式連續管餵小鼠π天。 管餵小鼠28天配合誘導氣喘模式試驗，如第二圖所示， 分別於模式第0天及第14天時腹腔注射卵白蛋白 (Ovalbumin ; 〇VA)抗原，同時於試驗模式第35〜38天時，以 5%的卵白蛋白溶液進行噴霧，誘發小鼠啤吸道過敏反應，負 控制組則以0.9 % normai saline取代卵白蛋白進行噴霧。、 實施例2_2血清特異性抗體IgE、IgG卜IgG2a測定 過敏f·生氣％之主要特徵，就是血清中賊抗體明顯高 無過敏性疾病之正常個體，另外，在誘導過敏性氣喘的模式: BALB/c^、鼠咖會產生抗料異性的抗體，目此在本實黯 測定代表TM途徑的啦特異性抗體，以及加途徑 1成、尬抗體。收集小鼠血液於室溫下靜置2—3小時： 漏释轉速離心20分鐘，收集上層血清，保存於嗍 200916110 待分析。 在96 well反應盤中置入10 pg/ml卵白蛋白、blocking buffer 300 pL/wel卜 avidin-peroxidase 100 pL/weU、適量稀釋之 待測小鼠血清100 ML/well’以及細胞激素二級抗體試劑 OVA-IgE、OVA-IgGi、OVA-IgGh於室溫下反應二小時，然後 加入ABTS 100 pL/wel卜於室溫下且避光反應適當時間後， 在以ELISA測定儀測定405 nm吸光值。實驗結果顯示，於誘 導過敏性氣喘過程期間’若銀取桂花萃取物連續28天，均能 顯著減少發生氣喘發作時IgE抗體的產生（p<0〇5)(第三圖 所示)。在第四圖及第五圖的結果中顯示，IgGi抗體及igG2a抗 體在反應期間内增加’ 並顯著高於正控制組，首先此結 果顯示本試驗誘導過敏性氣喘的模式成功，小鼠體内有過敏現 象之外，並明顯指出桂花萃取物可誘發小鼠體内的Thi免疫反 應途徑’因而使小鼠體内過敏性氣喘的特徵，〗班抗體，含量 顯著降低。由此實驗證實100 mg KFE/kg的低劑量以及1〇〇〇 mg KFE/kg高劑量桂花萃取物都具有抗氣喘反應的生理功 效。及高劑量 實施例2-2小鼠呼吸道阻力測定 本實驗方法為一項非侵入式測定小鼠呼吸道阻力之方 法所利用儀斋系列為Whole-body plethysmograph、 Preampllfier 及 P觀ure transducer 等 BUXCO 系列設備，在 非钕入式操作下，由低到高濃度methacholine (6.25, 12.5, 25, 50 mg/mL)刺激小鼠誘發氣管收縮，再記錄小鼠呼吸阻力值 200916110 (PEnh)測量小鼠呼吸道阻力的方法，其數值愈高代表呼吸阻力 就愈大。實驗結果顯示，隨著methacholine刺激濃度增加， 各組呼吸道阻力也隨之增加，各組實驗和正控制組相比較，均 能顯著降低呼吸道阻力。顯示預先管餵不同濃度桂花萃取物連 續28天，顯著減缓呼吸道過度反應現象（如第六圖戶斤示）。 實施例2-2小鼠肺氣管沖洗液中炎症細胞嗜酸性白血球 (Eosinophil)分析 小鼠犧牲後抽取肺氣管沖洗液，經離心後取得之部分細胞 妨細胞玻片染色’以計數發炎細胞在肺沖洗液細胞之總數 (如第七圖卿）。實驗結麵示f餵不㈤難桂花萃取物連續 Μ天，各實驗組與氣喘小鼠（正控制組)相比下，均能顯著降 低小鼠肺沖洗射嗜雜白血球發炎細胞增生，顯示桂花萃取 物可以降低氣喘反應下肺部發炎的現象。 本發明的優點 1.本貫驗材料是便宜的食品原料桂花， 合製成飲料或其他形式的食品產品 2.本實驗萃取方式簡單 花’並非昂貴的藥材，並適 3. 對生物體有明顯的保健作用 少氣喘的發作，Feed in a negative control group (0.9% normal saline), and the volume is! The body weight of mL/100g mice was attached to the esophagus (diameter 〇9 mm, length 50 mm) with a plastic needle cannula, and the mice were fed continuously for π days in a forced manner. Tubes were fed to mice for 28 days in combination with induction of asthma model test. As shown in the second panel, ovalbumin (Ovalbumin; 〇VA) antigen was intraperitoneally injected on day 0 and day 14 of the model, respectively, in test mode 35~38. At daytime, a 5% egg albumin solution was sprayed to induce an allergic reaction in the mouse suction channel, while a negative control group was sprayed with 0.9% normai saline instead of ovalbumin. Example 2_2 Serum-specific antibody IgE, IgG IgG2a The main characteristic of allergic f·angid% is the normal individual with high thief antibody in serum and no allergic disease. In addition, the mode of inducing allergic asthma: BALB/ c^, rat coffee will produce antibodies against the opposite sex, in order to determine the specific antibody that represents the TM pathway, and the addition of 1%, sputum antibody. The blood of the mice was collected and allowed to stand at room temperature for 2-3 hours: centrifuged at a leak release speed for 20 minutes, and the upper serum was collected and stored in 嗍 200916110 for analysis. Place 10 pg/ml ovalbumin, blocking buffer 300 pL/wel avidin-peroxidase 100 pL/weU in 96 well reaction plate, appropriate dilution of test mouse serum 100 ML/well' and cytokine secondary antibody reagent OVA-IgE, OVA-IgGi, OVA-IgGh were reacted at room temperature for two hours, then ABTS 100 pL/wel was added at room temperature and protected from light for a suitable period of time, and the absorbance at 405 nm was measured by an ELISA meter. The results of the experiment showed that the injection of osmanthus extract for 28 consecutive days during the induction of allergic asthma significantly reduced the production of IgE antibodies (p<0〇5) in the onset of asthma (Fig. 3). In the results of the fourth and fifth figures, it was shown that the IgGi antibody and the igG2a antibody increased during the reaction period and were significantly higher than the positive control group. First, the results showed that the mode of induction of allergic asthma was successful in the mouse. In addition to allergies, it is also apparent that the osmanthus extract can induce the Thi immune response pathway in mice', thus making the characteristics of allergic asthma in mice significantly reduced. This experiment confirmed that the low dose of 100 mg KFE/kg and the high dose of osmanthus extract of 1〇〇〇 mg KFE/kg have the physiological effects of anti-asthmatic reaction. And high-dose Example 2-2 Respiratory resistance measurement in mice This experimental method is a non-invasive method for measuring respiratory resistance in mice. The instrument is based on BUXCO series equipment such as Whole-body plethysmograph, Preampllfier and Pureure transducer. Under non-invasive operation, mice were stimulated by low to high concentration methacholine (6.25, 12.5, 25, 50 mg/mL) to induce tracheal contraction, and then the mouse respiratory resistance value 200916110 (PEnh) was measured to measure respiratory resistance of mice. The higher the value, the greater the respiratory resistance. The experimental results showed that with the increase of the methacholine stimulation concentration, the airway resistance of each group also increased. Compared with the positive control group, the experimental group and the positive control group could significantly reduce the airway resistance. It was shown that pre-tube feeding different concentrations of sweet-scented osmanthus extract for 28 consecutive days significantly slowed down the phenomenon of respiratory hyperreactivity (as shown in Figure 6). Example 2-2 Eosinophil Analysis of Inflammatory Cells in Mouse Lung Tracheal Washing Fluid After the sacrifice of the mouse, the lung tracheal irrigating solution was taken, and some cells obtained by centrifugation were stained with cell slides to count the inflammatory cells in the lungs. The total number of cells in the rinse solution (eg, Figure 7). The experimental results showed that f-fed (five) hard-scented osmanthus extracts continued for a few days. Compared with asthmatic mice (positive control group), the experimental groups could significantly reduce the proliferation of inflammatory cells in the lungs of mice. The extract can reduce the inflammation of the lungs under the asthmatic reaction. Advantages of the invention 1. The test material is a cheap food raw material osmanthus, which is combined into a beverage or other form of food product. 2. The simple extraction method of the experiment is not an expensive medicinal material, and is suitable for the organism. The health care effect has less asthmatic attacks,
4. 經由口服攝取"一 炎症狀，希望藉！ 200916110 綜上所述，本發明以桂花萃取物做為抗過敏性氣喘之產 品，具有產業上之利用價值。 【圖式簡單說明】 第一圖：本發明之桂花萃取物製造流程圖。 第二圖：本發明之餵食桂花萃取物及誘導氣喘模式之實驗流程 圖。 第三圖：本發明之桂花萃取物對BALB/c小鼠血清中特異性抗 體IgE含量之影響。 第四圖：本發明之桂花萃取物對BALB/c小鼠血清特異性抗體 IgG!含量之影響。 第五圖：本發明之桂花萃取物對BALB/c小鼠血清特異性抗體 OVA-IgGa含量之影響。 第六圖：本發明之魏萃取麟BALB/e錢呼吸道阻力之影 響。 ’、’ 第七圖本發明之桂|匕萃取物對BALB/c +鼠肺氣管沖洗液發 炎細胞嗜酸性白血球之影響。 114. Through oral intake " a symptom of inflammation, I hope to borrow! In summary, the present invention uses osmanthus extract as an anti-allergic asthma product, and has industrial use value. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a flow chart showing the manufacture of the sweet-scented osmanthus extract of the present invention. Fig. 2 is a flow chart showing the experimental procedure for feeding the sweet-scented osmanthus extract and inducing the asthmatic pattern of the present invention. Figure 3: Effect of the sweet-scented osmanthus extract of the present invention on the specific antibody IgE content in serum of BALB/c mice. Figure 4: Effect of the osmanthus extract of the present invention on the serum specific antibody IgG! content of BALB/c mice. Figure 5: Effect of the sweet-scented osmanthus extract of the present invention on the serum specific antibody OVA-IgGa content of BALB/c mice. Figure 6: The effect of the WELB/e money respiratory resistance of the Wei extraction Lin of the present invention. ‘,’ The seventh figure shows the effect of the scent of scented scorpion extract of the present invention on eosinophilic leukocytes of inflammatory cells in BALB/c + rat lung tracheal rinse. 11