1343815 5 傳統的中草藥已被使用來治療遺傳之過敏 及氣喘病。結果顯示，使用傳統的中草藥後，大部八 的一般性症狀均獲得改善，但少部分患者㈣為不明二原 因，無法治癒。有些草藥能在短期内顯現效果，且只有少 數的副作用，但對於預防疾病的長期療效則仍須更多、的二 學研究。因此，需要傳統中草藥的更進一步研究。下列文 獻指出草藥具有調節免疫反應的功能，但其中大部分仍有 明顯的不確定因子，並需要仔細的研究。 以美國專利第09/949,610號申請案為例，該發明揭 10露一種用於鼻炎的醫藥組合物，其包括：麥冬 〇phi〇P〇gon)、半夏（Tuber pinelUae)、甘草（Radix Glycyrrhizae)與西洋蔘（Radix Pancis Quinquef〇H)的水溶 液萃取物’以及Herba Tridacis procumbentis的50%乙醇萃 取物。該處方包含5種草藥’並具有消炎的良好效果。然 15而，由於該配方僅具有短期舒緩症狀之效果，因此不容易 控制它的長期療效。》 WO 02/78723 Α1揭露一種製備具補腎以及抗氣喘效 果之中草藥成分，該中草藥包括：紫蘇（Perilla Frutescens)、苦杏仁（Prunus Armeniaca)、甘草（Glycyrrhiza 20 Uralensis)、黃答（Scutellaria Baicalensis)、黃連（Coptis Chinesis)、款冬（Tusilago Farfara)、直立百部（Stemona Sessilifolia)、貝母（Fritllaria Cirrhosa)、參還毛虫弓 l(Pheretima Aspergillum)、補骨脂（Psoralea Corylifolia)、黨參 (Codonopsis Pilosula)、六列大麥（Hordeum Vulgara)、六曲 1343815 (Massa Fermentata MedicaIls)、五味子（sch_dra Chinensis)以及石膏（Gypsum)。由於這個處方包含15種不 同的草藥，因此草藥萃取物的岢質很難掌握。而且不容易 · 觀察處方中每個成分各自的藥理反應。 5 W〇 02/32440揭露一種治療氣喘的九裡香（Murraya koenigii)萃取物。 GB 2274059揭露一種可舒緩氣喘的醫藥組合物，其 成分中包含与藥。這些發明使用單—植物成分來治療或舒 緩急性的氣喘症狀，但均無法維持長期的療效。 隹 ,1〇 因此，提供一種更簡單的組合物及其製備方法，能夠 使有效成分在治療疾病之前不會變化，並能改善治療免疫 疾病的長期療效，以排除前述問題的方法是值得研究的。 . 有些具有抗發炎反應效果的中草藥，可能對治療免疫 . 失調疾病有效果。例如，早在西元二世紀以前，中國就有 . 15、以黃芩（Scutellaria Baicalensis)抗發炎的文獻紀錄，尤其是 作為治療濕熱證（hot&damp )類疾病如痢疾與腹瀉，的主 要成分之一。然而，文獻並無記載如何將其製成可長期保 · 存之均質粉末，以及應該如何使用來治療過敏性氣喘。因 此，本發明揭露一新穎製程，以参少的步驟一來製備包含黃 20芩（Scutellaria Baicalensis)及其他三種草藥萃取物之有效 成分，並藉由活體外及活體内實驗，顯示其良好之效果。 換句話說，黃芩（ScuteHaria Baica丨ensis)與其他三種草藥萃 ， 取物之組合可以提供延長數種活性成分穩定性之優點，並 顯不出對於抗過敏性氣喘的效果，以及降低藥物對人體的 8 1343815 毒性。 【發明内容】 本發明之目的係提供一種可緩和過敏性氣喘之醫藥 5 組合物，並可有效地治療病患的過敏性氣喘》 為達成前述之目的’本發明用來治療過敏性氣喘之醫 藥組合物包括山藥（Radix Dioscorea)、澤瀉（Rhizoma Alismatis)、茯茶（Poria cocos (schw) Wolf)以及黃答 (Scutellaria Baicalensis)的乙醇萃取物。山藥、澤丨寫、.获茶 10以及黃芩的較佳重量比為1 : 1-2 : 0.5-2 : 1-3。其中山藥 可選自許多不同之種類，而效果最好的是由申請人（工業 技術研九I5元的專豕）所採集並鑑定的Dioscorea opposita Thunb或Di〇scorea alata L.。這兩種山藥所得之萃取物， 由接下來的體外實驗證明，同樣具有抑制細胞分泌細胞激 15 素的能力，如IL-4與TNF-alpha » 本發明亦提供一種製備包括山藥、澤瀉、茯苓以及黃 今之醫藥組合物的方法。該方法包括下列步驟：用乙醇分 別=取山藥、澤离、获荼以及黃答，以產生山藥、澤渴、 茯7以及s今之乙醇萃取物；過濾並濃縮前述乙醇萃取 20得到濃縮之山藥、澤萬、获荼以及黃料取物：並 品合前述山藥、澤萬、茯荼以及黃答濃縮物。 本發明更提供另-種製備包含山藥、澤渴、获茶以及 黃答之水或乙醇萃取物之醫藥組合物。該方法包括下列步 驟，混合山藥、澤萬、获岑以及黃答，以產生-混合物； 9 1343815 限制。 實施例1 :醫藥組合物TCM-A之製備 一種醫藥組合物，係藉由下列五個步驟製得： (1) 取400公克的山藥（RadixDi〇sc〇rea)莖或根、、 5 300公克的茯令（卩〇1^。〇(：〇8(3(^评）〜〇1〇以及5的公克的 黃今（Scutellaria Baicalensis)’分別切碎成小片。 (2) 將、山藥與茯苓分別浸泡於2〇〇〇毫升水中，黃 今次泡於70°/❶乙醇中3〇分鐘。每種溶液分別以85加熱 60分鐘。之後分別得到山藥、茯苓以及黃芩的萃取物。 10 (3)萃取物冷卻後’將其分別以篩網過濾（約1〇〇 網目）。收集過濾液’並在真空條件下（30t〇rr)，以5〇至 6〇°C進行濃縮而獲致藥草濃縮液。 (4) 在將每種濃縮液冷凍乾燥之前，添加作為賦形 知|的足里敌基甲基纖維素（carb〇Xyl methylcellul〇Se)，然 15後將其分別製成顆粒狀粉末。 (5) 再將個別的粉末混合均勻，所形成的混合顆 粒，稱為TCM-A，並將其製成膠囊。 實施例2 :醫藥組合物TCM-Β之製備 20 一種醫藥組合物，可經由下列五個步驟製備： (1) 取400公克山藥的莖或根、4〇〇公克的澤瀉、3㈧ 公克的茯苓以及500公克的黃芩，分別切碎成小片。 (2) 山藥與茯苓分別浸泡於2000毫升之水中，澤填 次泡於50%乙醇申，而黃芩則另外浸泡於7〇%乙醇中各 25分鐘。每種溶液分別於饥加熱⑼分鐘，之後分別得到 11 山藥、澤瀉、茯苓以及黃芩的萃取物。 (3) 等萃取物冷卻後，將其分別以篩網過濾（約1〇〇 網目）成過濾液。個別之過濾液經收集後，在真空條件下 (3〇t〇rr)，以50至60它進行濃縮，得到各草藥之萃取 縮液。 (4) 於各濃縮液進行冷;東乾燥前，添加足量叛基甲基 纖維素（作為賦形劑），並分別製成顆粒狀。 (5) 將各草藥顆粒進行混合，形成混合之顆粒即為 TCM-B，並進行膠囊製成。 實施例3 :醫藥組合物TCM-C之製備 —種醫藥組合物，可經由下列步驟製備： 、（1)取公克山藥的莖或根、400公克的澤瀉、3〇〇 公克的茯苓以及500公克的黃芩，分別切碎成小片。 (2) 將前三種草藥一起浸泡於2〇〇〇毫升的5〇%乙醇 中，而黃芩另外浸泡於70°/〇乙醇中。乙醇溶液加熱3〇分鐘 後得到山藥、澤瀉、获荟以及黃芩的乙醇萃取物。 (3) 等萃取物冷卻後，將其以篩網過濾（約丨〇〇網 目），並混合形成一過濾液。該過濾液在真空條件下（30 ΓΓ)，以50至6CTC進行濃縮。然後在真空條件下，將濃 縮液以冷凍乾燥法製成粉末。 (4) 添加足量羧基曱基纖維素或磷酸鈣（Ca3(p〇4)2)(作 為賦形劑）至乾燥粉末中並混合均勻。將全部的混合物以冷 東乾燥法製成顆粒狀，並命名為TCM-C。 (5) 將顆粒於乾燥環境下製成膠囊，並充氮裝瓶。 1343815 實施例4 :醫藥組合物tcm-D之製備 一種醫藥組合物，可經由下列步驟製 5⑴取侧公克的山藥莖或根、働公克的叫· 公克的获荼以及500公克的黃答，分別切成小碎片·，再混 合成一混合物。 (2) 將前述四種草藥的混合物一起浸泡於6〇〇〇毫升 之50%^醇。將該乙醇溶液加& 6〇分鐘後，得到混合山 10藥、澤瀉、茯苓以及黃芩的乙醇萃取物。 (3) 等該萃取物冷卻後，將其以筛網過渡（約1〇〇 網目）。收集過濾液，並在真空條件下（3〇沁叮），以咒至 60°C進行濃縮。將獲得之濃縮液於真空下進行冷凍乾燥。 (4) 添加足量的羧基甲基纖維素或磷酸鈣 15 (Ca3(P〇4)2)(作為賦形劑）到乾燥粉末中，並混合均勻。將整 個/昆合物以冷/東乾燥法製成顆粒，並命名為Tcm-D。 實施例5 :活體外試驗 以下將測試幾種草藥抑制ILw4的效果。如圖2a、2b 20及圖3所示，清楚地顯示山藥、澤瀉以及黃芩可抑制IL_4、 TNF-alpha 以及 eotaxin 的釋放。 (A)抑制培養的細胞株產生細胞激素（IL_4, TNF-alpha ) 於實驗曰程中’草藥於細胞培養液中，稀釋成終點濃 25 度（0.5 pg/m卜5pg/ml或視所需之適當濃度配置）。分別測 13 1343815 量每種草藥組成物或其混合物對IL-4、TNF-alpha產生的 影響.，並測量半胱胺醢基白三烯素（cysteiny〗_leuk〇trienes， c-LTs)。 A1 :以EL-4細胞株評估IL_4 : 5 將老鼠胸腺細胞株EL-4培養於每種組合物或混合物 2小時’再以1 〇〇ng/m丨之A23 187以及PMA進行刺激。組 織培養液的樣本在細胞培養24小時後收取，並保存於攝氏 零下負70度直至進行分析試驗。 採用商品化可購得之ELISA檢驗套組（r&d Systems, 10 Minneapolis，Minn)，並依照其使用指示，進行細胞激素的 评估。簡言之，在含有預先佈放之抗細胞激素抗體的微量 滴定盤中，分別於每個滴定孔中加入1〇〇μ試驗溶液，再 加入依照實驗需求稀釋過的標準/樣本溶液1〇〇 μ1，培養於 室溫。2小時後，將該滴定盤以洗劑沖洗3次，二次抗體 15及偵測用抗體以稀釋劑稀釋為1:2〇〇後，加入1〇〇时於 每個滴定孔中，將滴定盤封緊，室溫培養2小時。之後分 別於每個滴定孔中加入以稀釋劑稀釋至i : 2〇〇之 streptavidin-HRP反應受質，於黑暗室溫下培養2〇分鐘。 每個滴定孔在將未結合於孔内之streptavidin HRp吸除並 20洗淨後，加入受質反應液（TMB)，於黑暗室溫下培養分 鐘，再分別加入50 μ12Ν之硫酸溶液以終止反應。以微^ 滴定盤讀取機於450nm波長下檢測其光學反鹿數據。 評估TNF-alpha的步驟與前述IL.4的方^完全相同， 僅將培養的細胞株由EL-4替換為THp—丨，而所評估之活性 14 1343815 係以抗TNF-alpha之抗體為之。 如圖2a所示，由於澤瀉以及黃芩之乙醇萃取物的抑制 作用，IL-4與TNF-alpha的表現量顯著地降低（分別為90% 的 TNF-alpha、17%的 IL-4 以及 78%的 TNF-alpha、47%的 5 IL-4)。在濃度0·2 mg/ml的條件下，山藥之乙醇萃取物分 別抑制40%的IL-4以及20%的TNF-alpha。大部分萃取物 抑制細胞激素的效果與其使用的劑量有關，而當細胞激素 的濃度如同細胞培養液般降低時，蛋白質分解即增加。至 少就某部分而言，如同草藥萃取物在體内具有免疫抑制效 10 果一樣，體外試驗中，細胞激素產量的降低亦起因於EL-4 與THP-1細胞株生長量的減少。 除了將細胞株改為THP-1，並以RPMI為培養液，其 ;他實驗步驟均與前述評估IL-4的相同。滴定孔中的上清液 包含TNF-alpha的抗體，並經由ELISA方法確定TNF-alpha 15 的分泌量。 A2 :以MTT法檢測每個滴定孔中各草藥之細胞毒性。 以EL-4細胞分解MTT的能力，評估各種組合物或混 合物的細胞毒性。MTT ( 3’， 5’-Dimethyl- thiazol-2yl-2,5-diphenyltetrazolium bromide)，是一種黃色 20 化合物，可被粒腺體酵素分解為紫色結晶產物（稱為 formazan )，並可提供或引發細胞的相容性或細胞毒性。 藉由前述方法評估IL-4、TNF-alpha c-LTs，可以得知 EL-4、THP-1與RBL-1細胞株的細胞毒性。將5 mg /ml 的 MTT ( Sigma Chemical Co., St. Louis, Μο·)溶於 pH.7.4 1343815 的峨酸鹽緩衝液（phosphate buffer saline，PBS)中，以製 備MTT濃縮液。在指定的條件下培養21小時後，將5 μΐ 的ΜΤΤ加入每一個培養滴定孔中。再經過4小時培養後， 加入DMSO以停止反應。以Molecular Device的微量滴定 5 盤讀取機，560 nm的波長量測樣品的光學密度。資料係為 測量樣品的光學密度與未處理的對照組的比例。 A3: 以RBL-1細胞株評估半胱胺醯基白三烯素 (cysteinyl-leukotrienes，c-LTs): 以 0.1 pg/ml 甲酸（Retinoic acid)刺激RBL-1 細胞株（大 10 鼠嗜鹼性淋巴球-1細胞）16小時，以活化細胞内LTC4合成 酶。然後將草藥組成混合物加入培養液中，並於37°C培養2 小時。隨後再加入10 μΜ的A23187，以刺激製造c-LTs。培 養15分鐘後，終止實驗，並收集上清液100 μΐ。細胞上清 液中c-LTs之濃度以一 ELISA檢驗套組（Cayman Chemical， 15 Michigan, USA)，依照其使用手冊進行評估。 A4 :以老鼠腹膜巨噬細胞評估TNF-alpha : 腹膜巨噬細胞係取自事先進行腹腔注射3% Brewer thioglycolate培養液之6-8週大的雄性Balb/c老鼠。7天 後，以冰的RPMI-1640培養液與10%FBS灌洗腹腔，收集 20 腹膜細胞，並將其培養於37°C，2小時。非附著性的細胞 、 將會被沖洗下來，並作為TNF-alpha試驗之用。在該試驗 中，一特殊之P-38 MAP激酶的抑制劑-SB203580,將作 為各試驗的正對照組（positive control )。如圖4a所示，由 SB203580引起的抑制反應，亦可做為細胞對LPS反應的 1343815 指標。 (B) 抑制人類BEAS-2B細胞分泌eotaxin 本實驗所使用的BEAS-2B細胞係取自美國菌種保存 中心（American Type Culture Collection, ATCC)。BEAS-2B 5 上皮細胞係分離自非死於癌症的屍體的人類支氣管上皮細 胞。 本實驗包括兩個部分，細胞毒性測試，以及抑制 eotaxin分泌測試。每一種醫藥組合物及混合物將會先進行 細胞毒性之測試，說明如下，並以細胞的存活百分比表示 10 之。如圖3 a所示，百分比越高，代表測試的物質對細胞而 言是無毒性的。通常，無毒性之草藥混合物的細胞存活率 高達80%以上者，該混合物將被用來進行抑制eotaxin分 泌之試驗。 在eotaxin分泌試驗中，BEAS-2B細胞以DMEM/F12 15 培養液培養於96孔滴定盤中，在37°C環境下培養一天。 BEAS-2B細胞培養於添加特定濃度的草藥混合物之培養 液中，37°C，2小時；再添加含100 ng/ml IL-13以及100 ng/ml TNF-α的PBS 20 μΐ，於37°C培養72小時，以刺激 BEAS-2B細胞。之後，收集上清液100 μΐ，以ELISA法檢 20 測eotaxin的含量。未添加任何草藥混合物或是垂體腺苷 酸環化酶胜肽(pituitary adenylate cyclase polypeptides, PACAP )之培養液，其細胞分泌etotain的量為基本值。添 加0.00 1 μΜ的PACAP所抑制eotaxin的量，則作為驗證實 驗正確性之正對照組。 17 1343815 圖3a的資料顯示，包括山藥、澤瀉、茯苓、黃芩的 TCM-B混合物0.5pg/ml，可以明顯抑制BEAS-2B分泌 eotaxin，同時並不會對細胞產生毒性作用。圖3b的數據 則顯示TCM-B對於EL-4細胞株分泌IL-4的抑制情形。此 5 抑制反應與劑量相關，最高劑量500pg/ml即對EL-4細胞 株的生長發生影響。以包含36%澱粉與1.9%磷酸鈣 (Ca3(P04)2)之TCM-B粉劑，測試EL-4細胞株分泌IL-4的影 響。此測試同時也用以檢驗一般賦形劑，如澱粉與磷酸鈣， 對於EL-4細胞株分泌IL-4是否發生影響。結果顯示，賦 10 形劑，如澱粉與磷酸鈣，對於EL-4細胞株分泌IL-4的現 象並沒有影響。含TCM-B與賦形劑的粉劑，對於EL-4細 胞株分泌IL-4的現象具有一致的抑制效果，如圖3c所示， 因此澱粉與磷酸鈣在草藥粉末製備成顆粒之過程中加入， 作為賦形劑。 15 圖4a顯示，50pg/ml濃度的TCM-B對於腹膜巨嗜細 胞分泌TNF-alpha的現象具抑制效果。但TCM-B的抑制效 果與等量的澤瀉萃取物相比，較不明顯。然而，如圖4b 所示，50pg/ml濃度的澤瀉萃取物對於腹膜巨噬細胞卻具 有一定程度的細胞毒性。在抑制BEAS-2B細胞分泌 20 eotaxin，增加細胞存活率的實驗中，也發生相同狀況。因 此，混合山藥、澤瀉、茯苓、黃芩之組合物TCM-B，不只 可以維持其抑制TNF-alpha分泌之效果，也可以降低 TCM-B對所培養之細胞株所可能產生之細胞毒性。 18 13438151343815 5 Traditional Chinese herbal medicines have been used to treat genetic allergies and asthma. The results showed that after the use of traditional Chinese herbal medicines, the general symptoms of most of them were improved, but a small number of patients (4) were unknown and could not be cured. Some herbs can show their effects in a short period of time with only a few side effects, but more long-term studies are needed to prevent the long-term effects of the disease. Therefore, further research on traditional Chinese herbal medicine is needed. The following literature indicates that herbal medicines have the function of modulating immune responses, but most of them still have significant uncertainties and require careful study. For example, U.S. Patent Application Serial No. 09/949,610, the disclosure of which is incorporated herein by reference in its entirety, the disclosure of the entire disclosure of the disclosure of the disclosure of the entire disclosure of Glycyrrhizae) and aqueous extracts of Radix Pancis Quinquef〇H and 50% ethanol extract of Herba Tridacis procumbentis. The prescription contains 5 herbs' and has good anti-inflammatory effects. However, since this formula has only a short-term soothing effect, it is not easy to control its long-term efficacy. WO 02/78723 Α1 discloses a herbal ingredient for preparing kidney and anti-asthmatic effects, including: Perilla Frutescens, Prunus Armeniaca, Glycyrrhiza 20 Uralensis, Scutellaria Baicalensis, Coptis Chinesis, Tusilago Farfara, Stemona Sessilifolia, Fritllaria Cirrhosa, Pheretima Aspergillum, Psoralea Corylifolia, Codonopsis Pilosula , six rows of barley (Hordeum Vulgara), six songs 1343815 (Massa Fermentata MedicaIls), Schisandra (sch_dra Chinensis) and gypsum (Gypsum). Because this prescription contains 15 different herbs, the enamel of herbal extracts is difficult to grasp. And it is not easy. • Observe the pharmacological response of each component in the prescription. 5 W〇 02/32440 discloses a Murraya koenigii extract for the treatment of asthma. GB 2274059 discloses a pharmaceutical composition which soothes asthma and which contains ingredients. These inventions use a single-plant component to treat or relieve acute asthma symptoms, but are unable to maintain long-term efficacy.隹,1〇 Therefore, it is worthwhile to provide a simpler composition and a preparation method thereof, which can make the active ingredient not change before the treatment of the disease, and can improve the long-term therapeutic effect of the immune disease, so as to eliminate the aforementioned problem. . Some Chinese herbal medicines with anti-inflammatory effects may be effective in treating immune disorders. For example, as early as the second century AD, there was a record of anti-inflammation in Scutellaria Baicalensis, especially as one of the main components of hot &le damp diseases such as dysentery and diarrhea. . However, the literature does not describe how it can be made into a homogeneous powder that can be preserved for a long time, and how it should be used to treat allergic asthma. Therefore, the present invention discloses a novel process for preparing an active ingredient comprising Scutellaria Baicalensis and three other herbal extracts in steps 1 and exhibiting good effects by in vitro and in vivo experiments. . In other words, the combination of ScuteHaria Baica丨ensis and the other three herbal extracts provides the advantage of extending the stability of several active ingredients, and shows no effect on anti-allergic asthma, as well as reducing the drug to the human body. 8 1343815 Toxic. SUMMARY OF THE INVENTION The object of the present invention is to provide a pharmaceutical composition for alleviating allergic asthma, and to effectively treat allergic asthma of a patient. To achieve the aforementioned object, the present invention is for treating allergic asthma. The composition includes the ethanol extracts of Radix Dioscorea, Rhizoma Alismatis, Poria cocos (schw) Wolf, and Scutellaria Baicalensis. The preferred weight ratio of yam, yam, and tea 10 and scutellaria is 1: 1-2 : 0.5-2 : 1-3. Among them, yam can be selected from many different types, and the best effect is Dioscorea opposita Thunb or Di〇scorea alata L., which is collected and identified by the applicant (Industrial Technology Research Institute I5 yuan). The extracts obtained from these two kinds of yam, as demonstrated by the following in vitro experiments, also have the ability to inhibit the secretion of cytokines by cells, such as IL-4 and TNF-alpha. The present invention also provides a preparation including yam, Alisma,茯苓 and the method of the pharmaceutical composition of Huang Jinzhi. The method comprises the steps of: taking yam, yttrium, sputum and yellow sputum respectively with ethanol to produce yam, thirst, sputum 7 and s ethanol extract; filtering and concentrating the aforementioned ethanol extract 20 to obtain concentrated yam , Zewan, obtained cockroaches and yellow material: and the above-mentioned yam, Zewan, sputum and yellow aloe concentrate. The present invention further provides a pharmaceutical composition for preparing a water or ethanol extract comprising yam, thirst, tea, and yellow. The method comprises the steps of mixing yam, Zewan, sputum and yellow yoke to produce a mixture; 9 1343815 limit. Example 1: Preparation of pharmaceutical composition TCM-A A pharmaceutical composition was prepared by the following five steps: (1) taking 400 g of radish (Radix Di〇sc〇rea) stem or root, 5 300 g The order of 、1^.〇(:〇8(3(^评)~〇1〇 and 5 grams of Scutellaria Baicalensis' is chopped into small pieces. (2) Will, Yam Soaked in 2 ml of water, respectively, and soaked in 70 ° / ❶ ethanol for 3 〇 minutes. Each solution was heated at 85 for 60 minutes. After that, extracts of yam, medlar and astragalus were obtained respectively. After the extract is cooled, it is filtered by a sieve (about 1 mesh). The filtrate is collected and concentrated under vacuum (30t rr) at 5 〇 to 6 ° C to obtain a concentrated herb. (4) Before lyophilizing each concentrate, add carb〇Xyl methylcellul〇Se as a shape, and then separate them into granules. (5) Mix the individual powders evenly, and form the mixed particles, called TCM-A, and make them into capsules. Example 2: Preparation of pharmaceutical composition TCM-Β 20 A pharmaceutical composition can be prepared by the following five steps: (1) taking 400 grams of yam stem or root, 4 gram of diarrhea, 3 (eight) gram of茯苓 and 500 grams of scutellaria, chopped into small pieces. (2) Yam and sputum were soaked in 2000 ml of water, respectively, and filled with 50% ethanol, while scutellaria was immersed in 7 〇% ethanol. Minutes. Each solution was heated for hunger (9) minutes, then 11 extracts of yam, Alisma, Poria and Astragalus were obtained. (3) After the extracts were cooled, they were separately filtered by mesh (about 1 mesh). The filtrate is collected, and the individual filtrates are collected and concentrated under vacuum (3〇t〇rr) at 50 to 60 to obtain the extract condensate of each herb. (4) Cold in each concentrate Before the east drying, a sufficient amount of ruthenylmethylcellulose (as an excipient) is added and separately granulated. (5) The granules of each herb are mixed to form a mixed granule which is TCM-B, and is carried out. Capsules made. Example 3: Preparation of pharmaceutical composition TCM-C - The pharmaceutical composition can be prepared by the following steps: (1) taking the stem or root of the gram yam, 400 grams of Alisma, 3 gram of sputum, and 500 grams of scutellaria, which are chopped into small pieces. The first three herbs were immersed in 2 ml of 5 〇% ethanol, and the scutellaria was additionally immersed in 70 ° / 〇 ethanol. The ethanol solution was heated for 3 〇 minutes to obtain yam, Alisma, sap and ethanol. (3) After the extract is cooled, it is filtered through a sieve (about mesh) and mixed to form a filtrate. The filtrate was concentrated under vacuum (30 Torr) at 50 to 6 CTC. The concentrate was then lyophilized to a powder under vacuum. (4) A sufficient amount of carboxymethyl cellulose or calcium phosphate (Ca3(p〇4) 2) (as an excipient) is added to the dry powder and mixed well. The entire mixture was pelletized by cold drying and designated TCM-C. (5) Capsules are granulated in a dry environment and filled with nitrogen. 1343815 Example 4: Preparation of pharmaceutical composition tcm-D A pharmaceutical composition can be obtained by the following steps: 5 (1) taking the side gram of the yam stem or root, the cockroach gram of the gram of the gram, and the 500 gram of the yellow gram, respectively Cut into small pieces and mix them into a mixture. (2) Soak a mixture of the aforementioned four herbs together in 6 ml of 50% alcohol. After the ethanol solution was added for < 6 minutes, an ethanol extract of mixed mountain 10, Alisma, sputum, and Astragalus was obtained. (3) After the extract is cooled, it is transitioned by a sieve (about 1 〇〇 mesh). The filtrate was collected and concentrated under vacuum (3 Torr) with a curse to 60 °C. The obtained concentrate was freeze-dried under vacuum. (4) Add a sufficient amount of carboxymethylcellulose or calcium phosphate 15 (Ca3(P〇4)2) (as an excipient) to the dry powder and mix well. The whole/kun compound was pelletized by a cold/east drying method and designated as Tcm-D. Example 5: In vitro test The effects of several herbs on the inhibition of ILw4 were tested below. As shown in Figures 2a, 2b, and 3, it is clearly shown that yam, Alisma, and Astragalus can inhibit the release of IL_4, TNF-alpha, and eotaxin. (A) Inhibition of cultured cell lines to produce cytokines (IL_4, TNF-alpha) In the course of the experiment, 'herbal in cell culture medium, diluted to a final concentration of 25 degrees (0.5 pg/m b 5pg/ml or as needed) The appropriate concentration configuration). The effect of each of the herbal compositions or their mixtures on IL-4 and TNF-alpha was measured by 13 1343815, and cysteine leukotrienes (cyliny _leuk〇trienes, c-LTs) were measured. A1: Evaluation of IL_4 by EL-4 cell line: 5 Mouse thymocyte cell line EL-4 was cultured in each composition or mixture for 2 hours' and then stimulated with 1 〇〇ng/m丨 of A23 187 and PMA. Samples of the tissue culture medium were collected 24 hours after cell culture and stored at minus 70 degrees Celsius until analysis. Commercially available ELISA test kits (r&d Systems, 10 Minneapolis, Minn) were used and cytokine assessments were performed according to their instructions. Briefly, in a microtiter plate containing pre-plated anti-cytokine antibodies, 1 μμ test solution is added to each titration hole, and a standard/sample solution diluted according to the experimental requirements is added. 11, cultured at room temperature. After 2 hours, the titration plate was rinsed 3 times with a lotion, and the secondary antibody 15 and the detection antibody were diluted to 1:2 稀释剂 with a diluent, and titrated in each titration hole when 1 加入 was added. The plate was sealed and incubated for 2 hours at room temperature. Thereafter, a streptavidin-HRP reaction substrate diluted to a ratio of i: 2 was added to each of the titration wells, and cultured at room temperature for 2 minutes in the dark. Each titration hole was aspirated and 20 washed in streptavidin HRp not bound to the well, and then added to the matrix reaction solution (TMB), and incubated at room temperature for several minutes, and then 50 μl of sulfuric acid solution was added to terminate the reaction. . The optical anti-stag data was detected at a wavelength of 450 nm using a microplate reader. The procedure for assessing TNF-alpha is identical to that of the aforementioned IL.4, only the cultured cell line is replaced by EL-4 to THp-丨, and the activity 14 1443815 evaluated is based on an antibody against TNF-alpha. . As shown in Figure 2a, the expression of IL-4 and TNF-alpha was significantly reduced due to the inhibition of Alisma orientale and Astragalus ethanol extract (90% TNF-alpha, 17% IL-4, and 78, respectively). % TNF-alpha, 47% of 5 IL-4). At a concentration of 0·2 mg/ml, the ethanol extract of yam inhibited 40% of IL-4 and 20% of TNF-alpha, respectively. The effect of most extracts on the inhibition of cytokines is related to the dose used, and when the concentration of cytokines is reduced as well as cell culture, protein breakdown increases. At least in part, as in the case of herbal extracts with immunosuppressive effects in vivo, the reduction in cytokine production in vitro was also due to a decrease in the growth of EL-4 and THP-1 cells. In addition to changing the cell line to THP-1 and using RPMI as the culture medium, the experimental procedures were the same as those for the evaluation of IL-4. The supernatant in the well was titrated with an antibody containing TNF-alpha, and the amount of TNF-alpha 15 secreted was determined by an ELISA method. A2: The cytotoxicity of each herb in each titration well was measured by the MTT method. The cytotoxicity of various compositions or mixtures was evaluated by the ability of EL-4 cells to decompose MTT. MTT ( 3', 5'-Dimethyl- thiazol-2yl-2, 5-diphenyltetrazolium bromide) is a yellow 20 compound that can be broken down into a purple crystalline product (called formazan) by a granular gland enzyme and can be supplied or triggered. Cell compatibility or cytotoxicity. The cytotoxicity of EL-4, THP-1 and RBL-1 cell lines can be known by evaluating IL-4 and TNF-alpha c-LTs by the aforementioned methods. 5 mg / ml of MTT (Sigma Chemical Co., St. Louis, Μο.) was dissolved in phosphate buffer saline (PBS) at pH 7.4 1343815 to prepare an MTT concentrate. After incubation for 21 hours under the specified conditions, 5 μM of hydrazine was added to each of the culture titration wells. After another 4 hours of incubation, DMSO was added to stop the reaction. The optical density of the sample was measured by a microplate of a Molecular Device with a 5-plate reader at a wavelength of 560 nm. The data is the ratio of the optical density of the measured sample to the untreated control. A3: Evaluation of cysteine-leukotrienes (c-LTs) by RBL-1 cell line: stimulation of RBL-1 cell line with 0.1 pg/ml formic acid (Retinoic acid) Lymphocytes-1 cells) for 16 hours to activate intracellular LTC4 synthetase. The herbal composition mixture was then added to the culture medium and incubated at 37 ° C for 2 hours. Then 10 μΜ of A23187 was added to stimulate the production of c-LTs. After 15 minutes of incubation, the experiment was terminated and the supernatant was collected 100 μM. The concentration of c-LTs in the cell supernatant was evaluated by an ELISA test kit (Cayman Chemical, 15 Michigan, USA) according to its manual. A4: Evaluation of TNF-alpha by rat peritoneal macrophages: The peritoneal macrophage cell line was obtained from male Balb/c mice 6-8 weeks old who had been intraperitoneally injected with 3% Brewer thioglycolate. After 7 days, the peritoneal cavity was lavaged with ice RPMI-1640 medium and 10% FBS, and 20 peritoneal cells were collected and cultured at 37 ° C for 2 hours. Non-adherent cells will be washed out and used as a TNF-alpha assay. In this assay, a special inhibitor of P-38 MAP kinase, SB203580, will serve as a positive control for each assay. As shown in Figure 4a, the inhibition reaction caused by SB203580 can also be used as an indicator of the 1343815 response of cells to LPS. (B) Inhibition of secretion of eotaxin by human BEAS-2B cells The BEAS-2B cell line used in this experiment was obtained from the American Type Culture Collection (ATCC). The BEAS-2B 5 epithelial cell line is isolated from human bronchial epithelial cells that are not dying from cancer. This experiment consists of two parts, a cytotoxicity test, and an inhibition of the eotaxin secretion test. Each of the pharmaceutical compositions and mixtures will be tested for cytotoxicity as described below and expressed as a percentage of cell survival. As shown in Figure 3a, the higher the percentage, the substance that is tested is non-toxic to the cells. Usually, the cell viability of a non-toxic herbal mixture is as high as 80% or more, and the mixture will be used for the test for inhibiting eotaxin secretion. In the eotaxin secretion assay, BEAS-2B cells were cultured in a 96-well titration dish in DMEM/F12 15 medium and cultured for one day at 37 °C. BEAS-2B cells were cultured in a medium supplemented with a specific concentration of herbal mixture at 37 ° C for 2 hours; then PBS 20 μΐ containing 100 ng/ml IL-13 and 100 ng/ml TNF-α was added at 37°. C was cultured for 72 hours to stimulate BEAS-2B cells. Thereafter, the supernatant was collected 100 μM, and the content of eotaxin was measured by ELISA. The culture medium containing no herbal mixture or pituitary adenylate cyclase polypeptides (PACAP) was added, and the amount of etotain secreted by the cells was a basic value. The amount of eotaxin inhibited by the addition of 0.001 μΜ of PACAP was used as a positive control group to verify the correctness of the experiment. 17 1343815 The data in Figure 3a shows that 0.5 μg/ml of TCM-B mixture including yam, Alisma, Poria and Astragalus can significantly inhibit the secretion of eotaxin by BEAS-2B without toxic effects on cells. The data in Figure 3b shows the inhibition of IL-4 secretion by IL-4 cells by TCM-B. The 5 inhibitory response was dose-related, and the highest dose of 500 pg/ml affected the growth of the EL-4 cell line. The effect of IL-4 cell line secretion of IL-4 was tested with a TCM-B powder containing 36% starch and 1.9% calcium phosphate (Ca3(P04)2). This test is also used to test whether general excipients, such as starch and calcium phosphate, affect IL-4 cell line secretion of IL-4. The results showed that the formulation of a 10-form agent such as starch and calcium phosphate had no effect on the secretion of IL-4 by the EL-4 cell line. The powder containing TCM-B and the excipient has a consistent inhibitory effect on the secretion of IL-4 by the EL-4 cell line, as shown in Fig. 3c, so the starch and the calcium phosphate are added in the process of preparing the granules of the herbal powder. As an excipient. 15 Figure 4a shows that TCM-B at a concentration of 50 pg/ml has an inhibitory effect on the secretion of TNF-alpha by peritoneal macrophages. However, the inhibitory effect of TCM-B was less pronounced than that of the equivalent amount of Alisma orientalis extract. However, as shown in Figure 4b, the Alisma orientale extract at a concentration of 50 pg/ml has a certain degree of cytotoxicity to peritoneal macrophages. The same situation occurred in experiments that inhibited the secretion of 20 eotaxin by BEAS-2B cells and increased cell viability. Therefore, the combination of yam, diarrhea, medlar, and astragalus TCM-B can not only maintain its effect of inhibiting TNF-alpha secretion, but also reduce the cytotoxicity of TCM-B against the cultured cell line. 18 1343815
Example 6. 活體内實驗 (A) 實驗動物 雄性Balb/c老鼠係來自國家實驗動物中心，並飼養 於無特定病原之環境。這些老鼠均為6-8週大。依照處理 5 方式不同，6隻老鼠分為一組，每組的老鼠均分開飼養。 重組之 Dermatophoides pteronyssinus group 2 ( Der p 2 )係 由蔡博士提供，而傳統中藥 A ( Traditional Chinese Medicine A，TCM-A，圖 7、8、¥與 10 中之、1?17A)以及Example 6. In vivo experiments (A) Experimental animals Male Balb/c mice are from the National Laboratory Animal Center and are maintained in a pathogen-free environment. These mice are 6-8 weeks old. According to the method of treatment 5, 6 mice were divided into one group, and the rats in each group were kept separately. The recombinant Dermatophoides pteronyssinus group 2 ( Der p 2 ) was provided by Dr. Cai, while Traditional Chinese Medicine A (TCM-A, Figures 7, 8, ¥ and 10, 1?17A)
I 傳統中藥 B ( Traditional Chinese Medicine B，TCM-B ’ 圖 10 7、8、9與10中之1217B)則來自台灣新竹之工研院生醫 中心。 (B) 引發過敏性氣管發炎反應 老鼠於分別第〇天與第7天，以腹腔注射的方式，注 射以 4 pg/0.062 ml 氧化紹（aluminum hydroxide，Α1(ΟΗ)3, 15 Whitehall Lab Ltd, Punchbowel, Australia)乳化之 1 pg/0· 1 ml Der p 2使其發生免疫反應《之後，以TCM餵食老鼠3 週。不同組別的老鼠，分別银食：地塞美松（Dexamethasome) (每鼠每天1 pg)，TCM-A (每鼠每天20 mg )，TCM-B (每 鼠每天20 mg)或是一般的生理食鹽水每鼠每天500 μΐ。 20 在第28與35天時，將老鼠以60 mg/kg劑量的苯巴比妥鈉 (sodium pentobarbital, Sigma Chemical Co., St. Louis, MO，USA)經由腹膜注射使輕微麻醉後，自氣管接種1 pg/50 μΐ的Der p 2。並於第二次接種後24小時，測量肺 功能，隨後將老鼠犧牲。 19 1343815 (c)測定肺功能 將每隻老鼠放於氣壓體積測量儀（Buxco electronics， Troy，NY )中。該測量儀包括有兩個空室：一個是主室， 或稱為動物室（内徑7.5公分，3.5公分高）；另一個則為參 5考室（内徑7.5公分，3.5公分高）。一壓力差轉移器被用來 偵測前述兩個空室的壓力差。該壓力差經由類比/數位訊 號轉換放大後’將訊號傳送至一載有BioSystem XA系統 (Buxco, Electronics，Troy，NY)之電腦，並進行樣品的呼 吸參數分析。與Hamelmann等人（1997 )的報告相似，增 10 強性間歇（enhanced pause，Penh) ’ 間歇（pause)，潮氣量 (tidal volume, Vt)，呼吸頻率（breathing frequency, f)， 最高吸氣量（peak inspiratory flow，PIF)，最高呼氣量（peak expiratory flow，PEF )，吸氣末間歇（end-inspiratory pause, EIP)以及呼氣末間歇（end-expiratory pause，EEP等參數 15 將可測得，結果如圖5所示。 將5毫升的食鹽水或乙醯丑曱基膽素（methach〇Hne， 1.56至50,00 mg/ml)溶液置於超音波霧化機（uUras〇nic nebulizer，DeVilbiss，Somerset, PA)中，可產生霧氣，並 經由三向管連接至氣壓體積測量儀的動物室。霧氣的平均 20大小約為3 μηι，依據不同製造商的規格，其大小範圍自1 至5 μηι。通常在15至20秒内，霧氣可以充滿空室。開始 時’先讓老鼠吸入食鹽水氣3分鐘，再測量其呼吸參數3 为知。然後’以乙醯丑甲基膽素替換食鹽水氣，並讓老鼠 吸入3分鐘。曝氣後’立即將空室内的霧氣排除。吸入乙 20 1343815 醯丑曱基膽素霧氣後，測量呼吸參數3分鐘。乙酿丑甲基 λ 膽素劑量·反應曲線係由低劑量至高劑量來表示。如圖5所 示，TCM-B與地塞美松可以改善肺功能以及過敏動物的免 疫發炎反應。每兩次曝氣之間，有15分鐘的間隔。數據係 5為平均值士SEM。不同組之間參數的差異，將分析其變數。 如果’不同組之間有顯著的差異，將以Newman-Keuls法 分析任兩組間的統計上的差異。曝氣於食鹽水或 乙醯丑曱 基膽素之前或之後的數值差異，將以成對t法分析。食鹽 水對照組與未實驗組之間的差異，將以非成對t法分析。 鲁 10若是P值小於0·〇5 ’該差異視為顯著。 (D)樣品收集與製備 測里肺功此後’支氣管肺泡灌洗（bronchoaive〇iar 4 laVage，BAL)將依下列方法進行。分2次注射1毫升消毒、 . 無内毒素之食鹽水，自氣管注射至鼠肺中。回收大約丨8 ‘ 15毫升的BAL沖洗液。該帶有氣體之BAL沖洗液在實驗前 保存於-70°C。於全白血球計數後，1〇〇毫升BAL沖洗液 經過細胞離心，以Liu染劑染色（T〇nyar Diagn〇stic Inc，I Traditional Chinese Medicine B (TCM-B 'Fig. 10, 12, 8 and 9 of 1217B) is from the Institute of Biomedical Sciences of HYI, Hsinchu, Taiwan. (B) Inducing allergic tracheal inflammatory response mice were injected intraperitoneally with 4 pg/0.062 ml of aluminum hydroxide (Α1(ΟΗ)3, 15 Whitehall Lab Ltd, on day 3 and day 7, respectively. Punchbowel, Australia) Emulsified 1 pg/0·1 ml Der p 2 to cause an immune response. After that, mice were fed with TCM for 3 weeks. Different groups of rats, silver food: Dexamethasome (1 pg per mouse per day), TCM-A (20 mg per mouse per day), TCM-B (20 mg per mouse per day) or general Physiological saline was administered 500 μM per mouse per day. 20 On days 28 and 35, mice were given a 60 mg/kg dose of sodium pentobarbital (Sigma Chemical Co., St. Louis, MO, USA) via a peritoneal injection for mild anesthesia, from the trachea Inoculate 1 pg/50 μΐ of Der p 2 . At 24 hours after the second vaccination, lung function was measured and the rats were sacrificed. 19 1343815 (c) Determination of lung function Each mouse was placed in a barometric volume meter (Buxco electronics, Troy, NY). The gauge consists of two empty chambers: one is the main chamber, or the animal chamber (7.5 cm inside diameter, 3.5 cm high); the other is the reference room (7.5 cm inside diameter, 3.5 cm high). A differential pressure diverter is used to detect the pressure difference between the two empty chambers. The pressure difference is amplified by analog/digital conversion and transmitted to a computer carrying the BioSystem XA system (Buxco, Electronics, Troy, NY) for sample parameter analysis. Similar to the report of Hamelmann et al. (1997), an increase in the intensity of pause (Penh) 'pause, tidal volume (Vt), respiratory frequency (f), maximum inspiratory volume (peak inspiratory flow, PIF), peak expiratory flow (PEF), end-inspiratory pause (EIP), and end-expiratory pause (EPP, etc.) The results are shown in Figure 5. A solution of 5 ml of saline or meth 醯 曱 胆 胆 (methach〇Hne, 1.56 to 50,00 mg/ml) was placed in a ultrasonic atomizer (uUras〇nic nebulizer). , DeVilbiss, Somerset, PA), can generate fog, and is connected to the animal chamber of the barometric volume measuring instrument via a three-way tube. The average size of the mist is about 3 μηι, depending on the manufacturer's specifications, the size ranges from 1 Up to 5 μηι. Usually within 15 to 20 seconds, the mist can fill the empty chamber. At the beginning, let the mouse inhale the saline gas for 3 minutes, and then measure the respiratory parameter 3 for the knowledge. Then, the acetaminophen Replace salt water and let the mouse Into 3 minutes. Immediately after aeration, the mist in the empty chamber was removed. After inhaling B 20 1343815 醯 曱 曱 胆 素 素 , , , 测量 测量 测量 测量 测量 测量 测量 测量 测量 测量 测量 测量 测量 测量 测量 测量 测量 测量 测量 测量 测量 测量 测量 测量 测量 测量 测量 测量 测量 测量 测量 测量The dose is expressed as a high dose. As shown in Figure 5, TCM-B and dexamethasone can improve lung function and immune inflammatory response in allergic animals. There is a 15-minute interval between each aeration. Data is 5 Mean SEM. Differences in parameters between different groups will be analyzed for their variables. If there is a significant difference between the different groups, the statistical difference between the two groups will be analyzed by Newman-Keuls method. Aeration in salt The difference in value before or after water or acetaminophen bilirubin will be analyzed by paired t method. The difference between the saline control group and the untested group will be analyzed by the unpaired t method. The value is less than 0·〇5 'The difference is considered significant. (D) Sample collection and preparation of lung function After this, 'bronchoaive〇iar 4 laVage (BAL) will be carried out according to the following method. Milliliter disinfection, . Endotoxin-free saline solution, Injection from the trachea into the lungs of the rats. Recover about 8 '15 ml of BAL rinse. The BAL rinse with gas was stored at -70 ° C before the experiment. After the whole white blood cell count, 1 ml of BAL rinse The solution was centrifuged and stained with Liu dye (T〇nyar Diagn〇stic Inc,
Taipei shien，Taiwan)並分別計算細胞。結果顯示於圖乂 · 與下面表一 ’ TCM-A與TCM-Β均可降低BAL沖洗液令有 2〇發炎反應的細胞數。血液樣本採自眶竇（〇rbiul sinus )， 而取得之血清在實驗前係保存於_7(rc ^ · 21 1343815 表1 樣品 細胞總數 (xl04/ml) 相對計數(xl〇4/ml) 巨噬細胞 淋巴細胞 嗜中性白 血球 啥伊紅性 白血球 控制組 14·3±4.3* 13.3±4.3* 1.0±0.0* 0 0 t理食鹽水 160.0+23.5 56.4+12.7 41.4 土 6.5 36.8±2.8 25.2±4.5 地塞美松 46A±2.2* 29.2±4A* 9.4±1.5* 6.6±1.0* 3.2±0.4* TCM-A 49.0±3·7* 25.8±2.5* 13.2±1·8* 6.0+0.7* 4.010.7* TCM-B 41.6±6.2* 21.2±2.6* 10.212.4* 6.0±1.2* 4.2±1.1* (E)測定具Der p 2專一性之IgG 1、IgG2a與IgE抗體 在實驗開始與結束前後，自眼窩（retro-orbital venous plexus)取血。藉由ELISA法，測定血清中抗Der p 2的 5 IgE、IgGl以及IgG2a的效價。將微量滴定盤（Nunc Lab, IL， USA)表面覆上濃度為0·5 pg/ml的100 μΐ Der p 2，置放 於4°C冰箱中隔夜。滴定盤以PBS-Tween-20 ( PBST)沖洗 三次，並保存於-70°C直到使用。添加老鼠血清以1:5稀釋 的IgE，1:100稀釋的IgGl，及1:5稀釋的IgG2a後，將滴 10 定盤置放於4°C中隔夜，在添加抗體（HRP-鍵結的山羊抗老 鼠的抗體，其中抗IgE與抗IgG2a的抗體為1:800稀釋， 抗 IgGl 抗體為 1:2000 稀釋，賭自 SouthernBiotech Assoc, Inc, Birmingham, AL, USA)前清洗 3 次。在 37°C 培養 1 小 時後，以PBST沖洗3次，再添加酵素反應受質ABTS 15 ( 2,2“-Azion-bis(3-ethylbenzothiazolin-6-sulfonic acid) diammonium salt, Bio-Rad, USA)。15 分鐘後，加入 50 μΐ 4N H2SO4以終止反應，並以多波長之分光光度計（A-5682, 22 1343815 SLT Lab Instruments, Salzburg，Austria)以 450nm 波長測 量光度。結果以ELIS A單位（EU )表示。一個EU定義為 血清稀釋後的倒數，所測得的光度值為1.0。該結果均落 在稀釋曲線的線性部分。為了確認再現性，在每次實驗中 5 將一個已知的血清作為標準值。圖7係為試驗血清中，IgE 的滴定濃度。 (F) 老鼠樣本中的細胞激素試驗 以商品購得之ELISA檢驗套組測量IFN- 7*與IL-5， 並使用老鼠的單株抗體，以決定細胞激素表面不同之抗原 10 決定位（epitopes )。最低的檢測範圍是10 pg/ml。結果如 圖8、9所示。 (G) 支氣管肺泡灌洗所得之淋巴球的培養，免疫螢光染色 以及流式細胞分析（flow cytometry analysis ) 依照Assenmacher等人（1994 )提出的方法，可以流 15 式細胞分析測量老鼠T助手細胞活化的細胞激素。雙染色 法被用來分析在CD4或CD8細胞IFN-T*與IL-5的表現。 來自周邊血液的白血球，係經過PMA ( 50 ng/ml )、 ionomycin( 2 μΜ)與 GolgiStop( Cytofix/Cytoperm Plus Cat No. 2076KK，Pharmingen, San Diego，CA ) 5 小時的刺激， 20 再以PBS沖洗2次。在室溫下，細胞以CD4-FITC或 CD8-FITC染色30分鐘，再加以清洗。細胞於室溫下，以 cytofix/cytoperm固定30分鐘，再以抗細胞激素之抗體染 色30分鐘，再沖洗之。將細胞重新懸浮於含有0.1 %疊氮 化鈉（sodium azide)之 0.5 ml PBS 中。以 Becton Dickinson 23 1343815 flow cytometry( Becton Dickinson, CA，USA )讀取螢光值。 每個樣本將分析2000個細胞。參照第1 〇圖，顯示Tcm_b 具有較佳調節血液中T細胞的能力。 (H)統計分析 5 結果係以算數平均數±SEM來表現。組別的差異係以Taipei shien, Taiwan) and calculate the cells separately. The results are shown in Figure 与. With Table 1 below, both TCM-A and TCM-Β can reduce the number of cells in the BAL rinse that cause 2 inflammatory responses. Blood samples were collected from the sinus sinus (〇rbiul sinus), and the obtained serum was stored in _7 before the experiment (rc ^ · 21 1343815 Table 1 total number of sample cells (xl04/ml) relative count (xl 〇 4 / ml) Phagocytic lymphocyte neutrophil erythrocyte leukocyte control group 14·3±4.3* 13.3±4.3* 1.0±0.0* 0 0 tfood saline 160.0+23.5 56.4+12.7 41.4 soil 6.5 36.8±2.8 25.2±4.5 Dexamethasone 46A±2.2* 29.2±4A* 9.4±1.5* 6.6±1.0* 3.2±0.4* TCM-A 49.0±3·7* 25.8±2.5* 13.2±1·8* 6.0+0.7* 4.010.7 * TCM-B 41.6±6.2* 21.2±2.6* 10.212.4* 6.0±1.2* 4.2±1.1* (E) Determination of Der p 2 specific IgG 1, IgG2a and IgE antibodies before and after the experiment Blood was taken from the retro-orbital venous plexus. The titer of 5 IgE, IgG1 and IgG2a against Der p 2 in serum was determined by ELISA. The surface of the microtiter plate (Nunc Lab, IL, USA) was coated with a concentration. 100 μΐ Der p 2 at 0·5 pg/ml was placed in a refrigerator at 4 ° C overnight. The titration plate was washed three times with PBS-Tween-20 (PBST) and stored at -70 ° C until use. Serum diluted 1:5 IgE, 1:100 diluted IgGl, and 1:5 diluted IgG2a, place 10 drops in 4 °C overnight, add antibody (HRP-bonded goat anti-mouse antibody, which is resistant IgE and anti-IgG2a antibodies were diluted 1:800, anti-IgG1 antibodies were diluted 1:2000, gambling from Southern Biotech Assoc, Inc, Birmingham, AL, USA) 3 washes. After incubation at 37 ° C for 1 hour, PBST Rinse 3 times, then add the enzyme reaction substrate ABTS 15 (2,2"-Azion-bis(3-ethylbenzothiazolin-6-sulfonic acid) diammonium salt, Bio-Rad, USA). After 15 minutes, add 50 μΐ 4N H2SO4. The reaction was terminated and the luminosity was measured at a wavelength of 450 nm using a multi-wavelength spectrophotometer (A-5682, 22 1343815 SLT Lab Instruments, Salzburg, Austria). The results are expressed in ELIS A units (EU). One EU is defined as the reciprocal after serum dilution and the measured photometric value is 1.0. This result falls in the linear part of the dilution curve. In order to confirm reproducibility, a known serum was used as a standard value in each experiment. Figure 7 is the titer concentration of IgE in the test serum. (F) Cytokine test in mouse samples IFN-7* and IL-5 were measured using commercially available ELISA test kits, and mouse monoclonal antibodies were used to determine antigenic epitopes on the surface of cytokine 10 (epitopes) ). The lowest detection range is 10 pg/ml. The results are shown in Figures 8 and 9. (G) Culture of lymphocytes obtained by bronchoalveolar lavage, immunofluorescence staining and flow cytometry analysis According to the method proposed by Assenmacher et al. (1994), it is possible to measure mouse T helper cells by flow cytometry. Activated cytokines. Double staining was used to analyze the expression of IFN-T* and IL-5 in CD4 or CD8 cells. White blood cells from peripheral blood were stimulated by PMA (50 ng/ml), ionomycin (2 μΜ) and GolgiStop (Cytofix/Cytoperm Plus Cat No. 2076KK, Pharmingen, San Diego, CA) for 5 hours, 20 rinsed with PBS 2 times. The cells were stained with CD4-FITC or CD8-FITC for 30 minutes at room temperature and washed. The cells were fixed with cytofix/cytoperm for 30 minutes at room temperature, stained with anti-cytokine antibody for 30 minutes, and rinsed. The cells were resuspended in 0.5 ml PBS containing 0.1% sodium azide. Fluorescence values were read with Becton Dickinson 23 1343815 flow cytometry (Becton Dickinson, CA, USA). Each sample will analyze 2000 cells. Referring to Figure 1, it is shown that Tcm_b has the ability to better regulate T cells in the blood. (H) Statistical analysis 5 Results are expressed as arithmetic mean ± SEM. Group differences
Mann-Whitiney U法來分析。若P值小於ο.”，則視為統 計上的顯著差異。 前述的例子著重於評估本發明之醫藥組合物的療 效。該配方可以抑制呼吸道之免疫性發炎反應，改善肺功 10能’降低血液中IgE含量，並防止氣喘發生。 雖然本發明係藉由最佳實施例說明内容，但在不背離 本發明之精神與範疇下，均可對本發明有種種改變及修 飾，以適用於種種用途與情況。換言之，對本發明中配方 的形式與細節之省略、修飾、減損、與改變，在不背離本 15發明之精神與範疇下，均可由熟習本項技藝者加以進行。 不須再進一步詳述’相信上述說明已能充分表達本發 明，他人可在就前案觀點而言，對組成本發明必要之特徵 不忽略的情形下’對本發明有種種改變。 20【圖式簡單說明】 圖1係一種造成過敏性氣喘的可能機制的示意圖（引自 加咖’ 402, 12_17(1999)，P.GH〇lt，c.Macabas，RAStumbles 與 P.D.Sly);其中IL-4係分泌自Th2細胞之主要細胞激素，與 重要的脂肪介質-白三烯素’同時在急、慢性過敏發炎反 24 1343815 應中扮演重要角色。 圖2a顯示以各種組合物抑制細胞株EL-4與THP-1分泌 IL-4與TNF-alpha的體外實驗結果圖。圖示下方文字中的 符號“aq”代表草藥的水溶液萃取物1符號“et”則是代 5 表草藥的乙醇萃取物； 圖2b顯示不同濃度TCM-B抑制RBL-1細胞分泌半胱胺 酿基白三稀素（cysteinyl-leukotrienes，c-LTs)及其細胞毒 性的體外實驗結果圖； 圖3a係以MTT實驗檢測不同濃度TCM-B抑制BEAS-2B 10 細胞分泌eotaxin及細胞存活率（細胞毒性）的體外實驗結果 圖； 圖3b係顯示以包含36%澱粉與1.9%磷酸鈣（Ca3(P04)2)之 TCM-B粉劑，測試EL-4細胞株分泌IL-4的影響。橫座標 顯示粉劑的濃度，賦形劑澱粉與磷酸鈣已事先測試過不干 15 擾EL-4細胞株分泌IL-4的現象； 圖3c係顯示使用包含TCM-B與賦形劑之草藥萃取粉劑， 對於抑制EL-4細胞株分泌IL-4的現象有均等的效果。 圖4a顯示各種組合物及TCM-B混合物抑制腹膜部位的巨 嗜細胞分泌TNF-alpha的體外實驗結果； 20 圖4b顯示以MTT實驗檢測各種組合物及TCM-B混合物 作用於腹膜部位之巨嗜細胞的細胞毒性；圖5顯示測定肺 功能時，壓力增加之訊號，（B)圖令Penh值從0.9升至 3.95，顯示出實驗用之過敏老鼠其肺功能衰退，是檢測老 鼠時一完全不同的參數（引用自E.Hamelmann and E.W· Gelfand 25 in Current Protocols in Immunology, John Wiley & Sons, Inc., edited by J. 25 1343815 E. Coligan, A.M.. Kruisbeek, D.H. Margulies, E. M. Shevach, and W. Strober, Vol. 3, Chapter 15,18.1-18.13(1999)) i 圖6顯示與正常生理食鹽水，TCM-B，及去除澤瀉萃取物 後之TCM-A相比，TCM-B可防止因乙醯丑曱基膽素 5 ( methacholine )之劑量從低到高，所造成的肺功能衰退； 圖7係在5組實驗老鼠血清中，IgE的滴定值； 圖8係在5組實驗老鼠之血清以及BAL沖洗液中，IL-5 的濃度； 圖9係在5組實驗老鼠之血清以及BAL沖洗液中，IFN-γ 10 的濃度；以及 圖10係在5組實驗老鼠中，TCM-B(1217B)提昇血液中調 節T細胞之能力的結果圖。 【圖號說明】 無 15Mann-Whitiney U method to analyze. If the P value is less than ο.", it is regarded as a statistically significant difference. The foregoing examples focus on evaluating the therapeutic effect of the pharmaceutical composition of the present invention. The formulation can suppress the immune inflammatory reaction of the respiratory tract and improve the lung function. The IgE content in the blood and the prevention of asthma. Although the present invention has been described in terms of the preferred embodiments, various modifications and changes can be made to the present invention without departing from the spirit and scope of the invention. In other words, the omission, modification, derogation, and alteration of the form and details of the present invention can be carried out by those skilled in the art without departing from the spirit and scope of the present invention. The above description has been able to fully express the present invention, and others may have various changes to the present invention in the case of the prior view that the features necessary for the present invention are not neglected. 20 [Simple description of the drawing] FIG. A schematic diagram of a possible mechanism for causing allergic asthma (cited from Kawai '402, 12_17 (1999), P.GH〇lt, c.Macabas, RAStumbles and PDSly); IL-4 is a major cytokine secreted from Th2 cells, and plays an important role in the acute and chronic allergic inflammation anti-24 1343815 with the important fatty medium leukotriene. Figure 2a shows various The composition inhibits the in vitro experiment results of IL-4 and THP-1 secreting IL-4 and TNF-alpha. The symbol "aq" in the text below represents the aqueous extract of the herb 1 symbol "et" is the generation 5 Ethanol extracts of herbal extracts; Figure 2b shows the results of in vitro experiments in which different concentrations of TCM-B inhibit the secretion of cysteamine-leukotrienes (c-LTs) and their cytotoxicity by RBL-1 cells; Figure 3a is a graph showing the results of in vitro experiments in which MTC assay detects different concentrations of TCM-B inhibiting the secretion of eotaxin and cell viability (cytotoxicity) in BEAS-2B 10 cells; Figure 3b shows the inclusion of 36% starch and 1.9% calcium phosphate (Ca3). (P04) 2) TCM-B powder, test the effect of IL-4 cell line secretion of IL-4. The abscissa indicates the concentration of the powder, the excipient starch and calcium phosphate have been tested beforehand. The phenomenon of IL-4 secretion by the strain; Figure 3c shows the use of containing TCM-B Herbal extract powder with excipients has an equal effect on inhibiting the secretion of IL-4 by EL-4 cell lines. Figure 4a shows that various compositions and TCM-B mixtures inhibit the secretion of TNF-alpha by macrophages at the peritoneal site. In vitro results; 20 Figure 4b shows the cytotoxicity of macrophages acting on the peritoneal site with various compositions and TCM-B mixtures by MTT assay; Figure 5 shows the signal of increased pressure when measuring lung function, (B) Penh values rose from 0.9 to 3.95, showing a decline in lung function in experimental allergic mice, a completely different parameter in detecting mice (cited from E. Hamelmann and EW·Gelfand 25 in Current Protocols in Immunology, John Wiley & Sons, Inc., edited by J. 25 1343815 E. Coligan, AM. Kruisbeek, DH Margulies, EM Shevach, and W. Strober, Vol. 3, Chapter 15, 18.1-18.13 (1999)) i Figure 6 shows normal TCM-B prevents lungs caused by low to high doses of methacholine, compared to TCM-A, which is physiological saline, TCM-B, and after removing Alisma orientalis extract. Functional decline; Figure 7 is in 5 groups IgE titer in mouse serum; Figure 8 is the concentration of IL-5 in the serum of 5 groups of experimental mice and BAL rinsing solution; Figure 9 is the IFN-γ in the serum of 5 groups of experimental mice and BAL rinsing solution. The concentration of 10; and Figure 10 is a graph showing the ability of TCM-B (1217B) to elevate the ability of T cells to regulate T cells in five groups of experimental mice. [Description of the figure] None 15