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氣喘之病理學定義認為是一種去上皮組織、嗜酸性球浸潤性 的慢性支氣管炎。其主要臨床表現為陣發性呼氣性之呼吸困難及 喘鳴。急性發作時之治療原則是以支氣管擴張劑等緩解支氣管之 收縮。然而只用支氣管擴張劑並未能完全治癒氣喘。遂有許多學 者提出氣喘發病機制中的新概念，認為氣道變應性炎症反應才是 引起氣道通氣障礙之主因。亦即遲發性氣喘反應的過程中產生炎 性介質及白血球浸潤所導致上皮細胞的損傷破壞、氣道神經末梢 曝露損傷 ，引起氣道高反應性(bronchial hyperresponsiveness， BHR)，終至不可逆性病理變化。雖然白三烯素(leukotriene)、組 織氨(hisyamine)、前列腺素(prostaglandins)等亦參與氣喘發病調 節，但並不能誘發氣喘約主要特徵-BHR。而氣喘已知之炎性介 質(inflammatory mediators)中作用最強、效應最廣介質是血小板 活化因子(platelet-activating factor，PAF)，幾乎所有炎性細胞都可 合成及釋放PAF，PAF不僅可引起氣道高反應性，並可促使嗜酸 性球釋放許多具有氣道上皮毒性之炎性介質及鹼性蛋白質(basic proteins)。
冬蟲夏草(Cordyceps sinensis(Berk.)Sacc)為一蟲生真菌，始載於本草從新，認為其具有保肺益腎，止血化痰，已癆嗽之作 用，臨床處方多為喘咳久嗽後期調養。傳統醫學應用上，視為補腎益肺之藥材，因此本論文選擇冬蟲夏草作為研究對象。而以 「抑制由PAF引起兔子血小板凝集」之體外試驗模式作為藥物活性篩選系統。體內篩選系統，則分別以「由PAF引發天竺鼠急性氣管高反應性」及「由ovalbumin引發天竺鼠雙相式氣喘反應」，試驗所篩出藥物對氣喘之治療。
結果發現，冬蟲夏草子實體的甲醇抽提物，經由矽膠管柱層析，依其極性大小粗分為六個部份，其中CS-EM-1對於由PAF引起紐西蘭大白兔血小板凝集具有呈劑量關係的抑制作用。急毒性試驗中，以肝功能作指標，將含2%CS-EM-1的飼料餵食ICR小鼠，並未影響肝中膽固醇及cyochrome p450含量。
「由PAF引發天竺鼠急性氣管高反應性」體內篩選系統，以Flow-volume loop方法評估肺功能變化，發現先給予CS-EM-1靜脈注射(0.5、1、2、4-10 mg/kg)，再投予PAF(25、50、100、200、500 ng/kg i.v.)之實驗組，不論在PEF，MFEF 75%、50%、25%FVC,VC皆比控制組之值高，均顯示出CS-EM-1對肺功能的保護作用。肺部病理組織檢查發現，雖均無氣道上皮組織脫落現象，然而兩組皆呈現有嗜酸性球、中性球之浸潤，不過，實驗組病理組織變化較控制組輕微。此外，CS-EM-1(0.5,1.0,0.2mg/kg)以劑量相關方式抑制PAF(30ng/kg)引起的急性相呼吸道收縮。
由ovalbumin引發天竺鼠雙相式氣喘反應模式中，實驗組天竺鼠在兩次敏感化(sensitization,1% ovalbumin)及challenge(4%ovalbumin)之前先予吸入CS-EM-1(0.5、0.5、3mg)，於七十二小時後以Acetylcholine進行激發試驗(provocation test)，同Flow-volume loop方法評估肺功能變化，發現實驗組PEF,MFFEF75%、50%、25% FVC,VC皆比控制組之值高，當換算成PD2oACh，比較兩組差異，發現實驗組降低百分之二十肺功能所需的Acetylcholine量皆大於控制組，其皆具有統計學上之意義。至於肺部病理組織學檢查所見:控制組嗜酸性球、中性球之浸潤、氣道上皮組織脫落現象遠比實驗組嚴重，且此兩組差異性較由PAF引發天竺鼠急性氣管高反應性大。
以上之結果皆初步證實:在動物模式中，冬蟲夏草中確實含 有可以治療氣喘之天然物，而其作用機轉之一可能是透過對血小 板活化因子的拮抗。
Twenty years ago, the pathogenesis of asthma was bronchial spasm causing paroxysmal dyspnea and expiratory wheezing. However, the use of bronchodilator only will not relieve the symptom completely. In recent years, people believe that bronchial inflammatory response plays the major role of bronchial airway hyperresponsiveness and destruction. The inflammatory mediators, infiltrated eosinophils and mononuclear cells (e.g. neutrophil, monocytes, T cells) damage of bronchial epithelial cells and irritation of nerve ending causes bronchial hyperresponsiveness and irreversible pathological change at late phase. Among the chemical mediators complicating the pathogenesis of asthma, platelete activating factor (PAF) has the most potent effect and the longest duration. Almost all the inflammatory cells may synthesize and release PAF. PAF will not only induce airway hyperresponsiveness directly, but also attract more eosinophils which will accumulate more eosinophils and release more PAF and harmful chemical mediators (e.g. major basic protein). Therefore, the purpose of this study will be to try to find a fraction of natural product which can inhibit PAF activity in vitro, and therapeutic effectiveness in animal models of asthma in vivo. Then it can be applied in the treatment of asthma in future.
Cordyceps smensis (CS) is a worm parasite fugus. In Chinese herbal medicine, it is believed that CS has the effect of protecting lung function, hemoptysis, and expectoration of sputum. It is usually applied in the management of wheezing cough, post-unremitting cough. In traditional medicine, it is beleved that CS is good for lungs. Therefore, in order to fractionate the crude components which can be used in the treatment of asthma, an in vitro inhibition of PAF-induced rabbit platelet aggregation and an in vivo amnimal models of PAF-induced acute airway hyperresponsiveness and ovalbumin induced dual asthmatic response in guinea pigs were established. Initially, we fractionated the crude methanolic extracts from fruiting bodies of CS using this in vitro inhibition of PAF-mduced rabbit platelet aggregation as our assay method. Briefly, the fruiting bodies were extracted by silica gel column chromatography. A fraction (CS-EM-1) (EtOAc: MeOH= 1:1, v/v) significantly inhibited the PAF-induced rabbit platelet aggregation with dose responsiveness. The acute toxicity test using male ICR mice showed there was no liver toxicity.
In the in vivo system, using flow-volume loop of forced expiration by plethysmograph and oscilloscope demonstrated that CS-EM-1 (0.5、1.0、2.0、4.0、10 mg/kg i.v.) inhibited 60% the decrease of pulmonary function including: PEF、MFEF75%、50%、25%FVC、VC, cuased by PAF (25、50、100、200、500 ng/kg) induced bronchial hyperresponsiveness. The histopathologic study demonstrated the inflammatory response including the eosinophils and neutrophils infiltration in the interstitial tissue and vessels and diffused congestion in the CS-EM-1 treated group was less than in the control group.
The ovalbumin-induced model was sensitized by 1% OA、2 ml、once a week、2 weeks and challenged by 4% OA、3ml、7 days after the second sensitization and CS-EM-1 was administrated by aerosol before the sensitization (0.5 mg) and challenge (3.0 mg). The provocation test was used to evaluate the airway response (late inhibited) to acetylcholine at 72 hours after the challenge. CS-EM-1 significantly decrease of PFT in the control group. The PD20ACh of PFT was also significantly increased in the CS-EM-1 treated group. Histologica study demonstrated that CS-EM-1 significantly improved the mononuclear cells and eosinophil infiltration in the interstitial tissue, blood vessels and sloughing of bronchial lining epithelium.
These results indicate that the fraction (CS-EM-1) bioactive natural products contained in Cordyceps sinensis may have therapeutic potential in the treatment of asthma and the effect of anti-PAF may be involved in the pharmacological mechanism.
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