揚波植物組織培養及藥理活性之研究

Studies on Tissue Culture and Pharmacological Activity of Buddleja asiatica Lour.

張靜雯(Ching-Wen Chang)

指導教授 ： 詹効松

朝陽科技大學/理工學院/生化科技產業研發碩士專班/碩士(2009年)

摘要

揚波（Buddleja asiatica Lour.）是馬錢科灌木植物，外用治療皮膚濕疹及無名腫毒，屬於臺灣民間常用且重要的皮膚科草藥。近年來，因濫用農藥，野生植株已日漸稀有，本研究首先研究利用植物組織培養技術進行揚波植物的量產性研究。試驗結果顯示，揚波成熟種子經1 %次氯酸鈉預處理5分鐘後，可提高室外播種小苗的發育速度，萌芽率約38.5 %；若無菌播種於MS培養基中，發芽率可顯著提升至接近100 %，MS鹽類不僅可顯著提升小苗的發育速度，而且也是限制性營養素；蔗糖在MS鹽類存在時，具有促進小苗發育的作用，但蔗糖單獨添加在僅含0.9 %瓊脂的培養基中，無法促進小苗發育；N6-benzylaminopurine（BA）會抑制揚波種子萌芽與小苗發育，α-Naphthaleneacetic acid（NAA）則可輕微緩解BA的抑制作用。揚波無菌苗的生長發育試驗中發現，BA及6-furfurylamino purine（kinetin）容易導致揚波無菌苗出現玻璃質化而快速死亡，NAA及Indole-3-acetic acid（IAA）幫助小苗生長的助益也不大，顯示揚波的生長不適合使用植物生長調節劑。揚波小苗或芽體培養於不含植物生長調節劑的MS培養基中，也容易出現玻璃質化而死亡，玻璃質化的發生原因可能是試管苗群聚密度太高或氣體交換不良所致；為了改善玻璃質化現象，本研究降以低無菌苗接種密度及使用不同的容器封口材料與封口方式，結果顯示，將培養容器以四層藥包紙封口且外覆石蠟膜，培養三週後，撕除石蠟膜使其透氣二週的處理，改善玻璃質化的效果最佳，小苗或芽體可健康成長與發育。小苗培養於僅含½~全量MS鹽類培養基中，再以藥包紙及石蠟膜封口二週並透氣一週的處理，可使根系生長正常且健壯，經移植於等比例的蛭石與培養土之混合介質中，並進行健化處理，植株生長健康。同時，利用不同極性之溶劑萃取及分離揚波乾燥葉之萃取物；結果發現，正丁醇萃取物（1.56 mg/ml）對DPPH自由基具有清除能力，顯示出良好的抗氧化作用，而且對金黃葡萄球菌（Staphylococcus aureus, ATTCC25923）的生長具有抑制能力，最低有效濃度為11.384 mg，此抑菌能力接近Vancomycin，顯示正丁醇萃取物具有開發為新抗生素的潛力。以上研究成果期能協助保護與開發臺灣本土自產生藥資源，並為建立臺灣醫藥保健產業做出貢獻。

關鍵字

玻璃質化 ； 大量繁殖 ； 抗氧化 ； 抗菌活性 ； 萌芽 ； 揚波

並列摘要

Buddleja asiatica is a well known and one of the most important traditional medicinal plant in Taiwan. It is used as a traditional medicine for the treatment of skin disease and an abortifacient. Due to overexploitation and usage of herbicide and insecticide, the plants have become very rare. In order to increase the population of this species, the tissue culture technology would offer unique opportunity to develop mass propagation of B. asiatica. The in vivo seedlings were achieved by the mature seeds treated with 1% sodium hypochlorite for 5 minutes than other treatment. For in vitro seedlings, the mature seeds were surface sterilized with 1% sodium hypochlorite and then inoculated into MS basal medium.
In vitro culture of B. asiatica showed vitrified growth when cultured on standard condition, resulting in poor survival rate of tissue culture derived plants. In order to improve the culture condition, the culture vessels were closed and sealed in different way. It was found that the best culture condition was achieved by the culture vessels covered with four layers of dispense paper and sealed with parafilm during the first three weeks and then the ventilation was achieved by removing the parafilm and continued the culture condition for another two weeks. This reduced the hyperhydricity rate and gave a good recovery of vitrified shoots. Among the different plant growth regulators (NAA, BA, and Kinetin) used, the maximum number of normal adventitious buds were observed at MS medium supplemented with 1mg/l of Kinetin. The healthy plantlets were transferred to pot containing soil and vermiculite in the ratio of 1:1 and the survival rate of 100% after one month of transplanting.
Antibacterial activity of Buddleja asiatica were tested against S. aureus strain ATTCC25923. The crude leaf extracts of B. asiatica was fractionated into five fractions soluble in hexane, chloroform, ethyl acetate, n-butanol and sterile water. The result showed that the minimum inhibitory concentration of n-butanol fractions was 11.384mg. The resulting, n-butanol fractions showed remarkable antibacterial activity and its potential as an antibacterial agent may be confirmed after further pharmacological studies. Moreover, we investigated the antioxidant activity of hexane, chloroform, ethyl acetate and n-butanol fractions. The resulting, n-butanol fractions showed higher (1.56mg/l) antioxidant activity than other fractions.

並列關鍵字

vitrification ； germination ； Buddleja asiatica ； anti-bacterial activity ； propagation ； anti-oxidation

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