TWI698245B - 用於提升昆蟲免疫力之組合物及方法 - Google Patents
用於提升昆蟲免疫力之組合物及方法 Download PDFInfo
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Abstract
本發明提供一種血桐製備物或萃取物用於製備提升昆蟲免疫力之組合物之用途，尤其針對蜜蜂。本發明亦提供一種用於提升昆蟲免疫力之方法，尤其針對蜜蜂，包含對該昆蟲投予一有效量的本發明之組合物。
Description
本發明係關於一種用於提升昆蟲免疫力之組合物及其方法，尤其係針對蜜蜂。
蜜蜂是對人類最有貢獻的昆蟲之一，除生產多樣具健康價值的副產品外，也是農業上重要的作物授粉者。因此，若能提升蜜蜂的免疫力，以提高飼養之蜜蜂之存活率（或防止其死亡），對相關產業將極有幫助。
在一方面，本發明提供一種血桐製備物或萃取物用於製備提升昆蟲免疫力之組合物之用途。
另一方面，本發明提供一種用於提升昆蟲免疫力之方法，包含對該昆蟲投予一有效量的如上述之組合物。
又一方面，本發明提供一種可作為提升昆蟲免疫力飼料或營養品之組合物，其包含一血桐製備物或萃取物。
再一方面，本發明提供一種用於防治昆蟲病害或死亡之方法，包含對該昆蟲投予一有效量的如上述之組合物。
應當理解的是，前文的一般性描述和以下的實施方式都只是例示性和說明性的，而不是對本發明的限制。
在一方面，本發明提供一種血桐製備物或萃取物用於製備提升昆蟲免疫力之組合物之用途。
另一方面，本發明提供一種用於提升昆蟲免疫力之方法，包含對該昆蟲投予一有效量的如上述之組合物。
又一方面，本發明提供一種可作為提升昆蟲免疫力飼料或營養品之組合物，其包含一血桐製備物或萃取物。
再一方面，本發明提供一種用於防治昆蟲病害或死亡之方法，包含對該昆蟲投予一有效量的如上述之組合物。
根據本發明，該組合物可使用任何習知方法或標準製程製備。在本發明之部分具體實施例中，該血桐萃取物係由包含下列步驟之方法製得：以一中高極性溶媒萃取乾燥血桐（較佳為葉及/或雄蕊），以獲得一第一萃取液；去除該第一萃取液中的不溶物，以獲得該血桐萃取物。根據本發明之一具體實施例，乾燥血桐葉以一中高極性溶媒（例如，醇類或丙酮）萃取，萃取液濃縮後以吸附性樹脂混合，由高至低用不同極性的溶媒（例如，水→甲醇®丙酮）依序沖提以去除不溶物，萃取液合併濃縮後，得到該血桐萃取物。
在其他具體實施例中，該血桐製備物係藉由將乾燥血桐（較佳為葉及/或雄蕊）磨碎為粉末狀而製得。
根據本發明實施例，本發明組合物特別有效於昆蟲免疫力之提升，尤其是蜜蜂。
由於蜜蜂瀕臨滅種之危機，本發明亦提供一種可作為提升蜜蜂免疫力飼料或營養品之組合物，其包含一血桐萃取物。
實施例
實例
1
：本發明之組合物之製備
乾燥血桐葉磨碎為粉末狀得組合物1。
乾燥血桐葉以一中高極性溶媒（例如，醇類或丙酮）萃取，萃取液濃縮後以吸附性樹脂混合，由高至低用不同極性的溶媒（例如，水→甲醇®丙酮）依序沖提以去除不溶物，萃取液合併濃縮後，得到該血桐萃取物，其包含20~25%單寧成份 (包括：galloylglucoses, galloylshikimic acids, galloylquinic acids, corilagin, punicafolin, furosin, terchebin, geraniin, etc.) 及20~25%類黃酮成份 (包括：propolin C、propolin D、 propolin F、propolin G…等)，該血桐萃取物可乾燥後直接使用或進一步混粉造粒後使用（作為組合物2使用或用於配製組合物2）。
實例
2
：本發明之組合物提升蜜蜂抗病力
A. 田間試驗：
實驗分弱勢蜂箱組與強勢蜂箱組進行，每組均由蜂農挑選六巢蜂勢接近的蜂箱，實驗組與對照組各三巢，弱勢蜂箱組的選擇條件為工蜂數量較少且蜂后產卵能力降低的蜂箱，強勢蜂箱組的選擇條件為蜂勢強盛且蜂王由同一蜂后所生之姊妹巢。營養補充劑以花粉餅為載體，實驗組餵食含有組合物1之特殊花粉餅，對照蜂箱餵食一般花粉餅，連續餵食一個月後，每巢隨機取樣24隻巢內蜂，檢測蜜蜂慢性麻痺病毒（Chronic Bee Paralysis Virus, CBPV）之感染率的差異。
1. 蜂慢性麻痺病毒CBPV之感染率變化
由各組取樣工蜂之分離中腸抽取Genomic RNA，再以Q-PCR檢測蜜蜂CBPV感染情形。分別觀察弱勢蜂箱組與強勢蜂箱組CBPV感染率的變化，弱勢蜂箱處理組在餵食特殊花粉餅一個月後CBPV的感染率由餵食前52.8±19.2%，大幅降低至27.8±14.7%，其中有兩個蜂箱CBPV降低比例更大於60%。在強勢蜂巢組中，餵食特殊花粉餅一個月後處理組蜂箱CBPV平均感染率由44.4±6.4%顯著下降至31.9±2.4%，與對照組蜂箱平均感染率50±8.3%相較仍明顯較低。CBPV是蜂場中普遍感染之病毒，試驗結果證實，食用含有組合物1之花粉餅的蜂群無論蜂勢強弱都可以明顯降低CBPV的感染率（參見圖1）。
2.蜜蜂免疫基因表現量的偵測
基因組學研究發現昆蟲有四條免疫訊號途徑, 包括Toll、Imd、JAK/STAT 和 JNK，本次試驗我們選擇研究最多的Toll途徑相關免疫基因作為標的，檢測各組取樣工蜂基因組RNA 中 toll、dif、relish等免疫基因表現量。實驗結果顯示，對照組蜜蜂之toll與dif等免疫基因表限量幾乎沒有變化，而實驗組蜜蜂的toll 基因表現量增強1.89~2.51倍（參見圖2）。證實組合物1能經由誘導蜜蜂免疫機制，降低CBPV感染情形。
B.實驗室試驗
為進一步證實田間試驗之結果，我們由蜂場帶回巢脾孵化新生蜜蜂，每次試驗均使用同一天羽化後3日齡之新生蜜蜂，以糖水為載體，不同濃度之如實例1所述之血桐萃取物分別配置於50% (w/v)蔗糖水（組合物2）。每組10隻，將試驗蜜蜂置於體積250 mL布丁杯中，於室溫環境中飼養(溫度26~30 °C；濕度50~60%)，每日提供1 mL糖水供蜜蜂自由取食。
1. 對蜜蜂生存率之影響
三種濃度(2 mg/mL、1 mg/mL、0.5 mg/ml)之血桐萃取物分別配置於50%蔗糖水中，對照組給予50%蔗糖水。實驗結果發現第16天時，各組存活率分別為：對照組(30%)、2 mg/mL組 (30%)、1 mg/mL組 (80%)、0.5 mg/mL組 (100%)，結果顯示在溫溼度與空間體積等不適於蜜蜂生長的逆境環境下餵食含濃度0.5 mg/mL血桐萃取物之組合物2，可顯著提升蜜蜂的存活率（參見圖3）。
2. 對蜜蜂免疫基因表現量與CBPV之影響
將上述實驗存活之3隻對照組蜜蜂與3隻0.5 mg/mL實驗組蜜蜂，進行CBPV與免疫基因toll、dif、pelle與myd88的表現量檢測。實驗結果如圖4所示。餵食0.5 mg/mL濃度營養補充劑的蜜蜂，其toll、dif、pelle與myd88等基因的表現量皆高於對照組蜜蜂2~3倍( toll表現量平均增加2.53倍、myd88表現量平均增加2.89倍、pelle表現量平均增加2.42倍、dif表現量平均增加2.48倍)。CBPV的檢測結果發現，對照組三隻工蜂中有二隻帶原CBPV，而實驗組蜜蜂體內皆無偵測到CBPV。綜合上述結果，確認實驗組蜜蜂免疫基因表現量的提升並非病原刺激誘導所至，而是食用組合物2誘導產生的結果。CBPV能經由蜂后垂直傳染給下一代，來自同一巢片同一時間羽化的新生蜜蜂，經過不同處理兩週後，體內CBPV感染率卻有極大差異，推測食用含血桐萃取物之糖水的蜜蜂可藉由誘導個體免疫基因表現提升免疫力進而抑制體內CBPV的增生。
實例
3
：本發明之組合物對提升蜜蜂抵抗農藥之功效
許多報告證實新尼古丁類藥劑會造成蜜蜂與多種生物的免疫系統損害 (Mason et al., 2013)，顯見免疫系統的損害可能是益達胺（農藥）造成蜜蜂死亡的重要原因之一。根據本發明之組合物對蜜蜂生存率與免疫基因調控之實驗結果，本實驗以容易接觸到農藥的外勤蜂作為試驗對象，餵食含0.5 mg/mL濃度血桐萃取物之糖水，觀察是否能增強蜜蜂對益達胺之耐受性。
實驗參考美國EPAOCSPP 850.3020:Honey Bee Acute Contact Toxicity Test之方法採表面施藥法 (Topical application)，益達胺原料藥以丙酮溶解稀釋，試驗濃度參考Suchail et al. (2000)之論文，以24小時內出現的兩個半致死劑量 (LD50) 6.7 ng/μL 與23.8 ng/μL作為試驗濃度，將蜜蜂冷凍迷昏後，以微量施藥器 (microapplicator)將益達胺溶液1 μg/bee 定量投予於蜜蜂前胸背板上，溶劑對照組則投予1 μg/bee的丙酮，每組10隻蜜蜂，藥物投予完畢後將蜜蜂移入恆溫生長箱中飼養，實驗組蜜蜂提供含0.5 mg/mL濃度血桐萃取物之糖水供其取食，對照組則餵食50%蔗糖水，24小時後觀察蜜蜂死亡隻數，死亡率校正公式為{1- (處理組死亡隻數/溶劑對照組死亡)}*100%。
實驗結果顯示，6.7 ng/μL益達胺處理組，食用含血桐萃取物之糖水之蜜蜂 24 小時平均死亡率為5.26%，明顯低於對照組19.30%；23.8 ng/μL益達胺處理組，餵食含血桐萃取物之糖水之蜜蜂24小時平均死亡率為17.54%，也顯著低於對照組的平均死亡率36.84%（參見圖5），顯示食用組合物2可以提高蜜蜂對益達胺的耐受性。
實例
4
：本發明之組合物提高健康蜂王的孵育率
依照合作蜂農慣行育王施作方法，於蜂王成長至15日齡時將王台帶回實驗室羽化，進行新生蜂王體重與健康程度評估。對照組蜂巢產出之蜂王平均體重為163±34.4mg，實驗組產出之蜂王平均體重178.1±25.4mg，統計結果顯示無顯著差異。同時評估不健康的新生蜂王佔各組羽化蜂王之比例，若新生蜂王有以下病徵：畸翅、舌吐出、無法羽化等則視為不健康。結果顯示，對照組蜂箱不健康蜂王比例為38.6%，而實驗組蜂巢不健康蜂王比例僅佔4.6%（參見圖6），因此，本發明之組合物在蜂王育成上顯示有提升健康蜂王產出的效果。
無
當結合附圖閱讀時，將更好地理解前述發明內容以及本發明的以下實施方式。在圖式中：
圖1顯示本發明之組合物能有效降低蜜蜂痲痺病毒(CBPV)感染率。
圖2顯示本發明之組合物能經由誘導蜜蜂免疫機制，降低蜜蜂痲痺病毒(CBPV)感染率。
圖3顯示本發明之組合物能延長蜜蜂生存天數。
圖4顯示本發明之組合物能藉由誘導個體免疫基因表現提升免疫力進而抑制體內CBPV的增生。
圖5顯示本發明之組合物可提高蜜蜂對益達胺的耐受性。
圖6顯示本發明之組合物可提高健康蜂王的育成率。
無
Claims (7)
- 一種血桐製備物或萃取物用於製備提升蜜蜂免疫力之組合物之用途。
- 如請求項1之用途，其中該組合物為一蜜蜂之藥物。
- 如請求項1之用途，其中該組合物可提高飼養蜜蜂的存活率，或防止死亡。
- 一種血桐製備物或萃取物用於製備提高蜜蜂存活率避免蜜蜂死亡之組合物之用途。
- 如請求項4之用途，其中該血桐製備物或萃取物係製成飼料或營養品餵食蜜蜂。
- 一種血桐製備物或萃取物用於製備防治蜜蜂免於病害之組合物之用途。
- 如請求項6之用途，其中該血桐製備物或萃取物係製成飼料或營養品餵食蜜蜂。
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