苦茶粕水萃液對神澤氏葉蟎之毒性探討

Study on antimite activity of aqueous extract from the tea seed dreg of Camellia oleifera Abel. against Tetranychus kanzawa Kishida.

游善植(Shan-Chih You)

指導教授 ： 顏才博

國立屏東科技大學/國際學院/熱帶農業暨國際合作系/碩士(2012年)

https://doi.org/10.6346/NPUST.2012.00005

摘要

神澤氏葉蟎（Tetranychus kanzawa Kishida）因生活史短及繁殖迅速，對多種化學農藥容易產生抗藥性，致使防治困難；發展低風險之植物源農藥佐以有害生物整合管理方法（Integrated Pest Management, IPM）為解決上述問題重要方法之一。油茶（Camellia oleifera Abel.）俗稱大果油茶，其茶籽榨取苦茶油後所剩的農業廢棄物稱苦茶粕，本研究欲評估苦茶粕萃取液作為植物源殺蟲劑之潛力，以提供另一種防治害蟎之選擇。此研究分別以水、50%乙醇、95%乙醇及丙酮萃取，對雌成蟎之毒性以水萃液效果最佳，並呈現緩效性的觸殺活性，處理72 h後各萃取液之LC50值由低至高為水萃取液（9.92 ± 0.57 mg/mL）、95%乙醇萃取液（LC50：20.35 ± 1.93 mg/mL）、50%乙醇萃取液（24.19 ± 3.35 mg/mL）及丙酮萃取液（25.85 ± 6.65 mg/mL）。水萃液低濃度（14.26 mg/mL）處理時，未死亡之葉蟎四肢抽搐，已無法正常取食，於高濃度（57.02 mg/mL）處理時則蟎體呈現腫脹及軟化死亡狀態。苦茶粕水萃液以正丁醇液相-液相層析分離後，其水可溶部呈現較高活性，顯示活性成份屬極性偏高之一端。經（逆向RP-18）管柱層析分離後，以A4次分離部對雌成蟎之毒性最高，15 mg/mL處理後120h之致死率為70.02 ± 1.51%，但效果並不比水可溶部高。由結果推斷苦茶粕之抗蟎活性成份並非單一組成，而可能是多種成份所產生的協同作用。進一步以LC-ESI-MS分離活性最高之A4次分離部，結果顯示其中成份組成複雜，且大多為大分子量之化合物（1202-1346），經比對與六種茶皂素類化合物分子量相符。抗氧化酵素試驗結果顯示，葉蟎之SOD與POD酵素於前期相較於控制組則呈現激活狀態，但48 h後反而受抑制。72 h時SOD酵素抑制率達100%，POD酵素則為89.68 ± 4.28%；而CAT酵素處理24 h之抑制率為27.29 ± 7.24%，但24 h後酵素含量逐漸上升，直至72 h之抑制率為-25.36 ± 12.04%，呈現激活狀態。由結果知，苦茶粕萃取液對葉蟎確實造成氧化逆境，並影響其抗氧化酵素調控，進而抑制葉蟎。

關鍵字

苦茶粕 ； 油茶（Camellia oleifera Abel.） ； 神澤氏葉蟎（Tetranychus kanzawa Kishida） ； 植物源殺蟲劑 ； 氧化逆境

並列摘要

Tetranychus kanzawa Kishida cause serious damage to crops due to their short lifecycle, high reproduction, and induced pesticides resistance. However, the traditional agrochemicals can cause serious environmental pollution. Therefore, it is necessary to develop environmental-friendly plant-source pesticides as part of the integrated pest management (IPM). Oil-tea seed dreg is the by-product after oil production from the seeds of Camellia oleifera Abel. The objective of this study was to evaluate the potential of oil-tea seed dreg as an alternative natural pesticide against T. kanzawa. The crude extracts (Aqueous, 50% EtOH, 95% EtOH, and acetone) from oil-tea seed dreg were used in the antimite activity test against T. kanzawa. The crude aqueous extract showed slow-toxicity and the highest lethal effect against female adult among crude extracts. The 50% lethal concentration (LC50) values of after 72 h, were 9.92 mg/mL for aqueous extrect, followed by 95% EtOH extract (20.35 ± 1.93 mg/mL), 50% EtOH extract (24.19 ± 3.35 mg/mL) and
acetone extract (25.85 ± 6.65 mg/mL). The aqueous extract at 14.26 mg/mL caused low mortality of the female adult mites, however, their feeding ability was lost due to epilepsy. At high concentration, swelling and softening of the bodies were evident. The aqueous extract was further partitioned with n-Butanol, producing n-Butanol and aqueous fractions. The aqueous fraction showed higher mortality than that of n-Butanol fraction against T. kansawai suggesting that its bioactive compounds were of high polarity. The LC50 values of n-Butanol fraction and aqueous fraction after 72 h was 12.44 mg/mL and 65.02 mg/mL, respectively. Furthermore, both fractions were separated by column chromatography and grouped into 5 sub-fractions by thin layer chromatography (TLC) in order to find out which sub-fraction containing active compounds. The mortality (70.02%) of T. kansawai treated with 15 mg/mL of A4 sub-fraction was significantly higher than other sub-fractions at the same concentration. However, the lethal activity was reduced after the fractions were separated. Therefore, it can be implied that a synergistic or additive effect of compounds existed in oil-tea seed dreg. As a result, the bioactive components were analyzed by LC-ESI-MS after HPLC isolation and purification. The molecular weights of components were similar with several tea saponins. In oxidative stress experiment, superoxide dismutase (SOD) and peroxidase (POD) were increased after 24 h then inhibited after 48 h. Moreover, after 72 h, the inhibition of SOD and POD were 100% and 89.68 ± 4.28%, respectively. On the contrary, after 24 h after treatment, inhibition of catalase (CAT) was 27.29 ± 7.24%; however, 72 h after treatment, enzyme activity became active (-25.36 ± 12.04%). As a result, the extract of oil-tea seed dreg performed effective antimite activity against T. kanzawa, and the induced oxidative stress may contribute to it’s effect.

並列關鍵字

oil-tea seed dreg ； Camellia oleifera Abel. ； Tetranychus kanzawa Kishida ； plant-source pesticide ； oxidative stress

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