以微胞毛細管電泳法檢測植物組織中的疏水性二次代謝物

Abstract

在分離電泳液中添加界面活性劑的微胞毛細管電泳技術已廣泛應用於帶電與不帶電分析物的檢測，然而受限分析物於分離電泳液中的溶解度，此技術無法運用於高疏水性、非水溶性的分析物檢測；是以本論文針對植物組織中可溶於水與非水溶性的疏水性植物二次代謝物分別提出快速萃取，其過濾後萃取液直接注入毛細管中的微胞毛細管電泳法開發。依研究對象物，本論文可分為茶品中的生物鹼與多元酚含量及熱帶藥用作物「青脆枝」中的抗癌藥物-喜樹鹼含量之微胞毛細管電泳法開發。 第一部分以80 oC、50 ml的熱水進行乾燥茶葉(500 mg)萃取10分鐘，其萃取液經孔徑0.45 um的硝化纖維膜進行過濾，再添加去離子水定量至100 ml。將茶湯樣品溶液注入於含pH 7.0, 10 mM磷酸緩衝溶液、4 mM硼酸緩衝溶液、45 mM SDS、0.5 %酒精的分離電泳液之融熔矽毛細管中(內徑50 um、總長47 cm)，並於毛細管兩端施與20 kV電壓進行分離，約在8分鐘內可得到高理論板數(>55,000)的7種兒茶素類分子、3種茶鹼類分子、茶胺酸、沒食子酸、維他命C與茶黃素之分離電泳圖；其樣品分子間解析度皆大於2、分離電流約為33 uA，而茶鹼類與兒茶素類分子的線性檢測範圍則落在2.5至100 ug ml-1 (S/N>5)之間。 從不同室內萎凋時間的部分發酵茶-包種茶之分離電泳圖中，可得知維他命C、兒茶素類分子會隨室內萎凋時間增長而減少，而沒食子酸則會隨室內萎凋時間增長而增加；藉由兒茶素類分子總量之減少百分比可計算出茶葉製程品管指標-發酵度。本研究同時以電位儀搭配白金電極、參考電極來量測不同室內萎凋時間的茶品之氧化還原電位，其氧化還原電位與不同室內萎凋時間的茶品發酵度呈現高度正相關。 第二部分則以高沸點(b.p. 189 oC)、高喜樹鹼溶解度、非質子性的Dimethyl Sulfoxide (DMSO)有機溶劑來直接加熱萃取(60 oC × 30 min)青脆枝中非極性、非水溶性的喜樹鹼(約1 ml DMSO有機溶劑萃取100 mg青脆枝)。由於DMSO有機溶劑有高介電常數(47.2)且會與水互溶之特性，因此並不會影響毛細管內的電滲透流的產生。將過濾後萃取液與緩衝溶液進行混合，可直接注入於含高DMSO濃度的分離電泳液(pH 8.6, 10 mM硼酸緩衝溶液、90 mM SDS、20 % DMSO)之融熔矽毛細管中(內徑75 um、總長34 cm)，並於毛細管兩端施與16 kV電壓進行分離，約在4.5分鐘內可得到高理論板數(>25,000)的喜樹鹼、9-甲氧基喜樹鹼、葫蘆巴鹼之分離電泳圖，其解析度大於2、分離電流約為77 uA；而喜樹鹼線性檢測範圍為5至400 ug ml-1 (S/N>5)。

Capillary electrophoresis (CE) is a convenient and rapid analytical method, but the usage is generally restricted in water soluble and charged compounds. Electrophoresis by the aid of surfactants added in the running buffer, namely micellar electrokinetic capillary electrophoresis (MEKC), has successfully expanded the application to uncharged compounds, but it still can not handle with highly hydrophobic and water-insoluble materials. In this dissertation, two direct-injection capillary electrophoresis procedures were developed for determining hydrophobic secondary metabolites in plant tissues: (1) polyphenols in tea infusions; and (2) the anti-cancer alkaloids, camptothecins (CPT), in Nothapodytes foetida, a tropical medicinal plant. The first approach used hot water (80 oC for 10 min) to extract dried tea leaves (500 mg / 50 ml); the extract was filtered through a 0.45 um nitrocellose filter membrane, filled up with deionized water to 100 ml and directly injected into the separation capillary (untreated fused silica, 47 cm in length, 50 um i.d.). The sample plug was separated in MEKC mode (20 kV, running buffer: 10 mM phosphate, 4 mM sodium tetraborate, 45 mM SDS and 0.5 % ethanol, pH 7.0) and detected with photodiode array (200, 266 nm, Agilent). Several important tea quality-relating chemicals including caffeine, theanine (the umami factor), vitamin C (the freshness factor) and polyphenols (namely tea catechins, the astringency factor) can be simultaneously determined within 8 min of migration time. The linear dynamic ranges for tea catechins were from 2.5 (S/N >8) to 100 ug ml-1. Using the rapid analytical method, the fermentation process (indoor withering) of pouching tea (a Taiwanese Oolong tea with mild fermentation) was monitored. From the electropherograms, tea catechins can be quantified and were found to be depleted during the indoor withering process. Therefore, not only the conventional quality factors such as theanine, we also successfully estimated the fermentation degrees during the tea making process. That is a crucial quality factor of tea industry but hard to obtain using traditional analytical strategy. The results were compared with the redox potential method developed in our department. The second approach dealt with a non-polar anti-cancer compound (camptothecin) in dried plant tissues; the chemical is water insoluble and therefore can not be analyzed by capillary electrophoresis even in MEKC mode. We are the first group to introduce an aprotic, water miscible organic solvent (dimethyl sulfoxide, DMSO) into the running buffer of CE to facilitate the separation (20 % DMSO, 90 mM SDS in 10 mM borate buffer, pH 8.60). DMSO is an excellent solvent with high dielectric constant (47.2) and the electroosmotic flow was therefore not hampered. Moreover, it possesses a high boiling point, so CPT can be easily extracted (c.a. 1 ml of DMSO for 100 mg of dried plant tissue) at elevated temperature (60 oC for 30 min) without using reflux apparatus. The filtered extract was directly injected into the separation capillary (untreated fused silica, 34 cm in length, 75 um i.d.) and analyzed in MEKC mode (16 kV, 369 nm). Within 5 min of migration, camptothecins were successfully separated and quantified with linear dynamic range from 5 to 400 ug ml-1.