薑科植物之葉片揮發性成分分析與利用

Abstract

本研究利用頂空固態微萃取法(HS-SPME)和氣相層析質譜儀(gas chromatograph / mass spectrometry，GC/MS)建立台灣薑科植物葉片的取樣及分析流程，再進行各品種葉片之香氣成分分析。先以月桃(Alpinia zerumbet (Pers.) Burtt & Smith)葉片5 種香氣主成分，α-pinene、Camphene、Limonene、Camphor及Caryophyllene確立分析流程為：取樣時間為早上 9 時至 12 時，樣品為新鮮葉片，取樣後不冷凍儲藏直接分析，以直徑 3 mm 打孔器取單一植株中間葉序之葉片，而單一葉片內靠近葉柄處葉圓片 8 片，置入 25ml 之頂空玻璃瓶，加入內標準品 n-tridecane、n-hexadecane，以 75℃ 乾熱器加熱 4 分鐘，SPME 纖維吸附 4 分鐘，之後插入注射孔以 250℃ 脫附 30 秒後開始進行香氣質譜分析。GC oven 升溫起始溫度為 30℃ 維持 3 分鐘，第一階段升溫為每分鐘升溫 15℃ 至 120℃，第二階段為每分鐘升溫 5℃ 至 200℃，再以每分鐘 25℃ 升溫至 300℃ 後維持 1 分鐘。 26 種台灣薑科植物葉片共分析得 126 種香氣成分。其中香氣種類最少的為閉鞘薑(Costus speciosus (Koenig) Smith)，僅含有 2 種香氣成分、最多的是呂宋月桃(Alpinia flabellate Ridly, 採自台中科博館)，含有 32 種香氣成分。 利用 R code cluster analysis 軟體建立群集分析，得到已知的20個台灣薑科種原之群聚樹狀圖。加上未檢定的6個種原，總共26個試驗樣品可分為三大群。採自蘭嶼(陳義男)及台中科博館的呂宋月桃(Alpinia flabellate Ridly)則分類在第一群；第二群為月桃屬中的UN 1 A. sp、 UN 2 A. sp、月桃、川上氏月桃(Alpinia kawakamii Hayata)、島田氏月桃(Alpinia shimadai Hayata)、角板山月桃(Alpinia mesanthera Hayata)、普萊氏月桃(Alpinia pricei Hayata)、屈尺月桃(Alpinia kusshakuensis Hayata)、七星月桃(Alpinia densespicata Hayata)、烏來月桃(Alpinia uraiensis Hayata)、UN 3 A. sp及UN 4 A. sp共 12 個種原；第三群為月桃屬中的山月桃(Alpinia intermedia Gagnep)、紅豆蔻(Alpinia galanga (L.) Sw )及紅花月桃(Alpinia purpurata (Vieill.) K. Schum )，薑黃屬中的二黃(Curcuma viridiflora Roxb)、莪朮(Curcuma zedoaria (Berg.)Rosc)、薑黃(Curcuma domestica Valet)、UN 5 C.sp，山奈屬中的三奈(Kaempferia galanga L.)，薑屬中UN 6 Z.sp，閉鞘薑屬中的閉鞘薑(Costus speciosus (Koenig) Smith)及蝴蝶薑屬中採自台大山地農場春陽分場及屏東的穗花山奈(Hedychium coronarium Koenig)共 12 個種原。 分別以水及95%乙醇萃取26種台灣薑科植物葉片，記錄其萃取率並測定萃取物之清除DPPH自由基能力及還原力。結果顯示，各種薑科植物之乙醇萃取率以三奈萃取率最高(205.67 mg/g)，紅花月桃最低(70.67 mg/g)；水萃取以屈尺月桃最高(82.33 mg/g)，三奈萃取率最低(6.33 mg/g)。另外清除 DPPH 自由基能力，各樣品水萃取物中，UN 1 A. sp (南投特生中心)對 DPPH自由基具最強之清除能力，IC50 值為0.412 mg/mL，較弱的二黃及莪朮，IC50 值為分別為11.441 mg/mL及9.852 mg/mL。乙醇萃取物中，紅花月桃具最強之清除能力，IC50 值為0.154 mg/mL，薑黃及UN 6 Z. sp較弱，IC50 值為分別為0.4517 mg/mL及0.3678 mg/mL。在還原力方面，各樣品水萃取物中以月桃最強(k值為0.920 ± 0.007 mL/mg)，最低的為二黃水萃取物(k值為0.032 ± 0.027 mL/mg)；各樣品乙醇萃取物中，以月桃最強(k值為1.480 ± 0.008 mL/mg)，最低的為UN 6 Z. sp 乙醇萃取物(k值為0.050 ± 0.002 mL/mg)。

This study utilized the headspace solid phase microextraction (HS-SPME) and gas chromatography-mass spectrometry (GC-MS) to establish procedure of sampling and analyzing for volatile leaf components of Zingiberaceae plants cultured in Taiwan. The standard operation procedure (SOP) for the analysis was established by analyzing five major ingredients (α-pinene, Camphene, Limonene, Camphor, Caryophyllene) of shell ginger (Alpinia zerumbet (Pers.) Burtt & Smith) leaf. The suggested SOP as follow: 8 discs of 3 mm diameter punched from the middle part, near the petiole of a fresh sample leave, between 9:00 to 12:00. Put discs in 25 mL vial immediately, heating 4 minutes at 75℃ after internal standards (n-tridecane and n-hexadecane) added, trapping 4 minutes by SPME. Volatile components analysis by chromatography-mass spectrometry (GC-MS) with the condition of inserting the 250℃injection holes for 30 seconds , maintained the initial GC oven temperature at 30℃ for 3 minutes, then rising 15℃/min to 120℃, 5℃/min to 200℃, and 25℃/min to 300℃ and maintained 1 minute. Analyzing leaves of 26 accessions in the ginger family totally have 126 volatile components. Costus speciosus (Koenig) Smith has only two components and Alpinia flabellate Ridly from Taichung Science Museum has thirty-two components. Using R code cluster analysis software, cluster tree can be obtained from identified 20 accessions. With another 6 unknown accessions, a total of 26 accessions were divided into three groups. Two accessions of Alpinia flabellate Ridly are classified as the first group, the second group including 12 accessions of Alpinia (zerumbet, kawakamii, shimadai , mesanthera, pricei, kusshakuensis, densespicata, uraiensis, UN 1, UN 2, UN 3 and UN 4) and the other 12 accessions as the third group, including Alpinia intermedia, A. galanga, A. purpurata, Curcuma viridiflora, C. zedoaria, C. domestica, C. sp. UN 5, Kaempferia galanga, Zingiber sp UN 6, Costus speciosus and 2 collections of Hedychium coronarium. Leaves of 26 accessions were extracted with water and 95% ethanol, after recording its extraction rate, the DPPH radical scavenging and reducing power of the extracts were measured. Water extraction rate of A. kusshakuensis (82.33 mg /g) is the highest and K. galanga (6.33 mg/g) is the lowest. 95% ethanol extraction rate of K. galanga (205.67 mg/g) is the highest and A. purpurata (70.67 mg/g) is the lowest. About DPPH radical scavenging capacity, water extracts of UN 1 A. sp has the strongest IC50 of 0.412 mg/mL, while C. viridiflora and C. zedoaria have weak IC50 of 11.441 mg/mL and 9.852 mg/mL, respectively. Ethanol extracts of A. purpurata has the strongest IC50 of 0.154 mg/mL while C. domestica and UN 6 Z. sp have weak IC50 of 0.4517 mg/mL and 0.3678 mg/mL, respectively. In terms of reducing power, aqueous extracts of A. zerumbet is with strong k value of 0.920 ± 0.007 mL/mg), the lowest is C. viridiflora with k value of 0.032 ± 0.027 mL/mg. The strongest k value of 1.480 ± 0.008 mL/mg is ethanol extracts of A. zerumbet, and ethanol extracts of UN 6 Z. sp has k value of 0.050 ± 0.002 mL/mg.