野山藥與杜莎藻中有效成分之分離與生物活性之探討

保健食品意指含有特定成分具有保健功效或調節生理機能之食品，世界上大部分之民族，尤其是中國，均有使用特殊食材或配料，經烹煮食用後，達到保健或治療疾病之傳統。時以至今，古人所使用之特殊食材或配料，就轉成現今所謂之特殊營養品、健康食品、機能性食品、膳食補充品等。隨著保健食品市場之日漸龐大，對保健食品中特定有效成分之定量與生物活性之評估，亦日見急迫。事實上保健食品中有效成分之含量與具有之生物活性往往隨著該植物之生長環境、採收季節、採集、提鍊與儲存過程，而有極大之變動。故本研究選擇山藥與杜式藻兩種保健食品素材，開發簡單與有效之高效能液相層析(HPLC)方法，對其特定之有效成分，進行分離與分析。山藥古稱薯蕷，原產於我國、日本及熱帶地區。為薯蕷科（Dioscoraceae）薯蕷屬（Dioscorea）多年生蔓性根莖類植物，其主要食用部位為地下之塊莖（擔根體）。山藥除了為人類糧食中澱粉及蛋白質之重要來源外，在傳統與保健醫藥方面，亦為備受矚目之藥用植物之一。尤其近年來研究指出山藥所含之皂素(saponins)成分，具有增強免疫細胞活性，促進干擾素(interferon)之誘生與T淋巴球細胞數之增加，細胞與動物實驗顯示具備抗腫瘤之作用，另可消除蛋白尿、抑制細胞突變及降低膽固醇等。本研究首次以製備型高效能液相層析(preparative HPLC)，分離野山藥中山藥皂素類之螺固醇皂苷 spirostanol glycosides，並以LC-MS, 1H- and 13C-NMR 決定其結構為. (25R)-spirost -5-en-3β-ol-3-O-α-L-rhamnopyranosyl-(1→2)-O-[β-D- glucopyranosyl -(1→4)]-β-D-glucopyranoside並比較野山藥中分離之螺固醇皂苷spirostanol glycosides與水解醣基後皂素diosgenin對癌細胞之生物活性，以MTT細胞活性測定顯示對癌細胞株Hep G2 之 IC50，螺固醇皂苷spirostanol glycosides與水解後皂素diosgenin 分別為9.02 及23.91 µM，HEK293細胞為13.21及27.31 µM，MCF7細胞為16.74及35.38 µM相較於一般之山藥皂素diosgenin野山藥萃取之螺固醇皂苷對於選取之癌細胞株之生長均有較大抑制作用。杜氏藻(Dunaliella, 又稱鹽藻)爲耐鹽的單細胞綠藻。藻體內能合成並積累比其他生物高得多的β-胡蘿蔔素，因此杜氏藻是生産β-胡蘿蔔素的良好天然資源，本研究發展出一種簡捷與有效之高效能液相層析(HPLC)方法，分離台灣養殖之杜氏藻(Dunaliella salina)中全反式與順式之類胡蘿蔔素(carotenoids)，以C30型管柱 (250×4.6 mm, 5 μm)為固定相，移動相選擇甲醇/氰甲烷/水=84/14/2和二氯甲烷，(75/25, v/v) ，以450nm紫外光為檢測波長。另外再以Trolox equivalent antioxidant capacity (TEAC)、reducing power和 2,2-diphenyl-2-picrylhydrazyl hydrate (DPPH) radical scavenging assay，檢測杜氏藻中類胡蘿蔔素之抗氧化能力，結果顯示杜氏藻萃取物中可分離出七種類胡蘿蔔素( carotenoids)且其含量為每克杜氏藻萃取物中含290.77mg 類胡蘿蔔素，所含之類胡蘿蔔素中又以全反式與9- 或9’-順式之β-類胡蘿蔔素(all-trans-β-carotene和 9-或9’-cis-β-carotene)為主要組成，每克中含 138.25mg，其他類胡蘿蔔素如 all-trans-lutein、all-trans-zeaxanthin、13-或13’-cis-β-carotene、all-trans-α-carotene 和 9-或9’-cis-α-carotene 則於每克杜氏藻萃取物中各佔有 6.55、11.27、4.95、2.69, 和 2.41 mg。所有之抗氧化能力檢測中杜氏藻萃取物比所有之全反式之α-carotene、lutein、zeaxanthin、β-carotene具有顯著較高之抗氧化活性，而此種現象應來自於杜氏藻萃取物中所含高量之9- 或9’-順式之類胡蘿蔔素。

關鍵字

Spirostanol glycoside ；山藥 (Dioscorea spp) ；細胞活性；杜氏藻；類胡蘿蔔素；抗氧化能力。

並列摘要

Functional foods can be defined as the foods contains special integrants which can improve the health condition and modulate biological function in human. The special benefit integrants in functional food verifies as the cultivated environment, process of extraction and restore. The goal of this research is to develop a dependable method to separate and determine the bioactive integrants in functional food. During recent years, steroidal saponins have attracted a growing interest owing to the wide range of their biological action on living organisms,including hemolytic,hypocholesterolemic,hypoglycemic, anti-thrombotic, anti-antineoplastic, antiviral and anti-canceractivities. Furostanol and spirostanol glycosides, two main steroidal saponins, have been found in many types of yams (Dioscorea species). Many reports indicated that steroidal saponins is one of the main bioactive compounds in yams (Dioscorea species). In this investigation, a spirostanol glycoside, (25R)-spirost-5-en-3β-ol 3-O-α-L-rhamnopyranosyl -(1→2)-O-[β-D-glucopyranosyl-(1→4)]-β-Dglucopyranoside,was isolated from wild yam (Dioscorea villosa) extract with reversed phase preparative HPLC. This was the first time found in yams (Dioscorea species). The structure was identified by LC-MS, 1H- and 13C-NMR. Cytostatic activities of the isolated saponin on Hep G2, HEK293 and MCF7 cells were estimated. Growth of these cells was inhibited as higher concentration of the compound was used. The IC50 values of the isolated spirostanol glycoside and its aglycon, diosgenin, were 9.02 and 23.91 μM for Hep G2 cells, 13.21 and 27.31 μM for HEK293 cells, and 16.74 and 35.38 μM for MCF7 cells, respectively. The isolated compound had significantly higher active intensity than diosgenin. Dunaliella salina is a type of unicellular and halophilic green biflagellate microalga contains abundant β-carotene, For the determination of carotenoids, the reversed-phase high performance liquid chromatography (RP-HPLC) has been used.A simple HPLC method with good separation efficiency was developed to determine all-trans and cis forms of carotenoids in Dunaliella salina cultivated in Taiwan. The analysis used a C30 column (250×4.6 mm, 5 μm) and an isocratic solvent system (flow rate = 1 mL/min) mixing methanol–acetonitrile–water (84/14/2, v/v/v) and methylene chloride, (75/25, v/v). Carotenoids were detected at 450 nm. Moreover, the antioxidant capacities of the algal carotenoid extract were also evaluated with Trolox equivalent antioxidant capacity (TEAC) assay, reducing power and 2,2-diphenyl-2-picrylhydrazyl hydrate (DPPH) radical scavenging assay. Results showed that 7 carotenoids in the algal extract could be separated simultaneously within 30 min and the total amount of them was 290.77 mg/g algae. The contents of all-trans-β-carotene and 9- or 9’-cis-β-carotene, the major carotenoids in the algae, were 138.25 and 124.65 mg/g algae, respectively. The contents of all-trans-lutein, all-trans-zeaxanthin, 13- or 13’-cis-β-carotene, all-trans-α-carotene and 9- or 9’-cis-α-carotene were 6.55, 11.27, 4.95, 2.69, and 2.41 mg/g algae, respectively. The algal carotenoid extract had significantly higher antioxidant activity than all-trans forms of α-carotene, β-carotene, lutein and zeaxanthin in all antioxidant assays. The cis forms of carotenoids, especially 9- or 9’-cis-β-carotene, might play crucial roles for the antioxidant capacities of the algal extract.

並列關鍵字

Dioscorea villosa ； spirostanol glycoside ； yam ； cytostatic activity ； Dunaliella salina ； Carotenoids ； Antioxidant capacity.

參考文獻

Chen, A. H. 1985. Applied studies on the saponins of Dioscorea I. Studies on the saponins of Dioscorea in Taiwan. J. Chin. Chem. Soc. 43: 79-85.

王祖興、劉新裕。2001。山藥萃取物質抗氧化性之評估。國科會專題研究計畫成果報告 (NSC89-2316-B-040-004) 台北。

徐煇妃、黃鵬。1997。山藥的營養及保健價值與食用法介紹。花蓮區農業專訊19:7-9

Wang, J. Y., Liu, S. Y., Son, L. M. and Kao, C. C. 1993. Genetic and agronomic variation among yam genotypes. J. Agric. Res. China. 42: 280-291.

Datta K, Datta SK, Datta PC. 1984. Pharmacognostic evaluation of potential yams Dioscorea. Journal ofEconomic and Taxonomic Botany. 5:181-196.

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野山藥與杜莎藻中有效成分之分離與生物活性之探討

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