薏仁麩皮中防癌及抗發炎活性成分之分離與鑑定

Abstract

薏苡為禾本科一年生草本植物，長期以來是被用於對抗腫瘤及發炎的傳統中藥，目前研究也陸續闡明薏苡的生理功效，且已證實含麩皮的糙薏仁比精白薏仁有較高的抗癌及抗發炎活性。綜合文獻結果推測，薏苡籽實中，特別是薏仁麩皮中一定含有某些抑癌、抗發炎活性成分，然而針對薏仁麩皮的抗癌、抗發炎活性成分尚未完全明瞭，故本研究基於探討食品抑癌、抗發炎作用之目的，將以活性區分為導向分離純化出薏仁麩皮中具有生理功效之成分。 研究結果發現薏仁麩皮甲醇萃取物乙酸乙酯區分層（ABM-EtOAc）對肺癌及腸癌細胞之生存力具有最顯著抑制的效果，並發現其可誘導癌細胞發生細胞週期改變和細胞凋亡。在抗發炎之研究發現，ABM-EtOAc及薏仁麩皮甲醇萃取物正丁醇區分層（ABM-BuOH）可抑制LPS所誘發之老鼠巨噬細胞RAW 264.7釋放促發炎細胞激素TNF-α及IL-1β，並且在50 μg/mL濃度之下之ABM-EtOAc及ABM-BuOH皆具有抑制人類肺癌細胞A549及人類腸癌細胞HT-29之COX-2蛋白表現的效果，而對常在型COX-1酵素並無顯著影響，其中尤以ABM-BuOH活性最佳。此結果顯示ABM-EtOAc及ABM-BuOH中一定含有一些抑癌、抗發炎活性成分，藉由促進癌細胞凋亡來達到抑制癌細胞生長，並且這些植物化學成分具有預防慢性發炎導致之癌病變功效。 進一步自ABM-EtOAc中以抑制肺癌及腸癌細胞生長為導向，純化鑑定出25個純化合物。純化合物中Coixspirolactam A、Coixspirolactam B、Coixspirolactam C鑑定為新化合物；5-Hydroxy-7-methoxy-4'-acetylisoflavone、Coixlactame、Coixspirolactone為天然物中首次分離之化合物；Caffeic acid、Protocatechuic acid、Chlorogenic acid、3-O-Coumaroyl-5β-sitostanol、3-O-Caffeoyl-5β-sitostanol、Methyl dioxindole-3-acetate及三萜類24,25-Oxidocycloartanol為首次由薏仁中分離出來之化合物。所分離出之純化合物之抑癌活性檢測結果顯示，異黃酮類5-Hydroxy-7-methoxy-4'-acetylisoflavone、三萜類24,25-Oxidocycloartanol、酚酸酯類3-O-Coumaroyl-5β-sitostanol、3-O-Feruloyl-5β-sitostanol、3-O-Caffeoyl-5β-sitostanol具有抑制此A549、HT-29、COLO 205癌細胞生長之作用，而酚酸類中只有Caffeic acid、Protocatechuic acid、Chlorogenic acid具有抑制三株癌細胞生長之效果。 另一方面，以抗發炎為活性導向，自ABM-BuOH中利用多種分離技術純化出9個與ABM-EtOAc不重複的純化合物，包括：1個Apocarotene：Loliolide、1個三萜類：3β-Hydroxyolean-18-en-28-oic acid、1個木酚素：Pinoresinol、1個酚酸酯類：(E)- methyl 3,4-dihydroxycinnamate、1個接有糖基的固醇類：β-Sitosteryl-3β-glucopyranoside、1個異黃酮：Biochanin A、3個類黃酮類化合物：5, 7-Dimethoxy-4'-hydroxyflavan、4', 5, 7-Trihydroxyflavone、Farrerol，此9個純化合物皆為薏苡中首次分離出來之化合物。

Adlay (Coix lachryma-jobi L. var. ma-yuen Stapf ) is an one year grass crop that has long been used as a traditional Chinese medicine to treat tumor and inflammation. Recent studies have continuously demonstrated the physiological effects of adlay and reveal that duhulled adlay posseses a higher anti-tumor and anti-inflammation activity than polished adlay. In summation of references, the adlay seeds contain some anti-tumor or anti-inflammation active compounds especially in the adlay bran. However, the active constituents involved in anti-tumor or anti-inflammation activity are still unknown. Therefore, the aim of our study is based on to discuss function of anti-tumor and anti-inflammation in food, to separate and identify the physiological effective constituents contained in adlay bran via active fractions. The study results show that the ethyl acetate fraction from adlay bran methanolic extract (ABM-EtOAc) had the highest ability to reduce survival rate of lung and colorectal carcinoma cell. It is also found that inhibition of cell cycle phase change, and cell apoptosis. Concerning anti-inflammation research, ABM-EtOAc and ABM-BuOH subfractions all inhibit LPS-induced TNF-α and IL-1β which released from RAW 264.7. Furthermore, ABM-EtOAc and ABM-BuOH subfractions inhibit COX-2 expression of A549 and HT29 at 50 μg/ml concentration, while constitutive COX-1 expression is not affected. Moreover, the ABM-BuOH subfraction is the best. Thus, these results indicate that ABM-EtOAc and ABM-BuOH subfractions must have some anti-tumor or anti-inflammation active compounds. They inhibit cancer cell growth by promoting apoptosis of cancer cell. The chemical compound of plant has preventative effects on chronic inflammation-related cancer. Furthermore, 25 pure compounds are isolated and identified by inhibiting growth of lung cancer and colon cancer. The coixspirolactam A, coixspirolactam B, and coixspirolactam C are newly identified compounds. Caffeic acid, protocatechuic acid, chlorogenic acid, 3-O-coumaroyl-5β-sitostanol, 3-O-caffeoyl-5β-sitostanol, methyl dioxindole-3-acetate and 24,25-oxidocycloartanol are first isolated from adlay. From the anti-tumor activity test, it shows that there are only Caffeic acid, Protocatechuic acid, and Chlorogenic acid have anti-tumor activity in phenolic acid. From another hand, via anti-inflammation activity, the nine pure compounds, which are not found in ABM-EtOAc, are isolated from ABM-BuOH using many different kinds of isolation technology. Including one Apocarotene: Loliolide, one triterpinoid: 3β-Hydroxyolean-18-en-28-oic acid, one lignan: Pinoresinol, one phenolic ester: (E)-methyl 3,4-dihydroxycinnamate, one sugar-link sterol: β-Sitosteryl-3β-glucopyranoside, one isoflavone: Biochanin A, three flavonoid: 5, 7-Dimethoxy-4'-hydroxyflavan, 4', 5, 7-Trihydroxyflavone (apigenin), Farrerol, all of the nine pure compounds are first isolated from adlay.