有色米及紫色稻葉生物活性物質分離純化及其功能特性

Isolation, purification and functional properties of bioactive substances from pigmented rice and black rice leaf

周珮可(Chorpaka Thepthanee)

指導教授 ： 廖遠東 ； 劉展冏

國立屏東科技大學/農學院/食品科學系所/博士(2022年)

https://doi.org/10.6346/NPUST202200028

摘要

有色米 (Oryza sativa L.) 為水稻之特殊品種，其葉片與種子富含多種色素成分，可呈現黑色、紫色或紅色等不同顏色。文獻指出，有色米為一種良好之天然抗氧化劑來源。本研究將黑米稻葉與四種分別產自泰國與台灣之紅色及黑色有色米，以水與不同濃度乙醇（25%、50%、75%及95%）進行萃取，分析其生物活性成分及抗氧化能力，並且分離黑米稻葉中有助於抗氧化、抗發炎與抗癌活性之酚類化合物。首先測定黑米稻葉與有色米各種溶劑萃取物之總酚（TPC）、總類黃酮（TFC）、總花青素（TAC）與總原花青素（TPAC）含量及抗氧化活性（DPPH、ABTS自由基清除能力與還原力）。實驗結果顯示，可獲得最佳抗氧化活性與植物化學成分之最適萃取條件分別為50％（黑色米）與75％乙醇（紅色米與黑米稻葉），其中黑米稻葉之75％乙醇萃取物（BRLEE）具有最高之TPC與TFC值（分別為303.54 mg GAE/g與405.23 mg QE/g），黑米稻葉及黑色米萃取物皆含有花青素，而原花青素僅存在於紅色米中。此外，BRLEE也具有最佳之抗氧化能力，因此將BRLEE使用不同極性之溶劑進行分餾，包括正己烷（Hex）、乙酸乙酯（EtOAc）與正丁醇（n-BuOH），其中n-BuOH與EtOAc分餾組之TPC與TFC含量最高，而n-BuOH與水相分餾組富含花青素，於DPPH、ABTS自由基清除能力及還原力試驗中顯示強大之抗氧化能力。於LPS誘導RAW 264.7巨噬細胞之抗發炎活性試驗中，BRLEE與EtOAc分餾組雖然對細胞因子TNF-α釋放之影響較小，但具有降低NO及其它促發炎細胞因子（PGE2、IL-6及IL-1β）生成之能力，且顯著抑制iNOS與COX-2蛋白表現量。另外，EtOAc分餾組也可顯著降低HepG2肝癌細胞之存活率，增加Sub-G1期之細胞比例且透過粒線體依賴性途徑誘導細胞凋亡，並使用UV-LC-MS/MS 分析 EtOAc 分餾組之生物活性成分、酚酸及類黃酮化合物。進一步將富含酚類之EtOAc分餾組藉由Sephadex LH-20管柱層析，分離得到五種亞分餾組，接著測定其抗氧化及抗癌活性。結果顯示，所有亞分餾組中，亞分餾組5（Sub5）之TPC與TFC含量皆最高，且具有最佳DPPH、ABTS自由基清除能力與還原能力，IC50值分別為3.23、31.95及72.74 µg/mL。而於抗癌活性試驗中發現，Sub5之酚類化合物相較於其他亞分餾組，更能有效抑制肝癌（HepG2）、乳腺癌（MCF-7）及結腸癌（Caco-2）細胞之存活率，尤其是對HepG2肝癌細胞，Sub5顯著增加Sub-G1期之細胞比例，並可能透過粒線體及死亡受體依賴性途徑，伴隨著PI3K/AKT及Erk訊號路徑之抑制來誘導細胞凋亡。而UV-LC-MS/MS 之分析結果表明，由黑米稻葉分離而得之Sub5中主要化合物為木犀草素、木犀草素-8-C-葡萄糖苷與木犀草素-7-O-葡萄糖苷。綜上所述，黑米稻葉副產物有望作為一種具抗氧化、抗發炎與抗癌能力之生物活性成分之來源，可能有助於機能性食品與新穎藥劑之開發。

關鍵字

黑米稻葉 ； 抗氧化活性 ； 細胞週期 ； 細胞凋亡

並列摘要

Pigmented rice (Oryza sativa L.) is a special type of rice with colors such as black, purple, and red in its seed or leaf. It has been reported to be a good source of natural antioxidants. The purposes of this study were to evaluate the effect of water and different concentrations of ethanol (25%, 50%, 75%, and 95%) extract on the phytochemical content and their antioxidant activity of four pigmented rice cultivars from Thailand and Taiwan and black rice leaf. Furthermore, isolated phenolic compounds from black rice leaf contribute strong antioxidant, anti-inflammatory and anticancer activities. Firstly, pigmented rice and black rice leaf were extracted with various solvents and then determined total phenolic (TPC), flavonoid (TFC), anthocyanins (TAC), and proanthocyanins (TPAC) contents, as well as antioxidant activities (DPPH and ABTS radicals scavenging activity and reducing power). The results showed that the optimal extraction condition for phytochemical contents and antioxidant activities recovery for red rice cultivars and black rice leaf were 75% ethanol, while black rice cultivars were 50% ethanol. Black rice leaf extracted with 75% ethanol (BRLEE) presented the highest TPC and TFC values (303.54 mg GAE/g and 405.23 mg QE/g, respectively). Moreover, black rice leaf and black rice extracts contained anthocyanin, whereas proanthocyanin was found only in red rice cultivars. Interestingly, the BRLEE showed higher antioxidant abilities than other solvent extracts and pigmented rice. Therefore, the BRLEE was collected to sequentially fractionated according to solvent polarity, including hexane (Hex), ethyl acetate (EtOAc), and n-butanol (n-BuOH). Among all the fractions, the n-BuOH and EtOAc fractions demonstrated the highest contents of TPC and TFC, while anthocyanins were found to be abundant in the n-BuOH and aqueous fractions, which also exhibited powerful antioxidant capacities according to DPPH and ABTS radical-scavenging assays and a reducing power assay. Regarding anti-inflammatory activity, BRLEE and EtOAc inhibited the production of NO and cytokine secretion (PGE2, IL-6, and IL-1β) but displayed less effect on TNF-α release in LPS-induced RAW 264.7 cells. They also significantly reduced iNOS and COX-2 protein expression. Additionally, the EtOAc fraction demonstrated the most significant activity against HepG2 liver carcinoma cells, inhibited cell growth, increased the Sub-G1 population, and induced apoptosis via mitochondrion-dependent mechanisms. The bioactive components, phenolic acids, and flavonoids from EtOAc fraction were also investigated using UV-LC-MS/MS. The phenolic-rich EtOAc fraction was further separated into five subfractions by Sephadex LH-20 column chromatography, and their antioxidant and anticancer activities were investigated. The results revealed that among all the subfractions, subfraction 5 (Sub5) showed the lowest IC50 values, 3.23, 31.95, and 72.74 µg/mL for DPPH, ABTS, and reducing power assays, respectively. The TPC and TFC contents were also superior for Sub5 compared to the other subfractions. The phenolic substances obtained from Sub5 could reduce the cell viability of liver (HepG2), breast (MCF-7), and colon (Caco-2) cancer cells more than other subfraction. Notably, Sub5 significantly increased the accumulation of cells in the Sub-G1 phase in HepG2 cells. Furthermore, it could trigger apoptosis cell death through mitochondrial and death receptor pathways accompanying by the inhibition of PI3K/AKT and Erk signaling pathways. The UV-LC-MS/MS analysis demonstrated that the main compounds in the Sub5 derived from black rice leaf were luteolin, luteolin-8-C-glucoside, and luteolin-7-O-glucoside. These findings highlight the possibility of using black rice leaves, a rice by-product, as a potential source of bioactive compounds with antioxidant, anti-inflammatory, and anticancer properties. Thus, it may be beneficial for use in functional foods and the pharmaceutical industry.

並列關鍵字

Black rice leaf ； Antioxidant activity ； Cell cycle ； Apoptosis

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