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標題: Antioxidant, anti-inflammatory, antimelanogenic and aoptotic effects of Nymphaea mexicana Zucc. flower
作者: 饒雅茜
Jao, Ya-Chien
關鍵字: Nymphaea mexicana Zucc. (yellow water lily);黃睡蓮;UV;antioxidation;inflammation;skin-whitening;apoptosis;UV;抗氧化性;發炎反應;美白;細胞凋亡
出版社: 食品暨應用生物科技學系所
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近年來全球暖化及氣候變遷已然成為不容忽視的議題。臭氧層的稀薄化加劇日光中紫外線 (ultraviolet, UV) 對人體健康之影響。過度曝曬於 UV 之下會造成皮膚紅斑、曬傷、發炎反應、DNA 氧化傷害、並刺激黑色素生成,甚至導致皮膚癌發生。許多文獻指出天然植物成分具有光化學防護作用 (photochemoprevention),可減少紫外線對皮膚的傷害。Nymphaea mexicana Zucc. 為睡蓮科(Nymphaeaceae) 水生植物,一般又稱為 yellow water lily,在台灣南部十分常見。目前已知睡蓮屬植物具有抗氧化、抗腫瘤及提升肝功能等生理活性。本研究針對三個主題探討黃睡蓮之:(一) 抗氧化與抗發炎能力及其中活性成分,(二) 抑制黑色素生成之皮膚美白潛力,(三) 誘發黑色素瘤細胞凋亡效應。
研究結果顯示:(一) 以總抗氧化力 (TEAC)、氧自由基吸收能力 (ORAC) 及胞內抗氧化力 (CAA) 評估黃睡蓮花水 (WENZ)、甲醇 (MENZ)、乙醇 (EENZ)、乙酸乙酯 (AENZ) 萃取物之抗氧化力,發現 WENZ 具有最佳抗氧化力。再者 WENZ 及 EENZ 均可有效抑制 Cu2+ 誘導 LDL 氧化生成共軛雙烯及丙二醛反應物,並降低相對電泳移動量 (relative electrophoretic mobility, REM)。且黃睡蓮花不同溶劑萃取物均可有效降低 H2O2 誘導大鼠淋巴球細胞之 DNA 氧化傷害。進一步評估其中活性成分,結果顯示 WENZ 含有較高總多酚及類黃酮含量,另外 HPLC 分析證實每克 WENZ 中含有 0.48 mmol gallic acid 以及 0.06 mmol naringenin。WENZ 另具有抗發炎能力,能有效降低 lipopolysaccharide (LPS) 所活化之 RAW 264.7 小鼠巨噬細胞之 nitric oxide (NO) 生成量;並且對於發炎指標蛋白 inducible nitric oxide synthase (iNOS) 及 cyclooxygenase-2 (COX-2) 蛋白質表現均有抑制效應。
(二)黃睡蓮花不同溶劑萃取物對於酪胺酸酶活性抑制率以 WENZ 效果最佳,IC50 約為 5.5 mg/mL。以酵素動力學評估發現,WENZ 屬於混合型抑制劑,能改變酵素與基質間親和力,且在四種萃取物中以 WENZ 與酵素結合能力較佳。在細胞實驗方面可知 WENZ 可顯著降低黑色素刺激生成荷爾蒙 (alpha-melanocyte stimulating hormone, alpha-MSH) 所誘導之胞內黑色素生成量以及酪酸酶活性;並可藉由抑制 alpha-MSH 所誘導之酪胺酸酶相關蛋白 (tyrosinase and tyrosinase-related protein-1, TRP-1) 以及轉錄因子(microphthalmia-associated transcription factor, MITF) 表現以抑制小鼠黑色素瘤細胞之黑色素生成,然而對於 tyrosinase-related protein-2 無顯著影響。(三) 誘發 B16F0 黑色素瘤細胞凋亡方面,WENZ 可造成 B16F0 細胞週期停滯於 sub-G1 phase 並提高早期凋亡及晚期凋亡細胞比例,促使粒線體膜電位下降。進一步評估其對於細胞凋亡機制影響,結果發現 WENZ 可提升 caspase-8 蛋白質表現,進而促使下游蛋白 Bid 斷裂形成 t-Bid。而在抗凋亡以及促凋亡蛋白表現上,WENZ 可提高促凋亡蛋白 Bax 表現量,並降低抗凋亡蛋白 Bcl-2 及 Bcl-XL 之蛋白質表現量;隨著 WENZ 處理時間增加,其 caspase-3 活性以及蛋白質表現量均隨之提高,並促使 PARP 斷裂。由以上實驗結果推測,WENZ可誘發小鼠黑色素瘤細胞走向細胞凋亡,是經由粒線體凋亡路徑。
綜合以上實驗結果,黃睡蓮花因富含酚類化合物而具有良好之抗氧化性,可有效抑制 LDL 氧化、減少 DNA 氧化損傷,更具有抗發炎能力。黃睡蓮花水萃取物另可做為混合型抑制劑以抑制酪胺酸酶活性,並透過 MITF 轉錄因子促使酪胺酸酶相關蛋白 (TYR, TRP-1) 表現量降低,以減少小鼠黑色素瘤細胞之黑色素生成。此外,黃睡蓮花水萃取物更可透過粒線體路徑誘發小鼠黑色素瘤細胞走向凋亡。本研究證實黃睡蓮花之抗氧化、抗發炎、美白以及抗皮膚癌功效,期望未來可做為黃睡蓮開發成多功能保健產品之重要佐證。

Global warming and climate change are becoming the greatest concern in recent years. Depletion of ozone layer intensifies the harmful effects caused by solar ultraviolet radiation (UV) and threatens human health. Excessive exposure to UV may cause erythema, sunburn, inflammation, DNA damage and melanogenesis, and even skin cancer. Numerous studies suggest that phytochemicals possess photochemopreventive activity, which can reduce the damage of UV. Nymphaea mexicana Zucc. is an aquatic plant which belongs to the family Nyphaeaceae and is commonly known as the yellow water lily. Nymphaea plants are widely distributed over southern Taiwan. Previous studies have demonstrated that Nymphaea plants have antioxidant, anti-tumorigenic and hepatoprotective capacities. In the present study, we focus on three topics of Nymphaea mexicana Zucc flower: (1) antioxidants, anti-inflammatory activities and bioactive compounds; (2) skin-whitening effects of antimelanogenesis, and (3) induction of apoptosis in mouse melanoma.
In the first part, the antioxidant, anti-inflammatory activities and bioactive compounds in water, methanol, ethanol, and ethyl acetate extracts derived from Nymphaea mexicana Zucc. flower (WENZ, MENZ, EENZ, and AENZ, respectively) were evaluated. Trolox equivalent antioxidant capacity (TEAC), oxygen-radical absorbance capacity (ORAC), and the cellular antioxidant activity (CAA) assays revealed that WENZ had the strongest antioxidant activity. WENZ and EENZ showed potent inhibitory effect on oxidative modification of low-density lipoprotein (LDL) upon Cu2+ stimulation, by decreasing conjugated diene, thiobarbituric acid reactive substances (TBARS) formation and reducing relative electrophoretic mobility (REM). Furthermore, In comet assay, all four extracts decreased DNA damage in H2O2-treated mouse lymphocytes. WENZ also had the highest phenolic and flavonoid contents. Gallic acid (0.48 mmol/g WENZ) and naringenin (0.06 mmol/g WENZ) were identified as the major components in WENZ by HPLC analysis. The anti-inflammatory effect of WENZ in lipopolysaccharide (LPS)-activated RAW 264.7 mouse macrophages was also investigated. Pre-treatmeny of cells with WENZ decreased nitric oxide (NO) formation, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein expression upon LPS stimulation.
In the second part, WENZ showed the best inhibitory capacity of tyrosinase activity with an IC50 value of 5.5 mg/mL among four extracts. According to enzyme kinetics analysis, WENZ was implicated as a mixed inhibitor of mushroom tyrosinase which could change the affinity between enzyme and substrate. Additionally, WENZ had the best affinity in binding to tyrosinase. In the in vitro cell model, WENZ significantly suppressed cellular melanin contents and tyrosinase activity induced by alpha-melanocyte stimulating hormone (a-MSH) in B16F0 cells. Moreover, WENZ lowered protein levels of tyrosinase, tyrosinase-related protein 1 (TRP-1) and microphthalmia-associated transcription factor (MITF) , but not lowered TRP-2 protein level in B16F0 mouse melanoma. In the third part, we evaluated effects of WENZ and its bioactive compound, gallic acid, on apoptosis induction and the possible underlying mechanism in B16F0 cells. Data indicated that cell cycle was arrested at sub-G1 phase, the apoptotic cells including early apoptotic and late apoptotic cells were increased and mitochondria membrane potential was significantly decreased by WENZ treatment in B16F0 cells. WENZ increased caspase-8, t-Bid, Bax and caspase-9 levels as well as decreased the expression of anti-apoptotic proteins, Bcl-2 and Bcl-XL. Furthermore, WENZ (80 ug/mL, 0-6 h) increased caspase-3 expression and activity in a time-dependent manner, leading to poly (ADP-ribose) polymerase (PARP) cleavage. Our present results suggest that the WENZ-induced mouse melanoma apoptosis is occurred via mitochondrial-modulated pathway.
In conclusion, the Nymphaea mexicana Zucc. flower was abundant in phenolic compounds, which inhibited LDL oxidation, decreased DNA damage and had anti-inflammatory activity. Besides, WENZ served as a mixed inhibitor of mushroom tyrosinase and suppressed melanogenesis through tyrosinase, TRP-1 and MITF pathway. Furthermore, WENZ induced mouse melanoma apoptosis via a mitochondria pathway. In this study, we demonstrated that the Nymphaea mexicana Zucc. flower possessed antioxidant, anti-inflammatory, antimelanogenic, and anti-skin cancer actions. This study would contribute to the development and application of a multifunctional health food, the Nymphaea mexicana Zucc. flower.
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