荷葉萃取物之抗氧化、抗發炎活性與成分及對血脂與體脂之影響

Abstract

動脈粥狀硬化所導致之心臟及心血管疾病等均位居我國十大死因。古書記載植物蓮各部位均是藥食兼用，特別是以荷葉「去瘀活血」的功效最受矚目。本研究目的擬分離鑑定荷葉中具抗氧化與抗發炎功效之活性化合物，並由高脂飲食倉鼠模式探討不同品種與劑量荷葉乙醇萃取物之調節血脂功能，及以高熱量飲食大鼠模式評估荷葉正丁醇區分層不易形成體脂肪之功能，對其機能性作更深入的探討，期能提高本土產植物蓮的應用價值。 在抗氧化活性方面，以抑制銅離子誘導人體離體低密度脂蛋白氧化、DPPH自由基清除能力配合抗氧化成分含量分析為生理活性導向模式，篩選荷葉甲醇萃取物及其溶劑區分層之抗氧化性，結果顯示荷葉甲醇萃取物之乙酸乙酯與正丁醇區分層具較佳抗氧化活性及含有較高含量抗氧化成分。進一步將此兩次區分層以矽膠、Sephadex LH-20管柱層析予以分離純化，共分得七個類黃酮類化合物分別為catechin (1)、quercetin (2) quercetin-3-O-glucopyranoside (3)、quercetin-3-O-glucuronide (4)、quercetin-3-O-galactopyranoside (5)、kaempferol-3-O-glucopyranoside (6)、myricetin-3-O-glucopyranoside (7)。以quercetin及帶醣基化合物較具抑制離體LDL氧化及抗氧化能力，myricetin-3-O-glucopyranoside (7)則具有最好之清除DPPH自由基能力，結果顯示出荷葉之抗氧化能力部分來自於其類黃酮類化合物，另外從荷葉具有清除超氧陰離子次區分層中分離鑑定出十二種化合物，包括五種前類胡蘿蔔素類、四種酚酸類、一種類黃酮類及兩種其他類化合物。 在抗發炎活性方面，荷葉總生物鹼萃取物與酚性、非酚性生物鹼區分層皆顯示出具有抑制內毒素誘導巨噬細胞之一氧化氮生合成作用，另外總生物鹼萃取物之DPPH與一氧化氮自由基之清除能力分別貢獻自酚性與非酚性生物鹼區分層，進一步以Sephadex LH-20管柱層析與製備型薄層層析後分離，由非酚性生物鹼區分層分離出四個aporphine類生物鹼化合物，包含有asimilobine (1)、roemerine (2)、lysicamine (3)、nuciferine (4)，由酚性生物鹼區分層分離出一個phenylisoquinoline類生物鹼化合物N-methylcoclaurine (5)。其中以asimilobine (1)與roemerine (2)具有顯著性抑制由 SNP所產生之一氧化氮自由基能力，僅有N-methylcoclaurine (5)具有DPPH自由基清除能力。N-methylcoclaurine (5) (EC50=6±1μM)亦能經由抑制iNOS蛋白質表現達到抑制一氧化氮生合成作用，並呈現一濃度效應，在非酚性生物鹼區分層中，roemerine (2) (EC50=21±4μM)與lysicamine (3)( EC50=25±5μM)對於iNOS與COX-2蛋白質表現與LPS組相較具有抑制之功效。 從調節血脂動物模式發現大憨蓮種與見蓮種能降低脂肪組織堆積及幫助總三酸甘油酯排除於糞便中，大憨蓮種亦能幫助膽固醇排除於糞便中，且桃園產荷葉中劑量組能降低AI(atherogenic index)值，高劑量組能降低總膽固醇濃度。在不易形成體脂肪功能評估模式下，結果顯示出餵食高熱量飲食十週後會造成體重與脂肪組織之累積，給予2.0%正丁醇區分層下可顯著性抑制脂肪組織之累積與降低血糖及胰島素之濃度，另外肝臟與血清中三酸甘油酯含量亦有顯著性降低。由微陣列資料顯示出荷葉正丁醇區分層抑制體內脂肪組織之累積與降低血清、肝臟中三酸甘油酯含量可能是經由活化脂肪酸氧化作用所造成，另外給予2.0%正丁醇區分層下改善因高熱量飲食下導致葡萄糖吸收不良與改善因肥胖導致高胰島素血症，與正調控glycolysis及insulin signalling途徑之基因相關。

Hypertension and cardiovascular diseases based on atherosclerosis are the leading cause of death in Taiwan. There are many reports about the healthy function of lotus leaf in old Chinese traditional herb book, especially for the function of regulating blood lipid. Therefore, this project aims to conduct bioassay-guided separation and identification of the effective constituents on the anti-oxidative and anti-immflamatory activities, and to understand its functional property. Furthermore, the high fat diet model was used to investigate the blood lipid modulatory effect of ethanolic extract of local lotus leaf in hamsters. Moreover, the high energy diet model was used to estimate anti-obesity effect of n-butanol fraction of local lotus leaf in SD rats.We expect to understand its functional property and to enhance the value of local lotus leaf. The DPPH scavenging effect, the inhibition of human low-density lipoprotein oxidation and antioxidative contents were employed for the activity-guided purification to identify the antioxidant components of lotus leaves (leaves of Nelumbo nucifera Gaertn.). The methanolic extract of lotus leaves (LLM) was separated into ethyl acetate (LLME), n-butanol (LLMB), and water (LLMW) fractions. LLME and LLMB exhibited greater capacity to scavenge DPPH radical, delayed LDL oxidation and had higher antioxidative contents than LLMW. Seven flavonoids were isolated from both fractions by column chromatography. On the basis of 1D- and 2D-NMR experiments, and MS data analyses, these compounds were identified as catechin (1), quercetin (2), quercetin-3-O-glucopyranoside (3), quercetin-3-O-glucuronide (4), quercetin-3-O-galactopyranoside (5), kaempferol-3-O-glucopyranoside (6), myricetin-3-O-glucopyranoside (7). Quercetin and its glycosides (compound 2-5) exerted potent inhibition of LDL oxidation, whereas myricetin-3-O-glucopyranoside (7) showed stronger DPPH scavenging activity. These results indicated that the antioxidant capacity of lotus leaves partially relevant to its flavonoids. However, We have identified twelve compounds from the ethyl acetate effective subfraction. They included 5 apocarotenoids, 4 phenolics, 1 flavonoids and 2 others. The alkaloid extracts from leaves of lotus, including total alkaloids (TAs), non-phenolic alkaloids (NPAs), and phenolic alkaloids (PAs) revealed inhibitory effects on nitric oxide (NO) production in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages. The direct scavenging effects on NO and DPPH radicals by TAs were respectively attributed to NPAs and PAs. Five alkaloids were isolated from both fractions by column chromatography and identified as asimilobine (1), roemerine (2), lysicamine (3), nuciferine (4), and N-methylcoclaurine (5). Asimilobine (1) and roemerine (2) exhibited potency as direct NO scavengers while N-methylcoclaurine (5) exerted the highest DPPH radical-scavenging effect. N-Methylcoclaurine (5) also inhibited NO production with an EC50 value of 6±1μM through suppression of inducible NO synthase expression in a concentration-dependent manner in LPS-induced RAW 264.7 macrophages. However, roemerine (2) and lysicamine (3) inhibited NO production (EC50 values of 21±4 and 25±5μM) as well as iNOS and COX-2 expressions. In the lipid modulatory model, the results showed that diets contained Dahan-lien and Kien-lien extract can reduce adipose tissue and increase fecal excretive cholesterol. Moreover, the diet contained Dahan-lien extract can increase fecal excretive triglyceride. Furthermore, the medium dose of diet contained Dahan-lien planted in Taoyuan can effectively decrease the AI value, and the high dose one can reduce total plasma cholesterol. In anti-obesity model, the results revealed that a high energy diet to SD rats for 10 weeks led to significant increases of body and adipose tissue weight. Ingestion of 2.0% LLEB inhibited the development of increased blood glucose, insulin concentration and abdominal fat mass, while plasma and hepatic triglyceride concentrations were significantly lower in the 2.0% LLEB group than in the HE group. The microarray analysis of hepatic mRNA indicated that 2.0% LLEB up-regulated the expression of gene related to fatty acid oxidation, probably resulting in the suppression of the elevating of triglyceride concentration and abdominal fat mass. Gene expression of glycolysis and insulin signaling was also up-regulated in the 2.0% LLEB group suggesting that administration of LLEB could ameliorate obesity-induced hyperinsulinemia as well as the decrease of blood glucose.