桑白皮萃取物之抗氧化活性及其對中波長紫外線輻射傷害之保護作用

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Title:	桑白皮萃取物之抗氧化活性及其對中波長紫外線輻射傷害之保護作用
Other Titles:	The Antioxidant Activities of Extracts from Root Bark of Morus alba and Its Protective Effects from the UVB Irradiation
Authors:	徐雅淳 Hsu, Ya-Chun
Contributors:	喬長誠;蘇正德 Chyau, Charng-Cherng;Su, Jeng-De 東海大學食品科學系
Keywords:	桑白皮;抗氧化;人類角質細胞株HaCaT;蛋白質體學
Date:	2008
Issue Date:	2011-03-11T06:07:47Z (UTC)
Abstract:	桑白皮（Root bark of Morus alba），為桑科（Moraceae）植物桑樹（Morus alba L.）去除栓皮的根皮。桑白皮在傳統療效上常被用來當作利尿、降壓、鎮靜的藥材，而根據文獻指出桑白皮因富含類黃酮及其衍生物，因此具有抗氧化、抗菌、抗發炎和抑制酪胺酸?等活性。本實驗首先以不同方法進行萃取，萃取方法包含甲醇萃取、水萃取、正己烷萃取以及三種不同條件的超臨界流體萃取（40 °C, 60 °C 與80 °C /5000psi，二氧化碳中含10 ?酒精修飾劑，分別以SFE-1、SFE-2以及SFE-3表示）。利用產率、總酚含量、總類黃酮含量、抑制酪胺酸?（Tyrosinase）活性以及抗氧化性實驗來比較這六種不同之萃取物特性。抗氧化實驗方面包含DPPH自由基清除能力、還原力和總抗氧化力分析。根據結果顯示，還原力、總抗氧化能力及抑制酪胺酸酵素等實驗，以甲醇萃取物的能力最佳。DPPH自由基清除能力實驗中，當甲醇萃取物濃度在10mg/mL時可達到90? DPPH自由基清除能力，以及濃度在10 mg/mL時，經30 mJ/cm2 UVB照射plasmid DNA之下，即可讓supercoiled DNA回復達 92.1%，綜合以上實驗結果，選用甲醇萃取物進行後續的細胞實驗。細胞實驗分成兩部分進行探討，第一部分針對甲醇萃取物對於人類皮膚角質細胞株HaCaT中觸? (catalase)、超氧歧化?（superoxide dismutase）和麩胱甘?轉化?（glutathione-S-transferase）等抗氧化酵素活性的影響，依據結果顯示桑白皮甲醇萃取物濃度在500 μg/mL時皆可有效的提高觸?（P ? 0.01）、超氧歧化?（P ? 0.001）和麩胱甘?轉化?（P ? 0.01）的酵素活性。第二部分則探討甲醇萃取物是否具有保護經中波紫外線（ultraviolet B, 290-320 nm）照射後的人類皮膚角質細胞株HaCaT中抗氧化酵素的作用，結果顯示桑白皮甲醇萃取物濃度在500 ?g/mL與經30 mJ/cm2UVB傷害的控制組相比時可有效的提高觸?（P ? 0.01）、超氧歧化?（P ? 0.001）和麩胱甘?轉化?（P ? 0.05）的酵素活性。桑白皮甲醇萃取物中成分的純化、分離以及鑑定部分，利用高效能液相層析儀以及離子阱質譜儀分析，初步鑑定為桑白皮甲醇萃取物含有cyclomorusin衍生物、moracin以及moracin之衍生物。此外利用二維電泳實驗來探討甲醇萃取物對於經UVB照射的HaCaT 細胞中蛋白質體表現的差異，二維電泳後，經由MALDI-Q-TOF進行蛋白質成分鑑定並與資料庫比對，已鑑定出二十二個蛋白質，已知其中十二個蛋白質具功能性或特性，且其中九個蛋白質具有正調控作用，包括Cyclin-dependent kinase inhibitor 1B、DNA-binding protein inhibitor ID-1、Geminin、Sulfotransferase 1A1、Malate dehydrogenase, mitochondrial precursor、Heterogeneous nuclear ribonucleoproteins A2/B1、Protein Wnt-2 precursor 、T-complex protein 1 subunit和Nuclear receptor ROR-alpha，三個蛋白質具有負調控作用，包括Serine/threonine/tyrosine-interacting-like protein 1、Protein FAM118A和Hypermethylated in cancer 1 protein，其中重要之蛋白質Cyclin-dependent kinase inhibitor 1B、Sulfotransferase 1A1和Malate dehydrogenase, mitochondrial precursor為與細胞內抗氧化活性有密切相關。綜合以上結果，認為桑白皮甲醇萃取物之抗氧化活性，具保護人類皮膚角質細胞株HaCaT免於自由基之傷害，且能提升其細胞內抗氧化酵素活性，其影響細胞內抗氧化酵素活性主要與細胞內Cyclin-dependent kinase inhibitor 1B、Sulfotransferase 1A1和Malate dehydrogenase, mitochondrial precursor等蛋白質之表現具相關性。 Root bark of mulberry tree (Morus species, Moraceae), also called ‘Sang Bai Pi’ in Chinese, has long been used as a folk medicine. It is an herb recorded in the Pharmacopoeia for removing heat from the lung, relieving asthma and inducing diuresis. Plants of this genus are known to be rich in flavonoids, a group of chemicals shown to have potent antiviral, antioxidant and tyrosinase inhibitory capabilities.In this study, the antioxidant effects of six different solvent extracts from four kinds of extract solvents, i.e. methanol, water, n-hexane and supercritical fluid (carbon dioxide containing 10% ethanol modifier and 5000 psi at 40 °C, 60 °C and 80 °C, repectively) were chosen and the effects of these solvents on antioxidant components from the root bark extract of Morus alba （RBMA）were investigated. Methanol extract showed with the highest free radical scavenging effect in 90.96% at 10 mg/mL of concentration and exhibited an effective capability on DPPH, total antioxidant activity, reducing power and lipid peroxidation inhibition, respectively, in a does-dependent manner. The extract possessed total phenols at the level of 12.06 ± 0.41 mg gallic acid equivalent/ 100 g and total flavonoids at the level of 65.35 ± 1.06 mg quercetin equivalent/100 g. Antioxidant enzyme activities of superoxide dismutase (SOD), catalase (CAT) and glutathione S-transferase (GST) in human keratinocyte HaCaT cells were increased effectively when extract was applied in concentrations of 250 to 500 μg/mL before the UVB irradiation. In chemical composition analysis, compositions from methanol extract of RBMA were isolated and identified by high performance liquid chromatography (HPLC) with photodiode array detector (PDA) and ion trap mass spectrometry in a negative or positive electrospray ionization (ESI) mode. Three compounds were tentatively identified as cyclomorucin-derivative, moracin and moracin-derivative, respectively. Furthermore, we used proteomic approach for evaluating the relative regulation in protein expressions after treatment with RBMA and 30mJ/cm2 UVB in HaCaT cell. Totally 22 spots were identified by using MALDI-Q-TOF after 2D-gel electrophoresis. In which, total of nine spots were found with up-regulation abilities in protein expression, including Cyclin-dependent kinase inhibitor 1B, DNA-binding protein inhibitor ID-1, Geminin、Sulfotransferase 1A1, Malate dehydrogenase, mitochondrial precursor, Heterogeneous nuclear ribonucleoproteins A2/B1, Protein Wnt-2 precursor, T-complex protein 1 subunit and Nuclear receptor ROR-alpha. In which, three spots with down-regulation abilities in protein expression, include Serine/threonine/tyrosine-interacting-like protein 1, Protein FAM118A and Hypermethylated in cancer 1 protein, indicated with the possible actor of extracts on cell metabolisms and differentiations. In conclusion, the studies showed a greatest antioxidant capability of RBMA extract prepared from the extract solvent of methanol comparing to that extract solvents of water, n-hexane and supercritical fluids. The antioxidant enzymes activities, i.e. SOD, CAT and GST in human HaCaT keratinocytes cells were found with an enhanced effect from methanol extract treated cells before the UVB irradiation, shown a protection effect of methanol extract from UVB damage. Moreover, the protection effect of methanol extract of RBMA from UVB treated HaCaT cells by using 2D-gel electrophoresis analysis was pointed out a significant relationship to the regulation of the protein expression regarding to the antioxidant enzymes.
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