【作者】 王芳；

【导师】 蒋跃明； 励建荣；

【作者基本信息】 浙江工商大学 ， 食品科学， 2008， 博士

【摘要】 我国具有丰富的桑叶资源;但传统的桑叶只用于养蚕,用途单一,出现了大量桑叶过剩现象。酪氨酸酶广泛存在于各种生物体内,是生物体内黑色素形成的关键酶;其抑制剂在医药领域用于治疗和预防黑色素瘤、黄褐斑等色素沉着性疾病,在化妆品领域用于人体肌肤的美白,在食品工业领域用于预防果蔬因褐变而失鲜变质,在农业领域因酪氨酸酶是昆虫赖以生存的关键酶而成为最有前景的生物杀虫药剂之一,因而酪氨酸酶抑制剂的开发有着广阔的应用前景。传统的酪氨酸酶抑制剂大多是化学合成,毒副作用明显。近年来,安全而有效的天然植物成为该类产品的发展方向;但有关桑叶中酪氨酸酶的抑制成分的研究还鲜见报道。本研究以浙江湖州产桑叶为试验材料,对桑叶中对酪氨酸酶活力具有抑制作用的成分进行了筛选,并对其作用机理进行了研究;同时,评价了其抗氧化能力和抑菌活性,并对相关活性成分的提取工艺进行了优化,以期实现桑叶的最大化利用。主要研究结果如下:1、在不同季节桑叶中,以秋季桑叶的醇提物对酪氨酸酶的抑制作用最强。以对酪氨酸酶的抑制率为指导,对秋季桑叶的醇提物依次用不同极性溶剂萃取、聚酰胺柱层析、硅胶柱层析、醇-水重结晶逐级分离,得到对酪氨酸酶活力具有显著抑制作用的成分,产率为0.025mg/g;其对酪氨酸酶的抑制作用属于可逆抑制。经过初步鉴定,该物质中含有黄酮类化合物。2、对桑叶中黄酮类物质的提取工艺进行了优化。超声波辅助提取、超高压提取和微波辅助提取可提高桑叶黄酮类化合物提取效果,其中超声波辅助提取法效果最好。确定优化条件为70%乙醇提取溶剂、1∶20料液比、200 W功率超声预处理10 min、70℃浸提1.5 h;桑叶黄酮提取得率为28.8 mg/g。经分离、结构鉴定,得到5种黄酮单体:芦丁（Rutin）、异槲皮甙（Isoquercitrin）、紫云英甙（Astragalin）、桑色素（Morin）和山奈酚（Kaempferol）。3、对所提取得到的黄酮类化合物对酪氨酸酶活力的影响进行了研究。桑叶黄酮类化合物表现出对酪氨酸酶单酚酶活性的抑制率(IC₅₀=200μg/mL)高于对酪氨酸酶二酚酶活性的抑制率(IC₅₀=300μg/mL);且桑叶总黄酮对酪氨酸酶的抑制作用显著高于各黄酮单体对酪氨酸酶的抑制作用,说明桑叶黄酮类化合物对酪氨酸酶的抑制作用存在各单体间的协同配伍作用。4、桑叶黄酮单体芦丁、异槲皮甙、紫云英甙、桑色素和山奈酚的混合物对酪氨酸酶的抑制机理研究表明,桑叶黄酮对酪氨酸酶的抑制效应属于可逆过程,说明桑叶黄酮和酶的结合导致酶活力受抑制,但并不导致酶的分子构象永久变化而失活。由桑叶黄酮的结构可推测,桑叶黄酮对酪氨酸酶的抑制作用与其分子中大量的还原性羟基与酪氨酸酶分子中Cu²⁺的络合有关,且其结构与酪氨酸酶底物的结构类似,均为对位苯酚的结构,可与酪氨酸酶竞争性结合,从而削弱了酪氨酸酶对酪氨酸及其系列氧化产物的催化氧化作用。桑叶黄酮对酪氨酸酶的单酚酶活性的抑制表现为既降低了其稳态酶活力,又延长了其迟滞时间;对酪氨酸酶的二酚酶活力表现为混合型抑制作用,说明桑叶黄酮可以和游离酶及酶-底物络合物结合;对游离酶的抑制常数（KI）和对酶-底物络合物的抑制常数（KIS）分别为176.01μg/mL和259.20μg/mL;KI＜KIS,说明底物对酶的抑制作用有明显的保护作用。5、由于酪氨酸酶抑制剂对酪氨酸酶抑制作用的机制主要有三个方面:结构与酶底物相似、络合了酪氨酸酶中的Cu²⁺以及清除了过氧自由基,终止了自由基链的引发,拮抗了氧对酪氨酸酶的激活,因而本研究还考察了桑叶黄酮的抗氧化能力。研究表明桑叶黄酮具有较强的抗氧化能力,说明其对酪氨酸酶的抑制作用与其能够终止自由基链的引发、拮抗氧对酪氨酸酶的激活密切相关。6、由于酪氨酸酶抑制剂主要应用的医药、化妆品、食品等领域,均需要抑制微生物的生长繁殖;所以对桑叶黄酮的抑菌活性进行了考察。试验结果表明桑叶黄酮具有一定的抑菌作用,其最小抑菌浓度（MIC）值分别为金黄色葡萄球菌0.0125 mg/mL、大肠杆菌0.025mg/mL、枯草芽孢杆菌0.05 mg/mL,有利于桑叶黄酮作为抑菌剂在相关领域的应用。更多还原

【Abstract】 Mulberry（Morus alba L.）leaves were collected from a commercial orchard in Huzhou,Zhejiang Province and then employed to select and identifiy tyrosinase inhibitors.The extraction efficiency of mulberry flavonoids was evaluated.Furthermore,the effect of mulberry flavonoids on tyrosinase activity in relation to their antioxidant abilities and antibacterial activities were investigated.The major results obtained in this study were as follows.1.The inhibitory extent of tyrosinase activity by mulberry leaves followed the harvest seasons,i.e.autumn＞summer＞spring.Mulberry leaves collected in autumn was extracted with 50%ethanolic,and then purified using different polar solvents,polyamide column chromatography,silica gel chromatography and recrystallization.The final extraction yield of the tyrosinase inhibitors from mulberry leaves was determined to be 0.025 mg/g.The major compounds of the mulberry extract were preliminarily identified as flavonoids.The inhibition of tyrosinase activity by mulberry extract exhibited a reversible effect. 2.The highest extraction yield of flavonoids from mulberry leaves was obtained by ultrasound assisted-extraction,followed by ultra high pressure extraction,microwave assisted-extraction,enzymatic assisted-extraction,alkali-solution plus acid-isolation and soxhlet extraction.The optimum ultrasound-assisted extraction conditions of flavonoids from mulberry leaves were determined to be 70%ethanol（solvent）,1:20 （material to solvent solution）,200 W（ultrasound power）and 10 min （ultrasound time）,following subsequent an extraction time of 1.5 h at 70℃,with an final extraction yield of 28.8 mg/g.Five flavonoids were purified and then identified as Rutin、Isoquercitrin,Astragalin、Morin and Kaempferol from the mulberry extract.3.Mulberry flavonoids exhibited a inhibitory effect on tyrosinase activity.The inhibitory effect of mulberry flavonoids on monophenolase activity was stronger than diophenolase activity of tyosinase.The 50% inhibition concentrations(IC₅₀)to monophenolase and diophenolase activities of tyosinase were 200 and 300μg/mL.It may be due to that the conformation of monophenolase was easier to change than that of diophenolase in the active sites of tyrosinase and a requirement of monophenolase for geometry and electronic effect was stricter than that of diphenolase,resulting in a decrease in the stability of the monophenolase.Furthermore,various flavonoid monomers showed a interaction effect on tyrosinase activity.4.The inhibition of the mixture of various mulberry flavonoid monomers on tyrosinase activity was reversible.The effects by combined flavonoid monomers did not result in a permenant change of conformation of tyrosinase but just cause the inhibition in the enzymatic activity.According to the flavonoid structure,the inhibitory effects of mulberry flavonoids on tyrosinase activity may be due to the complexation of OH present in the flavonoids and Cu²⁺present in the active sites of tyrosinase,or/and due to the similar structure of the flavonoids to the enzyme substrate tyrosinase.The mixture of various flavonoid monomers reduced the steady-state of and extended the lag time of monophenolase activity of tyosinase. While the mixture of various flavonoid monomers exhibited a mixed-type effect on diphenolase activity of tyosinase.The inhibition constants for the inhibitor binding with free enzyme（E）,KI and enzyme-substrate（ES） complex,KIS were estimated to be 176.01 and 259.20μg/mL, respectively.5.Flavonoids present in mulberry leaf exhibited a strong antioxidant ability,which were related to their inhibition of tyrosinase activity.6.Mulberry flavonoids had a distinct antimicrobial activity.The minimal inhibitory concentrations（MIC）of mulberry flavonoids against Staphylococcus.aureus,Escherichia coli and Bacillus subtilis were 0.0125,0.025 and 0.05 mg/mL,respectively.更多还原