【作者】 张永兵；

【导师】 岳永德； 汤锋；

【作者基本信息】 中国林业科学研究院 ， 生态学， 2009， 博士

【摘要】 为了推动我国丰富的竹叶资源的综合开发利用,本文以竹叶提取物对酪氨酸酶的影响作为评价指标,筛选高活性的竹种。采用活性追踪,利用现代色谱和波谱学手段,分析具有抑制酪氨酸酶活性的供试竹种的竹叶化学成分,分离并鉴定高活性化合物,进一步研究高活性化合物对黑色素瘤细胞的影响,为色素型皮肤病的治疗、化妆品、生物农药、食品添加剂等提供参考。主要研究结果如下:1.竹叶提取物对酪氨酸酶活性的影响在浓度为2g?L-1时,研究了34种竹叶提取物对酪氨酸酶的影响,发现毛环竹、翠竹、高节竹和阔叶箬竹等4种竹叶提取物对酪氨酸酶活性抑制性较强,这4种竹叶提取物和曲酸的IC50依次为: 7.88 g?L-1、6.83 g?L-1、5.24 g?L-1、4.93 g?L-1和0.08 g?L-1。2.阔叶箬竹竹叶中抑制酪氨酸酶活性成分的分离、鉴定通过活性追踪方法,采用波谱分析,从阔叶箬竹竹叶中分离并鉴定出对酪氨酸酶有抑制活性的化合物对羟基肉桂酸乙酯,其IC50=0.219g?L-1。分析其抑制机理,可知其抑制双酚酶是混合型抑制。3.阔叶箬竹竹叶的化学成分分析对具有抑制酪氨酸酶活性和经济价值较高的阔叶箬竹竹叶的化学成分进行分析,初步分离获得5种化合物包括苯甲酸、邻羟基苯甲酸、2-羟基-4-甲氧基-苯乙酮、7-羟基-4’-甲氧基-异黄酮和异鼠李素。4.对羟基肉桂酸乙酯对黑色素瘤细胞酪氨酸酶的抑制作用对羟基肉桂酸乙酯对黑色素瘤细胞酪氨酸酶的抑制作用测定结果表明,在添加药物24h后,从竹叶中提取的对羟基肉桂酸乙酯和化学合成的对羟基肉桂酸乙酯对酪氨酸酶的抑制差异显著,但48h和72h后,总体差异不显著。竹叶提取的对羟基肉桂酸乙酯和曲酸相比较:在添加药物24h和72h后,曲酸显著高于竹叶提取的对羟基肉桂酸乙酯;在添加药物48h后,两者差异不显著。同一浓度下的同一种药物总体上随着给药时间延长抑制率随之增加。给药时间相同,同一种药物随着浓度增加,抑制能力随之增强。在添加药物24h后,这三种药物对黑色素瘤细胞酪氨酸酶的抑制率较低,呈现一定的波动。给药48h后,曲酸、从竹叶提取的对羟基肉桂酸乙酯和化学合成的对羟基肉桂酸乙酯IC50分别为48.53 mg?L -1、75.75 mg?L -1和67.13 mg?L -1。给药72h后,曲酸、竹叶提取和化学合成的对羟基肉桂酸乙酯IC50分别为24.45 mg?L-1、61.95mg?L-1和56.06mg?L-1,这三种药物抑制能力从强到弱排序为:曲酸、化学合成的对羟基肉桂酸乙酯和竹叶提取的对羟基肉桂酸乙酯。随着给药时间延长,这三种药物的IC50均降低。5对黑色素瘤细胞增殖率的影响利用CCK-8试剂盒测定羟基肉桂酸乙酯对黑色素瘤细胞增殖的抑制率,发现在低浓度时,竹叶提取和化学合成的对羟基肉桂酸乙酯差异显著,但在高浓度时,它们差异就不显著。浓度在20mg?L-1时,曲酸显著高于化学合成的对羟基肉桂酸乙酯,但在5mg?L-1、10mg?L-1、50mg?L-1和100 mg?L-1等浓度下,两者的差异不显著。浓度在50mg?L-1和10 mg?L-1时,曲酸显著高于竹叶提取的对羟基肉桂酸乙酯,但在5mg?L-1、20mg?L-1和100mg?L-1等浓度下,两者的差异又不显著。从竹叶中提取的对羟基肉桂酸乙酯、化学合成的对羟基肉桂酸乙酯和曲酸的IC50分别为:30.262 mg?L-1、35.473 mg?L-1和19.325 mg?L-1,但它们的IC90比较接近,分别为117.51 mg?L-1、114.62 mg?L-1和115.54 mg?L-1。总体上,这三种药物在低浓度时差异显著;但在高浓度时,差异不显著。添加竹叶提取的和化学合成的对羟基肉桂酸乙酯、曲酸均可在一定程度上干扰细胞增殖,使细胞生长受到抑制。细胞生长受抑制和受伤害程度随着药物浓度增加和时间的延长而愈加严重。更多还原

【Abstract】 In order to promote the use of abundant bamboo resources in China, Some bamboo species with high inhibitory activity were selected through the effect of bamboo leaves extractive on the tyrosinase and some highly active compounds were seperated and identified by modern chromatography and spectroscopy. The analysis of the effect of the active compounds on the melanoma cells and the chemical composition of bamboo leaves with high inhibition will provid basic research for the treatment of skin pigment, cosmetics, biological pesticides, food additives and so on.The main results were as follows:1. The study for the tyrosinase inhibitory activity of extracts of the 34 kinds of bamboo leaves showed that Indoealamus latifotius, Phyollostachys promlnens,Sasa pygmaea and Phyllostachys meyeri exhibited stronger inhibition on the concentration of 2 g?L-1 and the IC50 of the aboved four bamboo leaves and kojic aicd were 7.88 g?L-1,6.83 g?L-1,5.24 g?L-1,4.93g?L-1 and 0.08 g?L-1.2. ethyl-p-hydroxycinnamate with higher inhibitory activity on tyrosinase was extracted from the leaves of Indoealamus latifotius through bioassay-guided and identified through modern spectroscopy.IC50 of ethyl-p-hydroxycinnamate was 0.22g?L-1. Ethyl-p-hydroxy- cinnamate exhibited mixed-competitive.3. The leaves of Indoealamus latifotius with higher inhibitory activity on tyrosianse and economic value were separated and five compounds were isolated:benzoic acid, 2-Hydroxy -benzoic acid, 2’-hydroxy-4’-methoxy-acetophenon,7-hydroxy-4’-methoxy isoflavone, Isorhamnetin.4. Effect of ethyl-p-hydroxycinnamate on the tyrosinase in melanoma cellsComparing with the ethyl-p-hydroxycinnamate extracted from bamboo and the chemosynthetic showed significant difference after adding drugs 24h but no significant difference affter adding drugs 48h and 72h.Comparing with the ethyl-p-hydroxycinnamate extracted from bamboo and kojic acid showed kojic acid was significant higher than the ethyl-p-hydroxycinnamate affter adding drugs 24 and 72h but no significant difference affter adding drugs 48h.The inhibition of every drug increased with the concentration and the deliver time in general.The inhibition of every drug increased with the concentration at the same deliver time. Adding drugs 24h later, inhibitiones ratio of above three drugs of above drugs on the tyrosinase of melanoma cells were relatively low and showed fluctuated. The IC50 of kojic acid and ethyl-p-hydroxycinnamate extracted from bamboo and the chemosynthetic were 48.53 mg?L-1,75.75mg?L-1 and 67.13mg?L-1 adding drug 48h later and 24.45 mg?L-1,61.95 mg?L-1 and 56.06 mg?L-1 after adding drug 72h. The order of the inhition of three drugs was kojic acid, ethyl-p-hydroxycinnamate extracted from the chemosynthetic and bamboo. The IC50 declined with the deliver time.5. Effect of ethyl-p-hydroxycinnamate on the appreciation rates of melanoma cellsThe determination of the inhibition on melanoma cells by CCK-8 showed significant difference when the concentrationes were 5mg?L-1,10mg?L-1 and 20 mg?L-1 comparing with the ethyl-p-hydroxycinnamate extracted from bamboo and the chemosynthetic but they were on the contrary when the concentriones were 50mg?L-1and 100 mg?L-1.The inhibition was higher than the chemosynthetic ethyl-p-hydroxycinnamate when the concentration was 20mg?L-1 but they were on the contrary when the concentriones were 5mg?L-1,10 mg?L-1 and 50 mg?L-1 and 100 mg?L-1. The inhibitiones were higher than the ethyl-p-hydroxycinnamate extracted from bamboo leaves when the concentration was 50mg?L-1 and 10mg?L-1, but they were on the contrary when the concentriones were 5mg?L-1,20 mg?L-1 and100mg?L-1.IC50 of the ethyl-p- hydroxycinnamate extracted from bamboo and the chemosynthetic ethyl-p-hydroxycinnamate and kojic acid were 30.262 mg?L-1,35.473 mg?L-1and19.325 mg?L-1. IC90 of three kinds of drug were closer and they were 117.51 mg?L-1,114.62 mg?L-1and 115.54 mg?L-1. In general, comparing with the aboved three drugs showed significant difference when the concentrationes were lower but no significant difference when the concentrationes were higher.Melanoma cells proliferation and growth were inhibited after adding ethyl-p-hydroxy- cinnamate extracted from bamboo leaves,the chemosynthetic ethyl-p-hydroxycinnamate and kojic acid and the extent of injury increased with concentration increasing and time.更多还原