南方红豆杉枝叶中药用抗癌活性物质含量

通过高效液相色谱（HPLC）,测定了人工栽培南方红豆杉枝叶中药用抗癌活性物质紫杉醇（taxol）、三尖杉宁碱（cephalomannine）和人工半合成紫杉醇的主要原料巴卡亭Ⅲ（baccatin Ⅲ）、10 去乙酰基巴卡亭Ⅲ（10-deacetylbaccatin Ⅲ,10-DAB）在一个生长季的含量变化.研究表明：从4月新枝叶萌发至11月枝叶基本停止生长,南方红豆杉枝叶中紫杉醇等药用活性物质含量季节性变化明显.其中紫杉醇和三尖杉宁碱含量的最高值都出现在5月;巴卡亭Ⅲ和10-去乙酰基巴卡亭Ⅲ含量的最高峰值分别出现在9月和4月.相关分析发现,南方红豆杉枝叶中4种药用活性物质之间有很好的相关性,其中紫杉醇与三尖杉宁碱含量呈显著正相关(P<0.05),三尖杉宁碱含量与10-去乙酰巴卡亭Ⅲ含量呈显著负相关(P<0.05).     

南方红豆杉人工引种及野生植株活性成分含量与分布

南方红豆杉是我国特有的濒危植物,因其具有抗癌药用价值而被过度采集,导致现存种群减少.采用超声辅助提取和HPLC检测人工引种及野生南方红豆杉植株中紫杉醇、7-木糖紫杉醇、三尖杉宁碱等活性成分含量,分析其在不同部位、年限和坡向的分布规律.结果表明:南方红豆杉人工引种后,植株中的紫杉醇、7-木糖紫杉醇和三尖杉宁碱含量变化明显,野生植株中紫杉醇含量显著高于人工栽培植株,约为栽培植株的1.8倍,其他2种物质含量以人工栽培略高.不同部位、冠层样品中3种活性物质含量差异显著,以茎皮、高冠层枝叶含量较高.不同年限南方红豆杉植株中紫杉醇、7-木糖紫杉醇和三尖杉宁碱含量以4年生植株较高,分别为0.08、0.91和0.32 mg·g-1.不同坡向植株中3种活性成分含量以阳坡植株较高,紫杉醇和7-木糖紫杉醇含量差异显著,三尖杉宁碱含量差异不显著.南方红豆杉中3种活性成分的积累与光照条件密切相关,栽培过程中适当增加光照有利于3种活性成分的积累.     

不同生境及采样部位对南方红豆杉活性成分含量的影响

采用HPLC法,对采自浙江、福建和江西7个产地以及不同坡向(阴坡和阳坡)、不同坡位(高、中、低坡位)的野生南方红豆杉[Taxus wallichiana var.mairei (Lemée et Lévl.)L.K.Fu et Nan Li]枝叶中紫杉醇、7-木糖紫杉醇和三尖杉宁碱含量进行了分析,并比较了南方红豆杉不同部位(茎皮和枝叶)和不同冠层中3种成分的含量;在此基础上对3种成分含量与环境因子(包括地理、气候及土壤因子)的相关性进行了分析.结果表明:采自阳坡的枝叶中3种成分的含量明显高于阴坡,不同坡向间紫杉醇和7-木糖紫杉醇含量差异显著(P＜0.05).采自不同坡位的枝叶中3种成分的含量均有一定差异,其中采自低坡位的枝叶中3种成分的含量均最高.不同产地间紫杉醇含量有极显著差异(P＜0.01)、7-木糖紫杉醇和三尖杉宁碱含量则有显著差异(P＜0.05);总体上看,采自福建建阳和江西景德镇的枝叶中3种成分的含量相对较高.南方红豆杉茎皮中3种成分的含量显著高于其枝叶;高冠层枝叶中3种成分的含量均高于低冠层枝叶,不同冠层间紫杉醇和7-木糖紫杉醇含量差异显著(P＜0.05).相关性分析结果表明:南方红豆杉中三尖杉宁碱含量与土壤中全N和土壤有机质含量呈极显著或显著的负相关性,与其他因子的相关性均不显著;而紫杉醇和7-木糖紫杉醇含量与所有因子均无显著的相关性;海拔与3种成分的含量均呈不显著的负相关性,提示低海拔可能有利于南方红豆杉中3种成分的合成及积累.     

分析紫杉醇与三尖杉宁碱的简易方法

以粒径单分散聚苯乙烯反相色谱填料为色谱柱固定相,即采用MKF-DK-RP型分析柱(300mm×7.8mm,8μm),流动相为乙腈-水溶液,流速为1.0mL/min,检测波长为229nm,建立了一种分析紫杉醇与三尖杉宁碱的简易方法。结果表明,本方法可使紫杉醇与三尖杉宁碱达到有效分离,且紫杉醇的检测浓度在0.0625~2.0mg/mL范围内线性关系良好(r=0.9999)。本方法简单,重复性好。     

细菌肥料Agr、PfPt和Mic对曼地亚红豆杉幼苗生长和次生代谢物含量的影响

运用单因素随机区组设计,在田间栽培条件下,对曼地亚红豆杉(Taxus media Rehder)1年生幼苗施用3种细菌肥料﹝放射性土壤杆菌肥料(Agr)、荧光假单胞菌肥料(PfPt)和微球菌肥料(Mic),浓度为2×107 CFU·mL-1,施肥2次﹞,对翌年生长期幼苗株高和冠幅的增长量变化以及枝叶中4种次生代谢物﹝紫杉醇、三尖杉宁碱、10-去乙酰紫杉醇和10-去乙酰基巴卡亭Ⅲ(10-DAB Ⅲ)﹞含量进行比较分析。结果表明：施用Agr、Mic和PfPt后的翌年11月份,曼地亚红豆杉幼苗株高和冠幅的增长量均大于CK(不施肥,对照)组,其中,施用PfPt后幼苗株高增长量最大,且显著高于CK组(P<0.05);施用Mic后幼苗的冠幅增长量最大,但与CK组间无显著差异(P>0.05)。施用Agr、Mic和PfPt后枝叶中紫杉醇、三尖杉宁碱和10-DABⅢ含量均显著高于CK组,其中,施用Mic后紫杉醇含量最高,施用PfPt后三尖杉宁碱和10-DAB Ⅲ含量最高,且总体上显著高于其他处理组;施用Mic后10-去乙酰紫杉醇含量最高,且显著高于CK组及其他处理组,而施用Agr和PfPt后10-去乙酰紫杉醇含量与CK组无显著差异。研究结果显示：施用Agr、Mic和PfPt均对曼地亚红豆杉幼苗生长以及枝叶中次生代谢物积累有一定的促进作用,但不同细菌肥料的促进效应存在差异,因此,在曼地亚红豆杉的栽培过程中应根据不同需求选择适宜的细菌肥料。     

代谢调节剂对紫杉醇及其相关化合物生物合成的影响

在诱导子添加的基础上研究了氯化氯代胆碱（CCC）和肉桂酸（CA）的浓度和时间因互对红豆杉悬浮培养细胞活力、三尖杉宁碱（Cephlomannine）和紫杉醇（Taxol）生物合成的影响,并进一步探讨了两者与前体的协同作用。实验表明：（1）氯化氯代胆碱、内桂酸对 细胞活力的影响均为：延滞期＞稳定期＞对数期;（2）d18加入氯化氯代胆碱10mg/L,肉桂酸20mg／L可使紫杉醇含量分别提高10,14倍;d18加入乞肥乞采抻填20mg/L可使三尖杉宁碱含量提高2倍,d12加入肉桂酸20mg/L可提高10倍;（3）40mg/L氯化氯代胆碱,20mg/L肉桂酸,5mg／L苯异丝氨酸,40mg/L甲瓦龙酸（MVA）内酯对提高紫杉醇产量较优;而10mg/L氯化氯代胆碱,20mg/L肉桂酸,10mg/L苯异丝氨酸,20mg/L 龙酸（MVA）内酯对三尖杉宁碱合成剂。     

南方红豆杉枝叶中6种紫杉烷类化合物含量季节变化

紫杉醇(taxol)是主要来源于红豆杉属植物的一种天然抗肿瘤药物,紫杉烷(taxane)是紫杉醇的代谢前体或支路代谢产物,同样具有开发成为抗肿瘤新药的潜质。本文采用高效液相色谱法研究了紫杉醇(Taxol)、10-去乙酰巴卡亭Ⅲ(10-DAB)、7-木-10-去乙酰紫杉醇(7-xyl-10-DAT)、10-去乙酰紫杉醇(10-DAT)、三尖杉宁碱(CE)和7-表-10-去乙酰紫杉醇(7-epi-10-DAT)6种紫杉烷类化合物在南方红豆杉枝叶中含量的季节变化,结果显示Taxol和10-DAT在8、9月份含量最低,10-DAB和7-xyl-10-DAT在8、9月份含量相对最高,Taxol含量季节变化和10-DAB呈负相关,与CE呈显著正相关。7-xyl-10-DAT含量季节变化和10-DAB、10-DAT分别呈正相关。本文为研究南方红豆杉中紫杉醇及相关紫杉烷的代谢、积累规律提供了依据,不但有助于阐明紫杉醇的生物合成的关键步骤及调控的生理机制,而且对红豆杉资源的深度开发具有指导意义。     

三种不同颜色果实的红豆杉树皮中紫杉醇含量比较研究

本研究比较了三种安徽宣城采集的不同颜色果实的红豆杉树皮中紫杉醇的含量。利用三氯甲烷料液比1:5,回流提取2 h,活性炭脱色后制备供试品溶液。采用HPLC法,Agilent HC-C18色谱柱(250 mm×4.6 mm,5μm),以甲醇-水-乙腈(24:30:36)为流动相,在227 nm波长下检测紫杉醇含量。检测发现红豆杉树皮中紫杉醇含量为0.000 564 mg/g,黄色果实红豆杉树皮中紫杉醇含量为0.000 785 mg/g,金黄色果实红豆杉树皮中紫杉醇含量为0.000 703 mg/g。安徽宣城发现的黄色果实红豆杉和金黄色果实红豆杉树皮中的紫杉醇含量比红豆杉高,初步明确了黄色果实红豆杉的药用价值,为进一步发掘其产品价值提供了依据。     

红豆杉低温半致死温度和低温胁迫下紫杉烷含量

通过测定低温胁迫下红豆杉电阻抗参数和紫杉烷含量变化,确定适合红豆杉低温半致死温度测定的电阻抗参数,探讨低温对红豆杉紫杉烷生物合成的影响,为扩大红豆杉引种范围及利用低温条件提高红豆杉中紫杉醇含量奠定了理论基础。以云南红豆杉、南方红豆杉(山西长治)、中国红豆杉、曼地亚红豆杉和东北红豆杉为材料,分别采用电导率法和电阻抗法测定上述树种低温半致死温度,并利用LC-MS法测定低温胁迫下红豆杉叶片中紫杉烷含量和脱落酸(abscisic acid, ABA)含量。结果表明,5种红豆杉中,中国红豆杉、南方红豆杉(山西长治)、东北红豆杉和曼地亚红豆杉均能耐受-14.668--9.106℃低温;云南红豆杉能耐受-8.802--3.521℃低温,高于其他4种红豆杉。电阻抗τ_m参数与电导法测定的5种红豆杉一年生枝低温半致死温度显著相关,相关系数为0.912。低温胁迫下,红豆杉一年生叶片中紫杉醇含量高于当年生叶片。随着处理温度的下降,云南红豆杉和东北红豆杉一年生叶片中紫杉醇含量显著增加,中国红豆杉当年生叶片中巴卡亭Ⅲ含量和10-去乙酰基紫杉醇含量显著增加。此外,红豆杉叶片中ABA含量随温度的...     

4种红豆杉培养细胞的生长和紫杉醇含量的比较(简报)

南方红豆杉、云南红豆杉、欧洲红豆杉和东北红豆杉细胞生长在45d的培养过程中基本至S型,在30d时生长速率最大,以后逐渐下降。随着细胞进入快速生长期,培养细胞中的紫杉醇含量也达到最大值,其中云南红豆杉含量最高（0.019％）,南方红豆杉含量居中（0.009％）,而欧洲红豆村和东北红豆杉含量最低（0.004％和0．006％）。     

The influence of ultraviolet radiation on the content of pharmacologically active taxoids in yew tissues

The aim of the work was the investigation of the influence of UV radiation on the taxoids contents in yew tissues. The experiment was performed using Taxus baccata var. Aurea Corr. twigs irradiated with UV C (lambda = 254 nm) and UV A (lambda = 366 nm) in comparison to control samples. Multistep procedure of sample preparation was applied before the analysis of paclitaxel and 10-DAB III: SPE using alumina - for purification from the chlorophylles, waxes and polar balasts as well as zonal micropreparative TLC on silica - for isolation of partially separated fractions. The quantitation of some taxoids in the isolated fractions was performed using RP-HPLC procedure in system C18/acetonitrile + water. The experiments with UV-A and especially UV-C radiation showed changes in concentrations of paclitaxel and its precursor-10-deacetylbaccatin III. The results can be utilized to increase the yield of the taxoids isolated for medicinal and practical purposes.     

远紫外辐射对紫杉幼苗针叶膜脂过氧化及内源保护系统的影响

实验室条件下用远紫外线(UV-BC)光源照射紫杉幼苗,随照射时间延长,针叶的离子渗出率、膜脂过氧化水平、组织自动氧化速率及H₂O₂含量显著增加,可溶性蛋白、抗坏血酸、类胡萝卜素和叶绿素含量下降,叶绿体光系统II电子传递活性显著下降,外源活性氧清除剂苯甲酸钠和抗坏血酸对针叶膜脂过氧化有抑制作用;甲基紫精和DDC对针叶膜脂过氧化有促进效果,远紫外线引起的紫杉伤害可能和针叶树的越冬光氧化伤害有类似之处.紫杉苗对紫外辐射的抗性远高于一般农作物.     

The effect of UV-B and UV-C radiation on Hibiscus leaves determined by ultraweak luminescence and fluorescence induction

The effects of UV-C (254 nm) and UV-B (280-320 nm) on chlorophyll fluorescence induction and ultraweak luminescence (UL) in detached leaves of Hibiscus rosa-sinensis L. were investigated. UL from leaves exposed to UV-B and UV-C radiation reached a maximum 72 h after irradiation. In both cases most of the light was of a wavelength over 600 nm. An increase in the percentage of long wavelength light with time was detected. UV radiation increased peroxidase activity, which also reached a maximum 72 h after irradiation. UV-B and UV-C both reduced variable chlorophyll fluorescence. No effect on the amount of chlorophyll or UV screening pigments was observed with the short-term irradiation used in this investigation.     

Seasonal changes in the concentrations of four taxoids in Taxus baccata L. during the autumn-spring period.

The concentrations of four common taxoids: baccatin III, paclitaxel, cephalomannine and 10-deacetylbaccatin III (10-DAB III) were measured in fresh needles and stems of Taxus baccata L. during the late autumn-spring period (November'96-April'97) which has not been investigated to date in this species. Baccatin III, paclitaxel and 10-DAB III were present on the surface of the twigs in concentrations of 8-26 micrograms/1000 g (fresh weight). Changes in the levels of baccatin III and paclitaxel inside the needles and stems showed similar trends over the investigated period. From November to March the total level of taxoids differed between the needles and stems, and were the same only in April. Total levels in fresh needles were stable from December to March. The highest concentrations of 10-DAB III in the whole analysed period in fresh stems were measured, as well as in the fresh needles except for samples collected in November and December when the levels of cephalomannine were higher. The concentrations of paclitaxel were usually the lowest. These results confirm that epigenetic factors--date of collection (and thus phyllogenesis) and kind of plant tissue--determine taxoid levels during the late autumn-spring period in T. baccata. The opposite patterns of changes for 10-DAB III and cephalomannine, especially in the fresh needles, suggest a possible role for 10-DAB III in the biosynthetic pathway to cephalomannine, a less polar taxoid with a side-chain at position C-13. As well, owing to the thermolability of taxoids, the influence of low temperatures in December and January could explain the highest observed concentrations of 10-DAB III in the fresh stems and needles, respectively.     

UV-C对紫杉针叶叶绿体膜脂过氧化及PSⅡ电子传递活性的影响

在实验室条件下,用12W·m^-2剂量的紫外线C(UV-C,254nm)辐射紫杉针叶离体叶绿体．结果表明。随辐射时间的延长,活性氧清除系统中类胡萝卜素(Car)、谷胱甘肽(GSH)含量和超氧化物歧化酶(SOD)活性有不同程度的下降;脂质过氧化产物丙二醛(MDA)含量和膜相对透性有不同程度的增加;光系统Ⅱ(PSⅡ)电子传递活性显著下降,这种下降与光合活性光(PAR)强度呈反比;叶绿素对UV-C辐射不敏感．根据以上结果推测,UV-C辐射诱导叶绿体膜脂过氧化是导致PSⅡ电子传递活性下降的原因之一．     

东北红豆杉可再生部位紫杉醇含量时空动态变化规律

利用ODS-C18反相高效液相色谱体系研究了东北红豆杉(Taxus cuspidata)叶、茎、果肉及种子中紫杉醇含量的时空动态变化。结果表明,东北红豆杉的4个部位均有紫杉醇存在,但存在的时间和部位具有较大差异。紫杉醇的含量最大值为0.043mg.g-1(FW)出现在10月份种子中,最小值0.001mg.g-1(FW)出现在11月份果肉中。叶与茎中紫杉醇的含量变化较为一致,叶和茎中紫杉醇含量在7月份达到最高,分别为0.032mg.g-1(FW)和0.029mg.g-1(FW),在8月份降至最低,分别为0.011mg.g-1(FW)和0.010mg.g-1(FW)。在新叶萌发期,老茎叶比新茎叶中紫杉醇的含量高约2.5倍。     

东北红豆杉可再生部位紫杉醇含量时空动态变化规律

利用ODS-C18反相高效液相色谱体系研究了东北红豆杉(Taxus cuspidata)叶、茎、果肉及种子中紫杉醇含量的时空动态变化。结果表明, 东北红豆杉的4个部位均有紫杉醇存在, 但存在的时间和部位具有较大差异。紫杉醇的含量最大值为0.043 mg.g-1 (FW)出现在10月份种子中, 最小值0.001 mg.g-1 (FW)出现在11月份果肉中。叶与茎中紫杉醇的含量变化较为一致, 叶和茎中紫杉醇含量在7月份达到最高, 分别为0.032 mg.g-1 (FW)和0.029 mg.g-1 (FW), 在8月份降至最低, 分别为0.011 mg.g-1 (FW)和0.010 mg.g-1 (FW)。在新叶萌发期, 老茎叶比新茎叶中紫杉醇的含量高约2.5倍。     

Action of UV radiation on the surface of mammalian immunocompetent cells. III. Ultrastructural changes in the glycocalyx of mouse lymphocytes

The outer perimembrane layer of murine spleen lymphocytes were studied with electron microscopy. Mice were treated with short-wave (254 nm) and long-wave (365 nm) ultraviolet radiation in isoeffective lethal doses. The other perimembrane layers were stained with Alcian blue and Ruthenium red. The 254 nm UV treatment decreased the level of dye--sorption by these perimembrane layers, whereas no such effect was obvious after the 365 nm UV treatment.     

Variation of taxane content in needles of Taxus x media cultivars with different growth characteristics

Needles from 17 different Taxus x media cultivars, belonging to 4 groups showing different growth characteristics, were analyzed using high performance liquid chromatography for their content of 10-deacetylbaccatin III, baccatin III, cephalomannine and paclitaxel (Taxol). The 4 Taxus x media cultivar groups were: 1.) medium to fast growing and upright form; 2.) slow growing and upright form; 3.) fast growing and spreading form; and 4.) slow growing and spreading form. The purpose of this study was to identify yew cultivars of fast growth rate, upright growth and high taxane content in their needles. The highest content of paclitaxel was found in 'Coleana' of group 1 (378 microg/g of the extracted dry weight). Three cultivars in group 1, 'Coleana', 'Stovekenii' and 'Hicksii', make good candidates for taxane extraction because of their high paclitaxel and 10-deacetylbaccatin III content, fast biomass accumulation and upright growing form. They are also good starting materials to develop alternative methods for the production of paclitaxel and its analogous compounds through modern biotechnology approaches.