Pharmacodynamics of frigid zone plant Taxus cuspidata S.et Z.against skin melanin deposition,oxidation,inflammation and allergy

Background:Taxus cuspidata S.et Z.is a precious species of frigid zone plant belonging to the Taxaceae family,which possesses anticancer,anti-inflammatory,hypoglycemic,and antibacterial pharmacological properties.While taxane extracted from Taxus chinensis has been reported to elicit antioxidant activities,whether Taxus cuspidata S.et Z.has skin-protective actions against injuries remained unknown.This study aims to explore the pharmacological effects of three Taxus extracts on skin melanin deposition,oxidation,inflammation,and allergy so as to provide new ideas for the prevention and treatment of various diseases related to skin damage.Methods:Skin melanin deposition was evaluated by measuring melanin content in the skin of guinea pigs by alkali lysis method.Antioxidant capacity was evaluated by measuring superoxide dismutase(SOD)concentration and glutathione(GSH)content in skin tissue homogenates of Kunming mice by SOD assay kit and micro reduced GSH assay kit.The quantitative real-time polymerase chain reaction(qRT-PCR)and western blotting were used to examine the levels of both SOD and recombinant glutathione peroxidase 4(GPX4).Skin inflammation was evaluated by xylene-induced ear swelling test and egg-white-induced paw swelling test in mice.In a mouse model of skin allergy induced by 4-aminopyridine(4-AP),allergy was determined by licking body counts and histamine concentrations in tissue homogenates using enzyme-linked immunosorbent assay(ELISA)kits.Two proinflammatory factors tumor necrosis factor(TNF)-αand interleukin(IL)-1βwere measured by qRT-PCR.Hematoxylin and eosin(HE)staining was conducted to assess the degree of skin lesion.Results:All three Taxus extracts including Taxus chinensis essential oil,Taxus chinensis extract and Taxus chinensis extract compound reduced the melanin deposits in the back skin relative to the non-treated control animals,of which Taxus chinensis essential oil produced the greatest effect.In contrast,the three Taxus extracts elevated SOD and GSH levels in the skin tissues,and the highest increase was seen with Taxus chinensis essential oil.Three Taxus extracts,especially Taxus chinensis essential oil,effectively reduce the rate of ear and paw swelling.All three Taxus extracts reduced the number of body licks,the levels of TNF-αand IL-1β,and the histamine content in tissue homogenates of mice and alleviated skin damage.Consistently,Taxus chinensis essential oil yielded the greatest magnitude of decreases.Conclusion:While all three Taxus extracts possessed the anti-skin melanin deposition,oxidation,and allergy properties,Taxus chinensis essential oil produced the superior effects.

A PRELIMINARY STUDY ON THE PHENOLOGY OF TAXUS CUSPIDATA SIEB.ET ZUCC.

In this paper, the phenomena of the life cycle of Taxus cuspidata Sieb. et Zucc., such as germination, leafing, flowing seed mature,new shoot growth etc. were observed and measured. As a result, the phenological regularity of Taxus cuspidata was found initially. At the same time,according to its five-day mean increment and height growth curve, the growing process of new shoot of Taxus cupidata was divided into three periods:slow period peak period and decline period. The paper supplied scientific data for the introduction of the rare and imininent variety, domestication,cultivating, expoiting, utilizing and protecting of Taxus cuspdata.

立地条件差异对天然东北红豆杉(Taxus cuspidata)种间竞争的影响

立地因子空间梯度特征是影响种间竞争的重要因素。通过研究主要竞争树种及竞争强度随海拔、坡向、坡位和坡度等立地因子变化的规律,揭示不同立地条件对天然东北红豆杉种间竞争的影响,为濒危东北红豆杉种群的保护与恢复提供理论指导。研究结果表明:东北红豆杉的种间竞争压力主要来自紫椴、臭冷杉、色木槭、红松、青楷槭、枫桦、花楷槭等7个树种,主要竞争树种以小径级个体居多。立地因子差异对种间竞争强度和主要竞争树种的排序有明显影响。东北红豆杉在阴坡受到的种间竞争压力是阳坡的3.27倍,主要树种的竞争强度显著高于阳坡,竞争树种的排序发生更迭;随着坡位的提升,东北红豆杉受到的种间竞争压力明显增加,主要竞争种类和强度也发生变化;坡度增加,种间竞争强度增加,主要竞争树种排序发生变化;海拔对种间竞争的影响总体不大,但影响主要竞争树种的竞争强度和排序。

天然东北红豆杉(Taxus cuspidata)种内和种间竞争

竞争是植物种内和种间关系的主要形式之一。通过对黑龙江穆棱东北红豆杉自然保护区的95株东北红豆杉对象木及980株竞争木的调查,运用Hegyi的单木竞争指数计算分析了东北红豆杉的种内和种间竞争强度。东北红豆杉的种内竞争强度不大,占总竞争的4%。竞争压力更多的来自于种间竞争,占总竞争的96%。与东北红豆杉竞争激烈的树种主要是冷杉、紫椴、色木槭和红松等地带性植被的优势种。随着东北红豆杉胸径的增大,所受到的竞争压力逐渐减小,胸径在20cm以前所受到的竞争压力最大,竞争强度与对象木胸径符合幂函数(CI=AD-B)关系。

东北红豆杉(Taxus cuspidata)细胞悬浮培养研究

用东北红豆杉的愈伤组织建立细胞悬浮系。培养研究发现，最适宜细胞生长的培养基仍为Ｂ５，最佳接种量应控制在２ｇ鲜重／瓶左右。在培养基中添加酪蛋白水解物比水解乳蛋白更利于悬浮细胞生物量的增加。培养基的ｐＨ值随细胞的培养周期而有相应的变化。

Optimization of RAPD Reaction System of Taxus cuspidate Sieb. et Zucc

[Objective] The research aimed to study the optimization conditions of RAPD reaction system of Taxus cuspidata.[Method] Using the leaves of Taxus cuspidata as materials,DNA was extracted by using modified CTAB method.The influences of template DNA concentration,primer concentration,dNTP concentration,Taq DNA polymerase amount and magnesium ion concentration on the amplification effects of RAPD of Taxus cuspidate were analyzed.[Result] Through analysis and comparison of various factors,the optimized reaction system was established as follows：in a total volume of 20 μl,containing 10 ng DNA,2.0 mmol/L Mg2＋,0.2 mmol/L dNTPs,15 pmol/μl random primer,1.0 U Taq polymerase,2 μl 10×buffer,and the supplementary ddH2O.[Conclusion] The research provided technical support for further discussion on the related studies with the sexual identification of Taxus cuspidate.

Study on the Browning in Cell Suspension Culture of Taxus cuspidata

[Objective] This study aimed to investigate the browning of T. cuspidata cells in suspension culture and provide the guidance for the cell suspension culture of T. cuspidata. [Method] T. cuspidata callus was used as experimental materials, to explore the effect of different medium, N/P ratio, pH, shaking speed, illumination time and light intensity and other factors on browning of T. cuspidata cells in suspension culture. [Result] Non-browning callus was transferred to 2MB5 medium （pH 7.0） for illumination culture at 22℃ under light intensity of 1 500 lx with shaking speed of 90 r/min for 24 h. Results showed that the cell browning was significantly inhibited. [Conclusion] This study laid the foundation for cell suspension culture of T. cuspidata and had important significance to the large-scale industrial production of paclitaxel.

东北红豆杉(Taxus cuspidata)大田扦插繁殖技术研究

大田插插繁殖东北红豆杉幼苗,是扩大繁殖东北红豆杉资源的最佳途径.经三年的田间实验,现已全部掌握了东北红豆杉的大田扦插繁殖技术,有的枝条在扦插当年即可获得成熟种子.

东北红豆杉(Taxus cuspidata)母树林基地管理系统的构建研究

东北红豆杉母树林基地管理系统由图像库、资料库、东北红豆杉资源贮量信息库和东北红豆杉母树林基地建设投入信息库组成 ,对规范东北红豆杉基地的管理有实际指导意义。

东北红豆杉(Taxus cuspidata)多倍体诱变初探

红豆杉(Taxus cuspidata)是一种天然珍稀抗癌药用植物,以东北红豆杉为材料,进行多倍体诱变研究,以期获得营养器官增大、药效成分提高的多倍体植株,改善因红豆杉资源有限造成紫杉醇稀缺的现状。采用改良初生分生组织处理法在0.2%秋水仙碱浓度下,不同处理时间(4d、6d、8d)处理东北红豆杉茎尖顶芽,对其进行多倍体诱变,并对诱变结果进行统计分析。结果表明,东北红豆杉的最佳诱变时间为6d和8d,诱变效果较好。与对照组相比,试验组生长受阻,叶形指数变小,叶色浓绿,叶片变厚且皱缩卷曲,具有明显的多倍体特征。

东北红豆杉(Taxus cuspidata)中四种紫杉烷类化合物抑制妇科肿瘤细胞增殖活性研究及其作用机制的初步探讨

通过四唑盐(MTT)比色法检测东北红豆杉中四种紫杉烷类化合物对人子宫颈癌细胞、人子宫内膜癌细胞、人卵巢透明癌细胞和人卵巢囊腺癌细胞增殖的影响,不仅探讨了这些化合物作用的构效关系,还初步探讨了其抑制肿瘤细胞增殖的作用机制。结果发现9,10-去乙酰紫杉宁对人子宫颈癌细胞、人子宫内膜癌细胞和人卵巢囊腺癌细胞的增殖均有明显的抑制作用,与紫杉宁和1β-羟基紫杉宁即使在100μmol/L的高浓度下,对五种妇科肿瘤细胞的增殖不显示抑制活性;流式细胞学检查发现9,10-去乙酰紫杉宁作用后的HeLa细胞出现凋亡现象。因此推测:1.C-9,10位的羟基和C-5位的肉桂酰基侧链是该类化合物抑制肿瘤细胞增殖所必需的;2.9,10-去乙酰紫杉宁有可能是通过诱导肿瘤细胞凋亡来实现其抑制HeLa细胞的增殖作用。

Population structure and distribution pattern of Taxus cuspidata in Muling region of Heilongjiang Province, China

An investigation was conducted on distribution pattern, site condition and population structure of yew Taxus cuspidata Sieb. et Zucc. in Muling Forest Bureau of Heilongjiang Province, China in April, 2005. Results showed that yew is mainly distributed under the main storey of natural mixed forest of conifer and broadleaf, the soil moisture content of the yew site is high （40%-60%）, the pH value of soil is relatively lower （4.7-5.5）, and that the population structure of wild yew is not rational, belonging to the degeneration population, which is one of the reasons leading to the population decline. Although the site conditions of Muling area are suitable for the growth of wild yew, the population of wild yew shows a decline tendency, due to the fact that the middle-sized adult yew trees have been cut, young yews are often grazed by wildlife, and that the trunks of adult yew tend to be hollow.

Effect of La,Ce on Taxus Cuspidata Cell Growth,Biosynthesis and Release of Taxol

The effect of La, Ce was firstly tested on growth of the Taxus(T.) cuspidata cell suspensions, biosynthesis and release of taxol. The results indicate that the growth pattern of T.cuspidata cells is altered significantly by adding high concentration of rare earth in the medium. The lag and exponential phases of cell growth are shortened, the stationary phase disappears and the biomass fluctuates periodically during the decline phase. The rare earth compounds added in the exponential phase obviously increase the taxol biosyntheis and release yields of T.cuspidata cells, and the supplement of carbon source in the medium containing rare earth is also favorable to taxol biosynthesis.

Effect of elicitors, precursors and metabolic inhibitors on paclitaxel production by Taxus cuspidata cell culture

In this study, using Taxus cuspidata as a raw material, we obtained stable high-yielding cell lines by subculturing and quantified paclitaxel content using ultra- sonic extraction combined with TLC-UV spectrophotom- etry. In single factor and multiple factors tests to optimize design and study the effects of elicitors, precursors, and metabolic inhibitors on paclitaxel production by Taxus cuspidata cells, paclitaxel production reached 4.32 mg/L when 100 μmol/L methyl jasmonate, 20 mg/L salicylic acid, 400 mg/L phenylalanine and 2 mg/L gibberellin （GA3） were added to the culture medium of suspension cells. When adding metabolic adjustment factors on the 7th day of culture, extra- and intracellular paclitaxel production was the highest at 4.855 mg/L, paclitaxel release rate was 10.48 %, fresh mass and paclitaxel production of cell increased, respectively, by 6.08 and 11.57 %. By controlling the anabolism of paclitaxel, paclitaxel yield was significantly improved.

The selection and stability analysis of stable and high Taxol-producing cell lines from Taxus cuspidata

In this study to screen for stable, high Taxolproducing cell lines（CL5, CL12, and CL21） of Taxus cuspidata, stem tissues were used to induce calli, which were then subcultured nine times to establish suspension cell cultures. From 97 cell lines obtained from conditioned cultures, 10 cell lines with high Taxol content were selected. Stability analyses on solid and liquid B5 media were then used to obtain lines that stably produced high levels of Taxol. Fresh biomass and Taxol production of the ninth generation became stable. Taxol content of selected CL5, CL12, and CL21 samples was 0.0448, 0.0477, and0.0428% of dry mass（DW）, respectively. Proliferation of CL5, CL12 and CL21 was 346.3, 382.5, and 409.2%,respectively. From work over about 2 years, the three cell lines appear suitable for mass production of Taxol,promoting the industrialisation and commercial-scale production of Taxol using cell culture.

Ce^(4+)-Induced Apoptosis of Taxus cuspidata Cells in Suspension Culture

The standard detection hallmarks of apoptosis of Taxus cuspidata cells in suspension culture with Ce 4+ were studied. The condensation and margination of chromatin were observed under the electron microscopy. DNA fragmentation ranged 'DNA ladder' on agarose gel electrophoresis. TdT mediated dUTP nick end labeling (TUNEL) analysis of the cells reveals that the nuclear DNA strand breaks can be identified by labeling free 3′ OH termini. These results suggest that Ce 4+ can induce apoptosis of Taxus cuspidata cells and also indicate that there is a certain relationship between apoptosis and secondary metabolite product Taxol.

Studies on Factors Affecting the in vitro Germination and Growth of Taxus cuspidata Embryos

Factors affecting the in vitro germination and growth of Taxus cuspidata embryos were examined. DCR medium was the best among 6 basal media tested; embryos from stage II seeds were the optimal developmental stage for in vitro germination; as the seeds approached maturity, germinability of embryos decrease. Embryos cultured in darkness tended to form callus and decrease the frequency of germination; inoculation method obviously affected frequency of embryo germination. Embryos cultured in optimal conditions developed into seedlings in 2 months.

Metabolites isolated from Penicillium HDS-Z-1E, an endophytic fungal strain isolated from Taxus cuspidata and their activation effect of catalase

Objective:To study the compounds isolated from Penicillium HDS-Z-1E,an endophytic fungal strain isolated from Taxus cuspidata and their activation effect of catalase(CAT).Methods:The chemical constituents were isolated from Penicillium HDS-Z-1E,by using silica gel,Sephadex LH-20 and HPLC.The structural elucidations of five metabolites were elucidated on the basis of spectroscopic including 1 H-NMR,13C-NMR,HMBC and HSQC.Their activation sites of catalase have been investigated by molecular docking.Results:Five metabolites,compounds(1–5)were isolated from Penicillium HDS-Z-1E and identified as 4-hydroxy-4-methyltetrahydro-2H-pyran-2-one(1),4-hydroxymethyl-5,6-dihydro-pyran-2-one(2),5,6-dihydro-2-oxo-2H-pyran-4-carboxylic(3),N-acetyl-hydrazinobenzoic acid(4),and methyl 2-(2,5-dihydroxyphenyl)acetate(5).Conclusion:Compound 3 is a new compound.Compounds 3 and 4 may have potential activators of catalase,providing a theoretical basis for the development of CAT activators.

穆棱东北红豆杉林空间结构特征与更新计数模型

为了解东北红豆杉(Taxus cuspidata)种群空间结构、分布格局及更新幼苗情况,以穆棱东北红豆杉国家级自然保护区东北红豆杉群落为研究对象,建立300 m×400 m标准地,开展树种记录、生长调查及空间定位,计算角尺度、大小比数、混交度等空间结构参数,分析东北红豆杉种群空间结构、分布格局和种间关系,分别建立并比较东北红豆杉更新苗木的泊松、负二项、零膨胀泊松和Hurdle泊松4种计数模型。结果表明(:1)东北红豆杉种群空间结构单元以随机分布、高混交度、高大小比数为主,相对频率为0.619 6(;2)东北红豆杉种群在0~100 m尺度呈现聚集分布(;3)东北红豆杉种群在0~150 m尺度均表现为正相关,幼苗与母树在不同尺度下表现出不同相关性,与其他优势种群相关性不高(;4)东北红豆杉幼苗在0~25 m尺度聚集分布,最优更新计数模型为负二项模型。

东北红豆杉在北方沿海地区迁地保护研究

【目的】本研究旨在分析东北红豆杉在北方沿海地区的迁地适应生长规律,为迁地保护提供支撑。【方法】本研究在烟台沿海防护林体系下设置四种不同立地条件,进行东北红豆杉迁地保护造林试验,分析基本物候规律、保存率、形态指标等。【结果】7年生红豆杉即可开花、结实,平均造林成活率均大于80%,以平原海防林过渡区最高,大于94%。综合苗木质量来看,牟平区酒馆村和莱山区冶头村表现最好,昆嵛山次之,沿海基干林最差。红豆杉在不同立地条件下高生长规律差异明显,其中丘陵区表现最好,连年增长量大于10 cm,沿海基干林带表现最差,出现了负增长。【结论】7年生东北红豆杉可在烟台沿海防护林体系完成整个植物生活史,迁地保护成功。不同立地条件下适应能力有显著差异,东北红豆杉在平原海防林过渡区、丘陵区的迁地效果较好。红豆杉苗木质量与造林地立地条件存在不同程度的关联性,苗木质量与土壤理化性质正相关。红豆杉耐旱不耐涝,海岸带风蚀作用严重影响红豆杉生长。