UPLC法同时测定曼地亚红豆杉中3个有效成分的含量

目的:建立UPLC法同时测定曼地亚红豆杉中10-脱乙酰基巴卡亭Ⅲ(10-DABⅢ)、三尖杉宁碱和紫杉醇的含量。方法:采用Acquity BEH Shield RP 18色谱柱(2.1 mm×100 mm,1.7μm),流动相为水(A)-乙腈(B),梯度洗脱(0～8 min,78%A;8～11 min,78%A→65%A;11～44 min,65%A),流速为0.2 mL.min-1,检测波长为227 nm,柱温25℃。结果:本方法可在44 min内完成10-DABⅢ、三尖杉宁碱和紫杉醇3个成分的色谱分析,且各成分色谱峰之间具有良好的分离度;10-DABⅢ、三尖杉宁碱、紫杉醇的线性范围分别为0.010～0.100μg(r=0.9996)和0.003～0.030μg(r=0.9995),0.080～0.800μg(r=0.9992)和0.001～0.010μg(r=0.9997),0.020～0.200μg(r=0.9997)和0.004～0.040μg(r=0.9998);平均加样回收率(n=9)分别为99.8%,97.8%,100.4%。结论:以10-DABⅢ、三尖杉宁碱和紫杉醇为目标成分时,5月份为5年生曼地亚红豆杉的最佳采收期。该方法灵敏度高、准确、简便快速,可用于曼地亚红豆杉的质量控制。

Productions of Taxol and Related Taxanes by Cell Suspension Cultures of Taxus yunnanensis

A high taxol yield cell line of Taxus yunnanensis Cheng et L. K. Fu keeps a high taxol_producing level after successive subcultures for more than eight years. In this study, eight taxanes were isolated from the suspension cell cultures of this cell line. Based on NMR and MS analyses, and comparison with literature data and standards, their structures were determined to be 2α,5α,10β_triacetoxy_14β_propionyloxy_4(20),11_taxadiene (1), 2α,5α,10β_triacetoxy_14β_(2′_methyl)_butyryloxy_4(20),11_taxadiene (2), 2α,5α,10β_14β_tetra_acetoxy_4 (20),11_taxadiene (3, taxuyunnanine C), 2α,5α,10β_triacetoxy_14β_(2′_methyl_3′_hydroxy)_butyryloxy_4(20),11_taxadiene (4, yunnanxane) and its 3′_epimer (5), baccatin Ⅳ (6), baccatin Ⅲ (7) and taxol (8), respectively. Among those compounds, 3, 5, 6 and 7 were reported to be isolated from the suspension cell cultures of T. yunnanensis for the first time. TLC and HPLC analyses indicated that the chemical constituents of the culture solution were similar to those of cultured cells. Moreover, the highest taxol content of this cell line reached 0.3% and the cell line could be applied for a large_scale culture.

Cell Apoptosis Induced by Oligosaccharide in Suspension Cultures of Taxus chinensis

Taxol production of Taxus chinensis (Pilger) Rehd. var. mairei (Lemee et Lévl.) Cheng et L. K. Fu induced by oligosaccharide from Fusarium oxysporum f. vasinfectum (Atkinson) Snyder et Hansen was studied in suspension cultures, and it was found that oligosaccharide triggered cell apoptosis. Under transmission electron microscope the following morphological changes were observed: cell shrinkage, condensation of cytoplasm, nuclear fragmentation, and the increase of high electron density bodies in vacuole in great quantity. In oligosaccharide_treated cells, agarose gel electrophoresis revealed that DNA was digested into oligonucleosomal fragments that were times of 200 bp appearing as DNA ladders. Control cells were in normal physiological state, they were intact, abundant in organelle and with integral nucleus DNA, and the rate of taxol biosynthesis in these cells was very low. After the oligosaccharide to the culture system, the defense system of cells was elicited and the secondary metabolism was strengthened, i.e. phenolics were accumulated in the medium, the activity of polyphenol oxidase (PPO) was increased quickly and secondary wall of cells was thickened. The activity of L _phenylalanine ammonia lyase (PAL), the critical enzyme of the phenylpropanoid pathway, was increased promptly 1 h after elicitation. The rate of taxol production was improved sharply and the maximal taxol concentration at 72 h was six times that of control. Appearance of cell apoptosis was accompanied with the highest concentration of taxol in suspension cultures.

Taxol Production in Suspension Cultures of Taxus chinensis as Affected by the Combination of Nutrient Feed with Dissolved Oxygen Control

Influence of the combination of nutrient feed with dissolved oxygen control on taxol production in suspension cultures of Taxus chinensis (Pilg.) Rehd. was investigated in bioreactors. Addition of feeding medium with 20 g/L sucrose on day 16 enhanced both the biomass and taxol production in 5-L bioreactors. Further investigation of the fed-batch cultures in a 20-L bioreactor showed that cultivation under a low dissolved oxygen tension (DOT) of 20% for the entire culture resulted in the highest taxol content of 0.98 mg/g DW, while the taxol production was lower than that with 40% and 60% DOT. Moreover, taxol accumulation was remarkably improved by the cultivation of cells initially under DOT of 60% for 20 d followed by changing the DOT to 20% for another 12 d. By combining the use of these two strategies, a maximum taxol content of 18.7 mg/L was obtained in a 20-L bioreactor.

Isolation and Identification of a Taxol-producing Endophytic Fungus Identified from Taxus media

[Objective] The aim was to isolate and identify a taxol-producing endophytic fungus from Taxus media. [Method] 32 strains of endophytic fungi were identified form the inner bark of T. media,and their fermentation products were detected by high performance liquid chromatography (HPLC). [Result] Through the screening,a strain of taxol-producing endophytic fungi M57 was obtained,which could produce 45-50 μg/L of taxol,and M57 was defined as Rhizopus sp. through morphological observation and 18S rDNA sequence analysis. [Conclusion] The finding of Rhizopus sp. M57 provided a promising strain for producing taxol with taxol-producing fungi fermentation process.

Improved Production of Paclitaxel from Suspension Culture of Taxus chinensis var. mairei by in situ Extraction with Organic Solvents

The production of paclitaxel from suspension culture of Taxus chinensis var,mairei was improved by in situ extraction with organic solvents to avoid feedback repression and product degradation.Oleic acid and dibutyl phthalate were proved to be suitable solvents .The optimal volumetric percentage of organic solvents in the culture medium was found to be around 8%,and the favorable time for their introduction was at the exponential phase of cell growth,Paclitaxel production with the in situ extraction was ca 3-fold of that without extraction.

A new 3, 11-cyclotaxane diterpenoid from Taxus media Rehd. cv. Hicksii

The aim of current study was to investigate the chemical constituents of the needles of Taxus media. The isolation and purification of components were achieved by a series of chromatography including silica gel, Sephadex LH-20, MCI and reversed- phase HPLC. Their structures were indentified based on 1D, 2D NMR, and mass spectral analysis. Seven taxane diterpenoids were isolated and identified as ll3,2tt,9ct,1013-tetrahydroxy-5ct-cinnamoyloxy-3,11-cyclotaxa-4（20）-en-13-one （1）, 2it, 7[3, 9ct, 1013, 13tt-pentaacetoxytaxa-4（20）, 11-dien-Sct-ol （2）, 2-deacetoxy-5-decinnamoyltaxinine J （3）, taxuspine F （4）, taxuspinanane K （5）, taxchin A （6）, and baccatin IV （7）. Among them, 1 is a new compound and compounds 2, 5, 6 and 7 are isolated from Taxus media for the first time.

INVESTIGATING SILVICULTURAL CHARACTERISTICS OF YEW （TAXUS BACCATA L.） STAND USING FULL-CALLIPERING IN NORTHERN FORESTS OF IRAN

In this research, some of site and silvicultural characteristics of Yew stand in northern forests of Iran was studied. For this purpose, compartment number 16 of district 4 Shir-ghalaye was selected. According to research purposes, stands with an area of 20 ha were chosen in center of the compartment and was separated from map. Due to restricted area of region and according to few number of Yew trees in the stand, the full-callipering method was conducted for measuring quantitative and qualitative characteristics. In the stand diameter at breast height （D.B.H.）, height and canopy diameter of all Yew trees were measured. The results showed that the diameter distribution curve and height distribution curve of Yew trees were similar to even-age forests curve. But according to average annual increment, mixture type of Yew trees in stand, diameter distribution of other species and low rate regeneration of Yew, it can be concluded that the studied Yew stand, is an uneven-age stand and because of low rate regeneration in previous years the number of young Yew trees were reduced and even-age forest curve was formed subsequently. The maximum height and diameter of Yew trees were recorded 26.8 m and 112 cm, respectively. Also, the number of trees per hectare and basal area per hectare of Yew trees were calculated 14.55 and 3.23 m2 respectively.