Taxus Chinensis Cell Cultures Research Articles

Elucidation of the Mechanism and Kinetics of Ultrasonic Extraction of Paclitaxel from Plant Cell Cultures of Taxus chinensis

Elucidation of the Mechanism and Kinetics of Ultrasonic Extraction of Paclitaxel from Plant Cell Cultures of Taxus chinensis

Kinetics, Mechanism, and Thermodynamics Studies of Vacuum Drying of Biomass from Taxus chinensis Cell Cultures

In this study, we investigated the kinetics, mechanism, and thermodynamics of vacuum drying and dehydration of plant cells of Taxus chinensis. The efficiency of vacuum drying increased with increases in the drying temperature (45–60°C). When the experimental data were fitted to typical kinetic models for drying, the Modified Page were the most appropriate. Thermodynamic parameters revealed the endothermic, irreversible, and spontaneous nature of the drying. The effective diffusion coefficient (3.6602 × 10−14−10.9810 × 10−14 m2/s) and mass transfer coefficient (2.2536 × 10−11−10.8110 × 10−11 m/s) increased with increasing drying temperature. The small Biot number (2.6167 × 10−2−4.1841 × 10−2) indicated that the process of mass transfer was externally controlled.

Adsorption Kinetics, Mechanism, Isotherm, and Thermodynamic Analysis of Paclitaxel from Extracts of Taxus chinensis Cell Cultures onto Sylopute

The adsorption characteristics of paclitaxel from extracts of Taxus chinensis cell cultures by the commercial adsorbent Sylopute were investigated using different parameters such as initial paclitaxel concentration, adsorption temperature, and contact time. The equilibrium adsorption data were best represented with the highest accuracy (r2 > 0.984, RMSD<0.0009) by the Langmuir isotherm. It was found that the adsorption capacity increased with increasing temperatures and the adsorption of paclitaxel onto Sylopute was favorable. The kinetic data fit the pseudo-second-order model well, and intraparticle diffusion played a dominant role in paclitaxel adsorption according to the intraparticle diffusion model. Thermodynamic parameters such as ΔH0, ΔS0, and ΔG0 were also calculated. The results indicate that the process of paclitaxel adsorption onto Sylopute is endothermic and nonspontaneous. The isosteric heat of adsorption, ΔHx (20–25 kJ/mol), was nearly a constant, even with variations in surface loading (qe: 3–9 mg/g), indicating a homogeneous surface.

Isotherm, Kinetic, and Thermodynamic Characteristics for Adsorption of 2,5-Xylenol onto Activated Carbon

The adsorption of the major tar compound, 2,5-xylenol, derived from the plant cell cultures of Taxus chinensis, onto activated carbon was examined at different initial 2,5-xylenol concentrations, durations, and temperatures. From the analysis of adsorption isotherms, the Langmuir isotherm model showed good fit to the equilibrium adsorption data. It was found that adsorption capacity decreased with increasing temperature, and the adsorption of 2,5-xylenol onto activated carbon was favorable. The obtained kinetic data for 2,5-xylenol adsorption with activated carbon agreed well with the pseudo-second-order kinetic model. By using intraparticle diffusion model, intraparticle diffusion and boundary layer diffusion did not play a dominant role in 2,5-xylenol adsorption. Thermodynamic parameters were calculated, which indicated that the adsorption was non-spontaneous, irreversible and exothermic nature. The isosteric heat of adsorption decreased with increase in surface loading, indicating a heterogeneous surface.

Development of a simultaneous extraction and acid hydrolysis process for recovery of paclitaxel from plant cell cultures

In this study, a microwave-assisted extraction (MAE)/acid hydrolysis simultaneous process was developed for recovering the anticancer agent paclitaxel from plant cell cultures of Taxus chinensis. The optimal pH of the extraction solution (90% aqueous methanol) for hydrolysis was 2.2 at fixed extraction time (6min), ratio of extraction solution to biomass (1:1, v/w), and extraction temperature (40°C). In the MAE/acid hydrolysis simultaneous process, the paclitaxel recovery was 2.2-fold higher than in the existing extraction methods. Regarding changes in the content of glycoside (7-xylosyl paclitaxel as sugar-binding paclitaxel) and paclitaxel depending on the inclusion of acid hydrolysis in the MAE process, the content of 7-xylosyl paclitaxel decreased after acid hydrolysis whereas the content of paclitaxel increased. Based on this result, it was confirmed that acid hydrolysis breaks down a glycosidic bond of glycoside (sugar-binding paclitaxel), and so the recovery of paclitaxel increases.

Preparative isolation of paclitaxel and related taxanes from cell cultures of Taxus chinensis using reversed-phase flash chromatography

In this study, paclitaxel, baccatin III, taxuyunnanine C and sinenxane C were successfully separated by reversed-phase flash chromatography on a manually packed C18 column from Taxus chinensis cell culture extract. The crude cell culture extract was first treated with Al2O3 column chromatography and then divided into two parts: fraction 1 and fraction 2. Ten milligrams of baccatin III and 19 mg of paclitaxel were obtained from 100 mg dried fraction 1. Fifty-two milligrams of taxuyunnanine C and 11 mg sinenxane C were obtained from 100 mg dried fraction 2. The purities of the four compounds were 98.02%, 98.53%, 98.93% and 98.76%, respectively. Their structures were characterised by using UV, MS and NMR. These results indicate that paclitaxel and related taxanes including baccatin III can be obtained from cell culture in a highly pure state using reversed-phase flash chromatography.

Effect of Zeta Potential on Fractional Precipitation for the Purification of Paclitaxel from Plant Cell Cultures of Taxus chinensis

Effect of Zeta Potential on Fractional Precipitation for the Purification of Paclitaxel from Plant Cell Cultures of Taxus chinensis

Identification and Quantification of Tar Compounds in Plant Cell Cultures of Taxus chinensis

In this study, the tar compounds derived from the plant cell cultures of Taxus chinensis were first identified and then quantified via gas chromatography/mass spectrometry (GC/MS) and gas chromatography (GC). 2-Picoline, 2,5-xylenol, acenaphthene, 1-methylnaphthalene and o-xylene were found to be the major tar compounds by biomass. These compounds were identified and confirmed by comparing their retention times with those of authentic compounds. Each compound also spiked with the pure standard. The contents of 2-picoline, 2,5-xylenol, acenaphthene, 1-methylnaphthalene, and o-xylene in biomass were 0.2512, 0.1586, 0.1240, 0.0942 and 0.0525 wt%, respectively. Liquid-liquid extraction and adsorbent treatment were able to remove 42% and 94% of the tars from biomass, respectivly. After hexane precipitation, all of the tars were perfectly removed.

Abscisic acid plays critical role in ozone‐induced taxol production of Taxus chinensis suspension cell cultures

Exposure to ozone induced a rapid increase in the levels of the phytohormone abscisic acid (ABA) and sequentially followed by the enhancement of Taxol production in suspension cell cultures of Taxus chinensis. The observed increases in ABA and Taxol were dependent on the concentration of ozone applied to T. chinensis cell cultures. To examine the role of ABA in ozone-induced Taxol production, we pretreated the cells with ABA biosynthesis inhibitor fluridone to abolish ozone-triggered ABA generation and assayed the effect of fluridone on ozone-induced Taxol production. The results showed that pretreatment of the cells with fluridone not only suppressed the ozone-triggered ABA generation but also blocked the ozone-induced Taxol production. Moreover, our data indicate that the effect of ABA on Taxol production of T. chinensis cell cultures is dose-dependent. Interestingly, the suppression of fluridone on ozone-induced Taxol production was reversed by exogenous application of low dose of ABA, although treatment of low dose ABA alone had no effect on Taxol production of the cells. Together, the data indicated that ozone was an efficient elicitor for improving Taxol production of plant cell cultures. Furthermore, we demonstrated that ABA played critical roles in ozone-induced Taxol production of T. chinensis suspension cell cultures.

Production of two intermediate taxoids, 2-hydroxy- 5α,10β-diacetoxytaxadiene and 2-hydroxy-5α,10β,14β- triacetoxytaxadiene, from Taxus chinensis cell culture

The production of taxuyunnanine C (Tc) (2α,5α,10β,14β-tetraacetoxytaxa-4(20),11(12)-diene) was significantly enhanced when Taxus chinensis cell cultures were treated with methyl jasmonate (MJA) or 2,3-dihydroxypropyl jasmonate (DHPJA), a newly synthesized jasmonate analog, in combination with in situ absorption. Along with the production of Tc, two intermediate taxoids were produced and purified. On the basis of liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) spectral data, the chemical structures of the two intermediate taxoids were found to be 2-hydroxy-5α,10β-diacetoxytaxadiene and 2-hydroxy-5α,10β,14β- triacetoxytaxadiene. This is the first study to report the production of these two taxoids using plant cell culture. Key words : Taxus chinensis , taxuyunnanine C, methyl jasmonate, 2,3-dihydroxypropyl jasmonate, in situ absorption, 2-hydroxy-5α,10β-diacetoxytaxadiene, 2-hydroxy-5α,10β,14β-triacetoxytaxadiene.

Chemo-Enzymatic Transformation of Taxanes and their Reversal Activity towards MDR Tumor Cells

Over 200 derivatives have been obtained through chemo-enzymatic transformation of the taxanes derived from cell cultures of Taxus chinensis. The reversal activity towards MDR tumor cells and cyto-toxicity of most these compounds were evaluated, and several derivatives exhibited powerful MDR reversal activity. The substrate-specificities of the two most important biotransformation reactions-C-7beta and 9alpha hydroxylations, were investigated and preliminarily concluded. In addition, some key intermediates in hypothetically biosynthetic pathway of taxoid were obtained. These results indicate that biotransformation combined with chemical transformation is a powerful approach to structural diversification of natural products, lead compound discovery and the study on the structure activity relationships of the derivatives. Moreover, biotransformation may mimic some steps in biosynthetic pathway of natural products and provide useful hints as to their biogenetic study.

Four new minor taxanes from cell cultures of Taxus chinensis

Four new minor taxanes (1–4) have been isolated from Ts-19 cell cultures of Taxus chinensis together with five known taxanes (5–9) by silica gel chromatography combined with semi-preparative HPLC chromatography. On the basis of the analyses of the chemical and spectroscopic (IR, MS, 1D, and 2D NMR) data, the structures of new compounds were elucidated as 5α-hydroxy-2α,10β-diacetoxy-14β-(3-hydroxy-2-methyl-butyryl)oxytaxa-4(20),11-diene (1), 2α,5α,10β-triacetoxy-14β-(2-hydroxy-propionyl)oxytaxa-4(20),11-diene (2), 2α,5α,10β-triacetoxy-14β-(2-hydroxy-3-methyl-butyryl)oxytaxa-4(20),11-diene (3), and 2α-benzoxy-4α,9α,10β,13α-tetraacetoxytax-11-ene (4), respectively. Compounds 1–5 were pharmacologically evaluated for their cytotoxicities against five human cancer cell lines (HCT-8, Bel-7402, BGC-823, A549, and A2780) and their reversing activity towards multi-drug resistance A549/taxol tumor cell line, and the results showed that all of the tested compounds exhibited very low cytotoxicities, while compound 4 possessed twice the reversing activity as that of verapamil at 10 μM.

Effect of pH on fractional precipitation for pre-purification of paclitaxel from plant cell cultures

Fractional precipitation is a simple, efficient method for pre-purifying paclitaxel from plant cell cultures of Taxus chinensis. The pH, a key process variable in fractional precipitation, was optimized in terms of the yield and purity of paclitaxel with a fixed methanol concentration in water (61.5%, v/v), paclitaxel content in the crude extract (0.5%, w/v), and storage time (14 hr). The maximum yield (~99%) and purity (~84.4%) of paclitaxel were obtained at pH 8.0 with potassium phosphate buffer. The precipitate from fractional precipitation was an amorphous state paclitaxel by analysis of XRD and SEM. Also, the particle size distribution of dried precipitate was a range of approximately 119-411 µm. The use of fractional precipitation in the pre-purification process allowed for rapid and efficient separation of paclitaxel from interfering compounds, and dramatically increased the yield and purity of the crude paclitaxel for subsequent purification steps.

Simultaneous Identification and Determination of Major Taxoids from Extracts of Taxus chinensis Cell Cultures

Liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI-MS/ MS) and high-performance liquid chromatography (HPLC) methods have been used to identify and quantify the major taxoids from extracts of Taxus chinensis cell cultures. Chromatography was carried out on a reverse phase C18 column with isocratic-mode elution. By analytically comparing LC/ESI-MS/MS of the extracts with that of the available reference substances and literature, six taxoids were identified as taxuyunnanine C (Tc, 1), yunnanxane (2), 2alpha,5alpha10beta-triacetoxy-14beta-propionyloxytaxa-4(20),11-diene (3), 2alpha,5alpha, 10beta-triacetoxy-14beta-(2-methyl)butyryloxytaxa-4(20),11-diene (4), taxol (5), and baccatin III (B III, 6), respectively. Among them, 2, 3 and 4 were assigned in the absence of the corresponding reference substances, and 3 and 4 were detected in this cell line for the first time. The identification was validated by NMR spectra. The precise quantification of 1 and 5 was made using HPLC. The limit of detection (LOD), 0.5 microg/ml for 5, 1.5 microg/ml for 1, and the linearity and accuracy of the quantitative method were evaluated, indicating a wide linear range and satisfactory accuracy. The amounts of other identified taxoids were calculated on the basis of comparison of the absolute response factors of similar structural substances. The proposed method provides a rapid, conventional and reliable tool to characterize and study cell lines for elucidating the taxane biosynthesis.

Large-scale purification of 13-dehydroxybaccatin III and 10-deacetylpaclitaxel, semi-synthetic precursors of paclitaxel, from cell cultures of Taxus chinensis

The taxane derivatives 13-dehydroxybaccatin III (13-DHB III) and 10-deacetylpaclitaxel (10-DAP) can be used as semi-synthetic precursors of paclitaxel. These precursors were isolated in a simple and economical procedure during purification of paclitaxel from plant cell culture extracts. No additional costs for cell culture or extraction by silica-gel low-pressure chromatography were incurred. Precipitation from dichloromethane/ n-hexane followed by HPLC on ODS and silica-gel resins resulted in paclitaxel of 99.5% purity with 80% overall yield. ODS low-pressure chromatography and THF/ n-hexane precipitation yielded 13-dehydroxybaccatin III at >99% purity with 87.1% overall yield, and ODS low-pressure chromatography alone yielded 10-deacetylpaclitaxel at >90% purity with 93.4% overall yield. These compounds are of sufficient purity for use in semi-synthesis of paclitaxel. Both compounds were obtained in high yield at >99.5% purity using ODS-HPLC. The procedures described allow the simultaneous purification of 13-dehydroxybaccatin III, 10-deacetylpaclitaxel, and paclitaxel with only minimal additional expense for reagents or equipment.

Nitric Oxide is Involved in Methyl Jasmonate-induced Defense Responses and Secondary Metabolism Activities of Taxus Cells

Methyl jasmonate (MeJA), a methyl ester of jasmonic acid (JA), is a well-established signal molecule in plant defense responses and an effective inducer of secondary metabolite accumulation in plant cell cultures such as the valuable anticancer diterpenoid taxol (paclitaxel) in Taxus spp. This work examines the involvement of nitric oxide (NO) in MeJA-induced plant defense responses and secondary metabolism in Taxus chinensis cell cultures. Exogenously supplied MeJA at 100 microM induced rapid production of NO in the Taxus cell cultures, reaching a maximum within 6 h of MeJA supply. Several other responses occurred concomitantly, including the production of hydrogen peroxide (H2O2), and the increases in intracellular malondialdehyde (MDA) content, lipoxygenase (LOX) and phenylalanine ammonium-lyase (PAL) activities. The MeJA-induced H2O2 production was suppressed by an NO donor, sodium nitroprusside (SNP), but enhanced by NO inhibitors, N (omega)-nitro-L-arginine (L-NNA) and 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (PTIO). In contrast, the MeJA-induced MDA, LOX and PAL were all enhanced by the NO donor but suppressed by the NO inhibitors. The NO inhibitors also suppressed MeJA-induced taxol accumulation. These results are suggestive of a role for NO as a signal element for activating the MeJA-induced defense responses and secondary metabolism activities of plant cells.

Purification, cloning, and functional expression of phenylalanine aminomutase: The first committed step in Taxol side-chain biosynthesis

The conversion of α-phenylalanine to β-phenylalanine is the first committed step in the biosynthesis of the C-13 side chain of Taxol. Thus, the novel enzyme responsible for this step, phenylalanine aminomutase (PAM), is of considerable interest for studies of Taxol biosynthesis and represents a potential target for genetic engineering. A method is described for purifying PAM from Taxus chinensis cell cultures. The purified enzyme has a K m of 1.1 mM, a V max of 110.1 μm/min/mg protein, a pH optimum of 7.5–8.0, and a denatured molecular weight of about 80 kDa. Peptide sequences derived from the purified protein were used to design and synthesize degenerate primers enabling the PCR synthesis of the PAM cDNA. The PAM cDNA encodes a protein of 687 amino acid residues with a deduced molecular weight of 75.3 kDa. The PAM cDNA was cloned and expressed in Escherichia coli, and PAM activity was demonstrated. As a gene symbol for the PAM enzyme, pam is proposed. Protein sequence alignments of PAM, phenylalanine ammonia-lyase (PAL), and histidine ammonia-lyase (HAL) sequences exhibit significant similarity providing insight into potential active site residues of PAM.

The role of lipoxygenase in elicitor-induced taxol production in Taxus chinensis cell cultures

The role of lipoxygenase (LOX) in the elicitor-induced taxol production of Taxus chinensis cell cultures was studied. Fungal elicitor F 5 treatment effectively increased LOX activity and taxol production. The LOX activity inhibitor phenidone could inhibit induced LOX activity and taxol production, suggesting that LOX pathway might be involved in elicitor-induced taxol production. Methyl jasmonate (MJ) also activated LOX activity and taxol production. Phenidone could inhibit LOX activity and taxol production challenged by MJ, suggesting that exogenous MJ may induce taxol production via activating the intracellular LOX pathway. For better understanding the role of LOX in T. chinensis, some biochemical features of LOX were also investigated. Results showed that at least 49.18% LOX was associated with membrane and the molecular weight of LOX was 93 kDa.

Efficient purification and morphology characterization of paclitaxel from cell cultures of Taxus chinensis

AbstractPaclitaxel was purified from cell cultures of Taxus chinensis by a combination of extraction, low‐pressure chromatography, precipitation and HPLC. A crude extract (purity 6.9%) was obtained by methanol extraction of plant cell cultures, followed by liquid–liquid extraction using dichloromethane. The extract was purified to greater than 32% with a 97% step yield by low‐pressure chromatography. After acetone/pentane precipitation, the resulting purity and step yield were 75.8% and 97.4%, respectively. High performance liquid chromatography steps, which were composed of an HPLC step with column‐packed ODS and an HPLC step with column‐packed silica, were applied to give over 99.5% purity with high yield. Amorphous paclitaxel with a fine particle size, which has a solubility advantage compared with the stable crystalline form, was obtained by dissolving in dichloromethane, followed by spray‐drying. Copyright © 2004 Society of Chemical Industry

Effects of absorbent treatment on the purification of paclitaxel from cell cultures of Taxus chinensis and yew tree

A new synthetic absorbent, sylopute was applied for the purification of paclitaxel from plant cell cultures and yew tree. There was selective absorption of impurities in the treatment of sylopute. Therefore, higher purity paclitaxel could be obtained simply in the following process with a higher yield than with active clay or activated carbon. The purity of crude paclitaxel after sylopute treatment followed by precipitation was increased up to 63%, while the purities for active clay and activated carbon were 57.7 and 40%, respectively. The overall yields from sylopute, active clay, and activated carbon were 80.2, 67.7, and 35.5%, respectively. The best result in removing impurities was obtained from the combination of sylopute treatment followed by 1st and 2nd precipitation processes. A similar result was observed in the case of absorbent treatment of crude extract from yew tree. Consequently, sylopute can be applied for production scale purification of other natural products as an alternative to synthetic absorbents, activated carbon, and other alternative processes.