Separation and purification of two taxanes and one xylosyl-containing taxane from Taxus wallichiana Zucc.: A comparison between high-speed countercurrent chromatography and reversed-phase flash chromatography.

10-Deacetylbaccatin III, an important semisynthetic precursor of paclitaxel and docetaxel, can be extracted from Taxus wallichiana Zucc. A process for the isolation and purification of 10-deacetylbaccatin III (1), baccatin III (2), and 7ß-xylosyl-10-deacetyltaxol (3) from the leaves and branches of Taxus wallichiana Zucc. via macroporous resin column chromatography combined with high-speed countercurrent chromatography or reversed-phase flash chromatography was developed in this study. After fractionation by macroporous resin column chromatography, 80% methanol fraction was selected based on high-performance liquid chromatography and liquid chromatography with mass spectrometry qualitative analysis. A solvent system composed of n-hexane, ethyl acetate, methanol, and water (1.6:2.5:1.6:2.5, v/v/v/v) was used for the high-speed countercurrent chromatography separation at a flow rate of 2.5 mL/min. The reversed-phase flash chromatography separation was performed using methanol/water as the mobile phase at a flow rate of 3 mL/min. The high-speed countercurrent chromatography separation produced compounds 1 (10.2 mg, 94.4%), 2 (2.1 mg, 98.0%), and 3 (4.6 mg, 98.8%) from 100 mg of sample within 110 min, while the reversed-phase flash chromatography separation purified compounds 1 (9.8 mg, 95.6%) and 3 (4.9 mg, 97.9%) from 100 mg of sample within 120 min.

Comparison of Pharmacokinetics and Biodistribution of 10-Deacetylbaccatin III after Oral Administration as Pure Compound or in Taxus chinensis Extract: A Pilot Study.

Taxanes are a class of bioactive compounds isolated from the Taxus species. 10-Deacetylbaccatin III is one of the popular taxane compounds with antitumor activity, but the pharmacokinetic profile of this compound remains elusive. Previously, we prepared the taxane fractions from the twigs and leaves of Taxus chinensis var. mairei containing 20.4 % 10-deacetylbaccatin III. This study aimed to investigate the pharmacokinetics of 10-deacetylbaccatin III and biodistribution, and explore the potential changes when it was administered in the form of taxane extracts. A simple, sensitive, and reliable liquid chromatography-tandem mass spectrometry method was developed and validated for the quantitative determination of 10-deacetylbaccatin III in biosamples. The results showed that 10-deacetylbaccatin III, after oral dosing, displayed a quick absorption into the blood and distribution into major organs. Oral administration of 10-deacetylbaccatin III in the form of taxane mixtures led to a 16-fold increase in the systemic exposure of pure 10-deacetylbaccatin III, with the AUC0-U in the plasma increasing from 25.75 ± 11.34 to 231.36 ± 70.12 µg h/L (p < 0.0001). Moreover, the concentrations of 10-deacetylbaccatin III in major tissues were significantly enhanced when given in taxane extracts. These findings revealed pharmacokinetic interactions in the taxane components from T. chinensis var. mairei, which contributed to an enhanced systemic exposure of pharmacologically active taxanes.

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.

[Chemical constituents of bark of Taxus chinensis var. mairei].

OBJECTIVE: To study the chemical constituents in the bark of Taxus chinensis var. mairei collected from southeast of China. METHODS: Chemical constituents were isolated and purified by column chromatography, Prep-TLC, and preparative HPLC. The structures were identified on the basis of 1D-and 2D-NMR spectral analysis. RESULTS: Twelve taxane diterpenoids were isolated from the bark of Taxus chinensis var. mairei grown in southeast of China. They were identified as: taxagifine (1), decinnamoyltaxagifine (2), 19-debenzoyl-19-acetyltaxinine M(3), 9-dihydro-13-acetyl-baccatin III (4), 7, 9-dideacetylbaccatin IV (5), 1,3-dihydro-taxinine (6), taxumairol C (7), taxezopidine J (8), 7-xylosyl-10-deacetyl-taxol A (9),10-deacetyltaxol (10), taxicin II (11), and 2alpha, 7beta, 10beta-triacetoxy-5alpha, 13alpha-dihydroxy-2 (3 --> 20) abeotaxa-4 (20), 11-dien-9-one (12). CONCLUSION: Compounds 1, 2, 4 - 6, 8, 9, 11 and 12 are obtained from this plant for the first time. Compound 7 is obtained from the bark of Taxus chinensis var. mairei for the first time.

A new taxane diterpenoid from Taxus chinensis var. mairei.

Taxadiene (3), a new taxane diterpenoid with an unusual hydroxy substituting at C-17, and six known compounds including two taxane diterpenoids (1 and 2) and four flavonoids (4-7) were isolated from the whole seedling of the Taxus chinensis var. mairei. Among them, compound 7 was isolated from T. chinensis var. mairei for the first time. Structures of these compounds were elucidated on the basis of spectroscopic data and by comparison with reported literature data.

Chemical studies on Taxus canadensis.

A series of new taxanes, 1-93, have been isolated, together with 37 known taxoids including Taxol(®) (paclitaxel) and cephalomannine, from the Canadian yew, Taxus canadensis (Taxaceae) in the past 30 years. These new taxoids possess various skeletons containing 5/7/6, 6/10/6, 6/5/5/6, 6/8/6, and 6/12 ring systems and six new taxanes with four novel skeletons, i.e., a taxane with a 6/6/8/6 ring system, a taxane with a [3.3.3] propellane skeleton, three taxanes with [3.3.3] [3.4.5] dipropellane sytems, as well as a novel taxane with a unique 5/5/4/6/6/6 hexacyclic skeleton, containing a unique [3.3.2] propellane, were isolated for the first time from natural sources. It should be emphasized that 13-acetyl-9-dihydrobaccatin III, a very useful starting material for the semisynthesis of Taxol(®) and Taxotere(®) , represents the most abundant taxane in the needles of this yew tree. These findings establish the above mentioned yew tree as significantly different from the remaining species. On the other hand, some chemical modifications on the taxanes isolated from this plant were carried out.

Synthesis, isolation, stereostructure and cytotoxicity of paclitaxel analogs from cephalomannine.

Four paclitaxel derivatives were afforded by preparative HPLC separation of two pairs of diastereoisomers, which were obtained by catalytic hydrogenation and epoxidation of the C-13 side-chain double bond of cephalomannine, a naturally occurring paclitaxel analog. The four paclitaxel derivatives were analyzed using NMR, CD spectroscopy, and side-chain hydrolysis in order to measure their optical rotations and GC characteristics. In this way, the stereoconfigurations of the products were determined. Evaluation of the compounds' activity indicated that they had differing cytotoxic activities: compound 5 had superior activity in BCG-823 tumor cells compared to paclitaxel, while compound 7 had superior activity in HCT-8 and A549 tumor cells compared to paclitaxel. These results indicate that the stereoconfiguration of the paclitaxel N-acyl side chain has a significant impact on its activity.

[Study on chemical constituents from flowers of Ailanthus altissima].

OBJECTIVE: To isolate and identify the chemical constituents from the flowers of Ailanthus altissima. METHODS: Macroporous adsorptive resins (DM130), Silica gel, Sephadex LH-20, ODS were employed for the isolation and purification of chemical constituents. The structures were identified on the basis of spectral data and physicochemical examination. RESULTS: Eight compounds were isolated and identified as follows: brevifolin (1), brevifolin carboxylic acid (2), methyl brevifolin carboxylate (3), ellagic acid (4), diethyl-2,2',3,3',4,4'- hexahydroxybiphenyl-6,6'-dicarboxylate (5), rutin (6), gallic acid (7), ethyl gallate (8). CONCLUSION: Compounds 1 -5 are isolated from this genus for the first time.

7,7''-Dimethoxyagastisflavone-induced apoptotic or autophagic cell death in different cancer cells.

7,7''-Dimethoxyagastisflavone (DMGF), a biflavonoid isolated from the needles of Taxus × media cv. Hicksii, was evaluated for its antiproliferative and antineoplastic effects in three human cancer cell lines. Interestingly, DMGF caused cell death via different pathways in different cancer cells. DMGF induced apoptosis, activated caspase-3 activity and changed the mitochondrial membrane potential in HT-29 human colon cancer cells. However, the apoptotic pathway is not the major pathway involved in DMGF-induced cell death in A549 human lung cancer cells and HepG2 human hepatoma cells. Treatment with 3-MA, an inhibitor of autophagy, significantly decreased DMGF-induced cell death in HepG2 and A549 cells, but did not affect DMGF-induced cell death in HT-29 cells. Following DMGF treatment, the HepG2 cells increased expression of LC3B-II, a marker used to monitor autophagy in cells. Thus, DMGF induced apoptotic cell death in HT-29 cells, triggered both apoptotic and autophagic death in A549 cells and induced autophagic cell death in HepG2 cells.

Analgesic and antiinflammatory activities of taxoids from Taxus wallichiana Zucc.

A study was conducted to identify constituents that might be responsible for analgesic and antiinflammatory conditions. Tasumatrol B, 1,13-diacetyl-10-deacetylbaccatin III (10-DAD) and 4-deacetylbaccatin III (4-DAB) were isolated from the bark extract of Taxus wallichiana Zucc. All the compounds were assessed for analgesic and antiinflammatory activities using an acetic acid-induced writhing model, a hot-plate test, a carrageenan-induced paw oedema model, a cotton-pellet oedema model and in vitro lipoxygenase inhibitory assay. All the compounds, especially tasumatrol B, revealed significant analgesic activity in comparison to a saline group based on an acetic acid-induced model. Similarly all of the test compounds, particularly tasumatrol B, showed significant antiinflammatory activity. However, all the compounds failed to exhibit any considerable activity in of the hot-plate test and the in vitro lipoxygenase inhibitory assay. This study has highlighted the potential of tasumatrol B to be further explored as a new lead compound for the management of pain and inflammation, one that has been discovered by scientific validation of the traditional medicinal use of T. wallichiana Zucc.

Novel taxa-4(20),12-diene and 2(3→20)abeotaxane from needles of Taxus canadensis.

A novel 6/8/6-membered taxane with a rare C-12(13)-double bond and rare 2(3→20)abeotaxane were isolated from the needles of Taxus canadensis. Their structures were characterized as 7ß,9α,10ß-triacetoxytaxa-4(20),12-diene-2α,5α,11ß-triol (1) and 2α,7ß,10ß-triacetoxy-5α-hydroxy-2(3→20)abeotaxa-4(20),11-diene-9,13-dione (2) on the basis of 1D and 2D spectroscopic data. 1 is the first example of a natural taxane without substitution at both C-13 and C-14.

[Chemical constituents of Ammopiptanthus mongolicus].

OBJECTIVE: To study the chemical constituents of aerial parts of Ammopiptanthus mongolicus. METHOD: Isolation and purification were carried out on silica gel, Sephadex LH-20 and HPLC column chromatography. The structures of the compounds were identified by physico-chemical properties and spectral analysis. RESULT: Nine compounds were isolated and identified as (+)-maackiain (1), brevifolin (2), 7-hydroxy-4'-methoxy isoflavanone (3), daidzein 4',7-diglucoside (4), genistein 4', 7-di-O-beta-D-glucoside (5), isolupalbigenin (6), ononin (7), beta-sitosterol (8), beta-daucosterol (9). CONCLUSION: Compounds 2, 4 - 6 were obtained from the genus Ammopiptanthus for the first time.

Analysis of biosynthetic fluctuations of cultured Taxus seedlings using a metabolomic approach.

Fluctuations in the biosynthesis of taxoids in 1-5 year old cultured seedlings of Taxus chinensis var. mairei were investigated using LC-IT-TOF-MS and a metabolomics approach. In the total ion chromatogram (TIC) of the extracts, 16 prominent peaks were observed. Ten compounds were identified by comparison of retention times and MS/MS spectra with those of reference compounds. An additional 6 taxoids were isolated by preparative HPLC and identified by comparison of their spectroscopic data with those reported in the literature. It was clarified that the relative concentrations of taxoids with 4(20) double bonds are high at early stages of cultivation. On the other hand, relatively higher amounts of 5-acetoxy taxoids oxidized at the 4- and 10- positions and taxoids having 5(20)-oxetane rings were found at later stages of cultivation. This approach provides practical information on the biosynthetic flow of taxoids in cultured yew seedlings.

A novel taxane 13-glucoside and other taxanes from the leaves of Taxus cuspidata.

Three new taxanes, 2 α,9 α,10 ß-triacetoxy-13 α-( ß-D-glucopyranosyloxy)taxa-4(20),11-dien-5 α-ol, 5 α,10 ß,13 α-triacetoxytax-11-ene-2 α,7 ß,9 α,20-tetraol, and 5 α,10 ß,13 ß-triacetoxy-2 α,7 ß-dihydroxy-2(3→20) abeotaxa-4(20),11-dien-9-one, were isolated from the leaves of the Japanese yew, Taxus cuspidata. Compound 1 is the first example of a taxane with 13-glycosidic linkage.

Molecular simulations of Taxawallin I inside classical taxol binding site of ß-tubulin.

A new taxoid Taxawallin I (1) along with two known taxoids (2-3) were isolated from methanolic bark extract of Taxus wallichiana Zucc. Structural characterization was confirmed by mass and NMR spectral techniques. Taxawallin I exhibited significant in-vitro anticancer activity against HepG2, A498, NCI-H226 and MDR 2780AD cancer lines. Tubulin binding assay was performed to assess its tubulin binding activity. Molecular docking analysis was performed to study the potential binding mode inside the taxol binding site of ß-tubulin.

Analysis of MS/MS fragmentation of taxoids.

The fragmentation pathways of seven types of taxoids were investigated by using a LC-MS/MS method, namely: (1) neutral taxoids with a C-4(20) double bond; (2) taxoids with a C-4(20) double bond and oxygenation at C-14; (3) 5-cinnamoyl taxoids with a C-4(20) double bond; (4) a basic taxoid with a C-4(20) double bond; (5) a taxoid with a C-4(20) epoxide; (6) taxoids with an oxetane ring; and (7) taxoids with an oxetane ring and a phenylisoserine C-13 side chain. Depending on the class of core structure and the substitution pattern, each taxoid gave either the molecular adduct ion [M+NH4]+ or [M+H]+. In the MS/MS, the molecular adduct ion gave characteristic product ions corresponding to the loss of water, acetic acid, benzoic acid, and cinnamic acid or the phenylisoserine group. These could reflect the difference of the substitutions and structural modifications and should be utilized for the structure elucidation oftaxoids by LC-MS.

Rapid qualitative and quantitative determination of seven valuable taxanes from various Taxus species by UFLC-ESI-MS and UFLC-DAD.

The distribution and level of yew constituents vary with species and tissues. In this study, a rapid and valid method incorporating ultra-fast liquid chromatography (UFLC) with MS and UV detection was developed for simultaneous determination of paclitaxel and its six semisynthesis precursors in needles and hair roots from various Taxus species. All target analytes could be identified by comparing their retention times as well as UV and MS spectra with authentic standards, while seven valuable taxanes in botanical samples can be rapidly determined by UFLC-DAD with excellent sensitivity. Analysis of more than one hundred yew samples from nine species showed significant variations in distribution and content of seven evaluated taxanes. Thus, different developmental schemes should be used for better utilization of various yew resources.

A comparative optical aggregometry study of antiplatelet activity of taxanes from Taxus cuspidata.

Platelets are highly reactive components of the circulatory system. The cytoskeleton of a platelet is an important structure for platelet aggregation as stimulated by several agonists. An anticancer agent, taxol, has been suggested to exert platelet anti-aggregating activity by stabilizing microtubules during the aggregation process. An activity-guided fractionation was performed with a methanol extract of the leaves and twigs of Taxus cuspidata to isolate taxanes with platelet anti-aggregating effects. Compounds 1 to 7 - taxinine (1), taxinine A (2), taxinine B (3), 2-deacetoxytaxinine B (4), taxacin (5), taxchinin B (6), and taxol (7) - were obtained as the antiplatelet components of this plant. These taxane compounds present the possibility of securing new antiplatelet compounds which differ from currently available antiplatelet agents in chemical structure and possibly in mechanisms of action. All compounds showed stronger inhibitory effects than acetylsalicylic acid (ASA) on platelet aggregation induced by arachidonic acid (AA) (IC(50): 14.4, 64.5, 35.5, 16.0, 21.9, 28.6 and 48.2 versus 63.0microM) or U46619 (IC(50): 34.8, 24.9, 36.2, 35.0, 46.9, 71.9 and 68.7 versus 340microM). Compounds 1, 3, 4 and 5, with a cinnamoyl group at the C(5) position, showed strong inhibitory effects against AA-induced aggregation compared to compound 2 (with an -OH group at C(5)) or compounds with an oxetane ring at C(4),(5), such as compounds 6 and 7. All of the seven compounds were 5-13-fold more strongly inhibitory than ASA against U46619-induced aggregation.

[Isolation and identification of chemical constituents from Taxus cuspidta Sibe et Zucc].

Three compounds were isolated from the extract of Taxus cuspidta Sibe et Zucc with the column chromatography on silica gel and preparative HPLC methods. Their structures were identified according to the physicochemical properties and spectral analysis, and they were identified as (E)-1-methoxy-2-O-(p-coumaroyl)-myo-inositol (1), 2-deacetoxy-7beta, 9a, 10beta-trideacetyltaxinine J (2) and (3aS, 4aR, 6S, 8S, 8aS, 9R, 10R, 10aS)-benz[f]azulene-6, 8, 9, 10 (3H)-terol, 3a, 4, 4a, 5, 6, 7, 8, 8a, 9, 10-decahydro-10a-(1-hydroxyl-1-methylethyl)-1, 8a-dimethyl-5-methylene (3). Among them, compound 1 was a new compound, and compounds 2, 3 were two novel natural products.