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Purification of Two Taxanes from Taxus cuspidata by Preparative High-Performance Liquid Chromatography

https://doi.org/10.3390/separations9120446

According to Zhang et al. [57], the yields of the two taxanes in the extract were quantified by high-performance liquid chromatography (HPLC, UltiMate 3000 Separations Module, Waltham, MA, USA). The gradient elution procedure was set as follows: Inertsil ODS-3 C18 column (250 mm × 4.6 mm, 5 μm); column temperature 30 °C. The detection wavelength was 227 nm; the flow rate was 0.8 mL/min; the injection volume was 10 μL; mobile phase A was purified water and B was acetonitrile. The gradient elution procedure was as follows: B increased from 40% to 50% at 0–10 min, and B increased from 50% to 53% at 10–13 min. B increased from 53% to 73% at 13–25 min, and at 25–30 min, B decreased from 73% to 40% for 10 min.

To generate the mixed standard curve, a total of 10 mg of paclitaxel and 10-DAT standard were accurately weighed and dissolved in methanol to a constant volume of 10 mL. Then, the samples were diluted with methanol to prepare solutions with different concentration gradients. The peak areas of standard solutions with different concentrations were measured by HPLC at 227 nm, and the high-performance liquid standard curves of two standards were drawn with the peak area as the ordinate and the standard concentration as the abscissa. The concentrations of two taxanes were calculated using the calibration curves (the concentration range of the calibration curves was 1.25–125 μg/mL). The total yields of the two main taxanes of Taxus cuspidata were calculated as follows:

Taxane yields = C × V/W

where C (μg/mL) is the total content of two main taxanes, V (mL) is the volume of extraction solution recovered, and W (g) is the weight of Taxus cuspidata.

2.4. Purification Experiment of Preparative HPLC

Preparative HPLC [58] (Hanbon Sci&Tech, Hangzhou, China) was used to collect two taxanes in Taxus cuspidata extract. The gradient elution procedure was set as follows: Hedera ODS-2 C18 column (250 mm × 20 mm, 5 μm, detection wavelength 227 nm). Then, according to the experimental scheme (single-factor and orthogonal experiments scheme), the parameters were set, including flow rate, injection volume, and column temperature. Mobile phase A was purified water and B was acetonitrile. Gradient elution procedure: B increased from 40% to 50% at 0–10 min, and B increased from 50% to 53% at 10–13 min. B increased from 53% to 73% at 13–25 min, and at 25–30 min, B decreased from 73% to 40% for 10 min.

Taxanes purity=Taxane yields in Taxus cuspidataTaxus cuspidata total amount × 100%Taxanes purity=Taxane yields in ����� �������������� ��������� total amount × 100%
Recovery rate of taxanes=Taxane yields after purificationTaxane yields before purification × 100%Recovery rate of taxanes=Taxane yields after purificationTaxane yields before purification × 100%

2.5. Single-Factor Experiments

As the two taxanes purity of Taxus cuspidata may be influenced by various factors, single-factor experiments were designed and carried out. The following factors, including flow rate, injection volume, and column temperature, were set according to the purity of the two products. The experimental range of different factors used in this study was: flow rate = 5–15 mL/min, injection volume = 0.5–2.5 mL (the extract concentration of Taxus cuspidata was 20 mg/mL), and column temperature = 20–40 °C.

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