| Comprehensive utilization of Chinese herbal medicine resources plays a crucial role on the sustainable development of the Chinese medicine industry. One of the important contents of Chinese medicinal material resources protection is to create the most value with limited resources. Taxus chinensis var. mairei is a vital extracting material of taxol and its precursor substances. A lot of waste in the production process had been produced due to the low content of such substances in needles. However, polyprenols are rich in the needles and waste which opened up a new way for the comprehensive utilization of Taxus needles. This article focuses on polyprenols from the needles of T. chinensis var. mairei, including structure identification, content change factors, isolation and purification, and anti-fibrotic activity, in order to provide theoretical basis on the comprehensive utilization and sustainable development of the needles of Taxus resources as well as provide model for reasonable utilization of other Chinese medicinal materials.(1) Taxus Polyprenols (TPs) extract was prepared by Soxhlet extraction and the analysis method TPs was established by high performance liquid chromatography and LC-MS. The specific analysis parameters of HPLC was as follows:XBridge RP, C18(5μm,4.6mm×250mm); column temperature was40℃; flow rate:1.5mL/min; injection volume:10μL; UV detection wavelength:210nm. Mobile phase:methanol (A) and isopropanol (B), gradient elution with0-5min,40%of B;5-35min,40%â†'75%B;36min, and40%B;50min,40%B. Analysis showed that the Polyprenols in T. chinensis var. mairei contained11homologues with two-family distribution.(2) The annual changes of TPs content were detected monthly. Studies have shown that TPs content in different plant age was basically in the same change trends, which was, continuously accumulating from initial growth and peaking in October or November (42.43mg/g), when the temperature decreased the concentration reduced while rising in the coming year. The change trend indicated TPs might be negatively correlated with the temperature which needed to be further studied. TPs contents in plants of different ages also varied at the same time that might be caused by different micro-environments. In addition, preliminary studies have shown that light intensity had a certain influence on TPs content that high light intensity was in favour of accumulating such of polyprenols substances.(3) The technology of polyprenols isolation and purification was conducted in order to provide a material basis for wider application of TPs. The TPs extracts was prepared by Soxhlet extraction and the purification technology of TPs was investigated by cold acetone dialysis, hydrolysis and silica gel column chromatography. Specific process parameters:for the Soxhlet extraction, under80℃for12h with800g needle power materials; for the cold acetone dialysis, under4-5℃for4h or overnight at room temperature; for the hydrolysis, under60℃for8h at240rpm/min with100g petroleum extraction and500mL5%NaOH-methanol solution (containing0.05%pyrogallol); for the silica gel column chromatography, sample amount of1:30, eluent petroleum ether and ethyl acetate19:1(v/v), elution flow rate of30mL/min. TPs prepared at this stage phase were used for animal experiments. In addition, the high-speed countercurrent chromatography (HSCCC) was used for separation and purification of the hydrolyzed TPs to provide a new way for polyprenols purification and the pre-conditions were explored.(4) The homologues of TPs were separated by the preparative HPLC on the basis of silica gel column chromatography separation. With the help of UV, IR,1H NMR and13C NMR spectral methods, the structures of polyprenol-17, polyprenol-18, polyprenol-21and polyprenol-22were identified, respectively. Combined with the analysis of LC-MS, the results showed that TPs contained homologue of polyprenol-14, polyprenol-15, polyprenol-16, polyprenol-17, polyprenol-18, polyprenol-19, polyprenol-20, polyprenol-21, polyprenol-22, polyprenol-23and polyprenol-24and belonged to beturaprenol (ω-t2-cn).(5) Animal experiments suggested that TPs had anti-fibrotic effects for alleviating the process of SD rat liver fibrosis induced by CCI4. The results showed that TPs could significantly improve the capacity to resist oxidation, inhibit hepatic stellate cell activation and reduce pro-fibrotic cytokines and protect liver cells. The effect might be achieved through inhibition of TGFβ/Samd2/3signaling pathway. Moreover, the cell experiment showed that the hepaprotective effects of TPs might due to its in vivo metabolite-dolicols which should be further investigated. |
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