| In the present study,simultaneous determination of paclitaxel,cephalomannine and 7-epi-10-deacetyl-taxol was established in the extract of Taxus chinensis var.mairei needles by HPLC.Enzyme-assisted ultrasound-and heat-reflux simultaneous extraction(EAU-HRE)of paclitaxel,cephalomannine and 7-epi-10-deacetyl-taxol from Taxus chinensis var.mairei needles.Extraction yields of paclitaxel,cephalomannine and 7-epi-10-deacetyl-taxol were used as indexes,the extraction conditions of them were optimized.At the same time,According to the different polarity of the flavonoids and taxanes,separation of flavonoids and taxanes from Taxus chinensis var.mairei needles using chloroform.Taxanes removing impurity in chloroform water phase after preliminary enrichment and purification,obtained crude extracts of flavonoids in the residual liquid of chloroform extract.In addition to,enrichment and separation of flavonoids in the enzyme-assisted ultrasound-and heat-reflux simultaneous residual extract of chloroform solutions by macroporous adsorption resins was studied and the purity of flavonoids and the separation effect of flavonoids monomer were used as indexes,the enrichment and separation conditions of them were optimized.Meanwhile,their antioxidant activities were preliminary evaluated.The optimal conditions for extraction,separation and enrichment of taxanes and flavonoids were established.Objective uing limited taxus chinensis resource to improve the resource comprehensive utilization by two-way research.1.The method on simultaneous determination of paclitaxel,cephalomannine and 7-epi-10-deacetyl-taxol from Taxus chinensis var.mairei needles by HPLC was established as follows:Chromatographic conditions:chromatographic column:Ecosil C18(4.6 mm?×250 mm.×5 ?m);The mobile phase was composed by methanol-acetonitrile-water(40:15:45,v/v/v);The UV detection wavelength applied was 227 nm and the column temperature was set 30?;the flow rate was 1mL/min;The injection volume was 10 ?L,may achieve a good symmetrical peakshape.2.Effect of enzyme induction on the contents of paclitaxel,cephalomannine and 7-epi-10-deacetyl-taxol from Taxus chinensis var.mairei needles were investigated.The technology on simultaneous extraction of paclitaxel,cephalomannine and 7-epi-10-deacetyl-taxol from Taxus chinensis var.mairei needles by EAU-HRE was builded for the first time and three kinds of taxanes combining quantitative analysis with qualitative analysis in Taxus chinensis var.mairei needles were analysed by HPLC method.The optimum process of enzyme preconditioning was confiremed for first time,and the main parameters affecting the contents were optimized as follows:(1)Enzyme type:cellulose(2)Concentration of pectinase:1 mg/mL(3)Preconditioning time:18 h(4)Preconditioning temperature:55 ?(5)Preconditioning pH:6-6.5(6)Reflux extraction temperature:65 ?(7)Liquid/solid ratio:27:1(8)Extraction time:42 min(9)Ultrasonic power:93 WUnder the best extration conditions of EAU-HRE,the extraction yields of paclitaxel,cephalomannine and 7-epi-10-deacetyl-taxol were respectively:0.0919 mg/g.0.04015 mg/g?0.0053 mg/g?0.0457 mg/g.Compared with the ultrasonic assisted extraction(UAE),heat reflux extraction(HRE),ultrasonic-assisted reflux extraction UHRE,enzymatic-assisted ultrasonic extraction(EAUE)enzymatic-assisted heat reflux extraction(EAUE)the extraction yields of paclitaxel,cephalomannine and 7-epi-10-deacetyl-taxol were in 1.52-3.75 times,1.69-3.01 times,1.58-2.42 times respectively.This method can save materials,enhance efficiency.3.Optimized the technology of flavonoids iolation and purification from the chloroform raffinate phase of Taxus and evaluation of its in antioxidant activity.With the desor:ption rateand purity of flavonoids as the indicstor.Six different,types macroporous adsorption resin including X-5,D101,AB-8,LSA-10,HPD400 and NKA-9 were used in The experiments onenrichment.The optimum the technology of flavonoids iolation and purification from the chloroform raffinate phase,and the main parameters affecting the contents were optimized as follows:(1)Type of macroporous adsorption resin:AB-8(2)Sample concentration:50 mg/mL(3)The adsorptionequilibrium time:5 h(4)Sample volume:70 mL(5)The conditions of isocratic elution:80%ethanol,8.5 BV(6)The conditions of gradient elution:10%ethanol 3 BV,20%ethanol 2.5 BV,30%ethanol 3 BV,40%ethanol 4.5 BV;50%ethanol 2 BVUnder the best isolation and purification conditions of isocratic elution,the purity of flavonoids was 17.73%,the purity of flavonoids was 4.91 folds for the original extract.Two flavonoids compounds were isolated by HPLC;In the three groups experimental of DPPH radical scavenging activity,ABTS+ scavenging activity and reducing power of chloroform raffinate phase of Taxus chinensis var.mairei needles.IC50 values of 3.67 mg/mL?0.2049 mg/mL respectively,FRAP value of 3.554 mg/mL.Enrichment of antioxidant capacity were higher than not concentrate.Under the best isolation and purification conditions of gradient elution,the purity of flavonoids was 23.82%,the purity of flavonoids was 6.60 folds for the original extract.Three flavonoids compounds were isolated by HPLC;In the three groups experimental of DPPH radical scavenging activity,ABTS+ scavenging activity and reducing power of chloroform raffinate phase of Taxus chinensis var.mairei needles.IC50 values of 3.17 mg/mL.0.1835 mg/mL respectively,FRAP value of 4.316 mg/mL.Enrichment of antioxidant capacity were higher than isocratic elution concentrate..In conclusion,in this paper,firstly,the HPLC established a rapid,sensitive and simple means for taxanes detection.Secondly,optimizing the best taxanes extraction technology,in order to realize high efficiency and material saving.Besides,optimizing flavonoids best separation enrichment technology and the preliminary evaluation of antioxidant activity for the extraction and separation of the other natural products laid the foundation of new practice.Implements the taxus chinensis resource comprehensive utilization,reasonable development,improve the economic value and social value. |
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