Study On Extraction And Purification Of Main Naphthoquinones And Diarylheptanes From Qinglongyi

Qinglongyi is the immature exocarp of Manchurian Walnut(Juglans mandshurica Maxim.).Research shows that it has anti-inflammatory,analgesic,anti-tumor and other pharmacological activities,especially the anti-tumor activity of Qinglongyi has have been used in clinical practice.Naphthoquinone and diarylheptane are the main active ingredients in Qinglongyi that exert anti-tumor activity,which present high research value.However,at present,the researches about Qinglongyi are mainly focused on the pharmacological activities of juglone,and there are few reports on the extraction and separation of other naphthoquinones and diaryheptanes.In this study,a high performance liquid chromatography(HPLC)method for the simultaneous determination of naphthoquinone and diaryheptane in Qinglongyi was established for the first time,and the active components were extracted by the integrated ultrasound-assisted ultra-turrax homogenization extraction technology,then the main active components were separated and purified by high-speed countercurrent chromatography.In this paper,the analytical determination,extraction and separation technology of the main active components from Qinglongyi were studied,which will provide an important scientific basis for the in-depth development and utilization of Qinglongyi resources.The results were as follows:1.A HPLC method was developed for the simultaneous analysis determination of two naphthoquinones(Regiolone and Juglone)and four diarylheptanes(Myricatomento-genin,Galeon,2-Oxatrycyclo[13.2.2.13,7]eicosa-3,5,7(20),15,17,18-hexaen-10-16-diol and Juglanin A)from Qinglongyi:Chromatographic column:Diamonsil C18V(5 ?m,250 mm×4.6 mm i.d.),injection volume 5 ?L,column temperature 30?,flow rate 1.0 mL/min;mobile phase:acetonitrile(A)-0.1%formic acid aqueous solution(B),gradient elution conditions:0-28 min,23-41%A;28-30 min,41%A;30-35 min,41-45%A;35-53 min,45%A;detection wavelength:254 nm(0-28 min)and 280 nm(28-53 min).Method validation demonstrated that the six target compounds showed a good linear relationship(R2>0.999)in the detection range.Besides,the limit of detection(LOD)and the limit of quantification(LOQ)were less than 0.83 ?g/mL and 1.61 p.g/mL,and the HPLC analysis method showed high sensitivity,good reproducibility,high precision and strong stability as well as high sample recovery yield(RSD<5%).The contents of regiolone,juglone,myricatomento-genin,galeon,2-oxatrycyclo[13.2.2.13,7]eicosa-3,5,7(20),15,17,18-hexaen-10-16-diol and juglanin A in test sample were 1.721 mg/g,0.457 mg/g,1.772 mg/g5 0.565 mg/g,2.156 mg/g,0.872 mg/g.2.An efficient extraction process,integrated ultrasound-assisted ultra-turrax homogenization extraction technology,was used to extract naphthoquinone and diarylheptane from Qinglongyi,and the optimal process parameters were determined:Homogeneous rate:8000 rpmUltrasonic power:250 WEthanol concentration:83%Liquid/solid ratio:21 mL/gExtraction time:13 minUnder the optimal extraction conditions,a total of six compounds were obtained,including regiolone,juglone,myricatomento-genin,galeon,2-oxatrycyclo[13.2.2.13,7]eicosa-3,5,7(20),15,17,18-hexaen-10-16-diol,juglanin A,the average extraction yields of six target compounds were 1.304 mg/g,0.308 mg/g,1.401 mg/g,0.413 mg/g,1.692 mg/g and 0.671 mg/g,respectively.3.The separation and purification of two naphthoquinones and four diarylheptanes from Qinglongyi by high-speed countercurrent chromatography were established:The first separation step:Solvent system:n-hexane:ethyl acetate:ethanol:water=8:7:7:4Host speed:850 rpmElution flow rate:2.0 mL/minStationary phase retention rate:83%Load:200 mgDetection wavelength:254 nm,280 nmSecond separation step:Solvent system:n-hexane:ethyl acetate:methanol:water=5:4:6:5Host speed:850 rpmElution flow rate:1.5 mL/minStationary phase retention rate:90%Detection wavelength:280 nmUnder the above optimized conditions,six compounds were obtained through two steps of countercurrent separations,including two naphthoquinones(Regiolone and Juglone)and four diaryheptanes(Myricatomento-genin,Galeon,2-Oxatrycyclo[13.2.2.13,7]eicosa-3,5,7(20),15,17,18-hexaen-10-16-diol and Juglanin A).The recovery yields of six compounds were all over 80%,and the purities of six compounds were over 95%.After the integrated ultrasound-assisted ultra-turrax homogenization extraction fellowed by high-speed countercurrent chromatography separation,six main naphthoquinones and diarylheptanes in Qinglongyi were obtained,including Regiolone,Juglone,Myricatomento-genin,Galeon,2-Oxatrycyclo[13.2.2.13,7]eicosa-3,5,7(20),15,17,18-hexaen-10-16-diol and Juglanin A.The ultimate yields of six compounds were 0.636 mg/g,0.331 mg/g,1.081 mg/g,0.371 mg/g,1.298 mg/g,0.558 mg/g,respectively.The ultimate recovery of six compounds were 36.98%?72.35%?60.99%?65.72%?60.19%?63.98%.This experiment studied the extraction,separation and purification process of naphthoquinone and diaryheptane in Qinglongyi,which will provide a theoretical basis for the rational development and in-depth utilization of Qinglongyi resources,meanwhile,it will also provide technical support for the efficient extraction,separation and purification secondary metabolites from other plants.