Analytical Methods Of Multiple Active Factors From Zizyphi Spinosi Semen

Nowadays,modem fast-paced lifestyles are putting more pressure on people.According to a World Health Organization（WHO）survey,27%of people worldwide have varying degrees of sleep problems.At the same time,the number of people suffering from insomnia,anxiety,depression and other diseases is increasing year by year,so the demand for tranquilizers is increasing.Although benzodiazepines are effective in treating insomnia,they are prone to adverse reactions such as dizziness,fatigue and drowsiness,and long-term use will also cause dependence.Zizyphi Spinosae Semen（ZSS）is the seed of jujube plant belonging to Rhamaceae.It has the effects of tranquilizing nerves and soothing nerves.It is a raw material of medicine and food homology.Therefore,it is more urgent and important to carry out in-depth research on the ZSS.In this study,an HPLC-MS-MS method was developed for the simultaneous determination of multiple active ingredients in ZSS.The separation was performed on an Agilent Poroshell 120 EC-C18（2.1 mm × 50 mm,4.0 μm）column with a mobile phase composed of methanol and 0.1%formic acid at a flow rate of 0.2 mL/min.In addition,column temperature and injection volume were 40℃ and 3μL.In electrospray ionization（ESI）and multiple reaction monitoring（MRM）mode,the quantitative determination of multiple active factors in ZSS was carried out.A good linear relationship between the peak area and the concentrations of magnoflorine,spinosin,6"’-feruloylspinosin,jujuboside A,jujuboside A1,jujuboside B and jujuboside B1 was obtained.The average recovery rates for them were 97.72%、98.15%、97.51%、97.49%、98.31%、97.67%and 97.66%,respectively,with relative standard deviation less than 2.0%.An HPLC-ELSD method was also developed for the simultaneous detection of betutin and betulinic acid in ZSS.The separation was performed on an Agilent Poroshell 120 EC-C₁₈（4.6 mm × 150 mm,4.0 μm）column with a mobile phase composed of methanol:water:acetic acid（88:12:0.05）at a flow rate of 1.0 mL/min for isocratic elution.In addition,column temperature and injection volume were 40℃ and 10 μL.The evaporation light temperature and carrier gas flow rate were 85℃ and 1.5 L/min.A good linear relationship between the peak area and the concentrations of betutin and betulinic acid was obtained.The average recovery rates for them were 96.94%and 97.68%,respectively,with relative standard deviation less than 2.0%.These two analysis methods have high sensitivity and good repeatability.In order to further use for the detection,the optimal extraction conditions for the extraction of various active ingredients in ZSS were established by L9（34）orthogonal test,the ratio of material to liquid was 1:30 g/mL,the volume fraction of ethanol was 50%,the extraction temperature was 80℃,and the extraction time was 3 h.The experimental results show that under these conditions,magnoflorine,spinosin,6"’-feruloylspinosin,jujuboside A,jujuboside A1,jujuboside B,jujuboside B1,betutin,betulinic acid in ZSS were relatively high,and their contents are（303.26 ± 3.53）μg/g,（905.55 ±3.93）μg/g,（547.77± 7.29）μ/g,（289.77± 4.38）μg/g,（92.86 ± 1.71）μg（189.33± 2.45）μg/g,（8.09± 0.16）μg/g,（115.00 ± 0.01）μg/g and（442.57 ± 0.02）μg/g;however,the contents of puerarin and sanjoinine E in ZSS were less,which did not reach the detection limit.Based on the establishment of analytical methods and the optimization of extraction conditions.This project uses the method of stir-fried processing to observe the change of the content of active ingredients by changing the degree of raw and ripening of ZSS.The experimental results show that the contents of various active ingredients in ZSS gradually decrease between 0～50 minutes with 150℃.And after 65 minutes,the ZSS begin to produce oil,and at 80 minutes,they show a wide range of oil.This study found changes in the active ingredient content of ZSS with different degrees of maturity,laying a foundation for future research on raw and fried ZSS products.