Screening And Separation Of Active Components Of Natural Products Based On Linear Gradient Countercurrent Chromatography

Lonicera japonica Thunb.leaves is the by-product of honeysuckle,the important medicinal and edible plants in China.In related research reports,it was believed that Lonicera japonica leaves had similar biological activity as honeysuckle.Lonicera japonica leaves contains a wide range of active ingredients and has a high value and potential for the separation of biologically active substances.As a new separation technology,countercurrent chromatography（CCC）is increasingly used in the separation of various biologically active components,and its separation mode has become more and more abundant.In these new separation modes,the gradient countercurrent chromatographic separation mode has a good separation effect for samples with a wide range of polarities,but still faces problems such as severe loss of stationary phase and excessive use of organic solvents at the same time.In order to solve these problems in the gradient countercurrent chromatography mode,to effectively separate the bioactive components with a wide range of polarities in the sample better,this experiment improved the existing gradient countercurrent chromatography technology and replaced the single pump with the binary pump mode,proposed a new linear gradient countercurrent chromatography mode by simulating the elution principle of HPLC.The experimental results showed that the linear gradient countercurrent chromatography technique proposed in this experiment can effectively and quickly separate biologically active components with the wide range of polarities,would not cause serious loss of stationary phase,and the amount of organic solvent used was also greatly reduced,which was very excellent for its development prospects.In addition,this paper also combined the rapid screening experiment of antiviral activity,rapid screening the antiviral activity of isolated substances,proposed the screening and separation method of natural product active ingredients by"linear gradient countercurrent chromatography rapid separation-segment collection-bioactivity rapid screening",which can be combined with various purification methods,makes the purification works more purposeful and efficient.In this paper,the antiviral activity experiment proved that Lonicera japonica leaves had good antiviral activity,the ethyl acetate extract and n-butanol extract were its major antiviral fractions.The research content is mainly divided into the following three parts:1.First chapter.Summarize the application history,research status and market development potential of Lonicera japonica leaves,discuss its feasibility in the development and production of food industry.Introduces the working principle,research progress,application advantages of countercurrent chromatography and gradient countercurrent chromatography.Introduce the action mechanism of neuraminidase and the principle of rapid screening experiments.2.Second chapter.A petroleum ether-ethyl acetate-water solvent system suitable for ethyl acetate extract and an ethyl acetate-n-butanol-water solvent system suitable for n-butanol extract were constructed for the polar characteristics of ethyl acetate extract and n-butanol extract,and doing countercurrent chromatography methodological experiments of them.The optimum linear gradient countercurrent chromatography operating parameters were determined for these two solvent systems.For the ethyl acetate extract,the best mode of operation was“FWD-OUT”with an optimum speed of 800 rpm and an optimum flow rate of 5.0 m L/min;For n-butanol extract,the best mode of operation was“FWD-OUT”with an optimum speed of 700rpm and an optimum flow rate of 3.0 m L/min.Taking the ethyl acetate extract and n-butanol extract of Lonicera japonica leaves as an example,the injection volume and gradient condition were optimized,the bioactive substances were successfully separated,and the materials separated by linear gradient countercurrent chromatography were collected in stages,detected into the HPLC for analysis.3.Third chapter.Through preliminary experiments on antiviral activity,the isolated substances collected by linear gradient countercurrent chromatography,each organic solvent extracts of Lonicera japonica leaves,and the positive control substances were rapidly screened for antiviral activity by the neuraminidase activity test.Each substance measures the IC₅₀ value of neuraminidase to determine the antiviral activity strength of them.Among them,the IC₅₀ value of ethanol extract of Lonicera japonica leaves to neuraminidase was 0.4411±0.0606 mg/m L,which proved that Lonicera japonica leaves had good antiviral activity;the ethyl acetate extract and n-butanol extract were the main part of antiviral activity in Lonicera japonica leaves,the IC₅₀ values for neuraminidase were 0.2133±0.0089 mg/m L and 0.1868±0.0155 mg/m L,respectively.By the substances isolated from ethyl acetate extract of Lonicera japonica leaves,the antiviral activity of Part B was the best.The IC₅₀ value of neuraminidase was 0.0652±0.0001 mg/m L;by the substances isolated from n-butanol extract of Lonicera japonica leaves,the antiviral activity of Part C was the best.The IC₅₀ value of neuraminidase was 0.1300±0.0076 mg/m L.