Isolation And Purification Of Different Components Of Polygonatum Sibiricum Before And After Processing

Polygonatum sibiricum has the functions of supplementing the middle and boosting Qi,moistening the heart and lung,and strengthening the muscles and bones,but,raw Polygonatum sibiricum is spicy,stimulates the throat,and triggers an allergic reaction,while "nine steaming and nine drying" Polygonatum sibiricum is sweet and has no irritation or sensitization."Nine steaming and nine drying" processing will change the chemical composition of Polygonatum sibiricum,remove allergenic substances,eliminate its toxicity and enhance its curative effect.At present,the determination of allergenic chemical substances and their isolation and purification are not thoroughly studied,and the biological activity and pharmacological studies of allergenic substances are lacking.In this project,ultrasound-assisted extraction combined with high performance liquid chromatography(HPLC)is used to obtain a complete HPLC chromatogram of Polygonatum sibiricum,differences in chemical composition are determined before and after the processing of "nine steaming and nine drying" and the combined chemical separation technology(macroporous resin combined with preparative chromatography)are used to separate and purify the dissimilar chemical substances,using the "four major spectra"(NMR,MS,ultra-violet spectrum,FTIR)to identify chemical structures.The results of the experiment are as follows:(1)The best solvent for ultrasound-assisted extraction is ethanol;(2)The analysis parameters of HPLC fingerprint are as follows:Ultimate XB-C18,5 ?m,4.6x250 mm column,the sample concentration is 0.0088 g/mL,the sample size is 20 uL,the detection wavelength is 281 nm,the column temperature is 35 ?,the flow rate is 1.0 mL/min,and the gradient elution procedure is 0-5 min 98%-70%A,5?20 min 70%?40%A,20?25 min 40%?25%A,25?30 min 25%?0%A,30?40 min 0%A;(3)The retention time of the major chemical components before and after the "nine steaming and nine drying" process is approximately 16.59 min,17.10 min,18.30 min,19.26 min,21.08 min,22.00 min,23.63 min,25.61 min;(4)The process parameters of D101 type macroporous resin in separating differentiation are as follows:1 Bv of 20%ethanol-water solution(the concentration of the extract is 0.5 g raw Polygonatum sibiricum/mL)is put into column,washing the colomn with 2 Bv of 20%ethanol-water,40%ethanol-water,60%ethanol-water,80%ethanol-water,100%ethanol solution at the room temperature step by step,each concentration of eluent is detected by HPLC,the differential chemical substances are mainly trapped in 80%ethanol-water elution,and the extract is used as raw material for preparing chromatography;(5)The analysis parameters of preparative chromatography are as follows:Ultimate XB-C18,10 ?m,21.2×250 mm column,the sample concentration is 0.02 g/mL,the sample size is 5 mL,the detection wavelength is 281 nm,the column temperature is 35 ?,the flow rate is 10 mL/min,and the gradient elution procedure is 0?70 min 70%A,70?115 min 60%A,sample collection time is 0?13 min,13?22 min,22?36 min,36?59 min,59?69 min,69?88 min,88?99 min,99?114 min;After HPLC analysis,the samples with chromatographic retention times of 99-114 min are pure substances and the corresponding retention time in HPLC fingerprints was approximately 23.63 min.At the same time,this substance is also the chemical substance with the highest content loss during the "nine steaming and nine drying" process;(6)Chemical structures are identified using the "four major spectra" and the chemical information of the component having the retention time of 99-114 min in preparative chromatography is as follows:name is 4',5,7-Trihydroxy-6,8-dimethylhomoisoflavanone,chemical formula is C18H18U5.In this project,HPLC is used to determine the differential chemical substances before and after the process of "nine steaming and nine drying",and the green combined separation technology is used to separate and purify the different substances.The process is simple and has low cost.These studies provide sufficient preparation for the determination of the sensitizing substances as well as the biological activity and pharmacological research.It's helpful to accelerate the development of the Chinese medicine's internationalization and modernization.