| Pigeon pea is a common food legume crops, the main ingredients are flavonoids and stilbenes. It is reported that the pharmacological effects of these compounds is very broad, including anti-inflammatory, anti-viral, anti-oxidation and protect nerve cells. In the present study, a high-pressure liquid chromatography (HPLC) method was developed for stimulation analysis of cajaninstilbene acid (CSA), pinostrobin (PI), pinostrbin chalcone (PIC) and longistyline C (LLC) from pigeon pea leaves. An efficient extraction method, negative-pressure cavition-aqueous two-phase extraction, was established for extraction of CSA, PI, PIC and LLC. The parameters of the extraction process were optimized. The results as follows:1. HPLC-UV analysis method for simultaneous determination of CSA, LLC, PI and PIC was established as follows:Chromatographic column:Phenomenex Gemini C18110A reverse column (250mm×4.6mm i.d.,5μm), flow rate1mL/min, column temperature30℃, detection wavelength287.6nm,341.1nm,298.3nm and258.0nm, injection volume5μL. A mobile phase was0.1%formic acid (A)-methanol (B). The following gradient elution program was used for separation:0-15min,77-80%(B);15-20min,80-90%(B);20-22min,90-100%(B).Under the decetion conditions, the linear relationships of target compounds were significant. R2of standard curve were all higher than0.9988. R.S.D of precision, repeatability and stability were less than5%. The recoveries were between97.36%and101.82%with R.S.D llower than4%.2. The NPCE process parameters of CSA, LLC, PI and PIC were optimized. The optimal parameters as follows:Solid/liquid ratio:1:50(g/g)Extraction time:3minNegative pressure:-0.07MPaExtraction temperature:30℃Ethanol concentration:80%(v/v)Under the above optimum conditions, the yield of CSA, LLC, PI and PIC from pigeon pea leaves were9.35mg/g,4.56mg/g,4.01mg/g and0.452mg/g. The contents of target compounds were2.254%,0.943%,0.906%and0.426%, significantly.3. Under the above optimum conditions of Negative pressure cavtation process, aqueous two-phase system was established and optimazed. The concrete parameters as follows:Concentration of ethanol:23%Concentration of (NH4)2SO4:21% Temperature:35℃Under the above optimum conditions, the extraction yield were10.175,5.162,4.925and0.523mg/g (n=3). The contents of target compounds were4.557%,2.204%,2.072%and0.873%, significantly. Compared with that extracted with ethanol, that extracted with ATPE were all improved more than2-folds.4. Comparision of antioxidant activities of crude extracts with different method:According to the DPPH, ABTS and FRAP assay, the extracts of NPC-ATPE had significant antioxidant activities with IC50of0.337mg/mL and TEAC of0.1511and0.2611mmol/g, respectively. The results demonstrated that NPC-ATPE could be an alternative method for natural compounds simultaneous extraction and enrichment in the single step.The results of this study fully proved NPC-ATPE were very applicable for efficient extraction and enrichement of flavonoid and stilbene compounds in pigeon pea leaves. It may provide valuable data for process design and pilot-scale as well as industrial scale-up applications, and as a potential eco-friendly technology, it is promising for extensive industry applications in the nature product extraction fields. |
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