Study On Extraction And Enrichment Of Main Flavonoids From Equisetum Palustre L

In the present study, deep eutectic solvents (DESs) as new extraction solvents, are proposed for the green and efficient extraction and separation of bioactive flavonoids from Equisetum palustre L. with negative pressure cavitation-assisted extraction (NPCE) combined with macroporous resin enrichment. In addition, the DES-NPCE extraction procedures were also optimized systematically. Our study focused on extraction, enrichment and separation of kaempferol-3,7-di-O-β-D-glucoside (KG), luteolin-7-O-β-D-glucoside (LG), quercetin-3-O-β-D-glucoside (QG), genkwanin-5-O-β-D-glucoside (GG) from Equisetum palustre L., developed a fast and efficient analysis method of HPLC, and established a rapid extraction process. The results as follows:1. An efficient HPLC method for determination of four flavonoids was developed as follow:The chromatographic column: Curosil-PFP column (5μm,250 mm ×4.6 mm i.d.); mobile phase:water (0.10% formic acid) (A)-methanol (B); column temperature: 30℃; detection wavelength: 349.4 nm, 341.1 nm, 344.2 nm, 330.4 nm; flow rate:1 mL/min; njection volume: 5 μL. The following gradient elution program was used for separation: 0-10 min,33-33% B; 10-12 min,33-36% B; 12-20 min,36-36% B; 20-22 min,36-44% B; 22-36 min,44-44% B; 36-40 min,44-51% B; 40-50 min,51%-75% B.2. The process was optimized by a Box-Behnken Design (BBD) and response surface methodology (RSM). The optimal parameters of negative pressure cavitation assisted deep eutectic solvent extraction process were as follows:Deep eutectic solvents (DESs):Choline chloride/betaine hydrochloride-ethylene glycol: 1/1/2 (mole ratio) DESs/water ratio:20%Negative pressure cavitation-assisted extraction method: Extraction pressure:-0.07 MPa Extraction temperature:63℃ Solvent to solid ratio:27:1 mL/g Extraction time:20 minBased on the above optimized conditions, the average extraction yield of KG, LG, QG, GG was 4.85 mg/g, 3.94 mg/g, 1.95 mg/g, 3.95 mg/g (n=3). Deep eutectic solvents-based negative pressure cavitation-assisted extraction process is better than 80% ethanol-based negative pressure cavitation-assisted extraction and deep eutectic solvents-based ultrasound-assisted extraction.3. In this work, the direct enrichment of four target flavonoids from DES solution were performed using macroporous adsorption resin NKA-9, AB-8, and HPD-826. It was found that among three kinds of macroporous adsorption resin, HPD-826 resin exhibited better performance. After one run treatment with HPD-826 resin, the contents of the four target flavonoids separated by HPD-826 resin were 67.76%, 64.40%, 89.79% and 79.17%, and the recovery yields were 3.51%, 2.71%, 1.87% and 3.34%, indicating HPD-826 resin could effectively enrich four target flavonoids from DES extraction solution.In the present study, the DES-NPCE was chosen to extract the flavonoids compounds from Equisetum palustre L.. DESs as a new kind of extraction solvent, possessed sustainability, biodegradability, pharmaceutical acceptable toxicity and high extractability for compounds with diverse polarity, and could be used as green solvents for the extraction of active components from plant materials. The developed procedures based on DES-NPCE and direct macroporous adsorption resin enrichment processes, could be an alternative and environmental method for the extraction and enrichment of natural products from plant sample matrices.