Study On Extraction And Enrichment Of Main Flavonoids From Sea Buckthorn Leaves

In the present study, the new and green extraction solvent-deep eutectic solvents (DESs) were used for the efficient and rapid extraction of five main flavonoids compounds (rutin, quercetin-3-O-glucoside, quercetin, kaempferol, isorhamnetin) from sea buckthorn (Hippophae rhamnoides) leaves. The extraction process conditions of deep eutectic solvent-based microwave-assisted extraction were optimized. The process of separation and purification of rutin, quercetin-3-O-glucoside, quercetin, kaempferol, isorhamnetin by macroporous resin and antioxidant activity of crude extracts were studied, respectively. An efficient, fast and accurate method was for the analysis of active constituents sea buckthorn leaves. Our study provided scientific guidance for reasonable development and efficient utilization of sea buckthorn leaves. The results were as follows:1. A HPLC method was developed for the simultaneous analysis and determination of utin, quercetin-3-O-glucoside, quercetin, kaempferol and isorhamnetin in sea buckthorn leaves, the chromatographic conditions as follows:Chromatographic column:KYA HIQ sil C18W column (250 mm × 4.6 mm i.d.,5 μm), flow rate 1.0 mL/min, column temperature 30 ℃, injection volume 5 μL, detection wavelength 254 nm and 370 nm. Mobile phase consisted of acetonitrile (A) and 0.4%phosphoric acid aqueous solution (B), gradient elution was performed as follows:0-7 min,10-15%A; 7-10 min, 15-17%A; 10-25 min,17-17%A; 25-27 min,17-18%A; 27-31 min,18-19%A; 31-34 min, 19-19%A; 34-38 min,19-26%A; 38-42 min,26-26%A; 42-55 min,26-50%A; 55-60 min, 50-10%A.2. The process of microwave-assisted deep eutectic solvent extraction was optimized by a Box-Behnken Design (BBD) and response surface methodology (RSM). The optimum parameters were as follows:Deep eutectic solvents (DESs):ChCl/1,4-butanediol= 1/3 (mol/mol.20%water content)Extraction temperature:64 ℃Extraction time:18 minLiquid/solid ratio:20:1 mL/gUnder the optimal parameters, the average extraction yields of rutin, quercetin-3-O-glucoside, quercetin, kaempferol and isorhamnetin were 8.967,1.809,9.107.0.448 and 0.489 mg/g, respectively. The deep eutectic solvent-based microwave-assisted extraction process is better than deep eutectic solvent-based ultrasound-assisted extraction and deep eutectic solvent-based hot reflux extraction.3. The optimum process for purification of flavonoids (rutin, quercetin-3-O-glucoside, quercetin, kaempferol and isorhamnetin) in sea buckthorn leaves from DESs was obtained, and the antioxidant activities were evaluated, the results were as follows:In this work, the direct enrichment of five main flavonoids from DES solution were performed using macroporous resin HPD-826, DM-130, D-3520, NKA-9, AB-8 and D101. It was found that among the six kinds of macroporous resin, AB-8 resin was the best in enrichment and separation of rutin, quercetin-3-O-glucoside, quercetin, kaempferol and isorhamnetin with recovery yields of 83.59%> 74.46%.84.99%.72.36%.77.85%. The antioxidant activity of extracts enriched by macroporous resin NKA-9, AB-8, D101 were assessed and compared. The results showed that AB-8 resin possessed better antioxidant activity by DPPH assay with IC50 value of 0.074 mg/mL, ABTS assay with TEAC value of 0.662 mmol/g Trolox, and reducing power test with IC50 value of 0.127 mg/mL.In this study, it was completed that the optimization of deep eutectic solvent microwave-assisted deep eutectic solvent extraction of five main flavonoids from sea buckthorns. The direct enrichment and separation of five main flavonoids from extracts were performed, and the antioxidant activities were evaluated. As a potential eco-friendly technology, it possesses important theoretical value for green and efficient extraction and enrichment of natural products. This study provides a scientific basis for the comprehensive development and utilization of sea buckthorn leaves.