| Litchi (Litchi chinensis Sonn.) is an important tropical to subtropical fruit. The litchiacreage and production of China rank first in the world. Since centralized harvest periodand intolerant storage of litchi, deep processing for promoting healthy and sustainabledevelopment of litchi industry is very important. Ancient records litchi has a tonic effect.To investgate the health effects material basis and bioactivity mechanism of litchi iscritical important to guide deep processing of litchi industry. Modern pharmacologicalstudies confirmed that litchi fruit has anti-oxidant anti-radioactive and hepatoprotectivefunctions. However, little is known on its bioactive substances. Our lab and abroadresearch showed similar results that litchi pulp is rich in polyphenols and has significantantioxidant activity. But still lack of awareness of the antioxidant activity of differentphenolics from litchi pulp. Modern Nutrition found that polyphenols regulate the miRNAexpression of lipid metabolism. As an important source of dietary polyphenols forsubtropical area residents the effects of litchi pulp polyphenols on lipid metabolism hasnot been reported. Therefore, the present study was to investigate the regulation lipidmolecular metabolism of litchi pulp phenolics in hyperlipidemic mice from the expressionlevels of miRNA after determine the optimum extraction conditions for litchi pulppolyphenols, to elucidate the major bioactive structure substances of phenolic compounds.The results have important implications for the promotion of litchi consumption,functional food deep processing and litchi industry sustainable development.1. Comparison of phenolic profiles extraction efficiency and cellular antioxidantactivities of litchi pulp extracts from different solvents Five different solvent mixtures were used to extract free phenolics from litchi pulp.Alkaline and acid hydrolysis were tested to hydrolysis bound phenolics from litchi pulpresidue. The antioxidant activities of litchi pulp extracts were evaluated using the oxygenradical absorbance capacity (ORAC) and cellular antioxidant activity (CAA) assays.Aqueous acetone extraction of litchi free phenolics exhibited the highest free phenolicscontent. The acid hydrolysis bound phenolics content of litchi pulp was2.0times higher than those obtained by alkaline hydrolysis. Aqueous acetone extraction exhibited the highest ORAC value. But for CAA, aqueous acetone extraction represented the same CAA value as aqueous methanol extraction. The bound phenolics obtained by acid hydrolysis exhibited2.6times ORAC and1.9times CAA values relative to that obtained by alkaline hydrolysis. Aqueous acetone solvent suitable for free phenolics extraction of litchi pulp, and acid hydrolysis exhibited better extraction efficency for its bound phenolics.2. Separation and purification of phenolic profiles in litchi pulp by macroporous resinThe static adsorption and desorption performance of eleven different polarities macroporous resins to litchi pulp total phenolics and total flavonoids were compared to select suitable resin for purification of phenolic compounds. The dynamic adsorption and desorption process parameters of macroporous resin of HPD-826were optimized. The phenolic compounds types and contents of litchi pulp were analyzed by HPLC. The results showed that HPD-826macroporous resin exhibited the best capability of adsorption and desorption of total phenolics and total flavonoids in litchi pulp. The optimal separating process parameters were as follows:the concentration of litchi pulp extract and the sampling rate were0.8mg/mL and3.0BV/h, respectively, and the elution concentration and flow velocity were95%ethanol and3.OB V/h, respectively. The contents of phenolic compounds of litchi pulp were deduced a little, but the phenolic profiles of litchi pulp were not changed after adsorption and desorption by HPD-826macroporous resin. Nine phenolic compounds,3,4dihydroxybenzoic acid, catechin, vanillic acid, caffeic acid, syringic acid, epicatechin,4-methylcatechol, ferulic acid and rutin, were preliminary identified by HPLC. The major phenolic profiles were4-methylcatechol, rutin and epicatechin. The percentage contribution of the three compounds to the total phenolic content was94.37%. In conclusion, HPD-826macroporous resin could be applied to purify total phenolics and total flavonoids in litchi pulp.3. Molecular mechanism of litchi pulp polyphenols hypolipidemic effectC57BL/6J male mice were divided into control group, high fat diet group and litchi pulp polyphenol group. Litchi pulp polyphenol group intake of high fat diet while intake by gavage a dose of500mg/kg?d of litchi pulp polyphenols separation and purification by macroporous resin. Ten weeks later, the levels of serum and liver lipid metabolism related indicators were analyzed by enzymatic assays. miRNA and mRNA were analyze using real-time quantitative PCR, and proteins were studied by Western blotting. Litchi pulp polyphenols represses miR-33and miR-122in the liver mice models that were induced by a high fat diet or a high fat diet plus dietary polyphenol extracts. Litchi pulp polyphenols promotes the expression of Abcal, miR-33target gene, and represses the expression of miR-122inderect target gene Fas. Besides, LPP improves fatty acid oxidation by enhance the expression of Cptla. These results show that LPP enhance the liver cholesterol efflux and improve HDL formation and reduce fatty acid synthesis and raise fatty acid oxidation. Therefore, the new molecular mechanism of LPP exert hypolipidemic effects in the liver could be considered the effects of LPP on the suppression of miR-33and miR-122.4. Structural elucidation and cellular antioxidant activity evaluation of major antioxidant phenolics in litchi pulpIn the present study, the major contributors to the antioxidant activity of fresh litchi pulp were identified and their cellular antioxidant activities were investigated. Aqueous acetone extracts of litchi pulp were fractionated on polyamide resin, and those fractions with the largest antioxidant and radical scavenging activities were selected using CAA and ORAC assays. Three compounds that were major contributors to the antioxidant activity in these fractions were obtained by reverse-phase preparative HPLC and identified as quercetin3-O-rutinoside-7-O-a-L-rhamnosidase (quercetin3-rut-7-rha), quercetin3-O-rutinoside (rutin) and (-)-epicatechin using NMR spectroscopy, HMBC, and ESI-MS spectrometry. The concentration of these three compounds were17.25,3.58and2.31mg per100g of litchi pulp fresh weight. The highest content of litchi fruit4-methylcatechol identified by HPLC was confirmed as quercetin3-rut-7-rha by spectroscopic. These results suggest that the highest content and strongest antioxidant activity component of litchi pulp was quercetin3-rut-7-rha. Thus, this compound of litchi pulp may exert the effects of antioxidant and regulation of lipid metabolism. This is the first report of the identification and cellular antioxidant activity of quercetin3-rut-7-rha from litchi pulp.The highlights of the present study could be summarized as following two points. Firstly, extraction and purification methods of free and bound phenolics of litchi pulp were established. The phenolic compounds quercetin-3-O-rutinose-7-O-a-L-rhamnoside was fistly isolated and identified from litchi pulp, and confirmed its as the major antioxidant component which could provide the material foundation to reveal the health effects of litchi. Secondly, Discovered and confirmed polyphenols of litchi pulp exert hypolipidemic effects and improve lipid metabolism. For the first time clear molecular mechanisms of litchi phenolics regulating lipid metabolism from the expression of miR-33, miR-122and miR-370and its target gene ABCA1, Fas, Cptla and protein.The topic of the present study elucidated the structural of major antioxidant phenolics in litchi pulp which could provides a scientific basis to reveal the health. protective effect of polyphenols structure-activity relationships.This study investgated the effects of plant polyphenol on the expression of miRNA and its target genes mRNA of lipid metabolism. This could provide new methods and new ideas to explore the molecular mechanisms of other fruits and vegetables biological activity. |
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