(Morella Rubra Sieb. Et Zucc.) Fruit Extracts and -Glucosidase Inhibitory
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molecules Article Purification of Flavonoids from Chinese Bayberry (Morella rubra Sieb. et Zucc.) Fruit Extracts and α-Glucosidase Inhibitory Activities of Different Fractionations Shuxia Yan 1,2, Xianan Zhang 1,2, Xin Wen 1,2, Qiang Lv 1,2, Changjie Xu 1,2, Chongde Sun 1,2 and Xian Li 1,2,* 1 Zhejiang Provincial Key Laboratory of Horticultural Plant Integrative Biology, Zhejiang University, Hangzhou 310058, China; [email protected] (S.Y.); [email protected] (X.Z.); [email protected] (X.W.); [email protected] (Q.L.); [email protected] (C.X.); [email protected] (C.S.) 2 Laboratory of Fruit Quality Biology, the State Agriculture Ministry Laboratory of Horticultural Plant Growth, Development and Quality Improvement, Zhejiang University, Hangzhou 310058, China * Correspondence: [email protected]; Tel.: +86-571-8898-2630; Fax: +86-571-8898-2224 Academic Editor: Nancy D. Turner Received: 6 July 2016; Accepted: 26 August 2016; Published: 31 August 2016 Abstract: Chinese bayberry (Morella rubra Sieb. et Zucc.) fruit have a diverse flavonoid composition responsible for the various medicinal activities, including anti-diabetes. In the present study, efficient simultaneous purification of four flavonoid glycosides, i.e., cyanidin-3-O-glucoside (1), myricetin-3-O-rhamnoside (2), quercetin-3-O-galactoside (3), quercetin-3-O-rhamnoside (4), from Chinese bayberry pulp was established by the combination of solid phase extract (SPE) by C18 Sep-Pak® cartridge column chromatography and semi-preparative HPLC (Prep-HPLC), which was followed by HPLC and LC-MS identification. The purified flavonoid glycosides, as well as different fractions of fruit extracts of six bayberry cultivars, were investigated for α-glucosidase inhibitory activities. The flavonol extracts (50% methanol elution fraction) of six cultivars showed strong α-glucosidase inhibitory activities (IC50 = 15.4–69.5 µg/mL), which were higher than that of positive control acarbose (IC50 = 383.2 µg/mL). Four purified compounds 1–4 exerted α-glucosidase inhibitory activities, with IC50 values of 1444.3 µg/mL, 418.8 µg/mL, 556.4 µg/mL, and 491.8 µg/mL, respectively. Such results may provide important evidence for the potential anti-diabetic activity of different cultivars of Chinese bayberry fruit and the possible bioactive compounds involved. Keywords: Morella rubra fruit; α-glucosidase inhibitory activity; flavonoid glycosides; purification 1. Introduction Diabetes is an endocrine disorder characterized by elevated blood glucose levels resulting from the defects in insulin-secretory response or resistance to insulin action [1]. One of the therapeutic methods for reducing postprandial hyperglycemia is to delay the absorption of carbohydrates by inhibiting the α-glucosidase activity [2,3]. So far, acarbose, voglibose, and miglitol are frequently used drugs to control hyperglycemia after meals. However, side effects of these compounds, such as flatulence, stomach ache, and diarrhea, are also apparent [4–6]. Natural plant products may provide good alternatives for new anti-diabetic compounds with high safety. Chinese bayberry (Morella rubra Sieb. et Zucc.) is an important subtropical fruit tree in the Myricaceae family and the fruit contains high nutritional and medicinal value [7–9]. Many bioactive compounds, such as cyanidin-3-O-glucoside (C3G) (1), myricetin-3-O-rhamnoside Molecules 2016, 21, 1148; doi:10.3390/molecules21091148 www.mdpi.com/journal/molecules Molecules 2016, 21, 1148 2 of 12 Molecules 2016, 21, 1148 2 of 11 (M3R) (2), quercetin-3-O-galactoside (Q3Gal) (3), quercetin-3-O-rhamnoside (Q3R) (4), and asquercetin-3- cyanidin-3-O-glucosideO-glucoside (Q3G) (C3G) have (1), been myricetin-3- identifiedO-rhamnoside in bayberry [(M3R)10]. Previously, (2), quercetin-3- our resultsO-galactoside showed (Q3Gal)that the ( C3G-rich3), quercetin-3- bayberryO-rhamnoside fruit extract (Q3R) from (4), the and ‘Biqi’ quercetin-3- (BQ) cultivarO-glucoside significantly (Q3G) have decreased been identified blood inglucose bayberry levels [10]. in Previously, streptozotocin our results (STZ)-induced showed that diabetic the C3G-rich mice bayberry [11]. Recently, fruit extract bayberry from fruitthe ‘Biqi’ extracts (BQ) cultivarfrom nine significantly cultivars weredecreased evaluated blood forglucose the glucose levels in consumption streptozotocin activities (STZ)-induced in HepG2 diabetic cells. mice Results [11]. Recently,showed thatbayberry bayberry fruit extracts cultivars from contained nine cultivars higher were contents evaluated of C3G for the and glucose three quercetinconsumption glycosides activities in HepG2 cells. Results showed that bayberry cultivars contained higher contents of C3G and three (Q3G, Q3Gal, and Q3R) showed higher glucose consumption activities than those cultivars with lower quercetin glycosides (Q3G, Q3Gal, and Q3R) showed higher glucose consumption activities than those contents of C3G and quercetin glycosides [12]. However, further characterization and comprehensive cultivars with lower contents of C3G and quercetin glycosides [12]. However, further characterization and investigation of these important bioactive compounds from bayberry were limited by the availability comprehensive investigation of these important bioactive compounds from bayberry were limited by the of pure compounds and relative high quantities required by further studies. So far, few efficient availability of pure compounds and relative high quantities required by further studies. So far, few efficient separation methods have been established for simultaneous purification of these compounds with separation methods have been established for simultaneous purification of these compounds with high recoverieshigh recoveries and high and yields. high yields. TheThe present present study study was was designed designed to develop to develop an effi ancient efficient isolation isolation method method to purify to C3G purify and C3Gthree flavonolsand three (M3R, flavonols Q3Gal, (M3R, and Q3R) Q3Gal, (Figure and 1) Q3R) from (Figure BQ pulp1) at from the same BQ pulp time, at using the samea combination time, using of solid a ® phasecombination extraction of solid(SPE) phase by C18 extraction Sep-Pak® (SPE) cartridges by C18 column Sep-Pak chromatographycartridges column and semi-preparative chromatography HPLC and (Prep-HPLC).semi-preparative α-Glucosidase HPLC (Prep-HPLC). inhibitoryα -Glucosidaseactivities and inhibitoryantioxidant activities activities and of antioxidant the purified activities flavonoid of glycosides,the purified fractions, flavonoid and glycosides, extracts of fractions, six bayberry and cultivars extracts were of six also bayberry evaluated. cultivars were also evaluated. FigureFigure 1. Chemical structures of fourfour mainmain flavonoidflavonoid glycosidesglycosides fromfrom ChineseChinese bayberrybayberry pulppulp extract: extract: cyanidin-3-cyanidin-3-OO-glucoside-glucoside (C3G) (C3G) (1); ( 1myricetin-3-); myricetin-3-O-rhamnosideO-rhamnoside (M3R) (M3R) (2); quercetin-3- (2); quercetin-3-O-galactosideO-galactoside (Q3Gal) ((Q3Gal)3); and quercetin-3- (3); and quercetin-3-O-rhamnosideO-rhamnoside (Q3R) (4). (Q3R) (4). 2. Results and Discussion 2. Results and Discussion 2.1. Purification of Four Flavonoid Glycosides from BQ Pulp 2.1. Purification of Four Flavonoid Glycosides from BQ Pulp A simple and efficient method by the combination of column chromatography and Prep-HPLC was A simple and efficient method by the combination of column chromatography and Prep-HPLC established for rapid isolation and purification of four flavonoid glycosides from BQ pulp. was established for rapid isolation and purification of four flavonoid glycosides from BQ pulp. Based on our preliminary experiment, the SPE column showed good isolation between C3G and three Based on our preliminary experiment, the SPE column showed good isolation between C3G and flavonols (M3R, Q3Gal, and Q3R). The dynamic adsorption and desorption tests of C3G were first carried outthree due flavonols to their (M3R,high quantity Q3Gal, (F andigure Q3R). 2). The dynamic leakage adsorption curves and of desorption C3G on the tests SPE of column C3G were were obtainedfirst carried based out on due the to volume their high of effluent quantity and (Figure the initial2). The concentration dynamic leakage of the curvessample of solution. C3G on The the initial SPE feedcolumn concentration were obtained of C3G based on on SPE the column volume was of effluent 1.18 mg/mL. and the Dynamic initial concentration adsorption and of the desorption sample experimentssolution. The showed initial feedthat the concentration breakthrough of C3Gvolume on SPEof C3G column on SPE was column 1.18 mg/mL. was about Dynamic 4 bed volume adsorption (BV) (Figureand desorption 2A), and experiments the majority showedof C3G thatabsorbed the breakthrough by SPE column volume was eluted of C3G by on 10% SPE methanol column was(with about 0.5% formic4 bedvolume acid) solution (BV) (Figure (Figure2A), 2B). and Therefore, the majority 10% ofme C3Gthanol absorbed aqueous by solution SPE column was used was elutedas the eluent by 10% in isocraticmethanol elution, (with 0.5%where formic complete acid) desorption solution (Figure was done2B). Therefore,with an elution 10% methanolvolume of aqueous about 120 solution mL (10 was BV) (Figureused as 2C). the eluentThe eluents in isocratic were elution,collected where as 10% complete fractions, desorption the SPE wascolumn done was with then an elutionwashed volume with 50% of methanolabout 120 aqueous mL (10 BV)solution,