Th the GPC software, applying KS-805 and KS-804 connection in series (Shodex Co., Tokyo, Japan). Gas chromatography (GC) was performed having a TRACE GC apparatus (Thermo Fisher Scientific, Waltham, MA, USA) equipped having a DB-624 column (length: 30 m 0.32 mm, thickness of liquid phase: 1.eight m, Agilent) for the determination of O-methyl group. GC-MS was analyzed having a TRACE DSQ apparatus (Thermo Fisher Scientific) equipped having a TR-5 column (length: 60 m 0.25 mm, thickness of liquid phase: 0.25 m, Thermo Fisher Scientific). The GC-MS temperature plan utilized for monosaccharide evaluation was 14098 at 2 /min, held for four min, rising to 214 at 4 /min, followed by a 1 /min gradient as much as 217 , held for 4 min, and finally to 250 at three /min held for four min; the methylation GC-MS system was 14080 at two /min, followed by a 1 /min gradient as much as 200 , and finally to 250 at 3 /min held for 5 min. (a) Extraction, isolation and purification Green tea leaves (2.five kg) had been extracted with 25 L water at 100 for two h. The residue was removed by filtration. The spent leaves were re-extracted below the exact same situations. The supernatant was combined and concentrated below decreased pressure. The crude TPS extract was obtained by means of precipitation by adding ethanol towards the concentrated resolution until the ethanol concentration reached 40 (40 ethanol precipitation fraction, TPS1, 102 g, yield 4.08 ) and 70 (70 ethanol precipitation fraction, TPS2, 53.2 g, yield 2.13 ), respectively. The precipitates had been collected by centrifugation (9829g, ten min), washed twice with 95 ethanol, and freeze-dried. The flow chart showing the process of isolating numerous fractions of polysaccharides is presented in Figure 1. TPS1 (5 g) was dissolved in 40 mL distilled water and centrifuged (43,540g, ten min).4,15-Isoatriplicolide methylacrylate The residue was dissolved in 20 mL distilled water once more and centrifuged (43,540g, ten min) to eliminate the residue.Omidenepag The supernatant was combined and loaded on a DEAE-cellulose column (50 cm 5 cm) pre-treated with 0.PMID:24834360 5 M NaOH, 0.5 M HCl and equilibrated with distilled water. The TPS1 was initially eluted with distilled water and then with 0.1, 0.two, 0.four and two.0 M of NaCl by stepwise increments. The fraction eluted with 0.two M NaCl (TPS1-2) was further fractionated on a High-Resolution SephacrylTM S-300 column (60 cm 2.six cm) and eluted with water to give two main fractions of TPS1-2a (18020 min) and TPS1-2b (24080 min). (b) Homogeneity and molecular weight Homogeneity and molecular weights from the isolated polysaccharides have been determined by high efficiency gel permeation chromatography (HPGPC), KS-805 and KS-804 column in serials, ID eight mm, and length 300 mm, (Shodex Co., Tokyo, Japan) [38]. The normal curve was established utilizing different pullulans with identified molecular weight (P-5, P-10, P-20, P-50, P-100, P-200, P-400 andInt. J. Mol. Sci. 2014,P-800, Shodex Co.). The column temperature was kept at 40.0 0.1 . NaCl 0.2 M was utilized as an eluant plus the flow rate was kept at 0.8 mL/min. All samples were prepared as two mg/mL options, and 20 L aliquot was injected for every single run. (c) Monosaccharide analysis The polysaccharide sample was hydrolyzed with two M TFA at 121 for 2 h. Soon after repeated evaporation with methanol to absolutely take away TFA, the residue was dissolved in 0.1 mL of distilled water and analyzed on a PEI-cellulose plate (Merck, Darmstadt, Germany), created with EtOAc-pyridine-AcOH-water 5:5:1:3 (v/v). The plate was visualized by spraying with O-phthalic a.
