With a TRACE GC apparatus (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a DB-624 column (length: 30 m ?0.32 mm, thickness of liquid phase: 1.8 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 system applied for monosaccharide evaluation was 140?98 at 2 /min, held for 4 min, growing to 214 at four /min, followed by a 1 /min gradient as much as 217 , held for four min, and finally to 250 at three /min held for 4 min; the methylation GC-MS system was 140?80 at 2 /min, followed by a 1 /min gradient as much as 200 , and finally to 250 at three /min held for 5 min. (a) Extraction, isolation and purification Green tea leaves (two.5 kg) were extracted with 25 L water at one hundred for 2 h. The residue was removed by filtration. The spent leaves were re-extracted under the identical situations. The supernatant was combined and concentrated below reduced stress. The crude TPS extract was obtained through precipitation by adding ethanol to the concentrated solution till the ethanol concentration reached 40 (40 ethanol precipitation fraction, TPS1, 102 g, yield 4.Ethyl 2-amino-5-methoxynicotinate web 08 ) and 70 (70 ethanol precipitation fraction, TPS2, 53.2 g, yield two.13 ), respectively. The precipitates have been collected by centrifugation (9829?g, 10 min), washed twice with 95 ethanol, and freeze-dried. The flow chart displaying the procedure of isolating numerous fractions of polysaccharides is presented in Figure 1. TPS1 (five g) was dissolved in 40 mL distilled water and centrifuged (43,540?g, ten min). The residue was dissolved in 20 mL distilled water once again and centrifuged (43,540?g, ten min) to take away the residue. The supernatant was combined and loaded on a DEAE-cellulose column (50 cm ?five cm) pre-treated with 0.five M NaOH, 0.five M HCl and equilibrated with distilled water. The TPS1 was initial eluted with distilled water after which with 0.1, 0.two, 0.four and 2.0 M of NaCl by stepwise increments. The fraction eluted with 0.2 M NaCl (TPS1-2) was additional fractionated on a High-Resolution SephacrylTM S-300 column (60 cm ?two.6 cm) and eluted with water to offer two important fractions of TPS1-2a (180?20 min) and TPS1-2b (240?80 min). (b) Homogeneity and molecular weight Homogeneity and molecular weights of the isolated polysaccharides had been determined by high functionality gel permeation chromatography (HPGPC), KS-805 and KS-804 column in serials, ID eight mm, and length 300 mm, (Shodex Co.1107658-78-5 Data Sheet , Tokyo, Japan) [38].PMID:24487575 The normal curve was established applying distinct 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 used as an eluant and also the flow price was kept at 0.8 mL/min. All samples were ready as 2 mg/mL options, and 20 L aliquot was injected for every run. (c) Monosaccharide evaluation The polysaccharide sample was hydrolyzed with two M TFA at 121 for two h. Immediately after repeated evaporation with methanol to absolutely eliminate TFA, the residue was dissolved in 0.1 mL of distilled water and analyzed on a PEI-cellulose plate (Merck, Darmstadt, Germany), developed with EtOAc-pyridine-AcOH-water 5:5:1:three (v/v). The plate was visualized by spraying with O-phthalic acid reagent and heating at 100 for 5 min [39]. The remaining hydrolysate was lowered by NaBH4 for 3 h at space temperat.
