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  • br Isolation and characterization of

    2020-08-12


    2.7. Isolation and characterization of green tea polysaccharide
    2.7.1. Isolation and fractionation of the polysaccharide from green tea The dried green tea leaves were obtained commercially from Huazhi
    Tea Co., Ltd. in China and was identified as grade six according to the Green Tea Quality Standard (GH016-84) of China. First, we used petro-leum ether and 99% ethanol in turn to remove lipids, most of the poly-phenols and monosaccharide at 70 °C and each for 1 h in the Soxhlet extractor. After the supernatant (petroleum ether and ethanol) was re-moved, the residues (200 g) were dried in air and then extracted with 800 ml of boiling distilled water at 100 °C for 60 min 3 times. The pooled extracts were filtered through sterilized gauze, condensed by a rotor vacuum evaporator and centrifuged at 6000 ×g for 30 min to remove water-insoluble materials. The supernatant was extracted with Sevag reagent (3:1, v/v) under a magnetic stirrer at room temperature for 30 min and three times to erase the protein. After centrifugation (10,000 ×g for min), the lower layer containing protein was discarded, and the upper layer was dialyzed (molecular weight cut-off, 8000 Da) against distilled water for 48 h followed by lyophilization to obtain the polysaccharide-enriched fraction, CGTP (26.5 g).
    CGTP (50 mg) were dissolved in distilled water and filtered through a 0.45 μm membrane, and then the solution was loaded onto a DEAE-Sepharose CL-6B Fast Flow (1.6 × 60 cm), on which was washed with equilibration buffer followed by successive elution with stepwise elu-tion of increasing NaCl solutions (distilled water, 0.3, and 1.0 M) at pH 7.0 at a flow rate of 3.0 ml/min. Each tube containing 8 ml was mon-itored, collected and combined by phenol-sulfuric L-Nicotine method [24]. The major polysaccharide fractions eluted with distilled water were pooled and further purified by gel filtration column chromatography of Sephacryl S-300 HR (2.0 × 100 cm). The column was eluted with 0.1 M NaCl solution at a flow rate of 1.0 ml/min. and one homogeneous fraction (test tubes 26–32) was pooled, dialyzed and freeze-dried, yield-ing one purified polysaccharide (GTP, 6.54).
    2.7.2. Determination of carbohydrate, protein, uronic acid contents
    The carbohydrate content of the purified sample was determined by the phenol‑sulfuric acid method as D-glucose equivalents [24]. The pro-tein content was evaluated with Bradford Protein Assay Kit (Beyotime Biotechnology, Shanghai, China) using bovine serum albumin as the standard [25]. The uronic acid content was determined using the carbazole–sulfuric acid method [26] using galacturonic acid to construct the standard curve. 
    2.7.3. Molecular weight determination
    The molecular weight of tea polysaccharides GTP was determined by high performance liquid chromatography on a Shimadzu HPLC appara-tus with TSK-GEL G-4000PW column (7.8 mm × 300 mm, Tokyo, Japan) and a Shimadzu refractive index detector (RID-10A). 10 μl of sample so-lution (1.0 mg/ml) was injected each run and the column was eluted with 0.7% Na2SO4 at a flow rate of 0.6 ml/min at 25 °C. Different known molecular weights of T-series Dextran (T-2000, T-500, T-70, T-40 and T-10) (Sigma Chemical Co. St. Louis, USA) were carried out in the same manner to plot the standard curve of the logarithm of relative molecular weight (MW) related to retention time (t). The relative mo-lecular weight of GTP was estimated by comparison with the retention time of monosaccharide reference standard.
    2.7.4. Monosaccharide composition analysis
    Monosaccharide composition analysis of GTP was conducted by gas chromatography (GC) analysis as described by Liu et al. [27]. Briefly, 10 mg of sample was hydrolyzed with 2M TFA at 100 °C for 2 h, filtered and conventionally converted into the alditol acetates as described pre-viously [28,29]. The final products were applied for the monosaccharide composition on an Agilent 7890 A GC with an HP-5 column (30 m × 320 × 0.25 μm) at a temperature program ranging 125–250 °C at a rate of 4 °C/min, Nitrogen carrier gas at 1 ml/min and flame ionization detector (FID) at 250 °C. The following sugars were used as references: D-man-nose, D-glucosamine, D-xylose, D-glucuronic acid, D-galacturonic acid, D-glucose, D-galactose, L-arabinose and L-fucose (Sigma Chemical Co. St. Louis, USA).
    2.8. In vitro antitumor assays
    The MTT assay was performed as same as Section 2.6.1, except that PC-3 cells were treated with different concentration of GTP (25, 50 and 100 μg/ml) for 48 h before the addition of MTT. Finally, after shaking the plate for 15 min, the OD value of each well was measured at the wavelength of 490 nm using a microplate reader.