Total Polysaccharides - Phenol Sulphuric Acid Assay Protocol for Dendrobium huoshanense

Total Polysaccharides - Phenol Sulphuric Acid Assay Species Dendrobium Reference Extraction: Zhu, H., Yi, X., Jia, S.S., Liu, C.Y., Han, Z.W., Han, B.X., Jiang, G.C., Ding, Z.F., Wang, R.L., & Lv, G.P. 2023. Optimization of Three Extraction Methods and Their Effect on the Structure and Antioxidant Activity of Polysaccharides inDendrobium huoshanense.Molecules,2824, 8019. https://doi.org/10.3390/molecules28248019 Phenol sulphuric acid assay: DuBois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. T., & Smith, F. 1956. Colorimetric method for determination of sugars and related substances.Analytical chemistry,283, 350356 Materials Reagents Ethanol 95% or Reagent alcohol Phenol ! CAUTION Irritating to eyes, respiratory system and skin. Use goggles, PPE, and work in a fume hood) Concentrated sulphuric acid ! CAUTION Extreme corrosive. Use goggles and PPE Glucose standard Reagent Setup 95 % Ethanol Prepare if necessary Phenol solution 5% w/w) Dissolve 5 g of phenol for every 95 g of DI water. Gently heat the mixture to improve dissolution. NOTE Prepare fresh solutions monthly as phenol oxidizes over time. Glucose standard solutions Prepare solutions with concentrations of 0100 mg/L See below for steps) Equipment Analytical balance Grinder or mortar and pestle Total Polysaccharides  Phenol Sulphuric Acid Assay 1 Sieves 4060 mesh) Drying oven Beakers (various sizes) Volumetric flasks 1 L, 25 mL Large test tubes with caps Test tube holder Filtration setup (filter paper, funnel) Falcon tubes 50 mL Pipettes 5 mL and 10 mL Pipette tips Centrifuge Water bath with temperature control Evaporator Glass cuvettes for spectrophotometer Vortex mixer Refrigerator Fine tip sharpie Labels Timer Equipment Setup UV-visible spectrophotometer Set to a wavelength of 490 nm. NOTE Switch the spectrophotometer on the previous day to ensure stabilisation Chemical requirements Use the table below to calculate required chemical quantities based on your sample count and number of standards. Chemical requirements Total Polysaccharides  Phenol Sulphuric Acid Assay 2 Total samples (#) Total standards Ethanol 95%  Extraction (mL) Phenol 5%  Assay (mL) Sulphuric acid - Assay (mL) Chemical 30 18 2880 48 240 Untitled Procedure Sample Preparation  Tissue Collection: Harvest plant samples and carefully remove all roots and leaves, retaining only the stems.  Washing: Rinse stem samples thoroughly with pure (deionized or distilled) water to remove any surface contaminants.  Drying: Place the cleaned stem samples in a drying oven at 60°C. Dry until samples reach constant weight (no further change in mass over repeated measurements). CRITICAL STEP The plants should be dried immediately after harvest, because the physiological activities in the fresh plants continues as polysaccharides are hydrolyzed for energy supply.  Grind the dried stems using a grinder or mortar and pestle.  Sieve the crushed material to obtain a uniform particle size (typically 4060 mesh unless specified otherwise). Hot Water Extraction Pre-treatment  Soak the dried Dendrobium stem powder with 60% ethanol in a beaker at room temperature overnight.  Centrifuge the contents at 4000g for 15 minutes and discard the supernatant.  Transfer the precipitate back to the beakers and dry them for 12 hours at 50 °C in an oven.  The dried pretreated powder can be milled again and stored in Falcon tubes until extraction. Extraction Day 1  Sample preparation Total Polysaccharides  Phenol Sulphuric Acid Assay 3 Weigh 0.25 grams of the pre-treated stem powder in a large test tube I used capped centrifuge tubes). Add 15 mL of distilled water to achieve a liquid-solid ratio of 601 (mL/g) and mix-well by vortexing. Cover the tubes with cling wrap to prevent steam escaping.  Hot water extraction Heat the mixture in the water bath at85 °Cfor60 minutes. Vortex the contents gently every 10 minutes. After extraction, allow the mixture to cool slightly. Ultrasonicate the mixture for 10 minutes at 20 °C.  Separation of extract Centrifuge the mixture at 5000g for 10 minutes to separate the supernatant containing the polysaccharides. For the Type 70i rotor, the RPM is 7000 Gently filter the supernatant into 50 mL Falcon tubes.  Precipitation of polysaccharides Pipette 2 mL of the initial extract solution into a 30 mL centrifuge tube. Add 10 mL of absolute ethanol/methanol to the extract (resulting in approximately 83% ethanol concentration for precipitation). Vortex and store the mixture at 4°C for 12 hours to precipitate the polysaccharides. Also place the centrifuge rotor at 4 °C for collection of crude polysaccharides. Day 2  Collection of crude polysaccharides Centrifuge the mixture at 10000g for 20 minutes at 4 °C to collect the precipitated polysaccharides. For the Type 70i rotor, this is 9900 rpm) Dry out the ethanol using the evaporator in a 40 °C water bath to get purified polysaccharides.  Dissolve the precipitated polysaccharides in 1015 mL hot 7080 °C DI water and transfer into 25 mL volumetric flasks and dilute to the mark 25 mL total volume). Mix thoroughly to create the purified sample solution.  Store the sample at 4 °C until the assay on the subsequent day. Phenol-Sulphuric Acid Assay Day 3 Preparation of Glucose Standards Total Polysaccharides  Phenol Sulphuric Acid Assay 4  Prepare 1 L stock solution of 100 mg/L concentration by diluting 100 mg 0.1 g) glucose with DI water in a volumetric flask.  Prepare a series of 25 mL glucose standard solutions (e.g., 0, 20, 40, 60, 80, 100 mg/L OR µg/mL by diluting the 100 mg/L glucose stock with distilled water. Dilution Table ID# Standard concentration (mg/L Stock concentration (mg/L Final Volume (mL) Volume of stock solution (mL) Volume of water (mL) 1 0 100 50 0 50 2 20 100 50 10 40 3 40 100 50 20 30 4 60 100 50 30 20 5 80 100 50 40 10 6 100 100 50 50 0  The standards can be stored at 4 °C for up to a week. Assay  Pipette 0.1 mL of the polysaccharide solution obtained in the previous step and transfer it to a stoppered test tube.  Add 0.9 mL of pure water into the test tubes to dilute the sample by a factor of 10.  Add 1 mL of a 5% phenol solution and mix well by vortexing.  Add 5 mL of concentrated sulfuric acid to the mixture and vortex gently. CAUTION The reaction is highly exothermic, and concentrated sulfuric acid can cause burns. Itʼs best to do this step in a fume hood. Work quickly after adding sulfuric acid, as the colour development happens rapidly.  Incubate the reaction mixture in a hot water bath at 75 °C for 10 minutes.  After the incubation period, allow the mixture to cool to room temperature. Calibration Curve  Prepare a blank-corrected calibration curve using glucose as a reference. Use DI water as a blank for calibration.  Measure the absorbance of the standard solutions at 490 nm using a UV-visible spectrophotometer. Total Polysaccharides  Phenol Sulphuric Acid Assay 5 Quantification of Polysaccharides  Measure the absorbance of the sample solutions at 490 nm ( ) using a UV-visible spectrophotometer.  Determine the concentration of polysaccharides based on the linear calibration plot of versus concentration.  Calculate the polysaccharide content using the following equation: Where, C is concentration of polysaccharides determined through spectrophotometer (mg/L, V is the volume of polysaccharide solution prepared after extraction used for the assay (mL), m is the weight of dendrobium stem powder in each sample (g), and D is the unit-less dilution factor (if diluted). References Extraction: Zhu, H., Yi, X., Jia, S.S., Liu, C.Y., Han, Z.W., Han, B.X., Jiang, G.C., Ding, Z.F., Wang, R.L., & Lv, G.P. 2023. Optimization of Three Extraction Methods and Their Effect on the Structure and Antioxidant Activity of Polysaccharides inDendrobium huoshanense.Molecules,2824, 8019. https://doi.org/10.3390/molecules28248019 Phenol sulphuric acid assay: DuBois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. T., & Smith, F. 1956. Colorimetric method for determination of sugars and related substances.Analytical chemistry,283, 350356 A 490 A 490 Polysaccharidecontent(mg/gdendrobiumdrymatter) = m× 103 C×V×D Total Polysaccharides  Phenol Sulphuric Acid Assay 6