Plant Lab Protocols / Methodology for Phenolics
Capsaicin
Capsaicin is a protoalkaloid which is responsible for the pungency of chillies. The quality of chilli fruits, extracts or oleoresins is determined by the capsaicin content.
Content
(i) Colorimetric Method
(ii) Spectrophotometric Method
(i) Colorimetric Method
Principle
The phenolic group in capsaicin reduces phosphomolybdic acid to lower acids of molybdenum. The resulting component is blue in color and is read at 650nm. The color intensity is directly proportional to the concentration of capsaicin.
Materials
» 0.4% Sodium Hydroxide
» 3% Phosphomolybdic Acid
» Dry Acetone (Add about 25g anhydrous sodium sulphate to 500mL acetone of analytical grade at least one day before use).
» Stock Standard Capsaicin Solution
Dissolve exactly 50mg capsaicin in 50mL of 0.4% sodium hydroxide solution (1,000mg/mL).
» Working Standard
Dilute 10mL of the stock standard to 50mL with 0.4% sodium hydroxide solution (200mg/mL).
Procedure
1.
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Weigh 0.5g dry chilli powder into a glass-stoppered test tube or volumetric flask.
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2.
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Pipette out 10mL dry acetone into the flask and shake it for 3h in a mechanical shaker. Let the contents settle down or centrifuge (10,000rpm for 10 min).
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3.
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Pipette out 1mL of the clear supernatant into a test tube and evaporate to dryness in a hot water bath.
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4.
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Dissolve the residue in 5mL of 0.4% sodium hydroxide solution.
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5.
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Add 3mL of 3% phosphomolybdic acid.
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6.
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Shake the contents and let stand for 1h.
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7.
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Filter the solution quickly into centrifuge tubes to remove any floating debris.
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8.
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Centrifuge at about 5,000rpm for 10-15 min.
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9.
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Transfer the clear blue colored solution directly into the cuvette and
read the absorbance at 650nm.
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10.
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Run a reagent blank along with the test samples.
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11.
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Prepare a standard graph using 0-200mg capsaicin simultaneously i.e., pipette out 0.2, 0.4, 0.6, 0.8 and 1mL of working standard solution and proceed as above.
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References
1. Quagliotti, L (1971) HorticRes 11 93.
2. Theymoli Balasubramanian, Raj, D, Kasthuri, R and Rengaswami, P (1982) Indian J Hortic 39 239.
(ii) Spectrophotometric Method
Principle
Capsaicin is extracted from red pepper (chillies) with ethyl acetate and made to react with ethyl acetate solution of vanadium oxychloride. Then it is read at 720nm. This method is sensitive and is useful to measure small quantities (less than 0.05%).
Materials
» Vanadium oxychloride (0.5%) in ethyl acetate
» Pure capsaicin (0.01%) in ethyl acetate (10mg in 100mL)
Procedure
1.
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Grind the sample well to pass through No. 40 sieve.
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2.
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Place 2g sample in a 100mL volumetric flask.
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3.
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Let it stand for 24h to extract (otherwise reflux the contents for 2.5h) and then make up to volume.
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4.
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Dilute 1mL of extract to 5mL with ethyl acetate.
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5.
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Add 0.5mL Vanadium oxychloride solution (just before reading) and shake.
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6.
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Read at 720nm in a spectrophotometer.
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7.
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Subtract the absorbance value.
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8.
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Prepare a standard curve using 0.5,1.0,1.5, 2.0 and 2.5mL of standard capsaicin solution containing 50, 100, 150, 200 and 250mg capsaicin respectively.
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Calculation
% capsaicin =
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mg capsaicin
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x
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100
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x
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100
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1000 x 1000
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1
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2
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References
1. Palacio, J J R (1977) JAOAC 60 970.
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