Methodology for Anti-nutritional Factor


Gossypol is a yellowish phenolic pigment distributed throughout the cotton plant but concentrated in cotton seed kernels, flowers and bark of roots. Its content varies widely depending upon species, variety, climatic and agronomic factors. It occurs in both free and bound forms of which the former is much more harmful. The occurrence of gossypol lowers the quality of products such as cotton seed oil, cake etc. However, it confers resistance to the cotton plant against a number of pests and diseases.

The method given below is simple and easy for the estimation of gossypol, colorimetrically.


Gossypol reacts with phloroglucinol under highly acidic condition to produce a reddish-brown product which is measured at 550nm.

» Ethanol(96%)
» Phloroglucinol Reagent
  Dissolve 5g phloroglucinol in 100mL of 80% ethanol.

Weigh 5g of fresh tissue, cut into small pieces and plunge into boiling 95% ethyl alcohol (15 - 20mL) for 5 min. Collect the extract by filtering. Repeat the extraction with residue and combine the extracts. Dilute the extract with 40% ethanol and adjust the extract with IN HC1 to pH 3.0. Mix the contents with 1.5 volumes of diethyl ether at 10°C using a separating funnel. Save the ether phase and wash with two changes of distilled water. Evaporate the ether extract in vacuo to dryness and redissolve the residue in a known volume of 95% ethanol.

Pipette out different aliquots (1,2mL) of the gossypol extract in ethanol in test tubes. Add 0.5mL of phloroglucinol reagent followed by 1mL of Cone. HC1 to each tube.
Incubate for 30 min with occasional shaking at room temperature.
Make the volume of solution to 10mL with 80% ethanol.
Read the absorbance of color at 550nm against a reagent blank.
Prepare a standard graph with gossypol acetate.


1. Bell, A A (1961) Phytopathol57 159.
2. Sood, D R and Singh, A (1973) Cotton Dev 3 15.