###### Abstract  
Cottonseed is one of the important by-products of the cotton crop. Researchers claim that cottonseed with less than 0.45% of gossypol is quite good for human consumption and animal feeding because it is a rich source of protein, edible oil, and energy. Total and free gossypols are the influencing parameters that reduce the edible nature of the cottonseed. In the present work, multiple quadratic regression models have been prepared to predict the reduction in the free and total gossypol percent. This response surface method (RSM)-based approach was applied to investigate the combined effect between input parameters such as acetone level, time of extraction, liquid-to-solid ratio (LSR), and the number of extraction cycles, whereas output responses are free and total gossypol reduction percentage. Analysis of Variance (ANOVA) has been performed to determine the highly significant parameter. The optimum combination of input parameters was determined using the RSM-based desirability approach, and confirmatory experiments were performed to validate the combination. Results revealed that the number of extraction cycles and liquid-to-solid ratio significantly affects the reduction of free and total gossypol levels. The values of r-square were found above 0.9, which indicates that the developed models are suitable and reliable for predicting free and total gossypol reduction percentage. cottonseed; gossypol; optimization; response surface methodology; toxicity +
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included as a protein source in animal feed, but higher replacement levels can lead to significantly decreased growth performance and mortality due to the presence of gossypol [5]. Gossypol is a polyphenolic compound that has a bulky hydrophobic bi naphthalene structure that can easily diffuse through the cell membrane and cause toxicity. The reactivity of phenolic hydroxyl groups and carbonyl groups makes gossypol chemically reactive. Gossypol is generally present in two forms, viz. free and bound gossypol. According to the American Oil Chemists Society (AOCS) (Ba 7-58-1986), the gossypol and its derivatives, which can be extracted using 70% aqueous acetone, are known as free gossypol [6]. Bound gossypol (BG) takes the form when cottonseed is processed, where gossypol reacts with the epsilon group of amino acids, especially lysine and arginine. Total gossypol (TG) (FG and BG) is defined as gossypol and its derivatives, which can hydrolyze and complexed with 3-amino-1-propanol in dimethyl formamide solution to form a diamino propanol complex (AOCS Ba 8-78) whereas BG is TG minus FG. U.S. Food and Drug Administration (FDA), 1974 set the limit for free gossypol in-human food products at 450 ppm [7]. While the United Nations Food and Agriculture Organization (FAO) and World Health Organization (WHO) has set limits for free and total gossypol at 600 ppm and 12,000 ppm, respectively [3]. Gossypol reduction (both FG and TG) up to safe limits may increase its importance and application as food and animal feed in India and other developing countries. Gossypol toxicity in cottonseed is the major concern that causes many problems in monogastric animals, such as decreased growth rate, fertility depression, internal organ abnormalities, feed conversion, and low protein digestibility. On the other hand, if gossypol is suitably extracted from cottonseed, it may be employed for various medicinal purposes such as anti-cancer, antiseptic, anti-fertility agents, and antiviral activity [8]. Several approaches have been performed for the reduction of gossypols such as pressure cooking [9], hydraulic pressing, screw pressing [10], solvent/alkali-salt extraction [3; 11; 12], liquid cyclone process [3; 13], microbial fermentation [14; 15; 16], and ultrasound technique for detoxification of gossypol from cottonseed meal [17]. Among all methods, the solvent-based removal of gossypol from cottonseed meal has been found commercially viable due to the dissolution of the solute (gossypol) in the acidified solvents [18; 19].