Effect of pH on a gossypol complex with phospholipids: Gossypol‐PE

Abstract

PE, one of the major phospholipids in oilseed soapstock, may react with gossypol to form Schiff bases. PE amounts to 20-30% of the phosphorus compounds in soapstock. In this report, the dependence on pH of the Schiff base products between PE and gossypol was investigated using a spectrophotometer, an HPLC equipped with an ELSD, and an LC-MS system. We observed that at pH 7 the Schiff reaction product and reactants were clearly detected by ELSD, absorption, and LC-MS spectra. The absorption spectra displayed the characteristic peak for the Schiff bases around 430-440 nm. The absorption spectra also indicated that the reaction was pH dependent. The reaction temperatures were 60 and 90°C. The LC-MS spectra supported the formation of Schiff bases as well as methyl ether derivatives of gossypol in alcohol at the elevated temperatures. The implications of these experimental findings are presented in this paper.Gossypol and phosphatides are extracted with TG when cottonseed meal and oil are produced by solvent extraction (1). By adding caustics to extracted oil, FFA, phosphatides, and gossypol are separated from oil, producing soapstock as a byproduct. Soapstock, separated from oil by centrifugation, is usually added back to animal feed (1). Cottonseed meal is one of the most popular feeds for ruminant animals.Gossypol reacts with protein [ε-amino groups of lysine (2)], and with alkyl-and phenylamines (3,4) to form bound gossypol. Because of its toxicity problem, the amount of free gossypol allowed in animal feed is lower than that of bound gossypol (3). Although gossypol reactions with phosphatides are believed to form a class of bound gossypol (5,6), experimental investigations to confirm the reaction in detail are scarce. In particular, the reversibility of the reaction and the effect of pH on the reaction have not been reported thus far. It is extremely important to investigate the reappearance of free gossypol by reversion of the reaction from the Schiff product, gossypol-phosphatide, because of the animals' physiological sensitivity to free gossypol (7). In general, PE amounts to up to 20-30% of phosphatides in oilseed soapstock (1). The dependence on pH of the Schiff product between PE and gossypol was investigated in this paper using a spectrophotometer, a highperformance liquid chromatograph equipped with an ELSD, and an LC-MS system. The reaction temperatures were room temperature (RT), 60 and 90°C. The formation of the Schiff base product was confirmed by mass spectra obtained from an atmospheric pressure chemical ionization (APCI) source. MATERIALS and METHODS Materials.A typical oilseed PE (1,2-di-9-octadecenoyl-snglycero-3-phosphatidylethanolamine; 99% pure with formula weight 716) extracted from soy (Avanti Lipids, Alabaster, AL), gossypol-acetic acid (GA) from cottonseed (89.61% gossypol by weight; SRRC, ARS, USDA, New Orleans, LA), and reagent-grade amines (isopropyl amine and aniline), HPLC solvents and reagents, and gossypol (G) (reagent grade; Sigma-Aldrich, St. Louis, MO) were used.Gossypol reaction...

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