Preparation of gossypol from cottonseed pigment glands

Summary Cottonseed pigment glands, produced from whole cottonseed meal and defatted cottonseed meal by the gland flotation process, have been investigated as a raw material for the production of gossypol. Methods based on the previously reported properties of gossypol and cottonseed pigment glands have been developed for the relatively rapid isolation of gossypol from pigment glands. Extraction of gossypol from pigment glands with acetone followed by precipitation of gossypol acetic acid from the extract was found to be the preferable method for obtaining pure gossypol in good yields. The precautions which must be applied in order to produce gossypol on a large scale by the acetone-acetic acid method are discussed together with methods for the purification and preservation of gossypol. Presented at the Fall Meeting of the American Oil Chemsits' Society, October 20–22, 1947. One of the laboratories of the Bureau of Agricultural and Industrial Chemistry, Agricultural Research Administration, U. S. Department of Agriculture.     

Gossyverdurin: A newly isolated pigment from cottonseed pigment glands

The constituents of cottonseed pigment glands were fractionated by the use of column chromatography with DEAE cellulose ion exchanger and silicic acid, and a new green pigment was isolated. The acute oral toxicity of the new pigment was determined using rats as experimental animals. The LD-50 value obtained was 0.66 g/kg of body weight indicating that the new pigment which was named gossyverdurin is the most toxic of any cottonseed pigment so far reported. Gossyverdurin showed absorption maxima at 250, 370, and 560 mμ. Reaction with para-anisidine under the conditions used for the determination of gossypol gave an absorption peak similar to that obtained with gossypol indicating that the new compound is structurally related to gossypol. In addition a second peak at 342 mμ appears on reaction with para-anisidine indicating important structural differences between gossypol and gossyverdurin.     

The acute oral toxicity of cottonseed pigment glands and intraglandular pigments

Acute oral toxicity studies were carried out on cottonseed pigment glands, gossypol, diaminogossypol and gossypurpurin using rats as experimental animals. It was found that both diaminogossypol and gossypurpurin are considerably less toxic than pure gossypol. The pigment glands were more toxic than gossypol and it was concluded that the toxicity of pigments cannot be accounted for entirely on the basis of their gossypol content. Administration of gossypol with cottonseed oil orSterculia foetida oil, both of which contain cyclopropene fatty acids, increased the toxicity very slightly over that found when gossypol was administered in corn oil. In the case of corn oil andSterculia foetida oil, the difference was statistically significant. Less gossypol was also found in the feces when the test dose was given in cottonseed oil orS. foetida oil indicating a possible effect of the cyclopropene fatty acids in increasing gossypol absorption.     

Effect of ultrasonic comminution on liquid classification of cottonseed protein and gossypol pigment glands

Slurries of pin-milled full-fat and flaked, extracted cottonseed were ultrasonically comminuted in hexane and liquid-classified using laboratory differential settling techniques. Sonication of full-fat cottonseed slurries increased the liquid-classified protein fraction recovery from 25.9% (nonsonicated control) to over 60%, while the protein content of the fraction remained basically constant at 67%, and free gossypol increased slightly from 0.027 to 0.032%. Sonication of flaked, solvent-extracted (fat-free), slurried cottonseed yielded a 28% classified fraction containing 72% protein and 0.032% free gossypol. Although it was demonstrated on laboratory scale only, ultrasonic comminution may make the price of edible cottonseed protein concentrates produced from glanded seed (via a process such as the liquid cyclone process) competitive with other edible protein products. Presented at the AOCS Annual Meeting, 1981, New Orleans.     

Extraction of lipids from cottonseed tissue: VI. Ultrastructural morphology of isolated pigment glands

Cottonseed pigment glands, isolated from the underflow fraction of the liquid cyclone process, were examined with an electron microscope. The glands were circumscribed by a layer of tangentially flattened cells. Subjacent to the flattened cells were partially lysed cells containing recognizable remnants of cell walls. Within the lumens or matrices of the glands were myriads of pigment spherules measuring 0.1–1.5 µ in diameter. These spherules remained within the glands, even though gland walls were ruptured mechanically. Since aggregates of pigment spherules devoid of gland walls were observed in the gland-rich fraction, it was concluded that maintenance of intact pigment glands was not a requisite for successful separation of gossypol from other cottonseed components with the liquid cyclone process. Avoiding dispersion of aggregated spherules from the glandular matrix was probably just as important as maintaining intact gland walls during mechanical separation of gossypol from other cottonseed components.     

Effect of long-term storage on acute oral toxicity and gossypol content of cottonseed pigment glands

Summary Six samples of cottonseed pigment glands and two samples of pure gossypol stored for more than four to nine years were re-evaluated for their acute oral toxicity in the rat and re-analyzed for gossypol content. There was no appreciable effect on the acute oral toxicity or gossypol content after these long storage periods.     

Effect of fractionation and treatment on the acute oral toxicity and on the gossypol and gossypurpurin content of cottonseed pigment glands

Summary Twenty-one preparations from cottonseed pigment glands were tested for their acute oral toxicity in 1,208 fasted rats and for their content of extractable gossypol and gossypurpurin. LD₅₀ studies on six samples of pure gossypol were performed on 167 fasted rats. There was no correlation between the toxicity of the various samples of cottonseed pigment glands and their extractable gossypol or gossypurpurin content. Samples containing very large amounts of extractable gossypol were less toxic than many samples with considerably lower extractable gossypol content. Various fractionation procedures carried out on the same lot of cottonseed pigment glands caused wide alterations in their toxicity, from the extreme of very marked toxicity for the water-soluble, acetone-soluble fraction (LD₅₀ ca. 700 mg./kg.) to no detectable toxicity for the acetone-insoluble residue (LD₅₀<6,000 mg./kg.). There was a decreased toxicity of subsequently prepared pigment glands with increased time of storage of the cottonseed in a silo. Storage of the pigment glands themselves at 7°C. however had little effect on their toxicity even after 26 and 32 months. The procedures causing greatest detoxification of cottonseed pigment glands, given in the order of increasing effectiveness, were: heating in the presence of water < extraction with ethanol < extraction with acetone. In the fasted rat the acute oral toxicity of pure gossypol was less than that of 17 preparations from cottonseed pigment glands having extractable gossypol contents ranging from as little as 24 to as much as 90%. Report of a study in which certain phase were carried on under the Research and Marketing Act of 1946. Presented at fall meeting, American Oil Chemists' Society, Chicago, Oct. 31, Nov. 1–2, 1949. This work was conducted with Swift and Company under a memorandum of understanding with the Bureau of Agricultural and Industrial Chemistry. The preparation of the samples of gossypol and cottonseed pigment glands and the analyses for gossypol and gossypurpurin content were carried out in the Southern Regional Research Laboratory; all of the animal experiments were performed in the Research Laboratories of Swift and Company. One of the laboratories of the Bureau of Agricultural and Industrial Chemistry, Agricultural Research Administration, U. S. Department of Agriculture.     

New information on the morphology of the gossypol pigment gland of cottonseed

Exploratory electron microscope studies of the cottonseed pigment gland demonstrate a complex internal structure in which discrete particles ranging in size from one micron to less than 0.2 micron diameter are held within a membranous meshlike network. This structure is extremely sensitive to water, and it may be the rapid swelling of the network which results in the explosive release of pigment particles from the gland on exposure to moisture. Pigment particles are spherical, of a wide range of diameters, and exhibit no birefringence in the gland or when extruded. Calculations of specific surface based on sizes of particles and density of purified gossypol (1.34) indicate surface area per gram of gossypol particles of the order of 8 square meters. Further work is indicated to determine details of the structure of the platelets, which constitute the wall of the gland, and to establish the relationship of gossypol and gossypurpurin to morphological features within the gland. One of the laboratories of the Southern Utilization Research and development Division, Agricultural Research Service, U.S. Department of Agriculture.     

The storage of cottonseed

Summary Treatment of moist cottonseed with ammonia prior to storage has been shown to reduce self-heating of the seed and the rate of formation of free fatty acids during storage. The color of oils obtained by solvent extraction from ammonia-treated cottonseed is considerably lighter than that of oils obtained from untreated cottonseed.     

The pigments of cottonseed

Summary A method for the purification of gossypol has been developed which yields material differing in optical properties from those previously reported for gossypol preparations. A colorimetric test for extractable gossypol has been described. It has been shown that cottonseed contains at least three pigments in addition to gossypol. Some of the properties of the three pigments have been reported. One of these newly detected pigments, gossypurpurin, has been shown to be the substance which, mixed with gossypol, constitutes the so-called “red gossypol” of Podol’skaja. The frequently reported water-dispersable blue pigment of cottonseed has been shown to be either a complex of gossypol, gossypurpurin and protein or a mixture of two protein-pigment complexes. Its dissociation into the two pigments and protein has been accomplished. Presented before the 34th Annual Meeting of the American Oil Chemists’ Society, New Orleans, Louisiana, May 12–14, 1943.     

Pigments of cottonseed. IV. Gossypurpurin,a purple pigment related to gossypol

Summary Gossypurpurin was prepared from gossypol via diaminogossypol, and its properties compared with gossypurpurin isolated from cottonseed pigment glands. A tentative molecular formula for synthetic gossypurpurin, C₃₀H₃₂O₇N, has been proposed on the basis of its elementary composition. The native pigment could not be obtained in the same degree of purity as the synthetic product and the analytical data could therefore not be brought into exact agreement for the two products. However solutions of both pigments in chloroform exhibit almost identical absorption spectra and identical antimony trichloride tests. Qualitative reactions seem to indicate that the functional groups of both native and synthetic gossypurpurin are identical, and the ready conversion of both products to gossypol upon contact with acid seems to indicate that their basic structures are similar. Presented before the 4th Southwest Regional Meeting of the American Chemical Society, Shreveport, La., December 9–10, 1948. One of the laboratories of the Bureau of Agricultural and Industrial Chemistry, Agricultural Research Administration, U. S. Department of Agriculture.     

Binding of gossypol under conditions of complete rupture of the pigment glands

Summary The “free” gossypol of cooked cottonseed meats is composed of gossypol and of “gossypol-like pigments,” which are soluble in 70% aqueous acetone. The “gossypol-like pigments” may account for 30 to 100% of the “free” gossypol as determined by the A.O.C.S. method. Thorough comminution, followed by prolonged cooking, results in reduction of all the pigments in cotton-seed meats. “Free” gossypol is not completely eliminated, but the gossypol level, as determined by the benzene transfer method, may be reduced to zero. “Gossypol-like pigments” are converted into gossypol by the action of hot aqueous hydrochloric acid. Good agreement is observed between the data for “total” gossypol, as determined through the use of the A.O.C.S. oxalic acid method and through the use of hot aniline. One of the laboratories of the Southern Utilization Research and Development Division, Agricultural Research Service, U. S. Department of Agriculture.