Pure Gossypol Research Articles

Micro-characterization of modified microemulsions loaded with gossypol, pure and extracted from cottonseed

Microemulsions (MEs) have gained increasing interest as carriers of hydrophobic bioactives in the last decades. However, it is still difficult to control the uptake and the release of bioactives directly extracted from plants. In this study, modified ME nanodroplets (nano-sized self-assembled liquids, NSSLs) were employed as extraction medium of gossypol, a toxic component of cottonseed. Loading was performed using both pure gossypol, and gossypol obtained by extraction from cottonseed. We achieved two goals: i) remove gossypol from cottonseed to obtain cotton-oil free of gossypol; and ii) extract gossypol directly into a nano-delivery vehicle for biomedical purposes. Structural and dynamical information on the unloaded and gossypol-loaded NSSL systems were obtained by self-diffusion nuclear magnetic resonance, SD-NMR, and spin-probe electron paramagnetic resonance (EPR) studies. The results showed that NSSL formed fluid water-in-oil (W/O) nano domains at the lowest water contents; a more viscous bicontinuous structure at comparable oil and water contents, and, finally, oil-in-water (O/W, micellar-like) at the higher concentration of water. These micellar-like structures were more fluid at the external hydrated surface, as demonstrated by SD-NMR, while the lipidic region tested by EPR revealed an increasing packing. In all these structures, gossypol mainly localized in the lipophilic region close to the water interface. Overall, SD-NMR and EPR provided complementary information, helping to clarify the structural properties of NSSLs formed at different water contents and their ability to incorporate gossypol also directly from cottonseed-NSSL mixtures.

1H‐ and 13C‐NMR, FTIR, UV‐VIS, ESI‐MS, and PM5 studies as well as emission properties of a new Schiff base of gossypol with 5‐methoxytryptamine and a new hydrazone of gossypol with dansylhydrazine

A new Schiff base of gossypol with 5-methoxytryptamine (GSTR) and a new hydrazone of gossypol with dansylhydrazine (GHDH) have been synthesized and studied by Fourier transform infrared (FTIR), 1H and 13C nuclear magnetic resonance (NMR), ultraviolet-visible (UV-VIS), electrospray ionization-mass spectroscopy (ESI-MS) as well as the parametric method PM5. The spectroscopic methods have provided clear evidence that GSTR exists in chloroform solution as an enamine-enamine tautomer, whereas GHDH is present in chloroform as a N-imine-N-imine tautomer. The fluorescence spectra of both compounds indicate that their quantum yield of fluorescence is increased by one or two orders of magnitude compared to that of pure gossypol. The ESI-MS spectra of the 1:1 mixtures of GSTR or GHDH with formic acid have demonstrated that both compounds exist as protonated monomers in the gas phase, whereas GHDH can also exist in a stable protonated dimeric structure. The structures of the stable tautomers are calculated and visualized using the PM5 semiempirical method. The intra- and intermolecular hydrogen bonds within these structures are discussed.

Supercritical carbon dioxide extraction of cottonseed with co‐solvents

AbstractExtraction of cottonseed lipids with supercritical carbon dioxide (SC‐CO2) was conducted with and without a cosolvent, ethanol or 2‐propanol (IPA). At 7000 psi and 80°C, the reduced pressure, temperature and density of SC‐CO2 was at 6.5, 1.17 and 1.85, respectively; the specific gravity was 0.87. Under these conditions, CO2 is denser than most liquid extraction agents such as hexane, ethanol and IPA. The extraction of cottonseed with SC‐CO2 gave a yield of more than 30% (moisture‐free basis). This is comparable to yields obtained by the more commonly used solvent, hexane. The crude cottonseed oil extracted by SC‐CO2 was visually lighter than refined cottonseed oil. This was substantiated by colorimetric measurements. No gossypol was detected in the crude oil. However, crude oil extracted by SC‐CO2, to which less than 5% of ethanol or IPA as co‐solvent was added, containedca. 200 ppm of gossypol, resulting in the typical dark color of cottonseed crude oil with gossypol. CO2 extracted a small amount of cottonseed phosphatides, about one‐third of that extracted by pure ethanol, IPA or hexane. A second extraction with 100% ethanol or IPA after the initial SC‐CO2 extraction produced a water‐soluble lipid fraction that contained a significant amount of gossypol, ranging between 1500 and 5000 ppm. Because pure gossypol is practically insoluble in water, this fraction is believed to be made up of gossypol complexed with polysaccharides and phosphatides.

Inhibition of aflatoxin production inAspergillus flavus by cotton ovule extracts

The effect of cotton ovule extracts onAspergillus flavus growth and aflatoxin biosynthesis was investigated. Extracts derived from either noninoculated cotton ovule tissue or cotton ovule tissue challenged withA. flavus inhibited aflatoxin (B1) synthesis by fungal cultures ofA. flavus. When added to fungal cultures in concentrations of 50 µg per mL of medium, extracts derived from fungus‐challenged ovule tissue inhibited aflatoxin synthesis by as much as 93%. The ED50 for this type of extract was 18 µg per mL of medium (P < 0.01; r2=0.46). Similar experiments with a noninoculated ovule extract (50 µg/mL medium) produced aflatoxin inhibition levels of up to 77%. The ED50 for noninoculated extracts was 35 µg per mL of medium (P < 0.01; r2=0.66). These extracts did not inhibit the growth ofA. flavus in culture. Cultures ofA. flavus that contained pure gossypol (10–50 µg/mL medium) also showed significantly reduced levels of aflatoxin production. Gossypol may account for the aflatoxin‐inhibitory activity observed in the extracts derived from noninoculated cotton ovule tissue.

High-performance liquid chromatographic method for the determination of free gossypol in chicken liver

A high-performance liquid chromatographic method for the determination of free gossypol in chicken liver at levels down to 0.5 ppm has been developed. Tissue was deproteinized with acetonitrile in presence of ascorbic acid and the filtrate was subjected to hydrolysis with hydrochloric acid. The liberated pure gossypol was partitioned into chloroform and analysed by gradient elution on a 10-μm C 18 column. The overall recovery was 83.5 ± 2.6%, with an overall relative standard deviation of 9%.

Determination of free and total gossypol by high performance liquid chromatography

AbstractExisting HPLC methods determine only pure gossypol whereas the official AOCS method determines both gossypol and other physiologically active gossypol‐like compounds that react with 3‐amino‐1‐propanol and aniline. The feed industry uses the official AOCS method, which is complex and produces results that do not correlate well among laboratories. HPLC methods were developed, using 3‐amino‐1‐propanol as a complexing agent, for the quantitative determination of free and total gossypol in cottonseed meal, oil, and ethanolic miscella. These methods are simple, sensitive, and provide reproducible results. In addition the use of toxic aniline is eliminated.

Analysis of gossypol by high performance liquid chromatography

Several high performance liquid Chromatographic methods for the analysis of gossypol in different kinds of sample were developed. (1) Pure gossypol: separation on C 18 column with MeOH/H 2O/CHCl 3 (70:30:40) containing 0.1% H 3PO 4 as mobile phase or on SO 3H column with MeOH/ citrate buffer (pH 6.3) (55:45) as mobile phase was recommended. With these systems, minute amounts of contaminants difficult to separate by other HPLC systems could be determined. (2) Plant material: acetone was selected as the extraction solvent. After evaporation of acetone from the extract, the residue was redissolved in 1% HOAc in CHCl 3. An aliquot of this solution was chromatographed and quantified by peak area method. The mean recovery of pure gossypol added to plant material was 91.1 ±1.1% (S.D.). (3) Plasma sample: a HPLC method with electrochemical detector was developed. A plasma sample with glutathione as protective agent and gossypol dimethyl ether as internal standard was introduced on to a C 18 pre-column. By using column-switching technique, a certain part of the eluate containing gossypol and gossypol dimethyl ether was subjected to a C 8 analytical column for further separation. MeOH/citrate buffer (pH 3.2) (80:20) was used as the mobile phase. The optimum potential for detection was + 0.6 V vs. Ag-AgCl. The assay sensitivity was 5ng/ml. This method is sensitive and selective, suitable for clinical pharmacokinetic studies.

Resolution of gossypol: Analytical and large‐scale preparative HPLC on non‐chiral phases

AbstractRacemic gossypol is resolved by HPLC of its amino acid ester Schiff's base derivatives on non‐chiral columns. This provides a rapid and accurate method for determination of optical purity on a small scale and, combined with hydrolysis and fractional crystallization, for the preparation of pure gossypol enantiomers on a multi‐gram scale.

Inhibition by (+) and (−) isomers of gossypol of testosterone release from mouse leydig cells [formula omitted

Racemic (+/−) mixtures of gossypol isomers exert an antifertility effect by inhibiting sperm motility and spermatogenesis. Purified (+) gossypol has been shown to be without these actions. In this study pure preparations of both (+) and (−) gossypol were found to inhibit, in a similar manner, both basal and LH-stimulated release of testosterone by isolated Leydig cells at concentrations down to 21 μM. It is possible that use of low doses of pure (−) gossypol could inhibit fertility with less endocrine side effects.

Effect of Gamma‐Radiation on Gossypol in Cottonseed Meal

ABSTRACTThe effect of gamma–radiation, in the range 5–30 KGy on free, total and pure gossypol was investigated in an attempt to reduce or eliminate gossypol from cottonseed meal using different solvent systems. Maximum reduction in gossypol concentration was attained at a dose of 25 KGy of gamma‐radiation with chloroform as a solvent, a spectrometric method was used for analysis. The reductions attained for pure, free and total gossypol were found to be 96%, 60% and 80%, respectively.

Antifertility effects of gossypol and its impurities on male hamsters

Recent papers reporting on the male antifertility effects of gossypol have been criticized due to the possible influence of impurities on efficacy and toxicity. Our laboratory has prepared purified gossypol and a fraction containing the total impurities. The effects of these two substances and impure gossypol on the fertility of male hamsters was evaluated. Only pure gossypol produced a decrease in fertility after six weeks of dosing. No effects on male fertility were observed in the other treatment groups. A full recovery of fertility in the pure gossypol group was observed four weeks after cessation of treatment. These data support the claim that gossypol is indeed an active antifertility agent for males. It is also clear that the impurities may have the ability to block or decrease the efficacy of gosypol.

Phagodeterrency Induced by Pure Gossypol and Leaf Extracts of a Cotton Strain with High Gossypol Content in the Larva of Spodoptera littoralis12

The effect of pure gossypol or of extracts of a high gossypol cotton strain on the feeding rate of larvae of Spodoptera littoralis Boisduval was investigated by the ‘Styropor method.’ Styropor (foamed polystyrene) lamellae were painted on one side with 5% sucrose and on the other side with a solution of 1, 0.5, 0.25, and 0.1% pure gossypol. One percent gossypol strongly suppressed feeding, and a negative correlation was found between the feeding rate on the lamellae and the concentration of gossypol. Larvae fed about half as much on lamellae painted with the leaf extract of a high-gossypol-content cotton strain (HG6-11-144) as on those painted with an exract of the glandless strain Acala 63-74.

Effect of Gossypol Source and Level on Chick Growth

Evidence that toxic effects of cottonseed pigment glands on young growing chickens is caused by free gossypol has been presented by Lillie and Bird (1950). Eagle and Davies (1957) have shown that at a dietary level equivalent to 0.1% free gossypol pigment glands give greater growth depression in rats than pure gossypol. However, this report contained no information on how frequently the diets were prepared in order to prevent destruction of gossypol.In this paper data are presented on the relative toxicities to young chickens of cottonseed meats (decorticated seed), cottonseed pigment glands, and pure gossypol when the materials were fed at levels to supply equal amounts of free gossypol. The diets were prepared frequently to minimize inactivation of gossypol. PROCEDUREGroups of 20 day-old New Hampshire or cross bred meat-type males were placed in separate decks of battery brooders at the start of each experiment. The first five experiments …

The acute oral toxicity of cottonseed pigment glands and intraglandular pigments

AbstractAcute 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.

Preparation of pure gossypol from dianilinogossypol

SummaryA simple method has been developed for the acid hydrolysis of dianilinogossypol, resulting in gossypol yields of about 86%. The gossypol may be readily purified by recrystallization as gossypol‐acetic acid from a mixture of ethyl ether and acetic acid. After removal of the acetic acid the gossypol may be crystallized either from a mixture of ether and light petroleum ether or from a mixture of ether, ethanol, and water. Crystallization from the latter mixture results in a yield of about 75% of gossypol, based on the amount of dianilinogossypol hydrolyzed, with a high degree of purity as is indicated by spectrophotometric and titrimetric methods.

Recovery of gossypol from cottonseed gums

SummaryA process has been developed for the isolation of pure gossypol from the gums obtained by water‐washings of crude hexane‐extracted cottonseed oil. Gums are heated with methyl ethyl ketone containing phosphoric acid to cleave gossypol‐phosphatide reaction products and are cooled to separate a ketone phase containing the gossypol from a phosphatide‐water phase. After concentration by distillation, gossypol is isolated from the methyl ethyl ketone concentrate by addition of glacial acetic acid to form the acetic acid addition compound of 92–94% purity. Two recrystallizations as the acetic‐acid complex produce gossypol acetic acid of 99% purity. The purified product can be dissociated by solution in dilute sodium carbonate, from which pure gossypol is recovered by acidification with mineral acid.Yield data from pilot‐plant experiments indicated that about 47% of the gossypol in gums is recovered as crude gossypol acetic acid. Depending on the degree of purification of the crude product, the over‐all yield of purified gossypol from gums will range from 41% for a product of 98% purity to 36% for a gossypol of 99% purity.

Production of gossypol from cottonseed gums. Preliminary cost study

SummaryBased on operations of a hypothetical gossypol‐extraction plant, it is estimated that crude gossypol‐acetic acid can be produced at a cost of $2.64 per pound at an annual production of 113,500 lbs.; that pure gossypol‐acetic acid can be produced at a cost of $3.80 per pound at an annual production of 100,200 lbs; and that pure gossypol will cost $5.55 per pound at an annual production of 81,000 lbs. By marketing the phosphatides instead of returning them to the gums, the cost of producing pure gossypol will be $3.91 per pound at an annual production of 81,000 lbs. These costs were estimated by assuming that Phase I of the process would be accomplished simultaneously with oil mill‐extraction operations, and Phases II and III during remainder of the season.It is readily apparent that gossypol or gossypol‐acetic acid as produced are not inexpensive chemicals and would probably have to be produced for specialized uses, such as pharmaceuticals and the like, where the cost of these chemicals would not be prohibitive. Should product evaluation research now under way establish specific uses for gossypol and its intermediates, a demand for sizeable quantities of these materials might result.

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

SummarySix 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.

High Levels of Free Gossypol in Hen Diets: Effects on Body Weight, Feed Consumption, and Egg Production

EVIDENCE has been presented by a number of investigators in recent years showing that the toxic effects of cottonseed meal are due to its free gossypol content. The experiments with growing chickens by Lillie and Bird (1950) are in agreement with the above statement; and the results obtained by Heywang, Denton and Bird (1949) and Heywang, Bird and Altschul (1950), in separate experiments, indicated that free gossypol in cottonseed fed to hens is responsible for the decreased hatchability of their eggs, and that gossypol in the feed is related to the discoloration of eggs. Unpublished experiments in this laboratory have shown that pure gossypol injected into laying hens in the form of an oil solution is highly toxic and eventually produces death. A number of papers have appeared in recent years on the role of gossypol in cottonseed in the nutrition of growing chickens and laying hens. However, little work . . .

Egg Production, Diet Consumption, and Live Weight in Relation to the Free Gossypol Content of the Diet

THE data of Heywang, Bird and Altschul (1950) show that pure gossypol had an appreciable adverse effect on egg weight and hatchability when it was mixed with a diet at the .024 percent level and a still greater adverse effect at the .036 percent level, but did not affect either at the .012 percent level.Gossypol is considered to exist in two forms—“free” and “bound”—in unprocessed cottonseed and cottonseed meal, with the bound form inactive and the free form resembling “pure” gossypol in that both are active. Because of the instability of pure gossypol (Heywang, Bird and Kupperman, 1952) raw decorticated cottonseed and cottonseed meals were used as sources of free gossypol by Heywang, Bird and Altschul (1954) in experiments on the dietary level at which free gossypol causes discolorations in egg yolks.Some of the eggs were examined for yolk discolorations soon after they were laid, while …