Stereoselective coupling of hemigossypol to form (+)-gossypol in moco cotton is mediated by a dirigent protein

The terpenoid gossypol, a secondary metabolite found in the cotton plant, is synthesized by a free radical dimerization of hemigossypol. Gossypol exists as an atropisomeric mixture because of restricted rotation around the central binaphthyl bond. The dimerization of hemigossypol is regiospecific in cotton. In the case of some moco cotton, the dimerization also exhibits a high level of stereoselectivity. The mechanism that controls this stereoselective dimerization is poorly understood. In this paper, we demonstrate that a dirigent protein controls this stereoselective dimerization process. A partially purified protein preparation from cotton flower petals, which by itself is unable to convert hemigossypol to gossypol, converts hemigossypol with a 30% atropisomeric excess into (+)-gossypol when combined with an exogenous laccase, which by itself produces racemic gossypol.     

In vitro inhibitory effect of gossypol from gossypol-acetic acid, and (+)- and (-)-isomers of gossypol on the growth of Edwardsiella ictaluri

AIM: This study was conducted to evaluate the toxic effect of gossypol from gossypol-acetic acid, and (+)- and (-)-isomers of gossypol on the growth of Edwardsiella ictaluri. METHODS AND RESULTS: Inhibitory effect of various concentrations of gossypol on the growth of E. ictaluri was determined. Bacterial recovery was performed by preincubation of bacteria in medium containing various concentrations of gossypol and subsequent activation of bacteria by inoculating on gossypol-free plates. Concentrations of racemic gossypol, (+)-gossypol and (-)-gossypol of 1.5 microg ml(-1) or higher significantly reduced the number of bacterial colonies compared with that of the control. The growth of E. ictaluri was completely inhibited on agar plates supplemented with 3 microg ml(-1), regardless of the forms of gossypol. The inhibitory effect of (+)-gossypol was higher than that of (-)-gossypol or gossypol-acetic acid. Recovery of E. ictaluri was <50% for all three forms of gossypol at concentrations of 5 microg ml(-1). Bacterial recovery remained relatively constant (6.5%) at gossypol concentrations from 10 to 100 microg ml(-1). Complete killing of E. ictaluri was not reached at gossypol levels up to 100 microg ml(-1). CONCLUSION: Gossypol-acetic acid, and (+)- and (-)-optical isomers have anti-bacterial effect against E. ictaluri. The results suggest the action is bacteriostatic rather than bactericidal. SIGNIFICANCE AND IMPACT OF THE STUDY: The therapeutic effect of gossypol against E. ictaluri may be useful in controlling enteric septicaemia of catfish.     

Terpenoid aldehyde formation and lysigenous gland storage sites in cotton: variant with mature glands but suppressed levels of terpenoid aldehydes

A new cotton variant with reduced levels of terpenoid aldehydes (sesquiterpenoids and sesterterpenoids (heliocides)) was isolated from the progeny of hemizygous cotton (Gossypium hirsutum cv. Coker 312) transformed with antisense (+)-delta-cadinene synthase cDNA. Southern analysis of leaf DNA digested with HindIII, Pst or KpnI restriction endonucleases did not detect any antisense cdn1-C1 DNA in the genome of the variant. The gossypol content in the seed of the variant was markedly lower than in the seed of T1 antisense plants. Eighty-nine percent of the variant seed had a 71.1% reduction in gossypol and the foliage of the variant plants showed a 70% reduction in gossypol and a 31% reduction in heliocides. Compared to non-transformed plants there was no reduction in the number of lysigenous glands in the seed of the variant. The cotton variant shows uncoupling of terpenoid aldehyde synthesis and gland formation. The cotton variant may have resulted from somaclonal variation occurring in the callus tissue during the transformation-regeneration process.     

Differential effects of (+)- and (-)-gossypol enantiomers on LDH-C4 activity of hamster spermatogenic epithelium in vitro

Tubular fragments (spermatogenic epithelium) from immature hamsters were isolated and cultured with low doses of (+)- and (-)-gossypol enantiomers. The activity of lactate dehydrogenase isoenzyme LDH-C4 was estimated as a marker for spermatogenic cell development and alpha-ketoisovalerate was used as the substrate. In the absence of gossypol, the specific activity of LDH-C4 in the tubular fragments was increased 40% during incubation for 48 h. This developmental increase was suppressed by gossypol. The specific activity of LDH-C4 in the tubular fragments was lowered by gossypol, after 48 h of culture in the presence of low doses of racemic gossypol (1-4 microM) and 1% fetal calf serum. In this in-vitro system the (-)-enantiomer but not the (+)-enantiomer of gossypol affected the LDH-C4 activity. This is in agreement with other reports showing that only the (-)-enantiomer induces infertility. The observed action of gossypol on LDH-C4 activity in the tubular fragments may reflect gossypol-induced degeneration of spermatogenic cells. The present in-vitro system can be used to estimate the actions of gossypol derivatives, other investigational antifertility agents, and toxic agents on the spermatogenic epithelium.     

The (+)- and (−)-gossypols potently inhibit human and rat 11β-hydroxysteroid dehydrogenase type 2

Gossypol has been proven to be a very effective male contraceptive. However, clinical trials showed that the major side effect of gossypol was hypokalemia. Gossypol occurs naturally as enantiomeric mixtures of (+)-gossypol and (−)-gossypol. The (−)-gossypol is found to be the active component of antifertility. 11β-Hydroxysteroid dehydrogenase 2 (11βHSD2) has been demonstrated to be a mineralocorticoid receptor (MR) protector by inactivating active glucocorticoids including corticosterone (CORT) in rats, and therefore mutation or suppression of 11βHSD2 causes hypokalemia and hypertension. In the present study, the potency of gossypol enantiomers was tested for the inhibition of 11βHSD1 and 2 in rat and human. Both (+) and (−)-gossypols showed a potent inhibition of 11βHSD2 with the half maximal inhibitory concentration (IC₅₀) of 0.61 and 1.33 μM for (+) and (−)-gossypols, respectively in rats and 1.05 and 1.90 μM for (+) and (−)-gossypols, respectively in human. The potency of gossypol to inhibit 11βHSD1 was far less; the IC₅₀ was ≥100 μM for racemic gossypol. The gossypol-induced hypokalemia is likely associated with its potent inhibition of kidney 11βHSD2.     

8-Hydroxy-(+)-delta-cadinene is a precursor to hemigossypol in Gossypium hirsutum

[(3)H](+)-delta-Cadinene and its 8-hydroxy derivative, prepared from (1RS)-[1-(3)H]FPP by the action of one and two recombinant enzymes, respectively, were infiltrated into cotyledons of bacterial blight-resistant cotton plants as they biosynthesized sesquiterpene phytoalexins in response to infection by Xanthomonas campestris pv. malvacearum. Following both treatments, tritium appeared in the HPLC fraction that contained hemigossypol. Hemigossypol was isolated from the cotyledons that had been treated with [(3)H](+)-8-hydroxy-delta-cadinene and was trimethylsilylated and purified. In two experiments, specific radioactivity of the hemigossypol derivative indicated that 5% and 10%, respectively, of the [(3)H](+)-8-hydroxy-delta-cadinene had been converted to hemigossypol.     

Inhibition of testicular LDH-X from laboratory animals and man by gossypol and its isomers

The inhibitory effect of (+)-, (-)-, (+/-)-gossypol and (+/-)-gossypol acetic acid upon testicular cytosolic LDH-X was measured in vitro. Gossypol acetic acid (0-100 mumol/l) inhibited LDH-X prepared from the testes of the mouse greater than rabbit greater than human greater than rat greater than hamster. There was no relationship between inhibition and in-vivo antifertility activity. LDH activity measured in vitro in serum of men and hamsters was unaffected by gossypol. Gossypol and its isomers were non-competitive inhibitors of human and hamster LDH-X with respect to the coenzyme NADH, competitive inhibitors of human LDH-X and noncompetitive-competitive inhibitors of hamster LDH-X with respect to the substrate alpha-ketobutyrate. Co-incubation with human serum albumin or poly-L-lysine but not lysine protected human and hamster LDH-X from gossypol.     

Production of (+)-5-deoxystrigol by Lotus japonicus root culture

Lotus japonicus roots, cultured in a modified B5 medium, produced and secreted germination stimulants that induced Striga hermonthica seed germination. The germination-inducing activity was detected both in the roots and the culture filtrate. Following bioassay-guided purification procedures, an active compound was isolated from hexane extracts of the roots and the culture filtrate. Based on chromatographic behaviour on HPLC, and 1H NMR, UV, MS and CD spectroscopic analyses, the germination stimulant was identified as (+)-5-deoxystrigol.     

Synthesis of gossypol atropisomers and derivatives and evaluation of their anti-proliferative and anti-oxidant activity

Gossypol 1, gossypolone 2, and a series of bis 3 and half Schiff's bases 4 of gossypol were synthesised and tested for anti-proliferative and anti-oxidant activity. (-)-Gossypol (-)-1 was the most potent inhibitor of the proliferation of the HPV-16 keratinocyte cell line (using an MTT viability assay) with a GI50 of 4.8 microM. The bis Schiff's base of (-)-gossypol with L-tyrosine ethyl ester (-)-3b was the most potent inhibitor of iron/ascorbate dependent lipid peroxidation (using the thiobarbituric acid test), with an IC50 of 11.7 microM, with (-)-gossypol being the next most potent of the series, with an IC50 of 13.1 microM. The results from these initial assays suggest that gossypol, as either a racemic mixture rac-1, or the individual atropisomers (-)-1 or (+)-1, has potential for the treatment of psoriasis.     

Optically active gossypol as a circular dichroism probe of interactions with serum albumins

The (+)-enantiomer of the polyphenolic binaphthyl gossypol, has been shown to be a useful CD probe of interactions with human and bovine serum albumin. (+)-Gossypol binds to albumin with same affinity as recemic (±)-gossypol, as shown by fluorescence quenching, and also displaces bilirubin from its albumin binding site. The CD characteristics of bound gossypol are different in the case of the two proteins.     

The absolute configuration of (-)-3-hydroxy-alpha-calacorene

3-Hydroxy-alpha-calacorene was identified in extracts from cold-shocked seedlings of cotton (Gossypium hirsutum L.) and kenaf (Hibiscus cannabinus L.), both of which are members of the Malvaceae family. (-)-3-Hydroxy-alpha-calacorene was isolated from Heterotheca inuloides Cass. (Asteraceae). HPLC on a chiral stationary phase column showed that the 3-hydroxy-alpha- calacorene from cotton and kenaf had the same relative configuration, while that from H. inuloides was of the opposite configuration. X-ray crystallographic analysis established the absolute configuration of the compound in H. inuloides as (8R)-(-)-3-hydroxy-alpha-calacorene.     

The effect of gossypol on the frequency of DNA-strand breaks in human leukocytes in vitro

Gossypol, a human antifertility agent isolated from the cotton plant, was found to induce a dose-dependent increase in the frequency of DNA-strand breaks in human leukocytes exposed to 2-40 micrograms/ml of the drug for 1 h in serum-free medium in vitro. DNA-strand breaks were studied by alkaline elution or alkaline unwinding of DNA followed by hydroxylapatite-chromatography. No decrease of gossypol-induced DNA-strand breaks was observed after post-treatment incubation times up to 24 h, whereas X-ray-induced DNA breaks disappeared within 2 h under the same incubation conditions. Cells exposed to gossypol in the presence of 10% fetal calf serum showed no or little increase of DNA breaks, suggesting that serum proteins inhibit the DNA-damaging activity of the drug. Both optical isomers of gossypol induced DNA-strand breaks. However, the effect of (-)-gossypol was only about half of that of (+)-gossypol and the racemic form. The induction and persistence of DNA-strand breaks by gossypol, as well as the reduction of this effect in the presence of serum should be considered in the evaluation of the potential in vivo genotoxicity of the drug.     

HPLC preparation of the chiral forms of 6-methoxy-gossypol and 6,6'-dimethoxy-gossypol

A concentrated mixture of gossypol, 6-methoxy-gossypol, and 6,6'-dimethoxy-gossypol was extracted from the root bark of St. Vincent Sea Island cotton with acetone. This extract was derivatized with R-(-)-2-amino-1-propanol to form diastereomeric gossypol Schiff's bases. Analytical-scale reverse-phase chromatography of these Schiff's bases produced six peaks, indicating separation of the enantiomeric forms of the three gossypol compounds. The elution order of the peaks was found to vary with the polarity of the mobile phase. The chromatography was scaled to a preparative level and was used to isolate each compound. After hydrolysis of the separated Schiff's bases, the original compounds were recovered by precipitation from solutions of diethyl ether, acetic acid, and water. Fifty injections yielded approximately 500 mg of each methoxy-gossypol enantiomer and 300 mg of each dimethoxy-gossypol enantiomer. Each compound was characterized for carbon and hydrogen content, optical rotation, UV-vis light absorption, and melting point. Standard curves were developed and were used to measure the concentration of each gossypol form in the root bark and dehulled seed of St. Vincent Sea Island cotton. In seed tissue, 48% of the gossypol compounds were methylated, and the (-)-optical form was found to be in a slight excess to the (+)-optical form (53-54%) for all three compounds. In root bark, 71% of the gossypol compounds were methylated, and the (+)-optical form was in excess to the (-)-optical form for all three compounds. However, in this tissue the extent of enantiomeric excess decreased with the degree of methylation, with 77% of the gossypol existing in the (+)-optical form and 59% of the 6,6'-dimethoxy-gossypol existing in the (+)-optical form.     

Determination of the absolute configuration and solution conformation of gossypol by vibrational circular dichroism

Vibrational circular dichroism (VCD) measurements and density functional theory (DFT) calculations were used to obtain the first definitive assignment of the absolute configuration for the polyphenolic binaphpthyl dialdehyde gossypol and a determination of the solution conformation in CDCl(3). VCD spectra recorded for the two resolved enantiomers are near mirror images and excellent agreement between the observed IR and VCD spectra and intensity calculations carried out at the DFT (B3LYP/6-31G*) level establish the absolute configurations of (+)-gossypol as P and (-)-gossypol as M, with two conformations in CDCl(3) solution that differ in isopropyl group orientation.     

Synthesis and anti-H5N1 activity of chiral gossypol derivatives and its analogs implicated by a viral entry blocking mechanism

A series of chiral gossypol derivatives and its analogs were synthesized and tested in vitro for their anti-H₅N₁ activity. Interestingly, (+)-gossypol derivatives and its analogs were more active against H₅N₁ than the corresponding (?)-gossypol derivatives and its analogs. Through a simple chemical modification with amino acids, less active chiral gossypol could be converted into more active derivatives, and most of chiral gossypol derivatives were more potent against H₅N₁ than 1-adamantylamine. With regard to the mechanism of action, chiral gossypol derivatives and its analogs might impair the virus entry step of cell infection, likely targeting to HA2 protein.     

棉叶中棉酚的快速提取与测定方法研究

棉酚(G)及其相关物甲氧基半棉酚(DHG)、半棉酚酮(HGQ)、半棉酚(HG)、杀实夜蛾素(H1-4)等是棉花中重要的抗虫性萜烯类次生物质.利用高效液相色谱法(HPLC)对棉酚及其相关物进行了分离,测定了棉叶中的棉酚含量,讨论了不同的提取方法和测定条件对结果的影响,给出了一套简便、快速的分析测试方法,同时与紫外-可见分光光度法(苯胺法)的结果进行了对比,认为对于棉花的抗虫性的研究来说,HPLC是比较适宜的方法.     

The Inheritance of Gossypol Level in Gossypium II: Inheritance of Seed Gossypol in Two Strains of Cultivated GOSSYPIUM BARBADENSE L

Two strains of cultivated Gossypium barbadense L., Sea Island AS-2 and Pima S-4, were used to study the effects of alleles at two loci on the production and/or storage of gossypol in mature embryos. The normal alleles, Gl(2) and Gl(3), are "native" to G. barbadense, whereas the mutant alleles, gl(2) and gl(3), were introduced from Gossypium hirsutum L. through backcrossing. Each strain was grown in three replications per trial, and one, Sea Island AS-2, was grown in three environments. Each experiment consisted of all possible crosses, including reciprocals, of the four true-breeding genotypes, plus parents. Additive effects accounted for more than 90% of the total genetic variance for seed gossypol level in all trials. Epistatic effects, though small, were frequently significant. In G. barbadense Gl(2) and Gl(3) were associated with the production of similar amounts of gossypol, whereas previous trials with cultivated varieties of G. hirsutum showed that Gl(2) was more than twice as expressive as Gl(3). The greater average productivity of seed gossypol in cultivated G. barbadense, as compared with G. hirsutum, was attributed to greater activity at the Gl(3) locus in the former species.     

Novel O-glycosidic gossypol isomers and their bioactivities

Novel glycosidic gossypol analogs, 7,7′-gossypol diglucoside tetraacetate GS1, 6,7′-gossypol diglucoside tetraacetate GS2, 7,7′-gossypol diglycoside GS1′, 6,7′-gossypol diglycoside GS2′ were obtained by the ultrasound-assisted reaction of the potassium salt of gossypol with 2,3,4,6-tetra-O-acetyl-α-d-glucopyranosyl bromide under PTC conditions and were fully characterized by 1D NMR (¹H NMR, ¹³C NMR, DEPT, 1D NOE), 2D NMR (HMBC, HMQC), FTIR, HRMS and HPLC. The anticancer activities, cytotoxic effects as well as anti-trypanosomal activities of these novel glycosidic gossypols were explored and suggest that gossypol glycosides could be used to develop new candidates for anticancer drugs and anti-trypanosomal agents.     

Differential binding of (+) and (-) gossypol to plasma protein and their entry into rat testis.

Concentrations of (+) and (-) gossypol were measured by high performance liquid chromatography after they were incubated with plasma proteins in vitro. The concentration of (-) gossypol decreased more than the concentration of (+) gossypol. A similar decrease in free gossypol concentrations in the blood plasma of rats was observed after intravenous infusion of gossypol enantiomers. The concentration of (-) gossypol was also found to be lower than the concentration of (+) gossypol at the blood-testis barrier. The biological effect of (-) gossypol probably results from its stereospecific binding to extra- and intracellular proteins in vivo and inhibition of the biological activity of some proteins.     

Changes in the Accumulation of [alpha]- and [beta]-Tubulin Isotypes during Cotton Fiber Development

The expression of [alpha]- and [beta]-tubulin proteins in developing fibers and several other tissues of cotton (Gossypium hirsutum, cv Texas Marker 1) have been analyzed by immunoblots of one- and two-dimensional gels utilizing anti-tubulin antibodies as probes. As a percentage of total protein, fibers had greater amounts of tubulin than did hypocotyls, roots, leaves, or cotyledons. Both [alpha]- and [beta]-tubulin, having apparent molecular masses of approximately 50 kD and isoelectric points between pH 5 and pH 6, were resolved on a single two-dimensional gel. Under the conditions used, [alpha]-tubulin was less acidic in the isoelectric focusing dimension and migrated slightly faster in the sodium dodecyl sulfate dimension than did [beta]-tubulin. Nine [alpha]-tubulin isotypes that formed two distinct groups were identified on immunoblots of two-dimensional gels. The three most abundant [alpha]-tubulin isotypes were common to all tissues examined. Seven distinct [beta]-tubulin isotypes were also identified. Although their level of accumulation differed, four of the [beta]-tubulin isotypes were common to all tissues. Preferential accumulation of isotypes was more apparent in fibers than in the other tissues examined. Two [alpha]-tubulin isotypes and two [beta]-tubulin isotypes showed preferential accumulation in 10- and 20-d postanthesis fibers, respectively.