Determination of gossypol solubility

To improve the extraction of gossypol from the cotton seeds, the solubility of gossypol in different solvents and at different concentrations of micelles was studied. Petroleum ether, hexane, and extraction benzol were used as solvents. Model micelles were prepared from the cotton oil free of gossypol; concentration of micelles ranged from 0 to 50%. Pure gossypol was added to the micelles in the amount sufficient for formation of saturated solution. The solution was then heated to a temperature close to the boiling point of the respective solvent for 10 minutes. The undissolved gossypol was then filtered off, washed and degreased with petroleum ether, dried and weighed. The amount of dissolved gossypol was determined by the difference between the initial and remaining amounts. Solubility of gossypol in the micelles was found to rise with the increase in the oil content. The solubility grew with the increase in the boiling point of a solvent. At micelles concentrations of 0-25%, solubility of gossypol increased in proportion to the increase in oil concentration. Further increase in the concentration resulted in marked rise in solubility, which reached 2.83% in the pure oil.     

超声提取-反相高效液相色谱法测定棉仁中游离棉酚

用超声提取对棉仁进行预处理,探讨了影响棉酚提取率的因素,确定其最佳提取条件为:料液比0.1∶15(g/mL),提取时间50min,超声功率90W。在最佳提取条件下,棉仁中游离棉酚的平均提取率为6.3282 mg/g。利用反相高效液相色谱(RP-HPLC)法测定棉仁中游离棉酚含量,使用SinoChrom ODS-BP C18色谱柱(200×4.6mm,5μm),流动相为甲醇-1%磷酸水溶液(体积比85∶15),流速为0.9mL/min,紫外检测波长为238nm。结果表明,棉酚在0.0044～0.0352mg/mL范围内与色谱峰面积呈良好线性关系,线性相关系数为0.9998,平均加标回收率在98.80%～99.03%之间。该法快速、灵敏、准确,适用于棉仁中游离棉酚的定量分析。     

Synthesis and anti-HIV-1 activity of the conjugates of gossypol with oligopeptides and D-glucosamine

A series of novel gossypol derivatives were synthesized and screened for their in vitro anti-HIV- 1I activity. The results showed that replacing the aldehyde groups of gossypol with certain oligopeptides and Dglucosamine not only reduced the cytotoxicity of gossypol derivatives but also enhanced their antiviral activity against HIV-1. Interestingly, D-glucosamine derivative of gossypol that lacked the COONa group also exhibited the same potent anti-HIV-1 activity as oligopeptide derivatives with the COONa group. These compounds blocked the entry of HIV-1ⅢB into target cell. which was similar to T20. Furthermore, the molecular docking analysis rationalized their anti-HIV-1 activity. The results also implied that certain oligopeptides and D-glucosamine were important moities to prepare gossypol derivatives as HIV- 1 entry inhibitors besides certain amino acids.     

液相色谱-串联质谱法测定食用植物油中的棉酚

建立了食用植物油中棉酚的液相色谱-串联质谱（LC-MS/MS）分析方法。待测物经无水乙醇涡旋振荡提取,C18色谱柱分离,以乙腈和0.1%（v/v）甲酸水溶液为流动相进行梯度洗脱,LC-MS/MS测定,外标法定量。方法的测定低限（S/N>10）为1 mg/kg;在添加浓度为1、2和200 mg/kg水平下,棉酚的加标回收率为87.4%~100%,相对标准偏差为3.9%~12.2%。结果表明,本方法灵敏度高,测定结果准确,回收率稳定,可用于食用植物油中棉酚残留的确证检测。     

Membrane-active properties and antiradical activity of gossypol and its derivatives

A novel asymmetric gossypol derivative was synthesized. The antioxidant activity of gossypol and certain of its derivatives at the aldehyde groups and the interaction of these compounds with model membranes were studied. It was shown that the antiradical and membrane activities of the gossypol derivatives were determined by the structure of the substituent and that gossypol and its derivatives were partially localized in the lipid bilayer and possibly induced formation of a new interdigitating phase. Published in Khimiya Prirodnykh Soedinenii, No. 3, pp. 265–266, May–June, 2008.     

Identification and quantification of gossypol in cotton by using packed micro-tips columns in combination with HPLC

Self-packed micro-tip columns containing a C₁₈-bonded silica stationary phase, based on the same principles as solid-phase extraction methods, were used to obtain gossypol and related sesquiterpenoid aldehyde-enriched fractions. The enriched metabolite fractions were then analyzed by optimized high-performance liquid chromatography (HPLC) with a C₁₈ column (4.6 mm×25 cm) eluted with the binary mobile phase acetonitrile–0.1% aqueous TFA solution (80:20). This method has proven to be highly reproducible. The precision and accuracy, as %RSD and %RME values, were determined to be less than 15% for the method. The minimum detection limit of gossypol was determined to be 10 ng (absolute gossypol). Absolute recovery was greater than 94% with a standard deviation of ±3.68%. This is a simple, fast, and cost-effective method for isolation, identification, and quantification of gossypol and related secondary metabolites. Comparative analysis of gossypol content was performed on different parts of the cotton plant (seeds, stems and leaves) of two different cultivars of Gossypium hirsutum L. (Acala_1517–70 and OR₁₉). The results indicate that the OR₁₉ cv naturally contains higher gossypol levels than the Acala cv. It was also found that treatment of leaves with a Verticillium dahliae-derived elicitor induced production of deoxyhemigossypol rather than gossypol.     

Molecular structures and stability constants of gossypol and its aza-derivative complexes with silver(I) cations studied by potentiometric, ESI MS, NMR, and AM1d semiempirical methods

Stability constants of complexes formed by gossypol and by ten of its Schiff bases with Ag (+) cations were determined by the potentiometric method. The potentiometric and ESI MS experiments indicate the formation of AgL (+) and Ag 2L (2+) complexes between the Schiff bases G1-G7 and Ag (+) cations as well as the formation of AgL (+), Ag 2L (2+), AgL 2 (+) and Ag 3L 2 (3+) complexes between the Schiff bases G8-G10 and Ag (+) cations. The highest stability constant was found for the AgL (+) complex of G8 Schiff base and the lowest one for the AgL (+) complex of G molecule. The (13)C NMR spectra of mixtures between G and AgClO 4 as well as G1-G10 and AgClO 4 indicate that the complexation of the Ag (+) cations is exclusively realized by the aldehyde-aldehyde tautomer of gossypol and by the enamine-enamine form of gossypol Schiff bases, respectively. We show that the main coordination sites for the Ag (+) metal cations are either the oxygen or the nitrogen atoms of the amine parts of the Schiff bases of gossypol. The energetically most favorable structures of the Ag (+) complexes with gossypol (G) or with the gossypol Schiff bases (G1-G10) were calculated and visualized by the AM1d method at an semiempirical level of theory.     

Extraction of gossypol

The conditions for the extraction of gossypol from cottonseed flakes and from isolated gossypol glands have been investigated. It has been established that the amount of gossypol extracted by hexane is affected mainly by the degree of stirring, the moisture content, the material, and the temperature. By steeping the flakes first with concentrated miscella and then with hexane it is possible to extract about 60–65% of the gossypol. It has been shown that hexane and miscella extract practically no gossypol either from dry or from moistened gossypol glands, and only acetone extracts it almost completely.Institute of the Chemistry of Plant Substances, Academy of Sciences of the Uzbek SSR, Tashkent. Translated from Khimiya Prirodnykh Soedinenii, No. 2, pp. 157–160, March–April, 1980.     

Sorption of Ammonia, Methylamine and Methanol by the P3 Polymorph of Gossypol. Synthesis of Unsymmetrical Monoamine Derivatives of Gossypol by a Solid-state Reaction

The P3 polymorph of gossypol has wide, empty channels andstrongly absorbs linear amines. One of the two gossypol aldehydegroups is located near the channel wall. This situation allowsreaction of the amines with half of a gossypol molecule,yielding unsymmetric monoaminoderivatives of gossypol in highyield and by a simple, solid-state method.     

High-performance liquid chromatography with electrochemical detection of gossypol in human plasma

A sensitive and selective high-performance liquid chromatographic method with electrochemical detection for the determination of gossypol in human plasma is described. Glutathione is used as a protective agent and gossypol dimethyl ether as an internal standard. Acetonitrile-treated protein-free plasma sample is first introduced on to a C18 pre-column for enrichment and clean-up. By using a column-switching technique, gossypol and the internal standard are subjected to further separation on a C8 analytical column, while the major interfering components are eliminated before entering the column. Methanol-0.1 M citrate buffer (pH 3.2) (80:20) is used as the mobile phase. The detector potential on the glassy carbon electrode is maintained at +0.6 V vs. an Ag-AgCl reference electrode. The linearity with human plasma ranged from 5 to 250 ng/ml. The absolute recoveries of gossypol and gossypol dimethyl ether were 91.3 and 97.5%, respectively, with a within-day precision of 2.5% and a day-to-day precision of 3.8%. The limit of detection is 5 ng/ml (signal-to-noise ratio = 3:1). The method is considered to be suitable for the clinical pharmacokinetic studies of gossypol.     

Chemiluminescence Determination of Gossypol by Oxidation with N‐Bromosuccinimide

A chemiluminescence (CL) phenomenon was observed when gossypol was injected into a reaction mixture of N‐bromosuccinimide (NBS) and alkaline dichlorofluorescein (DCF). Based on this phenomenon, a rapid and sensitive method for the determination of gossypol was established. Under the optimum conditions, the linear range was from 1.0 × 10^?9 to 1.0 × 10^?6 M. The detection limit was 1.0 × 10^?10 M. The method has been applied to the determination of gossypol in cottonseed, cottonseed oil, pharmaceutical and biological fluids with satisfactory results. The possible CL reaction mechanism was discussed briefly.     

Accelerated semimicro method of determining the amount of free gossypol in cotton seeds and the products of their processing

A semimicro method is proposed for determining the amount of free gossypol in liquid and solid materials using 70% aqueous acetone as the gossypol extractant.     

Supramolecular association of gossypol in the crystalline state

The natural physiologically active compound gossvpol easily forms clathrate compounds with almost all low-molecular organic substances. The clathrates and the polymorphs of gossypol with known structures are classified into 24 groups of isostructural crystals. They differ from each other in the type of molecular association of gossypol. All possible types of gossypol association with predominant intermolecular H-bonds are analyzed in detail. Zero-, one- and two- dimensional associates are formed; each type of associate, except the zero-dimensional one, is built from more than one system of interm olecular H-bonds. An attempt is made to find out the reasons for the diversity of the types of gossypol supramolecular association based on the specific features of the molecular structure of gossypol. Translated fromZhurnal Strukturnoi Khimii, Vol. 40, No. 5, pp. 849–871, September–October, 1999.     

Silica-gadolinium particles loaded with gossypol for simultaneous therapeutic effect and MRI contrast enhancement

Gossypol is a polyphenolic compound with a wide range of therapeutic properties. The incorporation of gossypol in a porous gadolinium-silica matrix was considered for both therapeutic effect and contrast in magnetic resonance imaging. The purpose of the study was to evaluate gossypol loading in silica-gadolinium particles prepared at different pH values, as a first step in designing new theranostic (therapeutic and diagnostic) compounds. Silica-gadolinium particles of 98SiO₂·2Gd₂O₃ (mol%) composition were prepared following the sol–gel route. The structure of the particles and their loading with gossypol were investigated by X-ray diffraction, dynamic light scattering, Brunauer–Emmett–Teller analysis, differential thermal analysis, Fourier transform infrared spectroscopy, electron paramagnetic resonance and X-ray photoelectron spectroscopy. All results confirmed the highest loading of gossypol on the surface of the particles synthesised at lower pH. The potential application in magnetic resonance imaging (MRI) of silica-gadolinium particles loaded with gossypol was tested through MRI measurements that showed improved contrast properties compared with the pristine silica-gadolinium particles.     

6,6′-Dimethoxygossypol: molecular structure,crystal polymorphism,and solvate formation

6,6′-Dimethoxygossypol (DMG) is a naturally occurring derivative of gossypol that is found in relatively high concentration in some Gossypium barbadense cotton varieties. Like gossypol, DMG forms an equimolar solvate with acetic acid, but it was not known if, like gossypol, the compound would also form clathrates with other molecules. To test for this, the compound was crystallized from different solvents. Four new structures of DMG were found that include two polymorphic and two solvated forms. The polymorphs include two monoclinic structures with P2₁/c and C2/c space groups (P1 and P2, respectively). Packing of the DMG molecules P1 is similar to packing of the gossypol molecules in the P1 polymorphic form of gossypol. The DMG molecules in P2 pack in a highly ordered arrangement that has not been previously observed among gossypol structures. DMG forms equimolar solvates with water (S1) and cyclohexanone (S2). Both structures are triclinic with P [`1]P \bar {1} space groups. The DMG molecules in S2 assemble in a manner that is similar to the gossypol molecules of gossypol–cyclohexanone (1:1), and the DMG molecules in S1 pack in a manner that is similar to the DMG molecules in DMG–acetic acid (1:1) as well as the gossypol molecules in gossypol–acetic acid (1:1). Although DMG is not as versatile a host compound as gossypol, it still forms solvates under many crystallization conditions. Consequently, some care is needed to be sure that one understands exactly which form is recovered when the compound is isolated.     

X-ray structural investigation of gossypol and its derivatives XXIII autoclathrates based on a mixed gossypol — 1,4-Dioxane matrix

Gossypol forms various complexes with the isomeric dioxanes. The clathrate with 1,4-dioxane is the only complex of gossypol in which the intrinsic symmetry of the gossypol molecule — the symmetry of a twofold axis — is retained. In this complex, two out of the three 1,4-dioxane molecules belonging to each gossypol molecule participate in the construction of a mixed H-bound gossypol -dioxane matrix, while the third molecule plays the part of guest, the guest molecules having no H-bonds with the host matrix and undergoing desolvation at 108–110°C.     

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-microns C18 column. The overall recovery was 83.5 +/- 2.6%, with an overall relative standard deviation of 9%.     

Study of reaction of gossypol and its imino derivatives with 2,2-diphenyl-1-picrylhydrazyl

Structures and state in solutions of natural polyphenol gossypol and four its imino derivatives, three of which were synthesized for the first time, were studied by IR and NMR spectroscopy, and by quantum chemistry. The reaction of these compounds with 2,2-diphenyl-1-picrylhydrazyl (DPPH) in ethanol was examined. The antioxidant activity of the studied compounds in the reaction with DPPH was evaluated using the values of the stoichiometric coefficients of reaction, EC₅₀, T ₁^2/DPPH and AE parameters. Gossypol hydrazones were shown to be 5–10 times more efficient, while Schiff base to be less efficient as antioxidants in comparison with gossypol itself. The influence of metal cations on the antioxidant activity of gossypol derivatives was studied.     

X-ray investigation of gossypol and its derivatives

The first four homologues of the carboxylic acid series and first two homologues of the monohydric alcohol series with gossypol give equimolar H-clathrates with the channel-type structure that are isostructural with gossypolacetic acid. Formic and acetic acids are capable of forming with gossypol a continuous series of solid substitution solutions. The desolvation of the unstable H-clathrates of carboxylic acids and monohydric alcohols form one and the same polymorph of gossypol. By x-ray structural analysis, the structures have been determined of two complexes of gossypol: an H-clathrate with methanol and a solid solution on the replacement of formic acid by acetic acid in a gossypol matrix.Institute of Bioorganic Chemistry, Uzbekistan Academy of Sciences, Tashkent. Translated from Khimiya Prirodnykh Soedinenii, No. 2, pp. 191–197, March–April, 1992.     

Gossypol exhibits a strong influence towards UDP-glucuronosyltransferase (UGT) 1A1, 1A9 and 2B7-mediated metabolism of xenobiotics and endogenous substances

Gossypol, the polyphenolic constituent isolated from cottonseeds, has been used as a male antifertility drug for a long time, and has been demonstrated to exhibit excellent anti-tumor activity towards multiple cancer types. The toxic effects of gossypol limit its clinical utilization, and enzyme inhibition is an important facet of this. In the present study, in vitro human liver microsomal incubation system supplemented with UDPGA was used to investigate the inhibition of gossypol towards UGT1A1, 1A9 and 2B7-mediated metabolism of xenobiotics and endogenous substances. Estradiol, the probe substrate of UGT1A1, was selected as representative endogenous substance. Propofol (a probe substrate of UGT1A9) and 3'-azido-3'-deoxythimidine (AZT, a probe substrate of UGT2B7) were employed as representative xenobiotics. The results showed that gossypol noncompetitively inhibits UGT-mediated estradiol-3-glucuronidation and propofol O-glucuronidation, and the inhibition kinetic parameters (K(i)) were calculated to be 34.2 and 16.4 μM, respectively. Gossypol was demonstrated to exhibit competitive inhibition towards UGT-mediated AZT glucuronidation, and the inhibition kinetic parameter (K(i)) was determined to be 14.0 μM. All these results indicated that gossypol might induce metabolic disorders of endogenous substances and alteration of metabolic behaviour of co-administered xenobiotics through inhibition of UGTs' activity.