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.

Natural Product Gossypol and its Derivatives in Precision Cancer Medicine.

Gossypol, a natural product extracted from the seed, roots, and stem of cotton, was initially used as a male contraceptive but was subsequently investigated as a novel antitumor agent. This review depicts the current status of gossypol and its derivatives as novel antitumor agents as well as presents their preparation and characteristics, especially of some gossypol Schiff bases, through quantitative and structural analysis. The main attractive target sites of gossypol and its derivatives are Bcl-2 family proteins containing the anti-apoptosis proteins Bcl-2 and Bcl-XL. The molecular mechanism of gossypol analogs not only involves cell apoptosis but also autophagy, cell cycle arrest, and other abnormal cellular phenomena. Gossypol and its derivatives exert antitumor effects on different cancer types in vitro and in vivo, and demonstrate synergistic effects with other chemo- and radio- therapeutic treatments. In addition, several nanocarriers have been designed to load gossypol or its derivatives in order to expand the range of their applications and evaluate their combination effects with other anti-tumor agents. This review may serve as a reference for the rational application of gossypol analogs as anti-tumor agents.

Preparation and evaluation of molecularly imprinted polymer for selective recognition and adsorption of gossypol.

Molecularly imprinted polymers (MIPs) were designed and prepared via bulk thermal polymerization with gossypol as the template molecule and dimethylaminoethyl methacrylate as the functional monomer. The morphology and microstructures of MIPs were characterized by scanning electron microscope and Brunauer-Emmett-Teller surface areas. Static adsorption tests were performed to evaluate adsorption behavior of gossypol by the MIPs. It was found that adsorption kinetics and adsorption isotherms data of MIPs for gossypol were fit well with the pseudo-second-order model and Freundlich model, respectively. Scatchard analysis showed that heterogeneous binding sites were formed in the MIPs, including lower-affinity binding sites with the maximum adsorption of 252 mg/g and higher-affinity binding sites with the maximum adsorption of 632 mg/g. Binding studies also revealed that MIPs had favorable selectivity towards gossypol compared with non-imprinted polymers. Furthermore, adsorption capacity of MIPs maintained above 90% after 5 regeneration cycles, indicating MIPs were recyclable and could be used multiple times. These results demonstrated that prepared MIPs could be a promising functional material for selective adsorption of gossypol.

Development of SECheM Concept for Isolation and Chemical Modification of Gossypol Directly from Cienfuegosia digitata.

INTRODUCTION: Gossypol is an axially chiral natural polyphenol classically extracted from the Malavaceae family. Nevertheless, its extraction and isolation from a plant can be quite complicated and extremely time-consuming since gossypol is known to be sensitive to degradation under solvents, high temperature and light action. Moreover, its purification over column chromatography is a challenging problem due to its ability to oxidise and the existence of various tautomer forms. OBJECTIVE: To develop an efficient "one-step" strategy for simultaneous extraction and semi-synthesis by short-circuiting critical gossypol isolation and purification steps. METHODOLOGY: Gossypol was first isolated from Cienfuegosia digitata roots, characterised (by 1D and 2D NMR) and quantified (by UV spectrophotometry). Thus, aniline was selected to test the "one-step" in situ trapping of freshly extracted gossypol leading to a Schiff base analogue. After screening solvents and extraction times on this model reaction, the "SECheM" (simultaneous extraction and chemical modification) concept was successfully extended to other amines, underlining the efficiency and the robustness of the strategy. RESULTS: After having shown that gossypol occurred as a major compound in C. digitata roots, different experimental procedures using Soxhlet extraction in the presence of aniline pointed out the best conditions for the SECheM concept (7 h of reaction and extraction time in ether as solvent). Ultimately, the concept has been generalised to 17 other amines. CONCLUSION: This is a report of the first semi-synthesis that allows: (1) "in situ" preparation of more stable gossypol Schiff base derivatives directly from ground plant material and (2) circumvention of gossypol extraction and purification problems. Copyright © 2017 John Wiley & Sons, Ltd.

Gossypol: phytoalexin of cotton.

Sesquiterpenoids are a class of 15-carbon secondary metabolites that play diverse roles in plant adaptation to environment. Cotton plants accumulate a large amount of sesquiterpene aldehydes (including gossypol) as phytoalexins against pathogens and herbivores. They are stored in pigment glands of aerial organs and in epidermal layers of roots. Several enzymes of gossypol biosynthesis pathway have been characterized, including 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) and farnesyl diphosphate synthase (FPS) that catalyze the formation of the precursor farnesyl diphosphate (FPP), (+)-δ-cadinene synthase (CDN) which is the first enzyme committed to gossypol biosynthesis, and the downstream enzymes of CYP706B1 and methyltransferase. Expressions of these genes are tightly regulated during cotton plants development and induced by jasmonate and fungi elicitors. The transcription factor GaWRKY1 has been shown to be involved in gossypol pathway regulation. Recent development of new genomic platforms and methods and releases of diploid and tetraploid cotton genome sequences will greatly facilitate the elucidation of gossypol biosynthetic pathway and its regulation.

Gossypolhemiquinone, a dimeric sesquiterpenoid identified in cotton (Gossypium).

The report that the cotton leaf perforator, Bucculatrix thurberiella, is one of the few insect herbivores to attack Gossypium thurberi prompted an investigation of the terpenoids present in the leaves of this wild species of cotton. Members of Gossypium produce subepidermal pigment glands in their leaves that contain the dimeric sesquiterpenoid gossypol as well as other biosynthetically related terpenoids. In addition to gossypol, a previously unknown dimeric sesquiterpenoid, gossypolhemiquinone (GHQ), was identified in trace amounts in G. thurberi, a member of the D genome. Other members of the D genome of Gossypium were subsequently found to contain this compound, but GHQ was not detected in commercial cotton cultivars. When fed to Helicoverpa zea in an artificial diet, GHQ delayed days-to-pupation, reduced pupal weights, and survival to adulthood to a lesser or equal extent than gossypol in comparison to the control diet. However, GHQ had a synergistic effect on survival and days-to-pupation when combined with gossypol at the highest dosage tested (0.18%; 15.5:84.5 GHQ:gossypol). Because gossypol exhibits anti-cancer activity, GHQ was also evaluated for its anti-cancer activity against the National Cancer Institute's 60-Human Tumor Cell Line Screen. Significant inhibitory activity against most of these cell lines was not observed, but the results may offer some promise against leukemia cancer cell lines.

Therapeutic potential of gossypol: an overview.

CONTEXT: Polyphenols are naturally occurring compounds found in fruits, vegetables, cereals, and beverages. Polyphenols occupy a unique place in biological science for their pharmacological properties. Gossypol is a polyphenolic compound that has attracted attention because of its biological effects. OBJECTIVE: Gossypol is reported to exhibit antifertility, antioxidant, anticancer, antivirus, antiparasitic, and antimicrobial properties and lower plasma cholesterol. These are summarized with attention to the mechanisms of activity. METHODS: This review summarizes the results of studies obtained in a comprehensive search of ScienceDirect, PubMed, Scirus, and Web of Science. RESULTS AND CONCLUSION: The results of these studies provide a comprehensive understanding of the biological action of gossypol and its potential for the prevention of and therapy for resistant tumors and chronic human diseases such as HIV, malaria, and psoriasis.

Synthesis, crystal structures and antibacterial activity studies of aza-derivatives of phytoalexin from cotton plant--gossypol.

Using Gossypol (GOS) extracted from cotton seeds, a series of its Schiff bases (1-18) and hydrazones (19-27) have been synthesized and evaluated for their antimicrobial activities against Gram-positive bacteria, Gram-negative bacteria and yeast-like organisms. Of the 27 aza-derivatives of gossypol, 11 have been found active against the microorganisms tested, similar to gossypol. Crystal structures of the new Schiff base (compound 7) and hydrazone (compound 25) of gossypol reveal the presence of two different tautomers within two types of the aza-derivatives studied. The newly synthesized aza-derivatives of gossypol are characterized by the FT-IR, NMR and MS methods.

Antifungal activity of alkyl and heterocyclic aza-derivatives of gossypol as well as their complexes with NaClO4 against Fusarium oxysporum f. sp. lupini.

Eight alkyl and six heterocyclic aza-derivatives of gossypol (2-15) have been synthesized using gossypol (1) extracted from Gossypium Herbaceum cottonseeds. The ability of gossypol aza-derivatives to form complexes with NaClO(4) has been investigated by electrospray ionisation (ESI) mass spectra recorded in the positive and negative ion detection modes. The gossypol aza-derivatives have been characterized by FT-IR, (1)H and (13)C NMR spectroscopic methods and subsequently tested for their antifungal properties against Fusarium oxysporum. Four alkyl aza-derivatives (2-5), present in the enamine-enamine tautomeric form, have shown activity comparable or higher than that of gossypol against this fungus. To improve the antifungal activity the complexes of the most active compounds 2-5 with NaClO(4) were prepared. Complexes of 2 and 5 with NaClO(4) have shown antifungal activity higher than that of the uncomplexed compounds.

Cytotoxic and antibacterial sesquiterpenes from Thespesia populnea.

Eight new sesquiterpenoids, named populene A-H (1-8), were isolated from dichloromethane extracts of the wood and dark heartwood of Thespesia populnea, together with 11 known compounds (9-19). Their structures were determined on the basis of spectroscopic analyses. The cytotoxic activity of isolated compounds was evaluated against four cancer cell lines: MCF-7, HeLa, HT-29, and KB. Mansonone E (11) and (+)-gossypol (18) showed significant activities. Their antibacterial properties against Bacillus subtilis, Staphylococcus aureus, and Enterococcus faecalis are also presented.

Rational design and real time, in-cell detection of the proapoptotic activity of a novel compound targeting Bcl-X(L).

Antiapoptotic Bcl-2-family proteins Bcl-2 and Bcl-X(L) have been recently validated as drug discovery targets for cancer. Here, by using a combination of molecular modeling, NMR-based structural analysis, fluorescence polarization assays, and cell-based assays, we have designed and characterized a novel proapoptotic compound targeting these proteins. Our compound, Apogossypol, is capable of binding and inhibiting Bcl-2 and Bcl-X(L) with high affinity and induces apoptosis of tumor cell lines. Mechanistic studies on the action of our compound were also performed via confocal microscopy that provided real-time detection of the interaction with Bcl-X(L) in intact cells. Finally, preliminary data on cells freshly isolated from patients affected by chronic lymphocytic leukemia strongly suggest potential applications of Bcl-2 antagonists as chemosensitizers in cancer therapy.

Preparation of enantiomeric gossypol by crystallization.

Large enantiomorphic crystals of gossypol-acetone (1:3) were grown from acetone solutions of rac-gossypol-acetic acid (1:1) at 4 degrees C. By controlling the initial gossypol concentration, crystallization time, and solution volume, single crystals were grown that weighed >50 mg, equivalent to >37 mg of enantiomeric gossypol. Even larger crystals were possible, but it was difficult to produce these reliably without contamination of the antipode. Essentially all of the acetone within the crystal form was removed by storing the crystals under vacuum for 3-4 days. By employing these techniques, gram quantities of enantiomeric gossypol were prepared in high chemical and optical purity. Based on measured and reported optical rotations, the optical purity of samples prepared by crystallization was greater than the optical purity of samples prepared by chromatographic separation of gossypol-amine diastereomers. The principal limitation of crystallization as a preparative method is the need to determine the chirality and purity of each product crystal. Nevertheless, the method competes favorably with preparative-scale chromatographic procedures.

Ion-channels in human sperm membrane and contraceptive mechanisms of male antifertility compounds derived from Chinese traditional medicine.

Ion channel plays a key role in maturation, capacitation and acrosome reaction of sperm. However, as it is difficult to record channel currents from a mature mammal sperm directly by patch-clamp technique, there were no basic data on the types and properties of the channels in human sperm until the method reconstituting the channels into bilayer was used. By reconstituting the channel proteins isolated from sperm membrane into phospholipid-forming bilayer, we have characterized several kinds of Ca2+-, Na+-, K+-, and Cl--permeable channels with different conductance and properties in human sperm membrane. To study the channels in spermatogenic cells is another approach to understand the ion-channels in mature sperm. The cell is used as a model to analyze the effects of male antifertility agents on Ca2+-channel. To date, several male contraceptives derived from Chinese traditional medicine have been attached worldwide interest, a lot of compounds have been purified from them, and the antifertility effects of some compounds were demonstrated. We studied the effects of gossypol and several compounds isolated from Tripterygium wilffordii on Ca2+ channel in mouse spermatogenic cells and found that each of them inhibited the channel and sperm acrosome reaction at a proximate concentration, suggesting that the inhibition of Ca2+ channels may be one mechanism of the antifertility effects of these contraceptives.

Isolation of (-)-gossypol from natural plant.

(-)-Gossypol has been isolated from cottonseed of G. barbadense L. Three Gossypium species were studied for the isolation. The study has also revealed a correlation between the optical activity of gossypol isolated and the species of cottonseed used. Gossypol isolated from the species G. barbadense is always found to be levorotatory caused by an excess of (-)-gossypol, whereas that from the species G. hirsutum is always dextrorotatory caused by an excess of (+)-gossypol. Results of most samples (cultivars) showed that optical purity of gossypol isolated from G. barbadense varied from 10-25%, while that from G. hirsutum varied from 10-20%. This is the first report of (-)-gossypol occurring naturally.

PIP: Both (+ and -)-gossypol and (+)-gossypol have been found to be naturally occurring, while no plant producing (-)-gossypol has to date been located. This study isolated (-)-gossypol from the cottonseed of Gossypium barbadense L. Cottonseed from G hirsutum L and G arboreum L was also investigated. The (-)-gossypol content was consistently higher than the (+) gossypol content for all 16 sample of G barbadense studied, while the (+)-gossypol content was higher in the 10 G hirsutum L samples and the 1 G arboreum L sample. The rotation property of gossypol was mainly correlated with the species of the cottonseed; the area where the cottonseed was cultivated had no effect. Gossypol isolated from G barbadense was always levorotatory as a result of the excess of (-)-gossypol, whereas that from the other 2 species studied was always dextrorotatory due to the excess of (+)-gossypol. The optical purity of gossypol isolated from G barbadense ranged from 10-25%, and that from G hirsutum varied from 10-20%.

Gossypol in cottonseed products: toxicology and inactivation.

The little attention given to the potential of cottonseeds as food for monogastric animals and man is due to the presence of toxic principles in them, especially gossypol. The injury to livestock associated with ingestion of cottonseeds results from the unfavorable physiological effect of gossypol per se, as well as from its adverse effects on the protein nutritive value of the food (mainly by rendering lysine metabolically unavailable). Various approaches have been employed to overcome these problems, by removing the gossypol glands, growing glandless varieties, extraction of gossypol with solvents, or through inactivation by heat processing, reacting with cations or with certain organic compounds. In this study the phospholipid fraction (gums) derived from soybeans, which is relatively rich in phospholipids containing a free amino group, was used with a view to lowering the tendency of gossypol to bind with lysine, and at the same time to inactivate the gossypol by converting it into a bound form. Dehulled and flaked cottonseeds were mixed with 5% soybean gums and 0.5% NaOH, in a laboratory cooker resembling the apparatus used in the cottonseed processing industry. The meat was cooked for different periods and at several temperatures. Later the oil was extracted with hexane and the meal was tested for free gossypol, "available lysine" and protein nutritive value, in comparison with a control batch treated similarly but without gums. The results proved the superiority of the meal treated with gums, as evidenced by all the above parameters.

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 gossypol.

PIP: Recent papers reporting on the male infertility 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 2 substances and impure gossypol on the fertility of male hamsters was evaluated. Only pure gossypol produced a decrease of fertility after 6 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 4 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 gossypol.

Tumor-inhibitory agent from Montezuma speciosissima (Malvaceae).

The petroleum ether extract of Montezuma speciosissima Sesse and Moc. demonstrated tumor-inhibiting properties in the P-338 lympocytic leukemia test system (3PS). The constituent responsible for this activity was shown to be a symmetrically substituted 2,2'-dinaphthol, identified as gossypol (C30H30O8).