Kristina Turner, M.S.

New Mexico State University,

Las Cruces, NM, USA

Master’s of Science, Biology (Microbiology), December 2006

Bachelor’s of Arts, Biology, December 2003
Bachelor’s of Arts, Foreign Languages (German, Russian minor), May 2000

The Polyphenolic Cotton Terpenoid Gossypol and Its Broad Bioactivity

1. Introduction
1.1 Effects of the Cotton Terpenoid Gossypol on Bacteria
1.1.1 The Effects of Saponins on Membrane Permeability and Possible
Enhancement in Uptake of Cationic Compounds such as Gossypol
into Gram-Negative Bacteria and Fungi
1.1.2 Effects of (+)- and (-)-Gossypol Enantiomers on the Gram-Negative
Bacteria Edwardsiella ictaluri
1.1.3 Gossypol-containing Cottonseed’s Use as Animal Fodder and Corresponding
Decrease in Incidence of Bacterial Infections in those Animals
1.2 Stereospecific Effects of Gossypol Enantiomers Against Human Cell Lines
1.3 New Gossypol Derivatives
1.4 Gossypol’s Effects as a Reversible Calcineurin Inhibitor
1.5 A Methyltransferase Capable of Decreasing Gossypol’s Activity
1.6 A Separation Method for Enantioresolving (+)- and (-)-Gossypol in
Multi-milligram Quantities
1.7 Gossypol’s Effectiveness Against Malaria-causing Plasmodium falciparum
and Related Disease-Causing Protozoa
1.8 Gossypol as a Retroviral Inhibitor of HIV-1
1.9 Eradication of Fecal Coliforms in a Bioreactor Conversion of Cotton Gin
Waste and Dairy Cattle Manure to Methane
1.10 Sequence Homology to Gossypol’s Synthase and Possible Insight into Gossypol’s
Remarkably Broad Activity
1.11 Literature Cited

1. Introduction

Gossypol is a polypenolic, aldehyde-containing constituent of cottonseed. The cotton

terpenoid gossypol is also found in the roots, leaves, and stems of Gossypium hirsutum, upland

cotton, and related species. Gossypol protects cotton from a wide variety of pests and pathogens.

Glandless cotton plants lacking gossypol are largely defenseless against insects, bacteria, fungi

and viruses. Gossypol has been investigated for a wide variety of applications. It has been
found to reduce the size of tumors, inhibit bacterial and fungal growth, have antiviral properties

as a retroviral inhibitor of HIV and influenza, inhibit the growth of malaria-causing Plasmodium

falciparum and toxoplasmosis-causing Toxoplasma gondii tachyzoites, have insecticidal uses,

and be effective as a male contraceptive.

The experiments described in the following chapters will concentrate on the antibacterial

effects of gossypol on the human pathogens Staphylococcus aureus and fecal coliforms such as

Escherichia coli O157:H7. Minimal Inhibitory Concentration (MIC) testing will be performed

using the NCCLS broth microdilution method on Staphylococcus aureus SH1000 and COL wild-

type strains. A bioreactor conversion of cotton gin waste and dairy cattle manure to methane

will also be performed to evaluate the effects of glanded cotton gin waste versus glandless

cotton gin waste on the survival of fecal coliforms.

1.1 Effects of the Cotton Terpenoid Gossypol on Bacteria

Tegos et al. (2002) tested gossypol and a panel of other plant antimicrobials against a

wide variety of plant pathogens and several important human pathogens. Gossypol was found to

have lower Minimal Inhibitory Concentrations (MICs) ranging from 1.95-3.91 µg/mL for the

gram-positive bacteria tested and much higher MICs ranging from 31.25-1000 µg/mL for the

gram-negative bacteria tested. This study suggested that plant antimicrobials have a lower

activity against gram-negative bacteria because they have a highly impermeable outer membrane

and a set of multidrug resistance pumps that expel amphipathic toxins across the outer

membrane. Plant antimicrobials are fairly effective against gram-positive bacteria, which have a

single membrane that is much more permeable and fewer multidrug resistance pumps than gram-

negative bacteria. A variety of plant antimicrobials were tested against a representative panel of

both gram-positive and gram-negative plant pathogens and a selection of several important
human pathogens. They also included mutant bacteria with either inhibited or disabled

multidrug resistance pumps. The activities of the plant antimicrobials against gram-negative

bacteria were greatly increased in the bacteria with disabled or inhibited multidrug resistance

pumps. A variety of plants produce multidrug resistance pump inhibitors in addition to their

amphipathic terpenoids. This could help to explain how plants can prevent infections by gram-

negative bacteria in addition to gram-positive bacteria, although their amphipathic cation

terpenoids are the major substrates of multidrug resistance pumps. This study suggests that plant

antimicrobials might be used as broad-spectrum antibiotics when used in conjunction with

multidrug resistance pump inhibitors. However, currently available multidrug resistance pump

inhibitors are not safe for systemic use in humans. Chelating agents such as EDTA have also

been added to surgical wound dressings to increase cell membrane permeability to amphipathic

cation antimicrobials such as gossypol, although this is limited to topical treatment.

1.1.1 The Effects of Saponins on Membrane Permeability and Possible

Enhancement in Uptake of Cationic Compounds such as Gossypol

into Gram-Negative Bacteria and Fungi

A review article by Kim Lewis (2001) suggests that plants may produce saponins to

increase the permeability of both eukaryotic cellular membranes. Plants make saponins that

specifically “extract” ergosterol from the membranes of eukaryotic cells. This is accomplished

by saponins, which are glycosilated sterols, forming a complex with ergosterol. Some saponins

have the ability to directly kill yeast or fungal pathogens. Destroying the permeability barrier

of a pathogen might be the plant’s solution of bypassing the numerous multidrug resistance

pumps, which normally are effective in extruding the amphipathic cations produced to kill

pathogenic bacteria and yeast. Lewis suggests that saponins should be tested for their ability to
potentiate the penetration of other antimicrobial compounds. Saponins are abundantly present in

many plant foods such as soy beans, indicating their low toxicity. Saponins could also be tested

in prokaryotic bacteria to see if they similarly increase permeability in membranes lacking

ergosterol. Increasing the permeability of prokaryotic cells would be an effective way of

bypassing their multidrug resistance pumps. This would increase their susceptibility to

amphipathic cations produced by plants as antimicrobials.

1.1.2 Effects of (+)- and (-)-Gossypol Enantiomers on the Gram-Negative

Bacteria Edwardsiella ictaluri

Catfish fed a diet of cottonseed have been observed to be more resistant to bacterial

infection. This led Yildrim-Aksoy et al. (2004) to test the antibacterial effectiveness of gossypol

against the gram-negative Enterobacteriaceae E. ictaluri. The study found that concentrations of

racemic gossypol, (+)-gossypol and (-)-gossypol of 1.5 µg/mL 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 µg/mL, 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 less than 50% for all three forms of gossypol at

concentrations of 5µg/mL. Bacterial recovery remained relatively constant (6.5%) at gossypol

concentrations from 100 to 100 µg/mL. Complete killing of E. ictaluri was not reached at

gossypol levels up to100 µg/mL. The study concluded that gossypol-acetic acid, and (+)- and
(-)-optical isomers have antibacterial effect against E. ictaluri. The results suggest the action is

bacteristatic rather than bactericidal. The study suggests that the therapeutic effect of gossypol

against E. ictaluri may be useful in controlling enteric septicaemia of catfish. E. ictaluri is a

gram-negative member of the Enterobacteriaceae that is considered one of the lesser human

pathogens and can cause enterocolitis, sepsis and wound infections.

1.1.3 Gossypol-containing Cottonseed’s Use as Animal Fodder and Corresponding

Decrease in Incidence of Bacterial Infections in those Animals

Yildrim-Aksoy et al. (2004) showed the (+)-gossypol enantiomer to be more effective in

killing E. ictaluri than the (-)-gossypol enantiomer. This is significant because the (-)-gossypol

enantiomer is more toxic to eukaryotes, while the (+)-gossypol enantiomer is much better

tolerated. Cottonseed is high in protein and often utilized as animal feed. Interest is high in

breeding cotton varieties with >95% (+)-gossypol enantiomer, since non-ruminant animals can

tolerate much higher quantities in their feed with fewer side-effects.

This study suggests that high (+)-gossypol enantiomer levels would still be beneficial in

preventing bacterial infections in the animals fed cottonseed primarily containing that

enantiomer. This would be a far better alternative to feeding large quantities of antibiotics to

stock animals, since fewer antibiotic-resistant bacteria would be put into the environment. Since

the (+)- and (-)-gossypol enantiomers have different breakdown pathways, any bacteria evolving

to be resistant to the (+)-gossypol enantiomer would be less likely to have cross-resistance to the

(-)-gossypol enantiomer. Stipanovic et al. (2005) have characterized the occurrence of (+)- and

(-)-gossypol in various wild cotton varieties and in Gossypium hirsutum Var. marie-galante

(Watt) Hutchinson. Knowing the enantiomer ratios of different cotton species could lead to the
breeding of cotton that could safely be fed to animals and reduce their contraction of bacterial

infections. The fact that (+)-gossypol does not induce the anti-fertility and toxicity effects that

(-)-gossypol induces, while still being effective against bacteria suggests that feeding high

protein cottonseed to animals that contains >95% (+)-gossypol would be safe. This also points

to the potential of (+)-gossypol being safely developed into a systemically administered

antibiotic.

1.2 Stereospecific Effects of Gossypol Enantiomers Against Human Cell Lines

Qui et al. (2002) illustrate the effects of gossypol against human cell lines. The study

was conducted because gossypol, a polyphenolic, aldehyde-containing constituent of cottonseed,

has produced partial responses (greater than 50% reduction in tumor size) in some patients with

advanced cancer and suppressed sperm as an anti-fertility agent for men. The researchers

utilized the random homozygous knockout approach of Li and Cohen to develop a cell line

resistant to killing by gossypol, but sensitive to methotrexate and doxorubicin. The cell line

showed stereospecific resistance to killing by (-) gossypol (ED50 4.9 µM) compared with wild

type (ED50 2.0 µM). The resistant and wild-type cells were equally sensitive to (+)-gossypol

(ED50 8.8 and 8.4 µM, respectively), methotrexate, and doxyrubicin. The study concluded that

gossypol affects cells by a stereospecific pathway for (-)-gossypol, possibly related to its

selective effects, and a nonstereospecific pathway for (+)-gossypol and higher concentrations of
(-)-gossypol. The study suggests that further knowledge about the stereospecific pathway may

lead to new chemotherapeutic drugs.

1.3 New Gossypol Derivatives

Dao et al. (2003) synthesized new dithiane and dithiolane aldehyde derivatives of

gossypol and gossypolone. This effort was undertaken to develop prodrugs to reduce the toxicity

of gossypol, while retaining its activity. The relative toxicity of these compounds against KB

cells, human epidermoid carcinoma cells of the mouth, was determined. As was expected by the

researchers, the chemistry was readily reversed in the presence of electrophiles, and the

possibility that these masking groups could be deprotected with NO- in physiological media was

tested. Their findings suggest that this class of analogues can be used as less-toxic prodrugs that

become activated near tumor cells surrounded by high concentrations of NO-. High

concentrations of NO- are also found in endosomes and are key in inducing conformational

changes to enveloped viruses such as influenza that promote fusion and uptake into host cells.

This fact suggests that dithiane derivatives of gossypol activated by high acid concentrations

could increase its effectiveness as a retroviral inhibitor against influenza and HIV, since the virus

particles would be exposed to gossypol only during key events in their life cycle and would have

less time to evolve resistance mutations specifically against gossypol. Also the dithiane

derivatives of gossypol are much less toxic than gossypol and would be better tolerated

systemically by humans.

Sabirova and Madaminov (2003) synthesized a new water-soluble derivative of gossypol

called mebavin that has been shown to possess anti-inflammatory properties similar to those of

gossypol. The main structure of mebavin is the same as gossypol, however the carbonyl groups
have been replaced with nitryl groups and a polyvinylpyrrolidone carrier has been attached.

Mebavin was tested for its anti-inflammatory activity in inbred lines of mice and rats, in which

adjuvant arthritis was induced. The study compared the effectiveness of mebavin, prednasol,

and their combinatory use in the treatment of adjuvant arthritis. Mebavin’s anti-inflammatory

effect was found to be comparable with prednasol, and their combinatory usage suggested

synergism and reduced the toxicity of mebavin. Gossypol is insoluble at pH lower than 8,

therefore the development of the new water-soluble derivative mebavin, could greatly increase

gossypol’s biological accessibility and potential usage.

1.4 Gossypol’s Effects as a Reversible Calcineurin Inhibitor

Baumgrass et al. (2001) show that gossypol is a novel Calcineurin inhibitor.

Calcineurin (CaN) is the only Ca2+/calmodulin-dependent protein Ser/Thr phosphatase, is

thought to be a key functional event for most cyclosporin A (CsA)- and tacrolimus (FK506)-

mediated biological effects. In addition to CaN inhibition, however, CsA and FK506 have

multiple biochemical effects because of their action in a gain-of-function model that requires

prior binding to immunophilic proteins. A small molecule library was screened for direct

inhibitors of CaN using CaN-mediated dephosphorylation of P33-labeled 19-residue

phosphopeptide substrate (RII phosphopeptide) as an assay found the polyphenolic aldehyde

gossypol to be a novel CaN inhibitor. Unlike CsA and FK506, gossypol does not need a

matchmaker protein for reversible CaN inhibition with an IC50 value of 15 µM. Gossypolone,

an analog of gossypol, showed improved inhibition of both RII phosphopeptide and p-

nitrophenyl phosphate desphorylation with an IC50 of 9 and 6 µM, respectively. Apogossypol

hexaacetate was inactive. Gossypol acts noncompetetively, interfering with the binding site for

the cyclophilin 18-CsA complex in CaN. Unlike CsA and FK506, gossypol does not inactivate

the peptidyl-prolyl-cis/trans-isomerase activity of immunophilins. Similar to CsA and FK506, T

cell receptor signaling induced by phorbol 12-myristate 13-acetate/ionomycin is inhibited by

gossypol in a dose-dependent manner, demonstrated by the inhibition of nuclear factor of

activated T cell (NFAT) c1 translocation from the cytosol into the nucleus and suppression of

NFAT-luciferase reporter gene activity. The fact that gossypol does not permanently inactivate

immunophilins suggests that its systemic use would be less harmful than cyclosporin A.

1.5 A Methyltransferase Capable of Decreasing Gossypol’s Activity

Liu et al. (1999) isolated a methyltransferase, produced upon infection of cotton stele

tissue with the pathogenic fungi Verticillium dahliae, which specifically methylates the 6-

position of desoxyhemigossypol to form desoxy hemigossypol-6-methyl ether with a Km value of

4.5 µM for desoxyhemigossypol and a Kcat/Km of 5.08 x 104 s-1 (mol/L)-1. The molecular mass of

the native enzyme is 81.4 kD and is dissociated into two subunits of 41.2 kD on sodium dodecyl

sulfate-polyacrylamide gel electrophoresis gels. The enzymatic reaction does not require Mg+2

and is inhibited 98% with 10 mM p-chloro-mercuribenzoate. Desoxyhemigossypol 6-methyl

ether leads to the biosynthesis of methylated hemigossypol, gossypol, hemigossypolone, and the

heliocides, which decreases their effectiveness as phytoalexins and insecticides.

1.6 A Separation Method for Enantioresolving (+)- and (-)-Gossypol in

 Multi-milligram Quantities

Cass and Oliveira (2002) were able to separate gossypol by using a chiral carbohydrate

carbamate phase under reversed-phase conditions. This allowed the researchers to separate out

gossypol into two phases, (+)-gossypol and (-)-gossypol. The biological activity of (-)-gossypol

has been shown experimentally to be far more active than (+)-gossypol. This gives a reason to

why there should be a good way to stereospecifically separate out gossypol. A column (200 x 7

mm ID) of cellulose Tris (3, 5-dimethyl-phenylcarbamate) coated onto naked silica (Hypersil,

particle size, 5 µm; pore size, 120 Angstroms) was used under reversed-phase condition with

recycle to enantioresolve multi-milligram quantities of gossypol enantiomers. The use of

amylose derivatives and the investigation on the influence of the acidity of the supports used for

the polysaccharide phases in the enantioresolution of gossypol was also discussed in the article

illustrating the experiment.

1.7 Gossypol’s Effectiveness Against Malaria-causing Plasmodium falciparum

and Related Disease-Causing Protozoa

Dando et al. (2001) found gossypol to be effective at inhibiting growth of Plasmodium

falciparum and Toxoplasma gondii tachyzoites, which cause malaria and toxoplasmosis,

respectively. Gossypol and its derivatives bind with the LDH enzyme of P. falciparum and T.

gondii to effectively inhibit tachyzoite growth. P. falciparum and T. gondii are obligate

intracellular protozoan parasites of the phylum Apicomplexa that cause life-threatening

infections of humans. P. falciparum causes the most severe cases of malaria, which is

responsible for killing millions of people annually worldwide, mostly in non-industrialized

tropical and subtropical nations, and is spread by bites of Aedes aegyptae and Aedes anopholes

mosquitos. T. gondii infection of an immunocompetent individual usually is asymptomatic,

however, infection of immunocompromised individuals or congenital infection of a fetus can

lead to debilitating or life-threatening illness. The phylum Apicomplexa also includes a number

of other important human pathogens, including Cryptosporidium, Eimeria and Trypanosoma

species.

1.8 Gossypol as a Retroviral Inhibitor of HIV-1

Keller et al. (2003) used a ligand-based computer-aided molecular modeling technique to

search for compounds capable of binding to non-nucleoside inhibitor binding pocket (NNIBP) of

the HIV-1 reverse transcriptase (RT) enzyme. Gossypol has long been used in studies against

HIV and influenza, in which it has been found to inhibit retroviral activity. Their findings

suggest that at least a part of gossypol’s anti-HIV activity is due to gossypol targeting the non-

nucleoside inhibitor binding pocket of HIV-1 reverse transcriptase.

1.9 Eradication of Fecal Coliforms in a Bioreactor Conversion of Cotton Gin

Waste and Dairy Cattle Manure to Methane

Riordan et al. (2003) performed a bioreactor conversion of cotton gin waste and dairy

cattle manure to generate methane production. An unexplained complete eradication of fecal

coliforms, including enterohemorhagic Escherichia coli O157:H7 was achieved. A possible

explanation is that the gossypol present in the cotton gin waste killed the fecal coliforms, since a

control of beta-cellulose and dairy cattle manure continued to contain fecal coliforms, although

in lesser amounts than at the initiation of the anaerobic digestion. These findings could lead to a

new composting procedure capable of utilizing two common agricultural wastes to eradicate

dangerous fecal coliforms before they can contaminate runoff to surface water and groundwater.

A repetition of this experiment is planned using glanded cotton containing gossypol, glandless

cotton containing very small amounts of gossypol, and beta-cellulose controls. Extractions of
(+) - and (-)-gossypol enantiomers and extraction of tannic acid from the glandless and glanded

cotton leaves and stems used in the experiment is planned to quantify the amounts of these cotton

terpenoids present.

1.10 Sequence Homology to Gossypol’s Synthase and Possible Insight into

Gossypol’s Remarkably Broad Activity

A BLASTP protein similarity search of NCBI’s non-redundant database using the

sequence of gossypol’s synthase in FASTA format resulted in a large number of similar

sequences with very low –e values as well as many more sequences with more distant homology.

Several of these distantly matching sequences were from Plasmodium, Cryptosporidium, and

Trypanosoma species, which might help to explain gossypol’s effectiveness against these

protozoa. Gossypol’s synthase also produced distant matches with proteins from pathogenic

bacteria such as Legionella pneumophila, which is the causative agent of Legionaire’s disease.

Many of the sequences closely matching gossypol’s synthase are those of synthases of terpenoids

from a wide variety of plant species including Melaleuca alternifolia (tea tree) which also have

antibacterial effects. A better understanding of the protein sequences conserved between these

terpenoids might provide insights into how these compounds function.

1.11 Literature Cited

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Cass, Q. B. and R. V. Oliveira. 2002. Separation of multi-milligram quantities

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