F. William Collins

Avenanthramides inhibit proliferation of human colon cancer cell lines in vitro.

A high intake of whole grain foods is associated with reduced risk of colon cancer, but the mechanism underlying this protection has yet to be elucidated. Chronic inflammation and associated cyclooxygenase-2 (COX-2) expression in the colon epithelium are causally related to epithelial carcinogenesis, proliferation, and tumor growth. We examined the effect of avenanthramides (Avns), unique polyphenols from oats with anti-inflammatory properties, on COX-2 expression in macrophages, colon cancer cell lines, and on proliferation of human colon cancer cell lines. We found that Avns-enriched extract of oats (AvExO) had no effect on COX-2 expression, but it did inhibit COX enzyme activity and prostaglandin E2 (PGE2) production in lipopolysaccharide-stimulated mouse peritoneal macrophages. Avns (AvExO, Avn-C, and the methylated form of Avn-C (CH3-Avn-C)) significantly inhibited cell proliferation of both COX-2-positive HT29, Caco-2, and LS174T, and COX-2-negative HCT116 human colon cancer cell lines, CH3-Avn-C being the most potent. However, Avns had no effect on COX-2 expression and PGE2 production in Caco-2 and HT29 colon cancer cells. These results indicate that the inhibitory effect of Avns on colon cancer cell proliferation may be independent of COX-2 expression and PGE2 production. Thus, Avns might reduce colon cancer risk through inhibition of macrophage PGE2 production and non-COX-related antiproliferative effects in colon cancer cells. Interestingly, Avns had no effect on cell viability of confluence-induced differentiated Caco-2 cells, which display the characteristics of normal colonic epithelial cells. Our results suggest that the consumption of oats and oat bran may reduce the risk of colon cancer not only because of their high fiber content but also due to Avns, which attenuate proliferation of colonic cancer cells.

Production of tranilast [N-(3′,4′-dimethoxycinnamoyl)-anthranilic acid] and its analogs in yeast Saccharomyces cerevisiae

Biological synthesis of therapeutic drugs beneficial for human health using microbes offers an alternative production strategy to the methods that are commonly employed such as direct extraction from source organisms or chemical synthesis. In this study, we evaluated the potential for yeast (Saccharomyces cerevisiae) to be used as a catalyst for the synthesis of tranilast and various tranilast analogs (cinnamoyl anthranilates). Several studies have demonstrated that these phenolic amides have antioxidant properties and potential therapeutic benefits including antiinflammatory, antiproliferative, and antigenotoxic effects. The few cinnamoyl anthranilates naturally produced in plants such as oats and carnations result from the coupling of various hydroxycinnamoyl-CoAs to anthranilic acid. In order to achieve the microbial production of tranilast and several of its analogs, we engineered a yeast strain to co-express a 4-coumarate/CoA ligase (4CL, EC 6.2.1.12) from Arabidopsis thaliana and a hydroxycinnamoyl/benzoyl-CoA/anthranilate N-hydroxycinnamoyl/benzoyltransferase (HCBT, EC 2.3.1.144) from Dianthus caryophyllus. This modified yeast strain allowed us to produce tranilast and 26 different cinnamoyl anthranilate molecules within a few hours after exogenous supply of various combinations of cinnamic acids and anthranilate derivatives. Our data demonstrate the feasibility of rapidly producing a wide range of defined cinnamoyl anthranilates in yeast and underline a potential for the biological designed synthesis of naturally and non-naturally occurring molecules.

Isolation of 4 -Acetyl-benzoxazolin-2-one (4-ABOA) and Diferuloylputrescine from an Extract of Gibberella Ear Rot-resistant Corn that Blocks Mycotoxin Biosynthesis, and the Insect Toxicity of 4 -ABOA and Related Compounds

Abstract Bioassay-directed fractionation of an extract of a gibberella ear rot-resistant corn hybrid resulted in the isolation of a fraction that inhibited mycotoxin production, but not growth, of Fusarium culmorum. The fraction contained diferuloylputrescine and a new allelochemical 4-acetyl-benzoxazolin-2-one (4-ABOA). This report describes the isolation of the bioactive fraction and tests of the effect of 4-ABOA on 3-acetyl deoxynivalenol (3ADON) and aflatoxin production. In addition, data are reported on the effects of 4-ABOA and related compounds on a number of insect species and some preliminary studies on the mechanism of insect toxicity. The effect of 4-ABOA on F. culmorum and Apergillus flavus was similar. Production of toxin was reduced without a concomitant reduction of growth. A 50% inhibition of 3ADON production occurred at ca. 4 μM and 0.12 mM for alfatoxin. 4-ABOA was slightly toxic to Spodoptera frugiperda.

Production of hydroxycinnamoyl anthranilates from glucose in Escherichia coli

BackgroundOats contain hydroxycinnamoyl anthranilates, also named avenanthramides (Avn), which have beneficial health properties because of their antioxidant, anti-inflammatory, and antiproliferative effects. The microbial production of hydroxycinnamoyl anthranilates is an eco-friendly alternative to chemical synthesis or purification from plant sources. We recently demonstrated in yeast (Saccharomyces cerevisiae) that coexpression of 4-coumarate: CoA ligase (4CL) from Arabidopsis thaliana and hydroxycinnamoyl/benzoyl-CoA/anthranilate N-hydroxycinnamoyl/benzoyltransferase (HCBT) from Dianthus caryophyllus enabled the biological production of several cinnamoyl anthranilates upon feeding with anthranilate and various cinnamates. Using engineering strategies to overproduce anthranilate and hydroxycinnamates, we describe here an entire pathway for the microbial synthesis of two Avns from glucose in Escherichia coli.ResultsWe first showed that coexpression of HCBT and Nt4CL1 from tobacco in the E. coli anthranilate-accumulating strain W3110 trpD9923 allowed the production of Avn D [N-(4′-hydroxycinnamoyl)-anthranilic acid] and Avn F [N-(3′,4′-dihydroxycinnamoyl)-anthranilic acid] upon feeding with p-coumarate and caffeate, respectively. Moreover, additional expression in this strain of a tyrosine ammonia-lyase from Rhodotorula glutinis (Rg TAL) led to the conversion of endogenous tyrosine into p-coumarate and resulted in the production of Avn D from glucose. Second, a 135-fold improvement in Avn D titer was achieved by boosting tyrosine production using two plasmids that express the eleven genes necessary for tyrosine synthesis from erythrose 4-phosphate and phosphoenolpyruvate. Finally, expression of either the p-coumarate 3-hydroxylase Sam5 from Saccharothrix espanensis or the hydroxylase complex HpaBC from E. coli resulted in the endogenous production of caffeate and biosynthesis of Avn F.ConclusionWe established a biosynthetic pathway for the microbial production of valuable hydroxycinnamoyl anthranilates from an inexpensive carbon source. The proposed pathway will serve as a platform for further engineering toward economical and sustainable bioproduction of these pharmaceuticals and other related aromatic compounds.

Isolation and characterization of 4-acetyl-benzoxazolin-2-one (4-ABOA), a new benzoxazolinone from Zea mays

Abstract The previously unreported 4-acetyl-benzoxazolin-2-one (4-ABOA, 1 ) was isolated from corn kernels. Its structure was determined by MS, NMR and X-Ray crystallography.

High Levels of Avenanthramides in Oat-Based Diet Further Suppress High Fat Diet-Induced Atherosclerosis in Ldlr–/– Mice

Oats, in addition to cholesterol-lowering properties, contain unique antioxidants called avenanthramides (Avns), which inhibit both inflammatory cytokines and adhesion molecules in endothelial cells in culture. This study evaluated the effects of Avns of oats on atherosclerosis in Ldlr-/- mice, one of the most commonly used atherosclerosis mouse models with their similar cholesterol distributions to humans. The Ldlr-/- mice were fed a low fat, high fat, high fat containing regular oat brans with low levels of Avns (HFLA), or high fat containing regular oat brans with high levels of Avns (HFHA) diet. After 16 weeks of intervention, blood cholesterol and extent of aortic lesions were evaluated. We found that both oat-based diets reduced high fat diet-induced atheroma lesions in the aortic valve (p < 0.01). Furthermore, the effects of oat-based diets are more profound in HFHA mice than mice fed HFLA. Total plasma cholesterol levels were similarly reduced in both oat-supplemented mice. We concluded that oat bran diets reduce atheroma lesions and higher levels of Avns further reduce aortic lesions compared to regular oat bran. These preliminary in vivo data indicate that consumption of oats bran, with high Avns, has demonstrable beneficial effects on prevention of cardiovascular disease.