EMBARGOED FOR RELEASE: Wednesday, Sept. 11, 2013, 8:30 a.m. Eastern Time Note to journalists: Please report that this research was presented at a meeting of the American Chemical Society. A press conference on this topic will be held Tuesday, Sept. 10, at 2:30 p.m. in the ACS Press Center, Room 211 in the Indiana Convention Center. Reporters can attend in person or access live audio and video of the event and ask questions at www.ustream.tv/channel/acslive.

Newswise — INDIANAPOLIS, Sept. 11, 2013 — The scientific spotlight focuses today on the healthful antioxidant substances in red wine, dark chocolate, olive oil, coffee, tea, and other foods and dietary supplements that are enticing millions of consumers with the promise of a healthier, longer life. The American Chemical Society, the world's largest scientific society, is holding a symposium on those substances during its 246th National Meeting & Exposition.

Reports in the symposium involve substances that consumers know best as "antioxidants," and that scientists term "phenolic derivatives." These ingredients, found naturally in certain foods and sold as dietary supplements, have been linked with health benefits that include reducing the risk of heart disease and cancer.

Entitled "Phenolic Derivatives for Food and Human Health," the symposium was held today. The meeting continues through Thursday in the Indiana Convention Center and downtown hotels. Thousands of scientists and others are expected for the meeting, which features almost 7,000 reports on new discoveries in science and other topics.

Among the topics:

  • The healthful effects of curcumin, found naturally in the spice, turmeric
  • Substances in dried ginger that kill cancer cells
  • How large intestine microbes interact with cranberry antioxidants in the diet
  • Using biotechnology to make more effective antioxidants

Abstracts in the symposium appear below.

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Antitumor activity of novel curcumin analogs in hepatocarcinoma (HepG2) cells

Curcumin, a natural polyphenol of turmeric, continues to receive considerable attention as potent antioxidant and anti-cancer agent. Numerous analogues of curcumin have been synthesized and screened for their improved biological activities. An assessment of anticancer activities of curcumin and its novel 15 analogues was conducted in vitro. Various biochemical and toxicological techniques were employed to assess the therapeutic action of the synthetic novel curcumin analogs in comparison to curcumin and Sorafenib™ (a clinically approved drug). Inhibition of the cancer cell (HepG2) proliferation assessed by MTS assay showed that compound 2-[(E)-3-(4-hydroxyphenyl)acryloyl]-6-[1-(4-hydroxyphenyl)meth-(E)-ylidene]-cyclohexanone exhibits the trongest anticancer activity compared to curcumin and Sorafenib™ (p<0.05). 2-[(E)-3-(3,4-Dimethoxyphenyl)acryloyl]-6-[1-(3,4-dimethoxyphenyl)meth-(E)-ylidene]cyclohexanone and curcumin showed the highest cytotoxic effects (p<0.05) in HepG2 cell line, analyzed by LDH release assay. All the curcumin analogues induced apoptosis in tumor cells by caspase-3 activation and DNA fragmentation. It was found that Sorafenib™, compounds 2-[(E)-(3-phenylacryloyl)]-5-[1-phenylmeth-(E)-ylidene]cyclopentanone and 2-((E)-3-p-tolylacryloyl)-6-[1-p-tolylmeth-(E)-ylidene]cyclohexanone induced the highest amount of apoptotic caspase-3 enzyme in cancer cells(p<0.05). Apoptosis in all drug candidates was further confirmed by florescence microscopy using annexin V and 7-ADD dyes. Flow cytometry was employed to analyze cell cycle and it was found that curcumin and its derivatives induced cell arrest at G0/G1 phase in cancer cells. Compounds 2-[(E)-3-(4-Chlorophenyl)acryloyl]-5-[1-(4-chlorophenyl)-meth-(E)-ylidene]-cyclopenta-none and 2-[(E)-(3-Phenylacryloyl)]-6-[1-phenylmeth-(E)-ylidene]cyclohexanone were found to have the highest proportion of cells in apoptotic G0/G1 phase. These effects were also accompanied by catalytic inhibition of human topoisomerase II by all analyzed curcumin analogues. In addition, toxicology study was conducted to investigate toxicity of curcumin derivatives using normal human and rat hepatocytes. It was found that all compounds except 2-((E)-3-p-tolylacryloyl)-6-[1-p-tolylmeth-(E)-ylidene]cyclohexanonewere less cytotoxic than Sorafenib™. The compound 2-[(E)-3-(4-Hydroxyphenyl)acryloyl]-6-[1-(4-hydroxyphenyl)meth-(E)-ylidene]-cyclohexanone with enhanced biological activity and non-toxic manifestations emerged as the strongest drug candidate. The novel curcumin analogue 2-[(E)-3-(4-hydroxyphenyl)acryloyl]-6-[1-(4-hydroxyphenyl)meth-(E)-ylidene]-cyclohexanone was further assessed for its molecular pathway involved in antitumor activity using RT-PCR technique.

In vitro fecal fermentation of cranberry A-type proanthocyanidins

In vitro fecal fermentation is a useful tool to elucidate the influence of dietary polyphenol interactions with colonic bacteria on gut function and health. Our work centers on enhancing the understanding of in vitro bacterial hydrolysis of cranberry proanthocyanidins (PAC). Batch and continuous fermentations were performed utilizing fecal inocula, obtained from three individuals, in a nutrient-rich anaerobic media supplemented with purified PAC. Protein content and fatty acid production measurements were evaluated to monitor bacterial fermentation. The addition of PAC slightly stimulated growth, relative to PAC deficient samples, and induced variations in the fatty acid profile, primarily in terms of relative concentrations. PAC utilization and metabolite generation were also investigated. As an ancillary benefit, the study assessed the stability of fecal inocula during storage, which is important when considering reproducibility. These results represent the first steps toward our primary goal of understanding PAC metabolite influence on innate immune function and inflammation.

Metabolites of alkylresorcinols as the exposure markers to reflect whole wheat consumption in human

An impressive body of evidence supports the notion that increasing consumption of whole grains and/or cereal bran is associated with lower risk of chronic diseases, such as cancer, obesity, diabetes, and heart diseases. Many epidemiological studies, however, have failed to generate consistent results on this topic due to a lack of accurate tools to assess dietary intake and internal dosage. In order to better understand the beneficial health effects of whole grains/cereal bran, biomarkers for their exposure and effects are needed. The bran fraction of the whole grain contains important bioactive phytochemicals and is the major source of cereal fiber. The metabolism of these phytochemicals may reflect inter-individual differences. Alkylresorcinols (ARs) are phenolic lipids found in high concentrations exclusively in the outer parts of wheat and rye grains among commonly consumed foods. ARs and its metabolites have been used as the exposure markers to reflect whole wheat/rye consumption. However, the metabolic profile of ARs is still unknown. We recently studied the metabolic profile of ARs in mice and in humans and have identified a unique AR metabolite that can serve as a more accurate exposure marker to reflect whole wheat/rye consumption.

Bioactive phenolic components in ginger for cancer prevention

Ginger, the rhizome of Zingiber officinale, has been utilized for thousands of years as a spice and crude drug. The major pharmacologically active components of ginger are gingerols and shogaols, which have both been implicated in chemoprevention over the past decade. Shogaols are the products of gingerols after thermal processing and are the primary constituents of dried ginger. Our group demonstrated that [6]-, [8]-, and [10]-shogaols exhibited significantly higher toxicity to HCT-116 human colon and H-1299 human lung cancer cells than [6]-, [8]-, and [10]-gingerols. We have also found that [6]-shogaol was more effective than [6]-gingerol in inhibiting 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced skin tumor promotion in mice. Our studies on the metabolism of [6]-shgaol indicated that it is extensively metabolized in cancer cells, in liver microsomes, and in vivo. Our results also found that most of the metabolites of [6]-shogaol remain bioactive and some of the metabolites even have greater activity than [6]-shogaol in cancer cells and in mice.

Enzymatic synthesis of novel biomolecules of phenolic lipids: A biotechnological approach 

There has been a growing interest in the use of nutraceuticals as food supplements as well as natural bio-ingredients in food industries. Phenolic compounds represent an important group that possesses antioxidant and many functional properties. On the other hand, the numerous health benefits of the ω-3 polyunsaturated fatty acids (PUFAs) have been recognized in the modulation of risk of a variety of diseases and disorders. The incorporation of phenolic compounds into triacylglycerols could potentially result in novel biomolecules, structured phenolic lipids, with enhanced anti-oxidative and functional properties. The synthesized phenolic lipids demonstrated radical scavenging activity, expressed as IC50, 3.7-fold higher than that of dihydroxyphenyl acetic acid, but compared to that of α-tocopherol. In addition, the experimental findings indicated that phenolic lipids exhibited enhanced antioxidant capacity (AOC) by 2.6- to 8-fold higher that of the edible oils. The experimental findings also showed that all synthesized phenolic lipids demonstrated an enhancement in the oxidative stability, when they were exposed to light, oxygen and high temperatures, as compared to that of the edible oils. The presented work will highlight the development of a novel biotechnological approach using enzymes for the production of selected phenolic lipids, using edible oils and endogenous phenolic extracts. The structural, anti-oxidative stability characteristics of these novel biomolecules will be presented.

Phenolics in pressurized fluid systems: From extraction to synthesis 

Phenolic acids found in various biomass have antioxidant and antimicrobial activities and their consumption have been associated with a lower incidence of cancer, heart disease, and diabetes. Research in my laboratory has focused on the use of pressurized fluids to extract these phenolic compounds to be used in various applications. Pressurized fluids, such as subcritical water and supercritical CO(SC-CO2) as "green" and environmentally friendly solvents, can be used for the extraction of phenolic compounds from biomass as well as for enzymatic reactions involving phenolic acids. Phenolic compounds were extracted from biomass using pressurized fluids in stainless steel reactors at different temperatures ranging from 100 to 240˚C, pressures of up to 20 MPa and times of up to 180 min. Extracts obtained after biomass treatment using pressurized fluids were evaluated for their phenolic content and antioxidant activity. Results indicated that the total phenolic content and antioxidant activity of the extract increased with extraction temperature. In addition, a phenolic acid was reacted with triglycerides of flax oil using lipase enzyme in SC-CO2 media in a laboratory-scale supercritical system at different temperatures ranging from 40 to 80˚C, pressures from 4 to 35 MPa and times of up to 53 h. Results have shown that SC-CO2 is a promising green solvent for the enzymatic synthesis of phenolic lipids. In addition, the use of selected phenolic compounds in milk as antioxidants was evaluated at temperatures of 60-120˚C, pressures of 100 – 600 MPa and times of up to 30 min using high pressure processing assisted by temperature. The use of selected phenolic compounds in milk retained valuable components, such as CLA. Results have shown that phenolic acids can be processed using pressurized fluids for different applications.

Transesterified tyrosol and hydroxytyrosol alkylesters from cuphea oil – antioxidant behavior in liposomes 

Tyrosol and hydroxytyrosol are the antioxidant molecules abundantly found in olive oil. Transesterification of tyrosol and hydroxytyrosol with cuphea oil results in medium chain alkyl esters with antioxidant properties. Membrane partitioning, antioxidant capacity and membrane location of these novel lipids were measured within phospholipid liposomes. Findings demonstrate that both novel lipids partition well within the membrane, possess similar antioxidant characteristics as their parent molecule and position themselves as typical aliphatic lipids. Our findings demonstrate that these two novel antioxidant lipids may serve as food additives that prevent spoilage through oxidation of fats and oils.

Antioxidant efficacy of feruloyl glycerols in model membranes 

Ferulic acid and its esters are known to be effective antioxidants. Ethyl ferulate was biocatalytically transesterified with triacylglycerols and long chain alcohols to form a series of lipid-based feruloyl esters: feruloylglycerol, diferuloylglycerol, feruloyldiacylglycerol, diferuloylacylglycerol, and octadecyl ferulate. The feruloylglycerol acted as a rapid antioxidant (50% reduction of DPPH radical < 5 min) in ethanol solutions and diferuloyl glycerol was an intermediate antioxidant (50% reduction of DPPH radical in 5–30 min). Partitioning behavior, bilayer depth, and antioxidant characteristics of the feruloylglycerols were determined in phospholipid vesicles. Each feruloyl glycerol derivative incorporated well into liposomes, proving to be non-destructive to the bilayer. Bilayer depth analysis placed ferulic acid, the feruloyl glycerols, and octadecyl ferulate at nearly the same bilayer depth. The feruloyl glycerols proved better antioxidants in the liposomes than octadecyl ferulate.

Biocatalytic synthesis, structural elucidation, and tyrosinase inhibition activity of long chain fatty acid acylated derivatives of phloridzin and isoquercitrin H.P.

Our present work describes a conventional method as well as microwave-assisted regioselective enzymatic acylation of quercetin-3-O-glucoside and phloretin-2'-glucoside with different long chain saturated and unsaturated fatty acids using immobilized lipase B from Candida antarctica (Novozyme 435®). The synthesized esters were analyzed by 1H NMR, 13C NMR spectroscopy. In this investigation, the microwave irradiation under solvent free condition has been found to be more efficient and economical than conventional conditions both, in terms of time and energy. Besides, this work describes the tyrosinase inhibition activities of the synthesized esters, using in vitro assays. Among all the phloridzin and isoquercitrin derivatives, the greatest potential for inhibition of tyrosinase activity (p≤0.05) was exhibited by the α-linolenic acid ester of isoquercitrin.

From phenolic acids to phenolipids: A strategy to improve antioxidant activity in emulsions and cells 

Nowadays, it is admitted that cancers are associated withoxidative stress caused by an imbalance between ROS and antioxidants. Since mitochondria are the major site of ROS production, they represent a prime subcellular target to deliver antioxidants. To target mitochondria and consequently to improve the antioxidant properties, we developed here a new strategy consisting in tailoring the hydrophobicity of phenolic compounds by the grafting of various aliphatic chains. This lipophilization has been achieved on rosmarinic acid and chlorogenic acid to obtain rosmarinate esters and chlorogenate esters respectively (from methyl to octadecyl). The incubation of these lipophilized antioxidants on cancerous ROS-overexpressing fibroblasts showed a cut-off effect of the alkyl chain length on the antioxidant activity with an optimal activity for critical chain length. Indeed, increasing the chain length leads to an increase of the antioxidant activity until a threshold is reached and beyond which any lengthening of the alkyl chain resulted in a significant decrease of antioxidant activity. Interestingly, we evidenced by confocal microscopy that esters have different locations in the subcellular compartments and that these locations are directly correlated to the type of alkyl chain grafted on the phenolic structure: mitochondria for medium chain esters and cytosolic location for long chain esters. Taken together, these results suggest that the penetration of antioxidant in mitochondria is size-dependant and that only a medium chain confers to the corresponding ester the adequate size and lipophilicity to enter the mitochondria.

Oxidative metabolism of curcumin: Products, mechanisms, and biological activity 

The dietary polyphenol curcumin shows great promise as an anti-cancer and anti-inflammatory agent. While many cellular targets of curcumin have been identified, the chemical mechanisms whereby these targets are affected remain unclear. Orally administered curcumin undergoes metabolic conjugation with glucuronic acid and reduction of the dienone double bonds. We have recently described oxidative transformation of curcumin in vitro leading to a dioxygenated bicyclopentadione derivative as the final product. In contrast to previous hypotheses on the reduced metabolites of curcumin, we proposed that the oxidative metabolites of curcumin are direct mediators of its biological effects.

We synthesized 14C-curcumin and used it to detect and isolate nine novel oxidized metabolites, including three intermediates of the reaction. These metabolites were identified by UV spectroscopy, mass spectrometry, and a combination of 1D and 2D NMR methods. Using the structural data from these molecules combined with H218O isotopic studies we propose a mechanism of oxidative transformation of curcumin through reactive quinone methide and epoxy intermediates. The final products incorporate two oxygen atoms and possess a common nucleophilic β-diketo moiety. We further showed that the phenolic glucuronide of curcumin undergoes the same enzymatic oxidative transformation in vitro to generate the bicyclopentadione-glucuronide.

In biological studies, using a recombinant enzyme in vitro, we showed that autoxidative activation of curcumin is required for its topoisomerase poisoning activity. Further, we detected the final bicyclopentadione product in human and mouse plasma after oral administration of curcumin, indicating oxidative transformation is prominent in vivo. These data support the hypothesis that oxidative metabolites of curcumin are direct mediators of some of its bioactivity. Altogether, our studies for the first time detail the major products of curcumin oxidative transformation and their mechanisms of formation.

Supported by NIH awards F31 AT007287, R01 AT006896

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American Chemical Society’s 246th National Meeting & Exposition