Polyphenol composition and antioxidant potential of Hibiscus esculentus L. fruit cultivated in Nigeria.

Consumption of certain fruits and vegetables is now widely associated with chemoprevention of degenerative diseases like cancer and cardiovacsular disorders because of their antioxidant components. Polyphenols, a heterogeneous group of compounds, are one of these constituents. Hibiscus esculentus L. (Family Malvaceae), commonly referred to as okro, okra, or lady's finger, is an important component of diet in Nigeria and other countries in sub-Saharan Africa. In this article, we describe the polyphenol composition and antioxidant potential of H. esculentus of Nigerian origin. Quercetin glucoside (quercetrin) and an unidentified flavonoid were detected. In vitro antioxidant assay of methanol extract of the fruits showed potent antioxidant/radical scavenging activities with 50% inhibitory concentration values of 25 and 43 microL when analyzed by the xanthine oxidase and 2-deoxyguanosine methods, respectively. These data suggest that H. esculentus, popular especially during the rainy season in Nigeria and many tropical West, Central, and Eastern African countries, is a good contributor to the antioxidant status and disease chemoprevention of people in these countries.

Evaluation of the polyphenol composition and antioxidant activity of African variety of Dacryodes edulis (G.Don) H.J Lam fruit.

Polyphenols are abundant micronutrients in our diet that have been credited with chemoprevention of diseases associated with oxidative stress. In this study, we investigated the whole ripened fruit of Dacryodes edulis (G.Don) H.J Lam, a multipurpose tree growing in West and Central Africa and other countries bordering the Gulf of Guinea, for polyphenol content as well as its antioxidant/radical scavenging capacity. Analysis of the methanol extract of the fruit by high-performance liquid chromatography coupled to an ultraviolet dual-array detector and mass-selective detector revealed the presence of catechol (9.27 mg/kg), gallate (10.40 mg/kg), methylgallate (0.88 mg/kg), ellagic acid (3.10 mg/kg), quercetin (0.21 mg/kg), and quercetin rhamnoside (0.76 mg/kg). The extract showed very high antioxidant potential (50% inhibitory concentration [IC(50)] = 14 microL), but a rather weak radical scavenging activity (IC(50) = 357 microL), when tested in vitro with the xanthine oxidase and 2-deoxyguanosine assay model systems, respectively. These results suggest that consumption of D. edulis could contribute to prevention of diseases that are related to oxidative stress.

Isolation, purification and identification of ellagic acid derivatives, catechins, and procyanidins from the root bark of Anisophyllea dichostyla R. Br.

The root bark of Anisophyllea dichostyla R. Br. is traditionally used in the Democratic Republic Congo for the treatment of several conditions such as anorexia, fatigue and intestinal infections. We have identified and quantitated several polyphenol antioxidants in the methanol extract of the root bark (120g). The polyphenol content (3.32g/kg) was predominantly ellagitannins (25%) and polyhydroxyflavan-3-ols (catechins and procyanidins, 75%) with 3'-O-methyl-3,4-methylenedioxo ellagic acid 4'-O-beta-d-glucopyranoside and (-)-epicatechin as the major species in each class. These two compounds and the following species were identified unequivocally by NMR spectroscopy: (+)-catechin, (-)-epicatechin 3-O-gallate, 3-O-methyl ellagic acid, 3,3'-di-O-methyl ellagic acid, 3'-O-methyl-3,4-methylenedioxo ellagic acid, 3'-O-methyl-3,4-methylenedioxo ellagic acid 4'-O-beta-d-glucopyranoside, and 3'-O-methyl ellagic acid 4-O-beta-d-xylopyranoside. The following additional compounds were purified by semi-preparative HPLC and tentatively identified on the basis of UV spectra, HPLC-ESI-MS and nano-ESI-MS-MS: (+)-catechin-3-O-beta-d-glucopyranoside, epicatechin-(4beta-->8)-catechin (procyanidin B(1)), epicatechin-(4beta-->8)-epicatechin (procyanidin B(2)), an (epi)catechin trimer, 3-O-methyl ellagic acid 4-O-beta-d-glucopyranoside, (-)-epicatechin 3-O-vanillate, 3,4-methylenedioxo ellagic acid 4'-O- beta-d-glucopyranoside, and 3,3'-di-O-methyl ellagic acid 4-O-beta-d-xylopyranoside. Fractionation of the raw extract by column chromatography on silicic acid yielded 10 fractions. In the hypoxanthine/xanthine oxidase antioxidant assay system, CC-9 which contained a range of polyphenols dominated by (-)-epicatechin-O-gallate proved to be the most potent antioxidant fraction (IC(50)=52 micro g/mL) in terms of ROS scavenging. In terms of XO inhibition CC-8, dominated by (epi)catechin trimer and which also contained appreciable amounts of 3'-O-methyl ellagic acid 4'-O-beta-d-xylopyranoside, as well as the catechins (+)-catechin-3-O-beta-d-glucopyranoside, epicatechin-(4beta-->8)-catechin (procyanidin B(1)), and (-)-epicatechin 3-O-gallate, proved to be the most potent (IC(50)=36 micro g/mL).

Contribution of linseed intake to urine and serum enterolignan levels in German females: a randomised controlled intervention trial.

Linseeds are a rich source of lignans, secondary plant substances which are suggested to possess chemopreventive effects inter alia with regard to breast cancer. In a randomised controlled trial 40 German women were informed about "5-a-day" and encouraged to increase their dietary intake of fruit and vegetables. Moreover 19 participants were randomly assigned to an intervention group supplemented with ground linseeds (20 g/d) over a 2-month period. Before and after intervention, urine and blood samples were collected after an overnight fast. Analysis was by intention-to-treat and the outcome parameters of interest were enterolignan concentrations. After linseed supplementation, enterolignan concentrations (mean) measured as their glucuronides by a newly developed high performance liquid chromatography electrospray mass spectrometry (HPLC-ESI-MS) in serum (122 nmol/l) as well as in urine (72 micromol/l) showed a significant increase (P<0.01) compared to pre-intervention values (47 nmol/l and 29 micromol/l). In the control group enterolignan levels were raised slightly but did not reach significance. Serum and urinary enterolignans of the whole collective showed a good pairwise correlation.

Analysis of enterolignan glucuronides in serum and urine by HPLC-ESI-MS.

A method involving the coupling of high-performance liquid chromatography with electrospray ionisation mass spectrometry (HPLC-ESI-MS) for the quantitative determination of the mammalian lignans enterolactone and enterodiol in human blood and urine has been developed. In contrast to techniques previously published, the method allows direct measurement of free enterolignans as well as their monoglucuronide conjugates in human biofluids with minimal sample preparation. Thereby the method is suitable for large-scale intervention, case-control and epidemiologic studies. Comprehensive, high-precision (1)H and (13)C nuclear magnetic resonance data (CD3OD as solvent) obtained at 11.7 T in combination with polarimetric data show that the major form of lignan precursor in the linseeds used is (-)-secoisolariciresinol diglucoside ((2R,3R)-2,3-bis(4'-hydroxy-3'-methoxy-benzyl)-1,4-butanediyl-bis-beta-d-glucopyranoside) which is transformed by human intestinal bacteria into (+)-enterodiol and (+)-enterolactone. However, these metabolites are mono-glucuronidated after absorption and are detected as (-)-enterodiol 3'-beta-d-glucuronide=(2R,3R)-2-(3'-O-(beta-d-glucopyranosyluronic acid)benzyl)-3-(3''-hydroxybenzyl)-butane-1,4,diol and (-)-enterolactone 3'-beta-d-glucuronide=(2R,3R)-2-(3'-O-(beta-d-glucopyranosyluronic acid)benzyl)-3-(3''-hydroxybenzyl)-beta-butyrolactone in blood and urine.

Characterization of alkyl phenols in cashew (Anacardium occidentale) products and assay of their antioxidant capacity.

In this study the content of anacardic acids, cardanols and cardols in cashew apple, nut (raw and roasted) and cashew nut shell liquid (CNSL) were analysed. The higher amounts (353.6 g/kg) of the major alkyl phenols, anacardic acids were detected in CNSL followed by cashew fibre 6.1 g/kg) while the lowest (0.65 g/kg) amounts were detected in roasted cashew nut. Cashew apple and fibre contained anacardic acids exclusively, whereas CNSL also contained an abundance of cardanols and cardols. Cashew nut (raw and roasted) also contained low amounts of hydroxy alkyl phenols. Cashew nut shell liquid was used for a basic fractionation of the alkyl phenol classes and the individual anacardic acids, major cardanols and cardols were purified to homogeneity from these fractions by semi-preparative HPLC and definitively identified by nano-ESI-MS-MS, GC-MS and NMR analyses. The hexane extracts (10 mg/ml) of all cashew products tested plus CNSL, displayed significant antioxidant capacity. Cashew nut shell liquid was the more efficient (inhibition=100%) followed by the hexane extract of cashew fibre (94%) and apple (53%). The antioxidant capacity correlated significantly (P<0.05) with the concentration of alkyl phenols in the extracts. A mixture of anacardic acids (10.0 mg/ml) showed the higher antioxidant capacity (IC50=0.60 mM) compared to cardols and cardanols (IC50>4.0 mM). The data shows that of these substances, anacardic-1 was by far the more potent antioxidant (IC50=0.27 mM) compared to cardol-1 (IC50=1.71 mM) and cardanol-1 (IC50>4.0 mM). The antioxidant capacity of anacardic acid-1 is more related to inhibition of superoxide generation (IC50=0.04 mM) and xanthine oxidase (IC50=0.30 mM) than to scavenging of hydroxyl radicals. At present a substantial amount of cashew fibre is mostly used in formulations of animal or poultry feeds. The data presented in this study, indicates that this waste product along with CNSL, both of which contain high contents of anacardic acids, could be better utilized in functional food formulations and may represent a cheap source of cancer chemopreventive agents.

Isolation and structure elucidation of phenolic antioxidants from Tamarind (Tamarindus indica L.) seeds and pericarp.

Although it is already known that Tamarind (Tamarindus indica L.) seeds contain phenolic substances, the individual components of the seeds have not been fully identified and quantitated, and in the case of Tamarind pericarp not reported. Therefore, major polyphenolic compounds were extracted using organic solvents and the metabolites were isolated by semi-preparative high performance liquid chromatography. Their structures were elucidated by liquid chromatography-electrospray-ionisation-mass spectrometry (LC-ESI-MS), nano-electrospray-ionisation mass spectrometry (ESI-MS), and where possible by gas chromatography-mass spectrometry (GC-MS) and 1H and 13C NMR. Quantitative analysis of polyphenolic compounds in Tamarind seeds and pericarp was conducted by analytical high performance liquid chromatography (HPLC), calculated against standard curves of authentic compounds. The yields of total phenolic compounds after Soxhlet extraction with methanol were 6.54 and 2.82 g/kg (dry weight) in the seeds and pericarp respectively. The profile (%) of polyphenolics in Tamarind pericarp was dominated by proanthcyanidins (73.4) in various forms (+)-catechin (2.0), procyanidin B2 (8.2), (-)-epicatechin (9.4), procyanidin trimer (11.3), procyanidin tetramer (22.2), procyanidin pentamer (11.6), procyanidin hexamer (12.8) along with taxifolin (7.4), apigenin (2.0), eriodictyol (6.9), luteolin (5.0) and naringenin (1.4) of total phenols, respectively. The content of Tamarind seeds comprised only procyanidins, represented (%) mainly by oligomeric procyanidin tetramer (30.2), procyanidin hexamer (23.8), procyanidin trimer (18.1), procyanidin pentamer (17.6) with lower amounts of procyanidin B2 (5.5) and (-)-epicatechin (4.8). Extraction of Tamarind pericarp and seeds using acetone:methanol:acetic acid gave only procyanidin oligomers, but in much higher yield and variety. The antioxidant capacities of the Soxhlet methanolic extracts were determined, and indicates that Tamarind may be an important source of cancer chemopreventive natural products in tropical regions.

Olives and olive oil in cancer prevention.

Epidemiologic studies conducted in the latter part of the twentieth century demonstrate fairly conclusively that the people of the Mediterranean basin enjoy a healthy lifestyle with decreased incidence of degenerative diseases. The data show that populations within Europe that consume the so-called 'Mediterranean diet' have lower incidences of major illnesses such as cancer and cardiovascular disease. Studies have suggested that the health-conferring benefits of the Mediterranean diet are due mainly to a high consumption of fibre, fish, fruits and vegetables. More recent research has focused on other important factors such as olives and olive oil. Obviously fibre (especially wholegrain-derived products), fruits and vegetables supply an important source of dietary antioxidants. What is the contribution from olives and olive oil? Apparently the potential is extremely high but epidemiologic studies rarely investigate consumption of these very important products in-depth, perhaps due to a lack of exact information on the types and amounts of antioxidants present. Recent studies have shown that olives and olive oil contain antioxidants in abundance. Olives (especially those that have not been subjected to the Spanish brining process) contain up to 16 g/kg typified by acteosides, hydroxytyrosol, tyrosol and phenyl propionic acids. Olive oil, especially extra virgin, contains smaller amounts of hydroxytyrosol and tyrosol, but also contains secoiridoids and lignans in abundance. Both olives and olive oil contain substantial amounts of other compounds deemed to be anticancer agents (e.g. squalene and terpenoids) as well as the peroxidation-resistant lipid oleic acid. It seems probable that olive and olive oil consumption in southern Europe represents an important contribution to the beneficial effects on health of the Mediterranean diet.

Isolation and structure elucidation of the major individual polyphenols in carob fibre.

Although it is already known that carob fibre contains several classes of polyphenolic substances, a comprehensive analysis of these has not been conducted to date. Therefore, the major polyphenolic compounds were extracted with organic solvents, and, following fractionation by normal-phase column chromatography on silicic acid, their structures were elucidated by liquid-chromatography electrospray-ionisation mass spectrometry (LC-ESI), nano-electrospray-ionisation mass spectrometry (ESI-MS), and gas-chromatography mass spectrometry (GC-MS). In addition, complete 1H and 13C NMR assignments were obtained for the isolated gallotannins 1,6-di-, 1,2,6-tri- and 1,2,3,6-tetra-O-galloyl-beta-D-glucose. Carob fibre was found to contain a rich variety of phenolic antioxidants. A total of 24 polyphenol compounds were identified with a yield of 3.94 g/kg (dry weight). The profile was dominated by gallic acid in various forms: free gallic acid (42% of polyphenols by weight), gallotannins (29%), and methyl gallate (1%), while simple phenols, mainly cinnamic acid, made up about 2% of the total. Flavonoids represented 26% of the polyphenols, and the major components were identified as the glycosides myricetin- and quercetin-3-O-alpha-L-rhamnoside (ca. 9% and 10%, respectively). These data indicate that carob fibre is rich in both amount and variety of phenolic antioxidant substances, and its inclusion in the diet may have chemopreventive properties.

Isolation, structure elucidation and antioxidant potential of the major phenolic and flavonoid compounds in brined olive drupes.

Because olives represent an important component of the Mediterranean diet, it is necessary to establish unequivocal identification and quantitation of the major potential antioxidant phenolic compounds they contain. The major phenolic antioxidants in two types of brined olives were isolated and purified by semi-preparative high performance liquid chromatography. Structural analysis was conducted using UV spectrophotometry, mass spectrometry and nuclear magnetic resonance spectroscopy. In particular, completely assigned 1H and 13C NMR data are presented and errors in literature data are corrected. The data show that tyrosol, hydroxytyrosol, 3-(3, 4-dihydroxyphenyl) propanoic acid (dihydrocaffeic acid), dihydro-p-coumaric acid (phloretic acid), the phenylpropanoid glucosides acteoside (verbascoside) and isoacteoside, along with the flavonoids luteolin and apigenin are major components of the phenolic fraction of brined black olives. Brined green olives contain only hydroxytyrosol and traces of other minor phenolics. Brined olives contain even higher concentrations of phenolic antioxidants than olive oil and may, therefore, be more important modulators of cancer chemopreventive activity.

Consumption of argan oil (Morocco) with its unique profile of fatty acids, tocopherols, squalene, sterols and phenolic compounds should confer valuable cancer chemopreventive effects.

The aim of this study was to evaluate the fatty acids, tocopherols, squalene, sterols and phenolic antioxidants in three types of argan oil (Moroccan food, Moroccan aesthetic and a French commercial variety) along with a basic comparison with extra virgin olive and sunflower oil. The fatty acid profiles in the argan oils were very similar, with oleic acid (43%) and linoleic acid (36%) and their respective monoacylglycerols predominating. The major vitamer identified was -tocopherol with a mean of 483+/-11 mg/kg, in contrast to -tocopherol, which is the major vitamer in olive (190+/-1 mg/kg) and sunflower oil (532+/-6 mg/kg). The squalene content of the argan oils was very similar with a mean of 313+/-4 mg/100 g, which is lower than that of the olive oil (499 mg/100 g) but significantly higher than in the sunflower oil (6 mg/100 g). In contrast to olive and sunflower oils in which -sitosterol is predominant, the major sterols detected in the argan oils were schottenol (mean 147+/-10 mg/kg) and spinasterol (mean 122+/-10 mg/kg). The only phenolic compounds other than the tocopherol vitamers which could be readily detected and quantitated were vanillic, syringic and ferulic (probably conjugated to glucose) acids along with tyrosol. In contrast to the extra virgin olive oil (793 mg/kg), the concentration of total phenolic compounds is extremely low (<5.0 mg/kg). Nevertheless, argan oil with its high content of the vitamer -tocopherol, squalene and oleic acid is likely to enhance the cancer prevention effects of the Moroccan diet.

Glutathione-S-transferase M1, M3, T1 and P1 polymorphisms and susceptibility to non-small-cell lung cancer subtypes and hamartomas.

Polymorphic glutathione-S-transferase (GST) genes causing variations in enzyme activity may influence individual susceptibility to lung cancer. In this case-control study (consisting of 389 Caucasian lung cancer patients, including 151 adenocarcinomas (ACs) and 172 squamous cell carcinomas (SCCs), and 353 hospital control subjects without malignant disease, genotype frequencies for GSTM1, GSTM3, GSTP1 and GSTT1 were determined by polymerase chain reaction (PCR)/ restriction fragment length polymorphism (RFLP)-based methods. While adjusted odds ratios (ORs) indicated no significantly increased risk for lung cancer overall due to any single GST genotype, the risk alleles for GSTM1, GSTM3 and GSTP1 conferring reduced enzyme activity were present at higher frequency in SCC than in AC patients. This is consistent with a reduced detoxification of carcinogenic polycyclic aromatic hydrocarbons (PAHs) from cigarette smoke that are more important for the development of SCC than for AC. An explorative data analysis also identified statistically significantly increased ORs for the combinations GSTT1 non-null and GSTP1 GG or AG for lung cancer overall (OR 2.23, CI 1.11-4.45), and for SCC (OR 2.69, CI 1.03-6.99). For lung cancer overall, and especially among SCC patients, the GSTT1 null genotype was underrepresented (SCC 11.2% v. control subjects 19%, P = 0.026, OR 0.57, CI 0.30-1.06). Additionally, in 28 patients with hamartomas, the GSTT1 null genotype was also protective (P = 0.013), while GSTP1 variant allele carriers were overrepresented (OR 2.48, CI 1.06-6.51). In conclusion, GST genotypes may act differently, either by detoxifying harmful tobacco carcinogens and/or by eliminating lung cancer chemopreventive agents. The latter role for GSTT1 would explain the observed lower risk of SCC and hamartoma associated with GSTT1 null. Further confirmatory studies are required.

Monitoring airborne genotoxicants in the rubber industry using genotoxicity tests and chemical analyses.

This research was designed to examine the presence of mutagenic/carcinogenic compounds in airborne pollutants in the rubber industry using an integrated chemical/biological approach. Inhalable airborne particulate matter (PM-10: <10 microm) was collected in four rubber factories using a high-volume sampler equipped with a cascade impactor for particle fractionation. The organic extracts of two different fractions (0.5-10 microm and <0.5 microm) were examined for mutagenicity with the Ames test and for in vitro DNA-damaging activity in human leukocytes by single-cell microgel electrophoresis (Comet assay). The extracts were also studied by gas chromatography/mass spectrometry (GC/MS) for polycyclic aromatic hydrocarbon (PAH) content. Nitrosamines in ambient air were sampled on cartridges and analysed by GC with a thermal energy analyser (TEA) detector. Airborne volatile genotoxins were monitored in situ using a clastogenicity plant test (Tradescantia/micronuclei test). The results showed that airborne particulates were mainly very fine (<0.5 microm) and that trace amounts of genotoxic nitrosamines (N-nitrosodimethylamine: 0.10-0.98 microg/m(3); N-nitrosomorpholine: 0.77-2.40 microg/m(3)) and PAH (total PAH: 0.34-11.35 microg/m(3)) were present in air samples. Some extracts, particularly those obtained from the finest fractions, were mutagenic with the Ames test and genotoxic with the Comet assay. In situ monitoring of volatile mutagens using the Tradescantia/micronuclei test gave positive results in two working environments. The results showed the applicability of this integrated chemical-biological approach for detecting volatile and non-volatile genotoxins and for monitoring genotoxic hazards in the rubber industry.

Relevance of N-acetyltransferase 1 and 2 (NAT1, NAT2) genetic polymorphisms in non-small cell lung cancer susceptibility.

The highly polymorphic N-acetyltransferases (NAT1 and NAT2) are involved in both activation and inactivation reactions of numerous carcinogens, such as tobacco derived aromatic amines. The potential effect of the NAT genotypes in individual susceptibility to lung cancer was examined in a hospital based case-control study consisting of 392 Caucasian lung cancer patients [152 adenocarcinomas, 173 squamous cell carcinomas (SCC) and 67 other primary lung tumours] and 351 controls. In addition to the wild-type allele NAT1*4, seven variant NAT1 alleles (NAT1*3, *10, *11, *14, *15, *17 and *22) were analysed. A new method based on the LightCycler (Roche Diagnostics Inc.) technology was applied for the detection of the polymorphic NAT1 sites at nt 1088 and nt 1095. The NAT2 polymorphic sites at nt 481, 590, 803 and 857 were detected by polymerase chain reaction-restriction fragment length polymorphism or LightCycler. Multivariate logistic regression analyses were performed taking into account levels of smoking, age, gender and occupational exposure. An increased risk for adenocarcinoma among the NAT1 putative fast acetylators [odds ratio (OR) 1.92 (1.16-3.16)] was found but could not be detected for SCC or the total case group. NAT2 genotypes alone appeared not to modify individual lung cancer risk, however, individuals with combined NAT1 fast and NAT2 slow genotype had significantly elevated adenocarcinoma risk [OR 2.22 (1.03-4.81)] compared to persons with other genotype combinations. These data clearly show the importance of separating different histological lung tumour subtypes in studies on genetic susceptibility factors and implicate the NAT1*10 allele as a risk factor for adenocarcinoma.

hOGG1 polymorphism and loss of heterozygosity (LOH): significance for lung cancer susceptibility in a caucasian population.

Oxidative damage is implicated in several chronic diseases including cancer. 8-Hydroxyguanine (8-oxoG) is one of the major promutagenic DNA lesions, which is produced by reactive oxygen species, causes G:C to T:A transversions and is excised by OGG1, an 8-oxoG specific DNA glycosylase/AP-Lyase. In a nested case-control study, gDNA from 105 Caucasian primary non-small cell lung cancer cases and 105 matched controls was screened for 6 possible new polymorphic sites in the human OGG1 gene, detected previously mainly in tumour tissue. The previously described Ser(326)Cys polymorphism was found to be common (allele frequency 0.22) in Caucasians. However, no major difference in Ser(326)Cys genotype distribution could be detected between cases and controls. Two 5;-end polymorphisms previously found in Japanese as well as Arg(131)Gln could not be detected in this population. An Ala(85)Ser polymorphism was found in 2 controls, whereas Arg(46)Gln was detected in only 1 case. As the hOGG1 gene is mapped (3p26.2) to a region frequently lost in primary lung tumours, the frequency of loss of heterozygosity (LOH) was investigated. Forty-three percent of the studied lung tumours exhibited loss of one of the hOGG1 alleles. The wt Ser(326) allele was not predominantly lost in our sample set, which suggests a minor role of this polymorphism in tumourgenesis. Our results show that LOH at the hOGG1 gene locus is a very common occurrence in lung tumourgenesis, possibly leading to increased mutational damage due to ROS in smokers. However, the hOGG1 polymorphisms studied are probably not major contributors to individual lung cancer susceptibility in Caucasians.

Identification of lignans as major components in the phenolic fraction of olive oil.

BACKGROUND: Because olive oil is an important component of the Mediterranean diet, it is necessary to establish unequivocal identification of the major potential antioxidant phenolic compounds it contains. METHODS: The major phenolic antioxidants in extra virgin olive oil were isolated and purified. Structural analysis was conducted using several spectroscopic techniques, including mass spectrometry and nuclear magnetic resonance (NMR). In particular, detailed (1)H and (13)C NMR data are presented, and several assignment errors in the literature are corrected. RESULTS: The data show for the first time that the lignans (+)-1-acetoxypinoresinol and (+)-pinoresinol are major components of the phenolic fraction of olive oils. These lignans, which are potent antioxidants, are absent in seed oils and virtually absent in refined virgin oils but are present at concentrations of up to 100 mg/kg (mean +/- SE, 41.53+/-3.93 mg/kg; range, 0.65-99.97 mg/kg) in extra virgin oils. As with the simple phenols and secoiridoids, there is considerable interoil variation in lignan concentrations. Foods containing high amounts of lignan precursors have been found to be protective against breast, colon, and prostate cancer. CONCLUSION: Lignans, as natural components of the diet, may be important modulators of cancer chemopreventive activity.

The antioxidant/anticancer potential of phenolic compounds isolated from olive oil.

In our ongoing studies on the chemoprevention of cancer we have a particular interest in the health benefits of the Mediterranean diet, of which olive oil is a major component. Recent studies have shown that extravirgin olive oil contains an abundance of phenolic antioxidants including simple phenols (hydroxytyrosol, tyrosol), aldehydic secoiridoids, flavonoids and lignans (acetoxypinoresinol, pinoresinol). All of these phenolic substances are potent inhibitors of reactive oxygen species attack on, e.g. salicylic acid, 2-deoxyguanosine. Currently there is growing evidence that reactive oxygen species are involved in the aetiology of fat-related neoplasms such as cancer of the breast and colorectum. A plausible mechanism is a high intake of omega-6 polyunsaturated fatty acids which are especially prone to lipid peroxidation initiated and propagated by reactive oxygen species, leading to the formation (via alpha,beta-unsaturated aldehydes such as trans-4-hydroxy-2-nonenal) of highly pro-mutagenic exocyclic DNA adducts. Previous studies have shown that the colonic mucosa of cancer patients and those suffering from predisposing inflammatory conditions such as ulcerative colitis and Crohn's disease generates appreciably higher quantities of reactive oxygen species compared with normal tissue. We have extended these studies by developing accurate high performance liquid chromatography (HPLC) methods for the quantitation of reactive oxygen species generated by the faecal matrix. The data shows that the faecal matrix supports the generation of reactive oxygen species in abundance. As yet, there is a dearth of evidence linking this capacity to actual components of the diet which may influence the colorectal milieu. However, using the newly developed methodology we can demonstrate that the antioxidant phenolic compounds present in olive oil are potent inhibitors of free radical generation by the faecal matrix. This indicates that the study of the inter-relation between reactive oxygen species and dietary antioxidants is an area of great promise for elucidating mechanisms of colorectal carcinogenesis and possible future chemopreventive strategies.

Phenolic compounds and squalene in olive oils: the concentration and antioxidant potential of total phenols, simple phenols, secoiridoids, lignansand squalene.

The aim of this study was to evaluate the phenolic antioxidant and squalene content in a range of olive and seed oils. A mean of 290 +/- 38 (SEM) mg squalene/100 g was detected. However, while there was a weak significant difference between extra virgin (424 +/- 21 mg/kg) and refined virgin (340 +/- 31 mg/100 g; P<0.05) olive oils, highly significant differences were evident between extra virgin olive oils (P<0.0001) refined virgin olive oils (P<0.0001) and seed oils (24 +/- 5 mg/100 g). While seed oils were devoid, on average, the olive oils contained 196 +/- 19 mg/kg total phenolics as judged by HPLC analysis, but the value for extra virgin (232 +/- 15 mg/kg) was significantly higher than that of refined virgin olive oil (62 +/- 12 mg/kg; P<0.0001). Appreciable quantities of simple phenols (hydroxytyrosol and tyrosol) were detected in olive oils, with significant differences between extravirgin (41.87 +/- 6.17) and refined virgin olive oils (4.72 +/- 215; P<0.01). The major linked phenols were secoiridoids and lignans. Although extra virgin contained higher concentrations of secoiridoids (27.72 +/- 6.84) than refined olive oils (9.30 +/- 3.81) this difference was not significant. On the other hand, the concentration of lignans was significantly higher (P<0.001) in extra virgin (41.53 +/- 3.93) compared to refined virgin olive oils (7.29 +/- 2.56). All classes of phenolics were shown to be potent antioxidants. In future epidemiologic studies, both the nature and source of olive oil consumed should be differentiated in ascertaining cancer risk.

Generation of reactive oxygen species by the faecal matrix.

BACKGROUND: Reactive oxygen species are implicated in the aetiology of a range of human diseases and there is increasing interest in their role in the development of cancer. AIM: To develop a suitable method for the detection of reactive oxygen species produced by the faecal matrix. METHODS: A refined high performance liquid chromatography system for the detection of reactive oxygen species is described. RESULTS: The method allows baseline separation of the products of hydroxyl radical attack on salicylic acid in the hypoxanthine/xanthine oxidase system, namely 2,5-dihydroxybenzoic acid, 2,3-dihydroxybenzoic acid, and catechol. The increased efficiency and precision of the method has allowed a detailed evaluation of the dynamics of reactive oxygen species generation in the faecal matrix. The data show that the faecal matrix is capable of generating reactive oxygen species in abundance. This ability cannot be attributed to the bacteria present, but rather to a soluble component within the matrix. As yet, the nature of this soluble factor is not entirely clear but is likely to be a reducing agent. CONCLUSIONS: The soluble nature of the promoting factor renders it amenable to absorption, and circumstances may exist in which either it comes into contact with either free or chelated iron in the colonocyte, leading to direct attack on cellular DNA, or else it initiates lipid peroxidation processes whereby membrane polyunsaturated fatty acids are attacked by reactive oxygen species propagating chain reactions leading to the generation of promutagenic lesions such as etheno based DNA adducts.

Olive-oil consumption and health: the possible role of antioxidants.

In the Mediterranean basin, olive oil, along with fruits, vegetables, and fish, is an important constituent of the diet, and is considered a major factor in preserving a healthy and relatively disease-free population. Epidemiological data show that the Mediterranean diet has significant protective effects against cancer and coronary heart disease. We present evidence that it is the unique profile of the phenolic fraction, along with high intakes of squalene and the monounsaturated fatty acid, oleic acid, which confer its health-promoting properties. The major phenolic compounds identified and quantified in olive oil belong to three different classes: simple phenols (hydroxytyrosol, tyrosol); secoiridoids (oleuropein, the aglycone of ligstroside, and their respective decarboxylated dialdehyde derivatives); and the lignans [(+)-1-acetoxypinoresinol and pinoresinol]. All three classes have potent antioxidant properties. High consumption of extra-virgin olive oils, which are particularly rich in these phenolic antioxidants (as well as squalene and oleic acid), should afford considerable protection against cancer (colon, breast, skin), coronary heart disease, and ageing by inhibiting oxidative stress.