Geographical differentiation of dried lentil seed (Lens culinaris) samples using diffuse reflectance Fourier transform infrared spectroscopy (DRIFTS) and discriminant analysis.

Diffuse reflectance Fourier transform infrared spectroscopy (DRIFTS) and discriminant analysis were used for the geographical differentiation of dried lentil seed (Lens culinaris) samples. Specifically, 18 Greek samples and nine samples imported from other countries were distinguished using the 2250-1720 and 1275-955 cm⁻¹ spectral regions. The differentiation is complete. The combination of DRIFTS and discriminant analysis enables simple, rapid, cheap and accurate differentiation of commercial lentil seeds in terms of geographical origin.

Probing the binding sites of resveratrol, genistein, and curcumin with milk ß-lactoglobulin.

We determined the binding sites of curcumin (cur), resveratrol (res), and genistein (gen) with milk ß-lactoglobulin (ß-LG) at physiological conditions. Fourier transform infrared spectroscopy, circular dichroism, and fluorescence spectroscopic methods as well as molecular modeling were used to determine the binding of polyphenol-protein complexes. Structural analysis showed that polyphenols bind ß-LG via both hydrophilic and hydrophobic contacts with overall binding constants of Kcurcumin-ß-LG = 4.4 (± .4) × 104 M⁻¹, Kresveratrol-ß-LG = 4.2 (± .2) × 104 M⁻¹, and Kgenistein-ß-LG = 1.2 (± .2) × 104 M⁻¹. The number of polyphenol molecules bound per protein (n) was 1 (cur), 1.1 (res), and 1 (gen). Molecular modeling showed the participation of several amino acid residues in polyphenol-protein complexation with the free binding energy of -12.67 (curcumin-ß-LG), -12.60 (resveratrol-ß-LG), and -10.68 kcal/mol (genistein-ß-LG). The order of binding was cur > res > gen. Alteration of the protein conformation was observed in the presence of polyphenol with a major reduction of ß-sheet and an increase in turn structure, causing a partial protein structural destabilization. ß-LG might act as a carrier to transport polyphenol in vitro.

Locating the binding sites of retinol and retinoic acid with milk ß-lactoglobulin.

ß-lactoglobulin (ß-LG) is a member of lipocalin superfamily of transporters for small hydrophobic molecules such as retinoids. We located the binding sites of retinol and retinoic acid on ß-LG in aqueous solution at physiological conditions, using FTIR, CD, fluorescence spectroscopic methods, and molecular modeling. The retinoid-binding sites and the binding constants as well as the effect of retinol and retinoic acid complexation on protein stability and secondary structure were determined. Structural analysis showed that retinoids bind strongly to ß-LG via both hydrophilic and hydrophobic contacts with overall binding constants of K (retinol-) (ß) (-LG )= 6.4 (± .6) × 10(6) M(-1) and K (retinoic acid-) (ß) (-LG )= 3.3 (± .5) × 10(6) M(-1). The number of retinoid molecules bound per protein (n) is 1.1 (± .2) for retinol and 1.5 (± .3) for retinoic acid. Molecular modeling showed the participation of several amino acids in the retinoid-protein complexes with the free binding energy of -8.11 kcal/mol for retinol and -7.62 kcal/mol for retinoic acid. Protein conformation was altered with reduction of ß-sheet from 59 (free protein) to 52-51% and a major increase in turn structure from 13 (free protein) to 24-22%, in the retinoid-ß-LG complexes, indicating a partial protein destabilization.

Antibacterial activities of essential oils from eight Greek aromatic plants against clinical isolates of Staphylococcus aureus.

Aromatic plants have been used widely to extend the shelf life of foods but at the same time research is undergoes for their properties as antibacterial agents in clinical use. Although there are promising results for the antimicrobial properties of various essential oils against environmental or food-isolated strains of Staphylococcus aureus, limited work has been done concerning these properties against clinical isolates of this pathogen. S. aureus is responsible for an increase number of nosocomial infections and at the same time exhibits increased resistance to synthetic agents. In this study, essential oils from eight aromatic plants common in Greece were isolated by hydrodistillation, analyzed by gas chromatography (GC) and GC/mass spectrometry (GC/MS) for their chemical components and tested for their antimicrobial activities against 24 clinical isolates of S. aureus. The methods used were disk diffusion and broth dilution in order to determine the Minimum Inhibitory Concentration (MIC). Our results showed that essential oils from Origanum vulgare and Origanum dictamnus were active against S. aureus when tested by disk diffusion, but exhibited increased MIC values (>256 mg/L) with the dilution method. In contrast, the reference strain NCTC 6571 showed to be extremely sensitive in most of the oils tested (MICs 0.25-32.0 mg/L) and resistant only to the essential oil from Ocimum basilicum. Therefore, there is no evidence of a potential clinical use for those essential oils and further research is needed in order to determine if they could substitute efficiently synthetic antibiotics or, perhaps be used in combination.

Milk ß-lactoglobulin complexes with tea polyphenols.

The effect of milk on the antioxidant capacity of tea polyphenols is not fully understood. The complexation of tea polyphenols with milk proteins can alter the antioxidant activity of tea compounds and the protein secondary structure. This study was designed to examine the interaction of ß-lactogolobulin (ß-LG) with tea polyphenols (+)-catechin (C), (-)-epicatechin (EC), (-)-epicatechin gallate (ECG) and (-)-epigallocatechin gallate (EGCG) at molecular level, using FTIR, CD and fluorescence spectroscopic methods as well as molecular modelling. The polyphenol binding mode, the binding constant and the effects of polyphenol complexation on ß-LG stability and secondary structure were determined. Structural analysis showed that polyphenols bind ß-LG via both hydrophilic and hydrophobic interactions with overall binding constants of KC-ß-LG=2.2 (±0.8)×10(3)M(-1), KEC-ß-LG=3.2 (±1)×10(3)M(-1), KECG-ß-LG=1.1 (±0.6)×10(4)M(-1) and KEGCG-ß-LG=1.3 (±0.8)×10(4)M(-1). The number of polyphenols bound per protein molecule (n) was 1.1 (C), 0.9 (EC), 0.9 (ECG) and 1.3 (EGCG). Molecular modelling showed the participation of several amino acid residues in polyphenol-protein complexation with extended H-bonding network. The ß-LG conformation was altered in the presence of polyphenols with an increase in ß-sheet and α-helix suggesting protein structural stabilisation. These data can be used to explain the mechanism by which the antioxidant activity of tea compounds is affected by the addition of milk.

An overview of structural features of DNA and RNA complexes with saffron compounds: Models and antioxidant activity.

Saffron is the red dried stigmas of Crocus sativus L. flowers and used both as a spice and as a drug in traditional medicine. Its numerous applications as an antioxidant and anticancer agent are due to its secondary metabolites and their derivatives (safranal, crocetin, dimethylcrocetin). In this work we are comparing the spectroscopic results and antioxidant activities of saffron components safranal, crocetin (CRT) and dimethylcrocetin (DMCRT) complexes with calf-thymus DNA (ctDNA) and transfer RNA (tRNA) in aqueous solution at physiological conditions Intercalative and external binding modes of saffron compounds to DNA and RNA were observed with overall binding constants of K(safranal)=1.24x10(3)M(-1), K(CRT)=6.20x10(3)M(-1) and K(DMCRT)=1.85x10(5)M(-1), for DNA adducts and K(safranal)=6.80x10(3)M(-1), K(CRT)=1.40x10(4)M(-1) and K(DMCRT)=3.40x10(4)M(-1) for RNA complexes. A partial B- to A-DNA transition occurred at high ligand concentrations, while tRNA remained in A-conformation in saffron-RNA complexes. The antioxidant activity of CRT, DMCRT and safranal was also tested by the DPPH (2,2-diphenyl-1-picrylhydrazyl) antioxidant activity assay and their IC(50) values were compared to that of well known antioxidants such as Trolox and Butylated Hydroxy Toluene (BHT). The IC(50) values were 95+/-1microg/mL for safranal and 18+/-1microg/mL for crocetin. The inhibition of DMCRT reached a point of 38.8%, which corresponds to a concentration of 40microg/mL.

Solid-phase microextraction/gas-chromatographic/mass spectrometric analysis of p-dichlorobenzene and naphthalene in honey.

Protection of honeycombs from the Wax moth, Galleria mellonella, involves the use of physical, biological or chemical control methods. As chemical control may result in residues in the extracted honey, the presence of p-dichlorobenzene and naphthalene residues was investigated by solid-phase microextraction (SPME) coupled to gas-chromatographic/mass spectrometry (GC/MS). The method was linear between 5 and 200 microg kg(-1) honey for p-dichlorobenzene and 1 and 200 microg kg(-1) for naphthalene. Limits of detection were 1 and 0.1 microg kg(-1), respectively, for p-dichlorobenzene and naphthalene, while relative standard deviations were 2.6 and 7.9%, respectively. Application of the method to 90 unifloral Greek honeys revealed that, in 25.6% of the samples, the concentration of either one of the pesticides exceeded the maximum residue level (MRL). Maximum concentrations were 163.03 microg kg(-1) honey for p-dichlorobenzene and 193.74 microg kg(-1) honey for naphthalene. Naphthalene was found in traceable amounts in 78.9% of the samples, but only 5.6% of them contained concentrations above the MRL, which indicates the use of pre-contaminated honeycomb foundations or built combs. Nevertheless, because naphthalene is naturally present in some plant species growing in Greece, the contribution of nectar from such a floral source should not be overlooked.

An overview of DNA and RNA bindings to antioxidant flavonoids.

In this report we are examining how the antioxidant flavonoids can prevent DNA damage and what mechanism of action is involved in the process. Flavonoids are strong antioxidants that prevent DNA damage. The anticancer and antiviral activities of these natural products are implicated in their mechanism of actions. We study the interactions of quercetin (que), kaempferol (kae), and delphinidin (del) with DNA and transfer RNA in aqueous solution at physiological conditions, using constant DNA or RNA concentration 6.25 mmol (phosphate) and various pigment/polynucleotide(phosphate) ratios of 1/65 to 1 (DNA) and 1/48 to 1/8 (tRNA). The structural analysis showed quercetin, kaempferol, and delphinidin intercalate DNA and RNA duplexes with minor external binding to the major or minor groove and the backbone phosphate group with overall binding constants for DNA adducts K(que) = 7.25 (+/-0.65) x 10(4) M(-1), K(kae) = 3.60 (+/-0.33) x 10(4) M(-1), and K(del) = 1.66 (+/-0.25) x 104 (-1) and for tRNA adducts K(que) = 4.80 (+/-0.50) x 10(4) M(-1), K(kae) = 4.65 (+/-0.45) x 10(4) M(-1), and K(del) = 9.47 (+/-0.70) x 10(4) M(-1). The stability of adduct formation is in the order of del>que>kae for tRNA and que>kae>del for DNA. Low flavonoid concentration induces helical stabilization, whereas high pigment content causes helix opening. A partial B to A-DNA transition occurs at high drug concentration, while tRNA remains in A-family structure. The antioxidant activity of flavonoids changes in order delphinidin>quercetin>kaempferol. The results show intercalated flavonoids can make them strong antioxidants to protect DNA from harmful free radical reactions.

DNA interaction with naturally occurring antioxidant flavonoids quercetin, kaempferol, and delphinidin.

Flavonoids are strong antioxidants that prevent DNA damage. The anticancer and antiviral activities of these natural products are implicated in their mechanism of actions. However, there has been no information on the interactions of these antioxidants with individual DNA at molecular level. This study was designed to examine the interaction of quercetin (que), kaempferol (kae), and delphinidin (del) with calf-thymus DNA in aqueous solution at physiological conditions, using constant DNA concentration (6.5 mmol) and various drug/DNA(phosphate) ratios of 1/65 to 1. FTIR and UV-Visible difference spectroscopic methods are used to determine the drug binding sites, the binding constants and the effects of drug complexation on the stability and conformation of DNA duplex. Structural analysis showed quercetin, kaempferol, and delphinidin bind weakly to adenine, guanine (major groove), and thymine (minor groove) bases, as well as to the backbone phosphate group with overall binding constants K(que) = 7.25 x 10(4)M(-1), K(kae) = 3.60 x 10(4)M(-1), and K(del) = 1.66 x 10(4)M(-1). The stability of adduct formation is in the order of que>kae>del. Delphinidin with a positive charge induces more stabilizing effect on DNA duplex than quercetin and kaempferol. A partial B to A-DNA transition occurs at high drug concentrations.

Determination of uronic acids in isolated hemicelluloses from kenaf using diffuse reflectance infrared fourier transform spectroscopy (DRIFTS) and the curve-fitting deconvolution method.

Hemicellulose samples were isolated from kenaf (Hibiscus cannabinus L.). Hemicellulosic fractions usually contain a variable percentage of uronic acids. The uronic acid content (expressed in polygalacturonic acid) of the isolated hemicelluloses was determined by diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) and the curve-fitting deconvolution method. A linear relationship between uronic acids content and the sum of the peak areas at 1745, 1715, and 1600 cm(-1) was established with a high correlation coefficient (0.98). The deconvolution analysis using the curve-fitting method allowed the elimination of spectral interferences from other cell wall components. The above method was compared with an established spectrophotometric method and was found equivalent for accuracy and repeatability (t-test, F-test). This method is applicable in analysis of natural or synthetic mixtures and/or crude substances. The proposed method is simple, rapid, and nondestructive for the samples.

New method for pollen identification by FT-IR spectroscopy.

A new methodology for identification of pollen was developed based on FT-IR spectroscopy. Pollen samples of twenty different plant species were collected and the diffuse reflectance infrared Fourier transform (DRIFTS) and KBr pellet spectra were recorded. Libraries of spectra were created. Spectra of unknown plant origin pollen were recorded and compared with those of the corresponding pollen library and the match value was measured automatically using the appropriate software (OMINC ver. 3.1). From the same pollen samples, microscopic slides were prepared and the photographs of the pollen grains were used as a second comparison method. Using light microscopy, the pollen identification is usually limited to the family or generic name, while FT-IR spectroscopy can distinguish species belonging to the same genus. This method is simple and fast, and when the DRIFTS technique is used the sample is not destroyed.

In vitro antibacterial, antifungal, and antioxidant activities of the essential oil and methanol extracts of herbal parts and callus cultures of Satureja hortensis L.

The present study was designated to evaluate the antimicrobial and antioxidant activities of the essential oil, obtained by using a Clevenger distillation apparatus, water soluble (polar) and water insoluble (nonpolar) subfractions of the methanol extracts from aerial parts of Satureja hortensis L. plants, and methanol extract from calli established from the seeds using Gamborg's B5 basal media supplemented with indole-3-butyric acid (1.0 ppm), 6-benzylaminopurine (N(6)-benzyladenine) (1.0 ppm), and sucrose (2.5%). The antimicrobial test results showed that the essential oil of S. hortensis had great potential antimicrobial activities against all 23 bacteria and 15 fungi and yeast species tested. In contrast, the methanol extract from callus cultures and water soluble subfraction of the methanol extract did not show antimicrobial activities, but the nonpolar subfraction had antibacterial activity against only five out of 23 bacterial species, which were Bacillus subtilis, Enterococcus fecalis, Pseudomonas aeruginosa, Salmonella enteritidis, and Streptococcus pyogenes. Antioxidant studies suggested that the polar subfractions of the methanol extract of intact plant and methanol extract of callus cultures were able to reduce the stable free radical 2,2-diphenyl-1-picrylhydrazyl to the yellow-colored diphenylpicrylhydrazine. In this assay, the strongest effect was observed for the tissue culture extract, with an IC(50) value of 23.76 +/- 0.80 microgram/mL, which could be compared with the synthetic antioxidant agent butylated hydroxytoluene. On the other hand, linoleic acid oxidation was 95% inhibited in the presence of the essential oil while the inhibition was 90% with the chloroform subfraction of the intact plant. The chemical composition of a hydrodistilled essential oil of S. hortensis was analyzed by gas chromatography (GC)/flame ionization detection (FID) and a GC-mass spectrometry system. A total 22 constituents representing 99.9% of the essential oil were identified by GC-FID analaysis. Thymol (29.0%), carvacrol (26.5%), gamma-terpinene (22.6%), and p-cymene (9.3%) were the main components.

Comparison of classical and ultrasound-assisted isolation procedures of cellulose from kenaf (Hibiscus cannabinus L.) and eucalyptus (Eucalyptus rodustrus Sm.).

A comparative study of classical and ultrasound-assisted extraction and purification of cellulose from kenaf (Hibiscus cannabinus L.) and eucalyptus (Eucalyptus rodustrus Sm.), has been conducted. The isolated cellulose samples were studied by diffuse reflectance infrared Fourier transform spectroscopy and 13C nuclear magnetic resonance (13C-NMR) spectroscopy and the crystallinity was also determined. The use of ultrasound decreased the total time of treatment, in addition the purity of the obtained cellulose was very high.

Enzymatic acylation of hydroxypropyl cellulose in organic media and determination of ester formation by diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy.

Esters were prepared by acylation of hydroxypropyl cellulose with fatty acid catalyzed by immobilized lipase from Candida antarctica in tert-butanol. The nature of the substrates used, the initial water activity of the system, and the molecular weight of the hydroxypropyl cellulose were investigated. Moreover, Fourier transform-infrared (FT-IR) spectroscopy was used for determination of ester content on hydroxypropyl cellulose. Specifically, a linear relationship was established between the peak height assigned to the absorption of the esterified carboxyl groups of the cellulose and the ester content. At optimum reaction conditions, the ester content on the hydroxypropyl cellulose was about 11%.

GC-MS analysis of essential oils from some Greek aromatic plants and their fungitoxicity on Penicillium digitatum.

The isolated essential oils from seven air-dried plant species were analyzed by gas chromatography-mass spectrometry (GC-MS). Thymus vulgaris (thyme), Origanum vulgare (oregano), and Origanumdictamus (dictamus) essential oils were found to be rich in phenolic compounds representing 65.8, 71.1, and 78.0% of the total oil, respectively. Origanum majorana (marjoram) oil was constituted of hydrocarbons (42.1%), alcohols (24.3%), and phenols (14.2%). The essential oil from Lavandula angustifolia Mill. (lavender) was characterized by the presence of alcohols (58.8%) and esters (32.7%). Ethers predominated in Rosmarinus officinalis (rosemary) and Salvia fruticosa (sage) essential oils, constituting 88.9 and 78.0%, respectively. The radial growth, conidial germination, and production of Penicillium digitatum were inhibited completely by oregano, thyme, dictamus, and marjoram essential oils at relatively low concentrations (250-400 microg/mL). Lavender, rosemary, and sage essential oils presented less inhibitory effect on the radial growth and conidial germination of P. digitatum. Conidial production of P. digitatum was not affected by the above oils at concentrations up to 1000 microg/mL. Apart from oregano oil, all essential oils were more effective in the inhibition of conidial germination than of radial growth. The monoterpene components, which participate in essential oils in different compositions, seem to have more than an additive effect in fungal inhibition.

FT-Raman spectroscopy - analytical tool for routine analysis of diazinon pesticide formulations.

FT-Raman spectroscopy based on band intensity and band area measurements, was used for the quantitative determination of diazinon in pesticide formulations. Bands at 554, 604, 631, 1562 and 2971 cm(-1) were used for calibration. Spectra were acquired by averaging 100 scans at a resolution of 4 cm(-1). Calibration curves were linear (correlation coefficients, 0.992-0.9992 and 0.99-0.999 for band intensity and band area measurements, respectively) in the range of 0.2-3.5 M for 554 and 2971 cm(-1), 0.3-3.5 M for 604 cm(-1), 0.6-3.5 M for 1562 cm(-1) and 1.0-3.5 M for 631 cm(-1) bands. Normalization of calibration curves against the 802 cm(-1) cyclohexane band improved their long term stability and minimized the effect of laser beam power fluctuations. No interference was found by commonly used surfactants and the proposed method was applied to the analysis of diazinon formulations. Results obtained compare well as indicated by the t-test, with those obtained by the HPLC reference method. Precision ranged between 0.2-7.8 and 0.1-7.2% RSD, (n=4) for band intensity and band area measurements, respectively. The proposed method is rapid, simple and safe, as toxic samples are analyzed 'as received' without sample pre-treatment, permiting the routine analysis of pesticides formulations.

Determination of saffron (Crocus sativus L.) components in crude plant extract using high-performance liquid chromatography-UV-visible photodiode-array detection-mass spectrometry.

The determination of saffron components in crude plant extracts by high-performance liquid chromatography-UV-visible photodiode-array detection on-line with mass spectrometry is described. The method is shown to be suitable for the determination of picrocrocin, the glycosidic precursor of safranal, safranal and flavonoids; it is the technique of choice for the analysis of crocetin glycosides (crocins) carrying one up to five glucoses and differentiation of their trans and cis isomers.

Inhibition of growth and induction of differentiation of promyelocytic leukemia (HL-60) by carotenoids from Crocus sativus L.

The effects of carotenoids of Crocus sativus L. (saffron) on cell proliferation and differentiation of HL-60 cells have been studied and compared with those of all-trans retinoic acid. Our results demonstrated that the doses inducing 50% inhibition of cell growth were 0.12 microM for all-trans retinoic acid (ATRA) and for carotenoids of saffron 0.8 microM for dimethylcrocetin (DMCRT), 2 microM for crocetin CRT and 2 microM for crocins (CRCs). At 5 microM, all these compounds induced differentiation of HL-60 cells, at 85% for ATRA, 70% for DMCRT, 50% for CRT and 48% for CRCs. In these experiments, leukemic cells were cultured for 5 days in the absence or in the presence of up to 5 microM ATRA or seminatural and natural carotenoids. Since retinoids have a potential application as chemopreventive agents in humans, their toxicity as an important limiting factor for their use in treatment should be extensively explored. The seminatural (DMCRT and CRT) and natural carotenoids (CRCs) of Crocus sativus L. are not provitamin A precursors and could therefore be less toxic than retinoids, even at high doses.

Separation of picrocrocin, cis-trans-crocins and safranal of saffron using high-performance liquid chromatography with photodiode-array detection.

High-performance liquid chromatography with photodiode-array detection was used to separate picrocrocin (bitter-tasting component, glucoside of safranal), cis/trans-crocins (carotenoids, glucosyl esters of crocetin) and safranal (flavour, monoterpene aldehyde) of saffron. All components of pure red Greek saffron were extracted from dried stigma with 50% methanol. These compounds were detected, separated collected and identified simultaneously using a Merck LiChroCART 125-4 Superspher 100 RP-18 (4 microns) column and as mobile phase a linear gradient from 20% to 100% acetonitrile in water in 20 min with a detection wavelength at 308 nm.

Possible role of water structure in biological magnesium systems.

Magnesium chloride-water solutions have been studied by Fourier Transform Infrared Spectroscopy (FT-IR) in the near infrared region, 5000-10,000 cm-1. The effect of the concentration of magnesium chloride and temperature on the solutions has been studied from the spectra and it is concluded that magnesium chloride modifies the structure of the bulk water. The important absorption bands of water at 5200 and 7020 cm-1 may be assigned to combination vibrations and overtones. They are shifted either by increasing the magnesium chloride concentration or the temperature. The hydrated magnesium ions, [Mg(H2O)6]2+, will most probably break important hydrogen bonds in the clusters of water (H2O)n, where n = 2, 3, 4, 5, 6.../forming new hydrogen bonds in the presence of hexa-aquated magnesium cations. FAB mass spectra also suggest the formation of hydrated magnesium cations, Mg (H2O)6(2+).