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 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.

Red Wine Age Estimation by the Alteration of Its Color Parameters: Fourier Transform Infrared Spectroscopy as a Tool to Monitor Wine Maturation Time.

Color, phenolic content, and chemical age values of red wines made from Cretan grape varieties (Kotsifali, Mandilari) were evaluated over nine months of maturation in different containers for two vintages. The wines differed greatly on their anthocyanin profiles. Mid-IR spectra were also recorded with the use of a Fourier Transform Infrared Spectrophotometer in ZnSe disk mode. Analysis of Variance was used to explore the parameter's dependency on time. Determination models were developed for the chemical age indexes using Partial Least Squares (PLS) (TQ Analyst software) considering the spectral region 1830-1500 cm-1. The correlation coefficients (r) for chemical age index i were 0.86 for Kotsifali (Root Mean Square Error of Calibration (RMSEC) = 0.067, Root Mean Square Error of Prediction (RMSEP) = 0,115, and Root Mean Square Error of Validation (RMSECV) = 0.164) and 0.90 for Mandilari (RMSEC = 0.050, RMSEP = 0.040, and RMSECV = 0.089). For chemical age index ii the correlation coefficients (r) were 0.86 and 0.97 for Kotsifali (RMSEC 0.044, RMSEP = 0.087, and RMSECV = 0.214) and Mandilari (RMSEC = 0.024, RMSEP = 0.033, and RMSECV = 0.078), respectively. The proposed method is simpler, less time consuming, and more economical and does not require chemical reagents.

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.

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.

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.

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.

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.

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.

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.

Effects of the active constituents of Crocus Sativus L., crocins, in an animal model of obsessive-compulsive disorder.

Crocins are among the active components of the plant Crocus Sativus L. C. Sativus L. and its constituents were effective in different models of psychiatric disorders including anxiety and depression. Obsessive-compulsive disorder (OCD) is a common psychiatric disorder defined by the presence of obsessive thoughts and repetitive compulsive actions. The non selective serotonin (5-HT) receptor agonist mCPP is known to induce OCD-like behavior (excessive self-grooming) in rodents and exacerbate symptoms in patients with OCD. The present study investigated whether or not crocins were able to counteract excessive self-grooming induced by mCPP (0.6 mg/kg, i.p.) in rats. Crocins (30 and 50mg/kg, i.p.) attenuated mCPP-induced excessive self-grooming. The present results also indicate that these effects of crocins on an animal model of OCD cannot be attributed to changes in locomotor activity. Our findings suggest that the active constituents of C. Sativus L. crocins might play a role in compulsive behavior and support a functional interaction between crocins and the serotonergic system.

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.

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.

Effects of the active constituents of Crocus sativus L., crocins, in an animal model of anxiety.

Crocus sativus L. is a plant cultivated in various parts of the world. Crocins are among the active components of Crocus sativus L. The present study was designed to investigate in the rat whether or not crocins possess anxiolytic properties. For this aim, the light/dark test was selected. Either crocins, at a dose which did not influence animals' motor activity (50mg/kg), or diazepam (1.5 mg/kg), significantly increased the latency to enter the dark compartment and prolonged the time spent in the lit chamber in the rats. Conversely, lower doses of crocins (15-30 mg/kg) did not substantially modify animals' behaviour. The present results indicate that treatment with these active constituents of Crocus sativus L. induce anxiolytic-like effects in the rat.

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.