Cutaneous Photoprotective Activity of a Short‐term Ingestion of High‐Flavanol Cocoa: A Nutritional Intervention Study

In vivo and in vitro studies have shown that prolonged oral administration of flavanol‐rich cocoas extracts have photoprotective effects. The aim of the present study was to assess the photoprotective activity of short‐time administration of a new variety of naturally selected cocoa extract rich in bioactive compounds. We selected a cocoa powder particularly rich in polyphenols, flavanols, caffeine, theobromine and theophylline. We then investigated, in 10 healthy subjects, the photoprotective effects of one week of daily oral administration of two doses of such powder. Phototesting with solar simulated radiation was performed at baseline and after cocoa supplementation. Visual assessment of the minimal erythema dose and spectrophotometric measurement of the “a” parameter were done after 24 h from the irradiation. Oral daily supplementation of 1 g of high‐flavanol cocoa was not effective but a one‐week administration of 4–6 g of cocoa induced a statistically significant increase in the visual erythema threshold and a significant reduction in the “a” parameter. We demonstrated that a new variety of naturally selected cocoa extract, even when administered for a short time, retains a dose‐dependent photoprotective effect. These results are also indicative of the fact that topical sunscreens could be supplemented by a specific diet.     

Low-magnesium, <Emphasis Type="Italic">trans</Emphasis>-cleavage activity by type III,tertiary stabilized hammerhead ribozymes with stem 1 discontinuities

Background  

Low concentrations of free magnesium in the intracellular environment can present critical limitations for hammerhead ribozymes, especially for those that are designed for intermolecular (trans) cleavage of a host or pathogen RNA. Tertiary stabilizing motifs (TSM's) from natural and artificial ribozymes with a "type I" topology have been exploited to stabilize trans-cleaving hammerheads. Ribozymes with "type II" or "type III" topologies might seem incompatible with conversion to trans-cleavage designs, because opening the loop at the end of stem 1 or stem 2 to accommodate substrate binding is expected to disrupt the TSM and eliminate tertiary stabilization.     

Characterization of a protease produced by a Trichoderma harzianum isolate which controls cocoa plant witches' broom disease

Background  

Several Trichoderma strains have been reported to be effective in controlling plant diseases, and the action of fungal hydrolytic enzymes has been considered as the main mechanism involved in the antagonistic process. However, although Trichoderma strains were found to impair development of Crinipellis perniciosa, the causal agent of cocoa plant witches' broom disease, no fungal strain is available for effective control of this disease. We have then undertaken a program of construction of hydrolytic enzyme-overproducing Trichoderma strains aiming improvement of the fungal antagonistic capacity. The protease of an indian Trichoderma isolate showing antagonistic activity against C. perniciosa was purified to homogeneity and characterized for its kinetic properties and action on the phytopathogen cell wall.     

The use of stable isotopes ratios for authentication of fruit juices

The determination of the content of stable isotopes, ¹⁸O and ²H, respectively, in juice water facilitates the distinction between authentic juices and juices made from concentrates by redilution with tap water. At the same time, the detection of C₄ cane or corn-derived sugar syrups in fruit juices which are produced from C₃ fruit types is thus facilitated by the characteristic differences in ¹³C/¹²C, expressed as δ ¹³C (‰) values due to photosynthetic CO₂ assimilation via the C₃₋, C₄₋, and crassulacean acid metabolism pathways. In this study, the quantitative determination of water added to an authentic juice, on the basis of δ ¹⁸O, and δ ²H values, respectively, was successfully performed. Also, the δ ¹⁸O, and δ ²H of juice water and δ ¹³C of the whole juice in 18 samples were also determined. The results obtained provided us with the possibility of distinguishing between authentic fruit juices and those obtained by redilution of concentrated fruit juices and the detection of C⁴ type added sugar.     

Chromatographic determination of D-amino acids as native constituents of vegetables and fruits

Summary Free D-amino acids (D-AA) were detected as native constituents in juices of vegetables (cultivars of cabbage, tomato, carrot, garlic) and fruits (organes, clementine, grapefruit, lemon, apples, pear, grapes) using gas chromatography (GC) or high-performance liquid chromatography (LC). For investigation by GC, AA enantiomers were converted into theirN(O)-pentafluoropropionyl 2-propyl esters and resolved on a Chirasil-L-Val capillary column. For determination by LC, precolumn derivatization of AA enantiomers usingo-phthaldialdehyde together with the chiral thiolsN-isobutyryl-L-cysteine orN-isobutyryl-D-cysteine and fluorescence detection of the diastereomeric isoindole derivatives, resolvable on an octadecylsilyl stationary phase, were used. D-Ala (0.6–3.8%) was detected in all freshly pressed plant juices usually in the highest relative amounts. Other D-AA detected were D-Asx (0.1–1.9%), D-Glx (0–1.3%), D-Ser (0–1.7%), D-Arg (0.4–1.2%, in grapes, orange, grapefruit, and clementine) and D-Leu and D-Val (1% in cabbage). Absolute amounts of native D-AA were totally 28–57 mol L^–1 in fruit juices, 14.5 mol L^–1 in a tomato juice and 8.5 mol L^–1 in a carrot juice.Presented in part as lecture at 3rd International Congress on Amino Acids, Peptides and Analogues, August 23–27, 1993, Vienna; and as posters at 31st Scientific Meeting of German Society of Nutrition, Giessen, March 17th and 18th, 1994 [19]; and at Analytica Conference, April 19–22, 1994, Munich [20].     

Detection of scopolin in the grapefruit (Citrus paradisi Macf.)

Ripe fruits of Citrus paradisi Macf. and their juices contain high concentrations of scopolin (β-glucopyranoside of 7-hydroxy-6-methoxycoumarin). The identity of the aglycon being scopoletin was proven analysing extracts of β-glucosidase-treated fruit preparations with TLC, HPLC and GC-MS techniques. The naturally occurring glycoside was identified as the glucoside of scopoletin comparing it with authentic scopolin. The intake of scopolin of either grapefruits or juices thereof leads to a renal excretion of its aglycon as a glucuronide. The excretion is complete within 24 h. An enzymatic hydrolysis of the glucuronide produces scopoletin (i.e. aglycon) which can be quantified by HPLC. Mass spectra revealed that scopoletin releases one methyl- and two carbonyl-groups. The molecule finally breaks into fragments of 51 and 69 atomic mass units. Received: 18 December 1996 / Revised: 28 January 1997 / Accepted: 16 February 1997     

Discrimination of the Traditional Chinese Medicine from Schisandra Fruits by Flash Evaporation-Gas Chromatography/Mass Spectrometry and Fingerprint Analysis

Flash evaporation-gas chromatography/mass spectrometry (FE-GC/MS) with 0.3 mg sample powder in a vertical microfurnace pyrolyzer at 300 °C was applied to analyze Schisandra fruits without any tedious pretreatment. In total, 80 compounds, 74 compounds of which were identified, were observed, including low-molecular-weight compounds, essential oils (especially terpenoids), fatty acids and esters, and lignans, with the relative standard deviations (RSDs) of the relative percent of peak areas less than 7.79 % (n = 5). 32 compounds of terpenoids and lignans were selected as fingerprint components, since they are the main bioactive constituents in Schisandra fruits. The standard characteristic fingerprints of S. chinensis fruits and S. sphenantherae fruits were established, based on the 32 fingerprint components of 11 genuine S. chinensis fruit samples and 9 genuine S. sphenantherae fruit samples. The discrimination of samples from different growing places was achieved by principle component analysis and hierarchical cluster analysis. Furthermore, a similarity evaluation method was developed to evaluate the quality of each Schisandra fruit sample on the basis of the 32 fingerprint components. The results proved that the FE-GC fingerprint combined with a chemometric approach is a simple, rapid, and effective method for the origin discrimination and quality control of Schisandra fruits.

     

Effect of gamma irradiation on microbiological,chemical, and sensory properties of fresh ashitaba and kale juices

Due to the popularity of health effects upon intake of fresh fruits and vegetables, the demand for fresh vegetables and fruit juices has rapidly increased. However, currently, washing is the only procedure for reducing contaminated microorganisms, which obviously limits the shelf-life of fresh vegetable juice (less than 3 days). In this study, we examined the effects of irradiation on the microbiological, chemical and sensory properties of ashitaba and kale juices for industrial application and possible shelf-life extension. Freshly made ashitaba and kale juices already had 2.3×10⁵ and 9.5×10⁴ CFU/mL, respectively. Irradiation of 5 kGy induced higher than 2 decimal reductions in the microbial level, which was consistently maintained during storage for 7 days under refrigerated conditions. Total content of ascorbic acid in vegetable juice decreased upon irradiation in a dose-dependent manner. However, the content of flavonoids did not change, whereas that of polyphenols increased upon irradiation. In sensory evaluation, the ashitaba and kale juices without irradiation (control) scored lower than the irradiated samples after 1 and 3 days, respectively. This study confirms that irradiation is an effective method for sterilizing fresh vegetable juice without compromising sensory property, which cannot be subjected to heat pasteurization due to changes in the bioactivities of the products.     

Optimization and validation of a methodology based on solvent extraction and liquid chromatography for the simultaneous determination of several polyphenolic families in fruit juices

A solvent extraction procedure of freeze-dried aliquots followed by the analysis of phenolic compounds by reversed-phase high-performance liquid chromatography (RP-HPLC) with photodiode array detection (DAD) has been developed for the analysis of polyphenolic compounds in fruit juices. This methodology is focussed on the characterization of fruit juices, mainly for quality control purposes. The effects of experimental variables, such as solvent composition and volume and time and temperature on extraction, have been studied. A unique gradient program for the separation of several phenolic classes (hydroquinones, hydroxybenzoic acids, flavan-3-oles, hydroxycinnamic acids, coumarins, flavanones, flavones, dihydrochalcones and flavonols) has been optimized, using standards of 55 commercially available phenolic compounds present in fruits, as well as representative real extracts from fruit juices. All phenolic compounds showed a high repeatability within-day (n=5) and between days (n=3) in peak area (RSD<8%) and excellent stability of their retention times. High precision was also observed in calibration slopes (RSD<8%). Detection limits ranged between 0.005 and 0.03 microg/mL for the different detected polyphenols. Complete recoveries (98-100%) were obtained for the majority of the phenolic structures of all representative phenolic families present in fruits. The method was successfully employed to measure diverse phenolic families in juices from 18 different fruits and consequently could be used for evaluate the quality of fruit juices.     

Identification of flavonoids and chlorogenic acids in elm fruits from the genus Ulmus and their antioxidant activity

Elm fruits were once an important food source in the years of famine. Research on the functional compounds in elm fruits was almost unavailable. In this study, we established an efficient high‐performance liquid chromatography method for the simultaneous separation of eight chlorogenic acids and 28 flavonoids in elm fruits for the first time. Total flavonoid contents ranged from 286 mg/100 g (Ulmus laciniata) to 1228 mg/100 g (U. pumila). High concentrations of rutin, quercetin 3‐O‐glucoside, and kaempferol derivatives were present in U. laevis, U. castaneifolia, and U. pumila, respectively. Furthermore, the fruit extracts of U. americana, U. castaneifolia, U. davidiana, and U. pumila showed higher antioxidant activity. These results suggest that fruits of these species can be used as bioresources for the extraction of the corresponding functional compounds. This work provides informative data and can be an important reference for future research on elm fruits as a renewed food resource.     

Non-invasive determination of glucose directly in raw fruits using a continuous flow system based on microdialysis sampling and amperometric detection at an integrated enzymatic biosensor

A non-destructive, rapid and simple to use sensing method for direct determination of glucose in non-processed fruits is described. The strategy involved on-line microdialysis sampling coupled with a continuous flow system with amperometric detection at an enzymatic biosensor. Apart from direct determination of glucose in fruit juices and blended fruits, this work describes for the first time the successful application of an enzymatic biosensor-based electrochemical approach to the non-invasive determination of glucose in raw fruits. The methodology correlates, through previous calibration set-up, the amperometric signal generated from glucose in non-processed fruits with its content in % (w/w). The comparison of the obtained results using the proposed approach in different fruits with those provided by other method involving the same commercial biosensor as amperometric detector in stirred solutions pointed out that there were no significant differences. Moreover, in comparison with other available methodologies, this microdialysis-coupled continuous flow system amperometric biosensor-based procedure features straightforward sample preparation, low cost, reduced assay time (sampling rate of 7 h⁻¹) and ease of automation.     

A general analytical strategy for the characterization of phenolic compounds in fruit juices by high-performance liquid chromatography with diode array detection coupled to electrospray ionization and triple quadrupole mass spectrometry

In the present study, a methodology based on liquid chromatography with diode array detection (HPLC/DAD) coupled to an electrospray ionization (ESI) interface and a triple quadrupole mass spectrometer for the simultaneous identification of phenolic compounds in fruit juices has been developed. 72 available phenolic compound standards from diverse families present in fruits have been studied in order to analyze their fragmentation pattern. As a result, a general strategy for the characterization of unknown phenolic compounds in fruit juices was designed: (i) taking into account its UV–visible spectrum and elution order, assign the unknown polyphenol to a polyphenol class, (ii) identify the quasi-molecular ion using positive and negative MS spectra, being supported by adducts generated with solvent or sodium and molecular complexes, (iii) determinate the pattern of glycosylation in positive mode using ESI(+)-CID MS/MS product ion scan experiments, selecting the quasi-molecular ion as precursor ion, and finally, (iv) study the identity of the aglycone through ESI(+)-CID MS/MS product ion spectra from the protonated aglycone, [Y₀]⁺. This strategy was successfully employed for the characterization of known and unknown phenolic compounds in juices from 17 different fruits.     

Fast determination of anthocyanins and free pelargonidin in fruits,fruit juices,and fruit wines by high‐performance liquid chromatography using a core–shell column

Anthocyanins are water‐soluble pigments that are liable for colors ranging from red to blue of most fruits, vegetables, and flowers. A novel and fast method was developed for the determination of five anthocyanins and free pelargonidin by high‐performance liquid chromatography coupled to photodiode array detection. A 10% formic acid and acetonitrile mixture was employed as mobile phase in the gradient elution mode. Mobile phase composition, column temperature, flow rate, injection volume, and column conditioning time were optimized by employing a stepwise strategy. Using a C₁₈ core–shell column (100 × 4.6 mm, 2.7 μm), the separation of six analytes was accomplished in less than 9.5 min with a run‐to‐run analysis time of 19 min. The developed method was validated with respect to linearity (r > 0.9999), limit of detection, limit of quantification, intra‐/interday precision (<2%), accuracy (98.6–104.4%), and specificity. Afterwards, the method was applied to the determination of anthocyanins present in 15 different samples including fruits, fruit juices, and fruit wines.     

DNA biosensors for the detection of aflatoxin producing <Emphasis Type="Italic">Aspergillus flavus</Emphasis> and <Emphasis Type="Italic">A. parasiticus</Emphasis>

Abstract  In this work, we report on the development of a DNA-based piezoelectric biosensor specific for the detection of an amplicon of the aflD gene of Aspergillus flavus and A. parasiticus. Expression of this gene is consistently correlated with a strain’s ability to produce aflatoxins that are considered very potent liver carcinogens in various animal species and humans. The DNA biosensor has been characterized with synthetic oligonucleotides and amplicons. Moreover, it has been applied to the analysis of real samples consisting of amplicons of DNA extracted from flours and feed contaminated with A. flavus and A. parasiticus. Graphical Abstract        

Determination of the Free Radical Scavenging Activity of Lycium Extracts

Lycium species growing in Turkey have not so far been studied sufficiently. For this reason, non-polar and polar extracts obtained from the fruits of Lycium barbarum L. and L. Ruthenicum Murray (Solanaceae) were assessed both in vitro for their potential as free radical scavenger crude extracts and their phenolic composition. Fruits of Lycium species were sequentially extracted with petroleum ether, ethyl acetate, methanol, n-butanol, and water in a Soxhlet extractor. All the extracts were assessed for the scavenging of the nitrogen-centered free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH^.) by in vitro method. Furthermore, the composition of each extract was investigated both in terms of its Folin-Ciocalteau reactive components and its qualitative content. The phenolic compounds within the extracts were determined as benzoic acid and hydroxycinnamic acid derivatives, flavonoids, and anthocyanins according to their retention time and UV spectral data by HPLC-DAD system.     

Comparative Study of the Chemical Compositions and Antioxidant Activities of Fresh Juices from Romanian Cucurbitaceae Varieties

Cucurbitaceae is a family of health-promoting plants due to their compounds with beneficial effects. The aim of this study was to analyze, for the first time, the chemical composition, the antioxidant activity and the metal chelating properties of fruit juices obtained from four different species of the Cucurbitaceae family cultivated in Romania, namely Momordica charantia, Cucumis metuliferus, Benincasa hispida and Trichosanthes cucumerina. The samples of juice were analyzed by high-performance liquid chromatography (HPLC) and all the four species displayed high levels of the two triterpenes, oleanolic and ursolic acids, and also in phenolic compounds, including catechin, (−)-epicatechin and gallic acid. The juices demonstrated significant antioxidant activity against the free radical 2,2-diphenyl-1-picrylhydrazyl (ranging from 20 to 95%,), a good iron binding ability (ranging from 7.45 ± 0.28% to 86.95 ± 0.97%) and also promising antioxidant potential against the ABTS radical (ranging from 4.97 to 32.60 μETx/mL juice). Our findings raise interesting questions for further research on Cucurbitaceae fruit juices and, consequently, their very good antioxidant potential suggests these fruits should be further explored for their protective effect against oxidative damage. This is the first time the chemical composition and antioxidant activities of fruit juices from these four Romanian Cucurbitaceae varieties have been investigated.     

Thermal behavior of different cocoa powder varieties and their physicochemical,phytochemical and microbiological characteristics

Four cocoa powder varieties processed in different European countries (Germany, Poland, Romania and Bulgaria) were subjected to physicochemical, phytochemical and microbiological analysis. The cocoa powders were extensively characterized by recording their pH and titratable acidity, respectively, the polyphenols and also the methylxantine derivatives content (theobromine and caffeine). The cocoa powders pH ranged between 5.37 and 8.23, while the titratable acidity was 3.2–4.3 miliequivalent (100 g)^?1 of cocoa powder. Their total polyphenols content ranged between 0.986?÷?2.003 g GAE/(100 g)^?1. The methylxanthine derivatives (theobromine and caffeine) were analyzed by the HPLC method and ranges of 0.992–1.174% for theobromine and 0.096–0.369% for caffeine were obtained. Thermal analysis (TG–DTA) combined with mass spectrometry (MS) elucidated the decomposition processes and the volatile substances (CO, CO₂, H₂O, NO, theobromine, caffeine). The thermal analysis revealed transformations in the cocoa powders composition: drying and water loss; decomposition of pectic polysaccharides; lipids, amino acids and proteins, crystalline phase transformations and carbonizations. The microbiological analysis tested the degree of preservation of the cocoa powders across time, specifically immediately after unwrapping and after 14 days.

     

Use of water activity characteristics enables a simplified approach for defining the reference moisture condition for FDA cocoa powder in-house reference material

The Food and Drug Administration uses water activity behavior characteristics when adjusting test portion mass to correct for the moisture condition of its cocoa powder in-house reference material. The cocoa powder’s moisture condition, and therefore weight, equilibrates according to the relative humidity (RH) of its surroundings. This process is predictable and defined by an isotherm. The reference values in the certificate of analysis are relative to the material’s condition at 30 % RH, which is assumed to be mid-range for typical laboratory settings. Since mass variations are relatively small within a 15–50 % RH range, the mass may be measured immediately after removing a test portion from a storage bottle and used without correction if a standard uncertainty of 0.7 % is acceptable for the mass. If greater accuracy is needed and the laboratory RH is known, a very simple and quick procedure can be used whereby the test portion is left open and exposed to the laboratory air overnight before weighing. After applying a correction, the standard uncertainty for mass measurement drops to 0.3 %.

The roles of aldehyde dehydrogenases (ALDHs) in the PDH bypass of Arabidopsis

Background  

Eukaryotic aldehyde dehydrogenases (ALDHs, EC 1.2.1), which oxidize aldehydes into carboxylic acids, have been classified into more than 20 families. In mammals, Family 2 ALDHs detoxify acetaldehyde. It has been hypothesized that plant Family 2 ALDHs oxidize acetaldehyde generated via ethanolic fermentation, producing acetate for acetyl-CoA biosynthesis via acetyl-CoA synthetase (ACS), similar to the yeast pathway termed the "pyruvate dehydrogenase (PDH) bypass". Evidence for this pathway in plants has been obtained from pollen.     

A three‐phase solvent extraction system combined with deep eutectic solvent‐based dispersive liquid–liquid microextraction for extraction of some organochlorine pesticides in cocoa samples prior to gas chromatography with electron capture detection

A sample pretreatment method based on the combination of a three‐phase solvent extraction system and deep eutectic solvent‐based dispersive liquid–liquid microextraction has been introduced for the extraction of four organochlorine pesticides in cocoa samples before their determination by gas chromatography‐electron capture detection. A mixture of sodium chloride, acetonitrile, and potassium hydroxide solution is added to cocoa bean or powder. After vortexing and centrifugation of the mixture, the collected upper phase (acetonitrile) is removed and mixed with a few microliters of N,N‐diethanol ammonium chloride: pivalic acid deep eutectic solvent. Then it is rapidly injected into deionized water and a cloudy solution is obtained. Under optimum conditions, the limits of detection and quantification were found to be 0.011‐0.031 and 0.036‐0.104 ng/g, respectively. The obtained extraction recoveries varied between 74 and 92%. Also, intra‐ (n = 6) and interday (n = 4) precisions were less than or equal to 7.1% for the studied pesticides at a concentration of 0.3 ng/g of each analyte. The suggested method was applied to determine the studied organochlorine pesticide residues in various cocoa powders and beans gathered from groceries in Tabriz city (Iran) and aldrin and dichlobenil were found in some of them.