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Inhibition of protein and lipid oxidation in liposomes by berry phenolics.

The antioxidant activity of berry phenolics (at concentrations of 1.4, 4.2, and 8.4 mug of purified extracts/mL of liposome sample) such as anthocyanins, ellagitannins, and proanthocyanidins from raspberry (Rubus idaeus), bilberry (Vaccinium myrtillus), lingonberry (Vaccinium vitis-idaea), and black currant (Ribes nigrum) was investigated in a lactalbumin-liposome system. The extent of protein oxidation was measured by determining the loss of tryptophan fluorescence and formation of protein carbonyl compounds and that of lipid oxidation by conjugated diene hydroperoxides and hexanal analyses. The antioxidant protection toward lipid oxidation was best provided by lingonberry and bilberry phenolics followed by black currant and raspberry phenolics. Bilberry and raspberry phenolics exhibited the best overall antioxidant activity toward protein oxidation. Proanthocyanidins, especially the dimeric and trimeric forms, in lingonberries were among the most active phenolic constituents toward both lipid and protein oxidation. In bilberries and black currants, anthocyanins contributed the most to the antioxidant effect by inhibiting the formation of both hexanal and protein carbonyls. In raspberries, ellagitannins were responsible for the antioxidant activity. While the antioxidant effect of berry proanthocyanidins and anthocyanins was dose-dependent, ellagitannins appeared to be equally active at all concentrations. In conclusion, berries are rich in monomeric and polymeric phenolic compounds providing protection toward both lipid and protein oxidation.

Antioxidants↗

Cutin composition of five finnish berries.

The raw cutin (i.e., extractive-free isolated cuticular membrane) fraction from Finnish berries, sea buckthorn (Hippophaë rhamnoides), black currant (Ribes nigrum), cranberry (Vaccinium oxycoccos), lingonberry (Vaccinium vitis-idaea), and bilberry (Vaccinium myrtillus), was depolymerized by NaOMe-catalyzed methanolysis. The composition of cutin monomers was determined by GC-(EI)MS analysis either as methyl esters or as TMSi esters, with OH groups derivatized to TMSi ethers. There was a notable difference in the degree of depolymerization, ranging from 6 to 47%. The extractive-free berry cuticle, that is, raw cutin, thus contains <50% polyester polymer cutin. The predominant cutin monomers were C(16) and C(18) omega-hydroxy acids with midchain functionalities, mainly epoxy and hydroxyl groups. Typically, the major compounds were 9,10-epoxy-18-hydroxyoctadecanoic acid, 10,16-dihydroxyhexadecanoic acid, 9,10,18-trihydroxyoctadecanoic acid, 9,10-epoxy-18-hydroxyoctadec-12-enoic acid, and 18-hydroxyoctadec-9-enoic acid. The amount of epoxyacids was rather high in sea buckthorn ( approximately 70%) and cranberry ( approximately 60%), compared with the other berries. The black currant cutin differed from that of the other berries with a significant portion of hydroxyoxohexadecanoic acid ( approximately 12% of total monomers). This investigation of the cuticular hydroxy acids of five Finnish berries is part of the exploitation of the northern natural resources related to the chemical composition, nutritional value, and sensory properties.

Finland↗

High molecular weight polysaccharides from black currant seeds inhibit adhesion of Helicobacter pylori to human gastric mucosa.

Several crude and purified polysaccharides from black currant seeds (Ribes nigrum L.) have been isolated, analysed and examined on their effects against Helicobacter pylori in in situ adhesion studies on sections of human gastric mucosa. After pre-treatment of Helicobacter pylori with 0.01 to 0.1 % solutions of the isolated raw polysaccharide (RPS), the epithelial binding of the bacteria was considerably reduced in a concentration-dependent manner, as compared with a non-treated control suspension. Preincubation of the mucosal sections with 0.1 % solutions did not result in a reduced binding of non-treated bacteria. An anion-exchange fraction of RPS eluted with 0.1 M phosphate buffer exhibited a comparable, concentration-dependent reduction of adhesion, whereas the water-eluted fraction was ineffective at the respective concentrations. Both subfractions consisted of similar 1,3-linked galactans, decorated with side chains possessing 1,4-galacturonic acid, galactose and arabinose residues. Molecular weight profiling by GPC revealed that the antiadhesive activity of the buffer eluate correlated with high molecular weight components ranging from about 1000 Da to 340 kDa, whereas the ones of the inactive water eluate had molecular weights of about 100 and 25 kDa, respectively. None of the active fractions revealed inhibitory effects on bacterial growth in vitro. We conclude that acidic, high molecular weight galactans are responsible for the antiadhesive qualities of black currant seed extracts and that these polymers are able to block Helicobacter surface receptors, thus inhibiting their interaction with specific binding factors located on human gastric epithelia.

Bacterial Adhesion↗

HPLC determination of the composition and stability of blackcurrant anthocyanins.

High-performance liquid chromatography with UV and mass spectrometry detectors are used to monitor the composition and stability of anthocyanins in blackcurrants harvested from different Ribes nigrum breeds at various ripeness phases. The highest amounts of pigments are found in overripe berries. The concentration of anthocyanins is higher in the berries of late blackcurrant breeds (Vakariai and Ben Alder). Delphinidin- 3-rutinoside is the dominant component in the reddish color berries (onset of ripening), and cyanidin-3-rutinoside is a major pigment in the black ones (ripe berries). Studies of the effect of temperature and light on the stability of the main pigments in blackcurrants show that aqueous solution prepared from a dry colorant is more stable when compared with the liquid water and ethanol extracts of coloring substances. Cyanidin-3-rutinoside is found to be the most thermally stable anthocyanin.

Anthocyanins↗

Fruit plant germplasm characterisation using molecular markers generated in RAPD and ISSR-PCR.

The genotypes of the strawberry (Fragaria x ananassa), apple (Malus domestica) and Ribes species (R. nigrum, R. rubrum and R. glossularia), maintained in our Institute's collection and used in breeding programs, were screened for DNA markers. Twenty primers for RAPD (among 60 tested) and seven for ISSR (among 10 tested) were chosen as creating polymorphic DNA bands differentiating the investigated genotypes. Based on those identity markers, the genetic distance between genotypes was determined, and their relatedness was estimated. In many cases, both RAPD- and ISSR-based genetic similarity confirmed relatedness connected with biological origin and with the place where the cultivar was developed. However, some diversity connected with the technique used for molecular marker generation was observed. Generally, the similarity values based on ISSR data were higher than those based on RAPD. Parallel study using two data sets seems to enable a reduction in the number of potential mistakes connected with each method's, technical limitations and ensures more precise relatedness determination.

DNA Fingerprinting↗

Thermal analysis of the plant encapsulation-dehydration cryopreservation protocol using silica gel as the desiccant.

The encapsulation-dehydration cryopreservation protocol is critically dependent upon the evaporative desiccation step, which must optimise survival with the retention of glass stability on sample cooling and rewarming. Desiccation is usually achieved evaporatively by drying in a sterile airflow. However, chemical desiccation using silica gel has advantages for laboratories that do not have environmental control and/or which are exposed to high relative humidities and risks of microbial contamination. This study characterised thermal profiles of silica gel-desiccated encapsulated shoot-tips of two Ribes species using Differential Scanning Calorimetry. For both species silica gel-desiccation at 16 degrees C for 5 h decreased bead water content from ca. 75 to 28% fresh weight (3.8 to 0.4 g x g(-1) dry weight); further desiccation (for 6 and 7 h) reduced the bead water content to 21% (0.3 g x g(-1) dry weight). These changes in water status altered the thermal properties of beads for both species. After 7 h desiccation over silica gel stable glass transitions were observed on both cooling and rewarming of beads containing meristems. Tg mid-point temperatures ranged from -78 to -51 degrees C (cooling) and from -88 to -54 degrees C (warming) [at cooling and warming rates of 10 and 5 degrees C min(-1), respectively] after 5 to 7 h silica gel-desiccation. Post-cryopreservation viability of both species was ca. 63%. Thermal analysis studies revealed that an encapsulation/dehydration protocol using silica gel as a desiccant should comprise a minimum 5 h drying (at 16 degrees C). This reduces bead moisture content to a critical point (ca. 0.4 g x g(-1) dry weight) at which stable glasses are formed on cooling and rewarming. It is concluded that silica gel has advantages for use as a desiccant for alginate-encapsulated plant meristems by promoting stable vitrification and is useful in laboratories and/or geographical locations where environmental conditions are not under stringent control.

Cell Survival↗

Antimicrobial evaluation of some medicinal plants for their anti-enteric potential against multi-drug resistant Salmonella typhi.

Screening was done of some plants of importance in the Ayurvedic system of traditional medicine used in India to treat enteric diseases. Fifty four plant extracts (methanol and aqueous) were assayed for their activity against multi-drug resistant Salmonella typhi. Strong antibacterial activity was shown by the methanol extracts of Aegle marmelos, Salmalia malabarica, Punica granatum, Myristica fragrans, Holarrhena antidysenterica, Terminalia arjuna and Triphal (mixture of Emblica of fi cinalis, Terminalia chebula and Terminalia belerica). Moderate antimicrobial activity was shown by Picorhiza kurroa, Acacia catechu, Acacia nilotica, Cichorium intybus, Embelia ribes, Solanum nigrum, Carum copticum, Apium graveolens, Ocimum sanctum, Peucedanum graveolens and Butea monosperma.

Anti-Bacterial Agents↗

Separation of gamma- and alpha-linolenic acid containing triacylglycerols by capillary supercritical fluid chromatography.

The separation of gamma- and alpha-linolenic acid containing triacylglycerols with an identical acyl carbon number and degree of unsaturation was obtained on capillary supercritical fluid chromatography using a 25% cyanopropyl-75% methylpolysiloxane stationary phase. The resolution of 1,3-dioleoyl-2-alpha-linolenoyl-sn-glycerol was 1.35 on a 10 m x 50 microns i.d. column, whereas the resolution was enhanced to 1.66 by combining two 10-meter columns in series. The difference in the position of double bonds in one linolenic acid residue of triacylglycerols resulted in two series of peaks in the separation of alpine currant (Ribes alpinum) and black currant (R. nigrum) seed oils. The use of the 10-meter column was found to be appropriate for the screening of the triacylglycerol profile in both seed oils studied.

Capillary Action↗

Current status of fertility control methods in India.

Approximately 48.2% of couples of 15 to 49 years of age practice family planning methods in India. Female sterilization accounts for 34.2%, with male sterilization declining from 3.4% in 1992-93 to 1.9% in 1998-99. Use of the condom increased to 3.1% from 2.4%. There is an urgent need for research to develop new contraceptive modalities especially for men and also for women and to make existing methods more safe, affordable and acceptable. Current efforts in India to develop a male contraceptive are mainly directed towards (i) development of antispermatogenic agents to suppress sperm production, (ii) prevention of sperm maturation, (iii) prevention of sperm transport through vas deferens or rendering these sperm infertile and (iv) prevention of sperm deposition. Research work in the field of prevention of sperm transport through vas deferens has made significant advances. Styrene maleic anhydride (SMA) disturbed the electrical charge of spermatozoa leading to acrosome rupture and consequent loss in fertilizing ability of sperm. A multicentre phase-III clinical trial using SMA is continuing and it is hoped that the SMA approach would be available in the near future as an indigenously developed injectable intra-vasal male contraceptive. The safety and efficacy of available oral contraceptives were evaluated. An indigenously developed oral contraceptive 'Centchorman', which is a nonsteroidal, weakly estrogenic but potently antiestrogenic, was found to be safe and effective and is now being marketed in India since 1991 as a 'once a week' pill. Cyclofem and Mesigyna have been recommended as injectable contraceptives with proper counselling and service delivery by Indian studies. It has been recommended that these injectable contraceptives be added to the existing range of contraceptive methods available in the National Family Planning Programme. Based on the Indian studies CuT 200 was also recommended. Studies have indicated the advantage of intrauterine devices (IUD); they are long acting, relatively easily removed and fertility returns rapidly after their removal. Recent studies have recommended CuT 200 for use up to 5 years. The combination of some plant products i.e. Embelia ribes, Borax and Piper longum has been found to be safe and effective as a female contraceptive and the results of phase-I clinical trials are encouraging. Research work is going on in the country in various areas with special reference to hormonal contraceptive - a three monthly injectable contraceptive, immuno-contraceptives, antiprogestins, etc.

Animals↗

Capillary supercritical fluid chromatography--atmospheric pressure chemical ionization mass spectrometry of gamma- and alpha-linolenic acid containing triacylglycerols in berry oils.

The effect of the gamma-linolenic acid (18:3n-6) residue on the elution of triacylglycerols on a 25% cyanopropyl-25% phenyl-50% methylpolysiloxane stationary phase was confirmed by using capillary supercritical fluid chromatography-atmospheric pressure chemical ionization mass spectrometry [cSFC-(APCI)MS]. The general elution rule on this stationary phase is that triacylglycerols having the same ACN + 2n value coeluted (ACN = acyl carbon number and n = combined number of double bonds in the acyl chains). The different effect of gamma- and alpha-linolenic acid residues on the retention of triacylglycerols and the use of cSFC-(APCI)MS allowed the study of the number of different linolenic acid residue isomer combinations in triacylglycerols with an identical ACN and degree of unsaturation. Stearidonic acid (18:4n=3) residue was found to have a similar effect on the retention behavior of triacylglycerols as that of gamma-linolenic acid residue. The abundance of the [M - RCOO]+ ion, formed by the loss of one fatty acid moiety of a triacylglycerol, was found to be clearly higher in the case of gamma-isomer of the linolenic acid than that of alpha-isomer in the identical regiospecific position. This indicates that the distance of the double bonds from the glycerol backbone in the acyl chain affects the stability of a triacylglycerol molecule in the (APCI)MS system. The triacylglycerol composition and the fatty acid combinations of triacylglycerols were found to be almost identical in black currant (Ribes nigrum) and alpine currant (R. alpinum) seed oils.

Chromatography, Liquid↗

Antispermatogenic effect of embelin, a plant benzoquinone, on male albino rats in vivo and in vitro.

Embelin, the active principle of the seeds of Embelia ribes Burm, has been isolated and the purity established. Daily subcutaneous administration of the compound at a dose of 20 mg/kg body weight to male albino rats for 15 or 30 days revealed an inhibition of: a) epididymal motile sperm count, b) fertility parameters such as pregnancy attainment and litter size, and c) the activities of the enzymes of glycolysis and energy metabolism. These changes were reversible, as seen after 15 and 30 days of recovery. Addition of embelin to epididymal sperm suspensions caused a dose- and duration-dependent inhibition of spermatozoal motility and the activities of the enzymes of carbohydrate metabolism. Light and scanning electron microscopy showed that both in vivo and in vitro treatment with the drug causes profound morphological changes in spermatozoa such as: a) decapitation of the spermatozoal head, b) discontinuity of the outer membranous sheath in the mid-piece and the tail region, and c) alteration in the shape of the cytoplasmic droplet in the tail.

Animals↗

Isoelectric protein purification in segmented immobilized pH gradients. Effect of salts on rate of contaminants' removal.

A method is described for keeping a constant salt background during protein purification in a segmented immobilized pH gradient. It is based on an external hydraulic flow replenishing the salt loss due to combined electric and diffusional mass transport (similar to the concept of Ribes' steady-state rheoelectrolysis). Such a minimum of ionic strength might be needed for proteins which tend to precipitate and aggregate at or in vicinity of the isoelectric point. However, it is found that any salt level in the sample feed (already at 1 mM concentration) deteriorates transport of non-isoelectric proteins, because of the much larger current fraction carried by the ions themselves as opposed to proteins. In addition, high salt levels in the sample reservoir might form cathodic and anodic ion boundaries, alkaline and acidic, respectively, which might hamper protein migration and even induce denaturation. Thus, when high salt backgrounds are needed in the sample feed, external pH control should be exerted, e.g. with a pH-stat. Three parameters influence protein transport in the segmented IPG chamber: (a) cross-sectional area of the Immobiline membranes; (b) delta pI between the isoelectric protein and the contaminants and (c) salt molarity in the sample reservoir. The first 2 show a positive, the last a negative correlation.

Electric Conductivity↗

Non-narcotic orally effective, centrally acting analgesic from an Ayurvedic drug.

Embelin, a p-quinone, is derived from Embelia ribes Burm. The analgesic effect of potassium embelate has been studied in rats and mice. The test drug was found to be effective by oral, i.m. and i.c.v. routes and the results compared well with morphine. Although potassium embelate acts centrally to produce analgesia, its effect is not antagonized by naloxone indicating a different central site of action. There is no precipitation of abstinence syndrome as observed with morphine. Peripheral site of action of the drug is ruled out as it lacks any demonstrable anti-inflammatory action. It can be concluded that high oral efficacy and non-narcotic properties of the test drug make it more acceptable than morphine. In addition, lack of any adverse effects, high therapeutic index and absence of abstinence syndrome confers a long term safety on potassium embelate for use as an analgesic.

Acetates↗

Application of sample disruption methods in the extraction of anthocyanins from solid or semi-solid vegetable samples.

Cassis, a commercial powder obtained from blackcurrant juice (Ribes nigrum L.), was used to optimize the anthocyanin extraction by matrix solid-phase dispersion (MSPD) and by sea sand disruption method (SSDM). The optimum eluent conditions were 5.0 mL of MeOH/H2O (1:1, v/v) at pH 2. The extracts were analyzed by HPLC with diode-array detection (DAD) and the extraction efficiency was evaluated by statistical comparison (analysis of variance, ANOVA-single factor) of the compounds peak areas. The extraction efficiency for the MSPD procedure was dependent on the chemical nature of the C18 material but the optimized procedure yielded results similar to those obtained by the inexpensive sea sand disruption method (SSDM). This procedure also compared favorably to a previously published liquid-liquid extraction method. The optimized sea sand method was then used in the anthocyanin extraction from red grapes (Vitis vinifera L.) and strawberries (Fragaria ananassa D.) yielding chromatographic profiles similar to those reported in the literature for these products.

Anthocyanins↗

Factors influencing audiovisual fission and fusion illusions.

Information processing in auditory and visual modalities interacts in many circumstances. Spatially and temporally coincident acoustic and visual information are often bound together to form multisensory percepts [B.E. Stein, M.A. Meredith, The Merging of the Senses, A Bradford Book, Cambridge, MA, (1993), 211 pp.; Psychol. Bull. 88 (1980) 638]. Shams et al. recently reported a multisensory fission illusion where a single flash is perceived as two flashes when two rapid tone beeps are presented concurrently [Nature 408 (2000) 788; Cogn. Brain Res. 14 (2002) 147]. The absence of a fusion illusion, where two flashes would fuse to one when accompanied by one beep, indicated a perceptual rather than cognitive nature of the illusion. Here we report both fusion and fission illusions using stimuli very similar to those used by Shams et al. By instructing subjects to count beeps rather than flashes and decreasing the sound intensity to near threshold, we also created a corresponding visually induced auditory illusion. We discuss our results in light of four hypotheses of multisensory integration, each advocating a condition for modality dominance. According to the discontinuity hypothesis [Cogn. Brain Res. 14 (2002) 147], the modality in which stimulation is discontinuous dominates. The modality appropriateness hypothesis [Psychol. Bull. 88 (1980) 638] states that the modality more appropriate for the task at hand dominates. The information reliability hypothesis [J.-L. Schwartz, J. Robert-Ribes, P. Escudier, Ten years after Summerfield: a taxonomy of models for audio-visual fusion in speech perception. In: R. Campbell (Ed.), Hearing by Eye: The Psychology of Lipreading, Lawrence Earlbaum Associates, Hove, UK, (1998), pp. 3-51] claims that the modality providing more reliable information dominates. In strong forms, none of these three hypotheses applies to our data. We re-state the hypotheses in weak forms so that discontinuity, modality appropriateness and information reliability are factors which increase a modality's tendency to dominate. All these factors are important in explaining our data. Finally, we interpret the effect of instructions in light of the directed attention hypothesis which states that the attended modality is dominant [Psychol. Bull. 88 (1980) 638].

Acoustic Stimulation↗

Chemopreventive effects of embelin and curcumin against N-nitrosodiethylamine/phenobarbital-induced hepatocarcinogenesis in Wistar rats.

The effects of embelin (50 mg/kg/day), a benzoquinone derivative of Embelia ribes, and the effects of curcumin (100 mg/kg/day), the active principle of Curcuma longa, against N-nitrosodiethylamine (DENA)-initiated and phenobarbital (PB)-promoted hepatocarcinogenesis were studied in Wistar rats. They were able to prevent the induction of hepatic hyper plastic nodules, body weight loss, increase in the levels of hepatic diagnostic markers, and hypoproteinemia induced by DENA/PB treatment. Hence, results of our study suggest the possible chemopreventive effects of embelin (EMB) and curcumin (CUR) against DENA/PB-induced hepatocarcinogenesis in Wistar rats.

Animals↗

Proteomic characterization of acidic aqueous extracts from Vicia faba L. pod valves identifies chitinase as a major co-extracted protein macromolecule.

Naturally acidic aqueous extracts from Vicia faba L. pod valves are being explored as sustainable, L-DOPA-oriented plant preparations. Pod valves represent an underutilized processing by-product reported to contain L-DOPA, a compound widely used in Parkinson's disease therapy, while acidic aqueous media may help preserve its physicochemical stability. However, the protein macromolecules co-extracted from V. faba pod valves under these conditions remain poorly characterized. This information is relevant because persistent plant proteins may influence extract composition, stability, susceptibility to degradation, and downstream processing requirements. Here, we characterized co-extracted V. faba protein macromolecules in aqueous pod-valve extracts prepared in ultrapure water or naturally acidic media, including 2% Phyllanthus emblica, 5% Punica granatum, and 2% Ribes rubrum. Protein profiles were first evaluated by SDS-PAGE and subsequently analyzed by nanoflow liquid chromatography coupled to high-resolution tandem mass spectrometry (nLC-MS/MS). Protein identifications were complemented with Gene Ontology annotation and a descriptive semi-quantitative assessment of relative protein representation across extraction media. Chitinase was the most represented V. faba-assigned protein macromolecule across the extracts, with additional highly represented proteins including glucan endo-1,3-beta-D-glucosidase, pathogenesis-related proteins, and polyphenol oxidase A1. These co-extracted proteins are mainly associated with plant defense, stress responses, cell-wall remodeling, and oxidative processing, suggesting that they may be relevant for extract quality attributes during handling and storage. This study provides a compositional proteomic reference for the co-extracted protein macromolecules present in acidic aqueous extracts from V. faba pod valves, supporting future studies on extract stability, processing optimization, and the development of standardized plant-based preparations.

Vicia faba↗

HPLC in reversed phase mode: Tool for investigation of kinetics of blackcurrant seed oil lipolysis in supercritical carbon dioxide.

Blackcurrant (Ribes nigrum) seed oil is rich in alpha- and gamma-linolenic acids, the latter in particular being of potential use in medicine. The enzymatic hydrolysis of the oil was carried out in supercritical carbon dioxide using lipase Lipozyme as catalyst and changes in the composition of acylglycerols were recorded. Mono-, di-, and triacylglycerols and free fatty acids were separated by non-aqueous high-performance liquid chromatography in reversed phase mode and detected by UV diode array and 1H NMR detectors. Lipozyme was found to exert low specificity to individual fatty acids in the hydrolysed oil.

Carbon Dioxide↗