The effect of enzyme treatment on polyphenol and cell wall polysaccharide extraction from the grape berry and subsequent sensory attributes in Cabernet Sauvignon wines.

Pectolytic enzyme maceration is common for producing red wines, but the effects on bitterness and astringency are not well understood. Glycan microarrays assessed polysaccharide diversity and with polyphenol analysis was correlated with sensory data on descriptors of astringency and their perceived levels in enzyme-crafted Cabernet Sauvignon wines. Enzyme use is shown to have no effect on bitterness, but enzyme-macerated wines are more astringent. The data suggests that pectolytic enzymes are much more pronounced in their effect on the cell wall matrix than the ripeness of the berries at harvest and subsequent sensory perception. Enzyme-macerated red wines showed higher levels of polyphenol which were more polymerized and galloylated. The polyphenol-rich wines were described as hard, chalky, grippy, grainy and dry. The non-enzyme wines had elevated levels of arabinogalactan protein and pectin epitopes (notably biomarker mAbs JIM8 and JIM13) with the wines being characterized as soft, fine and velvety.

The phenolic profile extracted from the desiccation-tolerant medicinal shrub Myrothamnus flabellifolia using Natural Deep Eutectic Solvents varies according to the solvation conditions.

Natural Deep Eutectic Solvents (NaDES) have been proposed as designer solvents for the green extraction of bioactive products from plants. Myrothamnus flabellifolia is a desiccation-tolerant medicinal shrub that has been widely studied for its phenolic properties; however, a NaDES-based approach for the extraction of phenolics has not been tested in this species. Our aim was thus to evaluate the extraction of phenolics from M. flabellifolia using four different NaDES with differing acidities using a non-targeted liquid chromatography-quantitative time-of-flight-tandem mass spectrometry (LC-QTOF-MS/MS) metabolomics approach. Anthocyanin pigments were quantified using targeted high-performance LC. Leaf material from M. flabellifolia was extracted in four different NaDES solutions (sucrose-fructose-glucose; proline-malic acid; sucrose-citric acid; and glucose-choline chloride), and the results were subjected to multivariate statistical analysis to evaluate the phenolic profiles of the different NaDES extracts. The NaDES were effective at extracting phenolic compounds from M. flabellifolia and also exhibited specificity in the suites of phenolics that they extracted, as indicated by principal component analysis. Using partial least squares-discriminant analysis, we were able to identify the phenolics that were most differentially abundant between the extracts, and a heatmap provided an indication of the types of phenolics that were extracted by the different NaDES. Furthermore, the NaDES also extracted several compounds not previously detected in M. flabellifolia using conventional organic solvents, demonstrating their use in compound discovery. The NaDES also differentially targeted anthocyanins, with the more acidic NaDES extracting higher quantities of anthocyanins and polymeric pigments. A green chemistry-based extraction technique using NaDES can thus effectively target phenolics in M. flabellifolia and offers a promising solution for future phytochemical investigations in medicinal plants using a highly efficient non-toxic solvent system that can be tailored to target particular compounds.

Metabolomic Profiling of the Desiccation-Tolerant Medicinal Shrub Myrothamnus flabellifolia Indicates Phenolic Variability Across Its Natural Habitat: Implications for Tea and Cosmetics Production.

The leaves and twigs of the desiccation-tolerant medicinal shrub Myrothamnus flabellifolia are harvested for use in traditional and commercial teas and cosmetics due to their phenolic properties. The antioxidant and pharmacological value of this plant has been widely confirmed; however, previous studies typically based their findings on material collected from a single region. The existence of phenolic variability between plants from different geographical regions experiencing different rainfall regimes has thus not been sufficiently evaluated. Furthermore, the anthocyanins present in this plant have not been assessed. The present study thus used an untargeted liquid chromatography-tandem-mass spectrometry approach to profile phenolics in M. flabellifolia material collected from three climatically distinct (high, moderate, and low rainfall) regions representing the western, southern, and eastern extent of the species range in southern Africa. Forty-one putative phenolic compounds, primarily flavonoids, were detected, nine of which are anthocyanins. Several of these compounds are previously unknown from M. flabellifolia. Using multivariate statistics, samples from different regions could be distinguished by their phenolic profiles, supporting the existence of regional phenolic variability. This study indicates that significant phenolic variability exists across the range of M. flabellifolia, which should inform both commercial and traditional cultivation and harvesting strategies.

Profiling the Hydrolysis of Isolated Grape Berry Skin Cell Walls by Purified Enzymes.

The unraveling of crushed grapes by maceration enzymes during winemaking is difficult to study because of the complex and rather undefined nature of both the substrate and the enzyme preparations. In this study we simplified both the substrate, by using isolated grape skin cell walls, and the enzyme preparations, by using purified enzymes in buffered conditions, to carefully follow the impact of the individual and combined enzymes on the grape skin cell walls. By using cell wall profiling techniques we could monitor the compositional changes in the grape cell wall polymers due to enzyme activity. Extensive enzymatic hydrolysis, achieved with a preparation of pectinases or pectinases combined with cellulase or hemicellulase enzymes, completely removed or drastically reduced levels of pectin polymers, whereas less extensive hydrolysis only opened up the cell wall structure and allowed extraction of polymers from within the cell wall layers. Synergistic enzyme activity was detectable as well as indications of specific cell wall polymer associations.

Following the compositional changes of fresh grape skin cell walls during the fermentation process in the presence and absence of maceration enzymes.

Cell wall profiling technologies were used to follow compositional changes that occurred in the skins of grape berries (from two different ripeness levels) during fermentation and enzyme maceration. Multivariate data analysis showed that the fermentation process yielded cell walls enriched in hemicellulose components because pectin was solubilized (and removed) with a reduction as well as exposure of cell wall proteins usually embedded within the cell wall structure. The addition of enzymes caused even more depectination, and the enzymes unravelled the cell walls enabling better access to, and extraction of, all cell wall polymers. Overripe grapes had cell walls that were extensively hydrolyzed and depolymerized, probably by natural grape-tissue-ripening enzymes, and this enhanced the impact that the maceration enzymes had on the cell wall monosaccharide profile. The combination of the techniques that were used is an effective direct measurement of the hydrolysis actions of maceration enzymes on the cell walls of grape berry skin.

Pectic-ß(1,4)-galactan, extensin and arabinogalactan-protein epitopes differentiate ripening stages in wine and table grape cell walls.

BACKGROUND AND AIMS: Cell wall changes in ripening grapes (Vitis vinifera) have been shown to involve re-modelling of pectin, xyloglucan and cellulose networks. Newer experimental techniques, such as molecular probes specific for cell wall epitopes, have yet to be extensively used in grape studies. Limited general information is available on the cell wall properties that contribute to texture differences between wine and table grapes. This study evaluates whether profiling tools can detect cell wall changes in ripening grapes from commercial vineyards. METHODS: Standard sugar analysis and infra-red spectroscopy were used to examine the ripening stages (green, véraison and ripe) in grapes collected from Cabernet Sauvignon and Crimson Seedless vineyards. Comprehensive microarray polymer profiling (CoMPP) analysis was performed on cyclohexanediaminetetraacetic acid (CDTA) and NaOH extracts of alcohol-insoluble residue sourced from each stage using sets of cell wall probes (mAbs and CBMs), and the datasets were analysed using multivariate software. KEY RESULTS: The datasets obtained confirmed previous studies on cell wall changes known to occur during grape ripening. Probes for homogalacturonan (e.g. LM19) were enriched in the CDTA fractions of Crimson Seedless relative to Cabernet Sauvignon grapes. Probes for pectic-ß-(1,4)-galactan (mAb LM5), extensin (mAb LM1) and arabinogalactan proteins (AGPs, mAb LM2) were strongly correlated with ripening. From green stage to véraison, a progressive reduction in pectic-ß-(1,4)-galactan epitopes, present in both pectin-rich (CDTA) and hemicellulose-rich (NaOH) polymers, was observed. Ripening changes in AGP and extensin epitope abundance also were found during and after véraison. CONCLUSIONS: Combinations of cell wall probes are able to define distinct ripening phases in grapes. Pectic-ß-(1,4)-galactan epitopes decreased in abundance from green stage to véraison berries. From véraison there was an increase in abundance of significant extensin and AGP epitopes, which correlates with cell expansion events. This study provides new ripening biomarkers and changes that can be placed in the context of grape berry development.

Profiling the main cell wall polysaccharides of grapevine leaves using high-throughput and fractionation methods.

Vitis species include Vitis vinifera, the domesticated grapevine, used for wine and grape agricultural production and considered the world's most important fruit crop. A cell wall preparation, isolated from fully expanded photosynthetically active leaves, was fractionated via chemical and enzymatic reagents; and the various extracts obtained were assayed using high-throughput cell wall profiling tools according to a previously optimized and validated workflow. The bulk of the homogalacturonan-rich pectin present was efficiently extracted using CDTA treatment, whereas over half of the grapevine leaf cell wall consisted of vascular veins, comprised of xylans and cellulose. The main hemicellulose component was found to be xyloglucan and an enzymatic oligosaccharide fingerprinting approach was used to analyze the grapevine leaf xyloglucan fraction. When Paenibacillus sp. xyloglucanase was applied the main subunits released were XXFG and XLFG; whereas the less-specific Trichoderma reesei EGII was also able to release the XXXG motif as well as other oligomers likely of mannan and xylan origin. This latter enzyme would thus be useful to screen for xyloglucan, xylan and mannan-linked cell wall alterations in laboratory and field grapevine populations. This methodology is well-suited for high-throughput cell wall profiling of grapevine mutant and transgenic plants for investigating the range of biological processes, specifically plant disease studies and plant-pathogen interactions, where the cell wall plays a crucial role.

Animal models for microbicide studies.

There have been encouraging recent successes in the development of safe and effective topical microbicides to prevent vaginal or rectal HIV-1 transmission, based on the use of anti-retroviral drugs. However, much work remains to be accomplished before a microbicide becomes a standard element of prevention science strategies. Animal models should continue to play an important role in pre-clinical testing, with emphasis on safety, pharmacokinetic and efficacy testing.

Inhibition of HIV-1 and M-MLV reverse transcriptases by a major polyphenol (3,4,5 tri-O-galloylquinic acid) present in the leaves of the South African resurrection plant, Myrothamnus flabellifolia.

A polyphenol-rich extract of the medicinal resurrection plant Myrothamnus flabellifolia was shown to inhibit viral (M-MLV and HIV-1) reverse transcriptases. Fractionation and purification of this extract yielded the major polyphenol, 3,4,5 tri-O-galloylquinic acid, as the main active compound. A sensitive, ethidium bromide based fluorescent assay, was developed and used to monitor the kinetics of M-MLV and HIV-1 reverse transcriptases in the presence and absence of 3,4,5 tri-O-galloylquinic acid. Kinetic monitoring of these enzymes in the presence of 3,4,5 tri-O-galloylquinic acid revealed non-competitive inhibition with IC(50) values of 5 µM and 34 µM for the M-MLV and HIV-1 enzymes, respectively. We propose that 3,4,5 tri-O-galloylquinic acid and related polymers have potential as indigenous drugs for anti-viral therapy.

Whither or wither microbicides?

After disappointing results from all efficacy trials conducted to date, the field of microbicides research now faces substantial challenges. Poor coordination among interested parties and the choice of nonvalidated scientific targets for phase III studies have hampered progress and created mistrust about the use of microbicides as a method to prevent HIV-1 sexual transmission. Although new promising strategies are available, there will need to be serious reappraisals of how decisions are made to advance the next generations of candidates into clinical trials, and the use of appropriate animal models in this process will be critical.

Entry inhibitor-based microbicides are active in vitro against HIV-1 isolates from multiple genetic subtypes.

Inhibitors of viral entry are under consideration as topical microbicides to prevent HIV-1 sexual transmission. Small molecules targeting HIV-1 gp120 (BMS-378806) or CCR5 (CMPD167), and a peptide fusion inhibitor (C52L), each blocks vaginal infection of macaques by a SHIV. A microbicide, however, must be active against multiple HIV-1 variants. We therefore tested BMS-C (a BMS-378806 derivative), CMPD167, C52L and the CXCR4 ligand AMD3465, alone and in combination, against 25 primary R5, 12 X4 and 7 R5X4 isolates from subtypes A-G. At high concentrations (0.1-1 microM), the replication of most R5 isolates in human donor lymphocytes was inhibited by >90%. At lower concentrations, double and triple combinations were more effective than individual inhibitors. Similar results were obtained with X4 viruses when AMD3465 was substituted for CMPD167. The R5X4 viruses were inhibited by combining AMD3465 with CMPD167, or by the coreceptor-independent compounds. Thus, combining entry inhibitors may improve microbicide effectiveness.

An overview of the biology of the desiccation-tolerant resurrection plant Myrothamnus flabellifolia.

BACKGROUND: Myrothamnus flabellifolia is unique as the only woody resurrection plant. It is an important plant in southern Africa because of its widespread occurrence and usage in African medicine and traditional culture. Many reports have investigated facets of its biology and the mechanisms associated with its desiccation tolerance. SCOPE: The general biology of the woody resurrection plant Myrothamnus flabellifolia is reviewed. The review focuses on the geography and ecology, systematic placement, evolution, morphology and reproductive ecology of M. flabellifolia as well as the wood anatomy and re-filling mechanism. In addition, the desiccation tolerance, ethnobotanical importance and medicinal properties of the plant are reviewed. Also, future research avenues are suggested, in particular the necessity to research the biogeography and systematics of the species and the role of the polyphenols present, as well as the molecular basis of the plant's desiccation tolerance.

Effect of a CCR5 inhibitor on viral loads in macaques dual-infected with R5 and X4 primate immunodeficiency viruses.

Human immunodeficiency virus type 1 (HIV-1) fusion with its target cells is initiated by sequential interactions between its envelope glycoprotein, CD4, and a co-receptor, usually CCR5 or CXCR4. Small molecules that bind to CCR5 and prevent its use by R5 HIV-1 strains are now being developed clinically as antiviral drugs. To test whether a block to CCR5 promotes the replication of viruses that enter cells via CXCR4 and are associated with accelerated disease progression, we administered a small molecule CCR5 inhibitor, CMPD 167, to three macaques dual-infected with both R5 (SIVmac251) and X4 (SHIV-89.6P) viruses. CMPD 167 caused a rapid and substantial (on average, 50-fold) suppression of R5 virus replication in each animal. In two of the animals, but not in the third, a rapid, transient, 8- to 15-fold increase in the amount of plasma X4 virus occurred. In neither animal was the increase in X4 viral load sustained throughout therapy, however. These observations may have relevance for the development of CCR5 inhibitors for treatment of HIV-1 infection of humans.