Use of grapevine cell cultures for the production of phytostilbenes of cosmetic interest

Plant cell cultures constitute pesticide-free sources for obtaining plant secondary metabolites or plant extracts. Additionally, they do not contain any fungal contaminants, mycotoxins or heavy metals providing to the consumer potential health benefits and justifying the development of this technology at an industrial scale. Significant production levels of these secondary metabolites can be obtained through the use of elicitors, which activate plant defense mechanisms. Resveratrol, a well-known grapevine polyphenolic compound which possesses potent antioxidant and antiaging activities as well as a protective action on skin, is a good example of such plant secondary metabolites. Resveratrol and its oligomeric derivatives are used by several companies of cosmetic products but their extraction from vine stems and similar vegetal sources remains difficult. Therefore grapevine cell suspensions could represent interesting systems for the large-scale bioproduction of those compounds. Here we present an update of the methods used for the production of phytostilbenes by using grapevine cell cultures and the results obtained.     

The effect of methyl jasmonate and light irradiation treatments on the stilbenoid biosynthetic pathway in Vitis vinifera cell suspension cultures

Grape stilbenes are a well-known family of plant polyphenolics that have been confirmed to have many biological activities in relation to health benefits. In the present study, we investigated the effect of methyl jasmonate (MeJA) elicitor at four different concentrations (25, 50, 100 and 200 μM) in combination or not with high-level light irradiation (10,000 LUX) on a cell line obtained from the pulp of Vitis vinifera cv. Shahani. Our results showed that the stilbene synthesis pathway is inhibited by high-light conditions. A concentration of 50 μM MeJA was optimum for efficient production and high accumulation of total phenolics and total flavonoids as well as total stilbenoids. Furthermore, we showed that there is a significant negative correlation between the production of these metabolites and cell growth. These data provide valuable information for the future scale-up of cell cultures for the production of these very high value compounds in bioreactor system.     

Plant cell culture technologies: A promising alternatives to produce high-value secondary metabolites

Plants have been used for its medicinal values since ancient time. The medicinal properties of plants are based on their phytochemical constituent particularly secondary metabolites which are produced in low amounts by plants. Secondary metabolites have been used as medicines, flavors, colors, and fragrances. In recent time, these natural compounds are gaining enormous attention in pharmaceutical, cosmetics, and nutraceutical industries and are regarded economically valuable products. The production of plant secondary metabolites in plant is largely dependent on the plant species, environmental factors and geographical regions. In addition, the main challenges in their mass production is reported to be the quality and quantity issues during their synthesis. Therefore, enthusiasm has grown for increasing the production of secondary metabolites by employing in vitro plant cell culture technology and bioengineering methods. Such technological advancement, has led to production of a huge number of medicinal herbs and high-value secondary metabolites that are mostly used in pharmaceuticals, cosmetics and nutraceuticals industries. The current mini-review article focuses on applications of plant cell culture system for the production secondary metabolites and recent techniques used to improve metabolite contents. Furthermore, our review emphasizes safety issues of plant cell culture derived products.     

Strategies for ascertaining the interference of phase II metabolites co‐eluting with parent compounds using LC–MS/MS

LC‐MS/MS is currently the most selective and efficient tool for the quantitative analysis of drugs and metabolites in the pharmaceutical industry and in clinical assays. However, phase II metabolites sometimes negatively affect the selectivity and efficiency of the LC‐MS/MS method, especially for the metabolites that possess similar physicochemical characteristics and generate the same precursor ions as their parent compounds due to the in‐source collision‐induced dissociation during the ionization process. This paper proposes some strategies for examining co‐eluting metabolites existing in real samples, and further assuring whether these metabolites could affect the selectivity and accuracy of the analytical methods. Strategies using precursor‐ion scans and product‐ion scans were applied in this study. An example drug, namely, caffeic acid phenethyl ester, which can generate many endogenous phase II metabolites, was selected to conduct this work. These metabolites, generated during the in vivo metabolic processes, can be in‐source‐dissociated to the precursor ions of their parent compounds. If these metabolites are not separated from their parent compounds, the quantification of the target analytes (parent compounds) would be influenced. Some metabolites were eluted closely to caffeic acid phenethyl ester on LC columns, although long columns and relatively long elution programs were used. The strategies can be utilized in quantitative methodologies that apply LC‐MS/MS to assure the performance of selectivity, thus enhancing the reliability of the experimental data.     

Role of pretreatment and conditioning processes on toxicity of lignocellulosic biomass hydrolysates

The Department of Energy’s Office of the Biomass Program has set goals of making ethanol cost competitive by 2012 and replacing 30% of 2004 transportation supply with biofuels by 2030. Both goals require improvements in conversions of cellulosic biomass to sugars as well as improvements in fermentation rates and yields. Current best pretreatment processes are reasonably efficient at making the cellulose/hemicellulose/lignin matrix amenable to enzymatic hydrolysis and fermentation, but they release a number of toxic compounds into the hydrolysate which inhibit the growth and ethanol productivity of fermentation organisms. Conditioning methods designed to reduce the toxicity of hydrolysates are effective, but add to process costs and tend to reduce sugar yields, thus adding significantly to the final cost of production. Reducing the cost of cellulosic ethanol production will likely require enhanced understanding of the source and mode of action of hydrolysate toxic compounds, the means by which some organisms resist the actions of these compounds, and the methodology and mechanisms for conditioning hydrolysate to reduce toxicity. This review will provide an update on the state of knowledge in these areas and can provide insights useful for the crafting of hypotheses for improvements in pretreatment, conditioning, and fermentation organisms.     

Ecosystem consequences of enhanced solar ultraviolet radiation: secondary plant metabolites as mediators of multiple trophic interactions in terrestrial plant communities

The potential role of ultraviolet-B (UV-B)-induced secondary plant metabolites as mediators of multiple trophic responses in terrestrial ecosystems is considered through review of the major classes of secondary metabolites, the pathways for their biosynthesis, interactions with primary and secondary consumers and known UV effects on their induction. Gross effects of UV-B radiation on plant growth and survival under realistic spectral balances in the field have been generally lacking, but subtle changes in carbon allocation and partitioning induced by UV-B, in particular production of secondary metabolites, can affect ecosystem-level processes. Secondary metabolites are important in plant-herbivore interactions and may affect pathogens. They act as feeding or oviposition deterrents to generalists and nonadapted specialists, but adapted specialists are stimulated to feed by these same compounds, which they detoxify and often sequester for use against their predators. This provides a route for tritrophic effects of enhanced UV-B radiation whereby herbivory may be increased while predation on the herbivore is simultaneously reduced. It is in this context that secondary metabolites may manifest their most important role. They can be the demonstrable mechanism establishing cause and effect at higher trophic levels because the consequences of their induction can be established at all trophic levels.     

UPLC-MS-Based Metabolomic Study of Streptomyces Strain HCCB10043 Under Different pH Conditions Reveals Important Pathways Affecting the Biosynthesis of A21978C Compounds

The A21978C family of compounds includes precursors of daptomycin, an important antibiotic for the treatment of diseases infected by Gram-positive resistant bacteria. Focusing on these valuable compounds, the differences in metabolites obtained with or without pH control in their producing strain Streptomyces parvus HCCB10043 were investigated by comparative metabolomics analysis based on UPLC-TOF-MS technology. According to principal component analysis, there were fourteen biomarker compounds selected under the two pH culture conditions. The ten known compounds were divided into two types: a glycoside family participating in the primary metabolism (daidzein, glycitein, genistein, and soyasaponin Bb) and a peptide family of secondary metabolites (valistatin, bestatin, 3-amino-2-hydroxy-4-phenylbutanoylvalylisoleucine, and arylomycins A2, A4, and A5). Through orthogonal partial least squares-discriminant analysis, three compounds, soyasaponin Bb and arylomycins A2 and A4 were identified as the most relevant compounds to A21978C1-3 production, the glycolytic pathway, and the NRPS synthesis pathway. The competitive relationship between arylomycin and A21978C was verified. These results have demonstrated the usefulness of the metabolomic strategy based on UPLC-MS in studying significant metabolic changes in actinomycetes. Moreover, this metabolomic strategy can provide new ideas and guidance for the regulation and improvement of secondary metabolites production.     

Applications of stable,nonradioactive isotope tracers in in vivo human metabolic research

The human body is in a constant state of turnover, that is, being synthesized, broken down and/or converted to different compounds. The dynamic nature of in vivo kinetics of human metabolism at rest and in stressed conditions such as exercise and pathophysiological conditions such as diabetes and cancer can be quantitatively assessed with stable, nonradioactive isotope tracers in conjunction with gas or liquid chromatography mass spectrometry and modeling. Although measurements of metabolite concentrations have been useful as general indicators of one''s health status, critical information on in vivo kinetics of metabolites such as rates of production, appearance or disappearance of metabolites are not provided. Over the past decades, stable, nonradioactive isotope tracers have been used to provide information on dynamics of specific metabolites. Stable isotope tracers can be used in conjunction with molecular and cellular biology tools, thereby providing an in-depth dynamic assessment of metabolic changes, as well as simultaneous investigation of the molecular basis for the observed kinetic responses. In this review, we will introduce basic principles of stable isotope methodology for tracing in vivo kinetics of human or animal metabolism with examples of quantifying certain aspects of in vivo kinetics of carbohydrate, lipid and protein metabolism.     

Bioaccumulation of heavy metals on adapted <Emphasis Type="Italic">Aspergillus foetidus</Emphasis>

The development of the organisms extracellular and intracellular mechanisms for the uptake of heavy metals were conducted by using the natural detoxification strategies of the organism to toxicity. Aspergillus foetidus was used as a test case organism to examine these processes. Aspergillus foetidus was adapted to multi-metals (Al, Co, Cr, Cu, Fe, Mg, Mn, Ni and Zn) by a sequential method for tolerance development. The detoxification strategies of A. foetidus occurred by two mechanisms. The first mechanism is the production of extracellular metabolites that is capable of adsorbing and precipitating the metal ions on the cell surface. The second mechanism for the detoxification of metals is the intracellular binding of heavy metals to thiol containing compounds such as GSH and sequestering these metal–thiol complexes into sub-cellular compartments or vacuoles. These detoxification strategies resulted in adapted organisms with tolerance to multi-heavy metals concentrations and significantly higher metal uptake with adaptation.     

Sonochemistry I. Effects of ultrasounds on heterogeneous chemical reactions – a useful tool to generate radicals and to examine reaction mechanisms

Application of ultrasound to a system that contains at least one liquid phase produces microscopic bubbles in the liquid which undergo periodic expansions and contractions. Some of these microbubbles eventually destabilize and collapse violently, generating temperatures in the thousands of degrees Kelvin and pressures in the hundreds of atmospheres. This phenomenon, known as cavitational implosion, favors the production of free solvent radicals that react amongst themselves and with other substrates in the system. In addition, ultrasound accelerates reactions that involve single electron transfers but seems to have no effect on reactions that proceed via ionic mechanisms for reasons that remain unclear. In practical terms, ultrasound allows the synthesis of novel compounds as well as the improved preparations of standard compounds. Sonication is more than just more efficient stirring. The high temperatures produced on cavitation, both in the cavity and at the interface, could lead to molecular combustion of the substrate and of the solvent to form radical species which could then initiate reactions.     

Strategy of using microsome-based metabolite production to facilitate the identification of endogenous metabolites by liquid chromatography mass spectrometry

One of the challenges in metabolomic profiling of complex biological samples is to identify new and unknown compounds. Typically, standards are used to help identify metabolites, yet standards cannot be purchased or readily synthesized for many unknowns. In this work we present a strategy of using human liver microsomes (HLM) to metabolize known endogenous human metabolites (substrates), producing potentially new metabolites that have yet to be documented. The metabolites produced by HLM can be tentatively identified based on the associated substrate structure, known metabolic processes, tandem mass spectrometry (MS/MS) fragmentation patterns and, if necessary, accurate mass measurements. Once identified, these metabolites can be used as references for identification of the same compounds in complex biological samples. As a proof of principle, a total of 9 metabolites have been identified from individual HLM incubations using 5 different substrates. Each metabolite was used as a standard. In the analysis of human urine sample by liquid chromatography MS/MS, four spectral matches were found from the 9 microsome-produced metabolite standards. Two of them have previously been documented as endogenous human metabolites, the third is an isomer of a microsome-metabolite and the fourth compound has not been previously reported and is also an isomer of a microsome-metabolite. This work illustrates the feasibility of using microsome-based metabolism to produce metabolites of endogenous human metabolites that can be used to facilitate the identification of unknowns in biological samples. Future work on improving the performance of this strategy is also discussed.     

Metabolic engineering is key to a sustainable chemical industry

The depletion of fossil fuel stocks will prohibit their use as the main feedstock of future industrial processes. Biocatalysis is being increasingly used to reduce fossil fuel reliance and to improve the sustainability, efficiency and cost of chemical production. Even with their current small market share, biocatalyzed processes already generate approximately US$50 billion and it has been estimated that they could be used to produce up to 20% of fine chemicals by 2020. Until the advent of molecular biological technologies, the compounds that were readily accessible from renewable biomass were restricted to naturally-occurring metabolites. However, metabolic engineering has considerably broadened the range of compounds now accessible, providing access to compounds that cannot be otherwise reliably sourced, as well as replacing established chemical processes. This review presents the case for continued efforts to promote the adoption of biocatalyzed processes, highlighting successful examples of industrial chemical production from biomass and/or via biocatalyzed processes. A selection of emerging technologies that may further extend the potential and sustainability of biocatalysis are also presented. As the field matures, metabolic engineering will be increasingly crucial in maintaining our quality of life into a future where our current resources and feedstocks cannot be relied upon.     

Complementary use of molecular and element-specific mass spectrometry for identification of selenium compounds related to human selenium metabolism

The aim of this paper is to give an overview of analytical data on the identification of selenium compounds in biological samples with relevance for selenium metabolism. Only studies applying the combination of element-specific inductively coupled plasma mass spectrometry as well as molecular electrospray mass spectrometry detection have been included. Hence, selenium compounds are only considered identified if molecular mass spectra obtained by analysis of the authentic biological sample have been provided. Selenium compounds identified in selenium-accumulating plants and yeast are included, as extracts from such plants and yeast have been widely used for examination of the cancer-preventive effect of selenium in cell lines, animal models and human intervention trials. Hence, these selenium compounds are available for absorption and further metabolism. Identification of selenium metabolites in simulated gastric and intestinal juice, intestinal epithelial tissue, liver and urine is described. Hence, selenium metabolites identified in relation to absorption, metabolism and excretion are included.     

Effects of Pulsed Electric Field on Secondary Metabolism of <Emphasis Type="Italic">Vitis vinifera</Emphasis> L. cv. Gamay Fréaux Suspension Culture and Exudates

Plant cell cultures provide a large potential for the production of secondary metabolites. Through the application of different physical and chemical cell stress factors, we investigated the production of the secondary metabolites in plant cell cultures. The effects of pulsed electric field (PEF) and ethephon on growth and secondary metabolism, particularly anthocyanins and phenolic acids synthesis, were investigated by using suspension culture of Vitis vinifera L. cv. Gamay Fréaux as a model system. Anthocyanins were measured by spectrophotometer and extracellular phenolic acids were determined by high-performance liquid chromatography. The compounds were identified by liquid chromatography–mass spectrometry and nuclear magnetic resonance. After the treatments with PEF and ethephon, the concentrations of anthocyanins and phenolic acids in cell culture were higher than in the control, without loss of biomass. The combination of PEF treatment and ethephon improved secondary metabolites formation. Production levels of extracellular phenolic acids, 3-O-glucosyl-resveratrol were increased by PEF and ethephon treatments. The results show that PEF induced a defense response of plant cells and may have altered the cell/membrane’s dielectric properties. PEF, an external stimulus or stress, is proposed as a promising new abiotic elicitor for stimulating secondary metabolites biosynthesis in plant cell cultures.     

Computing and Interpreting Specific Production Rates in a Chemostat in Steady State According to the Luedeking-Piret model

The Luedeking–Piret model is an empirical relationship which is very widely used in cell cultures to evaluate specific production rates of some products (metabolites or others). It constitutes a very common method of calculation as much in fundamental as in applied research and especially for designing and optimizing industrial processes in very varied fields. However, this model appears to be frequently deficient and has to be greatly adapted, practically, one might say, for each individual case. Obviously, this is a very great drawback, requiring a great deal of time spent on it and one that greatly lessens the ‘universality’ of the model. This work reveals that it is possible to give the initial Luedeking–Piret model a much more general scope. The used method revealed metabolic switches that have never been suspected until now. Confirmation of the method would certainly give a precious general tool both to optimize production processes and to increase understanding of some physiological states of cells in chemostat.     

Use of liquid chromatography quadrupole time-of-flight mass spectrometry in the elucidation of transformation products and metabolites of pesticides. Diazinon as a case study

Liquid chromatography (LC) coupled to hybrid quadrupole time-of-flight (QTOF) mass spectrometry (MS) is a useful analytical tool in the elucidation and confirmation of transformation products (TPs)/metabolites of pesticides with a wide range of polarity, in both environmental and biological samples. Firstly, the versatility of LC allows the determination of very distinct TPs/metabolites as chromatographic conditions can be easily changed and optimized depending on the analytical problem. Secondly, the mass accuracy provided by the TOF analyser allows the assignment of a highly probable empirical formula for each compound and the differentiation between nominal isobaric compounds. Finally, the possibility of performing MS/MS spectra with accurate mass measurements can been used for the final characterization of the TPs/metabolites detected and for the differentiation of isomeric compounds. In this study, the insecticide diazinon was used as model compound, and its photodegradation and metabolism have been investigated by LC-QTOF-MS. On one hand, environmental spiked water was irradiated with a mercury lamp for 9 days, sampling 3-mL aliquots approximately every 12 h. On the other hand, both in vitro and in vivo metabolism experiments were carried out with different substrate concentrations and incubation times. After centrifugation, and protein precipitation in the in vitro and in vivo studies, 50-μL aliquots of both environmental and biological samples were directly injected into the LC electrospray ionization QTOF system. The most important transformation processes were found to be hydrolysis of the ester moiety, hydroxylation in the aromatic ring or in one of the alkylic groups, oxidation of the sulfur atom on the P=S cleavage or a combination of these processes, with the highest number of compounds being found in the photodegradation study. Very polar compounds, such as diethyl phosphate and diethyl thiophosphate, were detected after direct injection of the aqueous sample, which was feasible owing to the characteristics of the LC. In MS mode, mass errors were below 3 mDa, leading to an empirical formula for each compound. MS/MS spectra with accurate mass were used for the final elucidation of the compounds detected.     

Solid phase microextraction as a powerful alternative for screening of secondary metabolites in actinomycetes

Actinobacteria are one of the most promising producers of medically and industrially relevant secondary metabolites. However, screening of such compounds in actinobacteria growth demands simple, fast, and efficient extraction procedures that enable detection and precise quantification of biologically active compounds. In this regard, solid phase microextraction (SPME) emerges as an ideal extraction technique for screening of secondary metabolites in bacteria culture due to its non‐exhaustive, minimally invasive, and non‐destructive nature: its integrated sample preparation workflow; balanced coverage feature; metabolism quenching capabilities; and superior cleanup, as well as its versatility in configuration, which enables automation and high throughput applications. The current work provides a comparison of micro‐scale and direct immersion SPME (DI‐SPME) for screening of secondary metabolites, describes the optimization of the developed DI‐SPME method, and introduces the developed technique for mapping of target secondary metabolites as well as its direct coupling to mass spectrometry for such applications. The optimized DI‐SPME method provided higher amounts of extracted ions and intensity signals, yielding superior extraction and desorption efficiency as compared with micro‐scale extraction. Studied compounds presented stability on the coating for 24 h at room temperature. The DI‐SPME mapping approach revealed that lysolipin I and the lienomycin analog are distributed along the center and edges of the colony, respectively. Direct coupling of SPME to MS provided a similar ions profile as SPME‐LC‐MS while enabling a significant decrease in analysis time, demonstrating its suitability for such applications. DI‐SPME is herein presented as an alternative to micro‐scale extraction for screening of secondary metabolites in actinobacteria solid medium, as well as a feasible alternative to DESI‐IMS for mapping of biologic radial distribution of secondary metabolites and cell life cycle studies. Lastly, the direct coupling of DI‐SPME to MS is presented as a fast, powerful technique for high throughput analysis of secondary metabolites in this medium.     

Chemical characterization of leaves and calli extracts of Rosmarinus officinalis by UHPLC-MS

Rosmarinus officinalis L. (Lamiaceae) is an aromatic plant widely popular mainly due to its uses in traditional medicine as an anti-inflammatory, diuretic and antimicrobial, as well as in the prevention and treatment of diseases. These biological activities are mainly related to the presence of phenolic and terpenic compounds. This work reports a chemical profile analysis of extracts from leaves and calli of rosemary obtained by both pressurized liquid extraction and maceration. Chemical profiles were determined on calli extracts of 3, 6, 9, and 15 days of culture; chemical characterization and quantification of compounds was carried out using ultrahigh performance liquid chromatography-mass spectrometry. A total of 53 metabolites were identified in callus and 47 compounds in leaf extracts, of which 25 correspond to phenolic compounds, mainly flavonoids and flavones, 13 terpenes that include phenolic terpenes and one diterpenolactone, two glycosides which correspond to 6-O-caffeoyl-β-D-fructofuranosil-(2→1)-α-D-glucopyranoside and primulaverin, an aromatic compound identified as fenantrenone and a growth regulator 12-hydroxy jasmonic acid. These results showed that undifferentiated rosemary cells accumulate the same compounds identified mainly in highly specialized tissues such as leaves. The plant cell culture supply the possibility of developing biotechnological processes to obtain compounds of commercial interest.