Toward Arabidopsis thaliana hydrophilic metabolome: assessment of extraction methods and quantitative 1H NMR

Our goal was to establish the hydrophilic metabolome of heterotrophic Arabidopsis thaliana cells grown in suspension, a cellular model of plant sink tissues. Water‐soluble metabolites were extracted using four protocols: perchloric acid, boiling ethanol, methanol and methanol/chloroform (M/Chl). They were detected and quantified using ¹H nuclear magnetic resonance (NMR) spectroscopy at 400 MHz. Extraction yields and reproducibility of the extraction methods were investigated. The effects of cell harvest protocol, cell grinding and lyophilization and storage conditions on the measured metabolic profiles were also studied. These quantitative studies demonstrated for the first time that the four extraction protocols commonly used do lead to quite similar molecular compositions as analyzed by ¹H NMR. The M/Chl method proved effective and reliable to prepare series of physiologically significant extracts from plant cells for ¹H NMR analysis. Reproducibility of the detected metabolome was assessed over long periods of time by analyzing a large number of separate extracts prepared from independent cultures. Larger variations in the NMR metabolite profiles could be correlated to changes in physiological parameters of the culture medium. Quantitative resolved ¹H NMR of cell extracts proved to be robust and reliable for routine metabolite profiling of plant cell cultures.     

Contributions to the biotechnolgical production of sweeteners from stevia rebaudiana bertoni. III. Accumulation of secondary metabolites by means of a precursor and by elicitation of cell cultures

Gibberellin A₃ fed to cell suspension cultures of Stevia rebaudiana showed a fast conversion to stevioside. The product was detected within one day after gibberellin addition and achieved its maximum concentration after one week. However, using special production media (without precursor or elicitor), stevioside was produced only two to seven weeks after inoculation [1]. Elicitation of suspension cultures was performed with Stevia specific and non-specific fungi and with yeast extract. Although production of some secondary metabolites was induced, stevioside was not synthesized.     

Effects of plant-growth-promoting rhizobacteria and arbuscular mycorrhizal fungus on plant growth,stevioside, NPK,and chlorophyll content of Stevia rebaudiana

Stevia rebaudiana Bertoni is a unique medicinal plant which is mostly utilized as a sugar substitute for diabetic patients. In this research, regenerated plantlets of stevia in tissue culture is transferred to pots in greenhouse and inoculated with plant-growth-promoting rhizobacteria (PGPRs) (Bacillus polymixa, Pseudomonas putida, and Azotobacter chroococcum) and arbuscular mycorrhizal fungus (AMF) (Glomus intraradices). The results showed that in comparison to control, inoculation with a single microorganism, significantly increased root and shoot biomass as well as stevioside, chlorophyll, and NPK content in plants. However, such increased effects have been found to be further enhanced significantly due to dual compatible mixtures of inoculants resulting from their strong synergistic relationships among themselves. All growth parameters recorded the highest in 60-days-old plants in the treatment of Glomus+Azotobacter and followed with Glomus+Bacillus and Azotobacter+Pseudomonas treatments, respectively. Furthermore, high-performance liquid chromatography (HPLC) chromatograms revealed that the highest stevioside content have been produced in same treatments. Triple treatments had less positive effects compared to dual inoculations. Probably competence between microorganisms in triple inoculations has reduced their efficiency. Thus, suitable combination of mycorrhizal fungi and PGPR as biotic elicitors can enhance growth and stevioside content in tissue culture-regenerated plantlets of stevia.     

Downstream processing of stevioside and its potential applications

Stevioside is a natural sweetener extracted from leaves of Stevia rebaudiana Bertoni, which is commercially produced by conventional (chemical/physical) processes. This article gives an overview of the stevioside structure, various analysis technique, new technologies required and the advances achieved in recent years. An enzymatic process is established, by which the maximum efficacy and benefit of the process can be achieved. The efficiency of the enzymatic process is quite comparable to that of other physical and chemical methods. Finally, we believe that in the future, the enzyme-based extraction will ensure more cost-effective availability of stevioside, thus assisting in the development of more food-based applications.     

Enzyme-assisted extraction of bioactives from plants

Demand for new and novel natural compounds has intensified the development of plant-derived compounds known as bioactives that either promote health or are toxic when ingested. Enhanced release of these bioactives from plant cells by cell disruption and extraction through the cell wall can be optimized using enzyme preparations either alone or in mixtures. However, the biotechnological application of enzymes is not currently exploited to its maximum potential within the food industry. Here, we discuss the use of environmentally friendly enzyme-assisted extraction of bioactive compounds from plant sources, particularly for food and nutraceutical purposes. In particular, we discuss an enzyme-assisted extraction of stevioside from Stevia rebaudiana, as an example of a process of potential value to the food industry.     

Enrichment of the rebaudioside A concentration in Stevia rebaudiana extract with cyclodextrin glycosyltransferase from Bacillus licheniformis DSM 13

Stevia rebaudiana is a sweet herbaceous perennial plant, which is frequently used in the preparation of plant-based sweeteners. The demand for such sweeteners continues to increase due to purposeful nutrition and modern-day metabolic syndromes. More than 20 types of steviol glycosides provide a sweet taste, which are more than 300 times sweeter than sucrose. They are formed of two main components, namely stevioside and rebaudioside A. Only a handful of studies have dealt with Stevia rebaudiana leaf extracts, the conversion of pure stevioside into the preferred rebaudioside A is more common. The aim of this study was to enrich the rebaudioside A content of Stevia rebaudiana leaf extract using enzymatic bioconversion by applying fermented cyclodextrin glycosyltransferase from Bacillus licheniformis DSM13. Two differently processed plant materials, namely dried and lyophilized Stevia rebaudiana plants, were extracted and compared. Following the bioconversion, the rebaudioside A content was on average doubled. The maximum increase was fivefold with a 70–80% conversion of the stevioside.     

Phytochemical Investigations and In Vitro Bioactivity Screening on Melia azedarach L. Leaves Extract from Nepal

Melia azedarach is a common tree used in the traditional medicine of Nepal. In this work, leaves were considered as source of bioactive constituents and composition of methanol extract was evaluated and compared with starting plant material. Flavonoid glycosides and limonoids were identified and quantified by HPLC-DAD-MSⁿ approaches in dried leaves and methanolic extract, while HPLC-APCI-MSⁿ and GC/MS analysis were used to study phytosterol and lipid compositions. β-Sitosterol and rutin were the most abundant constituents. HPLC-APCI-MSⁿ and HPLC-DAD-MSⁿ analysis revealed high levels of phytosterols and flavonoids in methanolic extract accounting 9.6 and 7.5 % on the dried weight, respectively. On the other hand, HPLC/MSⁿ data revealed that limonoid constituents were in minor amount in the extract <0.1 %, compared with leaves (0.7 %) indicating that degradation occurred during extraction or concentration procedures. The methanol extract was subjected to different bioassays, and antioxidant activity was evaluated. Limited inhibitory activity on acetyl and butyryl cholinesterase, as well as on amylase were detected. Moreover, tyrosinase inhibition was significant resulting in 131.57±0.51 mg kojic acid equivalents/g of dried methanol extract, suggesting possible use of this M. azedarach extract in skin hyperpigmentation conditions. Moderate cytotoxic activity, with IC₅₀ of 26.4 μg/mL was observed against human ovarian cancer cell lines (2008 cells). Our findings indicate that the Nepalese M. azedarach leaves can be considered as valuable starting material for the extraction of phenolics and phytosterols, yielding extracts with possible cosmetic and pharmaceutical applications.     

Overexpression of SrUGT76G1 in Stevia alters major steviol glycosides composition towards improved quality

Steviol glycosides (SGs) are extracted from Stevia leaves for use as a natural sweetener. Among SGs, stevioside is most abundant in leaf extracts followed by rebaudioside A (Reb A). However, Reb A is of particular interest because of its sweeter and more pleasant taste compared to stevioside. Therefore, the development of new Stevia varieties with a higher Reb A to stevioside ratio would be desirable for the production of higher quality natural sweeteners. Here, we generated transgenic Stevia plants overexpressing Stevia UDP‐glycosyltransferase 76G1 (SrUGT76G1) that is known to convert stevioside to Reb A through 1,3‐β‐d ‐glucosylation in vitro. Interestingly, by overexpressing SrUGT76G1, the Reb A to stevioside ratio was drastically increased from 0.30 in wild‐type (WT) plants up to 1.55 in transgenic lines without any significant changes in total SGs content. This was contributed by a concurrent increase in Reb A content and a decrease in stevioside content. Additionally, we were able to find an increase in the Reb C to dulcoside A ratio in transgenic lines. Using the glutathione S‐transferase‐tagged SrUGT76G1 recombinant protein for an in vitro glucosyltransferase assay, we further demonstrated that Reb C can be produced from the glucosylation of dulcoside A by SrUGT76G1. Transgenic Stevia plants having higher Reb A to stevioside ratio were visually indistinguishable from WT plants. Taken together, our results demonstrate that the overexpression of SrUGT76G1 in Stevia is an effective way to generate new Stevia varieties with higher proportion of the more preferred Reb A without compromising on plant development.     

Contributions to the biotechnological production of sweeteners from stevia rebaudiana bertoni. II. Induction of stevioside accumulation in cell cultures by variation of the nutrient medium and the analysis of small amounts of stevioside

Cell cultures of Stevia rebaudiana in general do not contain stevioside when grown on their basal nutrient medium. No stable and prolonged stevioside accumulation was achieved by varying the medium components. However, in some cases a transient state was observed where more stevioside was synthesized than catabolized. The dynamic behaviour of product synthesis which led to accumulation of stevioside during only a short period was demonstrated. For accurate determination of very small amounts of stevioside, a combined TLC-HPLC method was used [1].     

The quantification of ellagic acid in the crude extract of Phyllanthus amarus Schum. & Thonn. (Euphorbiaceae)

Introduction – Phyllanthus amarus Schum. & Thonn. (Euphorbiaceae), already well known for its antiviral, antihyperglycaemic and antihepatotoxic effects, is also investigated for its antimalarial activity. The major constituent of the crude extract of the whole plant was isolated and identified in this research to be ellagic acid, for which antiplasmodial activity already has been reported. Objective – Because of the potential of the plant and the interesting properties of ellagic acid, an analytical method can be useful for the standardisation of the extracts to allow further biological and pharmacological investigations. In order to obtain an easily performable and inexpensive method, an HPLC analysis was developed and validated. Methodology – The samples were dissolved in DMSO, ultrasonicated for 15 min, and diluted with 50% methanol. Analysis was performed using water and methanol containing 0.06% TFA and the peaks were detected at 254 nm. Results – Ellagic acid showed a linear relationship in the range of 1.74–20.91 µg/mL and a single‐point calibration was allowed. The method was shown to be precise with respect to time (RSD of 1.84%, 3 days, n = 6) and concentration (RSD of 2.54%, 3 levels, n = 6). The overall mean content of ellagic acid was 2.06%. A recovery experiment was performed and it showed an accuracy of 100.4%. Conclusion – Based on the obtained results, it can be concluded that the newly developed method is suitable for its purpose, namely the determination of ellagic acid in the crude extract of P. amarus. Copyright © 2010 John Wiley & Sons, Ltd.     

INTRA‐CELLULAR AND EXTRA‐CELLULAR TOXINS IN THE RED TIDE DINOFLAGELLATE,GYMNODINIUM BREVE

The marine dinoflagellate, Gymnodinium breve (Davis), produces several neurotoxins that cause neurotoxic shellfish poisoning (nsp), massive fish kills and respiratory irritation in marine mammals and humans. The common method for discerning toxic levels of G. breve for public health advisories is enumeration of live cells in a given water mass. In this study, laboratory cultures, as well as natural blooms, were added to a stirred ultra‐filtration cell concentrator to separate viable cells containing intra‐cell toxins from ambient water containing extra‐cell toxins. Methods were validated using various mixtures of lysed and whole G. breve laboratory culture. Extractions and recovery of brevetoxins were done using a C‐18 bonded‐phase glass fiber extraction disc eluted with methanol. Total PbTx toxin concentrations were quantified by HPLC/UV using a C‐18 column and an 85:15 methanol:water (1 ml min^?1) isocratic elution at 215 nm. This method of separation and extraction was subsequently applied to water samples collected during natural blooms along two different areas of the Florida Gulf coast. The results indicated that early stages of G. breve blooms contained primarily intra‐cell toxins with extra‐cell toxins increasing as the bloom progressed, even though very few viable G. breve cells were present. This suggests that enumeration of cells alone may be insufficient and additional toxin quantitation is necessary.     

Extraction of chlorophyll a from freshwater phytoplankton for spectrophotometric analysis

The efficiency of pigment extraction forms the crux of the spectrophotometric analysis of chlorophyll a. The alcoholic solvents, methanol and ethanol, proved to be superior to acetone and acetone with DMSO. Homogenisation and sonication did not improve the extraction in the alcoholic solvents. Boiling at 100°C had an adverse effect whereas complete extraction of the pigments was obtained at the solvents boiling point and allowing the samples to stand for 24 h in the dark.     

基于天然低共熔溶剂的甜叶菊中甜菊糖绿色提取方法及优化

尝试利用天然低共熔溶剂(NADES)提取甜叶菊(Stevia rebaudiana)中的甜菊糖, 探索一种高效、绿色和环保的甜菊糖提取新方法。以甜叶菊干叶为原料, 对照传统提取溶剂水, 以甜菊糖中甜菊苷和莱鲍迪苷A的提取浓度作为指标, 筛选出最优的NADES提取配方, 然后通过Box-Behnken响应面法对NADES提取甜叶菊中甜菊糖的工艺条件进行筛选优化。结果表明, 提取效率最高的NADES配方为1,2-丙二醇:甘油:水=8:1:1 (v/v/v), 提取的甜菊苷浓度为2.59 mg∙mL^-1, 比水提取高16.40%, 提取的莱鲍迪苷A浓度为1.06 mg∙mL^-1, 比水提取高12.62%; 通过响应面法得到最优提取条件: 提取时间90分钟, 提取温度60°C, 超声功率为80 J∙s^-1, 预测甜菊苷提取浓度为3.49 mg∙mL^-1, 莱鲍迪苷A提取浓度为1.43 mg∙mL^-1, 与实验验证值(甜菊苷浓度为3.48 mg∙mL^-1, 莱鲍迪苷A浓度为1.42 mg∙mL^-1)接近。在最优条件下, 甜菊苷提取浓度比初始条件提高了34.36%, 莱鲍迪甘A提取浓度比初始条件提高了33.96%。NADES绿色环保, 且提取效率高于传统溶剂, 可用于甜叶菊中甜菊糖的绿色提取; 同时, 该提取方法可为后续推广至其它大宗经济植物类天然产物的绿色工业生产提供参考。     

Sample purification using a C18-bonded reversed-phase cartridge for the quantitative analysis of corticosteroids in adrenal cell cultures by high-performance liquid chromatography or gas chromatography—mass spectrometry

Quantitative extraction and subsequent purification of small biological samples often involve cumbersome procedures. We have devised a short and efficient method for the quantitative extraction of the corticosteroid and the 20α reduced steroid series from culture medium containing 20% sera in a single, pure fraction with separation from cholesterol. Passage through a C₁₈-bonded reversed-phase Sep-Pak^® cartridge of the acidified culture medium and subsequent extraction of the steroid fraction with methanol yields a single fraction containing all steroids in 90% recovery and reduced quantities of cholesterol down to 30%. The extract can then be used without further purification for quantitative analysis by high-performance liquid chromatography or derivatized and analyzed by gas chromatography and gas chromatography—mass spectrometry.     

Bioprocess considerations for production of secondary metabolites by plant cell suspension cultures

Plant cell culture provides a viable alternative over whole plant cultivation for the production of secondary metabolites. In order to successfully cultivate the plant cells at large scale, several engineering parameters such as, cell aggregation, mixing, aeration, and shear sensitivity are taken into account for selection of a suitable bioreactor. The media ingredients, their concentrations and the environmental factors are optimized for maximal synthesis of a desired metabolite. Increased productivity in a bioreactor can be achieved by selection of a proper cultivation strategy (batch, fed-batch, two-stageetc.), feeding of metabolic precursors and extraction of intracellular metabolites. Proper understanding and rigorous analysis of these parameters would pave the way towards the successful commercialization of plant cell bioprocesses.     

A robust sampling approach for identification and quantification of methyl jasmonate in leaf tissue of oilseed rape for analysis of early signaling in sclerotinia sclerotiorum resistance

Introduction – Methyl jasmonate (MJA), which is a natrual hormonal regulator, is thought to be essential for the regulation of systemic defence responses. The information about MJA levels in plant tissues is helpful for the study of the disease resistance mechanism and genetically engineered cultivars with increased resistance. Therefore, the quantification of MJA levels in plant tissues by means of a sensitive and reliable method is of interest. Objective – Development of a film extraction method coupled with GC for determination of methyl jasmonate in leaf tisssue of oilseed rape for analysis of early signalling in sclerotinia sclerotiorum resistance. Methodology – A robust polydimethylsiloxane film was prepared and used for extraction of MJA in leaf tissues. By using in‐solution extraction mode, optimum extraction efficiency was achieved with methanol–water (1 : 5, v/v) as extraction medium at 40°C for 60 min. Results – Under the optimal conditions, a detection limit of 0.2 ng/mL was achieved. Excellent reproducibility was found over a linear range of 1–1000 ng/mL. MJA in leaves infected by sclerotinia sclerotiorum was determined, with the results showing that basal levels of MJA (15 ng/g) were present in noninfested controls, but increased to 313 ng/g 10 h after fungal attack. Conclusion – The film extraction method is a simple, rapid and inexpensive sampling technique for determination of endogenous MJA in plant tissues that can be applied to most plants. Copyright © 2009 John Wiley & Sons, Ltd.     

A method for the medium-term storage of plant tissue samples at room temperature and successive cycles of DNA extraction

Based on the protocol originally described by Stein et al. (2001), we have developed a method that allows for medium-term conservation at room temperature of wheat (Triticum aestivum) tissue samples to use for DNA extraction. DNA quality was suitable for analysis by PCR and Southern hybridization, even after 2 months of storage at room temperature. This method allows successive DNA re-extractions from a previously extracted sample and maximization of the DNA yield that can be recovered from precious samples. This method has applications for conservation of leaf samples and management of DNA extraction. Our method can help improve data recovery in many plant molecular genetics research projects.     

A simple method for the extraction and quantification of photopigments from Symbiodinium spp.

We have developed a simple, mild extraction procedure using methanol which, when coupled with HPLC analysis and diode array detection (DAD), can be used to quantify the major photopigments found in cultured Symbiodinium spp. Extracts were prepared by suspending, fresh or frozen (− 70 °C), wet cell pellets in methanol and sonicating or not sonicating the cell suspensions before soaking the cells for 2 h in an ice bath. To assist the soaking process, cell suspensions were vortex mixed at 30 min intervals. After soaking, 0.5 M ammonium acetate buffer was added (1 part buffer to 9 parts methanol) before suspensions were stored over night at − 20 °C. Greater than 92% the recoverable pigment was obtained in the initial extraction of the four major photopigments, chlorophyll c, peridinin, diadinoxanthin, and chlorophyll a. Neither sonication nor freezing substantially increased the recovery of photopigments extracted with methanol. Extraction by other commonly used solvents such as acetone or acetone:water with or without freezing and sonication were less effective.     

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% ¹. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient ². Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K¹⁵NO₃ as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest ³. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control ⁴.     

Efficient enzymatic production of rebaudioside A from stevioside

Stevioside and rebaudioside A are the chief diterpene glycosides present in the leaves of Stevia rebaudiana. Rebaudioside A imparts a desirable sweet taste, while stevioside produces a residual bitter aftertaste. Enzymatic synthesis of rebaudioside A from stevioside can increase the ratio of rebaudioside A to stevioside in steviol glycoside products, providing a conceivable strategy to improve the organoleptic properties of steviol glycoside products. Here, we demonstrate the efficient conversion of stevioside to rebaudioside A by coupling the activities of recombinant UDP-glucosyltransferase UGT76G1 from S. rebaudiana and sucrose synthase AtSUS1 from Arabidopsis thaliana. The conversion occurred via regeneration of UDP-glucose by AtSUS1. UDP was applicable as the initial material instead of UDP-glucose for UDP-glucose recycling. The amount of UDP could be greatly reduced in the reaction mixture. Rebaudioside A yield in 30?h with 2.4?mM stevioside, 7.2?mM sucrose, and 0.006?mM UDP was 78%.