Hemin Modified Carbon Fat Electrode for Analyzing Antimalarial Endoperoxide Artemisinin in Artemisia annua

A hemin bulk modified carbon electrode with Adeps neutralis (solid fat) as binder was developed for the determination of antimalarial endoperoxide artemisinin in plant matrix. The hemin modified electrode showed significant catalytic activity for the electrochemical reduction of artemisinin at about ?380 mV vs. Ag/AgCl in phosphate buffer solution of pH 7 by using cyclic and differential pulse voltammetry. Under optimized conditions strict linearity between artemisinin concentration and height of the cathodic catalytic current peak was observed in 4.8×10^?6–7.8×10^?5 M concentration range (R=0.9991) when using differential pulse voltammetry. The detection limit was calculated as 1.4×10^?6 M of artemisinin. The developed electroanalytical device is suitable for the determination of artemisinin in Artemisia annua extracts.     

Single-Step Preparative Extraction of Artemisinin from Artemisia annua by Charcoal Column Chromatography

Activated charcoal column chromatography was successfully applied for preparative extraction of artemisinin with high-purity from Artemisia annua L. in one single run for the first time in this study. The adsorption kinetics study showed that powdery activated charcoal had high adsorption speed for artemisinin. The experimental adsorption data fitted well using the Langmuir adsorption model. The optimal parameters for the extraction of artemisinin were obtained using a column packed with activated charcoal: the loading amount, 1 g extract/5 g activated charcoal; the elution mode, CH₂Cl₂?CMeOH (2:5, v/v) for 5 BV (bed volume) after the removal of some impurities with CH₂Cl₂?CMeOH (1:10, v/v). With this optimized condition, artemisinin was finally obtained with purity at 95.2% and the recovery at 72.3%. The developed technique would provide a feasible large-scale method for the extraction of artemisinin in pharmaceutical industry.     

Biotechnological Approaches for Production of Artemisinin,an Anti-Malarial Drug from Artemisia annua L.

Artemisinin is an anti-malarial sesquiterpene lactone derived from Artemisia annua L. (Asteraceae family). One of the most widely used modes of treatment for malaria is an artemisinin-based combination therapy. Artemisinin and its associated compounds have a variety of pharmacological qualities that have helped achieve economic prominence in recent years. So far, research on the biosynthesis of this bioactive metabolite has revealed that it is produced in glandular trichomes and that the genes responsible for its production must be overexpressed in order to meet demand. Using biotechnological applications such as tissue culture, genetic engineering, and bioreactor-based approaches would aid in the upregulation of artemisinin yield, which is needed for the future. The current review focuses on the tissue culture aspects of propagation of A. annua and production of artemisinin from A. annua L. cell and organ cultures. The review also focuses on elicitation strategies in cell and organ cultures, as well as artemisinin biosynthesis and metabolic engineering of biosynthetic genes in Artemisia and plant model systems.     

Heat and Mass Transfer in a Tank Reactor Stirred by Gaseous Jets

A spherical tank reactor stirred by gaseous jets has been developed in order to study the activity and selectivity of a catalytic or a reactive solid. In the present work, mass and heat transfer from a sphere suspended in the reactor are studied.The mass transfer study was carried out by using naphthalene spheres of different diameters and varying the air flow rate. A correlation of the experimental results is developed and compared with those in the literature.A heat transfer study from a heated sphere was also carried out. The natural convection effect on the experimental results is analyzed and these are correlated in terms of the effective Reynolds number.     

Strategies to Overcome Oxygen Transfer Limitations During Hairy Root Cultivation of Azadiracta indica for Enhanced Azadirachtin Production

The vast untapped potential of hairy root cultures as a stable source of biologically active chemicals has focused the attention of scientific community toward its commercial exploitation. However, the major bottleneck remains its successful scale-up. Due to branching, the roots form an interlocked matrix that exhibits resistance to oxygen transfer. Thus, present work was undertaken to develop cultivation strategies like optimization of inlet gas composition (in terms of % (v/v) O₂ in air), air-flow rate and addition of oxygen vectors in the medium, to curb the oxygen transfer limitations during hairy root cultivation of Azadirachta indica for in vitro azadirachtin (a biopesticide) production. It was found that increasing the oxygen fraction in the inlet air (in the range, 20–100% (v/v) O₂ in air) increased the azadirachtin productivity by approximately threefold, to a maximum of 4.42 mg/L per day (at 100% (v/v) O₂ in air) with respect to 1.68 mg/L per day in control (air with no oxygen supplementation). Similarly, increasing the air-flow rate (in the range, 0.3–2 vvm) also increased the azadirachtin productivity to a maximum of 1.84 mg/L per day at 0.8 vvm of air-flow rate. On the contrary, addition of oxygen vectors (in the range, 1–4% (v/v); hydrogen peroxide, toluene, Tween 80, kerosene, silicone oil, and n-hexadecane), decreased the azadirachtin productivity with respect to control (1.76 mg/L per day).     

Artemisinin evaluation in Romanian Artemisia annua wild plants using a new HPLC/MS method

Artemisinin, a sesquiterpene lactone from Artemisia annua L., has received considerable attention in the last few decades as a potent antimalarial drug. Artemisinin has rather low toxicity; it is effective against drug-resistant Plasmodium species and against cerebral malaria. This study reports the development of a rapid and sensitive assay for the quantification of artemisinin in A. annua by reversed phase HPLC/MS. In the selected optimal experimental conditions, artemisinin exhibited a well-defined chromatographic peak with a retention time of 2 ± 0.2 min. The chromatographic signal shows a linear dependence with artemisinin concentration, enabling the use of this signal for artemisinin quantification according to the following regression equation: y = 2665.40x - 14697.61. The correlation coefficient (R(2)) was 0.9989. For every concentration within the range of the standard curve (0.1-2 μg mL(-1)), accuracy was between 95 and 104%. Artemisinin content in Romanian A. annua wild plants varies between 0.17 and 0.21% (dry weight basis).     

Production of the Biopesticide Azadirachtin by Hairy Root Cultivation of Azadirachta indica in Liquid-Phase Bioreactors

Batch cultivation of Azadirachta indica hairy roots was carried out in different liquid-phase bioreactor configurations (stirred-tank, bubble column, bubble column with polypropylene basket, and polyurethane foam disc as root supports) to investigate possible scale-up of the A. indica hairy root culture for in vitro production of the biopesticide azadirachtin. The hairy roots failed to grow in the conventional bioreactor designs (stirred tank and bubble column). However, modified bubble column reactor (with polyurethane foam as root support) configuration facilitated high-density culture of A. indica hairy roots with a biomass production of 9.2 g l^?1dry weight and azadirachtin yield of 3.2 mg g^?1 leading to a volumetric productivity of azadirachtin as 1.14 mg l^?1 day^?1. The antifeedant activity in the hairy roots was also evaluated by no choice feeding tests with known concentrations of the hairy root powder and its solvent extract separately on the desert locust Schistocerca gregaria. The hairy root powder and its solvent extract demonstrated a high level of antifeedant activity (with an antifeedant index of 97 % at a concentration of 2 % w/v and 83 % at a concentration of 0.05 % (w/v), respectively, in ethanol).     

Simultaneous Nonchiral Determination of Artemisinin and Arteannuin B in Artemisia annua Using Circular Dichroism Detection

A new method was performed using on-line coupling of nonchiral reversed-phase liquid chromatography (RP-LC) to circular dichroism (CD) spectroscopy for simultaneous determination of artemisinin and arteannuin B in crude plant extracts of Artemisia annua. Analysis was carried out on an LC–CD system equipped with an Agilent TC-C₁₈ column (4.6 mm I.D. × 200 mm L, 5 μm) using gradient of acetonitrile. The method was validated to be practicable and reliable at alterable wavelength in the ranges of 220–420 nm as desired. LOD and LOQ of artemisinin and arteannuin B were 0.08, 0.26 and 0.31, 1.02 μg mL⁻¹, respectively. It was more sensitive than conventional LC–UV and comparatively cheaper than LC–MS in analysis of TCM.

     

Temperature Dynamics and Start-up of a Continuous Flow Stirred Tank Polymerisation Reactor

Experimental results have been obtained for the potassium persulphate initiated polymerisation of acrylamide in a continuous flow stirred tank reactor. Variables investigated included monomer and initiator concentrations and feed inlet temperature and flowrate. Results were compared with a theoretical model and satisfactory agreement obtained for steady state temperature and conversion. During the start-up period, during which temperature measurements only were made, comparison with theory showed that the induction periods observed experimentally under certain conditions were not predicted. Allowance for heat transfer from the reactor to the surroundings has been shown to be important.     

Heterotrophic Cultivation of Euglena gracilis in Stirred Tank Bioreactor: A Promising Bioprocess for Sustainable Paramylon Production

Paramylon is a valuable intracellular product of the microalgae Euglena gracilis, and it can accumulate in Euglena cells according to the cultivation conditions. For the sustainable production of paramylon and appropriate cell growth, different bioreactor processes and industrial byproducts can be considered as substrates. In this study, a complex medium with corn steep solid (CSS) was used, and various bioreactor processes (batch, fed batch, semicontinuous and continuous) were performed in order to maximize paramylon production in the microalgae Euglena gracilis. Compared to the batch, fed batch and repeated batch bioprocesses, during the continuous bioprocess in a stirred tank bioreactor (STR) with a complex medium containing 20 g/L of glucose and 25 g/L of CSS, E. gracilis accumulated a competitive paramylon content (67.0%), and the highest paramylon productivity of 0.189 g/Lh was observed. This demonstrated that the application of a continuous bioprocess, with corn steep solid as an industrial byproduct, can be a successful strategy for efficient and economical paramylon production.     

Optimized Extraction of Artemisinin from Artemisia annua L. and Corroborated Quantitative Analysis Using High-Performance Thin-Layer Chromatography

JPC – Journal of Planar Chromatography – Modern TLC - An optimized method for the extraction and quantification of artemisinin using high-performance thin-layer chromatography (HPTLC)...     

Effect of Agitation and Aeration Rates on Chitinase Production Using Trichoderma virens UKM1 in 2-l Stirred Tank Reactor

Shrimps have been a popular raw material for the burgeoning marine and food industry contributing to increasing marine waste. Shrimp waste, which is rich in organic compounds is an abundant source of chitin, a natural polymer of N-acetyl-D-glucosamine (GluNac), a reducing sugar. For this respect, chitinase-producing fungi have been extensively studied as biocontrol agents. Locally isolated Trichoderma virens UKM1 was used in this study. The effect of agitation and aeration rates using colloidal chitin as control substrate in a 2-l stirred tank reactor gave the best agitation and aeration rates at 200 rpm and 0.33 vvm with 4.1 U/l per hour and 5.97 U/l per hour of maximum volumetric chitinase activity obtained, respectively. Microscopic observations showed shear sensitivity at higher agitation rate of the above system. The oxygen uptake rate during the highest chitinase productivity obtained using sun-dried ground shrimp waste of 1.74 mg of dissolved oxygen per gram of fungal biomass per hour at the kappaL a of 8.34 per hour.     

Bifurcation Phenomena for an Oxidation Reaction in a Continuously Stirred Tank Reactor I. Adiabatic Operation

We investigate the steady-state multiplicity exhibited by the reaction of a fuel/air mixture in a continuously stirred tank reactor. The chemical mechanism used is a modification of a scheme due to Sal'nikov. We consider four cases; corresponding to the choice of fuel fraction, inflow temperature, inflow pressure, or precursor decay rate as the primary bifurcation parameter. From the perspective of fire-retardancy, the case when the fuel fraction is varied is the most important. In this case the steady-state diagrams provide a basis for a systematic investigation into the effectiveness of gas-phase active fire retardants.     

Comparison of Vinyl Acetate - Butyl Acrylate Emulsion Copolymerizations Conducted in a Continuous Pulsed Sieve Plate Column Reactor and in a Batch Stirred Tank Reactor

In this work, vinyl acetate/butyl acrylate emulsion copolymerizations carried out in a continuous tubular reactor (pulsed sieve plate column, PSPC) were compared to those conducted in a semibatch stirred tank reactor under similar operational conditions. In order to minimize the compositional drift along the PSPC, reactions were carried out with different numbers (2, 3 and 4) of lateral feed streams of the more reactive monomer (butyl acrylate). For comparison, fed batch reactions were conducted with the same number of intermittent shot additions of butyl acrylate, at the corresponding batch times. Both systems (tubular and semibatch) with distributed feeding of more reactive monomer are able to reduce composition drift thus providing more uniformity in copolymer composition. In addition, the tubular reactor presents much better control of temperature than the tank reactor, which is important to achieve higher productivity.     

Enhanced Production of a Thermostable Carbonic Anhydrase in Escherichia coli by Using a Modified NEXT Tag

Carbonic anhydrase (CA) is an ultrafast enzyme that catalyzes the reversible conversion of carbon dioxide (CO₂) to bicarbonate. CA is considered to be a green catalyst for enzyme-based CO₂ capture and utilization. In particular, the CA of Thermovibrio ammonificans (taCA) has attracted increasing attention as a highly stable enzyme. However, the poor solubility and the low expression level in Escherichia coli have hampered further utilization of taCA. In a recent study, these limitations were partly resolved by using a small solubility-enhancing fusion tag named NEXT, which originates from the N-terminal extension of Hydrogenovibrio marinus CA. In this study, the NEXT tag was engineered by adding small peptides to the N terminus to further increase the production yield of NEXT-tagged taCA. The addition of ng3 peptide (His-Gly-Asn) originating from the N-terminal sequence of Neisseria gonorrhoeae CA improved the expression of NEXT-taCA, while the previously developed translation-enhancing element (TEE) and Ser-Lys-Ile-Lys (SKIK) tag were not effective. The expression test with all 16 codon combinations for the ng3 sequence revealed that the change in translation initiation rate brought about by the change in nucleotide sequence was not the primary determinant for the change in expression level. The modified ng3-NEXT tag may be applied to increase the production yields of various recombinant proteins.     

原油乳状液破乳的动态法研究

The stability of different crude oil emulsions from ASP flooding production well is studied by the method of a stirred tank. The demulsifying of crude-oil emulsion by different demulsifying agents is discussed. The breakage of the crude oil emulsions from the well PO11 by the different types and concentrations of the emulsifying agents is also discussed. The breaking mechanism of the demulsifying agent is described microscopically.     

Production of L(+)-Lactic Acid Using Acid-Adapted Precultures of <Emphasis Type="Italic">Rhizopus arrhizus</Emphasis> in a Stirred Tank Reactor

Cultivations of filamentous fungi in stirred tank reactors (STRs) to produce metabolites are often limited by insufficient mixing and mass transfer because of the formation of mycelial clumps inside the reactors. This study developed an acid-adapted preculture approach to control the morphology of filamentous Rhizopus arrhizus in a STR, consequently to enhance the production yield and productivity of L(+)-lactic acid efficiently using waste potato starch as substrate. Using the acid-adapted precultures as inoculum, the morphology of R. arrhizus was maintained as large clumps, coalesced loose small pellets, and freely dispersed small pellets. The highest lactic acid concentration of 85.7 g/L with a yield of 86% was obtained in association with the formation of coalesced loose small pellets. The results indicate that the use of the acid-adapted precultures as inoculum is a promising approach for lactic acid production in STRs.     

Enhanced Aryltetralin Lignans Production in Linum Adventi-Tious Root Cultures

Lignans are the main secondary metabolites synthetized by Linum species as plant defense molecules. They are also valuable for human health, in particular, for their potent antiviral and antineoplastic properties. In this study, the adventitious root cultures of three Linum species (L. flavum, L. mucronatum and L. dolomiticum) were developed to produce aryltetralin lignans. The effect of two elicitors, methyl jasmonate and coronatine, on aryltetralin lignans production was also evaluated. The adventitious root cultures from L. dolomiticum were obtained and analyzed for the first time and resulted as the best producer for all the aryltetralins highlighted in this system: Podophyllotoxin, 6-methoxypodophyllotoxin and 6-methoxypodophyllotoxin-7-O-β-glucoside, the last showing a productivity of 92.6 mg/g DW. The two elicitors differently affected the production of the 6-methoxypodophyllotoxin and 6-methoxypodophyllotoxin-7-O-β-glucoside.     

In Vitro Azadirachtin Production by Hairy Root Cultivation of <Emphasis Type="Italic">Azadirachta indica</Emphasis> in Nutrient Mist Bioreactor

Azadirachtin, a well-known biopesticide is a secondary metabolite conventionally extracted from the seeds of Azadirachta indica. The present study involved in vitro azadirachtin production by developing hairy roots of A. indica via Agrobacterium rhizogenes-mediated transformation of A. indica explants. Liquid culture of hairy roots was established in shake flask to study the kinetics of growth and azadirachtin production. A biomass production of 13.3 g/L dry weight (specific growth rate of 0.7 day⁻¹) was obtained after 25 days of cultivation period with an azadirachtin yield of 3.3 mg/g root biomass. To overcome the mass transfer limitation in conventionally used liquid-phase reactors, batch cultivation of hairy roots was carried out in gas-phase reactors (nutrient spray and nutrient mist bioreactor) to investigate the possible scale-up of A. indica hairy root culture. The nano-size nutrient mist particles generated from the nozzle of the nutrient mist bioreactor could penetrate till the inner core of the inoculated root matrix, facilitating uniform growth during high-density cultivation of hairy roots. A biomass production of 9.8 g/L dry weight with azadirachtin accumulation of 2.8 mg/g biomass (27.4 mg/L) could be achieved in 25 days of batch cultivation period, which was equivalent to a volumetric productivity of 1.09 mg/L per day of azadirachtin.