Critical Assessment of Analytical Techniques in the Search for Biomarkers on Mars: A Mummified Microbial Mat from Antarctica as a Best-Case Scenario.

The search for biomarkers of present or past life is one of the major challenges for in situ planetary exploration. Multiple constraints limit the performance and sensitivity of remote in situ instrumentation. In addition, the structure, chemical, and mineralogical composition of the sample may complicate the analysis and interpretation of the results. The aim of this work is to highlight the main constraints, performance, and complementarity of several techniques that have already been implemented or are planned to be implemented on Mars for detection of organic and molecular biomarkers on a best-case sample scenario. We analyzed a 1000-year-old desiccated and mummified microbial mat from Antarctica by Raman and IR (infrared) spectroscopies (near- and mid-IR), thermogravimetry (TG), differential thermal analysis, mass spectrometry (MS), and immunological detection with a life detector chip. In spite of the high organic content (ca. 20% wt/wt) of the sample, the Raman spectra only showed the characteristic spectral peaks of the remaining beta-carotene biomarker and faint peaks of phyllosilicates over a strong fluorescence background. IR spectra complemented the mineralogical information from Raman spectra and showed the main molecular vibrations of the humic acid functional groups. The TG-MS system showed the release of several volatile compounds attributed to biopolymers. An antibody microarray for detecting cyanobacteria (CYANOCHIP) detected biomarkers from Chroococcales, Nostocales, and Oscillatoriales orders. The results highlight limitations of each technique and suggest the necessity of complementary approaches in the search for biomarkers because some analytical techniques might be impaired by sample composition, presentation, or processing. Key Words: Planetary exploration-Life detection-Microbial mat-Life detector chip-Thermogravimetry-Raman spectroscopy-NIR-DRIFTS. Astrobiology 17, 984-996.

Laboratory to pilot scale: Microwave extraction for polyphenols lettuce.

Microwave hydrodiffusion and gravity (MHG) technique has been applied to pilot-scale solvent-free microwave extraction (SFME) of polyphenols from Lettuce sativa. Following the dictates of green extraction and with the aim to save time and energy, the lab-scale knowledge on SFME was exploited for the development of a pilot-scale process. The investigation entailed the optimization of all main parameters (temperature, time, extracted water volume, etc.) and we showed that the polyphenols composition profile under SFME was similar to the classic methods though a bit lower in total content. The energy consumption in the optimized procedure (30min) was 1W/g of fresh matrix.

Comparison of microwave-assisted and heat reflux extraction techniques for the extraction of ten major compounds from Zibu Piyin Recipe using ultra high performance liquid chromatography with tandem mass spectrometry.

Microwave-assisted extraction and efficient ultra-high performance liquid chromatography with triple quadrupole mass spectrometry were previously used to quickly extract and simultaneously quantify ginsenoside Rf, Ro, and Rd, 20(S)-ginsenoside-Rg2 , 20(R)-ginsenoside-Rg2 , tanshinone IIA, cryptotanshinone, dihydrotanshinone I, lithospermic acid, and osthole from Zibu Piyin Recipe. We here showed that heat reflux extraction provides higher extraction efficiency of these target compounds but is more time consuming. Chromatographic separation was achieved on an Agilent ZORBAX RRHD Eclipse Plus C18 column with a gradient mobile phase consisting of water/0.5% formic acid and acetonitrile at a flow rate of 0.2 mL/min, and detection was performed by positive and negative ion multiple-reaction monitoring mode. All analytes showed good linearity (r, 0.9989-0.9999) within the test range, with a limit of detection of 0.002-0.180 µg/mL. The overall intra- and interday variations of the ten compounds were ≤2.9%, and the accuracy was evaluated using a recovery test at three concentrations and was in the range 97.61-103.18% (RSD ≤ 4.25%). The analytical results showed remarkable differences in the concentrations of the ten compounds extracted from Zibu Piyin Recipe by microwave-assisted extraction and heat reflux extraction. These findings provide important information for determining the quality of Zibu Piyin Recipe.

Smashing Tissue Extraction of Five Lignans From the Fruit of Schisandra chinensis.

Schisandra chinensis is one of the most famous herbal medicines in China, Korea and Japan. It has been widely used as a tonic, sedative, anti-aging and astringent agent. Lignans are one of its main bioactive components. The classical methods for extracting lignans, however, were tedious and energy-consuming. With the aim to develop an effective extraction method of lignans, the smashing tissue extraction (STE) technique was adopted and optimized in this study. Extraction conditions of STE have been optimized by the response surface methodology based on the Box-Behnken design. Results showed that 75% aqueous ethanol was the optimal extraction solvent, and the other optimal conditions were as follows: extraction voltage of 180 V, extraction time of 1 min, solid-liquid ratio of 1 : 19 and sample particle size of 120 mesh. Under these optimized conditions, the total content of the five lignans (Schisandrol A, Schisantherin A, Deoxyschisandrin, Schisandrin B and Schisandrin C) in S. chinensis collected from Baishan City located in the northeast of China was 13.89 ± 0.014 mg/g, which was well matched with the value predicted by the model. Other techniques, including heat reflux, Soxhlet, ultrasonic-assisted and microwave-assisted extraction, were further compared. Results suggested that STE had the highest extraction efficiency of lignans with the shortest time. It indicates that the approach proposed in this study is a simple and efficient technique for the extraction of lignans in S. chinensis.

Effects of ultrahigh pressure extraction on yield and antioxidant activity of chlorogenic acid and cynaroside extracted from flower buds of Lonicera japonica.

The present study was designed to establish and optimize a new method for extracting chlorogenic acid and cynaroside from Lonicera japonica Thunb. through orthogonal experimental designl. A new ultrahigh pressure extraction (UPE) technology was applied to extract chlorogenic acid and cynaroside from L. japonica. The influential factors, including solvent type, ethanol concentration, extraction pressure, time, and temperature, and the solid/liquid ratio, have been studied to optimize the extraction process. The optimal conditions for the UPE were developed by quantitative analysis of the extraction products by HPLC-DAD in comparison with standard samples. In addition, the microstructures of the medicinal materials before and after extraction were studied by scanning electron microscopy (SEM). Furthermore, the extraction efficiency of different extraction methods and the 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activities of the extracts were investigated. The optimal conditions for extracting chlorogenic acid and cynaroside were as follows: ethanol concentration, 60%; extraction pressure, 400 MPa; extraction time, 2 min; extraction temperature, 30 °C; and the solid/liquid ratio, 1 : 50. Under these conditions, the yields of chlorogenic acid and cynaroside were raised to 4.863% and 0.080%, respectively. Compared with other extraction methods, such as heat reflux extraction (HRE), ultrasonic extraction (UE), and Sohxlet extraction (SE), the UPE method showed several advantages, including higher extraction yield, shorter extraction time, lower energy consumption, and higher purity of the extracts. This study could help better utilize L. japonica flower buds as a readily accessible source of natural antioxidants in food and pharmaceutical industries.

Comparison of microwave, ultrasound and accelerated-assisted solvent extraction for recovery of polyphenols from Citrus sinensis peels.

Peel of Citrus sinensis contains significant amounts of bioactive polyphenols that could be used as ingredients for a number of value-added products with health benefits. Extraction of polyphenols from the peels was performed using a microwave-assisted extraction (MAE) technique. The effects of aqueous acetone concentration, microwave power, extraction time and solvent-to-solid ratio on the total phenolic content (TPC), total antioxidant activity (TAA) (using DPPH and ORAC-values) and individual phenolic acids (IPA) were investigated using a response surface method. The TPC, TAA and IPA of peel extracts using MAE was compared with conventional, ultrasound-assisted and accelerated solvent extraction. The maximum predicted TPC under the optimal MAE conditions (51% acetone concentration in water (v/v), 500 W microwave power, 122 s extraction time and 25 mL g(-1) solvent to solid ratio), was 12.20 mg GAE g(-1) DW. The TPC and TAA in MAE extracts were higher than the other three extracts.

Process optimization for extraction of carotenoids from medicinal caterpillar fungus, Cordyceps militaris (Ascomycetes).

Natural carotenoids have attracted great attention for their important beneficial effects on human health and food coloring function. Cordyceps militaris, a well-known edible and medicinal fungus, is a potential source of natural carotenoids. The present study aimed to optimize the process parameters for carotenoid extraction from this mushroom. The effects of different methods of breaking the fungal cell wall and organic solvents were studied by the one-factor-at-a-time method. Subsequently, the process parameters including the duration of the extraction time, the number of extractions, and the solvent to solid ratio were optimized by using the Box-Behnken design. The optimal extraction conditions included using an acid-heating method to break the cell wall and later extracting three times, each for a 1 h duration, with a 4:1 mixture of acetone: petroleum ether and a solvent: solid ratio of 24:1. The carotenoid content varied from 2122.50 to 3847.50 µg/g dry weights in different commercially obtained fruit bodies of C. militaris. The results demonstrated that the C. militaris contained more carotenoid content in its fruit bodies than other known mushrooms. Stability monitoring by HPLC demonstrated that the carotenoids could be stored at 4°C for 40 d. It is suggested that the carotenoid content should be considered as the quality standard of commercial products of this valued mushroom. These findings will facilitate the exploration of carotenoids from C. militaris.

Innovative assistant extraction of flavonoids from pine (Larix olgensis Henry) needles by high-density steam flash-explosion.

High-density steam flash-explosion (HDSF) was first employed to extract flavonoids from pine needles. The HDSF treatment was performed at a steam pressure of 0.5-2.0 MPa for 20-120 s. Scanning electron microscopy and high-performance liquid chromatography combined with photodiode-array detection and electrospray ionization mass spectrometry (HPLC-DAD-ESI-MS) were used to characterize the morphological changes and analyze flavonoids of pine needles before and after HDSF treatment. Our results indicated that, after steam explosion at 1.5 MPa for 60 s, the flavonoids extracted reached 50.8 rutin equivalents mg/g dry weight, which was 2.54-fold as that of the untreated sample. HDSF pretreatment caused the formation of large micropores on the pine needles and production of particles, as well as the removal of wax layers. Compared to microwave-assisted, ultrasound-assisted, and solvent extraction, HDSF pretreatment took only 30 min to reach a maximum yield of 47.0 rutin equivalents mg/g flavonoids extract after pine needles were treated at 1.5 MPa for 80 s. In addition, after HDSF treatment, the aglycones were 3.17 times higher than that of untreated pine needles, while glycosides were lower by 57% (in HPLC-DAD individuals' sum) due to hydrolysis of flavonoids glycosides. It can be concluded that HDSF is a practical pretreatment for extraction of flavonoids and conversion in the healthy food and pharmaceutical industries.

Optimisation of ultrasound-assisted enzymatic extraction of arabinoxylan from wheat bran.

Arabinoxylan, the major dietary fibre component of wheat bran, is important from both technological and nutritional points of view. In this study, ultrasound-assisted enzymatic extraction technology was first employed to extract arabinoxylan from wheat bran. The process for extraction of arabinoxylan was optimised using response surface methodology, employing a five-level, five-variable central composite rotatable design. The optimum extraction conditions were as follows: raw material concentration, 50g/l, enzyme dose, 4.5g/l, extraction temperature, 50°C; extraction time, 70min; and ultrasonic power, 180W. Under the above mentioned conditions, the experimental yield was 142.6±0.17mg/g of wheat bran, which is well matched with the predictive yield. Ultrasound increased the efficiency of enzymatic treatment with higher extraction yield.

Disposable pipette extraction for the simultaneous determination of biperiden and three antipsychotic drugs in human urine by GC-nitrogen phosphorus detection.

BACKGROUND: Disposable pipette extraction with reversed-phase sorbent is proposed for the fast and simple GC determination of the antipsychotic drugs chlorpromazine, olanzapine, clozapine and biperiden - an anticholinergic drug - in human urine. The method was validated and successfully applied to postmortem urine samples. The analytical run was 11 min and lidocaine was used as the internal standard. RESULTS: The developed method showed good linearity, over the range of 0.34 to 5 ng/µl, for all compounds. The within-day and between-day precision and accuracy assays revealed values ≤15%, while the recoveries ranged from 85 to 120%. LOD and LOQ ranged from 0.28 to 0.42 ng/µl and from 0.85 to 3.58 ng/µl, respectively. CONCLUSION: The developed method is a user-friendly technique, which is simple and fast.

Preparative divergent flow IEF without carrier ampholytes for separation of complex biological samples.

Efficient separation method is a crucial part of the process in which components of highly complex biological sample are identified and characterized. Based on the principles of recently newly established electrophoretic method called divergent flow IEF (DF IEF), we have tested the DF IEF instrument which is able to operate without the use of background carrier ampholytes. We have verified that during separation and focusing of sample consisting of high numbers of proteins (yeast lysate and wheat flour extract), the pH gradient of preparative DF IEF can be created by autofocusing of the sample components themselves without any addition of carrier ampholytes. In DF IEF, the proteins are separated, desalted and concentrated in one step. The fractions of yeast lysate sample, collected at the DF IEF output and subjected to gel IEF, contained the zones of proteins gradually covering the pI values from 3.7 to 8.5. In our experimental arrangement, the highest number of proteins has been found in fractions with pI values around 5.3 as detected by polyacrylamide gel IEF with CBB staining. During DF IEF, the selected protein bands have been concentrated up to 16.8-fold.