Name: lethality bioassay using artemia salina l.
Uploaded: 2022-10-11T13:40:00
Description: Watch this Scientific Journal Video about Lethality Bioassay Using Artemia salina L. at JoVE.com

In memory of&#160;Professor Amilcar Roberto.
This work was financially supported by Fundac&#807;a&#771;o para a Cie&#770;ncia e a Tecnologia (FCT, Portugal) under projects UIDB/04567/2020 and UIDP/04567/2020 attributed to CBIOS and PhD grant SFRH/BD/137671/2018 (Vera Isca).

1. Equipment preparation
<ol>
	<li>Acquire commercially available hatching equipment. Select a suitable place to set up the hatching equipment (Figure 2A). Place the funnel-shaped container in the black support (included in the set) and turn the funnel in a suitable direction to see the level mark and the tap.</li>
	<li>To make hand-made migration equipment, cut the top of two 0.5 L (5.8 cm diameter) plastic bottles to obtain a final height of 12 cm. Create a hole of 1.5 c...

<ol>
	<li>Ntungwe, N. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artemia+species:+An+important+tool+to+screen+general+toxicity+samples.">Artemia species: An important tool to screen general toxicity samples.</a> Current Pharmaceutical Design. 26 (24), 2892-2908 (2020).</li><li>Cragg, G. M., Newman, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+products:+A+continuing+source+of+novel+drug+leads.">Natural products: A continuing source of novel drug leads.</a> Biochimica et Biophysica Acta (BBA) - General Subjects. 1830 (6), 3670-3695 (2013).</li><li>Ntungwe, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=General+toxicity+screening+of+Royleanone+derivatives+using+an+artemia+salina+model.">General toxicity screening of Royleanone derivatives using an artemia salina model.</a> Journal Biomedical and Biopharmaceutical Research. 18 (1), 114 (2021).</li><li>Seca, A., Plant Pinto, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=secondary+metabolites+as+anticancer+agents:+Successes+in+clinical+trials+and+therapeutic+application.">secondary metabolites as anticancer agents: Successes in clinical trials and therapeutic application.</a> International Journal of Molecular Sciences. 19 (1), 263 (2018).</li><li>Calixto, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+natural+products+in+modern+drug+discovery.">The role of natural products in modern drug discovery.</a> Anais da Academia Brasileira de Ci&#234;ncias. 91 (3), 1-7 (2019).</li><li>Mandrell, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+zebrafish+chorion+removal+and+single+embryo+placement:+optimizing+throughput+of+zebrafish+developmental+toxicity+screens.">Automated zebrafish chorion removal and single embryo placement: optimizing throughput of zebrafish developmental toxicity screens.</a> Journal of Laboratory Automation. 17 (1), 66-74 (2012).</li><li>Zhang, Y., Mu, J., Han, J., Gu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+brine+shrimp+larvae+lethality+microwell+test+method.">An improved brine shrimp larvae lethality microwell test method.</a> Toxicology Mechanisms and Methods. 22 (1), 23-30 (2012).</li><li>Dom&#237;nguez-Villegas, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=antioxidant+and+cytotoxicity+activities+of+methanolic+extract+and+prenylated+flavanones+isolated+from+leaves+of+eysehardtia+platycarpa.">antioxidant and cytotoxicity activities of methanolic extract and prenylated flavanones isolated from leaves of eysehardtia platycarpa.</a> Natural Product Communications. 8 (2), 177-180 (2013).</li><li>Hamidi, M. R., Jovanova, B., Panovska, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxicological+evaluation+of+the+plant+products+using+Brine+Shrimp+(Artemia+salina+L.)+model.">Toxicological evaluation of the plant products using Brine Shrimp (Artemia salina L.) model.</a> Macedonian Pharmaceutical Bulletin. 60 (01), 9-18 (2014).</li><li>Libralato, G., Prato, E., Migliore, L., Cicero, A. M., Manfra, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+toxicity+testing+protocols+and+endpoints+with+Artemia+spp.">A review of toxicity testing protocols and endpoints with Artemia spp.</a> Ecological Indicators. 69, 35-49 (2016).</li><li>Mendes Hacke, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytotoxicity+of+cymbopogon+citratus+(DC)+Stapf+fractions,+essential+oil,+citral,+and+geraniol+in+human+leukocytes+and+erythrocytes.">Cytotoxicity of cymbopogon citratus (DC) Stapf fractions, essential oil, citral, and geraniol in human leukocytes and erythrocytes.</a> Journal of Ethnopharmacology. 291, 115147 (2022).</li><li>Thangapandi, V., Pushpanathan, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+Artemia+salina+and+Artemia+fransiscana+bioassays+for+toxicity+of+Indian+medicinal+plants.">Comparison of the Artemia salina and Artemia fransiscana bioassays for toxicity of Indian medicinal plants.</a> Journal of Coastal Life Medicine. 2 (6), 453-457 (2014).</li><li>Syahmi, A. R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+oral+toxicity+and+brine+shrimp+lethality+of+Elaeis+guineensis+Jacq.,+(Oil+Palm+Leaf)+methanol+extract.">Acute oral toxicity and brine shrimp lethality of Elaeis guineensis Jacq., (Oil Palm Leaf) methanol extract.</a> Molecules. 15 (11), 8111-8121 (2010).</li><li>Sasidharan, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+toxicity+impacts+of+Euphorbia+hirta+L+extract+on+behavior,+organs+body+weight+index+and+histopathology+of+organs+of+the+mice+and+Artemia+salina.">Acute toxicity impacts of Euphorbia hirta L extract on behavior, organs body weight index and histopathology of organs of the mice and Artemia salina.</a> Pharmacognosy Research. 4 (3), 170 (2012).</li><li>Libralato, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+case+of+Artemia+spp.+in+nanoecotoxicology.">The case of Artemia spp. in nanoecotoxicology.</a> Marine Environmental Research. 101, 38-43 (2014).</li><li>Okumu, M. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artemia+salina+as+an+animal+model+for+the+preliminary+evaluation+of+snake+venom-induced+toxicity.">Artemia salina as an animal model for the preliminary evaluation of snake venom-induced toxicity.</a> Toxicon: X. 12, 100082 (2021).</li><li>Rajabi, S., Ramazani, A., Hamidi, M., Naji, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artemia+salina+as+a+model+organism+in+toxicity+assessment+of+nanoparticles.">Artemia salina as a model organism in toxicity assessment of nanoparticles.</a> DARU Journal of Pharmaceutical Sciences. 23 (1), 20 (2015).</li><li>Svensson, B. -. M., Mathiasson, L., M&#229;rtensson, L., Bergstr&#246;m, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artemia+salina+as+test+organism+for+assessment+of+acute+toxicity+of+leachate+water+from+landfills.">Artemia salina as test organism for assessment of acute toxicity of leachate water from landfills.</a> Environmental Monitoring and Assessment. 102 (1), 309-321 (2005).</li><li>Banti, C., Hadjikakou, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+toxicity+with+brine+shrimp+assay.">Evaluation of toxicity with brine shrimp assay.</a> Bio-Protocol. 11 (2), 3895 (2021).</li><li>Pecoraro, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artemia+salina:+A+microcrustacean+to+assess+engineered+nanoparticles+toxicity.">Artemia salina: A microcrustacean to assess engineered nanoparticles toxicity.</a> Microscopy Research and Technique. 84 (3), 531-536 (2021).</li><li>Lillicrap, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+approaches+to+vertebrate+ecotoxicity+tests+in+the+21st+century:+A+review+of+developments+over+the+last+2+decades+and+current+status.">Alternative approaches to vertebrate ecotoxicity tests in the 21st century: A review of developments over the last 2 decades and current status.</a> Environmental Toxicology and Chemistry. 35 (11), 2637-2646 (2016).</li><li>Ribeiro, I. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeasts+as+a+model+for+assessing+the+toxicity+of+the+fungicides+Penconazol,+Cymoxanil+and+Dichlofulanid.">Yeasts as a model for assessing the toxicity of the fungicides Penconazol, Cymoxanil and Dichlofulanid.</a> Chemosphere. (10), 1637-1642 (2000).</li><li>Armour, C. D., Lum, P. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+drug+to+protein:+using+yeast+genetics+for+high-throughput+target+discovery.">From drug to protein: using yeast genetics for high-throughput target discovery.</a> Current Opinion in Chemical Biology. 9 (1), 20-24 (2005).</li><li>Modarresi Chahardehi, A., Arsad, H., Lim, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+as+a+successful+animal+model+for+screening+toxicity+of+medicinal+plants.">Zebrafish as a successful animal model for screening toxicity of medicinal plants.</a> Plants. 9 (10), 1345 (2020).</li><li>Fischer, I., Milton, C., Wallace, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxicity+testing+is+evolving.">Toxicity testing is evolving.</a> Toxicology Research. 9 (2), 67-80 (2020).</li><li>de Ara&#250;jo, G. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+methods+in+toxicity+testing:+the+current+approach.">Alternative methods in toxicity testing: the current approach.</a> Brazilian Journal of Pharmaceutical Sciences. 50 (1), 55-62 (2014).</li><li>Toussaint, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+method+to+measure+the+sensitivity+of+yeast+cells+to+genotoxic+agents+in+liquid+cultures.">A high-throughput method to measure the sensitivity of yeast cells to genotoxic agents in liquid cultures.</a> Mutation Research/Genetic Toxicology and Environmental Mutagenesis. 606 (1), 92-105 (2006).</li><li>Horzmann, K. A., Freeman, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+waves:+New+developments+in+toxicology+with+the+zebrafish.">Making waves: New developments in toxicology with the zebrafish.</a> Toxicological Sciences. 163 (1), 5-12 (2018).</li><li>Avdesh, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regular+care+and+maintenance+of+a+zebrafish+(Danio+rerio)+laboratory:+An+introduction.">Regular care and maintenance of a zebrafish (Danio rerio) laboratory: An introduction.</a> Journal of Visualized Experiments. (69), e4196 (2012).</li><li>Cunliffe, V. T., N&#252;sslein-Volhard, C., Dahm, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Zebrafish: A Practical Approach. , (2002).</li><li>Sitarek, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insight+the+biological+activities+of+selected+Abietane+Diterpenes+isolated+from+Plectranthus+spp.">Insight the biological activities of selected Abietane Diterpenes isolated from Plectranthus spp.</a> Biomolecules. 10 (2), 194 (2020).</li><li>Matias, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytotoxic+activity+of+Royleanone+Diterpenes+from+Plectranthus+madagascariensis+Benth.">Cytotoxic activity of Royleanone Diterpenes from Plectranthus madagascariensis Benth.</a> ACS Omega. 4 (5), 8094-8103 (2019).</li><li>Garcia, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Royleanone+derivatives+from+Plectranthus+spp.+as+a+novel+class+of+P-glycoprotein+inhibitors.">Royleanone derivatives from Plectranthus spp. as a novel class of P-glycoprotein inhibitors.</a> Frontiers in Pharmacology. 11, (2020).</li></ol>

During the last years, the scientific community has increased its attention toward alternative models for toxicity screenings21. Beside A. salina lethality bioassay, other methodologies are usually performed for the evaluation of sample tolerability and include vertebrate bioassays (such as rodents), invertebrates (such as zebrafish), in vitro methods using yeast strains or cells, and in silico methods22,23,...

Natural products from plants, animals, or microorganisms have been a growing area of interest over the years in the development of new bioactive molecules because of their varied range of biological and pharmacological activities1. However, the associated side effects, drug resistance, or inadequate specificity of the agents, especially when used as anticancer drugs, represent the major factors that can lead to ineffective treatment1,2.
Over the last few decades, several plant-derived cytotoxic agents have been discovered, some of them used as anticancer agents1,2,3. In this context, paclitaxel is reported as one of the best-known and most active chemotherapeutic drugs of natural origin3,4. Currently, it is estimated that more than 35% of all medicines on the market are derived from or are inspired by natural products5. The potential high toxicity of these compounds requires consideration during all of the study phases, since different types of contaminants or even metabolic components of the plant itself can cause toxic effects. For this reason, pharmacological and toxicological profiles should be undertaken in the preliminarily phase, to assess the biological activity and safety of new potential plant-based treatments. To evaluate the toxicity of new bioactive samples, invertebrate animals can be considered as the best models to study. They demand minimal ethical requirements and allow preliminary in vitro assays, to prioritize the most promising products for the next round of testing in vertebrates1,6.
Commonly known as brine shrimp, A. salina is a small halophilic invertebrate belonging to the genus Artemia (family Artemiidae, order Anostraca, subphylum Crustacea; Figure 1). In marine and aquatic saline ecosystems, brine shrimps play an important nutritional role as they feed on microalgae and are constituents of the zooplankton used to feed fish. Moreover, their larvae (known as nauplii) are widely used in the assessment of general toxicity during preliminary studies1,3,7.
Artemia spp. are widely used in lethality studies and are also a convenient starting point for toxicity assessments, by tracking the toxicity of potentially bioactive compounds based on their ability to kill nauplii grown in the laboratory1,8. For this reason, the use of A. salina gained attraction in general toxicity studies, because it is a very efficient and easy-to-use method, compared to other tests on animal models9.
Owing to their simple anatomy, tiny size and short life cycle, a vast number of invertebrates can be studied in a single experiment. As such, they combine genetic amenability and low-cost compatibility with large-scale screenings1. In this context, the use of brine shrimp in a general toxicity assay shows several advantages, such as fast growth (28-72 h is needed from hatching to the first results), cost-effectiveness, and long shelf-life of commercial eggs, that can be used all year round3,10. On the other hand, since invertebrates have a primitive organ system and lack an adaptive immune system, they do not represent a perfect and reliable model for human cells1.
However, it provides a preliminary evaluation method for the general toxicity of selected samples. Since it is widely used as a lethality assay, it can provide provisional indications about the toxic effects of potential anticancer agents. It is often also used to obtain feedback about the general toxicity of compounds endowed with any other biological activities for which it is essential to show the lowest mortality rate possible among the Artemia shrimps.
In an ongoing study from our group, different extracts from Plectranthus species showed antioxidant and antimicrobial activities (unpublished results). In parallel, isolated compounds were obtained by purification of the extracts and were then chemically modified. The extracts, pure compounds, and semisynthetic derivatives were then tested in terms of general toxicity. In this context, the present work aims to give an overview of the use of the Artemia lethality bioassay for the evaluation of general toxicity and potential cytotoxic activity of bioactive extracts and isolated compounds from different plants of the genus Plectranthus11.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64472/64472fig01.jpg" /> 
Figure 1: Artemia salina under the microscope. Newly hatched nauplii of A. salina as seen under the microscope (magnification 12x). <a href="https://www.jove.com/files/ftp_upload/64472/64472fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>24-well plates</td>
			<td>Thermo Fisher Scientific, Denmark</td>
			<td>174899</td>
			<td>Thermo Scientific Nunc Up Cell 24 multidish</td>
		</tr>
		<tr>
			<td>Aluminium foil</td>
			<td>Albal</td>
			<td>-</td>
			<td>Can be purchased in supermarket</td>
		</tr>
		<tr>
			<td>Artemio Set</td>
			<td>JBL GmbH and Co. KG, D-67141, Neuhofen Germany</td>
			<td>61066000</td>
			<td>Can be purchased in pet shops</td>
		</tr>
		<tr>
			<td>Binocular microscope</td>
			<td>Ceti, Belgium</td>
			<td>&#160;1700.0000</td>
			<td>Flexum-24AED, 220-240 V, 50 Hz</td>
		</tr>
		<tr>
			<td>Bottles</td>
			<td>-</td>
			<td>-</td>
			<td>0.5 L Diameter: 5.8 cm; Height: 12 cm</td>
		</tr>
		<tr>
			<td>Brine shrimp cysts</td>
			<td>JBL GmbH and Co. KG, D-67141, Neuhofen Germany</td>
			<td>3090700</td>
			<td>Can be purchased in pet shops</td>
		</tr>
		<tr>
			<td>Brine shrimp salt</td>
			<td>JBL GmbH and Co. KG, D-67141, Neuhofen Germany</td>
			<td>3090600</td>
			<td>Can be purchased in pet shops</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>VWR chemicals</td>
			<td>CAS: 67-68-5</td>
			<td>&#160;99% purity</td>
		</tr>
		<tr>
			<td>Discartable tips</td>
			<td>Diamond</td>
			<td>F171500</td>
			<td>Volume range: 100 - 1000 &#181;L</td>
		</tr>
		<tr>
			<td>Eppendorf microtubes</td>
			<td>BRAND</td>
			<td>7,80,546</td>
			<td>Microtubes, PP, 2 mL, BIO-CERT&#160;PCR QUALITY</td>
		</tr>
		<tr>
			<td>Erlenmeyer flask</td>
			<td>VWR chemicals</td>
			<td>4,47,109</td>
			<td>volume: 100 mL</td>
		</tr>
		<tr>
			<td>Glass beaker</td>
			<td>Normax</td>
			<td>3.2111654N</td>
			<td>Volume: 1000 mL</td>
		</tr>
		<tr>
			<td>Gloves</td>
			<td>Guantes Luna</td>
			<td>GLSP3</td>
			<td>-</td>
		</tr>
		<tr>
			<td>GraphPad Prism</td>
			<td>GraphPad Software, San Diego, CA, USA</td>
			<td>-</td>
			<td>GraphPad Prism version 5.00 for Windows, www.graphpad.com, accessed on 5 February 2021; commercial statistical analysis software</td>
		</tr>
		<tr>
			<td>Home-made A. salina Grower</td>
			<td>&#160;-</td>
			<td>&#160;-</td>
			<td>Home made: two plastic bottles connected by a hose</td>
		</tr>
		<tr>
			<td>Hot glue</td>
			<td>Parkside</td>
			<td>PHP500E3</td>
			<td>230 V, 50 Hz, 25 W</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Heidolph Instruments, Denmark&#160;</td>
			<td>&#160;-</td>
			<td>One Heidolph Unimax 1010 equipment and one Heidolph Inkubator 1006</td>
		</tr>
		<tr>
			<td>Light</td>
			<td>Roblan</td>
			<td>SKYC3008FE14</td>
			<td>LED light bulb</td>
		</tr>
		<tr>
			<td>Micropipettes</td>
			<td>VWR chemicals</td>
			<td>613-5265</td>
			<td>Volume range: 100 - 1000 &#181;L</td>
		</tr>
		<tr>
			<td>Potassium dichromate (K2Cr2O7)</td>
			<td>VWR chemicals</td>
			<td>CAS: 7778-50-9</td>
			<td>&#160;99% purity</td>
		</tr>
		<tr>
			<td>Pump ProAir a50</td>
			<td>JBL GmbH and Co. KG, D-67141, Neuhofen Germany</td>
			<td>&#160;-</td>
			<td>Included in the Artemio Set+1 kit</td>
		</tr>
		<tr>
			<td>Rubber tube</td>
			<td>-</td>
			<td>-</td>
			<td>1.3 cm outer and 0.9 cm inner diameter</td>
		</tr>
		<tr>
			<td>Stirring rod</td>
			<td>VWR chemicals</td>
			<td>441-0147</td>
			<td><img alt="Equation 1" src="/files/ftp_upload/64472/64472eq02.jpg" style="margin: auto;" /> 6 mm, 250 mm</td>
		</tr>
		<tr>
			<td>Termometer</td>
			<td>VWR chemicals</td>
			<td>620-0821</td>
			<td>0 - 100 &#176;C</td>
		</tr>
	</tbody>
</table>

lethality bioassay using artemia salina l.

Natural products have been used since ancient times to produce medicines. Nowadays, there are plenty of chemotherapeutic drugs obtained from natural sources and used against a plethora of diseases. Unfortunately, most of these compounds often display systemic toxicity and adverse effects. In order to better evaluate the tolerability of selected potentially bioactive samples, brine shrimp (Artemia salina) is generally used as a model in lethality studies. The A. salina test is based on the ability of the studied bioactive compounds to kill the microcrustaceans in their larval stage (nauplii). This method represents a convenient starting point for cytotoxicity studies, as well as for the general toxicity screening of synthetic, semisynthetic, and natural products. It can be considered a simple, quick, and low-cost assay, compared to many other assays (in vitro cells or yeast strains, zebrafish, rodents) generally suitable for the aforementioned purposes; moreover, it can be easily performed even without any specific training. Overall, A. salina assay represents a useful tool for the preliminary toxicity evaluation of selected compounds and the bio-guided fractionation of natural product extracts.

The general toxicity of some natural products recently studied by our group was evaluated through the brine shrimp lethality bioassay. Four extracts (Pa- P. ambigerus; Pb- P. barbatus; Pc- P. cylindraceus; and Pe- P. ecklonii) from Plectranthus genus, known for their antioxidant activity (unpublished results), were tested. Additionally, two natural compounds (1 and 2) obtained from Plectranthus spp., and three semi-synthetic derivatives (3, 4, 5; ...

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Lethality Bioassay Using Artemia salina L.

This work aims to evaluate and review the Artemia salina lethality bioassay procedure, also identified as brine shrimp lethality assay. This simple and cheap method gives information about the general toxicity (considered as a preliminary toxicity evaluation) of samples, namely, natural products.

Lethality Bioassay Using Artemia salina L.

Natural products have been used since ancient times to produce medicines. Nowadays, there are plenty of chemotherapeutic drugs obtained from natural ...

This protocol is a very useful tool for a preliminary general toxicity evaluation on different types of samples, such as extracts, fractions, and compounds from different natural or synthetic sources. The main advantage of our protocol is that it's very easy and cheap and can be used on many samples at the same time. Moreover, the handmade equipment improves the reliability of the site.In this technique stage, we do a screening to select the samples based on their preliminary general toxicity profile that will undergo further specific tests, such as expansive cell lines or even in-vivo tests. This method is mainly exploited in medicinal and other products chemistry fields to screen biotic samples. Our protocol can also be used for other kinds of samples such as nanosystems.While performing for the first time, be patient because this needs fine observation skills and a lot of attention to all the parameters reported Marcia Filipe and Vera Isca, the PhD students from our lab will help to demonstrate this procedure. Begin by placing the funnel shaped container in the black support and turning the funnel in a suitable direction to see the level mark and the tap. To make the handmade migration equipment cut the top of two 0.5 liter plastic bottles to obtain a final height of 12 centimeters.Create a hole of 1.5 centimeter diameter on one side and seven centimeters from the bottom of each bottle and insert a 13 centimeter rubber tube between the two openings. Seal the openings with hot glue and leave them to dry for 15 minutes. Put the bottles on a flat surface and fill them with water to verify there is no leakage.Add 28 grams of the salt to 800 milliliters of tap water to prepare the artificial salt solution, or brine shrimp salt at a concentration of 35 grams per liter. Mix it with a stirring rod until all the salt is thoroughly dissolved. To prepare the samples dissolve a suitable amount of each extract and compound dimethylsulfoxide, or DMSO, at a final concentration of 10 milligrams per milliliter.Then dilute 10 microliters of each sample in a new micro centrifuge tube using 990 microliters of the artificial saline solution to obtain a final concentration of 0.1 milligrams per milliliter. Under a fume hood in an Erlenmeyer flask, prepare a solution of potassium dichromate in distilled water at a concentration of one milligram per milliliter. Fill the hatching vessel with the medium up to the 500 milliliter mark and add one spoon, or approximately 0.5 grams of brine shrimp cysts to the salt solution.Close the container. Place a lamp pointing directly toward the equipment and turn it on. Attach the air supplier system to the connector placed on top of the equipment and turn the pump on.Brine shrimp cysts hatch in the artificial salt solution under vigorous aeration, continuous lighting, and stable temperature. After 24 to 48 hours, when the eggs have hatched, turn the air pump off and wait until the nauplii are separated from the empty egg cases. To separate the unhatched eggs from the alive nauplii, open the outlet tap at the bottom and discharge the content of the funnel in one of the containers of the handmade migration equipment container.Ensure that the solution containing the nauplii and the residual unhatched eggs is below the tube level. Place the equipment in the incubator for four hours, then in the second container add the residual salt solution above the height of the tube. Cover the container with nauplii and the residual unhatched eggs using aluminum foil and place the lamp on the second container with just the salt solution.The brine shrimp will be attracted by the light and migrate from one container to another, leading to efficient separation between eggs and alive artemia. From the harvesting container, collect 900 microliters of saline solution containing 10 to 15 nauplii and place the solution in one well of a 24 well plate. All the samples are tested in quadruplicates.Add 100 microliters of each negative control. Positive control, the artificial salt solution, and each of the samples to the wells and incubate the plate under illumination for 24 hours. After 24 hours, register the number of dead larvae in each well under a binocular microscope at 12 x magnification.Add 100 microliters of potassium dichromate solution to induce the death of the remaining living larvae and wait for six hours. Count the total dead larva in each well under a microscope and determine the mortality rate according to the equation shown on the screen. Perform all the tests in triplicates and calculate the standard deviations.Finally, express the results as the mean of three independent experiments each with internal quadruplicates plus, minus standard deviations. The structure of the selected compounds extracted from Plectranthus species and obtained by semi-synthesis are shown in this figure. A newly hatched nauplii of Artemia Salina as seen under the microscope is shown in this image.This graphical image represents the mortality rate of Artemia Salina after 24 hours of exposure to four methanol extracts of Plectranthus species at 0.1 milligrams per milliliter. All the extracts were acceptable in terms of general toxicity using this assay. Here salt corresponds to the salt solution or blank.Potassium dichromate was used as a positive control and DMSO was used as a negative control. All the tested extracts showed very encouraging results with very low values of mortality percentages comparable to those registered for the blank and the negative control. The mortality rate of Artemia Salina after 24 hours of exposure to the five pure compounds at 0.1 milligrams per milliliter is presented in this figure.From this data, it is evident that only five shows minimal toxicity using this assay You need to remember three steps in particular, hatching, migration and collection. Consequently, we have other shrimp separated from and the numbers to work with. The preliminary site is a simple and low cost model channel toxicity compared to other sites like cell culture or zebra fish.Further, more expensive cytotoxicity sites can then be exploited.

lethality-bioassay-using-artemia-salina-l

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Chemistry

Large Scale Non-targeted Metabolomic Profiling of Serum by Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS)

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to investigate metabolism. The approach offers an unbiased and in-depth analysis that can enable the development of diagnostic tests, novel therapies, and further our understanding of disease processes. The inherent chemical diversity of the metabolome creates significant analytical challenges and there is no single experimental approach that can detect all metabolites. Additionally, the biological variation in individual metabolism and the dependence of metabolism on environmental factors necessitates large sample numbers to achieve the appropriate statistical power required for meaningful biological interpretation. To address these challenges, this tutorial outlines an analytical workflow for large scale non-targeted metabolite profiling of serum by UPLC-MS. The procedure includes guidelines for sample organization and preparation, data acquisition, quality control, and metabolite identification and will enable reliable acquisition of data for large experiments and provide a starting point for laboratories new to non-targeted metabolite profiling by UPLC-MS.

Large Scale Non-targeted Metabolomic Profiling of Serum by Ultra Performance Liquid Chromatography-Mass Spectrometry UPLC-MS

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to investigate metabolism. This article outlines a typical workflow utilized for non-targeted metabolite profiling of serum including sample organization and preparation, data acquisition, data analysis, quality control, and metabolite identification.

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to ...

Covalent Binding of BMP-2 on Surfaces Using a Self-assembled Monolayer Approach

Bone morphogenetic protein 2 (BMP-2) is a growth factor embedded in the extracellular matrix of bone tissue. BMP-2 acts as trigger of mesenchymal cell differentiation into osteoblasts, thus stimulating healing and de novo bone formation. The clinical use of recombinant human BMP-2 (rhBMP-2) in conjunction with scaffolds has raised recent controversies, based on the mode of presentation and the amount to be delivered. The protocol presented here provides a simple and efficient way to deliver BMP-2 for in vitro studies on cells. We describe how to form a self-assembled monolayer consisting of a heterobifunctional linker, and show the subsequent binding step to obtain covalent immobilization of rhBMP-2. With this approach it is possible to achieve a sustained presentation of BMP-2 while maintaining the biological activity of the protein. In fact, the surface immobilization of BMP-2 allows targeted investigations by preventing unspecific adsorption, while reducing the amount of growth factor and, most notably, hindering uncontrolled release from the surface. Both short- and long-term signaling events triggered by BMP-2 are taking place when cells are exposed to surfaces presenting covalently immobilized rhBMP-2, making this approach suitable for in vitro studies on cell responses to BMP-2 stimulation.

We describe a method for accomplishing efficient immobilization of BMP-2 on surfaces. Our approach is based on the formation of a self-assembled monolayer to achieve the covalent binding of BMP-2 via its free amine residues. This method is a useful tool to study signaling at the cell membrane.

Bone morphogenetic protein 2 (BMP-2) is a growth factor embedded in the extracellular matrix of bone tissue. BMP-2 acts as trigger of mesenchymal cell ...

Dependence of Laser-induced Breakdown Spectroscopy Results on Pulse Energies and Timing Parameters Using Soil Simulants

The dependence of some LIBS detection capabilities on lower pulse energies (&lt;100 mJ) and timing parameters were examined using synthetic silicate samples. These samples were used as simulants for soil and contained minor and trace elements commonly found in soil at a wide range of concentrations. For this study, over 100 calibration curves were prepared using different pulse energies and timing parameters; detection limits and sensitivities were determined from the calibration curves. Plasma temperatures were also measured using Boltzmann plots for the various energies and the timing parameters tested. The electron density of the plasma was calculated using the full-width half maximum (FWHM) of the hydrogen line at 656.5 nm over the energies tested. Overall, the results indicate that the use of lower pulse energies and non-gated detection do not seriously compromise the analytical results. These results are very relevant to the design of field- and person-portable LIBS instruments.

LIBS detection capabilities on soil simulants were tested using a range of pulse energies and timing parameters. Calibration curves were used to determine detection limits and sensitivities for different parameters. Generally, the results showed that there was not a significant reduction in detection capabilities using lower pulse energies and non-gated detection.

The  dependence of some LIBS detection capabilities on lower pulse energies (&lt;100  mJ) and timing parameters were examined using synthetic silicate ...

High-pressure Sapphire Cell for Phase Equilibria Measurements of CO2/Organic/Water Systems

The high pressure sapphire cell apparatus was constructed to visually determine the composition of multiphase systems without physical sampling. Specifically, the sapphire cell enables visual data collection from multiple loadings to solve a set of material balances to precisely determine phase composition. Ternary phase diagrams can then be established to determine the proportion of each component in each phase at a given condition. In principle, any ternary system can be studied although ternary systems (gas-liquid-liquid) are the specific examples discussed herein. For instance, the ternary THF-Water-CO2 system was studied at 25 and 40&#160;&#176;C and is described herein. Of key importance, this technique does not require sampling. Circumventing the possible disturbance of the system equilibrium upon sampling, inherent measurement errors, and technical difficulties of physically sampling under pressure is a significant benefit of this technique. Perhaps as important, the sapphire cell also enables the direct visual observation of the phase behavior. In fact, as the CO2 pressure is increased, the homogeneous THF-Water solution phase splits at about 2 MPa. With this technique, it was possible to easily and clearly observe the cloud point and determine the composition of the newly formed phases as a function of pressure.
The data acquired with the sapphire cell technique can be used for many applications. In our case, we measured swelling and composition for tunable solvents, like gas-expanded liquids, gas-expanded ionic liquids and Organic Aqueous Tunable Systems (OATS)1-4. For the latest system, OATS, the high-pressure sapphire cell enabled the study of (1) phase behavior as a function of pressure and temperature, (2) composition of each phase (gas-liquid-liquid) as a function of pressure and temperature and (3) catalyst partitioning in the two liquid phases as a function of pressure and composition. Finally, the sapphire cell is an especially effective tool to gather accurate and reproducible measurements in a timely fashion.

High-pressure Sapphire Cell for Phase Equilibria Measurements of CO2/Organic/Water Systems

The high pressure sapphire cell apparatus is a unique tool to study, without sampling, phase behavior under a wide range of pressures. Using a cathetometer, very precise volume measurements can be recorded to measure liquid expansion and phase composition. Thus, this synthetic method enables the study of (1) phase equilibria of multi-component mixtures and (2) the partition behavior of catalyst or model compounds as a function of pressure.

The high pressure sapphire cell apparatus was constructed to visually determine the composition of multiphase systems without physical sampling. ...

The Use of Gas Chromatography to Analyze Compositional Changes of Fatty Acids in Rat Liver Tissue during Pregnancy

Gas chromatography (GC) is a highly sensitive method used to identify and quantify the fatty acid content of lipids from tissues, cells, and plasma/serum, yielding results with high accuracy and high reproducibility. In metabolic and nutrition studies GC allows assessment of changes in fatty acid concentrations following interventions or during changes in physiological state such as pregnancy. Solid phase extraction (SPE) using aminopropyl silica cartridges allows separation of the major lipid classes including triacylglycerols, different phospholipids, and cholesteryl esters (CE). GC combined with SPE was used to analyze the changes in fatty acid composition of the CE fraction in the livers of virgin and pregnant rats that had been fed various high and low fat diets. There are significant diet/pregnancy interaction effects upon the omega-3 and omega-6 fatty acid content of liver CE, indicating that pregnant females have a different response to dietary manipulation than is seen among virgin females.

Pregnancy leads to significant changes to the fatty acid composition of maternal tissues. Lipid profiles can be obtained via gas chromatography to allow identification and quantification of fatty acids in individual lipid classes among rats fed various high and low fat diets during pregnancy.

Gas chromatography (GC) is a highly sensitive method used to identify and quantify the fatty acid content of lipids from tissues, cells, and plasma/serum, ...

HPLC Measurement of the DNA Oxidation Biomarker, 8-oxo-7,8-dihydro-2&#8217;-deoxyguanosine, in Cultured Cells and Animal Tissues

Oxidative stress is associated with many physiological and pathological processes, as well as xenobiotic metabolism, leading to the oxidation of biomacromolecules, including DNA. Therefore, efficient detection of DNA oxidation is important for a variety of research disciplines, including medicine and toxicology. A common biomarker of oxidatively damaged DNA is 8-oxo-7,8-dihydro-2&#39;-deoxyguanosine (8-oxo-dGuo; often erroneously referred to as 8-hydroxy-2&#39;-deoxyguanosine (8-OH-dGuo or 8-oxo-dG)). Several protocols for 8-oxo-dGuo measurement by high pressure liquid chromatography with electrochemical detection (HPLC-ED) have been described. However, these were mainly applied to purified DNA treated with pro-oxidants. In addition, due to methodological differences between laboratories, mainly due to differences in analytical equipment, the adoption of published methods for detection of 8-oxo-dGuo by HPLC-ED requires careful optimization by each laboratory. A comprehensive protocol, describing such an optimization process, is lacking. Here, a detailed protocol is described for the detection of 8-oxo-dGuo by HPLC-ED, in DNA from cultured cells or animal tissues. It illustrates how DNA sample preparation can be easily and rapidly optimized to minimize undesirable DNA oxidation that can occur during sample preparation. This protocol shows how to detect 8-oxo-dGuo in cultured human alveolar adenocarcinoma cells (i.e., A549 cells) treated with the oxidizing agent KBrO3, and from the spleen of mice exposed to the polycyclic aromatic hydrocarbon dibenzo(def,p)chrysene (DBC, formerly known as dibenzo(a,l)pyrene, DalP). Overall, this work illustrates how an HPLC-ED methodology can be readily optimized for the detection of 8-oxo-dGuo in biological samples.

HPLC Measurement of the DNA Oxidation Biomarker, 8-oxo-7,8-dihydro-2&amp;#8217;-deoxyguanosine, in Cultured Cells and Animal Tissues

The goal of this protocol is the detection of the DNA oxidation marker, 8-oxo-7,8-dihydro-2&#39;-deoxyguanosine (8-oxo-dGuo) by HPLC-ED, in DNA from cultured cells or animal tissues.

Oxidative stress is associated with many physiological and pathological processes, as well as xenobiotic metabolism, leading to the oxidation of ...

Improved Heterojunction Quality in Cu2O-based Solar Cells Through the Optimization of Atmospheric Pressure Spatial Atomic Layer Deposited Zn1-xMgxO

Atmospheric pressure spatial atomic layer deposition (AP-SALD) was used to deposit n-type ZnO and Zn1-xMgxO thin films onto p-type thermally oxidized Cu2O substrates outside vacuum at low temperature. The performance of photovoltaic devices featuring atmospherically fabricated ZnO/Cu2O heterojunction was dependent on the conditions of AP-SALD film deposition, namely, the substrate temperature and deposition time, as well as on the Cu2O substrate exposure to oxidizing agents prior to and during the ZnO deposition. Superficial Cu2O to CuO oxidation was identified as a limiting factor to heterojunction quality due to recombination at the ZnO/Cu2O interface. Optimization of AP-SALD conditions as well as keeping Cu2O away from air and moisture in order to minimize Cu2O surface oxidation led to improved device performance. A three-fold increase in the open-circuit voltage (up to 0.65 V) and a two-fold increase in the short-circuit current density produced solar cells with a record 2.2% power conversion efficiency (PCE). This PCE is the highest reported for a Zn1-xMgxO/Cu2O heterojunction formed outside vacuum, which highlights atmospheric pressure spatial ALD as a promising technique for inexpensive and scalable fabrication of Cu2O-based photovoltaics.

Improved Heterojunction Quality in Cu2O-based Solar Cells Through the Optimization of Atmospheric Pressure Spatial Atomic Layer Deposited Zn1-xMgxO

Here we present a protocol for synthesizing Zn1-xMgxO/Cu2O heterojunctions in open-air at low temperature via atmospheric pressure spatial atomic layer deposition (AP-SALD) of Zn1-xMgxO on cuprous oxide. Such high quality conformal metal oxides can be grown on a variety of substrates including plastics by this cheap and scalable method.

Atmospheric pressure spatial atomic layer deposition (AP-SALD) was used to deposit n-type ZnO and Zn1-xMgxO thin films onto p-type thermally oxidized Cu2O ...

Characterization, Quantification and Compound-specific Isotopic Analysis of Pyrogenic Carbon Using Benzene Polycarboxylic Acids (BPCA)

Fire-derived, pyrogenic carbon (PyC), sometimes called black carbon (BC), is the carbonaceous solid residue of biomass and fossil fuel combustion, such as char and soot. PyC is ubiquitous in the environment due to its long persistence, and its abundance might even increase with the projected increase in global wildfire activity and the continued burning of fossil fuel. PyC is also increasingly produced from the industrial pyrolysis of organic wastes, which yields charred soil amendments (biochar). Moreover, the emergence of nanotechnology may also result in the release of PyC-like compounds to the environment. It is thus a high priority to reliably detect, characterize and quantify these charred materials in order to investigate their environmental properties and to understand their role in the carbon cycle.
Here, we present the benzene polycarboxylic acid (BPCA) method, which allows the simultaneous assessment of PyC&#39;s characteristics, quantity and isotopic composition (13C and 14C) on a molecular level. The method is applicable to a very wide range of environmental sample materials and detects PyC over a broad range of the combustion continuum, i.e., it is sensitive to slightly charred biomass as well as high temperature chars and soot. The BPCA protocol presented here is simple to employ, highly reproducible, as well as easily extendable and modifiable to specific requirements. It thus provides a versatile tool for the investigation of PyC in various disciplines, ranging from archeology and environmental forensics to biochar and carbon cycling research.

Characterization, Quantification and Compound-specific Isotopic Analysis of Pyrogenic Carbon Using Benzene Polycarboxylic Acids BPCA

We present the benzene polycarboxylic acid (BPCA) method for assessing pyrogenic carbon (PyC) in the environment. The compound-specific approach uniquely provides simultaneous information about the characteristics, quantity and isotopic composition (13C and 14C) of PyC.

Fire-derived, pyrogenic carbon (PyC), sometimes called black carbon (BC), is the carbonaceous solid residue of biomass and fossil fuel combustion, such as ...

Nanosponge Tunability in Size and Crosslinking Density

We describe a protocol for the synthesis of linear polyesters containing pendant epoxide functionality and their incorporation into a nanosponge with controlled dimensions. This approach begins with synthesis of a functionalized lactone which is key to the pendant functionalization of the resulting polymer. Valerolactone (VL) and allyl-valerolactone (AVL) are then copolymerized using ring-opening polymerization. Post-polymerization modification is then used to install an epoxide moiety on some or all of the pendant allyl groups. Epoxy-amine chemistry is employed to form nanoparticles in a dilute solution of both polymer and small molecule diamine crosslinker based on the desired nanosponge size and crosslinking density. Nanosponge sizes can be characterized by transmission electron microscopy (TEM) imaging to determine the dimension and distribution. This method provides a pathway by which highly tunable polyesters can create tunable nanoparticles, which can be used for small molecule drug encapsulation. Due to the nature of the backbone, these particles are hydrolytically and enzymatically degradable for a controlled release of a wide range of hydrophobic small molecules.

This article describes a process for tuning the size and crosslinking density of covalently crosslinked nanoparticles from linear polyesters containing pendant functionality. By tailoring synthesis parameters (polymer molecular weight, pendant functionality incorporation, and crosslinker equivalents), a desired nanoparticle size and crosslinking density can be achieved for drug delivery applications.

We describe a protocol for the synthesis of linear polyesters containing pendant epoxide functionality and their incorporation into a nanosponge with ...

Enzymatic Cascade Reactions for the Synthesis of Chiral Amino Alcohols from L-lysine

Amino alcohols are versatile compounds with a wide range of applications. For instance, they have been used as chiral scaffolds in organic synthesis. Their synthesis by conventional organic chemistry often requires tedious multi-step synthesis processes, with difficult control of the stereochemical outcome. We present a protocol to enzymatically synthetize amino alcohols starting from the readily available L-lysine in 48 h. This protocol combines two chemical reactions that are very difficult to conduct by conventional organic synthesis. In the first step, the regio- and diastereoselective oxidation of an unactivated C-H bond of the lysine side-chain is catalyzed by a dioxygenase; a second regio- and diastereoselective oxidation catalyzed by a regiodivergent dioxygenase can lead to the formation of the 1,2-diols. In the last step, the carboxylic group of the alpha amino acid is cleaved by a pyridoxal-phosphate (PLP) decarboxylase (DC). This decarboxylative step only affects the alpha carbon of the amino acid, retaining the hydroxy-substituted stereogenic center in a beta/gamma position. The resulting amino alcohols are therefore optically enriched. The protocol was successfully applied to the semipreparative-scale synthesis of four amino alcohols. Monitoring of the reactions was conducted by high performance liquid chromatography (HPLC) after derivatization by 1-fluoro-2,4-dinitrobenzene. Straightforward purification by solid-phase extraction (SPE) afforded the amino alcohols with excellent yields (93% to &#62;95%).

Chiral amino alcohols are versatile molecules for use as scaffolds in organic synthesis. Starting from L-lysine, we synthesize amino alcohols by an enzymatic cascade reaction combining diastereoselective C-H oxidation catalyzed by dioxygenase followed by cleavage of the carboxylic acid moiety of the corresponding hydroxyl amino acid by a decarboxylase.

Amino alcohols are versatile compounds with a wide range of applications. For instance, they have been used as chiral scaffolds in organic synthesis. ...