Name: employing pressurized hot water extraction (phwe) to explore natural products chemistry in the undergraduate laboratory
Uploaded: 2018-11-07T13:00:00
Description: Watch this Scientific Journal Video about Employing Pressurized Hot Water Extraction PHWE to Explore Natural Products Chemistry in the Undergraduate Laboratory at JoVE.com

The authors acknowledge the School of Natural Sciences - Chemistry, University of Tasmania and the School of Chemistry, The University of Sydney for financial support. B.J.D. and J.J. thank the Australian Government for Research Training Program Scholarships.

NOTE: It is advisable that all procedures are performed in a fume hood. Students must wear appropriate personal protective equipment at all times in the laboratory and the safety data sheets (SDS) associated with each reagent must be consulted before use.
1. PHWE of cloves: isolation of eugenol and acetyleugenol
<ol>
	<li>Extraction of eugenol and acetyleugenol from cloves
	<ol>
		<li>Place coarsely ground cloves (12.5 g) in a 250-mL beaker.</li>
		<li>Add s...

<ol>
	<li>Newman, D. J., Cragg, G. M. <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+as+Sources+of+New+Drugs+from+1981+to+2014.">Natural Products as Sources of New Drugs from 1981 to 2014.</a> Journal of Natural Products. 79, 629-661 (2016).</li><li>Barnes, E. C., Kumar, R., Davis, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+isolated+natural+products+as+scaffolds+for+the+generation+of+chemically+diverse+screening+libraries+for+drug+discovery.">The use of isolated natural products as scaffolds for the generation of chemically diverse screening libraries for drug discovery.</a> Natural Product Reports. 33, 372-381 (2016).</li><li>DeCorte, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Underexplored+Opportunities+for+Natural+Products+in+Drug+Discovery.">Underexplored Opportunities for Natural Products in Drug Discovery.</a> Journal of Medicinal Chemistry. 59, 9295-9304 (2016).</li><li>Bucar, F., Wube, A., Schmid, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+product+isolation+-+how+to+get+from+biological+material+to+pure+compounds.">Natural product isolation - how to get from biological material to pure compounds.</a> Natural Product Reports. 30, 525-545 (2013).</li><li>Sticher, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+product+isolation.">Natural product isolation.</a> Natural Product Reports. 25, 517-554 (2008).</li><li>Just, J., Deans, B. J., Olivier, W. J., Paull, B., Bissember, A. C., Smith, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+Method+for+the+Rapid+Extraction+of+Natural+Products:+Efficient+Isolation+of+Shikimic+Acid+from+Star+Anise.">New Method for the Rapid Extraction of Natural Products: Efficient Isolation of Shikimic Acid from Star Anise.</a> Organic Letters. 17, 2428-2430 (2015).</li><li>Caprioli, G., Cortese, M., Cristalli, G., Maggi, F., Odello, L., Ricciutelli, M., Sagratini, G., Sirocchi, V., Tomassoni, G., Vittori, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+espresso+machine+parameters+through+the+analysis+of+coffee+odorants+by+HS-SPME-GC/MS.">Optimization of espresso machine parameters through the analysis of coffee odorants by HS-SPME-GC/MS.</a> Food Chemistry. 135, 1127-1133 (2012).</li><li>Just, J., Jordan, T. B., Paull, B., Bissember, A. C., Smith, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Practical+isolation+of+polygodial+from+Tasmannia+lanceolata:+a+viable+scaffold+for+synthesis.">Practical isolation of polygodial from Tasmannia lanceolata: a viable scaffold for synthesis.</a> Organic Biomolecular Chemistry. 13, 11200-11207 (2015).</li><li>Just, J., Bunton, G. L., Deans, B. J., Murray, N. L., Bissember, A. C., Smith, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+of+Eugenol+from+Cloves+Using+an+Unmodified+Household+Espresso+Machine:+An+Alternative+to+Traditional+Steam+Distillation.">Extraction of Eugenol from Cloves Using an Unmodified Household Espresso Machine: An Alternative to Traditional Steam Distillation.</a> Journal of Chemical Education. 93, 213-216 (2016).</li><li>Deans, B. J., Bissember, A. C., Smith, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Practical+Isolation+of+Asperuloside+from+Coprosma+quadrifida+via+Rapid+Pressurised+Hot+Water+Extraction.">Practical Isolation of Asperuloside from Coprosma quadrifida via Rapid Pressurised Hot Water Extraction.</a> Australian Journal of Chemistry. 69, 1219-1222 (2016).</li><li>Deans, B. J., Just, J., Chetri, J., Burt, L. K., Smith, J. N., Kilah, N. L., de Salas, M., Gueven, N., Bissember, A. C., Smith, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pressurized+Hot+Water+Extraction+as+a+Viable+Bioprospecting+Tool:+Isolation+of+Coumarin+Natural+Products+from+Previously+Unexamined+Correa+(Rutaceae).">Pressurized Hot Water Extraction as a Viable Bioprospecting Tool: Isolation of Coumarin Natural Products from Previously Unexamined Correa (Rutaceae).</a> ChemistrySelect. 2, 2439-2443 (2017).</li><li>Deans, B. J., Olivier, W. J., Girbino, D., Bissember, A. C., Smith, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+of+carboxylic+acid-containing+diterpenoids+from+Dodonaea+viscosa+via+pressurised+hot+water+extraction.">Extraction of carboxylic acid-containing diterpenoids from Dodonaea viscosa via pressurised hot water extraction.</a> Fitoterapia. 126, 65-68 (2018).</li><li>Deans, B. J., Kilah, N. L., Jordan, G. J., Bissember, A. C., Smith, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arbutin+Derivatives+Isolated+from+Ancient+Proteaceae:+Potential+Phytochemical+Markers+Present+in+Bellendena,+Cenarrhenes+and+Persoonia+Genera.">Arbutin Derivatives Isolated from Ancient Proteaceae: Potential Phytochemical Markers Present in Bellendena, Cenarrhenes and Persoonia Genera.</a> Journal of Natural Products. 81, 1241-1251 (2018).</li><li>Deans, B. J., Tedone, L., Bissember, A. C., Smith, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phytochemical+profile+of+the+rare,+ancient+clone+Lomatia+tasmanica+and+comparison+to+other+endemic+Tasmanian+species+L.+tinctoria+and+L.+polymorpha.">Phytochemical profile of the rare, ancient clone Lomatia tasmanica and comparison to other endemic Tasmanian species L. tinctoria and L. polymorpha.</a> Phytochemistry. 153, 74-78 (2018).</li><li>Deans, B. J., Skierka, B., Karagiannakis, B. W., Vuong, D., Lacey, E., Smith, J. A., Bissember, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Siliquapyranone:+a+Tannic+Acid+Tetrahydropyran-2-one+Isolated+from+the+Leaves+of+Carob+(Ceratonia+siliqua)+by+Pressurised+Hot+Water+Extraction.">Siliquapyranone: a Tannic Acid Tetrahydropyran-2-one Isolated from the Leaves of Carob (Ceratonia siliqua) by Pressurised Hot Water Extraction.</a> Australian Journal of Chemistry. 71, (2018).</li><li>Olivier, W. J., Kilah, N. L., Horne, J., Bissember, A. C., Smith, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ent-Labdane+Diterpenoids+from+Dodonaea+viscosa.">ent-Labdane Diterpenoids from Dodonaea viscosa.</a> Journal of Natural Products. 79, 3117-3126 (2016).</li><li>Rihak, K. J., Bissember, A. C., Smith, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polygodial:+A+viable+natural+product+scaffold+for+the+rapid+synthesis+of+novel+polycyclic+pyrrole+and+pyrrolidine+derivatives.">Polygodial: A viable natural product scaffold for the rapid synthesis of novel polycyclic pyrrole and pyrrolidine derivatives.</a> Tetrahedron. 74, 1167-1174 (2018).</li><li>Ntamila, M. S., Hassanali, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+Oil+of+Clove+and+Separation+of+Eugenol+and+Acetyl+Eugenol.">Isolation of Oil of Clove and Separation of Eugenol and Acetyl Eugenol.</a> Journal of Chemical Education. 53, 263 (1976).</li><li>Still, W. C., Kahn, M., Mitra, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+chromatographic+technique+for+preparative+separations+with+moderate+resolution.">Rapid chromatographic technique for preparative separations with moderate resolution.</a> Journal of Organic Chemistry. , 2923-2925 (1978).</li></ol>

The classical procedure for isolating eugenol from cloves by steam distillation has been part of the intermediate chemistry laboratory program at the University of Sydney for decades but was modernized to employ PHWE methodology in 2016 (Figure 1).9,18 This provided a number benefits. Firstly, utilizing household espresso machines in the laboratory environment immediately fascinated and engaged students by illustrating the applicatio...

The isolation and identification of natural products are of fundamental importance to the scientific community and society more generally.1 Bioprospecting, the search for valuable organic molecules found in nature,remains an indispensable process in the discovery of new drug leads and potential therapeutic agents. It is estimated that, from 1981-2014, ~75% of all approved small molecule pharmaceutical drugs were natural products, natural product-derived or natural product-inspired.1 Furthermore, natural products possess enormous structural and chemical diversity. For this reason, they also represent valuable chemical scaffolds that can be directly used in organic synthesis or in the development of chiral ligands and catalysts.2,3
Traditionally, relatively time-intensive procedures such as maceration, Soxhlet extraction, and steam distillation have been the mainstay of research focused on the isolation of secondary metabolites from plants.4 More modern extraction techniques, including accelerated solvent extraction, have focused on reducing extraction times and establishing greener protocols.4,5 In 2015, an original pressurized hot water extraction (PHWE) method was reported.6 This technique employed an unmodified household espresso machine to facilitate the rapid and particularly efficient extraction of shikimic acid from star anise. Espresso machines have been specifically designed and engineered to extract organic molecules from appropriately ground coffee beans. To achieve this, these instruments heat water at temperatures up to 96 &#176;C and at pressures of typically 9 bar.7 With this in mind, it is perhaps not surprising that espresso machines can be utilized to efficiently extract natural products from a range of plant material.
Subsequent studies involving a variety of terrestrial plant species have demonstrated the capacity of this PHWE technique to efficiently extract natural products across a relatively broad polarity range.6,8,9,10,11,12,13,14,15 Furthermore, compounds containing somewhat sensitive functional groups, such as aldehydes, epoxides, glycosides, and potentially epimerizable stereogenic centers were typically unaffected by the extraction process. The complementary nature of this technique relative to more traditional methods has also been demonstrated.12,16 This PHWE method has also been employed to isolate multi-gram quantities of natural products, which have been used to prepare novel natural product derivatives and in complex molecule synthesis more generally.8,11,17
It was identified that this new PHWE method could serve as a useful teaching tool that could be incorporated in the undergraduate laboratory. This primarily derives from the rapid nature of this technique relative to the traditional extraction methods that are often incompatible with the time constraints associated with undergraduate laboratory experiments. Consequently, this technique supplanted the traditional undergraduate chemistry laboratory experiment focused on the extraction of eugenol from cloves employing steam-distillation at the University of Tasmania.9,18 Since that time, variations of this experiment have been adopted by other universities and a modified experiment focusing on the PHWE of cloves now features in the undergraduate chemistry laboratory program at the University of Sydney (vide infra).
In order to demonstrate the practicality and feasibility of employing this new PHWE approach for teaching purposes, two protocols are presented as part of this study. The first part of this report highlights an experiment on the PHWE of eugenol and acetyleugenol from cloves which is part of the second-year undergraduate laboratory program at the University of Sydney (Figure 1). This experiment serves to introduce students to natural products chemistry while developing fundamental practical skills. The second part features an experiment on the PHWE of the endemic Australian plant species Correa reflexa which is part of the third-year undergraduate laboratory program at the University of Tasmania (Figure 2). This experiment is designed to simulate natural products bioprospecting and reinforce core laboratory techniques.11

<table border="0" cellpadding="0" cellspacing="0" style="width:1069px;" width="1070">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">espresso machines</td>
			<td style="width:167px;">Breville/Sunbeam</td>
			<td style="width:520px;">Breville espresso machine model 800ES / Sunbeam EM3820 Caf&#233; Espresso II</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">rotary evporators</td>
			<td style="width:167px;">Buchi and Heidolph</td>
			<td style="width:520px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">cloves (plant material)</td>
			<td>Dijon Food Pty Ltd</td>
			<td></td>
			<td>Cloves must be ground in a food processor for students.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Correa reflexa (plant material)</td>
			<td>sample obtained in Tasmania</td>
			<td>Sample collected from mature shrubs in the Thomas Crawford Reserve at the University of Tasmania</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">sand</td>
			<td style="width:167px;">Ajax</td>
			<td style="width:520px;">1199</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">ethanol</td>
			<td>Redoc Chemicals</td>
			<td>E95 F3</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">hexanes</td>
			<td>Ajax</td>
			<td>251</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">magnesium sulfate</td>
			<td>Ajax</td>
			<td>1548</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">diethyl ether</td>
			<td>Merck</td>
			<td>1009215000</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">silica on aluminium TLC plates</td>
			<td>Merck</td>
			<td>1055540001</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">eugenol</td>
			<td>Merck</td>
			<td>1069620100</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">eugenyl acetate</td>
			<td>Aldrich</td>
			<td>W246905</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">acetone</td>
			<td>Redox Chemicals</td>
			<td>Aceton13</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">cyclohexane</td>
			<td>ChemSupply</td>
			<td>CA019</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">silica gel 60</td>
			<td>Trajan</td>
			<td>5134312</td>
			<td style="width:167px;">40 - 63um (230-400mesh)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Congo red paper</td>
			<td>ChemSupply</td>
			<td>IS070-100S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">32% hydrochloric acid</td>
			<td>Ajax</td>
			<td>256</td>
			<td></td>
		</tr>
	</tbody>
</table>

employing pressurized hot water extraction (phwe) to explore natural products chemistry in the undergraduate laboratory

A recently developed pressurized hot water extraction (PHWE) method which utilizes an unmodified household espresso machine to facilitate natural products research has also found applications as an effective teaching tool. Specifically, this technique has been used to introduce second- and third-year undergraduates to aspects of natural products chemistry in the laboratory. In this report, two experiments are presented: the PHWE of eugenol and acetyleugenol from cloves and the PHWE of seselin and (+)-epoxysuberosin from the endemic Australian plant species Correa reflexa. By employing PHWE in these experiments, the crude clove extract, enriched in eugenol and acetyleugenol, was obtained in 4-9% w/w from cloves by second-year undergraduates and seselin and (+)-epoxysuberosin were isolated in yields of up to 1.1% w/w and 0.9% w/w from C. reflexa by third-year students. The former exercise was developed as a replacement for the traditional steam distillation experiment providing an introduction to extraction and separation techniques, while the latter activity featured guided-inquiry teaching methods in an effort to simulate natural products bioprospecting. This primarily derives from the rapid nature of this PHWE technique relative to traditional extraction methods that are often incompatible with the time constraints associated with undergraduate laboratory experiments. This rapid and practical PHWE method can be used to efficiently isolate various classes of organic molecules from a range of plant species. The complementary nature of this technique relative to more traditional methods has also been demonstrated previously.

PHWE of cloves. When attempting to perform the liquid-liquid extraction step, students often encountered emulsions (the addition of brine was typically not effective). At this stage, students were instructed to allow the mixture to stand in the separating funnel while they explored the effects of eluent composition on the separation of eugenol and acetyleugenol by TLC. It should be noted that hexane can be substituted with either heptane or dichloromethane in the liquid-liquid extraction ...

Watch this Scientific Journal Video about Employing Pressurized Hot Water Extraction PHWE to Explore Natural Products Chemistry in the Undergraduate Laboratory at JoVE.com

Employing Pressurized Hot Water Extraction PHWE to Explore Natural Products Chemistry in the Undergraduate Laboratory

Here, we employ a pressurized hot water extraction (PHWE) method, which utilizes an unmodified household espresso machine to introduce undergraduates to natural products chemistry in the laboratory. Two experiments are presented: PHWE of eugenol and acetyleugenol from cloves and PHWE of seselin and (+)-epoxysuberosin from the Australian plant Correa reflexa.

Employing Pressurized Hot Water Extraction (PHWE) to Explore Natural Products Chemistry in the Undergraduate Laboratory

A recently developed pressurized hot water extraction (PHWE) method which utilizes an unmodified household espresso machine to facilitate natural products ...

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