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.
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.
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 °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
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
2. PHWE of Correa reflexa : isolation of seselin and (+)-epoxysuberosin
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 step.9 Students were allocated a TLC solvent ratio of acetone and cyclohexane and provided with pure standards of eugenol and acetyleugenol and then performed TLC analysis (Figure 3). Their results were tabulated on a whiteboard, and the effects of solvent composition on the retention factor (Rf) and the optimum eluent were considered in a group discussion (Table 1). The optimum solvent compositions identified by students typically ranged from 5-20% acetone/cyclohexane with a ΔRf between 0.1-0.2.
Following TLC eluent optimization, students returned to their eugenol extractions. The crude clove extract (consisting mainly of eugenol and acetyleugenol) was isolated in 4-9% w/w. In the second session of this experiment, students exploited the different acid-base properties of the two major organic molecules to separate them by liquid-liquid extraction. Typically, eugenol was isolated in a yield of 45-65% w/w of the crude extract while acetyleugenol was isolated in a yield of 5-10% w/w of the crude extract. Students then utilized the optimized eluent (identified as outlined above) to determine the success of their liquid-liquid extraction by comparison of their extracts to the pure reference samples by TLC (Figure 3). Students also analyzed their crude clove extract, and their purified eugenol and acetyleugenol samples by performing Fourier-transform infrared (FTIR) spectroscopy.9 Solvent or water peaks were occasionally observed in IR spectra due to poorly executed work-up procedures (or poor sample preparation).
Advanced students committed approximately half of their isolated crude oil to the liquid-liquid extraction described above and subjected the other portion to flash column chromatography (more information is provided in the supporting information). Although completing the liquid-liquid extraction and flash column chromatography steps in a single four-hour session may appear rather ambitious this was achievable for most of the advanced students undertaking this experiment. The complete separation of eugenol from acetyleugenol by flash column chromatography was rarely achieved due to their close retention factors (Figure 4). However, students were generally able to collect a few fractions containing pure eugenol. Advanced students were then asked to comment on the two different purification techniques as part of their report.
PHWE of Correa reflexa. Students performed the PHWE of Correa reflexa with minimal assistance of the laboratory instructor. During the liquid-liquid extraction step emulsions typically formed and students often were required to allow the mixture to stand in the separatory funnel (~0.25 h) with periodic agitation of the mixture with a glass rod. The chromatographic purification of the crude extract was comfortably completed within the four-hour laboratory session by students. Seselin and (+)-epoxysuberosin were isolated in yields of up to 1.1% w/w and 0.9% w/w, respectively and isolated samples of both compounds were analyzed by 1H and 13C NMR and FTIR spectroscopy (Figure 2). While students undertook the FTIR spectroscopy experiments and prepared samples for NMR spectroscopy, lab technicians performed NMR spectroscopy experiments. The results obtained by students were consistent with previously published work.11
Although this has not been presented in this report, in practice, this experiment also features a second part that challenges students to perform the extraction a plant species that has not been studied employing PHWE (more information is provided in the supporting information).
Figure 1. PHWE of cloves.9 Please click here to view a larger version of this figure.
Figure 2. PHWE of Correa reflexa.11 Please click here to view a larger version of this figure.
Figure 3. A representative TLC plate prepared by a student. (10% acetone/ cyclohexane elution). Lane 1 (E): eugenol standard; lane 2 (crude): crude clove extract; lane 3 (A): acetyleugenol standard). Please click here to view a larger version of this figure.
acetone/ cyclohexane (%v/v) | mean acetyl-eugenol Rf | acetyl-eugenol Rf (σ) | mean eugenol Rf | eugenol Rf (σ) | mean ΔRf | number of TLC analyses |
0 | 0.06 | 0.08 | 0.04 | 0.06 | 0.02 | 12 |
5 | 0.34 | 0.11 | 0.27 | 0.09 | 0.07 | 15 |
10 | 0.45 | 0.07 | 0.34 | 0.05 | 0.12 | 20 |
20 | 0.51 | 0.07 | 0.41 | 0.06 | 0.10 | 20 |
30 | 0.58 | 0.10 | 0.49 | 0.12 | 0.10 | 19 |
40 | 0.63 | 0.08 | 0.56 | 0.08 | 0.07 | 16 |
50 | 0.76 | 0.08 | 0.73 | 0.08 | 0.03 | 17 |
60 | 0.77 | 0.13 | 0.73 | 0.15 | 0.04 | 12 |
70 | 0.84 | 0.13 | 0.81 | 0.13 | 0.03 | 11 |
80 | 0.90 | 0.06 | 0.87 | 0.08 | 0.02 | 10 |
90 | 0.88 | 0.06 | 0.87 | 0.05 | 0.01 | 11 |
100 | 0.87 | 0.13 | 0.86 | 0.14 | 0.02 | 6 |
Table 1. Table of retention factors outlining the effect of eluent composition on Rf.
Figure 4. Representative TLC plates prepared by students. Left: A TLC plate analyzing the outcome of the liquid-liquid extraction step (10% acetone/ cyclohexane elution). Lane 1 (E): eugenol reference standard; lane 2 (LEB): eugenol-containing organic extract; lane 3 (A): acetyleugenol reference standard; lane 4 (LEN): acetyleugenol-containing organic extract. Right: A TLC plate analyzing the outcome of flash column chromatography step (10% acetone/ cyclohexane elution). Numbers on the TLC plate relate to the test-tube fraction number. Please click here to view a larger version of this figure.
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 application of a non-classical, alternative method to effect a traditional scientific study. In addition, this new method reduced the time taken to complete the extraction and enabled the incorporation of additional exercises into this new iteration of the experiment. Specifically, this allowed thin-layer chromatography (TLC) to be introduced (and flash column chromatography for advanced students).
The experiment focusing on the PHWE of cloves was designed as an introductory laboratory experience for second-year undergraduate chemistry students and for this reason it features expository teaching methods.9 This more prescriptive, recipe-style procedure allows students with somewhat limited experience in organic chemistry to efficiently complete the extraction of eugenol from cloves. In this experiment, concepts such as acid-base extraction of acidic compounds, utilizing TLC to identify suitable eluent composition for chromatography, and the use of a rotary evaporator are introduced or reinforced by a combination of on-line pre-lab video training and in-person demonstrations. In complementary components undertaken during the two allocated sessions, students in the advanced stream of intermediary chemistry also separated eugenol and acetyleugenol by column chromatography and determined the identity of the extracted components using TLC. In the second session, students could critically compare the two separation methods. In general, students were able to complete the overall experiment within the allocated two four-hour periods with minimal instruction.
The experiment focusing on the PHWE and isolation of seselin and (+)-epoxysuberosin from Correa reflexa was developed for more experienced students third-year undergraduate chemistry students. Notably, this learning exercise was a result of a study originating in the research laboratory.11 The first iteration of the experiment was incorporated into the third-year undergraduate chemistry laboratory program at the University of Tasmania in 2015. After two years of revisions and re-evaluation, this experiment was performed by a third-year undergraduate class for the third time in 2017.
This experiment was specifically designed as a guided-inquiry-based activity that strives to simulate some of the approaches employed in natural products research laboratories and features minimal written instructions. This is a student-directed learning experience and the laboratory instructor plays a key role in assisting students as they work through the experiment by providing direction as required. In this experiment, students develop key laboratory skills in chromatography and employ NMR spectroscopy to perform structure elucidation. This laboratory experience reinforces the concept of bioprospecting which is presented to students in the classroom and this can be extended to studies on previously unstudied plant material to provide a more representative experience of natural products bioprospecting. C. reflexa is an endemic Australian plant species, however, this sample can be substituted for appropriate leaf material from other terrestrial plant species in this experiment.
The authors have nothing to disclose.
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.
espresso machines | Breville/Sunbeam | Breville espresso machine model 800ES / Sunbeam EM3820 Café Espresso II | |
rotary evporators | Buchi and Heidolph | ||
cloves (plant material) | Dijon Food Pty Ltd | Cloves must be ground in a food processor for students. | |
Correa reflexa (plant material) | sample obtained in Tasmania | Sample collected from mature shrubs in the Thomas Crawford Reserve at the University of Tasmania | |
sand | Ajax | 1199 | |
ethanol | Redoc Chemicals | E95 F3 | |
hexanes | Ajax | 251 | |
magnesium sulfate | Ajax | 1548 | |
diethyl ether | Merck | 1009215000 | |
silica on aluminium TLC plates | Merck | 1055540001 | |
eugenol | Merck | 1069620100 | |
eugenyl acetate | Aldrich | W246905 | |
acetone | Redox Chemicals | Aceton13 | |
cyclohexane | ChemSupply | CA019 | |
silica gel 60 | Trajan | 5134312 | 40 – 63um (230-400mesh) |
Congo red paper | ChemSupply | IS070-100S | |
32% hydrochloric acid | Ajax | 256 |