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

Summary

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.

Abstract

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.

Introduction

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

Protocol

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

1. Extraction of eugenol and acetyleugenol from cloves
   1. Place coarsely ground cloves (12.5 g) in a 250-mL beaker.
   2. Add sand (12.5 g) to the clove grinds and mix well.
   3. Collect a portafilter (sample compartment) and load the basket with the entire clove-sand mixture. Lightly compress the sample with the tamper.
      NOTE: Do not compress the mixture too much or fluid will not flow through.
   4. Position the portafilter into the espresso machine and place a clean 250-mL beaker beneath it. Add a 30% ethanol/H₂O solution to the water tank of the espresso machine if it is less than half full.
   5. Use the espresso machine to collect 100 mL of the extract.
      NOTE: Consult an instructor if the machine appears to be clogged.
   6. Allow the portafilter to finish dripping and then remove it from the espresso machine.
      CAUTION: The grinds and surrounding metal areas will be hot.
   7. Using a spatula, remove the clove grinds from the portafilter and discard into the waste bin.
   8. Rinse out the residual solids from the portafilter with H₂O under a tap in the sink and return it for the next person to use.
   9. Cool the clove extract in an ice bath until the temperature has reduced to at least 30 °C.
   10. Place the extract into a 250-mL separatory funnel, add 30 mL of hexane and shake gently.
   11. Place the separating funnel in a ring clamp fitted to a retort stand and allow the aqueous and organic layers to separate then collect the aqueous (lower) layer back into the 250-mL beaker.
       NOTE: It can take to 10 minutes for the layers to separate. Students are advised to carry out solvent optimization of TLCs while waiting for the first separation to occur (see steps 1.2).
   12. Transfer the organic (top) layer (which contains the product) to a clean 250-mL conical flask, and then pour the bottom (aqueous) layer back into the separatory funnel.
   13. Extract the aqueous layer a further two times with hexane (2x 30 mL).
   14. Combine the organic (top) layers into the same flask after each extraction.
   15. After the third liquid-liquid extraction, pour the combined organic extract into the separatory funnel, and wash with 100 mL of H₂O by shaking vigorously. Collect the organic (top) layer into a clean 250-mL conical flask and dry by adding MgSO₄ and swirling the flask.
   16. Filter the ensuing mixture through fluted filter paper contained in a glass funnel into a pre-weighed 250-mL round-bottom flask.
       NOTE: The solid residue (hydrated MgSO₄) can be discarded in the waste.
   17. Evaporate the solvent (hexane) from the collected filtrate using a rotary evaporator (water bath temperature: 60 °C, vacuum pressure: 350 mbar) and re-weigh the flask containing the resultant oil.
2. Optimization of thin-layer chromatography (TLC) solvent system
   NOTE: As a group, students will be assigned a solvent system from 100:0 acetone:cyclohexane to 0:100 acetone:cyclohexane by the instructor to identify the ratio that provides the maximum resolution of eugenol from acetyleugenol. 
   1. Obtain a TLC reference solution of pure eugenol and acetyleugenol.
      NOTE: The necessary TLC reference solutions of pure eugenol and acetyleugenol were prepared by lab technicians prior to the lab session.
   2. On a TLC plate, mark a baseline ~1.5 cm from the bottom with a soft pencil. Mark off three equally spaced points.
   3. Use a TLC spotter to spot one drop of pure eugenol TLC reference solution in one lane, one spot of pure acetyleugenol TLC reference solution in the third lane and a spot of each in the second lane (the co-spot).
   4. Check for the presence of eugenol and acetyleugenol on the TLC plate by viewing the plate under a UV lamp (254 nm) in the TLC viewing cabinet.
      NOTE: There should be small (1-2 mm wide) black spots on the plate where the TLC reference solutions were spotted. If there are no spots or the spots appear faint, apply another spot of the appropriate TLC solution until a black spot is observed under UV light.
   5. Add 10 mL of the allocated solvent mixture to a clean, dry TLC jar.
      NOTE: Ensure the solvent height in the jar does not exceed ~1 cm.
   6. Using tweezers, place the prepared TLC plate into the TLC jar. Close the lid of the jar.
      NOTE: The solvent must lie below the baseline of the TLC plate.
   7. Allow the solvent to travel up the TLC plate. Once the solvent is ~1 cm from the top of the plate, remove the TLC plate from the jar with tweezers and mark the line of the solvent front with a pencil.
   8. Allow the solvent to evaporate from the TLC plate (~1 min) then view the TLC plate under a UV lamp (254 nm). Using a pencil, circle the black spots observed on the TLC plate.
   9. Calculate the retention factor (R_f) of eugenol and acetyleugenol by dividing the distance travelled by the compound by the distance travelled by the solvent.
   10. Calculate the difference between the R_f values for eugenol and acetyleugenol (ΔR_f).
   11. Share the results with the rest of the class. Record the retention values acquired by other students with other solvent ratios.
   12. Identify which solvent system will be best to analyze the crude eugenol solution and subsequent purification steps.
       NOTE: The best TLC solvent ratio will provide the greatest separation between eugenol and acetyleugenol denoted by the largest ΔR_f value. If ΔR_f is plotted against solvent composition, the graph should resemble a bell curve.
3. Separation of eugenol and acetyleugenol by liquid-liquid extraction
   1. Add hexane (10 mL) to the crude eugenol-containing extract obtained from step 1.1.17, and pour the ensuing solution into a 250-mL separatory funnel.
   2. Rinse the round-bottom flask with hexane (10 mL) and add this to the separatory funnel.
   3. Extract the hexane solution with 3 M aqueous NaOH (2 x 25 mL) via liquid-liquid extraction. Collect and combine the aqueous bottom layers in a 250-mL conical flask from each extraction. Collect the organic layer in a 50-mL conical flask and dry by adding MgSO₄ and swirling the flask.
      CAUTION: NaOH is corrosive. Avoid any contact with skin.
      NOTE: Acetyleugenol remains in the organic layer, while eugenol is now in the alkaline aqueous extract (bottom layers).
   4. Retain the organic layer (organic solution A) for later TLC analysis.
   5. Swirl the conical flask that contains the alkaline aqueous fraction from step 1.3.3 in an ice-water bath and slowly add 10 M aqueous HCl until a white emulsion is formed; check its acidity with Congo red paper, using a pipette to transfer a drop of the solution onto the pH paper (it should turn blue).
      CAUTION: HCl is corrosive. Avoid any contact with skin. Addition of HCl can cause vigorous bubbling, HCl should be added carefully, keeping the conical flask on ice.
      NOTE: A total of 20-30 mL of HCl (10 M of an aqueous solution) will be required.
   6. Extract the milky aqueous emulsion with hexane (2 x 30 mL), using liquid-liquid extraction in a 250 mL separating flask. Make sure that the temperature of the aqueous extract is at room temperature or below before adding the hexane. Combine the two hexane extracts into a clean 100 mL conical flask.
      NOTE: The eugenol will now be in the combined organic (top) layers (organic solution B).
   7. Add MgSO₄ to dry organic solution B.
   8. Analyze organic solution A, organic solution B, pure eugenol TLC reference and acetyleugenol TLC reference by TLC using the optimized TLC solvent ratio identified in the previous session.
   9. Filter organic solution A and B through fluted filter paper into separate pre-weighed 250-mL round-bottom flasks. Discard the solid residue (hydrated MgSO₄) in the waste.
   10. Remove the solvent from the round-bottom flasks using a rotary evaporator (water bath temperature: 60 °C, vacuum pressure: 350 mbar).
   11. Add diethyl ether (5 mL) to each round-bottom flask and transfer the purified acetyleugenol (organic solution A)  and eugenol (organic solution B) into an unlabeled, preweighed vial using a funnel.
   12. Rinse the flask with further diethyl ether (5 mL) into the vial. Evaporate the solvent using a rotary evaporator (water bath temperature: 50 °C, vacuum pressure 800 mbar) with a vial attachment. Record the yield and label the vial appropriately. 
   13. Analyze organic solution A, organic solution B, pure eugenol TLC reference and acetyleugenol TLC reference by TLC using the optimized TLC solvent ratio identified in the previous session.
       NOTE: Aqueous solutions can be poured down the sink for disposal. Hexane and ether waste should be disposed of in the non-chlorinated organic waste bottles.

2. PHWE of Correa reflexa : isolation of seselin and (+)-epoxysuberosin

1. Session 1. PHWE of Correa reflexa
   1. Grind Correa reflexa leaves (10 g) in an electric spice grinder and then transfer the ground plant material to a 250-mL beaker.
      NOTE: Grinding should take 20-30 seconds.
   2. Add ~ 2 g of coarse sand to the beaker containing plant material.
   3. Mix and pack into the basket of the portafilter (sample compartment). Compress the sample with the tamper.
      NOTE: Do not pack the sample too tightly.
   4. Add ~300 mL of 35% ethanol/H₂O solution to the espresso machine tank.
   5. Position the portafilter into the espresso machine and place a clean 250-mL beaker beneath it.
   6. Collect ~100 mL of extract, wait for ~1 minute and then collect a further 100 mL.
      CAUTION: The machine and extracts will be hot at this point.
   7. Cool this mixture in ice bath and evaporate the ethanol using a rotary evaporator (water bath temperature: ~40 °C).
   8. Transfer the aqueous extract to a separatory funnel and extract with ethyl acetate (4x 50 mL).
      NOTE: Time may be needed to allow emulsions to separate between extractions.
   9. Combine the organic extracts, dry by adding MgSO₄ and swirling the flask, filter using a sintered glass funnel, and evaporate using a rotary evaporator (water bath temperature: ~35 °C) to provide the crude extract.
   10. Obtain an ¹H nuclear magnetic resonance (NMR) spectrum (see the instructor for assistance).¹¹
   11. Perform TLC analysis of the crude extract to determine an appropriate solvent system to isolate the compounds that have been extracted.
       NOTE: TLC analysis is performed by analogy to procedures outlined in step 1.2.
2. Session 2. Separation of seselin and (+)-epoxysuberosin by flash column chromatography^11,19
   NOTE: The following protocol involves the use of flash column chromatography for the separation of organic compounds. Please consult an instructor to demonstrate how to pack a flash silica gel column.
   1. Place the column (~30 mm in diameter) in a clamp fitted to a retort stand. Place a 100-mL conical flask underneath the column.
   2. Fill the column with silica gel (60 μm flash grade) to a level of ~10 cm and then add hexanes (~100 mL) to the column.
   3. Place a glass stopper in the column, remove the column from the clamp and shake to obtain a slurry. Place the column in the clamp and then allow the mixture to settle.
   4. Open the tap of the column and, using a gas adaptor attached to a compressed air line, empty the column to leave ~2 mm of solvent above the bed of silica gel. Remove the gas adaptor then close the tap.
   5. Use the hexanes collected in the conical flask (~5 mL) to wash down any silica gel from the walls of the column with a Pasteur pipette fitted with a rubber septum.
   6. Repeat step 2.2.4 then add a small layer of sand (~1 cm) to the column.
   7. Add dichloromethane (~ 1mL) to the flask containing the crude extract from step 2.1.9. Carefully load the ensuing solution onto the column using a Pasteur pipette fitted with a rubber septum. Open the tap of the column and allow the sample to adsorb onto the silica gel.
   8. Repeat step 2.2.7 a further two times.
   9. Carefully add hexanes (~20 mL) to the column. Repeat step 2.2.4.
   10. Carefully add (~180 mL of a 15% ethyl acetate/hexanes solution). Open the tap of the column and, using a gas adaptor attached to a compressed air line, empty the column to leave ~2 mm of solvent above the bed of silica gel, collecting the fractions into 10-mL test tubes.
       NOTE: This will allow seselin to be isolated.
   11. Combine the test tube fractions containing seselin in a 250-mL round bottom flask and evaporate using a rotary evaporator (water bath temperature: ~35 °C).
       NOTE: TLC analysis is used to determine this and is performed by analogy to procedures outlined in step 1.2.
   12. Carefully add (~75 mL of a 25% ethyl acetate/hexanes solution). Open the tap of the column and, using a gas adaptor attached to a compressed air line, empty the column to leave ~2 mm of solvent above the bed of silica gel, collecting the fractions into 10-mL test tubes.
       NOTE: This will allow (+)-epoxysuberosin to be isolated.
   13. Combine the test tube fractions containing (+)-epoxysuberosin in a 250-mL round bottom flask and evaporate using a rotary evaporator (water bath temperature: ~35 °C).
       NOTE: TLC analysis is used to determine this and is performed by analogy to procedures outlined in step 1.2.
   14. Samples of the isolated compounds are analyzed using NMR spectroscopy.¹¹
       NOTE: NMR spectroscopy experiments are performed by a lab technician.

Results

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 ...

Discussion

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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