Extraction and Analysis of Terpenes/Terpenoids

Zuodong Jiang1, Chase Kempinski1, Joe Chappell2

1 These authors contributed equally to this work, 2 Department of Pharmaceutical Sciences, University of Kentucky, Lexington, Kentucky
Publication Name:  Current Protocols in Plant Biology
Unit Number:   
DOI:  10.1002/cppb.20024
Online Posting Date:  June, 2016
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Terpenes/terpenoids constitute one of the largest classes of natural products, due to the incredible chemical diversity that can arise from the biochemical transformations of relatively simple prenyl diphosphate starter units. All terpenes/terpenoids comprise a hydrocarbon backbone generated from various length prenyl diphosphates (a polymer chain of prenyl units). Upon ionization (removal) of the diphosphate group, the remaining allylic carbocation intermediates can be coaxed down complex chemical cascades leading to diverse linear and cyclized hydrocarbon backbones that can be further modified with a wide range of functional groups (e.g., alcohols or ketones) and substituent additions (e.g., sugars or fatty acids). Because of this chemical diversity, terpenes/terpenoids have great industrial uses as flavors, fragrances, high grade lubricants, biofuels, agricultural chemicals, and medicines. The protocols presented here focus on the extraction of terpenes/terpenoids from various plant sources and have been divided into extraction methods for terpenes/terpenoids with various levels of chemical decoration—from relatively small, nonpolar, volatile hydrocarbons to substantially large molecules with greater physical complexity due to chemical modifications. © 2016 by John Wiley & Sons, Inc.

Keywords: hydrocarbons; isoprenoid; polarity; substituent groups; terpene; terpenoid; volatile

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Table of Contents

  • Introduction
  • Basic Protocol 1: Extraction and Analysis of a Non‐Polar Terpene
  • Alternate Protocol 1: Extraction and Analysis of a Volatile Terpene
  • Basic Protocol 2: Extraction and Analysis of a Terpene with Modifications
  • Alternate Protocol 2: Extraction and Analysis of Terpenes Decorated with Large Groups
  • Basic Protocol 3: Extraction and Analysis of Polar Terpenoids
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Extraction and Analysis of a Non‐Polar Terpene

  • Plant material (e.g., tobacco leaf)
  • Hexane
  • Ethyl acetate
  • 85:15 (v/v) hexane:ethyl acetate
  • (−)‐α‐cedrene (Sigma‐Aldrich, cat. no. 22133)
  • Hexadecane (Sigma‐Aldrich, cat. no. 442679)
  • Silica gel, 60 Å pore size, 230‐400 mesh (Sigma‐Aldrich, cat. no. 227196)
  • Scale with an accuracy of ±0.1 mg
  • Liquid nitrogen
  • Glass vials with PTFE sealed lids (Kimble‐Chase, cat. no. 60940A24)
  • Mortar and pestle
  • Glass Pasteur pipets (Fisherbrand, cat. no. 13‐678‐20A)
  • Glass serological pipets
  • Glass wool
  • Gaseous nitrogen
  • GC‐MS, e.g., Agilent 6890a GC equipped with a HP5‐MS column (30‐m × 0.250‐mm × 0.25‐μm) coupled to an Agilent 5975C Mass Spectral Detector or Flame Ionization Detector (FID)
  • HPLC, e.g., Waters 2695 equipped with a Nomura Chemical Develosil 60‐3 250 × 20‐mm column and a Waters 2696 photodiode array detector

Alternate Protocol 1: Extraction and Analysis of a Volatile Terpene

  Additional Materials (see also protocol 1)
  • Transgenic tobacco plant that emits volatile sesquiterpines (Wu et al., )
  • Tenax resin 20‐35 mesh, 150 mg (Sigma‐Aldrich, cat. no. 11049‐U)
  • Gas tight, glass chamber with two ports capable of attaching tubing
  • Compressed or house provided air 300 to 500 ml/min
  • Vacuum line, 300 to 500 ml/min

Basic Protocol 2: Extraction and Analysis of a Terpene with Modifications

  • Chloroform
  • Tobacco plant cell culture
  • Hexane
  • Cyclohexane
  • Acetone
  • Vanillin indicator reagent (see recipe)
  • Pear‐shaped collection flasks
  • Separatory funnel
  • Rotoevaporator
  • Glass serological pipets
  • Glass Pasteur pipets (Fisherbrand, cat. no. 13‐678‐20A)
  • TLC sheets, silica gel 60 F 254 (Millipore, cat. no. 105735)
  • TLC chromatography tank
  • Glass atomizer (must be connected to compressed air or nitrogen)
  • Spray box (to catch excess TLC indicator reagent)

Alternate Protocol 2: Extraction and Analysis of Terpenes Decorated with Large Groups

  Additional Materials (see also Basic Protocols protocol 11 and protocol 32)
  • 20 to 100 mg plant tissue
  • Ethanol
  • 33% (w/v) potassium hydroxide, in water
  • 1 μg/μl 5‐α‐cholestane (Sigma‐Aldrich, cat. no. C8003)
  • Deionized water
  • Hexane
  • Pyridine
  • MSTFA–1% TMCS (Thermo Scientific, cat. no. TS‐48915)
  • Scale accurate to within ±0.1 mg
  • Heat block set at 50°C
  • Glass vials, 14‐ml, with PTFE cap (e.g., Kimble Chase, cat. no. 60940A24)
  • Glass serological pipets
  • Glass Pasteur pipets (Fisherbrand, cat. no. 13‐678‐20A)
  • GC vials
  • Glass GC vial inserts

Basic Protocol 3: Extraction and Analysis of Polar Terpenoids

  • Chloroform
  • Methanol
  • 1 mM ammonium acetate, pH 5.5
  • β‐artemether (Sigma‐Aldrich, cat. no. A9361)
  • HPLC, e.g., Waters Alliance 2695 equipped with two pumps (pump 1: 1 mM ammonium acetate buffer, pH 5.5; pump 2: methanol) and an Waters XTerra MS C 18 5 μm guard column (10‐mm × 2.1‐mm) coupled to an Alltech Ultrasphere C 18 IP 5 μm column (150‐mm × 2.1‐mm) with a LC Packings ACUrate ICP‐04‐20 post‐column splitter sending a quarter of the column eluate into the LC‐MS
  • ESI Q‐TOF MS/MS, e.g., Ultima MS with ESI source operating in positive mode with a capillary voltage of 2.7 kV, a source temperature of 130°C, and a desolvation temperature of 300°C; nitrogen as the desolvation gas with a flow rate of 500 liters/hr; MS/MS conducted using argon (0.9 bar) for collision; cone voltage set for 40 V and a 7 eV collision energy is optimal for artemisinin
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Literature Cited

Literature Cited
  Chemat, F., Abert‐Vian, M., and Fernandez, X. 2012. Microwave‐assisted extraction of essential oils and aromas. In Microwave‐Assisted Extraction of Bioactive Compounds. (F. Chemat and G. Cravotto, eds.) pp. 53‐68. Springer, New York. doi: 10.1007/978‐1‐4614‐4830‐3.
  Croom, E., Pace, R., Paletti, A., Sardone, N., and Gray, D. 2008. Single‐laboratory validation for the determination of terpene lactones in Ginkgo biloba dietary supplement crude materials and finished products by high‐performance liquid chromatography with evaporative light‐scattering detection. J. AOAC Int. 90:647‐658.
  Dewick, P.M. 2009. Medicinal natural products: A biosynthetic approach (Third Ed.). John Wiley & Sons, Ltd., Chichester, U.K. doi: 10.1002/9780470742761.
  Ding, C., Chen, E., Zhou, W., and Lindsay, R.C. 2004. A method for extraction and quantification of Ginkgo terpene trilactones. Anal. Chem. 76:4332‐4336. doi: 10.1021/ac049809a.
  Du, M. and Ahn, D.U. 2002. Simultaneous analysis of tocopherols, cholesterol, and phytosterols using gas chromatography. J. Food Sci. 67:1696‐1700. doi: 10.1111/j.1365‐2621.2002.tb08708.x.
  Kawamura, F., Kikuchi, Y., Ohira, T., and Yatagai, M. 1999. Accelerated solvent extraction of paclitaxel and related compounds. J. Nat. Prod. 62:244‐247. doi: 10.1021/np980310j.
  Kempinski, C., Jiang, Z., Bell, S., and Chappell, J. 2015. Metabolic engineering of higher plants and algae for isoprenoid production. Adv. Biochem. Eng. Biotechnol. 148:161‐199. doi: 10.1007/10_2014_290.
  Kim, G.‐J. and Kim, J‐H. 2015. Development of a simultaneous extraction and acid hydrolysis process for recovery of paclitaxel from plant cell cultures. Process Biochem. 50:279‐284. doi: 10.1016/j.procbio.2014.11.009.
  Kodama, H., Fujimori, T., and Katō, K. 1981. Isolation of a new terpene glucoside, 3‐hydroxy‐5,6‐epoxy‐β‐ionyl‐β‐d‐glucopyranoside from flue‐cured tobacco. Agric. Biol. Chem. 45:941‐944. doi: 10.1271/bbb1961.45.941.
  Lapkin, A., Plucinski, P.K., and Cutler, M. 2006. Comparative assessment of technologies for extraction of artemisinin. J. Nat. Prod. 69:1653‐1664. doi: 10.1021/np060375j.
  Niehaus, T.D., Okada, S., Devarenne, T.P., Watt, D.S., Sviripa, V., and Chappell, J. 2011. Identification of unique mechanisms for triterpene biosynthesis in Botryococcus braunii. Proc. Natl. Acad. Sci. U.S.A. 108:12260‐12265. doi: 10.1073/pnas.1106222108.
  Oh, H.J., Jang, H.R., Jung, K.Y., and Kim, J.H. 2012. Evaluation of adsorbents for separation and purification of paclitaxel from plant cell cultures. Process Biochem. 47:331‐334. doi: 10.1016/j.procbio.2011.11.004.
  Pawliszyn, J. 2012. Solid‐phase microextraction in perspective. In Handbook of Solid Phase Microextraction, pp. 1‐12. Elsevier, New York.
  Rodriguez, G.A. 2001. Extraction, isolation, and purification of carotenoids. Curr. Protoc. Food Anal. Chem. 00:F2.1.1‐F2.1.8. doi: 10.1002/0471142913.faf0201s00.
  Schwab, W., Davidovich‐Rikanati, R., and Lewinsohn, E. 2008. Biosynthesis of plant‐derived flavor compounds. Plant J. 54:712‐732. doi: 10.1111/j.1365‐313X.2008.03446.x.
  Shelar, D. and Shirote, P. 2011. Nature product in drug discovery: back to future. Biomed. Pharmacol. J. 4:141‐146. doi: 10.13005/bpj/272.
  Sun, Y., Li, W., Fitzloff, J.F., and van Breemen, R.B. 2005. Liquid chromatography/electrospray tandem mass spectrometry of terpenoid lactones in Ginkgo biloba. J. Mass Spectrom. 40:373‐379. doi: 10.1002/jms.795.
  Taura, K., Yamamoto, S., Hayashi, S., and Shioya, S. 2013. Enhanced paclitaxel production by addition of water‐soluble 5‐aminolevulinic acid and in situ extraction with lauryl alcohol in a suspension callus culture. Solvent Extr. Res. Dev. Jpn. 20:65‐70. doi: 10.15261/serdj.20.65.
  Taylor, K.L., Brackenridge, A.E., Vivier, M.A., and Oberholster, A. 2006. High‐performance liquid chromatography profiling of the major carotenoids in Arabidopsis thaliana leaf tissue. J. Chromatogr. A 1121:83‐91. doi: 10.1016/j.chroma.2006.04.033.
  Theodoridis, G., Laskaris, G., de Jong, C.F., Hofte, A.J.P., and Verpoorte, R. 1998. Determination of paclitaxel and related diterpenoids in plant extracts by high‐performance liquid chromatography with UV detection in high‐performance liquid chromatography‐mass spectrometry. J. Chromatogr. A 802:297‐305. doi: 10.1016/S0021‐9673(97)01174‐6.
  Van Nieuwerburgh, F.C.W., Vande Casteele, S.R.F., Maes, L., Goossens, A., Inzé, D., Van Bocxlaer, J., and Deforce, D.L.D. 2006. Quantitation of artemisinin and its biosynthetic precursors in Artemisia annua L. by high performance liquid chromatography‐electrospray quadrupole time‐of‐flight tandem mass spectrometry. J. Chromatogr. A 1118:180‐187. doi: 10.1016/j.chroma.2006.03.121.
  Wu, S., Schalk, M., Clark, A., Miles, R.B., Coates, R., and Chappell, J. 2006. Redirection of cytosolic or plastidic isoprenoid precursors elevates terpene production in plants. Nat. Biotechnol. 24:1441‐1447. doi: 10.1038/nbt1251.
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