Laboratory Experiment pubs.acs.org/jchemeduc
Nature’s Antidepressant for Mild to Moderate Depression: Isolation and Spectral Characterization of Hyperforin from a Standardized Extract of St. John’s Wort (Hypericum perforatum) Bopha Chrea, Juliette A. O’Connell, Orlando Silkstone-Carter, John O’Brien, and John J. Walsh* School of Pharmacy and Pharmaceutical Sciences, Trinity College Dublin, Dublin 2, Ireland S Supporting Information *
ABSTRACT: St. John’s wort (Hypericum perforatum) is a medicinal plant that has been used throughout history to treat depression. Its active constituent, hyperforin, inhibits neuronal uptake of monoamines and has been shown to be effective in the treatment of mild to moderate depression. However, hyperforin is highly unstable, sensitive to the effects of heat, light, oxygen, and lipophilic solvents. Despite this extensive degradation potential, this laboratory experiment has been optimized to allow upper-level undergraduate students studying nature’s medicines to isolate purified hyperforin from a standardized preparation by employing a number of techniques, such as purging mobile phases with nitrogen gas to remove oxygen and using amber sample tubes to protect purified fractions from light. Using these conditions, hyperforin is stable in a methanolic solution, and students take advantage of this factor during the isolation. Students acquire skills in the isolation and stabilization of a highly unstable molecule through use of flash column chromatography and carry out structure elucidation using a variety of spectroscopic methods. The experiment can be conducted over one three-hour laboratory period. A crossword puzzle to assess student learning following the experiment enables a comprehensive and engaging education, allowing appreciation of the journey of medicinal plant from bench to bedside. KEYWORDS: Upper-Division Undergraduate, Laboratory Instruction, Organic Chemistry, Hands-On Learning/Manipulatives, Chromatography, Drugs/Pharmaceuticals, NMR Spectroscopy, Natural Products, Thin Layer Chromatography, Plant Chemistry
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t. John’s wort (Hypericum perforatum) (SJW) has become an increasingly popular herbal alternative used in the treatment of depression. This nature’s medicines laboratory experiment details the isolation of the main active constituent, hyperforin (Figure 1). Students conducting this experiment
hydrogen bonding interactions. Detailed spectroscopic analysis by 1- and 2-D NMR, MS, and IR spectroscopy are used to confirm the structure via assignment of 1H and 13C resonances. Students gain a more complete knowledge of SJW as a medicinally important plant through a student-led seminar. Groups of 2−3 students are assigned a topic, prepare 2−3 slides per topic, and present their findings to their peers. Their education culminates in assessment by means of a crossword puzzle. The experiment complements the many other laboratory-based experiments on the isolation of nature’s medicines, including lovastatin from Monascus purpureus,1 valtrate from Centranthus ruber,2 galantamine from Leucojum aestivum,3 parthenolide from Tanacetum parthenium,4 quinine from Cinchona calisaya,5 shikimic acid from star aniseed,6 curcumin from turmeric,7 and thiarubrine A from Ambrosia artemisiifolia.8
Figure 1. Structure of hyperforin, the main active constituent of St. John’s wort.
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BACKGROUND SJW has been used as a natural remedy for over a millennium. The first documented use was by Hippocrates (ca. 460−370 B.C.E.) for treatment against demonic possessions.9 Later, Greek and Roman doctors used SJW for wound healing and
gain valuable skills and knowledge needed to work with an unstable, active constituent of a medicinal plant. Various isolation methodologies are employed in purifying stabilized hyperforin from St. John’s wort with minimal degradation products. Hyperforin is unstable in the lipophilic solvent (pentane) required to purify the crude extract but is stable for weeks in methanol, a solvent that allows for stability via © 2014 American Chemical Society and Division of Chemical Education, Inc.
Published: January 7, 2014 440
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interpretation of a variety of 1- and 2-D NMR spectroscopic data. The NMR spectroscopic data provided to the students include 1-D 1H and 13C spectra, as well as 2-D proton− hydrogen (HH), proton−carbon (HSQC), and long-range proton−carbon correlation (HMBC) techniques. Mass spectra and infrared spectra are also provided to students to strengthen the information gathered from the 1- and 2-D NMR spectra. At the end of the program, an hour-long student-led seminar provides a comprehensive education to students on all aspects of SJW.
relief from menstrual cramps. Currently SJW extracts have become one of the most well-known and best selling natural alternatives to traditional medicines in the treatment of depression.10 In 2002, 12% of U.S. adults reported to having used SJW within the last 12 months.11 It continues to be one of the most widely prescribed antidepressants in Germany, with more than 2.7 million prescriptions written each year.12 SJW is currently marketed as Kira (LI160), Pirkia (WS 5570), SC27 from New Chapter, Neuroplant AKTIV-WS 5570, Amoryn and sold in Canada under Jamieson and Webber companies. Hyperforin is the primary active constituent of SJW responsible for its antidepressant profile,13,14 inhibiting neuronal reuptake of monoamines (e.g., norepinephrine, serotonin, dopamine), thus effectively increasing neurotransmission at the level of the synapse. Recent reviews provide strong evidence that hypericum extracts are both efficacious and tolerable for the treatment of mild to moderate depression and major depression.15,16 Concerns have been raised, however, about the potential of SJW to interact with other drugs through the induction of cytochrome P450 enzymes (particularly CYP3A4 and also CYP2C9) or P-glycoprotein (P-gp). These interactions reduce efficacy of comedications by both increasing the expression and drug efflux function of P-gp and enhancing the rate of oxidative metabolism of these drugs.13 In vitro investigations indicate induction of CYP3A4 is mediated through an interaction of pregnane X receptor (PXR) with hyperforin.17,18 Hyperforin is an extremely unstable molecule, and only recently has an enantioselective total synthesis been devised.19 It undergoes a variety of oxidation reactions and is very sensitive to the effects of heat and light.20,21 A number of degradation products have been identified, which for the most part are oxidation products such as furohyperforins and pyranohyperforins.21,22 The drive to produce stable forms of the active constituent have led to synthesis of aristoforin,20 hyperforin acetate, and numerous salts, such as an imipraminehyperforin combination and dicyclohexylamine salt.23
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EXPERIMENTAL SECTION In the first week, hyperforin is extracted from a tablet of a standardized extract of Hypericum perforatum. One tablet (∼450 mg, Kira) is crushed, extracted sequentially with methanol and diethyl ether, and centrifuged. The supernatant is reduced in volume and the resulting residue reconstituted in a minimum amount of dichloromethane to allow for easy transfer onto a flash column. The flash column is prepared as described in the Supporting Information; all solvents are purged with nitrogen gas to exclude oxygen and minimize the possibility of oxidative degradation. A gradient mobile phase system is used consisting of pentane, pentane/ethyl acetate (40:1), and finally pentane/ ethyl acetate (20:1). Fractions (approximately 1.0 mL) are collected in amber sample tubes containing 2−3 drops of methanol to stabilize purified hyperforin. Fractions containing pure hyperforin are identified using silica gel TLC with pentane/ethyl acetate (10:1) as mobile phase and visualization with vanillin/H2SO4. Anisaldehyde/H2SO4 as spray reagent is also equally effective for visualization. The fractions containing pure hyperforin are combined, reduced to dryness in vacuo, and weighed to determine yield. Details of the procedure are in the Supporting Information. In the second week, a wide variety of 1- and 2-D NMR spectra are used to confirm that the substance isolated was hyperforin. This analysis includes examination of 1H NMR, 13C NMR, DEPT 135, H−H COSY, HMBC, and HSQC spectra. Assignment of all signals is available in the Supporting Information. Students analyzed a mass spectrum and IR spectrum of hyperforin provided to them.
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EXPERIMENTAL OVERVIEW The practical is designed for upper-level organic chemistry undergraduate students and has been completed easily by groups (typically 2 groups of 15 students) of third-year pharmacy students over one three-hour laboratory session, with an additional two-hour workshop on structure assignment. Throughout the course of the experiment, students develop the skills required for (1) extraction of the highly unstable molecule hyperforin in a stabilized form from a commercial standardized preparation of St. John’s wort using the appropriate solvent, (2) isolation of fractions containing pure hyperforin through the use of flash column chromatography, (3) identification of fractions containing the pure compound using thin-layer chromatography (TLC), and (4) calculation of yield achieved from the isolation. As hyperforin is an extremely unstable molecule subject to oxidation, thermal decomposition, and photodegradation,20,21 tips are provided to optimize the isolation of pure hyperforin and reduce the quantity of degradation products. These precautions include exclusion of oxygen by purging mobile phases with nitrogen gas, using amber sample vials to protect the hyperforin-rich fractions from light, and using methanol as a solvent to extract and stabilize the molecule. In the two-hour workshop on spectroscopic assignment, students confirm the structure of hyperforin through the
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HAZARDS Laboratory coats must be worn at all times in the laboratory. Eye protection must be worn while dealing with active chemical spray and materials. Protective gloves must be worn when handling chemicals and samples. Dichloromethane, methanol, vanillin, and anisaldehyde are toxic if inhaled, swallowed, or absorbed through skin. Exercise great care when handling. Use in a fume hood. Prevent contact with skin and clothes. Ethyl acetate, pentane, and diethyl ether are extremely flammable and are toxic to the eyes, skin, and respiratory system. Avoid inhaling vapors. Use in a fume hood. Ultraviolet light (UV) radiation can cause severe damage to the eyes. Do not look directly into the UV light source. Use silica gel and spray reagents in a fume hood. Treat with the utmost care and attention.
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RESULTS The average yield of hyperforin isolated was 3.0 ± 1.0 mg. However, care must be taken to ensure that the column is not overloaded with the extract. Studies on the assignment of its 1H 441
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and 13C resonance values are conducted during the course of the workshop session, which serves as the ideal platform for interactive dialogue with the students as they work through the various 1- and 2-D spectra obtained on hyperforin. A mass spectrum displayed the (M − H+)− at m/z at 535.3820 for hyperforin as the abundant ion. Analysis of an infrared spectrum of hyperforin revealed interesting structural features of the molecule. The functional groups were identified at characteristic absorption peaks, and assignment of the O−H stretching vibration, the C−O bending vibration and the C−H bending of the CH2 and CH3 groups were of most interest. One important absorbance peak was the one identified at 1723 cm−1 for the 1,3-diketone in the moleculea very distinctive functional feature of hyperforin.
(2) Doyle, A. M.; Reilly, J.; Murphy, N.; Kavanagh, P. V.; O’Brien, J. E.; Walsh, M. S.; Walsh, J. J. Nature’s Sedative: Isolation and Structural Elucidation of Valtrate from Centranthus ruber. J. Chem. Educ. 2004, 81, 1486−1487. (3) Halpin, C. M.; Reilly, C.; Walsh, J. J. Nature’s Anti-Alzheimer’s Drug: Isolation and Structure Elucidation of Galantamine form Leucojum aestivum. J. Chem. Educ. 2010, 87, 1242−1243. (4) Walsh, E. L.; Ashe, S.; Walsh, J. J. Nature’s Migraine Treatment: Isolation and Structure Elucidation of Parthenolide from Tanacetum parthenium. J. Chem. Educ. 2012, 89, 134−137. (5) Carroll, A. M.; Kavanagh, D. J.; McGovern, J . R.; Reily, J. R.; Walsh, J. J. Nature’s Chiral Catalyst and Anti-Malarial: Isolation and Structural Elucidation of Cinchonine and Quinine from Cinchona calisaya. J. Chem. Educ. 2012, 89, 1578−1581. (6) Payne, R.; Edmonds, M. Isolation of Shikimic Acid from Star Aniseed. J. Chem. Educ. 2005, 82, 599−600. (7) Anderson, A. M.; Mitchell, M. S.; Mohan, R. S. Isolation of Curcumin from Turmeric. J. Chem. Educ. 2000, 77, 359−360. (8) Reyes, J.; Morton, M.; Downum, K.; O’Shea, K. Isolation and Spectral Analysis of Naturally Occuring Thiarubrine A. J. Chem. Educ. 2001, 78, 781−783. (9) Asch, S. S. Depression and Demonic possession: The Analyst as Exorcist. Hillside J. Clin. Psychiatry 1985, 7, 149−164. (10) Howland, R. H. Update on St. John’s Wort. J. Psychosoc. Nurs. Ment. Health Serv. 2010, 48, 20−4. (11) Barnes, P. M.; Powell-Griner, E.; McFann, K.; Nahin, R. L. Complementary and Alternative Medicine Use Among Adults: United States, 2002. Adv. Data 2004, 343, 1−19. (12) Hennessy, M.; Kelleher, D.; Spiers, J. P.; Barry, M.; Kavanagh, P.; Back, D.; Mulcahy, F.; Feely, J. St. John’s Wort increases expression of P-glycoprotein: Implications for drug interactions. Br. J. Clin. Pharmacol. 2002, 53, 75−82. (13) Laakmann, G.; Schule, C.; Baghai, T.; Kieser, M. St. John’s wort in Mild to Moderate Depression. Pharmacopsychiatry 1998, 1, 54−59. (14) Leuner, K.; Kazanski, V.; Muller, M.; Essin, K.; Henke, B.; Gollasch, M.; Harteneck, C.; Muller, W. E. Hyperforin a Key Constituent in St. John’s Wort Specifically Activates TRPC6 Channels. FASEB J. 2007, 21, 4101−4111. (15) Kasper, S.; Caraci, F.; Forti, B.; Drago, F.; Aguglia, E. Efficacy and tolerability of Hypericum extract for the treatment of mild to moderate depression. Eur. Neuropsychopharmacol. 2010, 20, 747−765. (16) Linde, K.; Berner, M. M.; Kriston, L. St. John’s Wort for Major Depression. Cochrane Database Syst. Rev. 2008, 4, CD000448. (17) Vollmer, J.; Rosenson, J. Chemistry of St. John’s Wort: Hypericin and Hyperforin. J. Chem. Educ. 2004, 81, 1450−1456. (18) Moore, L.; Goodwin, B.; Jones, S.; Wisely, G. B.; Serabjit-Singh, C. J.; Wilson, T. M.; Collins, J. L.; Kliewer, S. A. St. John’s Wort Induces Hepatic Drug Metabolism Through Activation of the Pregnane X Receptor. Proc. Natl. Acad. Sci. U. S. A. 2000, 97, 7500−7502. (19) Sparling, B. A.; Moebius, D. C.; Shair, M. D. Enantioselective total synthesis of hyperforin. J. Am. Chem. Soc. 2013, 135, 644−7. (20) (a) Gartner, M.; Müller, T.; Simon, J.; Giannis, A.; Sleeman, J. Aristoforin, a Novel Stable Derivative of Hyperforin, Is a Potent Anticancer Agent. Chem. Biochem. 2005, 6, 171−177. (b) Bilia, A. R.; Bergonzi, M. C.; Morgenni, F.; Mazzi, G.; Vincieri, F. F. Evaluation of chemical stability of St. John’s wort commercial extract and some preparations. Int. J. Pharm. 2001, 213, 199−208. (21) Ang, C. Y. W.; Hu, L.; Heinze, T. M.; Cui, Y.; Freeman, J. P.; Kozak, K.; Luo, W. Instability of St. John’s wort (Hypericum perforatum L.) and Degradation of Hyperforin in Aqueous solutions and Functional Beverage. J. Agric. Food. Chem. 2004, 52, 6156−6164. (22) Shan, M.; Hu, L.; Chen, Z. Three New Hyperforin Analogues from Hypericum perforatum. J. Nat. Prod. 2001, 64, 127−130. (23) Bombardelli, E.; Morazzoni, P. Hyperforin derivatives, use thereof and formulations containing them. U.S. Patent 6,656,510 B2, Dec 2, 2003.
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ASSESSMENT Students submitted a detailed report; during the experiment they were assessed on their ability to prepare a flash column, the quality of their TLC results, the appearance of the column at the end of the experiment, and on the yield of hyperforin isolated. In the student-led seminar, groups of students prepared slides and presented them to their peers to gain a more complete, bench to bedside, knowledge of St. John’s wort for the treatment of depression. Typically, groups of 2−3 students prepared 2−3 slides per topic and presented their findings to their peers (see the Supporting Information). Topics were randomly assigned to each group of students. Students were then assessed on their knowledge by means of a crossword puzzle (see the Supporting Information). Morning groups completed the across clues, whereas afternoon groups completed the down clues. The annual end-of-year examination on this topic contained questions based on structure assignment, biosynthesis of hyperforin, pharmacological properties, drug interactions, and clinical use of St John’s wort. It was apparent from the quality of answers provided to these questions that students in general had a very clear appreciation of the subject matter.
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ASSOCIATED CONTENT
S Supporting Information *
A student handout, including questions, notes for the instructor, answers to the student questions, spectra, tabulated spectral data and the crossword for student assessment. This material is available via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*J. J. Walsh. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Joe R. Reilly for validation of the hyperforin isolation protocol, Brian Talbot for recording the HRMS data, and Thomas McMurry for NMR discussions on hyperforin.
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REFERENCES
(1) Naziri, M. M.; Samat, F. D.; Kavanagh, P. V.; Walsh, J. J. Nature’s Cholesterol Lowering Drug: Isolation and Structure Elucidation of Lovastatin from Red Yeast Rice-containing Dietary Supplements. J. Chem. Educ. 2012, 89, 138−140. 442
dx.doi.org/10.1021/ed300800f | J. Chem. Educ. 2014, 91, 440−442
