HPLC-Based Activity Profiling for GABA_A Receptor Modulators in Searsia pyroides Using a Larval Zebrafish Locomotor Assay^[*]

Abstract

A dichloromethane extract from leaves of Searsia pyroides potentiated gamma aminobutyric acid-induced chloride currents by 171.8 ± 54% when tested at 100 µg/mL in Xenopus oocytes transiently expressing gamma aminobutyric acid type A receptors composed of α ₁ β ₂ γ ₂s subunits. In zebrafish larvae, the extract significantly lowered pentylenetetrazol-provoked locomotion when tested at 4 µg/mL. Active compounds of the extract were tracked with the aid of HPLC-based activity profiling utilizing a previously validated zebrafish larval locomotor activity assay. From two active HPLC fractions, compounds 1 – 3 were isolated. Structurally related compounds 4 – 6 were purified from a later eluting inactive HPLC fraction. With the aid of ¹H and ¹³C NMR and high-resolution mass spectrometry, compounds 1 – 6 were identified as analogues of anacardic acid. Compounds 1 – 3 led to a concentration-dependent decrease of pentylenetetrazol-provoked locomotion in the zebrafish larvae model, while 4 – 6 were inactive. Compounds 1 – 3 enhanced gamma aminobutyric acid-induced chloride currents in Xenopus oocytes in a concentration-dependent manner, while 4 – 6 only showed marginal enhancements of gamma aminobutyric acid-induced chloride currents. Compounds 2, 3, and 5 have not been reported previously.

Key words

Searsia pyroides - Anacardiaceae - GABA_A receptor - zebrafish larvae - Xenopus oocytes - pentylenetetrazole - HPLC-based activity profiling

^* Dedicated to Professor Dr. Max Wichtl in recognition of his outstanding contribution to pharmacognosy research.

• References

• 1 Uusi-Oukari M, Korpi ER. Regulation of GABAA receptor subunit expression by pharmacological agents. Pharmacol Rev 2010; 62: 97-135
• 2 Kash TL, Jenkins A, Kelley JC, Trudell JR, Harrison NL. Coupling of agonist binding to channel gating in the GABAA receptor. Nature 2003; 421: 272-275
• 3 Barrera NP, Edwardson JM. The subunit arrangement and assembly of ionotropic receptors. Trends Neurosci 2008; 31: 569-576
• 4 Rudolph U, Knoflach F. Beyond classical benzodiazepines: novel therapeutic potential of GABAA receptor subtypes. Nat Rev Drug Discov 2011; 10: 685-697
• 5 Möhler H. GABA A receptors in central nervous system disease: anxiety, epilepsy, and insomnia. J Recept Signal Transduct Res 2006; 26: 731-740
• 6 Potterat O, Hamburger M. Concepts and technologies for tracking bioactive compounds in natural product extracts: generation of libraries, and hyphenation of analytical processes with bioassays. Nat Prod Rep 2013; 30: 546-564
• 7 Potterat O, Hamburger M. Combined use of extract libraries and HPLC-based activity profiling for lead discovery: potential, challenges, and practical considerations. Planta Med 2014; 80: 1171-1181
• 8 Kim HJ, Baburin I, Khom S, Hering S, Hamburger M. HPLC-based activity profiling approach for the discovery of GABAA receptor ligands using an automated two microelectrode voltage clamp assay on Xenopus oocytes. Planta Med 2008; 74: 521-526
• 9 Rueda DC, Raith M, De Mieri M, Schöffmann A, Hering S, Hamburger M. Identification of dehydroabietic acid from Boswellia thurifera resin as a positive GABAA receptor modulator. Fitoterapia 2014; 99: 28-34
• 10 Rueda DC, De Mieri M, Hering S, Hamburger M. HPLC-based activity profiling for GABAA receptor modulators in Adenocarpus cincinnatus . J Nat Prod 2014; 77: 640-649
• 11 Rueda DC, Schöffmann A, De Mieri M, Raith M, Jähne EA, Hering S, Hamburger M. Identification of dihydrostilbenes in Pholidota chinensis as a new scaffold for GABAA receptor modulators. Bioorg Med Chem 2014; 22: 1276-1284
• 12 Schramm A, Ebrahimi SN, Raith M, Zaugg J, Rueda DC, Hering S, Hamburger M. Phytochemical profiling of Curcuma kwangsiensis rhizome extract, and identification of labdane diterpenoids as positive GABAA receptor modulators. Phytochemistry 2013; 96: 318-329
• 13 Khom S, Strommer B, Schöffmann A, Hintersteiner J, Baburin I, Erker T, Schwarz T, Schwarzer C, Zaugg J, Hamburger M. GABAA receptor modulation by piperine and a non-TRPV1 activating derivative. Biochem Pharmacol 2013; 85: 1827-1836
• 14 Rueda DC, Zaugg J, Quitschau M, Reich E, Hering S, Hamburger M. Discovery of GABAA receptor modulator Aristolactone in a commercial sample of the Chinese herbal drug “Chaihu” (Bupleurum chinense roots) unravels adulteration by nephrotoxic Aristolochia manshuriensis roots. Planta Med 2012; 78: 207-210
• 15 Kim HJ, Baburin I, Zaugg J, Ebrahimi SN, Hering S, Hamburger M. HPLC-based activity profiling-discovery of sanggenons as GABAA receptor modulators in the traditional Chinese drug Sang bai pi (Morus alba root bark). Planta Med 2012; 78: 440-447
• 16 Zaugg J, Ebrahimi SN, Smiesko M, Baburin I, Hering S, Hamburger M. Identification of GABAA receptor modulators in Kadsura longipedunculata and assignment of absolute configurations by quantum-chemical ECD calculations. Phytochemistry 2011; 72: 2385-2395
• 17 Zaugg J, Khom S, Eigenmann D, Baburin I, Hamburger M, Hering S. Identification and characterization of GABAA receptor modulatory diterpenes from Biota orientalis that decrease locomotor activity in mice. J Nat Prod 2011; 74: 1764-1772
• 18 Zaugg J, Eickmeier E, Ebrahimi SN, Baburin I, Hering S, Hamburger M. Positive GABA(A) receptor modulators from Acorus calamus and structural analysis of (+)-dioxosarcoguaiacol by 1D and 2D NMR and molecular modeling. J Nat Prod 2011; 74: 1437-1443
• 19 Zaugg J, Eickmeier E, Rueda DC, Hering S, Hamburger M. HPLC-based activity profiling of Angelica pubescens roots for new positive GABAA receptor modulators in Xenopus oocytes. Fitoterapia 2011; 82: 434-440
• 20 Yang X, Baburin I, Plitzko I, Hering S, Hamburger M. HPLC-based activity profiling for GABAA receptor modulators from the traditional Chinese herbal drug Kushen (Sophora flavescens root). Mol Divers 2011; 15: 361-372
• 21 Zaugg J, Baburin I, Strommer B, Kim HJ, Hering S, Hamburger M. HPLC-based activity profiling: discovery of piperine as a positive GABAA receptor modulator targeting a benzodiazepine-independent binding site. J Nat Prod 2010; 73: 185-191
• 22 Li Y, Plitzko I, Zaugg J, Hering S, Hamburger M. HPLC-based activity profiling for GABAA receptor modulators: a new dihydroisocoumarin from Haloxylon scoparium . J Nat Prod 2010; 73: 768-770
• 23 Moradi-Afrapoli F, Ebrahimi SN, Smiesko M, Hamburger M. HPLC-based activity profiling for GABA_A receptor modulators in extracts – validation of an approach utilizing a larval zebrafish locomotor assay. J Nat Prod 2017;
• 24 Afrikanova T, Serruys ASK, Buenafe OE, Clinckers R, Smolders I, de Witte PAM, Crawford AD, Esguerra CV. Validation of the zebrafish pentylenetetrazol seizure model: locomotor versus electrographic responses to antiepileptic drugs. PLoS One 2013; 8: e54166
• 25 Baraban SC, Taylor MR, Castro PA, Baier H. Pentylenetetrazole induced changes in zebrafish behavior, neural activity and c-fos expression. Neuroscience 2005; 131: 759-768
• 26 Jäger A, Knap D, Nielsen B, Stafford GI, van Staden J. Searsia species with affinity to the N-methyl-D-aspartic acid (NMDA) receptor. S Afr J Bot 2012; 78: 312-314
• 27 Marchetti C, Gavazzo P, Stafford GI, van Staden J. South African plants used in traditional medicine to treat epilepsy have an antagonistic effect on NMDA receptor currents. J Ethnopharmacol 2011; 137: 382-388
• 28 Pedersen ME, Baldwin RA, Niquet J, Stafford GI, van Staden J, Wasterlain CG, Jäger AK. Anticonvulsant effects of Searsia dentata (Anacardiaceae) leaf extract in rats. Phytother Res 2010; 24: 924-927
• 29 Stafford GI, Pedersen ME, van Staden J, Jäger AK. Review on plants with CNS-effects used in traditional South African medicine against mental diseases. J Ethnopharmacol 2008; 119: 513-537
• 30 Svenningsen AB, Madsen KD, Liljefors T, Stafford GI, van Staden J, Jäger AK. Biflavones from Rhus species with affinity for the GABAA/benzodiazepine receptor. J Ethnopharmacol 2006; 103: 276-280
• 31 Frost DJ, Gunstone FD. The PMR analysis of non-conjugated alkenoic and alkynoic acids and esters. Chem Phys Lipids 1975; 15: 53-85
• 32 ACD/Structure Elucidator, version 16.1.1, Advanced Chemistry Development, Inc., Toronto, ON, Canada.
• 33 Schultz DJ, Olsen C, Cobbs GA, Stolowich NJ, Parrott MM. Bioactivity of anacardic acid against Colorado potato beetle (Leptinotarsa decemlineata) larvae. J Agric Food Chem 2006; 54: 7522-7529
• 34 Jie LK, Marcel SF. Analysis of conjugated linoleic acid esters by nuclear magnetic resonance spectroscopy. Eur J Lipid Sci Technol 2001; 103: 628-632
• 35 Malik MA, OʼBrien P, Tuna F, Pritchard R, Buchweishaija J, Kimambo E, Mubofu EB. The synthesis, spectroscopy and X-ray single crystal structure of catena-[(µ-anacardato)-copper(II)-bipyridine][Cu₂{(µ-O₂CC₆H₃(o-OH)(o-C₁₅H₃₁)}₄-(NC₅H₅)₂]. Dalton Trans 2013; 42: 14438-14444
• 36 Choi YH, Choi HK, Peltenburg-Looman A, Lefeber AW, Verpoorte R. Quantitative analysis of ginkgolic acids from Ginkgo leaves and products using 1H-NMR. Phytochem Anal 2004; 15: 325-330
• 37 Chen J, Zhang YH, Wang LK, Sucheck SJ, Snow AM, Hecht SM. Inhibitors of DNA polymerase β from Schoepfia californica . Chem Commun 1998; 24: 2769-2770
• 38 Kubo I, Kim M, Naya K, Komatsu S, Yamagiwa Y, Ohashi K, Sakamoto Y, Hirakawa S, Kamikawa T. Prostaglandin synthetase inhibitors from the African medicinal plant Ozoroa mucronata . Chem Lett 1987; 16: 1101-1104
• 39 Gény C, Rivière G, Bignon J, Birlirakis N, Guittet E, Awang K, Litaudon M, Roussi F, Dumontet V. Anacardic acids from Knema hookeriana as modulators of Bcl-xL/Bak and Mcl-1/Bid interactions. J Nat Prod 2016; 79: 838-844
• 40 Akhtar MN, Lam KW, Abas F, Ahmad S, Shah SAA, Choudhary MI, Lajis NH. New class of acetylcholinesterase inhibitors from the stem bark of Knema laurina and their structural insights. Bioorg Med Chem Lett 2011; 21: 4097-4103
• 41 Spencer GF, Tjarks LW, Kleiman R. Alkyl and phenylalkyl anacardic acids from Knema elegans seed oil. J Nat Prod 1980; 43: 724-730
• 42 Westerfield M. The Zebrafish Book. A Guide for the Laboratory Use of Zebrafish (Danio rerio), 5th edition. Eugene: University of Oregon Press; 2007