HPLC-based Activity Profiling – Discovery of Sanggenons as GABA_A Receptor Modulators in the Traditional Chinese Drug Sang bai pi (Morus alba Root Bark)

• Abstract
• Introduction
• Materials and Methods
• General experimental procedures
• Plant material
• Extraction
• HPLC activity profiling
• Preparative isolation of compounds
• Expression and functional characterization of GABA_A receptors
• Two-microelectrode voltage clamp studies
• Screening of extract library and profiling for GABA_A receptor modulating activity
• Data analysis and statistics
• Results and Discussion
• Supporting information
• Acknowledgments
• References

Abstract

EtOAc extracts from two batches of Morus alba root bark (Sang bai pi) potentiated γ-aminobutyric acid (GABA)-induced chloride influx in Xenopus oocytes, which transiently expressed GABA_A receptors of the subunit composition α ₁ β ₂ γ _2S. With the aid of HPLC-based activity profiling of the extract from the first batch, activity was traced to a peak subsequently identified as sanggenon G (3). The second batch had a different phytochemical profile, and HPLC-based activity profiling led to the identification of sanggenon C (4) and a stereoisomer of sanggenon D (2) as positive GABA_A receptor modulators. The structurally related compound kuwanon L (1) was inactive. The sanggenons represent a new scaffold of positive GABA_A receptor modulators.

Introduction

Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian central nervous system. It mediates fast synaptic inhibition resulting from increased chloride influx through the γ-aminobutyric acid type A receptor (GABA_A receptor), a member of the ligand-gated ion channel superfamily. GABA_A receptors are heteropentamers assembled from different combinations of a total of 19 subunit isoforms (α _1–6, β _1–3, γ _1–3, δ, ε, π, ρ _1–3 and θ _1–3), which results in distinct GABA_A receptor subtypes. These subtypes differ in their biological and pharmacological function and the most common receptor subtype is composed of 2 α ₁, 2 β ₂, and 1 γ ₂ subunits [1], [2].

Enhancement of GABAergic neuronal inhibition through GABA_A receptors underlies the therapeutic action of the benzodiazepines which are clinically used drugs in the treatment of anxiety disorders (generalized anxiety disorder, panic attacks, phobias), sleep disorders (insomnia), epilepsy, muscle spasms, spastic disorders, and intoxication and withdrawal from alcohol [3], [4]. However, therapy is associated with severe unwanted side effects including reduced coordination, cognitive impairment, increased accident proneness, dependence, and abuse liability. Subtype-selective GABA_A receptor ligands are expected to overcome these undesired effects [5], [6]. However, synthetic drug candidates in development derive from only a few scaffolds and therefore represent limited structural diversity [5].

Natural products have been a major source of novel drug leads [7], [8]. A number of GABA_A receptor ligands from natural origins have been identified over the past two decades, including flavonoids, polyacetylenes, monoterpenes, diterpenes, neolignans, and β-carbolines [9], [10]. In an attempt to identify natural products with new scaffolds for the target, we recently screened a library of 982 plant and fungal extracts with the aid of an automated fast perfusion system and a two-microelectrode voltage clamp assay using Xenopus oocytes, which transiently expressed GABA_A receptors of the desired subunit composition. The EtOAc extracts of two different batches of the traditional Chinese medicinal (TCM) herb Sang bai pi (root bark of Morus alba L., Moraceae) significantly enhanced the GABA-induced chloride ion current.

Sang bai pi is a well-known TCM drug and has been used mainly as an expectorant, detumescent, and diuretic to treat cough, asthma, facial edema, and dysuria [11]. Analgesic, diuretic, antitussive, antiedemic, sedative, anticonvulsive, and hypotensive activities have been described [12]. The drug has been extensively studied from a phytochemical perspective, and a wide range of compounds such as Diels-Alder type adducts, benzofuran derivatives, and stilbenes have been identified [13].

Efficient identification of pharmacologically active natural products in highly complex mixtures such as plant extracts requires rapid and generally applicable deconvolution strategies. HPLC-based activity profiling is a miniaturized and highly effective approach for rapid localization and dereplication of active molecules [14]. This approach has been successfully established and has been used in our labs in combination with various cell-based and biochemical assays [15], [16], [17]. We recently developed and validated an HPLC-based activity profiling approach for the discovery of new GABA_A receptor modulators [18], which we subsequently employed to identify new scaffolds for the target [19], [20], [21], [22], [23], [24]. Here, we report on the identification of sanggenon C (4), sanggenon G (3), and a stereoisomer of sanggenon D (2) ([Fig. 1]) as positive GABA_A receptor modulators, with the aid of the HPLC-based activity profiling approach.

Materials and Methods

General experimental procedures

NMR spectra including ¹H, COSY, HSQC, and HMBC were recorded with a Bruker Avance III spectrometer operating at 500 MHz. A 1-mm TXI-microprobe with a z-gradient was used for ¹H-detected experiments. ¹³C-NMR spectra were recorded with a 5-mm BBO-probe head with a z-gradient. Spectra were analyzed using Bruker TopSpin 2.1 software. Quasimolecular ions were obtained from an HPLC/DAD/ESIMS system including an Agilent HP 1100 system coupled to an Esquire 3000 Plus ion trap mass spectrometer (Bruker Daltonics). Data acquisition and processing were performed with Bruker Hystar 3.0 software. Further spectroscopic data were acquired on a Hewlett Packard 8453 Photometer (UV), an AVIV CD Spectrometer Model 62ADS (CD), and a Perkin Elmer polarimeter (model 341) equipped with a 10-cm microcell (OR). The optical rotation for the Na D line (589 nm) was extrapolated from the lines of an Hg lamp using the Drude equation [25].

SunFire™ C18 (3.5 µm, 3.0 × 150 mm i. d.) and SunFire™ Prep C18 (5 µm, 10 × 150 mm i. d.) columns were used for analytical and semipreparative HPLC, respectively.

HPLC-grade MeCN (Scharlau Chemie S. A.) and H₂O were used for HPLC separations. HPLC solvents contained 0.1 % HCOOH for analytical separations. DMSO-d ₆ (100 Atom%) for NMR was purchased from Armar Chemicals. Solvents used for extraction, open column chromatography, and MPLC were of technical grade and purified by distillation.

Plant material

Batches of Morus alba root bark were purchased from Peter Weinfurth GmbH (Batch I) and Yong Quan GmbH (Lot. Nr. 0601401; Batch II). The identity was checked by one of us (H.-J. K.) by comparison of macroscopic and microscopic characteristics with the corresponding monograph of the Chinese Pharmacopoeia [26]. Voucher specimens (Batch I: 00218; Batch II: 0 601 401) are deposited at the Division of Pharmaceutical Biology, University of Basel.

Extraction

The dried plant samples were ground with a Retsch ZM1 mill. Extracts for the initial screening were prepared with an ASE 200 extraction system with a solvent module (Dionex) by consecutive extraction with DCM, EtOAc, and MeOH (1 cycle of 5 min for each solvent). The extraction pressure was 120 bar and the temperature was set at 70 °C.

For preparative isolation, 65 g (Batch I) and 200 g (Batch II) of powdered root bark were extracted at room temperature with 3 × 0.4 L and 2 L of EtOAc, respectively, for 3 days. Extracts were concentrated at a reduced pressure to yield 2.37 g (Batch I) and 6.67 g (Batch II) of dry residue.

HPLC activity profiling

Microfractionation for activity profiling was performed using a Waters SunFire™ Prep C18 (5 µm, 10 × 150 mm i. d.). HPLC separations for time-based and peak-based fractionation were carried out with MeCN and H₂O, at a flow rate of 7 mL/min. The gradient profile was as follows: 10 % to 100 % MeCN in 30 min, followed by 100 % MeCN for 5 min. HPLC chromatograms were recorded at UV 254 nm. The extract was dissolved in DMSO at a concentration of 20 mg/mL, and aliquots of 500 µL (corresponding to 10 mg of extract) were injected. The column effluent was collected into test tubes (90 s per fraction) for time-based microfractionation. Fractionation was started 3 min after injection. Peak-based fractionation for the extract (Batch I) was performed under the same HPLC conditions as above. A total of 11 fractions were collected on the basis of the HPLC chromatogram in the selected time window (16.5 to 19.5 min). After evaporation of the solvent, all fractions were redissolved in 1 mL of MeOH, evenly distributed into 2 vials, and dried under N₂ gas to yield two sets of fractions for bioassay and analytical purposes.

Preparative isolation of compounds

For the EtOAc extract of batch I, a portion (2.3 g) of extract was separated using open column chromatography on silica gel (Merck, 40–63 µm; 3.5 × 27 cm). A step gradient with mixtures of CHCl₃-MeOH [15 : 1 (800 mL) → 13 : 1 (280 mL) → 12 : 1 (390 mL) → 10 : 1 (330 mL) → 9 : 1 (200 mL) → 5 : 1 (240 mL)] followed by MeOH 100 % (300 mL) afforded 17 fractions. Fractions were analyzed by TLC [silica gel; CHCl₃-MeOH (0.1 % HCOOH); detection with vanillin-sulfuric acid reagent]. Fractions 11–14 (Rf 0.32–0.25) were further separated by semipreparative HPLC. Fraction 13 (22.5 mg) was purified using MeCN-H₂O (42 : 58 → 70 : 30 in 30 min; flow rate 2 mL/min) to afford sanggenon G (3) (6.5 mg, t _R 21.87 min, > 95 % [¹H NMR]) [30]. Fraction 14 (32.0 mg) was separated with MeCN-H₂O (20 : 80 → 70 : 30 in 30 min; flow rate 4 mL/min) to afford additional sanggenon G (3) (12.2 mg, t _R 24.57 min, > 95 % [¹H NMR]). Fraction 12 (42.5 mg) was separated using MeCN-H₂O (30 : 70 → 50 : 50 in 30 min; flow rate 4 mL/min) into 7 subfractions. Subfraction 6 (7.2 mg, t _R 21.87 min) was further purified on semipreparative HPLC using MeCN (0.1 % HCOOH)-H₂O (0.1 % HCOOH) (30 : 70 → 70 : 30 in 30 min; flow rate 2 mL/min) to afford 2 (6 mg, t _R 29.04 min, > 95 % [¹H NMR]). An aliquot (35 mg) of fraction 11 (total 77.7 mg) was separated by semipreparative HPLC using MeCN-H₂O (30 : 70 → 50 : 50 in 30 min; flow rate 4 mL/min) to afford kuwanon L (1) (16.3 mg, t _R 21.83 min, ∼ 85 % [¹H NMR]).

For the EtOAc extract of batch II, a portion (6.18 g) of extract was separated using open column chromatography on silica gel (Merck, 40–63 µm; 3.5 × 25 cm). Step gradient mixtures of n-hexane-EtOAc-MeOH [7 : 5 : 1 (1300 mL) → 6 : 6 : 1.2 (660 mL) → 4 : 8 : 1.6 (340 mL)] followed by MeOH 100 % (500 mL) afforded 12 fractions. Fractions were analyzed by TLC [silica gel, CHCl₃-MeOH (+ 0.1 % HCOOH) (6 : 1); detection with vanillin-sulfuric acid reagent]. Fractions 7 (380 mg; Rf 0.26–0.40) and 9 (330 mg; Rf 0.19–0.36) were purified on Sephadex LH-20 (1.5 × 50 cm and 1.5 × 45 cm, respectively) using MeOH to afford a sanggenon C containing subfraction (180 mg; Rf 0.28–0.42), and compound 2 (140 mg, Rf 0.33, ∼ 90 % [¹H NMR]), respectively. Sanggenon C (4) (59 mg, t _R 19.94 min, > 95 % [¹H NMR]) was purified from an aliquot (67.3 mg) of the above-mentioned subfraction, by semipreparative HPLC (MeCN-H₂O gradient 45 : 55 → 80 : 20 in 30 min; flow rate 2 mL/min).

Expression and functional characterization of GABA_A receptors

Preparation of oocytes from Xenopus laevis as well as injection and expression of cRNA for α ₁ β ₂ γ _2S GABA_A receptor subtypes were performed as previously described [18], [27].

Two-microelectrode voltage clamp studies

Electrophysiological experiments were performed by the two-microelectrode voltage clamp method making use of a TURBO TEC-03X amplifier (npi electronic GmbH) at a holding potential of − 70 mV and pCLAMP 10 data acquisition software (Molecular Devices). Currents were low-pass filtered at 1 kHz and sampled at 3 kHz. The bath solution contained 90 mM NaCl, 1 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂ and 5 mM HEPES (pH 7.4). Voltage recording and current injecting microelectrodes were filled with 2 M KCl and pulled to have resistances between 0.5 and 2 MΩ.

Screening of extract library and profiling for GABA_A receptor modulating activity

Screening of extracts and microfractions in an automated fast perfusion system with Xenopus oocytes have been previously reported [18], [28]. Briefly, aliquots from the liquid library (10 mg/mL extract in DMSO) were diluted with saline bath solution to a concentration of 100 µg/mL and then mixed with 1 mM GABA stock solution to an effective concentration of GABA that induces 3–10 % of the maximal response (EC_3–10). Microfractions collected from the semipreparative HPLC separations were dissolved in 30 µL DMSO, and mixed with 2.97 mL of bath solution containing a GABA EC_3–10. From all samples and controls (GABA EC_3–10 with 1 % DMSO), 100 µL were applied to the oocyte at a perfusion speed of 300 µL/s. DMSO (1 %) had a negligible effect on the control GABA-induced chloride current (− 2.7 ± 1.4 %, n = 3) which was statistically not significant from zero (p > 0.05, [Fig. 2]). Midazolam (Sigma; purity ≥ 98 %) was used as a positive control. At 1 µM, midazolam potentiated I_GABA by 271.2 ± 15.6 % (n = 3, [Fig. 2], see previously published study by Khom et al. [27] for comparison).

Data analysis and statistics

Enhancement of the GABA-induced chloride current (I_GABA) was defined as I_{(GABA + Comp)}/I_GABA − 1, where I_{(GABA + Comp)} is the current response in the presence of a given compound, and I_GABA is the control GABA-induced chloride current. Concentration-response curves were generated and the data were fitted by nonlinear regression analysis using Origin Software (OriginLab Corporation). Data were fitted to the equation 1/(1+(EC₅₀/[compound]^nH), where EC₅₀ is the concentration of the compound that increases the amplitude of the GABA-evoked current by 50 %, and n _H is the Hill coefficient.

Data are given as mean ± SE (n = number of experiments) and statistical significance was calculated using the t-test by ANOVA. Differences were considered to be significant at p < 0.05.

Results and Discussion

The EtOAc extracts from two different batches of Morus alba enhanced the GABA-induced chloride ion current (I_GABA) by 107.2 % ± 2.8 % (n = 2, batch I) and 57.2 % ± 34.4 % (n = 2, batch II) when tested at a concentration of 100 µg/mL in the initial screening. The I_GABA modulation by the two batches was statistically not significantly different (p > 0.05). The extracts were then submitted to HPLC-based activity profiling [18]. Aliquots of 10 mg of extract were separated by semipreparative HPLC using gradient elution, and time-based microfractions (90 s each) were collected and subjected to the bioassay. HPLC chromatograms and corresponding activity profiles for the two extracts are shown in [Fig. 3] (batch I) and [Fig. 4] (batch II). Online UV and MS data of the extracts revealed two differing compound profiles, which also resulted in distinctly different activity profiles ([Figs. 3] and [4]).

Batch I showed a strong enhancement of I_GABA in the time window 16.5 to 19.5 min, corresponding to fractions 10 (297.5 % ± 2.5 %, n = 2) and 11 (580.3 % ± 28.6 %, n = 3), whereas virtually no activity was found in the other fractions ([Fig. 3 A, B]). The chromatogram in the region of activity was highly complex, and activity could not be assigned to particular peaks. Therefore, the critical time window was submitted to peak-based microfractionation. Despite a fairly complex chromatogram in this time window, the activity profile showed a single distinctive activity peak corresponding to the HPLC peak at t _R = 17.43 min ([Fig. 3 C, D]). The mean I_GABA enhancement by 631.8 % ± 108.2 % (n = 2, e), 72.6 % ± 6.6 % (n = 2, f), 134.6 % ± 11.1 % (n = 2, i) and 68.2 % ± 8.2 % (n = 2, k) of the four most active subfractions is illustrated in [Fig. 3 E].

Subfraction a contained the major peak and inactive compound 1. Spectral data including CD measurements were compared with the literature, and 1 could be unambiguously identified as 2S,3′′R,4′′R,5′′S-Kuwanon L [29]. Compound 3 corresponded to the active peak in fraction e. LC-UV and ESI-MS data, and off-line microprobe NMR spectra, were compared with reported data [30]. The compound was subsequently isolated in a preparative scale to afford a pale yellow powder with λ_max (MeOH) at 203 and 286 nm. ESI-MS (negative and positive ion modes) spectra of 3 showed quasimolecular ions at m/z 693 [M – H]⁻, 695 [M + H]⁺ and 717 [M + Na]⁺, respectively, indicating a molecular mass of 694. 1D and 2D NMR spectra showed signals typical to a 1′′,3′′,4′′,5′′-substituted cyclohexene ring identical to Diels-Alder type adducts from M. alba with a trans-trans configuration. The substituents attached to the cyclohexene were identified as a C6-substituted 2′,4′,5,7-tetrahydroxyflavanone moiety at C3′′, a 2,4-dihydroxyphenyl moiety at C-5′′, a 2,4-dihydroxybenzyl moiety at C-4′′, and a 4-methyl-pent-3-en-1-yl moiety at C-1′′. Hence, the structure of 3 was identical to sanggenon G. Up till now, the absolute configuration of the cyclohexene ring of sanggenon G had not been unambiguously established [31]. Since the CD spectra of 1 and 3 were highly congruent ([Fig. 5]), the previously suggested absolute configuration 3′′R,4′′R,5′′S of the cyclohexene moiety of 3 was confirmed. The configuration at C-2 was established by comparison with the CD spectra of kuwanon G and 2S-flavanones. Kuwanon G [31] is another trans-trans-Diels-Alder type adduct bearing a 3′′R,4′′R,5′′S-cyclohexene moiety without additional stereocenters. Its CD spectrum was very similar to 3, with the exception of a negative Cotton effect at ∼ 310 nm appearing as a shoulder of the negative minimum at ∼ 290 nm [31]. The spectrum of 3 showed no shoulder in this region ([Fig. 5]). This difference can be explained by the positive Cotton effect of 2S-flavanones at ∼ 330 nm [32], which overlaps with the contribution of the cyclohexene moiety [29]. Hence, the absolute configuration of 3 could be assigned as 2S,3′′R,4′′R,5′′S.

HPLC-based activity profiling of the EtOAc extract of batch II revealed two active fractions (209.4 % ± 26.4 % and 1050.0 % ± 25.0 % potentiation of I_GABA, n = 3, [Fig. 4 A]) corresponding to peaks eluting in the time windows 15.0 to 16.5 (fraction 9) and 18.0 to 19.5 min (fraction 11). Two major peaks at t _R = 16.31 (identical with compound 2 in batch I) and 18.77 min (compound 4) in the chromatogram ([Fig. 4 B]) were identified as being responsible for the activity.

Compounds 2 and 4 were subsequently isolated at a preparative scale and obtained as pale yellow amorphous powders. The UV and ESI-MS spectra of 2 and 4 were similar with λ_max (MeOH) at 204/203, 284/285, and 310/311 nm, respectively. Both showed quasimolecular ions at m/z 707 [M – H]⁻ and 731 [M + Na]⁺ corresponding to a molecular mass of 708 g/mol. However, off-line microprobe ¹H-NMR spectra of 4 showed typical coupling constants corresponding to a cis-trans configuration of the methylcyclohexene ring (J _{4′′,3′′} = 6.7 Hz, J _{4′′,5′′} = 9.5 Hz) resulting from an endo-Diels-Alder addition [13], whereas 2 showed a trans-trans configuration in the cyclohexene ring as in 1 and 3. Hence, the molecular constitution of 2 was similar to that of sanggenon D, while 4 corresponded to sanggenon C [33], [34], [35]. Detailed spectral data are given in the Supporting Information. The CD spectrum and optical rotation of 4 ([Fig. 5] and Supporting Information) were identical with previously reported data [31], [36]. Hence the absolute configuration was established as 2R,3S,3′′S,4′′R,5′′S. In contrast to 4, the CD spectrum of 2 did not completely match with the literature for sanggenon D [31]. However, it closely matched with the spectrum of kuwanon X, another 3′′S,4′′R,5′′S-Diels-Alder type adduct lacking additional stereocenters. Apart from confirming the covalent structure of 2, our NMR data clearly indicated a trans-trans configured cyclohexene ring. Hence, we corroborated the 3′′S,4′′R,5′′S configuration in 2, but further experiments are needed to determine the absolute configuration of the remaining stereocenters at C-2 and C-3.

Compounds 2–4 were subsequently applied to oocytes expressing α ₁ β ₂ γ _2S GABA_A receptors at 1, 3, 10, 30 and 100 µM. The corresponding dose-response curves for I_GABA enhancement are shown in [Fig. 6]. They displayed statistically comparable potencies (the EC₅₀ values ranging from 13 to 17 µM, p > 0.05) with maximal I_GABA potentiation ranging from 716 % to 730 % (see legend of [Fig. 6] for details).

Interestingly, application of the saturating concentrations of 2–4 (30 µM) induced chloride currents in the absence of GABA ([Fig. 7]). The current amplitudes induced by sanggenons C, D, and G were not significantly different (p > 0.05) and did not exceed 5 % of the maximal I_GABA induced by a saturating GABA concentration (1 mM).

The isolated sanggenons (2–4) represent new scaffolds of GABA_A receptor ligands. Structurally related kuwanon L (1) was inactive. Given the limited set of molecules, it is not possible to derive structure-activity information. However, the attachment position of the substituted cyclohexenyl residue of the flavanone moiety (compare active 3 vs. inactive 1) seems important for activity. Potencies and efficiencies of the sanggenons are comparable with isoprenylated flavonoids (e.g., 8-lavandulyl flavanoids from Sophora flavescens [23] or simple flavones such as wogonin from Scutellaria baicalensis [37]). In contrast, the C5′-C8 biflavone amentoflavone acts as a negative GABA_A receptor modulator inhibiting the GABA-induced chloride current in vitro [38], [39]. Further experiments should be conducted to explore the binding site of the sanggenons and their potential for GABA_A receptor subtype specificity. This may eventually lead to a better understanding of the structural features responsible for either positive or negative modulatory activity of various flavonoids [40].

Supporting information

Optical rotation, circular dichroism, UV, mass spectrometry data, and ¹H and ¹³C NMR shifts (¹³C from HSQC and HMBC) of compounds 1–4 are available as Supporting Information.

Acknowledgments

The authors thank Dr. I. Plitzko for recording NMR spectra, Ms T. Schulthess for technical assistance with the UV and CD spectrometer, and Dr. M. Quitschau for valuable discussions on structure elucidation. Financial support was provided from the Korea Research Foundation funded by the Korean Government (MOEHRD) (Grant No. KRF-2006–352-E0026 to H. K.), from FWF grant 15914 (to S. H.), and the Swiss National Science Foundation grant 205321–116157/1 (to M. H.). Contributions from the Swiss National Science Foundation (Projects 31600–113109), the Steinegg-Foundation and the Stutz-Stiftung, both Herisau, and the Fonds zur Förderung von Lehre und Forschung, Basel, for the purchase of an NMR spectrometer are gratefully acknowledged.

Conflict of Interest

The authors declare no conflict of interest.

References

• 1 Olsen R W, Sieghart W. International union of pharmacology. LXX. Subtypes of gamma-aminobutyric acid(A) receptors: classification on the basis of subunit composition, pharmacology, and function. Update.  Pharmacol Rev. 2008;  60 243-260
  Search in Google Scholar
• 2 Olsen R W, Sieghart W. GABA(A) receptors: subtypes provide diversity of function and pharmacology.  Neuropharmacology. 2009;  56 141-148
  Search in Google Scholar
• 3 Rudolph U, Knoflach F. Beyond classical benzodiazepines: novel therapeutic potential of GABAA receptor subtypes.  Nat Rev Drug Discov. 2011;  10 685-697
  Search in Google Scholar
• 4 Tan K R, Rudolph U, Luscher C. Hooked on benzodiazepines: GABAA receptor subtypes and addiction.  Trends Neurosci. 2011;  34 188-197
  Search in Google Scholar
• 5 Mohler H. The rise of a new GABA(A) pharmacology.  Neuropharmacology. 2011;  60 1042-1049
  Search in Google Scholar
• 6 Rudolph U, Mohler H. GABA-based therapeutic approaches: GABAA receptor subtype functions.  Curr Opin Pharmacol. 2006;  6 18-23
  Search in Google Scholar
• 7 Newman D J, Cragg G M. Natural products as sources of new drugs over the last 25 years.  J Nat Prod. 2007;  70 461-477
  Search in Google Scholar
• 8 Butler M S. Natural products to drugs: natural product-derived compounds in clinical trials.  Nat Prod Rep. 2008;  25 475-516
  Search in Google Scholar
• 9 Johnston G A R, Hanrahan J R, Chebib M, Duke R K, Mewett K N. Modulation of ionotropic GABA receptors by natural products of plant origin.  Adv Pharmacol. 2006;  54 286-316
  Search in Google Scholar
• 10 Tsang S Y, Xue H. Development of effective therapeutics targeting the GABA(A) receptor: naturally occuring alternatives.  Curr Pharm Des. 2004;  10 1035-1044
  Search in Google Scholar
• 11 Chang H-M, But P P-H. Pharmacology and applications of Chinese Materia Medica. Singapore: World Scientific Publishing Co. Pte. Ltd.; 1987: 1022
  Search in Google Scholar
• 12 Tang W, Eisenbrand G. Handbook of Chinese medicinal plants. Chemistry, pharmacology, toxicology. Weinheim: Wiley-VCH; 2011: 777-781
  Search in Google Scholar
• 13 Nomura T, Hano Y. Isoprenoid-substituted phenolic compounds of moraceous plants.  Nat Prod Rep. 1994;  11 205-218
  Search in Google Scholar
• 14 Potterat O, Hamburger M. Natural products in drug discovery – concepts and approaches for tracking bioactivity.  Curr Org Chem. 2006;  10 899-920
  Search in Google Scholar
• 15 Danz H, Stoyanova S, Wippich P, Brattstroem A, Hamburger M. Identification and isolation of the cyclooxygenase-2 inhibitory principle in Isatis tinctoria.  Planta Med. 2001;  67 411-416
  Search in Google Scholar
• 16 Dittmann K, Gerhaeuser C, Klimo K, Hamburger M. HPLC-based activity profiling of Salvia miltiorrhiza for MAO A and iNOS inhibitory activities.  Planta Med. 2004;  70 909-913
  Search in Google Scholar
• 17 Adams M, Zimmermann S, Kaiser M, Brun R, Hamburger M. A protocol for HPLC-based activity profiling for natural products with activities against tropical parasites.  Nat Prod Commun. 2009;  4 1377-1381
  Search in Google Scholar
• 18 Kim H J, Baburin I, Khom S, Hering S, Hamburger M. HPLC-based activity profiling approach for the discovery of GABA(A) receptor ligands using an automated two microelectrode voltage clamp assay on Xenopus oocytes.  Planta Med. 2008;  74 521-526
  Search in Google Scholar
• 19 Zaugg J, Baburin I, Strommer B, Kim H J, Hering S, Hamburger M. HPLC-based activity profiling: discovery of piperine as a positive GABA(A) receptor modulator targeting a benzodiazepine-independent binding site.  J Nat Prod. 2010;  73 185-191
  Search in Google Scholar
• 20 Zaugg J, Eickmeier E, Ebrahimi S N, 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
  Search in Google Scholar
• 21 Zaugg J, Eickmeier E, Rueda D C, 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
  Search in Google Scholar
• 22 Zaugg J, Khom S, Eigenmann D, Baburin I, Hamburger M, Hering S. Identification and characterization of GABA(A) receptor modulatory diterpenes from Biota orientalis that decrease locomotor activity in mice.  J Nat Prod. 2011;  74 1764-1772
  Search in Google Scholar
• 23 Yang X, Baburin I, Plitzko I, Hering S, Hamburger M. HPLC-based activity profiling for GABA(A) receptor modulators from the traditional Chinese herbal drug Kushen (Sophora flavescens root).  Mol Divers. 2011;  15 361-372
  Search in Google Scholar
• 24 Li Y, Plitzko I, Zaugg J, Hering S, Hamburger M. HPLC-based activity profiling for GABA(A) receptor modulators: a new dihydroisocoumarin from Haloxylon scoparium.  J Nat Prod. 2010;  73 768-770
  Search in Google Scholar
• 25 Fluegge J. Grundlagen der Polarimetrie. Berlin: De Gruyter-Verlag; 1970
  Search in Google Scholar
• 26 Pharmacopoeia of the People's Republic of China. English edition. Beijing: China Medical Science Press; 2010: 282
  Search in Google Scholar
• 27 Khom S, Baburin I, Timin E N, Hohaus A, Sieghart W, Hering S. Pharmacological properties of GABA(A) receptors containing gamma1 subunits.  Mol Pharmacol. 2006;  69 640-649
  Search in Google Scholar
• 28 Baburin I, Beyl S, Hering S. Automated fast perfusion of Xenopus oocytes for drug screening.  Pflugers Arch. 2006;  453 117-123
  Search in Google Scholar
• 29 Hano Y, Shinkichi S, Kohno H, Nomura T. Absolute configuration of Kuwanon L, a natural Diels-Alder type adduct from the Morus root bark.  Heterocycles. 1988;  27 75-81
  Search in Google Scholar
• 30 Fukai T, Hano Y, Fujimoto T, Nomura T. Structure of sanggenon G, a new Diels-Alder adduct from the Chinese crude drug “Sang Bai Pi” (Morus root barks).  Heterocycles. 1983;  20 611-615
  Search in Google Scholar
• 31 Hano Y, Shinkichi S, Nomura T, Iitaka Y. Absolute configuration of natural Diels-Alder type adducts from the Morus root bark.  Heterocycles. 1988;  27 2315-2325
  Search in Google Scholar
• 32 Gaffield W. Circular dichroism, optical rotatory dispersion and absolute configuration of flavanones, 3-hydroxyflavanones and their glycosides.  Tetrahedron. 1970;  26 4093-4108
  Search in Google Scholar
• 33 Nomura T, Fukai T, Hano Y, Uzawa J. Structure of sanggenon D, a natural hypotensive Diels-Alder adduct from Chinese crude drug “Sang-Bai-Pi” (Morus root barks).  Heterocycles. 1982;  17 381-389
  Search in Google Scholar
• 34 Nomura T, Fukai T, Hano Y, Uzawa J. Structure of sanggenon C, a natural hypotensive Diels-Alder adduct from Chinese crude drug “Sang Bai-Pi” (Morus root barks).  Heterocycles. 1981;  16 2141-2148
  Search in Google Scholar
• 35 Hano Y, Kanzaki R, Fukai T, Nomura T. Revised structure of sanggenon A.  Heterocycles. 1997;  45 867-874
  Search in Google Scholar
• 36 Shi Y-Q, Fukai T, Ochiai M, Nomura T. Absolute structures of 3-hydroxy-2-prenylflavanones with an ether linkage between the 2′- and 3-positions from moraceous plants.  Heterocycles. 2001;  55 13-20
  Search in Google Scholar
• 37 Hui K M, Huen M S, Wang H Y, Zheng H, Sigel E, Baur R, Ren H, Li Z W, Wong J T, Xue H. Anxiolytic effect of wogonin, a benzodiazepine receptor ligand isolated from Scutellaria baicalensis Georgi.  Biochem Pharmacol. 2002;  64 1415-1424
  Search in Google Scholar
• 38 Hansen R S, Paulsen I, Davies M. Determinants of amentoflavone interaction at the GABAA receptor.  Eur J Pharmacol. 2005;  519 199-207
  Search in Google Scholar
• 39 Hanrahan J R, Chebib M, Davucheron N L M, Hall B J, Johnston G A R. Semisynthetic preparation of amentoflavone: a negative modulator at GABAA receptors.  Bioorg Med Chem Lett. 2003;  13 2281-2284
  Search in Google Scholar
• 40 Hanrahan J R, Chebib M, Johnston G A. Flavonoid modulation of GABA(A) receptors.  Br J Pharmacol. 2011;  163 234-245
  Search in Google Scholar
• 41 Khom S, Baburin I, Timin E, Hohaus A, Trauner G, Kopp B, Hering S. Valerenic acid potentiates and inhibits GABA(A) receptors: molecular mechanism and subunit specificity.  Neuropharmacology. 2007;  53 178-187
  Search in Google Scholar

1 These authors contributed equally to this work.

Prof. Matthias Hamburger

Department of Pharmaceutical Sciences
University of Basel

Klingelbergstrasse 50

4056 Basel

Switzerland

Phone: +41 (0) 6 12 67 14 25

Fax: +41 (0) 6 12 67 14 74

Email: matthias.hamburger@unibas.ch

References

• 1 Olsen R W, Sieghart W. International union of pharmacology. LXX. Subtypes of gamma-aminobutyric acid(A) receptors: classification on the basis of subunit composition, pharmacology, and function. Update.  Pharmacol Rev. 2008;  60 243-260
  Search in Google Scholar
• 2 Olsen R W, Sieghart W. GABA(A) receptors: subtypes provide diversity of function and pharmacology.  Neuropharmacology. 2009;  56 141-148
  Search in Google Scholar
• 3 Rudolph U, Knoflach F. Beyond classical benzodiazepines: novel therapeutic potential of GABAA receptor subtypes.  Nat Rev Drug Discov. 2011;  10 685-697
  Search in Google Scholar
• 4 Tan K R, Rudolph U, Luscher C. Hooked on benzodiazepines: GABAA receptor subtypes and addiction.  Trends Neurosci. 2011;  34 188-197
  Search in Google Scholar
• 5 Mohler H. The rise of a new GABA(A) pharmacology.  Neuropharmacology. 2011;  60 1042-1049
  Search in Google Scholar
• 6 Rudolph U, Mohler H. GABA-based therapeutic approaches: GABAA receptor subtype functions.  Curr Opin Pharmacol. 2006;  6 18-23
  Search in Google Scholar
• 7 Newman D J, Cragg G M. Natural products as sources of new drugs over the last 25 years.  J Nat Prod. 2007;  70 461-477
  Search in Google Scholar
• 8 Butler M S. Natural products to drugs: natural product-derived compounds in clinical trials.  Nat Prod Rep. 2008;  25 475-516
  Search in Google Scholar
• 9 Johnston G A R, Hanrahan J R, Chebib M, Duke R K, Mewett K N. Modulation of ionotropic GABA receptors by natural products of plant origin.  Adv Pharmacol. 2006;  54 286-316
  Search in Google Scholar
• 10 Tsang S Y, Xue H. Development of effective therapeutics targeting the GABA(A) receptor: naturally occuring alternatives.  Curr Pharm Des. 2004;  10 1035-1044
  Search in Google Scholar
• 11 Chang H-M, But P P-H. Pharmacology and applications of Chinese Materia Medica. Singapore: World Scientific Publishing Co. Pte. Ltd.; 1987: 1022
  Search in Google Scholar
• 12 Tang W, Eisenbrand G. Handbook of Chinese medicinal plants. Chemistry, pharmacology, toxicology. Weinheim: Wiley-VCH; 2011: 777-781
  Search in Google Scholar
• 13 Nomura T, Hano Y. Isoprenoid-substituted phenolic compounds of moraceous plants.  Nat Prod Rep. 1994;  11 205-218
  Search in Google Scholar
• 14 Potterat O, Hamburger M. Natural products in drug discovery – concepts and approaches for tracking bioactivity.  Curr Org Chem. 2006;  10 899-920
  Search in Google Scholar
• 15 Danz H, Stoyanova S, Wippich P, Brattstroem A, Hamburger M. Identification and isolation of the cyclooxygenase-2 inhibitory principle in Isatis tinctoria.  Planta Med. 2001;  67 411-416
  Search in Google Scholar
• 16 Dittmann K, Gerhaeuser C, Klimo K, Hamburger M. HPLC-based activity profiling of Salvia miltiorrhiza for MAO A and iNOS inhibitory activities.  Planta Med. 2004;  70 909-913
  Search in Google Scholar
• 17 Adams M, Zimmermann S, Kaiser M, Brun R, Hamburger M. A protocol for HPLC-based activity profiling for natural products with activities against tropical parasites.  Nat Prod Commun. 2009;  4 1377-1381
  Search in Google Scholar
• 18 Kim H J, Baburin I, Khom S, Hering S, Hamburger M. HPLC-based activity profiling approach for the discovery of GABA(A) receptor ligands using an automated two microelectrode voltage clamp assay on Xenopus oocytes.  Planta Med. 2008;  74 521-526
  Search in Google Scholar
• 19 Zaugg J, Baburin I, Strommer B, Kim H J, Hering S, Hamburger M. HPLC-based activity profiling: discovery of piperine as a positive GABA(A) receptor modulator targeting a benzodiazepine-independent binding site.  J Nat Prod. 2010;  73 185-191
  Search in Google Scholar
• 20 Zaugg J, Eickmeier E, Ebrahimi S N, 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
  Search in Google Scholar
• 21 Zaugg J, Eickmeier E, Rueda D C, 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
  Search in Google Scholar
• 22 Zaugg J, Khom S, Eigenmann D, Baburin I, Hamburger M, Hering S. Identification and characterization of GABA(A) receptor modulatory diterpenes from Biota orientalis that decrease locomotor activity in mice.  J Nat Prod. 2011;  74 1764-1772
  Search in Google Scholar
• 23 Yang X, Baburin I, Plitzko I, Hering S, Hamburger M. HPLC-based activity profiling for GABA(A) receptor modulators from the traditional Chinese herbal drug Kushen (Sophora flavescens root).  Mol Divers. 2011;  15 361-372
  Search in Google Scholar
• 24 Li Y, Plitzko I, Zaugg J, Hering S, Hamburger M. HPLC-based activity profiling for GABA(A) receptor modulators: a new dihydroisocoumarin from Haloxylon scoparium.  J Nat Prod. 2010;  73 768-770
  Search in Google Scholar
• 25 Fluegge J. Grundlagen der Polarimetrie. Berlin: De Gruyter-Verlag; 1970
  Search in Google Scholar
• 26 Pharmacopoeia of the People's Republic of China. English edition. Beijing: China Medical Science Press; 2010: 282
  Search in Google Scholar
• 27 Khom S, Baburin I, Timin E N, Hohaus A, Sieghart W, Hering S. Pharmacological properties of GABA(A) receptors containing gamma1 subunits.  Mol Pharmacol. 2006;  69 640-649
  Search in Google Scholar
• 28 Baburin I, Beyl S, Hering S. Automated fast perfusion of Xenopus oocytes for drug screening.  Pflugers Arch. 2006;  453 117-123
  Search in Google Scholar
• 29 Hano Y, Shinkichi S, Kohno H, Nomura T. Absolute configuration of Kuwanon L, a natural Diels-Alder type adduct from the Morus root bark.  Heterocycles. 1988;  27 75-81
  Search in Google Scholar
• 30 Fukai T, Hano Y, Fujimoto T, Nomura T. Structure of sanggenon G, a new Diels-Alder adduct from the Chinese crude drug “Sang Bai Pi” (Morus root barks).  Heterocycles. 1983;  20 611-615
  Search in Google Scholar
• 31 Hano Y, Shinkichi S, Nomura T, Iitaka Y. Absolute configuration of natural Diels-Alder type adducts from the Morus root bark.  Heterocycles. 1988;  27 2315-2325
  Search in Google Scholar
• 32 Gaffield W. Circular dichroism, optical rotatory dispersion and absolute configuration of flavanones, 3-hydroxyflavanones and their glycosides.  Tetrahedron. 1970;  26 4093-4108
  Search in Google Scholar
• 33 Nomura T, Fukai T, Hano Y, Uzawa J. Structure of sanggenon D, a natural hypotensive Diels-Alder adduct from Chinese crude drug “Sang-Bai-Pi” (Morus root barks).  Heterocycles. 1982;  17 381-389
  Search in Google Scholar
• 34 Nomura T, Fukai T, Hano Y, Uzawa J. Structure of sanggenon C, a natural hypotensive Diels-Alder adduct from Chinese crude drug “Sang Bai-Pi” (Morus root barks).  Heterocycles. 1981;  16 2141-2148
  Search in Google Scholar
• 35 Hano Y, Kanzaki R, Fukai T, Nomura T. Revised structure of sanggenon A.  Heterocycles. 1997;  45 867-874
  Search in Google Scholar
• 36 Shi Y-Q, Fukai T, Ochiai M, Nomura T. Absolute structures of 3-hydroxy-2-prenylflavanones with an ether linkage between the 2′- and 3-positions from moraceous plants.  Heterocycles. 2001;  55 13-20
  Search in Google Scholar
• 37 Hui K M, Huen M S, Wang H Y, Zheng H, Sigel E, Baur R, Ren H, Li Z W, Wong J T, Xue H. Anxiolytic effect of wogonin, a benzodiazepine receptor ligand isolated from Scutellaria baicalensis Georgi.  Biochem Pharmacol. 2002;  64 1415-1424
  Search in Google Scholar
• 38 Hansen R S, Paulsen I, Davies M. Determinants of amentoflavone interaction at the GABAA receptor.  Eur J Pharmacol. 2005;  519 199-207
  Search in Google Scholar
• 39 Hanrahan J R, Chebib M, Davucheron N L M, Hall B J, Johnston G A R. Semisynthetic preparation of amentoflavone: a negative modulator at GABAA receptors.  Bioorg Med Chem Lett. 2003;  13 2281-2284
  Search in Google Scholar
• 40 Hanrahan J R, Chebib M, Johnston G A. Flavonoid modulation of GABA(A) receptors.  Br J Pharmacol. 2011;  163 234-245
  Search in Google Scholar
• 41 Khom S, Baburin I, Timin E, Hohaus A, Trauner G, Kopp B, Hering S. Valerenic acid potentiates and inhibits GABA(A) receptors: molecular mechanism and subunit specificity.  Neuropharmacology. 2007;  53 178-187
  Search in Google Scholar

1 These authors contributed equally to this work.

Prof. Matthias Hamburger

Department of Pharmaceutical Sciences
University of Basel

Klingelbergstrasse 50

4056 Basel

Switzerland

Phone: +41 (0) 6 12 67 14 25

Fax: +41 (0) 6 12 67 14 74

Email: matthias.hamburger@unibas.ch

• Supplementary Material