Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

1 Contents lists available at ScienceDirect 2 3 4 5 6 journal homepage: www.elsevier.com/locate/jep 7 8 9 10 11 12 13 A 14 15 Manuel Candelario a,1, Erika Cuellar a,1, Jorge Mauricio Reyes-Ruiz b, Narek Darabedian c, 16 c b a b,d,n Q1 Zhou Feimeng , Ricardo Miledi , Amelia Russo-Neustadt , Agenor Limon 17 a 18 Biological Sciences Department, California State University, Los Angeles, CA, United States b Department of Neurobiology & Behavior, University of California, Irvine, CA, United States 19 c Department of Chemistry and Biochemistry, California State University, Los Angeles, CA, United States 20 d Department of Psychiatry and Human Behavior, University of California, Irvine, CA, United States 21 22 23 art ic l e i nf o a b s t r a c t 24 25 Article history: Ethnopharmacological relevance: Withania somnifera (WS) has been traditionally used in Ayurvedic 26 Received 25 February 2015 medicine as a remedy for debility, stress, nervous exhaustion, insomnia, loss of memory, and to enhance 27 Received in revised form cognitive function. This study provides an empirical evidence to support the traditional use of WS to aid 22 May 2015 28 in mental process engaging GABAergic signaling. Accepted 30 May 2015 29 Aim of the study: We evaluated the effect of aqueous WS root extract (aqWS), and its two main 30 components, withaferin A and withanolide A, on the main inhibitory receptors in the central nervous Keywords: system: ionotropic GABAA receptors. 31 Synaptic receptors Materials and methods: The pharmacological activity of aqWS, withaferin A and withanolide A, was 32 Extrasynaptic receptors tested on native rat brain GABAA channels microtransplanted into Xenopus oocytes and GABAρ1 receptors 33 Ashwagandha heterologously expressed in oocytes. The GABAergic activity of aqWS compounds was evaluated by the 34 GABAergic signaling two-electrode voltage-clamp method and the fingerprint of the extract was done by LC–MS. GABA 35 Results: Concentration-dependent inward ion currents were elicited by aqWS in microtransplanted 36 oocytes with an EC50 equivalent to 4.7 mg/mL and a Hill coefficient (nH) of 1.6. The GABAA receptor 37 antagonist bicuculline blocked these currents. Our results show that aqWS activated inotropic GABAA 38 channels but with lower efficacy compared to the endogenous agonist GABA. We also demonstrate for 39 first time that aqWS is a potent agonist of GABAρ1 receptors. GABAρ1 receptors were 27 fold more 40 sensitive to aqWS than GABAA receptors. Furthermore, aqWS activated GABAρ1 receptors eliciting 41 maximum currents that were no significantly different to those produced by GABA (paired t-test; p¼ 0.533). The differential activity on GABAA and GABA ρ1 receptors and the reported lack of significant 42 GABA presence in WS root extract indicates that the GABAergic activity of aqWS is not mediated by 43 GABA. WS main active components, witaferin A and withanolide A, were tested to determine if they were 44 responsible for the activation of the GABA receptors. Neither compound activated GABAA nor GABAρ1 45 receptors, suggesting that other constituent/s in WS are responsible for GABAA receptor mediated 46 responses. 47 Conclusions: Our results provide evidence indicating that key constituents in WS may have an important 48 role in the development of pharmacological treatments for neurological disorders associated with 49 GABAergic signaling dysfunction such as general anxiety disorders, sleep disturbances, muscle spasms, 50 and seizures. In addition, the differential activation of GABA receptor subtypes elucidates a potential 51 mechanism by which WS accomplishes its reported adaptogenic properties. 52 & 2015 Published by Elsevier Ireland Ltd. 53 54 67 55 68 1. Introduction 56 69 57 70 Withania somnifera (WS), also known as Ashwagandha, is an herb 58 71 traditionally used in the Nepali and Hindi indigenous medical 59 72 n Corresponding author at: 2226 Gillespie Neuroscience Research Facility, system, Ayurveda, for its adaptogenic properties. Adaptogens refer 60 73 Department of Psychiatry and Human Behavior, University of California, Irvine, to substances that increase the “state of non-specific resistance to 61 74 CA 92697, United States. Tel.: þ 1 949 824 9058. stressors of the everyday life,” have normalizing action and are E-mail address: [email protected] (A. Limon). 62 75 1 relatively non-toxic (Lazarev, 1958; Lazarev et al., 1959). WS roots are Q2 76 First authors listed in alphabetical order. 63 64 77 http://dx.doi.org/10.1016/j.jep.2015.05.058 65 78 0378-8741/& 2015 Published by Elsevier Ireland Ltd. 66

Journal of Ethnopharmacology

Direct evidence for GABAergic activity of Withania somnifera on mammalian ionotropic GABA and GABAρ receptors

Please cite this article as: Candelario, M., et al., Direct evidence for GABAergic activity of Withania somnifera on mammalian ionotropic GABAA and GABAρ receptors. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.05.058i

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M. Candelario et al. / Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

used in the Ayurvedic medicinal system to treat general conditions like debility, stress, nervous exhaustion, insomnia, loss of memory, and to enhance cognitive function (Sivarajan and Balachandran, 1994; Warrier et al., 1996; Usmanghani et al., 1997; Kuboyama et al., 2014). Double-blind, placebo-controlled and randomized clinical studies have shown that WS has beneficial precognitive effects on bipolar disorder (Chengappa et al., 2013), and reduction of stress and anxiety measured by the Beck Anxiety Inventory that support its traditional use (Cooley et al., 2009). These results are also supported by studies in animal models reporting that WS has anticonvulsive, anti-stress, anxiolytic properties, as well as aiding in health conditions involving insomnia. All together, the traditional and clinical use of WS suggest that the WS effects are associated with potential GABAergic action (Kulkarni et al., 1993; Sharma, 1999; Bhattacharya et al., 1997, 2000). Indeed, recent studies have shown that methanolic WS root extracts induced inward currents in gonadotropin releasing hormone neurons that were blocked in the presence of the GABAA antagonist, picrotoxin (Bhattarai et al., 2010). Previous reports have shown that oral administration of withanolides isolated from WS reverse oxidation, prevent neuritic atrophy, and help remove amyloid-β peptides from the brain in mice (Kuboyama et al., 2006; Bhattacharya et al., 1995; Sehgal and Steinmann, 2012). Much of this activity seems to be dependent on the C28-steroidal lactones withanolide A (WLA) and withaferin A (WA). However, it is not known whether these withanolides are also responsible for the putative activation of GABA receptors. And whether an aqueous infusion of WS, the way WS is traditionally used, has any effect on GABA receptors. GABA is the most important inhibitory neurotransmitter in the vertebrate CNS and is involved in a variety of physiological and pathological processes such as sleep, mood, anxiety, epilepsy, and cognitive disorders (Martínez-Delgado et al., 2010). In the mammalian brain, many GABA effects are mediated by the activation of ionoptropic GABAA receptors in the cellular membrane. GABAA receptors located in the synapse mediate phasic inhibition, while those located extrasynaptically are known to regulate the resting potential of neurons by activating tonic currents in these locations (Farrant and Nusser, 2005). Notably, a number of clinically important drugs affecting cognition, stress and mood, as benzodiazepenes, neurosteroids, barbiturates, and anesthetics, are known to modulate these receptors. The GABAA receptor is a pentameric protein complex that forms a channel with high permeability to chloride ions. The subunit composition of the pentameric complex determines their sensitivity to GABA and dictates their pharmacological properties. For example, the α1 subunit of the GABAA receptor has been associated with the sedative effects of benzodiazepenes, while both, α2 and α3 subunits have been suggested to mediate anxiolytic activity and muscle relaxing effects. The α5 subtype has been associated with cognitive processes (Olsen and Sieghart, 2009). GABAA receptors, composed entirely of ρ subunits, have pharmacologically distinct properties to other GABAA receptors. GABAρ receptors are more sensitive to GABA, have a low rate of desensitization, a five-fold increase in mean channel open time and are insensitive to bicuculline, a GABAA antagonist (Martínez-Delgado et al., 2010). In addition to their unique pharmacological profile, stimulation of GABAρ receptors has been linked to PKA activated anti-apoptotic effects in primary cultured rat hippocampal neurons (Yang et al., 2008). In the presence of the GABAρ receptor agonist, cis-4aminocrotonic acid (CACA), hippocampal neurons were protected from amyloid-β-enhanced glutamate neurotoxicity (Yang et al., 2008). This protective effect was blocked by the GABAρ receptor specific antagonist (1,2,5,6-tetrahydropyridine-4-yl) methylphosphinic acid (TPMPA). Because of the role that GABAA and GABAρ subtype receptors play in physiological and neuropathological conditions associated

to stress, they are sensible targets of adaptogenic compounds; therefore, these receptors were used to evaluate the potential GABAergic bioactivity of aqWS and its main constituents, WLA and WA.

2. Materials and methods 2.1. Herbal material used W. somnifera (L.) Dunal is a species in the genus Withania and belonging to the family Solanaceae listed in the www.theplantlist. org and the World Checklist of Selected Plant Families (WCSP). WS is known by several names: English name is Winter Cherry, Latin name is Withania somnifera and Sanskirt name is Ashwagandha. For this study we used root powder (particle size o 0.85 mm) from WS organically grown in India and imported for distribution by Banyan Botanicals located in Albuquerque, NM, USA. Batch: 417063. WS from Banyan Botanicals is certified by New Mexico's Department of Agriculture, with quality control testing for organoleptic properties and presence of contaminants. Quality control data is the following: Test Method Specification Result TPC USP (2021; 2022) o 10,000,000 CFU/g 520,000 CFU/g Total coliforms AOAC 991.14 o 10,000 CFU/g o 100,000 CFU/g o 100,000 CFU/g 2000 CFU/g Arsenic (As) ICP-MS r 0.01 mg/d 0.118 ppm Cadmium (Cd) ICP-MS r 0.006 mg/d 0.020 ppm Lead (Pb) ICP-MS r 0.02 mg/d 0.185 ppm Mercury (Hg) ICPMS r 0.02 mg/d 0.004 ppm Organoleptic QC-010 See Organoleptic Spec. Complies Identity FTIR Banyan Method. 2.2. Aqueous WS root extract preparation Three commonly used preparations in traditional Ayurvedic medicine were tested for biological activity: 1) boiled decoction, 2) cold dissolved solution and 3) hot infusion. The stock solutions of each preparation were prepared at their highest concentration possible and serial dilutions from the stock in Ringer's solution (see below) were applied to receptor-expressing oocytes to test for activity. The stock boiled decoction was prepared by boiling diH2O (10 mL), adding WS root powder (2000 mg), and continuing to boil while adding diH2O in 10 mL increments. Total time of boiling for this preparation was 5 min. This preparation has limited solubility as most of the added diH2O was absorbed by the WS powder and became a paste. The available supernatant was collected after nonsoluble WS components were removed by centrifugation (2  10,000g) and filtration (0.80 mm). The stock dissolved solution was prepared by adding WS root powder (2000 mg) into room temperature diH2O (10 mL) and left overnight at 4 1C to allow water soluble components of WS to diffuse into solution. The supernatant was collected after centrifugation and filtration using the protocol described above. Stock hot infusion was prepared by boiling diH2O and then adding fresh WS root powder (200 mg/ mL). The solution was then covered and allowed to brew for 20 min. The supernatant was collected by centrifugation followed by filtration as mentioned above. We did not dehydrate the supernatant for any of the different preparations to avoid potential loss of activity and to study better the bioactivity of traditional forms of WS preparations. After testing dilutions of all three stock preparations, it was found that hot infusion showed the largest activity and was therefore used as the mode for aqWS preparation (Supplemental Fig. 1). For the rest of the experiments we used centrifuged and filtered supernatant from fresh, daily prepared, aqWS stock (200 mg/mL; WS root powder in diH2O) as our starting material. Serial dilutions of the aqWS stock in Ringer's solution were used for the experiment in this study.

Please cite this article as: Candelario, M., et al., Direct evidence for GABAergic activity of Withania somnifera on mammalian ionotropic GABAA and GABAρ receptors. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.05.058i

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2.3. Chemicals For the electrophysiological experiments withaferin A (WA; PubChem CID: 265237), withanolide A (WLA; PubChem CID: 11294368), GABA and bicuculline were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Kainic acid was purchased from Tocris Biosciences (UK). WA was reconstituted in ethanol (1 mg/mL) to make a 2.12 mM stock solution. Similarly, WLA was prepared by reconstituting in ethanol (0.5 mg/mL) to make a 1.06 mM stock solution. WLA and WA stocks were kept at 20 1C until use. Working solutions were made freshly every day by diluting the stock in Ringer's solution to concentration of 40 μM. The effects of the vehicles on the responses were also evaluated. For the Liquid Chromatography–Mass Spectrometry (LC–MS) experiments, hydrochloric acid (HCl), formic acid (FA), dichloromethane (DCM), methanol (MeOH), acetonitrile (ACN), and 0.42 μm syringe filters were obtained from Thermo Fisher Scientific Inc. (Pittsburgh, PA). 2.4. Preparation and microtransplantation of rat cortex Membranes The Xenopus oocyte two-electrode voltage clamp model system was used in our study. This model system allows for the testing of drugs/compounds on functionally active neurotransmitter receptor channels microtransplanted from native brain tissue. Oocytes from Xenopus laevis were prepared as described previously (Miledi et al., 2006; Limon et al., 2012). Native membranes containing GABA receptors were isolated from rat brain cortex. Briefly, adult Sprague Dawley rats were euthanized following procedures in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and IACUC at University of California, Irvine (IACUC: 1998-1388). Next, brains were surgically removed; the cortex was isolated and frozen by immersion in liquid nitrogen. Membrane preparations for microtransplantation experiments were prepared as described elsewhere (Limon et al., 2008). 50 nL of this preparation (3.4 mg/mL of protein) were injected into excised and defolliculated stage V–VI Xenopus oocytes.

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induced current (in nA), and k is the slope of the curve, was fitted to the experimental data (SigmaPlot 11). Since the identity and concentration of the bioactive substances are not known the x-axis of concentration/response curves represents each dilution as the Log (1/ DFaqWS); e.g. 1:10 dilution is Log(1/10) or  1. Experimental data are shown as mean7SEM unless otherwise stated. 2.7. Sample preparation for LC–MS analysis Water extraction was prepared by boiling 2000 mg of WS root powder in 10 mL of deionized H2O. The resulting solution was then centrifuged and the supernatant was collected. MeOH extraction was conducted in a similar manner except with 10 mL of MeOH. After the supernatant collection MeOH was boiled off and the resulting residue was dissolved in 5 mL ACN. DCM extraction was conducted on the residue obtained from the MeOH extraction, and the residue was dissolved with 100 mL of 50/50 (V/V) HCl/ dichrolomethane (DCM) and 10% HCl. The DCM layer was collected using a separation funnel. This was followed by boiling off DCM. Finally, the resulting residue was suspended in 1 mL ACN. All samples were filtered with a 0.42 μm syringe filter before LC–MS analyses. A Thermo Fisher Acella UHPLC system (Thermo Fisher Scientific, San Jose, CA), coupled to an Exactive Mass Spectrometry (Thermo Fisher Scientific, San Jose, CA), was used for the separation and compound identification. Separation was performed using an Aeris Widepore XB-C18 column (3.6 mm, 2.1 mm  150 mm, 200 Å) from Phenomenex (Torrance, CA) at a flow rate of 0.5 mL/min at 40 1C and eluted species were detected at 254 nm. The mobile phase contained H2O and ACN with 0.1% FA (V/V). Chromatographic separation was achieved with the following gradient 0 min – 5% B, 2 min – 5% B, 30 min – 45% B, 31 min – 95% B, 34 min – 95% B, 35 min – 5%, and 39 min – 5%. Electrospray ionization–MS was conducted in positive ion mode at a spray voltage of 4 kV and capillary temperature was set at 400 1C. The sheath, auxiliary, and sweep gases were set to 50, 10, and 1 psi, respectively. Aliquots of 20 μL were used for analysis of species extracted with water, MeOH, and DCM.

2.5. Heterologous expression of homomeric GABA p1 receptor Channels in Xenopus Oocytes 3. Results To test the activity of aqWS on GABAρ receptors, porcine GABAρ1 subunit cDNA (Reyes-Ruiz et al., 2014) was used as template to generate synthetic RNA using mMessage mMachine (Ambion, Grand Island, New York, USA) and injected into Xenopus oocytes at a concentration of 0.1 mg/mL. After 24 to 48 h, injected oocytes were used for pharmacological analyses of aqWS. 2.6. Electrophysiology and data analysis After microinjection of Xenopus oocytes with cell membrane preparations or cRNA, electrophysiological procedures previously described by Miledi, 1982 and Miledi et al., 2002, were conducted. Between day 1 and day 7 post-injection, oocytes were placed in a recording chamber (volume 0.1 mL) and continuously perfused (5–10 mL/min) by gravity with Ringer's solution [115 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 5 mM Hepes (pH 7.4)] at room temperature (19–21 1C). Membrane currents were then recorded from the oocytes voltage-clamped at  80 mV and using two glass microelectrodes filled with 3 M KCl. Data acquisition and analyses were performed with WinEDR version 2.3.8 Strathclyde Electrophysiology software (John Dempster, Glasgow, UK). Concentration/response curves were generated as previously reported (Limon et al., 2010). Briefly, a logistic equation of the form I(x)¼Imin þ(Imax  Imin)/[1þ (x/EC50)\widehatk], where x is the concentration of substance (GABA in M or aqWS in 1/DFaqWS (DF¼ dilution factor of aqWS)), I is the amplitude of the substance-

3.1. Functionally active mammalian Receptors in Xenopus system The goal of our study was to test the hypothesis that aqWS has GABAergic activity on mammalian GABAA receptors and therefore support its traditional use as anxiolytic and adaptogen. First, we evaluated whether Xenopus oocytes expressed endogenous GABA and glutamate receptors. Application of GABA (1 mM) and kainate (100 μM) elicited no responses in non-injected oocytes indicating the lack of endogenous GABA and glutamate receptors in noninjected oocytes. While the same concentrations of GABA and kainate applied to oocytes injected with rat cortex membrane preparations elicited ion currents of 2447 45 nA (n ¼4) and 657 25 nA (n ¼4) (Supplemental Fig. 2), confirming the integration of functional receptor channels in injected oocyte membranes. 3.2. aqWS GABAergic activity The effects of aqWS root extract were tested upon confirmation of functionally microtransplanted receptors. Application of aqWS, diluted in a range from 1:10 to 1:1000, consistently generated smooth inward currents in a dose dependent manner. The most concentrated application elicited currents that had a maximum response of 56 78 nA (n¼ 5). Fig. 1 shows that a 1:43 dilution of aqWS (Hill ¼1.6; n ¼ 5) was able to elicit 50% of the maximal response (EC50). According to our preparation method, this

Please cite this article as: Candelario, M., et al., Direct evidence for GABAergic activity of Withania somnifera on mammalian ionotropic GABAA and GABAρ receptors. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.05.058i

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dilution corresponds to an aqueous hot infusion of 4.7 mg/mL of WS root powder. In non-injected oocytes, aqWS only elicited small inward currents of 77 2 nA (n¼ 7) at the highest concentration tested (1:10 dilution) indicating that naïve Xenopus oocytes have a small native response to aqWS that should be considered when comparing responses in that range of concentration. To test whether the currents elicited by aqWS were mediated by the activation of GABAA receptors, we evaluated the effects of bicuculline, a GABAA antagonist. For this, 1:30 aqWS dilution was applied to the oocyte and once the aqWS-elicited current reached its maximum value we co-applied 10 mM bicuculline. Bicuculline reduced aqWS-elicited currents by 98.75% (37 7 22 vs. 3 72 nA; before and during bicuculline co-application, respectively; n ¼5) (Fig. 2). A comparison of the membrane responses elicited by aqWS relative to the endogenous agonist GABA shows that aqWS has lower efficacy on GABAA receptors (Fig. 3). In the same oocytes, application of 1:10 aqWS dilution generated a maximum current of 40 79 nA which is only 21% of the current elicited by 1 mM GABA (187 729 nA; n ¼7). To explore potential temporal interactions we evaluated the effect of aqWS pre-application on the responses elicited by GABA. Fig. 4 shows that pre-application of 1:10 aqWS activated GABA channels (29.64 75.83 nA) and then produced a reversible antagonism of the GABA response. GABA responses after aqWS were 88% smaller (20.39 78.88 nA) than the response elicited by GABA before aqWS application (165.407 23.44 nA). This result indicates that the lower efficacy of aqWS has a complex negative modulatory activity of responses elicited by GABA.

The concentration response curve gave an EC50 of 1:1176 aqWS dilution (equivalent to 0.17 mg/mL) and nH of 3.7 (Fig. 5B). As control, a concentration response curve for the GABA response was also done and showed an EC50 of 0.84 mM (Hill ¼3.3) (Fig. 5D) (n ¼5). Our EC50 for GABA in GABAρ1 receptors is in agreement with the published range of 0.8–2.2 mM for homomeric GABAρ channels (Martínez-Delgado et al., 2010). It was interesting to observe that aqWS elicited inward currents of similar amplitude as those elicited by GABA on GABAρ1 receptors (Fig. 6). The maximal current elicited by high concentrations

3.3. aqWS activity on GABAρ1 receptor We also investigated whether aqWS was bioactive on GABA receptors with different pharmacologies. For this, aqWS effects were evaluated on homomeric GABAρ1 receptors heterologously expressed in Xenopus oocytes. As Fig. 5 shows, GABAρ1 receptors were activated by highly diluted aqWS solutions. Perfusion of 1:3000 aqWS dilution elicited clear inward currents with the distinctive kinetic activation profile of GABAρ1 receptors (Fig. 5A).

Fig. 2. Antagonism of aqWS current by bicuculline. (A) Current responses elicited by aqWS in oocytes microtransplanted with brain membranes were blocked by 10 mM bicuculline. (B) Submaximal aqWS concentration (1:30 dilution) elicited responses of 377 10 nA on microtransplanted oocytes. Bicuculline reduced aqWS currents to 37 2 nA: a reduction of 92% (n¼ 5).

Fig. 1. Concentration-dependent response of aqWS in rat cortex membrane injected oocytes. (A) Representative trace of concentration-dependent inward currents elicited by aqWS on non-injected oocytes (top), or in oocytes injected with rat cortex membranes (bottom). (B) Semi-logarithmic plot of current induced by aqWS dilutions on rat cortex membrane receptors (dark circles) shows a mean maximum response of 56 7 7 nA. Because the identity and concentration of the bioactive substances are not known, here and in the following figures where dilutions of aqWS were used, the x-axis of concentration/response curves represents the Log (1/DFaqWS) where DF is the dilution factor, e.g. 1:10 dilution is Log(1/10) or  1. The EC50 value of aqWS is equivalent to a 1:43 dilution or an estimated value of 4.7 mg/mL (Hill¼ 1.6; n¼ 5). The solid curves are the fit of the Hill equation to experimental data. Application of aqWS on non-injected oocytes (clear squares) produced smaller responses with a mean maximum response of 77 2 nA (n¼7).

Please cite this article as: Candelario, M., et al., Direct evidence for GABAergic activity of Withania somnifera on mammalian ionotropic GABAA and GABAρ receptors. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.05.058i

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was elicited with a 1:1176 aqWS dilution. The differential affinity and efficacy of aqWS on GABAA receptor channel subtypes further suggest that the constituent or constituents responsible for GABAergic activity is not GABA itself. 3.4. WA and WLA activity on GABAA receptors

Fig. 3. aqWS efficacy on GABAA receptors is lower than that for GABA. Maximal concentration of aqWS (1:10 dilution equivalent to 20 mg/mL) on microtransplanted oocytes elicited membrane responses that were 21% lower than membrane responses elicited by maximal concentration of GABA (1 mM; n¼ 7).

In our last set of experiments, we tested the hypothesis that a known component found in aqWS root could be responsible for eliciting the GABAergic activity seen in the results presented above. Therefore, rat cortex membrane injected oocytes, and oocytes expressing GABAρ1 receptors were perfused with WA or WLA along with their respective ethanolic vehicles. Neither WA nor WLA activated GABAA or GABAρ1 receptors, suggesting that other constituent/s are responsible for activating the observed GABA currents. On oocytes injected with rat cortex membrane preparations, WA (40 mM) and WLA (40 mM) elicited small outward currents, 6 73 nA and 5 72 nA (n ¼5), comparable to that of their vehicles (Fig. 8B). A paired t-test showed there was no significant difference between the currents elicited by WA relative to its vehicle (p ¼0.32) or WLA and its respective vehicle (p ¼0.57). Similarly, when GABAρ1 receptors were perfused with WA (40 mM) and WLA (40 mM) small outward currents of 6 72 nA (n ¼5) were elicited. The observed current changes were no different than those produced by their respective vehicles (paired t-test value of WA vs. vehicle p¼ 0.32 and WLA vs. vehicle p ¼0.60) (Fig. 8C). 3.5. LC–MS analyses of aqWS constituents

Fig. 4. aqWS temporal interaction with GABA. Oocytes injected with rat cortex membranes were tested sequentially with GABA and aqWS. Responses to 1 mM GABA (165.40 7 23.44 nA; n¼ 5) were reduced when GABA receptors were first activated by 1:10 dilution of aqWS. aqWS elicited a maximal response of 29.647 5.83 nA (n¼ 5) and 1 mM GABA, applied immediately after aqWS, elicited a response of 20.39 7 8.88 nA (n ¼5). Notice that combined responses of aqWS and post-aqWS GABA does add up to the initial GABA response. After 5 min wait GABA responses recovered eliciting a maximal response of 138.92 7 27.48 nA (n¼3).

of aqWS (1:300 or 1:30 dilutions) was 5258 7987 nA while concentrations of 10 mM or 30 mM GABA, tested in the same oocytes, elicited maximum currents of 4787 71282 nA (n ¼5). A paired t-test (p ¼0.533) confirms that mean maximal currents elicited by aqWS are no different relative to those elicited by GABA. Therefore aqWS has similar efficacy on GABAρ1 receptors as GABA itself. Of note, in these sets of experiments the most concentrated aqWS, (1:10 dilution), elicited a small response in non-injected oocytes (n ¼10) that had mean maximum response of 9.65 72.49 nA (data not shown). This non-specific current is less than 0.2% of the maximal GABA-mediated current and does not affect our comparison of aqWS and GABA response. Because whole WS root was used, the affinity of aqWS vs. GABA on either GABAA or GABAρ1 receptors cannot be compared through EC50 values until the component/s in aqWS that activates GABA receptors are identified. Nonetheless, a comparison of current changes induced by aqWS on oocytes expressing GABAA and GABAρ1 receptors shows that aqWS has higher affinity for GABAρ1 receptors over other microtransplanted GABAA receptors (Fig. 7). While 50% of the maximal response of GABAA receptors was elicited by a 1:43 aqWS dilution, 50% of GABAρ1 maximal response

In attempt to identify the active compound(s) we conducted LC– MS. Our aqWS root extract analysis resulted in over 100 different compounds as shown in Fig. 9. We hypothesized that withanolides are responsible for the biological activity, and monitored ions of withanolide B (455.27920 m/z), withaferin A/withanolide A/withanone (471.27412 m/z), 27-hydroxywithanone/withastramonolide (487.26903 m/z), Physagulin D (621.36332 m/z), withanoside V/withanoside VI (767.42123 m/z), and withanoside IV (783.41615 m/z). Single ion monitoring (SIM) resulted in multiple peaks being generated for a given compound, and a more in-depth analysis of the MS spectra showed that they are fragments from higher m/z species. For example, as seen in Supplemental Fig. 3 (spectra corresponding to the purple arrow in Fig. 9) contains two m/z values at 471.27328, and 488.29968. The m/z of 471.27412 m/z correlates to withaferin A/ withanolide A/withanone but 488.30206, which differs by 17.02640 in m/z, shows that the precursor contains a protonated amino group R-NH3þ (theoretical difference of 17.02600 m/z). Overall all the peaks observed between 12 to 30 min are withanolide derivatives. The high abundances of withanolides derivatives are consistent with results from other studies (Kumar et al., 2010; Bolleddula et al., 2012). A comprehensive identification of all of the withanolide derivatives is not possible because to our knowledge no databases are available for MS/MS spectra and structures. We compared our aqWS root extract with that from the methanol extraction. As seen in Fig. 9 (middle) the peaks before 12 min decreased, and new peaks appeared after 30 min, but the withanolide region from 12 to 30 min in the methanol extraction is essentially identical to that in the aqueous extraction. Therefore, we concluded that at least one of the withanolides derivatives is responsible for activating GABAA channels. To ensure that the withanolide derivatives are not overlapping peaks and contain amine groups, we conducted a DCM wash. Amine groups would have been protonated and migrate towards the aqueous layer, and withanolides should stay within the DCM layer. Many of the peaks in the withanolide region were removed, but some peaks were enriched. The most notable enrichments are marked with ●, ▼, and ◆ in Fig. 9 (bottom). The ● corresponds to withanolide B

Please cite this article as: Candelario, M., et al., Direct evidence for GABAergic activity of Withania somnifera on mammalian ionotropic GABAA and GABAρ receptors. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.05.058i

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Fig. 5. aqWS activation of GABAρ1 receptors. Representative trace of concentration-dependent currents elicited by aqWS (A) and GABA (C) on oocytes expressing GABAρ1 receptors. Perfusion with aqWS elicited maximum currents of 5258 7 987 nA with an EC50 value equivalent to a 1:1176 dilution or an estimated value of 174 μg/mL (Hill ¼3.7). Graphs (B) and (D) show a semi-logarithm plot for responses to aqWS and GABA in the same oocytes, respectively (n¼ 5). GABA elicited maximum currents of 4786 7 1282 nA with an EC50 value of 0.84 mM (Hill¼ 3.3). Data is normalized to maximal response in each oocyte and expressed as response % (response/max response x 100).

Fig. 6. aqWS and GABA has similarly efficacy on GABAρ1 receptors. In GABAρ1 expressing oocytes, aqWS elicited inward currents of similar amplitude relative to GABA (n¼ 5). GABA and aqWS maximal responses were obtained from their respective concentration-response curves.

(455.27961 m/z), and ▼ corresponds to three peaks at 19.14 (m/ z¼ 471.27376), 19.24 (m/z ¼471.27312), and 19.52 min (m/ z¼ 471.27424), which were identified as withaferin A, withanolide A, and withanone. We detected 27-hydroxywithanone and withastramonolide (noted by ◆) at 14.19 (m/z ¼ 487.26915) and 14.45 min (m/z ¼ 487.26912), but UV–vis spectrometric detection is not sufficiently sensitive to detect all species. To structurally identify the withanolide derivatives within aqWS root extract, each peak in the LC would have to be separated on a larger scale with an extremely large amount of crude sample for structural analysis. This is beyond the scope of this study.

Fig. 7. aqWS affinity comparison between GABAρ1 and native GABAA receptors. aqWS had a higher affinity to GABAρ1 receptors (triangles) than to GABAA receptors (circles) microtransplanted from rat cortex membranes (n¼ 5). The larger affinity for GABAρ1 receptors is shown through a low excitatory concentration EC50 value of 0.17 mg/mL (Hill¼ 3.7) vs GABAA rat cortex membranes which showed an EC50 value of 4.7 mg/mL (Hill¼ 3.81).

4. Discussion 4.1. aqWS effects on ionotropic GABAA receptors W. somnifera root extract has been used in traditional Indian medicine as an adaptogen with memory enhancing and anti-stress properties. It has also been used to treat convulsive disorders. Yet the molecular mechanisms by which the herbal extracts' multiple

Please cite this article as: Candelario, M., et al., Direct evidence for GABAergic activity of Withania somnifera on mammalian ionotropic GABAA and GABAρ receptors. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.05.058i

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Fig. 8. No response by WA or WLA on rat cortex membrane or GABAρ1 cRNA injected oocytes. (A) Sample traces of membrane currents elicited by GABA (1 mM), used as control, withanolide compounds and their ethanolic vehicles. (B) On rat cortex membrane injected oocytes, WA (40 mM) and WLA (40 mM) produced small outward currents (6 7 3 nA and 57 2 nA) similar to that of their respective vehicles (8 7 4 nA and 67 1 nA) (n¼ 5). A paired t-test showed there was no significant difference between the currents elicited by WA and its vehicle (p ¼ 0.32) or WLA and its respective vehicle (p ¼ 0.57). (C) Bar plot showing responses to GABA (10 μM), WA (40 mM) and WLA (40 mM) on oocytes expressing GABAρ1 receptors (n¼ 5). There was no significant difference between the currents produced by vehicle (8 7 2 nA) and WA (6 7 2 nA) (p¼ 0.32) or vehicle 57 1 nA and WLA 6 7 2 nA (p¼0.60). Responses in (B) and (C) were normalized to maximum of their respective GABA control response.

Fig. 9. HPLC separation of major compounds present in WS extracts. (A) HPLC chromatogram of species extracted with H2O (top), MeOH (middle), and DCM (bottom). Single ion monitoring resulted in multiple peaks being generated for a given compound. Chromatographic peak at 20.40 min of H2O extracts (denoted by ▀) represents withaferin A/withanolide A/withanone and their derivatives (See further information in Supplemental Fig. 3). The chromatographic peaks at 26.60, 19.20, and 14.40 min of DCM extracts (denoted by by ●, ▼, and ◆) represent withanolide B, withaferin A/withanolide A/withanone and 27-hydroxywithanone/ withastramonolide, respectively.

components mitigate these effects are unknown. In our study we demonstrate that the aqueous extract of WS, prepared in the way which it has traditionally been utilized, has differential GABAmimetic activity on GABAA and GABAρ receptors. For our first set of experiments, we recorded the activity of aqWS on oocytes injected with rat cortex membrane preparations. This method relies on the property of lipidic membranes to form vesicles in aqueous

solutions, and the ease with which these vesicles fuse with the oocyte's plasma membrane. Once fused the microtransplanted receptors are exposed and available to pharmacological experimentation. It is important to keep in mind that the microtransplanted receptors are a representation of the mosaic of receptors found natively in the adult male rat cortex and consequently are representative of potential aqWS targets in vivo. Specifically, with this method most transplanted receptors are of the ionotropic type (Limon et al., 2008, 2012). Since GABAA receptors with the subunit stoichiometry α1β2/3γ2 arrangement have the highest relative abundance, comprising about 43% of the total of GABAA receptors in the rat brain (McKernan and Whiting, 2008; Pirker et al., 2000), these receptors are partially responsible for the GABA and aqWS responses on microtransplanted oocytes In agreement with previous findings on methanolic extracts of WS (Bhattarai et al., 2010), aqWS also generated inward currents in a concentration-dependent manner. The fact that bicuculline blocked most of aqWS current clearly indicates that aqWS has agonist activity on GABAA receptors. The small remaining response after bicuculline administration may be due to native receptor activation that was observed also in non-injected oocytes. By comparing maximal inward currents generated by aqWS and GABA it is possible to observe that aqWS root extract efficacy on microtransplanted GABAA receptors was only 20% of the efficacy of the endogenous neurotransmitter GABA; moreover, aqWS reduced the maximal response elicited by GABA suggesting that aqWS possess compound(s) with partial agonist activity of GABAA receptors that may have antagonistic properties in vivo. However, because the identity and concentration of active compounds it is not known we cannot discard the possibility that the concentration of the active compounds is not enough to produce a maximal effect equivalent to that produced by GABA. Future identification of the compounds responsible for these effects will help to determine the pharmacodynamic properties of these compounds and their receptors isoform specificity.

Please cite this article as: Candelario, M., et al., Direct evidence for GABAergic activity of Withania somnifera on mammalian ionotropic GABAA and GABAρ receptors. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.05.058i

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4.2. aqWS effects on GABAρ1 receptors The protein subunits that make up the GABAA and GABAρ1 receptor subfamilies show up to 36% optimized sequence identity overall and up to 73% sequence identity in the membrane spanning regions. This difference in identity is the source of some of the functional differences observed between these receptors (Johnston, 2002). Initially found mainly in the retina, it has been recently shown that GABAρ receptors can also be found in the hippocampus and olfactory bulb, albeit at small levels (MartínezDelgado et al., 2010). GABAρ receptors are also responsible for generating a tonic hyperpolarizing conductance that modulates the excitability and integration of synaptic activity: processes important for learning and memory (Johnston, 2002). In addition, stimulation of GABAρ receptors can protect cells from cell death through the activation of intercellular PKA-mediated anti-apoptotic signaling cascade (Yang et al., 2008). Our data suggests that WS may have additional neuroprotective effects through activation of GABAρ receptor channels, supporting the traditional use of WS as an adaptogen. Interestingly, aqWS showed higher affinity to GABAρ1 over GABAA receptors. GABAρ1 receptor subtypes are of interest because they are spatially and functionally distinct from other GABAA receptors. Our results indicate that aqWS has similar pharmacological efficacy on GABAρ1 than the endogenous agonist GABA. Furthermore, the Hill coefficient (Hill ¼3.7) of the expressed GABAρ1 receptors (homomeric assembly of five ρ1 subunits, each subunit with its own GABA binding pocket (Amin and Weiss, 1996)) indicates a higher binding cooperativity compared to that of GABAA receptors (Hill ¼ 1.6) which only has two binding sites for GABA at the interface of the α-β subunits (Olsen and Sieghart, 2009). Our findings seem to be in good standing with Hill coefficients between 3 and 4 of the GABA ρ1 receptor (Yang et al., 2006), and Hill coefficients of 1 and 2 for GABAA receptors (Seeburg et al., 1990). Interestingly, the differential activation of GABA receptors seems to partly describe the mechanism by which WS exerts its adaptogenic properties.

responsible for the effects on GABA receptors. Future studies should provide the identity of the compound(s) responsible of aqWS effects on GABA receptors. 4.4. Xenopus system as model to study medicinal plant activity The present study focuses on the activity of aqWS root on GABAA receptors but the Xenopus oocyte system provides a model in which to test its effects on other receptor channel types such as AMPA and cholinergic receptors (Bernareggi et al., 2007, 2011). Data presented is one in a series of experiments that will be carried out to characterize aqWS response on multiple receptor families. The Xenopus oocytes system is ideal for investigating the activity of plant-derived substances on neurotransmitter receptors because of the ability to provide a rapid, and high yielding model of information on functional, and pharmacological properties and its minimal amounts of protein required for testing (Limon et al., 2011). Biophysical studies are done directly on mammalian native receptors that are still embedded in their original lipids and with their own cohort of associated proteins. In addition, the pharmacological properties of traditional plant substances can be tested on receptors of interest through the heterologous expression of specific receptor subtypes of preferred stoichiometry. This approach will: 1) quickly advance the determination of a pharmacological profile on medicinal plants that are inherently complex and may be comprised of multiple active compounds and 2) aide in the identification of novel substances derived from plants used in traditional medicinal systems for the treatment of CNS disorders.

Acknowledgments Funding for this project was partially provided by the CSULA Los Angeles Basin Bridges to the PhD Program and the Grant CN13-613, to AL and RM, from the University of California Institute for Mexico and the United States (UC-MEXUS).

4.3. Potential active compounds in aqWS Differential effects of aqWS on GABA receptors suggest that some of its constituents may provide important scaffold molecules for the development of new GABAergic tools and, pharmacological treatments for neurological disorders associated with GABAergic pathways. WA and WLA are two constituents of WS root that have received much attention because of their effects on the CNS. They have been reported to have neuroprotective properties. After testing both compounds on GABAA and GABAρ1 receptors, our results indicate that WA and WLA are not responsible for the GABAergic effects of WS root reported in this and other studies. This opens up the question of which key constituents in WS are responsible for activating the GABAergic system. To date a total of 16 metabolites have unambiguously been detected in the aqueous fraction of the root extracts (Chatterjee et al., 2010). These include fatty acids, organic acids, amino acids, sugars, and sterol-based compounds. Although the neurotransmitter GABA was present in WS leaves (16.74 mg/g of dry weight (DW) of WS aqueous extracts) it was only detected in trace amounts in root aqueous extracts (Chatterjee et al., 2010). The lack of significant GABA presence in root aqueous extracts and the differential activity on GABAA and GABAρ1 indicate that the GABAergic activity of aqWS is not mediated by GABA. Our aqWS root HPLC and LC_MS extract analysis resulted in over 100 different compounds with potential activity on GABAA and GABAρ1 receptors. A comparison of HPLC chromatograms of aqueous and methanolic extracts, both known to produce GABAmimetic actions, suggests that at least one of the withanolide derivatives, found in the 12 to 30 min region, is

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Please cite this article as: Candelario, M., et al., Direct evidence for GABAergic activity of Withania somnifera on mammalian ionotropic GABAA and GABAρ receptors. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.05.058i

Direct evidence for GABAergic activity of Withania somnifera on mammalian ionotropic GABAA and GABAρ receptors.

Withania somnifera (WS) has been traditionally used in Ayurvedic medicine as a remedy for debility, stress, nervous exhaustion, insomnia, loss of memo...
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