Selective Interactions of Valeriana officinalis Extracts and Valerenic Acid with [3H]Glutamate Binding to Rat Synaptic Membranes

Although GABA neurotransmission has been suggested as a mechanism for Valeriana officinalis effects, CNS depression can also be evoked by inhibition of ionotropic (iGluR) and metabotropic glutamate receptors (mGluR). In this study, we examined if aqueous valerian extract interacted with glutamatergic receptors. Freshly prepared aqueous valerian extract was incubated with rat cortical synaptic membranes in presence of 20 nM [3H]Glutamate. Aqueous valerian extract increased [3H]Glutamate binding from 1 × 10−7 to 1 × 10−3 mg/mL. In the presence of (2S,1′S,2′S)-2-(Carboxycyclopropyl)glycine (LCCG-I) and (2S,2′R,3′R)-2-(2′,3′-Dicarboxycyclopropyl)glycine (DCG-IV), Group II mGluR agents, valerian extract markedly decreased [3H]Glutamate binding, while (2S)-2-amino-3-(3,5-dioxo-1,2,4-oxadiazolidin-2-yl) propanoic acid) (quisqualic acid, QA), Group I mGluR agonist, increased [3H]Glutamate binding. At 0.05 mg/mL aqueous valerian extract specifically interacted with kainic acid NMDA and AMPA receptors. Valerenic acid, a marker compound for Valeriana officinalis, increased the [3H]Glutamate binding after 1.6 × 10−2 mg/mL, and at 0.008 mg/mL it interacted only with QA (Group I mGluR). The selective interactions of valerian extract and valerenic acid with Group I and Group II mGluR may represent an alternative explanation for the anxiolytic properties of this plant.


Introduction
Valeriana officinalis L., s.l. (Valerianaceae family) is a medicinal plant used in complementary and alternative medicine for its sedative and anxiolytic properties [1,2]. Valerian's effects on the central nervous system have been well documented and attributed to many of it active compounds: valepotriates, baldrinals, valerenic acid, valerenal and valeranone, and other constituents in the essential oils [1,[3][4][5][6][7][8]. Albeit the anxiolytic properties of valerian have been demonstrated in animals [9,10], there are no sufficient studies in humans [6]. Consequently, the therapeutic properties of Valeriana officinalis have yet to be conclusively demonstrated [6,9,11].
"Anxiety and stress-related diseases are a group of disorders that have in common excessive or inappropriate brain excitability within crucial brain circuits" [12]. The actions of glutamate, the major excitatory neurotransmitter, are mediated by two types of receptors: (1) ionotropic receptors (iGluR): NMDA, AMPA, and kainate receptors and (2) metabotropic receptors (mGluR) which are comprised of three groups (I, II, and III). Decreasing excitatory neurotransmission in the CNS by modulating glutamate receptors is an alternative approach to produce anxiolysis and sedation.
The present study investigated the interaction of Valeriana officinalis aqueous extract with the glutamatergic receptors. We also examined the interaction of valerenic acid and isoborneol, two constituents present in the extracts. To accomplish this objective, receptor-binding assays were performed using rat cortical synaptic membranes in the presence of fresh valerian extracts and both types of glutamate receptor ligands.

Valerenic Acid.
A 10 mM stock solution was prepared using EtOH 95%. The dilutions used for the assays were freshly prepared with 50 mmTris-HCl/100 mM KCl buffer before each experiment.

Isoborneol.
A 65 mM stock solution was prepared using EtOH 95%. The dilutions used for the assays were freshly prepared with EtOH 70% before each experiment.

Cerebral Cortex Synaptic Membranes.
Cerebral cortex synaptic membranes were purchased from Analytical Biological Services, Inc. (Wilmington, DE). They report that these were prepared as follows: female rats of approximately two months of age were decapitated and the brain promptly removed. The cortex was dissected and homogenized (1 : 10 w/v) in ice-cold 10 mM TRIS-HCl buffer pH 7.4. The homogenate was centrifuged twice at 2,500 g for 10 min. The resulting supernatant was centrifuged at 12,500 g for 20 min. The pellet was washed twice with ice-cold 10 mM TRIS-HCl buffer pH 7.4 (1 : 10 w/v) and centrifuged at 12,500 g for 20 min. The pellet (synaptical membrane, P2) was resuspended in 10 mM TRIS-HCl buffer pH 7.4 and freeze thawed at least three times before been stored at −80 • C until used. Protein concentration was determined using the   groups were tested for significance using one way analysis of variance followed by Tukey-Kramer multiple comparisons test, with P < .05. Statistics for the experimental group versus total binding were not shown for clarity.   Valerian (10 mg/mL) did not interact with AMPA or KA. The same assay was performed with 0.008 mg/mL of valerenic acid, and it showed (Figure 2(b)) that valerenic acid decreased the [ 3 H]Glutamate binding more than 40%. However, valerenic acid did not change the effects of iGluR ligands on [ 3 H]Glutamate binding. In contrast, Figure 2(c) shows that isoborneol (0.0008 mg/mL) only interacted with NMDA. At higher concentrations (1 mg/mL) isoborneol interacted with AMPA and KA.

[ 3 H]Glutamate Displacement Curves for Valerian and
As shown in Figure 3( (Figure 3(a)-Insert) confirmed a marked decrease in the [ 3 H]Glutamate binding when valerian extracts (0.001 mg/mL) were in presence of DCG-IV (28%) and EGLU (38%), a highly selective Group II metabotropic glutamate receptor agonist and antagonist, respectively.
Most of the Valerian effects described above are consistent with the enhancement of GABAa-mediated transmission [7,[22][23][24]. Cavadas and colleagues showed that valerian extracts bind to GABA a receptors [4], while other researchers found that valerian is a partial agonist of the 5-HT(5a) receptor [8] and promotes cell proliferation in the hippocampus of "depressive" rats [25]. Moreover, recent studies suggest that different components within the valerian extract mediate the activation of adenosine receptors [26,27]. Indeed, many studies have been done to determine the interaction of valerian with different receptors, but none of them examined valerian-glutamate receptor interaction.
Our [ 3 H]Glutamate displacement curve in presence of Valeriana officinalis demonstrated that valerian extracts increase glutamate binding from 8 × 10 −7 to 1 × 10 −1 mg/mL reaching a maximum 3 [H]Glutamate binding at 1 × 10 −3 mg/mL (160%). Therefore, at these physiological attainable concentrations (8×10 −7 -1×10 −1 mg/mL) valerian potentiates 3 [H]Glutamate binding. In contrast, different findings were obtained for valerenic acid and isoborneol. The [ 3 H]Glutamate displacement curves and the receptor selectivity obtained for valerian and its constituents, for instance, are very different from each other, suggesting that these constituents are not the compounds responsible for the increase in 3 [H]Glutamate binding observed with valerian.
The glutamatergic system plays an important role in anxiety pathogenesis [28]. It is suggested that the physiological and behavioral responses associated with anxiety are regulated by a balance between the inhibition produced by GABA and the excitation caused by glutamate. Treatments used to decrease excitability in neurons from the amygdala are achieved by increasing GABA neurotransmission. Alternatively, treatments to decrease the excitability can be obtained by decreasing the excitatory glutamatergic transmission. Therefore, decreasing excitatory neurotransmission in the CNS, by modulating the response produced by the glutamatergic receptors, is an alternative approach to produce anxiolysis and sedation. The glutamate receptors found in the amygdala produce excitatory and inhibitory actions, and the degree of ionotropic and metabotropic activation is an important factor to determine the amygdala cell excitability. Thus, the modulation of glutamate actions mediated by iGluR and mGluR represent a feasible alternative to treat anxiety states [12,29].
Compounds that decrease glutamatergic transmission via blockade of NMDA have been reported to produce anxiolytic and antidepressant like actions in animal tests and models [30,31]. Our group demonstrated that valerian extract had modest inhibitory effects on [ 3 H]MK-801 binding, an indicator of NMDA-valerian interaction [32]. In 2004, Malva and colleagues reported that by decreasing neuronal network excitability through AMPA permeable Ca 2+ receptors, valerian preparations could contribute to neuroprotection and may be of therapeutic use in preventing glutamate-mediated degeneration related to aging or neurodegenerative disorders [33]. Now, our study confirms that in the presence of iGluR agonists, valerian extracts interact with KA and NMDA (0.05 mg/mL and 10 mg/mL, resp.). While isoborneol exhibited significant interactions with all iGluR, valerenic acid did not interact with iGluR agonists.
Many studies suggest that metabotropic glutamate receptors are involved in anxiety [12,[34][35][36]. For this reason, we performed receptor-binding assays with valerian extracts in presence of different types of metabotropic glutamate receptors. In this study, we demonstrated that valerian extracts, in presence of mGluR ligands, exhibited significant interaction with QA (Group I mGluR) and LCCG-I (Group II mGluR). Valerenic acid selectively interacts with QA. However, isoborneol interacts with all mGluR receptors. Our results clearly demonstrated that Valeriana offiicinalis and it constituents (valerenic acid and isoborneol) interact with Group I and II mGluR, which supports previous studies showing the role of Group I and II mGluR in anxiety [35,37].
The pharmacological effects of Valeriana officinalis extracts in [ 3 H]Glutamate binding are not conventional and easy to understand. They could reflect complex interactions (agonist/antagonist) or synergism occurring in the complex mixture of phytochemical constituents. The biphasic [ 3 H]Glutamate displacement curve obtained in presence of valerian represents an example of the interactions that occur in an extract which could not be evident when single constituents are studied in isolation. This study exclusively used receptor-binding assays to determine ligand-receptor interactions, but functionalities studies are being conducted to characterize them.
Our results confirm the hypothesis of valerian interaction with the glutamatergic receptors, suggesting a possible mechanism by which valerian extracts produce their effects. Val-mGluR interactions may represent a novel alternative for the treatment of anxiety. Further studies with fractions of valerian extract should be done to evaluate the interactions between a group of compounds and their effects on receptorbinding selectivity.