Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2011, Article ID 891819, 7 pagesdoi:10.1155/2011/891819Research ArticleAqueous and Ethanolic Valeriana officinalis Extracts Changethe Binding of Ligands to Glutamate ReceptorsLisa M. Del Valle-Mojica, Jos e M. Cordero-Hern andez,Giselle Gonz alez-Medina, Igmeris Ramos-V elez, Nairimer Berr os-Cartagena,Bianca A. Torres-Hern andez, and Jos e G. Ort zDepartment of Pharmacology and Toxicology, School of Medicine, University of Puerto Rico, Medical Sciences Campus,P.O. Box 365067, San Juan, 00936-5067 Puerto Rico, USACorrespondence should be addressed to Lisa M. Del Valle-Mojica, lisadelvalle@yahoo.comReceived 12 June 2009; Revised 6 July 2010; Accepted 27 October 2010Copyright 2011 Lisa M. Del Valle-Mojica et al. This is an open access article distributed under the Creative CommonsAttribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work isproperly cited.Theeects of twovalerianextracts(aqueousandhydroalcoholic)wereinvestigatedthrough[3H]Glutamate([3H]Glu)and[3H]Fluorowillardine ([3H]FW) receptor binding assays using rat synaptic membranes in presence of dierent receptor ligands.In addition, the extract stability was monitored spectrophotometrically. Both extracts demonstrated interaction with ionotropicglutamate receptors (iGluRs). However, the extracts displayed considerable dierences in receptor selectivity. The hydroalcoholicextractselectivelyinteractedwithquisqualicacid(QA), groupImetabotropicglutamatereceptor(mGluR)ligand, whiletheaqueous extract did not alter the binding of QA. The stability of the extracts was examined during several weeks. Freshly preparedextractinhibited3860%of[3H]FWbinding(AMPA). After10days, theaqueousextractinhibited85%of[3H]FWbindingwhile the hydroalcoholic extract markedly potentiated (200%) [3H]FW binding to AMPA receptors. Thus, our results showed thatfactors such as extraction solvent and extract stability determine the selectivity for glutamate receptor (GluR) interactions.1. IntroductionValeriana ocinalis, commonly known as Valerian, is one ofapproximately250ValerianspeciesfromtheValerianaceaefamily, and it is used for the preparation of phytomedicineswithsedative, anxiolytic, andspasmolyticproperties. Thetherapeuticbenets of Valerianhavealways beenincon-troversy due to the inconsistent clinical results [13].The discrepancies among clinical trials may be due tomethodological limitationsanddierencesintheValerianpreparations (method of extraction and extraction solvent).Manystudies usewater as theextractionsolvent becausetraditionally natural product preparations (i.e., tea) areprepared in water. Aqueous extracts of Valerian have dierentspectrum of active constituents than extracts that have beenprocessed with ethanol [4].The specic mechanism(s) of action responsible for thepharmacological eectsof Valerianroot extractshavenotbeenfullyelucidatedalthoughit is thought that Valerianroot extracts stimulate the GABA transmission [2, 3, 510].On the other hand, few studies have investigated the eectsof Valeriana ocinalis in the excitatory glutamate-mediatedneurotransmission [11].Basedonthesestudies, thepossibleeectsof Valerianextracts on glutamate-mediated (excitatory) neurotransmis-sionwereassessedusingreceptor bindingassays withratsynaptic membranes in presence of aqueous and hydroalco-holic Valerian extracts and glutamate receptors ligands (MK-801 for NMDA, AMPA or [3H]Fluorowillardiine for AMPA,kainicacidforKA, andquisqualicacid(QA)forGroupImetabotropic glutamate receptors (mGluRs)).2. Materials and Methods2.1. Chemicals. L-[2,3,4-3H]-Glutamic acid (60 Ci/mmol)and [3H]Fluorowiillardiine (80 Ci/mmol) were obtainedfromAmerican Radiolabeled Chemicals, Inc (St. Louis,MO). AMPA (-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid) was obtainedfromTocris Cookson, Inc.(Ellisville, MO). PotassiumChloridewas purchasedfrom2 Evidence-Based Complementary and Alternative MedicineQA KA AMPA MK801 Total NSH2O+versus GluR ligand020406080100120Bound(AVGSEM)(%)+++++++++(a)QA KA AMPA MK801 Total NSEtOH+versus GluR ligand020406080100120Bound(AVGSEM)(%)+++++++++ Valerian+ Valerian(b)Figure 1: Eects of dierent fresh Valerian extracts (1 mg/mL) on [3H]Glutamate binding to rat synaptic membranes in the presence of1 mM glutamate receptor (GluR) ligands. (a) The aqueous extract decreases the eects of the NMDA (MK-801), AMPA, and Kainate (KA),but it did not alter the eects of quisqualic acid (QA), a group I metabotropic glutamate receptor (mGluR) ligand. (b) The hydroalcoholicextract also decreases the eects of NMDA, AMPA, and KA. In contrast, Valerian reverses the inhibitory eects of QA. +agonist versus agonist+ Valerian, P < .05;++P < .01;+++P < .001.MathesonColeman&Bell (Norwood, OH). UniverSol ESwas obtained from MP Biomedicals (Solon, OH). All otherreagents were obtained from Sigma Chemical Company (St.Louis, MO).2.2. Valerian Extracts. Organically grown and certied Vale-riandrypowderedroots(Lot. 1111H-OUP), harvestedin2004, wereobtainedfromPacicBotanicals (LLCGrantsPass, Oregon). Commercial Valerian. Natures ResourcesExtract (Lot. LD11282N) and Natures Resource Herb (Lot.MG11258) were purchased from a local pharmacy. Valerianwasextractedinultrapurewaterorethyl alcohol (EtOH)70%(1 : 10 w/v)at 23C, stirredfor1 hour, andlteredthrough a 12.5 cm Whatman Qualitative no. 1 lter. Aliquotswere centrifuged at 6,700 g to remove particulates and storedat 4C.2.3. Cerebral Cortex Synaptic Membranes. Analytical Biolog-icalServices, Inc. (Wilmington, DE)preparedthesynapticmembranes as follows: female rats of approximately twomonths of age were decapitatedandthe brainpromptlyremoved. The cortex was dissected and homogenized(1 : 10 w/v)inice-cold10 mMTris-HClbuerpH7.4.Thehomogenate was centrifuged twice at 2,500 g for 10 min. Theresulting supernatant was centrifuged at 12,500 g for 20 min.The pellet was washed twice with ice-cold 10mMTris-HCl buerpH7.4(1 : 10 w/v)andcentrifugedat12,500 gfor 20 min. The pellet (synaptical membrane, P2) wasresuspendedin10 mMTris-HCl buerpH7.4andfreezethawedat least threetimes beforebeingstoredat 80Cuntil used. Protein concentration was determined using theBradfordassay[12]usingbovineserumalbumin(BSA)asreference standard.2.4. Receptor Binding Assays2.4.1. [3H]Glutamate Binding. Receptor bindingcompeti-tion assays were done using synaptic membranes of cerebralcortex from Analytical Biological Services, Inc. (Wilmington,DE). Thereactionwas initiatedbytheadditionof tissue(100 g protein) to tubes containing 1 mM of (MK-801 forNMDA, AMPA, kainic acid for KA, and quisqualic acid (QA)for Group I metabotropic glutamate receptors (mGluR) and20 nM[3H]GlutamicAcid([3H]Glu)inanal volumeof500 L of 50 mM Tris HCl/100 mM KCl buer, pH 7.4). Thenonspecic binding was determined in the presence of 1 mMnonradioactive glutamate. Total binding was determined inthe presence of Valerian extracts in dierent solvents (H2Oand EtOH) in order to do competition studies. All sampleswereincubatedonice(04C) for 40 minutes. Theassaywas stoppedby centrifugationfor 30 minat 6,764 g; thesupernatant was extracted and the pellet washed two timeswith 1 mL of ice-cold buer. The pellet was resuspended in500 L of buer. Radioactivity of the samples was quantiedinaBeckmanLS6500MultipurposeScintillationCounterwith 1 mL of UniverSol ES scintillation cocktail. Results areshown as percentage of total binding (SEM).2.4.2. [3H]Fluorowillardine Binding. Assays were done usingsynaptic membranes of cerebral cortex fromAnalyticalBiological Services, Inc. (Wilmington, DE). The reaction wasinitiated by the addition of tissue (100 g protein) to tubescontaining 15 nM [3H] Fluorowillardine ([3H]FW) in a nalvolumeof 500 Lof 50 mMTrisHCl/100 mMKCl buer,pH 7.4. Nonspecic binding was determined in the presenceof10 mMglutamate. Total bindingwasdeterminedinthepresence of Valerian extracts in dierent solvents (H2O andEvidence-Based Complementary and Alternative Medicine 3[3H]FW30 20 10 0(days)0100200300Bound(AVGSEM)(%)EtOHH2OFigure2: Changes in3[H]Fluorowillardine binding withtime.Initially, a 60% of inhibition is observed with the aqueous extractswhile the hydroalcoholic extract inhibits only a 38%. However, withtime, the inhibitory eects of the aqueous extract increase to 85%whereasthehydroalcoholicextract markedlypotentiates(200%)3[H]Fluorowillardine ([3H]FW) binding to AMPA receptors.EtOH) in order to do competition studies. All samples wereincubatedonice (04C) for 40 minutes. The assay wasstoppedbycentrifugationfor30 minat6,764 g. Then, thesupernatantwasremovedandthepelletwashedtwotimeswith 1 mL of ice-cold buer. The pellet was resuspended in500 L of buer. Radioactivity of the samples was quantiedina BeckmanLS 1800 counter with1 mLof UniverSolscintillation cocktail. Results are shown as percentage of totalbinding (SEM).2.5. Wavelength Scans. Spectrophotometric analyses weredonewithaBeckmanDUSeries 500Spectrophotometer(Fullerton, CA). Avolumeof 1 mLof 10 mg/mLValerianextract was placedinElkayUltra-VuDisposableCuvettesfrom Elkay Products, Inc. (Shrewsbery, MA). The scans weredone from 190 nm to 320 nm in steps of 1.0 nm.2.6. Statistical Analysis. Dataareexpressedasmeanvaluesthe standard error of the mean (SEM) of at leastthree experiments. The dierences between the experimentalgroups were tested for signicance using one way analysis ofvariancefollowedbyTukey-KramerMultipleComparisonsTest, withP < .05. Statistics for theexperimental groupversus total binding are not shown for clarity.3. Results3.1. Extraction Solvents Change Ligand Binding to GlutamateReceptors. Figure 1shows the eects of dierent Valerianextracts (1 mg/mL) on [3H]Glutamate binding to rat synap-tic membranes in the presence of 1 mMGluR ligands(NMDA, AMPA, KA, or QA). The aqueous extract (a)decreasestheeectsoftheNMDA(MK-801), AMPA, andkainate (KA), but it did not alter the eects of quisqualic acid(QA), a Group I metabotropic glutamate receptor (mGluR)ligand. The hydroalcoholic extract (b) has similar eects asthe aqueous extracts onNMDA, AMPA, andKAtreatedmembranes. However, the hydroalcoholic extract potentiatesthe eects of QA, suggesting a strong eect ongroupImGluR.Similarly, the eects of aqueous and hydroalcoholicextractsonAMPAwereevaluatedbybindingassaysusing[3H]FW. Initially, theaqueousandhydroalcoholicextracts(Figure 2) have 60% and 38% inhibitory eects on [3H]FWbinding, respectively. However, withtime (4weeks), theaqueous extract inhibits [3H]FW to 85% while the hydroal-coholic extract markedly increases [3H]FW binding (200%)to AMPA receptors.3.2. Extract Stability Alters Ligand Binding to GlutamateReceptor. Theeect of twoValeriancommercial products(preparedas aqueous extracts) was evaluatedonAMPAbinding ([3H]FW). Figure 3 shows the eect of timein[3H]FWbinding. (a) At time zero, ValerianNaturesResource Extract and Natures Resource Herb inhibit[3H]Fluorowillardinebinding. (b) After aperiodof time(42 days (Extract) or 39 days (Herb)), the extracts loose theinhibitoryeects.Furtherintime(48days(Extract)or45days (Herb)), they potentiate [3H]Fluorowillardine binding.3.3. Spectrophotometric Analysis of Aqueous andHydroal-coholic Extracts. Thespectraof dierent Valerianextracts(10 mg/mL) preparedinH2OandEtOHwere evaluatedspectrophotometricallyasshowninFigure 4.TheinsertinFigure 4showsalistofwavelengthandonlytwo, 292and298 nm, demonstrate signicant dierences.4. DiscussionValerianis a well-recognizedplant usedinthe folkloricmedicine for its sedative and anxiolytic properties [2].Numerous studies evaluate the use of Valerian root extractstoinduceandimprovesleepquality[1315]whileothersevaluate Valerian anxiolytic eects [1618]. In humans,Valerian extracts reduced latency to fall asleep as eectivelyas small doses of benzodiazepines [19]. Although Valeriansanxiolytic properties are well documented in mice and rats[20], thereisnoscienticevidencethatcouldsupporttheanxiolytic properties of the plant in humans, due to intrinsicmethodological problems such as variation in Valeriandosesandpreparations, numberofpatients, andlengthoftreatment [1].4 Evidence-Based Complementary and Alternative Medicine1 0 1 2Log [valerian] (mg/mL)020406080100120140160180Bound(AVGSTD)(%)0 days42 days48 days(a)Valerian Natures Resource Extract1 0 1 2Log [valerian] (mg/mL)020406080100120140160180Bound(AVGSTD)(%)0 days39 days45 days(b)Valerian Natures Resource HerbFigure3: Eectof timein3[H]FluorowillardinebindingfromtwoValeriancommercial products. (a)Attimezero, aqueousextractsof NaturesResourceExtractandNaturesResourceHerbinhibit[3[H]]Fluorowillardinebinding. (b)Afteraperiodof time(42days(Extract) or 39 days (Herb)), the extracts loose the inhibitory eects. Further in time (48 days (Extract) or 45 days (Herb)), they potentiate[3[H]]Fluorowillardine binding.325 300 275 250 225 200Wavelength ()00.250.50.7511.251.51.7522.25ODValerian EtOHValerian H2OEtOHH2Oversus H2O327 298 292 280 254 207 20500.511.522.5ODFigure 4: Spectra and absorbance from aqueous and hydroalcoholic Valerian extracts (10 mg/mL). Signicant dierences at 292 and 298 nmwere observed. H2O versus EtOH, P < .05;++P < .01;+++P < .001.It is not fully understood which constituents of Valerianaocinalisareresponsibleforthepharmacological actions,butmanymanufacturersstandardizeValerianpreparationsaccordingtothe valerenic acidcontent as recommendedby the German Commission E. Studies suggested thatvalerenicacidistheprincipal activecomponent responsi-blefor Valerians anxiolyticproperties [21, 22]. However,other Valerianconstituents suchas borneol, lignans, andavonoids (hesperidin, linarin, and 6-methylapigenin) havealso been found to have anxiolytic and sedative activity [2327]. Moreover, other Valerian species with low valerenic acidconcentration (e.g., Valeriana edulis and Valeriana sitchensis)have similar pharmacological proles [19, 2830]. Thus,there is no evidence showing that valerenic acid is exclusivelyEvidence-Based Complementary and Alternative Medicine 5Critical factors that aect Valerians pharmacological propertiesExtractionsolventExtractionstabilityAqueousextractEthanolicextract[3H]glutamate binding: NMDA, AMPA, and KA= QA (mGluR I)[3H]glutamate binding: NMDA, AMPA, and KA QA (mGluR I)[3H]FW binding:(inhibits) after 30 days[3H]FW binding: (potentiates) after 30 days[3H]FW binding:0 days: (inhibition)3942 days: loose inhibition4548 days: potentiationUV spectra:signicant dierences at292 and 298 nmFigure 5: Summary of the aqueous and ethanolic Valerian extract eects in receptor binding assays and spectrophotometric absorbance.responsible for the sedative and anxiolytic eects attributedto Valeriana ocinalis.Circostaandcolleagues [31] conductedthebiologicaland chemical characterization of two Valerian extracts (aque-ousandhydroalcoholic)andfoundremarkabledierencesinthecontentof valepotriates. Similarresultswerefoundby Occhiutoet al. in2008[32]. Bothstudies, fromthesame group of researchers, revealed that the phytochemicalanalysis of the hydroalcoholic extract and the aqueous extractwas similarinthevalerenicacidcomposition(aqueousextractsbeinghalf of thehydroalcoholicextract content),buttheconcentrationofthevalepotriateswasmuchlowerin the aqueous extract than in the hydroalcoholic extract. Inthe biological assays, Circosta and colleagues [31] observeda lower percent of animals with arrhythmias when thehydroalcoholic andnot the aqueous extracts were givenat adoseof 50 mg/kg(80%versus60%, resp.). Similarly,Occhiuto et al. [32] reported that the concentration causing50%inhibitionoftheamplitudeofcontractions(uterore-laxant eect) was 29.5 3.40 and 68.7 5.20 g/mL for thehydroalcoholic and aqueous extracts, respectively. Therefore,thebiological analysis bythesestudies demonstratedthatthe Valerian extracts possess coronary dilatory, hypotensive,andbronchodilatoryproperties as well as relaxingeectsonhumanmyometrial muscleat dierent concentrations;the hydroalcoholic extracts being always more potent. Thesedierences inpotencyof thehydroalcoholicandaqueousextracts may be related to dierences in the extraction solventandprocedure, rawmaterial storage conditions, growingtechniques, harvesting conditions, and age of the plant,among other factors.Previously, modest eects of commercial aqueous Vale-rian extracts on NMDA receptor [3H]MK-801 bindinghave beenreported[33]. Similarly, Malvaandcolleagues[11] reportedAMPA-mediatedneuroprotective eects ofValerian. Our experiments show that aqueous and hydroal-coholic extracts fromValeriana ocinalis have dierentbiological activity as demonstratedby the GluRbindingassays. Aqueous and hydroalcoholic Valerian extracts interactwith NMDA, AMPA, and KA receptors. Moreover, Valerianextracts eects on dierent receptors change with timeas seenwithAMPAbinding assays. Onthe other hand,the aqueous extracts didnot have eect inQAwhile astronginteractionwithgroupI mGluRis observedwiththehydroalcoholicextracts(seeFigure 5). Previousresultsfromour lab(not shown) haddemonstratedthat whenvalerenicacidwasused, asapurecompound, inthesametypeofexperiments, asimilarresponse(potentiation)wasobtained. These results suggestedthat the hydroalcoholicValerian extract reverses the inhibitory eects of QAprobablyduetoahighercontent of valerenicacid. Furtherstudiesshouldbedonetoverifythevalerenicacidcontentinthedierent extracts.Previous studies by dierent researchers suggest thatmGluRmay have animportant role inanxiety [3436].Inparticular, thereisevidenceinvolvinggroupIImGluR(mGlu2/3) and group I mGluR (mGlu5) receptor agonists aspotential anxiolytic agents [37, 38]. This mGluR role couldsuggest apossiblemechanismbywhichValerianextractsproduce their eects. A more detailed study of these Valerianinteractions with mGluR is being examined by our lab.Navarreteetal. (2006)[29]usedHPLCanddetectionat dierent wavelengths toidentify Valeriancomponents(i.e., chlorogenicacid, lignans, avonoids, valerenicacids,and valepotriates) in dierent species. Similarly, we screenedfor possible spectrographic dierences between the aqueousandhydroalcoholic extracts. Aqualitative analysis of theabsorbance spectra (at dierent wavelengths) of the extracts6 Evidence-Based Complementary and Alternative Medicinerevealedsignicantdierencesat292and298 nm.Furtherstudiesshouldbedoneinordertodeterminetherelationbetween spectra changes and binding selectivity.Asurvey made by ConsumerLab.comreported that4 of 17 Valerian products had no detectable Valeriancompounds, four had half of the expected levels, twohadleadcontamination, andonehadcadmiumcontam-ination(http://www.consumerlab.com)[39]. Thisparticu-lar situationoccurredwithcommercial natural productsmanufactured by various processes using dierent solvents.Inaddition, the lowstability of the compounds, mainlyvalepotriates, could result in the absence of these compoundsinthe dierent pharmaceutical dosage forms since theymay decompose during the manufacturing process or uponstorage [40].Products containing Valerian have managed to keep theirplaceinthenatural productsmarket, inspiteof thelackof science basedevidence for its ecacy andthe strongcompetitionwithsyntheticdrugssuchasbenzodiazepines.Inconsistentandvariablepharmacologicaleectsofplant-derivedproductsareoneof thechallengesof thenaturalproductsresearch.Consequently,adrugderivedfromnat-ural productscouldbeconsideredasarational drugonlyif it is standardizedandproventomeet thestandards ofquality (prove identity, purity, etc.). Biological screening ofplant extractsinsteadof analysisof individual compoundmay be a useful tool in clarifying Valerian pharmacologicaleects because of possible synergismas a mechanismofaction(s)of variousactivecompounds. Thepresent studydemonstrates that Valerian extracts interact with the GluR,and factors such as the extraction solvent and stability of theextractsarecritical todeterminechangesinselectivityforGluR interaction.AbbreviationsAMPA: Alpha-amino-3-hydroxy-5-methylisox-azole-4-propionic acidBSA: Bovine serum albuminEtOH: Ethyl alcohol[3H]FW: [3H]Fluorowillardine[3H]Glu: [3H]GlutamateGluR: Glutamate receptorsGroup ImGluR: Metabotropic glutamate receptor (1/5)iGluR: Ionotropic glutamte receptorKA: Kainic acidmGluR: Metabotropic glutamate receptorsNMDA: N-methyl-D-aspartic acidQA: Quisqualic acid, (2S)-2-amino-3-(3,5-dioxo-1,2,4-oxadiazolidin-2-yl)propanoic acid).AcknowledgmentsThis work was supported in part by the Research Centers forMinority Institution (RCMI/NIH Grant no. G12 RR03051),Institutional Minority Biomedical Research Support (MBRS-RISE)Programat theUniversityof PuertoRico, MedicalSciences Campus (Grant no. 2 R25 GM061838-05), andPuerto Rico Louis Stokes Alliance for Minority Participation(PR-LSAMP) Programat the University of Puerto Rico(Grant no. HRD-0601843).References[1] L. S. Miyasaka, A. N. Atallah, and B. G. O. Soares, Valerian foranxietydisorders,CochraneDatabaseofSystematicReviews,no. 4, Article ID CD004515, 2006.[2] P. J. Houghton, The scientic basis for the reputed activity ofvalerian, Journal of Pharmacy and Pharmacology, vol. 51, no.5, pp. 505512, 1999.[3] A. Diaper and I. Hindmarch, Adouble-blind, placebo-controlled investigation of the eects of two doses of a valerianpreparation on the sleep, cognitive and psychomotor functionof sleep-disturbedolderadults,PhytotherapyResearch, vol.18, no. 10, pp. 831836, 2004.[4] V. Schulz, R. H ansel, M. Blumenthal, and V. E. 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