Abstract!

Valeriana officinalis L. is a popular herbal treat-ment for mild sleep disorders. Clinical and non-clinical studies found contradictory results for va-lerian extracts and single constituents regardingthe influence on sleep parameters. It was the aimof this study to investigate the sedative effects of avalerian root extract. Therefore, locomotor activ-ity and core body temperature were recorded inmale mice using radiotelemetry. A 70% ethanolicextract prepared from the roots of V. officinalis(s. l.) and some of its single constituents, valerenicacid, linarin, and apigenin, were tested for effectson locomotion and body temperature over 180minutes after oral administration. The extractwas tested in a dose range of 250–1000mg/kg,and only a dose of 1000mg/kg valerian extract

showed a mild short-term sedative effect with re-duced locomotor activity between 66–78minminutes after administration. Paradoxically, anincreased activity was observed after 150 min-utes after gavage. A dose of 1mg/kg valerenic acidproduced an intermittent stimulation of activity.However, a mild short-term sedative effect wasfound for linarin at 12mg/kg and apigenin at1.5mg/kg. Considering the cumulative locomotoractivity over the observation period of 180min, itis concluded that neither the extract nor one ofthe compounds had considerable sedative effects.More precisely, the observed short-term changesin activity pattern indicate that valerian extractas well as the flavonoids linarin and apigenin arerather effective to reduce sleep latency than to actas a sleep-maintaining agent.

Telemetry as a Tool to Measure Sedative Effectsof a Valerian Root Extract and Its Single Constituentsin Mice

Authors Nicholas K. Chow1, Michael Fretz2, Matthias Hamburger2, Veronika Butterweck1

Affiliations 1 Department of Pharmaceutics, College of Pharmacy, University of Florida, Gainesville, FL, USA2 Institute of Pharmaceutical Biology, Department of Pharmaceutical Sciences, University of Basel, Basel, Switzerland

Key wordsl" Valeriana officinalis L.l" Valerianaceael" linarinl" valerenic acidl" apigeninl" telemetry

received Sept. 8, 2010revised Nov. 3, 2010accepted Nov. 5, 2010

BibliographyDOI http://dx.doi.org/10.1055/s-0030-1250589Published online December 10,2010Planta Med 2011; 77: 795–803© Georg Thieme Verlag KGStuttgart · New York ·ISSN 0032‑0943

CorrespondenceDr. Veronika ButterweckDepartment of PharmaceuticsCollege of PharmacyUniversity of FloridaPO Box 100494Gainesville FL 32610USAPhone: + 13522737859Fax: + 13522737854butterwk@cop.ufl.edu

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Introduction!

An estimated 10 to 15% of the adult populationsuffers from chronic insomnia (≥ 1 month) andan additional 25 to 35% from transient or occa-sional insomnia (< 1 month) [1]. These high prev-alence rates connected with pharmacologicaltreatments cause significant economic and clini-cal costs. Traditionally, preparations obtainedfrom the roots of Valeriana officinalis L. (Valeria-naceae) are used as an herbal remedy because oftheir putative sleep promoting effects. In mostcountries, valerian preparations are a marketedover-the-counter product with remarkable suc-cess. One reason for this is that herbal productsand dietary supplements are often considered asmore natural and safer than prescription or othernonprescription drugs and therefore are verypopular [2].Not only are extracts from Valeriana officinalisL. the best known, but also the most studied herb-al treatment for insomnia. Clinical studies of its

Cho

sleep-inducing and sleep-promoting effects arecontradictory [2–7]. Although sedative and anx-iolytic effects have been observed in nonclinicalstudies [8,9], literature provides insufficient evi-dence to imply beneficial effects on sleep.Several compounds have been isolated and iden-tified from valerian root preparations, but it isstill unclear which of them are responsible forthe recorded activities. Some of the active com-pounds found in commonly used extracts are thesesquiterpenic acids, especially valerenic acid,which was recently identified as a GABAA recep-tor modulator [8,10]. Recently, sedative or sleep-enhancing properties were detected for the fla-vonoids: linarin (acacetin-7-O-rutinoside) [11],6-methylapigenin (4′,5,7-trihydroxy-6-methyl-flavone) [12,13], and hesperidin (2S(−)-7-rham-noglucosyl-hesperetin) [12]. More recently, lig-nan derivatives, in particular 4′-O-beta-D-gluco-syl-9-O-(6′′-deoxysaccharosyl)olivil found in V.officinalis, were shown to bind the adenosine A1

receptor, which is linked to sleep induction [14].

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In animal studies, sleep parameters are optimally evaluatedunder familiar and undisturbed conditions where usual routineis not interrupted. Therefore, the measurement of sleep-inducingeffects in nonclinical studies is complicated by handling-relateddisturbances or unfamiliar measuring instruments. For this rea-son, changes in locomotor activity are used as a surrogate param-eter to quantify the effect of valerian root, and experiments areconducted with methods such as open field [15], rotarod test[16], and elevated plus maze [8,9]. But these approaches requirespecial apparatuses, equipped with a particular measuring sys-tem (e.g., photocells and detectors, infrared beams and sensors,electromagnetic field, or video cameras). Moreover, long-termstudies in animals are not possible using these methods, andtherefore results are inadequate for the investigation of sleep-in-ducing effects (plus handling effects which cause stress). Sleep-wake cycle and body temperature are strongly influenced byeach other [17]. While core temperature in humans is rapidly ris-ing in the morning and falling in the evening, activity and bodytemperature in mice are subject to an inversed circadian rhythm.It is known that individuals normally fall asleep when core bodytemperature is decreasing [18]. Thus, it is indicated to consultboth, activity and core temperature, to evaluate possible effectson sleep.The present study represents an innovative, objective approachto measure sedative effects with minimized handling-relatedstress and remote data collection. The animals can be left undis-turbed during the recording period and long-term data acquisi-tion is possible. Most importantly, effects on locomotion can di-rectly be quantified. For the first time, radiotelemetry was usedto investigate a 70% ethanolic extract of Valeriana officinalis L. inmice. Additionally, selected constituents with reported positivepharmacological activity, namely valerenic acid, linarin, and api-genin [8,10,11,19], were tested. Locomotor activity and bodytemperature were recorded and analyzed for possible acute andmedium-term sedative effects.

Material and Methods!

Extract and single compoundsValerian extract was manufactured by Finzelberg GmbH & Co.KG, with 70% ethanol as the solvent and a drug:extract ratio(DER) of 3–6:1 (91% native extract, batch #: 08016674). Its con-tent of sesquiterpenic acids, calculated as valerenic acid, was0.32% (w/w). Valerenic acid (purity ≥ 98.5%) was purchased fromPhytolab GmbH & Co. Linarin (purity ≥ 98.5%) and apigenin (pu-rity ≥ 99%) were purchased from Indofine Chemical Company,Inc.

Chemicals and drugsMidazolam hydrochloride 50mg/10mL solution was purchasedfrom Bedford Laboratories™. Zolpidem tablets (5mg) were pur-chased from Henry Schein, Inc. and comminuted by mortar andpestle for preparation as a suspension for oral administration.Caffeine (purity ≥ 99%) was purchased from Sigma-Aldrich, Inc.

Drug preparationAll preparations for administrationwere made on the morning ofthe experimental run. Compounds for oral administration weredissolved, suspended or diluted in a vehicle of deionized watercontaining 0.5% propylene glycol (Fisher Scientific, Inc.). Com-pounds were administered by feeding needle in a volume of

Chow NK et al. Telemetry as a… Planta Med 2011; 77: 795–803

10mL/kg in the following concentrations: midazolam 5mg/kg,zolpidem 5mg/kg, caffeine 5mg/kg, valerian extract 250–1000mg/kg, valerenic acid 0.5–5.0mg/kg, linarin 2.0–12mg/kg,and apigenin 1.5–6.0mg/kg. The concentration of each com-poundwas chosen based on published literature. Control animalsonly received vehicle of the same volume.

AnimalsNon-fasted male C57BL/6J mice (supplied by Harlan SpragueDawley, Inc.) weighing 22–30 g (between 8–12 weeks old) weresingly caged in a temperature controlled (20 ± 2°C) quiet roomand maintained on a nonreversed 12-h light/dark cycle (lightson at 6 a.m. and lights off at 6 p.m.). The animals had unlimitedaccess to food (Harlan Teklad LM-485, standard diet) and water.From the day of the E-Mitter implantation, all mice were handleddaily before the treatment in order to reduce potential handlingstress. Each mouse was considered available for experimentalruns after a minimum of 7 days post-transmitter implantation.Animals were randomly assigned to the different treatmentgroups (n = 8 per group). Experiments were conducted between9 a.m. and 1 p.m. All animal experiments were performed ac-cording to the policies and guidelines of the Institutional AnimalCare and Use Committee (IACUC) of the University of Florida,Gainesville, USA (NIH publication #85-23), study protocol #200801768 (approved on 09/25/2008).

G2 E-Mitter implantationThe surgical implantation of the G2 E-Mitters was performedunder aseptic conditions according to the University of Florida,Animal Care Services Guidelines for Telemetry Implants andGuidelines for Survival Surgery in Mice. Before surgery, meloxicam(Sigma-Aldrich, Inc.) 1mg/kg was administered orally for analge-sia, and anesthesia was maintained by 1–3% isoflurane deliveredby nose cone. Twenty-four hours before implantation, the E-Mit-ters were sterilized with 2.4% activated glutaraldehyde (Cidex®,Johnson & Johnson, Inc.). The abdomen was opened by making a2-cm incision along the linea alba. The intestines and colon weregently reflected to allow insertion of the E-Mitter into the ab-dominal cavity along the sagittal plane, placing it in front of thecaudal arteries and veins, but dorsal to the digestive organs. Thepositioning is important to acquire accurate temperature data. Inorder to prevent migration of the E-Mitter within the peritonealcavity, transmitters were anchored to the abdominal cavity wallby the silastic suture sleeve. The peritoneal incision was closedusing 4/0 caliber monofilament polypropylene nonabsorbablesutures with reverse cutting needle in a simple interrupted stitchpattern. The skin incisionwas closed with a series of 9-mm surgi-cal staples whichwere removed 7 days after surgery. Rehydrationat the end of the surgery was assured by subcutaneous adminis-tration of sterile isotonic saline (10mL/kg body weight). Woundrecovery was checked regularly.

Telemetry systemA data acquisition system, the VitalView® from Mini Mitter, aRespironics Company (Series 4000), was used to acquire animalbody temperature and activity data. VitalView® is a softwareand hardware system specially designed for controlling data col-lection in laboratory monitoring of physiological parameterswithout the need for animal handling and prior habituation tothe test environment or the presence of the experimenter. E-Mit-ters capture energy from the field of radio waves emitted by coilsof the ER-4000 Energizer/Receiver, allowing the E-Mitter devices

Fig. 1 Changes in locomotor activity (A,C,E) andin body temperature (B,D,F) after oral administra-tion of midazolam (5mg/kg), zolpidem (5mg/kg),caffeine (5mg/kg), or vehicle. Values are expressedas mean ± SEM, n = 8 per group. Zero min repre-sents time of dosing.

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to be free from battery requirements. The signal returned to thereceiver consists of sequences of pulses whose period is depen-dent on temperature. The VitalView® system records the averagerate and the receiver converts this pulse frequency into a serialbit stream using calibration values specific to each device. Forthe quantification of locomotor activity, movement of the im-planted E-Mitter results in fluctuations in signal strength, whichare detected by the ER-4000 Receiver via telemetry and noted asactivity counts. The total number of these counts during a sam-pling period interval is recorded. After a minimum 5-day wash-out period following administration of the test substance, eachmouse was administered its matched control vehicle for a com-parison run. At least two corresponding control values werematched with every test value. On the day of the experiment,the cages were placed on the receivers at least one hour beforedosing. At 9:29 a.m. EST, dosing was started for the first four miceand at 9:35 a .m. EST for the second four mice. To assure and con-trol constant environmental conditions, experiment runs werealways accompanied by control animals.

Statistical analysisAll statistical procedures were calculated with GraphPad Prism®

statistical software package, version 5.00 (GraphPad Software,Inc). Locomotor activity and body temperature data were col-lected in 6-minute intervals and are displayed as mean ± SEM.Comparative cumulative locomotor activity between groups wasanalyzed using two-way analysis of variance (ANOVA) followedby Bonferroniʼs post hoc test. Additionally, temperature and ac-tivity pattern differences between control and each treatmentgroup were evaluated using the unpaired two-tailed t-test. Aprobability level of p < 0.05 was set as statistically significant.

Results!

l" Fig. 1A shows the depressant effect of midazolam (5mg/kg,p.o.) and illustrates the rapid onset of actionwithin the first min-utes. Locomotor activity was significantly lower at 12 minutes(control: 104.86 ± 16.48, midazolam: 17.36 ± 4.55; p < 0.001,two-way ANOVA) and 18 minutes (control: 109.45 ± 20.85, mid-azolam: 24.73 ± 11.33; p < 0.001, two-way ANOVA) after adminis-tration. As shown in l" Fig. 1B, body temperature showed a sig-

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Table 1 Cu-mulative loco-motor activityafter oral ad-ministration oftest com-pounds.

Treatment Group Cumulative activity

[0–180min]

Control 1242 ± 73

Caffeine 5mg/kg 1622 ± 97*

Midazolam 5mg/kg 829 ± 66**

Zolpidem 5mg/kg 951 ± 87*

Control 1286 ± 75

Valerian extract 250mg/kg 1305 ± 112

Valerian extract 500mg/kg 1398 ± 158

Valerian extract 1000mg/kg 1367 ± 108

Control 1405 ± 97

Valerenic acid 0.5mg/kg 1232 ± 175

Valerenic acid 1.0mg/kg 1221 ± 152

Valerenic acid 2.0mg/kg 1268 ± 97

Valerenic acid 5.0mg/kg 1282 ± 171

Control 1303 ± 59

Linarin 2.0mg/kg 1254 ± 130

Linarin 6.0mg/kg 1335 ± 134

Linarin 12.0mg/kg 1197 ± 151

Control 1173 ± 73

Apigenin 1.5mg/kg 1026 ± 194

Apigenin 3.0mg/kg 1185 ± 124

Apigenin 6.0mg/kg 1195 ± 126

Values are expressed as mean ± SEM, two-way analysis of variance

(ANOVA) followed by Bonferroniʼs post hoc test, * p < 0.05;

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nificant drop of 0.78°C 12 minutes after dosing (control: 37.19 ±0.15°C, midazolam: 36.41 ± 0.18°C; p < 0.05, two-way ANOVA).The mean body temperature for the initial 30 minutes was signif-icantly lower in the midazolam group compared to vehicle (con-trol: 37.13 ± 0.09°C, midazolam: 36.66 ± 0.12°C; p < 0.05, un-paired two-tailed t-test).Similarly to midazolam, zolpidem (5mg/kg, p.o.) decreased theactivity in the first 30 minutes (l" Fig. 1C). Mean body tempera-ture at 12 minutes after administration was significantly lowerin the zolpidem group (35.96 ± 0.13°C) than in the control group(36.43 ± 0.07°C; p < 0.01, unpaired two-tailed t-test) (l" Fig. 1D).The total activity for 180 minutes after administration of 5mg/kg caffeine was increased to 126% compared to the control group(l" Fig. 1E). More precisely, locomotion was significantly stimu-lated only during the initial 30 minutes with a high significanceat 12 minutes (control: 72.40 ± 10.86, caffeine: 176.00 ± 31.76;p < 0.001), 18 minutes (control: 42.71 ± 10.38, caffeine: 146.43 ±36.90; p < 0.001) and 24 minutes (control: 30.09 ± 7.23, caffeine:140.29 ± 32.96; p < 0.001) indicating a rapid onset of action. Bodytemperature was not considerably affected (l" Fig. 1F). The cu-mulative locomotor activity of midazolam, zolpidem, and caf-feine are shown in l" Table 1.l" Fig. 2A and C show no major effect on locomotor activity afteroral administration of 250 and 500mg/kg valerian extract, re-

Fig. 2 Changes in locomotor activity (A,C,E) andin body temperature (B,D,F) after oral administra-tion of valerian extract (250, 500, and 1000mg/kg,respectively) or vehicle. Values are expressed asmean ± SEM, n = 8 per group. Zero min representstime of dosing.

** p < 0.01

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Fig. 3 Changes in locomotor activity (A,C,E,G)and in body temperature (B,D,F,H) after oral ad-ministration of valerenic acid (0.5, 1.0, 2.0, and5.0mg/kg, respectively) or vehicle. Values are ex-pressed as mean ± SEM, n = 8 per group. Zero minrepresents time of dosing.

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spectively, and body temperature remained mostly unaffected inthese concentrations (l" Fig. 2B,D). In a higher concentration of1000mg/kg,valerianextractdidnot induceasignificantdifferencein the overall locomotor activity over 180minutes (l" Table 1), butaltered the pattern of activity with nonsignificant short-term de-creases between 60 and 90 minutes (l" Fig. 2E) and increases inthe extract group between 132 and 150 minutes. Body tempera-ture remained unchanged during the whole recording period ex-cept with a nonsignificant increase between 132 and 180minutes(control: 35.68 ± 0.05°C, extract: 35.96 ± 0.10°C) (l" Fig. 2F).No major effects were observed for valerenic acid in a low con-centration (0.5mg/kg) (l" Fig. 3A,B). One mg/kg of valerenic acidconsiderably altered the locomotion pattern (l" Fig. 3C). After aprominent increase at 78 minutes, a subsequent decrease oc-curred. Between 138 and 180 minutes, activity was significantlylower in the valerenic acid group. Simultaneously, body temper-ature was significantly decreased by 0.34°C during the same pe-riod (l" Fig. 3D; control: 35.82 ± 0.09°C, valerenic acid: 35.48 ±0.10°C; p < 0.05, unpaired two-tailed t-test). In higher concentra-

tions (2mg/kg and 5mg/kg, respectively), valerenic acid did notinduce any statistically significant changes in locomotion(l" Fig. 3E,G) or body temperature (l" Fig. 3F,H). Overall, valerenicacid had no effects on the cumulative activity from 0–180min(l" Table 1).l" Fig. 4A and 4B illustrate that linarin at a concentration of 2mg/kg did not considerably affect locomotion or body temperature.After oral administration of 6mg/kg linarin, a significant increasein locomotor activity during the initial 30 minutes compared tovehicle was observed.l" Fig. 4C shows the significantly higher ac-tivity values at 12 minutes (control: 72.40 ± 10.86, linarin:150.20 ± 35.00; p < 0.05), 18minutes (control: 42.71 ± 10.38, linar-in: 152.80 ± 41.04; p < 0.001) and 24 minutes (control: 30.09 ±7.23, linarin: 110.80 ± 40.42; p < 0.01). Body temperature was notaffected over the whole observation period (l" Fig. 4B). In a doseof 12mg/kg, linarin significantly reduced locomotor activity be-tween 60 and 132 minutes (l" Fig. 4E) whereas no effect on bodytemperature was observed (l" Fig. 4F). Considering the effects on

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Fig. 4 Changes in locomotor activity (A,C,E) andin body temperature (B,D,F) after oral administra-tion of linarin (2.0, 6.0, and 12.0mg/kg, respec-tively) or vehicle. Values are expressed as mean ±SEM, n = 8 per group. Zero min represents time ofdosing.

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the cumulative activity from 0–180min, linarin had no signifi-cant effect (l" Table 1).Oral administration of 1.5mg/kg apigenin induced a significantreduction in locomotor activity (l" Fig. 5A). The evaluation indi-cates significantly lower activity between 90 and 120 minutesafter gavage. The overall activity during 180 minutes did not sig-nificantly differ between the groups after oral administration of3mg/kg apigenin (l" Fig. 5C). However, locomotion was slightlylowered between 84 and 114 minutes. Locomotion pattern wasalso not considerably altered after administration of 6mg/kg api-genin (l" Fig. 5E). However, the total cumulative activity re-mained unaffected (l" Table 1). No considerable effect on bodytemperature was observed for all concentrations (l" Fig. 5B,D,F).

Discussion!

It is generally acknowledged that the quality of physiologicalmeasurements collected from conscious unstressed animals pos-sesses the highest validity and best reflects the normal state ofthe animal [20–22]. The radiotelemetry system therefore repre-sents an adequate method and has proven its justification inmodern pharmaceutical research thanks to a minimum of han-dling-related or restraint stress to the animal [17,23,24]. In lightof the above, the present study was interested in possible seda-tive effects of a 70% ethanolic valerian extract and some of its sin-

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gle constituents (valerenic acid, linarin, and apigenin), which areassumed to be mainly responsible for valerianʼs activity.The outcomes of the positive (midazolam, zolpidem) and nega-tive (caffeine) controls are supported by existing literature dataand validate the used method [23,25–29].The effects of valerian root on sleep parameters are only sparselyinvestigated in animals. Wagner et al. [30] observed a sedative ac-tion of a fresh valerian tincture at 1000mg/kg p.o. in mice,whereas an aqueous extract induced a slight decrease in sponta-neous motility at 10mg/kg p.o. Similar findings are reported byLeuschner et al. [15] who found pronounced sedative propertiesafter oral administration of 20 and 200mg/kg of an aqueous alka-line extract. While assessing these results, it must be taken intoconsideration that statistical specification or significance is ab-sent in these studies [15,30]. Additionally, enhancing of barbitu-rate sleeping time is demonstrated in several studies [15,31] andconsidered to be evidence for sedation. But its relevance for prov-ing sedative properties remains unclear since barbiturates havebeen shown to interfere with CYP P450 enzymes [32], and there-fore an increased sleeping time due to interactions of valerianwith the barbiturate metabolism in mice cannot be excluded.Tokunaga et al. [33] reported significant shortening of sleep la-tency in rats after oral administration of 1000mg/kg valerian ex-tract without affecting total time of wakefulness, non-REM sleep,and REM sleep. These results are not supported by the presentstudy, which did not find any significant effect on total cumula-

Fig. 5 Changes in locomotor activity (A,C,E) andin body temperature (B,D,F) after oral administra-tion of apigenin (1.5, 3.0, and 6.0mg/kg, respec-tively) or vehicle. Values are expressed as mean ±SEM, n = 8 per group. Zero min represents time ofdosing.

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tive activity over 180 minutes. However, 1000mg/kg altered theactivity pattern of the mice and induced a nonsignificant inter-mittent drop in activity (66–78min). This short-term depressanteffect is not sufficient to ascribe positive effects on sleep, since astimulating activity was observed thereafter. In contrast, 250mg/kg and 500mg/kg did not affect locomotor activity at all. Hatte-sohl et al. [9] drew a similar conclusion since they reported no ef-fect on locomotion for several valerian extract preparations afteracute and subacute (19 days) oral administration.Temperature data indicate that valerian extract produces a milddose-dependent hyperthermic effect in higher doses. This find-ing is contradictory to Hendriks et al. [34] who reported a hypo-thermic effect for the essential oil of V. officinalis and several con-stituents, including valerenic acid. In contrast, Hiller and Zetler[31] did not observe any influence on body temperature after ad-ministration of 50 and 100mg/kg ethanolic valerian extract (i.p.),whereas diazepam produced a significant hypothermic effect. Nomarked effects on temperature have been observed in thepresent study. In conclusion, considering our results on locomo-tor activity and body temperature obtained with telemetry, itseems unlikely that valerian has any significant sedative actions.None of the tested concentrations of valerenic acid did signifi-cantly lower the activity over 180minutes. However, at a concen-tration of 1mg/kg the activity pattern was considerably altered.A short-lasting increase was observed between 78 and 102 min-utes after administration followed by a reduction in locomotion

between 138 and 180 minutes. These findings are in line with re-sults from recent studies. Benke et al. [8] reported no indicationfor sedative activity for 10mg/kg p.o. and 30mg/kg i.p. (elevatedplus-maze). A similar conclusion was drawn by Fernandez et al.[11]. They observed no in vivo effects in mice at low doses up to15mg/kg i.p. (sodium thiopental-induced sleeping time andholeboard test). In contrast, Hendriks et al. [34] found valerenicacid to decrease motility of mice at a concentration of 50mg/kgi.p. and higher. The same authors reported in a later study a de-crease in rotarod performance in mice (100mg/kg i.p.) and adose-related increase in pentobarbital-induced sleeping time(50 and 100mg/kg i.p.) [16]. Furthermore, a significant decreaseof rectal temperature in mice at concentrations of 100, 200, and400mg/kg was observed. Paradoxically, after administration of50mg/kg, a mild increase in body temperature occurred [16].Overall, the authors concluded that this compound has centraldepressant properties. However, considering the extremely highand unphysiological doses, the relevance of these results has tobe questioned.In the present study, linarin induced only at a higher concentra-tion (12mg/kg) a significant reduction of locomotor activity be-tween 60 and 132 minutes; however, when considering the cu-mulative activity, no significant changes were detected. There-fore, the findings of Fernandez et al. [11], could partly be con-firmed. The authors observed depressant effects at doses of 4and 7mg/kg i.p. in the holeboard test. Furthermore, 7 and

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14mg/kg i.p. significantly prolonged the thiopental-inducedsleeping time in mice. Hence it was concluded that linarin pos-sesses sedative and sleep-enhancing activity. In contrast to thefindings of Fernandez et al. [11] we found an initial stimulatingeffect after oral administration of 6mg/kg linarin.While in the present study all substances were administered or-ally, Fernandez et al. [11] used intraperitoneal injection. It islikely that the different routes of administration caused differenteffects on locomotor activity. Consequentially, results obtainedusing different routes of administration should not be compareddirectly.In the present study, apigenin showed mild sedative effects atlow concentrations. Locomotor activity was significantly reducedbetween 90 and 120 minutes (1.5mg/kg) after gavage. The ob-served effect was indeed short-lasting since after 120minutes ac-tivity began to rise to control levels. In higher doses, apigenin didnot significantly reduce activity or body temperature. Viola et al.[35] suggested apigenin to act as a benzodiazepine partial agonistand to possess anxiolytic activity at low doses (3mg/kg). In thesame study, sedative effects were only observed after i.p. admin-istration of high doses (30 and 100mg/kg) in mice (holeboardand locomotor activity test), but not for doses up to 10mg/kgi.p. Similarly, Avallone et al. [36] reported potent sedative proper-ties for high doses (25 and 50mg/kg i.p.) in rats as well. However,in most of the previously reported studies apigeninwas tested foranxiolytic properties and not particularly for sedation. Therefore,the presented results reveal for the first time the potential bene-ficial effect of apigenin on sleep.In summary, the present findings indicate that the radioteleme-try system is suitable for the investigation of sleep-modifying ef-fects. Considering all aspects, it is concluded that neither the ex-tract nor one of the compounds had significant sedative activitiesover 180min. However, positive short-term sedative effects werefound for valerian extract 1000mg/kg, linarin 12mg/kg, and api-genin 1.5mg/kg whereas valerenic acid (0.5, 1, 2, and 5mg/kg)had no significant effect on locomotor activity or body tempera-ture.The fact that mild sedative effects of the extract and the singlecompounds lasted only for a very short period suggests an inter-esting pharmacokinetic profile probably pointing to a short half-life of all compounds. Since pharmacokinetic data about valeriancompounds are only sparsely available, this assumption presentlyis speculative. However, pharmacokinetic data are an importantissue to link data with pharmacological assays. Our findings, ob-tained with telemetric recordings of locomotor activity and bodytemperature, do not support the use of valerian root for the treat-ment of sleep disorders in general. However, the observed short-term effects presented for the flavonoids linarin and apigenin in-dicate that these compounds could be effective to reduce sleep la-tency rather than to act as sleep-maintaining agents. Future stud-ies therefore should focus on the investigation of other flavonoidsfrom valerian as sleep-inducing agents.

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