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Extracts of Valeriana officinalis L. s.l. show anxiolytic and antidepressant effects but neither sedative nor myorelaxant properties
ARTICLE IN PRESS
Phytomedicine 15 (2008) 2–15 www.elsevier.de/phymed
Extracts of Valeriana officinalis L. s.l. show anxiolytic and antidepressant effects but neither sedative nor myorelaxant properties Miguel Hattesohla,, Bjo¨rn Feistelb, Hartwig Sieversc, Romanus Lehnfeldc, Mirjam Heggera, Hilke Winterhoffa a
Department of Pharmacology and Toxicology, Universita¨tsklinikum Mu¨nster, Westfa¨lische Wilhelms-Universita¨t Mu¨nster, Domagkstr. 12, 48149 Mu¨nster, Germany b Finzelberg GmbH & Co. KG, Koblenzer Str. 48-56, 56626 Andernach, Germany c PhytoLab GmbH & Co. KG, Dutendorfer Str. 5-7, 91487 Vestenbergsgreuth, Germany
Abstract Extracts of Valeriana officinalis L. s.l. are used for treating mild sleep disorders and nervous tension. Despite intensive research efforts, the pharmacological actions accounting for the clinical efficacy of valerian remain unclear. Thus, it was the aim of this study to evaluate CNS-related effects of different valerian extracts using behavioral paradigms (mice and rats). Following oral administration two commercially available preparations (extraction solvents: 45% methanol m/m and 70% ethanol v/v), a 35% ethanolic v/v extract and a refined extract derived from it (patented special extract phytofin Valerian 368) were tested for sedative (locomotor activity, ether-induced anaesthesia) and anxiolytic (elevated plus maze) activity. Using the forced swimming and the horizontal wire test the latter two extracts were additionally tested for antidepressant and myorelaxant properties. Up to maximum dosages of 500 or 1000 mg/kg bw none of the valerian extracts displayed sedative effects. Neither spontaneous activity was reduced nor the duration of ether-induced narcosis was prolonged. In contrast, results obtained in the elevated plus maze test revealed a pronounced anxiolytic effect of the 45% methanolic and 35% ethanolic extract as well as of phyotofin Valerian 368 in a dose range of 100–500 mg/kg bw. Additionally and different from its primary extract (35% ethanolic extract) phytofin Valerian 368 showed antidepressant activity in the forced swimming test after subacute treatment. Myorelaxant effects were not observed in dosages up to 1000 mg/kg bw. Due to these findings it is proposed that not sedative but anxiolytic and antidepressant activity, which was elaborated particularly in the special extract phytofin Valerian 368, considerably contribute to the sleep-enhancing properties of valerian. r 2007 Elsevier GmbH. All rights reserved. Keywords: Valeriana officinalis L.s.l.; Elevated plus maze; Anxiolytic; Sedative; Locomotor activity; Antidepressant; Myorelaxant
Introduction
Corresponding author. Tel.:+49 251 8355514; fax: +49 251 8355501. E-mail address: [email protected] (M. Hattesohl).
0944-7113/$ - see front matter r 2007 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2007.11.027
Extracts derived from roots of Valeriana officinalis L. s.l. are used for the treatment of mild sleep disorders and nervous tension. Only recently the therapeutic efficacy of valerian has been confirmed by the European Medicines Evaluation Agency in an actual monograph
ARTICLE IN PRESS M. Hattesohl et al. / Phytomedicine 15 (2008) 2–15
(Committee on Herbal Medicinal Products (HMPC/ EMEA), 2006). Valerian preparations contained in commercially available products are extracted with water, water/methanol or water/ethanol mixtures. In several placebo-controlled clinical studies an improvement of sleep-related parameters following treatment with aqueous or ethanolic extracts was demonstrated. For instance, acute administration of an aqueous extract reduced sleep latency (Balderer and Borbely, 1985; Leathwood et al., 1982). Using subacute treatment regimes, an extract made with 70% v/v ethanol affected different sleep parameters in a positive manner as well (Donath et al., 2000; Vorbach et al., 1996). Furthermore an improvement of sleep quality comparable to treatment with 10 mg oxazepam was observed for the same ethanolic extract in two comparative studies (Dorn, 2000; Ziegler et al., 2002). It is commonly assumed that efficacy of valerian preparations in sleep disorders could be due to putative sedative properties of these extracts. However, results of studies which tested valerian for the proposed sedative effects and its influence on vigilance in healthy volunteers are contradictory to this hypothesis (Gutierrez et al., 2004; Hallam et al., 2003; Kuhlmann et al., 1999). None of the tested ethanolic extracts (70% v/v) revealed sedative properties since neither psychomotor nor cognitive nor subjective parameters were affected significantly in these trials. Many efforts have been made to elucidate the pharmacological profile of different valerian extracts as well as of isolated constituents. In vivo studies provided inconsistent results concerning sedative, anticonvulsant, myorelaxant and other effects indicating central mechanisms of action. For instance, extracts made with 70% ethanol varied in their capability to affect motor activity in mice. While Hiller and Zetler (1996) observed no effect locomotor activity was weakly reduced in a study of Wagner et al. (1980). Testing an aqueous alkaline extract, Leuschner et al. (1993) observed a reduction in spontaneuos activity as well. Other in vivo experiments revealed further pharmacological effects of common valerian extracts like a significant shortening of sleep latency in sleep-disturbed rats after treatment with a 70% ethanolic preparation (Shinomiya et al., 2005) or anticonvulsant properties using a 70% ethanolic or an aqueous alkaline extract (Hiller and Zetler, 1996; Leuschner, et al., 1993). Furthermore Hiller and Kato (1996) showed a pronounced anxiolytic effect of the ethanolic extract STEI Val using the elevated plus maze test. Taking into account that type of extract, doseregimes, the method of application and the used ethological paradigms varied between all studies it becomes obvious that precise conclusions on pharmacological actions of valerian may hardly be drawn. Moreover some studies preferred intraperitoneal admin-
3
istration, an application mode which is of questionable relevance for orally administered plant extracts. In trying to determine the active constituents of valerian, many single compounds have been tested in distinct in vivo experimental procedures. There is certain evidence that valerenic acid and flavonoids like hesperidin and 6-methylapigenin may contribute to central effects of extracts derived from Valeriana wallichii. There is no evidence, however, that both flavonoids occur in detectable amounts also in Valeriana officinalis. Valerenic acid was shown to possess anticonvulsant properties (Hiller and Zetler, 1996) while hesperidin proved to be sedative since locomotor activity was reduced and thiopental-induced sleeping time was prolonged (Marder et al., 2003). Additionally, in the latter study 6-methylapigenin was shown to be active in the elevated plus maze procedure. Whilst evaluating these results it must be considered that all studies used intraperitoneal injection as the route of administration. Valepotriates, which were also discussed to be determinant for central actions of valerian (Andreatini and Leite, 1994; Andreatini et al., 2002), are detectable at trace levels or not at all in recent drug preparations (ESCOP, 2003; Schulz and Ha¨nsel, 2004). Other compounds that were often associated with pharmacological actions of valerian, like the essential oil or single components of it (Hendriks et al., 1981), were mostly tested in dosages clearly exceeding the range that actually can be reached when administering any extract preparation. Thus, for an evaluation of the pharmacological profile of valerian it does not seem valuable to include results from those studies. In vitro studies testing extracts or isolated constituents provided certain evidence for an involvement of particularly the GABAA-system in the psychotropic effects of valerian (Balduini and Cattabeni, 1989; Bodesheim and Ho¨lzl, 1997; Cavadas et al., 1995; Dietz et al., 2005; Fernandez et al., 2004, 2005; Ferreira et al., 1996; Marder, et al., 2003; Mennini and Bernasconi, 1993; Ortiz et al., 1999, 2006; Riedel et al., 1982; Santos et al., 1994a–c; Simmen et al., 2005; Wasowski et al., 2002; Yuan et al., 2004). In addition, the adenosinesystem may also account for central actions since in some studies an interaction of valerian extracts as well as of an olivil derivate at the adenosine A1-receptor was observed (Balduini and Cattabeni, 1989; Mu¨ller et al., 2002; Schumacher et al., 2002). However, despite intensive research efforts, the pharmacological actions accounting for the proved efficacy of valerian in mild sleep disorders remain unclear. Thus, it was the aim of the present study to evaluate the pharmacological profile of extracts prepared with different solvents including both, commercially available extracts and newly developed extracts, by using in vivo paradigms for detecting sedative, anxiolytic, antidepressant and myorelaxant properties.
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Materials and methods Chemicals, plant material and treatment of animals Diazepam was purchased from ratiopharm (Ulm, Germany), N6-Cyclopentyladenosine (CPA) and Caffeine from Sigma-Aldrich GmbH (Taufkirchen, Germany). All tested valerian preparations, VAL SE 35E, VAL TE 35E, the patented special extract phytofin Valerian 368 and the commercially available extracts VAL H 70E and VAL H 45 M were provided by Finzelberg (Andernach, Germany) and derived from dried, cutted roots of Valeriana officinalis L. s.l. (Ph. Eur.) by percolation with water, ethanol/water 35% or 70% v/v or methanol/water 45% m/m as shown in Table 1. The extraction was carried out by exhaustive percolation at a temperature of 50 1C. After vacuum evaporation the obtained soft extracts were spray-dried under addition of 25% or 30% maltodextrine (Ph. Eur.) respectively to avoid agglomeration. Phytofin Valerian 368 was prepared from the soft extract VAL SE 35E using column adsorption technique. Therefore the soft extract was resolved in water until a percentage of indissoluble compounds of 10% (w/w) was achieved. After separating the precipitate by filtration the remaining dilution was applied to a column containing the polymeric adsorbent AMBERLITEs XAD-4 (Rohm and Haas Company, Philadelphia, USA) as packing material. This non ionic crosslinked Divinylbenzene Copolymer adsorbs hydrophobic molecules of low molecular weight (like valerenic acids) from polar solvents. Finally the purified aqueous phase that passed the column was evaporated again and transformed into a dry extract by spray drying and adding 30% maltodextrine. Due to the usage of solvents of different polarity and to differences in subsequent processing the valerian
Table 1.
preparations showed distinctions regarding the native drug:extract ratio and the amount of valerenic acids that were determined by HPLC (Table 1). The DC-chromatogramm in Fig. 1 demonstrates a clear decline of lipophilic constituents in the extract phytofin Valerian 368 compared to its primary extract VAL SE 35E (Fig. 1). None of the preparations contained valepotriates. For animal experiments diazepam and all extracts were dissolved in tap water and administered orally (p.o.). Except when testing for an influence on the duration of ether-induced narcosis diazepam was injected intraperitoneally (i.p.). N6-Cyclopentyladenosine (CPA) and caffeine were prepared with saline and applied intraperitoneally (i.p.). All test solutions were given in a volume of 10 ml/kg bodyweight.
Fig. 1. TLC-fingerprint chromatogram on polar constituents in valerian extracts acc. Finzelberg method after derivatization with Anisaldehyd–Sulfur acid-mix in white light (eluent:dichloroethane (50):acetic acid (25):methanol (15):water (10), v/v/v/v).
Specifications of valerian extracts
Type of extract
Code name
Extraction solvent
Notes to extracts
Drug: extract ratio
Maltodextrine (%)
Total valerenic acids (%)
Soft extract
VAL SE 35E
Ethanol 35% (v/v)
Primary extract
2–4:1
–
0.29
Purified, spraydried and powdered special extract
Phytofin Valerian 368 (patented)
Ethanol 35% (v/v)
Prepared from VAL SE 35E by column adsorption technique
3–5:1
30
0.13
Spray-dried, powdered extracts
VAL TE 35E
Ethanol 35% (v/v)
Prepared from VAL SE 35E
2–4:1
30
0.29
VAL H 45M
Methanol 45% (m/m)
Represents standard quality
5–8:1
25
0.38
VAL H 70M
Ethanol 70% (v/v)
Represents standard quality
3–6:1
30
0.31
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The indicated dosages of extract preparations refer to the native plant extract.
Animals Female NMRI mice (18–20 g, Charles River Laboratories, Sulzfeld, Germany) were housed in groups of six animals per cage, male Sprague-Dawley rats (150–170 g, Charles River Laboratories, Sulzfeld, Germany) in groups of two. All animals were kept in a 12 h light/dark cycle, with lights off at 6:00 p.m., at a constant temperature of 2571 1C and with free access to food (Altromin 1324, Altromin, Lage, Germany) and tap water. For experimental testing the animals were randomly assigned to control or treatment groups. Experiments were carried out after the animals had adapted to the laboratory conditions for at least 1 week. The procedures used comply with the European Community’s Council Directive of 24 November 1986 (86/609/EEC) and were officially approved by the local committee on animal care (Regierungspra¨sident Mu¨nster, AC/2004).
Locomotor activity Activity experiments were carried out with female NMRI mice (n ¼ 8/group) by using infrared (IR) technique. The apparatus containing 16 macrolon cages was placed in a sound attenuated room and darkened by a roller blind during measurement. For experimental testing mice were singly introduced into the cages and initially allowed to adapt to experimental conditions for 30 min. Immediately after treatment (p.o. or i.p.) locomotor activity was recorded continuously by passive infrared (IR) sensors. The number of IR-impulses was registered accumulatively within consecutive intervals of 10 min (counts per 10 min) over a total period of 2 h. A reduced locomotor activity represents a sedative effect whereas stimulant properties are indicated by an increased motility.
Ether-induced anaesthesia An influence of test compounds on the duration of ether-induced narcosis was tested in female NMRI mice (n ¼ 12–15 per group) 30 or 60 min after treatment (p.o. or i.p.). For anesthetisation 3.5 ml diethylether were given into a 5 l glass tank. After a vaporisation period of 1 min six animals were simultaneously placed into that tank for 2 min. Subsequently mice were taken out of the ether atmosphere and sleeping time was determined by registration of the time until the righting reflex was completely recovered. A prolongation of ether-induced narcosis is caused by central depressant drugs.
5
Elevated plus maze The procedure, first described by Pellow et al. (1985) and Lister (1987) as a behavioral model to measure anxiety in rats and mice respectively, was conducted with female NMRI mice (n ¼ 12–14 per group) in a sound attenuated room under high illumination. The elevated plus maze consisted of respectively two black-surfaced open (30 cm long and 5 cm wide) and enclosed arms (30 cm long and 5 cm wide with walls of 15 cm height) that extended from a central platform (5 cm 5 cm). Open and enclosed arms were arranged opposite to each other and the whole maze was elevated to a height of 40 cm. 60 min after treatment mice were placed in the central square of the maze, facing one of the enclosed arms. During a 5 min test period animals were allowed to freely explore the environment. Behavior was recorded by video camera and the time mice spent in the open and closed arms as well as the number of open and closed arm entries was evaluated afterwards by a blinded observer. An entry was defined as placement of all four paws into an arm. An increase in the percentage of time spent in the open arms (topen arms/topen+closed arms 100) and in the percentage of open arm entries (nopen arms/nopen+closed arms 100) indicate an anxiolytic effect. To avoid the phenomenon of one trial tolerance (File et al., 1990) each animal was only used once. After each trial the test arena was cleaned carefully with water containing detergents.
Forced swimming test (FST) including additional open field test The forced swimming test, first published by (Porsolt et al., 1977, 1978) as a paradigm to detect compounds with antidepressant activity, was performed with male Sprague-Dawley rats. The animals (n ¼ 12 per group) received pre-treatment with the test solutions (p.o.) two times daily (8:00 a.m., 20:00 p.m.) over a period of 16 days. One day before the test the rats experienced a 15 min lasting pretest session, in which the animals were singly introduced into a plexiglass cylinder that contained a 17–21 cm (depending on the bodyweight) high column of water at 2571 1C. Experimental testing was conducted 24 h later by exposing the rats to the same test conditions for 5 min 5 h after last drug administration. Behavior was recorded by video camera. The cumulative time rats persisted in an immobile position (despair behavior) was evaluated afterwards by a blinded observer. A rat was judged to be immobile whenever it remained floating in the water in contrast to motor activities that represented attempts to escape from the situation. The time rats spent making small movements to keep the head above the water was assigned to time of
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immobility. A shortened immobility time indicates an antidepressant effect. To exclude false positive results due to compoundrelated stimulating effects on motility, locomotor activity was determined 2 days (day 14 of drug treatment) before FST performance by using the open field paradigm. The open field test, first described by Hall (1934), was carried out in a sound attenuated room with a round arena (diameter: 70 cm, height of surrounding wall: 30 cm) whose grey base area was divided in 18 equal sections. Rats were placed individually in the center of the arena and behavior was video recorded for 5 min. Afterwards a blinded observer scored the number of line crossings (crossings per 5 min) as an index of locomotor activity. After each trial the test arena was washed carefully with detergent solution. FST and open field experiments were carried out between 1:00 and 5:00 p.m.
Horizontal wire test To detect potential myorelaxant effects the horizontal wire test according to the method published by Boissier and Simon (1960) was used. The test apparatus consisted of a steel bar (2 mm diameter, 15 cm length) that was fixed in horizontal position 20 cm above the ground. In a first session an appropriate collective of female NMRI mice was selected. Mice that, when lifted by the tail and allowed to grasp the wire with both forepaws, failed to grasp the steel bar with at least one hindpaw within 5 sec were excluded from further experiments. Immediately after oral treatment the selected animals (n ¼ 10 per group) were tested for myorelaxant activity consecutively every 10 min over a period of 1 h. Mice that had lost the ability to grasp the wire with at least one hindpaw were considered to exhibit impaired muscle strength. Results are expressed as percentage of impaired animals.
Data analysis and statistics All statistical procedures were performed by using the STATVIEW statistical software package, version 5.0 (SASR, USA). Except in the case of motility experiments data analysis was carried out by analysis of variance (ANOVA) with the Fisher-PLSD post hoc test for multiple comparisons. Data obtained from activity experiments were statistically evaluated by using unpaired Student t-test. All data are expressed as MEANS 7SEM. Only the values gained from horizontal wire experiments are indicated as percentages. Statistical significance was set at po0.05 (*po0.05, **po0.01, ***po0.001).
Results Locomotor activity Results of motility experiments are shown in Table 2 and Fig. 2. The benzodiazepine diazepam reduced locomotor activity significantly in a dose dependent manner. In both dosages the effect occurred 20 min after gavage while 4 mg/kg bw evoked a more pronounced and longer lasting reduction of motor activity. In contrast, dosages of 3.5 and 5 mg/kg bw of the stimulant caffeine produced a significant increase in locomotor activity starting 30 min after intraperitoneal injection. As expected the higher dose revealed a more distinct as well as persistent effect. Contrary to these reference compounds neither the valerian preparations VAL SE 35E and phytofin Valerian 368 nor the commercially available extract VAL H 70E showed significant changes in locomotor activity in dosages of 100, 250, 500 or 1000 mg/kg bw within the test period of 2 h immediately after oral application. Only the methanolic extract VAL 45 M led to an alteration of motility. 90 min after oral administration of 100 mg/kg bw an increased activity was observed. However, higher doses of the same extract (250, 500 mg/kg bw) did not show stimulant properties.
Ether-induced anaesthesia As shown in Table 3 and Fig. 3 diazepam prolonged the duration of ether-induced narcosis significantly 30 min after intraperitoneal injection, with a pronounced effect at the higher dose (1.5 mg/kg bw). An extended sleeping time was also observed after administration of N6-Cyclopentyladenosine. While 0.2 mg/kg bw of the selective agonist at the adenosine A1 receptor led to a significant prolongation, 0.1 mg/kg bw only tend to result in an increased sleeping time. However, the lowest dose (0.05 mg/kg bw) of this reference did not affect this parameter. While the central depressant drugs, diazepam and CPA, produced an extension of ether-induced anaesthesia, none of the valerian preparations influenced sleeping time significantly 30 or 60 min after gavage of 100, 250 and 500 mg/kg bw respectively.
Elevated plus maze Diazepam (2 mg/kg bw), that was used as reference in all elevated plus maze tests, always exhibited anxiolytic activity after gavage since the percentage of time spent in the open arms as well as the percentage of open arm entries was significantly increased in each experiment (Figs. 4–7).
ARTICLE IN PRESS M. Hattesohl et al. / Phytomedicine 15 (2008) 2–15
Table 2.
7
Locomotor activity
Treatment
Minutes after application 10
20
30
60
90
120
27537162 22487151 18317359 15807184
23817183 12067283** 20337283 100731***
2145794 9027235*** 20137414 77720***
12097249 8777328 12077448 171748***
9027297 4397252 12747548 15.4711***
29722 72772 18267470 2497146***
19607370
14307350
11027329
5267352
7917471
5877391
25837249 15517318
22887389 18627279
24277315* 14777320
17327373* 4847330
16117464 5837322
11567445 3677221
19407405
21667245
26217151***
22237185***
20117214***
14987302***
VAL SE 35E (p.o.) Control 21387244 100 mg/kg 24757270 Control 22857189 250 mg/kg 25967136 Control 25277271 500 mg/kg 23947248
20907229 21127254 19337213 14837137 18667227 22057371
20397207 18527300 17167200 20737297 16367394 18407427
14347347 14927287 9877332 10737304 10967442 15937379
7117271 7567384 8727329 7737356 5277455 2717246
4457249 2987271 5947334 5897345 7267423 3207204
Phytofin Val. 368 Control 100 mg/kg Control 500 mg/kg Control 1000 mg/kg
(p.o.) 23127115 24327188 23927140 24197224 22627184 20157276
23667152 23817170 23447389 24547319 21697244 19697384
25437189 21677258 20567322 24567281 22027212 18527405
21287292 21707305 15107271 18647265 15717310 11307447
11767424 17327345 10357341 15677472 13177304 11347440
5437328 10717446 2497119 11437438 10237429 5137213
VAL H 70E (p.o.) Control 23897276 100 mg/kg 20257242 Control 19827209 250 mg//kg 25207292 Control 24267210 500 mg/kg 24587195
20557244 19557235 16737224 22167165 23127261 19147343
18797322 15347199 16957253 22977438 18797153 18617304
11737353 5847247 7557348 16117555 10987502 12007468
5307264 9627310 6107399 11197471 3197317 6187277
6527383 3827220 6397297 4027207 5727335 7867290
VAL H 45M (p.o) Control 22757305 100 mg/kg 18987209 Control 26437316 250 mg/kg 24257282 Control 23247103 500 mg/kg 25807326
20247218 16877100 24207369 22417322 19967185 23107376
18517208 21797211 23017383 21977336 20207308 24597367
10287330 15337181 13127328 15757472 14557286 18147339
4197208 12417209* 9637303 12187490 16857483 6567347
6047242 5257322 3027202 8377480 6767551 4577210
Diazepam (p.o.) Control 3 mg/kg Control 4 mg/kg Caffeine (i.p.) Control (saline) 3.5 mg/kg Control (saline) 5 mg/kg
Values are means7SEM expressed in counts per 10 min of 8 animals in each group. *po0.05, **po0.01 and ***po0.001 vs. corresponding control group, Student’s t-test.
An increased exploration of the open arms was also observed 60 min after oral administration of the valerian preparation VAL TE 35E, that was prepared with 35% v/v ethanol (Fig. 4). While the percentage of time in the open arms was increased significantly at doses of 100 and 500 mg/kg bw, the parameter concerning the number of open arm entries was affected without
statistical significance. A dosage of 250 mg/kg bw hardly caused an increase of time spent in the open arms and did not influence the ratio of open and closed arm entries. The special extract phytofin Valerian 368, that was derived from the soft extract VAL SE 35E using column absorption technique, exhibited a distinctive anxiolytic
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Table 3.
3000 2500
Duration (s) of ether-induced anaesthesia
Treatment
2000 1500
Minutes after application 30
60
Diazepam (i.p.) Control 1 mg/kg Control 1.5 mg/kg
9779 210719*** 124712 388768***
– – – –
CPA (i.p.) Control 0.05 mg/kg 0.1 mg/kg 0.2 mg/kg
105712 10379 129711 147714*
– – – –
VAL TE 35E (p.o.) Control 100 mg/kg 500 mg/kg 1000 mg/kg
107716 119723 109716 107712
9079 89713 10479 88711
Phytofin Val. 368 (p.o.) Control 100 mg/kg 500 mg/kg 1000 mg/kg
103710 11579 115711 97713
80711 92710 9577 8878
VAL H 70E Control 100 mg/kg 250 mg/kg 500 mg/kg
8876 8077 8377 9676
124715 99710 128711 13877
Val H 45 M (p.o.) Control 100 mg/kg 250 mg/kg 500 mg/kg
90710 82713 63711 7875
107713 89713 83713 87710
1000 500
A
counts per 10 minutes
0 3000 2500 2000 1500 1000 500
B
0 3000 2500 2000 1500 1000 500
C
0 0
**
* * ** **
10 20 30 40 50 60 70 80 90 100 110 120 min after application
Fig. 2. Locomotor activity (mean7S.E.M. counts per 10 min) of NMRI mice (8 per group) after oral application of the test solutions: J water, K A: VAL H 70E 500 mg/kg bw, K B: phytofin Valerian 386 1000 mg/kg bw, K C: diazepam 3 mg/kg bw. Data of these experiments is taken from Table 2 which presents results of further experiments. **po0.05, ***po0.001 vs. corresponding control group, Student’s t-test.
effect in the EPM procedure after oral application (Fig. 5). The ratio of open and closed arms regarding time and number of entries was significantly increased in favour of the open arms after treatment with 100 and 250 mg/kg bw. Although mice treated with the highest dose (500 mg/kg bw) showed anxiolytic-like behavior as well, open arm exploration was less pronounced in comparison to the lower dose levels. Only the percentage of open arm entries was affected with statistical significance. Thus 100 and 250 mg/kg bw appeared to prove maximal effective. Considering the results gained with the commercially available extracts, only the preparation extracted with 45% methanol m/m (VAL H 45 M) showed anxiolytic activity (Fig. 6). 250 mg/kg bw of this extract led to a significant increase of the percentage of time spent in the open arms and of open arm entries, while both lower dosages (25 and 100 mg/mg bw) only tended to result in an enhancement of these parameters. The preparation VAL H 70E, that was obtained using 70% ethanol v/v as extraction solvent, left these parameters unaffected within the same tested dose range of 25, 100 and 250 mg/kg bw (Fig. 7).
Values are means7SEM expressed in seconds (duration of sleeping time) of 12–15 animals in each group.*po0.05, **po0.01 and ***po0.001 vs. control group, post-hoc Fisher-PLSD.
Forced swimming test including additional open field test The special extract phytofin Valerian 368 proved to be antidepressant since immobility time, as shown in Fig. 8A, was significantly decreased in the FST 16 days after daily treatment with 2 125 mg/kg bw (8:00 a.m. and 20:00 p.m.). 2 50 mg/kg bw had a similar effect, but statistical significance was not reached. A negligible prolongation of immobility time was observed at the lowest dose level (2 12.5 mg/kg bw). In contrast, extract VAL TE 35E, which was used as base material for phytofin Valerian 368, showed no effect on this parameter at the tested dose level of 2 125 mg/kg bw per day. The difference between fraction and extract at this dosage reached statistical significance.
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To verify if the observed changes in immobility times may be due to unspecific, compound-related effects on locomotor activity, animals were tested in an open field for alterations in activity 2 days before the FST procedure. Since none of the treatments induced significant changes in locomotor activity (Fig. 8B), the pronounced reduction of immobility time after treatment with phytofin Valerian 368 (2 125 mg/kg bw) can be considered as a result of an antidepressant property of this preparation.
duration of anaesthesia [s]
Horizontal wire test As shown in Fig. 9A and B neither the extract VAL TE 35E nor the special extract phytofin Valerian 368 showed myorelaxant activity in the horizontal wire test in dosages up to 1000 mg/kg bw. Similar to the control group, none of the valerian treated mice failed to grasp the steelbar with at least one hind paw within a time period of 1 h. By contrast, treatment with 2 mg/kg bw diazepam led to a distinct impairment of test performance. Impairment occurred 10 min after administration, reached its maximum after 20 min, diminished afterwards and was hardly detectable at the end of the procedure.
* **
240
9
200
*
160
Discussion
120
The present study evaluated the pharmacological profile of different extracts derived from Valeriana officinalis L. s.l. including two commercially available extracts and the newly developed preparations VAL SE 35E and phytofin Valerian 368. Therefore tests for sedative, anxiolytic, antidepressant as well as for myorelaxant properties were conducted in rodents. None of the different extracts revealed sedative properties after acute administration. Neither the valerian preparations VAL SE 35E and special extract phytofin Valerian 368 nor the commercially available extracts VAL H 70E and VAL 45 M induced a reduction of locomotor activity or a prolongation of ether-induced anaesthesia. Since the tested dosages were up to
80 40 0
ctrl.
1
ctrl. 0.2
ctrl. 1000 mg/kg BW
water / saline (p.o. / i.p.) VAL H 70E (p.o.)
ctrl. 500
CPA (i.p.)
Diazepam (i.p.)
VAL H 45M (p.o.)
ctrl. 500
phytofin Val. 368 (p.o.)
Fig. 3. Mean (7 S.E.M., 12–15 NMRI mice per group) duration of sleeping time 30 min after application of the test solutions. This figure constitutes an exemplary composition of data derived from 5 experiments which are in full detail presented in Table 3; *po0.05, ***po0.001 vs. corresponding control group, post-hoc Fisher-PLSD.
45
35 30 25
*
*
20 15
35 30 25 20 15
10
10
5
5
0
ctrl.
2
* **
40 open arm entries [%]
40 time on open arms [%]
45
* **
100
250
500
mg/kg BW water
diazepam
0
ctrl.
2
100 250 500 mg/kg BW
VAL TE 35E
Fig. 4. Mean (7 S.E.M., 12–14 NMRI mice per group) percentage time spent on open arms and percentage open arm entries in the EPM 60 min after oral application of the test solutions. *po0.05, ***po0.001 vs. corresponding control group, Fisher-PLSD post hoc test.
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30
30
20
25
* **
open arm entries [%]
25 time on open arms [%]
* **
* **
* **
15 10
* **
15
*
10 5
5 0
* ** 20
ctrl.
2
100
250
0
500
ctrl.
2
water
100
250
500
mg/kg BW
mg/kg BW diazepam
phytofin Val. 368
Fig. 5. Mean (7 S.E.M., 12–14 NMRI mice per group) percentage time spent on open arms and percentage open arm entries in the EPM 60 min after oral application of the test solutions. *po0.05, ***po0.001 vs. corresponding control group, Fisher-PLSD post hoc test.
**
25 20 15 10
*
25 20 15 10 5
5 0
**
30
** open arm entries [%]
time on open arms [%]
30
0 ctrl.
2
25
100
250
mg/kg BW water
diazepam
ctrl.
2
25 100 mg/kg BW
250
VAL H 45M
Fig. 6. Mean (7 S.E.M., 12–14 NMRI mice per group) percentage time spent on open arms and percentage open arm entries in the EPM 60 min after oral application of the test solutions. *po0.05, **po0.01 vs. corresponding control group, Fisher-PLSD post hoc test.
1000 mg/kg bw and, considering the motor experiments, a 2 h test period with consecutive measurements was chosen, a sedative action of valerian seems to be unlikely. Additionally, even after subacute treatment (19 days) with phytofin Valerian 368 and VAL H 70E (2 500 mg/kg bw daily) an alteration of motor activity and of duration of sleeping time indicating a sedative action could not be observed (data not shown). The absence of sedation is partially contradictory to results obtained in other studies using similar behavioral paradigms. Wagner, et al. (1980) showed a valerian
tincture (DAB 7) to slightly reduce locomotor activity in mice after gavage of 1000 mg/kg bw and, after a storage period of 9 month, also of 100 mg/kg bw. When assessing these results, the absence of statistical specifications must be taken into consideration. Similar difficulties occur with the study of Leuschner, et al. (1993) that showed a maximum reduction of 28.6% and 35.9% in motor activity 120 min after oral administration of 20 and 200 mg/kg bw of an aqueous alkaline extract, since no information regarding statistical significance is given.
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50
50
* **
* **
40
40 open arm entries [%]
time on open arms [%]
11
30
20
10
30
20
10
0
0 ctrl.
2
25 100 mg/kg BW
250
water
ctrl.
2
25 100 mg/kg BW
250
VAL H 70E
diazepam
Fig. 7. Mean (7 S.E.M., 12–14 NMRI mice per group) percentage time spent on open arms and percentage open arm entries in the EPM 60 min after oral application of the test solutions. ***po0.001 vs. corresponding control group, Fisher-PLSD post hoc test.
180
140
120 *
100 80 60 40
100 80 60 40 20
20 0
120
#
140
line crossings / 5 min
duration of immobility [s]
160
ctrl.
25
100
250
250
0
mg /kg BW water
phytofin Val. 368
ctrl.
25
100 250 250 mg/kg BW
VAL TE 35E
Fig. 8. Mean (7 S.E.M., 12 Sprague-Dawley rats per group) duration of immobility in the forced swimming test (A) and number of line crossings per 5 min in the open filed test (B) after subacute, oral treatment (FST ¼ 16 days, OFT ¼ 14 days, 2 daily) with the test solutions. *po0.05 vs. corresponding control group, #po0.05 vs. phytofin Valerian 368 (250 mg/kg bw), Fisher-PLSD post hoc test.
Although in some studies an extended anestheticinduced sleeping time after administration of valerian preparations or isolated constituents like hesperidin was demonstrated (Hiller and Zetler, 1996; Leuschner, et al., 1993; Marder, et al., 2003), the relevance with regard to sedative properties of valerian remains unclear. All these experiments were carried out using barbiturates for anesthetisation. Since barbiturates have been proven to interfere with CYP P-450 enzymes (Kakinohana et al.,
1998) an increased sleeping time due to interactions of valerian with the barbiturate metabolism in mice cannot be excluded. Though in men no relevant interactions of valerian with CYP 3A4, 1A2, 2D6, 2E1 enzymes were shown (Donovan et al., 2004; Gurley et al., 2005), interactions might nevertheless occur in rodents due to differences in metabolism. Considering results obtained in clinical trials investigating valerians effect on vigilance, psychomotor and
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A mg/kg BW
40
diazepam 2 mg/kg
500
1000
30 20 10
VAL TE 35E
100
impaired mice [%]
50
water
0 0
10
B mg/kg BW
diazepam 2 mg/kg
500
20 10
water phytofin Val. 368
1000
30
60
100
impaired mice [%]
40
20 30 40 50 minutes after application
0 0
10
20 30 40 50 minutes after application
60
Fig. 9. Percentage of impaired NMRI mice (10 per group) in the horizontal wire test after oral application of water, diazepam and VAL TE 35E (A) or phytofin Valerian 368 (B).
cognitive functions as well as on hang over phenomenons, it can be ascertained that findings of our in vivo experiments (locomotor activity, ether-induced anaesthesia) comply with these data. In four out of six of those studies in healthy volunteers no hints on sedating properties of common valerian preparations were observed after acute and (partially) long-term treatment using dosages up to 1800 mg (Glass et al., 2003; Gutierrez, et al., 2004; Hallam, et al., 2003; Kuhlmann, et al., 1999). In contrast, Gerhard et al. (1996) reported, a slight but significant decrease in vigilance in a group treated with a valerian syrup. However, it has to be annotated that this study has been conducted without blinding the syrup group. In a second study an increase in self-rated tiredness after acute treatment with 1200 mg of a valerian extract was shown (Schulz et al., 1998), but this dosage clearly exceeds the recommended dose level and statistical analysis of these data was not performed. Moreover, valerian-induced changes in quantitative EEG, which were similar to placebo treatment in many aspects, were interpreted as a hint on sedative properties of the extract. Contrarywise, the authors themselves admit a lack of specificity of the EEG method, since changes in EEG profile do not seem to be valid predictors of sedation, tranquilisation or anxiolysis. To summarize, considering all results obtained in clinical trials and in in vivo pharmacological experiments there is hardly evidence of sedative activity of common
valerian preparations. Against this background, the commonly reported interference of valerian extracts or of isolated constituents with the adenosine A1 receptor or binding sites at the GABAA receptor may not play a decisive role in mediating sleep-enhancing effects of valerian in vivo. In accordance with published data (Karcz-Kubicha et al., 2003; Mandryk et al., 2005; Patterson et al., 2005) it was demonstrated in our own studies that GABAA- or adenosine A1-agonistic compounds like diazepam and CPA respectively reduce locomotor activity and/or extend the duration of narcosis as a consequence of the expected central depression. Due to the distinct comorbidity between sleep disorders and psychiatric diseases like anxiety or mood disorders (Schramm et al., 1995) we carried out behavioral experiments in mice and rats to test valerian for anxiolytic and antidepressant activity that may account for its proved sleep-enhancing properties in humans. Using the elevated plus maze test the ethanolic extract VAL TE 35E and the special extract phytofin Valerian 368 that was derived from it, as well as the commercially available methanolic extract VAL H 45M displayed a pronounced anxiolytic activity. In contrast, the also commonly used ethanolic extract VAL H 70E was not shown to be active in this test procedure. The maximum effect of VAL TE 35E and phytofin Valerian 368 seems to be reached at a dosage of 100 mg/kg bw, using higher dose levels exploration of the open arms was not further increased. Interestingly, 500 mg/kg bw of the special extract were less active compared to the lower dose levels. This phenomenon could be due to an (inverted) u-shaped dose response curve that is already known for other compounds (Borsini, 1995; Gries et al., 2005). Anxiolytic-like effects of valerian or of isolated constituents have previously been observed by Hiller and Kato (1996) and Marder et al. (2003) that showed the ethanolic extract STEI Val and the flavonoid 6-Methylapigenine to be active in the elevated plus maze paradigm. Additionally, several observations indicate a putative efficacy of valerian preparations in the treatment of anxiety and stress-related symptoms. In a comparative study a 4 week treatment with a 70% v/v ethanolic extract led, amongst others, to a reduction of the Hamilton-scores similar to the oxazepam treated group (Dorn, 2000). Furthermore, after a treatment period of 1 week using the same extract a diminished response to mental stress under laboratory conditions was reported by Cropley et al. (2002). Particularly with regard to clinical findings using ethanolic extracts in which repeatedly an initial lack of efficacy associated with a time-dependent development of sleep-enhancing properties (Donath, et al., 2000; Kamm-Kohl and Jansen, 1984; Vorbach, et al., 1996) was observed, we tested VAL TE 35E and phytofin
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Valerian 368 for putative antidepressant activity. A lack of efficacy at the beginning of treatment is well known in the treatment of mood disorders with synthetic antidepressant compounds (Aktories et al., 2005). In contrast to its primary extract VAL TE 35E, phytofin Valerian 368 reduced immobility time in the forced swimming test after subacute treatment. Due to the absence of compound-related alterations in motor activity the effect can be considered as a result of an antidepressant property of this extract. This additional antidepressant effect is obviously a result of the manufacturing process (column adsorption) that led to a diminished content of lipophilic compounds and can be arised from an alteration regarding the quantitative composition of phytofin Valerian 368. The potential to impair psychomotor functions is one of the most common side effects of widely-used sleep aids (Aktories, et al., 2005). To verify the effect of our extracts on muscle strength we used the horizontal wire test in mice. None of the tested extracts (VAL TE 35E and phytofin Valerian 368) showed myorelaxant activity in dosages up to 1000 mg/kg bw. Against the background that enhanced GABAA-ergig transmission, the known mechanism for benzodiazepines, is associated with a loss of muscle strength, these findings affirm that valerian-induced CNS-related effects might not be caused by interference with the GABAA receptor.
Conclusion Our investigations are partially contradictory to the common view on the pharmacological activity of valerian preparations. The clinical efficacy of valerian is generally accepted, and mostly sedation is assumed to account for improvement of nervousness and sleep disorders. However, in our investigations neither commonly used (45% methanol m/m, 70% ethanol m/m) nor newly prepared extracts (35% ethanol v/v, special extract phytofin Valerian 368) derived from Valeriana officinalis L. s.l show sedative activity in mice, since locomotor activity and the duration of ether-induced anaesthesia were not affected after acute or subacute treatment. These observations are in sound accordance with clinical studies that predominantly failed to reveal a valerian-induced influence on vigilance, psychomotor and cognitive performance. Instead, the special extract phytofin Valerian 368, its 35% v/v ethanolic primary extract VAL SE 35E and the commercially available 45% methanolic (m/m) preparation were proved to exhibit a pronounced anxiolytic effect in the elevated plus maze paradigm following acute administration. Furthermore, after a pretreatment period of 16 days only phytofin Valerian 368 showed activity in the forced swimming test indicating anti-
13
depressant properties, whereas its primary extract was not effective. Obviously the observed antidepressant and anxiolytic activity may not be mediated by lipophilic constituents of low molecular weight, e.g. valerenic acids, since these compounds were removed from phytofin Valerian 368 to a considerable extent. Moreover, results obtained in the horizontal wire test after gavage of the 35% ethanolic extract and its derived special extract phytofin Valerian 368 justify the exclusion of myorelaxant properties of these preparations. It is suggested that instead of sedative effects, anxiolytic as well as antidepressant properties of valerian, effects in particular demonstrated for the special extract phytofin Valerian 368, may account for the clinically proved sleep-enhancing effects of valerian. Considering these findings, a retrospective analysis of clinical data in respect of a possible correlation between psychiatric disorders and efficacy of valerian in treating mild sleep disorders may appear reasonable.
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Phytomedicine 15 (2008) 2–15 www.elsevier.de/phymed
Extracts of Valeriana officinalis L. s.l. show anxiolytic and antidepressant effects but neither sedative nor myorelaxant properties Miguel Hattesohla,, Bjo¨rn Feistelb, Hartwig Sieversc, Romanus Lehnfeldc, Mirjam Heggera, Hilke Winterhoffa a
Department of Pharmacology and Toxicology, Universita¨tsklinikum Mu¨nster, Westfa¨lische Wilhelms-Universita¨t Mu¨nster, Domagkstr. 12, 48149 Mu¨nster, Germany b Finzelberg GmbH & Co. KG, Koblenzer Str. 48-56, 56626 Andernach, Germany c PhytoLab GmbH & Co. KG, Dutendorfer Str. 5-7, 91487 Vestenbergsgreuth, Germany
Abstract Extracts of Valeriana officinalis L. s.l. are used for treating mild sleep disorders and nervous tension. Despite intensive research efforts, the pharmacological actions accounting for the clinical efficacy of valerian remain unclear. Thus, it was the aim of this study to evaluate CNS-related effects of different valerian extracts using behavioral paradigms (mice and rats). Following oral administration two commercially available preparations (extraction solvents: 45% methanol m/m and 70% ethanol v/v), a 35% ethanolic v/v extract and a refined extract derived from it (patented special extract phytofin Valerian 368) were tested for sedative (locomotor activity, ether-induced anaesthesia) and anxiolytic (elevated plus maze) activity. Using the forced swimming and the horizontal wire test the latter two extracts were additionally tested for antidepressant and myorelaxant properties. Up to maximum dosages of 500 or 1000 mg/kg bw none of the valerian extracts displayed sedative effects. Neither spontaneous activity was reduced nor the duration of ether-induced narcosis was prolonged. In contrast, results obtained in the elevated plus maze test revealed a pronounced anxiolytic effect of the 45% methanolic and 35% ethanolic extract as well as of phyotofin Valerian 368 in a dose range of 100–500 mg/kg bw. Additionally and different from its primary extract (35% ethanolic extract) phytofin Valerian 368 showed antidepressant activity in the forced swimming test after subacute treatment. Myorelaxant effects were not observed in dosages up to 1000 mg/kg bw. Due to these findings it is proposed that not sedative but anxiolytic and antidepressant activity, which was elaborated particularly in the special extract phytofin Valerian 368, considerably contribute to the sleep-enhancing properties of valerian. r 2007 Elsevier GmbH. All rights reserved. Keywords: Valeriana officinalis L.s.l.; Elevated plus maze; Anxiolytic; Sedative; Locomotor activity; Antidepressant; Myorelaxant
Introduction
Corresponding author. Tel.:+49 251 8355514; fax: +49 251 8355501. E-mail address: [email protected] (M. Hattesohl).
0944-7113/$ - see front matter r 2007 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2007.11.027
Extracts derived from roots of Valeriana officinalis L. s.l. are used for the treatment of mild sleep disorders and nervous tension. Only recently the therapeutic efficacy of valerian has been confirmed by the European Medicines Evaluation Agency in an actual monograph
ARTICLE IN PRESS M. Hattesohl et al. / Phytomedicine 15 (2008) 2–15
(Committee on Herbal Medicinal Products (HMPC/ EMEA), 2006). Valerian preparations contained in commercially available products are extracted with water, water/methanol or water/ethanol mixtures. In several placebo-controlled clinical studies an improvement of sleep-related parameters following treatment with aqueous or ethanolic extracts was demonstrated. For instance, acute administration of an aqueous extract reduced sleep latency (Balderer and Borbely, 1985; Leathwood et al., 1982). Using subacute treatment regimes, an extract made with 70% v/v ethanol affected different sleep parameters in a positive manner as well (Donath et al., 2000; Vorbach et al., 1996). Furthermore an improvement of sleep quality comparable to treatment with 10 mg oxazepam was observed for the same ethanolic extract in two comparative studies (Dorn, 2000; Ziegler et al., 2002). It is commonly assumed that efficacy of valerian preparations in sleep disorders could be due to putative sedative properties of these extracts. However, results of studies which tested valerian for the proposed sedative effects and its influence on vigilance in healthy volunteers are contradictory to this hypothesis (Gutierrez et al., 2004; Hallam et al., 2003; Kuhlmann et al., 1999). None of the tested ethanolic extracts (70% v/v) revealed sedative properties since neither psychomotor nor cognitive nor subjective parameters were affected significantly in these trials. Many efforts have been made to elucidate the pharmacological profile of different valerian extracts as well as of isolated constituents. In vivo studies provided inconsistent results concerning sedative, anticonvulsant, myorelaxant and other effects indicating central mechanisms of action. For instance, extracts made with 70% ethanol varied in their capability to affect motor activity in mice. While Hiller and Zetler (1996) observed no effect locomotor activity was weakly reduced in a study of Wagner et al. (1980). Testing an aqueous alkaline extract, Leuschner et al. (1993) observed a reduction in spontaneuos activity as well. Other in vivo experiments revealed further pharmacological effects of common valerian extracts like a significant shortening of sleep latency in sleep-disturbed rats after treatment with a 70% ethanolic preparation (Shinomiya et al., 2005) or anticonvulsant properties using a 70% ethanolic or an aqueous alkaline extract (Hiller and Zetler, 1996; Leuschner, et al., 1993). Furthermore Hiller and Kato (1996) showed a pronounced anxiolytic effect of the ethanolic extract STEI Val using the elevated plus maze test. Taking into account that type of extract, doseregimes, the method of application and the used ethological paradigms varied between all studies it becomes obvious that precise conclusions on pharmacological actions of valerian may hardly be drawn. Moreover some studies preferred intraperitoneal admin-
3
istration, an application mode which is of questionable relevance for orally administered plant extracts. In trying to determine the active constituents of valerian, many single compounds have been tested in distinct in vivo experimental procedures. There is certain evidence that valerenic acid and flavonoids like hesperidin and 6-methylapigenin may contribute to central effects of extracts derived from Valeriana wallichii. There is no evidence, however, that both flavonoids occur in detectable amounts also in Valeriana officinalis. Valerenic acid was shown to possess anticonvulsant properties (Hiller and Zetler, 1996) while hesperidin proved to be sedative since locomotor activity was reduced and thiopental-induced sleeping time was prolonged (Marder et al., 2003). Additionally, in the latter study 6-methylapigenin was shown to be active in the elevated plus maze procedure. Whilst evaluating these results it must be considered that all studies used intraperitoneal injection as the route of administration. Valepotriates, which were also discussed to be determinant for central actions of valerian (Andreatini and Leite, 1994; Andreatini et al., 2002), are detectable at trace levels or not at all in recent drug preparations (ESCOP, 2003; Schulz and Ha¨nsel, 2004). Other compounds that were often associated with pharmacological actions of valerian, like the essential oil or single components of it (Hendriks et al., 1981), were mostly tested in dosages clearly exceeding the range that actually can be reached when administering any extract preparation. Thus, for an evaluation of the pharmacological profile of valerian it does not seem valuable to include results from those studies. In vitro studies testing extracts or isolated constituents provided certain evidence for an involvement of particularly the GABAA-system in the psychotropic effects of valerian (Balduini and Cattabeni, 1989; Bodesheim and Ho¨lzl, 1997; Cavadas et al., 1995; Dietz et al., 2005; Fernandez et al., 2004, 2005; Ferreira et al., 1996; Marder, et al., 2003; Mennini and Bernasconi, 1993; Ortiz et al., 1999, 2006; Riedel et al., 1982; Santos et al., 1994a–c; Simmen et al., 2005; Wasowski et al., 2002; Yuan et al., 2004). In addition, the adenosinesystem may also account for central actions since in some studies an interaction of valerian extracts as well as of an olivil derivate at the adenosine A1-receptor was observed (Balduini and Cattabeni, 1989; Mu¨ller et al., 2002; Schumacher et al., 2002). However, despite intensive research efforts, the pharmacological actions accounting for the proved efficacy of valerian in mild sleep disorders remain unclear. Thus, it was the aim of the present study to evaluate the pharmacological profile of extracts prepared with different solvents including both, commercially available extracts and newly developed extracts, by using in vivo paradigms for detecting sedative, anxiolytic, antidepressant and myorelaxant properties.
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Materials and methods Chemicals, plant material and treatment of animals Diazepam was purchased from ratiopharm (Ulm, Germany), N6-Cyclopentyladenosine (CPA) and Caffeine from Sigma-Aldrich GmbH (Taufkirchen, Germany). All tested valerian preparations, VAL SE 35E, VAL TE 35E, the patented special extract phytofin Valerian 368 and the commercially available extracts VAL H 70E and VAL H 45 M were provided by Finzelberg (Andernach, Germany) and derived from dried, cutted roots of Valeriana officinalis L. s.l. (Ph. Eur.) by percolation with water, ethanol/water 35% or 70% v/v or methanol/water 45% m/m as shown in Table 1. The extraction was carried out by exhaustive percolation at a temperature of 50 1C. After vacuum evaporation the obtained soft extracts were spray-dried under addition of 25% or 30% maltodextrine (Ph. Eur.) respectively to avoid agglomeration. Phytofin Valerian 368 was prepared from the soft extract VAL SE 35E using column adsorption technique. Therefore the soft extract was resolved in water until a percentage of indissoluble compounds of 10% (w/w) was achieved. After separating the precipitate by filtration the remaining dilution was applied to a column containing the polymeric adsorbent AMBERLITEs XAD-4 (Rohm and Haas Company, Philadelphia, USA) as packing material. This non ionic crosslinked Divinylbenzene Copolymer adsorbs hydrophobic molecules of low molecular weight (like valerenic acids) from polar solvents. Finally the purified aqueous phase that passed the column was evaporated again and transformed into a dry extract by spray drying and adding 30% maltodextrine. Due to the usage of solvents of different polarity and to differences in subsequent processing the valerian
Table 1.
preparations showed distinctions regarding the native drug:extract ratio and the amount of valerenic acids that were determined by HPLC (Table 1). The DC-chromatogramm in Fig. 1 demonstrates a clear decline of lipophilic constituents in the extract phytofin Valerian 368 compared to its primary extract VAL SE 35E (Fig. 1). None of the preparations contained valepotriates. For animal experiments diazepam and all extracts were dissolved in tap water and administered orally (p.o.). Except when testing for an influence on the duration of ether-induced narcosis diazepam was injected intraperitoneally (i.p.). N6-Cyclopentyladenosine (CPA) and caffeine were prepared with saline and applied intraperitoneally (i.p.). All test solutions were given in a volume of 10 ml/kg bodyweight.
Fig. 1. TLC-fingerprint chromatogram on polar constituents in valerian extracts acc. Finzelberg method after derivatization with Anisaldehyd–Sulfur acid-mix in white light (eluent:dichloroethane (50):acetic acid (25):methanol (15):water (10), v/v/v/v).
Specifications of valerian extracts
Type of extract
Code name
Extraction solvent
Notes to extracts
Drug: extract ratio
Maltodextrine (%)
Total valerenic acids (%)
Soft extract
VAL SE 35E
Ethanol 35% (v/v)
Primary extract
2–4:1
–
0.29
Purified, spraydried and powdered special extract
Phytofin Valerian 368 (patented)
Ethanol 35% (v/v)
Prepared from VAL SE 35E by column adsorption technique
3–5:1
30
0.13
Spray-dried, powdered extracts
VAL TE 35E
Ethanol 35% (v/v)
Prepared from VAL SE 35E
2–4:1
30
0.29
VAL H 45M
Methanol 45% (m/m)
Represents standard quality
5–8:1
25
0.38
VAL H 70M
Ethanol 70% (v/v)
Represents standard quality
3–6:1
30
0.31
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The indicated dosages of extract preparations refer to the native plant extract.
Animals Female NMRI mice (18–20 g, Charles River Laboratories, Sulzfeld, Germany) were housed in groups of six animals per cage, male Sprague-Dawley rats (150–170 g, Charles River Laboratories, Sulzfeld, Germany) in groups of two. All animals were kept in a 12 h light/dark cycle, with lights off at 6:00 p.m., at a constant temperature of 2571 1C and with free access to food (Altromin 1324, Altromin, Lage, Germany) and tap water. For experimental testing the animals were randomly assigned to control or treatment groups. Experiments were carried out after the animals had adapted to the laboratory conditions for at least 1 week. The procedures used comply with the European Community’s Council Directive of 24 November 1986 (86/609/EEC) and were officially approved by the local committee on animal care (Regierungspra¨sident Mu¨nster, AC/2004).
Locomotor activity Activity experiments were carried out with female NMRI mice (n ¼ 8/group) by using infrared (IR) technique. The apparatus containing 16 macrolon cages was placed in a sound attenuated room and darkened by a roller blind during measurement. For experimental testing mice were singly introduced into the cages and initially allowed to adapt to experimental conditions for 30 min. Immediately after treatment (p.o. or i.p.) locomotor activity was recorded continuously by passive infrared (IR) sensors. The number of IR-impulses was registered accumulatively within consecutive intervals of 10 min (counts per 10 min) over a total period of 2 h. A reduced locomotor activity represents a sedative effect whereas stimulant properties are indicated by an increased motility.
Ether-induced anaesthesia An influence of test compounds on the duration of ether-induced narcosis was tested in female NMRI mice (n ¼ 12–15 per group) 30 or 60 min after treatment (p.o. or i.p.). For anesthetisation 3.5 ml diethylether were given into a 5 l glass tank. After a vaporisation period of 1 min six animals were simultaneously placed into that tank for 2 min. Subsequently mice were taken out of the ether atmosphere and sleeping time was determined by registration of the time until the righting reflex was completely recovered. A prolongation of ether-induced narcosis is caused by central depressant drugs.
5
Elevated plus maze The procedure, first described by Pellow et al. (1985) and Lister (1987) as a behavioral model to measure anxiety in rats and mice respectively, was conducted with female NMRI mice (n ¼ 12–14 per group) in a sound attenuated room under high illumination. The elevated plus maze consisted of respectively two black-surfaced open (30 cm long and 5 cm wide) and enclosed arms (30 cm long and 5 cm wide with walls of 15 cm height) that extended from a central platform (5 cm 5 cm). Open and enclosed arms were arranged opposite to each other and the whole maze was elevated to a height of 40 cm. 60 min after treatment mice were placed in the central square of the maze, facing one of the enclosed arms. During a 5 min test period animals were allowed to freely explore the environment. Behavior was recorded by video camera and the time mice spent in the open and closed arms as well as the number of open and closed arm entries was evaluated afterwards by a blinded observer. An entry was defined as placement of all four paws into an arm. An increase in the percentage of time spent in the open arms (topen arms/topen+closed arms 100) and in the percentage of open arm entries (nopen arms/nopen+closed arms 100) indicate an anxiolytic effect. To avoid the phenomenon of one trial tolerance (File et al., 1990) each animal was only used once. After each trial the test arena was cleaned carefully with water containing detergents.
Forced swimming test (FST) including additional open field test The forced swimming test, first published by (Porsolt et al., 1977, 1978) as a paradigm to detect compounds with antidepressant activity, was performed with male Sprague-Dawley rats. The animals (n ¼ 12 per group) received pre-treatment with the test solutions (p.o.) two times daily (8:00 a.m., 20:00 p.m.) over a period of 16 days. One day before the test the rats experienced a 15 min lasting pretest session, in which the animals were singly introduced into a plexiglass cylinder that contained a 17–21 cm (depending on the bodyweight) high column of water at 2571 1C. Experimental testing was conducted 24 h later by exposing the rats to the same test conditions for 5 min 5 h after last drug administration. Behavior was recorded by video camera. The cumulative time rats persisted in an immobile position (despair behavior) was evaluated afterwards by a blinded observer. A rat was judged to be immobile whenever it remained floating in the water in contrast to motor activities that represented attempts to escape from the situation. The time rats spent making small movements to keep the head above the water was assigned to time of
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immobility. A shortened immobility time indicates an antidepressant effect. To exclude false positive results due to compoundrelated stimulating effects on motility, locomotor activity was determined 2 days (day 14 of drug treatment) before FST performance by using the open field paradigm. The open field test, first described by Hall (1934), was carried out in a sound attenuated room with a round arena (diameter: 70 cm, height of surrounding wall: 30 cm) whose grey base area was divided in 18 equal sections. Rats were placed individually in the center of the arena and behavior was video recorded for 5 min. Afterwards a blinded observer scored the number of line crossings (crossings per 5 min) as an index of locomotor activity. After each trial the test arena was washed carefully with detergent solution. FST and open field experiments were carried out between 1:00 and 5:00 p.m.
Horizontal wire test To detect potential myorelaxant effects the horizontal wire test according to the method published by Boissier and Simon (1960) was used. The test apparatus consisted of a steel bar (2 mm diameter, 15 cm length) that was fixed in horizontal position 20 cm above the ground. In a first session an appropriate collective of female NMRI mice was selected. Mice that, when lifted by the tail and allowed to grasp the wire with both forepaws, failed to grasp the steel bar with at least one hindpaw within 5 sec were excluded from further experiments. Immediately after oral treatment the selected animals (n ¼ 10 per group) were tested for myorelaxant activity consecutively every 10 min over a period of 1 h. Mice that had lost the ability to grasp the wire with at least one hindpaw were considered to exhibit impaired muscle strength. Results are expressed as percentage of impaired animals.
Data analysis and statistics All statistical procedures were performed by using the STATVIEW statistical software package, version 5.0 (SASR, USA). Except in the case of motility experiments data analysis was carried out by analysis of variance (ANOVA) with the Fisher-PLSD post hoc test for multiple comparisons. Data obtained from activity experiments were statistically evaluated by using unpaired Student t-test. All data are expressed as MEANS 7SEM. Only the values gained from horizontal wire experiments are indicated as percentages. Statistical significance was set at po0.05 (*po0.05, **po0.01, ***po0.001).
Results Locomotor activity Results of motility experiments are shown in Table 2 and Fig. 2. The benzodiazepine diazepam reduced locomotor activity significantly in a dose dependent manner. In both dosages the effect occurred 20 min after gavage while 4 mg/kg bw evoked a more pronounced and longer lasting reduction of motor activity. In contrast, dosages of 3.5 and 5 mg/kg bw of the stimulant caffeine produced a significant increase in locomotor activity starting 30 min after intraperitoneal injection. As expected the higher dose revealed a more distinct as well as persistent effect. Contrary to these reference compounds neither the valerian preparations VAL SE 35E and phytofin Valerian 368 nor the commercially available extract VAL H 70E showed significant changes in locomotor activity in dosages of 100, 250, 500 or 1000 mg/kg bw within the test period of 2 h immediately after oral application. Only the methanolic extract VAL 45 M led to an alteration of motility. 90 min after oral administration of 100 mg/kg bw an increased activity was observed. However, higher doses of the same extract (250, 500 mg/kg bw) did not show stimulant properties.
Ether-induced anaesthesia As shown in Table 3 and Fig. 3 diazepam prolonged the duration of ether-induced narcosis significantly 30 min after intraperitoneal injection, with a pronounced effect at the higher dose (1.5 mg/kg bw). An extended sleeping time was also observed after administration of N6-Cyclopentyladenosine. While 0.2 mg/kg bw of the selective agonist at the adenosine A1 receptor led to a significant prolongation, 0.1 mg/kg bw only tend to result in an increased sleeping time. However, the lowest dose (0.05 mg/kg bw) of this reference did not affect this parameter. While the central depressant drugs, diazepam and CPA, produced an extension of ether-induced anaesthesia, none of the valerian preparations influenced sleeping time significantly 30 or 60 min after gavage of 100, 250 and 500 mg/kg bw respectively.
Elevated plus maze Diazepam (2 mg/kg bw), that was used as reference in all elevated plus maze tests, always exhibited anxiolytic activity after gavage since the percentage of time spent in the open arms as well as the percentage of open arm entries was significantly increased in each experiment (Figs. 4–7).
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Table 2.
7
Locomotor activity
Treatment
Minutes after application 10
20
30
60
90
120
27537162 22487151 18317359 15807184
23817183 12067283** 20337283 100731***
2145794 9027235*** 20137414 77720***
12097249 8777328 12077448 171748***
9027297 4397252 12747548 15.4711***
29722 72772 18267470 2497146***
19607370
14307350
11027329
5267352
7917471
5877391
25837249 15517318
22887389 18627279
24277315* 14777320
17327373* 4847330
16117464 5837322
11567445 3677221
19407405
21667245
26217151***
22237185***
20117214***
14987302***
VAL SE 35E (p.o.) Control 21387244 100 mg/kg 24757270 Control 22857189 250 mg/kg 25967136 Control 25277271 500 mg/kg 23947248
20907229 21127254 19337213 14837137 18667227 22057371
20397207 18527300 17167200 20737297 16367394 18407427
14347347 14927287 9877332 10737304 10967442 15937379
7117271 7567384 8727329 7737356 5277455 2717246
4457249 2987271 5947334 5897345 7267423 3207204
Phytofin Val. 368 Control 100 mg/kg Control 500 mg/kg Control 1000 mg/kg
(p.o.) 23127115 24327188 23927140 24197224 22627184 20157276
23667152 23817170 23447389 24547319 21697244 19697384
25437189 21677258 20567322 24567281 22027212 18527405
21287292 21707305 15107271 18647265 15717310 11307447
11767424 17327345 10357341 15677472 13177304 11347440
5437328 10717446 2497119 11437438 10237429 5137213
VAL H 70E (p.o.) Control 23897276 100 mg/kg 20257242 Control 19827209 250 mg//kg 25207292 Control 24267210 500 mg/kg 24587195
20557244 19557235 16737224 22167165 23127261 19147343
18797322 15347199 16957253 22977438 18797153 18617304
11737353 5847247 7557348 16117555 10987502 12007468
5307264 9627310 6107399 11197471 3197317 6187277
6527383 3827220 6397297 4027207 5727335 7867290
VAL H 45M (p.o) Control 22757305 100 mg/kg 18987209 Control 26437316 250 mg/kg 24257282 Control 23247103 500 mg/kg 25807326
20247218 16877100 24207369 22417322 19967185 23107376
18517208 21797211 23017383 21977336 20207308 24597367
10287330 15337181 13127328 15757472 14557286 18147339
4197208 12417209* 9637303 12187490 16857483 6567347
6047242 5257322 3027202 8377480 6767551 4577210
Diazepam (p.o.) Control 3 mg/kg Control 4 mg/kg Caffeine (i.p.) Control (saline) 3.5 mg/kg Control (saline) 5 mg/kg
Values are means7SEM expressed in counts per 10 min of 8 animals in each group. *po0.05, **po0.01 and ***po0.001 vs. corresponding control group, Student’s t-test.
An increased exploration of the open arms was also observed 60 min after oral administration of the valerian preparation VAL TE 35E, that was prepared with 35% v/v ethanol (Fig. 4). While the percentage of time in the open arms was increased significantly at doses of 100 and 500 mg/kg bw, the parameter concerning the number of open arm entries was affected without
statistical significance. A dosage of 250 mg/kg bw hardly caused an increase of time spent in the open arms and did not influence the ratio of open and closed arm entries. The special extract phytofin Valerian 368, that was derived from the soft extract VAL SE 35E using column absorption technique, exhibited a distinctive anxiolytic
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Table 3.
3000 2500
Duration (s) of ether-induced anaesthesia
Treatment
2000 1500
Minutes after application 30
60
Diazepam (i.p.) Control 1 mg/kg Control 1.5 mg/kg
9779 210719*** 124712 388768***
– – – –
CPA (i.p.) Control 0.05 mg/kg 0.1 mg/kg 0.2 mg/kg
105712 10379 129711 147714*
– – – –
VAL TE 35E (p.o.) Control 100 mg/kg 500 mg/kg 1000 mg/kg
107716 119723 109716 107712
9079 89713 10479 88711
Phytofin Val. 368 (p.o.) Control 100 mg/kg 500 mg/kg 1000 mg/kg
103710 11579 115711 97713
80711 92710 9577 8878
VAL H 70E Control 100 mg/kg 250 mg/kg 500 mg/kg
8876 8077 8377 9676
124715 99710 128711 13877
Val H 45 M (p.o.) Control 100 mg/kg 250 mg/kg 500 mg/kg
90710 82713 63711 7875
107713 89713 83713 87710
1000 500
A
counts per 10 minutes
0 3000 2500 2000 1500 1000 500
B
0 3000 2500 2000 1500 1000 500
C
0 0
**
* * ** **
10 20 30 40 50 60 70 80 90 100 110 120 min after application
Fig. 2. Locomotor activity (mean7S.E.M. counts per 10 min) of NMRI mice (8 per group) after oral application of the test solutions: J water, K A: VAL H 70E 500 mg/kg bw, K B: phytofin Valerian 386 1000 mg/kg bw, K C: diazepam 3 mg/kg bw. Data of these experiments is taken from Table 2 which presents results of further experiments. **po0.05, ***po0.001 vs. corresponding control group, Student’s t-test.
effect in the EPM procedure after oral application (Fig. 5). The ratio of open and closed arms regarding time and number of entries was significantly increased in favour of the open arms after treatment with 100 and 250 mg/kg bw. Although mice treated with the highest dose (500 mg/kg bw) showed anxiolytic-like behavior as well, open arm exploration was less pronounced in comparison to the lower dose levels. Only the percentage of open arm entries was affected with statistical significance. Thus 100 and 250 mg/kg bw appeared to prove maximal effective. Considering the results gained with the commercially available extracts, only the preparation extracted with 45% methanol m/m (VAL H 45 M) showed anxiolytic activity (Fig. 6). 250 mg/kg bw of this extract led to a significant increase of the percentage of time spent in the open arms and of open arm entries, while both lower dosages (25 and 100 mg/mg bw) only tended to result in an enhancement of these parameters. The preparation VAL H 70E, that was obtained using 70% ethanol v/v as extraction solvent, left these parameters unaffected within the same tested dose range of 25, 100 and 250 mg/kg bw (Fig. 7).
Values are means7SEM expressed in seconds (duration of sleeping time) of 12–15 animals in each group.*po0.05, **po0.01 and ***po0.001 vs. control group, post-hoc Fisher-PLSD.
Forced swimming test including additional open field test The special extract phytofin Valerian 368 proved to be antidepressant since immobility time, as shown in Fig. 8A, was significantly decreased in the FST 16 days after daily treatment with 2 125 mg/kg bw (8:00 a.m. and 20:00 p.m.). 2 50 mg/kg bw had a similar effect, but statistical significance was not reached. A negligible prolongation of immobility time was observed at the lowest dose level (2 12.5 mg/kg bw). In contrast, extract VAL TE 35E, which was used as base material for phytofin Valerian 368, showed no effect on this parameter at the tested dose level of 2 125 mg/kg bw per day. The difference between fraction and extract at this dosage reached statistical significance.
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To verify if the observed changes in immobility times may be due to unspecific, compound-related effects on locomotor activity, animals were tested in an open field for alterations in activity 2 days before the FST procedure. Since none of the treatments induced significant changes in locomotor activity (Fig. 8B), the pronounced reduction of immobility time after treatment with phytofin Valerian 368 (2 125 mg/kg bw) can be considered as a result of an antidepressant property of this preparation.
duration of anaesthesia [s]
Horizontal wire test As shown in Fig. 9A and B neither the extract VAL TE 35E nor the special extract phytofin Valerian 368 showed myorelaxant activity in the horizontal wire test in dosages up to 1000 mg/kg bw. Similar to the control group, none of the valerian treated mice failed to grasp the steelbar with at least one hind paw within a time period of 1 h. By contrast, treatment with 2 mg/kg bw diazepam led to a distinct impairment of test performance. Impairment occurred 10 min after administration, reached its maximum after 20 min, diminished afterwards and was hardly detectable at the end of the procedure.
* **
240
9
200
*
160
Discussion
120
The present study evaluated the pharmacological profile of different extracts derived from Valeriana officinalis L. s.l. including two commercially available extracts and the newly developed preparations VAL SE 35E and phytofin Valerian 368. Therefore tests for sedative, anxiolytic, antidepressant as well as for myorelaxant properties were conducted in rodents. None of the different extracts revealed sedative properties after acute administration. Neither the valerian preparations VAL SE 35E and special extract phytofin Valerian 368 nor the commercially available extracts VAL H 70E and VAL 45 M induced a reduction of locomotor activity or a prolongation of ether-induced anaesthesia. Since the tested dosages were up to
80 40 0
ctrl.
1
ctrl. 0.2
ctrl. 1000 mg/kg BW
water / saline (p.o. / i.p.) VAL H 70E (p.o.)
ctrl. 500
CPA (i.p.)
Diazepam (i.p.)
VAL H 45M (p.o.)
ctrl. 500
phytofin Val. 368 (p.o.)
Fig. 3. Mean (7 S.E.M., 12–15 NMRI mice per group) duration of sleeping time 30 min after application of the test solutions. This figure constitutes an exemplary composition of data derived from 5 experiments which are in full detail presented in Table 3; *po0.05, ***po0.001 vs. corresponding control group, post-hoc Fisher-PLSD.
45
35 30 25
*
*
20 15
35 30 25 20 15
10
10
5
5
0
ctrl.
2
* **
40 open arm entries [%]
40 time on open arms [%]
45
* **
100
250
500
mg/kg BW water
diazepam
0
ctrl.
2
100 250 500 mg/kg BW
VAL TE 35E
Fig. 4. Mean (7 S.E.M., 12–14 NMRI mice per group) percentage time spent on open arms and percentage open arm entries in the EPM 60 min after oral application of the test solutions. *po0.05, ***po0.001 vs. corresponding control group, Fisher-PLSD post hoc test.
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30
30
20
25
* **
open arm entries [%]
25 time on open arms [%]
* **
* **
* **
15 10
* **
15
*
10 5
5 0
* ** 20
ctrl.
2
100
250
0
500
ctrl.
2
water
100
250
500
mg/kg BW
mg/kg BW diazepam
phytofin Val. 368
Fig. 5. Mean (7 S.E.M., 12–14 NMRI mice per group) percentage time spent on open arms and percentage open arm entries in the EPM 60 min after oral application of the test solutions. *po0.05, ***po0.001 vs. corresponding control group, Fisher-PLSD post hoc test.
**
25 20 15 10
*
25 20 15 10 5
5 0
**
30
** open arm entries [%]
time on open arms [%]
30
0 ctrl.
2
25
100
250
mg/kg BW water
diazepam
ctrl.
2
25 100 mg/kg BW
250
VAL H 45M
Fig. 6. Mean (7 S.E.M., 12–14 NMRI mice per group) percentage time spent on open arms and percentage open arm entries in the EPM 60 min after oral application of the test solutions. *po0.05, **po0.01 vs. corresponding control group, Fisher-PLSD post hoc test.
1000 mg/kg bw and, considering the motor experiments, a 2 h test period with consecutive measurements was chosen, a sedative action of valerian seems to be unlikely. Additionally, even after subacute treatment (19 days) with phytofin Valerian 368 and VAL H 70E (2 500 mg/kg bw daily) an alteration of motor activity and of duration of sleeping time indicating a sedative action could not be observed (data not shown). The absence of sedation is partially contradictory to results obtained in other studies using similar behavioral paradigms. Wagner, et al. (1980) showed a valerian
tincture (DAB 7) to slightly reduce locomotor activity in mice after gavage of 1000 mg/kg bw and, after a storage period of 9 month, also of 100 mg/kg bw. When assessing these results, the absence of statistical specifications must be taken into consideration. Similar difficulties occur with the study of Leuschner, et al. (1993) that showed a maximum reduction of 28.6% and 35.9% in motor activity 120 min after oral administration of 20 and 200 mg/kg bw of an aqueous alkaline extract, since no information regarding statistical significance is given.
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50
50
* **
* **
40
40 open arm entries [%]
time on open arms [%]
11
30
20
10
30
20
10
0
0 ctrl.
2
25 100 mg/kg BW
250
water
ctrl.
2
25 100 mg/kg BW
250
VAL H 70E
diazepam
Fig. 7. Mean (7 S.E.M., 12–14 NMRI mice per group) percentage time spent on open arms and percentage open arm entries in the EPM 60 min after oral application of the test solutions. ***po0.001 vs. corresponding control group, Fisher-PLSD post hoc test.
180
140
120 *
100 80 60 40
100 80 60 40 20
20 0
120
#
140
line crossings / 5 min
duration of immobility [s]
160
ctrl.
25
100
250
250
0
mg /kg BW water
phytofin Val. 368
ctrl.
25
100 250 250 mg/kg BW
VAL TE 35E
Fig. 8. Mean (7 S.E.M., 12 Sprague-Dawley rats per group) duration of immobility in the forced swimming test (A) and number of line crossings per 5 min in the open filed test (B) after subacute, oral treatment (FST ¼ 16 days, OFT ¼ 14 days, 2 daily) with the test solutions. *po0.05 vs. corresponding control group, #po0.05 vs. phytofin Valerian 368 (250 mg/kg bw), Fisher-PLSD post hoc test.
Although in some studies an extended anestheticinduced sleeping time after administration of valerian preparations or isolated constituents like hesperidin was demonstrated (Hiller and Zetler, 1996; Leuschner, et al., 1993; Marder, et al., 2003), the relevance with regard to sedative properties of valerian remains unclear. All these experiments were carried out using barbiturates for anesthetisation. Since barbiturates have been proven to interfere with CYP P-450 enzymes (Kakinohana et al.,
1998) an increased sleeping time due to interactions of valerian with the barbiturate metabolism in mice cannot be excluded. Though in men no relevant interactions of valerian with CYP 3A4, 1A2, 2D6, 2E1 enzymes were shown (Donovan et al., 2004; Gurley et al., 2005), interactions might nevertheless occur in rodents due to differences in metabolism. Considering results obtained in clinical trials investigating valerians effect on vigilance, psychomotor and
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A mg/kg BW
40
diazepam 2 mg/kg
500
1000
30 20 10
VAL TE 35E
100
impaired mice [%]
50
water
0 0
10
B mg/kg BW
diazepam 2 mg/kg
500
20 10
water phytofin Val. 368
1000
30
60
100
impaired mice [%]
40
20 30 40 50 minutes after application
0 0
10
20 30 40 50 minutes after application
60
Fig. 9. Percentage of impaired NMRI mice (10 per group) in the horizontal wire test after oral application of water, diazepam and VAL TE 35E (A) or phytofin Valerian 368 (B).
cognitive functions as well as on hang over phenomenons, it can be ascertained that findings of our in vivo experiments (locomotor activity, ether-induced anaesthesia) comply with these data. In four out of six of those studies in healthy volunteers no hints on sedating properties of common valerian preparations were observed after acute and (partially) long-term treatment using dosages up to 1800 mg (Glass et al., 2003; Gutierrez, et al., 2004; Hallam, et al., 2003; Kuhlmann, et al., 1999). In contrast, Gerhard et al. (1996) reported, a slight but significant decrease in vigilance in a group treated with a valerian syrup. However, it has to be annotated that this study has been conducted without blinding the syrup group. In a second study an increase in self-rated tiredness after acute treatment with 1200 mg of a valerian extract was shown (Schulz et al., 1998), but this dosage clearly exceeds the recommended dose level and statistical analysis of these data was not performed. Moreover, valerian-induced changes in quantitative EEG, which were similar to placebo treatment in many aspects, were interpreted as a hint on sedative properties of the extract. Contrarywise, the authors themselves admit a lack of specificity of the EEG method, since changes in EEG profile do not seem to be valid predictors of sedation, tranquilisation or anxiolysis. To summarize, considering all results obtained in clinical trials and in in vivo pharmacological experiments there is hardly evidence of sedative activity of common
valerian preparations. Against this background, the commonly reported interference of valerian extracts or of isolated constituents with the adenosine A1 receptor or binding sites at the GABAA receptor may not play a decisive role in mediating sleep-enhancing effects of valerian in vivo. In accordance with published data (Karcz-Kubicha et al., 2003; Mandryk et al., 2005; Patterson et al., 2005) it was demonstrated in our own studies that GABAA- or adenosine A1-agonistic compounds like diazepam and CPA respectively reduce locomotor activity and/or extend the duration of narcosis as a consequence of the expected central depression. Due to the distinct comorbidity between sleep disorders and psychiatric diseases like anxiety or mood disorders (Schramm et al., 1995) we carried out behavioral experiments in mice and rats to test valerian for anxiolytic and antidepressant activity that may account for its proved sleep-enhancing properties in humans. Using the elevated plus maze test the ethanolic extract VAL TE 35E and the special extract phytofin Valerian 368 that was derived from it, as well as the commercially available methanolic extract VAL H 45M displayed a pronounced anxiolytic activity. In contrast, the also commonly used ethanolic extract VAL H 70E was not shown to be active in this test procedure. The maximum effect of VAL TE 35E and phytofin Valerian 368 seems to be reached at a dosage of 100 mg/kg bw, using higher dose levels exploration of the open arms was not further increased. Interestingly, 500 mg/kg bw of the special extract were less active compared to the lower dose levels. This phenomenon could be due to an (inverted) u-shaped dose response curve that is already known for other compounds (Borsini, 1995; Gries et al., 2005). Anxiolytic-like effects of valerian or of isolated constituents have previously been observed by Hiller and Kato (1996) and Marder et al. (2003) that showed the ethanolic extract STEI Val and the flavonoid 6-Methylapigenine to be active in the elevated plus maze paradigm. Additionally, several observations indicate a putative efficacy of valerian preparations in the treatment of anxiety and stress-related symptoms. In a comparative study a 4 week treatment with a 70% v/v ethanolic extract led, amongst others, to a reduction of the Hamilton-scores similar to the oxazepam treated group (Dorn, 2000). Furthermore, after a treatment period of 1 week using the same extract a diminished response to mental stress under laboratory conditions was reported by Cropley et al. (2002). Particularly with regard to clinical findings using ethanolic extracts in which repeatedly an initial lack of efficacy associated with a time-dependent development of sleep-enhancing properties (Donath, et al., 2000; Kamm-Kohl and Jansen, 1984; Vorbach, et al., 1996) was observed, we tested VAL TE 35E and phytofin
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Valerian 368 for putative antidepressant activity. A lack of efficacy at the beginning of treatment is well known in the treatment of mood disorders with synthetic antidepressant compounds (Aktories et al., 2005). In contrast to its primary extract VAL TE 35E, phytofin Valerian 368 reduced immobility time in the forced swimming test after subacute treatment. Due to the absence of compound-related alterations in motor activity the effect can be considered as a result of an antidepressant property of this extract. This additional antidepressant effect is obviously a result of the manufacturing process (column adsorption) that led to a diminished content of lipophilic compounds and can be arised from an alteration regarding the quantitative composition of phytofin Valerian 368. The potential to impair psychomotor functions is one of the most common side effects of widely-used sleep aids (Aktories, et al., 2005). To verify the effect of our extracts on muscle strength we used the horizontal wire test in mice. None of the tested extracts (VAL TE 35E and phytofin Valerian 368) showed myorelaxant activity in dosages up to 1000 mg/kg bw. Against the background that enhanced GABAA-ergig transmission, the known mechanism for benzodiazepines, is associated with a loss of muscle strength, these findings affirm that valerian-induced CNS-related effects might not be caused by interference with the GABAA receptor.
Conclusion Our investigations are partially contradictory to the common view on the pharmacological activity of valerian preparations. The clinical efficacy of valerian is generally accepted, and mostly sedation is assumed to account for improvement of nervousness and sleep disorders. However, in our investigations neither commonly used (45% methanol m/m, 70% ethanol m/m) nor newly prepared extracts (35% ethanol v/v, special extract phytofin Valerian 368) derived from Valeriana officinalis L. s.l show sedative activity in mice, since locomotor activity and the duration of ether-induced anaesthesia were not affected after acute or subacute treatment. These observations are in sound accordance with clinical studies that predominantly failed to reveal a valerian-induced influence on vigilance, psychomotor and cognitive performance. Instead, the special extract phytofin Valerian 368, its 35% v/v ethanolic primary extract VAL SE 35E and the commercially available 45% methanolic (m/m) preparation were proved to exhibit a pronounced anxiolytic effect in the elevated plus maze paradigm following acute administration. Furthermore, after a pretreatment period of 16 days only phytofin Valerian 368 showed activity in the forced swimming test indicating anti-
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depressant properties, whereas its primary extract was not effective. Obviously the observed antidepressant and anxiolytic activity may not be mediated by lipophilic constituents of low molecular weight, e.g. valerenic acids, since these compounds were removed from phytofin Valerian 368 to a considerable extent. Moreover, results obtained in the horizontal wire test after gavage of the 35% ethanolic extract and its derived special extract phytofin Valerian 368 justify the exclusion of myorelaxant properties of these preparations. It is suggested that instead of sedative effects, anxiolytic as well as antidepressant properties of valerian, effects in particular demonstrated for the special extract phytofin Valerian 368, may account for the clinically proved sleep-enhancing effects of valerian. Considering these findings, a retrospective analysis of clinical data in respect of a possible correlation between psychiatric disorders and efficacy of valerian in treating mild sleep disorders may appear reasonable.
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