20(S)-protopanaxadiol, an active ginseng metabolite, exhibits strong antidepressant-like effects in...

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20(S)-protopanaxadiol, an active ginseng metabolite, exhibits strongantidepressant-like effects in animal tests

Changjiang Xu a, Jijun Teng a, Weidong Chen a, Qiang Ge a, Zhiqi Yang a, Chunying Yu a,Zirong Yang a, William Jia a,b,⁎a Shanghai Innovative Research Centre of Traditional Chinese Medicine, Shanghai, Chinab Brain Research Centre, University of British Columbia, Vancouver, BC, Canada V6T 2B5

a b s t r a c ta r t i c l e i n f o

Article history:Received 8 February 2010Received in revised form 10 July 2010Accepted 12 July 2010Available online 18 July 2010

Keywords:DepressionGinsengGinsenosidesPMCPost metabolism compound

Ginseng has beenused formood adjustment in traditional Chinesemedicine for thousands of years. Our previousstudy has shown that, total ginsenosides, themajor pharmacologically functional ingredients of ginseng, possessantidepressant activity. In the present study, we hypothesized that an intestinal metabolite of ginseng, 20(S)-protopanaxadiol (code name S111), as a post metabolism compound (PMC) of ingested ginsenosides, may beresponsible for the antidepressant activity of ginseng. To test this hypothesis, antidepressant-like activity of orallygiven S111 was measured in animal tests including tail suspension test, forced swimming test and rat olfactorybulbectomy depression model. In all those tests, S111 demonstrated antidepressant-like activity as potent asfluoxetine. S111 treated bulbectomy animals had higher levels ofmonoamine neurotransmitters in the brain andin vitro reuptake assay showed that S111 had a mild inhibitory effect. Furthermore, S111 but not fluoxetinesignificantly reduced brain oxidative stress and down-regulated serum corticosterone concentration inbulbectomy animals. No disturbance to central nervous system (CNS) normal functions were found in S111treated animals. These results suggest that the ginseng activemetabolite S111 is a potential antidepressant. Sincethe monoamine reuptake activity of this compound is rather weak, it remains to be investigated whether itsantidepressant-like effect is by mechanisms that are different from current antidepressants. Furthermore, thisstudyhas demonstrated that postmetabolism compounds (PMCs) of herbmedicines such as S111maybeanovelsource for drug discovery from medicinal herbs.

© 2010 Elsevier Inc. All rights reserved.

1. Introduction

Clinical depression is a mood disorder that is the leading cause ofdisability and suicide. According to the World Health Organization,depression is currently the 2nd cause of disability adjusted life yearsin the age category 15–44 years for both sexes combined, affecting121 million people worldwide and it will be at the 2nd place by 2020calculated for all ages (WHO, 2010). While antidepressants fortreatment of major depression are widely available, they are oftenineffective (Berton and Nestler, 2006). In addition, side effects of thosemedications are common and could be severe in some cases (Ernst,2009; Grunze, 2008; Papakostas, 2010; Salisbury et al., 2009;

Schweitzer et al., 2009). Thus, more effective antidepressants withless side effects are needed.

A number of animal tests are being used to investigate mechanismsof depressive diseases and to screen potential new antidepressants. Tailsuspension test (TST) (Steru et al., 1985) and forced swimming test(FST) (Porsolt et al., 1977) are two most commonly used. They bothreflect a depressive behaviour generally considered as despair orhopelessness. Both tests are thought to be highly predictable forantidepressant effects in humans (Fuchs and Fliugge, 2006; McArthurand Borsini, 2006). Rat olfactory bulbectomy model (Leonard, 1984)creates a dysfunction of the cortical–hippocampal–amygdala circuit,which contributes to the regulation of mood, learning and memory(Song and Leonard, 2005). This circuit may also be dysfunctional inpatients with major depression (Dannlowski et al., 2009; Frodl et al.,2008; Kronenberg et al., 2009; Paparrigopoulos et al., 2008; vanEijndhoven et al., 2009). Biochemically, it has been well documentedthat depression is associated with reduced levels of brain monoamineneurotransmitters, such as 5-hydroxytryptamine (5-HT) and norepi-nephrine (NE) (Sulser and Brodie, 1962), which have led to thedevelopment of numerous antidepressants with a commonmechanismof upregulating the levels of those neurotransmitters in the brainthrough selective inhibition on their reuptake (Lanni et al., 2009; Lopez-

Progress in Neuro-Psychopharmacology & Biological Psychiatry 34 (2010) 1402–1411

Abbreviations: PMC, post metabolism compound; CNS, central nervous system; TST,tail suspension test; FST, forced swimming test; 5-HT, 5-hydroxytryptamine; NE,norepinephrine; HPA, hypothalamic–pituitary–adrenal; PPDs, protopanaxadiols; PPTs,protopanaxatriols; GI, gastrointestinal; HPLC, high performance liquid chromatogra-phy; ESI-MS, electrospray ionization mass; NMR, nuclear magnetic resonance; OB,olfactory bulbectomy; 5-HTP, L-5-hydroxytryptophan; LD, L-Dopa; SOD, superoxidedismutase; MDA, Malondialdehyde; ACTH, adrenocorticotropic hormone.⁎ Corresponding author. Shanghai Innovative Research Centre of Traditional Chinese

Medicine, Shanghai, China.E-mail address: [email protected] (W. Jia).

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Munoz and Alamo, 2009). However, monoamine depletion may not bethe sole cause of depressive diseases (Chen and Skolnick, 2007), currentunderstanding of the etiologic mechanism of clinical depressionincludes dysregulation of the hypothalamic–pituitary–adrenal (HPA)axis (Carroll et al., 1976; Miura et al., 2008; Pariante and Lightman,2008) and stress (Miura et al., 2008; Selye, 1955) as well as disorders ofneurotrophic factors and neuroplasticity (Duman, 2002; Shirayama etal., 2002; Brunoni et al., 2008; Yulug et al., 2009). Hence, compoundswith new mechanisms of action may be useful as candidates of newclasses of antidepressants.

Ginseng has been used as an herbal medicine for thousands ofyears. One of the clinical indications for ginseng is antidepression andantianxiety according to traditional Chinese medicine (Kennedy andScholey, 2003). In recent years, ginseng has also been widely used inwestern world for mood improvement and general well-being. Tworandomized and controlled clinical trials have reported improve-ments in mood and anxiety for postmenopausal women (Tode et al.,1999; Wiklund et al., 1999). As an herbal medicine, ginseng is oftengiven orally as aqueous extraction. It has been believed that the majorpharmacological active ingredients of ginseng are ginsenosides, whichare saponin-like compounds. There are two major ginsenosides inginseng, protopanaxadiols (PPDs) and protopanaxatriols (PPTs), bothhave good solubility in water (Chu and Zhang, 2009). Our previouswork has demonstrated that total ginsenosides when given orally areantidepressant demonstrated in both forced swimming test andchronicmild stressmodels of depression (Dang et al., 2009). However,it has been shown that most native ginsenosides are poorlyabsorbable (Han et al., 2006; Hasegawa et al., 1996). Further, whileginsenosides are known for their neuroprotection in various animalmodels (Bae et al., 2004; Lee et al., 2006; Lian et al., 2005; Lim et al.,1997; Park et al., 2005; Yoshikawa et al., 2008; Zhang and Liu, 1996),their in vitro effective concentrations are N10 μM (~4 μg/ml) (Kim etal., 2000; Liao et al., 2002; Radad et al., 2004), which is substantiallybeyond its in vivo bioavailability. One explanation for the abovediscrepancy in ginsenoside pharmacology is that they may act as pro-drugs when they are taken orally and it is their metabolites that areresponsible for the in vivo effect. Indeed, ingested ginsenosidesundergo extensive metabolism in the gastrointestinal tract anddeglycosylated into active metabolites by intestinal bacteria ofmicroflora (Hasegawa et al., 1997; Hasegawa et al., 1996; Qian et al.,

2006). Those metabolites have better bioavailability by crossingintestine-blood and blood–brain barriers (Hasegawa et al., 1997;Hasegawa et al., 1996; Tawab et al., 2003).

We postulated that some ginsenoside intestinal metabolites maybe responsible for antidepressant activity of observed total ginseno-sides. Further, we hypothesized that a novel class of drug candidatesnamed post metabolism compounds (PMC) may be developed fromcompounds present in the gastrointestinal (GI) tract, blood or targetorgans after ingestion of medicinal herbs that are clinically efficacious.In the present study, we reported that purified 20 (S)-protopanax-adiol (code name S111), an aglycon ginsenoside found in the intestineafter ingestion of ginseng, possesses novel antidepressive-like effectin all animal tests with a potency equal or better than fluoxetine.

2. Materials and methods

2.1. Chemicals and drugs

S111 was provided by Shanghai Innovative Research Centre ofTraditional Chinese Medicine. The compound was a white powderwith MW=460.69 at the purity of 98% verified with high perfor-mance liquid chromatography (HPLC). Chemical structure of S111 isshown in Fig. 1.

2.2. Preparation and characterization of S111

S111 was prepared by hydrolysis of leaf saponins of Panaxnotoginseng (Burk) with a mixed solution of sodium ethoxide and n-Butanol at 100 °C for 72 h. The insoluble residue was extracted withethylacetate and separated with silica gel column chromatography.The compound structure was characterized by spectroscopic analysiswith both ESI-MS and 13C NMR. Themass spectrumwas obtainedwithThermo LCQ advantage MAX spectrometer (Thermo, USA) andshowed that the m/z value of PPD is 459.69[M−1]−. The 13C NMRspectrumwere obtained using a VarianMercury VX-300 spectrometer(300 MHz) (Varian, USA) equipped with 5 mm ASW probe (Varian,USA) operating at 25 °C, and determined using 60 mg S111 dilutedwith 0.5 ml of CDCl3. The chemical shift values of S111 were47.66 ppm, 26.79 ppm, 53.47 ppm, 74.05 ppm, 27.31 ppm and34.75 ppm and were assigned to C-13, C-16, C-17, C-20, C-21 and C-

Fig. 1. Structure of S111 and its mass spectrum.

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22, respectively. Thus, the configuration of C-20 on S111 is assigned SConfiguration.

Other drugs utilized in the experiments were purchased fromvarious sources. Fluoxetinewas from Eli Lilly, USA; Perchloric acidwasfromDongfang Chemical Factory (Tianjin, China). Estazolamwas fromSiyao (Changzhou, China). Noradrenaline Bitartras (Na) and Adren-aline (Adr) were from the National Institute for The Control ofPharmaceutical and Biological Products (Beijing, China). The 5-hydroxytryptamine was from Sigma (USA). Pentobarbital was fromNational Medicines Corporation Ltd (Shanghai, China). Most chemi-cals except specifically mentioned were purchased from FlukaChemicals (Australia). The radioactive labelled [14C]-5-HT (3.7 MBq/ml) and [3H]-Norepinephrine (3.7 MBq/ml) were purchased fromDuPont New England Nuclear (USA).

2.3. Animals and behaviour tests

Male Swiss mice weighing 18–22 g and male Sprague–Dawley(SD) rats weighing 200–250 g were all purchased from Institute ofExperimental Animals (Chengdu, China). The animals were housed ingroups except mentioned specifically under controlled conditions(23.5±1.5 °C, under a 12-h light–dark cycle with lights on at 6:00 a.m., food and water ad libitum). The animals were allowed to adapt tolaboratory conditions for at least 1 week before starting the experi-ments. All experiments were performed during the light phase of thelight–dark cycle. All possible steps were taken to avoid animals'suffering at any stage of the experiments. The procedures wereapproved by the Institutional Animal Care Committee of ShanghaiInnovative Research Centre of Traditional Chinese Medicine and werecarried out in accordance with the European Council Directive ofNovember 24th, 1986 (86/609/EEC).

2.3.1. Tail suspension test (TST)Tail suspension test was performed as previously described (Steru

et al., 1985; Yi et al., 2009). Briefly, mice were given S111 or fluoxetineby gavage. Sixty minutes after the last drug administration, theanimals were suspended by the tail from a ledge with adhesive tape(approximately 1 cm from the tip of the tail). The distance betweenthe tip of the nose of themouse and the floorwas approximately 5 cm.Animals were partitioned to avoid interference during the test.Immobility was defined as the absence of movement and was scoredover a 5-min trial by an observer blinded to the drug treatment.

2.3.2. Forced swimming test (FST)The test was performed using the method by Porsolt et al. (Porsolt

et al., 1977). Sixty minutes after the last drug administration, eachmouse was placed in an open cylindrical container (total volume of2500 ml, 20 cm height and 14 cm diameter) filled with 10 cm of water(25 °C). Each mouse was judged to be immobile as absence of motionexcept that required to keep head above water. Duration ofimmobility of last 4 min in the total 6 min of swimming time wasrecorded by a blinded experimenter. No hindlimb abduction wasfound in animals after the treatment and the animals were constantlywatched for no contact between their paws and the base of thecylinder during the FST (Lucki et al., 2001).

2.3.3. Olfactory bulbectomy depression modelBilateral olfactory bulbectomy (OB) (Leonard, 1984) was carried

out under chloral hydrate anaesthesia. After anaesthesia, a midlineincision was made from approximately 1 cm posterior and 1 cmanterior to bregma. Burr holes of 2 mm in diameter were drilledthrough the skull at approximately 7 mm anterior to bregma and1.5 mm on either side of the midline. The olfactory bulbs were thengently removed by suction with care taken not to damage the cortex.The holes were filled with dental cement and the wound was closedwith surgical thread under aseptic conditions. The sham operated

animals underwent the same surgical procedure but without olfactorybulbectomy. Animals were returned to their home-cage and received40,000 U/day penicillin for three consecutive days. The animals weregiven 6 days to recover following surgery prior to drug administrationand behaviour tests. During this period, the rats had handling daily bythe experimenter to eliminate any aggressiveness that wouldotherwise arise.

2.3.4. Step-down passive avoidance test (Archer et al., 1984)The OB and sham control rats with or without the drug treatment

were subjected to step-down passive avoidance test (Redmond et al.,1999). The step-down test apparatus (Model No. DTT-2, Institute ofMateria Medica, Chinese Academy of Medical Sciences, Beijing, China)consisted of a Plexiglas box (50 cm×50 cm×50 cm) with a stainlesssteel grid floor. The rods were 0.8 cm apart and connected to theterminals of a shock generator delivering 5 Hz, 36 V square wavepulses. A rubber platform (6.5 cm in diameter and 4.5 cm high) wasplaced in a corner of the grid floor. In the training session, rats werefirst placed individually inside the apparatus free running (withoutelectric shock) for 3 min to acclimatize to the environment. Theywerethen placed on the rubber platform. For the next 5 min, the electricshock was immediately delivered when the rat stepped down fromthe platform and placed all its paws on the grid floor. The electricshocks were continuously given until the animals jumped back to theplatform. The number of times the animal received foot shock wasrecorded as number of errors. Animals were returned to their home-cage immediately after the session. In the test session (24 h later), theanimals were placed on the platform again. The latency and number ofstep-down were recorded for 5 min (no shocks were given during thetest session).

2.3.5. Step-through passive avoidance testThe Step-through test apparatus (Model No.DCS-2, Institute of

Materia Medica, Chinese Academy of Medical Sciences, Beijing, China)consisted of two boxes (25×15×20 cm for each). The frontilluminated (40-W bulb) chamber (white box) was connected by aguillotine door to the rear dark chamber equipped with a grid floor.Animals were placed individually into the illuminated chamber of theapparatus and allowed to explore the boxes for 5 min and then takenout. The rear dark chamber was then electrified (36 V, approximately0.2–0.4 mA, 1 s) and the animals were placed back into theilluminated chamber. The latency of each animal to get the electricshock and times of errors were recorded during this training session(5 min). The animals were placed in the illuminated chamber again onday 2 for a 5-min trial session and the latency and errors wererecorded (no shocks were given during the test session).

2.3.6. Sweet-water consumption testSweet-water consumption was measured on the 13th and 20th

day after operation (7th and 14th day after drug treatment). Thewater was first removed 24 h before and two bottles of 200 ml each(one filled with water and the other one containing 1.0% sucrose)were then given and continuously available to rats for 24 h. Bothnormal and sweet-water intakes were measured (Becker et al., 2008).

2.3.7. L-5-hydroxytryptophan (5-HTP) induced trembling and L-Dopa(LD) induced running/jumping models

Body trembling and increased locomotor activity can be inducedby high levels of 5-HTP and LD, respectively. This has been used to testwhether a drug could increase the levels of 5-HT or dopamine (Majand Pawlowski, 1975; Dolphin et al., 1976; Gronan, 1975). Swiss micewere randomly divided into groups of 10 mice each. Three differentdoses of S111 were orally administrated in mice 60 min prior to i.p.injection of either 5-HTP (200 mg/kg) or LD (200 mg/kg). Both 5-HTPand LD were at the maximum subthreshold dose determined bypreliminary tests that did not induce the trembling or running/

1404 C. Xu et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 34 (2010) 1402–1411


jumping behaviour when applied alone. Percentages of the animalsshowed trembling in 5-HTP groups and increased spontaneouslocomotion (running and jumping) in LD groups were determinedin 30 min immediately following the i.p injections. The results wereanalyzed by Fisher's exact test (Table 1).

2.3.8. Locomotor activity measurementLocomotor activity was measured with a 36-point infrared ray

passive sensor system (model No. ZZ-6, Taimeng Tech Ltd. Chengdu).To reduce the potential effect of food intake on animal's locomotoractivity, mice were restricted with food but not water for 12 h beforetests. Animals were placed in a chamber of locomotor activitymeasurement device to adjust the environment for 1 min followedby recording for 5 min. The results were analyzed with Student t test.

2.4. Biochemical measurements

2.4.1. Measurement of brain superoxide dismutase (SOD),Malondialdehyde(MDA) and serum corticosterone levels

The whole rat brain was washed with ice-cold 0.9% sodiumchloride solution and homogenized. The homogenate was centrifugedat 12,000×g for 15 min at 4 °C. The supernatants were used tomeasure SOD and MDA according to the manufacturer's instructionwith SOD assay kit (Biovision, USA) and MDA detection kit (Biomol,USA), respectively. Samples of rat blood were stored in sterile plastictubes for overnight at 4 °C and centrifuged at 12,000×g for 10 min.The supernatants (sera) were collected and stored at −80 °C.Concentrations of corticosterone in sera were measured by ratcorticosterone ELISA kit (Diagnostic Systems Laboratories, Inc, USA)as instructed by the manufacturer's manual.

2.4.2. Measurement of monoamine neurotransmitters in rat brainThe frontal cortex and hippocampus were rapidly removed from

the brains of rats immediately after the completion of behaviour testsand then stored at −80 °C until use. NE and 5-HT were measured byfluorescence assay originally described by Curzon and Green (1970)and Jacobowitz and Richardson (1978) and then modified by Wu andLIU (1987). Briefly, brain tissue was homogenized in 10 volumes ofice-cold n-butanol. After brief centrifugation, the supernatant wasdiluted with 2-time volume of n-heptane followed by 5 s vortex.Monoamines were contained in the aqueous phase. For 5-HT, 0.1 mLsamples of the above aqueous phase were transferred into a test tubefollowed by adding 0.6 mL of 0.004% O-Phthalaldehyde in 10 N HCl.After brief mixing, the reaction was incubated in boiling water for15 min. The fluorescence was measured with a spectrophotofluo-rometer (Perkin–Elmer 3000) at 355 nm (excitation) and 475 nm(emission), respectively. For NE and DA, EDTA Disodium, iodine,alkaline sodium sulfite and 5 N acetic acid were sequentially addedinto the above aqueous samples followed by incubation in boiling

water for 5 min. Read NE fluorescence immediately at 385/475 and DAfluorescence at 322/370. The fluorescent readings were convertedinto concentrations (ng/g tissue) using the following formula:

Concentration ng= gð Þ = f sample–blankð Þ standard ngð Þð Þ× totalvolumeof n−butanol for extractionð Þg= f standard−sampleð Þ tissueweight gð Þð Þ× volumeof n−butanolusedformeasurementð Þg

2.4.3. In vitro monoamine reuptake assaysNormal SD rats were decapitated. The hippocampi and hypothe-

lami were quickly dissected on an ice-cold plate, weighted andplunged in tubes containing 9 volumes of ice-cold 0.32 mol/L sucrose(Thomas et al., 1987). The tissues were homogenized and centrifugedat 3420 rpm (10 min, 4 °C). For measuring 5-HT reuptake, samplealiquots of 200 μl supernatant (containing crude synaptosomes, 10aliquotes per group) were each preincubated for 5 min (37 °C) with800 μl of oxygenated (5%/95% CO2/O2 for 30 min) Krebs buffercontaining 120 mM NaCl, 25 mM NaHCO3, 10 mM glucose, 5 mMKCl, 1.2 mM MgCl2, 0.05 mM EDTA, 1.3 mM CaCl2, 1 mM NaH2PO4,0.1 mM ascorbic acid, and 0.06 mM pargyline (glucose, ascorbic acidand pargyline were made fresh every day) followed by adding62.5 nmol/L 14C-5-HT or 3H-NE (DuPond, USA) and 20 μl fluoxetine orS111 of designed concentrations. For control samples, 20 μl of salinewas added instead. The samples further incubated for another 5 minat 3H-NE. Uptake was also measured at 0 °C for one group of controlsamples containing 62.5 nmol/L 14C-5-HT or 3H-NE, 200 μl samplealiquot, 800 μl Krebs buffer and 20 μl saline to correct for passivetransport. The uptake assays were stopped by the addition of coldbuffer through Whatman GF/C glass fiber filters. The tubes and thefilters were then washed with the buffer, and entrapped radioactivitywas counted by liquid scintillation. Protein concentrations wereassessed using bovine serum albumin as standard. The amount ofreuptake was calculated as DPM (37 °C)–DPM (0 °C).

2.5. Statistical analysis

SPSS was used for all statistical analysis. Values are expressed asmean±SEM. Student t test was used to assess significance whencomparing between groups. For TST and FST experiments a two-wayANOVAwas used (single or repeated dosing and 3 levels of doses). One-way ANOVAwas used for the rest experiments except for specified ones,where Student t testwasperformed. ForANOVA results, plannedcontrasttesting was used to test relationships hypothesized a priori betweengroups. p values (two-tailed) b0.05 were considered significant.

Table 1Animals and dose regimes.

TEST Animals (10/group)

Drugs and dosing (mg/kg×time)

Fluoxetine S111

Tail suspension test (single dose) Male Swiss mice 18×1 3.75×1 7.5×1 15×1Tail suspension test(multi-dose) Male Swiss mice 18×10 3.75×10 7.5×10 15×10Force swimming test (single dose) Male Swiss mice 18×1 3.75×1 7.5×1 15×1Force swimming test(multi-dose) Male Swiss mice 18×10 3.75×10 7.5×10 15×10Passive avoidance tests after OB Male SD Rat 16×14 3.33×14 6.67×14 13.3×14Sweet-water consumption test after OB Male SD Rat 16×14 3.33×14 6.67×14 13.3×14Biochemical measurements after OB Male SD Rat 16×14 3.33×14 6.67×14 13.3×145-HTP and L-Dopa potentiation tests Male Swiss mice N/A 3.8×1 7.5×1 15×1Locomotor activity measurement Male SD Rat 18×1 3.8×1 7.5×1 15×1



3. Results

3.1. S111 induces antidepressant-like effect behaviours: tail suspensiontest and forced swim test

To investigate whether S111 has a potential as an antidepressant,we first conducted two classical depression behaviour tests: the tailsuspension test (TST) and forced swimming test (FST).

Three different doses of S111 were administrated by gavage eitheronce or once daily for 10 days. The TST was performed 60 minfollowing the last dosing. Mice in a positive control group wereadministrated with fluoxetine (18 mg/kg). Fluoxetine of single orrepeated doses both significantly reduced immobile time (Fig. 2A).Single dose of 15 mg/kg S111 was also effective, showing lessimmobile time (85.40±20.21 s, pb0.05) than those in controlgroup (108.40±27.45 s). For lower doses of S111, only those micetreated with repeated doses were significantly less immobile than thecontrols, suggesting an accumulated effect of low dose S111treatment.

Both single- and repeated doses of S111 treated mice exhibitedsignificantly less immobility than control mice in the FST (Fig. 2B).These results are consistent with those of the TST, suggesting thatS111 may possess antidepressant-like activity nearly as potent asfluoxetine.

3.2. Antidepressant-like effect of S111 on behaviours of olfactory bulbectomyrats

To further verify possible antidepressant-like effect of S111, wehave tested it on olfactory bulbectomy (OB) rats that exhibitdepression behaviours with more complex characteristics (Leonard,1984) (Song and Leonard, 2005). S111 of the similar doses as abovewere orally administrated daily for 14 days starting 6 days after thebulbectomy. The behaviour tests described below were performed24 h after the last administration of S111.

3.2.1. Sweet-water consumption testOne of the commonly used measurements for depression is

consumption of sweet-water of modeled animals (Sampson et al.,1991). Depressed animals typically lose their preference to sweet-water, showing a decreased intake of sweet-water. As shown in Fig. 3,OB rats displayed a continuous reduction in amount of sweet-waterintake following the operation. By the 13th days after the operation,there was a substantially reduced sweet-water intake by OB animalsalthough it had not reached statistical significance from the amount ofsham operated animals due to large variations within the group. By the20th day after the olfactory bulbectomy, there was a progressivelysignificant reduction in sweet-water consumption by OB animals. Incontrary, OB animals treated with either fluoxetine (16.0 mg/kg) orS111 (13.3 mg/kgor 6.7 mg/kg) consumed similar levels of sweet-wateras the sham operated animals. Thus, treatment with fluoxetine or S111remained sweet-water preference of animals with olfactory bulb lesion.

3.2.2. Passive avoidance taskIt was reported that OB animals are deficient in learning and

memory, demonstrated by elongated latency time in passive-avoidance tests. To demonstrate the effect of S111 on learning andmemory in OB animals, animals were first evaluated by passiveavoidance tests of both “step-down” and “step-through” at day 7 post-OB operation. Rats with significantly higher rates of errors than shamoperated animal group (more than 30%) were then randomly groupedfor drug treatment. Following 14 days oral administration of variousdoses of S111 or 16 mg/kg fluoxetine, both tests were performed asdescribed in Materials and methods. The results are shown in Fig. 4.While OB animals without any treatment exhibited severe deficiencyin learning and memory, demonstrated by significantly shorter

Fig. 2. Antidepressant-like effect of S111 on mouse tail suspension and forcedswimming tests. Animals were orally given fluoxetine) or S111 either single dose orrepeated doses (once daily for 10 days) before the tests. No significant difference wasfound between fluoxetine and S111 treated groups. The data are shown as averages of10 animals in each group. * pb0.05, ** pb0.01, ***pb0.001.

Fig. 3. Antidepressant-like effect of S111 on sweet-water consumption test in olfactorybulbectomy (OB) rats. Animals were orally given either fluoxetine or S111 forconstitutive 14 days starting on day 6 after OB. Each group contains 10 animals. Therewas no statistical significance among all the groups on day 13 post-OB. OB animalswithout any treatment showed significantly reduced sweet-water preference by day 20post-OB (▲ pb0.05) while animals treatment with either fluoxetine (16 mg/kg) or S111(13.3 mg/kg and 6.7 mg/kg) had significantly higher sugar-water consumption than OBonly group (* pb0.05). No significant difference was found between fluoxetine andS111 treated groups.



latency time for entering the wrong chamber, OB animals treated withfluoxetine (16 mg/kg) or high (13.3 mg/kg) and medium (6.6 mg/kg)doses of S111 performed significantly better and statistically had nodifference with that of sham operated animals.

3.3. S111 inhibits monoamine neurotransmitter reuptake

The above results clearly showed that S111 possesses a strongantidepressant-like effect. Next, we wanted to explore the possiblemechanisms of action by S111. As most of antidepressants currentlyon the market are based on inhibition of reuptake of monoamineneurotransmitters, we asked whether the S111 has the similar effect.

We first measured concentrations of Noradrenaline (NE) and 5-hydroxytryptamine (5-HT) in the hippocampus and cortex of OBanimals with or without treatment. As shown in Fig. 5, OB animals hadsignificantly reduced levels of bothNE and5-HT in thehippocampus and

cortex. Animals treated with S111 at 13.33 mg/kg and 6.67 mg/kg dailyfor 14 days had similar levels of NE and 5-HT as sham operated animalsand are significantly higher than OB rats without treatment in the twobrain regions. Interestingly, although daily treatment with fluoxetine(16 mg/kg) also effectively elevated 5-HT levels in both the hippocam-pus and cortex, it had no effect on the NE concentrations in the OB rats.

To further understand the mechanism of S111 causing higher levelsof NE and 5-HT in OB animals, we conducted in vitro monoaminereuptake assaysusinghomogenized tissues fromnormal rat striatum forNE and hypothalamus NEfor 5-HT. Fig. 6 demonstrated that fluoxetineFig. 4. Antidepressantlike effect of S111onpassive avoidance tests inolfactorybulbectomy

(OB) rats. S111 caused protection in cognitive functions in OB animals were demonstratedby step-down and step-through passive avoidance tests. OB animals treated withfluoxetine or S111 for 14 days (20 days post-OB) showed significantly improved cognitivefunctions demonstrated by significantly longer latencies than OB only animals (* pb0.05,** pb0.01, *** pb0.001). The latency of OB only animals was significantly shorter thansham operated control animals (▲▲ pb0.01, ▲▲▲ pb0.0001). No significant differencewas found between fluoxetine and S111 treated groups. Each group has 10 animals.

Fig. 5. S111 prevented OB induced reduction in brain levels of monoamine neurotrans-mitters. Brains of OB animals with or without S111 treatment were dissected on day 20post-OB (14 days after S111 treatment). Concentrations of Noradrenaline and 5-HT weremeasured asdescribed in theMaterials andmethods. Valuesare averagesof10animals.OBcaused significant reduction in concentrations of the monoamine in the brain, ▲▲▲pb0.0001). Fluoxetine and S111 both increased levels of brain 5-HT in OB animals to nearthe control levels. But only S111 had significant effect in restoring levels of brainnoradrenaline (Student t test, * pb0.05, ** pb0.01, *** pb0.001). No significant differencewas found between control and S111 treated groups.

Fig. 6. S111 inhibited monoamine reuptake in the homogenized rat striatum andhypothalamus tissues for NE and 5-HT, respectively. Each data point was the average of8 animals (Student t test, * pb0.05, ** pb0.01, *** pb0.001).



(1ug) inhibited 5-HT and NE reuptake by 33.7% and 22.9%, respectively.S111 was also effective in blocking reuptake of both neurotransmittersin a concentration-dependent fashion.

Inhibitory effect of S111 on monoamine reuptake was furtherdemonstrated by its potentiation in L-5-hydroxytryptophan (5-HTP)induced trembling (Maj and Pawlowski, 1975) and L-Dopa (LD)induced running/jumping models (Dolphin et al., 1976; Gronan,1975). Our preliminary tests had determined that 200 mg/kg i.p.injection of 5-HTP or LD did not cause trembling or running whenapplied alone. S111 (15 mg/kg) given alone orally also had no sucheffect. When S111 was co-administrated with the subthreshold dosesof 5-HTP (200 mg/kg) or LD (200 mg/kg), trembling or running/jumping behaviour appeared in substantial portions of animals(Fig. 7), suggesting that S111 may mildly increase in vivo monoaminelevels that potentiates the effects of administrated subthreshold 5-HTP or LD.

3.4. S111 reduces brain oxidative stress and down-regulates serumcorticosterone concentration in OB rats

Since previous study has shown that ginseng extracts or individualginsenosides were anti-oxidative in cell cultures (Lopez et al., 2007;Naval et al., 2007) and suppressed stress in animal models (Ichikawaet al., 2006; Takahashi et al., 1992; Cheng et al., 1987; Kim et al., 1970;Yuan et al., 1989), we speculated that the metabolite of ginsenosideS111 may also possess the similar function. To test this hypothesis,brain levels of superoxide dismutase (SOD) and Malondialdehyde(MDA) as well as serum levels of corticosterone were measured in OBrats with or without treatment by day 20 post-operation (14 daysafter treatment). As shown in Fig. 8, OB rats had a reduced brain SODactivity (pb0.01) and increased levels of MDA (pb0.05). The serumcorticosterone level in those animals were also significantly higherthan sham operated animals (pb0.001). In contrast, OB rats treatedwith all the three doses of S111 had near normal levels of brain SODand MDA. The serum corticosterone levels in those animals with S111were also normal. Interestingly, fluoxetine had no effect on any of thethree measurements in OB rats.

3.5. S111 does no change CNS excitability

Finally, we asked whether S111 causes any abnormality in basicactivity of the brain. We first measured locomotor activity of micetreated with S111 of various doses. As shown in Fig. 9A, except thegroup treated with Estazolam (0.6 mg/kg), a positive control withknown sedative activity, there was no change in spontaneous activity

of all groups treated with S111, measured by locomotor activity 1 hafter drug administration. We further examined effect of S111 onPentobarbital (i.p. 40 mg/kg) induced sleep in normal mice bymeasuring the durations of sleeping and induction (Fig. 9B). Again,Estazolam treatment prior to Pentobarbital significantly elongated thesleeping time (pb0.001) and shortened the induction period(pb0.01) while S111 had no effect on those parameters. Takentogether, S111 has no effect on normal brain activity both at awake orsleeping status.

4. Discussion

The present study evaluated potential antidepressant-like effect ofS111, an active metabolite of ginseng. Our results showed that S111was effective in both tail suspension and forced swimming, the twomost classic depression behaviour models. It was also able to reversedepressive behaviour caused by olfactory bulbectomy in ratsdemonstrated by step-down and step-through learning, memorytests and sweet-water consumption measurement. In addition, wealso showed that S111 elevated concentrations of NE and 5-HT in thebrain of OB rats and S111 is probably a weak monoamine reuptakeinhibitor. Unlike fluoxetine, S111 was also able to increase the level ofSOD, decrease the level of MDA in the brain and suppress the serumcorticosterone concentration in the OB rats. Finally, S111 does notseem to interfere with normal brain activity demonstrated bylocomotor activity and Pentobarbital induced sleep.

In the present study, OB rats demonstrated significant deficienciesin sweet-water consumption test and passive avoidance tasks.

Fig. 7. S111 potentiates 5-HTP-induced trembling and L-Dopa-induced rotation. 5-HTP(200 mg/kg), L-Dopa (200 mg/kg) or S111 (15 mg/kg) treated alone did not induce anytrembling or rotation in animals. The same dose of 5-HTP or L-Dopa caused significantincreases in number of animals showing trembling or rotation when co-administratedwith S111 at 15 mg/kg and 7.5 mg/kg. Each group had 10 animals. * pb0.05. Nosignificant difference was found between fluoxetine and S111 treated groups.

Fig. 8. S111 prevented bulbectomy induced changes in brain superoxide dismutase(SOD) and Malondialdehyde (MDA) as well as serum corticosterone levels. Each datapoint was the average of 10 animals.▲ pb0.05,▲▲ pb0.01,▲▲▲ pb0.0001, OB animalscompared with sham operated animals; * pb0.05, ** pb0.01, S111 treated OB animalscompared with OB only animals. No significant difference was found betweenfluoxetine and S111 treated groups.



Biochemically, OB rats showed reduced concentrations of monoam-inergic neurotransmitters in the brain, which are all in consistencewith previous studies on this model (Song and Leonard, 2005). Itmight be worthwhile to point out that, unlike tail suspension andforced swimming models, effect of antidepressants on OB model is tocorrect pre-existing depression-like behaviours. Like many antide-pressant drugs, S111 demonstrated excellent effect on reversing thedeficiency of OB animals at both behaviour and biochemical aspects.

The mechanism of action of S111 on its antidepressant-like effectremainsunclear. However, it seems less likely thatmonoamine reuptakeinhibition is themainmechanism of S111. In vitroNE and 5-HT reuptakeinhibition assay clearly showed thatfluoxetinewasmore potent (Fig. 6).The molecular weight of S111 and fluoxetine are 460.69 and 345.79,respectively. Ten micrograms, the highest concentration of S111 testedin in vitro reuptake assay produced less inhibitory effect on reuptakethan1 μgoffluoxetine for both5-HTandNE. Bymolar concentration, thepotency of inhibitingmonoamine reuptake forfluoxetinewas 7.5-fold ofthat of S111. Since the in vitro reuptake assay is the most directmeasurement on interaction between the drug and those reuptake sites,our results indicate that S111 is only a weak inhibitor on monoaminereuptake sites. This couldbe verifiedbymeasuringaffinities of S111withthose reuptake sites using binding assays of labelled S111. Despiteweaker inhibition on monoamine reuptake, S111 is more effective thanfluoxetine in maintaining the NE levels in both the hippocampus andfrontal cortex of OB rat. Thismay suggest that inhibition on NE reuptakeby fluoxetine at the current dose was not sufficient to restore the NElevel in those regions of the OB rat brain and significantly elevated NElevel by S111 in the OB rat may be attributed to a novel mechanism ofaction by S111. Despite the weaker inhibition on monoamine reuptake,S111 exhibited strong antidepressant-like activity in behaviour tests orbiochemical analysis in OB animals that is equal to or evenmore potentthan fluoxetine. The discrepancy between the ability of S111 oncorrection of OB induced deficiency but less effective in inhibition ofmonoamine reuptake suggest an additional mechanism of action forantidepressantpropertyof S111. It appears thatS111was able to act on atarget in theOBbrain that is upstream in regulating levels ofmonoamineneurotransmitters, which resulted in preserving the levels of both 5-HTandNE in thebrain. Onepossiblemechanism is that it regulates the anti-oxidative system and systemic corticosterone concentration as demon-strated on OB animals (Fig. 8). Numerous studies have shown thatginseng as well as individual ginsenosides are potent anti-oxidativeagents (Deng and Zhang, 1991; Feng et al., 1987; He et al., 2008; Huonget al., 1998; Lee et al., 1998; Li et al., 1999; Lin et al., 2008; Samukawa etal., 2008). In particular, ginseng extracts have shown strong effects onreducing MDA and increasing SOD levels in various animal models,

including ischemia in the brain (Kim et al., 2009; Shah et al., 2005) andheart (Zheng and Min, 2008), mouse chronic fatigue syndrome in thebrain (Singh et al., 2002), CCl4-induced injury in the liver (Chang et al.,2007), and glutamate toxicity in the lung (Shen et al., 2007). While it isnot clearhow theantioxidant system in thebrain involves in depression,there have been reports on reduced antioxidant activity in peripheralblood of depression patients, including higher levels of MDA andreduced levels of SOD (Bilici et al., 2001; McDaniel et al., 1995; Ozcan etal., 2004; Peet et al., 1998). It has been well documented thatoverproduction of reactive oxygen species results in destruction ofphospholipids and neurotoxicity (Block, 2008; Nakamura and Lipton,2009), which may contribute to pathological conditions of the brain insevere depression patients (Sheline, 2003; Swaab et al., 2005). Ourresults demonstrated that S111 is able to reverse the reduced activity ofthe antioxidant system in the brains of OB animals.

Ginseng is also known for its anti-stress effect (Bhattcharyya and Sur,1999; Rai et al., 2003). However, the effect of ginseng or ginsenosides onlevels of systemic corticosterone has been less clear. Some early studiessuggested that ginseng extracts either orally or intraperitoneallyadministrated caused an increase in systemic corticosterone concentra-tion in normal animals. This upregulation of plasma corticosterone wasmediated by the hypothalamus causing increased release of ACTH (Hiaiet al., 1979). However, total ginsenosides or ginsenoside intestinalmetabolites injected intracerebrally but not intraperitoneally couldinhibit intracerebroventricular injection itself induced elevation ofplasma corticosterone level in mice (Kim et al., 2003a, 1998). Mostinterestingly, while i.p. injection of total ginsenosides raised mouseplasma corticosterone levels, pretreating mice with the same dose oftotal ginsenosides i.p. could attenuate immobilization stress-inducedincrease in plasma corticosterone (Kim et al., 2003b). Total ginsengextracts but not Ginkgo biloba inhibit serum corticosterone levels inchronically stressed rats (Rai et al., 2003). Similarly, total ginsenosidesalso inhibited an increase of serum corticosterone induced by heat-stressin mice (Yuan et al., 1989). Taken together, it seems that ginsenosidesmay regulate systemic corticosterone levels bi-directionally. It upregu-lates plasma corticosterone levels under normal condition but sup-presses stress-induced elevation of corticosterone levels. The latter is inconsistence with our findings in OB rats. On the other hand, fluoxetinehas been shown to dose-dependently stimulate corticosterone release insocially isolated mice (Hendrie et al., 2003). Long-term fluoxetine wasbehaviourally anxiolytic on stressed rats but could not inhibit stress-induced elevation of plasma corticosterone level (Zhang et al., 2000). InOB rats, fluoxetine was not effective on elevated corticosterone levels(Marcilhac et al., 1999), which is in consistence with our currentobservations. Therefore, effect of S111 on regulation of corticosterone

Fig. 9. S111 does not alter normal brain excitability. A, locomotor activity of animals treated with Estazolam (Estaz), fluoxetine (Fluox) and S111; B, effects of Estazolam (Estaz),fluoxetine (Fluox) or S111 on sleeping time induced by pentobarbital (i.p. 40 mg/kg); C, effects of Estazolam (Estaz), fluoxetine (Fluox) or S111 on induction time for the sleepinduced by pentobarbital. ** pb0.01, *** pb0.0001 compared with untreated control animals (Cont).



release may differentiate it from existing antidepressants and rendersfurther investigation for its potential as a novel antidepression drugcandidate.

The present study is the first, to our knowledge to reveal that thepharmacological activity of mood regulation of ginseng used bytraditional Chinese medicine for thousands of years may be mediatedby ginsenoside GI metabolites, such as S111. The unique character-istics of S111 discovered by the present study and excellent safetyprofile demonstrated by both acute and chronic toxicology studies(data not shown) suggest this compound may be a promising newclass of antidepressants with a novel pharmacological property that isdistinguishable with current antidepressants.

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20(S)-protopanaxadiol, an active ginseng metabolite, exhibits strong antidepressant-like effects in...

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