Chinese Journal of Natural Medicines  2017, Vol. 15Issue (8): 584-596  DOI: 10.3724/SP.J.1009.2017.00584

Cite this article as: 

Chandra Sekhar Y, Phani Kumar G, Anilakumar K.R. Terminalia arjuna bark extract attenuates picrotoxin-induced behavioral changes by activation of serotonergic, dopaminergic, GABAergic and antioxidant systems[J]. Chinese Journal of Natural Medicines, 2017, 15(8): 584-596.

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Phani Kumar G, Tel: 0821-2579485, E-mail:

Article history

Received on: 11-Aug.-2016
Available online: 20 Aug., 2017
Terminalia arjuna bark extract attenuates picrotoxin-induced behavioral changes by activation of serotonergic, dopaminergic, GABAergic and antioxidant systems
Chandra Sekhar Y , Phani Kumar G , Anilakumar K.R     
Applied Nutrition Division, Defence Food Research Laboratory, DRDO, Mysore-570011, Karnataka, India
[Abstract]: Stress and emotion are associated with several illnesses from headaches to heart diseases and immune deficiencies to central nervous system. Terminalia arjuna has been referred as traditional Indian medicine for several ailments. The present study aimed to elucidate the effect of T. arjuna bark extract (TA) against picrotoxin-induced anxiety. Forty two male Balb/c mice were randomly divided into six experimental groups (n=7): control, diazepam (1.5 mg·kg-1), picrotoxin (1 mg·kg-1) and three TA treatemt groups (25, 50, and 100 mg/kg). Behavioral paradigms and PCR studies were performed to determine the effect of TA against picrotoxin-induced anxiety. The results showed that TA supplementation increased locomotion towards open arm (EPM) and illuminated area (light-dark box test), and increased rearing frequency (open field test) in a dose dependent manner, compared to picrotoxin (P < 0.05). Furthermore, TA increased number of licks and shocks in Vogel's conflict. PCR studies showed an up-regulation of several genes, such as BDNF, IP3, D2L, CREB, GABAA, SOD, GPx, and GR in TA administered groups. In conclusion, alcoholic extract of TA bark showed protective activity against picrotoxin in mice by modulation of genes related to synaptic plasticity, neurotransmitters, and antioxidant enzymes.
[Key words]: Anxiety     Terminalia arjuna     Gene expression     Picrotoxin     Neurotransmitters     LC-MS    

Stress plays a primary role in the pathogenesis of several neuropsychiatric and neurodegenerative disorders, such as anxiety, depression, Alzheimer's disease, Parkinson's diseases, dementia, schizophrenia, epilepsy, and hyper activity disorder [1]. According to World Health Organization (WHO), anxiety is one of the most prevailing psychological problems among stress-induced neurological disorders characterized by disproportionate emotions, heightened tension, worthless worries, and misappropriated fear [2]. Several subtypes of anxiety include general anxiety disorder (GAD), panic disorder, phobias, social anxiety disorder (SAD), post-traumatic stress disorder (PTSD), obsessive-compulsive disorder (OCD), and separation anxiety disorder [1-2].

Imbalance of neurotransmitters is responsible for the pathogenesis of several psychosomatic disorders. Among various neurotransmitters are serotonergic, noradregic, dopaminergic, and GABAergic systems that play important role in pathophysiology of anxiety [3]. Moreover, a recent gene expression study has revealed the role of multitude of genes responsible for the anxiety like disorders and their role in the neuronal signaling pathway [3]. SRIs (serotonin reuptake inhibitors), SNRIs (serotonin and nor epinephrine reuptake inhibitors) and other related drugs increase rate of neuronal proliferation through activation of cAMP-CREB cascade [4]. Some antidepressant drugs directly modulate NMDA receptors and influence the mechanism of neuroplasticity at synaptic level [5]. Several pre-clinical studies have elucidated the putative molecular mechanism of antidepressants on modulation of glutamatergic neurotransmission pathway, up-regulation of cAMP signal transduction pathway via cAMP-PKA-CREB cascade, up regulation of MAPK cascade, and expression of brain derived neuro factor (BDNF) [6].

Currently, the most widely prescribed medications for anxiety disorders are SRIs, SNRIs, and benzodiazepines (BZDs). SRIs and SNRIs are classified as antidepressants and commonly used to treat both anxiety and depression. BZDs are classified as sedatives and generally prescribed to treat anxiety or insomnia. Current management of anxiety disorders has self-effacing value; many patients do not respond to the treatment or are unable to tolerate pharmacological approaches and cognitive-behavioral therapy [7]. Almost all these drugs used to minimize the anxiety disorder often produce common side effects, like dry mouth, tiredness, drowsiness, dizziness, gastrointestinal complaints, obesity, headache, insomnia, lack of coordination, and nightmares [8]. Thus a quest for safer and alternative source of such drugs would be an important step in providing herbal or plant-based natural therapy to overcome the side effects of synthetic drugs. Yet, the evidences to recommend the use of herbal medicines in the treatment of several behavioral illnesses are still insufficient. In addition, more clinical research is needed to establish safety and efficacy of herbal drugs [9]. A recent survey reveals that about 63.9% of the studied population endorses the use of alternative medicine (including herbal) and this trend is similar throughout the world [10]. Authors suggest that predominant users are willing to try new and alternative therapy without abandoning the conventional treatment. With this background, we speculated that, in the quest for new drugs that ameliorate anxiety disorders, Terminalia arjuna, a plant selected in the current study, might play an important role in future, owing to its interesting medicinal properties.

Terminalia arjuna (Roxb. ex DC.) Wight and Arn. belongs to the family Combretaceae and is known as Arjuna in traditional Indian medicine, i.e., Ayurveda. Bark of the plant is well known for its medicinal importance as cardio-protective agent [11]. T. arjuna bark is frequently used to treat hypertension, ischemic heart diseases, and hypercholesterolemia and it is believed to possess anti-microbial, anti-mutagenic, anthelmintic, anti-fertility and anti-HIV properties [12]. Arjuna bark is also been reported to be used for the treatment of biliousness and sores and as an antidote to poisons, and further believed to have the ability to cure hepatic, congenital, venereal, and viral diseases [13]. Bark extract of T. arjuna has been reported to play a significant role as a cardiac stimulant in the treatment of hypercholesterolemia, heart failure, and atherosclerosis [14]. Previous studies on this plant have shown that the bark extract contains bioactive triterpenes such as arjunic acid, arjunolic acid, arjungenin, arjunetin, and arjunosides Ⅰ-Ⅳ [11]. Arjunolic acid extracted from T. arjuna bark is reported to possess multifunctional therapeutic applications, such as cardio-protective, anti-inflammatory, anti-diabetic, anti-tumour, anti-oxidant, anti-asthmatic, anti-microbial, insecticidal, and anti-coagulant activities [15]. Sinha et al [16] have reported that arjunolic acid attenuates protection against nephrotoxicity. Alcoholic extract of T. arjuna bark constitutes polyphenols, flavones, proanthocyanidins, and tannins, which possess significant antioxidant activity [17-18]. Subramanium et al [19] have reported the toxicological studies of the alcoholic extracts of T. arjuna bark up to the dose of 2 000 mg·kg·bwt.-1 by oral administration to Balb/c mice and found no toxic effects. Hence, the doses of the present study (25-100 mg·kg·bwt.-1) falls within the safety limits.

The present study was designed to assess the anxiolytic properties of alcoholic extract of T. arjuna bark in picrotoxin (PTX)-induced experimental mouse model. Picrotoxin, a prototypic antagonist of GABAA receptor and the primary mediator of inhibitory neurotransmission, enhances excitatory responses in the central nervous system, attenuates mood elevation, and generates anxiety [20-21]. Some established etiological models were used in the present study to explore the effect of TA alcoholic extract on animal behavioral responses to novel environmental stimuli. The present study also elaborated the changes in neurotransmitter levels related to PTX-induced anxiety.

Materials and Methods Bark materials and extraction

Bark samples of Terminalia arjuna (Roxb. ex DC.) Wight and Arn. were collected from Mysore, India, and authenticated by professor Alka chaturvedi, Department of Botany, RTM Nagpur University, Nagpur, India. Shade dried bark samples were pulverized and defatted with diethyl ether overnight, and the sample (1 kg) was extracted by soxhlet distillation unit with 100% ethyl alcohol (5 Liters) for 72 h, followed by flash evaporation under reduced pressure (Heidolph, Germany). The yield of the lyophilized extract (TA) was 104 g/kg dry bark powder.

Analysis of total polyphenols, flavonoids, and condensed tannins

Total polyphenols, flavonoids, and condensed tannins were measured spectrophotometrically [18]. Total polyphenols were determined by Folin-Ciocalteu reagent. An aliquot of the sample was mixed with 0.5 mL of Folin-Ciocalteu reagent and 4.5 mL of sodium carbonate solution, and incubated for 30 min at 40 ℃. Absorbance was measured at 765 nm and expressed as mg·g-1 gallic acid equivalents. Total flavonoid content was determined by AlCl3 solution. Briefly, 0.5 mL of AlCl3 solution was added to the equal amount of sample. The absorbance was measured at 420 nm after an incubation of 55 min and expressed as mg·g-1 rutin equivalents. Condensed tannins were measured by vanillin-HCl method as follows: vanillin (1.5 mL; 4% W/V in methanol) and HCl (0.75 mL; 37%) were added to the sample (0.25 mL) and the mixture was allowed to stand for a period of 30 min. The absorbance was measured at 500 nm and total tannin was expressed as mg·g-1 catechin equivalents.

LC-MS analysis for polyphenolic compounds

Terminalia arjuna bark alcoholic extract (TA) was analyzed for polyphenol detection using LC-MS-TOF and the chromatographic conditions are tabulated in Table 1. Agilent G6530 Q-TOF with Agilent Jet Stream source was used for the identification of phenolic components. The QTOF data were processed by molecular feature extraction (MFE) algorithm and Metlin database to identify the compounds and their empirical formula. The predicted structures were drawn based on the empirical formula generated and also based on the MSC software.

Table 1 Analytical conditions for LC-MS-IT-TOF
Determination of bioactive triterpenoids from TA by HPLC

The bark extract was qualitatively analyzed by HPLC (JASCO HPLC system) using a reverse phase C18 column (150 mm × 4.5 mm) as described by Verma et al [22]. All standards and samples were dissolved in methanol and filtered through micro syringe filter (0.22 μm), and the injection volume was 20 μL. Separation was achieved with a two pump gradient program for pump A―phosphate buffer and pump B―acetonitrile as follows: initially 30% B, then increased gradually to 60% B until 18 min; increased gradually 60%-85% B up to 22 min; and washed the column for 8 min with 30% B. Flow rate was maintained at 0.8 mL·min-1 and the detection wavelength was 205 nm with a UV detector, the absorption maxima close to all the compounds. For identification of the triterpenoids, following standards were used: arjunic acid, arjunolic acid, arjungenin and arjunetin, which were procured from Natural Remedies Private ltd, Bangalore, India (Purity: > 95%).


Animal studies were conducted according to the institute animal ethical committee regulations approved by the Committee for the Purpose of the Control and Supervision of Experiments on Animals (CPCSEA) with the IAEC approval No. 28/1999/CPCSEA. Balb/c mice weighing 25-30 g were selected from the stockpile colony, Defence Food Research Laboratory (DFRL), Mysore, India, and housed in an acryl fibre cage in a temperature controlled room (23 ± 2 ℃). Animals were maintained in 12 h/12 h light/dark cycle with an access to standard pellet 'chow' feed and pure water ad libitum.


Forty two male Balb/c mice were randomly divided into the following six experimental groups: control group, positive control group, negative control group and three treatment groups. Different doses of alcoholic extract of TA were orally administered to the mice groups separately such as, TA-25, TA-50 and TA-100 (25, 50, and 100 mg·kg-1, respectively) for a period of 21 days. Positive control group received diazepam (DIZ, 1.5 mg·kg-1, p.o.) for the same duration; control and negetive control (PTX group) received equal amount of distilled water orally during the experimental period. Except the control group, all the mice received PTX (1.0 mg·kg-1; i.p.) on the 21st day, thirty minutes before the behavioral test. Distilled water was used as vehicle for both PTX and diazepam treatments. Once the behavioral test was concluded, animals were sacrificed under mild anesthesia; brain samples were collected and stored at -80 ℃ until analysis. Schematic diagram of the experimental design is given in Fig. 1.

Figure 1 Schematic diagram of the experimental design
Open field test

The open field test (OFT) is generally used to observe the exploratory behavior and locomotary activity in test animals [23]. Open field area was made up of plain plexiglas and consisted of square arena 30 cm × 30 cm × 15 cm and the floor of the arena was divided into twenty equal squares. Individual mice were placed in the middle of the open field and behavioral parameters were recorded for 5 min. The following observations were recorded using ANY-maze software (ver.4.98, Stoelting Co., New Delhi, India): (1) locomotion (ambulation): number of grids crossed from the central position; (2) rearing: number of times the mouse stands on its hind legs; (3) leaning: number of times the mouse stands on forelimbs; and (4) grooming: number of times the mouse 'washes' itself by licking, wiping, combing or scratching.

Elevated plus maze test

Elevated plus maze (EPM) test is the most frequently used animal model for assessment of the anxiolytic activity. The elevated plus maze consisting of two open arms (30 cm × 5 cm) and two closed arms (30 cm × 5 cm) across each other respectively with 25-cm walls was placed 60 cm above the ground level. A rim of plexiglas (0.5 cm in height) surrounded the perimeter of the open arms to provide additional grip and thus prevent the mice falling off. Each animal was placed in the center of the maze, facing one of the closed arms [24]. Behavioral stimuli were recorded on the basis of number of entries and time spent in closed or open arms of EPM for a duration of 5 min, using ANY-maze software (Stoelting Co, New Delhi, India).

Light-dark paradigm

The light-dark (LD) exploration test is commonly used in murine model of anxiety. The test apparatus was consisted of two compartments, a large illuminated compartment (30 cm × 30 cm) made of clear plastic walls and was illuminated by bright light (500 lx), while the other compartment was made of black plastic (15 cm × 30 cm), not illuminated but covered by a black roof. The two compartments were separated by a partition with an opening (4 cm × 4 cm) through which the animal could pass from one compartment to the other [25]. The spontaneous exploratory behavior in response to novel environments and light were recorded for 5 min, using ANY-maze software (ver.4.98, Stoelting Co., New Delhi, India).

Vogel's conflict test

The Vogel's conflict test is a simple and reliable conflict procedure to verify the anxiolytic activity [26]. The entire apparatus had a stainless steel grid floor covered by plexi glass box (38 cm × 38 cm) and a water bottle with a metal drinking tube was fitted into it. An anxiometer circuit was connected between the drinking tube and the grid floor of the apparatus, so that the mouse completed the circuit whenever it licked the tube. Shock was administered to the feet of the animal by switching the connections to the drinking tube and grids from the anxiometer to a shocker. The mice were water deprived for 24 h and placed in the apparatus to record number of licks and shocks delivered within a span of 3 min.

RNA isolation and cDNA conversion

Brain tissue samples were placed in a vial containing RNA later and stored at -80 ℃ until analysis. Total RNA was extracted from about 300 mg of tissue by TRIzol reagent (Sigma Aldrich, Bangalore, India), as described the manufacturer's protocol. RNA samples were treated with RQ1 RNase-free DNase (Applied Bio-systems, Bangalore, India) to remove DNA contamination. The concentration of total RNA was measured with spectrophotometer (NanoDrop, Thermo Fisher, Bangalore, India) and the OD260/OD280 ratio was tested for the purity of RNA. The integrity, quantity, and purity of the RNA yield were also checked by electrophoresis. The cDNA strand was synthesized using high capacity cDNA converting kit (Applied Bio systems) in one vial procedure containing a total reaction of 20 μL having 3.2 μL of distilled H2O, 0.8 μL of dNTP's, 2 μL of the 10 × buffer, 2 μL of the random primers, 1 μL of reverse transcriptase, 1 μL of RNAse Inhibitor, and 10 μL of RNA sample. The sample was incubated following the manufacturer's protocol.

Semi-quantitative PCR

Ten different genes were targeted to check the modulation of gene expressions in response to PTX-induced anxiety, including brain derived neurotrophic factor (BDNF), CRE-binding protein (CREB), gamma-amino butyric acid receptor A (GABAA), 5-hydroxytryptamine receptor 1A (5-HT1A), dopamine receptor 2L (D2L), inositol trisphosphate (Ip3), superoxide dismutase (SOD), glutathione reductase (GR), and glutathione peroxidase (GPx). Forward and reverse primers of the targeted genes are listed in Table 2. PCR amplification was performed as a 20 μL of reaction with 6 μmol·L-1 of each forward and reverse primers (Table 2), 2 μL of 10X PCR buffer, 1.5 mmol·L-1 of MgCl2, 80 μmol·L-1 of each dNTP's, and 1 unit of Taq polymerase (Sigma-Aldrich, Bangalore, India) in a Master cycler thermal cycler (Eppendorf, Hamburg, Germany). The PCR program was consisted of initial denaturation at 94 ℃ for 4min, 30 cycles of denaturation at 94 ℃ for 45 s, annealing at 57 ℃ to 71 ℃ (subject to primer; Table 2) for 30 s, and extension at 72 ℃ for 8 min. PCR amplicons were resolved on 1% agarose gel and visualized under a UV transilluminator. (Taurus Incorporation, Bangalore, India).

Table 2 Primers used for the targeted genes
Analysis of gene expression of GABAA receptor by RT-PCR

The following primers were used: Forward 5′-AAAGCG TGGTTCCAGAAAA-3′, Reverse 5′-GCTGGTTGCTGTAG GAGCAT-3′ (GABAA). The quantitative real-time PCR (qRT-PCR) experiment for GABAA expression study was performed using SsoFast Eva Green (Bio-Rad, Bangalore, India) as per the manufacture's instruction. GAPDH was used as common housekeeping gene expressed in brain tissues. The total reaction mixture was 20 μL which contained 5 μL of cDNA, 10 μL of reaction buffer, 2 μL of primers, and 3 μL of RNase-free water. The cycling conditions were holding at 50 ℃ for 2 min, 95 ℃ for 10 min, followed by 40 cycles of 95 ℃ for 15 s, and 60 ℃ for 1 min. For no template control (NTC) reactions, 5 μL of cDNA was replaced with 5 μL of RNase-free water. Melting curve analysis of amplification products was performed at the end of each PCR reaction to confirm that a single PCR product was detected. All the reactions were carried out in triplicate. The amount of targeted gene in brain was obtained by the 2-ΔΔCt method after being normalized against control (GAPDH) [27].

Western bolt analysis

Brain tissue samples were processed for SDS-PAGE followed by Western blotting for the expression analysis of antioxidant enzymes, viz., catalase and glutathione reductase (GR). Tissue samples were homogenized with HEPES buffer (pH 7.4) and were centrifuged at 10 000 r·min-1 for 20 min. The supernatant was estimated for total protein by Lowry et al [28]. Protein from each sample (50 μg) was separated on 12% SDS-PAGE and transferred onto a nitrocellulose membrane using an electro blotting apparatus (Bio-Rad, USA). The membrane was blocked in 5% defatted milk solution at 4 ℃ for 24 h, probed by individual primary antibody (1 : 1 000), and incubated at room temperature for 3 h. The membrane was washed four times with PBST and PBS for 15 min, followed by incubation for 2 h in HRP conjugated rabbit anti-mouse secondary antibody (Sigma, India; 1 : 10 000). The membranes were washed again and developed using an enhanced chemiluminescent method (luminal and p-coumaric acid, Sigma). Developed membranes were exposed onto X-ray film and the band intensity was captured [29].

Statistical analysis

The data are expressed as means ± standard deviation of the mean (SD). The data were analysed using Tukey HSD one way ANOVA. Differences at P < 0.05 were considered to be statistically significant.

Results Total polyphenols, flavonoids and condensed tannins

Spectrophotometric analysis showed that the alcoholic extract of TA was rich in bioactive phytochemicals, such as polyphenols, flavonoids, and condensed tannins. Polyphenols were 430 ± 18 mg·g-1 gallic acid equivalents; flavonoids were 11 ± 0.6 mg·g-1 rutin equivalents; and condensed tannins were 152 ± 21 mg·g-1 tannic acid equivalents.

Bioactive triterpenoids

Three bioactive triterpenoids were targeted for the HPLC studies and the results are shown in Fig. 2. These bioactive triterpenoids were quantified against respective standard curves and their retention times, showing the following results: arjunetin (12.9 min; 751 ± 11 μg·g-1), arjungenin (17.36 min; 966 ± 21 μg·g-1), and arjunic acid (23.89 min; 1280 ± 30 μg·g-1).

Figure 2 HPLC analysis of triterpenoids. A: HPLC of standard peaks; B: HPLC of triterpenoids from TA bark compared to standards
Bioactive polyphenols

Identification of major polyphenols was carried out on their average mass, m/z and score under mass spectrometry (Fig. 3). Major compounds were galic acid, gallocatechin, procyanidin, epigallo-catechin, catechin, epigalocatechingalate, epicatechin-3-O-gallate, vanillylmandelic acid, xylofuanoside, ellagic acid mannopyranoside, and ellagic acid (Table 3).

Figure 3 LC-MS analysis. A: Total ion current (TIC) chromatogram from the HPLC separation of polyphenols present in TA; B: HPLC-UV-Vis chromatogram at 280 nm of TA
Table 3 Characterization and quantification of major polyphenols in TA using LC-MS
Effects of TA in the elevated plus maze

Administration of PTX, a GABA antagonist, decreased the movement of animals towards the open arms of EPM, compared to the control group (P < 0.05). There was a significant increase in the frequency of animal entries to open arm, time spent in the open arm, and time spent in the central position by the supplementation of TA and, positive control (DIZ) (P < 0.05). However, there was no significant difference observed among the different treatment groups, i.e. TA-25, TA-50 and TA-100 (Fig. 4).

Figure 4 Effects of TA on elevated plus maze (EPM). A: Open arm entries (%); B: Time spent in open arm (%); C: Time spent in central position (%); D: Time spent in closed arm (%). Con: control group; PTX: picrotoxin (1 mg·kg-1); DIZ: diazepam (1.5 mg·kg-1); TA 25, TA 50 and TA 100: TA treatment with 25, 50 and 100 mg·kg-1, respectively. Values are expressed as mean ± SD; n = 7 in each group, #P < 0.05 vs Con; *P < 0.05 vs PTX
Effects of TA in the light-dark box conflict

The treatment with TA-25 did not alter the parameters in light-dark conflict. Higher concentrations of the treatment, i.e. TA-50 and TA-100 showed significant positive difference (P < 0.05), compared to PTX group in the light avoidance paradigm, for instance, number of crossings, time spent in the light area, and rearing frequency (Fig. 5I).

Figure 5 Effects of TA on light/dark box and open filed conflicts. Ⅰ: Light/ dark box conflict, A) Total no. of crossings, B) Time spent in light box in seconds C) Total rearing frequency. Ⅱ: Open field test (OFT), A) Locomotion in seconds, B) Time spent in central position in seconds C) Total rearing frequency. Values are expressed as mean ± SD; n = 7 in each group, #P < 0.05 vs Con; *P < 0.05 vs PTX. Con: control group; PTX: picrotoxin (1 mg·kg-1); DIZ: diazepam (1.5 mg·kg-1); TA 25, TA 50 and TA 100: TA treatment with 25, 50 and 100 mg·kg-1, respectively
Effects of TA in the open field conflict

Picrotoxin administration had negative affect on the locomotion of the animal in the open field conflict. As shown in Fig. 5II, supplementation of TA and DIZ showed a markedly significant difference in locomotor activity of the mice (P < 0.05). The TA treatment (25-100 mg·kg-1) produced a dose-dependent increase in the total locomotion.

Effects of TA in the Vogel's conflict

To confirm the anxiolytic like effect of TA, the Vogel conflict test was performed. Supplementation of TA at higher concentrations (TA-50 and TA-100) significantly (P < 0.05) increased the number of punished licks and shocks in the Vogel's conflict. However, TA-25 treatment did not show any significant change in the number of punished shocks (Fig. 6). As expected, DIZ significantly increased the number of punished licks and shocks (P < 0.05).

Figure 6 Effects of TA on Vogel's conflict. A: Total no. of licks; B: Total no. of shocks. Values are expressed as mean ± SD; n = 7 in each group, #P < 0.05 vs Con; *P < 0.05 vs PTX. Con: control group; PTX: picrotoxin (1 mg·kg-1); DIZ: diazepam (1.5 mg·kg-1); TA 25, TA 50 and TA 100: TA treatment with 25, 50 and 100 mg·kg-1, respectively
Effects of TA on modulation of gene expressions

Fig. 7 shows the effects of TA on modulation of expression of genes related to the anxiety like disorders. The study was focused on mode of expression of various genes involved in the neurotransmitter mechanism, viz., GABAergic, serotonergic and dopamergic systems, and antioxidant system such as GABAA, 5-HT1A, D2L, Ip3, BDNF, CREB, SOD, GR, and GPx. It was observed that PTX negatively regulated all the studied genes. Supplementation of TA up-regulated the expression of several genes, such as BDNF, IP3, CREB, GABAA, SOD, GPx, and GR, and down-regulated 5HT1Aexpression. The regulation of gene expression by TA supplementation was found to be dose dependent (25-100 mg·kg-1) with exception of CREB, 5-HT1A and D2L genes. Administration of DIZ (1.5 mg·kg-1; p.o.) reversed the PTX effects by bringing back the mRNA levels to that of control group. Western blotting was used to confirm the role of TA on antioxidant enzymes, like GR and CAT, and significant protective effects were found, compared to that of the PTX group (Fig. 8).

Figure 7 Effects of TA on modulation of gene expressions. Ⅰ: Gel images; Ⅱ: Densitometric analysis of gel images. GAPDH: Glyceraldehyde 3-phosphate dehydrogenase, BDNF: brain derived neurotrophic factor, IP3: inositol triphosphate, CREB-CRE: binding protein, GABAA: γ-amino butyric acid receptor A, 5-HT1A: 5-hydroxy tryptamine receptor 1A, D2L: dopamine receptor 2L, SOD: superoxide dismutase, GPx: glutathione peroxidise, GR: glutathione reductase, Con: control group, PTX: picrotoxin (negative control), DIZ: diazepam (positive control), TA 25, TA 50 and TA 100: TA treatment groups 25, 50 and 100 mg·kg-1, respectively
Figure 8 Western blot analysis. Ⅰ: Western blot images; Ⅱ: Densitometric analysis of western blot images. GAPDH: Glyceraldehyde 3-phosphate dehydrogenase, GR: glutathione reductase, CAT: catalase, Con: control group; PTX: picrotoxin (negative control); DIZ: diazepam (positive control); TA 25, TA 50 and TA 100: TA treatment groups 25, 50, and 100 mg·kg-1, respectively. #P < 0.05 vs Con; *P < 0.05 vs PTX
Effects on the mRNA expression of GABAA in brain

Quantitative RT-PCR was employed to check the mRNA expression of GABAAgene in the brain. Compared to the PTX group, the mice from TA treated groups showed increased expression of GABAA.The relative expression levels of mRNA of targeted gene are shown in Fig. 9.

Figure 9 Gene expression of GABAA receptor by RT-PCR. Values are expressed as mean ± SD; n = 7 in each group, #P < 0.05 vs Con; *P < 0.05 vs PTX. Con: control group, PTX: picrotoxin (1 mg·kg-1), DIZ: diazepam (1.5 mg·kg-1), TA 25, TA 50 and TA 100: TA treatment with 25, 50, and 100 mg·kg-1, respectively

Changes in social and emotional behavior of an individual reflect the decision-making process in the brain. Several pre-clinical experimental models have been developed to evaluate the behavioral pharmacology of anxiety-like disorders [30]. Open field paradigm (OF) is commonly used to assess anxiety-related behavior in experimental animals such as locomotion, motivation, exploration, habituation and spatial learning [30]. In the present study, PTX group showed decreased locomotion and increased grooming activity in the experimental mice. However, TA supplementation showed an increase in the central locomotion and time spent in the central part of the open field device in a dose-dependent manner.

Anxiolytic drugs diminish animal aversion to the open arms of EPM and promote the exploration thereof. On the other hand, the voluntary movement of the animal towards closed arms of the EPM is linked with hormonal and behavioral changes. It has been suggested that the avoidance of the open arms of the EPM is a sign indicating increased anxiety, which is caused by fear of open spaces (agoraphobia) [31]. When compared to PTX group, TA supplemented group showed a tendency of spending significant increased time in the open arms. Light-dark paradigm was used in the present study to test the conflict between leaving a familiar dark area to explore a non-familiar bright area (Passive avoidance test). In the present study, DIZ and TA supplementation increased the time spent by the mice in the light compartment and increased the number of transitions between light-dark compartments. The effects of supplementation of TA and DIZ on mice were in line with the activity of other anxiolytic drugs like desipramine, amitriptyline (anti-depressants), phenelzine (monoamine oxidase inhibitor), benzodiazepines, paroxetine, fluoxetine and mianserin [32-33]. Previous reports on GABAergic agents like chlordiazepoxide and benzodiazepines have found increaed licks and shocks in water deprived rats [34]. In the present study, PTX induced anxiety created fear in mice, as observed by the decreased number of licks and shocks. However, TA reversed the PTX-induced behavior and acted as a GABAergic agent by motivating the animals to drink.

Phytochemical analysis of the Terminalia arjuna bark showed the presence of high quantity of polyphenols, flavonoids, condensed tannins, and triterpenoids. In the present study, LC-MS analysis demonstrated that the TA bark extract was rich in several polyphenolic compounds that may possess anxiolytic activity. Polyphenols identified in the present study like catechin, epicatechin, gallocatechin, epigallo-catechin, epigalocatechin galate and (+)-epicatechin-3-O-gallate were previously reported with protective role in neuronal pathways (serotonergic, dopaminergic, and GABAergic systems) [35-36]. Further, our HPLC results provided information regarding presence of bioactive triterpenoids, viz., arjunic acid, arjungenin and arjunetin. Previous investigation showed that these triterpenoids have cardioprotective, anti-cancerous, and hepatoprotective properties [37]. Alcoholic extract of T. arjuna contained several other phytochemicals, like arjunoglycoside Ⅰ-Ⅲ, arjunoside Ⅰ-Ⅳ , tomentosic acid, oleanolic acid, methyloleanolate, and maslinic acid, which may not directly affect the pathophysiological processes of anxiolytic activity. However, these phytochemicals may modify the absorption, distribution, metabolism, and excretion of bioactive constituents [38].

Oxidative stress is the most vulnerable mechanism in the development and progression of anxiety and other neurological disorders. Our previous report has suggested that TA extract can act as a potent antioxidant agent and eliminates several free radicals, such as O2-, HO-, H2O2, and NO [18]. The present gene expression studies on antioxidant enzymes revealed that PTX decreased SOD, GPx and GR levels and TA down-regulated free radical generation at molecular level and reversed the trend of antioxidant enzyme expressions.

Several reports have indicated that sub-convulsive doses of PTX induce anxiogenic effects such as increase in heart rate, increase plasma catecholamine levels, changes in behavioral conflicts in the elevated plus maze, open field, light dark and social interaction [39-40]. Our observations in behavioral models were further substantiated by the modulation of neurotransmitters levels. Drugs that can interact with postsynaptic monoaminergic receptors may possess complementary mechanisms of anxiolytic properties. Monoamine receptors modulate the cyclic-AMP pathway by increasing cAMP levels, which leads to the activation of the gene transcription factor cAMP responsive element-binding protein (CREB). Previous studies have reported that CREB-dependent BDNF expression is linked to pathophysiology of anxiety, and that the decreased CREB function might be operative in maintaining high anxiety levels [41-42]. Present mRNA expression data were in line with these previous studies: PTX treatment might be involved in signal transduction of cAMP pathway and consequently the expression of CREB was down-regulated. However, known anxiolytic drug (DIZ) and TA treatment groups reversed this CREB expression pattern.

Brain derived neuro factor (BDNF) is in controlling emotions by regulating the plasticity and integrity of neurons in brain circuits [41]. Previous studies have shown considerable evidence supporting BDNF role in the pathogenesis of anxiety [41]. The persistent down-regulation of BDNF expression observed in the brain tissue of PTX treated mice in the present study was thus likely related to the anxiety that was confirmed by the behavioral paradigms like, elevated plus-maze test, light-dark test, and exploratory open field test. The anxiolytic effect of TA may be mediated by increasing expression of the brain-derived neurotrophic factor (BDNF), since all three doses of TA significantly up-regulated the BDNF expression. The increased expression of BDNF might be related to the atrophy caused by PTX-induced stress in hippocampal CA3 neurons [42].

Dopamine (DA) is a neurotransmitter that exerts mood elevation effects on postsynaptic neurons through interaction with dopamine receptors (D2L). Previous studies [43] have revealed that ROS have an adverse effect on modulation of dopaminergic system. In the present study, the D2L expression levels were drastically reduced upon PTX, which was reversed by DIZ treatment. However, the mRNA expression studies showed that TA significantly up-regulated the D2L levels. Excessive ROS may metabolize DA via monoamine oxidase resulting in production of H2O2 and dihydroxyphenylacetic acid, which further may lead to non-enzymatic hydroxylation, resulting in production of 6-hydroxydopamine [43]. 6-hydroxydopamine is intrinsically unstable and is converted into p-quinone, finally leading to apoptosis via caspase-8 pathway. Previous reports have proven that glutathione S-transferase (GST) and glutathione reductase (GR) enzymes might alter the toxic effects of 6-hydroxydopamine through primary antioxidant system of brain [43]. This may be one possible mechanism in the present study, supporting the elevated m-RNA expression levels of glutathione reductase and consequent up-regulation of dopamine levels.

Clinical and pre-clinical studies have suggested that serotonergic neurotransmission may be involved in the wide range of psychiatric disorders, such as anxiety, depression, post-traumatic stress, schizophrenia, anorexia nervosa, obsessive-compulsive disorder, and attention-deficit disorder [44]. There are at least 14 different types of serotonin receptors that have been identified, and among them 5HT1A receptor is thought to play an important role in the etiology of anxiety [45]. However, there is a contradiction about the exact role of 5-HT neurotransmission; some reports support both anxiogenic and anxiolytic roles of 5-HT in anxiety/depression like disorders [46]. Previous findings on 5-HT action in animal behavior models have revealed that 5-HT reuptake blockers, 5-HT synthesis precursor, 5-HTP, and some 5-HT receptor agonists impair same kind of behavior [46]. In the present study, 5-HT1A receptor expression was down-regulated by TA supplementation, and decreased activation of 5-HT1A may be suppressed anxiety like behavior through multiple neurotransmitter pathways and neuro circuits including GABAergic, glutamergic, noradrergic and dopaminergic transmission (Fig. 10).

Figure 10 Schematic representation of possible interactions underlying the anxiolytic properties of TA against PTX-induced neurodegeneration

Several receptors have been identified that are coupled to the inositol phospholipid system, including cholinergic, adrenergic, serotonergic, histaminergic, glutaminergic, and dopaminergic receptors [47]. IP3 plays a major role in patients with anxiety, depression, attention deficit hyperactivity disorder, and related CNS disorders [48]. The results of the present study confirmed that changes in IP3 expression levels were coupled with neurotransmitter systems. Behavioral and electrophysiological evidence substantiates that GABA receptor role in neuro-physiological modulation [49]. Earlier post-mortem studies have shown that low brain levels of GABA in rodents exhibit anxiety like behavior [50]. In the present study, TA treatment and DIZ enhanced the anxiolytic activity via positive modulation of the GABAA receptor.


Therapeutic drugs derived from medicinal plants may have possible remedy with minimum side effects in the treatment of anxiety-like disorders. The results obtained in the present study suggested that alcoholic extract of TA possessed an anxiolytic like activities in behavioral paradigms of mice model. Terminalia arjuna bark alcoholic extract was rich with many bioactive compounds and had a major role in scavenging the reactive oxygen species. Gene expression results of the present study support the effect of TA extract on positive modulation of genes related to seratogenic, GABAergic and dopamergic systems under anxiety-like conditions. However, further clinical investigations are needed to confirm TA as an anxiolytic drug.


We are thankful to Director, DFRL, DRDO and Mysore for his kind support and keen interest.

Clement Y, Chapouthier G. Biological bases of anxiety[J]. Neurosci Biobehav Rev, 1998, 22(5): 623-633. DOI:10.1016/S0149-7634(97)00058-4
Nutt DJ. Overview of diagnosis and drug treatments of anxiety disorders[J]. CNS Spectrums, 2005, 10(1): 49-56. DOI:10.1017/S1092852900009901
Nikolaus S, Antke C, Beu M, et al. Cortical GABA, striatal dopamine and midbrain serotonin as the key players in compulsive and anxiety disorders-results from in vivo imaging studies[J]. Rev Neurosci, 2010, 21(2): 119-139.
Malberg JE, Eisch AJ, Nestler EJ, et al. Chronic anti-depressant treatment increases neurogenesis in adult rat hippocampus[J]. J Neurosci, 2000, 20(24): 9104-9110.
Miller AL. Botanical influences on cardiovascular disease. Alternative medicine review[J]. J Clin Therapeutics, 1998, 3(6): 422-431.
Manji HK, Quiroz JA, Sporn J, et al. Enhancing neuronal plasticity and cellular resilience to develop novel, improved therapeutics for difficult-to-treat depression[J]. Biol Psychiatry, 2003, 53(8): 707-742. DOI:10.1016/S0006-3223(03)00117-3
Kendall PC. Empirically supported psychological therapies[J]. J Consult Clin Psychol, 1998, 66(1): 3-6. DOI:10.1037/0022-006X.66.1.3
Goldberg HL. Buspirone hydrochloride: a unique new anxiolytic agent; pharmacokinetics, clinical pharmacology, abuse potential and clinical efficacy[J]. Pharmacother: J Hum Pharmacol Drug Ther, 1984, 4(6): 315-321. DOI:10.1002/j.1875-9114.1984.tb03385.x
Wong AH, Smith M, Boon HS. Herbal remedies in psychiatric practice[J]. Arch Gen Psychiatry, 1998, 55(11): 1033-1044. DOI:10.1001/archpsyc.55.11.1033
Hasan SS, Ahmed SI, Bukhari NI, et al. Use of com-plementary and alternative medicine among patients with chronic diseases at outpatient clinics[J]. Complement Ther Clin Pract, 2009, 15(3): 152-157. DOI:10.1016/j.ctcp.2009.02.003
Jain S, Yadav PP, Gill V, et al. Terminalia arjuna a sacred medicinal plant: phytochemical and pharmacological profile[J]. Phytochem Rev, 2009, 8(2): 491-502. DOI:10.1007/s11101-009-9134-8
Dwivedi S. Terminalia arjuna Wight Arn.-a useful drug for cardiovascular disorders[J]. J Ethnopharmacol, 2007, 114(2): 114-129. DOI:10.1016/j.jep.2007.08.003
Kumar DS, Prabhakar YS. On the ethnomedical sig-nificance of the arjuna tree, Terminalia arjuna (Roxb.) Wight & Arnot[J]. J Ethnopharmacol, 1987, 20(2): 173-190. DOI:10.1016/0378-8741(87)90086-9
Kapoor D, Vijayvergiyab R, Dhawan V. Terminalia arjuna in coronary artery disease: Ethnopharmacology, pre-clinical, clinical and safety evaluation[J]. J Ethnopharmacol, 2014, 155(2): 1029-1045. DOI:10.1016/j.jep.2014.06.056
Hemalatha T, Pulavendran S, Balachandran C, et al. Arjunolic acid: A novel phytomedicine with multi-functional therapeutic applications[J]. Indian J Exp Biol, 2010, 48(3): 238-247.
Sinha M, Manna P, Sil PC. Terminalia arjuna protects mouse hearts against sodium fluoride-induced oxidative stress[J]. J Med Food, 2008, 11(4): 733-740. DOI:10.1089/jmf.2007.0130
Packer L, Rimbach G, Virgili F. Antioxidant activity and biologic properties of a procyanidin-rich extract from pine (Pinus maritima) bark, pycnogenol[J]. Free Radic Biol Med, 1999, 27(5-6): 704-724. DOI:10.1016/S0891-5849(99)00090-8
Kumar GP, Navya K, Ramya EM, et al. DNA damage protecting and free radical scavenging properties of Terminalia arjuna bark in PC-12 cells and plasmid DNA[J]. Free Radic Antiox, 2013, 3(1): 35-39. DOI:10.1016/j.fra.2013.04.001
Subramaniam S, Subramaniam R, Rajapandian S, et al. Anti-atherogenic activity of ethanolic fraction of Terminalia arjuna bark on hypercholesterolemic rabbits[J]. Evid Based Complement Alternat Med, 2011, 2011: 487916.
Dombrowski A, Fernandes LH, Andreatini R. Picrotoxin blocks the anxiolytic-and panicolytic-like effects of sodium valproate in the rat elevated T-maze[J]. Eur J Pharmacol, 2006, 537(1): 72-76.
Carro-Juarez MI, Rodríguez-Landa JF, Rodríguez-Peña ML, et al. The aqueous crude extract of Montanoa frutescens produces anxiolytic-like effects similarly to diazepam in Wistar rats: Involvement of GABAA receptor[J]. J Ethnopharmacol, 2012, 143(2): 592-598. DOI:10.1016/j.jep.2012.07.022
Verma SC, Jain CL, Padhi MM, et al. Microwave extraction and rapid isolation of arjunic acid from Terminalia arjuna (Roxb. ex DC.) stem bark and quantification of arjunic acid and arjunolic acid using HPLC-PDA technique[J]. J Sep Sci, 2012, 35(13): 1627-1633. DOI:10.1002/jssc.201200083
Asano Y. Characteristics of open field behaviour of Wistar and Sprague-Dawley rats[J]. Exp Anim, 1986, 35(4): 505-508. DOI:10.1538/expanim1978.35.4_505
Yemitan OK, Adeyemi OO. Anxiolytic and mus-cle-relaxant activities of Dalbergia saxatilis[J]. West Afr J Pharmacol Drug Res, 2004, 19(1): 42-46.
Costall B, Jones BJ, Kelly ME, et al. Exploration of mice in a black and white test box: validation of a model of anxiety[J]. Pharmacol Biochem Behav, 1989, 32(3): 777-785. DOI:10.1016/0091-3057(89)90033-6
Garg DV, Dhar VJ, Sharma A, et al. Experimental model for antianxiety activity: a review[J]. Pharmacol Online, 2011(1): 394-404.
Livak KJ, Thomas DS. Analysis of relative gene ex-pression data using real-time quantitative PCR and the 2-ΔΔCT method[J]. Methods, 2001, 25(4): 402-408. DOI:10.1006/meth.2001.1262
Lowry OH, Rosebrough NJ, Farr AL, et al. Protein measurement with the Folin phenol reagent[J]. J Boil Chem, 1951, 193(1): 265-275.
Jayaraj R, Anand T, Rao PL, et al. Activity and gene expression profile of certain antioxidant enzymes to microcystin-LR induced oxidative stress in mice[J]. Toxicol, 2006, 220(2-3): 136-146. DOI:10.1016/j.tox.2005.12.007
Bhattacharya SK, Bhattacharya, Chakrabarti A. Adaptogenic activity of Siotone, a poly herbal formulation of Ayurvedic rasayanas[J]. Indian J Exp Biol, 2000, 38(2): 119-128.
Santos FJBD, Lima SGD, Cerqueira GS, et al. Chemical composition and anxiolytic-like effects of the Bauhinia platypetala[J]. Rev Bras Farmacogn, 2012, 22: 507-516. DOI:10.1590/S0102-695X2012005000018
Crawley J, Goodwin FK. Preliminary report of a simple animal behavior model for the anxiolytic effects of benzodiazepines[J]. Pharmacol Biochem Behav, 1980, 13(2): 167-170. DOI:10.1016/0091-3057(80)90067-2
Cadogan AK, Wright IK, Coombs I, et al. Repeated paroxetine administration in the rat produces an anxiolytic profile in the elevated X-maze and a decreased 3H-ketanserin binding[J]. Neurosci Lett, 1992, 42: S8.
Cooper SJ. Benzodiazepine mechanisms and drinking in the water-deprived rat[J]. Neuropharmacol, 1982, 21(8): 775-780. DOI:10.1016/0028-3908(82)90064-8
Johnston Graham AR. Flavonoid nutraceuticals and ionotropic receptors for the inhibitory neurotransmitter GABA[J]. Neurochem Int, 2015, 89: 120-125. DOI:10.1016/j.neuint.2015.07.013
Adachi N, Tomonaga S, Tachibana T, et al. (-)-Epigallocatechin gallate attenuates acute stress responses through GABAergic system in the brain[J]. Eur J Pharmacol, 2006, 531(1): 171-175.
Patil UH, Gaikwad DK. Medicinal profile of a scared drug in Ayurveda: Crataeva religosa[J]. J Pharm Sci Res, 2011, 3(1): 923-929.
Andrea JD, Fred M, Denis B, et al. Human metabolism of dietary flavonoids: identification of plasma metabolites of quercetin[J]. Free Radic Res, 2001, 35(6): 941-952. DOI:10.1080/10715760100301441
File SE, Lister RG. Do the reductions in social interaction produced by picrotoxin and pentylenetetrazole indicate anxiogenic actions?[J]. Neuropharmacol, 1984, 23(7): 793-796. DOI:10.1016/0028-3908(84)90113-8
Bazyan AS, Van Luijtelaar G. Neurochemical and behavioral features in genetic absence epilepsy and in acutely induced absence seizures[J]. ISRN Neurol, 2013, 2013: 875-834.
Duman RS, Monteggia LM. A neurotrophic model for stress-related mood disorders[J]. Biol Psychiatry, 2006, 59(12): 1116-1127. DOI:10.1016/j.biopsych.2006.02.013
McEwen BS. Glucocorticoids, depression, and mood disorders: structural remodeling in the brain[J]. Metabol, 2005, 54(5): 20-23. DOI:10.1016/j.metabol.2005.01.008
Vadnal R, Parthasarathy L, Parthasarathy R. Role of inositol in the treatment of psychiatric disorders[J]. CNS Drugs, 1997, 7(1): 6-16. DOI:10.2165/00023210-199707010-00002
Livak KJ, Thomas DS. Analysis of relative gene ex-pression data using real-time quantitative PCR and the 2-ΔΔCT method[J]. Methods, 2001, 25(4): 402-408. DOI:10.1006/meth.2001.1262
Haleem DJ. Extending therapeutic use of psy-chostimulants: Focus on serotonin-1A receptor[J]. Prog Neuropsychopharma-col Biol Psychiatry, 2013, 46(10): 170-180.
Graeff FG, Guimaraes FS. Role of 5-HT in stress, anxiety, and depression[J]. Pharmacol Biochem Behav, 1996, 54(1): 129-141. DOI:10.1016/0091-3057(95)02135-3
Tang L, Todd RD, OMalley KL. Dopamine D2 and D3 receptors inhibit dopamine release[J]. J Pharmacol Exp Ther, 1994, 270(2): 475-479.
Vadnal R, Parthasarathy L, Parthasarathy R. Role of inositol in the treatment of psychiatric disorders[J]. CNS Drugs, 1997, 7(1): 6-16. DOI:10.2165/00023210-199707010-00002
Puia G, Mienville JM, Matsumoto K, et al. On the putative physiological role of allopregnanolone on GABAA receptor function[J]. Neuropharmacol, 2003, 44(1): 49-55. DOI:10.1016/S0028-3908(02)00341-6
Guillot PV, Chapouthier G. Inter male aggression, GAD activity in the olfactory bulbs and Y chromosome effect in seven inbred mouse strains[J]. Behav Brain Res, 1998, 90(2): 203-206. DOI:10.1016/S0166-4328(97)00110-1