Effect of Hydroalcoholic Extract of Centella Asiatica and its Synergy with N–Acetyl Cysteine on Marble– Burying Behavior in Mice: Implications for Obsessive– Compulsive Disorder

Research Article

Austin J Pharmacol Ther. 2016; 4(2).1083.

Effect of Hydroalcoholic Extract of Centella Asiatica and its Synergy with N–Acetyl Cysteine on Marble– Burying Behavior in Mice: Implications for Obsessive– Compulsive Disorder

Shivani Sharma1 and Girdhari Lal Gupta2*

1Department of Pharmacy, Jaypee University of Information Technology, India

2Department of Pharmacology, School of Pharmacy and Technology Management, India

*Corresponding author: Girdhari Lal Gupta, Department of Pharmacology, School of Pharmacy and Technology Management, SVKM’s NMIMS University, V. L. Mehta Road, Vile Parle (W), Mumbai - 400056, Maharashtra, India

Received: May 25, 2016; Accepted: July 18, 2016; Published: July 28, 2016


Traditionally, Centella asiatica Lin. (CA) whole plant is used as anti– compulsive, memory enhancing, anxiolytic and antidepressant effects. However, no scientific validation is available on the anti–compulsive potential of the plant. To reduce the doses of CA and N–acetyl cysteine combination study was also carried out to target two different neurotransmitters. This study was carried out to examine the effect of CA whole plant hydroalcoholic extract and its combination with N–acetyl cysteine on marble–burying behavior test. Mice from different groups were administered with vehicle, CA (50, 100, 200 or 500 mg/kg), N–Acetyl–L–Cysteine (NAC) (25, 50, 100 or 200 mg/kg) and fluoxetine (5, 10 or 20 mg/kg). One hour after CA and thirty minutes after NAC or fluoxetine administration, individual mice was subjected to marble burying test for thirty minutes and actophotometer for ten minutes. CA (200 and 500 mg/kg, p.o.), NAC (100 and 200 mg/kg, p.o.) and fluoxetine (10 and 20 mg/ kg, p.o.) significantly inhibited marble–burying behavior in mice. Moreover, in separate experiments, co–treatment with sub–effective dose of both CA (50 or 100 mg/kg, p.o.) and NAC (25 or 50 mg/kg, p.o.) significantly decreased marble–burying behavior at doses, which either of these alone could not protect the behavior. All dose levels of CA, NAC and fluoxetine showed insignificant changes in locomotor activity in actophotometer. Our results reveal that Centella asiatica possesses anti–compulsive activity and co–administration of CA with nutraceutical N–acetyl cysteine also show synergistic effects in reducing obsessive–compulsive behavior in mice.

Keywords: Centella asiatica; Fluoxetine; Obsessive–compulsive disorder


Obsessive–compulsive disorder (OCD) is a mental disorder characterized by persistent intrusive thoughts (obsessions) and the expression of ritualistic repetitive behaviors (compulsion). The patient spends greater than one hour time each day in symptoms like doubting, checking, washing associated with OCD [1]. The World Health Organization has identified OCD as one of the top 10 disabling illnesses by lost income and decreased quality of life [2]. Financial cost associated with health care in the USA estimated to be around $10.6 billion per annum [3]. Lifetime prevalence in India and USA has been estimated 0.6% and 2–3% respectively [4,5]. The pathogenesis of OCD involved glutamatergic and serotonergic pathway dysregulation in orbito frontal cortex, anterior cingulate cortex and caudate nucleus [6]. Treatment of OCD with first–line selective serotonin reuptake inhibitors have also been reported with limited benefit and associated side effects [7]. Therefore, it is always desired worldwide to opt for safer and effective nutraceuticals [8] and plant–derived anti-compulsives mentioned in the traditional medical systems.

Centella asiatica Lin. (Umbelliferae/Apiaceae family) is a perennial creeping herb. Since ancient times, it has been used as anti–OCD, memory enhancing, anxiolytic, antidepressant, strength promoting, wound healing, immune booster, anti–epileptic and anti–stress herbal medicine. However, no scientific validation is available on the anti– compulsive potential of the plant. The main active principles of CA are the triterpenoids glycosides such as asiaticoside, madecassoside (asiaticoside A) and asiatic acid [9,10]. Moreover, total triterpenes of CA has already been reported to increase serotonin contents in rat brain [9], which was also taken as the strategy for selection of the plant first time to reverse the depletion of serotonin content in OCD.

N–Acetyl–L–Cysteine (NAC) has been evaluated in a diversity of neuropsychiatric disorders, including OCD, schizophrenia and bipolar disorder and appears to have evidence of both safety and tolerability. NAC has been reported for attenuating glutamatergic neurotransmission [11]. NAC augmentation in SSRI treatment– resistant obsessive–compulsive disorder has also been reported. Therefore, the present study was undertaken to investigate the effects of CA alone and its combinations with NAC to target two different neurotransmitter system in marble–burying behavior of mice– a well–accepted model of obsessive–compulsive disorder.

Materials and Methods

Plant material

Dried whole plant of Centella asiatica (CA) was purchased from Natural Remedies, Bangalore (Batch Number: ERD–40). The plant was coarsely powdered with a mechanical grinder and subjected to defatting with petroleum ether (60°–80 °C). The defatted air dried powder was extracted by Soxhlet apparatus using 70% v/v of ethanol at 50 °C for 48 hours. The solvent was evaporated under reduced pressure by rotary evaporator (Heidolph, Germany) to obtain a semisolid mass, which was further lyophilized (New Brunswick) for 24 hours. The obtained dry powder was stored at –20 °C till further use. The percentage extractive value was also calculated in terms of air–dried weight of plant material.


For standardization of CA extract, active marker compounds like asiatic acid, asiaticoside and madecassoside plus asiaticoside B were purchased from Natural Remedies, Bangalore. Parachlorophenylalanine, NAC, HPLC grade phosphoric acid and acetonitrile were purchased from Sigma Aldrich, USA. Fluoxetine was obtained from Cadila Pharmaceuticals Limited, India.

Standardization of CA extract by high performance liquid chromatography (HPLC)

Whole plant hydroalcoholic extract of CA was standardized for their triterpene composition like asiatic acid, asiaticoside and madecassoside plus asiaticoside B by high–performance liquid chromatography [10]. Each triterpene marker compound asiatic acid, madecassoside plus asiaticoside B and asiaticoside were diluted with HPLC grade methanol to obtain concentration of 200 μg/ml. HPLC system (Shimadzu, LC 2010 CHT), photodiode array detector (Shimadzu), Phenomenex– Luna 5μm (250 mm × 4.6 mm) C18 column and a personal computer with Empower software were used for analysis at detection wavelength 210 nm. The flow rate 1.5 ml/min of mobile phase having 0.05% phosphoric acid and 100% acetonitrile was maintained during separation. For sample preparation, hydroalcoholic CA extract 200 mg was dissolved in 50 ml of methanol by ultrasonic. Marker compounds and sample were filtered through a 0.45 μm nylon membrane filter paper thereafter three injections of 20 μl were performed. The standard curve was prepared by plotting peak area against concentration of marker compounds and concentrations of triterpenes in the sample were estimated from the standard curve.


Swiss male albino mice weighing 25 to 30 g were housed separately in groups of five per cage under controlled light (12:12 hrs light: dark cycle), relative humidity of 75 ± 5% and temperature (25 ± 2 °C) environment and behavioral assessment was conducted during the light cycle. Water and basal diet (Ashirwad, Chandigarh, India) were provided ad libitum. All procedures were carried out after due approval and under strict compliance with ethical principles and guidelines of the Institutional Animal Ethical Committee constituted as per CPCSEA (1716/PO/a/13/CPCSEA). All the experiments were carried out in a noise free room. Separate groups (n=6) were used for each set of experiment.

Preparation of test drugs

Parachlorophenylalanine (PCPA)–a serotonin depleting agent, was dissolved in 0.9% w/v sodium chloride. CA was suspended in 2% w/v of gum acacia in distilled water. The stock solution contained 100 mg/ml of CA. NAC and fluoxetine were dissolved in distilled water. PCPA was administered by the intraperitoneal route. All drugs except PCPA were administered via the oral route.

Selection of dose

The doses of CA extract, NAC were determined from previous studies and based on our another acute toxicity study, which were carried out according to Organisation for Economic Co–operation and Development (OECD) test guidelines 423 (Unpublished data). The hydroalcoholic extract of CA and NAC were found to be safe up to 2000 mg/kg. There was no mortality of animals in any of the groups used in different experimental design (data not shown).

Marble–burying behavior test

The anti–compulsive effect was assessed by the marble–burying behavior due to high predictive and good face validity. In brief, twenty small marbles of glass (diameter 10 mm), were arranged on the bedding evenly spaced in four rows of five. Individual mouse from different group was placed in separate plastic cages (21×38×14 cm) containing 5 cm thick sawdust bedding. After 30 min exposure to the marbles, mice were removed, and unburied marbles were counted. A marble was considered ‘buried’ if its two–third size was covered with sawdust. The total number of marbles buried was considered as an index of obsessive–compulsive behavior [12,13].

Locomotor activity tests

Motor activity was also assessed in all groups of mice using actophotometer (Techno, Lukhnow) for the duration of 10 min. Actophotometer operates on photoelectric cells that are connected in circuit with a digital counter. A count is recorded when the movement of animal cuts off the beam of light falling on the photocell. Every time the central arena was wiped out with the wet cotton and removed the fecal pellets [12].

Experimental design

Each experimental group had a separate set of mice being allotted randomly. All subjects were experimentally naïve at the beginning of each study. In all groups, mice were pretreated with PCPA– a serotonin depleting agent, once daily at a dose of 300 mg/kg for three consecutive days and 24h thereafter all drug treatment was given. Mice from different groups were administered with vehicle (2% w/v gum acacia in distilled water, p.o., n=6), CA (50, 100, 200 or 500 mg/ kg, p.o., n=6 per group), NAC (25, 50, 100 or 200 mg/kg, p.o., n=6 per group) and fluoxetine (5, 10, 20 mg/kg, p.o., n=6 per group). One hour after CA and thirty minutes after NAC or fluoxetine administration, individual mouse was subjected to marble burying test for thirty minutes and in actophotometer for ten minutes. In combination studies, CA (50 or 100 mg/kg, p.o., n=6 per group) was administered 30 min prior to NAC (25 or 50 mg/kg, p.o., n=6 per group) and 30 min thereafter individual mouse was subjected to marble burying test and actophotometer test. The doses of PCPA and fluoxetine were based on previous reports [14].

Statistical analysis

All data were expressed as mean ± SEM (n=6) and analyzed by one–way analysis of variance (ANOVA), followed by post hoc Student Newman–Keuls test. The groups treated with extract, NAC and fluoxetine were compared with vehicle group. The groups treated with combination of CA and NAC were compared with the respective CA group. Differences were considered to be significant at P<0.05.


The percentage yield of the hydroalcoholic CA extract was 24% w/w. Figure 1 shows the HPLC chromatograms of CA extract (A) and marker compounds (B). On HPLC analysis for quality assurance, whole plant hydroalcoholic extract of CA showed the presence of asiaticoside (6.3% w/w), madecassoside plus asiaticoside B (6.1% w/w) and asiatic acid (2.2 % w/w).