Effect of hydroalcoholic extract of leaves of Colocasia esculenta on marble-burying behavior in mice: Implications for obsessive–compulsive disorder

Abstract

Context: Over the past decades, the inhibition of spontaneous burying of glass marbles by mice has been used as an index of anxiolytic drug action in the so-called marble-burying test. Although Colocasia esculenta Linn. (Araceae), commonly known as elephant ear (English), possesses several medicinal properties, little is known for its use in neurological activity.

Objective: The current research evaluated the anti-obsessive–compulsive disorder (anti-compulsive) activity of the hydroalcoholic extract of leaves of Colocasia esculenta (HECE) for the first time using the marble-burying behavior test in mice.

Materials and methods: In the present study, the effect of HECE (25 and 50 mg/kg) intraperitoneally (i.p.) was examined using the marble-burying behavior test, which is an animal model of obsessive compulsive disorder (OCD), using Swiss albino mice.

Results and discussion: The acute toxicity studies showed that the LD₅₀ value of the HECE in mice was 1000 mg/kg by i.p. route. The effect of HECE (25 and 50 mg/kg, i.p.) was characterized by significant reduction in the number of buried marbles as compared with the control group (p < 0.001). The effect of HECE was comparable with that of fluoxetine (5 mg/kg, i.p.) – a reference standard drug used in the treatment of obsessive–compulsive disorder (p < 0.001). Fluoxetine and HECE do not produce any overt motor dysfunction.

Conclusions: The results of the study for the first time show that the plant possesses anti-compulsive activity, confirming the traditional claims. Future research should focus on the identification and the anti-compulsive activity of the constituents from this plant.

• Fluoxetine
• marble-burying behavior
• preliminary phytochemical screening

Introduction

Obsessive–compulsive disorder (OCD) is characterized by persistent ritualistic thoughts (compulsions), which are ego-diatomic and associated with seemingly purposeful behaviors (compulsions) (Rasmussen & Eisen, 1992). Its co-morbidity with major depression is often evident, and it is considered as an anxiety disorder that is refractory to benzodiazepines (Bartz & Hollander, 2006). Its lifetime prevalence rate is about 1–3% (Rasmussen & Eisen, 1992). Only potent serotonin reuptake inhibitors (SSRIs) are consistently effective in patients of obsessive–compulsive disorder (El Mansari & Blier, 2006).

Marble-burying behavior of mice simulates some aspects of obsessive–compulsive behavior; therefore, it is often used to screen anti-compulsive drugs due to high predictive and good face validity (Joel, 2006). In both natural and laboratory conditions, rats and mice spontaneously use available bedding material to bury unpleasant sources of discomfort present in their home environment (Archer et al., 1987). Burying behavior consists of forward shoving the diggable material over the source of aversion using the snout and forepaws to avoid and protect from the localized threat (Poling et al., 1981). This characteristic behavior, which is usually directed toward several classes of harmful and noxious objects such as food associated with unpleasant tasting, small predators such as scorpions, dead conspecifics or electrified prod, is described as a defensive behavior reflecting the anxiety state of animals (Londei et al., 1998; Treit et al., 1981; Wilkie et al., 1979). However, as the defensive nature of marble-burying behavior is still actively debated, the mouse marble-burying test has been used as a screening model for the detection of anxiolytics.

Colocasia esculenta Linn. (Araceae), commonly known as elephant ear in English, is a tall herb found throughout the hotter parts of India and Ceylon. It has been used traditionally as an expectorant, astringent, appetizer, otalgia, laxative, demulcent, and to treat internal hemorrhages (Anonymous, 2005). The plant is also useful as a nerve tonic, in cases of inflamed glands, piles, and diarrhea (Kirtikar & Basu, 2005). Cyanoglucoside from C. esculenta has been implicated as an aggravating factor in neurological disorders (Grindley et al., 2002). Although the plant is traditionally claimed to possess diverse pharmacological actions including neuropharmacological activity, only the anti-inflammatory (Shah et al., 2007) and anticancer activities (Brown et al., 2005) have been studied. Considering the available information and folklore use of the plant, the present study was designed to evaluate the anti-compulsive effects of the hydroalcoholic extract of leaves of Colocasia esculenta (HECE) using the marble-burying behavior test in mice.

Materials and methods

Collection of plant materials

Leaves of C. esculenta were purchased from a local market in July 2008. The plant was identified and authenticated by Prof. P. J. Parmar, Botanical Survey of India, Jodhpur, India. A voucher specimen (SU/DPS/Herb/05) of the same has been deposited in the Department of Pharmaceutical Sciences, Saurashtra University, Rajkot, for future reference.

Preparation of plant extract

Leaves were shade-dried, moderately ground by electric grinder and macerated with 50% ethanol (w/w) for 7 d with intermittent shaking. On the 8th day, the macerate was filtered through muslin cloth and the solvent was completely removed under reduced pressure to give the hydroalcoholic extract (yield 9.8% w/w). The extract was stored in a refrigerator and prepared freshly in sodium carboxymethyl cellulose (SCMC) solution just before the experiments. The HECE was subjected to phytochemical investigations (Trease & Evans, 2008).

Experimental animals

Adult male albino Swiss mice (22–25 g) were group housed (n = 6) in polypropylene cages and placed in a climate-controlled central animal house having temperature 22 ± 2 °C, relative humidity 60 ± 5%, and a 12 h light/dark cycle (lights on at 08:00 h and off at 20:00 h). Mice were fed standard rodent chow (Amrut, Pranav Agro Industries, Baroda, Gujarat, India) and water ad libitum. Mice were acclimatized to laboratory conditions for 7 d before carrying out the experiments. All the experiments were carried in a noise-free room between 08:00 and 15:00 h. A separate group (n = 6) of mice was used for each set of experiments and each animal was used only once. All the protocols were approved (SU/DPS/IAEC/9003) by the Institutional Animal Ethics Committee (IAEC) of the Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA), Ministry of Environment and Forests, Government of India.

Drugs

Fluoxetine was obtained as a gift sample by Torrent Research Centre, Ahmedabad, Gujarat, India. Drug solutions were freshly prepared, and their doses are expressed in terms of free bases. The solvents used were of analytical grade.

Administration of drugs

HECE were suspended in 0.5% SCMC in distilled water and administered via intraperitoneal (i.p.) route. Fluoxetine was dissolved in 0.9% saline. HECE was administered at dose levels of 25 and 50 mg/kg. Control group animals received only vehicle (0.5% SCMC, 1 ml/kg) and standard group animals received fluoxetine as the reference standard drug (5 mg/kg, i.p.; Selective Serotonin Reuptake Inhibitor (SSRI)). All the drugs as well as extracts were freshly prepared and administered 30 min prior to test.

Acute toxicity study

The acute toxicity study was performed as per the method described by Litchfield and Wilcoxon (1949) and the LD₅₀ value was calculated accordingly. Briefly, the HECE in the dose range of 10–1600 mg/kg was administered intraperitoneally to different groups of mice (n = 10). The animals were examined every 30 min up to a period of 3 h and then, occasionally for an additional period of 4 h, and finally, overnight mortality was recorded. All tests on mice were performed at two-dose levels 25 and 40 mg/kg, body weight corresponding to 10 and 20% of LD₅₀ value (250 mg/kg, i.p.), respectively, in order to assess the anti-compulsive activity of HECE.

Assessment of marble-burying behavior

The anti-compulsive effect was assessed by widely used model of studying the marble-burying behavior of mice (Njung'e & Handley, 1991a,b; Uday et al., 2007). In brief, each mouse was individually placed in a plastic cage (21 × 38 × 14 cm³), containing 5 cm thick sawdust bedding. Twenty small glass marbles (diameter 10–12 mm) were arranged on the bedding evenly spaced in four rows. After 30 min exposure, the number of unburied marbles was counted. A marble covered at least two-third (2/3) of its size by saw dust was considered as “buried”. The total number of marbles buried was considered as an index of obsessive–compulsive behavior.

Statistical analysis

All data are expressed as mean ± S.E.M. (n = 6) and analyzed using one-way ANOVA, followed by the Student Newman–Keuls test. The groups treated with HECE and fluoxetine were compared with the respective vehicle group. p Values < 0.001 were considered statistically significant.

Results

Effect on marble-burying behavior in mice

HECE (25 and 50 mg/kg, i.p.) dose-dependently reduced the marble-burying behavior in mice (Table 1), and the reduction was significant (p < 0.001) compared with the control group. Fluoxetine (5 mg/kg) also showed significant (p < 0.001) reduction in the number of buried marbles (Table 1).

Table 1. Effect of hydroalcoholic extract of C. esculenta leaves and fluoxetine on marble-burying behavior in mice^a.

Groups	Dose (mg/kg, i.p.)	No. of marbles buried
Control	–	13.20 ± 1.828
Fluoxetine	05	3.80 ± 0.683***
HECE	25	7.60 ± 0.678**
HECE	50	6.00 ± 0.896***

^aValues are expressed as mean ± S.E.M. (n = 6).

**p < 0.01, ***p < 0.001, compared with control (one-way ANOVA followed by the Student–Newman–Keuls test).

Discussion

This is the first report to show that HECE can modulate the compulsive behavior. These studies were carried out by employing an animal model, wherein marble-burying behavior of mice is considered as the marker index of compulsive behavior, which is characteristically evident in OCD (Ichimaru et al., 1995; Londei et al., 1998; Njung'e & Handley, 1991a). OCD is a neuropsychiatric disorder characterized by recurrent thoughts and/or repetitive behavior that causes marked distress and interferes with normal functioning (Rasmussen & Eisen, 1992). Although obsessive–compulsive disorder is classified as an anxiety disorder, widely prescribed anxiolytic agents such as benzodiazepines are generally ineffective in relieving obsessive–compulsive disorder symptoms. Clinical reports indicate that SSRIs and clomipramine are effective in reducing obsessive–compulsive disorder symptoms (Gorman & Kent, 1999). The marble-burying behavior test has been suggested as a useful model for evaluating anti-obsessive–compulsive disorder drugs because no change in the intensity of the marble-burying behavior occurred during repeated testing (this is considered as compulsive behavior) and antidepressants such as tricyclics and SSRIs suppress marble-burying behavior (Millan et al., 2001; Njung’e & Handley, 1991b). Marble-burying behavior is an unconditioned species-specific defensive reaction in rodents, which is not associated with physical danger, and does not habituate upon repeated testing (Njung'e & Handley, 1991a). In male mice, it is markedly attenuated by acute administration of SSRI and tricyclic antidepressants (Greist et al., 2003; Ichimaru et al., 1995). These observations suggest that the burying behavior in male mice models is the compulsive behavior rather than anxiety (Gyertyan, 1995; Millan et al., 2001). Therefore, this paradigm was employed in the present investigations. The present study revealed that HECE (25 and 50 mg/kg, i.p.) reduced marble-burying behavior, and the effect was comparable with that of fluoxetine (5 mg/kg, i.p.).

According to pathophysiology, OCD has been linked to abnormalities with the neurotransmitter serotonin, although it could be either a cause or an effect of these abnormalities. Serotonin is thought to have a role in regulating anxiety. To send chemical messages from one neuron to another, serotonin must bind to the receptor sites located on the neighboring nerve cell. It is hypothesized that the serotonin receptors of OCD sufferers may be relatively under stimulated condition. This suggestion is consistent with the observation that many OCD patients benefit from the use of SSRIs, a class of antidepressant medications that allow for more serotonin to be readily available to other nerve cells (BBC Science, 2006). So, it can be assumed that HECE may have an identical effect on SSRI or some inhibitory effect on serotonergic neurotransmission.

In the present study, phytochemical screening of HECE revealed the presence of flavonoids, steroids and β-sitosterol, so the mechanism of anti-compulsive action of HECE may be due to the involvement of any of these phytoconstituents in serotonergic neurotransmission. Moreover, steroidal compounds present in the HECE are able to cross blood–brain barrier due to their lipophilic nature, so it can be assumed that such compounds might also be responsible for eliciting anti-compulsive activity at molecular level in the central nervous system (brain) (Librowaski et al., 2000).

Conclusion

This is the first report to show that HECE dose-dependently attenuated the marble-burying behavior in mice, and the effect was comparable to that shown by fluoxetine, a reference standard drug. The present study concludes that the HECE shows anti-compulsive effect in a dose-dependent manner. Phytochemical screening of the extract has shown the presence of flavonoids, β-sitosterol, and steroids, which may account for biological activities. Similarly, identification and isolation of compound(s) responsible for the activity could be used as prototype(s) for designing new substances with anti-OCD activity. Further, major active components and precise anti-compulsive mechanisms need to be identified.

Acknowledgements

We are grateful to the Head, Department of Pharmaceutical Sciences, Saurashtra University, Rajkot, Gujarat, India, for providing the facilities during the course of this study. Special thanks to Professor P. J. Parmar, Botanical Survey of India, for identification and authentication of the plant. A gift sample of fluoxetine by Torrent, Ahmedabad, India, is gratefully acknowledged.

Declaration of interest

The authors report that they have no conflicts of interest. The authors alone are responsible for the content and writing of the paper.