Anxiolytic activity of methanol leaf extract of Achyranthes aspera Linn in mice using experimental models of anxiety.

OBJECTIVE: To study the anxiolytic activity of methanol extract of Achyranthes aspera Linn (Amaranthaceae).
MATERIALS AND METHODS: Male Swiss albino mice were used. Methanolic extract of Achyranthes aspera (MEAA) was administered in the doses of 100, 300 and 600 mg/kg p.o. Hole board (HB), open field (OF), elevated plus maze (EPM) and light/dark exploration (LDE) tests were used for determination of anxiolytic activity.
RESULTS: The methanolic extract of Achyranthes aspera significantly increased the number and duration of head poking in HB test. The extract also significantly increased the time spent and the number of entries in open arm in EPM. In LDE test, the extract produced significant increase in time spent and number of crossings and decreased the duration of immobility in light box. In OFT, the extract showed significant increase in number of rearing, assisted rearing and the squares crossed.
CONCLUSION: In the present study, MEAA exhibited anxiolytic activity which might be attributed to its phyto-constituents viz. alkaloid, steroid and triterpenes. Since Achyranthes aspera is ubiquitous and abundantly grown, it could be a fairly economical therapeutic agent for management of anxiety disorders.

Introduction

Anxiety-related disorders such as generalized anxiety, panic, obsessive-compulsive disorder, phobias or post-traumatic stress are the most common mental illness and a major cause of disability in the world. Mental disorders have been found to be common, with over a third of people in most countries reporting them with sufficient criteria to be diagnosed at some point in their life.[1] Although benzodiazepines are among the first line of anxiolytic drugs with well-known benefits, their side effects are prominent, including sedation, muscle relaxation, anterograde amnesia and physical dependence.[2]

Despite a phenomenal development of modern drug industry, medicinal plants still constitute an important part of pharmacopoeias in both the developed and developing countries. These plants are important elements of traditional medicine in virtually all cultures. North East India is considered as one of the “hotspots” of biodiversity in India and out of the 1500 species of medicinal plants available in India, almost 350 species are found in Assam. However, many of these traditionally used plants have not yet been studied scientifically and can be developed as potential drugs after scientific validation.

Achyranthes aspera Linn. (Prickly Chaff flower) (Amaranthaceae), locally known as Apang, is an annual, biennial, lower portion perennial erect under shrub or rather stiff herb growing up to 0.3 to 1.0 m in height. It grows throughout the world in tropical and warmer regions. Yunani doctors and local quakes use the stem, leaves and fruits as a remedy for piles, renal dropsy, pneumonia, cough, kidney stone, skin eruptions, snake bite, gonorrhea, dysentery etc. The plant has antibacterial,[3] anti-inflammatory,[4] and abortifacient activity.[5] A. aspera extract elevates thyroid hormone level and is also reported to have anticoagulant, antiarthritic, antitumor and antihepatocarcinogenic activity.[6]

In our previous study, we have reported antidepressant,[78] wound healing,[9] and antinociceptive[10] activity of this plant. The present study is undertaken to investigate the anxiolytic activity of methanol leaf extract of Achyranthes aspera in laboratory animals, since this action is not reported so far.

Materials and Methods

Plant material

The leaves of Achyranthes aspera Linn were collected during the months of February-June, 2009, from the medicinal garden of the Department of Pharmacology, College of Veterinary Science, Khanapara, Guwahati and were identified by Taxonomist Dr.S.C.Nath of NEIST, Jorhat, Assam. A voucher specimen(no.AAU/CVSc/PHT/01) has been deposited in the Herbarium of Botanical Survey of India, Shillong, Meghalaya.

Preparation of methanol extract

Fresh leaves of Achyranthes aspera were cleaned and shade dried in clean, dust free environment, ground and stored in an air-tight container. About 250 g of leaves were soaked in 1000 ml of methanol for 72 h in separate beakers. The mixture was stirred every 18 h using a sterile glass rod. The solvent was filtered every 3rd day using muslin cloth and Whatman's filter paper No 1. The filtrate obtained was concentrated in rotary evaporator (Equitron, Roteva) at 5060°C under reduced pressure leaving a dark brown residue. The methanol extract of Achyranthes aspera (MEAA), thus obtained was transferred to a Petri dish and kept over water bath (50°C-) until the solvent gets completely evaporated. It was stored at 4°C for future use. Recovery was 6.89% (w/w).

Phytochemical screening

Qualitative phytochemical screening of MEAA was carried out as per standard method.[11]

Animals and treatment regimes

Healthy adult male Swiss albino mice (20–30 g) were used for the study of anxiolytic activity and adult female Swiss albino mice (20–30 g) and Wistar rats (120–130 g) were used for acute toxicity study. This was done in order to avoid the influence of ovarian hormone fluctuations across the estrous cycle in female mice. The behavioral observations took place in sound proof rooms at the same period of the day to reduce the confounding influence of diurnal variation in spontaneous behavior.[12] The animals were housed in a group of six per cage and maintained under standard environmental conditions: 25 ± 2°C temperature, 12:12 h light and dark cycle, and 45–55% relative humidity, with free access to food and water ad libitum. Food (not water) was withdrawn 6 h before and during the experiment. All the experiments were carried out during the light period (0800:1600 h) of the day.

Acute toxicity study was carried out according to the Organization of Economic Corporation Development (OECD) guidelines No. 425. MEAA was administered orally in doses of 100, 200, 400, 800, 1000 and 2000 mg/kg to female mice (n=3) and the percentage mortality was recorded for a period of 24 h. During the first 1 h after the drug administration, the mice were observed for any gross behavioral change and the parameters observed were hyperactivity, grooming, convulsions, sedation, loss of righting reflex, respiration, salivation, urination and defecation.[13]

Based on the above toxicity study, direct limit test was done. Initially a particular dose, determined on the basis of the above study, was administered to a single female rat and the rat was observed for 48 h with close surveillance up to initial 4 h (same as in case of the first rat). After 48 h (of the second administration), same dose was administered to two more female rats and observation was done as for the previous rats. No morphological alteration was observed during the 14 days of observation. The weight of the animals was recorded on 7th and 14th day.

The experimental animals were randomly allocated into five groups of six animals each. Group I served as vehicle treated control, Groups. II, III and IV received MEAA p.o. at 100, 300 and 600 mg/kg, respectively and Group V received the standard drug diazepam (1 mg/kg i.p.). The doses were selected on the basis of the preliminary screening. The study was conducted in accordance with the ethical rules on animal experimentation, approved by Institutional Animal Ethics Committee, College of Veterinary Science, Khanapara (Registration number- 770/03/ac/ CPCSEA/FVSc, AAU/ IAEC/06/21).

Drugs

Diazepam (Ranbaxy Laboratories Ltd, Mumbai, India) was used as standard anxiolytic agent. Methanol was procured from Merck (Mumbai, India). All the chemicals and solvents were of analytical grade.

Hole board test

The hole board apparatus consisted of a wooden chamber (40 × 40 × 25 cm3) with 16 holes (each of 3 cm diameter) evenly distributed on the floor. The apparatus was elevated to a height of 25 cm from the ground so that the mice could peep through the holes. The mice were treated with MEAA (100, 300 and 600 mg/kg, p.o.), diazepam (1 mg/kg, i.p.) or distilled water (p.o.) 30 min prior to test and kept in the apparatus. The numbers and the duration of head poking were recorded during the 5 min observation period.[14]

Open field test

The apparatus consisted of a wooden box (60 × 60 × 30 cm3) with the floor divided into 16 squares (15 × 15 cm2). The apparatus was illuminated with a 40-W lamp suspended 100 cm above. Mice were treated with MEAA (100, 300 and 600 mg/kg, p.o.), diazepam (1 mg/kg, i.p.) or vehicle (p.o.). After 30 min, they were placed individually in one of the corner squares. The number of rearing, assisted rearing (forepaws touching the wall of the apparatus) and squares traveled were counted for 5 min.[1415]

Elevated plus maze test

The plus maze apparatus consisted of two open arms (35 × 5 cm2) crossed with two closed arms (35 × 5 × 20 cm3). The arms were connected together with a central square of 5 × 5 cm2. The apparatus was elevated to the height of 25 cm in a dimly illuminated room. Mice were treated with MEAA (100, 300 and 600 mg/kg, p.o.), diazepam (1mg/kg i.p.) or vehicle 30 min before being placed individually in the center of the apparatus, facing the closed arm. The time spent and the numbers of entries in both the open and the closed arms were recorded for 5 min. An entry was defined as having all four paws within the arm.[16]

Light/dark exploration test

The apparatus consisted of two boxes (25 × 25 × 25 cm3) joined together. One box was made dark by covering its top with plywood, whereas a 40-W lamp illuminated the other box. The light source was placed 25 cm above the open box. The mice were treated with MEAA (100, 300 and 600 mg/kg, p.o.), diazepam (1 mg/kg, i.p.) or vehicle 30 min before being placed individually in the center of the lit box and four behavioral events i.e. number of crossings to light compartment, time spent in light box, time spent in dark box and transfer latency were recorded for the next 5 min.[17]

Statistical analysis

The statistical analysis of data was done by one-way analysis of variance using the SPSS software (version 11.5). P<0.01 was considered as highly significant.

Results

The phytochemical screening of MEAA showed presence of alkaloids by Wagner's and Dragendroff's test; steroids by Salkowski's and Lieberman–Burchardt's test and triterpenes by Salkowski's and Lieberman–Burchardt's test.

Acute toxicity studies

Oral administration of MEAA up to 2 g/kg did not produce any toxic effect in the mice. No mortality was observed and the extract was found to be safe at the given dose.

There was significant and dose-dependent increase in the number and duration of head poking after administration of MEAA (100, 300 and 600 mg/kg, p.o.) compared to the control group and the results were comparable with the standard drug diazepam [Figure 1a and b].

Figure 1

Effect of MEAA (100, 300 and 600 mg/kg p.o.) on (a) head poking and (b) duration (s) of head pokes in hole board test (n=6). Values are expressed as mean ± SEM. Means bearing same superscript do not differ significantly

In the open field test, there was significant (P<0.01) increase in rearing, assisted rearing and number of squares traveled in the MEAA treated (100 to 600 mg/kg p.o.) as well as the standard group, compared to the control group. The numbers of assisted rearing in the groups treated with MEAA at 100 and 300 mg/kg was comparable with the standard and at 600 mg/kg, the count was significantly (P<0.01) higher than the standard group. The number of squares traveled also significantly (P<0.01) increased when MEAA was administered orally at 300 and 600 mg/kg, indicating a dose-dependent anxiolytic activity of the plant extract [Figure 2a–c].

Figure 2

Effect of MEAA (100, 300 and 600 mg/kg p.o.) on (a) rearing, (b) assisted rearing and (c) squares traveled in open field test (n=6). Values are expressed as mean ± SEM. Means bearing same superscript do not differ significantly

In the elevated plus maze test, there was significant (P<0.01) increase in the number of entries and the time spent in the open arm in MEAA (100 to 600 mg/kg, p.o.) and diazepam treated groups, whereas there was a significant (P<0.01) decrease in the time spent in the close arm compared to the control group. Numbers of entries into the open arm in MEAA treated animals with 600 mg/kg dose were higher than the standard drug [Figure 3a–d].

Figure 3

Effect of MEAA (100, 300 and 600 mg/kg p.o.) on (a) time spent (s) in open arm, (b) time spent (s) in closed arm, (c) entries in open arm and (d) entries in closed arm in elevated plus maze test (n=6). Values are expressed as mean ± SEM. Means bearing same superscript do not differ significantly

Light/Dark exploration test

In the light/dark exploration test, the time spent (s) in lighted box, numbers of crossings and transfer latency increased significantly (P < 0.01) and the time spent in dark box decreased significantly (P < 0.01) in MEAA (300 and 600 mg/kg p.o.) and diazepam treated groups compared to the control group. The values of MEAA (at 600 mg/kg p.o. treated group) were comparable with the standard treated group for all the parameters under study [Figure 4a–d].

Figure 4

Effect of MEAA (100, 300 and 600 mg/kg p.o.) on (a) time spent (s) in light box, (b) time spent (s) in dark box, (c) number of crossings and (d) transfer latency time (s) in light/dark exploration test (n=6). Values are expressed as mean ± SEM. Means bearing same superscript do not differ significantly

Discussion

Hole-board test indicated that the head-dipping behavior was sensitive to changes in the emotional state of the animal, and suggested that the expression of an anxiolytic state may be reflected by an increase in head-dipping behavior.[18] In our study, MEAA significantly (P< 0.01) increased the numbers and duration of head poking compared to the control group.The open field test also showed that after administration of MEAA (100-600 mg/kg) or diazepam (1 mg/kg), there was a significant increase in the number of rearing, assisted rearing and number of squares traveled compared to the control group which might be attributed to the anxiolytic activity of the plant extract. The EPM test is based on a premise where the exposure to an open arm of EPM evokes an approach-avoidance conflict that is considerably stronger than that evoked by the exposure to an enclosed arm.[19] The decrease in aversion to the open arm is the result of an anxiolytic effect, expressed by an increase in time spent and entries into the open arm. The primary index is spatiotemporal in nature, it is reduced by anxiolytic drugs and can be increased by anxiogenic compounds.[20] In case of MEAA, an increase in the time spent in open arm than that of closed arm was observed along with more number of entries. In light-dark test, animals always try to spend more time in dark compartment compared to light box out of fear of exposure to the new environment. Transitions have been reported to be an index of activity exploration because of habituation over time and the time spent in each compartment are reflection of aversion.[21] The administration of MEAA showed significant increase in the time spent in the lighted box, number of crossing and the time latency, with decrease in time spent in the dark box.

Nepeta cataria, used in European folk medicine, contains an anxiolytic component linalool, which is a monoterpenoid.[22] MEAA contains triterpenes as one of the phytoconstituents, which might be responsible for its anxiolytic action. In another study, we have reported antidepressant like activity of Achyranthes aspera using forced swimming models in rat and mice[7] and modified forced swimming model. The antidepressant activity of Achyranthes aspera was believed to be mediated through noradrenergic mechanism as they behaved like noradrenergic reuptake inhibitor desmethyl imipramine and not like selective serotonin reuptake inhibitor (SSRI), citalopram.[8]

In conclusion, the present study hypothesizes that MEAA exhibited anxiolytic activity in all the animal models and may have possibly exerted its effect through diverse mechanisms, possibly due to the presence of different phytoconstituents like alkaloids, steroids and triterpenes present therein. Further studies on isolation and fractionation of the active components from the leaf of Achyranthes aspera and on its mechanism of action are strongly recommended.