Evaluation of anxiolytic effect of Medicago sativa in mice

Abstract

Context: Medicago sativa Linn. (Leguminosae) has a long tradition of use as an Ayurvedic and Homoeopathic medicine in a variety of central nervous system (CNS) disorders. Traditionally, M. sativa is used to improve the memory, as a rejuvenator, antidiabetic, antioxidant, anti-inflammatory, and in CNS disorders. Despite a long tradition of use, no systematic phytochemical and pharmacological work has been carried out on this potential plant. M. sativa was subjected to preliminary anti-anxiety screening studies, with a view to ascertain the verity of its traditional use as an anxiolytic.

Objective: Various extracts, viz., petroleum ether, chloroform, methanol and aqueous extract from the aerial parts of M. sativa was subjected to preliminary anti-anxiety screening studies, with a view to ascertain the truth on evidence of its traditional use as an anxiolytic.

Materials and methods: The aerial parts of the plant were extracted using solvents in order of increasing polarity, viz., petroleum ether (60–80°C), chloroform, methanol and distilled water. All the crude extracts were evaluated for anti-anxiety activity in mice using elevated plus-maze apparatus. Diazepam was used as the standard drug.

Results: Among all extracts, only the methanol extract exhibited significant (p < 0.05) anti-anxiety activity by increasing the average time spent, and number of entries in open arms at a dose of 100 mg/kg in mice with respect to the vehicle treated control as well as the standard (2 mg/kg).

Conclusion: These results suggest that administration of M. sativa exerts anxiolytic effect on mice, and it could serve as a new approach for the treatment of anxiety.

Keywords::

• Anxiety
• elevated plus-maze test
• phenolics
• flavonoids

Introduction

A survey by the World Health Organization reported that about 450 million people suffer from a mental or behavioral disorder, yet only a small minority of them receives even the most basic treatment (WHO, 2003). This amounts to 12.3% of the global burden of disease, and will rise to 15% by 2020 (Reynolds, 2003; WHO, 2004). Anxiety disorders are among the most common mental, emotional, and behavioral problems (Olatunji et al., 2007; Kessler & Wang, 2008). These affect one-eighth of the total population worldwide, and have become a very important area of research interest in psychopharmacology (WHO, 2004). In addition to the high prevalence, anxiety disorders account for major expenditure for their management, and have a substantial negative impact on quality of life (Olatunji et al., 2007).

Major drug classes for the treatment for anxiety disorders are benzodiazepines and selective serotonin-reuptake inhibitors (Kunovac & Stahl, 1995). All drug classes currently used are associated with side effects like hepatotoxicity, insomnia and muscle relaxation which impair normal daily life (Lader & Morton, 1991). These considerations implicate the search for new anxiolytic compounds with less side effects and a wider safety margin.

Herbal therapies could be considered as alternative/complementary medicines. The use of herbal medications by physicians in Asia and Europe is becoming very common and researchers are exploring the traditional remedies to find a suitable cure for these ‘mind affecting diseases’ (Rabbania et al., 2003). In addition, the search for novel pharmacotherapy from medicinal plants for psychiatric illnesses has progressed significantly in the past decade. This is reflected in the large number of herbal medicines whose psychotherapeutic potential has been assessed in a variety of animal models. These studies have provided useful information for the development of new pharmacotherapies from medicinal plants for use in clinical psychiatry.

Medicago sativa Linn. (Leguminosae), commonly called alfalfa, is a widely cultivated, environmentally tolerant forage crop (Mitchell, 1983). M. sativa has a long tradition of use as an Ayurvedic and Homoeopathic medicine in variety of central nervous system (CNS) disorders (Inamul, 2004). Traditionally, M. sativa is used to improve the memory, to cure kidney pain, cough, as a rejuvenator, antidiabetic, antioxidant, anti-inflammatory, anti-asthmatic, antimicrobial, galactagogue, and in CNS disorders (Finkler, 1985; BHMA, 1996; Inamul, 2004). The plant contains many important substances including saponins, sterols, coumarins, flavonoids, phenolics, vitamins, proteins, minerals, and other nutrients (Duke, 1985; Kundan & Anupam, 2011a).

Pharmacological studies revealed that saponins isolated from M. sativa have been used as hypocholesterolemic and antiatherosclerotic (Khaleel et al., 2005; Kundan & Anupam, 2011a). It is beneficial in diabetes (Alison & Peter, 1997), cardiovascular complications (Reilly, 1989), convalescence and debility (Mills, 1994), and also used as a tonic after blood loss and during anemia. In our previous work on M. sativa, we have shown that the plant exhibited significant antioxidant and neuroprotective effects against ischemia and reperfusion insult in mice (Kundan & Anupam, 2011b).

The present study was designed to evaluate the anxiolytic effect with treatment of methanol extract of M. sativa in mice using elevated plus-maze model of anxiety.

Materials and methods

Animal

Swiss albino mice of either sex (20–25 g) were employed in the present study. The animals were maintained at the Animal House, L.R. Institute of Pharmacy under standard environmental conditions, and allowed standard laboratory feed and water ad libitum. All animals used in the study were naive to the elevated plus-maze test. The approval from the Institutional Animal Ethical Committee of L.R. Institute of Pharmacy, Solan was obtained before carrying out biological studies.

Plant material, preparation of extracts and their phytochemical screening

Aerial parts (leaves and stems) of the plant were procured from Himalaya Herbs Stores, Saharanpur, India. The identity of the plant was confirmed through Dr. H.B. Singh, Scientist F, Head of Raw Material Herbarium and Museum (RHMD), National Institute of Science Communication and Information Resources (NISCAIR), New Delhi, India (Ref. No. NISCAIR/RHMD/Consult/2008-09/1170/202, dated 25th Feb. 2009).

Dried plant materials were pulverized using a mechanical grinder. Powdered material was subjected to successive Soxhlet extraction by solvents in increasing order of polarity, viz, petroleum ether (60–80°C), chloroform and methanol. Before each extraction, the powdered material was dried in hot air-oven below 50°C. Finally, marc was digested at 50°C with distilled water for 24 h to obtain the aqueous extract. Each extract was evaporated to dryness in vacuum. Thereafter, the resulting extract was reduced in rotary vacuum evaporator (Buchi™, Postfach, Switzerland) (40°C), freeze-dried and stored at 4°C until further use in the experiment. Extracts were weighed and percentage was calculated in terms of the air-dried weight of the plant material. All the four extracts were dissolved in respective solvents, and were screened for different classes of phytoconstituents (Farnsworth, 1966).

Vehicle and standard drug

Distilled water + Tween 80 (5%) were used as vehicle (control) for preparing the suspension of various test doses of different extracts of M. sativa. Diazepam (2 mg/kg, p.o.) was used as a standard anxiolytic drug.

Elevated plus-maze test

The plus-maze apparatus consisting of two open arms (16 × 5 cm) and two closed arms (16 × 5 × 12 cm) having an open roof, with the plus-maze elevated (25 cm) from the floor was used to observe anxiolytic behavior in animals (Kulkarni & Reddy, 1996). Test extracts were administered orally, using a tuberculin syringe fitted with oral canula. The dose administration schedule was adjusted so that each mouse was having its turn on the elevated plus-maze apparatus 60 min after the administration of the dose. Each mouse was placed at the centre of the elevated plus-maze with its head facing the open arms. During this 5-min experiment, the behavior of the mouse was recorded as: (i) the number of entries into the open arms, and (ii) average time spent by the mouse in the open arms (average time = total time spent in open arms/number of entries in open arms). During the entire experiment, the animals were allowed to socialize. Every precaution was taken to ensure that no external stimuli could invoke anxiety in the animals. Similar observations were recorded for the standard group as well as the control group.

Standardization of extract

Determination of total phenolic and flavonoid content

The total phenol and flavonoid content of methanol extract of M. sativa were estimated according to the method described by Lin and Tang (2007). The total phenol content was expressed in milligrams of gallic acid equivalents per gram of extract. Whereas, the total flavonoid content was expressed in milligrams of rutin equivalents per gram of extract.

Statistical analysis

All data are expressed as mean ± SEM. Statistically significant differences between groups were calculated by the application of an analysis of variance (ANOVA) followed by post hoc Tukey’s multiple range tests. The groups treated with extract were compared with the respective control (vehicle) group; p values <0.05 were considered statistically significant.

Results

Extract yield, total phenolic and flavonoid content

Yields of petroleum ether, chloroform, methanol and water extracts of the dried aerial parts of the plant M. sativa are detailed in Table 1. The total phenolic and flavonoid content of methanol extract of M. sativa were estimated to be 13.71 ± 0.60 mg gallic acid equivalents/g of dry extract, and 8.72 ± 0.52 mg rutin equivalents/g of dry extract, respectively.

Table 1.  Yield of different extracts of M. sativa.

Extracts	Yield (% w/w, dry weight basis)
Petroleum ether	1.88
Chloroform	2.62
Methanol	9.32
Aqueous	6.74

Phytochemical screening

Table 2 shows the classes of phytoconstituents of different extracts of M. sativa. Phytochemical screening indicates the presence of mainly flavonoids and polyphenols in methanol extract of M. sativa.

Table 2.  Results of phytochemical screening of various extracts of M. sativa.

Class of phytoconstituents	M. sativa
	Pet. ether extract	Chloroform extract	Methanol extract	Aqueous extract
Alkaloids	^−	^−	^−	+
Glycosides
Coumarins	^−	^−	^−	^−
Flavonoids	^−	^−	++	+
Saponins	^−	^−	^−	++
Sterols/steroids	^−	+	+	^−
Carbohydrates	^−	++	++	^−
Tannins/polyphenols	^−	^−	++	+
Proteins	^−	^−	^−	^−
Amino acids	^−	^−	+	+
Fats/oil	++	^−	^−	^−
Vit-C	^−	^−	+	^−

Evaluation of anxiolytic activity of various extracts of M. sativa

Relative anxiolytic activity profiles (the mean number of entries in open arms, and the mean time spent by the mice in open arms after oral administration) of different doses (50, 100 or 200 mg/kg) of petroleum ether, chloroform, methanol and water extracts of M. sativa, and diazepam (2 mg/kg) and the control (vehicle) are shown in Table 3.

Table 3.  Effect of different extracts of M. sativa on mice using elevated plus-maze model.

Treatment	Dose (mg/kg)	Number of entries in open arms	Average time spent in open arms
Control	Vehicle	4.02 ± 0.53*	5.05 ± 0.61*
Diazepam (standard)	2	10.01 ± 0.46^#	16.45 ± 0.40^#
Petroleum ether extract	50	3.89 ± 0.53*	3.56 ± 0.45*
	100	3.98 ± 0.49*	3.80 ± 0.82*
	200	4.01 ± 0.66*	3.92 ± 0.65*
Chloroform extract	50	4.05 ± 0.86*	3.98 ± 0.55*
	100	4.09 ± 0.79*	4.02 ± 0.82*
	200	4.00 ± 0.89*	3.76 ± 0.69*
Methanol extract	50	7.05 ± 0.64*^#	10.01 ± 0.49*^#
	100	9.85 ± 0.82^#	16.20 ± 0.79^#
	200	5.59 ± 0.76*	4.05 ± 0.52*
Aqueous extract	50	3.90 ± 0.63*	3.75 ± 0.69*
	100	4.20 ± 0.96*	4.22 ± 0.56*
	200	4.02 ± 0.88*	4.50 ± 0.64*

All data are expressed as mean ± SEM (n = 10).

^#p < 0.05 vs. control; *p < 0.05 vs. standard.

Discussion

In the present study, the anti-anxiety activity of M. sativa was evaluated in animal models of anxiety. The Elevated plus-maze (EPM) stands as one of the most popular in vivo animal tests currently in use. The test was further validated as an animal model of anxiety on pharmacological, physiological and behavioral grounds (Carobrez & Bertoglio, 2005). The EPM is usually employed as a pre-clinical screening to test new anxiolytic drugs. An anxiolytic agent increases the frequency of entries into the open arms and increases the time spent in open arms of the EPM. The model was chosen as it is effective, cheap, simple, less time consuming, requires no preliminary training to the mice and does not cause much discomfort to the animals while handling. The model is principally based on the observations that the exposure of animals to an elevated and open maze results in approach-avoidance conflict which is manifested as an exploratory-cum-fear drive. The fear due to height (acrophobia) induces anxiety in the animals when placed on the elevated plus-maze. The ultimate manifestation of anxiety and fear in the animals is exhibited by decrease in motor activity, which is measured by the time spent by the animal in the open arms. In the present study, diazepam, a standard anxiolytic drug used clinically, is also employed in behavioral pharmacology as a reference compound for inducing anxiolytic effect.

Dried petroleum ether, chloroform, methanol and water extracts of M. sativa, separately suspended in a suitable vehicle, were administered orally to mice, and the activity was compared with that observed in the control group as well as with the group treated with the standard anxiolytic drug diazepam. Complete manifestation of anxiety in mice of the control group is evident from the minimum mean time spent in the open arms of elevated plus-maze by these animals. Among the extracts tested, maximum anxiolytic activity was observed in the standardized methanol extract at the dose of 100 mg/kg which was at par with that of diazepam as is evident from statistical equivalence between the results of this dose and that manifested by diazepam. However, the activity decreased at higher doses, which might be due to sedation.

In the present study, phytochemical screening showed presence of mainly flavonoids and polyphenols in methanol extract of M. sativa.

Earlier reports on the chemical constituents of plants and their pharmacology suggest that plants containing flavonoids, phenolics and tannins possess activity against many CNS disorders including anxiety (Bhatacharya & Satyan, 1997; Une et al., 2001). In the present study, phenolic and flavonoid contents of methanol extract of M. sativa were identified and quantified. Thus, it is possible that the anxiolytic action of M. sativa could be due to the presence of phenolic and flavonoid contents in the extract. However, further studies are needed to ascertain this anxiolytic effect.

Presently, the authors are involved in anxiolytic-directed-fractionation of the methanol extract of M. sativa with a view to characterize the bioactive constituent(s).

These observations suggest that M. sativa may be a clinically viable protective agent against a variety of CNS disorders such as anxiety.

Declaration of interest

The authors are grateful to the L.L.R. Educational Trust, which runs the L.R. Institute of Pharmacy, Solan, Himachal Pradesh, India, for providing funds to carry out the present investigation. The authors alone are responsible for the content and writing of the paper.