Effect of Ethanol Extract Leaves of Nymphaea Lotus (Water Lily) on Fear and Anxiety Behaviour in Mice

The aim of this study was to investigate the effect of the leave extract of Nymphaea lotus on neuro-behaviour using mice as experimental model on fear/anxiety. Twenty seven mice weighing between 20-29 g were randomly selected into A, B and C groups for the study. Prior to the assessment of these neurobehavioral parameters, the phytochemical screening of the plant and the LD50 were analyzed. The elevated plus maze was used to access anxiety/fear related behaviours. Our findings showed that N. lotus extract reduces fear/anxiety related behavior, this was so because, the time spent in the open arm was increased significantly (p<0.01 respectively) in the mice that were treated with the leave extract, the head dips showed similar results. However, the number of fecal boles, frequency of SAP and close arm duration were decreased in the mice treated groups compared to control (p<0.01 and p<0.05 respectively. In conclusion, the leaves of Nymphaea lotus may be responsible for the reduced level of anxiety and fear we found in the treated mice.


Introduction
Nymphaea lotus is a perennial plant that grows up to 45 cm in height; whose leaves float in water [1]. They are easily recognized by their aquatic habit, floating leaves, flamboyant flowers, several petals, many stamens and carpels and endospermous seeds with small embryo [2] This plant is mostly found in the Northern part of Nigeria. The plant contains many pharmacological and chemical important compounds which have been useful in the field of medicine [3]. Despite the immense technological advancement in modern medicine, many people still rely on traditional medicine and healing practices for their daily health practices [4,5]. This is owned to the fact that modern medicine leave resultant side effects coupled with the high cost in acquiring synthetic drugs. Moreover, there is an increasing incidence of neurobehavioral disorders among people of all ages. The side effects of many neuro-cognition enhancing drugs have left many individuals and patients with irreversible neurological damage [6]. This debilitating situation has led to the investigation of the medicinal properties of N. lotus. The validation of the therapeutic efficacy of N. lotus will provide novel means for the management/treatment of neurobehavioural disorders such as anxiety disorders [7][8][9].

Animal care
Twenty six (26) Swiss white mice were used in the study. The animals weighing between 20-29 g were housed in the Department of Physiology, Gregory University, Uturu for one week and later transported to the Department of Physiology, Abia state university, were they were kept for 14 day in a clean and well ventilated compartment for the animal to acclimatize before the experiment was carried out. The animals were feed properly and allowed drinking water adlibitum

Experimental design
The Twenty-six (26) Swiss mice were divided into three groups. A, B and C of 10 mice. Animals in group A received normal rodent chow and served as the control, group B and group C which serve as the low and high dose treated animals received 200 mg and 400 mg/kg of the leave extracts for the number of days that the experiment lasted.

Ethical approval
All authors hereby declare that principle of Laboratory animal care was followed. All animals have been examined and approved by the appropriate ethics committee.

Experimental procedure
• Each mouse was picked from its tail and then placed in the centre square between the open and close arm of the apparatus. • Between each trial the apparatus is cleaned using a cotton wool soak in 70% ethyl alcohol in order to remove olfactory cues. • Each mouse spent five minutes after which each mouse was removed from the apparatus and returned into their home cages.

Research Article
Behaviors that were scored included: open arm entries (frequency and duration), closed arm entries (frequency and duration), head dipping, frequency of Stretch attend posture, grooming frequency, rearing; urination and defecation (number of fecal bole produced).    Figure 3 shows the close arm duration for the various groups, starting from the control, and then the treated groups (low and high dose). Their values were 147.88 ± 4.17; 127.33 ± 4.10 and 127.33 ± 2.19 seconds respectively. The close arm duration for the treated mice were significantly lower (P<0.001) compared to control. However, there was no significant difference among the treated groups.

Frequency of head dips
The frequency of head dips for the experimental animals were 9.88 ± 1.91; 20.44 ± 0.92/5 mins and 20.88 ± 2.40/5 min for mice fed control, low dose diet and high dose diet of N. lotus. The head dips for the mice administered the plant extract were higher when compared to the control (P<0.001) (Figure 4).

Frequency of grooming
Grooming frequency values for the mice fed control, low dose and high dose treated were 2.55 ± 0.72; 2.44 ± 0.72 and 2.44 ± 0.41/5 mins respectively. Grooming frequency did not differ among the groups (Figure 6).

Discussion
The findings in the elevated plus maze shows that the treated mice turned out to be less fearful and anxious than the control because the treated mice spent more time in the open arm indicating absence of fear and anxiety [10]. The animals treated with doses of the extract also significantly spent less time in the arm that was closed, also indicating absence of anxiety/fear, the animals perhaps did not feel frightened from falling off the platform, thereby showing what the leaves of N. lotus can do, on the treated mice [11][12][13][14]. Findings from the elevated plus maze also showed that the animals treated with N. lotus extract had significantly more head dips [15]. Defecation frequency was significant less in the treated mice when compared to control, also indicating less anxiety and fear related behavior [16]. The amygdala control the level of Fear and anxiety mostly and the hypothalamus [17]. Stimulation of the amygdala for instance is associated with fear and feeling of terror in the animals [18]. Therefore, it is likely that the leaves of Nymphaea lotus contain a lot of chemical compounds, such as cardiac glycosides which has been reported to decrease contraction of the heart [19]. The leaves of Nymphaea lotus could be responsible for the anxiolytic properties found in the experimental animals my causing a decrease in the excitability of the cells of the amygdala, thereby increasing the level to which it response to stimuli, thus reducing the level of fear/ anxiety in the experimental animals [20,21]. Conclusion N. lotus reduces the level of fear/anxiety in animals. The plant also has low toxicity and reduced side effects, therefore pharmaceutical industries can use this plant in producing safe and efficient drugs for people with anxiety disorders. Based on the findings from this study, one can rightly say that N. lotus tends to have a significant effect on the nervous system by reducing the level of anxiety in animals. Therefore, cardiac glycoside which has been reported to decrease heart contraction and one of the constituents in N. lotus may be responsible for the decrease in anxiety/fear related behaviors in the experimental mice.