Oral Supplementation of Bauhinia variegata affects the Behavior of Albino Mice in a Gender Specific Manner

Present study was designed to investigate the effects of 300 mg/ml solvent/Kg of body weight of Bauhinia variegata’s leaf extract on selective aspects of albino mice behavior in a gender specific manner. Seven week old female and male albino mice were used as experimental animals. Mice were orally supplemented either with 300 mg/ml solvent/Kg of body weight Bauhinia variegata’s leaf extract or with saline solution (0.9% NaCl) for 17 days. Behavioral observations were made by applying a series of neurological tests (Rota rod, Elevated plus maze, Light and dark box, Open field, Novel object and Morris water maze test). It was observed that Bauhinia variegata’s leaf extract supplementation improved neuromuscular co-ordination in female albino mice during rota rod test (P = 0.002), had more rotations (P = 0.02) and clockwise rotations (P = 0.01) during plus maze test and had more stretch attend reflex (P = 0.005) than control group. During the second trial of novel object test, Bauhinia variegata’s leaf extract treated male albino mice approached old object A (P = 0.04) and spend more time with object A (P = 0.05) as compared to control group. Open filed, light dark test and probe trial of Morris water maze test performances remain unaffected in both genders (P > 0.05) when compared between 300 mg/ml solvent/Kg body weight of Bauhinia variegata’s leaf extract treated and untreated albino mice of both genders. We concluded that applied dose of Bauhinia variegata’s leaf extract has the potential to improve neuromuscular co-ordination in female albino and should be explored further as potential treatment for neuromuscular problems.


INTRODUCTION
M emories are ability of an individual to record event, information and retain them over short or long period of time, recall the same whenever needed (Joshi and Parle, 2006) Nootropics agents are known to augment the cognitive dexterity (Whitehouse, 2015). Various drugs are known to have detrimental effect on learning and memory. Nootropic agents are used primarily for improving memory, mood, and behavior. However, the resulting sideeffects associated with these agents have limited their use (Whitehouse, 2015). Thus, it is necessary to explore the utility of traditional medicines in the treatment of various cognitive disorders. Traditional medicines deal with use of plants and plant products for maintenance of good health. This indigenous form of medicinal system uses the active O n l i n e

F i r s t A r t i c l e
Dhake, 2011). The present work is focused to evaluate effect of 300 mg/ml solvent/Kg of body weight of Bauhinia variegata's leaf extract on neuromuscular coordination, exploratory and locomotory behavior in adult albino mice.

Subjects
Seven weeks old albino mice were used as experimental animals in order to demonstrate the effect of B. variegate leaf extract on selected behavioral aspects of albino mice in a gender specific manner. Animals were reared at the animal facility of Bio Park at Bahauddin Zakariya University Multan, Pakistan following the colony maintenance conditions as described by Aftab et al. (2018). Animals were kept in locally manufactured small rodent cages filled with wood chips. Standard mouse diet and water were available ad labium. Room temperature was maintained at 22±1ºC. The light/dark rhythm was maintained at 14:10. The room was illuminated with artificial light at an intensity of about 200 Watt from 8am to 6pm.
All the experimental protocols and mouse handling procedures were approved by the ethical committee of the Institute of Pure and Applied Biology, Bahauddin Zakariya University Multan, Pakistan.

Preparation of Bauhinia variegata leaves extract
The leaves of Bauhinia variegata plant were collected from various localities in Multan and thoroughly washed with distilled water and air dried under shade for 15-20 days. The completely dried plant leaves were ground well in to a fine powder in an herbal grinder and sieved to have particle size of 50-300 mm. The powder was stored in an air tight polythene bag at room temperature before extraction. 300 gm of B. variegata leaves powder was used to prepare the leaf extract following the Zahra et al. (2015). The crude extract was stored in dried and airtight container and stored in refrigerator at 4ºC till further use.

Experimental design
Leaf extract of B. variegata (300 mg) was dissolved in 1ml of distilled water to prepare the working solution. Albino mice were weighed and orally administered either with 300 mg B. variegata leaf extract /ml solvent/Kg body weight (N = 14) or with 0.9% saline solution [Otsuka,Pakistan (N = 14)].

Assessment of neurofunction
Dose were applied for 10 consecutive days and a series of neurological tests including Rota rod, light dark box, open field, elevated plus maze, novel object and Morris water maze test were conducted on next 7 consecutive days to determine the effect of B. variegata's leaf extract on adult female and male albino mice behavior. Doses were administered during neurological testing, at least 30 minutes prior to start of each test.

Rota rod
Rota Rod is used to test the balance and neuromuscular coordination of an animal. Rota Rod test was performed by using a locally manufactured apparatus comprised of rotating drum with acceleration of 40 rpm. Test was conducted following Sunyer et al. (2007).

Light and dark box
The light/dark test is based on the innate aversion of rodents to illuminated areas and on the natural exploratory behavior of rodents in response to new environment and illumination which is a mild stressor for rodents (Bourin and Hascoet, 2003). The test was conducted as we have previously reported by Zahra et al. (2015).

Open field test
Open field test is used to assess locomotory and exploratory behaviour of an animal (Prut and Belzung, 2003). The test was performed following Weitzdoerfer et al. (2015).

Elevated plus maze
Elevated plus maze test is used to assess the anxiety related behaviors in rodents (Walf and Frye, 2015). The test was conducted following Zahra et al. (2015).

Novel object test
Novel object recognition is a form of memory task that doesn't rely on spatial cues and is used to judge the recognition memory by measuring its liking for novel object (Broadbent et al., 2009;Ennanceur and Delacour, 1988). The test was performed following Zhanga et al. (2012).

Morris water maze test
The Morris Water Maze has been used extensively to study strain differences in spatial learning in mice (Upchurch and Wehner, 1988). The test was conducted following Ullah et al. (2017).

Statistical analysis
All the data was expressed as mean ± standard deviation (SD) statistical package Minitab (Version 17, USA) was used for the analysis of results. Probability (P) values less than 0.05 were considered to be significantly O n l i n e

Rota rod test
Statistical analysis of the Rota rod test revealed that female albino mice treated with 300 mg/ml solvent/ Kg body weight of Bauhinia variegata leaf extract spent significantly more time on rotating rod as compared to their saline treated control group (P = 0.002) (Fig. 1). On the other hand, rota rod reflex did not varied significantly when compared between male mice treated with 300 mg/ ml solvent/Kg body weight (P = 0.77).

Elevated plus maze test
Analysis of the elevated plus maze data for female albino mice revealed that Female mice supplemented with dose (300 mg/ml solvent/Kg body weight) had more rotations (P = 0.02) and clockwise rotations (P = 0.01) than their saline treated control group (Table I). On the other hand, all the studied parameters varied non significantly (P > 0.05) when compared between saline treated and male mice supplemented for 300 mg/ml solvent/Kg body weight of Bauhinia variegata leaf extract (Table I).

Light dark box test
Analysis of the results revealed that all the studied parameters of light and dark test varied non-significantly (P > 0.05) for both genders when compared between plant extract treated and control group (Table II).

Open field test
Analysis of the data revealed that the applied dose of Bauhinia variegata leaf extract (300 mg/ml solvent/ Kg body weight) had no influence on studied parameters of open field as all open field parameters varied non significantly (P > 0.05) when compared between leaf extract treated and untreated albino mice of both genders; female and male, indicating that Bauhinia variegata is not potent to influence the exploratory and locomotory behavior of albino mice (Table III).

Novel object test
Analysis of the data revealed that during novel object test (trial-1) female albino mice treated with 300 mg/ml solvent/Kg body weight had more stretch attend reflex (P = 0.005) as compared to their control group (Table IV). All other studied parameters varied non-significantly (P > 0.05) when compared between plant extract treated and untreated animal of both genders (Table IV).
When results of novel object test (trial 2) were analyzed, it was observed that although leaf extract treated females spent more time with the novel object (P = 0.16) but all the studied parameters varied nonsignificantly when compared between Bauhinia variegata leaf extract treated and untreated female albino mice (Table IV). Analysis of the data indicated that during the second trial of novel object test male albino mice treated with 300 mg/ml solvent/Kg body weight of Bauhinia variegata leaf extract approached old object A (P = 0.04) and spend more time with object A (P = 0.05) as compared to control group. All other studied parameters varied non-significantly (P > 0.05) when compared between Bauhinia variegata leaf extract treated and untreated female albino mice (Table IV).

Morris water maze test Acquisition phase
Analysis of results revealed that male mice treated with 300 mg/ml solvent/Kg body weight of Bauhinia variegata leaf extract reached the platform significantly earlier on training day 3(P = 0.01) than their saline treated control group (Data not shown here). Analysis of results revealed that average swimming speed of Bauhinia variegata leaf extract treated female albino mice was significantly less on training day 3 (P = 0,02) and 4 (P = 0.04) when compared with their control group (Data not shown here). The time mobile for male albino mice treated with 300 mg/ml solvent/Kg body weight of Bauhinia variegata leaf extract was significantly higher (P = 0.01) on training day 3 when compared with the control group. Analysis of results revealed that time immobile for Bauhinia variegata leaf extract treated female albino mice was significantly less on training day 2 (P = 0.009) and 3 (P = 0.02) as compared to their control group. All other studied parameters varied non significantly (P > 0.05 when compared between Bauhinia variegata leaf extract treated and untreated mice of both genders. Analysis of the results revealed that all studied parameters for probe trial varied non significantly (P > 0.05)  when compared between Bauhinia variegata leaf extract treated and untreated mice of both genders.

DISCUSSION
Medicinal plants, as potential source of therapeutic aids, have attained significance in health system, for both humans and animals, all over the world not only in diseased condition but also for maintaining proper health (Verma and Singh, 2008). Plants are the most precious resource for an extensive range of derivative metabolites utilized as medicine, agrochemicals, biopesticides, food additives and flavours (Al-Snafi, 2013).The genus Bauhinia belongs to the family Caesalpiniaceae (formally Leguminosae) and several members of this genus like Bauhinia manca, Bauhinia divaricata, Bauhinia purpurea and Bauhinia variegata are known for their medicinal importance (Vasconcelos et al., 2000;Khan et al., 2012).

O n l i n e F i r s t A r t i c l e
A. Ullah et al. Bauhinia variegata Linn is conventionally used for the treatment of tumors, bronchitis and leprosy and it is an anti hepatotoxic agent (Rajkapoor et al., 2003;Kumar et al., 2011;Marasani et al., 2013;Rajani and Ashok, 2009). This study was conducted to investigate the effect of Bauhinia variegata leaf extract (300mg/ ml solvent/ Kg body weight) on behavior of adult albino mice of both genders. As limited information is available in literature regarding the effect of Bauhinia variegata leaf extract on the behavior of albino mice, present study was designed to demonstrate the effect of two different doses of ethanolic leaf extract of Bauhinia variegata on behavior of albino mice in a gender specific manner. The rota rod test is designed to assess motor coordination, balance and equilibrium and is used to evaluate the pharmacological action of pscychotropic agents on the central and peripheral nervous system (Sunyer et al., 2007;Shiotsuki et al., 2010;Iqbal et al., 2015). Impairment of rota rod performance has been thought to reflect, at least in a part, a behaviorally depressive state. However, it is well known that riding time on rota rod is also decreased by relaxation or weakness of muscle or motor dysfunction (Amos et al., 2001). Analysis of our results indicated that applied dose of Bauhinia variegata (300 mg/ml solvent/ Kg body weight) had significantly improved the muscular activity in female albino mice as compared to their saline treated controls (Fig. 1). Interesting, the same doses did not affected the time spent by male albino mice on rotating rod indicating a gender specific effect of ethanolic leaf extract of Bauhinia variegata on the muscular strength and neuromuscular coordination in adult albino mice. Our results are in agreement with those reported by Jatav et al. (2014) as they had demonstrated that flavonoid-rich fraction of Bauhinia variegata (200 mg/Kg) significantly decreased the fall of time as compared to Diazepam (1 mg/ Kg, ip) indicating improved muscular activity in Wistar albino rats.
Elevated plus maze is one of the important methods that help to assess state of anxiety in animals (Espejo, 1997). It is also used to determine status of learning and short term memory (Kulkarni and Dhir, 2007). Analysis of our data revealed interesting results. It was observed that ethanolic leaf extract treatment at the dose of 300 mg/ ml solvent /Kg body weight significantly improved the short memory of female albino mice as evident from their elevated plus maze test (Table I).
Interestingly, the same dose did not affect the short term memory of male albino mice indicating a gender specific response of Bauhinia variegata leaf extract on elevated plus maze test results of adult albino mice ( Table  I). The presence of antioxidants like terpenoids, flavonols (Kaempferol, Quercetin), coumarins (Scopoletin and Scopolin), steroids and catchol in Bauhinia variegata has probably improved the memory and learning in albino mice.
Our results are in agreement with those of Shah and Goyal (2011) as they have also reported that the animals treated with flavonoid-rich fraction of Bauhinia variegata (400 mg/Kg) showed a significant decrease in transfer latency of rats as compared to the control group, indicating cognitive enhancement effect (Jatav et al., 2014) has also reported that the rats treated with flavonoid rich fraction of Bauhinia variegata showed nootropic effect in terms of significant increase in the time spent in enclosed arm than open arm after their training sessions. This was found comparable to the standard drug and very significant as compared to the control group. Pritipadma et al. (2015) has recently reported that the methanolic extract of Bauhinia racemosa administered orally in mice at two different doses of 150 mg/Kg and 300 mg/Kg were able to increase the time spent and the number of arm entries in the open arms of the elevated plus-maze indicating improved short term memory. Santos et al. (2012) has reported that 50 mg/Kg ethanolic extract of Bauhinia platypetala has anxiolytic effect and increase the exploration in male Swiss mice where the dose was administered intraperitoneally supporting our findings. It has been reported that the aerial non woody parts of B. variegata contain flavonoids that have neuro-protective effect and have the ability to protect neurotoxins induced damage and also have the capability to repress neuro inflammation, supportive in efficient learning, increase memory and cognitive role (Rao et al., 2008). These flavonoids act together with lipid and protein kinase signaling pathways in the brain and promote neuronal survival and have useful effect on cerebro-vascular blood flow (Vauzour et al., 2008). So the improvements in neuofunctioning in adult albino mice can be attributed with the presence of antioxidants like terpenoids, flavonols (Kaempferol, Quercetin), coumarins (Scopoletin and Scopolin), steroids and catchol in Bauhinia variegata assumed to improve the memory and learning in mice (Bouwknecht and Paylor, 2002).
The light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. This test is also used to test the exploratory behavior in rodents (Bourin and Hascoet, 2003). Our light and dark box test results again indicated gender specific response as only 300 mg/ml solvent/Kg body weight of Bauhinia variegata leaf extract was the dose that significantly improved the parameters associated with exploratory behavior in male albino mice (Table II). The same dose did not affect the exploratory behavior in female albino mice.
Similar results were documented by Shah and Goyal

O n l i n e F i r s t A r t i c l e
Bauhinia variegata and Behavior of Albino Mice 7 (2011) as they have reported that exposure to methanolic extract of Bauhinia racemosa resulted in increased time spent by mice in the illuminated side of the light-dark test along with significant increase in nose poking (Rearing) in comparison with control animals. Davey et al. (2011) had also reported that indicate that methanolic extract of Bauhinia racemosais an effective anxiolytic agent in mice. These results indicate the Bauhinia racemosa and Bauhinia variegata has similar chemical composition as they have selectively positive impact on rodent behavior. Open field test is one of the most widely used test in animal psychology experiments. It is used to test the emotionality of rodents, is used to measure general locomotor activity qualitatively and quantitatively and to measure willingness to explore in rodents (Stanford, 2007). Analysis of our results indicated that the applied dose remains unaffected for both the genders indicating the exploratory and locomotory behavior of male and female albino mice is not influenced by Bauhinia variegata leaf extract application for 17 days (Table III). Our results are in agreement with those of Santos et al. (2012) as they have reported that the exposure to ethanolic leaf extract of Bauhinia platypetala did not induce any change in Swiss adult mice behavior during the open field test. Cavalcanti et al. (2011) had also reported that the aquous extract (5.0g/ Kg) of Bauhinia forficate supplementation resulted in decrease in locomotion frequency and increase immobility during open field test. Sathya et al. (2011) who had reported reduced mobility in mice and rats upon treatment with alcoholic leaf extracts of B. tomentosa (200 mg/Kg and 400 mg/Kg) and B. forficata (5 g/Kg) respectively. These results indicate the Bauhinia platypetala, Bauhinia forficate, Bauhinia tomentosa and Bauhinia variegata leaf extract composition has variation as they are elective different response in rodents during neurological testing.
The Novel object recognition test has been introduced by Ennanceur and Delacour (1988) and can be regarded as a spontaneous Delayed-Non-Matching-to-Sample test. The test is based on a spontaneous behavior: the main assumption at the base of this test is that access to novelty (e.g. an object or an environment) can elicit approach behaviors in animals (Gaskin et al., 2010). This apparent 'unconditioned preferencefor novelty has been used in the NOR test in order to study memory functions, assessing the ability of animals to recognize a novel object in a familiar environment, because they maintain a representation of those is more familiar stored in memory (Aggleton et al., 2010). Analysis of our results indicates that the applied dose (300 mg/ml solvent/Kg body weight) of Bauhinia variegata did not affect the object recognition ability of both male and female albino mice (Table IV). Our results are contradictory to De-Laine et al. (2011) as they had reported that estrous rats spent a significantly greater percentage of time with the novel object than did diestrous rats. The difference in results is probably due to two reasons. First is the De-Laine et al. (2011) used rats as experimental subject while albino mice were used in our study. Two different species may show different behavioral response. Secondly, we did not account the estrous or nonestrous phase of animals during our study. De-Laine et al. (2011) had also reported that adolescent rodents have been characterized as showing higher novelty-seeking and risktaking behavior than adults, potentially due to selection pressures that have favored a willingness to engage with novelty around the time of dispersal.
In conclusion, we have conducted a study to demonstrate the effects of 300 mg/ml solvent/Kg body weight of ethanolic leaf extract of Bauhinia variegata on behavior of male and female albino mice. It was observed that applied dose significantly improved the rota rod test performance in females and can be used for them to improve muscular functioning. Female albino mice displayed improved elevated plus maze and light-dark box performance when supplemented with 300 mg/ml solvent/Kg body weight of Bauhinia variegata ethanolic leaf extract for 17 days. Open field, Novel object test and Morris water maze results remains unaffected in both the genders following application of 300 mg/ml solvent/Kg body weight of Bauhinia variegata leaf extract.

Statement of conflict of interest
Authors declare that they do not have conflict of interest of any sort with any one.