Behavioural assessment of Mikania micrantha Kunth roots in Wistar albino rats

The present study was designed to evaluate the adaptogenic activity of methanolic and aqueous extract of roots of Mikania micrantha kunth in Wistar albino rats using different experimental models such as anoxia stress tolerance, swimming endurance and immobilisation stress. The plant was subjected to preliminary phytochemical screening. The parameters like anoxia stress tolerance and swimming endurance time were recorded. The estimation of biochemical marker levels and determination of organs weight were carried out in immobilisation stress model. These activities are tested at oral doses of extract at 250 and 500 mg/kg and diazepam 2 mg/kg was used for comparison. Preliminary phytochemical screening revealed the presence of flavonoids, steroids and tannins. Pretreatment with methanolic extract showed increase in anoxia stress tolerance time and swimming endurance time. There was a dose dependant significant reduction in biochemical parameters like serum glucose, cholesterol and blood urea nitrogen levels exhibited methanolic extract treated animals. The stress induced increase in weight of liver, adrenal gland and decrease in weight of spleen were significantly reversed by the methanolic extract at higher dose. The results from the study indicated that methanolic extract of M. micrantha roots possessed significant antistress activity.


INTRODUCTION
Stress can be defined as the sum of all the reactions of the body, which disturb the normal physiological condition and results in a state of threatened homeostasis.Normally stress induced changes are compensatory, self limiting and adaptogenic in nature.However in higher animals when stress events of any nature (physical, chemical, biological and emotional) over certain 'threshold' limits occur, the changes become rather irreversible.It leads to altered homeostasis and exhaustion, manifesting itself in the pathologic form of stress induced disease and maladjustment.There is no treatment in modern drug therapy for stress related diseases.The available techniques for increasing endurance performance include physical training for endurance work, yogic and meditation practices, supplementation of nutraceuticals and intervention by adaptogens.Present study will provide a scientific base for experimental research on Indian herb for stress related diseases.The term 'Adaptogen' denotes an agent that improves adaptation capacity of the organism during stress and "Antistress" agent is a pharmacological word for the same, meaning an agent which nullifies or prevents the effects of stress and improves adaptation (Sharma and Arora, 2006).
Mikania micrantha Kunth (Asteraceae) is found in the tropics of America and Asia, and is widely known as guaco, the plant is a branched, extensively scrambling and twining slender-stemmed vine.Mikania comprises about 300 identified species, but only 20 of them have been studied.It is used to treat fever, rheumatism, influenza and respiratory diseases.Terpenes such as mikanolide are the major constituents isolated from plants of this genus (Facey et al., 2010).In the present study, an attempt has been made to investigate the adaptogenic activity using aqueous and methanolic extract of roots of the plant M. micrantha in view of reported adaptogenic activity of other species of Mikania namely Mikania cordata (Bishayee and Chatterjee, 1994).

Collection and identification of the plant materials
M. micrantha roots were collected from Kottayam, Kerala, India during the month of March, 2011 and were authenticated by Mr. Joby Paul, Botanist, School of Environmental Sciences, Mahatma Gandhi University, Kottayam, Kerala (Voucher No. 1461).

Preparation of extracts
Extraction of roots of M. micrantha was carried out using methanol by hot continuous extraction method using soxhlet apparatus.500 g of dried roots were taken, size reduced, extracted with 2 L of methanol in the round bottom flask and extraction was continued for few hours.The extract obtained was collected and concentrated by gentle heating.The concentrated extract was then weighed and stored.Thus total methanolic extract is obtained.Aqueous extraction was carried out with the remaining marc by reflex method.The marc was packed in a round bottom flask and refluxed for 2 h using a reflex condenser.The extract was then concentrated to dry residue by heating.Percentage yield of methanolic and aqueous extracts were found to be 7.7 and 3.8% w/w.

Preliminary phytochemical analysis
The preliminary phytochemical studies were performed for testing different chemical constituents present in methanolic and aqueous extracts using standard methods (Kokate, 1994;Khandelwal, 2004).

Selection of animals
Healthy adult Wistar albino rats, weighing about 150 to 220 g obtained from the registered Animal house of University College of Pharmacy, MG University, Kottayam were used for the study.The study protocol was approved by the Institutional Animal Ethical Committee, University College of Pharmacy, Cheruvandoor Campus (001/MPH/UCP/CVR/12).All the animals were housed individually in polypropylene cages, maintained under standard husbandry conditions (12 h light and dark cycles, room temperature Ittiyavirah and Sajid 449 and 45 to 55% relative humidity).They had been given standard pellet diet and water ad libitum throughout the course of the study.

Acute toxicity studies (OECD Guidelines 423)
The acute toxicity studied was carried out in female albino rats by "acute toxic class method" (OECD guidelines 423).The animals were fasted overnight and extracts of the herb M. micrantha suspended in 0.5% Na.Carboxy methyl cellulose (CMC) was administered starting at 2000 mg/kg p.o, food was withheld for next 3 to 4 h.The animals were observed continuously for body weight, any changes in skin and fur, eyes , behavior pattern and also signs of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma every 30 min for the next 3 h and finally death after 24 h.

Anoxia stress tolerance test
Albino Wistar rats of either sex weighing 150 to 220 g were selected and divided into 5 groups of six each as Group I: Control (received only vehicle CMC 0.5% w/v p.o.), Group II: Methanolic extract of M. micrantha root (MEMMR 250 mg/kg p.o.); Group III: MEMMR (500 mg/kg p.o.); Group IV: Aqueous extract of M. micrantha root (AEMMR 500 mg/kg p.o.); and Group V: diazepam (2 mg/kg p.o.).Animals were treated as shown for 3 weeks.At the end of 1st, 2nd and 3rd week that is, on 7th, 14th and 21st day, 1 h after the treatment.Stress was induced by placing each animal individually in the hermetic vessel of 1 L capacity to record anoxia tolerance time.The time duration of entry of the animal into the hermetic vessel and the appearance of the first convulsion was taken as time of anoxia tolerance (Pawar and Shivakumar, 2011).

Swimming endurance test
Albino Wistar rats of either sex weighing 150 to 220 g were selected and divided into five groups of six each as mentioned.The rats were subjected to swimming stress by keeping them in propylene tank of dimension (37 × 37 × 30 cm), filled with water to a height of 25 cm.Extracts were given to rats, once daily for period of 7 days.On 8th day the rats were allowed to swim till complete exhaustion and the endpoint was taken when the animal started drowning.The mean swimming time for each group was calculated (Kothiyal and Ratan, 2011).

Immobilisation stress
Adult male albino rats of 150 to 220 g were selected and divided into 6 groups of 6 animals each as Group I: Negative control (unstressed, untreated); Group II: Positive control (Stressed, received vehicle); Group III: MEMMR (250 mg/kg p.o.); Group IV: MEMMR (500 mg/kg p.o.); Group V: AEMMR (500 mg/kg p.o.); Group VI: diazepam (2 mg/kg p.o.).The treatment was made as stated for 10 days, 1 h before the exposure of stress.Stress was induced by immobilizing rats with head down, supine position by fixing the forelimbs and hind limbs to a wooden board inclined at an angle of 60°, daily 2 h for a period of 10 days.The animals were sacrificed at the end of specified period and blood was collected by retro-orbital for estimation of biochemical parameters such as, serum glucose, cholesterol and blood urea nitrogen (BUN).The weight of organs, such as liver, spleen and adrenal glands after washing with alcohol was recorded per 100 g body weight of animal (Pawar and Shivakumar, 2011).

Statistical analyses
The statistical analyses were performed using 'Graph Pad Prism 6' Software by one way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test.All data were expressed as mean ± standard error of mean (SEM), P < 0.05 was considered as statistically significant.

Preliminary phytochemical analysis
The preliminary phytochemical studies were performed for testing different phytochemical constituents present in methanolic and aqueous extracts of M. micrantha.The observations showed the presence of alkaloids, flavonoids, steroids, tannins and phenolics, which were found to be more in methanolic extract.

Acute toxicity studies
The methanolic and aqueous extracts of the plant M. micrantha was found to be safe up to 2,000 mg/kg body weight by oral route.After 24 h, animals were found well tolerated; there was no mortality and no signs of toxicity.The extracts were found to be safe, so the two dose levels that is, 250 and 500 mg/kg body weight were selected for the present study.

Anoxia stress tolerance time
The results obtained from the anoxia stress tolerance test was expressed as mean ± SEM.Anoxia stress tolerance time was significantly (P < 0.05) enhanced on 7th, 14th and 21st day in MEMMR (500 mg/kg) and diazepam (2 mg/kg) treated groups.There was increased anoxia tolerance time seen after 2nd and 3rd week of MEMMR (250 mg/kg) treated group but not statistically significant with result obtained on 7th day.However the effect of AEMMR (500 mg/kg) on anoxia stress tolerance time in rats was not statistically significant at the end of 1st, 2nd and 3rd week of treatment (Table 1 and Figure 1).

Swimming endurance test
The swimming endurance time was significantly (P < 0.05) enhanced on 8th day in MEMMR (250 mg/kg), MEMMR (500 mg/kg) and diazepam (2 mg/kg) treated groups when compared to the stressed group (Table 2 and Figure 2).

Immobilisation stress
Effect on biochemical parameters: The immobilisation stress caused marked increase in biochemical parameters such as glucose, cholesterol and blood urea nitrogen in stressed group when compared to the control group.This stress induced elevated level of biochemical parameters such as, serum glucose, cholesterol and blood urea nitrogen were significantly reversed in MEMMR 500 mg/kg treated groups whereas AEMMR failed to reverse the elevated levels of biochemical parameters significantly (Table 3).
Effect on organ weight: Weight of liver and adrenal gland was increased, while weight of spleen was reduced in stressed group when compared to unstressed group.Pretreatment with MEMMR extract at high dose significantly (P < 0.01) reduced the weight of the liver, adrenal gland and increased the weight of the spleen.However the MEMMR extract at low dose (250 mg/kg) and AEMMR (500 mg/kg) failed to protect the immobilisation stress induced changes in organ weight such as liver, adrenal gland and spleen (Table 4).

DISCUSSION
In the present investigation, methanolic and aqueous extracts of M. micrantha has been evaluated for the antistress (adaptogenic) activity against different types of stress: anoxia, swimming endurance and immobilisation models.Diazepam, a benzodiazepine anxiolytics was used for the comparison.Diazepam is reported to possess a non-specific anti-stress activity involving the mesocortical dopamine system and the norepinephrine and 5HT levels of whole brain and hypothalamus.It is proposed that this effect is produced through an enhancement of GABAergic neurotransmission (Kenjale   et al., 2007).
In anoxia stress tolerance model, depletion of oxygen in hermetic vessel leads to convulsions in animals, and pretreatment with methanolic extract of M. micrantha had increased the duration of stress tolerance indicating their adaptogenic/anti-stress activity.This effect may be due to that fact that; during stress the methanolic extract of M. micrantha was capable of increasing succinate dehydrogenase (SDH) in the brain (Pawar and Shivakumar, 2011).This enzyme is responsible for utilization and conservation of energy in the cellular system of the organism, which helps adaptive processes during stress.Adaptogens producing beneficial effects in stress are believed to act by increasing non-specific resistance.
In case of swimming endurance test, MEMMR exhibited significant antistress activity as indicated by increase in swimming endurance time.There are reports that plasma levels of adrenaline and noradrenaline are enhanced during stress induced by swimming endurance test.In addition, monoamine oxidase (MAO) levels in the brain are reportedly decreased during stress (Debnath et al., 2011).The swim endurance test results indicate clearly that the methanolic extract of M. micrantha has the properties whereby it increases the physical endurance as well as the overall performance in rats and possessed significant anti-stress activity.It may be possibly normalizing the plasma level of catecholamine and MAO.
The immobilisation stress caused marked increase in biochemical parameters such as glucose, cholesterol and blood urea nitrogen in stressed group when compared to the control group.In the present study, a significant hyperglycemia was observed with immobilization stress model.Under stressful conditions, cortisol in human and corticosterone in rats will be secreted by adrenal cortex.Hyper secretion of cortisol helps the maintenance of internal homeostasis through the process of gluconeogenesis and lipogenesis (Debnath et al., 2011).Methanolic extract of M. micrantha significantly reduced hyperglycemia probably by reducing the hyperactivity of adrenal cortex and also by maintenance of homeostatic mechanism in immobilization stress animals.
The mechanism by which stress rises serum cholesterol is likely to be related to the enhanced activity of hypothalamo-hypophyseal axis (HPA) resulting in liberation of catecholamines and corticosteroids.This could lead to increase in blood cholesterol level since epinephrine is known to mobilise lipids from adipose tissues.The increase in release of catecholamines leads to elevated levels of glucose and BUN (Tsigos and Chrousos, 2002).This stress induced elevated level of biochemical parameters were significantly reversed in MEMMR 500 mg/kg treated groups.
Adrenal glands and liver weights were significantly increased in immobilization stress models.Stress induces adreno-medullary response in man to release adrenaline which in turn stimulates β 2 receptors on the pituitary gland.It leads to greater release of adrenocorticotropic hormone (ACTH) that can stimulate the adrenal medulla as well as cortex resulting in further release of adrenaline and increase in weight of adrenal gland to a greater extent.Cortisol increases mRNA levels in liver cells, this lead to increase in weight of liver.Spleen constricts to release more blood cells (RBC) during stress.So its weight decreases during stress (Pawar and Shivakumar, 2011).This stress induced changes of organs weight were significantly reversed by the methanolic extract at higher dose 500 mg/kg.
Literature survey indicates that flavonoids, triterpenes and tannins were reported to possess variety of pharmacological activities including antistress activity (Chethan et al., 2012).In this present investigation, preliminary phytochemical screening on MEMMR gave positive tests for flavonoids, steroids and tannins, this might be the reason for significant adaptogenic property.
The result from the study showed an increase in duration of anoxia tolerance and swimming endurance time in rats treated with methanolic extract.The reversal of immobilization stress induced changes in biochemical parameters and organs weight were also exhibited in alcoholic extract treated groups.So the results suggest the adaptogenic activity of the plant M. micrantha, hence it can be categorized as plant adaptogen.The results are encouraging to pursue further studies on the bioactivity guided fractionation of these extracts to isolate and characterize probable bioactive molecule responsible for ant-stress activity.

Figure 1 .Figure 2 .
Figure 1.Effect of M.micrantha on anoxia stress tolerance time in rats.Values are expressed as Mean ± SEM (n=6), analysed by one-way ANOVA followed by Dunnett's post hoc test, *Represents statistical significance vs. control (p<0.05).

Table 2 .
Effect of M.micrantha on swimming endurance time.

Table 3 .
Effect of M. micrantha on immobilization stress induced changes in biochemical parameters.

Table 4 .
Effect of M. micrantha on immobilization stress induced changes in organ weight.