Evaluation of anxiolytic potential of Cichorium Intybus methanolic root extracts

: Cichorium Intybus L. is a medicinally important plant that belongs to the family Asteraceae. The plant root is used as anti-hepatotoxic, antiulcerogenic, antiinflammatory, appetizer, digestive, stomachic, liver tonic, cholagogue, cardiotonic, depurative, diuretic, emmenagogue, febrifuge, alexeteric and also as tonic. The present study was carried to screen the anxiolytic potential of Cichorium Intybus L methanolic root extracts by using different animal models like actophotometer, rotarod, staircase, holeboard, elevated plus maze tests . In the experiment the extracts found to decrease the anticipatory anxiety of the animal without significant alteration in motor coordination. Efficacy may be attributed to the presence of different phytochemicals like coumarins, flavonoids in the extracts.

diabetes, dysmenorrhoea, impotence, insomnia, splenitis and tachycardia (Duke, J.A., 1983.). Inulin is used to replace fat or sugar and reduce the calories of food. It is suitable for consumption by diabetics (Niness, K.R., 1999.)and is also used in inulin clearance test to measure glomerular filtration rate-GFR ( The sesquiterpene lactones such as lactucin and lactucopicrin were isolated from chicory and reported for its antibacterial and antimalarial activity (Bischoff,2004). Therefore, we undertook the study to evaluate the anxiolytic potential of Cichorium Intybus root extractsby using different animal models and studying the effect of the plant on their exploratory behavior.

Materials and methods:
Collection of plant material: The dried roots of Cichorium Intybus are collected from the Chicory Factory, located in Ananthapur District. The plant material was authentified a specimen was kept in the department for future referances. The roots were cleaned, shade dried, coarse powdered, and subjected to extraction with methanol by using Soxhlet's apparatus.

Preparation of ethanolic extract:
The plant material was cut into small pieces and powdered coarsely and then placed in the extraction thimble. The 100grams of chicory root powder was placed in an extraction chamber which was suspended above the flask containing the solvent methanol and below a condenser. The flask was heated and the methanol evaporated and moved into the condenser where it was converted into a liquid that trickled into the extraction chamber containing the plant material. The extraction chamber was designed so that when the solvent surrounding the sample exceeded a certain level it overflowed and trickled back down into the boiling flask. At the end of the extraction process, the flask containing the methanol extract was removed and methanol was evaporated by heating mantle.
Drugs: Diazepam inj. i.p, (Ranbaxy laboratories limited, Boiser, Thane, India) was used as a reference drug. It was diluted using saline before use. Different doses of Cichorium Intybus flowers extract were prepared by dissolving with 1% of gum acacia in distilled water. All solutions were prepared freshly on the test days and administered orally.

Treatments:
The animals were divided into 3 groups with 8 animals each. All the treatments were given orally 30 minutes prior to the experiment. Group 1 is vehicle control, Group 2 is Standard drug: Diazepam(2mg/kg), Group 3 is Test: Cichorium Intybus extract (500mg/kg), Animals: Healthy adult Swiss albino mice (20-25 g) were used for the study of anxiolytic activity . Experimental animals were purchased from Mahaveer Enterprises Ltd, Hyd. 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. Mice were treated with Diazepam (2 mg/kg, i.p.), Cichorium Intybus extract for15days. 30 min after treatment with diazepam and 1 h after treatment with control and/ or extracts, the following tests were performed.

Pharmacological screening:
General behavioural profile Evaluation of general behavioural profile was performed by the method of Irwin S. The animals were under observation for behavioral changes if any, at 30 minutes interval in the first hour and at one hour intervals for next 6 h.  Cichorium intybus methanolic extract at a dose of 500mg/kg affected spontaneous activity, irritability gait and touch responses. They also produce moderate or slight depression relating to awareness and alertness animals. However, the righting reflex corneal reflex and vocalization remain normal throughout the study process. Above studies indicates that there is a mild sedation action of the extract on the animals. All values are Mean± SEM of n= 6, One way ANOVA followed by Brown-Forsythe test, Bartlett's test, ** p <0.05. Ns-non significant when compared to control( 0.5% sodium CMC) treated animal.
In the current study methanolic extract of chicory root had shown significant (p<0.05) decrease in no.of rearings when compared to control which indicates anxiolytic potential of extract. And also shown significant (p < 0.05) increase in no. of steps ascended by mice is seen with administration of methanolic extract treated animals as compared to standard. All values are Mean± SEM of n= 6, One way ANOVA followed by Brown-Forsythe test, Bartlett's test, ** p <0.05. Ns-non significant when compared to control( 0.5% sodium CMC) treated animal.
The methanolic chicory root extract has shown significant (p<0.05) increase in immobility time when compared to control group of animals. But less significant (p<0.05) when compared to diazepam. All values are Mean± SEM of n= 6, One way ANOVA followed by Brown-Forsythe test, Bartlett's test , * p <0.05, ** p<0.01, Ns-non significant when compared to control( 0.5% sodium CMC) treated animal.
In the present study, methanolic extract of chicory root at dose 500 mg/kg significantly (p<0.05) decreased headdip counts as compare to the control. All values are Mean± SEM of n= 6, One way ANOVA followed by Brown-Forsythe test, Bartlett's test , * p <0.05, ** p<0.01, Ns-non significant when compared to control( 0.5% sodium CMC) treated animal.
The mice treated with methanolic chicory root extract has shown significant(p< 0.05) decrease in locomotor activity scores when compared to control. But scores are less significant as compared to standard. All values are Mean± SEM of n= 6, One way ANOVA followed by Brown-Forsythe test, Bartlett's test , * p <0.05, ** p<0.01, Ns-non significant when compared to control( 0.5% sodium CMC) treated animal. Incase of rotarod test mice treated with methanolic chicory root extract has shown significant (p< 0.05) increase in immobility time is recorded when compared with control. This effect is less significant (p<0.05) when compared to standard.

Discussions
The loss of irritability, reactivity and grooming indicates anxiolytic effect of the extracts on the animal. Irritability is an extension of restlessness and in higher degree it is demonstrated by aggressiveness. The test extract suppress the normal irritability when expose to stressful external stimuli. The gait of the animal indicates the control disturbance or neuromuscular blocker upon administration of the test drug some animals show bit staggering gait. Above studies indicates that there is a mild sedation action of the extract on the animals. The loss of irritability, reactivity and grooming indicates an anxiolytic effect of the extracts on the animal. The elevated plus-maze is a well-established animal model for testing anxiolytic drugs (Dawson 1995). It represents a forced exploration, i.e., no non-stressed situation, since all parts of the situation are novel The results showed that chicory at 500mg/kg could increase the time in the light area and the number of entries into the open arms, suggesting again that chicory possesses anxiolytic properties. It at the same time, the entries of closed arms was not affected by the administered chicory. The evaluation of certain components of behaviour of mice such as curiosity or exploration has been attempted by Boissier et al. (1964) and Boissier and Simon (1964). The "hole-board" test provides a simple method for measuring the response of an animal to an unfamiliar environment and is widely used to assess emotionality, anxiety and,/or responses to stress in animals. In the present study, methanolic extract of chicory root at dose 500 mg/kg significantly (p<0.05) decreased head -dip counts as compare to the control. These results indicate the significant anxiolytic potential of chicory extracts in this paradigm. They do not alter spontaneous behaviour of the animals at the chosen dosage regimen. The stair case test, one of the exteroceptive model chosen for the current study was originally described by Thiebot et al., and subsequently used by several workers conforming the utility of this test in the successful and rapid assessment of anxiolytic activity. When (test animal) introduced into novel environment, rodents experience a conflict between anxiety and exploratory behaviour manifesting as increased vigilance and behavioural activity. Steps ascended is believed to reflect exploratory or locomotor activity, while rearing recorded as number of rearing in observation duration (5minutes) is an index of anxiety. In the present study methanolic extract of chicory root had shown significant (p<0.05) decrease in no.of rearing and increase in no. of steps ascended by mice when compared to control which indicates anxiolytic potential of extract. The locomotor activity can be an index of wakefulness (alertness) of mental activity (Arora 2011). Most of the CNS drugs influence the locomotor activities in animals. The locomotor activity (horizontal activity) can be easily measured using actophotometer which operates on photoelectric cells which are connected in circuit with a counter. When the beam of light falling on the photocell is cut off by the animal, a count is recorded. In locomotor activity test (Actophotometer) the mice treated with methanolic chicory root extract has shown significant(p< 0.05) decrease in locomotor activity scores when compared to control. But scores are less significant as compared to standard. The results indicates that the extract possess anxiolytic potential. Rotarod test is used to evaluate the activity of drugs interfering with motor coordination. In 1956, Dunham and Miya suggested that the skeletal muscle relaxation induced by a test compound could be evaluated by testing the ability of mice or rats to remain on a revolving rod. (Deacon 2013)An anxiolytic drug usually shows skeletal muscle relaxation property together with taming or calming effect which reduces anxiety and tension. The loss of muscle-grip strength is an indication of muscle relaxation. In case of rotarod test mice treated with methanolic chicory root extract has shown significant (p< 0.05) increase in immobility time is recorded when compared with control. It indicates that extract shows anxiolytic effect. Plants containing tannins, sterols, flavonoids etc. are reported to have anxiolytic activity and preliminary phyto -chemical screening of chicory root revealed the presence of sterols, tannins, flavonoids and carbohydrates in the chicory roots. Therefore, the anxiolytic activity of root chicory is may be due to the presence of tannins, sterols, flavonoids etc. However, further investigations are required to isolate the phytoconstituents responsible for anxiolytic activity and to find their mechanism of action.As described by several authors and accepted universally, a novel environment created anxiety in these test animals, which reflected in its behaviour typical of a state of anxiety and conflict. It was demonstrated that the administration of the methanolic extract of chicory root in mice was able to induce anxiolytic effects, without modifying significantly the spontaneous motor activity.

Conclusion:
From the above study, it was concluded that Cichorium Intybus root extract possess significant anxiolytic effect. These findings give a scientific rationale for the future work of Cichorium Intybus