D. Kumar* and M. Dhobi1

Department of Pharmaceutical Sciences and Drug Research, Punjabi University, Patiala-147 002, Punjab, India
1University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh-160 014, India

*Corresponding Author:
D. Kumar
Department of Pharmaceutical Sciences and Drug Research, Punjabi University, Patiala-147 002, Punjab, India
E-mail: jindalpu@gmail.com
Date of Submission 08 July 2016
Date of Revision 01 April 2017
Date of Acceptance 08 October 2017
Indian J Pharm Sci 2017;79(6): 1022-1025  

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DOI: 10.4172/pharmaceutical-sciences.1000322

Abstract

Previous studies from our laboratory demonstrated that the antianxiety and antioxidant activities of the roots of the blue variety of Clitoria ternatea were greater compared to the white variety. An attempt has been made to investigate the antianxiety and antioxidant activities of aerial parts of this plant using elevated plus maze and 2,2-diphenyl-1-picrylhydrazyl assay, respectively. Methanol extract of stems and leaves of the blue variety (50, 100 or 200 mg/kg, p.o.), diazepam (2 mg/kg, p.o.) and control (vehicle) were separately evaluated for antianxiety activity. The stem extract (200 mg/kg) and leaf extract (100 or 200 mg/kg) exhibited significant antianxiety activity with respect to control and standard. The remaining doses did not exhibit any antianxiety activity. Mice treated with the blue variety leaf extract, showed decresed number of entries from 6.20 to 4.60 when the dose was increased from 100 to 200 mg/kg indicating a possibility of decreased locomotor activity, which could be due to cental nervous system depressant activity. In case of blue variety stem extract, the average number of entries increased from 3.20 to 5.80 as the dose was increased from 100 to 200 mg/kg. The IC50 values of ascorbic acid, blue variety stem extract and leaf extract were found to be 20.01, 264.32 and 251.13 µg/ml, respectively. None of the plant parts showed activity close to that of ascorbic acid. Finally, it can be concluded that aerial parts of blue variety of C. ternatea exhibited mild antianxiety and antioxidant activities.

Keywords

Anxiety, antioxidant, Clitoria ternatea, diazepam, DPPH

Clitoria ternatea L. (Butterfly pea; Papilionaceae) has long tradition of use in the treatment of asthma, fever, inflammation and mental disorders [1-3]. The plant is mainly distributed in Andaman Islands at an altitude of 1500 m [4]. C. ternatea has been reported to contain flavonoids like norneolignans, kaempferol, quercetin, myricetin and triterpenoids like taraxerol, taraxerone [5-7]. The plant has been reported to possess anticonvulsant, antistress, antidepressant and tranquilizing properties [8,9].

Previous studies from our laboratory reported a greater antianxiety and antioxidant activities from the roots of the blue variety of C. ternatea compared to the white variety [10]. Preliminary phytochemical screening of methanol extract of roots, stems and leaves showed presence of steroids, triterpenoids, flavonoids and alkaloids. A survey of literature revealed that these phytoconstituents are responsible for antianxiety and antioxidant activities. Thus, it was planned to investigate antianxiety and antioxidant activities on stems and leaves of blue variety of C. ternatea. The stems and leaves of blue variety of C. ternatea were collected during the month of June 2010 from the cultivated plants at herbal garden and medicinal plants garden, Panjab University, Chandigarh. The samples were further authenticated at the Forest Research Institute, Dehradun, vide certificate number 765/2006- bot/15-1. The methanol extract of stems and leaves (2 kg each) of blue vareity of C. ternatea were extracted separately with methanol (Laboratory Grade; E Merck, Delhi, India) as per procedure described in our laboratory [10].

Anxiolytic activity was investigated using well established model, i.e., elevated plus maze (EPM) model [11]. Laboratory Animal Centre A-strain (LACA) mice were procured from the Central Animal House of Panjab University, Chandigarh. The animal model was approved by the Institutional Animal Ethics Committee of Panjab University, Chandigarh (IAEC/97, dated 24-03-2011). The experimental animals were divided into 3 groups of control, standard and test. The control group animals received only vehicle (2 % Tween 80, p.o.); the standard group animals received diazepam as standard drug (2 mg/kg, p.o.) for comparison and test group animals received the test substances (50, 100 and 200 mg/kg, p.o.). The results were expressed as mean±SD. The inter group variation was measured by one way analysis of variance (ANOVA) followed by Tukey’s test.

The EPM model was used in present investigations because it is cost effective, easy to operate, less time consuming, and require no preliminary training to the mice and do not cause much discomfort to the animals while handling. The model is principally based on the phobia due to height [12]. The percentage yields of methanol extract of blue variety stems and blue variety leaves were found to be 8.3 and 16.9 % w/w, respectively. It is evident from Table 1 that blue variety stems and blue variety leaves possessed mild anxiolytic activity. The blue variety stems showed mild anxiolytic activity, which was dose dependent. Blue variety stems showed it’s best mild response at dose 200 mg/kg and average time spent in open arms was increased from 3.66 s in control group to 10.08 s in treated animals. The activity of stems and leaves of blue variety at a dose of 50, 100 and 200 mg/kg was significant with respect to diazepam. In comparison to control, methanol extract of stems and leaves showed significant activity except 50 mg/kg dose and 100 mg/kg of blue variety stems. A dip in activity at a higher dose in case of blue variety leaves extract showed the CNS depressant activity. The behavioural changes were also observed in the treated animals. The animals treated with 100 mg/kg dose of blue variety leaves showed an average number of entries 6.20 in open arms. The number of entries decreased when the dose was increased from 100 to 200 mg/kg indicating a possibility of decreased locomotor activity, which could further be related to CNS depressant activity. In case of blue variety stems, the average number of entries increased from 3.20 to 5.80 as the dose was increased from 100 to 200 mg/kg. The antioxidant activity was investigated using well established in vitro DPPH assay [13]. The IC50 values of ascorbic acid (Analytical Grade; Hi-media Laboratories Pvt. Ltd, Mumabi, India) blue variety stems and blue variety leaves were found to be 20.01, 264.32 and 251.13 μg/ml, respectively (Tables 2-4). None of the plant parts showed activity close to the ascorbic acid (IC50=20.01 μg/ml) and confirmed mild antioxidant activity. From the results, it can be concluded that aerial parts of blue variety of C. ternatea exhibited mild antianxiety and antioxidant activities.

Group Dose
(mg/kg)		 Meann number of entries in open arm ±SD Meann time spent in open arms (s)±SD
Control - 1.60±0.54a 3.66±0.73a
Diazepam 2 10.60±1.14* 24.96±4.37*
Stems 50
100
200		 1.50±0.57a
3.20±1.30a
5.80±1.48*a		 3.20±0.34a
7.64±1.89a
10.08±2.51*a
Leaves 50
100
200		 2.70±0.95a
6.20±1.14*a
4.60±2.16*a		 4.47±0.54a
12.52±3.95*a
11.06±1.29*a

Table 1: Anxiolytic Activity of stems and leaves of C. Ternatea blue variety using EPM model

Concentration (µg/ml) Absorbance (mean±SD) Percent inhibition
12 0.2507±0.005 6.07
15 0.1961±0.003 26.52
18 0.1542±0.004 42.22
21 0.1237±0.004 53.65
24 0.0728±0.002 72.72
27 0.0365±0.002 86.32
30 0.0072±0.0005 97.30

Table 2: Percent Inhibition of DPPH At Different Concentrations of Ascorbic Acid

Concentration (µg/ml) Absorbance (mean±SD) Percent inhibition
50 0.2120±0.001 15.06
150 0.1650±0.003 33.80
250 0.1260±0.001 49.51
350 0.0952±0.002 61.85
450 0.0456±0.003 81.73
550 0.0229±0.002 90.82

Table 3: Percent Inhibition of DPPH by Methanol extract of blue varietystems of C. Ternatea

Concentration (µg/ml) Absorbance (mean±SD) Percent inhibition
50 0.2281±0.003 22.34
150 0.1770±0.002 39.73
250 0.1462±0.005 50.22
350 0.1090±0.002 62.8
450 0.0700±0.003 76.16
550 0.0431±0.002 85.32

Table 4: Percent Inhibition of DPPH by methanol extract of blue varietyleaves of C. Ternatea

Acknowledgements

The financial support provided by the University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh for the present research work is duly acknowledged.

Conflict of interest

The authors declare that they have no conflict of interest.

Financial support and sponsorship

Nil.

References