Anxiolytic activity of Evolvulus alsinoides and Convulvulus pluricaulis in rodents

Pharmaceutical Biology, 2009; 47(5): 444–451

R E S E A R C H A R T I C L E

Anxiolytic activity of Evolvulus alsinoides and Convulvulus pluricaulis in rodents

Alok Nahata, U.K. Patil, and V.K. Dixit

Department of Pharmaceutical Sciences, Dr. Hari Singh Gour University, Sagar 470 003, Madhya Pradesh, India

Address for Correspondence: Professor V.K. Dixit, Department of Pharmaceutical Sciences, Dr. Hari Singh Gour University, Sagar 470 003, M.P., India. Tel.: +917582264582; Fax: +917582264163; E-mail: [email protected]

(Received 14 January 2008; revised 04 April 2008; accepted 04 April 2008)

Introduction

Modern lifestyles have resulted in stress-related disorders, and various approaches, for example, yoga, meditation and anti-stress drugs, are used to coun-teract aversive stress effects. Plant drugs have come to the rescue to mankind in many ailments and may offer satisfactory solutions to stress-induced perturbations. The importance of plants acting on the central nerv-ous system (CNS) has been reviewed (Carlini, 2003), emphasizing the role of adaptogens from plant origins. Rasayana is a clinical speciality of Ayurveda which prevents disease and counteracts the aging process by optimization of homeostasis and thereby rejuvenat-ing the body (Auddy et al., 2003). Medhya rasayana is a

category of rasayana which rejuvenates, maintains, and potentiates intellect and memory.

Shankhpushpi is considered as “medhya rasay-ana” in Ayurvedic texts. Shankhpushpi of Ayurvedic Pharmacopoeia of India consists of the whole plant of Convulvulus pluricaulis Choisy (Convulvulaceae) (syn; Convulvulus microphyllus Sieb. ex Spreng) (MHFW, 2001a). Plants other than Convuluvulus pluricaulis are used as sources of drug in different parts of the country and Evolvulus alsinoides Linn. (EA) (Convulvulaceae) is also used as shankhpushpi by some practitioners. Other plants, e.g., Clitorea ternatea Linn. (Papilionaceae) and Canscora decussata Schult. (Gentianaceae) are also used as Shankhpushpi by some practitioners (Gupta et al., 2005). Whatever is the source, the drug

ISSN 1388-0209 print/ISSN 1744-5116 online © 2009 Informa UK LtdDOI: 10.1080/13880200902822596

AbstractShankhpushpi is a popular medicinal plant in the Ayurvedic system of medicine for treating mental disor-ders. Convulvulus pluricaulis Choisy. (Convulvulaceae) and Evolvulus alsinoides Linn. (Convulvulaceae) are used as Shankhpushpi by Ayurvedic practitioners. Ethanol extract of the aerial parts of both these drugs was evaluated for central nervous system (CNS) activity. The ethanol extract was fractionated into ethyl acetate and aqueous fractions and was tested in experimental models employing rats and mice. Elevated plus maze test, open field exploratory behavior and rotarod performance experiments were undertaken to observe influence on CNS. The extracts were also studied for their in vitro antioxidant potential to correlate their anxiolytic activity. In the elevated plus maze, ethyl acetate fractions of both the drugs at 100 mg/kg per oral showed an anxiolytic effect as evidenced by increase in the time spent in open arms and the number of open arm entries, compared to the control group. The open field exploratory behavior was also increased on administration of the ethyl acetate fractions (100 mg/kg p.o.) of both the drugs. The ethyl ace-tate fractions at doses of 200 mg/kg p.o. significantly reduced the neuromuscular coordination indicative of the muscle relaxant activity at a higher dose in both the drugs. The aqueous fractions of both the drugs were devoid of the above pharmacological actions at similar doses. Diazepam (1 mg/kg i.p.) was used as a standard in all the animal models studied. The present study provides scientific support for the anxiolytic and antioxidant activities of extracts of Evolvulus alsinoides and Convulvulus pluricaulis and substantiates the traditional claims for the usage of these drugs in stress-induced disorders.

Keywords: Evolvulus alsinoides; Convulvulus pluricaulis; elevated plus maze; neuromuscular coordination; antioxidant activity; exploratory behavior

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Anxiolytic activity of Evolvulus alsinoides and Convulvulus pluricaulis in rodents 445

finds use for its therapeutic effect on CNS disorders like insanity, epilepsy, nervous debility and memory enhancement (Chatterjee, 1990). Many formula-tions containing shankhpushpi as a single drug or in combination with other drugs are available in Indian markets and shankhpushpi is vigorously advertised for memory enhancement in print and electronic media in India.

To explore the scientific basis for the use of shankhpushpi, it was considered worthwhile to investi-gate various plants, which are used as shankhpushpi for their action on CNS. E. alsinoides is an important plant that has been well documented in Ayurveda for its ther-apeutic values. The plant is found throughout India, in the plains and to 6,000 ft in the Himalayas. Recent phar-macological studies on leaves and the whole plant of E. alsinoides have indicated anti-ulcer (Asolkar et al., 1992), immunomodulatory (Lilly et al., 2003; Ganju et al., 2003), cytoprotective (Bhatnagar et al., 2000), adaptogenic and antiamnesic properties (Siripurapu et al., 2005) and in vitro experiments (Auddy et al., 2003) have revealed the antioxidant properties of E. alsinoides. Presence of water-soluble alkaloids betaine and evol-vine has been reported. Evolvine (C

12H

17NO

2) has been

isolated and characterized as hydrochloride, oxalate, carbonate and mercury salt. A sterol and stearic, oleic and linolenic acids have also been isolated from the fatty residue. A good quantity of magnesium phosphate has been obtained from the aqueous extract (Varadan et al., 1958). Some proteins, amino acids, and phenolic com-pounds are reported in E. alsinoides (Handa & Kapoor, 1999).

Convulvulus pluricaulis is a prostrate, sub-erect, spreading, hairy, perennial herb with a woody root-stock, found throughout India on plains (Gupta et al., 2005). Upadhyay (1986) studied the therapeutic role of Ayurvedic herbs in mental disorders and classified C. pluricaulis as a brain tonic. C. pluricaulis has been reported to exhibit antidiabetic (Alam et al., 1990), anti-anxiety (Dandiya, 1990), tranquilizing (Handa, 1994) and anti-ulcer properties (Sairam et al., 2001). C. plu-ricaulis is also reported to have hypolipidemic activities (Chaturvedi et al., 1995) and is useful in hyperthyroidism (Panda & Kar, 2001). In phytochemical investigations on C. pluricaulis, an alkaloid (shankhpushpine), fla-vonoids, and inorganic salts (e.g. potassium chloride) have been reported. Two bases have been isolated. Base A (C

5H

11N0

2) depressed the blood pressure of an

anesthetized dog, stimulated the isolated rat ileum and had temporary inhibitory action on pithed frog’s heart. Base B (C

5H

9NO

2) was devoid of such pharmacological

action (Rakhit & Basu, 1958).The present study investigated the effects of E. alsi-

noides and C. pluricaulis, both of which are regarded as

shankhpushpi acting on the central nervous system in rodents. Furthermore, the extracts were studied for their in vitro antioxidant potential to correlate their anxiolytic activity.

Materials and methods

Plant material

Aerial parts of E. alsinoides and C. pluricaulis were col-lected from Bhapel, a village in Sagar district, Madhya Pradesh, India, in the months of January to March 2006. The herbs were identified by Dr. Pradeep Tiwari, Department of Botany, Dr. Hari Singh Gour University, Sagar and preserved with voucher specimen numbers HBD/12030 and HBD/11015 for E. alsinoides and C. pluricaulis respectively in the herbarium of the institute.

Preparation of extracts

Aerial parts of E. alsinoides and C. pluricaulis were shade-dried at room temperature. The shade-dried plant material was coarsely powdered and subjected to extrac-tion with petroleum ether in a Soxhlet apparatus. The defatted marc of both the drugs was subjected to etha-nol (95%) extraction. The ethanol extract was suspended in distilled water and fractionated with ethyl acetate (Sigma Aldrich, St. Louis, MO, 98% pure) to get the ethyl acetate soluble fraction and the aqueous fraction. These fractions were utilized for the neuropharmacological investigation. Extraction was done as per the Ayurvedic Pharmacopoeia of India (MHFW, 2001b). All chemicals used for the purpose were of analytical grade.

Chromatographic studies of extracts

Thin-layer chromatographyOut of the various solvent systems tried, chloroform: methanol: toluene (8:1:1) gave the best resolution (number of spots E. alsinoides = 14, C. pluricaulis = 11)

The detecting reagent was anisaldehyde in sulphuric acid followed by heating at 110°C for 5 min.

High Performance Thin Layer Chromatography (HPTLC) studies

Selection of HPTLC platesPrecoated and preactivated TLC plates (E. Merck No. 5548) of silica gel 60 F

254 + 366, with the support of alu-

minum sheets having thickness of 0. 1 mm and size 20 × 20 cm, were cut smaller according to required dimensions (Agrawal et al., 2004).

446 Alok Nahata et al.

Sample preparationTwo grams of ethanol extract of EA and CP was weighed accurately and dissolved in 20 mL of ethanol. Solution was then refluxed for 30 min on a water bath at 60–70°C.

The extract was cooled, filtered and finally volume made 20 mL with ethanol.

Application of sampleThe extract samples were applied in the form of a band using CAMAG LINOMAT IV, an automatic sample application device, maintaining a band width 9 mm, space 9 mm, 15 μL/sec. The quantity of sample applied was 5–10 μL (Chopra et al., 2006).

HPTLC developmentThe following mobile phase was selected experimentally:Chloroform: Glacial acetic acid: Methanol: Water (60:32:12:8).

The plates were developed by placing in a presatu-rated tank ( 12 cm height) with the mobile phase for 2 h. The plates were dried by evaporating the solvent either at room temperature or by spraying hot air by air dryer. The HPTLC densitograms are shown in Figures 1 and 2.

AnimalsSprague-Dawley rats (180-200 g) and Swiss albino mice (25-30 g) of either sex were used for the study. The ani-mals were housed in groups of six in polypropylene cages, under standard laboratory conditions of tem-perature (25 ± 2°C), lighting (0800–2000 h) and relative humidity (50 ± 5%). The animals had free access to stand-ard pellet chow (Brooke Bond-Lipton, India) and water. The animals were acclimatized for a minimum period of 7 days. Experiments were conducted between 0900 and 1400 h. The experimental protocol was approved by the Institutional Animals Ethics Committee (IAEC) and care of laboratory animals was taken as per CPCSEA guidelines (Reg. No. 379/01/ab/CPCSEA).

Drugs and chemicalsDiazepam (Calmpose, 10 mg/2 mL injection, Ranbaxy Laboratories, India) was used as the reference drug in all the animal models studied. It was dissolved in normal saline for i.p. injection. For in vitro antioxidant activity riboflavin, nitro blue tetrazolium (NBT) and l-ascorbic acid were purchased from Sigma Aldrich, St. Louis, MO. EDTA was obtained from Himedia.

Administration of the extractsSuspensions of the ethyl acetate and aqueous fractions were prepared in distilled water using Tween 80 (0.2% v/v) as the suspending agent. The extracts were admin-istered in a dose of 100 and 200 mg/kg to rats by oral route, 45 min before the test procedures. Control groups were given only the vehicle (0.2% v/v Tween 80 solu-tion) in volume equivalent to that of the plant extracts.

100

50

00.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

Wavelength: 254 nm

12

3 4 56 78

9

1011

[Rf]

Figure 2. HPTLC densitogram of ethanolic extract of Convolvulus pluricaulis in chloroform:glacial acetic acid:methanol:water (60:32:12:8).

Figure 1. HPTLC densitogram of ethanolic extract of Evolvulus alsi-noides in chloroform:glacial acetic acid:methanol:water (60:32:12:8).

120

100

80

60

40

20

00.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

Wavelength: 254 nm

12 3 4

56

7

8

910

[Rf]

CP-Aqueous

CP-Ethyl Acetate

EA-Ethyl Acetate

CP-Ethanolic

EA-Ethanolic

Ascorbic acid

EA-Aqueous

0 20 40 60EC 50

Figure 3. Effects of test extracts on superoxide radical scavenging activity. EC50 values were computed and compared.

Anxiolytic activity of Evolvulus alsinoides and Convulvulus pluricaulis in rodents 447

Assessment of anxiolytic activity

Elevated plus maze testAnxiolytic activity was evaluated using the elevated plus maze (Pellow et al., 1985). The elevated plus maze (EPM) consisted of two open arms (50 × 10 cm) crossed with two closed arms (50 × 10 × 40 cm). The arms were connected together with a central square (10 × 10 cm). The apparatus was elevated to a height of 70 cm in a dimly illuminated room. The animals were divided into six groups containing six animals each. The ethyl acetate and aqueous fractions (100 mg/kg p.o.) of E. alsinoides and C. pluricaulis were administered 45 min before trial on the EPM in separate groups of animals. Control groups were given only the vehicle (0.2% v/v Tween 80 solu-tion) in volume equivalent to that of the plant extracts. Diazepam (1 mg/kg i.p.) was used as the reference drug for comparison. One hour post-administration, each rat was placed individually at the center of the elevated maze. The number of entries in the open and closed arm of the elevated maze during a period of 5 min and the duration of stay in the open and the closed arm were noted (Soman et al., 2004).

Open field testThis test was performed following the method described by Bronstein (1972). The open field apparatus was made of plywood and consisted of squares (61 × 61 cm). The entire apparatus was painted black except for 6 mm thick white lines, which divided the floor into 16 squares. The open field was lit by a 40 W bulb focusing on to the field from a height of about 100 cm. The entire room, except the open field was kept dark during the experiment. Each animal was centrally placed in the test apparatus for 5 min and the following behavioral aspects were noted:

Ambulation: this was measured in terms of the 1. number of squares crossed by the animal;

Rearings: number of times the animal stood on its 2. hind limbs;

Self-grooming: number of times the animal groomed 3. facial region, and licked/washed/scratched various parts of its body;

Activity in center: number of central squares crossed 4. by the animal; and,

Fecal droppings: number of fecal droppings excreted 5. during the period.

The rats were divided into 6 groups containing six animals each. The ethyl acetate and aqueous fractions (100 mg/kg p.o.) of E. alsinoides and C. pluricaulis were adminis-tered 45 min before trial on the open field apparatus in individual groups of animals. Control groups were given only the vehicle (0.2% v/v Tween 80 solution) in volume

equivalent to that of the plant extracts. Diazepam (1 mg/kg i.p.) was used as the standard drug for comparison.

Neuromuscular coordination–rotarodThe effect on motor coordination was assessed using a rotarod apparatus (Dunham & Miya, 1957). A day before the test, mice were trained to remain on the rotating rod ( 3 cm in diameter, 40 rpm). On the test day, mice were tested on the rotarod before and 45 min after the administration of vehicle, diazepam (1 mg/kg i.p.) or the ethyl acetate and aqueous fractions of E. alsinoides and C. pluricaulis (100 and 200 mg/kg p.o.). The number of seconds each mouse remained on the rotating wheel was recorded before and after the administration. The percentage reduction in the motor coordination was calculated from the readings obtained.

In vitro antioxidant activity – assay for superoxide radical scavenging activityThe assay was based on the capacity of the samples to inhibit blue formazan formation by scavenging the superoxide radicals generated in riboflavin-light-NBT system (Beauchamp & Friedovich, 1971). The reaction mixture contained 50 mM phosphate buffer (pH 7.6), 20 µg riboflavin, 12 mM EDTA, 0.1 mg/3mL NBT, added in that sequence. The reaction was started by illuminating the reaction mixture with different concentration of sam-ples. Immediately after illumination, the absorbance was measured at 590 nm and EC

50 (effective concentration,

required to inhibit NBT reduction by 50%) values were calculated from the dose-inhibition curves by graphical method (Sokmen et al., 2005). Ascorbic acid was used as positive control (Bagul et al., 2005). The ethanol extracts of the drugs as well as their ethyl acetate and aqueous fractions were evaluated for their free radical scavenging activity by this method (Figure 3).

Statistical analysis

The data were expressed as mean ± SEM and statistically analyzed using one-way ANOVA followed by Dunnet’s test. P < 0.05 was considered to be statistically significant. F and P values and degrees of freedom were calculated for the readings obtained.

Results

Elevated plus maze test

The oral administration of the ethyl acetate fractions of E. alsinoides and C. pluricaulis (100 mg/kg p.o.) produced a significant increase (P < 0.5) in the time spent in the open arms as well as the number of entries in the open arm of

448 Alok Nahata et al.

the elevated plus maze indicating the anxiolytic activity of the drugs. Vehicle treated rats spent 27.33 ± 18. 39 s in the open arm. Rats treated with ethyl acetate fractions of E. alsinoides and C. pluricaulis spent 97.33 ± 3. 51 s and 95.66 ± 9. 95 s in the open arm respectively. The number of open arm entries also increased to 8.16 ± 1.57 and 8.50 ± 2.07 for E. alsinoides and C. pluricaulis, respectively, as compared to the vehicle (1.00 ± 0.44). The aqueous fractions (100 mg/kg p.o.) did not cause any significant change in the above parameters. The administration of diazepam (1.0 mg/kg i.p.) significantly increased (P < 0.01) the number of entries as well as the duration of stay in the open arms, as compared to the extract treated groups. F values were also calculated and found to be extremely significant. The results are given in Table 1.

Open field test

The oral administration of ethyl acetate fractions sig-nificantly increased the ambulatory activity (P < 0.5 for E. alsinoides and P < 0.01 for C. pluricaulis), rearings (P < 0.01 for E. alsinoides and P < 0.001 for C. pluricaulis), self-groomings (P < 0.5 for both the drugs) and activity in center (P < 0.01 for E. alsinoides and P < 0.001 for C.

pluricaulis). The open field fecal droppings were signifi-cant in the case of E. alsinoides and comparable to vehi-cle in the case of C. pluricaulis. Diazepam also induced significant anxiolytic activity and the effects were found to be highly significant (P < 0.001) and more than that of the extracts. The aqueous fractions did not produce significant changes, indicating a lack of anxiolytic activ-ity. F values were calculated and found to be extremely significant. The results are shown in Table 2.

Neuromuscular coordination–rotarod

Diazepam (1 mg/kg i.p.) and the ethyl acetate fractions (200 mg/kg p.o.) of E. alsinoides and C. pluricaulis caused significant reduction in the time spent on the rotarod, compared to control group (P < 0.001). The reduction in the performance on the rotarod before and after the experiment was 53.56 ± 6.76% and 69.56 ± 4.23% for groups treated with E. alsinoides and C. pluricaulis ethyl acetate fractions (200 mg/kg, p.o.) and 78.65 ± 3.79% for the diazepam-treated group as compared to the vehicle-treated control group (4.04 ± 1.33%). The ethyl acetate fractions of E. alsinoides and C. pluricaulis were devoid of significant effect on motor coordination at

Table 2. Effect of test fractions on open field exploratory behavior in rats.

Treatment Ambulation Rearings Self grooming Activity in center Fecal droppings

Vehicle 31.50 ± 5.30 1.50 ± 0.56 4.16 ± 0.74 3.00 ± 0.36 5.00 ± 1.03

Diazepam(1 mg/kg i.p.) 79.00 ± 6.40*** 10.16 ± 0.30*** 9.83 ± 0.94*** 9.83 ± 0.65*** 0.83 ± 0.40**

EAEA (100 mg/kg p.o.) 53.83 ± 4.92* 5.83 ± 0.79** 7.66 ± 0.88* 5.50 ± 0.22** 1.83 ± 0.47*

EAAqs(100 mg/kg p.o.) 35.83 ± 3.64 4.16 ± 0.90 5.00 ± 0.96 3.00 ± 0.36 4.67 ± 0.71

CPEA (100 mg/kg p.o.) 58.00 ± 4.10** 8.33 ± 0.88*** 8.16 ± 0.47* 7.33 ± 0.66*** 2.00 ± 0.51

CPAqs (100 mg/kg p.o.) 37.33 ± 3.16 4.5 ± 0.42 5.16 ± 0.47 8.66 ± 0.33 5.00 ± 0.63

One way ANOVA F = 14.585 F = 20.791 F = 8.262 F = 31.089 F = 8.110

P < 0.0001 P < 0.0001 P < 0.0001 P < 0.0001 P < 0.0001

*P < 0.5, **P < 0.01, ***P < 0.001 compared to vehicle. Df = 5,35. One way ANOVA followed by Dunnett’s test. All values are Mean ± SEM (n = 6).EAEA, E. alsinoides ethyl acetate extract; EAAqs, E. alsinoides aqueous extract; CPEA, C. pluricaulis ethyl acetate extract; CPAqs, C. pluricaulis aqueous extract.

Table 1. Effect of test fractions on exploratory activity of the rats in elevated plus maze apparatus.

Treatment

Time spent (sec) No. of entries

Open arm Closed arm Open arm Closed arm

Vehicle 27.33 ± 18.30 274.33 ± 16.73 1.00 ± 0.44 2.00 ± 0.44

Diazepam (1mg/kg i.p.) 131.77 ± 31.72** 168.83 ± 31.72** 9.00 ± 2.63* 5.00 ± 0.93

EAEA(100 mg/kg p.o.) 97.33 ± 3.51* 202.67 ± 3.52* 8.16 ± 1.57* 3.00 ± 0.36

EAAqs (100 mg/kg p.o.) 51.66 ± 6.48 248.67 ± 6.67 1.66 ± 0.33 2.83 ± 0.40

CPEA(100 mg/kg p.o.) 95.66 ± 9.95* 203.33 ± 9.93* 8.50 ± 2.04* 5.16 ± 1.16

CPAqs(100 mg/kg p.o.) 46.33 ± 13.62 253.67 ± 13.62 2.33 ± 0.49 3.83 ± 0.79

One way ANOVA F = 5.561 F = 5.854 F = 6.170 F = 2.799

P < 0.0001 P < 0.0007 P < 0.005 P < 0.0344

*P < 0.5, **P < 0.01 compared to vehicle. Df (Degree of Freedom) = 5,35. One way ANOVA followed by Dunnett’s test. All values are Mean ± SEM (n = 6).EAEA, E. alsinoides ethyl acetate extract; EAAqs, E. alsinoides aqueous extract; CPEA, C. pluricaulis ethyl acetate extract; CPAqs, C. pluricaulis aqueous extract.

Anxiolytic activity of Evolvulus alsinoides and Convulvulus pluricaulis in rodents 449

100 mg/kg oral dose. The same was the case with the aqueous fractions (100 and 200 mg/kg, p.o.) of E. alsi-noides and C. pluricaulis. These results indicate the muscle relaxant activity of the ethyl acetate fractions of E. alsinoides and C. pluricaulis at the higher dose of 200 mg/kg. Surprisingly, this is the first report about the muscle relaxant activity of both drugs. F and P values came out to be highly significant indicating the accuracy of the results. The results are shown in Table 3.

Antioxidant activity–superoxide radical scavenging activity

The assay was based on the capacity of the samples to inhibit blue formazan formation by scavenging the superoxide radicals generated in riboflavin-light-NBT system. The total ethanol extract and its ethyl acetate and aqueous fractions exhibited significant antioxidant activity, comparable to that of ascorbic acid, used as a positive control. The best superoxide radical scaveng-ing activity was shown by the total extract followed by the ethyl acetate and aqueous fractions in both E. alsi-noides and C. pluricaulis (Figure 3). These preparations inhibited the development of color, produced during the reaction of superoxide with NBT by 79.97%, 74.82%, and 68.45% (E. alsinoides ethanol, ethyl acetate and aqueous fractions, respectively) and 88.77%, 77.95%, and 54.39% (C. pluricaulis ethanol, ethyl acetate, and aqueous frac-tions respectively). Moreover, the samples of total extract and its ethyl acetate and aqueous fractions suppressed superoxide radical release in a dose-dependent manner. Inhibition of NBT-reduction by superoxide in the pres-ence of tested preparations increased with the increase of their concentrations.

Discussion

The question of reliability and validity is of prime impor-tance in establishing experimental paradigms of prac-tical predictable value. These factors assume further importance when animal models of human behavior and its perturbations are being used. The paradigms used in the present study have been subjected to thor-ough critical appraisal and validated as animal models of anxiety (Treit, 1971; File, 1985, 1988; Lister, 1990; Kumar et al., 2000). Thus, in the open field and similar tests, when the animals are taken from their home cage and placed in a novel environment, they express their anxiety and fear by a decrease in ambulation, rearings, and other exploratory behaviors. Likewise, the elevated plus maze test is based on the principle that exposure to the maze leads to an approach conflict which is considerably stronger than that evoked by exposure to the enclosed part of the maze (Pellow et al., 1985). All these behaviors are increased by anxiogenic agents and attenuated by anxiolytics under identical experimental conditions (Kumar et al., 2000).

The findings of the present study indicate that ethyl acetate fractions of E. alsinoides and C. pluricaulis caused significant anxiolysis in the elevated plus maze test. Diazepam (1.0 mg/kg i.p.) significantly increased (P < 0.01) the number of entries as well as the duration of stay in the open arms, indicating anxiolytic activity. The ethyl acetate fraction of both the drugs was found to be effective. The aqueous fraction was devoid of such response in both the cases. Thus both the drugs came out to be good anxiolytics using EPM.

Anxiolytics have been shown to increase the open field ambulation, rearings, self-groomings and activity in the center, and a decrease in the number of fecal droppings is also apparent as a result of their administration. The anxiety induced by the open field conditions is attenu-ated by anxiolytic drugs (Kumar et al., 2000). The ethyl acetate fractions of both E. alsinoides and C. pluricaulis have shown sufficient anxiolytic activity. The aqueous fractions did not produce significant changes. C. plu-ricaulis proved to be a better anxiolytic than E. alsinoides in this model.

Vale et al. (1999) affirmed that the absence of interfer-ence with the motor coordination in the rotative bar dis-cards the possibility of a muscular relaxing effect. In the present work, the ethyl acetate fractions of E. alsinoides and C. pluricaulis were devoid of significant effect on motor coordination at 100 mg/kg oral dose. The same was the case with the aqueous fractions (100 and 200 mg/kg p.o.) of E. alsinoides and C. pluricaulis. The ethyl acetate fractions of E. alsinoides and C. pluricaulis at the higher dose of 200 mg/kg were found to produce a significant reduction in the rotarod performance. Therefore, the

Table 3. Effect of test fractions on rotarod performance.

Treatment % Reduction in performance (sec)

Vehicle 4.04 ± 1.33

Diazepam (1mg/kg, i.p.) 78.65 ± 3.79 ***

EAEA (100 mg/kg, p.o.) 13.53 ± 3.08

EAEA (200 mg/kg, p.o.) 53.56 ± 6.76***

EAAqs (100 mg/kg, p.o.) 9.38 ± 2.01

EAAqs (200 mg/kg, p.o.) 4.82 ± 1.30

CPEA (100 mg/kg, p.o.) 13.35 ± 5.37

CPEA (200 mg/kg, p.o.) 69.56 ± 4.23***

CPAqs (100 mg/kg, p.o.) 8.05 ± 1.94

CPAqs (200 mg/kg, p.o.) 4.46 ± 1.95

One way ANOVA F = 65.482

Df = 9,59

P < 0.0001

***P<0.001 compared to vehicle. One way ANOVA followed by Dunnett’s test. All values are Mean ± SEM (n = 6).EAEA, E. alsinoides ethyl acetate extract; EAAqs- E. alsinoides aqueous extract; CPEA, C. pluricaulis ethyl acetate extract; CPAqs, C. pluricaulis aqueous extract.

450 Alok Nahata et al.

observed reduction of the motor activity may be related to the muscle relaxant effect of the extracts.

Further, in the present study, the extracts were studied for their in vitro antioxidant potential to cor-relate their anxiolytic activity. This investigation of the total ethanol extract and its fractions was performed in a non-enzymatic system–NBT, riboflavin, and light. The assay was based on the capacity of the samples to inhibit blue formazan formation by scavenging the superoxide radicals generated in the riboflavin-light-NBT system. Superoxide radical is known to be very harmful to cellular components as a precursor of more reactive oxygen species (Halliwell & Gutteridge, 1985). Free radicals and other reactive oxygen species are con-sidered to be important causative factors in the devel-opment of diseases of aging such as neurodegenerative diseases, cancer and cardiovascular diseases (Auddy et al., 2003). The total ethanol extract and its ethyl ace-tate and aqueous fractions exhibited significant anti-oxidant activity, comparable to that of ascorbic acid, used as a positive control. Preliminary phytochemical studies on the drugs showed the presence of flavonoids in the ethanol extract. This is in accordance with the findings that the antioxidant properties of flavonoids were affected mainly via scavenging of superoxide anions (Haslem, 1966; Rice Evans et al., 1977). Thus the antioxidant potential of the extracts throws light on the cytoprotective effects of the extracts and usage in stress-induced disorders.

Thus, the present investigations on E. alsinoides and C. pluricaulis have led us to conclude that both the drugs possess significant anxiolytic effects, have effects on the neuromuscular coordination, and possess significant antioxidant potential.

Acknowledgements

The authors are grateful to the Director, B.R. Nahata Smriti Sansthan – Contract Research Centre, Mandsaur, Madhya Pradesh India for granting permission to carry out the in vivo studies. Alok Nahata is thankful to the University Grants Commission, New Delhi for providing a junior research fellowship.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

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