Evaluation of Anti-Anxiety activity of Alcoholic Extract of Cyndon dactylon Linn. in Experimental Animal Models

S. Haja Sherief*, S. Sindhura, S. Anusha, P. Jaya Preethi, S. Sengotuvelu, T. Sivakumar

Department of Pharmacy, Nandha College of Pharmacy and Research Institute, Koorapalayam Piruvu, Erode, Tamil Nadu, India 638052

ABSTRACT:

The present paper deals with evaluation of anti-anxiety activity of extract of Cyndon dactylon linn (200mg/kg and 400mg/kg P.O) on male mice using various paradigms of anxiety Cynodon dactylon commonly known as Durva is considered as a sacred herb by the Hindus and is used in religious rites. It is widely used by the people of India as a traditional medicine for diarrhea, dysentery, catarrhal opthalmia, dropsy, etc. Despite a long tradition of use, systematic phytochemical and pharmacological work has been carried out on this potential plant. Extraction was carried out with soxhlet apparatus using different solvent. Phytochemical screening showed presence alkaloids, tannins, carbohydrates, fixed oils, glycosides, flavanoids in methanolic extract of Cyndon dactylon linn thus, these constituents might be responsible for anxiolytic potential and was subjected to preliminary anti-anxiety screening studies, with a view to ascertain the verity of its traditional use as an anxiolytic.. The anti-anxiety activity was done by using elevated plus maze test, open field test, rotarod test, despair swim test, locomotar activity in rats,and then compared with the standard drug and control. In elevated plus maze, extract ( 200mg/kg and 400mg/kg) had shown a dosedependent increase in time spent and number of entries into open arm compared to control group. The number of central squares, peripheral squares crossed and rearings were stepped up significantly in open field paradigm. The treated groups had shown accession in time spent in light compartment, number of crossings, latency compared to control group in light dark exploration test.The alcoholic extract of Cyndon dactylon possesses significant facilitation of anti-anxiety without affecting motor coordination.

Key words: Cyndon dactylon (Linn), Phytochemical screening, Anti-anxiety activity.

1. INTRODUCTION:

Anxiety is a psychological and physiological state which is characterized by somatic, emotional, cognitive, and behavioural components. The root meaning of the word anxiety is 'to vex or trouble'; in either presence or absence of psychological stress, anxiety can create feelings of fear, worry, nervousness, and dread. Anxiety is considered to be a normal reaction to a stressor. It may help an entity to deal with a demanding situation by prompting them to cope with it. When anxiety becomes excessive, it may fall under the category of an anxiety disorder.

Anxiety is a generalized mood condition that can occur without an identifiable triggering stimulus. As such, it is renowned from fear, which is an appropriate emotional response to a perceived threat. Additionally, fear is related to the unambiguous behaviours of escape and avoidance, whereas anxiety is related to situations perceived as uncontrollable or unavoidable. Another view defines anxiety as "a future-oriented mood state in which one is ready or prepared to attempt to cope with upcoming negative events", fear and anxiety were said to be differentiated into four domains: (1) duration of emotional experience, (2) temporal focus, (3) specificity of the threat, and (4) motivated direction^1-5.

Cynodon dactylon belonging to the Family Poaceae, grow on either acid or alkaline soils and can survive under flood conditions or drought^6-8,. Literature showed that the plant posses anti inflammatory, anti diabetic, anti arrhymthic, diuretic⁹, anti oxidant¹⁰, immuno modulatory¹¹,anti convuslant¹². The present deals with anti anxiety of the aerial parts of Cynodon dactylon.

2. MATERIALS AND METHODS:

2.1 PLANT MATERIAL

The plant Cyndon dactylon (Linn) (family Poaceae) is widely found throughout India. It is available in and around our college campus and the collected plants were authenticated by botanist (BSI/SRC/5/23/2012-13/Tech).Dr .G.V.S. Murthy joint director, botanical survey of India, southern circle, TNAU Campus, Coimbatore-3. All the experimental procedures and protocols used in this study were reviewed by institutional animal ethics committee of Nandha College of pharmacy proposal number (NCP/IAEC/No.03/2012-2013) and were in accordance with the guidelines of the IACE.

2.2 EXTRACTION:

350gms of powdered leaf material was evenly packed in the Soxhlet apparatus. It was then extracted with chloroform, Methanol and aqueous successively. The aqueous extraction was carried out by maceration process. Then hot continuous extraction was carried out for about 24 hrs with the remaining solvents. The solvent is removed by distillation then they were cooled and placed in a dissector to remove the excess moisture. The dried extract were packed in airtight containers.¹³

2.3 PHYTOCHEMICAL SCREENING:

The chemical tests were performed for testing different chemical groups present in all the extracts¹⁴ and were shown in Table no.1. The phytochemical constituents present are alkaloids, glycosides, carbohydrates, flavonoids, tannins, fixed oils.

TABLE NO 1 PHYTOCHEMICAL ANALYSIS:

Phytoconstituents	Presence of phytoconstituents
Alkaloids	+ ve
Glycosides	+ve
Flavonoids	+ve
Terpenoids	-ve
Fixed oils	+ve
Carbohydrates	+ve
Proteins and amino acids	-ve
Tannins	+ve
Saponins	-ve

+ve = present, -ve = absent

2.5 PHARMACOLOGICAL SCREENING:

2.5.1 ELEVATED PLUS MAZE:

The elevated plus maze was made of 2 open arms

(35×5cms) and 2 closed arms (35×5×20 cm s). They were arranged perpendicular to each other with small central square (5× 5) between arms. The maze was hiked up to 25cms from the floor in adimly lit room. All the four groups were given respective treatment and after 45 min, mice were individually placed in centre square face neither one of the open arms. The number of entries into open arm, closed arm, latency time, time spent in open and closed arm were recorded for period of 5 min. An entry into an arm is noted when the mice with its four paws cross the demarcation of respective arm.^15-17

Table no 2 Number of entries to closed arm

Sl no	Group	Dose	Mean SEM entris to closed arm
1	Control (normal Saline)	1ml/kg	9.46±0.81
2	Test	200mg/kg	5.58±0.17^**
3	Test	400mg/kg	4.51±0.29**
4	Standard (Diazepam)	5mg/kg	4.25±0.25**

Values are mean ± SEM; n=6 in each group.*P<0.05, **P<0.01, ***P<0.001 when compared th control group; One-way ANOVA followed by post Dunnett's Multiple comparison Test.

Table no 3 Time spent in open arms

S. no	Group	Dose	Percentage time spent in open arms
1	Control (normal Saline)	1ml/kg	5.23±0.25
2	Test	200mg/kg	9.33±0.38**
3	Test	400mg/kg	12.36±0.57**
4	Standard (diazepam)	5mg/kg	13.73±0.46**

Values are mean ± SEM; n=6 in each group.*P<0.05, **P<0.01, ***P<0.001 when compared with control group; One-way ANOVA followed by post Dunnett's Multiple comparison Test.

2.5.2 mCPP INDUCED ANXIETY IN RATS:

mCCP is a metabolite (1-(3-chlorphenyl) piperazine) of antidepressant drug trazadone, which has been classified as 5HT agonist. It has been shown to produce anxiety in man and rats. Locomotion and Hypophagia can be studied under this model.

LOCOMOTION STUDY:

Male Wister albino rats (200-250g) were used for the study. Test compound or vehicles were administered orally 1 h or i.p. 30 min before the locomotion test. Trazadone was injected i.p. in a dose of 7mg / kg 20 min before the test. Thereafter the animals were placed individually in an automated locomotor activity cages and locomotion was recorded for 10 min.^18-20

Table no 4 Locomotor action

S. No	Groups	Dose	Before drug administration	After drug administration
1	Control (normal-saline)	1ml/ kg	441.3±9.43	523.3± 6.22
2	Test+ trazadone	200 mg/kg	443.2±5.45	317.2± 5.37**
3	Test+ trazadone	400 mg/kg	458.5±13.09	215.8± 6.23***
4	Standard (Diazepam)	5mg/ kg	457.5±12.57	191.5± 5.07***

Values are mean ± SEM; n=6 in each group. ***P<0.001, **P<0.01,*P<0.05 when compared before and after treatment. Statistics was performed using One-way ANOVA followed by post Dunnett's Multiple Comparison Test.

2.5.3 OPEN FIELD TEST:

A dark colored wooden box(60×60×30cms) with its floor carved into 16equal sized squares (15×15cms) and 40w lamp appended 100cms above for illumination was used. Before dropping the individual rat in one of the corner of the box (i.e. 45min prior) respective groups were administered with respective treatment (Normal saline, diazepam1mg/kg i.p, ECD 200mg/kg and 400mg/kg) and then record number of central squares crossed peripheral squares cross end and number of rearing for 5min.²¹

Table no 5 No of squares travelled and no of rearings

S.No	Groups	Dose	No. of rearings	No. of squares travelled
1.	Control (normal-saline)	1ml/kg	34.0±2.25	142.6±4.21
2.	Test	200mg/kg	25.83±1.80**	113.7±3.21**
3.	Test	400mg/kg	20.33±1.33**	85.83±3.21**
4.	Standard (Diazepam)	5mg/kg	18.33±1.20**	61.67±3.23***

2.5.4 DESPAIR SWIM TEST:

Male Wistar albino rats (150-180g) were used. Rats were individually forced to swim inside a vertical Plexiglass cylinder. Rats when placed first time in the cylinder were initially highly active, vigorously swimming in circles, trying to climb the wall or diving to the bottom. After 2-3 min this activity was going to subside and gets interspersed with phases of immobility or floating of increasing duration. After 5-6min immobility reaches a plateau where the animals become immobile for 80% of time. They were removed and dried. After 24 hrs they were again placed in cylinder individually and the immobility time was measured for 5 min. Test drug and standard drug were administered 1 hr prior to testing.²²

2.5.5 ROTAROD:

Rota rod apparatus consisted of a base platform and an iron rod of 3 cm diameter and 30cm length, with a non-slippery surface. This rod was divided into two equal sections by two disks, thus enabling two rat to walk on the rod at the same time at the speed of 25rpm. Animals (albino rats) of either sex were taken. They were fasted for 12 hr before starting the experiment. Animals were divided in to 4 groups; each group consists of six animals. Group I receives vehicle, Group II receives Standard (Diazepam 2mg/kg) and Group III, IV receives extracts at 200mg/kg, 400mg/kg body weight. The animals were trained to remain for 5min on the rod rotating at a speed of 25rpm on the previous day of experiment. On the next day either vehicle or extracts were administered orally and

their ability to remain on the rotating rod was assessed before and after 30minutes of the oral administration. The fall off time from the rotating rod was noted for each animal.²³

Table no 6 measurment of immobility

S. No	Groups	Dose	Pre-treatment	1 hr	2 hr	4 hr
1	Control (Normal saline)	1ml/kg	127.5±1.78	127.8± 2.97	130.5± 2.57	127.3± 3.05
2	Test	200mg/kg	110.3± 1.36*	100.3± 1.78**	91.3± 1.28**	86.0± 1.53**
3	Test	400mg/kg	107.8± 5.87*	94.2± 1.70**	88.8± 2.09**	81.5±0.76***
4	Standard (Diazepam)	5mg/kg	125.3± 3.67	90.5± 1.73**	86.7± 1.91**	79.5± 0.76***

Table no 7 measurment of muscle grip strength

S. No	Groups	Dose	Before-treatment	After-treatment
1	Control Normal saline)	1ml/kg	292.7±5.21	272.8 ±4.98
2	Test + haloperidol	200mg/kg	276.3± 6.53	59.33±2.90***
3	Test + haloperidol	400mg/kg	268.0±4.99*	46.5±1.85***
4	Standard(haloperidol +Diazepam)	1mg/kg, 5mg/kg	279.7±3.61	39.33±1.71***

3. DISCUSSION:

Anxiolytic compounds, by decreasing anxiety, increase the open arm exploration time as well as the number of entries into the open arm. With respect to our findings, in contrast to that of diazepam, the extracts cause an increase in the number of entries into the open arm.

The neurotoxicity of any compound is easily reflected as change in movements and hence locomotor study test is usually recommended. C. dactylon 200 and 400 mg/kg possess significant (*P<0.05) locomotor activity in actophotometer. However, the mechanism of this action has not been investigated here.

In the open field test, forced confrontational situations induce anxiety behaviour in rodents. In such a situation, rodents spontaneously prefer the periphery of the apparatus and enter less in the central parts of the open filed. Indeed, mice and rats walk close to the walls, a behaviour called thigmotaxis . An increase in central locomotion or in time spent in the central part of the device without modification of total locomotion is interpreted as an anxiolytic effect . In the present study, C. dactylon (100 mg/kg and 200 mg/kg) increased the time spent in central part of the device without increasing the total number of squares crossed whereas in diazepam (1 mg/kg) and C. dactylon (400mg/kg), there is an increase in the time spent in central part of the device with a decrease in the total number of squares crossed which may be due to sedative effect.

It is well established that the despair swim test (forced swim test) is sensitive to drugs which enhance adrenergic transmission, particularly in rats. In modified FST, when mice are forced to swim in a limited space, they quickly abandon swimming and stand still. Although all antidepressant drugs reduce immobility in the FST, two distinct active behavioral patterns are produced by pharmacologically selective antidepressant drugs. Antidepressants that selectively inhibit norepinephrine uptake reduce immobility and selectively increase climbing without affecting swimming. On the other hand serotonin reuptake inhibitors also reduce immobility but increase swimming instead of climbing. The present results showed that the alcoholic extract of Cyndon dactylon was effective in producing significant antidepressant and anti-anxiety effects in despair swim test in mice.

One of the important pharmacological actions of anxiolytics of benzodiazepine class of drug is muscle relaxant property. The skeletal muscle relaxation together with taming or calming effect reduces anxiety. The loss of muscle grip is an indication of muscle relaxation. This effect can be easily studied in animals using the inclined plane or rotarod apparatus. The difference in the fall of time from rotating rod between the control and diazepam treated animals is taken as an index of muscle relaxation. The plant extract showed skeletal muscle relaxation.

4. CONCLUSION:

The plant Cyndon dactylon (Linn) family Poaceae is taken for the present study. Aerial parts were selected and they are extracted. Phytochemical tests were done and the plant contains alkaloids, flavonoids, carbohydrates, glycosides, tannins and fixed oils.

The results suggest that the alcoholic extract of Cyndon dactylon possesses significant facilitation of anti-anxiety without affecting motor coordination.

The above studies have been concluded that the alcoholic extract of leaves of Cyndon dactylon exhibits anxiolytic effect in experimental rats. Further investigation should be made to elucidate the mechanism of Cyndon dactylon and its role in anxiolytic activity and other CNS effects. The mechanism of action is yet to be investigated but may be due to the effect of flavonoids present in the aerial parts of the plant.

5. ACKNOWLEDGMENTS:

The authors are grateful to the Chairman V. Shanmugan and S. Nandha Kumar Pradeep, Secretary of Nandha College of Pharmacy and Research Institute, Erode, Tamil Nadu, India for their assistance.

6. REFERENCES:

1. R.J. Rodgers et al “Behaviorally selective effects of neuroactive steroids to treat anxiety in mice” Science direct-Pharmacology-Biochemistry and Behaviour, Jan 1998:59:1

2. S. Akhondzadeh et al “Effect of Passion flower in the treatment of generalized anxiety; a pilot double-blind randomized controlled trail with oxazepam. Journal of clinical pharmacy and therapeutics, Jan 2002

3. Turner RA. Screening procedure in Pharmacology. Volume I. 1^st ed. Academic press; 1972.

4. Text Book of Drug Discovery and Evaluation, Vogel, 2^nd Edition.

5. Text Book of Screening Methods in Pharmacology, Parmar.

6. Amrita Asthana et al., has done work on Pharmacological Perspectives of Cyndon dactylon. Research Journal of Pharmaceutical, Biological and Chemical Sciences ISSN: 0975-8585.

7. Kumara et al., has carried work on Pharmacological actions and medicinal applications of the plant Cyndon dactylon. International Journal of Herbal Drug Research, Vol I, Issue 1, 1-7, 2011.

8. Jolly C.I et al., have done work on Pharmacognosy of Aerial Parts of Cyndon dactylon. Ancient Science of Life Vol. No XIX (3and4) January, February, March, April 2000.

9. Shivalinge Gowda K.P et al., had Study on the Diuretic Activity of Cyndon dactylon root stalk Extract in Albino Rats. Research J. Pharm. and Tech. 2(2): April.-June. 2009.

10. Saradha Devi K. M et al., has carried work on Hepatic antioxidative potential of ethyl acetate fraction of Cyndon dactylon in Balb/c mice. Journal of Medicinal Plants Research Vol. 5(6), pp. 992-996, 18 March, 2011

11. Santhi R. et al., has done work on Efficacy of Cyndon dactylon for immunomodulatory activity. Drug Invention Today 2010,2(2),112-114.

12. Vipin K. Garg1 et al., has done work on Anticonvulsant activity of ethanolic extract of Cyndon dactylon . Pelagia Research Library Der Pharmacia Sinica, 2011, 2 (2): 86-90.

13. Brijesh Kumar Sharma “Extraction techniques for Herbal Drugs Part-1” 2011-05- 31.

14. Sumit Gangwar et al., has carried work on Pharmacognostical and Phytochemical Investigation of Cyndon dactylon L. Pers. leaves. Current Pharma Research ISSN: 2230-7842 CPR 2(1), 2011, 419-426.

15. Brett RR, Pratt JA (1990) Chronic handling modifies the anxiolytic effect of diazepam in the elevated plus-maze. Eur J Pharmacol 178:135–138

16. Corbett R, Fielding St, Cornfeldt M, Dunn RW (1991) GABA mimetic agents display anxiolytic-like effects in the social interaction and elevated plus maze procedures. Psychopharmacology 104:312–316

17. File SE, Mabbutt PS, Hitchcott, PH (1990) Characterisation of the phenomenon of “one-trial tolerance” to the anxiolytic effect of chlordiazepoxide in the elevated plus-maze. Psychopharmacology 102:98–101.

18. Bilkei-Gorzo A, Gyertyan I, Szabados T (1996) mCPP-induced anxiety – A potential new method for screening anxiolytic drugs. Neurobiology 4:253–255

19. Bilkei-Gorzo A, Gyertyan I, Levay G (1998) mCPP-induced anxiety in the light-dark box in rats – A new method for screening anxiolytic activity. Psychopharmacology 136:291–298

20. Curzon G, Gibson EL, Kennedy AJ, Kennett GA, Sarna GS, Whitton P (1991) Anxiogenic and other effects of mCPP, a 5-HT1C agonist. In: Briley M, File SE (eds) New Concepts in Anxiety. McMillan Press Ltd., London, pp154–167

21. sai sampath et al anxiolytic effect of ethanolic extract of Oxalis corniculata. L. in mice. International journal of Pharmaceutical Sciences (vol2/issue3/jul-sep 2011).

22. Santosh.P, Venugopal.R, Nilakash.A, Kunjbihari.S. Antidepressant activity of methanolic extract of Passiflora foetida leaves in mice. International Journal of Pharmacy and Pharmaceutical Sciences: 2010 November; 3(1): 112-115

23. Shilpa et al evaluation of anti-anxiety activity of successive extract of Samanea samanlinn. International Journal of Pharmaceutical Sciences.

Received on 11.09.2012

Modified on 02.10.2012

Accepted on 09.10.2012

Research J. Science and Tech. 4(5): September –October, 2012: 197-202