Central Nervous System Depressant Activity of Aqueous Extract of Leaves of Azadirachta indica Linn in Mice

 

P. Thamarai Selvi*, M. Senthil Kumar,  T. Yaswanth, E. Adiyaman, P.T. Anusha

Department of Pharmacology, Annai Veilankanni’s Pharmacy College, Chennai-15, India

*Corresponding Author E-mail: tthamarai_pharma@yahoo.com.

 

ABSTRACT:

Aim of the study to investigate the effects of Acetone extract of Azadirachta indica on central nervous system and possibility to use it as folk medicine. The Acetone extract of Azadirachta indica was soxhlat extracted with Acetone and concentrated. The effect of Acetone extract on CNS was studied by using analgesic activity and sedative-hypnotic activity. Preliminary phytochemical evaluation of extract was also carried out. The extract (100mg and 200mg/kg, i.p) showed significant (p<0.001) potentiation of reaction time to thermal stimulus. It also showed significant (p<0.01). Potentiation o phenobarbitone induced sleeping time to control.

 

KEYWORDS: Azadirachta indica, Analgesic activity, Sedative-hypnosis.

 


INTRODUCTION:

Currently available antipsychotics are associated1 with variety of autonomic, endocrine, allergic, haematopoietic, and neurological side effects. As a result there is a high prevalence of usage of complementary and alternative medicines for treatment of psychiatric disorders. In the search for new therapeutic products for the treatment of neurological disorders. Medicinal plant research worldwide has progressed constantly, demonstrating the pharmacological effectiveness of different plant species in a variety of animal models. Azadirachta indica Linn (Meliaceae) commonly known as neem is a large tree upto 8m high with almost straight trunk, distributed throughout the India2, Pakistan, Bangladesh and Srilanka. The medicinal utilities have been described, especially for leaf, fruit and bark. It is used for the treatment of rheumatism, chronic syphilitic sores and indolent ulcer, skin infection, blood morbidity, biliary afflictions, itching, skin ulcers, burning sensations3 has also been evident.

 

Previous phytochemical studies reveales the presence of Triterpenes: β-sitosterol, stigmasterol, Nimbidinin, Nimbidin4, Nimbin, Azadirachtin, Flavonol glycosides- Quercitin, Kaempferol, Feliantriol, Salanin, Cyclic tri and tetra sulphides. Several investigations have proposed that this plant possess antipyretic5, antiulcer6, immunostimulant7, antimalarial8, hepatoprotective9, anti-inflammatory10 and antidiabetic11 effects.

 

Generally plants possess many pharmacological actions since they contain numerous constituents of active chemicals in it. Based on the above criteria this study was designed to evaluate the activity of acetone extract on central nervous system in mice.

 

MATERIALS AND METHODS:

Collection and authentification of plant material:

The plant materials were collected from local areas in Chennai, Tamilnadu and the plant material was identified and authenticated by resident botanist Prof. Dr. P. Jayaraman Plant Anatomy Research Centre (PARC), Chennai. A voucher specimen was submitted at Annai Veilankanni’s Pharmacy College, Chennai. Reg No; PARC/2011/860.

 

Preparation of Aqueous extract12:

450gm of leaf extracted with chloroform water by double maceration for 48hrs. The extract was filtered through          mucelin cloth. The filtrate was evaporated to dryness in vaccum and kept in a refrigerator.

Preparation of crude extract:

The crude extract of Azadirachta indica was   freshly prepared every day by dissolving in distilled water in order to obtain the desired concentration before the oral administration via intra gastric tube once daily. Each animal should receive the same volume of substance in order to avoid from the confounding error due to different in volume.

 

Animals::

Wistar albino mice (25-35g) breed in central animal house facility of the institute were used. They were housed under standard conditions maintained on a 12 h light/dark cycle and hand free access to food and water up to the time of experimentation. The mice were acclimatised to the laboratory environment 1 h before the experiments. All the experiments were conducted during the light periods (08.00-16.00 h). During the experiments animals were free access to water only. IAEC NO: 793/03/C/CPCSEA.

 

Drug-chemicals::

Phenobarbitone sodium (Sigma, UK) dissolved in saline(40mg/kg) and used accordingly. Pentazocine (5mg/kg).

 

Phytochemical Screening13:

The extract was subjected to preliminary phytochemical screening by the methods previously described by Kokate and Jayaraman J.

 

Acute Toxicity Study:

The procedure was followed as per OECD 423 guidelines. The extract was administered orally at a dose 2000 mg/kg body weight to different groups of mice and observed for signs of behavioral, Neurological toxicity and mortality 14 days.

 

Phenobarbitone induced sleeping time:

Mice were given a single i.p dose of aqueous and aqueous extract of Azadirachta indica 100 and 200 mg/kg body weight. The control animal received the saline. Those treatments were carried out 30min before challenging the animal with i.p injection of Phenobarbitone (40mg/kg). The latency of the loss of righting reflex and the total sleep time [the time between the loss of recovery of the righting reflex] were determined for each mouse as stated previously. The mouse was considered as being awake if it could right itself (return to upright position). Once mice right itself, it was placed on its back once more and allowed to right a second time for confirmation14.

 

Hot Plate Method15:

The parameter evaluated was the latency time for paw licking and jumping response after exposure on surface of hot plate. The standard used was pentazocine (10mg/kg i.p). The hot plate temperature was kept at 500± 10C and the cut off time was 20 sec26.

 

 

Statistical Analysis:

 The data were expressed as mean ± standard error mean (SEM). The significance of differences among the groups was assessed using one way analysis of variance (ANOVA). The test was followed by Dunnett’s ‘t’-test, p values less than 0.05 were considered as significance.

 

RESULTS:

Phytochemical Screening:

 The preliminary phytochemical analysis of AEAI showed that the plant contains carbohydrates, flavanoid, phenols, proteins, saponin but, steroids, gums and mucilage, and tannins were absent.

 

Acute toxicity Study:

Acute oral toxicity studies revealed the nontoxic nature of AEAI. There was no morbidity observed or any profound toxic reactions found at a dose of 2000 mg/Kg p.o. which indirectly pronouns the safety profile of the plant extract.

 

Phenobarbitone Induced Sleeping Time:

Aqueous extract of Azadirachta indica leaves produce dose dependent increase in     phenobarbitone induced sleeping time (p<0.01). The aqueous extract significantly  increases the sleep duration by 0.13min, 0.15min respectively when compared to saline  treated control i.e.., 0.32 (p<0.05).

\

Group               

Dose mg/kg

(i.p)

Mean Onset OF Sleep (min) ± SEM

Mean Duration Of Sleep (min) ± SEM

 

Control

5mg/kg

0.52 ± 0.009

0.32 ± 0.009

AEAI 100

100mg/kg

0.59 ± 0.016

0.416 ± 0.026**

AEAI 200

200mg/kg

0.498 ± 0.007*

0.426 ± 0.007**

AEAI- Aqueous extract of Azadirachta indica

One way ANOVA followed by Dunnet’s test. Values are mean ± S.E.M, n=5 in each group *p<0.05, **p<0.01, when compared to control.

 

Aqueous extract of azadirachta indica leaves produces dose dependent increase in the pain threshold time(p<0.01) when compared to saline treated control group. The standard drug Pentazocine also increases the pain threshold level significantly(p<0.001).


Treatment

Dose i.p (mg/kg)

Mean reaction time in seconds

15 min

30 min

60 min

90 min

120 min

Control

5ml/kg

2.62 ± 0.203

2.95 ± 0.203

3.18 ± 0.244

3.39 ± 0.189

3.77 ± 0.206

Pentazocine

5mg/kg

4.76 ± 0.113

5.60 ± 0.09

5.30 ± 0.947

5.51 ± 0.102

5.69 ± 0.09

AEAI 100

100mg/kg

3.37 ± 0.185

3.52 ± 0.147

3.76 ± 0.133

3.96 ± 0.123

4.13 ± 0.122

AEAI 200

200mg/kg

3.69 ± 0.136

3.85 ± 0.156

4.03 ± 0.145

4.23 ± 0.149

4.39 ± 0.16

One way ANOVA followed by Dunnet’s test. Values are mean ± S.E.M, n=5 in each group *p<0.05, **p<0.01, when compared to control.

 

 


DISCUSSION:

The aqueous extract of Azadirachta indica from the present study produce substantial depressant effects on central nervous system. The central action of the leaf extract was shown its ability to induce sleep, its effects 0n phenobarbitone16 sleeping time and analgesic activities. The extract potentiates the barbiturate sleeping time in a dose dependant manner indicating a pharmacological action. The increase in the dose of extract from 100 to 200mg/kg in phenobarbitone treated rats resulted in increased duration of sleep by 0.13 min and 0.15 min. Barbiturates are known as CNS depressants. Extract from other plants have also been observed to potentiate the sleeping time of barbiturates and other CNS depressants. The extract (200 mg/kg) on its own was observed to induce sleep within 10 to 15 min of administration. This is an indication of sedative and depressant action on the central nervous action and agrees with similar experiments in mice and rats using other plant extracts. The depressant activity of the extract may be attributed in part to an action on the cerebral mechanism involved in the regulation of sleep. The result of this study also shows that the leaf extract of Azadirachta indica induced some analgesic activity17 in rats. The plant produced analgesia when pain was induced with heat. The extract doses (100 and 200 mg/kg) increased the time taken for paw licking and jumping response significantly. Pentazocine (10 mg/kg) increased the time of paw licking when compared with the extract. The analgesic superiority of pentazocine over the extract is expected since pentazocine is a narcotic analgesic used to elevate deep seated pain.

 

The data present in our study suggest that A.indica may have some CNS depressant activity which may be due to analgesic and Sedative-hypnotic property. This study provides experimental support for medicinal traditional use of this plant in nervous disorder.

 

ACKNOWLEDGEMENT:

The authors are thankful to Dr. M. Senthil Kumar, Principal and Head of the Department of Pharmaceutics Annai Veilankanni’s Pharmacy College, for his encouragement in carrying out this work.

 

REFERENCE:

1.     Zhang ZJ. Therapeutic effect of herbal extract and constituents in animal models of psychiatric disorders. Life science 2004;75;1659-1699.

2.       Wealth of India. Raw materials. Publication and information directorate, CSIR, New Delhi. 1985 pp.418.

3.       Kritikar KR., Basu BD and ICS Indian Medicinal Plants. Lalit Mohan Basu publication, 1981;2nd edn, Vol II; pp. 1707-1708.

4.       Pillai, N. R. and Santhakumari, G., Planta Med., 1981, 43, 59–63.

5.       Tidjani, M. A., Dupont, C. and Wepierre, J., Planta Med. Phytother., 1989, 23, 259–266.

6.       Bandyopadhyay, U., Chatterjee, R. and Bandyopadhyay, R., USPatent 5,730,986, 1998;      corresponding to Indian Patent 1100/ Del/95.

7.       Upadhyay, S. N., Dhawan, S., Garg, S. and Talwar, G. P., Int. J.Immunopharmacol., 1992, 14, 1187–1193.

8.       Badani, L., Deolankar, R. P., Kulkarni, M. M., Nagsampgi, B. A.and Wagh, U. V., Indian J. Malariol., 1987, 24, 111–117.

9.       Bhanwra, S., Singh, J. and Khosla, P., Indian J. Physiol.Pharmacol., 2000, 44, 64–68.

10.    Murthy, S. P. and Sirsi, M., Indian J. Physiol. Pharmacol., 1958,2, 456–460.

11.    Chakraborty, T., Uerotta, L. and Poddar, G., Phytother. Res.,1989, 3, 30–32.

12.    Akindele AJ, Adeyemi OO. Anxiolytic and sedative effects of Byrsocarpus  coccineus schum, and thonn.extract. International journal of applied research in natural products  vol(3)1.pp-28-36.march-ap-2010.

12.    Dr.C.K. kokate, Purohit A.P, Gokhale S.B. Text Book Of Pharmacognosy And  Phytochemistry   39th edition Vol-1 pp 14.1-14.5.

13.    Rivera F, Gervaz E, Sera P. Toxicological studies of the aqueous extract from Achyroclime sapureioides (LAM). DC (MARCELA). J Ethnopharmacol., 2004;95: 362-389.

14.    Emamghoreishi M, Heidari-Hamedani G.  Sedative –Hypnotic activity of extracts and essential oil of coriander seeds. Iran J Med Sci March 2006; Vol no;1 p.no-23.

15.    Khairnar Pranit P, Pawar jayashree C,Chaudhari sanjay R. Asian journal of   Pharmaceutical and clinical Research. 2010 Vol:3, Issue 4, p p:0974-2441. 

16.    Asuzu, I.U. and I. Abu bakar, 1995. The effect of Icaciana Trichata  Tuber on the nervous system. Phytother. Res., 9: 21-25.

17. Turner, R.A., 1965. Screening methods in pharmacology. Vol. 1, Academy press, New York, pp: 106.

 

 

 

Received on 07.07.2012       Accepted on 14.08.2012     

© Asian Pharma Press All Right Reserved

Asian J. Pharm. Res. 2(3): July-Sept. 2012; Page 97-99