Introduction

Azadirachta indica A. Juss (Neem) has been widely used as an antimalaria in Nigeria and the West Coast of Africa as anti-inflammatory agent and insect repellent with antifeeding properties (Egunjobi, 1976; Bhanwra, 2000)

Neem is now widely distributed in many countries of the world by cultivation.  It was also demonstrated that no plant material showed greater activity against a broad spectrum of insect pest species than the neem tree (Ekong, 1967). Several compounds were isolated from the seeds of neem one of these,

azadirachtin, was found to both repel and disrupt the growth and reproduction of many destructive insect species. The stem and leaf extracts have been shown by Okpanyi, 1977; Bhanwra 2000; Subapriya, 2005) to have antipyretic, anti-inflammatory and analgesic

effects. The antibacterial, anti-viral, and anti-inflammatory actions of this plant justify its use in the treatment of fevers, malaria and skin diseases. Due to the therapeutic and pesticidal properties of Azadirachta indica  the extracts of its various parts has been the subject of extensive investigations leading to the isolation of interesting constituents.  The constituents of Azadirachta indica includes a wide range of terpenoids bioactive limonoids and flavonoids amidst other chemical

constituents.  The objective of this study is to assess the effect of Azadirachta indica (neem) on the heart using rat as animal model.

Materials and Methods

 Plant Material

Fresh leaves of Azardirachta indica were obtained from the local plant in Ado-Ekiti, Nigeria and identified in the Department of Plant Science and Forestry Herbarium, University of Ado-Ekiti, Nigeria.

Animal grouping

Twenty rats of both sexes (3 months old) weighing between 100-200g were obtained from the Animal breeding Unit of Anatomy and Physiology department, University of Ibadan, Ibadan, Nigeria.  The animals were divided equally into four groups of five animals each.  Rats in group one served as control.  They were fed with pellets and water ad libitum.

Administration of extract

Fresh leaves of Neem were sun dried and made into powdery form. Aqueous extract of the leaves was prepared by soaking 6g of the powdered leaves in 100ml of distilled water (6%^w/_v).  The resulting suspension was left overnight at room temperature (37⁰C) Thereafter, the suspension was filtered.  The filtrate was administered orally to the rats at a dose of 100mg/kg body weight.  Rats in group two were given only one dose, those in group three were administered two doses at 24h intervals, while those in group four were administered three doses at 24h intervals and were kept alive for four days.  The control group (Group one) was given distilled water, instead of the extract.  All the experimental rats were closely examined for signs of restlessness, excitement, intoxication and other behavioural changes.

 Collection of tissue

The rats were bled while under anaesthesia, into a clean dry beaker and serum was prepared as described by (Akanji and Nlumanze, 1987) after the completion of each dose regimes. They were sacrificed and dissected while still under anaesthesia by cervical dislocation.  Heart was removed into ice-cold 0.25M sucrose solution, washed free of blood and weighed. It was later cut into tiny pieces with a sterile blade into ice-cold 0.25M Sucrose solution. The homogenates were kept frozen overnight (-20⁰C) prior to enzyme assay. This was to ensure the maximum release of those enzyme systems located in the cell organelles (Ngaha et al, 1989).

Determination of Enzyme Activities

Alkaline phosphatase (ALP), alanine transa-minase (ALT) and aspartate transaminase (AST) activities were determined using the appropriate buffer systems

Spectrophotometric  method of (Kings, 1960) was used  to measure alanine and aspartate transaminase.  Alanine transaminase was measured by monitoring the concentration of pyruvate hydrazone formed into 2,4- dintrophenyl hydrazine at 546nm. Aspartate  transaminase  was measured by monitoring the concentration of oxaloacetate hydrazone formed with 2,4…dinitrophenyl hydrazine at 546nm.  Alkaline phosphatase activity was determined by measuring the p-nitrophenol  liberated form p-nitrophenyl phosphate at 400nm ( Wright et al, 1972 ).

Results and Discussion

Table 1: Effect of neem leaf extract on some enzyme activities (m/l) in rat heart.

Enzyme activities are expressed as specific activity m/l± standard deviation (SD).

Statistical significance was tested using student’s t-test, compared with control value (P<0.05)

Table 2: Effect of neem leaf extract on some blood serum enzyme activities (m/l) in rat heart.

Statistical significance was tested by student’s t-test (P<0.05)

Table 1 shows the changes in the activities of aspartate transaminase (AST), alanine transaminase (ALT) alkaline phosphatase (ALP) in the heart of rat following administration of aqueous extract from neem leaf when compared with control.  Aspartate transaminase and alkaline phosphatase activities reduced in the heart (P<0.05) after the administration of the first dose when compared with control animals.  The activities of these enzymes (AST and ALP) in the heart recovered on the third day Alanine transaminase on the other hand increased appreciably after the administration of the first dose on the first day. The activity of ALT was found to decrease in the heart from the third day after the animals have received second dose of the extract. The reduction continued to the seventh day. The significant reduction of alkaline phosphatase activity in the heart on the third day (Table 1) may be attributed to the destruction of the plasma membrane and hence efflux of cytoplasmic content of the enzyme into extracellular space.  This report is confirmed by corresponding increase in ALP activity noticed in the serum on the same day after the administration of the second dose.  (Table 2).

Coodly, 1970 reported that enzyme activities present in the serum (or plasma) represent an “overflow” from their tissues of origin.  The reduction of aspartate transaminase activity (AST) does not show a significant effect on the serum rather they may be inactivated at the cellular level or as a result of the enzyme being inhibited by the extract.  It is possible that low serum values may be due to released enzymes not getting into the serum due to inhibition of enzyme molecules insitu (Akanji and Nlumanze, 1987).

The recovery of the activities of the enzymes (AST and ALP) on the third day after administration of the second dose (Table 1) indicates denovosynthesis of the enzyme molecules.

High serum levels of the enzymes (AST, ALT and ALP) on the seventh day is an indication of a diseased state.  This is confirmed by the significant reduction of the enzyme activities in the heart (Table 1). This report revealed the effect of the high dosage of the extract on the heart, which implies that the heart is exposed to insult with chemical compounds which result in cell damage (i.e plasma membrane) hence efflux of the enzyme into extracellular environment.

References

1. Akanji, M.A. and Nlumanze, S.E. (1987) Alkaline Phosphatase activities following repeated Suramin administration in some rat tissues in relationship to their functions. Phamacol. Toxicol, 6:182-183.
2. Bandyopadhyay, U; Biswas, K; Sengupta, A; Moitra P; Dutta,P; Sarka, D; Genguly, C.K. and Banerjee, R.K. (2004).  Clinical Studies on the effect of neem (Azadirachta indica) bark extract on gastric secretion and gastro duodenal ulcer.  Lifesci 75(24):2867 –2878.
3. Bhanwra, S(2000). Effect of Azadirachta indica (neem) leaf aqueous extract on paracetamol induced liver damage in rats. Indian J. Physiol and Pharmacol, 44: 64-68
4. Dreyer, M. (1987) Field and laboratory trials with simple neem products as protectants against pests of vegetable and field crops in Togo.  In: Proc 2^nd Int Neem conference Rauischalzhausen, 1983.  H Schmutterer and K.P.S. Ascher (eds) GTZ. Eschbron; 435 –444
5. Egunjobi L.O. (1976) Nematocidal effect of A indica extract against Pratytenchus brachyurus Nematologia 22(2): Pp 125 –135.
6. Ekong D.E.U. (1967) Chemistry of Meliacin (Limonoids): The structure of nimbolide, a new meliacin from A indica chem. Commun 6, (16) Pp 808.
7. Ngaha, E.O; Akanji, M.A. and Madelsolumno, M.A.(1989) Studies on correlation between chloroquine induced tissue damage and serum changes in the rat.  Experimentia 45: 143-149.
8. Nwafor, S.V; Akah, P.A; Okoli, C.O; Onyirioha, A.C. and Nworu, C.S (2003).  Interaction between Chloroquine Sulphate and aqueous extract of Azadirchita indica A. (Juss) (Meliaceae) in rabbits. Acta pharm. 53: 305 –311.
9. Iwu, M.M; Obidoa, O and Anazodo, M (1986) Biochemical mechanism of the antimalaria activity of Azadirachta indica leaf extract. Pharmacological Res. Comm. 18: 81-91.
10. Kings E.J. (1960). Transaminases Assay. J. Canad. Med. Assoc. 31: 376 –380
11. Okpanyi S.N. (1977) Anti-inflammatory effect of A. indica:  In prospective in Medicinal plant. Research today. Book of Abstract, 3^rd Symposium on Medicinal plant. Pub. Drug Research and production unit.  University of Ife Pp 88.
12. Subapriya, R.R. and Nagini, S (2005).  Medicinal properties of neem leaves. Curr. Med. Chem. 5(2):149-156
13. Wright P.J; Leather wood, P.D; and Plummer D.T. (1972).  Enzymes in rat urine: Alkaline Phosphatase.  Enzymologia 42: 317 – 327.