In the present study, the toxicity and IGR effect of neem (Azadrechta indica A. Juss.) fruit seed extract (RB-a) and neem fruit coatings extract (RB-b) were compared against the 4th instar larvae of malaria vector mosquito Anopheles stephensi Liston (Orangi Town wildstrain). The neem fruit coatings extract was found more effective and better IGR than neem fruit seed extract. The LC50 value of RB-a and RB-b was 784 and 290 ppm, respectively. In the case of IGR effect 50% morphological effects produced by RB-a and RB-b were at 300 and 100 ppm, respectively. Hence fruit coatings extract (RB-b) was fractionated into EtOAc, RB-b 'A', RB-b 'N' PE 'S', PE 'I', 'ES' and 'EI' fraction. The LC50 value of these fractions was found to be 165, 142, 43, 159, 154, 106 and 15 ppm, respectively. The fraction 'ES' was sub fractionated into 13 VLC-fraction (A-M). The LC50 of these was found to be 100(A), 200(13), 150(C), (D,E,F & G subfractions were not assayed as such because they were mixture of various compounds) 215(H), 107(1), 136(J), 212(K), 66(L) and 182(M) ppm, respectively. The VLC-fraction 'L' was the most effective fraction, therefore it was further subfractionated, till the pure compounds were isolated using TLC and HPLC. Total sixteen pure compounds were isolated from the neem fruit coatings extract (RB-b), they are (1) azadiradione (2) nimbocinol, (3) 17 β-hydroxynimbocinol (4) azadirone (5) deoxygedunin, (6) gedunin (7) α-nimolactone (8) β -nimolactonc (9) 14,15-epoxyazacliraclione, (10) desfuranoazacliradione (11) meliacinin, (12) azadironic acid, (12a) methylester of azadioronic acid, (13) limocin-A, (14) limocin-B, 115) desfurano-6a hydroxyazadiradione and (16) 22,23-dihydronimocinol. The LC50 value of these pure compounds was found to be 15,30, 15, 10, 150, 120, 60, 45, 18, 37, 13, 4.47, 2.8, 19, 19, 43, and 60 ppm, respectively. The decreasing sequence of their toxicity was 2.8 (derivative of AZA) >4.4 7(AZA) 10 Azad> 13(MCN)>15(Azadd) = 17 β -Hydro> 18(Epoxy)>19(LA)=(L-B)> 30(Nimb) >37 (Des)> 43(Des-6a.H)>45(β-Nimo)>60(aNimo)=(22,23-Dihyd)> 120 (Ged) and >150 (Deoxyg).
The morphological (IGR) effect of neem fruit coatings (RB-b) and its selected fractions and compounds were also carried out, they are RB-b 'A', RB-b 'N', 'ES'-fraction, epoxyazadiradione, azadiradione, meliacinin and azadironic acid. The 50% IGR effect caused by above fractions and pure compounds was found at 60, 25, 37, 5, 3, 2.5 and 0.5 ppm, respectively. The decreasing sequence of the effect was 0.5(AZA)> 2.5(MCN)> 3(Azadd)>5(Epoxy»25(RB-b'N)>37('ES') and >60(RB-b'A.'). The results of all above fractions of Neem fruit coatings extract (RB-b) and pure compounds isolated from RB-b extract were compared with permethrin (PMN) 25EC a synthetic pyrethroid, used as standard. The LC50 value of PMN was found to be 0.120 ppm where as 50% IGR effects caused by PMN were at the dose of 0.0019 ppm. All above neem fractions as well as pure compounds were less effective (toxic) as compared to permethrin.
The effect of neem fruit coats (RB-b), selected fractions from RB-b extract and pure compounds from RB-b extract, on the activity of GOT, GPT and ACh was also taken into consideration, including the mother extract (RB-b) and permethrin (PMN), used as standard. They are RB-b extract, 'ES'-fraction, RB-b 'N'-fraction, epoxyazadiradione, azadiradione, meliacinin, azadironic acid and permethrin.
The % inhibition of GOT by the above fractions and pure compounds was 46.51, 54.37, 62.31, 66.99, 67.93, 68.54, 80.86 and C) 1.52% respectively. Where as in check it was 4.76%. The % inhibition of GPT by the above fractions and pure compounds was 45.22, 57.44, (,2.43,72.18,77.13,78.34,87.26 and 92.68% respectively, where as in check it was 13.92%. The % inhibition of cholinesterase by the above fractions and compounds was 29.52, 41.24, 52.28, 57.68, 60.42, '61.0 1, 69.99 and 82.30%, respectively, where as in check it was 15.50%. The % inhibition in control for all was nil.
The extraction, isolation of fractions and compounds was done by the ;author and structure determination of the pure compounds was done by Dr. M. Rasheed using IR, NMR, GC-Mass and etc, at International Centre for Chemical Sciences, WHO Collaborating Centre, University of Karachi, Karachi-Pakistan.