Title of Invention	"EFFICIENT PROCESS FOR THE PREPARATION OF NEEM BASED REDUCED AZADIRACHTIN(S) PESTICIDES"
Abstract	A process for the preparation of tetrahydroazadirachtin, and/or dihydroazadirachtin concentrates from azadirachtin concentrates is described. It is characterized by catalytic reduction of azadirachtin under either of ambient or near ambient conditions of temperature and pressure, under continuous stirring, to yield the claimed product(s) in 80 to 100% yields.

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Description of invention The following specification particularly describes and ascertains the nature of invention and the manner in which it is to be performed Field of invention This invention falls in drug category and relates to a process for preparation of reduced azadirachtin(s) pesticides characterized by reduction of the double bond(s) in azadirachtin molecule to yield respectively di- and/or tetrahydro derivative(s) of azadirachtin. The reduced derivatives of azadirachtin are more stable and as effective pesticides as azadirachtin against various agricultural and other pests. Background of invention Literature is replete with examples of a large number of compounds exhibiting insecticidal, insect feeding deterrent, growth inhibitory, antifungal and nematicidal activity. Several of these have been banned or restricted for use due to their harmful effects on non-target organisms, and ecosystem. There are others against which a number of insect pests have developed resistance. In quest for ecologically sound pest control chemicals, plant based biopesticides such as azadirachtin, isolated from neem (Azadirachta indica A. Juss, Meliaceae) seed kernel, have attracted worldwide attention. Being of natural origin, azadirachtin is considered as an environmentally safe alternative to synthetic pesticides Following bioactivity-guided fractionation, azadirachtin with astonishing antifeedant activity against the desert locust, was isolated from neem seed kernel (Butterworth, J.H. and Morgan, E.D. 1968, J. Chem. Soc. Chem. Comm. 23-24; J. Chem. Soc., Perkins Trans 1972: 2545 -2550). The procedure for the isolation of azadirachtin comprised of several steps (Ubel, E.C.; Warthen, O.D. and Jacobson, M. J Liq. Chromatogr. 1979, 2: 875-882; Schroeder, D.R. and Nakanishi, K. J. Nat. Prod. 1987, 50: 241-244). Pure azadirachtins have been isolated using direct preparative HPLC using one or more of reverse phase HPLC columns (Govindachari, T.R.; Sandhya, G. and Ganesh Raj, S. P. J. Chromatogr. 1990, 513: 389-391; Govindachari, T.R.; Suresh. G.; Gopalkrishanan, G. Phytoparasitica, 1998, 26: 109-116; Yamasaki, R.B.; Klocke, J.A.; Lee, S.M.; Stone, G.A.; and Darlington, M.V. J Chromatogr. 1986, 356: 220-226), and medium pressure liquid chromatography (Sharma, V.; Walia, S.; Parmar, B. S.; Kumar, J. andNair, M. G. J. Agric. Food Chem. 2003, 51: 3966-3972). At present, a large number of neem extracts and (or) oil-based products are commercially available throughout the world. These products are standardized and valued based on their azadirachtin content. The products, however, suffer from serious defects due to instability of azadirachtin(s) and other meliacins under both storage and field conditions. Consequently, these are not able to sustain their efficacy for long under practical use conditions. The instability of azadirachtin has been attributed mainly to the unsaturated double bonds in the tigloyl and dihydrofuran moiety. There have been global efforts to reduce these moieties to overcome the problem of instability. The present invention reports an efficient process for the preparation of reduced azadirachtin(s) comprising of dihydroazadirachtin, tetrahydroazadirachtin or mixture of dihydroazadirachtin and tetrahydroazadirachtin. Prior Art Azadirachtins are the most complex and highly oxidized limonoids having several oxygen functionalities. The instability of azadirachtin has been, in general, assigned in part to the presence of ester functions and unsaturation in tiglate and enol ether moieties in the molecule. Their sensitivity to acid, base, moisture, heat, and light has therefore necessitated stabilization to improve both shelf life and field bioactivity. In past, several efforts have been made to stabilize the molecule through the use of antioxidants/ uv-screens or convert it to more stable products through structure modification, derivatization and related chemical studies. Hydrogenation of the olefinic moieties is, therefore, crucial as the resultant reduced products are more stable to both heat and light. Moreover reduction of unsaturated moieties did not have any detrimental impact on the biological activity (Ley, S. V.; Sautafianos, D.; Blaney, W.M and Sirnmonds, M.S.J. Tetrahedron Letters 1987, 28: 221-224; Ley, S.V.; Anderson, J. C.; Blaney, W.M.; Jones, P.S.; Lidert, Z.; Morgan, E.D.; Robinson, N.G.; Santafmo, D.; Simmonds, M.S.J. and Toogood, P.L. Tetrahedron. 1989, 45: 5175- 5192; Ley, S.V.; Denholm, A.A.; Wood, A. Nat. Prod. Rep. 1993, 10: 109-157; Simmonds, M. S. J., Blaney, W. M., Ley, S. V., Anarson, J. and Toogood, P. L. Entomol. Exp. Applic. 1990, 55: 169-181). As per literature reports, azadirachtin is resistant to hydrogenation at atmospheric pressure under a variety of conditions but at higher pressures, it generally yields di- or tetrahydroazadirachtin. It has been reported that azadirachtin can be selectively reduced using palladium on charcoal to 22,23-dihydroazadirachtin (Bilton, J. N.; Broughton, H.B.; Jones, P.S.; Ley; S.V.; Lidert; Z.; Morgan, E. D.; Rzepa, H.S.; Sheppard, R.N.; Slawin, A.M.Z and Williams, D.J. Tetrahedron 1987, 43: 2805-2815); or 2', 3', 22,23-tetrahydroazadirachtin (Ley, S.V.; Denholm, A.A.; Wood, A. Nat. Prod. Rep. 1993, 10: 109-157) at elevated hydrogen pressure. In these reports hydrogenation was accomplished in the presence of catalysts such as palladised charcoal, platinum oxide, nickel boride and/or Raney nickel at 1 to 60 atmospheric pressure, more preferably at 2 to 4 atmospheric pressure to obtain hydrogenated crude extract mixture comprising of reduced or partially reduced azadirachtinoids and other constituent meliacins. Preparation of antifeedant compositions comprising dihydroazadirachtin and tetrahydroazadirachtin with retained biological activity, and enhanced stability have also been reported (Klocke, J, A. and Yamasaki, R.B., 1991, U.S.Patent no. 5,001,149; and Klocke, J, A.; Lee, S.M. and Yamasaki, R.B. 1991, U.S.Patent no. 5,047, 242). In these preparations, reduced azadirachtins were prepared by hydrogenation of azadirachtin in the presence of platinum oxide and palladium over alumina. According to these inventions platinum oxide (PtOi) at 5 atmosphere hydrogen pressure yielded dihydroazadirachtin while palladium based catalyst furnished tetrahydroazadirachtin at elevated pressure of at least 10 atmospheres. The method reported in US patent no. 4,943,434 (Lidert, Z. (1990) and EP 0311284 (Syn. US Patent 4,943,434) describes a process for the preparation of insecticidal hydrogenated neem (Azadirachta indicd) seed kernel extract wherein the substrate material comprised of methanolic solution of ethyl acetate extract of defatted neem seed kernel instead of azadirachtin power concentrate of varying purity. Resultant hydrogenated extract predominantly comprised of 5-50% or preferably 15-50% by weight of dihydroazadirachtin. Formation of tetrahydroazadirachtin or mixture of dihydroazadirachtin and tetrahydroazadirachtin has not been described in any of the examples described therein. Hydrogenation has been attempted at 2-4 atmospheric pressure, or more preferably at elevated pressure (40-55 psi) of hydrogen. In one of the examples described in EP 0311284, the hydrogenated product formed at 40 psi contained only 25% dihydroazadirachtin. Further, in none of the examples, hydrogenation has been attempted between 1 to 2 atmospheric pressure of hydrogen to yield dihydroazadirachtin and tetrahydroazadirachtin in 85-95% yield. Reduced azadirachtins exhibit longer residual life than non-hydrogenated extracts. These have been found effective to combat insect and fungal infestation in horticultural and agricultural crops, mosquito control and/or veterinary health. Like azadirachtin, dihydroazadirachtin and tetrahydroazadirachtin are functionally identical in thek IGR and antifeedant activity. These have also been found effective both as nematicide and fungicide. Dihydroazadkchtin has been registered in US as a technical powder and an end-use product for both in-door and outdoor applications. Being ecologically sound, it has been exempted from the requirement of a tolerance for its residues in or on all raw agricultural commodities when applied as an IGR and/or antifeedant at 20 g or less of the active ingredient per acre. There is no report describing an exclusive formation of dihydroazadkachtin, tetrahydroazadkachtin or mixture of dihydroazadkachtin and tetrahydroazadkachtin at ambient to less than two atmospheric pressure of hydrogen. The improvised process described in this invention relates to the manufacture of neem based stable reduced azadkachtins pesticides under ambient to less than two atmospheric pressure of hydrogen. Object of invention An object of the invention is to prepare stable reduced azadkachtin(s) from technical azadkachtin concentrates under near ambient reaction conditions. The invention According to this invention, there is provided a process for the preparation of reduced azadkachtins pesticides with completely or partially hydrogenated olefinic bonds from azadirachtin concentrates of 20-100% strength, and wherein the process is characterized by reduction of azadirachtin (1) in the presence of a catalyst at temperature up to 60°C and hydrogen pressure from 1 to 5 atmospheres under continuous stirring for 2 to 10 hours followed by filtration/centrifugation to yield dihydroazadirachtin (2) and/or tetrahydroazadirachtin (3) concentrates in 85 to 95% yield. Azadkachtin-A powder concentrate (20-100%) is obtained from alcoholic (preferably ethanolic) extract of defatted neem seed kernel. The hydrogenation reaction is carried out in presence of either of palladium, platinum or nickel based catalysts. In case of palladium based catalysts, palladium is adsorbed on asbestos, charcoal, alumina or calcium carbonate. The nickel-based catalyst is an activated form of catalyst derived especially from nickel or nickel-aluminum alloy. (Structure Removed) Fig. 1. Structures of azadkachtin-A (1), dihydroazadkachtin-A (2) and tetrahydroazadkachtin-A (3) The hydrogenation is carried out at hydrogen pressures ranging from ambient to five atmospheres. Temperatures varying from ambient to 60° C can be employed. The reduction is carried out under continuous stirring for periods ranging from two to ten hours depending upon the parameters employed. The invention can be utilized to prepare pesticidal composition(s) comprising a compound of formula 2 and (or) 3 as active ingredient together with a carrier, diluent, or surfactant and (or) other formulant(s). Suitable solid diluents such as pumice, gypsum, kaolin, bentonite, talc etc, liquid diluents such as ketonic as well as aliphatic and aromatic solvents, along with suitable swelling agent, dispersing agent or emulsifying agent, can be used to prepare various solid and liquid preparations such as powders, emulsifiable concentrates, suspension concentrates, solutions etc. The composition may include a mixture of compounds of formulae 2 and/or 3 and/or other ingredients including another pesticide e.g. an insecticide, fungicide, nematicide etc. or adjuvants such as synergist etc. While various compositions may preferably contain from 1 to 20% by weight of active ingredient, those applied after dilution may contain from 0.001 to 0.1% by weight of the active ingredient at the final application stage. The composition may conveniently be applied at an application rate of 1 to 500 g a. i. per hectare. The compound(s) have been found to be active in combating pests such as insects, fungi and nematodes in agricultural and horticultural crops as well as for the control of pests of public and veterinary health. The objects of the present invention are achieved by applying the tetrahydroazadirachtin or mixture of tetrahydroazadirachtin and dihydroazadirachtin to the crops in controlling the proliferation of larval, pupal and adult stages of pests infesting agricultural/horticultural crops and commodities. These compounds can be applied alone or in succession with other environment friendly synthetic pesticides under the umbrella of integrated pest management programme. Examples The following examples illustrate the preparation of reduced azadirachtin(s) such as dihydroazadirachtin, tetrahydroazadirachtin and/or mixture of dihydroazadirachtin and tetrahydroazadirachtin; spectral characterization of azadirachtins; and bioefficacy in controlling the proliferation of pests in the agricultural crops and commodities. While examples I relate to the preparation of dihydroazadirachtin, mixture of dihydroazadirachtin and tetrahydroazadirachtin; example II relates to preparation of tetrahydroazadirachtin respectively. Example III describes the JH NMR, MS and LC-MS analysis of dihydroazadirachtin and tetrahydroazadirachtin. The example IV relates to the use of compounds of the invention as insect growth inhibitors, insect antifeedant, nematicidal, and as antifungal agent. Example I Preparation of reduced azadirachtins This example demonstrates the preparation of partially or completely reduced azadirachtin-A concentrate in which one or both the unsaturated moieties (tigloyl and dihydrofuran segments) of the azadirachtin molecule have been reduced. The typical hydrogenation procedure for the preparation of reduced azadirachtins is described as follows. A solution of azadirachtin powder concentrate (35 to 100%, 2 to 10 g), and nickel, platinum or palladium based catalyst (0.5 to 5 g) in aliphatic alcohol(s) or ethyl acetate (100 to 500 ml) was charged in a reaction vessel (preferably glass or glass lined) of 1 litre capacity and the contents continuously stirred (200-600 rpm) in hydrogen gas atmosphere (1-5 atmospheres) at a temperature ranging from 10 to 60°C. A reaction time of 2-8 hours was needed for complete conversion of azadirachtin to dihydroazadirachtin or mixture of both dihydroazadirachtin and tetrahydroazadirachtin. The progress of hydrogenation was monitored by periodical withdrawal of the samples from the reaction vessel and their subsequent analysis by LC. After aza-A has been completely converted to either dihydroazadirachtin, and/or tetrahydroazadirachtin, the stirring was stopped and the product filtered through a bed of Celite. The filtrate was then concentrated under reduced pressure to obtain the product(s) (yield 85 to 95 %). The sample was filtered and analyzed by HPLC using RP-18 column (250 * 5 mm), PDA detector, and methanol-water (1:1) as solvent system at a flow rate of 0.75 ml per minute. After 4 to 6 hrs of hydrogenation, the resultant hydrogenated mixture furnished two peaks (Rt 12.231 and 15.411) in the LC-MS spectrum, which were attributed to dihydroaza-A and tetrahydroaza-A. After 8 hrs of hydrogenation, the resultant product gave only one peak (Rt 15.4 min) and was identified as dihydroazadirachtin by LC-MS. Example II Preparation of tetrahydroazadirachtin This example demonstrates the preparation of completely reduced tetrahydroazadirachtin-A concentrate in which both the unsaturated moieties (tigloyl and dihydrofuran segments) of the azadirachtin molecule have been saturated. The reaction was repeated as in Example 1 using azadirachtin enriched concentrate of 60-100 per cent purity and employing 0.5 to 5.0 g of Raney nickel, freshly prepared nickel catalyst, or palladium over charcoal or alumina. The reaction was monitored by analyzing the sample(s) by HPLC. When azadirachtin and the intermediate product dihydroazadirachtin is no more detected and are completely converted to tetrahydroazadirachtin, the reaction was stopped and the product processed as above to obtain tetrahydroazadirachtin (yield > 85%). Reaction takes more preferably 8 to 10 hrs for completion depending upon quantity of catalyst employed. Example III (Spectral Characterization) 1H NMR Spectral characterization of azadirachtin-A, dihydroazadirachtin-A and tetrahydroazadirachtin-A The structures of azadirachtin A and reduced azadirachtins were confirmed by comparison of their 'H-NMR spectral data with the literature values (Rembold et al 1986, 3rd Intern. Neem Conf.. Nairobi, pp 149-160; Govindachari, T.R.; Sandhya, G.; Ganesh Raj, S.P. (1992) J. Natural Products 55: 396-401; Yamasaki, R.B. and Klocka, J.A. (1987). J Agric Food Chem. 35: 467-471). The significant difference in the 1H NMR spectrum of azadirachtin-A, dihydroaza-A and tetrahydroaza-A is listed in Table 1. 1H NMR spectrum of dihydroazadirachtin-A and tetrahydroaza-A clearly showed the differences in the chemical shifts of protons attached to carbons that have been affected by the hydrogenation. The comparison of 'H NMR spectral data revealed that two doublet peaks at 8 5.05 and 6.46 assigned to C22 and C23 bond in the dihydrofuran segment of azadirachtin-A were shifted upfield at 8 2.1(m) and 8 3.95 (m) in dihydroaza-A, Table 1. Significant differences in the 'H NMR peaks of aza-A and tetrahydroaza-A (Table Removed) and at 8 2.1 (m) and 3.8- 4.1 (m) in tetrahydroaza-A. Further, the dihydrotigloyl moiety in tertrahydroaza-A was characterized by an additional multiplet (8 1.78) corresponding to C-2' proton. This peak was otherwise absent in aza-A. Further a one-proton multiplet at 8 6.93 (C- 3' proton) in azadirachtin-A was shifted upfield as a two-proton multiplet at 8 1.45. Similarly 4' and 5'- methyl protons that appeared as double quartet (8 1.78 and 1.85) in azadirachtin remained unaffected in dihydroaza-A, and shifted upfield at 8 0.95 and 1.16 as triplet and multiplet in tetrahydroaza-A. Except for some minor differences, the chemical shifts for other tetrahydroaza-A protons were the same as aza-A. Mass spectral characterization of azadirachtins A The ESI-MS of azadirachtin-A showed a molecular ion peak at m/z 721 [MH]+ corresponding to its molecular formula C-35H44O16 (Table 2). The major fragment ion peaks originating as a result of the elimination of one molecule each of neutral water (18 amu), acetic acid (60 amu) and tiglic acid (100 amu) from the parent molecular ion, indicated the presence of hydroxyl, acetyl and tigloyl functions in the molecule (Sharma, V.; Walia, S.; Parmar, B. S.; Kumar, J. and Nair, M. G. (2003) J. Agric. Food Chem. 51: 3966-3972.). The fragment ion peak (m/z 703) formed as a result of the loss of first H2O molecule, underwent elimination of second H2O, and tiglic acid molecules yielding major characteristic fragment ions at m/z 685 and m/z 603 respectively. A minor peak located at m/z 661 originated possibly as a result of the loss of AcOH moiety from the protonated molecular (MH)+ ion peak. The major fragment ion (m/z 685) underwent further elimination of water and tiglic acid in the third and fourth step to form corresponding fragment ions at m/z 667 and 567. Alternatively, loss of tiglic acid (100 amu) and water molecule (18 amu) from the fragment ion m/z 685 yielded corresponding fragment ions at m/z 585 and 567, respectively. Similarly the fragment ion (m/z 603) formed as a result of the loss of tiglic acid in the second step underwent Table 2. Positive ion electrospray mass spectral peaks of azadirachtin-A (Table Removed) successive loss of two water molecules creating fragment ions at m/z 585 and 567. The mass spectrum also exhibited two important fragment ions at m/z 625 and 507 formed as a result of the loss of acetic acid molecules from the fragment ion m/z 685 and 567, respectively. The ESI-MS, further showed two characteristic sodiated and potassiated peaks at m/z 743 and 759 which were attributed to [M+ Na]+ and {M+K]+, 23, and 39 mass units higher than that of the parent molecular ion peak. The intensity of sodiated peak (4.5 %) was far greater than the potassiated peak (3.125 %). The electron impact mass spectrum (EI-MS) of aza-A showed a minor molecular ion (M+) peak at m/z 720 and a significant proton abstracted peak at m/z 719. Successive losses of H2O and -CH3 units from the molecular ion yielded two prominent fragments at m/z 702 and 687 (Table 3). As a result of the further loss of H2O unit from the fragment ion m/z 687, a prominent peak emerged at m/z 669 (base peak) that after losing a methylene moiety produced another fragment ion at m/z 655. The peak recorded at m/z 195 originated as a result of the cleavage of the bond joining decalin and dihydrofuranacetal parts of the molecule. Further, peaks at m/z 587 and 642 were attributed to the loss of tiglic acid and CO2 molecule from the fragment ion m/z 687. Table 3. El mass spectral peaks of azadirachtin-A (Table Removed) Mass Fragmentation pattern of dihydroaza-A and tetrahydroaza-A The major fragment ions located in the ESI-mass spectrum of dihydroaza-A and tetrahydroaza-A El are given in Table 4 and 5. The ESI-MS of dihydroaza-A in the positive ion mode exhibited a molecular ion peak at m/z 723 (M+H), a base peak at m/z 705 [(M+H)+- H2O] and a major quasimolecular ion peak at m/z 745 M+Na)+. The peaks located at 705.1, 687.2 and 669 were attributed to the loss of one, two and three water molecules respectively from the molecular ion. The ESI-MS of tetrahydroaza-A showed the presence of a molecular ion peak at m/z 725 (MH+) corresponding to its molecular formula C35H48O16. It differed from azadirachtin-A in having four additional hydrogen atoms (4 amu). The protonated molecular ion peak (MH+) readily lost three successive neutral water molecules to form fragment ions at m/z 707, 689 and 671. The fragment ion at m/z 707 and 689 further loses a neutral dihydro tiglic acid moity (102 amu) to create corresponding two fragment ions at m/z 605 and 587. Characteristically significant two quasimolecular adduct ion peaks located at m/z 747.38 and 763.33 were attributed to (M+Na)+ and (M+K)+ ions which were typical of a tetrahydroaza-A molecule. The former peak (m/z 763.33) was incidentally the base ion peak. The conspicuous absence of the molecular ion peak at m/z 722 (723 in positive mode) in the mass spectrum was indicative of the absence of any trace of dihydroaza-A in the sample. Table 4. Positive ion electro-spray mass spectral peaks of dihydroaza-A (Table Removed) (Table Removed) Table 5. Positive ion electro-spray mass spectral peaks of dihydroaza-A The major fragment ions located in the EI-MS of tetrahydroaza-A is given in Table 6. In this mode the molecular ion peak (M+) of tetrahydroazadirachtin-A was recorded at m/z 724 with relative abundance of 88%. The molecular ion underwent further loss of water, methanol or acetic acid molecule to form fragment ions at m/z 706 (M-H2O)+, 692 (M-CH3OH) +, and 664 (M-ACOH) +, respectively (Table 16). The elimination of acetic acid molecule from the molecular ion (m/z 664) accounted for the base peak Table 6. El mass spectral peaks of tetrahydroazadirachtin-A (Table Removed) The successive loss of two water units from the third most abundant ion at m/z 692 yielded to two fragment ions at m/z 674 and 658. Besides the usual peaks originating as a result of the loss of neutral water, methanol or acetic acid molecules, two additional fragment ion peaks at m/z 141 and 125 possibly emerged as a result of the cleavage of the tetrahydrofuran moiety in the molecule. Interestingly, molecular ion or any of the major fragment ions did not produce any fragment ion peak corresponding to the loss of tiglic acid moiety. Example IV (Bioassay) Bioactivity of azadirachtin-A, dihydroazadirachtin-A and tetrahydroaza-A concentrates against Spodoptera litura and Helicoverpa armigera In a comparative study of the antifeedant and growth regulatory activities, reduced azadirachtins display a higher degree of activity against Spodoptera litura and Helicoverpa armigera and other pests. Amounts required for pest control is governed by the nature of the pest, its infestation level, use form, and environmental conditions. The compounds have been additionally found effective against phytophagous fungi, Rhizoctonia solani and Sclerotium rolfsii and the root knot nematode Melidogynae incognita. Insect antifeedant activity Antifeedant activity of aza-A concentrates (20 %, 60 % and 90 %), dihydroazadirachtin concentrate (60%), and tetrahydroaza-A concentrates (60 %, 90 %) against Spodoptera litura and Helicoverpa armigera in the no choice bioassay after 24 and 48 hours of the treatment is given in Table 7. The results revealed that Spodoptera litura is reasonably sensitive in terms of antifeedancy to all the test concentrations. After 24 hrs of the treatment and feeding on treated leaves, tetrahydroazadirachtin-A (90%) was most effective showing antifeedant index (AI50) of 0.005 % followed by dihydroaza-A concentrate (60%) (Al50 0.009 %), azadirachtin-A (90 %) (Also 0.011%), tetrahydroazadirachtin-A (60 %) (AI 50 0.013 %), azadirachtin-A (60 %) (AI 50 0.022 %) and azadirachtin-A (20 %) (AI 50 0.033 %). Similarly after 48 hours, tetrahydroaza-A (90%) with AI50 of 0.008% was most effective followed by dihydroaza-A concentrates (60%) with AI 50 of 0.015 percent. After 24 hours of feeding, H. armigera larvae treated with test concentrations of various azadirachtin concentrates consumed less portion of the leaves as food compared to the control. While all the aza-A and tetrahydroaza-A concentrates in general exhibited almost similar level of activity, the most active tetrahydroaza-A (90%) displayed Also level of 0.0014 percent. Rest of the concentrates with Table 7. Antifeedant activity (Also) of azadirachtin-A and tetrahydroazadirachtin-A concentrates against Spodoptera litura and Helicoverpa armigera (Table Removed) different levels of aza and/or tetrahydroaza content exhibited Al50 in the range of 0.0025- 0.0030 %. After 48 hours of the treatment, tetrahydroaza-A concentrates (60 %, 90 %) were found more active than azadirachtin concentrates. While tetrahydroaza-A concentrates (60 %, 90 %) exhibited AI50 of 0.003 and 0.0015 percent, aza-A concentrates (20 %, 60 % and 90 %) showed AI50 of 0.0084, 0.0067 and 0.0049 percent, respectively. Insect growth regulatory activity Insect growth regulatory activity of aza-A concentrates (20 %, 60 % and 90 %) dihydroaza-A (60%), and tetrahydroaza-A concentrates (60 %, 90 %) against Spodoptera litura and Helicoverpa armigera is given in Table 8. The test compounds were applied through foliar application enabling insect to feed on treated plants. In terms of IC50 values, Aza-A concentrate (90 %) was found most effective (IC50 0.027 %) against mature Spodoptera litura larvae followed by tetrahydroaza-A (90 %) (IC50 0.035 %); dihydroaza-A (60 %) (IC50 0.057 %); tetrahydroaza-A (60 %) (IC50 0.059 %); aza-A (20 %) (IC50 0.070 %) and aza-A (60 %) (IC50 0.099 %). Treatment of H. armigera larvae with aza-A and tetrahydroaza-A concentrates caused growth inhibition, malformation and mortality in a dose dependent manner. Overall the reduced azadirachtins were slightly more active than their corresponding counterparts indicating that hydrogenation besides providing stability, retained bioactivity. In terms of IC50 values, Aza-A concentrate (90 %) was most active (IC50 0.041 %) followed by tetrahydroaza-A (90 %) (IC50 0.043 %); tetrahydroaza-A (60 %) (IC50 0.081 %); and dihydroaza A (60%) (IC50 0.082%). Table 8. Growth inhibition (IC50, %) of azadirachtin and tetrahydro-azadirachtin concentrates against Spodoptera litura and Helicoverpa armigera (Table Removed) Percent inhibition of larval growth following ingestion of food (leaves treated with aza-A and tetrahydroaza-A concentrates) by S. litura and H. armigera revealed that the effect of tetrahydroazadirachtin was superior to that of aza-A concentrate of the same strength. Out of the two tetrahydroaza-A concentrates (90 % and 60 %), tetrahydroaza-A (90 %) was most effective, and this in turn was more active than the corresponding aza-A and dihydroaza-A concentrate suggesting that hydrogenation of the unsaturated moieties produced no adverse effect on the biological activity, rather to some extent, it enhanced the activity. Antifungal and nematicidal activity Antifungal activity of azadirachtin dihydroazadirachtin and tetrahydroazadirachtin concentrates showed the same trend of fungicidal activity. (Table 9). Of the three azadirachtin concentrates, azadirachtin-A (90%) exhibiting ED50 of 93.6 and 104.8 ppm against S. rolfsii and R. solani respectively was found to be the most active. Tetrahydroazadirachtin (90%) was most effective (ED50, 102.2 ppm) against the test fungus Rhizoctonia solani followed by azadirachtin (90%) (ED50 104.8 ppm), and dihydroazadirachtin (ED50116.6 ppm) All the test concentrations of azadirachtin-A, dihydroazadirachtin, and tetrahydroazadirachtin-A were also found effective against the reniform nematode Rotylenchulus reniformis after 72h of treatment. These exhibited significant nematicidal activity. (EC50 119.1 to 193.9 ppm). At 200 ppm concentration, different azadirachtin and tetrahydroazadirachtin concentrates were also active against Caenorhabditis elegans nematode. The test compounds after 24h evoked mortality response ranging from 56 and 62%. Table 9. Antifungal and nematicidal activity of aza-A, dihydroaza-A and tetrahydroaza-A concentrates against the phytophagous fungi R. solani, S. rolfsii and the reniform nematode R. reinformis (After 24 h of application) (Table Removed) I/We claim:- 1. A process for preparation of reduced azadirachtin(s) pesticides from the azadirachtin concentrates of 20-100% strength characterized by reduction of azadirachtin of the formula (1) in the presence of a catalyst as herein described at temperatures up to 60°C and hydrogen pressure from 1 to 5 atmospheres under continuous stirring for 2 to 12 hours followed by filtration/centrifugation to yield dihydroazadirachtin of the formula (2) and/or tetrahydroazadirachtin of the formula (3) concentrates in 85 to 95% yield. 2. A process as claimed in claim 1 wherein azadirachtin concentrates as employed in the preparation of reduced derivatives comprising dihydroazadirachtin or mixture of dihydroazadirachtin and tetrahydroazadirachtin contains azadirachtin-A content of 40% or above. 3. A process as claimed in claim 1 wherein azadirachtin concentrates as employed in the preparation of tetrahydroazadirachtin contains azadirachtin-A content of 60% or above. 4. A process as claimed in claim 1 wherein catalyst used to achieve reduction of azadirachtin is one or more of various catalysts such as palladium on silica, alumina, charcoal or calcium carbonate; metal oxide such as platinum oxide, nickel boride; or nickel based catalysts derived from nickel-aluminum alloy, at a preferred concentration of 1 to 50 percent w/w of azadirachtin. 5. A process as claimed in claim 1 wherein the temperature of reduction reaction is preferably between 10-40°C. 6. A process as claimed in claim 1 wherein the pressure of reduction reaction is preferably slightly positive hydrogen pressure between 1-2 atmospheres. 7. A process as claimed in claim 1 wherein the reduction mixture is continuously stirred at preferably 200-600 revolutions per minute to ensure thorough mixing of the reactants. 8. A process as claimed in claim 1 wherein the reaction mixture is continuously stirred for a preferable period of 4-10 hours, till the required conversion is achieved. 9. A process as claimed in claim 1 wherein a quantitative conversion of azadirachtin of the formula (1) into dihydroazadirachtin of the formula (2) and/or tetrahydroazadirachtin of the formula (3) is achieved at the preferred conversion yield of 85 to 95 per cent of the claimed product. 10. Process as claimed in any of the above claims and as substantiated in the body of application to yield reduced azadirachtin(s) pesticides.

Documents:

1126-del-2003-abstract.pdf

1126-del-2003-claims.pdf

1126-del-2003-correspondence-others.pdf

1126-del-2003-correspondence-po.pdf

1126-del-2003-description (complete).pdf

1126-del-2003-form-1.pdf

1126-del-2003-form-19.pdf

1126-del-2003-form-2.pdf

1126-del-2003-form-3.pdf

1126-del-2003-form-4.pdf

1126-del-2003-form-5.pdf