Title of Invention

AN INSECTICIDAL FORMULATION

Abstract

ABSTRACT The present invention disclosed an insecticidal formulation using inclusion complex of neem seed kernel extract (NSKE) containing azadirachtin-A as described in co-pending application no. 316 /Mas/99 comprising: a solution of inclusion complex containing cyclodextrin and neem seed kernel extract containing predominantly azadirachtin-A in water in the range of 0.1 to 1000ppm with respect to azadirachtin-A, and if desired, a pre-determined quantity of sun screen agents or salicylic acid or its derivatives either alone or in combination for enhancing the shelf life and in plant defense mechanism against pathogen to have multiple effect.

Full Text

This invention relates to insecticidal formulation using inclusion complex of neem seed kernel extract (NSKE) containing azadirachtin-A described in our co-pending application no. /Mas/99 BACKGROUND: Azadirachtin, a tetrauortriterpenoid isolated from the seed kernels of the Indian neem tree Azadirachta indica is known to be active on more than 200 economically important species of insects. It often causes moult inhibition, growth retardation, antifeedancy, sterilisation of the ovaries and reduced fecundity in adults. It is active at ppm levels and the physiological concentration required to ehcit growth retardation varies from species to species, between 1-100ppm. Azadirachtin containing insecticides first act as oral poisons. Death of the target insect is dose dependent, usually occurring a few days after application but in extreme cases the larvae may live upto several weeks without undergoing metamorphosis. Azadirachtin-A can be obtained in 99% purity by repeated chromatography but it is not as active as NSKE (containing azadirachtin-A Neem seed kernel extract containing azadirachtin-A is very sparingly soluble in water and several surfactants are used to enhance its solubility in insecticidal formulations. Achieving complete solubility in water without altering the insecticidal activity has not been attempted so far. Considering this point, we have developed a process for preparing a water soluble NSKE containing azadirachtin-A, fulfilling the requirements of a true eco-friendly biopesticide. Cyclodextrins are cyclic oligosaccharides consisting of glucose units fused together as a ring. This arrangement of the glucose units makes the molecule appear like a truncated cone. The primary and secondary hydroxyl groups of the glucose units are present on the exterior of the cyclodextrin molecule thus making the exterior part hydrophilic, whereas the interior of the cyclodextrin molecule is hydrophobic. This amphiphatic property of cyclodextrin makes it an ideal molecule for solubiUsing water insoluble compounds by forming inclusion complexes, a, p and y-cyclodextrins are the three naturally occurring forms consisting of 6,7 and 8 glucose units respectively. Among these, )3-cyclodextrin is the most abundant and commonly used one. Several synthetic derivatives of cyclodextrins such as methyl, propyl, isopropyl, hydroxy methyl, hydroxy ethyl, hydroxy propyl and sulfo alkyl, have also been used to enhance their solubility in water. Natural cyclodextrins and their derivatives are being used in the pharmaceutical industry for solubiUsing sparingly water soluble drugs to enhance their bioavability. Some of the drugs like Piroxicam, Lonmiel, Nitropen, Pansporin-T etc., are commercially available in cyclodextrin encapsulated form also. PRIOR ART: Biopesticides of plant origin have several advantages over their synthetic counterparts. Azadirachtin-A is the best-characterized candidate both biologically and chemically among known plant derived insecticides. The exact mode of action of this compound in insects is still not fully understood, even though it is implicated as an antihormonal substance that blocks the release of the aeurohemal substance from the carpus cardiacum into the hemolymph. Although azadirachtin has very potent insecticidal activity, it is highly unstable in aqueous medium and extremely sensitive to sunlight. A number of insecticidal formulations containing azadirachtin such as Neem gold. Fortune Aza, Margosan O, Neemazal F, etc. are available in the market. All these commercial formulations contain several surfactants and a solvent like ethanol, methanol, turpentine, isopropanol, polyethylene glycol, etc. as a carrier. The use of these carriers and surfactants in biopesticidal formulations containing azadirachtin has not sufficiently enhanced the shelf life of (he formulations. For these reasons, the object of this invention was to develop a stable neem seed kernel extract preparation containing azadirachtin-A that is completely water soluble, possessing the following characteristics: ♦ The carrier molecule for solubilisation should be economical, safe, biodegradable and freely soluble in water. ♦ Should not reduce the insecticidal activity of the parent molecule upon solubilisation ♦ Should contain in addition to azadiracbtin-A, other limonoids which are also known to enhance the insecticidal activity either direcfly or through synergism. ♦ Should not have any phytotoxicity. To achieve the said objective this invention provides a process for the preparation of a water soluble inclusion complex of neem seed kernel extract (NSKE) containing azadirachtin-A in a carrier molecule comprising: - adding neem seed kernel extract containing azadirachtin-A to the carrier molecule of the kind as herein described, the molar ratio of the azadirachtin-A to the carrier molecule ranging from 1:0.5 to 1:20. - Stirring the above mixture, - Filtering the above mixture to obtain a water soluble inclusion complex of neem seed kernel extract and - If desired, freeze/ spray drying the said water soluble complex of neetn seed kernel extract to get a free flowing pale cream powder. The above inclusion complex of neem seed kernel extract containing azadirachtin- A and the process of preparing the same is the subject matter of our co-pending application no. /Mas/99. In other words the said inclusion complex is a mixture of cyclodextrin and neem seed kernel extract containing predominantly azadirachtin-A. The object of the present invention is to make an insecticidal formulation, which is totally eco-friendly and devoid of the presence of any organic solvents or surfactants. The instant invention is in respect of insecticidal formulation using inclusion complex of neem seed kernel extract (NSKE) containing azadirachtin-A comprising: - a solution of inclusion complex containing cyclodextrin and neem seed kernel extract containing predominantly azadirachtin-A in water in the ratio of 0.1 to 1000ppm with respect to azadirachtin-A. - if desired, a pre-determined quantity of sun screen agents or salicyhc acid or its derivatives either alone or in combination for enhancing the shelf life and in plant defense mechanism against pathogen to have multiple effect. The sunscreen agents are hydroquinone and anacardic acid. The salicylic acid ranges from 0.001% to 25% based on azadirachtin-A content in the inclusion complex ranging from 1% to 50% (w/w) The salicylic acid, hydroquinone, anacardic acid are complexed in cyclodextrin with the ratio of salicylic acid /hydroquinone to cyclodextrin ranging from 1:0.5 to 1:20. The sunscreen agent ranges from 0.001% to 25% based on azadirachtin-A content in the inclusion complex ranging from 1% to 50% (w/w). Inclusion complex of neem seed kernel extract (NSKE) contains azadirachtin-A of 0.01% to 96% purity. Initial experiments on the solubilisation of azadirachtin-A (96 % pure) in p-methyl cyclodextrin indicated that 1:1 water soluble complex is formed. However, when NSKE containing azadirachtin-A in the range of 10 to 40% (w/w) was extracted with 1 molar equivalent of p-methyl cyclodextrin corresponding to the azadirachtin-A content, it was observed that there was no selective extraction of azadirachtin-A. In fact, other limonoids present in NSKE were also encapsulated. Therefore, larger amount of cyclodextrin had to be used to maximize extraction and it was found that a 1:4 molar ration of azadirachtin-A to p-methyl cyclodextrin appeared to be optimum, when extractability and economy was considered. When P-cyclodextrin was used instead of p-methy] cyclodextrin for solubilising NSKE, it was observed that some of the cyclodextrin also remain in the residue due to relatively poor solubility of P-cyclodextrin in water. Such a cyclodextrin inclusion complex of neem seed kernel extract containing azadirachtin-A and other limonoids obtained by the process mentioned in this invention was found to be highly water soluble. Insecticide formulations using the solubilised NSKE mentioned above incorporating sunscreen agents like hydroquinone and anacardic acid have been prepared for enhancing the shelf life of the product. Salicylic acid (or its derivatives), which has been implicated in plant defence mechanism against pathogen has also been included in the formulation to have multiple effects. The bioactivity stuthes of the complexes and formulations done on Spodoptera litura and Helicoverpa armigera larvae showed that the inclusion complex was active at concentration ranging from 1-100 ppm in leaf disc assays. The first instar larvae treated with solubilised neem seed extract containing 25 ppm of azadirachtin-A have prolonged postembryonic development in comparison to other treatments (Table 1). The larvae of the solublised NSKE treated group never survived beyond the third instar and their growth indices were comparatively lower than all other treatments. In the case where cyclodextrin alone was used as control, the normal development took place with out any pupal mortality, eventhough there was some delay in pupal emergence. All pupae emerged to normal adults which reproduced normally, indicating that cyclodextrin as such does not contribute for the observed effects of the complex. At a dosage of 100 ppm, the developmental profile of the larvae was similar to that of 25 ppm but the growth indices were considerably lower than the latter (Table 2). Total mortality of the solublised complex treated group was achievable within 3 days. Hence, the bioactivity stuthes clearly indicate that there is no reduction in the insecticida! activity of the complexed product both at lethal (25-100ppm) and sublethal (1-10 ppm) doses as compared to that of the uncomplexed NSKE (Figure I of the accompanying drawing). Further, no phytotoxicity was observed under laboratoiy conditions, when leaf surfaces were sprayed with the water soluble NSKE complex/formulation containing azadirachtin-A (1-100 ppm). Thus an effective water soluble powder of the insecticidal formulation has been prepared for the first time. The formulation did not show any deterioration in the quality, azadirachtin content and insecticidal activities at least for a storage period of 6 months. The invention will now be described with reference to the following examples: Example 1: Insecticidal liquid spray formulation using water soluble NSKE inclusion complex: To 1000 mL of water, 14.7 g of water soluble NSKfi inclusion complex containing 6.8% azadirachtin-A (w/w) was added and stirred to get a clear solution containing 1000 ppm of azadirachtin-A (w/w). This stock solution was used for bio-assays at concentrations ranging from 1 to 100 ppm by direct dilution with water. Example 2: Insecticidal formulation using water soluble NSKE inclusion complex: To 100 g of water soluble NSKE inclusion complex containing 6.8% azadirachtin -A (w/w) was added 500 mL aqueous solution containing salicylic acid (0.6S g) and hydroquinone (0.68 g) [each 1/10* (w/w) basis of azadirachtin-A], and stirred for 30 min at 3TC. The ingrethents were filtered and freeze/spray dried to obtain a powder containing 6.7% of azadirachtin-A and 0.67% (w/w) of each of the additives viz. Salicylic acid and hydroquinone. Example 3: Preparation of 1:1 molar ratio inclusion complex of salicylic acid in p-methyl cyclodextrin; To a solution of P-methyl cyclodextrin (47.74 g) in 150 mL of water, 5 g of salicylic acid was added. The contents were shaken and freeze/spray dried to give 52.74 g of the complex. Salicylic acid content in the complex was found to be 9.5% (w/w). Example 4: Preparation of 1:1 molar ratio inclusion complex of hydroquinone in p- methyl cyclodextrin: To a solution of P-methyl cyclodextrin (59.18 g) in 150 mL of water, 5 g of hydroquinone was added. The contente were shaken and freeze/spray dried to give 64.18 g of the complex. Hydroquinone content in the complex was found to be 7.8% (w/w). Example 5: Insecticidal formulation using the water soluble NSKE inclusion complex and P-methyl cyclodextrin solubilised additives [each 1/10* (w/w) basis of azadirachtin-A]: p-methyl cyclodextrin-salicyhc acid complex (7.2 g, containing 0.68 g of salicylic acid, from Example 3) and P-methyl cyclodextrin-hydroquinone complex (8.8 g, containing 0.68g of hydroquinone, from Example 4) were added to 100 g of water soluble NSKE inclusion complex containing 6.8% azadirachtin-A (w/w) and blended thoroughly. The powder thus obtained contains 6.66% (w/w) of azadirachtin-A and 0.66% (w/w) of each of the additives viz. Salicylic acid and hydroquinone. Example 6: Insecticidal formulation using the water soluble NSKE inclusion complex and P-methyl cyclodextrin solubilised additives leach 1/10' (w/w) basis of azadirachtin-AJ: Salicylic acid (0.68 g) and hydroquinone (0.68 g) were added to a 25 mL solution of p-methyl cyclodextrin (14.67 g) and stirred for 30 min. to the resultant clear solution was added 100 g of water soluble NSKE mclusion complex containing 6.8% azadirachtin-A (w/w) dissolved in 500 mL of water and stirred for 30 min at 37° C. The solution obtained was filtered and freeze/spray dried to obtain a powder containing 6.66% (w/w) of azadirachtin-A and 0.66% (w/w) of each of the additives viz. Salicylic acid ana hydroquinone. Example 7: Insecticidal formulation using the water soluble NSKE inclusion complex and P~methyl cyclodextrin sotubilised additives [each 1/10' (w/w) basis of azadirachtin-A): This formulation was made in the same lines as that given in Example 6, except that anacardic acid was used instead of hydroquinone as the sunscreen agent. We claim: 1. Insecticidal fornnulation using inclusion complex of neem seed kernel extract (NSKE) containing azadirachtin-A comprising: a solution of inclusion complex containing cyclodextrin and neem seed kernel extract containing predominantly azadiractin-A in water in the range of 0.1 to 1000 ppm with respect of azadirachtin-A, and if desired, a predetermined quantity of sun screen agents or salicylic acid or its derivatives either alone or in combination for enhancing the shelf life of the insecticidal formulation 2. Insecticidal formulation as claimed in claim 1 wherein the sunscreen agents are hydroquinone and anacardic acid. 3. Insecticidal formulation as claimed in claim 1 wherein salicylic add ranges from 0.001% to 25% based on azadirachtin-A content In the inclusion complex ranging from 1% to 50% (w/w). 4. Insecticidal formulation as claimed in claim 1 wherein sunscreen agent ranges from 0.001% to 25% based on azadirachtin-A content in the inclusion complex ranging from 1% to 50% (w/w). 5. Insecticidal formulation as claimed in claim 1 wherein salicylic acid, hydroquinone and anacardic acid are complexed in cyclodextrin with the ratio of salicylic acid/ hydroquinone to cyclodextrin ranging from 1:0.5 to 1:20. 6. Insecticidal formulation as claimed in claim 1 wherein inclusion complex of neem seed kernel extract (NSKE) contains azadirachtin-A of 0.01% to 96% purity. 7. Insecticidal formulation using inclusion complex of neem seed kemel extract (NSKE containing azadirachtin -A substantially as herein described with reference to the foregoing examples and accompanying drawings.

Documents:

314-mas-1999 abstract duplicate.pdf

314-mas-1999 abstract.pdf

314-mas-1999 claims duplicate.pdf

314-mas-1999 claims.pdf

314-mas-1999 correspondence others.pdf

314-mas-1999 correspondence po.pdf

314-mas-1999 description (complete) duplicate.pdf

314-mas-1999 description (complete).pdf

314-mas-1999 drawings.pdf

314-mas-1999 form-1.pdf

314-mas-1999 form-19.pdf

314-mas-1999 form-26.pdf