Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2001-09-14
2003-10-21
Wilson, James O. (Department: 1623)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C514S054000, C514S058000, C536S102000, C536S103000, C536S123100, C536S127000, C536S128000
Reexamination Certificate
active
06635757
ABSTRACT:
This application is a 371 of PCT/IN99/00009 filed Mar. 18, 1999.
This invention relates to a process for the preparation of water soluble cyclodextrin) inclusion complex of neem seed kernel extract containing azadirachtin-A and its use in insecticidal formulation.
BACKGROUND OF THE INVENTION
Azadirachtin, a tetranortriterpenoid 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 elicit growth retardation varies from species to species, between 1-100 ppm. 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 is obtained in 99% purity by repeated chromatography but it is not as active as NSKE (containing azadirachtin-A<70%).
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 oligosacclarides consisting or 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 arc 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 solubilising water insoluble compounds by forming inclusion complexes. &agr;, &bgr; and &ggr;-cyclodextrins are the three naturally occurring forms consisting of 6, 7 and 8 glucose units respectively. Among these, &bgr;-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 alky, have also been used to enhance their solubility in water.
Natural cyclodextrins and their derivatives are being used in the pharmaceutical industry for solubilising sparingly water soluble drugs to enhance their bioavailability. Some of the drugs like Piroxicam, Lonmiel, Nitropen, Pansporin-T etc., are commercially available in the cyclodextrin encapsulated form also.
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 neurohemal substances 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 the formulations.
Patent Number WO-A1-921609 discloses a method for the preparation of a storable azadirachtin-rich insecticide extracting crushed neem seed kernel with water, extracting the aqueous phase with an organic solvent, concentrating azadirachtin contained solvent to produce a concentrate. Patent number EP-A1-0581469 disclosed the method for preparing high purity neem seed kernel extract by loading aqueous neem seed extract macroporous polymeric adsorbent followed by desorption using solvent such as alcohol, an ester or ethyl acetate, methanol or methyl isobutylketone. Patent no. EP-A1-0581467 relates to a process for preparing storage stable neem seed extract by dissolution of crude neem seed extract in a polar solvent and removal of impurities by precipitation and/or treatment of the extract with an oxidizing agent.
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 substance used for solubalisation should be economical, safe, biodegradable and freely soluble in water.
Should not reduce the insecticidal activity of the parent molecules upon solubilisation.
Should contain in addition to azadirachtin-A, other limonoids which are also known to enhance the insecticidal activity either directly or through synergism.
Should not have any phytotoxiety.
Further object of the invention is to make an insecticidal formulation, which is ecofriendly and devoid of presence of any organic solvents or surfactants.
To achieve the first objective, this invention provides a process for the preparation of a water soluble inclusion complex of the neem seed kernel extract (NSKE) containing azadirachtin-A in cyclodextrin comprising:
adding neem seed kernel extract containing 0.01% to 96% of azadirachtin-A by wt. to cyclodextrin, the molar ratio of azadirachtin-A to the cyclodextrin 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 neem seed kernel extract to get a free flowing pale cream powder.
Cyclodextrin, may be natural cyclodextrins or their synthetic derivatives or row combination thereof. The said natural cyclodextrins are &agr;, &bgr;, &ggr; cyclodextrins and the synthetic derivatives are methyl, propyl, isopropyl, hydroxymethyl, hydroxyethyl, hydroxypropyl and sulpho alkyl derivatives. The said synthetic derivative is a &bgr;-methylcyclodextrin [(2,6-di-O-methyl)-&bgr;-cyclodextrin].
Initial experiments on the solubilisation of azadirachtin-A (96% pure) in &bgr;-methyl cyclodextrin indicated that a 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 &bgr;-methyl cyclodextrin corresponding to the azadirachtin-A content, it as 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 ratio of azadirachtin-A to &bgr;-methyl cyclodextrin appeared to be optimum, when extractability and economy was considered. When &bgr;-cyclodextrin was used instead of &bgr;-methyl cyclodextrin for solubilising NSKE, it was observed that some of the cyclodextrin also remain in the residue due to relatively poor solubility of &bgr;-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.
To achieve the second objective, this invention provides an insecticidal formulation comprising:
a solution of inclusion complex containing cyclodextrin and neem seed kernel extract containing azadirachtin-A in water in the ratio of 0.1 to 1000 ppm with respect to azadirachtin-A, and
if desired, 0.001% to 25% of sun screen agents or salicylic
Kumble Sandeep Prabhu
Ramadoss Candadai Seshardi
Rao Alapati Srinivasa
Rao Pillarisetti Venkata Subba
SambasivamAnnadurai Ramaswamy
Lewis Patrick
Sidley Austin Brown & Wood LLP
Vittal Mallya Scientific Research Foundation
Wilson James O.
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