Process of preparing purified azadirachtin in powder form from neem seeds and storage stable aqueous composition containing azadirachtin

This invention relates to a process of preparing storage stable aqueous Azadirachtin containing Azadirachtin A to provide a predominant pesticidal composition which comprises the steps of diluting 50,000-100,000 ppm Azadirachtin with 25,000 to 50,000 Azadirachtin A weight/volume with ethanol water solvent in a ratio in the range 100-70:0-30 volume/volume to produce 2,000-40,000 ppm Azadirachtin containing 1,000-20,000 ppm of Azadirachtin A weight/volume. This is followed by emulsifying of the diluted Azadirachtin with non-ionic and non-toxic emulsifying agents in an amount ranging from 0.2 to 30% and neem oil 20-50% volume/volume. The pH of the emulsified diluted Azadirachtin is adjusted to 3.5 to 6.0 by adding an alkali solution. 1-2.5% of a sunscreen like p.aminobenzoic acid or its esters and 1-10% oleic acid volume/volume are added to create a micro-emulsion for stabilizing the composition with a good bio-efficacy.

BACKGROUND OF THE RELATED ART
 Several power and effective synthetic insecticides have been used to
 protect food and fiber crops with varied success for many years now. More
 recently, there has been a great deal of controversy about the effect of
 these on the environment and some of the insecticides which have been in
 extensive use have been banned. It is likely that other insecticides could
 be banned but which are still in use are considered to be potentially
 harmful to the environment but are required to be used for lack of other
 alternatives.
 As a result, a search has been going on for "botanical pesticides" which
 are environmentally friendly. These are compositions which would deter
 insects or other pests but would have no or minimal harmful effect on the
 environment, particularly to humans who sometimes are at the end of the
 food chain and may thus suffer bio-accumulation.
 Presently an agent known to protect crops from pests is Azadirachtin which
 is a natural product found in the seeds of the neem tree (Azadirachtin
 indica A. Juss). This has gained a lot of importance all over the world as
 the most environmentally safe pesticide. The neem tree is found in great
 abundance in India and also is distributed in other countries like
 Pakistan, Bangladesh, Burma, Thailand and Malaysia and is also found in
 Africa.
 Several Azadirachtin isomers, derivatives and related compounds have been
 studied. From such studies it is evident that Azadirachtin A is the most
 important compound which has the desired insecticidal property.
 Azadirachtin A has been extracted from neem seeds and is found to have an
 anti-feedant property (which deters insects from feeding on plants) and
 growth regulation potency against such pests. It is readily applied by
 coating seeds or by applying a spray to the crops themselves.
 Various methods of extracting Azadirachtin or such similar principles have
 been described in Indian patents 153415, 172150, 173327, 173328, 173449,
 173989, 173996, 173997 and 173998 and U.S. Pat. Nos. 4,556,562, 5,391,779,
 5,372,817, 5,352,677, 5,298,251, 5,281,618, 5,229,007 and 5,124,349.
 Azadirachtin is a known agent, but stable Azadirachtin A has not been
 purified to a higher content through a process which enriches the content
 to about 60-75% of the total Azadirachtin content without the use of
 laborious columns. The commercial uses of Azadirachtin A have been based
 on its stability and purification and nowhere in the above patents is a
 process for purifying and enriching Azadirachtin A described.
 U.S. Pat. No. 5,736,145, upon which the present application relies for its
 teaching of a successful method of extracting Azadirachtin A from neem
 seeds is incorporated herein by reference.
 SUMMARY OF THE INVENTION
 It is therefore a principal object of this invention to prepare
 Azadirachtin A rich extract from total Azadirachtin, with emphasis on a
 stable and purified form.
 To achieve the objective, this invention provides a process of preparing
 storage stable aqueous Azadirachtin containing Azadirachtin A predominant
 pesticidal composition, which comprises the steps of:
 diluting 50,000-100,000 ppm Azadirachtin with 25,000 to 50,000 Azadirachtin
 A weight/volume with ethanol water solvent in a ratio 100-70:0-30
 volume/volume to 2000-40000 ppm Azadirachtin containing 1000-20000 ppm of
 Azadirachtin A weight/volume;
 emulsifying the diluted Azadirachtin with non-ionic and non-toxic
 emulsifying agents as herein described in an amount ranging from 0.2 to
 30% and neem oil 20-50% volume/volume;
 adjusting the pH of emulsified diluted Azadirachtin to 3.5 to 6.0 by adding
 alkali solution; and
 adding 1-2.5% of a sunscreen comprising one of p.aminobenzoic acid or its
 esters and 1-10% oleic acid volume/volume, to create a micro-emulsion for
 stabilizing the composition with a good bio-efficacy.
 Preferred non-ionic and non-toxic emulsifying agents used in the process
 are sorbitan esters, ethoxylated and propoxylated mono or diglycerides,
 acetylated mono or diglycerides, lactylated mono, or diglycerides citric
 acid esters of mono or diglycerides, sugar esters, polysorbates and
 polyglycerol esters. A preferred emulsifier is polyoxyethylene sorbitan
 monolaurate which is sold under the name "Tween 20 (R)"
 The alkali solution preferably used in the process is NH.sub.4 OH, and the
 emulsifying agent preferably used is 15-25% volume/volume of the
 composition.
 An oleic acid, preferably 1% volume/volume of the composition, is added to
 create a micro-emulsion for stability with a good bio-efficacy.
 DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
 The step of forming neem kernels pellets involves decortication of the seed
 either manually or using a decorticator, i.e., a known mechanism for
 shelling nuts.
 The extraction of 80% enriched Azadirachtin A without oozing oil is carried
 out using acetone and water solvent (hereinafter referred to as the
 "extracting agent") in the ratio 90; 10 at 80.degree. C. by involving
 multiple passes of the acetone and water solvent through the neem kernels.
 This involves, for example, passing a batch of the extracting agent through
 the kernels and recovering it and then passing another batch of the
 extracting agent through the kernels again, and continuing this process
 until a total of 3 to 5 passes occur. The extract is filtered using an
 ordinary filter paper to remove clay, sand and some scum. The extract is
 then washed several times with dichloromethane. The dichloromethane
 extract is pooled, and moisture removed therefrom with anhydrous sodium
 sulphate. Dichloromethane is removed, distilled and recovered.
 Azadirachtin enriched in Azadirachtin A is stuck on the walls of the
 container and is scraped and collected. Dichloremethane is used in the
 process as it is an environmentally friendly solvent.
 The process requires diluting of 50,000-100,000 ppm Azadirachtin with
 25,000 to 50,000 Azadirachtin A weight/volume. The step of diluting to
 form an emulsion comprises adding dilutent, preferably ethanol:water in
 the range 100-70:0-30, and an emulsifying agent along with oleic acid
 volume/volume comprising from about 2,000 to about 40,000 ppm Azadirachtin
 comprising 1,000-20,000 ppm of Azadirachtin A weight/volume usually from
 about 20% to about 25% neem oil. Ethanol:water is preferably in the ratio
 70:30 volume/volume.
 It is highly preferred to include an emulsifying agent in the composition
 so that the Azadirachtin and neem oil and any other ingredients are kept
 uniformly distributed in the composition. The percentage of the
 composition which is the emulsifier normally depends on the emulsifier
 which is used and the emulsifying agent that is the active ingredient is
 used in an amount ranging from about 0.2% to about 25% volume/volume.
 Preferred emulsifying agents are those normally utilized in foods, and
 include sorbitan esters, ethoxylated and propoxylated mono or
 diglycerides, acetylated mono. or diglycerides, lactylated mono. or
 diglycerides citric acid esters of mono. or diglyerides, sugar esters,
 polysorbates and polyglycerol esters. The preferred emulsifier is
 polyoxyethylene sorbitan monolaurate which is solid under the name "Tween
 20 (R)".
 It is preferred to include oleic acid at a level for example of about 1-10%
 volume/volume to aid microemulsion preparation and about 1% volume/volume
 of a sunscreen like P. Amino benzoic acid.
 The instant invention will now be described with reference to the following
 examples:

EXAMPLE I
 3 kg of decorticated kernal collected from Tamil Nadu, India, is used in
 the extraction. Acetone/water in the ratio of 90:10 volume/volume is used
 for extracting, The extracts are pooled. A minimum of three extractions is
 required at the elevated temperatures mentioned above, with an optimum of
 80.degree. C. as the inner jacket temperature. The first extract is
 filtered and an equal volume of Dichloromethane is added and extracted.
 Three extracts of Dicholoromethane of the aqueous extract are pooled.
 Anhydrous sodium sulphate is added, and water if any in excess is removed.
 The dichloromethane is distilled and recovered. The enriched Azadirachtin
 A thus obtained is used further for formulation. To the extract is added
 Tween 20 (R) (consisting of water and polyoxyethylene sorbitan
 monolaurate) to obtain a diluted extract containing 3,000 ppm Azadirachtin
 and 20% neem oil weight/volume. To the resulting emulsion is added
 ammonium hydroxide to adjust the pH to 4.0. To this is added 1% p-amino
 benzoic acid or its esters and 1% oleic acid volume/volume for
 microemulsion purposes. This formulation is an agent against a wide
 spectrum of pests and is shelf stable for more than 8 weeks after
 formulation. A product containing 2,000 ppm Azadirachtin A weight/volume
 minimum and having a pH of 4.0 maintained under normal shelf life
 conditions (without refrigeration or addition of sunscreen) was found to
 have retained 65% of its potency even two years later.
 EXAMPLE II
 Processing is carried out as in Example 1, except that extraction and
 separation of extract from neem kernels is carried out by centrifuging.
 The resulting product is effective against a wide spectrum of pests.