Title of Invention

PESTICIDE FORMULATION CONTAINING AZADIRACHTIN (NOT LESS THEN 300 PPM) AND SALANIN IN A FORMULATED PRODUCT WITH NEEM OIL

Abstract The invention relates to a process for the preparation of a storage stable Neem oil based Emulsion concentrate formulated pesticide, comprising of neem tetranortriterpeniods and neem limonoids including Azadirachtin not less than 300 ppm. These linnonoids are impregnated into neem oil and made into an emulsion concentrate with not less than 300 ppm Azadlrachtin and satanin and having atieast 3.90 % by volume of the aportic solvent and less than SO % and not more than 8.00 % percent of ethoxytated vegetable oil containing long chain fatty alcohol series from C8 to C18 by volume and which can enter into a stable oil water emulsion without degrading azadlrachtin content The preparation of pesticide formulation comprises the process of steps in sequence nannely preparing neem seeds from neem fruits, neem seed kernels from the neem seeds, neem oil from the neem seeds, an azadirachtin concentrate extract containing at least 300 ppm of azadirachtin by solvent extraction of the neem seed kernels and thereafter purifying by reverse osnnosls. Then the oil density of the neem cril is reduced by controlled thermic process, emulsified and then dosing the emulsified neem oil with the azadirachtin concentrate extract and further stabilizing agents and US/ screens are added to yield a pesticide containing at least 300 ppm of azadirachtin.
Full Text OBJECT QF THE INVENTION
The object of the invention Is to aim at extraction of azadlrachtin from neem seed kernel, In a very simplified but efficient manner which has highest level of azadlrachtin and without allowing mycotoxins contamination, and consequently to prepare neem oil based emulsion concentrate, a stable pesticide containing mostiy the iimonoids of neem as it occurs in nature in the neem seed, and hence fit to be called as neem based pesticide.
In addition, the Invention also aims at extracting the constituents of custard
apple fh3m immature fruits and leaves as well as fix>m mature seeds using the desired solvents and dousing It Into a neem seed kernel extract In the course of the preparation of neem oil emulsion concentrate without affecting the pesticidal properties of the llmanoids of neem leading to a botanical pesticide, a combination of neem and custard apple whereby the pesticidal potency of neem as well as of custand apple are exploited for the benefit of controllirtg pests which posses mere susceptibility to either neem or custard apple and hence more order of pests can be controlled through combination.
The first object of the Invention is to form storage stable neem oil emulsion which contains a desired amount Azadlrachtin to be an effiective pesticide and also combine these with the extracts of custard apple to exploit the pestlcidal properties of custard apple in the immature fruit stage, seed stage as well as In tjhe leaves and thereby producing a totally natural and effective herbal pesticide containing the characteristics of neem and custard apple leading a preparation of neutral pesticide which benefit nrK>ne number of target pests in the process of pest control.
It Is another object of the invention to extract neem oil as well as Azadlrachtin simultaneously from the neem seeds.

The other object of the invention includes^ to extract out the constituents of custard apple from its immature ^its^ immature seeds and leaves and dousing it in the neem oil preparation to enhance or improve the pesticidal properties of neem In combination with the synergistic constituents of custard apple extracts.
It is anotlier object of the invention to form environmentally safe pesticide taking advantage of the pesticide characteristics found in plants like neem tree and custard apple in their specil^c parts like neem seeds as well as In the Immature fruits, mature seeds and leaves of Annona Squmosa and Annona Reticulata -comn^nly referred to as Custard Apple and developing a herbal based combination pesticide, which In no way Inhibits the main objective of inventing an environmentally saf5e pesticide a combination pesticide.
It is another object of the invention to form an pesticide which contains neem oil, desired amount of Azadlrachtin emulsifying agent and desired preparation of extracts from immature fruits of Annona Squmosa and Annona Reticulata.
As per the invention, neem oil as well as Azadirachtin are sinruiltaneousiy extracted from the neem seeds and the extracts of custard apple Anr»na Squmosa and Annona Reticulata from their Inrimature frirfts, seeds and leaves, extracted separately and doused in the preparation at appropriate tinne as described in the process of the preparation of neem oil emulsion based pesticides.
The Invention has been described and illustrated with pnaferences and
ranges In the foregoing paras, much variation therein may be done by those skilled In the art without departing from the sport and &x>pe of the Invention claims.
Rjrther to produce it In a nrwst economical and Inexpensive nnethods, with minimal chemlcallsation and to produce through simple techniques which are more natural than chemical and at the same time extract the iimonoids of neem seed

with high yields of pofient tetranortripenoids Including azadlrachtin (an antlfeedaant iimonoid) with high oil content as high as 85.00% and further the potency of such pesticide so produced is also enhanced to a great extent without affecting the basic concept of safe and environment freely pesticide through the deployment of the extracts of ciBtard apple obtained fhsm the immature fruits and seeds and leaves of Annona Squmosa and Annona Reticulata pnaductjon of a combinab'on pesticide of hert)al base which is safe, environment friendly pesticide and further that which enhanced the pesticide potential of the resultant pesticide and results in greater bioefficacy by increasing the control capability of more number of target pests.
The Invention herein described relates to a process for preparing a neem based emulsifying concentrats containing azadlrachtin not less than 300 ppm, neem oil more than 85%, aportSc solvent by volume 3.90 % and less than SO % and not nrwre than S,00 % percent of ethoxylated vegetable oil containing long chain fatty alcohol series ttiom CS to CIS by volume and which can enter Into a stable oil water emulsion without degrading azadlrachtin content along with addition of not more than 5% of the extracts fhDm custard apple Immature fruits, seeds and leaves without degrading the efficacy of Azadlrachtin contained under the same time having Azadlrachtin synergisticism with neem linrx}noids facilitating the enhancement of pesticidal potency and thereby leading to Azadlrachtin combination pesticide with high bio-efTicacy standards without affecting the safe and environment frieraliy nature of neem based combination pesticides. This formulation formulated by the process of this invention has found its application and usage as a pest control formulation in Agriculture, and in Veterinary with higher bioefficacy and poteru:y to control more number of target pests.
There are a number of chemical, broad-spectrum pesticide formulations, systemic and contact, available for the control of Insect pests that attack and

damage food crops and economically im|X)rtant crops. Tliese chemical pesticides aim at pest eradication and kill the pests and insects and in this process all the insects good and bad insects are killed and in addition they not only affect the ecosystem but also pollute the atmosphere with chemical, kills the natural enemies to pests and other beneficial microorganisms and pollute the water we drink, air we breathe and food we eat. The pests or the insects also develop resistance to the pesticide in the coarse of time on continuous usage. Hence^ a need for pest control agent which are biodegradable that which are ecofriendly, biodegradable, that does not affect the beneficial organisms, non-toxic to ecosystem and human beir^js and at the same time effective against the target pest or insect is fait and the answer is this invention at least partiy If not fully.
The Pesticidal plant that has drawn the attention of many scientists, researchers, and industrialist all over the world the Neem {Aadirachta indica A JLSS) is widely spread in India and Pakistan and some parts of South Bast Asia. The neem tree and its various parts (Ijaaves, Seeds, Bark etc.,) have (bund Its application in various fields like food, medicine, and cosmetics and in agriculture. Out of the various parts of the neem tree the neem seeds In different forms Utoa neem oil, neem cake, neem seed extracts have found wide application in various fields. In agriculture the Neem seeds and its various forrr« are used as nitrogen fertilizer, pesticide, fungicides, nematicides etc., the usage of neem s^xl is more advisable than usage of other parts like green leaves, bark and not because these will affect the life if the neem tree where as neem seed wUl not
Similarly, the additive from the satellite plant used in tif^s {^reparation is a natural combination pesticide viz., Annona Squmosa and Arnxina Reticulata only referred to as custard apple also known as plants with pesticidal properties. Many scientific refererKes indicate that the various parts of tNs plant have pesticidal property. The extracts of its different parts viz.. Immature fruits, seeds and leaves have been scientifically found to have insecticidal property. The aqueous extracts of

different parts of custard apple were tried in a crude form in the control of a number of pests that commonly aflfects diflferent agrlcuituraj crops. It is well known that custard apple is used as a common fruits in tropical countries Including mid an southem parts of India. There are a number of references and literature available to prove that it has pesticidal property.
There are a number of nsferences and literatures available. (Schmutterer et Ai 19eO;Schmutt5erer amd Ascher, 1984, 1987; Jacobson 1966^1988; Randhawa and Parmar 1993) Neem seed is a storehouse of over 100 tetrenortritserpenoids and diverse isoprenoids (Devakumar and Sukh Dev 1993) with Insectlcldal pno|3erty.
The most important and biologically proven active corstituent of neem seeds is the Azadirachtin. Several isomers of azadirachtin have been identified and out of the isomers the most potent and active component is the Azadirachtin A
There are a number of articles reporting the insect antifeedant and insect growth inhibitory properties of azadirachtin for a variety of insect pests (Butterworth J.H. and Morgan, ED., J.Chem.Soc.Chem. Cummuns. 23 1968; Leushner, K, IMaturwissenschaften, 59,217,1972; Ruscoe,C.N.E, Nature, lond., 236,466,1972 Schmutterer, H and Rembold, H.Z, Angew. Ent 2,179-188, 1980; Wartherv J.D.Jr., ARMNE-4 USOAr SEA, Agricuiturat review and maiuiais, 1979; Kubo, I and klocke, J.A., Agricultural and Biological Chemistry, 46,1951, 1982; Champagne, D.E, Kbul, O., Isman, M,B., Scudder, G.E and Towers, GH.N., phytochemlstry, 31, 377,1992).
There are a number of articles contributing insecticidat property to the extracts from seeds, immature fruits and leaves of Annona Sqi.«nnosa and Annona Reticulata, The constituents of custard apple extracts from Immature fruits, mature seeds and leaves have also been reported to be safe and have no mammalian toxicity.

Azadlrachtin has also been reported to be Non-Mutagenic (jacooson^ivi., proceedings of the first International Neem conference, Rottach Egem, 33, 1980; in natural pesticides ftiom the neem tree, Azadirachta indica A.Juss, Schmutberer, H., Ascher, K.R.S., German agency for technical cooperatiorv Eshbon\ Qemnan, 1981) and It appears to have no apparent mammalian toxlcity( Nakashini, K, Recent advances in phytochemistry,5,283, 1975 ; Morgan, ED., Proceedings of the Rrst international neem conference, Rottach Egern, 43, 1980; in Natural pesticides from neem tree, Azadirachta indica A.Juss, Schmutfcerer, H., Ascher, KR.S., Rembold, H., Eds., German Agency for Technical cooperation, Eschbom, Germany 1981).
It has also been reported that neem products with or without azadlrachtin has shown very good control over 300 Insect pests and Infections by nematodes viruses and some pathogenic fungi (Devakun^r .C, and Parmar. B,S., 1993). The other timonoids or the bitter principle such as Azadlrachtin, Salanin, Meliarttrlol, deacetylsatanin, and nimbtn contained In the neem oil are also responsible for the pesticldal activity of neem oil (Vimal O.P and Naphade, K.T., 1980 Jour, Sole. Ind. Res. Vol. 39, P 197-211)
Though Individually neem and custard apple have been reported to have varied Insectlcldal properties so far no attempts have been made to understand the efRcacy of the combination, viz., neem oil with custard apple extracts In the production of a safe and eco friendly pestjclde towards the conljol of agricultural pests.
Plant based pesticides are normally preferred due to Its shorter persistence in the atnrKisphere, biodegradable, non-toxiclty to the ecosystem, atmosphere, human beings, and beneficial microorganism, harmless to birds, fish, and low toxicity to flora and fauna. Neem oil, extracted finm neem seeds of the neem tree, is known for Its high pesticldal value. Due to Its low coverage, low evaporation capacity from

the leaves^ and as it sticks to the surface of the leaves it stays in the leaves for a long time and turns toxic to the plants^ hence neem oil spray per say is neither efHcient nor preferable to neem oil based EC. formulation with Annona sps. Additives.
The pesticides produced with combination of neem and custard apple extracts 3S ref^msd above has t^en found to be non-toxic, aco-friendiy and efficient as p^ control agent with increased bio-efficacy with synergicism with r«em and without any hazardous effects to mammals and non polluting the atmosphere.
i-lence from ti^ above studies, It is obvious that a2!adirachtin is considered to be a promising environmentally compatible, non-toxic Insect pest control molecule for plant protsctioa This molecule in Its pure form has not conne to commercial use because it is expensive to isolate it In a pure form fix>m the ne&n seed/kernel extract and It is a very complex molecule for an economical chemical synthesis. Azadlrachtin has also been found to degrade rapidly due to environmental factors such as UV radiation in sunlight, heat, air moisture, acidity and enzymes present In foliar surfaces (Sundaram, K M. S and Curry, J., Journal of Pesticide Science, 41, 129, 1994).
Ttiese negativities of the Azadirachtin wNch may express itself under unfavourable agro-climatic condition, is neutralized by this formulation. The Bio-efUcacy aspects is further ei^ianced through the addition of the extracts of custard apple obtained fr-om immature fruits, mature seeds and teaves of the Annona Squnxisa and Annona Reticulata plants which extract has synergicism with other linrx3noids of neem and does not in any way neither fundamentally alter the toxicological properties of the ncwim preparation not its eco-friendly nature.

A scan through the literature indicates ra such prior art There are so much idea as expressed to produce a combination pesticide with [email protected] and ei^Eird dpp]% seem to have occurred in the past even through individual plants like custard apple ones we studied extensively for its pesticidal properties.
In the prior art^ Butter worth and i^rgan (Butter worth, J. H. arxi Morgan, E D., J. Insect Physiol., 17, 969, 1971) prepared azadirachtin from neem seeds by extraction with ethanol, partitioning of the concentrate of ethanot extract between methanol and light petroleum, chromatography of partition product, fham methanol phase leading to azadirachtin containing fractions, partitioning again by preparative layer chromatography of azadirachtin containing fractions resulting in azadirachtin (1.5 g.). The product obtained in this process (76 g.) would be economical to obtailn but it would be viscous and oily due to ttie presence of water soluble compounds and therefore, it is not suitable for preparing good formulations,- and the steps Involved in the process are too many and makes it cumbersome and not simple for commercial application in large scale and therefore requires further simplification in processing by less expensive less cumt>ersome techniques for the preparation of a ready to use product enriched in azadirachtin. (Moreover the solvent used is only ettianol and it has been reported by many that 100 % extraction of the amdirachtin from tt« neem seed Is not possible and a combination of solvents is always recommended for better extraction of azadirachtin from the neem seeds)
In the prior art; Uebel, Warthen, Jr. and Jacobson (Uebel, E C, Waitheii, if. J. D. and Jacobson, M, J. Uq. Chromatogr., 2, 878, l^f) hiVe liOlfSed azadirachtin from neem seeds/kernels (4a 2 kg.) Invotving grinding neem seeds/k»mels in hexane in a Waring blender, filtration of the homog^naeg W glv« a residue marc, soxhiet extraction of powdered marc so obtained with acetone for 24 h, washing of acetone extract with hexane, watjer, and hexane, «nd trTeatmentof washed acetone extract with 70/30, 75/25, methanot/water, treatment of 70/30, 75/25 nrwthanol/water soluble parts with 75/25, diethyl ether/acetone to give



Neemgold containing 300 ppm azadirachtin and Neemazal F but these preparation are ^ndamentally diffierent from the expressed in tNs invention.
The procedure consists of the following stieps; (1), ground fwaom seed
Icemeis were first extracted in a soxhiet with petroleum ether to remove fotty matter; (2), the extraction was continued with solvents such as MIBK and MTB or acetone or MeOH or AZT or MEK or butanoi for 10 h.; (3), the residue from AZT extract after removal of the solvent was treated with medianoi; (4), the methanol soluble portion from sbsp 3, was subjected to liquid-iiquid extraction with methanol 50% and light petroleum giving rise to AZT-VR-NR in a yield of 1-1.S% which is expected to be enriched in a2adirachtin.
In the broadest scope the invention nalates to a pnocess which netates to environmentally safe and storage stable, neem oil emulsion, which emulsion contain required amount of a2adirachtin.
In the present invention azadirachtin Is extracted fix»m neem seeds alongwith neem oil. In addition, the constituents c^ tiie neem caloa and the neem seed kernels are extracted in as is done elsewiiens in otiier inventions tiirough liquid phase of the solvent by penoolating into the gn>und bio-mass of the neem seed kernels but here, the liquid phase of the solvent is converted Into a gaseous state and these vapourised state of the solvent In a gaseous oondltton is allowed to pass through the ground bkimass where Xhet vapours condense and reconverted to a state of liquid phase again and thereby percolates efRcientty Into the dough of the biomass and in the process of condensation acts as a liquid solvent percolates freely and dissolves the limonoids of the neem. The percolatton Initkilty In a gaseous state and subsequently into a liquid state makes the process of extraction nmre effective then the liquid state solvent extractkm procedure alor¥3 as Ntherto employed by other in such similar processes. The lipids and lipo proteins in the neem oil protect the limorKiid azadirachtin from UV degradatioa

In addition. In this invention the pesticidal potency of custard apple from the


as to have optimal limonoids content in general and Azadirachtin in particular to have the desired pestfddaj prapertles.


aiid Stored carefully. The neem oil obtained may contains Aflatoxin, alongwith ^ttyacids and terpenoids, l^enoe the oil is tested and Aflatxndn if present within limits Is neutralized or the oil Is rejected.
iiL pRePAfan^ nsEM QiL
a. Neutratisation of neem oil : The said neem oil is then suttfecDed to chemical
neutralization for the removal of iAflatoxin.
b. I'redpltation of fetty acids : The said neem oil contairring fatty adds
(saturated and unsaturated) alongwith otiier terpenoids is subjected te
precipitation. The free ^tty acids are made to precipitate in lye solutton. Tt>e
amount of iye solution added to the said neem oil for pnadpiteting the said
free fottty acid is calculated by detemnining the percenta;^ c^ said ft«e fatty
acid in the oil. This process is done to matoe the said neem oil less dense
lighter and to separate the said fatty acids without subjecting it to vtrith any
ctiemical pncxiess or using any organic solvents like hexane, petroleum ether
or benasene. The said pr«cipitetion process is r«peatBd over a number of
tjmes until all fatty acids is separated from the neem oil.
c Separation by filtration : Sui^jecting the said resutting mix comprising of said
neutral neem oil and said predpitoted fatty add to filtration process by means of a filter cloth i.e. passing tlie said oil through the filter cloth. In this prcx»ss the predpitated fatty adds, wNch are the solid portion of the mix, are removed from the liquid portion of the said mix to get a seml-pura neutral neem oil with lower density.
d. Separation by oentrifugation : The seminnire neutral neem oil writh raduoed density is then suf;)ected to centtifugation to separate the remalhlr^ fetty adds and to obtein desired neutral less dense neem r^eem oil.

e. Addition of preservatives : Such as Sodium Benaoate singly or in association with such oth»- pnesravatives to the neem oil to maintain it at the same lev jyf- PREPARING AZADIRACHTIN CONCENTRATE EXTPACT
a. The said neem cake separated through crusNng of the said seeds Is crushed
to a particle size fit enough for making soft dough, giving minin^l time for soatdng I.e particle siase being 60 mesh.
b. The said neem cake te then further gn>und from particle siae of 60 mesh into
powder. Ti^ ground f)owder is termed as Neem cake powder.
c This ne«m caloe powder is soaioed in water at the rate of 10 kgs in 20 litres of water tbrlZ hrs to yieM soal^ neem caked powder a
d. The said IMeem seeds are deccntteated and tiw kernels is separated.
e. The sakf kernel Is then soaked in ¥»ter with electrical conductivity normafly
not greater than 0.05 m mhos cm-^ and such that 10 kgs are soaked in 20 litres of water and kept overnight or till tiie kernel becomes soft; which is normally 12 to 14 hours approximately.
f. The said soft kerrt^ is grourvl in a grinder U!rtng one or more polar solvent to
yield a neem kernel dough C.
g. Neem kamel dough C and soaked neem cake powder B ar« mbead in equal
proportion and blended to form a dough D. This blend of 1:1 of neem seed

kernel dough C and neem cake powder B forming the dough D Is taken Into hot vapour percuiator system having pomus bottom and air tight lid.
h. To separate the residual oil and azadirachtin, one or more polar solvcHit vapour is passed through the dough D which condenses and perwilatws and extracts the soluble terpenoids E The purity of vapour of polar solvent that penetrates is alnx>st 100 Vtt. f=br every 10 kikis of dough mix D^ 30 litres of 99% polar solvent is used for vapour condensation.
i. To remove the excess solvent from the extract the tedinique of vaccum vapourizatkNi process is employed with e Gommon known system of vapourization at a temperature, wNch Is adjusted to suit the point of vaporization of individual polar solvent If for examnpie the polar solvent used Is ethanol It Is normally AO^ to 60^, temperature is adJustod to recover the solvent ethanol. The resuRant residue is a semi-pure torpenolds efxtract (F) of neem.
j. Concentrating the semi-pure tjerpenoids extract F with condensation and by means of cold vaccum evaporator process so as to yield extract G
k. Mixing the extract G with non-polar solvents at the tamperatune range between 30-70 degrees such as to separata the liquid phase containing the azadirachtin and other terperxiids lh>m the organte phase. The lk|uid phase atKl the organic phase together form the firvil slurry H.
I. After the solvent extraction, the residue dough miidure is futthBT subjected
to filtration for separating solid arxi liquid. The filtration may be carried out following two methodologies :

m. In the first nnethod the residue dough nnixture is subjected to centrifugation with an industrial oentrlfuge at 6000 rpm for an hour or mora and the solid and liquid piiases are separated.
n. In the second method the nasidua dough mixture is padoad in HHBT bags and subjected to pressure in a hydraulic press and the liquid portion is separated
fix>m thte solids under high pressure. Nomnally pressure employed In the hydradic press is 100 - 110 psi.
o. The sakJ iiquid phase obtained in dxher case i.e, (i) oenblfiigation at 6000
rpm or (ii) In hydraulic press will yield a new terpenoids concentrate. This slurry further processed through different solvent extraction herein above described in preceding paras to yield the thick liquid portion I.
p. The H, Either individualiy or a mbctuns thenaof so obtained will be processed
In different batclies and the resultant mixtiK^ are mixed i30 yield a tNck slirryJ.
q. Saki final portkm J containing the tarpenoids indufino aadHmcHin and
salanin as the nrvijor constituents in associatton with otihter synergistic compounds is ftvtfier conomtrated by passing the resultant thkk Rquld through a membrane filter following the process of Reverse osmosis filtration through membrane filters. In this process the solutions Is sut^cted to filtration of diffienent compounds with different containing different nnolecular weight So die compounds are separated on the basis of their molecular weight The separation is so done that compounds having molecular weight > 1000 are separated firom the compounds having molecular wc^hts
720. Therefore the solvent: portion containing ail tlie compounds having molecular weight r. Purifying the said thick solution K containing the lower molecdar weight compounds including azadlrachtin is done by passing the portion K through filters and columns and allowing it to perc»iate through the solid material and the filtrate having Azadlrachtin in varying concentration ranging upto 25% and above azadlrachtin is collected. Azadlrachtin concentrate L is the Azadlrachtin extradL obtained through mentioned diflerenl: syst3E»ns and process.
V. PREPARING BTTRACT OF CUSTARD APPLE
a. The immatune fruits of Annona Squmosa and Annona RedculaAa ana ooOeded
and washed in water containing fungicides like Dinltn> DIbromo propane 1^3 dioi f and shade dried to remove the surface moisture, but not allowing the fruits to whither.
b. The seeds of custard apple (Annona Squmosa and Annona Redculata) are
collected by crushing matured fruits in water through wooden agiCBtors
without injuring die seeds. The pulp is separated from the seeds through
v^ater washings, filtrBtlon, methodologies and the seeds are ooliected and sun
dried. After sun drying, the seeds are cleaned by winnowing. The seeds are
drifxi. Natural (eaves of custard a(^[>(e are coifected and sun dried and
puh/erized.
c. The immature fruits, the seeds and (eaves are mixed and crushed. The
crushing is done using power driven wooden crushers (rotary crushers) or
grinding stone. The proportion is 1:1 of inwnatwie fruits and seeds by weight
and the crushed leaf powder are added to desired {avei wMch is standardtaed
by eyes which Is done by experience.

d. Dough is obtained through crusNng. The said dough is then soaked in water
20K^s of the dough In 20 Litres of water for 3 hours. The said dough is
flirtfier ground in a grinder, if needed, using one or mona polar solvents to
make it to a semi solid portion.
e. The preparation of immature fhrits, inrHfnature seeds and the leaves is in the
ratio of 1:1:0.Z normally
f. The dough is taken into a hcA vapour penoolator where they have portxjs
botbom and ease out heat
g. To separabe the constituents from the dough one or more polar solvent
vapours are passed through the dough wNch condenses and percolates and
extracts tiie constituerrts. The vapour of polar solvent is almost 100%. Fbr
every 10 i^s. of dough mix 30 litres of 99% polar solvent is used fbr vapour
condensate.
h. The vapour condensate percolates and produces an inlijslon, whk:h is collected.
i. The dough containing the polar solvent after the solvent extnactton, the residue is further subjected to filtration fbr separating the solid and liquid, which is done in two methods.
(a) In the first method, the residue dough mixture Is sufatjectad to centrifligatton with a natural centrifuge at a speed of 6000 rpm fbr an hour and the solid and liquid phases are separatad,
(b) In the second method, the residue dough mbcture is packed In filter bags and using and hydraulk: press the lk|ukt phase is squeezed out under high pressuns. I\brmally, the pnassur« used in tfia hydrauRc press is 100 to 110 psi. When using a hydraulic press, if by chance any soikt partkJe contaminates the infusion that is a liquid phase;, it is separatBd either through « prooass of fHtnation and sedimertatlon or through centrifugation.

j. The liquid phase obtained in either case is processed through diAiansnt solvent extractions, if needed.
tc Further, the solvent extractton is put through vaccum vapoufBtk>n process wherein more than 90% of the solvent and mixtures thereof Is recovered. The slurry that is left over is called the extract of custard apple, i^ch is used in the preparation of the coml>fnation pesticide^ viz., neem oii custard api:^ preparBtion. (M)
1/7. EMUtSIFICATIQN QF NSSM QIL
a. Tne prepared neem oil A is subjected to reduction in density through a
thermic process in a specialfssed vessel and to dilution with mineral oils Ifke Anomax (liice Aromax and other mineral oil of p^noleum origin), Edioxylation of the vegetable oil.
b. Emiisificatjon of the said oil A is done with ionic and non-ionic emulsillws of
vegetable origin, s^>arately or in combination. The appropriate selectton of
emuisffier \s done to arrive at a HLB value of the emulsifier ranges between
11 to 12. The emulsifier is added to the neutrad oH proporttonately and
churned for 3 hours at 6CX)0 rpm. The time for diuming will be varied
depending upon the emuisification and it may be less or more than 3 hours.
c. Addition of custard apple extract (M) during emuteiflcationr meams adding of
ethoxylated vegetable oils containing fotty alcohois in desined proportion described heroin earlier.
d. The iA/stafaiiiaar and the stabiUanlion of eitharsyi«helkchamicai or natural
origin like Para Amino Bera»ic Add, palm sugar* and molasses, (SteriliaEsd)
are added proportionately and stirred in an emulsifier tank to acNeva the
emuisification oii (N).

VIX. DOSING a, FORHULATZOU OF FIHAL COMCOtTiJATE
The dosing of the emulsified oil (N) with azadirachdn oonoantratB extract L until desired strengths of 300 ppm of azadirachtin is achieved in ttie enudsion concentrate.
Now ttie pnscess for nnaking a neenn Oil-based enudsion conoentratie containing at least 300 ppm of azadirschtin will be described in sequence of steps as per the Invention :-
A process for nnaking a neem Oil-based emulsion concentrate contaii^ng at least 300 ppm of azadiraciitin as per the invention, comprising the steps of, in order:
a. preparing neem seeds from neem fniits,
b. preparing neem seed loemeis from the neem seeds,
c. preparing neem oil from the neem seeds,
d. pr^Mirfng an azadirachtin conoantrate extract containing at least 2KX>
ppm of azadirachtin by solvent extraction of the neem seed kernels
and thereafter purifying by reverse osnnosis,
e. reducing an oil density of the neem oil by thermic processing under
controlled conditions,
f. emulsifying the neem oil,
g. dosing the emulsified neem oil with tfie azadirachtin concentrate
extract to yield a pesticide containing at iMtst 300 ppm of
azadirachtin.
As per the inventiorv a process for making a neem Oil-based emulsion concentrate containing at least 300 ppm of azadirachtin, wherein the said process further comprises the step of adding stabilizing agents and UV screens to the

pesticide to yield the naem oil-based emulsion conoentnies coniainino at leaat 300 Dom azadirachtia
■ ■
As per the invention^ a process for nnaldng a neem Oil-based emulsion concentrate containing at kiast 300 ppm of azadirachtin, wherun the said preparing neem seeds from neem fruits comprises the st^TS of, in order:
a. depuiping of fruits to separate the seed^
b. coating the seeds with anbfungal and antibactBrial material, and
c drying the seeds in open air or at a controlled tamperatave to a predetermined moisture level.
As per the invention, a process for matdng a neem Oil-based emulsion concentrate containing at least 300 ppm of azadirachOn, wherein tlie said prepanation of neem oil from neem seeds comprises the steps of, in onder:
a. cold crushing of dry seeds in wooden crushens, and
b. adding palm sugar as a separating agent to sepaiate the neem oil
from a neem cake.
As per the invention, a process for maidng a neem Oil-based emulsion concentrate containing at least 300 ppm of azadirachtin, whensin the said preparation of neem CNI from neem seeds further comprises the steps of, in order :
il) separating ^tty acids from the neem oil by employing a lye solution to precipitate the fatty acids,
iil) separating and filtering the neem oil and the preciplteted flatty adds,
iv) separating a low density oil portion of the neem oil, and
v) adding preservatives.
As per tlie invention, a process for making a neem Oil-based enrud^on concentrate conteining at least 300 ppm of azadirachtin, wherein the said

preparation of an azadiraciKJn concentrate extract oontairring at least 300 ppm of azsdirachtin by solvent extnactjon of the n&em seed kemeis and thefeaftsr puriiying by reverse osmosis, comprises the steps of, in order :
j) crusNng a neem cai obtained neem caice particles to a powder, ii) separately soaking the prepared seeds and the powder in water for 12
to 14 hours or until the seed low'nels are softened. Hi) grinding the soaked seeds and soaked powder to obtain a dough using
a polar solvent^ iv) passing polar solvent vapours through the dough to condense and to
percolate soluble terpenoids to obtain a first condensate, v) vaporizing excess polar solvent fhom the first condensate, vi) condensing tlie first condensate by cold vacuum evaporator procera, vii) filtrating the first condensate at 30-70*C, vlll) extracting a Ikiuki portion containing azadirachtin and other
triberpenoids from an organk potk>n of the first condensate using a
non-polar solvent; Ix) separating solid and lk:|uid piiases of tiie dough by mechanteal nneans, x) concentrating the liquid phase of ix) wliich oontains the terpenoids to
a slurry, xi) extracting a second condensate from a second biomass residue of tiie
dough by using individual and/or a combination of solvents in an
effective amount thereof, xit) concentrating the second condensate to obtain an extract with a
concentration of terpenokls by solvent extraction, and wiierein the
extract fiattier conrq;>rises a concentration of Azadirachtin from 10 to
25%, xiii) Golfecting and nnixing of the first and second condensates to yieU a
semi- pure slurry,

xiv) filtering the semi-pure slurry by a reverse osmosis process to separate compounds with a nrwiecular weight xv) purifying the filtered slurry to obtain a filtrate having an Azadlrachtin content in the extract of at least 20%;
fis per the invention, a process for maldng a neem Ot-based emulsion concentrate containing at leaat 300 ppm of aaadirachtfn, wherein the said reducing an oil den^ty of the neem oil by themiic processing under controlled conditions comprises the steps of, In order:
a. reducing the oil density of the neem oil by a oonCroUed thermic treatment of
the oil vvhensin the thermic process is carried out in specially designed
b. diluting the neem oil by adding rr^neral diluents and/or othar vegetable oils
with lower densities tfian that of tiie neem oil,
c adding emiisiliers under constant stirring and agitation fbr 3 hours «^ 6000
rpm, d. additHI an fi»ctract from custanJ apple wNch is obtained by extracting a
crushed biomass of immature custard apple fruits, seeds and matured
leaves,
As per the invention a process fbr maldng a neem Oli>based enxilslon concentrate containing at least 300 ppm of aasadlFachtiiv wherein the said emulsifying the neem oil comprises stirring neem oil by agitation arvl dosing thereof with the Azadlrachtin extract until the neem oil contains a concentrations of at least 300 ppm Azadlrachtin,
As per the invention, a process fbr making a neem Oil>based emulsion concentrate containing at leaat 300 ppm of azadlrachtin, wherein dosing the

emulsified neem oil with the azadlrachtin concentrate extract to yield a pesticide containing at least 300 ppm of azadiracbtin comprises of stapS; in order :
a. adding stabilizing agents from natural and/or synthetic sounoes either alone
or in combination to the emulsified neem oil, and
b. adding LA/ Screening agents to the emulsified neem oil to ofatein the neem oil
based emission concentrate.
As per the Inventfon, a process for making a neem Oihbased emulsion concentrate coffining at least 300 ppm c^ asadirachtirv wherein the antifungal material used in OMiting neem seeds is chosen from the gn>up consisting of Trfchoderma Vfrfde^ Trtehoderma Hamatum, Trfchoderma Harzfanum and rrtfxtune thereof are in the ratio of 1:1:1.
fis per the invention, a pro«ss for making a neem Oii-based envision concentrate containing at least 300 ppm of azadlrachtin, wiier^n the chemicals used in the said neutralization process are chosen fhim the group consisting of Sodiivn chloride CaMum Chkwld^ Silica dioodde^ Aluminum hydroxide and mixtures thereof The said neutralization process as per the invention erT^?k>y5 1% sodium chloride, 2% calcium cNoride, 66% silica dioxide and 12% aluminum hydroxide.
As per the invention, a process for making a neem OU-based emulsion concentrate containing at least 300 ppm of azadlrachtin, wherein at least one of the chemicals are in a mixture comprising 98% Sodium Chloride, 96% Cakium CNoride, 90% Silica dioxide, and 95% Aluminium hydroxide.
As per the inventton, a process for making a neem Oii>bBsed emulston concentrate containing at least 300 ppm of azadlrachtin, wherein the said lye solution is a 48% caustic soda solution and a quantum of NaOH or the lye solution

Is added in an effective amount thereof. The said lye solution comprises 30 litres of fis per the invention, a pncxaess fbr matdng a neem Oil-based emulsion concentrate containing at least 300 ppm of asBdirechtinr wherein the said polar solvent is chosen fhom the grnup consisting of methanol, ethanol, melhyt ethyl ketone, methyl-tertiary-butyl-ether and mixture thereof, wherein the polar scrfvent is employed fbr grinding in preparing the dough and wherein the polar soivcwit vapours are employed in the percolation process.
As per the invention, a process fbr making a neem Oil-based emulston concentrate containing at leaet 300 ppm of azsdirachdn, wherein tfie said polar solvent vapours are In a ratio of 1:1:1:1 consisting of methanol; Ethanol; Methyl ethyl ketone; methyl-tertlary-butyi-ether in the ratio 1:1:1:1 when used in the percolation process.
As per the invention, a process fbr making a neem Oil-based emulsion concentrate containing at (eaat 300 ppm of azadlrachtinf wherein the said non-polar solvent is chosen fixNm the group ccH^dsting of hexane, p^roieum ether, and dichloro methane and mixtures thereof.
As per the inventkm, a process tor making a neem Oli-based emulston concentrate contairang at least 300 ppm of azadlrachdiv wheran the said mechanical means comprise cenbifuging or separating by a hydraulic squeezing process tiie residual dough mixture into the liquid and the soik) phases.
As per the invention, a process fbr making a neem Oil-based emulston GonoentratB containing at least 300 ppm of azadirachtin, wherein the said residual dough mixture is centriAjged at 6000 rpm fbr at least one hour.

As per the invention, a process for making a neem Oil-based emulsion concentrate containing «t least 300 ppm of azadirBchUn, whanain the said hydraulic squeezing pnacess comprises applying a pressura of 100 - 110 psi to separate the liquid portion from the remaining neem seed l«emel biomass which contains both liquid and solid portions.
It is tD be mentioned that various changes and modification wMch will be apparent to those skilled in the art^ without departing fhom the spirit and scope of present invention without diminishing the contertts of invention, are also to be covered in this inventton.


V
WE CLAIM
1. A process for making a neem Oil-based emulsion concentrate containino at least 300 ppm of azadirachting comprising die steps of, in order:


4. The process of claim 1, wherein steps c) comprises the steps of, in onder


viil) extracting a liquid portion containing azadiractin and other


iv. adding an extract fram custard apple which is obtained by extracting a


14. The prcxess of claim 5, wherein the lye solution is a caustic soda solution.
15 The process of dtaim S, wherein the lye solution is a 48^ caustic soda
solution and a quantum of I^OH or the lye solution is added in an effective amotffit thereof.
16 The process of claim 6, wherein the polar solvent is chosen fhoMn the gnxip
consisting of methanol, ethanol, methyl ethyl ketone , m^ihyl-tertfary-butyl-
ether and mixture thereof, wherein the polar solvent is employed for grinding
in preparing the dough.
17. The iMTKess cf daim 6> wherein the polar solvent vapours are chosen from the group consisting of methanol, ethanoi. M^:hyl ethyl ketone, methyi-trattary-butyl -ether or mixture thereof, wherein tdie polar solvent vapours are employed in the percolation process.
la The pnxess of daim 6, wherein tlie polar solvent vapours are In a nHio of 1:1:1:1 consisting of methanol; Ethanoi; Mdhyt i^iiyi ketone; methyt-tertiary-butyi-ether in the ratio 1:1:1:1 when used in the percolation process.
19. The process of daim 6, wfierein the non-polar solvent Is chosen from the group consisting cf hexane, petroleum either, and dIcNoro methane and
mixtures thereof.
2a The process of daim S, wherein the mechanical means comprise centrlAiging or separating by a hydraulic squeezing process the residual dough mixture into the liquid and the solid phases.

21. The process of claim 20, wherein the residual dough mixture Is cenCrtfuQed at 6000 rpm for at least one hour.
2Z The process of claim 20, wherein the hydraulic squeezing process corr^nlses applying a pnsssuns of 100 - 110 psf to separate the tfqiM portion from the nemabning neem seed kernel biomass which oontabv both liquid and soMd
portions.
23. The pnxess of claim 13, wherein the neutralization pnxess employs 1%
sodium chloride, 2% calcium chloride, 66% silica dioxide and 12% aluminum
hydroxide.
24. The pixicess of claim 14, wherein the lye solution comprises 30 litres of 40%
NBOH soludon for 1000 litres of neem oM having 10% of free ftrt

Documents:

116-mas-2002 abstract duplicate.pdf

116-mas-2002 abstract.pdf

116-mas-2002 claims duplicate.pdf

116-mas-2002 claims.pdf

116-mas-2002 correspondence others.pdf

116-mas-2002 correspondence po.pdf

116-mas-2002 description (complete).pdf

116-mas-2002 description (provisional) duplicate.pdf

116-mas-2002 form-1.pdf

116-mas-2002 form-19.pdf

116-mas-2002 form-26.pdf

116-mas-2002 form-3.pdf

116-mas-2002 form-5.pdf


Patent Number 198109
Indian Patent Application Number 116/MAS/2002
PG Journal Number 08/2007
Publication Date 23-Feb-2007
Grant Date 03-Feb-2006
Date of Filing 12-Feb-2002
Name of Patentee T. STANES & COMPANY LIMITED
Applicant Address 8/23-24, RACE COURSE ROAD, COIMBATORE 641 018
Inventors:
# Inventor's Name Inventor's Address
1 SANTHANAM RAMARETHINAM EXECUTIVE DIRECTOR/PROJECTS 8/23-24,RACE COURSE ROAD, COIMBATORE-641 018
PCT International Classification Number A01N65/00
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA