Title of Invention	"AN IMPROVED PROCESS FOR THE PREPARATION OF HIGH YIELDING TECHNICAL DRY AMORPHOUS, OIL-FREE POWDER CONTAINING 10 TO 17% AZADIRACHTIN"
Abstract	An improved process for the preparation of high yielding technical dry amorphous oil-free powder containing 10-17% azadiractin from Azadirachta indica seed kernel, which comprises grinding seed kernels of "Azadirachta indica to fine powder, defatting the obtained powder using non-polar solvent, extracting the marc thus obtained by polar solvent, filtering and concentrating the filtrate by conventional methods, incorporating of molecular sieves of the kind as defined herein to the concentrate filtrate, cooling at a temperature ranging from 0° to 10° C for a period of 8-12 hours, precipitating the said filtrate by adding non-polar solvent, filtering and drying the precipitate to obtain powder with yield ranging between 1.1 -1.7% by weight of seed kernels, the process is characterized in using simple steps and molecular sieves of mesh size ranging from 4A° to 5A°. Reference has been made to patent application no. AU-9215646,661482, DE-4109473, 59206503 Es, US - 5695763, EP - 579624.

Title of Invention

"AN IMPROVED PROCESS FOR THE PREPARATION OF HIGH YIELDING TECHNICAL DRY AMORPHOUS, OIL-FREE POWDER CONTAINING 10 TO 17% AZADIRACHTIN"

Abstract

An improved process for the preparation of high yielding technical dry amorphous oil-free powder containing 10-17% azadiractin from Azadirachta indica seed kernel, which comprises grinding seed kernels of "Azadirachta indica to fine powder, defatting the obtained powder using non-polar solvent, extracting the marc thus obtained by polar solvent, filtering and concentrating the filtrate by conventional methods, incorporating of molecular sieves of the kind as defined herein to the concentrate filtrate, cooling at a temperature ranging from 0° to 10° C for a period of 8-12 hours, precipitating the said filtrate by adding non-polar solvent, filtering and drying the precipitate to obtain powder with yield ranging between 1.1 -1.7% by weight of seed kernels, the process is characterized in using simple steps and molecular sieves of mesh size ranging from 4A° to 5A°. Reference has been made to patent application no. AU-9215646,661482, DE-4109473, 59206503 Es, US - 5695763, EP - 579624.

Full Text	The present invention relates to an improved Process for the preparation of high yielding technical dry amorphous powder containing 10 to 17% azadirachtin. More particularly this invention relate to the process for the preparation of technical dry amorphous powder free from oil in high yield. Several extraction methods have been adapted by the scientists in India and aboard to obtain azadirachtin in pure form and crude powder form. The following are the few important methods reported in the literature. 1. Nakanishi (1987) J. Nat. Products, 50 : 241-244; wherein 2 kg seed kernels were defatted in hexane (oil 1050 ml was discarded), defatted marc was extracted in 95% ethanol. 185 g ethanol extract was partitioned with petroleum ether (oil 47 g discarded) and 95% methanol water. 138 g polar extracts was partitioned with water and ethyl acetate (water soluble proteins, subjected to vacuum liquid chromatography. 13 g azadirachtin enriched fraction was crystallised from carbon tetrachloride. 8.5 g crude azadirachtin was subjected to flash chromatography to obtain 5 g 98% pure azadirachtin. The drawbacks are: 1. The process involves six steps, making it very tedious and time consuming 2. The procedure requires the use of five solvents. Some of the solvents are costly which enhances the total cost of the product. 3. Percentage of azadirachtin in crude powder is not reported. 2. Zanno et al. (1975), J. Am. Chem. Soc. 97 : (1975-1977); wherein Defatted marc was extracted with 95% ethanol, the extract was partitioned first with ethyl acetate/water and then methanol/water. Resultant polar mixture was twice chromatographed over silica gel eluting first with ether-acetone mixture followed by chloroform-ethyl acetate mixture. Crystallisation of crude powder and percentage of azadirachtin in crude is not reported. Preparative TLC was done to obtain pure azadirachtin (98%). The drawbacks are: 1. The procedure involves use of seven solvents which makes the procedure expensive. 2. The process involves eight steps making it very tedious. 3. The process include the use of silica gel chromatography making the process tedious. 3. Butterworth and Morgan (1968), J.Chem. Soc. chem Comm. : 23-24; wherein 1 kg seeds were defatted with hexane, extracted with 95% ethanol and the extract was then partitioned between ethyl acetate and methanol: benzene. Residue obtain on the removal of ethyl acetate was partitioned between petroleum ether and 90% methanol. Petroleum layer was discarded and residue obtain on removal of methanol was subjected to silica gel chromatography followed by preparative TLC to obtain pure azadirachtin (98%). Crystallisation of crude powder and concentration of azadirachtin is not reported. The drawbacks are: 1. Two time partitioning was done followed by silica gel chromatography makes the procedure tedious. 2. The process involves the use of five solvent which makes method costly. 3. The process involves seven steps, makes the process lengthy. 4. Siddiqui et al. (1992), J. Nat. Products 55 : 303-310; wherein Fresh unruptured, ripe fruits of neem (10 kg) were repeatedly percolated with ethanol at room temperature. Removal of solvent from combined extract under reduced pressure gave a dark green residue, which was partitioned between ethyl acetate and water. The ethyl acetate layer was repeatedly extracted with 1% sodium hydroxide to separate the acidic impurities from the neutral constituents. The residue obtained, on removal of solvent from ethyl acetate layer was partitioned between 50% ethanol and petroleum ether (1:1). The upper layer was treated wit* charcoal and freed of solvent in vacuum. The residue thus obtained was divided into petroleum ether soluble and petroleum ether insoluble fraction. Petroleum ether insoluble fraction was subjected to preparative TLC to obtain azadirachtin along with naheedin. Crystallisation of crude mass was not attempted. The drawbacks are: 1. Six solvents have been used in the process making it costly. 2. The process involves eight steps which makes it lengthy and tedious. 3. Use of charcoal makes it cumbersome. 4. Crystallization of crude was not attempted. 5. Rengasamy et al. (1993), World Neem Conference, India, 1993; wherein Kernel powder (200 g) was soxhlet-extracted successively with hexane and ethanol by a modified method of Schroeder and Nakanishi (1987). Hexane extract on concentration yielded an oil. The ethanol layer on concentration yield a semi solid. It was dissolved in ethanol and analysed for azadirachtin content by HPLC. The ethanol concentrate was dissolved in ethanol and partitioned with hexane (6x50ml) to remove traces of oil. The ethanol fraction was suspended in water and extracted with ethyl acetate. The ethyl acetate fraction was dried over sodium sulphate and concentrated. The concentrated mass was subjected to silica gel chromatography using 150 ml ethyl acetate for elution. The drawbacks are: 1. The process involves the use of four solvents making the method costly. 2. There are five steps in the method which makes it lengthy tedious. 3. Crystallization of crude was not attempted. AU-9215646 AU-661482 DE^109473 DE-59206503Es US-5695763 EP-579624 Wo-9216109 JP-65Q596 ES-2088134 1. Extraction with water and centrifuge was used to separate the solid to make the solid free from azadirachtin. 2. Aqueous extract was added to an organic solvent which is not fully miscible with water and which has a great solubility for aza. than o water separation of aza. containing solvent. 3. Concentration of the solvent containing aza. and 1-20 times the vol. of liquid hydrocarbon. 4. Aza. containing ppt. allowed to settle down and is separated and surf actant added turbidity. Concentration of aza. in ppt was not reported. drawback: Water has been used for the extraction which has disadvantage as it effect the cone, in the final product and many steps are involves AU-9670281 CA-2188110 JP-9255684 1. Disintegration of neem seed kernel into powder. 2. Extraction with ethanol or methanol (both optionally aqueous). 3. Concentrating the extract and stirring the concentrate with 60-80 petroleum ether or hexane. 4. Stirring the denser phase with a water immiscible organic solvent and water. 5. Concentrating the organic phase, 60-80 petroleum ether or hexane was added. 6. Filtering and drying the ppt. gave a powder containing 10-19% aza. drawback: Multiple steps are in the process. * WO-9S02962 AU-9474031 US-1541 Preparation of a concentrate (A) containing azadirachtin comprises mixing a (I) containing plant with a non-polar solvent in which (1) has low solubility to produce a (l)-contg. ppte. drawback: Starting material is neem oil EP-617119 AU-9466335 JP-6298614 US-53977I EP-617119 US-5503837 1. Solvent are chosen to be miscible to form a homogeneous mixture 2. Non-polar solvent e.g. Pentane, heptane, nonane, decane, petroleum ether or mix of the above. 3. Polar solvent is methanol ethanol, n-or, i-proh, n-s or th butanol, bezyl alcohol, or their mix. Most pref. combination is hexane with ethanol. 4 Solvent limits are 10-90% of ether: most pref. 60% hydrocarbon and 40% polar solvent. 5. The extract was stripped of solvents to provide a neem extract mix. contg.aza and oil, addition of low polarity solvent ppt. the aza. asa solid with aza. content as in advantage. The neem oil recovered by evaporation of the filtrate.The extract was chilled to split the phases most preferred to below 5c These are sepd and each evapd to yield their prods, and oil. drawback: Multiple steps are involved in this process and process is tedious. Us-5397571 1. Single counter extn. of neem seeds with miscible co-solvent mix. of a polar solvent and non polar aliphatic hydrocarbon, solvent. 2. Preeip. of Ad. by low polarity solvent of solid more than 10 (more than 20%) wt. azadirachtin prefer solvent (pentane, decane, chloropentane, chloroform) and 90-10% wt polar solvent {methanol, butanol, benzyl alcohol etc. drawback: Precipitation by stirring and Yield not reported. Technical azadirachtin (10-25%) contains azadirachtin along with other meliacin compounds such as nimbin, salanin, nimbolide, nimbinene, deacetylsalianin, deacetylnimbine, epinimbine, deacetylnimbin. More than 50 neem base pesticide formulations containing azadirachtin have been developed in India and aboard (parmar and Ketkar, 1996) but only the following formulations have been registered under the Insecticides Act (1968) for the control of insects, pests of arhar, gram, cotton, brinjal, cabbage, jute, potato, tobacco etc. Besides, a number of entrepreneurs are coming up for registration of their neem based products for the control of pests. 1. Neem gold : Kernel extract (Azadirachtin 1500 ppm) 2. Azadirachttn EC : Oil based 300 ppm azadirachtin 3. Godrej Achook: Oil base 300 ppm azadirachtin 4. Margocide CK 200% EC : Azadirachtin and its 15 derivatives, kernel base, containing azadirachtin 1500 ppm 5. Margocide OK 80% EC : Oil based, contains azadirachtin 300 ppm 6. RD-9 Replin : Oil base, containing 300 ppm azadirachtin. 7. Rakshak: Extract base, containing 1500 ppm azadirachtin There is a considerable emphasis on the development of neem pesticides base on "azadirachtin" which is the most widely evaluated neem compound. However, it may not be advisable to develop a product based on pure azadirachtin atone because of the following reasons : 1. There is hardly any evidence of the antagonistic effect of one neem compound on the other. Literature survey reveals that under many situations, crude or semi-purified extracts of seeds give similar results as compared to pure azadirachtin and in fact, sometime, crude extracts; perform better than azadirachtin alone base preparations. 2. There is always a danger of development of resistance in insects against; a product containing a single active molecule. 3. Greater technical skills are required to produce very high concentrations of azadirachtin in technical grade material which also leads to marked escalation in cost. The main object of the present invention is to provide An improved method for the preparation of high yieding technical dry amorphous powder containing 10-17 % azadirachtin. Which obviates the drawback as detailed above. Another object of the present invention is to reduce the multiple steps. Still another object of the present invention is not to use water which effects the cone, at the end product Yet another object of the present invention is to prepare dry powder in high yield wNch contains aza. in more than 10% cone, which has a long shelf life. Yet another object of the present invention is to eliminate the use of column Accordingly the present invention provides an improved process for the preparation of high yielding technical dry amorphous oil-free powder containing 10-17% azadiractin from Azadirachta indica seed kernel , which comprises grinding seed kernels of Azadirachta indica to fine powder , defatting the obtained powder using non-polar solvent, extracting the marc thus obtained by polar solvent, filtering and concentrating the filtrate by conventional methods , incorporating of molecular sieves of the kind as defined above to the concentrate filtrate , cooling at a temperature ranging from 0° to 10 ° C for a period of 8 -12 hours , precipitating the said filtrate by adding non-polar solvent, filtering and drying the precipitate to obtain powder with yield ranging between 1.1-1.7 % by weight of seed karnel , the process is characterized in using simple steps and molecular sieves of mesh size ranging from 4 A° to 5A°, to the said filtrate to obtain the precipitate followed by continuous stirring filtering and drying of powder with yield ranging between 1.1-1.7% by weight. In an embodiment of the present invention wherein the defatting of the powder neem kernels is effected by using non-polar solvent such as petroleum ether, hexane, pentane, benzene and mixture thereof. In anotfier embodiment of the present invention wherein the extraction of the defatted marc is effected with polar solvent such as ethanol, methanoi, chloroform, ethylacetate, acetonitrile, acetone and mixture thereof. In yet another embodiment of the present invention wherein the filtration and concentration is effected by the know methods like distillation, heating on a water bath, or rotary evaporator. In yet another embodiment of the present invention is where in the incorporation of molecular sieves of mesh size ranging from 4Ao to 5Ao is effected by keeping it at temperature 0 to 10 for 8 to 12 hours. In still another embodiment of the present invention is wherein the precipitation is effected by non-polar solvent such as petroleum ether, hexane, pentane, benzene and mixture thereof. In stilt another embodiment of the present invention is wherein the yield of powder is ringing from 1.1 to 1.7 %. Details of the process 500 gm seeds kernels were ground to fine powder and defatted by simple washing with hexane or by soxhlet till the complete removal of oil. Defatted mark was taken in container fitted with mechanical stirrer and chloroform was added such that 2" layer of the solvent remained above the solid mass (approx. 1 L or more solvent was required for the first extracting). Whole mass was stirred for 7-8 hrs. The mass was allowed to settled down and filtered using bochner funnel. The extraction was repeated two more times. Extraction was done in soxhiet also which prevents loss of solvent. Soxhiet extraction was done for 7-10 hrs. The combine filtrate was concentrated in a rotary evaporator to about 300 ml and molecular sieves mesh size ranging from 4A or 5A (2-3 gm) was incorporated in the solution the stopper of the flask replaced tightly and kept for several hrs. or overnight and precipitated by 60-80 petroleum ether or hexane. About 7gm crude powder was obtain by this method. Reported yield was 2.1 gm powder from 500 gm seed kernels. If the seed kernels are of pure quality than by this methodology crude powder obtain would contain more than 10 to 17% azadirachtin. It is therefore advisable to procure the best quality of neem seeds in order to obtain good percentage of azadirachtin in dry powder. The following examples are given by way of illustration of the present invention and therefore should not be construed to limit the scope of the present invention. Example 1. 500 gm seeds kernels were ground to fine powder and defatted by simple washing with hexane or by soxhiet till the complete removal of oil. Defatted mark was taken in container fitted with mechanical stirrer and ethyte acetate was added such that 2 layer of the solvent remained above the solid mats (approx. 1 L or more solvent was required for the first extracting). Whole mass was stirred for 7-8 hrs. The mass was allowed to settled down and filtered using bochner funnel. The extraction was repeated two more times. Extraction was done in soxhiet also which prevents loss of solvent. Soxhiet extraction was done for 7-10 hrs. The combine filtrate was concentrated in a rotary evaporator to about 300 ml and molecular sieves mesh size ranging from 4Ao or 5A> was incorporated in the solution the stopper of the flask replaced tightly and kept for several hrs. or overnight and precipitated by 60-80 petroleum eflier or hexane. Crude powder (0.4 % yield) was obtain by this method. Example 2. 500 gm seeds kernels were ground to fine powder and defatted by simple washing with hexane or by soxhiet till the complete removal of oil. Defatted mark was taken in container fitted with mechanical stirrer and acetonitrite was added such that 2 layer of the solvent remained above the solid mass (approx. 1 L or more solvent was required for the first extracting). Whole mass was stirred for 7-8 hrs. The mass was allowed to settled down and filtered using bochner funnel. The extraction was repeated two more times. Extraction was done in; soxhiet also which prevents loss of solvent. Soxhiet extraction was done for 7-10 hrs. The combine filtrate was concentrated in a rotary evaporator to about' 300 ml and molecular sieves mesh size ringing from 4Ao or 5Ao (2-3 gm) was? incorporated In the solution the stopper of the flask replaced tightly and kept for several hrs. or overnight and precipitated by 60-80 petroleum ether or hexane.Sticky mass was obtained. Which was triturated with hexane to get solid (0.41 % yield). Novelty & Advantage : The novelty of the present invention over the priorart method reducing the multiple steps. Water has not been used as it affect the concentration and yield of the end product (dry powder). Molecular sieves removes moisture to give instant precipitation. Two solvent system has been used which are recycled with in the process. This process eliminate the use of column. This process give a crude powder in dry amorphous form in high yield (1.7 % yield by weight). Reported yield was 0.4%. Dry powder is completely free from oil and can be used directly in formulation as insecticides. Preparation of powder involves less steps column has not been used. Two solvents are used therefore it is cost effective powder in high yield and more stable (shelf life six months) than pure azadirechtin. We claim: 1. An improved process for the preparation of high yielding technical dry amorphous oil-free powder containing 10-17% azadiractin from Azadirachta indica seed kernel , which comprises grinding seed kernels of Azadirachta indica to fine powder, defatting the obtained powder using non-polar solvent, extracting the marc thus obtained by polar solvent, filtering and concentrating the filtrate by conventional methods , incorporating of molecular sieves of the kind as defined herein to the concentrate filtrate , cooling at a temperature ranging from 0° to 10 ° C for a period of 8 -12 hours , precipitating the said filtrate by adding non-polar solvent, filtering and drying the precipitate to obtain powder with yield ranging between 1.1 -1.7 % by weight of seed kernels , the process is characterized in using simple steps and molecular sieves of mesh size ranging from 4 A° to 5A° , 2. An improved process as claimed in claim 1 wherein the non-polar solvent used is selected from petroleum ether , hexane , pentane , benzene and mixture thereof. 3. An improved process as claimed in claim 1 and 2 wherein the polar solvent used is selected from ethanol, methanol , chloroform , ethylacetate, acetonitrile , acetone and mixture thereof. 4. An improved process as claimed in claim 1 to 3 wherein the conventional methods used for filtration and concentration are methods such as distillation, heating on water bath or rotary evaporation. 5. An improved process for the preparation of high yielding technical dry amorphous oil-free powder containing 10-17% azadiractin from Azadirachta indica seed kernel substantially as here in described with reference to the examples.

Full Text

The present invention relates to an improved Process for the preparation of high yielding technical dry amorphous powder containing 10 to 17% azadirachtin.
More particularly this invention relate to the process for the preparation of technical dry amorphous powder free from oil in high yield.
Several extraction methods have been adapted by the scientists in India and aboard to obtain azadirachtin in pure form and crude powder form. The following are the few important methods reported in the literature.
1. Nakanishi (1987) J. Nat. Products, 50 : 241-244; wherein 2 kg seed
kernels were defatted in hexane (oil 1050 ml was discarded), defatted marc was
extracted in 95% ethanol. 185 g ethanol extract was partitioned with petroleum
ether (oil 47 g discarded) and 95% methanol water. 138 g polar extracts was
partitioned with water and ethyl acetate (water soluble proteins, subjected to
vacuum liquid chromatography. 13 g azadirachtin enriched fraction was
crystallised from carbon tetrachloride. 8.5 g crude azadirachtin was subjected to
flash chromatography to obtain 5 g 98% pure azadirachtin.
The drawbacks are:
1. The process involves six steps, making it very tedious and time
consuming
2. The procedure requires the use of five solvents. Some of the
solvents are costly which enhances the total cost of the product.
3. Percentage of azadirachtin in crude powder is not reported.
2. Zanno et al. (1975), J. Am. Chem. Soc. 97 : (1975-1977); wherein
Defatted marc was extracted with 95% ethanol, the extract was partitioned first
with ethyl acetate/water and then methanol/water. Resultant polar mixture was twice chromatographed over silica gel eluting first with ether-acetone mixture followed by chloroform-ethyl acetate mixture. Crystallisation of crude powder and percentage of azadirachtin in crude is not reported. Preparative TLC was done to obtain pure azadirachtin (98%). The drawbacks are:
1. The procedure involves use of seven solvents which makes the
procedure expensive.
2. The process involves eight steps making it very tedious.
3. The process include the use of silica gel chromatography making
the process tedious.
3. Butterworth and Morgan (1968), J.Chem. Soc. chem Comm. : 23-24; wherein 1 kg seeds were defatted with hexane, extracted with 95% ethanol and the extract was then partitioned between ethyl acetate and methanol: benzene. Residue obtain on the removal of ethyl acetate was partitioned between petroleum ether and 90% methanol. Petroleum layer was discarded and residue obtain on removal of methanol was subjected to silica gel chromatography followed by preparative TLC to obtain pure azadirachtin (98%). Crystallisation of crude powder and concentration of azadirachtin is not reported. The drawbacks are:
1. Two time partitioning was done followed by silica gel
chromatography makes the procedure tedious.
2. The process involves the use of five solvent which makes method
costly.
3. The process involves seven steps, makes the process lengthy.
4. Siddiqui et al. (1992), J. Nat. Products 55 : 303-310; wherein Fresh
unruptured, ripe fruits of neem (10 kg) were repeatedly percolated with ethanol
at room temperature. Removal of solvent from combined extract under reduced
pressure gave a dark green residue, which was partitioned between ethyl
acetate and water. The ethyl acetate layer was repeatedly extracted with 1%
sodium hydroxide to separate the acidic impurities from the neutral constituents.
The residue obtained, on removal of solvent from ethyl acetate layer was
partitioned between 50% ethanol and petroleum ether (1:1). The upper layer
was treated wit* charcoal and freed of solvent in vacuum. The residue thus
obtained was divided into petroleum ether soluble and petroleum ether insoluble
fraction. Petroleum ether insoluble fraction was subjected to preparative TLC to
obtain azadirachtin along with naheedin. Crystallisation of crude mass was not
attempted.
The drawbacks are:
1. Six solvents have been used in the process making it costly.
2. The process involves eight steps which makes it lengthy and
tedious.
3. Use of charcoal makes it cumbersome.
4. Crystallization of crude was not attempted.
5. Rengasamy et al. (1993), World Neem Conference, India, 1993; wherein
Kernel powder (200 g) was soxhlet-extracted successively with hexane and
ethanol by a modified method of Schroeder and Nakanishi (1987). Hexane
extract on concentration yielded an oil. The ethanol layer on concentration yield
a semi solid. It was dissolved in ethanol and analysed for azadirachtin content by HPLC.
The ethanol concentrate was dissolved in ethanol and partitioned with hexane (6x50ml) to remove traces of oil. The ethanol fraction was suspended in water and extracted with ethyl acetate. The ethyl acetate fraction was dried over sodium sulphate and concentrated. The concentrated mass was subjected to silica gel chromatography using 150 ml ethyl acetate for elution. The drawbacks are:
1. The process involves the use of four solvents making the method
costly.
2. There are five steps in the method which makes it lengthy tedious.
3. Crystallization of crude was not attempted.
AU-9215646 AU-661482 DE^109473 DE-59206503Es US-5695763 EP-579624 Wo-9216109 JP-65Q596 ES-2088134
1. Extraction with water and centrifuge was used to separate the solid to
make the solid free from azadirachtin.
2. Aqueous extract was added to an organic solvent which is not fully
miscible with water and which has a great solubility for aza. than
o
water separation of aza. containing solvent.
3. Concentration of the solvent containing aza. and 1-20 times the vol. of
liquid hydrocarbon.
4. Aza. containing ppt. allowed to settle down and is separated and surf
actant added turbidity. Concentration of aza. in ppt was not reported.
drawback: Water has been used for the extraction which has disadvantage as it effect the cone, in the final product and many steps are involves AU-9670281 CA-2188110 JP-9255684
1. Disintegration of neem seed kernel into powder.
2. Extraction with ethanol or methanol (both optionally aqueous).
3. Concentrating the extract and stirring the concentrate with 60-80
petroleum ether or hexane.
4. Stirring the denser phase with a water immiscible organic solvent and
water.
5. Concentrating the organic phase, 60-80 petroleum ether or hexane
was added.
6. Filtering and drying the ppt. gave a powder containing 10-19% aza.
drawback: Multiple steps are in the process.
* WO-9S02962 AU-9474031 US-1541 Preparation of a concentrate (A)
containing azadirachtin comprises mixing a (I) containing plant with a non-polar solvent in which (1) has low solubility to produce a (l)-contg. ppte. drawback: Starting material is neem oil
EP-617119 AU-9466335 JP-6298614 US-53977I EP-617119 US-5503837
1. Solvent are chosen to be miscible to form a homogeneous mixture
2. Non-polar solvent e.g. Pentane, heptane, nonane, decane, petroleum
ether or mix of the above.
3. Polar solvent is methanol ethanol, n-or, i-proh, n-s or th butanol, bezyl
alcohol, or their mix. Most pref. combination is hexane with ethanol.
4 Solvent limits are 10-90% of ether: most pref. 60% hydrocarbon and
40% polar solvent.
5. The extract was stripped of solvents to provide a neem extract mix.
contg.aza and oil, addition of low polarity solvent ppt. the aza.
asa solid with aza. content as in advantage. The neem oil recovered
by evaporation of the filtrate.The extract was chilled to split the phases most preferred to below 5c
These are sepd and each evapd to yield their prods, and oil.
drawback: Multiple steps are involved in this process and process is tedious. Us-5397571
1. Single counter extn. of neem seeds with miscible co-solvent mix. of a
polar solvent and non polar aliphatic hydrocarbon, solvent.
2. Preeip. of Ad. by low polarity solvent of solid more than 10 (more than
20%) wt. azadirachtin prefer solvent (pentane, decane,
chloropentane, chloroform) and 90-10% wt polar solvent {methanol,
butanol, benzyl alcohol etc.
drawback: Precipitation by stirring and Yield not reported.
Technical azadirachtin (10-25%) contains azadirachtin along with other meliacin compounds such as nimbin, salanin, nimbolide, nimbinene, deacetylsalianin, deacetylnimbine, epinimbine, deacetylnimbin. More than 50 neem base pesticide formulations containing azadirachtin have been developed in India and aboard (parmar and Ketkar, 1996) but only the following
formulations have been registered under the Insecticides Act (1968) for the control of insects, pests of arhar, gram, cotton, brinjal, cabbage, jute, potato, tobacco etc. Besides, a number of entrepreneurs are coming up for registration of their neem based products for the control of pests.
1. Neem gold : Kernel extract (Azadirachtin 1500 ppm)
2. Azadirachttn EC : Oil based 300 ppm azadirachtin
3. Godrej Achook: Oil base 300 ppm azadirachtin
4. Margocide CK 200% EC : Azadirachtin and its 15 derivatives, kernel
base, containing azadirachtin 1500 ppm
5. Margocide OK 80% EC : Oil based, contains azadirachtin 300 ppm
6. RD-9 Replin : Oil base, containing 300 ppm azadirachtin.
7. Rakshak: Extract base, containing 1500 ppm azadirachtin
There is a considerable emphasis on the development of neem pesticides base on "azadirachtin" which is the most widely evaluated neem compound. However, it may not be advisable to develop a product based on pure azadirachtin atone because of the following reasons :
1. There is hardly any evidence of the antagonistic effect of one neem
compound on the other. Literature survey reveals that under many
situations, crude or semi-purified extracts of seeds give similar results as
compared to pure azadirachtin and in fact, sometime, crude extracts;
perform better than azadirachtin alone base preparations.
2. There is always a danger of development of resistance in insects against;
a product containing a single active molecule.
3. Greater technical skills are required to produce very high concentrations of azadirachtin in technical grade material which also leads to marked escalation in cost. The main object of the present invention is to provide An improved
method for the preparation of high yieding technical dry amorphous powder
containing 10-17 % azadirachtin. Which obviates the drawback as detailed
above.
Another object of the present invention is to reduce the multiple steps. Still another object of the present invention is not to use water which effects the cone, at the end product
Yet another object of the present invention is to prepare dry powder in high yield wNch contains aza. in more than 10% cone, which has a long shelf life.
Yet another object of the present invention is to eliminate the use of
column
Accordingly the present invention provides an improved process for the preparation of high yielding technical dry amorphous oil-free powder containing 10-17% azadiractin from Azadirachta indica seed kernel , which comprises grinding seed kernels of Azadirachta indica to fine powder , defatting the obtained powder using non-polar solvent, extracting the marc thus obtained by polar solvent, filtering and concentrating the filtrate by conventional methods , incorporating of molecular sieves of the kind as defined above to the concentrate filtrate , cooling at a temperature ranging from 0° to 10 ° C for a period of 8 -12 hours , precipitating the said filtrate by adding non-polar solvent, filtering and drying the precipitate to obtain powder with yield ranging between 1.1-1.7 % by weight of seed karnel , the process is characterized in using simple steps and molecular sieves of mesh size ranging from 4 A° to 5A°,
to the said filtrate to obtain the precipitate followed by continuous stirring filtering and drying of powder with yield ranging between 1.1-1.7% by weight.
In an embodiment of the present invention wherein the defatting of the powder neem kernels is effected by using non-polar solvent such as petroleum ether, hexane, pentane, benzene and mixture thereof.
In anotfier embodiment of the present invention wherein the extraction of the defatted marc is effected with polar solvent such as ethanol, methanoi, chloroform, ethylacetate, acetonitrile, acetone and mixture thereof.
In yet another embodiment of the present invention wherein the filtration and concentration is effected by the know methods like distillation, heating on a water bath, or rotary evaporator.
In yet another embodiment of the present invention is where in the incorporation of molecular sieves of mesh size ranging from 4Ao to 5Ao is effected by keeping it at temperature 0 to 10 for 8 to 12 hours.
In still another embodiment of the present invention is wherein the precipitation is effected by non-polar solvent such as petroleum ether, hexane, pentane, benzene and mixture thereof.
In stilt another embodiment of the present invention is wherein the yield of powder is ringing from 1.1 to 1.7 %.
Details of the process
500 gm seeds kernels were ground to fine powder and defatted by simple washing with hexane or by soxhlet till the complete removal of oil. Defatted mark was taken in container fitted with mechanical stirrer and chloroform was added
such that 2" layer of the solvent remained above the solid mass (approx. 1 L or more solvent was required for the first extracting). Whole mass was stirred for 7-8 hrs. The mass was allowed to settled down and filtered using bochner funnel. The extraction was repeated two more times. Extraction was done in soxhiet also which prevents loss of solvent. Soxhiet extraction was done for 7-10 hrs. The combine filtrate was concentrated in a rotary evaporator to about 300 ml and molecular sieves mesh size ranging from 4A or 5A (2-3 gm) was incorporated in the solution the stopper of the flask replaced tightly and kept for several hrs. or overnight and precipitated by 60-80 petroleum ether or hexane. About 7gm crude powder was obtain by this method. Reported yield was 2.1 gm powder from 500 gm seed kernels.
If the seed kernels are of pure quality than by this methodology crude powder obtain would contain more than 10 to 17% azadirachtin. It is therefore advisable to procure the best quality of neem seeds in order to obtain good percentage of azadirachtin in dry powder.
The following examples are given by way of illustration of the present invention and therefore should not be construed to limit the scope of the present invention.
Example 1.
500 gm seeds kernels were ground to fine powder and defatted by simple washing with hexane or by soxhiet till the complete removal of oil. Defatted mark was taken in container fitted with mechanical stirrer and ethyte acetate was added such that 2 layer of the solvent remained above the solid mats (approx. 1
L or more solvent was required for the first extracting). Whole mass was stirred for 7-8 hrs. The mass was allowed to settled down and filtered using bochner funnel. The extraction was repeated two more times. Extraction was done in soxhiet also which prevents loss of solvent. Soxhiet extraction was done for 7-10 hrs. The combine filtrate was concentrated in a rotary evaporator to about 300 ml and molecular sieves mesh size ranging from 4Ao or 5A> was incorporated in the solution the stopper of the flask replaced tightly and kept for several hrs. or overnight and precipitated by 60-80 petroleum eflier or hexane. Crude powder (0.4 % yield) was obtain by this method.
Example 2.
500 gm seeds kernels were ground to fine powder and defatted by simple washing with hexane or by soxhiet till the complete removal of oil. Defatted mark was taken in container fitted with mechanical stirrer and acetonitrite was added such that 2 layer of the solvent remained above the solid mass (approx. 1 L or more solvent was required for the first extracting). Whole mass was stirred for 7-8 hrs. The mass was allowed to settled down and filtered using bochner funnel. The extraction was repeated two more times. Extraction was done in; soxhiet also which prevents loss of solvent. Soxhiet extraction was done for 7-10 hrs. The combine filtrate was concentrated in a rotary evaporator to about' 300 ml and molecular sieves mesh size ringing from 4Ao or 5Ao (2-3 gm) was? incorporated In the solution the stopper of the flask replaced tightly and kept for several hrs. or overnight and precipitated by 60-80 petroleum ether or hexane.Sticky mass was obtained. Which was triturated with hexane to get solid (0.41 % yield).
Novelty & Advantage : The novelty of the present invention over the priorart method reducing the multiple steps. Water has not been used as it affect the concentration and yield of the end product (dry powder). Molecular sieves removes moisture to give instant precipitation. Two solvent system has been used which are recycled with in the process. This process eliminate the use of column. This process give a crude powder in dry amorphous form in high yield (1.7 % yield by weight). Reported yield was 0.4%. Dry powder is completely free from oil and can be used directly in formulation as insecticides. Preparation of powder involves less steps column has not been used. Two solvents are used therefore it is cost effective powder in high yield and more stable (shelf life six months) than pure azadirechtin.

We claim:
1. An improved process for the preparation of high yielding technical dry
amorphous oil-free powder containing 10-17% azadiractin from Azadirachta
indica seed kernel , which comprises grinding seed kernels of Azadirachta indica
to fine powder, defatting the obtained powder using non-polar solvent, extracting
the marc thus obtained by polar solvent, filtering and concentrating the filtrate by
conventional methods , incorporating of molecular sieves of the kind as defined
herein to the concentrate filtrate , cooling at a temperature ranging from 0° to 10 °
C for a period of 8 -12 hours , precipitating the said filtrate by adding non-polar
solvent, filtering and drying the precipitate to obtain powder with yield ranging
between 1.1 -1.7 % by weight of seed kernels , the process is characterized in
using simple steps and molecular sieves of mesh size ranging from 4 A° to 5A° ,
2. An improved process as claimed in claim 1 wherein the non-polar solvent used is
selected from petroleum ether , hexane , pentane , benzene and mixture thereof.
3. An improved process as claimed in claim 1 and 2 wherein the polar solvent used
is selected from ethanol, methanol , chloroform , ethylacetate, acetonitrile ,
acetone and mixture thereof.
4. An improved process as claimed in claim 1 to 3 wherein the conventional
methods used for filtration and concentration are methods such as distillation,
heating on water bath or rotary evaporation.

5. An improved process for the preparation of high yielding technical dry amorphous oil-free powder containing 10-17% azadiractin from Azadirachta indica seed kernel substantially as here in described with reference to the examples.

Documents:

1973-del-1998-abstract.pdf

1973-del-1998-claims cancelled.pdf

1973-del-1998-claims.pdf

1973-del-1998-complete specification (granted).pdf

1973-del-1998-correspondence-others.pdf

1973-del-1998-correspondence-po.pdf

1973-del-1998-description (complete).pdf

1973-del-1998-form-1.pdf

1973-del-1998-form-2.pdf

1973-del-1998-form-3.pdf

1973-del-1998-form-9.pdf

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Patent Number

189274

Indian Patent Application Number

1973/DEL/1998

PG Journal Number

4/2003

Publication Date

25-Jan-2003

Grant Date

15-Jan-2004

Date of Filing

10-Jul-1998

Name of Patentee

COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH

Applicant Address

RAFI MARG, NEW DELHI-110001, INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	BINA SINGH	DIVISION OF AGRICULTURAL CHEMICALS INDIA AGRICULTURAL RESEARCH INSTITUTE NEW DELHI-110012, INDIA.

PCT International Classification Number

A61K 35/78

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA