Title of Invention

"AN IMPROVED PROCESS FOR THE PREPARATION OF TECHNICAL GRADE AZADIRACHTIN".

Abstract

An improved process for preparation of technical grade azadirachtin, characterized in four step process of preparation 0.21-0.42% oil-free powder of neem containing 27-40% azadirachtin from non aqueous extract of seed kernel of Azadiracta indica (neem), the said process comprising the step of: (i) obtaining filtrate of said Azadiracta seed kernel powder by mechanical stirring in «powdef by mechanical stirring in polar solvent and concentrating in rotary evaporator, (ii) centrifuging the obtained filtrate, diluted with non polar solvent, under low temperature at range 4-5°C, filtering and concentrating by the method as defined above, (iii) adding molecular sieve having mesh size 4-5 A0 to the filtrate obtained at step (ii) and diluting with less polar solvent, cooling the filtrate at a temperature 0-10° C for period 8-12 hours (iv) precipitating with non-polar solvent, filtering and drying by conventional method such as herein described to get the desired technical grade azadirachtin powder.

Full Text

The present invention relates to an improved process for preparation of technical grade azadirachtin More particularly it provides an improved process for the preparation of technical azadirachtin as dry amorphous powder completely free from oil by low temperature centrifuge which is useful for formulation as insecticide. 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. Sop. 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 azadirachtinjn 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 Whereini 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 with 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 byHPLC. 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-4109473 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 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-9502962 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-9456335 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. 6. 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. Precip. 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. . _ Azadirachtin EC : Oil based 300 ppm azadirachtin 3. Godrej Achook: Oil base 300 ppm azadirachtin 4. Margocide CK 200% EEC : Azadirachtin and its 15 derivatives, kernel " if 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 alone 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 process for the preparation of technical dry amorphous powder containing 27-40% 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 affects the concentration at the end product. Yet another object of the present invention is to prepare dry powder which is completely free from oil by use of centrifuge at low temp. Yet another object of the present invention is to eliminate the use of column. Yet another object of the present invention is to prepare powder without obtaining the sticky mass by use of molecular sieves and continuous stirring. Accordingly the present invention provides an improved process for preparation of technical grade azadirachtin , characterized in four step process of preparation 0.21-0.42 % oil-free powder of neem containing 27-40% azadiractin from non aqueous extract of seed kernel of Azadiracta indica (neem) , the said process comprising the step of: (i) obtaining filtrate of said Azadiracta seed kernel powder by mechanical stirring in polar solvent and concentrating in rotary evaporator , (ii) centrifuging the obtained filtrate , diluted with non polar solvent, under low temperature at range 4 -5 ° C , filtering and concentrating by the method as defined above , (in) adding molecular sieve having mesh size 4 -5 A to the filtrate obtained at step (ii) and diluting with less polar solvent , cooling the filtrate at a temperature 0-10 ° C for period 8-12 hours (iii) precipitating with non-polar solvent , filtering and drying by conventional method such as herein described to get the desired technical grade azadiractin powder. In an embodiment of the present invention wherein the extraction of powder is effected by polar solvent such as ethanol, methanol, chloroform, ethylacetate, acetonitrile, acetone and mixture thereof. In another embodiment of the present invention wherein defatting of polar extract is effected by non-polar solvent such as petroleum ether, hexane, pentane, benzene and mixture thereof. In yet another embodiment of the present invention wherein the centrifuge is effected at low temperature, to remove solid and make the polar extract free from oil. In yet another embodiment of the present invention is wherein the less polar solvent used are selected from ethyl acetate, acetonitrile, chloroform and mixture thereof. In still another embodiment of the present invention is wherein incorporation of molecular sieves mesh size ranging from 4Ao or 5Ao is effected to make the solvent free from moisture by cooling it at temperature ranging between OoC to 10X for 8 to 12 hours. In still another embodiment of the present invention is wherein precipitation is effected by non-polar solvent such as petroleum ether, hexane, heptane, pentane and mixture thereof by continuous stirring to obtain dry powder. In still another embodiment of the present invention is wherein the yield ranging between 0.21 % to 0.42 %. Details of the process 500- gm seeds kernels were ground to fine powder and taken in a container fitted with mechanical stirrer, ethanol or methanol (approx. 1 L. or more was required for the first extraction). Whole mass was stirred for 8-10 hrs. The mass was allowed to settle down and filtered using buchner funnel. The extraction was repeated two more times. The combine filtrate was cone, in rotary evaporator to about 600 ml and non-polar solvent hexane was added and kept in fridge for some time and centrifuge at low temperature ranging between 4oC-5oC filtrate and concentrated in rotary evaporator ethyl acetate was added (approx. 1L or more) and thick dark mass was discarded and ethyl acetate portion was filtered to get a clear solution it was concentrated and molecular sieves mesh size ranging from 4A>or 5Ao was incorporated in the solution and cooling was done at temperature 0oC - 10oC by keeping it in fridge and precipitated by 60-80 petroleum ether or hexane by continuous stirring to get dry amorphous powder completely free from oil. If the seed kernels are of pure quality than by this methodology crude powder obtain would contain 27 - 40 % 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 taken in a container fitted with mechanical stirrer, ethanol (approx. 1 L. or more was required for the first extraction). Whole mass was stirred for 8-10 hrs. The mass was allowed to settle down and filtered using buchner funnel. The extraction was repeated two more times. The combine filtrate was cone, in rotary evaporator to about 600 ml and non-polar solvent hexane was added and kept in fridge for some time and centrifuge at low temperature 4c-5c filtrate and cone, in rotary evaporator acetone was added (approx. 1L or more) and thick dark mass was discarded and ethyl acetate portion was filtered to get a clear solution it was concentrated and molecular sieves 4A0or 5Ao was incorporated in the solution and cooling at temperature ranging between 0oC -10 oC and precipitated by 60-80 petroleum ether or hexane by continuous stirring to get dry amorphous powder (0.42 % yield). Example 2 500 gm seeds kernels were ground to fine powder and taken in a container fitted with mechanical stirrer, methanol (approx. 1 L. or more was required for the first extraction). Whole mass was stirred for 8-10 hrs. The mass was allowed to settle down and filtered using buchner funnel. The extraction was repeated two more times. The combine filtrate was cone, in rotary evaporator to about 600 ml and non-polar solvent hexane was added and kept in fridge for some time and centrifuge at low temperature 4c-5c filtrate and cone, in rotary evaporator ethyl acetate was added (approx. 1L or more) and thick dark mass" was discarded and ethyl acetate portion was filtered to get a clear solution it was concentrated and molecular sieves 4A0or 5Ao was incorporated in the solution and cooling at temperature 0 -10 oC and precipitated by 60-80 petroleum ether or hexane by continuous stirring-to get dry amorphous powder completely free from oil (0.31 % yield). Example 3 500 gm seeds kernels were ground to fine powder and defatted with hexane using soxhlet and defatted marc was taken in a container fitted with mechanical stirrer, chloroform (approx. 1 L. or more was required for the first extraction). Whole mass was stirred for 8-10 hrs. The mass was allowed to settle down and filtered using buchner funnel. The extraction was repeated two more times. The combine filtrate was concentrated in rotary evaporator to about 100 and precipitated by non-polar solvent filtering and drying to get dry powder (0.31% yield). Example 4 . 500 gm seeds kernels were ground to fine powder and taken in a container fitted with mechanical stirrer, ethyl acetate (approx. 1 L. or more was required for the first extraction). Whole mass was stirred for 8-10 hrs. The mass was allowed to settle down and filtered using buchner funnel. The extraction was repeated two more times. The combine filtrate was cone, in rotary evaporator to about 200 ml and molecular sieves 4A0or 5Ao was incorporated in the solution and kept in fridge for several hrs. or over night and precipitated by 60-80- petroleum ether or hexane by continuous stirring to get sticky mass which was triturated with hexane to get dry powder (0.41 % yield). Novelty & Advantage: The novelty of the present invention over the priorart method is low temperature centrifuge give the dry powder completely free from oil containing 27 - 40 % azadirachtin. Water has not been used as it affects the concentration and yield of the end product (dry powder). Molecular sieves remove moisture to give instant precipitation. All the three solvent system which has been used are recycled within the process. This process eliminate the use of column. Precipitation by stirring gave the light yellow amorphous powder without obtaining sticky mass. Dry powder is completely free from oil and can be used directly in formulation as insecticides. The process is economical and industrially simple giving high degree of extraction of azadirachtin as dry powder which is completely free form oil because of low temperature centrifuge column and water has not been used which makes the process tedious. We Claim: 1. An improved process for preparation of 'technical grade azadirachtin , characterized in four step process of preparation 0.21-0.42 % oil-free powder of neem containing 27-40% azadiractin from non aqueous extract of seed kernel of Azadiracta indica ( neem), the said process comprising the step of: (i) obtaining filtrate of said Azadiracta seed kernel powder by mechanical stirring in polar solvent and concentrating in rotary evaporator, (ii) centrifuging the obtained filtrate , diluted with non polar solvent, under low temperature at range 4 -5 ° C , filtering and, concentrating by the method as defined above, (iii) adding molecular sieve having mesh size 4 -5 A ° to the filtrate obtained at step (ii) and diluting with less polar solvent , cooling the filtrate at a temperature 0-10 ° C for period 8-12 hours (iii) precipitating with non-polar solvent , filtering and drying by conventional method such as herein described to get the desired technical grade azadiractin powder. 2.1 An improved process as claimed in claim 1 wherein the polar solvent used is selected from ethanol ..methanol, chloroform, ethylacetate , acetonitrile and mixture thereof. 3.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, selected: from ethyle acetate, acetonitrile , chloroform and mixture thereof. 5. An improved process as claimed in claim 1 wherein the preferable non-polar solvent used for precipitation is selected from petroleum ether, hexane ,pentane and mixture thereof. 6. An improved process for preparation of technical grade azadirachtin substantially as herein defined with reference to the examples .

Documents:

1972-del-1998-abstract.pdf

1972-del-1998-claims.pdf

1972-del-1998-complete-spacification(granted).pdf

1972-del-1998-correspondence-others.pdf

1972-del-1998-correspondence-po.pdf

1972-del-1998-description (complete).pdf

1972-del-1998-form-1.pdf

1972-del-1998-form-2.pdf