Title of Invention	"SYNERGISTIC INSECTICIDAL AND LARVICIDAL BOTANICAL COMPOSITIONS"
Abstract	A novel insecticidal and larvicidal botanical synergistic composition is disclosed comprising neem (Azadirachta indica) and kabuli keekar (Prosopis juliflora) plant pods extracts. Neem is known for it"s insecticidal activities but kabuli keekar does not have any insecticidal activity of it"s own. However, when kabuli keekar pod extract was used in composition with neem kernel extract, it showed a synergist effect on the insecticidal and larvicidal activities of the neem and the insecticidal and larvicidal activities of the neem were remarkably enhanced due to this synergistic interaction.

Title of Invention

"SYNERGISTIC INSECTICIDAL AND LARVICIDAL BOTANICAL COMPOSITIONS"

Abstract

A novel insecticidal and larvicidal botanical synergistic composition is disclosed comprising neem (Azadirachta indica) and kabuli keekar (Prosopis juliflora) plant pods extracts. Neem is known for it"s insecticidal activities but kabuli keekar does not have any insecticidal activity of it"s own. However, when kabuli keekar pod extract was used in composition with neem kernel extract, it showed a synergist effect on the insecticidal and larvicidal activities of the neem and the insecticidal and larvicidal activities of the neem were remarkably enhanced due to this synergistic interaction.

Full Text	TITLE: Synergistic insecticidal and larvicidal botanical compositions. FIELD OF INVENTION: This invention relates to synergistic action of Kabuli Keekar with neem for enhancement of insecticidal and larvicidal action of neem against agricultural and household insects. More particularly, this invention relates to an insecticidal composition comprising the extracts of Neem {Azadirachta indica) seeds and Kabuli Keekar (Prosopis juliflora) pods having synergistic effect to produce enhanced insecticidal activity of the composition. The Kabuli Keekar producing synergist action is abundantly available plant and has no insecticidal activity of its own but it significantly increases insecticidal activity of neem. The composition of the present invention is produced from naturally occurring plants and it does not comprise any synthetic ingredient, therefore, it is environment friendly and free of all the side effects associated with synthetic insecticides like hazards to human, animals and the environment. The compositions can be used for insect control in agriculture, household and public health sectors. The present invention provides effective compositions for controlling insects in organic farming (chemical pesticides free farming). The compositions of the present invention will help farmers to control insects in organic farming and thus increasing the quality and yield of crop products. BACKGROUND OF THE INVENTION: This invention relates to a botanical insecticidal composition to control various agricultural and household insects and larvae. In preferred embodiments, the invention relates more particularly to synergistic botanical insecticide adapted to increase the mortality of insects and larvae and reduce the action time. Insects have always been a problem for the human civilization. It is well known that insects directly and indirectly affect the health and food cycle of the human being and animals. The losses caused by pests to agriculture include less yield and inferior quality of food and fibre products. The insects like mosquitoes are vectors of pathogenic microbes which infects human beings and cause diseases like malaria, plague, dengue etc. Pesticides provide rapid and effective means of controlling pests. A large number of synthetic pesticides are being used in agriculture against different pests like insects, weeds, fungi, rodents etc. In public health sector, the pesticides are being used at large scale for mosquito control. The widespread use of synthetic pesticides cause environmental contamination and hazards to non-target organisms. Persistent organic pesticides leave toxic reduces in food products. The surface and ground water sources get contaminated by leaching and transportation of pesticides into the streams. Commercially available insecticides, including those available for agricultural and home use, commonly comprise active synthetic ingredients which are not only toxic to the target insects, but, if used in relatively confined environments, can be present in sufficient concentration to also be toxic to humans, animals, household pets, plants and to the environment. Due to environmental contamination and residue problems, the Environmental Protection Authorities and environment activists are trying to promote effective alternatives to synthetic pesticides worldwide. In United States of America The Environment Protection Agency (EPA) regulates the use of potentially toxic ingredients in pesticidal compositions. The EPA and national regulating agencies like Indian Central Insecticides Board have deregulated some substances offering minimum risk in normal use. Attempts to use insecticidal compositions having deregulated essential oils such as cedar oil, cinnamon oil, citronella oil, clove oil, garlic oil, lemongrass oil, rosemary oil and thyme oil as the active ingredient had limited success due to the reasons like high cost and inadequate efficacy. With the rising concern about environmental safety, the use of botanical pesticides is rapidly gaining popularity worldwide as these are safe to human, non target organisms and environment. Among the others, Neem (Azadirachta spp.) is widely used as insecticide for controlling household and agricultural insects. Neem contains a mixture of several bioactive compounds having insecticidal and insects feeding inhibition properties. For the last two decades, the use of neem is being promoted in the country for various advantages like good bio-efficacy, availability, bio-degradability and safety. The use of neem has been started on all crops due to residue problems associated with the use of synthetic pesticides. Azadirachtin A (normally simply referred to as azadirachtin) is present throughout the Neem tree (Azadirachta Indica) and in significant quantities of up to 0.8% in the seed and is well known for its potentially wide ranging beneficial properties including insecticidal properties. In addition, several hundred compounds have been identified in the Neem Tree, many occurring in the seed including the 12 main triterpenoids which include nimbin, salannin and 3-tigloylazadirachtol (also known as azadirachtin B). Several of these compounds have been shown to have beneficial pharmaceutical and insecticidal properties. In the prior art, methods are known in which the neem seed (or other azadirachtin source) is processed, including solvent extraction steps to obtain a crude solid extract containing the azadirachtin; typically such known processes (e.g. U.S. Pat. No. 5,856,526) result in a product containing normally up to 15% azadirachtin and occasionally 25-30%. In order to isolate pure azadirachtin A (e.g. above 90% purity), chromatographic methods are normally used. Conventionally the neem oil and water extract of neem kernels is used for insect control. However, with the increase in the use of neem, ifs availability is declining and thus the cost of neem plant parts like wood, leaves, seed etc. is also increasing. Therefore, to reduce the cost of neem application and to enhance the biological activity of neem, there has been a need of some more economical natural compliment which is non-toxic, safe to human, animals and environment and can be used to improve the insecticidal activities of the neem, thereby reducing the quantitative requirements of neem parts like seed, leaves, wood etc in the insecticide and as a result reducing the cost of the product. OBJECTIVES OF THE INVENTION: In the preferred embodiment, primary objective of this invention is to provide a natural synergistic insecticidal composition comprising "neem" and a "synergist" which can improve the insecticidal activities of the neem. Another objective of this invention is to provide a synergistic insecticidal composition possessing no toxic effects on human beings and animals and no hazardous effects on the environment. Further objective of this invention is to provide a synergistic insecticidal composition only comprising the plant extracts without any synthetic ingredients. Another objective of this invention is to provide an economical natural synergistic insecticidal composition which can be used safely and on mass scale by the public at large for insect control in agriculture, household and public health sectors. Another objective of this invention is to provide effective compositions for controlling insects in organic farming (chemical pesticides free farming) SUMMARY OF THE INVENTION: The insecticidal activities of the neem are conventionally known in the prior art. Neem fruits, barks, leaves and seeds have been used for various purposes against agricultural and household insects and larvae due to their insecticidal activities. Whereas, the kabuli keekar (Prosopis juliflora) plant or it's parts like pods do not have any insecticidal activity of their own. When the extract of kabuli keekar (Prosopis juliflora) pods were used in different concentrations in a composition containing extract of neem seeds as the other constituent, the resulting composition showed a remarkable synergistic effect. The insecticidal activities of the composition, compared to pure neem seed extract, were remarkably increased. This increase in the insecticidal activity of the composition is due to synergistic effect being produced by the constituents of the composition. The bio-efficacy of the composition was found to be better than the efficacy of pure neem. The composition of the invention not only has the enhanced insecticidal and larvicidal activity, but it also inhibits the growth of larvae. Therefore, the kabuli keekar (Prosopis juliflora) having no insecticidal or larvicidal activity of it's own, significantly increased the insecticidal and larvicidal activity of the neem due to synergistic effect. Aqueous extracts of neem (Azadirachta indica) seeds and kabuli keekar (Prosopis juliflora) pods were obtained by conventional methods and they were mixed together in different concentrations. The insecticidal and larvicidal activities of the various compositions were studied. Various examples hereinafter described and results obtained in various studies described hereinafter showed that kabuli keekar (Prosopis juliflora) had enhanced the insecticidal and larvicidal activities of neem (Azadirachta indica). This enhancement of insecticidal and larvicidal activities of the insecticidal composition of the present invention was due to synergistic interaction between the kabuli keekar (Prosopis juliflora) pods extract and neem (Azadirachta indica) seed (kernel) extract. DETAILED DESCRIPTION OF THE INVENTION: In the present invention a novel insecticidal composition has been disclosed. The insecticidal composition of the invention is obtained from Neem (Azadirachta indica) and kabuli keekar (Prosopis chilensis or Prosopis juliflora) plant extracts. The insecticidal activity of the composition is produced due to synergistic effect of both the extracts. Neem (Azadirachta indica) is a tree in the mahogany family Maliaceae. All parts of Neem including seeds, leaves, flowers and bark have medicinal and insecticidal properties. In the present invention, decorticated seeds (kernels) of the neem (Azadirachta indica) have been used. The insecticidal activities of the neem seed extract are known in the prior art. Various methods of extraction of the neem seed extracts are also known in the prior art. The seeds (kernel) of the neem contain a complex secondary metabolite azadirachtin. Various other compounds which are present in the seed are 1-Alpha- methoxy-1,2-dihydroazadiradione, 22, 23-Dihydro-23-Beta-Methoxy-Azadirachtin, Azadirachtin, Azadirone, Epoxyazadiradione, Fat, Gedunin, Meldenin, Meliantriol, Nimbidin, Nimolicinol, Resin, Sulfur, Vepinin, 2-Beta-diepoxy-azadiradione, 3-Desacetysalannin, 3-Tigloylazadirachtol, 4-Epinimbin, Azadiradione 6-Acetyl-nimbandiol, 6-Desacetylnimbinene., 7-Acetylneotrichilenone, 7-Desacetyl-7-benzoyl-azadiradione, 7-Desacetyl-7-benzoylepoxy-azadiradione, Desacetyl-Gedunin, 17-Beta-Hydroxyazadiradion and 17-Epiazadiradione. The kabuli keekar (Prosopis juliflora) is a shrub or small tree abundantly found in India and other parts of the world. The genus Prosopis belongs to the family leguminosae. The leaves are bipinnate and flowers are small, yellowish in dense spikes. The bark is greyish and the pods are yellow. 10-25 cm long and 8-15 mm broad, straight or falcate, flat or cylindrical, often with transverse depression between the seeds, 10-30 seeds are present in a pod and ovoid flattened, 7mm x 3 mm hard, yellowish brown and shiny. Various chemical constituents have been isolated from the kabuli keekar(Prosop/s juliflora) plant which include flavones glycoside patulitrin(l) 3, 5, 6, 3, 4-pentamethoxy-7-hydroxy flavone, alkaloids like juliflorine, julifloricine and julifloridine, keto alcohol, triterpedoid glycoside, ethanolic extract, tannin, polystyrene etc. Chemical and nutritional studies on Prosopis juliflora beans have showed that it comprises protein 10-15%, fat 2-35, crude fibre 20-30%, sucrose 21% and reducing sugars 2-6%. Kernels contain 38% protein, 3% fat and 9% crude fibres. The present invention comprises an insecticidal composition comprising the extracts of Neem {Azadirachta indica) seeds and Kabuli Keekar (Prosopis juliflora) pods. The insecticidal and larvicidal activity of neem is conventionally known in the prior art. Neem fruits, barks, leaves and seeds have been used for various purposes against agricultural and household insects and larvae due to their insecticidal activities. The kabuli keekar (Prosopis juliflora) plant or it's parts like pods do not have any insecticidal or larvicidal activity of their own. However, when the extract of said kabuli keekar (Prosopis juliflora) pods were used in different concentrations in a composition containing extract of said neem seeds as the other constituent, the resulting composition showed a remarkable synergistic effect and the insecticidal as well as larvicidal activities of the composition, due to said neem seed extract were remarkably increased. Various tests and studies have proved that when taken alone, the kabuli keekar(Prasop/s juliflora) pods have no insecticidal and / or larvicidal activity. The insecticidal and larvicidal activities of the neem (Azadirachta indica) seed extracts were also studied alone. When the compositions of the present invention were studied and the insecticidal and larvicidal activities of the composition were recorded using the same method as used for the constituents separately, it was found that the insecticidal and larvicidal activity of the resulting composition remarkably increased. This increase in the insecticidal and larvicidal activity of the composition was due to synergistic effect being produced by the constituents of the composition. The bio-efficacy of the composition was found to better than the efficacy of pure neem. The composition of the invention not only has the enhanced insecticidal and larvicidal activity, but it also inhibits the growth of larvae. Therefore, the kabuli keekar (Prosopis juliflora) having no insecticidal and/or larvicidal activity of it's own, significantly increased the insecticidal and larvicidal activity of the neem due to synergistic effect. In the forthcoming examples abbreviation NKP has been used for neem (Azadirachta indica) seed (kernel) powder extract and KP has been used for kabuli keekar (Prosopis juliflora) pods extract, only for defining and explaining the invention. The following compositions having different concentrations of NKP and KP were used in the experiments for studying the insecticidal and larvicidal activities: Pure neem seed (kernel) powder extract (NKP) Pure kabuli keekar pods powder extract (KP) NKP + KP in the ratio of 50:50 NKP + KP in the ratio of 75:25 NKP + KP in the ratio of 25:75 Control - Placebo. The Following three methods were adopted for making aqueous extracts of KP and NKP and performing the bio-efficacy experiments; METHOD 1: Soaking the NKP and KP in water individually and then mixing the aqueous extracts In this method 1% aqueous extract of neem kernel powder was prepared by soaking 10 g of powdered NKP (sieved through 36 mesh sieve) in 1000 ml of water for overnight, followed by filtering through a fine muslin cloth. The remains on the cloth were squeezed into the beaker. Similarly 1% aqueous extract of KP (sieved through 36 mesh sieve) was prepared. Both the extracts were then mixed in different ratios like 1:1, 3:1 and 1:3 and bioefficacy experiments were performed. Example 1: Synergistic Effect on Bio-efficacy of NKP and KP on mosquito larvae. These studies were performed on Mosquito larvae. In this study the inventors reported the potential synergistic effects of the kabuli keekar pods powder extract (KP) to enhance the larvicidal activity of neem kernel aqueious extract (NKAE). The role of KP in the enhancement of insecticidal activity of NKAE on larval instars of Anopheles Stephens! and mixed culture of Culex quinquefasciatus, Anopheles stephensi and Aedes aegypti was investigated under laboratory conditions was studied. The experiments for larvicidal activity were conducted in triplicates at 25 ± 1° C, 70 ± 5% RH. 20 mosquito larvae (3rd larvae instar) of required species were taken in 500 ml beaker containing 250ml aqueous extract of the required concentration of the material to be studied. Mortality of larvae was calculated after 48 hours. Table 1: Synergistic effect on bioefficacy of KP & NKP on mosquito larvae (Table Removed) The results in Table 1 showed that larvicidal activity produced by pure NKP extract was only 40% whereas, pure KP solution had very less larvicidal activity, at par with control i.e. without any larvicidal solution. The larvicidal activity of the composition was maximum i.e., 70% for the extract containing NKP and KP in 1:1 ratio, which was much higher than the sum total of individual larvicidal activities of NKP and KP. Therefore, it was established that this enhanced larvicidal activity of the composition was due to synergistic effect of KP extract on NKP extract. Example 2: Synergistic Effect on Bio-efficacy of KP and NKP on Spodoptera litura pupae These studies were performed on Spodoptera litura pupae. The experiments for larvicidal activity were conducted at 25 ± 1° C and 60-70 % RH. Percent reduction in number of viable pupae was observed after 7 days. Table 2: Synergistic effect on Bio-efficacy of KP and NKP on Spodoptera litura pupae (Table Removed) The above results showed that 1 % NKP produced 100% reduction in number of viable pupae. While 1 % KP produced only 10% reduction in number of viable pupae, which is at par with control i.e. without any larvicidal solution. When the dose of NKP is reduced to half i.e., 0.5% and 0.5% KP is added to it, 80% reduction In number of viable pupae is observed. This results shows that by addition of KP, the half dose of NKP produces 80% activity compared to full dose of NKP. Therefore, it was established that this activity of the composition is due to synergistic effect of KP extract on NKP extract. METHOD 2: Mixing KP and NKP in powder form in required ratio and then soaking in water for three hours In this method NKP and KP powders were mixed in different ratios like 1:1, 3:1 and 1:3. The 1% aqueous extracts of NKP+KP mixtures were prepared by soaking 10g of mixture in 1000 ml water for three hours followed by filtering through a fine muslin cloth. The remains on the cloth were squeezed into the beaker. The 1% aqueous extracts of pure NKP and pure KP were also prepared by same procedure. The bioefficacy experiments were performed with these freshly prepared extracts. Example 3 Synergistic Effect on Bio-efficacy of KP and NKP on mosquito larvae. The same procedure as described in Example 1 was followed for bioefficacy experiment Table 3: Synergistic Effect on Bio-efficacy of KP and NKP on mosquito larvae. (Table Removed) The above results showed that larvicidal activity produced by pure NKP extract was 40%, whereas, pure KP solution had very less larvicidal activity at par with control i.e. without any larvicidal solution. The larvicidal activity of the composition was maximum (60%) for the suspension containing NKP and KP in 3:1 ratio, which was much higher than the individual larvicidal activities of NKP and KP. Therefore, it was established that this enhanced larvicidal activity of the composition was due to synergistic effect of KP extract on NKP extract. METHOD 3: Mixing KP and NKP in powder form in required ratio and then soaking in water for 24 hours In this method NKP and KP powders were mixed in different ratios like 1:1, 3:1 and 1:3 and then 1% aqueous extracts were prepared by soaking 10g of mixture in 1000 ml water for 24 hours followed by filtering through a fine muslin cloth. The remains on the cloth were squeezed into the beaker. The 1% aqueous extracts of pure NKP and pure KP were also prepared by same procedure. The bioefficacy experiments were performed with these freshly prepared extracts. Example 4 Synergistic Effect on Bio-efficacy of of KP and NKP on mosquito larvae. The same procedure as described in Example 1 was followed for bioefficacy experiment Table 4: Synergistic Effect on Bio-efficacy of KP and NKP on mosquito larvae. (Table Removed) The above results showed that larvicidal activity produced by pure NKP extract was 40% whereas, pure KP extract had very less larvicidal activity at par with control i.e. without any larvicidal solution. Results of the above table show that the larvicidal activity of the composition was maximum for the solution containing NKP and KP in 3:1 ratio, which is at par with individual larvicidal activities of NKP Therefore, it was established that if method No. 3 is adopted for extraction, 25% of NKP may be replaced by KP to produce equivalent bioefficacy. Example 5: Synergistic Effect on Bio-efficacy of KP and NKP on Pieris brassicae (Cauliflower) The extraction procedure described in method No. 3 was adopted, in place of 1% extracts, NKP 2.5%, NKP 3.75% , NKP 5%, and KP 5% and NKP 2.5%+ KP2.5% extracts were used in the experiments. Mortality of first instar larvae (N=20; replications 3) was recorded after 48 hours of release. Table 5: Laboratory studies on Pieris brassicae (Cauliflower) (Table Removed) It is clear from table 5 that pure KP shown mortality rate at par with control i.e., without any insecticidal solution. The mortality with pure NKP -5% was 93 %. The mortality increased to 100% with the composition comprising NKP 2.5% + KP 2.5% The results of the study on Pieris brassicae larvae again establish that KP has synergistic effect on larvicidal activity of NKP. Example 6: Synergistic Effect on Bio-efficacy of of KP and NKP on Helicoverpa armigera (Gram pod) The procedure described in Example 5 was adopted for doing the bioefficacy studies on Helicoverpa armigera (Gram pod) Table 6: Results on mortality of Helicoverpa armigera (Gram pod) larvae (N=20) (Table Removed) It is clear from table 6 that control and pure KP solution produced 0% mortality. The mortality with pure NKP -2.5 % & 5% after 7 days was 75 %. The mortality increased to 100% with the composition comprising NKP 2.5% + KP 2.5% . The results of the study on Helicoverpa armigera again established that KP has synergistic effect on larvicidal activity of NKP. Table 7: Results of choice feeding experiment: (Table Removed) The above results show that the damage to gram pod was maximum i.e., 20% in case of control and KP-5%. The damage was minimum i.e., 0% in case of NKP 5%. In the case of NKP 2. 5% + KP 2.5 %, the damage is only 10%, which is better than NKP 3.75%, which shows that NKP at 2.5% level gives the better result compared to NKP 3.75% level, if KP 2.5% is added to it. This established that KP has synergistic effect on insecticidal activity of NKP. The results obtained in various studies described hereinabove have established that kabuli keekar (Prosopis juliflora) had enhanced the insecticidal and larvicidal activities of neem (Azadirachta indica) in the present invention. This enhancement of insecticidal and larvicidal activities of the insecticidal composition of the present invention was due to synergistic interaction between the kabuli keekar {Prosopis juliflora) pods extract and neem (Azadirachta indica) seed (kernel) extract. The composition of the present invention is natural (botanical) insecticidal composition obtained from the naturally occurring plant based substances and is devoid of any synthetic ingredients. Therefore it is non-polluting and does not poses any toxic side effect or hazardous effects on human, animals and to the environment. The kabuli keekar plants are found abundantly in the country .therefore these plant based materials are available at very low cost. The use of economical kabuli keekar as synergist also reduces the cost of neem application. The compositions can be used for effective insect control in agriculture, household and public health sectors. The present invention provides effective compositions for controlling insects in organic farming (chemical pesticides free farming). The compositions of the present invention can be used by the farmers to control insects in organic farming and thereby increasing the product quality and yield. Claim: 1. A synergistic insecticidal and larvicidal botanical composition comprising neem (Azadirachta indica) and kabuli keekar (Prosopis juliflora) plant extracts. 2. A synergistic insecticidal and larvicidal botanical composition as claimed in claim 1, wherein the concentration of said neem (Azadirachta indica) extract is between 25% to 75% by weight. 3. A synergistic insecticidal and larvicidal botanical composition as claimed in claim 1, wherein the concentration of said kabuli keekar (Prosopis juliflora) extract is between 25% to 75% by weight. 4. A synergistic insecticidal and larvicidal botanical composition as claimed in any of the claims 1 to 3, wherein the ratio of said neem (Azadirachta indica) extract to said kabuli keekar (Prosopis juliflora) extract (NKP: KP) is 3 :1. 5. A synergistic insecticidal and larvicidal botanical composition as claimed in any of the claims 1 to 3, wherein the ratio of said neem (Azadirachta indica) extract to said kabuli keekar (Prosopis juliflora) extract (NKP: KP) is 1 :1. 6. A synergistic insecticidal and larvicidal botanical composition as claimed in claim 1, wherein the said neem (Azadirachta indica) extract is obtained from neem (Azadirachta indica) seeds or kernel. 7. A synergistic insecticidal and larvicidal botanical composition as claimed in claim 1 wherein said kabuli keekar (Prosopis juliflora) extract is obtained from kabuli keekar (Prosopis juliflora) pods. 8. A synergistic insecticidal and larvicidal botanical composition as claimed in claim i, which can be used against agricultural and household insects and larvae. 9. A method of using the synergistic insecticidal and larvicidal botanical composition of claim 1 comprising the steps of: a. taking the dry powders of said neem (NKP) and said kabuli keekar (KP) and mixing them in the required ratio; b. soaking desired quantity of the said mixture thus obtained in step (a) in water for 3/24 hours; c. filtering the said soaked mixture through fine muslin cloth; d. collecting the said freshly prepared extract and using against insects and larvae as per the requirement. 10. A method of using the synergistic insecticidal and larvicidal botanical composition of claim 1 comprising the steps of: a. taking dry powder extract of said neem (NKP) and said kabuli keekar (KP) and soaking them separately in water for overnight; b. filtering the said soaked extracts of step (a) through a fine muslin cloth; c. collecting freshly prepared aqueous extracts obtained in step (b), separately; d. mixing the said aqueous extracts in desired ratio and using the said obtained aqueous mixture against insects and larvae as per requirement.

Full Text

TITLE: Synergistic insecticidal and larvicidal botanical compositions.
FIELD OF INVENTION:
This invention relates to synergistic action of Kabuli Keekar with neem for enhancement of insecticidal and larvicidal action of neem against agricultural and household insects. More particularly, this invention relates to an insecticidal composition comprising the extracts of Neem {Azadirachta indica) seeds and Kabuli Keekar (Prosopis juliflora) pods having synergistic effect to produce enhanced insecticidal activity of the composition. The Kabuli Keekar producing synergist action is abundantly available plant and has no insecticidal activity of its own but it significantly increases insecticidal activity of neem.
The composition of the present invention is produced from naturally occurring plants and it does not comprise any synthetic ingredient, therefore, it is environment friendly and free of all the side effects associated with synthetic insecticides like hazards to human, animals and the environment. The compositions can be used for insect control in agriculture, household and public health sectors. The present invention provides effective compositions for controlling insects in organic farming (chemical pesticides free farming). The compositions of the present invention will help farmers to control insects in organic farming and thus increasing the quality and yield of crop products.
BACKGROUND OF THE INVENTION:
This invention relates to a botanical insecticidal composition to control various agricultural and household insects and larvae. In preferred embodiments, the invention relates more particularly to synergistic botanical insecticide adapted to increase the mortality of insects and larvae and reduce the action time.
Insects have always been a problem for the human civilization. It is well known that insects directly and indirectly affect the health and food cycle of the human being and animals. The losses caused by pests to agriculture include less yield and inferior quality of food and fibre products. The insects like mosquitoes are vectors of pathogenic microbes which infects human beings and cause diseases like malaria, plague, dengue etc.
Pesticides provide rapid and effective means of controlling pests. A large number of synthetic pesticides are being used in agriculture against different pests like insects, weeds, fungi, rodents etc. In public health sector, the pesticides are being used at large scale for mosquito control.
The widespread use of synthetic pesticides cause environmental contamination and hazards to non-target organisms. Persistent organic pesticides leave toxic reduces in food products. The surface and ground water sources get contaminated by leaching and transportation of pesticides into the streams.
Commercially available insecticides, including those available for agricultural and home use, commonly comprise active synthetic ingredients which are not only toxic to the target insects, but, if used in relatively confined environments, can be present in sufficient concentration to also be toxic to humans, animals, household pets, plants and to the environment.
Due to environmental contamination and residue problems, the Environmental Protection Authorities and environment activists are trying to promote effective alternatives to synthetic pesticides worldwide. In United States of America The Environment Protection Agency (EPA) regulates the use of potentially toxic ingredients in pesticidal compositions. The EPA and national regulating agencies like Indian Central Insecticides Board have deregulated some substances offering minimum risk in normal use.
Attempts to use insecticidal compositions having deregulated essential oils such as cedar oil, cinnamon oil, citronella oil, clove oil, garlic oil, lemongrass oil, rosemary oil and thyme oil as the active ingredient had limited success due to the reasons like high cost and inadequate efficacy.
With the rising concern about environmental safety, the use of botanical pesticides is rapidly gaining popularity worldwide as these are safe to human, non target organisms and environment. Among the others, Neem (Azadirachta spp.) is widely used as insecticide for controlling household and agricultural insects. Neem contains a mixture of several bioactive compounds having insecticidal and insects feeding inhibition properties. For the last two decades, the use of neem is being promoted in the country for various advantages like good bio-efficacy, availability, bio-degradability and safety. The use of neem has been started on all crops due to residue problems associated with the use of synthetic pesticides.
Azadirachtin A (normally simply referred to as azadirachtin) is present throughout the Neem tree (Azadirachta Indica) and in significant quantities of up to 0.8% in the seed and is well known for its potentially wide ranging beneficial properties including insecticidal properties. In addition, several hundred compounds have been identified in the Neem Tree, many occurring in the seed including the 12 main triterpenoids which include nimbin, salannin and 3-tigloylazadirachtol (also known as azadirachtin B). Several of these compounds have been shown to have beneficial pharmaceutical and insecticidal properties.
In the prior art, methods are known in which the neem seed (or other azadirachtin source) is processed, including solvent extraction steps to obtain a crude solid extract containing the azadirachtin; typically such known processes (e.g. U.S. Pat.
No. 5,856,526) result in a product containing normally up to 15% azadirachtin and occasionally 25-30%. In order to isolate pure azadirachtin A (e.g. above 90% purity), chromatographic methods are normally used. Conventionally the neem oil and water extract of neem kernels is used for insect control.
However, with the increase in the use of neem, ifs availability is declining and thus the cost of neem plant parts like wood, leaves, seed etc. is also increasing.
Therefore, to reduce the cost of neem application and to enhance the biological activity of neem, there has been a need of some more economical natural compliment which is non-toxic, safe to human, animals and environment and can be used to improve the insecticidal activities of the neem, thereby reducing the quantitative requirements of neem parts like seed, leaves, wood etc in the insecticide and as a result reducing the cost of the product.
OBJECTIVES OF THE INVENTION:
In the preferred embodiment, primary objective of this invention is to provide a natural synergistic insecticidal composition comprising "neem" and a "synergist" which can improve the insecticidal activities of the neem.
Another objective of this invention is to provide a synergistic insecticidal composition possessing no toxic effects on human beings and animals and no hazardous effects on the environment.
Further objective of this invention is to provide a synergistic insecticidal composition only comprising the plant extracts without any synthetic ingredients.
Another objective of this invention is to provide an economical natural synergistic insecticidal composition which can be used safely and on mass scale by the public at large for insect control in agriculture, household and public health sectors.
Another objective of this invention is to provide effective compositions for controlling insects in organic farming (chemical pesticides free farming)
SUMMARY OF THE INVENTION:
The insecticidal activities of the neem are conventionally known in the prior art. Neem fruits, barks, leaves and seeds have been used for various purposes against agricultural and household insects and larvae due to their insecticidal activities.
Whereas, the kabuli keekar (Prosopis juliflora) plant or it's parts like pods do not have any insecticidal activity of their own.
When the extract of kabuli keekar (Prosopis juliflora) pods were used in different concentrations in a composition containing extract of neem seeds as the other constituent, the resulting composition showed a remarkable synergistic effect. The insecticidal activities of the composition, compared to pure neem seed extract, were remarkably increased.
This increase in the insecticidal activity of the composition is due to synergistic effect being produced by the constituents of the composition. The bio-efficacy of the composition was found to be better than the efficacy of pure neem. The composition of the invention not only has the enhanced insecticidal and larvicidal activity, but it also inhibits the growth of larvae. Therefore, the kabuli keekar (Prosopis juliflora) having no insecticidal or larvicidal activity of it's own, significantly increased the insecticidal and larvicidal activity of the neem due to synergistic effect.
Aqueous extracts of neem (Azadirachta indica) seeds and kabuli keekar (Prosopis juliflora) pods were obtained by conventional methods and they were mixed together in different concentrations. The insecticidal and larvicidal activities of the various compositions were studied.
Various examples hereinafter described and results obtained in various studies described hereinafter showed that kabuli keekar (Prosopis juliflora) had enhanced the insecticidal and larvicidal activities of neem (Azadirachta indica). This enhancement of insecticidal and larvicidal activities of the insecticidal composition of the present invention was due to synergistic interaction between the kabuli keekar (Prosopis juliflora) pods extract and neem (Azadirachta indica) seed (kernel) extract.
DETAILED DESCRIPTION OF THE INVENTION:
In the present invention a novel insecticidal composition has been disclosed. The insecticidal composition of the invention is obtained from Neem (Azadirachta indica) and kabuli keekar (Prosopis chilensis or Prosopis juliflora) plant extracts. The insecticidal activity of the composition is produced due to synergistic effect of both the extracts.
Neem (Azadirachta indica) is a tree in the mahogany family Maliaceae. All parts of Neem including seeds, leaves, flowers and bark have medicinal and insecticidal properties.
In the present invention, decorticated seeds (kernels) of the neem (Azadirachta indica) have been used. The insecticidal activities of the neem seed extract are known in the prior art. Various methods of extraction of the neem seed extracts are also known in the prior art.
The seeds (kernel) of the neem contain a complex secondary metabolite azadirachtin. Various other compounds which are present in the seed are 1-Alpha-
methoxy-1,2-dihydroazadiradione, 22, 23-Dihydro-23-Beta-Methoxy-Azadirachtin, Azadirachtin, Azadirone, Epoxyazadiradione, Fat, Gedunin, Meldenin, Meliantriol, Nimbidin, Nimolicinol, Resin, Sulfur, Vepinin, 2-Beta-diepoxy-azadiradione, 3-Desacetysalannin, 3-Tigloylazadirachtol, 4-Epinimbin, Azadiradione 6-Acetyl-nimbandiol, 6-Desacetylnimbinene., 7-Acetylneotrichilenone, 7-Desacetyl-7-benzoyl-azadiradione, 7-Desacetyl-7-benzoylepoxy-azadiradione, Desacetyl-Gedunin, 17-Beta-Hydroxyazadiradion and 17-Epiazadiradione.
The kabuli keekar (Prosopis juliflora) is a shrub or small tree abundantly found in India and other parts of the world. The genus Prosopis belongs to the family leguminosae. The leaves are bipinnate and flowers are small, yellowish in dense spikes. The bark is greyish and the pods are yellow. 10-25 cm long and 8-15 mm broad, straight or falcate, flat or cylindrical, often with transverse depression between the seeds, 10-30 seeds are present in a pod and ovoid flattened, 7mm x 3 mm hard, yellowish brown and shiny.
Various chemical constituents have been isolated from the kabuli keekar(Prosop/s juliflora) plant which include flavones glycoside patulitrin(l) 3, 5, 6, 3, 4-pentamethoxy-7-hydroxy flavone, alkaloids like juliflorine, julifloricine and julifloridine, keto alcohol, triterpedoid glycoside, ethanolic extract, tannin, polystyrene etc.
Chemical and nutritional studies on Prosopis juliflora beans have showed that it comprises protein 10-15%, fat 2-35, crude fibre 20-30%, sucrose 21% and reducing sugars 2-6%. Kernels contain 38% protein, 3% fat and 9% crude fibres.
The present invention comprises an insecticidal composition comprising the extracts of Neem {Azadirachta indica) seeds and Kabuli Keekar (Prosopis juliflora) pods.
The insecticidal and larvicidal activity of neem is conventionally known in the prior art. Neem fruits, barks, leaves and seeds have been used for various purposes against agricultural and household insects and larvae due to their insecticidal activities.
The kabuli keekar (Prosopis juliflora) plant or it's parts like pods do not have any insecticidal or larvicidal activity of their own.
However, when the extract of said kabuli keekar (Prosopis juliflora) pods were used in different concentrations in a composition containing extract of said neem seeds as the other constituent, the resulting composition showed a remarkable synergistic effect and the insecticidal as well as larvicidal activities of the composition, due to said neem seed extract were remarkably increased.
Various tests and studies have proved that when taken alone, the kabuli keekar(Prasop/s juliflora) pods have no insecticidal and / or larvicidal activity. The insecticidal and larvicidal activities of the neem (Azadirachta indica) seed extracts were also studied alone. When the compositions of the present invention were studied and the insecticidal and larvicidal activities of the composition were recorded
using the same method as used for the constituents separately, it was found that the insecticidal and larvicidal activity of the resulting composition remarkably increased.
This increase in the insecticidal and larvicidal activity of the composition was due to synergistic effect being produced by the constituents of the composition. The bio-efficacy of the composition was found to better than the efficacy of pure neem. The composition of the invention not only has the enhanced insecticidal and larvicidal activity, but it also inhibits the growth of larvae. Therefore, the kabuli keekar (Prosopis juliflora) having no insecticidal and/or larvicidal activity of it's own, significantly increased the insecticidal and larvicidal activity of the neem due to synergistic effect.
In the forthcoming examples abbreviation NKP has been used for neem (Azadirachta indica) seed (kernel) powder extract and KP has been used for kabuli keekar (Prosopis juliflora) pods extract, only for defining and explaining the invention.
The following compositions having different concentrations of NKP and KP were used in the experiments for studying the insecticidal and larvicidal activities:
Pure neem seed (kernel) powder extract (NKP)
Pure kabuli keekar pods powder extract (KP)
NKP + KP in the ratio of 50:50
NKP + KP in the ratio of 75:25
NKP + KP in the ratio of 25:75
Control - Placebo.
The Following three methods were adopted for making aqueous extracts of KP and NKP and performing the bio-efficacy experiments;
METHOD 1: Soaking the NKP and KP in water individually and then mixing the aqueous extracts
In this method 1% aqueous extract of neem kernel powder was prepared by soaking 10 g of powdered NKP (sieved through 36 mesh sieve) in 1000 ml of water for overnight, followed by filtering through a fine muslin cloth. The remains on the cloth were squeezed into the beaker. Similarly 1% aqueous extract of KP (sieved through 36 mesh sieve) was prepared. Both the extracts were then mixed in different ratios like 1:1, 3:1 and 1:3 and bioefficacy experiments were performed.
Example 1:
Synergistic Effect on Bio-efficacy of NKP and KP on mosquito larvae.
These studies were performed on Mosquito larvae. In this study the inventors reported the potential synergistic effects of the kabuli keekar pods powder extract (KP) to enhance the larvicidal activity of neem kernel aqueious extract (NKAE). The role of KP in the enhancement of insecticidal activity of NKAE on larval instars of Anopheles Stephens! and mixed culture of Culex quinquefasciatus, Anopheles stephensi and Aedes aegypti was investigated under laboratory conditions was studied.
The experiments for larvicidal activity were conducted in triplicates at 25 ± 1° C, 70 ± 5% RH. 20 mosquito larvae (3rd larvae instar) of required species were taken in 500 ml beaker containing 250ml aqueous extract of the required concentration of the material to be studied. Mortality of larvae was calculated after 48 hours.
Table 1: Synergistic effect on bioefficacy of KP & NKP on mosquito larvae

(Table Removed)
The results in Table 1 showed that larvicidal activity produced by pure NKP extract was only 40% whereas, pure KP solution had very less larvicidal activity, at par with control i.e. without any larvicidal solution.
The larvicidal activity of the composition was maximum i.e., 70% for the extract containing NKP and KP in 1:1 ratio, which was much higher than the sum total of individual larvicidal activities of NKP and KP.
Therefore, it was established that this enhanced larvicidal activity of the composition was due to synergistic effect of KP extract on NKP extract.
Example 2:
Synergistic Effect on Bio-efficacy of KP and NKP on Spodoptera litura pupae
These studies were performed on Spodoptera litura pupae. The experiments for larvicidal activity were conducted at 25 ± 1° C and 60-70 % RH. Percent reduction in number of viable pupae was observed after 7 days.
Table 2: Synergistic effect on Bio-efficacy of KP and NKP on Spodoptera litura pupae

(Table Removed)
The above results showed that 1 % NKP produced 100% reduction in number of viable pupae. While 1 % KP produced only 10% reduction in number of viable pupae, which is at par with control i.e. without any larvicidal solution.
When the dose of NKP is reduced to half i.e., 0.5% and 0.5% KP is added to it, 80% reduction In number of viable pupae is observed. This results shows that by addition of KP, the half dose of NKP produces 80% activity compared to full dose of NKP. Therefore, it was established that this activity of the composition is due to synergistic effect of KP extract on NKP extract.
METHOD 2: Mixing KP and NKP in powder form in required ratio and then soaking in water for three hours
In this method NKP and KP powders were mixed in different ratios like 1:1, 3:1 and 1:3. The 1% aqueous extracts of NKP+KP mixtures were prepared by soaking 10g of mixture in 1000 ml water for three hours followed by filtering through a fine muslin cloth. The remains on the cloth were squeezed into the beaker. The 1% aqueous extracts of pure NKP and pure KP were also prepared by same procedure. The bioefficacy experiments were performed with these freshly prepared extracts.

Example 3
Synergistic Effect on Bio-efficacy of KP and NKP on mosquito larvae.
The same procedure as described in Example 1 was followed for bioefficacy experiment
Table 3: Synergistic Effect on Bio-efficacy of KP and NKP on mosquito larvae.

(Table Removed)
The above results showed that larvicidal activity produced by pure NKP extract was 40%, whereas, pure KP solution had very less larvicidal activity at par with control i.e. without any larvicidal solution.
The larvicidal activity of the composition was maximum (60%) for the suspension containing NKP and KP in 3:1 ratio, which was much higher than the individual larvicidal activities of NKP and KP.
Therefore, it was established that this enhanced larvicidal activity of the composition was due to synergistic effect of KP extract on NKP extract.
METHOD 3: Mixing KP and NKP in powder form in required ratio and then soaking in water for 24 hours
In this method NKP and KP powders were mixed in different ratios like 1:1, 3:1 and 1:3 and then 1% aqueous extracts were prepared by soaking 10g of mixture in 1000 ml water for 24 hours followed by filtering through a fine muslin cloth. The remains on the cloth were squeezed into the beaker. The 1% aqueous extracts of pure NKP and pure KP were also prepared by same procedure. The bioefficacy experiments were performed with these freshly prepared extracts.
Example 4
Synergistic Effect on Bio-efficacy of of KP and NKP on mosquito larvae.
The same procedure as described in Example 1 was followed for bioefficacy experiment
Table 4: Synergistic Effect on Bio-efficacy of KP and NKP on mosquito larvae.

(Table Removed)
The above results showed that larvicidal activity produced by pure NKP extract was 40% whereas, pure KP extract had very less larvicidal activity at par with control i.e. without any larvicidal solution.
Results of the above table show that the larvicidal activity of the composition was maximum for the solution containing NKP and KP in 3:1 ratio, which is at par with individual larvicidal activities of NKP
Therefore, it was established that if method No. 3 is adopted for extraction, 25% of NKP may be replaced by KP to produce equivalent bioefficacy.
Example 5:
Synergistic Effect on Bio-efficacy of KP and NKP on Pieris brassicae (Cauliflower)
The extraction procedure described in method No. 3 was adopted, in place of 1% extracts, NKP 2.5%, NKP 3.75% , NKP 5%, and KP 5% and NKP 2.5%+ KP2.5% extracts were used in the experiments. Mortality of first instar larvae (N=20; replications 3) was recorded after 48 hours of release.
Table 5: Laboratory studies on Pieris brassicae (Cauliflower)

(Table Removed)
It is clear from table 5 that pure KP shown mortality rate at par with control i.e., without any insecticidal solution. The mortality with pure NKP -5% was 93 %. The mortality increased to 100% with the composition comprising NKP 2.5% + KP 2.5% The results of the study on Pieris brassicae larvae again establish that KP has synergistic effect on larvicidal activity of NKP.
Example 6: Synergistic Effect on Bio-efficacy of of KP and NKP on Helicoverpa armigera (Gram pod)
The procedure described in Example 5 was adopted for doing the bioefficacy studies on Helicoverpa armigera (Gram pod)
Table 6: Results on mortality of Helicoverpa armigera (Gram pod) larvae (N=20)

(Table Removed)
It is clear from table 6 that control and pure KP solution produced 0% mortality. The mortality with pure NKP -2.5 % & 5% after 7 days was 75 %. The mortality increased to 100% with the composition comprising NKP 2.5% + KP 2.5% . The results of the
study on Helicoverpa armigera again established that KP has synergistic effect on larvicidal activity of NKP.
Table 7: Results of choice feeding experiment:

(Table Removed)
The above results show that the damage to gram pod was maximum i.e., 20% in case of control and KP-5%. The damage was minimum i.e., 0% in case of NKP 5%. In the case of NKP 2. 5% + KP 2.5 %, the damage is only 10%, which is better than NKP 3.75%, which shows that NKP at 2.5% level gives the better result compared to NKP 3.75% level, if KP 2.5% is added to it. This established that KP has synergistic effect on insecticidal activity of NKP.
The results obtained in various studies described hereinabove have established that kabuli keekar (Prosopis juliflora) had enhanced the insecticidal and larvicidal activities of neem (Azadirachta indica) in the present invention. This enhancement of insecticidal and larvicidal activities of the insecticidal composition of the present invention was due to synergistic interaction between the kabuli keekar {Prosopis juliflora) pods extract and neem (Azadirachta indica) seed (kernel) extract.
The composition of the present invention is natural (botanical) insecticidal composition obtained from the naturally occurring plant based substances and is devoid of any synthetic ingredients. Therefore it is non-polluting and does not poses any toxic side effect or hazardous effects on human, animals and to the environment. The kabuli keekar plants are found abundantly in the country .therefore these plant based materials are available at very low cost. The use of economical kabuli keekar as synergist also reduces the cost of neem application.
The compositions can be used for effective insect control in agriculture, household and public health sectors. The present invention provides effective compositions for controlling insects in organic farming (chemical pesticides free farming). The compositions of the present invention can be used by the farmers to control insects in organic farming and thereby increasing the product quality and yield.

Claim:
1. A synergistic insecticidal and larvicidal botanical composition comprising neem (Azadirachta indica) and kabuli keekar (Prosopis juliflora) plant extracts.
2. A synergistic insecticidal and larvicidal botanical composition as claimed in claim 1, wherein the concentration of said neem (Azadirachta indica) extract is between 25% to 75% by weight.
3. A synergistic insecticidal and larvicidal botanical composition as claimed in claim 1, wherein the concentration of said kabuli keekar (Prosopis juliflora) extract is between 25% to 75% by weight.
4. A synergistic insecticidal and larvicidal botanical composition as claimed in any of the claims 1 to 3, wherein the ratio of said neem (Azadirachta indica) extract to said kabuli keekar (Prosopis juliflora) extract (NKP: KP) is 3 :1.
5. A synergistic insecticidal and larvicidal botanical composition as claimed in any of the claims 1 to 3, wherein the ratio of said neem (Azadirachta indica) extract to said kabuli keekar (Prosopis juliflora) extract (NKP: KP) is 1 :1.
6. A synergistic insecticidal and larvicidal botanical composition as claimed in claim 1, wherein the said neem (Azadirachta indica) extract is obtained from neem (Azadirachta indica) seeds or kernel.
7. A synergistic insecticidal and larvicidal botanical composition as claimed in claim 1 wherein said kabuli keekar (Prosopis juliflora) extract is obtained from kabuli keekar (Prosopis juliflora) pods.
8. A synergistic insecticidal and larvicidal botanical composition as claimed in claim i, which can be used against agricultural and household insects and larvae.
9. A method of using the synergistic insecticidal and larvicidal botanical composition of claim 1 comprising the steps of:
a. taking the dry powders of said neem (NKP) and said kabuli keekar (KP) and
mixing them in the required ratio;
b. soaking desired quantity of the said mixture thus obtained in step (a) in water
for 3/24 hours;
c. filtering the said soaked mixture through fine muslin cloth;
d. collecting the said freshly prepared extract and using against insects and
larvae as per the requirement.
10. A method of using the synergistic insecticidal and larvicidal botanical
composition of claim 1 comprising the steps of:

a. taking dry powder extract of said neem (NKP) and said kabuli keekar (KP)
and soaking them separately in water for overnight;
b. filtering the said soaked extracts of step (a) through a fine muslin cloth;
c. collecting freshly prepared aqueous extracts obtained in step (b),
separately;
d. mixing the said aqueous extracts in desired ratio and using the said
obtained aqueous mixture against insects and larvae as per requirement.

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Patent Number

271924

Indian Patent Application Number

1627/DEL/2009

PG Journal Number

11/2016

Publication Date

11-Mar-2016

Grant Date

10-Mar-2016

Date of Filing

04-Aug-2009

Name of Patentee

INSTITUTE OF PESTICIDE FORMULATION TECHNOLOGY

Applicant Address

SECTOR-20, UDYOG VIHAR NH-8, GURGAON-122016, INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	BHAVANA SRIVASTAVA	INSTITUTE OF PESTICIDE FORMULATION TECHNOLOGY, SECTOR 20, UDYOG VIHAR, NH-8, GURGAON-122016, INIDA.
2	PHOOL KUMAR PATANJALI	INSTITUTE OF PESTICIDE FORMULATION TECHNOLOGY, SECTOR 20, UDYOG VIHAR, NH-8, GURGAON-122016, INIDA.
3	AMRISH AGRAWAL	INSTITUTE OF PESTICIDE FORMULATION TECHNOLOGY, SECTOR 20, UDYOG VIHAR, NH-8, GURGAON-122016, INIDA.
4	VISHAL JHELUM	INSTITUTE OF PESTICIDE FORMULATION TECHNOLOGY, SECTOR 20, UDYOG VIHAR, NH-8, GURGAON-122016, INIDA.

PCT International Classification Number

A01N 65/08

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA