Combination of phytochemicals to potentiate the effect of pesticides

Abstract

The present invention relates to a pesticidal composition comprising an effective amount of phytochemical(s), which optionally combined with commercially known pesticide and agrochemically acceptable excipients or vehicle, and a process for the preparation thereof. The present invention provides a pesticidal composition, which results in enhanced insecticidal efficacy, lower effective doses, multi-target pest control and decreases the possibility of developing the resistance in insects.

Description

[0001]COMBINATION OF PHYTOCHEMICALS TO POTENTIATE THE EFFECT OF PESTICIDES FIELD OF THE INVENTION The present invention relates to a combination of phytochemicals to potentiate the effect of pesticides. More particularly, the present invention relates to a pesticidal composition comprising an effective amount of phytochemical(s), which optionally combined with commercially known pesticide and process for the preparation thereof. BACKGROUND OF THE INVENTION Insects and pests are abundant species on the Earth and mostly they survive on plant tissues as their nutrition that creates a considerable agricultural loss and lowers the crop quantity. Further these insects / pests create damage not only quantitatively but also qualitatively. Increased resistance to pesticides now became a major problem in agriculture; as generations pass, resistant genotypes become more common in insects, lowering the overall effectiveness of the pesticide. Conventional pesticides are inadequate to cope with this problem. Traditional pesticides are less effective because of an increase in resistant insect populations caused by the overuse of these pesticides. This necessitates the development of new, long term pest management strategies. Reference may be made to the article “Biological Reviews, 94(1), pp.135–155” reviews that the effectiveness of pesticides is threatened by the evolution of resistant pathogens, weeds and insect pests. Pesticides are mostly novel synthetic compounds, and yet target species are often able to evolve resistance soon after a new compound is introduced. Therefore, pesticide resistance provides an interesting case of rapid evolution under strong selective pressures. Phytochemicals are plant-based bioactive compounds produced by plants for their protection. They can be derived from various sources such as whole grains, fruits, vegetables, nuts, and herbs. So many phytochemicals are known in the literature till date.The phytochemicals in the formulation are obtained from biological sources and are non-toxic to humans at lower concentration. Single phytochemical formulation alone also can act as insecticidal agent and can act synergistically to increase the pesticide activity. This combination can be used with a wide range of pesticides. Combination of multiple molecules reduces the possibility of emergence of resistance in insects. Reference may be made to the article “Journal of Agriculture and Food Research Volume 9, September 2022, 100345” reviews a botanical pesticides, phytochemical composition and importance in agricultural production. It further reports that botanical pesticides are efficacious inmanaging different crop pests, inexpensive, easily biodegraded, have varied modes of action; their sources are easily available and have low toxicity to non-target organisms. It is required to come up with an effective pesticide that can withstand with these problems and which can reduce the chance of emergence of resistance in insects. Along with this, there should not be human harm by usage of pesticide and it should be effective even in less amount. OBJECTIVES OF THE INVENTION Main objective of the present invention is to provide a pesticidal composition comprising a combination of phytochemical(s), which optionally combined with commercially known pesticide. Another objective of the present invention is to provide a process of preparation of said pesticidal composition comprising an effective amount of phytochemical(s), which optionally combined with commercially known pesticide. SUMMARY OF THE INVENTION Accordingly, in order to accomplish an objective, the present invention provides a pesticidal composition comprising an effective amount of phytochemical(s), which optionally combined with commercially known pesticide. In an embodiment, the present invention provides a pesticidal composition comprising: i. at least two phytochemical agents; ii. an agrochemically acceptable excipient or vehicle; wherein the at least two phytochemical agents are selected from the group consisting of a phenolic compound, a flavonoid, an alkaloid, a curcuminoid, an isoflavone, and a carotenoid or any of combination thereof. In another embodiment of the present invention, the pesticidal composition optionally comprises a pesticidal compound / agent. In another embodiment of the present invention, the composition comprises at least three phytochemical agents or at least four phytochemical agents. In another embodiment of the present invention, the flavonoid is selected from the group consisting of gossypetin, herbacetin, isoquercitrin, myricetin, naringenin, rutin and quercetin or any of combination thereof; In another embodiment of the present invention, the phenolic compound is selected from the group consisting of eugenol, gallic acid, ferulic acid, caffeic acid, chlorogenic acid, thymol, carvacrol, ellagic acid, vanillic acid, syringic acid, p-coumaric acid, protocatechuic acid or any of combination thereof; In another embodiment of the present invention, the alkaloid is selected from the group consisting of coptisine, palmatine, jatrorrhizine and berberine or any of combination thereof; In another embodiment of the present invention, the curcuminoid is selected from the group consisting of demethoxycurcumin, resveratrol and curcumin or any of combination thereof; In another embodiment of the present invention, the isoflavone is selected from the group consisting of genistein, daidzein, glycitein, formononetin, biochanin A, prunetin, puerarin, calycosin, ononin or any of combination thereof; and In another embodiment of the present invention, the carotenoid is selected from the group consisting of β-carotene, α-carotene, lutein, zeaxanthin, lycopene, astaxanthin, violaxanthin, neoxanthin, canthaxanthin, fucoxanthin, capsanthin or any of combination thereof. In another embodiment of the present invention, the pesticidal compound / agent is selected from the group consisting of aldrin, dieldrin, chlordane, dichlorodiphenyltrichloroethane, endrin, heptachlor, mirex, toxaphene, abamectin, ivermectin, doramectin, eprinomectin, selamectin, moxidectin and emamactin. In another embodiment of the present invention, the agrochemically acceptable excipient or vehicle comprises a solvent. In another embodiment of the present invention, the solvent is selected from dimethylsulfoxide and water or combination thereof. In another embodiment of the present invention, the amount or concentration of the at least two phytochemical agents is in the range of 25-75% w / v. In another embodiment of the present invention, the amount or concentration of the pesticidal compound / agent is in the range of 5-20% w / v. In another embodiment of the present invention, the rest is the agrochemically acceptable excipient or vehicle to make up 100% w / v of the composition. Another embodiment of the present invention provides a pesticidal composition which comprises: a) flavonoid, b) phenolic compound, c) alkaloid, d) curcuminoid, e) pesticidal compound / agent; and f) agrochemically acceptable excipient or vehicle. In another embodiment of the present invention, the amount or concentration of the flavonoid in the composition is in the range of 5-30% w / v. In another embodiment of the present invention, the amount or concentration of the alkaloid in the composition is in the range of 5-30% w / v. In another embodiment of the present invention, the amount or concentration of the curcuminoid in the composition is in the range of 5-30% w / v. In another embodiment of the present invention, the amount or concentration of the phenolic compound in the composition is in the range of 0.005-0.04% w / v. In another embodiment of the present invention, the amount or concentration of the commercially known pesticidal compound / agent in the composition is in the range of 5- 20% w / v. In another embodiment of the present invention, the agrochemically acceptable excipient or vehicle is added to make up 100% w / v of the composition. Another embodiment of the present invention provides a pesticidal composition which comprises: a) rutin, b) quercetin, c) eugenol, d) berberine, e) curcumin, f) pesticidal compound / agent; and g) agrochemically acceptable excipient or vehicle. Another embodiment of the present invention provides a process for the preparation of said pesticidal composition comprising: i. mixing at least two phytochemical agents in agrochemically acceptable excipient or vehicle under stirring at temperature in the range of 25-35 ℃ to obtain a 1st mixture; ii. adding and mixing commercially known pesticidal compound / agent in said 1st mixture of step i) under stirring at temperature in the range of 25-35 ℃ to obtain 2nd mixture; and iii. adding agrochemically acceptable excipient or vehicle to obtain the pesticidal composition. Another embodiment of the present invention provides the process for preparing stock solution with these phytochemicals either separately or in combination comprises of dissolving the phytochemical in DMSO solvent and diluting it with tap water. BRIEF DESCRIPTION OF THE DRAWINGS FIG.1 illustrates the percentage survival (a) and Body weight (b) of insects fed on castor leaves of each treatment (N=84). FIG.2 illustrates Castor leaves eaten by insects on the fifth day. Leaves were sprayed with a) 0.1% DMSO (v / v) b) 10µg / ml rutin c) 10µg / ml Quercetin d) 10µg / ml Berberine e) 10µg / ml Curcumin f) 0.1 µl / ml Eugenol g) Combination of phytochemicals. FIG.3 illustrates the images of insects on the third day fed on leaves sprayed with a) 1µl / ml DMSO b)10µg / ml rutin c) 10µg / ml Quercetin d) 10µg / ml Berberine e) 10µg / ml Curcumin f) 0.01 µl / ml Eugenol g) Combination of phytochemicals. FIG.4 illustrates the percentage survival (a) and Body weight (b) of insects fed on castor leaves of each treatment (N=84) . FIG.5 illustartesCastor leaves eaten by insects on the third day. Leaves were sprayed with (a) 0.2% DMSO (b) 0.2% DMSO (v / v) and 10 ppm Emamactin (c) 0.2% DMSO, and a combination of phytochemicals (d) 0.2% DMSO, 10 ppm Emamactin and a combination of phytochemicals. FIG.6 illustrates the images of insects on the third day fed on leaves sprayed with (a) 0.2% DMSO (b) 0.2% DMSO and 10 ppm Emamactin (c) 0.2% DMSO, and a combination of phytochemicals (d) 0.2% DMSO, 10 ppm Emamactin and a combination of phytochemicals. DETAILED DESCRIPTION OF THE INVENTION The present invention provides a pesticidal composition comprising an effective amount of phytochemical(s), which optionally combined with commercially known pesticide. In an embodiment, the present disclosure provides a pesticidal composition comprising: i. at least two phytochemical agents; ii. commercially known pesticidal compound / agent; and iii. agrochemically acceptable excipient or vehicle; wherein the at least two phytochemical agents are selected from phenolic compound, flavonoid, alkaloid, curcuminoid, isoflavone, and carotenoid or any of combination thereof. In an embodiment, the composition comprises at least three phytochemical agents. In an embodiment, the composition comprises at least four phytochemical agents. In an embodiment, the at least three phytochemical agents are selected from phenolic compound, flavonoid, alkaloid, curcuminoid, isoflavone, and carotenoid or any of combination thereof. In an embodiment, the at least four phytochemical agents are selected from phenolic compound, flavonoid, alkaloid, curcuminoid, isoflavone, and carotenoid or any of combination thereof. In an embodiment, the flavonoid is selected from gossypetin, herbacetin, isoquercitrin, myricetin, naringenin, rutin and quercetin or any of combination thereof. In an embodiment, the phenolic compound is selected from eugenol, eugenol, gallic acid, ferulic acid, caffeic acid, chlorogenic acid, thymol, carvacrol, ellagic acid, vanillic acid, syringic acid, p-coumaric acid, protocatechuic acid or any of combination thereof. In an embodiment, the alkaloid is selected from coptisine, palmatine, jatrorrhizine and berberine or any of combination thereof. In an embodiment, the curcuminoid is selected from demethoxycurcumin, resveratrol and curcumin or any of combination thereof. In an embodiment, the isoflavone is selected from genistein, daidzein, glycitein, formononetin, biochanin A, prunetin, puerarin, calycosin, ononin or any combination thereof. In an embodiment, the carotenoid is selected from β-carotene, α-carotene, lutein, zeaxanthin, lycopene, astaxanthin, violaxanthin, neoxanthin, canthaxanthin, fucoxanthin, capsanthin or any combination thereof. In an embodiment, the commercially known pesticidal compound / agent is selected from aldrin, dieldrin, chlordane, dichlorodiphenyltrichloroethane (DDT), endrin, heptachlor, mirex, toxaphene, abamectin, ivermectin, doramectin, eprinomectin, selamectin, moxidectin and emamactin or any of combination thereof. In an embodiment, the agrochemically acceptable excipient or vehicle comprises a solvent, wherein the solvent is selected from dimethylsulfoxide and water or combination thereof. In an embodiment, the amount or concentration of the at least two phytochemical agents is in the range of 25-75% w / v. In an embodiment, the amount or concentration of the commercially known pesticidal compound / agent is in the range of 5-20% w / v. In an embodiment, the rest is the agrochemically acceptable excipient or vehicle to make up 100% w / v of the composition. In an embodiment, the amount or concentration of the at least two phytochemical agents is in the range of 30-75% w / v. In an embodiment, the amount or concentration of the at least two phytochemical agents is in the range of 35-75% w / v. In an embodiment, the amount or concentration of the at least two phytochemical agents is in the range of 40-75% w / v. In an embodiment, the amount or concentration of the at least two phytochemical agents is in the range of 45-75% w / v. In an embodiment, the amount or concentration of the at least two phytochemical agents is in the range of 50-75% w / v. In an embodiment, the amount or concentration of the at least two phytochemical agents is in the range of 55-75% w / v. In an embodiment, the amount or concentration of the at least two phytochemical agents is in the range of 30-70% w / v. In an embodiment, the amount or concentration of the at least two phytochemical agents is in the range of 30-65% w / v. In an embodiment, the amount or concentration of the at least two phytochemical agents is in the range of 30-60% w / v. In an embodiment, the amount or concentration of the at least two phytochemical agents is in the range of 30-55% w / v. In an embodiment, the amount or concentration of the at least two phytochemical agents is in the range of 30-50% w / v. In an embodiment, the amount or concentration of the at least two phytochemical agents is in the range of 30-45% w / v. In an embodiment, the amount or concentration of the at least two phytochemical agents is in the range of 30-40% w / v. In an embodiment, the amount or concentration of the flavonoid in the composition is in the range of 5-30% w / v. In an embodiment, the amount or concentration of the flavonoid in the composition is in the range of 7.5-25% w / v. In an embodiment, the amount or concentration of the flavonoid in the composition is in the range of 7.5-20% w / v. In an embodiment, the amount or concentration of the flavonoid in the composition is in the range of 10-20% w / v. In an embodiment, the amount or concentration of the alkaloid in the composition is in the range of 5-30% w / v. In an embodiment, the amount or concentration of the alkaloid in the composition is in the range of 7.5-25% w / v. In an embodiment, the amount or concentration of the alkaloid in the composition is in the range of 7.5-20% w / v. In an embodiment, the amount or concentration of the alkaloid in the composition is in the range of 10-20% w / v. In an embodiment, the amount or concentration of the curcuminoid in the composition is in the range of 5-30% w / v. In an embodiment, the amount or concentration of the curcuminoid in the composition is in the range of 7.5-25% w / v. In an embodiment, the amount or concentration of the curcuminoid in the composition is in the range of 7.5-20% w / v. In an embodiment, the amount or concentration of the curcuminoid in the composition is in the range of 10-20% w / v. In an embodiment, the amount or concentration of the phenolic compound in the composition is in the range of 0.005-0.04% w / v. In an embodiment, the amount or concentration of the phenolic compound in the composition is in the range of 0.005-0.03% w / v. In an embodiment, the amount or concentration of the phenolic compound in the composition is in the range of 0.005-0.02% w / v. In an embodiment, the amount or concentration of the phenolic compound in the composition is in the range of 0.01-0.02% w / v. In an embodiment, the amount or concentration of the commercially known pesticidal compound / agent in the composition is in the range of 5-18% w / v. In an embodiment, the amount or concentration of the commercially known pesticidal compound / agent in the composition is in the range of 5-15% w / v. In an embodiment, the amount or concentration of the commercially known pesticidal compound / agent in the composition is in the range of 7.5-15% w / v. In an embodiment, the amount or concentration of the commercially known pesticidal compound / agent in the composition is in the range of 10-20% w / v. In an embodiment, the pesticidal composition comprises: a) flavonoid, b) phenolic compound, c) alkaloid, d) demethoxycurcumin, resveratrol or curcumin, e) commercially known pesticidal compound / agent; and f) agrochemically acceptable excipient or vehicle. In an embodiment, the amount or concentration of the flavonoid in the composition is in the range of 5-30% w / v. In an embodiment, the amount or concentration of the alkaloid in the composition is in the range of 5-30% w / v. In an embodiment, the amount or concentration of the curcuminoid in the composition is in the range of 5-30% w / v. In an embodiment, the amount or concentration of the phenolic compound in the composition is in the range of 0.005-0.04% w / v. In an embodiment, the amount or concentration of the commercially known pesticidal compound / agent in the composition is in the range of 5-20% w / v. In an embodiment, the pesticidal composition comprises: a) gossypetin, herbacetin, isoquercitrin, myricetin, naringenin, or rutin, b) quercetin, c) eugenol, d) coptisine, palmatine, jatrorrhizine or berberine, e) demethoxycurcumin, resveratrol or curcumin, f) commercially known pesticidal compound / agent; and g) agrochemically acceptable excipient or vehicle. In an embodiment, the pesticidal composition comprises: a) rutin, b) quercetin, c) eugenol, d) berberine, e) curcumin, f) commercially known pesticidal compound / agent; and g) agrochemically acceptable excipient or vehicle. In an embodiment, the amount or concentration of the rutin in the composition is in the range of 5-30% w / v. In an embodiment, the amount or concentration of the quercetin in the composition is in the range of 5-30% w / v. In an embodiment, the amount or concentration of the berberine in the composition is in the range of 5-30% w / v. In an embodiment, the amount or concentration of the curcumin in the composition is in the range of 5-30% w / v. In an embodiment, the amount or concentration of the eugenol in the composition is in the range of 0.005-0.04% w / v. In an embodiment, the amount or concentration of the commercially known pesticidal compound / agent in the composition is in the range of 5-20% w / v. In an embodiment, the present disclosure provides a process of preparation of the pesticidal composition, comprising: i. mixing at least two phytochemical agents in agrochemically acceptable excipient or vehicle under stirring at temperature in the range of 25-35 ℃ to obtain a 1stmixture; ii. adding and mixing commercially known pesticidal compound / agent in said 1stmixture of step i) under stirring at temperature in the range of 25-35 ℃ to obtain 2ndmixture; and iii. optionally adding agrochemically acceptable excipient or vehicle to obtain the pesticidal composition. In an embodiment of the present invention the phytochemcials are selected from Polyphenols, Flavonoids, alkaloids, curcuminoids, Isoflavones, Carotenoids and the like. Particularly, Flavonoids such as Gossypetin, Herbacetin, Isoquercitrin, Myricetin, Naringenin, rutin and quercetin, alkaloids such as Coptisine, Palmatine, Jatrorrhizineand berberine and curcuminoids such as Demethoxycurcumin, Resveratroland curcumin and the like, Secondary metabolites. The commercially know pesticide is selected from aldrin, dieldrin, chlordane, DDT, endrin, heptachlor, mirex, toxaphene, Abamectin, Ivermectin, Doramectin, Eprinomectin, Selamectin, Moxidectin and Emamactin or combinations thereof. In particularly useful embodiment, the phytochemicals are selected from rutin, berberin, eugenol, quercetin, and curcumin or a combination of all and wherein commercially known pesticide is emamectin. In an embodiment, the present invention provides a pesticidal composition comprising an effective amount of phytochemical(s), which optionally combined with commercially known pesticide, wherein the composition of pesticides in pesticidal composition is rutinin the range of 20-30%, berberin in the range of 20-30%, eugenol in the range of 0.01-0.04%, quercetin in the range of 20-30%, and curcumin in the range of 20-30% with different combinations or a combination of all and wherein commercially known pesticide is emamectin. The pesticidal composition further comprises of suitable solvents selected from DMSO (0.1 % v / v) and water. In particularly useful embodiment, the pesticidal composition comprises of 10% rutin, 10% berberin, 0.01% eugenol, 10% quercetin, and 10% curcumin. Further the composition optionally may contain emamectin pesticide of 10 mg per 1 ml of DMSO. Berberine has a wide range of biological activities, engineered arabidopsis plants produce more berberine. In comparison to unmodified plants, the engineered plants showed higher levels of berberine and much lower growth and mortality rates in diamondback moth larvae. This shows that berberine could be an environmentally beneficial alternative to chemical pesticides. Curcumin, another natural substance with insecticidal activity, further strengthens the case for exploring botanical alternatives. Curcumin, the brilliant yellow ingredient in turmeric, shows potential as a natural pesticide. It inhibits insect growth and behavior, acting as a repellant, pesticide, and growth modulator. Eugenol is another aromatic compound found in clove oil that can serve as an insecticide against Thrips nymphs (T. erytreae), a harmful plant pest. Interestingly, Eugenol's insecticidal activity increased over time, with mortality rates rising within 48 hours of application. Naturally occurring flavonoids, such as Rutin and Quercetin, are also possible bioinsecticides as it plays a potential role to control the grasshopper species Calliptamusabbreviatus. Another embodiment of the present invention provides a process for the preparation of said pesticidal composition comprising an effective amount of phytochemical(s), which optionally combined with commercially known pesticide. The process for preparing stock solution with these phytochemicals either separately or in combination comprises of dissolving the phytochemical in DMSO solvent and diluting it with tap water. More particularly, the process for preparing different stock solutions of these above-said phytochemials optionally with commercially known pesticide and solvents comprises of dissolving each rutin, quercetin, berberine, curcumin and eugenol independently or in combinations in DMSO solvent and further adding tap water to make the final solutions, respectively; optionally mixing the solution of pesticide in DMSO and water with the above prepared solutions of phytochemicals. In an aspect of an embodiment, the present invention provides that by combining five well- studied phytochemicals (berberine, curcumin, rutin, quercetin, and eugenol), significant insecticidal effects against Spodopterafrugiperda larvae at lower concentrations than previously reported for individual components are achieved. This synergistic effect suggests exciting possibilities for reducing the environmental impact of pesticides. The efficacy of the combinations have been increased when it is combined with well-known insecticide, emamectin, provides up possibilities to more integrated pest management strategies. EXAMPLES The following examples illustrate the nature of the invention and are provided for illustrative purposes only and should not be construed to limit the scope of the invention. Materials All the phytochemcials utilized are commercially sourced. DMSO, Rutin, and Quercetin (Merck, Sigma-Aldrich, IN), Berberine (BLD pharma, India), Curcumin (BLD pharma, India), Eugenol (BLD pharma, India). All the phytochemicals utilized in the present invention Rutin, Quercetin, Berberine, and Curcumin were obtained in powder form. Eugenol was procured in its liquid form as it is naturally an essential oil component. Example 1: Preparation of stock solutions Solution A: mixing 100 µL of DMSO and 100 mL of tap water. Solution B-E: dissolving 10 mg of each rutin, quercetin, berberine and curcumin independently in 700 µL DMSO, further adding DMSO to make the final volume up to 1 mL and further adding 100 µL of this solution to 100 mL of tap water to make the final solutions B, C, D and E, respectively. Solution F: adding 10 µL of eugenol into 990 µL DMSO and further adding 100 µL of this solution to 100 mL of tap water to make the final solution. Solution G: mixing 10 mg of each rutin, quercetin, berberine, and curcumin and dissolving in 700 µL DMSO, further adding 10 µL of eugenol in DMSO to raise the volume up to 1 mL to afford solution G1; followed by mixing 100 µL of solution G1 with 100 mL of tap water to make the final solution G. Solution H: mixing 200 µL of DMSO with 100 mL of tap water. Solution I: dissolving 10 mg emamectin in 1 mL DMSO and mix the solution by pipetting to afford solution I1; followed by mixing 100 µL of solution I1 with 100 µL of DMSO and 100 mL of tap water to make the final solution I. Solution J: mixing 100 µL of solution G1, 100uL of DMSO, and 100 mL of tap water to make the final solution J. Solution K: Add 100 µL of solution G1 and 100 µL of solution I1 to 100 mL of tap water to make the final solution K. Example 2: Insect bioassay to check the insecticidal effect of individual components of the combination Detached-leaf feeding insect bioassay was performed using second instar larvae of Spodoptera frugiperda fed on a diet of castor leaves. Castor leaves were cut into uniform discs of 3cm radius and placed in Petri plates. These leaves were separated into seven sets and sprayed with solutions A (0.1% DMSO), B (10µg / ml rutin), C (10µg / ml quercetin), D (10µg / ml berberine), E (10µg / ml curcumin), F (0.1 µl / ml eugenol) or G (combination of phytochemicals) and left to dry for 15 mins. Once dried, twenty-one S. frugiperda larvae each were fed on leaves sprayed with solutions A, B, C, D, E, F, and G respectively. These leaves were replaced by fresh leaves sprayed with the same solution every day. The assay was carried out over four days and the mortality and body weight of the larvae are recorded on each day. Example 3: Insect bioassay to check the insecticidal effect of combination along with emamectin Detached-leaf feeding insect bioassay was performed using second instar larvae of Spodopterafrugiperda and castor leaves as previously described. The castor leaves were sprayed with solutions H (0.2% DMSO), I (10 ppm emamectin), J (combination of phytochemicals), or K (10 ppm emamectin and combination of phytochemicals) and left to dry for 15 mins. Once dried, twenty-one S. frugiperdalarvae each were fed on leaves sprayed with solutions H, I, J, and K respectively. These leaves were replaced by fresh leaves sprayed with the same solution every day. The assay was carried out for three days and the mortality and body weight of the larvae were recorded on each day. Insecticidal effect of the phytochemical combination The percentage of survival and body weight of insects are recorded across all five days of the detached-leaf bioassay to compare the insecticidal effects of the individual phytochemicals (groups A-F) to that of the combination (group G). The survival percentage of insects on the fifth day of the bioassay are found to be 90.47%, 100%, 90.47%, 66.66%, 90.47%, 61.90% and 33.33% for groups A (DMSO), B (rutin), C (quercetin), D (berberine), E (curcumin), F (eugenol) and G (combination of phytochemicals) respectively. The survival percentage (FIG.1a) and body weight (FIG. 1b) of insects are lower in the group treated with the combination of phytochemicals than in the groups treated with the individual components of the phytochemical combination. The images of the leaves eaten by the insects on the fifth day are shown in FIG.2 and the images of insects on the fifth day are shown in FIG.3. The concentration of DMSO used is 0.1% (v / v), which showed about 9.5% mortality. Toxicity of DMSO against Drosophila has been reported to have a LC50 concentration of 0.42% (v / v).(Cvetković et al.,Biol Nyssana, 6(2), pp. 91–95, 2015). Rutin used at a concentration of 10 µg / ml (0.001%) does not show any insecticidal activity, nor does it have an effect on the insect body weight. Rutin and quercetin have been previously reported to have an effect on the survival rate of grasshoppers when used at concentrations higher than 0.01%.(Huang, Zheng and Wang, Insects, 15(2), p. 95, 2024) Insects fed on leaves sprayed with 10 µg / ml (0.001%) quercetin and curcumin, both show similar trends in body weight and survivability as those fed on DMSO sprayed leaves. Investigation on curcuminoids have found a dosage of 50 µg produced a 45% growth inhibition in Dysdercuskoenigii.(Roy, Chakraborty and ParthasarathiNandy,American Scientific Research Journal for Engineering, Technology, and sciences, 7(1), pp.31–43, 2014). It is shown that both 10 µg / ml berberine and 0.1 µl / ml (0.01%) eugenol treatments have had a significant effect in survivability and body weight of insects. The anti-insect effects of berberine have been reported. They have reported an EC50 value in the range of 2-4 µg / ml for different Leishmaniaspecies and a value of 10µg / ml for Plasmodium falciparum.(Li et al., Journal of Asian natural products research, 20(2), pp. 148–162, 2018) Similarly, mortality in Triozaerytreaeis reported when exposed to eugenol at concentrations greater than 0.4% (v / v), which is much greater than the 0.01% concentration that is used in the present invention.(Sousa et al.,Journal of Natural Pesticide Research, 1, p.100004, 2022) Two-way ANOVA reveals a significant effect of treatments on body weight. Dunnett's post hoc test indicates no significant difference in body weight between the control group (DMSO) and the rutin, quercetin, berberine, or curcumin groups (all p > 0.05). However, insects in the eugenol group (show two-star significance, whichindicates a statistically significant difference between Eugenol and the control DMSO group, with a p-value likely less than 0.01 but greater than 0.001. Eugenol treatment appears to reduce body weight compared to the control.) and the combination of thephytochemicals group (showing four- star significance, which implies a highly significant difference between the combination group and the control. The p-value here is likely less than 0.0001, indicating a very strong effect.) have significantly lower body weights compared to the control group (DMSO). However, when compared to each individual phytochemical, the combination of phytochemicals has the highest effect on insect survival and body weight. The concentrations of all components of the phytochemicals are lower than that of the effective concentrations as reported in previous studies. Hence, they have had little effect on the growth and survival of the insects. However, when the combination is prepared with the same concentration of the individual phytochemicals, there is a significant increase in the growth and survival of the insects. Effect of the phytochemical combination along with emamectin When the effects of the combinations of phytochemicals are studied along with that of a known pesticide, emamectin, the survival percentage of insects in groups H (DMSO), I (emamectin), J (Combination of phytochemicals), and K (emamectin and combination of phytochemicals) are found to be 85.71%, 80.95%, 57.14% and 47.61 respectively. (FIG. 4 a) A similar trend is observed while comparing the insect body weight across 2 days. The insects in the group fed on leaves sprayed with solution K, which is a mix of emamectin and the phytochemical combination, showed the lowest survival percentage as well as the lowest body weight (FIG.4 b) over the period of the assay. The images of the leaves eaten (FIG.5) and of the insects (FIG.6) on the third day are shown. Emamectin which is a commercial pesticide has a LC50 value of 0.269 μg / mL against Helicoverpaarmigera (Dagar and Kumar, 2018). Two-way ANOVA reveals a significant main effect of treatment on insect body weight. Dunnett's post hoc test indicated a significant difference (four-star significance for each treatment) in body weight between the control group (DMSO) and all other treatment groups (Emamectin, Combination of phytochemicals, and Emamectin + Combination of phytochemicals). However, to isolate the effect of the combined treatment, the effect of emamectin and the combination of phytochemicals together is greater than that of either of their effect. ADVANTGAES OF THE INVENTION a) Traditional pesticides are becoming less effective due to rising insect resistance. The present invention provides a synergistic phytochemical combination as an alternative insecticidal strategy. b) The present invention results in enhanced insecticidal efficacy, lower effective doses, multi-target pest control and decreases the possibility of developing the resistance in insects. c) The present invention avoids synthetic chemical residues and is safer for non-target organisms. d) Phytochemicals based pesticide is environment friendly as it breaks down easily into non-toxic degradation products which are not harmful to beneficial insects and wildlife.

Claims

AMENDED CLAIMS received by the International Bureau on 18 December 2025 (18.12.2025)1. A pesticidal composition comprising: i. at least two phytochemical agents selected from the group consisting of a phenolic compound, a flavonoid, an alkaloid, a curcuminoid, an isoflavone, and a carotenoid or any of combination thereof; ii. an agrochemically acceptable excipient or vehicle; wherein the flavonoid is selected from the group consisting of gossypetin, herbacetin, isoquercitrin, myricetin, naringenin, rutin and quercetin or any of combination thereof; the phenolic compound is selected from the group consisting of eugenol, gallic acid, ferulic acid, caffeic acid, chlorogenic acid, thymol, carvacrol, ellagic acid, vanillic acid, syringic acid, p-coumaric acid, protocatechuic acid or any of combination thereof; the alkaloid is selected from the group consisting of coptisine, palmatine, jatrorrhizine and berberine or any of combination thereof; the curcuminoid is selected from the group consisting of demethoxycurcumin, resveratrol and curcumin or any of combination thereof; the isoflavone is selected from the group consisting of genistein, daidzein, glycitein, formononetin, biochanin A, prunetin, puerarin, calycosin, ononin or any of combination thereof; and the carotenoid is selected from the group consisting of P-carotene, a-carotene, lutein, zeaxanthin, lycopene, astaxanthin, violaxanthin, neoxanthin, canthaxanthin, fucoxanthin, capsanthin or any of combination thereof.

2. The pesticidal composition as claimed in claim 1, wherein the composition optionally comprises a pesticidal compound / agent.

3. The pesticidal composition as claimed in claim 1, wherein the composition comprises at least three phytochemical agents or at least four phytochemical agents.

4. The pesticidal composition as claimed in claim 2, wherein the pesticidal compound / agent is selected from the group consisting of aldrin, dieldrin, chlordane, dichlorodiphenyltrichloroethane, endrin, heptachlor, mirex, toxaphene, abamectin, ivermectin, doramectin, eprinomectin, selamectin, moxidectin and emamactin.

5. The pesticidal composition as claimed in claim 1, wherein the agrochemically acceptable excipient or vehicle comprises a solvent, wherein the solvent is selected from dimethyl sulfoxide and water or combination thereof.

6. The pesticidal composition as claimed in claim 1, wherein amount or concentration of the at least two phytochemical agents is in the range of 25- 75% w / v; amount or concentration of the pesticidal compound / agent is in the range of 5-20% w / v; and rest is the agrochemically acceptable excipient or vehicle to make up 100% w / v of the composition.

7. The pesticidal composition as claimed in claim 1, wherein the pesticidal composition comprises: a) flavonoid, b) phenolic compound, c) alkaloid, d) curcuminoid, e) pesticidal compound / agent; and f) agrochemically acceptable excipient or vehicle.

8. The pesticidal composition as claimed in claim 7, wherein amount or concentration of the flavonoid in the composition is in the range of 5-30% w / v; amount or concentration of the alkaloid in the composition is in the range of 5-30% w / v; amount or concentration of the curcuminoid in the composition is in the range of 5-30% w / v;amount or concentration of the phenolic compound in the composition is in the range of 0.005-0.04% w / v; amount or concentration of the pesticidal compound / agent in the composition is in the range of 5-20% w / v; and wherein the agrochemically acceptable excipient or vehicle is added to make up 100% w / v of the composition.

9. The pesticidal composition as claimed in claim 1, wherein the pesticidal composition comprises: a) rutin, b) quercetin, c) eugenol, d) berberine, e) curcumin, f) pesticidal compound / agent; and g) agrochemically acceptable excipient or vehicle.

10. A process of preparation of the pesticidal composition as claimed in claim 1, comprising: i. mixing at least two phytochemical agents in agrochemically acceptable excipient or vehicle under stirring at temperature in the range of 25-35 °C to obtain a 1stmixture; ii. adding and mixing pesticidal compound / agent in said 1stmixture of step i) under stirring at temperature in the range of 25-35 °C to obtain 2ndmixture; and iii. adding agrochemically acceptable excipient or vehicle to obtain the pesticidal composition.[0001][0002]STATEMENT UNDER ARTICLE 19 (1 )[0003]With reference to the search report and written opinion of ISA / IN, the applicant has amended claims to address the novelty and inventive step objections.[0004]1. The Applicant submits that claim 1 has been amended by adding the features of claim 4.[0005]2. The Applicant submits that claim 4 has been deleted accordingly.[0006]3. The Applicant submits that claims have been renumbered accordingly.[0007]The Applicant undertakes that no new subject matter has been added in claims and the amended claims do not go beyond disclosure of international application as-filed.

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