AU2011375107B2 - An anti-termite biological formulation and a process for the preparation thereof - Google Patents

Abstract

A biological formulation comprising: 10 to 60% of an aqueous seed extract of cuminum cyminum, 5-30% of essential oil of curcuma aromatica and 10-45% of secondary metabolites isolated from Pseudomonas aeruginosa along with not more than 12% of an inert carrier.

Description

WO 2013/024486 PCT/IN2011/000844 1 FIELD OF INVENTION The present invention relates to an anti-termite biological formulation and a process for the preparation thereof. BACKGROUND OF THE INVENTION: There are still a number of commercial anti-termite products on the market currently. The various chemicals have been used for anti-termite treatment, such as those derived from organic phosphor, azole, carbamate, naphthalene, and several pyrethroids, including pennethrin, cypennethrin, bifenthrin pyrethroid, fipronil, imidacloprid, chlorfenapyr, metallic salts such as mercuric chloride, copper chloride, zinc chloride, ferrous sulphate, arsenic trioxide, wood tar creosote, and coal creosote etc. All of the foregoing are known toxic chemicals, and as such are declining in use. Particularly these are synthetic termite repellents and are still not so much acceptable and have unpleasant odour, can damage wood, plastic and synthetic rubber, applying a chemical termiticide to a structure's, soil, wooden materials often, leads to soil and water contamination. Moreover, the chemical repellents are not safe for public health, they lack widespread acceptance owing to the incomplete protection provided, lack of persistence, cosmetic disadvantages. As with chemicals used for termite treatments, very few less toxic alternatives exist. Accordingly, there is a substantial need for less toxic alternatives for preventing and treating termite infestation.

WO 2013/024486 PCT/IN2011/000844 2 Typically an anti-termite formulation comprises a liquid or solid carrier material for the volatilizable/non-volatilizable components. The carrier enables more efficient transport and distribution, such as by spreading on the surface. The carrier material may also serve to depress or elevate the vapour pressure of the volatilizable component, so as to increase, moderate or sustained volatilization of that component. Thus, the provision of an anti termite formulation which is effective against termites tends to require the optimization of blends of volatilizable and water soluble components. Some anti-termite formulations advantageously employ volatilizable components which demonstrate synergistic effects in their anti-termite properties. These effects are rarely predictable, especially in multi-component formulations and formulations where other factors need to be taken into account, such as acceptability for surface application, olfactory response ofia human user, colour and stability, among other factors. Owing to the lack of persistence, there is currently not an acceptable anti-termite formulation available. Moreover, there is a need for safe, easy to apply, new and improved anti termite formulation for the protection from termites that causes serious substantial damage to residential and commercial buildings, plants, wood and wood objects or other cellulosic material, of the structure and its contents. In addition to termiticide formulation being non-toxic and non irritating to humans, a further requirement of an anti-termite formulation is environmental acceptability.

WO 2013/024486 PCT/IN2011/000844 3 Therefore, the development of alternative control measure against termites in an ecofriendly environment is highly essential. Keeping view on this point, it is urgently required to prepare a safe, ecofriendly biological based termiticide formulation as protection measure against termites. In view of the above, a promising approach in the need of the day to find out a natural, ecofriendly, and non toxic in nature, for preparing biological formulation. It is therefore; in our study we have prepared a biological based anti-termite formulation which is nontoxic, ecofriendly and biodegradable in the environment with no adverse effects, easy and safe to use with acceptable fragrance. U.S.Patent 2007/7282212 discloses a method of controlling termites comprising thiamethoxam as an active ingredient with at least one insecticide mixing partner selected from fipronil and lambda-cyhalothrin; and an adjuvant. WO/2004/029017 discloses an anti-termite, waterproofing agents; binders; strengtheners; antifouling agents; antimicrobial agents; and/or biocides, which comprises of water insoluble quaternary ammonium compounds as an active ingredient.

WO 2013/024486 PCT/IN2011/000844 4 Japanese Patent 2006/JP2006347894 discloses an anti-termite agent comprising 2-aryl-4-alkylimidazole, 2-phenyl-4-methylimidazole, 2 alkylimidazole, 2-methylimidazole, or the like as an active ingredient. The anti-termite agents are suitable for use for treating xylem or soil. The anti termite agent can be caused to be brought into contact with termites or eaten by termites. U.S. Patent 1993/5196407 to Luzian, et al., the invention relates to an aqueous composition for preserving wood and wood materials, comprising a combination of fungicides selected from the group consisting of ca-[[2-(4 chlorophenyl)ethyl] -a-(1,1 -dimethylethyl)- 1 H- 1,2,4-triazol-l-ethanol and other 1- [[2-(2,4-dichlorophenyl-4-propyl-1,3dioxolan-2-yl}methyl}-1H 1,2,4-triazole, a fungicidal carbamate; namely 3-iodo-2-propynylbutyl carbamate or methyl benzimidazol-2-ylcarbamate; a stabilizer, 2, 2, 4 trimethyl-1,3-pentadiol monoisobutyrate a stabilizer and a diluent, and optionally, an insecticide, an emulsifier, a wetting agent and an organic chemical binder or fixing agent. US Patent 1975/3858346 discloses the Control of subterranean termites in buildings is carried out by impregnating at least the exposed lower timbers of the building hexachlorocyclopentadiene dimmer as an active ingredient in a liquid organic solvent carrier.

WO 2013/024486 PCT/IN2011/000844 5 WO/2011/027362 discloses a herbal formulation that consists active extracts of Azadirachta indica, Pongamia pinnata and genus of Acorus, Allium, and Vitex, sesame oil infused with peel of genus Citrus, pitch, and cashew nut shell liquid -by product of cashew industry, along with suitable carrier solvents and emulsifiers, which in cumulative effect controls termites, white ants, wood borers and wood decaying fungi. U.S. Patent No. 5089483, issued Feb, 18, 1992, shows a composition for antitermite agent for timber, the invention contains alkyldimethylamine tetraborate as an effective component; the compound can be applied as it is or in a form of preparation generally be used in a form of emulsion with water or as an oil agent by dissolving in an appropriate organic solvent. Biological deterioration is a main factor that degrades the durability of wooden houses (Takahashi et al, 2000). Among all factors leading to biodegradation, termites are most damaging to wooden structure worldwide. It is also known that termites cause damage to a variety of materials ranging from paper fabrics to even non-cellulosic materials such as asbestos, asphalt bitumen, and metal foils (Ampong et al, 1979). Damage to wooden structures and other cellulosic materials attributed to termites has been estimated to exceed $3 billion annually worldwide ((Scheffrahn et al, 1990).

WO 2013/024486 PCT/IN2011/000844 6 This destruction due to termites is not limited to wooden structures alone many commercial termiticides used to combat these pests are potential environmental contaminants and carcinogens. For example, the most common method of termite infestation prevention, which involves applying a chemical termiticide to a structure's peripheral grounds, often, leads to soil and water contamination when applied improperly. Accordingly, it is desirable to find new means for controlling termites which do not pose such potential hazards. There are still a number of commercial repellent, pesticide or termiticide products on the market currently. These are typically contact poisons and are effective in killing a wide variety of insects. Many of the commercial pesticides carry a warning label that they are hazardous to humans, and state that protective gloves should be used while handling. The potential hazards of using a product with chemical termiticide suggests that there exists a great need for a comparably termiticide product that is not dangerous to its users. The excessive use of chemicals even is a serious environmental concern as target insects develop resistance. A need still remains though for a product which is effective against termites and at the same time is non-toxic to humans, biodegradable and environmentally safe. The above references are merely intended to be representative of the general nature of prior art compositions in the anti-termite field which utilize fairly exotic active ingredients which may typically potential toxicants, environmental pollutants, skin irritants or the like.

H:\kll\Introvn\NRPortbl\DCC\KLL\801787_ .docx-16/07/2015 6A Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

H:\kll\Introvn\NRPortbl\DCC\KLL\801787_ .docx-16/07/2015 7 ASPECTS OF THE INVENTION: An aspect of the present invention is to provide a biological formulation for termites. Another aspect of the present invention is to provide a process for preparing biological formulation for termites. Still another aspect of the present invention is to provide a non-toxic and environment friendly formulation for termites. Further aspect of this invention is to provide a simple and easily obtainable biological formulation for termites. Still further aspect of this invention is to provide a biological formulation with superior properties. Still another aspect of this invention is to provide a process for the stable biological based anti-termite formulation.

WO 2013/024486 PCT/IN2011/000844 8 BRIEF DESCRIPTION OF THE INVENTION: According to this invention there is provided a biological formulation comprising: 10 to 60% of an aqueous seed extract of cuminum cyminum, 5-30% of essential oil of curcuma aromatica and 10-45% of secondary metabolites isolated from Pseudomonas aeruginosa alongwith not more than 12% of an inert carrier. DETAILED DESCRIPTION OF THE INVENTION: Plant and Microorganism could be an important source for development of environmentally safe and economically viable termiticide because they constitute a potential source of bioactive compounds / secondary metabolites and typically free from harmful effects. The essential oil extracted from plant sources of Curcuma aromatica, aqueous seed extract of a plant Cuminum cyminum, and secondary metabolites isolated from Pseudomonas aeruginosa were found effective as termiticide and termite repellent in the present investigation. The termiticide and termite repellent compositions of the invention can be used to repel and kill termites from a wood and wood objects, plants, soil, residential and commercial buildings, or other cellulosic material, and include, as active ingredients i.e. an essential oil, aqueous seed extract of a plant, metabolite isolated from microorganism and polysorbate 80 was used as solvent in the formulation to increase the persistency and smooth spreading of the anti-termite formulation, for application through spray on termite affected surfaces.

WO 2013/024486 PCT/IN2011/000844 9 No skin irritation has been noticed with the active compounds or with vehicles according to the invention. a) Prior Art of essential oil for Curcuma aromatica Salisb. (Wild turmeric, jungle Haldi) oil: * C. aromatica Salisb is a perennial tuberous herb with annulate, aromatic yellow rhizome which is internally orange-red in color " Leaves are elliptic or lanceolate-oblong, caudate-acuminate, 30 60cm long, petioles as long or even longer, bracts ovate, recurved, more or less tinged with red or pink. * Flowers are pink, lip yellow, obovate, deflexed, sub-entire or obscurely three lobed. " Fruits are dehiscent, globose, 3-valved capsules. The plant is also described by Gamble and Kirtikar and Basu. * Distributed throughout the Tropical belt, found wild throughout India and its *cultivation is largely confined to India and Southeast Asia and Indonesia. " Rhizomes of turmeric (Curcuma aromatica) are used in combination with astringents and aromatics for bruises, sprains, hiccough, bronchitis, cough, leucoderma and skin eruptions. " Oil of Curcuma aromatica is used for treatment of early stage of cervix cancer WO 2013/024486 PCT/IN2011/000844 10 * Rhizomes of Curcuma aromatica yield 6.1% essential oil. Essential oil contains a -and - b-curcumene, d-camphene and p-methoxy cinnamic acid. The colouring matter is curcumin and the active constituents of oil are curcumol and curdione. * C. aromatica rhizomes contain ar-curcumene (18.6%), beta curcumene (25.5%) and xanthorhizol (25.7%) (Zwaving and Bos, 1992). e Essential oil from rhizomes showed anthelmintic, antifungal and antimicrobial activity. b) Cuminum cyminum L. (Cumin, Cumino, Cumino Aigro, Comino, Jeera, Comino Romano): o Cumin is a strong aromatic dried ripe fruit (seed) of Cuminum cyminum L. It belongs to the Apiaceae family (parsley family). The cumin plant grows to 30-50 cm (0.98-1.6 ft) tall and is harvested by hand. o It is an herbaceous annual plant, with a slender branched stem 20-30 cm tall. The leaves are 5-10 cm long, pinnate or bipinnate, thread-like leaflets. e The flowers are small, white or pink, and borne in umbels. The fruit is a lateral fusiform or ovoid achene 4-5 mm long, containing a single seed.

WO 2013/024486 PCT/IN2011/000844 11 " Cultivation of cumin requires a long, hot summer of 3-4 months, with daytime temperatures around 30'C (86'F); it is drought-tolerant, and is mostly grown in Mediterranean climates. It is grown from seed, sown in spring, and needs fertile, well-drained soil. e The dried fruit or seed is crushed and distilled immediately to obtain the essential oil. Steam distillation is usually carried out. The oil recovery ranges from 2.5 to 4.5%. Older seeds yield less oil. " Cumin oil contain -pinene (14.3-19.7%), p-cymene (2.7-6.0%), terpinene (11.5-16.3%), cuminaldehyde (20.0-22.4%), p-menth 1,3-dien-7-al (11.1-13.5%), p-menth-1,4dien-7-al (23.6-24.3%), cuminyl alcohol (3.7-72.2)%, sabinene (3.38%), benzyl cinnamate (4.47%), famesol (2.98%), (Joseph et al, 2000). c) Pseudomonas aeruginosa: e Is a common bacterium found in soil, water, skin flora, and most man-made environments throughout the world. o It thrives not only in normal atmospheres but also in hypoxic atmospheres, and has, thus, colonized many natural and artificial environments. o It is a Gram-negative, aerobic, rod-shaped bacterium with unipolar motility (Ray et al, 2004).

WO 2013/024486 PCT/IN2011/000844 12 * P. aeruginosa secretes a variety of pigments, including pyocyanin (blue-green), pyoverdine (yellow-green and fluorescent), and pyorubin (red-brown) (Raney et al, 1954). * P. aeruginosa is often preliminarily identified by its pearlescent appearance and grape-like or tortilla-like odor in vitro. Definitive clinical identification of P.aeruginosa often includes identifying the production of both pyocyanin and fluorescein, as well as its ability to grow at 42'C. P. aeruginosa is capable of growth in diesel and jet fuel, where it is known as a hydrocarbon-using microorganism (or "HUM bug"), causing microbial corrosion. * Although classified as an aerobic organism, P. aeruginosa is considered by many as a facultative anaerobe, as it is well adapted to proliferate in conditions of partial or total oxygen depletion. This organism can achieve anaerobic growth with nitrate as a terminal electron acceptor, and in its absence, it is also able to ferment arginine by substrate-level phosphorylation (Whiteley et al, 2007, Han et al, 1984). * Pseudomonas aeruginosa simultaneously produced secondary metabolites, 86% produced 2-alkyl-4-hydroxyquinolines, 75% produced 2-alkyl-4-hydroxyquinolines, 75% produced rhamnolipids, and 58% produced phenazines, including pyocyanin (Hastie et al, 1984).

H:\kll\Introvn\NRPortbl\DCC\KLL\801787_ .docx-16/07/2015 13 - P. aeruginosa secretes numerous proteins and secondary metabolites (Britigan et al, 2003). The present invention proposes anti-termite formulation for spray application as one of the simplest, economical, nontoxic and effective anti-termite formulation against termites. It is an aspect of the present invention to provide an anti-termite formulation that is safe, long-lasting, effective and pleasant. The formulation compositions are more substantive to termite infected surfaces and are thus more effective than prior repellent, pesticide or termiticide product compositions. The disclosed invention is a biological formulation for treating wood to preserve the appearance and condition of the treated wood from termite. The formulation, as defined in the present invention, also effectively controls termites in agriculture fields. The present formulation avoids the problems of previously mentioned chemical pesticides, termiticide and termite repellents, which can harm the environment and are hazardous to handle, by using ingredients which are easily accessible and environmentally safe and when used in the manner described here are lethal to termites. Polysorbate-80 an inert carrier in which essential oil of Curcuma aromatica, aqueous seeds extract of Cuminum cyminum and secondary metabolites isolated from Pseudomonas aeruginosa as the sole anti-termite agents are disposed.

WO 2013/024486 PCT/IN2011/000844 14 Accordingly the disclosed formulation may further comprise of Polysorbate 80 as an emulsifier and nonionic surfactant. The Polysorbate-80 may be used to enhance the solubility and/or stabilize the formulation, which regarded as safe for agricultural and household uses. The present inventors attempted to formulate a spray based formulation of aqueous seeds extract of Cuminum cyminum, essential oil of Curcuma aromatica, and secondary metabolites isolated from Pseudomonas aeruginosa in Polysorbate-80 an acceptable inert carrier. A formulation obtained by mixing essential oils of Curcuma aromatica, aqueous seeds extract of Cuminum cyminum and -secondary metabolites isolated from Pseudomonas aeruginosa with Polysorbate-80 as inert carrier base, however, has been found to have good absorbability with termite infected surfaces which is preferred in terms of stability, substantive to various surfaces, provides increased effective lifetimes with acceptable carrier. According to this invention, there is provided a biological formulation comprising by volume of aqueous seeds extract of Cuminum cyminum, essential oil of Curcuma aromatica, and secondary metabolites isolated from Pseudomonas aeruginosa and Polysorbate-80 was used as carrier. All the described ingredients are active against the targeted organism and exhibit cumulative effect when applied as combined formulation. The synergistic effect of the described combined formulation acts much better than the effect when each ingredient is applied individually.

WO 2013/024486 PCT/IN2011/000844 15 The synergistic formulation of the present invention is further illustrated in the following, non-limiting Examples. The Examples are illustrative of various embodiments only and do not limit the claimed invention regarding the materials, conditions, weight ratios, process parameters and the like recited herein. The examples used herein are intended merely to facilitate an understanding of ways in which the formulation herein may be practiced and to further enable those of skill in the art to practice the embodiments herein. Accordingly, the examples should not be construed as limiting the scope of the formulation herein. According to this invention, there is provided a formulation comprising (a) 10 to 60% of aqueous seeds extract of Cuminum cyminum, (b) 05 to 30% of essential oil of Curcuma aromatica, (c) 10 to 45% of secondary metabolites isolated from Pseudomonas aeruginosa, (d) Not more than 12% of Polysorbate-80, and It should be understood that in the present specification and the appended claims, all percentages showing the concentrations of the essential oil, aqueous extract and secondary metabolite with Polysorbate-80 (inert carrier) of the formulation are based on the volume. The ingredients of the formulation of this invention and a method for its preparation will now be described in detail.

WO 2013/024486 PCT/IN2011/000844 16 The Polysorbate-80/Tween-80 used in the formulation of this invention includes a nonionic surfactant and emulsifier derived from polyethoxylated sorbitan and oleic acid, and is often used as an emulsifier and excipient that is used to stabilize aqueous formulations, as a well regard pharmaceutically acceptable ingredients. The aqueous extract of Cuminum cyminum as a major ingredient of the formulation of this invention denotes aqueous extract obtained with distilled water and grinded Cuminum cyminum seeds in a ratio of 2:1 (water: blended Cuminum cyminum seeds) and was then left steeping for 18-24 h preferably overnight and filtered using Whatman No. 1 paper. It accounts for 10 to 60% by volume, preferably 30 to 50% by volume, more preferably 40 to 50% by volume, of the formulation. As required, the formulation of this invention may contain a pharmacologically effective substance which is essential oil of Curcuma aromatica itself proven showed antifungal and antimicrobial activity. (Asolkar et al, 1992). It accounts for 05 to 30% by volume, preferably 10 to 30% by volume, more preferably 20 to 25% by volume, of the formulation. In addition to the above ingredients, as required, the formulation of this invention may further contain secondary metabolites isolated from Pseudomonas aeruginosa. The P.aeruginosa strain identified and named DRL- 1 was isoloated from 100 soil sample collected from rhizosphere WO 2013/024486 PCT/IN2011/000844 17 region of termite infested tea plants in different locations of Assam-India using King's B medium. The fermentation was performed under fixed parameters; temperature 28'C, rpm-120, pH-7.0 for 96 hours. At the end of the fermentation the culture was centrifuged at 11, 000 rpm for about 15-20 minutes and then filtered using Whatman No. 1 paper to get the cell free filterate for further studies, which accounts for 10 to 45% by volume, preferably 10 to 30% by volume, more preferably 15 to 20% by volume, of the formulation. The product may be formulated by following the steps written flow: The formulation of this invention can be prepared by method For example, it can be formulated by adding essential oil of Curcuma aromatica the oil-soluble ingredients with Polysorbate-80/Tween-80, fully mixing them at a room temperature to form a component A, separately dissolving the other ingredient the aqueous seeds extract of Cuminum Cyminum and secondary metabolites isolated from Pseudomonas aeruginosa the water soluble ingredients at a room temperature to form a component B, then adding the component B in small portions to the component A with stirring at room temperature to form an intermediate emulsion, with continuing addition of the component B with stirring results in final formulation. The formulation suitable for spray can be prepared by first formulating the oil and water phases separately and mixing the two together. The oil phase contains about 10 to 60 percent by volume of the essential oil of Curcuma aromatica preferably added to the oil phase slowly, with stirring, at room temperature.

WO 2013/024486 PCT/IN2011/000844 18 The addition of low levels of stabilizing ingredients in the water phase has been shown to be helpful. Since the formulation of this invention has excellent storage stability of essential oil and water soluble ingredients, which proves to be effectively blending of oil in the conveying medium in turn provides long lasting effective lifetimes for the control of termites; when applied to a termite affected locus and prevent wood, wood structures, plants, soil, residential and commercial buildings, or other cellulosic material from attack of termites, with no adverse effects, easy and safe to use with acceptable carrier. This invention proposes a process for preparing the highly effective biological anti-termite formulation containing essential oil of Curcuma aromatica, aqueous seeds extract of Cuminum cyminum and secondary metabolites isolated from Pseudomonas aeruginosa based formulation, which was found to give longer protection time of >08 months in field conditions against termites. Further it is nontoxic, biodegradable and safe for human use on skin of exposed body parts. It is to be understood that the process of the present invention has been found to be economical and nontoxic to human and other higher animals.

WO 2013/024486 PCT/IN2011/000844 19 EXAMPLES: An anti-termite formulation in accordance with the present invention was formed as follows: Formula Active Ingredients Concentration (%) Cuminum cyminum (aqueous extract) 40 Curcuma aromatica oil 30 Secondary metabolites from Pseudomonas aeruginosa 20 Polysorbate-80 10 It can be formulated by adding essential oil of Curcuma aromatica the oil soluble ingredients with Polysorbate-80/Tween-80, with continuous stirring them at a room temperature to form a component 'A', separately dissolving the other ingredient the aqueous seed extract of Cuminum cyminum and secondary metabolites isolated from Pseudomonas aeruginosa, the water soluble ingredients at a room temperature to form a component 'B', then adding the component B in small portions to the component 'A' with stirring at room temperature to form an intermediate emulsion, with continuing addition of the component 'B' with stirring results in final formulation. The formulation suitable for spray can be prepared by first formulating the oil and water phases separately and mixing the two together.

WO 2013/024486 PCT/IN2011/000844 20 It is clear from the above preparation (Formula) listed above that the biological anti-termite spray of the invention have been found improved active ingredients stability. The following example illustrates the preparation of the formulation suitable for spray of this invention. Example: The individual essential oil of the plant Curcma aromatica, aqueous seed extract of Cuminum cyminum and secondary metabolites isolated from Pseudomonas aeruginosa have the application as anti-termite agents. Before preparing the spray based formulation of biological anti-termite, the active ingredients are tried against termites individually under laboratory conditions, as testing to determine the percentage mortality of termites on filter paper treated with individual active constituents. Individually, the aqueous extract of Cuminum cyminum seeds, oil of curcuma aromatica, and secondary metabolites isolated from Pseudomonas aeruginosa exhibited some activity against targeted pest (Table 1), but the claimed product exhibited much increased mortality and knock down effect than active ingredients showed individually. This experiment confirms the existence of synergistic activity among the used active ingredients (Table 2). Moreover getting encouraging results under laboratory conditions, the herbal spray formulation was further evaluated in field conditions, which proves to give extended protection time against termites.

WO 2013/024486 PCT/IN2011/000844 21 The biological anti-termite spray based formulation for application on the surfaces of termite affected wood, wood structures, plants, soil, commercial buildings, or other cellulosic material developed in the present invention provided protection for more than 08 months in field conditions (Table 4). The amount of the herbal spray formulation to be applied on termite affected surfaces varies depending upon the termite concentration. Percentage of usage may vary according to the severity of damage if already done previously. Generally, a suitable amount of it is applied to the affected surfaces two times more preferably required protection for single application only. This invention proposes a process for preparing the highly effective biological anti-termite formulation containing aqueous seeds extract of Cuminum cyminum, essential oil of Curcuma aromatica, and secondary metabolites isolated from Pseudomonas aeruginosa based formulation, which was found to give longer protection time of more than 08 months in field conditions against termites in single application. Further it is nontoxic, biodegradable and safe for human use.

WO 2013/024486 PCT/IN2011/000844 22 Table 1: Laboratory Evaluation of Antitermitic Activity with individual ingredient Used In Formulation: Ingredients in spray Concentration formulation 5% 10% 20% 30% 40% 50% (%)Termite Mortality Curcuma aromatica 12.0 17.0 23.0 30.0 30.0 30.0 oil Aqueous seeds extract 37.0 44.0 52.0 61.0 69.0 69.0 of Cuminum cyminum Secondary 22.0 25.0 39.0 39.0 40.0 40.0 metabolites from Pseudomonas Control Polysorbate- 0.0 0.0 0.0 0.0 0.0 0.0 80 *Average of 3 replicates **Intensity of termites: 30 per replicate WO 2013/024486 PCT/IN2011/000844 23 Table 2: Laboratory Evaluation of Antitermitic Activity with Final Formulation Containing All Active Ingredients at Optimized Concentration: Biological Antitermitic formulation (%) Termite Mortality with all ingredients (Final Formulation) 98.0 - 100.0 Control (Polysorbate-80) 0.0 *Average of 3 replicates **Intensity of termites: 30 per replicate The aqueous seeds extract of Cuminum cyminum 40%, essential oil .of Curcuma aromatica 30%, and secondary metabolites isolated from Pseudomonas aeruginosa 20% are optimized based on the laboratory evaluation performances of individual active ingredients used in biological anti-termite formulation. At above optimized concentration exhibited the best result. There was no significant difference in the percentage mortality beyond the optimized concentration of active ingredients tested under laboratory conditions. Therefore, biological anti-termite formulation at above given concentration was taken as the standard rather than higher concentration keeping in view the cost factor of the formulation. The present composition and process provides an effective anti-termite formulation contained in a unique emulsion matrix. The resulting material is environmentally and cosmetically appealing, stable over time and contains WO 2013/024486 PCT/IN2011/000844 24 no chemical active ingredients. The present formulations used on surfaces of termite affected wood, wood structures, plants, soil, residential and commercial buildings, or other cellulosic material by an individual through spray to give a thin covering, in the conventional manner which has very effective anti-termite properties. The invention further is illustrated by the following examples, which are not intended to be limiting in any way. Example: The following examples are given by way of illustration for the working of the invention in actual practice and therefore, these should not be limiting the scope of the present invention. A) The protection time against the termites obtained were showed in Table 3, the responses varied in a dose-dependent manner for each ingredient providing 100% mortality, further the field evaluation experiment confirms the existence of synergistic activity among the used active ingredients. The invention (refer-Formula) evaluated against termites under field conditions. The results with antitermitic formulation of the present invention obtained were very much encouraging, provided WO 2013/024486 PCT/IN2011/000844 25 protection for more than 08 months with 100% mortality in field conditions (Table 4). The studies were carried out at Borjuli Tea Estate, Rangapara, 44 Assam Rifles, HQ Lokhra, and Solmara Cantt. Area of District-Sonitpur (Assam)-India respectively. Table 3: Field Evaluation of Protection Time against Termite with Individual Ingredient Used In Formulation: Ingredients in spray Concentration formulation 5% 10% 20% 30% 40% 50% Termite Protection Time (In Months) Curcuma aromatica oil <0.5 >0.5 >1.0 >1.0 >1.0 >1.0 Aqueous seeds extract <1.0 >2.0 >3.0 >4.0 >5.0 >5.0 of Cuminum cyminum Secondary metabolites <1.0 >1.0 >3.0 >3.0 >3.0 >3.0 from Pseudomonas aeruginosa Control Polysorbate-80 0.0 0.0 0.0 0.0 0.0 0.0 WO 2013/024486 PCT/IN2011/000844 26 Table 4: Field Evaluation of Protection Time against Termite with Final Formulation Containing All Active Ingredients at Optimized Concentration: Biological Antitermitic formulation Termite Protection Time (In Months) with all ingredients (Final Formulation) >8.0 Control (Polysorbate-80) 0.0 B) The process of the extraction of essential oil of Curcuma aromatica, seeds extract of Cuminum cyminum and secondary metabolites from Pseudomonas aeruginosa is well known and can be performed by a person skilled in this art. C) The plants are available in plenty in North Eastern region of India. In addition; the essential oil is available as much as required, from the essential oil is available as much as required, from the essential oil extraction industry in NE region. The essential oils are being used as insects repellent by the community since long.

Claims (7)

1. A biological formulation comprising: 10 to 60% of an aqueous seed extract of Cuminum cyminum,

5-30% of essential oil of Curcuma aromatica and 10-45% of secondary metabolites isolated from Pseudomonas aeruginosa along with not more than 12% of an inert carrier. 2. The formulation as claimed in claim 1, wherein the said inert carrier is polysorbate-80. 3. The formulation as claimed in claim 1 or claim 2, wherein the formulation is water soluble. 4. The formulation as claimed in any one of claims 1 to 3, wherein 30 to 50% by volume of aqueous seed extract of Cuminum cyminum is used. 5. The formulation as claimed in any one of claims 1 to 4, wherein 40 to 50% by volume of aqueous seed extract of Cuminum cyminum is used.

6. The formulation as claimed in any one of claims 1 to 5, wherein 10 to 30% by volume of essential oil of Curcuma aromatica is used.

7. The formulation as claimed in any one of claims 1 to 6, wherein 20 to 25% by volume of essential oil of Curcuma aromatica is used.

8. The formulation as claimed in any one of claims 1 to 7, wherein 10 to 30% by volume of the secondary metabolites isolated from Pseudomonas aeruginosa H:\kll\Introvn\NRPortbl\DCC\KLL\801787_ .docx-16/07/2015 28 is used.

9. The formulation as claimed in any one of claims 1 to 8, wherein 15 to 20% by volume of the secondary metabolites isolated from Pseudomonas aeruginosa is used.

10. The formulation as claimed in any one of claims 1 to 9, wherein the aqueous extract is obtained from distilled water and grinded Cuminum cyminum seeds in a ratio of 2:1.