Title of Invention	"A HERBAL INSECTICIDAL COMPOSITION"
Abstract	The present invention relates to a herbal insecticidal composition of hexane extract of the dry fruit of Solanum nigrum and a process of preparation of the extract for controlling the mosquito larvae.

Full Text

Field of the invention: The present invention relates to a herbal insecticidal composition. The present invention further relates to a novel herbal composition useful as an insecticide. The composition comprises an extract of Solarium nigrum. The invention also provides a method for controlling mosquito larvae. Background of the invention: Insects are known to be carriers of diseases. Mosquitoes in particular act as vectors of diseases such as malaria, encephalitis, dengue, haemorrhagic fever, yellow fever, etc. The number of people who succumb to these diseases is rising by the day and hence, the health-hazard caused by insects is assuming alarming proportions. Currently, the market is flooded with various insect repellent compositions that claim to keep mosquitoes particularly, from biting and thereby transmitting viruses to the human. Presently for larval control of mosquitoes synthetic organic insecticides and malaria oil (distillate from oil refinery) areused. Most of these are insecticides and likely to affect the health and environment in the long run. As for other products, US Patent No. 6,355,264 discloses a composition that contains oil of citronella, an emollient, an alkyl benzoate and an alcohol-containing vehicle. This composition is for topical administration. This composition does contain some synthetic ingredient, hence it is not a purely herbal composition. Clearly, there is a need to develop a herbal composition for controlling hsects, especially mosquitoes. Towards this end, the Applicant conducted detailed studies on the plant Solanum nigrum, which is widely grown in India. The plant is known for its medicinal properties and commonly used as an anti-dysentric, diuretic, laxative and anti-spasmodic. S.P. Singh et al., reportedin Current Science, Vol. 81, No.12, 25 Dec 2001 that the leaf extract of Solanum nigrum exhibits larvicidal effect on Anopheles culicifacies Giles SpeciesA, a vector that transmits malaria. However, the efficacy of aqueous extract of leaf for the three mosquito species of three genera, Anopheles culicifacies species A, Culex quinquefasciatus and Aedes aegypti, was in the range of 270-320 ppm. Whereas the present claim with hexane extract of dried fruit for these three species was 12-18 ppm, and was 17-22 fold less than the reported concentrations of aqueous crude extract of leaf. The Applicants, during their investigation worked on dried fruits of Solanum nigrum and tested their insecticidal activity. Based on these studies is proposed a herbal composition for controlling mosquitoes. Objects of the invention: The main object of the invention is to provide a herbal composition useful for controlling mosquitoes. Another object is to provide a method for controlling mosquitoes. Statement description of the invention: An embodiment of the present invention relates to a herbal insecticidal composition for controlling insect larvae, the said composition comprising: (i) 100-150 ppm of hexane extract of the fruit of Solanum nigrum (ii) and 200-300 ml of water as carrier. Detailed description of the invention: Accordingly, the invention provides a herbal composition useful as an insecticide comprising a hexane extract obtained from dried fruit of Solanum nigrum together with an acceptable carrier. The amount of hexane extract in the composition may be about lOOmg to 150 mg% wt/vol (100 to 150 ppm/mg/1000 ml water).. The "hexane extract" as used herein is an extract obtained via a special process as described herein in example 3. The carrier that may be used in the composition is water. The amount of carrier in the composition may be about 200-300 ml especially 250 ml. The Applicant during their study, tested various parts of Solanum nigrum for its larvicidal activity. The aqueous extract of the fruit, flowers, stem were more or less equitoxic to larvae of mosquitoes as the crude aqueous leaf extract. (LC50 for aqueous leaf extract was in the range of 270-320 ppm for different species while the same for aqueous extract of dried fruit was 208 to 360 ppm). However, to their surprise, the hexane extract of the fruit was found to be 17-22 times more effective than the aqueous leaf extract. As an example, the hexane extract was tested against various larvae of mosquitoes. The results are as under:- (Table Removed) * Singh etal, 2001. The above test shows that hexane extract obtained from the fruit of Solatium irigrum is at least 13 fold more efficacious than aqueous extract of dried fruit or the aqueous leaf extract of the same plant and was as high as 39 fold for Anopheles stephensi. The reasons for this surprising phenomenon could be many. One may be that plants accumulate bioactive chemicals differentially in different parts such as leaves, fruits, flowers, roots, etc: hence, extracts of some parts are more effective than other parts. Generally, an extract of a given plant/part of a plant contains different ingredients that may exhibit the larvicidal efficacy. The solvent used for extraction is equally important, as this is the medium into which the bioactive principles of the given plant will be extracted. The plant products comprise of both polar and non-polar constituents and the extraction of these components will depend on certain physical properties of the solvent to achieve the maximum retention of the products. Aqueous extract can only retain the polar compounds while organic solvents retain non-polar compounds and insecticidal compounds are generally non-polar in nature. Organic solvent (e.g. hexane, acetone, petroleum ether, methanol, ethanol, etc.) may thus concentrate the bioactive insecticidal compounds and render the extract relatively free of impurities and more effective. Thus, even the solvent used for extraction plays a critical role and influences the activity exhibited by the extract. The following table illustrates how different plant extracts (in different solvents) differ in their activity: (Table Removed) Further, the same extract may have different effect on different types of insects. See table below: (Table Removed) From the above tables, it is clear that from the activity exhibited by an extract of one part of the plant, the activity of other parts of the same plant cannot be predicted. Therefore, it was after much study and investigations that the Applicants found to their surprise that the hexane extract is more efficacious as an insecticide or larvicide, for controlling mosquitoes. This hexane extract is prepared as under: crushing dried fruits of Solatium nigrum to obtain fine powder, heating the powder with hexane, separating the residue and collecting the supernatant as desired hexane extract. According to the invention, the hexane extract so prepared may be combined with a carrier such as water to obtain a composition effective against mosquito larvae. The composition of the invention may act as an effective insecticide against various larvae of mosquitoes. Fig. 1 sets out the comparative larvicidal efficacy (LCso) of aqueous extract of leaf, aqueous extract of dried fruit and hexane extract of fruit. It is clear from Fig. 1 that the hexane extract is highly effective as compared to other extracts. Thus, the composition of the invention may be formulated as a spray, cream suspension, or any other physical form as a larvicidal agent/formulation against mosquito vectors. In an embodiment, the invention provides a method for controlling mosquitoes comprising the step of exposing mosquitoes to the composition of the invention for a period of about 24 hours. At the end of this period, it is observed that most of the mosquitoes are killed at a given dose. The invention is now illustrated by the following examples which should not be construed to limit the scope in any manner: Example 1; Preparation of hcxane extract. Fruits of Solarium nigrum were washed, sterilized, dried and reduced to the form of a powder. These particles may be introduced into a cartridge made of porous cellulose cardboard. The cartridge is introduced into a "Soxhlet"-type extractor: this extractor is equipped at its base with a round-bottomed flask containing hexane heated to boiling point (69.degree. C.). The solvent vapors pass through a diversion, are condensed in the reflux condenser and fall back in the liquid state into the cartridge, progressively immersing the latter. After total immersion, the solvent, laden with plant extracts, flows out by syphoning into the starting round-bottomed flask. The process is carried on continuously, the liquid in the round-bottomed flask being increasingly laden with plant extracts. The extraction is carried out for 12 hours. The hexane fractions are then evaporated to dryness under reduced pressure and used in further experiments. Example 2: Preparation of hexane extract; The fruits of Solatium nigrum may be were washed, sterilized, dried and reduced to the form of a powder. Weighed quantity of the powder was dispersed in appropriate quantity of n-hexane If desired the dispersion containing Solatium nigrum fruit powder & n-hexane may be lightly heated and a homogenous liquid may be obtained. Example 3: Preparation of composition of invention Hexane extract of the seeds was made as described. 25 gms of the above dried fruit powder was mixed with 250 ml of n-hexane (10% w/v) in a narrow-mouth brown glass bottle with airtight stopper and was left at room temperature for overnight. Next day, the mixture was stirred on a magnetic stirrer for one hour and filtered with Whatniann paper. The residue after filtration was remixed with n-Hexane as above (10% w/v) and procedure of extraction repeated. The filtrates from the above processes were allowed to dry at room temperature. The concentrated gummy extract was mixed with acetone and used for larval bioassays. Example 4: Efficacy of the hexane extract To test the efficacy of the hexane extract, the Standard WHO test (WHO, 1981) was employed with slight modification in the test procedure. Fourth-stage larvae of different mosquito species were put into 500 ml glass beaker containing 250 ml of the test solution (composition of the carrier and hexane extract) each of concentration 0.781 ppm to 150 ppm (Mg/litre). Each beaker had 25 mosquito larvae. A totale of 150-1800 mosquito larvae of different species belonging to three genera were used as follows: Aqueous exgtract of dried fruit: An. Culicifacies species A - 250 larvae An. Culicifacies species C - 1800 larvae An. Stephensi - 1800 larvae Cx. quinquefasciatus - 1800 larvae Ae. Aegypti - 300 larvae Aqueous extract of leaves An. Culicifacies species A - 150 larvae CX. quinquefasciatus -150 larvae Ae. Aegypti - 150 larvae Mortality data for mosq2uito larvae were recorded after 24 hours. The observed 100% mortality in laboratory bioassays was more or less at uniform concentrations and varied for a given type of extracts namely for hexane extract of dried fruit, aqueous extract of dried fruit and of leaf in different mosquito species. It was observed that hexane extract of Solarium nigrum caused 100% mortality in different species, An. Culicifacies species A - 100 ppm. An. Culicifacies species C-100 ppm, An. Stephensi - 100 ppm, Cx. Quinquefasciatus - 100 ppm and Ae. Aegypti - 150 ppm (96% at 100 ppm). Under laboratory conditions, whereas 100% mortality An. Culicifacies species C-1000 ppm, An. Culicifacies species A-1000 ppm, An. Stephensi - 1000 ppm, Cx. Quinquefasciatus - 1000 ppm and Ae. Aegypti - 2000 ppm. was observed for aqueous extract of fruit and 100% mortality was observed. An. Culicifacies species A-2000 ppm. Cx. Quinquefasciatus - 2000 ppm and Ae. Aegypti - 2000 ppm for aqueous extract of leaves. The Table 1 referred earlier demonstrates the comparative efficacy of the hexane extract of dried fruit against its aqueous extract in anopheline species. (Table Removed) Hexane extract was found more effective against An. Stephensi with 39 fold increased efficacy compared to aqueous extract. While against An. Culicifacies species A and C it was respectively 13 fold and 28 fold. Example 5; Efficacy of the hexane extract in various mosquito species: Results of the efficacy of the aqueous extract of the dried fruit against five mosquito species revealed that An. Culicifacies species A has registered the least LCso. value of 208.5 ppm among the five species tested while it was maximum for Ae. Aegypti and was 359 ppm. The efficacy of the aqueous extract against the five species was in the order An. Culicifacies species A (208.5 ppm) > An. Stephensi (242.5 ppm) > An. Culicifacies species C (251.7 ppm) > Cx. Quinquefasciatus (337.2 ppm) > Ae. Aegypti (359 ppm) (Table 1) On the contrary hexane extract of seeds was relatively more effective. An. Stephensi has registered the lowest LCso value of 6.25 ppm among the five species tested while it was maximum for Ae. Aegypti (17.63 ppm). The efficacy of the hexane extract against the five species was in the order An. Stephensi (6.25 ppm) > An. Culicifacies species C (9.04 ppm) > Cx. Quinquefasciatus (12.25 ppm) > Ae. Aegypti (17.63 ppm). The calculated LC50 values indicated increased efficacy of the hexane extract of dried fruit and was 13-39 folds more effective than aqueous extract. It was respectively 13 fold more effective against An. Culicifacies species A, 20 fold against Ae. Aegypti, 28 fold against An. Ciilicifacies species C and Cx. Quinquefasciatus and 39 fold against An. Stephensi. Hexane extract was found comprehensively effective against five mosquito vector species of three genera and the observed LCso value was less than 20 ppm and could be a prospective biorational alternative molecule for organic insecticides. However, more studies are needed to assess the field efficacy to validate the above laboratory results. Such studies with natural plant extracts are all the more important, as this candidate insecticide molecules are easily biodegradable and are environment friendly. We Claim: 1. A herbal insecticidal composition for controlling insect larvae, the said composition comprising: i. 100-150 ppm of hexane extract of the fruit of Solanum nigrum ii. And 200-300 ml of water as carrier 2. A composition as claimed in claim 1, wherein the amount of hexane extract is 100 ppm. 3. A composition as claimed in claim 1, wherein the amount of carrier is 250 ml. 4. A process for preparing the composition as claimed in claim 1 for controlling the insect larvae, the steps consisting of: i) Crushing dried fruits of Solanum nigrum to obtain fine powder ii) Heating the crushed powder of step (i) in the ration of 10% w/v at 69°C with hexane iii) separating the residue and drying the supernatant as desired to obtain hexane extract iv) Mixing the hexane extract with water to obtain the herbal insecticidal composition 5. A herbal insecticidal composition substantially such as herein described.

Documents:

1195-DEL-2004-Abstract-(05-05-2009).pdf

1195-del-2004-abstract.pdf

1195-DEL-2004-Claims-(05-05-2009).pdf

1195-DEL-2004-Claims-(11-05-2009).pdf

1195-DEL-2004-Claims-(13-05-2009).pdf

1195-del-2004-claims.pdf

1195-DEL-2004-Correspondence Others-(06-03-2012).pdf

1195-del-2004-Correspondence Others-(11-04-2012).pdf

1195-DEL-2004-Correspondence-Others-(05-05-2009).pdf

1195-DEL-2004-Correspondence-Others-(11-05-2009).pdf

1195-del-2004-correspondence-others.pdf

1195-DEL-2004-Description (Complete)-(05-05-2009).pdf

1195-del-2004-description (complete).pdf

1195-DEL-2004-Form-1-(05-05-2009).pdf

1195-del-2004-form-1.pdf

1195-del-2004-form-13-(05-05-2009).pdf

1195-del-2004-form-18.pdf

1195-DEL-2004-Form-2-(05-05-2009).pdf

1195-del-2004-form-2.pdf

1195-DEL-2004-Form-26-(05-05-2009).pdf

1195-del-2004-form-3.pdf

1195-del-2004-form-5.pdf

1195-del-2004-GPA-(11-04-2012).pdf