DESC:FORM-2

THE PATENTS ACT, 1970
&
THE PATENTS RULES, 2003

COMPLETE SPECIFICATION

Ocimum gratissimum essential oil based biopesticide formulation for the control of uzi fly Exorista sorbillans Wiedemann (Diptera:Tachinidae)

BULBULI KHANIKOR and DIPSIKHA BORA
DEPT. OF ZOOLOGY, GAUHATI UNIVERSITY,
GUWAHATI-781014
ASSAM, INDIA

The following specification particularly describes and ascertains the nature of this invention and the manner in which it is to be performed
FIELD OF THE INVENTION

The present invention mainly relates to the identification of petroleum ether extract and essential oil of Ocimum gratissimum L. as the efficacious product against the silkworm parasitoid uzi fly, Exorista sorbillans. It also relates to the chemical characterization of the most active extracted fraction against the silkworm parasitoid uzi fly, Exorista sorbillans. It further relates to preparation of biopesticide formulations based on essential oil of Ocimum gratissimum and its active ingredients for the control of silkworm parasitoid uzi fly, Exorista sorbillans.

BACKGROUND OF THE INVENTION

Uzi fly Exorista sorbillans is an endoparasitoid of some lepidopteran insects and creates major damage to silkworm crops. The incidence of Uzi infestation in commercial silkworms with regard to mullberry silkworm, Bombyx mori (Kumar, 1987), tasar silkworm, Antheraea mylitta Drury (Rath, 1966), eri silkworm, Samia cynthia ricini Boisduval (Ghosh, 1949; Sarkar, 1988) and muga silkworm, Antheraea assama (Bharali, 1968) was reported at different times by different scientists. The flies spend their larval stages within the body of the silkworm and either kill the larvae or render the silk cocoons unreelable. The threat posed by this fly infestation became a national concern since 1980. There are reports of uzi fly infestation of silkworm from all silk producing countries of the world. It causes 20-80% crop loss depending upon the season of the year. Different methods including physical, mechanical, chemical and biological control have been suggested to control uzi menace from time to time. Use of herbal product in sericulture field for controlling pests and pathogens are preferable as use of chemical pesticides highly affect the silkworms when targeting the pests and pathogens. Herbal products are eco-friendly and have less effect on non-target organisms. Therefore, extracts and essential oil of Ocimum gratissimum derived from leaves of the plant was chosen to control the fly population.
Ocimum gratissimum L. (Lamiales:Lamiaceae) is a perennial herb with a long history of traditional medicinal uses in many countries in the world especially in Africa and Asia. A wide range of chemical compounds including alkaloids, flavonoids, terpenoids etc. have been isolated from this species. Extracts and essential oils of this plant have been reported to possess medicinal, bactericidal, fungicidal, nematicidal and insecticidal activities. It has been used safely in food, flavor and fragrance industry. It is a potential candidate of pharmaceutical industry and having report of low mammalian toxicity. The plant has been already exploited for the control and management of many insect pests and parasitoids.
There are few reports in the non-patent literature on the extraction of Ocimum gratissimum. Among them the Okoli et. al, [C. O. Okoli, A. C. Ezike, O. C. Agwagah, and P. A. Akah “Anticonvulsant and anxiolytic evaluation of leaf extracts of Ocimum gratissimum, a culinary herb” Pharmacognosy Res. 2010 Jan-Feb; 2 (1): 36–40] which reported the Anticonvulsant and anxiolytic activities of leaf extracts and fraction of Ocimum gratissimum L. There is also a report on the antibacterial activity of Ocimum gratissimum leaf extract to Staphylococcus aureus, Escherichia coli, Salmonella typhi and Shigella flexneri in Yakubu et al., 2009 [Yakubu, S. E., ELLA, E. E. and Eluogu, O. O. (2009). Preliminary investigation into the antibacterial activity of Ocimum gratissimum leaf extract to Staphylococcus aureus, Escherichia coli, Salmonella typhi and Shigella flexneri. Biological and Environmental Science Journal for the Tropic Vol. 6(4): 132 – 135]. But there is no report of efficacy of extracts and essential oil of Ocimum gratissimum against silkworm parasitoid uzi fly, Exorista sorbillans (Diptera: Tachinidae).
We have reported the toxicity of essential oil compounds against Exorista sorbillans (Diptera: Tachinidae), a parasitoid of silkworm in Khanikor and Bora 2011 [Khanikor, B. and Bora, D. Toxicity of essential oil compounds against Exorista sorbillans (Diptera: Tachinidae), a parasitoid of silkworm African Journal of Biotechnology 10(85), pp. 19807-19815, 28 December, 2011] and also in chapter 8 of Pesticides – Advances in Chemical and Botanical Pesticides by Bora et al., 2012 [Dipsikha Bora, Bulbuli Khanikor and Hiren Gogoi Plant Based Pesticides: Green Environment with Special Reference to Silk Worms at http://dx.doi.org/10.5772/47832] as Plant Based Pesticides: Green Environment with Special Reference to Silk Worms.
The US Patent No. 5688509 A discloses an environment friendly invention describing controlled release insect repellent device and a method for repelling insects from food, tobacco or other consumable items. The repellent compound was chosen from the group consisting of essential oils and active ingredients of essential oils. These were almond bitter oil, anise oil, basil oil, bay oil, caraway oil, cardamom oil, cedar leaf oil, caley oil, chamomile oil, cinnamon leaf oil, citronella oil, clove oil, coriander oil, cumin oil, dill oil, eucalyptus oil, fennel oil, ginger oil, grape fruit oil, lemon oil, lime oil, mint oil, parsley oil, pepper oil, rose oil, spearmint oil, sweet oil, orange oil, thyme oil, turmeric oil, oil of wintergreen, citronellal, methyl salicylate, ethyl salicylate, propyl salicylate, citronellol, safrole and D- limonene. The substrate was chosen from the group consisting of paper, paperboard, corrugated boxes, and medium of corrugated boxes, plastics, plastic sheeting, cloth and metals. The controlled release agent was chosen from the group consisting of latex, resins, solution acrylics, polyvinyl resins, natural gums, polyethylene waxes, wax emulsions, polymeric printing inks, polymeric aqueous foams, adhesives, polymeric protective coatings, primers and natural resin formulations. The inventor suggested the device as a safe and beneficial device to be used against insects. EP 1845786 A1 discloses 15 formulations of biopesticides containing microcapsulated essential oils. Each formulation comprised at least one encapsulated essential oil like Citronella oil and a non-volatile vehicle like non volatile oil sesame oil , cotton seed oil etc. US 20120128648 A1 discloses an invention describing isolation and characterization of biopesticide composition and biopesticide formulations which are capable of serving as effective bio control agent and or pest management agents and also able to serve as biomarkers in research studies. Bioassay was conducted against Helicoverpa armigera. The U.S. Patent No. 7230033 B2 discloses an invention describing efficacy of aqueous formulation of nootkatone (5%) and carvacrol (5%) against Ixodes scapularis Say and Amblyomma americanum L. (Acari : Ixodidae). Arora et al., 2012 [Sumitra Arora, Ashok K. Kanojia, Ashok Kumar, Navin Mogha and Vikrant Sahu. Biopesticide formulation to control tomato lepidopteran pest menace. Current Science. 102(7), pp. 1051-1057. 10 April, 2012] proposed an indigenous biopesticide formulation to control tomato lepidopteran pest menace. The ingredients of the formulation was Phyllanthus emblica fruit (4%), Curcuma zedooria (6%), Potassium aluminum sulphate dodecahydrate (5%), Allium cepa bulb (3.5%), Allium sativum bulb (4%), Calotropis procera (5%), fresh cow-dung extract (3%), Lycopersion esculentum leaf extract (6%), Ferula narthexboiss (2%), Azadirachta indica leaves (5.5%), Ocimum canum (4%), Cow urine (52%).
None of the prior arts report or disclose the efficacious product against silkworm parasitoid uzi fly, Exorista sorbillans. Therefore, the present inventors have developed efficacious extract from Ocimum gratissimum against silkworm parasitoid uzi fly, Exorista sorbillans. Also, prepared the biopesticide formulation based on essential oil of Ocimum gratissimum and its active ingredient(s) for using as a biocontorl agent of silkworm parasitoid uzi fly, Exorista sorbillans.

OBJECTS OF THE INVENTION

The primary object of the present invention is to get the extract from Ocimum gratissimum as a biocontrol agent for control of silkworm parasitoid uzi fly, Exorista sorbillans.

Another object of the present invention is to get the essential oil of Ocimum gratissimum for studying the efficacy against silkworm parasitoid uzi fly, Exorista sorbillans.

The further object of the present invention is Chemical characterization of the most active fraction(s).

The yet another object of the present invention is for preparation of efficacious herbal product which is eco-friendly and have less effect on non-target organisms as biopesticide formulation based on the extracts of Ocimum gratissimum for control of silkworm parasitoid uzi fly, Exorista sorbillans.

STATEMENT OF INVENTION
Process for extraction of petroleum ether extract of Ocimum gratissimum as an efficacious product for control of silkworm parasitoid uzi fly, Exorista sorbillans comprising:
selection, washing and shade drying of fresh leaves of Ocimum gratissimum,
extraction with absolute ethanol
Further fractionation of ethanol extract with the solvents selected from the group of petroleum ether, chloroform, butanol and water.
Process for extraction of essential oils as an efficacious product for control of silkworm parasitoid uzi fly, Exorista sorbillans comprising of:
selection, washing and cutting of fresh leaves of herbs selected from Ocimum gratissimum, Ocimum sanctum, Eucalyptus maculata, Calistemon linearis and peel of Citrus sinensis into small pieces;
extraction of essential oil;
heating &
Collection of the essential oil.
The extraction is carried with water (hydrodistillation method) using Clevenger type apparatus. The essential oil floating in the oil collection area after 4 hours of heating was collected after draining the bottom water layer and further separated with petroleum ether and ethyl acetate solvent system (5:1) wherein the F2 fraction acts as an active fraction for control of silkworm parasitoid uzi fly, Exorista sorbillans. Product obtained is an efficacious product for control of silkworm parasitoid uzi fly, Exorista sorbillans. Product is the essential oil of Ocimum gratissimum as an efficacious product for control of silkworm parasitoid uzi fly, Exorista sorbillans.
Biopesticide formulation as an efficacious product for control of silkworm parasitoid uzi fly, Exorista sorbillans comprises of:
an effective amount of essential oil of Ocimum gratissimum with or without one or more of the essential oils selected from Ocimum sanctum, Eucalyptus maculata, Calistemon linearis, Citrus sinensis; and
synthetic gum as a control release agent.
The essential oils &/essential oil compounds selected are carvacrol, thymol, eugenol, eucalyptol and citral and the effective amount of selected essential oils&/essential oil compounds is in the ratio of 1:1:1:1:1.
Process for preparation of biopesticide formulation as an efficacious product for control of silkworm parasitoid uzi fly, Exorista sorbillans comprising of:
extraction of essential oils as claimed in claims 1 to 5;
Mixing the effective amount of extracted essential oil of Ocimum gratissimum optionally with one or more of the essential oils &/essential oil compounds selected from Ocimum sanctum, Eucalyptus maculata, Calistemon linearis, Citrus sinensis along with the synthetic gum to form the biopesticide formulation.

SUMMARY OF THE INVENTION

Process for the extraction of petroleum ether extract from Ocimum gratissimum for control of the silkworm parasitoid uzi fly, Exorista sorbillans comprised of : a) Selection of fresh leaves; b) Shade drying of fresh leaves c) preparation of ethanolic, hydro alcoholic and water extract and their bioassay against E. sorbillans d) Based on bioassay result, further fractionation of crude ethanolic extract was carried out by using solvents i.e. petroleum ether, chloroform, butanol and water in successive series. e) Bioassay of each solvent extract was carried out by preparing 5% concentration of each extract in acetone against uzi fly using contact residual method.
From the result of comparative efficacy test, as petroleum ether extract was found as promising product to be used against uzi fly with LC50 value at 5.38% essential oil was extracted from the leaves of Ocimum gratissimum.
The essential oil obtained was dried by using anhydrous sodium sulphate (Merck). GC-MS analysis of essential oil of Ocimum gratissimum showed the possible presence of the following compounds in the oil: alpha-phellandrene (RT 8.33); bicycle[3.1.0] hex-2-ene, 2-methyl- (RT 8.33); bicycle[3.1.0]hexane,4-methyl-1-(RT8.33); (+)-2-carene(RT8.68); cyclohexene, 1-methyl-4- (1-methylethyl) (RT 8.68); 1,3-cyclohexadiene,1-methyl-4-(1-(RT 8.68); benzene,1-ethyl-2,3-dimethyl (RT 8.75);benzene,1-methyl-4-(1-methyl ethyl (RT 8.75); benzene, 4-ethyl-1,2-dimethyl-(RT 8.75); 3-cyclohexen-1-ol,4-methyl-1(1-m(RT 10.27); benzenemethanol, 4- (1,1-dimethylet (RT 10.27); copaene (RT 10.65); alpha- cubebene (RT 10.65); ylangene (RT 10.65); thymol (RT 11.02); phenol,2-methyl-5-(1-methylethyl) (RT 11.02); phenol, diethyl-(RT 11.02); thymol (RT 10.78); phenol,2-methyl-5- (1-methylethyl) (RT 10.78); phenol,2-(1,1-dimethylethyl)- (RT 10.78); benzene, 2-methoxy-1,3,5-trimethyl (RT 11.19); phenol,2,3,5,6-tetramethyl- (RT 11.19); benzene, 2-methoxy-1,3,4-trimethyl (RT 11.19); silane, trimethylphenyl- (RT 11.24); phenol,2,3,5,6-tetramethyl-(RT 11.24); thymol (RT 11.24).
e) Bioassay of essential oil of O. gratissimum was carried out against E. sorbillans using contact residual method and the sub lethal concentration (LC50) value was found to be at 0.42%. From the result it was revealed that the essential oil of the plant was more potential than the petroleum ether extract. f) Further fractionation of essential oil was carried out with the help of preparative thin layer chromatography technique using petroleum ether and ethyl acetate (5:1) as solvent system. Two fractions F1 and F2 were separated and after elution, bioassay of each fraction was carried out against uzi fly E. sorbillans using contact residual method. g) F2 fraction of the oil was found as the active fraction of O. gratissimum oil. h) GC-MS analysis of the active fraction (F2) from extract of essential oil of Ocimum gratissimum showed the presence of possible compounds i.e. thymol, (RT 10.3) or phenol 2-methyl- 5-(1-methylethyl)- (RT 10.3); oxamide, n- butyl-N-( 4- tert butylphenyl)- (RT, 16.2 & 21.7) and or oxamide-N-( 4- tert butylphenyl)-N-(1-methylpropyl)- (RT, 16.2 & 21.7). The major compound in the active fraction was either Thymol or phenol 2-methyl- 5-(1-methylethyl) - i.e. carvacrol (RT 10.3) that might be the active compound (s) for the potential activity of the active fraction of the oil against E. sorbillans.
Biopesticide formulations were prepared based on essential oil of Ocimum gratissimum. Fumigant mode of application was applied in all the cases. Synthetic gum(GripFix Adhesive Paste) was used as the control release agent. The efficacy of biopesticide formulation was tried to improve by incorporating essential oil of some other aromatic plants viz. Ocimum sanctum, Eucalyptus maculata, Calistemon linearis, Citrus sinensis which have been reported effective against many flies and found effective against uzi fly E. sorbillans in our earlier pilot studies. A total of six formulations were prepared. Of these, Formulation M which was a mixture of equal amount of essential oil of O. gratissimum, O. sanctum, E. maculata, C. linearis, C. citratus was found potential against E. sorbillans. Further formulations comprising essential oil compounds (Sigma) were prepared based on major constituent compound of active fraction of essential oil of O. gratissimum i.e. carvacrol [(phenol 2-methyl- 5-(1-methylethyl) -]. Two formulations (C1 & MII) were prepared. One comprised (C1) carvacrol (5µl) in synthetic gum (5ml) as control release agent in 500ml volume of air and the other formulation (MII) comprised equal mixture of carvacrol (1 µl) and some of the major constituent compound of essential oil of O. gratissimum, O. sanctum, E. maculata, C. linearis, C. citratus in synthetic gum (5ml) as control release agent in 500ml volume of air. The compounds were thymol (1ul), eugenol (1µl), eucalyptol (1µl), citral (1µl). The formulation MII consisting of mixture of compounds was found more effective than C1 i.e. the formulation comprised single compound. To assess the effect of air volume against a given dose, MII formulation was kept in 250ml and 1000ml volume of air besides 500 ml volume of air. The difference of effect in terms of percent mortality of uzi fly with increase volume of air with respect to same dose of the formulation was not apparent at 24h. But decreased percentage of mortality of uzi fly was recorded with increased air volume (from 250ml to 1000ml) at early hours (3h-6h).

DETAILED DESCRIPTION OF THE INVENTION
Collection and Preparation of plant extracts: Plant specimen was identified in Botanical Survey of India, Shillong (Ref.No. BSI/ERC/2010/AKV/181) and the herbarium was submitted in the Department of Life Sciences, Dibrugarh University, Assam.
Preparation of crude plant extracts: Leaves of Ocimum gratissimum was collected from Dibrugarh district of Assam, washed to remove dust, shade dried and ground to powder and preserved in refrigerator at 40 C for future use until extraction. Crude extract of leaf powder was prepared in hot water, alcohol water (50:50) and absolute ethanol as solvents. Crude extracts of alcohol-water (50:50) and absolute ethanol (350ml for each) were prepared by dissolving 150g of ground leaves for 72 h and crude extracts of hot water was prepared by boiling 150 g leaf powder in distilled water (350ml) for 1h and kept for 72 h. Extracts were filtered by using Whatman Filter Paper No 1 after 72 h and the solvents were removed under vacuum below 400C.
Further fractionation of ethanol extract: Ethanol extract of the leaves of Ocimum gratissimum was further fractionated by a series of solvents(Merck) on the basis of polarity namely petroleum ether, chloroform, butanol and water. Initially ethanol extract was prepared by dissolving shade dried leaf material of the selected plants for 72h in absolute ethanol. Then the extracts were filtered by Whatman No1 filter paper and solvent was evaporated in vacuum below 400C. The dried ethanolic extract was then taken in a 2.5 liter separating funnel and petroleum ether was poured and shaked for 30 minutes. It was then allowed to settle for 3h and the supernatant was decanted and filtered. The residue was extracted thrice and the filtrate was taken as petroleum ether extract. Similarly the chloroform, butanol and water extracts were prepared and stored in refrigerator for bioassay.
Collection and preparation of plant essential oils: Fresh leaves of the selected plant was collected from areas under Dibrugarh district of Assam, washed to remove dust, cut into small pieces and subjected for oil extraction by hydrodistillation method using Clevenger type apparatus (Clevenger, 1928). Two hundred grams (200g) of fresh leaves of selected plants were put in 5 L round bottomed flask and was filled with double distilled water up to two third marks of the flask. Electrical heating mantle was used to heat(40-500C) the water. The essential oil floating in the oil collection area after 4 hours of heating was collected after draining the bottom water layer.
Measurement of yield of essential oils and storage
The percentage (v/w) of oil yield was calculated by using the following formula:

% of oil yield = Recovery of the Oil x 100
Weight of the plant

The essential oil extracted was dried over anhydrous sodium sulphate (Na2SO4) stored in sealed screw cap vial in deep freezer (-200C) for bioassay and analysis.
Insect culture: Exorista sorbillans Wiedemann (Diptera: Tachinidae): Maggots of Exorista sorbillans (uzi flies) were brought from Govt. Sericulture Farm of Assam and kept in wire-netted wooden box (size: 30cm x 30cm). The newly emerged adult flies were maintained on 20% honey in a well ventilated room under natural light and temperature conditions (Max: 32°C; Min: 190C).
Bioassay with aqueous, hydroalcohol and ethanol extract against E. sorbillans: Three day old adult uzi flies were taken for bioassay. Bioassay was done by contact residual film technique (Finney, 1971) using 10 percent concentration of crude hot water, hydroalcohol and ethanol extracts prepared by using the same respective solvent in which the extract was initially prepared. 1.5ml of prepared solution of each extract was applied into 110mm Whatman No.1 filter paper which was kept in a petri dish and was allowed to dry in room temperature (19 - 32°C, RH 56 -82%). The treated petriplates were covered by 110mm diameter glass funnel. The stem of the funnel was wrapped with nylon net and thus aeration was provided to insects during treatment hours. For each experiment a control was maintained where only the respective solvent was applied.
Bioassay with fractionated ethanol extract against E. sorbillans: Five percent concentration of extracted petroleum ether fraction, chloroform fraction, butanol and water fractions prepared in the same respective solvents were used to determine the effective solvent fraction of crude ethanolic extract against uzi fly. Observation was made at 1, 3, 6, 24, 30, 48 hours from the time of application. 1.5ml of 5% concentration of each solution was applied into Whatman (110 mm) filter paper which was pasted in a petri dish and was allowed to dry in room temperature (19 - 32°C, RH 56 - 82%). The treated petri plates were covered by glass funnel of 110mm diameter. The stem of the funnel was wrapped with nylon net and thus aeration was provided to insects during treatment hours. For each experiment control was maintained where only the respective solvent was applied. Bioassay was carried out by contact residual film technique.
Bioassay with effective solvent extract against E. sorbillans: The fractionated ethanolic extract which gave more than 50% death of E. sorbillans after 24h of treatment at 5% concentration was considered for further bioassay to get sub lethal dose. For the purpose, different concentrations (0.01 to 15% at geometrically uniform interval) of the effective extract was prepared and subsequent bioassay against uzi fly was carried out by contact residual film technique to determine LC50 value of the most effective solvent extract. Bioassay was carried out by contact residual film technique.
Bioassay with essential oil of selected plants: Bioassay by using essential oil of Ocimum gratissimum against E. sorbillans was carried out by contact residual film technique. Essential oil solutions of O. gratissimum were prepared in acetone. 1.5 ml oil solution was applied to 110 mm Whatman No.1 filter paper which was kept in a petri dish and was allowed to dry for 5-7 minutes in room temperature (19 - 32°C, RH 56 - 82%). The treated petriplates were covered by 110 mm diameter glass funnel. The stem of the funnel was wrapped with nylon net and thus aeration was provided to insects during treatment hours. For control, only acetone solvent at an amount of 1.5ml was applied to Whatman No.1 Filter paper against each test concentration. Third day old adult uzi flies were subjected to treatments. Three replications, each containing 10 insects were maintained for each treatment. Different concentrations of oil (0.01 to 0.5%) were prepared for determining LC50 value of essential oil of O. gratissimum. Lethal time for the essential oil against E. sorbillans was calculated by applying 1µl of crude essential oil topically, on thorax of the third day old uzi flies with the help of micropipette.
FTIR (Fourier Transfer Infrared Spectrograph) analysis of active fraction of petroleum ether extract: The bioassay guided active fractions of petroleum ether extract and essential oil of the experimental plant was dissolved in chloroform. Spectra were taken in neat mode using Shimadzu Prestige-21 FTIR spectrophotometer.

GC (Gas chromatograph) analysis of essential oil of the plant: For determining the compounds present in essential oil of O.gratissimum GC analysis was done by using Varian CP-3800. The conditions applied were: Detector FID 250 0C, Injector 250 0C. Column ovens were 400 for 1 min, @ 30 C till 1100 for 3 min, @ 50C till 2600 C for 5 min Carrier gas was nitrogen (80 PSI). Air (O2) pressure 60 PSI, hydrogen (H2) 40 PSI. Flow rate 1µl/min.
GC-MS (Gas chromatography- mass spectroscopy) analysis of essential oil and active fraction of essential oil of Ocimum gratissimum: For determining the compounds present in essential oil and active fraction of essential oil of O. gratissimum GC-MS analysis was done by using Jeol, Accu TOK GCV JMS-T100GCV MODEL. Column was HP5, 30 meter long, 0.25mm ID and 0.25nm film thickness. The conditions applied were: Injector temperature 250 0C; Interface temperature 2800C. Column program was 800C - 1 min, Hold 50C/Min- 2500C-10 Min, Hold-300C/Min- 2800C. Carrier gas was Helium. Flow rate 1ml/min. Injection volume 0.2µl. Split ratio 1:100.
TLC (Thin layer chromatography) separation of essential oil of Ocimum gratissimum: For initial screening, three solvent systems comprising of petroleum ether (boiling point 40-600C) and ethyl acetate at the ratio of 10:1, 7:1 and 5:1 were prepared to differentiate individual spots of the essential oils in aluminium TLC sheet (Merck). Finally, fractionation of oils was done in preparative TLC plate using the solvent system comprising of petroleum ether and ethyl acetate (5:1) and different fractions were collected. Solvent was removed by distillation with temperature set at 400C.
Bioassay with different fractions of essential oil obtained from TLC of the essential oil of Ocimum gratissimum on E.sorbillans: Bioassay of each individual fraction obtained from TLC was carried out by contact residual film technique using acetone (Merck) as solvent.
Preparation of bioformulation from essential oils and bioassay against E. sorbillans: Based on essential oil of O.gratissimum, initially six formulations were prepared and tested on 3rd day old adult E. sorbillans. Essential oil of some other locally available plants namely Ocimum sanctum (Lamiaceae), Eucalyptus maculata (Myrtaceae), Callistemon linearis (Myrtaceae), Citrus sinensis (Rutaceae) were considered to add as ingredient of formulation on the basis of their efficacy obtained in our earlier pilot studies. The prepared formulations were 5:0G, 4:1GOs, 4:1GEm, 4:1GCl, 4:1GCs and M. Synthetic gum (GripFix Adhesive Paste) was used as control release agent. Fumigant mode of application was chosen for bioassay studies of bioformulations against uzi fly. Freshly prepared formulation was poured into plastic cap (cap of 15ml screw cap vial) and wrapped by muslin cloth from upper side and placed in conical flask of 500ml capacity. The muslin cloth prevented the flies to come in direct contact of the formulation but allowed the vapor to come out and spread uniformly in the enclosed chamber. After keeping the formulation containing cap in the conical flask, 10 numbers of flies were released and the flask was sealed with the help of aluminium sheet and tightened by rubber band to keep the air blocked inside the flask. Response of the flies in terms of knock down and mortality was recorded from 1minute to 24hours at successive time interval. The formulation 5:0G comprised 5ul essential oil of O. gratissimum in total of 5ml synthetic gum. The formulation 4:1GOs comprised 4ul essential oil of O. gratissimum and 1ul of O. sanctum in total of 5ml synthetic gum. The formulation 4:1GEm comprised of 4ul essential oil of O. gratissimum and 1ul of E. maculata in total of 5ml synthetic gum. The formulation 4:1GCl comprised of 4ul essential oil of O. gratissimum and 1ul of C. linearis in total of 5ml synthetic gum. The formulation 4:1GCs comprised of 4ul essential oil of O. gratissimum and 1ul of C.sinensis in total of 5ml synthetic gum and the formulation M comprised of equal amount of essential oil of Ocimum gratissimum (1ul), Ocimum sanctum(1ul), Eucalyptus maculata(1ul), Callistemon linearis(1ul), Citrus sinensis(1ul) in 5ml of synthetic gum.

Preparation of bioformulation from essential oil compounds and bioassay against E. sorbillans: Based on the efficiencies of formulations prepared from crude essential oil, further formulations were prepared by selecting essential oil compounds which have been reported and identified as major constituent compound of the concerned plant essential oils. These were Carvacrol, Thymol, Eugenol, Eucalyptol and Citral. Carvacrol and Thymol are the constituent compounds of O.gratissimum, Eugenol is the constituent compound of O.sanctum, eucalyptol is the constituent compound of both E. maculata, and C.linearis, citral is the constituent compound of C.sinensis. Two formulations were prepared taking these compounds. Fumigant mode of application was used for bioassay studies. Synthetic gum(GripFix Adhesive Paste) was used as control release agent and plastic cap was used as substrate of the formulation where the prepared formulation was poured and after covering by muslin cloth placed carefully in conical flask. Formulation MII was prepared by mixing equal amount of Carvacrol (1µl), Thymol (1µl), Eugenol (1 µl), Eucalyptol (1µl) and Citral (1µl) into a total of 5ml synthetic gum. Three separate set of this MII formulation was then kept in conical flask of three volumes i.e. 250ml, 500ml and 1000ml capacities. The conical flask having three volume capacities would show the effect of air volume with respect to given doses of insecticide after fumigant mode of application. Flies were then subjected to get exposure of the formulation and mortality data were recorded against time interval. Another formulation C1 was prepared by mixing 5ul of carvacrol into 5ml of synthetic gum and the prepared formulation was kept in conical flask having500ml capacity.
RESULTS:
10% concentrations of cold water, hot water, hydroalcohol and ethanol extracts of O. gratissimum were prepared and bioassay was carried out by contact residual film technique to determine the active extract for further study. When adults of E. sorbillans were subjected to crude hot water, hydroalcohol and ethanol extracts, percent mortality of flies at 24h was recorded 13.33±3.33, 6.67 ±3.33 and 15±5.01 respectively. The ethanol extract was the most effective of all (Fig.7).
The ethanolic extract of O.gratissimum was further fractionated in eleutropic series of solvents namely petroleum ether, chloroform, butanol and water. 5% concentration of each solvent extract was used in bioassay experiments. The petroleum ether extract of O.gratissimum caused 6.67±3.34, 13.33±5.77, 70±17.34, 86.68±13.35 and 96.67±3.34% mortality after 3h, 6h, 24h, 30h and 48h respectively (Fig.8). Chloroform extract caused 20±0 and 20.83±8.34% at 30 and 48h respectively. Butanol extract caused 14.50±3.92 and 15.40±4.82 respectively at 30 and 48h and water extract caused no mortality of uzi fly. Regression plot was drawn using mortality percentage against time interval at 5% concentration of petroleum ether extract of O.gratissimum. Result showed that LT50 (Lethal time 50) of O.gratissimum 20.86h (Y=2.45+2.27X) (Fig.10).
Out of the four extracts the petroleum ether extract was found most potential and so the extract was chosen to determine their sublethal concentration (LC50) against E.sorbillans. To determine LC50 value, 0.03, 0.06, 0.125, 0.50, 0.75, 1.0, 2.5, 5.0, 7.5, 10, 12.5, 15% concentrations of petroleum ether extract were applied against E.sorbillans by contact residual film technique. Concentrations were chosen on the basis of preliminary examination. The calculated LC50 was 5.38% (Y=3.49+2.06X; R.Sq= 0.980) (Fig.11). From probit analysis 95% confidence interval was recorded as 2.058 to 2.338 with Z-value as 30.735. The chi square value was 553.53 with 37 degrees of freedom (P<0.05) (Table.1).
As the O. gratissimum plant is aromatic and contains good quantity of essential oil which has been reported effective against different insect pest including dipteran flies by many investigators and as the nonpolar fraction of extract i.e. petroleum ether extract was found effective we further focused on essential oil of the plant for bioassay against E.sorbillans. Percentage of essential oil yield from fresh leaves of O.gratissimum was 0.63±0.09 percent.
Based on pilot experiments, 0.01, 0.03, 0.06, 0.125, 0.25 and 0.5% concentration of essential oil of O.gratissimum was chosen to determine LC50 value against E. sorbillans by contact residual film technique. The LC50 value of O. gratissimum was 0.42% (Y=6.07+2.81X; R.Sq= 0.941) (Fig.12). From probit, 95% confidence interval was recorded as 2.672 - 3.325 with Z-value at 17.996. The chi square value was 185.26 with p<0.05 (Table 1). The lethal time recorded after topical application of 1µl essential oil of O. gratissimum on E.sorbillans was recorded as 0 .51±0.2 minute which was more effective than other competent essential oil (Table 2). Early knock down and subsequent rapid recovery of uzi fly was observed at contact residual mode of application of essential oil of O.gratissimum compared to other competent essential oils.
FTIR analysis of petroleum ether extract of O.gratissimum showed absorption peak at 3471.87 indicating –NH stretching, at 2966.87 indicating –CH stretching, at 1734.01 indicating –C=O stretching and at 1462.04 indicating –CH asymmetric bending (Fig.1).
FTIR analysis of essential oil of O.gratissimum showed absorption peak at 3527.80 indicating presence of OH or NH stretching, 2962.66 indicating –CH stretching, 1618.28, 1581.53, 1510.26, 1450.26 indicating aromatic system and absorption peak at 1153.43 indicating presence of -C-O-C stretching (Fig.2).
GC-MS analysis of the essential oil of O.gratissimum showed the presence of following possible compounds: alpha-phellandrene (RT 8.33); bicycle[3.1.0] hex-2-ene, 2-methyl- (RT 8.33); bicycle[3.1.0]hexane,4-methyl-1-(RT 8.33); (+)-2-carene(RT 8.68); cyclohexene,1-methyl-4-(1-methylethyl) (RT 8.68); 1,3-cyclohexadiene,1-methyl-4-(1-(RT 8.68); benzene,1-ethyl-2,3-dimethyl (RT 8.75); benzene,1-methyl-4-(1-methylethyl (RT 8.75); benzene, 4-ethyl-1,2-dimethyl-(RT 8.75); 3-cyclohexen-1-ol,4-methyl-1(1-m(RT 10.27); benzenemethanol,4-(1,1-dimethylet(RT 10.27); copaene (RT 10.65); alpha-cubebene (RT 10.65); ylangene (RT 10.65); thymol (RT 11.02); phenol,2-methyl-5-(1-methylethyl) (RT 11.02); phenol, diethyl-(RT 11.02); thymol (RT 10.78); phenol,2-methyl-5-(1-methylethyl) (RT 10.78); phenol,2-(1,1-dimethylethyl)- (RT 10.78); benzene, 2-methoxy-1,3,5-trimethyl (RT 11.19); phenol,2,3,5,6-tetramethyl- (RT 11.19); benzene, 2-methoxy-1,3,4-trimethyl (RT 11.19); silane, trimethylphenyl- (RT 11.24); phenol,2,3,5,6-tetramethyl-(RT 11.24); thymol (RT 11.24) (Fig.3).
In the preparative thin layer chromatography, two fractions (F1, F2) were separated from the essential oil of O.gratissimum at solvent system comprising of petroleum ether and ethyl acetate (5:1). Effective fraction was determined by conducting bioassay of each fraction against 3rd day old uzi fly by using contact residual film method. F2 fraction of O.gratissimum was found effective against uzi fly. GC-MS analysis of the F2 fraction of the oil showed presence of the following possible compounds: thymol or phenol,2-methyl-5-(1-methylethyl)- (RT=10.3 min; molecular weight 150 , molecular formula C10H14O); oxamide, n-butyl-N’-(4-tert-butylphenyl)- (RT=21.7 min; molecular weight 276, molecular formula C16H24N2O2); oxamide,N-(4-tert-butylphenyl)-N’(1-methylpropyl)- (RT=21.7 min; molecular weight 276 , molecular formula C16H24N2O2) (Fig.4, 5). The major peak was at retention time 10.3 which was detected as either Thymol or phenol,2-methyl-5-(1-methylethyl)- (also known as carvacrol) that might be the active compound of essential oil fraction (F2) of the plant showing adulticidal activity against uzi fly. From the results, it can be conferred that petroleum ether extract, essential oil and constituent of essential oil thymol and /or carvacrol are potential candidate for controlling uzi fly Exorista sorbillans.
Six formulations were prepared based on essential oil of O. gratissimum and tested against adults of E.sorbillans. These were 5:0 G, 4:1 GOs, 4:1GEm, 4:1GCl, 4:1GCs and M. The formulation M comprising of equal amount of essential oil of Ocimum gratissimum, Ocimum sanctum, Eucalyptus maculata, Callistemon linearis, Citrus sinensis showed best performance with maximum six deaths of the uzi flies against 0-2 deaths caused by the other formulations (Table 4; Fig.13).
Based on the efficiencies of formulations prepared from crude essential oil, another set of formulations was prepared by selecting essential oil compounds which have been reported and identified as major constituent compound of the tested plant essential oils. These were Carvacrol, Thymol, Eugenol, Eucalyptol and Citral. In one formulation equal amount (1µl) of each essential oil compound was added to a total of 5 ml synthetic gum. The freshly prepared formulation (MII) was then carefully kept in conical flask of 500ml volume. Flies were exposed to the formulation and mortality was recorded at different time interval. For testing the efficacy of single compound a formulation was prepared by adding 5 µl of carvacrol to 5 ml of synthetic gum. The prepared formulation (CI) was kept in conical flask having 500 ml capacity and flies were exposed to the formulation to evaluate its fumigant effect on flies. The results showed that the formulation (MII) prepared from mixing of selected essential oil compounds gave 60% mortality of flies after 6 h of treatment and 100% mortality after 24h of exposure period at 500 ml of air volume. Thus it revealed that the formulation (MII) prepared from constituent essential oil compounds was more effective than the formulation (M) prepared from selected crude essential oils. Formulation (CI) prepared from single essential oil compound at equal dose was less effective than the formulation (MII) prepared from mixing of selected essential oil compounds. The result showed that the formulation CI comprising single compound carvacrol gave 30% mortality at 24 h of exposure period whereas formulation MII comprising equal mixture of essential oil compounds gave 100% mortality at the same exposure period (Fig.14)
To study the effect of volume of air on efficacy of the formulation, the formulation (MII) comprising mixture of compounds were kept in flasks of different capacities such as 250 ml, 500 ml and 1000 ml .The result showed that the efficiency of the formulation reduced with increase of air volume. It was apparent at 6 h after exposure where mortality of flies decreased from 70% to 30% with increase of air volume from 250 ml to 1000 ml (Fig.15).

Table 1: LC50 (Lethal concentration 50) of petroleum ether extract and essential oil of selected plants when applied against E.sorbillans (CRF)
Sl No. Extract/Oil Mode of application LC50
(%) Regression Equation R2 Value Chi sq. value D.F. Z-value 95% Confidence interval
1 O.gratissimum petroleum ether extract Contact residual 5.38 Y=3.49+2.06X 0.980 553.53 37 30.74 2.058-2.338
2 O.gratissimum oil Contact residual 0.42 Y=6.07+2.81X 0.941 185.26 16 17.20 2.672-3.325

Table 2: Lethal time of essential oil of six plants against E. sorbillans on topical application at dose 1µl/insect
Sl.No. Plant (Oil) LT (Minute±SE)
1. Ocimum gratissimum 0.51±0.20
2. Ocimum sanctum 4±0.58
4. Citrus reticulata 3.11±0.07
5. Cymbopogon citratus 5.25±0.14
6. Eucalyptus globulus 105±8.67

Table 3: Result of GC-MS analysis (Library search) of essential oil of O.gratissimum

Sl.No RT Area% Compound Name(Library/ID) Ref Qual
1 8.33 3.05 Alpha-Phellandrene 6698 90
Bicyclo[3.1.0]hex-2-ene, 2-methyl- 65773 78
Bicyclo[3.1.0]hexane, 4-methyl-1- 6650 43
2. 8.68 3.12 (+)-2-Carene 6599 98
Cyclohexene, 1-methyl-4-(1-methyle 65783 97
1,3- Cyclohexadiene, 1-methyl-4-(1- 6620 97
3. 8.75 38.41 Benzene, 1-ethyl-2, 3-dimethyl- 65556 81
Benzene, 1-methyl-4-(1-methylethyl 65535 81
Benzene, 4-ethyl-1,2-dimethyl- 65569 81
4. 10.27 2.61 3-Cyclohexen-1-ol, 4-methyl-1(1-m 67103 83
3-Cyclohexen-1-ol, 4-methyl-1(1-m 67093 50
Benzenemethanol, 4-(1,1-dimethylet 13530 18
5. 10.65 0.14 Copaene 23943 96
Alpha-Cubebene 23919 96
Ylangene 23969 80
6. 10.72 11.02 Thymol 66820 94
Phenol, 2-methyl-5-(1-methylethyl) 9788 90
Phenol, diethyl- 9816 72
7. 10.78 34.42 Thymol 66820 76
Phenol, 2-methyl-5-(1-methylethyl) 9788 76
Phenol, 2 -(1,1-dimethylethyl)- 9824 72
8. 11.19 1.79 Benzene, 2-methoxy-1,3,5-trimethyl 66823 59
Phenol, 2,3,5,6-tetramethyl- 66825 59
Benzene, 2-methoxy-1,3,4-trimethyl 9866 58
9. 11.24 5.44 Silane, trimethylphenyl- 66768 47
Phenol, 2,3,5,6-tetramethyl- 66825 40
Thymol 9862 30
RT? Retention time; Library/ID=C: DATABASE\NBS75K.L; Ref? Reference;
Qual? Quality of identification

Examples of Biopesticide formulation:
Table 4: Fumigant effect of different formulations of essential oil (500 ml air) against E. sorbillans

1m
(k) 1m
(d) 15m
(k) 15m
(d) 30m
(k) 30m
(d) 1h
(k) 1h
(d) 3h
(k) 3h
(d) 18h
(k) 18h
(d) 24h
(k) 24h
(d)
Con 0 0 1 0 1 0 0 0 0 0 4 0 4 0
M(1:1:1:1:1) 2 0 2 0 3 0 3 0 3 0 4 1 4 6
4:1GOs 1 0 2 0 1 0 2 0 2 0 3 0 3 0
4:1GEm 3 0 2 0 3 0 2 0 2 0 6 1 8 2
4:1GCl 1 0 2 0 1 0 0 0 2 0 1 0 3 0
4:1GCs 3 0 2 0 4 0 7 0 6 0 8 0 8 0
5:0 G 1 0 4 0 6 0 0 0 0 0 3 0 3 0
k-> knock down; d-> death
Con-> Control (5ml gum)
M(1:1:1:1:1)-> Formulation containing mixture of essential oil of O. gratissimum, O sanctum, Eucalyptus maculata, Calistemon linearis, Citrus sinensis at a ratio of 1 µl each in total of 5 ml gum.
4:1GOs -> Formulation containing 4µl essential oil of O. gratissimum and 1 µl of O. sanctum in total of 5 ml gum.
4:1GEm -> Formulation containing 4 µl essential oil of O. gratissimum and 1 µl of E. maculata in total of 5 ml gum.
4:1GCl -> Formulation containing 4 µl essential oil of O. gratissimum and 1 µl of C. linearis in total of 5 ml gum.
4:1GCs -> Formulation containing 4 µl essential oil of O. gratissimum and 1 µl of C.sinensis in total of 5 ml gum.
5:0G - > Formulation containing 5 µl essential oil of O. gratissimum in total of 5 ml gum.

Table. 5 Fumigant effect of formulation MII & CI against E.sorbillans exposed to 250 ml, 500 ml, 1000 ml air volume having Carvacrol : Citral : Eucalyptol : Eugenol : Thymol (1:1:1:1:1) & only Carvacrol (5µl):
Sample Total no. Mortality
1h 3h 6h 18h 24h
250 ml 500 ml 1 L CI
500 ml 250 ml 500 ml 1 L CI
500 ml 250 ml 500 ml 1 L CI
500 ml 250 ml 500 ml 1 L CI
500 ml 250 ml 500 ml 1 L CI
500 ml
Control 1 10 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Control 2 10 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Control 3 10 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Replica 1 10 0 0 0 0 6 1 2 0 8 5 2 0 10 9 8 0 10 10 10 2
Replica 2 10 0 0 0 0 4 2 2 0 6 7 4 0 10 9 10 1 10 10 10 4
Replica 3 10 0 0 0 0 5 2 2 0 7 6 3 0 10 9 9 1 10 10 10 3
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWING
Fig. 1: IR spectrum of effective petroleum ether extract of Ocimum gratissimum.
Fig. 2: IR spectrum of essential oil of Ocimum gratissimum.
Fig. 3: MS spectra of essential oil of O. gratissimum.
Fig. 4: Chromatogram of effective oil fraction of O. gratissimum.
Fig. 5: MS spectra of effective oil fraction of O. gratissimum.
Fig. 6: MS spectra of effective essential oil fraction of O. gratissimum and possible structures in comparison with literature (RT=10.3 min).
1. Thymol, 2. Phenol, 2-methyl-5-(1-methylethyl)-
Fig. 7: Effect of crude extracts of O. gratissimum on E. sorbillans.
Fig. 8: Effect of different solvent extracts (5% concentration) of O. gratissimum on E. sorbillans.
Fig. 9: Effect of essential oil (0.1 % concentration) of O. gratissimum on E. sorbillans.
Fig. 10: Regression plot of percent mortality of E.sorbillans against time of petroleum ether extract of O. gratissimum (5% concentration).
Fig. 11: Regression plot of probit mortality of E.sorbillans against log concentration of petroleum ether extract of O.gratissimum
Fig. 12: Regression plot of probit mortality of E.sorbillans against log concentration of essential oil of O. gratissimum.
Fig. 13 Fumigant effect of different formulations of essential oil on E. sorbillans against time interval.
k-> knock down; D-> death.
Fig.14. Fumigant effect (500ml air volume) of formulations (MII & CI) of essential oil compounds against E. sorbillans in terms of percent mortality vs. time interval.
(MII-> formulation prepared from mixture of selected essential oil compounds,
CI-> formulation prepared from single essential oil compound i.e. carvacrol).
Fig.15. Effect of air volume on efficacy of formulations in terms of percent mortality of E. sorbillans.
(MII-> formulation prepared from mixture of selected essential oil compounds,
250ml, 500ml, 1000ml -> air volume).
Application No. 1202 /KOL/2013 Sheet 1 of 7
BULBULI KHANIKOR and DIPSIKHA BORA

Fig. 1

Fig. 2

Dated this 17th day of October 2014 SHARANABASAVA
PATENT AGENT FOR APPLICANTS

Application No. 1202 /KOL/2013 Sheet 2 of 7
BULBULI KHANIKOR and DIPSIKHA BORA

Fig. 3

Dated this 17th day of October 2014 SHARANABASAVA
PATENT AGENT FOR APPLICANTS

Application No. 1202 /KOL/2013 Sheet 3 of 7
BULBULI KHANIKOR and DIPSIKHA BORA

Fig. 4

Fig. 5
Dated this 17th day of October 2014 SHARANABASAVA
PATENT AGENT FOR APPLICANTS

Application No. 1202 /KOL/2013 Sheet 4 of 7
BULBULI KHANIKOR and DIPSIKHA BORA

Fig. 6

Dated this 17th day of October 2014 SHARANABASAVA
PATENT AGENT FOR APPLICANTS

Application No. 1202 /KOL/2013 Sheet 5 of 7
BULBULI KHANIKOR and DIPSIKHA BORA

Fig. 7 Fig. 8

Fig. 9

Dated this 17th day of October 2014 SHARANABASAVA
PATENT AGENT FOR APPLICANTS

Application No. 1202 /KOL/2013 Sheet 6 of 7
BULBULI KHANIKOR and DIPSIKHA BORA

Fig. 10

Fig. 11

Fig. 12

Dated this 17th day of October 2014 SHARANABASAVA
PATENT AGENT FOR APPLICANTS
Application No. 1202 /KOL/2013 Sheet 7 of 7
BULBULI KHANIKOR and DIPSIKHA BORA

Fig. 13

Fig.14

Fig. 15

Dated this 17th day of October 2014 SHARANABASAVA
PATENT AGENT FOR APPLICANTS

,CLAIMS:We Claim,
1. Process for extraction of petroleum ether extract of Ocimum gratissimum as an efficacious product for control of silkworm parasitoid uzi fly, Exorista sorbillans comprising:
a. selection, washing and shade drying of fresh leaves of Ocimum gratissimum,
b. extraction with absolute ethanol
c. Further fractionation of ethanol extract with the solvents selected from the group of petroleum ether, chloroform, butanol and water.
2. Process for extraction of essential oils as an efficacious product for control of silkworm parasitoid uzi fly, Exorista sorbillans comprising of :
a. selection, washing and cutting of fresh leaves of herbs selected from Ocimum gratissimum, Ocimum sanctum, Eucalyptus maculata, Calistemon linearis and peels of Citrus sinensis into small pieces;
b. extraction of essential oil;
c. heating (40-500C)&
d. Collection of the essential oil.
3. Process as claimed in claim 2 wherein the extraction is carried with water (hydrodistillation method) using Clevenger type apparatus.
4. Process as claimed in claim 2 wherein the essential oil floating in the oil collection area after 4 hours of heating was collected after draining the bottom water layer and further separated with petroleum ether and ethyl acetate solvent system (5:1) wherein the F2 fraction acts as an active fraction for control of silkworm parasitoid uzi fly, Exorista sorbillans .
5. Product obtained accordingly by the process as claimed in any of the preceding claims as an efficacious product for control of silkworm parasitoid uzi fly, Exorista sorbillans.
6. Product as claimed in claim 5 wherein it is the essential oil of Ocimum gratissimum as an efficacious product for control of silkworm parasitoid uzi fly, Exorista sorbillans.
7. Biopesticide formulation as an efficacious product for control of silkworm parasitoid uzi fly, Exorista sorbillans comprises of:
a. an effective amount of essential oil of Ocimum gratissimum with or without one or more of the essential oils selected from Ocimum sanctum, Eucalyptus maculata, Calistemon linearis, Citrus sinensis; and
b. synthetic gum as a control release agent.
8. Biopesticide formulation as claimed in claim 7 wherein the essential oils &/essential oil compounds selected are carvacrol, thymol, eugenol, eucalyptol and citral.
9. Biopesticide formulation as claimed in claim 7 or 8 wherein the effective amount of selected essential oils&/essential oil compounds is in the ratio of 1:1:1:1:1.
10. Process for preparation of biopesticide formulation as an efficacious product for control of silkworm parasitoid uzi fly, Exorista sorbillans comprising of :
a. extraction of essential oils as claimed in claims 1 to 5;
b. Mixing the effective amount of extracted essential oil of Ocimum gratissimum optionally with one or more of the essential oils &/essential oil compounds selected from Ocimum sanctum, Eucalyptus maculata, Calistemon linearis, Citrus sinensis along with the synthetic gum to form the biopesticide formulation.
Dated this 17th day of October 2014 SHARANABASAVA
PATENT AGENT FOR APPLICANTS