Title of Invention

A METHOD OF EXTRACTION OF AN ESSENTIAL OILS FROM CYMBOPOGON WINTERIANUS PLANTS USING CLEISEN APPARATUS, FOR PROTECTION AGAINST PEST

Abstract

A method of extracting an essential oils from Cymbopogon winterianns plant using Clevenger apparatus for protection against pests, comprising collecting Cymbopogon winterianus, small pieces taken into a round bottom flask, which is fitted with Clevener apparatus for heating to which water is added and heated at 80°C for 8 hrs until water vapour starts passing through the condenser and circulation of oil mixture starts separating on the top of the water, resultant oil and water mixture allowed to fractional distillation to separate and collect the oil.

Full Text

Form 2 THE PATENTS ACT,1972 (39 of 1970) Complete specification (See Section 10) Title : A method of extraction of an essential oils from Cymbopogon winterianus plant using Clevenger and Cleisen apparatus, for protection against pests. Address: 1) Dr Samiron Phukan Global Enterprise Infotech solutions 412B, Acme Plaza, Opp. Sangam Cinema, Andheri (East), Mumbai-400059 2) Dr. M.C.Kahta Dept. of Biotechnology Gauhat University Guwahati-781014, The following specifiation particularly describes the nature of the invention and the manner in which it is performed. Title : A method of extraction of an essential oils from Cymbopogon winterianus plant using Clevenger and Cleisen apparatus, for protection against pests. Field of Invention; The present invention relates to a method of extracting essential oils from Cymbopogon winterianus plant using Clevenger apparatus, for protection against pests in the field of biopesticides, the extraction which has potency against to a mosquitoes. Background In Zimbabwe the farmers used the extracts of native plants Swartiza madagascariensis and Datura stramonium as efffective fungicide and insecticides respectively. Similarly, though originated in Yugoslovia, the plant Chrysanthemum cinerariaefolium, popular as pyrethrum has been grown in Kenya basically as a source of important insecticides, which is prepared from its dried flowers. Probably, the most successful use of a plant product as an insecticide is that of the pyrethroids. The insecticidal properties of the several Chrysanthemum species were known for centuries in Asia. Even today, powders of the dried flowers of these plants are sold as insecticides. After elucidation of the chemical structures of the six terpenoid esters (pyrethrins) responsible for the insecticidal activity of these plants, many synthetic analogs have been patented and marketed. Synthetic pyrethroids have better photo stability and are generally more active than their natural counterparts. Another plant terpenoid, camphene was a very successful herbicide in its polyhalogenated form. Sold as toxaphrenereg., this product was the leading insecticide in the United States before it was removed from the market. Although this product was a mixture of over two hundred chlorinated forms of camphene, certain specific compounds in the mixture were found to be much more active than the mixture oh a unit weight basis. Many other terpenoids have been demonstrated to have insecticidal or other insect-inhibiting activities. For instance, azadirachtin and other terpenoids of the limonoid group from the families Meliaceae and Rutaceae are potent growth inhibitors of several insect-species. The present invention is a new and novel no similar patent or publication are available. Objects of the present invention: An object of the present invention is to isolate the active ingredients of the oil of Cymbopogon winterianus and its indivisual effect. Another object of the present invention is to the formulation of the above compound which can be used for an effective biopesticide. Further object of the present invention is to the investigation and to identify the compounds and recommend it as as effective formulation, which can be used as a potential biopesticides against mosquitoes. Description of the Invention; The present invention is direted to a method of extracting essential oils and isolating indivisual compound(s) such as geraniol , citronelal, limonene from Cymbopogon winterianus plant using Clevenger apparatus and Cleisien apparatus for protection against pest, comprising, the following steps, collecting Cymbopogon winterianus leaves which are cutted into small pieces and taken in to a round bottom flask and heated at 800 c using water for around 8hrs in the a Clevenger apparatus which is fitted in the round bottom flask, followed by fractional distillation using Clevenger apparatus. Example 1: A Fresh material (1.0 kg) of Cymbopogon winterianus was cut into small pieces and taken in a 5 Its round bottom flask to which 2 Its of water was added and then heated over a 5 Its heating mantle; where in a Clevenger apparatus was fitted in the flask. After 1 hr the water vapour started passing through the condenser and circulation of water oil mixture started. The oil was started separating on the top of the water. The heating was continued for 8 hrs. The essential oil (lOOgm) extracted from the plant was taken in a fractional distillation apparatus Cleisen apparatus (250ml) fitted with 2 ft long column packed with glass beads and distillation head with thermometer pocket and condenser with receiving adapter connecting to a vacuum pump. The oil was heated on a water bath under reduced pressure and the vapour temperature was measured and different fractions(6 only) were collected during the distillation. GC/MS . GC AND HPLC Analysis Of The Extracts And Essential Oil: GC/MS experiment conditions: Model of the instrument: (Fission MD800 GC MS model) Oven Temperature: Programed at 100°C for lmin to 200°C at the rate of 10°C held for 5mins then to 250°C at the rate of 15°C per minutes and held for 8 mins Injector temperature :200°C Interface : 250°C Source :200°C Flow rate :lm/mt Split ratio : 1:25 Column :Fused silica capillary column and linked with 5% Phenylmethylsilicone Ionization source : Electron impact ionization (EI) at 70eV. The peaks obtained in the total ion current chromatography were studied separately for identification of the component for the mass fragmentation pattern and then compared the same with standard library present in the data station. GC conditions for analysis of the oil fractions: Instrument model: HP. Inlet temperature: 200°C Detector : 260°C Oven condition : Ramp °C Hold time °C/min (min) 25 60 1 25 100 1 25 150 1 15 180 1 15 200 2 15 240 4 Column : 5%Phenyldimethylpolysilicone(HP5MS &HP-5 trace Analysis) The major peaks obtained were identified by comparing with the retention time of the authentic samples. Parameters tested for estimating the potency of the plant: a) Larval mortality b) Larval pupation c) Adult emergence For larval mortality the time period was at the interval of 1 hr, 6hrs, 12rhs, 24hrs, 48hrs, &72hrs and for larval pupation and adult emergence the record was taken after 6 days and 8 days respectively. Allethrin which is used as commercial pesticide was taken as reference compound Laboratory Culture And Maintenance Of Mosquitoes: The mosquitoes mat were subjected to bioassay were cultured and maintained in the Environmental Biotechnology Laboratory, of the Depatment of Biotechnology, Gauhati Universty. The authentic eggs of A. aegypti and C. quinquefasciatus were obtained from the standard culture maintained by Regional Medical Research Center for N.E. region , Indian Council of Medical Research (ICMR), Dibrugarh for last over eight years. Individual egg rafts generally hatch between 24 and 36hrs. Larvae were fed with dog biscuit powder (1:1). Initially a small amount of food was provided but as the size of the larvae increased the quantity was also increased proportionately. The water was changed on every alternate days to avoid contamination and larval death. The density of 1 larvae/cm3 was found ideal for proper development. At room temperature (28° ± 2° C), the larval development was observed within 6-8 days. Pupae were transferred to bowls containing tap water. Pupal duration was 24 hrs-48 hrs. On emergence, adults were transferred to cages made of wooden frame of size (30 x 30x 40 cm ) and nylon mesh . They were kept at temperature of 28° ± 2° C and fed with blood of Guinea pig. The males were fed with glucose soaked in cotton pads. Bowls containing tap water were placed in cages for egg laying. In this way a self-perpetuating colony was established and was maintained fro the entire period of study, which provides the required number of adults and larvae for the bioassays. Methods Used For Bioassays: For testing the bioefficacy of the plants against mosquitoes a slight modification of the standard procedure as recommended by WHO (WHO/VBC/81.807) was followed. Larval mortality: For a test of one extract, sufficient amount of larvae were collected from the field in order that 300 individuals of the same species could be selected ; they were in their late 3rd instar or early 4th instar and were retained in water in which they were collected until selected for testing. Any larvae showing abnormalities for example a fuzzy appearance due to presence of parasites on the body surface was discarded. Lots of 20-25 larvae were distributed in each of the 12 small beakers each containing 25ml of water. Their transfer was affected either by means of a strainer, or by means of a dropper; during the process they were rinsed tightly in clean water. Into each of the 12 beakers approximately 7.5-10cm in diameter; 225ml of water is placed. The vessels were such that the depth of the water 2.5 and 7.5cm. Distilled water was used. The average temperature of water used was approximately25°C. The test concentration was prepared by pouring the appropriate amount of standard insecticide solution in each of the glass vessels and stirring vigorously for 30 sec. with a glass rod. The concentrations were prepared as 2000ppm, lOOOppm, 500ppm, 250ppm, lOOppm ,50ppm ,25ppm and lOppm for the oil. Within 15-30mins of the preparation of the test concentrations the mosquito larvae were added to them by tipping the test contents of the beaker into the vessels. The mortality counts were taken after lhr, 6hrs, 12 hrs, 24hrs, 48hrs and 72hrs. In recording the mortality counts for each concentration, the moribund and the dead larvae in both replicates were combined. Dead larvae were those that cannot be induced to move when they are probed with a needle in the siphon or the cervical region. Moribund larvae are those incapable of rising to the surface (within a reasonable period of time) or of showing the characteristic diving reaction when the water was disturbed. The larvae that have pupated during the test are discarded. If more than 10% of the control larvae pupate in the course of the experiment the test is discarded. Test with the control mortality of over 20% are unsatisfactory and in such case the test was repeated. When 5 replicates were performed with the same population of mosquito larvae, adequate data became available for constructing a base line susceptibility. The results were recorded. Larval pupation and adult emergence: After the recording of the data of larva the surviving larvae were transferred to Barraud cages and kept till 8th day for observing the growth regulatory activities i.e. percentage of pupae formation and percentage of adult emergence. Statistical analysis: The LC50 is calculated with the help of NCSS computer program. FINDINGS OF THE INVESTIGATION 1. The oil of C. winterianus recorded toxicity with LC50 value of 46.88ppm and 11.62ppm for A aegypti and C. quinquefasciatus respectively. 2. The oil was fractionated into its major compounds to yield six fractions and their individual efficacy was studied. The Fraction V of the oil exhibited the highest toxicity with LC50 value of 12ppm followed by Fraction II with LC50 .value of 13.04ppm against A. aegypti.. Against C. quinquefasciatus, Fraction II showed the highest toxicity with LC50 value of l0.l0ppm followed by that of FractionV with 14.03ppm. Fraction I too showed some promise with LC50 value of 24.31 ppm . These three fractions were subjected to GC analysis for identification of the compounds by taking their standard retention time. They were identified as geraniol, limonene and citronellal. Further, the combined effect of geraniol, limonene and citronellal were studied to evaluate their toxicity and to estimate whether their combination could be more effective than their individual effect(Table 3 and 4). It was found that a combination of 19ppm geraniol and 6ppm of citronellal proved to be the most effective combination having a mortality of 76% in 24 hrs against A. aegypti and 66% against C. quinquefasciatus. The combination of limolene and citronellal however did not show any effective toxicity against both the species of mosquitoes. However, the combination of geraniol and limonene showed a moderate toxicity with 96 % mortality at a combination of 25ppm of both geraniol and limonene after 24%hrs against A. aegypti (Table 3) and 100% mortality at 12ppm of geraniol and 38ppm of limonene against C. quinquefasciatus. Table 1 : LCso values of different fractions of essential oil of C.winterianus on the larvae of A. aegypti Treatment LCso (ppm) Fraction I 31.89 Fraction II 13.04 Fraction III 51.00 Fraction IV 51.09 Fraction V 12 Fraction VI 94.86 Table 2 : LC50 values of different fractions of essential oil of C.winterianus on the larvae of C.quinquefasciatusi Treatment LC50 (ppm) Fraction I 24.31 Fraction II 10.10 Fraction III 45.15 Fraction IV 74.00 Fraction V 14.03 Fraction VI 47.84 Table 3: Effect of combination of different potent fractions of C. winterianus on the larvae of A. aegypti Treatment % Mortality fin hrs) & Conc(ppm) 1 6 12 24 48 72 Control 00 00 00 00 00 00 Ger.Lim 25 25 100 100 100 100 100 100 12 38 20 30 40 95 100 100 38 12 00 20 20 40 40 40 12.5 12.5 46 46 50 50 50 50 6 19 20 30 30 36 30 30 19 6 00 00 05 05 05 05 5 5 00 00 00 20 20 20 Ger. Cit 25 25 100 100 100 100 100 100 12 38 100 100 100 100 100 100 38 12 100 100 100 100 100 100 12.5 12.5 46 46 80 100 100 100 6 19 40 40 76 76 76 76 19 6 40 40 76 76 76 76 5 5 5 15 15 15 15 15 Lim Cit 25 25 20 36 36 36 36 36 12 38 10 26 26 26 26 26 38 12 00 18 18 46 46 46 12.5 12.5 00 00 06 26 26 26 6 19 00 00 00 20 20 20 19 6 00 00 00 00 00 00 5 5 00 00 00 00 00 00 Table 4 : Effect of combination of different potent fractions of C. winterianus on the larvae of C. quinquefasciatus Treatment % Mortality finHrs) & Conc(ppm) 1 6 12 24 48 72 Control 00 00 00 00 00 00 Ger. Lim. 25 25 10 0 100 100 100 100 100 12 38 20 34 46 100 100 100 38 12 00 20 20 35 40 40 12.5 12.5 40 40 50 50 50 50 6 19 20 20 36 36 40 40 19 6 00 00 06 06 06 06 5 5 00 00 00 10 10 10 Ger. Cit. 25 25 10 0 100 100 100 100 100 12 38 10 100 100 100 100 100 0 38 12 10 0 100 100 100 100 100 12.5 12.5 40 40 75 100 100 100 6 19 36 36 66 66 66 66 19 6 40 40 70 70 70 70 5 5 6 16 16 16 16 16 Lim. Cit. 25 25 20 30 30 30 30 30 12 38 10 26 26 26 26 26 38 12 00 10 10 40 40 40 12.5 12.5 00 00 06 30 30 30 6 19 00 00 00 16 16 16 19 6 00 00 00 00 00 00 5 5 00 00 00 00 00 00 We claim: 1. A method of extracting an essential oils from Cymbopogon winterianns plant using Clevenger apparatus for protection against pests, comprising collecting Cymbopogon winterianus, small pieces taken into a round bottom flask, which is fitted with Clevener apparatus for heating to which water is added and heated at 80°C for 8 hrs until water vapour starts passing through the condenser and circulation of oil mixture starts separating on the top of the water, resultant oil and water mixture allowed to fractional distillation to separate and collect the oil. 2. A method as calim in 1, wherein fractional distillation is carried out under reduced pressure and temperature using Cleisen Apparatus, 3. A method of extracting an essential oils from Cymbopogon winteranus plant using Clevenger apparatus for protection against pest such as herein described with reference to an example Dated this Day of 10th Feb 2004 Signature: (Dr. SPhukan)

Documents:

187-mum-2004-abstract(complete)-(17-2-2004).pdf

187-mum-2004-abstract(granted)-(14-6-2007).pdf

187-mum-2004-cancelled pages(1-4-2004).pdf

187-mum-2004-cancelled pages(9-8-2010).pdf

187-mum-2004-claims(amanded)-(1-4-2004).pdf

187-mum-2004-claims(granted)-(14-6-2007).pdf

187-mum-2004-claims(granted)-(17-02-2004).doc

187-mum-2004-claims(granted)-(17-02-2004).pdf

187-mum-2004-correspondence 1(1-4-2004).pdf

187-MUM-2004-CORRESPONDENCE 19-6-2008.pdf

187-mum-2004-correspondence 2(19-7-2007).pdf

187-MUM-2004-CORRESPONDENCE(1-4-2009).pdf

187-MUM-2004-CORRESPONDENCE(28-1-2010).pdf

187-mum-2004-correspondence(4-5-2010).pdf

187-MUM-2004-CORRESPONDENCE(IPO)-(1-4-2009).pdf

187-mum-2004-correspondence(ipo)-(10-8-2010).pdf

187-mum-2004-correspondence(ipo)-(14-06-2007).pdf

187-mum-2004-correspondence(ipo)-(23-7-2007).pdf

187-mum-2004-correspondence(ipo)-(5-4-2010).pdf

187-mum-2004-description(complete)-(17-2-2004).pdf

187-mum-2004-description(granted)-(14-6-2007).pdf

187-MUM-2004-FORM 1 19-6-2008.pdf

187-mum-2004-form 1(17-02-2004).pdf

187-mum-2004-form 1(19-06-2008).pdf

187-MUM-2004-FORM 13 19-6-2008.pdf

187-mum-2004-form 13(19-06-2008).pdf

187-mum-2004-form 19(17-02-2004).pdf

187-mum-2004-form 2(complete)-(17-2-2004).pdf

187-mum-2004-form 2(granted)-(14-6-2007).pdf

187-mum-2004-form 2(granted)-(17-02-2004).doc

187-mum-2004-form 2(granted)-(17-02-2004).pdf

187-mum-2004-form 2(title page)-(complete)-(17-2-2004).pdf

187-mum-2004-form 2(title page)-(granted)-(14-6-2007).pdf

187-mum-2004-form 3(16-02-2004).pdf

187-MUM-2004-OTHER DOCUMENT(28-1-2010).pdf

187-mum-2004-power of authority(16-03-2004).pdf

187-MUM-2004-PUBLICATION REPORT 19-6-2008.pdf