Title of Invention

A METHOD FOR THE PREPARATION OF A BIOPESTICIDE FROM THE ROOTS OF DECALEPIS HAMILTONII"

Abstract

This invention relates to a method for the preparation of a biopesticide from the roots of Decalpis hamiltom which comprises (i) preparing powder from clean dry roots of D.hamiltoni, (ii) extracting powdered roots using supercritical carbondioxide/carbon-dioxide and polar and non-polar solvents such as herein described, at a temperature in the range of 40-80°C and at a pressure in range of 100-160 bars for a period of 24-72 hours, getting a creamish white, waxy material from the extract useful as biopesticide.

Full Text

The present invention relates to a method for the preparation of a biopesticide from the roots of Decalpis hamiltoni. The plant Decaleptis hamiltonii is a climbing shrub distributed in the Deccan region and Western Ghats. The roots are marketed in South India where it is pickled with lime or used as such as rechreded by Cherian Jacob, 1937 (An unrecorded economic product Dacalepis hamiltonii Wight & Arn. Family Asclepiadaceae. Charian Jacob, Madras Agric. J., 1937, 25 , 176). Murti and Seshadri,1941 have reported that the root has a strong aromatic odour with a vanillin-like note which is organoleptically acceptable ( A study of the chemical components of the repts of Decalepis hamiltonii Part I. P B R Murthy and Seshadri, T R , Proc. Ind. Acad.Sci., 1941, 13A, 221) also described in the Wealth of India, 1976 ( Wealth of India. India Raw Materials & Industrial Products, Vol.X ; Sp-W, 1976, Publications & Information Directorate, CSIR, New Delhi , 399). There' is no literature on the insecticidal activity the root of D.hamiltonii nor any traditional piractice exploiting the insecticidal potential of roots of D.hamiltonii. Biopesticides are recogized as an environmentally-friendly alternative to the ssynthetic organic pesticides. There is an urgent need for environmentally-safe pesticides that are easy-to-use, non-texic to man and animal life and biodegradable (Dale MJ, 1996. A review of plant materials used for controlling insect pests of stored products. Bulletin 65. Natural Resources Institute,U.K.). The traditional storage of food grains in rural areas and at the domestic level involves mixing with neerti leaves/turmeric powder/ash or treating grain with vegetable oils. Vegetable oils are expensive and also turn rancid imparting an off-flavour to the produce. Naem leaf has insect repellent properties but does not prevent build-up of insect infestation in stored grain as reported by Pereira and Wohlgemuth, 1982 ( Neem ( Azadirachta indica A. Juss) of West African origin as a protectant of stored maize. John Pereira and R.Wohlgemuth, Zeit. ange. Ent.,1982, 94, 208-214) In the organized sector,residual insecticides and/or fumigants are used. Residual insecticides are toxic and leave residues on the stored produce. In view of the stringent regulations regarding safety and residue.levels,the number of residual insecticides that can be used in the premises for grain storage is very limited. The fumigants are suited for large-scale storage of grains but require functionally air-tight conditions, expensive equipment and trained personnel to carry out the fumigation. The number of fumigants that can be used for stored grain are very limited. The main object of the present invention is to provide a method for the preparation of an active fraction from the roots of D.hamiltonii useful as a biopesticide which obviates the drawbacks as detailed above. Another object of the present invention is to prepare a biopesticide from the roots of Decalepis hamiltonii that can be used to treat packaging materials to prevent the entry of insect pests and also to protect dried medicinal plants in storage from insect pests. Yet another objective is to provide a biopesticide that is non-toxic to man and animals which obviates the drawbacks associated with current practices as detailed above. The novelty of this patent proposal is that it is also possible to obtain the active or near; active fractions from the roots of Decalepis hamiltonii obtained by in vitro culture methods employing known nutrient media and growth conditions. Accordingly the present invention provides a method for the preparation ot a biopesticide from the roots of Decaljytii hamiltoni which comprises (i) preparing powder from clean dry roots of D. hmiltoni , (ii) extracting powdered roots using supercritical carbon-dioxide/carbon-dicxide and polar and non-polar solvents such as herein described , at a temperature in the range of 40;80 ° C and at a pressure in range of 100 - 160 bars for a period oi 24 -72 hours , getting a creamish white, waxy material from the extract useful as biopesticide . In another embodiment of the invention the solvent used for the extraction may be polar and non-polar solvents such as supercritical carbon dioxide, chloroform, alkanol, steam and mixture thereof. Extraction may be carried out using supercritical carbon dioxide at a temperature in the range of 40-80°C, at a pressure in the range of -100-160 bars for a period in the range of 24 to 72 hours. In yet another embodiment of the present invention, the insecticidal powder/ extract can be employed with other known compatible active agents like insecticides, synergists etc. As used herein,the term "biaesticidal", is intended to encompass insect repellent, insecticidal , Tarvicidal ,ovicidal properties from the root of Decalepis hamiltonii. Root includes apart from the naturally grown root of the plant, tissues obtained by growing in lan artificial nutrient medium containing macro-and micronutrients, specific growth factors under controlled temperature and photoperiodic conditions. Detailed Description I) Starting material (Raw material) : a) The roots of Decalepis hamiltonii were cleaned off extraneous matter and soil by washing with cold water (10 - 15°C) at least four to five times. b) The iroot material were sliced/diced/ chipped/maceratsf/sonicated. The material obtained from a) and b) were then subjected to the following process/s given below. II) Drying : A critical requirement in the crying process is avoidance of direct, intense sunlight, air blowers and harsh temperatures. These conditions reduce the fnsecticidal potency of the final product. , a) The processed material obtained in I were dried in shade in a warm place away from direct sunlight / in a dehumidifier (15-40°C) / hot air oven (35-50°C). b) Alternatively, the processed material obtained from I was subjected to Accelerated Freeze Drying at a vacuum of 0.05-0.1 mbar betfeen -60 to -75°C and finally to room temperature. c) By yet another alternative step,the processed material obtained from I were sibjected to lyophi1ization at 0.1-0,3 mbar and between -40 %3 -45°C for 24-72 hours.The condensate byproduct obtained during lyophi1ization was used directly or in suitaWe insecticidal / flavouring formulations or was further Ifophilized at 0.1 to 0.3 mbar and temperature of -40 to -45PC and then used directly/ formulated. Alternatively, the condensate byproduct was extracted in chloroform and the extract free of chloroform formulated for specific use. III) Powdering : In the powdering process, care ii taken to avoid generation of excessive heat as this redmied the potency of the final product. The processed material obtained from I and II were powdered in a Hammermi 1 l/cryogrinder/manual/ mechanical pounder and passed through a sieve to obtain^ 20-100 mesh size particles. IV) Extraction : a) The processed material obtained, from III was extracted by supercritical extraction with carbon-di-oxide or carbon-di-oxide - solvent mixtures- Extraction was effected between 40-80°C and pressure of 100-160 bars; with the help of 4 L High Pressure Extractor. The extract so obtained exhibited insecticidal activity after purification. b) The processed materiel obtained' from III was extracted with various polar and non-polar solvents that included chloroform, alkanol, steam, hexane,' petroleum ether, ethyl acetate and mixtures thereof at room"temperature or in Soxhlet's apparatus. The solvents were recovered by evaporation at ambient or reduced pressure' and the residues from these exhibited insecticidal activity and suitably formulated for various applications. c) The processed material obtained from III was subjected to extraction methods employing steam to obtain insecticidal fraction/s for formulations. The active fraction from the roots of Decalepis hamiltonii useful as biopesticide as obtained in III and IV may be used as such or suitably formulated in a manner, by one skilled in the art. The following examples are provided to illustrate the principles of this invention. Variations and changes may be made by one skilled in the art, without departing from the spirit of the invention. All farts and percentages are by weight unless otherwise indicated. The following examples are given by way of illustration of the present invention only and should not be construed to limit the scope of the present invention. Example 1, 1 kg of roots of Decalepis hamiltonii were cleaned off extraneous matter and soil with 2 L of cold water (12°C) at least four times. The roots were sliced and were dried in shade in a warm place away from direct sunlight. The dried roots were powdered in a machanical pounder and passed through a sieve to obtain 80 mesh size particles.The processed material obtained from above was mixed at 5% and 10% with whole wheat (11.5% M.e). Adult Sitophilus oryzae was inoculated on the treated and control wheat. There was 97% and 100% mortality repecttvely in 72 hours. The root powder was also found to be toxic to adults of the red flour beetle Tribolium castaneum Herbst, lesser grain borer Rhizopertha dominica F., spice beetle Stegobium paniceum L., tobacco beetle Lasioderma serrH^orne Fabricius. Mixing the processed plant powder with Stored grain at 2-8% (w/w) affords protection for at least t months. Example 2 The powdered root obtained from Decalepis hamiltonii as mentioned in Example 1 was subjected to Accelerated Freeze Drying (AFD) at a vacuum of around 0.075 "mbar at -60°C and finally to room temperature. The root was powdered in a mechanical pounder and passed through a sieve to obtain 80 mesh size particles. ' 200 g of the powdered root was extracted with supercritical carbon-di-oxide at 160 bar and 40°C for a period of 8 hours with a 4 L High Pressure Extractor of M/s Nova Werke AG, Switzerland. An extract of 7 g of creamish white, waxy material was obtained. This' purified fraction from the supercritical extract of the root was mixed with wheat grains at 500 ppm and inoculated with adult Si tophi 1 us oryzae. There was 75% mortality in 72 hours. The F1 generation was reduced by around 85% compared to the control. Example 3 200 g of the powdered material obtained from Decalepis hamiltonii as mentioned in Example 2 was extracted with supercritical carbon-di-oxide and methanol (LR grade) at 160 bar and 40°C for a period of 8 hours with a 4 L High Pressure Extractor of M/s Nova Werke AG, Switzerland. An extract of 14 g of dark yellow,waxy material was obtained. This purified fraction from the supercritical extract/steam distillate of the root was coated over glass. The extracts were found to be toxic to adults of the ric$ weevil sitophilus oryzae L., red flour beetle Tribolium castaneum Herbst. , lesser grain borer Rhizopertha dominica F.,spice beetle Stegobium paniceum L. , tobacco beetle Lasioderma serricorne Fabricius. The ED50 (Effective Deposit for 50% mortality on glass) value after 15 hours, for a few insect pests are given below : (Table Removed) With Stegobium paniceum (spice beetle) at a deposit of 19 µg/cm2,the mortality was 100 %. With Lasioderma serricorne (tobacco beetle) at 19 µg/cm2 -and 38 µg/cm2, the mortality was 92% and 100% respectively. Example 4 500 g of the powdered material obtained from sterile in vitro cultured tissues of Decalepis hamilltonii transformed with LMG-150 strain of Agrobacterium rhizogenes and grown on Lloyd and McCown Medium (1981) 25 ± 20°C, in dark at 50 rpm for 21 days was extracted with supercritical carbon-di-oxide and methanol (LR grade) at 160 bar and 40°C for a period of 8 hours with a 4 L High Pressure Extractor of M/s Nova Werke AG, Switzerland . An extract of 6 g of yellow, waxy material was obtained. The purified fraction from the root extract gave 35% mortality of adult sitophilus oryzae in 72 hours. Advantages : The main advantages of the present invention are : i) A non-toxic biopesticide to protect food-grains and their products in storage from insect, infestation, ii) Application of the active fraction to packaging materials to prevent the sentry of insect pests, iii) The active fraction can be used as spray or powder in storage/ residential premisses as a safe prophylactic treatment, iv) Can be used to protect dried medicinal plants from insect attack in storage, v) The avoidance of toxic residues on grain meant for consumption by man and livestock, as the root which is itself a food material can be used to protect food material from insect infestation. We Claim : 1. A method for the preparation of a biopesticide from the roots of Decalpis hamiltoni which comprises (i) preparing powder from clean dry roots of D. hamiltoni , (ii) extracting powdered roots using supercritical carbon-dioxide/carbon-dioxide and polar and non-polar solvents such as herein described , at a temperature in the range of 40-80° C and at a pressure in range of 100 -160 bars for a period of 24 -72 hows , getting a creamish white, waxy material from the extract useful as biopesticide . 2. A method as claimed in claim 1 wherein drying of roots is effected by air drying, oven drying , freeze drying . 3. A method as claimed in claim I and 2 wherein polar and non-polar solvents used for extraction are selected from chloroform, alkanol, steam, hexane, petroleum ether, ethyl acetate and mixture thereof. 4. A method for the preparation of a biopesticide from the roots of Decalpis hamiltoni substantially as herein described with reference to the examples .

Documents:

1301-del-1998-abstract.pdf

1301-del-1998-claims.pdf

1301-del-1998-complete specification (granted).pdf

1301-del-1998-correspondence-others.pdf

1301-del-1998-correspondence-po.pdf

1301-del-1998-description (complete).pdf

1301-del-1998-form-1.pdf

1301-del-1998-form-2.pdf

1301-del-1998-form-3.pdf

1301-del-1998-form-9.pdf