Title of Invention	A METHOD FOR EXTRACTION OF BIOACTIVE COMPOUNDS FROM IPOMOEA CORNEA PALANT USING SOLVENTS FOR PROTECTION AGAINST PESTS.
Abstract	A method of extracting bioactive compounds from Ipomea cornea by fractional solvent extraction method and study the effect of each extracts against pest. The leaves of I.cornea were powdered to 120 mesh and was subjected to series of fractional extraction by solvents of different polarity viz. Hexane, Toulene, Choloform, Acetone, Methanol and water at room temperature each for 72 hrs followed by condensation of the extract in vacuo. The concentrated extract was subjected to Bioassay against mosquitoe larvae and its bioactivity was studied.

Title of Invention

A METHOD FOR EXTRACTION OF BIOACTIVE COMPOUNDS FROM IPOMOEA CORNEA PALANT USING SOLVENTS FOR PROTECTION AGAINST PESTS.

Abstract

A method of extracting bioactive compounds from Ipomea cornea by fractional solvent extraction method and study the effect of each extracts against pest. The leaves of I.cornea were powdered to 120 mesh and was subjected to series of fractional extraction by solvents of different polarity viz. Hexane, Toulene, Choloform, Acetone, Methanol and water at room temperature each for 72 hrs followed by condensation of the extract in vacuo. The concentrated extract was subjected to Bioassay against mosquitoe larvae and its bioactivity was studied.

Full Text	Form 2 THE PATENTS ACT, 1972 (39 of 1970) Complete specification (See Section 10) Title : A method for extraction of bioactive compounds from Ipomoea cornea plant using solvents for protection against pests. Address: 1) Dr Samiron Phukan Global Enterprise Infotech solutions 412B, Acme Plaza, 0pp. Sangam Cinema, Andheri (East), Mumbai-400059 2) Dr. M.C.Kalita Dept. of Biotechnology Gauhat University Guwahati-781014, The following specification particularly describes the nature of the invention and the manner in which it is performed. GRANTED 17-12-2004 ORIGINAL Title : A method the extraction of bioactive compounds from Ipomoea cornea plant using solvents for protection against pests. Field of Invention; The present invention relates to a method of extracting bioactive compounds from Ipomoea cornea plant using solvents extraction method, for protection against pests in the field of biopesticides; the extraction which has potency against mosquitoes. Background In Zimbabwe the farmers use 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 the natural counterparts. Another plant terpenoid, camphene is 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 on 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 method of extracting bioactive compounds from Ipoema cornea using solvents fro protection against pests. Objectives of the present invention: An objective of the present invention is to study the bioefficacy of Ipomoea cornea against pest. Another objective of the present invention is to study the nature of the compounds which can be used for an effective biopesticide against pest. Description of the Invention; The present invention is directed to a method of extracting bioactive compounds from Ipomea cornea by fractional solvent extraction method and study the effect of each extracts against pest. The leaves of I.cornea were powdered to 120 mesh and was subjected to series of fractional extraction by solvents of different polarity viz. Hexane, Toulene, Choloform, Acetone, Methanol and water at room temperature each for 72 hrs followed by condensation of the extract in vacuo. Example 1: The plant materials (leaves of I. cornea) were dried in shade and powdered to 120 mesh. The dried powder was then subjected to successive extraction in various solvents in order of their polarity i.e from non-polar to polar viz. hexane, toulene, choloroform, acetone , methanol and water at room temperature for 72 hrs and then condensed in vacou. The condensed dried extract was then subjected to bioassay as mentioned below. 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. • AUethrin which is used as commercial pesticide was taken as reference compound Laboratory Culture And Maintenance Of Mosquitoes: The mosquitoes that were subjected to bioassay were cultured and maintained in the Environmental Biotechnology Laboratory, of the Department of Bioteclinology, 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 ahernate days to avoid contamination and larval death. The density of 1 larvae/cm3 was found ideal for proper development. At room temperature (28° ± 20 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 3 Ox 40 cm ) and nylon mesh . They were kept at temperature of 28° ± 20 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 culture maintained in the laboratory of Environmental Biotechnology, Gauhati University in order that 300 individual of the same species were selected ; they were in their late 3rd instar or early 4rd 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 effected 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 7.5-1Ocm 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 and 50ppm for the solvent extracts and even at lower concentration i..e 25ppm and 10ppm 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 thr, 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 camiot 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 aqueous extract of I .cornea exhibited toxicity against Aedes aegypti with LC50 value of 0.33%. 2. The Toulene extract exhibits 100% inhibition of pupal development 3. Against Culex quinquefasciatus it exhibited only 50%) pupal development at 10%) concentration in its aqueous extract The significant findings. of the present study is that we used toluene in place of benzene - which exhibited promising results with regards to its growth regulatory activity. We claim: 1. A method the extracting bioactive compounds from Ipomoea cornea by fractional solvent extraction for protection against pest comprising the following steps; The leaves of /. cornea were powdered to 120 mesh and was subjected to series of fractional extraction by solvents of different polarity viz. Hexane, Toulene, Choloform, Acetone, Methanol and water at room temperature each for 72 hrs followed by condensation of the extract in vacuo. 2. A method as claim in 1, wherein toluene is used as a potential solvent for extraction in place of benzene. 3. A method of extracting bioactive compounds from Ipomoea cornea for protection against pest such as herein described in reference with an example. Dated this Day of 23rd June 2004 Signature : ( S.Phukan)

Full Text

Form 2
THE PATENTS ACT, 1972
(39 of 1970)
Complete specification
(See Section 10)
Title : A method for extraction of bioactive compounds from Ipomoea cornea plant using solvents for protection against pests.
Address: 1) Dr Samiron Phukan
Global Enterprise Infotech solutions 412B, Acme Plaza, 0pp. Sangam Cinema, Andheri (East), Mumbai-400059
2) Dr. M.C.Kalita
Dept. of Biotechnology Gauhat University Guwahati-781014,
The following specification particularly describes the nature of the invention and the manner in which it is performed.

GRANTED
17-12-2004

ORIGINAL

Title : A method the extraction of bioactive compounds from Ipomoea cornea plant using solvents for protection against pests.
Field of Invention;
The present invention relates to a method of extracting bioactive compounds from Ipomoea cornea plant using solvents extraction method, for protection against pests in the field of biopesticides; the extraction which has potency against mosquitoes.
Background
In Zimbabwe the farmers use 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 the natural counterparts.
Another plant terpenoid, camphene is 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 on 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 method of extracting bioactive compounds from Ipoema cornea using solvents fro protection against pests.
Objectives of the present invention:
An objective of the present invention is to study the bioefficacy of Ipomoea cornea against pest.
Another objective of the present invention is to study the nature of the compounds which can be used for an effective biopesticide against pest.
Description of the Invention;
The present invention is directed to a method of extracting bioactive compounds from Ipomea cornea by fractional solvent extraction method and study the effect of each extracts against pest. The leaves of I.cornea were powdered to 120 mesh and was subjected to series of fractional extraction by solvents of different polarity viz. Hexane, Toulene, Choloform, Acetone, Methanol and water at room temperature each for 72 hrs followed by condensation of the extract in vacuo.
Example 1:
The plant materials (leaves of I. cornea) were dried in shade and powdered to 120 mesh. The dried powder was then subjected to successive extraction in various solvents in order of their polarity i.e from non-polar to polar viz. hexane, toulene, choloroform, acetone , methanol and water at room temperature for 72 hrs and then condensed in vacou. The condensed dried extract was then subjected to bioassay as mentioned below.

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. • AUethrin which is used as commercial pesticide was taken as reference compound
Laboratory Culture And Maintenance Of Mosquitoes:
The mosquitoes that were subjected to bioassay were cultured and maintained in the Environmental Biotechnology Laboratory, of the Department of Bioteclinology, 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 ahernate days to avoid contamination and larval death. The density of 1 larvae/cm3 was found ideal for proper development. At room temperature (28° ± 20 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 3 Ox 40 cm ) and nylon mesh . They were kept at temperature of 28° ± 20 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 culture maintained in the laboratory of Environmental Biotechnology, Gauhati University in order that 300 individual of the same species were selected ; they were in their late 3rd instar or early 4rd 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 effected 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 7.5-1Ocm 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 and 50ppm for the solvent extracts and even at lower concentration i..e 25ppm and 10ppm 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 thr, 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 camiot 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 aqueous extract of I .cornea exhibited toxicity against Aedes aegypti with LC50 value of 0.33%.
2. The Toulene extract exhibits 100% inhibition of pupal development
3. Against Culex quinquefasciatus it exhibited only 50%) pupal development at 10%) concentration in its aqueous extract
The significant findings. of the present study is that we used toluene in place of benzene - which exhibited promising results with regards to its growth regulatory activity.

We claim:
1. A method the extracting bioactive compounds from Ipomoea cornea by fractional solvent extraction for protection against pest comprising the following steps; The leaves of /. cornea were powdered to 120 mesh and was subjected to series of fractional extraction by solvents of different polarity viz. Hexane, Toulene, Choloform, Acetone, Methanol and water at room temperature each for 72 hrs followed by condensation of the extract in vacuo.
2. A method as claim in 1, wherein toluene is used as a potential solvent for extraction in place of benzene.
3. A method of extracting bioactive compounds from Ipomoea cornea for protection against pest such as herein described in reference with an example.
Dated this Day of 23rd June 2004
Signature : ( S.Phukan)

Documents:

737-mum-2004-abstract(17-12-2004).pdf

737-mum-2004-abstract-(17-12-2004).doc

737-mum-2004-cancelled page(17-12-2004).pdf

737-mum-2004-claim(granted)-(17-12-2004).pdf

737-mum-2004-claims(granted)-(17-12-2004).doc

737-mum-2004-correspondence(17-12-2004).pdf

737-mum-2004-correspondence(ipo)-(29-10-2004).pdf

737-mum-2004-form 1(09-07-2004).pdf

737-mum-2004-form 19(10-09-2004).pdf

737-mum-2004-form 2(granted)-(17-12-2004).doc

737-mum-2004-form 2(granted)-(17-12-2004).pdf

737-mum-2004-form 3(09-07-2004).pdf

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Patent Number

205769

Indian Patent Application Number

737/MUM/2004

PG Journal Number

40/2008

Publication Date

03-Oct-2008

Grant Date

10-Apr-2007

Date of Filing

09-Jul-2004

Name of Patentee

PHUKAN, S.

Applicant Address

GLOBAL ENTERPRISE INFOTECH SOLUTIONS, 412B ACME PLAZA, ANDHERI (E), MUMBAI.

Inventors:

#	Inventor's Name	Inventor's Address
1	PHUKAN, S.	GLOBAL ENTERPRISE INFOTECH SOLUTIONS, 412B ACME PLAZA, ANDHERI (E), MUMBAI 400059.
2	KALITA M.C.	DEPARTMENT OF BIOTECHNOLOGY, GAUHATI UNIVERSITY, GAUHATI-781014

PCT International Classification Number

A 61 K 3/00

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA