Title of Invention

A PROCESS FOR OBTAINING ECDYSTEROID FROM CARYOPHY LLACEAE ,

Abstract

ABSTRACT A process for obtaining ecdysteroid from Carvophyllaceace This invention relates to a process for preparation of ecdysteroid for administration into silk worms so as to provide a uniform maturation of the silk worm, thereby enable fester spinning activity in the silk worms. The process comprises at least a single step of extraction with ethanol or methanol, condensing, removing the solvent, dissolving the concentrated residue in water, filtering, partitioning with equal volume of chloroform, concentrating by refluxing with n-butanol, removing n-butanol, drying residue, refluxing with methanol or ethanol and removing solvent from the filtrate.

Full Text

FIELD OF INVENTION This invention relates to a process for obtaining ecdysteroid from Caryophyllaceae. EXISTING STA TE-OF-ART In normal practice of silkworm rearing, farmers are forced to pick and mount the larvae as and when they mature especially during cooler seasons. As the maturation and spinning activities in a given silkworm colony is not uniform due to various intrinsic reasons viz., temperature, nature of the breed etc., the mounting process sometimes extends up to 3-4 days. In Kashmir (temperate region) the mounting period prolongs up to 8-9 days. This involves a lot of time, labour and extra mulberry leaf and also ends up in some marketing difficulties or extended grainage operation where seeds are produced. Apart from this, unforeseen shortage of mulberry leaf or a sudden outbreak of disease leaves no option to the farmers but to desert their crop at very close to the end thereby incurring huge loss. These problems could be avoided to a larger extent or could be minimized if a hormone treatment is given during silkworm rearing as it controls the maturation events and spinning activities. As such, it is not known to combat the quite common problems of irregular maturation and non-synchronized spinning activity in a silkworm crop. In the present system, a farmer has to examine the silkworm rearing bed continuously once one or two worms mature in a colony, pick the worms as and when they mature, mount it for cocoon formation and feed the immature worms. This process may extend up to 4 days and even up to 5 days in cooler seasons. Thus it takes on time, manpower, and expenditure for increasing the rearing temperature, extra mulberry leaf and other related problems as already explained. OBJECTS OF THE INVENTION An object of this invention is to propose a process for obtaining ecdysteroid from Caryophyllaceae for administering into silkworms so as to provide a uniform maturation of the silkworm. Another object of this invention is to propose a process for obtaining ecdysteroid from Caryophyllaceae for administering into silkworms which provides a faster spinning activity. Still object of this invention is to propose a process for obtaining ecdysteroid from Caryophyllaceae for administering into silkworms which is cost effective. DESCRIPTION OF INVENTION According to this invention there is provided a process A process for obtaining ecdysteroid from Caryophyllaceae wherein the said ecdysteroid is an insect hormone for treatment of silk worms for their uniform and advanced maturation and ecdysteroid is prepared by a process comprising the steps of extracting from air dried whole plants with organic solvent at least three times, selecting organic solvent from methyl alcohol and ethyl alcohol, extraction being carried out by a method selected from soxhlet extraction, hot percolation in the said organic solvent, boiling in the said organic solvent with reflux condensing, boiling in organic solvent in the presence of water with reflux condensing, or boiling simply in water with reflux condensing; followed by removing the solvent from the combined condensed extract thus obtained at temperature of 55-60°C; dissolving the crude concentrate thus obtained in water; subjecting the aquous extract thus prepared to filtration; partitioning the filtrate with equal volume of chloroform; extracting the aquous extract with n-butanol; removing n-butanol under vacuum at temperature below 75°C; drying the residue and refluxing it with said organic solvent, rejecting the insoluble residue and removing the solvent from the filtrate; obtaining ecdysteroid of the present invention. In accordance with one embodiment of this invention the step of extraction is carried out by soxhlet extraction with a solvent selected from methanol or ethanol, the solvent from the condensed extract being removed at a temperature not exceeding 55 to 60°C,the concentrated residur being dissolved in water and the extract filtered. Jn accordance with another embodiment the step of extraction is carried out by hot percolation of Caryophyllaceae in a solvent selected from methanol or ethanol, such a step of extraction being repeated several times removing the solvent from the condensed extract at a temperature not exceeding 55 to 60°C, the concentrated residue being dissolved in water and the extract filtered. In accordance with the present invention, the isolated and characterized ecdysteroid is from a plant Caryophyllaceae. The process consists of extraction of said compound from dried whole plants with hot methanol or ethanol. The concentrated residue is dissolved in water. The aqueous extract is partitioned with chloroform and concentrated in n-butanol. Butanol is removed from the extract and the residue is refluxed with methanol/ethanol. The ecdysteroid content in the extract is quantified by TLC and HPLC. This bioactive extract is administered to silkworm. This practice helps in synchronizing/advancing the spinning activities in silkworm, which enables the farmers in saving labour, time, mulberry leaf and even sometimes the cocoon crop itself. The Caryophytlaceae is boiled in a solvent selected from ehtanol or methanol with reflux condensing repeating such a step of extraction and the combined extract being filtered and partially concentrated. The step of extraction comprises in boiling Caryophyllaceae in the presence of water and solvent selected from methanol or ethanol with reflux condensing, said step of extraction being repeated several times and the confined mass filtered. The process of extraction comprises in subjecting Caryophyllaceae to repeated steps of boiling with water with refluxed condensing, the extract being then subjected to the step of filtration to obtain a filtrate containing ecdysteroid. The applicants have established that judicious administration of insect moulting hormone, ecdysterone can accelerate the maturation events and synchronize spinning activities in silkworm, Bombyx mori L. EXTRACTION PROCESS Example No J.l The process consists of extraction of ecdysteroid from dried whole plants by soxhlet extraction with methanol or ethanol for 24 hours. The solvent from the condensed extract is removed at a temperature not more than 55-60°C. The concentrated residue is dissolved in water. Extract was then filtered. The filtrate is partitioned with equal volume of chloroform. The water layer is tested through TLC and quantified with HPLC and preserved. Example No. 1,2 The process consists of extraction of ecdysteroid from dried whole plants by hot percolation with methanol or ethanol for 60-70 hours. The extraction is repeated 3-4 times. The solvent from the condensed extract is removed at a temperature not more than 55-66°C. The concentrated residue is dissolved in water. The combined extract is then filtered. The filtrate is partitioned with equal volume of chloroform. The aqueous extract is extracted with n-butanol repeatedly. Butanol is removed from the extract under vacuum below 750C. The residue after drying was refluxed with methanol/ethanol. The insoluble residue is rejected. Removal of methanol or ethanol provides bioactive extract. Exampfe No. 1.3 The process consists of boiling the dried whole plants directly with methanol or ethanol in a flask with reflux condensing for 4-5 hours. The extraction is repeated 3-4 times. The combined extract is filtered and frnrtinlly ■—n—nfrnt—' The filtrate is tested through TLC for the presence of ecdysteroid. After confirmation, the active ingredient is quantified through HPLC. Example f.4 The process consists of boiling the dried whole plants directly with water: methanol or ethano! (4:1) for 4-5 hours with reflux condensing. The extraction is repeated 3-4 times and the confined mass is filtered. The filtrate is tested through TLC for the presence of ecdysteroid. After confirmation, the active ingredient is quantified through HPLC Example 1.5 The process consists of boiling the dried whole plants directly with water for 4-5 hours with refluxed condensing. The extraction is repeated 3-4 "times and filtered. The filtrate is tested through TLC for the presence of ecdysteroid. After confirmation, the active ingredient is quantified through HPLC and the extract is preserved for further use. Application on silkworm: The bioassay trials shows that phylinica is effective in inducing advanced or uniform maturation activities when administration is done orally @ 100-110 ml containing 2.5-2.8 mg of phylinica mixed with 1-1.1 kg of leaf and fed to 1000-1100 silkworms as a single dose. The extract is first sprayed on to mulberry leaf, mixed properly and then fed. For early or advanced maturation, the administration is to be done any time after 72-75 h in fifth instar. For uniform or synchronized maturation, the administration is to be done on the on set of spinning i.e., when one or two silkworms start spinning. Use of phylinica during these occasions would be extremely beneficial to farmers. On phylinica application for uniform spinning, the duration of maturation activity and mounting process gets shortened to about 24-30 h from about 72-96 h depending on the season without affecting the cocoon quality. This helps in synchronizing all post cocoon activities and saving labour and mulberry leaf On the contrary, when it is used for advanced spinning, the larval feeding period itself is shortened from about 6-7 days to about 5-6 days. But the shortage in larval feeding period is reflected marginally in the cocoon characters, the advantage being an unaffected cocoon crop, which would otherwise have been lost due to disease or leaf shortage. Example 2.1 In December 1999, phylinica was tested on bivoltine silkworm hybrid, CSR2 X CSR5. Phylinica was administered at the onset of spinning. About 97% larvae matured within 1 24 h of administration, whereas, in control only 42% matured within 24 h. Example 2.2 In March 2000, phylinica was again tested by using the same silkworm hybrid. About 97% larvae were matured within 24 h of administration and in control, it was only 66%. WE CLAIM: 1. A process for obtaining ecdysteroid from Caryophyllaceae wherein the said ecdysteroid is an insect harmone for treatment of silk worms for their uniform and advanced maturation and ecdysteroid is prepared by a process comprising the steps of extracting from air dried whole plants with organic solvent at least three times, selecting organic solvent from methyl alcohol and ethyl alcohol, extraction being carried out by a method selected from soxhlet extraction, hot percolation in the said organic solvent, boiling in the said organic solvent with reflux condensing, boiling in organic solvent in the presence of water with reflux condensing, or boiling simply in water with reflux condensing; followed by removing the solvent from the combined condensed extract thus obtained at temperature of 55-60°C; dissolving the crude concentrate thus obtained in water; subjecting the aquous extract thus prepared" to filtration; partitioning the filtrate with equal volume of chloroform; extracting the aquous extract with n-butanol; removing n-butanol under vacuum at temperature below 75°C; drying the residue and refluxing it with said organic solvent, rejecting the insoluble residue and removing the solvent from the filtrate; obtaining ecdysteroid of the present invention. 2. A process as claimed in calim 1 wherein said soxhlet extraction using said organic solvent is carried out for 24 hours. 3. A process as claimed in claim 1 wherein said extraction by hot percolation using said organic solvent is carried out for 60-70 hours. 4. A process as claimed in claim 1 wherein said extraction by boiling in organic solvent is carried out for 4-5 hours. 5. A process as claimed in claim 1 wherein said extraction by boiling in the said organic solvent in the presence of water is carried out for 4-5 hours, taking organic solvent and water in the ratio of 4:1. 6. A process as claimed in claim 1 wherein said extraction by boiling in water is carried out for 4-5 hours. 7. A process for obtaining of ecdysteroid for hormone treatment of silk worms, from plant family Caryophyllaceae, as substantially herein described and illustrated.

Documents:

0475-mas-2001 abstract.pdf

0475-mas-2001 claims.pdf

0475-mas-2001 correspondence-others.pdf

0475-mas-2001 correspondence-po.pdf

0475-mas-2001 description (complete).pdf

0475-mas-2001 form-1.pdf

0475-mas-2001 form-26.pdf

0475-mas-2001 form-4.pdf