Title of Invention

A PROCESS FOR THE EXTRACTION OF 3-(B GLUCOSIDO) INDOLE

Abstract

ABSTRACT A process for extracting 3- ghicosido) indole comprising obtaining plant/plant part containing 3-( - glucosido) indole, inactivating the plant enzyme, mdimulsin in the plant/plant part in a manner as herein described for preventing degradation of 3- - glucosido) indole and extracting the 3-( -glucosido) indole into the broth, treating the broth with an alkali to adjust the pH of the said broth to a value between 7 to 11, clarifying and sterilizing the broth in a manner as herein described, passing the sterilized clarified broth through a column/bed of sterilized anion exchange resin with exchangeable hydroxyl or chloride for removing the anions, passing the sterilized broth through a column bed of sterilized column bed cation exchange resin with exchangeable hydronium or alkali cation, concentrating the de-ionized 3- -glucosido) indole by passing it over sterile absorbent column, eluting 3-( -glucosido) indole from the absorbent column with organic solvents and recovering the said organic solvent by distillation or evaporation.

Full Text

ORIGINAL 1028/MUMNP/2000 THE PATENTS ACT, 1970 COMPLETE SPECIFICATION Section 10 "A process for the extraction of 3-\ glucosido) indole" Dr. Bosco Maria Agnelo Henriques, an Indian national, of 1-21 Stone Castle, Mandalpeshwar, Borivili(West), Mumbai-400 103, India The following specification particularly describes and ascertains the nature of this invention and the manner in which it is to be performed: GRANTED 25-4-2005 FIELD OF INVENTION: The present invention relates to a process for the extraction of 3-( glucosido) indole. BACKGROUND: Indigo has been known as a natural blue color used for dyeing of fabric. The most primitive indigo dyeing process involved clipping the article to be dyed into 3-( - glucosido) indole-containing fresh leaf extract and then oxidizing die article by airing it. This process required fresh leaves, which were not always available, and was difficult to control. Hence the leaves were fermented and the ferment then oxidized to produce indigo which could be stored, easily transported and then applied using another fermentation process to reduce and solubilize the dye. This was the basis of the ancient indigo dyeing industry. Later chemical reductants replaced the fermentative processes to reduce and apply the dye. Several other methods for obtaining 3-( - glucosido) indole are known. Some of these may be illustrated in brief: Dried leaves of woad (which retain their green colour) were powdered and extracted with cold alcohol. The alcohol was removed from this green liquid by distillation which turned brown during this process. The brown liquid was separated from the gelatinous precipitate by filtration and further evaporated to form a syrup. The syrup was dissolved in alcohol and excess aether was added to it and agitated. It was allowed to settle and the phases were separated. The aether was distilled and water added to the brown syrupy residue. The yellow solution containing 3-( - glucosido) indole was filtered free of the brown residue. (Schunck E, Phil. Mag. 1855) Pounded woad leaves were extracted with cold alcohol as above. To the green alcoholic solution, an alcoholic solution of lead acetate was added. A pale green precipitate was formed. Addition of a little ammonia completed the precipitation. The precipitate was washed on a filter with cold alcohol to remove excess reagents and green colouring matter. The precipitate was suspended in water and carbon dioxide passed through it. After the liquid acquired a yellow colour it was filtered and hydrogen sulphide was bubbled through it to remove ail traces of lead. The liquid was filtered and evaporated to yield a yellow syrup containing 3-(P - glucosido) indole. (Schunck E, Phil. Mag. 1855) Plant material containing 3-( - glucosido) indole was extracted in hot water and the solution evaporated to dryness. 3-( - glucosido) indole was extracted into methyl alcohol. /Ether was added to precipitate non-nitrogenous impurities and purified supernatant liquid was evaporated and residue dissolved in minimal water. On standing, 3-( - glucosido) indole was crystallized out. (Hoogewerff and ter Meulin, Proc. K. Acad Weiensch. 1900). Plant (1kg) was repeatedly extracted with acetone (4'L). The green solution was filtered and acetone was distilled till 150 mL remained behind. To this 1.5L light petroleum was added and a yellowish-brown viscous precipitate was formed which was washed with light petroleum until no colouring matter was extracted. The residue was dissolved in water and the solution was freed of particulate matter by decantation or filtration. The solution clarified by agitating with aether and the residual aether was removed under reduced pressure. The 3-(P - glucosido) indole-containing liquid was further treated with sodium carbonate to neutralize plant acids and placed in a vacuum dessicator over NaOH. Crystals of 3-((3 - glucosido) indole were formed on both sides of the beaker. (Perkin and Bloxam, J. Chem. Soc. 1907). Leaves were introduced into boiling water having a temperature of at least 85°C. The broth was drained off and the leaves squeezed to remove residual liquid. The broth was thereafter treated with calcium hydroxide to precipitate tannins and other impurities. The broth was thereafter concentrated by evaporating the water, acetone was added to extract the 3- - glucosido) indole into the organic solvent. The supernatant acetone was decanted and the acetone was evaporated. The 3-( - glucosido) indole was dissolved in a minimum quantity of water and was cooled on ice to crystallize the 3-{ -glucosido) indole hydrate These crystals were dissolved in alcohol and the solution was treated with animal charcoal to remove coloured impurities. 3- - glucosido) indole was precipated with benzene. The alcohol-to-benzene step can be repeated to get 3-1 - glucosido) indole of purer quality. (Amin BA, PUSANo. 5,1919). The draw back of aforementioned processes are that they are time consuming, not environmental friendly due to the wasteful use of large amounts of various organic solvents and the yield of the 3-i - glucosido) indole is very low and of variable quality. Also the 3-( - glucosido) indole prepared could not be stored as it could be degraded by microbial action. The object of the present invention is therefore to overcome the above disadvantages. To achieve the said object, the present invention provides for process for extracting 3-( glucosido) indole comprising: - procuring plant/plant part containing 3- glucosido) indole, - inactivating the enzyme indimulsin in the plant/plant part in a manner as herein described for preventing degradation of 3-( - glucosido) indole and extracting the 3-( - glucosido) indole into the broth, - treating the broth with an alkali to adjust the pH of the said broth to a value between 7 to 11, - clarifying and sterilizing the broth in a manner as herein described, - passing the sterilized clarified broth through a column/bed of sterilized anion exchange resin with exchangeable hydroxyl or chloride for removing the anions, - passing the sterilized broth through a column bed of sterilized column bed cation exchange resin with exchangeable hydronium or alkali cation, - concentrating the de-ionized 3-( - glucosido) indole by passing it over sterile absorbent column, - eluting 3-( - glucosido) indole from the absorbent column with organic solvents and recovering the said organic solvent by distillation or evaporation. The plant enzyme is inactivated either in a batch fashion or a continuous pfocess. In a batch process, the leaves/plant containing 3-\ - glucosido) indole is introduced into a steaming vessel and super-heated water of an alkaline pH is rapidly introduced into the vessel. Said steam should be passed through the vessel to ensure the temperature of the leaves never falls below 85° C. The ratio of the leaves of the water (w/w) is between 40% and 75%. After 3-10 minutes, the broth is drained out and the residual broth is removed by pressing the plant material or rotating it in a centrifuge like device. The broths are mixed together. To improve the recovery, this process can be repeated. Micro-wave radiation may be used as a continuos process to inactivate the enzyme. In this process, the leaves plant containing 3-( - glucosido) indole are immersed in a minimal amount of water and passed in a micro-wave generator of appropriate energy and strength to rapidly raise the tissue temperature to above 90 degrees Celsius. After 3-10 minutes, the broth is drained out and the residual broth is removed by pressing the plant material or rotating it in a centrifuge like device. The broths are mixed together. To improve the recovery, this process can be repeated. The alkali used for adjusting the pH of the broth is selected from sodium hydroxide or potassium hydroxide or calcium hydroxide and broth is clarified by centrifugation or by filtration. The temperature of the steam used for sterilizing the clarified broth is approximately 121 degree Celsius for approximately 20 minutes. The clarified broth may also be sterilized by filtering it through a micro filter (less than 0.22 microns). The organic solvent used for eluting 3-C. - glucosido) indole from the absorbent column is selected from alcohol or acetone or any other solvent or mixture thereof in which 3-( - glucosido) indole is soluble. The 3-i - glucosido) indole may be further sterilized by dissolving it in a minimum amount of alkaline water (pH of final solution between 7.5-10.5) and sterilized with the help of moist heat (121°C 15 min) or filtering it through a sterile micro ( The sterile 3-(r - glucosido) indole is packaged in sterile containers and stored for use. The 3- - glucosido) indole obtained by the process can not only be easily packaged m sterile containers and stored for further use but it is also environment friendly. The said hyrodxyl or chloride ion may be obtained from any inorganic base or inorganic salt. The hydronium ion or alkali cation may be obtained from any inorganic acid or inorganic salt. 3-( - glucosido) indole can be used interalia for dyeing fibers, as a reagent in biochemical reactions, immunoassays and genetic screening methods. The above-described discussion of this invention is also directed primarily to preferred embodiments and practices thereof. It will be readily apparent to those skilled in the art that further changes and modifications in the actual implementation of the concepts described herein can readily be made without departing from the spirit and scope of the invention as defined by the following claims. We claim: 1. A process for extracting 3-( - glucosido) indole comprising: procuring plant/plant part containing 3- • glucosido) indole, inactivating the enzyme indimulsin in the plant/plant part in a manner as herein described for preventing degradation of 3-( -glucosido) indole and extracting the 3-( - glucosido) indole into the broth, treating the broth with an alkali to adjust the pH of the said broth to a value between 7 to 11, clarifying and sterilizing the broth in a manner as herein described, passing the sterilized clarified broth through a column/bed of sterilized anion exchange resin with exchangeable hydroxyl or chloride for removing the anions, passing the sterilized broth through a column bed of sterilized column bed cation exchange resin with exchangeable hydronium or alkali cation, concentrating the de-ionized 3- (glucosido) indole by passing it over sterile absorbent column, eluting 3-( - glucosido) indole from the absorbent column with organic solvents and recovering the said organic solvent by distillation or evaporation. 2. A process as claimed in claim 1 wherein said plant enzyme is inactivated either by batch or a continuous process. 3. A process as claimed in claim 1 wherein alkali used is selected from sodium hydroxide, potassium hydroxide or calcium hydroxide. 4. A process as claimed in claim 1 wherein clarification of broth is done by centrifugal or filtration process. 5) A process as claimed in claim 1 wherein said organic solvents used for eluting may be either selected from alcohol or acetone 6) A process as claimed in claim 1 wherein said hydroxyl or chloride ion is obtained from any organic base or inorganic salt. 7) A process as claimed in claim 1 wherein the hydronium ion or alkali cation is obiained from any organic base or inorganic salt. 8) A process TOT extracting 3-( glucosido) indole substantially as herein described. Dated this 15 thday of November 2001 Of Anand and Anand advoeates Agents for the Applicants

Documents:

1028-mum-2000-abstract(25-04-2005).doc

1028-mum-2000-abstract(25-04-2005).pdf

1028-mum-2000-cancelled pages(25-04-2005).pdf

1028-mum-2000-claims(granted)-(25-04-2005).doc

1028-mum-2000-claims(granted)-(25-04-2005).pdf

1028-mum-2000-correspondence(25-04-2005).pdf

1028-mum-2000-correspondence(ipo)-(14-10-2004).pdf

1028-mum-2000-form 1(16-11-2000).pdf

1028-mum-2000-form 19(19-05-2004).pdf

1028-mum-2000-form 2(granted)-(25-04-2005).doc

1028-mum-2000-form 2(granted)-(25-04-2005).pdf

1028-mum-2000-form 3(16-11-2001).pdf

1028-mum-2000-form 3(25-04-2005).pdf

1028-mum-2000-form 5(16-11-2001).pdf

1028-mum-2000-other(16-11-2000).pdf