Title of Invention	"A PROCESS FOR THE ISOLATION OF PODOPHYLLETOXIN AND 4-DEMETHYLPODOPHYLLOTOXIN FROM THE MARC OF PODOPHYLLIN (PODOPHYLLETOXIN RESIN)."
Abstract	A process for the isolation of a mixture of podophyllotoxln and 4-demethylpodophyllotoxin from podophyllin (podophyllotoxln resin) which comprises treating powdered rhizomes of penodi with polar solvents, extracting with chlorinated solvents to produce residue separating the said residue (marc) by known methods, hydrolysing the marc so obtained with conventional polar solvents such as aliphatic alcohols in the presence of an enzyme emulsion (-glucosidase), at a pH in the range of 4 to 7 w at a temperature in the range of 30° to 37° C for 3 to 6 hours extrating the mixture with known chlorinated hydrocarbons & treating with neutral alumina to get a mixture of podophyllotoxln and 4 -demetnylpodophyllotoxin.

Title of Invention

"A PROCESS FOR THE ISOLATION OF PODOPHYLLETOXIN AND 4-DEMETHYLPODOPHYLLOTOXIN FROM THE MARC OF PODOPHYLLIN (PODOPHYLLETOXIN RESIN)."

Abstract

A process for the isolation of a mixture of podophyllotoxln and 4-demethylpodophyllotoxin from podophyllin (podophyllotoxln resin) which comprises treating powdered rhizomes of penodi with polar solvents, extracting with chlorinated solvents to produce residue separating the said residue (marc) by known methods, hydrolysing the marc so obtained with conventional polar solvents such as aliphatic alcohols in the presence of an enzyme emulsion (-glucosidase), at a pH in the range of 4 to 7 w at a temperature in the range of 30° to 37° C for 3 to 6 hours extrating the mixture with known chlorinated hydrocarbons & treating with neutral alumina to get a mixture of podophyllotoxln and 4 -demetnylpodophyllotoxin.

Full Text	This invention relates to A process for the isolation of podophyllotoxin and 4'-demethylpodophyllototoxin from the Marc of podophyliin (Podophyllotoxin resin). Podophyllotoxin since long has been known to possess medicinal properties and it is obtained fmm P-emodi Wall. This crystalline substance was first isolated by Podwyesotzki- V: {Pharmokologische Studien uber Podophyllum pellatum. Arch., exp. Pathol. Phamacol- 13,29(1880)] The alcohol soluble and water insoluble portion of podophyllum is a complex mixture containing most of biological activity of original root. It is an alkaloid and usually called podophyliin but has also been named as podophyllum resin. The research on this drug was done on many fronts like pharmacology, biochemistry, cytology and clinical medicine by Kelly, M.G. : ( J. National Cancer Institute 14, 967 (1954). The drug was studied in many clinical conditions other than Condylowa acuminatum including disease of skin due to infectious agents, non specific dermatoses, metabolic diseases, benign growth & malignant new growths. Podophyliin has been used in the treatment of cancer by private practioners In US prior to 1897 Hartwell J.L. [The chemistry of Podophyliin, Progress \n the Chemistry of Organic Natural Products. XV, 87, (1958)]. It was reported by Hartwell, J.L: [Cancer Research 7, 716 (1947)] for the first time that podophyliin had destructive effect on Cancer cells of experimental animals. It was demonstrated by King, L: Science 104, 244 (1946) that podophyliin produced its therapeutic effect by acting like colchicine as a mitotic poison. Podophyliin was so popular in the earlier days of last century as a cathartic and cholagogue that it was included in the first U.S.Pharmacopia (1820) & was listed until the twelfth revision published in 1942 [Pharmacopia of the United States of America, 1st Ed. (1820): 4th revision (1863); 12th revision (1942); 15th revision (1955), New York.: US. Pharmacopeia] Convension.]. Both American & Indian podophyllum were included in British Pharmacopoeia of 1948 but were dropped later from 1953 edition [British Pharmacopoeia, London: The Pharmaceutical press (1948, 1953)]. American podophyllum has been listed at one time or other in most Europeon, South American & Asian pharmacopoeias; [Hirch. B : Universal Pharmakopoe, 2 Vols. Gottingen: Vandenhoeck und Ruperecht (1902)]. The Chemistry and antineoplastic activity of many constituents of podophyllin were actively investigated at National Cancer Institute, USA by Hartwell J.L. in the 1940's [Jardine, I.: Podophyllotoxins, Anticancer agents based on Natural Products Models, Academic Press, 319, 1980)]. Its structure was proposed independently by Borche, W., [Justus Liebigs Ann. Chem. 494,126 (1932)]. Soath, E.,[ Ber. Dtsch. Chem. Ges, 65, 1536 (1932)] and was revised by Hartwell, J.L. {J. Am. Chem. Soc. 73,2909 (1961)}. color. It has characteristic odour, a bitter taste & is irritating to eyes & mucous membrane. Besides podophyllotoxin and 4'-demethylpodophyllotoxin, podophyllin has other constituents e.g. kaempferol, quercetin, podophyllotoxin-1 -O-p-D-glucopyranoside, and 4-demethylpodophyllotoxln-1 -O-p-D-glucopyranoside and tannins, Stoll, A.,[ Die Isolierung Von Podophyllotoxin - glucoside ausdem indischen Podophyllin emodii Mitt. Uber mitosehemmeude Naturstoffe. Helv. Chim. Acta 37, 7747(1954)]. Extraction of podophyllin with chloroform yields a residue which upon recrystallisation from benzene:methanol & hexane gives a 9:1 mixture of podophyllotoxin & 4'-demethylpodophyllotoxin (30 to 40% based upon resin). The remaining chloroform insoluble portion is called the Marc which consists of quercetin, kampferol, tannins, podophyllotoxin-1-0-ß-D-glucopyranoside and 4'-demethylpodophyllotoxin-1-0-ß-D-glucopyranoside. The introduction of anticancer drug Etoposide & Teniposide has created a demand for podophyllotoxin, the natural tetralin lignan (Podophyllotoxin) is used for the semi synthesis of these drugs Mugia, F.H.; [Etoposide VP-16 current status & New Developments, Academic Press, Orlando (1984)]. Since the total synthesis of podophyllotoxin is still uneconomic, future production of these drugs will depend upon either systematic cultivation of Podophyllum plants or the use of tissue culture techniques to obtain podophyllotoxins. Alternatively improvement in extraction techniques or new sources of these compounds need to be exploited. The main object of the present invention is to provide a method for the isolation of a mixture of podophyllotoxin & 4'demethylpodophyllotoxin from the enzymatic hydrolysis of the Marc which consists of kaempferol, quercetin, podophyllotoxin-1-0-ß-D-glucopyranoside and 4'- emethylpodophyllo- toxin 1-0-ß-D-glucopyranoside which is left after extraction of resin. Based upon earlier reports by Stoll.A., [J.Am.Chem. Soc. 76, 3103 (1954)] regarding the solubility of podophyllum glycosides in water, attempts were made to hydrolyse aqueous solution in which alcoholic extract has been poured with emulsin (ß-glucosidase) which gave podophyllotoxin and 4'-demethylpodophyllotoxin in the yield of (0-1%). Our method of isolation of podophyllotoxin and 4'- demethylpodophyllotoxin according to this invention is quite simple and involve isolation of the compounds from waste material of podophyllotoxin extraction process. The process consists of dissolving the marc in hot methanol and filtration. The filtrate is poured in water & emulsin (C- glucosidase) enzyme added to it. The mixture is kept at room temperature with stirring & the resulting slurry is repeatedly extracted with chloroform at room temperature which upon concentration & recrystallisation gives a mixture of podophyllotoxin & 4'- demethylpodophyllotoxin in the ratio of 4:1 (total yield 0.6%) of Podophyllum roots/rhizomes. The process of the present invention increases the overall yield of the mixture of podophvllotoxin and 4'- demethylpodophyllotoxin. Accordingly the present invention provides a process for the isolation of podophyllotoxin and 4'-demethylpodophyllototoxin from the Marc of podophyllin (Podophyllotoxin resin) which comprises treating powdered rhizomes of p. emodi with polar solvents, then extracting with chlorinated solvents to produce residue, separating the residue (marc) by known methods, hydrolysing the marc so obtained with polar solvents such as aliphatic alcohols in the presence of an enzyme emulsin )p-glucosidase). at a pH in the range of 5 to 7 at a temperature in the range of 30° to 37°C for 3 to 6 hours extracting the mixture with chlorinated hydrocarbons and treating with neutral alumina to get a mixture of podophyllotoxin and 4'-demethylpodophyllotoxin. The polar solvents used are selected from ethanol, methanol, isopropanol, propanol. The chlorinated solvent used is selected from dichloromethane, chloroform, carbon tetrachloride and dichloroethane. The process of the present invention is further illustrated with the help of following examples. However this should not limit the scope of the invention. Example 1 Air dried powdered rhizomes/roots (100 g) of P. emodi were extracted with methanol (500 ml) in a soxhiet for 15 hr. The extract concentrated under vacuum and poured in water (500 ml) with constant stirring. The water removed by decantation & resin is washed with water (3x200 ml) and (podophyllin) dried under vacuum at room temperature.lt gives light-brown colored powder (9.5 g). It was extracted with chloroform (3x50 ml) which upon concentration and repeated crystallization from benzene: methanol gave a mixture of podophyllotoxin & 4'-demethylpodophyllotoxin (3.40 g). It was finally purified by treatment with neutral alumina. The marc (chloroform insoluble residue 4 g) was dissolved in (25 ml) of hot ethanol/methanol, filtered and poured into the flask having 400 ml of water and pH adjusted to 5 with dil. HCI Emulsin (p-glucosidase, 0.3 g) in 20 ml water was added to the solution & the mixture was stirred for 6 hrs at 34-37T°C.The reaction was heated on water both for 10 minutes, cooled and the contents were extracted with chloroform (3x30 ml). The extract upon concentration yielded a semi solid yellowish mass which comprises of kaemferol, quercetin, podophyllotoxin & 4'demethylpodophyllotoxin (1.2 g). This was dissolved in methanol (30 ml) and solution refluxed with neutral alumina (3x2 g) which absorbs kaempferol & quercetin. A clear solution was obtained which comprises of podophyllotoxin & 4'-demethylpodophyllotoxin (0.6 g) In the ratio of (4:1). There is no reference in literature which Indicates the usage of marc for the Isolation of these compounds. Example 2 Air dried powdered roots/rhizomes of P. emodi (1 kg) are extracted with methanol (5 L) in a soxhlet extractor for 20 hrs. at refluxing temperature. The extract concentrated under vacuum and poured in ice cold water (7 L) with constant stirring. The water removed by decantation. The resin is washed with water (3xlL) at room temperature. It is dried at room temperature and extracted with ethyl acetate which upon concentration and repeated crystallisation with benzene methanol gives a mixture of podophyllotxln & 4'-demethylpodophyllotoxin in the ratio of 9:1 (35 g) and the undissolved light brown colored material called marc (40-45 g). Marc is dissolved in methanol (250 ml), filtered and the solvent removed under vacuum. The semi solid thus obtained was added to water (5L) with constant stirring and emulsin (ß- glucosidase, 5g) added to it. The mixture was constantly stirred and kept at 35 to 37°C. The reaction was heated on a steam bath for 15 min. cooled and the contents extracted with chloroform (3x500 ml) to give yellow mass (12.5 g). This was dissolved in methanol (250 ml) and solution refluxed with neutral alumina (3x50 g). Methanol was removed under vacuum and the solid thus obtained was crystallised from benzene methanol to give a mixture of two podophyllotoxin and 4'-demethylpodophyllotoxin in the ratio of 4:1. Example 3 Air dried powdered roots/rhizomes of P.emodi (1 kg) are extracted with ethanoi (5 L) in a soxhlet extraction apparatus for 20 hr. at refluxing temperature.The extract concentrated under vacuum and poured in ice cold water (7 L) with constant stirring.The water removed by decantation. The resin is washed with water (3xlL) and air dried at room temperature when it gives light brown colored powder (98.0 g). It is extracted with chloroform (3xlL) at reflux temperature which upon concentration with ethyl acetate-benzene gives a mixture of and repeated crystallisation with ethyl acetate-benzene gives a mixture of podophyllotoxin and 4'-demethylpodophyllotoxin in the ratio of 9:1 (38 g) and the undissolved light brown colored material called marc (45 g), Marc is dissolved in ethanoi (250 ml), filtred and solvent removed. The semi solid thus obtained was added to water (5 L) and pH adjusted to 6 by adding buffer. It was constantly stirred. To it added emulsin (10 g) and the mixture stirred for 4 hrs. while maintaining the temperature in the range of 35 to 37°C. The reaction contents were extracted with chloroform (5x300 ml). The extract upon concentration gave a yellow mass (12 g). This was dissolved in methanol (250 ml) and solution refluxed with neutral alumina (3xl5 g). Methanol was removed under vacuum & solid thus obtained was crystallised from benzene methanol gave light cream colored solid (7 g).HPLC analysis showed it to be a mixture of two compounds namely a) podophyllotoxin, b)4'-demethylpodophyllotoxin in the ratio of 4:1. The process of the present invention increases the overall yield of the desired products. Based upon earlier reports by Stoll, A., [J.Am.Chem.Soc. 76, 3103 (1954)] regarding the solubility of podophyllum glycosides in water.attempts were made to hydrolyse aqueous solution in which alcoholic extract has been poured) under enzymatic conditions which yielded the podophyllotoxin & 4'-demethylpodophyllotoxin in the yield of (0-1%). Our method of isolation of podophyllotoxin and 4'-demethylpodophyllotoxin is quite simple & involve isolation of required compounds from waste material of podophyllotoxin extraction process. Advantages: 1. Single organic solvents are used for the extraction. 2. No use of lead acetate is made to remove tannins. 3. No repeated column chromatography is performed in order to isolate the glucosides. 4. The Isolation of a mixture of podophyllotoxin and 4'-demethylpodophyllotoxin has been achieved from marc which is a We Claim: 1. A process for the isolation of podophyllotoxin and 4'-demethylpodophyllototoxin from the Marc of podophyllin (Podophyllotoxin resin) which comprises treating powdered rhizomes of p. emodi with polar solvents, then extracting with chlorinated solvents to produce residue, separating the residue (marc) by known methods, hydrolysing the marc so obtained with polar solvents such as aliphatic alcohols in the presence of an enzyme emulsin )p-glucosidase), at a pH in the range of 5 to 7 at a temperature in the range of 30° to 37°C for 3 to 6 hours extracting the mixture with chlorinated hydrocarbons and treating with neutral alumina to get a mixture of podophyllotoxin and 4'-demethylpodophyllotoxin. 2. A process as claimed in claim 1 wherein the polar solvent such as ethanol, methanol, propanol, isopropanol is used. 3. A process as claimed in claims 1 & 2 wherein the chlorinated solvent used is selected from dichloromethane, chloroform, carbon tetrachloride and dichloroethane. 4. A process for the isolation of podophyllotoxin and 4'- demethylpodophyllototoxin from the Marc of podophyllin (Podophyllotoxin resin) substantially as herein described with reference to the examples.

Full Text

This invention relates to A process for the isolation of podophyllotoxin and 4'-demethylpodophyllototoxin from the Marc of podophyliin (Podophyllotoxin resin).
Podophyllotoxin since long has been known to possess medicinal properties and it is obtained fmm P-emodi Wall. This crystalline substance was first isolated by Podwyesotzki- V: {Pharmokologische Studien uber Podophyllum pellatum. Arch., exp. Pathol. Phamacol- 13,29(1880)] The alcohol soluble and water insoluble portion of podophyllum is a complex mixture containing most of biological activity of original root. It is an alkaloid and usually called podophyliin but has also been named as podophyllum resin. The research on this drug was done on many fronts like pharmacology, biochemistry, cytology and clinical medicine by Kelly, M.G. : ( J. National Cancer Institute 14, 967 (1954). The drug was studied in many clinical conditions other than Condylowa acuminatum including disease of skin due to infectious agents, non specific dermatoses, metabolic diseases, benign growth & malignant new growths. Podophyliin has been used in the treatment of cancer by private practioners In US prior to 1897 Hartwell J.L. [The chemistry of Podophyliin, Progress \n the Chemistry of Organic Natural Products. XV, 87, (1958)]. It was reported by Hartwell, J.L: [Cancer Research 7, 716 (1947)] for the first time that podophyliin had destructive effect on Cancer cells of experimental animals. It was demonstrated by King, L: Science 104, 244 (1946) that podophyliin produced its therapeutic effect by acting like colchicine as a mitotic poison. Podophyliin was so popular in the

earlier days of last century as a cathartic and cholagogue that it was included in the first U.S.Pharmacopia (1820) & was listed until the twelfth revision published in 1942 [Pharmacopia of the United States of America, 1st Ed. (1820): 4th revision (1863); 12th revision (1942); 15th revision (1955), New York.: US. Pharmacopeia] Convension.]. Both American & Indian podophyllum were included in British Pharmacopoeia of 1948 but were dropped later from 1953 edition [British Pharmacopoeia, London: The Pharmaceutical press (1948, 1953)]. American podophyllum has been listed at one time or other in most Europeon, South American & Asian pharmacopoeias; [Hirch. B : Universal Pharmakopoe, 2 Vols. Gottingen: Vandenhoeck und Ruperecht (1902)].
The Chemistry and antineoplastic activity of many constituents of
podophyllin were actively investigated at National Cancer Institute, USA by
Hartwell J.L. in the 1940's [Jardine, I.: Podophyllotoxins, Anticancer agents
based on Natural Products Models, Academic Press, 319, 1980)]. Its
structure was proposed independently by Borche, W., [Justus Liebigs Ann.
Chem. 494,126 (1932)]. Soath, E.,[ Ber. Dtsch. Chem. Ges, 65, 1536 (1932)]
and was revised by Hartwell, J.L. {J. Am. Chem. Soc. 73,2909 (1961)}.

color. It has characteristic odour, a bitter taste & is irritating to eyes & mucous membrane. Besides podophyllotoxin and 4'-demethylpodophyllotoxin, podophyllin has other constituents e.g. kaempferol, quercetin,

podophyllotoxin-1 -O-p-D-glucopyranoside, and 4-demethylpodophyllotoxln-1 -O-p-D-glucopyranoside and tannins, Stoll, A.,[ Die Isolierung Von Podophyllotoxin - glucoside ausdem indischen Podophyllin emodii Mitt. Uber mitosehemmeude Naturstoffe. Helv. Chim. Acta 37, 7747(1954)]. Extraction of podophyllin with chloroform yields a residue which upon recrystallisation from benzene:methanol & hexane gives a 9:1 mixture of podophyllotoxin & 4'-demethylpodophyllotoxin (30 to 40% based upon resin). The remaining chloroform insoluble portion is called the Marc which consists of quercetin, kampferol, tannins, podophyllotoxin-1-0-ß-D-glucopyranoside and 4'-demethylpodophyllotoxin-1-0-ß-D-glucopyranoside.
The introduction of anticancer drug Etoposide & Teniposide has created a demand for podophyllotoxin, the natural tetralin lignan (Podophyllotoxin) is used for the semi synthesis of these drugs Mugia, F.H.; [Etoposide VP-16 current status & New Developments, Academic Press, Orlando (1984)]. Since the total synthesis of podophyllotoxin is still uneconomic, future production of these drugs will depend upon either systematic cultivation of Podophyllum plants or the use of tissue culture techniques to obtain podophyllotoxins. Alternatively improvement in extraction techniques or new sources of these compounds need to be exploited.
The main object of the present invention is to provide a method for the isolation of a mixture of podophyllotoxin & 4'demethylpodophyllotoxin from the enzymatic hydrolysis of the Marc which consists of kaempferol,

quercetin, podophyllotoxin-1-0-ß-D-glucopyranoside and 4'-
emethylpodophyllo- toxin 1-0-ß-D-glucopyranoside which is left after extraction of resin. Based upon earlier reports by Stoll.A., [J.Am.Chem. Soc. 76, 3103 (1954)] regarding the solubility of podophyllum glycosides in water, attempts were made to hydrolyse aqueous solution in which alcoholic extract has been poured with emulsin (ß-glucosidase) which gave podophyllotoxin and 4'-demethylpodophyllotoxin in the yield of (0-1%)*.
Our method of isolation of podophyllotoxin and 4'-
demethylpodophyllotoxin according to this invention is quite simple and
involve isolation of the compounds from waste material of podophyllotoxin
extraction process. The process consists of dissolving the marc in hot
methanol and filtration. The filtrate is poured in water & emulsin (C-
glucosidase) enzyme added to it. The mixture is kept at room
temperature with stirring & the resulting slurry is repeatedly extracted
with chloroform at room temperature which upon concentration &
recrystallisation gives a mixture of podophyllotoxin & 4'-
demethylpodophyllotoxin in the ratio of 4:1 (total yield 0.6%) of
Podophyllum roots/rhizomes. The process of the present invention increases
the overall yield of the mixture of podophvllotoxin and 4'-
demethylpodophyllotoxin.
Accordingly the present invention provides a process for the isolation of podophyllotoxin and 4'-demethylpodophyllototoxin from the Marc of

podophyllin (Podophyllotoxin resin) which comprises treating powdered rhizomes of p. emodi with polar solvents, then extracting with chlorinated solvents to produce residue, separating the residue (marc) by known methods, hydrolysing the marc so obtained with polar solvents such as aliphatic alcohols in the presence of an enzyme emulsin )p-glucosidase). at a pH in the range of 5 to 7 at a temperature in the range of 30° to 37°C for 3 to 6 hours extracting the mixture with chlorinated hydrocarbons and treating with neutral alumina to get a mixture of podophyllotoxin and 4'-demethylpodophyllotoxin.
The polar solvents used are selected from ethanol, methanol, isopropanol, propanol.
The chlorinated solvent used is selected from dichloromethane, chloroform, carbon tetrachloride and dichloroethane.
The process of the present invention is further illustrated with the help of following examples. However this should not limit the scope of the invention.
Example 1
Air dried powdered rhizomes/roots (100 g) of P. emodi were extracted with methanol (500 ml) in a soxhiet for 15 hr. The extract concentrated under vacuum and poured in water (500 ml) with constant stirring. The water

removed by decantation & resin is washed with water (3x200 ml) and (podophyllin) dried under vacuum at room temperature.lt gives light-brown colored powder (9.5 g). It was extracted with chloroform (3x50 ml) which upon concentration and repeated crystallization from benzene: methanol gave a mixture of podophyllotoxin & 4'-demethylpodophyllotoxin (3.40 g). It was finally purified by treatment with neutral alumina. The marc (chloroform insoluble residue 4 g) was dissolved in (25 ml) of hot ethanol/methanol, filtered and poured into the flask having 400 ml of water and pH adjusted to 5 with dil. HCI Emulsin (p-glucosidase, 0.3 g) in 20 ml water was added to the solution & the mixture was stirred for 6 hrs at 34-37T°C.The reaction was heated on water both for 10 minutes, cooled and the contents were extracted with chloroform (3x30 ml). The extract upon concentration yielded a semi solid yellowish mass which comprises of kaemferol, quercetin, podophyllotoxin & 4'demethylpodophyllotoxin (1.2 g). This was dissolved in methanol (30 ml) and solution refluxed with neutral alumina (3x2 g) which absorbs kaempferol & quercetin. A clear solution was obtained which comprises of podophyllotoxin & 4'-demethylpodophyllotoxin (0.6 g) In the ratio of (4:1).
*There is no reference in literature which Indicates the usage of marc for the Isolation of these compounds.

Example 2
Air dried powdered roots/rhizomes of P. emodi (1 kg) are extracted
with methanol (5 L) in a soxhlet extractor for 20 hrs. at refluxing temperature.
The extract concentrated under vacuum and poured in ice cold water (7 L)
with constant stirring. The water removed by decantation. The resin is
washed with water (3xlL) at room temperature. It is dried at room temperature
and extracted with ethyl acetate which upon concentration and repeated
crystallisation with benzene methanol gives a mixture of podophyllotxln &
4'-demethylpodophyllotoxin in the ratio of 9:1 (35 g) and the undissolved light
brown colored material called marc (40-45 g). Marc is dissolved in methanol
(250 ml), filtered and the solvent removed under vacuum. The semi solid thus
obtained was added to water (5L) with constant stirring and emulsin (ß-
glucosidase, 5g) added to it. The mixture was constantly stirred and kept at 35 to 37°C. The reaction was heated on a steam bath for 15 min. cooled and the contents extracted with chloroform (3x500 ml) to give yellow mass (12.5 g). This was dissolved in methanol (250 ml) and solution refluxed with neutral
alumina (3x50 g). Methanol was removed under vacuum and the solid thus obtained was crystallised from benzene methanol to give a mixture of two podophyllotoxin and 4'-demethylpodophyllotoxin in the ratio of 4:1.

Example 3
Air dried powdered roots/rhizomes of P.emodi (1 kg) are extracted with ethanoi (5 L) in a soxhlet extraction apparatus for 20 hr. at refluxing temperature.The extract concentrated under vacuum and poured in ice cold water (7 L) with constant stirring.The water removed by decantation. The resin is washed with water (3xlL) and air dried at room temperature when it gives light brown colored powder (98.0 g). It is extracted with chloroform (3xlL) at reflux temperature which upon concentration with ethyl acetate-benzene gives a mixture of and repeated crystallisation with ethyl acetate-benzene gives a mixture of podophyllotoxin and 4'-demethylpodophyllotoxin in the ratio of 9:1 (38 g) and the undissolved light brown colored material called marc (45 g), Marc is dissolved in ethanoi (250 ml), filtred and solvent removed. The semi solid thus obtained was added to water (5 L) and pH adjusted to 6 by adding buffer. It was constantly stirred. To it added emulsin (10 g) and the mixture stirred for 4 hrs. while maintaining the temperature in the range of 35 to 37°C. The reaction contents were extracted with chloroform (5x300 ml). The extract upon concentration gave a yellow mass (12 g). This was dissolved in methanol (250 ml) and solution refluxed with neutral alumina (3xl5 g). Methanol was removed under vacuum & solid thus obtained was crystallised from benzene methanol gave light cream colored solid (7 g).HPLC analysis showed it to be a mixture of two compounds namely a) podophyllotoxin, b)4'-demethylpodophyllotoxin in the ratio of 4:1.

The process of the present invention increases the overall yield of the desired products. Based upon earlier reports by Stoll, A., [J.Am.Chem.Soc. 76, 3103 (1954)] regarding the solubility of podophyllum glycosides in water.attempts were made to hydrolyse aqueous solution in which alcoholic extract has been poured) under enzymatic conditions which yielded the podophyllotoxin & 4'-demethylpodophyllotoxin in the yield of (0-1%).
Our method of isolation of podophyllotoxin and 4'-demethylpodophyllotoxin is quite simple & involve isolation of required compounds from waste material of podophyllotoxin extraction process.
Advantages:
1. Single organic solvents are used for the extraction.
2. No use of lead acetate is made to remove tannins.
3. No repeated column chromatography is performed in order to isolate the glucosides.
4. The Isolation of a mixture of podophyllotoxin and 4'-demethylpodophyllotoxin has been achieved from marc which is a

We Claim:
1. A process for the isolation of podophyllotoxin and 4'-demethylpodophyllototoxin from the Marc of podophyllin (Podophyllotoxin resin) which comprises treating powdered rhizomes of p. emodi with polar solvents, then extracting with chlorinated solvents to produce residue, separating the residue (marc) by known methods, hydrolysing the marc so obtained with polar solvents such as aliphatic alcohols in the presence of an enzyme emulsin )p-glucosidase), at a pH in the range of 5 to 7 at a temperature in the range of 30° to 37°C for 3 to 6 hours extracting the mixture with chlorinated hydrocarbons and treating with neutral alumina to get a mixture of podophyllotoxin and 4'-demethylpodophyllotoxin.
2. A process as claimed in claim 1 wherein the polar solvent such as ethanol, methanol, propanol, isopropanol is used.
3. A process as claimed in claims 1 & 2 wherein the chlorinated solvent used is selected from dichloromethane, chloroform, carbon tetrachloride and dichloroethane.

4. A process for the isolation of podophyllotoxin and 4'-
demethylpodophyllototoxin from the Marc of podophyllin
(Podophyllotoxin resin) substantially as herein described with reference to the examples.

Documents:

2793-del-1997-abstract.pdf

2793-del-1997-claims.pdf

2793-del-1997-complete specification (granted).pdf

2793-del-1997-correspondence-others.pdf

2793-del-1997-correspondence-po.pdf

2793-del-1997-description (complete).pdf

2793-del-1997-form-1.pdf

2793-del-1997-form-2.pdf

« Previous Patent

Next Patent »

Patent Number

186736

Indian Patent Application Number

2793/DEL/1997

PG Journal Number

43/2001

Publication Date

27-Oct-2001

Grant Date

07-Jun-2002

Date of Filing

30-Sep-1997

Name of Patentee

COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH

Applicant Address

RAFI MARG,NEW DELHI-110001, INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	SURINDER MOHAN ANAND	REGIONAL RESEARCH LABORATORY (CSIR),JAMMU,INDIA.
2	RANDHIR SINGH KAPIL	REGIONAL RESEARCH LABORATORY (CSIR),JAMMU,INDIA.
3	SATINDER MOHAN JAIN	REGIONAL RESEARCH LABORATORY (CSIR),JAMMU,INDIA.

PCT International Classification Number

A61K 35/78

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA