Title of Invention	"AN IMPROVED PROCESS FOR THE SIMULTANEOUS EXTRACTION & SEPARATION OF CAMPTOTHECIN &9-METHOXYCAMPTOTHECIN FROM STEM OF MAPPIA FOETIDA"
Abstract	An improved process for the simultaneous extraction & separation of camptothecin & 9-methoxycamptothecin having a general formula I wherein R=H or OMe from stem of Mappia foetida which comprises : (a) defatting the stems of the said plant by conventional methods, (b) extracting the said defatted part in soxhlet apparatus using an organic solvent such as acetone, methanol and ethyl acetate. (c) Concentrating the extract and then treating with glacial acetic acid and separating residue and filtrate by known methods, (d) Recovering camptothecin from residue by known method and 9-methoxycamptothecin from filtrate by conventional solvent partitioning.

Title of Invention

"AN IMPROVED PROCESS FOR THE SIMULTANEOUS EXTRACTION & SEPARATION OF CAMPTOTHECIN &9-METHOXYCAMPTOTHECIN FROM STEM OF MAPPIA FOETIDA"

Abstract

An improved process for the simultaneous extraction & separation of camptothecin & 9-methoxycamptothecin having a general formula I wherein R=H or OMe from stem of Mappia foetida which comprises : (a) defatting the stems of the said plant by conventional methods, (b) extracting the said defatted part in soxhlet apparatus using an organic solvent such as acetone, methanol and ethyl acetate. (c) Concentrating the extract and then treating with glacial acetic acid and separating residue and filtrate by known methods, (d) Recovering camptothecin from residue by known method and 9-methoxycamptothecin from filtrate by conventional solvent partitioning.

Full Text	This invention relates to an improved process for the simultaneous extraction & separation of camptothecin and 9-methoxycamptothecin from Mappia foetida. Both these compounds have the general formula I as shown in the drawing accompanying this specification and possess significant antitumour and antileukemic properties (M. E. Wall et. al., /. Am. Chem. Soc, 88,3888, 1966; s. P. Gunasekara et. al., /. Nat. Prod., 42, 475, 1979). However, camptothecin wherein R=H is much more active than 9-methoxycamptothecin wherein R=OMe and the former is a major alkalaid from the stems of Mappia foetida which contains 9- methoxycamptothecin as a minor constituent. The structural modifications of these two compounds can improve their activity The polarities of camptothecin wherein R=H and 9-methoxycamptothecin wherein R=OMe are very close and so their quantitative separation is a problem. Previously they were separated by column chromatography (T. uS. Wu et. al., Phytochemistry, 39,383, 1995), preparative thin layer chromatography (S. P. Gunasekara et. al., /. Nat. Prod., 42,475, 1979) or by precipitating the alkaloids from the concentrated extract of the plant material followed by subsequent crystallization of the major alkaloid, camptothecin and chromatographic separation of the minor alkaloid, 9-methoxycamptothecin from the mother liquor (T. R. Govindachari et. a!., Phytochemistry, 11,3529, 1972). The systematic column chromatography is not suitable for the large-scale separation of the alkaloids as the column would be eluted with large quantities of different solvents and each fraction of the elutes would be monitored properly. For preparative thin layer chromatography a large number of chromatographic plates would be needed and the required time for adsorption and subsequent recovery of the compounds would be high. Moreover, due to close polarity of the compounds both the processes ( column chromatography and thin layer chromatography) would not be efficient by single operation and repeated chromatography would be required. The column would be eluted with mixtures of different solvents to generate different fractions. The quantitative recovery of the solvents from all the eluates is also a problem. The other process involved the precipitation of the alkaloidal portion from the crude concentrated extract keeping on the ice-chest for several days (7-10 days). However, the crude extract is tarry/gummy in nature and the quantitative precipitation of the alkaloids from this extract is difficult. As a result, the yield would be diminished. The yield of camptothecin ranges from 0.06-0.08%. Moreover, the arrangement of ice-chest for several days for each operation would be required. The total time needed for the separation of large quantity of alkaloids (for each batch 7-10 days) would also be high. The object of the present invention is to provide an improved process for the simultaneous separation of camptothecin and 9- methoxycamptothecin obviates the drawbacks of known processes as detailed above. Accordingly the present invention provides an improved process for the simultaneous extraction & separation of camptothecin and 9-methoxycamptothecin having a general formula I as shown in the drawing accompanying this specification wherein R=H or OMe from stem of Mappia foetida which comprises : a) defatting the stems of the said plant by conventional methods. b) extracting the said defatted part in soxhlet apparatus using an organic solvent such as acetone, methanol and ethyl acetate. c) concentrating the extract and then treating with glacial acetic acid and separating residue and filtrate by known methods. d) recovering camptothecin from residue by known method and 9-methoxycamptothecin from filtrate by conventional solvent partitioning. In an embodiment of the present invention the defatting may be effected by soxhleting powdered stem part using solvent such as hexane, petroleum ether for 10 to 12 hr. at 60- 80 ° C. In another embodiment of the invention extraction in step (b) may be effected for 45 to 50 hrs. The solvent used for solvent partitioning may be such as dichloromethane, chloroform. The present investigation has led to develop an improved process for the separation of camptothecin and 9- methoxycamptothecin from the stems of Mappia foetida, which are highly avaiiabie and can be easily collected. The process is suitable for large-scale separation and quantitative isolation of these two compounds. The time required is minimum (3-4 days) and the overall process is economic as the solvents required for extraction of the plant materials and separation of alkaloids can be recycled . As a result, the abundant indigenous plant, Mappia foetida can be conveniently used for the isolation of its two important anticancer constituents, camptothecin and 9-methoxycamptothecin. The process of the present invention is illustrated by the following examples. However, these should not limit the scope of the present invention. Example 1 The stems of Mappia foetida (5 kg) were powdered and at first extracted with a Soxhlet apparatus using hexane (30 lit) for 12 hr. The defatted materials were then extracted thrice with the Soxhlet apparatus using acetone. Each extraction was continued for 16 hr with 30 lit of acetone. The combined acetone extract was concentrated with a rotavapour. The solid material was churned with glacial acetic acid (150 ml) for 1 hr and filtered. The insoluble portion was then washed with glacial acetic acid ( 50 ml) to yield 6.5 gm of pure camptothecin (98% pure). The acetic acid soluble portion was partitioned between water (250 ml) and CHCI3 (250 ml). The CHCI3 layer was separated. The aqueous portion was again extracted with CHCI 3 (250 ml). The combined CHCI 3 soluble fraction was washed with water (250 ml) and then concentrated and completely dried. The dry mass was again churned with CHCI 3 (100 ml) and filtered. The filtrate was concentrated to yield 9-methoxycamptothecin with 78% purity. Further purification of the compound was achieved by absorbing the compound on silica gel (100 gm) in a column and washing the column with 250 ml of hexane-EtOAc (1:1) with subsequent recovery of the compound by elution with EtOAc (500 ml). Finally, the crystallization of the compound from EtOAc afforded 98% pure 9- methoxycamptothecin (1.1 gm). Example 2 The powdered stems (7 kg) of Mappia foetida were defatted with hexane (40 lit) using a Soxhiet apparatus for 12 hr. The plant materials were then extracted with acetone (40 lit) for 15 hr with the same soxhiet apparatus. The extraction with acetone was repeated twice more keeping the conditions similar. The total acetone extract was concentrated with a rotavapour to afford a solid mass, which was churned with glacial acetic acid (200 ml) for I hr. The insoluble material was filtered and washed with glacial acetic acid (75 ml) to obtain pure camptothecin (10.5 gm, 95% pure). The filtrate was partitioned between water (300 ml) and CHC13 (300 ml). The CHC13 portion was separated and the aqueous portion was again extracted twice with CHC13 (2X200 ml). The total CHC13 extract was washed twice with water (2X200 ml), concentrated under reduced pressure and then completely dried to a solid mass. The solid was shaken well with CHC13 (150 ml) and filtered. The concentration of the filtrate afforded 9-methoxycamptothecin (80% pure). For further purification the compound was absorbed on silica gel (100 gm) in a column. The column was at first washed with hexane-EtOAc ( I: I) (400 ml) and then eluted with EtOAc ( I lit) to recover 9-methoxycamptothecin which was then crystallized from EtOAc to produce 97% pure compound (1.5 gm). Example 3 The powdered stems (10 kg) of Mappia foetida were Soxhleted with hexane (55 lit) for 10 hr. The defatted plant materials were again soxhleted thrice (for 15 hr in each case) with acetone (60 lit in each case). The total acetone extract was concentrated with a rotavapour to a solid mass. The solid mass was churned with glacial acetic acid (300 ml) for I hr, filtered and the residue was thoroughly washed with glacial acetic acid (120 ml) to give 98% pure camptothecin (14 gm). The filtrate was shaken with water (400 ml) and CHCI 3 (400 ml). The CHCI 3 layer was separated and the aqueous portion was re-extracted thrice with CHCI 3 (3X250 ml). The total CHCI 3 soluble portion was washed twice with water (2X250 ml) and then concentrated and dried to a solid mass. The solid mass was then churned with CHCI 3 (250 mi), filtered and the filtrate was concentrated to give 80% pure 9-methoxycamptothecin. The compound was more purified by absorbing it on silica gel (150 gm) in a column. The column was at first washed with 500 ml hexane-EtOAc (1:1) and then the compound was recovered by eluting with EtOAc (1.2 lit). The compound was crystallized from EtOAc to give 97% pure 9-methoxycamptothecin (2.3 gm). The process of the present invention has following advantages: The process is suitable for the simultaneous separation of camptothecin and 9- methoxycamptothecin in larger quantities from the stems of Mappia foetida. The separation can be achieved in minimum time (single operation within 3-4 days) with quantitative yields (camptothecin: 1.3-1.5gm/kg of the sterns and 9-methoxycamptothecin: 200- 230 mg/kg of the sterns) and high purity (97-98%). In this process no systematic column chromatography or preparative thin layer chromatography is required. All the solvents used in the process can be recycled to make the process economic. We Claim : 1. AM improved process for the simultaneous extraction & separation of camptothecin & 9- methoxycamptothecin having a general formula I as shown in the drawing accompanying this specification wherein R=H or OMe from stem of Mappia foetida which comprises : a) defatting the stems of the said plant by conventional methods, b) extracting the said defatted part in soxhlet apparatus using an organic solvent such as acetone, methanol and ethyl acetate. c) concentrating the extract and then treating with glacial acetic acid and separating residue and nitrate by known methods, d) recovering camptothecin from residue by known method and 9-methoxycamptothecin from filtrate by conventional solvent partitioning. 2. An improved process as claimed in claim 1 wherein defatting is effected by soxhleting powdered stems part using hexane or petroleum ether for 10 to 12 hr at 60-80 °C. 3. An improved process as claimed in claims 1 and 2 wherein the extraction in step (b) is effected for 45 to 48 hrs. 4. An improved process as claimed in claims 1 to 3 wherein camptothecin is recovered by washing and drying the residue obtained in step ( d). 5. An improved process as claimed in claims 1 to 4 wherein solvent partitioning is done using water and chloroform or water and dichloromethane. 6. An improved process for the simultaneous extraction & separation of camptothecin & 9-methoxycamptothecin having general formula I as defined in claim 1 substantially as herein described with reference to the exampes.

Full Text

This invention relates to an improved process for the simultaneous extraction & separation of camptothecin and 9-methoxycamptothecin from Mappia foetida. Both these compounds have the general formula I as shown in the drawing accompanying this specification and possess significant antitumour and antileukemic properties (M. E. Wall et. al., /. Am. Chem. Soc, 88,3888, 1966; s. P. Gunasekara et. al., /. Nat. Prod., 42, 475, 1979). However, camptothecin wherein R=H is much more active than 9-methoxycamptothecin wherein R=OMe and the former is a major alkalaid from the stems of Mappia foetida which contains 9- methoxycamptothecin as a minor constituent. The structural modifications of these two compounds can improve their activity
The polarities of camptothecin wherein R=H and 9-methoxycamptothecin wherein R=OMe are very close and so their quantitative separation is a problem. Previously they were separated by column chromatography (T. uS. Wu et. al., Phytochemistry, 39,383, 1995), preparative thin layer chromatography (S. P. Gunasekara et. al., /. Nat. Prod., 42,475, 1979) or by precipitating the alkaloids from the concentrated extract of the plant material followed by subsequent crystallization of the major alkaloid, camptothecin and chromatographic separation of the minor alkaloid, 9-methoxycamptothecin from the mother liquor (T. R. Govindachari et. a!., Phytochemistry, 11,3529, 1972). The systematic column chromatography is not suitable for the large-scale separation of the alkaloids as the column would be eluted with large quantities of different solvents and each fraction of the elutes would be monitored properly. For preparative thin layer chromatography a large number of chromatographic plates would be needed and the required time for adsorption and subsequent recovery of the compounds would be high. Moreover, due to close polarity of the compounds both the processes ( column chromatography and thin layer chromatography) would not be efficient by single operation and repeated chromatography would be required. The column would be eluted with mixtures of different solvents to generate different fractions.
The quantitative recovery of the solvents from all the eluates is also a problem. The other process involved the precipitation of the alkaloidal portion from the crude concentrated extract keeping on the ice-chest for several days (7-10 days). However, the crude extract is tarry/gummy in nature and the quantitative precipitation of the alkaloids from this extract is difficult. As a result, the yield would be diminished. The yield of camptothecin ranges from 0.06-0.08%. Moreover, the arrangement of ice-chest for several days for each operation would be required. The total time needed for the separation of large quantity of alkaloids (for each batch 7-10 days) would also be high.
The object of the present invention is to provide an improved process for the simultaneous separation of camptothecin and 9- methoxycamptothecin obviates the drawbacks of known processes as detailed above.
Accordingly the present invention provides an improved process for the simultaneous extraction & separation of camptothecin and 9-methoxycamptothecin having a general formula I as shown in the drawing accompanying this specification wherein R=H or OMe from stem of Mappia foetida which comprises : a) defatting the stems of the said plant by conventional methods.
b) extracting the said defatted part in soxhlet apparatus using an organic solvent such as acetone, methanol and ethyl acetate.
c) concentrating the extract and then treating with glacial acetic acid and separating residue and filtrate by known methods.
d) recovering camptothecin from residue by known method and 9-methoxycamptothecin from filtrate by conventional solvent partitioning.
In an embodiment of the present invention the defatting may be effected by soxhleting powdered
stem part using solvent such as hexane, petroleum ether for 10 to 12 hr. at 60- 80 ° C.
In another embodiment of the invention extraction in step (b) may be effected for 45 to 50 hrs.
The solvent used for solvent partitioning may be such as dichloromethane, chloroform.
The present investigation has led to develop an improved process for the separation of
camptothecin and 9- methoxycamptothecin from the stems of Mappia foetida, which are highly
avaiiabie and can be easily collected. The process is suitable for large-scale separation and
quantitative isolation of these two compounds. The time required is minimum (3-4 days) and the
overall process is economic as the solvents required for extraction of the plant materials and
separation of alkaloids can be recycled . As a result, the abundant indigenous plant, Mappia
foetida can be conveniently used for the isolation of its two important anticancer constituents,
camptothecin and 9-methoxycamptothecin.
The process of the present invention is illustrated by the following examples. However, these
should not limit the scope of the present invention.
Example 1 The stems of Mappia foetida (5 kg) were powdered and at first extracted with a Soxhlet apparatus using hexane (30 lit) for 12 hr. The defatted materials were then extracted thrice with the Soxhlet apparatus using acetone. Each extraction was continued for 16 hr with 30 lit of acetone. The combined acetone extract was concentrated with a rotavapour. The solid material was churned with glacial acetic acid (150 ml) for 1 hr and filtered. The insoluble portion was then washed with glacial acetic acid ( 50 ml) to yield 6.5 gm of pure camptothecin (98% pure). The acetic acid soluble portion was partitioned between water (250 ml) and CHCI3 (250 ml). The CHCI3 layer
was separated. The aqueous portion was again extracted with CHCI 3 (250 ml). The combined CHCI 3 soluble fraction was washed with water (250 ml) and then concentrated and completely dried. The dry mass was again churned with CHCI 3 (100 ml) and filtered. The filtrate was concentrated to yield 9-methoxycamptothecin with 78% purity. Further purification of the compound was achieved by absorbing the compound on silica gel (100 gm) in a column and washing the column with 250 ml of hexane-EtOAc (1:1) with subsequent recovery of the compound by elution with EtOAc (500 ml). Finally, the crystallization of the compound from EtOAc afforded 98% pure 9- methoxycamptothecin (1.1 gm).
Example 2 The powdered stems (7 kg) of Mappia foetida were defatted with hexane (40 lit) using a Soxhiet apparatus for 12 hr. The plant materials were then extracted with acetone (40 lit) for 15 hr with the same soxhiet apparatus. The extraction with acetone was repeated twice more keeping the conditions similar. The total acetone extract was concentrated with a rotavapour to afford a solid mass, which was churned with glacial acetic acid (200 ml) for I hr. The insoluble material was filtered and washed with glacial acetic acid (75 ml) to obtain pure camptothecin (10.5 gm, 95% pure). The filtrate was partitioned between water (300 ml) and CHC13 (300 ml). The CHC13 portion was separated and the aqueous portion was again extracted twice with CHC13 (2X200 ml). The total CHC13 extract was washed twice with water (2X200 ml), concentrated under reduced pressure and then completely dried to a solid mass. The solid was shaken well with CHC13 (150 ml) and filtered. The concentration of the filtrate afforded 9-methoxycamptothecin (80% pure). For further purification the compound was absorbed on silica gel (100 gm) in a column. The column was at first washed with hexane-EtOAc ( I: I) (400 ml) and then eluted with EtOAc ( I lit) to recover 9-methoxycamptothecin which was then crystallized from EtOAc to produce 97% pure compound (1.5 gm).
Example 3 The powdered stems (10 kg) of Mappia foetida were Soxhleted with hexane (55 lit) for 10 hr. The defatted plant materials were again soxhleted thrice (for 15 hr in each case) with acetone (60 lit in each case). The total acetone extract was concentrated with a rotavapour to a solid mass. The solid mass was churned with glacial acetic acid (300 ml) for I hr, filtered and the residue was
thoroughly washed with glacial acetic acid (120 ml) to give 98% pure camptothecin (14 gm). The filtrate was shaken with water (400 ml) and CHCI 3 (400 ml). The CHCI 3 layer was separated and the aqueous portion was re-extracted thrice with CHCI 3 (3X250 ml). The total CHCI 3 soluble portion was washed twice with water (2X250 ml) and then concentrated and dried to a solid mass. The solid mass was then churned with CHCI 3 (250 mi), filtered and the filtrate was concentrated to give 80% pure 9-methoxycamptothecin. The compound was more purified by absorbing it on silica gel (150 gm) in a column. The column was at first washed with 500 ml hexane-EtOAc (1:1) and then the compound was recovered by eluting with EtOAc (1.2 lit). The compound was crystallized from EtOAc to give 97% pure 9-methoxycamptothecin (2.3 gm).
The process of the present invention has following advantages:
The process is suitable for the simultaneous separation of camptothecin and 9-
methoxycamptothecin in larger quantities from the stems of Mappia foetida.
The separation can be achieved in minimum time (single operation within 3-4 days) with
quantitative yields (camptothecin: 1.3-1.5gm/kg of the sterns and 9-methoxycamptothecin: 200-
230 mg/kg of the sterns) and high purity (97-98%).
In this process no systematic column chromatography or preparative thin layer chromatography is
required. All the solvents used in the process can be recycled to make the process economic.

We Claim :
1. AM improved process for the simultaneous extraction & separation of camptothecin & 9-
methoxycamptothecin having a general formula I as shown in the drawing accompanying this
specification wherein R=H or OMe from stem of Mappia foetida which comprises :
a) defatting the stems of the said plant by conventional methods,
b) extracting the said defatted part in soxhlet apparatus using an organic solvent such as acetone, methanol and ethyl acetate.
c) concentrating the extract and then treating with glacial acetic acid and separating residue and nitrate by known methods,
d) recovering camptothecin from residue by known method and 9-methoxycamptothecin from filtrate by conventional solvent partitioning.

2. An improved process as claimed in claim 1 wherein defatting is effected by soxhleting powdered stems part using hexane or petroleum ether for 10 to 12 hr at 60-80 °C.
3. An improved process as claimed in claims 1 and 2 wherein the extraction in step (b) is effected for 45 to 48 hrs.
4. An improved process as claimed in claims 1 to 3 wherein camptothecin is recovered by washing and drying the residue obtained in step ( d).
5. An improved process as claimed in claims 1 to 4 wherein solvent partitioning is done using water and chloroform or water and dichloromethane.

6. An improved process for the simultaneous extraction & separation of camptothecin & 9-methoxycamptothecin having general formula I as defined in claim 1 substantially as herein described with reference to the exampes.

Documents:

117-del-1998-abstract.pdf

117-del-1998-claims.pdf

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

117-del-1998-correspondence-others.pdf

117-del-1998-correspondence-po.pdf

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

117-del-1998-drawings.pdf

117-del-1998-form-1.pdf

117-del-1998-form-2.pdf

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Patent Number

187517

Indian Patent Application Number

117/DEL/1998

PG Journal Number

19/2002

Publication Date

11-May-2002

Grant Date

22-Nov-2002

Date of Filing

16-Jan-1998

Name of Patentee

COUNCIL OF SCIENTIFIC & INDUSTRIAL RESEARCH

Applicant Address

RAFI MARG,NEW DELHI-110001,INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	PURUSHOTHAM MADHUSUDHAN	CHEMICAL TECHNOLOGY,HYDERABAD 500007,ANDHRA PRADESH,INDIA.
2	JHILLU SINGH YADAV	CHEMICAL TECHNOLOGY,HYDERABAD 500007,ANDHRA PRADESH,INDIA.
3	GUNTURU ANJANI	CHEMICAL TECHNOLOGY,HYDERABAD 500007,ANDHRA PRADESH,INDIA.
4	BISWANATH DAS	CHEMICAL TECHNOLOGY,HYDERABAD 500007,ANDHRA PRADESH,INDIA.
5	ALISALA KASHINATHAM	CHEMICAL TECHNOLOGY,HYDERABAD 500007,ANDHRA PRADESH,INDIA.

PCT International Classification Number

C07C - 49/00

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA