Title of Invention	"AN IMPROVED PROCESS FOR ISOLATION OF CAMPTOTHECIN FROM NOTHAPODYTES FOETIDA"
Abstract	The present invention relates to an improved and economical process for the isolation of anticancer agent Camptothecin from the twigs and stem of Nothapodytes foetida using hot extraction. 20(S) Camptothecin is an alkaloid, which contains a pentacyclic ring system that includes a pyrrolo [3,4-b] quinoline moiety (ring A, B and C), the pyridone ring D and a chiral centre at C-20 in ring E.

Full Text

Field of Invention The present invention relates to an improved and economical process for the isolation of anticancer agent " Camptothecin" from the twigs and stem of Nothapodytes foetida. 20(S) Camptothecin (1) is an alkaloid, which contains a pentacyclic ring system that includes a pyrrolo [3,4-b] quinoline moiety (ring A, B and C), the pyridone ring D and a chiral centre at C-20 in ring E. Background of Invention Camptothecin is one of the most impressive anticancer molecule of the recent years because it is the first compound found to directly block the topoisomerase (Topo-I), a DNA replication enzyme, thus stopping cell division. It was originally isolated from a rare Chinese plant Camptotheca acuminata Decne (Nyssacea) by Prof. Wall and co-workers in 1966 under the natural anticancer agent screening programme, carried out by the National Cancer Institute (NCI), USA. Because of the noteworthy activities of camptothecin towards L 1210 in mice and walker 256 tumor in rats, camptothecin has been a molecule of great interest from the time of its initial isolation, but due to low solubility and high toxicity, its therapeutic utility was restricted for a long time in various parts of the world. However, at the same time it was used in China for the treatment of liver carcinoma and tumors of head and neck. Recently scientist around the world carried out tremendous work on the chemical transformation of camptothecin into analogs having potential anticancer activities, better solubility and less toxicity. Finally, success has been achieved and two camptothecin derived drugs, Topotecan (Hycamtin) and Irinotecan (CPT-II, Camptosar) have been approved by the FDA for the treatment of ovarian, lung and colorectal cancers. 9-Nitrocamptothecin (Orathecin) another camptothecin derived drug is expected to receive FDA approval for pancreactic cancer treatment soon. Simultaneously 9-Aminocamptothecin (9-AC) has also been introduced in clinical trials because it exhibited curative ability against human colon carcinoma and strong antitumor activity against solid tumor xenographts. There are 12 other camptothecin derived drugs, which showed promising results and are in clinical trials. Camptothecin anologs have also been demonstrated to be potent antiviral, anti-HIV agents and chemosterilants. Thus, camptothecin will have broader uses and world wide demand of camptothecin (CPT) will dramatically increase. Presently, CPT production relies primarily on the extracts from Camptotheca acuminata Decaisne. Although trees of C. acuminata grow fast but since many parts of this tree are being used for the extraction of camptothecin, C. acuminata is becoming endangered in many parts of the world with special reference to China. In India camptothecin is being isolated from various parts of Indian Nothapodytes foetida (formerly Mappia foetida) Miers (Icacinaceae) in about 0.01 - 0.15%. Nothapodytes foetida is a small tree abundant in western ghats of India. The literatures indicates many reports on distribution, isolation, characterization and biological activities of camptothecin and its various derivatives. Now a days camptothecin is mainly isolated from the roots of Nothapodytes foetida. To isolate lKg of camptothecin, more than 1 OOOKg of roots are required, thus leading to uprooting or destruction of several thousands of plants. This destruction method of camptothecin isolation from the roots of N. foetida is the biggest draw back of the existing processes, which prompted us to find out a non-destructive method of camptothecin isolation and develop an easy and economical process for the isolation of this important anticancer agent, so that it can be brought under the reach of common masses. On going through the literature, it was recorded that an isolation procedure for camptothecin has been reported from the stem of Mappia foetida syn Nothapodytes foetida ("Alkaloids of Mappia foetida", Govindachari and Visvanathan, 1972, Phytochenmistry, 11, 3529-3531). The process involves defatting of the powdered stem twice with hexane followed by successive cold extraction thrice with Me2CO and MeOH. Extracts are combined separately, concentrated under vacuum and are left in ice chest for a week to give a greenish white solid, which on triangular crystallization results in the isolation of camptothecin in 0.11 % yield. Drawbacks of the Prior Arts The method described above suffers from a number of disadvantages. The biggest disadvantage of the above process is the poor yield of camptothecin (0.11%) when compared with ours (0.15%). The second disadvantage of the above method is that it uses cold percolation process where, plant material is left over night in a solvent for each percolation, hence for complete extraction (twice with hexane, thrice with Me2CO and twice with MeOH) of plant material at least seven days are required. The third disadvantage of the above method is that it requires more solvent, more electricity, more manpower and more time, thus resulting in an expensive and time taking process for the isolation of camptothecin from the twigs and stem of N.foetida. Objects of the Invention The main object of the present invention is to provide an improved process for the isolation of camptothecin from the twigs and stem of N. foetida. Another object of the present invention is to avoid use of tedious and time taking extraction and purification processes for the isolation of camptothecin from the twigs and stem of N. foetida. Still another object of the present invention is to provide an economical process for the isolation of camptothecin from the twigs and stem of N.s foetida. Still another object of the invention is to provide a process that achieved the defatting and complete extraction of plant material with the hot soxhlet extraction in only one day. Still another object of the present invention is to provide a process that results in 36% yield advantage of camptothecin to that of prior art process. Still another object of the present invention is to provide a process that uses 2-7 timeless amount of solvents, electricity, man power and time to that of prior art process. Accordingly the present invention provides an improved process for the isolation of camptothecin from from the twigs and stem of N. foetida, which comprises of drying, grinding and hot defattening of N.foetida twigs and stems with light petroleum fraction selected rom petroleum ether, hexane, benzene, toluene and dichloromethane followed by successive hot extraction with two solvents (one at a time) selected from CH2C12, CHC13, EtOAc, ether, acetone, MeOH, EtOH and CH3CN etc; removal of solvents under vacuum at 35-40°C, precipitation and filtration of crude extracts give camptothecin upto 0.15% of yield. In an embodiment of the present invention a varied range of defattening solvents, petroleum ether, hexane, benzene, toluene and dichloromethane can be used. In another embodiment of the present invention a varied range of solvents, chloroform, ethylacetate, ether, acetone, methanol, ethanol, acetonitrile and n-butanol can be used for the extraction of plant material. In yet another embodiment of the present invention the purification of camptothecin takes less time and is inexpensive. In yet another embodiment of the present invention the defatting and complete extraction of plant material is achieved with the of hot soxhlet extraction in only one day. In yet another embodiment of the present invention the process results in 36%/yield advantage of camptothecin to that of prior art process. In yet another embodiment of the present invention the process uses 2-7 times less amount of solvents, electricity, man power and time to that of prior art process. Detailed Description of the Invention The following examples are given by way of illustration of the present invention and should not be construed to limit the scope of present invention. Example 1 The powdered twigs and stem (lKg) of N. foetida were first cold defatted with petroleum ether (bp 40-60°C) in a percolator. Further extraction of defatted material was carried out with EtOAc and later by acetone. Removal of the solvent was carried out under vacuum at 35-40°C. Both EtOAc and Me2CO extracts on precipitation and filtration thrice resulted in the isolation of camptothecin in 0.085% yield. Example 2 The powdered twigs and stem (800g) were first hot defatted with hexane in a soxhlet extractor. Further extraction of the defatted material was successively carried out with CHC13 and CH3CN. Removal of the solvent was carried out under vacuum at 35-40°C. Both CHC13 and CH3CN extracts on precipitation and filtration twice resulted in the isolation of camptothecin in 0.095% yield. Example 3 The powdered twigs and stem (2Kg) were first cold defatted with petroleum ether (bp 40-60°C) in a percolator. Further extraction of the defatted material was carried out first with acetone and later on with EtOH. Removal of the solvents was carried out under vacuum at 35-40°C. Both acetone and EtOH extracts on repeated precipitation and filtration thrice resulted in the isolation of camptothecin in 0.09% yield. Example- 4 The powdered twigs and stem (1.5Kg) were first hot defatted with hexane in soxhlet extractor. Further extraction of the defatted material was successively carried out with EtOAc and MeOH. Both EtOAc and MeOH extracts on precipitation and filtration thrice resulted in the isolation of camptothecin in 0.10% yield. Advantages 1. The present process uses simple precipitation and filtration methods for the purification of camptothecin, which are easy, less time taking and inexpensive, while in prior art process, extracts are kept in ice-chest for a week and uses tedious triangular crystallization for the isolation of camptothecin. 2. The other major advantage of our process is that with the use of hot soxhlet extraction, defatting and complete extraction of the plant material is achieved in only one day, while defattening and extraction of plant material in prior art process requires more than seven days. 3. The main advantage of our process is that it results in 36% yield advantage of camptothecin to that of prior art process. 4. The other advantages of our process are that it uses 2-7 times less amount of solvents, electricity, man power and time to that of prior art process. We claim: 1. An improved process for the isolation of camptothecin from N. foetida, which comprises of drying, grinding and hot defatting of N. foetida twigs and stems with light petroleum fraction selected from petroleum ether, hexane, benzene, toluene and dichloromethane followed by successive hot extraction with two solvents (one at a time) selected from CH2C12, CHC13, EtOAc, ether, acetone, MeOH, EtOH and CH3CN as herein described, removal of solvents under vacuum at 35-40°C, precipitation and filtration of crude extracts gives camptothecin with 0.15% yield. 2. An improved process for isolation of camptothecin from N. foetida substantially as herein described with reference to the examples accompanying this specification.

Documents:

746-del-2003-abstract.pdf

746-del-2003-claims cancelled.pdf

746-del-2003-claims.pdf

746-del-2003-complete specification (granted).pdf

746-del-2003-correspondence-others.pdf

746-del-2003-correspondence-po.pdf

746-del-2003-description (complete).pdf

746-del-2003-form-1.pdf

746-del-2003-form-19.pdf

746-del-2003-form-2.pdf

746-del-2003-form-3.pdf

746-del-2003-petition-137.pdf

746-del-2003-petition-138.pdf