Abstract:
The present invention relates to an insecticidal enriched extract (biopesticide) isolated from the stem of  Nothapodites foetida  (Wight) Sleumer (formerly  Mappia foetida  (Miers) useful for the insect free storage and transport of grains and seed. The insecticidal activity of the enriched extract (biopesticide) of the invention is ascribed to compound/s other than camptothecin. The invention further relates to the process for preparation thereof, containing Compounds of general formula 1 and formula 2 (General formula 1) wherein R1=CH 3 , CH 2 OAC and wherein R2=COOH, or (I) and R3=H. OH. (Formula 2)

Description:
FIELD OF THE INVENTION 
       [0001]    The present invention relates to an insecticidal compound of general formula 1 and formula 2 isolated from the stem of  Nothapodites foetida  (Wight.) Sleumer (formerly  Mappia foetida  (Miers)) useful for the insect free storage and transport of grains and seed. Particularly, present invention further relates to insecticidal enriched extract (biopesticide) isolated from the stem of  Nothapodites foetida  (Wight.) Sleumer (formerly  Mappia foetida . More particularly, the insecticidal activity of the enriched extract (biopesticide) of the invention is ascribed to compound/s other than camptothecin. The present invention further relates to the process for the extraction thereof. 
       BACKGROUND OF THE INVENTION 
       [0002]    Even though every plant species has an inbuilt unique chemical complex structure that protects it from/provides resistance against pests/insects; there remains a need for external pesticides that will protect the plant against those pests which are resistant to inbuilt resistance of plant. 
         [0003]    Currently, synthetic insecticides are used to protect stored grain/seed from insect infestation. Synthetic insecticides are manufactured at least in part using synthetic chemicals created from other chemicals. Synthetic insecticides are undesirable because of ecological and toxic effects of the chemicals. They are not permitted for use on organically produced grain/seed. The continued use of synthetic insecticides has led to a large number of insect pests developing resistance to the insecticides. As a result, these insecticides have to be withdrawn or larger quantities need to be used, exacerbating the problem. The problem of insect resistance is also indicated with  Bacillus thuringiensis , the most widely used and intensively studied microbial insecticide. 
         [0004]    Biopesticide offer an answer to the above problems, as they are eco-friendly, non-persistent, safer to use and the chances of insect pests developing resistance is unlikely as the insecticidal activity is normally due to multi-component active ingredients. 
         [0005]    Thus, alternative to synthetic pesticides are pesticides derived from natural materials such as animals, plants, bacteria, and certain minerals. 
         [0006]    Preferable, biopesticides are natural plant products that belong to the so called secondary metabolites that include thousands of alkaloids, terpenoids, phenolics and minor secondary metabolites. Biopesticides have usually no known function in photosynthesis, growth or other basic aspects of plant physiology; however, their biological activity against insect pests, nematodes, fungi and other organisms is well documented. 
         [0007]    Reference may be made to the use of plants and minerals as traditional protectants of stored products, wherein the traditional method of mixing sand with stored grain is documented. However, since the sand particles are used to block the inter granular spaces and act as a physical barrier to the movement of insect pests, large quantities of sand have to be used which is unhygienic and also necessitates sieving off the sand before using the grain. [Golob, P and Webley, D. J., (1980) G138, Chatham, UK, Natural Resources Institute] 
         [0008]    The use of diatomaceous earth based formulations for the protection of stored grain in the USA and Australia is documented. However, the drawbacks are that the treatment inhibits the free flow of grain and also damages grain handling equipment. More recently, health problems to workers have restricted its use considerably. [Frank H. A., Grain protectants: Current status and prospects for the future, J. Stored Prod. Res. 32(4)293-302 1996] 
         [0009]    Use of mineral dusts for protection against insect pests with special reference to cereal grains, in Grain Sanitation wherein activated clay is used as a nontoxic grain protectant is documented. [Eds. S. K. Majumder &amp; J. S. Venugopal, Academy of Pest Control Sciences, Mysore, India, 1969, pp. 81-96]. Diatomaceous dusts have also been used as grain protectants. However, the treatment is not effective on grain with high moisture content or in regions with high relative humidity as in coastal areas as reported by Mevis I, Ch. Ulrichs (2001), J. Stored Prod. Res. 37. pp. 153-164. Action of amorphous diatomaceous earth against different stages of the stored product pests  Triboliumconfusum, Tenebriomolitor, Sitophilusgranarius  and  Plodiainterpunctella  are also disclosed. 
         [0010]    Reference may also be made to Studies on plant oils as surface protectant against pulse beetle  Callosobruchus chinensis  (L.) in chickpea,  Cicer arietinum  L. [Singal, S. K. and Singh. Z, 1990, Tropical Pest Management, 36.314-316] wherein the traditional practice of mixing stored grain with vegetable oils is documented. However, the drawbacks are that the oils apart from being expensive also turn rancid and may impart an off-flavour to the grain as shown by Pereira, J. and Wholgemuth, R. 1983, J. Stored Prod. Res. 19, 57-62. The effect of six vegetable oils as protectants of cowpeas and Bambara groundnut against infestation by  Callosobruchus maculatusis  also reported. 
         [0011]    References may be made to Journals Efficacy of deltamethrin against storage insects in rice and wheat under FCI&#39;s storage system. (Pesticides pg 39-43) and toxicity of deltamethrin, chlorpyriphos, methyl etrifos, malathion and fluvalinate against  Sitophilus oryzae  and  Trogoderma granarium . Ind. J. Ent. 56(4). 322-325 by Yadav et al., wherein the organophosphate insecticide DDVP is used in insecticide formulations along with malathion for the control of insect pests in grain storage. However, insects have become resistant to DDVP and malathion and these are being phased out. DDVP is known to be carcinogenic. Currently, while malathion is being replaced with deltamethrin, there is no replacement for DDVP which acts in the vapor phase. 
         [0012]    References may be made to patents IN192851 and IN199867 by Pereira et. al. describe a process for the preparation of a formulation useful for the insect free storage of cereals using roots of  Decalepis hamiltonii  Wight. and Arn. and its extracts mixed with substituted phenols wherein substituted phenols were mixed with the dried root powder/solvent extracts of the roots of  Decalepis hamiltonii  to provide a stable, synergistic formulation for the storage of insect free cereals. However, the formulation is not effective against the red flour beetle,  Tribolium castaneum  Herbst. There is need to develop new insecticides of biological origin (biopesticides) that are biodegradable and do not leave residues, which are also species specific and do not harm non target organisms. 
         [0013]      Nothapodytes nimmoniana  (Syn:  N. foetida, Mappia foetida ) (Family: Icacinaceae) is gaining international importance due to their pharmacological and curative properties. The wood-extract of this tree is used in the treatment of cancer. It is believed that Camptothecin (CPT) is the third most important alkaloid sought after by the pharmaceutical companies around the world. Few developments on pesticide/herbicide and insecticidal properties of  Nothapodytes nimmoniana  extracts are enlisted in below references: 
         [0014]    References may be made to patent JP61200902, provides a novel herbicide containing 9-methoxycamptothecin, etc., extracted from vegetables as active components, exhibiting excellent herbicidal effect to various weeds and free from phototoxicity to important crops such as corn, wheat, rice, soybean, cotton, etc. The said herbicide contains, as active component, 9-methoxycamptothecin separated from  Mappia foetida  (a plant of Icacinaceae family) and reported to have carcinostatic and antileukemic activities, or an extract containing 9-methoxycamptothecin extracted from a plant. 
         [0015]    The extraction of the above active component is carried out by conventional process for the extraction of natural component, e.g. by the extraction with an organic solvent such as methanol, acetone, ethyl acetate, etc., following concentration, etc. The herbicide is effective against broad-leaved weeds such as white-bird&#39;s-eye, smooth pigweed, green amaranth, etc., gramineous weeds such as barnyard grass, green foxtail, etc., cyperaceous weeds such as yellow-cyperus, etc. 
         [0016]    References may be made to patent U.S. Pat. No. 6,893,668 (corresponding Indian Patent Application No. 746/DEL/2003) provides a process for the isolation of Camptothecin from the twigs and stem of  N. foetida , which comprises of drying, grinding  N. foetida  twigs and stems, subjecting the dried and ground  N. foetida  twigs and stems to hot defatting with a light petroleum fraction followed by successive sequential hot extraction with two solvents, removing the solvents under vacuum to obtain crude extracts of the plant material, precipitating and filtering the crude plant extracts to obtain Camptothecin with up to 0.15% yield. 
         [0017]    References may be made to patent CN101243796 (WO2009/111950) which relates to a composition of pesticide and the manufacturing method, belonging to botanical pesticide which is characterized in that the composition comprises an active constituent with 0.1 to 2.0 percent of the total weight. The constituent is single compound or the mixture of compounds which accords to the general formula I, wherein R 1  is Na and R 2  is H or OH. 
         [0000]    
       
                 
         
             
             
         
       
     
         [0018]    The composition of pesticide adopts the ring opening camptothecin salt as the insecticidal active compound, wherein, the camptothecin salt is high in activity and can be dissolved in water. 
         [0019]    References may be made to patent US20020018762 relates to insect control with Camptotheca products, particularly, termiticides, baits, preservatives, and termite-resistant products. Natural or synthetic Camptothecin and/or one of its natural or synthetic analogs are applied to deter and/or eliminate termites, while flavonoids are applied to attract termites. CPTs and/or CPTs-containing plant matter is processed with cellulose products to produce termite-resistant products and structures. 
         [0020]    Further “Insecticidal Activity of Camptothecin against  Nilaparvata lugens, Brevicoryne brassicae , and  Chilo suppressalis ” by Jianyi Ma, et. al. discloses that greenhouse tests showed that 0.2% Camptothecin Emulsifiable Concentrate (EC) has strong contact toxicity to three agricultural pests in the following descending order  Nilaparvata lugens  Stahl,  Brevicoryne brassicae  L.,  Chilo suppressalis  Walker. 
         [0021]    Camptothecin isolated from the stem of  Nothapodytes foetida  is known to possess anti-cancer properties, which is currently in demand for anticancer treatment. Therefore, the present inventors have evaluated the  Nothapodytes foetida  stem extract that is substantially free from camptothecin for its other biological activities and surprising found that the stem extract of  Nothapodytes foetida  is found to be an effective biopesticide for the insect free storage of stored grain and seed. 
         [0022]    Accordingly, the present inventors have developed a biopesticide from the stem of  Nothapodites foetida , that can be used to protect stored grain/seed and also store organically produced grain/seed from insect infestation. The said biopesticide being of biological origin is eco-friendly and less toxic to non-target organisms. 
       OBJECTIVE OF THE INVENTION 
       [0023]    Main objective of the present invention is to provide insecticidal compound of general formula 1 and formula 2 isolated from the stem of  Nothapodites foetida  (Wight.) Sleumer (formerly  Mappia foetida  (Miers)) useful for the insect free storage and transport of grains and seed. 
         [0024]    Another objective of the present invention is to provide insecticidal enriched extract isolated from the stem of  Nothapodites foetida  (Wight.) Sleumer. 
         [0025]    Another objective of the present invention is to provides insecticidal activity of the enriched extract (biopesticide) of the invention, ascribed due to compound/s other than camptothecin. 
       SUMMARY OF THE INVENTION 
       [0026]    Accordingly, present invention provides compound of general formula 1 and formula 
         [0000]    
       
                 
         
             
             
         
       
     
         [0027]    wherein R1=CH 3 , CH 2 OAc and wherein R2=COOH, or 
         [0000]    
       
                 
         
             
             
         
       
     
         [0028]    and R3=H, OH. 
         [0000]    
       
                 
         
             
             
         
       
     
         [0029]    In an embodiment of the present invention, representative compounds of general formula 1 comprising following compounds: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0030]    In another embodiment of the present invention, said compounds are useful for the protection of stored grains and seed from insect infestation. 
         [0031]    Yet another embodiment of the present invention, the said compounds are non-toxic to non-target insect pests like aphids, thrips, diamondback moth, tobacco caterpillar, gram pod borer, bihar hairy caterpillar, potato tuber moth, tea mosquito, red spider mite and Mexican beetle. 
         [0032]    Yet another embodiment of the present invention, the said compounds are active against stored product insect pests namely the red flour beetle,  Tribolium castaneum , lesser grain borer,  Rhizopertha dominica , pulse beetle,  Callosobruchus chinensis , almond moth,  Ephestia cautella , rice moth,  Corcyra cephalonica.    
         [0033]    Yet another embodiment of the present invention, Insecticidal composition comprising compound of general formula 1 or 2 either alone or in combination thereof optionally along pharmaceutically acceptable additives useful for the protection of stored grains and seed from insect infestation. 
         [0034]    Yet another embodiment of the present invention, the additives are selected from the group consisting of diluents, solvents, surfactants and carriers. 
         [0035]    Yet another embodiment of the present invention, the said composition is in the form of spray, dust, powder, tablet and pellets. 
         [0036]    Yet another embodiment of the present invention, the said composition is effective for more than 100 days with a reduction in F1 progeny of the infestants. 
         [0037]    Yet another embodiment of the present invention, the said composition exhibits 100% mortality of rice weevil  Sitophilus oryzae , lesser grain borer  R. dominica  and  Callosobruchus chinensis.    
         [0038]    Yet another embodiment of the present invention, process for the preparation of compounds of general formula 1 and formula 2 from the stem of  N. foetida  comprising the steps of:
       i. drying the stem of the  Nothapodites foetida  in shade followed by cutting into small pieces and pulverizing;   ii. extracting the powder of step [i] with methanol and designating the extract as S1;   iii. extracting the residue of step [ii] with methanol and designating the extract as S2;   iv. extracting the residue of step [iii] with methanol and designating the extract as S3;   v. stripping off methanol from extracts 1, 2 and 3 as obtained in step (ii) (iii) and (iv) respectively, pooling and defatting the residue in petroleum ether;   vi. optionally, combining the extracts 1, 2 and 3 as obtained in step (ii), (iii) and (iv) respectively to yield an extract designated as S4;   vii. stripping off methanol from extract 4 as obtained in step (vi) followed by defatting thereof in petroleum ether to obtain extract S5 and defatted residue S6;   viii. partitioning the defatted residues S6 as obtained in step [v] and/or [vii] in n-butanol designated as S7 and in water designated as S8;   ix. stripping off the solvent to obtain the insecticidal enriched bioactive extract;   x. removing camptothecin by column chromatography (CC) to obtain fraction D1-D26;   xi. subjecting fraction D1-D26 to CC using successively methyl cynate and chloroform (2:8) and then gradient of methanol and chloroform to obtain D24, D25, D18, D21 and D4;   xii. subjecting fraction D18, D21 and D4 to CC as obtained in step (xi) in gradient of MeCN: chloroform to obtain fraction D18:2, D21:2 and D4:2 contained pure phthalate of general formula 2;   xiii. separating fraction D24 as obtained in step (xi) using gradient of methanol in chloroform followed by methanol wash to obtain fraction D24:12 contained compound campptothecanoid 1 of general formula 1;   xiv. separating fraction D25 as obtained in step (xi) using gradient of methanol in chloroform to obtain fraction D25:4-5 contained compounds camptothecanoids 2 and 3 of general formula 1.       
 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0053]      FIG. 1  represents  1 H NMR of camptothecanoid 1. 
           [0054]      FIG. 2  represents  13 C NMR of camptothecanoid 1. 
           [0055]      FIG. 3  represents Distortionless Enhancement by Polarization Transfer (DEPT) of camptothecanoid 1. 
           [0056]      FIG. 4  represents  1 H NMR of camptothecanoid 2. 
           [0057]      FIG. 5  represents DEPT of camptothecanoid 2. 
           [0058]      FIG. 6  represents  13 C NMR of camptothecanoid 2. 
           [0059]      FIG. 7  represents  1 H NMR of camptothecanoid 3. 
           [0060]      FIG. 8  represents  13 C NMR of camptothecanoid 3. 
           [0061]      FIG. 9  represents DEPT of camptothecanoid 3. 
           [0062]      FIG. 10  represents  1 H NMR of Phthalate. 
           [0063]      FIG. 11  represents  13 C NMR of Phthalate. 
           [0064]      FIG. 12  represents DEPT of Phthalate. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0065]    The present invention describes insecticidal activity of compound of general formula 1 isolated from the stem of  Nothapodites foetida , containing compound/s substantially free from camptothecin that is used to protect stored and under transit grain/seed and also to store and transport organically produced grain/seed from insect infestation. 
         [0066]    The insecticidal enriched extract (biopesticide) from the stem of  Nothapodites foetida , containing compound/s other than camptothecin that is used to protect the grains during transport also. 
         [0067]    The biopesticide compound of general formula 1 and formula 2 described in the current invention is non-toxic to non-target insect pests and active against stored product insect pests namely the red flour beetle,  Tribolium castaneum , lesser grain borer,  Rhizopertha dominica , pulse beetle,  Callosobruchus chinensis , almond moth,  Ephestia cautella , rice moth,  Corcyra cephalonica ; and not active against aphids, thrips, diamondback moth, tobacco caterpillar, gram pod borer, Bihar hairy caterpillar, potato tuber moth, tea mosquito, red spider mite, Mexican beetle. Thus, the insecticidal extract is specific to the stored product insect pests. 
         [0068]    The insecticidal enriched extract (biopesticide) using wheat treatment technique (defined in example 1) is found to give 100% mortality of rice weevil  Sitophilus oryzae  and lesser grain borer  Rhizopertha dominica . The treatment is found to be effective for more than 100 days with a reduction in F1 progeny. In yet another aspect, the active insecticidal enriched extract is formulated into a free flowing dust formulation with the addition of inactive additives like diluents, solvents, surfactants and carriers. The dust formulation is used to protect stored wheat from insect attack for more than 6 months causing 100% insect mortality and reduced F1 progeny. (first generation of insects.) 
         [0069]    The insecticidal activity of the extract is attributed to its low camptothecin concentration and the other contents of the extract. 
         [0070]    The pulverized stem is extracted from methanol, 1:1 methanol:water and water to detect the insecticidal activity of the extracts and found that the methanolic extract is found to be good over hydro-methanolic extract and water extract. Due to this, present invention discloses a process for preparation of insecticidal enriched extract (biopesticide) from the stem of  Nothapodites foetida , which comprises;
       1. Extracting the dried stem powder repeatedly with methanol only;   2. Stripping off methanol from all the combined extract and then extracting residue in Petroleum ether to remove fatty material;   3. Partitioning the residue from the above step with n-butanol and water; and   4. Stripping off the n-butanol solvent to obtain the insecticidal enriched extract;   5. Isolating compound of general formula 1 and formula 2 from insecticidal enriched extract       
 
       DEFINITIONS AND ABBREVIATIONS 
       [0000]    
       
         CPT: Camptothecin 
         S1: Methanol extract of powder. 
         S2: Methanol extract of residual powder after removal of S1. 
         S3: Methanol extract of residual powder after removal of S2. 
         S4: Combined methanol extracts (S1+S2+S3) contains 0.262% of CPT. 
         S5: Pet ether extract of S4 containing the fatty fraction and devoid of CPT. Labeled as 
         S4—A in bioassays. 
         S6: Defatted fraction of S4 i.e. residue of S4 after extraction with Pet ether Labeled as S4-B containing 0.272% CPT. 
         S7: n-butanol soluble fraction of S6 i. e. S6 was separated in n-butanol:water Labeled as S4-B1 containing 1.168% CPT. 
         S7A from S1 directly (% Camptothecin: 5.0763) 
         S7B from S2 directly (% Camptothecin: 6.037) 
         S7C from S3 directly (% Camptothecin: 3.849) 
         S8: Water soluble fraction of S6 separated from n-butanol fraction as explained in 
         S7 containing devoid of CPT. 
         S4—Methanol extract (with 4.89% camptothecin) 
         T1—Methanol:water(1:1) extract 
         T2—Water extract 
         “Substantially Free of Camptothecin” refers to the extract containing less than about 12.5 ppm of the Camptothecin content. Preferably, 10 ppm of the Camptothecin content. More preferably the 5 ppm. 
         F1 Progeny refers to first generation of insects. 
         Progeny/adult day refers to generation. 
       
     
       EXAMPLE 
       [0096]    The following examples are given by way of illustration therefore should not construed to limit the scope of the invention. 
       Example 1 
       [0097]    The stem of the  Nothapodites foetida  plant material was shade dried, cut into small pieces and pulverized. The powder was extracted in three solvent systems and the extracts labeled as follows: 
         [0000]    
       
         
               
               
               
             
           
               
                   
                   
               
               
                   
                 Solvent system 
                 Extract 
               
               
                   
                   
               
             
             
               
                   
                 Methanol 
                 S4 
               
               
                   
                 Methanol:water (1:1) 
                 T1 
               
               
                   
                 Water 
                 T2 
               
               
                   
                   
               
             
          
         
       
     
         [0098]    The bioefficacy of the extracts were screened against  S. oryzae  and  R. dominica  on wheat at 1000 ppm. Required quantity of the extract dissolved in 6 ml of the solvent was pipetted over 60 gm wheat, taken in a 500 ml beaker. The grain was stirred with a glass rod to obtain uniform application. The control grain received the solvent alone. The treated grain was spread on petridish and kept overnight (12 hrs) for the solvent to evaporate. The 60 gm grain was divided into three lots of 20 gm each, taken in test tubes 25 mm diameter and 95 mm long. 
         [0099]    Fifteen unsexed adults, 1-2 weeks old, of the rice weevil,  Sitophilus oryzae  and the lesser grain borer,  Rhizopertha dominica  were seeded into the tubes separately. The tubes were covered with muslin held by rubber bands. The mortality of the test insects was recorded after 1, 3, 7, 14 and 21 days. After the last count, the insects were discarded and the grain incubated to obtain the F1 progeny. The number of emerging progeny was recorded every second day till the emergence was complete. The progeny/adult day was calculated by Kazmaier et. al method. The progeny/adult day corrects for the reduction in the number of progeny due to adult mortality. A significant reduction in the progeny/adult day compared to the control indicates Insect Growth Regulating (IGR) activity, which is presented in Tables 1, 2 and 3. 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Bioefficacy of  Nothapodites foetida  extracts against  S. oryzae   
               
             
          
           
               
                   
                   
                   
                 Progeny/adult day 
               
             
          
           
               
                   
                   
                 % mortality 
                 Control 
                 Treated 
               
               
                   
                   
               
             
          
           
               
                   
                 S4 
                 74 
                 0.855 
                 0.016 
               
               
                   
                 T1 
                 42.43 
                 0.855 
                 0.034 
               
               
                   
                 T2 
                 00.00 
                 1.030 
                 0.660 
               
               
                   
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Bioefficacy of  Nothapodites foetida  extracts against  R. dominica   
               
             
          
           
               
                   
                   
                 Progeny/adult day 
               
             
          
           
               
                   
                 % mortality 
                 Control 
                 Treated 
               
               
                   
               
             
          
           
               
                 S4 
                 53.33 
                 0.785 
                 0.020 
               
               
                 T1 
                 44.00 
                 0.785 
                 0.033 
               
               
                 T2 
                 36.00 
                 0.939 
                 0.184 
               
               
                   
               
             
          
         
       
     
         [0100]    Conclusion: The extract S4 was the most active against both the tested insects. 
         [0000]    
       
         
               
             
               
               
             
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Bioefficacy of  Nothapodites foetida  extracts against  C. chinensis   
               
             
          
           
               
                 Treatment 
                 Avg. No. of Progeny* 
               
               
                   
               
             
          
           
               
                 Solvent Control 
                 101.75 
               
               
                 S4 
                 00.00 
               
               
                 T1 
                 02.25 
               
               
                   
               
               
                 *Average of four replicates. 
               
             
          
         
       
     
         [0101]    Avg. No. of Progeny is average number of individuals emerged while Progeny/adult day is a ratio term. From Tables 1, 2 and 3, it is seen that all the three extracts were active. Apart from adult mortality, there was a significant reduction in the progeny/adult day indicating a strong Insect Growth Regulating activity (IGR) of the three extracts and S4 was found to be the most active extract. 
       Example 2 
       [0102]    The stem of the plant material was shade dried, cut into small pieces and pulverized. Since, methanol was the best solvent of the three solvent systems assayed, the powder was extracted with methanol and designated as Extract 1 (S1). The residual powder was extracted again with methanol and the extract designated as Extract 2 (S2). The residual powder was extracted again with methanol and the extract designated as Extract 3 (S3). 
         [0103]    All the three extracts were stripped of methanol individually (S4) and the residue extracted in petroleum ether (S5) to remove the fatty material present in the extracts. 
         [0104]    The defatted residue (S6) was partitioned in n-butanol and water. The n-butanol fraction (S7) was stripped of the solvent to obtain: 
         [0000]    S7A from S1 directly (% Camptothecin: 5.0763)
 
S7B from S2 directly (% Camptothecin: 6.037)
 
S7C from S3 directly (% Camptothecin: 3.849)
 
         [0105]    These three fractions were assayed against  S. oryzae  and  R. dominica  as in Example 1. The results are presented in Table 4. 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Bioefficacy of fractions S7A, S7B and S7C against  S. oryzae  and  
               
               
                 
                   R. dominica 
                 
               
             
          
           
               
                   
                   
                 % mortality* 
                 No. adults emerged 
               
             
          
           
               
                 Treatment 
                 (ppm) 
                 
                   S. 
                   oryzae 
                 
                 
                   R. 
                   dominica 
                 
                 
                   S. 
                   oryzae 
                 
                 
                   R. 
                   dominica 
                 
               
               
                   
               
             
          
           
               
                 Blank 
                 00 
                 00.00 
                 00.00 
                 212 
                 171 
               
               
                 control 
                   
                   
                   
                   
                   
               
               
                 S7A 
                 50 
                 11 
                 43 
                 00 
                 00 
               
               
                 S7B 
                 50 
                 29 
                 37 
                 00 
                 00 
               
               
                 S7C 
                 50 
                 46 
                 46 
                 00 
                 00 
               
               
                 S7A 
                 100 
                 39 
                 59 
                 00 
                 00 
               
               
                 S7B 
                 100 
                 63 
                 76 
                 00 
                 00 
               
               
                 S7C 
                 100 
                 55 
                 65 
                 00 
                 00 
               
               
                 S7A 
                 200 
                 77 
                 84 
                 00 
                 00 
               
               
                 S7B 
                 200 
                 86 
                 91 
                 00 
                 00 
               
               
                 S7C 
                 200 
                 100 
                 100 
                 00 
                 00 
               
               
                 S7A 
                 500 
                 100 
                 100 
                 00 
                 00 
               
               
                 S7B 
                 500 
                 100 
                 100 
                 00 
                 00 
               
               
                 S7C 
                 500 
                 100 
                 100 
                 00 
                 00 
               
               
                   
               
               
                 *% mortality at the end of 21 days. With 200 and 500 ppm, 100% mortality was obtained within 14 days. 
               
             
          
         
       
     
         [0106]    Fraction S7C contained 3.85% Camptothecin as determined by HPLC. Fraction S7C at 50 ppm and 100 ppm on wheat would be equivalent to 1.993 ppm and 3.986 ppm of Camptothecin respectively. 
         [0107]    Camptothecin obtained from Sigma Aldrich was assayed on wheat against  S. oryzae  and  R. dominica  as in example 1. The results are presented in table 5. 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 Bioefficacy of Camptothecin treated on wheat against rice weevil, 
               
               
                   Sitophilus oryzae  and lesser grain borer,  Rhizopertha dominica .* 
               
             
          
           
               
                   
                   
                 No. days for 100%  
                   
               
               
                 Treatment (ppm) 
                 Mortality (%) 
                 kill 
                 No. of F1 progeny 
               
               
                   
               
             
          
           
               
                 100 
                 100 
                 3 
                 00 
               
               
                 75 
                 100 
                 7 
                 00 
               
               
                 50 
                 100 
                 14 
                 00 
               
               
                 25 
                 100 
                 21 
                 00 
               
               
                 12.50 
                 00 
                 — 
                 00 
               
               
                 6.25 
                 00 
                 — 
                 00 
               
               
                   
               
             
          
         
       
     
         [0108]    At 12.50 ppm, there was no adult mortality but 100% inhibition of F1 progeny. However, with fraction S7C at 100 ppm and 50 ppm equivalent to 3.986 ppm and 1.993 ppm respectively of Camptothecin, there was adult mortality and 100% inhibition of F1 progeny with  S. oryzae.    
         [0109]    From the above experiment, it can be concluded that there is/are toxic principle/s in the extracts other than Camptothecin, that is/are acting as effective components of biopesticide/s over camptothecin. 
       Example 3 
       [0110]    Fraction S7A as obtained in example 2 was separated by column chromatography using silica gel as stationary phase and methanol:chloroform gradient as mobile phase into 6 fractions designated as E1, E2, E3, E4, E5 and E6. The six fractions as obtained were assayed on wheat against  S. oryzae  and  R. dominica  as described in example 1. The results are presented in Table 6 and Table 7. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                 Bioefficacy of S7A fractions against rice weevil,  Sitophilus oryzae   
               
             
          
           
               
                   
                 25 ppm (in brackets, 
                   
                   
                   
               
               
                   
                 amount of CPT in 
               
               
                   
                 ppm is reported) 
                 50 ppm 
                 100 ppm 
                 200 ppm 
               
             
          
           
               
                   
                 % 
                 % 
                 Avg. No. 
                 % 
                 Avg. No. 
                 % 
                 Avg. No. 
                 % 
                 Avg. No. 
               
               
                 Fraction 
                 CPT 
                 mort. 
                 emerge 
                 mort. 
                 emerged 
                 mort. 
                 emerged 
                 mort. 
                 emerged 
               
               
                   
               
             
          
           
               
                 E1 
                 0.1542 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 E2 
                 18.8903 
                 18.3 (4.72)  
                 35 
                 96.6 (9.44) 
                 7.5 
                 98.3 (18.88) 
                 7.0 
                  95 (37.76) 
                 4.7 
               
               
                 E3 
                 3.1209 
                 8.3 (0.78)  
                 79 
                 18.3 (1.56) 
                 51.5 
                 18.3 (3.12)  
                 20.5 
                 18.3 (6.24)  
                 13.2 
               
               
                 E4 
                 0.1409 
                 5.0 (0.035) 
                 114 
                  3.3 (0.07) 
                 115 
                 5.0 (0.14) 
                 119 
                 5.0 (0.28)  
                 92 
               
               
                 Control (solvent 
                   
                 1.6 
                 145 
                 1.6 
                 145 
                 1.6 
                 145 
                 1.6 
                 145 
               
               
                 control for E1 to E4) 
               
               
                 E5 
                 0.0330 
                 8.3 (0.008) 
                 150 
                  5.0 (0.016) 
                 135 
                  5.0 (0.032) 
                 125 
                 5.0 (0.064) 
                 136 
               
               
                 E6 
                 0.6430 
                 3.3 (0.161) 
                 129 
                  1.6 (0.232) 
                 120 
                  5.0 (0.464) 
                 121 
                 5.0 (0.928) 
                 83 
               
               
                 Control (water 
                   
                 1.6 
                 172 
                 1.6 
                 172 
                 1.6 
                 172 
                 1.6 
                 172 
               
               
                 control for E5 and E6) 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 7 
               
             
             
               
                   
               
               
                 Bioefficacy of S7A fractions against lesser grain borer,  Rhozopertha dominica   
               
             
          
           
               
                   
                 25 ppm 
                 50 ppm 
                 100 ppm 
                 200 ppm 
               
             
          
           
               
                   
                 % 
                 Avg. No. 
                 % 
                 Avg. No. 
                 % 
                 Avg. No. 
                 % 
                 Avg. No. 
               
               
                 Fraction 
                 mort. 
                 emerged 
                 mort. 
                 emerged 
                 mort. 
                 emerged 
                 mort. 
                 emerged 
               
               
                   
               
             
          
           
               
                 E1 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 E2 
                 75.0 
                 4.7 
                 81.6 
                 0.0 
                 90 
                 0.0 
                 91.6 
                 0.0 
               
               
                 E3 
                 20.0 
                 1.25 
                 23.3 
                 0.5 
                 18.3 
                 0.75 
                 33.3 
                 0.0 
               
               
                 E4 
                 65.0 
                 12.0 
                 71.6 
                 3.7 
                 81.6 
                 4.5 
                 95.0 
                 3.2 
               
               
                 E5 
                 11.6 
                 30 
                 10.0 
                 23.2 
                 11.6 
                 26.2 
                 15.0 
                 15.5 
               
               
                 E6 
                 11.6 
                 18 
                 100 
                 2.7 
                 90.0 
                 0.2 
                 95.0 
                 0.5 
               
               
                 Control 
                 3.3 
                 74.3 
                 3.3 
                 74.3 
                 3.3 
                 74.3 
                 3.3 
                 74.3 
               
               
                   
               
             
          
         
       
     
         [0111]    All the fractions exhibited toxicity to the test insects and there was a significant reduction in progeny. 
       Example 4 
       [0112]    Fraction E4 and E6 as obtained in example 3 were further fractionated by preparative HPLC using RP-18 column and acetonitrile: water gradient as mobile phase for E4 and methanol: water gradient for E6 into 5 and 4 fractions respectively and designated as E4-A, E4-B, E4-C, E4-D, E4-E and E6-A, E6-B, E6-C, E6-D respectively. 
         [0113]    The fractions as obtained were assayed against  S. oryzae  and  R. dominica  as described in Example 1. The results are presented in Table 8 and Table 9. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 8 
               
             
             
               
                   
               
               
                 Bioefficacy of E4 and E6 fractions against rice weevil,  Sitophilus oryzae   
               
             
          
           
               
                   
                 12.5 ppm 
                 25 ppm 
                 50 ppm 
                 100 ppm 
               
             
          
           
               
                   
                 % 
                 Avg. No. 
                 % 
                 Avg. No. 
                 % 
                 Avg. No. 
                 % 
                 Avg. No. 
               
               
                 Fraction 
                 mort. 
                 emerged 
                 mort. 
                 emerged 
                 mort. 
                 emerged 
                 mort. 
                 emerged 
               
               
                   
               
             
          
           
               
                 E4-A 
                 0.0 
                 119 
                 1.6 
                 139.5 
                 0.0 
                 100 
                 0.0 
                 95.24 
               
               
                 E4-E 
                 5.0 
                 197 
                 5.0 
                 132.7 
                 8.3 
                 145.5 
                 8.3 
                 137.7 
               
               
                 E6-A 
                 0.0 
                 136 
                 3.3 
                 122.7 
                 0.0 
                 95.7 
                 0.0 
                 89.5 
               
               
                 E6-C 
                 0.0 
                 162 
                 0.0 
                 159.7 
                 1.6 
                 151.7 
                 0.0 
                 112.5 
               
               
                 E6-D 
                 1.6 
                 192 
                 1.6 
                 193 
                 5.0 
                 108 
                 6.6 
                 94.2 
               
               
                 Control 
                 1.6 
                 134.7 
                 5.0 
                 181.5 
                 3.3 
                 171.7 
                 3.3 
                 171.7 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 9 
               
             
             
               
                   
               
               
                 Bioefficacy of E4 and E6 fractions against 
               
               
                 lesser grain borer,  Rhozopertha dominica   
               
             
          
           
               
                   
                 100 ppm 
                 50 ppm 
                 25 ppm 
                 12.5 ppm 
               
             
          
           
               
                   
                 % 
                 Avg. No. 
                 % 
                 Avg. No. 
                 % 
                 Avg. No. 
                 % 
                 Avg. No. 
               
               
                 Fraction 
                 mort. 
                 emerged 
                 mort. 
                 emerged 
                 mort. 
                 emerged 
                 mort. 
                 emerged 
               
               
                   
               
             
          
           
               
                 E4-A 
                 98.3 
                 0.026 
                 98.3 
                 0.143 
                 98.3 
                 0.184 
                 98.3 
                 0.186 
               
               
                 E4-B 
                 98.3 
                 0.049 
                 91.6 
                 0.084 
                 91.6 
                 0.136 
                 88.3 
                 0.198 
               
               
                 E4-C 
                 95.0 
                 0.049 
                 83.3 
                 0.14 
                 83.3 
                 0.107 
                 78.3 
                 0.274 
               
               
                 E4-D 
                 96.0 
                 0.109 
                 91.6 
                 0.176 
                 91.6 
                 0.50 
                 88.3 
                 0.319 
               
               
                 E4-E 
                 96.6 
                 0.046 
                 88.3 
                 0.164 
                 88.3 
                 0.249 
                 81.6 
                 0.219 
               
               
                 E6-A 
                 98.3 
                 0.029 
                 95 
                 0.089 
                 73.3 
                 0.042 
                 66.6 
                 0.084 
               
               
                 E6-B 
                 91.6 
                 0.014 
                 91.6 
                 0.044 
                 90.0 
                 0.048 
                 90.0 
                 0.068 
               
               
                 E6-C 
                 96.6 
                 0.042 
                 90.0 
                 0.037 
                 88.3 
                 0.054 
                 78.3 
                 0.044 
               
               
                 E6-D 
                 96.6 
                 0.039 
                 95.0 
                 0.016 
                 85.0 
                 0.075 
                 85.0 
                 0.168 
               
               
                 Control 
                 10.00 
                 0.107 
                 10.00 
                 0.107 
                 10.00 
                 0.107 
                 10.00 
                 0.107 
               
               
                   
               
             
          
         
       
     
       Example 5 
       [0114]    Extract S4 as defines above was extracted in petroleum ether and a fat fraction S5 [S4-A] and defatted fraction S6 [S4-B] were obtained. The S6 fraction was partitioned in n-butanol: water to obtain n-butanol soluble S7[S4-B1] and water soluble S8 [S4-B2] fractions. 
         [0115]    Extract T1 was extracted in petroleum ether and a fat fraction T1-A and defatted fraction T1-B were obtained. The T1-B fraction was partitioned in n-butanol: water to obtain n-butanol soluble T1-B1 and water soluble T1-B2 fractions. 
         [0116]    The three fractions so obtained were assayed at 1000 ppm on wheat against  S. oryzae  and  R. dominica  and presented in Table 10 and Table 11. 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 10 
               
             
             
               
                   
               
               
                 Bioefficacy of Fractions of S4 and T1 tested against rice weevil 
               
               
                 
                   Sitophilus oryzae 
                 
               
             
          
           
               
                   
                 % Mortality 
                 No. 
                 Progeny/adult 
               
               
                 Treatment 
                 (at week) 
                 F 1  Progeny 
                 day 
               
               
                   
               
             
          
           
               
                 Control (Acetone) 
                 00.00 
                 96 
                 0.33 
               
               
                 S5 (S4-A) 
                 49.00 3   
                 29 
                 0.12 
               
               
                 S6 (S4-B) 
                   100 2   
                 00 
                 0.00 
               
               
                 Control (Methanol) 
                 00.00 
                 138 
                 0.43 
               
               
                 S7 (S4-B1) 
                   100 2   
                 01 
                 0.02 
               
               
                 (T1-B1) 
                   100 1   
                 00 
                 0.00 
               
               
                 Control (Water) 
                 00.00 
                 185 
                 0.57 
               
               
                 S8 (S4-B2) 
                 83.00 3   
                 12 
                 0.066 
               
               
                 (T1-B2) 
                 46.00 3   
                 51 
                 0.19 
               
               
                   
               
               
                   1, 2 and 3 Mortality obtained after 1, 2 and 3 weeks respectively. 
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 11 
               
             
             
               
                   
               
               
                 Bioefficacy of fractions of S4 and T1 against lesser grain borer, 
               
               
                 
                   Rhizopertha dominica 
                 
               
             
          
           
               
                   
                   
                 No. 
                   
               
               
                   
                 % Mortality 
                 F 1  Progeny 
                 Progeny/adult 
               
               
                 Treatment 
                 (at week) 
                 (Avg) 
                 day 
               
               
                   
               
             
          
           
               
                 Control (Acetone) 
                 9.99 
                 138 
                 0.406 
               
               
                 S4-A 
                 44.31 
                 6.5 
                 0.03 
               
               
                 * S4-B 
                 — 
                 — 
                 — 
               
               
                 Control (Methanol) 
                 20 
                 137.5 
                 0.49 
               
               
                 S4-B1 
                 92.85 
                 02.0 
                 0.016 
               
               
                 T1-B1 
                 100 
                 00.0 
                 0.00 
               
               
                 Control (Water) 
                 13.33 
                 172.5 
                 0.585 
               
               
                 S4-B2 
                 27.07 
                 09.0 
                 0.034 
               
               
                 T1-B2 
                 35.53 
                 17.75 
                 0.073 
               
               
                   
               
             
          
         
       
     
         [0117]    These fractions exhibited 100% mortality and very significantly reduced Progeny/adult day. 
       Example 6 
       [0118]    The active fraction S7 (S4-B1) was formulated into a dust formulation. 50 gm of the active fraction was mixed with 900 gm of precipitated silica. To this mixture, 50 gram of diluent, in this case, china clay, was added. The mixture was thoroughly mixed until all of the active fraction was absorbed into the additives to give a free flowing powder without caking to form lumps to provide 5% Dust Formulation (5D). 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 12 
               
             
             
               
                   
               
               
                 Bioefficacy of dust formulation of active extract on wheat and green gram. 
               
             
          
           
               
                 Treatment 
                 % mortality of test insects 
                 Progeny/adult day 
               
             
          
           
               
                 (ppm) 
                 
                   S. oryzae 
                 
                 
                   R. dominica 
                 
                 
                   C. chinensis 
                 
                 
                   S. oryzae 
                 
                 
                   R. dominica 
                 
                 
                   C. chinensis 
                 
               
               
                   
               
             
          
           
               
                 0.00 
                 0.00 
                 03.33 
                 29.99 
                 0.58 
                 0.45 
                 01.41 
               
               
                 200 
                 100 
                 100 
                 100 
                 00.00 
                 00.00 
                 00.00 
               
               
                 100 
                 100 
                 100 
                 100 
                 00.09 
                 00.08 
                 00.05 
               
               
                 50 
                 100 
                 100 
                 48.32 
                 00.19 
                 00.08 
                 00.36 
               
               
                   
               
             
          
         
       
     
         [0119]    Conclusion: The dust formulation was toxic to the test insects and reduced the progeny 
         [0120]    of  S. oryzae  and  R. dominica  by &gt;80% and that of  C. chinensis  by &gt;90%. The insecticidal activity of the fraction so obtained from the stem of  N. foetida  according to the invention is due to the presence of more than one insecticidal component. Thus the chances on insect pests developing resistance are remote as it is exhibiting additive effect due to multiple active components, 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
             
           
               
                 TABLE 13 
               
             
             
               
                   
               
               
                 Toxicity of Bio-pesticide formulation 5D 
               
             
          
           
               
                   
                 LD 50   
                   
               
               
                 Criteria 
                 mg/kg 
                 Comment 
               
               
                   
               
             
          
           
               
                 Acute oral toxicity study in Swiss 
                 &gt;5000 
                 GHS (Globally 
               
               
                 albino mouse 
                   
                 Harmonized System) 
               
               
                   
                   
                 Category: Unclassified 
               
               
                 Acute oral toxicity study in rat 
                 &gt;5000 
                 GHS Category: Unclassified 
               
               
                 Acute dermal toxicity to rat 
                 &gt;2000 
                 NA 
               
               
                 Primary skin irritation study in rabbit 
                 — 
                 Non irritant to skin 
               
               
                 Mucous membrane irritation study in 
                   
                 Non irritant to mucous 
               
               
                 rabbit 
                   
                 membrane 
               
               
                   
               
             
          
         
       
     
       Example 7 
       [0121]    To study contribution of compounds other than camptothecin (CPT) towards insecticidal active, from active product (145 g, S4B1) camptothecin was removed by column chromatography (CC) and rest of the product was separated into 26 broad fractions (D1-D26) and assayed for their insect control activity. Fraction D4 exhibited toxicity to adults of the rice weevil,  S. oryzae . Since no other fraction exhibited toxicity to both the test insect species, it appears that the insect toxicity exhibited by the extract S4B1 may be the result of combined effect of activities of the compounds in the extract. 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 14 
               
             
             
               
                   
               
               
                 Bio-efficacy of D4. 
               
             
          
           
               
                   
                 
                   Sitophilus oryzae 
                 
                 
                   Rhizopertha dominica 
                 
               
             
          
           
               
                   
                 % 
                   
                 Progeny/ 
                 % 
                   
                 Progeny/ 
               
               
                 Sample No 
                 mor- 
                 Av 
                 adult 
                 mor- 
                 Av. 
                 adult 
               
               
                 D4 
                 tality 
                 progeny 
                 days 
                 tality 
                 Progeny 
                 days 
               
               
                   
               
             
          
           
               
                 Control 
                 19.99 
                 129.5 
                 0.441 
                 1.66 
                 161 
                 0.513 
               
               
                 12.5 ppm     
                 54.96 
                 118.5 
                 0.459 
                 4.99 
                 141 
                 0.454 
               
               
                 25 ppm 
                 78.3 
                 106.25 
                 0.460 
                 23.33 
                 80.75 
                 0.286 
               
               
                 50 ppm 
                 59.99 
                 105.25 
                 0.431 
                 3.33 
                 97.25 
                 0.316 
               
               
                 100 ppm  
                 26.66 
                 104.75 
                 0.373 
                 1.66 
                 77.5 
                 0.247 
               
               
                   
               
               
                 Values are averages of four replicates. 
               
             
          
         
       
     
         [0122]    Fractions D18, D21, D24 &amp; D25 were very active in reducing the F1 progeny (IGR activity). Adjacent fractions of D17, D22 &amp; D26 exhibited reduced activity indicating reduced titres of the active compounds. However none of these exhibited toxicity to the test insects. 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 15 
               
             
             
               
                   
               
               
                 Fractions exhibiting IGR activity in the test insects. 
               
             
          
           
               
                   
                 
                   Sitophilus oryzae 
                 
                 
                   Rhizopertha dominica 
                 
               
             
          
           
               
                   
                 % 
                   
                 Progeny/ 
                 % 
                   
                 Progeny/ 
               
               
                   
                 mor- 
                 Av 
                 adult 
                 mor- 
                 Av. 
                 adult 
               
               
                 Sample No 
                 tality 
                 progeny 
                 days 
                 tality 
                 Progeny 
                 days 
               
               
                   
               
             
          
           
               
                 D18 
               
             
          
           
               
                 Control 
                 0.0 
                 211.5 
                 0.663 
                 3.33 
                 129 
                 0.421 
               
               
                 12.5 ppm     
                 1.66 
                 195.5 
                 0.577 
                 3.33 
                 93.25 
                 0.275 
               
               
                 25 ppm 
                 1.66 
                 216.5 
                 0.632 
                 3.33 
                 82.75 
                 0.244 
               
               
                 50 ppm 
                 0.0 
                 147 
                 0.426 
                 10 
                 20 
                 0.064 
               
               
                 100 ppm  
                 1.66 
                 92.25 
                 0.344 
                 15 
                 6.5 
                 0.020 
               
             
          
           
               
                 D-20 
               
             
          
           
               
                 Control 
                 01.66 
                 193.0 
                 0.615 
                 1.66 
                 144.5 
                 0.463 
               
               
                 12.5 ppm     
                 4.99 
                 244.25 
                 0.788 
                 0 
                 157.75 
                 0.500 
               
               
                 25 ppm 
                 3.33 
                 331.75 
                 1.064 
                 0 
                 116.0 
                 0.367 
               
               
                 50 ppm 
                 3.33 
                 292.0 
                 0.948 
                 1.66 
                 81.25 
                 0.258 
               
               
                 100 ppm  
                 1.66 
                 306.25 
                 0.977 
                 0 
                 25.5 
                 0.079 
               
             
          
           
               
                 D-21 
               
             
          
           
               
                 Control 
                 01.66 
                 193.0 
                 0.615 
                 1.66 
                 144.5 
                 0.463 
               
               
                 12.5 ppm     
                 3.33 
                 254 
                 0.814 
                 3.33 
                 23.5 
                 0.0747 
               
               
                 25 ppm 
                 0.0 
                 146.25 
                 0.464 
                 0 
                 19.25 
                 0.060 
               
               
                 50 ppm 
                 6.66 
                 118.5 
                 0.392 
                 1.66 
                 8.5 
                 0.0265 
               
               
                 100 ppm  
                 14.99 
                 64.25 
                 0.230 
                 1.66 
                 2.25 
                 0.0067 
               
             
          
           
               
                 D-22 
               
             
          
           
               
                 Control 
                 01.66 
                 193.0 
                 0.615 
                 1.66 
                 144.5 
                 0.463 
               
               
                 12.5 ppm     
                 0 
                 328 
                 1.040 
                 0 
                 97.5 
                 0.309 
               
               
                  25ppm 
                 8.33 
                 226 
                 0.746 
                 0 
                 64 
                 0.226 
               
               
                 50 ppm 
                 0 
                 252.25 
                 0.800 
                 0 
                 46.75 
                 0.139 
               
               
                 100 ppm  
                 1.66 
                 223.25 
                 0.712 
                 1.66 
                 37.25 
                 0.118 
               
             
          
           
               
                 D24 
               
             
          
           
               
                 Control 
                 0 
                 211.5 
                 0.663 
                 3.33 
                 129 
                 0.421 
               
               
                 12.5 ppm     
                 0 
                 151.5 
                 0.439 
                 10.0 
                 21.25 
                 0.062 
               
               
                 25 ppm 
                 1.66 
                 141.25 
                 0.411 
                 20.0 
                 16.5 
                 0.055 
               
               
                 50 ppm 
                 0 
                 145.5 
                 0.421 
                 3.33 
                 7.0 
                 0.020 
               
               
                 100 ppm  
                 3.33 
                 106.75 
                 0.320 
                 13.33 
                 3.75 
                 0.011 
               
             
          
           
               
                 D-25 
               
             
          
           
               
                 Control 
                 0 
                 261.25 
                 0.829 
                 0.0 
                 113.25 
                 0.359 
               
               
                 12.5 ppm     
                 1.66 
                 214.25 
                 0.683 
                 0 
                 59.5 
                 0.060 
               
               
                 25 ppm 
                 1.66 
                 175.25 
                 0.559 
                 1.66 
                 19.0 
                 0.024 
               
               
                 50 ppm 
                 0 
                 125.0 
                 0.396 
                 1.66 
                 7.75 
                 0.024 
               
               
                 100 ppm  
                 1.66 
                 56.5 
                 0.180 
                 3.33 
                 5.0 
                 0.026 
               
             
          
           
               
                 D-26 
               
             
          
           
               
                 Control 
                 0 
                 261.25 
                 0.829 
                 0.0 
                 113.25 
                 0.359 
               
               
                 12.5 ppm     
                 0 
                 244.5 
                 0.776 
                 3.33 
                 105.75 
                 0.341 
               
               
                 25 ppm 
                 0 
                 219.25 
                 0.696 
                 3.33 
                 92.5 
                 0.301 
               
               
                 50 ppm 
                 1.66 
                 217.5 
                 0.694 
                 3.33 
                 95.25 
                 0.308 
               
               
                 100 ppm  
                 0 
                 208.7 
                 0.662 
                 0 
                 95.25 
                 0.188 
               
               
                   
               
               
                 Values are averages of 4 replicates. 
               
             
          
         
       
     
         [0123]    From D4 (fraction with insecticidal activity) Pthalate was isolated. 
         [0124]    From D18, D21 (fraction exhibiting IGR activity) Pthalate was isolated. 
         [0125]    From D24 (fraction exhibiting IGR activity)—Campthothecanoid 1 was isolated. 
         [0126]    From D25 (fraction exhibiting IGR activity)—Campthothecanoids 2 and 3 were isolated. 
         [0127]    These compounds have not been individually assessed for their insecticidal or IGR activities. 
       Example 8 
     Isolation of Phthalate 
       [0128]    Fractions D18, D21 and D4 contained phthalate. Fraction D18 and D21 were both subjected separately to CC using successively MeCN: chloroform 2:8 and then gradient of methanol: chloroform from 2 to 10% to collect nine fractions. Fraction D4 was subjected to CC in gradient of MeCN: chloroform from 1 to 20% to collect fourteen fractions. Fractions D18:2, D21:2 and D4:2 contained pure phthalate (1.1 g). 
       Isolation of Camptothecanoids 
       [0129]    Fraction D24:12 (78 mg) was separated by Medium Pressure Liquid Chromatography (MPLC) using gradient of methanol in chloroform from 3 to 25% followed by methanol wash to collect twenty six fractions. Combined fractions 14 to 16 were subjected to preparative TLC in 15% methanol: chloroform and then 20% methanol: chloroform to isolate campptothecanoid 1 (10.2 mg). 
         [0130]    Fraction D25:4-5 (180 mg) was separated by MPLC using gradient of methanol in chloroform from 0 to 25% followed by methanol wash to collect twenty two fractions. Fractions 3, 4, 5, 12 and 13 were combined (100 mg) and subjected to CC in gradient of methanol in chloroform from 5 to 25% followed by methanol wash to collect sixteen fractions. Fractions 10-11 and 12-15 contained camptothecanoids 2 and 3. They were separated and purified by preparative TLC in 20% methanol: chloroform to isolate camptothecanoids 2 and 3. 
         [0131]    Fraction 14-17 (60 mg) from D25:4-5 contained camptothecanoid 2 which was subjected to CC in gradient of methanol in chloroform from 5 to 25% followed by methanol wash to collect fifteen fractions. From fractions 11-12 camptothecanoid 2, which was isolated by preparative TLC in 15% methanol: chloroform. Fractions 13 and 14-15 contained camptothecanoids 2 and 3. They were separated and purified by preparative TLC in 20% methanol: chloroform to isolate camptothecanoids 2 and 3. Fraction 18-21 from D25:4-5 contained camptothecanoid 3 which was purified by preparative TLC in 20% methanol: chloroform. In total 80 mg and 60 mg of camptothecanoid 2 and 3 were isolated. 
       Example 9 
       [0132]      
         [0000]    
       
         
               
             
               
               
               
             
               
               
             
               
               
               
             
           
               
                 TABLE 16 
               
             
             
               
                   
               
               
                 Bioassay of Camptothecanoid 1, 2 and 3 and Phthalate: 
               
             
          
           
               
                   
                 XTT assay  Mycobacterium   
                 MTT assay against  
               
               
                   
                   tuberculosis  Ra 
                 THP 1 cell line 
               
             
          
           
               
                 Compound* 
                 % Inhibition 
               
               
                   
               
             
          
           
               
                 Camptothecanoid 1 
                 48.32 
                 70.87 
               
               
                 Camptothecanoid 2 
                 88.42 
                 87.48 
               
               
                 Camptothecanoid 3 
                 58.96 
                 90.20 
               
               
                 Phthalate 
                 45.61 
                 20.15 
               
               
                 Paclitaxel 
                 — 
                 61.00 
               
               
                 Isoniazid 
                 0.05** 
                 — 
               
               
                   
               
               
                 *inhibition at 100 μg/ml, 
               
               
                 **IC 90  in μg/m1 
               
             
          
         
       
     
       Example 10 
     Anti-Mycobacterial Activity 
       [0133]      M. tuberculosis  H37Ra (ATCC 25177) cells, obtained from MTCC (Chandigarh, India), were grown to logarithmic phase (O.D, 0.595-1.0) in a defined medium ( M. pheli  medium) under aerobic conditions in a shaker incubator (Thermo Electron Corporation Model 481) maintained at 150 rpm and 37° C. After growth, the culture was sonicated for 2 min using water bath sonicator. Sonicated cells were used for inoculation in micro plate wells. 250 l of culture containing ˜1×10 5  cells/ml was added to each well of 96 well plates. Camptothecanoid 1, 2 and 3 and Phthalate, 2.5 l, dissolved in DMSO (Sigma), were added to the wells to attain a final concentration of 100 g/ml respectively for screening. Then, the plate was incubated in a CO 2  incubator at 37° C. The plate was taken out on the 8th day of incubation to measure the viable cell counts. The optical density of the culture was measured before addition of XTT (Sigma) at 470 nm which was served as a blank for the MIC calculations. XTT, 200 M, was added and incubated for 20 min at 37° C. after shaking for 1 min. After 20 min of incubation, 60 M Menadione (Sigma) was added and incubated at 37° C. for 40 min after mixing of 1 min. Finally, the optical density of the suspension was measured at 470 nm by using microplate reader. Isoniazid (Sigma) was used as a positive control. 
       Example 11 
     Anti Proliferative Activity-MTT Cell Proliferation Assay on Human Thp-1 Cell Line 
       [0134]    Camptothecanoid 1, 2 and 3 and Phthalate were tested for their inhibitory effect on THP-1 cells. About 10,000 cells were taken per well in 96-well tissue culture plates and treated with test samples at 100 μg/ml for 72 h. Vehicle control (DMSO, 1%) and positive control (Paclitaxel, 100 μg/ml) was run simultaneously. Cell proliferation was assessed with 10 μl from 5 mg/ml stock solution of tetrazolium salt (MTT) dissolved in cell culture medium and subsequently incubated for additional 1 h at 37° C., 5% of CO 2  and 95% humidity in incubator. The violet coloured formazan crystals formed were solubilized in 200 μl of isopropanol and incubated for another 4 h. The optical density was read on a micro plate reader (Spectramax plus384 plate reader, Molecular Devices Inc) at 490 nm filter against a blank prepared from cell-free wells. Absorbance given by cells treated with the carrier DMSO alone was taken as 100% cell growth (Table 2). 
       Example 12 
     Activity Data of Fractions Containing Compounds of General Formula 1 and Formula 2 
       [0135]    Fractions NCL49-D24 and NCL49-D25 containing camptothecanoids 2 and 3 displayed potent insect regulatory activity against  Rhizopertha dominica  and good activity against  Sitophilus oryzae.    
         [0136]    Similarly fraction NCL49-D21 containing phthalate displayed potent insect regulatory activity against  Rhizopertha dominica  and good activity against  Sitophilus oryzae . 
         [0000]    
       
         
               
               
               
             
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
             
           
               
                   
                 TABLE 17 
               
             
             
               
                   
                   
               
               
                   
                 
                   Sitophilus oryzae 
                 
                 
                   Rhizopertha dominica 
                 
               
             
          
           
               
                   
                 % 
                   
                 Progeny/ 
                 % 
                   
                 Progeny/ 
               
               
                 Sample 
                 mor- 
                 Av 
                 adult 
                 mor- 
                 Av. 
                 adult 
               
               
                 Code No 
                 tality 
                 progeny 
                 days 
                 tality 
                 Progeny 
                 days 
               
               
                   
               
             
          
           
               
                 Pthalate (D-21) 
               
             
          
           
               
                 Control 
                 01.66 
                 193.0 
                 0.615 
                 1.66 
                 144.5 
                 0.463 
               
               
                 12.5 ppm     
                 3.33 
                 254 
                 0.814 
                 3.33 
                 23.5 
                 0.0747 
               
               
                 25 ppm 
                 0.0 
                 146.25 
                 0.464 
                 0 
                 19.25 
                 0.060 
               
               
                 50 ppm 
                 6.66 
                 118.5 
                 0.392 
                 1.66 
                 8.5 
                 0.0265 
               
               
                 100 ppm  
                 14.99 
                 64.25 
                 0.230 
                 1.66 
                 2.25 
                 0.0067 
               
             
          
           
               
                 Camptothecanoids - 1 (D24) 
               
             
          
           
               
                 Control 
                 0 
                 211.5 
                 0.663 
                 3.33 
                 129 
                 0.421 
               
               
                 12.5 ppm     
                 0 
                 151.5 
                 0.439 
                 10.0 
                 21.25 
                 0.062 
               
               
                 25 ppm 
                 1.66 
                 141.25 
                 0.411 
                 20.0 
                 16.5 
                 0.055 
               
               
                 50 ppm 
                 0 
                 145.5 
                 0.421 
                 3.33 
                 7.0 
                 0.020 
               
               
                 100 ppm  
                 3.33 
                 106.75 
                 0.320 
                 13.33 
                 3.75 
                 0.011 
               
             
          
           
               
                 Camptothecanoid - 2 &amp; 3 (D-25) 
               
             
          
           
               
                 Control 
                 0 
                 261.25 
                 0.829 
                 0.0 
                 113.25 
                 0.359 
               
               
                 12.5 ppm     
                 1.66 
                 214.25 
                 0.683 
                 0 
                 59.5 
                 0.060 
               
               
                 25 ppm 
                 1.66 
                 175.25 
                 0.559 
                 1.66 
                 19.0 
                 0.024 
               
               
                 50 ppm 
                 0 
                 125.0 
                 0.396 
                 1.66 
                 7.75 
                 0.024 
               
               
                 100 ppm  
                 1.66 
                 56.5 
                 0.180 
                 3.33 
                 5.0 
                 0.026 
               
               
                   
               
               
                 Values are averages of 4 replicates. 
               
             
          
         
       
     
       Advantages of the Invention 
       [0137]    The insecticidal enriched extract (biopesticide) prepared from the stem of  Nothapodites foetida  (Wight) Sleumer (formerly  Mappia foetida  (Miers) is
       useful for the insect free storage and transport of grains and seed.   non-toxic to non-target organisms.   biodegradable and hence has good safety profile.   has multiple active ingredients and hence has less prone for the development of resistance.   acts with multiple mechanisms (insecticidal, growth inhibition) and hence less prone for the development of resistance.   insecticidal activity is ascribed to compound/s other than camptothecin an anticancer drug.