Title of Invention


Abstract A method of preparing a botanical pesticide from seeds of Annona squamosa characterized in that (i) defatting seed powder of A. squamosa with defatting solvent(s); (ii) treating the residue cake obtained in step (i) with a polar solvent; (iii) filtering the extract of step (ii); (iv) concentrating the filtered extract of step (iii); (v) partitioning the concentrated extract of step (iv) with a suitable extracting solvent; (vi) adding suitable dehydrating agent in extract of step (v); (vii) completely drying the extract of step (vi); (viii) dissolving the residue of step (vii) in a suitable extracting solvent; (ix) further defatting extract of step (viii) with defatting solvent; (x) drying completely/partially the extract of step (ix); (xi) dissolving the residue of step (x) in a suitable polar solvent and (xii) purifying by passing through a column as described herein before.
Full Text Original
(39 of 1970)
(See section 10; rule 13)

NORTH MAHARASHTRA UNIVERSITY P.B. No. 80, Umavinagar, Jalgaon-425001 (MS) Indian University
The following specification particularly describes the nature of this invention and the manner in which it is to be performed.
13 JUL 2004

The present invention is related to a botanical pesticide obtained from seeds of Annona squamosa. It mainly involved serial solvent extraction of seeds, efficacy of this extract on primary post-harvest pest of pulses, Callosobruchus chinensis and polyphagous pre-harvest pest, Helicoverpa armigera.
BACKGROUND OF THE INVENTION Pulses are important and economical sources of proteins in west African and south Asian diets. Due to vegetarian dietary habit of majority of Indian population pulses are mainly consumed as a source of proteins. Accordingly, India is the largest producer of pulses accounting for 27-28% of the global production. Still, India has to import pulses due to (i) ever increasing population, (ii) less production per hectare, (iii) stagnation in production since last four decades (in the range of 12-14 MMT since 1960 to 2000) and (iv) prone to heavy pre- and post-harvest losses due to various insect pests. Pre- harvest losses are due to insects, weeds or diseases afflicting the crop when it is still in the field. Insects belonging to 150 different species are pests of 10 different crops of pulses. Cutworms, caterpillars, aphids and fly maggots act as leaf miners, stem borers and pod borers causing damage at various stage of crop's life. The gram pod borer (H. armigera), also called as the American cotton boll worm, has a polyphagous feeding habit. It is a serious pest of 181 plant species, including chickpea, (Cicer arietinum), pigeon pea (Cajanus cajan), tomato (Lycopersicon esculentum), okra (Abelmoschus esculentus) and cotton (Gossypium species). The larval stage is the most devastating, extending for about two weeks after which it enters the soil and pupates for one week to

emerge as a moth. Adults lay eggs on the tender parts of the plant from which the larvae hatch out. The feeding of the larvae either results in direct destruction of the flower or a reduction of fiber content in the boll. Chickpea is the third most important crop of the world, providing high quality protein in a vegetarian diet, used as a feed for livestock and mainly contributes to soil nitrogen (Mendki P.S., 2002. Ph.D. Thesis, North Maharashtra University, Jalgaon (MS) India). India alone produces 75% of the total worlds supply of chickpea. This crop is prone to heavy infestation by H. armigera. A single larva of H. armigera damages several chickpea pods per day leading to severe losses in crop yield (Harsulkar, A.M. Giri, A.P., Patankar, A.G., Gupta, V.S., Sainani, M.N., Ranjekar, P.K. and Deshpande, V.S. 1999. Plant Physiol, 121, 1-10).
During storage, seeds of nearly all legumes are vulnerable to attack by seed beetles, both in the field and in storage. Common insect pests of stored pulses are Acanthoscelides obtectus, Callosobruchus chinensis, C. maculatus, C. analis, Sitophilus oryzae and Zabrotes subfasciatus. Pulse beetle C. chinensis, a primary pest of pulses during storage, causes substantial economic losses. It is a notorious pest of green gram, Bengal gram and red gram. Severe infestation reduces quality and germinability to under 20 % after four months only and as much as 90% loss within 6 months of storage. This pest causes maximum damage during February to August with the larvae feeding inside the grain to consume its entire endosperm. Infested grains develop fungal infections and as a result emit foul odour.
Various chemical pesticides like synthetic pyrethroids are being used for controlling pre-harvest pod-borers and post-harvest beetles. Additionally, fumigants like aluminium phosphide and methyl bromide are also being used for combating the attack of various

stored grain pests. As many as 30% losses are caused by these insect pests. Of which, Lepidopteran insect pests (pre-harvest) cause about 60% losses and Coleopteran insect pests (post-harvest) are responsible for remaining 40% losses. Chemical pesticides, no doubt, have played the major role as a safeguard against different insect pests in green revolution but their non-judicious use over a long period has caused several drawbacks like (i) non-permissible levels of their residues in soil and water due to their non¬biodegradable nature, (ii) development of resistance by various insect pests like pod borer and red flour beetle due to continuous exposure and (iii) resurgence of new pests. To overcome these drawbacks, certain alternative strategies are urgently required to combat the attack of these insect pests at different stages of crop produce and storage. In this category, biopesticides, derived from microorganisms (viruses, bacteria and fungi) and plants emerged as the best possible option. Over 2000 plant species belonging to some 60 plant families are known to exhibit insecticidal activities. Naturally occurring plant materials or products derived from such plant materials are termed as botanical insecticides. Botanical insecticides were extensively used (1800 to 1940) until the discovery of synthetic insecticide DDT. They may be crude preparations of plant parts such as powders or dusts (neem leaf dust, pyrethrum flower dusts etc.), aqueous or organic solvent extracts, used as liquid concentrates or as dusts with carriers (Weinzierl, R.A. 1998.Biological and biotechnological control of insect pests (Rechcigl, J.E. and Rechcigl, N. A. (Eds.) 101-123).
The potential of several secondary metabolites in plants to disrupt specific physiological mechanism involved in nutrition, reproduction, metamorphosis and behavior of insects could be exploited in the control of insect pests as an eco-friendly alternative to the

conventional pesticide use. In this study, we have specifically investigated Annona squamosa L. (Family: Annonaceae), an evergreen plant, native of West Indies and also found in tropical and subtropical Asia and Africa for controlling the attack of pre- and post-harvest insect pests of pulses.
Family Annonaceae consists approximately of 119 species, of which, 7 species including A. cherimola (cherimoya), A. muricata (soursop) and A. squamosa (sweetsop, custard apple) are grown for domestic/commercial purposes. A. squamosa is a small partially deciduous tree up to 6 m in height. It has 15-20 cm long leathery, soft, oval leaves. The flowers are approximately 2 cm long, greenish and fleshy. The fruit is subspherical, grayish beige, up to 10 cm in diameter containing 20-28 or more, black seeds surrounded by white, sweet and juicy flesh. The fruits are a good source of carbohydrates, vitamin C and minerals such as calcium, phosphorus and potassium.
The average life of the tree is about 15 years with a fruit yield of about 50-100 kg/tree. Annona is easy to cultivate, requires comparatively little care and does not suffer from serious pests and diseases. The fruits are consumed fresh in India, China and Thailand but when processed they can be used to prepare drinks, fermented liquors and ice-creams. The seed kernels contain non-drying oil (14-49 %) with a saponification index of 186.40 and is a proposed substitute for peanut oil in the manufacturing of soap. It is known to possess excellent insecticidal activity too.
The leaves of A. squamosa are effective against filarial vector mosquito, Culex quinquefasciatus. The leaves yield oil rich in terpenes and sesquiterpenes which is mainly used in perfumes. Chemical compounds including flavonoids, alkaloids and acetogenins have been extracted from various parts of the tree. These secondary metabolites of

Annona have been reported to possess insecticidal and antimicrobial properties. Chemical constituents of the bark, root, seeds and stems include aporphine alkaloids (anonaine, norcorydine, corydine, glaucine etc.). Annonaceous acetogenins having a single terra hydrofuran ring and adjacent bis tetrahydrofuran moiety isolated from the seeds of Annona squamosa showed insecticidal activities (Chinese Patent CN 1,083,060). Japanese patent (JP 10,101,517) describes ether extracts of seeds of A. squamosa as moth proofing agents for fiber products against Tinia pellionella and Antherenus nerbasci. US Patent. (US 2003/0050336 Al) describes a novel compound, isosquamocin obtained from this plant and the insecticidal composition. It is reported that out of the different seed extracts of A. squamosa, only the oily portion extracted with organic solvents possessed appreciable activity against certain stored grain pests. The prior art thus describes that (i) solvent extracts of seeds and leaves of Annona squamosa possesses important insecticidal activities against a number of agriculturally significant insect pests and (ii) various solvent extracts yielded annonin/squamocin as active principle(s). In the light of the above facts, our objective was to explore the use of Annona squamosa, particularly its seeds, for pre- and post-harvest preservation of pulses.
SUMMARY OF THE INVENTION Accordingly, a biopesticide for controlling pre- and post-harvest targeted insect pests of pulses, viz. H. armigera and C. chinensis has been prepared. It comprises a serial solvent seed extract of Annona squamosa. The biopesticide exhibited promising contact/touch, oviposition deterrent and ovicidal activities against C. chinensis. At the same time it has exhibited feeding deterrent, ovicidal and insect growth regulatory activity against H. armigera.

Fig. 1 represents the ovicidal activity of the biopesticide of A. squamosa against H. armigera.
Serial solvent extraction of seeds was carried out by the following method (a) Step 1:
Air-dried powdered seeds (lOOg) of A. squamosa were defatted (4 x 0.1 L) with defatting solvent(s) and filtered. The solvent washings were collected, combined and vacuum evaporated to separate oil. (b) Step 2: The residue cake was extracted in a polar solvent, (c) Step 3: The polar solvent was filtered. The residue cake was discarded, (d) Step 4: The polar solvent layer was evaporated to 50 ml (Buchi, Switzerland consisting of a Rotavapor R 124, a vacuum pump, Vac V-500 and a water bath B-480). (e) Step 5: The polar solvent layer was partitioned with a suitable extracting solvent (4 x 50 ml), (f) Step 6: The polar layer was discarded while extracting solvent was dried on sodium sulphate, (g) Step 7: Extracting solvent was evaporated to dryness, (h) Step 8: The residue, thus obtained, was dissolved in yet another extracting solvent, (i) Step 9: Further partitioned with defatting solvent, (j) Step 10: The extracting solvent was again evaporated to dryness, (k) Step 11: The residue was finally dissolved in a polar solvent referred to as biopesticide and used for further studies. (1) Step 12: The extract thus obtained was also subjected to column cleanup to selectively retain the compound(s) of interest and remove other interfering plant pigments. The sintered column (15 cm x 0.8 cm i.d) consisted of florisil (5 cm) at the bottom and anhydrous sodium sulphate (1 cm) at the top. The column was preconditioned with ethyl acetate (10 ml), which was then loaded with 1 ml of the crude extract and eluted with 15 ml ethyl acetate. This elute, containing the

probable active principle(s), was evaporated to dryness and the residue was dissolved in
Statistical analysis of all the data obtained was accomplished with an analysis of variance
(ANOVA) using Indostat software. It should be clearly understood that the methods and
compositions referred to herein are illustrative only and are not intended to have any
limitations on the scope of the invention.
EXAMPLES EXAMPLE 1 Rearing conditions of Callosobruchus chinensis and Helicoverpa armigera Uninfested and untreated healthy green gram (Vigna radiata L.) Wilczek were procured from the local market just after harvest. Culture of Callosobruchus chinensis was identified by an expert entomologist and subsequently grown on these pulses in the laboratory throughout the experimental studies. Rearing of insects was done with a standardized procedure using food grade plastic jars (Sunpet, India) having 50 g of green gram as a nutritional source, infested with 15 pairs of insects in each jar. Average daily temperature was 30±2°C and relative humidity was 60-70%. Helicoverpa armigera larvae were reared on a basal or supplemental diet or collected from chickpea or cotton fields.
Insecticidal activity against post-harvest beetle, C. chinensis
To demonstrate insecticidal activity against C. chinensis, biopesticide at different doses
were sprayed on a Petri plate preloaded with a Whatman filter paper no.l followed by
addition of 25 adult C. chinensis. The plates were kept at room temperature and the effect

of seed extract on pulse beetle was monitored every 24h for 96h. Each set was run in
triplicate and repeated three times.
In the direct contact application method (Table 1), almost 30% and 80% insects were
found dead after 24 h of exposure at lowest (2 mg/ml) and highest (10 mg/ml) doses,
respectively. Besides moderate mortality, after 24 h at lower doses, severely impaired
mobility in insects was also observed which within next 120 h were found dead.
Further, it was noted that, at the dose of more than 2 mg/ml, adult longevity was also
severely affected as C. chinensis, which remained ataxic were found to be dead within
next 120 h. There was no adverse effect on solvent and master control insects. In doses of
biopesticide ranging from 6-10 mg/ml, all the insects showed severely impaired mobility
within few hours of exposure, followed by paralysis and death. In insects these are the
results of nerve poisoning. Our results suggest that the seed extract has contact poison
effect on C. chinensis. No mortality was observed in both, solvent as well as master
control sets.
Table 1
Mortality profile of C. chinensis by direct contact application method with various doses of biopesticide from Annona squamosa

Treatments (mg/ml) Adult mortality of C. chinensis (% )
24 h 48 h 72 h 96 h
Master control 0.00 0.00 0.00 0.00
Solvent control 0.00 0.00 0.00 0.00
2 29.33 ±1.71 45.32 ± 1.65 48.00 ±1.00 69.32 ±1.62
4 40.00 ± 3.00 42.66 ±1.74 46.66 ±1.68 65.33 ±3.45
6 46.66 ±1.68 58.66 ±1.77 66.64 ±1.68 77.33 ± 1.70
8 66.65 ±1.71 77.32 ±3.45 78.66 ±1.71 94.64 ±4.56
10 81.33 ±1.57 92.00 ± 3.00 97.33 ±1.74 98.66 ± 1.74
SE 1.05 0.99 1.08 1.23
SED 1.47 1.44 1.04 1.77
CD 3.09 2.97 3.21 3.69
Insects (25) were placed in Petri plates containing filter papers treated with various concentrations of seed extract. Solvent control (alcohol) and master control (distilled water) were applied at the rate of I ml. All the sets were run in triplicate with 25 insects taken in each replicate and repeated thrice. S.E.= Standard Error Difference, C.D.= Critical Difference (P=0.05) Statistical analysis was done on an Indostat software by ANOVA.

Application of biopesticide on green gram to evaluate oviposition deterrent and
ovicidal effect on C. chinensis
Biopesticide was applied on green gram to demonstrate oviposition deterrent activity
against the post harvest pest of pulses, C. chinensis. Various doses of seed extract ranging
from 2, 4, 6, 8 and 10 mg/ml were prepared to check oviposition deterrent activity. Green
gram (250 g) was added to 750 ml capacity jars. Doses of biopesticide (2 ml each) were
slowly applied on green gram in each jar by a micropipette with continuous shaking to
allow uniform coating on the grains. The jars were kept open for 6 h during which the
solvent was allowed to evaporate. Five pairs of C. chinensis were introduced to each jar.
Solvent control was also monitored simultaneously. After 48 h, all live/dead insects were
removed from the jars. Delay in insect emergence, number of live insects and number of
grains damaged in 25 g sample was monitored.
In this example, biopesticide was also applied on green gram to demonstrate ovicidal
activity. In 750 ml capacity food grade plastic jars, green gram (250 g each) was added.
Five pairs of insects were added in each jar and allowed to lay eggs for 48 h. All the
insects were removed after initial oviposition. Doses of 2, 4, 6, 8 and 10 mg/ml (2 ml)
were applied by the method described earlier on the previously infested grains.
Simultaneously, solvent control (only methanol) and master control (without any
treatment) were also run. All the jars were kept open for 6 h for evaporation of the
solvent. Different parameters like effect on size and shape of eggs, delay in insect
emergence, number of live insects and number of grains damaged in 25 g of sample was
then monitored. The set was carried out in triplicate and repeated thrice.

Table 2 depicts the results of actual application of biopesticide on green gram before and
after oviposition to demonstrate oviposition deterrent and ovicidal activities, respectively.
The oviposition and ovicidal activities were found to be dose dependent. This may be due
to the irritant effect of the seed extract, which might be causing depression of
reproductive activity.
Table 2
Mean number of grains damaged and adults emerged upon addition of biopesticide
before and after oviposition

Treatments (mg/ml) Application of seed extract before oviposition Application of seed extract after oviposition
Mean number of grains damaged Mean number of adults emerged Mean number of grains damaged Mean number of adults emerged
Master control 110 ±14.01 108 ±13.50 151 ±13.65 138.66 ±11.23
Solvent control 108 ±14.29 99.00 ±12.12 131.00 ±16.09 140.66 ±14.57
2 18.00 ±2.64 10.33 ±2.08 10.33 ±1.52 9.00 ±1.73
4 5.00 ±1.00 5.33 ±0.57 0.00 0.00
6 0.00 0.00 0.00 0.00
8 0.00 0.00 0.00 0.00
10 0.00 0.00 0.00 0.00
SE 3.63 3.28 3.84 3.40
SED 5.14 4.64 5.43 4.81
CD 10.67 9.62 11.27 9.98
aster control = No treatment, ral her distilled wat er was used.
Solvent control = alcohol. Both treatment as well as solvent control was applied at the rate of 2 ml and the sets were carried out in triplicate and repeated thrice. S.E. = Standard Error Difference, CD. = Critical Difference (P = 0.05). Statistical analysis was done on an Indostat software by ANOVA.
At higher doses C. chinensis could not lay eggs, but there was no effect on size and shape
of the eggs laid at lower doses as compared to control. The results are significant because
almost all the stored grain insects have a higher rate of multiplication. A single insect can

consume 35 mg of food during its metamorphosis from egg to adult (Rajendran, S. 1999. J. Food Sci. Technol, 36, 283-300). Therefore within one season of storage, they may destroy 10-15% grains, partially damage others and contaminate the rest with undesirable odours. Initial mortality started above 4 mg/ml and increased steadily upto 10 mg/ml, where almost all insects were found dead. No/less egg laying was observed from 10 mg/ml while the number of eggs in other treatments (2-4 mg/ml) and alcohol control were significant. These results suggested that the extract has oviposition deterrent activity. The data can be corroborated with results of the insecticidal assay. Further, when biopesticide (2-10 mg/ml) was applied on green gram after oviposition similar trend was observed (Table 2). Oviposition is affected by the roughness or wrinkling of the seed and hardness of the seed and its odour. In fact, the ovicidal activity was found to be stronger than oviposition deterrent activity as number of insects emerging at 2 mg/ml was found to be less in former than in latter (Table 2). While studying ovicidal activity, the hatching of eggs was also found to be dose dependent. It was noted that in treated sets eggs could not mature at higher doses compared with controls. There was also a delay in metamorphosis (from egg to adult) of C. chinensis at lower doses. On an average it takes about 28-30 days for complete metamorphosis while at lower doses it took 36-40 days for complete metamorphosis. Thus, promising contact/touch, oviposition deterrent and ovicidal activities of the biopesticide against post-harvest pulse beetle were obtained.

EXAMPLE 4 Diet contamination bioassay against H. armigera
The next important aspect was to control pre-harvest pest(s) of pulses. For this purpose (i) Helicoverpa armigera was identified as a target pest on the basis of its polyphagous and voracious feeding habit on economically important pulse crops and most importantly its development of resistance against certain synthetic/chemical pesticides and (ii) at the same range of doses more than 90% activity was obtained against pulse beetle therefore different bioassays against H. armigera were carried out.
The development of pest-resistant transgenic plants expressing genes of proteinase/amylase inhibitors and use of non-proteinaceous secondary metabolites of plant origin are now being explored as an alternative approach for combating the attack of H. armigera (Harsulkar, A.M., Giri, A.P., Gupta, V.S., Sainani, M.N., Deshpande, V.V., Patankar, A.G. and Ranjekar, P.K. 1998. Electrophoresis, 19, 1397-1402; Nakaguchi, T., Arakawa, T., Philo, J.S., Wen, J., Ishimoto, M. and Yamaguchi, H. 1997. J. Biochem., 121, 350-354; Koul, O., Shankar, J.S., Mehta, N., Taneja, S.C., Tripathi, A.K. and Dhar, K.L. 1997. J. Appl. Entomol, 121, 245-248)
Aqueous extract of Gnidia glauca Gilg. and Toddalia asiatica Lam. exhibited antifeedant activity while Ocimum sanctum, Lantana camara and Curcuma longa possess growth inhibitory activity against gram pod borer H. armigera. Turmeric rhizome extract exhibited ovicidal action against H. armigera (Chowdhury, H., Walia, S. and Saxena, V. 2000. Pest Management Science, 56, 1086-1092). Plant based pesticides do not lead to high selection pressure approach executed by synthetic pesticides. The consortium of non-proteinaceous secondary metabolites further reduces the chances of development of

resistance by insect pest (Kotkar, H.M., Mendki, P.S., Sadan, S., Jha, S.R., Upasani, S.M.
and Maheshwari, V.L. 2002. Pest Manag. Sci., 58, 33-37). A. squamosa biopesticide was
tested for its activity against the third instar larvae of H. armigera. To evaluate the
bioefficacy, various assays were employed viz. diet contamination method, leaf dip
method and ovicidal assay.
The invention on hand is illustrated by means of control and reference control (A. indica
extract) tests, which are set out in the following tables. These tests are laboratory tests,
for which the third instar larvae of H. armigera were fed on an artificial diet comprising
gram flour, yeast powder, ascorbic acid, sorbic acid, streptomycin sulphate, agar agar,
biopesticide (3 mg/g of dry weight) and water. The individual larva was allowed to feed
in small plastic cups containing moist filter papers to maintain moisture. The experiment
was carried out at 25 ± 2 C, relative humidity of 75 ± 2% and 16 : 8 LD photoperiod. The
larval growth was assessed as a function of weight gained by the individual larva. Larval
mortality, if any, was also recorded. The set was monitored until the emergence of adults.
Initial observations were noted at an interval of 24 h.
Table 3
Annona - induced growth inhibition and toxicity on third instar of//, armigera in
the diet contamination method
Treatment % Mortality (during ecdysis) % Adult emergence
A. squamosa 43.33 56.66
Control 333 96.66
Significant larval mortality and reduction in adult emergence of H. armigera was
obtained as compared with the control (without any treatment). Larvae fed on control diet
(without any treatment) showed normal growth and on diet containing serial solvent seed
extract showed stunted growth as a result of drastically reduced food intake. Thus

confirming feeding deterrent activity in the seed extract. Earlier results using the acetone
seed extract of A. squamosa have shown that it resembled the action of juvenile hormone
analogues as it caused developmental retardation, prolonged juvenile life and deformities
in the resultant adults of the red cotton bug Dysdercus koenigii (Bhagawan, C.N., Reddy,
K.D. Sukumar, K. 1992. Ind. J. Exptl. Biol. 30, 908-912). Though significant
morphological deformities were not observed in the adults of//, armigera treated with A
squamosa seed extract, adults were found to be dead after first few hours of emergence.
This may be probably the consequence of growth inhibitory action accompanied with
some covert morphogenetic effects. Similarly, 6 P hydroxy gedunin isolated from A.
indica and dichloroethane and methanol extracts of Melia dubia exhibited growth
inhibitory activity against H. armigera and S. litura (Koul, O., Jain, M., Sharma, V. 2000.
Ind J. Exptl. Biol. 38 (1): 63-68).
Leaf Dip Bioassay against H. armigera to demonstrate feeding deterrent activity of
the biopesticide
Further tests were carried out to demonstrate the feeding deterrent activity of the
biopesticide using the leaf dip bioassay. For this purpose, I) individual chickpea leaves
were weighed, ii) the third instar larvae of H. armigera were also weighed individually
after starving them for 6 h iii) allowed to feed on A. squamosa biopesticide and A. indica
extract treated chickpea leaves for 48 h and were transferred to fresh untreated leaves
until pupation. All the indices relating to consumption, digestion and utilization of leaves
were calculated. There was almost 3-fold reduction in the consumption of leaves by the
treated larvae. This decrease in the total food consumed by a single larva resulted in a
decrease in weight of the faeces voided. The number of feeding days was also reduced to

8.41 as compared with the control where the larvae continued to feed for 11.13 days. These results indicate that the seed extract of A. squamosa has a pronounced feeding deterrent activity on the larvae of H. armigera.
In a similar experiment, biopesticide was checked for its ovicidal (Sheet 1 of 1) effect on the eggs of H. armigera. Fresh eggs were treated with the biopesticide by an automizer and placed in a Petriplate pre loaded with moist filter paper to maintain humidity. Control sets did not receive any treatment. Each treatment was replicated thrice using 30 eggs per replication. Observations were recorded until egg hatching. A. squamosa treatment exhibited good ovicidal activity as compared with the control. On an average, 55.55 % eggs hatched from A. squamosa treated sets while 51.10 % and 93.33 % hatching was observed in A. indica and control sets respectively. The results are an average of 3 independent observations ± SD.
EXAMPLE 6 Effect of the biopesticide on germination of treated green gram Germination is an important parameter to check the inhibitory effects, if any, of the preservative chemical in use. Therefore, effect of application of biopesticide on the germination of green gram was noted. Green gram seeds (10) after application of the highest dose of seed extract (10 mg/ml) used in all the experiments was allowed to dry at room temperature. These treated seeds were sowed in earthen pots and regularly watered sufficiently to allow germination. Similarly, healthy untreated seeds and seeds treated with the solvent were set up as controls. Number of germinated seeds was counted upon transformation into healthy seedlings, which was expressed as percent germination. Each set was carried out in triplicate and repeated thrice. These laboratory pot assays suggested

that there was no effect on the germination as 100% transformation of seeds into healthy seedlings was obtained.
We claim:
1. A method of preparing a botanical pesticide from seeds of Annona squamosa
characterized in that
(i) defatting seed powder of A. squamosa with defatting solvent(s);
(ii) treating the residue cake obtained in step (i) with a polar solvent;
(iii) filtering the extract of step (ii);
(iv) concentrating the filtered extract of step (iii);
(v) partitioning the concentrated extract of step (iv) with a suitable
extracting solvent;
(vi) adding suitable dehydrating agent in extract of step (v);
(vii) completely drying the extract of step (vi);
(viii) dissolving the residue of step (vii) in a suitable extracting solvent;
(ix) further defatting extract of step (viii) with defatting solvent;
(x) drying completely/partially the extract of step (ix);
(xi) dissolving the residue of step (x) in a suitable polar solvent and
(xii) purifying by passing through a column as described herein before.
2. A process as claimed in claim 1 (i) wherein the defatting solvents are selected from the list of defatting solvents like hexane and petroleum ether.
3. A process as claimed in claim 1 and claim 2, the defatting solvent is preferably hexane.
4. A process as claimed in claim 1 (ii) wherein the residue cake obtained after discarding the fat layer is treated with a primary alcohol.
5. A process as claimed in claim 1 and 4 wherein the primary alcohol is methanol.
6. A process as claimed in claim 1 (iii) wherein the alcoholic extract is filtered using Whatman filter paper No. 1 to remove suspended particulate matter.
7. A process as claimed in claim 1 (iv) wherein the alcoholic extract is concentrated upto about 10 times using vacuum evaporation.
8. A process as claimed in claim 1 (v) wherein the concentrated extract is partitioned with dichloromethane to selectively retain active principles.
9. A process as claimed in claim 1 (vi) wherein the dehydrating agent is sodium sulphate.
10. A process as claimed in claim 1 (vii) & (x) wherein the dichloromethane extracted layer is dried using vacuum evaporation upto dryness.

11. A process as claimed in claim 1 (viii) wherein the dried residue is dissolved in acetonitrile to further selectively retain the active principles.
12. A process as claimed in claim 1 (ix) wherein the acetonitrile containing active principles are further defatted using hexane.
13. A process as claimed in claim 1 (ix) wherein the completely or partially dried residue is dissolved in methanol.
14. A method of preparing botanical pesticide from seeds of A. squamosa and its effectiveness in the management of pre- and post-harvest pests as substantially described with accompanying examples 1-6 and drawing.
Signature of Applicant Registrar
farth Maharashtra Universlh




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Patent Number 207131
Indian Patent Application Number 245/MUM/2004
PG Journal Number 43/2008
Publication Date 24-Oct-2008
Grant Date 23-May-2007
Date of Filing 27-Feb-2004
Applicant Address P.O.NO. 80, UMAVINAGAR, JALGAON 425001(MS)
# Inventor's Name Inventor's Address
PCT International Classification Number B01D 11/02
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA