Screening Of Bioactive Components Of Azadirachta Indica A. Juss. And Their Efficacy Against The Red Flour Beetle Tribolium Castaneum (Herbst)

Abstract

The chloroform and methanol extracts of the leaves, flower, root bark, root wood, seed, stem bark and stem wood of Azadirachta indica were tested against Tribolium castaneum adults through residual film assay. According to the intensity of activity observed through mortality of the adult beetles the potentiality of the chloroform extracts could be arranged in a descending order of seed (107.0412μg/cm2) > leaf (113.3073μg/cm2) > stem wood (177.580μg/cm2) > root wood (192.5573μg/cm2) > stem bark (244.4488 μg/cm2) > flower (259.3435 μg/cm2) > root bark (480.3277 μg/cm2) and for the methanol extracts, seed (222.3965 μg/cm2) > root wood (418.4427μg/cm2) > leaf(447.2792μg/cm2) > stem bark (457.6257μg/cm2) > stem wood (492.0781μg/cm2) > flower (752.3578 μg/cm2) > root bark (1011.733 μg/cm2) for 96h of exposure. Due to prolongation of exposure no alteration of the results was observed other than proportional increase in the number of mortality. However, mortality was observed just within 24h of exposure, which is very special potentiality in dose-mortality experiments. All the chloroform and methanol extracts of the flower, leaves, root bark, root wood, seed, stem bark and stem wood of A. indica have been applied against the larvae of T. castaneum for the detection of their biological activity (including lethality, prolongation of larval instars, causing deformity in body, abnormality in any of the biological parameters). According to the intensity of activity against he 1st instar larvae of chloroform extract the result could be arranged in the following order: seed> root wood > stem bark > stem wood> flower. In case of methanol extracts, the results were as follows: stem wood > root bark > seed > root wood>flower>stem bark>leaf. For 2nd instars, the results were leaf flower> root wood> stem bark> root bark > seed > stem wood for chloroform extract after 72 hours respectively. For the methanol extracts the results were as follows: leaf > flower> root wood> root bark > stem wood > seed > stem bark after 72 hours respectively. In case of 3rd instars larvae the results were stem bark > root wood> stem wood > seed > flower> root bark> leaf for chloroform extract after 72 hours respectively. For the methanol extracts the results were stem bark > root wood> seed >stem wood > flower> root bark> leaf for after 72 hours respectively. For the 4th instars larvae against the chloroform extracts the results were as follows: root wood > root bark> seed >flower > leaf > stem wood >stem bark after 72 hours respectively. For the methanol extracts, the results were flower > root bark> seed >root wood > leaf > stem wood >stem bark respectively. The larval mortality showed a possibility of raising toxicity by the magnification of the amount of ingestion of the treated food. Besides mortality of the larvae and abnormality in changing instars, as well as differences in size were also observed. The number of death has been increased just proportional to that of the age of the larvae, which indicates the increase in volume of food intake by the larvae as well. All the test extracts of leaves, flower, seed, root bark, root wood, stem bark and stem wood of A. indica collected in chloroform and methanol showed repellent activity against adult beetles of T. castaneum. The F values have been established were 51. 03662, 253.5068, 43.04438, 83.58911, 75.79346, 75.94017, 64.50964, 50.44838, 25.82928, 61.28114, 45.56164, 34.5519, 35.75216 and 21.5157 for the analysis between doses and 6.778143, 3.007724, 5.447409, 3.835164, 1.400522, 1.856993, 5.669432, 4.258362, 5.590989, 0.876118, 4.630108, 3.285364, 1.990562 and 0.989226 for the analysis between time interval for seed, stem wood, stem bark, root wood, root bark, flower and leaves of Chloroform and Methanol extracts respectively. Among the tested CHCl3 and MeOH extracts all the rest offered repellency at 0.01% level of significance (P<0.001) According to the intensity of repellency the result could be arranged in a descending order: In case of chloroform extract stem bark >root wood> seed >flower> stem wood> leaf> root bark and for the methanol extracts seed> stem wood>stem bark> root bark> root wood> flower> leaf extract. The cytotoxic effect of the above mentioned extracts was also found promising. The seed extract was found to offer the highest mortality of the nauplii, while the LC50 values were 520.1635, 24.50645 and 5.942745ppm for the chloroform extracts; 906.5301, 61.17362 and 18.24789ppm for the methanol extracts for 30 min, 24h and 48h of exposures respectively. The LC50 values for the stem bark extract were 1042.544, 196.883 and 24.53654ppm for the chloroform extracts; 6030.069, 167.7432 and 34.2457ppm for the methanol extracts. The LC50 values for the stem wood extract were 3711.381, 94.12271 and 45.16339ppm for the chloroform extracts; 1641.063, 92.75699 and 48.30029ppm for the methanol extracts. The LC50 values for the flower extract were 933.4176, 67.70986 and 26.04309ppm for the chloroform extracts; 18450.49, 113.4081 and 24.50362ppm for the methanol extracts. For the leaf extract the LC50 values were 3476.365, 101.4525 and 51.38413ppm for the chloroform extracts; 9577.411, 455.9743 and 160.1078ppm for the methanol extracts. The LC50 values for the root bark extracts were 987.7583, 28.04569 and 23.26771ppm for the chloroform extracts; 1030.155, 57.71285 and 26.29665ppm for the methanol extracts. The LC50 values for the root wood extracts were 838.2706, 36.47875 and 8.40184ppm for the chloroform extracts; 5187.234, 82.83993 and 23.38707ppm for the methanol extracts for 30 min, 24h and 48h of exposures respectively. According to the intensity of activity the results of the extracts against the brine shrimp nauplii could be arranged in the following order: seed > root wood >root bark> stem bark> flower>stem wood> leaf for the chloroform extract and seed > root wood > flower> root bark> stem bark> stem wood> leaf for the methanol extracts. The antibacterial activity of A. indica extractives collected in CHCl3 and MeOH were tested against 14 bacteria (6 Gram-positive bacteria) S. aureus, B. cereus, B. megaterium, B. subtilis, S. lutea, S.-ß -haemolyticus and (8 Gram-negative bacteria) S. typhi, S. dysenteriae, S. shiga, S. sonnei, S. boydii, E. coli, P. aeruginosa and Proteus sp. at concentrations of 50 and 200 µg/disc along with a standard antibiotic, ciprofloxacin 30 µg/disc. The seed (chloroform) extract S. aureus, B. cereus, B. megaterium, B. subtilis, S. lutea , S. typhi, S. dysenteriae, S. shiga, S. boydii, E. coli and Proteus sp. were responsive with inhibition zones 06,13, 11, 10, 10, 12, 14, 12, 10, 13, 13 and 12 mm for 50 and 200 µg/disc application and for the methanol extract S. aureus, B. cereus, B. subtilis, S. lutea, S. typhi, S. dysenteriae, S. shiga, S. boydii, E. coli and Proteus sp. were responsive with inhibition zones11, 10, 12, 09, 12, 11, 09, 10,12, 05 and 10 mm respectively for the same doses; while the inhibition zones for the standard Ciprofloxacin 30µg/disc were 30, 28, 28, 30, 28, 30, 30, 29, 29, 28, 28, 28,28 and 28 mm for the above mentioned test agents respectively. For the stem bark (chloroform) extract only S. aureus, B. megaterium, B. subtilis, S. lutea, S.-ß –haemolyticus, S. typhi, S. dysenteriae, S. boydii, E. coli and P. aeruginosa were responsive with inhibition zones 12, 11, 10, 11, 12, 13, 12, 11, 12, 13mm and 7mm for 200 and 50 µg/disc application and for the methanol extract S. aureus, B. megaterium, B. subtilis, S. lutea, S.-ß –haemolyticus, S. typhi, S. dysenteriae, S. boydii, E. coli and P. aeruginosa were responsive with inhibition zones 10, 09, 10, 10, 11, 12, 13, 10, 12, 11mm and 08, 10, 09mm respectively for the same doses; while the inhibition zones for the standard Ciprofloxacin 30 µg/disc were 30, 30, 28, 30, 28, 30, 29, 29, 29, 28, 28, 29, 29 and 29 mm for the above mentioned test agents respectively. The stem wood extract (chloroform) was responsive to S. aureus, B. cereus , B. megaterium, B. subtilis, S.- ß – haemolyticus, S. typhi, S. dysenteriae, S. boydii, E. coli and Proteus sp. with inhibition zones 10, 08, 10, 12, 10, 12, 10, 09, 10 and 10 mm for 200 µg/disc application and for the methanol extract S. aureus, B. megaterium, B. subtilis, S.- ß -haemolyticus, S. typhi, S. dysenteriae, E. coli and Proteus sp. with inhibition zones 08, 09, 11, 07, 10, 09, 07 and 10mm respectively for the same doses (Table ); while the inhibition zones for the standard Ciprofloxacin 30µg/disc were30, 30, 30, 30, 32, 30, 30, 32, 30, 30, 30, 31, 30 and 30 mm for the above mentioned test agents respectively. The flower extract (chloroform) was responsive to B. cereus, B. subtilis, S.- ß -haemolyticus, S. typhi, S. dysenteriae, S. boydii , S. shiga, E. coli and P. aeruginosa with inhibition zones 12, 11, 13, 13, 12, 12, 11, 14 and 12mm for 200 µg/disc application and the methanol extract was responsive to B. subtilis, S.- ß -haemolyticus, S. typhi, S. dysenteriae, S. boydii, E. coli and P. aeruginosa with inhibition zones 10, 12, 11, 10, 11, 10, 13 and 10mm respectively for the same doses; while the inhibition zones for the standard Ciprofloxacin 30µg/disc were 30, 30, 30, 30, 32, 30, 32, 30, 30, 30, 31, 32, 32 and 30 mm for the above mentioned test agents respectively. The leaf (chloroform) extracts were responsive to S. aureus, B. megaterium, S.-ß-haemolyticus, S. typhi, S. boydii, S. lutea, E. coli and P. aeruginosa with inhibition zones 12, 11, 12, 12, 12, 10, 12 and 10 mm for 200 µg/disc application and the methanol extract was responsive to S. aureus, B. megaterium, S. typhi, S. lutea, S. boydii, E. coli and P. aeruginosa and with inhibition zones 09, 10, 11, 10, 12, 10 and 09 mm for the same doses; while the inhibition zones for the standard Ciprofloxacin 30 µg/disc were 30, 30, 32, 30, 30, 32, 32, 30, 30, 30, 32, 32, 32 and 30 mm for the above mentioned test agents respectively. In case of the root bark extract (chloroform) B. cereus, B. megaterium, B. subtilis, S.- ß -haemolyticus, S. typhi, S. shiga, S. boydii, E. coli and Proteus sp. were responsive with inhibition zones 13, 12, 13, 13, 13, 12, 09, 12 and 13mm for 200 µg/disc application, and for the methanol extract B. cereus, B. megaterium, B. subtilis, S.- ß –haemolyticus, S. typhi, S. shiga and E. coli were responsive with inhibition zones 11,10,12, 10,11,09 and 11mm for the same doses; while the inhibition zones for the standard Ciprofloxacin 30 µg/disc were 30, 30, 32, 30, 30, 32, 30, 30, 30, 32, 30, 32, 30 and 30mm for the above mentioned test agents respectively. The root wood (chloroform) extracts were responsive to S. aureus, B. megaterium, B. subtilis, S.-ß-haemolyticus, S. typhi, S. dysenteriae, S. sonnei, S. boydii, E. coli and Proteus sp. with inhibition zones 12, 13, 12, 13, 14, 13, 13, 12, 13 and 10 mm for 200 µg/disc application, and the methanol extract was responsive to S. aureus, B. megaterium, S. lutea, S.- ß –haemolyticus, S. shiga, S. dysenteriae, S. boydii, E. coli and P. aeruginosa with inhibition zones 10,11, 11, 09, 12, 10, 12, 10, 11 and 09 mm for the same doses; while the inhibition zones for the standard Ciprofloxacin 30 µg/disc were 30, 30, 30, 32, 30, 30, 28, 29, 29, 30, 28, 30, 30 and 28mm for the above mentioned test agents respectively. Among all the CHCl3 and MeOH extracts only CHCl3 extracts of the seed and the root wood were subjected to evaluate the minimum inhibition zones. The MIC value of the chloroform extract of the seed was 128µg/ml against B. cereus, 64µg/ml against S.- ß –haemolyticus and 32µg/ml against S. dysenteriae. The MIC values of the chloroform extract of root wood were 128µg/ml against S. - ß –haemolyticus; 64µg/ml against B. megaterium and 32µg/ml against S. typhi. Antifungal activity of the A. indica extractives collected in chloroform and methanol were tested against six pathogenic fungi F. vasinfectum, A. fumigatus, A. niger, A. flavus, C. albicans and P. notatum at concentrations of 50 and 200µg/disc along with a standard Nystatin (50µg/disc). For the flower extract (chloroform) A. flavus, A. niger, P. notatum and C. albicans were responsive with inhibition zones 13, 12, 12, 11mm and for the methanol extract 10, 10, 14 and 10mm for 200 µg/disc application, while the inhibition zones for the standard nystatin 50 µg/disc were 20, 20, 22, 22, 21 and 20mm for the above mentioned test agents respectively. In case of the leaf extracts (chloroform) A. niger, A. flavus, P. notatum and C. albicans were responsive with inhibition zones 14, 13, 12, 12mm and for the MeOH extract 12, 11, 10, 11mm for 200 µg/disc application, while the inhibition zones for the standard nystatin 50µg/disc were 20, 20, 22, 22, 20 and 20mm for the above mentioned test agents respectively. For the root bark extracts (chloroform) A. niger, A. flavus, C. albicans and P. notatum were responsive with inhibition zones 14, 13, 10, 11mm and for the methanol extract 13, 11, 08, 10mm for 200 µg/disc application; while the inhibition zones for the standard nystatin 50 µg/disc were 20, 20, 23, 23, 22 and 20mm for the above mentioned test fungi. In case of the root wood extract (CHCl3) A. niger, A. flavus, C. albicans and P. notatum were responsive with inhibition zones15, 13, 13, 12mm and for the MeOH extract 12, 11, 12, 11mm for 200 µg/disc application; while the inhibition zones for the standard nystatin 50µg/disc were 20, 20, 23, 23, 22 and 20 mm for the above mentioned test agents respectively. In case of the seed extract (chloroform) only A. niger, A. flavus, C. albicans and P. notatum were responsive with inhibition zones 16, 14, 12,11mm and for the methanol extract 13, 12, 10, 09 mm for 200 µg/disc; while the inhibition zones for the standard nystatin 50 µg/disc were 23, 24, 24, 24, 23 and 23mm for the above mentioned test fungi respectively. For the stem bark extract (CHCl3) only A. niger, A. flavus, C. albicans and P. notatum were responsive with inhibition zones 13, 12, 13, 11mm and for the MeOH extract 12, 10, 11, 10mm for 200 µg/disc application; while the inhibition zones for the standard nystatin 50 µg/disc were 24, 24, 25, 25, 23 and 23 mm for the above mentioned test agents respectively. For the stem wood extract (CHCl3) only A. niger, A. flavus, C. albicans and P. notatum were responsive with inhibition zones 13, 12, 13, 12mm and for the MeOH extract 12, 10, 11, 10mm for 200 µg/disc application; while the inhibition zones for the standard nystatin 50 µg/disc were 23, 24, 25, 25, 23 and 23 mm for the above mentioned test agents respectively. The leaf, stem bark, root wood and seed of A. indica shows alkaloids, carbohydrates, flavanoids, glycosides, phenol, protein, resins, saponnins, tannins and sterols of different solvents. The phytochemical screening was performed with chloroform and methanol extracts of A. indica. Through activity guided chromatographic fractionation two bioactive compounds have been isolated from the leaf extracts of the test plant A. indica. The CHCl3 extract of the leaf yiel ded 2 compounds, i) Quercetin3-ß-D- glucosi cde.i ci) ß-sitosterol The MIC values of the pure compound A1 were 32µg/ml against B. cereus, 16µg/ml against S. - ß –haemolyticus and 64µg/ml against S. dysenteriae and of the compound A2 were 64µg/ml against B. cereus, 64µg/ml against S. - ß –haemolyticus and 32µg/ml against S. dysenteriae.