Extraction Procedures for Meliacins
The extraction method is also critical for the realistic assessment of meliacin content and
biological activity. As far as the extraction of azadirachtin was concerned, 95% ethanol was the
best followed by 85% aqueous methanol while acetone performed the least160. The extraction
with ethanol was best achieved in three cycles, wherein the second cycle afforded maximum
quantity of azadirachtin. In yet another study45 on the evaluation of different solvents for their
efficiency and selectivity in the extraction of azadirachtin from neem kernels, it was shown that
methanol, water, methylethyl ketone and the azeotropic mixture of methyl tertiarybutyl ether and
methanol (AZT) were the most efficient. The azeotropic mixture (AZT) showed the best selectivity
while the highest degree of purification and enrichment of azadirachtin and other highly active
substances was achieved by a liquid-liquid partition of the aqueous extract with methyl-tertiary
butyl ether.
The extraction method is also critical for the realistic assessment of meliacin content and
biological activity. As far as the extraction of azadirachtin was concerned, 95% ethanol was the
best followed by 85% aqueous methanol while acetone performed the least160. The extraction
with ethanol was best achieved in three cycles, wherein the second cycle afforded maximum
quantity of azadirachtin. In yet another study45 on the evaluation of different solvents for their
efficiency and selectivity in the extraction of azadirachtin from neem kernels, it was shown that
methanol, water, methylethyl ketone and the azeotropic mixture of methyl tertiarybutyl ether and
methanol (AZT) were the most efficient. The azeotropic mixture (AZT) showed the best selectivity
while the highest degree of purification and enrichment of azadirachtin and other highly active
substances was achieved by a liquid-liquid partition of the aqueous extract with methyl-tertiary
butyl ether.
Another important consideration in the extraction and isolation process of pure neem oil is the
stability and shelf life of the extractives and formulations. It was shown that 30% of azadirachtin
decomposed within 24 h when left in chloroform 163 and therefore, the influence of the pH of
the solvent or medium is critical in such case. The effect of sunlight on the crude materials and
extracts should also be borne in mind. It has been reported that on exposure to sunlight, rapid
decrease in the antifeeding potency of azadirachtin was noticed'5O
stability and shelf life of the extractives and formulations. It was shown that 30% of azadirachtin
decomposed within 24 h when left in chloroform 163 and therefore, the influence of the pH of
the solvent or medium is critical in such case. The effect of sunlight on the crude materials and
extracts should also be borne in mind. It has been reported that on exposure to sunlight, rapid
decrease in the antifeeding potency of azadirachtin was noticed'5O
Isolation of azadirachtin is tedius, involving most of the following techniques: partition between solvents, column chromatography,
preparative thin layer chromatography (tlc) flash chromatography, preparative high-pressure liquid chromatography (hplc) etc. Butterworth
and Morgan29 isolated 1.51 g from 2 kg seeds by extraction with ethanol, partition between solvents followed by chromatography on floridin
earth and finally by multiple elution preparative tlc. Zanno et al., 163 carried, out two partitions of the ethanolic extractives of seed kernels
first between ethyl acetate and water, and then between methanol and hexane. The resultant polar mixture was twice chromatographed
over silica gel using ether -acetone elution mixture followed by CHCI3-EtOAc elution mixture. Pure sample was then obtained by
preparative tic. A simplified procedurel24' involved first defatting of neem seed kernels with hexane before ethanol extraction. The ethanolic
extractive was partitioned sequentially with hexane and water to get rid of adhering fats, sugars and proteins respectively. The resultant
residue in ethyl acetate was first filtered through a bed of silica gel before submitting to a vacuum liquid chromatography, crystallisation
from CCl4 and finally flash chromatography to yield azadirachtin of 70-80 per cent purity. The USDA orocedurel55 for the larger scale
isolation of >90% pure azadirachtin involved extraction of defatted marc with acetone and washing of acetone extract sequentially with
hexane, water and hexane, two partitioning steps with 70:30 and 75:25 methanol-water each and ether-acetone (25:75) followed by florosil
column chromatography. The crude azadirachtin was finally purified by open reverse phase, column chromatography employing phase
bonded C-IO' Florisil and methanol-water (I: l) as eluting medium. Another procedure161 introduced dichloromethane partition to obtain an
azadirachtin enriched fraction which was then subjected to sequential normal phase and reverse phase flash and preparative high
performance liquid chromatography to obtain 98% pure sample with 29 per cent overall yield efficiency. Direct purification of azadirachtin by
submitting alcoholic extractives of defatted seed kernels to preparative hplc54 and extension, of Schroeder and Nakanishi procedure for the
isolation of other 55-57 novel congeners have also been reported.55.57
preparative thin layer chromatography (tlc) flash chromatography, preparative high-pressure liquid chromatography (hplc) etc. Butterworth
and Morgan29 isolated 1.51 g from 2 kg seeds by extraction with ethanol, partition between solvents followed by chromatography on floridin
earth and finally by multiple elution preparative tlc. Zanno et al., 163 carried, out two partitions of the ethanolic extractives of seed kernels
first between ethyl acetate and water, and then between methanol and hexane. The resultant polar mixture was twice chromatographed
over silica gel using ether -acetone elution mixture followed by CHCI3-EtOAc elution mixture. Pure sample was then obtained by
preparative tic. A simplified procedurel24' involved first defatting of neem seed kernels with hexane before ethanol extraction. The ethanolic
extractive was partitioned sequentially with hexane and water to get rid of adhering fats, sugars and proteins respectively. The resultant
residue in ethyl acetate was first filtered through a bed of silica gel before submitting to a vacuum liquid chromatography, crystallisation
from CCl4 and finally flash chromatography to yield azadirachtin of 70-80 per cent purity. The USDA orocedurel55 for the larger scale
isolation of >90% pure azadirachtin involved extraction of defatted marc with acetone and washing of acetone extract sequentially with
hexane, water and hexane, two partitioning steps with 70:30 and 75:25 methanol-water each and ether-acetone (25:75) followed by florosil
column chromatography. The crude azadirachtin was finally purified by open reverse phase, column chromatography employing phase
bonded C-IO' Florisil and methanol-water (I: l) as eluting medium. Another procedure161 introduced dichloromethane partition to obtain an
azadirachtin enriched fraction which was then subjected to sequential normal phase and reverse phase flash and preparative high
performance liquid chromatography to obtain 98% pure sample with 29 per cent overall yield efficiency. Direct purification of azadirachtin by
submitting alcoholic extractives of defatted seed kernels to preparative hplc54 and extension, of Schroeder and Nakanishi procedure for the
isolation of other 55-57 novel congeners have also been reported.55.57
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