Azadirachtin extraction process

Details Azadirachtin extraction process Azadirachtin extraction process

2000-01-14

2001-11-06

Tate, Christopher R. (Department: 1651)

Drug, bio-affecting and body treating compositions

Plant material or plant extract of undetermined constitution...

Containing or obtained from azadirechta

C424S405000, C514S453000, C549S354000

Reexamination Certificate

active

06312738

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to a process for the recovery of azadirachtijn and/or similar insecticidal compounds from neem seeds.
BACKGROUND ART
It is known that the seeds and other parts of the neem tree (
Azaderachta indicia
and related species) contain natural pesticidal compositions. The main active pesticidal composition is azadirachtin which is a tetraniortriterpenoid that causes feeding inhibition and growth inhibition in a variety of organisms including insects, mites and neniatodes. It is possible that there are a number of similar insecticidal compounds present in neem extracts that partition with the azadirachtini. As used in this specification, the term azadirachtin is taken to include all insecticidal terpenoids present in neen extracts that partition with azadirachtin.
In recovering azadirachtin from neem seeds it is necessary to separate the active constituent from other materials including the other triterpenes, the oil. fibre and other insoluble materials. and water soluble constituents such as sugars and water soluble proteins. These separation procedures are complicated by the fact that azadirachtin is susceptible to hydrolysis in water and to heat degradation.
One conventional method for extracting azadirachtin from neem seeds involves the use of three organic solvents and two liquid/liquid partition steps. Firstly, the neem seeds are pressed to remove the majority of the neem oil. The resulting expeller cake is then extracted with methanol. The methanol extracts a wide range of substances including azadirachtin and the other triterpenies, diterpenies, the residual oil, and some polysaccharides and proteins. To produce a powder from the methanolic supernatant, the extract must undergo a number of clean-up steps.
In the first clean-up step, the supernatant is concentrated and partitioned against hexane, or a similar non-polar solvent, to remove the oils and diterpenes. The hexane is then driven off in a still and the resulting oil is collected. The second clean-up step involves taking the de-oiled supernatant and driving off the methanol. The resulting tar is then resolved in ethyl acetate, or a similar solvent, and partitioned against water to remove the polysaccharides and water soluble proteins. The ethyl acetate which contains the azadirachtin and other triterpenes is then evaporated to produce an azadirachtin rich powder.
There are a number of problems associated with this conventional method, with the major problem being cross-contamination of the solvents used in the process and the resulting variation in product quality. While good quality azadirachtin powder can be produced when first using the process with fresh solvents. after a number of cycles problems with cross-contamination do arise. While hexane and methanol are normally considered essentially immiscible, in a multi-component system, such as is created during the extraction of azadirachtin described above, the hexane and methanol do demonstrate some miscibility. The change in the polarity of the solvents allows oils to be carried through the hexane partition and so creates difficulties in producing a non-oily or flowable powder. A more serious problem is any occurrence of cross-contamination between the methanol and ethyl acetate. If the methanol is not removed in the drying step after the hexane partition, cross-contamination with ethyl acetate occurs. This contamination of the ethyl acetate with methanol causes azadirachtin to be carried over into the water in the ethyl acetate/water partition or in the worst case prevents a partition forming at all.
A further problem with the conventional method for extracting azadirachtin is the production of waste water with a high biological oxygen demand (BOD). In many countries, the release of waste water with high BOD is not permitted and requires the installation of a relatively expensive water treatment facility.
An alternative azadirachtin extraction process is described in Australian patent no 661482 to Trifolio-M GmbH, Herstellung Und Vertrieb Hochreinier Biosubstanzen. In this alternative process, the neem seed is pressed or crushed to remove the majority of the oil and the expeller cake is extracted with warm water. The warm water extraction removes the azadirachtin, some of the more polar triterpenes, the majority of the polysaccharides and water soluble proteins, and a slight amount of the more polar oily compounds. The aqueous supernatant is partitioned against a solvent of intermediate polarity, such as ethyl acetate or dichloromethane. with the azadirachtin partitioning into the organic layer. The organic layer can then be concentrated under vacuum and the azadirachtin precipitated through the addition of a nioni-polar solvent, such as hexanie or petroleum ether. While high yields of azadirachtin powder are produced using this alternative process, the process still results in the discharge of an aqueous solution loaded with polysaccharides and proteins which, in some countries, will require treatment to meet environmental standards. Further, the precipitation step where a non-polar solvent is added to the concentrated supernatant results in cross-contamination with its attendant problems and expense of requiring purification of the solvents prior to their re-use in the extraction process.
SUMMARY OF THE INVENTION
According to a first aspect, the present invention consists in a process for the production of all azadirachtin rich aqueous solution comprising the steps of:
(i) de-oiling ground or otherwise comminuted neem seeds to remove a first proportion of the oil content thereof;
(ii) extracting the de-oiled neem seeds with a polar organic solvent, in which azadirachtin is soluble but in which polysaccharides and proteins are not substantially soluble, to form an organic solution of azadirachtin and the remaining oil;
(iii) partitioning the organic solution against water to form an aqueous solution of azadirachtin and the remaining oil; and
(iv) separating the remaining oil from the aqueous solution to form an azadirachtini rich aqueous solution.
In one embodiment, the process can further comprise the steps of:
(v) extracting the aqueous solution with a polar organic solvent, in which azadirachtin is soluble but in which polysaccharides and proteins are not substantially soluble, to form an organic solution of azadirachtin; and
(vi) evaporating the organic solvent to form an azadirachtin rich powder.
The neem seeds are preferably first dried to a moisture content of from 1 to 10% by weight, more preferably 2 to 4% by weight, and then pressed to produce an expeller cake of seeds from which has been removed a substantial proportion of the oil content of the seeds. Preferably, the pressing is done at a low speed to reduce the temperature build up in the neem seeds during pressing and so minimise heat degradation of the azadirachtin. The pressing is. however, preferably conducted so that at least 70% by weight, more preferably 80% by weight, and most preferably 90% by weight of the oil is removed from the seeds during the pressing process.
The polar organic solvent used to extract the substantially de-oiled expeller cake in step (ii) is preferably selected from the following: ethyl acetate. methyl acrylate, butanol, 2-butanone, butyl carbitol, ethylene glycol monio bhityl ether, ethyl formate, propyl formate, furan, tetrahydirofturan. isopherone, 2,4-dihydroxy 4-methyl pentanone, 2,4-dimethyl pentanone, dibutyl phthalate, dipentyl phthalate, 1,2-epoxy propane. dibutyl sebacate, chloroform, dimethyl sulphide, ethanthiol, ethyl chloride. pentachloroethane, trichloroethene, 1-bromopentane or another solvent with a similar dielectric constant and mixtures thereof.
Ethyl acetate is preferred and can be used in the process as it is not polar enough to extract the polysaccharides and proteins from the expeller cake but is sufficiently polar to extract the azadirachtin, the other triterpenes, and any residual oil from the expeller cake. The ratio of expeller cake to ethyl acetate in the extraction can be 1:1. The extractio

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