Simple and effective manufacturing process for large scale...

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2002-06-07

2003-04-08

Lambkin, Deborah C. (Department: 1626)

Organic compounds -- part of the class 532-570 series

Organic compounds

Heterocyclic carbon compounds containing a hetero ring...

Reexamination Certificate

active

06545167

ABSTRACT:

OBJECT OF THE INVENTION
The present invention relates to an economically beneficial, hence, inexpensive, method which is simple avoiding labor intensive techniques, such as HPLC or column chromatography, for large-scale isolation of Nimbin. Specifically, the invention relates to a method of using a combination of alcohol solubilization, organic partitioning and crystallization to obtain highly purified Nimbin from Neem kernels.
BACKGROUND OF THE INVENTION
Nim, Neem or Nimba (
Melia azadiracta, Melia indica
) is native to India and is cultivated to a large extent because of its medicinal properties. These therapeutic properties are so highly valued to the indigenous population that the nim tree plays the role of “village dispensary” in many rural areas (Siddiqui and Mitra,
J Sci Indus Res
(India) 4:5 (1945)). Its twigs are used as a toothbrush for general oral hygiene and the leaves and bark are employed for a variety of uses including wound healing and treatment of various skin ailments (Siddiqui and Mitra, (1945)).
The first report of the pesticidal properties of Neem appeared around 1927 when Mann and Burns (
Agr J India
(Calcutta) 22:325 (1927)) observed during the locust cycle of 1926-27 that adult locusts did not feed on Neem leaves. This was followed by Chopra (
Rept of Dept of Agr
(Punjab) Pt. 2, 1:67 (1928)) who treated the extact of Neem leaves as contact poison on grub weevils.
Since then, a number of publications describing the various activities from different parts of the Neem tree have appeared. For example, Neem oil has shown antifeedant activity against
Nephotettix virescens
(leafhopper of rice). (
Neem Newsletter
1:28 (1984)).
Azadirachta Idica (Neem) seed kernels contain the active constituents which have interested a number of workers since Chaterjee and Sen (
Ind J Med Res
(1920) 8:356) reported the isolation of margosic acid in 1919. The investigation of the bitter principles continued often with conflicting results. (See, e.g., Siddiqui,
Curr Sci
(India) 11:278 (1942)). In 1923, Wetson et al. (
J Soc Chem Ind
(1923) 42:387) isolated from the soap lye of the oil, a sulfur-free crystalline acid margoso-picrin (yield 0.12 to 0.017% oil and 0.15 to 0.24% amorphous bitter acids). Sen and Bennerjee (
J Ind Chem SOC
(1931) 8:773) noted the isolation of a sulfur-containing acidic bitter principle from aqueous extracts of the oil. Quadrat-I-Khuda et al. (
J Ind Chem SOC
(1940) 17:189) reported the isolation of a sulfur-containing essential oil and an amorphous, water-soluble bitter principle from an aqueous extract of the oil previously distilled to remove steam-volatile products. Industrial methods for isolation of such bitter principles have been reported to give yields of approximately 1.2%.
Neem seeds contain close to 100 molecules of which 84 have been identified. Nimbin was first isolated by Siddiqui in 1942 but its structural elucidation was accomplished by Narayanan et al. (
Chem
&
Ind
(1962) 1238;
Chem
&
Ind
(1964) 322;
Chem
&
Ind
(1964) 324;
J Chem
(1964) 2:108) and Harris et al. (
Tetrahedron
(1968) 24:1517) in the sixties. Merck Index (12
th
ed.) (1996) describes Nimbin as the first bitter principle from various parts of nim tree,
Azadirachta indica,
Juss., having a melting point of 205° C.
On account of the medicinal and antifeedant uses of nim, increased efforts have been made by different investigators to isolate the active ingredients which account for the above properties. In view of the growing importance of Neem kernel and its constituents as commercial products, the present inventors teach a method of isolating one of the active principles in a more efficient and practical manner which comports with industrial production.
SUMMARY OF THE INVENTION
The object of the present invention is to provide a simplified method for large-scale extraction of pure Nimbin from Neem kernel powder comprising extracting Neem kernel powder with alcoholic solvents. In a related aspect, the solvents can include methanol, ethanol and isopropanol.
The invention also envisages concentrating the alcoholic extract under vacuum, to include separating sugars and starches by partitioning between water and chlorinated hydrocarbon layers. Such chlorinated hydrocarbons include but are not limited to dichioromethane, dichioroethane and trichloroethylene. The method also comprises filtering the chlorinated hydrocarbon extract, stripping and precipitating the extract with a hydrocarbon solvent (e.g., hexane, heptane, octane). Moreover, the hydrocarbon extract is stripped, suspended and stirred in twice the volume of hydrocarbon. The mixture then can be cleared (e.g., by centrifugation), and the resulting solids suspended in 3 times the volume of alcohol where the material is stirred, centrifuged, dried and re-crystallized twice with alcohol and activated carbon.
Another object of the present invention is to provide a process for the preparation of essentially 98.50% pure Nimbin from pulverized Neem kernels. In a related aspect, the Nimbin obtained by the present method has a melting point of 210-212° C. and is tasteless.
The resulting compound may be formulated into many different compositions, such as lotions, sprays, cremes and the like for use on humans, animals and vegetation.
Other features and objects of the present invention will become apparent from the following description.
DETAILED DESCRIPTION OF THE INVENTION
The method employed for the isolation of the bitter products does not involve the initial saponification of the oil and is based on the difference in the solubilities of the various constituents in diluted alcohol and other non-miscible organic solvents such as ether, petroleum ether and ethyl acetate. Apart from the fact that such a method ensures the isolation of the constituents in a native form, the method has the advantage of yielding a comparatively purified oil which can be used for industrial purposes (e.g., photo-degradation products of Nimbin have insecticidal activity).
According to the invention, Neem kernels are pulverized and extracted with alcoholic solvents including, but not limited to, methanol, ethanol or isopropanol. Methanol is a preferred alcohol. Alcohol is added at approximately 1-5 times the volume by weight of kernel powder at about 25-50° C. for between about 2 hours to about 24 hours under agitation. In a preferred embodiment, the ratio of alcohol to kernel powder is 2, 3 or 4, preferably 2.5. In a related aspect, the incubation temperature is about 30° C., 40° C., 45° C. or 47° C. In a preferred embodiment, the temperature is 40° C. The incubation can occur for 3, 5, 7, 9, 11, 13, 15 or more hours. In a related aspect, longer incubation times are not necessary and 2, 4, 6 or 8 hours of incubation can be sufficient. A preferred incubation range is 4 to 6 hours. In one embodiment, when the incubation temperature is 40° C., the temperature is reduced to 30° C. before separation of the phases. In a related aspect, the temperature is brought down to about 25° C. and treated to separate the liquid and solid phases.
The solid phase can be re-extracted with an excess of about 1.5-4 times the volume of alcohol by original weight of kernel powder under the similar conditions. A preferred alcohol is methanol. It is preferred that the same alcohol is used for both extractions. A preferred ratio of alcohol to kernel powder is 1.5. The solid phase is stripped of solvent and stored.
Both liquid phases are pooled and solvent concentrated to approximately 5% of the original volume. A suitable means of concentration is evaporation, the liquid being passed successively through falling film and wiped film evaporators under reduced pressure (600 mm Hg). The resultant gummy mass containing Neem limonoids in addition to sugars, starches and lipids is partitioned between water and a chlorinated hydrocarbon solvent. Suitable chlorinated hydrocarbon solvents include but are not limited to, methylene dichloride, ethylene dichloride and trichloroethylene. In a preferred embodiment, ethylene dichloride and water are used to extract

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