Organic compounds -- part of the class 532-570 series – Organic compounds – Carboxylic acids and salts thereof
Reexamination Certificate
2001-12-19
2004-03-02
Killos, Paul J. (Department: 1625)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carboxylic acids and salts thereof
Reexamination Certificate
active
06700014
ABSTRACT:
TECHNICAL FIELD
The present invention relates generally to a process for extracting oleanolic acid. More specifically, the present invention relates to processes for extracting oleanolic acid from plant materials with non-halogenated, polar to medium polar solvents.
BACKGROUND
Oleanolic acid (3-&bgr;-hydroxyoleanan-12-en-28-oic acid) is a pentacyclic triterpene acid which is ubiquitously distributed throughout the plant kingdom. Oleanolic acid has a number of biological properties of interest. Specifically, oleanolic acid has been shown to have cytotoxicity against human tumor cell lines. Oleanolic acid is also known to have anti-fungal, anti-malarial, anti-carcinogenic, anti-inflammatory and anti-hepatotoxic activity. Further, oleanolic acid has also been shown to inhibit human topoisomerase I and II-&agr;. Finally, oleanolic acid has also been known to inhibit HIV-1 protease.
Consequently, oleanolic acid is of increasing interest in pharmaceutical research circles because it may have distinct pharmaceutical properties of its own or provide a basis for the development of new pharmaceutical drugs by partial structure variation.
Because it is difficult to synthesize and extract from plant materials, oleanolic acid is expensive. Depending on the purity, oleanolic acid can cost from $300 to $400 per gram (purity grade approximately 97%) and up to $900 per gram (purity grade 99%). Accordingly, there is a need for an efficient method that will result in the obtaining of oleanolic acid in a pure form but at a low cost.
Oleanolic acid has been recovered for use of a food additive (i.e., in a non-pure form) from olive skins. In low concentration, it has also been applied in non-pure form to remove the metallic aftertaste sensation of artificial sweeteners.
Oleanolic acid has also been recovered in non-purified forms from grape skins in connection with the study of the wax that covers grape skins in order to enhance the drying rate of the grapes. Specifically, Radler and Horn, “The Composition of Grape Cuticle Wax,” Aust. J. Chem., pp. 1059-69 (1965) teach the extraction of the grape skin wax with a light petroleum fraction. Radler and Horn disclose no process for the purification or isolation of oleanolic acid that may or may not be contained in the grape skin wax. However, a later article, Radler, “The Surface Waxes of Sultana Vine,” Aust. J. Biol. Sci., pp. 1045-56 (1965) discloses the extraction of the surface waxes with chloroform. A similar extraction is also disclosed in Grncarevic and Radler, “A Review of Surface Lipids of Grapes and Their Importance in the Drying Process,” Am. J. Enol. Vitic. (22), pp. 80-86 (1971).
The use of petroleum, similar solvents and chloroform in an extraction process for obtaining oleanolic acid from plant material is disadvantageous because chloroform is toxic, carcinogenic and dangerous to handle. Further, light petroleums, such as hexane, are not desirable because these very non-polar solvents do not selectively extract oleanolic acid but, instead, extract additional unwanted materials such as waxes, long-chain aldehydes and long-chain alcohols which are difficult to separate from the target compound oleanolic acid. Other non-polar solvents such as benzene, toluene, cyclohexane, and THF, are toxic and/or carcinogenic. Further, none of the above-described processes have been developed into a method for obtaining oleanolic acid from plant material in a relatively pure form.
Thus, due to the potential of oleanolic acid as a pharmaceutical agent and due to the high cost of oleanolic acid in a pure or relatively pure form, there is a need for an improved process for generating pure or relatively pure oleanolic acid in a more economical and safer fashion than current known processes.
SUMMARY OF THE DISCLOSURE
In satisfaction of the aforenoted needs, a process for extracting oleanolic acid from plant materials is disclosed. In the disclosed process, an initial extraction step is carried out with a non-halogenated polar to medium polar solvent. Specifically, plant material is provided which contains oleanolic acid. This plant material which is preferably dried, and, if applicable, seeds and stems are removed as the non-halogenated polar to medium polar solvent would extract seed oil as well as other semi-polar substances from the stems which would be difficult to separate from the oleanolic acid in the later processing steps. The extraction step with the non-halogenated polar to medium polar solvent results in a crude extract that comprises oleanolic acid.
The oleanolic acid is then removed from the solvent. Specifically, the oleanolic acid may then be crystallized out of the oversaturated solution to form a precipitate. The non-halogenated polar to medium polar solvent is removed from the precipitate by conventional means. Optionally, the precipitated oleanolic acid is then purified in different steps.
The oleanolic acid may be precipitated out of the solution by cooling the solution, adding an additional solvent such as water to the solution, or removing solvent by vacuum evaporation until the precipitate is formed.
The subsequent purification step can comprise dissolution of the precipitate in fresh non-halogenated polar to medium polar solvent and re-crystallization of the precipitate which will have a higher purity of oleanolic acid.
Subsequent re-crystallization can also involve dissolving the precipitate in fresh non-halogenated polar to medium polar solvent, adding controlled amounts of water until the oleanolic acid precipitates, separating the oleanolic acid precipitate from the resulting organic solvent/water solution resulting in increased purity of the oleanolic acid. Polar components such as tannins, phenolics and other impurities have a tendency to remain in the aqueous/organic solvent phase.
The subsequent purification can also comprise washing of the crystalline precipitate with small amounts of fresh solvent, washing the precipitate with a polar solvent or washing the precipitate with both a non-polar and a polar to medium polar solvent.
Preferably, for all steps including extraction and purification, the non-halogenated polar to medium polar solvent is ethanol due to its non-toxicity, abundance and low cost. Ethanol has a Snyder polarity index of 5.2. For purposes of this application, polar to medium polar solvents will be defined as those having a Snyder polarity index of greater than 2.5. On the other hand, non-polar solvents will hereinafter be defined as those non-polar solvents having a Snyder polarity index of less than 2.5. Other non-halogenated polar to medium polar solvents suitable for the initial extraction step and subsequent purification steps include (with the Snyder polarity index given in parenthesis) e.g., isopropanol (4.3), n-propanol (4.3), methanol (6.6), diethylether (2.8), acetone (5.4), acetonitrile (6.2), methyl tertiary butyl ether (MTBE), and ethylacetate (4.3).
Non-halogenated solvents are preferred due to their general low cost, lower toxicity and ease of disposal.
If a non-polar solvent is used in the purification process, a suitable non-polar solvent one petroleum ether fractions (C
5
-C
7
), or n-hexane cyclohexane. Hexane has a Snyder polarity index of zero or close to zero.
Other suitable preparative purification steps include the use of column chromatography such as medium pressure liquid chromatography (MPLC) and preparative high performance liquid chromatography (HPLC).
Another purification step could include dissolving the oleanolic precipitate in a solvent and passing the resulting solution through one or more membranes.
One plant material which is abundant and relatively rich in oleanolic acid is grapes, grape pomace or still more specifically, grape skins. Accordingly, a suitable process according to this disclosure includes providing a material that comprises grape skins that has been either sun dried, air dried or freeze dried. Mixing a polar to medium polar solvent with the material, extracting a solution comprising the alcohol solvent and oleanolic acid from the material, cooling the solution
E. & J. Gallo Winery
Killos Paul J.
Marshall & Gerstein & Borun LLP
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