The present invention provides a process for purifying the cinnamoyl-C-glycoside, 8-C-β-D-[2-O-(E)-cinnamoyl]glycopyranosyl-2- [(R)-2-hydroxy]propyl-7-methoxy-5-methyl-chromone. 8-C-β-D-[2'-O- (E)-cinnamoyl]glycopyranosyl-2-[(S) -2-hydroxy]propyl-7-methoxy-5-methylchromone, referred to herein as the "540 compound." In one embodiment of the present invention the 540 compound is purified by extraction from a decolorizing agent with an organic solvent. The extracted product can be purified by high pressure liquid chromatography. In a second embodiment crude 540 compound, which has not been treated with a decolorizing agent, is purified by passage over neutral alumina or sephadex.
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1. A method for purifying 8-C-β-D-[2-O-(E)-cinnamoyl]glycopyranosyl-2-[(R)-2-hydroxy]propyl-7-methoxy-5-methyl-chromone 8-C-β-D-[
(a) contacting a decolorizing agent, which has been obtained from the processing of a slurry of an aloe leaf extract, with an organic solvent to extract the absorbed 540 compound from the decolorizing agent into the organic solvent, thereby forming a mixture of decolorizing agent and organic solvent containing the 540 compound; and (b) separating the decolorizing agent from the organic solvent to yield a solution highly enriched in compound 540.
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2. The method of
(c) purifying the solution obtained in step (b) by high pressure liquid chromatography (HPLC).
3. The method of
4. The method of
5. The method of
6. The method of
7. The method of
8. The method of
9. The method of
10. The method of
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12. The method of
(c) concentrating the solution obtained in step (b); (d) adding water to the concentrated residue to form an aqueous solution; and (e) extracting the aqueous solution with an organic solvent.
13. The method of
14. A method for purifying compound 540 isolated from the aloe plant comprising the steps of:
(a) applying a solution of crude aloe extract to a pad of neutral alumina; and (b) eluting the extract with an organic solvent.
15. The method of
16. The method of
17. A method for purifying compound 540 isolated from the aloe plant comprising the steps of:
(a) applying a solution of crude aloe extract to a Sephadex, column; and (b) eluting the extract with an organic solvent.
18. The method of
19. The method of
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The present invention relates generally to the purification of an anti-inflammatory and epithelial growth factor-inhibiting compound isolated from the Aloe barbadensis plant. Specifically, this invention describes the purification of the cinnamoyl-C-glycoside,
The present invention includes a novel process for the purification of the cinnamoyl-C-glycoside, 8-C-β-D-[2-O-(E)-cinnamoyl]glycopyranosyl-2-[(R)-2-hydroxy]propyl-7-methoxy-5-methyl-chromone 8-C-β-D-[
The 540 compound exhibits potent anti-inflammatory activity as measured by in vivo assays. This compound has also been shown to inhibit EGF-induced DNA synthesis in in vitro testing in epithelial cell lines. The isolation and structural determination of the 540 compound and its activity are described in greater detail in related U.S. application Ser. No. 08/391,139, entitled Cinnamoyl-C-Glycoside Chromone Isolated from Aloe barbadensis, filed Feb. 21, 1995, which is incorporated herein by reference.
Several commercial processes employed in the processing of slurries from aloe leaf extracts utilize activated carbon (also referred to as activated charcoal) to improve the color of the extracts. Treatment with activated carbon is particularly common with so-called "whole-leaf processing", wherein the entire leaf is ground and processed. A series of experiments on aloe extracts, which had been isolated from the Aloe barbadensis plant, followed by treatment with activated carbon led to the discovery that certain activity associated with the 540 compound was not present in the extracts after treatment with the activated carbon. This led to the discovery that the 540 compound is absorbed by activated carbon. (See, U.S. application Ser. No. 08/391,139, entitled Cinnamoyl-C-Glycoside Chromone Isolated from Aloe barbadensis, filed Feb. 21, 1995). A key feature in the method of the first embodiment of the present invention is the extraction of compound 540 from decolorizing agents, such as activated charcoal, using an organic solvent, preferably an alkyl alcohol, such as ethanol and methanol or mixtures containing methanol or ethanol. A particularly attractive aspect of this procedure is the use of spent (waste) activated charcoal or decolorizing agent from commercial processes as the starting material.
In a second embodiment of the present invention crude aloe extract, isolated from the leaf rind of the aloe plant is purified by column chromatography without first being treated with a decolorizing agent. This method cuts out a step in the processing of the extracts and provides a fast and simple method of purification, which is ideally suited for large scale production.
In the first embodiment of the present invention, described in Example 1, a decolorizing agent, isolated from the processing of a slurry prepared from aloe leaf extracts, is treated with an organic solvent to extract the absorbed 540 compound. In a preferred embodiment the slurry from which the decolorizing agent is isolated is prepared from the whole leaf or the leaf rind of the Aloe barbadensis plant. Because the majority of the 540 compound is present in the leaf rind or latex, in the most preferred embodiment the slurry is prepared from the leaf rind or latex. In the preferred embodiment the decolorizing agent is activated charcoal, however any of the synthetic resins which are currently used as decolorizing agents, such as, AMBERLITE® resin (e.g. XAD-2 resin) can also be used.
The organic solvent used to extract the 540 compound from the decolorizing agent can be selected from an alkyl alcohol, such as methanol or ethanol, or can be selected from other organic solvents or mixtures containing MeOH or EtOH typically used in extraction processes, such as acetone. Because the decolorizing agent is wet water soluble organic solvents are preferred. The use of water insoluble solvents, such as methylene chloride, requires that the decolorizing agent be dried prior to extraction, which becomes extremely costly on a large scale. Additionally, methanol and ethanol were found to be more efficient at extracting the 540 compound from the decolorizing agent than acetone (as determined by analytical HPLC) (See Examples 1 and 3). In the most preferred embodiment, therefore, the organic solvent is an alkyl alcohol selected from the group consisting of methanol or ethanol or mixtures containing MeOH or EtOH. The organic solvent acts both to extract the 540 compound from the decolorizing agent and to regenerate the decolorizing agent for further use in the processing of aloe leaf extracts.
The extraction can be conducted within a temperature range of ambient temperature to the boiling point of the solvent being used. The preferred temperature range is 40°C-60°C C.
In another embodiment of the invention, described in Examples 2 and 3, the organic extract is concentrated and the residue is dissolved in water. The aqueous solution is then extracted with an organic solvent. This step serves to remove a number of impurities, particularly the anthraquinones, that are present in the crude extract and remain in the aqueous layer. In the preferred embodiment the organic solvent is selected from the group consisting of: n-butanol, ethyl acetate or methylene chloride. In the most preferred embodiment methylene chloride is used. Following extraction from water, the 540 compound is approximately 30-35% pure. Finally, preparative high pressure liquid chromatography (HPLC) may be used to further purify the 540 compound. The 540 compound is isolated in greater than 95% purity by collecting the appropriate elution volume from HPLC.
The present invention therefore includes a method for purifying the 540 compound isolated from either whole leaf aloe extract or leaf rind extract comprising the steps of: (1) contacting a decolorizing agent, which has been isolated from the processing of aloe leaf extracts, with an organic solvent to extract the 540 compound from the decolorizing agent into the organic solvent, thereby forming a mixture of decolorizing agent and organic solvent containing the 540 compound; and (2) subsequently filtering the mixture so as to separate the decolorizing agent from the organic solvent.
Optionally, the method comprises an additional step (3) wherein the 540 compound isolated from step (2) is concentrated, dissolved in water and further extracted with an organic solvent, such as, n-butanol, ethyl acetate or methylene chloride to remove any water soluble impurities that may be present.
Alternatively, after step (2) or after step (3), the resultant organic phase can be concentrated and further purified by preparatory HPLC.
In a second embodiment of the present invention crude aloe extract isolated from the leaf rind is purified by passage over Sephadex® (Example 6) or neutral alumina (Example 5) without first being treated with a decolorizing agent. In the preferred embodiment the 540 compound is further purified using alumina. Neutral alumina removes all of the acidic impurities from the crude product, including any phenolic compounds and yields a product that is approximately 64% pure. Additionally, the procedure is simple and fast and ideally suited for large scale production.
The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention.
Extraction of the 540 Compound from Activated Carbon using Methanol and Acetone (room temperature).
Five hundred grams of freeze-dried, spent activated carbon from whole leaf production was placed in a 2 liter beaker. One and one half liters of methanol was added and the slurry was stored at room temperature for 2 hours. After 2 hours, the slurry was filtered through 0.5 micron cellulose filter paper. The filtrate was stripped of methanol under vacuum and the residue subjected to HPLC analysis. The procedure was then repeated using acetone as the solvent.
Analytical HPLC Analysis
Column: ODS-1, 4 mm×250 mm, 5 μ particle size
Temperature: Ambient
Mobile Phase: 55% methanol/45% water
Flow Rate: 0.70 ml/min
Detector Wavelength: 205 nm
Injection Volume: 10-20 μl
Sensitivity: 0.20 AUFS
Retention Time: Approximately 16-17 min.
HPLC analysis shows that methanol is more effective at extracting the 540 compound from the decolorizing agent than acetone. An illustrative HFLC spectrum of the 540 compound is depicted in FIG. 1.
Isolation of the 540 Compound from Activated carbon 40°C C.
Five hundred grams of dried, spent activated carbon from whole leaf processing was placed in a 4 liter beaker. One and one-half liters of methanol was added to the beaker and the slurry was stirred for 1 hour at 40°C C. The slurry was then filtered through 0.5 micron filter paper, using 250 ml of fresh methanol as a wash. The methanol was evaporated under vacuum and the residue was dissolved in 500 ml of distilled water. The aqueous solution was then extracted with methylene chloride (3×100 ml). The methylene chloride extracts were combined and evaporated and 20 ml of methanol was added to the residue and the slurry was centrifuged. The above procedure was then repeated using acetone in place of methanol.
Analytical HPLC analysis of both extracts (using the conditions set forth in Example 1) indicated that a number of the impurities, specifically the anthraquinones, had been removed from the crude extract. The product isolated was approximately 30-35% pure as determined by HPLC.
Both pools were combined and further purified by preparative HPLC to yield the 540 compound in greater than 95% purity.
Preparative HPLC.
Column: ODS-1, 22 mm×250 mm, 10 1 μ particle size
Mobile Phase: 45% methanol/55% water
Temperature: Ambient
Flow Rate: 15 ml/min
Detector Wavelength: 205 nm
Sensitivity: 0.05 AUFS
Product eluted between 20-24 minutes. The solvent was evaporated and the residue lyophilized. Structural analysis was then performed on the lyophilized product which confirmed the characterization of compound 540. The structural elucidation was accomplished by use of nuclear magnetic resonance (1H) spectroscopy and mass spectrometry, evaluation of ultraviolet absorbance, melting point and thin-layer chromatographic behavior. The results were as follows:
Analytical data and methodology:
Melting Point: sublimes>90°C C.
Thin-layer chromatography:
TLC plate: Silica
Solvent system: ethyl acetate/methanol/water (100:13:3, v/v/v)
Rf: 0.69
UV Spectrum
Solvent: CH3OH
λmax (log e): 246 nm (4.22), 254 nm (4.27), 284 nm (4.36)
Proton NMR analysis:
Instrument: General Electric model QE 300 spectrometer
Frequency: 300 mHz
Solvent: CD3OD
Reference: CH3OH, 330 ppm
Spectral data: 1.30 (3H, d, 6 Hz) (2-γ-methyl); 2.74 (3H, s) (5-methyl); 2.82 (2H, dd, 14 Hz, 6.5 Hz) (2-α-methylene); 3.42-3.50 (1H, m) (5'-H); 3.61 (1H, dd, 10 Hz) (4'-H); 3.70-3.80 (2H, m) (3'6'a-H); 3.89 (3H, s) (7-methoxy); 4.00 (1H, m) (6'b-H); 4.37 (1H, m) (2-β-carbionl); 5.21 (1H, d, 10 Hz) (1'-H); 5.73 (1H, dd, 10 Hz) (2'-H); 6.13, (1H, s) (3-H); 6.27 (1H, d, 16 Hz) (cinnamoyl β-H); 6.81 (1H, s) (6-H); 7.37 (2H, m) (cinnamoyl m-H); 7.45 (1H, d, 16 Hz) (cinnamoyl β-H); 7.50 (3H, m) (cinnamoyl o-, p-H)
Mass spectral analysis:
Instrument Finnigan MAT 4615 quadrupole mass spectrometer
Ionization: electron impact (70 eV)
Source temperature: 160°C C.
Sample introduction: direct insertion probe
Sample heating: ballistic; peak evaporation at approximately 250°C C.
Spectral data (relative to intensity in parenthesis): m/z 540 (12), [M]+; m/z 522 (5), [M-H2O]+([M-18]+); m/z 496 (4), [M--CH3CHO]+(*) ([M-44]+); m/z410 (16), [M-cinnamoyl]+(**) ([M-130]+); m/z 392 (10), [M-(130+18)]+; m/z 376 (4), [M-(18+cinnamate)]+(**) ([M-(18+146]+; m/z 366 (8), [M-(130+44)]+; m/z 343 (18), [M-(18+147+33)]+(***) ; m/z 277 (50), [M-(130+133)]+(****); m/z 259 (100), [M-(130+18+133)]+; m/z 233 (92), [M-(130+44+133)]+; m/z 193 (73), [M-(130+84(*****)+133)]+; m/z 131 (40), [cinnamoyl]+; m/z 103 (33), [C6H5CHCH]+
Extraction of the Crude 540 Compound from Water.
One hundred grams of methanol eluate from spent activated carbon from whole leaf processing, as described in Example 2, was dissolved in 1500 ml of distilled water. The solution was divided into three equal portions, each of which was extracted with one the following solvents (3×10 ml): ethyl acetate, n-butanol and methylene chloride. The organic extracts were analyzed by HPLC, which indicated that methylene chloride was the most effective of the three solvents for extraction of the 540 compound from water.
Extraction of the 540 Compound from Activated Carbon using Ethanol and Acetone (40°C C.).
Fifty grams of dried spent activated carbon from whole leaf processing was placed in a 500 ml beaker. Acetone (200 ml) was added and the mixture was stirred at 40°C C. for one hour. The resultant slurry was filtered through 0.5 micron cellulose filter paper and the cake washed with 100 ml of fresh acetone. The acetone was stripped and the sample freeze-dried. This procedure was repeated from the beginning with another 50 grams of spent activated carbon using ethanol. The yield from the acetone elution was 1.34 grams and the yield from the ethanol elution was 5.24 grams.
Purification of the 540 Compound using Alumina.
Neutral alumina oxide (A1203) (600 g), purchased from Sigma (Type WN-3), was placed in a Büchner funnel. A methanolic solution of compound 540 from the rind, which had not been treated with a decolorizing agent (400 ml of a 14.68% solution as measured by HPLC) was applied to the alumina and eluted with 2 liters of methanol, which was collected as follows:
| Elution | Dry | Purity of | ||
| Volume | Weight (g) | Compound 540 (%) | ||
| Fraction 1 | 0-50 | ml | 0.350 | 100 |
| Fraction 2 | 51-100 | ml | 0.461 | 96.77 |
| Fraction 3 | 101-1000 | ml | 7.6 | 64.86 |
| Fraction 4 | 1001-2000 | ml | 0 | |
Purity was measured by HPLC analysis of the eluent. (See FIG. 2). HPLC operating conditions were the same as described in Example 1, except the detector wavelength was 297 nm and flowrate was 1.0 ml/min.
Purification of the 540 Compound on a Sephadex Column.
Sephadex (200 g, LH-20, 30-100 μm mesh, purchased from Pharmacia Biotech Co., California) was packed into a 5.0×40 cm chromatography column. A methanolic solution of compound 540 (10 g in 25 ml methanol) was applied to the column and eluted with a 1:1 solution of methanol and water at a flow rate of 5-6 ml/min. (The 540 compound used in this example was obtained by ethanol extraction of activated carbon and was approximately 16% pure as determined by analytical HPLC). After 150 ml of solvent had eluted, 50 ml fractions were collected and analyzed by HPLC. Fractions 7-11 were combined, concentrated and lyophilized to yield 633 mg of compound 540 (35.8% pure)
Jia, Qi, Padmapriya, Abeysinghe, Waller, Todd
| Patent | Priority | Assignee | Title |
| Patent | Priority | Assignee | Title |
| 4966892, | Jun 05 1908 | CARRINGTON LABORATORIES, INC | Processes for preparation of aloe products products produced thereby and compositions thereof |
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