Purification of cinnamoyl-C-glycoside chromone

Details Purification of cinnamoyl-C-glycoside chromone Purification of cinnamoyl-C-glycoside chromone

1999-10-07

2002-06-25

Khare, Devesh (Department: 1623)

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

Organic compounds

Carbohydrates or derivatives

C536S004100, C536S124000, C536S127000

Reissue Patent

active

RE037771

ABSTRACT:

FIELD OF INVENTION
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,
8-C-&bgr;-D-[2-O-(E)-cinnamoyl]glycopyranosyl-2-[(R)-2-hydroxy]propyl-7-methoxy-5-methyl-chromone.

8
-
C
-
&bgr;
-
D
-
[
2
′
-
O
-(
E
)-
cinnamoyl]glycopyranosyl
-
2
-[(
S
)-
2
-
hydroxy]propyl
-
7
-
methoxy
-
5
-
methylchromone,
which has a molecular weight of 541 Daltons, and is referred to herein as the “540 compound.”
BACKGROUND OF THE INVENTION
The aloe plant is an intricate plant which contains many biologically active substances. (Cohen et al. in Wound Healing/Biochemical and Clinical Aspects, 1st ed. W B Saunders, Philadelphia (1992)). Studies have shown that these biologically active substances are located in three separate sections of the aloe leaf—a clear gel fillet located in the center of the leaf, in the leaf rind or cortex of the aloe leaf and in a yellow fluid contained in the pericyclic cells of the vascular bundles, located between the leaf rind and the internal gel fillet. Historically, aloe products have been used in dermatological applications for the treatment of burns, sores and other wounds. These uses have stimulated a great deal of research on identifying compounds from aloe plants that have clinical activity, especially anti-inflammatory activity. (See, e.g., Grindlay and Reynolds (1986) J. of Ethnopharmacology 16:117-151; Hart et al. (1988) J. of Ethnopharmacology 23:61-71).
A variety of methods are currently used for the isolation and purification of products from the aloe plant. In conventional prior art methods the entire aloe leaf is crushed to produce an aloe vera juice, which is then purified by various steps of filtration and stabilization, to yield a purified mixture of compounds. See, McAnnalley, U.S. Pat. No. 4,966,892, entitled Processes for Preparation of Aloe Products, Products Produced Thereby and Composition Thereof, for a detailed explanation of conventional prior art methods. As explained by McAnnalley, conventional methods fail to take into account that the three separate segments of the aloe leaf—the gel fillet, leaf rind and yellow fluid or latex—have varied compositions containing compounds which may be inconsistent with the intended use of the final composition. McAnnalley describes an improved method of purification wherein the various segments of the aloe leaf are first separated prior to processing.
In other more recent methods, the purification of individual compounds from various aloe plants is described. For example, Holdsworth describes a purification scheme for two Aloe chromones—Aloesin and Aloesone—using chromatography followed by counter current extraction. (Holdsworth (1972) Chromones in Aloe Species, Part I-Aloesin. PM 19(4): 322-325; Holdsworth (1972) Chromones in Aloe Species, Part II Aloesone, PM 22(1): 54-58. Speranza and coworkers describe the purification and identification of a number of Aloe chromones using a combination of droplet counter current chromatography and flash chromatography (Speranza et al. (1986) Phytochemistry 25: 2219-2222); and a combination of flash chromatography and high pressure liquid chromatography (HPLC) (Speranza et al. (1985) Phytochemistry 24:1571-1573; Gramatica et al. (1982) Tetrahedron Letters 23:2423-2424).
In one process for isolating the 540 compound, gel or rind slurry of the Aloe barbadensis plant is dialyzed, suspended in ethanol, agitated and centrifuged. (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). The ethanol is stripped from the supernatant, and the resultant product extracted with a mixture of chloroform and water. The chloroform phase is evaporated and lyophilized, and then taken up in ethanol. Use of this process indicated retained activity, however, the process failed to yield compound 540 of sufficient purity.
Neither this process nor any of the other reported prior art processes for purification of aloe compounds, provide a commercially viable process for the purification of compound 540.
SUMMARY OF THE INVENTION
The present invention includes a process for isolating and purifying the cinnamoyl-C-glycoside,
8-C-&bgr;-D-[2-O-(E)-cinnamoyl]glycopyranosyl-2-[(R)-2-hydroxy]propyl-7-methoxy-5-methyl-chromone

8
-
C
-
&bgr;
-
D
-
[
2
′
-
O
-(
E
)-
cinnamoyl]glycopyranosyl
-
2
-[(
S
)-
2
-
hydroxy]propyl
-
7
-
methoxy
-
5
-
methylchromone
from the aloe plant. In one embodiment of the present invention, the method comprises: 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; separating the decolorizing agent from the organic solvent to yield a solution highly enriched in compound 540. Optionally, the 540 compound is further purified using reversed phase high pressure liquid chromatography (HPLC) or by conventional chromatographic methods, such as column chromatography.
In a second embodiment of the present invention crude extracts of the 540 compound, isolated from the leaf rind of the aloe plant, are purified by column chromatography. This method of purification comprises: purifying the crude methanol or ethanol extract by passage over neutral alumina or sephadex.
The present invention provides a commercially viable process for the purification of compound 540 having desirable physiological activity.


REFERENCES:
patent: 4966892 (1990-10-01), McAnnalley
Gramatica et al. (1982) Tetrahedron Letters 23:2423-2424.
Hart et al. (1988) J. of Ethnopharmacology 23:61-71.
Holdsworth (1972)Chromones in Aloe Species, Part II—Aloesone, PM 22(1):54-58.
Holdsworth (1972)Chromones in Aloe Species, Part I—Aloesin, PM 19(4):322-325.
Speranza et al. (1985) Phytochemistry 24:1571-1573.
Speranza et al. (1986) Phytochemistry 25:2219-2222.
Okamura et al., “Three Chromone Components from Aloe Vera Leaves”, Phytochemistry, vol. 43, No. 2, pp. 495-498, 1996.*
Hutter et al., Journal of Natural Products, vol. 59; 541-543, (1996).

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