Food or edible material: processes – compositions – and products – Processes – Separating a starting material into plural different...
Reexamination Certificate
1998-12-21
2002-03-26
Wilson, James O. (Department: 1623)
Food or edible material: processes, compositions, and products
Processes
Separating a starting material into plural different...
C426S425000, C426S429000, C426S478000, C514S733000, C514S736000
Reexamination Certificate
active
06361815
ABSTRACT:
The present invention relates to trihydroxystilbenes and the glycosylated derivatives thereof. The present invention also relates to methods for isolation and purification of these products using reverse phase liquid chromatography and a method for converting glycosylated to aglycone product. The present invention further relates to treatment of diseases using compounds of the invention.
Resveratrol, 3,4′,5-trihydroxystilbene, was first isolated from grape leaves (Inghim, T. L.,
Phytochem.,
15 (1979) (1976)). Inghim characterized the structure of resveratrol using chemical methods. Resveratrol has following chemical structure in which both R
1
and R
2
are hydrogen.
Where &bgr;-glc is
O
-&bgr;-D-glucose
Trihydroxystilbenes and derivatives thereof derivatives are reported to have medicinal properties including anti-leukemic and anti-tumor activities. For example, plant material containing resveratrol has been used as an herbal medication for treatment of hyperlupemia and liver diseases in China and Japan for many centuries (Kimura, K. M. et al.,
Shoygakugaku Zasshi,
83, 35-58 (1981)). Subsequent experiments with purified trans-resveratrol demonstrate that the many biologically useful functions, including modulation of hepatic cholesterol synthesis, inhibition of lypooxygenase activity (Kimura, Y. et al.,
Biochem. Biophys. Acta.
834, 275 (1985)), inhibition of anaphylactoid (Ragazy, E., et al.,
Pharmacol. Ref. Commun.,
79, 20 (1988)), and protection of lypoproteins against oxydative and free radical damage (Frankel, E. N., et al.
Lancet,
1, 1017 (1979)).
Recent literature reports indicate that the extract derived from
Cassia quinquangulata Rich
(
neguminasae
) collected in Peru is a potent inhibitor of cyclooxygenase (COX) (Kudo, T. et al.,
Gann,
71, 260 (1980), Pollard, M. et al.,
Cancer Lett.,
21, (1983), Waddell, W. R. et al.,
Am. J. Surg.,
157, 175 (1989), Thun, M. J. et al.,
N. Engl. J.Med.,
325, 1593 (1991)).
In the following discussion, ED
50
represents effective dosage for 50% inhibition.
In a recent article, Meishiang Jang reported that resveratrol inhibits the hydroperoxidase activity of COX-1, ED
50
=3.7 &mgr;M, and also hydroperoxidase activity of COX-2, ED
50
-85 &mgr;M (Jang, M. et al.,
Science,
275 218 (1997). This inhibitory activity is relevant to cancer therapy and prevention because COX catalyzes the conversion of arachidonic acid to pro-inflammatory substances such as prostaglandins, which can stimulate tumor cell growth and suppress immune responses (Plescia, O. J. et al.,
Proc. Nat. Acad. Sci. USA.,
72, (1975)).
3,4′5-Trihydroxystilbene (Resveratrol) has also been found to inhibit certain events associated with tumor growth. For instance, resveratrol inhibits the free radical formation, ED
50
=27 &mgr;M, when human promyclocytic leukemia cells were treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) (Shama, S. et al.
Cancer Res.
54 5848 (1994). Moreover, Jang et al. investigated the anti-inflammatory activity of resveratrol. In the carrageenan-induced model of inflammation in rats, resveratrol significantly reduced pedal edema both in the acute phase (3 to 24 hours) and the chronic phase (24 to 144 hours). The edema-suppressing activity of resveratrol was greater than that of phenylbutazone and similar to that of indomethacin. Jang et al. also investigated the effect of resveratrol in a mouse mammary gland culture model of carcinogenesis. Resveratrol, in a dose-dependent manner, inhibited the development of DMBA-induced preneoplastic lesions (ED
50
=3.1 &mgr;M). (Jang, M. et al.,
Science,
275, 218 (1997)).
There has recently been an increase in interest in resveratrol and analogous compounds as a result of epidemiological data showing a lower incidence of mortality due to cardiovascular damage in populations with a high-calorie high-lipid diet, but whose diet also includes red wine, as compared to populations who had a lower calorie consumption and lower percentage of lipids, but whose diet does not include red wine (Seigneur, M. et al.
J. Appl. Card.,
5, 215 (1990); Siemann, E. H. and Creasy, L. L.,
Am. J Enol. Vitic.
43, 49 (1992); Renaud, S. and De Lorgeril, M.,
Lancet,
339, 1523 (1992); Scharp, D., Lancet. 341 27 (1993)).
Investigations have revealed that resveratrol effectively possesses many pharmacological activities which can potentially explain the protective effects of red wine at the cardiovascular level (Frankel, E. N. et al.,
Lancet,
341 454 (1993)). In addition, resveratrol has proved capable of promoting the formation of nitroxides which have a vasodilatory action and inhibit platelet aggregation induced by collagen or ADP (Fitzpatrick, D. et al.,
Am. J Physiol.,
265 (
Heart Circ. Physiol.
), 34 774 (1993)).
Plant materials that are natural sources for resveratrols include
Vitis vinifera
and
Polygonum cuspidatum
(Huzhang). The concentration of resveratrol in
P. cuspidatum
is much higher than in
V. vinifera.
The procedures currently practiced for isolating resveratrols from plant materials are very difficult and low yielding normal phase chromatographic procedures that also use chlorinated solvents which are toxic to humans and can damage the environment.
The isolation of resveratrols from natural sources represents a potential reliable source of supply. The present invention provides an isolation and purification technique which provides high yields and low cost of production of resveratrol and related compounds.
SUMMARY OF THE INVENTION
By the present invention, products containing a stilbene fraction, compositions containing these products, and reverse phase liquid chromatography processes for isolating and purifying these products from plant material are identified.
The present invention provides a first product having a solids content of at least about 60% wherein the solids include at least about 10% by weight of a stilbene fraction and a process for making the product that includes the step of contacting a plant material with an alcohol and obtaining the product from the alcohol after contacting.
Also provided is a second product obtained by mixing the first product with a pharmaceutically acceptable processing excipient and drying the resulting mixture.
The present invention further provides a third product made up of at least about 20% by weight of a mixture of trihydroxystilbenes and mono-&bgr;-D-glycosylated trihydroxystilbenes and a composition of the third product with an aqueous solvent. According to the present invention, the composition including the third product is made by an MD-1 reverse phase liquid chromatography process.
Similarly, the present invention provides fourth products made up of at least about 30% by weight of a stilbene fraction including trihydroxystilbenes and mono-&bgr;-D-glycosylated trihydroxystilbenes and compositions of these fourth products with aqueous solvents. Fourth products are made using an MD-2 process starting with a composition containing the third product in which a polyamide resin is the stationary phase. A composition containing the third product is concentrated to form a loading concentrate that is loaded onto an MD-2 column, optionally using a washing elution volume, followed by elution with one or more MD-2 elution volumes of an aqueous solvent, especially a mixture of an alcohol and water to make an MD-2 effluent that is a composition containing a fourth product. The effluent is collected in toto or as gradient fractions collected by fractionate collection. Fourth products are obtained by removing aqueous solvent from an MD-2 effluent, however collected.
The present invention also provides fifth products that are made up of at least about 60% of a stilbene fraction. A fifth product can be at least about 85% by weight mono-&bgr;-D-glycosylated trihydroxystilbenes or at least about 85% aglycone thereof. Also provided are compositions including fifth products and an aqueous solvent. Fifth products are made by an MD-3 process in which the stationary phase is silica gel based. The starting material for an
He Kan
Kim Calvin Hyungchan
Zheng Bo Lin
Zheng Qun Yi
Dechert
Owens, Jr. Howard V.
Pure World Botanicals, Inc.
Wilson James O.
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