PRODUCTS COMPRISING TRIHYDROXYSTILBENES AND DERIVATIVES THEREOF AND METHODS FOR THEIR MANUFACTURE AND USE

Abstract: Provided are products including trihydroxystilbenes and glycosylated derivatives thereof. Also provided are compositions containing these products with an aqueous solvent, particularly an alcohol-water mixture, and reverse phase chromatographic methods for isolating and purifying the compositions from plant materials. The products have biological activity, including anti-tumor activity.

Description: PRODUCTS COMPRISING TRIHYDROXΥSTILBENES AND DERIVATIVES THEREOF AND METHODS FOR THEIR MANUFACTURE AND USE 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., Phvtochem.. 15 (1979) (1976)). Inghim characterized the stnicture of resveratrol using chemical methods. Resveratrol has following chemical stnicture in which both Ri and R 2 are hydrogen. (ra j-resveratrol (1) or trans- c j-resveratrol (4) or cis- piceid (2 or 3) piceid (5 or 6) 1 R,=R 2 =H 4 R,=R 2 =H 2 R,=β-glc, R 2 =H 5 Rι=β-glc, R 2 =H 3 R,=H, R 2 =β-glc 6 R,=H, R : =β-glc Where β-glc is 0-β-D-glucose. Trihydroxystilbenes and derivatives thereof derivatives are reported to have medicinal properties including anti-leukcmic 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 actiuty (Kimura. Y et al . Diochem Biophys Acta . 834. 275 (1985)), inhibition of anaphylactoid (Ragazy , E , et al . Pharmacol Ref Commun . 79, 20 (1988)), and protection of Kpoproteins against oxydative and free radical damage (Frankel, E.N , et al. Lancet. 1, 1017 (1979)) Recent literature reports indicate that the extract denved from Cassia cjitinquangitlata Rich (negttmmasae) collected in Peru is a potent inhibitor of cyclooxygenase (COX) (Kudo, T et al , Gann. T V, 260 (1980), Pollard, M et al , Cancer Lett . 2 \, (1983), Waddell, W R et al , Am J Sure . 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, ED50 = 3 7 μM, and also hydroperoxidase activity of COX-2, ED 50 - 85 μM (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-Trihydroxystιlbene (Resveratrol) has also been found to inhibit certain events associated with tumor growth. For instance, resveratrol inhibits the free radical formation, ED 50 = 27 μM, when human promyelocytic leukemia cells were treated with 12-0-tetradecanoylphorbol-13-acetate (TPA) (Shama, S et al Cancer Res 54 5848 (1994) Moreover, Jang et al. investigated the anti-infiammatory 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 μM). (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 Physio 265 (Heart Circ. Phvsiol.). 34 774 (1993)). Plant materials that are natural sources for resveratrols include Vitis vinifera and Polygonum cuspidatitm (Huzhang). The concentration of resveratrol in P. cuspidatitm 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. T e 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 u ith 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-β-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-l reverse phase liquid chromotography 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-β-D- glycosylated trihydroxystilbenes and compositions of these fourth products with aqueous solvents. Fourth products are made using an MD-2 process starting w ith 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-β- 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 MD-3 process is an effluent, especially a gradient fraction, from an MD-2 process. The effluent is concentrated to form a loading concentrate that is eluted through the MD-3 column and can be followed by a washing elution volume. The MD-3 column is then eluted with one or more elution volumes of an aqueous solvent. Each elution volume can consist of one or more discrete gradient volumes, each made up of a different aqueous solvent, or the composition of each elution volume can vary linearly, exponentially, logarithyrnically', hyφerbolically, or stepwise during elution of the elution volume. The effluent from a first MD-3 process is a composition containing a fifth product. The effluent may be collected in toto or fractionate collected as gradient fractions. Fifth products are obtained by removing aqueous solvent from effluent or gradient fractions of a first MD-3 process. A gradient fraction of a first MD-3 process is the starting material for a cold crystallization process to make a fifth product that contains at least about 85% by weight mono-β-D-glycosylated trihydroxystilbene. The gradient fraction of a first MD-3 process is concentrated to a solids content of at least about 20g/L and then diluted with water. The resulting mixture is cooled to less than about 0°C to foπn a slurry from which such fifth product can be isolated, washed, and then dried. Similarly, the present invention provides a second MD-3 process for making compositions containing sixth products that are at least about 70% by weight, trihydroxystilbenes. Sixth products are isolated by removing aqueous solvent from the compositions. Starting material for a second MD-3 process is an MD-2 gradient fraction that has been fractionate collected. The MD-2 gradient fraction is concentrated to a loading concentrate having a solids content of at least about 7 g/L. The MD-3 column is eluted with first and second MD-3 elution volumes that are made up of aqueous solvent. Either or both MD-3 elution volumes can be made up of gradient volumes that include different aqueous solvents or the composition of the aqueous solvent of either or both elution volumes may be varied linearly, exponentially, logarithymically, hyperbolically, or stepwise during elution of the respective elution volume. Effluents corresponding to the respective elution volumes or gradient volumes can be collected in toto or fractionate collected. Sixth products are obtained by removing aqueous solvent from the effluent or gradient fractions of a second MD-3 process, however collected. An effluent or gradient fraction of a second MD-3 process is a starting material for making a sixth product that is at least about 85% by weight, 3,4',5-trihydroxy-trans-stilbene in which the effluent or gradient fraction is concentrated to a concentrated composition and twice contacting this concentrated composition with separate extraction volumes of a volatile polar organic solvent (e.g., ethyl acetate), combining the extraction volumes, and removing the volatile polar organic solvent to obtain the 85% product. An alternative process for making the 85% product is provided in which an elution volume or gradient fraction from a second MD-3 process is evaporated to dryness, the residue so formed dissolved in water at a temperature greater than 0°C to form a solution, the solution cooled to less than about 0°C to form crystals, and separating the crystals from supernatant to obtain the 85% sixth product. Tlie present invention also provides a third MD-3 process for making a composition containing a seventh product that includes at least 50% by weight of 3,4', 5- trihydroxy-c/ ' -'-stilbene. Starting material for a third MD-3 process is a fractionate collected gradient fraction of a second MD-2 process. The fractionate collected gradient fraction of a second MD-2 process is concentrated to a solids content of at least about 7 g/L to form a loading concentrate that is eluted through an MD-3 column. The third MD-3 process further includes the steps of eluting the MD-3 column with first, second, and third elution volumes that are made up of aqueous solvent. Each of the elution volumes can be made up of two or more gradient volumes in which the aqueous solvent has the same or a different composition. The elution volumes or gradient volumes result in MD-3 effluents of a third MD-3 process. The elution volumes or gradient volumes are collected to toto or fractionate collected. The effluents of the third MD-3 elution volume of a third MD-3 process, or gradient fractions thereof, are compositions containing the seventh product of the present invention. The seventh products of the present invention are obtained by removing aqueous solvent from the effluent resulting from the third MD-3 elution volume of a third MD-3 process, or gradient fractions thereof. A process for converting a mono-β-D-glycosylated trihydroxystilbene to the corresponding aglycone is also provided. The process includes the steps of providing a solution or suspension of a glycosolated trihydroxystilbene, contacting the solution or suspension with HC1 at a total concentration between 0.01 and 0.02 g/ml, and refluxing the acidified solution or suspension for about 10 to about 200 minutes. The corresponding aglycone is isolated from the reaction mixture by techniques as are known in the art. The converting process can be carried out under a blanket of inert gas, for example, nitrogen. DEFINITIONS Alcohol. As used herein, the term alcohol refers to a lower aliphatic alcohol, in particular one selected from the group consisting of methanol, ethanol. the isomeric propanols, the isomeric butanols, the isomeric pentanols, and the isomeric hexanols. Aqueous Solvent. As used herein, the term aqueous solvent refers to water or a polar organic solvent that is miscible with water in all proportions from 1 :99 to 99: 1. Examples of polar organic solvents that are, or can be used, as components of an aqueous solvent, as that term is herein used, includes but is not limited to methanol, ethanol, isopropanol, n-propanol, acetone, and acetonitrile. Other suitable polar organic solvents are known to the skilled artesian. Column Volume. As used herein, column volume refers to the volume of the space defined by the inner surface of the chromatography column or chamber that surrounds the RPLC stationary phase. Column volume is abbreviated herein as CV. Composition. As used herein, the term composition is a slurry, suspension, dispersion, or especially a solution of a material, normally solid at room temperature, in an aqueous solvent. Examples of compositions include but are not limited to loading elutions, loading concentrates, and effluents or gradient fractions from any reverse phase liquid chromatography process of the present invention. Fractionate Collecting. When used in connection with an effluent or a gradient effluent, or a gradient fraction, the term fractionate collecting denotes that the effluent or gradient effluent or gradient fraction or gradient subfraction is segregated into at least two portions or aliquots. MD-l Column. An MD-l column is a reverse phase liquid chromatography column of any size in which the stationary phase is a crosslinked copolymer of a vinyl aromatic compound, for example styrcne, cross linked with a polyvinyl aromatic compound, for example divinylbcnzene, wherein the stationary phase has a mean surface area of at least about a 400 nr/g, preferably 800 nr/g, and a porosity of at least about 0.55 ml/ml, preferably at least 0.58 ml/ml. The mean diameter of the particles comprising the stationary phase is between about 490μ and 700μ. The dipole moment of the crosslinked polymer comprising the stationary phase is less than about 0.5. Water is used for conditioning an MD- l column. MD-2 Column. An MD-2 column is a reverse phase liquid chromatography column of any size in which the stationary phase is a polyamide resin. As used herein, polyamide resin is a polymer of a lactam or a copolymer of a diamine and a dicarboxylic acid (or of the salt formed between the diacid and the diamine). Examples of polyamide resins include poly(caprolactam) and poly(hexamethylene adipamide). An alcohol water mixture comprising 10 vol-% methanol is used to condition an MD-2 column. MD-3 Column. An MD-3 column is a reverse phase liquid chromatography column of any size in which the stationary phase is silica gel based reverse phase particles having C 8 to C|_, alkane moieties or cyano moieties bonded to its surface. A suitable material is WP Octadecyl reverse phase media available from J.T. Baker, Phillipsburg, New Jersey (Cat. #7248-2). An MD-3 column is conditioned with a mixture of methanol and water comprising about 20 vol-% methanol. Percent Solid. As used herein, the quantity percent solids refers to the weight of a nominally solid composition comprising an aqueous solvent that remains after the aqueous solvent is removed. Unless otherwise indicated, the quantity percent solid is expressed as the ratio of the weight of the composition remaining after removal of aqueous solvent divided by the weight of the composition before removal of the aqueous solvent, multiplied by 100. A nominally solid composition is a composition that does not flow under its own weight at room temperature. Pharmaceutically acceptable processing excipients. Pharmaceutically acceptable processing excipients are pharmaceutically acceptable organic or inorganic carrier substances that do not react or otherwise interfere with biologically active components of pharmaceuticals or neutriceuticals and which assist in processing the biologically active components, or products containing them, into a form convenient for administering the biologically active components to an animal, including a human. Many such pharmaceutically acceptable processing excipients are known in the art. Among these, tricalcium phosphate and maltodextrin are particularly preferred. SPA. As used herein SDA refers to specially denatured alcohol. See U.S. Pharmacopoeia. Solids Component. The portion of a slurry , suspension, dispersion or solution is an aqueous solvent that remains after the aqueous solvent is removed. Synonymous with solids portion. Solids Content. As used herein, the term solids content quantifies the portion of a solution, slurry, suspension, or dispersion in an aqueous solvent that remains when the aqueous solvent is removed and is expressed in units of grams of solid remaining per liter of solution or slurry and is abbreviated g/L. Stilbene Fraction. Stilbene fraction refers collectively to the constituents or components of a material especially a solids component, that consists essentially of 1,2- diphenylethenes and substituted 1,2-diphenylethenes, where either or both of the phenyl rings can bear one or more substituents. Volume Percent. As used herein the term volume percent, abbreviated vol-%, is used to describe the composition of an aqueous solvent. The vol-% of a component represents the ratio of the volume of the component added to a composition to the total of the volumes of all components added to the composition times 100. The volume percent of an aqueous solvent can be easily calculated at the time it is formulated or it can be determined later using standard techniques, for example, GL chromatography using suitable reference mixtures. In one embodiment, the present invention provides a first product that comprises at least about 60%, preferably at least about 65%, by weight solids which solids comprise at least about 10%, preferably at least about 12%, by weight of a stilbene fraction. According to the present invention, the first product can be obtained by providing a solid plant material, preferably V. vinifera, more preferably P. cuspidatitm, which plant material has been cut or ground to pieces having an average volume from about 0.001 mm 3 to about 15 mm 3 , and contacting the plant material with an aqueous solvent, preferably an alcohol-water mixture comprising about 75 volume percent (vol-%) SDA. The contacting may be by any suitable means as are known in the art; for example, percolation, vat extraction, counter current extraction, and the like. The first product can then be obtained by removing aqueous solvent or a component thereof from the resulting composition. In this or any embodiment, aqueous solvent can be removed by any of the means as are known in the art such as evaporation, distillation, and lyophilization, to mention a few. The first product can be used to prepare a second product that can be directly administered to an animal, including a human, and which second product has a stilbene fraction amounting to at least about 8% by weight and at least one pharmaceutically acceptable processing excipient. According to one embodiment of the present invention, the second product is made by slurrying the first product in water and homogenizing the slurry with one or more pharmaceutically acceptable processing excipients. A Silverson Model 14 RT-A homogenizer, Silverson Corporation, East Longmeadow, MA is suitable for this purpose. The homogenized mixture is then dried by spray drying or vacuum drying. According to another embodiment of the invention, the first product is also useful as a starting material for preparation of products having a stilbene fraction of at least about 20% by weight or for the preparation of tπhydrowstilbenes and gly cosylated deπvatu cs thereof by employing reverse phase liquid chromatography processes In reverse phase liquid chromatography (RPLC) as practiced in embodiments of the present invention, the column packing (stationary phase, or adsorbent) is non-polar, typicalh having a dipole moment of 3 or less. Silica gel that has been treated to provide it with a bonded surface layer that is paraffinic in nature is an example of a stationary phase for reverse phase chromatography. Silica gels having permanently bonded C 8 to Cm alky 1 groups are commercially available as a stationary phase Reverse phase liquid chromatography columns are eluted with eluents of decreasing polarity which causes the more polar compounds loaded on a column to elute first. Reverse phase liquid chromatography stationary phases of organic material are also known. Polymers of vinyl aromatic compounds, for example styrene, that are heavily crosslinked with polyviny c aromatic hydrocarbons, for example diviny lbenzene, can be used as stationary phases for reverse phase liquid chromatography These organic polymeric stationary phases are made by processes that yield small, extremely rigid, macroreticular particles Highly crosslinked acrylic polymers are also useful as stationary phases for reverse phase liquid chromatography. Suitable stationary organic phases are commercially available. For example, styrenic and acrylic stationary phases are available from the Rohm and Haas Company, Philadelphia, PA, under the trade name Amberhte® Styreneic stationary phases are also available under the trade name Amberchrom® from Tossohass, Montgomeryville, PA Polyamide resins (e g nylons), polyester resins, and phenolic resins are also useful stationary phases for the reverse phase chromatography processes of the present invention Many polar organic solvents are suitable eluents for reverse phase liquid chromatography Lower alcohols, such as methanol, ethanol, and propanol. as well as nitπles such as acetonitπle, are used as organic eluents Lower aliphatic ketones such as acetone methyl ethyl kctone, and diethyl ketone, as well as cyclic ethers such as tctrahvdrofiiran, can also be used. Dimethyl formamide, dimethyl sulfoxide, and alkyl esters of acetic acid such as ethvl acetate can also be used. Mixtures of such solvents in various proportions can be used when it is desired to elute or wash the column with solvents of varying polarity, from high to low relative polarity. Applicants have found that mixtures of water and an alcohol, for example, methanol, ethanol, n-propanol, iso-propanol, n-butanol, and n-and sec-hexanol, are particularly useful as mobile phases or eluents for separating and purifying stilbene compounds, especially those obtained from plant material. The RPLC processes of the present invention are advantageously carried out using an eluent of variable composition, that is a so-called gradient eluent. The limits of concentration of gradient eluents are determined by the concentration of polar organic solvent necessary to elute products from the stationary phase and by the requirement that the polar organic solvent be miscible to form a single phase at the required concentration. In certain embodiments of the present invention the initial alcohol concentration is 10 volume percent (10 vol-%) or less and is increased as separation and purification proceeds. The reverse phase liquid chromatography systems used to practice the present invention may be either preparative or analytical. Preparative columns require larger loading capacity and are typically larger in size. Flow rates of the eluent are adjusted according to the column dimensions, the degree of separation desired, the particle size of the stationary phase, and the back pressure in the column. The separation is typically carried out at 20°C to 30°C. However, a temperature up to about 45°C can be used. The separation may be carried out at high pressure (500-200 psi) or moderate pressures (100-500 psi) or, preferably, at lower pressures (10-100 psi). With regards to the dimensions of the reverse phase liquid chromatographic column, the loading of the column, the temperature, and flow rate, one skilled in the art will know to vary these parameters based primarily upon practical considerations known in the art. The product to be chromatographically treated is generally provided as a solution or suspension in an aqueous solvent. Preferably, the aqueous solvent is a mixture of an alcohol and water having a volume percent alcohol between about 5 vol-% and about 20 vol-%, as determined by known methods, for example gas chromatography. The concentration of product in the solution or suspension to be chromatographically treated is also varied according to the particular embodiment, but is generally between about 0.1 and about 10 g/L. Preferably, the concentration of the product to be treated is such that column loading is between about 1 g L and 12 g/L. The reverse phase liquid chromatography column can be conditioned by eluting the column with a conditioning volume of a conditioning liquid, preferably an aqueous solvent. The conditioning volume is preferably between about 1 and about 10 column volumes. The product to be treated is loaded onto the conditioned stationary phase of the reverse phase chromatography column by means of a solution, a slurry, or, a loading concentrate obtained by evaporating an aqueous solvent, preferably alcohol, from a composition containing the product. Loading of the column is accomplished by eluting the solution, slurry, or loading concentrate through the column. Preferably, elution of the solution, slurry, or loading concentrate is followed by elution with a washing elution volume comprising an aqueous solvent having the same composition as the aqueous solvent of the solution, slurry, or loading concentrate used to load the column stationary phase. The washing elution volume, when one is used, is preferably between about 1 and about 10 column volumes. A further embodiment of the present invention provides a third product from an MD-l reverse phase liquid chromatography process having at least about 20% by weight and more preferably at least about 24% by weight of a mixture of trihydroxystilbenes and mono-β- D-glycosylated trihydroxystilbenes. In a preferred embodiment of the MD- l process, the first product of the present invention is slurried in an aqueous solvent, preferably a mixture comprising between about 3 vol-% and about 7 vol-% alcohol, preferably methanol The first product is loaded onto an MD- l column support by eluting the slurry through the MD-l column and can be followed by a washing elution volume including an aqueous solvent that is preferably a mixture of alcohol and water having between about 5 vol-% and about 20 vol-% alcohol. preferably methanol. A composition including the third product can be eluted from the loaded MD-l column stationary phase with a first MD-l elution volume to produce a first MD-l effluent. The first MD-l elution volume includes an aqueous solvent, preferably a mixture of alcohol and water having between about 70 vol-% and about 80 vol-%, preferably about 75 vol- %, of an alcohol, preferably a methanol. Aqueous solvent can be removed from the first MD-l effluent composition by any suitable means as discussed above, to obtain the third product. Yet another embodiment of the present invention provides a fourth product having at least about 30% by weight of a stilbene fraction having a mixture of trihydroxystilbenes and mono-β-D glycosylated trihydroxystilbenes. The fourth product can be obtained by a first MD-2 reverse phase liquid chromatography process. Starting material for a first MD-2 process is a first MD-2 loading concentrate having a third product of the present invention in an aqueous solvent, preferably a mixture of alcohol and water comprising not more than about 20 vol-% alcohol, preferably methanol. The MD-2 loading concentrate can be made by removing sufficient aqueous solvent from the first MD-l effluent resulting from the first MD-l elution volume so that the solids content of the first MD-2 loading concentrate is at least about 10 g/L, preferably at least about 13 g/L. The third product is loaded onto an MD-2 column stationary phase by eluting the first MD-2 loading concentrate through the MD-2 column which can be followed by a washing elution volume. The MD-2 column is then eluted with a first MD-2 elution volume to make a first MD-2 effluent In one embodiment, the first MD-2 elution volume is a mixture of an alcohol, preferably methanol, and water having at least about 60 vol-% and preferably at least about 70 vol-% alcohol, more preferably at least about 75 vol-% alcohol. In another embodiment, a second MD-2 process, which includes the steps of the first MD-2 process, the MD-2 column is eluted with a first MD-2 elution volume of a second MD-2 process that includes at least a first gradient volume and a second gradient volume, both of which are mixtures of an alcohol, preferably methanol and water, and both of, which can be divided into any number of subgradient volumes. In a preferred embodiment, the first gradient volume includes between about 20 vol-% and about 40 vol-%, preferably at least about 30 vol- %, of an alcohol, preferably methanol, and the second gradient volume includes between about 70 vol-% to about 80 vol-%, preferably at least about 75 vol-%, of an alcohol, preferably methanol. In those embodiments of the second MD-2 process in which the first MD-2 elution volume of a second MD-2 process has first and second gradient volumes, the effluent that results from elution of the first and second gradient volumes can be fractionate collected and segregated into first and second gradient fractions, respectively, of the second MD-2 process that are compositions containing specific embodiments of the fourth product of the present invention. Furthermore, either gradient fraction can itself be fractionate collected to obtain gradient sub fractions. In preferred embodiments of the second MD-2 process in which the first gradient volume is a mixture of alcohol and water having about 30 vol-% methanol and the second gradient volume is a mixture of alcohol and water having about 70 vol-% methanol, the first gradient fraction is a composition including a fourth product of the present invention that includes at least about 40% by weight of a stilbene fraction that includes at least about 90% mono-β-D-glycosylated trihydroxystilbenes and the second gradient fraction is a composition also including a fourth product of the present invention including at least about 30% by weight of a stilbene fraction that has at least about 80% trihydroxystilbenes, preferably 3,4 " ,5- trihydroxystilbenes. The respective fourth products can be obtained by removing alcohol-water mixture from the respective gradient fractions. Another embodiment of the present invention provides a third MD-2 process for making a composition that includes a stilbene fraction that has at least about 80% and preferably at least about 90% by weight mono-β-D-glycosylated-3,4',5-trihydroxystilbcne. In a third MD-2 process, a third product is loaded onto an MD-2 column stationary phase by means of an MD-2 loading concentrate. The MD-2 column is eluted with a first MD-2 elution volume of a third MD-2 process. In a preferred embodiment, the first MD-2 elution volume of a third MD- 2 process is an MD-2 gradient elution volume including a mixture of alcohol and water the composition of which can be varied linearly, exponentially, logarithmically, parabolically, step- wise, or according to any combination of the foregoing. The MD-2 effluent is fractionate collected to obtain one or more compositions, each of which contains a fourth product. Another embodiment of the present invention provides a fifth product that has at least about 60%, preferably at least about 65%, of a stilbene fraction containing at least about 90% by weight of mono-β-D-glycosylated-3,4\5-trihydroxy-trøm'~stilbene. This embodiment of the fifth product of the present invention can be made in a first MD-3 reverse phase chromatography process. Starting material for this first MD-3 reverse phase chromatography process is a loading concentrate made by removing sufficient aqueous solvent from the segregated first gradient fraction of the second MD-2 process or a segregated fraction of the third MD-2 process that includes a stilbene fraction that has at least about 50% of mono-β-D- glycosylated-3,4'5-trihydroxystilbenes, so that the loading concentrate has a solids content of at least about 3 g/L. In preferred embodiments in which the first gradient volume of the second MD-2 elution volume is a mixture of alcohol and water, the loading concentrate preferably has not more than about 5% alcohol. The loading concentrate is eluted through an MD-3 column to load the column stationary phase and, in preferred embodiments, is followed by a washing elution that is an aqueous solvent, preferably a mixture of alcohol and water having about 5 vol- % alcohol, preferably methanol, and the volume of the loading elution corresponds to about 0.5 to about 10 column volumes. The MD-3 column is then eluted with a first MD-3 elution volume of the first MD-3 process to obtain a first MD-3 effluent of a first MD-3 process that is fractionate collected to obtain a first fraction of a first MD-3 effluent of a first MD-3 process and a second fraction of a first MD-3 effluent of a first MD-3 process. In preferred embodiments the first fraction of the first MD-3 effluent of a first MD-3 process amounts to about 0.5 to about 3, preferably about 1.5, column volumes and the second fraction of the first MD-3 effluent of a first MD-3 process amounts to between about 0.5 and about 3 column volumes, preferably 1 column volume. The fifth product that has at least about 60% of a stilbene fraction comprising at least about 90% mono-β-D-glycosylated-3,4',5-trihydroxy- t/ww-stilbene can be obtained by removing the aqueous solvent from the fractionate collected first MD-3 effluent of a first MD-3 process. In another embodiment, the present invention provides an evaporative crystallization process for making a fifth product containing at least about 85% and preferably at least about 90% by weight 3,4',5-trihydroxy-tro/7_-stilbene-3-β-mono-D-glucoside. The starting point for the evaporative crystallization process is fractionate collected first MD-3 effluent of a first MD-3 process, preferably a second fraction of a first MD-3 effluent of a first MD-3 process that is fractionate collected after a first fraction of a first MD-3 effluent of a first MD-3 process amounting to 0.5 to about 3 column volumes is collected. The second fraction of a first MD-3 effluent of a first MD-3 process is evaporated to between about 0.1 and about 0.2 times its original volume and cooled, preferably to 4°C or below, to form crystals that are a fifth product containing at least about 85% 3,4',5-trihydroxy-/r<7/7.y-stilbene-3-β-mono-D-glucoside. In another embodiment, the present invention provides a sixth product having at least about 70% and preferably at least about 75% of a stilbene fraction including at least about 70% by weight of 3,4',5-trihydroxy-trfl/w-stilbene. The sixth product can be prepared by a second MD-3 process. The starting material for the second MD-3 process is the second gradient fraction of the second MD-2 process. Aqueous solvent is removed from the second MD-2 gradient fraction of the second MD-2 process to form a loading concentrate having a solids content of at least about 7 g/L. The loading concentrate is eluted through an MD-3 column and, in preferred embodiments, can be followed by a washing elution volume including an aqueous solvent, preferably a mixture of alcohol and water including between about 10 vol-% and about 20 vol-% alcohol, preferably methanol. The MD-3 column is then eluted with a first MD-3 elution volume of a second MD-3 process having first and second gradient volumes. The first gradient volume of the first MD-3 elution volume of the second MD-3 process is preferably an aqueous solvent that is preferably a mixture of alcohol and water having between about 35 vol-% and about 45 vol-%, preferably 40 vol-%, of an alcohol, preferably methanol, and elutes a first MD-3 gradient fraction of a second MD-3 process and is followed by elution with the second MD-3 gradient volume of a first MD-3 elution volume of the second MD-3 process that includes an aqueous solvent, preferably an alcohol-water mixture having between about 50 vol- % and about 60 vol-%, preferably about 65 vol-%, of an alcohol, preferably methanol, to elute a second MD-3 gradient fraction of the second MD-3 process. The sixth product of the present invention can be obtained by removing the aqueous solvent from the second MD-3 gradient fraction of the second MD-3 process. In other embodiments, the present invention provides a sixth product that includes at least about 85% and preferably at least about 90% /rαm--resvcratrol (3,4',5- trihydroxy-/rø/7.y-stilbcne) which can be obtained by an extraction process. In one embodiment, the extraction process includes removing aqueous solvent from the second MD-3 gradient -20- fraction of a second MD-3 process to attain a solids content of at least about 1.5g/L and twice contacting the so concentrated second MD-3 gradient volume with one or more extraction volumes, each preferably 0.5 to 2 times the volume of the so concentrated second MD-3 gradient volume, of a polar organic solvent, preferably ethyl acetate. The extraction volumes are combined and the polar organic solvent is removed to obtain the sixth product having at least about 85% by weight tra -resveratrol. Yet another embodiment of the present invention is a crystallization process for making the substantially colorless product which comprises removing the aqueous solvent from the second MD-3 gradient volume of a first MD-3 elution volume of a second MD-3 process, dissolving the resulting solid at T>10°C in methanol, cooling to T<0"C form crystals of the substantially colorless product and recovering the crystals of substantially colorless product by conventional means. In another embodiment, the invention provides a partition crystallization process for making a sixth product that contains at least about 80% and preferably at least about 85% 3,4 , ,5-trihydroxy-trø , -stilbene. Starting point for the partition crystallization process is a second MD-3 gradient fraction of a second MD-3 process. The second MD-3 gradient fraction is concentrated under vacuum to 0.35 to 0.40 times its original volume and a solid concentration of at least about 1,5 g/L. The concentrated gradient fraction is contacted with a polar organic solvent, preferably ethyl acetate. Preferably, the volume of the polar organic solvent used is between bout 0.75 and about 0.85 time the volume of the concentrated second gradient fraction. In preferred embodiments, the gradient fraction is contacted serially with two separate volumes of polar organic solvent and the volumes are combined. The polar organic solvent, from single or multiple contactings, are evaporated to dryness to yield a sixth product having at least 80% 3,4',5-trihydroxy-tra/7. y -stiIbene. -21- Yet another embodiment of the present invention provides a seventh product that has at least about 50% and preferably at least about 55% more preferably at least about 60%, by weight of a stilbene fraction that includes at least about 50% by weight of 3,4',5-trihydroxy- -stilbene. The seventh product of the present invention can be prepared by a third MD-3 reverse phase liquid chromatography process. Starting material for the third MD-3 process is a second MD-2 gradient fraction of a second MD-2 process from which aqueous solvent is removed to form a loading concentrate having a solid content of at least 7 g/L. The loading concentrate thus formed is eluted through a conditioned MD-3 column. In preferred embodiments, elution of the loading concentrate can be followed by elution of a washing elution volume including an aqueous solvent, preferably a mixture of alcohol and water having between about 5 vol-% and about 20 vol-% alcohol, preferably methanol. The washing elution volume, when used, is followed by first and second MD-3 elution volumes of the third MD-3 process to produce, respectively, first and second effluents of the third MD-3 process. The first MD-3 elution volume of the third MD-3 process can include an aqueous solvent of a particular composition and can have two or more gradient volumes that when fractionate collected, result in two or more gradient fractions of a first effluent of the third MD-3 process. The first and second MD-3 elution volumes of the third MD-3 process include aqueous solvents which, in preferred embodiments, are mixtures of alcohol and water. The first MD-3 elution volume of the third MD-3 process preferably includes a mi.xture of alcohol and water comprising up to about 70% alcohol, preferably methanol. In one embodiment, the first MD-3 elution volume of the third MD-3 process comprises first and second gradient volumes of a first MD-3 elution volume of a third MD-3 process that are mixtures of alcohol, preferably methanol, and water wherein the first MD-3 gradient volume of a first MD-3 elution volume of a third MD-3 process has between about 35 vol-% and about 45 \ol-%, preferably about 40 vol-%, alcohol and the second gradient volume of a first MD-3 elution volume of a third MD-3 process compiises between about 50 vol-% and about 60 vol-% preferably about 55 vol-%, alcohol In other embodiments, the first elution volume of the third MD-3 process is a gradient elution volume and includes an aqueous solvent the composition of which is varied over the course elution of the first elution volume of a third MD-3 process according to a predetermined program The program may be linear, exponential, logarithmic, hyperbolic, step-wise, or a combination of the foregoing For example, if the aqueous solvent is a mixture of alcohol and water, the volume percent alcohol can be varied from about 20 vol-% to about 60 vol-% during elution of the first MD-3 elution volume of the third MD-3 process The volume of this first MD-3 elution volume of the third MD-3 process is from about 1 to about 12 column volumes, preferably less than about 8 column volumes The second elution volume of the third MD-3 process is preferably a mixture of an alcohol, preferably methanol, and water including between about 80 vol-% and about 90 vol- %, preferably about 75 vol-% of alcohol The seventh product can be obtained by collecting a second MD-3 effluent of a third MD-3 process eluted by the second MD-3 elution volume of the third MD-3 process and removing the aqueous solvent therefrom The second MD-3 effluent of a third MD-3 process eluted by the second MD-3 elution volume of a third MD-3 process can be fractionate collected When the second MD-3 effluent of a third MD-3 process is fractionate collected, it mav be collected in any number of fractions In a preferred embodiment, the second effluent of the third MD-3 process is fractionate collected in two fractions The first fraction of the second effluent of the third MD-3 process preferably amounts to between about 0 5 and about 1 column volume The second fraction of the second effluent of the third MD-3 process preferably amounts to between about 0 5 and 2 0 column volumes and is a composition including the seventh product of the present invention In the following examples, "% alcohol" indicates the volume percent (vol-%) of alcohol in an alcohol - water mixture. Analysis of stilbene fractions was performed used HPLC on a Hewlett Packard Series 1 100 HPLC using an ODS H persil column. EXAMPLE 1 This example illustrates an MD-l process. The dried ground roots of Huzhang (Polygonum cuspidatitm) was extracted three times by percolation with 75% ethanol. The ethanol extract was concentrated at reduced pressure to a brown gummy semisolid (called Native Extract and abbreviated NE). The temperature during evaporation was kept between 35°C and 40 and the pressure was kept between 15-25 mm. About 1.4 kg of the NE (wet solid, Lot No. 7-1752) were dissolved in 4.9 L MeOH at 45°C and were stirred for 30 min. 44.1 L H;0 were added to the mixture (to yield 10% MeOH). The resulting mixture was loaded onto a water-conditioned, 4 in. x 35 in. MD-l column (containing Amberlite® resin, XAD-16HP). The column was eluted with a washing elution volume (2 CV of 10% MeOH) and then eluted with a first MD-l elution volume (7.7 CV of 75% MeOH) to obtain a first MD-l effluent. The first MD-l effluent (the best pool of fractions) from the MD-l column was concentrated from 126.4 L (75% MeOH ) to 35.5 L (19.5% MeOH) at 45°C for 1.5 hrs under vacuum in a still to form an MD-2 loading concentrate. Analysis indicated that the solids recovery is quantitative and the stilibene fraction of the solid component of the first MD-l effluent amounts to 24.2%. EXAMPLE 2 This example illustrates a second MD-2 process. The MD-2 loading concentrate from Example 1 was loaded onto a 10% MeOH conditioned, 4" x 49" MD-2 column having a polyamide resin as the stationary phase. The column was gradient eluted with a first gradient volume of a first MD-2 elution volume of a second MD-2 process (8 CV of 35% MeOH) and a second gradient volume of a first MD-2 elution volume of a second MD-2 process (6 CV of 75% MeOH). The first and second gradient fractions from 35% MeOH and 75% MeOH gradient volumes were fractionate collected. The solids component of first gradient fraction had a stilbene fraction of 44.4%, of which 60% was mono-β-D-glycosylated trihydroxy-t π -stilbene. The solids component of the second gradient fraction had a stilbene fraction of 35% of which 86% was trihysroxtstilbenes. EXAMPLE 3 This example illustrates removal of aqueous solvent from a composition. The alcohol - water mixture was removed from the second gradient fraction of Example 2 using a rotary evaporator (Biichi Rotavapor Model R-187 ) followed by drying in an tray vacuum oven. About 2L of the second gradient fraction (BZ 1-57-3 resveratrol pool, 75% MeOH, 35.% total stilbene, 86% thereof trr y-resveratrol and w-resveratrol) were rotary evaporated to 555 ml (approx. 10% MeOH) at 32°C and 70-1 10 psi for 7 hours. About 30 ml of the resulting solution was transferred onto a metal dish and dried in a vacuum oven at 42°C, 30" water vacuum for 15 hours. About 281.4 mg of dried fourth (resveratrol) product were obtained. EXAMPLE 4 This example illustrates making a sixth product of the present invention by a second MD-3 process. 20 L of a second MD-2 gradient fraction from a second MD-2 process (BZ1- 57-3: 16.1% trα/w-resveratrol, 8.4 g; 13.9% cώ-resveratrol, 7.2 g; 1.4% tm -piceid, 0.7 g; 3.6% -piceid, 1.8 g; total stilbene, 18.2 g; 75% MeOH) were rotary evaporated to form an MD-3 loading concentrate (6.28L, approx. 20% MeOH) that was eluted through a conditioned (20% MeOH) 4" x 52" MD-3 column (C18 bonded silica gel) to load the stationary phase of the column. The column was gradient eluted with 3.7 CV of a first MD-3 elution volume of a -25- second MD-3 process (40% MeOI I) followed by 1.5 CV of a second MD-3 elution volume of a second MD-3 process (55% MeOH) that resulted in 1.5 CV of a second MD-3 effluent of a second MD-3 process. The best pool of /ra -resveratrol was fractionate collected as the first 1.1 CV of the second MD-3 effluent of a second MD-3 process, which contained a sixth product having a stilbene fraction that was 74.7% trihydroxy-iTw/.y-stilbene. Thus, 86.2% of the trihydroxy-trø '-stilbenes present in the second gradient volume of the second MD-2 process were recovered. EXAMPLE 5 This example illustrates a partition crystallization process for purification of trihydroxy-trø -stilbene. About 15 L of the above second MD-3 effluent of the second MD-3 process of Example 4 (resveratrol best pool BZ1-60-13-17, 75% MeOH - water, 9.723g 74.7% trihydroxy-/rø/«- stilbene) were rotary evaporated to a volume of 5.7 L (approx. 33% MeOH). The solution was twice extracted with 0.79 times its volume of ethyl acetate. Ethyl acetate layers from each extraction were then combined and evaporated to dryness. About 7.8 g of dry solids containing 6.7 g 82% trihydroxy-tm/7.ϊ-stilbene (fra '-resveratrol) were obtained. The solids were crystallized in 40 ml of methanol at -10°C. About 5.8 g of colorless crystal (90% purity, 81.1% recovery) were obtained. EXAMPLE 6 This example illustrates making a fifth product by a first MD-3 process. About 37 L (75 g solid) of a first gradient subfraction of a first gradient fraction of a second MD-2 process (BZl-57-l:34.15% /røm'-resveratrol, 22 g; 0.49% c/.y-resveratrol, 0.31 g; 0.25% //røw-piceid, 0.16 g; 4.54% -piceid, 2.9 g: 39.4% total stilbene, 29.4 g: in 35% MeOH) was rotary evaporated to 24.7 L (5% MeOH) and was loaded onto a I LO conditioned 4 " ' 5 x 52" MD-3 column (C-18 bonded silica gel). The column was eluted with a first MD-3 elution -26- volume of a first MD-3 process (3.6 CV of 30% MeOH). The resulting first MD-3 effluent of a first MD-3 process was fractionate collected. The best pool of /ro/w-piceid was fractionate collected between 1.68 CV and 2.20 CV (total of 0.52 CV) of the first MD-3 effluent of a first MD-3 process. The content of trø/w-piceid in the stilbene fraction of the solid component of the best pool was 66% and 94% of the Irans-piccid loaded onto the MD-3 column was recovered. EXAMPLE 7 This E.xample illustrates evaporative crystallization to increase the purity of Crøm'-piceid. About 9L of above best pool of trørø-piceid fractions fractionate collected from the first MD-3 effluent of the first MD-3 process of Example 7 (BZ 1 -64-6-8, 30% MeOH, 31.47 g of 65.7% trαm--piceid) were concentrated (rotary evaporation) to 1.3L, chilled and crystallized at 4°C (73 ml cold H 2 0/g crystal), and filtered with #5 Whatman paper. The peach- colored crude crystal was washed with 100 ml of cold H 0 three times to remove the color completely. This procedure yielded 18 g of colorless tro --piceid crystals of 90% purity; with 80% recovery of the trπm'-piceid loaded onto the MD-3 column. EXAMPLE 8 The following Example illustrates making the third product by an MD-l process. About 1.5kg (wet wt, 0.95kg dry wt.) of NE (Lot # 7-1752, 5.9% tra '-piceid, 0.96% -piceid, 2.1 % t/W7.y-resveratrol, 2.9% m-resveratrol, 1 1.9% total stilbene) was dissolved in 3.5L of SDA (95% EtOH) and made up to 35 L with H 2 0 to make a 10% SDA solution. The solution was loaded onto a 15 x 99cm MD-l column (Amberlite® polystyrenic resin, XAD-16HP, 17.5 L per column volume). Prior to use, the column was conditioned with 1 CV of 10% SDA at 7.2g stilbcne/L resin. The column was eluted with a washing elution volume ( 1 CV of 10% SDA) and then eluted with a first MD-l elution volume (3 CV of 90% SDA) to produce a first MD-l effluent. The first MD- l effluent was fractionate collected ( 1/2 CV per fraction) and the later 2'/ 2 CV (CK2-25-3-4) were pooled and collected as a composition containing the third product. The solids component of the combined later 2'Λ fractionate collected pool amounted to 424.2 g having a 25.5% stilbene fraction. EXAMPLE 9 The following example illustrates a first MD-2 process for making a fourth product having 40% of a stilbene fraction. About 44.4 L of 90% SDA containing a third product from a MD-l process (319 g solid, 27% total stilbene, 13% trø/w-piceid, 2.3% m-piceid, 4.7% /r -resveratrol, 5.6% cw-resveratrol) was evaporated to 9.6 L of 35% SDA, using a Bϋchi Rotavapor R-187, to form a first MD-2 loading concentrate. About 7 L of the first MD-2 loading concentrate were diluted to 9.8 L with H 2 0 to bring the SDA content to 25 vol-%. The diluted loading concentrate was loaded onto a 10 x 117cm water-conditioned MD-2 column (polyamide, 9.6L/CV). The loading was 8.9 g stilbene/L of stationary phase. The column was eluted with a washing elution volume (4 CV of 20% SDA) and was eluted with a first MD-2 elution volume of a first MD-2 process (4 CV of 75% SDA) to make a first MD-2 effluent. SDA - water mixture was removed from the first MD-2 effluent. 195g of solid third product having a 40% stilbene fraction were obtained (22% tra/i. y -piceid, 2.4% m-piceid, 5.7% tra/w-resveratrol, 9.4% cw-resveratrol). Approximately 90% of the stilbenes in the loading concentrate were recovered. EXAMPLE 10 2 g NE (first product from Polygonum cuspidatitm, lot # 7-1752, 5.9% trans- piceid, 1 % c/s-piceid, 2.1% trα -resveratrol, 2.9% .y-resveratrol, 1 1.9% total stilbene) were mixed with 5 ml of concentrated hydrochloric acid and 95 ml of Dl-water (5% HC 1). The solution was refluxed with agitation at 80"C under nitrogen for 60 minutes. /rα/?.y-Resveratrol (0.13 g) was isolated from the solution. Thus, 81.3% of all trihydroxystilbenes were converted to //W7. y -resveratrol (/rø -3,4 , ,5-trihydroxystilbene). EXAMPLE 1 1 The following example illustrates conversion of a β-D-glycosylated stilbene to the aglycone. 200 mg of 90% trø/15-piceid (3,4'5-/rfl/ι.y-trihydroxystilbene -3-β-mono-D- glucoside, BZ 1-69-1) was mixed with 5 ml of concentrated hydrochloric acid and 95 ml water (5% HC1 v/v) and refluxed with agitation at 100°C for 90 minutes to yield 1 14g (57% yield) trø -resveratrol (tra/ι_--3,4',5-trihydroxystilbene). EXAMPLE 12 The following example describes an In vivo study that showed that the levels of DNA synthesis in HL-60 cells were 87%, 89%, and 79%inhibited, respectively, when cells are treated with 80 μg/ml concentrations of mixtures containing third products having 30% resveratrol, 40% stilbene, and 40% piceid, respectively. RNA synthesis levels were also inhibited by the aforementioned compounds at a concentration of 80 μg/ml by 93%, 91%, and 81 %, respectively. The amount of 3 [H 2 0] released as a byproduct of DMBA metabolism was inhibited by percentages of 69%, 70%, and 58% of the three previously mentioned compounds respectively, also at a concentration of 80 μg/ml. Test results are given in the Tables below. Table 1. Inhibitory Effect of a Third Product (30% Resveratrol) on the Synthesis of DNA in HL-60 Cells. Composition Containing 3 rd [ 3 H] Thymidine Incorporation into DNA Percent Product (30%-Resveratrol) (cpm) Inhibition ML 0 32880 ± 432 — 2.5 23166 ± 3178 30 5.0 14575 ± 1 165 56 10.0 10856 ± 1004 67 20.0 6307 ± 828 81 40.0 5762 ± 652 83 80.0 4328 ± 337 87 Tlie third product used in this experiment was obtained from the second gradient faction of a second MD-2 process according to the method of Example 2. HL-60 (5 x 10 5 cells) in 1 ml RPMI medium were incubated with various concentration of 30% resveratrol at 37"C in a 5% C0 atmosphere incubator for 90 minutes. [ H] thymidine incorporation into DNA was determined. Table 2. Inhibitory Effect of a Third Product (40% Piceid) on the Synthesis of DNA in HL-60 Cells. [ 3 H] Thymidine Incorporation into DNA Percent Composition Containing 3 rd (cpm) Inhibition Product (40% Piceid) (μg) 0 32880 ± 432 — 2.5 22733 ± 864 31 5.0 21266 ± 206 35 10.0 19740 ± 1234 40 20.0 16507 ± 864 50 40.0 10704 ± 1058 67 8TJL0 7194 ± 746 78 The third product used in this Example was obtained from the first gradient fraction of a second MD-2 process according to the method of Example 2. HL-60 (5 x 10 5 cells) in 1 ml RPMI medium were incubated with various concentration of 30% resveratrol at 37°C in a 5% C0 atmosphere incubator for 90 minutes. [ 3 H] thymidine incorporation into DNA was determined. Table 3. Inhibitory Effect of a Third Product (40% Stilbene) on the Synthesis of DNA in HL-60 Cells. Composition Containing Third [ 3 H] Thymidine Incorporation into DNA Percent Product (40% Stilbene (cpm) Inhibition Fraction) 0 32880 ± 432 — 2.5 18123 ± 2037 45 5.0 15483 ± 631 53 10.0 14410 ± 144 56 20.0 7888 ± 86 76 40.0 5388 ± 360 84 80.0 3775 ± 137 89 -30- The third product used in this Example was obtained from the first elution volume of a first MD- 2 process. HL-60 (5 x 10 s cells) in 1 ml RPMI medium was incubated with various concentration of 30% resveratrol at 37"C in a 5% C0 2 atmosphere incubator for 90 minutes. [ 3 H] thymidine incorporation into DNA was determined. EXAMPLE 13 This Example illustrates the anti tumor properties of a second product. Table 4. Effect of A second Product (8% Stilbene Fraction) and Sulforphane on DMBA-induced Mammary Carcinogenesis in Rats. Female Sprague-Dawley rats were given AIN 76 A diet or AIN 76 A diet containing 1 % of a second product (approximately 0.08% resveratrol) at 2 weeks before oral administration of 7, 12- dimethylbenz[a]anthracene (DMBA), and continuing until the end of the experiment. Sulforaphane was orally administered by gavage (50 mg / kg body weight in 0.1 ml corn oil) to rats once a day at 46 th , 47 th , 48 ,h , 49 th , 50 th and 5 I s ' days of their age. DMBA (8 mg / rat) in 1 ml corn oil was intubated to rats at 50 days old. Palpable mammary tumors were counted every week after DMBA administration. EXAMPLE 14 The following Example illustrates the anti-tumor properties against various human tumor cell lines exhibited by various products of the present invention according to the results of in vitro 6-cell line clinical test. The tests were performed according to the MTT assay (See, Mosmann, T., J. Immuπ. Met , 65, 55(1983). Cells were planted in 96 well flat bottom plates with low evaporation lids. Three cell lines per plate were seeded in 0.2 ml medium per well. Each cell line was planted at the optimum concentration for its particular growth rate: H-29 and A-549, 5000 c/ml, MCF-7, 15000 c/well, KB and similar cells, 2500 c/ml, P388 and similar cells, 15000 c/ml. Products were tested at various dilutions (at least ten) to determine the ED 50 Table 5. 6-Cell Line Clinical Test Results. Sample Lot// Kidney Prostrat Pancreat Lung Breast Colon Description A-478 e ic A-549 MCF-7 HT-29 ED 50 PC-3 PACA-2 ED 50 ED50 ED 50 (μg/ml) ED 50 ED 50 (μg/ml) (μg/ml) (μg/ml) (μg/ml) (μg/ml) Second 7-1752 276.61 14.42 45.32 77.86 521.37 41.65 Product, 8% Stilbene Fraction Fifth Product, BZ1- 34.95 80.5* 40.98 35.77 100 79.20 90% Piceid 069-1 Sixth Product, BZ1- 7.40x10 " 100 3.55 3.26x10 " 4.23 1 66 Recryst., 90%, 67-2 ' 1 Resveratrol Fourth CK1- 19.57 100.00 7.1 ! 3.55 77.96 18.01 Product, First 100-3 MD-2 Process, 40%, Stilbene Fraction Doxorubicin 3.59x10- 2.81x10 " 5.22x10 " 3.16x10 " 1 07x1 2 20x10 " HCL o- 1 (Control)

Claims: 1 claim 1. A first product having a solids content of at least about 60% wherein the solids comprise at least about 10% by weight of a stilbene fraction wherein the stilbene fraction comprises trihydroxy-/rø -stiblenes, trihydroxy cis-stilbenes, mono-β-D-glycosylated trihydroxy trans- stilbenes, and mono-β-D-glycosylated trihydroxy -stilbenes. 2. A second product comprising the first product of claim 1 and at least one pharmaceutically acceptable processing excipient and having a stilbene fraction of at least about 8% by weight. 3. A third product comprising at least about 20% by weight of a mixture of trihydroxystilbenes and mono-β-D-glycosylated trihydroxystilbenes. 4. A composition comprising the third product of claim 3 and an aqueous solvent wherein the concentration of all stilbenes in the composition is at least about 3 g/L. 5. A fourth product comprising at least about 30% by weight of a stilbene fraction comprising trihydroxystilbenes and mono-β-D-glycosylated trihydroxystilbenes. 6. The fourth product of claim 5 comprising at least about 40% of a stilbene fraction wherein the stilbene fraction comprises at least about 90% mono-β-D-glycosylated 3,4', 5- trihydroxystilbenes. 7. A composition comprising the product of claim 6 and an aqueous solvent wherein the concentration of the mono-β-D-glycosylated 3,4',5-trihydroxystilbenes in the composition is at least about 0.5 g/L. 8. The composition of claim 7 wherein the aqueous solvent comprises a mixture of an alcohol and water comprising between about 30 vol-% and about 40 vol-% alcohol. The composition of claim 8 wherein the alcohol is methanol. 10. The composition of claim 8 wherein the alcohol is ethanol. 1 1. The fourth product of claim 5 wherein the stilbene fraction comprises at least about 80% trihydroxystilbenes. 12. A composition comprising the product of claim 1 1 and an aqueous solvent wherein the concentration of trihydroxystilbenes in the composition is at least about 0.7 g/L. 13. The composition of claim 12 wherein the aqueous solvent comprises a mixture of an alcohol and water comprising between about 70 vol-% and about 80 vol-% alcohol. 14. The composition of claim 12 wherein the alcohol is methanol. 15. The composition of claim 12 wherein the alcohol is ethanol. 16. A composition comprising the product of claim 5 and an aqueous solvent wherein the concentration of all stilbenes in the composition is at least about 0.6 g/L. 17. The composition of claim 16 wherein the aqueous solvent comprises a mixture of an alcohol and water comprising between about 45 vol-% and 55 vol-% alcohol. 18. The composition of claim 17 wherein the alcohol is methanol. 19. The composition of claim 17 wherein the alcohol is ethanol. 20. A fifth product comprising at least about 60% of a stilbene fraction wherein the stilbene fraction comprises at least about 90% mono-β-D-glycolsy ated-3,4',5-trihydroxy-/ , -stilbenes. 21. A composition comprising the fifth product of claim 20 and an aqueous solvent wherein the concentration of the fifth product in the composition is at least about 2.0 g/L. 22. The composition of claim 21 wherein the aqueous solvent comprises a mixture of an alcohol and water comprising between about 25 vol-% and about 35 vol-% alcohol. 23. The composition of claim 22 wherein the alcohol is methanol. 24. The composition of claim 22 wherein the alcohol is ethanol. 25. The fifth product of claim 20 comprising at least about 85% by weight mono-β-D- glycosylated trihydroxy-trαm'-stilbene. 26. A sixth product comprising at least about 70% by weight trihydroxy-trøm-stilbenes. 27. The sixth product of claim 26 comprising at least about 85% by weight 3,4',5- trihydroxy-tra/ry-stilbene. 28. A composition comprising the product of claim 26 and an aqueous solvent wherein the concentration of trihydroxy-trnny-stilbenes is at least about 0.4 g/L. 29. The composition of claim 24 wherein the aqueous solvent comprises a mixture of an alcohol and water comprising between about 50 vol-% and 60 vol-% alcohol. 30. The composition of claim 29 wherein the alcohol is methanol. 31. The composition of claim 29 wherein the alcohol is ethanol. 32. A seventh product comprising at least about 50% by weight of 3,4',5-trihydroxy-c7_-- stilbene. 33. A composition comprising the product of claim 32 and an aqueous solvent wherein the concentration of trihydroxy-c7.y-stilbenes in the composition is at least about 0.2 g/1. 34. The composition of claim 33 wherein the aqueous solvent comprises a mixture of an alcohol and water comprising between about 80 vol-% and 90 vol-% alcohol. 35. The composition of claim 34 wherein the alcohol is methanol. 36. The composition of claim 34 wherein the alcohol is ethanol. 37. A process for making the first product of claim 1 comprising the steps of: a) providing pieces of a plant material containing a stilbene fraction, b) contacting the plant material with an alcohol to form a slurry, c) separating the alcohol from the slurry, and d) evaporating the alcohol to obtain the first product. 38. The process of claim 37 wherein the plant material is Polygonum cuspidatitm. 39. The process of claim 37 wherein the plant material is Vttis vinifera. 40. A process for making the second product of claim 2 comprising the steps of: a) providing the first product of claim 1, b) making a slurry of the first product in water, c) homogenizing the slurry with one or more pharmaceutically acceptable processing excipients, and d) drying the product of step c to obtain the second product. 41. The process of claim 40 wherein the drying is spray drying. 42. The process of claim 40 wherein the drying is vacuum drying. 43. An MD-l process for making the composition of claim 4 comprising the steps of: a) providing a slurry in an aqueous solvent of the first product of claim 1, which first product comprises at least about 8% by weight, relative to the weight of all solids, of a stilbene fraction, b) loading the slurry onto an MD-l column having a stationary phase, optionally using a washing elution volume, and c) eluting the composition of claim 4 with a first MD- l elution volume comprising a aqueous solvent. 44. Tlie MD-l process of claim 43 wherein the stationary phase comprises a copolymer of styrene and divinylbenzene. 45. The process of claim 43 wherein the aqueous solvent of the first MD-l elution volume comprises a mixture of an alcohol and water comprising between about 70 vol-% and about 80 vol-% alcohol. 46. The process of claim 43 wherein the alcohol is methanol. 47. The process of claim 43 wherein the alcohol is ethanol. 48. A process for making the third product of claim 3 comprising the step of removing aqueous solvent from the product of step c of claim 43. 49. A first MD-2 process for making the composition of claim 16 comprising the steps of: a) providing the composition of claim 4, b) removing aqueous solvent from the composition of claim 4 to form a loading concentrate having a solids content of at least about 13 g L, c) loading the concentrate of step c onto an MD-2 column having a stationary phase, optionally using a washing elution volume, and d) eluting a first MD-2 effluent of a first MD-2 process with a first MD-2 elution volume of a first MD-2 process comprising an aqueous solvent wherein the first MD-2 effluent comprises the composition of claim 12. 50. The MD-2 process of claim 49 wherein the stationary phase is selected from the group consisting of poly(caprolactam) and poly(hexamethylene adipamide). 51. The process of claim 49 wherein the aqueous solvent of the first MD-2 elution volume of a first MD-2 process is an alcohol-water mixture comprising at least about 70 vol-% alcohol. 52. The process of claim 49 wherein the alcohol is methanol. 53. The process of claim 49 wherein the alcohol is ethanol. 54. The process of claim 49 wherein the aqueous solvent of the first MD-2 elution volume of the first MD-2 process comprises an alcohol - water mixture comprising at least about 70% alcohol. 55. A second MD-2 process comprising the steps of a first MD-2 process of claim 49 wherein the first MD-2 elution volume of the first MD-2 process comprises: (1) a first MD-2 gradient volume comprising an aqueous solvent that comprises an alcohol - water mixture comprising at least about 30% alcohol and that elutes a first MD-2 gradient fraction, followed by (2) a second MD-2 gradient volume that comprises an aqueous solvent comprising an alcohol - water mixture comprising at least about 70% alcohol that elutes a second MD-2 gradient fraction. 56. A process for making the fourth product of claim 5 comprising the step of removing the aqueous solvent from a first MD-2 effluent of claim 49. 57. A process for making the composition of claim 7 comprising the steps of fractionate collecting first and second gradient fractions of claim 55, which first gradient fraction is the composition of claim 7. 58. The process of claim 57 wherein the first gradient fraction is fractionate collected to obtain gradient subtractions of a first gradient fraction. 59. A process for making the fourth product of claim 6 comprising the step of removing aqueous solvent from the first gradient fraction of claim 57. 60. A process for making the composition of claim 12 comprising fractionate collecting first and second MD-2 gradient fractions from the first MD-2 effluent that results upon elution of the first MD-2 elution volume of the first MD-2 process of claim 54 wherein the second MD-2 gradient fraction is the composition of claim 12. 61. The process of claim 60 wherein the second gradient fraction is fractionate collected to obtain gradient subfractions of the second gradient fraction. 62. A process for making the product of claim 1 1 comprising the step of removing the aqueous solvent from the second MD-2 gradient fraction fractionate collected according to the process of claim 60. 63. A third MD-2 process for making the compositions of claims 7 and 12 comprising the steps of: a) providing the composition of claim 4, b) removing aqueous solvent from the composition of claim 4 to form a loading concentrate having a solids content of at least about 13 g/L, c) loading the loading concentrate of step b onto an MD-2 column, optionally using a washing elution volume comprising an aqueous solvent, d) eluting a second MD-2 effluent with an MD-2 gradient elution volume of a third MD-2 process comprising an aqueous solvent, wherein the composition of the aqueous solvent of the MD-2 gradient elution volume is varied linearly, exponentially, logarithmically, hyperbolically, or step-wise during elution of the gradient elution volume, to produce an effluent, and e) fractionate collecting the effluent to obtain gradient fractions that are the compositions of claims 7 and 12. 64. The third MD-2 process of claim 63 wherein the aqueous solvent of the second MD-2 elution volume is a mixture of an alcohol and water wherein the alcohol content is varied between about 30 vol-% and about 60 vol-% alcohol during elution with the gradient elution volume. 65. The third MD-2 process of claim 64 wherein the alcohol is methanol. 66. The third MD-2 process of claim 64 wherein the alcohol is ethanol. 67. A process for making the fourth product of claim 6 comprising the step of removing aqueous solvent from the second MD-2 effluent of claim 61. 68. A first MD-3 process for making the composition of claim 21 comprising the steps of. a) providing the composition of claim 7, -40- b) removing aqueous solvent to form a loading concentrate having a solids content of at least about 3 g/L, c) loading the concentrate of step b onto an MD-3 column having a stationary phase, optionally using a washing elution volume comprising an aqueous solvent, d) eluting the MD-3 column with a first MD-3 elution volume of the first MD-3 process, which elution volume comprises and aqueous solvent, to form a first MD-3 effluent of a first MD-3 process, and e) fractionate collecting the first MD-3 effluent of the first MD-3 process to segregate at least first and second MD-3 fractions of a first MD-3 process, wherein the second MD-3 fraction of a first MD-3 process is the composition of claim 12. 69. The MD-3 process of claim 68 wherein the stationary phase is C| 8 . 70. The MD-3 process of claim 68 wherein the stationary phase is C 8 . 71. The process of claim 68 wherein the aqueous solvent comprises a mixture of alcohol and water comprising between about 25 vol-% and about 30 vol-% alcohol. 72. The process of claim 71 wherein the alcohol is methanol. 73. The process of claim 71 wherein the alcohol is ethanol. 74. A process for making the fifth product of claim 20 comprising the step of removing the aqueous solvent from the second MD-3 fraction of a first MD-3 effluent of a first MD-3 process of claim 68. 75. A process for making the product of claim 25 comprising the steps of: a) concentrating the second MD-3 fraction of a first MD-3 process of claim 46 by removing aqueous solvent until the volume of the fifth effluent is less than about one-fifth of its original volume and the solids content of the concentrated second MD-3 fraction of a first MD-3 process is at least about 20 g/L, b) adding 1.5 to 2.0 L of water per liter of concentrated second MD-3 fraction of a), c) cooling to less than about 0°C to form a slurry, d) separating the solid from the slurry, and e) optionally washing the solid with water having a temperature less than about 25°C. f) drying the solid. 76. A second MD-3 process for making the composition of claim 28 comprising the steps of: a) providing the second MD-2 gradient fraction of a first MD-2 effluent fractionate collected according to claim 50, b) concentrating the fractionate collected second MD-2 gradient fraction by removing aqueous solvent to a solids content of at least about 7 g/L, c) loading the concentrated fractionate collected second MD-2 gradient fraction of step b onto an MD-3 column, optionally using a washing elution volume, d) eluting the column with a first MD-3 elution volume of a second MD-3 process, which elution volume comprises and aqueous solvent to elute a first MD-3 effluent of a second MD-3 process, and e) eluting the column with a second MD-3 elution volume of a second MD-3 process to elute a second MD-3 effluent of a second MD-3 process that is the composition of claim 28. 77. The process of claim 76 wherein at least one of the first and second MD-3 elution volumes is a gradient elution volume comprising an aqueous solvent wherein the composition of the aqueous solvent is varied linearly, exponentially, logarithmically, hyperbolically, or step-wise during elution of the elution volume to form an effluent and fractionate collecting the effluent. 78. A process for making the sixth product of claim 26 comprising the step of removing the aqueous solvent from the second MD-3 effluent of the second MD-3 process of claim 76. 79. A process for making the product of claim 27 comprising the steps of: a) providing the composition of claim 25, b) concentrating the composition of step a by removing aqueous to make a concentrated composition having a solids content of at least about 1.5g l, c) twice contacting the concentrated composition of step b with an extraction volume of a volatile polar organic solvent, and d) combining the extraction volumes of volatile polar organic solvent of step c and removing the volatile polar organic solvent to obtain the product of claim 23. 80. A process for purifying a sixth product of claim 26 comprising the steps of: a) providing the composition of claim 28, b) removing the aqueous solvent from the composition of step a to obtain a solid residue, c) dissolving the residue of step b in methanol at a temperature greater than 0°C to form a solution, d) cooling the solution of step c to a temperature less than about 0°C, whereby crystals are formed, and e) separating the crystals of step d from supernatant to obtain a purified sixth product. 81. A third MD-3 process for making the composition of claim 33 comprising the steps of: a) providing the second MD-2 gradient fraction fractionate collected according to claim 55, b) concentrating the fractionate collected second MD-2 gradient fraction by removing aqueous solvent until the solids content is at least about 7 g/L, c) loading the concentrated second MD-2 gradient fraction of step b onto an MD-3 column, optionally using a washing elution volume, d) eluting the column with a first elution volume of a third MD-3 process, which elution volume comprises an aqueous solvent, to elute a first MD-3 effluent of a third MD-3 process, c) eluting the column with a second MD-3 elution volume of a third MD-3 process, which elution volume comprises an aqueous solvent volume to elute a second MD-3 effluent of a third MD-3 process , f) eluting the column with a third MD-3 elution volume of a third MD-3 process, which elution volume comprises an aqueous solvent, to elute a third MD-3 effluent of a third MD-3, and g) fractionate collecting the third MD-3 effluent of a third MD-3 process to segregate a first gradient fraction of the third MD-3 effluent of a third MD-3 process and a second gradient fraction of the third MD-3 effluent of a third MD- 3 process, whereby such second gradient fraction is the composition of claim 18. 82. The process of claim 81 wherein at least one of the first, second or third elution volumes of a third MD-3 process is a gradient elution volume comprising an aqueous solvent wherein the composition of the aqueous solvent is varied linearly, exponentially, logarithmically, hyperbolically, or step-wise during elution of the elution volume to form an effluent and further comprising the step of fractionate collecting the effluent. 83. The process of claim 82 wherein the aqueous solvent comprises water and methanol or ethanol. 84. A process for making the seventh product of claim 32 comprising the step of removing the polar oxygenated solvent from the second gradient fraction of a third MD-3 effluent fractionate collected according to claim 81. 85. A process for converting a mono-β-D-glycosylated trihydroxystilbene component of a stilbene fraction to the corresponding trihydroxystilbene component comprising the steps of: 1) providing a solution or suspension of a product having a stilbene fraction in a mixture of alcohol and water comprising about 35% alcohol, 2) acidifying the solution or suspension with sufficient HC1 to bring the concentration of HCL in the solution to between about 0.01 and 0.02 g/ml, and 3) refluxing the acidified solution for about 10 to about 200 minutes. 86. The process of claim 85 wherein the alcohol is methanol. „ 0 -44- 87. The process of claim 85 wherein the alcohol is ethanol. 88. The process of claim 85 wherein the HC1 is 38% aqueous HC1. 89. A method of treating or preventing cancer in an animal comprising administering an effective amount of the first product of claim 1 to an animal. 90. A method of treating or preventing cancer in an animal comprising administering an effective amount of the second product of claim 2 to an animal. 91. A method of treating or preventing cancer in an animal comprising administering an effective amount of the product of claim 3 to an animal. 92. A method of treating or preventing cancer in an animal comprising administering an effective amount of the fourth product of claim 5 to an animal . 93. A method of treating or preventing cancer in an animal comprising administering an effective amount of the product of claim 6 to an animal. 94. A method of treating or preventing cancer in an animal comprising administering an effective amount of the sixth product of claim 14 to an animal.

Inventors: HE KAN, KIM CALVIN HYUNGCHAN, ZHENG BO LIN, ZHENG QUN YI, HE, KAN, KIM, CALVIN, HYUNGCHAN, ZHENG, BO, LIN, ZHENG, QUN, YI
Applicants: PURE WORLD BOTAN INC, PURE WORLD BOTANICALS, INC.
Application year: 1999
Main classification: A61K
IPC codes: C07D471, C07D498, C07D513