US007897791 B2 (12) United States Patent (10) Patent No.: US 7,897,791 B2 Seeram et al. (45) Date of Patent: *Mar. 1, 2011 (54) PURIFICATIONS OF POMEGRANATE OTHER PUBLICATIONS ELLAGTANNINS AND THEIR USES Aviram, M., et al., “Pomegranate juice consumption inhibits serum THEREOF angiotensin converting enzyme activity and reduces systolic blood pressure.” (2001) Atherosclerosis, 158: 195-198. (75) Inventors: Navindra P. Seeram, Kingston, RI (US); Cerda, B., et al., “Evaluation of bioavailability and metabolism in the David Heber, Los Angeles, CA (US) rat of punicalagin, an antioxidant polyphenol from pomegranate juice.” (2003) Eur, J. Nutr., 42:18-28. (73) Assignee: The Regents of the University of Cerda, B., et al., “Repeated oral administration of high doses of the California, Oakland, CA (US) pomegranate elagitannin punicalaginto rats for 37 days is not toxic.” (2003) J. Agric. Food Chem. 51:3493-3501. (*) Notice: Subject to any disclaimer, the term of this Doig, A., et al., “Isolation and structure elucidation of punicalagin, a patent is extended or adjusted under 35 toxic hydrolysable tannin, from Terminalia oblongata.” (1990) J. U.S.C. 154(b) by 0 days. Chem. Soc. Perkin Trans. I, 2317-2321. EL-Toumy, S., et al., “Two ellagitannins from Punica granatum This patent is Subject to a terminal dis heartwood.” (2002) Phytochemistry, 61:971-974. claimer. Filippich, L., et al., “Hepatotoxic and nephrotoxic principles in Terminalia oblongata.” (1991) Research in Veterinary Science, Appl. No.: 12/647,229 50:17O-177. (21) Gil, M., et al., “Antioxidant activity of pomegranate juice and its Filed: Dec. 24, 2009 relationship with phenolic composition and processing.” (2000) J. (22) Agric. Food Chem., 48:4581-4589. Mayer, et al., “Punicalagin and punicalin, two tannins from pome (65) Prior Publication Data granate peel.” (1977) Liebigs Ann. Chem, 1976-1986. US 2010/O1 7386O A1 Jul. 8, 2010 Negi, P. et al., “Antioxidant and antimutagenic activities of pome granate peel extracts.” (2003) Food Chemistry, 80:393-397. Related U.S. Application Data Scalbert, A., et al., “Absorption and metabolism of polyphenols in the gut and impact on health.” (2002) Biomed. Pharmacother, 56:276 (60) Division of application No. 1 1/395,447, filed on Mar. 282. 30, 2006, now Pat. No. 7,638,640, which is a continu Tanaka; et al., “Punicafolin, an Ellagitannin from the Leaves of ation-in-part of application No. PCT/US2005/009337, Punica granatum'. Phytochemistry (1985), 24 (9): 2075-2078. filed on Mar. 21, 2005. Tanaka, T., et al., “Tannins and related compounds XL. Revision of the structures of punicalin and punicalagin, and isolation and (60) Provisional application No. 60/556,322, filed on Mar. characterizatin of 2-O-Galloylpunacalin from the bark of Punica 24, 2004. granatum L.” (1986) Chem. Pharm. Bull., 34(2): 650-655. Tanaka, T., et al., “Tannins and related compounds. XLL. Isolation (51) Int. C. and characterization of novel ellagitannins, punicacorteins A, B, C CO7D 49.3/00 (2006.01) and D, and punigluconin from the bark of punica granatum L.” AOIK 43/04 (2006.01) (1986) Chem. Pharm. Bull., 34 (2):656-663. AOIN 43/42 (2006.01) Vidal, A., et al., “Studies on the toxicity of Punica granatum . AOIN 43/16 (2006.01) (Punicaceae) whole fruit extracts.” (2003) Journal of (52) U.S. Cl. ....................... 549/278: 514/292; 514/453; Ethnopharmacology, 89:295-300. 514/456; 514/27 * cited by examiner (58) Field of Classification Search ................. 549/278; 514/27, 292, 453, 456 Primary Examiner Nizal S Chandrakumar See application file for complete search history. (74) Attorney, Agent, or Firm—Bozicevic, Field & Francis LLP: Pamela J. Sherwood (56) References Cited (57) ABSTRACT U.S. PATENT DOCUMENTS 6,387,418 B1 5, 2002 Aviram et al. Compositions of purified and biologically active ellagitan 6,641,850 B1 1 1/2003 Aviram et al. nins are provided by separation from pomegranate husk using 6,977,089 B1 12/2005 Aviram et al. a method of extraction and purification using a solid poly 7,638,640 B2 * 12/2009 Seeram et al. .............. 549,278 meric adsorbent and the uses of the said compounds. FOREIGN PATENT DOCUMENTS JP 61100517 A 5, 1986 13 Claims, 4 Drawing Sheets U.S. Patent Mar. 1, 2011 Sheet 1 of 4 US 7,897,791 B2 5.0 100 15.0 0.00 25.0 30,0 350 4.O.O 45 Minute 5.0 O.O 15.0 2O.O 25.0 3O.O 35.0 400 45 Minute 5.O 10.0 15,0 20.0 25.0 3O.O 35.0 40.O 45 Minute U.S. Patent Mar. 1, 2011 Sheet 2 of 4 US 7,897,791 B2 U.S. Patent Mar. 1, 2011 Sheet 3 of 4 US 7,897,791 B2 Elagic acid (4) (mz301) Punicalagin (1) (miz1083) Gallagic acid (3) (miz601) Punicalin (2) (mz781) Figure 3A U.S. Patent Mar. 1, 2011 Sheet 4 of 4 US 7,897,791 B2 7812 | 5 O 30 a. 20 5 549.1 575.2 538 a 3932 1064.5 3374 930.9 988.9 O -- el, DeCD D D O D D 300 400 500 600 700 800 900 1000 1100 1200 Figure 3B US 7,897,791 B2 1. 2 PURIFICATIONS OF POMEGRANATE pounds, the development of inexpensive, high throughput ELLAGTANNINS AND THEIR USES methods of purificationis of particular interest. THEREOF SUMMARY OF THE INVENTION CROSS REFERENCE TO RELATED Compositions of purified and biologically active ellagitan APPLICATION(S) nins, specifically including punicalagin and gallagic acid are provided, as well as methods for producing Such composi This application is a divisional non-provisional patent tions and their uses. Ellagitannins are separated from other application of Ser. No. 1 1/395,447 (now allowed) filed before 10 pomegranate natural products by a method of extraction and the U.S. Patent & Trademark Office (USPTO) on Mar. 30, purification using a series of solvent-solvent extractions and 2006, which is in turn a continuation-in-part application of chromatography with solid polymeric adsorbents. Included PCT application No. PCT/US2005/009337 filed before the in the compositions of the invention are extracts comprising USPTO receiving office on Mar. 21, 2005, which claims gallagic acidata concentration of at least about 0.1% or more, 15 up to Substantially pure gallagic acid. Also included in the priority to U.S. provisional patent application of Ser. No. compositions of the invention are extracts comprising puni 60/556,322 filed before the USPTO on Mar. 24, 2004. This calagin at a concentration of at least about 0.1% or more, up divisional application incorporates by reference in its entirety to Substantially pure punicalagin. Compositions are also pro each of the foregoing patent applications (i.e., non-provi vided comprising elagic acid and ellagic acid glycosides at a sional patent application of Ser. No. 1 1/395,447; continua concentration of at least about 5% or more. In another tion-in-part application of PCT application no. PCT/US05/ embodiment, compositions are provided comprising antho 09337; and U.S. provisional patent application of Ser. No. cyanins and anthocyanidins at a concentration of at least 60/556,322. about 0.1%, about 5%, or more. Extracts comprising these compounds, and extracts comprising total pomegranate tan GOVERNMENT RIGHT(S) 25 nins, are useful for a variety of applications, including phar maceutical, neutraceutical, cosmetic, and food uses. In one embodiment of the invention, dried pomegranate This invention was made with Government support of peels are ground and Suspended with an alcohol Such as Grant No. AT000151 awarded by the National Institutes of methanol. A rapid polymeric adsorbent column is used to Health. The government has certain rights in this invention. 30 purify pomegranate tannins from an aqueous or alcoholic Recent research has shown that pomegranate (Punica extract, which extract is derived from a source of elagitan granatum L.) juice and pomegranate extracts exhibit potent nins such as a variety of plant extracts, including Punica biological properties attributable to the presence of polyphe granatum L., and the like. The tannins are adsorbed to the nols known as elagitannins. These hydrolysable tannins are columns, and followed by cleansing, gravity evacuation, and present in high levels in pomegranates and include punicala 35 vacuum aspiration of fluid from the column, the adsorbed gin anomers commonly referred to as punicalagins punicalin tannins are eluted from the adsorbent solid and the solvent gallagic acid and elagic acid Pomegranate elagitannins have removed in vacuo to yield a powder of highly concentrated been identified as the active antioxidant compounds respon total tannins comprising a high percentage (e.g., 80%) of sible for protecting low-density lipoprotein cholesterol from punicalagin anomers, as well as a significant percentage of oxidation in Vivo, a key step in the pathogenesis of athero 40 ellagic acid. Additional chromatography steps may be per Sclerosis. The punicalagins are key compounds responsible formed to provide for highly purified compositions of gal for the antioxidant properties of pomegranate extracts. How lagic acid or punicalagin. ever, research has shown that the synergistic combination of The present invention also provides a method for produc punicalagins with other minor pomegranate elagitannins, ing a mixture comprising ellagitannins from pomegranate. 45 The method may include the use of enzymes Such as extrac specifically punicalin and gallagic acid, results in Superior tase and pectinase. The method may include the steps of biological activities. Unfortunately, there are no methods cur providing an aqueous solution containing the ellagitannins rently available for rapid and large scale production of these from the pomegranate; removing the elagitannins onto a minor pomegranate elagitannins. This invention proposes a resin Surface from the aqueous solution; eluting the resin method to produce ellagitannins from pomegranates and out 50 Surface with a eluant to remove the ellagitannins from the lines their uses.