Isolation and Purification of Polymethoxyflavones as Substrates for

Chapter 14

Isolation and Purification of Polymethoxyflavones as Substrates for Efficacy Studies Downloaded by UNIV OF CALIFORNIA SAN DIEGO on July 22, 2016 | http://pubs.acs.org Publication Date: September 1, 2008 | doi: 10.1021/bk-2008-0987.ch014

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Shiming Li , Chih-Yu Lo , Slavik Dushenkov , and Chi-Tang Ho

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Department of Food Science, Rutgers University, 65 Dudley Road, New Brunswick, NJ 08901 WeIIGen Inc., 63 Dudley Road, New Brunswick, NJ 08901 2

Polymethoxyflavones (PMFs) isolated from members of the citrus genus are of particular interest in that they exhibit a wide spectrum of biological activities. Due to the current interest in the exploration of the beneficial health properties accociated with citrus-derived PMFs, there has been an increased interest in clinical trials amed at determining efficacy parameters.. Unfortunately, the supply of pure PMFs available for these in vivo studies is a limiting factor because of the difficulties related to large scale isolation of the required PMFs. As a result of this, an efficient and large scale separation process for the PMFs was needed. In this paper, we discuss the newly developed preparatve methods for efficient and large scale isolation of PMFs from sweet orange (Citrus sinensis) peel. These procedures employ chiral liquid chromatography and supercritical chromatography (SFC) technology.

© 2008 American Chemical Society

Ho et al.; Dietary Supplements ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

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Downloaded by UNIV OF CALIFORNIA SAN DIEGO on July 22, 2016 | http://pubs.acs.org Publication Date: September 1, 2008 | doi: 10.1021/bk-2008-0987.ch014

212 There are numerous research reports concerning polymethoxyflavone's (PMFs) biological activities, including anti-inflammatory, anti-carcinogenic, and anti-atherogenic properties (1-6). The majority of the bioactivity studies of PMFs were performed in vitro, and these procedures require only small amounts (milligram scale) of materials as investigational product. Animal and Clinical trials such as pharmacokinetics, safety, metabolism, and efficacy studies utilizing specific PMFs have rarely been done, primarily because of the difficulties associated with isolation of suitable quantities of these unique PMF materials. Although some PMFs are commercially available, the cost is often too high to permit these in vivo studies to be performed.. For example, 3,5,6,7,8,3',4heptamethoxyflavone has been reported to exhibit in vitro potent anti-tumor activity and also to be a chemopreventive agent against nitric oxide carcinogenesis (7,8). However, more in-depth in vitro investigations and efficacy studies of 3,5,6,7,8,3',4-heptamethoxyflavone have not been initiated, because of its limited availability and high cost ($300/mg). An efficacy study in animals of lower species (mice, rats etc.) could easily consume grams of 3,5,6,7,8,3',4'-heptamethoxyflavone at a price of $300,000/g. It could theoretically cost billions of dollars for clinical trial studies using this compound, a cost that puts this material beyond the pratical range for commercial applications. To make these biologically interesting PMFs available for the pharamaceutical and nutrition industries, a lower cost procedure was required, which will be described in the subsequent text

Analysis and Identification Method Earlier studies related to the properties of PMFs have focused on the analysis and identification of PMFs in a variety of plant families, especially those from the citrus genus and a Variety of analytical methods have been applied to characterize different PMFs in citrus plants. For example, using Gas Chromatography (GC) with a coated capillary column, Gaydou, et al. separated and identified 27 PMFsfromthree industriallyderived types of orange peel oils (9). In this study, they also found that sinesetin, nobiletin, and 3,5,6,7,8,3',4'-heptamethoxyflavone are characteristic of orange peel oils, and are among the most abundant PMFs. The other three PMFs identified, tangeretin, tetra-O-methylscutellarein, and 3,5,6,7,3',4'hexamethoxyflavone, were found to be less abundant in the three samples they tested. Gas Chromatography/Mass Spectrometry (GC-MS) is another technique that was first used to charcaterize the PMFs extant in sweet orange, tangeretin and grapefruit (10). There were six predominant PMFs in cold pressed orange peel oils separated during this study and hydroxylated PMFs were identified in



213 tangeretin oil. Monohydroxylated pentamethoxyflavone (M* = 388 amu), previously identified in tangeretin was also present in both orange and grapefruit oils, but only at lower concentrations. Due to its ease of use, robustness, and ability to process both volatile and non-volatile compounds, high performance liquid chromatography (HPLC) has become popular for citrus-derived PMF analysis and identification. The high resolution and accurate quantification of citrus PMFs and other related natural products by HPLC has been reviewed (11). HPLC has also been used for simultaneous separations of PMFs and flavone glycosides (12) as well as the simultaneous determination of four PMFs (nobiletin, tangeretin, 3,5,6,7,8,3',4'-heptamethoxyflavone, and 5-demethylnobiletin) plus four other compounds (glycyrrhizin, hesperidin, honokiol and magnolol) from a multi-component traditional Chinese medicine (13). The detection of flavonoid compositions in 42 species and cultivars of the citrus genus by HPLC has also been described (14). It is reported that reverse phase HPLC coupled with mass spectrometry (MS) yielded enhanced identifications of PMFs (tangeretin and nobiletin) and other flavonoids (naringin, isonaringin, hesperidin, neohesperidin, hesperitin and naringenin) (75) and also enabled the qualitative and quantitative determination of the flavonoid content of extracts of Greek navel sweet orange peel (16). Additionally, the application of HPLCMS/NMR (Nuclear Magnetic Resonance) was used as a complementary analytical tools for the reliable identification of PMFs in residuesfrommolecular distillation of cold preesed peel oils of Citrus sinensis (17). Supercritical Fluid Chromatography (SFC) was also applied in the analysis of PMFs. It has been reported that the SFC method is both rapid and quantitative (18,19). Milligram quantities of six PMFs were isolatedfromsweet orange peel using a SFC procedure (19). However, both HPLC and SFC are purely analytical methods used solely for the purpose of individual PMF identification.

Isolation and Preparation Procedures for PMFs Purification and isolation procedures for PMFs were of little interest until recently when a separation method of PMFs based on high-speed countercurrent chromatography was reported (20). Although this method was able to isolate some PMFs in multi-milligram quantities, it was laborious and timeconsuming, which in turn limited its scalability and application for larger separations. As a result, the PMF supply for potential in vivo and clinical trial study remained very limited. The first reported large scale separation of PMFs was a nobiletin isolation (27) procedure based on preparative, chiral HPLC. The cold pressed orange peel is first passed through a silica-gel column to remove orange peel oils and to



214 divide the PMFs into six groups. Group V, mainly containing nobiletin and 5,6,7,4'-tetramethoxyflavone, is then loaded onto a Welk-0 chiral column (Regis Welk-O 1 R,R 450 gram column). Using a mixture ethanol and hexanes as eluting solvents, more than 2 grams of nobiletin can be obtained from one preparation within 45 minutes. The application of this chiral preparative HPLC method not only opened a new era of nobiletin research for in vitro and in vivo studies because of the availability of PMFs in large amount. Secondarily, it demonstrated the general application of the utility of chiral preparative HPLC columns for PMF isolation. This is the first successful application of chiral chromatography in separating PMFs, and it demonstrated the technique's ablity to resolve these closely related PMFs which exhibit a high degree of chemical structural and physical property similarity More general separation methods, useful for isolation of large quantities of PMFs, were recently developed using supercritical fluid chromatography (SFC) (22). Following a number of attempts to purify PMFsfromsweet orange (citrus sinenesis) we developed an efficient and scalable SFC method for the large scale separation of four common PMFs. This process has the potential application to become the predominant technology for large scale PMF isolation. This SFC technology has a number of advantages over the other separation techniques in cost effectiveness, time efficiency, ease of automation. This is the first reported SFC application for the preparative separation of PMFs. It is also of significance because it has not only provided an efficient and large scale preparation of PMFs, but has also explored a new application of the SFC technology in the field of PMF research. The four PMFs isolated in this study were nobiletin, tangeretin, 3,5,6,7,8,3',4'-heptamethoxyflavone, and 5,6,7,4tetramethoxyflavone.

Summary Analytical techniques for PMF separation and identification have been successfully developed and employed by various research groups interested in PMFs. These techniques were not able to provide quantities suitable to provide sufficient investigational product for preclinical and clinical studies related to efficacy and bioavailabilty of PMFs. However, to meet the demand of gram or kilogram required, efficient and scalable separation methods of PMFs have been developed employing chiral preparative HPLC chromatography and preparative SFC separation techniques.

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Isolation and Purification of Polymethoxyflavones as Substrates for

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