Food Phytochemicals for Cancer Prevention II - ACS Publications

Chapter 30

Development of Stability-Indicating Analytical Methods for Flaxseed Lignans and Their Precursors R. Κ. Harris, J . Greaves, D. Alexander, T. Wilson, and W. J. Haggerty

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Midwest Research Institute, 425 Volker Boulevard, Kansas City, M O 64110

Analytical method development was undertaken to identify and quantify lignan and lignan precursors in flaxseed powder. Experi ments indicated that the most successful modes for extracting the lignans from the flaxseed powder involved a hexane defatting process followed by extraction with either methanol or dioxane: ethanol (1:1, v:v). High performance liquid chromatography (HPLC) and thin layer chromatography (TLC) assays were performed on flax seed powder extracts. Ultimately, six components from these extracts were separated and identified by HPLC versus phenolic standards. In descending order of abundance, the identified lignans were: gallic acid, ferulic acid, chlorogenic acid, coumaric acid, secoisolariciresinol diglucoside (SDG) and 4-hydroxybenzoic acid. The concentrations of these phenolics ranged from 0.1 to 12.0 μg/g, with only traces of 4-hydroxybenzoic acid. The flaxseed lignans identified remain stable when stored at -20°C for nine months.

Nutritional research has indicated a relationship between diet and chronic diseases (1-4). In addition, certain plant derived chemicals (phytochemicals) have shown inhibitory activity toward mutagens and carcinogens (5). These reports have increased interest in the anticarcinogenic activity of a wide variety of phytochemicals. Of particular interest are those found in vegetables and cereal grains. For example, there is a positive correlation in higher cancer incidence (colon, rectal, and mammary) when humans or other mammals consume large amounts of fat and red meat (6). The same study found this correlation is reversed, however, with diets containing large amounts of cereals. Cancer incidence has been shown to be lower in countries where the diets are vegetarian or semi vegetarian (7-10). Close review of these studies suggests these benefits are not totally due to fiber content alone, but may also be caused by other substances associated with the fiber. One source of these effects may be the presence of certain lignans and/or their precursors. Lignans demonstrate a broad spectrum of biological activity. They include antitumor, antimitotic, and antiviral

0097-6156/94/0547-0295$06.00/0 © 1994 American Chemical Society

Ho et al.; Food Phytochemicals for Cancer Prevention II ACS Symposium Series; American Chemical Society: Washington, DC, 1994.


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FOOD PHYTOCHEMICALS II: TEAS, SPICES, AND HERBS

activities. They also inhibit enzymes, interfere with nucleic acid synthesis, and have shown other physiological effects. Two reviews on these effects are found in recent publications (11,12). They have also been investigated as potential anticancer agents (17-19). Phenolic compounds such as caffeic, ferulic, coumaric, gallic, and syringic acids, commonly found in grains, have exhibited marked inhibitory effects in the presence of carcinogens (13). Plant phenolics can occur as monomers or dimers, or they can react with other chemical units to form esters or glycosides. Examples of dimeric phenolics are diferulic, ellagic, guaiaretic, and nordihydroguaiaretic acids. An example of a phenolic ester found in oats is chlorogenic acid formed from caffeic and quinic acids. Ellagic acid (a dimer of gallic acid) isolated from various green plants and diferulic acid from wheat are lignans that exhibit remarkable inhibitory activity. For example, studies with benzo[a]pyrene indicate that ellagic acid destroys the diol-epoxide of benzo[a]pyrene, making it incapable of attacking D N A in a cell (14). Caffeic acid and ferulic acid, both hydroxycinnamic acids, inhibit the formation of nitrosamines (75). Lignans are "plant products of low molecular weight formed primarily by the oxidative coupling of p-hydroxyphenylpropene units" (76). They are believed to be chemical intermediates for lignin, a natural polymer and a major constituent of plants. The monomeric units, which form lignans, are primarily the hydroxy- and hydroxy-methoxy derivatives of cinnamic and benzoic acids. Cinnamic, caffeic, coumaric, ferulic, and sinapic acids represent the cinnamic acid group; while benzoic, hydroxy benzoic, protocatechuic, vanillic, and syringic acids compose the benzoic acid group. These phenolic acid monomers are commonly found in cereal grains (20) and are "biochemically...the building blocks for most of the other phenolics in plants" (13). One lignan relevant to this project is 2,3-bis(3-methoxy-4-hydroxybenzyl)butane-l,4-diol. Its common name is secoisolariciresinol (I). (Refer to Figure 1 for the structures of the lignans discussed in this paragraph.) Until a decade ago, lignans were thought to occur only in higher plants. In 1980, however, Setchell (27-22), Stitch (23), and their coworkers discovered the first mammalian lignans. These lignans are structurally related to I and are named enterodiol (II) and enterolactone (III). In 1982, Axelson (24) reported very convincing evidence that the likely precursor for both II and III was the diglucoside (la) of I. Later, Booriello et al. (25) reported that the in vivo conversion of I to II and III is caused by facultative anaerobes within the gut of the human or animal species. They also observed that matairesinol could also be a source for III. The importance of large amounts of both II and III as human metabolites and their role in mitigating breast and other cancers in humans has been vigorously studied since their discovery (26-29). Recently, Thompson (30) reported using in vitro bacterial techniques to screen for the production of II and III from 68 common foods. These studies found that flaxseed flour produced greater amounts (from 75 to 800 times) of the mammalian lignans than the other food sources. This increasing evidence for using flaxseed in the diet as a method for preventing certain cancers has led to increased activity in evaluating flaxseed in human test diets. Isolation Procedures for Secoisolariciresinol Preventing the formation of artifacts during the extraction process is an important factor which needs to be considered when devising an extraction procedure. Ayres


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and Loike (12a) discuss some of the factors to be considered when milling, extracting, and purifying plant extracts. For example, high temperatures should be avoided. In addition, acids and bases can alter lignans through hydrolysis, rearrangement, condensations, and decomposition. Obviously, any alteration of the lignans or their chemical precursors during extraction would interfere in establishing the true stability of the lignans in the flaxseed powder.

II Enterodiol

III Enterolactone

Figure 1. Structures of plant and mammalian lignans. A literature review for solvent extraction methods for I or I a (SDG) revealed only one citation for flaxseed. In 1956, Bakke and Klosterman (33) isolated la from flaxseed. Since then, no other isolation procedures for I or la from flaxseed have been reported. The review did reveal, however, that I or its derivatives have been isolated from the fruit, stems, leaves, needles, resin, and wood of 13 other plant species. Secoisolariciresinol (I) was found in 10 species. The other species contained a diferulic acid ester, a monoglycoside, and a bisrhamnoside of I, respectively. It is believed that plant lignans are formed biosynthetically by the union of two phenylpropane units or cinnamic acid residues (12b). This mechanism is known as the Shikimic Acid-Cinnamic Acid pathway. Thus, it is possible for chemicals occurring in this pathway to be found as intermediates or as decomposition products in flaxseed extracts. Isolation Procedures for Flaxseed Lignans and Precursors The flaxseed powder received by us from the National Cancer Institute was not defatted. We evaluated extraction of the flaxseed powder with various organic



298


solvents such as methanol, ethanol, acetone, ethyl acetate, and acetonitrile. The initial extraction method, performed at room temperature, utilizing ethanol containing 1% hydrochloric acid (v:v) yielded chromatograms with the greatest number of peaks. It became apparent, however, that acidic solvents could (as previously discussed) and were altering the analytical profiles and interfering with the goals of the project. Therefore, acidic or basic solution extractions were not utilized for future extractions. Experimentation with extraction procedures revealed that the powder requires defatting prior to analysis. Without prior removal of the linseed oil, it was not possible to obtain reproducible analytical results. The defatting of the flaxseed powder was achieved by overnight (16 hr) Soxhlet extraction using high purity hexane. Portions of bulk flaxseed powder (20 g each) were weighed into Whatman cellulose thimbles and extracted with 150 mL of solvent. After the defatted flaxseed powder (DFFSP) air dried for several hours, the samples were then suitable for further solvent extractions. Extraction of DFFSP was performed with various solvents or mixed solvents. Solvent mixtures included ratios of methanol and chlorinated solvents which produced similar chromatographic profiles. The solvents which gave the best results were high purity methanol and 95% ethanol: 1,4-Dioxane (1:1, v:v). A l l extractions were performed in duplicate. A typical extraction procedure was performed by weighing out 10 g of DFFSP into 250-mL short-necked, round-bottom boiling flasks. The sample flasks were then charged with 100 mL extraction solvent and Teflon® boiling chips. After the samples refluxed for 8 hr, the cooled mixtures were filtered and collected. The filtered samples were reduced in vacuo at 65°C to a syrup, transferred to 5-mL volumetric flasks using small amounts of methanol, and then brought to volume with methanol. The samples were then transferred to 10-mL amber serum vials, capped, and stored at 4°C until the chromatographic analyses were performed. Stability Indicating Analytical Methods for Flaxseed Lignans and Their Precursors In order to assess the anticancer effects of flaxseed phytochemicals in diets, it is important that suitable analytical methods are available. The methods should be confirmatory and quantitative if measurement of the phytochemical's stability in food matrices is desired. Our approach for flaxseed chemicals was to develop chromatographic techniques capable of monitoring changes in the amounts of the phytochemicals at the parts-per-million levels and, when possible, to confirm the identity of the chemical(s) using mass spectrometry (MS). We have developed thin layer chromatography (TLC) and high pressure liquid chromatography (HPLC) methods to monitor and quantitate the presence of lignans and their precursors in flaxseed. The T L C method was useful for monitoring the chemical profile of flaxseed when extracted by various solvents. The HPLC method also can be used to monitor and quantitate the lignans and their precursors. Table I lists the estimated lignan levels in fresh and aged (~9 months at ~-20°C) flaxseed powder. The instrumentation and conditions for the H P L C procedure used to quantitate the lignans and the T L C parameters used to verify the H P L C data are given below. Example chromatograms of flaxseed extract, spiked with phenolic standards and unspiked, are shown in Figures 2 and 3.


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HARRIS ET AL.


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Thin Layer Chromatography Parameters Plate: HP Keiselgel 60F (254 nm) Solvent: chloroform/methanol/acetic acid (90/10/1, v/v)


Standards Chlorogenic acid Gallic acid 4-Hydroxybenzoic acid Coumaric acid Ferulic acid Secoisolariciresinol diglucoside

Rf value 0.01 0.17 0.57 0.59 0.70 0.71

High Performance Liquid Chromatography Parameters Varian 5000 pump, Waters 440 U V detector at 280 nm, Waters Wisp 710B autosampler, integration by Nelson Analytical Interface and software. Column:Zorbax Cs, 7 μηι Mobile phase: (A) Deionized water/acetic acid (99/1, v/v); (B) Methanol Injection volume: 25 μΐ. Gradient: Time (min) % A %B 0 100 0 5 90 10 20 90 10 70 0 100 72 100 0 85 100 0 Phenolic acid Gallic acid 4-Hydroxybenzoic acid Chloroenic acid Coumaric acid Ferulic acid Secoisolariciresinol diglucoside

Retention Time (min) 4.4 9.2 17.2 20.2 29.4 34.2 & 35.7 (doublet)

Table I. Quantitation of Lignans and Their Precursors in Defatted Flaxseed Powder ^g/g) by HPLC

Lignan standard Chlorogenic Coumaric Ferulic Gallic 4-OH-Benzoic SDG (la)

Ethanol/dioxane Fresh (10 g) Aged (10 g) 15.1

14.9

—

—

—

—

19.9

18.6

—

—

2.5

3.2

Methanol Fresh (10 g) Fresh (5 g) Aged (5 g) 12.5 2.0 7.6 3.4 trace 0.7

7.5

6.0

—

—

10.9 2.8 trace 0.7

11.4 1.9 —

0.9

Note: The estimated levels for all of the lignans have been corrected for the defatted flaxseed powder. The estimated levels are for raw flaxseed powder.


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302


The identity of the chemicals was based on retention time matching with pure standards. T L C indicated the presence of the same lignan standards. More definitive identification experiments, such as HPLC/MS, however, should be used to confirm these results. These data demonstrate that, for those lignans and lignan precursors present in the flaxseed, there is little difference in lignan concentrations between aged and fresh flaxseed powder. In addition, increasing the solvent to solute ratio by using only 5 g portions of flaxseed, instead of 10 g portions, did not significantly enhance the extraction efficiency for any of the lignans. Bakke and Klosterman (31) utilized a rigorous extraction and degradation process to isolate the diglucoside of SDG from flaxseed. They noted that the SDG existed in a polymeric form. Fong and colleagues at the University of Illinois in Chicago have used a similar procedure to successfully isolate bulk quantities of secoisolariciresinol I. Therefore, it is not surprising that the quantitative estimates of this lignan in the extracts using less rigorous methods were lower than might be expected. The HPLC method used to quantitate the lignans in the above table required a programmed gradient of watenacetic acid (99:1) to 100% methanol over 70 minutes. We have developed a Coupled Column Chromatography Particle Beam (CCC-PB) HPLC/mass spectrometry (MS) technique that is capable of identifying eluting peaks at very low levels. The optimum solvent for efficient transfer of an analyte through the PB/MS interface is methanol or acetonitrile with a volatile buffer sometimes added. Using water in the mobile phase can seriously degrade overall performance of PB-LC/MS. As the aqueous concentration is increased in the mobile phase, the sensitivity of the PB technique begins to deteriorate. Frequently, a 50:50 solution of water/methanol will reduce the overall sensitivity by a factor of 10 to 100. Coupled column chromatography allows the use of any mobile phase composition for the primary separation of an analyte. The primary separation can be performed on any type of L C column with any desired solvent (including water) flow rate or buffer necessary to separate an analyte from possible interferences. The primary separation L C column is coupled to a secondary L C column via a switching valve that is manually or electronically controlled. After proper calibration, a portion of the eluent from the primary column is "heart-cut" or diverted by a switching valve to a secondary column. The analyte of interest is then flushed off the secondary column and directed to the PB-MS system with more compatible solvents (i.e. methanol or acetonitrile) and flow rates (0.4 mL/min). The C C C techniques combined with L C / M S have greatly extended the range of applications successfully performed at our institute. Several lignan standards that are found in flaxseed powder were used to develop a prototype method capable of identifying 1 μg on a column using this technique. With this technique, we are confident that the C C C - P B - H P L C / M S method will be useful as a confirmatory procedure. Initial experiments, while promising, indicated that a more robust "cleanup" technique is required before the technique is adopted as a routine method to identify the lignans. Discussion and Considerations for Future Work The analytical procedures employed to establish lignan concentrations for this program indicated lower than expected concentrations of the lignans and their precursors, the phenolic acids. These lower concentrations, however, are most likely due to the extraction procedures used, which were selected primarily to



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prevent degradation of the lignans to monomeric species. Comparison of concentra tions of lignans in aged flaxseed powder with fresh powder indicated very little change in the characteristic lignan fingerprint during ~9 months storage at -20°C. Because identification of the phenolic acid lignan precursors and the secoisolariciresinol derivatives was based on retention time matching with neat standards by both H P L C and T L C , further identification with an improved compound-specific method such as HPLC/MS analysis should be utilized for flaxseed powder extracts. In addition, the flaxseed powder extracts should be evaluated after hydrolysis experiments in order to establish the presence of higher concentrations of the polymeric lignans. Due to the success of the T L C experiments, methods employing high performance T L C (HPTLC) may allow for a larger number of samples to be analyzed in a shorter period of time. One of the major advantages that HPTLC could provide is the potential of analyzing the flaxseed powder extractions without employing a defatting procedure. Supercritical fluid extraction (SFE) techniques could possibly provide improved defatting or extraction of lignans or lignan precursors, without contri buting to hydrolysis problems. The SFE technique has been successfully applied to extraction of cinnamon from chewing gum; pesticides from alfalfa, onions, almonds, oranges, strawberries, and lettuce; caffeine from coffee; and cholesterol from eggs (32). Modifications of similar techniques may be applicable to flaxseed powder and other physiologically functional foods. Another follow-up series of experiments could encompass the character ization of flaxseed powder after its use in complex food matrices. Baked products with incorporated flaxseed should be analyzed before and after the cooking process to identify the presence or absence of the lignans or lignan precursors such as the phenolic acids. After the product has baked at elevated temperatures in the presence of other matrices, greater quantities of the phenolic monomeric species may be present. Additionally, depending upon the matrix of the food product, natural enzymatic activity or hydrolytic processes may increase the concentrations of lignans with greatest physiological interest (e.g. secoisolariciresinol diglucoside, enterolactone, and enterodiol). Summary Analytical procedures employed to establish lignan concentrations indicated lower than expected concentrations of the lignans and their precursors, the phenolic acids. The six lignans and phenolic acid components found in flaxseed extracts were: gallic acid, ferulic acid, chlorogenic acid, coumaric acid, secoisolariciresinol diglu coside (SDG) and 4-hydroxybenzoic acid. The concentrations of these phenolics ranged from 0.1 to 12.0 μg/g, with only traces of 4-hydroxybenzoic acid. These lower concentrations, however, are most likely due to the extraction procedures used, which were selected primarily to prevent degradation of the lignans and establish stability. The concentration of the lignans present in the aged flaxseed powder as compared to fresh flaxseed demonstrated very little change in the characteristic lignan fingerprint during ~9 months storage at -20°C. Future experi mentation will focus on utilizing supercritical fluid extraction as an alternative method for isolating lignans from the flaxseed powder for analysis.



304 Acknowledgements

The authors wish to thank Ms. Jill Guthrie of Midwest Research Institute for her contributions to the mass spectrometry work discussed in this article. This project was funded by the National Cancer Institute, Division of Cancer Prevention and Control, Diet and Cancer Branch, under Contract NO1-CN-05281-01 and the Victor E. Speas Foundation of Kansas City, Missouri.


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28. Setchell, K. D. R.; Adlercreutz, H. in The Role of the Gut Flora in Toxicity and Cancer; 1988; Chapter 14, p 316. 29. Adlercreutz, H. Front. Gastrointest. Res. 1988, 14, 165. 30. Thompson, L. U.; Robb, P.; Serraino, M.; Cheung, F. Nutr. and Cancer 1991, 16,43. 31. Bakke, J. E.; Klosterman, H. J. Proceedings of the North Dakota Academy of Science 1956, 10, 18. 32. Costelli, P. Lab Tech '91: Industrial and Environmental Laboratory Technology Conference and Exhibitions; Atlantic City, NJ; 1991. 1993


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