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PHYTOCHEMICAL ANALYSIS. VOL. 2, 225-229 ( 19’91)

Quantitative Determination by High Performance Liquid Chromatography and Microspectrofluorimetry of Phenolic Acids in Maize Grain A. Sen, S. S. Miller and J. T. Amason* Departments of Biology and Biochemktry, University of Ottawa, Ottawa KIN 6N5, Canada

R. G. Fulcher Department of Food Scicoce and Nutrition, University o f M~nnesota,St. Paul, Minnesota 55108, USA

A method for improved recoveries of phenolic acids in maize grain extracts and their quantification by high performance liquid chromatography (HPLC) is described. A quantitative imaging technique to determine mean ferulic acid autofluorescence of ground grain samples was found to be correlated (r=O.86, a = 14, P = 0.05) with HPLC analysis of ferulic acid in a wide range of different maize genotypes. The advantage of the imaging method is that it reduces procedures required and analysis time considerably.

Keywords: Phenolics; ferulic acid; p-coumaric acid; maize; fluorescence; HPLC.

INTRODUCTION

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Benzoic and cinnamic acids are secondary compounds that are widely distributed in plants. These phenolic acids occur in the free form and as esters (RibereauGayou, 1972; Krygier et al., 1982; Sosulski et al., 1982), or in an insoluble form complexed to cell wall carbohydrates (Fincher and Stone, 1986) or other substances (Collins, 1987). For several years the influence of cinnamic acids on the organoleptic characteristics of food, as well as factors contributing to colour in food products, has generated interest in these compounds in the food industry (Maga and Lorenz, 1973). Apart from their potential physiological and biochemical role in plants, numerous examples of phenolic acids as fungistatic, phytoalexic, phytohormonal, bacteriostatic and allelopathic agents, have been reported (see reviews by Swain, 1977; Harborne, 1977, 1980; Brown, 1981). Our interest in these compounds arises from their effects on phytophagous insects. In particular, the variation in total as well as individual phenolic acid contents in cultivars of maize correlates significantly and negatively with susceptibility towards the stored grain pests, Sitophilus zearnais and Prostephanus truncatus (Serratos et nl., 1987; Classen et al., 1990). For this reason knowledge of the phenolic acid content of a cultivar can be a good indicator of its storage qualities when protection from insects is poor. There is also recent research interest in the phenolic acid content of grain for monitoring bran in milling operations (Pussayanawin et al., 1988). Because of the importance of assessing maize varieties for phenolic acid content, an extraction procedure for a high performance liquid chromatographic (HPLC) method was developed to provide better yields of phenolics. In addition, a new quantitative imaging method was applied t o maize which reduced analysis * Author to whom correspondence should he addressed. 0958-0344/9 1/050225-05 $05.oo 0 1991 by John Wiley & Sons, Ltd

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time from hours to minutes. In this method fluorescence due t o phenolic acids in a ground but otherwise untreated maize sample was determined using a scanning microspectrophotometer. A comparison of the methods was made to determine if the fluorescence imaging technology could be used reliably for rapid scanning of samples for phenolic acids. EXPERIMENTAL HPLC analysis. HPLC analytical equipment included a

Perkin-Elmer LC 480 Diode Array Scan detector equipped with a Perkin-Elmer LC250 pump. Phenolic acids were separated on a 4.6 X 250 mm, 5 pm particle size CI8reversed

phase column (Altex, Ultrasphere) and a Lichrosorb C18 precolumn. The mobile phase was an isocratic mixture of methanol and 10 mM citrate buffer, pH 5.4 (20: 80). The flow rate was 1.0 mLlmin and detection was at 280 nm. Aliquots (100 pL) were collected from samples and dried under nitrogen, and then redissolved in methanol (1 mL). All samples and standards were filtered through Nylon 66 (0.45 pm) filters and injected into a fixed volume 20 pL loop. Comparison of acid and base hydrolysis. Various methods using either base or acid hydrolysis have been described to determine the relative proportions of various phenolic acids Received 27 November 1990 Accepted 25 May 1991

226

A . SEN E T A L .

in cereal grains (Krygier et at., 1982; Sosulski et af., 1982; Pussayanawin and Wetzel, 1987). These were assessed with ground whole kernels of corn.

Comparison of base hydrolysis procedures. Several land races (identified by accession number) from the International Center for Wheat and Maize Improvement (CIMMYT) collection were chosen because of their wide variation in phenolic content. A modern hybrid is shown for comparison (Ritchies). These were evaluated for their total phenolic content following the extraction procedure outlined in Sosulski et al. (1982) as well as a modification of Classen et al. (1990) as described below. Maize grain (I g), finely ground in a coffee grinder, was hydrolysed with 2 N N a O H (50 mL) under nitrogen. Sample was contained in a 250 mL Erlenmeyer flask capped with a rubber septum. After purging with nitrogen, the sample was shaken for 4 h. The sample was then acidified to p H 2.0 by dropwise addition of 6 N HCI. The slurry was centrifuged at 550 x g for 30 min, and the supernatant decanted to a separatory funnel. The supernatant was extracted with ethyl acetate ( 5 x 50 mL), allowing it to settle for 5 min between extractions. The ethyl acetate phase was saved and the aqueous layer returned to the pellet, vortexed and recentrifuged for another 30 min. The supernatant was extracted with ethyl acetate (5 x 50 mL) as before. Finally, distilled water (50 mL) was added to the pellet, which was vortexed and the resulting slurry was again partitioned five times against ethyl acetate. The emulsion that formed due to starch gelatinization was removed by adding more volumes of the solvent, thereby enabling complete recovery of the organic phase. All ethyl acetate layers were combined and dried over anhydrous sodium sulphate. The extract was evaporated at 35°C in a Rotavapor and the residue dissolved in methanol (1 mL). Standards of ferulic and p-coumaric acids were purchased from Sigma Chemical Co., St. Louis, Missouri. To calculate percent recovery of phenolic acids by either acid or base hydrolysis, 1 mg of ferulic and p-coumaric acid standards were treated with 2 N N a O H for 4 h at room temperature or with 0.2 N H2S04(35 mL) for 30 min at 100 "C, as outlined in Pussayanawin and Wetzel (1987). These samples were extracted into ethyl acetate, evaporated and dissolved in MeOH (10mL) which was prepared for HPLC analysis. Recoveries were determined for the trans isomers. Mirrospectrofluorometry. Autofluorescence of ferulic acid was used to quantitate total ferulic acid in maize grain. Small amounts (1-2 g) of finely ground maize flour, prepared in a Udy mill and passed through a 1 mm sieve, were put on a microscope slide and then compressed using another slide to give a flattened top surface with a uniform depth of about 23 mm. These samples were placed on the scanning stage of a Carl Zeiss UMSP 80 microspectrofluorimeter (Plant Research Centre, Agriculture Canada, Ottawa) equipped with an epi-illuminating condensor, H B O 100-W mercury illuminator, a 3651418 nm fluorescence filter combination and controlled by a Hewlett-Packard minicomputer. The objective lens was a 1OX Neofluar and a uranyl glass standard was used as as full-scale fluorescence intensity standard by setting the intensity at 60%. Using the APAMOS software, diagonal corners were marked on the compressed maize flour samples to generate a rectangle. The selected field to be scanned had 4368 points (pixels) at 30 pm steps and the software generated data in terms of mean relative fluorescence intensity. Three replicates were done for each maize population.

RESULTS AND DISCUSSION With the reverse phase system using isocratic conditions (20:80, methanol: 10 mM citrate buffer, pH 5.4) previously applied to ferulic acid in wheat (Pussayanawin and Wetzel, 1987), good separation and symmetrical peaks of phenolic acids in all hydroysed maize extracts were obtained. A typical chromatogram for a maize extract (Fig. 1) reveals significant amounts of pcoumaric acid as well as ferulic acid. Sinapic acid can also be detected by this method but was present only in trace quantities in maize extracts. Peaks with relative retention times corresponding to p-coumaric and transferulic acids were detected in all cultivars of maize and the UV spectra of the peaks corresponded to those of standard compounds. The application of the HPLC method improves analysis time and avoids the derivatization required for gas chromatographic (GC) methods (Classen et a f . 1990). The recovery of standards was high following 4 h base hydrolysis compared to acid hydrolysis (Table 1). In the base hydrolysis, the recovery of trans-p-coumaric acid averaged 81 2.3% while the recovery of transferulic acid was significantly higher (87 1.1%) (P=0.05). Due to the presence of a double bond in the side-chain, phenolic acids are known partially to convert to their cis analogues in the presence of UV light. Although hydrolyses were performed in an atmosphere of nitrogen to prevent oxidation, and in spite of precautions taken to protect from UV light, there was about 2.4 and 4.5% conversion of trans to cis isomers of trans-ferulic and trans-p-coumaric acid standards, respectively, following base hydrolysis, even when samples were refrigerated and analysed within 24 h after extraction. Trans-cis isomerizations were lower (0.51.8%) in acid-hydrolysed standards. Pussayanawin and Wetzel(l987) suggested that extraction of phenolics by acid hydrolysis provides a more stable environment f o r undissociated phenolic acids, whereas in basic solutions they may be more susceptible to oxidation or to cistrans rearrangement. Since recovery of standard phenolic acids was poor

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RETENTION TIME (min 1 Figure 1. HPLC chromatogram of a maize grain extract following base hydrolysis; (A) cis-ferulic acid, (B)p-coumaric acid, (C) trans-ferulic acid

QUANTITATIVE DETERMINATION AND MICROSPECrROFLUORIMETRY OF PHENOLIC ACIDS IN MAIZE GRAIN 227

Table 1. Recovery of phenolic acid standards added to maize flour following acid or base hydrolysis" % Recovery Compound

Acid hydrolysis

trans-Ferulic acid 56 2 4.3 p-Coumaric acid 49 k 5.1 "Values are HPLC means f SD of five replicates.

Base hydrolysis

87 f3.6 81 ? 7.5

following acid hydrolysis, different extraction procedures involving base hydrolysis were evaluated using eight populations of maize. The procedure of Sosulski et a/. (1982), previously applied only to debranned corn flour, involves the fractionation of phenolic constituents into free, soluble and insoluble bound phenolics. In extracts of whole grain of the maize populations, only traces of free and soluble phenolic esters (0.51.2% of the total) were observed. Each of the maize cultivars had a characteristic quantitative pattern. Insoluble bound ferulic and p-coumaric acids were the only phenolic acids detected in all the cultivars of maize with ferulic acid and accounted for almost the total phenolics present. The population M-5 had the highest levels of trans-ferulic acid, while Puebla-463 displayed the highest concentrations of trans-p-coumaric acid. Morelos-52 had the lowest levels of both trans-ferulic and trans-p-coumaric acid. These results are in agreement with those reported in literature for other cereal grains where cinnamic acids and their derivatives generally occur bound rather than in the free form. Phenolic acids are considered to be present as carbohydrate esters. Ferulic acid may also be bound by an amide linkage (Van Sumere et ul., 1975). El-Basyouni and Towers (1964) suggested ferulic acid as a biosynthetic precursor of lignin biosynthesis and an alcoholinsoluble enzyme ester of hydrocinnamic acids as the active intermediate. Thus, in grasses, ferulic and p coumaric acids are found esterified to the hemicellulosic components and core lignin in cell walls, but ferulic acid is predominantly associated with the polysaccharide fraction while p-coumaric acid is attached to the core lignin (Atsuski et al., 1984; Azuma et al., 1985). In mature forage leaves, there is an increase in the p coumaric to ferulic acid ratio which is probably due to the reduction in hemicellulosic synthesis or concentration and increase in core lignin in the cell wall that occurs during maturation (Jung and Shalita-Jones, 1990). It is not certain if such changes occur with maturity in cereal grains. However, enzymatic hydrolysis of cereal grain cell walls shows the presence of

feruloyl moieties esterified usually with an arabinoxyIan component (Smith and Hartley, 1983). 2 - 0 - ( 5 ' 0 trans - feruloyl- (3 - L - arabinofuranosyl) - D - xylopyranose (FAX), a widespread component of cereal cell walls, has been identified in preparations of wheat endosperm and aerial parts of wheat, barley and ryegrass. Following treatment of barley straw cell walls with cellulase, Mueller-Harvey (1986) isolated and identified for the first time a trans-p-coumaroyl group linked to arabinose and xylose (0-[5-O-(trans-p-coumaroyl)a - L - arabinofuranosyl - (1 3) - 0 - (3 - D - xylopyranosyl(1- 4)-~-xylopyranose(PAXX). This ester linkage is similar to the FAXX reported in sugarcane bagasse (Kato et al., 1983) and maize cell walls (Kato and Nevins, 1985). It has been suggested that cross-linking of arabinoxylans by oxidative coupling of ferulic acid polysaccharide esters may occur in uivo during the deposition of cell wall hemicelluloses in the developing kernel (cf. Collins, 1987; Fincher and Stone, 1986). A comparison of the extraction methods for total phenolic acids of Sosulski et al. (1982), Classen et al. (1990) and the new method (Table 2) shows that the new method yields 20-30% more ferulic acid. The differences are striking and it appears that the pellet, neglected after initial centrifugation in both previous methods, contains appreciable amounts of phenolic acids, and extracting it with ethyl acetate releases cellwall-bound phenolic acids. In addition, reduction of particle size as followed in the present study, results in greater extraction of phenolic acids. In wheat leaves, Scalbert et al. (1985) demonstrated the release of etherified or other linkages by reducing particle size. Release of PAXX also depended on the extent of grinding (Mueller-Harvey et al., 1990). The UMSP80 microspectrofluorimeter allows rapid scanning of a large field of maize flour at fixed emission and excitation wavelengths for detection of ferulate autofluorescence. Figure 2 shows scans of relative intensity of a compressed sample of maize flour versus horizontal displacement. Regions of high and low intensity are indicated by peaks and valleys. The software allowed rapid determination of mean fluorescence in multiple scans of this field. Figure 3 demonstrates a linear relationship between total ferulic acid obtained by HPLC and by microspectrofluorimetry mean relative fluorescence values (in 14 samples from five different populations of maize representing a group with a wide range of ferulic acid content). A significant correlation (r = 0.86) exists between the mean absorbance values and results

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Table 2. Total HPLC ferulic acid (FA) and p-coumaric acid (PCA) content (pg/gpb in maize populations following base hydrolysis following different methods Maize type

Sosulski ef a/. (1982) FA PCA

Classen ef a/ (1990) FA PCA

1181 f 2 3 . 1 433515.6 1255f37.8 Nayarit 185 1584 f41.8 542 A 26.1 1526 k 32.4 Mexico-55 1179536.6 449523.6 1099k28.5 Ritchies 1647 f 33.8 579 5 32.4 1542 f 28.6 Puebla 463 1244f32.8 1326f36.7 527527.4 Mexico-182 1364f33.1 476525.9 1417k39.4 Yucatan-7 1091 k42.6 1158A34.8 466f32.4 Morelos-52 1639 133.3 523k 25.7 1691 k 34.4 Mexico-5 "Values are means 5SD of three replicates. bValues corrected for trans-cis isomerization.

512k29.6 491 f24.7 371516.8 5 3 6 f 31.3 514f41.2 417f36.1 328238.1 466 5 28.6

Present method FA PCA

1544f26.9 1825 f 32.6 1417f51.2 2008A 47.2 1589f26.7 1788523.5 1359f31.1 2041 f43.1

598f31.4 731 f24.3 528f18.0 755 f 28.6 650f31.6 682f28.3 519f26.7 674 f32.6

A. SEN E T A L .

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FERULIC ACID ( v g / g ) Figure 3. Relative fluorescence Intensity of maize populations versus total ferulic acid (yglg) as determined by HPLC (r=O.86, n=14, P=O.O5).

Figure 2. Quantitative imaging analysis of maize flour samples showing relative fluorescence intensity due to ferulic acid. The top trace shows a single scan of fluorescence intensity (vertical axis) versus horizontal displacement along the sample (horizontal axis). The lower trace shows multiple scans used to generate mean fluorescence values using image analysis software.

obtained using HPLC. A higher correlation between the two methods (r=0.97) has been reported in modern elite cultivars of wheat (Pussayanawin et al., 1988). This is perhaps because the microspectrophotometer method is highly specific for ferulate autofluorescence and wheat contains primarily ferulic acid while maize also contains p-coumaric acid in significant concentrations. Also the land races of maize provide a much more rigorous test of the quantitative imaging method since they are highly variable in colour and other characteristics that might be expected to influence the results. The fluorescence method using the microspectrophotometer is simple, requires a small amount of sam-

ple and, since data acquisition for a single sample takes only 2-3 min, it provides a good alternative for rapid screening of large numbers of samples, in contrast to the several hours required in chemical techniques involving hydrolysis, extraction and derivatization (in the case of GC). While the quantitative imaging method was not found to be highly precise in this situation, it should be adequate for a variety of screening procedures. For example, we are currently using the method to screen exotic maize germplasm for potential sources of insect resistance. The method could also be used to select moderately high levels of phenolic acids in elite maize germplasm in an attempt to improve the insect resistance of grain. Acknowledgements This research was supported by grants from the International Development Research Centre of Canada and the Natural Sciences and Engineering Research Council of Canada (Strategic program). We are grateful to Agriculture Canada for access to the microspectrophotometer and to CIMMYT for maize germplasm. Technical assistance was provided by J. Gale and K. Collingwood.

REFERENCES Atsuski, K., Azuma, J. and Koshijima, T. (1984). Lignin-carbohydrate complexes and phenolic acids in Bagasse. Holzforsch. 38, 141-149. Azuma, J., Nomura, T. and Koshijima, T. (1985). Lignin-carbohydrate complexes containing phenolic acids isolated from the culms of Bamboo. Agric. Bid. Chem. 49, 2661-2669. Brown, S. (1981). Coumarins. In The Biochemistry of Plants, Vol. 7, Secondary Plant Products (Conn, E. E., ed.), pp. 269-300. Academic Press, New York. Classen, D., Arnason, J. T., Serratos, J. A., Lambert, J. D. H., Nozzolillo, C. and Philogene, B. J. R. (1990). Correlation of phenolic acid content of maize to resistance to Sitophilus zeamais, the maize weevil in CIMMYT's collections. J. Chem. Ecol. 16,301-315. Collins, F. W. (1987). Oat phenolics: Structure, occurrence and function. In Oats: Chemistry and Technology (Webster, F. H., ed.), pp. 227-295, AACC, Minnesota. El-Basyouni, S. and Towers, G. H. N. (1964). The phenolic acids in wheat. I. Changes during growth and development. Can. J. Biochem. 42,203-210. Fincher, G. 6. and Stone, 6. H. (1986). Cell walls and their components in cereal grain technology. Adv. Cer. Sci. Tech. 8,207-295. Harborne, J. B. (1977).Variation in and functional significance of

phenolic conjugation in plants. Rec. Adv. fhytachem. 12, 457-474. Harborne, J. 6. (1980). Plant phenolics. Enc. Plant Physiology, New Series, Vol. 8, (Bell, E. A. and Charlwood, 8. V., eds) pp. 329-402. Springer-Verlag, Berlin. Jung, H.-J. G. and Shalita-Jones, S. C. (1990). Variation in the extractibility of esterified p-coumaric and ferulic acid from forage cell walls. J. Agric. Food Chem. 38, 397-402. Kato, Y. and Nevins, D. J. (1985). Isolation and identification of ~ - ( 5 - O - f e r u ~ o y ~ - a - ~ - a r a b i n o f u r a n o s y ~ ) - ( l ~ 3 ) - ~ - ~ - ~ - x y pyranosyl-(I+4)-~-xylopyranose as a component of Zea shoot cell walls. Carbohydr. Res. 137, 139-150. Kato, A., Azuma, J. and Koshijima, T. (1983).A new feruloylated trisaccharide from Bagasse. Chem. Lett. 137-140. Krygier, K., Sosulski, F. and Hogge, L. (1982). Free, esterified and insoluble-bound phenolic acids. 1. Extraction and purification procedure. J. Agric. Food Chem. 30,330-334. Maga, J. A. and Lorenz, K. (1973). Taste threshold values for phenolic acids which can influence flavor properties of certain flours, grains and oilseeds. Cereal Sci. Today 18, 326-328. Mueller-Harvey, I., Hartley, R. D., Harris, P. J. and Curzon, E. H. (1986). Linkage of p-coumaroyl and feruloyl groups to cellwall polysaccharides of barley straw. Carbohydr. Res. 148, 71-85.

QUANTITATIVE DETERMINATION AND MICROSPECTROFLUORIMETRY OF PHENOLIC ACIDS IN MAIZE GRAIN 229 Pussayanawin, V. and Wetzel, D. L. (1987). High performance liquid chromatography determination of ferulic acid i n wheat milling fraction as a measure of bran contamination. J. Chromatogr. 391, 243-255. Pussayanawin, V., Wetzel, D. L. and Fulcher, R. G. (1988). Fluorescence detection and measurement of ferulic acid in wheat milling fractions by microscopy and HPLC. J. Agric. Food Chem. 36,515-520. Ribereau-Gayou, P. (1972). Plant Phenolics. Oliver and Boyd, Edinburgh. Scalbert, A,, Monties, B., Lallemeand, J.-Y., Guillet, E. and Roland, C. (1985). Ether linkage between phenolic acids and lignin linkage fractions from wheat straw. Phytochemistry 24, 1359-1362. Serratos, A,, Arnason, J. T., Nozzolillo, C., Lambert, J. D. H., Philogene, B. J. R., Fulcher, G., Davidson, K., Peacock, L.,

Atkinson, J. and Morand, P. (1987). The factors contributing to resistance of exotic maize populations t o maize weevil Sitophilus zeamais., J. Chem. Ecol. 13, 751-762. Smith, M. M. and Hartley, R. D. (1983). Occurrence and nature of ferulic acid substitution of cell wall polysaccharides in graminaceous plants. Carbohydr. Res., 118, 65-80. Sosulski, F., Krygier, K. and Hogge, L. (1982). Free, esterified and insoluble bound phenolic acids. 3. Composition of phenolic acids in cereal and potato flour. J. Agric. Food Chem. 30, 337-340. Swain, T. (1977). Secondary compounds as protective agents. Ann. Rev. Plant Physiol. 28, 479-501. Van Sumere, C. F., Albrecht, J., Dedoner, A,, De Pooter, H. and Pe, I . (1975). Plant proteins and phenolics. In The Chemistry and Biochemistry of Plant Proteins. (Harborne, J. B., ed.), pp. 211-264. Academic Press, London.