Antioxidant potential of ferulic acid

FreeRadical Biology& Medicine, Vol. 13, pp. 435-448, 1992 Printed in the USA. All fights reserved.

0891-5849/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd.

Review Article ANTIOXIDANT

POTENTIAL

OF FERULIC

ACID

ERNST GgAF Tastemaker, 1199 Edison Drive, Cincinnati, OH 45216, U.S.A.

(Received 17 February 1992; Revised 13 April 1992; Accepted 30 April 1992) Abstract--Ferulic acid is a ubiquitous plant constituent that arises from the metabolism of phenylalanine and tyrosine. It occurs primarily in seeds and leaves both in its free form and covalently linked to lignin and other biopolymers. Due to its phenolic nucleus and an extended side chain conjugation, it readily forms a resonance stabilized phenoxy radical which accounts for its potent antioxidant potential. UV absorption by ferulic acid catalyzes stable phenoxy radical formation and thereby potentiates its ability to terminate free radical chain reactions. By virtue of effectively scavenging deleterious radicals and suppressing radiation-induced oxidative reactions, ferulic acid may serve an important antioxidant function in preserving physiological integrity of cells exposed to both air and impinging UV radiation. Similar photoprotection is afforded to skin by ferulic acid dissolved in cosmetic lotions. Its addition to foods inhibits lipid peroxidation and subsequent oxidative spoilage. By the same mechanism ferulic acid may protect against various inflammatory diseases. A number of other industrial applications are based on the antioxidant potential of ferulic acid. Keywords--Coniferic acid, Ferulic acid, Food preservative, Free radical, Iron chelation, 7-Oryzanol, Phenolic antioxidant, UV absorber

a dibasic acid, which is consistent with its currently known structure. During the following 60 years, hardly any additional research was carried out on this plant constituent. In 1891 a report 2 appeared on the isolation of ferulic acid from Pinus laricio Poir. Finally in 1925 it was chemically synthesized by the amine-catalyzed condensation of vanillin with malonic acid. 3'4 By 1927 the chemistry of ferulic acid was fairly well understood. 5 The cis and trans isomers were separated in 19576 and the stereochemistry of ferulic acid was unequivocally ascertained by carbon-13 nuclear magnetic resonance (NMR) spectroscopy in 19767 and confirmed by X-ray crystallographic analysis in 1988. 8 Despite its ubiquity in plants and its early isolation and identification 126 years ago, there still is a paucity of published information on the antioxidant properties of ferulic acid. During the past 10 years, several hundred publications have appeared describing its allelochemical properties, occurrence, metabolism in plants, physiological role during lignification, soil levels, utilization by microorganisms, analytical methodology, and various industrial applications, but no systematic studies have been carried out to determine the mechanism of radical scavenging, the relationship be-

INTRODUCTION

In 1866 Hlasiwetz and Barth in Innsbruck, Austria, isolated protocatechuic acid and resorcinol from the commercial resin of Ferula foetida, an umbelliferous fennel-like plant. 1 At the same time they obtained a light yellow precipitate upon the addition of divalent lead to an alcoholic resin solution. They washed the precipitated lead salt with alcohol, reconstituted the free acid, and determined its composition as C~0H~004 from an elemental analysis. They named this compound Ferulas/iure or ferulic acid and further characterized its various metal salts and base-catalyzed reaction products. Furthermore, they termed it Address correspondence to: Ernst Graf. Ernst Grafis Manager of Food Science in the Technology Center at Tastemaker in Cincinnati, Ohio. He received his undergraduate education in Switzerland and earned his PhD degree in Biochemistry from the University of Minnesota in 1981. His primary academic research interests include the chemistry and biochemistry of activated oxygen species and their effects on biological and food systems. He is particularly interested in the effects of radical scavengers and iron chelating agents on oxidative reactions. He has authored a book and over a dozen publications on various aspects of phytic acid, another naturally occurring antioxidant. At Tastemaker he is directing new product research in the area of industrial food science and technology. In his spare time he likes to travel, exercise, write, cook, and camp with his family. 435

436

E. GRAF COOH

COOH

OCH 3 OH

Ferulic Acid

OH OCH 3

Isoferulic Acid

Fig. 1. Structure of ferulic and isoferulic acids.

tween stereochemistry and antioxidant potential, and its photoprotective role in plant cells. The present review attempts to selectively summarize the literature on this widely distributed plant antioxidant and to provide some direction for future research. STRUCTURE AND CHEMICAL PROPERTIES

Structure Ferulic acid is the common name for 3-(4-hydroxy-3-methoxyphenyl)-2-propenoic acid. Other names include 3-methoxy-4-hydroxycinnamic acid, caffeic acid 3-methyl ether, and coniferic acid. Plants also produce isoferulic acid or 3-hydroxy-4-methoxycinnamic acid, as shown in Figure 1. Ferulic acid isolated from plants usually exists as the trans isomer. The structure elucidation was carried out by carbon-13 NMR spectroscopy using partial pH titration for independent carbon assignments; 7 it was confirmed by X-ray crystallography.8 During storage of cis- or trans-ferulic acid in water at room temperature, stow isomerization over about 2 weeks results in an equilibrium ratio of 23% cis and 77% trans. 9

phenolate anion. The high degree of resonance stabilization of the phenolate anion across the entire conjugated molecule markedly increases its acidity relative to similar phenolic acids.l~ Trans-ferulic acid strongly absorbs in the UV range with absorption maxima at 284 nm (log ~ = 4.18) and 307 nm (log ~ = 4.19) in aqueous solution, pH 6.0 (Ref. 12). It also exhibits strong fluorescence. 13 Ferulic acid is fairly stable when dissolved in organic solvents. In water, however, it may undergo slow thermal decarboxylation to form 4-vinylguaiacol. One hour heating at pH 4.0 and 100°C resulted in a 5.2% decarboxylation. 14 Iron catalyzes its oxidation to a dilactone. 5 A more detailed description of the organic chemistry of ferulic acid is published in Beilstein)

Isolation and separation Ferulic acid was isolated from plant bark or resins by alcoholic lead precipitation and recrystallization from hot water as the free acid.1 It can easily be separated from similar plant phenolic acids by gas chromatography of silylated derivatives. Retention times (s) at 210°C of six silylated cinnamic acid derivatives were as follows: cis-ferulic, 206; cis-caffeic, 256; cissinapic, 304; trans-ferulic, 336; trans-caffeic, 407; trans-sinapic, 522. Similar resolution for the separation of cis-trans isomers of cinnamic acid derivatives was achieved using two-dimensional thin-layer chromatography,~5 paper chromatography,6 near-horizontal long-bed chromatography, ~6 electrophoresis in a fused silica capillary tube, 17 thin-layer reflectometry, 18 and high-pressure liquid chromatography (HPLC). 15'19-21 The trans isomer of ferulic acid is immediately visible under UV light, while the cis isomer requires several seconds of irradiation before becoming visible. 22 HPLC elution of phenolic acids can be monitored using a UV or fluorescence detector.~5

Physical, chemical, and spectroscopic properties

Synthesis

Cis-ferulic acid does not crystallize but instead forms a yellow oil with a UV maximum at 316 nm in ethanol. Colorless orthorhombic needles of trans-ferulic acid can be crystallized from hot water. 1° Its molecular weight is 194 with a melting point of 174°C. It is soluble in ethanol, ethyl acetate, and hot water, moderately soluble in ether, and only sparingly soluble in benzene and petroleum ether. Ferulic acid is a strong dibasic acid. The first proton dissociation produces the carboxylate anion, while the second proton dissociation generates a

Knoevenagel condensations for the synthesis of cinnamic acid and its oxy-defivatives such as ferulic acid were reported as early as 1925. 3,4The first systematic analytical study of the factors influencing the synthesis of ferulic acid was published in 1932, when Dalal and Dutt 23 showed that malonic acid condensed very easily with aromatic and aliphatic aldehydes in the presence of small quantities of pyridine and piperidine. The reaction took place in pyfidine alone, but small quantities of piperidine or other secondary or tertiary amines greatly accelerated the reaction, producing a-unsaturated monocarboxylic acids with

Ferulic acid

rapid loss of carbon dioxide. The condensation of malonic acid with vanillin in the presence of quinoline at 80°C for 8 h produced ferulic acid with a theoretical yield of 50.3%. Vorsatz improved the yield to 73% by carrying out the condensation at room temperature for 3 weeks. 24 Additional minor modifications of the malonic acid condensation with vanillin were publishedfl 5 A second route for synthesizing ferulic acid is the Perkin condensation of vanillin with acetic anhydride.~° Vanillin, acetic anhydride, and anhydrous sodium acetate were heated for 5 h at 150-170°C. The reaction was stopped with cold water, the precipitate filtered, ether extracted, and recrystallized from hot water.

437

CH3° . ~ / HO" w

Cycloartenyl Ferulate

OCCURRENCE

Ferulic acid occurs in rice, 26 wheat, 27-31 barley, 27'32 oat, 33 s o r g h u m , 34 forage, 35 t r e e b a r k , m,36-39 poplar

buds, 4° roasted coffee,4~ tomatoes, 42 asparagus, 43,44 olives, 45berries, 46 peas, 47 vegetables, 48 citrus fruits and leaves, ~5"49 and m a n y other plants. In seeds ferulic acid is generally localized in the bran fraction where it accounts for the intense autofluorescence of such tissues as wheat aleurone layers28'29 and wheat and barley scutella. 27 In wheat it is the major phenolic acid, 3° with typical levels of 50 and 500 u/g in white flour and ground whole wheat, respectively. 3~ Much of the ferulic acid occurs as esters in many plants. 35-37'5° It is covalently conjugated with monoand disaccharides, 5~ plant cell wall polysaccharides, 52 glycoproteins, 53 lignin, 54-56 betacyanins, 5~ and other insoluble carbohydrate biopolymers of cell walls) v-61 Cyclodimers of ferulic acid may occur by photodimerization and have been isolated from cell walls of n u m e r o u s plants. 58,62-65 Ferulic acid has also been identified as a lipid moiety. Structural tissues such as wood, bark, and pine needles contain long-chain n-alkyl ferulates, 66 whydroxy fatty acid ferulates, 67 and a ferulate ester of (4S,5S,7S,8S)- 1( 10)-guaiene-8,10-diol. 68 Steryl ferulates were isolated from maize, corn, wheat, rye, and triticale 69'7° and from rice bran oil, where they occur at a concentration of 1.47 to 1.97%. 7t Steryl ferulates were first discovered in rice bran oil in 1954. 72 As a result of extensive animal experimentation, this substance was regarded as a novel growthpromoting vitamin. Since it was isolated from rice bran oil (Orysae Sativa L.) and contained a hydroxyl group, it was conveniently named oryzanol. Subsequent studies revealed that oryzanol is not a single

24-Methylenecycloartanyl Ferulate Fig. 2. Structure of cycloartenyl and 24-methylenecycloartanyl ferulate.

compound but instead comprises a variety of ferulic acid esters called a-, 13-, and 7-oryzanol. Of these, 3,oryzanol has been the best characterized. The triterpene alcohol components of a typical 7-oryzanol consist primarily of cycloartenol and 24-methylenecycloartanol (see Fig. 2), but they also include other minor sterols, such as campestanol, stigmastanol, flsitosterol, cycloartanol, and cholesterol. This rice bran oil extract, 7-oryzanol, is of substantial commercial significance in Japan as a food and medical antioxidant, especially when used in synergy with a-tocopherol. 73 7-Oryzanol markedly inhibits the oxidation of rice bran oil, TM malondialdehyde generation during iron-mediated microsomal lipid peroxidation, 75 and formation ofdienes during peroxidation of linoleic acid by UV irradiation, v5 Due to its excellent emulsifying properties and high UV absorption, 7-oryzanol also is the active ingredient in various cosmetic preparations like skin creams, suntan lotions, and cosmetic soaps. 73 Furthermore, ~,-oryzanol, particularly cycloartenyl and 24-methylenecycloartanyi ferulate (Fig. 2), are effective agents in the treatment of arteriosclerosis. 76 Methods are now available for labeling steryl ferulates with carbon-14 (Refs. 77, 78) for research purposes. At the same time, a process has been developed for their large-scale industrial manufacture. 79

HO~

OH Vanillin

~

CHO

OCH3

Caffeic Acid

OH

COOH

~--

Cinnamaldehyde

CHO

COOH

Coniferyl Alcohol

OH

OCH3

CH2OH

Ferulic Acid

OH

COOH

I~

3

Lignins

Sinapic Acid

~H30~CH OH

OH

Curcumin

OH

c.3o~~.~ " "T "~ "°OH3

Fig.3. Metabolicpathwaysof ferulicacidformationin plants.

A Coumarin

0

p-Coumaric Acid

Tyrosine

- -

OH

COOH

Cinnamic Acid

COOH

OH

NH 2

COOH

~

Phenylalanine

'L~NH2

COOH

Ferulic acid METABOLISM AND PHYSIOLOGICAL FUNCTION

Plants Figure 3 illustrates the principal pathway for the biosynthesis of ferulic acid in plants. The reactions are initiated by phenylalanine and tyrosine ammonia lyases, two plant enzymes that convert phenylalanine and tyrosine to trans-cinnamate and p-coumarate, respectively. Ferulic acid derives from p-coumaric acid through hydroxylation followed by methylation with methionine acting as the methyl donor, 54catalyzed by s-adenosyl-L-methionine:caffeic acid 3-o-methyltransferase, s° Subsequent oxidation and methylation reactions give rise to additional di- and trihydroxy cinnamate derivates that become the starting materials for the formation of lignin. 56'81 Fluorinated analog82 and ferulic acid labeled with tritium s3 or carbon14 (Ref. 78) are often employed to follow their biochemical fate during the lignification process. Oxidative degradation of ferulic acid leads to vanillin and guaiacol, s4 Other alterations of the propenoic acid side chain may produce additional classes of phenylpropanoid structures, such as flavonoids. A few rare metabolites of ferulic acid have also been reported, such as methyl ferulate in rice brain oil 85 and 5-hydroxyferulic acid in zea mays and hordeum vulgare. 86 Most of the compounds shown in Figure 3 impart characteristic flavors and fragrances to many spices and other plants. As mentioned earlier, much of the ferulic acid in plant tissue is conjugated with other molecules (e.g., esters or amides are formed via a ferulic acid-coenzyme A thioester intermediate). A recent r e v i e w 87 describes the biosynthetic pathways of various hydroxycinnamic acid conjugates with special emphasis on their localization and molecular metabolic regulation. Several physiological roles of ferulic acid have been proposed. It crosslinks vicinal pentosan chains, arabinoxylans, and hemicelluloses in cell walls, which increases wall extensibility during cell elongation, gs Ferulate-mediated crosslinking renders these cell components insoluble 57"63's9and lowers their susceptibility to hydrolytic degradation by endosperm enzymes during germination. 28Therefore, ferulic acid has been suggested to act as a germination inhibitor, 9° presumably by limiting substrate availability. A recent study described avian repellent properties of ferulic acid, 9~ which may imply a defensive role of cinnamic acid derivatives against natural predators. Ferulic acid also increased the host's resistance to maize weevil infestation in m a i z e 92 and aphids in c o w p e a s . 93

Several plant phenolics, including ferulic acid,

439

have been shown to promote the reductive release of ferritin iron as determined by the spectroscopic measurement of the ferrous-ferrozine complex at 562 nm. 94 This chemical reactivity of liberating iron from phytofen'itin may represent an important process in plant mineral metabolism and a possible physiological function of ferulic acid in plant cells. Ferulic acid in soil and mulch has also been shown to exhibit an allelopathic function in plants by regulating plant growth via root interaction. 95'%This allelochemical transfer from soil to host plant may play an important role during wheat crop rotation and also during the self-preservation of plants in arid regions. However, the major physiological role of ferulic acid is likely to be its potent antioxidant function.

Microorganisms Various microorganisms including bacteria, yeasts, and molds can grow on a medium containing ferulic acid as the sole carbon and energy s o u r c e 97'98 converting up to 99.5% of the total ferulic acid to vanillic acid. Some strains did not metabolize vanillic acid any further while most others, such as Fusarium, completely degraded it to CO2 and H 2 0 (Ref. 99). Bacillus subtilis utilized ferulic acid and its intermediatespvanillin, vanillic acid, and protocatechuic acid--as sole carbon source, l°° A Penicillium sp. demethylated ferulic acid to caffeic acid and oxidized the side chain to form protocatechuic acid, which was further catabolized via the ortho-fission pathway.t°l A fungus of the genus Pycnoporus (basidiomycetes) was found to metabolize ferulic acid to vanillin. 1°2 This bioconversion was harnessed for the industrial production of natural vanillin, a common flavor constituent. Ferulic acid metabolism was also studied in cultures of two micromycetes. ~°3 Both fungi decarboxylated ferulic acid to 4-vinylguaiacol and then metabolized it further to vanillin, vanillic acid, vanillyl alcohol, methoxyhydroquinone, guaiacol, hydroxyquinolcatechol, and pyrogallol. Microbial degradation of ferulic acid to vanillic acid and other substituted phenols occurs during the fermentation of rice and is a recognized source of many important flavor compounds in fermented products, such as sake, soy, koji, and moromi) °4 For example, 4-ethylphenol and 4-ethylguaiacol, the important flavor components of soy, are formed by yeast from p-coumaric acid and ferulic acid, respectively. During the past 10 years, a vast body of knowledge in the metabolism of ferulic acid by microorganisms has accumulated. Specific enzymes were identified,

440

E. GRAF

such as a ferulic acid esterase from Streptomyces olivochromogenes 1°5 and from Schizophyllum commune, 1°6 exact pathways were elucidated, and metabolic regulation was well characterized. However, a detailed description of the microbial ferulate metabolism is outside the scope of this review article.

acid derives most of its antioxidant potential from its radical scavenging ability. Second, ferulic acid may partially act as an antioxidant by virtue of strongly mitigating the harmful effects of UV radiation. Each antioxidant mechanism will be discussed in detail.

ANTIOXIDANT CHEMISTRY

As has been thoroughly reviewed in this journal, 107 transition metals, particularly iron, play a crucial role in oxygen radical reactions and subsequent oxidative damage to biological materials. These reactions often involve iron-catalyzed activation of oxygen to form superoxide anion radical, hydrogen peroxide, and highly reactive hydroxyl radical via the Fenton reaction and Haber-Weiss cycle. As demonstrated by ~HN M R relaxation and spectroscopic azide titration studies, iron-catalyzed formation of "OH strictly requires the availability of at least one free iron coordination site through which the redox shuttle can occur.~°8 By virtue of occupying all aquo coordination sites, a few chelating agents have the ability to completely block the catalytic activity of iron. One such rare chelating agent is phytic acid, a natural plant antioxidant.'°9 In emulsions and biological systems composed of hydrophobic membranes surrounding aqueous media, chelating agents may further act as antioxidants through metal sequestration. Removal of the catalyst and of reactive ferryl and perferryl ions from the lipid substrates may effectively reduce the degree of in situ oxidation. In view of the dibasic acidity of ferulic acid due to its carboxyl and phenolic hydroxyl group, it appears a potential candidate for chelating iron. However, the addition of 0.25 mM Fe 3+ to 5 mM ferulic acid in 100 mM NaHCO3, pH 8.0, caused an immediate flocculant precipitate resembling that of iron hydroxide (Graf, unpublished results). Therefore, ferulic acid is unlikely to form an iron chelate. Upon closer inspection of the spatial configuration of ferulic acid, this observation is consistent with the planarity of the entire molecule. Even if it did form a polymolecular complex involving several ferulates clustered around iron, this complex probably would still contain some coordination water and participate in redox transfer reactions. In fact, Fe 3+ has been shown to rapidly oxidize ferulic acid dissolved in aqueous ethanol to a dilactone, which was identified as 1,4-dioxo-3,6-bis-[4hydroxy-3-methoxy-phenyl]-3,3a,6,6a-tetrahydroi H,4H-furo[3,4-c]furan (Ref. 5). Heavy lanthanides, however, do form complexes with ferulic acid. 11° From these results it becomes evident that ferulic

Radical scavenging Ferulic acid (5 raM) provided 70.9% inhibition of in vitro lipid peroxidation in rat brain homogenates as determined by malondialdehyde generation, l jl Caffeic acid was approximately 1000-fold more effective, whereas cinnamic acid exhibited virtually no antioxidant activity at all. A similar relationship was observed during the oxidation of Ghee, 112 as demonstrated in Figure 4. The antioxidant potential increases with the number of phenolic hydroxyl groups (Fig. 3), cinnamic acid being the weakest and caffeic acid being the most potent. In contrast to these studies, one recent article reported that ferulic acid, unlike p-coumaric and caffeic acids, scavenged superoxide anion radical and also inhibited lipid peroxidation induced by superoxide; the effect was similar in magnitude to that observed with superoxide dismutase. ~13 Most published results demonstrate a dramatic increase in the antioxidant potential upon the hydroxylation ofcinnamic acid (cf. Fig. 4). Whereas cinnamic acid exhibited very little activity, p-coumaric and ferulic acids strongly suppressed the oxidation of Ghee and of brain tissue. From these observations it becomes evident that most of the antioxidant potential of ferulic acid is derived from its ability to effectively terminate radical chain reactions by the following mechanism: Any reactive radical colliding with ferulic acid easily abstracts a hydrogen atom to form a phenoxy radical. As shown in Figure 5, this radical is highly resonance stabilized since the unpaired electron may be present not only on the oxygen but it can be delocalized across the entire molecule. Additional stabilization of the phenoxy radical is provided by the extended conjugation in the unsaturated side chain. This resonance stabilization accounts for the ease of the formation of the phenoxy radical and its consequent lack of reactivity; therefore, this phenoxy radical is unable to initiate or propagate a radical chain reaction, and its most probable fate is a collision and condensation with another radical, including another ferulate radical to yield the dimer curcumin. Such coupling may lead to a host of products, all of which still contain phenolic hydroxyl groups capable of radical scavenging. Both thionyl chloride and oxalyl chlo-

Ferulic acid

441

1.5 ~r

E ~D

1

CO

X O

0.5

0 Control

Cinnamic Acid

Ferulic Acid

p-Coumaric Acid

Caffeic Acid

Fig. 4. Effect ofcinnamic acid derivatives on the peroxidation of Ghee during storage for 30 d at 37°C. Redrawn from Ref. 112.

ride catalyze rapid polycondensation to poly(ferulic acid).t14, lls The presence of a second phenolic hydroxyl group substantially enhances the radical-scavenging activity due to additional resonance stabilization and o-quinone formation. Abstraction of a hydrogen atom from caffeic acid forms a stable phenoxy radical that can easily donate another hydrogen atom to rearrange into the o-quinone. Each H" abstraction leads to the termination of a radical chain reaction. Therefore, the lack of superoxide anion radical scavenging by caffeic acid as reported earlier t 13 seems to be in conflict with the proposed radical mechanism. Methoxylation of p-coumaric acid to form ferulic acid somewhat destabilizes the phenoxy radical, which also slightly impairs its antioxidant potential. Nevertheless, with its phenolic hydroxyl group and the extended side chain conjugation, ferulic acid is an efficacious radical scavenger. Acetylation of ferulic acid greatly reduces its antioxidant activity. ~6 This further corroborates the aforementioned mechanism, in which a radical chain reaction is terminated by the abstraction of a hydrogen atom from the phenolic lay-

COOH

COOH

droxyl group to form a resonance stabilized phenoxy radical.

UV Absorption Due to the high degree of conjugated unsaturation, ferulic acid is a strong UV absorber. Its absorption of UV radiation initiates phenoxy radical formation leading to cis-trans isomerization. 9 The most likely mechanism invokes initial generation of a phenoxy radical followed by addition of a hydrogen atom to the last structure in Figure 5, which forms a mixture of cis- and trans-ferulic acid. This UV absorption has been demonstrated to inhibit other free radical reactions. 9 As shown in Figure 6, sinapic acid significantly inhibited the rate of cis-trans isomerization of ferulic and caffeic acids by virtue of exhibiting the highest cis-trans isomerization rate of the three cinnamic acid derivatives. 9 In this example, the compound which is converted most rapidly acts as an inhibitor to the isomerization of the other compounds, presumably by terminating radical chain reactions through the provision of stable UV-generated radicals.

COOH

COOH

"~ O"

0

0

COOH

"OCHa

O

Fig. 5. Resonance stabilization of ferulic acid radical.

OCH3 O

E. GRAF

442

30

20

10 E 0

._~

0 30

.O ~-J ci=

E o

ii

to Sinapic Acid [ ] Ferulic Acid .

,

NI

IN

20

0

60

300 650 Incubation Time (hours)

I/

Fig. 6. The formation of cis isomers of ferulic, caffeic, and sinapic acids in aqueous solution. (A) Acids stored individually. (B) Acids stored together. Redrawn from Ref. 9.

The proposed mechanism of antioxidant function through UV absorption is further supported by the inhibitory effect of 5 mM ferulic acid and campesteryl ferulate on diene formation during the peroxidation oflinoleic acid by UV irradiation, as shown in Figure 7. 75 Campesteryl ferulate at a concentration of 50 mM

reduced the dienb content to 22% and 30% of the control after 15 and 30 h of incubation at 30°C, respectively. At high concentrations, ferulic acid may also protect other light-sensitive compounds against oxidative damage by attenuating the amount of UV radiation impinging on the dissolved molecules. Whereas this effect remains undetectable in aqueous model solutions, UV attenuation by ferulic acid may become significant in opaque biological solids, such as seeds, leaves, and others. Similarly, the UV-initiated generation of stable ferulic acid radicals leading to isomerization or termination of other radical chain reactions probably plays only a minor antioxidant role in aqueous test solutions, which explains the large differences in antioxidant potentials of cinnamic, ferulic, and caffeic acid despite their similar UV-absorbing properties. However, this UV-catalyzed radical-scavenging mechanism may become very important in biological materials of limited light permeability. The hypothesis linking biological antioxidant potential to the UV absorption and subsequent generation of stable radicals of ferulic acid is consistent with the observation that the highest levels of ferulic acid and similar cinnamate derivatives notoriously occur in leaves, seeds, and other plant organs of large surface to volume ratios. Ferulic acid is likely to impart tremendous antioxidant protection to several cell layers located immediately below the surface that are exposed to UV penetration. And finally, topical application of ferulic acid to human skin is expected to provide some photoprotection. Indeed, ferulic acid constitutes the active ingredient in many cosmetic skin lotions.

40 [ ] Control -~

•

Ferulic Acid

"~: 30

[ ] Campesteryl Ferulate

O

tO

20

U.

c• 1 ~5

0

~

1

3 Incubation Time (hours)

5

Fig. 7. Diene formation during UV-induced linoleic acid peroxidation. Diene content was monitored at 30°C by measuring the absorption at 233 nm. Redrawn from Ref. 75.

Ferulic acid ANTIOXIDANT APPLICATIONS

Food preservative Ferulic acid was first used in Japan in 1975 to preserve oranges and to inhibit the autoxidation of linseed oil.~t 7 Phenolic compounds also stabilized lard ~8 and soybean oil,ll9 especially in the presence of added copper or iron. Mixtures of ferulic acid and amino acids or ferulic acid and dipeptides, such as glycylglycine or alanylalanine, exerted a synergistic inhibitory effect on the peroxidation oflinoleic acid; also, 0.05% ferulic acid in combination with 0.5% glycine completely inhibited the oxidation of biscuits stored at 30°C for 40 d. 12°A synthetic derivative of ferulic acid, tocopherol ferulate, proved to be a much superior antioxidant for fats and oils as evaluated by peroxide value, m The putative strong antioxidant potential of several food proteins, such as soybean protein, defatted soybean flour, casein, maize gluten, and zein could be ascribed to contaminant phenolic compounds, t22"t23 Ferulic acid and other cinnamic acid derivatives also accounted for the antioxidant activity in tomato extracts. 42 Similarly, lipids extracted from grasses and leaves showed strong antioxidant behavior due to their phenolic contaminants) 24 Virgin olive oil also contains large quantities of phenolic compounds and a high degree of antioxidant potential that is destroyed during the refining process,4S perhaps due to the loss of synergistic antioxidant effects of other components. 125 During cooking of food, thermal decarboxylation of ferulic acid may produce 4-vinylguaiacol, ~4'26 the main contributor of off-flavors in many cooked products. For example, the generation of 4-vinylguaiacol from ferulic acid also occurs during the manufacture of beer 126and accounts for the development of off-flavors in stored orange juice) 5 Extreme care must be exercised to minimize the formation of 4-vinylguaiacol in products containing ferulic acid.

Skin protection Ferulic acid constitutes the active ingredient in many skin lotions and sunscreens designed for photoprotection) 27-~3° A hair cream composition also included ferulic acid to prevent alopecia, seborrhea, and pruritis, t3~ Some Japanese textile makers use it for the manufacture of golf wear with UV-absorption properties) 32 As elaborated on earlier, the high UV absorbancy and radiation-initiated antioxidant potential of ferulic acid confer excellent photoprotection to UVsensitive biological materials.

443 ADDITIONAL APPLICATIONS

Ferulic acid has been claimed to lessen the side effects of chemo- and radiotherapy of carcinomas by increasing the natural immune defense) 33 Due to its natural antioxidant potential, ferulic acid exhibited strong antiinflammatory properties in a carrageenaninduced rat paw edema model and other systems. 134,~35The same antioxidant properties also account for the inhibition of chemically induced carcinogenesis in rats ~36 and tumor promotion in mouse skin L37 and are believed to explain its antidiarrheal mechanism.~38 A review of ancient Chinese herbal drugs revealed ferulic acid to be one of the active constituents for the treatment of cardiovascular diseases) 39 Both ferulic acid and its steryl esters, y-oryzanol, exhibit strong hypocholesterolemic and antiatherogenic properties. 14°,t41 A recent feeding study at the University of Lowell in Massachusetts using cynomolgous monkeys reported the unique ability of rice bran oil to cause a 40% reduction in total cholesterol and to decrease LDL, or low-density lipoprotein, by 30% without affecting HDL, or high-density lipoprotein) 42 The significant reducing effects of rice bran on total cholesterol and LDL were confirmed by the results of three independent human clinical trials at the University of California-Davis Medical Center, the Human Ecology Department at Louisiana State University, and the CSIRO Division of Human Nutrition in Australia. These benefits can be traced directly to the unsaponifiable fraction of rice bran oil, primarily 3,-oryzanol, which is virtually absent in bran products derived from other seeds. A cosmetic containing anthocyanin-type pigments from tulip flowers utilized ferulic acid to stabilize the rouge against oxidative discoloration. 143 Naturally occurring ferulic acid in wheat has been shown unequivocally to affect the dough rheology and cause dough breakdown during bread making by interacting with the sulfhydryl groups of gluten via a free radical mechanism) 44-147 A combination of ferulic acid and tetramethylpyrazine had a synergistic inhibitory effect on the spontaneous movement of rat uterus in situ. ~4s REGULATORY STATUS AND COMMERCIAL AVAILABILITY

FDA status In the United States, ferulic acid is currently not GRAS (Generally Recognized as Safe) and it lacks FDA (Food and Drug Administration) or FEMA (Flavor and Extract Manufacturers' Association) ap-

444

E. G ~ F

proval and therefore cannot be used as a food additive, cosmetic, or pharmaceutical. However, ferulic acid is approved in several other countries, such as Japan, where it is added to food items as an antioxidant and where it constitutes the active ingredient in a variety of cosmetics and pharmaceutical preparations. Also, in the United States and most European countries, numerous medicinal essences and natural extracts of herbs, coffee, vanilla beans, spices, and other botanicals are selected for their high content of ferulic acid and other phenolic compounds and added to a food as an FDA-approved antioxidant concoction. No acute or chronic side effects of ferulate ingestion or topical application have ever been reported.* Therefore, increasing evidence for its health benefits is likely to inspire future clinical trials and a change in its FDA status within the next 5 to 10 years.

Commercial availability Commercial utilization of ferulic acid has been limited by its availability and cost. Most recently, however, the Tsuno Food Industrial Company--with the aid of the Industrial Technology Center of Wakayama Prefecture--developed a novel cost-effective process for the large-scale extraction and purification of natural ferulic acid from the byproduct of rice bran salad oil refining.132 Tsuno processes approximately 150,000 tons of rice bran annually, which generates 7500 tons of byproduct. Naturally derived ferulic acid sharply contrasts the cost-prohibitive synthetic counterpart produced by the Knoevenagel condensation of vanillin with malonic acid. The availability of inexpensive industrial quantities of ferulic acid is likely to stimulate both basic research into the antioxidant properties of ferulic acid and development of additional food, cosmetic, medical, and veterinary applications. RESEARCH P R O S P E C r S

A fundamental understanding of the free radical chemistry of ferulic acid and related cinnamic acid * Steryl ferulates---r-oryzanol--have been recently indicated by the California Food and Drug Branch to constitute a reproductive toxicant at dietary levels above 0.3 mg per serving per day (Anonymous. Gamma-oryzanol embargoed in California. Health Food Business, January 1992, page 14). Companies whose products exceed the threshold may face charges of adulteration. However, no other literature supports the claims made in this internal Food and Drug Branch toxicology report. Any valid conclusions concerning the clinical effects of "r-oryzanol on the reproductive organs in humans will require publication of the aforementioned government investigation in a peer-reviewed journal, extensive future experimentation using molecular, cellular, and animal models, and welldesigned human epidemiological studies.

derivatives will be essential to the development of any future antioxidant applications. Some of the recommended directions will include a thorough electron spin resonance spectroscopic investigation of the phenoxy radical, an analysis of the scavenging potential for a whole series of activated oxygen species, a study of the relationship between antioxidant function and the stereochemistry of structural isomers and related phenolic compounds, and a characterization of the antioxidant potential of ferulic acid in nonaqueous media, such as organic solvents, semisolid emulsions, and lipid membranes. Furthermore, it would be valuable to understand the antioxidant potential of ferulic acid immobilized on synthetic polymers or biological materials, such as lignin. Of more academic interest will be a basic understanding of the physiological function ofphotoprotection by ferulic acid in plants. Is the high UV absorption and radiation-induced radical-scavenging potential in low water systems as proposed in the current review article essential to the survival of some plant tissues? Could the same mechanism be exploited to develop several medical applications based on the photoprotective effect of ferulic acid? In addition to providing photoprotection simply during tanning, ferulic acid may also alleviate some of the symptoms of hereditary or acquired porphyrias, conditions under which blood porphyrin levels are elevated and its urinary excretion is enhanced. In one type of congenital porphyria, large quantities of uroporphyrin I are excreted due to a deficiency of the cosynthetase that is required for the formation ofprotoporphyrin IX. Free porphyrins are also deposited under the skin and cause severe photosensitivity that might be partially relieved by an ointment containing ferulic acid or 3,-oryzanol. This hypothesis may warrant controlled clinical trials aimed at testing the therapeutic effects of topically applied ferulic acid and its esters on photosensitive disorders, such as severe porphyrias. Ferulic acid may also prove beneficial in the treatment of burn and abrasive wounds. There will be similar applications in the animal health care industry. Furthermore, ferulic acid may exert protective effects during UV sterilization of other biological materials. Another application based on the photoprotection of ferulic acid may be the formulation of an ophthalmologic solution for the prevention of cataract in humans exposed to high UV radiation, such as in individuals living at high altitudes. The lens is unique in lacking the ability for tissue regeneration, and a gradual increase in opacity due to lens protein oxidation is generally irreversible. High UV radiation may potentiate this slow free-radical-mediated oxidation and ac-

Ferulic acid celerate cataract f o r m a t i o n leading to a decline in visual acuity. Existing knowledge in the m e c h a n i s m o f oxidative cataract d e v e l o p m e n t merits a t h o r o u g h investigation o f the p h o t o p r o t e c t i v e effects o f ferulic acid on lens protein p o l y m e r i z a t i o n in vitro a n d in an a n i m a l model. Various researchers have suggested several additional health effects o f ferulic acid, such as its inhibition ofatherosclerosis, hypercholesterolemia, a n d cardiovascular disease. A c o n c e r t e d effort should be initiated by b o t h industrial a n d a c a d e m i c laboratories to c o n d u c t m o r e in vivo tests a n d to assess the clinical validity o f the hypotheses p r o p o s e d in this review. T h e present article, together with the recent availability o f inexpensive industrial quantities o f ferulic acid, is likely to stimulate basic research into the antioxidant properties o f ferulic acid, the d e v e l o p m e n t o f additional food, cosmetic, medical, a n d veterinary applications, a n d a petition to the F D A for a critical review o f its regulatory status.

SUMMARY Ferulic acid is a ubiquitous plant constituent that arises f r o m the m e t a b o l i s m o f p h e n y l a l a n i n e a n d tyrosine. It occurs primarily in seeds a n d leaves b o t h in its free f o r m a n d covalently linked to lignin a n d other biopolymers. D u e to its phenolic nucleus a n d an ext e n d e d side chain conjugation, it readily f o r m s a reson a n c e stabilized p h e n o x y radical which a c c o u n t s for its p o t e n t antioxidant potential. U V absorption by ferulic acid catalyzes stable p h e n o x y radical f o r m a tion a n d thereby potentiates its ability to terminate free radical chain reactions. By virtue o f effectively scavenging deleterious radicals a n d suppressing radiat i o n - i n d u c e d oxidative reactions, ferulic acid m a y serve an i m p o r t a n t antioxidant f u n c t i o n in preserving physiological integrity o f cells exposed to b o t h air a n d impinging U V radiation. Similar p h o t o p r o t e c t i o n is afforded to skin by ferulic acid dissolved in cosmetic lotions. Its addition to foods inhibits lipid peroxidation a n d subsequent oxidative spoilage. By the same m e c h a n i s m , ferulic acid m a y protect against various i n f l a m m a t o r y diseases. A n u m b e r o f other industrial applications are based o n the a n t i o x i d a n t potential o f ferulic acid.

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