Inhibition of Polyphenol Oxidase by Phenolic Compounds - American

Chapter 25

Inhibition of Polyphenol Oxidase by Phenolic Compounds A. J. McEvily, R. Iyengar, and A. T. Gross

Downloaded by UNIV OF GUELPH LIBRARY on May 14, 2012 | http://pubs.acs.org Publication Date: October 1, 1992 | doi: 10.1021/bk-1992-0506.ch025

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Several inhibitors of polyphenol oxidase (PPO) were isolated from a plant extract. The inhibitors were purified and their relative potencies determined using commercially-available mushroom PPO in an in vitro assay system. Structural elucidation of the inhibitors showed them to be novel, plant secondary metabolites closely related to other phenolic compounds known to play important roles in flowering plants. Structure/activity relationships have been studied using synthetic analogs, several of which are potent PPO inhibitors. In addition to the in vitro studies, certain of these compounds inhibit enzymatic browning (melanosis) in foods such as shrimp, potatoes and apples as well as beverages such as grape juice. The inhibitors are water-soluble, stable, effective at low concentration, and have potential as functional alternatives to sulfites for the inhibition of melanosis. The chemical nature of the PPO inhibitors, kinetic studies, safety data, and results obtained on foods are discussed.

Enzymatic Browning Browning or melanosis of foods and beverages is a cosmetic discoloration which has a negative impact on the appearance, consumer acceptability, commercial value, and in certain cases such as wines, the organoleptic properties of the food system. In most foods, the browning process has two components: non-enzymatic and enzymatic. Since the compounds described herein inhibit enzymatic browning, only this component will be addressed. The causative agent which initiates the formation of brown pigments is the enzyme, polyphenol oxidase (PPO). This enzyme is endogenous to most foods and browning is not necessarily an indication of microbial contamination or spoilage. PPO is a mixed function oxidase which catalyzes the hydroxylation of monophenols to diphenols and in a second step, the oxidation of colorless diphenols to highly colored 0-quinones. The o-quinones react spontaneously with other o-quinones and with many constituents of foods such as proteins, amino acids, reducing sugars, etc. to form high molecular weight polymers which precipitate yielding the dark pigments characteristic of browned foods. The most widespread methodology used in the food and beverage industries for control of browning is the addition of sulfiting agents to foods susceptible to 0097-6156/92/0506-0318$06.00/0 © 1992 American Chemical Society

In Phenolic Compounds in Food and Their Effects on Health I; Ho, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.


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browning. Sulfites are currently used to inhibit melanosis (blackspot) in shrimp, browning of potatoes, mushrooms, apples, and other fruits and vegetables, as well as to reduce formation of polyphenols polymers which can lead to off-flavors in juices and wines. The major effect of sulfites on enzymatic browning is to reduce the oquinones produced by PPO catalysis to the less reactive, colorless diphenols thereby preventing the non-enzymatic condensations to high molecular weight pigments. Sulfites may also function as antimicrobial agents when used in sufficient concentration. Although sulfites are very effective in the inhibition of both enzymatic and nonenzymatic reactions, there are several negative attributes associated with their use in foods and beverages. Sulfites are known to cause adverse health effects especially in sensitive individuals such as asthmatics. Several deaths have resulted due to consumption of sulfited foods among this highly sensitive group. According to the Food Safety and Applied Nutrition Health Hazard Evaluation Board of FDA "a fourounce serving of shrimp containing 90 ppm sulfites presents an acute life-threatening hazard to health in sulfite sensitive individuals" (1). Sulfites can liberate sulfur dioxide gas and in enclosed areas such as the holds offishingvessels, sulfur dioxide vapors have led to several deaths among fishermen (2). Sulfites are highly reactive, nonspecific reducing agents which are consumed during browning inhibition necessitating the use of relatively high concentrations. Also, in certain foods sulfite residuals are high enough to have a negative effect on the taste of the treated product. For more information on the use of sulfiting agents and associated healthrisks,the reader is referred to an excellent review by Taylor et al. (3). In recent years, the Food and Drug Administration has banned sulfites for use on salad bars, moved to ban their use on fresh, peeled potatoes (4), increased surveillance and seizure of imported products with undeclared or excessive sulfite residuals (5), and has set specific limits on allowable sulfite residuals in certain foods. The negative connotations associated with sulfited foods has led to decreased consumer acceptance. According to the National Coalition of Fresh Potato Processors: "There are no substitutes" for sulfites available (6); however, the adverse health effects, increased regulatory scrutiny, and lack of consumer acceptance of sulfited foods have created the need for a practical, functional sulfite alternative. Isolation and Characterization of Browning Inhibitors from Fig Certain protease preparations especially ficin, the protease from fig (Ficus sp.) latex, appear to function as anti-browning agents in a host of food systems (7,8). The protease preparations employed in these studies were only partially purified, hence, the possibility existed that a non-protease component of the preparation was responsible for the observed browning inhibition. Indeed, preparations of heatinactivated ficin andfiglatex ultrafiltered to remove ficin were equally effective in PPO inhibition as the preparation containing active protease (9,10). Partially-purified ficin preparations (Enzyme Development Corporation, New York, NY) were extracted with deionized water, centrifuged, and the supernatant was ultrafiltered using a YM5 (5,000 molecular weight cut-off) membrane (Amicon, Beverly, MA). The ultrafiltrate was concentrated by lyophilization, resolubilized in 2 mM sodium phosphate, pH 6.5, then subjected to conventional cation exchange liquid chromatography on SP-Sephadex (Pharmacia, Piscataway, NJ). The column was developed with a 0 to 0.2 M sodium chloride gradient and fractions were analyzed for absorbance at 214 and 280 nm, conductivity, and inhibition of commercially-available mushroom PPO (Sigma Chemical Co., St. Louis, MO) in a spectrophotometric assay (70). Three absorbance peaks associated with peaks of PPO inhibition were observed indicating the existence of at least three potential PPO inhibitors. A linear increase in inhibition was seen in the chromatogram due to increasing sodium chloride R


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PHENOLIC COMPOUNDS IN FOOD AND THEIR EFFECTS ON HEALTH I

concentration which was used to elute bound materials. Sodium chloride is known to be a weak inhibitor of PPO activity (77); however, discrete and measurable peaks of PPO inhibition were observed above this background. The fractions containing significant absorbance and inhibition peaks were combined into three separate pools for further fractionation. The three inhibitor poolsfromSP-Sephadex chromatography were concentrated by lyophilization and further fractionated by reverse phase HPLC. One of these pools had not adsorbed to the SP-Sephadex and thus was further fractionated on DEAE-Sephadex (Pharmacia, Piscataway, NJ) prior to the HPLC procedure. The major absorbance peak in each HPLC run was concentrated and tested as an inhibitor of PPO. Subsequent analyses showed the recovered compounds to be nearly homogeneous preparations of three distinct inhibitors. The HPLC procedure was reproduced on a preparative scale and the recovered inhibitors were analyzed by methods such as mass spectrometry, *H NMR, C Ν MR, and FTIR. Based on the resultant analytical data, the three purified inhibitors were found to be 4-substituted resorcinols and are novel, plant secondary metabolites (72). The compounds, shown in Figure 1, were identified as 2,4-m^ydroxym^ydrocinnamic acid (I), 2,4-dihydroxydihydrocinnamoyl putrescine (Π), and ^w-(2,4-dihydroxydihydrocinnamoyl)-sperntidine (ΠΙ). The structures of I and II were confirmed by total synthesis. Compounds II and ΙΠ are novel whereas the synthesis of I in vitro had been described previously (73-75). However, I had not been previously isolated from a natural source. In subsequent research, compound I has also been isolated from the edible figfruit,in addition to the fig latex from which the ficin preparation had been derived (unpublished results). R

R


R

1 3

ο

Figure 1. Structures of the naturally occurring browning inhibitorsfromfig latex: (I) 2,4-dihyckoxydihy(kocinnamic acid; (Π) 2,4-dihydroxydihydrocinnamoyl putrescine; and (ΙΠ) Z?w-(2,4-dihydroxydihydrocinnamoyl)spermidine.


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Ato-substitutedputrescine derivative, Z?w-(2,4-dihydroxydihydrocinnamoyl)-putrescine was produced in a secondary reaction during the in vitro synthesis of II (Figure 2).


ο

IV Figure 2. Structure of synthetic browning inhibitor to-(2,4dihydroxydihydrocinnamoyl)-putrescine (IV). It is interesting to note the structural relationship between the amine derivative, II, and hydroxycinnamic acid amides (HCAs) such as caffeoyl putrescine (Figure 3). HCAs are known to have important physiological roles in the flowering of certain plants. ο

OH

Caffeoyl putrescine Figure 3. Structures of naturally occuring browning inhibitor from fig latex 2,4-dihydroxydihydrocinnamoyl putrescine (Π) and the HCA, caffeoyl putrescine. Screening of 4-Substituted Resorcinols as PPO Inhibitors The I50 values for the naturally occurring inhibitors and compound IV were determined using mushroom PPO in the in vitro assay system (10). The I50 is defined as the inhibitor concentration at which 50% inhibition of PPO activity is obtained. The results are presented in Table I. Since the resorcinol moiety was common to the natural inhibitors, commercially-available, synthetic 4-substituted resorcinols were screened for efficacy


322


Table I. I50 values for 4-substituted resorcinols as PPO inhibitors Compound


I II III IV

I50, μΜ 25 5 5 5

as PPO inhibitors. Initial studies were performed to determine the I50 of the synthetic resorcinol derivatives to obtain information on their relative potencies. The results are summarized in Table Π. Resorcinol is a poor inhibitor with an I50 in the millimolar range; however, substitutions in the 4-position yield decreased I values. The lowest values are obtained with hydrophobic substituents in the 4-position such as 4-hexyl-, 4-dodecyl- and 4-cyclohexylresorcinol with I50 values of 0.5, 0.3, and 0.2, respectively. 50

Table II. I50 values for synthetic 4-subsituted resorcinols as PPO inhibitors

R Hexanoyl Carboxyl Ethyl Hexyl Dodecyl Cyclohexyl

150, μΜ

750 150 0.8 0.5 0.3 0.2

Resorcinol derivatives with substitutions in the 5-, 2-, and 1,3-positions were also evaluated as PPO inhibitors. 5-Substituted resorcinols exhibited an inhibitory trend analogous to that seen with 4-substituted resorcinols: hydrophobic substituents of increasing chain length yielded inhibitors with decreasing I50 values (Table ΠΙ). Although the 5-substituted resorcinols appeared to be effective PPO inhibitors in vitro, their use in food applications was not pursued due to the toxic and irritant properties associated with these compounds. Substitutions in the 2- or 1,3-positions led to greatly increased I50 values relative to resorcinol (Table IV). These compounds exhibited only low levels of PPO inhibition even at the limit of their respective solubilities.


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The use of 4-hexylresorcinol as a browning inhibitor was focused on due to its low I50UI the spectrophotometric assay system, positive pren'minary results from tests on foods, and the fact that this compound has a long, safe history of human use in over-the-counter drugs. 4-Hexylresorcinol is the active ingredient in Sucrets lozenges at 2.5 mg/2.5 g lozenge. Also, the numerous toxicological studies on 4hexylresorcinol which are the subject of a recent review (16) show this compound to be safe and, therefore, a potential candidate for food use. R


Table III. I50 values for 5-substituted resorcinols as PPO inhibitors R

150. μΜ

R

1000 250 350 7

Methyl Pentyl Heptyl Pentadecyl

Table IV. I50 values for 2- and 1,3-subsituted resorcinols as PPO inhibitors

I50, mM

R

R

Methyl

>70

Methyl

>5


324



Applications in Foods Several 4-substituted resorcinols described herein inhibit PPO in vitro and have potential for widespead application in the food and beverage industries on products that exhibit problematic browning. Preliminary research shows that 4-substituted resorcinols have several advantages over sulfites for use on foods. Among others, these include: 1) these compounds are specific, potent polyphenol oxidase inhibitors allowing use at much lower concentrations than sulfites; 2) 4-substituted resorcinols do not "bleach" pigments as excess sulfites can, therefore, use of excessive concentrations is not encouraged; and 3) the 4-substituted resorcinols are more chemically stable relative to sulfites. The initial food application targetted for intensive investigation was the prevention of shrimp melanosis (blackspot). The efficacy of 4-hexylresorcinol in maintaining the high quality of landed shrimp has been shown in both laboratory and field trials under a variety of process conditions (17,18). This highly potent inhibitor is substantially more effective than bisulfite on a weight-to-weight basis, should prove to be competitive with bisulfite on a cost basis, and will require no changes in the on board or ex-vessel handling of the shrimp product. 4-Hexylresorcinol is a water soluble, stable compound which is non-toxic, non-mutagenic, and non-carcinogenic and is Generally Recognized As Safe (GRAS) for use in the prevention of shrimp melanosis (16). The use of 4-hexylresorcinol as a processing aid for the inhibition of shrimp melanosis has no negative impact on taste, texture, or color of the treated shrimp product due to very low residuals (

Inhibition of Polyphenol Oxidase by Phenolic Compounds - American

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