Radical Scavenging and Antioxidative Properties of Phenolic

Chapter 14

Radical Scavenging and Antioxidative Properties of Phenolic Compounds in Relation to Their Chemical Structure

Downloaded by UNIV OF CINCINNATI on February 18, 2015 | http://pubs.acs.org Publication Date: January 15, 2000 | doi: 10.1021/bk-2000-0754.ch014

K.

Bungert and K. Eichner Institut für Lebensmittelchemie der Universität Münster, Gorrensstrasse 45, D-48149 Münster, Germany

Many foods of plant origin, especially tea, coffee and cocoa, contain higher amounts of polyhydroxyphenols which show antioxidative effects in vitro and in vivo prolonging the shelf life of foods and protecting human health. It is shown that there are clear relationships between the antioxidative properties of different polyhydroxyphenols and their reducing and radical scavenging effects. The radical scavenging properties of phenolic compounds were determined by reaction with the stable radical 2,2-diphenyl-1-picrylhydrazyl. It turned out that phenolic compounds with at least one hydroxy or phenoxy group in o-position to the phenolic hydroxy group are showing radical scavenging and reducing properties due to their proton-donating activities. There are clear relationships between the chemical structures of phenolic compounds and the degree of their radical scavenging effect. To this catechol and quercetin were the most effective, followed by cinnamic acid derivatives like ferulic acid, sinapic acid and caffeic acid, whereas benzoic acid derivatives were less effective. These compounds are showing the same sequence in their antioxidative activity in lard. In methyllinoleate-water emulsions the antioxidative effect of phenolic compounds increases with decreasing droplet size and increasing total surface of the fatty phase.

© 2000 American Chemical Society

In Caffeinated Beverages; Parliment, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2000.

119


120

Caffeine containing beverages like tea, coffee and cocoa drinks are rich in polyphenols which have the capability to prevent cancer and cardiovascular diseases based on their radical-scavenging and antioxidative activity (1 - 5). Polyphenols are recognized as in vitro as well as in vivo antioxidants prolonging the shelf life of foods and protecting human health (3,4). On the other side, in biological systems like fruits and vegetables, also in tea, coffee and cocoa, polyphenols are oxidized by the enzyme polyphenol oxidase causing enzymatic browning (6). The most important phenolic compounds in plants can be divided into phenolic carboxylic acids (benzoic acid and cinnamic acid derivatives like chlorogenic acid) and flavonoid compounds like catechins and quercetin. Figure 1 shows the main representatives of phenolic compounds in foods of plant origin. Hydroxy-benzoic acid derivatives can be found in spices (7) and in smaller amounts in fruits and vegetables (8). Hydroxycinnamic acid derivatives like caffeic acid (3,4-dihydroxy-cinnamic acid) or chlorogenic acids (esters between caffeic acid and quinic acid) are present in vegetables and fruits (8, 9). Glycosides of flavons and flavonols like quercetin are occurring in a great variety of fruits, vegetables and spices (8), whereas catechins are present in many fruits and spices (10, 11), in tea and cocoa. Tea contains several derivatives of catechin (12, a high portion of it being esterified with gallic acid), chlorogenic acids (caffeoyl- and pcoumaroylquinic acids,13), 3-galloylquinic acid (1g/100g dry matter,14) and theogallin (15) with a total amount of 20 - 30 % of dry matter. In coffee mainly chlorogenic acids (5-caffeoyl-quinic acid being the main representative) are present (16), whereas in cocoa catechins are prevailing. During roasting the chlorogenic acid content in coffee decreases by 60 % and more. On the other side, during roasting of cocoa a much smaller amount of catechins is lost. It is well known that cocoa because of its content of catechin and epicatechin shows antioxidative properties used in products like nougat, milk crumb or milk chocolate. It turned out that alcoholic extracts of defatted cocoa or cocoa husks exert antioxidative effects on unsaturated oils (17) which can mainly be attributed to their epicatechin content. It is also well known that the antioxidative effect of polyhydroxyphenols increases dependent on the amount of OH- or methoxy groups in the o- or pposition (18). These groups have electron shifting properties (negative sigma values according to the Hammett equation) thus stabilizing the intermediate phenoxy radicals formed within radical chain oxidative reactions; in this way the radical scavenging and antioxidative activity of polyhydroxyphenols is increased. In order to establish structure-activity relationships of polyhydroxyphenols as shown in Figure 1 the reducing, radical scavenging and antioxidative properties of selected phenolic compounds shall be investigated.



OH

OH

2- OH 4-OH 3.4-di-OH 3- OCH3, 4-OH 3,4,5-tri-OH 3.5- di-OCH3, 4-OH

ο

Η Η OH Η OH OH

R1 R2

(Flavonols: X = OH) (Flavones: X = H )

Kaempferol Quercetin Myricetin

OH

Sinapic acid

o-Coumarie acid p-Coumaric acid Caffeic acid Ferulic acid

COOH

Figure 1. Chemical structures of different classes of polyhydroxyphenols

Catechin, Epicatechin: R= Η Gallocatechin, Epigallocatechin: R = OH

Catechins

HO

Salicylic acid p-Hydroxybenzoic acid Protocatechuic acid Vaniiiic acid Gallic acid Syringic acid

COOH


122

Reductive, Radical Scavenging and Antioxidative Properties of Polyhydroxy-phenols in Homogeneous Systems


Methods It was demonstrated by Crowe et al. (19) that the overall reducing capacity of milk and milk products can be measured by the potassiumferricyanide method, where it is reduced to ferrocyanide giving a blue color after addition of Fe-lll ions. Because ferricyanide shows stoichiometrical reactions with cysteine and ascorbic acid, the reaction can be calibrated as to the amount of reducing equivalents measured; as expected, one mole of cysteine yielded one reducing equivalent, one mole of ascorbic acid two reducing equivalents. For analyzing the radical scavenging effect of phenolic antioxidants they were reacted with the stable radical 2,2-diphenyM-pikrylhydrazyl (DPPH) (Fig.2) showing a blue color. After taking up a hydrogen atom from a hydrogen-donating (reducing) compound, it is transferred to the colorless 2,2-diphenyl-1-pikrylhydrazine (DPPHH) in a stoichiometrical reaction (20). DPPH reacts with cysteine, ascorbic acid, tocopherol and polyhydroxyphenols, but not with glucose or phenolic compounds with only one OH-group (20, 21). Figure 3 shows the absorption spectra of DPPH and DPPHH. The reaction can be followed up by measuring the decrease of the absorption at 525 nm (absorption maximum of DPPH). The reaction takes place in ethanolic solution within 60 min at room temperature. The antioxidative effects of different phenolic compounds were measured by using a modified Swift test at 120 °C (rancimat method) (18). This method is based on a conductometric measurement of volatile acids (mainly formic acid) emerging after the induction period as by-products of fat oxidation. For comparing the antioxidative effects of different phenolic compounds, alcoholic solutions of these compounds were mixed thoroughly with molten lard in amounts of 1 mmol/kg. After that the solvent was evaporated and air passed through aliquots of the lard at 120 °C into a conductivity cell. After the induction period the conductivity increases steeply. There is a good correlation between the induction period measured and the storage stability of fats and oils at room temperature (22).

Results The antioxidative effect of polyhydroxyphenols is based on their hydrogen donating effect, by which they inactivate free radicals and interrupt the radical chain of lipid oxidation (23). The question was, if there are structure-related connexions between their antioxidative effect and their reducing and radical scavenging effect. As it is shown in Figure 4, monohydroxy-benzoic acids and dihydroxybenzoic acids with the OH-groups in m-position do not exert any reducing or



123

2,2-Diphenyl-1 -picrylhydrazyl ( D P P H ) Figure 2. Chemical structure of 2,2diphenyl-1-picrylhydrazyl



300

1.4

500 Wavelength (nm)

Figure 3. Absorption spectra of 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2-dipheny!-1-picryl-hydrazine (DPPHH)

400


625

ta 4±


0

1

2

3

4

5

6

7

ο

2

3

4

5 M o l D P P H / m o l antioxidant

1

Figure 4. Reducing and radical scavenging properties of hydroxybenzoic acid derivatives

M o l ferricyanide/mol antioxidant (HB = hydroxybenzoic acid, DHB = dihydroxybenzoic acid)

o-HB m-HB p-HB 3.5- DHB 2.6- DHB 2,4-DHB 2,4,6-THB Vanillic acid Syringic acid 3.4- DHB 2.5- DHB 2,3-DHB Gallic acid


6


126

radical scavenging effect. In the presence of one ore two electron-shifting methoxy groups in o-position to the OH-group there is an increase of both effects from vanillic acid to syringic acid. There is a further increase in the reducing and radical scavenging properties, if there are two or three hydroxy groups in o- or p-position. Gallic acid shows the strongest reducing capability. It is remarkable that the dihydroxy-benzoic acids and gallic acid have higher reducing and radical scavenging effects than it should be expected from a stoichiometrical point of view. Figure 5 shows the induction periods for the oxidation of lard in the presence of different hydroxybenzoic acids. It can be seen that monohydroxy- and dihydroxy-benzoic acids with the OH-group in m-position have no or almost no antioxidative effects. This is also the case with vanillic and syringic acid (having one or two methoxy groups in o-position), whereas they clearly show radical scavenging effects. The antioxidative effect really starts with a second OH-group in o- or pposition, gallic acid with three hydroxy groups in o-position having the highest activity; on the other side, 2,3-dihydroxy-benzoic acid has the lowest effect, possibly because of steric hindrance. Figure 6 demonstrates the reducing and radical scavenging effects of different hydroxy-cinnamic acids. Similar to the monohydroxy-benzoic acids, o-, m- and p-coumaric acid show neither reducing nor radical scavenging capacities. One methoxy group in oposition (ferulic acid) introduces reducing as well as radical scavenging properties which are enhanced by a second methoxy group (sinapic acid). As expected from the foregoing results, a second OH-group in o-position (caffeic acid) greatly increases both effects. Apparently, esterification of the carboxy group of caffeic acid with quinic acid (chlorogenic acid) does not change these properties. Figure 7 shows a very clear relationship between the antioxidative activities of the above mentioned hydroxy-cinnamic acids and their radical scavenging effects. They have a higher antioxidative potential than the respective hydroxy-benzoic acids. In Figure 8 the reducing and radical scavenging effects of the flavonoids catechin and quercetin are demonstrated. Catechin having o-dihydroxy groups in the B-ring shows a certain similarity to caffeic acid also having odihydroxy groups. Quercetin which has an OH-group at C3 being in a vinylogical position to the OH- groups in the B-ring has stronger reducing and hydrogen-donating properties than catechin which is comparable to propyl gallate. On the other hand, tocopherol, BHT, BHA and ascorbyl palmitate show smaller effects than catechin and quercetin. Tocopherol and ascorbyl palmitate show the expected reducing capacity of two reducing equivalents per mole. As shown in Figure 9, there again is a good correlation betwen the radical scavenging effects and the antioxidative effects of all compounds listed in the Figure. It becomes clear that generally phenolic compounds with two or three OH-groups in o-position (catechin, quercetin, propyl gallate) have a stronger antioxidative effect than ascorbyl palmitate, BHT, BHA and tocopherol with only one phenolic OH-group. The antioxidative capacity of quercetin may be influenced to a certain extent by its heavy metal-chelating properties caused by the OH-group in C3 position in neighbourhood to the carbonyl function in C4. Hydration of the double bond between C2 and C3 or glycosidation of the OH-group in the C3 position reduces the antioxidative potential of quercetin significantly.



10

9.3

1.08

2.92

2.05

0.87

0.72

1.48

0.83

1

0.73

0.65

0.57

0.62

0.57

1 2 3 4 5 6 Mol DPPH/mol antioxidant

Figure 5. Antioxidative and radical scavenging properties of hydroxybenzoic acid derivatives

Rancimat induction period (h) (HB = hydroxybenzoic acid, DHB = dihydroxybenzoic acid)

0 1 2 3 4 5 6 7 8 9

None hi o-HB • m-HB • p-HB • 3.5- DHB m 2.6- DHB • • 2,4-DHB • 1 2,4,6-THB mm Vanillic acid m Syringic acid m 3.4- DHBtmrnm 2.5- DHB• • • • 2,3-DHB p i Gallic acid



0

2

3

4

5

M o l ferricyanide/mol antioxidant

1

6

1

2

3

4

5 M o l DPPH/mol antioxidant

0

Figure 6. Reducing and radical scavenging properties of cinnamic acid derivatives

o-Coumaric acid m-Coumaric acid p-Coumaric acid Ferulic acid Sinapic acid Caffeic acid Chlorogenic acid


6

7

In Caffeinated Beverages; Parliment, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2000. 3

4

5

6

7

8

9

Rancimat induction period (h)

2

^ ^ 10

4.05

4.17

2.37

0.97

0.63

0.42

0.62

0.57

M o l D P P H / m o l antioxidant

Figure 7. Antioxidative and radical scavenging properties of cinnamic acid derivatives

•

•

None hi o-Coumaric acid m-Coumaric acid • p-Coumaric acid Ferulic acid L i Sinapic acid • • • ι Caffeic acid Chlorogenic acid ^ ^ ^ ^ ^ ^


ta


Catechin Quercetin BHT BHA Propyl gallate Tocopherol Asc. palmitate 2

3

4

5

6

7 0

2

3

4

5 M o l D P P H / m o l antioxidant

1

Figure 8. Reducing and radical scavenging properties of some natural and synthetic antioxidants

M o l ferricyanide/mol antioxidant

1


6


1

3

4

5

6

7

8

9

Rancimat induction period (h)

2

10

2

3

4

5 M o l DPPH/mol antioxidant

1

Figure 9. Antioxidative and radical scavenging properties of some natural and synthetic antioxidants

0

None Catechin Quercetin BHT BHA Propyl gallate Tocopherol Asc.palmitate


6

132


Antioxidative Properties of Polyhydroxyphenols in Heterogeneous Systems ft is of great practical interest how polyhydroxyphenols exert their antioxidative effect in heterogeneous systems like oil/water emulsions. Therefore methyllinoleate (ML)/water model emulsions (3,4 mmol ML/I) using Tween 20 (1 g/l) as an emulsifier were prepared. Since the solubility of phenolic acids like caffeic acid or gallic acid in the oil or water phase strongly depends on the pH value, the influence of pH on the antioxidative activity of polyhydroxyphenols was investigated. Figure 10 shows the induction periods for different polyhydroxyphenols (0,1 mmol/f each) in ML/water emulsions at different pH values. The induction periods were determined at 30 °C by measuring the oxygen uptake of the emulsions with the Warburg technique. It becomes clear that the antioxidative effects of e.g. ferulic, sinapic or caffeic acid strongly increase by lowering the pH value because of their increased solubility in the fat phase at lower pH values. It is remarkable that polyphenols like gallic acid, caffeic acid or chlorogenic acid showing a strong antioxidative effect in homogeneous systems have a relatively low antioxidative potential in the heterogeneous system, because they are highly soluble in water and part of their antioxidative effect may become effective only at the contact surface between the oil phase and the water phase. Of great influence on the rate of oxidation is the droplet size of the fatty phase in the emulsions. The droplet sizes were adjusted by addition of different amounts of the emulsifier Tween 20 in the range between 0,1 g/i and 10 g/l. The average droplet sizes dependent on the amount of emulsifier were determined by transmission measurements of the emulsions. It turned out that at an emulsifier concentration of under 1 g/l the droplet size was higher than 1 μΐη, whereas at a concentration of 10 g/l the emulsion appeared transparent corresponding to a droplet size of less than 0,05 μηι. Figure 11 shows the induction periods of the above described ML emulsions at pH 5,5 and different concentrations of Tween 20 in the absence and presence of antioxidants, it can be seen that without antioxidants the induction periods decrease with increasing emulsifier concentrations, because the overall surface of the fatty phase increases with decreasing droplet sizes leading to a better access of oxygen to the substrate. On the other side, in the presence of catechin, which is more soluble in water, the induction periods increase with increasing emulsifier concentrations, because catechin mainly becomes effective at the boarder surface between the fatty phase and the water phase; therefore the antioxidative effect of catechin increases with decreasing droplet size and increasing contact surface between the two phases. Based on the same principle, the prooxidative effect of heavy metal ions becomes more pronounced at higher emulsifier concentrations. However, in the presence of tocopherol, contrary to catechin, the induction periods decrease with decreasing droplet size of the fatty phase, because the concentration of the fat soluble tocopherol in the fatty phase is independent of the droplet size. Therefore, in this case only the increase of the total surface of the fatty phase improving the access of oxygen thus decreasing the induction period remains effective.



133

None Syringic acid Gallic acid Femlic acid Sinapic acid Caffeic acid Chlorogenic acid Catechin Quercetin

-

•

PH7.5

•SSHI

υ

pH 5,5

!•

H

PH 3,5

200

400

600

800

1000

1200

Induction period (h) Figure 10. Antioxidative properties of some phenolic antioxidants methyllinoleate emulsions dependent on the pH value

Figure 11. Antioxidative properties of a water soluble and a water insoluble antioxidant in methyllinoleate emulsions dependent on the emulsifier concentration


134

Conclusions


The relationship between the chemical structure of polyhydroxyphenols and their antioxidative effect has been pointed out. There are clear correlations between the antioxidative effect of phenolic compounds and their reducing and radical scavenging properties. Therefore, the efficiency of phenolic antioxidants present in foods of plant origin can very easily and quickly be screened by reaction with the stable radical 2,2diphenyl-1pikrylhydrazyl. In oil/water emulsions the solubility of polyhydroxyphenols in the fat and water phase and the droplet size of the fatty phase determining the contact area between both phases play an important role with regard to their antioxidative effect.

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Radical Scavenging and Antioxidative Properties of Phenolic

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