FreeRadwaIBwlogy & Medwme, Vol 9, pp 111-115, 1990
0891-5849/90
Pnnted m the USA All rightsreserved
© 1990PergamonPressplc
÷
$3 00+ O0
Original Contribution HYDROXYL
RADICAL OF
AND ANTIPSORIATIC ACTIVITY A C I D DERIVATIVES
SCAVENGING BENZOIC
REINER F HASELOFF,*t INGOLF E BLASIG,:~HANS MEFFERT,§ and BERND EBERT* *Academyof Sciences of the GDR, Central Institute of Molecular Biology, Robert-Roessle-Str 10, Berhn, 1115, :~Academyof Sciences of the GDR, Institute of Drug Research, Berhn, §HumboldtUniversity, Schoolof Medicine (Chant6), Berlin, G D R (Recetved 27 November 1989, Revtsed 26 March 1990, Accepted 25 Aprtl 1990)
Abstract--The hydroxyl ra&cal scavenging and antipsoriatic activity of a number of hpophihc and hydrophlhc benzoic acid denvatives was investigated To quantify antloxldatwe effects, a newly introduced test system based on the diminution of the ESR signal of DMPO-OH (generated by Fenton's reagent) by the tested compounds was apphed It was found that the in vitro antlOXldatlve (toward hydroxyl radical) activity of benzoic acid esters decreases with increasing chain length whereas the antlpsoriatic actwity increases This effect is discussed in terms of a larger hpophdlcity of long-chain esters Propyl gallate was found to be the most active OH scavenger since it is some orders of magmtude more efficient than "model" antlOXldants hke ot-tocopherol or mannitol The highest antipsonatic actlwty was exhibited by hydroxy benzoic acid decyl ester Keywords--Hydroxyl ra&cal scavenging activity, Antlpsoriatic activity, ESR, rivatives, Free radicals
Spin trapping, Benzoic acid de-
pounds hke manmtol, propyl gallate, or a-tocopherol are often tested to standardize these systems The present study a~ms to investigate the antlpsorlat~c and hydroxyl radical scavenging activity of benzoic a o d derivatives Psoriasis is a common and longstandmg skin &sease for which no causal treatment ~s yet avadable One of its basic biochemical features is an enormously increased arachldomc acid pathway which is mmated by oxygen ra&cals, accompamed by the generation of large amounts of these species, and results m accumulation of large amounts of leukotnenes 6 7 Although superoxlde is considered to be the primary oxygen species formed, the main damaging effect should be due to the more reactive hydroxyl radical The arachldonlc acid pathway has received much attention because ~t contains many of the components needed to estabhsh the abnormaht~es of psoriatic skin 8 Once empirically introduced for psoriasis therapy, antmx~dants are thought to mteract with the excessive radical and leukotnene production 9 Based on the general knowledge of the mechamsms of the antloxldatlve action of phenols and benzols, l° this paper presents a systematic Investigation of the hydroxyl radical scavenging and antlpsonatlc activity
INTRODUCTION
Ant~oxldant systems provide effloent defense agamst oxidative stress m biological systems There are different mechamsms of ant~ox~datwe action, for example, ra&cal scavenging, recombination of ra&cals, chelation of transition metal ~ons, or electron transfer with formation of stable products Hydroxyl radical scavenging ~s one of the crucml elements of antlox~dative activity because the reactivity of these radicals is extremely high Under various pathological con&t~ons, such as inflammation and allergy, l lschemm2 and reperfuslon,3 or radiation damage,4 the natural defense mechamsms such as superoxlde dlsmutase/catalase, reduced glutathlone are not sufficient to prevent radical-reduced injury On the other hand, free radicals are important m the photodynamlc therapy of some kinds of cancer 5 One approach m the therapy of several &seases is the administration of antloxldatlve systems including hydroxyl radical scavengers In vitro test systems are used for evaluation of antlox~dant capacity, and comtAuthor to whom correspondence should be addressed 111
112
R F HAS1-LOFFet al
t II-I[-N~ ?N+I >
~)
go[l~r
orld
R~ = I~2=
ester~s R 3 = OH
b) p-hydroxy benzorc acid esfeF R~ = R3= H R2=
OH
~) m-hydroxy benzoic ocld esters /
P~,R~ = OH R~-
H
I F,' Fig 1 Structure ot the benzoic acid derivatives
of denvatwes of benzoic acid with different numbers of hydroxy groups and different chain length of the ester, which are, in part,~l known to have antmxldanve acnvlty The hydroxyl radical scavenging capacity was evaluated m a newly Introduced test system which allows mvesnganon and comparison of hpophfllC and hydrophfllC as well as acl&c and alkahne compounds M A T E R I A L S AND M E T H O D S
Hydroxyl radicals were generated by Fenton's reagent consisting of 0 05 mM FeSO4 and 0 1 mM H202 m the presence of 1 mM 5,5-dlmethylpyrrohne- l-oxide (DMPO*, solutions prepared according to Floyd'2), 14 mM HCI, 93 mM NaC1, 1 24 M pyndme, and the ant~oxldant In a concentration w~thm and around the hnear part of the concentration response curve determined in preceding experiments Chemicals pyndme (for spectroscopy, Merck, Darmstadt). butylated hydroxytoluene--BHT--(Calblochem-Behrmg, La Jolla, CA), D,L-c~-tocopherol (under Argon, research grade, SERVA, Heidelberg), mannltol (BDA Chemicals. L t d ) If not otherwise stated, chemicals were of analytical reagent grade The benzoic acid denvauves (Fig 1) were provided by G Tomaschewsky (Department of Chemistry, Humboldt University, Berhn) Its concentration in the test system was checked by UV absorption spectroscopy ESR spectra were recorded on a Vanan E 3 at room temperature under the followmg standard condmons scan range 10 mT, modulation amphtude 0 1 mT, microwave power 20 mW For the mvesngatlon of antlpsonatlc activity a proven method was used l~ 20 patients (ages 19-38, 9 male, 11 female) suffering from stable plaque-type psoriasis were included m that study after having given
full-reformed consensus Small skm lesions (1-2 cm m dmmeter and all of them exhlbmng the symptoms of the same stage of disease) were treated daily with 2 % (v/v) anttoxldant resulting m 12 mg annoxldant/ cm 2 of treated skin surface "Pure ointment and a strong topical comcosterold (flUOClnoloneacetomde. Flucmar R) served as controls Following ointment apphcat~on the test s~tes were covered with alummmm chambers 14 Before and 2, 4, 7, 9, and 11 days after starting the experimental treatment, the stage of each particular test s~te was evaluated according to the cnterm of Wemstem et al ~ Briefly, each of the three phenomena, erythema, infiltration, and desquamatton, were fit into a 4-point scale The resulting "psoriasis a c h w t y " ranges from 9 (maximal erythema, mfiltranon and scahng) to 0 (no psoriasis symptoms at all) When corrected by the control (pure omtment), the term "mean antlpsonanc efficacy" (MAPE) represents the percentage of dechne of psonanc symptoms compared to fluocmoloneacetonlde
RESULTS
In the mvesngatlons presented here, special interest was given to a series of substances with different cham length and different numbers and posmons of the hydroxy groups The results concerning their hydroxyl radical scavenging and antlpsonattc acnvtty are summanzed m Table 1
dFor practical reasons, the chmcal effect ts based on the volume and the weight of the substances apphed On a molar basts, the efficacy of the substances with higher molecular weight is even larger compared to the substances with lower molecular weight
Investigation of antmx~dants
Table 1 Hydroxyl Radical Scavenging and Antlpsonatlc Actlvlty of Benzotc Acid Denvatxves Compound Galhc acid p r o w l ester butyl ester octyl ester decyl ester tetradecyl ester heptadecyl ester p-hydroxy benzoic a o d methyl ester propyl ester -n-butyl ester -tso-butyl ester heptyl ester decyl ester m-hydroxy benzoic acid propyl ester decyl ester Manmtol Butylated hydroxytoluene c(-tocopherol
ECso*(~M)
MAPE**(%)
2 20 55 50 65 65 23 29 20 23 30 500 14 100 1300 1500 1650
n d 8 3 33 3 50 0 58 3 75 0 61 5 71 5 n d n d n d 91 7 66 7 93 3 n d n d n d
*The ECso-valuegives the concentration for reduction of the DMPO-OH ESR signal intensity on 50%, each individual value represents the mean of at least three mdependent determmanons of the concentratmn response curve **MAPE meananapsonatlc efficacy (percentage of dechne of psoriatic symptoms compared to fluocmoloneacetomde)after rune treatments under occlusmn (n d not determined) For statlsncal treatment, Wdcoxon's pmred rank sum test and hnear regression were used, respectively Except for gallic actd decyl/ tetradecyl ester and m-hydroxy benzoic acid propyl ester, all differences within the series of compounds (galhc aod esters, p-hydroxy benzoic acid esters mhydroxy benzotc acid esters) were slgmflcant (p < 0 01) Correlation coeffioents galhc acid esters, r = 0 897, p-hydroxy benzolt, acid esters r = 0 948 m-hydroxy benzoic esters, r = 1 00
Hydroxyl radtcal scavengmg m vttro
In Figure 2, the spectrum of DMPO-OH generated m the described system is shown (there Is a small mplet with aN = 1 39 mT superimposed on DMPO-OH, it originates from the trapping of products of the decomposmon of DMPO m some charges of the spm trap) The maximum intensity reached 2 rain after lnmatlon of the reaction was used for the evaluation of the OH scavenging actwlty In the presence of antloxl-
113
(*/,)
i
i
|
i
6
5
4
3
- tg c
(MI
Fig 3 Decrease of ESR-slgnal intensity in the presence of antloxtdants (100%, no antloxldant present), o, propyl gallate, x, m-hydroxy benzoic acid propyl ester, + , m-hydroxy benzoic acid decyl ester, O, p-hydroxy benzoic acid decyl ester, each m&vldual pomt represents the mean of at least 3 measurements
dants, the concentration of DMPO-OH decreased depending on the concentration of these antloxldants, as depicted m Figure 3 The curves show a slgmoldal shape, the efficacy of the substances is quantified by an ECs0-value representing the concentration necessary for 50% &mmutlon of the ESR signal intensity taken from the approximately hnear part of the graph From the ECso-values it follows that galhc acid propyl ester (propyl gallate) is the most effectwe of the investigated substances, with an ECso about three orders of magmtude lower than found for manmtol, BHT, or ct-tocopherol In both groups of compounds a tendency of decreasing hydroxyl ra&cal scavenging capacity with increasing chain length was observed There were only shght &fferences in hydroxyl radIcal scavenging capacity between benzoic acid derivatives with different numbers and posmons of the hydroxy groups Galhc acid esters with short chain lengths (n -< 3) are more active than those of hydroxy benzoic acid (independently of the posmon of the hydroxy group) The differences in activity between the esters of gallic and hydroxy benzoic a o d with medium cham lengths (n < 10) are neghglble, whereas galhc acid esters with longer chain lengths are more effective than those of hydroxy benzoic acid with respect to the hydroxyl radical scavenging activity
Anttpsortattc acttvzty 1 mT I )
Fig 2 ESR spectrum of DMPO-OH generated by a Fenton-type reaction (0 05 mM Fe2SO4, 0 1 mM H202, 1 mM DMPO, 14 mM HCI, 93 mM NaCI, 1 24 M pyndme), hyperfine sphttmg constants, as = an = 1 4 8 m T
It was found that the antlpsorlatlc actwlty of the substances increases with increasing chain length The denvatwes of hydroxy benzoic acid are more actwe than those of gallic acid as can be seen by comparison of the actwmes of the decyl esters (MAPE = 50 0 for the galhc acid denvatxve, MAPE = 91 7 [93.3] for the benzoic acid denvatwe[s])
R F H~SElOFVetal
114 DISCUSSION
In the present study, a possible mechamsm of interaction of the chosen compounds on the molecular level as well as their biological effect m the treatment of psoriasis was investigated fn general, the combinatron of phenomenologlcal and molecular approach warrants a more detailed characterlzatmn of the propert~es of the substances Regarding the mvest~gatmn of the hydroxyl radical scavenging actlwty it can be stated that spin trapping using DMPO is one of the most reliable methods for detection and ldentlflcanon of hydroxyl radicals D M P O - O H is sufficiently stable. and the rate constant for ~ts formatmn is high compared to other adducts (e g , DMPO-O2 )~5 Thus, the method is a powerful tool for the evaluation of hydroxyl radical scavenging, that ~s, anttoxldatlve capacity of different compounds The apphcatmn of pyndme and hydrochloric acid m this test system otfers the advantage of pH stablhzatmn and guarantees favorable conditions concerning the mvestlgatmn of substances which are usually soluble in water or hplds It was found that, in agreement with earher results. ~ propyl gallate ~s a very strong antmxldant since it xs about three orders of magnitude more efficient than BHT, o~-tocopherol, or mannitol In this context, the problem of the mechanism of ~ts action artses In the test system applied here, the antloxldatlve actlvlty has been evaluated by the amount of spin adduct formed m the competition between the spin trap and the antmx~dant There ~s no poss~bdlty to ddferentlate between the hydroxyl radical scavenging act~vlty and the abd~ty to decrease the rate of formanon of OH (for example, by chelatmn of Fe 2÷ or catalytic/peroxldat~c activity) However, m addmonal experiments with some compounds including propyl gallate, no reaction with hydrogen peroxide was observed, and m the micromolar concentration range Fe z" chelatmn ~s not expected to exert a dominating effect (ECso lpropyl gallate]
0891-5849/90
Pnnted m the USA All rightsreserved
© 1990PergamonPressplc
÷
$3 00+ O0
Original Contribution HYDROXYL
RADICAL OF
AND ANTIPSORIATIC ACTIVITY A C I D DERIVATIVES
SCAVENGING BENZOIC
REINER F HASELOFF,*t INGOLF E BLASIG,:~HANS MEFFERT,§ and BERND EBERT* *Academyof Sciences of the GDR, Central Institute of Molecular Biology, Robert-Roessle-Str 10, Berhn, 1115, :~Academyof Sciences of the GDR, Institute of Drug Research, Berhn, §HumboldtUniversity, Schoolof Medicine (Chant6), Berlin, G D R (Recetved 27 November 1989, Revtsed 26 March 1990, Accepted 25 Aprtl 1990)
Abstract--The hydroxyl ra&cal scavenging and antipsoriatic activity of a number of hpophihc and hydrophlhc benzoic acid denvatives was investigated To quantify antloxldatwe effects, a newly introduced test system based on the diminution of the ESR signal of DMPO-OH (generated by Fenton's reagent) by the tested compounds was apphed It was found that the in vitro antlOXldatlve (toward hydroxyl radical) activity of benzoic acid esters decreases with increasing chain length whereas the antlpsoriatic actwity increases This effect is discussed in terms of a larger hpophdlcity of long-chain esters Propyl gallate was found to be the most active OH scavenger since it is some orders of magmtude more efficient than "model" antlOXldants hke ot-tocopherol or mannitol The highest antipsonatic actlwty was exhibited by hydroxy benzoic acid decyl ester Keywords--Hydroxyl ra&cal scavenging activity, Antlpsoriatic activity, ESR, rivatives, Free radicals
Spin trapping, Benzoic acid de-
pounds hke manmtol, propyl gallate, or a-tocopherol are often tested to standardize these systems The present study a~ms to investigate the antlpsorlat~c and hydroxyl radical scavenging activity of benzoic a o d derivatives Psoriasis is a common and longstandmg skin &sease for which no causal treatment ~s yet avadable One of its basic biochemical features is an enormously increased arachldomc acid pathway which is mmated by oxygen ra&cals, accompamed by the generation of large amounts of these species, and results m accumulation of large amounts of leukotnenes 6 7 Although superoxlde is considered to be the primary oxygen species formed, the main damaging effect should be due to the more reactive hydroxyl radical The arachldonlc acid pathway has received much attention because ~t contains many of the components needed to estabhsh the abnormaht~es of psoriatic skin 8 Once empirically introduced for psoriasis therapy, antmx~dants are thought to mteract with the excessive radical and leukotnene production 9 Based on the general knowledge of the mechamsms of the antloxldatlve action of phenols and benzols, l° this paper presents a systematic Investigation of the hydroxyl radical scavenging and antlpsonatlc activity
INTRODUCTION
Ant~oxldant systems provide effloent defense agamst oxidative stress m biological systems There are different mechamsms of ant~ox~datwe action, for example, ra&cal scavenging, recombination of ra&cals, chelation of transition metal ~ons, or electron transfer with formation of stable products Hydroxyl radical scavenging ~s one of the crucml elements of antlox~dative activity because the reactivity of these radicals is extremely high Under various pathological con&t~ons, such as inflammation and allergy, l lschemm2 and reperfuslon,3 or radiation damage,4 the natural defense mechamsms such as superoxlde dlsmutase/catalase, reduced glutathlone are not sufficient to prevent radical-reduced injury On the other hand, free radicals are important m the photodynamlc therapy of some kinds of cancer 5 One approach m the therapy of several &seases is the administration of antloxldatlve systems including hydroxyl radical scavengers In vitro test systems are used for evaluation of antlox~dant capacity, and comtAuthor to whom correspondence should be addressed 111
112
R F HAS1-LOFFet al
t II-I[-N~ ?N+I >
~)
go[l~r
orld
R~ = I~2=
ester~s R 3 = OH
b) p-hydroxy benzorc acid esfeF R~ = R3= H R2=
OH
~) m-hydroxy benzoic ocld esters /
P~,R~ = OH R~-
H
I F,' Fig 1 Structure ot the benzoic acid derivatives
of denvatwes of benzoic acid with different numbers of hydroxy groups and different chain length of the ester, which are, in part,~l known to have antmxldanve acnvlty The hydroxyl radical scavenging capacity was evaluated m a newly Introduced test system which allows mvesnganon and comparison of hpophfllC and hydrophfllC as well as acl&c and alkahne compounds M A T E R I A L S AND M E T H O D S
Hydroxyl radicals were generated by Fenton's reagent consisting of 0 05 mM FeSO4 and 0 1 mM H202 m the presence of 1 mM 5,5-dlmethylpyrrohne- l-oxide (DMPO*, solutions prepared according to Floyd'2), 14 mM HCI, 93 mM NaC1, 1 24 M pyndme, and the ant~oxldant In a concentration w~thm and around the hnear part of the concentration response curve determined in preceding experiments Chemicals pyndme (for spectroscopy, Merck, Darmstadt). butylated hydroxytoluene--BHT--(Calblochem-Behrmg, La Jolla, CA), D,L-c~-tocopherol (under Argon, research grade, SERVA, Heidelberg), mannltol (BDA Chemicals. L t d ) If not otherwise stated, chemicals were of analytical reagent grade The benzoic acid denvauves (Fig 1) were provided by G Tomaschewsky (Department of Chemistry, Humboldt University, Berhn) Its concentration in the test system was checked by UV absorption spectroscopy ESR spectra were recorded on a Vanan E 3 at room temperature under the followmg standard condmons scan range 10 mT, modulation amphtude 0 1 mT, microwave power 20 mW For the mvesngatlon of antlpsonatlc activity a proven method was used l~ 20 patients (ages 19-38, 9 male, 11 female) suffering from stable plaque-type psoriasis were included m that study after having given
full-reformed consensus Small skm lesions (1-2 cm m dmmeter and all of them exhlbmng the symptoms of the same stage of disease) were treated daily with 2 % (v/v) anttoxldant resulting m 12 mg annoxldant/ cm 2 of treated skin surface "Pure ointment and a strong topical comcosterold (flUOClnoloneacetomde. Flucmar R) served as controls Following ointment apphcat~on the test s~tes were covered with alummmm chambers 14 Before and 2, 4, 7, 9, and 11 days after starting the experimental treatment, the stage of each particular test s~te was evaluated according to the cnterm of Wemstem et al ~ Briefly, each of the three phenomena, erythema, infiltration, and desquamatton, were fit into a 4-point scale The resulting "psoriasis a c h w t y " ranges from 9 (maximal erythema, mfiltranon and scahng) to 0 (no psoriasis symptoms at all) When corrected by the control (pure omtment), the term "mean antlpsonanc efficacy" (MAPE) represents the percentage of dechne of psonanc symptoms compared to fluocmoloneacetonlde
RESULTS
In the mvesngatlons presented here, special interest was given to a series of substances with different cham length and different numbers and posmons of the hydroxy groups The results concerning their hydroxyl radical scavenging and antlpsonattc acnvtty are summanzed m Table 1
dFor practical reasons, the chmcal effect ts based on the volume and the weight of the substances apphed On a molar basts, the efficacy of the substances with higher molecular weight is even larger compared to the substances with lower molecular weight
Investigation of antmx~dants
Table 1 Hydroxyl Radical Scavenging and Antlpsonatlc Actlvlty of Benzotc Acid Denvatxves Compound Galhc acid p r o w l ester butyl ester octyl ester decyl ester tetradecyl ester heptadecyl ester p-hydroxy benzoic a o d methyl ester propyl ester -n-butyl ester -tso-butyl ester heptyl ester decyl ester m-hydroxy benzoic acid propyl ester decyl ester Manmtol Butylated hydroxytoluene c(-tocopherol
ECso*(~M)
MAPE**(%)
2 20 55 50 65 65 23 29 20 23 30 500 14 100 1300 1500 1650
n d 8 3 33 3 50 0 58 3 75 0 61 5 71 5 n d n d n d 91 7 66 7 93 3 n d n d n d
*The ECso-valuegives the concentration for reduction of the DMPO-OH ESR signal intensity on 50%, each individual value represents the mean of at least three mdependent determmanons of the concentratmn response curve **MAPE meananapsonatlc efficacy (percentage of dechne of psoriatic symptoms compared to fluocmoloneacetomde)after rune treatments under occlusmn (n d not determined) For statlsncal treatment, Wdcoxon's pmred rank sum test and hnear regression were used, respectively Except for gallic actd decyl/ tetradecyl ester and m-hydroxy benzoic acid propyl ester, all differences within the series of compounds (galhc aod esters, p-hydroxy benzoic acid esters mhydroxy benzotc acid esters) were slgmflcant (p < 0 01) Correlation coeffioents galhc acid esters, r = 0 897, p-hydroxy benzolt, acid esters r = 0 948 m-hydroxy benzoic esters, r = 1 00
Hydroxyl radtcal scavengmg m vttro
In Figure 2, the spectrum of DMPO-OH generated m the described system is shown (there Is a small mplet with aN = 1 39 mT superimposed on DMPO-OH, it originates from the trapping of products of the decomposmon of DMPO m some charges of the spm trap) The maximum intensity reached 2 rain after lnmatlon of the reaction was used for the evaluation of the OH scavenging actwlty In the presence of antloxl-
113
(*/,)
i
i
|
i
6
5
4
3
- tg c
(MI
Fig 3 Decrease of ESR-slgnal intensity in the presence of antloxtdants (100%, no antloxldant present), o, propyl gallate, x, m-hydroxy benzoic acid propyl ester, + , m-hydroxy benzoic acid decyl ester, O, p-hydroxy benzoic acid decyl ester, each m&vldual pomt represents the mean of at least 3 measurements
dants, the concentration of DMPO-OH decreased depending on the concentration of these antloxldants, as depicted m Figure 3 The curves show a slgmoldal shape, the efficacy of the substances is quantified by an ECs0-value representing the concentration necessary for 50% &mmutlon of the ESR signal intensity taken from the approximately hnear part of the graph From the ECso-values it follows that galhc acid propyl ester (propyl gallate) is the most effectwe of the investigated substances, with an ECso about three orders of magmtude lower than found for manmtol, BHT, or ct-tocopherol In both groups of compounds a tendency of decreasing hydroxyl ra&cal scavenging capacity with increasing chain length was observed There were only shght &fferences in hydroxyl radIcal scavenging capacity between benzoic acid derivatives with different numbers and posmons of the hydroxy groups Galhc acid esters with short chain lengths (n -< 3) are more active than those of hydroxy benzoic acid (independently of the posmon of the hydroxy group) The differences in activity between the esters of gallic and hydroxy benzoic a o d with medium cham lengths (n < 10) are neghglble, whereas galhc acid esters with longer chain lengths are more effective than those of hydroxy benzoic acid with respect to the hydroxyl radical scavenging activity
Anttpsortattc acttvzty 1 mT I )
Fig 2 ESR spectrum of DMPO-OH generated by a Fenton-type reaction (0 05 mM Fe2SO4, 0 1 mM H202, 1 mM DMPO, 14 mM HCI, 93 mM NaCI, 1 24 M pyndme), hyperfine sphttmg constants, as = an = 1 4 8 m T
It was found that the antlpsorlatlc actwlty of the substances increases with increasing chain length The denvatwes of hydroxy benzoic acid are more actwe than those of gallic acid as can be seen by comparison of the actwmes of the decyl esters (MAPE = 50 0 for the galhc acid denvatxve, MAPE = 91 7 [93.3] for the benzoic acid denvatwe[s])
R F H~SElOFVetal
114 DISCUSSION
In the present study, a possible mechamsm of interaction of the chosen compounds on the molecular level as well as their biological effect m the treatment of psoriasis was investigated fn general, the combinatron of phenomenologlcal and molecular approach warrants a more detailed characterlzatmn of the propert~es of the substances Regarding the mvest~gatmn of the hydroxyl radical scavenging actlwty it can be stated that spin trapping using DMPO is one of the most reliable methods for detection and ldentlflcanon of hydroxyl radicals D M P O - O H is sufficiently stable. and the rate constant for ~ts formatmn is high compared to other adducts (e g , DMPO-O2 )~5 Thus, the method is a powerful tool for the evaluation of hydroxyl radical scavenging, that ~s, anttoxldatlve capacity of different compounds The apphcatmn of pyndme and hydrochloric acid m this test system otfers the advantage of pH stablhzatmn and guarantees favorable conditions concerning the mvestlgatmn of substances which are usually soluble in water or hplds It was found that, in agreement with earher results. ~ propyl gallate ~s a very strong antmxldant since it xs about three orders of magnitude more efficient than BHT, o~-tocopherol, or mannitol In this context, the problem of the mechanism of ~ts action artses In the test system applied here, the antloxldatlve actlvlty has been evaluated by the amount of spin adduct formed m the competition between the spin trap and the antmx~dant There ~s no poss~bdlty to ddferentlate between the hydroxyl radical scavenging act~vlty and the abd~ty to decrease the rate of formanon of OH (for example, by chelatmn of Fe 2÷ or catalytic/peroxldat~c activity) However, m addmonal experiments with some compounds including propyl gallate, no reaction with hydrogen peroxide was observed, and m the micromolar concentration range Fe z" chelatmn ~s not expected to exert a dominating effect (ECso lpropyl gallate]
