Phytochemistry, Vol. 31, No. 6, Pp. 1947-1950, 1992 Printedin Great Britain.
0
003 l-9422/92 $5.00 + 0.00 1992 PergamonPress Ltd
ANTIOXIDANT ACTIVITY OF FLAVONOIDS FROM SIDERITIS JAVALAMBRENSZS JOSE LUIS RIOS, SALVADOR MAREZ,
MIGUEL PAYA
and
MARIA JOSE ALCARAZ*
Departamento de Farmacologia, Facultad de Farmacia, Avda. Blasco Ibliiez 13,460lO
Valencia, Spain
(Received in revised form 26 November 1991) Key Word Index-Sideritis jaualambrensis; Labiatae; flavonoids; antioxidant activity; superoxide scavengers.
Abstract-Five flavonoid glycosides, 4’-0-methylisoscutellarein 7-0-[6”‘-acetylallopyranosyl(1+2)glucopyranoside], 3’-hydroxy-4’-0-methylisoscutellarein 7-04’-0-methylisoscutellarein 7-0-allopyranosyl(l+2)glucopyranoside, [6”‘-acetylallopyranosyl(l+2)glucopyranoside], isoscutellarein 7-0-[6”‘-acetylallopyranosyl(l-t2)glucopyranoside] and hypolaetin-8-glucoside have been isolated from Sideritis jaualambrensis aerial parts and identified by standard methods. These glycosides have been tested for their antioxidant properties alongside other 7,tGsubstituted flavonoids using FeSOJcysteine-induced microsomal lipid peroxidation. Superoxide scavenging activity was assessed in the nitroblue tetrazolium test. Among this series of flavonoids, hypolaetin-8glucoside is the most potent inhibitor of nonenzymic lipid peroxidation. The antiperoxidative activity of these flavonoids may be related to their superoxide scavenging ability.
INTRODUCTION
Sideritis javalambrensis Pau (Labiatae)
is an endemic species growing in Sierra Javalambre (Teruel, Spain). Previous studies have demonstrated the anti-inflammatory activity of the n-hexane extract and isolated compounds, such as ent- 16-hydroxy- 13-epi-manoyl oxide [l, 21 and esters of tyrosol [2]. On the other hand, S. jaoalambrensis contains other constituents endowed with biological activity and belonging to a different chemical class. Thus, we isolated the flavonoid aglycones, cirsiliol, cirsimaritin, 8-methoxycirsilineol, sideritoflavone and xanthomicrol [S], present in other species of Sideritis, and studied their anti-inflammatory properties [4], to assess their antiperoxidative activity. As a result, 8methoxycirsilineol and cirsimaritin were found to inhibit microsomal lipid peroxidation [3]. Lipid peroxidation is a key process in many pathological states, which can be modulated by flavonoids in a number of ways [S-l 11. Accordingly, we isolated a series of flavonoid glycosides from S. javalambrensis and examined their effects on non-enzymic lipid peroxidation and superoxide scavenging. RESULTS AND DISCUSSION
Isolation of jIavonoids
Defatted plant material was extracted with methanol. The concentrated methanol extract was suspended in water and fractionated into diethyl ether, ethyl acetate and butanol. The flavonoids present in the ‘butanol extract of S.- javalambrensis were isolated by CC and DCCC and identified by comparison of their spectral data with literature data [ 12,131. Thus, we have isolated *Author to whom correspondence should he addressed.
for the first time in this species five flavone glycosides: 4’-0-methylisoscutellarein 7-0-[6”‘-acetylallopyranosyl(1-+2)glucopyranoside] (I), 4’-0-methylisoscutellarein 7-0-allopyranosyl( 1-r2)glucopyranoside (2), 3’-hydroxy4’-0-methylisoscutellarein 7-0-[6”‘-aceiylallopyranosyl(1+2)glucopyranoside] (3), isoscutellarein 7-0[6”‘-acetylallopyranosyl( 1+2)glucopyranoside] (4) and hypolaetin-8glucoside (5). These compounds are present in other species of Sideritis [14] and Stackys [13]. Some minor constituents have been identified by HPLC-DAD in chromatographic fractions: caffeic acid and its esters with quinic acid, chlorogenic acid and isochlorogenic acid. Antiperoxidatiue actioity
The five glycosides isolated from S. jaualambrensis were tested as inhibitors of non-enzymic lipid peroxidation using FeSO, and cysteine as inducers. We have also included the aglycone 7,8_dihydroxyflavone and the glycoside gossypin to allow a better comparison of their antiperoxidative activity with those of compounds possessing the same pattern of substitution (Table 1). In this series of 7,8-dihydroxyl derivatives a number of conclusions on the structure-activity relationship can be drawn, confirming and extending our previous observations on other flavonoids [3]. As can be seen in Table 2, 7,8-dihydroxyflavone, 5 and gossypin have an I&, in the PM range, comparable to synthetic antioxidants. These potent flavonoids possess a free catcchol group at positions 3’4’ or 7,8. The participation of the 3’,4’-catechol group in the antiperoxidative properties of flavonoids has been established in previous work [3, 8, 93, while the relevance of the 7,8-catechol group is apparent because of the potent inhibitory effects exerted by the parent compound 7,8_dihydroxyflavone. The introduction of a free hydroxyl at C-3 is slightly detrimental, as can be deduced
1947
J. L. RIOSet al.
1948 Table 1. Structures of flavonoids tested
R3 7,gDihydroxyflavone
1 2 3 4
5 Gossypin AAGe-
H H H H
R5
R’ Rs OH OH H OH OAAG OH OH OAG OH OH OAAG OH
H H OH
OH OAAG OH OH OH oc OH OH OG
R”’
R’
H
H
H H OH H OH OH
OMe OMe OMe OH OH OH
interesting to note that all flavonoids acted as superoxide scavengers (Figs l-4 and Table 2). Most of the compounds showed similar inhibitory potencies for lipid peroxidation and superoxide scavenging. There are two exceptions: 7&dihydroxyflavone, which probably interacts with peroxidative processes at different levels and 2, less active than expected on lipid peroxidation. Thus, superoxide scavenging may be one of the mechanisms by which flavonoids inhibit lipid peroxidation. We have previously demonstrated the inhibitory effects of 5 and gossypin on inflammation and arachidonic acid metabolism [4]. In this respect the antioxidant activity shown by these flavonoids may participate in their antiinflammatory properties, since radical-induced actions play a central role in inflammatory conditions. It is known that lipid peroxides promote arachidonic acid metabolism [IS] and redox agents, such as phenolic derivatives can inhibit oxidation of arachidonic acid by Slipoxygenases [16]; thus antioxidant and free radical scavenging flavonoids could prevent generation of inflammatory mediators.
acetylallopyranosyl (1-2) glucopyranose.
EXPERIMENTAL
AG=allopyranosyl (l-2) glucopyranose.
Plant material. Sideritis jaualambrensis Pau was collected in Sierra Javalambre (Teruel, Spain) and a voucher specimen is
G=ghJcose.
166 -
Table 2. Inhibitory potencies (IC,,) of flavonoids on nonenzymic lipid peroxidation and superoxide scavenging
60. 60. 70
Flavonoid 7,8-Dihydroxyflavone 1
2 3 4 5 Gossypin
Lipid peroxidation 6.0 33.3 >loo 19.7 52.1 1.3 7.4
Superoxide scavenging 37.0 19.4 33.6 18.6 25.7 6.4 4.3
I&,, PM. The synthetic antioxidant propyl gallate showed an IC,, =4.4 PM for inhibition of lipid peroxidation and an IC,, = 7.7 pM for superoxide scavenging.
by comparing 5 with the corresponding flavonol glucoside, gossypin. The influence of glycosylation deserves special attention. O-Glycosylation at C-7 has been reported as weakly negative on activity [3]. We have now observed that compounds having the 6”‘-acetylallopyranosyl(1-*2)glucopyranoside group are also active, although with a reduced potency. Interestingly, the presence of the 6”‘-acetyl group increases the antiperoxidative activity (1 versus 2). On the other hand, O-glycosylated flavonoids at C-8 (5 and gossypin) exhibit a high potency. Flavonoids are reducing agents able to interact with free radical species; this is probably responsible for their antioxidant properties. Their mechanism of action can be related to inhibition of the initiation of lipid peroxidation acting as oxygen radical scavengers or by interaction with peroxyl radicals leading to termination of chain reactions [3,7]. The precise mechanism of action of the flavonoids included in this study is not known; nevertheless it is
80 9‘
lN”l6lTlON
so40
.
30
.
20
1
10 04
0
20
40
60
CONCENTRATION
60
100
@M,
Fig. 1. Effects of 1 and 2 on lipid peroxidation and superoxide scavenging Results show mean of n=6. Standard deviations have been omitted for simplicity. Values are significant at least at P
0
003 l-9422/92 $5.00 + 0.00 1992 PergamonPress Ltd
ANTIOXIDANT ACTIVITY OF FLAVONOIDS FROM SIDERITIS JAVALAMBRENSZS JOSE LUIS RIOS, SALVADOR MAREZ,
MIGUEL PAYA
and
MARIA JOSE ALCARAZ*
Departamento de Farmacologia, Facultad de Farmacia, Avda. Blasco Ibliiez 13,460lO
Valencia, Spain
(Received in revised form 26 November 1991) Key Word Index-Sideritis jaualambrensis; Labiatae; flavonoids; antioxidant activity; superoxide scavengers.
Abstract-Five flavonoid glycosides, 4’-0-methylisoscutellarein 7-0-[6”‘-acetylallopyranosyl(1+2)glucopyranoside], 3’-hydroxy-4’-0-methylisoscutellarein 7-04’-0-methylisoscutellarein 7-0-allopyranosyl(l+2)glucopyranoside, [6”‘-acetylallopyranosyl(l+2)glucopyranoside], isoscutellarein 7-0-[6”‘-acetylallopyranosyl(l-t2)glucopyranoside] and hypolaetin-8-glucoside have been isolated from Sideritis jaualambrensis aerial parts and identified by standard methods. These glycosides have been tested for their antioxidant properties alongside other 7,tGsubstituted flavonoids using FeSOJcysteine-induced microsomal lipid peroxidation. Superoxide scavenging activity was assessed in the nitroblue tetrazolium test. Among this series of flavonoids, hypolaetin-8glucoside is the most potent inhibitor of nonenzymic lipid peroxidation. The antiperoxidative activity of these flavonoids may be related to their superoxide scavenging ability.
INTRODUCTION
Sideritis javalambrensis Pau (Labiatae)
is an endemic species growing in Sierra Javalambre (Teruel, Spain). Previous studies have demonstrated the anti-inflammatory activity of the n-hexane extract and isolated compounds, such as ent- 16-hydroxy- 13-epi-manoyl oxide [l, 21 and esters of tyrosol [2]. On the other hand, S. jaoalambrensis contains other constituents endowed with biological activity and belonging to a different chemical class. Thus, we isolated the flavonoid aglycones, cirsiliol, cirsimaritin, 8-methoxycirsilineol, sideritoflavone and xanthomicrol [S], present in other species of Sideritis, and studied their anti-inflammatory properties [4], to assess their antiperoxidative activity. As a result, 8methoxycirsilineol and cirsimaritin were found to inhibit microsomal lipid peroxidation [3]. Lipid peroxidation is a key process in many pathological states, which can be modulated by flavonoids in a number of ways [S-l 11. Accordingly, we isolated a series of flavonoid glycosides from S. javalambrensis and examined their effects on non-enzymic lipid peroxidation and superoxide scavenging. RESULTS AND DISCUSSION
Isolation of jIavonoids
Defatted plant material was extracted with methanol. The concentrated methanol extract was suspended in water and fractionated into diethyl ether, ethyl acetate and butanol. The flavonoids present in the ‘butanol extract of S.- javalambrensis were isolated by CC and DCCC and identified by comparison of their spectral data with literature data [ 12,131. Thus, we have isolated *Author to whom correspondence should he addressed.
for the first time in this species five flavone glycosides: 4’-0-methylisoscutellarein 7-0-[6”‘-acetylallopyranosyl(1-+2)glucopyranoside] (I), 4’-0-methylisoscutellarein 7-0-allopyranosyl( 1-r2)glucopyranoside (2), 3’-hydroxy4’-0-methylisoscutellarein 7-0-[6”‘-aceiylallopyranosyl(1+2)glucopyranoside] (3), isoscutellarein 7-0[6”‘-acetylallopyranosyl( 1+2)glucopyranoside] (4) and hypolaetin-8glucoside (5). These compounds are present in other species of Sideritis [14] and Stackys [13]. Some minor constituents have been identified by HPLC-DAD in chromatographic fractions: caffeic acid and its esters with quinic acid, chlorogenic acid and isochlorogenic acid. Antiperoxidatiue actioity
The five glycosides isolated from S. jaualambrensis were tested as inhibitors of non-enzymic lipid peroxidation using FeSO, and cysteine as inducers. We have also included the aglycone 7,8_dihydroxyflavone and the glycoside gossypin to allow a better comparison of their antiperoxidative activity with those of compounds possessing the same pattern of substitution (Table 1). In this series of 7,8-dihydroxyl derivatives a number of conclusions on the structure-activity relationship can be drawn, confirming and extending our previous observations on other flavonoids [3]. As can be seen in Table 2, 7,8-dihydroxyflavone, 5 and gossypin have an I&, in the PM range, comparable to synthetic antioxidants. These potent flavonoids possess a free catcchol group at positions 3’4’ or 7,8. The participation of the 3’,4’-catechol group in the antiperoxidative properties of flavonoids has been established in previous work [3, 8, 93, while the relevance of the 7,8-catechol group is apparent because of the potent inhibitory effects exerted by the parent compound 7,8_dihydroxyflavone. The introduction of a free hydroxyl at C-3 is slightly detrimental, as can be deduced
1947
J. L. RIOSet al.
1948 Table 1. Structures of flavonoids tested
R3 7,gDihydroxyflavone
1 2 3 4
5 Gossypin AAGe-
H H H H
R5
R’ Rs OH OH H OH OAAG OH OH OAG OH OH OAAG OH
H H OH
OH OAAG OH OH OH oc OH OH OG
R”’
R’
H
H
H H OH H OH OH
OMe OMe OMe OH OH OH
interesting to note that all flavonoids acted as superoxide scavengers (Figs l-4 and Table 2). Most of the compounds showed similar inhibitory potencies for lipid peroxidation and superoxide scavenging. There are two exceptions: 7&dihydroxyflavone, which probably interacts with peroxidative processes at different levels and 2, less active than expected on lipid peroxidation. Thus, superoxide scavenging may be one of the mechanisms by which flavonoids inhibit lipid peroxidation. We have previously demonstrated the inhibitory effects of 5 and gossypin on inflammation and arachidonic acid metabolism [4]. In this respect the antioxidant activity shown by these flavonoids may participate in their antiinflammatory properties, since radical-induced actions play a central role in inflammatory conditions. It is known that lipid peroxides promote arachidonic acid metabolism [IS] and redox agents, such as phenolic derivatives can inhibit oxidation of arachidonic acid by Slipoxygenases [16]; thus antioxidant and free radical scavenging flavonoids could prevent generation of inflammatory mediators.
acetylallopyranosyl (1-2) glucopyranose.
EXPERIMENTAL
AG=allopyranosyl (l-2) glucopyranose.
Plant material. Sideritis jaualambrensis Pau was collected in Sierra Javalambre (Teruel, Spain) and a voucher specimen is
G=ghJcose.
166 -
Table 2. Inhibitory potencies (IC,,) of flavonoids on nonenzymic lipid peroxidation and superoxide scavenging
60. 60. 70
Flavonoid 7,8-Dihydroxyflavone 1
2 3 4 5 Gossypin
Lipid peroxidation 6.0 33.3 >loo 19.7 52.1 1.3 7.4
Superoxide scavenging 37.0 19.4 33.6 18.6 25.7 6.4 4.3
I&,, PM. The synthetic antioxidant propyl gallate showed an IC,, =4.4 PM for inhibition of lipid peroxidation and an IC,, = 7.7 pM for superoxide scavenging.
by comparing 5 with the corresponding flavonol glucoside, gossypin. The influence of glycosylation deserves special attention. O-Glycosylation at C-7 has been reported as weakly negative on activity [3]. We have now observed that compounds having the 6”‘-acetylallopyranosyl(1-*2)glucopyranoside group are also active, although with a reduced potency. Interestingly, the presence of the 6”‘-acetyl group increases the antiperoxidative activity (1 versus 2). On the other hand, O-glycosylated flavonoids at C-8 (5 and gossypin) exhibit a high potency. Flavonoids are reducing agents able to interact with free radical species; this is probably responsible for their antioxidant properties. Their mechanism of action can be related to inhibition of the initiation of lipid peroxidation acting as oxygen radical scavengers or by interaction with peroxyl radicals leading to termination of chain reactions [3,7]. The precise mechanism of action of the flavonoids included in this study is not known; nevertheless it is
80 9‘
lN”l6lTlON
so40
.
30
.
20
1
10 04
0
20
40
60
CONCENTRATION
60
100
@M,
Fig. 1. Effects of 1 and 2 on lipid peroxidation and superoxide scavenging Results show mean of n=6. Standard deviations have been omitted for simplicity. Values are significant at least at P
