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Antioxidant activity of isocytisoside and extracts of Aquilegia vulgaris
Fitoterapia 76 (2005) 476 – 480 www.elsevier.com/locate/fitote
Short report
Antioxidant activity of isocytisoside and extracts of Aquilegia vulgaris Marek Murias a,*, Jadwiga Jodynis-Liebert a, Irena Matlawskab, Wieslawa Bylkab a
b
Department of Toxicology, Poznan˜ University of Medical Sciences, Dojazd 30, 60-631 Poznan˜, Poland Department of Pharmacognosy, Poznan˜ University of Medical Sciences, Sieroca 10, 61-777 Poznan˜, Poland Received 2 August 2004; accepted in revised form 26 April 2005
Abstract Two extracts (ethyl acetate and ethanol) and isocytisoside obtained from Aquilegia vulgaris were tested for their antioxidant and free radical scavenging activity in vitro. Inhibition both non-enzymatic (IC50: 150–219 Ag/ml) and enzymatic (IC50: 23–60 Ag/ml) microsomal lipid peroxidation was observed, the extracts being more active than isocytisoside. The substances tested appeared to be weak hydroxyl radical scavengers, showed very low TEAC values and moderate iron chelation ability. However, all preparations at the concentration 25 Ag/ml inhibited superoxide anion formation at the range 47–68%. Despite of the lack of a potent free radical scavenging ability the substances tested demonstrated significant antioxidant activity. Relationship between this parameter and the content of phenolic groups was noticed. D 2005 Elsevier B.V. All rights reserved. Keywords: Aquilegia vulgaris; Microsomal lipid peroxidation; Free radical scavenging; Iron chelation
1. Plant material Aquilegia vulgaris L. (Ranunculaceae) stems and leaves were collected in the Botanic Garden of A. Mickiewicz University, Poznan, Poland in June 2001. A voucher specimen (No. KF1262001) is deposited in the authors’ laboratory. * Corresponding author. Tel./fax: +48 61 8470721. E-mail address: [email protected] (M. Murias). 0367-326X/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2005.04.004
M. Murias et al. / Fitoterapia 76 (2005) 476–480
477
2. Use in traditional medicine The aerial parts are used against liver and bile duct disorders, especially for the treatment of jaundice, scurvy, neurosis, dermatitis and as a diaphoretic agent [1]. The herb is a component of the immunostimulating preparation Padma 28 and homeopathic drugs [1]. Preliminary investigation showed that extracts of A. vulgaris as well as isocytisoside could protect against liver injury caused by CCl4 [2].
3. Previously isolated classes of constituents Stems and leaves: flavonoids [3–5], phenolic acids [6], tannins, anthocyanins and alkaloids [7].
4. New isolated constituent Isocytisoside (4V-methoxy-5,7-dihydroxyflavone 6-C-glucopyranoside) [3].
5. Tested material Ethanol extract (EE, yield: 24% from air-dried leaves and stems) [2], ethyl acetate extract (EAE) [prepared after extraction with boiling methanol, treatment with hot water, deflating with ethyl ether (discarded) and extraction with ethyl acetate, yield: 3%]. Isocytisoside (IST) [3]. HPLC analysis showed that the two extracts contained 1.5% and 5% of IST, respectively. Besides, the extracts contained: isocytisoside 7-O-glucoside, isoorientin, orientin, isovitexin 4V-O-glucoside, apigenin 7-O-rutinoside, apigenin 7-O-glucoside and apigenin. Additionally, the ethanol extract contained phenolic acids: caffeic, ferulic, p-coumaric, resorcylic, p-hydroxybenzoic, vanillic, sinapic and chlorogenic.
6. Studied activity Inhibition of microsomal lipid peroxidation stimulated with: Fe2+/ascorbate, Fe3+/ADP/ NADPH, CCl4/NADPH, assayed by TBARS test [8,9]. Determination of hydroxyl radical scavenging activity is when hydroxyl radicals generated in Fenton reaction cause a degradation of deoxyribose and formation of TBARS; any compound which can scavenge hydroxyl radical causes decrease in TBARS formation [10]. Determination of superoxide radical scavenging activity is an effect of substances tested on nitroblue tetrazolium (NBT) reduction in the hypoxanthine/xanthine oxidase system [11]. TEAC (Trolox equivalent antioxidant capacity) assay is based on the ability of the antioxidant to scavenge the blue-green coloured ABTSS+ [2,2V-azinobis (3-ethyl-
478
M. Murias et al. / Fitoterapia 76 (2005) 476–480
Table 1 Antioxidant activity of the A. vulgaris extracts and of isocytisoside on microsomal lipid peroxidation IC50 [Ag/ml]a
Sample 2+
IST EAE EE a-Tocopherolb
3+
Fe /ascorbate
Fe /ADP/NADPH
CCl4/NADPH
217.8 F 37.6 105.5 F 4.5 218.9 F 7.5 9.8 F 0.6
55.6 F 5.8 22.6 F 2.3 32.1 F 5.3 6.8 F 1.2
60.3 F 4.3 45.3 F 4.7 56.7 F 2.8 10.3 F 2.3
IST—isocytisoside, EAE—ethyl acetate extract, EE—ethanol extract. a Mean F SD. b Positive control.
benzothiozoline-6-sulphonic acid)ammonium salt] radical cation relative to the ABTSS+ scavenging ability of Trolox (water-soluble analogue of vitamin E) [12]. Iron chelation is when ferrozine binds Fe2+, forming a complex with a high extinction coefficient; any compound which can chelate Fe2+ causes the decrease in the absorbance of this complex [13]. Determination of phenol groups is based on Folin-Ciocalteu reagent [14]. Data analysis in all experiments were run in triplicate and averaged. Statistical analysis was performed by one-way analysis of variance followed by Dunnett’s test for multiple comparisons. IC50 was calculated from the concentration–effect regression lines.
7. Results Presented in Tables 1–3.
8. Conclusions The tested materials showed antioxidant activity as demonstrated in microsomal lipid peroxidation assay. In the system dependent on Fe2+/ascorbate (non-enzymatically Table 2 Scavenging effects of the A. vulgaris extracts and of isocytisoside on superoxide anion and hydroxyl radical Sample
Inhibition of NBT reduction (A 550 nm)a
Extent of deoxyribose degradation (A 532 nm)b
IST EAE EE SODc 10 U/ml Mannitolc 10 mM Control
0.49 F 0.07* 0.30 F 0.03* 0.34 F 0.04* 0.08 F 0.01*
0.331 F 0.012* (18%) 0.342 F 0.011* (15%) 0.340 F 0.028* (16%)
a
0.93 F 0.01
Concentration of sample: 25 Ag/ml, mean F SD. Concentration of sample: 100 Ag/ml, mean F SD. c Positive control, SOD—superoxide dismutase. * P b 0.001 as compared with the control value.
b
(47%) (62%) (68%) (91%)
0.152 F 0.002* (62%) 0.404 F 0.014
M. Murias et al. / Fitoterapia 76 (2005) 476–480
479
Table 3 Phenolic groups, TAEC values and iron chelation of the A. vulgaris extracts and of isocytisoside Sample
Phenolic groups (AM/mg)a
TEACa
Iron chelation %
IST EAE EE EDTAd
4.52 F 0.20 12.74 F 0.80 8.11 F 0.06
0.081 F 0.006b 0.321 F 0.018c 0.149 F 0.001c
46.7 F 3.6 51.2 F 6.3 69.2 F 4.9 100
a b c d
Mean F SD. Value expressed in mM. Values calculated for 1 g of dry mass. Positive control.
stimulated) ethyl acetate extract was the most potent. Enzymatic lipid peroxidation stimulated by CCl4/NADPH was almost equally inhibited by isocytisoside and extracts. The greatest antioxidant activity of substances tested was demonstrated in Fe3+/ADP/ NADPH stimulated system (Table 1). Hence, it could be concluded that isocytisoside and extracts can be classified as chain-breaking antioxidants since they inhibit NADPHdependent lipid peroxidation, the process involving generation of a great amount of ROOS radicals [15]. The tested materials appeared to be rather weak hydroxyl radical scavengers, since they decrease deoxyribose degradation by 15–18% (Table 2). Both extracts and isocytisoside inhibited superoxide radical at a concentration 25 Ag/ml by 63%, 68% and 47%, respectively (Table 2). Higher scavenging activity of extracts could be due to the presence of other compounds acting as potent superoxide scavengers, for example isoorientin [16]. Tested materials showed low TEAC values and moderate activity of iron chelation. The most active Fe2+ chelator was ethanol extract, 70% in comparison with EDTA (Table 3). The highest content of phenolic groups, which show the ability to neutralize lipid radicals by donating hydrogen atoms, was found in the ethyl acetate extract (Table 3).There was a relationship between the content of phenolic groups in this extract and its antioxidant activity. Ethyl acetate extract was the most potent inhibitor of microsomal lipid peroxidation in all systems tested. It can be suggested that demonstrated previously hepatoprotective properties of A. vulgaris may be ascribed, at least in part, to the antioxidant activity of its constituents. Acknowledgements This work was supported by a research grant of the Polish State Committee for Scientific Research (No. 835/PD5/2001/20). References [1] PDR for herbal medicines, second ed. NJ7 Medicinal Economics Company; 2000. p. 211. [2] Adamska T, Myynarczyk W, Jodynis-Liebert J, Bylka W, Matyawska I. Phytother Res 2003;17:691. [3] Bylka W, Matyawska I. Acta Pol Pharm 1997;54:331.
480 [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16]
M. Murias et al. / Fitoterapia 76 (2005) 476–480 Bylka W, Matyawska I. Acta Pol Pharm 1997;54:334. Bylka W. Acta Pol Pharm 2001;54:237. Szaufer-Hajdrych M, Drost-Karbowska K, Kowalewski Z. Acta Pol Pharm 2002;59:57. Hansell R, Keller K, Rimpler H, Schneider G, Handbuch der Pharmaceutische Praxis, Bd. 4 Drogen A-D. Berlin: Springer Verlag, p. 312. Sanz MJ, Ferrandiz ML, Cejudo M, Terencio MC, Gil B, Bustos G, et al. Xenobiotica 1994;24:689. Haraguchi H, Ishikawa H, Sanchez Y, Ogura T, Kubo Y. Bioorg Med Chem 1997;5:865. Halliwell B, Gutteridge JMC, Aruoma OJ. Anal Biochem 1987;165:215. Ko FN, Liao CH, Kuo YH, Lin YL. Biochim Biophys Acta 1995;1258:145. Tyrakowska B, Soffers AE, Szymusiak H, Boeren S, Boersma MG, Leman˜ska K, et al. Free Radic Biol Med 1999;27:1427. Lopes GKB, Schulma HM, Hermes-Lima M. Biochim Biophys Acta 1999;1472:142. Sharma HM, Hanna AN, Kauffinan EM, Newman HAI. Free Radic Biol Med 1995;18:687. Sevanian A, Nordenbrand K, Kim E, Emster L, Hochstein P. Free Radic Biol Med 1990;8:145. Ko FN, Chu CC, Lin CN, Chang CC, Teng CM. Biochim Biophys Acta 1998;1389:90.
Short report
Antioxidant activity of isocytisoside and extracts of Aquilegia vulgaris Marek Murias a,*, Jadwiga Jodynis-Liebert a, Irena Matlawskab, Wieslawa Bylkab a
b
Department of Toxicology, Poznan˜ University of Medical Sciences, Dojazd 30, 60-631 Poznan˜, Poland Department of Pharmacognosy, Poznan˜ University of Medical Sciences, Sieroca 10, 61-777 Poznan˜, Poland Received 2 August 2004; accepted in revised form 26 April 2005
Abstract Two extracts (ethyl acetate and ethanol) and isocytisoside obtained from Aquilegia vulgaris were tested for their antioxidant and free radical scavenging activity in vitro. Inhibition both non-enzymatic (IC50: 150–219 Ag/ml) and enzymatic (IC50: 23–60 Ag/ml) microsomal lipid peroxidation was observed, the extracts being more active than isocytisoside. The substances tested appeared to be weak hydroxyl radical scavengers, showed very low TEAC values and moderate iron chelation ability. However, all preparations at the concentration 25 Ag/ml inhibited superoxide anion formation at the range 47–68%. Despite of the lack of a potent free radical scavenging ability the substances tested demonstrated significant antioxidant activity. Relationship between this parameter and the content of phenolic groups was noticed. D 2005 Elsevier B.V. All rights reserved. Keywords: Aquilegia vulgaris; Microsomal lipid peroxidation; Free radical scavenging; Iron chelation
1. Plant material Aquilegia vulgaris L. (Ranunculaceae) stems and leaves were collected in the Botanic Garden of A. Mickiewicz University, Poznan, Poland in June 2001. A voucher specimen (No. KF1262001) is deposited in the authors’ laboratory. * Corresponding author. Tel./fax: +48 61 8470721. E-mail address: [email protected] (M. Murias). 0367-326X/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2005.04.004
M. Murias et al. / Fitoterapia 76 (2005) 476–480
477
2. Use in traditional medicine The aerial parts are used against liver and bile duct disorders, especially for the treatment of jaundice, scurvy, neurosis, dermatitis and as a diaphoretic agent [1]. The herb is a component of the immunostimulating preparation Padma 28 and homeopathic drugs [1]. Preliminary investigation showed that extracts of A. vulgaris as well as isocytisoside could protect against liver injury caused by CCl4 [2].
3. Previously isolated classes of constituents Stems and leaves: flavonoids [3–5], phenolic acids [6], tannins, anthocyanins and alkaloids [7].
4. New isolated constituent Isocytisoside (4V-methoxy-5,7-dihydroxyflavone 6-C-glucopyranoside) [3].
5. Tested material Ethanol extract (EE, yield: 24% from air-dried leaves and stems) [2], ethyl acetate extract (EAE) [prepared after extraction with boiling methanol, treatment with hot water, deflating with ethyl ether (discarded) and extraction with ethyl acetate, yield: 3%]. Isocytisoside (IST) [3]. HPLC analysis showed that the two extracts contained 1.5% and 5% of IST, respectively. Besides, the extracts contained: isocytisoside 7-O-glucoside, isoorientin, orientin, isovitexin 4V-O-glucoside, apigenin 7-O-rutinoside, apigenin 7-O-glucoside and apigenin. Additionally, the ethanol extract contained phenolic acids: caffeic, ferulic, p-coumaric, resorcylic, p-hydroxybenzoic, vanillic, sinapic and chlorogenic.
6. Studied activity Inhibition of microsomal lipid peroxidation stimulated with: Fe2+/ascorbate, Fe3+/ADP/ NADPH, CCl4/NADPH, assayed by TBARS test [8,9]. Determination of hydroxyl radical scavenging activity is when hydroxyl radicals generated in Fenton reaction cause a degradation of deoxyribose and formation of TBARS; any compound which can scavenge hydroxyl radical causes decrease in TBARS formation [10]. Determination of superoxide radical scavenging activity is an effect of substances tested on nitroblue tetrazolium (NBT) reduction in the hypoxanthine/xanthine oxidase system [11]. TEAC (Trolox equivalent antioxidant capacity) assay is based on the ability of the antioxidant to scavenge the blue-green coloured ABTSS+ [2,2V-azinobis (3-ethyl-
478
M. Murias et al. / Fitoterapia 76 (2005) 476–480
Table 1 Antioxidant activity of the A. vulgaris extracts and of isocytisoside on microsomal lipid peroxidation IC50 [Ag/ml]a
Sample 2+
IST EAE EE a-Tocopherolb
3+
Fe /ascorbate
Fe /ADP/NADPH
CCl4/NADPH
217.8 F 37.6 105.5 F 4.5 218.9 F 7.5 9.8 F 0.6
55.6 F 5.8 22.6 F 2.3 32.1 F 5.3 6.8 F 1.2
60.3 F 4.3 45.3 F 4.7 56.7 F 2.8 10.3 F 2.3
IST—isocytisoside, EAE—ethyl acetate extract, EE—ethanol extract. a Mean F SD. b Positive control.
benzothiozoline-6-sulphonic acid)ammonium salt] radical cation relative to the ABTSS+ scavenging ability of Trolox (water-soluble analogue of vitamin E) [12]. Iron chelation is when ferrozine binds Fe2+, forming a complex with a high extinction coefficient; any compound which can chelate Fe2+ causes the decrease in the absorbance of this complex [13]. Determination of phenol groups is based on Folin-Ciocalteu reagent [14]. Data analysis in all experiments were run in triplicate and averaged. Statistical analysis was performed by one-way analysis of variance followed by Dunnett’s test for multiple comparisons. IC50 was calculated from the concentration–effect regression lines.
7. Results Presented in Tables 1–3.
8. Conclusions The tested materials showed antioxidant activity as demonstrated in microsomal lipid peroxidation assay. In the system dependent on Fe2+/ascorbate (non-enzymatically Table 2 Scavenging effects of the A. vulgaris extracts and of isocytisoside on superoxide anion and hydroxyl radical Sample
Inhibition of NBT reduction (A 550 nm)a
Extent of deoxyribose degradation (A 532 nm)b
IST EAE EE SODc 10 U/ml Mannitolc 10 mM Control
0.49 F 0.07* 0.30 F 0.03* 0.34 F 0.04* 0.08 F 0.01*
0.331 F 0.012* (18%) 0.342 F 0.011* (15%) 0.340 F 0.028* (16%)
a
0.93 F 0.01
Concentration of sample: 25 Ag/ml, mean F SD. Concentration of sample: 100 Ag/ml, mean F SD. c Positive control, SOD—superoxide dismutase. * P b 0.001 as compared with the control value.
b
(47%) (62%) (68%) (91%)
0.152 F 0.002* (62%) 0.404 F 0.014
M. Murias et al. / Fitoterapia 76 (2005) 476–480
479
Table 3 Phenolic groups, TAEC values and iron chelation of the A. vulgaris extracts and of isocytisoside Sample
Phenolic groups (AM/mg)a
TEACa
Iron chelation %
IST EAE EE EDTAd
4.52 F 0.20 12.74 F 0.80 8.11 F 0.06
0.081 F 0.006b 0.321 F 0.018c 0.149 F 0.001c
46.7 F 3.6 51.2 F 6.3 69.2 F 4.9 100
a b c d
Mean F SD. Value expressed in mM. Values calculated for 1 g of dry mass. Positive control.
stimulated) ethyl acetate extract was the most potent. Enzymatic lipid peroxidation stimulated by CCl4/NADPH was almost equally inhibited by isocytisoside and extracts. The greatest antioxidant activity of substances tested was demonstrated in Fe3+/ADP/ NADPH stimulated system (Table 1). Hence, it could be concluded that isocytisoside and extracts can be classified as chain-breaking antioxidants since they inhibit NADPHdependent lipid peroxidation, the process involving generation of a great amount of ROOS radicals [15]. The tested materials appeared to be rather weak hydroxyl radical scavengers, since they decrease deoxyribose degradation by 15–18% (Table 2). Both extracts and isocytisoside inhibited superoxide radical at a concentration 25 Ag/ml by 63%, 68% and 47%, respectively (Table 2). Higher scavenging activity of extracts could be due to the presence of other compounds acting as potent superoxide scavengers, for example isoorientin [16]. Tested materials showed low TEAC values and moderate activity of iron chelation. The most active Fe2+ chelator was ethanol extract, 70% in comparison with EDTA (Table 3). The highest content of phenolic groups, which show the ability to neutralize lipid radicals by donating hydrogen atoms, was found in the ethyl acetate extract (Table 3).There was a relationship between the content of phenolic groups in this extract and its antioxidant activity. Ethyl acetate extract was the most potent inhibitor of microsomal lipid peroxidation in all systems tested. It can be suggested that demonstrated previously hepatoprotective properties of A. vulgaris may be ascribed, at least in part, to the antioxidant activity of its constituents. Acknowledgements This work was supported by a research grant of the Polish State Committee for Scientific Research (No. 835/PD5/2001/20). References [1] PDR for herbal medicines, second ed. NJ7 Medicinal Economics Company; 2000. p. 211. [2] Adamska T, Myynarczyk W, Jodynis-Liebert J, Bylka W, Matyawska I. Phytother Res 2003;17:691. [3] Bylka W, Matyawska I. Acta Pol Pharm 1997;54:331.
480 [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16]
M. Murias et al. / Fitoterapia 76 (2005) 476–480 Bylka W, Matyawska I. Acta Pol Pharm 1997;54:334. Bylka W. Acta Pol Pharm 2001;54:237. Szaufer-Hajdrych M, Drost-Karbowska K, Kowalewski Z. Acta Pol Pharm 2002;59:57. Hansell R, Keller K, Rimpler H, Schneider G, Handbuch der Pharmaceutische Praxis, Bd. 4 Drogen A-D. Berlin: Springer Verlag, p. 312. Sanz MJ, Ferrandiz ML, Cejudo M, Terencio MC, Gil B, Bustos G, et al. Xenobiotica 1994;24:689. Haraguchi H, Ishikawa H, Sanchez Y, Ogura T, Kubo Y. Bioorg Med Chem 1997;5:865. Halliwell B, Gutteridge JMC, Aruoma OJ. Anal Biochem 1987;165:215. Ko FN, Liao CH, Kuo YH, Lin YL. Biochim Biophys Acta 1995;1258:145. Tyrakowska B, Soffers AE, Szymusiak H, Boeren S, Boersma MG, Leman˜ska K, et al. Free Radic Biol Med 1999;27:1427. Lopes GKB, Schulma HM, Hermes-Lima M. Biochim Biophys Acta 1999;1472:142. Sharma HM, Hanna AN, Kauffinan EM, Newman HAI. Free Radic Biol Med 1995;18:687. Sevanian A, Nordenbrand K, Kim E, Emster L, Hochstein P. Free Radic Biol Med 1990;8:145. Ko FN, Chu CC, Lin CN, Chang CC, Teng CM. Biochim Biophys Acta 1998;1389:90.