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Antioxidant and free radical scavenging activity of flavonol glycosides from different Aconitum species
Journal of Ethnopharmacology 86 (2003) 63–67
Antioxidant and free radical scavenging activity of flavonol glycosides from different Aconitum species Alessandra Braca a,∗ , Gelsomina Fico b , Ivano Morelli a , Francesco De Simone c , Franca Tomè b , Nunziatina De Tommasi c a
c
Dipartimento di Chimica Bioorganica e Biofarmacia, Università di Pisa, Via Bonanno, 33, 56126 Pisa, Italy b Dipartimento di Biologia, Università di Milano, Via Celoria, 26, 20133 Milano, Italy Dipartimento di Scienze Farmaceutiche, Università di Salerno, Via Ponte Don Melillo, 84084 Fisciano (SA), Italy Received 3 March 2002; received in revised form 7 January 2003; accepted 23 January 2003
Abstract Bioassay-guided fractionation by 1,1-diphenyl-2-dipicrylhydrazyl (DPPH) radical scavenging test of polar extracts of some Italian Aconitum species (A. napellus subsp. tauricum, A. napellus subsp. neomontanum, A. paniculatum, A. vulparia) led to the isolation of 13 flavonol glycosides: quercetin 3-O-(6-trans-caffeoyl)--glucopyranosyl-(1 → 2)--glucopyranoside-7-O-␣-rhamnopyranoside (1), kaempferol 3O-(6-trans-caffeoyl)--glucopyranosyl-(1 → 2)--glucopyranoside-7-O-␣-rhamnopyranoside (2), quercetin 3-O-(6-trans-p-coumaroyl)-glucopyranosyl-(1 → 2)--glucopyranoside-7-O-␣-rhamnopyranoside (3), kaempferol 3-O-(6-trans-p-coumaroyl)--glucopyranosyl-(1 → 2)--glucopyranoside-7-O-␣-rhamnopyranoside (4), quercetin 7-O-(6-trans-caffeoyl)--glucopyranosyl-(1 → 3)-␣-rhamnopyranoside-3O--glucopyranoside (5), kaempferol 7-O-(6-trans-caffeoyl)--glucopyranosyl-(1 → 3)-␣-rhamnopyranoside-3-O--glucopyranoside (6), kaempferol 7-O-(6-trans-p-coumaroyl)--glucopyranosyl-(1 → 3)-␣-rhamnopyranoside-3-O--glucopyranoside (7), kaempferol 3-O-(2 -acetyl)galactopyranoside (8), kaempferol 3-O--(2 -acetyl)galactopyranoside-7-O-␣-arabinopyranoside (9), quercetin 3-O--(2 -acetyl) galactopyranoside-7-O-␣-arabinopyranoside (10), quercetin 3,7-di-O-␣-rhamnopyranoside (11), kaempferol 3,7-di-O-␣-rhamnopyranoside (12) and quercetin 3-O--glucopyranoside-7-O-␣-rhamnopyranoside (13). Their antioxidant activity (AA) was determined by measuring free radical scavenging activity by DPPH test and the coupled oxidation of -carotene and linoleic acid assay. The results showed that 5 is the most active compound in the DPPH free-radical scavenging test (IC50 1.9 M) while in the coupled oxidation of -carotene and linoleic acid assay compound 1 has the highest inhibitory ratio after 1 h (58.9%). Some structure–activity relationships on the AA were obtained. © 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Antioxidant; Aconitum species; Flavonol glycosides; Free radical
1. Introduction Among naturally occurring phenolic compounds, flavonoids have gained a particular interest because of their broad pharmacological activity (Di Carlo et al., 1999). Putative therapeutic effects of many traditional medicines may be ascribed to the presence of flavonoids. Recently, the most important reported biological property of flavonoids is due to their antioxidant activity (AA) by scavenging oxygen radicals and inhibiting peroxidation (Hanasaki et al., 1994). The potential value of such antioxidants prompted investigators to study new flavonoids to improve the treatment of various diseases. Moreover, the list of newly discovered
∗ Corresponding
author. Fax: +39-050-43321. E-mail address: [email protected] (A. Braca).
flavonoids is constantly growing due to the enormous structural diversity associated with these compounds. Aconitum genus is mainly characterised by the presence of diterpene alkaloids and for this reason its roots have long been used as Oriental folk medicine against gout, neuralgia, articular rheumatism, and cardiac failure (Bisset, 1981; Schauenberg and Paris, 1977). In the last years, the study of Aconitum genus was directed to the other secondary metabolites such as flavonoids. The flavonoidic composition of several oriental species of Aconitum have been reported (Wang et al., 1994; Jeong et al., 1997; Lim et al., 1999), but there have not been any studies on the flavonoids content of European entities. In this context, we conducted a bioassay-guided fractionation, using DPPH antioxidant assay of polar extracts of some Italian Aconitum species: A. napellus subsp. tauricum, A. napellus subsp. neomontanum, A. paniculatum and A.
0378-8741/03/$ – see front matter © 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0378-8741(03)00043-6
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A. Braca et al. / Journal of Ethnopharmacology 86 (2003) 63–67
vulparia to obtain 13 pure flavonol glycosides that were successively tested by the radical scavenging activity on DPPH test (Cuendet et al., 1997) and the coupled oxidation of -carotene and linoleic acid (Igile et al., 1994; Pratt, 1992).
2. Materials and methods 2.1. Plant material, extraction and isolation of flavonol glycosides The flowers of Aconitum species were collected from different Italian Alps during the late summer of 1998: A. napellus subsp. tauricum in Passo di Maniva, Brescia, 2000 m above sea level, A. napellus subsp. neomontanum in Val di Peio, Trento, 1200 m above sea level, A. paniculatum and A. vulparia in Val di Rabbi, Trento, 1200 m above see level. The voucher specimens were deposited at the herbarium of Dipartimento di Biologia, Università di Milano (A. napellus subsp. tauricum no. An-206; A. napellus subsp. neomontanum no. An-201/2; A. paniculatum no. Ap-101; A. vulparia no. Al-101). The determination of the plants has been made according to Flora d’Italia (Pignatti, 1982). The dried powdered flowers of A. napellus subsp. tauricum, A. napellus subsp. neomontanum and A. paniculatum were extracted and fractionated as reported in our previous works (Fico et al., 2000, 2001a,b) to obtain, respectively, quercetin 3-O-(6-trans-caffeoyl)--glucopyranosyl-(1 → 2)-glucopyranoside-7-O-␣-rhamnopyranoside (1), kaempferol 3-O-(6-trans-caffeoyl)--glucopyranosyl-(1 → 2)-glucopyranoside-7-O-␣-rhamnopyranoside (2), quercetin 3O-(6-trans-p-coumaroyl)--glucopyranosyl-(1 → 2)--glucopyranoside-7-O-␣-rhamnopyranoside (3) and kaempferol 3-O-(6-trans-p-coumaroyl) -  - glucopyranosyl-(1 → 2)-glucopyranoside-7-O-␣-rhamnopyranoside (4) from A. napellus subsp. tauricum; quercetin 7-O-(6-trans-caffeoyl)-glucopyranosyl-(1 → 3)-␣-rhamnopyranoside - 3 - O--glucopyranoside (5), kaempferol 7-O-(6-trans-caffeoyl)-glucopyranosyl-(1 → 3) - ␣-rhamnopyranoside-3 - O - -glu-
copyranoside (6) and kaempferol 7-O-(6-trans-p-coumaroyl)--glucopyranosyl-(1 → 3)-␣-rhamnopyranoside-3-O-glucopyranoside (7) from A. napellus subsp. neomontanum; kaempferol 3-O--(2 -acetyl)galactopyranoside (8), kaempferol 3-O--(2 -acetyl)galactopyranoside-7-O-␣-arabinopyranoside (9) and quercetin 3-O--(2 -acetyl)gala-
Fig. 1. Bioassay-oriented fractionation of MeOH extract of A. napellus subsp. tauricum (IC50 is reported in parenthesis).
Fig. 4. Bioassay-oriented fractionation of MeOH extract of A. vulparia (IC50 is reported in parenthesis).
Fig. 2. Bioassay-oriented fractionation of MeOH extract of A. napellus subsp. neomontanum (IC50 is reported in parenthesis).
Fig. 3. Bioassay-oriented fractionation of MeOH extract of A. paniculatum (IC50 is reported in parenthesis).
A. Braca et al. / Journal of Ethnopharmacology 86 (2003) 63–67
ctopyranoside-7-O-␣-arabinopyranoside (10) from A. paniculatum (see Figs. 1–4). Powdered air-dried flowers of A. vulparia (42 g) were extracted at room temperature with n-hexane, CHCl3 , CHCl3 –MeOH (9:1) and MeOH, each solvent for three times to give 0.3, 0.6, 0.5 and 6 g of residues, respectively. The methanolic extract was chromatographed on Sephadex LH-20 (Pharmacia Fine Chemical Co., Ltd) using MeOH as eluent to give 30 fractions of 12 ml, combined together into 10 fractions according to TLC separation (silica gel plates in n-BuOH–CH3 COOH–H2 O (60:15:25)). Fractions 6–8 were submitted to RP-HPLC on a C18 -Bondapak column (30 cm × 7.8 mm, flow rate 2.5 ml/min) with MeOH–H2 O (45:55) to yield, respectively, quercetin 3,7-di-O-␣-rhamnopyranoside (11), kaempferol 3,7-di-O-␣-rhamnopyranoside (12) and quercetin 3-O--glucopyranoside-7-O-␣-rhamnopyranoside (13). The structural identification of all the isolated compounds was determined by mono and bidimensional NMR techniques as well as by MS analysis.
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30 min (UV, Perkin-Elmer-Lambda 11 spectrophotometer) and the percent inhibition activity was calculated (Cuendet et al., 1997). IC50 values denote the concentration of sample required to scavenge 50% DPPH free radicals. All tests were run in triplicate and averaged. 2.3. Autoxidation of β-carotene Oxidation of linoleic acid was measured by the method described by Pratt (1992). Quantities of linoleic acid (20 mg) and Tween 20 (200 mg) were placed in a flask, and a solution of 2 mg of -carotene in 10 ml of CHCl3 was added. After removal of CHCl3 , 50 ml of distilled water saturated with oxygen for 30 min were added. Aliquots (200 l) of each compound, dissolved in ethanol to a 15 g/ml solution, were added to each flask with shaking. Samples without test compounds were used as blanks, and a sample with 2,6-di-tert-butyl-4-methoxyphenol (BHT, Aldrich Chemical Co., Gillingham, Dorset, UK) was used as a control substance. Samples were subjected to oxidation by placing in an oven at 50 ◦ C for 3 h. The absorbance was read at 470 nm at regular intervals. The antioxidant activity was expressed as AA and calculated with the equation: A0 − A t AA = 100 1 − A00 − A0t where, A0 : absorbance at the beginning of the incubation with compound; At : absorbance at time t with compound;
1 2 3 4 5 6 7 8 9 10 11 12 13 Glc, glucose;
R1
R2
R3
Glc(1 → 2)Glc(1 → 6)caffeoyl Glc(1 → 2)Glc(1 → 6)caffeoyl Glc(1 → 2)Glc(1 → 6)t-p-coumaroyl Glc(1 → 2)Glc(1 → 6)t-p-coumaroyl Glucose Glucose Glucose Gal(1 → 2)acetyl Gal(1 → 2)acetyl Gal(1 → 2)acetyl Rhamnose Rhamnose Glucose Rha, rhamnose; Gal, galactose.
Rhamnose Rhamnose Rhamnose Rhamnose Rha(1 → 2)Glc(1 → 6)caffeoyl Rha(1 → 2)Glc(1 → 6)caffeoyl Rha(1 → 2)Glc(1 → 6)t-p-coumaroyl H Arabinose Arabinose Rhamnose Rhamnose Rhamnose
OH H OH H OH H H H H OH OH H OH
2.2. Scavenging activity to DPPH radical The potential AA of extracts, fractions and pure compounds was determined on the basis of the scavenging activity of the stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical. Aliquots of 30 l of a methanolic solution containing each pure compound were added to 3 ml of 0.004% MeOH solution of DPPH. Absorbance at 517 nm, against a blank of methanol without DPPH, was determined after
A00 : absorbance at beginning of the incubation without compound; A0t : absorbance at time t without compound. Each experiment was performed in three replicates.
3. Results and discussion All extracts from A. napellus subsp. tauricum, A. napellus subsp. neomontanum, A. paniculatum and A. vulparia were tested by DPPH radical scavenging activity but only the
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Table 1 Antioxidant activities of compounds 1–13 and standards in DPPH and autoxidation of -carotene assays Compound
1 2 3 4 5 6 7 8 9 10 11 12 13 Quercetinb Rutinb BHTb a b
DPPH (IC50 , M)a
9.0 4.5 9.8 19.0 1.9 2.6 20.0 25.5 82.0 41.5 3.0 65.2 3.5 2.5 3.9
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.04 0.05 0.05 0.70 0.01 0.04 0.80 0.70 0.90 1.90 0.04 0.07 0.03 0.01 0.03
DPPH (IC50 , g/ml) 8.4 4.1 9.0 17.1 1.8 2.4 18.0 12.5 51.0 26.5 1.8 37.7 2.1 8.5 2.4 –
AA (min) 60
120
58.9 36.1 25.5 7.4 46.3 48.8 7.5 31.5 45.4 43.0 41.3 18.2 16.0 – – 63.9
71.5 22.7 12.8 4.6 40.1 39.6 0.9 33.2 38.1 35.6 33.0 9.0 17.4 – – 62.5
The values are mean ± S.D. (n = 3). Used as controls; BHT, 2,6-di-tert-butyl-4-methoxyphenol.
polar residues showed moderate actions. Bioassay-guided fractionation of the methanolic extract of A. napellus subsp. tauricum led to the isolation of pure compounds 1–4. The methanolic extract was purified by Sephadex LH-20 to obtain 16 fractions. Fractions 14 and 16 showed the highest activity in the DPPH assay with respect to some other non-flavonoidic fractions with IC50 2.0 g/ml and IC50 2.6 g/ml, respectively; nevertheless fraction 7 was active but at higher concentration than 14 and 16 (see Fig. 1). From the methanolic extracts of A. napellus subsp. neomontanum, compound 5–7 were isolated in the same way. Fraction 12 exhibited the strongest activity in the DPPH test (IC50 4.2 g/ml); this fraction, by separation on RP-HPLC, yielded the pure flavonol glycosides (see Fig. 2). The methanolic extract of A. paniculatum was chromatographed on Sephadex LH-20 to obtain 13 fractions: fraction 6 appeared to possess greater AA than the other fractions (IC50 9.5 g/ml). Fraction 6 was successively purified by RP-HPLC to yield pure compounds 8–10 (see Fig. 3). Finally, from the methanolic extract of A. vulparia were obtained flavonol glycosides 11–13 (see Fig. 4). The pure flavonol glycosides 1–13 were successively tested by the radical scavenging activity on DPPH test and the coupled oxidation of -carotene and linoleic acid. The DPPH test is a non-enzymatic method currently used to provide basic information on the reactivity of compounds to scavenge free radicals. Quercetin and rutin were used as reference compounds. As shown in Table 1, the most active compounds with quercetin as aglycon were 5, 11, and 13. The quercetin derivative 5 showed the highest activity (IC50 1.9 M), being a more potent antioxidant than quercetin, having in its structure the presence of two o-dihydroxyl groups (one in the ring B of the aglycon and the other in
the aromatic acyl group) important for radical scavenging activity (Rice-Evans et al., 1996). The difference between compounds 5 and 1 was that the last one had the caffeoylglycosidic substituent linked at C-3 instead of C-7, so that the minor activity of 1 (IC50 9.0 M) was probably due to the steric hindrance between the C-3 substituent and the ring B of the flavonol. Compound 3 (IC50 9.8 M) was slightly less active than 1 because of the presence of the p-coumaroyl group instead of the caffeoyl moiety linked at C-3, being the p-coumaroyl group less efficient for the radical scavenging activity (Rice-Evans et al., 1996). Compound 10 was poorly active for the presence of an acetyl group instead of an aromatic one linked at C-3. Among the kaempferol derivatives, the most active was compound 6 (IC50 2.6 M) having in its skeleton the presence of a caffeoyl ester. Similarly to the quercetin derivatives, the shift of the glycosidic ester moiety from the C-7 position to the C-3 reduced the activity and the compounds with the p-coumaroyl moiety (4 and 7) were less active than the one with the caffeoyl group (2). All other kaempferol derivatives with an acetyl group instead of an aromatic ester did not reduce significantly the free radical. The structure–activity relationship conclusions for the radical scavenging activity of compounds 1–13 agreed with those reported in the literature: the o-dihydroxy structure conferred higher stability to the radical form and participated in the electron delocalisation (Pietta, 2000; Rice-Evans et al., 1997; Rice-Evans et al., 1996). Thus, the caffeoyl derivatives apparently had a higher radical scavenging ability than the p-coumaroyl ester compounds consistent with the chemical criteria applied to diphenolics. From this conclusion, was clear that the most active compounds had in their structure an ester of caffeic acid because the caffeoyl moiety enhanced the activity. Our relationship confirmed that O-glycosylation at C-3 had a slight negative influence on the activity, also showed by the reference compounds quercetin and rutin; besides, the presence of a big substituent at C-3 reduced the activity probably for steric hindrance. The effect of antioxidant pure compounds 1–13 on the coupled oxidation of -carotene and linoleic acid was examined. Membrane lipids are rich of unsaturated fatty acids that are most susceptible to oxidative processes. Especially, linoleic acid and arachidonic acid are the target of lipid peroxidation. It is generally thought that the inhibition of lipid peroxidation by antioxidants may be due to their free radical scavenging activities. Superoxide indirectly initiates lipid peroxidation because superoxide anion acts as a precursor of singlet oxygen and hydroxyl radical (Gao et al., 2000). Hydroxyl radical eliminates hydrogen atoms from the membrane lipid which results in lipid peroxidation. The inhibition of the above flavonol glycosides against the coupled oxidation of -carotene and linoleic acid was, therefore, tested using the method described by Pratt (1992). The values of AA measured at t = 60 and 120 min, employing bleaching of -carotene as a model system, are reported in Table 1. BHT, an antioxidant widely used to protect food,
A. Braca et al. / Journal of Ethnopharmacology 86 (2003) 63–67
was tested as reference compound. Compounds 1, 5 and 6 showed the highest activity having a caffeoyl moiety in their structures. Particularly, 1 had a similar action in respect to the reference compound BHT, while 5 and 6 were less active than BHT, but with an activity that remained high after 2 h. Kaempferol derivatives without caffeoyl group in the molecule were practically inactive. The conclusions drawn for the radical scavenging DPPH activity seemed to be the same of this last test: o-dihydroxylation contibuted markedly to the AA of flavonoids, so that the derivatives with caffeoyl group possessed the more potent action. Flavonoids have been shown to scavenge various reactive oxygen species and have been implicated as inhibitors of lipid peroxidation (Mora et al., 1990). Herein, we demonstrate the ability of some flavonol glycosides obtained from Aconitum species to scavenge peroxyl radicals. Therefore, further studies of the mechanistic properties of flavonoids are of potential importance in understanding and preventing reactive oxygen species-linked diseases. The scavenging of peroxyl radicals is a key step in the prevention of lipid peroxidation by breaking the chain of propagation of free-radical reactions. Active flavonoids obtained from dry extracts of Aconitum species flowers may be an alternative to the more toxic synthetic antioxidants as additives in pharmaceutical and cosmetic preparations and/or for the treatment of pathological conditions derived from lipid peroxidation. The results of the present work also suggested that the activity against gout, neuralgia, rheumatism and cardiac failure of Aconitum species could be explained, at least in part, by presence of antioxidant flavonol glycosides.
References Bisset, N.G., 1981. Arrow poisons in China. Part II. Aconitum–botany, chemistry, and pharmacology. Journal of Ethnopharmacology 4, 247– 336. Cuendet, M., Hostettmann, K., Potterat, O., 1997. Iridoid glucosides with free radical scavenging properties from Fagraea blumei. Helvetica Chimica Acta 80, 1144–1152.
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Di Carlo, G., Mascolo, N., Izzo, A.A., Capasso, F., 1999. Flavonoids: old and new aspects of a class of natural therapeutic drugs. Life Science 65, 337–353. Fico, G., Braca, A., Bilia, A.R., Tomè, F., Morelli, I., 2000. New flavonol glycosides from the flowers of Aconitum paniculatum. Journal of Natural Products 63, 1563–1565. Fico, G., Braca, A., Bilia, A.R., Tomè, F., Morelli, I., 2001a. New flavonol glycosides from the flowers of Aconitum napellus ssp. tauricum. Planta Medica 67, 287–290. Fico, G., Braca, A., De Tommasi, N., Tomè, F., Morelli, I., 2001b. Flavonoids from Aconitum napellus subsp. neomontanum. Phytochemistry 57, 543–546. Gao, J., Igarashi, K., Nukina, M., 2000. Three new phenylethanoid glycosides from Caryopteris incana and their antioxidative activity. Chemical and Pharmaceutical Bulletin 48, 1075–1078. Hanasaki, Y., Ogawar, S., Fukui, S., 1994. The correlation between active oxygens scavenging and antioxidative effects of flavonoids. Free Radical Biology and Medicine 16, 845–850. Igile, G.O., Oleszek, W., Jurzysta, M., Burda, S., Fafunso, M., Fasanmade, A.A., 1994. Flavonoids from Vernonia amygdalina and their antioxidant activities. Journal of Agriculture and Food Chemistry 42, 2445–2448. Jeong, H.J., Whang, W.K., Kim, I.H., 1997. New flavonoids from the aerial parts of Aconitum chiisanense. Planta Medica 63, 329–334. Lim, C.E., Park, J.H., Park, C.W., 1999. Flavonoid variation of the Aconitum jaluense complex (Ranunculaceae) in Korea. Plant System Evolution 218, 125–131. Mora, A., Paya, M., Rios, J.L., Alcaraz, M.J., 1990. Structure–activity relationships of polymethoxyflavones and other flavonoids as inhibitors of non-enzymic lipid peroxidation. Biochemical Pharmacology 40, 793–797. Pietta, P.G., 2000. Flavonoids as antioxidants. Journal of Natural Products 63, 1035–1042. Pignatti, S., 1982. Flora d’Italia, vol. I. Edagricole, Bologna, pp. 285–288. Pratt, D.E., 1992. Natural antioxidants from plant material. In: Huang, M.T., Lee, C.Y. (Eds.), Phenolic Compounds in Food and Their Effects on Health. II. Antioxidants and Cancer Prevention. ACS, Washington, DC, pp. 54–71. Rice-Evans, C., Miller, N.J., Paganga, G., 1996. Structure–antioxidant activity relationship of flavonoids and phenolic acid. Free Radical Biology and Medicine 20, 933–956. Rice-Evans, C., Miller, N.J., Paganga, G., 1997. Antioxidant properties of phenolic compounds. Trends in Plant Science 2, 152–159. Schauenberg, P., Paris, F., 1977. Guide to Medicinal Plants. Lutterworth Press, Guilford and London. Wang, W.K., Oh, I.S., Lee, M.T., Kim, I.H., 1994. Flavonoids from the aerial parts of Aconitum jaluense for album. Saengyak Hakhoechi 25, 336–341.
Antioxidant and free radical scavenging activity of flavonol glycosides from different Aconitum species Alessandra Braca a,∗ , Gelsomina Fico b , Ivano Morelli a , Francesco De Simone c , Franca Tomè b , Nunziatina De Tommasi c a
c
Dipartimento di Chimica Bioorganica e Biofarmacia, Università di Pisa, Via Bonanno, 33, 56126 Pisa, Italy b Dipartimento di Biologia, Università di Milano, Via Celoria, 26, 20133 Milano, Italy Dipartimento di Scienze Farmaceutiche, Università di Salerno, Via Ponte Don Melillo, 84084 Fisciano (SA), Italy Received 3 March 2002; received in revised form 7 January 2003; accepted 23 January 2003
Abstract Bioassay-guided fractionation by 1,1-diphenyl-2-dipicrylhydrazyl (DPPH) radical scavenging test of polar extracts of some Italian Aconitum species (A. napellus subsp. tauricum, A. napellus subsp. neomontanum, A. paniculatum, A. vulparia) led to the isolation of 13 flavonol glycosides: quercetin 3-O-(6-trans-caffeoyl)--glucopyranosyl-(1 → 2)--glucopyranoside-7-O-␣-rhamnopyranoside (1), kaempferol 3O-(6-trans-caffeoyl)--glucopyranosyl-(1 → 2)--glucopyranoside-7-O-␣-rhamnopyranoside (2), quercetin 3-O-(6-trans-p-coumaroyl)-glucopyranosyl-(1 → 2)--glucopyranoside-7-O-␣-rhamnopyranoside (3), kaempferol 3-O-(6-trans-p-coumaroyl)--glucopyranosyl-(1 → 2)--glucopyranoside-7-O-␣-rhamnopyranoside (4), quercetin 7-O-(6-trans-caffeoyl)--glucopyranosyl-(1 → 3)-␣-rhamnopyranoside-3O--glucopyranoside (5), kaempferol 7-O-(6-trans-caffeoyl)--glucopyranosyl-(1 → 3)-␣-rhamnopyranoside-3-O--glucopyranoside (6), kaempferol 7-O-(6-trans-p-coumaroyl)--glucopyranosyl-(1 → 3)-␣-rhamnopyranoside-3-O--glucopyranoside (7), kaempferol 3-O-(2 -acetyl)galactopyranoside (8), kaempferol 3-O--(2 -acetyl)galactopyranoside-7-O-␣-arabinopyranoside (9), quercetin 3-O--(2 -acetyl) galactopyranoside-7-O-␣-arabinopyranoside (10), quercetin 3,7-di-O-␣-rhamnopyranoside (11), kaempferol 3,7-di-O-␣-rhamnopyranoside (12) and quercetin 3-O--glucopyranoside-7-O-␣-rhamnopyranoside (13). Their antioxidant activity (AA) was determined by measuring free radical scavenging activity by DPPH test and the coupled oxidation of -carotene and linoleic acid assay. The results showed that 5 is the most active compound in the DPPH free-radical scavenging test (IC50 1.9 M) while in the coupled oxidation of -carotene and linoleic acid assay compound 1 has the highest inhibitory ratio after 1 h (58.9%). Some structure–activity relationships on the AA were obtained. © 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Antioxidant; Aconitum species; Flavonol glycosides; Free radical
1. Introduction Among naturally occurring phenolic compounds, flavonoids have gained a particular interest because of their broad pharmacological activity (Di Carlo et al., 1999). Putative therapeutic effects of many traditional medicines may be ascribed to the presence of flavonoids. Recently, the most important reported biological property of flavonoids is due to their antioxidant activity (AA) by scavenging oxygen radicals and inhibiting peroxidation (Hanasaki et al., 1994). The potential value of such antioxidants prompted investigators to study new flavonoids to improve the treatment of various diseases. Moreover, the list of newly discovered
∗ Corresponding
author. Fax: +39-050-43321. E-mail address: [email protected] (A. Braca).
flavonoids is constantly growing due to the enormous structural diversity associated with these compounds. Aconitum genus is mainly characterised by the presence of diterpene alkaloids and for this reason its roots have long been used as Oriental folk medicine against gout, neuralgia, articular rheumatism, and cardiac failure (Bisset, 1981; Schauenberg and Paris, 1977). In the last years, the study of Aconitum genus was directed to the other secondary metabolites such as flavonoids. The flavonoidic composition of several oriental species of Aconitum have been reported (Wang et al., 1994; Jeong et al., 1997; Lim et al., 1999), but there have not been any studies on the flavonoids content of European entities. In this context, we conducted a bioassay-guided fractionation, using DPPH antioxidant assay of polar extracts of some Italian Aconitum species: A. napellus subsp. tauricum, A. napellus subsp. neomontanum, A. paniculatum and A.
0378-8741/03/$ – see front matter © 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0378-8741(03)00043-6
64
A. Braca et al. / Journal of Ethnopharmacology 86 (2003) 63–67
vulparia to obtain 13 pure flavonol glycosides that were successively tested by the radical scavenging activity on DPPH test (Cuendet et al., 1997) and the coupled oxidation of -carotene and linoleic acid (Igile et al., 1994; Pratt, 1992).
2. Materials and methods 2.1. Plant material, extraction and isolation of flavonol glycosides The flowers of Aconitum species were collected from different Italian Alps during the late summer of 1998: A. napellus subsp. tauricum in Passo di Maniva, Brescia, 2000 m above sea level, A. napellus subsp. neomontanum in Val di Peio, Trento, 1200 m above sea level, A. paniculatum and A. vulparia in Val di Rabbi, Trento, 1200 m above see level. The voucher specimens were deposited at the herbarium of Dipartimento di Biologia, Università di Milano (A. napellus subsp. tauricum no. An-206; A. napellus subsp. neomontanum no. An-201/2; A. paniculatum no. Ap-101; A. vulparia no. Al-101). The determination of the plants has been made according to Flora d’Italia (Pignatti, 1982). The dried powdered flowers of A. napellus subsp. tauricum, A. napellus subsp. neomontanum and A. paniculatum were extracted and fractionated as reported in our previous works (Fico et al., 2000, 2001a,b) to obtain, respectively, quercetin 3-O-(6-trans-caffeoyl)--glucopyranosyl-(1 → 2)-glucopyranoside-7-O-␣-rhamnopyranoside (1), kaempferol 3-O-(6-trans-caffeoyl)--glucopyranosyl-(1 → 2)-glucopyranoside-7-O-␣-rhamnopyranoside (2), quercetin 3O-(6-trans-p-coumaroyl)--glucopyranosyl-(1 → 2)--glucopyranoside-7-O-␣-rhamnopyranoside (3) and kaempferol 3-O-(6-trans-p-coumaroyl) -  - glucopyranosyl-(1 → 2)-glucopyranoside-7-O-␣-rhamnopyranoside (4) from A. napellus subsp. tauricum; quercetin 7-O-(6-trans-caffeoyl)-glucopyranosyl-(1 → 3)-␣-rhamnopyranoside - 3 - O--glucopyranoside (5), kaempferol 7-O-(6-trans-caffeoyl)-glucopyranosyl-(1 → 3) - ␣-rhamnopyranoside-3 - O - -glu-
copyranoside (6) and kaempferol 7-O-(6-trans-p-coumaroyl)--glucopyranosyl-(1 → 3)-␣-rhamnopyranoside-3-O-glucopyranoside (7) from A. napellus subsp. neomontanum; kaempferol 3-O--(2 -acetyl)galactopyranoside (8), kaempferol 3-O--(2 -acetyl)galactopyranoside-7-O-␣-arabinopyranoside (9) and quercetin 3-O--(2 -acetyl)gala-
Fig. 1. Bioassay-oriented fractionation of MeOH extract of A. napellus subsp. tauricum (IC50 is reported in parenthesis).
Fig. 4. Bioassay-oriented fractionation of MeOH extract of A. vulparia (IC50 is reported in parenthesis).
Fig. 2. Bioassay-oriented fractionation of MeOH extract of A. napellus subsp. neomontanum (IC50 is reported in parenthesis).
Fig. 3. Bioassay-oriented fractionation of MeOH extract of A. paniculatum (IC50 is reported in parenthesis).
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ctopyranoside-7-O-␣-arabinopyranoside (10) from A. paniculatum (see Figs. 1–4). Powdered air-dried flowers of A. vulparia (42 g) were extracted at room temperature with n-hexane, CHCl3 , CHCl3 –MeOH (9:1) and MeOH, each solvent for three times to give 0.3, 0.6, 0.5 and 6 g of residues, respectively. The methanolic extract was chromatographed on Sephadex LH-20 (Pharmacia Fine Chemical Co., Ltd) using MeOH as eluent to give 30 fractions of 12 ml, combined together into 10 fractions according to TLC separation (silica gel plates in n-BuOH–CH3 COOH–H2 O (60:15:25)). Fractions 6–8 were submitted to RP-HPLC on a C18 -Bondapak column (30 cm × 7.8 mm, flow rate 2.5 ml/min) with MeOH–H2 O (45:55) to yield, respectively, quercetin 3,7-di-O-␣-rhamnopyranoside (11), kaempferol 3,7-di-O-␣-rhamnopyranoside (12) and quercetin 3-O--glucopyranoside-7-O-␣-rhamnopyranoside (13). The structural identification of all the isolated compounds was determined by mono and bidimensional NMR techniques as well as by MS analysis.
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30 min (UV, Perkin-Elmer-Lambda 11 spectrophotometer) and the percent inhibition activity was calculated (Cuendet et al., 1997). IC50 values denote the concentration of sample required to scavenge 50% DPPH free radicals. All tests were run in triplicate and averaged. 2.3. Autoxidation of β-carotene Oxidation of linoleic acid was measured by the method described by Pratt (1992). Quantities of linoleic acid (20 mg) and Tween 20 (200 mg) were placed in a flask, and a solution of 2 mg of -carotene in 10 ml of CHCl3 was added. After removal of CHCl3 , 50 ml of distilled water saturated with oxygen for 30 min were added. Aliquots (200 l) of each compound, dissolved in ethanol to a 15 g/ml solution, were added to each flask with shaking. Samples without test compounds were used as blanks, and a sample with 2,6-di-tert-butyl-4-methoxyphenol (BHT, Aldrich Chemical Co., Gillingham, Dorset, UK) was used as a control substance. Samples were subjected to oxidation by placing in an oven at 50 ◦ C for 3 h. The absorbance was read at 470 nm at regular intervals. The antioxidant activity was expressed as AA and calculated with the equation: A0 − A t AA = 100 1 − A00 − A0t where, A0 : absorbance at the beginning of the incubation with compound; At : absorbance at time t with compound;
1 2 3 4 5 6 7 8 9 10 11 12 13 Glc, glucose;
R1
R2
R3
Glc(1 → 2)Glc(1 → 6)caffeoyl Glc(1 → 2)Glc(1 → 6)caffeoyl Glc(1 → 2)Glc(1 → 6)t-p-coumaroyl Glc(1 → 2)Glc(1 → 6)t-p-coumaroyl Glucose Glucose Glucose Gal(1 → 2)acetyl Gal(1 → 2)acetyl Gal(1 → 2)acetyl Rhamnose Rhamnose Glucose Rha, rhamnose; Gal, galactose.
Rhamnose Rhamnose Rhamnose Rhamnose Rha(1 → 2)Glc(1 → 6)caffeoyl Rha(1 → 2)Glc(1 → 6)caffeoyl Rha(1 → 2)Glc(1 → 6)t-p-coumaroyl H Arabinose Arabinose Rhamnose Rhamnose Rhamnose
OH H OH H OH H H H H OH OH H OH
2.2. Scavenging activity to DPPH radical The potential AA of extracts, fractions and pure compounds was determined on the basis of the scavenging activity of the stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical. Aliquots of 30 l of a methanolic solution containing each pure compound were added to 3 ml of 0.004% MeOH solution of DPPH. Absorbance at 517 nm, against a blank of methanol without DPPH, was determined after
A00 : absorbance at beginning of the incubation without compound; A0t : absorbance at time t without compound. Each experiment was performed in three replicates.
3. Results and discussion All extracts from A. napellus subsp. tauricum, A. napellus subsp. neomontanum, A. paniculatum and A. vulparia were tested by DPPH radical scavenging activity but only the
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Table 1 Antioxidant activities of compounds 1–13 and standards in DPPH and autoxidation of -carotene assays Compound
1 2 3 4 5 6 7 8 9 10 11 12 13 Quercetinb Rutinb BHTb a b
DPPH (IC50 , M)a
9.0 4.5 9.8 19.0 1.9 2.6 20.0 25.5 82.0 41.5 3.0 65.2 3.5 2.5 3.9
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.04 0.05 0.05 0.70 0.01 0.04 0.80 0.70 0.90 1.90 0.04 0.07 0.03 0.01 0.03
DPPH (IC50 , g/ml) 8.4 4.1 9.0 17.1 1.8 2.4 18.0 12.5 51.0 26.5 1.8 37.7 2.1 8.5 2.4 –
AA (min) 60
120
58.9 36.1 25.5 7.4 46.3 48.8 7.5 31.5 45.4 43.0 41.3 18.2 16.0 – – 63.9
71.5 22.7 12.8 4.6 40.1 39.6 0.9 33.2 38.1 35.6 33.0 9.0 17.4 – – 62.5
The values are mean ± S.D. (n = 3). Used as controls; BHT, 2,6-di-tert-butyl-4-methoxyphenol.
polar residues showed moderate actions. Bioassay-guided fractionation of the methanolic extract of A. napellus subsp. tauricum led to the isolation of pure compounds 1–4. The methanolic extract was purified by Sephadex LH-20 to obtain 16 fractions. Fractions 14 and 16 showed the highest activity in the DPPH assay with respect to some other non-flavonoidic fractions with IC50 2.0 g/ml and IC50 2.6 g/ml, respectively; nevertheless fraction 7 was active but at higher concentration than 14 and 16 (see Fig. 1). From the methanolic extracts of A. napellus subsp. neomontanum, compound 5–7 were isolated in the same way. Fraction 12 exhibited the strongest activity in the DPPH test (IC50 4.2 g/ml); this fraction, by separation on RP-HPLC, yielded the pure flavonol glycosides (see Fig. 2). The methanolic extract of A. paniculatum was chromatographed on Sephadex LH-20 to obtain 13 fractions: fraction 6 appeared to possess greater AA than the other fractions (IC50 9.5 g/ml). Fraction 6 was successively purified by RP-HPLC to yield pure compounds 8–10 (see Fig. 3). Finally, from the methanolic extract of A. vulparia were obtained flavonol glycosides 11–13 (see Fig. 4). The pure flavonol glycosides 1–13 were successively tested by the radical scavenging activity on DPPH test and the coupled oxidation of -carotene and linoleic acid. The DPPH test is a non-enzymatic method currently used to provide basic information on the reactivity of compounds to scavenge free radicals. Quercetin and rutin were used as reference compounds. As shown in Table 1, the most active compounds with quercetin as aglycon were 5, 11, and 13. The quercetin derivative 5 showed the highest activity (IC50 1.9 M), being a more potent antioxidant than quercetin, having in its structure the presence of two o-dihydroxyl groups (one in the ring B of the aglycon and the other in
the aromatic acyl group) important for radical scavenging activity (Rice-Evans et al., 1996). The difference between compounds 5 and 1 was that the last one had the caffeoylglycosidic substituent linked at C-3 instead of C-7, so that the minor activity of 1 (IC50 9.0 M) was probably due to the steric hindrance between the C-3 substituent and the ring B of the flavonol. Compound 3 (IC50 9.8 M) was slightly less active than 1 because of the presence of the p-coumaroyl group instead of the caffeoyl moiety linked at C-3, being the p-coumaroyl group less efficient for the radical scavenging activity (Rice-Evans et al., 1996). Compound 10 was poorly active for the presence of an acetyl group instead of an aromatic one linked at C-3. Among the kaempferol derivatives, the most active was compound 6 (IC50 2.6 M) having in its skeleton the presence of a caffeoyl ester. Similarly to the quercetin derivatives, the shift of the glycosidic ester moiety from the C-7 position to the C-3 reduced the activity and the compounds with the p-coumaroyl moiety (4 and 7) were less active than the one with the caffeoyl group (2). All other kaempferol derivatives with an acetyl group instead of an aromatic ester did not reduce significantly the free radical. The structure–activity relationship conclusions for the radical scavenging activity of compounds 1–13 agreed with those reported in the literature: the o-dihydroxy structure conferred higher stability to the radical form and participated in the electron delocalisation (Pietta, 2000; Rice-Evans et al., 1997; Rice-Evans et al., 1996). Thus, the caffeoyl derivatives apparently had a higher radical scavenging ability than the p-coumaroyl ester compounds consistent with the chemical criteria applied to diphenolics. From this conclusion, was clear that the most active compounds had in their structure an ester of caffeic acid because the caffeoyl moiety enhanced the activity. Our relationship confirmed that O-glycosylation at C-3 had a slight negative influence on the activity, also showed by the reference compounds quercetin and rutin; besides, the presence of a big substituent at C-3 reduced the activity probably for steric hindrance. The effect of antioxidant pure compounds 1–13 on the coupled oxidation of -carotene and linoleic acid was examined. Membrane lipids are rich of unsaturated fatty acids that are most susceptible to oxidative processes. Especially, linoleic acid and arachidonic acid are the target of lipid peroxidation. It is generally thought that the inhibition of lipid peroxidation by antioxidants may be due to their free radical scavenging activities. Superoxide indirectly initiates lipid peroxidation because superoxide anion acts as a precursor of singlet oxygen and hydroxyl radical (Gao et al., 2000). Hydroxyl radical eliminates hydrogen atoms from the membrane lipid which results in lipid peroxidation. The inhibition of the above flavonol glycosides against the coupled oxidation of -carotene and linoleic acid was, therefore, tested using the method described by Pratt (1992). The values of AA measured at t = 60 and 120 min, employing bleaching of -carotene as a model system, are reported in Table 1. BHT, an antioxidant widely used to protect food,
A. Braca et al. / Journal of Ethnopharmacology 86 (2003) 63–67
was tested as reference compound. Compounds 1, 5 and 6 showed the highest activity having a caffeoyl moiety in their structures. Particularly, 1 had a similar action in respect to the reference compound BHT, while 5 and 6 were less active than BHT, but with an activity that remained high after 2 h. Kaempferol derivatives without caffeoyl group in the molecule were practically inactive. The conclusions drawn for the radical scavenging DPPH activity seemed to be the same of this last test: o-dihydroxylation contibuted markedly to the AA of flavonoids, so that the derivatives with caffeoyl group possessed the more potent action. Flavonoids have been shown to scavenge various reactive oxygen species and have been implicated as inhibitors of lipid peroxidation (Mora et al., 1990). Herein, we demonstrate the ability of some flavonol glycosides obtained from Aconitum species to scavenge peroxyl radicals. Therefore, further studies of the mechanistic properties of flavonoids are of potential importance in understanding and preventing reactive oxygen species-linked diseases. The scavenging of peroxyl radicals is a key step in the prevention of lipid peroxidation by breaking the chain of propagation of free-radical reactions. Active flavonoids obtained from dry extracts of Aconitum species flowers may be an alternative to the more toxic synthetic antioxidants as additives in pharmaceutical and cosmetic preparations and/or for the treatment of pathological conditions derived from lipid peroxidation. The results of the present work also suggested that the activity against gout, neuralgia, rheumatism and cardiac failure of Aconitum species could be explained, at least in part, by presence of antioxidant flavonol glycosides.
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