Biochemical Systematics and Ecology 32 (2004) 239–243 www.elsevier.com/locate/biochemsyseco
Flavonoids from Lychnophora passerina (Asteraceae): potential antioxidants and UV-protectants Patricia Chicaro a, Ernani Pinto b, Pio Colepicolo c, Joa˜o Luis Callegari Lopes d, Norberto Peporine Lopes d, a
b
Departamento de Quı´mica, Faculdade de Filosofia Cieˆncias e Letras de Ribeira˜o Preto, Universidade de Sa˜o Paulo, CEP 14.040-903, Ribeira˜o Preto, SP, Brazil Departamento de Ana´lises Clı´nicas e Toxicolo´gicas, Faculdade de Cieˆncias Farmaceˆuticas, Universidade de Sa˜o Paulo, Av. Prof. Lineu Prestes 580, 05508-900, Sa˜o Paulo, Brazil c Departamento de Bioquı´mica, Instituto de Quı´mica, Universidade de Sa˜o Paulo, CP 26077, 05599-970, Sa˜o Paulo, Brazil d Departamento de Fisica e Quı´mica, Faculdade de Cieˆncias Farmaceˆuticas de Ribeira˜o Preto, Universidade de Sa˜o Paulo, Av. do Cafe´ s/n, 14040-903, Ribeira˜o Preto, SP, Brazil Received 14 March 2003; accepted 12 August 2003
Abstract Five flavonoids, kaempferol, apigenin, luteolin, quercetin and tiliroside and two sesquiterpenes lactones, were isolated from Lychnophora passerina. Tiliroside, found in the genus Lychnophora, can be used for chemical standardization and its presence in high amounts, relative to other flavonoids, may suggest that it is an antioxidant as well as being a UV-protectant. # 2003 Elsevier Ltd. All rights reserved. Keywords: Antioxidant activity; Ecological function; Flavonoids; UV-block
1. Subject and source Lychnophora passerina (Asteraceae, Vernonieae) is one of the 34 species of this Brazilian endemic genus (Robinson, 1999). This species is typical of the cerrado, an arid region located in the central part of Brazil, 1000 m above sea level, which is
Corresponding author. Tel.: +55-16-6024168; fax: +55-16-6332960. E-mail address:
[email protected] (N.P. Lopes).
0305-1978/$ - see front matter # 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2003.08.003
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covered with bushes (Leita˜o Filho and Semir, 1979). L. passerina was collected in Serra do Cipo´, Brazil, April 1998, and was identified by Dr. Joa˜o Semir. A voucher specimen has been deposited in the herbarium of Botany (Lopes-138), University of Campinas, Brazil. 2. Previous work The genus Lychnophora is endemic to the Brazilian ‘‘campus rupestris’’. This vegetation is a special kind of cerrado that occurs in high altitudes, where hydroalcoholic extracts of many species are popularly used as anti-inflammatory and analgesic agents (Lopes, 2001). Sesquiterpenoids, diterpenoids, triterpenoids, steroids and flavonoids are the major compounds that occur in the Lychnophora genus. Sesquiterpene lactones are characteristic sesquiterpenoids from Vernonieae and belong to the goyazensolide, eremantholide, guaianolide and eudesmanolide types (Borella et al., 1992). Polyacetylenes and lignans have also been reported for some species (Borsato et al., 2000). The chemotaxonomic importance of furanoheliangolide sesquiterpene lactones is known (Meragelman et al., 1998; Passreiter et al., 1999), and recently the compounds centratherin and goyazensolide showed high anti-inflammatory activity in vitro (Ru¨ngler et al., 1999), corroborating the popular medicinal uses of the extracts. Previous investigations of the aerial parts of L. passerina resulted in the isolation of the terpenoids, such as, taraxasterol, a-amyrin, lupeol, and their respective acetates, a-humulene, costunolide, dihydrocostuslactone, eremantine, 11b,13-dihydroeremantine, goyazensolide and the steroid stigmasterol (Bohlmann et al., 1981; Oliveira et al., 1996). 3. Present study In this paper, we describe the isolation and chemical characterization of flavonoids from L. passerina as well as of the sesquiterpene lactones often found in this genus. In addition, the antioxidant activity of the flavonoids is investigated. 3.1. Extraction, isolation and identification Leaves (100 g) were dried, pulverized and extracted with ethanol. The extract (9.5 g) suspended in methanol–water (7:3) was filtered on Celite. The filtrate was extracted with dichloromethane (3 50 ml), and after solvent elimination produced 6.0 g of the dichloromethane fraction. The methanolic fraction was concentrated under vacuum to yield 2.6 g. The apolar extract (6.0 g) was chromatographed over silica gel G-60 (classical column) using a gradient starting with hexane/ethyl acetate 8:2 with the polarity increased gradually with ethyl acetate. The fractions acquired were fractionated by preparative TLC (hexane/ ethyl acetate, 7:3), yielding 15-deoxygoyazensolide (14.9 mg) and goyazensolide (31.5 mg).
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The absence of flavonoids in the previous reports is unexpected in view of their important ecological function as antioxidants and in UV screening pigment formation (Rice Evans et al., 1996; Weisshaar and Jenkins, 1998; Rozema et al., 2002). In natural conditions, L. passerina is exposed to high light intensity and grows in a dry climate. The methanolic extract of the leaves (2.6 g) was resuspended with MeOH and fractionated by Sephadex LH-20 (MeOH 100%), producing fractions identified as luteolin (3.0 mg), kaempferol (4.9 mg), apigenin (1.9 mg), quercetin (3.0 mg) and tiliroside (140 mg). Spectral data (UV, 1H and 13C NMR) of all flavonoids and sesquiterpene lactones isolated are identical to those previously described (Vichnewski et al., 1976, 1989; Markham et al., 1978; Kaouadji, 1990; Tomczyk et al., 2002). 3.2. Flavonoid antioxidant activity assay The antioxidant activity was determined following procedures previously described by Pryor et al. (1993) and Foti et al. (1996). The method consists of measuring the ability of the flavonoid to protect linolenic acid peroxidation in micelles of sodium dodecyl sulfate in phosphate buffer (0.01 M, pH 7.4). Briefly, to induce the peroxidation, 10 ll of 2,20 -azobis(2-amidinopropane) dihydrochloride in water v were added to 2 ml of micellar suspension of linolenic acid maintained at 50 C inside the spectrophotometer (234 nm). After the initiator addition, a spectrum was recorded at 15 min. Then, scalar amounts of flavonoid solutions (1–20 ll at 1.25, 6.25, 12.5, 18.75, 25 lM) were added and the kinetics followed. The slope of the linear plot of absorbance vs. time after the addition of flavonoids gave dA=dt. From the plot of dA=dt vs. flavonoid concentrations, the slope S was obtained and the relative antioxidant efficiency (RAE) value was calculated (S value determined for an a-tocopherol solution was considered to be 100%). The amounts of luteolin and apigenin obtained were insufficient to perform the assays. Table 1 shows the results found for the methanolic extract, quercetin, kaempferol and tiliroside as well as their percentage yield. Table 1 Relative antioxidant efficiency for flavonoids and the methanolic extract from Lichnophora passerina Source
Extract yield (%)
Relative antioxidant efficiency (%)
a-Tocopherola Methanolic extractc Quercetin Kaempferol Tiliroside
Not determinedb 100 0.11 0.19 5.38
100 1.7 72.1 13.1 7.2
a
a-Tocopherol was the control and considered to have 100% efficiency. Used as control, therefore not determined. c The methanolic extract was also tested and its total weight (2.6 g) used for the yield calculations (% of the ethanolic extract). b
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4. Ecological significance Lychnophora passerina is found only in the Brazilian cerrado, where high incidence of light and UV as well as a long period of drought are regional characteristics. This harsh environment can promote stressful conditions for plants. In general, to adapt to such an environment, plants possess mechanisms to protect themselves against the deleterious effects of the stressful conditions (Tevini et al., 1991). An effective mechanism is to accumulate pigments, such as carotenoids, chlorophylls and flavonoids (Kootstra, 1994; Tevini et al., 1991). Besides their antioxidant activity, flavonoids, more specifically flavonol glycosides, can absorb UV-B light. They accumulate after an inductive light treatment in their glycosylated form in the vacuoles of epidermal cells (Weisshaar and Jenkins, 1998). Our findings showed that quercetin and kaempferol, present in the ethanolic extract, were efficient antioxidants in the present assay (72.1% and 13.1%, respectively), corroborating the findings of Rice Evans et al. (1996). Although tiliroside did not show high activity against free radicals in SDS micelles of linolenic acid when compared to quercetin, its high amount in L. passerina (5.4% of the ethanolic extract) can compensate for its low activity. Also, since flavonoids can absorb UVB, the high tiliroside concentration in the ethanolic extract indicates that this natural product might be more important as a UV blocker. Moreover, the sesquiterpene lactones 15-deoxygoyazensolide and goyazensolide were also identified in L. passerina, reinforcing the importance of this class of compound as taxonomic markers in this genus. Acknowledgements This study was further financed by the Fundac¸a˜o para o Amparo e a Pesquisa do Estado de Sa˜o Paulo (FAPESP), Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES) and Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq). The authors thank Dr. Paul Gates (Cambridge University) for helpful comments. References Bohlmann, F., Mu¨ller, L., King, R.M., Robinson, H., 1981. A guaianolide and other constituents from Lychnophora species. Phytochemistry 20, 1149–1151. Borella, J.C., Lopes, J.L.C., Leita˜o Filho, H.F., Semir, J., Diaz, J.G., Herz, W., 1992. Eudesmanolides and 15-deoxygoyasenzolide from Lychnophora pseudovillosissima. Phytochemistry 31, 692–695. Borsato, M.L., Grael, C.F., Souza, G.E., Lopes, N.P., 2000. Analgesic activity of the lignans from Lychnophora ericoides. Phytochemistry 55, 809–813. Foti, M., Piattelli, M., Baratta, M.T., Ruberto, G., 1996. Flavonoids, coumarins, and cinnamic acids as antioxidants in a micellar system. Structure–activity relationship. J. Agric. Food Chem. 44, 497–501. Kaouadji, M., 1990. Acylated and non-acylated kaempferol monoglycosides from Platanus acerifolia buds. Phytochemistry 29, 2295–2297. Kootstra, A., 1994. Protection from UV-B induced DNA damage by flavonoids. Plant Mol. Biol. 26, 771–774.
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