South African Journal of Botany 113 (2017) 318–323
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Antioxidant activities and phenolic profile of Baccharis trimera, a commonly used medicinal plant from Brazil S.M. Sabir a,⁎, M.L. Athayde b, A.A. Boligon b, J.B.T. Rocha c a b c
Department of Chemistry, University of Poonch, Rawalakot, Azad Kashmir, Pakistan Phytochemical Research Laboratory, Department of Industrial Pharmacy, Federal University of Santa Maria, Build 26, room 1115, Santa Maria CEP 97105-900, Brazil Departmento de química, Centro de Ciências Naturais e Exatas, Universidade Federal de Santa Maria, Santa Maria CEP 97105-900, Brazil
a r t i c l e
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Article history: Received 10 May 2017 Received in revised form 30 August 2017 Accepted 11 September 2017 Available online xxxx Edited by J Grúz Keywords: Antioxidant activity Baccharis trimera Gallic acid Lipid peroxidation HPLC analysis
a b s t r a c t The infusions of Baccharis trimera (Asteraceae) are traditionally used in Brazil as a tea to treat liver disorders. It is one of the major constituents in herbal formulations used for the treatment of liver disorders and other diseases. The present study was therefore, aimed to evaluate the potential in vitro and in vivo antioxidant activities and phenolic profile of Baccharis trimera. High performance liquid chromatography coupled with DAD analysis indicated that the gallic acid, rutin and quercetin were the main phenolic compounds present in aqueous extract. The aqueous extract showed inhibition against thiobarbituric acid reactive species (TBARS), induced by different prooxidants (10 μM FeSO4 and 5 μM sodium nitroprusside) in rat liver, brain and phospholipid homogenates from egg yolk. Moreover, the free radical scavenging activities of the extract was determined by the quenching of DPPH (IC50, 415.2 ± 15.2 μg/ml) and hydroxyl radicals in deoxyribose assays. The administration of extract at 100 mg/kg and 250 mg/kg dose significantly reduced the lipid peroxidation, increased the catalase activity and enhanced the levels of ascorbic acid and reduced glutathione in the liver of mice. Thus, oxidative stress in brain and liver could be managed/prevented by dietary intake of Baccharis trimera. © 2017 SAAB. Published by Elsevier B.V. All rights reserved.
1. Introduction Excessive reactive oxygen species (ROS) production can over match cellular antioxidant defenses leading to deleterious condition which is called oxidative stress which has been responsible for the initiation and progression of several human diseases via modification of DNA, proteins and lipids (Finkel and Holbrook, 2000). Earlier studies has shown that Fe2+ increases lipid peroxidation by the breaking down of hydrogen and lipid peroxides which are formed by the Fenton reaction (Yamaguchi et al., 2000). Sodium nitroprusside is an antihypertensive drug and it acts by relaxing smooth vascular muscle (Halliwell and Gutteridge, 1999). However, SNP has been found to be cytotoxic by releasing cyanide and/or nitric oxide (NO). NO is involved in the pathophysiology of disorders such as stroke, trauma, and seizure disorders. NO could act independently or in cooperation with other ROS (Oboh et al., 2007). Studies have shown the potential toxicity of sodium nitroprusside in the brain (Terwel et al., 2000; Dominiak et al., 2016). In addition, more recent studies have demonstrated a potential protective effect of Fe(II) against lead-induced oxidative stress (Ferreira et al., 2017) and SNP against hepatic but not cerebral oxidative stress (Wrobel et al., 2017). Studies have shown that the use of polyphenolic compounds found in tea, fruits and vegetables is associated with low ⁎ Corresponding author. E-mail address:
[email protected] (S.M. Sabir).
https://doi.org/10.1016/j.sajb.2017.09.010 0254-6299/© 2017 SAAB. Published by Elsevier B.V. All rights reserved.
risk of degenerative diseases (Hertog et al., 1993). Consequently, there is huge interest in edible plants that contain antioxidants and health-promoting phytochemicals. One of such plant is Baccharis trimera (Less) DC (Asteraceae), known as carqueja in Brazil, is a wide spread plant species used as tea in South America. Infusions, decoctions, and tinctures of its aerial parts are used in popular medicine in Brazil. Several bioactive compounds such as diterpenic lactones, sesquiterpenes, flavonoids, saponins, tannins, phenolic compounds and essential oils have been described for the species, whose principal active constituent is carquejol (Simões et al., 1998). This plant has been popularly used in the treatment of liver and gastrointestinal tract illnesses (Soicke and Leng-Peschlow, 1987), inflammatory processes and diabetes (Oliveira et al., 2005). Moreover, it showed anti-hyperglycemic activity in mice in vitro. It is cytotoxic against human cellular carcinoma, antispasmodic, anti-inflammatory, analgesic in mice, antibacterial and antiviral in vitro (Michele et al., 2007). Several recent articles have demonstrated the antioxidant effect of B. trimera (Pádua et al., 2010; Pádua et al., 2013; De Araújo et al., 2016; Lívero and Acco, 2016; Lívero et al., 2016). Certain flavonoids, such as silymarin in milk thistle, have shown liverprotective properties and are used for many liver conditions in herbal medicine systems. Carqueja is rather like the South American version of milk thistle. The hepatoprotective effects of crude ethyl acetate extract of B. trimera against carbon tetrachloride (CCl4)-induced liver damage have been already reported (Ames et al., 1997). In this study
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we have evaluated the antioxidant and phenolic profile of B. trimera, which is used as herbal tea by the local communities in Brazil. 2. Materials and methods
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2.5. Determination of total flavonoids The total flavonoid content as quercetin equivalents/g extract was based on the method of Kosalec et al. (2004).
2.1. Chemicals
2.6. Test animals
Thiobarbituric acid (TBA), malonaldehyde-bis-dimethyl acetal (MDA), 2,2-diphenyl-1-picrylhydrazyl (DPPH), quercetin, rutin, gallic acid, and phenanthroline were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sodium nitroprusside (SNP) was obtained from Merck (Darmstadt, Germany) and Iron (II) sulfate from Reagen (Rio de Janeiro, RJ, Brazil).
All animal procedures were in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the University Federal Santa Maria Ethical Council (UFSM 10067). Wistar male rats (200–250 g) and age (3 months) from our own breeding colony were used for in vitro studies. For in vivo studies male albino mice weighing 25–32 g (2–3 months) were used. The animals were kept in separate cages with access to water and food ad libitum, in a room with controlled temperature (22 °C ± 3) and in 12 h light/dark cycle with lights turn on at 7:00 am.
2.2. Preparation of plant extract The whole plant was collected close to the campus of Federal University of Santa Maria (RS, Brazil) during October–November, 2009, identified by a taxonomist and a voucher specimen (SMDB 11.115) was deposited at the Herbarium of University Federal Santa Maria, Department of Biology. The leaves of the plant (25 g) were ground and soaked in boiling water (500 ml) for 15 min, allowed to cool and filtered using Whatman filter paper No. 1. The obtained residue was further extracted twice and finally the whole extract was concentrated using a rotary evaporator (50 °C). The extract weight and percentage yield were found to be 3.5 g (14%). Serial dilutions were made to obtain the desired concentration of plant for the experiments. For HPLC analysis, 0.25 g of dried sample was extracted with hot water (3 × 25 ml), filtered and concentrated to dryness at reduced pressure (45 °C). 2.3. Phytochemical characterization of the extract The dried hot water extract of B. trimera was dissolved in HPLC grade methanol (1 mg/ml), filtered through a 0.45 μm membrane filter and subjected for qualitative and quantitative analysis by using Shimadzu HPLC system, Prominence Auto-Sampler (SIL-20A), equipped with Shimadzu LC-20 AT reciprocating pumps connected to the degasser DGU 20A5 with integrator CBM 20A, UV–VIS detector DAD SPD-M20A and Software LC solution 1.22 SP1. Reverse phase chromatographic analyses were carried out under isocratic conditions using C18 column (4.6 mm × 250 mm) packed with 5 μm diameter particles. For the separation of phenolics the mobile phase composition methanol:acetonitrile:water (40:15:45) containing 1% acetic acid was used. The mobile phase was filtered through a 0.45 μm millipore membrane filter and then degassed by an ultrasonic bath prior to use. Standard curves were prepared by using gallic acid (y = 1.09x104c526,314, r2 = 0.997), rutin (y = 1.92 × 10008.85c-16,949, r2 = 0.999) and quercetin (y = 3.01 × 10017.6c-235,135, r2 = 0.996). Data was monitored at 257 nm for the detection of phenolic acids and flavonoids. Quantification was carried out by the integration of the peak using external standard method. The flow rate was 1 ml/min and the injection volume of aqueous extract was 10 μl. Results were obtained by comparison of retention times and DAD-UV spectra (λmax = 257 nm) with those of the reference standards. All chromatographic operations were carried out at ambient temperature and in triplicate.
2.7. Production of TBARS from animal tissues Production of TBARS was determined using a modified method (Ohkawa et al., 1997). The rats were anesthetized with diethyl ether and then sacrificed by decapitation. Liver and brain were quickly removed and placed on ice. Tissues (1:10, w/v) were homogenized in cold 100 mM Tris buffer pH 7.4 (1:10 w/v) and centrifuged at 1000 × g for 10 min. The homogenates (100 μl) were incubated with or without 50 μl of the various freshly prepared oxidants (iron and sodium nitroprusside) and different concentrations of the plant extracts together with an appropriate volume of deionized water to give a total volume of 300 μl at 37 °C for 1 h. The color reaction was carried out by adding 200, 500 and 500 μl each of the 8.1% Sodium dodecyl sulfate (SDS), acetic acid (pH 3.4) and 0.6% TBA, respectively. The reaction mixtures, including those of serial dilutions of 0.03 mM standard MDA, were incubated at 97 °C for 1 h. The absorbance was read after cooling the tubes at a wavelength of 532 nm in a spectrophotometer. 2.8. Production of TBARS from phospholipid Production of TBARS from phospholipid was determined using the method of (Ohkawa et al., 1997) with slight modifications. One g of egg yolk was extracted with 100 ml of hexane-isopropanol (3:2) and filtered. The solution was dried in a rotary evaporator at 60 °C until it is pastured. The remaining procedure was the same as mentioned above except that the color reaction was carried out without adding SDS. The tubes were cooled and 2 ml of n-butanol was finally added and centrifuged at 1000 ×g. The organic layer (supernatant) was collected and the absorbance was read at 532 nm in a spectrophotometer. 2.9. DPPH radical scavenging Scavenging of the stable DPPH radical (ethanolic solution of 0.25 mM) was assayed in vitro (Hatano et al., 1988) and the absorbance was measured at 517 nm. Percent inhibition was calculated from the control. Ascorbic acid was used as a standard compound in DPPH assay. 2.10. Metal chelating activity The ability of the aqueous extract to chelate Fe2+ was determined using a modified method (Puntel et al., 2005) at 510 nm.
2.4. Determination of phenolics content
2.11. Antioxidant potential assay
The total phenol content was determined by following the method of Singleton et al. (1999). The mean of three readings was used and the total phenol content was expressed in milligram of gallic acid equivalents/g extract.
The total antioxidant potential of the extracts was estimated using the phosphomolybdenum reduction assay (Prieto et al., 1999). The reducing capacity of the extract was expressed as the ascorbic acid equivalents.
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2.12. In vivo antioxidant activity
3. Results and discussion
The 15 mice in group of three were divided into three groups comprising five mice in each group. Group 1(control) received distilled water only. Group II (plant group) received hot water extract at a dose of 100 mg/kg dose of extract 1. Group III (plant group) received hot water extract at a dose of 200 mg/kg dose of extract 2. All the treatments were instilled orally by means of a gastric tube and on the eighth day of experiment all animals were sacrificed under mild ether anesthesia. The liver was quickly removed, placed on ice and homogenized in seven volumes of NaCl (0.9%). Catalase (CAT) activity was estimated by following the breakdown of hydrogen peroxide according to the method of Aebi and Bergmyer (1983). Ascorbic acid was measured by the method of Natelson (1963). Lipid peroxidation was measured in terms of TBARS following the thiobarbituric acid method (Ohkawa et al., 1997). Non protein thiol content was measured in liver homogenate as determined by Jollow et al. (1974).
3.1. Isolation and identification of antioxidants from B. trimera
2.13. Data analysis The results were expressed as mean ± SD. The data were analyzed statistically by one way ANOVA and different group means were compared by Duncan's multiple range test (DMRT); p b 0.05 was considered significant in all cases. The software package, Statistica was used for analysis of data.
In order to identify the compound, that may be responsible for the antioxidant activity of hot water extract, the phenolic compound profile was also determined by means of HPLC method. The extract obtained from leaves presented a chemical profile composed of several phenolic compounds. However, gallic acid, rutin and quercetin were identified through their retention times (tR) i.e. about 3.28, 5.52 and 14.48 mins. for 1, 2, 3 respectively (Fig. 1a). Total phenolic content as gallic acid equivalent was found to be 101 ± 4.1 mg/g of crude extract. Whereas, the flavonoid content as quercetin equivalent was found to 35 ± 1.9 mg/g of crude extract. HPLC quantitative analysis revealed the presence of gallic acid (45.8 ± 7.2 mg/g) as a major compound whereas, rutin (15.1 ± 2.5 mg/g) and quercetin (5.1 ± 1.4 mg/g) relatively showed minor contribution (Table 1). The retention times of these peaks were matched to those of the reference standards gallic acid (Fig. 1b), rutin (Fig. 1c) and quercetin (Fig. 1d). From the HPLC phenolic profile of B. trimera, we observed that there were different peaks representing the phenolic compounds. However, the instrumental analysis revealed the presence of gallic acid as a major compound and two important flavonoids, rutin and quercetin which are well recognized as potential antioxidants and free radical scavengers and inhibit lipid peroxidation via the scavenging of free radicals and metal chelation (Robert et al., 2008). This is the first report which shows the presence of gallic acid and rutin in the aqueous extract of B. trimera. The earlier studies have shown high content of flavonoids (20%) in
Fig. 1. HPLC phenolic profile of B. trimera aqueous extract from leaves and standard phenolics. The detection wavelength was 257 nm (a) Representative HPLC chromatogram of extract. Peaks: (1) gallic acid (2) rutin and (3) quercetin (b) HPLC chromatogram of standard gallic acid (c) HPLC chromatogram of standard rutin (d) HPLC chromatogram of standard quercetin.
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Table 1 Content of phenolics and flavonoids in aqueous extract of leaves of B. trimera. B. trimera compounds
Composition
Total phenolics (*GAEA mg/g) Total flavonoids (*QuerB mg/g) Gallic acid (mg/g) Rutin (mg/g) Quercetin (mg/g)
101 ± 4.1 35 ± 1.9 45.8 ± 7.2 15.1 ± 2.5 5.1 ± 1.4
*GAEA is gallic acid equivalent, *QuerB is quercetin equivalent.
B. trimera leaves including quercetin, luteolin, nepetin, apigenin, and hispidulin (Soicke and Leng-Peschlow, 1987).
3.2. In vitro and in vivo assays The present study was designed to investigate the antioxidant activity of B. trimera. Lipid peroxidation in rat liver was induced with iron (10 μM) and sodium nitroprusside (5 μM) and the antioxidant effect of B. trimera extract was determined. There was statistically significant increase in the formation of TBARS in ferrous sulfate (81%) and SNP (79%) induced liver homogenate compared to the basal or normal (Fig. 2a). However, treatment with B. trimera caused a concentration dependent inhibition of TBARS production and brought the values close to the basal level (Fig. 2a). Fig. 2b shows the interaction (inhibition) of the plant extract with Fe(II)- and SNP-induced lipid peroxidation in rat's brain. The results revealed that incubation of the cerebral tissue in the presence of 10 μM Fe(II) and 5 μM SNP caused 80% and 75.5% increases in lipid peroxidation which was significantly decreased by the treatment with extract. Egg yolk phospholipids underwent rapid non-enzymatic peroxidation when incubated in the presence of ferrous sulfate and SNP (Fig. 2c). The exposure of phospholipid to iron (10 μM) and sodium nitroprusside (5 μM) elevated lipid peroxidation by 76% and 71% respectively. Treatment with aqueous extract significantly reduced lipid peroxidation in phospholipid in a concentration dependent manner near to basal values (Fig. 2c). Here we have used pro-oxidant agents that induce lipid peroxidation by different mechanisms. Free iron can cause neurotoxicity (Bostanci and Bagirici, 2008) via stimulation of Fenton reaction (Fraga and Oteiza, 2002). The increased lipid peroxidation in the presence of Fe (II) could be attributed to the fact that Fe (II) can catalyze one-electron transfer reactions that generate ROS, such as the reactive OHo which is formed from H2O2 through the Fenton reaction. Iron also results in decomposition of lipid peroxides, thus generating peroxyl and alkoxyl radicals, which favors the propagation of lipid oxidation (Zago et al., 2000). Sodium nitroprusside is an antihypertensive drug which acts by relaxation of vascular smooth muscle; consequently it dilates peripheral arteries and veins. However, earlier studies have shown that photo degradation of SNP ultimately produces NO, [(CN)5− Fe]3+ and [(CN)4− Fe]2+ species (Bates et al., 1990). Nitric oxide is a molecule that is regarded as a universal neuronal messenger in the central nervous system and is involved in pathophysiology of disorders such as Alzheimer's and Parkinson's diseases, stroke, trauma and seizures etc. (Bolanos and Almeida, 1990). The protections offered by the aqueous extract in rat liver and brain as well as on phospholipid homogenates confirms the antioxidant activity of the extract and indicates its therapeutic use in the accidental toxicities resulting from the potential overload of iron and SNP. Indeed the distinct antioxidant activities of plant extracts could indicate that they were acting via distinct mechanism. Although this can be the case, plant extracts could be inhibiting a common final (or downstream) pathway in polyunsaturated fatty acids peroxidation. Thus, we cannot exclude that a single mechanism is involved in the anti-oxidant of the tested extract. The extract of B. trimera contains rutin and quercetin which are well known flavonoids and the observed decrease of lipid peroxidation here may due to these flavonoids (Pereira et al., 2003).
Fig. 2. The inhibitory effect of aqueous extract of B. trimera on ferrous sulfate and sodium nitroprusside (SNP) induced lipid peroxidation. TBARS production was induced with 10 μM Fe(II) and 5 μM SNP in tissues and phospholipid for 60 min (control). (a) inhibitory effect of B. trimera on ferrous sulfate and sodium nitroprusside (SNP) induced lipid peroxidation in rat liver (b) inhibitory effect of B. trimera on ferrous sulfate and sodium nitroprusside (SNP) induced lipid peroxidation in rat brain (c) inhibitory effect of B. trimera on ferrous sulfate and sodium nitroprusside (SNP) induced lipid peroxidation in phospholipids homogenates. Basal indicates tissue without the prooxidant. Control includes tissue with the pro-oxidants (Fe and SNP). Values represent the means of three separate experiments in duplicate ±SD. Values in figures which share different letters are significantly (p b 0.05) different from each other by DMRT.
The radical scavenging activity of the extract was tested against two important in vitro models of free radicals namely DPPH and hydroxyl radicals (Fig. 3). The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical has
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Fig. 4. Iron chelating ability of the extract assessed by reaction with 1,10-phenanthroline. Values are mean ± SD (n = 3), Bars with different letters are significantly different from each other by DMRT.
Fig. 3. Antioxidant and free radical scavenging activities of aqueous extract of B. trimera (a) DPPH radical scavenging activity of the extract and ascorbic acid. AA, standard ascorbic acid at a concentration of 30 μg/ml. (b). scavenging of hydroxyl radical generated by the reaction of Fe(II) + H2O2 in deoxyribose degradation assay. Values are mean ± SD (n = 3), Bars with different letters are significantly different from each other by DMRT.
been widely used to test the free radical scavenging ability of various natural products and has been accepted as a model compound for free radicals originating in lipids (Hatano et al., 1989; Yasuda et al., 2000). The role of an antioxidant is to remove free radicals. The aqueous extract showed higher efficiency in eliminating DPPH radical with an IC50 value of 415 ± 12.1 μg/ml which was less compared to the reference ascorbic acid (IC50 = 28.4 ± 1.25 μg/ml) (Fig. 3a). The effect of aqueous extract on Fe(II) dependent deoxyribose damage is shown in Fig. 3b. The results revealed that the extract was capable of reducing deoxyribose (an important constituent of DNA) damage at all concentrations at least by its ability to chelate iron which was greater than 50% at the extract concentration of 100 μg/ml (Fig. 3). Scavenging of hydroxyl radical is an important antioxidant activity because of very high reactivity of the hydroxyl radical which enables it to react with a wide range of biomolecules such as sugars, amino acids, lipids and nucleotides. The ability of the B. trimera extract to quench the hydroxyl radical generated in the deoxyribose assay can be directly related to the prevention and propagation of the process of lipid peroxidation by scavenging the active oxygen species thus reducing the rate of chain reaction at least in part by chelating and deactivating iron ions. The plant extracts also chelate Fe2 + (Fig. 4). This result, however, is in agreement with the Fe2 +induced lipid peroxidation (Fig. 1), phenolic content (Table 1), and antioxidant activity of the extracts, suggesting that Fe chelation may be one of the possible mechanisms through which antioxidant phytochemicals from leaves of B. Trimera prevent lipid peroxidation in tissue
by forming a complex with Fe, thus preventing the initiation of lipid peroxidation. Furthermore, the reducing power of the extractable phytochemicals from B. Trimera expressed as ascorbic acid equivalent (AAE) is presented in Fig. 5. The total antioxidant activity of the extract (equivalent to ascorbic acid) was found to be 119 ± 2.5 μg/ml at maximal concentration (100 μg/ml) and was increased with increasing concentrations of extract (Fig. 5). Allhorn et al. (2005) reported that the reducing property can be a novel antioxidant defense mechanism due to the ability of the antioxidant compound to reduce transition metals. Therefore, the higher reducing ability of B. Trimera extract may have contributed to the higher protective effect observed. The results of in vivo antioxidant activities are shown in Table 2. It is indicated that the aqueous extract possess antioxidant activity as it reduced the lipid peroxidation, increased the catalase activity and enhanced the levels of ascorbic acid in the liver of mice. An increase in intracellular thiol based antioxidant GSH (reduced glutathione) was also observed. GSH serves to detoxify the damaging radicals either by directly scavenging them or by acting as a co substrate in the glutathione peroxidase (GPx)-catalyzed reduction of hydrogen peroxide and lipid peroxides. Consequently, after in vivo administration, the extract of B. trimera had a much complex antioxidant pattern than observed in vitro. In addition to chelating iron and potentially interacting with hydroxyl radicals, the flavonoid components of B. trimera could activate antioxidant responsive elements (ARE) or the electrophileresponsive element (EpRE), which in turn increase the antioxidant
Fig. 5. Total antioxidant activity of B. trimera leaves measured by phosphomolybdenum assay. Values are mean ± SD (n = 3), all the tested concentrations are significantly different from each other by DMRT.
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Table 2 In vivo antioxidant activities of hot water extract of Baccharis trimera (BT) in mice liver. Groups
Treatments (mg/kg)
TBARS (nmole/g·tissue)
CAT (IU/mg·tissue)
NPSH (μmol/g·tissue
Vitamin C (μg/g·tissue)
Control BT BT
Distilled water 100 mg/kg 250 mg/kg
455 ± 5.1a 425 ± 4.5b 380 ± 7.2c
0.20 ± 0.02a 0.31 ± 0.04a 0.51 ± 0.02b
61.9 ± 7.1a 71.2 ± 4.5b 85 ± 6.5c
0.51 ± 0.04a 0.65 ± 0.02b 0.81 ± 0.08c
Values represent the means ± SD (n = 5). Values in figures which share different letters are significantly (p b 0.05) different from each other by DMRT.
capacity of different types of cells (Tanigawa et al., 2007; Kim et al., 2017; Qiu et al., 2017). 4. Conclusions In conclusion, the aqueous extract of B. trimera possess anti-lipid peroxidative and free radical scavenging activities which may be associated with its high medicinal use as a functional food and effectiveness in the treatment of different diseases among which the liver disease is most important. The scavenging activities observed against DPPH and hydroxyl radicals, as well as the protective activities against lipid peroxidation, lead us to propose B. trimera leaf as a promising natural source of antioxidants suitable for application in food and pharmaceutical fields and in the prevention of free radical-mediated diseases. Acknowledgments SM Sabir thanks CNPq and TWAS for the doctoral fellowship. J.B.T.R. is the recipient of a CNPq research fellowship and acknowledges the financial support of FINEP “Rede Instituto Brasileiro de Neurociência (IBNNet)” # 01.06.084200, CNPq, CAPES/SAUX/PROAP, VITAE Foundation and FAPERGS (01.06.084200). References Aebi, H.E., Bergmyer, H.O., 1983. Catalase Methods Enzymology. Academic Press, New York, p. 2. Allhorn, M., Klapyta, A., Åkerström, B., 2005. Redox properties of the lipocalin α1-microglobulin: reduction of cytochrome c, hemoglobin, and free iron. Free Radical Biology and Medicine 38 (5), 557–567. Ames, B.N., Shigenaga, M.K., Hagen, T.M., 1997. Oxidants, antioxidants and the degenerative diseases of aging. Proceedings of the National Academy of Sciences 90, 7915–7922. Bates, J.N., Baker, M.T., Guerra, R., Harrison, D.G., 1990. Nitric oxide generation from nitroprusside by vascular tissue. Biochemical Pharmacology 42, S157–S165. Bolanos, J., Almeida, A., 1990. Roles of nitric oxide in brain hypoxia-ischemia. Biochimica et Biophysica Acta 1411, 415–436. Bostanci, M.O., Bagirici, F., 2008. Neuroprotective effect of ami-noguanidine on iron-induced neurotoxicity. Brain Research Bulletin 76, 57–62. De Araújo, G.R., Rabelo, A.C.S., Meira, J.S., Rossoni-Júnior, J.V., de Castro-Borges, W., GuerraSá, R., Batista, M.A., Silveira-Lemos, D., Souza, G.H.B., Brandão, G.C., Chaves, M.M., Costa, D.C., 2016. Baccharis trimera inhibits reactive oxygen species production through PKC and down-regulation p47phox phosphorylation of NADPH oxidase in SK Hep-1 cells. Experimental Biology and Medicine 242, 333–343. Dominiak, A., Wilkaniec, A., Wroczyński, P., Jęśko, H., 2016. Protective effects of Selol against sodium nitroprusside-induced cell death and oxidative stress in PC12 cells. Neurochemical Research 41, 3215–3226. Ferreira, M.C., Zucki, F., Duarte, J.L., Iano, F.G., Ximenes, V.F., Buzalaf, M.A., Oliveira, R.C., 2017. Influence of iron on modulation of the antioxidant system in rat brains exposed to lead. Environmental Toxicology 32, 813–822. Finkel, T., Holbrook, N.J., 2000. Oxidants, oxidative stress and the biology of aging. Nature 408, 239–247. Fraga, C.G., Oteiza, P.I., 2002. Iron toxicity and antioxidant nutrients. Toxicology 80, 23–32. Halliwell, B., Gutteridge, J.M.C., 1999. Free Radicals in Biology and Medicine. 3rd ed. Oxford University Press, Oxford. Hatano, T., Kagawa, H., Yasuhara, T., Okuda, T., 1988. Two new flavonoids and other constituents in licorice root; their relative astringency and radical scavenging effects. Chemical and Pharmaceutical Bulletin 36, 2090–2097. Hatano, T., Edmatsu, R., Hiramatsu, M., Mori, A., Fujita, Y., Yasuhara, T., Yoshida, T., Okuda, T., 1989. Effects of the interaction of tannins with coexisting substances. VI effect of tannins and related polyphenols on superoxide anion radicals and on DPPH. Chemical and Pharmaceutical Bulletin 37, 2016–2021. Hertog, M.G.L., Hollman, P.C.H., Van, P.B., 1993. Content of potentially anticarcinogenic flavonoids of tea infusions, wines and fruit juices. Journal of Agricultural and Food Chemistry 41, 1242–1246.
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