Revista Brasileira de Farmacognosia 25 (2015) 219–227 www.sbfgnosia.org.br/revista Original Article Analysis of flavonoids in Rubus erythrocladus and Morus nigra leaves extracts by liquid chromatography and capillary electrophoresis a a b c Luciana R. Tallini , Graziele P.R. Pedrazza , Sérgio A. de L. Bordignon , Ana C.O. Costa , d e a,∗ Martin Steppe , Alexandre Fuentefria , José A.S. Zuanazzi a Departamento de Produc¸ ão de Matéria Prima, Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil b Centro Universitário La Salle, Canoas, RS, Brazil c Departamento de Ciência e Tecnologia de Alimentos, Universidade Federal de Santa Catarina, Florianópolis, SC, Brazil d Departamento de Produc¸ ão e Controle de Medicamentos, Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil e Departamento de Análises, Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil a a b s t r a c t r t i c l e i n f o Article history: This study uses high performance liquid chromatography and capillary electrophoresis as analytical tools Received 15 January 2015 to evaluate flavonoids in hydrolyzed leaves extracts of Rubus erythrocladus Mart., Rosaceae, and Morus Accepted 30 April 2015 nigra L., Moraceae. For phytochemical analysis, the extracts were prepared by acid hydrolysis and ultra- Available online 17 June 2015 sonic bath and analyzed by high performance liquid chromatography using an ultraviolet detector and by capillary electrophoresis equipped with a diode-array detector. Quercetin and kaempferol were iden- Keywords: tified in these extracts. The analytical methods developed were validated and applied. Quercetin and HPLC-UV −1 −1 kaempferol were quantified in R. erythrocladus, with 848.43 ± 66.68 ␮g g and 304.35 ± 17.29 ␮g g , UPLC-DAD/MS −1 respectively, by HPLC-UV and quercetin, 836.37 ± 149.43 ␮g g , by CE-DAD. In M. nigra the quan- CE-DAD −1 −1 tifications of quercetin and kaempferol were 2323.90 ± 145.35 ␮g g and 1446.36 ± 59.00 ␮g g , Rubus erythrocladus −1 −1 ± ␮ ± ␮ Morus nigra respectively, by HPLC-UV and, 2552.82 275.30 g g and 1188.67 99.21 g g , respectively, by CE- Flavonoids DAD. The extracts were also analyzed by ultra-performance liquid chromatography coupled with a diode-array detector and mass spectrometer (MS), UPLC-DAD/MS. © 2015 Sociedade Brasileira de Farmacognosia. Published by Elsevier Editora Ltda. All rights reserved. Introduction Studies have shown that Rubus leaves demonstrate antioxi- dant (Venskutonis et al., 2007; Martini et al., 2009), anticancer The fruits of Rubus (Rosaceae) and Morus (Moraceae) have been (Durgo et al., 2012), gastrointestinal (Rojas-Vera et al., 2002), extensively studied around the world because of their beneficial antiangiogenic (Liu et al., 2006), antithrombotic (Han et al., 2012), effects on health. Rubus, popularly known as the genus of rasp- hypoglycemic (Jouad et al., 2002), antimicrobial (Panizzi et al., berries and blackberries, is widely distributed around the world, 2002; Thiem and Goslinska, 2004; Martini et al., 2009; Ostrosky both as wild and cultivated species (Deighton et al., 2000). Morus et al., 2011) and anxiolytic activities (Nogueira et al., 1998; is known as the genus of mulberry, and is found from temper- Nogueira and Vassilieff, 2000). For the leaves of Morus, there are ate regions to subtropical regions of the Planet (Gundogdu et al., studies reporting antioxidant (Katsube et al., 2010; Choi et al., 2011). In Brazil, plants belonging to the genus Morus are listed by 2013), anticancer (Dat et al., 2010; Skupien et al., 2008), hypo- the government as interesting to research because of their use in glycemic (Volpato et al., 2011; Chung et al., 2013), anti-obesity (Oh folk medicine, and their potential for use as phytotherapics (Portal et al., 2009; Tsuduki et al., 2013), antimicrobial (Omidiran et al., da Saúde, 2013). However, Rubus and Morus are often mistaken in 2012) and vasodilation activities (Xia et al., 2008). the differentiation, due to the similarity of their fruits. Also, there Flavonoids derived from quercetin and/or kaempferol were is a lot of information about the fruits of these species, but little reported in extracts of Rubus leaves analyzed by TLC (Tzouwara- information about their leaves. Karayanni and Philianos, 1982), HPLC (Venskutonis et al., 2007; Martini et al., 2009; Durgo et al., 2012; Gudej and Tomczyk, 2004) and NMR (Panizzi et al., 2002; Han et al., 2012; Gudej, 2003). There are no studies about analysis of Rubus leaves by CE, and the species ∗ erythrocladus has never been studied before. In Morus leaves, sec- Corresponding author. E-mail: [email protected] (J.A.S. Zuanazzi). ondary metabolites derived from quercetin and/or kaempferol have http://dx.doi.org/10.1016/j.bjp.2015.04.003 0102-695X/© 2015 Sociedade Brasileira de Farmacognosia. Published by Elsevier Editora Ltda. All rights reserved. 220 L.R. Tallini et al. / Revista Brasileira de Farmacognosia 25 (2015) 219–227 been identified by HPLC (Katsube et al., 2010; Thabti et al., 2012; was performed on a C18 reverse phase column (Acquity UPLC beh, Choi et al., 2013), and by NMR (Dat et al., 2010; Katsube et al., 2010), 50.0 × 2.1 mm, 1.7 ␮m). The software program Mass Lynx v.4.1 and there are two studies on the chemical composition of Morus was used for the analysis and data acquisition and the program alba by CE (Suntornsuk et al., 2003; Chu et al., 2006). Acquity UPLC Columns Calculator v.1.1.1 was used to adjust the High performance liquid chromatography is a classic method HPLC method to the UPLC method. used for the separation and quantification of secondary metabolites Mobile phase A consisted of water with 0.1% (v/v) of formic acid in plant extracts but sometimes it can be an expensive analyti- and acetonitrile B with 0.1% (v/v) of formic acid. The gradient was cal tool (Merken and Beecher, 2000). Capillary electrophoresis is 0 min 50% of B, 0–0.09 min 60% of B, 0.09–0.38 min 80% of B and characterized by high separation efficiency and good compromise 0.38–0.80 min 95% of B. Column temperature was maintained at ◦ −1 between analysis time and satisfactory characterization of pheno- 25 C, flow rate was 0.294 ml min , injection volume was 1.0 ␮l lic compounds in plants. The low operational cost and the small and detection was between 200 and 400 nm. residue generation make this technique an attractive option for the development of analytical methods for phytochemical analysis CE conditions (Carrasco-Pancorbo et al., 2006). The aim of this study was to develop HPLC-UV and CE-DAD ana- The CE assays were conducted in a capillary electrophoresis lytical methods for the evaluation of quercetin and kaempferol system (Agilent Technologies, model 7100, Palo Alto, CA, USA) in Rubus erythrocladus Mart. and Morus nigra L. leaves extracts. equipped with a diode array detector, temperature-control device ◦ The results were statistically analyzed using validation parameters (maintained at 25 C) and data acquisition and analysis software ® and the methods were applied to determine the amount of these supplied by the manufacturer (HP ChemStation ). Before the first −1 flavonoids in these samples. run, the capillary column was sequentially rinsed with 1 mol l NaOH (30 min) and water (30 min). Between runs, the capillary was Experimental flushed for 1 min with background electrolyte (BGE). At the begin- ning of each day the capillary was conditioned by flushing with Samples −1 1 mol l NaOH (20 min), followed by a 20 min flush with deion- ized water and an electrolyte solution (20 min). Between runs, the Leaves of Rubus erythrocladus Mart., Rosaceae, were collected at −1 capillary was reconditioned with 1 mol l NaOH (2 min), deionized Embrapa – Temperate Climate (Pelotas, Brazil) and leaves of Morus water (1 min) and electrolyte solution (2 min). At the end of each nigra L., Moraceae, were collected in Porto Alegre (Brazil). Vouchers −1 working day, the capillary was rinsed with 1 mol l NaOH (10 min) have been deposited in the Herbarium – UFRGS, Federal University and water (10 min). of Rio Grande do Sul (179856 and 176765, respectively). The sam- Separations were conducted in an uncoated fused-silica ples were dried at room temperature for two weeks and kept in the capillary 48.5 cm in length (40 cm effective length × 50 ␮m dark. I.D. × 375 ␮m O.D.). Direct UV detection was set at 210 nm, and the ◦ temperature was maintained at 25 C. The standards and samples Chemicals and materials were introduced to the capillary with a hydrodynamic pressure of 50 mbar, for 3 s. The separation voltage applied was 30 kV under Quercetin (purity ≥ 98%) and kaempferol (purity ≥ 97%) were normal polarity. The optimized BGE used in the proposed method obtained from Sigma (USA) and methylparaben from Fluka (USA). −1 was comprised of 20 mmol l (pH 9.2) and 10% (v/v) acetonitrile, HPLC-grade acetonitrile and methanol were purchased from and methylparaben was used as internal standard, diluted to obtain Merck (Germany). Throughout the study, deionized and ultra- −1 a final concentration of 45.46 ␮g ml . pure water were used. All other chemicals were analytical grade. Dichloromethane and hydrochloric acid were obtained from Synth Standard preparation and calibration curves (Brazil), trifluoroacetic acid from Vetec (Brazil), and ethyl acetate, sodium hydroxide and sodium tetraborate decahydrate from Merck The standard stock solutions were prepared by dissolving 1 mg (Germany). of each compound in 1 ml of methanol. These solutions were stored ◦ in dark glass bottles at 4 C. Working standard solutions were HPLC conditions freshly prepared by dissolving a suitable amount of the above solutions with methanol before injection.