JOURNAL OF MEDICINAL FOOD J Med Food 18 (7) 2015, 830–834 # Mary Ann Liebert, Inc., and Korean Society of Food Science and Nutrition DOI: 10.1089/jmf.2014.0089 Phytochemical Characterization, Antimicrobial Activity, and Antioxidant Potential of Equisetum hyemale L. (Equisetaceae) Extracts Geisiany M. de Queiroz,1 Fla´vio A.S. Politi,1 Edvaˆnio R. Rodrigues,1 Tatiana M. Souza-Moreira,1 Raquel R.D. Moreira,2 Ca´ssia R.P. Cardoso,3 Lourdes C. Santos,3 and Rosemeire C.L.R. Pietro1 Departments of 1Drugs and Medicines and 2Natural Active Principles and Toxicology, School of Pharmaceutical Sciences of Araraquara, UNESP-Univ Estadual Paulista, Araraquara, Brazil. 3Department of Chemistry of Natural Products, Institute of Chemistry of Araraquara, UNESP-Univ Estadual Paulista, Araraquara, Brazil. ABSTRACT Equisetum hyemale species is considered a medicinal plant used in the form of infusions to combat infectious or inflammation diseases and also diuretic effects, presenting several compounds related to these actions. In previous studies different species of Equisetum showed several phenolic compounds. The objective of this study was, for the first time, based on phytochemistry analysis to evaluate the antioxidant and antimicrobial activity. The 70% ethanolic and methanolic extracts of E. hyemale were characterized by spectrophotometric and high-performance liquid chromatography with pulsed am- perometric detector analyses, as well as its antioxidant potential based on the scavenger activity of 2,2-diphenyl-1-pi- crylhydrazyl (DPPH). In addition was verified the antimicrobial activity by broth microdilution technique against bacteria and fungi. The extracts showed phytochemical similarity, which demonstrated the presence of phenolic compounds, the scav- enging activity for free radicals was about 30% and was observed better antifungal activity against dermatophyte fungi, with minimum inhibitory concentration and minimum fungicidal concentration of 0.62 mg/mL to Trichophyton rubrum and Mi- crosporum canis. The extracts exhibits great potential to therapeutic applications or product development, since both possess antifungal activity and antioxidant action associated with little difference in their phytochemical composition. KEY WORDS: antimicrobial activity antioxidant effect Equisetum hyemale medicinal plant analysis MIC phytochemistry INTRODUCTION rations for cosmetic purposes there is enamel that uses water- soluble extract of Equisetum arvense in the formulation and quisetum hyemale, popularly known as horsetail, is that proved to be effective in strengthening the nails, re- used commonly to prepare infusions to combat infec- E ducing brittleness and surface roughness of brittle nails, tious diseases in the kidney or urinary tract and presents being able to improve the appearance of psoriatic nails.8 This diuretic effects. There are other reports showing some background justifies new studies aiming at the antioxidant properties related to E. hyemale such as hyperlipidemic, and antimicrobial potential of E. hyemale extracts, based on antimicrobial, hepatoprotective, antioxidant, and anti- their phytochemical constitution, to verify its potential as a inflammatory activities.1,2 Previous studies identified kaemp- medicinal agent. ferol,3 glycosil flavonoids,4 silica,5 quercetin,6 and ethyl 7 palmitate in the essential oil. The Equisetum genus has been MATERIALS AND METHODS widely chemically and biologically characterized in differ- ent regions due to folk use and the commerce of derivative Plant material 6 products with this species in its formulations. Currently The material of E. hyemale was collected in March 2009, there are several products on the world market using extracts of ‘‘Horto de Plantas Medicinais e To´xicas de Araraquara,’’ of species of this genus. Among the phytochemical prepa- Sa˜o Paulo, Brazil and the identification was performed by Downloaded by Shanghai Information Center for Life Sciences, CAS from online.liebertpub.com at 10/29/17. For personal use only. Prof. Dr. Marco Antoˆnio de Assis. The voucher specimen was deposited in the UNESP Herbarium (Rio Claro, SP) Manuscript received 26 May 2014. Revision accepted 29 October 2014. with number 51670 HRCB. The aerial parts of E. hyemale Address correspondence to: Rosemeire C.L.R. Pietro, PhD, Department of Drugs and were dried in an oven with air circulation at 40°C until Medicines, School of Pharmaceutical Sciences of Araraquara, UNESP-Univ Estadual Paulista, Rodovia Araraquara-Jau´, km 1, Araraquara 14801-902, SP, Brazil, E-mail: stabilization and then pulverized. Eighty grams of plant [email protected] material was submitted to the exhaustive percolation using 830 EQUISETUM HYEMALE PHYTOCHEMICAL AND ACTIVITIES 831 ethanol (70%) and methanol as solvents.9 The extracts ob- Escherichia coli (ATCC 25922), Bacillus subtilis (ATCC tained were concentrated under vacuum, lyophilized, and 9392), Pseudomonas aeruginosa (ATCC 23212), and Can- stored in desiccators. dida albicans (ATCC 64548). The dermatophytes fungi Trichophyton rubrum, Trichophyton mentagrophytes, and Analyses by high-performance liquid chromatography Microsporum canis (clinical isolates) were kindly provided with pulsed amperometric detector by Prof. Dr. Ana Marisa Fusco Almeida (Mycology La- boratory, School of Pharmaceutical Sciences of Araraquara, The chromatographic profile of the 70% ethanolic extract UNESP). Determination of minimum inhibitory concentra- was established using a Jasco liquid chromatography tion (MIC) against dermatophyte fungi, aerobic bacteria, equipped with a PU-2089 quaternary solvent pump, a MD- and yeast C. albicans were performed using the microdilu- 2010 PAD, and a Rheodyne 7725 sample injector with a 20 tion technique in 96-well plates, according to the protocols lL sample loop. The analytical column was a Phenomenex M38-A, M7-A7, M27-A3, respectively, of the Clinical and Synergi Hydro RP18 (250 · 4.6 mm i.d.; 4 lm) equipped Laboratory Standards Institute11–14 with modifications. with a Phenomenex security guard column (4.0 · 2.0 mm Stock solutions of the extracts were dissolved in DMSO i.d.). The mobile phase was composed by water (eluent A) (50 mg/mL) and evaluated at concentrations between 1.25 to and methanol (eluent B), both containing 0.05% of tri- 0.12 mg/mL, except for oral microorganisms in which fluoroacetic acid, with 5–100% B gradient. The flow rate concentrations tested ranged from 2.00 to 0.09 mg/mL, was 1.0 mL/min and the total run time was 55 min. The using DMSO as control solvent (1:5, v/v). Ampicillin was EZChrom Elite Data System software was used for both the used as positive control against bacteria and amphotericin B operation of detector and for data processing. Standard against yeast and fungi. The yeast growth used RPMI-1640, substances were obtained from Sigma (caffeic acid, incubated at 35°C for 48 h under constant agitation at chlorogenic acid, and rutin, 99.5% purity). 150 rpm. To filamentous fungi, RPMI-1640 medium sup- plemented with glucose was used, incubated at 28°C under Determination of total flavonoids contents constant agitation at 100 rpm for 7 days. MICs of bacteria The determination of total flavonoids was performed by a were determined as the lowest concentration presenting blue colorimetric method based on the formation of a complex coloration after 2 h of incubation with 0.01% Resazurin flavonoid–aluminum.10 All determinations were made in solution. MIC of yeast was determined as the lowest con- triplicate and the values were calculated from a calibration centration in which the cells were stained after 3 h of incu- curve obtained with the quercetin standard. The final results bation with 2% solution of 2,3,5-triphenyl tetrazolium. MIC were expressed as milligrams of equivalents in quercetin per of fungi was determined as the lowest concentration of ex- gram of extract (mgEQ/g). The reading was performed at tract in which there was no development of microorganisms. 430 nm in a Shimadzu-1603 spectrophotometer. To prepare Minimum bactericidal concentration (MBC) and minimum the samples, the extracts were diluted (10.0 mg in 20.0 mL fungicidal concentration (MFC) were determined as the MeOH 80% v/v) and aliquots of each solution were added to lowest concentration in which there was no development of 2.0 mL of AlCl3. (6H2O) in MeOH 2% (v/v), completing to colonies after subculturing samples from the microplates on final volume of 4.0 mL in MeOH solution 80% (v/v). Both agar of each microorganism in appropriate medium. extracts were analyzed in concentrations 125, 150, 175, 200, 225, and 250 lg/mL. The concentration of the standard for Statistical analysis the calibration curve ranged from 0.25 to 4.0 lg/mL. All experiments were carried out in triplicate, determin- ing the average and standard deviation of the results. The Evaluation of the ability of free radical scavenging determination of total flavonoids and the ability of scav- The potential antioxidant activity of extracts was deter- enging free radicals were submitted to analysis of variance mined based on the radical scavenging activity of 2,2- (ANOVA) by Tukey test, with P < .05. diphenyl-1-picrylhydrazyl (DPPH). The tests were performed with E. hyemale extracts obtained at concentrations of 25, RESULTS AND DISCUSSION 50, and 100 lg/mL in methanol. The positive control was The yield was 12.16 g (15.16%) to 70% ethanolic extract ascorbic acid (vitamin C) at the same concentrations. To and 7.40 g (9.23%) to methanolic extract. The spectrum of 1 mL of sample solution was added 2.5 mL of 0.004% DPPH gallic acid, tannic acid, and the samples of E. hyemale in methanol. The solutions were kept in the dark for 30 min presented absorbance profiles between 200 and 250 nm and at room temperature and after the reading was done at 250–300 nm, similar to phenolic compound used as refer- 517 nm in a Shimadzu-1603 spectrophotometer. The blank ence, suggesting the presence of this class of metabolites solution consisted of methanol and the negative control of Downloaded by Shanghai Information Center for Life Sciences, CAS from online.liebertpub.com at 10/29/17.