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 40C 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 35C 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 28C 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. For personal use only. (data not showed). The identification of these compounds on 1 mL of methanol and 2.5 mL of DPPH. 70% ethanolic extract was performed with the aid of PDA detector scanning, in the spectral range of 200–600 nm. The Microorganisms and antimicrobial activity analytical high performance liquid chromatography with The strains used were: Staphylococcus aureus (ATCC pulsed amperometric detector chromatogram recorded at 25923), Staphylococcus epidermidis (ATCC 12228), 255 nm with its respective UV spectra, highlighting the 832 QUEIROZ ET AL.

Table 1. Antimicrobial Activity and Quantification of Total Flavonoids of 70% Ethanolic and Methanolic Extracts of E. hyemale

70% ethanolic Methanolic extract (mg/mL) extract (mg/mL) CF (mgEQ/g)

Microorganisms Concentration 70% ethanolic Methanolic MIC MBC/MFC MIC MBC/MFC (lg/mL) extract extract S. aureus > 1.25 > 1.25 > 1.25 > 1.25 125 9.15a 8.34a S. epidermidis > 1.25 > 1.25 > 1.25 > 1.25 150 9.66a 9.09a B. subtilis > 1.25 > 1.25 > 1.25 > 1.25 175 9.71a 10.17a E. coli > 1.25 > 1.25 > 1.25 > 1.25 200 10.28a 9.65a P. aeruginosa > 1.25 > 1.25 > 1.25 > 1.25 225 10.00a 9.51a C. albicans > 1.25 > 1.25 > 1.25 > 1.25 250 10.02a 9.20a T. rubrum 1.25 1.25 0.62 0.62 Average – SD 9.80 – 0.39 9.33 – 0.61 T. menthagrophytes 1.25 > 1.25 1.25 1.25 M. canis 1.25 1.25 0.62 0.62

Ampicillin and Amphotericin B showed MIC in the concentration range of 0.002–0.007 and 0.001–0.005 mg/mL for bacteria and fungi, respectively. Values followed by the same superscript letters did not show statistically significant differences by Tukey’s test (P < .05). CF, concentration of total flavonoids (mgEQ/g) expressed in milligrams of equivalents in quercetin per gram of extract; MBC, minimum bactericidal concentration; MFC, minimum fungicidal concentration; MIC, minimum inhibitory concentration; SD, standard deviation.

peaks with retention time of 20.5, 21.0, and 22.5 min. These abilities. Therefore, it has been used as anti-inflammatory, compounds were identified by comparing the peaks of their anticarcinogenic, and photoprotective agents in various bi- UV spectra with those of the available standards and com- ological systems.18 pared with the references in the literature. The chlorogenic Both extracts showed antioxidant activity above 30%, acid and caffeic acid have characteristic bands at 280– showing no significant difference between them in the 290 nm15,16 and the rutin have bands at 266 and 350 nm.17 concentrations tested (Fig. 1). Previous studies concerning The same results were observed in the analysis of the me- the scavenger activities of Equisetum arvense, Equisetum thanolic extract. The determination of total flavonoids ramosissimum L., and Equisetum telmateia L. showed high

showed that both extracts presented *0.9% of flavonoid scavenger abilities against DPPH, NO, and OH radicals compounds (Table 1) and did not present differences sta- and also high total antioxidant capacity according to the tistically significant by ANOVA. The results show the fluorescence recovery after photobleaching method.19 The similarity between the phytochemical compositions of these methanolic extract of E. hyemale, using DPPH method, two extracts, presenting in its composition the presence of exhibited a percentage of antiradical activity of 24.29% at phenolic compounds. Phenols are compounds comprising a the concentration of 100 lg/mL,2 lower than the results wide variety of molecules represented in the majority of obtained in the present study. The pharmaceutical, cos- secondary metabolite classes. Flavonoids have structures metic, and food industries search for novel compounds that provide antioxidant properties and radical scavenging extracted from plant species that can be utilized as

FIG. 1. Antioxidant activity of the vitamin C and 70% ethanolic and me- thanolic extracts of E. hyemale. Values followed by same superscript letters did not show statistically significant differences by Tukey’s test (P < .05). Downloaded by Shanghai Information Center for Life Sciences, CAS from online.liebertpub.com at 10/29/17. For personal use only. EQUISETUM HYEMALE PHYTOCHEMICAL AND ACTIVITIES 833

antioxidant sources. The literature suggests that the AUTHOR DISCLOSURE STATEMENT horsetail extracts could be used as natural antioxi- No competing financial interests exist. dants based on its phytochemical composition.6 Horsetail extract is used in medicinal preparations, globally mar- keted for several therapeutic purposes.20 Another product, REFERENCES named Eviprostat , used to treat benign prostatic hyper- 1. Xu CF, Bian XY, Qu SM, You LH, Qi ZM, Cheng W, Liu XJ, plasia, presents one component which was extracted from Liu WZ, Ren SJ: Effect of Equisetum hyemale on experimental E. arvense. According to studies in rats, the removal of free hyperlipemia in rats and its toxic test. Zhongguo Zhong Yao Za radicals caused by this component contributes to the anti- Zhi 1993;18:52–53. inflammatory action of Eviprostat involving their thera- 2. Park EY, Jeon H: Antioxidant and anti-inflammatory activities of 21 peutic effect. Equisetum hyemale. Nat Prod Sci 2008;14:239–243. 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