Phytochemical Characterization,Antimicrobial Activity,and Antioxidant Potential of Equisetum hyemale L. (Equisetaceae) Extracts

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 amperometric detector analyses, as well as its antioxidant potential based on the scavenger activity of 2,2-diphenyl-1-picrylhydrazyl (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 scavenging 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 Microsporum 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.

Introduction

E quisetum hyemale , popularly known as horsetail, is used commonly to prepare infusions to combat infectious diseases in the kidney or urinary tract and presents diuretic effects. There are other reports showing some properties related to E. hyemale such as hyperlipidemic, antimicrobial, hepatoprotective, antioxidant, and anti-inflammatory activities.^1,2 Previous studies identified kaempferol,³ glycosil flavonoids,⁴ silica,⁵ quercetin,⁶ and ethyl palmitate in the essential oil.⁷ The Equisetum genus has been widely chemically and biologically characterized in different regions due to folk use and the commerce of derivative products with this species in its formulations.⁶ Currently there are several products on the world market using extracts of species of this genus. Among the phytochemical preparations for cosmetic purposes there is enamel that uses water-soluble extract of Equisetum arvense in the formulation and that proved to be effective in strengthening the nails, reducing brittleness and surface roughness of brittle nails, being able to improve the appearance of psoriatic nails.⁸ This background justifies new studies aiming at the antioxidant and antimicrobial potential of E. hyemale extracts, based on their phytochemical constitution, to verify its potential as a medicinal agent.

Materials and Methods

Plant material

The material of E. hyemale was collected in March 2009, of “Horto de Plantas Medicinais e Tóxicas de Araraquara,” São Paulo, Brazil and the identification was performed by Prof. Dr. Marco Antônio de Assis. The voucher specimen was deposited in the UNESP Herbarium (Rio Claro, SP) with number 51670 HRCB. The aerial parts of E. hyemale were dried in an oven with air circulation at 40°C until stabilization and then pulverized. Eighty grams of plant material was submitted to the exhaustive percolation using ethanol (70%) and methanol as solvents.⁹ The extracts obtained were concentrated under vacuum, lyophilized, and stored in desiccators.

Analyses by high-performance liquid chromatography with pulsed amperometric detector

The chromatographic profile of the 70% ethanolic extract was established using a Jasco liquid chromatography equipped with a PU-2089 quaternary solvent pump, a MD-2010 PAD, and a Rheodyne 7725 sample injector with a 20 μL sample loop. The analytical column was a Phenomenex Synergi Hydro RP18 (250×4.6 mm i.d.; 4 μm) equipped with a Phenomenex security guard column (4.0×2.0 mm i.d.). The mobile phase was composed by water (eluent A) and methanol (eluent B), both containing 0.05% of trifluoroacetic acid, with 5–100% B gradient. The flow rate was 1.0 mL/min and the total run time was 55 min. The EZChrom Elite Data System software was used for both the operation of detector and for data processing. Standard substances were obtained from Sigma (caffeic acid, chlorogenic acid, and rutin, 99.5% purity).

Determination of total flavonoids contents

The determination of total flavonoids was performed by a colorimetric method based on the formation of a complex flavonoid–aluminum.¹⁰ All determinations were made in triplicate and the values were calculated from a calibration curve obtained with the quercetin standard. The final results were expressed as milligrams of equivalents in quercetin per gram of extract (mgEQ/g). The reading was performed at 430 nm in a Shimadzu-1603 spectrophotometer. To prepare the samples, the extracts were diluted (10.0 mg in 20.0 mL MeOH 80% v/v) and aliquots of each solution were added to 2.0 mL of AlCl₃. (6H2O) in MeOH 2% (v/v), completing to final volume of 4.0 mL in MeOH solution 80% (v/v). Both extracts were analyzed in concentrations 125, 150, 175, 200, 225, and 250 μg/mL. The concentration of the standard for the calibration curve ranged from 0.25 to 4.0 μg/mL.

Evaluation of the ability of free radical scavenging

The potential antioxidant activity of extracts was determined based on the radical scavenging activity of 2,2-diphenyl-1-picrylhydrazyl (DPPH). The tests were performed with E. hyemale extracts obtained at concentrations of 25, 50, and 100 μg/mL in methanol. The positive control was ascorbic acid (vitamin C) at the same concentrations. To 1 mL of sample solution was added 2.5 mL of 0.004% DPPH in methanol. The solutions were kept in the dark for 30 min at room temperature and after the reading was done at 517 nm in a Shimadzu-1603 spectrophotometer. The blank solution consisted of methanol and the negative control of 1 mL of methanol and 2.5 mL of DPPH.

Microorganisms and antimicrobial activity

The strains used were: Staphylococcus aureus (ATCC 25923), Staphylococcus epidermidis (ATCC 12228), Escherichia coli (ATCC 25922), Bacillus subtilis (ATCC 9392), Pseudomonas aeruginosa (ATCC 23212), and Candida albicans (ATCC 64548). The dermatophytes fungi Trichophyton rubrum, Trichophyton mentagrophytes, and Microsporum canis (clinical isolates) were kindly provided by Prof. Dr. Ana Marisa Fusco Almeida (Mycology Laboratory, School of Pharmaceutical Sciences of Araraquara, UNESP). Determination of minimum inhibitory concentration (MIC) against dermatophyte fungi, aerobic bacteria, and yeast C. albicans were performed using the microdilution technique in 96-well plates, according to the protocols M38-A, M7-A7, M27-A3, respectively, of the Clinical and Laboratory Standards Institute^11

–14 with modifications.

Stock solutions of the extracts were dissolved in DMSO (50 mg/mL) and evaluated at concentrations between 1.25 to 0.12 mg/mL, except for oral microorganisms in which concentrations tested ranged from 2.00 to 0.09 mg/mL, using DMSO as control solvent (1:5, v/v). Ampicillin was used as positive control against bacteria and amphotericin B against yeast and fungi. The yeast growth used RPMI-1640, incubated at 35°C for 48 h under constant agitation at 150 rpm. To filamentous fungi, RPMI-1640 medium supplemented with glucose was used, incubated at 28°C under constant agitation at 100 rpm for 7 days. MICs of bacteria were determined as the lowest concentration presenting blue coloration after 2 h of incubation with 0.01% Resazurin solution. MIC of yeast was determined as the lowest concentration in which the cells were stained after 3 h of incubation with 2% solution of 2,3,5-triphenyl tetrazolium. MIC of fungi was determined as the lowest concentration of extract in which there was no development of microorganisms. Minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC) were determined as the lowest concentration in which there was no development of colonies after subculturing samples from the microplates on agar of each microorganism in appropriate medium.

Statistical analysis

All experiments were carried out in triplicate, determining the average and standard deviation of the results. The determination of total flavonoids and the ability of scavenging free radicals were submitted to analysis of variance (ANOVA) by Tukey test, with P<.05.

Results and Discussion

The yield was 12.16 g (15.16%) to 70% ethanolic extract and 7.40 g (9.23%) to methanolic extract. The spectrum of gallic acid, tannic acid, and the samples of E. hyemale presented absorbance profiles between 200 and 250 nm and 250–300 nm, similar to phenolic compound used as reference, suggesting the presence of this class of metabolites (data not showed). The identification of these compounds on 70% ethanolic extract was performed with the aid of PDA detector scanning, in the spectral range of 200–600 nm. The analytical high performance liquid chromatography with pulsed amperometric detector chromatogram recorded at 255 nm with its respective UV spectra, highlighting the peaks with retention time of 20.5, 21.0, and 22.5 min. These compounds were identified by comparing the peaks of their UV spectra with those of the available standards and compared with the references in the literature. The chlorogenic acid and caffeic acid have characteristic bands at 280–290 nm^15,16 and the rutin have bands at 266 and 350 nm.¹⁷ The same results were observed in the analysis of the methanolic extract. The determination of total flavonoids showed that both extracts presented ∼0.9% of flavonoid compounds (Table 1) and did not present differences statistically significant by ANOVA. The results show the similarity between the phytochemical compositions of these two extracts, presenting in its composition the presence of phenolic compounds. Phenols are compounds comprising a wide variety of molecules represented in the majority of secondary metabolite classes. Flavonoids have structures that provide antioxidant properties and radical scavenging abilities. Therefore, it has been used as anti-inflammatory, anticarcinogenic, and photoprotective agents in various biological systems.¹⁸

Table 1.

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

	70% ethanolic extract (mg/mL)		Methanolic extract (mg/mL)			CF (mgEQ/g)
Microorganisms	MIC	MBC/MFC	MIC	MBC/MFC	Concentration (μg/mL)	70% ethanolic extract	Methanolic extract
S. aureus	>1.25	>1.25	>1.25	>1.25	125	9.15^a	8.34^a
S. epidermidis	>1.25	>1.25	>1.25	>1.25	150	9.66^a	9.09^a
B. subtilis	>1.25	>1.25	>1.25	>1.25	175	9.71^a	10.17^a
E. coli	>1.25	>1.25	>1.25	>1.25	200	10.28^a	9.65^a
P. aeruginosa	>1.25	>1.25	>1.25	>1.25	225	10.00^a	9.51^a
C. albicans	>1.25	>1.25	>1.25	>1.25	250	10.02^a	9.20^a
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.

Both extracts showed antioxidant activity above 30%, showing no significant difference between them in the concentrations tested (Fig. 1). Previous studies concerning the scavenger activities of Equisetum arvense, Equisetum ramosissimum L., and Equisetum telmateia L. showed high scavenger abilities against DPPH, NO, and OH radicals and also high total antioxidant capacity according to the fluorescence recovery after photobleaching method.¹⁹ The methanolic extract of E. hyemale, using DPPH method, exhibited a percentage of antiradical activity of 24.29% at the concentration of 100 μg/mL,² lower than the results obtained in the present study. The pharmaceutical, cosmetic, and food industries search for novel compounds extracted from plant species that can be utilized as antioxidant sources. The literature suggests that the horsetail extracts could be used as natural antioxidants based on its phytochemical composition.⁶ Horsetail extract is used in medicinal preparations, globally marketed for several therapeutic purposes.²⁰ Another product, named Eviprostat^®, used to treat benign prostatic hyperplasia, presents one component which was extracted from E. arvense. According to studies in rats, the removal of free radicals caused by this component contributes to the anti-inflammatory action of Eviprostat involving their therapeutic effect.²¹

FIG. 1.

Antioxidant activity of the vitamin C and 70% ethanolic and methanolic extracts of E. hyemale. Values followed by same superscript letters did not show statistically significant differences by Tukey's test (P<.05).

Some chemical analysis showed that E. hyemale contains phenolic compounds,²² for example, the flavonoids,²³ and there are reports that certain flavonoids show some anticancer activity.²⁴ In a study done by Li et al., ²⁵ they observed that the 50% ethanolic extract of E. hyemale presented cytotoxic activity against cancer cells (L1210), and a low level of toxicity to healthy cells (peripheral blood mononuclear cell) from mice, it can be potentially useful as an agent for the prevention or treatment of cancer. Another recent study done by Jin et al.,²⁶ isolated a new phenyl glycoside of 70% ethanol extract of the aerial parts of E. hyemale, which showed that some compounds present in this extract possess significant hepatoprotective effect.

Several studies have demonstrated that Equisetum sp. extracts presented antimicrobial activity in higher concentrations by different analyses.^27
–29 However, no other previous study evaluated extracts of E. hyemale against dermatophytic fungi, configuring this work as a pioneer. The results of MIC, MBC, and MFC for both extracts tested in this work were higher than the highest concentration tested against all bacterial strains and C. albicans. Moreover, the MIC and MFC for dermatophyte fungi ranged between 62 and 125 μg/mL (Table 1). Studies related to the antimicrobial activity of the E. hyemale showed a variety of results due to the different methods employed, concentrations, and microorganisms tested. Analyzing a series of investigations of biological activity performed with the genus Equisetum in the literature, it is possible to observe that the species of this genus are popularly used in several geographic regions. It seems that this species could represent a potential source of antimicrobial agents independent of being used in the future as an antiseptic, disinfectant, or other therapeutic agent. According to some authors, the ideal MIC for plant extracts should be lower than 100 μg/mL.³⁰ Although our results showed concentrations above this limit, they are more significant when compared with literature data for extracts of E. hyemale obtained from other regions. On the other hand, besides the resistant dermatophytes tested, the extracts showed good results, with MIC ranging between 0.62–1.25 mg/mL. To improve and to verify what the mechanism of action of these samples are, additional studies must be performed searching for enriched fractions or pure compounds with a biocidal potential.

Footnotes

Acknowledgments

This study was supported by CAPES and PADC-UNESP.

Author Disclosure Statement

No competing financial interests exist.

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