Antimicrobial Activity of Lippia Species from the Brazilian Semiarid Region Traditionally Used as Antiseptic and Anti-Infective Agents

Lippia origanoides Kunth, Lippia alnifolia Schauer, and Lippia thymoides Martius and Schauer are shrubs used in the traditional Brazilian medicine as antiseptics, as well as in the treatment of infectious diseases. This study was designed to investigate the antibacterial and antifungal activities of the methanolic extracts of these species, as new potential sources of antimicrobial drugs. The antimicrobial activity of methanolic extracts was investigated against resistant yeasts and bacteria by agar disk diffusion. Then, the MIC determination of the most active species and its fractions in hexane, dichloromethane, ethyl acetate, and water was performed. By the agar diffusion assay, all species were active against at least two microorganisms, giving evidence to support their use in the popular medicine. L. origanoides leaves exhibited the widest antimicrobial action, inhibiting the growth of two Gram-positive bacteria and two yeasts; this activity was also confirmed by the MIC evaluation. The fractionation of L. origanoides crude extracts improved the activity in spectrum and intensity. The results obtained in this study indicate that L. origanoides may be a promising alternative in the treatment of bacterial and fungal infections and in the seeking of new antimicrobial drugs.


Introduction
The development and spread of antimicrobial resistance is a worldwide concern, due to the negative impacts on public health [1]. Therefore, there is a growing need for novel drugs against bacterial, fungal, and viral infections. The traditional knowledge on the therapeutic potential of plants has been attracting scientific interest seeking new ways to control and treat many diseases caused by microorganisms [2][3][4].
The genus Lippia has a great number of medicinal species, such as L. origanoides Kunth, L. alnifolia Schauer, and L. thymoides Martius and Schauer, which are frequently used in folk medicine for the treatment of microbial diseases [5]. In Brazil, L. origanoides is popularly known as "salva-de-Marajó and "alecrim d' Angola. " The leaf infusions are used as a general antiseptic for the mouth, throat, and wounds, for the treatment of baby colic, diarrhea, indigestion, flatus, heartburn, nausea, vaginal discharges, menstrual complaints, 2 Evidence-Based Complementary and Alternative Medicine and fever [5,6]. L. alnifolia is also known as "alecrimdo-mato, " "pedrécio, " and "alecrim-de-vaqueiro. " The leaves are employed as a topic antiseptic against dermatitis and dandruff, as well as an oral antiseptic and in vaginal infections [7,8]. Aerial parts of L. thymoides, known as "alecrim-domato, " are used in the treatment of skin infections [7].
Although the traditional use of these plants, studies supporting the antibiotic activity of L. alnifolia and L. thymoides are not found in the literature. Antimicrobial activity has been reported to L. origanoides, though it was related to the leaf essential oils [7,[9][10][11]. Therefore, a bioprospecting study was designed to investigate the antimicrobial activities of L. alnifolia, L. origanoides, and L. thymoides extracts from the Brazilian semiarid region against bacteria and yeasts, in order to select the most active species.

Extracts Preparation.
The air dried and powdered stems, leaves, and flowers were macerated in methanol for 10 days at room temperature. Extraction was repeated at least five times, and the filtrates of all portions were combined. The solvent was removed by evaporation under vacuum on reduced pressure at 40-45 ∘ C using a rotary evaporator. Water residues were removed by lyophilization and packed in a glass pot to store in a refrigerator.

Fractions Preparation.
The dry methanolic crude extracts (20 g) were resuspended in a methanol : water solution (1 : 1) and extracted with hexane (3 × 500 mL). After methanol removal by evaporation, the residual aqueous solution was subjected to a sequential partition with dichloromethane (3 × 500 mL) and ethyl acetate (3 × 500 mL). The solvent of each fraction, including the final aqueous fraction, was removed as described above.

Agar Disk Diffusion Method (ADD). A screening for antimicrobial activity was performed by Agar Disk Diffusion method, according to the Clinical and Laboratory Standards
Institute [12], with some modifications. Methanolic solutions of the extracts at 200 mg⋅mL −1 were sterilized by filtration through a 0.22 m membrane filter. Filter paper disks (ø 6 mm) were impregnated with 5 L of each extract solution (1 mg of crude extract/disk), and the methanol was evaporated at room temperature for 2 h. The microorganisms were grown on Agar Type 1 Himedia-RM 666 and Mueller Hinton Himedia-M391 broth. After 18 h (bacteria) and 36 h (yeast), the inoculum was adjusted to 5 × 10 5 UFC⋅mL −1 and 1.5 × 10 8 UFC⋅mL −1 , respectively, in saline solution at 0.45%. Microorganism suspensions (100 L) were uniformly spread in Petri plates containing Mueller-Hinton Agar. The paper disks impregnated with the samples were placed on the surface of the agar. The plates were incubated at 28 ∘ C/48 h and 37 ∘ C/18 24 h for yeasts and bacteria, respectively. Inhibition zones (mm) were measured, and a positive result was considered with halos larger than 6.5 mm (disk diameter included). Nystatin (10 g/disk), erythromycin, and chloramphenicol (30 g/disk) were used as positive controls against yeasts and bacteria. The tests were done in triplicate.

Minimum Inhibitory Concentration (MIC) Tests.
The determination of the Minimum Inhibitory Concentration was performed as described in the CLSI [13,14], with modifications. The extracts or fractions (42 mg) solubilized in DMSO 50% were sterilized by filtration through a 0.22 m membrane filter. In 96-well plates, 95 L of extracts solution and 95 L of Mueller-Hinton broth (2X concentrated) were conditioned in the first well, and the seriated dilutions were carried out in all subsequent wells. The range of evaluated extract concentration was from 21.05 mg⋅mL −1 to 0.01 mg⋅mL −1 . Cultures of 18 h (bacteria) and 36 h (yeast) were collected to saline solution 0.45%, and 5 L of microorganism suspension at 9 × 10 6 UFC⋅mL −1 and 5 × 10 5 UFC⋅mL −1 (bacteria and yeasts, resp.) was added in each well. The microplates were incubated at 28 ∘ C/48 h for yeasts and 37 ∘ C/24 h for bacteria. An aqueous solution of 2,3,5-triphenyltetrazolium chloride (50 L at 5 mg⋅mL −1 ) for yeasts or rezasurin (30 L at 0,1 mg⋅mL −1 ) for bacteria was added in all wells. The result was read after 3 h of incubation, and red wells were considered an indication of microbial growth. The MIC was defined as the lowest concentration in which there was no visible growth after incubation. Controls with nystatin for yeasts (10 mg⋅mL −1 ) and chloramphenicol (20 mg⋅mL −1 ) for bacteria were made. Controls of the microbial strains viability, sample sterility, and water were also performed. The MIC of the DMSO solution in water (50%) was also determined. All tests were done in triplicate. Plant extracts with MICs ≤2.631 mg⋅mL −1 were considered active antimicrobial agents.

Phytochemical Screening.
A phytochemical screening was performed on all fractions of L. origanoides by thin layer chromatography, according to the procedure described in the TLC Atlas "Plant Drug Analysis" [15].
Evidence-Based Complementary and Alternative Medicine 3

Results and Discussion
The disk diffusion assay results are summarized in Table 1. All extracts have shown antimicrobial activity, inhibiting at least one of the evaluated microorganisms, as for the flowers and stems extracts of L. thymoides. A wide spectrum antimicrobial activity was noted with L. origanoides leaves, inhibiting the growth of two S. aureus strains, in addition to C. albicans and C. parapsilosis. S. aureus CCMB262 was the most sensitive strain concerning theantibacterial effect of the extracts (seven out of eight extracts were active), whereas E. coli and P. aeruginosa were the least sensitive microorganisms (all extracts inactive). In general, the Gram-positive bacteria were the most sensitive to the tested methanolic extracts, with inhibition zones of 6.9 to 12.3 mm. This higher resistance of Gram-negative bacteria was expected, due to the polysaccharide outer membrane with a lower permeability and efflux pumps. This system prevents the accumulation of the antimicrobial agent inside the cell, thus impeding the substance from reaching its target or the concentration becoming lethal to the cell [16].
Despite the low permeability of the fungal cell walls, composed of 1,3--glucan and chitin polymers [17], an anticandidal activity was observed in L. alnifolia flowers, as well as in L. origanoides leaves. C. parapsilosis was the most sensitive yeast, which was inhibited by two out of eight extracts.
L. origanoides was selected for further assays (fractionation by liquid-liquid partition, MIC determination, and phytochemical screening) because of its wide spectrum of antimicrobial activity. The MIC determination (Table 2) 4 Evidence-Based Complementary and Alternative Medicine  showed that leaf crude extracts also inhibited the microbial growth of all evaluated Gram-positive bacteria and yeasts (MIC ranging from 2.631 to 0.658 mg⋅mL −1 ), in agreement with the antimicrobial results by the previous ADD test. However, stem methanol extracts, although inactive in the ADD assay, showed action against S. aureus CCMB263 and C. albicans, in the latter with MIC values similar to those obtained by the leaf extracts. The reason for the difference in sensitivity between the two methods might be related to diffusion properties of the antimicrobial compounds from the extracts into the agar and adsorption in the paper disk [18]. The fractions from leaves and stems exhibited varying degrees of antimicrobial action, with hexane and ethyl acetate fractions from leaves showing the highest antibacterial effect against S. aureus (MIC-0.329 mg⋅mL −1 ). An enhanced activity against all microorganisms was observed with the fractionation, including against E. coli, to which the extracts were considered inactive. Dichloromethane fractions from leaves and stems, as well as the ethyl acetate fraction from leaves, were the most efficient against E. coli (MIC-0.658 mg⋅mL −1 ). The best result against C. albicans was obtained by the dichloromethane fraction from the stems (MIC-0.658 mg⋅mL −1 ), a noteworthy result considering that invasive candidiasis is a leading cause of mycosis-mortality [19].
The results of phytochemical screening of L. origanoides fractions showed the presence of terpenes, steroids, coumarins, saponins, flavonoids, and phenolic acids in stems and leaves, while alkaloids were absent (Table 3). These phytoconstituents, common in Lippia [5], are recognized as antimicrobial agents against several bacteria and yeasts [20]. Terpenes were the predominant metabolites in the hexane fractions and flavonoids in ethyl acetate fractions. These constituents may be the responsible for the improved antibacterial activity in hexane and ethyl acetate fractions.

Conclusions
The antimicrobial activity by ADD gives preliminary scientific evidence to the traditional use of L. alnifolia, L. origanoides, and L. thymoides as antiseptic agents and in the treatment of infectious diseases. Although the results of this study suggest the antibacterial potential of three evaluated species, the L. origanoides leaves exhibited the widest antimicrobial action being a promising alternative in the treatment of bacterial and fungal infections. As the fractionation of L. origanoides crude extracts improved the activity in spectrum and intensity, further investigations are in progress to isolate and characterize the active metabolites.