Biological properties of medicinal plants: a review of their antimicrobial activity

Plants have been used for thousands of years to flavor and conserve food, to treat health disorders and to prevent diseases including epidemics. The knowledge of their healing properties has been transmitted over the centuries within and among human communities. Active compounds produced during secondary vegetal metabolism are usually responsible for the biological properties of some plant species used throughout the globe for various purposes, including treatment of infectious diseases. Currently, data on the antimicrobial activity of numerous plants, so far considered empirical, have been scientifically confirmed, concomitantly with the increasing number of reports on pathogenic microorganisms resistant to antimicrobials. Products derived from plants may potentially control microbial growth in diverse situations and in the specific case of disease treatment, numerous studies have aimed to describe the chemical composition of these plant antimicrobials and the mechanisms involved in microbial growth inhibition, either separately or associated with conventional antimicrobials. Thus, in the present work, medicinal plants with emphasis on their antimicrobial properties are reviewed.


INTRODUCTION
The use of plants for treating diseases is as old as the human species. Popular observations on the use and efficacy of medicinal plants significantly contribute to the disclosure of their therapeutic properties, so that they are frequently prescribed, even if their chemical constituents are not always completely known. All over the globe, especially in South American countries, the use of medicinal plants has significantly supported primary health care (1). From 250 to 500 thousand plant species are estimated to exist on the planet, and only between 1 and 10% are used as food by humans and other animals (2). Brazil has the world's highest biodiversity, accounting for over 20% of the total number of known species. This country presents the most diverse flora, with more than 55 thousand described species, which corresponds to 22% of the global total. Such biodiversity is followed by a wide acceptance of the medicinal plant use (3). Most of the Brazilian population (80%) consumes only 37% of the commercially available drugs and depend almost exclusively on medicines of natural origin (4). Thus, phytotherapics entered the market promising a shorter and cheaper production, since basic requirements to use medicinal plants do not involve strict quality control regarding safety and efficacy compared to the other types of drugs (5).
Infectious diseases represent an important cause of morbidity and mortality among the general population, particularly in developing countries. Therefore, pharmaceutical companies have been motivated to develop new antimicrobial drugs in recent years, especially due to the constant emergence of microorganisms resistant to conventional antimicrobials. Apparently, bacterial species present the genetic ability to acquire and transmit resistance against currently available antibacterials since there are frequent reports on the isolation of bacteria that are known to be sensitive to routinely used drugs and became multiresistant to other medications available on the market (6,7). Consequently, common strategies adopted by pharmaceutical companies to supply the market with new antimicrobial drugs include changing the molecular structure of the existing medicines in order to make them more efficient or recover the activity lost due to bacterial resistance mechanisms (8).
On the other hand, given the search for new antimicrobials, those of plant origin must be emphasized since Brazil possess such great biodiversity among which a large number of plants have been used for diverse purposes and tested throughout the globe for hundreds of years by different populations.

ANTIMICROBIAL PROPERTIES OF MEDICINAL PLANTS
The antimicrobial actions of "carqueja" (Baccharis trimera Less.) decoction on grampositive (Staphylococcus aureus and Streptococcus uberis) and gram-negative (Salmonella gallinarum and Escherichia coli) bacterial strains were evaluated and it was found that the former microorganisms are more sensitive to this herb than the latter, which corroborates previous studies (9). Similarly, antimicrobial assays with plant extracts used in Asia (Ruta graveolens and Zingiber officinale) revealed an inhibitory capacity against Bacillus cereus strains (10).
In another study, the inhibitory activity of concentrates from 14 Brazilian plants against methicillin-resistant Staphylococcus aureus (MRSA) strains was analyzed (11). The substances that demonstrated inhibitory activity were ethanol extract and its fractions (n-hexane, water, chloroform, dichloromethane, ethyl acetate and n-butanol) from Punica granatum fruit (pomegranate) and parts of T. avellanedae wood (purple trumpet tree). The greatest activities were found in the ethyl acetate fraction from P. granatum and hexane and chloroform fractions from T. avellanedae.
As to the common yarrow (Achillea millefolium), its essential oil (obtained from stem and leaves) presents higher antimicrobial activity than its respective extracts (methanol extract separated by chloroform into parts that were not all soluble). The oils prevented the growth of Streptococcus pneumoniae, Clostridium perfringes and Candida albicans and slightly inhibited Mycobacterium smegmatis, Acinetobacter lwoffii and Candida krussei (12) A study evaluated the effects of some plant extracts (aqueous and 40% hydroalcoholic) against bacteria found in the oral cavity of dogs (13). It found that standard S. aureus strain and isolated Streptococcus oralis and Streptococcus mitis strains were sensitive to extracts from garlic (Allium sativum), "espinheira santa" (Maytenus ilicifolia) and guava tree leaves (Psidium guajava).
Similarly, antibacterial properties against Staphylococcus aureus were found in chamomile. The phenolic compounds present in its ethanol extract are responsible for this activity (14). It was also reported that the aqueous extract from the artichoke (Cynara scolymus) and the ethanol extracts (80%) from both artichoke and "macela" (Achyrocline satureioides) inhibited the growth of Bacillus cereus, B. subtilis, Pseudomonas aeruginosa and S. aureus (14). In Argentina, terpene compounds (eugenol, geraniol, thymol and carvacrol) derived from essential oils of native plants showed inhibitory effects on MRSA (15).
In another work, essential oils from 28 plants were tested against ETEC (enterotoxigenic E. coli) and EPEC (enteropathogenic E. coli) serotypes, and the results indicated that palmarosa (Cymbopogon martinii), a highly common plant in Brazil, presents a wide spectrum of action against three ETEC and two EPEC serotypes, whereas Java citronella grass (Cymbopogon winterianus) inhibited one EPEC and two ETEC serotypes (16). The concentration responsible for the microbial inhibition varied between 100 and 500 µg/mL, while other plants caused inhibition only at higher concentrations.
In a study carried out in an indigenous community, it was reported that the hydroalcoholic extracts from Vernonia polyanthes ("assa-peixe"), Aristolochia triangularis ("cipó mil-homens"), Tabebuia avellanedae (purple trumpet tree) and Stryphnodendron adstringens ("barbatimão") presented a significant antimycobacterial effect and that a local beverage, similar to rum (with an ethanol content of 30%), was employed to prepare the extracts (18).
Furthermore, the results of a recent study described a potent inhibitory activity of Vernonia polyanthes extract against Leishmania strains (19). However, its concentrate had no antifungal action under the same conditions. Similarly, Baccharis dracunculifolia oil ("alecrim-do-campo") at a 10-µL dose prevented microbial growth of E. coli, S. aureus and P. aeruginosa in antimicrobial assays (20).
The essential oils from Pelargonium graveolens (geranium) present low values of minimum inhibitory concentration against B. cereus (0.36 mg/mL), B. subitilis (0.72 mg/mL) and S. aureus (0.72 mg/mL), whereas Origanum vulgare (oregano) oils also show antimicrobial activity against the same bacteria, in addition to E. coli; however, in the latter, a concentration of 0.35 mg/ mL is required to inhibit B. subitilis whereas 0.70 mg/mL is necessary to inhibit the other bacteria (21).
In a recent study carried out at the Department of Microbiology and Immunology, Botucatu Biosciences Institute, UNESP, tests were performed utilizing extracts from A. sativum (bulbs), Z. officinale (rhizomes), Caryophyllus. aromaticus (flower buds), C. citratus (leaves), P. guajava (leaves) and M. glomerata (leaves) against Enterococcus sp., E. coli, S. aureus and Salmonella. The extracts from garlic (A. sativum) and ginger (Z. officinale) presented the most intense activity against gram-negative bacteria; for garlic, concentrations ranged from 1.38 to 1.61 mg/mL while for ginger it was 6.97. Grampositive strains were more susceptible to guava extracts at concentrations between 0.77 and 1.74 mg/mL, and to clove extracts at concentrations from 0.46 to 1.24 mg/mL (22).
Costa et al. (23) tested the inhibitory capacity of essential oils from Croton zehntneri (wild cinnamon) leaves against Shigella flexneri, Salmonella Typhimurium, E. coli, S. aureus and Streptococcus β-hemolyticus strains and found antimicrobial activity against all bacteria, except Salmonella. Moreover, the inhibitory action against S. flexneri was highly significant, with a minimal inhibitory concentration of 25 µg/mL (23).
Remarkable results using the disc methodology were established in tests on the leaf, phloem and latex of Croton urucurana ("urucuana") against the bacteria Enterococcus faecalis, S. aureus, Staphylococcus epidermidis, Streptococcus pyogenes, E. coli, Klebsiella pneumoniae, P. aeruginosa, Salmonella Typhimurium and S. flexneri (24). It was found that latex inhibited all tested bacteria except E. coli and showed strong activity against K. pneumoniae and P. aeruginosa (0.125 to 1 mg/disc), whereas leaf hexane extract presented the widest spectrum against gramnegative bacteria and strong action against K. pneumoniae and P. aeruginosa (0.25 mg/disc). The dichloromethane extract was active only against S. pyogenes (0.5 mg/disc), whereas hydroalcoholic extract acted against gram-positive bacteria and revealed a potent action against Salmonella Typhimurium (0.5 to 1 mg/disc). On the other hand, the ethyl acetate extract was inactive. As to phloem, the hexane and dichloromethane extracts were active against S. aureus, S. epidermidis and P. aeruginosa at concentrations from 0.5 to 1 mg/ disc; chloroform extract was the most potent against E. faecalis, S. aureus, S. pyognes and K. pneumoniae, at concentrations ranging from 0.25 to 1 mg/disc, but was ineffective against S. flexneri and Salmonella Typhimurium. Furthermore, ethyl acetate extract presented an antimicrobial effect only against K. pneumoniae and S. epidermidis, whereas 75% ethanol extract had a wide activity against E. faecalis, S. pyogenes and P. aeruginosa strains; for both extracts the concentration on the disc was between 0. 25  The ethanol extract from Hyptis martiusii suppressed the growth of E. coli and MRSA strains (at concentrations between 128 and 512 µg/mL) and was more effective when compared with gentamicin and methicillin (27). Likewise, the ethanol extract and essential oil from Myrtus communis (myrtle) presented antibacterial effect against B. subtilis and S. aureus, but not against E. coli (28). Extracts from the peel of Punica granatum fruit (pomegranate) were inhibitory against 38 S. aureus strains (29).
Although there are numerous previous studies on medicinal uses of natural products, their applicaton as food additive has only been reported in recent years. Thus, assays with Listeria monocytogenes, S. aureus, E. coli and Salmonella Enteritidis strains isolated from food and essential oils from leaves of oregano, thyme, basil, marjoram, lemongrass, ginger rhizomes and clove flower buds were performed (31). These bacterial strains were susceptible to the oils, among which lemongrass was the most effective against gram-positive microorganisms, followed by clove and ginger; the concentrations (%v/v) were respectively 0.05, 0.09 and 0.09. In addition, thyme and clove were the most effective against gram-negative bacteria at the concentration of 0.10%v/v. The potential use of these essential oils from aromatic plants to conserve meat and meat derivatives deserves further research.
The antibacterial activity of essential oils from oregano (Origanum vulgare) against multiresistant bacteria -including E. coli, E. faecalis, Acinetobacter baumannii, K. pneumoniae, P. aeruginosa and MRSA -was analyzed by Costa et al. (33). Concentrations of at least 0.125% showed higher antimicrobial efficiency on the studied bacterial strains, although P. aeruginosa was found to be more resistant to the essential oil because it was only inhibited at the concentration of 0.5% (33 (34).
In another study, essential oils from rosemary (R. officinalis), clove (Caryophyllus aromaticus L.), ginger (Z. officinalis), lemongrass (C. citratus), peppermint (M. piperita) and cinnamon (Cinnamomum zeilanicum Blume) were tested against S. aureus and E. coli strains. The oils showed some antimicrobial action; ginger essential oil was the most efficient against S. aureus while cinnamon and clove were the most effective against E. coli at 0.09%v/v (35). Additionally, the cyclohexane extract of Monodora myristica fruit and the ethyl acetate extract from the stem bark of Albizia gummifera were effective in inhibiting C. albicans and Candida krusei (36).
As in previous studies, the antimicrobial activity of essential oils from oregano (Origanum vulgare), thyme (Thymus vulgaris), marjoram (Origanum majorana) and basil (Ocimun basilicum) was evaluated in meat products against strains of L. monocytogenes and Salmonella Enteritidis (37). It was reported that all oils showed antibacterial activity and that, particularly, gram-positive bacteria were more susceptive to them.
Based on the aforementioned data, it is possible to conclude that the literature on testing antimicrobial activity of plant products is broad, including an increasing number of publications per year. Therefore, it is difficult to integrate all those numerous studies on the antimicrobial action of plant products into the present review; a multidisciplinary approach to this theme is increasingly required.

INTERACTION BETWEEN NATURAL PRODUCTS AND ANTIMICROBIAL DRUGS
In addition to the antimicrobial action of plant extracts and essential oils, a synergism between conventional antimicrobial drugs and products obtained from medicinal plants has also been reported. Possible interactions among medications are frequently observed, which has motivated researchers to test such possibilities. However, it must be emphasized that interactions between synthetic and natural drugs depend on several factors including pharmacokinetics and employed doses, since combinations confirmed in vitro may not have the same effect on humans (38). Nevertheless, innumerous studies on this particular theme can be found in the literature.
Synergism assays between terpenes and penicillin against MRSA and E. coli revealed a synergistic effect produced by the combination of carvone and penicillin whereas an antagonistic effect between thymol and penicillin was detected against MRSA strains. Regarding E. coli, synergism was present among penicillin, eugenol and thymol; however, terpene and myrcene only revealed an antagonistic effect (15).
The synergistic effect between plant extracts -clove, jambul (Syzygium cumini), pomegranate and thyme -and some antimicrobial drugs was also evaluated. Garlic extract, when combined with ampicillin, revealed some effects on Klebsiella pneumoniae whereas Proteus sp. growth was inhibited by an association between clove extract and tetracycline. These extracts presented antimicrobial activity, even against microorganisms resistant to antibiotics, thus acting either separately or in association with antibiotics used in conventional therapy (6) In another study on the interactions between natural products and drugs, synergism was discovered between pairs involving one of eight plant extracts and one of 13 antimicrobial drugs. Furthermore, synergism was also detected in at least two drugs with ginger and 11 drugs with lemongrass and clove when tested against S. aureus strains (17). On the other hand, synergism was not observed when this type of study was carried out using E. coli strains; instead, antagonistic reactions were detected (39).
Essential oils from Conyza bonariensis (horseweed), Lippia sidoides (rosemarypepper), Plectranthus amboinicus (mint) and Eucalypthus citriodora (eucalyptus) were studied by Oliveira et al. (40), who aimed to verify synergism of ampicillin, cephalothin, chloramphenicol, gentamicin and tetracycline against S. aureus, S. epidermidis, P. aeruginosa and E. coli strains. Ampicillin, cephalothin and tetracycline presented synergistic interactions with the oils whereas gentamicin mostly had antagonistic interactions. Some of the synergisms detected in the study were with L. sidoides oil, which improved the antimicrobial effect of ampicillin and cephalothin against S. aureus and S. epidermidis; while C. bonariensis had synergistic action with ampicillin, cephalothin, chloramphenicol and tetracycline against S. epidermidis; and P. amboinicus, associated with ampicillin and cephalothin had synergistic action when tested against S. aureus and S. epidermidis.
Synergism was also detected in in vitro assays testing interactions between Pelargonium graveolens oil and norfloxacin against S. aureus and B. cereus, confirming the capacity of that oil to increase the antimicrobial activity of this antibiotic (21).
Fungi have also been evaluated for synergism between natural products and antifungals.
There were reports of synergism between ketoconazole and Agastache rugosa essential oil against Blastischizomyces capitatus and between Pelargonium graveolens essential oil and amphotericin B plus ketoconazole on strains of Aspergillus sp. (41,42).
Synergism using different concentrations of commercial oils derived from Melaleuca alternifolia, Thymus vulgaris, M. piperita and R. officinalis with ciprofloxacin against S. aureus and K. pneumonia, and with amphotericin B against C. albicans strains was observed while R. officinalis had a predominantly antagonistic profile in combination with with the tested drugs against S. aureus and C. albicans strains (43). Synergism was also verified against K. pneumoniae while M. alternifolia had a higher level of antagonism than synergism. T. vulgaris presented antagonism against all studied pathogens, and M. piperita revealed synergism against S. aureus and antagonism against C. albicans. Regarding K. pneumonia, the results indicated antagonism or synergism according to the concentration assayed.
Probst (44) reported the antimicrobial action of clove (C. aromaticus), ginger (Z. officinale), peppermint (M. piperita) and cinnamon (C. zeylanicum) essential oils against S. aureus and E. coli. Moreover, the interactions of these plants with ethanol extracts of propolis, ginger and mint were synergistic when tested against S. aureus whereas against E. coli only cloves showed synergism (44).
Synergism between the essentials oils of cinnamon (C. zeylanicum), lemon grass (C. citratus), peppermint (M. piperita), ginger (Z. officinale), clove (C. aromaticus) and rosemary (R. officinalis) and eight antimicrobial drugs (chloramphenicol, gentamicin, cefepime, tetracycline, sulfazotrim, cephalothin, ciprofloxacin and rifampicin) was tested by the Kirby and Bauer method against strains of S. aureus and E. coli. The highest rates of synergism were between lemon grass and the eight drugs tested followed by mint and seven drugs (except cefepime against S. aureus). Against E. coli, only rosemary associated with three drugs and lemon grass with two antimicrobials revealed synergism (45).
The interaction of Surinam cherry (Eugenia uniflora), "alecrim-do-campo" (Baccharis dracunculifolia), "assa-peixe" (V. polyanthes) and chamomile (M. chamomilla) with conventional antimicrobial drugs against strains of S. aureus was reported and the results are presented in Table 1 (46). In the same study, their interactions against E. coli were not relevant, since they presented only three cases of synergism and five of antagonism.
Thus, studies on the interactions between natural products and antimicrobial drugs have also multiplied in recent years, indicating the importance of elucidating those types of interactions, which can be either favorable, such as in synergism, or harmful, as in antagonism. However, such associations, even if beneficial, will not necessarily be used in therapy against infectious diseases, since further studies are still required, especially in vivo studies and research on the toxicity of these products to humans.  S  I  S  I  I  I  I  I  Gentamicin  S  I  S  I  I  I  S  I  Tetracycline  S  I  S  I  I  S  I  I  Sulfazotrin  S  I  S  I  S  I  S  I  Ciprofloxacin  S  I  S  I  S  I  S  I  Cefepime  S  I  S  I  I  I  I  I  Chloramphenicol  S  I  S  I  I  I  I  I  Rifampicin  S  I  S  I  S  S  I  I Synergism was considered positive when p < 0.05; S: synergism; I -indifferent. Source: Silva (46).

ANTIMICROBIAL ACTIVITY MECHANISMS OF NATURAL PRODUCTS
Most plants contain several compounds with antimicrobial properties for protection against aggressor agents, especially microorganisms. The chemical structures of some antimicrobial compounds, according to Cowan (2), obtained from plants are shown in Figure 1.
Active compounds found in some plants have antiseptic action; for example, thyme has thymol and carvacrol, clove has eugenol and isoeugenol, and oregano has carvacrol and terpinenol-4. In some cases, terpenes from essences that are soluble in water have higher antibacterial power than others (47).
The sites or structures of the bacterial cell that are considered targets for action by the components of natural products are illustrated in Figure 2. The action mechanisms of natural compounds are related to disintegration of cytoplasmic membrane, destabilization of the proton motive force (PMF), electron flow, active transport and coagulation of the cell content. Not all action mechanisms work on specific targets, and some sites may be affected due to other mechanisms (48).
permeable (52). Their structure disintegrates the external membrane of gram-negative bacteria, releasing lipopolysaccharides (LPS) and increasing the permeability of the cytoplasmic membrane to ATP. The presence of magnesium chloride does not influence this action, suggesting a chelating mechanism of different cations on the external membrane (53).

Eugenol
Different concentrations of eugenol may inhibit the production of amylase and protease by B. cereus. Furthermore, cell wall degradation and cell lysis were also reported (54).

p-Cymene
A precursor of carvacrol, this hydrophobic compound provokes greater swelling of the cytoplasmic membrane compared to carvacrol (55).

Carvone
When tested at concentrations higher than its minimum inhibitory concentration, carvone dissipates gradient pH and cell membrane potential. The growth of E. coli, Streptococcus thermophilus and Lactococcus lactis may decrease according to the concentrations of carvone, suggesting that it acts by disturbing the general metabolic status of the cell (56).

Cinnamaldehyde
Cinnamaldehyde is known to inhibit E. coli and Salmonella Typhimurium growth at concentrations similar to those of carvacrol and thymol. However, it neither disintegrates the outer membrane nor weakens the intracellular ATP (53). Its carbonyl group has affinity for proteins, preventing the action of decarboxylase amino acids on E. aerogenes (57).
Lastly, Table 2 includes the main action mechanisms of plant antimicrobials according to the groups already mentioned.

FINAL REMARKS
Since ancient times, plants have been used by several communities to treat a large number of diseases, including infections. Numerous studies on the pharmacology of medicinal plants have been accomplished, since they Important characteristics responsible for the antimicrobial action of essential oils include hydrophobic components that allow the participation of lipids from the bacterial cell membrane, which disturbs cell structures and make them more permeable (49).
Chemical compounds from essential oils also act on cytoplasmic membrane proteins (47). Cyclic hydrocarbons act on ATPases, enzymes known to be located at the cytoplasmic membrane and surrounded by lipid molecules. In addition, lipid hydrocarbons may distort the lipid-protein interaction, and the direct interaction of lipophilic compounds with hydrophobic parts of the protein is also possible (50). Some essential oils stimulate the growth of pseudo-mycelia, evidencing that they may act on enzymes involved in the synthesis of bacterium structural components (51).
Several compounds and their mechanisms of action on microorganisms are listed below.

Carvacrol and thymol
The structure of thymol is similar to that of carvacrol; however, they differ as to the location of the hydroxyl group in the phenolic ring. Both substances seem to make the membrane constitute a potential source for the production of new medicines and may enhance the effects of conventional antimicrobials, which will probably decrease costs and improve the treatment quality. However, several plants may present antagonistic effects during antibiotic therapy.
An important aspect comprises the search for new compounds that have antimicrobial action and synergism with currently available antimicrobial drugs, since bacteria resistant to conventional medicines are increasingly frequent; consequently, medicinal plants constitute an alternative for infection treatment.
The antimicrobial activity of plants was proven by various examples, in the form of both essential oils and extracts. Thus, this property can be a promising ally in the development of medicines necessary to combat the increasing number of bacterial strains that become resistant to conventional antibiotics. Therefore, given that the literature on tests for the antimicrobial action of plant products is broad, including an increasing number of publications per year, it is highly difficult to relate the countless reports on the antimicrobial action of these products in this review article about a subject of such a great complexity, which requires a multidisciplinary approach.