Antimicrobial and Antioxidant Activities of Some Plant Extracts

Infectious diseases are the world’s leading cause of premature deaths, killing almost 50,000 people every day. In recent years, drug resistance to human pathogenic bacteria has been commonly reported from all over the world (N’guessan et al., 2007). The abusive and indiscriminate use of antimicrobial compounds over many years is the main factor responsible for the appearance of the phenomenon of bacterial resistance to such compounds (Andremont, 2001). With increased incidence of resistance to antibiotics, natural products from plants could be interesting alternatives (Lu et al., 2007; Mbwambo et al., 2007). Some plant extracts and phytochemicals are known to have antimicrobial properties, and can be of great significance in therapeutic treatments. In the last few years, a number of studies have been conducted in different countries to demonstrate such efficacy (BenoitVical et al., 2006; Senatore et al., 2007; Singh et al., 2007). On the other hand, free radicals are known to be the major cause of various chronic and degenerative diseases. Oxidative stress is associated with pathogenic mechanisms of many diseases including atherosclerosis, neurodegenerative diseases, cancer, diabetes and inflammatory diseases, as well as aging processes. It is defined as an imbalance between production of free radicals and reactive metabolites, so-called oxidants, and it also includes their elimination by protective mechanisms, referred to as antioxidative systems. This imbalance leads to damage of important biomolecules and organs with potential impact on the whole organism. Antioxidants can delay, inhibit or prevent the oxidation of oxidizable materials by scavenging free radicals and diminishing oxidative stress (Durackova, 2010; Reuter et al., 2010). Natural antioxidants have been studied extensively for decades in order to find compounds protecting against a number of diseases related to oxidative stress and free radical-induced damage. To date, many plants have been claimed to pose beneficial health effects such as antioxidant properties (Kaur & Arora, 2009; Newman & Cragg 2007). According to World Health Organization (WHO), 65 80% of the world populations rely on traditional medicine to treat various diseases (Kaur & Arora, 2009). The WHO recommends

Phytochemicals as Nutraceuticals -Global Approaches to Their Role in Nutrition and Health 22 research into the use of the local flora for therapeutic purposes, with the intention of reducing the number of people excluded from effective therapy in the government health systems, which could constitute an economically viable alternative treatment of several diseases, especially in developing countries (Gonçalves et al., 2005;WHO, 2002). The potential of higher plants as source for new drugs is still largely unexplored. Among the estimated 250,000 -500,000 plant species, only a small percentage has been investigated phytochemically and the fraction submitted to biological or pharmacological screening is even smaller (Mahesh & Satish, 2008). In this scenario, the screening of plant extracts has been of great interest to scientists for the discovery of new drugs effective in the treatment of several diseases, and about 20% of the plants or their extracts in the world have been submitted to biological or pharmacological tests (Rayne & Mazza, 2007;Suffredini et al., 2004). The phytochemical research based on ethnopharmacological information is considered an effective approach in the discovery of new agents from higher plants (Chen et al., 2008;Duraipiyan, 2006). Thus, in this study, methanol extracts of different parts of 70 species, most of them commonly used in Brazil for treating conditions likely to be associated with microorganisms, were evaluated for their antimicrobial and antioxidant activity. Furthermore, a phytochemical screening of the bioactive extracts was performed.

Plant material
Specimens of 70 species (Table 1) were collected in Juiz de Fora, Minas Gerais, Brazil. A voucher specimen was deposited at the Herbarium Leopoldo Krieger (CESJ) of Federal University of Juiz de Fora.

Preparation of plant extracts
The dried parts of the plant (50 g each) were powdered and macerated with methanol (3 x 200 mL) for five days at room temperature. After evaporation of the solvent under reduced pressure, the respective methanol extracts were obtained. All the extracts were kept in tightly stoppered bottles under refrigeration (4 °C) until used for the biological testing and phytochemical analysis.

Reducing power assay
The reducing power was determined by the method of Oyazu (1986), based on the chemical reaction of Fe(III) to Fe(II). Ten mg of each sample were mixed with potassium phosphate buffer (0.2 M, pH 6.6) (2.5 mL) and potassium ferricyanide (10 g/L) (2.5 mL). The mixture was incubated at 50 °C for 20 min. A 2.5 mL aliquot of 10% trichloroacetic acid was added to the mixture, which was then centrifuged at 3.000 g for 10 min. The upper layer of the solution (2.5 mL) was mixed with distilled water (2.5 mL) and 0.1% FeCl 3 (0.5 mL), and the absorbance was measured at 700 nm. Ascorbic acid was used as reference material. All tests were performed in triplicate. Increase in absorbance of the reaction indicated the reducing power of the samples. A higher absorbance indicated a higher reducing power. EC 50 (effective concentration) values (g/mL) were calculated and indicate the effective concentration at which the absorbance was 0.5 for reducing power.

β-carotene -linoliec acid assay
In this assay, antioxidant capacity is determined by measuring the inhibition of the volatile organic compounds and the conjugated diene hydroperoxides arising from linoleic acid oxidation (Dapkevicius et al., 1998). A stock solution of -carotene/linoleic acid mixture was prepared as follows: 50 µL of -carotene (10 mg/mL) in chloroform (HPLC grade), 20 µL linoleic acid, 200 µL Tween 40 and 1 mL of chloroform was added. Chloroform was completely evaporated using a vacuum evaporator. Then, 30 mL of distilled water saturated with oxygen (30 min 100 mL/min) were added with vigorous shaking, and 250 µL of the reactive mixture and 10 µL of the extracts (40 µg/mL) were added in a microplate and incubated at 45 °C to accelerate oxidation reactions and start the bleaching of -carotene. The absorbance readings were taken immediately at intervals of 15 min for 120 min in spectrophotometer at 470 nm (Duarte-Almeida et al., 2006). The same procedure was repeated with the antioxidant flavonoid quercetin as positive control, and a blank. After this incubation period, absorbances of the mixtures were measured at 490 nm. Antioxidative capacities of the extracts were expressed as percentage inhibition (1)

Serial dilution assay for determination of the minimal inhibitory concentration (MIC)
The MIC of each extract was determined by using the broth microdilution techniques for bacteria and yeasts, respectively (Bouzada et al., 2009;NCCLS, 2002

Phytochemical studies
A portion of each extract that was subjected for the biological screening was used for the identification of the major secondary metabolites employing the protocols described by Matos (1997). Briefly, the extract (1 mg/mL) was submitted to the following identification reactions: The characterization for tannins was performed by gelatin, iron salt and lead acetate reactions. Triterpenoids and sterols were investigated by Liebermann-Burchard reagent and the alkaloids analysis was done by precipitation reactions with the reagents of Dragendorff, Bouchardat, Mayer and Bertrand. For the research of flavonoids, the reactions www.intechopen.com Phytochemicals as Nutraceuticals -Global Approaches to Their Role in Nutrition and Health 30 of Shinoda and aluminum chloride were employed and the presence of saponins was determined by the formation of foam.

Statistical analysis
DPPH, reducing power and -carotene/linoleic acid assays were carried out in triplicates. The results were expressed as mean ± standard deviation (SD). All statistical analysis were conducted using Graph Pad Prism software.

Results and discussion
The paper describes the antimicrobial and antioxidant activities and the phytochemical profile of some methanol extracts belonging to Brazilian traditional medicinal plants, most of them commonly used for treating conditions likely to be associated with microorganisms.
The major classes of phytocompounds of the bioactive extracts are presented in Table 2.

Plant species
Part tested a Phytocompounds b Al     (Table 3). Infections still cause about one-third of all deaths worldwide and are the leading cause of death, mainly because of disease in developing countries.
S. sonnei, a gram-negative bacterium, is a significant cause of gastroenteritis in both developing and industrialized countries (Boumghar-Bourtchai et al., 2008). People infected with Shigella develop diarrhoea, fever and stomach cramps starting a day or two after they are exposed to the bacterium. It is typically associated with mild self-limiting infection (DeLappe et al., 2003). Recently, there has been a rise in strains resistant to multiple antibiotics. P. aeruginosa, an increasingly prevalent opportunistic human pathogen, is the most common gram-negative bacterium found in nosocomial infections. Three of the more informative human diseases caused by P. aeruginosa are bacteremia in severe burn victims, chronic lung infection in cystic fibrosis patients, and acute ulcerative keratitis in users of extended-wear soft contact lenses (Lyczak et al., 2000). S. aureus is a gram-positive bacterium that commonly colonises human skin and mucosa (e.g. inside the nose) without causing any problems. However, if either of these is breached due to trauma or surgery, S. aureus can enter the underlying tissue, creating its characteristic local abscess lesion, and if it reaches the lymphatic channels or blood can cause septicaemia (Harris et al., 2002 (Calderone & Fonzi, 2001).Cryptococcus neoformans is an encapsulated basidiomycete yeast responsible for disseminated infections in immunosuppressed patients. Meningoencephalitis and pneumonia are the most frequent visceral presentations of the disease, but other rare presentations have been reported (Braga et al., 2007;Charlier-Woerther et al., 2011). Some of the most active species had already been studied for their antimicrobial effects elsewhere. The essential oil of different parts of B. segetum presented antifungal activity (Nascimento et al., 2008). Flavonoids isolated from the leaves of T. grandifolia demonstrated antifungal activity against the phytopathogenic fungus Cladosporium cucumerinum (Kuster et al., 2009). Dichlorometane extract of A. australe showed positive results against Bacillus subtilis, Micrococcus luteus, Listeria monocytogenes and S. aureus (Vivot et al., 2007). Antimicrobial efficacy of flavonoids and crude alkaloids of L. camara was found against C. Albicans, Proteus mirabilis, S. aureus, E. coli, and Trichophyton mentagrophytes (Sharma & Kumar, 2009). The iridoid isolated from A. cathartica presented fungitoxicity against some dermatophytes that causes dermatomycosis (Tiwari et al., 2002). The wound healing activity of this specie has also been tested, and it presented significant results in tests in vivo (Nayak et al., 2006). Flavonoids from A. cotula flowers showed interesting antimicrobial activity against both gram-negative and gram-positive microorganisms (Quarenghi et al., 2000). Quercetin isolated from the ethyl acetate extract of A. brasiliana presented antibacterial action against S. aureus (Silva et al., 2011). Antimicrobial activity of aqueous and hydroalchoolic fractions from R. rosifolius leaves showed activity against E. coli, S. aureus, P. aeruginosa and C. albicans (Mauro et al., 2002) and B. trimera was active against S. aureus and E. coli (Avancini et al., 2000). The antifungal activity of the leaf oil of S. chilensis was assayed by paper disk agar diffusion test and showed that human pathogenic dermatophytes were very sensitive (Vila et al., 2002). The crude hydroalcoholic extract of S. cumini was active against Candida krusei and against multi-resistant strains of P. aeruginosa, K. pneumoniae and S. aureus (de Oliveira et al., 2007). However, antimicrobial activity for C. desvauxii, S. scabra, A. floribunda, S. multijuga, S. glaziovii, C. ingrata, C. marianus, C. pachystachya and T. majus were reported here for the first time. Preliminary phytochemical analysis revealed that almost all the antimicrobial extracts showed flavonoids and tannins in their chemical composition (Table 2). Flavonoids are a broad class of plant phenolics that are known to possess antimicrobial activity, essentially by enzyme inhibition of DNA gyrase (Cushnie & Lamb, 2005). The mode of tannins antimicrobial action may be related to their ability to inactivate microbial adhesions, enzymes, cell envelope transport protein, etc. They also complex with polysaccharides (Ya et al., 1988). Condensed tannins have been determined to bind cell walls of ruminal bacteria, preventing growth and protease activity (Jones et al., 1994). However, the extracts tested also contain triterpenoids, sterols, saponins and alkaloids. Saponins are known to interact with cell membranes, increasing permeability and producing cell damage (Francis et al., 2002). In this sense, saponins may be involved in antimicrobial properties. The mechanism of action of some alkaloids is attributed to their ability to intercalate with DNA (Phillipson & O`Neill, 1989). The antimicrobial activity of triterpenes and sterols may be related to lipophilic components of plant extracts. This components increase permeability and loss of cellular components, and a change variety of enzyme systems, including those involved in the production of cellular energy and synthesis of structural components, inactivating or destroying genetic material (Bagamboula et al., 2004;Kim et al., 1995). The antioxidant hability of the extracts was also measured.
Natural antioxidants have been studied extensively for decades in order to find compounds protecting against a number of diseases related to oxidative stress and free radical-induced damage. Antioxidants are believed to play a very important role in the body defense system against reactive oxygen species (ROS), which are the harmful byproducts generated during normal cell aerobic respiration (Gutteridge & Halliwell, 2000). There is a number of assays designed to measure overall antioxidant activity/reducing potential, as an indication of host total capacity to withstand free radical stress. DPPH assay is very convenient for the screening of large numbers of samples of different polarity because of its high throughput. It evaluates the ability of antioxidants to scavenge free radicals. These antioxidants donate hydrogen to free radicals, leading to non-toxic species and therefore to inhibition of the propagation of lipid oxidation. Hydrogen-donating ability is an index of primary antioxidants (Lugasi et al., 1998). Among all extracts, 24 showed an outstanding antioxidant activity with IC 50 ≤ 10 µg/mL. Cecropia pachystachya, Tibouchina mutabilis, Cupania oblongifolia, and Myrcia splendens were the most active (IC 50 ≤ 3 µg/mL) (  The total antioxidant activity of the extracts is constituted by individual activities of each of the antioxidant compounds. Moreover, these compounds render their effects via different mechanisms such as radical scavenging, metal chelating activity, inhibition of lipid peroxidation, quenching of singlet oxygen, and so on to act as antioxidants. Even if a sample exhibits high activity with one method, it does not always show similar good results with all other methods. Therefore, it is essential to evaluate samples accurately by several methods. Hence, the antioxidant activity for those extracts was also evaluated by reducing power and -carotene/linoleic acid assays. The reducing ability of a compound generally depends on the presence of reductants, which exhibited antioxidative potential by breaking the free radical chain, by donating a hydrogen atom. Antioxidant action of the reductones is based on the breaking of free radicals chain by the donation of a hydrogen atom. Reductones are believed not only to react directly with peroxides, but also prevent peroxide formation by reacting with certain precursors (Jamuna et al. 2010). The results found using this assay showed an outstanding antioxidant property of C. pachystachya, T. mutabilis, C. oblongifolia, and M. splendens and suggested that compounds present in those extracts were good electron and hydrogen donors, and could terminate the radical chain reaction by converting free radicals into more stable products. When employing -carotene/linoleic acid assay, the more active inhibitors of -carotene bleaching were C. pachystachya, T. mutabilis and B. orellana which showed values greater than 75% of inhibition. Interestingly, C. oblongifolia and M. splendens were not so effective in quenching -carotene. It is well known that the value of this method appears to be limited to less polar compounds. They exhibit stronger antioxidative properties in emulsions because they concentrate at the lipid:air surface, thus ensuring high protection of the emulsion itself. On the other hand, polar antioxidants remaining in the aqueous phase are more diluted and are thus less effective in protecting the lipid (Koleva et al., 2002). It is well known that plants which possess antioxidative and pharmacological properties are related to the presence of phenolic compounds, specially phenolic acids and flavonoids . Antioxidant activity had also been detected for C. pachystachya (Aragão et al., 2010) and B. orellana (Chisté et al., 2011). For T. mutabilis, C. oblongifolia and M. splendens, the antioxidant capacities were reported here for the first time. Polyphenolic compounds such as flavonoids and tannins found in the extracts (Table 2) are considered to be the major contributors to the antioxidant activity of medicinal plants. The antioxidant activities of polyphenols were attributed to their redox properties, which allow them to act as reducing agents, hydrogen donators and singlet oxygen quenchers, as well as their metal chelating abilities (Vladimir-Knezevic et al., 2011). It would seem that a great part of the extracts tested in this study for antimicrobial activity does not possess antioxidant effects (Table 3 and 4).

Conclusion
The results obtained represent a worthwhile expressive contribution to the characterization of antimicrobial and antioxidant activity of plant extracts of traditional medicinal plants from Brazilian flora and justify, in part, the popular uses of some of these species.

Acknowledgment
The authors are grateful to Fundação de Amparo a Pesquisa do Estado de Minas Gerais (FAPEMIG) and the Universidade Federal de Juiz de Fora (UFJF)/Brazil for financial Phytochemicals are biologically active compounds present in plants used for food and medicine. A great deal of interest has been generated recently in the isolation, characterization and biological activity of these phytochemicals. This book is in response to the need for more current and global scope of phytochemicals. It contains chapters written by internationally recognized authors. The topics covered in the book range from their occurrence, chemical and physical characteristics, analytical procedures, biological activity, safety and industrial applications. The book has been planned to meet the needs of the researchers, health professionals, government regulatory agencies and industries. This book will serve as a standard reference book in this important and fast growing area of phytochemicals, human nutrition and health.

How to reference
In order to correctly reference this scholarly work, feel free to copy and paste the following:  Bouzada (2012). Antimicrobial and Antioxidant Activities of Some Plant Extracts, Phytochemicals as Nutraceuticals -Global Approaches to Their Role in Nutrition and Health, Dr Venketeshwer Rao (Ed.), ISBN: 978-953-51-0203-8, InTech, Available from: http://www.intechopen.com/books/phytochemicals-as-nutraceuticals-global-approaches-to-their-role-innutrition-and-health/antimicrobial-and-antioxidant-activities-of-some-plant-extracts