Study of the best technique to extract compounds from olea europaea and acacia dealbata with bioactivity

Dissertation for Master Degree in Bioengineering i Study of the best technique to extract compounds from Olea europaea and Acacia dealbata with bioactivity Dissertation for Master Degree in Bioengeneering Specialization in Biological Engineering Helena Manuel de Azevedo Ferreira José June, 2013 Supervisors: Prof. Manuel Simões and Prof. Vera Homem Dissertation for Master Degree in Bioengineering i Dissertation for Master Degree in Bioengineering ii Supervisors Manuel Simões Vera Homem Dissertation for Master Degree in Bioengineering iii Dissertation for Master Degree in Bioengineering iv ‘‘The future is an opportunity.’’ J. F. Ware Dissertation for Master Degree in Bioengineering i Dissertation for Master Degree in Bioengineering i ACKNOWLEDGMENTS Aos meus pais, à minha irmã e aos meus avós. Ao João. À Carolina, à Joana, à Inês e ao Nelson. À Rute, à Sandra, à Mónica, à Ana, à Sara e a todos os meus companheiros de muitas horas de água. Ao Prof. Manuel Simões, à Prof. Vera Homem, à Paula, à Sílvia, à Ana Abreu e a todos os que trabalham nos laboratórios 201 e 007. Dissertation for Master Degree in Bioengineering ii Dissertation for Master Degree in Bioengineering iii ABSTRACT Plants are used with several purposes, like in medicine, decoration, food and as sustainable resource of chemical products. Medicine is increasingly receptive to their use. The interaction between antibiotic exposure and the transmission of resistance within and between individuals results in a worrying antibacterial resistance. The compounds extracted from plants have different mechanisms of action from antibiotics and can have an important role in the treatment of resistant microbial strains. The main bioactive compounds from plants are phenolics and polyphenols, which involve simple phenols, phenolic acids, quinones, flavones, flavonoids, flavonols, tannins and coumarins, terpenoids and essential oils, alkaloids, lectins and polypeptides. To obtain these compounds there are some methods that are usually used, like solidliquid extraction, Soxhlet, microwave assisted extraction and superfluid critical extraction. The main purpose of this study was to investigate the methods of extraction of two plants in order to obtain the best extracts with higher bioactive properties, particularly, antimicrobial and antioxidant. Extracts from Olea europaea (olive) and Acacia dealbata (mimosa), obtained by solid-liquid extraction, ultrasounds extraction, Soxhlet and micro-wave extraction, were tested in their antimicrobial and antioxidant activity. Different solvents (methanol, ethanol, acetone, dichloromethane, hexane and water) were tested in solid-liquid and ultrasounds extraction. In relation to the extraction yield, methanol and water were considered the best solvents to solid-liquid and ultrasound extractions. Comparing all the techniques used in the current study, Soxhlet and micro-wave were the methods that were able to achieve the best extraction efficiency. For olive leaves the efficiency was 12.3 ± 1.2% and 10.7 ± 0.8% and for mimosa leaves it was 13.4 ± 1.4 %and 11.9 ± 1.8%, respectively. It was concluded that mimosa and olive can produce extracts with similar antimicrobial and antioxidant activities. Ethanol proved to be the best solvent to extract compounds with antimicrobial activity, whereas to extract compounds with antioxidant properties acetone proved to be the best solvent. Soxhlet and micro-wave extractions were the best techniques to extract compounds with antimicrobial activity, whereas to extract compounds with antioxidant activity any method was highlighted. Dissertation for Master Degree in Bioengineering x Dissertation for Master Degree in Bioengineering xi FIGURES LIST Figure 1 - Structure of three typical phenolic acids (adapted from Huang et al., 2009). ............. 8 Figure 2. Structure of 1,4-benzoquinone or cyclohexadienedione, the prototypical member of the quinones. ................................................................................................................................ 9 Figure 3. Structures of (a) flavones and (b) flavonoids (adapted from Huang et al., 2009). ........ 9 Figure 4 - Structure of the oligomeric proanthocyanidins (adapted from Huang et al., 2009). . 11 Figure 5. Structure of a simple coumarin. ................................................................................... 11 Figure 6. Conventional Soxhlet extractor (Luque de Castro and Priego-Capote, 2010). ............ 14 Figure 7. Leaves and flower of Acacia dealbata. ........................................................................ 20 Figure 8. Leaves of Olea europaea. ............................................................................................. 20 Figure 9. QSI assessment in (a) olive leave and (b) methanol extract of olive leaves. ............... 40 Figure A.1. Linear regression for antioxidant activity estimated by ABTS method……………..…….III Figure A.2. Linear regression for antioxidant activity estimated by DPPH method……………........III Dissertation for Master Degree in Bioengineering xii Dissertation for Master Degree in Bioengineering xiii TABLES LIST Table 1. Main solvents used for active compounds extraction (from Cowan, 1999). ................ 17 Table 2. Extraction efficiency (%) per extraction technique and solvent used with both olive and mimosa leaves. ..................................................................................................................... 23 Table 3. Antibacterial activity of olive leaves extracts for solid-liquid extraction and for the selected solvents against S. aureus and E. coli – diameter of inhibition (mm). .......................... 31 Table 4. Antibacterial activity of olive leaves extracts at 5 mg/mL for solid-liquid extraction and for the selected solvents against S. aureus and E. coli - diameter of inhibition (mm). .............. 32 Table 5. Antibacterial activity of olive leaves extracts for ultrasounds extraction and for the selected solvents against S. aureus and E. coli – diameter of inhibition (mm). .......................... 32 Table 6. Antibacterial activity of olive leaves extracts at 5 mg/mL for ultrasounds extraction and for the selected solvents against S. aureus and E. coli - diameter of inhibition (mm). ....... 33 Table 7. Antibacterial activity of mimosa leaves extracts for solid-liquid extraction and for the selected solvents against S. aureus and E. coli – diameter of inhibition (mm). .......................... 34 Table 8. Antibacterial activity of mimosa leaves extracts at 5 mg/mL for solid-liquid extraction and for the selected solvents against S. aureus and E. coli - diameter of inhibition (mm)......... 34 Table 9. Antibacterial activity of mimosa leaves extracts for ultrasounds extraction and for the selected solvents against S. aureus and E. coli – diameter of inhibition (mm). .......................... 35 Table 10. Antibacterial activity of mimosa leaves extracts at 5 mg/mL for ultrasounds extraction and for the selected solvents used against S. aureus and E. coli - diameter of inhibition (mm)............................................................................................................................ 35 Table 11. Antibacterial activity of olive and mimosa leaves extracts for Soxhlet and micro-wave extraction against S. aureus and E. coli - diameter of inhibition (mm). ...................................... 36 Table 12. Antibacterial activity of olive and mimosa leaves extracts at 5 mg/mL for Soxhlet and micro-wave extraction against S. aureus and E. coli - diameter of inhibition (mm). .................. 36 Table 13. Classification of the antimicrobial potential of antibiotic-extract dual combination against S. aureus as antagonism (-), indifference (+), additive (++) and synergy (+++). ............. 38 Table 14. Classification of the antimicrobial potential of antibiotic-extract dual combination against E. coli as antagonism (-), indifference (+), additive (++) and synergy (+++). .................. 38 Table 15. Quorum-sensing inhibition for olive leaves and olive leaves extracts. ....................... 39 Table 16. Quorum-sensing inhibition for mimosa leaves and mimosa leaves extracts. ............. 40 Table 17. Antioxidant activity estimated by DPPH and ABTS of olive leaves extracts at 5 mg/mL for solid-liquid extraction and for the selected solvents (expressed in TE/g fresh mass). ......... 41 Dissertation for Master Degree in Bioengineering xiv Table 18. Antioxidant activity estimated by DPPH and ABTS of olive leaves extracts at 5 mg/mL for ultrasounds extraction and for the selected solvents (expressed in TE/g fresh mass). ........ 41 Table 19. Antioxidant activity estimated by DPPH and ABTS of olive leaves extracts at 5 mg/mL for extraction techniques with water (expressed in TE/g fresh mass). ...................................... 42 Table 20. Antioxidant activity estimated by DPPH and ABTS of olive leaves extracts at 5 mg/mL for solid-liquid extraction and for the selected solvents (expressed in TE/g fresh mass). ......... 43 Table 21. Antioxidant activity estimated by DPPH and ABTS of mimosa leaves extracts at 5 mg/mL for ultrasounds extraction and for the selected solvents (expressed in TE/g fresh mass). ..................................................................................................................................................... 43 Table 22. Antioxidant activity estimated by DPPH and ABTS of mimosa leaves extracts at 5 mg/mL for extraction techniques with water (expressed in TE/g fresh mass). .......................... 44 Dissertation for Master Degree in Bioengineering xv GLOSSARY ABTS 2,2-azinobis (3-ethyl-benzothiazoline-6sulfonic acid) CFU Colony-forming unit DMSO Dimethyl sulfoxide DPPH 2,2-diphenyl-1-picrylhydrazyl HIV Human immunodeficiency virus LBA Luria-Bertani Agar MAE Micro-wave assisted extraction MHA Mueller-Hinton Agar MIC Minimum inhibitory concentration QS Quorum-sensing QSI Quorum-sensing inhibition RSV Respiratory syncytial virus SFE Supercritical fluid extraction SPSS Statistical Package for the Social Sciences TE Trolox Equivalent Dissertation for Master Degree in Bioengineering xiv Dissertation for Master Degree in Bioengineering 1 CHAPTER 1 - Work Outline 1.1 Background Plants have been used with several purposes, like in medicine, decoration, food and as sustainable resource of chemical products. Medicine is one of the areas where plants are becoming a great subject of study, being increasingly receptive to their use. Actually, the evolution of bacterial resistance to antibiotics is becoming more and more an issue of concern. A promising alternative could be the antimicrobial compounds extracted from plants, which have different mechanisms of action from antibiotics and can have an important role in the treatment of resistant microbial strains (Abreu et al., 2012). The search for new anti-infective agents (including vaccines) could also be answered by plant extracts. There are studies that have been reported that extracts from plants are effective in treating febrile illnesses, sleeping sickness, wounds, diarrhea, reproductive and liver problems, circulatory and respiratory problems and parasitic infections. Therefore, they have a great power in health care (Chah et al., 2006; Obi et al., 2006). Their main compounds are alkaloids, lectins, polypeptides, phenolics, polyphenols, terpenoids and essential oils, which have recognized antimicrobial and antioxidant properties (Cowan, 1999; Karou, 2005; Gallo, 2010). To obtain the extracts from plants there are several techniques that can be used. Solid-liquid, Soxhlet, supercritical fluid and micro-wave assisted extractions are some examples (Aziz, 2006; Tatke and Jaiswal, 2011). 1.2 Objectives The main purpose of this study was to investigate the methods of extraction of two plants in order to obtain the best extracts with higher bioactive properties, particularly, antimicrobial and antioxidant. Extracts from Olea europaea (olive) and Acacia dealbata (mimosa), obtained by solid-liquid extraction, ultrasounds extraction, Soxhlet and micro-wave extraction were tested for their antimicrobial and antioxidant activities. Different solvents (methanol, ethanol, acetone, dichloromethane, hexane and water) were tested in solid-liquid and ultrasounds extraction. Many studies about Olea europaea demonstrate its antimicrobial and antioxidant properties, suggesting that it can Dissertation for Master Degree in Bioengineering 2 be used in diverse applications. Therefore, the results obtained with the extracts from this plant were taken as reference values. About Acacia dealbata, an invasive plant, information on the extracts bioactivity is lacking. The antimicrobial activity of the extracts was assessed against two bacteria. S. aureus, a Gram-positive bacterium, and E. coli, a Gram-negative bacterium, were chosen for this study since they are considered two of the most clinical significant bacteria involved in drug-resistant infections (Simões et al., 2008). Antimicrobial properties were evaluated with the extracts alone and together with two antibiotics, erythromycin and tetracycline. Thereby, it was possible to observe if these combinations are benefic or not. Moreover, the leaves and extracts of plants were also tested for their capacity of inhibiting quorum-sensing (QS). The antioxidant activity was studied by two methods: using 2,2-diphenyl-1picrylhydrazyl (DPPH) and 2,2-azinobis (3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS). The goal of both tests was to evaluate the capacity of the redox molecules from the plants extracts to scavenge the free radicals. The main objective of this work was to verify what was the type of extraction which allowed to obtain extracts with the highest antimicrobial and antioxidant activities, in order to maximize the extraction yield of the bioactive products with potential medicinal application. 1.3 Thesis Organization This thesis is divided in 6 Chapters and respective subchapters. It starts with a background of the theme in Chapter 1, where the main goals of the study are presented. Chapter 2 is dedicated to a literature review about the plants and their relevance in diverse applications, focusing on the use of plant extracts in the clinical setting. The main bioactive compounds from plants are briefly described, as well as the main methods to extract them and the relation between the extraction solvent and the compounds extracted. Chapter 3 provides information on the assessment of the extraction efficiency of Olea europaea and Acacia dealbata extracts, using different techniques and extraction solvents. The goal of this work was to find the type of extraction and the solvent which caused the higher efficiency. Dissertation for Master Degree in Bioengineering 3 Chapter 4 shows the data from the bioactivity assessment of Olea europaea and Acacia dealbata extracts. Extracts were tested for their antimicrobial potential, alone and together with antibiotics, and for their antioxidant activities. This chapter aims to evaluate the antimicrobial and antioxidant activities of the plant extracts and to conclude about the best extraction method that produces an extract with high bioactive properties. Finally, Chapter 5 presents the main conclusions about this study and gives suggestions for future research. Dissertation for Master Degree in Bioengineering 10 activities in vitro, being able to scavenge a wide range of reactive species (Hollman and Katan, 2000). Flavonoid compounds exhibit inhibitory effects against multiple viruses, like human immunodeficiency virus (HIV) (Critchfield et al., 1996) and respiratory syncytial virus (RSV) (Barnard et al., 1993). Although there are several studies about the topic, there is no clear predictability for the degree of hydroxylation and toxicity to microorganisms relatively to the flavonoids and flavones. Flavonoids are the most common pigments, together with chlorophyll and carotenoids, and they generally occur in plants as glycosylated derivatives. Two of the most important functions of the flavonoids are the catalytic action in the light phase of photosynthesis and the regulation of the ion channels involved in phosphorylation (Pietta et al., 1999). 2.2.3.4 Tannins Tannins are natural and water-soluble compounds with molecular weights ranging from 500 to 4000 g/mol (Cai et al., 2004) and they are present in many plant foods (Chung et al., 1998). Tannins are a group of polymeric phenolic substances capable of tanning leather or precipitating gelatin from solution (astringency). They are found in almost every plant part, like bark, wood, leaves, fruits and roots (Scalbert, 1991). There are two groups of tannins. One is the hydrolysable tannins; in this group, tannins are based on gallic acid. The other group is the condensed tannins, also called proanthocyanidins, which are derived from flavonoids monomers. Tannins are commonly found combined with alkaloids, polysaccharides and, particularly, proteins (Han et al., 2007). The antimicrobial action of tannins is probably due to their ability to inactivate microbial adhesins, enzymes and cell envelop transport proteins (Cowan, 1999). Tannins also have antimutagenic, anticarcinogenic and antioxidant activities (Chung et al., 1998). Oligomeric proanthocyanidins (Figure 4) are considered to be the most potent antioxidants in tannins and they are frequently used in health care and cancer treatments (Huh et al., 2004). Dissertation for Master Degree in Bioengineering 11 Figure 4 - Structure of the oligomeric proanthocyanidins (adapted from Huang et al., 2009). 2.1.3.5 Coumarins Coumarins (Figure 5) are phenolic substances made of fused benzene and αpyrone rings (Kennedy and Thornes, 1997) and they are responsible for the characteristic odor of hay (Hoult and Payá, 1996). Warfarin is an important coumarin which has antimicrobial properties (Rice et al., 2003). Figure 5. Structure of a simple coumarin. These compounds are obtained by the cyclization of cis-ortho-hydroxicinnamic, belonging to the phenolics with the basic skeleton of C6 + C3, and this cyclization forms lactones (Cai et al., 2004). Coumarins are present in plants in the free form and as glycosides (Fresco et al., 2006). Cai et al. (2006) characterized these compounds with a great chemical diversity, mainly differing in the degree of oxygenation of their benzopyrane moiety. Major coumarin constituents include simple hydroxilcoumarins, furocoumarins, isofurocoumarins, pyranocoumarins, bicoumarins and di-hydro-isocoumarins (Surveswaran et al., 2007; Cai et al., 2003). Studies reported that these compounds have antimicrobial, anti-inflammatory, antioxidant, anticoagulation, antiestrogenic and sedative activity (Paramjeet et al., 2012). Huang et al. (2009) described the medicinal herbs where they found coumarins, specifically Umbelliferae, Asteraceae, Convolvulaceae, Leguminosae, Magnoliaceae, Oleaceae, Rutaceae, and Ranunculaceae, such as simple coumarins from Artemisia Dissertation for Master Degree in Bioengineering 12 annua, furocoumarins from Angelica sinensis, pyranocoumarins from Citrus aurantium and isocoumarins from Agrimonia pilosa. 2.2.4 Terpenoids and essential oils Essential oils are secondary metabolites that are highly enriched in compounds based on an isoprene structure (Cowan, 1999). The general chemical structure of essential oils is C10H6 and they are called terpenes; they occur as diterpenes, triterpenes, tetraterpenes, hemiterpenes and sesquiterpenes. Terpenoids are these compounds containing additional elements, usually oxygen (Cowan, 1999). Camphor is a monoterpene and farnesol and artemisn are sesquiterpenoids. Terpenoids are active against bacteria, fungi, viruses and protozoa (Cowan, 1999). It is speculated that the mechanism of antimicrobial action of terpenes involves membrane disruption by the lipophilic compounds (Cowan, 1999). Terpenoids and essential oils have antidiarrhoeal activity. They inhibit release of autocoids and prostaglandins (Tiwari et al., 2011). Terpenes are the most numerous and structurally diverse plant natural products. Then, these compounds offer much potential in an array of industrial and medicinal applications (Zwenger and Basu, 2008). They have a complex nomenclature. The single isoprene unit (five carbon molecule) represents the most basic class of terpenes, the hemiterpenes. A terpene is an isoprene unit bonded with a second isoprene and it is also called a monoterpene (C10) (Zwenger and Basu, 2008). Some important terpenes are menthol and pyrethrins (insecticides), limonene and digitoxigenin (Croteau et al., 2000), besides the most known, the rubber. Rubber is a polyterpene, composed of repeating subunits of isoprene (Zwenger and Basu, 2008). 2.3 Extraction of the phytochemicals compounds There are several methods of extraction that can be used to obtain the plants compounds. Solid-liquid, Soxhlet, supercritical fluid and micro-wave assisted extractions are some examples (Aziz, 2006; Tatke and Jaiswal, 2011; Patil and Shettigar, 2010). Dissertation for Master Degree in Bioengineering 13 2.3.1 Solid-liquid extraction The first step of this type of extraction is the contact of an appropriate solvent with the product to be treated for a certain period of time. This enables the transfer of the soluble constituent or solute to the solvent. The second step is the separation of the solid phase from the liquid. The recovery of the solute and solvent are also included in the complete process. This is done by another operation such as evaporation or distillation (Aziz, 2006). To increase the extraction efficiency of the compounds it is possible to vary some parameters, such as the solvent, the temperature and the agitation. Simeonov and Koleva (2012) published a study wherein by solid-liquid extraction they extracted tannins (with 70% ethanol and water) from Geranium sanguineum L. and studied the extraction kinetics, the influence of solid phase particle size and the liquid-solid ratio on extraction rate. Also, using a solid-liquid extraction, Simeonov et al. (2011) extracted from Tribulus terrestris furostanal saponins. They used methanol as the extraction solvent. Wongkittipong et al. (2004) used the same type of extraction for leaves and stems of Andrographis paniculata in ethanol-water solvent (0, 60, 70 e 80% in ethanol) in order to obtain andrographolide, a diterpenoid lactone. Mirela et al. (2007) extracted the terpenic and phenolic compounds by solid-liquid extraction from Olea europaea, using ethanol as solvent. 2.3.2 Soxhlet The sample is placed in a thimble-holder (usually a filter paper inside the main chamber of the apparatus) that is gradually filled with condensed fresh extraction solvent from a distillation flask. When the solvent reaches the overflow level, a siphon aspirates the solute from the thimble-holder and unloads it back to the distillation flask. Therefore, the extracted analytes are carried into the bulk liquid (Luque de Castro and Priego-Capote, 2010). After extraction the solvent is removed, typically by means of a rotary evaporator, yielding the extracted compound. The non-soluble portion of the extracted solid remains in the thimble, and is usually discarded (Jensen, 2007). Figure 6 represents a conventional Soxhlet extractor, where is indicated the position of the heat source, the distillation flask, the sample, the siphon, the extractor and the condenser. Dissertation for Master Degree in Bioengineering 14 Figure 6. Conventional Soxhlet extractor (Luque de Castro and Priego-Capote, 2010). The main advantages of this method are to be a very simple methodology that requires little training and can extract more sample mass than most of the latest techniques (such as supercritical-fluid extraction). Besides that, the sample is repeatedly in contact with fresh portions of the extractant, which facilitates the displacement of the transfer equilibrium. Also, the sample throughput can be increased by performing several simultaneous extractions in parallel, which is facilitated by the low cost of the basic equipment (Luque de Castro and Priego-Capote, 2010). Lastly, no filtration is required after the process. Ahmad et al. (2009) described the Soxhlet extraction with methanol and nhexane from Elephantopus scaber L. and the study shows that methanol is better in terms of extraction yield. The different yields of extracts might be influenced by the polarities of solvents (Romdhane and Gourdon, 2001). This happens for all parts of the plant. 2.3.3 Microwave assisted extraction (MAE) A microwave device is composed of four major components. The microwave generator, also called magnetrom, is responsible for generation of microwaves. The gave wide is used to direct the propagation of microwave from the source to the microwave cavity. The applicator is where the sample holder and the sample are placed. The last component is the circulator, which regulates the movement of microwaves only in the forward direction (Tatke and Jaiswal, 2011). There are two microwave systems for extraction: closed extraction vessels/multimode microwave ovens and focused microwave ovens. The first is brought by controlled pressure and temperature. As the name suggests, in the focused microwave Dissertation for Master Degree in Bioengineering 15 ovens only the part of the extraction vessel containing the sample is focused for irradiation with microwave. This process can also be named as microwave assisted Soxhlet or solvent extraction (Mandal et al., 2007). This process is based on exposing the analytes to the solvent through cell rupture (Jyothi et al., 2010). With this method, the plants can contain microscopic traces of moisture that serves as the target for the microwave heating. Due to microwave heating, the moisture is heated up inside the plant cell, evaporates and generates tremendous pressure on the cell, because of the swelling of the plant cell. The pressure pushes the cell wall from inside, stretching and ultimately rupturing it (Mandal et al., 2007), thus the exudation of active constituents from the ruptures cells occurs, hence increasing the yield of phytoconstituents (Tatke and Jaiswal, 2011). There are some factors which affect MAE, namely the solvent nature and the volume, extraction time, microwave power, temperature and matrix characteristics (Mandal et al., 2007). MAE methods require shorter time, less solvent and provide higher extraction rates and with lower cost (Gallo et al., 2010). Gallo et al. (2010) described the MAE of phenolic compounds from Cinnamomum zeylanicum, Coriandrum sativum, Cuminum cyminum and Crocus sativus. In this experiment, the efficiency of extraction of bioactive compounds obtained with the microwave extraction process was in general about four times higher than that resulting from sonication extraction. Earlier, Pan et al. (2003) had already shown that MAE has more efficiency than conventional techniques in the extraction of tea polyphenols and tea caffeine. On the other hand, Waksmundzka-Hajnos et al. (2004) concluded the MAE is not an appropriate method for furanocoumarin recovery. They studied the optimal conditions for the extraction of furanocoumarins from fruits of Archangelica officinalis and determined that some compounds, like imperatorin and phellopterin, may be transformed during pressurized MAE. 2.3.4 Supercritical fluid extraction (SFE) In this method a gas, usually CO2, is compressed into a dense liquid and it is pumped through a cylinder containing the material to be extracted. The extract-laden liquid is pumped into a separation chamber where the extract is separated from the gas and the gas is recovered for re-use. It is very easy to recover the supercritical solvent after extraction, it is only necessary to adjust the pressure and/or the temperature Dissertation for Master Degree in Bioengineering 16 (Doughari, 2012). One advantage of this process is the fact that the final extract has virtually no solvent residues left in it (gas evaporates completely) (Patil, 2010). SFE has been increasingly used in the extraction of plant volatile components and essential oils (Pourmortazavi and Hajimirsadeghi, 2007). There are several studies with supercritical fluids to extracts compounds from plants. Fadel et al. (1999) made a research performed with eucalyptus which demonstrates the differences that exist between the composition and functional properties of extracts obtained with SFE and hydrodistillation. Supercritical fluid extracts had a higher content of sesquiterpenes and oxygenated compounds (Fadel et al., 1999). 2.3.5 Ultrasounds extraction This type of extraction utilizes acoustic cavitation to cause molecular movement of solvent and sample (Jerman et al., 2010). Cavitation forces are the result of the propagation of ultrasound pressure (Knorr et al., 2002). In the case of plants, bubbles can explosively collapse and generate localized pressure causing plant tissue rupture and improving the release of intracellular substances into the solvent (Knorr et al., 2002). There are two commons devices for ultrasounds extraction. The more widely used is the bath system but the one which offers more advantages is the probe system. This provides directly cavitation in the solution, being more efficient (Priego-Capote and Luque de Castro, 2004). Jerman et al. (2010) studied the extraction of phenolic compounds of olive fruit (Olea europaea) with ultrasounds extraction. The authors used a high intensity probe sonication. They studied different parameters (sonication time, temperature, solvent composition and extraction steps) in order to optimize the extraction. It was concluded that the method used was more efficient in comparison to ultrasound bath and agitation. 2.4 Extraction solvent For all of these techniques a solvent extraction is needed. The extraction solvents can be various and the choice is influenced by what is pretended with the extract and what are the targeted compounds to be extracted (Das et al., 2010). The low toxicity, ease of evaporation at low heat, promotion of rapid physiologic absorption of the extract and inability to cause the extract to complex or disassociate are the main characteristics Dissertation for Master Degree in Bioengineering 17 to be a good solvent. The factors that affect the choice of the solvent are, briefly, the quantity of phytochemicals to be extracted, rate of extraction, diversity of different compounds existing in the initial matrix, diversity of inhibitory compounds extracted, ease of subsequent handling of the extracts, toxicity of the solvent in the bioassay process and potential health hazard of the extractants (Eloff, 1998). Then, the type of solvent is essential on the successful determination of biologically active compounds from plant material (Tiwari et al., 2011). Table 1 shows the main solvents used to extract the different compounds, according to Cowan (1999). Table 1. Main solvents used for active compounds extraction (Cowan, 1999). Water Ethanol Methanol Ether Acetone Chloroform Anthocyanins Tannins Anthocyanins Alkaloids Phenols Terpenoids Starches Polyphenols Terpenoids Terpenoids Flavonols Flavonoids Tannins Polyacetylenes Saponins Coumarins Saponins Flavonols Tannins Fatty acids Terpenoids Terpenoids Xanthoxyllines Polypeptides Sterols Totarol Lectins Alkaloids Quassinoids Lactones Flavones Phenones Polyphenols Water is an universal solvent and it is mainly used to extract compounds with antimicrobial activity. However, organic solvents extracts give more consistent antimicrobial activity than water extracts. Water soluble flavonoids, such as anthocyanins, have no antimicrobial activity and water soluble phenolics are only important as antioxidant compound (Dask et al., 2010). Acetone dissolves many hydrophilic and lipophilic components from plants, is volatile and has low toxicity, which makes it an useful extractant. It is very used for antimicrobial studies, where more phenolic compounds are required to be extracted (Tiwari et al., 2011; Dask et al., 2010). Ethanolic extracts have high amounts of polyphenols, which means they are more efficient in cell wall and seeds degradation which have nonpolar character and cause polyphenols to be released from cells (Dask et al., 2010). Methanol is more polar Dissertation for Master Degree in Bioengineering 18 than ethanol but it has a cytotoxic nature, which makes it unsuitable for extraction in certain studies (Tiwari et al., 2011). The composition of the plant extract is influenced by the temperature, solvent extracting power, extraction time and method adapted for the extraction (Ksouri et al., 2009). This variability is due to the different affinities of these compounds for solvent extraction and specialty to the polarity of the molecules constituting of the solvent (Hayouni et al., 2007). Therefore, it is necessary to optimize the solvent/concentration to utilize in order to obtain the highest possible yield of the desired compounds. Dissertation for Master Degree in Bioengineering 19 CHAPTER 3 – Evaluation of Extraction Efficiency of Olea europaea and Acacia dealbata 3.1 Introduction Man has used plants since the start of humankind. In the course of time, plants became a useful source of disease cure and health improvement across various human communities (Vinatoru, 2001). Compounds of plants, such as alkaloids, phenolic and terpenoids, have currently recognized antimicrobial and antioxidant properties (Cowan, 1999; Karou, 2005; Gallo, 2010). The selection of the proper extraction method is very important for the qualitative and quantitative studies of bioactive compounds from plants. The different techniques of extraction have all the same goals in the process: to extract targeted bioactive compounds from complex plant samples, to increase sensitivity of bioassay by increasing the concentration of targeted compounds, to convert the bioactive compounds into a more suitable form for detection and separation and to provide a strong and reproducible method that is independent of variations in the sample matrix (Smith, 2003). The extracting power of different solvents in use and the application of heat and/or mixing are the basis of most techniques (Azmir et al., 2013). Soxhlet and solid-liquid extraction belong to the conventional or classic techniques of extraction. Nowadays, the major challenges of conventional extractions are longer extraction time, requirement of costly and high purity solvents, evaporation of the huge amount of solvent, low extraction selectivity and thermal decomposition of thermo labile compounds (Luque de Castro and Garcia-Ayuso, 1998). To overcome these limitations, new techniques are been introduced, as micro-wave and ultrasound extractions (Azmir et al., 2013). This chapter aims to select the solvent and extractions conditions in order to achieve the maximum recovery of bioactive compounds from selective plants, Olea europaea and Acacia dealbata. Dissertation for Master Degree in Bioengineering 26 Dissertation for Master Degree in Bioengineering 27 CHAPTER 4 – Analysis of the Bioactivity of Olea europaea and Acacia dealbata Extracts 4.1 Introduction Plants extracts can be a promising alternative to current antimicrobials, particularly antibiotics, taking in account the evolution of bacterial resistance (Abreu et al., 2012). Many compounds of plants have recognized antimicrobial and antioxidant properties. Giving some examples, alkaloids have been exploited as pharmaceuticals, stimulants, narcotics and poison due to their potent biological activity (Ziegler and Facchini, 2008) and they have good antimicrobial activity against several microorganisms (Karou et al., 2005). Phenolic compounds have antioxidant activity mainly due to their redox properties, which allow them to act as reducing agents, hydrogen donors and singlet oxygen quenchers (Gallo et al., 2010) and some phenolic acids and analogs also exhibit antibacterial, antifungal, antiviral, antimutagenic and anti-inflammatory activities (Silici et al., 2007; Chaubal et al., 2005; Larrosa et al., 2006). Tannins have antimicrobial (Cowan, 1999), antimutagenic, anticarcinogenic and antioxidant activity (Chung et al., 1998). Studies also reported that coumarins have antimicrobial, anti-inflammatory, antioxidant, anticoagulation, antiestrogenic and sedative activity (Paramjeet et al., 2012). The analysis of the bioactivity of the phytochemicals compounds is very important to understand their potential applications in clinical settings. The antioxidant activity of the natural compounds is related to the three major groups: vitamins, phenolics and carotenoids (Halliwell, 1996). Temple (2000) reported that the frequent consumption of natural oxidants is associated with a lower risk of cardiovascular disease and cancer, which indicates their potential application. Also, the efficacy of antimicrobial effects of antibiotics can be improved by combining them with extracts of plants against several pathogens, such as S. aureus and E. coli (Adwan and Mhanna, 2008). Besides that, the combination between antibiotics and phytochemical compounds can help in the prevention of the development of microbial resistance (Sakharkar et al., 2009). Dissertation for Master Degree in Bioengineering 28 This chapter aims to evaluate the antimicrobial and antioxidant activities of Olea europaea and Acacia dealbata extracts, complementing the previous chapter on the role of selection of an extraction method and solvent to maximize the biological effects of the plant extracts. 4.2 Material & Methods 4.2.1 Extracts The extracts from Olea europaea and Acacia dealbata previously obtained were used in this chapter. 4.2.2 Bacterial Strains The bacteria used in this study were obtained from the Spanish Type Culture Collection (CECT): the Gram-negative bacterium Escherichia coli (CECT 434), the Gram-positive bacterium Staphylococcus aureus (CECT 976) and the Gram-negative Chromobacterium violaceum (ATCC 12472). E. coli and S. aureus were distributed over the surface of Mueller-Hinton Agar (MHA, Merck) and incubated for 24 h at 27 ± 3 ºC and C. violaceum was distributes over the surface of Luria-Bertani Agar (LBA, Merck) and incubated for 24 h at 27 ± 3 ºC. 4.2.3 Antimicrobial Activity Assessment Antimicrobial activity was tested using a modification of the disc diffusion method originally described by Bauer et al. (1966). Bacteria were grown overnight and the turbidity was adjusted to match 0.5 in McFarland standards with sterile saline (Spectrometer VWR V-1200). Petri dishes with 90 mm of diameter were prepared with approximately 20 mL of MHA. Sterile filter paper discs (6 mm of diameter) impregnated with 10 µL of the samples were placed on the agar plate seeded with the respective bacteria. The plates were incubated at 37 ˚C for 24 h. Discs impregnated with DMSO were used as negative control. After incubation, the diameter in mm of the inhibitory zones around the discs was recorded (Saavedra et al., 2010). 4.2.4 Antioxidant Activity Assessment Assays used to estimate the antioxidant capacity of the plants mostly include ABTS (Leong and Shui, 2002) and DPPH (Gil et al., 2002). Dissertation for Master Degree in Bioengineering 29 4.2.4.1 ABTS Assay A stock solution included 7.4 mM ABTS solution (Sigma-Aldrich) and 2.6 mM potassium persulfate solution (Sigma-Aldrich). Then these two stock solutions were mixed in equal quantities and allowed to react for 12 h at room temperature in the dark. The resulting solution was diluted by mixing 1 mL of this solution with 60 mL of methanol to obtain an absorbance of 1.10 ± 0.02 units at 734 nm. The samples are then mixed with the prepared solution (1:20 V/V) and allowed to react for 2 h in a dark condition. The absorbance was taken at 734 nm (Spectrometer VWR V-1200). The standard curve was linear between 25 and 800 µM Trolox (Sigma-Aldrich) and the results are expressed in µM Trolox equivalents (TE)/g fresh mass (Thaipong et al., 2006). 4.2.4.2 DPPH Assay A stock solution included 0.024 mg/mL DPPH solution (Sigma-Aldrich) in methanol. The working solution was prepared by mixing 10 mL of the stock solution with 45 mL of methanol to obtain an absorbance of 1.10 ± 0.02 units at 515 nm. The samples (150 µL) are then mixed with the working solution (2850 µL) and allowed to react for 24 h in a dark condition. The absorbance was taken at 515 nm (Spectrometer VWR V-1200). The standard curve was linear between 25 and 800 µM Trolox and the results are expressed in µM TE/g fresh mass (Thaipong et al., 2006). 4.2.5 Antibiotics-Extracts Dual Combination Assay To study the antimicrobial effects of the extracts with antibiotics, the extract (dissolved in DMSO) was inserted in MHA medium (at a final concentration of 5000 µg/mL) after autoclaved and cooled. Suspensions were prepared mixing bacteria from an overnight culture with sterile saline solution in order to obtain a final cell turbidity matching 0.5 McFarland standards (Spectrometer VWR V-1200). The cellular suspension was poured over hardened MHA/extracts plates using a sterilized cotton swab and allowed to set. Antibiotic discs containing erythromycin (15 µg/disc) and tetracycline (30 µg/disc) (Sigma-Aldrich), according to the Clinical Laboratory Standards Institute standards (CLSI, 2005), were placed on the surface of the plates and then the plates were incubated at 37 ˚C for 48 h (Saavedra et al., 2010). The zones of grown inhibition were measured after 24 and 48 h (CLSI, 2005). Dissertation for Master Degree in Bioengineering 30 4.2.6 Antibiotics-Extracts Dual Combination Classification The effect of dual combinations of antibiotics and extracts was classified according to Saavedra et al. (2010): • antagonism (-) if [inhibition halo – (antibiotic inhibition halo + extract inhibition halo)/2] < 0; • indifference (+) if 0 ≤ [inhibition halo – (antibiotic inhibition halo + extract inhibition halo)/2] < antibiotic inhibition halo or extract inhibition halo; • additive (++) if antibiotic inhibition halo < [inhibition halo – (antibiotic inhibition halo + extract inhibition halo)/2] < 2 × antibiotic inhibition halo or extract inhibition halo; • synergy (+++) if inhibition halo > 3 × antibiotic inhibition halo or extract inhibition halo. For the classification was selected the highest inhibition halos caused by the antibiotic or phytochemical application for each condition tested. 4.2.7 Quorum-sensing Inhibition (QSI) Chromobacterium. violaceum was grown overnight in LB broth at 30 ˚C with shaking. To test the plant leaves, they were placed in LB agar plates and overlain with 5 mL of LBA soft (tempered at 45 ˚C) containing 106 CFU/mL of the C. violaceum. Then the plates were incubated at 30 ºC for 24 h and, after this, the color was examined. To test the extracts of the plants the cell suspension was prepared in order to obtain 106 CFU/mL of the C. violaceum (by mixing with LB broth). Sterile filter paper discs (6 mm of diameter) impregnated with 10 µL of the extracts were placed on LB agar plates seeded with 100 µL of the respective bacterium. Then the plates were incubated at 30 ºC for 24 h and, after this, the color was examined (McLean et al., 2004). 4.2.8 Statistical Analysis The statistical analysis of the results obtained was performed as described in section 3.2.5. Dissertation for Master Degree in Bioengineering 31 4.3 Results and Discussion 4.3.1 Antimicrobial Activity Assessment Olive leaves – solid-liquid and ultrasounds extractions Regarding to olive leaves extracted with solid-liquid extraction, activity for almost all solvents was found, as indicated in Table 3. Concentrations of the plant extracts were not standardized, ranging from 48.9 mg/mL for hexane to 254.1 mg/mL for acetone. Concentrations depend on the volume of DMSO used to dissolve the dry extracts. Table 3. Antibacterial activity of olive leaves extracts for solid-liquid extraction and for the selected solvents against S. aureus and E. coli – diameter of inhibition (mm). Solvents Water Methanol Ethanol Acetone Dichloromethane Hexane Extract concentration (mg/mL) 150.3 100.5 84.9 254.1 253.2 48.9 S. aureus 0.0±0.0 16.7±1.2 25.3±2.3 22.7±1.2 8.7±0.6 11.3±1.2 E. coli 0.0±0.0 16.7±1.2 16.7±1.2 14.7±1.2 0.0±0.0 12.0±2.0 The extraction with water did not show activity against any microorganism. Several studies support that the olive leaves in the aqueous extracts have antimicrobial activity against pathogenic bacteria (Aliabadi et al., 2012; Keskin et al., 2012). Therefore, maybe, if some conditions were different, for example if the extraction time was superior, the results would have been different for these extracts. The extraction with dichloromethane only showed activity against S. aureus. All the other extracts presented activity for both bacteria. Ethanol and acetone were the solvents that caused the highest inhibition halo (p<0.05) against S. aureus and the differences between these solvents were not significant (p>0.05). For these solvents the activity was superior against S. aureus (p<0.05). According to Cowan (1999), ethanol and acetone are highly efficient in the extraction of phenolic compounds. Concerning the effects of the extracts obtained against E. coli, there were no significant differences between the antimicrobial activity of methanol, ethanol, acetone and hexane (p>0.05). The fact that the antimicrobial activity is higher against S. aureus is supported by Simões et al. (2008). This species is Gram-positive and the permeability of the cell wall can make it more susceptible and, for that reason, does not restrict the penetration Dissertation for Master Degree in Bioengineering 32 of antimicrobials. However, this only happens with the extracts obtained with ethanol, acetone and dichloromethane. For methanol and hexane extracts there was no significant differences on the effects caused by the plant extracts on both Gram-positive and Gram-negative bacteria (p>0.05). In order to have more consistent results on the relation between the solvent used and antimicrobial effects, the concentrations of the extracts were standardized and tested at 5 mg/mL. This concentration is also more appropriated to use the extracts in medicine. The results are shown in Table 4. Table 4. Antibacterial activity of olive leaves extracts at 5 mg/mL for solid-liquid extraction and for the selected solvents against S. aureus and E. coli - diameter of inhibition (mm). Solvents Water Methanol Ethanol Acetone Dichloromethane Hexane S. aureus 0.0±0.0 0.0±0.0 10.7±0.6 0.0±0.0 0.0±0.0 0.0±0.0 E. coli 0.0±0.0 0.0±0.0 11.0±0.0 0.0±0.0 0.0±0.0 0,0±0.0 With lower concentrations the results were very different. All the extracts lost their antimicrobial activity, except the sample extracted with ethanol. However, this extract also lost some activity against both species, since the inhibition halo decreased 58% against S. aureus (p<0.05) and 52% against E. coli (p<0.05). However, with a concentration of 5 mg/mL the activity of this extract did not change depending on the Gram-type of the bacteria. These results indicate that it is necessary a concentration relatively high of olive leaves extract to have antimicrobial activity. It would be interesting to discover the minimal concentration of the extract to obtain activity The results on the antimicrobial activity of olive leaves extract obtained by ultrasounds extraction are shown in Table 5. The concentrations of the extracts range from 59.6 mg/mL (with methanol) to 239.3 mg/mL (with acetone). Table 5. Antibacterial activity of olive leaves extracts for ultrasounds extraction and for the selected solvents against S. aureus and E. coli – diameter of inhibition (mm). Solvents Water Methanol Ethanol Acetone Dichloromethane Hexane Concentration (mg/mL) 123.4 59.6 168.6 239.3 93.4 161.3 S. aureus 11.3±1.2 18.7±1.2 27.3±2.3 18.7±1.2 0.0±0.0 15.0±1.4 E. coli 9.3±0.6 16.7±1.2 16.7±1.2 14.0±2.0 0.0±0.0 11.3±1.2 Dissertation for Master Degree in Bioengineering 33 Ethanol, with its ability to extract phenolic compounds (Cowan, 1999), seems to be the best solvent to extract olive leaves, since its activity causes the higher inhibition halo against S. aureus (p<0.05). The sample extracted with dichloromethane did not show activity against both S. aureus and E. coli. For water, methanol, acetone and hexane the results did not significantly differ for both bacteria (p>0.05). Once again, for ethanol, S. aureus is the bacterium more susceptible to the antimicrobials (p<0.05). The exterior membrane of E. coli, with a set of outer membrane proteins, which are powerful barriers to the antimicrobials, can explain the lower inhibition halos (Simões et al., 2008). As for the solid-liquid extraction, the concentrations of the extracts were standardized to 5 mg/mL and the results of the antimicrobial activity assessment are presented in Table 6. Table 6. Antibacterial activity of olive leaves extracts at 5 mg/mL for ultrasounds extraction and for the selected solvents against S. aureus and E. coli - diameter of inhibition (mm). Solvents Water Methanol Ethanol Acetone Dichloromethane Hexane S. aureus 9.3±0.0 0.0±0.0 10.7±0.6 9.3±0.6 0.0±0.0 0.0±0.0 E. coli 0.0±0.0 0.0±0.0 10.7±0.6 10.7±0.6 0.0±0.0 0.0±0.0 In this case, extracts with methanol and hexane did not show antimicrobial activity. Extracts with water and dichloromethane only demonstrate activity against S. aureus and, as already said, this is the bacterium more susceptible to the plant extracts. For extracts with ethanol and acetone the results were similar for both bacteria and there were no significant differences (p>0.05). No solvent extraction excelled in this assay: results are similar for water, ethanol, acetone and dichloromethane against S. aureus (p>0.05) and for ethanol and acetone against E. coli (p>0.05). Comparing to higher concentrations, extracts performed with methanol and hexane lost their activity with the decrease of concentration. The same happened with the extract obtained with water which lost activity against E. coli. Differences were not observed by decreasing the concentration for extracts with water against S. aureus and with acetone against E. coli (p>0.05). Ethanol was more efficient in extract antimicrobial compounds with a high concentration (p<0.05) and the same happened with extracts performed with acetone, but only against S. aureus. Dissertation for Master Degree in Bioengineering 34 Mimosa leaves – solid-liquid and ultrasounds extractions The results for mimosa leaves extract from solid extraction are presented in this section. The concentrations of the extracts ranged from 100.2 mg/mL (with dichloromethane) and 253.3 mg/mL (with ethanol) on a first approach (Table 7), having been later standardized to 5 mg/mL (Table 8). Table 7. Antibacterial activity of mimosa leaves extracts for solid-liquid extraction and for the selected solvents against S. aureus and E. coli – diameter of inhibition (mm). Solvents Water Methanol Ethanol Acetone Dichloromethane Hexane Concentration (mg/mL) 106.3 140.5 253.3 190.4 100.2 145.7 S. aureus 0.0±0.0 16.7±1.2 28.0±2.0 12.0±0.0 0.0±0.0 11.3±1.2 E. coli 0.0±0.0 16.7±1.2 13.3±1.2 10.0±0.0 0.0±0.0 10.0±0.0 Table 8. Antibacterial activity of mimosa leaves extracts at 5 mg/mL for solid-liquid extraction and for the selected solvents against S. aureus and E. coli - diameter of inhibition (mm). Solvents Water Methanol Ethanol Acetone Dichloromethane Hexane S. aureus 0.0±0.0 0.0±0.0 10.0±1.0 0.0±0.0 0.0±0.0 0.0±0.0 E. coli 0.0±0.0 0.0±0.0 9.3±0.6 0.0±0.0 0.0±0.0 0.0±0.0 With the extracts at higher concentrations the results were similar to olive leaves extract. Ethanol was the only solvent that caused different activities depending on the bacteria (p<0.05). It also seems that ethanol is the best solvent to extract antimicrobial compounds against S. aureus (p<0.05). With extracts of water and dichloromethane no activity was found against the microorganisms. Extracts with methanol, acetone and hexane did not express differences between both bacteria (p>0.05). For the test with the extracts at 5 mg/mL only the ethanol showed activity against the bacteria, which proves its good ability to extract the antimicrobials compounds from plants. However, its activity was less efficient with the decreased concentration against both bacteria (p<0.05). Relatively to the ultrasounds extraction, the results are shown in Table 9 (concentrations of the extracts range from 19.0 mg/L with hexane to 181.5 with methanol) and Table 10 (concentrations at 5 mg/mL). Dissertation for Master Degree in Bioengineering 35 Table 9. Antibacterial activity of mimosa leaves extracts for ultrasounds extraction and for the selected solvents against S. aureus and E. coli – diameter of inhibition (mm). Solvents Water Methanol Ethanol Acetone Dichloromethane Hexane Concentration (mg/mL) 93.4 181.5 129.1 27.5 24.4 19.0 S. aureus 10.0±1.8 9.3±0.6 25.3±2.3 15.0±0.0 0.0±0.0 12.0±0.0 E. coli 0.0±0.0 16.7±1.2 16.7±1.2 16.7±1.2 0.0±0.0 12.0±0.0 Table 10. Antibacterial activity of mimosa leaves extracts at 5 mg/mL for ultrasounds extraction and for the selected solvents used against S. aureus and E. coli - diameter of inhibition (mm). Solvents Water Methanol Ethanol Acetone Dichloromethane Hexane S. aureus 10.0±1.8 0.0±0.0 10.0±1.0 0.0±0.0 0.0±0.0 0.0±0.0 E. coli 0.0±0.0 0.0±0.0 9.7±0.6 0.0±0.0 0.0±0.0 0.0±0.0 Once again the extract with dichloromethane did not show activity against the bacteria for any concentration. In relation to the extract with water the decrease of the concentrations did not cause changes in the antimicrobial activity: antimicrobial activity was found against S. aureus but not against E. coli. Ethanol demonstrated to be the best solvent and, once again, its activity decreased with the decrease of the concentration (p<0.05). The extract performed with ethanol was again more efficient against S. aureus than against E. coli (p<0.05), but only for the higher concentration. Extracts with acetone and hexane at higher concentrations did not present differences against the Gram-positive and Gram-negative bacteria (p>0.05). With the extracts at 5 mg/mL, the extracts with methanol, acetone, dichloromethane and hexane did not show antimicrobial activity. Water and ethanol presented similar results against S. aureus (p>0.05). Regarding to the sample extracted with methanol at 181.5 mg/mL, a higher antimicrobial activity was found against E. coli than against S. aureus (p<0.05), which contradicts what Simões et al. (2008) reported. However, this result can be supported by the previous findings of Taguri et al. (2006). The authors said that there is no relation between the Gram-type and the antimicrobial activity and that it depends on bacterial species, mode of action of the phytochemicals and its physico-chemical properties. Dissertation for Master Degree in Bioengineering 42 For both techniques, the extract obtained with acetone is the one with the highest antioxidant ability, for DPPH and ABTS (p<0.05). This confirms the studies of Cowan (1999) and Gallo et al. (2010). These authors stated that acetone is one of the main solvents used to extract phenolic compounds from plants and these compounds have a high antioxidant activity. Acetone dissolves many hydrophilic and lipophilic compounds from plants, which makes it a very useful solvent (Eloff, 1998). For both extractions, in relation to the extract with lower antioxidant power, methanol is the solvent which was less efficient to extract compounds with antioxidant properties, as assessed by both DPPH as ABTS assays. Relatively to the ultrasounds extraction, methanol and ethanol originated the extracts with less antioxidant activity (p>0.05) with the method of ABTS. The antioxidant activity of plant extracts is strongly dependent on the nature of the extracting solvent, due to the presence of several antioxidant compounds of diverse chemical characteristics and polarities. Polar solvents are most frequently employed for the recovery of polyphenols, which are one of the main groups of compounds more responsible for antioxidant activity (Peschel et al., 2006). Actually, ethanol, acetone, ethyl acetate, methanol and aqueous mixtures of them have been extensively used to extract compounds from plants and plant-based foods with antioxidant properties, such as broccoli and rosemary (Peschel et al., 2006: Abdille et al., 2005). By this way, it was not expected that methanol and ethanol provided the extracts with lower antioxidant activities. Table 19 presents the results of antioxidant activity for olive leaves extracts with water for every types of extraction. Table 19. Antioxidant activity estimated by DPPH and ABTS of olive leaves extracts at 5 mg/mL for extraction techniques with water (expressed in TE/g fresh mass). Water Extractions SolidLiquid Ultrasounds Soxhlet Micro-wave DPPH (TE/g fresh mass) 686.5±6.9 677.9±4.0 738.6±5.5 740.8±10.8 ABTS (TE/g fresh mass) 414.8±5.9 459.2±11.9 450.4±6.9 482.3±9.7 Dissertation for Master Degree in Bioengineering 43 With the method of DPPH, differences between the types of extraction were not found: all the extracts had similar antioxidant potential (p>0.05). The same did not happened with the ABTS method; the solid-liquid extraction proved to be less efficient in extracting antioxidant compounds. Mimosa leaves The acetone extracts proved to be those with higher antioxidant activity, as indicated in Table 20 and Table 21. This fact is probably due to the presence of phenolic compounds. Table 20. Antioxidant activity estimated by DPPH and ABTS of olive leaves extracts at 5 mg/mL for solid-liquid extraction and for the selected solvents (expressed in TE/g fresh mass). Methanol Ethanol Acetone Dichloromethane Hexane DPPH (TE/g fresh mass) 739.8±6.9 312.2±8.8 782.9±4.7 225.8±11.8 84.4±6.0 ABTS (TE/g fresh mass) 474.8±4.3 514.2±6.9 607.9±5.9 539.2±12.9 392.9±7.5 Table 21. Antioxidant activity estimated by DPPH and ABTS of mimosa leaves extracts at 5 mg/mL for ultrasounds extraction and for the selected solvents (expressed in TE/g fresh mass). Methanol Ethanol Acetone Dichloromethane Hexane DPPH (TE/g fresh mass) 730.8±4.8 477.21±12.8 770.1±6.6 44.4±4.5 134.4±8.7 ABTS (TE/g fresh mass) 451.1±6.6 534.2±14.9 607.9±7.9 502.3±10.8 464.8±5.9 Acetone and methanol were the best solvents to extract antioxidant compounds with the DPPH method, for both extractions. In several studies, methanol is considered the best solvent to extract antioxidant compounds (Sultana et al., 2009). With the ABTS method, dichloromethane joined to acetone as the best solvents, in solid-liquid extraction, (p<0.05). With ultrasounds extractions only acetone was highlighted as the best solvent. For extracts with water, the highest values of antioxidant ability for DPPH were found for solid-liquid, ultrasounds and Soxhlet extraction (p<0.05). Assays with ABTS did not show significant differences between the types of extraction (p>0.05). Results are presented in Table 22. Dissertation for Master Degree in Bioengineering 44 Table 22. Antioxidant activity estimated by DPPH and ABTS of mimosa leaves extracts at 5 mg/mL for extraction techniques with water (expressed in TE/g fresh mass). Water Extractions SolidLiquid Ultrasounds Soxhlet Micro-wave DPPH (TE/g fresh mass) 765.8±8.9 756.5±4.2 698.6±12.0 595.1±2.1 ABTS (TE/g fresh mass) 356.7±5.9 387.3±5.5 338.6±9.1 387.3±9.8 To finalize the study about the antioxidant properties of the studied plants, it is possible to conclude that both of them can produce extracts with antioxidant activity. Several studies have already reported the antioxidant activity of olive leaves extract, such as Fitó et al. (2007), who reported the antioxidant properties of the extracts and the corresponding health benefits such as cardioprotective and chemopreventive effects. The presence of oleuropein and phenolic compounds is an important factor for antioxidant capacity of olive leaf extracts (Lee et al., 2009). The magnitude of the antioxidant power depends on the extraction solvent used. For both plants, acetone was the solvent that originated the extracts with higher antioxidant potential as assessed by both methods (DPPH and ABTS), which is probably due to its ability to extract phenolic compounds. Phenolic compounds have antioxidant activity mainly due to their redox properties, which allow them to act as reducing agents, hydrogen donors and singlet oxygen quenchers (Gallo et al., 2010). According to Martysiak-Zurowska and Wenta (2012), DPPH method has lower sensitivity than ABTS, probably because the DPPH method has more limitations. ABTS radical is reactive towards most antioxidants and it is soluble in both aqueous and organic solvents (Cano et al., 2000), while DPPH is more restrictive. However, in the current study, DPPH assay obtained in most cases the highest values. For that reason, DPPH method was considered a more useful method in the assessment of antioxidant activity witholive and mimosa leaves extracts. Despite the observed differences between the values of DPPH and ABTS methods, the most important information to retain is that, almost always, both assays ranked the extracts in a similar order. Both gave acetone extracts as the most effective free radical scavenger and this evidence is actually more relevant than knowing the exact chemical reactivity of each sample (Wooton-Beard et al., 2011). Dissertation for Master Degree in Bioengineering 45 Comparing the plants used in the current study, differences were not observed between the antioxidant activities for the extracts with higher antioxidant properties (extracts of acetone) (p>0.05). Then, olive and mimosa extracts have similar ability to scavenge free radicals. Lou et al. (2012) studied the antioxidant properties of Morus alba L. (mulberry fruits) and they found values between 75 µM TE/g fresh mulberry fruits and 240 µM TE/g fresh mulberry fruits for ABTS method and 95 µM TE/g fresh mulberry fruits and 180 µM TE/g fresh mulberry fruits for DPPH method, which indicates that olive and mimosa have higher antioxidant activity than mulberry fruits. In fact, the values of antioxidant activity in the current study are extremely high comparing with other studies. Wang et al. (1996) studied the antioxidant activity of 12 fruits and they obtained from 1 µM TE/g for melon to 15 µM TE/g for strawberry. 4.4 Conclusions This work allowed to conclude that Olea euopaea and Acacia dealbata extracts have antimicrobial and antioxidant activities. Typically, ethanol was the solvent that could extract compounds with more antimicrobial properties. This means that it was apparently very efficient in the extraction of phenolic compounds (Cowan, 1999). On the contrary, extracts with dichloromethane were rarely efficient against the bacteria, which allow to conclude that this solvent was not a good option. In most cases, antimicrobial activity of the extracts was superior against S. aureus, which suggest the susceptibility of the bacterium due to the permeability of the cell wall (Simões et al., 2008). With the decreasing of the extracts concentration, extracts lost some activity, and, in some cases, they suffered complete loss of activity. Comparing all the extracts performed with water, it was found that Soxhlet and micro-wave extractions are those for which the extracts have higher antimicrobial activity. The combination between the plant extracts and antibiotics did not prove to be beneficial. No plant showed positive effects in bacterial killing when combined with tetracycline and erythromycin. Dissertation for Master Degree in Bioengineering 46 Extracts with water and hexane from both plants had antimicrobial activity against C. violaceum but they were not efficient in QSI. Antimicrobial activity of water extracts was superior than that of hexane extracts. Leaves and methanol extracts did not obtained antimicrobial activity against C. violaceum and they were not efficient in QSI. In relation to antioxidant activity, it was concluded that acetone was the best solvent to extract compounds with antioxidant properties. DPPH method achieved the highest values in most cases, whereby it was considered the method more useful in determination of antioxidant activity of olive and mimosa extracts. However, both methods ranked the extracts almost always in the same order. Comparing with previous studies, it was found that olive and mimosa leave extracts have much higher antioxidant activity than some fruits, like melon and strawberry. Any method excelled in the extraction of antioxidant compounds. Dissertation for Master Degree in Bioengineering 47 CHAPTER 5 – Conclusions and Perspectives for Future Work Differences found between the results of bioactivity of Olea europaea extracts in previous studies and the present study can be explained by the preparation of the extracts. Usually, the extracts come from powdered leaves and are boiled or autoclaved and in this study the extracts were obtained from fresh leaves. Besides that, the choice of extraction solvent, crop origin, harvesting time and climate may influence the leaf composition, which can influence the bioactivity of extracts (Sudjana et al., 2008). In relation to the extraction yield, methanol and water were considered the best solvents to solid-liquid and ultrasound extractions. Despite the advantages reported in the literature about ultrasound extraction, solid-liquid extraction allowed to obtain extracts with similar extraction efficiency. Comparing all the techniques used in the current study, Soxhlet and micro-wave were the methods that were able to achieve the best extraction efficiency. It can be concluded that mimosa and olive leaves can provide extracts with antimicrobial and antioxidant activities. Ethanol proved to be the best solvent to extract compounds with antimicrobial activity, whereas for extract compounds with antioxidant properties acetone proved to be the best solvent. Dichloromethane was considered a weakest solvent to extract antimicrobial compounds. This happened for both mimosa and olive leaves. It was also concluded that olive and mimosa extracts have similar bioactivity. Soxhlet and micro-wave extractions were the best techniques to extract compounds with antimicrobial activity, whereas to extract compounds with antioxidant activity no method was highlighted. In most cases, extracts were more efficient against S. aureus than E. coli, suggesting the higher susceptibility of S. aureus due to the permeability of the outer layer of the cell. It was also demonstrated that, by decreasing the extract concentration,, the antimicrobial activity also decreases (in some cases the extract lost completely its activity at 5 mg/mL). As so, it would be interesting to find what is the minimal inhibitory concentration for each extract. The combination of extracts of olive and mimosa with tetracycline and erythromycin against E. coli and S. aureus was not benefic and the extract leaves and the leaves themselves were not efficient on QSI. To conclude, no extraction solvent was the best in all of the parameters tested. 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