Phenolic Compounds and Antimicrobial Activity of Olive (Olea europaea L.) Leaves

The olive (Olea europaea L.) is an evergreen tree requires chilling for fruiting. It is mostly grown for oil extraction and it holds numerous biological and medicinal values (Kumar and Sharma, 2016).
 
Olive tree (Olea europaea L.) is one of the most important fruit trees in Mediterranean countries, where they cover 8 million ha, accounting for almost 98% of the world crop (Guinda et al., 2004). (Olea europaea L.) is widely studied for its alimentary use, whereas the leaves are important for their secondary metabolites such as the secoiridoid compounds oleacein and oleuropein, the former responsible for hypotensive activity (Hansen et al., 1996). Several reports have shown that olive leaf extract has the capacity to lower blood pressure in animals (Samuelsson, 1951) and increase blood flow in the coronary arteries (Zarzuelo et al., 1991), relieve arrhythmia and prevent intestinal muscle spasms (Garcia et al., 2000).
       
Fungal pathogens are mainly responsible for the decay of fruits and vegetables during the postharvest period (Pathak, 1997; Debjani et al., 2018). Aspergillus, Fusarium and Penicilliumare are responsible for spoilage of many foods and causes decay on stored fruits damaged by insects, animals, early splitsand mechanical harvesting. Apart from causing diseases in plants, many species of Aspergillus, Penicillium and Alternaria can also synthesize mycotoxins (Rojas et al., 2004, Agrios, 1997, Alkooranee et al., 2020). The main aim of this work was to evaluate the antifungal properties of the extract of olive leaves against phytopathogens that cause several diseases in olive, such as Aspergillus solani, Aspergillus niger, Penicillium digitatum and Mucor hiemalis.
       
In this paper, HPLC was used to evaluate the qualitative composition of the phenolic compounds in olive leaves with the aim to identify new compounds and we evaluated the extracts’ antimicrobial activity against bacteria and fungi.

Plant materials
 
The olive leaves (Sigoise variety of olive fruit) were collected from Tlemcen (West of Algeria), in autumn and dried away from direct sunlight. Dried plant material was then crushed into a mortar and stored at very low temperature until further use.
 
Extraction of phenolic compounds 
 
The dried powder of olive leave (10 g) was extracted in triplicate, using EtOH (96% v/v) at room temperature, under stirring. The aqueous suspension of the concentrated EtOH extract was evaporated to dryness and used for all investigations (Kukic et al., 2008).
 
High performance liquid chromatography (HPLC)
 
Total phenolics analyses of methanolic extract of infected olive leaves were carried out using Jasco HPLC, consisting of a pump (PU-2089 Plus) and UV detector model UV-2077 Plus with ChromNAV on a XBridge analytical column (RP-C18: 5 µm, 4.6×150 mm) (Waters Inc. USA) with gradient solvent system and parameter condition as shown in Table 1. The chromatograms were observed at wavelengths of 254, 270, 280 and 329 nm. All the analyses were carried out at sample concentration of 1 mg/ml and injection volume of 20 µl.
 

 
Pathogenic fungi
 
Four fungal isolates causing olive rot. Aspergillus niger, Aspergillus solani, Penicillium digitatum and Mucor hiemalis were isolated directly from rotten Olea europaea fruits. All isolated fungal species were transferred to sterilized triplicate 9 cm Petri dishes containing fresh potato dextrose agar medium (PDA: potato 200, dextrose 20 g and agar 15 g/L in distilled water at 25^oC) in the presence of a quantity of lactic acid (25%) to stop the growth of bacteria. The plates were incubated at 25±2^oC for 8 days, in darkness. The developing fungal colonies were purified and identified up to the species level by microscopic examination through the help of published materials (Barnett, 2006).
 
Antifungal assay
 
The antifungal activity of essential oil and extracts was tested using the radial growth technique (Zambonelli et al., 1996, Bajpai et al., 2007). Appropriate volumes of the essential oil and extracts were added to the PDA medium immediately before it was poured into the Petri dishes (9.0 cm diameter) (at 40-45^oC) to obtain a series of concentrations (0.01 to 5500 μg/mL). Each concentration was tested in triplicate. The discs of mycelial felt (0.5 cm diameter) of the plant pathogenic fungi, taken from 8-day-old cultures on PDA plates, were transferred aseptically to the center of Petri dishes. Amphotericin B was used as a reference fungicide. The treatments were incubated at 27^oC in the dark. Colony growth diameter was measured after the fungal growth in the control treatments had completely covered the Petri dishes.
 
Antibacterial activity
 
Growth inhibition activities for sample extracts against: Staphylococcus aureus, Bacillus cereus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumonia, Enterobacter cloacae, Proteus mirabilis and Salmonella typhimurium were tested using disc diffusion method (Berghe et al., 2001). The suspension of bacteria of about 1.5×10⁶ CFU/ml. Colony forming units per milliliter obtained following a 0.5 McFarland turbidity standard, which was standardized by adjusting the optical density to 0.1 at 600 nm (JENWAY 6405UV/Vis spectrophotometer) (Tereschck et al., 1997). One milliliter of inoculums suspensions were used to inoculate by flooding the surface of Mueller-Hinton Agar plates. Excess liquid was air dried under a sterile hood. Dried extracts were dissolved in DMSO at the concentration 25, 30 and 50 mg/ml for aqueous methanol extract and aqueous acetone extract and 10, 15 and 20 mg/ml of ethyl acetate fraction, butanolic fraction. After, sterilized discs (Whatman N°1, 6 mm diameter) were impregnated with 5 µl of each extract (equivalent to 125, 150 and 250 µg/disc for aqueous methanol extract and aqueous acetone extract, respectively and equivalent to 50, 75 and 100 µg/disc for ethyl acetate fraction and butanolic fraction, respectively) and placed on the agar surface. DMSO was used as a negative control. The plates were left for 30 min at room temperature to allow the diffusion of extractand then they were incubated at 37°C for 24 h. Antibacterial activity was evaluated by measuring the diameter of the inhibition zone and presented in millimeters.

Phenolic compounds in olive leaves
 
The data (retention time, λ max in the visible regionand tentative identification) obtained for the phenolic compound peaks in the HPLC-DAD analysis are presented in Table 1 and Fig 1. HPLC studies point to five phenolic compounds determined in olive leaves extracts: Ascorbic acid (Rt=1.964 min, maximum absorbance at 243 nm), Verbascoside (Rt=16.23 min, maximum absorbance at 251 nm), Oleuropein (Rt=16.95 min, maximum absorbance at 242 nm), Rutin (Rt=18.535 min, maximum absorbance at 250 nm) and catechin (Rt=19,549 min, maximum absorbance at 252 nm).
       
The retention time and absorption spectrum (obtained by means of a UV/vis DAD) were identical to those obtained for the corresponding standards. The HPLC analysis of the studied sample revealed different chemical profiles, in which five phenolic compounds were identified and quantified: oleuropein, ascorbic acid,rutin, catechinand verbascoside (Fig 1, Table 1). All these compounds were previously reported to occur in olive leaf except ascorbic acid (Benavente-Garcia et al., 2000; Meirinhos et al., 2005; Pereira et al., 2007).
 
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The differences found in the phenolic composition are not surprising, considering that a different extractive method was applied (Romero et al., 2004). According to the literature, these compounds are present in the olive fruit (Blekas et al., 2002; Romero et al., 2004; Pereira et al., 2006). The phenolics content of olive depends on several factors, such as cultivar (Vinha et al., 2005; Esti et al., 1998), climate (Salvador et al., 2001), irrigation regimes (Romero et al., 2002), degree of ripeness of the fruit (Gutierrez et al., 2005) and elaboration process (Romero et al., 2004; Das et al., 2021).
 
Antibacterial and antifungal activities
                                               
The inhibitory effects of olive leaf extracts were evaluated against eight four bacteria: Staphylococcus aureus, Bacillus cereus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumonia, Enterobacter cloacae, Proteus mirabilis and Salmonella typhimurium and against four fungi: Aspergillus niger, Aspergillus solani, Penicillium digitatum and Mucorhiemalis. The results obtained from assays of antibacterial activity at different concentrations of olive leaf extracts by the radial growth technique are reported in Table 2 and 3.
 

 

       
The results indicate that the inhibition of the mycelial growth of each strain was significantly influenced by the extracts concentration. This study revealed the significant antimicrobial activity of olive leaf extracts.
       
Some researchers have also demonstrated that biocompounds present in olive products, such as oleuropein (Furneri et al., 2002; Battinelli et al., 2006) and hydroxytyrosol (Furneri et al., 2002) and aliphatic aldehydes (Battinelli et al., 2006), inhibit or delay the rate of growth of a range of bacteria and microfungi. In this study, the antimicrobial activity of extracts of olive leaves was evaluated against fungi isolated from olive and bacteria.
       
The response for each microorganism tested was different. The mycelial growth of colonies in the presence of the extracts of olive leaves showed that it effectively controlled all the fungi tested. This efficiency can be explained by the presence of active molecules that inhibited the growth of the five phytopathogenic fungi. Several authors have attributed the antifungal capacity of olive (Pereira et al., 2006). Oleuropein and hydroxytyrosol have shown antimicrobial activity against Salmonella spp., Vibrio spp. and Staphylococcus aureus (Pereira et al., 2006).
       
In addition, some reports (Ruiz-barba et al., 1991; Marsilio et al., 1998) have shown that some phenolic substances of olive trees may inhibit the growth of bacteria, such as Lactobacillus plantarum, Leuconostoc mesenteroides and fungi like Phytophthora (Delrio et al., 2003). Similarly, the phenolic metabolism of the olive tree is considered as a plant-response to the infection by Verticilliumdahliae (Daayf, 1993).
       
The chemical composition of olive leaf extracts impacted the antimicrobial effects observed. In fact, the mode of action of phenolics has been shown to be concentration dependent (Battinelli et al., 2006; Cowan, 1999). Additionally, the antimicrobial action of these compounds is well-known and is related to their ability to denature proteins, which in general renders them to be classified as surface-active agents (Denyer et al., 1998). These results are important against several pathogenic microorganisms resistant to a number of phytochemicals.

This study strongly suggests that some phenolic compounds present in olive leaves play a role in the natural defense mechanism, as it has been established for other phenolic secondary metabolites in different plant materials infected by pathogens. Regarding the part of the evaluation of the antibacterial activity of olives in vitro, it turns out that the bactericidal effect varies considerably depending on the nature and concentration of the polyphenol. Overall, olives are a source of valuable natural bio-phenols, endowed with remarkable antibacterial activity, which can serve in the pharmaceutical, food and agriculture industries.

None.