INTRODUCTION:

Plants are potential reservoir of antibiotics in many countries [1]. In developing countries, about sixty to ninety percent of people use plant-derived medicine. Traditionally, unprocessed herbal extracts are utilized as antibiotic for the cure of human infectious [1-3]. Plants contain a broad diversity of phytochemicals including tannins, terpenoids, alkaloids and flavonoids found in vitro to have antimicrobial activities [4,5]. Herbal extracts' mode of effect and efficacy, in many situations still needs to be validated, these products mediate main host reaction [6,7]. Global prevalence of infectious illnesses caused by bacteria is a main community health problem [3,8].

Bacteria including Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and Proteus vulgaris cause many human infections [9,10]. New emergence of antibacterial resistance and related toxicity issues, decrease the utilization of antibacterial therapy [11] and is prompting importance in research of the antimicrobial capacity of plants against resistant strains due to similarity in safety and efficacy [1].

FIG (Ficus carica ) is from the family Moraceae which is considered a bygone plant on earth. Ficus carica constitutes the largest genera of medical plants with about 750 species of woody plants, trees, and shrubs. Plant parts like fruits, bark, seeds, shoots, leaves and latex are therapeutically important [12]. The fig is a nutritional food and used in manufacturing products. It is rich in vitamins, mineral elements, water, and fats. Fresh and dried figs are rich in calcium, fiber and polyphenols [13,14]. It also contain phenolic compounds, such as proanthocyanidins, while tea and red wine, considered as great sources of phenolic composite, have phenols less than fig [15]. Fruit, root and leaf are used in old medicine to heal many diseases like gastrointestinal (indigestion, colic, loss of appetite and diarrhea), respiratory (sore throats, coughs, and bronchial problems) and cardiovascular disorders and as anti-inflammatory [16,17].

Olives (Olea europaea ) is a part of the family Oleaceae. It is an evergreen tree, height range between 3.65 to 6.09 meters, with hard branches, and a gray bark. Olea europaea products were used broadly in traditional therapy in India, Europe, Arabia peninsula, Mediterranean area, tropical and subtropical regions as hypotensive, diuretic, emollient and for urinary tract infections [18]. Olive oil represents an important component of the Mediterranean diet whose intake is greatly increasing in developed and developing countries for its known healing effects.

Several phytochemical compounds have been obtained and investigated from all organs of the plant such as glycosides, secoiridoids, flavonoids and poly-unsaturated fatty acids [19]. O. europaea possess antihypertensive [20], vasodilator [21], antimicrobial [22], hypolipidemic [23], antioxidant and anti-diabetic activities [24]

Pseudomonas aeruginosa:

Pseudomonas aeruginosa is a negative gram stained, rod-shaped bacteria that lead to infections in humans, plants and animals [25]. It forms biofilms on wet surfaces such as those of rocks and soil [26,27] Pseudomonas aeruginosa as an opportunistic human pathogen, it causes hospital acquired infections during patients stay in hospitals with another illness or condition or patients having a debilitated immune system. It causes infections in any section of the body. P. aeruginosa is the main cause of bacteremia in burned patients, urinary-tract infections in patients with catheter, and hospital-acquired pneumonia in patients on respirators [28] It is also the predominant cause of morbidity and mortality in cystic fibrosis patients, whose abnormal airway epithelia allow long-term colonization of the lungs by P. aeruginosa.

Escherichia coli

Escherichia coli is a Gram- negative, facultative anaerobic, rod-shaped, coliform bacterium. It is found in the lower part of the intestine of human and warm- blooded animals [29,30]. Escherichia coli colonize the human gastrointestinal tract immediately after birth and stay there for decades. Although E. coli considered part of the normal flora, but some of the population have the ability to induce disease. Disease causing E.coli divided into three major sub-groups: commensals or nonpathogenic, pathogenic causing intestinal infections and extra-intestinal pathogenic E.coli. The extra-intestinal pathogenic bacteria cause urinary tract infections in human and animals, and others responsible for neonatal meningitis and septicemia [31].

Staphylococcus aureus

Staphylococcus aureus is a Gram-positive spherical bacteria found in small grape-like clusters produce yellow colored colonies. They are facultative anaerobes that can survive aerobic breathing or lactic acid fermentation of glucose [32]. It is part of the intestinal flora of the body, mostly present in the nose, lungs, bronchi and skin [33]. Staphylococcus aureus is not always pathogenic, but considered the major pathogenic bacteria in human causes a range of infections vary from minor issues such as minor skin infections to severe toxin mediated diseases [34]. Most common infections caused by Staphylococcus species; impetigo, toxic shock syndrome (TSS), bacteremia, endocarditis, folliculitis, furuncle (boils), osteomyelitis and food poisoning. A lot of species of Staphylococcus have the ability to form biofilms which invade parts or all of medical catheters, heart valves, stents, shunts, prostheses [35].

The aim of the study is to investigate the in vitro antibacterial and synergistic activities of aqueous crude concentrates of the leaves of Ficus carica (figs) and Olea europaea (olives) against Pseudomonos aureginosa, Escherichia coli and Staphylococcus aureus bacteria.

MATERIALS AND METHODS:

Preparation of aqueous extracts:

Freshly picked leaves of the Olea europaea (Olive leaves) and Ficus carica (fig) collected from private garden. The leaves washed, dried by air and powdered using an electrical mill. The powdered plant stored in the laboratory in a dark, well closed jar at ambient temperature till use.

Preparation of fig aqueous extract using decoction method:

Twenty grams of dried powder of fig leaves placed in 100 ml of distilled water and boiled for about 15-20 minutes or until the color of the mixture change to dark green. The mixture cooled and filtered by using two layers of guaze then Whatman no.1 filter paper. The final concentration of aqueous extract was 20% (w/v). Different concentrations were prepared (2.5%, 5%, 10%, 20%) w/v.

Preparation of olive leaves aqueous extract by decoction method:

The extract was prepared by weighing 20 grams of olive leaves powder and placed in a beaker containing 100 milliliters of distilled water. The mixture boiled for 15-20 minutes or until the color of the mixture change to dark green. The decoction cooled and filtered using two layers of guaze then Whatman no.1 filter paper. The final concentration of aqueous extract was 20% (w/v) [36]. Four concentrations made from the extract (2.5%, 5%, 10%, 20%) w/v.

Phytochemical investigation of fig and olive leaves:

Phytochemical screening of fig and olive leaves aqueous extracts done by the following tests [37,38,39]:

1 Detection of saponins: Foam test

2 Detection of glycosides: Lieberman's test

3 Detection of flavonoids: Alkaline potassium hydroxide test

4 Detection of alkaloids: Dragendroff's test

5 Detection of polyphenols and tannins: Ferric chloride test

6 Detection of coumarine compounds: alcoholic sodium hydroxide

7 Detection of oleuropein: Thin layer chromatography and Ferric chloride spray

In vitro antimicrobial investigation of olive and fig aqueous extracts:

Twenty four hours colonies cultivated on agar used to prepare the bacterial suspension with turbidity of 0.05 McFarland (equal to 1.5×108) CFU/ml. Well diffusion assay used to evaluate the water extracts on Muller- Hinton agar [40]. The water concentrates sterilized by 0.22µm Millipore filter. The bacteria used for in vitro antimicrobial assay of the water concentrates were Staphylococcus aureus (Gram +ve), Escherichia coli (Gram - ve) and Pseudomonos aureginosa ( Gram -ve) supplied by Consumer Protection Center for Market Research, Baghdad, Iraq. Positive control was Amoxicillin (30µg) for all bacteria. The Muller- Hinton agar plates seeded with bacterial strains under aseptic condition. A six millimeter diameter wells were made using a sterile cork borer and 50µl of each concentration of Olea europea extract (2.5%, 5%, 10%, 20%) introduced separately into the wells, the same steps were done for the Ficus carica extract.

For the assessment of synergy, a mixture of the concentrations of both extracts was used. The plates of bacterial strains incubated overnight at 37°C. After the incubation period, the diameters of inhibition zones were measured [41]. The experiment was repeated 3 times and the mean values of inhibitory zones recorded.

Statistical Analysis

The data was statistically analyzed by Statistical Analysis System (SAS) 2012 program to show the effect of different concentrations individually and in combination of fig and olive water leaf concentrates on antibacterial activity and Least Statistical Difference (LSD) test was used for statistical significance at (P≤ 0.05) [42].

RESULTS:

Phytochemical investigation:

The phytochemical investigation tests of the secondary metabolites of Olea europea (olive) and Ficus carica (Fig) showed positive results for all the tested phytochemicals except a negative result for oleurupein in fig leaf extract as it is shown in Table 1.

Table 1: Phytochemical screening results of Olea europea and Ficus carica aqueous leave extracts

Phytochemical	*Olea europea*	*Ficus carica*
Alkaloids	Positive	Positive
Tannins	Positive	Positive
Saponin	Positive	Positive
Glycosides	Positive	Positive
Flavonoids	Positive	Positive
Phenolic	Positive	Positive
Coumarine	Positive	Positive
Oleuropein	Positive	Negative

The antibacterial activity of Olea europea (olive) and Ficus carica (fig) aqueous crude extracts

All bacterial strains showed a variable sensitivity to both individual extracts at 10% and 20% concentrations in contrast to the positive control (amoxicillin30 µg). The lower concentrations (2.5% and 5%) did not show antibacterial activity. Combination of 10% and 20% concentrations showed a significant synergy for P.aeurginosa, E. coli and S.aureus with an LSD value of (5.259), (5.071) and (3.893) respectively. The results summed up in Table 2 and Figure 1.

Table (2): Antibacterial activity of Olea europea (olive) and Ficus carica (figs) extracts Aqueous Extract

Aqueous Extract	*S.aureus*	*E.coli*	*P.aeruginosa*
Fig (2.5%)	NA	NA	NA
Fig (5%)	NA	NA	NA
Fig (10%)	4mm	8mm	7mm
Fig (20%)	9mm	10mm	9mm
Olive (2.5%)	NA	NA	NA
Olive (5%)	NA	NA	NA
Olive (10%)	3mm	7mm	6mm
Olive (20%)	8mm	12mm	7mm
Olive (10%) + Fig (10%)	6mm	NA	5mm
Olive (20%) + Fig (20%)	8mm	10mm	10mm
Positive control (Amoxicillin 30μg)	14mm	19mm	21mm
Negative Control	NA	NA	NA
LSD Value	3.893*	5.071*	5.259*

NA=No Activity; (P˂ 0.05)*

Figure 1: In vitro antibacterial assay of olive and fig leaves aqueous extracts against S. aureus, E. coli and P.seudomonos bacteria on Muller- Hinton agar plates

DISCUSSION:

The increasing resistance that is formed by bacteria against antibiotics led to many studies and attempts to develop new antibacterial agents from plant derived active secondary metabolites as an alternative to the current antibiotics. Traditionally, solvent such as water is used for extraction in herbal medicine, being a non-toxic, cheaper and an accessible solvent for most people. Many studies showed that methanol and ethanol extracts of these leaves are better solvents for extraction than water [43]. In the present study, water was used for extraction of phytochemicals from figs and olives and investigating antibacterial effect in vitro. Lower concentrations of (2.5% and 5%) of both fig and olive extracts have no antibacterial effect on tested bacteria. This disagrees with a study done by Aliabadi et. al. that showed concentration of (5%) of olive aqueous extract inhibited the growth of S.aureus and E.coli. [44]. At a concentration of (10%) of fig extract, the Gram- negative bacteria (P.aeruginosa and E.coli) showed a greater inhibition zones (7mm and 8mm) respectively than Gram- positive bacteria (S.aureus) with an inhibition zone of (4mm). These results are inconsistent with a study done by Kamal et.al. [45] that showed a greater inhibitory activity against S. aureus (8mm) than E.coli (6mm) using (10%) fig aqueous extract. While (20%) fig extract exhibited a greater inhibitory effect against E.coli (10mm) than S. aureus (9mm) and P. aeruginosa (9mm) but when compared relatively to the positive control inhibition zones (19mm, 14mm and 21mm respectively), S.aureus was more sensitive to fig extract than other tested bacteria. These results agree with a study done by Al-Askari et. al. [46] that show (20%) fig leaves aqueous extract have a better effect against Gram positive than Gram negative bacteria.

Olive leaves aqueous extract (10%) showed an inhibition for E. coli, P.aeureginosa and S. aureus with a zone of (7mm, 6mm,, and 3mm) respectively. These results disagree with the concentration but agree with the antibacterial activity of a research conducted by Markin et.al. that reports water extract of olive leaves of a lower concentration (0.6% w/v) inhibited the growth of E. coli, P. aerugenosa and S. aureus in a 3 hour exposure [47]. The results also disagree with a study done by Alhamed that showed similar results to the this study at a higher concentration of (50%) [48]. At (20%) of olive leaf extract showed a great inhibition for E. coli compared to the positive control. As the concentration of the extracts increase, the growth rate of tested bacteria decrease and this agree with Pereira et. al. [49].

In this study, the synergistic effect studied against test bacteria by combining different concentrations of water extracts of fig and olives. Synergy describes the combined effects that are greater than the sum of their individual effect [50]. Significant antibacterial inhibition results obtained for all bacteria and are in the following order P.aeruginosa, E.coli and S aureus for (10%) and (20%) combinations, combinations of lower concentrations (2.5%) and (5%) were tested as well but the results were not encouraging as summarized in Table 1.

As far as our knowledge, no previous studies been conducted concerning the synergy of aqueous leaves extracts of figs and olives. Phytochemical tests showed the existence of phenolic and polyphenols in both leaves of fig and olive aqueous extracts. Phenols and polyphenols are varying group of compounds occur widely in many different plants including olives [51].

Research studies on bioactive compounds revealed single phenolic compounds or their combination resulted in growth inhibition of different bacterial strains [52]. The phenolic compounds found in olives that exhibited antibacterial effect is oleuropein and this result is consistent with a study by Sousa et. al. [53]. The extracting solvents used may have an impact on the concentrations of phytochemicals extracted.

In the present study, water used as an extracting solvent, that may have extracted low concentrations of phytochemicals that explains the lack of antibacterial activity at low concentrations but a better activity at higher concentrations. Because polyphenols are very polar compounds, highly- polar solvents e.g. water and non-polar solvents e.g.chloroform and hexane are not suitable for extracting phenolic compounds in high percentage. The use of water together with other organic solvents like alcohol makes it a better solvent to ensure the ideal conditions for extracting polyphenols [54]. This explains the outcome of the current study, combining extracts of both fig and olive leaves showed synergism due to an increase in phenolic compounds from both plants.

The study concludes, the combination of crude aqueous leaf extracts of figs and olives is highly recommended to induce synergism for better antibacterial effect. These findings are useful in developing novel antibiotics to fight resistance of bacteria that is threatening mankind around the world. This finding indeed testifies to the truth of Allah's words in the Holy Qur'an who mentioned the use of the two plants together for their benefits; “By the fig and the olive, by Mount Sinai, by this secure town.

We certainly created man in the best form, then we relegate him to the lowest of the low, except those who have faith and do righteous deeds. There will be an everlasting rewards for them” [55]

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 19.07.2019 Modified on 25.08.2019

Research J. Pharm. and Tech 2020; 13(3):1198-1203.

DOI: 10.5958/0974-360X.2020.00221.8