Introduction

Infectious diseases are among the main causes of morbidity and mortality affecting the world, (Ahmad & Beg, 2001; Daneshmand et al., 2013a; Alzaid et al., 2020). Drug-resistant bacteria and fungi have entangled the therapy of infectious illnesses. Faced with an increasing antibiotic-resistant, the discovery of new antimicrobial compounds is a major concern for several types of research (Taramian et al., 2019; Usman, et al., 2019). Antimicrobial herbal compounds are considered as the potential medical resources (Daneshmand et al., 2013b). Zizyphus jujuba Mill. (Rhamnaceae family) commonly known as jujube or Chinese red date has several medicinal properties, it is used as foods and remedies for insomnia, fever, diarrhea, wounds, and ulcers (Abd-alrahman et al., 2013). In previous phytochemical studies of jujube fruits, the results have highlighted a lot of their biological impacts, including the anticancer, anti-inflammatory, sedative, immunostimulating, antioxidant, antimicrobial and hepatoprotective; these effects are mainly related to the richness of this species in various constituents, including Alkaloids (Zizyphine C, D, and E) flavonoids (quercetin, catechin, rutin), terpenoids (Betulinic acid), amino acids (proline, aspartic acid, and glutamic acid), lipids (oleanolic acid, betulonic acid, oleanonic acid, palmitic acid, oleic acid) sugars (glucose, galactose, and sucrose) and polysaccharides (Jiang et al., 2007; San & Yildirim, 2010; Daneshmand et al., 2013a; Elaloui et al., 2014; Elaloui et al., 2015; Damiano et al., 2017; Adjdir et al., 2019b).  In Algeria different parts of Z. jujube are widely used in traditional medicine, however as far as we know, no reports exist on antimicrobial activity of fruits. Hence, the present study has been established to evaluate the antibacterial and antifungal activity of dried pulp fruits extracts of Z. jujuba harvested from Tlemcen in the North of Algeria.

Materials and Methods

Plant Material

Fruits of Z. jujuba were collected in September during the fruit maturation period from Tlemcen city in western Algeria (Latitude: 34° 53′ 24″ Nord, 1° 19′ 12″ Ouest; Altitude: 715m). Botanical identification of the plant was authenticated in the lab of Ecology and Ecosystems Management, Department of Biology, University of Tlemcen, Algeria. In the laboratory, fruits were cleaned and dried at room temperature in the shadow.

Preparation of Plant Extracts

Extracts of Z. jujuba Mill. were obtained by maceration at room temperature: 10 g of dried fruits in 300 ml of solvent: distilled water for the aqueous extract (Aq); acetone–water and methanol-water mixtures (70/30 v/v) to prepare the extracts EA and EM extracts, respectively. The aqueous (Aq) and acetone-water (EA) extracts underwent fractionation by liquid-liquid extraction using ethyl acetate and n-butanol. The fractions recovered from the acetone-water extract were Acet₂ and n-but₂; those of the aqueous extract were Acet₁ and n-but₁, respectively.

RP-HPLC-PDA Analysis of Phenolic Acids and Flavonoids

Separation and identification of phenolic compounds of the hydro-acetone extract EA of Z. jujube and the ethyl acetate fractions Acet₁ and Acet₂ were carried out by RP-HPLC-PDA technique (Adjdir et al., 2019a; El-Haci et al., 2020). Using a binate pump transmission method and an Eclipse ODS Hypersil C18 column (150 mm ´ 4.6 µm), RP-HPLC-PDA investigation of phenolic compositions was carried out on a Perkin Elmar Flexar system. The portable phase included resolvent A- Acetic acid (2%) and B- Acetonitrile. The gradient elution system was: 5 min with 10 % of B; 25 min with 90 % of B and 15 min of linear gradient from 90 % to 100% of B, then, 15 min have included equilibration. The outflow extent was 1 ml/min. The chromatograms were controlled at 280 nm. The compositions’ diagnosis and maximum transfers were performed according to their confinement durations as well as correlating with criteria applied.

Assessment of the Antibacterial Function

The Clinical and Laboratory Standards Institute (CLSI) recommended Two approaches that were applied; the disk dispersion and the microdilution approach. Only extracts that have shown important results by the disk-method have been evaluated for their activity by the microdilution method. In this study, five various American Type Culture Collection (ATCC) reference pathogenic bacteria and three fungal strains were utilized. Gram-negative types: Escherichia coli ATCC 25933, Acinetobacter baumanni ATCC19606, Gram-positive species: Staphylococcus aureus ATCC 25923, Bacillus subtilis ATCC 6633, Bacillus cereus ATCC 11778, and fungal strains Candida albicans ATCC 10231, 26790 and IP 444.

The Disk Diffusion Method

Every essence was liquefied in dimethyl sulfoxide (DMSO) in order to obtain 1000 mg/mL ultimate condensation. Gentamicin (Gent 10μg/mL) along with amphotéricin B (AmB 10μgmg/mL) served as the positive control antibacterial and antifungal. The dimness of the suspensions was regulated at 0.5 McFarland criteria corresponding to 108 CFU/mL. The Petri dishes injected with the suspension regulated at Mueller-Hinton agar by an ear stick. After dehydrating, the antiseptic filter paper disk (6 mm diameter) was subsequently saturated with 10 μL of every essence. Petri dishes were nurtured at 37 °C, for 18-20 hours. To investigate the sterile or antifungal property, the area of development prevention encompassing the disks was measured.

Specification of Minimum Inhibitory Concentration (MIC)

The inoculums got ready and the entire bacterial suspensions were regulated at 0.5 McFarland standard dimness. Sequential subtilizations in Muller Hinton broth were prepared to achieve an ultimate condensation of 5.104CFU /ml by well. In the mentioned experiment, sterile 96 well microplates were utilized. Then, the essences were transmitted to each microplate well, with concentrations ranged from 110 to 450 mg/ml, and the microplates have been nurtured at 35±2 ºC, for 16 to 20 hours. The development of the microorganisms in the microdilution wells, as diagnosed by the unaided eye is completely inhibited by the lowest condensation of the essences called the MIC.

Results and Discussion

Recognition of Phenolic Compounds

Chromatographic evaluation by RP-HPLC-PDA indicated in the crude hydro-acetone extract EA and in the ethyl acetate fractions Acet₁ and Acet₂ of Z. jujuba fruits in the presence of gallic acid and quercetin, rutin was only discovered in Acet2 (Figure 1). In a previous study, these extracts revealed a high content in polyphenols, between 50.96 and 94.70 mg gallic acid equivalent per g extract, and in flavonoids from 75.73 to 427.33 mg catechin equivalent per g extract (Adjdir et al., 2019b). In addition, the total phenolic value of fruits methanolic essences from fifteen selected jujube genotypes of Turkey varies between 42 and 40 mg gallic acid equivalent/g DW (Kamiloglu et al., 2009). According to the literature, Gao et al. (2012) have determined a high total phenolic content, in Z. jujube harvested in china, ranged from 275.6 to 541.8 mg of GAE/100 g FW and a dozen phenolic compositions: gallic acid, cinnamic acid, chlorogenic acid, caffeic acid, ferulic acid, ellagic acid, catechin, epicatechin, rutin, and quercetin. Elaloui et al. (2015) have also shown in Z. jujube pulp from Tunisia phenolic compounds especially rutin and chlorogenic acid.

Antibacterial and Antifungal Activity

The growth inhibition zones of the tested extracts, measured by disk diffusion method, have shown that most extracts present a slight antimicrobial activity with an inhibition zone not exceeding 6.5 mm diameter (Table 1), except for S. aureus which was sensitive to EA (8mm), Acet₂ (7mm), n-but₁ (9mm) and n-but₂ (9mm). The minimum inhibitory concentration results (Table 2) show high MIC values ranged between 450 and 110 mg/mL.  E. coli and S. aureus are the most sensitive strains for all extracts. No particular antifungal activity was observed against C. albicans strains (Table 3). On the contrary, several studies have demonstrated the antimicrobial effect of the extracts fruits and other parts from Z. jujube. The ethanolic extract of the fruit is an inhibitor of pediatric infectious strains, S. aureus (MIC=2.26mg/mL), C. albicans (MIC=2.35mg/mL), and A. fumigatus (2.86mg/mL), respectively (Daneshmand et al., 2013b). Snakin-Z, a new cationic peptide (31 amino acids) derived from Z. jujuba fruits has broad-spectrum antimicrobial activity against bacteria, S. aureus (MIC=28.8µg/mL), E. coli (MIC=13.6 µg/mL), and fungi C. albicans (MIC=8.23µg/mL) (Daneshmand et al., 2013a). The hydro-ethanolic extract of seeds inhibits S. aureus (41.25µg/mL; 10mm), E. coli (52.5µg/mL; 17.5mm), and K. pneumonia (42.5µg/mL; 17mm) (Abd-alrahman et al., 2013). Moreover, alphitolic acid a triterpenoid identified from leaves is a significant antibiofilm against S. mutans, a causative agent of human dental caries (Damiano et al., 2017). In the literature, several studies have proved the inhibitory effect of phenolic compounds and flavonoids on microbial strains; it has been reported that Gram-positive bacteria are hypersensitive to herb essences than Gram-negative due to hydrophobic lipopolysaccharide in the outer membrane that protects various factors (Ferreira et al., 2012; Mezni et al., 2015).

Table 1. Mean Inhibition Zone Diameter (mm) of Ziziphus jujuba Mill. Fruit Extracts on Tested Bacteria by Disk Diffusion Method

(-): No activity; ND: no determined,

Table 2. The MIC Values (mg/mL) of Z. jujuba Mill. Fruit Extracts against Bacteria

(-): No activity

Table 3. Mean Inhibition Zone Diameter (mm) of Z. jujuba Mill. Fruit Extracts on C. albicans Strain by Disk Diffusion Method

(-): No activity