Introduction
The therapeutic applications of medicinal plants grown in Tunisian have increased; thus, a better understanding of their biological activities has become necessary.1 Medical plants have become crucial therapeutic agents and valuable raw active compounds have been used for manufacturing many traditional and modern treatments.2 Indeed, a majority of natural antioxidants are derived from plant materials and have been known to benefit human health because they have antidiabetic, antiproliferative, and antimicrobial activities.3 These antioxidants have been given particular focus because of their ability to reduce cholesterol levels and ischemic damage.4
Among the different Ziziphus plants in Tunisia, Ziziphus spina-christi (Z. spina-christi), known as ‘sidra’, is habitually grown in Tunisia’s oases, due to its greater pivoting root growth. This green tree belongs to the Rhamnaceae family and can continue to grow for over 900 years.5Z. spina-christi leaves are considered to have antiinflammatory, hypoglycemic, hypotensive, antinociceptive, and antitumoral effects.6–10 A previous study identified that different Ziziphus spina-christi organs from Tunisian regions contain phenolic compounds1 and the plant’s roots are the most studied plant part.11 However, there is no information on whether Z. spina-christi methanolic leaf extracts contain a primordial component, such as rutins. These components, known as vitamin P, are the most common flavonol glycol-sides and have potential to be developed as new medicines. This flavonol glyco-sides have antioxidant, cytoprotective, vasoprotective, anti-inflammatory, antibacterial, antiviral, antiulcerogenic, neuroprotective, cardioprotective, hepatoprotective, and nephroprotective properties.12 The leaf extracts contain flavonoids, alkaloids, saponins, lipids, proteins, and free sugar.
In Tunisia, the biological activities of Ziziphus spina-christi leaves should be further studied for their anti-inflammatory, hypoglycemic, hypotensive, and antinociceptive effects.13 The present study aimed to investigate the antioxidant, antimicrobial, and insecticidal activities of leaf extracts.
Methods
Plant materials
Z. spina-christi leaves (Fig. 1) were obtained from Metlaoui, Degueche, Tozeur, and Nefta of Tunisia in April 2018.
The Z. spina-christi trees were identified by a specialist at the National Institute of Researches on Rural Engineering, Water and Forests, and a voucher specimen them was deposited at the Herbarium of INRGREF (Tunisia). Leaves were collected and dried at room temperature for 15 days in a dry and airy environment. The dried leaves were ground using a mill equipped with a grid with a hole size of 1.00 mm in diameter. The generated powders were stored in plastic bags in the dark until chemical analysis. The leaf powder samples (1 g each) were macerated for 30 min in 10 ml of methanol (70%). The extracts were filtered through Whatman N°1 filter paper, pooled, and concentrated under a vacuum.
Chemical reagents
Folin-ciocalteu, phenol, 2, 2-diphenyl-1-picrylhydrazyl (DPPH), gallic acid, catechin, sodium carbonates, hydrochloric acid, rutin standard, and all solvents were purchased from Sigma-Aldrich (St. Louis, MO, USA). All solvents and reagents used were of the highest purity.
Total phenol, flavonoid, and tannin contents
The total phenolic content in the leaf extracts was determined using the Folin-Ciocalteu method.14 Briefly, 0.5 mL of a methanolic solution was mixed with 2.5 mL of Folin- Ciocalteu reagent and 2 mL of sodium carbonate (75 g/L) solution. After incubation for 30 min, the absorbance was measured at 765 nm using a Shimadzu 1600-UV spectrophotometer. The same procedure was repeated using gallic acid as a standard. Total phenols of each fraction, expressed in mg GAE/g dry weight (DW), were measured using the regression equation of a calibration curve y = 0.0114x + 0.518, r2 = 0.9932. The flavonoid contents were measured by mixing 1 mL of AlCl3 and 1 mL of plant extract, and incubated at room temperature for 40 min. Absorption was measured at 420 nm using a Shimadzu UV-1600 spectrophotometer. The results are expressed as mg quercetin equivalent per gram dry weight (mg QE/g DW). All experiments were carried out in triplicate. In contrast, the condensed tannins were tested calorimetrically as described by Earp et al.15 The condensed tannins in the methanolic extracts are expressed as mg catechin equivalents per g of dry weight (mg CE. g−1 DW).
LC-ESI-MS analysis
Quantitative analysis of phenolic compounds was performed using the LC-MS 2020 system (Shimadzu, Tokyo, Japan) associated with an electro-spray ionization source (ESI). Briefly, practicals of 3 µm and pore size of 130 Å were eluted using a glass capillary C18 column (150×4.6 mm) (Phenomenex, Torrance, CA, USA). The mobile phase was set with a flow rate of 6 mL/min, and contained 0.5% formic acid in H2O, v/v (solvent A) and acetonitrile (solvent B). The gradient elution was 90:10 (A: B, v/v) during 0–5 min, then it decreased to 86:14 (A: B, v/v) (during 5–7 min), and was held at 86:14 (A: B, v/v) from 7–17 min, before falling from 86:14 to 75:25 (A: B, v/v) during 17–19 min (linear gradient). Finally, the elution was held at 75:25 (A: B, v/v) and decreased to 10:90 (A: B, v/v) (linear gradient) during 19–24 min; 25–31 min held at 10:90; 31–32 min from 10:90 to 90:10; and 32–40 min held at 90:10 (reequilibration step). The injection volume was 20 µL at 40 °C temperature. High-purity (>99%) nitrogen was used as a nebulizer and an auxiliary gas. The spectra were monitored in selected ion monitoring (SIM) mode and processed using Shimadzu Lab Solutions LC-MS software. The mass spectrometer was operated in negative ion mode with a capillary voltage of −3.5 V, a dry gas flow rate of 12 L/min, a nebulizing gas flow rate of 1.5 L/min, a block source temperature of 400 °C, a dissolving line temperature of 250 °C, a 1.2 V voltage detector, and the full scan spectra from 50 to 2,000 m/z. Rutin was identified by comparing the retention time with those of the standard, and the content was calculated using the calibration curve by plotting the peak area against the concentration of the respective standard. The data were reported with convergence limits in triplicate.
Biological activities
Antioxidant activity
The in vitro antioxidant activity of leaf extract samples was examined using the 2, 2-diphenyl-1-picrylhydrazyl radical, DPPH method.9 The different concentrations of oil extracts (2.5 to 50.0 mg/mL) were prepared and incubated (V/V) at 25 °C for 30 min with standard or DPPH solution (2.36 mg in 100 mL of ethanol (70%)). The absorbance was measured at 734 nm. The concentration for 50% inhibition (IC50) was estimated.16 Tests were carried out in triplicate. The percent inhibition of the DPPH radicals by the samples was calculated according using the following formula:
Scavenging effect (%)=A0−A1A0×100,
where A0 is the absorbance of the blank sample; and A1 is the absorbance in the presence of the sample.Antimicrobial activity
Agar diffusion method
The methanol extracts of Z. spina-christi leaves were dissolved in sterile water. Each extract solution was diluted to 50 µg/mL and sterilized by filtration through a 0.2 µm pore size filter. The antibacterial and antifungal activities were determined using the agar well diffusion method as described by Thakur et al.17 The tested pathogens included Streptococcus agalactiae, Staphylococcus aureus, and Escherichia coli. Three Candida species – Candida albicans, Candida sake, and Candida parapsilosis – were included. The broth microdilution assay with sterile Mueller-Hinton media (BioRad, France) was used for bacterial strains and yeast malt extract agar (Bio-Rad, France) was used for antifungal tests. A fresh cell suspension (0.1 mL) adjusted to 107 CFU/mL for bacteria and 105 cells/mL for fungi was inoculated onto the surface of agar plates. The agar plates were punched with wells at a 6-mm diameter and the wells were filled with 30 µL of the extract dilution and culture medium. Negative control wells were filled with 30 µL of sterile water. The plates were allowed to stand for 40 min at 4 °C to permit extract diffusion and then incubated at 37 °C for 24 h for bacteria and at 48 h for yeast. Antibacterial and antifungal activities were evaluated by measuring the zones of inhibition (clear zone around the well) against the microorganisms. All tests were repeated three times.18
Determination of minimum inhibitory concentration (MIC)
The MIC of each extracted sample was determined using the microdilution broth method. MIC values were estimated visually by the absence of turbidity as previously reported by Khémiri et al.19
Insecticidal activity
Insecticidal assays of plant extracts were carried out at the National Agricultural Research Institute of Tunisia using Tribolium castaneum as a model insect. Experiments were accomplished in 25 ± 1 °C and 65 ± 5% relative humidity in the dark. Ten insects of mixed sex at 7–10 days were used for bioassay tests according to the method of Pascual-Villalobos and Robledo.20
Insects were treated with Z. spina-christi extracts (2 mg extract or control), transferred to Petri dishes, and kept at room culture at 25 ± 1 °C. The number of dead insects was observed and recorded every six hours. The percent of dead insects was calculated using the Abbott’s21 formula:
Mortality (%)=Mt−Mc100−Mc×100
with Mt = % mortality in treated and Mc = % mortality in controls.The median lethal concentrations (LC50 and LC95) were calculated according to the Probit method.22
Statistical analysis
Results were statistically analyzed using STATISTICA (Statsoft, 1998). Each data presented was mean of three replicates (±standard deviation). Multiple mean comparisons were performed using the Student-Newman Keuls test with a significance level of p< 0.05.
Results
Phenolic content of Z. spina-christi leaf extracts
The contents of total phenolic (TPC), total flavonoids (TFC), and condensed tannin (CT) in Z. spina-christi leaf extracts from three Tunisian provenances are shown in Table 1.
Table 1Biochemical composition of Z. spina-christi leaves
| Provenance | TPC (mg GAE/g DW) | TFC (mg QE/g DW) | CT (mg CE/g DW) |
|---|
| Metlaoui | 6.08 ± 0.06a | 2.22 ± 0.03b | 0.925 ± 0.003a |
| Tozeur | 8.16 ± 0.05b | 4.42 ± 0.03c | 0.82 ± 0.007b |
| Dgueche | 8.04 ± 0.04b | 1.96 ± 0.01a | 1.62 ± 0.001c |
Identification and quantification of phenolic compounds in Ziziphus spina-christi leaf extracts
LC-MS analyses identified that rutin was the major flavonoid compound present in Z. spina-christi leaf extracts (Fig. 2) and other phenolic compounds varied among these provenances. The rutin values varied from 1.3 ± 0.005 µg/mL to 3 ± 0.005 µg/mL. The leaf extracts from Tozeur provenance exhibited the highest rutin (Table 2). The contents were more important than those of Ziziphus jujuba leaf extracts (1.91 mg/g).
Table 2Rutin amounts (µg/mL) in Z. spina-christi leaf extracts
| Provenance | RT | Quantity (µg/mL) |
|---|
| Metlaoui | 15.85 ± 0.03 | 2 ± 0.005 |
| Tozeur | 16.06 ± 0.05 | 3 ± 0.005 |
| Dgueche | 15.93 ± 0.004 | 1.3 ± 0.005 |
Biological activities of Ziziphus spina-christi
Phenolic compounds from Tunisian Z. spina-christi, particularly from its roots have antibacterial, antifungal, and antioxidant properties.23 The most identified phenolic compound was epicatechin, which can protect organisms against oxidative stress caused by reactive oxygen and nitrogen species.11
Antioxidant activity
The results indicated that the reduction ability of DPPH radical was determined by the decrease in absorbance induced by plant antioxidants. The scavenging effects of each extract sample on the DPPH radical are expressed as IC50 values in Table 3. The IC50 values were 0.125 mg/mL (Tozeur provenance) and 1 mg/mL (Dgueche provenance), respectively. The radical scavenging properties of natural products are frequently linked with their ability to shape stable radicals. Superior antioxidant activity was observed from leaf extracts from Tozeur and Metlaoui provenances.
Table 3Inhibitory concentrations of Z. spina-christi leaf extracts using the free radical scavenging activity (DPPH) method
| Provenances | Metlaoui | Tozeur | Dgueche |
| DPPH radical-scavenging (mg/ml) | 0.5 ± 0.00b | 0.125 ± 0.01a | 1 ± 0.02c |
The results indicated that all leaf extracts of Z. spina-christi from different Tunisian provenances had high antioxidant activity. Statistical analyses revealed that this composition varied significantly among the extracts from different provenances (Fig. 3). The leaf extracts from Tozeur provenance exhibited the highest values.
Antimicrobial activity
Overall, the leaf extracts from Tozeur provenance had superior antibacterial potential against the tested bacteria species (Fig. 4). The results indicated that the tested extracts were able to inhibit Streptococcus agalactiae with inhibitory zone varying between 19.5–27.5 mm and the highest activtiy was obtained by the extract from Tozeur provenance. The Gram-negative bacteria species belonging to E. coli were sensitive to Dgueche and Tozeur extracts with 13.5 and 11.5 mm inhibitory zones, respectively. Only extracts from the Tozeur provenance were able to inhibit the growth of Staphylococcus aureus with an inhibitory zone diameter 16 mm (Fig. 5). Numerous reports have shown that antibacterial potential is related to the bacteria strain species tested, and the composition of the extract can be influenced by many factors such as geographical region, plant organs, season, extraction methods, and solvent.
The antifungal activities of three extracts were analyzed using three Candida species. The results indicated the highest antimicrobial potential of the extract from Tozeur provenance, which was the only extract that effectively inhibited the growth of two Candida clinical species – Candida albicans and Candida sake – with a high zone of inhibition of 11.5 mm and 21 mm, respectively. There was no extract tested to inhibit the growth of Candida parapsilosis specie. The extract from Dgueche provenance was effective against Candida albicans specie with an inhibitory zone diameter of 12 mm (Fig. 5).
The same MIC value of 25 µg/mL was obtained for antifungal activity as well as for antibacterial activity against Streptococcus agalactiae specie. The extract from Tozeur had superior CMI values of approximately 6.25 µg/mL; the extract from Tozeur had potent antifungal activity (Table 4).
Table 4MIC comparison of various extracts against bacteria and Candida expressed in µg/mL
| Extracts | MIC (µg/mL)
|
|---|
| Candida albicans | Candida sake | Streptococcus agalactiae | Eschcherchia coli | Staphylococcus aureus |
|---|
| Tozeur | 25 | 25 | 25 | 6, 25 | 6, 25 |
| Dguech | 25 | – | 25 | 25 | 25 |
| Metlaoui | 25 | 25 | 25 | 25 | 25 |
Insecticidal activity
The insecticidal activities of different doses (1 mL, 500 µL, and 200 µL) of three extracts against Tribolium castaneum (T. castaneum) were evaluated. Treatment with individual extracts from Degueche, Tozeur, and Metlaoui at 500 µL for 72 h caused T. castaneum mortality of 60.66%; 77.33%, and 100%, respectively (Fig. 6).
Discussion
Our data indicate that Ziziphus spina-christi leaf extracts from different provenances have varying biological activities. This variability may reflect the influence of geographical conditions (humidity, temperature, and altitude), soil-growth conditions, and origin and period of leaf harvest.23 A previous study reported that the total phenolic content of Z. spina-christi leaf extracts differs significantly among the various extraction methods (ethanolic, aqueous extracts).24 In addition, Z. spina-christi leaf extracts contain higher levels of phenolic compounds than that in Z. jujuba leaf extracts, with values of 3.97 and 6.04 mg GAE/g, respectively.25
Rutin was identified as the major flavonoid compound in Z. spina-christi leaf extracts. Our data are in disagreement with a previous report that identified luteolin and quercetin as the major compounds in Z. spina-christi leaf extracts.9 These compositions differ among the specie. In fact, chlorogenic acid was identified as the major phenolic compound in Z. lotus, while caffeic and salviolinic acids were detected in Z. mauritiana samples.26 Another study reported a correlation between total phenol, flavonoid, and condensed tannin contents and antioxidant activities.27 Thus, our findings support the application of this kind of extract in pharmacology and cosmetic industries.
The results revealed that rutin was the richest compound in the leaf extracts from Tozeur. However, we did not detect quercetin in the extracts, but we did detect antifungal activity of these Z. spina-christi leaf extracts.18 In addition, we detected strong antibacterial activities of these extracts against Staphylococcus aureus and E. coli, and the activities were stronger than a recent report.28
A previous study reported that Ziziphus leaf extracts contain alkaloids, saponins, tannins, glycosides, flavonoids, and terpenoids.29 In our study, we provide evidence that the Z. spina-christi leaf extracts contain the major compound of rutin, which had potent antibacterial and antifungal activities because the amount of rutin in the extracts correlated with the antibacterial and antifungal activities. The efficiency of the specific phytochemical composition in the extracts was also confirmed by comparing the ethanolic with methanolic extracts.30 More importantly, the MIC values from our studies ranged from 25–6.5 µg /mL, which were more efficient than that in a previous report.29
We also observed that the Z. spina-christi leaf also had high insecticidal activities, although a previous study reported that the n-hexane and ethyl acetate fraction in Zizyphus oxyphylla had moderate insecticidal activity against Callosobruchus analis.31 To the best of our knowledge, our data study is the first report on the insecticidal activity of the Z. spina-christi leaf extracts, supporting the potential application of these extracts for industrial use.
Future directions
In this study, we sought to optimize our experiments in order tovaloriz e our Z. spina-christi extracts and demonstrate their potential use as synthetic insecticides or natural antioxidants in the cosmetics industry. We hope also to substantiate the therapeutic uses of the extracts and to enhance Z. spina-christi fruit trees growth in gardens and fields.
Conclusions
The present study highlights the importance of Z. spina-christi leaf extracts in medicinal and pharmaceutical industries. Due to their richness of phenolic contents, these extracts may be used as antioxidant, insecticidal, and allelopathic agents. Z. spina-christi leaves may be a good candidate for food and pharmaceutical supplement formulations, as well as industrial uses.
Abbreviations
- CFU:
colony forming units
- CT:
condensed tannin
- DPPH:
2, 2-diphenyl-1-picrylhydrazyl
- IC50:
concentration for 50% inhibition
- MIC:
minimum inhibitory concentration
- TPC:
total phenolic
- TFC:
total flavonoids
Declarations
Acknowledgement
The authors acknowledge the technical assistance of the Research Group of the INRGREF Institute, Tunis.
Data sharing statement
No additional data are available.
Funding
None.
Conflict of interest
The authors have no conflicts of interest related to this publication.
Authors’ contributions
Conceived of the presented idea (EM and AL), carried out the experiment (ME, BE, SH, JBJ, RBN), contributed to the interpretation, discussed the results and co-wrote the manuscript (ME, BE, contributed to English revision of the manuscript (ME, BE and RDC), contributed to statistical analysis (BS), contributed to the final manuscript (YA and AL)