Antioxidant and Biological Activities of Acacia saligna and Lawsonia inermis Natural Populations

Acacia saligna and Lawsonia inermis natural populations growing in Northern Saudi Arabia might be a valuable source of polyphenols with potent biological activities. Using high-performance liquid chromatography–diode array detection (HPLC-DAD), several polyphenols were detected tentatively in considerable amounts in the methanolic leaf extracts of A. saligna and L. inermis. A. saligna mainly contained rutoside, hyperoside, quercetin 3-glucuronide, gallic acid and p-coumaric acid, whereas those of L. inermis contained apigenin 5-glucoside, apigetrin and gallic acid. Strong antioxidant activities were found in the leaf extracts of both species due to the presence of hyperoside, quercetin 3-glucuronide, gallic acid, isoquercetin, p-coumaric acid, quercitrin and rutoside. A. saligna and L. inermis leaf extracts as well as hyperoside, apigenin 5-glucoside, and quercetin 3-glucuronide significantly reduced reactive oxygen species accumulation in all investigated cancer cells compared to the control. Methanolic leaf extracts and identified polyphenols showed antiproliferative and cytotoxic activities against cancer cells, which may be attributed to necrotic cell accumulation during apoptotic periods. Antibacterial activities were also found in both species leaf extracts and were twice as high in A. saligna than L. inermis due to the high composition of rutoside and other polyphenols. Finally, strong antifungal activities were detected, which were associated with specific phenols such as rutoside, hyperoside, apigenin 5-glucoside and p-coumaric acid. This is the first study exploring the polyphenolic composition of A. saligna and L. inermis natural populations in northern Saudi Arabia and aiming at the detection of their biological activities.


Introduction
Natural populations of medicinal plants are considered as important sources of natural compounds that may have bio/pharmacological activities. Large portions of the global population are reliant on these botanical remedies as traditional or alternative medicine, while others consume or utilize these

Analyses of Phenolic Compounds
The qualitative and quantitative estimations were performed by high-pressure liquid chromatography (HPLC) analysis using a Merck-Hitachi liquid chromatograph (LaChrom Elite, Berlin, Germany) with an L-2455 diode array detector (DAD). The used analytical column was Purospher RP-18e solid phase column (250 × 4 mm; 5 µm, Merck, Kenilworth, NJ, USA). As the eluent for the gradient program, we used A-methanol and B-a 0.5% acetic acid and methanol mixture 1:4 (v/v). The solvent ratio changed over time as follows: 0-20 min, 100% B; 20-35 min, 100-80% B; 35-55 min, 80-60% B; 55-70 min, 60-0% B; 70-75 min, 75-90 min, 100% B. The flow rate was 1 mL/min and the sample injection volume was 10 µL. Temperature was maintained at 25 • C. The DAD analytical waveband ranged from 200 to 400 nm. Quantitative analysis was conducted at 254 nm. The HPLC validation was carried out earlier [28,29]. Compounds contained in extracts were identified by comparing their retention times and UV spectra with standards. Quantitative measurements were conducted using calibration curves ( Figure S1).
In the DPPH experiments, 5 mL of 0.004% methanolic DPPH solution was added to methanolic leaf extracts serial concentrations, then incubated for 30 min at room temperature in the dark. Finally, the absorbance was measured at 517 nm. The DPPH-free radical inhibition was determined as follows: The percentage inhibition of antioxidant activity (IAA) was calculated in triplicate: where (AB 5170nm ) C and (AB 517nm ) s are Abs.517 nm of the control and sample, respectively. The inhibition concentration of each sample was compared with that of the BHT (butylated hydroxytoluene) as a positive control and blank.
In the β-carotene-bleaching assay, the mixture was prepared by dissolving the β-carotene (0.5 mg) in chloroform (1 mL), then adding linoleic acid (25 µL) and Tween 40 (200 mg). A vacuum evaporation was used to remove the chloroform; finally, the distilled water was added (100 mL) and followed by vigorous shaking. The mixture (2.5 mL) was added to serial concentrations of leaf extracts, then incubated for 48 h at room temperature, and the absorbance was measured at 470 nm.
In the FRAP assay, aliquots (100 µL) of leaf extracts/Trolox (Sigma-Aldrich, Berlin, Germany) were added to the prepared FRAP reagent (3 mL), then mixed vigorously and incubated for 30 min at 37 • C. The calibration procedure of FRAP was conducted by applying serial dilutions of Trolox (0-0.5 mmol/L), as standard. The absorbance was determined at 593 nm for FRAP. All antioxidant experiments were conducted in triplicates and repeated thrice.

Detection Intercellular ROS Accumulation
To assay the capacity of A. saligna and L. inermis methanolic extracts as well as identified polyphenols to reduce the intracellular levels of ROS in HeLa, Jurkat, T24, and MCF-7 cancer cells, the fluorogenic dye H 2 DCF-DA was used. Following passive diffusion into the cells, H 2 DCFDA was deacetylated using esterases into the nonfluorescent compound. That compound was oxidized by ROS and converted to the highly fluorescent 2 ,7 -dichlorofluorescein (DCF) [35]. Cells were grown in 96-well plates at a density of 1 × 10 4 cells per well for 24 h before experiments; then, the culture medium was changed on 10 µM H 2 DCFDA (Sigma Aldrich, St. Louis, MO, USA) in serum-free medium (MEM) and incubated for 45 min before applying treatments. Then, cancer cells were exposed to plant extracts and identified polyphenol DPPH IC 50 values. Cells treated with 1 mM hydrogen peroxide (H 2 O 2 ) were considered as a positive control. The DCF fluorescence was measured after 90 min using a microplate reader FilterMax F5 (Thermo Fisher Scientific, Waltham, MA, USA) at 485 nm.

Anticancer Activities
Antiproliferative and cytotoxic effects of the leaf extracts of A. saligna and L. were tested against HeLa, HT-29, Jurkat, MCF-7 and HEK-293 (normal human cells) [5,13,31,36]. To study the antiproliferative effects on cell viability, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used. Leaf extracts were solubilized in DMSO (1%) then added to standard media (MEM) containing (10% FBS, 0.1 mM non-essential amino acids, 17.8 mM NaHCO 3 , and 1 mM sodium pyruvate) and cancer cells in 75 cm 2 flasks. The cancer cells were prepared in microtiter plates at 4 × 10 −4 cells per µL in 270 µL medium for 48 h (37 • C, 5% CO 2 ). Leaf extract serial concentrations were used until final concentrations of 50, 100, 200, 300 and 400 µg/mL were reached. Then, a washing procedure was performed using PBS. The MTT was dissolved in PBS then added (12 mM) to the medium. Finally, isopropanol (0.04 N HCl) was mixed with the MTT solution, and the mixture was incubated at room temperature for 40 min. A negative control (untreated) and positive couple (vinblastine sulfate and taxol) were used. To calculate the inhibition activity percentage (IAA) obtained from measuring the absorbance at a 570 nm wavelength, the following equation was used: where AB is the absorbance and (AB 570nm ) C and (AB 570nm ) s are Abs.570 nm of the control and sample, respectively. The percentage of viable cells was plotted against the extract concentration (µg mL −1 ) to determine the IC 50 . The IC 50 amounts were applied in the flow cytometry assay to study the cytotoxic activities of A. saligna and L. inermis leaf extracts; then, the apoptotic cell populations were determined (FACScan, New York, NY, USA) [5,13,36,37]. The assay was based on monitoring phosphatidylserine translocation in the Annexin [38]. In viable cells, the phosphatidylserine is located in the inner layer of the plasma membrane. Early apoptosis starts when the phosphatidylserine translocates from the inner to the outer layer of the plasma membrane, and apoptotic cells are reflected by measuring the binding of Annexin V-FITC to external phosphatidylserine. The flow cytometer data were presented in quadrants as percentages: lower left, viable cells; upper left, necrotic cells; lower right, early apoptotic cells; and upper right, late apoptotic cells.

Antibacterial Effect
Bacterial isolates of Listeria monocytogenes (clinical isolate), Escherichia coli (ATCC 35210), Staphylococcus aureus (ATCC 6538), Bacillus cereus (ATCC 14579), Micrococcus flavus (ATCC 10240) and Pseudomonas aeruginosa (ATCC 27853) were used in this experiment. A microtiter plate-based protocol (micro-dilution) was used following the procedure in [14,[39][40][41], by preparing serial concentrations of A. saligna and L. inermis extracts that were mixed with bacterial inoculum at 1.0 × 10 4 CFU in each well with 100 µL tryptic soy broth per well, then incubated for one day at 37 • C in a rotary shaker. The minimum inhibitory concentration (MIC) was defined as the lowest concentration that caused no visible growth by a binocular microscope. The minimum bactericidal concentration (MBC) was determined using the serial subculturing of extracts (2 µL), and the minimal concentration causing the elimination of 99.5% of each inoculum was considered as the MBC value. The optical density (OD) was determined using a 655 nm wavelength. A positive control (streptomycin) at 0.01-10 mg/mL was used alongside the negative one (DMSO, 1%).

Antioxidant Effects
A. saligna and L. inermis methanolic extracts showed antioxidant activities comparable to the identified polyphenols, as shown in Figure 2. A. saligna showed significantly higher antioxidant activity than L. inermis as measured by different assays (DPPH, B-carotene bleaching and FRAP). In A. saligna, the identified polyphenols including hyperoside, quercetin 3-glucuronide, gallic acid, isoquercetin, p-coumaric acid and quercitrin showed strong antioxidant activities, with quercetin 3-glucuronide having the lowest IC 50 value. In L. inermis, identified polyphenols including apigetrin and apigenin 5-glucoside showed strong antioxidant effects. Quercetin 3-glucuronide, gallic acid and p-coumaric acid antioxidant activities were comparable to antioxidant standards.

Antioxidant Effects
A. saligna and L. inermis methanolic extracts showed antioxidant activities comparable to the identified polyphenols, as shown in Figure 2. A. saligna showed significantly higher antioxidant activity than L. inermis as measured by different assays (DPPH, B-carotene bleaching and FRAP). In A. saligna, the identified polyphenols including hyperoside, quercetin 3-glucuronide, gallic acid, isoquercetin, p-coumaric acid and quercitrin showed strong antioxidant activities, with quercetin 3glucuronide having the lowest IC50 value. In L. inermis, identified polyphenols including apigetrin and apigenin 5-glucoside showed strong antioxidant effects. Quercetin 3-glucuronide, gallic acid and p-coumaric acid antioxidant activities were comparable to antioxidant standards.

MTT Assay and Flow Cytometry
The MTT assay was employed to determine the antiproliferative activities of methanolic leaf extracts and the identified polyphenols against an array of cancer cells ( Figure 3). Strong antiproliferative effects were exhibited by A. saligna and L. inermis leaf extracts as well as the identified polyphenols against all cells except the normal cells of HEK-293 (showing IC50 values > 400 μg mL −1 ). A. saligna showed significantly higher antiproliferative activities than L. inermis. Strong antiproliferative activities were detected when using identified polyphenols including rutoside, hyperoside, quercetin, quercetin 3-glucuronide and p-coumaric acid against cancer cells.
The cytotoxic activities of A. saligna and L. inermis leaf extracts as well as hyperoside, apigenin 5-glucoside and quercetin 3-glucuronide were investigated by monitoring phosphatidylserine translocation in the annexin assay ( Figure 4). In viable cells, the phosphatidylserine is located in the inner layer of the plasma membrane. Early apoptosis starts when the phosphatidylserine translocates from the inner to the outer layer of the plasma membrane, and apoptotic cells are reflected by measuring the binding of Annexin V-FITC to external phosphatidylserine. The flow cytometry showed the apoptotic cell accumulation following 48 h of exposure in the upper and lower-right quadrant. The cytotoxic activities of A. saligna and L. inermis leaf extracts as well as major identified  50 in µg/mL). Identified polyphenol (rutoside, hyperoside, quercetin 3-glucuronide, gallic acid, isoquercetin, p-coumaric acid, quercitrin, apigetrin and apigenin 5-glucoside) antioxidant effects were also added. Values are expressed as mean ± standard deviation. TEAC: Trolox equivalent antioxidant capacity. Different letters among group of columns indicate significant differences (p ≤ 0.05).

MTT Assay and Flow Cytometry
The MTT assay was employed to determine the antiproliferative activities of methanolic leaf extracts and the identified polyphenols against an array of cancer cells ( Figure 3). Strong antiproliferative effects were exhibited by A. saligna and L. inermis leaf extracts as well as the identified polyphenols against all cells except the normal cells of HEK-293 (showing IC 50 values > 400 µg mL −1 ). A. saligna showed significantly higher antiproliferative activities than L. inermis. Strong antiproliferative activities were detected when using identified polyphenols including rutoside, hyperoside, quercetin, quercetin 3-glucuronide and p-coumaric acid against cancer cells.
The cytotoxic activities of A. saligna and L. inermis leaf extracts as well as hyperoside, apigenin 5-glucoside and quercetin 3-glucuronide were investigated by monitoring phosphatidylserine translocation in the annexin assay ( Figure 4). In viable cells, the phosphatidylserine is located in the inner layer of the plasma membrane. Early apoptosis starts when the phosphatidylserine translocates from the inner to the outer layer of the plasma membrane, and apoptotic cells are reflected by measuring the binding of Annexin V-FITC to external phosphatidylserine. The flow cytometry showed the apoptotic cell accumulation following 48 h of exposure in the upper and lower-right quadrant. The cytotoxic activities of A. saligna and L. inermis leaf extracts as well as major identified polyphenols were confirmed. The cell death was induced by the leaf extracts hyperoside, quercetin 3-glucuronide (Q3G) and apigenin 5-glucoside (A5G). polyphenols were confirmed. The cell death was induced by the leaf extracts hyperoside, quercetin 3-glucuronide (Q3G) and apigenin 5-glucoside (A5G).

ROS Accumulation
A. saligna and L. inermis leaf extracts as well as hyperoside, apigenin 5-glucoside and quercetin 3-glucuronide significantly reduced the ROS accumulation in all investigated cancer cells compared to controls using the intercelleular ROS accumulation assay of H 2 DCFDA fluorescence ( Figure 5). The highest reduction of ROS was achieved using apigenin 5-glucoside (A5G) in Jurkat, T24 and MCF-7 following 90 min of incubation. H 2 O 2 showed the highest levels of ROS accumulation in all cancer cells.

ROS Accumulation
A. saligna and L. inermis leaf extracts as well as hyperoside, apigenin 5-glucoside and quercetin 3-glucuronide significantly reduced the ROS accumulation in all investigated cancer cells compared to controls using the intercelleular ROS accumulation assay of H2DCFDA fluorescence ( Figure 5). The highest reduction of ROS was achieved using apigenin 5-glucoside (A5G) in Jurkat, T24 and MCF-7 following 90 min of incubation. H2O2 showed the highest levels of ROS accumulation in all cancer cells.

Antibacterial Activities of A. saligna and L. inermis Leaf Extracts
A. saligna and L. inermis extracts showed antibacterial effects against different bacteria (Table 2)

Antibacterial Activities of A. saligna and L. inermis Leaf Extracts
A. saligna and L. inermis extracts showed antibacterial effects against different bacteria ( Table 2). A. saligna showed two-fold higher antibacterial activities than L. inermis against all bacteria. The most sensitive bacteria were E. coli and S. aureus (showing the lowest IC 50 values), while the most resistant were L. monocytogenes and M. flavus. Identified polyphenols including rutoside, apigenin 5-glucoside and p-coumaric acid showed strong antibacterial activities against all bacteria except hyperoside, quercetin and quercetin 3-glucuronide, showing moderate to low antibacterial effects.

Antifungal Effects of Leaf Extracts
A. saligna and L. inermis extracts showed strong antifungal effects against most fungi studied (Table 3). A. saligna showed two to three-fold higher activity (lower IC 50 values) than L. inermis. Rutoside, hyperoside, apigenin 5-glucoside and p-coumaric acid showed the highest antifungal effects among the studied polyphenols.
The methanolic leaf extracts of L. inermis growing in Northern Saudi Arabia investigated in the current study contained high amounts of apigetrin, at 1180.9 mg/100 g DW; apigenin 5-glucoside, at 596.3 mg/100 g DW; as well as gallic acid, at 81.0 mg/100 g DW (Table 1 and Figure 1B). Phenolic compounds, including flavonoids, tannins, coumarins, and naphthoquinones, are particularly abundant ingredients of L. inermis leaf extracts [22]. Mikhaeil et al. [44] studied the Egyptian L. inermis leaf extracts and detected p-coumaric acid from phenolic acids and, similar to our samples, apigenin and apigetrin (cosmosiin) from flavonoids. The apigenin derivative 4 -methoxyapigenin (linarigenin, linarisenin) was also detected by Mahmoud et al. in L. inermis leaf extracts of Egyptian origin [45]. Gallic acid was confirmed by other scientists from Iran [46]. Moreover, the presence of p-coumaric acids was reported by Mikhaeil et al. [44], but this compound was not detected in the Saudi Arabian henna leaf extract.
Leaf extracts of A. saligna and L. inermis showed strong antioxidant effects using different assays. A. saligna showed higher antioxidant effects compared to L. inermis. The higher antioxidant effects of A. saligna can mainly be attributed to the polyphenolic composition of leaves, including hyperoside, quercetin 3-glucuronide, gallic acid, p-coumaric acid, quercitrin and rutoside. These polyphenols showed strong antioxidant activities when examined individually. In agreement with the current study, a previous investigation on polyphenols showed that hyperoside has comparable antioxidant activities to other polyphenols, such as caffeic and ellagic acids [47]. Quercetin, as a flavonoid glycoside, is common in plants and in the human diet, reduces ROS production and may stimulate THP-1 acute monocytic leukemia in vitro [48]. Previous investigation found that quercetin has antioxidant and antiproliferative activities against RAW264.7 cancer Cells [49]. In the current study, we found strong quercetin 3-glucuronide antioxidant activities using different assays, and this is the first study exploring the antioxidant and biological activities of this quercetin derivative. Gallic acid (3,4,5-trihydroxybenzoic acid) is a known triphenolic compound that has strong antioxidant activities, as found in this study [50]. It was found that p-coumaric acid is a strong antioxidant, which is in agreement with previous investigations [51,52]. In L. inermis, moderate antioxidant activities were found, and these were attributed to the polyphenolic composition of leaf extracts including apigenin 5-glucoside. Apigenin has in vitro and in vivo antioxidant effects, as well as anti-mutagenic and anti-inflammatory effects, by inhibiting the cell cycle, inducing apoptosis and diminishing oxidative stress [53]. This is the first study to investigate the antioxidant and biological activities of the apigenin-derivative apigenin 5-glucoside and to show strong antioxidant activities.
Few studies have investigated the antioxidant activities of Acacia species; for example, Egyptian A. saligna flower water extracts showed weak antioxidant and antibacterial activities but had good antifungal activity against Penicillium chrysogenum using the disc diffusion method [18]. In another study, the Egyptian Acacia nilotica and Acacia seyal leaf extracts showed higher antioxidant activities than Acacia laeta extracts [19]. To our best knowledge, this is the first investigation on the antioxidant activities of Saudi A. saligna. In Tunisian L. inermis, the butanolic fraction showed strong antioxidant activities, and these activities were attributed to phenolic glycosides including 1,2,4-Trihydroxynaphthalene-1-O-β-d-glucopyranoside [23]. In another study, Tunisian L. inermis leaf and seed aqueous extracts showed antioxidant activities, but no active compounds were identified [24]. The Iranian L. inermis aqueous leaf extracts (Henna leaves) showed diverse antioxidant activities associated with ecotypes [25].
MTT and flow cytometry assays revealed that A. saligna and L. inermis leaf extracts have antiproliferative and cytotoxic activities against different cancer cells. These activities were attributed to the polyphenolic composition including rutoside, hyperoside, quercetin, quercetin 3-glucuronide and p-coumaric acid. Rutoside (rutin) has known antiproliferative and cytotoxic activities against different cancer cells [54]. A previous investigation showed that hyperoside has anticancer activity against lung cancer cells by upregulating FoxO1 via CCAT1 [55]; additionally, it showed an inhibitory effect against SW620 human colorectal cancer cells via the induction of the p.53 signaling pathway and apoptosis [56]. Apigenin (4 ,5,7-trihydroxyflavone) is a common component of the human diet, especially in fruits, vegetables and herbs, with known antiproliferative and proapoptotic bioactivity [57]. The current study is the first to report on the antiproliferative and cytotoxic activities of the apigenin derivative apigenin 5-glucoside.
Gallic acid showed strong antiproliferative activities against the different cancer cells examined in this study, which is in agreement with previous investigations [50]. Gallic acid obtained from mango peels has antiproliferative effects against human colon adenocarcinoma cells and mouse connective cells, which can be attributed to its antioxidant activities [50]. Quercetin is recognized as an antiproliferative factor against specific cancer cells and has been shown to reduce the activity of specificity protein 1 and minimize the proliferation of human carcinoma HepG2 cells [57,58]. Quercetin has antiproliferative activities against prostate cancer cells, which can be attributed to its synergy with other polyphenols in green tea [58]. In addition, quercetin has cytotoxic activity against lung cancer cells [59]. In the current study, quercetin 3-glucuronide (quercetin derivative) was shown for the first time to exhibit antiproliferative and cytotoxic activities against different cancer cells. Cytotoxic spirostane saponin and biflavonoid glycoside were identified in the leaves of Egyptian A. saligna and showed anticancer activities against HepG2 cancer cells [42]. A previous investigation on the aerial parts of Acacia species (A. salicina, A. laeta, A. hamulosa, and A. tortilis) from the eastern region of Saudi Arabia (approximately 700-1000 km from the Riyadh region) revealed that A. laeta and A. hamulosa have cytotoxic activities against HepG2 and breast cancer cell line [20]. In a previous investigation, leaf aqueous and methanolic extracts of Indian L. inermis showed significant antioxidant activities that inhibited Cr (VI)-induced cytotoxicity in MDA-MB-435S breast cancer cells, and these activities were associated with phenolic compounds including gallic acid and p-hydroxybenzoic acid [26].
A. saligna and L. inermis leaf extracts had antibacterial effects against most bacteria investigated. A. saligna exhibited two-fold higher antibacterial activities than L. inermis against all bacteria. The most sensitive bacteria were E. coli and S. aureus. Identified polyphenols (rutoside, apigenin 5-glucoside and p-coumaric acid) showed comparable antibacterial activities to the standards. A previous investigation on Egyptian A. saligna showed that flower water extracts had weak antibacterial activities but good antifungal activity against Penicillium chrysogenum using the disc diffusion method [18]. In another investigation, A. saligna from Egyptian methanolic leaf extract exhibited no antibacterial activities against most bacteria examined [17]. Another study on the aerial parts of other Acacia species revealed that A. laeta and A. tortilis have antimicrobial activities against S. aureus, E. coli, and P. aeruginosa [20]. Tunisian L. inermis leaf and seed aqueous extracts showed antimicrobial activities, but no active compounds were identified [24]. Iranian L. inermis aqueous leaf extracts showed strong antibacterial activities against S. aureus, Streptococcus agalactiae, B. cereus, Corynebacterium pseudotuberculosis, Klebsiella pneumonia, E. coli and Salmonella enterica serovar typhi [25]. However, no active compounds were identified. The antibacterial activities of A. saligna and L. inermis found in this study are attributed to the moderate to high contents of polyphenols such as rutoside, apigenin 5-glucoside and p-coumaric acid. Rutoside (rutin) has known antibacterial activities against E. coli, Proteus vulgaris, Shigella sonnei, P. auruginosssa and B. subtilis [54]. Apigenins such as aglycone have antibacterial activity against E. coli and P. aeruginosa [60]; however, this is the first report on the wide-spectrum biological activities of apigenin 5-glucoside. P-coumaric acid exhibited antibacterial activity against B. cereus and Salmonella typhimurium when applied with niacin [61]. In the current study, we found moderate antibacterial activities of hyperoside, quercetin and quercetin 3-glucuronide. Quercetin showed moderate antibacterial activities against S. aureusm, E. coli and P. aeruginosa in previous investigations [62,63].
A. saligna and L. inermis extracts showed strong antifungal effects against most fungi studied (Table 3). A. saligna showed two to three-fold higher activity (lower IC 50 values) than L. inermis. Rutoside, hyperoside, apigenin 5-glucoside and p-coumaric acid showed the highest antifungal effects among the studied polyphenols.
A. saligna and L. inermis extracts showed strong antifungal effects against the studied fungi. A. saligna showed two to three-fold higher activity than L. inermis. The identified polyphenols including rutoside, hyperoside, apigenin 5-glucoside and p-coumaric acid showed the highest antifungal activities, explaining the activities of leaf extracts. A previous investigation on Egyptian A. saligna showed that flower water extracts had good antifungal activity against Penicillium chrysogenum using the disc diffusion method [18]. A study on the aerial parts of other Acacia species revealed that A. laeta and A. tortilis have antifungal activities against C. albicans [20]. Chinese Acacia confuse seeds contained a protein showing strong antifungal activities against Rhizoctonia solani [64]. Apigenin 5-glucoside showed strong antifungal activities against C. albicans in the current study, which were comparable or greater than those of apigenin and its derivatives found previously [65]. These antifungal activities were attributed to the reduction of intra and extracellular reactive oxidative species as a mechanism of antifungal action. The current study is the first comprehensive illustration of the fungicidal activities of the apigenin-derivative apigenin 5-glucoside. Rutoside has antifungal activities against C. albicans [54]. A previous investigation showed that hyperoside had antifungal activities against Alternaria alternata, Pestalotia guepinii, Fusarium avenaceum, Drechslera sp. and Epicoccum nigrum [66]. P-coumaric acid showed antifungal effects against Botrytis cinerea [67]. Quercetin has antifungal activities against C. albicans [68][69][70]. However, this is the first report on the antifungal activities of apigenin 5-glucoside.

Conclusions
To the best of our knowledge, this is the first study to explore the polyphenol composition and biological activities of methanolic leaf extracts of natural A. saligna and L inermis populations from northern Saudi Arabia. Several polyphenols were tentatively identified in the leaf extracts including rutoside, hyperoside, gallic acid, quercetin 3-glucuronide and p-coumaric acid in A. saligna and apigetrin, apigenin 5-glucoside and gallic acid in L. inermis. Indeed, further analyses are needed to better understand the complete composition of the studied plants. The MTT and flow cytometry assays revealed that A. saligna and L. inermis leaf extracts had antiproliferative and cytotoxic activities against different cancer cells. These activities were attributed to the polyphenolic composition, including rutoside, hyperoside, quercetin, quercetin 3-glucuronide and p-coumaric acid, and necrotic cell accumulation during apoptotic periods. Leaf extracts of A. saligna and L. inermis showed strong antioxidant effects using different assays. A. saligna showed higher antioxidant effects compared to L. inermis. The higher antioxidant effects of A. saligna can mainly be attributed to the polyphenolic composition of leaves, including hyperoside, quercetin 3-glucuronide, gallic acid, p-coumaric acid, quercitrin and rutoside. A. saligna and L. inermis leaf extracts had antibacterial effects against most bacteria investigated. A. saligna had twice higher antibacterial activities than L. inermis against all bacteria. The most sensitive bacteria were E. coli and S. aureus. The identified polyphenols (rutoside, apigenin 5-glucoside, and p-coumaric acid) showed comparable antibacterial activities to the standards. Both species exhibited antifungal activities, which were attributed to their polyphenol composition and associated with specific polyphenols including rutoside, hyperoside, apigenin 5-glucoside and p-coumaric acid. The current study is the first to report on the antiproliferative, cytotoxic, antibacterial and antifungal activities of the apigenin-derivative apigenin 5-glucoside.