INTRODUCTIONRadical oxygen species lead to cell damage, whichcan induce genetic instability responsible for manypathological processes. This damage can be repairedby some natural compounds, e.g. radical scavengersand powerful protective antioxidants [1]. Kitagishi et al.proved that medicinal herbs could one day becomea promising therapeutic means of cancer therapy [2].According to Dayani et al., antioxidant, antiinflammatory,and anti-apoptotic properties of plantsand their derivatives make them good radioprotectorsagainst the mutagenic action of X-rays [3].Phytotherapy relies on medicinal plants and theiractive compounds. Rhamnus alaternus L. (Rhamnaceaefamily), also called imlilesse or safir in the North ofAlgeria, is well known for its biological properties [4].Zeouk and Bakheti reported that a decoction of theaerial part of the R. alatrenus leaves and branches hasbeen widely used in traditional medicine to lower bloodpressure and treat hepatitis, icterus, musculoskeletaldisorders, and gastrointestinal diseases. They also serveas a cataplasm for skin infections [5].Previous findings proved that R. alaternus extractspossess potential antioxidant, cytotoxic antimutagenic,antigenotoxic, and antimicrobial activities [5–8]. Intheir bibliographic review, Nekkaa et al. focused onthe phytochemical and pharmacological properties ofR. alaternus [4]. Its leaf extracts were rich in flavonoids,197Gadouche L. et al. Foods and Raw Materials. 2022;10(2):196–205tannins, and anthocyanins, which explains theirpotential antigenotoxic and antimutagenic activity.Bhouri et al. isolated kaempferol 3-O-b-isorhamninosideand rhamnocitrin 3-O-b-isor-hamninosidefrom R. alternus leaves [9]. These flavonoidsare effective free radical scavengers and potentantigenotoxics. However, they can induce apoptosis inhuman lymphoblastoid cells by the extrinsic apoptoticmechanism including DNA fragmentation, PARPcleavage, and active caspase-3 and caspase-8 [10].Oligomer flavonoid extract from R. alaternus leavesproved to have a good potential for alternativeantimelanoma therapies [11].Although some plant remedies have welldocumentedprotective effects and alleviate manydiseases, cytotoxicity studies are very important fordeveloping new drugs. Gadouche et al. described thetoxic effect of Aristolochia longa L. and Calycotomespinosa L. on the blood cells and concluded thatit should be studied on cancer cells [12]. Naturalantioxidants can even protect human organism againstthe cytotoxic and mutagenic effects of xenobiotics.In this research, we analyzed the genotoxic andDNA damage protecting activity of R. alaternusleaf extract by using the Allium cepa assay with azidesodium as a mutagen agent.STUDY OBJECTS AND METHODSPlant material. The research featured Rhamnusalaternus eu-alaternus L., a subspecies of Rhamnusalaternus L. The samples were collected in the Bissaforest located in the north of the Chlef province(Algeria). This species of Algerian flora was identifiedby Dr. Belhacine, a botanist from the Chlef University[13].The R. alaternus leaves were dried in the dark for 10days. After that, they were ground into a fine powderand kept in an airtight container, and 10 g of the drypowder was macerated in 100 mL of petroleum ether for24 h with stirring. The mix was filtered on Whatman No.1 paper. The maceration included 100 mL of methanol.After filtration, the marc was evaporated in a rotaryevaporator at 39°C. The extract obtained was stored at4°C until use [14].Quantitative analysis and antioxidantactivity. The polyphenols were assayed accordingto the method developed by Raafat and Samy[15]. The amount of total polyphenols wasdetermined spectrophotometrically using the Folin-Ciocalteu reagent and deduced from a calibrationcurve established with gallic acid (0–1 mg/mL).The results were expressed in mg of gallic acidequivalent per g of dry matter (mg GAE/g of dry matter).The mix included 250 μL of Folin Ciocalteu’s phenolreagent, 50 μL of each concentration prepared fromstock solution, and 500 μL of 20% Na2CO3 aqueoussolution. After vortexing, the solution was adjustedwith 5 mL of distilled water. After 30 min of incubation,the absorbance was measured at 765 nm. The sameprocedure was carried out with the extract obtainedfrom R. alaternus leaves.The flavonoid content was assayed according to themethod developed by Hmid et al. [16]. After 1 mL ofextract was added to 1 mL of 2% aluminum chloride,the absorbance was determined at 430 nm after 10 minof incubation. Quercetin served as calibration curvestandard and was established from the concentration of40 μg/mL of stock solution. Total flavonoids content inthe extract was expressed as mg quercetin equivalentsper g of sample (mg EQ/g of dry matter).The DPPH assay followed the method described byBurits and Bucar [17]. The R. alaternus extract had thefollowing concentrations: 0.2, 0.4, 1.0, and 2.0 mg/mL.We mixed 50 μL of each concentration with 5 mL of0.004% DPPH. The absorbance was taken at 517 nmafter 30 min of incubation. The results were comparedto ascorbic acid, which was used as standard antioxidantand handled under the same conditions. The percentageof inhibition and IC50 were calculated according toSharififar et al. [18]. The percentage inhibition wascalculated using the following equation:IC50 is the concentration of extract required for 50%inhibition of DPPH. It was calculated using a linearregression analysis.The β-carotene-linoleic acid assay was performedaccording to the method described by Kartal et al. [19].The emulsion included 0.5 mg of β-carotene, 1 mLof chloroform, 25 μL of linoleic acid, and 200 mg oftween 40. The chloroform was eliminated in a rotaryevaporator under vacuum, and 100 mL of distilledoxygen-saturated water was added to the emulsion.Subsequently, 350 μL of the extract at a concentrationof 2 mg/mL was mixed with 2.5 mL of the emulsion.After 48 h of incubation, the absorbance was registeredat 490 nm and compared with that obtained withbutylohydroxytoluene (BHT), which served as astandard antioxidant and was prepared under the sameconditions. The inhibition percentage of bleaching(I, %) was measured for each assay using the followingequation:Allium cepa assay. The Allium cepa assay wasperformed according to Tedesco and Laughinghouse withsome modifications [20]. The onion bulbs were kept ina culture medium that included 60 mg/L of CaSO4, 60mg/L of MgSO4, 96 mg/L of NaHCO3, and 4 mg/L ofKCl. They were incubated at 25°C for 72 h until theroots reached 2 cm. Seven onion bulbs were utilized foreach treatment as follows:Sample 1: culture medium + distilled water;Sample 2: culture medium + sodium azide(50 mg/mL);Sample 3: culture medium + sodium azide (100 mg/mL);198Gadouche L. et al. Foods and Raw Materials. 2022;10(2):196–205Sample 4: culture medium + methanolic extract(50 mg/mL);Sample 5: culture medium + methanolic extract(100 mg/mL);Sample 6: culture medium + sodium azide(100 mg/mL) + methanolic extract (50 mg/mL);Sample 7: culture medium + sodium azide(100 mg/mL) + methanolic extract (100 mg/mL).The effect of the different treatments on the growth(cm) of the A. cepa roots was measured at differenttime intervals: 0, 24, 48, and 72 h. In parallel, the rootswere tested for color, shape, and stiffness. After eachtime interval, the roots were collected for microscopicobservation of the meristem cells and stored in 70%ethanol for later use. The roots were fixed in acetic acidand ethanol solution (1:3) for 24 h. After triple rinsingwith distilled water, the roots were hydrolyzed withHCl (1N) and incubated in a hot water bath at 60°C for10 min. After the hydrolysis, the roots were rinsedonce again in distilled water and stained with 2%acetic carmine in a hot water bath at 60°C for 10 min.After incubation, the terminal meristem cells of thecolored roots were cut with a scalpel under a binocularmagnifier. The meristem regions were crushedmanually between blade and coverslip to visualize thechromosomes and the different stages of cell division.Meristem cells were counted for each sample andtested for normal or abnormal cell division in search formutations. The mitotic index and the rate of aberrantcells of each bulb were calculated by the followingformula [21]:Statistical analysis. The experimental data wereanalyzed using the ExcelSTAT software. The researchalso included the ANOVA variance analysis, followed bythe Tukey’s test. The statistically highly significant valuewas P < 0.001.RESULTS AND DISCUSSIONThe total phenol content in Rhamnus alaternus L.leaves was 32.6 ± 1.82 mg GAE/g DM, and the totalflavonoid content was 27.58 ± 0.01 mg EQ/g DM. Themethanolic extract of R. alaternus leaves demonstrateda moderate efficiency against free radicals emittedby linoleic acid (50.71 ± 4.17%). Its capacity to beatfree radical of DPPH (I% = 80.39 ± 2.33%, IC50 =0.74 ± 0.30 mg/mL) was close to that of ascorbic acid, i.e.96.80 ± 9.98% with IC50 = 0.37 ± 1.1 mg/mL (Table 1).Plants are excellent indicators of the cytotoxic,cytogenetic, and mutagenic effects of environmentalchemicals. They can serve as an alternative for detectingpossible genetic damage in cells [22]. Genotoxicitystudies were carried out by the Allium cepa assay. Thismethod provides a convenient in vivo model to evaluatecell cycle alterations induced by mutagens [20].The A. cepa roots treated with distilled water had nomorphological change: the growth rate was good, thecolor was whitish, and the roots were rigid and bulky.However, the roots treated with sodium azide (50 and100 mg/mL) changed the color and shape of the roots, aswell as reduced their rigidity (+very brittle) and growthrate.The methanolic leaf extract of R. alaternus hadno negative effect on the morphology. The samplesdemonstrated good growth, strong rigidity, and whitishcolor. Their morphology was comparable to the controlroots. The roots incubated in both methanolic extractand sodium azide had a phenotype close to the controlroots. They were better preserved than the roots treatedonly with sodium azide. The roots of this sample showedgood growth, and the color was comparable to that of thecontrol roots (Fig. 1).Table 2 shows a highly significant decrease in thegrowth of the A. cepa roots treated with sodium azideat two concentrations (50 and 100 mg/mL) at three timeintervals. The data obtained from the sample treatedwith 50 mg/mL of sodium azide after 48 h was foundinsignificant (P < 0.001).The roots treated with the methanolic extract ofR. alaternus leaves showed highly significant growth(P < 0.001) after 24 and 48 h. The roots reached 8 cmafter 72 h (P < 0.001) and were longer than those treatedwith distilled water (7 cm).The difference in length for the antigenotoxicitytest was highly significant after 48 and 72 h andnot significant after 24 h. The roots demonstrated aclearly significant improvement in the diameter afterParameter Leaves of R. alaternus Ascorbic acid ButylatedhydroxytoluenePolyphenol, mg GAE/g dry matterFlavonoids, mg QE/g dry matterDPPH, % (R. alaternus extract concentration= 1 mg/mL)IC50, mg/mLβ-carotene bleaching, % (R. alaternus extractconcentration = 2 mg/mL)32.60 ± 1.8227.58 ± 0.0180.39 ± 2.330.74 ± 0.3050.71 ± 4.17––96.80 ± 9.980.37 ± 1.10–––––98.84 ± 1.69Table 1 Total phenolic content and total flavonoid content of Rhamnus alaternus leaves, DPPH inhibition, IC50, and % bleachingof β-carotene199Gadouche L. et al. Foods and Raw Materials. 2022;10(2):196–205Figure 1 Morphological aspects of Allium cepa roots: (a) control group; (b) sodium azide (50 mg/mL); (c) sodium azide(100 mg/mL); (d) methanolic extract (50 mg/mL); (e) methanolic extract (100 mg/mL); (f) sodium azide (100 mg/mL) + methanolicextract (50 mg/mL); (g) sodium azide (100 mg/mL) + methanolic extract (100 mg/mL)a b c d e f g72 h. It was 0.26 and 0.13 cm respectively for the twoextract concentrations.Microscopy revealed no abnormalities ordisturbances in mitotic division: chromosome integritymaintained its high mitotic index (69.76 ± 7.01%),and no chromosomal aberrations were registered(Fig. 2, Table 2).The microscopy of the roots stained with 2% aceticcarmine after treatment with two concentrations ofsodium azide revealed several chromosomal anomalieswith disruption of all the stages of cell division(Figs. 3 and 4, Table 2). Several cells containedC-mitosis, S-mitosis, chromosomal breaks, bridges,and uneven distribution of chromosomes, which led todisturbed anaphases, metaphases, and telophases. Theseanomalies were caused by both concentrations of azide;however, they were much more severe at 100 mg/mL ofsodium azide.Table 2 ΔL – differences in length of the Allium cepa roots before and after each treatment, % mitotic index, and chromosomalaberrationsTreatment Time, h ΔL, cm Mitotic index, % Chromosomalaberrations, %Control 02448722.66 ± 0.560.57 ± 0.050.84 ± 0.090.70 ± 0.24 69.76 ± 7.010Sodium azide (50 mg/mL) 02448722.36 ± 0.480.50 ± 0.11**0.94 ± 0.10–0.54 ± 0.31**34.48 ± 10.505.03 ± 1.51**Sodium azide (100 mg/mL) 02448722.84 ± 0.47–1.12 ± 0.18**–0.40 ± 0.16**–0.03 ± 0.12** 29.25 ± 8.507.84 ± 2.41**Methanolic extract (50 mg/mL) 02448722.93 ± 0.191.09 ± 0.21**0.90 ± 0.07**0.46 ± 0.15 69.54 ± 14.50.43 ± 0.53Methanolic extract (100 mg/mL) 02448722.80 ± 0.471.87 ± 0.04**1.37 ± 0.09**1.05 ± 0.02 73.66 ± 9.41 0.29 ± 0.49Sodium azide (100 mg/mL) + methanolicextract (50 mg/mL)02448722.71 ± 0.380.11 ± 0.05–0.33 ± 0.12**0.26 ± 0.18** 51.77 ± 14.44 2.55 ± 1.98Sodium azide (100 mg/mL) + methanolicextract (100 mg/mL)02448722.47 ± 0.570.53 ± 0.08–0.10 ± 0.07**0.13 ± 0.09**52.34 ± 8.123.34 ± 2.82ΔL – mean difference in length of Allium cepa roots before and after treatment**P ≤ 0.001200Gadouche L. et al. Foods and Raw Materials. 2022;10(2):196–205Figure 3 Various anomalies caused by sodium azide at 50 mg/mL (100×): (a) binucleated cell; (b) disturbed telophase; (c) disturbedanaphase; (d) normal anaphase; (e), (f) disturbed metaphaseFigure 4 Chromosomes of Allium cepa roots treated with 100 mg/mL of sodium azide (100×): (a) binucleated cells; (b)chromosomal break, chromosomal bridge; (c), (e) disrupted (uneven) anaphase; (d) prophase, chromosomal bridge; (f) C-mitosisFigure 2 Normal mitotic divisions of Allium cepa meristem cells (100×): (a) interphase, prophase, and metaphase; (b) start ofanaphase; (c), (e), (f) anaphase; (d) telophasea b c de fa b c de fa b c de f201Gadouche L. et al. Foods and Raw Materials. 2022;10(2):196–205Chromosomal aberrations increased together withthe concentration of sodium azide. The genotoxic effectwas most severe at 100 mg/mL. Both concentrations ofsodium azide reduced the mitotic index, which meantthat sodium azide blocked cell division. On the otherhand, the number of chromosomal aberrations grewtogether with sodium azide concentration. They wererepresented mainly by C-mitosis, chromosomal bridgesand breaks, and nuclear lesions of binucleate types.Therefore, sodium azide was an aberration inducer(Figs. 3 and 4, Table 2).Sodium azide produced a cytotoxic effectwhich led to poor growth and length narrowing. Itsmitodepressive effect decreased mitotic activityand increased chromosomic abnormality incidence.Indeed, chemical agents are recognized as factorsinvolved in the structural and numerical modificationsof chromosomes. As a result, they cause defects inchromosome segregation, abnormal DNA replication,and DNA breaks. These chromosomal aberrationsresult from clastogenic and aneugenic effects [23]. Thisstudy confirmed the genotoxic effect of sodium azide.According to Al-Qurainy et al., sodium azide is amutagenic metabolite that damages DNA by substitutingone base pair with another [24]. Indeed, the shorterlength of A. cepa roots treated with sodium azidecould be explained by the mitodepressive effect causedby the apoptosis of meristem cells. Other samplesdemonstrated evolution of the normal length, probably,due to the resumption of mitosis.Sodium azide induced the development ofchromosome bridges in the meristem cells ofA. cepa roots. According to Neelamkavil and Thoppil,the chromosomal aberrations and nuclear lesions inA. cepa root meristems treated with bleaching powderindicated a genotoxic effect, which confirms thatsodium azide is genotoxic [25]. The clastogenic effectssuggest that bleaching powder caused chromosome andchromatin breaks, which, in return, led to abnormalchromosome number, stickiness, breakage, and reunionof chromosome, as well as to bridges during mitoticdivision [26, 27].The mitotic index was higher in the roots treatedwith two concentrations of the R. alaternus extractthan in those treated with distilled water. Therefore,the extract induced cell division and, subsequently,produced a genoprotective effect. Moreover, thenumber of cells in division was high with traceschromosomal aberrations also proven by a markedroot length. The samples treated with 50 mg/mL ofR. alaternus methanolic extract had pycnotic nucleiand chromosomal breaks (Figs. 5 and 6, Table 2). Thisfinding confirms the conclusion made by Ben Ammaret al., who experimented with methanolic, petroleumether, chloroform, and aqueous extracts of R. alaternusleaves and registered no mutagenicity, which means thatR. alaternus is a promising antimutagenic [28].The antigentoxic effect showed that the mitoticindex was close to that of the control. It had a moderatechromosomal aberration percentage, chromosomalbridges and breaks, and a lower C-mitosis (Figs. 7and 8, Table 2).A quantitative analysis of the R. alaternus methanolicextract revealed a lot of polyphenols and flavonoidsand thus a prominent antioxidant effect. This antioxidanteffect might be the cause of the continuous cell division,mitoprotective activity, and a good DNA protection.Perron et al. tested 12 polyphenolic compounds,which demonstrated a 100% ability to inhibit DNA damage[29]. The polyphenolic compounds had hydroxylradicals in their chemical structures, which preventedoxidative DNA damage.On the other hand, Silva et al. showed that flavonoidshave special DNA repair mechanisms that enablethem to reduce and repair DNA strand breaks inducedby oxidative stress [30]. Therefore, polyphenols areeffective protectors against oxidative DNA damage.Figure 5 Chromosomes of Allium cepa roots treated with 50 mg/mL of Rhamnus alaternus leaf extract (100×): (a) prophase; (b) endof interphase; (c) telophase; (d) pycnotic nucleus; (e) metaphase; (f) anaphasea b c de f202Gadouche L. et al. Foods and Raw Materials. 2022;10(2):196–205Figure 6 Chromosomes of Allium cepa roots treated with 100 mg/mL of Rhamnus alaternus leaf extract (100×): (a), (b), (f)telophase/cytodiuresis; (c) anaphase, prophase, and metaphase; (d) anaphase with chromosome breaks; (e) chromosome bridge withan isolated chromosomea b c de fFigure 7 Chromosomes of Allium cepa roots treated with 50 mg/mL of methanolic extract and 100 mg/mL of sodium azide (100×):(a) several prophases; (b) several prophases; (c), (f) metaphase; (d) anaphase; (e) telophase start of prophasea b c de fFigure 8 Chromosomes of Allium cepa roots treated with 50 mg/mL of methanolic extract and 100 mg/mL of sodium azide (100×):(a) binucleated; (b) metaphase, anaphase; (c) several prophases; (d) prophase; (e) prophase, metaphase, and anaphase.a b c de f203Gadouche L. et al. Foods and Raw Materials. 2022;10(2):196–205CONCLUSIONMedicinal plants contain a lot of secondarymetabolites with beneficial therapeutic and pharmacologicalproperties, which deserve extensive research.Rhamnus alaternus L. proved to be an effective antioxidantand mitoprotector that can boost the developmentof pharmacognosy and produce new herbal drugsfor the pharmaceutical industry. The genoportectiveeffect of R. alaternus leaf extract could be a source ofnew cancer drugs and protect human genome from theside effects of chemical treatment.CONTRIBUTIONL. Gadouche conceived and designed the analysis,performed the biological experiments, and wrote thepaper. A. Zidane and K. Zerrouki contributed to the dataanalysis and revised the paper. A. Ababou performedthe statistical analysis. I. Bachir Elazaar, D. Merabet,W. Henniche, and S. Ikhlel performed the biologicalexperiments. All the authors revised the manuscript forpublication.CONFLICT OF INTERESTThe authors declare that there was no potentialconflict of interests regarding the publication of thisarticle.