INTRODUCTIONMedicinal plants are an important pool of moleculeswith therapeutic potential for drug innovation [1].According to Estella et al., vulgarization of traditionalherbal remedies is confronted with many predicamentsdue to the lack of information on their therapeutic andtoxicological properties to guarantee their rationaluse [2]. According to Calixto [3], plants containhundreds of phytotherapeutic agents with adverse effectsand some of them are very toxic if inappropriately used.In fact, Kharchoufa et al. have identified more than89 toxic medicinal plants used as treatment in the North-Eastern region of Morocco [4]. These plants containtoxic compounds: alkaloids followed by glucosides,terpenoids, proteins, and phenolics. Their toxicity canlead to serious adverse reactions or interactions withother plants. On the other hand, a misidentificationof plants can lead to a toxicity that may also resultfrom an uncontrolled or excessive use [5]. Therefore,before formulating and marketing a herbal medicine,appropriate scientific studies are essential, includingthose into pharmacological properties, toxicity, and sideeffects [6].Algeria has more than 3000 species belongingto several botanical families distributed all alongthe Mediterranean, Saharan, and tropical regions.Calycotome spinosa (L.) belongs to the Fabaceae family,Aristolochia longa belongs to the Aristolochiaceaefamily, and Myrtus communis belongs to the Myrtaceaefamily [7]. Algeria is the only country that hostsboth species, M. communis (L.) in the North andMyrtus novelli in the South [8]. For several centuries,380Gadouche L. et al. Foods and Raw Materials, 2021, vol. 9, no. 2, pp. 379–386M. communis has been used in folk medicine astreatment for many diseases due to its broad spectrumof pharmacological and therapeutic effects [9]. Speciesof Aristolochia are known for their toxicity and posepotential health risk associated with their content ofaristolochic acids [10].Since these species are widely used in the littoralzone of Algeria in traditional and folk medicine,there is a need for research into their toxicity. In thiscontext, we aimed to evaluate the hemolytic effect onhuman erythrocyte cells induced by aqueous extractsof M. communis (Rayhan), A. longa (Bereztem), andC. spinosa (Guendoul). The last two species have beenrarely studied.STUDY OBJECTS AND METHODSMedicinal plants Myrtus communis (L.), Calycotomespinose (L.), and Aristolochia longa (L.) were collectedin many areas in the littoral of Algeria (March 2018),namely Damous (Tipaza), Benni Haoua (Chlef), andBissa (Chlef) (Fig. 1). These species were identifiedby Dr. Belhacini, a teacher and researcher at HassibaBenbouali University of Chlef (Algeria).Preparation of aqueous extracts. For each specieswe used dried and powdered plant aerial parts accordingto traditional use in these areas, namely leaves forM. communis and A. longa and leaves and flowers forC. spinosa. Aqueous extracts were prepared by adecoction of the plant material. In particular, 10 g of theplant material was boiled with 100 mL of distilled waterfor 15 min and then the solution was filtered and driedat 39°C.Phytochemical screening. Phytochemical tests wereperformed on 5% infusion to detect certain secondarymetabolites according to Takaidza et al. and Behbahaniet al. [11, 12].Determination of total phenol contents. A mixtureof 250 μL of Folin Ciocalteu phenol reagent, 50 μL ofthe sample, and 500 μl of 20% Na2CO3 was prepared.The volume was adjusted to 5 mL with distilled waterwhile shaking vigorously. After 30 min incubation,absorbance was read at 765 nm. A calibration curve ofgallic acid (0–1 mg/mL) was done in parallel. The resultswere expressed in mg of gallic acid equivalent/g of drymatter (mg EAG/g DM) [13].Determination of total flavonoid contents. Theflavonoid assay was performed according to the methodof Hmid et al. [14]. 1 mL of each extract was mixed with1 mL of 2% AlCl3. After 10 min incubation, absorbancewas read at 430 nm. The flavonoid concentrationswere calculated using a calibration curve establishedwith quercetin (0–40 μg/mL) and expressed in mg ofquercetin equivalent/g of dry matter (mg EQ/g DM).Hemolytic activity determination. A phosphatebuffered saline (PBS) solution with pH = 7.4 wasprepared by mixing the following compounds inappropriate concentrations: Na2HPO4 (10 Mm), K H2PO4(1.8 Mm), KCl (2.7 Mm), and NaCl (137 Mm) [15].A concentration range for each extract (M. communis,C. spinose, and A. longa) was prepared by dilutingin PBS: 25, 50, 100, and 200 mg/mL. An erythrocytesuspension was prepared from the blood of a healthydonor in a heparin tube. After centrifugation at2400 rpm for 10 min, the plasma was removed andthe pellet was washed twice with PBS and then filledup with the same volume of plasma removed. Theerythrocyte suspension was diluted 20 times with PBS.Erythrocyte hemolysis assay. The hemolytic effecttest of the species studied was carried out accordingto the method described by Haddouchi et al. and Guo-Xiang and Zai-Qun [16, 17]. We mixed 2950 μL of theerythrocyte suspension with 50 μL of aqueous extractfor each species in a hemolysis tube. The operationwas repeated three times for each concentration. Thetubes were incubated at 37°C for one hour. During thisperiod, 500 μL of each test was taken every 15 min(in 15, 30, 45, and 60 min) and added to 1.5 mL of PBSand then centrifuged again at 2400 rpm for 10 min. Theabsorbance of the hemoglobin leak in the supernatantwas read at 548 nm against a blank containing PBS.A negative control tube was prepared under the sameexperimental conditions, 2950 μL of the erythrocytesuspension and 50 μL of the PBS buffer solution. Onthe other hand, a total hemolysis tube was preparedFigure 1 1 – Myrtus communis; 2 – Calycotome spinose; 3 – Aristolochia longa381Gadouche L. et al. Foods and Raw Materials, 2021, vol. 9, no. 2, pp. 379–386containing 250 μL of the erythrocyte suspension and4750 μL of distilled water. Each test was repeatedthree times. The hemolysis rate of various extracts wascalculated as a percentage (%) of total hemolysis after15, 30, 45, and 60 min of incubation, according to thefollowing formula:% Hemolysis=A(extract at 60 min)-A(negative controlat 60 min)/A(Total hemolysis at 60 min) (1)Statistical analysis. Statistical analysis was doneby One Way ANOVA. The data obtained were analyzedusing the student’s t-test. A P value less than 0.01 wasconsidered statistically significant.RESULTS AND DISCUSSIONPhytochemical screening. The phytochemicalscreening allowed us to highlight the presence of somesecondary metabolites (saponosides, tannins, alkaloids,flavonoids, and anthocyanins). The phytochemicaltests carried out on the infused flowers and leaves ofthe selected plants are shown in Table 1. The resultsobtained after shaking the infusion for 15 min showedthat Myrtus communis and Aristolochia longa wererich in saponosides because the foam was greater than1 cm. In the Calycotome spinosa leave and flowerinfusion, the foam was unstable in the order of a fewmm. The appearance of the orange and pink color afterthe addition of isoamyl alcohol indicated the presenceof flavones in the C. spinosa leave and flower infusion.The purplish pink color indicated the presence offlavonones in the leaves of M. communis. In the A. longainfusion, the result was negative. The precipitate in theC. spinosa and M. communis infusions, which werepreviously acidified with sulfuric acid, after addingsome drops of the Mayer reagent indicated the presenceof alkaloids. However, the test was negative for A. longa.The appearance of a pink and red coloration after addingammonia to the HCl-infused A. longa and C. spinosaindicated the presence of anthocyanins. However, thissecondary metabolite was absent in the M. communisleave infusion.Contents of total polyphenols and flavonoids.The amount of polyphenols in the dry matter wasexpressed in mg gallic acid equivalent (mg EAG/gMS) and determined by the equation: y = 0.940x + b;R2 = 0.981. T he a mount of flavonoids i n t he d ry m atterwas expressed in mg of quercetin equivalent (mg EQ/gMS) and determined by the equation: y = 0.055x + b;R2 = 0.996 (Table 2). The total polyphenol content inthe dry matter was 234.89 ± 0.80, 283.68 ± 0.60, and346.27 ± 2.00 mg GEA/g for C. spinosa, A. longa,and M. communis, respectively. The content of totalflavonoids in the dry matter was 10.50 ± 0.03, 34.86 ±0.06, and 31.02 ± 0.19 mg EQ/g for A. longa, C. spinose,and M. communis, respectively (Table 2).Hemolytic activity. In the negative control tube(tube containing only PBS and erythrocyte suspension),the hemolysis rate was constant and did not exceed2.77 ± 0.35% after one hour of incubation. On theTable 1 Results of phytochemical screening tests for Myrtus communis, Calycotome spinose, and Aristolochia longaSamples Saponoside Test Tanin Test Flavonoid Test Anthocyanin Test Alkaloid testMyrtuscommunis (leaves)Foam > 1cm Catechin tannins Flavonones – +Calycotome spinose(leaves and flowers)Foam 0.2 cm – Flavones – +++Aristolochia longa(leaves)Foam > 1,8 cm Catechin tannins +/– + –“+” Present, “++” Moderate presence, “+++” High presence, “+/–” Presence not evident, “–” Absent382Gadouche L. et al. Foods and Raw Materials, 2021, vol. 9, no. 2, pp. 379–386other hand, a total hemolysis of red blood cells wasclearly observed in the total hemolysis tube. Indeed, werecorded a hemolysis rate that reached 99.86 ± 10.32% at60 min.For the aqueous extract of the M. communis leaves,we observed a significantly low hemolysis rate duringthe first 15 minutes (P < 0.01). The hemolysis rateswere 5.07 ± 0.21, 7.85 ± 1.20, and 12.57 ± 2.89% forthe concentration of 25 mg/mL; 6.04 ± 1.90 (P < 0.01),6.46 ± 0.77, and 20.42% for 50 mg/mL; 8.05 ± 1.41,11.32 ± 5.72, and 19.51 ± 6.71 for 100 mg/mL; and12.01 ± 0.21, 12.22 ± 0.26, and 20.14 ± 3.11% (P < 0.01)for 200 mg/mL, respectively, compared to totalhemolysis (Fig. 2).For C. spinosa, we found a significant increasein hemolysis rates over time (15, 30, 45, 60 min).Also, the rates were considerably higher with higherconcentrations of the extract. For the concentrationsof 25 and 50 mg/mL, hemolysis rates ranged between6.52 ± 3.78 and 17.12 ± 1.50%, as well as 7.50 ± 2.95and 22.36 ± 2.12%, respectively. However, a significanthemolytic effect was recorded in 100 (45 min) and200 mg/mL (15, 45, and 60 min) of the C. spinosaextract. This rate increased from 8.14 ± 1.23% at 15 minto 23.61 ± 8.94% at 60 min in the presence of a100 mg/mL concentration and from 24.44 ± 3.95% at15 min to 34.86 ± 5.05% at 60 min in the presence of a200 mg/mL concentration (Fig. 3).For the extract of the A. longa leaves (Fig. 4), wefound an increase in hemolysis rates over time (15,30, 45, and 60 min). As the concentration increased,the percentage of hemolysis increased as well. Atconcentrations of 25 and 50 mg/mL, a hemolysispercentage ranged from 5 (15 min) to 6.71% (60 min)and from 7.22 (15 min) to 18.47% (60 min), respectively.The hemolysis rate was significant at 15 and 45 min(P < 0.01).On the other hand, we observed an importanthemolytic effect of the A. longa aqueous extract atconcentrations of 100 and 200 mg/mL. This rate wentTable 2 Polyphenol and flavonoid content in Myrtuscommunis, Calycotome spinose, and Aristolochia longaSpecies Polyphenolsmg GEA/g DMFlavonoidsmg EQ/g DMMyrtus communisCalycotome spinosaAristolochia longa346.27 ± 2.00234.89 ± 0.80283.68 ± 0.6031.02 ± 0.1934.86 ± 0.0610.50 ± 0.03TH: Total Hemolysis. NC: Negative Control.The means of 3 replicates. P < 0.01. ** significantFigure 2 Hemolytic effect of four concentrationsof Myrtus communis extract at 15, 30, 45, and 60 minTime, minHemolysis, %TH: Total Hemolysis. NC: Negative Control.The means of 3 replicates. P < 0.01. ** significantFigure 3 Hemolytic effect of four concentrationsof Calycotome spinosa extract at 15, 30, 45, and 60 minTime, minHemolysis, %TH: Total Hemolysis. NC: Negative Control.The means of 3 replicates. P < 0.01. ** significantFigure 4 Hemolytic effect of four concentrationsof Aristolochia longa extract at 15, 30, 45, and 60 minTime, minHemolysis, %383Gadouche L. et al. Foods and Raw Materials, 2021, vol. 9, no. 2, pp. 379–386from 22.12 ± 1.95 (15 min) to 40.23 ± 9.13% (60 min)and from 41.71 ± 0.75 (15 min) to 68.75 ± 6.11% (60 min),respectively. This increase in hemolytic effect remainedinferior to total hemolysis.Hemolytic effect of the plants studied at 60 min.Fig. 5 shows the evolution of the hemolytic effect or theleakage of Hb after 60 min for the A. longa, C. spinose,and M. communis extracts at four concentrations (25, 50,100, and 200 mg/mL) in a PBS buffer medium (pH 7.4)containing an erythrocyte suspension incubated at 37°C,compared to a negative control tube (PBS + suspension)and a total hemolysis tube (distilled water + suspension).TH: Total Hemolysis. NC: Negative Control. Themeans of 3 replicates. P < 0.01. ** significantThe M. communis species showed a significantly lowhemoglobin leakage rate compared to the other species,as well as a 20.14 ± 3.11% total hemolysis. This specieshad a lesser effect on the cell membrane of erythrocytes(P < 0.01). However, C. spinosa caused a significantintermediate leakage of hemoglobin, compared toM. communis and A. longa, at 200 mg/mL (60 min),namely in the range of 34.86 ± 5.06% (P < 0.01).Nevertheless, the most important cytotoxic effect onred cells was produced by the aqueous extract of theA. longa leaves, where the leakage rate was 68.75 ±6.11% at 200 mg/mL (60 min) and close to totalhemolysis (P > 0.01), indicative of the species’ hightoxicity. These results are phenotypically observable inthe supernatant.For millennia, humans have been searching for drugsin barks, seeds, fruit organs and other parts of plantsto heal themselves and alleviate pain [18]. Nowadays,several studies have been conducted on plants to createnew drugs and, to some extent, to evaluate their toxicityand identify their components. Polyphenolic compoundsof M. communis L. extracts are grouped in three majorchemical classes: phenolic acids, tannins, and flavonoids[19]. Our results of the phytochemical screening ofC. spinosa are consistent with those reported by Cherfiaet al., who identified polyphenols, flavonoids, alkaloids,tannins, and saponosides [20]. The Aristolochia speciesare a source of various active compounds such asaristolochic acid, alkaloids (aporphines, protoberberines,protopines), quinolines, amides, chlorophylls, terpenoids,lignans, flavonoids, tetralones, and steroids [21].The polyphenol and flavonoid contents that wefound in the M. communis leaves were higher thanthose obtained by Bouaziz et al., who reported157.70 ± 2.65 mg EAG/g MS and 2.64 ± 0.22 mg EQ/gof dry matter [22]. In another study, the hydromethanolicextract of C. spinosa leaves had a polyphenol content of228.42 ± 8.86 and a flavonoid content of 4.87 ± 0.12 [23].According to Djeridane et al., the methanolic extract ofA. longa contained 1.47 ± 0.20 mg/g EAG polyphenolsand 0.81 ± 0.02 mg/g EQ flavonoids [24]. Our resultswere in agreement with Merouani et al., who found396.88 ± 8.86 mg/g EAG polyphenols and 9.92 ±0.23 mg/g EQ flavonoids [25].Plants contain toxic compounds in high doses,which makes the evaluation of their hemolytic powerindispensable for their correct use in traditional therapy,as well as for choosing the right mode of administrationand preserving the integrity of membranes. Accordingto Haddouchi et al., the hemolysis test should beperformed even if a plant has a powerful antioxidantpower, since its use in traditional medicine and inpharmacological preparations will be impossible in thepresence of their hemolytic effect, which is an indicatorof cytotoxicity [16]. “Free radicals induce severaleffects on erythrocytes, such as hemolysis, fluidityof the membrane, changes in morphometry and lipidperoxidation, among others. Erythrocytes potentiallypromoting the oxidative process are extremely sensitiveto oxidative damage because of the polyunsaturatedfatty acid content in their cell membranes and their highcontent of oxygen and hemoglobin” [26].Many secondary metabolites were revealed inour extracts that may be a cause of cytotoxicity. Wefound a major lysis of red blood cells treated withC. spinosa, which was more prominent when treatedwith Aristolchia, testifying to severe toxicity. We foundFigure 5 Hemolytic effect of four concentrations of Myrtus communis, Calycotome spinose,and Aristolochia longa extracts at 60 min60 minHemolysis, %384Gadouche L. et al. Foods and Raw Materials, 2021, vol. 9, no. 2, pp. 379–386very few studies on A. longa and C. spinosa, trying toexamine a relationship between the extracts’ chemicalcomposition and toxicity.According to Bissinger et al., saponins – a secondarymetabolite identified in aqueous extracts of the plantsunder our study – may lead to the stimulation ofhemolysis as well as to suicidal erythrocyte death [27].Alkaloids are present in many plants which may be toxicand affect human health [28]. Mahdeb et al. reportedthat alkaloids are capable of disrupting the permeabilityof the membranes of erythrocytes [29].As stated by Galati and O’Brien, many adverseeffects were associated with dietary polyphenolconsumption or exposures such as hemolyticanemia [30]. The authors added that before using thesepolyphenols for therapy, they need to be assessed forsafety.According to Grollman et al., the toxicity ofAristolochia longa is due to a toxin that is a majorcomponent of all Aristolochia species, namelythe aristolochic acid responsible for nephropathicsyndromes, although the therapeutic use of Aristolochiahas rarely taken into account its intrinsic toxicitybefore [31]. These findings corroborate the study ofTouiti et al., which showed that Aristolochia longa wasincriminated in nephrotoxicity [32].CONCLUSIONSome herbs used in traditional therapy in highdoses can reveal toxic properties and harm humanhealth. It appears essential to determine their hemolyticcapacity as a marker of toxicity for rational adaptationto traditional therapy. We found that Aristolochia longaand Calycotome spinosa caused significant lyses ofred blood cells and a potent leakage of hemoglobin.Therefore, these species cannot be used withoutcontrol as a therapeutic or pharmacological tool totreat diseases. Furthermore, it is important to performantitumoral tests on cancer cells with these plantextracts or their chemical compounds to develop anticancerdrugs.CONFLICT OF INTERESTThe authors declare no conflict of interest.CONTRIBUTIONL. Gadouche conceived and designed the analysis,performed the biological experiments, and wrotethe paper. A. Zidane and K. Zerrouki contributed todata analysis and revised the paper. K. Azouni andS. Bouinoune performed the biological experiments. Allthe authors revised the manuscript for publication.