The Lawsonia inermis leaves were harvested in January 2020 at the National Center for Scientific and Technological Research (CNRST) botanical garden. A CNRST botanist authenticated the samples and the specimens, and the specimens were deposited under the reference number 8734. The samples were washed thoroughly with water, dried in the shade, and ground to powder with a mechanical grinder. The powders were put in freezer bags and stored away from light at room temperature for later use.

Seven ATCC microbial strains including two Gram-negative bacilli (Escherichia coli ATCC 25922; Pseudomonas aeruginosa ATCC 27653), three Gram-positive cocci (Staphylococcus aureus ATCC 25923; Streptococcus pyogenes ATCC 19615; Streptococcus agalactiae ATCC 13813) and two fungal strains (Candida albicans ATCC 90028; Candida tropicalis ATCC 750) were used. These microbial strains were purchased from LGC standard distributor ATCC in South Africa.

Mueller Hinton (MH) agar (Liofilchem, lot: 032720504) was used for the isolation and susceptibility testing of S. aureus and Gram-negative bacilli (E. coli and Pseudomonas aeruginosa). Chocolate agar (GC Medium: Liofilchem, lot: 071320502) + Isovitalex (GC + IVx) were used for the isolation and antibiogram of Streptococci (S. pyogenes and S. agalactiae). Sabouraud agar was used to isolate and test the susceptibility of fungal strains (C. albicans and C. tropicalis). MH (Liofilchem, lot: 011822506), Sabouraud (Liofilchem, lot: 07280501), and Brain Heart Broth (BHI) (Liofilchem, lot: 110420503) were used for the determination of Minimum Inhibitory Concentration (MIC).

All solvents used were of analytical grade. Ethanol, n-hexane, and ethyl acetate were purchased from CARLO ERBA (Val de Reuil Cedex, France). One-butanol and dichloromethane were purchased from Honeywell (Riedel-de Haën Chemicals Seeize GmbH, Germany). Dimethyl sulfoxide (DMSO) (≥99.7%), vanillin (≥ 99%), sodium carbonate, ferric chloride, aluminum chloride (≥ 98%), 1,3,5-Triphenyltetrazolium Chloride (TTC) and Folin-Ciocalteu from Sigma-Aldrich (Laborchemikalien GmbH, Germany); MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) from EMD (Millipore Corp MW 414.3, Lot 3727633; USA). All the following standards were purchased from Sigma-Aldrich (St. Louis): Gallic acid, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), Catechin, Quercetin, and Ascorbic acid. The water was of Milli-Q quality (Sartorius Arium^® Pro).

One hundred grams of sample powder was macerated at room temperature (25 ± 2˚C) for 24 h with 500 mL of 96˚ ethanol. During maceration, the mixture was shaken in closed flasks using a shaker (Modal T25 Digital Ultra Turrax) and filtered using a Buchner funnel and Whatman No.5 filter paper. The filtrate was concentrated under reduced pressure with an evaporator (Heidolph Rotacool, Allemagne Type: Laborota 4003) at 40˚C. The concentrated extract was dried at 40˚C in an oven, and the dry residue was weighed and stored at 4˚C until use.

The ethanolic extract was fractionated by liquid partitioning using a separatory funnel with organic solvents of increasing polarity: hexane, dichloromethane, ethyl acetate, and butanol. 5 g of each crude ethanol extract was initially dissolved in 25 mL of distilled water and extracted with 25 mL of each organic solvent (hexane, dichloromethane, ethyl acetate, and butanol successively). The experiment was carried out three times with each solvent. The organic phases were collected and concentrated at 40˚C using a rotary evaporator. Finally, the aqueous fraction was frozen and lyophilized. The different fractions were stored at 4˚C.

The phytochemical analysis of the ethanolic extract and its derived fractions was carried out using standard protocols and high-performance thin-layer chromatography (HPTLC).

Previous protocols [4] have been used to identify phytochemicals in plant extracts, including alkaloids, coumarins, flavonoids, tannins, triterpenes, steroids, saponosides, etc.

Phytochemical screening of L. inermis leaf extracts was carried out on 200 mm ⨯ 100 mm HPTLC 60 F254 silica gel plates (Merck, Darmstadt, Germany) according to the method described by Koala et al., [17] . Briefly, 5 µL of each sample was applied to the plate reference line (8 mm from the down border) using a semi-automatic plate spotter Linomat V (CAMAG, Muttenz, Switzerland). The distance between the two extracts on the reference line was 3.4 mm. The distances between the left and right edges of the plate were 20 mm. A continuous flow rate of 100 nL/s was used for the application. The plates were placed in a glass chamber previously saturated with 10 mL of mobile phase vapor for 30 minutes. The mobile phase in the twin troughs of the chamber’ consisted of:

Ethyl acetate-formic acid-acetic acid-water 100:11:11:26, v/v/v/v, for flavonoids, ethyl acetate-methanol-water-chloroform 18:2.4:2.1:6, v/v/v/v, for tannins, sterols, and triterpenes, n-hexane-ethyl acetate 20:4, v/v, for sterols and triterpenes and ethyl acetate-petroleum ether 10:20, v/v, for saponosides. The developed plates were then dried with a hairdryer and revealed by spraying Neu’s reagent for flavonoids, 2% iron III trichloride for tannins, Lieberman Burchard reagent for sterols and triterpenes and anisaldehyde sulphuric acid for saponosides. The developed plates were heated at 110˚C for five minutes for flavonoids, sterols, and triterpenes), at 100˚C for two minutes for tannins, and at 100˚C for five to ten minutes for saponosides.

Interpreting of results

The phytochemical profile of the ethanolic extracts of L. inermis was carried out on a gas chromatograph, Agilent 6890N, equipped with an autosampler coupled to an Agilent capillary column mass spectrometer, HP5 MS (30 m long, 0.25 mm diameter and 0.25 μm layer thickness). The system is controlled by a computer equipped with Chemstation data acquisition and processing software. Helium N60, with a purity of 99.9999%, was used as the carrier gas at a constant flow rate of 1 ml/min. The oven temperature was set at 70˚C for 5 min with increases of 20˚C/min to 120˚C for 5 min, 10˚C/min to 200˚C and 5˚C/min to 310˚C for the 5 min. An aliquot of 1µl of the sample was injected into the column at 270˚C in splitless mode. The ionization energy of 69.9 eV was used, and electron ionization is involved. The mass range is 30 to 550 atomic mass units (amu), the inlet line temperature was 270˚C and the source temperature was 230˚C. The execution time of the GC program was 42.50 minutes.

Identification of compounds

Molecular identification was performed using an internal library (National Institute Standard and Technique (NIST Version-Year 2004). The relative percentage of each component was calculated by comparing its average peak area with the total area.

Each sample’s total phenolic content (TPC) was determined using the Folin-Ciocalteu spectrophotometric method previously described by Koala et al. [17] with slight modifications. The reagent phosphomolybdate and sodium tungstate were reduced during the oxidation of phenols in alkaline media to a mixture of tungsten blue and molybdenum.

Briefly, 25 μL of diluted extract (1 mg/mL) was mixed with 125 μL of Folin-Ciocalteu reagent (FCR) solution (0.2 N). After 5 minutes of incubation at room temperature, 100 μL of a saturated sodium carbonate solution (7.5% in water) was added to the mixture. After 60 minutes of incubation in the dark at 37˚C, the absorbance of the resulting blue hue was measured using a SHIMADZU UV-Vis spectrophotometer at 760 nm. Using the calibration curve equation Y = 0.0516x + 0.0673, R² = 0.9936, the total phenolic content of the extract was calculated. Data are reported as milligrams of gallic acid equivalents (GAE) per gram of dry weight. Each measurement was performed in triplicate (n = 3).

The total flavonoid content (TFC) was determined using the aluminum trichloride method and quercetin as a reference compound according to previous methods [4] [17] . Briefly, the crude ethanolic extract was dissolved in methanol at room temperature at 1 mg/ml and filtered. Quercetin was used as a standard and a calibration curve was produced using concentrations ranging from 0.001 to 0.5 mg/mL of a quercetin solution. Then, 100 µL of the extract solution was mixed with 100 µL of a 2% methanol aluminum trichloride (AlCl₃) solution. After one hour of incubation at room temperature, the absorbance of the supernatant was measured at 415 nm using a spectrophotometer. The TFC of the extract was obtained by relating the absorbance reading to the standard curve equation y = 0.0151x + 0.0593, R² = 0.9992. The total flavonoid content was expressed as mg quercetin equivalents per gram of dry weight. All measurements were performed in triplicate (n = 3).

The identification and quantification of triterpenes, sterols and polyphenolic compounds from L. inermis leaf extracts were carried out using the high-performance liquid chromatography method equipped with an HPLC-DAD diode array detector (Agilent, model 1100 series; serial number DE23917296) as described by [21] . The separation was carried out with an analytical Eclipse X08-C18 column (250 × 4.6 mm; 5 µm, Agilent, USA). The diode array detector was set at 200 - 400 nm acquisition range.

The test was run in isocratic mode. The eluent consisting of 0.5% acetic acid (v/v) in methanol, acetonitrile, and water (70/25/5, v/v/v) was filtered through a membrane filter of 0.45 μm (Grosseron, ref: 0700817) and degassed by an ultrasonic bath before use. The sample extract and standard were dissolved in the mobile phase and also filtered as the eluent. Ursolic acid and three sterols were detected at 220 and 250 nm, respectively. The flow rate was 1 mL/minute, and the injection volume was 50 μL.

The eluent consisting of 1% acetic acid (v/v) in methanol, acetonitrile and water (40/15/45, v/v/v) was filtered and degassed as before. The diluted ethanolic extract, fractions and standard solutions were filtered through a 0.45 μm membrane. Flavonoids were detected at 365 nm, caffeic and sinapic acids at 327 nm and gallic, ellagic and vanillic acids at 271 nm. The flow rate was 0.5 mL/minute, and the injection volume was 10 μL.

Individual phenolic compounds were quantified using the calibration curves of the respective reference compounds. Twelves standards (Sigma-Aldrich, Berin, Germany), including a triterpene (ursolic acid, R² = 0.9987), three sterols (β-sitosterol, R² = 0.9998; stigmasterol, R² = 0.9914; campesterol, R² = 0.9998), three flavonoids (rutin, R² = 0.9928; quercetin, R² = 0.9994; and kaempferol, R² = 0.9978) and five phenolic acids (gallic acid, R² = 0.9842, vanillic acid, R² = 0.9982; ellagic acid, R² = 0.9959; cafeic acid, R² = 0.9961 and sinapic acid, R² = 0.9954) were used. For this purpose, each standard’s stock solutions (1000 µg/mL) was diluted to concentrations of 6.25 to 500µg/mL. The chromatographic peaks of the extracts were confirmed by comparing their retention times and UV spectra to those of the standards. The limit of detection (LOD) and limit of quantification (LOQ) were calculated based on the standard deviation of the original calibration curve values (σ) and slope (S) using three independent analytical curves according to ICH guidelines. The following equations were used: LOD = 3.3 σ /S, LOQ = 10 σ /S [21] .

The antimicrobial activity of Lawsonia inermis leaf fractions was assessed using the disk diffusion method and the microdilution method described by Youl et al. [4] .

Petri dishes containing 20 mL of appropriate agar medium were inoculated with an 18 - 24 h culture of bacterial strains from the nutrient broth. The inoculum was adjusted to approximately 10⁸ UFC/mL with sterile saline solution. The fractions were solubilized in DMSO at 100 mg/mL. Twenty-five microliters of each sample were applied separately to sterile filter paper disks (Whatman No 1; 6 mm in diameter), and placed on the surface of the inoculated medium. Antibiotics (ciprofloxacin 5 µg, erythromycin 15 µg, and nystatin 100 IU) were used as positive control and diluted DMSO as negative control. A swab-inoculated Petri dish was used as a control for bacterial growth. The inoculated Petri dishes were placed in the oven according to the strain incubation conditions ( Table 1 ). All tests were performed in triplicate.

The microdilution method (NCCLS, 2000) on a 96-well microplate was carried out for fractions presenting an inhibition zone ≥ 10 mm. Fresh colonies of each test strains were inoculated into sterile MH, Sabouraud and Brain Heart Infusion (BHI) broths and incubated according to the growth conditions of each strain. The overnight culture was appropriately diluted in broth to obtain a viable count of approximately 10⁶ UFC/mL. Each fraction’s dilution (0.025-25 mg/mL) was prepared from stock solutions. To each well of the microplate containing 100 µL of inoculum, 100 µL of diluted fraction was added.

DMSO (1/5) and Tween 80 (1/10) were used as negative controls; the growth of each strain (inoculum alone without fraction) was also monitored throughout the experiment. The microplates covered with their lids were packed in plastic bags and incubated (5% CO₂, 37˚C) for 24 to 48 hours. Then, 25 µL of triphenyltetrazolium chloride (TTC) or MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution at 2 mg/mL was added to each well and the plates were incubated at 37˚C. The indicator solution changes from clear to pink (TTC) or from yellow to purple or dark blue (MTT).

The indicator solution changing from clear to pink (TTC) or yellow to purple or dark blue (MTT) respectively attests to bacterial or fungal activity; however, it remains clear when microbial growth is inhibited.

The MIC was defined as the lowest fraction concentration which gave visible inhibition of the growth of the test strain. The MIC was defined as the lowest fraction concentration giving visible inhibition of the growth of the test strain. The minimum bactericidal concentration (MBC) was determined by spreading 10 µL of contents of transparent wells showing no visible growth onto appropriate agar plates and incubating according to the required conditions. The MBC was defined as the lowest extract concentration at which 99.99% of the bacteria present in the inoculum were killed. These concentrations correspond to less than 100 CFU per 10 µL of the contents of the well at concentrations below the MIC. The tests were performed in triplicate on the same microplate for each sample, and the experiment was repeated three times.

Bacterial sensitivity to fractions was interpreted based on the criteria established by Ponce et al. [22] and their and their efficacy was assessed based on the scale proposed by Kuete [23] .

An inhibition diameter ≥10 mm on the microorganisms tested: fraction selected for the microdilution test.

The BMC/MIC ratio was used to classify the fractions [24] :

The strain is considered tolerant to the extract if the BMC/MIC ratio is ≥ 32.

The results of the qualitative phytochemical screening of the crude ethanolic extract of L. inermis are summarized in Table 2 and Figure 1 . The presence of steroidal and triterpene glycosides (saponosides), tannins, reducing compounds, anthraquinones and flavonoids was noted in the ethanolic extract of the plant ( Table 2 ). Anthocyanins, alkaloids and cardenolides were not detected.

Under UV/366 nm and white lights, the high-performance thin-layer chromatography profile ( Figure 1 ) showed many different colored spots (orange, yellow, blue, green, pink, purple) which were revealed using specific indicators, as indicated in the methodology.

Sterols, Triterpenes and saponosides

L. inermis leaves contained sterols and triterpenes including oleanane, ursane and lupine types ( Figure 1(a) and Figure 1(b) ) and saponosides [17] .

Flavonoids

Favonoids including flavonols flavones, ethylated flavone, isoflavones, flavanones chalcones flavanols and aurones ( Figure 1(c) ) were highly present in the ethanolic extract of the leaves of L. inermis.

Tannins

Figure 1(d) presents the chromatographic profile of the plant’s tannins (condensed and hydrolysable tannins).

Li: 96% ethanolic extract of Lawsonia inermis leaves. (-) not detected, (+) presence.

Table 3 and Figure 2 show the phytochemical profile of the crude ethanolic extract of L. inermis by GC-MS. Twenty-seven peaks corresponding to various phytochemical compounds were identified. The proportion and retention time of compounds were also given.

Mw: Molecular weight, RT: Retention time.

The total phenolic (TPC) and flavonoids (TFC) contents of L. inermis leaves are presented in Figure 3 . TPC was expressed as gallic acid equivalent (mg of gallic acid equivalent per g of extract in dry weight (mg GA/g DW)) while TFC was expressed in mg of quercetin equivalents/g of dry weight of extract (QE/g DW). The results were 84.53 ± 0.3 mg GAE/g DW and 40.47 ± 0.21 mg QE/g DW for TPC and TFC, respectively.

Triterpene and sterol compounds

The triterpene and sterol compounds identified and quantified by HPLC-DAD are presented in Table 4 . Ursolic acid and β-sitosterol were the most detected. The crude ethanol extract of leaves (FeLi), hexane fraction (FHLi) and dichloromethane fraction (FDLi) of L. inermis contain ursolic acid (around 43.14 g/100g DW, 13.9 g/100g DW and 0.68 g/100g DW), respectively. In addition, β-sitosterol is detected in FeLi (56.7 mg/100g DW), FHLi (10.55 g/100g DW), FDLi (106.1 mg/100g DW) and butanolic fraction (FBLi) (357.4 mg/100g DW). Stigmasterol and campesterol were only detected in FDLi (0.95 g/100g DW) and FeLi (24.16 mg/100g DW) respectively.

Phenolic compounds

The results of the quantitative analysis of polyphenols compounds of ethanolic extracts of L. inermis leaves are presented in Table 5 . The ethanolic leaf extracts and its derived fractions contained phenolic acids such as caffeic acid, gallic acid, vanillic acid and ellagic acid. Flavonoids were also detected in the ethanolic extract (Rutin = 3.24 g/100g and Quercetin = 0.63 g/100g); in the dichloromethane fraction (FDLi, (Rutin = 15.73 g/100g) and in the aqueous fraction (FAqLi (Rutin = 227.06 mg/100g). All extracts showed their richness in phenolic acids and the highest amounts (caffeic acid = 37.65 g/100g DW, gallic acid = 8.62 g/100g DW, vanillic acid = 22.70 g/100g DW and ellagic acid = 8.04 g/100g DW) were recorded with the ethyl acetate fraction (FAeLi).

Values are presented in mean ± standard deviation (n = 3); Nd = no detected, FeLi: 96% ethanolic extract of L. inermis leaves, FHLi: hexane fraction, FDLi: dichloromethane fraction, FAeLi: ethyl acetate fraction, FAqLi: aqueous fraction, FBLi: butanolic fraction.

Values are presented in mean ± standard deviation (n = 3); nd = no detected, FeLi: 96% ethanolic extract of L. inermis leaves, FHLi: hexane fraction, FDLi: dichloromethane fraction, FAeLi: ethyl acetate fraction, FAqLi: aqueous fraction, FBLi: butanolic fraction.

Five fractions from the ethanolic extract of L. inermis were tested for their antimicrobial activity against a panel of Gram-positive cocci, Gram-negative bacilli and fungi. The antimicrobial activity expressed by the diameters of the inhibition zones and the minimum inhibitory concentrations are summarized in Table 6 and Table 7 . The hexane (FHLi), dichloromethane (FDLi), ethyl acetate (FAeLi) and butanol (FBLi) fractions displayed significant antibacterial activity against microorganisms (Ø ranged from 8 mm to 24 mm) ( Table 6 ). The most active ethyl acetate and dichloromethane fractions showed broad-spectrum antimicrobial activities against P. aeruginosa, Gram-positive cocci and fungi. FHLi inhibited only fungal strains at 100 mg/mL (Ø = 11 ± 0 mm and 20 ± 1 mm) from a standardized inoculum of 10⁸ CFU/mL of C. albicans and C. tropicalis, respectively. FBLi, on the other hand, only inhibited Gram-positive cocci with inhibition zone diameters of 11 ± 0.52 mm (S. aureus), 12 ± 0.52 mm (S. agalactiae) and 13 ± 0.67 mm (S. pyogenes).

By the microdilution method, FDLi was the most effective with a MIC value of 8.4 mg/mL, 0.52 mg/mL, 0.16 mg/mL and 0.23 mg/mL on standardized inoculum (10⁶ CFU/mL) of P. aeruginosa, S. aureus, S. agalactiae and S. pyogenes, respectively. FAeLi showed moderate to low activity on the strains tested (MIC varying from 0.33 mg/mL to 4.1 mg/mL). Additionally, FHLi exhibited moderate to weak fungicidal activity against C. tropicalis and C. albicans, respectively; with no growth at concentrations of 0.66 mg/mL and 1.02 mg/mL for the two strains. Gram-positive cocci were the most sensitive (lowest MIC) to L. inermis extracts and fractions.

Values are presented in mean ± standard deviation (n = 3); NC: negative control (DMSO 1/5); PC: positive control (Erythromycin 15 µg for gram-positive cocci; Ciprofloxacin 5µg for gram-negative bacilli and Nystatin 100UI for fungi); FHLi: Hexane fraction of leaves; FDLi: Dichloromethane fraction of leaves; FAeLI: Ethyl Acetate fraction of leaves; FBLi: Butanol fraction of leaves; Pa: Pseudomonas aeruginosa; Ec: Escherichia coli; Sa: Staphylococcus aureus; Sp: Streptococcus pyogenes; Sag: Streptococcus agalactiae; Ca: Candida albicans; Ct: Candida tropicalis.

FHLi: Hexane fraction of leaves; FDLi: Dichloromethane fraction of leaves; FAeLI : Ethyl Acetate fraction of leaves; FBLi : Butanol fraction of leaves; Pa: Pseudomonas aeruginosa; Ec: Escherichia coli; Sa: Staphylococcus aureus; Sp: Streptococcus pyogenes; Sag: Streptococcus agalactiae; Ca: Candida albicans; Ct: Candida tropicalis.