Anti-biofilm, anti-quorum sensing potential, cytotoxicity, and UPLC-UV/DAD- MS/MS/QTOF profiling of Prosopis africana (Guill. & Perr.) Taub. leaves and stems: benefits of a traditional medicine in dental care

Anti-biofilm, anti-quorum sensing potential, cytotoxicity, and UPLC-UV/DAD- MS/MS/QTOF profiling of Prosopis africana (Guill. & Perr.) 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Taub. leaves and stems: benefits of a traditional medicine in dental care Bance Alimata, Rouamba Ablassé, Compaoré Moussa, Compaoré Eli, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7348474/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Background Prosopis africana (Guill. & Perr.) Taub. is traditionally used in folk medicine in Burkina Faso for oral diseases. Leaves and stems are used in rural areas to treat dental caries, and the bark is used to treat green diarrhea in infants. In the context of a better understanding of the herbal drug and its bioactivity or toxicity, the present study deals with the chemical profiling of the different botanical parts used in phytomedicine. The impact of herbal medicine on various factors contributing to oral infections and caries, and specifically as anti-biofilm and anti-quorum sensing properties has also been little investigated. Methods The anti-biofilm effect of methanolic extracts of leaves and stems of P. africana was evaluated on Streptococcus mutans, Staphylococcus aureus, and Pseudomonas aeruginosa , as well as the anti-quorum sensing effect on Chromobacterium CV026 and Pseudomonas aeruginosa . The cytotoxicity of the leaves and stems extracts was also evaluated. The chemical composition of the extracts was characterized by UPLC-UV/DAD-MS 2 /ESI-QTOF analysis. Results The extracts (100µg/ml), without affecting cells viability, significantly reduced the biofilm formation of S. mutans with the best inhibition rates of 56.7% and 47.6% for stem and leaf extracts respectively. Inhibition rates of 49.03%, 40.2%, and 46.7% were obtained for pyocyanin, elastase, and rhamnolipids respectively, with leaf extracts. No cytotoxic effect on gingival cells was observed for stem extract. UPLC-UV-MS 2 analysis identified sixteen compounds among which mainly polyphenols and alkaloids. They could be related to the activities. Conclusion The present study provides evidence of efficacy and basic scientific justification for the use of P. africana in the treatment of dental caries. Prosopis africana UPLC-UV/DAD-ESI-MS/MS anti-biofilm anti-quorum sensing dental care polyphenols alkaloids Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Microbial communities living in an extracellular polymeric matrix that they produce themselves, called bacterial biofilms, are responsible for over 60% of microbial infections in humans. The transformations in gene expression and the activation of numerous extracellular communication pathways during biofilm formation often result in increased pathogenicity and bacterial virulence. [ 1 ]. Surprisingly, the antimicrobial resistance of a biofilm becomes thousand times higher than a free planktonic biota. Quorum Sensing (QS) is a mode of bacterial communication between species and intra-species, based on the production of small mediator molecules called autoinducers, which are produced during bacterial growth. QS is the mechanism used by cariogenic Gram-negative and Gram-positive bacteria to synchronize and regulate the expression of several genes depending on cell density [ 2 ]. Physiological processes that affect the production of virulence factors [ 3 ], can be characterized by the emission of bioluminescence [ 4 ], the production of pigments [ 5 ], the formation of biofilm and motility [ 6 , 7 ]. In Gram-negative bacteria, QS involves the production of small signaling molecules called N-acyl homoserine lactones (AHLs) [ 8 ]. AHLs are generally produced by an enzyme, a synthase, and diffuse passively across the cell membrane [ 9 ]. When their concentration in the external environment reaches a critical threshold, generally at high cell density, AHLs invade the cell, bind and activate specific intracellular receptors [ 10 ]. Activated receptors bind to promoters of targeted genes and regulate their transcription [ 11 ]. QS and biofilm are therefore linked because QS controls the formation and maturation of biofilm in several types of bacteria like Pseudomonas aeruginosa PAO1 [ 12 ]. A buccal biofilm contains hundreds of different oral bacteria responsible of serious diseases in the oral cavity. In addition, virulence in response to drastic changes in the biofilm's microenvironment can be spread systemically and cause significant infections in other organs. [ 13 ]. The presence of Streptococcus mutans, Staphylococcus aureus , and Pseudomonas aeruginosa in supragingival and subgingival biofilm can cause dental caries. Dental plaque, composed of Gram-positive and Gram-negative bacteria, can adhere to the surface of teeth, proliferate, and produce lactic acid, leading to the demineralization of enamel and dentin. The limitations of conventional antibiotic therapy, as well as the increasing drug resistance, have led to an urgent need to find other approaches to treat infections related to oral biofilm. Previous studies have shown that plant drugs or molecules isolated from medicinal plants have properties that inhibit dental biofilm formation. These properties include a reduction in the adhesion of bacteria to dental plaque, a key step in the initiation and progression of dental caries. Prosopis africana (Guill. & Perr.) Taub (fabaceae) is a tree widely used in Burkina Faso to treat traditionally oral and dental infections. Ethnomedicinal surveys conducted in the south-central region reported the use of stems and leaves of P. africana in the treatment of dental caries. Also, the barks are used to treat diarrhea in young children. Antibacterial properties against Escherichia coli, Staphylococcus aureus, Streptococcus mutans, Klebsiella have also been reported [ 14 , 15 ]. However, its anti-biofilm and anti-quorum sensing properties have been poorly investigated. We report here the anti-biofilm, the anti-quorum sensing activity, and the cytotoxicity of the methanolic leaves and stem bark extracts of Prosopis africana. Material and Methods Plant material and extraction A collection guided by traditional practitioners in the province of Zoundweogo (11° 34'60''N; 1°0'00'') allowed to harvest bark and leaves of Prosopis africana . The identification of the plant material was carried out by Dr. Souleymane COMPAORE junior researcher of botany from the plant biology and ecology laboratory of the Life and Earth Sciences Training and Research Unit (UFR/SVT) of the Université Joseph KI-ZERBO. The wild plant was harvested on public land. A herbarium was deposited at the Herbarium of the UFR/SVT under the identification code No. 6852. The barks and stems were dried under laboratory conditions before to pulverize. The powders were kept cool at 4°C for the subsequent extraction of the plant drug. One hundred grams (100 g) of the leaves and stems powders were macerated in one liter of methanol (MeOH) during 24 hours. The methanolic maceration was filtered using Whatman No. 1 paper and concentrated under reduced pressure. The concentrate was placed in the Speedvac (SpeedVac ThermoFischer®) until complete dryness to yield two crude methanolic extracts (Leaves Methanolic Extract (LME), Stems Methanolic Extract (SME). Bacterial Strains The bacteriology laboratory of the Muraz Center in Bobo Dioulasso (Burkina Faso) and the plant biotechnology laboratory of the Université Libre de Bruxelle (Belgium) respectively provided the standard strains ( Streptococcus mutans ATCC 25175 and Staphylococcus aureus ATCC 43300 methicillin-resistant) and the wild strains ( Pseudomonas aeruginosa PAO1 and Chromobacterium vio-laceum CV026). S. aureus and S. mutans strains were stored at -80°C in Brain Heart Infusion liquid medium while P. aeruginosa and C. violaceum strains were inoculated in Luria Bertani medium containing 50% glycerol until use. . Determination of the minimum inhibitory concentration (MIC) The minimum inhibitory concentration of crude methanolic extracts (Leaves Methanolic extract (LME), Stems Methanolic extract (SME)) was determined using the 96-well microplate method [ 14 ]. A range of extracts was carried out on a sterile microplate from a stock concentration of 4 mg/mL dissolved in 5% DMSO to get a final volume of 100 µL for each well. Then, 100 µL of the prepared inoculum in the corresponding culture medium were added to each well. DMSO medium (2.5%) was used as DMSO growth control and negative medium control, and DMSO-free medium as a bacterial growth control and negative medium control. The microplates were then incubated at 37° C for 24 h. At the final point 30 µL of iodonitrotetrazolium (INT) (2 mg/mL) were added to each well. 30 min after wereition of INT, MIC of the extracts was deduced regarding the color changes observation. Pink staining of INT indicates the presence of microbial growth in the wells. Indeed, the MIC is defined as the concentration of the first well of the range of dilutions devoid of bacterial growth [ 16 ] Determination of the minimum bactericidal concentration (MBC) The minimum bactericidal concentration of an extract or a pure compound corresponds to the lowest concentration capable of killing more than 99.9% of the initial bacterial inoculum ( i.e. less than 0.01% of survivors). The range of concentrations produced to determine the MIC in a liquid medium was used to determine the MBC of the extracts. Samples in the control tube (2.5% DMSO) and each of the tubes devoid of bacterial growth were deposited in a "streak" on LB-agar/BHI-agar medium previously prepared and poured into Petri dishes. Plates were incubated for 24 h at 37°C. The MBC was deduced as being the first bacteria-free dish. The tests were carried out in triplicate [ 16 ]. Inhibition of biofilm formation The anti-biofilm activity of the crude methanolic extracts (LME, SME) was evaluated according to the method previously described [ 17 ]. For this purpose, sterile 96-well microplates were used. Extracts and reference (salicylic acid) were prepared at the concentration of 1 mg/mL in DMSO (1%) to have a final concentration of 100 µg/mL in each well. After incubating the plates during 24 h at 37°C, absorbances at λ600 nm were read to ensure that the extracts did not affect bacterial growth compared to the negative control (1% DMSO). Planktonic bacteria were removed with the supernatant, and the biofilms were thoroughly washed three times with distilled water and 2ml of methanol were used to fixed the adhered bacteria. After 15 min of incubation at 37°C, the methanol was removed, and the formed biofilm was quantified with crystal violet. Crystal violet (0.1% in water) was added to each well (200 µL/well) for 30 min at room temperature. The excess of crystal violet was removed, and 1 ml of distilled water was used to wash the stained biofilms. The crystal violet fixed by the biofilm bacteria was solubilized with acetic acid (33%) and the absorbance was measured at λ590 nm. The biofilm/bacterial growth ratio (OD 590 nm/OD 600 nm) was determined. The percentage of inhibition was calculated using the following formula: Inhibition (%) = [(Abs control – Abs extract)/Abs control] x100 Anti-quorum sensing activity Violacein production inhibition test by Chromobacterium violaceum CV026 Inhibition of violacein production in Chromobacterium violaceum CV026 by all crude extracts (LME, SME) was tested by direct assay of culture medium according to protocols reported Choo et al. (2006). Violacein production was induced in C. violaceum CV026 by adding N-hexanoyl-L-homoserine lactone (HHL) (5 µM final concentration) in the presence or absence of the extracts (100µg/mL final concentration). C. violaceum CV026 was first grown in the Luria Bertani medium for 24 h (30°C ;175 rpm) and the diluted CV026 was next introduced into 12-well plates. The extracts or the reference salicylic acid dissolved in DMSO 1% were added to have a final concentration of 100 µg/mL, in the presence of C6-HSL (final concentration 10 µM). Plates were incubated for 24 h at 30°C with shaking at 175 rpm. Bacterial growth was first evaluated by measuring bacterial turbidity at 600 nm and the produced violacein was then quantified using a spectrophotometer. Briefly, the solution was vigorously vortexed for 30 S to solubilize the violacein and the supernatant was removed. The pellet was dissolved using 100% DMSO. The violacein contained in the supernatant was recovered by centrifugation (7000 rpm; 10 min) and 200 µL were introduced into microplate wells for measuring the absorbance at 585 nm. The percentage of inhibition was calculated against the negative control (DMSO 1%) (18). The ratio of OD 585 nm to OD 600 nm was determined as: Inhibition (%) = [(Abs control – Abs Extract)/Abs control] x100 Pyocyanin production inhibition test in Pseudomonas aeruginosa PAOI The pyocyanin production inhibition assay was performed for the crude methanolic extracts (LME, SME). After 18 h incubation in a 24-well plate at 37°C with shaking at 175 rpm, the bacterial suspension was gently homogenized and 800 µl was transferred to a 1.8 mL Eppendorf tube, were then centrifuged at 16000 × g for 1 min. 200 µL of the remaining suspension was directly transferred to a flat-bottom microtiter plate to measure absorbance at 600 nm, corresponding to the turbidity of the suspension. Then, 700 µL of supernatant from each sample after centrifugation was taken and transferred to 2 mL Eppendorf tubes; 600 µL of chloroform was added to each tube and the mixture was vortexed for a few seconds to extract pyocyanin. After centrifugation, two phases were formed and 500 µL of the lower phase, which contains pyocyanin, was transferred to a new 1.8 mL Eppendorf tube. Then, 250 µL of a 0.2 N hydrochloric acid solution was added to each tube to extract again the pyocyanin. The use of an acidic phase results in a color shift of pyocyanin from turquoise blue to pink. In nature, pyocyanin was blue, but it has been shown that in an acidic medium, it changes its structural configuration and turns pink [ 19 ]. After centrifugation, two phases appear again with pyocyanin in the upper phase. Finally, 200 µL of this upper phase was recovered and the absorbance of this solution was measured at 360 nm to quantify the pyocyanin produced by the bacteria [ 18 ]. The experiments were repeated three times (n = 3). The percentage of inhibition was calculated by the following formula: Inhibition (%) = [(Abs control – Abs Extract)/Abs control] x100 Elastase inhibition test in Pseudomonas aeruginosa PAO1 The elastase inhibition test was performed with the methanolic crude extracts (LME, SME) and fractions. Elastase activity performed on P. aeruginosa PAO1 in the absence or presence of extract was measured by the Elastin-Congo Red method [ 20 ]. This method was based on the properties of the elastase produced by the bacteria to degrade the elastin. P. aeruginosa PAO1 was incubated in LB medium with extracts (final concentration of 100 µg/mL) for 18 hours (37°C; 175 rpm) and the tubes were centrifuged for 5 min (3000 ; 24°C). Two hundred and fifty microliters (250 µL) of congo red elastin at 5 mg/mL dissolved in Tris-HCl buffer (0.1 M Tris-HCl pH 8;1 mM CaCl 2 ) was added to 750 µL of supernatant contained in each Eppendorf tube. The mixture was incubated at 37°C for 16 h at 200 rpm. During this incubation phase, elastin degradation results in solubilization of congo red, yielding a red solution while the undegraded Elastin-Congo Red complex was insoluble in water [ 18 ]. The reaction mixture was centrifuged at 3000 g for 10 min to separate the non-soluble portion and the absorbance of 200 µL of supernatant was measured at λ495 nm to estimate the elastin activity [ 21 ]. The percentage of inhibition was calculated as follows: Inhibition (%) = [(Abs control – Abs Extract) / Abs control] x100 Rhamnolipid inhibition test in Pseudomonas aeruginosa PAO1 The rhamnolipid inhibition test was performed with crude methanolic extracts (LME, SME). For rhamnolipid extraction, P. aeruginosa PAO1 grew at 37°C with shaking at 175 rpm for 18 h in a 5 mL LB medium in the presence of extract (100 µg/mL) or DMSO (1%). Bacterial cultures were then collected in Eppendorf tubes and centrifuged (3000 g at room temperature for 5 min). One milliliter of bacteria-free supernatant was recovered and filtered through an Ultrafree® CL-Millipore filter, and the pH of the supernatant was adjusted to 2.3 ± 0.2 with HCl 1N. The rhamnolipids were extracted from the supernatant with 4 mL of ethyl acetate (EtOAc) on the vortex for a few seconds. After centrifugation, the upper phase, which contains the rhamnolipids, was transferred to a new 30 mL tube. This extraction procedure was repeated three times. All the collected organic solvents were finally removed by evaporation under vacuum centrifugation. Quantification of rhamnolipids was performed by the methylene blue complexation method according to the literature with some modifications [ 22 ]. Briefly, 4 ml of chloroform were used to dissolve the rhamnolipid produced by the bacteria in each extract treatment and contacted with a methylene blue solution consisting of 200 µL of 1 g/L methylene blue reagent and 4.9 mL of distilled water that was adjusted to pH 8.6 ± 0.2 by adding 50 mM borax buffer (15 µL). After vigorous shaking for 4 min, the mixture was allowed to stand for 15 min to allow complexation of methylene blue to rhamnolipids. One milliliter of the lower chloroform phase turning blue was transferred to a new Eppendorf tube and vortexed with 500 µL of 0.2 N HCl, allowing the transfer of the complex methylene blue into the acid phase. Finally, 200 µL of the acidic phase was transferred to a 96-well microplate and the absorbance measured at λ638 nm [ 18 ]. The percentage of inhibition was calculated by the following formula: Inhibition (%) = [(Abs control – Abs sample) / Abs control] x100 Cytotoxicity Assay The cytotoxicity study was performed for LME and SME. Gingival cells were grown and maintained in a humidified incubator with 5% CO 2 at 37°C in Petri dishes in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum, 1% penicillin-streptomycin, and 1% L-Glutamine [ 23 ]. Cultures were subjected to medium changes and passages or transplants when cells were at the confluence. All manipulations were performed under highly sterile conditions in a laminar flow hood. After observation under a reverse-phase microscope (Jenco USA), gingival cells were seeded on sterile 96-well microplates in 100 µL of complete culture medium at 2.104 cells/well. The microplates were then incubated for 24 h to ensure adequate cell adhesion. The crude methanolic extracts from the leaves and stems were dissolved in DMSO and diluted in the culture medium to a concentration of 1% DMSO. A range of dilutions was performed for concentrations from 500 µg/mL to 15.625 µg/mL. 100 µL of each extract was then contacted with the cells in the wells and the plates returned to incubation for 72 h. The blank contained only the culture medium and the control contained cells with the culture medium, without extract. The cell suspension in the presence of hydrogen peroxide (H 2 O 2 ) (50 µL, 300 mM) served as the positive control, and 1% DMSO served as the negative control. Phytochemical Analysis: Ultra-High-Performance Liquid Chromatography Coupled with High-Resolution Tandem Mass Spectrometry (UHPLC/MS-MS) Extracts were analyzed using Agilent Technologies Accurate-Mass Q-TOF LCMS 6530, with LC 1290 Infinity system. The separation was carried out at 40°C using a 120 EC-C18 column (3.0*100 mm*2.7 µm; Agilent Poroshell). Each sample (10 mg. mL − 1 ) was injected (3 µL), and the column was eluted at 0.7 mL.min − 1 with a solvent gradient using solvent A (water with formic acid 0.4% (v/v)) and solvent B (acetonitrile). The proportion of solvent B increased, step by step, from 10 to 36% during 4.5 min; 36% to 100% during 4 min, followed by 2 min isocratic phase with 100% solvent B; back from 100% to 10% in 0.5 min and equilibration at 10% solvent B during 3 min until the end of the run at 14 min. Mass analyses were made in positive and negative modes, with the nebulization gas (Nitrogen) at a flow of 10 L.min − 1 and 40 psg pressure. The capillary tension was 3000 V and gaz ionization energy of 100 eV. Analyses included QC (Qualitative Control) and blank samples, which were regularly injected between runs, giving a total of 8 blanks and 8 QCs. QC was prepared by mixing 100 µL of each sample prepared at 10 mg. mL − 1 . Moreover, to facilitate the identification of specific compounds samples were analyzed with tandem mass spectrometry (MS/MS). MS/MS analyses were performed by collision-induced dissociation (CID) with a collision energy of 20 eV. Chromatograms were explored with MassHunter Qualitative Analysis B.07.00 software (Agilent® Technologies), and the Semetl1.0.2020.11.10 version 2 in-house database containing the analyzed by UHPLC-MS/MS of 400 commercial standards in positive and negative mode. Compounds identifications were performed by comparison of experimental data to that of SemetI database (in house database) and to the existing literature. Statistical analysis The statistical analysis was performed by using Graph Pad Prism version 6 software. The results were expressed as mean of three independent repeated experiments ± Mean Standard Error. One-way analysis of variance (ANOVA) followed by the post-test Dunnett’s was used to measure the degree significance of the results. The extracts were compared with the negative control and the value of p < 0.05, p < 0.01, p 0.250 mg/mL) and MBC (> 1mg/mL) values evaluated allow the selection of a sub-inhibitory concentration for the bioassay on all strains (Table 1 ). Table 1 Minimum inhibitory concentration and minimal bactericidal concentration of methanolic extract of leaves and stem bark of Prosopis Africana. Extracts LME (mg/mL) SME (mg/mL) Strains MIC MBC MIC MBC Streptococcus mutans ATCC 25175 0.5 1 1 > 1 Staphylococcus aureus ATCC 43300 1 > 2 1 > 2 Pseudomonas aeruginosa PAOI 0.5 1 2 > 2 Chromobacterium violaceum CV 026 1 > 2 0.5 1 MIC: Minimum inhibitory concentration, MBC: minimal bactericidal concentration, LME: methanolic extract of leaves, SME: stem bark Inhibition of biofilm production As shown in Fig. 1 , the methanolic extracts from the leaves and stems of P. africana , at a concentration of 100 µg/mL affected neither the viability nor the growth of Pseudomonas aeruginosa PAO1, Staphylococcus aureus, Streptococcus mutans. Overall, compared to salicylic acid (SA), all extracts significantly reduced biofilm formation of Pseudomonas aeruginosa PAO1 with respective inhibition percentages of 32.8 ± 5.3 for LME and 36.3 ± 4.6. for SME. Concerning Staphylococcus aureus , biofilm formation was inhibited with respective inhibition percentages of 31.8 ± 1.9% for LME; 40.4 ± 4.6% for SME. For Streptococcus mutans , biofilm formation was inhibited with respective inhibition percentages of 47.6 ± 0.8% for LME and 56.7 ± 0.6% for SME. Effect of extracts on violacein production in Chromobacterium violaceum CVO26 As shown in Fig. 2 , methanolic extracts of P. africana significantly reduced violacein production compared to 1% DMSO. At the final concentration of 100 µg/mL, the extracts did not affect either viability or bacterial growth of C. violaceum CV026. For LME, the inhibition of violacein was 37.9 ± 3.7% and for SME the inhibition was 42.6 ± 1.4%. Effect of extracts on pyocyanin, rhamnolipid, and elastase production in P. aeruginosa PAO1 The effect of P. africana extracts at the final concentration of 100 µg/mL was evaluated on the production of pyocyanin, elastase, and rhamnolipids (Fig. 3 ) which are the main virulence factors excreted by P. aeruginosa PAO1 and whose production was under the control of the QS in dental biofilm. As shown in Fig. 3 , the extracts at the final concentration tested of 100 µg/mL significantly inhibited the production of pyocyanin, and rhamnolipids compared to the control (DMSO 1%) without affecting bacterial growth. Pyocyanin inhibition was marked by methanolic leaf extract (LME 49.03 ± 0.03%). The reduction of elastase was marked by both extracts (LME 40.2 ± 0.05%; SME 39.6 ± 0.014). For rhamnolipids, the percentage of inhibition of methanolic leaf extract (LME) was 46.70 ± 0.02% and that of methanolic stem extract (SME) was 39.60 ± 0.01%. This reduction of virulence factors by the extracts implies a probable interference with the regulatory mechanism of pyocyanin production that depends on QS in dental biofilm formation. Cytotoxic effect of extracts on gingival cells The cytotoxic effect of the crude methanolic extracts of leaves and stems on gingival cell viability was determined after 72 h following the MTT colorimetric cytotoxicity test. The methanolic stem extract (SME) showed no adverse effect on gingival cell viability compared to the DMSO control (Fig. 4 ). In contrast, crude leaf extract (LME) had a significant cytotoxic effect on gingival cells compared to DMSO starting from 15.625 µg/mL (Fig. 4 ). Identification of compounds of interest by HPLC/MS-MS in methanolic extracts of leaves and stems of Prosopis africana Tables 2 and 3 provide informations on the characterized compounds in both methanolic extract LME and SME. Comparing chromatograms in both positive- and negative modes ESI–MS experiments, a first group of forteen polar compounds were detected between 2.00 and 7.00 min. They were better ionized in negative mode than in postive one. A second group was eluted between 7.00 and 13.00 min, corresponding to less polar molecules that were better ionized in negative mode. To complete identifications the UV spectra was also used. This data combination was compared to published data and to the in-house database SemetI. As an example, compound 11 in LME was identified as follows, and the same approach was used to characterized the chemical composition of both extracts. The UV spectrum showed the following maxima of absorption: 222, 260, 300sh et 348 nm suggesting a polyhydroxylated flavonoid like a 3- O -substituted myricetin derivative (Mabry et al ., 2012). The pos-ESI-MS spectrum exhibited a pseudo-molecular ion at m/z 465.0998 u corresponding to [M + H] + (Fig. 5 ). The ions at m/z 487.0848 [M + Na] + and 951.1756 u [2M + Na] + in ESI + , together with the ion in ESI - at m/z 464.0899 u [M-H] - confirmed a molecular weight of 464 uma. A fragment at m/z 319.0425 in ESI + (and m/z 316.0243 u in ESI - ) showed out the loss of 146 u fragment, corresponding to the loss of a desoxyhexosyl unit. Altogether these data confirmed the formula of C 21 H 20 O 12 (with a score of 99,64%) (Fig. 5 ). Compared to published data, this analysis led to a presumed identification of compound 11 to myricetin 3- O -rhamnoside or myricitrin (Sharifi-Rad et al. , 2019). The same method allowed us to identify thirteen other compounds in LME, among which 2,4,6-trihydroxyphenyl β-D-glucopyranoside, procyanidin A, catechin 7- O -glycoside, procyanidin dimer digallate, glucopyranosyl- O -tyrosine, myricetin-3- O -galactoside, myricitrin, phloridzin, and quercitrin. Most of the identified compounds were flavonoids. Similarly, SME (methanolic stem extract) was analyzed and nine compounds were characterized such as galloylquinic acid, digalloylshikimic acid, 3,4-digalloylquinic acid, 1,3-digalloylquinic acid, prosopinin, pinelic acid, and spectalin. Table 2 Identification by UHPLC-ESI-QTOF of the main compounds of the methanolic leaves extract (LME) of Prosopis africana N o Rt (min) UV (nm) m/z (M - H) − ΔM ppm m/z (M - H) − m/z MS 2 (fragment) m/z (M + H) + ΔM ppm m/z (M + H) + m/z MS 2 (fragment) Formule Annotation (SemetI) or proposal Confidence level a Reference 1 2.733 224, 286 303.0728 -2.13 141.0187 [M-H-C 6 H 10 O 5 ] − 305.0672 2.48 327.0672 [M + Na] + 631.1464 [2M + Na] + 143.0331 [M + H-C 6 H 10 O 5 ] + C 12 H 16 O 9 2,3,5-trihydroxyphenyl glucoside 2b [ 24 ] 2 3.260 224, 278–279 451.1244 0.41 289.0728 [M-H-C 6 H 10 O 5 ] − 453.1374 3.72 475.1199 [M + Na] + 291.0851 [M + H-C 6 H 10 O 5 ] + 273.0747 [M + H-C 6 H 12 O 6 ] + C 21 H 24 O 11 catechin 5- O or 7- O - glycoside 3 [ 25 ] 3 3.455 224, 276 593.1314 595.1433 3.67 617.1245 [M + Na] + 1211.2554 [2M + Na] + C 30 H 26 O 13 derivative of Procyanidin A 3 [ 26 ] 4 3.355 224, 276 593.1299 0.28 1187.2746 [2M-H] − 447.0706 [M-H-146] − 595.1425 2.50 617.1238 [M + Na] + 1211.2554 [2M + Na] + C 30 H 26 O 13 procyanidin derivative 3 [ 26 ] 5 3.763 224, 278 593.1305 -0.73 595.1430 - 617.1240 [M + Na] + . C 30 H 26 O 13 procyanidin derivative 3 [ 26 ] 6 3.947 223, 284 - - 325.0923 [M-H 2 O] − 163.0395 [M-H 2 O-C 6 H 11 O 5 ] − 344.1327 3.76 365.0617 [M + Na] + 183.0306 [M + H-C 6 H 11 O 5 ] + 165.0535 [M + H-C 6 H 11 O 6 ] + C 15 H 21 NO 8 Glucopyranosyl- O -tyrosine 2b [ 27 ] 7 4.177 223, 284 881.1936 -0.17 1764.3936 [2M] − 603.1352 ; 577.1352 451.1245 ; 440.0914 289.0714 883.2047 3.74 1788.3812 [2M + H + Na] + C 45 H 38 O 19 dimer procyanidin digallate – type B 3 [ 28 ] 8 4.367 224, 278 577.1351 0.09 498.1257 ; 435.1299 579.1472 4.32 C 30 H 26 O 12 procyanidin derivative 3 [ 26 ] 9 5.053 224, 262, 302 sh, 357 479.0827 0.86 - 481.0965 2.43 503.0782 [M + Na] + 983.1650 [2M + Na] + 319.0436 [M + H-C 6 H 10 O 5 ] + C 21 H 20 O 13 myricetin 3- O -hexoside 1 2a [ 26 ] 10 5.132 224, 258, 304 sh, 354 479.0825 1.28 316.0221 [M-H-C 6 H 10 O 5 ] − 481.0979 -0.49 503.0796 [M + Na]+ 983.1667 [2M + Na] + 319.0445 [M + H-C 6 H 10 O 5 ] + C 21 H 20 O 13 myricetin-3- O -galactoside 2 (isomer) 2a [ 26 ] 11 5.627 222, 260, 300sh, 348 463.0872 1.95 317.0636 [M-H-C 6 H 10 O 4 ] − 465.1023 1.08 487.0834 [M + Na]+ 651.1789 [2M + Na]+ 319.0427 [M + H-C 6 H 10 O 4 ] + C 21 H 20 O 12 myricitrin 2b [ 29 ] 12 6.05 224, 280 435.1305 -1.73 313.0931 [M-H-C 8 H 9 O] − 273.0774 [M-H-C 6 H 10 O 5 ] − 151.0401 ; 121.0292 - - - C 21 H 24 O 10 phloridzin 2a [ 29 ] 13 6.427 256, 346 447.0949 -3.93 301.0357 [M-H-C 6 H 10 O 4 ] + 449.1066 2.4 919.1845 [2M + Na]+ 303.0490 [M + H-C 6 H 10 O 4 ] + C 21 H 20 O 11 quercitrin 2b [ 30 ] a according to Schymansky et al., 2014. Table 3 Identification by UHPLC-ESI-QTOF of the main compounds of the methanolic stems extract (SME) of Prosopis africana . N° t R (min) UV (nm) m/z (M - H) − ΔM ppm m/z (M - H) − m/z MS 2 (Fragment) m/z (M + H) + ΔM ppm m/z (M + H) + m/z MS 2 (fragment) Formula Annotation or proposal Confidence level a References 1 1.905 222. 272 343.0677 -1.87 687.1442 [2M-H] − 345.0810 0.98 153.,0171 [M + H-C 7 H 12 O 6 ] + 327.0683 122.0222 C 14 H 16 O 10 galloylquinic acid 3 [ 31 ] 3 3.360 222, 274 495.0810 -3.5 343.0683 [M-H-C 7 H 5 O 4 ] − 191.0564 [M-H-2(C 7 H 5 O 4 )] − 169.0144 497.0907 -2.52 519.0732 [M + Na]+ 479.0808 [M + H-H 2 O]+ 309.0583 153.0167 C 21 H 20 O 14 digalloylshikimic acid isomer 1 3 (32) 4 3.458 222, 276 495.0782 -0.82 343.0683 [M-H-C 7 H 5 O 4 ] − 191.0556 [M-H-2(C 7 H 5 O 4 )] − 169.0138 497.0905 4.19 479.0790 [M + H-H 2 O] + 309.0590 [M + H-C 7 H 12 O 6 ] + 153.0169 [M + H-C 7 H 12 O 6 -C 7 H 10 O 4 ] + C 21 H 20 O 14 digalloylquinic acid isomer 1 3 [ 33 ] 4’ 3.557 222, 275 495.0795 - 343.0676 [M-H-C 7 H 12 O 6 ] − 191.0545 [M-H-2(C 7 H 12 O 6 )] − 169.0130 497.0939 -2.7 519.0733 [M + Na] + 479.0809 [M + H-H 2 O] + C 21 H 20 O 14 digalloylquinic acid isomer 2 3 [ 33 ] 9 9.045 - - - - 314.2693 -1.58 298.2743 278.2460 C 18 H 35 NO 3 prosopinine isomeric isoprosopinin 3 [ 34 ] 10 9.2 - - - - 314.2693 -1.61 298.2742 C 18 H 35 NO 3 prosopinine isomeric isoprosopinin 3 [ 34 ] 11 9.327 - - - - 314.2698 -2.55 296.2561 278.2460 262.2521 C 18 H 35 NO 3 prosopinine isomeric isoprosopinin 3 [ 34 ] 12 10.242 - 329.2340 -2.13 - - - - C 18 H 34 O 5 pinellic acid 2b (34] 13 11.223 - - - 342.2994 1.42 324.2883 [M + H-18]+ 306.2780 [M + H-2*18]+ C 20 H 39 NO 3 7-hydroxy-spectaline 2b [ 34 ] a according to Schymansky et al., 2014 Discussion Prosopis africana was a plant used in traditional medicine and known in the treatment of dental caries and dental hygiene (35). It was also used in the treatment of childhood diarrhea, ulcer, wounds, dermatoses, and gonococcal disease [ 36 ]. All these diseases were influenced by bacterial biofilm [ 37 ]. The antibacterial activity of the plant against several strains such as E. coli, S . aureus, S. mutans, K. pneumoniae, P. aeruginosa has also been reported [ 36 ]. Bacteria biofilm formation is responsible for the development of antibiotic resistance [ 38 ]. Indeed, bacteria surround themselves with extracellular polymers (glycocalyx) which constitute a physical barrier, thus limiting the access of antibiotics to the interior of the cells. The methanolic extracts of leaves and stems of P. africana showed in this study a potential inhibitor of biofilm formation by S. mutans ATCC, S. aureus ATCC, P. aeruginosa PAO1 without inhibiting bacterial growth. These finding showed that the inhibition of biofilm formation by the extracts is in no way associated with a bactericidal or bacteriostatic effect, but rather with a interference of the QS system [ 38 ]. The effect of extracts could be explained by the fact that they act on the mechanism of dental biofilm either by preventing bacteria from adhering to dental surfaces, or they would prevent cell divisions that lead to the formation of a truly three-dimensional, structurally and functionally well-organized bacterial network that ensure the integrity, resistance, and nutrition of the biofilm or on the formation of the exogenous pellicle that can create favorable micro-niches for stable adhesion of bacteria [ 39 ]. Extracts could act on extracellular polymeric substances (EPS). According to previous studies, EPS can be considered ''the home'' of biofilm cells within which they organize their lives [ 40 ]. It consists mostly of water but also polysaccharides, nucleic acids, and proteins, of which extracellular enzymes retained in the form of consolidated polysaccharide complexes, participate in its formation and structuration [ 41 , 42 ]. The properties of P. africana extracts to quench the dental biofilm controlled by the quorum sensing could justify the use of P. Africana in the folklore medicine to treat oral infections [ 37 ]. The reduction of violacein production was therefore a consequence of interference of the extracts with the Quorum Sensing mechanisms of C. violaceum CV026. The effect of P. africana extracts at the final concentration of 100 µg/ml was evaluated on the production of pyocyanin, rhamnolipids, and elastases, which are the main virulence factors excreted by P. aeruginosa PAO1 and whose production was under the control of the QS in dental biofilm. The reduction of virulence factors by the extracts implies a probable interference with the regulatory mechanism of pyocyanin production which depends on the QS in the formation of the dental biofilm. The results of the cytotoxic effect of the methanolic crude extracts of stems and leaves showed that the methanolic extract of stems under our experimental conditions did not induce any cytotoxicity on gingival cells. The inhibitory effect of the crude methanolic leaf extract could be explained by the abundant presence of alkaloids in the leaves and whose cytotoxicity was demonstrated previously [ 43 ]. UHPLC/UV/MS-MS analysis characterized in the methanolic extract of leaves and stems 2,4,6-trihydroxyphenyl β-D-glucoside, procyanidin A, catechin 7- O -glycoside, procyanidin dimer digallate, glucosyl- O -tyrosine, myricetin-3- O -galactoside, myricitrin, phloridzin and quercitrin, galloylquinic acid, digalloylshikimic acid, 3,4-digalloylquinic acid, 1,3-digalloyl quinic acid, prosopinin, pinelic acid, and spectalin. Most of these compounds were known to have anti-biofilm and anti-quorum sensing properties such as reported for catechin, galloyl shikimic acid, galloyl quinic acid, procyanidin, and quercitrin [ 44 ]. Indeed, these compounds could support the medical applications of Prosopis africana since the formation and structure of biofilm contribute greatly to the establishment of oral infections by increasing the properties of resistance to antibacterials [ 45 ]. They are therefore important building blocks for the development of new therapeutic products against biofilm encapsulated pathogens by disrupting the biofilm structure, thereby increasing the exposure of the pathogen to antibiotics. QS systems and biofilm are therefore new targets for the development of novel antibacterial strategies to inhibit the production of virulence factors by pathogenic bacteria [ 46 ]. Conclusion The present study whose general objective was to evaluate the anti-biofilm and anti-quorum sensing properties of methanolic extracts of leaves and stems of Prosopis africana , also allowed for evaluating the cytotoxicity of these extracts after phytochemical analyses. The evaluation of the anti-biofilm and anti-quorum sensing properties showed very appreciable effects of the extracts. The cytotoxicity study showed that the methanolic extracts of P. africana do not induce any cytotoxicity on gingival cells. Phytochemical analysis by HPLC/UV/MS 2 allowed the characterization of flavonoid glycosides, tannin derivatives (catechin, procyanidin, gallic acid derivatives) and alkaloids (prosopinin, pinelic acid, and spectalin). The biological effects of the extracts demonstrated in this study, as well as their chemical composition, may justify the use of Prosopis africana in the treatment of oral affections, as a mouthwash, especially since they are of low toxicity. Abbreviations Abs Absorbance AHLs N-acyl homoserine lactones B.H.I. Brain Heart Infusion broth DMEM Dulbecco's Modified Eagle Medium DMSO DiMethyl SulfOxide ESI-QTOF Electrospray Ionization-Quadrupole Time Of Flight INT IodoNitroTetrazolium L.B. Luria-Bertani broth LME Leaves Methanolic Extract MBC Minimum Bactericidal Concentration SME Stems Methanolic Extract MIC Minimum Inhibition Concentration MS Mass Spectrometry OD Optical Density QC Qualitative Control QS Quorum Sensing UPLC-UV/DAD Ultrahigh Performance Liquid Chromatography coupled to DAD UV-detector Declarations Ethics approval and consent to participate Not applicable Consent for publication Not applicable Availability of data and materials All data were presented in the present manuscript Competing interests The authors declare no conflicts of interest. Funding Not applicable Authors' contributions B.A. Writing- Original draft, data curation, methodology, investigation; R.A . data curation, methodology, supervision and validation; C.E . data curation, methodology; C.M. data curation, methodology; B.W. data curation, methodology; O.N. data curation, methodology, supervision and validation; H.E data curation, supervision, investigation; methodology, writing - review & editing; K.M . data curation, supervision, investigation, methodology; D.M . data curation, supervision, investigation, methodology, writing - review & editing Acknowledgements The authors are thankful to Mrs. Marijolène. Rey, Dr. Serge Michalet, Dr Isabelle Kerzaon, members of the CESN, for their support in processing the LC/MSMS experiment. References Szafrański SP, Winkel A, Stiesch M. The use of bacteriophages to biocontrol oral biofilms. J Biotechnol. 2017;250:29–44. Hasan S, Danishuddin M, Adil M, Singh K, Verma PK, Khan AU. 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07:22:26","extension":"xml","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":151830,"visible":true,"origin":"","legend":"","description":"","filename":"8ac2a5b3e80a400785f66c238a1479d61structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7348474/v1/0fdc712900ad5d759cb227f5.xml"},{"id":93012431,"identity":"4663b88a-47ea-42e2-97d7-7d0a8cab1d6b","added_by":"auto","created_at":"2025-10-08 07:22:27","extension":"html","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":159881,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7348474/v1/545fcf4ee488fbec24da7a2f.html"},{"id":93012412,"identity":"6c629653-69d7-43e2-82b0-934cf7acc887","added_by":"auto","created_at":"2025-10-08 07:22:26","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":70487,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of \u003cem\u003eP. africana\u003c/em\u003e extracts on biofilm formation of cariogenic bacteria. ns: not significant compared to the positive control. *P\u0026lt;0.05; **P\u0026lt;0.01; *** P\u0026lt;0.001; **** P \u0026lt;0.0001 versus Salicylic acid. LME: Leaves methanolic extract; SME: Stem Methanolic Extract\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7348474/v1/3b646349f43ab0ff8ba13f64.png"},{"id":93012413,"identity":"d1eacb38-8aa1-4bcd-8820-eafe65c088ab","added_by":"auto","created_at":"2025-10-08 07:22:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":27863,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of extracts on violacein production in \u003cem\u003eChromobacterium violaceum\u003c/em\u003e CVO26. ns: not significant compared to the positive control. *P\u0026lt;0.05; **P\u0026lt;0.01; *** P\u0026lt;0.001; **** P \u0026lt;0.0001 \u003cem\u003eversus\u003c/em\u003e Salicylic acid. LME: Leaves methanolic extract; SME: Stem Methanolic Extract\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7348474/v1/680959308aacc82be476189c.png"},{"id":93014290,"identity":"17ff08d1-9cc4-4117-83c7-baa2735e8dfe","added_by":"auto","created_at":"2025-10-08 07:38:26","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":72852,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of extracts on virulence factors. ns: not significant compared to the positive control. *P\u0026lt;0.05; **P\u0026lt;0.01; *** P\u0026lt;0.001; **** P \u0026lt;0.0001 \u003cem\u003eversus\u003c/em\u003e Salicylic acid. LME: Leaves methanolic extract; SME: Stem Methanolic Extract\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7348474/v1/880a9d3eef14add0097e49c9.png"},{"id":93012419,"identity":"51b4d3e6-e1fc-48f6-aaef-c462cda08bad","added_by":"auto","created_at":"2025-10-08 07:22:26","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":99290,"visible":true,"origin":"","legend":"\u003cp\u003eCytotoxicity of the methanol extracts of leaves (LME) and stems bark (SME) of \u003cem\u003eP. africana. \u003c/em\u003ens: not significant, *P\u0026lt;0.05, **P\u0026lt;0.01, ***P\u0026lt;0.0001 significant difference compared to the vehicle (DMSO 1%)\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7348474/v1/a76ce6448708797254a933a8.png"},{"id":93012417,"identity":"8f7c9b72-8a44-477f-90e7-2ec43fc929a8","added_by":"auto","created_at":"2025-10-08 07:22:26","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":91450,"visible":true,"origin":"","legend":"\u003cp\u003eESI\u003csup\u003e+\u003c/sup\u003e and ESI\u003csup\u003e-\u003c/sup\u003e mass spectra and UV spectrum of compound \u003cstrong\u003e11\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7348474/v1/a3f94ce9f396f4154d7cc533.png"},{"id":93014916,"identity":"4ed4e801-08b2-4cd8-815b-12d28da82607","added_by":"auto","created_at":"2025-10-08 07:46:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1852617,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7348474/v1/893c9c98-f228-4fec-98d9-b3a4eb9e6a70.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Anti-biofilm, anti-quorum sensing potential, cytotoxicity, and UPLC-UV/DAD- MS/MS/QTOF profiling of Prosopis africana (Guill. \u0026 Perr.) Taub. leaves and stems: benefits of a traditional medicine in dental care","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMicrobial communities living in an extracellular polymeric matrix that they produce themselves, called bacterial biofilms, are responsible for over 60% of microbial infections in humans. The transformations in gene expression and the activation of numerous extracellular communication pathways during biofilm formation often result in increased pathogenicity and bacterial virulence. [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Surprisingly, the antimicrobial resistance of a biofilm becomes thousand times higher than a free planktonic biota.\u003c/p\u003e\u003cp\u003eQuorum Sensing (QS) is a mode of bacterial communication between species and intra-species, based on the production of small mediator molecules called autoinducers, which are produced during bacterial growth. QS is the mechanism used by cariogenic Gram-negative and Gram-positive bacteria to synchronize and regulate the expression of several genes depending on cell density [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Physiological processes that affect the production of virulence factors [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], can be characterized by the emission of bioluminescence [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], the production of pigments [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], the formation of biofilm and motility [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In Gram-negative bacteria, QS involves the production of small signaling molecules called N-acyl homoserine lactones (AHLs) [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. AHLs are generally produced by an enzyme, a synthase, and diffuse passively across the cell membrane [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. When their concentration in the external environment reaches a critical threshold, generally at high cell density, AHLs invade the cell, bind and activate specific intracellular receptors [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Activated receptors bind to promoters of targeted genes and regulate their transcription [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. QS and biofilm are therefore linked because QS controls the formation and maturation of biofilm in several types of bacteria like \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e PAO1 [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. A buccal biofilm contains hundreds of different oral bacteria responsible of serious diseases in the oral cavity. In addition, virulence in response to drastic changes in the biofilm's microenvironment can be spread systemically and cause significant infections in other organs. [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The presence of \u003cem\u003eStreptococcus mutans, Staphylococcus aureus\u003c/em\u003e, and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e in supragingival and subgingival biofilm can cause dental caries. Dental plaque, composed of Gram-positive and Gram-negative bacteria, can adhere to the surface of teeth, proliferate, and produce lactic acid, leading to the demineralization of enamel and dentin. The limitations of conventional antibiotic therapy, as well as the increasing drug resistance, have led to an urgent need to find other approaches to treat infections related to oral biofilm.\u003c/p\u003e\u003cp\u003ePrevious studies have shown that plant drugs or molecules isolated from medicinal plants have properties that inhibit dental biofilm formation. These properties include a reduction in the adhesion of bacteria to dental plaque, a key step in the initiation and progression of dental caries. \u003cem\u003eProsopis africana\u003c/em\u003e (Guill. \u0026amp; Perr.) Taub (fabaceae) is a tree widely used in Burkina Faso to treat traditionally oral and dental infections. Ethnomedicinal surveys conducted in the south-central region reported the use of stems and leaves of \u003cem\u003eP. africana\u003c/em\u003e in the treatment of dental caries. Also, the barks are used to treat diarrhea in young children. Antibacterial properties against \u003cem\u003eEscherichia coli, Staphylococcus aureus, Streptococcus mutans, Klebsiella\u003c/em\u003e have also been reported [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. However, its anti-biofilm and anti-quorum sensing properties have been poorly investigated. We report here the anti-biofilm, the anti-quorum sensing activity, and the cytotoxicity of the methanolic leaves and stem bark extracts of \u003cem\u003eProsopis africana.\u003c/em\u003e\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003ePlant material and extraction\u003c/h2\u003e\u003cp\u003eA collection guided by traditional practitioners in the province of Zoundweogo (11\u0026deg; 34'60''N; 1\u0026deg;0'00'') allowed to harvest bark and leaves of \u003cem\u003eProsopis africana\u003c/em\u003e. The identification of the plant material was carried out by Dr. Souleymane COMPAORE junior researcher of botany from the plant biology and ecology laboratory of the Life and Earth Sciences Training and Research Unit (UFR/SVT) of the Universit\u0026eacute; Joseph KI-ZERBO. The wild plant was harvested on public land. A herbarium was deposited at the Herbarium of the UFR/SVT under the identification code No. 6852. The barks and stems were dried under laboratory conditions before to pulverize. The powders were kept cool at 4\u0026deg;C for the subsequent extraction of the plant drug. One hundred grams (100 g) of the leaves and stems powders were macerated in one liter of methanol (MeOH) during 24 hours. The methanolic maceration was filtered using Whatman No. 1 paper and concentrated under reduced pressure. The concentrate was placed in the Speedvac (SpeedVac ThermoFischer\u0026reg;) until complete dryness to yield two crude methanolic extracts (Leaves Methanolic Extract (LME), Stems Methanolic Extract (SME).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eBacterial Strains\u003c/h3\u003e\n\u003cp\u003eThe bacteriology laboratory of the Muraz Center in Bobo Dioulasso (Burkina Faso) and the plant biotechnology laboratory of the Universit\u0026eacute; Libre de Bruxelle (Belgium) respectively provided the standard strains (\u003cem\u003eStreptococcus mutans\u003c/em\u003e ATCC 25175 and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e ATCC 43300 methicillin-resistant) and the wild strains (\u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e PAO1 and \u003cem\u003eChromobacterium vio-laceum\u003c/em\u003e CV026). \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eS. mutans\u003c/em\u003e strains were stored at -80\u0026deg;C in Brain Heart Infusion liquid medium while \u003cem\u003eP. aeruginosa\u003c/em\u003e and \u003cem\u003eC. violaceum\u003c/em\u003e strains were inoculated in Luria Bertani medium containing 50% glycerol until use.\u003c/p\u003e\u003cp\u003e. \u003cb\u003eDetermination of the minimum inhibitory concentration (MIC)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe minimum inhibitory concentration of crude methanolic extracts (Leaves Methanolic extract (LME), Stems Methanolic extract (SME)) was determined using the 96-well microplate method [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. A range of extracts was carried out on a sterile microplate from a stock concentration of 4 mg/mL dissolved in 5% DMSO to get a final volume of 100 \u0026micro;L for each well. Then, 100 \u0026micro;L of the prepared inoculum in the corresponding culture medium were added to each well. DMSO medium (2.5%) was used as DMSO growth control and negative medium control, and DMSO-free medium as a bacterial growth control and negative medium control. The microplates were then incubated at 37\u0026deg; C for 24 h. At the final point 30 \u0026micro;L of iodonitrotetrazolium (INT) (2 mg/mL) were added to each well. 30 min after wereition of INT, MIC of the extracts was deduced regarding the color changes observation. Pink staining of INT indicates the presence of microbial growth in the wells. Indeed, the MIC is defined as the concentration of the first well of the range of dilutions devoid of bacterial growth [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/p\u003e\n\u003ch3\u003eDetermination of the minimum bactericidal concentration (MBC)\u003c/h3\u003e\n\u003cp\u003eThe minimum bactericidal concentration of an extract or a pure compound corresponds to the lowest concentration capable of killing more than 99.9% of the initial bacterial inoculum (\u003cem\u003ei.e.\u003c/em\u003e less than 0.01% of survivors). The range of concentrations produced to determine the MIC in a liquid medium was used to determine the MBC of the extracts. Samples in the control tube (2.5% DMSO) and each of the tubes devoid of bacterial growth were deposited in a \"streak\" on LB-agar/BHI-agar medium previously prepared and poured into Petri dishes. Plates were incubated for 24 h at 37\u0026deg;C. The MBC was deduced as being the first bacteria-free dish. The tests were carried out in triplicate [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eInhibition of biofilm formation\u003c/h3\u003e\n\u003cp\u003eThe anti-biofilm activity of the crude methanolic extracts (LME, SME) was evaluated according to the method previously described [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. For this purpose, sterile 96-well microplates were used. Extracts and reference (salicylic acid) were prepared at the concentration of 1 mg/mL in DMSO (1%) to have a final concentration of 100 \u0026micro;g/mL in each well. After incubating the plates during 24 h at 37\u0026deg;C, absorbances at λ600 nm were read to ensure that the extracts did not affect bacterial growth compared to the negative control (1% DMSO). Planktonic bacteria were removed with the supernatant, and the biofilms were thoroughly washed three times with distilled water and 2ml of methanol were used to fixed the adhered bacteria. After 15 min of incubation at 37\u0026deg;C, the methanol was removed, and the formed biofilm was quantified with crystal violet. Crystal violet (0.1% in water) was added to each well (200 \u0026micro;L/well) for 30 min at room temperature. The excess of crystal violet was removed, and 1 ml of distilled water was used to wash the stained biofilms. The crystal violet fixed by the biofilm bacteria was solubilized with acetic acid (33%) and the absorbance was measured at λ590 nm. The biofilm/bacterial growth ratio (OD 590 nm/OD 600 nm) was determined. The percentage of inhibition was calculated using the following formula:\u003c/p\u003e\u003cp\u003eInhibition (%) = [(Abs control \u0026ndash; Abs extract)/Abs control] x100\u003c/p\u003e\n\u003ch3\u003eAnti-quorum sensing activity\u003c/h3\u003e\n\u003cp\u003e\u003cb\u003eViolacein production inhibition test by\u003c/b\u003e \u003cb\u003eChromobacterium violaceum\u003c/b\u003e \u003cb\u003eCV026\u003c/b\u003e\u003c/p\u003e\u003cp\u003eInhibition of violacein production in \u003cem\u003eChromobacterium violaceum\u003c/em\u003e CV026 by all crude extracts (LME, SME) was tested by direct assay of culture medium according to protocols reported Choo \u003cem\u003eet al.\u003c/em\u003e (2006). Violacein production was induced in \u003cem\u003eC. violaceum\u003c/em\u003e CV026 by adding N-hexanoyl-L-homoserine lactone (HHL) (5 \u0026micro;M final concentration) in the presence or absence of the extracts (100\u0026micro;g/mL final concentration). \u003cem\u003eC. violaceum\u003c/em\u003e CV026 was first grown in the Luria Bertani medium for 24 h (30\u0026deg;C ;175 rpm) and the diluted CV026 was next introduced into 12-well plates. The extracts or the reference salicylic acid dissolved in DMSO 1% were added to have a final concentration of 100 \u0026micro;g/mL, in the presence of C6-HSL (final concentration 10 \u0026micro;M). Plates were incubated for 24 h at 30\u0026deg;C with shaking at 175 rpm.\u003c/p\u003e\u003cp\u003eBacterial growth was first evaluated by measuring bacterial turbidity at 600 nm and the produced violacein was then quantified using a spectrophotometer. Briefly, the solution was vigorously vortexed for 30 S to solubilize the violacein and the supernatant was removed. The pellet was dissolved using 100% DMSO. The violacein contained in the supernatant was recovered by centrifugation (7000 rpm; 10 min) and 200 \u0026micro;L were introduced into microplate wells for measuring the absorbance at 585 nm. The percentage of inhibition was calculated against the negative control (DMSO 1%) (18).\u003c/p\u003e\u003cp\u003eThe ratio of OD 585 nm to OD 600 nm was determined as:\u003c/p\u003e\u003cp\u003eInhibition (%) = [(Abs control \u0026ndash; Abs Extract)/Abs control] x100\u003c/p\u003e\u003cp\u003e\u003cb\u003ePyocyanin production inhibition test in\u003c/b\u003e \u003cb\u003ePseudomonas aeruginosa\u003c/b\u003e \u003cb\u003ePAOI\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe pyocyanin production inhibition assay was performed for the crude methanolic extracts (LME, SME). After 18 h incubation in a 24-well plate at 37\u0026deg;C with shaking at 175 rpm, the bacterial suspension was gently homogenized and 800 \u0026micro;l was transferred to a 1.8 mL Eppendorf tube, were then centrifuged at 16000 \u0026times; g for 1 min. 200 \u0026micro;L of the remaining suspension was directly transferred to a flat-bottom microtiter plate to measure absorbance at 600 nm, corresponding to the turbidity of the suspension. Then, 700 \u0026micro;L of supernatant from each sample after centrifugation was taken and transferred to 2 mL Eppendorf tubes; 600 \u0026micro;L of chloroform was added to each tube and the mixture was vortexed for a few seconds to extract pyocyanin. After centrifugation, two phases were formed and 500 \u0026micro;L of the lower phase, which contains pyocyanin, was transferred to a new 1.8 mL Eppendorf tube. Then, 250 \u0026micro;L of a 0.2 N hydrochloric acid solution was added to each tube to extract again the pyocyanin. The use of an acidic phase results in a color shift of pyocyanin from turquoise blue to pink. In nature, pyocyanin was blue, but it has been shown that in an acidic medium, it changes its structural configuration and turns pink [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. After centrifugation, two phases appear again with pyocyanin in the upper phase. Finally, 200 \u0026micro;L of this upper phase was recovered and the absorbance of this solution was measured at 360 nm to quantify the pyocyanin produced by the bacteria [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The experiments were repeated three times (n\u0026thinsp;=\u0026thinsp;3). The percentage of inhibition was calculated by the following formula:\u003c/p\u003e\u003cp\u003eInhibition (%) = [(Abs control \u0026ndash; Abs Extract)/Abs control] x100\u003c/p\u003e\u003cp\u003e\u003cb\u003eElastase inhibition test in\u003c/b\u003e \u003cb\u003ePseudomonas aeruginosa\u003c/b\u003e \u003cb\u003ePAO1\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe elastase inhibition test was performed with the methanolic crude extracts (LME, SME) and fractions. Elastase activity performed on \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 in the absence or presence of extract was measured by the Elastin-Congo Red method [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. This method was based on the properties of the elastase produced by the bacteria to degrade the elastin. \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 was incubated in LB medium with extracts (final concentration of 100 \u0026micro;g/mL) for 18 hours (37\u0026deg;C; 175 rpm) and the tubes were centrifuged for 5 min (3000 ; 24\u0026deg;C). Two hundred and fifty microliters (250 \u0026micro;L) of congo red elastin at 5 mg/mL dissolved in Tris-HCl buffer (0.1 M Tris-HCl pH 8;1 mM CaCl\u003csub\u003e2\u003c/sub\u003e) was added to 750 \u0026micro;L of supernatant contained in each Eppendorf tube. The mixture was incubated at 37\u0026deg;C for 16 h at 200 rpm. During this incubation phase, elastin degradation results in solubilization of congo red, yielding a red solution while the undegraded Elastin-Congo Red complex was insoluble in water [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The reaction mixture was centrifuged at 3000 \u003cem\u003eg\u003c/em\u003e for 10 min to separate the non-soluble portion and the absorbance of 200 \u0026micro;L of supernatant was measured at λ495 nm to estimate the elastin activity [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The percentage of inhibition was calculated as follows:\u003c/p\u003e\u003cp\u003eInhibition (%) = [(Abs control \u0026ndash; Abs Extract) / Abs control] x100\u003c/p\u003e\u003cp\u003e\u003cb\u003eRhamnolipid inhibition test in\u003c/b\u003e \u003cb\u003ePseudomonas aeruginosa\u003c/b\u003e \u003cb\u003ePAO1\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe rhamnolipid inhibition test was performed with crude methanolic extracts (LME, SME). For rhamnolipid extraction, \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 grew at 37\u0026deg;C with shaking at 175 rpm for 18 h in a 5 mL LB medium in the presence of extract (100 \u0026micro;g/mL) or DMSO (1%). Bacterial cultures were then collected in Eppendorf tubes and centrifuged (3000 \u003cem\u003eg\u003c/em\u003e at room temperature for 5 min). One milliliter of bacteria-free supernatant was recovered and filtered through an Ultrafree\u0026reg; CL-Millipore filter, and the pH of the supernatant was adjusted to 2.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 with HCl 1N. The rhamnolipids were extracted from the supernatant with 4 mL of ethyl acetate (EtOAc) on the vortex for a few seconds. After centrifugation, the upper phase, which contains the rhamnolipids, was transferred to a new 30 mL tube. This extraction procedure was repeated three times. All the collected organic solvents were finally removed by evaporation under vacuum centrifugation. Quantification of rhamnolipids was performed by the methylene blue complexation method according to the literature with some modifications [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Briefly, 4 ml of chloroform were used to dissolve the rhamnolipid produced by the bacteria in each extract treatment and contacted with a methylene blue solution consisting of 200 \u0026micro;L of 1 g/L methylene blue reagent and 4.9 mL of distilled water that was adjusted to pH 8.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 by adding 50 mM borax buffer (15 \u0026micro;L). After vigorous shaking for 4 min, the mixture was allowed to stand for 15 min to allow complexation of methylene blue to rhamnolipids. One milliliter of the lower chloroform phase turning blue was transferred to a new Eppendorf tube and vortexed with 500 \u0026micro;L of 0.2 N HCl, allowing the transfer of the complex methylene blue into the acid phase. Finally, 200 \u0026micro;L of the acidic phase was transferred to a 96-well microplate and the absorbance measured at λ638 nm [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The percentage of inhibition was calculated by the following formula:\u003c/p\u003e\u003cp\u003eInhibition (%) = [(Abs control \u0026ndash; Abs sample) / Abs control] x100\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eCytotoxicity Assay\u003c/h2\u003e\u003cp\u003eThe cytotoxicity study was performed for LME and SME. Gingival cells were grown and maintained in a humidified incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C in Petri dishes in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum, 1% penicillin-streptomycin, and 1% L-Glutamine [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Cultures were subjected to medium changes and passages or transplants when cells were at the confluence. All manipulations were performed under highly sterile conditions in a laminar flow hood.\u003c/p\u003e\u003cp\u003eAfter observation under a reverse-phase microscope (Jenco USA), gingival cells were seeded on sterile 96-well microplates in 100 \u0026micro;L of complete culture medium at 2.104 cells/well. The microplates were then incubated for 24 h to ensure adequate cell adhesion. The crude methanolic extracts from the leaves and stems were dissolved in DMSO and diluted in the culture medium to a concentration of 1% DMSO. A range of dilutions was performed for concentrations from 500 \u0026micro;g/mL to 15.625 \u0026micro;g/mL. 100 \u0026micro;L of each extract was then contacted with the cells in the wells and the plates returned to incubation for 72 h. The blank contained only the culture medium and the control contained cells with the culture medium, without extract. The cell suspension in the presence of hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) (50 \u0026micro;L, 300 mM) served as the positive control, and 1% DMSO served as the negative control.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003ePhytochemical Analysis: Ultra-High-Performance Liquid Chromatography Coupled with High-Resolution Tandem Mass Spectrometry (UHPLC/MS-MS)\u003c/h3\u003e\n\u003cp\u003eExtracts were analyzed using Agilent Technologies Accurate-Mass Q-TOF LCMS 6530, with LC 1290 Infinity system. The separation was carried out at 40\u0026deg;C using a 120 EC-C18 column (3.0*100 mm*2.7 \u0026micro;m; Agilent Poroshell). Each sample (10 mg. mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was injected (3 \u0026micro;L), and the column was eluted at 0.7 mL.min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e with a solvent gradient using solvent A (water with formic acid 0.4% (v/v)) and solvent B (acetonitrile). The proportion of solvent B increased, step by step, from 10 to 36% during 4.5 min; 36% to 100% during 4 min, followed by 2 min isocratic phase with 100% solvent B; back from 100% to 10% in 0.5 min and equilibration at 10% solvent B during 3 min until the end of the run at 14 min. Mass analyses were made in positive and negative modes, with the nebulization gas (Nitrogen) at a flow of 10 L.min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 40 psg pressure. The capillary tension was 3000 V and gaz ionization energy of 100 eV. Analyses included QC (Qualitative Control) and blank samples, which were regularly injected between runs, giving a total of 8 blanks and 8 QCs. QC was prepared by mixing 100 \u0026micro;L of each sample prepared at 10 mg. mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Moreover, to facilitate the identification of specific compounds samples were analyzed with tandem mass spectrometry (MS/MS). MS/MS analyses were performed by collision-induced dissociation (CID) with a collision energy of 20 eV. Chromatograms were explored with MassHunter Qualitative Analysis B.07.00 software (Agilent\u0026reg; Technologies), and the Semetl1.0.2020.11.10 version 2 in-house database containing the analyzed by UHPLC-MS/MS of 400 commercial standards in positive and negative mode. Compounds identifications were performed by comparison of experimental data to that of SemetI database (in house database) and to the existing literature.\u003c/p\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eThe statistical analysis was performed by using Graph Pad Prism version 6 software. The results were expressed as mean of three independent repeated experiments\u0026thinsp;\u0026plusmn;\u0026thinsp;Mean Standard Error. One-way analysis of variance (ANOVA) followed by the post-test Dunnett\u0026rsquo;s was used to measure the degree significance of the results. The extracts were compared with the negative control and the value of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 was considered significant, very significant. The experiments were carried out in triplicate (n\u0026thinsp;=\u0026thinsp;3) and the results were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eThe MIC (\u0026gt;\u0026thinsp;0.250 mg/mL) and MBC (\u0026gt;\u0026thinsp;1mg/mL) values evaluated allow the selection of a sub-inhibitory concentration for the bioassay on all strains (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eMinimum inhibitory concentration and minimal bactericidal concentration of methanolic extract of leaves and stem bark of \u003cem\u003eProsopis Africana.\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eExtracts\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eLME (mg/mL)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003eSME (mg/mL)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eStrains\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMIC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMBC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMIC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eMBC\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eStreptococcus mutans\u003c/em\u003e ATCC 25175\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eStaphylococcus aureus\u003c/em\u003e ATCC 43300\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e PAOI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eChromobacterium violaceum\u003c/em\u003e CV 026\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003eMIC: Minimum inhibitory concentration, MBC: minimal bactericidal concentration, LME: methanolic extract of leaves, SME: stem bark\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eInhibition of biofilm production\u003c/h2\u003e\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the methanolic extracts from the leaves and stems of \u003cem\u003eP. africana\u003c/em\u003e, at a concentration of 100 \u0026micro;g/mL affected neither the viability nor the growth of \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e PAO1, \u003cem\u003eStaphylococcus aureus, Streptococcus mutans.\u003c/em\u003e Overall, compared to salicylic acid (SA), all extracts significantly reduced biofilm formation of \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e PAO1 with respective inhibition percentages of 32.8\u0026thinsp;\u0026plusmn;\u0026thinsp;5.3 for LME and 36.3\u0026thinsp;\u0026plusmn;\u0026thinsp;4.6. for SME. Concerning \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, biofilm formation was inhibited with respective inhibition percentages of 31.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9% for LME; 40.4\u0026thinsp;\u0026plusmn;\u0026thinsp;4.6% for SME. For \u003cem\u003eStreptococcus mutans\u003c/em\u003e, biofilm formation was inhibited with respective inhibition percentages of 47.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8% for LME and 56.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6% for SME.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eEffect of extracts on violacein production in\u003c/b\u003e \u003cb\u003eChromobacterium violaceum CVO26\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, methanolic extracts of \u003cem\u003eP. africana\u003c/em\u003e significantly reduced violacein production compared to 1% DMSO. At the final concentration of 100 \u0026micro;g/mL, the extracts did not affect either viability or bacterial growth of \u003cem\u003eC. violaceum\u003c/em\u003e CV026. For LME, the inhibition of violacein was 37.9\u0026thinsp;\u0026plusmn;\u0026thinsp;3.7% and for SME the inhibition was 42.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4%.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eEffect of extracts on pyocyanin, rhamnolipid, and elastase production in\u003c/b\u003e \u003cb\u003eP. aeruginosa PAO1\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe effect of \u003cem\u003eP. africana\u003c/em\u003e extracts at the final concentration of 100 \u0026micro;g/mL was evaluated on the production of pyocyanin, elastase, and rhamnolipids (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) which are the main virulence factors excreted by \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 and whose production was under the control of the QS in dental biofilm. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, the extracts at the final concentration tested of 100 \u0026micro;g/mL significantly inhibited the production of pyocyanin, and rhamnolipids compared to the control (DMSO 1%) without affecting bacterial growth. Pyocyanin inhibition was marked by methanolic leaf extract (LME 49.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03%). The reduction of elastase was marked by both extracts (LME 40.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05%; SME 39.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.014). For rhamnolipids, the percentage of inhibition of methanolic leaf extract (LME) was 46.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02% and that of methanolic stem extract (SME) was 39.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01%. This reduction of virulence factors by the extracts implies a probable interference with the regulatory mechanism of pyocyanin production that depends on QS in dental biofilm formation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eCytotoxic effect of extracts on gingival cells\u003c/h2\u003e\u003cp\u003eThe cytotoxic effect of the crude methanolic extracts of leaves and stems on gingival cell viability was determined after 72 h following the MTT colorimetric cytotoxicity test. The methanolic stem extract (SME) showed no adverse effect on gingival cell viability compared to the DMSO control (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). In contrast, crude leaf extract (LME) had a significant cytotoxic effect on gingival cells compared to DMSO starting from 15.625 \u0026micro;g/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eIdentification of compounds of interest by HPLC/MS-MS in methanolic extracts of leaves and stems of\u003c/b\u003e \u003cb\u003eProsopis africana\u003c/b\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eTables\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e provide informations on the characterized compounds in both methanolic extract LME and SME. Comparing chromatograms in both positive- and negative modes ESI\u0026ndash;MS experiments, a first group of forteen polar compounds were detected between 2.00 and 7.00 min. They were better ionized in negative mode than in postive one. A second group was eluted between 7.00 and 13.00 min, corresponding to less polar molecules that were better ionized in negative mode. To complete identifications the UV spectra was also used. This data combination was compared to published data and to the in-house database SemetI. As an example, compound \u003cb\u003e11\u003c/b\u003e in LME was identified as follows, and the same approach was used to characterized the chemical composition of both extracts. The UV spectrum showed the following maxima of absorption: 222, 260, 300sh et 348 nm suggesting a polyhydroxylated flavonoid like a 3-\u003cem\u003eO\u003c/em\u003e-substituted myricetin derivative (Mabry \u003cem\u003eet al\u003c/em\u003e., 2012). The pos-ESI-MS spectrum exhibited a pseudo-molecular ion at \u003cem\u003em/z\u003c/em\u003e 465.0998 u corresponding to [M\u0026thinsp;+\u0026thinsp;H] \u003csup\u003e+\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The ions at \u003cem\u003em/z\u003c/em\u003e 487.0848 [M\u0026thinsp;+\u0026thinsp;Na]\u003csup\u003e+\u003c/sup\u003e and 951.1756 u [2M\u0026thinsp;+\u0026thinsp;Na] \u003csup\u003e+\u003c/sup\u003e in ESI\u003csup\u003e+\u003c/sup\u003e, together with the ion in ESI\u003csup\u003e-\u003c/sup\u003e at \u003cem\u003em/z\u003c/em\u003e 464.0899 u [M-H]\u003csup\u003e-\u003c/sup\u003e confirmed a molecular weight of 464 uma. A fragment at \u003cem\u003em/z\u003c/em\u003e 319.0425 in ESI\u003csup\u003e+\u003c/sup\u003e (and \u003cem\u003em/z\u003c/em\u003e 316.0243 u in ESI\u003csup\u003e-\u003c/sup\u003e) showed out the loss of 146 u fragment, corresponding to the loss of a desoxyhexosyl unit. Altogether these data confirmed the formula of C\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e20\u003c/sub\u003eO\u003csub\u003e12\u003c/sub\u003e (with a score of 99,64%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Compared to published data, this analysis led to a presumed identification of compound \u003cb\u003e11\u003c/b\u003e to myricetin 3-\u003cem\u003eO\u003c/em\u003e-rhamnoside or myricitrin (Sharifi-Rad \u003cem\u003eet al.\u003c/em\u003e, 2019).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe same method allowed us to identify thirteen other compounds in LME, among which 2,4,6-trihydroxyphenyl β-D-glucopyranoside, procyanidin A, catechin 7-\u003cem\u003eO\u003c/em\u003e-glycoside, procyanidin dimer digallate, glucopyranosyl-\u003cem\u003eO\u003c/em\u003e-tyrosine, myricetin-3-\u003cem\u003eO\u003c/em\u003e-galactoside, myricitrin, phloridzin, and quercitrin. Most of the identified compounds were flavonoids.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eSimilarly, SME (methanolic stem extract) was analyzed and nine compounds were characterized such as galloylquinic acid, digalloylshikimic acid, 3,4-digalloylquinic acid, 1,3-digalloylquinic acid, prosopinin, pinelic acid, and spectalin.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eIdentification by UHPLC-ESI-QTOF of the main compounds of the methanolic leaves extract (LME) of \u003cem\u003eProsopis africana\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"13\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eN\u003csup\u003eo\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003csub\u003eRt (min)\u003c/sub\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eUV (nm)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003em/z\u003c/em\u003e\u003c/p\u003e\u003cp\u003e(M - H)\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eΔM ppm\u003c/p\u003e\u003cp\u003em/z\u003c/p\u003e\u003cp\u003e(M - H)\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cem\u003em/z\u003c/em\u003e MS\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003cp\u003e(fragment)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cem\u003em/z\u003c/em\u003e\u003c/p\u003e\u003cp\u003e(M\u0026thinsp;+\u0026thinsp;H)\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eΔM ppm\u003c/p\u003e\u003cp\u003e\u003cem\u003em/z\u003c/em\u003e\u003c/p\u003e\u003cp\u003e(M\u0026thinsp;+\u0026thinsp;H)\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u003cem\u003em/z\u003c/em\u003e MS\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003cp\u003e(fragment)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\"\u003e\u003cp\u003eFormule\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c11\"\u003e\u003cp\u003eAnnotation (SemetI)\u003c/p\u003e\u003cp\u003eor\u003c/p\u003e\u003cp\u003eproposal\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c12\"\u003e\u003cp\u003eConfidence level \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c13\"\u003e\u003cp\u003eReference\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2.733\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e224, 286\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e303.0728\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-2.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e141.0187 [M-H-C\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e]\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e305.0672\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e2.48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e327.0672 [M\u0026thinsp;+\u0026thinsp;Na]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\u003cp\u003e631.1464 [2M\u0026thinsp;+\u0026thinsp;Na]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\u003cp\u003e143.0331 [M\u0026thinsp;+\u0026thinsp;H-C\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eC\u003csub\u003e12\u003c/sub\u003eH\u003csub\u003e16\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e2,3,5-trihydroxyphenyl glucoside\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e2b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e2\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3.260\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e224, 278\u0026ndash;279\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e451.1244\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e289.0728 [M-H-C\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e]\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e453.1374\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e3.72\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e475.1199 [M\u0026thinsp;+\u0026thinsp;Na]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\u003cp\u003e291.0851 [M\u0026thinsp;+\u0026thinsp;H-C\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\u003cp\u003e273.0747 [M\u0026thinsp;+\u0026thinsp;H-C\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eC\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e24\u003c/sub\u003eO\u003csub\u003e11\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003ecatechin 5-\u003cem\u003eO\u003c/em\u003e or 7-\u003cem\u003eO\u003c/em\u003e- glycoside\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3.455\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e224, 276\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e593.1314\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e595.1433\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e3.67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e617.1245 [M\u0026thinsp;+\u0026thinsp;Na]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\u003cp\u003e1211.2554 [2M\u0026thinsp;+\u0026thinsp;Na]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eC\u003csub\u003e30\u003c/sub\u003eH\u003csub\u003e26\u003c/sub\u003eO\u003csub\u003e13\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003ederivative of Procyanidin A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e4\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3.355\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e224, 276\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e593.1299\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1187.2746 [2M-H]\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003c/p\u003e\u003cp\u003e447.0706 [M-H-146]\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e595.1425\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e2.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e617.1238 [M\u0026thinsp;+\u0026thinsp;Na]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\u003cp\u003e1211.2554 [2M\u0026thinsp;+\u0026thinsp;Na]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eC\u003csub\u003e30\u003c/sub\u003eH\u003csub\u003e26\u003c/sub\u003eO\u003csub\u003e13\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003eprocyanidin derivative\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e5\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3.763\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e224, 278\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e593.1305\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-0.73\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" 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colname=\"c12\"\u003e\u003cp\u003e2b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e7\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e4.177\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e223, 284\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e881.1936\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-0.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1764.3936 [2M]\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003c/p\u003e\u003cp\u003e603.1352\u0026nbsp;; 577.1352\u003c/p\u003e\u003cp\u003e451.1245\u0026nbsp;; 440.0914\u003c/p\u003e\u003cp\u003e289.0714\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e883.2047\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e3.74\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e1788.3812 [2M\u0026thinsp;+\u0026thinsp;H\u0026thinsp;+\u0026thinsp;Na]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eC\u003csub\u003e45\u003c/sub\u003eH\u003csub\u003e38\u003c/sub\u003eO\u003csub\u003e19\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003edimer procyanidin digallate \u0026ndash; type B\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR28\" 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[M-H-C\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e]\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e481.0979\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e-0.49\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e503.0796 [M\u0026thinsp;+\u0026thinsp;Na]+\u003c/p\u003e\u003cp\u003e983.1667 [2M\u0026thinsp;+\u0026thinsp;Na]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\u003cp\u003e319.0445 [M\u0026thinsp;+\u0026thinsp;H-C\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eC\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e20\u003c/sub\u003eO\u003csub\u003e13\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003emyricetin-3-\u003cem\u003eO\u003c/em\u003e-galactoside 2 (isomer)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e2a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e11\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e5.627\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e222, 260, 300sh, 348\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e463.0872\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.95\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e317.0636 [M-H-C\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e]\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e465.1023\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e487.0834 [M\u0026thinsp;+\u0026thinsp;Na]+\u003c/p\u003e\u003cp\u003e651.1789 [2M\u0026thinsp;+\u0026thinsp;Na]+\u003c/p\u003e\u003cp\u003e319.0427 [M\u0026thinsp;+\u0026thinsp;H-C\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eC\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e20\u003c/sub\u003eO\u003csub\u003e12\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" 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[M-H-C\u003csub\u003e8\u003c/sub\u003eH\u003csub\u003e9\u003c/sub\u003eO]\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003c/p\u003e\u003cp\u003e273.0774 [M-H-C\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e]\u003csup\u003e\u0026minus;\u003c/sup\u003e151.0401\u0026nbsp;; 121.0292\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eC\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e24\u003c/sub\u003eO\u003csub\u003e10\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003ephloridzin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e2a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e13\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e6.427\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e256, 346\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e447.0949\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-3.93\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e301.0357 [M-H-C\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e449.1066\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e2.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e919.1845 [2M\u0026thinsp;+\u0026thinsp;Na]+\u003c/p\u003e\u003cp\u003e303.0490 [M\u0026thinsp;+\u0026thinsp;H-C\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eC\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e20\u003c/sub\u003eO\u003csub\u003e11\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003equercitrin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e2b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e according to Schymansky et al., 2014.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eIdentification by UHPLC-ESI-QTOF of the main compounds of the methanolic stems extract (SME) of \u003cem\u003eProsopis africana\u003c/em\u003e.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"14\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c14\" colnum=\"14\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eN\u0026deg;\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003et\u003c/em\u003e\u003csub\u003eR (min)\u003c/sub\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eUV (nm)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003em/z\u003c/em\u003e\u003c/p\u003e\u003cp\u003e(M - H)\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eΔM ppm\u003c/p\u003e\u003cp\u003e\u003cem\u003em/z\u003c/em\u003e\u003c/p\u003e\u003cp\u003e(M - H)\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cem\u003em/z\u003c/em\u003e MS\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003cp\u003e(Fragment)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cem\u003em/z\u003c/em\u003e\u003c/p\u003e\u003cp\u003e(M\u0026thinsp;+\u0026thinsp;H) \u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eΔM ppm\u003c/p\u003e\u003cp\u003e\u003cem\u003em/z\u003c/em\u003e\u003c/p\u003e\u003cp\u003e(M\u0026thinsp;+\u0026thinsp;H) \u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u003cem\u003em/z\u003c/em\u003e MS\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003cp\u003e(fragment)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\"\u003e\u003cp\u003eFormula\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c11\"\u003e\u003cp\u003eAnnotation or\u003c/p\u003e\u003cp\u003eproposal\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c12\"\u003e\u003cp\u003eConfidence level\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c14\" namest=\"c13\"\u003e\u003cp\u003eReferences\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.905\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e222. 272\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e343.0677\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-1.87\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e687.1442 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colname=\"c13\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c14\" namest=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.360\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e222, 274\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e495.0810\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-3.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e343.0683 [M-H-C\u003csub\u003e7\u003c/sub\u003eH\u003csub\u003e5\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e]\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003c/p\u003e\u003cp\u003e191.0564 [M-H-2(C\u003csub\u003e7\u003c/sub\u003eH\u003csub\u003e5\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e)]\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003c/p\u003e\u003cp\u003e169.0144\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e497.0907\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e-2.52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e519.0732 [M\u0026thinsp;+\u0026thinsp;Na]+\u003c/p\u003e\u003cp\u003e479.0808 [M\u0026thinsp;+\u0026thinsp;H-H\u003csub\u003e2\u003c/sub\u003eO]+\u003c/p\u003e\u003cp\u003e309.0583\u003c/p\u003e\u003cp\u003e153.0167\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eC\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e20\u003c/sub\u003eO\u003csub\u003e14\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003edigalloylshikimic acid isomer 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c14\" namest=\"c13\"\u003e\u003cp\u003e(32)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e4\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.458\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e222, 276\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e495.0782\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-0.82\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e343.0683 [M-H-C\u003csub\u003e7\u003c/sub\u003eH\u003csub\u003e5\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e]\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003c/p\u003e\u003cp\u003e191.0556 [M-H-2(C\u003csub\u003e7\u003c/sub\u003eH\u003csub\u003e5\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e)]\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003c/p\u003e\u003cp\u003e169.0138\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e497.0905\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e4.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e479.0790 [M\u0026thinsp;+\u0026thinsp;H-H\u003csub\u003e2\u003c/sub\u003eO]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\u003cp\u003e309.0590 [M\u0026thinsp;+\u0026thinsp;H-C\u003csub\u003e7\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\u003cp\u003e153.0169\u003c/p\u003e\u003cp\u003e[M\u0026thinsp;+\u0026thinsp;H-C\u003csub\u003e7\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e-C\u003csub\u003e7\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eC\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e20\u003c/sub\u003eO\u003csub\u003e14\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003edigalloylquinic acid isomer 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c14\" namest=\"c13\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e4\u0026rsquo;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.557\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e222, 275\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e495.0795\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e343.0676 [M-H-C\u003csub\u003e7\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e]\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003c/p\u003e\u003cp\u003e191.0545 [M-H-2(C\u003csub\u003e7\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e)]\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003c/p\u003e\u003cp\u003e169.0130\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e497.0939\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e-2.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e519.0733 [M\u0026thinsp;+\u0026thinsp;Na]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\u003cp\u003e479.0809 [M\u0026thinsp;+\u0026thinsp;H-H\u003csub\u003e2\u003c/sub\u003eO]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eC\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e20\u003c/sub\u003eO\u003csub\u003e14\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003edigalloylquinic acid isomer 2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c14\" namest=\"c13\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e9\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e9.045\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e314.2693\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e-1.58\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e298.2743\u003c/p\u003e\u003cp\u003e278.2460\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eC\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e35\u003c/sub\u003eNO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003eprosopinine isomeric\u003c/p\u003e\u003cp\u003eisoprosopinin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c14\" namest=\"c13\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e10\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e9.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e314.2693\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e-1.61\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e298.2742\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eC\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e35\u003c/sub\u003eNO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003eprosopinine isomeric\u003c/p\u003e\u003cp\u003eisoprosopinin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c14\" namest=\"c13\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e11\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e9.327\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e314.2698\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e-2.55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e296.2561\u003c/p\u003e\u003cp\u003e278.2460\u003c/p\u003e\u003cp\u003e262.2521\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eC\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e35\u003c/sub\u003eNO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003eprosopinine isomeric\u003c/p\u003e\u003cp\u003eisoprosopinin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c14\" namest=\"c13\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e12\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e10.242\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e329.2340\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-2.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eC\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e34\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003epinellic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e2b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c14\" namest=\"c13\"\u003e\u003cp\u003e(34]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e13\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e11.223\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e342.2994\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1.42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e324.2883 [M\u0026thinsp;+\u0026thinsp;H-18]+\u003c/p\u003e\u003cp\u003e306.2780 [M\u0026thinsp;+\u0026thinsp;H-2*18]+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eC\u003csub\u003e20\u003c/sub\u003eH\u003csub\u003e39\u003c/sub\u003eNO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e7-hydroxy-spectaline\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e2b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c14\" namest=\"c13\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"14\"\u003e\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e according to Schymansky et al., 2014\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cem\u003eProsopis africana\u003c/em\u003e was a plant used in traditional medicine and known in the treatment of dental caries and dental hygiene (35). It was also used in the treatment of childhood diarrhea, ulcer, wounds, dermatoses, and gonococcal disease [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. All these diseases were influenced by bacterial biofilm [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The antibacterial activity of the plant against several strains such as \u003cem\u003eE. coli, S\u003c/em\u003e. \u003cem\u003eaureus, S. mutans, K. pneumoniae, P. aeruginosa\u003c/em\u003e has also been reported [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eBacteria biofilm formation is responsible for the development of antibiotic resistance [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Indeed, bacteria surround themselves with extracellular polymers (glycocalyx) which constitute a physical barrier, thus limiting the access of antibiotics to the interior of the cells. The methanolic extracts of leaves and stems of \u003cem\u003eP. africana\u003c/em\u003e showed in this study a potential inhibitor of biofilm formation by \u003cem\u003eS. mutans ATCC, S. aureus ATCC, P. aeruginosa\u003c/em\u003e PAO1 without inhibiting bacterial growth. These finding showed that the inhibition of biofilm formation by the extracts is in no way associated with a bactericidal or bacteriostatic effect, but rather with a interference of the QS system [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The effect of extracts could be explained by the fact that they act on the mechanism of dental biofilm either by preventing bacteria from adhering to dental surfaces, or they would prevent cell divisions that lead to the formation of a truly three-dimensional, structurally and functionally well-organized bacterial network that ensure the integrity, resistance, and nutrition of the biofilm or on the formation of the exogenous pellicle that can create favorable micro-niches for stable adhesion of bacteria [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Extracts could act on extracellular polymeric substances (EPS). According to previous studies, EPS can be considered ''the home'' of biofilm cells within which they organize their lives [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. It consists mostly of water but also polysaccharides, nucleic acids, and proteins, of which extracellular enzymes retained in the form of consolidated polysaccharide complexes, participate in its formation and structuration [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe properties of \u003cem\u003eP. africana\u003c/em\u003e extracts to quench the dental biofilm controlled by the quorum sensing could justify the use of \u003cem\u003eP. Africana\u003c/em\u003e in the folklore medicine to treat oral infections [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The reduction of violacein production was therefore a consequence of interference of the extracts with the Quorum Sensing mechanisms of \u003cem\u003eC. violaceum\u003c/em\u003e CV026. The effect of \u003cem\u003eP. africana\u003c/em\u003e extracts at the final concentration of 100 \u0026micro;g/ml was evaluated on the production of pyocyanin, rhamnolipids, and elastases, which are the main virulence factors excreted by \u003cem\u003eP. aeruginosa PAO1\u003c/em\u003e and whose production was under the control of the QS in dental biofilm. The reduction of virulence factors by the extracts implies a probable interference with the regulatory mechanism of pyocyanin production which depends on the QS in the formation of the dental biofilm.\u003c/p\u003e\u003cp\u003eThe results of the cytotoxic effect of the methanolic crude extracts of stems and leaves showed that the methanolic extract of stems under our experimental conditions did not induce any cytotoxicity on gingival cells. The inhibitory effect of the crude methanolic leaf extract could be explained by the abundant presence of alkaloids in the leaves and whose cytotoxicity was demonstrated previously [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eUHPLC/UV/MS-MS analysis characterized in the methanolic extract of leaves and stems 2,4,6-trihydroxyphenyl β-D-glucoside, procyanidin A, catechin 7-\u003cem\u003eO\u003c/em\u003e-glycoside, procyanidin dimer digallate, glucosyl-\u003cem\u003eO\u003c/em\u003e-tyrosine, myricetin-3-\u003cem\u003eO\u003c/em\u003e-galactoside, myricitrin, phloridzin and quercitrin, galloylquinic acid, digalloylshikimic acid, 3,4-digalloylquinic acid, 1,3-digalloyl quinic acid, prosopinin, pinelic acid, and spectalin. Most of these compounds were known to have anti-biofilm and anti-quorum sensing properties such as reported for catechin, galloyl shikimic acid, galloyl quinic acid, procyanidin, and quercitrin [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Indeed, these compounds could support the medical applications of \u003cem\u003eProsopis africana\u003c/em\u003e since the formation and structure of biofilm contribute greatly to the establishment of oral infections by increasing the properties of resistance to antibacterials [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. They are therefore important building blocks for the development of new therapeutic products against biofilm encapsulated pathogens by disrupting the biofilm structure, thereby increasing the exposure of the pathogen to antibiotics. QS systems and biofilm are therefore new targets for the development of novel antibacterial strategies to inhibit the production of virulence factors by pathogenic bacteria [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe present study whose general objective was to evaluate the anti-biofilm and anti-quorum sensing properties of methanolic extracts of leaves and stems of \u003cem\u003eProsopis africana\u003c/em\u003e, also allowed for evaluating the cytotoxicity of these extracts after phytochemical analyses. The evaluation of the anti-biofilm and anti-quorum sensing properties showed very appreciable effects of the extracts. The cytotoxicity study showed that the methanolic extracts of \u003cem\u003eP. africana\u003c/em\u003e do not induce any cytotoxicity on gingival cells. Phytochemical analysis by HPLC/UV/MS\u003csup\u003e2\u003c/sup\u003e allowed the characterization of flavonoid glycosides, tannin derivatives (catechin, procyanidin, gallic acid derivatives) and alkaloids (prosopinin, pinelic acid, and spectalin).\u003c/p\u003e\u003cp\u003eThe biological effects of the extracts demonstrated in this study, as well as their chemical composition, may justify the use of \u003cem\u003eProsopis africana\u003c/em\u003e in the treatment of oral affections, as a mouthwash, especially since they are of low toxicity.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAbs\u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Absorbance\u003c/p\u003e\n\u003cp\u003eAHLs \u0026nbsp;N-acyl homoserine lactones\u003c/p\u003e\n\u003cp\u003eB.H.I.\u0026nbsp;\u0026nbsp;Brain Heart Infusion broth\u003c/p\u003e\n\u003cp\u003eDMEM \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Dulbecco's Modified Eagle Medium\u003c/p\u003e\n\u003cp\u003eDMSO\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;DiMethyl SulfOxide\u003c/p\u003e\n\u003cp\u003eESI-QTOF\u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Electrospray Ionization-Quadrupole Time Of Flight\u003c/p\u003e\n\u003cp\u003eINT \u0026nbsp; \u0026nbsp;\u0026nbsp;IodoNitroTetrazolium\u003c/p\u003e\n\u003cp\u003eL.B.\u0026nbsp; \u0026nbsp; \u0026nbsp;Luria-Bertani broth\u003c/p\u003e\n\u003cp\u003eLME\u0026nbsp; \u0026nbsp;\u0026nbsp;Leaves Methanolic Extract\u003c/p\u003e\n\u003cp\u003eMBC \u0026nbsp;\u0026nbsp;Minimum Bactericidal Concentration\u003c/p\u003e\n\u003cp\u003eSME\u0026nbsp; \u0026nbsp;\u0026nbsp;Stems Methanolic Extract\u003c/p\u003e\n\u003cp\u003eMIC\u0026nbsp; \u0026nbsp;\u0026nbsp;Minimum Inhibition Concentration\u003c/p\u003e\n\u003cp\u003eMS\u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Mass Spectrometry\u003c/p\u003e\n\u003cp\u003eOD\u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Optical Density\u003c/p\u003e\n\u003cp\u003eQC\u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Qualitative Control\u003c/p\u003e\n\u003cp\u003eQS \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Quorum Sensing\u003c/p\u003e\n\u003cp\u003eUPLC-UV/DAD \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Ultrahigh Performance Liquid Chromatography coupled to DAD UV-detector\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data were presented in the present manuscript\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB.A.\u003c/strong\u003e Writing- Original draft, data curation, methodology, investigation; \u003cstrong\u003eR.A\u003c/strong\u003e. data curation, methodology, supervision and validation; \u0026nbsp;\u003cstrong\u003eC.E\u003c/strong\u003e. data curation, methodology; \u003cstrong\u003eC.M.\u003c/strong\u003e data curation, methodology; \u003cstrong\u003e\u0026nbsp;B.W.\u003c/strong\u003e data curation, methodology; \u003cstrong\u003eO.N.\u0026nbsp;\u003c/strong\u003edata curation, methodology, supervision and validation; \u003cstrong\u003eH.E\u0026nbsp;\u003c/strong\u003e data curation, supervision, investigation; methodology, writing - review \u0026amp; editing; \u003cstrong\u003eK.M\u003c/strong\u003e. data curation, supervision, investigation, methodology; \u003cstrong\u003eD.M\u003c/strong\u003e. data curation, supervision, investigation, methodology, writing - review \u0026amp; editing\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors are thankful to Mrs. Marijol\u0026egrave;ne. Rey, Dr. Serge Michalet, Dr Isabelle Kerzaon, members of the CESN, for their support in processing the LC/MSMS experiment.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eSzafrański SP, Winkel A, Stiesch M. The use of bacteriophages to biocontrol oral biofilms. J Biotechnol. 2017;250:29\u0026ndash;44.\u003c/li\u003e\n \u003cli\u003eHasan S, Danishuddin M, Adil M, Singh K, Verma PK, Khan AU. Efficacy of E. officinalis on the cariogenic properties of streptococcus mutans: A novel and alternative approach to suppress Quorum-sensing mechanism. PLoS One. 2012;7(7):1\u0026ndash;12.\u003c/li\u003e\n \u003cli\u003eTeresa R, Iglewski BH. MINIREVIEW Bacterial Quorum Sensing in Pathogenic Relationships. Society. 2000;68(9):4839\u0026ndash;49.\u003c/li\u003e\n \u003cli\u003eVisick KL, Foster J, Doino J, McFall-Ngai M, Ruby EG. Vibrio fischeri lux genes play an important role in colonization and development of the host light organ. 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Mucin-Pseudomonas aeruginosa interactions promote biofilm formation and antibiotic resistance. Mol Microbiol. 2006;59(1):142\u0026ndash;51.\u003c/li\u003e\n \u003cli\u003eH\u0026oslash;iby N, Bjarnsholt T, Givskov M, Molin S, Ciofu O. Antibiotic resistance of bacterial biofilms. Int J Antimicrob Agents. 2010;35(4):322\u0026ndash;32.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-complementary-medicine-and-therapies","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcam","sideBox":"Learn more about [BMC Complementary Medicine and Therapies](https://bmccomplementmedtherapies.biomedcentral.com/)","snPcode":"","submissionUrl":"","title":"BMC Complementary Medicine and Therapies","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Prosopis africana, UPLC-UV/DAD-ESI-MS/MS, anti-biofilm, anti-quorum sensing, dental care, polyphenols, alkaloids","lastPublishedDoi":"10.21203/rs.3.rs-7348474/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7348474/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003e\u003cem\u003eProsopis africana\u003c/em\u003e (Guill. \u0026amp; Perr.) Taub. is traditionally used in folk medicine in Burkina Faso for oral diseases. Leaves and stems are used in rural areas to treat dental caries, and the bark is used to treat green diarrhea in infants. In the context of a better understanding of the herbal drug and its bioactivity or toxicity, the present study deals with the chemical profiling of the different botanical parts used in phytomedicine. The impact of herbal medicine on various factors contributing to oral infections and caries, and specifically as anti-biofilm and anti-quorum sensing properties has also been little investigated.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eThe anti-biofilm effect of methanolic extracts of leaves and stems of \u003cem\u003eP. africana\u003c/em\u003e was evaluated on \u003cem\u003eStreptococcus mutans, Staphylococcus aureus, and Pseudomonas aeruginosa\u003c/em\u003e, as well as the anti-quorum sensing effect on \u003cem\u003eChromobacterium\u003c/em\u003e CV026 and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e. The cytotoxicity of the leaves and stems extracts was also evaluated. The chemical composition of the extracts was characterized by UPLC-UV/DAD-MS\u003csup\u003e2\u003c/sup\u003e/ESI-QTOF analysis.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eThe extracts (100\u0026micro;g/ml), without affecting cells viability, significantly reduced the biofilm formation of \u003cem\u003eS. mutans\u003c/em\u003e with the best inhibition rates of 56.7% and 47.6% for stem and leaf extracts respectively. Inhibition rates of 49.03%, 40.2%, and 46.7% were obtained for pyocyanin, elastase, and rhamnolipids respectively, with leaf extracts. No cytotoxic effect on gingival cells was observed for stem extract. UPLC-UV-MS\u003csup\u003e2\u003c/sup\u003e analysis identified sixteen compounds among which mainly polyphenols and alkaloids. They could be related to the activities.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eThe present study provides evidence of efficacy and basic scientific justification for the use of \u003cem\u003eP. africana\u003c/em\u003e in the treatment of dental caries.\u003c/p\u003e","manuscriptTitle":"Anti-biofilm, anti-quorum sensing potential, cytotoxicity, and UPLC-UV/DAD- MS/MS/QTOF profiling of Prosopis africana (Guill. \u0026amp; Perr.) Taub. leaves and stems: benefits of a traditional medicine in dental care","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-08 07:22:21","doi":"10.21203/rs.3.rs-7348474/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-09T10:51:08+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-06T13:31:57+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-01T20:56:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"171438138393155469143163831148817362075","date":"2025-09-25T07:13:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"215833526635394626351837970235054614023","date":"2025-09-24T18:36:51+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-24T17:56:53+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-22T12:09:42+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-15T11:09:10+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Complementary Medicine and Therapies","date":"2025-09-15T10:45:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-complementary-medicine-and-therapies","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcam","sideBox":"Learn more about [BMC Complementary Medicine and Therapies](https://bmccomplementmedtherapies.biomedcentral.com/)","snPcode":"","submissionUrl":"","title":"BMC Complementary Medicine and Therapies","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"494ce5ec-d402-4cd3-85e8-92a6a8c595fe","owner":[],"postedDate":"October 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-10-29T15:23:10+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-08 07:22:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7348474","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7348474","identity":"rs-7348474","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}