<?xml version="1.0" encoding="UTF-8"?><!DOCTYPE article  PUBLIC "-//NLM//DTD Journal Publishing DTD v3.0 20080202//EN" "http://dtd.nlm.nih.gov/publishing/3.0/journalpublishing3.dtd"><article xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink" dtd-version="3.0" xml:lang="en" article-type="research article"><front><journal-meta><journal-id journal-id-type="publisher-id">AiM</journal-id><journal-title-group><journal-title>Advances in Microbiology</journal-title></journal-title-group><issn pub-type="epub">2165-3402</issn><publisher><publisher-name>Scientific Research Publishing</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="doi">10.4236/aim.2023.134010</article-id><article-id pub-id-type="publisher-id">AiM-124194</article-id><article-categories><subj-group subj-group-type="heading"><subject>Articles</subject></subj-group><subj-group subj-group-type="Discipline-v2"><subject>Biomedical&amp;Life Sciences</subject></subj-group></article-categories><title-group><article-title>
 
 
  Anti-Biofilm, Anti-Quorum Sensing, and Anti-Proliferative Activities of Methanolic and Aqueous Roots Extracts of &lt;i&gt;Carica papaya&lt;/i&gt; L. and &lt;i&gt;Cocos nucifera&lt;/i&gt; L.
 
</article-title></title-group><contrib-group><contrib contrib-type="author" xlink:type="simple"><name name-style="western"><surname>Wendkouni</surname><given-names>Leila Marie Esther Belem-Kabré</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="corresp" rid="cor1"><sup>*</sup></xref></contrib><contrib contrib-type="author" xlink:type="simple"><name name-style="western"><surname>Vincent</surname><given-names>Ouédraogo</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author" xlink:type="simple"><name name-style="western"><surname>Bagora</surname><given-names>Bayala</given-names></name><xref ref-type="aff" rid="aff3"><sup>3</sup></xref></contrib><contrib contrib-type="author" xlink:type="simple"><name name-style="western"><surname>Alimata</surname><given-names>Bancé</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author" xlink:type="simple"><name name-style="western"><surname>Estelle</surname><given-names>Ouédraogo</given-names></name><xref ref-type="aff" rid="aff3"><sup>3</sup></xref></contrib><contrib contrib-type="author" xlink:type="simple"><name name-style="western"><surname>Boubacar</surname><given-names>Yaro</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author" xlink:type="simple"><name name-style="western"><surname>Lazare</surname><given-names>Belemnaba</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author" xlink:type="simple"><name name-style="western"><surname>Moussa</surname><given-names>Compaoré</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author" xlink:type="simple"><name name-style="western"><surname>Martin</surname><given-names>Kiendrébeogo</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author" xlink:type="simple"><name name-style="western"><surname>Noufou</surname><given-names>Ouédraogo</given-names></name><xref ref-type="aff" rid="aff4"><sup>4</sup></xref></contrib></contrib-group><aff id="aff2"><addr-line>Laboratory of Biochemistry and Applied Chemistry (LABIOCA), Doctoral School of Science and Technology, Joseph KI-ZERBO University, Ouagadougou, Burkina Faso</addr-line></aff><aff id="aff1"><addr-line>Department of Traditional Medicine and Pharmacopoeia-Pharmacy, Health Sciences Research Institute, Ouagadougou, Burkina Faso</addr-line></aff><aff id="aff3"><addr-line>Laboratory of Molecular Biology and Genetics (LABIOGENE), Doctoral School of Science and Technology, Joseph KI-ZERBO University, Ouagadougou, Burkina Faso</addr-line></aff><aff id="aff4"><addr-line>International Research Laboratory: Environnement, Sant&amp;amp;eacute;, Soci&amp;amp;eacute;t&amp;amp;eacute;s (IRL 3189, ESS) CNRST/CNRS/UCAD/UGB/USTTB, Ouagadougou, Burkina Faso</addr-line></aff><pub-date pub-type="epub"><day>07</day><month>04</month><year>2023</year></pub-date><volume>13</volume><issue>04</issue><fpage>165</fpage><lpage>180</lpage><history><date date-type="received"><day>10,</day>	<month>February</month>	<year>2023</year></date><date date-type="rev-recd"><day>7,</day>	<month>April</month>	<year>2023</year>	</date><date date-type="accepted"><day>10,</day>	<month>April</month>	<year>2023</year></date></history><permissions><copyright-statement>&#169; Copyright  2014 by authors and Scientific Research Publishing Inc. </copyright-statement><copyright-year>2014</copyright-year><license><license-p>This work is licensed under the Creative Commons Attribution International License (CC BY). http://creativecommons.org/licenses/by/4.0/</license-p></license></permissions><abstract><p>
 
 
  <b>Objective:</b> This study focused on the antibacterial and anti-proliferative activity of extracts from 
  Carica papaya and 
  Cocos nucifera roots. 
  <b>Methodology:</b> The minimum inhibitory concentration and the minimum bactericidal concentration of the extracts on 
  Escherichia coli, 
  Pseudomonas aeruginosa, 
  Streptococcus mutans, and 
  Staphylococcus aureus were deduced by the microdilution method. The anti-biofilm activity was determined on all four strains and anti-quorum sensing activity by inhibition of violacein production in 
  Chromobacterium violaceum. Anti-proliferative activity on prostate cultured cancer cells was evaluated by MTT assay. Sterols and triterpenes were also assayed in this study. 
  <b>Results:</b> The methanolic extract of 
  Carica papaya showed the best anti-biofilm effect with a percentage inhibition of 66.10 &#177; 1.79. The methanolic extract of 
  Cocos nucifera had the strongest inhibition on the production of quorum sensing (61.42 &#177; 0.28). In addition, the methanolic extract of 
  Cocos nucifera roots showed the best cytotoxic effect on prostate cancer LNCaP cell lines (IC
  <sub>50</sub> = 26.98 &#177; 2.6 μg/mL) and 
  Carica papaya on the PC-3 lines (IC
  <sub>50</sub> = 127.20 &#177; 5.99 μg/mL). The extracts were also rich in triterpenes and sterols. 
  <b>Conclusion:</b> This study provides support for the ethnomedical use of 
  Carica papaya and 
  Cocos nucifera roots as an antimicrobial and anticancer.
 
</p></abstract><kwd-group><kwd>Triterpenes and Sterols Content</kwd><kwd> Antibacterial</kwd><kwd> Biofilm</kwd><kwd> Quorum Sensing</kwd><kwd> Anti-Proliferative</kwd><kwd> Medicinal Plants</kwd></kwd-group></article-meta></front><body><sec id="s1"><title>1. Introduction</title><p>The upsurge of resistant microbial pathogenic strains and cancer has become a global public health concern [<xref ref-type="bibr" rid="scirp.124194-ref1">1</xref>] [<xref ref-type="bibr" rid="scirp.124194-ref2">2</xref>] . Indeed, antimicrobial resistance is recognized as one of the top ten growing public health threats, currently estimated to account for more than 700,000 deaths annually. If proper care is not taken, this figure could rise substantially to 10 million by 2050, thus leading our planet to the post-antibiotic era [<xref ref-type="bibr" rid="scirp.124194-ref3">3</xref>] . Likewise, the development of resistance to chemotherapy continues to be the main barrier in the treatment of cancer patients [<xref ref-type="bibr" rid="scirp.124194-ref4">4</xref>] . Consequently, there is a significant need for newer agents with low susceptibility to common drug resistance mechanisms to improve response rates and potentially extend survival [<xref ref-type="bibr" rid="scirp.124194-ref4">4</xref>] . Over the years, nature has been a source of medicinal agents. The extraction, isolation, and identification of plants containing phytochemicals have led to the discovery of new therapeutics in the research and development sector of the pharmaceutical industry [<xref ref-type="bibr" rid="scirp.124194-ref3">3</xref>] . Therefore, since ancient times a plethora of plant extracts and active biological compounds have been widely identified and documented for the treatment of infectious and cancerous diseases [<xref ref-type="bibr" rid="scirp.124194-ref5">5</xref>] [<xref ref-type="bibr" rid="scirp.124194-ref6">6</xref>] [<xref ref-type="bibr" rid="scirp.124194-ref7">7</xref>] [<xref ref-type="bibr" rid="scirp.124194-ref8">8</xref>] . Thus, curcumin from Curcuma Longa L. (Zingiberaceae) has been used in the treatment of infectious diseases. Taxol a very potent chemotherapeutic compound derived from the bark of Taxus brevifolia (Taxaceae) is very active in the treatment of cancer. Morphine from Papaver somniferum L. (Papaveraceae) is a powerful analgesic [<xref ref-type="bibr" rid="scirp.124194-ref3">3</xref>] [<xref ref-type="bibr" rid="scirp.124194-ref9">9</xref>] . Furthermore, plants offer a unique and renewable resource for the discovery of new therapeutically active biomolecules thanks to the structural and biological properties of their constituents [<xref ref-type="bibr" rid="scirp.124194-ref10">10</xref>] . In the World Health Organization “Traditional Medicine Strategy” for the period 2014-2023, it is recognized that traditional medicines are often underestimated in terms of the role they can play in health care systems. She further states that medicinal plants may in some countries be the main source of health care or even the only health service available, especially for rural populations [<xref ref-type="bibr" rid="scirp.124194-ref11">11</xref>] . The main advantages of using plant-derived medicine are that they are safer than synthetic alternatives, providing therapeutic benefits and affordable treatment [<xref ref-type="bibr" rid="scirp.124194-ref5">5</xref>] [<xref ref-type="bibr" rid="scirp.124194-ref12">12</xref>] .</p><p>This study is a continuation of our previous work on the energy capacity, antioxidant and anti-inflammatory properties of C. papaya and C. nucifera root extracts [<xref ref-type="bibr" rid="scirp.124194-ref13">13</xref>] . Therefore, in the present report, we set out to study the effect of plant extracts on biofilm formation on a few bacterial strains, namely Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus mutans, as well as their ability to interfere with the bacterial quorum sensing system. The cytotoxicity of the extracts on prostate cancer cell lines and the total content of triterpenes and sterols were also provided.</p></sec><sec id="s2"><title>2. Material and Methods</title><sec id="s2_1"><title>2.1. Study Area</title><p>This study was carried out between from October, 2019 to November, 2020 at the Department of traditional medicine of Research Institute of Health Sciences, the Laboratory of Applied Biochemistry and Chemistry of the University Joseph KI-ZERBO; and the Pietro Annigoni Biomolecular Research Center (Ouagadougou, Burkina Faso).</p></sec><sec id="s2_2"><title>2.2. Standards and Reagents</title><p>All chemicals and reagents used to carry out the experiments were of analytical grade.</p><p>N-hexanoyl-L-homoserine lactone (HHL), Crystal violet, iodonitrotetrazolium chloride (INT), salicylic acid, agar agar, Brain Heart Infusion (BHI) broth and Luria-Bertani (LB) broth, blue trypan, 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and Phosphate buffered saline PBS were purchased from Sigma&#174; (St Louis, USA). Dimethyl sulfoxide (DMSO), methanol, glycerol, hydrochloric acid, isopropanol, acetic acid, and ethanol were procured from Prolabo (France). Culture medium RPMI-1640, Penicillin, and streptomycin were purchased from Invitrogen (Oslo, Norway); and Fetal calf serum from Biowest, Nuaill&#233;, France.</p></sec><sec id="s2_3"><title>2.3. Plant Material</title><p>The Fresh roots of Carica papaya were collected in September of 2018 from the area of Dedougou/Burkina Faso. The plant was taxonomically authenticated and a voucher specimen bearing the number T4316 was deposited at the Plant Biology and Ecology Laboratory of the University Joseph KI-ZERBO, Burkina Faso. Fresh roots of Cocos nucifera were provided by a tradipractician and were also authenticated.</p></sec><sec id="s2_4"><title>2.4. Preparation of the Plant’s Extracts</title><p>The roots of two plants were shade-dried at room temperature. Roots powder was obtained by a blade crusher. Two extracts (aqueous and methanolic) of each plant were prepared from the powdered roots.</p><p>The aqueous maceration was obtained by dispersing 50 g of each powder in 500 mL of distilled water. After 24 hours at room temperature, the extract was filtered, freeze at −54˚C and freeze-dried under pressure reduced to 0.151 mbar at −49˚C during 72 hours.</p><p>To prepare the methanolic maceration, the same method was used. Thereafter, the extract was filtered, concentrated using a rotary vacuum evaporator, and put in an oven until completely dry.</p></sec><sec id="s2_5"><title>2.5. Dosage of Triterpenes and Sterols</title><p>The total content of triterpenes and sterols was determined colorimetrically using the procedure described by Chang et al. [<xref ref-type="bibr" rid="scirp.124194-ref14">14</xref>] with slight modifications. 30 μL of vanillin—glacial acetic acid 5% solution was mixed with 20 μL of total genins previously extracted (5 mg/mL) and dissolved in methanol. 100 μL of perchloric acid was then added. The mixture was placed in a water bath at 60˚C for 45 min, then cooled in an ice water bath for a few minutes. After the addition of 450 &#181;L of glacial acetic acid, the absorbance of each sample solution was measured using a spectrophotometer (BioTeck instruments, USA) at 548 nm against a curve of ursolic acid (y = 0.1259x + 0.0653; R<sup>2</sup> = 0.99) for the triterpenes (<xref ref-type="fig" rid="fig1">Figure 1</xref>). The absorbance was also measured at 640 nm against a cholesterol calibration curve (y = 0.0744x − 0.0056; R<sup>2</sup> = 0.99) for sterols (<xref ref-type="fig" rid="fig2">Figure 2</xref>).</p><p>The analyzes were carried out in triplicate and the results were evaluated respectively in mg Ursolic Acid Equivalent per g of extract (mg UAE/g extract) and mg Cholesterol Equivalent per g of extract (mg CE/g extract).</p></sec><sec id="s2_6"><title>2.6. Antimicrobial Testing</title><sec id="s2_6_1"><title>2.6.1. Bacterial Strains Used in Assays and Growth Conditions</title><p>The pathogens used for the experiment were: Escherichia coli ATCC (American Types Collection Culture) 25922, Pseudomonas aeruginosa PAO1, Staphylococcus aureus ATCC 43300, Streptococcus mutans ATCC 25175, and Chromobacterium violaceum CV026. The strains Escherichia coli, Pseudomonas aeruginosa, and Chromobacterium violaceum were grown in LB broth medium while Staphylococcus aureus and Streptococcus mutans were grown in BHI liquid medium. All strains were grown at 37˚C except CV026 grown at 30˚C.</p></sec><sec id="s2_6_2"><title>2.6.2. Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)</title><p>The MIC values of the extracts were determined using the 96-well plate method according to Eloff [<xref ref-type="bibr" rid="scirp.124194-ref15">15</xref>] . To do this, a range of dilutions of the extracts was carried out on a sterile microplate from a stock of 2 mg/mL final concentration. To 100 &#181;L of each dilution was added 100 &#181;L of inoculum (10<sup>6</sup> CFU/mL) prepared in the corresponding culture. The microplates were then incubated for 24 hours at 37˚C after which 30 μL of p-iodonitrotetrazolium (INT) (2 mg/mL) was added to each well. 30 min after the addition of INT in the dark, the MIC of the extracts was visibly deduced. Living microorganisms reduce the INT by producing a pink color [<xref ref-type="bibr" rid="scirp.124194-ref16">16</xref>] .</p><p>The MBC was determined using samples from the MIC microplate. A loopful of inoculum was collected from the well with the concentration before the well that had been read as the MIC. They were then streaked onto agar medium. The Petri dishes were incubated for 24 hours at 37˚C. The CMB was visibly deduced as being the first dish devoid of bacteria [<xref ref-type="bibr" rid="scirp.124194-ref17">17</xref>] .</p></sec><sec id="s2_6_3"><title>2.6.3. Biofilm Formation Using Crystal Violet Assay</title><p>Anti-biofilm activity of C. papaya and C. nucifera extracts on the strains (Escherichia coli, Streptococcus mutans, Pseudomonas aeruginosa, Staphylococcus aureus) was carried out according to crystal violet method on 96-well plates, based on Vandeputte et al [<xref ref-type="bibr" rid="scirp.124194-ref18">18</xref>] . An appropriate dilution (100 μL) of each strain overnight culture was added to the corresponding culture medium supplemented (80 &#181;L) with 20 μL of extract, salicylic acid (reference substance), or DMSO (100 μg/mL, final concentration). After incubation of the plates, the absorbances were read at 600 nm to follow the bacterial growth compared to the negative control (DMSO). The supernatant from each well was then removed and the biofilms were gently washed with distilled water, fixed with methanol for 15 min, and dried. 0.1% crystal violet (in water) was added to each well and the plates were incubated for 30 min. After removing the crystal violet, the wells were rinsed with distilled water and 200 μL of acetic acid (33% in water) was added to dissolve the crystal violet. The absorbances of the solution were read at 590 nm and the biofilm/bacterial growth ratio (590 nm/600 nm) was determined. The percentage inhibition of the extracts was calculated by the following formula:</p><p>% Inhibition = [(Ac − At)/Ac] &#215; 100</p><p>Ac: absorbance of negative control; At: absorbance of the tests (extracts, reference).</p></sec><sec id="s2_6_4"><title>2.6.4. Violacein Production in Chromobacterium Violaceum Assay</title><p>C. violaceum CV026 was used to determine anti-quorum sensing (QS) activity by quantifying QS-controlled violacein production. Thus, the extracts were evaluated by their ability to inhibit the production of violacein in C. violaceum according to the method described by Choo et al. [<xref ref-type="bibr" rid="scirp.124194-ref19">19</xref>] adapted by Ouedraogo et al [<xref ref-type="bibr" rid="scirp.124194-ref20">20</xref>] . Briefly, C. violaceum was grown for 24 h at 30˚C. CV026 was diluted and introduced into a 12-well plate, then the extracts and reference (salicylic acid) were added to have a final concentration of 100 μg/mL in the presence of HHL. The plates were incubated for 24 hours at 30˚C. Bacterial turbidity was measured at 600 nm to assess bacterial growth, then 1 mL from each well was taken and introduced into tubes to quantify the violacein. The solution was vigorously vortexed to solubilize the violacein then the supernatant was removed and 1 mL of DMSO was added to the pellet. After centrifugation, 200 μL of the supernatant containing the violacein are introduced into a 96-well microplate. The production of violacein was quantified by measuring the absorbance at 585 nm and the percent inhibition was calculated against the negative control (DMSO). The ratio between 585 nm and 600 nm was also determined.</p></sec></sec><sec id="s2_7"><title>2.7. Antiproliferative Activity</title><sec id="s2_7_1"><title>2.7.1. Cancer Cell Lines and Culture Conditions</title><p>The cell lines used were adherent LNCaP (androgen-sensitive) and PC-3 (androgen-resistant) prostate cancer cells supplied to CERBA/LABIOGENE laboratories by the GReD Laboratory (University Clermont-Auvergne, France). The LNCaP (Lymph Node Cancer of the Prostate) and PC-3 cell lines were cultured and maintained at 37˚C in a humidified incubator with 5% CO<sub>2</sub> in 75 cm<sup>2</sup> cell culture flasks, in RPMI-1640 (Roswell Park Memorial Institute) medium supplemented with 10% Fetal Calf Serum, 1% Penicillin-Streptomycin and 1% L-Glutamine.</p></sec><sec id="s2_7_2"><title>2.7.2. Cytotoxicity Assay</title><p>The MTT assay was used to measure cell survival. Briefly, 10,000 cells/mL were seeded for 24 h in a 96-wells microplate. After 24 h, the methanolic extracts of Carica papaya and Cocos nucifera were dissolved in complete culture medium RPMI-1640 (1% DMSO) and a dilution range was achieved of concentrations ranging from 500 &#181;g/mL to 1.95 &#181;g/mL. 50 &#181;L of each extract was then brought into contact with the cells in the wells and the plates were incubated again. After 72 h incubation, the number of living cells was measured as described by Bayala et al. [<xref ref-type="bibr" rid="scirp.124194-ref21">21</xref>] using a microplate reader type Bio-Rad 11885 at 490 nm. Data are the results of three independent experiments for each cell line. Growth inhibition was calculated as follows:</p><p>% Inhibition = 100 − [(Extract absorbance − blank absorbance)/(Control absorbance − blank absorbance)] &#215;100.</p></sec></sec><sec id="s2_8"><title>2.8. Statistical Analysis</title><p>The data were expressed as Mean &#177; Standard Error of Mean (SEM). The statistical analysis was carried out according to one-way ANOVA analysis followed by Dunnett’s test compared to the control and different extracts on Graph Pad Prism software version 6.0. The level of significance was accepted at p &lt; 0.05.</p></sec></sec><sec id="s3"><title>3. Results</title><sec id="s3_1"><title>3.1. Total Triterpenes and Sterols Contents</title><p>The total triterpenes and sterols contents of methanolic and aqueous extracts were showed in <xref ref-type="table" rid="table1">Table 1</xref>. The methanolic extracts exhibited higher levels.</p></sec><sec id="s3_2"><title>3.2. Total Minimum Inhibitory Concentration and Minimum Bactericidal Concentration</title><p>MIC and MBC values of C. papaya and C. nucifera extracts on tested strains were recorded in <xref ref-type="table" rid="table1">Table 1</xref>. The Minimum Inhibitory Concentration of the extracts was greater than 2 mg/mL except for the methanolic extracts on Staphylococcus aureus which were 2 mg/mL. The MBC of the extracts on all the studied strains is greater than 2 mg/mL.</p><table-wrap id="table1" ><label><xref ref-type="table" rid="table1">Table 1</xref></label><caption><title> Content of extracts in total triterpenes and sterols</title></caption><table><tbody><thead><tr><th align="center" valign="middle" >Extracts</th><th align="center" valign="middle" >Total Triterpenes mg UAE/g extract</th><th align="center" valign="middle" >Total Sterols mg CE/g extract</th></tr></thead><tr><td align="center" valign="middle" >CN M</td><td align="center" valign="middle" >349.56 &#177; 14</td><td align="center" valign="middle" >242.3 &#177; 5</td></tr><tr><td align="center" valign="middle" >CN A</td><td align="center" valign="middle" >159.8 &#177; 11</td><td align="center" valign="middle" >132.8 &#177; 6.2</td></tr><tr><td align="center" valign="middle" >CP M</td><td align="center" valign="middle" >343.3 &#177; 4.6</td><td align="center" valign="middle" >245.2 &#177; 8.3</td></tr><tr><td align="center" valign="middle" >CP A</td><td align="center" valign="middle" >179.6 &#177; 16</td><td align="center" valign="middle" >144.02 &#177; 8.3</td></tr></tbody></table></table-wrap><p>Legend: UAE: Ursolic Acid Equivalent, CE: Cholesterol Equivalent, CN M: C. nucifera methanolic extract, CN AQ: C. nucifera aqueous extract, CP M: C. papaya methanolic extract, CP AQ: C. papaya aqueous extract.</p><table-wrap id="table2" ><label><xref ref-type="table" rid="table2">Table 2</xref></label><caption><title> MIC and MBC of C. nucifera and C. papaya extracts on Escherichia coli, Pseudomonas aeruginosa, Streptococcus mutans and Staphylococcus aureus</title></caption><table><tbody><thead><tr><th align="center" valign="middle"  rowspan="2"  >Extracts</th><th align="center" valign="middle" >Strains</th><th align="center" valign="middle" >MIC</th><th align="center" valign="middle" >MBC</th></tr></thead><tr><td align="center" valign="middle" ></td><td align="center" valign="middle"  colspan="2"  >mg/mL</td></tr><tr><td align="center" valign="middle" >CN M, CN A, CP M, CP A</td><td align="center" valign="middle" >E. Coli P. aeruginosa S. mutans</td><td align="center" valign="middle" >&gt;2</td><td align="center" valign="middle" >&gt;2</td></tr><tr><td align="center" valign="middle" >CN M, CP M CN A, CP A</td><td align="center" valign="middle" >S. aureus</td><td align="center" valign="middle" >2 &gt;2</td><td align="center" valign="middle" >&gt;2 &gt;2</td></tr></tbody></table></table-wrap><p>Legend: CN M: C. nucifera methanolic extract, CN AQ: C. nucifera aqueous extract, CP M: C. papaya methanolic extract, CP AQ: C. papaya aqueous extract.</p></sec><sec id="s3_3"><title>3.3. Inhibition of Extracts on Biofilm Formation</title><p>The effect of extracts from the roots of C. papaya and C. nucifera at a concentration of 100 μg/mL on the biofilm formation of Escherichia coli, Streptococcus mutans, Staphylococcus aureus, Pseudomonas aeruginosa was evaluated after 24 hours of growth. At this concentration, the extracts did not affect bacterial growth as shown in <xref ref-type="fig" rid="fig3">Figure 3</xref>(a), <xref ref-type="fig" rid="fig3">Figure 3</xref>(c), <xref ref-type="fig" rid="fig4">Figure 4</xref>(a), and <xref ref-type="fig" rid="fig4">Figure 4</xref>(c). Unlike DMSO used as a control, the extracts reduced the formation of the biofilm of the strains studied (<xref ref-type="fig" rid="fig3">Figure 3</xref>(b), <xref ref-type="fig" rid="fig3">Figure 3</xref>(d), <xref ref-type="fig" rid="fig4">Figure 4</xref>(b), and <xref ref-type="fig" rid="fig4">Figure 4</xref>(d)). The methanolic extract of C. nucifera had a better inhibition on gram-positive bacteria (52.72% inhibition of the biofilm formed by Pseudomonas aeruginosa) and the methanolic extract of C. papaya on gram-negative (66.10% of inhibition of the biofilm formed by Streptococcus mutans). The salicylic acid used as a reference substance was in general more active compared to the extracts or was not significant (inhibition of C. nucifera on P. aeruginosa).</p></sec><sec id="s3_4"><title>3.4. Effect of Extract on Violacein Production</title><p>The effect of the extracts on violacein production was evaluated at the final concentration of 100 μg/mL. As shown in <xref ref-type="fig" rid="fig5">Figure 5</xref>(a), the viability, and growth of</p><disp-formula id="scirp.124194-formula2"><graphic  xlink:href="//html.scirp.org/file/1-2271906x5.png?20230407180432935"  xlink:type="simple"/></disp-formula><p>Legend: OD: optical density, CN M: C. nucifera methanolic extract, CN AQ: C. nucifera aqueous extract, CP M: C. papaya methanolic extract, CP AQ: C. papaya aqueous extract. ****p &lt; 0.05 Extracts/Salicylic acid vs DMSO.</p><p><xref ref-type="fig" rid="fig3">Figure 3</xref>. Effect of C. nucifera, C. papaya extracts, and Salicylic acid on biofilm formation of two Gram-positive bacteria. (a) Effects on E. coli growth; (b) Effects on Biofilm formed by E. coli; (c) Effects on P. aeruginosa growth; (d) Effects on Biofilm formed by P. aeruginosa.</p><p>C. violaceum CV026 were not influenced by the presence of extracts. C. violaceum strain has insufficient capacity to produce the quorum detectable by self-inducers (homoserine-lactones). Thus, the addition of homoserine-lactone in the growth medium allowed the production of violacein. Then, the reduction in the production of violacein was the consequence of the interference of the extract with the mechanisms of quorum Sensing of C. violaceum CV026 (<xref ref-type="fig" rid="fig5">Figure 5</xref>(b)). All extracts reduced the production of violacein with a better inhibition recorded by the methanolic extract of C. nucifera (61.42%).</p></sec><sec id="s3_5"><title>3.5. Anti-Proliferative Effects on Prostate Cancer Cell Lines</title><p>The different IC<sub>50</sub> of the methanolic extracts of C. papaya and C. nucifera corresponding to the results of the anti-proliferative activity tests are given in <xref ref-type="table" rid="table3">Table 3</xref>. The extracts exhibited anti-proliferative activity on prostate cancer cell lines LNCaP and PC-3. C. nucifera demonstrated the best activity on LNCAP, either an IC<sub>50</sub> of 26.98 &#177; 2.6 &#181;g/mL and C. papaya on PC-3 with an IC<sub>50</sub> of 127.20 &#177; 5.99 &#181;g/mL.</p><p><xref ref-type="fig" rid="fig6">Figure 6</xref> shows the viability of LNCaP and PC-3 cells according to the concentrations of each extract used.</p></sec></sec><sec id="s4"><title>4. Discussion</title><p>Medicinal plants have been used as a possible source of new classes of antibiotics with new modes of action and represent an alternative to treat infections caused by resistant microbes [<xref ref-type="bibr" rid="scirp.124194-ref22">22</xref>] . C. papaya and C. nucifera are two medicinal plants used in Burkina Faso for the management of infectious diseases and with an anti-inflammatory component such as cancer. In addition, in vitro antioxidant and</p><table-wrap id="table3" ><label><xref ref-type="table" rid="table3">Table 3</xref></label><caption><title> IC<sub>50</sub> of methanolic extracts of C. nucifera and C. papaya on the viability of LNCaP and PC-3 cells in prostate cancer</title></caption><table><tbody><thead><tr><th align="center" valign="middle"  rowspan="2"  >Extracts</th><th align="center" valign="middle" >PC-3 cell lines</th><th align="center" valign="middle" >LNCaP cell lines</th></tr></thead><tr><td align="center" valign="middle"  colspan="2"  >IC<sub>50</sub> (&#181;g/mL)</td></tr><tr><td align="center" valign="middle" >Cocos nucifera</td><td align="center" valign="middle" >195.19 &#177; 1.24</td><td align="center" valign="middle" >26.98 &#177; 2.6</td></tr><tr><td align="center" valign="middle" >Carica papaya</td><td align="center" valign="middle" >127.20 &#177; 5.99</td><td align="center" valign="middle" >332.12 &#177; 4.43</td></tr></tbody></table></table-wrap><p>Legend: IC<sub>50</sub>: Inhibitory concentration 50; Values are expressed as mean values &#177; standard deviation. n = 3 independent experiments.</p><p>anti-inflammatory properties of extracts from these plants have been demonstrated previously [<xref ref-type="bibr" rid="scirp.124194-ref13">13</xref>] [<xref ref-type="bibr" rid="scirp.124194-ref23">23</xref>] . The same study showed that the extracts are rich in polyphenols, tannins, and flavonoids [<xref ref-type="bibr" rid="scirp.124194-ref13">13</xref>] . It emerges from this study that the extracts of C. nucifera and C. papaya are rich in triterpenes and sterols (<xref ref-type="table" rid="table1">Table 1</xref>). The lipophilic nature of terpenes offers cytotoxic activities against a wide range of organisms ranging from bacteria to selected organisms. They are used in herbal medicine against infections [<xref ref-type="bibr" rid="scirp.124194-ref24">24</xref>] .</p><p>Biofilm formation is a major problem factor in many areas, ranging from industrial corrosion and biofouling to chronic and nosocomial infections [<xref ref-type="bibr" rid="scirp.124194-ref25">25</xref>] . Indeed, many strains of uropathogens can form a biofilm (physical barrier against antimicrobial agents) allowing them to avoid the immune system and increase their resistance to recommended drugs [<xref ref-type="bibr" rid="scirp.124194-ref26">26</xref>] [<xref ref-type="bibr" rid="scirp.124194-ref27">27</xref>] . The results of this study showed that the extracts resulted in the formation of biofilm on the resistant strains used (<xref ref-type="fig" rid="fig3">Figure 3</xref> and <xref ref-type="fig" rid="fig4">Figure 4</xref>). The eradication of biofilms is of great importance in the fight against infection [<xref ref-type="bibr" rid="scirp.124194-ref27">27</xref>] . The ability to inhibit the biofilm of E. coli by the ethanolic extract of the leaves of Carica papaya over a range of concentrations was shown in the study conducted by Hastuty [<xref ref-type="bibr" rid="scirp.124194-ref28">28</xref>] . Coconut water has an anti-biofilm activity of P. aeruginosa [<xref ref-type="bibr" rid="scirp.124194-ref29">29</xref>] . The tannins, flavonoids, and triterpenes in the extracts are known for their ability to disrupt the biofilm of bacteria by interfering with its mechanism [<xref ref-type="bibr" rid="scirp.124194-ref28">28</xref>] [<xref ref-type="bibr" rid="scirp.124194-ref30">30</xref>] . Biofilms are associated with microbial infections and their formation can be regulated by quorum sensing [<xref ref-type="bibr" rid="scirp.124194-ref30">30</xref>] . Because of continuing emergence and spread of multidrug-resistant bacteria, an antipathogenic strategy to combat bacterial infections through the disruption of quorum sensing controlled virulence factors has received increased attention [<xref ref-type="bibr" rid="scirp.124194-ref31">31</xref>] . In the present study, one of the best-known cases of quorum-sensing-regulated phenotypes, pigment production by the bio-reporter strain of C. violaceum was used to screen the four extracts for their potential for inhibiting quorum sensing (<xref ref-type="fig" rid="fig5">Figure 5</xref>). Quorum sensing is a recently discovered chemical communication system that enhances bacterial survival, as a group allowing resident bacteria to assume specialized roles vital for the regulation of intra- and inter-bacterial genes, and for maintaining bacterial colonies intact [<xref ref-type="bibr" rid="scirp.124194-ref31">31</xref>] [<xref ref-type="bibr" rid="scirp.124194-ref32">32</xref>] . Violacein production by C. violaceum was significantly inhibited by the extracts in the present study. The decrease in the production of violacein is therefore the consequence of interference of the extracts with the mechanisms of QS. The effect of the extracts could be due to the polyphenols they contain and in particular to the flavonoids known for their anti-QS potential [<xref ref-type="bibr" rid="scirp.124194-ref33">33</xref>] [<xref ref-type="bibr" rid="scirp.124194-ref34">34</xref>] . In general, most of the extracts showed no antibacterial activity within the limit of the maximum concentration used which was 2 mg/mL (<xref ref-type="table" rid="table2">Table 2</xref>).</p><p>This might be due to the effects of the extracts’ compounds in limited molecular target areas, which could involve only in quorum sensing signaling of the bacteria [<xref ref-type="bibr" rid="scirp.124194-ref19">19</xref>] [<xref ref-type="bibr" rid="scirp.124194-ref31">31</xref>] . Thus, our result indicates the need for a thorough examination of plants by identifying the active compounds and the mechanism of quorum sensing actions by the active compound. The methanolic extracts demonstrated a better antibacterial effect than the aqueous extracts which could be due to the difference in the contents of the phytochemical compounds highlighted. In addition, the extracts are more active against gram-positive bacteria (S. mutans, S. aureus) than gram-negative bacteria (E. coli, P. aeruginosa). The peptidoglycan layer present in gram-positive bacteria could be the target of phytochemicals contained in the extracts [<xref ref-type="bibr" rid="scirp.124194-ref35">35</xref>] .</p><p>Cytotoxicity was estimated using the MTT assay, which is a routine test assessing cell viability and proliferative activity through cell enzyme activity [<xref ref-type="bibr" rid="scirp.124194-ref36">36</xref>] . Human prostate adenocarcinoma cell lines, such as LNCaP (androgen-dependent), and PC-3 (androgen-independent) are among the most often used in vitro experiments [<xref ref-type="bibr" rid="scirp.124194-ref37">37</xref>] . The extracts exhibited a detrimental effect on cancer cell viability in a concentration-dependent manner, unlike the DMSO negative control which did not affect the cells (<xref ref-type="fig" rid="fig6">Figure 6</xref>). According to the classification of the plant screening program of the National Cancer Institute of the United States, the extract of C. nucifera has good cytotoxic potential on the viability of LNCaP cells of prostate cancer with an IC<sub>50</sub> = 26. 98 &#177; 2.6 μg/mL (<xref ref-type="table" rid="table3">Table 3</xref>). Furthermore, the extracts of C. papaya and C. nucifera show a moderate effect on the PC-3 lines. According to Bayala et al. [<xref ref-type="bibr" rid="scirp.124194-ref38">38</xref>] , the cytotoxic effects in this study could be attributed in part to the antioxidant potency of the extracts demonstrated in the previous studies. Indeed, the cytotoxic and anti-proliferative effect of triterpenes, tannins, flavonoids, and saponins highlighted in the extracts has been proven [<xref ref-type="bibr" rid="scirp.124194-ref38">38</xref>] [<xref ref-type="bibr" rid="scirp.124194-ref39">39</xref>] .</p></sec><sec id="s5"><title>5. Conclusion</title><p>This study demonstrated that extracts from the roots Carica papaya and Coco nucifera are remarkable antibacterial agents against the biofilm formed by Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus mutans strains, and while inhibiting the signal molecules, which form the basis of the bacterial communication mechanism. Cocos nucifera extract has therefore shown a good cytotoxic effect and could be further investigated in order also to develop an anticancer phytomedicine. Those data provide scientific data and show that the folkloric uses of the roots of Carica papaya and Cocos nucifera to treat certain health problems could have a scientific basis.</p></sec><sec id="s6"><title>Acknowledgements</title><p>The authors are thankful to the Laboratory of Applied Biochemistry and Chemistry (University Joseph KI-ZERBO); Pietro Annigoni Biomolecular Research Center (CERBA); and the Department of traditional medicine of Research Institute of Health Sciences (IRSS).</p></sec><sec id="s7"><title>Authors’ Contributions</title><p>WLME B-K performed the tests, analyzed the results, and wrote the manuscript. VO and AB contributed to the achievement of antibiofilm and anti-quorum activity. BB guided antiproliferative test. EO contributed to the performance of antiproliferative activity. BY contributed to perform the phytochemical dosage. LB, MC and MK reviewed the final version. NO supervised the study, read and approved the final version of the manuscript.</p></sec><sec id="s8"><title>Conflicts of Interest</title><p>The authors declare no conflicts of interest regarding the publication of this paper.</p></sec><sec id="s9"><title>Cite this paper</title><p>Belem-Kabr&#233;, W.L.M.E., Ou&#233;draogo, V., Bayala, B., Banc&#233;, A., Ou&#233;draogo, E., Yaro, B., Belemnaba, L., Compaor&#233;, M., Kiendr&#233;beogo, M. and Ou&#233;draogo, N. (2023) Anti-Biofilm, Anti-Quorum Sensing, and Anti-Proliferative Activities of Methanolic and Aqueous Roots Extracts of Carica papaya L. and Cocos nucifera L. Advances in Microbiology, 13, 165-180. https://doi.org/10.4236/aim.2023.134010</p></sec></body><back><ref-list><title>References</title><ref id="scirp.124194-ref1"><label>1</label><mixed-citation publication-type="other" xlink:type="simple">Ferlay, J., Colombet, M., Soerjomataram, I., Mathers, C., Parkin, D., Pi&amp;ntilde;eros, M., Znaor, A. and Bray, F. 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