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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Pilot Study with regard to the Wound Healing Activity of Protein from <italic>Calotropis procera</italic> (Ait.) R. Br.</title><author><name sortKey="Perumal Samy, Ramar" sort="Perumal Samy, Ramar" uniqKey="Perumal Samy R" first="Ramar" last="Perumal Samy">Ramar Perumal Samy</name></author><author><name sortKey="Chow, Vincent T K" sort="Chow, Vincent T K" uniqKey="Chow V" first="Vincent T. K." last="Chow">Vincent T. K. Chow</name></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">22973400</idno><idno type="pmc">3437960</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3437960</idno><idno type="RBID">PMC:3437960</idno><idno type="doi">10.1155/2012/294528</idno><date when="2012">2012</date><idno type="wicri:Area/Pmc/Corpus">003E09</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">003E09</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Pilot Study with regard to the Wound Healing Activity of Protein from <italic>Calotropis procera</italic> (Ait.) R. Br.</title><author><name sortKey="Perumal Samy, Ramar" sort="Perumal Samy, Ramar" uniqKey="Perumal Samy R" first="Ramar" last="Perumal Samy">Ramar Perumal Samy</name></author><author><name sortKey="Chow, Vincent T K" sort="Chow, Vincent T K" uniqKey="Chow V" first="Vincent T. K." last="Chow">Vincent T. K. Chow</name></author></analytic><series><title level="j">Evidence-based Complementary and Alternative Medicine : eCAM</title><idno type="ISSN">1741-427X</idno><idno type="eISSN">1741-4288</idno><imprint><date when="2012">2012</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>We provide the scientific basis for the use of <italic>Calotropis procera</italic> for treating skin
and wound infections in traditional medicine. The aqueous extract of stem-bark of <italic>C. procera</italic> exhibited more pronounced potent antimicrobial activity. Calo-protein was purified and identified from the most-active aqueous extracts of <italic>C. procera</italic> and showed broad-spectrum activity. Calo-protein inhibited the growth of <italic>S. aureus</italic> and <italic>E. aerogenes</italic> effectively at 25 <italic><italic>μ</italic></italic>g/ml concentration. Mice topically treated with Calo-protein revealed significant wound healing after 14 days comparable to fusidic acid (FA) as positive control. This protein was devoid of cytolytic effect even at higher concentrations on skin cells after 24 h. Further investigation of this Calo-protein of <italic>C. procera</italic> on bacterial inhibition may provide a better understanding of the scientific basis and justification for its use in traditional medicine.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Frei, Cr" uniqKey="Frei C">CR Frei</name></author><author><name sortKey="Makos, Br" uniqKey="Makos B">BR Makos</name></author><author><name sortKey="Daniels, Kr" uniqKey="Daniels K">KR Daniels</name></author><author><name sortKey="Oramasionwu, Cu" uniqKey="Oramasionwu C">CU Oramasionwu</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Pichereau, S" uniqKey="Pichereau S">S Pichereau</name></author><author><name sortKey="Rose, We" uniqKey="Rose W">WE Rose</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Klevens, Rm" uniqKey="Klevens R">RM Klevens</name></author><author><name sortKey="Morrison, Ma" uniqKey="Morrison M">MA Morrison</name></author><author><name sortKey="Nadle, J" uniqKey="Nadle J">J Nadle</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Moet, Gj" uniqKey="Moet G">GJ Moet</name></author><author><name sortKey="Jones, Rn" uniqKey="Jones R">RN Jones</name></author><author><name sortKey="Biedenbach, Dj" uniqKey="Biedenbach D">DJ Biedenbach</name></author><author><name sortKey="Stilwell, Mg" uniqKey="Stilwell M">MG Stilwell</name></author><author><name sortKey="Fritsche, Tr" uniqKey="Fritsche T">TR Fritsche</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Driscoll, P" uniqKey="Driscoll P">P Driscoll</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Li, J" uniqKey="Li J">J Li</name></author><author><name sortKey="Chen, J" uniqKey="Chen J">J Chen</name></author><author><name sortKey="Kirsner, R" uniqKey="Kirsner R">R Kirsner</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Bowler, Pg" uniqKey="Bowler P">PG Bowler</name></author><author><name sortKey="Duerden, Bi" uniqKey="Duerden B">BI Duerden</name></author><author><name sortKey="Armstrong, Dg" uniqKey="Armstrong D">DG Armstrong</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Cullum, N" uniqKey="Cullum N">N Cullum</name></author><author><name sortKey="Nelson, Ea" uniqKey="Nelson E">EA Nelson</name></author><author><name sortKey="Flemming, K" uniqKey="Flemming K">K Flemming</name></author><author><name sortKey="Sheldon, T" uniqKey="Sheldon T">T Sheldon</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ku Isec Tepes, N" uniqKey="Ku Isec Tepes N">N Kučišec-Tepeš</name></author><author><name sortKey="Bejuk, D" uniqKey="Bejuk D">D Bejuk</name></author><author><name sortKey="Kosuta, D" uniqKey="Kosuta D">D Košuta</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Frank, C" uniqKey="Frank C">C Frank</name></author><author><name sortKey="Bayoumi, I" uniqKey="Bayoumi I">I Bayoumi</name></author><author><name sortKey="Westendorp, C" uniqKey="Westendorp C">C Westendorp</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Blaser, M" uniqKey="Blaser M">M Blaser</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Macdonald, J" uniqKey="Macdonald J">J MacDonald</name></author><author><name sortKey="Asiedu, K" uniqKey="Asiedu K">K Asiedu</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kumar, B" uniqKey="Kumar B">B Kumar</name></author><author><name sortKey="Vijayakumar, M" uniqKey="Vijayakumar M">M Vijayakumar</name></author><author><name sortKey="Govindarajan, R" uniqKey="Govindarajan R">R Govindarajan</name></author><author><name sortKey="Pushpangadan, P" uniqKey="Pushpangadan P">P Pushpangadan</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="De Smet, Pagm" uniqKey="De Smet P">PAGM de Smet</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Poonam, K" uniqKey="Poonam K">K Poonam</name></author><author><name sortKey="Singh, Gs" uniqKey="Singh G">GS Singh</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Mahady, Gb" uniqKey="Mahady G">GB Mahady</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Engel, Lw" uniqKey="Engel L">LW Engel</name></author><author><name sortKey="Straus, Se" uniqKey="Straus S">SE Straus</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Jeevan Ram, A" uniqKey="Jeevan Ram A">A Jeevan Ram</name></author><author><name sortKey="Bhakshu, Lm" uniqKey="Bhakshu L">LM Bhakshu</name></author><author><name sortKey="Venkata Raju, Rr" uniqKey="Venkata Raju R">RR Venkata Raju</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kumar, Vl" uniqKey="Kumar V">VL Kumar</name></author><author><name sortKey="Chaudhary, P" uniqKey="Chaudhary P">P Chaudhary</name></author><author><name sortKey="Ramos, Mv" uniqKey="Ramos M">MV Ramos</name></author><author><name sortKey="Mohan, M" uniqKey="Mohan M">M Mohan</name></author><author><name sortKey="Matos, Mpv" uniqKey="Matos M">MPV Matos</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Tour, N" uniqKey="Tour N">N Tour</name></author><author><name sortKey="Talele, G" uniqKey="Talele G">G Talele</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="De Freitas, Cdt" uniqKey="De Freitas C">CDT de Freitas</name></author><author><name sortKey="Lopes, Jl" uniqKey="Lopes J">JL Lopes</name></author><author><name sortKey="Beltramini, Lm" uniqKey="Beltramini L">LM Beltramini</name></author><author><name sortKey="De Oliveira, Rs" uniqKey="De Oliveira R">RS de Oliveira</name></author><author><name sortKey="Oliveira, Jt" uniqKey="Oliveira J">JT Oliveira</name></author><author><name sortKey="Ramos, Mv" uniqKey="Ramos M">MV Ramos</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Thankamma, L" uniqKey="Thankamma L">L Thankamma</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kanojiya, S" uniqKey="Kanojiya S">S Kanojiya</name></author><author><name sortKey="Madhusudanan, Kp" uniqKey="Madhusudanan K">KP Madhusudanan</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Moustafa, Amy" uniqKey="Moustafa A">AMY Moustafa</name></author><author><name sortKey="Ahmed, Sh" uniqKey="Ahmed S">SH Ahmed</name></author><author><name sortKey="Nabil, Zi" uniqKey="Nabil Z">ZI Nabil</name></author><author><name sortKey="Hussein, Aa" uniqKey="Hussein A">AA Hussein</name></author><author><name sortKey="Omran, Ma" uniqKey="Omran M">MA Omran</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Matthew, Km" uniqKey="Matthew K">KM Matthew</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Bauer, Aw" uniqKey="Bauer A">AW Bauer</name></author><author><name sortKey="Kirby, Wm" uniqKey="Kirby W">WM Kirby</name></author><author><name sortKey="Sherris, Jc" uniqKey="Sherris J">JC Sherris</name></author><author><name sortKey="Turck, M" uniqKey="Turck M">M Turck</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Harborne, Jb" uniqKey="Harborne J">JB Harborne</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Saratha, V" uniqKey="Saratha V">V Saratha</name></author><author><name sortKey="Subramanian, S" uniqKey="Subramanian S">S Subramanian</name></author><author><name sortKey="Sivakumar, S" uniqKey="Sivakumar S">S Sivakumar</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Elsinghorst, Ea" uniqKey="Elsinghorst E">EA Elsinghorst</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Samy, Rp" uniqKey="Samy R">RP Samy</name></author><author><name sortKey="Ignacimuthu, S" uniqKey="Ignacimuthu S">S Ignacimuthu</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ahmad, I" uniqKey="Ahmad I">I Ahmad</name></author><author><name sortKey="Beg, Az" uniqKey="Beg A">AZ Beg</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Parabia, Fm" uniqKey="Parabia F">FM Parabia</name></author><author><name sortKey="Kothari, Il" uniqKey="Kothari I">IL Kothari</name></author><author><name sortKey="Parabia, Mh" uniqKey="Parabia M">MH Parabia</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Freitas, Cdt" uniqKey="Freitas C">CDT Freitas</name></author><author><name sortKey="Oliveira, Js" uniqKey="Oliveira J">JS Oliveira</name></author><author><name sortKey="Miranda, Mra" uniqKey="Miranda M">MRA Miranda</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Magalhaes, Hif" uniqKey="Magalhaes H">HIF Magalhães</name></author><author><name sortKey="Ferreira, Pmp" uniqKey="Ferreira P">PMP Ferreira</name></author><author><name sortKey="Moura, Es" uniqKey="Moura E">ES Moura</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Soares De Oliveira, J" uniqKey="Soares De Oliveira J">J Soares de Oliveira</name></author><author><name sortKey="Pereira Bezerra, D" uniqKey="Pereira Bezerra D">D Pereira Bezerra</name></author><author><name sortKey="Teixeira De Freitas, Cd" uniqKey="Teixeira De Freitas C">CD Teixeira de Freitas</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lima Filho, Jv" uniqKey="Lima Filho J">JV Lima-Filho</name></author><author><name sortKey="Patriota, Jm" uniqKey="Patriota J">JM Patriota</name></author><author><name sortKey="Silva, Afb" uniqKey="Silva A">AFB Silva</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Rasik, Am" uniqKey="Rasik A">AM Rasik</name></author><author><name sortKey="Raghubir, R" uniqKey="Raghubir R">R Raghubir</name></author><author><name sortKey="Gupta, A" uniqKey="Gupta A">A Gupta</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Hernandez, V" uniqKey="Hernandez V">V Hernandez</name></author><author><name sortKey="Recio, Mdc" uniqKey="Recio M">MDC Recio</name></author><author><name sortKey="Manez, S" uniqKey="Manez S">S Manez</name></author><author><name sortKey="Prieto, Jm" uniqKey="Prieto J">JM Prieto</name></author><author><name sortKey="Giner, Rm" uniqKey="Giner R">RM Giner</name></author><author><name sortKey="Rios, Jl" uniqKey="Rios J">JL Rios</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Parrotta, Ja" uniqKey="Parrotta J">JA Parrotta</name></author></analytic></biblStruct></listBibl></div1></back></TEI><pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">Evid Based Complement Alternat Med</journal-id><journal-id journal-id-type="iso-abbrev">Evid Based Complement Alternat Med</journal-id><journal-id journal-id-type="publisher-id">ECAM</journal-id><journal-title-group><journal-title>Evidence-based Complementary and Alternative Medicine : eCAM</journal-title></journal-title-group><issn pub-type="ppub">1741-427X</issn><issn pub-type="epub">1741-4288</issn><publisher><publisher-name>Hindawi Publishing Corporation</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">22973400</article-id><article-id pub-id-type="pmc">3437960</article-id><article-id pub-id-type="doi">10.1155/2012/294528</article-id><article-categories><subj-group subj-group-type="heading"><subject>Research Article</subject></subj-group></article-categories><title-group><article-title>Pilot Study with regard to the Wound Healing Activity of Protein from <italic>Calotropis procera</italic> (Ait.) R. Br.</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Perumal Samy</surname><given-names>Ramar</given-names></name><xref ref-type="corresp" rid="cor1">*</xref></contrib><contrib contrib-type="author"><name><surname>Chow</surname><given-names>Vincent T. K.</given-names></name></contrib></contrib-group><aff id="I1">Infectious Diseases Programme, Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597</aff><author-notes><corresp id="cor1">*Ramar Perumal Samy: <email>rperumalsamy@yahoo.co.uk</email></corresp><fn fn-type="other"><p>Academic Editor: Fatma U. Afifi</p></fn></author-notes><pub-date pub-type="ppub"><year>2012</year></pub-date><pub-date pub-type="epub"><day>29</day><month>8</month><year>2012</year></pub-date><volume>2012</volume><elocation-id>294528</elocation-id><history><date date-type="received"><day>5</day><month>3</month><year>2012</year></date><date date-type="rev-recd"><day>16</day><month>5</month><year>2012</year></date><date date-type="accepted"><day>28</day><month>5</month><year>2012</year></date></history><permissions><copyright-statement>Copyright © 2012 R. Perumal Samy and V. T. K. Chow.</copyright-statement><copyright-year>2012</copyright-year><license xlink:href="https://creativecommons.org/licenses/by/3.0/"><license-p>This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</license-p></license></permissions><abstract><p>We provide the scientific basis for the use of <italic>Calotropis procera</italic> for treating skin
and wound infections in traditional medicine. The aqueous extract of stem-bark of <italic>C. procera</italic> exhibited more pronounced potent antimicrobial activity. Calo-protein was purified and identified from the most-active aqueous extracts of <italic>C. procera</italic> and showed broad-spectrum activity. Calo-protein inhibited the growth of <italic>S. aureus</italic> and <italic>E. aerogenes</italic> effectively at 25 <italic><italic>μ</italic></italic>g/ml concentration. Mice topically treated with Calo-protein revealed significant wound healing after 14 days comparable to fusidic acid (FA) as positive control. This protein was devoid of cytolytic effect even at higher concentrations on skin cells after 24 h. Further investigation of this Calo-protein of <italic>C. procera</italic> on bacterial inhibition may provide a better understanding of the scientific basis and justification for its use in traditional medicine.</p></abstract></article-meta></front><body><sec id="sec1"><title>1. Introduction</title><p><italic>Staphylococcus aureus</italic> is a leading cause of skin and soft-tissue infections worldwide [<xref rid="B1" ref-type="bibr">1</xref>]. <italic>S. aureus</italic> infections are increasingly caused by methicillin-resistant <italic>S. aureus</italic> (MRSA) that has developed resistance to <italic>β</italic>-lactam antibiotics. MRSA is a major problem in healthcare settings [<xref rid="B2" ref-type="bibr">2</xref>], with reported incidence rate of invasive MRSA infections of 31.3 per 100,000 individuals, and 20% of these infections resulting in death [<xref rid="B3" ref-type="bibr">3</xref>]. World-wide, the rate of methicillin resistance 35.9% is high for surgical [<xref rid="B4" ref-type="bibr">4</xref>], chronic (7.4 million) [<xref rid="B5" ref-type="bibr">5</xref>], and traumatic wounds (1.5 million). Wound healing is a complex process that involves various inflammatory, proliferative, and remodeling phases [<xref rid="B6" ref-type="bibr">6</xref>]. In particular, chronic wounds are major concerns for patient. Since they affect a large number of patients and may reduce their life span [<xref rid="B7" ref-type="bibr">7</xref>]. Chronic wounds such as leg ulcers, diabetic foot ulcers, and sores are common in both acute and community healthcare settings [<xref rid="B8" ref-type="bibr">8</xref>]. Chronic wounds may be infected by bacteria such as <italic>Streptococcus pyogenes, Enterococcus faecalis, Staphylococcus aureus/MRSA </italic>[<xref rid="B9" ref-type="bibr">9</xref>],<italic> Pseudomonas aeruginosa, Enterobacter aerogenes,</italic> and <italic>Escherichia coli, </italic>while fungi and viruses may also cause skin and wound infections [<xref rid="B10" ref-type="bibr">10</xref>].</p><p>Many bacteria have developed resistance due to the abuse use of antibiotics, while the existing drugs have also caused serious adverse effects in humans [<xref rid="B11" ref-type="bibr">11</xref>]. Various preventive and treatment options of wounds are available [<xref rid="B8" ref-type="bibr">8</xref>]. However, drugs capable of promoting the wound repair process are still limited. Other considerations include the higher cost for producing synthetic drugs and the various side effects associated with their use [<xref rid="B12" ref-type="bibr">12</xref>]. To overcome these issues, the search for alternative agents from plants used in traditional medicine is justified. World-wide, research is conducted to identify new potent, nontoxic wound-healing agents from medicinal plants. Plants are important potential sources of drugs for the biomedical or pharmaceutical industry. Countries such as India and China have rich resources of valuable medicinal plants for the treatment of wound and burns [<xref rid="B13" ref-type="bibr">13</xref>]. Approximately 80% of the world population still relies on traditional medicine for the treatment of common diseases [<xref rid="B14" ref-type="bibr">14</xref>, <xref rid="B15" ref-type="bibr">15</xref>]. Medicinal plants offer significant potential for the development of novel antibacterial therapies and adjunct treatments [<xref rid="B16" ref-type="bibr">16</xref>]. Plant-derived drugs serve as prototypes to develop more effective and less toxic medicines. In previous studies, few attempts were made to confirm the antimicrobial activity of indigenous medicinal plants [<xref rid="B17" ref-type="bibr">17</xref>, <xref rid="B18" ref-type="bibr">18</xref>]. Various extracts of medicinal plants were shown to possess antimicrobial activity against <italic>Staphylococcus aureus </italic>[<xref rid="B18" ref-type="bibr">18</xref>].</p><p><italic>Calotropis procera</italic> (Ait.) R. Br. is well known for its toxic as well as medicinal properties. The milk weed has been found to be effective in the treatment of leprosy, fever, menorrhagia, malaria, and snake bites. Previous investigation study demonstrated various biological activities of <italic>C. procera</italic> such as anti-inflammatory potential in rats [<xref rid="B19" ref-type="bibr">19</xref>, <xref rid="B20" ref-type="bibr">20</xref>], and its osmotin proteins exert antifungal activity [<xref rid="B21" ref-type="bibr">21</xref>]. <italic>C. procera</italic> latex administered to rats revealed toxic, wound healing, and pain-killing effects [<xref rid="B22" ref-type="bibr">22</xref>]. Chemical compounds in the latex are calotropagenin glycosides/derivatives [<xref rid="B23" ref-type="bibr">23</xref>], cardenolides, flavonoids, and saponins [<xref rid="B24" ref-type="bibr">24</xref>]. However, the <italic>C. procera</italic> stem bark has hitherto not been well studied completely. Hence, in the present study <italic>C. procera</italic> was evaluated and further characterized for antimicrobial activity and wound-healing potential in mouse model.</p></sec><sec id="sec2"><title>2. Materials and Methods</title><sec id="sec2.1"><title>2.1. Ethnomedicinal Survey</title><p>The stem bark of <italic>Calotropis procera</italic> (Ait.) R. Br. (family Asclepiadaceae) plant was collected in May 2004, from the Thiruthani, Tiruvallur district, (near Chennai), Tamil Nadu, India. Plant was identified by a taxonomist with help of Matthew [<xref rid="B25" ref-type="bibr">25</xref>]. Voucher specimen was prepared and stored at Entomology Research Institute, India. </p></sec><sec id="sec2.2"><title>2.2. Preparation of Extracts</title><p>The stem bark was collected in the field and cleaned by a sterile muslin cloth, cut into small pieces by sterile razor blade, and stored in a sterile polythene bags. All the parts were dried under shade at ambient temperature (31°C) and ground to small powdery granules by electric blender (Preethi, Chennai, India). Using a soxhlet apparatus, the shade-dried and powdered plant material (200 g of each) was extracted with 1000 mL of hexane for 10 h, and successive extracts were done using different organic solvents such as ethyl acetate, dichloromethane, and methanol individually and then finally extracted twice with 600 mL of sterile distilled water (H<sub>2</sub>O) using a shaking water bath at 65°C for 3 h. All the collected extracts were filtered using Whatman number 1 filter paper and evaporated with a rotary evaporator (Buchi, Labortechnik AG, Switzerland) and freeze dryer (lyophilized) to obtain the crude extracts. The dried crude extracts were stored at 4°C for antimicrobial assays [<xref rid="B18" ref-type="bibr">18</xref>].</p></sec><sec id="sec2.3"><title>2.3. <italic>In Vitro</italic> Antimicrobial Activity</title><p>The standard bacterial cultures of Gram-negative (<italic>Escherichia coli, Enterobacter aerogenes, Proteus vulgaris,</italic> and <italic>Proteus mirabilis)</italic> and Gram-positive (<italic>Pseudomonas aeruginosa</italic> and <italic>Staphylococcus aureus) </italic>bacterial strains were obtained from the Department of Microbiology, National University of Singapore, Singapore. The strains were stored at −70°C, subcultured on 20 mL Mueller Hinton (MH) agar plates (pH 7.4), and incubated overnight at 37°C prior to use. The antimicrobial property was tested using the disc-diffusion method [<xref rid="B26" ref-type="bibr">26</xref>]. Five young colonies of each strain of bacteria taken from their respective cultures grown overnight on MH agar plates (Oxoids, UK) were suspended in 5 mL of sterile saline (0.9%), and the density of the suspension adjusted to approximately 3 × 10<sup>8</sup> colony forming unit (CFU). The swab was used to inoculate the dried surface of MH agar plate by streaking four times over the surface of the agar and rotating the plate approximately 90°C to ensure an even distribution of the inoculums. The medium was allowed to dry for about 3 min before adding a 6 mm diameter sterile paper disc (Beckton Dickson, USA) on the surface. Each disc was tapped gently down onto the agar to provide a uniform contact. 100 <italic>μ</italic>g/mL of each plant extract (lyophilized aqueous residues) was weighed and dissolved in 1 mL of water, and 20 <italic>μ</italic>L of the extracts (containing 100 <italic>μ</italic>g of residues) were applied on each disc (3 replicates). The sterile blank disc served as a normal control. The antimicrobial activity of the extracts on the clinical isolates was determined in comparison with the reference antibiotic (chloramphenicol 30 <italic>μ</italic>g/disc), which was used as a positive control. The plates were incubated at 37°C for 24 h, and the inhibition zones measured and calculated. </p></sec><sec id="sec2.4"><title>2.4. Phytochemical Screening of Extracts</title><p>The phytochemical screening was done on the extracts using the chemical method previously reported for the detection of secondary metabolites [<xref rid="B27" ref-type="bibr">27</xref>]. </p></sec><sec id="sec2.5"><title>2.5. Extraction and Isolation</title><p>The most active aqueous extract of stem bark of <italic>C. procera</italic> was used for the purification of antimicrobial agents [<xref rid="B27" ref-type="bibr">27</xref>]. The residue 0.5 gm was dissolved completely in 5 mL of 50 mM Tris-HCl (pH 7.4), after centrifugation at 12,000 rpm for 15 min, and clear solution was filtered by a membrane filter (0.22 <italic>μ</italic>m). Sample 1 mL diluted (1 : 5 ratio) in 4 mL of Tris-HCl and injected into a Superdex G-75 column linked to high-performance liquid chromatography (HPLC AKTA, Denmark) resolved five fractions (P1–P5). Fraction (1 mL) was monitored at 280 nm and collected. All the fractions were screened against bacteria, of which fraction (P2) exerted higher antimicrobial activity. The most active fraction (P2) was further separated by C18 reverse-phase (RP)-HPLC column, which gave four fractions (CF-1–CF-4). Resultant separation of active fraction (CF-1) displayed maximum inhibitory effect against bacteria that was eluted by C8 RP-HPLC column, final yields of the fraction purity were determined by matrix-assisted laser desorption ionization-time of flight/mass (MALDI)-TIF/MS and designated as “Calo-protein.” The concentrated protein was then tested (100 <italic>μ</italic>g/mL) against bacteria <italic>in vitro</italic>.</p></sec><sec id="sec2.6"><title>2.6. Animals</title><p>Eight-week-old male Swiss Albino mice, body weight ranging from 25–30 gm, were obtained from the NUS animal breeding center Sembawang, Singapore. All animals were kept in individual cages (Darzon, Laboratory, USA) with standard laboratory conditions with dark/light cycle (24 ± 2.0), food pellet and water ad libitum used in wound healing model of experiments.</p><sec id="sec2.6.1"><title>2.6.1. Mice Model of Wound</title><p>Mice were divided into three groups, each group consists of three mice each (<italic>n</italic> = 3), respectively, (IACUC Protocol number 691/04). The dorsal skin of the mice was marked by permanent marker and then cleaned with 70% ethyl alcohol (Merck, Germany) for surface sterilization before shaved by sterile surgical blade [<xref rid="B28" ref-type="bibr">28</xref>]. The animals were anesthetized (75 mg/kg of ketamine + 0.1 mg/kg of medetomidine) and full thickness of 16 mm (8 × 8 mm) for excision wound created and 50 <italic>μ</italic>L of bacteria (1 × 10<sup>5</sup> colony forming units per mL). Group I infected mice wound received only saline served as a control, Group II infected mice wound treated with 5 mg/kg, body weight of Calo-protein mixed with aqueous cream (ICM Pharma Pte Ltd., Kallang Place, Singapore) formulations were topically applied on the dorsal side of the mice, Group III infected mice applied 5 mg/kg, b.w of fusidic acid (FA) sodium salts (Sigma Co., St. Louis, USA) served as an antibiotic control. The wounds were left open without any dress and kept individually in separate cages. The wound area monitored and measured every day up to 14 days (2-week time). </p></sec><sec id="sec2.6.2"><title>2.6.2. Measurement of Wound Area</title><p>Treatment with Calo-protein and standard drug were performed by topical application on the wound surface up to 4 days. The wound areas were traced on millimeter in diameter (mm<sup>2</sup>) by tracer paper (Merck Co., Germany) immediately after the wound created and every day until the healing was completed. The percentage of wound reduction in the wound size was calculated according to the following formula:
<disp-formula id="eq1"><label>(1)</label><mml:math id="M1"><mml:mtable><mml:mtr><mml:mtd><mml:mtext>Wound</mml:mtext><mml:mi> </mml:mi><mml:mtext>contraction</mml:mtext><mml:mi> </mml:mi><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:mi>%</mml:mi></mml:mrow><mml:mo>)</mml:mo></mml:mrow><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:msub><mml:mrow><mml:mi>W</mml:mi></mml:mrow><mml:mrow><mml:mi>c</mml:mi></mml:mrow></mml:msub><mml:mo>−</mml:mo><mml:msub><mml:mrow><mml:mi>W</mml:mi></mml:mrow><mml:mrow><mml:mi>t</mml:mi></mml:mrow></mml:msub></mml:mrow><mml:mrow><mml:msub><mml:mrow><mml:mi>W</mml:mi></mml:mrow><mml:mrow><mml:mi>c</mml:mi></mml:mrow></mml:msub></mml:mrow></mml:mfrac><mml:mo>×</mml:mo><mml:mn mathvariant="normal">100</mml:mn><mml:mo>,</mml:mo></mml:mtd></mml:mtr></mml:mtable></mml:math></disp-formula>where <italic>W</italic><sub><italic>c</italic></sub> is the wound area immediately after wound creation as a control (<italic>c</italic>), <italic>W</italic><sub><italic>t</italic></sub> is the wound area on day of treatment (<italic>t</italic>).</p></sec><sec id="sec2.6.3"><title>2.6.3. Histological Evaluation</title><p>The experiment was terminated after 14 days and the wound removed by surgically for histopathological examination. The tissues were cut and trimmed down and processed in dehydration in alcohol series 50–90% for 30 min each, 100% ethyl alcohol (I/II), and histoclear 100% (I) for 60 min each, and 100% histoclear (II) for overnight. The decalcification was done with molted wax at 55°C for 1 h in each jar. The blocks were prepared by using wax, 5 <italic>μ</italic>m thick sections were stained with haematoxylin and eosin (H&E) and imaged by Olympus Light microscope (Olympus America Inc., PA, USA).</p></sec><sec id="sec2.6.4"><title>2.6.4. Measurement of Collagen</title><p>Healed tissues of the wounds were cut, weighed (100 mg), and homogenized with 1 mL of T-PER buffer (Thermo Scientific, IL, USA). The homogenates were centrifuged at 10,000 rpm for 10 min at 4°C. The supernatant collected and collagen content in the Calo-protein, FA-treated, and wound control mice were determined by ELISA using mouse antitype I collagen assay kit (Chondrex, Inc., Redmond, WA, USA) as per the manufactures instructions.</p></sec></sec><sec id="sec2.7"><title>2.7. Cytotoxicity Assay</title><p>The cytotoxic potential of different crude extracts of <italic>C. procera </italic>was evaluated against human macrophage (U-937) cell lines by XTT assay at 2000, 1000, 500, 250, 100, 50, 25, and 12.5 <italic>μ</italic>g/mL [<xref rid="B29" ref-type="bibr">29</xref>]. The cell proliferation assayed in 96-well microtitre plates, confluent cells (5 × 10<sup>6</sup> cells per well) were incubated with diverse extracts for 24 h and the inhibitory concentrations recorded. Further, cytotoxic and cytolytic effects of purified protein tested on human skin fibroblast cells (HEPK) were evaluated by measuring the release of lactate dehydrogenase (LDH) enzyme using a cytotoxicity detection kit (Roche Mannheim, Germany) at various concentrations (1000–0.001 <italic>μ</italic>g/mL). After 24 h, 200 <italic>μ</italic>L of aliquot of the cell supernatant obtained from each 96-well plate was used for the quantification of cell death and cell lysis, based on the measurement (450 nm). LDH activity was released from the cytosol of damaged cells into the supernatant. The assay was performed in triplicate (<italic>n</italic> = 3), percentage cell proliferation and cytolytic effects were calculated.</p></sec><sec id="sec2.8"><title>2.8. Statistical Analysis</title><p>The data presented as mean ± standard deviation (SD), each concentration tested three replicates (<italic>n</italic> = 3), inhibitory concentrations millimeter (mm) in diameter. The percentage reduction of wound data was compared using one-way analysis of variance (ANOVA) followed by Dunnett's tests. Values with <italic>P</italic> less than *<italic>P</italic> > 0.01, and **<italic>P</italic> > 0.05 compared with the control group were considered as statistical significance.</p></sec></sec><sec id="sec3"><title>3. Results </title><sec id="sec3.1"><title>3.1. Ethnobotanical Survey and Application in Healthcare</title><p>This study attempts to provide the scientific basis for the traditional beliefs of this herbal remedy. We surveyed the traditional medical knowledge on <italic>C. procera </italic>from the local practitioners for preparing home remedies for their routine healthcare (Figures <xref ref-type="fig" rid="fig1">1(a)</xref>, <xref ref-type="fig" rid="fig1">1(b)</xref>, <xref ref-type="fig" rid="fig1">1(c)</xref>, and <xref ref-type="fig" rid="fig1">1(d)</xref>). In Tamil Nadu, decoction of the stem bark is traditionally used for the treatment of skin diseases, while the aerial parts of <italic>C. procera</italic> are used for treating fever, pain, muscular spasm, and so forth. The milky latex wet with a clean cloth was applied mainly on affected areas of cut wounds, thorn injuries, and inflamed swellings. The botanical/family name, yield of extracts, and their activities are provided.</p></sec><sec id="sec3.2"><title>3.2. <italic>In Vitro</italic> Antimicrobial Activity</title><p><xref ref-type="table" rid="tab1">Table 1</xref> shows the <italic>in vitro</italic> antimicrobial, cytotoxic, and phytochemical properties of organic and aqueous extracts of stem bark of <italic>C. procera</italic> as a traditional medicinal plant. Among the tested extracts, ethyl acetate, methanol, and aqueous extracts displayed high antimicrobial activity against the tested bacteria. The methanol and aqueous extracts revealed relatively broad-spectrum activity against bacteria at 100 <italic>μ</italic>g/mL concentration. Aqueous extracts exhibited more pronounced antimicrobial activity with the largest inhibitory zones of 28 mm diameter compared to the standard chloramphenicol antibiotic. Overall, methanol and aqueous extracts of <italic>C. procera </italic>exerted a broad activity against both Gram-negative and Gram-positive bacteria. However, the hexane and dichloromethane extracts exhibited weaker activity against the tested bacteria, while the hexane, ethyl acetate, and dichloromethane extracts did not exhibit any effect against <italic>E. coli</italic> and <italic>P. aeruginosa</italic>.</p><p>The most active aqueous crude extract of <italic>C. procera</italic> was purified by superdex G-75 (<xref ref-type="fig" rid="fig2">Figure 2(a)</xref>), reverse-phase HPLC columns C18 (<xref ref-type="fig" rid="fig2">Figure 2(b)</xref>). The final fraction was designated as Calo-protein (<xref ref-type="fig" rid="fig2">Figure 2(c)</xref>), the purity of its molecular weight of the isolated Calo-protein determined by MALDI-TOF/MS (<xref ref-type="fig" rid="fig2">Figure 2(d)</xref>). The Calo-protein was tested against bacteria at a wide range of concentrations (100–6.25 <italic>μ</italic>g/mL). The purified Calo-protein showed broad-spectrum activity against tested bacteria (<xref ref-type="fig" rid="fig2">Figure 2(c)</xref>). However, the Calo-protein inhibited the growth of <italic>S. aureus </italic>and<italic> E. aerogenes</italic> effectively at 25 <italic>μ</italic>g/mL concentrations. It showed largest inhibitory zones (30 mm) equal to chloramphenicol (31 mm). Even at the lowest concentration of Calo-protein, the growth of <italic>S. aureus, E. aerogenes, P. vulgaris, P. mirabilis, </italic>and<italic> E. coli</italic> was effectively inhibited. <italic>P. aeruginosa</italic> was weakly inhibited by Calo-protein at all the tested concentrations. Finally, the purified protein from the <italic>C. procera</italic> stem bark exerted the strongest antimicrobial activity against <italic>S. aureus</italic> than the crude extracts (<xref ref-type="fig" rid="fig3">Figure 3</xref>).</p></sec><sec id="sec3.3"><title>3.3. Phytochemical Screening</title><p>Phytochemical screening of the stem bark of <italic>C. procera</italic> indicated the presence of various secondary metabolites such as alkaloids, flavonoids, tannins, coumarins, anthraquinones, saponins, cardiac glycosides, sterols, and teriterpenes. The crude extracts of this plant are relatively rich in glycosides, alkaloids, tannins, sterols, and terpenes which may inhibit the growth of organisms.</p></sec><sec id="sec3.4"><title>3.4. Cytotoxicity of Crude Extracts</title><p>The hexane, ethyl acetate, and dichloromethane crude extracts of <italic>C. procera</italic> showed toxicity to human macrophages (U-937) to 250 and 500 <italic>μ</italic>g/mL, However, methanol and aqueous extracts were less toxic up to >2000 <italic>μ</italic>g/mL, whereas the lower concentrations (100–12.5 <italic>μ</italic>g/mL) were devoid of toxic effects and morphological changes of cells (<xref ref-type="table" rid="tab1">Table 1</xref>). However, varied toxic effects were observed in the <italic>C. procera</italic> crude extracts in a dose-dependent manner compared to control cells.</p></sec><sec id="sec3.5"><title>3.5. Cytotoxic Effect of Protein</title><p>Normal human skin fibroblast (HEPK) cells were exposed to Calo-protein to assay for cytotoxicity varying concentrations. However, this protein did not exert any toxic effect on skin cells even at higher concentrations (1000 <italic>μ</italic>g/mL) (<xref ref-type="fig" rid="fig4">Figure 4(a)</xref>). Furthermore, the Calo-protein did not display any cytolytic effects at all the tested concentrations after 24 h compared with control cells (<xref ref-type="fig" rid="fig4">Figure 4(b)</xref>). The purified bioactive protein of <italic>C. procera</italic> did not show toxic effects on skin cells up to 1000 <italic>μ</italic>g/mL. Light microscopic images of human skin fibroblasts were exposed to the Calo-protein at varying doses (1000–0.001 <italic>μ</italic>g/mL). There was no alteration in the control cells (<xref ref-type="fig" rid="fig4">Figure 4(c)</xref>). Whereas the lower dose (100 <italic>μ</italic>g/mL) of protein did not affect the cell morphology (<xref ref-type="fig" rid="fig4">Figure 4(d)</xref>), but the higher dose of protein (1000 <italic>μ</italic>g/mL) showed some changes on HEPK cells after 24 h (Figures <xref ref-type="fig" rid="fig4">4(e)</xref> and <xref ref-type="fig" rid="fig4">4(f)</xref>).</p></sec><sec id="sec3.6"><title>3.6. Wound Healing Effect of Calo-Protein</title><p>Significant wound healing activity was recorded in the mice treated with Calo-protein after 14 days compared to control (Figures <xref ref-type="fig" rid="fig5">5(a)</xref> and <xref ref-type="fig" rid="fig5">5(b)</xref>). There was a considerable reduction in wound area from day 4 onwards in treated mice compared to mice receiving fusidic acid. The treatment with Calo-protein accelerated the rate of wound closure or repair much faster than the control mice. In the histopathological examination, there was notably less noticeable infiltration of inflammatory cells, greatly increased blood vessel formation, and proliferation of cells by the Calo-protein. Interestingly, there was full thickness of reepithelialization in the epidermis, well-organized granular layer, compared to the control. The wounds of animal treated with Calo-protein showed full-thickness of epidermal regeneration that covered completely the wounded area. The epidermis was thick and disorganized especially compared with the adjacent normal skin and complete epithelialization, vascularization, and hair follicles formation were observed in treated mice (Figures <xref ref-type="fig" rid="fig5">5(c)</xref>, <xref ref-type="fig" rid="fig5">5(d)</xref>, <xref ref-type="fig" rid="fig5">5(e)</xref>, and <xref ref-type="fig" rid="fig5">5(f)</xref>). This protein exerted a positive impact on wound healing by enhanced cellular proliferation, granular tissue formation and epithelialization, and early dermal and epidermal regeneration. In addition, topical application of the Calo-protein of mice pronounced in more collagen content than the FA-treated mice versus control mice (Figures <xref ref-type="fig" rid="fig5">5(g)</xref>, <xref ref-type="fig" rid="fig5">5(h)</xref>, and <xref ref-type="fig" rid="fig5">5(i)</xref>).</p></sec></sec><sec id="sec4"><title>4. Discussion</title><p>Our findings confirmed the antimicrobial property of <italic>C. procera</italic> used in traditionally medicine for the treatment of skin and wound-related disorders. We previously reported the antimicrobial activity [<xref rid="B30" ref-type="bibr">30</xref>] of certain traditional medicinal plants used in Tamil Nadu, India. Of these, the aqueous extracts of <italic>C. procera, </italic>exhibited showed the most significant antimicrobial effect against <italic>S. aureus </italic>and <italic>E. aerogenes</italic>. Interestingly, the growth of several wound causing bacterial was controlled by organic and aqueous extracts. Out of 5 different extracts that were examined, 3 extracts were failed to show any antimicrobial effect against <italic>E. coli </italic>and <italic>P. aeruginosa </italic>at the tested concentrations. This negative effect may be attributed to the different climatic and edaphic factors influencing the secondary metabolites. Another study reported the lack of antimicrobial compounds inadequate concentrations in the extracts [<xref rid="B31" ref-type="bibr">31</xref>].</p><p>Both the organic and aqueous extracts showed antimicrobial activity against wound-causing bacteria. Notably, the aqueous extract of <italic>C. procera</italic> exerted comparatively stronger effect against <italic>S. aureus, E. aerogenes,</italic> and <italic>P. mirabilis</italic>. In addition, the inhibitory activities of <italic>C. procera </italic>extracts were comparable to that of the reference antibiotic. Previously, it was reported that the apical twigs and latex of <italic>C. procera</italic> produced greatest inhibition zones against <italic>S. aureus </italic>[<xref rid="B32" ref-type="bibr">32</xref>]. Furthermore, we demonstrated the high antimicrobial potency of the <italic>C. procera </italic>extract against all the tested bacteria may be due to the high content of glycosides and various proteins present in the aqueous extracts [<xref rid="B23" ref-type="bibr">23</xref>, <xref rid="B24" ref-type="bibr">24</xref>]. The differences in the antimicrobial activity of various extracts may be directly related to the diversity of compounds accumulated in <italic>C. procera</italic>: proteins [<xref rid="B21" ref-type="bibr">21</xref>], calotropagenin glycosides [<xref rid="B23" ref-type="bibr">23</xref>], cardenolides, flavonoids, and saponins [<xref rid="B24" ref-type="bibr">24</xref>] thus corroborating our results. Such compounds can bind the Gram-negative bacteria to form a heavy soluble complex on the cell surface that subsequently disturbs the interaction between bacteria and cell receptors. </p><p>Previously, Freitas et al. [<xref rid="B33" ref-type="bibr">33</xref>] studied the enzymatic activities and protein profile of latex of the <italic>C. procera</italic>. The <italic>C. procera</italic> protein showed broad-spectrum activity against tested bacteria at 100–6.25 <italic>μ</italic>g/mL concentrations. We showed that the Calo-protein inhibited the growth of <italic>S. aureus </italic>and<italic> E. aerogenes,</italic> and it was equivalent to chloramphenicol. Even at the lowest dose of protein, the growth of <italic>S. aureus, E. aerogenes, P. vulgaris, P. mirabilis, </italic>and<italic> E. coli</italic> was inhibited effectively. The ethyl acetate and dichloromethane crude extracts of <italic>C. procera</italic> showed toxicity to human macrophage (U-937) cells at 250 and 500 <italic>μ</italic>g/mL doses than the methanol and aqueous extracts. However, the pure protein failed to show toxic effects on human skin (HEPK) cells at doses of up to 1000 <italic>μ</italic>g/mL. Magalhães et al. [<xref rid="B34" ref-type="bibr">34</xref>] studied that the various organic extracts of the stem of <italic>C. procera</italic> and found that ethyl acetate and acetone extracts displayed higher cytotoxic potential against tumor cells (IC 0.8–4.4 mL), but the methanolic extract was weakly cytotoxic. Similarly, laticifer proteins (LPs) obtained from the latex of <italic>C. procera </italic>displayed considerable cytotoxic effects (IC 0.42–1.36 <italic>μ</italic>g/mL) on SF295 and MDA-MB435 cells. In addition, LP was shown to inhibit DNA synthesis and to target DNA topoisomerase I triggering apoptosis in cancer cells [<xref rid="B35" ref-type="bibr">35</xref>].</p><p><statement id="head1"><title>Wound Healing Effect of Calo-Protein</title><p>The latex of <italic>C. procera</italic> has been used in traditional medicine to treat different inflammatory diseases. The anti-inflammatory activity of latex proteins has been well documented using different inflammatory models [<xref rid="B36" ref-type="bibr">36</xref>]. Wound healing events involve different phases such as preventing blood loss, inflammation, epithelial repair such as proliferation [<xref rid="B7" ref-type="bibr">7</xref>], mobilization, migration, differentiation, tissue remodeling, and collagen deposition. The acute inflammatory phase involved in wound healing is accompanied by the synthesis of extracellular matrix that is remodeled to the formation of scar tissue, connective tissue, collagenization, and acquisition of wound strength [<xref rid="B37" ref-type="bibr">37</xref>]. Our mouse model revealed enhanced rate of wound contraction than the control. The mice treated with 5 mg/kg Calo-protein showed favorable results compared with other groups. The treated wound exhibited marked dryness of wound margins with tissue regeneration at 14 days. The healing potential of <italic>C. procera </italic>on dermal wounds in guinea pigs has been documented [<xref rid="B37" ref-type="bibr">37</xref>]. In this study, the topical application of Calo-protein constituent accelerated wound repair, with full-thickness coverage of the wound area by organized epidermis. The wound healing potential of Calo-protein is corroborated by documented anti-inflammatory effect of the <italic>C. procera </italic>plant in rats [<xref rid="B19" ref-type="bibr">19</xref>, <xref rid="B20" ref-type="bibr">20</xref>]. The Calo-protein led to potent wound contraction comparable with FA treatment. In addition, it had favorable effects on inflammation and wound healing. </p><p>Histological examination showed that increased cellular infiltration in treated cases may be due to the chemotactic effect enhanced by the extract that attracts inflammatory cells towards the wound site. Augmented cellular proliferation may be due to the mitogenic activity of the Calo-protein that may have contributed significantly to the repair process. Dermal regeneration in treated mice also implied that the protein exerted a positive impact on cellular proliferation, granular tissue formation and epithelization. The final stage of the healing process involves collagen deposition and remodeling within the dermis [<xref rid="B38" ref-type="bibr">38</xref>]. Thus, the MT staining clearly supported the wound repair process in Calo-protein-treated mice that showed more pronounced collagen accumulation than the control. Several types of secondary metabolites and active compounds isolated from plants have been documented in animal models as the active principles for influencing wound healing. The latex of <italic>C. gigantean</italic> was previously shown to have wound healing effects in rats comparable to those of nitrofurazone, and the extract contains high content of glycosides, flavonoids, phenolic, and triterpenoid compounds with antimicrobial and antioxidant properties [<xref rid="B28" ref-type="bibr">28</xref>]. In our study, Calo-protein isolated from the aqueous extract of <italic>C. procera</italic> possesses wound-healing property against bacterial infections. It was previously reported that calactin, mudarin, and calotropain are active constituents of <italic>C. procera</italic> latex [<xref rid="B39" ref-type="bibr">39</xref>], with antimicrobial and antioxidant properties.</p></statement></p></sec><sec id="sec5"><title>5. Conclusions</title><p>The therapeutic potency of <italic>C. procera</italic> demonstrated that the wound-healing activity of this protein was effective for inhibition of bacterial pathogens. We conclude that the Calo-protein from the <italic>C. procera</italic> stem bark has potential to be developed into new therapeutic agent(s) for wound healing against bacterial infections.</p></sec></body><back><ack><title>Acknowledgments</title><p>The authors are grateful to the Academic Research Fund (ARF), National University of Singapore for the financial support (Grant no. R-181 000 078 122). They also thank the Department of Microbiology, NUS for providing the bacterial cultures used in this study.</p></ack><glossary><title>Abbreviations</title><def-list><def-item><term>Calo-p:</term><def><p><italic>Ca</italic><italic>lo</italic><italic>tr</italic><italic>op</italic><italic>is</italic>
<italic>procera</italic> protein</p></def></def-item><def-item><term>XTT:</term><def><p>(2,3-Bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide)</p></def></def-item><def-item><term>FA:</term><def><p>Fusidic acid (sodium salts)</p></def></def-item><def-item><term>MRSA:</term><def><p>Methicillin resistant <italic>Staphylococcus</italic>
<italic>aureus</italic></p></def></def-item><def-item><term>MH:</term><def><p>Mueller Hinton agar</p></def></def-item><def-item><term>CFU:</term><def><p>Colony forming unit </p></def></def-item><def-item><term>CHL:</term><def><p>Chloramphenicol</p></def></def-item><def-item><term>HPLC:</term><def><p>High-performance liquid chromatography</p></def></def-item><def-item><term>CF:</term><def><p><italic>Ca</italic><italic>lo</italic><italic>tr</italic><italic>op</italic><italic>is</italic>
<italic>procera</italic> fraction</p></def></def-item><def-item><term><italic>W</italic><sub><italic>c</italic></sub>:</term><def><p>Wound control</p></def></def-item><def-item><term><italic>W</italic><sub><italic>t</italic></sub>:</term><def><p>Wound treatment</p></def></def-item><def-item><term>T-PER:</term><def><p>Tissue protein extraction reagent</p></def></def-item><def-item><term>ELISA:</term><def><p>Enzyme-linked immunoabsorbance assay</p></def></def-item><def-item><term>U-937:</term><def><p>Human macrophage</p></def></def-item><def-item><term>HEPK:</term><def><p>Human skin fibroblast</p></def></def-item><def-item><term>LDH:</term><def><p>Lactate dehydrogenase enzyme</p></def></def-item><def-item><term>SBE:</term><def><p>Stem-bark extract</p></def></def-item><def-item><term>LP:</term><def><p>Latex proteins.</p></def></def-item></def-list></glossary><ref-list><ref id="B1"><label>1</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Frei</surname><given-names>CR</given-names></name><name><surname>Makos</surname><given-names>BR</given-names></name><name><surname>Daniels</surname><given-names>KR</given-names></name><name><surname>Oramasionwu</surname><given-names>CU</given-names></name></person-group><article-title>Emergence of community-acquired methicillin-resistant <italic>Staphylococcus aureus</italic> skin and soft tissue infections as a common cause of hospitalization in United States children</article-title><source><italic>Journal of Pediatric Surgery</italic></source><year>2010</year><volume>45</volume><issue>10</issue><fpage>1967</fpage><lpage>1974</lpage><pub-id pub-id-type="other">2-s2.0-77957757077</pub-id><pub-id pub-id-type="pmid">20920714</pub-id></element-citation></ref><ref id="B2"><label>2</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Pichereau</surname><given-names>S</given-names></name><name><surname>Rose</surname><given-names>WE</given-names></name></person-group><article-title>Invasive community-associated MRSA infections: epidemiology and antimicrobial management</article-title><source><italic>Expert Opinion on Pharmacotherapy</italic></source><year>2010</year><volume>11</volume><issue>18</issue><fpage>3009</fpage><lpage>3025</lpage><pub-id pub-id-type="other">2-s2.0-78649381207</pub-id><pub-id pub-id-type="pmid">20955116</pub-id></element-citation></ref><ref id="B3"><label>3</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Klevens</surname><given-names>RM</given-names></name><name><surname>Morrison</surname><given-names>MA</given-names></name><name><surname>Nadle</surname><given-names>J</given-names></name><etal></etal></person-group><article-title>Invasive methicillin-resistant Staphylococcus aureus infections in the United States</article-title><source><italic>Journal of the American Medical Association</italic></source><year>2007</year><volume>298</volume><issue>15</issue><fpage>1763</fpage><lpage>1771</lpage><pub-id pub-id-type="other">2-s2.0-35349019173</pub-id><pub-id pub-id-type="pmid">17940231</pub-id></element-citation></ref><ref id="B4"><label>4</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Moet</surname><given-names>GJ</given-names></name><name><surname>Jones</surname><given-names>RN</given-names></name><name><surname>Biedenbach</surname><given-names>DJ</given-names></name><name><surname>Stilwell</surname><given-names>MG</given-names></name><name><surname>Fritsche</surname><given-names>TR</given-names></name></person-group><article-title>Contemporary causes of skin and soft tissue infections in North America, Latin America, and Europe: report from the SENTRY Antimicrobial Surveillance Program (1998–2004)</article-title><source><italic>Diagnostic Microbiology and Infectious Disease</italic></source><year>2007</year><volume>57</volume><issue>1</issue><fpage>7</fpage><lpage>13</lpage><pub-id pub-id-type="other">2-s2.0-33845422285</pub-id><pub-id pub-id-type="pmid">17059876</pub-id></element-citation></ref><ref id="B5"><label>5</label><element-citation publication-type="other"><person-group person-group-type="author"><name><surname>Driscoll</surname><given-names>P</given-names></name></person-group><article-title>Wound prevalence and wound management, in clinical practice, market data, medtech, surgery, wound management</article-title><comment>April 14, 2009</comment></element-citation></ref><ref id="B6"><label>6</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Li</surname><given-names>J</given-names></name><name><surname>Chen</surname><given-names>J</given-names></name><name><surname>Kirsner</surname><given-names>R</given-names></name></person-group><article-title>Pathophysiology of acute wound healing</article-title><source><italic>Clinics in Dermatology</italic></source><year>2007</year><volume>25</volume><issue>1</issue><fpage>9</fpage><lpage>18</lpage><pub-id pub-id-type="other">2-s2.0-33846627240</pub-id><pub-id pub-id-type="pmid">17276196</pub-id></element-citation></ref><ref id="B7"><label>7</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Bowler</surname><given-names>PG</given-names></name><name><surname>Duerden</surname><given-names>BI</given-names></name><name><surname>Armstrong</surname><given-names>DG</given-names></name></person-group><article-title>Wound microbiology and associated approaches to wound management</article-title><source><italic>Clinical Microbiology Reviews</italic></source><year>2001</year><volume>14</volume><issue>2</issue><fpage>244</fpage><lpage>269</lpage><pub-id pub-id-type="other">2-s2.0-0035071319</pub-id><pub-id pub-id-type="pmid">11292638</pub-id></element-citation></ref><ref id="B8"><label>8</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Cullum</surname><given-names>N</given-names></name><name><surname>Nelson</surname><given-names>EA</given-names></name><name><surname>Flemming</surname><given-names>K</given-names></name><name><surname>Sheldon</surname><given-names>T</given-names></name></person-group><article-title>Systematic reviews of wound care management: (5) beds, (6) compression, (7) laser therapy, therapeutic ultrasound, electrotherapy and electromagnetic therapy</article-title><source><italic>Health Technology Assessment Journal</italic></source><year>2001</year><volume>5</volume><issue>9</issue><fpage>1</fpage><lpage>221</lpage></element-citation></ref><ref id="B9"><label>9</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kučišec-Tepeš</surname><given-names>N</given-names></name><name><surname>Bejuk</surname><given-names>D</given-names></name><name><surname>Košuta</surname><given-names>D</given-names></name></person-group><article-title>Characteristics of war wound infection</article-title><source><italic>Acta Medica Croatica</italic></source><year>2006</year><volume>60</volume><issue>4</issue><fpage>353</fpage><lpage>363</lpage><pub-id pub-id-type="other">2-s2.0-33749546233</pub-id><pub-id pub-id-type="pmid">17048790</pub-id></element-citation></ref><ref id="B10"><label>10</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Frank</surname><given-names>C</given-names></name><name><surname>Bayoumi</surname><given-names>I</given-names></name><name><surname>Westendorp</surname><given-names>C</given-names></name></person-group><article-title>Approach to infected skin ulcers</article-title><source><italic>Canadian Family Physician</italic></source><year>2005</year><volume>51</volume><issue>10</issue><fpage>1352</fpage><lpage>1359</lpage><pub-id pub-id-type="other">2-s2.0-27744502607</pub-id><pub-id pub-id-type="pmid">16250422</pub-id></element-citation></ref><ref id="B11"><label>11</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Blaser</surname><given-names>M</given-names></name></person-group><article-title>Antibiotic overuse: stop the killing of beneficial bacteria</article-title><source><italic>Nature</italic></source><year>2011</year><volume>476</volume><issue>7361</issue><fpage>393</fpage><lpage>394</lpage><pub-id pub-id-type="other">2-s2.0-80052065175</pub-id><pub-id pub-id-type="pmid">21866137</pub-id></element-citation></ref><ref id="B12"><label>12</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>MacDonald</surname><given-names>J</given-names></name><name><surname>Asiedu</surname><given-names>K</given-names></name></person-group><article-title>WAWLC: world alliance for wound and lymphedema care</article-title><source><italic>Wounds</italic></source><year>2010</year><volume>22</volume><issue>3</issue><fpage>55</fpage><lpage>59</lpage><pub-id pub-id-type="other">2-s2.0-77950971475</pub-id><pub-id pub-id-type="pmid">25901830</pub-id></element-citation></ref><ref id="B13"><label>13</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kumar</surname><given-names>B</given-names></name><name><surname>Vijayakumar</surname><given-names>M</given-names></name><name><surname>Govindarajan</surname><given-names>R</given-names></name><name><surname>Pushpangadan</surname><given-names>P</given-names></name></person-group><article-title>Ethnopharmacological approaches to wound healing-exploring medicinal plants of India</article-title><source><italic>Journal of Ethnopharmacology</italic></source><year>2007</year><volume>114</volume><issue>2</issue><fpage>103</fpage><lpage>113</lpage><pub-id pub-id-type="other">2-s2.0-35148854626</pub-id><pub-id pub-id-type="pmid">17884316</pub-id></element-citation></ref><ref id="B14"><label>14</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>de Smet</surname><given-names>PAGM</given-names></name></person-group><article-title>Herbal remedies</article-title><source><italic>New England Journal of Medicine</italic></source><year>2002</year><volume>347</volume><issue>9009</issue><fpage>2046</fpage><lpage>2056</lpage><pub-id pub-id-type="other">2-s2.0-0037137560</pub-id><pub-id pub-id-type="pmid">12490687</pub-id></element-citation></ref><ref id="B15"><label>15</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Poonam</surname><given-names>K</given-names></name><name><surname>Singh</surname><given-names>GS</given-names></name></person-group><article-title>Ethnobotanical study of medicinal plants used by the Taungya community in Terai Arc Landscape, India</article-title><source><italic>Journal of Ethnopharmacology</italic></source><year>2009</year><volume>123</volume><issue>1</issue><fpage>167</fpage><lpage>176</lpage><pub-id pub-id-type="other">2-s2.0-64249165124</pub-id><pub-id pub-id-type="pmid">19429357</pub-id></element-citation></ref><ref id="B16"><label>16</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Mahady</surname><given-names>GB</given-names></name></person-group><article-title>Medicinal plants for the prevention and treatment of bacterial infections</article-title><source><italic>Current Pharmaceutical Design</italic></source><year>2005</year><volume>11</volume><issue>19</issue><fpage>2405</fpage><lpage>2427</lpage><pub-id pub-id-type="other">2-s2.0-21044443338</pub-id><pub-id pub-id-type="pmid">16026296</pub-id></element-citation></ref><ref id="B17"><label>17</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Engel</surname><given-names>LW</given-names></name><name><surname>Straus</surname><given-names>SE</given-names></name></person-group><article-title>Development of therapeutics: opportunities within complementary and alternative medicine</article-title><source><italic>Nature Reviews Drug Discovery</italic></source><year>2002</year><volume>1</volume><issue>3</issue><fpage>229</fpage><lpage>237</lpage><pub-id pub-id-type="other">2-s2.0-0036491604</pub-id><pub-id pub-id-type="pmid">12120507</pub-id></element-citation></ref><ref id="B18"><label>18</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Jeevan Ram</surname><given-names>A</given-names></name><name><surname>Bhakshu</surname><given-names>LM</given-names></name><name><surname>Venkata Raju</surname><given-names>RR</given-names></name></person-group><article-title><italic>In vitro</italic> antimicrobial activity of certain medicinal plants from Eastern Ghats, India, used for skin diseases</article-title><source><italic>Journal of Ethnopharmacology</italic></source><year>2004</year><volume>90</volume><issue>2-3</issue><fpage>353</fpage><lpage>357</lpage><pub-id pub-id-type="other">2-s2.0-0842326394</pub-id><pub-id pub-id-type="pmid">15013201</pub-id></element-citation></ref><ref id="B19"><label>19</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kumar</surname><given-names>VL</given-names></name><name><surname>Chaudhary</surname><given-names>P</given-names></name><name><surname>Ramos</surname><given-names>MV</given-names></name><name><surname>Mohan</surname><given-names>M</given-names></name><name><surname>Matos</surname><given-names>MPV</given-names></name></person-group><article-title>Protective effect of proteins derived from the <italic>Latex of Calotropis procera</italic> against inflammatory hyperalgesia in monoarthritic rats</article-title><source><italic>Phytotherapy Research</italic></source><year>2011</year><volume>25</volume><issue>9</issue><fpage>1336</fpage><lpage>1341</lpage><pub-id pub-id-type="other">2-s2.0-80052277032</pub-id><pub-id pub-id-type="pmid">21328619</pub-id></element-citation></ref><ref id="B20"><label>20</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Tour</surname><given-names>N</given-names></name><name><surname>Talele</surname><given-names>G</given-names></name></person-group><article-title>Anti-inflammatory and gastromucosal protective effects of <italic>Calotropis procera</italic> (Asclepiadaceae) stem bark</article-title><source><italic>Journal of Natural Medicines</italic></source><year>2011</year><volume>65</volume><issue>3-4</issue><fpage>598</fpage><lpage>605</lpage><pub-id pub-id-type="other">2-s2.0-79960442853</pub-id><pub-id pub-id-type="pmid">21400248</pub-id></element-citation></ref><ref id="B21"><label>21</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>de Freitas</surname><given-names>CDT</given-names></name><name><surname>Lopes</surname><given-names>JL</given-names></name><name><surname>Beltramini</surname><given-names>LM</given-names></name><name><surname>de Oliveira</surname><given-names>RS</given-names></name><name><surname>Oliveira</surname><given-names>JT</given-names></name><name><surname>Ramos</surname><given-names>MV</given-names></name></person-group><article-title>Osmotin from Calotropis procera latex: new insights into structure and antifungal properties</article-title><source><italic>Biochimica et Biophysica Acta</italic></source><year>2011</year><volume>1808</volume><issue>10</issue><fpage>2501</fpage><lpage>2507</lpage><pub-id pub-id-type="other">2-s2.0-79961101434</pub-id><pub-id pub-id-type="pmid">21798235</pub-id></element-citation></ref><ref id="B22"><label>22</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Thankamma</surname><given-names>L</given-names></name></person-group><article-title>Hevea latex as a wound healer and pain killer</article-title><source><italic>Current Science</italic></source><year>2003</year><volume>84</volume><issue>8</issue><fpage>971</fpage><lpage>972</lpage><pub-id pub-id-type="other">2-s2.0-4444378525</pub-id></element-citation></ref><ref id="B23"><label>23</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kanojiya</surname><given-names>S</given-names></name><name><surname>Madhusudanan</surname><given-names>KP</given-names></name></person-group><article-title>Rapid Identification of calotropagenin glycosides using high-performance liquid chromatography electrospray ionisation tandem mass spectrometry</article-title><source><italic>Phytochemical Analysis</italic></source><year>2012</year><volume>23</volume><issue>2</issue><fpage>117</fpage><lpage>125</lpage><pub-id pub-id-type="other">2-s2.0-79958036173</pub-id><pub-id pub-id-type="pmid">21656605</pub-id></element-citation></ref><ref id="B24"><label>24</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Moustafa</surname><given-names>AMY</given-names></name><name><surname>Ahmed</surname><given-names>SH</given-names></name><name><surname>Nabil</surname><given-names>ZI</given-names></name><name><surname>Hussein</surname><given-names>AA</given-names></name><name><surname>Omran</surname><given-names>MA</given-names></name></person-group><article-title>Extraction and phytochemical investigation of Calotropis procera: effect of plant extracts on the activity of diverse muscles</article-title><source><italic>Pharmaceutical Biology</italic></source><year>2010</year><volume>48</volume><issue>10</issue><fpage>1080</fpage><lpage>1090</lpage><pub-id pub-id-type="other">2-s2.0-77957062424</pub-id><pub-id pub-id-type="pmid">20690894</pub-id></element-citation></ref><ref id="B25"><label>25</label><element-citation publication-type="book"><person-group person-group-type="author"><name><surname>Matthew</surname><given-names>KM</given-names></name></person-group><source><italic>The Flora of Tamil Nadu Carnatic</italic></source><year>1981–1983</year><issue>Part I–III</issue><publisher-loc>Madras, India</publisher-loc><publisher-name>Diocesan Press</publisher-name></element-citation></ref><ref id="B26"><label>26</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Bauer</surname><given-names>AW</given-names></name><name><surname>Kirby</surname><given-names>WM</given-names></name><name><surname>Sherris</surname><given-names>JC</given-names></name><name><surname>Turck</surname><given-names>M</given-names></name></person-group><article-title>Antibiotic susceptibility testing by a standardized single disk method.</article-title><source><italic>American Journal of Clinical Pathology</italic></source><year>1966</year><volume>45</volume><issue>4</issue><fpage>493</fpage><lpage>496</lpage><pub-id pub-id-type="other">2-s2.0-0013902668</pub-id><pub-id pub-id-type="pmid">5325707</pub-id></element-citation></ref><ref id="B27"><label>27</label><element-citation publication-type="book"><person-group person-group-type="author"><name><surname>Harborne</surname><given-names>JB</given-names></name></person-group><source><italic>Phytochemical Methods</italic></source><year>1976</year><publisher-loc>New York, NY, USA</publisher-loc><publisher-name>Chapman & Hall</publisher-name></element-citation></ref><ref id="B28"><label>28</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Saratha</surname><given-names>V</given-names></name><name><surname>Subramanian</surname><given-names>S</given-names></name><name><surname>Sivakumar</surname><given-names>S</given-names></name></person-group><article-title>Evaluation of wound healing potential of calotropis igantea latex studied on excision wounds in experimental rats</article-title><source><italic>Medicinal Chemistry Research</italic></source><year>2010</year><volume>19</volume><issue>8</issue><fpage>936</fpage><lpage>947</lpage><pub-id pub-id-type="other">2-s2.0-78650719297</pub-id></element-citation></ref><ref id="B29"><label>29</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Elsinghorst</surname><given-names>EA</given-names></name></person-group><article-title>Measurement of invasion by gentamicin resistance</article-title><source><italic>Methods in Enzymology</italic></source><year>1994</year><volume>236</volume><fpage>405</fpage><lpage>420</lpage><pub-id pub-id-type="other">2-s2.0-0027992183</pub-id><pub-id pub-id-type="pmid">7968625</pub-id></element-citation></ref><ref id="B30"><label>30</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Samy</surname><given-names>RP</given-names></name><name><surname>Ignacimuthu</surname><given-names>S</given-names></name></person-group><article-title>Antibacterial activity of some folklore medicinal plants used by tribals in Western Ghats of India</article-title><source><italic>Journal of Ethnopharmacology</italic></source><year>2000</year><volume>69</volume><issue>1</issue><fpage>63</fpage><lpage>71</lpage><pub-id pub-id-type="other">2-s2.0-0033991405</pub-id><pub-id pub-id-type="pmid">10661885</pub-id></element-citation></ref><ref id="B31"><label>31</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Ahmad</surname><given-names>I</given-names></name><name><surname>Beg</surname><given-names>AZ</given-names></name></person-group><article-title>Antimicrobial and phytochemical studies on 45 Indian medicinal plants against multi-drug resistant human pathogens</article-title><source><italic>Journal of Ethnopharmacology</italic></source><year>2001</year><volume>74</volume><issue>2</issue><fpage>113</fpage><lpage>123</lpage><pub-id pub-id-type="other">2-s2.0-0035130819</pub-id><pub-id pub-id-type="pmid">11167029</pub-id></element-citation></ref><ref id="B32"><label>32</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Parabia</surname><given-names>FM</given-names></name><name><surname>Kothari</surname><given-names>IL</given-names></name><name><surname>Parabia</surname><given-names>MH</given-names></name></person-group><article-title>Antibacterial activity of solvent fractions of crude water decoction of apical twigs and latex of <italic>Calotropis procera</italic> (Ait.) R. Br</article-title><source><italic>Natural Product Radiance</italic></source><year>2008</year><volume>7</volume><issue>1</issue><fpage>30</fpage><lpage>34</lpage><pub-id pub-id-type="other">2-s2.0-44949192770</pub-id></element-citation></ref><ref id="B33"><label>33</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Freitas</surname><given-names>CDT</given-names></name><name><surname>Oliveira</surname><given-names>JS</given-names></name><name><surname>Miranda</surname><given-names>MRA</given-names></name><etal></etal></person-group><article-title>Enzymatic activities and protein profile of latex from Calotropis procera</article-title><source><italic>Plant Physiology and Biochemistry</italic></source><year>2007</year><volume>45</volume><issue>10-11</issue><fpage>781</fpage><lpage>789</lpage><pub-id pub-id-type="other">2-s2.0-35148886814</pub-id><pub-id pub-id-type="pmid">17888673</pub-id></element-citation></ref><ref id="B34"><label>34</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Magalhães</surname><given-names>HIF</given-names></name><name><surname>Ferreira</surname><given-names>PMP</given-names></name><name><surname>Moura</surname><given-names>ES</given-names></name><etal></etal></person-group><article-title><italic>In vitro</italic> and <italic>in vivo</italic> antiproliferative activity of calotropis procera stem extracts</article-title><source><italic>Anais Of Academia Brasileira de Ciências</italic></source><year>2010</year><volume>82</volume><issue>2</issue><fpage>407</fpage><lpage>416</lpage><pub-id pub-id-type="other">2-s2.0-77954187425</pub-id></element-citation></ref><ref id="B35"><label>35</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Soares de Oliveira</surname><given-names>J</given-names></name><name><surname>Pereira Bezerra</surname><given-names>D</given-names></name><name><surname>Teixeira de Freitas</surname><given-names>CD</given-names></name><etal></etal></person-group><article-title><italic>In vitro</italic> cytotoxicity against different human cancer cell lines of laticifer proteins of <italic>Calotropis procera</italic> (Ait.) R. Br</article-title><source><italic>Toxicology in Vitro</italic></source><year>2007</year><volume>21</volume><issue>8</issue><fpage>1563</fpage><lpage>1573</lpage><pub-id pub-id-type="other">2-s2.0-36049006862</pub-id><pub-id pub-id-type="pmid">17604595</pub-id></element-citation></ref><ref id="B36"><label>36</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lima-Filho</surname><given-names>JV</given-names></name><name><surname>Patriota</surname><given-names>JM</given-names></name><name><surname>Silva</surname><given-names>AFB</given-names></name><etal></etal></person-group><article-title>Proteins from latex of <italic>Calotropis procera</italic> prevent septic shock due to lethal infection by <italic>Salmonella enterica serovar Typhimurium</italic></article-title><source><italic>Journal of Ethnopharmacology</italic></source><year>2010</year><volume>129</volume><issue>3</issue><fpage>327</fpage><lpage>334</lpage><pub-id pub-id-type="other">2-s2.0-77953340117</pub-id><pub-id pub-id-type="pmid">20371281</pub-id></element-citation></ref><ref id="B37"><label>37</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Rasik</surname><given-names>AM</given-names></name><name><surname>Raghubir</surname><given-names>R</given-names></name><name><surname>Gupta</surname><given-names>A</given-names></name><etal></etal></person-group><article-title>Healing potential of <italic>Calotropis procera</italic> on dermal wounds in Guinea pigs</article-title><source><italic>Journal of Ethnopharmacology</italic></source><year>1999</year><volume>68</volume><issue>1-3</issue><fpage>261</fpage><lpage>266</lpage><pub-id pub-id-type="other">2-s2.0-0032692214</pub-id><pub-id pub-id-type="pmid">10624886</pub-id></element-citation></ref><ref id="B38"><label>38</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Hernandez</surname><given-names>V</given-names></name><name><surname>Recio</surname><given-names>MDC</given-names></name><name><surname>Manez</surname><given-names>S</given-names></name><name><surname>Prieto</surname><given-names>JM</given-names></name><name><surname>Giner</surname><given-names>RM</given-names></name><name><surname>Rios</surname><given-names>JL</given-names></name></person-group><article-title>A mechanistic approach to the <italic>in vivo</italic> anti-inflammatory activity of sesquiterpenoid compounds isolated from <italic>Inula viscose</italic></article-title><source><italic>Planta Medica</italic></source><year>2001</year><volume>67</volume><issue>8</issue><fpage>726</fpage><lpage>731</lpage><pub-id pub-id-type="pmid">11731914</pub-id></element-citation></ref><ref id="B39"><label>39</label><element-citation publication-type="book"><person-group person-group-type="author"><name><surname>Parrotta</surname><given-names>JA</given-names></name></person-group><source><italic>Healing Plants of Peninsular India</italic></source><year>2001</year><publisher-loc>Wallingford, UK</publisher-loc><publisher-name>CAB International</publisher-name></element-citation></ref></ref-list></back><floats-group><fig id="fig1" orientation="portrait" position="float"><label>Figure 1</label><caption><p> The giant milkweed <italic>Calotropis procera </italic>growing as a spreading shrub or small tree has a simple stem with only few branches. (a, b) Large, dark-green leaves in opposite pairs along smooth stem. (c) Beautiful waxy white flowers have deep purple spots or blotches at the base of each five petals. (d) It produces a fleshy fruit with inflated pod containing several brown seeds with white long silky hair. It exudes a milky white latex when cut or broken. St: stem, f: flower, fp: fruit pod, and wl: waxy leaf surface.</p></caption><graphic xlink:href="ECAM2012-294528.001"></graphic></fig><fig id="fig2" orientation="portrait" position="float"><label>Figure 2</label><caption><p>(a) The most active stem bark extract was separated by Superdex G-75 column and resolved into five fractions (P1–P5). (b) The active fraction (P2) was further eluted and gave fractions CF-1–CF-F4. (c) The final pure fraction was separated by reverse-phase HPLC column (C8) using CF-1 fraction and designated as Calo-protein (Clo-p) of <italic>C. procera. </italic>Calo-p was tested for <italic>in vitro </italic>antimicrobial efficacy at various dose and in wound healing studies in mouse model. (d) The molecular mass of the Calo-protein was determined by MALDI-TOF/MS. </p></caption><graphic xlink:href="ECAM2012-294528.002"></graphic></fig><fig id="fig3" orientation="portrait" position="float"><label>Figure 3</label><caption><p>Antimicrobial properties of purified protein from the most active <italic>C. procera </italic>extract tested against bacteria that cause skin infections. (a–f) Results are expressed as the mean ± SD (<italic>n</italic> = 3) of the inhibition zones (diameter around the discs). The original size of disc was 6 millimeter in diameter. The bacterial growth inhibitory efficacy was compared with chloramphenicol used as a positive control.</p></caption><graphic xlink:href="ECAM2012-294528.003"></graphic></fig><fig id="fig4" orientation="portrait" position="float"><label>Figure 4</label><caption><p>Cytotoxic effect of Calo-protein from the stem bark of <italic>C. procera </italic>assayed on human skin cells and their morphological changes observed for 24 h. (a) Calo-protein was tested against skin cell up to 1000 <italic>μ</italic>g/mL concentrations for 24 h. There were no toxic effects recorded at high concentrations. (b) The protein did not produce severe cytolytic effects after 24 h exposure of the plant protein <italic>in vitro</italic>. Light microscopic images showing the morphological changes of human skin fibroblast cells were exposed to the Calo-protein obtained from the stem bark of <italic>C. procera </italic>tested at varying doses (1000–0.001 <italic>μ</italic>g/mL). (c) There was no alteration in the control cells, (d) while lower dose (100 <italic>μ</italic>g/mL) of protein did not affect the cell morphology (e, f), the higher dose of protein (1000 <italic>μ</italic>g/mL) showed some changes on HEPK cells after 24 h. Untreated cells served as a control (ctrl).</p></caption><graphic xlink:href="ECAM2012-294528.004"></graphic></fig><fig id="fig5" orientation="portrait" position="float"><label>Figure 5</label><caption><p>Wound-healing activity by <italic>C. procera </italic>protein was determined in a mouse model. (a) Reduction of wound area was compared with antibiotic (FA) up to 14 days. (b) Percentage of wound healing effect of Calo-protein was compared with the control group. (c, d) The protein treatment showed full thickness of reepithelialization in the epidermis, well-organized granular layer, compared to the wound control. (e, f) Masson's Trichrome (MT) staining showed less deposition of collagen in the wound control mice after 14 days. (g–i) Biochemical analysis showed that protein treated enhanced more collagen synthesis than the FA and WCtrl after 14 days. ep: epidermis, de: dermis, ct: cortex, ne: neutrophils, mf: muscle fiber, and co: collagen.</p></caption><graphic xlink:href="ECAM2012-294528.005"></graphic></fig><table-wrap id="tab1" orientation="portrait" position="float"><label>Table 1</label><caption><p>Antimicrobial activity of different extracts of stem-bark of <italic>C. procera </italic>tested on Gram-positive and Gram-negative bacteria at 100 <italic>μ</italic>g/mL concentration and their cytotoxic potential was evaluated against human macrophage (U-937) cell lines by XTT assay at 2000, 1000, 500, 250, 100, 50, 25, and 12.5 <italic>μ</italic>g/mL doses.</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="1" colspan="1">Crude extracts</th><th align="center" rowspan="1" colspan="1">Phytochemical screening</th><th align="center" rowspan="1" colspan="1">Yield</th><th align="center" rowspan="1" colspan="1"><italic>E. coli </italic></th><th align="center" rowspan="1" colspan="1"><italic>E. aerogenes</italic></th><th align="center" rowspan="1" colspan="1"><italic>P. vulgaris </italic></th><th align="center" rowspan="1" colspan="1"><italic>P. mirabilis </italic></th><th align="center" rowspan="1" colspan="1"><italic>P. aeruginosa </italic></th><th align="center" rowspan="1" colspan="1"><italic>S. aureus </italic></th><th align="center" rowspan="1" colspan="1">Cytotoxicity (<italic>μ</italic>g/mL)</th></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">(1) Hexane</td><td align="center" rowspan="1" colspan="1">Flovanoids</td><td align="center" rowspan="1" colspan="1">5.36 gm</td><td align="center" rowspan="1" colspan="1">—</td><td align="center" rowspan="1" colspan="1">11 ± 0.94</td><td align="center" rowspan="1" colspan="1">7 ± 0.1</td><td align="center" rowspan="1" colspan="1">9 ± 0.06</td><td align="center" rowspan="1" colspan="1">—</td><td align="center" rowspan="1" colspan="1">8 ± 0.12</td><td align="center" rowspan="1" colspan="1"><250</td></tr><tr><td align="left" rowspan="1" colspan="1">(2) Ethyl acetate</td><td align="center" rowspan="1" colspan="1">Steroids</td><td align="center" rowspan="1" colspan="1">3.42 gm</td><td align="center" rowspan="1" colspan="1">—</td><td align="center" rowspan="1" colspan="1">18 ± 0.06</td><td align="center" rowspan="1" colspan="1">17 ± 0.27</td><td align="center" rowspan="1" colspan="1">15 ± 0.2</td><td align="center" rowspan="1" colspan="1">—</td><td align="center" rowspan="1" colspan="1">25 ± 0.06</td><td align="center" rowspan="1" colspan="1"><500</td></tr><tr><td align="left" rowspan="1" colspan="1">(3) Dichloromethane</td><td align="center" rowspan="1" colspan="1">Polyphenols</td><td align="center" rowspan="1" colspan="1">4.78 gm</td><td align="center" rowspan="1" colspan="1">—</td><td align="center" rowspan="1" colspan="1">12 ± 0.4</td><td align="center" rowspan="1" colspan="1">8 ± 0.23</td><td align="center" rowspan="1" colspan="1">11 ± 0.48</td><td align="center" rowspan="1" colspan="1">—</td><td align="center" rowspan="1" colspan="1">13 ± 0.21</td><td align="center" rowspan="1" colspan="1"><500</td></tr><tr><td align="left" rowspan="1" colspan="1">(4) Methanol</td><td align="center" rowspan="1" colspan="1">Terpenoids</td><td align="center" rowspan="1" colspan="1">5.98 gm</td><td align="center" rowspan="1" colspan="1">14 ± 0.31</td><td align="center" rowspan="1" colspan="1">19 ± 0.2</td><td align="center" rowspan="1" colspan="1">23 ± 0.4</td><td align="center" rowspan="1" colspan="1">20 ± 0.6</td><td align="center" rowspan="1" colspan="1">8 ± 0.12</td><td align="center" rowspan="1" colspan="1">27 ± 0.06</td><td align="center" rowspan="1" colspan="1">>2000</td></tr><tr><td align="left" rowspan="1" colspan="1">(5) Aqueous</td><td align="center" rowspan="1" colspan="1">Glycosides; Tannins; saponins</td><td align="center" rowspan="1" colspan="1">7.15 gm</td><td align="center" rowspan="1" colspan="1">11 ± 0.14</td><td align="center" rowspan="1" colspan="1">22 ± 0.7</td><td align="center" rowspan="1" colspan="1">25 ± 1.3</td><td align="center" rowspan="1" colspan="1">20 ± 0.12</td><td align="center" rowspan="1" colspan="1">9 ± 0.23</td><td align="center" rowspan="1" colspan="1">28 ± 0.15</td><td align="center" rowspan="1" colspan="1">>2000</td></tr><tr><td align="left" rowspan="1" colspan="1">(6) Chloramphenicol</td><td align="center" rowspan="1" colspan="1">Standard antibiotic</td><td align="center" rowspan="1" colspan="1">30 <italic>μ</italic>g</td><td align="center" rowspan="1" colspan="1">21 ± 0.34</td><td align="center" rowspan="1" colspan="1">24 ± 0.3</td><td align="center" rowspan="1" colspan="1">23 ± 0.12</td><td align="center" rowspan="1" colspan="1">26 ± 0.23</td><td align="center" rowspan="1" colspan="1">10 ± 0.35</td><td align="center" rowspan="1" colspan="1">29 ± 0.12</td><td align="center" rowspan="1" colspan="1">—</td></tr></tbody></table><table-wrap-foot><fn><p>Clinical isolates of Gram-positive and Gram-negative bacteria were used. Values are mean ± SD of three replicates (<italic>n</italic> = 3), inhibition zones in diameters (mm) produced by the extract around the discs. Size of disc was 6 mm. Absence of bacterial inhibition denoted by (—), that is, no inhibition zone around the disc by crude extract of plants.</p></fn></table-wrap-foot></table-wrap></floats-group></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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