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<meta http-equiv="Content-Type" content="text/html;charset=UTF-8"><base href="https://ejabf.journals.ekb.eg/article_351686.html"><div style="background:#fff;border:1px solid #999;margin:-1px -1px 0;padding:0;"><div style="background:#ddd;border:1px solid #999;color:#000;font:13px arial,sans-serif;font-weight:normal;margin:12px;padding:8px;text-align:left">This is the html version of the file <a href="https://ejabf.journals.ekb.eg/article_351686.html"><font color=blue>https://ejabf.journals.ekb.eg/article_351686.html</font></a>.<br> Google automatically generates html versions of documents as we crawl the web.</div></div><div style="position:relative"><html> <head> <meta http-equiv="Content-Type" content="text/html; charset=UTF-8"> <meta name="Author" content="ahmed youssef"> <meta name="CreationDate" content="D:20240424212419+03'00'"> <meta name="Creator" content="Microsoft® Word 2010"> <meta name="ModDate" content="D:20240424212419+03'00'"> <meta name="Producer" content="Microsoft® Word 2010"> <title>In vitro Assessment of Antischistosomal Effect of the Marine Hyrtios sp. Sponge Extract against Schistosoma mansoni Adult Worms</title> </head><body bgcolor=#ffffff vlink="blue" link="blue"> <table border=0 width=100%><tr><td bgcolor=eeeeee align=right><font face=arial,sans-serif><a name=1><b>Page 1</b></a></font></td></tr></table><font size=3 face="Times"><span style="font-size:14px;font-family:Times"> <div style="position:absolute;top:303;left:135"><nobr>Egyptian Journal of Aquatic Biology & Fisheries</nobr></div> <div style="position:absolute;top:322;left:135"><nobr>Zoology Department, Faculty of Science,</nobr></div> <div style="position:absolute;top:341;left:135"><nobr>Ain Shams University, Cairo, Egypt.</nobr></div> <div style="position:absolute;top:360;left:135"><nobr>ISSN 1110 –6131</nobr></div> <div style="position:absolute;top:379;left:135"><nobr>Vol. 28(2): 799–823 (2024)</nobr></div> <div style="position:absolute;top:398;left:135"><nobr>www.ejabf.journals.ekb.eg</nobr></div> </span></font> <font size=3 color="#0d0d0d" face="Times"><span style="font-size:16px;font-family:Times;color:#0d0d0d"> <div style="position:absolute;top:438;left:155"><nobr><i><b>In vitro </i>Assessment of Antischistosomal Effect of the <font color="#000000">Marine </font><i><font color="#000000">Hyrtios </font></i><font color="#000000">sp. Sponge</font></b></nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:459;left:262"><nobr><b>Extract <font color="#0d0d0d">against </font><i><font color="#0d0d0d">Schistosoma mansoni </font></i><font color="#0d0d0d">Adult Worms</font></b></nobr></div> </span></font> <font size=3 color="#0d0d0d" face="Times"><span style="font-size:14px;font-family:Times;color:#0d0d0d"> <div style="position:absolute;top:500;left:143"><nobr><b>Samir A. I. El-Abbassy, <font color="#000000">Donia N. A. El-Hawary</font>, Gihan M. El‑Khodary, Aml Z. Ghoneim,</b></nobr></div> <div style="position:absolute;top:519;left:392"><nobr><b>Salwa A. El-Saidy</b><font color="#000000">*</font></nobr></div> <div style="position:absolute;top:538;left:135"><nobr>Zoology Department, Faculty of Science, Damanhour University, Damanhour, Egypt</nobr></div> </span></font> <font size=2 face="Times"><span style="font-size:7px;font-family:Times"> <div style="position:absolute;top:553;left:301"><nobr><b>*<font style="font-size:12px">Corresponding Author: </font></b><font color="#0000ff" style="font-size:12px"><a href="mailto:s.elsaidy@sci.dmu.edu.eg">s.elsaidy@sci.dmu.edu.eg</a></font></nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:1170;left:171"><nobr><b>INTRODUCTION</b></nobr></div> </span></font> <font size=3 color="#0d0d0d" face="Times"><span style="font-size:16px;font-family:Times;color:#0d0d0d"> <div style="position:absolute;top:1211;left:171"><nobr>Schistosomiasis (bilharzia) is a highly common parasitic disease among humans</nobr></div> <div style="position:absolute;top:1232;left:135"><nobr>worldwide caused by the trematode worm of the genus <i>Schistosoma </i><b>(McManus <i>et al</i>.,</b></nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:616;left:115"><nobr><b>ARTICLE INFO</b></nobr></div> <div style="position:absolute;top:596;left:500"><nobr><b>ABSTRACT</b></nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:12px;font-family:Times"> <div style="position:absolute;top:636;left:115"><nobr><b>Article History:</b></nobr></div> <div style="position:absolute;top:653;left:115"><nobr>Received: March 1, 2024</nobr></div> <div style="position:absolute;top:671;left:115"><nobr>Accepted: April 11, 2024</nobr></div> <div style="position:absolute;top:688;left:115"><nobr>Online: April 24, 2024</nobr></div> <div style="position:absolute;top:706;left:110"><nobr><i><b>_______________</b></i></nobr></div> <div style="position:absolute;top:731;left:115"><nobr><b>Keywords</b>:</nobr></div> </span></font> <font size=3 color="#0d0d0d" face="Times"><span style="font-size:12px;font-family:Times;color:#0d0d0d"> <div style="position:absolute;top:748;left:115"><nobr><i>Hyrtios </i>sponge extract,</nobr></div> <div style="position:absolute;top:766;left:115"><nobr><i>Schistosoma mansoni,</i></nobr></div> <div style="position:absolute;top:783;left:115"><nobr>Phytochemicals,</nobr></div> <div style="position:absolute;top:800;left:115"><nobr>Antioxidant capacity,</nobr></div> <div style="position:absolute;top:817;left:115"><nobr>Antischistosomal activity,</nobr></div> <div style="position:absolute;top:835;left:115"><nobr>Scanning electron</nobr></div> <div style="position:absolute;top:852;left:115"><nobr>microscope</nobr></div> <div style="position:absolute;top:636;left:327"><nobr>Schistosomiasis is a prevalent epidemic disease in Egypt. As a result of the</nobr></div> <div style="position:absolute;top:653;left:296"><nobr>exclusive use of praziquantel (PZQ) in the treatment of schistosomiasis, the parasite</nobr></div> <div style="position:absolute;top:671;left:296"><nobr>may become resistant to the medication. Accordingly, there has been a growing interest</nobr></div> <div style="position:absolute;top:688;left:296"><nobr>in research with the aim of developing novel antischistosomal medicines from natural</nobr></div> <div style="position:absolute;top:705;left:296"><nobr>sources. The marine <i>Hyrtios </i>sponge exhibits bioactive compounds, qualifying it to</nobr></div> <div style="position:absolute;top:722;left:296"><nobr>explore novel drugs. Hence, the current study aimed to assess the <i>in vitro </i>activity of</nobr></div> <div style="position:absolute;top:740;left:296"><nobr>the <i>Hyrtios </i>sponge extract (HSE) against schistosomiasis through the detection of</nobr></div> <div style="position:absolute;top:757;left:296"><nobr>mortality and tegumental alterations in <i>S. mansoni </i>adult worms. The mortality rates of</nobr></div> <div style="position:absolute;top:774;left:296"><nobr><i>S. mansoni </i>worms exceeded 50% after 2 and 3 hours of exposure to a concentration of</nobr></div> <div style="position:absolute;top:791;left:296"><nobr>200µg/ ml of HSE. After 24 hours, mortality rates surpassed 50% at concentrations of</nobr></div> <div style="position:absolute;top:809;left:296"><nobr>both 100 and 200µg/ ml. Additionally, after 36 and 48 hours, mortality rates reached</nobr></div> <div style="position:absolute;top:826;left:296"><nobr>above 50% across concentrations ranging from 60 to 200µg/ ml. Furthermore, after</nobr></div> <div style="position:absolute;top:843;left:296"><nobr>48h, the mortality rate of <i>S. mansoni </i>worms reached 100% at a 200µg/ ml</nobr></div> <div style="position:absolute;top:860;left:296"><nobr>concentration. After exposing the <i>S. mansoni </i>worms to HSE for 24h, the lethal</nobr></div> <div style="position:absolute;top:878;left:296"><nobr>concentration values (LC<font style="font-size:7px">50 </font>and LC<font style="font-size:7px">90</font>) were 115.40 and 241.19µg/ ml, respectively.</nobr></div> <div style="position:absolute;top:895;left:296"><nobr>These lethal concentrations became 59.36 for LC<font style="font-size:7px">50 </font>and 144.11µg/ ml for LC<font style="font-size:7px">90 </font>after</nobr></div> <div style="position:absolute;top:912;left:296"><nobr>48h of exposure. Scanning electron microscopy revealed morphological changes in</nobr></div> <div style="position:absolute;top:929;left:296"><nobr>both male and female worms at concentrations of 100 and 200μg/ ml following a 48-h</nobr></div> <div style="position:absolute;top:947;left:296"><nobr><i>in vitro </i>treatment of <i>S. mansoni </i>worms with the HSE compared to the control group.</nobr></div> <div style="position:absolute;top:964;left:296"><nobr>Morphological abnormalities in male worms included swollen or flattened bodies, self-</nobr></div> <div style="position:absolute;top:981;left:296"><nobr>coiling contractions, tegument shrinkage, invaginations, erosion, deformed</nobr></div> <div style="position:absolute;top:998;left:296"><nobr>gynechophoric canal, distorted suckers, and smooth tubercles with blebs between them.</nobr></div> <div style="position:absolute;top:1016;left:296"><nobr>The female body displayed morphological alterations, including swollen body,</nobr></div> <div style="position:absolute;top:1033;left:296"><nobr>shrinkage of the ventral sucker, a pitted tegument with corrugation, folds,</nobr></div> <div style="position:absolute;top:1050;left:296"><nobr>invaginations, wrinkles, erosions, and blebs in addition to the absence of body spines.</nobr></div> <div style="position:absolute;top:1067;left:296"><nobr>The death of <i>S. mansoni </i>worms caused by HSE exposure could be associated with its</nobr></div> <div style="position:absolute;top:1085;left:296"><nobr>effect on their tegumental surface and may be related to the existence of different</nobr></div> <div style="position:absolute;top:1102;left:296"><nobr>antioxidant phytochemicals like alkaloids, saponins, phenolics, and flavonoids in this</nobr></div> <div style="position:absolute;top:1119;left:296"><nobr>extract. In conclusion, HSE has an effective antischistosomal impact and can be</nobr></div> <div style="position:absolute;top:1136;left:296"><nobr>considered an adjuvant medication for PZQ in <i>S. mansoni </i>infection. </nobr></div> </span></font> <div style="position:absolute;top:1363;left:0"><hr><table border=0 width=100%><tr><td bgcolor=eeeeee align=right><font face=arial,sans-serif><a name=2><b>Page 2</b></a></font></td></tr></table></div><font size=3 color="#0d0d0d" face="Times"><span style="font-size:12px;font-family:Times;color:#0d0d0d"> <div style="position:absolute;top:1420;left:387"><nobr><b>El-Abbassy <i><font color="#000000">et al.</font></i><font color="#000000">, 2024</font></b></nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:12px;font-family:Times"> <div style="position:absolute;top:1420;left:135"><nobr>800</nobr></div> </span></font> <font size=3 color="#0d0d0d" face="Times"><span style="font-size:16px;font-family:Times;color:#0d0d0d"> <div style="position:absolute;top:1475;left:135"><nobr><b>2020; Llanwarne & Helmby, 2021)</b>. In Egypt, schistosomiasis is still one of the most</nobr></div> <div style="position:absolute;top:1496;left:135"><nobr>prevalent epidemic diseases although various control programs exert many attempts to</nobr></div> <div style="position:absolute;top:1516;left:135"><nobr>get rid of this infection <b>(Abdella <i>et al., </i>2024)</b>. The most widespread type of</nobr></div> <div style="position:absolute;top:1537;left:135"><nobr>schistosomiasis among the Egyptian Nile Delta's rural inhabitants is <i>S. mansoni </i><b>(Ahmed</b></nobr></div> <div style="position:absolute;top:1558;left:135"><nobr><i><b>et al.</i>, 2021)</b>. Schistosomiasis principally affects children, and its long-term infection</nobr></div> <div style="position:absolute;top:1578;left:135"><nobr>causes significant health problems, including anemia, child growth impairment, fatigue,</nobr></div> <div style="position:absolute;top:1599;left:135"><nobr>and impaired mental abilities <b>(King & Dangerfield-Cha, 2008; Ezeamama <i>et al</i>., 2018)</b>.</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:1620;left:171"><nobr>Schistosomiasis control exclusively depends on chemotherapy through a single</nobr></div> <div style="position:absolute;top:1641;left:135"><nobr>anthelmintic praziquantel (PZQ) medicine <b>(<font color="#212121">Kura </font><i><font color="#212121">et al</font></i><font color="#212121">., 2022)</font></b><font color="#212121">. </font>Intensive and exclusive</nobr></div> <div style="position:absolute;top:1661;left:135"><nobr>use of a single drug to treat schistosomiasis for decades may eventually give rise to</nobr></div> <div style="position:absolute;top:1682;left:135"><nobr>legitimate worries that the disease may become resistant to PZQ <b>(Cioli <i>et al</i>., 2014;</b></nobr></div> <div style="position:absolute;top:1703;left:135"><nobr><b>Abou-El-Naga <i>et al., </i>2019)</b>. Therefore, <font color="#0d0d0d">significant attention has been paid to developing</font></nobr></div> <div style="position:absolute;top:1723;left:135"><nobr>new, safe, and effective antischistosomal medications mostly derived from natural</nobr></div> <div style="position:absolute;top:1744;left:135"><nobr>sources.</nobr></div> <div style="position:absolute;top:1765;left:171"><nobr>Marine invertebrates are rich sources of marine natural products with various</nobr></div> <div style="position:absolute;top:1785;left:135"><nobr>medicinal benefits to improve human health <b>(De Zoysa, 2012)</b>. The isolated chemicals or</nobr></div> <div style="position:absolute;top:1806;left:135"><nobr>prepared extracts from marine invertebrates have demonstrated a broad range of</nobr></div> <div style="position:absolute;top:1827;left:135"><nobr>therapeutic properties, including anticoagulant <b>(Karthikeyan <i>et al., </i>2012)</b>, anthelmintic</nobr></div> <div style="position:absolute;top:1848;left:135"><nobr><b>(Mona <i>et al., </i>2012)</b>, anti-inflammatory <b>(Senthilkumar & Kim, 2013)</b>, antiprotozoa</nobr></div> <div style="position:absolute;top:1868;left:135"><nobr><b>(Lhullier <i>et al., </i>2020)</b>, antiviral, antimicrobial <b>(Mona <i>et al., </i>2021)</b>, anticancer <b>(Singh <i>et</b></i></nobr></div> <div style="position:absolute;top:1889;left:135"><nobr><i><b>al., </i>2021)</b>, antioxidant <b>(<font color="#212121">Rasyid & Putra, 2023)</font></b>, and other medicinal effects. Therefore,</nobr></div> <div style="position:absolute;top:1910;left:135"><nobr>marine invertebrates became a trend for discovering more novel treatments for diseases</nobr></div> <div style="position:absolute;top:1930;left:135"><nobr>affecting humans.</nobr></div> <div style="position:absolute;top:1951;left:171"><nobr>Among marine invertebrates, sponges are the most promising source of biologically</nobr></div> <div style="position:absolute;top:1972;left:135"><nobr>active compounds and are used for exploring novel medications <b>(<font color="#0d0d0d">Perdicaris </font><i>et al., </i>2013;</b></nobr></div> <div style="position:absolute;top:1992;left:135"><nobr><b>Mehbub <i>et al</i>., 2014)</b>. <i>Hyrtios </i>sponge <b>(</b>class, Demospongiae; order, Dictyoceratida;</nobr></div> <div style="position:absolute;top:2013;left:135"><nobr><b>Duchassaing & Michelotti, 1864) </b>is reported to be a plentiful source of various kinds of</nobr></div> <div style="position:absolute;top:2034;left:135"><nobr>bioactive secondary metabolites of different classes <b>(Shady <i>et al</i>., 2017a, b)</b>. The</nobr></div> <div style="position:absolute;top:2055;left:135"><nobr>prominent bioactive compound classes from this genus include macrolides <b>(Kobayashi <i>et</b></i></nobr></div> <div style="position:absolute;top:2075;left:135"><nobr><i><b>al</i>., 1993)</b>, alkaloids <b>(Salmoun <i>et al</i>., 2002; Utkina, 2009; Youssef <i>et al</i>., 2013)</b>,</nobr></div> <div style="position:absolute;top:2096;left:135"><nobr>sesterterpenes <b>(Youssef <i>et al., </i>2002; Qiu <i>et al</i>., 2004; Mahidol <i>et al</i>., 2009)</b>,</nobr></div> <div style="position:absolute;top:2117;left:135"><nobr>sesquiterpenes <b>(Longeon <i>et al</i>., 2011)</b>, flavonoids, and saponins <b>(Wardany & Nirmala,</b></nobr></div> <div style="position:absolute;top:2137;left:135"><nobr><b>2023)</b>. Numerous studies have indicated that bioactive compounds isolated from <i>Hyrtios</i></nobr></div> <div style="position:absolute;top:2158;left:135"><nobr>are shown to exhibit pharmacological activities, including antioxidant <b>(Utkina, 2009)</b>,</nobr></div> <div style="position:absolute;top:2179;left:135"><nobr>anticancer <b>(Lim <i>et al</i>., 2014)</b>, anti-inflammatory <b>(Muthiyan <i>et al</i>., 2018)</b>,</nobr></div> </span></font> <font size=3 color="#0d0d0d" face="Times"><span style="font-size:16px;font-family:Times;color:#0d0d0d"> <div style="position:absolute;top:2200;left:135"><nobr>antitrypanosomal <b>(Shady <i>et al.</i>, 2018)</b>, <font color="#000000">anti-Alzheimer </font><b><font color="#000000">(Nabil-Adam </font><i><font color="#000000">et al</font></i><font color="#000000">., 2020)</font></b><font color="#000000">, and</font></nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:2220;left:135"><nobr>antiplasmodial activities <b>(Mahfur <i>et al</i>., 2022). </b>Despite the previous studies, <font color="#0d0d0d">there </font>have</nobr></div> <div style="position:absolute;top:2241;left:135"><nobr>yet to be any relevant reports <font color="#0d0d0d">on </font>the anthelmintic effect of <i>Hyrtios </i>marine sponge.</nobr></div> <div style="position:absolute;top:2262;left:135"><nobr>Therefore, the current study aimed to explore the <i>in vitro </i>activity of the methanolic</nobr></div> <div style="position:absolute;top:2282;left:135"><nobr>extract prepared from the <i>Hyrtios </i>sponge against schistosomiasis by detecting mortality</nobr></div> <div style="position:absolute;top:2303;left:135"><nobr>and morphological alterations of <i>S. mansoni </i>adult worms. </nobr></div> </span></font> <div style="position:absolute;top:2551;left:0"><hr><table border=0 width=100%><tr><td bgcolor=eeeeee align=right><font face=arial,sans-serif><a name=3><b>Page 3</b></a></font></td></tr></table></div><font size=3 color="#0d0d0d" face="Times"><span style="font-size:11px;font-family:Times;color:#0d0d0d"> <div style="position:absolute;top:2607;left:135"><nobr><b>Antischistosomal Effect of <font color="#000000">the Marine </font><i><font color="#000000">Hyrtios </font></i><font color="#000000">Sponge Extract </font>against <i>Schistosoma mansoni </i>Worms</b></nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:14px;font-family:Times"> <div style="position:absolute;top:2608;left:791"><nobr>801</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:2663;left:177"><nobr><b>MATERIALS AND METHODS</b></nobr></div> </span></font> <font size=3 color="#0d0d0d" face="Times"><span style="font-size:16px;font-family:Times;color:#0d0d0d"> <div style="position:absolute;top:2705;left:135"><nobr><b>1. <i>Hyrtios </i>sponge collection </b></nobr></div> <div style="position:absolute;top:2740;left:171"><nobr>Marine sponge <i>Hyrtios </i>sp. was collected from Hurghada, Egypt, in February 2022</nobr></div> <div style="position:absolute;top:2761;left:135"><nobr>(Fig. 1). Sampling was done using snorkeling and self-contained underwater breathing</nobr></div> <div style="position:absolute;top:2781;left:135"><nobr>apparatus (SCUBA) diving technique at a depth between 13 and 20m. Freshly collected</nobr></div> <div style="position:absolute;top:2802;left:135"><nobr>samples were washed with sterilized artificial seawater to remove sediments and</nobr></div> <div style="position:absolute;top:2823;left:135"><nobr>impurities, immediately frozen, and kept till usage at –20°C. </nobr></div> <div style="position:absolute;top:3200;left:135"><nobr><b>Fig. 1. a: </b>A black round ball shaped of <i>Hyrtios </i>marine sponge with scattered oscula</nobr></div> <div style="position:absolute;top:3220;left:135"><nobr>(white arrows) and spongin fibrous skeleton. <b>b: </b>Google map showing the sampling site</nobr></div> <div style="position:absolute;top:3241;left:135"><nobr>for collection of <i>Hyrtios </i>sponge (Hurghada indicated by red dot) on the Red Sea coast,</nobr></div> <div style="position:absolute;top:3262;left:135"><nobr>Egypt</nobr></div> <div style="position:absolute;top:3304;left:135"><nobr><b>2. Preparation of the methanolic crude <i>Hyrtios </i>sponge extract </b></nobr></div> <div style="position:absolute;top:3339;left:171"><nobr>The extraction process was conducted following the maceration method, as reported</nobr></div> <div style="position:absolute;top:3360;left:135"><nobr>in studies by <b>Purushottama <i>et al. </i>(2009) </b>and <b>Hutagalung <i>et al. </i>(2014)</b>. In brief, sponge</nobr></div> <div style="position:absolute;top:3380;left:135"><nobr>tissues were lyophilized for two days at room temperature (25– 27°C), cut into small</nobr></div> <div style="position:absolute;top:3401;left:135"><nobr>pieces, and pulverized until they became powder. The grounded sample was then soaked</nobr></div> <div style="position:absolute;top:3422;left:135"><nobr>in absolute methanol as a solvent (1: 20 <i>w/ v</i>) for 5h with genital stirring at room</nobr></div> <div style="position:absolute;top:3443;left:135"><nobr>temperature. A vacuum rotary evaporator operating at 40°C was used to evaporate the</nobr></div> <div style="position:absolute;top:3463;left:135"><nobr>solvent after the solution had undergone filtration through Whatman Filter Paper No. 1.</nobr></div> <div style="position:absolute;top:3484;left:135"><nobr>Following the lyophilization of the residual solvent, the dried <i>Hyrtios </i>sponge extract</nobr></div> <div style="position:absolute;top:3505;left:135"><nobr>(HSE) was preserved at –20°C for additional biochemical tests. </nobr></div> </span></font> <div style="position:absolute;top:3739;left:0"><hr><table border=0 width=100%><tr><td bgcolor=eeeeee align=right><font face=arial,sans-serif><a name=4><b>Page 4</b></a></font></td></tr></table></div><font size=3 color="#0d0d0d" face="Times"><span style="font-size:12px;font-family:Times;color:#0d0d0d"> <div style="position:absolute;top:3796;left:387"><nobr><b>El-Abbassy <i><font color="#000000">et al.</font></i><font color="#000000">, 2024</font></b></nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:12px;font-family:Times"> <div style="position:absolute;top:3796;left:135"><nobr>802</nobr></div> </span></font> <font size=3 color="#0d0d0d" face="Times"><span style="font-size:16px;font-family:Times;color:#0d0d0d"> <div style="position:absolute;top:3851;left:135"><nobr><b>3. Determination of bioactive compounds in <i>Hyrtios </i>sponge extract </b></nobr></div> <div style="position:absolute;top:3887;left:135"><nobr><i><b>3.1. Total phenolics</b></i></nobr></div> <div style="position:absolute;top:3922;left:171"><nobr>The Folin-Ciocalteu procedure was utilized to evaluate the extract's total content of</nobr></div> <div style="position:absolute;top:3943;left:135"><nobr>phenolics <b>(Taga <i>et al</i>., 1984)</b>. Two milliliters of 2% Na2CO3 solution were combined</nobr></div> <div style="position:absolute;top:3964;left:135"><nobr>with 100µl of either the sample or gallic acid (GA) standard. Subsequently, 100µl of 50%</nobr></div> <div style="position:absolute;top:3984;left:135"><nobr>Folin-Ciocalteu reagent was added to the mixture. This mixture was <font color="#000000">preserved at room</font></nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:4005;left:135"><nobr>temperature <font color="#0d0d0d">for a period of 30min. A spectrophotometer was utilized to indicate the</font></nobr></div> </span></font> <font size=3 color="#0d0d0d" face="Times"><span style="font-size:16px;font-family:Times;color:#0d0d0d"> <div style="position:absolute;top:4026;left:135"><nobr>absorbance of the solution at 750nm wavelength. By comparing the results to the GA</nobr></div> <div style="position:absolute;top:4047;left:135"><nobr>calibration curve, the total content of phenolics was calculated as milligrams of gallic</nobr></div> <div style="position:absolute;top:4067;left:135"><nobr>acid equivalent (GAE) per gram of extract.</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:4103;left:135"><nobr><i><b>3.2. Total flavonoids</b></i></nobr></div> </span></font> <font size=3 color="#0d0d0d" face="Times"><span style="font-size:16px;font-family:Times;color:#0d0d0d"> <div style="position:absolute;top:4139;left:171"><nobr>The extract's total content of flavonoids was estimated following the method of</nobr></div> <div style="position:absolute;top:4159;left:135"><nobr><b>Zhishen <i>et al. </i>(1999)</b>. Firstly, one ml of sample was combined with 4ml of distilled water</nobr></div> <div style="position:absolute;top:4180;left:135"><nobr>(dis. H<font style="font-size:10px">2</font>O) and 0.3ml of NaNO<font style="font-size:10px">2 </font>(1: 20). Secondly, after 5min, 3ml of AlCl<font style="font-size:10px">3 </font>(1: 10) was</nobr></div> <div style="position:absolute;top:4201;left:135"><nobr>added and allowed to stand for a period of 6min. Finally, 2ml of 1.0M NaOH was added</nobr></div> <div style="position:absolute;top:4221;left:135"><nobr>to the solution, and the volume was increased by adding dis. H<font style="font-size:10px">2</font>O to 10ml. After</nobr></div> <div style="position:absolute;top:4242;left:135"><nobr>thoroughly mixing the solution, a spectrophotometer was used to measure the solution's</nobr></div> <div style="position:absolute;top:4263;left:135"><nobr>absorbance at 510nm against a blank. Quercetin was employed as a standard solution. By</nobr></div> <div style="position:absolute;top:4284;left:135"><nobr>comparing the total flavonoid concentration to the reference calibration curve, it was</nobr></div> <div style="position:absolute;top:4304;left:135"><nobr>calculated as milligrams of quercetin equivalent (QE) per gram of extract.</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:4340;left:135"><nobr><i><b>3.3. Total alkaloids</b></i></nobr></div> </span></font> <font size=3 color="#0d0d0d" face="Times"><span style="font-size:16px;font-family:Times;color:#0d0d0d"> <div style="position:absolute;top:4376;left:171"><nobr>The estimation of the total content of alkaloids in the extract was based on the</nobr></div> <div style="position:absolute;top:4396;left:135"><nobr>reaction between alkaloids in the extract and acetic acid forming a salt, and the liberation</nobr></div> <div style="position:absolute;top:4417;left:135"><nobr>of alkaloids was stimulated with ammonia <b>(Harbone, 1973)</b>. About 0.2ml of 10% acetic</nobr></div> <div style="position:absolute;top:4438;left:135"><nobr>acid in ethanol was mixed with 5mg of extract, and the mixture was permitted to stand</nobr></div> <div style="position:absolute;top:4459;left:135"><nobr>overnight. The filtrate was concentrated in a water bath to be a quarter of its initial</nobr></div> <div style="position:absolute;top:4479;left:135"><nobr>volume. The filtrate was gradually mixed with concentrated NH<font style="font-size:10px">4</font>OH until precipitation</nobr></div> <div style="position:absolute;top:4500;left:135"><nobr>occurred. The entire solution was left to settle overnight, and finally, the collected</nobr></div> <div style="position:absolute;top:4521;left:135"><nobr>precipitate was washed with 0.1M NH<font style="font-size:10px">4</font>OH and filtered. The total content of alkaloids was</nobr></div> <div style="position:absolute;top:4541;left:135"><nobr>calculated as milligrams per gram of extract after the alkaloid residue was weighed and</nobr></div> <div style="position:absolute;top:4562;left:135"><nobr>dried.</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:4598;left:135"><nobr><i><b>3.4. Total saponins </b></i></nobr></div> </span></font> <font size=3 color="#0d0d0d" face="Times"><span style="font-size:16px;font-family:Times;color:#0d0d0d"> <div style="position:absolute;top:4633;left:171"><nobr>The quantification of the total content of saponins in the extract occurred following</nobr></div> <div style="position:absolute;top:4654;left:135"><nobr>the method of <b>Espinoza <i>et al. </i>(2021)</b>. About 0.8ml of the extract was hydrolyzed in an</nobr></div> <div style="position:absolute;top:4675;left:135"><nobr>aliquot using an equivalent amount of 6N HCl for a period of 2h at 110°C and then</nobr></div> <div style="position:absolute;top:4695;left:135"><nobr>cooled for 5min in an ice-cold water bath. Then, after neutralizing it with an ammonia</nobr></div> <div style="position:absolute;top:4716;left:135"><nobr>solution, it was centrifuged for 5min at 3000 ×g. After extracting the supernatant, 3ml of</nobr></div> <div style="position:absolute;top:4737;left:135"><nobr>ethyl acetate was added for every 5ml sample. After that, the fractions were mixed and</nobr></div> <div style="position:absolute;top:4758;left:135"><nobr>filtrated on a bed of anhydrous Na<font style="font-size:10px">2</font>SO<font style="font-size:10px">4</font>. About 250µl of this final extract was shaken</nobr></div> <div style="position:absolute;top:4779;left:135"><nobr>vigorously with 1000µl of the reagent mixture (glacial CH₃COOH/H₂SO₄, 1:1 <i>v/ v</i>) for a</nobr></div> <div style="position:absolute;top:4799;left:135"><nobr>period of 30sec and, and then heated at 60°C in a water bath for 30min to develop light </nobr></div> </span></font> <div style="position:absolute;top:4927;left:0"><hr><table border=0 width=100%><tr><td bgcolor=eeeeee align=right><font face=arial,sans-serif><a name=5><b>Page 5</b></a></font></td></tr></table></div><font size=3 color="#0d0d0d" face="Times"><span style="font-size:11px;font-family:Times;color:#0d0d0d"> <div style="position:absolute;top:4983;left:135"><nobr><b>Antischistosomal Effect of <font color="#000000">the Marine </font><i><font color="#000000">Hyrtios </font></i><font color="#000000">Sponge Extract </font>against <i>Schistosoma mansoni </i>Worms</b></nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:14px;font-family:Times"> <div style="position:absolute;top:4984;left:791"><nobr>803</nobr></div> </span></font> <font size=3 color="#0d0d0d" face="Times"><span style="font-size:16px;font-family:Times;color:#0d0d0d"> <div style="position:absolute;top:5039;left:135"><nobr>purple color. The absorbance of the cooled mixture was estimated at a specific</nobr></div> <div style="position:absolute;top:5060;left:135"><nobr>wavelength of 527nm. Total saponin content was quantified as milligrams of sapogenin</nobr></div> <div style="position:absolute;top:5081;left:135"><nobr>equivalent (SE) per gram of extract.</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:5116;left:135"><nobr><b>4. <font color="#0d0d0d">Determination of antioxidant capacity of </font><i><font color="#0d0d0d">Hyrtios </font></i><font color="#0d0d0d">sponge extract using 2,2-</font></b></nobr></div> </span></font> <font size=3 color="#0d0d0d" face="Times"><span style="font-size:16px;font-family:Times;color:#0d0d0d"> <div style="position:absolute;top:5137;left:135"><nobr><b>diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay</b></nobr></div> <div style="position:absolute;top:5172;left:171"><nobr>The antioxidant capacity of <i>Hyrtios </i>extract was evaluated using the 2,2-diphenyl-1-</nobr></div> <div style="position:absolute;top:5193;left:135"><nobr>picrylhydrazyl (DPPH) radical scavenging assay, which was suggested by <b>Brand-</b></nobr></div> <div style="position:absolute;top:5214;left:135"><nobr><b>Williams <i>et al. </i>(1995) </b>and <b>Molyneux (2004)</b>. Ascorbic acid concentrations ranging from</nobr></div> <div style="position:absolute;top:5235;left:135"><nobr>0.1 to 0.5mg/ ml were prepared to establish a standard curve. In brief,</nobr></div> <div style="position:absolute;top:5255;left:135"><nobr>1.0ml of 0.2Mm DPPH in methanol (0.0078 g/100 ml) was prepared and added to 1ml of</nobr></div> <div style="position:absolute;top:5276;left:135"><nobr>the extract (5mg/ ml) and/or standard solution at varying concentrations. The mixture was</nobr></div> <div style="position:absolute;top:5297;left:135"><nobr>incubated in the dark at an ambient temperature for 30min. Subsequently, the absorbance</nobr></div> <div style="position:absolute;top:5317;left:135"><nobr>(Abs) of the resulting reduction in DPPH color intensity was determined at 517nm</nobr></div> <div style="position:absolute;top:5338;left:135"><nobr>utilizing a spectrophotometer. The DPPH radical-scavenging activity was determined as</nobr></div> <div style="position:absolute;top:5359;left:135"><nobr>the percentage of DPPH inhibition using the subsequent equation:</nobr></div> <div style="position:absolute;top:5395;left:237"><nobr>DPPH inhibition % = (Abs <font style="font-size:10px">Control</font><font color="#365f91" style="font-size:12px">– </font>Abs <font style="font-size:10px">Sample</font>)/Abs <font style="font-size:10px">Control </font>× 100</nobr></div> <div style="position:absolute;top:5430;left:171"><nobr>For DPPH control, all reagents were added except the tested sample. The calibration</nobr></div> <div style="position:absolute;top:5451;left:135"><nobr>curve was employed to express the antioxidant capacity as milligrams of ascorbic acid</nobr></div> <div style="position:absolute;top:5472;left:135"><nobr>equivalent (AAE) per gram of dried extract.</nobr></div> <div style="position:absolute;top:5508;left:135"><nobr><b>5. <i>In vitro </i>screening of antischistosomal activity of <i>Hyrtios </i>sponge extract</b></nobr></div> <div style="position:absolute;top:5543;left:171"><nobr>Adult <i>S. mansoni </i>worms of Egyptian strain were obtained from <i>Schistosoma</i></nobr></div> <div style="position:absolute;top:5564;left:135"><nobr>Biological Supply Center, Theodor Bilharz Research Institute, Embaba, Giza, Egypt. The</nobr></div> <div style="position:absolute;top:5584;left:135"><nobr>used culture medium was Roswell Park Memorial Institute (RPMI-1640) medium</nobr></div> <div style="position:absolute;top:5605;left:135"><nobr>(Sigma, USA) enhanced by sterilizing antibiotics (100µg/ ml of streptomycin and 100U/</nobr></div> <div style="position:absolute;top:5626;left:135"><nobr>ml of penicillin; Sigma, USA), 2g/ L glucose, 0.39g/ L glutamate, 20g/ L NaHCO<font style="font-size:10px">3</font>, and</nobr></div> <div style="position:absolute;top:5647;left:135"><nobr>20% fetal calf serum (Gibco, USA) as a nutrient agent.</nobr></div> <div style="position:absolute;top:5667;left:171"><nobr>The antischistosomal activity of the methanolic crude HSE was evaluated <i>in vitro </i>on</nobr></div> <div style="position:absolute;top:5688;left:135"><nobr>adult <i>S. mansoni </i>worms according to the method of <b>Melek <i>et al. </i>(2012) </b>and <b>Abou El-</b></nobr></div> <div style="position:absolute;top:5709;left:135"><nobr><b>Nour and Fadladdin (2021)</b>. The known weight of the HSE was dissolved in dis. H<font style="font-size:10px">2</font>O to</nobr></div> <div style="position:absolute;top:5729;left:135"><nobr>obtain a stock solution from which the desired concentrations (20, 40, 60, 80, 100, and</nobr></div> <div style="position:absolute;top:5750;left:135"><nobr>200µg/ ml) were prepared. Adult worms incubated with medium and dis. H<font style="font-size:10px">2</font>O served as a</nobr></div> <div style="position:absolute;top:5771;left:135"><nobr>negative control, and the reference drug PZQ (0.2µg/ ml) was utilized as a positive</nobr></div> <div style="position:absolute;top:5791;left:135"><nobr>control. Under sterile conditions, eight adult <i>S. mansoni </i>worms of both sexes were added</nobr></div> <div style="position:absolute;top:5812;left:135"><nobr>to the culture media in each well of a flat-bottom 24-well tissue culture plate and</nobr></div> <div style="position:absolute;top:5833;left:135"><nobr>preserved at 37°C in a moist condition containing 5% CO<font style="font-size:10px">2 </font>for 2h for adaptation before</nobr></div> <div style="position:absolute;top:5854;left:135"><nobr>the addition of treatments. After that, 1.0ml of PZQ and/or different tested extract</nobr></div> <div style="position:absolute;top:5874;left:135"><nobr>concentrations were added to each well in triplicate and re-incubated for 48h. The adult</nobr></div> <div style="position:absolute;top:5895;left:135"><nobr>worms were monitored at different intervals (1, 2, 3, 24, 36, and 48h). After each</nobr></div> <div style="position:absolute;top:5916;left:135"><nobr>incubation period, the viability and mortality of worms were checked under a</nobr></div> <div style="position:absolute;top:5936;left:135"><nobr>stereomicroscope and recorded. Worms that remained motionless for one minute were</nobr></div> <div style="position:absolute;top:5957;left:135"><nobr>classified as dead. The antischistosomal activity of HSE was calculated as the number of</nobr></div> <div style="position:absolute;top:5978;left:135"><nobr>dead worms relative to the total number of worms and compared with the negative and </nobr></div> </span></font> <div style="position:absolute;top:6115;left:0"><hr><table border=0 width=100%><tr><td bgcolor=eeeeee align=right><font face=arial,sans-serif><a name=6><b>Page 6</b></a></font></td></tr></table></div><font size=3 color="#0d0d0d" face="Times"><span style="font-size:12px;font-family:Times;color:#0d0d0d"> <div style="position:absolute;top:6172;left:387"><nobr><b>El-Abbassy <i><font color="#000000">et al.</font></i><font color="#000000">, 2024</font></b></nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:12px;font-family:Times"> <div style="position:absolute;top:6172;left:135"><nobr>804</nobr></div> </span></font> <font size=3 color="#0d0d0d" face="Times"><span style="font-size:16px;font-family:Times;color:#0d0d0d"> <div style="position:absolute;top:6227;left:135"><nobr>positive controls. The <font color="#000000">extract lethal concentrations for 50 and 90% mortality of the</font></nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:6248;left:135"><nobr>exposed <i>S. mansoni </i>adult worms <font color="#0d0d0d">(LC</font><font color="#0d0d0d" style="font-size:10px">50 </font><font color="#0d0d0d">and LC</font><font color="#0d0d0d" style="font-size:10px">90</font><font color="#0d0d0d">, respectively) were evaluated using</font></nobr></div> </span></font> <font size=3 color="#0d0d0d" face="Times"><span style="font-size:16px;font-family:Times;color:#0d0d0d"> <div style="position:absolute;top:6269;left:135"><nobr>linear regression equations at all-time intervals.</nobr></div> <div style="position:absolute;top:6305;left:135"><nobr><b>6. Scanning electron microscopic examination of <i>Schistosoma mansoni </i>adult worms</b></nobr></div> <div style="position:absolute;top:6340;left:171"><nobr><i>S</i>. <i>mansoni </i>adult worms were rinsed multiple times in 9% saline. Then, they were</nobr></div> <div style="position:absolute;top:6360;left:135"><nobr>primarily fixed in 4% paraformaldehyde (PFA) + 2.5% glutaraldehyde solution and were</nobr></div> <div style="position:absolute;top:6381;left:135"><nobr>kept overnight at 4°C. After that, the worms were submerged in a phosphate buffer for</nobr></div> <div style="position:absolute;top:6402;left:135"><nobr>15min to remove any remaining fixative residue. Post-fixation of worms occurred in 1%</nobr></div> <div style="position:absolute;top:6423;left:135"><nobr>osmium tetroxide phosphate buffer for one hour. Then, the worms were given two rounds</nobr></div> <div style="position:absolute;top:6443;left:135"><nobr>of washing in the phosphate buffer before being dehydrated using ethyl alcohol (30, 40,</nobr></div> <div style="position:absolute;top:6464;left:135"><nobr>and 50%) for 15min each. After being left in 70% ethanol for 30min, the worms were</nobr></div> <div style="position:absolute;top:6485;left:135"><nobr>cleaned twice in 80, 90, and 100% ethanol, mounted on copper stubs holders, and</nobr></div> <div style="position:absolute;top:6505;left:135"><nobr>covered with gold using a sputter coater. Finally, the worms were examined and</nobr></div> <div style="position:absolute;top:6526;left:135"><nobr>microphotographed utilizing a scanning electron microscope (SEM) (Jeol–JSM 6510 LV)</nobr></div> <div style="position:absolute;top:6547;left:135"><nobr>at Mansoura University <b>(Mona <i>et al., </i>2012)</b>.</nobr></div> <div style="position:absolute;top:6583;left:135"><nobr><b>7. Statistical analysis of data</b></nobr></div> <div style="position:absolute;top:6627;left:171"><nobr>All experiments were conducted in triplicate, and the resulting data were presented</nobr></div> <div style="position:absolute;top:6647;left:135"><nobr>as means ± standard deviations (SD). This study utilized linear regression analysis to</nobr></div> <div style="position:absolute;top:6668;left:135"><nobr>estimate LC<font style="font-size:10px">50 </font>and LC<font style="font-size:10px">90 </font>of HSE against <i>S. mansoni </i>adult worms. The one-way analysis of</nobr></div> <div style="position:absolute;top:6685;left:135"><nobr>variance (ANOVA) test and Tukey’s test for multiple comparisons were conducted to</nobr></div> <div style="position:absolute;top:6710;left:135"><nobr>analyze the obtained results using the Statistical Package for the Social Sciences (IBM-</nobr></div> <div style="position:absolute;top:6730;left:135"><nobr>SPSS Version 20.0) software. The differences were statistically significant at <i>P</i>≤ 0.05</nobr></div> <div style="position:absolute;top:6751;left:135"><nobr><b>(Kirkpatrick & Feeney, 2012)</b>.</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:6787;left:177"><nobr><b>RESULTS </b></nobr></div> <div style="position:absolute;top:6829;left:135"><nobr><b>1. Characterization of the methanolic crude <i>Hyrtios </i>sponge extract </b></nobr></div> <div style="position:absolute;top:6849;left:171"><nobr>Phytochemical analysis of HSE indicated the presence of a high level of total</nobr></div> <div style="position:absolute;top:6870;left:135"><nobr>alkaloid content at a concentration of 370± 10mg/ g of extract, followed by total contents</nobr></div> <div style="position:absolute;top:6890;left:135"><nobr>of saponins, phenolics, and flavonoids at concentrations of 311.68± 5.23mg SE/ g of</nobr></div> <div style="position:absolute;top:6911;left:135"><nobr>extract, 5.19± 0.32mg GAE/ g of extract, and 2.36± 0.16mg QE/ g of extract,</nobr></div> <div style="position:absolute;top:6932;left:135"><nobr>respectively. Moreover, the <font color="#0d0d0d">antioxidant capacity </font>of this extract <font color="#0d0d0d">was 101.70± 6.72mg</font></nobr></div> </span></font> <font size=3 color="#0d0d0d" face="Times"><span style="font-size:16px;font-family:Times;color:#0d0d0d"> <div style="position:absolute;top:6952;left:135"><nobr>AAE/ g of extract (Table 1).</nobr></div> <div style="position:absolute;top:6994;left:135"><nobr><b>Table 1. </b><font color="#000000">Phytochemicals and antioxidant capacity of the methanolic crude </font><i><font color="#000000">Hyrtios</font></i></nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:7015;left:135"><nobr>sponge extract (HSE)</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:14px;font-family:Times"> <div style="position:absolute;top:7045;left:127"><nobr><b>Parameter</b></nobr></div> <div style="position:absolute;top:7045;left:613"><nobr><b>Concentration</b></nobr></div> <div style="position:absolute;top:7064;left:127"><nobr>Total phenolics (mg GAE/g of extract)</nobr></div> <div style="position:absolute;top:7064;left:626"><nobr>5.19 ± 0.32</nobr></div> <div style="position:absolute;top:7083;left:127"><nobr>Total flavanoids (mg QE/g of extract)</nobr></div> <div style="position:absolute;top:7083;left:626"><nobr>2.36 ± 0.16</nobr></div> <div style="position:absolute;top:7102;left:127"><nobr>Total alkaloids (mg/g of extract)</nobr></div> <div style="position:absolute;top:7102;left:634"><nobr>370 ± 10</nobr></div> <div style="position:absolute;top:7121;left:127"><nobr>Total saponins (mg SE/g of extract)</nobr></div> <div style="position:absolute;top:7121;left:618"><nobr>311.68 ± 5.23</nobr></div> <div style="position:absolute;top:7140;left:127"><nobr>Antioxidant capacity (mg AAE/g of extract)</nobr></div> <div style="position:absolute;top:7140;left:618"><nobr>101.70 ± 6.72</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:12px;font-family:Times"> <div style="position:absolute;top:7160;left:135"><nobr>Each value is the mean of 3 replicates ± SD. </nobr></div> </span></font> <div style="position:absolute;top:7303;left:0"><hr><table border=0 width=100%><tr><td bgcolor=eeeeee align=right><font face=arial,sans-serif><a name=7><b>Page 7</b></a></font></td></tr></table></div><font size=3 color="#0d0d0d" face="Times"><span style="font-size:11px;font-family:Times;color:#0d0d0d"> <div style="position:absolute;top:7359;left:135"><nobr><b>Antischistosomal Effect of <font color="#000000">the Marine </font><i><font color="#000000">Hyrtios </font></i><font color="#000000">Sponge Extract </font>against <i>Schistosoma mansoni </i>Worms</b></nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:14px;font-family:Times"> <div style="position:absolute;top:7360;left:791"><nobr>805</nobr></div> </span></font> <font size=3 color="#0d0d0d" face="Times"><span style="font-size:16px;font-family:Times;color:#0d0d0d"> <div style="position:absolute;top:7415;left:128"><nobr><b>2. Screening of antischistosomal activity of the methanolic crude <i>Hyrtios </i>sponge</b></nobr></div> <div style="position:absolute;top:7436;left:170"><nobr><b>extract </b></nobr></div> <div style="position:absolute;top:7468;left:171"><nobr>As shown in Table (2), there is a complete absence of mortality in the <i>S. mansoni</i></nobr></div> <div style="position:absolute;top:7489;left:135"><nobr>adult worms of the control group and 20μg/ ml HSE group. PZQ at a 0.2μg/ ml</nobr></div> <div style="position:absolute;top:7510;left:135"><nobr>concentration eliminated all worms at 3, 24, 36, and 48h (<i>P</i>˂ 0.001). The rise in the</nobr></div> <div style="position:absolute;top:7530;left:135"><nobr>mortality rate of <i>S. mansoni </i>adult worms depended on the increasing HSE concentration</nobr></div> <div style="position:absolute;top:7551;left:135"><nobr>and longer exposure time. The HSE, ranging from a concentration of 60µg/ ml after 2</nobr></div> <div style="position:absolute;top:7572;left:135"><nobr>hours to 200µg/ ml after 48 hours, showed significant mortality percentages (<i>P</i>≤ 0.001)</nobr></div> <div style="position:absolute;top:7593;left:135"><nobr>of adult <i>S. mansoni </i>worms compared to the control group. The <i>S. mansoni </i>adult worm’s</nobr></div> <div style="position:absolute;top:7613;left:135"><nobr>mortality of more than 50% occurred after 2 and 3h of exposure to HSE at 200µg/ ml,</nobr></div> <div style="position:absolute;top:7634;left:135"><nobr>after 24h of exposure to HSE at 100 and 200µg/ ml, and after 36 and 48h of exposing to</nobr></div> <div style="position:absolute;top:7655;left:135"><nobr>HSE at concentrations ranged from 60 to 200µg/ ml. The HSE achieved potent <i>in vitro</i></nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:7675;left:135"><nobr>schistosomicidal <font color="#0d0d0d">activity on the adult worms of </font><i><font color="#0d0d0d">S. mansoni </font></i><font color="#0d0d0d">(</font><i><font color="#0d0d0d">P< </font></i><font color="#0d0d0d">0.001) with 100%</font></nobr></div> </span></font> <font size=3 color="#0d0d0d" face="Times"><span style="font-size:16px;font-family:Times;color:#0d0d0d"> <div style="position:absolute;top:7696;left:135"><nobr>mortality at 200µg/ ml after 48h in comparison to the control group. </nobr></div> <div style="position:absolute;top:7717;left:171"><nobr>On 24 and 48h post-treatment, the mortality percentage of <i>S. mansoni </i>adult worms</nobr></div> <div style="position:absolute;top:7738;left:135"><nobr>fluctuated between its maximum values (68.44 and 100%, respectively) at the highest</nobr></div> <div style="position:absolute;top:7758;left:135"><nobr>applied concentration (200µg/ ml), and the minimum values (33.33 and 37.50%,</nobr></div> <div style="position:absolute;top:7779;left:135"><nobr>respectively) at 40µg/ ml. The values of LC<font style="font-size:10px">50 </font>and LC<font style="font-size:10px">90 </font>for the HSE on the adult <i>S.</i></nobr></div> <div style="position:absolute;top:7799;left:135"><nobr><i>mansoni </i>worms were estimated by the linear regression equation, as presented in Table</nobr></div> <div style="position:absolute;top:7820;left:135"><nobr>(2). The HSE exhibited LC<font style="font-size:10px">50 </font>values of 115.40 and 59.36µg/ ml after 24 and 48h of</nobr></div> <div style="position:absolute;top:7841;left:135"><nobr>exposure time, respectively. The LC<font style="font-size:10px">90 </font>values of HSE against the <i>S. mansoni </i>adult worms</nobr></div> <div style="position:absolute;top:7862;left:135"><nobr>decreased dramatically with the increasing exposure time; they were 241.19 and</nobr></div> <div style="position:absolute;top:7882;left:135"><nobr>144.11µg/ ml after 24 and 48h of exposure time, respectively.</nobr></div> <div style="position:absolute;top:7918;left:135"><nobr><b>3. <i>In vitro </i>morphological changes of male and female <i>Schistosoma mansoni </i>adult</b></nobr></div> <div style="position:absolute;top:7939;left:135"><nobr><b>worms</b></nobr></div> <div style="position:absolute;top:7974;left:171"><nobr>SEM examination of the adult worms of <i>S. mansoni </i>obtained from the control group</nobr></div> <div style="position:absolute;top:7995;left:135"><nobr>illustrated that the adult male of <i>S. mansoni </i>was thicker, cylindrical, and shorter than the</nobr></div> <div style="position:absolute;top:8016;left:135"><nobr>adult female. The male had a ventral gynaecophoric canal where the female was located</nobr></div> <div style="position:absolute;top:8037;left:135"><nobr>(Fig. 2a). Body of the male had three distinguished regions; the anterior region, which</nobr></div> <div style="position:absolute;top:8057;left:135"><nobr>comprises of oral sucker, ventral sucker, and a tegumental area connecting them, the</nobr></div> <div style="position:absolute;top:8078;left:135"><nobr>middle region, and the posterior region. The male <i>S. mansoni </i>adult worm had an oval-</nobr></div> <div style="position:absolute;top:8099;left:135"><nobr>shaped oral sucker at the top of the body anterior region with three notable regions; a thin</nobr></div> <div style="position:absolute;top:8119;left:135"><nobr>rim, a large inner zone, and the mouth. The ventral sucker was round and consisted of a</nobr></div> <div style="position:absolute;top:8140;left:135"><nobr>rim, inner zone, and central zone. The tegument of the body anterior region had different</nobr></div> <div style="position:absolute;top:8161;left:135"><nobr>shapes of papillae, including hemispherical papillae (Fig. 2b). The outer margin of the</nobr></div> <div style="position:absolute;top:8181;left:135"><nobr>gynaecophoral canal had tiny spines (Fig. 2c), and its inner surface appeared as folds with</nobr></div> <div style="position:absolute;top:8202;left:135"><nobr>numerous spines (Fig. 2d). Moreover, the middle part of male body exhibited sponge-like</nobr></div> <div style="position:absolute;top:8223;left:135"><nobr>tegument covered with many large spiny tubercles and macule papillae on the wrinkled</nobr></div> <div style="position:absolute;top:8244;left:135"><nobr>tegument around them (Fig. 2e). Tegument of the body posterior region was consisted of</nobr></div> <div style="position:absolute;top:8264;left:135"><nobr>spiny tubercles and macule papillae between them (Fig. 2f). </nobr></div> </span></font> <div style="position:absolute;top:8491;left:0"><hr><table border=0 width=100%><tr><td bgcolor=eeeeee align=right><font face=arial,sans-serif><a name=8><b>Page 8</b></a></font></td></tr></table></div><font size=3 color="#0d0d0d" face="Times"><span style="font-size:12px;font-family:Times;color:#0d0d0d"> <div style="position:absolute;top:8548;left:387"><nobr><b>El-Abbassy <i><font color="#000000">et al.</font></i><font color="#000000">, 2024</font></b></nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:12px;font-family:Times"> <div style="position:absolute;top:8548;left:135"><nobr>806</nobr></div> </span></font> <font size=3 color="#0d0d0d" face="Times"><span style="font-size:16px;font-family:Times;color:#0d0d0d"> <div style="position:absolute;top:8603;left:135"><nobr><b>Table 2. </b><font color="#000000">The percentage of dead adult worms of </font><i><font color="#000000">Schistosoma mansoni </font></i><font color="#000000">after </font><i><font color="#000000">in vitro</font></i></nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:8624;left:135"><nobr>exposure to 0.2µg/ ml of praziquantel (PZQ) and different concentrations of <i>Hyrtios</i></nobr></div> <div style="position:absolute;top:8644;left:135"><nobr>sponge extract (HSE) during different incubation periods in <font color="#0d0d0d">comparison </font>with the control</nobr></div> <div style="position:absolute;top:8665;left:135"><nobr>group</nobr></div> <div style="position:absolute;top:8697;left:76"><nobr><b>Group</b></nobr></div> <div style="position:absolute;top:8687;left:147"><nobr><b>Concentration</b></nobr></div> <div style="position:absolute;top:8708;left:174"><nobr><b>(µg/ml)</b></nobr></div> <div style="position:absolute;top:8687;left:278"><nobr><b>Percentage of dead adult worms of <i>S. mansoni </i>after different incubation</b></nobr></div> <div style="position:absolute;top:8708;left:518"><nobr><b>periods (h)</b></nobr></div> <div style="position:absolute;top:8744;left:313"><nobr><b>1 h</b></nobr></div> <div style="position:absolute;top:8744;left:423"><nobr><b>2 h</b></nobr></div> <div style="position:absolute;top:8744;left:522"><nobr><b>3 h</b></nobr></div> <div style="position:absolute;top:8744;left:612"><nobr><b>24 h</b></nobr></div> <div style="position:absolute;top:8744;left:706"><nobr><b>36 h</b></nobr></div> <div style="position:absolute;top:8744;left:795"><nobr><b>48 h</b></nobr></div> <div style="position:absolute;top:8783;left:74"><nobr>Control</nobr></div> </span></font> <font size=3 color="#0d0d0d" face="Times"><span style="font-size:16px;font-family:Times;color:#0d0d0d"> <div style="position:absolute;top:8790;left:149"><nobr>Distilled <font color="#000000">water 0.00 ± 0.00</font></nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:10px;font-family:Times"> <div style="position:absolute;top:8786;left:363"><nobr>a</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:8790;left:392"><nobr>0.00 ± 0.00</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:10px;font-family:Times"> <div style="position:absolute;top:8786;left:473"><nobr>a</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:8780;left:511"><nobr>0.00 ±</nobr></div> <div style="position:absolute;top:8801;left:515"><nobr>0.00</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:10px;font-family:Times"> <div style="position:absolute;top:8797;left:547"><nobr>a</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:8780;left:605"><nobr>0.00 ±</nobr></div> <div style="position:absolute;top:8801;left:610"><nobr>0.00</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:10px;font-family:Times"> <div style="position:absolute;top:8797;left:641"><nobr>a</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:8780;left:699"><nobr>0.00 ±</nobr></div> <div style="position:absolute;top:8801;left:704"><nobr>0.00</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:10px;font-family:Times"> <div style="position:absolute;top:8797;left:735"><nobr>a</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:8780;left:788"><nobr>0.00 ±</nobr></div> <div style="position:absolute;top:8801;left:793"><nobr>0.00</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:10px;font-family:Times"> <div style="position:absolute;top:8797;left:824"><nobr>a</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:8833;left:85"><nobr>PZQ</nobr></div> <div style="position:absolute;top:8833;left:191"><nobr>0.2</nobr></div> <div style="position:absolute;top:8833;left:281"><nobr>0.00 ± 0.00</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:10px;font-family:Times"> <div style="position:absolute;top:8828;left:363"><nobr>a</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:8822;left:408"><nobr>33.33 ±</nobr></div> <div style="position:absolute;top:8843;left:400"><nobr>11.54</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:10px;font-family:Times"> <div style="position:absolute;top:8839;left:441"><nobr>cd***</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:8822;left:513"><nobr>100 ±</nobr></div> <div style="position:absolute;top:8843;left:507"><nobr>0.00</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:10px;font-family:Times"> <div style="position:absolute;top:8839;left:539"><nobr>f***</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:8822;left:607"><nobr>100 ±</nobr></div> <div style="position:absolute;top:8843;left:601"><nobr>0.00</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:10px;font-family:Times"> <div style="position:absolute;top:8839;left:632"><nobr>e***</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:8822;left:701"><nobr>100 ±</nobr></div> <div style="position:absolute;top:8843;left:695"><nobr>0.00</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:10px;font-family:Times"> <div style="position:absolute;top:8839;left:726"><nobr>e***</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:8822;left:791"><nobr>100 ±</nobr></div> <div style="position:absolute;top:8843;left:784"><nobr>0.00</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:10px;font-family:Times"> <div style="position:absolute;top:8839;left:815"><nobr>e***</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:8970;left:85"><nobr>HSE</nobr></div> <div style="position:absolute;top:8875;left:194"><nobr>20</nobr></div> <div style="position:absolute;top:8875;left:281"><nobr>0.00 ± 0.00</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:10px;font-family:Times"> <div style="position:absolute;top:8871;left:363"><nobr>a</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:8875;left:392"><nobr>0.00 ± 0.00</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:10px;font-family:Times"> <div style="position:absolute;top:8871;left:473"><nobr>a</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:8864;left:511"><nobr>0.00 ±</nobr></div> <div style="position:absolute;top:8885;left:515"><nobr>0.00</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:10px;font-family:Times"> <div style="position:absolute;top:8881;left:547"><nobr>a</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:8864;left:605"><nobr>0.00 ±</nobr></div> <div style="position:absolute;top:8885;left:610"><nobr>0.00</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:10px;font-family:Times"> <div style="position:absolute;top:8881;left:641"><nobr>a</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:8864;left:699"><nobr>0.00 ±</nobr></div> <div style="position:absolute;top:8885;left:704"><nobr>0.00</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:10px;font-family:Times"> <div style="position:absolute;top:8881;left:735"><nobr>a</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:8864;left:788"><nobr>0.00 ±</nobr></div> <div style="position:absolute;top:8885;left:793"><nobr>0.00</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:10px;font-family:Times"> <div style="position:absolute;top:8881;left:824"><nobr>a</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:8917;left:194"><nobr>40</nobr></div> <div style="position:absolute;top:8917;left:281"><nobr>0.00 ± 0.00</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:10px;font-family:Times"> <div style="position:absolute;top:8913;left:363"><nobr>a</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:8907;left:408"><nobr>12.50 ±</nobr></div> <div style="position:absolute;top:8927;left:414"><nobr>0.00</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:10px;font-family:Times"> <div style="position:absolute;top:8923;left:445"><nobr>ab</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:8907;left:518"><nobr>25 ±</nobr></div> <div style="position:absolute;top:8927;left:506"><nobr>0.00</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:10px;font-family:Times"> <div style="position:absolute;top:8923;left:538"><nobr>b***</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:8907;left:601"><nobr>33.33 ±</nobr></div> <div style="position:absolute;top:8927;left:600"><nobr>7.21</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:10px;font-family:Times"> <div style="position:absolute;top:8923;left:632"><nobr>b***</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:8907;left:695"><nobr>37.50 ±</nobr></div> <div style="position:absolute;top:8927;left:694"><nobr>0.00</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:10px;font-family:Times"> <div style="position:absolute;top:8923;left:726"><nobr>b***</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:8907;left:784"><nobr>37.50 ±</nobr></div> <div style="position:absolute;top:8927;left:784"><nobr>0.00</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:10px;font-family:Times"> <div style="position:absolute;top:8923;left:815"><nobr>b***</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:8959;left:194"><nobr>60</nobr></div> <div style="position:absolute;top:8949;left:297"><nobr>17.50 ±</nobr></div> <div style="position:absolute;top:8969;left:297"><nobr>4.33</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:10px;font-family:Times"> <div style="position:absolute;top:8965;left:328"><nobr>b***</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:8949;left:408"><nobr>28.33 ±</nobr></div> <div style="position:absolute;top:8969;left:405"><nobr>2.88</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:10px;font-family:Times"> <div style="position:absolute;top:8965;left:436"><nobr>bc***</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:8949;left:518"><nobr>35 ±</nobr></div> <div style="position:absolute;top:8969;left:506"><nobr>4.33</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:10px;font-family:Times"> <div style="position:absolute;top:8965;left:538"><nobr>c***</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:8949;left:612"><nobr>35 ±</nobr></div> <div style="position:absolute;top:8969;left:600"><nobr>4.33</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:10px;font-family:Times"> <div style="position:absolute;top:8965;left:632"><nobr>b***</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:8949;left:695"><nobr>58.33 ±</nobr></div> <div style="position:absolute;top:8969;left:695"><nobr>7.21</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:10px;font-family:Times"> <div style="position:absolute;top:8965;left:726"><nobr>c***</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:8949;left:795"><nobr>70 ±</nobr></div> <div style="position:absolute;top:8969;left:784"><nobr>8.66</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:10px;font-family:Times"> <div style="position:absolute;top:8965;left:815"><nobr>c***</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:9001;left:194"><nobr>80</nobr></div> <div style="position:absolute;top:8991;left:297"><nobr>27.38 ±</nobr></div> <div style="position:absolute;top:9012;left:297"><nobr>2.06</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:10px;font-family:Times"> <div style="position:absolute;top:9007;left:329"><nobr>c***</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:8991;left:408"><nobr>34.52 ±</nobr></div> <div style="position:absolute;top:9012;left:405"><nobr>5.15</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:10px;font-family:Times"> <div style="position:absolute;top:9007;left:436"><nobr>cd***</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:8991;left:506"><nobr>41.06 ±</nobr></div> <div style="position:absolute;top:9012;left:504"><nobr>3.08</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:10px;font-family:Times"> <div style="position:absolute;top:9007;left:535"><nobr>cd***</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:8991;left:601"><nobr>47.61 ±</nobr></div> <div style="position:absolute;top:9012;left:601"><nobr>4.12</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:10px;font-family:Times"> <div style="position:absolute;top:9007;left:632"><nobr>c***</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:8991;left:695"><nobr>60.71 ±</nobr></div> <div style="position:absolute;top:9012;left:695"><nobr>3.09</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:10px;font-family:Times"> <div style="position:absolute;top:9007;left:726"><nobr>c***</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:8991;left:784"><nobr>73.80 ±</nobr></div> <div style="position:absolute;top:9012;left:784"><nobr>2.06</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:10px;font-family:Times"> <div style="position:absolute;top:9007;left:815"><nobr>c***</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:9043;left:189"><nobr>100</nobr></div> <div style="position:absolute;top:9033;left:297"><nobr>34.52 ±</nobr></div> <div style="position:absolute;top:9054;left:294"><nobr>5.15</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:10px;font-family:Times"> <div style="position:absolute;top:9049;left:326"><nobr>cd***</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:9033;left:408"><nobr>34.52 ±</nobr></div> <div style="position:absolute;top:9054;left:405"><nobr>5.15</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:10px;font-family:Times"> <div style="position:absolute;top:9049;left:436"><nobr>cd***</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:9033;left:506"><nobr>47.61 ±</nobr></div> <div style="position:absolute;top:9054;left:504"><nobr>4.12</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:10px;font-family:Times"> <div style="position:absolute;top:9049;left:535"><nobr>de***</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:9033;left:601"><nobr>54.76 ±</nobr></div> <div style="position:absolute;top:9054;left:601"><nobr>4.12</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:10px;font-family:Times"> <div style="position:absolute;top:9049;left:632"><nobr>c***</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:9033;left:695"><nobr>73.80 ±</nobr></div> <div style="position:absolute;top:9054;left:694"><nobr>2.06</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:10px;font-family:Times"> <div style="position:absolute;top:9049;left:726"><nobr>d***</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:9033;left:784"><nobr>86.90 ±</nobr></div> <div style="position:absolute;top:9054;left:784"><nobr>1.03</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:10px;font-family:Times"> <div style="position:absolute;top:9049;left:815"><nobr>d***</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:9086;left:189"><nobr>200</nobr></div> <div style="position:absolute;top:9075;left:297"><nobr>41.06 ±</nobr></div> <div style="position:absolute;top:9096;left:297"><nobr>3.08</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:10px;font-family:Times"> <div style="position:absolute;top:9092;left:328"><nobr>d***</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:9075;left:408"><nobr>50.59 ±</nobr></div> <div style="position:absolute;top:9096;left:403"><nobr>11.33</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:10px;font-family:Times"> <div style="position:absolute;top:9092;left:443"><nobr>d***</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:9075;left:506"><nobr>54.76 ±</nobr></div> <div style="position:absolute;top:9096;left:506"><nobr>4.12</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:10px;font-family:Times"> <div style="position:absolute;top:9092;left:538"><nobr>e***</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:9075;left:601"><nobr>68.44 ±</nobr></div> <div style="position:absolute;top:9096;left:600"><nobr>5.14</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:10px;font-family:Times"> <div style="position:absolute;top:9092;left:632"><nobr>d***</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:9075;left:695"><nobr>82.14 ±</nobr></div> <div style="position:absolute;top:9096;left:694"><nobr>6.18</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:10px;font-family:Times"> <div style="position:absolute;top:9092;left:726"><nobr>d***</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:9075;left:791"><nobr>100 ±</nobr></div> <div style="position:absolute;top:9096;left:784"><nobr>0.00</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:10px;font-family:Times"> <div style="position:absolute;top:9092;left:815"><nobr>e***</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:9117;left:178"><nobr>F-ratio</nobr></div> <div style="position:absolute;top:9117;left:300"><nobr>122.95</nobr></div> <div style="position:absolute;top:9117;left:415"><nobr>24.57</nobr></div> <div style="position:absolute;top:9117;left:509"><nobr>401.18</nobr></div> <div style="position:absolute;top:9117;left:603"><nobr>206.37</nobr></div> <div style="position:absolute;top:9117;left:697"><nobr>310.07</nobr></div> <div style="position:absolute;top:9117;left:787"><nobr>507.98</nobr></div> </span></font> <font size=3 color="#0d0d0d" face="Times"><span style="font-size:16px;font-family:Times;color:#0d0d0d"> <div style="position:absolute;top:9139;left:176"><nobr><i>p</i>-value</nobr></div> <div style="position:absolute;top:9139;left:309"><nobr>0.00</nobr></div> <div style="position:absolute;top:9139;left:419"><nobr>0.00</nobr></div> <div style="position:absolute;top:9139;left:518"><nobr>0.00</nobr></div> <div style="position:absolute;top:9139;left:612"><nobr>0.00</nobr></div> <div style="position:absolute;top:9139;left:706"><nobr>0.00</nobr></div> <div style="position:absolute;top:9139;left:796"><nobr>0.00</nobr></div> <div style="position:absolute;top:9171;left:179"><nobr>Linear</nobr></div> <div style="position:absolute;top:9191;left:166"><nobr>regression</nobr></div> <div style="position:absolute;top:9212;left:172"><nobr>equation</nobr></div> <div style="position:absolute;top:9171;left:278"><nobr>y = 0.239x +</nobr></div> <div style="position:absolute;top:9191;left:304"><nobr>0.132</nobr></div> <div style="position:absolute;top:9212;left:291"><nobr>R² = 0.76</nobr></div> <div style="position:absolute;top:9171;left:396"><nobr>y = 0.253x</nobr></div> <div style="position:absolute;top:9191;left:408"><nobr>+ 5.597</nobr></div> <div style="position:absolute;top:9212;left:401"><nobr>R² = 0.81</nobr></div> <div style="position:absolute;top:9171;left:495"><nobr>y = 0.253x</nobr></div> <div style="position:absolute;top:9191;left:506"><nobr>+ 12.81</nobr></div> <div style="position:absolute;top:9212;left:500"><nobr>R² = 0.68</nobr></div> <div style="position:absolute;top:9160;left:616"><nobr>y =</nobr></div> <div style="position:absolute;top:9181;left:596"><nobr>0.318x +</nobr></div> <div style="position:absolute;top:9202;left:608"><nobr>13.30</nobr></div> <div style="position:absolute;top:9222;left:594"><nobr>R² = 0.74</nobr></div> <div style="position:absolute;top:9171;left:683"><nobr>y = 0.375x</nobr></div> <div style="position:absolute;top:9191;left:694"><nobr>+ 20.83</nobr></div> <div style="position:absolute;top:9212;left:688"><nobr>R² = 0.64</nobr></div> <div style="position:absolute;top:9160;left:800"><nobr>y =</nobr></div> <div style="position:absolute;top:9181;left:779"><nobr>0.472x +</nobr></div> <div style="position:absolute;top:9202;left:791"><nobr>21.98</nobr></div> <div style="position:absolute;top:9222;left:777"><nobr>R² = 0.67</nobr></div> <div style="position:absolute;top:9244;left:185"><nobr>LC<font style="font-size:10px">50</font></nobr></div> <div style="position:absolute;top:9244;left:300"><nobr>208.65</nobr></div> <div style="position:absolute;top:9244;left:410"><nobr>175.50</nobr></div> <div style="position:absolute;top:9244;left:509"><nobr>146.99</nobr></div> <div style="position:absolute;top:9244;left:603"><nobr>115.40</nobr></div> <div style="position:absolute;top:9244;left:702"><nobr>77.78</nobr></div> <div style="position:absolute;top:9244;left:791"><nobr>59.36</nobr></div> <div style="position:absolute;top:9265;left:185"><nobr>LC<font style="font-size:10px">90</font></nobr></div> <div style="position:absolute;top:9265;left:300"><nobr>376.01</nobr></div> <div style="position:absolute;top:9265;left:410"><nobr>333.60</nobr></div> <div style="position:absolute;top:9265;left:509"><nobr>305.09</nobr></div> <div style="position:absolute;top:9265;left:603"><nobr>241.19</nobr></div> <div style="position:absolute;top:9265;left:697"><nobr>184.45</nobr></div> <div style="position:absolute;top:9265;left:787"><nobr>144.11</nobr></div> </span></font> <font size=3 color="#0d0d0d" face="Times"><span style="font-size:12px;font-family:Times;color:#0d0d0d"> <div style="position:absolute;top:9307;left:135"><nobr>PZQ: praziquantel, HSE: <i>Hyrtios </i>sponge extract, LC<font style="font-size:7px">50</font>: Lethal concentration for 50% mortality of the</nobr></div> <div style="position:absolute;top:9324;left:135"><nobr>exposed <i>S. mansoni </i>adult worms, LC<font style="font-size:7px">90</font>: Lethal concentration for 90% mortality of the exposed <i>S. mansoni</i></nobr></div> <div style="position:absolute;top:9341;left:135"><nobr>adult worms, y: % Mortality value, and x: Concentration of the HSE. Each value is the mean of 3 replicates</nobr></div> <div style="position:absolute;top:9358;left:135"><nobr>± SD. Means in the same column with different superscript letters <font style="font-size:7px">(a, b, c, d, e, and f) </font>are significantly different. <font style="font-size:7px">*</font></nobr></div> <div style="position:absolute;top:9376;left:135"><nobr>Significant at <i>P</i>-value≤ 0.05, <font style="font-size:7px">** </font>Significant at <i>P</i>-value≤ 0.01, and <font style="font-size:7px">*** </font>Significant at <i>P</i>-value≤ 0.001 (one-way</nobr></div> <div style="position:absolute;top:9393;left:135"><nobr>ANOVA).</nobr></div> </span></font> <font size=3 color="#0d0d0d" face="Times"><span style="font-size:16px;font-family:Times;color:#0d0d0d"> <div style="position:absolute;top:9426;left:189"><nobr>Regarding female adult <i>S. mansoni </i>obtained from the control group, SEM</nobr></div> <div style="position:absolute;top:9447;left:135"><nobr>examination indicated that its body was cylindrical and elongated. The adult female's</nobr></div> <div style="position:absolute;top:9467;left:135"><nobr>body was partitioned into three regions, as in males. The anterior part consisted of the</nobr></div> <div style="position:absolute;top:9488;left:135"><nobr>oral sucker, ventral sucker, and the connecting tegumental area (Fig. 3a). Oral sucker was</nobr></div> <div style="position:absolute;top:9509;left:135"><nobr>triangular with three regions; a thin rim, a large inner zone, and mouth. It had several</nobr></div> <div style="position:absolute;top:9529;left:135"><nobr>spines oriented toward the mouth cavity. Hemispherical papillae were found around the</nobr></div> <div style="position:absolute;top:9550;left:135"><nobr>oral sucker (Fig. 3b). Ventral sucker was circular, consisted of a rim, inner zone, and </nobr></div> </span></font> <div style="position:absolute;top:9679;left:0"><hr><table border=0 width=100%><tr><td bgcolor=eeeeee align=right><font face=arial,sans-serif><a name=9><b>Page 9</b></a></font></td></tr></table></div><font size=3 color="#0d0d0d" face="Times"><span style="font-size:11px;font-family:Times;color:#0d0d0d"> <div style="position:absolute;top:9735;left:135"><nobr><b>Antischistosomal Effect of <font color="#000000">the Marine </font><i><font color="#000000">Hyrtios </font></i><font color="#000000">Sponge Extract </font>against <i>Schistosoma mansoni </i>Worms</b></nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:14px;font-family:Times"> <div style="position:absolute;top:9736;left:791"><nobr>807</nobr></div> </span></font> <font size=3 color="#0d0d0d" face="Times"><span style="font-size:16px;font-family:Times;color:#0d0d0d"> <div style="position:absolute;top:9791;left:135"><nobr>central zone, and had several spines with consistent shape and size (Fig. 3c).</nobr></div> <div style="position:absolute;top:9812;left:135"><nobr>Hemispherical and macule papillae spread over the dorsal surface of the tegument (Fig.</nobr></div> <div style="position:absolute;top:9832;left:135"><nobr>3d). The posterior part of the body had several spines and hemispherical papilla (Fig. 3e).</nobr></div> <div style="position:absolute;top:9853;left:171"><nobr>After 48h of <i>in vitro </i>exposure of adult worms to the HSE at 100 and 200μg/ ml</nobr></div> <div style="position:absolute;top:9874;left:135"><nobr>concentrations, distinct morphological changes appeared in both sexes of <i>S. mansoni</i></nobr></div> <div style="position:absolute;top:9894;left:135"><nobr>adult worms compared to the control group. The morphological alterations were clear in</nobr></div> <div style="position:absolute;top:9915;left:135"><nobr>the male adult <i>S. mansoni </i>worms after exposure to 100µg/ ml of HSE since they appeared</nobr></div> <div style="position:absolute;top:9936;left:135"><nobr>to have abnormalities, including intensive contraction of the swelling body and the</nobr></div> <div style="position:absolute;top:9957;left:135"><nobr>gynechophoric canal <font color="#ff0000">(Fig. 4a). </font>Severe shrinkage and invaginations in the worm tegument</nobr></div> <div style="position:absolute;top:9977;left:135"><nobr>were quite evident, particularly in the first part of the dorsal side of the body (Fig. 4b). A</nobr></div> <div style="position:absolute;top:9998;left:135"><nobr>self-coiling phenomenon and deformation of the gynechophoric canal could also be</nobr></div> <div style="position:absolute;top:10019;left:135"><nobr>observed after exposure to HSE at 100µg/ ml (Fig. 4c). Swelling of suckers was observed</nobr></div> <div style="position:absolute;top:10039;left:135"><nobr>to the extent that makes the mouth opening scarcely recognizable (Fig. 4d). The</nobr></div> <div style="position:absolute;top:10060;left:135"><nobr>tegumental tubercles became swollen with complete loss of spines and presence of blebs</nobr></div> <div style="position:absolute;top:10081;left:135"><nobr>between them (Fig. 4e). Moreover, collapsed tubercles appeared on male tegument, and</nobr></div> <div style="position:absolute;top:10101;left:135"><nobr>the sub- tegumental tissues were exposed as a result of extensive erosion in the</nobr></div> <div style="position:absolute;top:10122;left:135"><nobr>integument (Fig. 4f).</nobr></div> <div style="position:absolute;top:10143;left:171"><nobr>As a result of exposure of the male <i>S. mansoni </i>adult worms to HSE at a</nobr></div> <div style="position:absolute;top:10164;left:135"><nobr>concentration of 200µg/ ml, severe body alterations occurred. The body became flattened</nobr></div> <div style="position:absolute;top:10184;left:135"><nobr>and smooth with a loss of tubercles and spines (Fig. 5a). The marked distortion of the</nobr></div> <div style="position:absolute;top:10205;left:135"><nobr>suckers, like the flattening of the ventral sucker, was also recorded (Fig. 5b). The</nobr></div> <div style="position:absolute;top:10226;left:135"><nobr>gynechophoric canal was distorted, seemed open, and devoided of its ridges (Fig. 5c).</nobr></div> <div style="position:absolute;top:10246;left:135"><nobr>Tegumental sloughing appeared on lateral side of the gynechophoric canal (Fig. 5d).</nobr></div> <div style="position:absolute;top:10267;left:135"><nobr>Most parts of the tegument were collapsed and/or ruptured tubercles were arranged in</nobr></div> <div style="position:absolute;top:10288;left:135"><nobr>irregular rows with no spines (Fig. 5e, f).</nobr></div> <div style="position:absolute;top:10308;left:171"><nobr><i>In vitro </i>treatment with the HSE at 100µg/ ml resulted in observable morphological</nobr></div> <div style="position:absolute;top:10329;left:135"><nobr>changes on the female <i>S. mansoni </i>adult worms, like severe shrinkage of ventral sucker,</nobr></div> <div style="position:absolute;top:10350;left:135"><nobr>tegumental pits with corrugation, tegumental folds including invaginations, and</nobr></div> <div style="position:absolute;top:10371;left:135"><nobr>tegumental sloughing (Fig. 6). At the same time, HSE at 200µg/ ml caused destructive</nobr></div> <div style="position:absolute;top:10391;left:135"><nobr>impacts on the female <i>S. mansoni </i>adult worms, including swelling of the whole body,</nobr></div> <div style="position:absolute;top:10412;left:135"><nobr>abnormal oral sucker surrounding with blebs, marked shrinkage of ventral sucker with</nobr></div> <div style="position:absolute;top:10433;left:135"><nobr>pulling it inside. Furthermore, severe alterations in the tegumental architecture, like</nobr></div> <div style="position:absolute;top:10453;left:135"><nobr>wrinkling, invaginations, folding, erosions, and tegumental blebs with the absence of</nobr></div> <div style="position:absolute;top:10474;left:135"><nobr>spines, were recorded (Fig. 7). </nobr></div> </span></font> <div style="position:absolute;top:10867;left:0"><hr><table border=0 width=100%><tr><td bgcolor=eeeeee align=right><font face=arial,sans-serif><a name=10><b>Page 10</b></a></font></td></tr></table></div><font size=3 color="#0d0d0d" face="Times"><span style="font-size:12px;font-family:Times;color:#0d0d0d"> <div style="position:absolute;top:10924;left:387"><nobr><b>El-Abbassy <i><font color="#000000">et al.</font></i><font color="#000000">, 2024</font></b></nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:12px;font-family:Times"> <div style="position:absolute;top:10924;left:135"><nobr>808</nobr></div> </span></font> <font size=3 color="#0d0d0d" face="Times"><span style="font-size:14px;font-family:Times;color:#0d0d0d"> <div style="position:absolute;top:11646;left:135"><nobr><b>Fig. 2. </b>Scanning electron micrographs (SEM) of the male adult worms of <i>Schistosoma mansoni</i></nobr></div> <div style="position:absolute;top:11665;left:135"><nobr>from the control group showing: <b>(a) </b>Copulation with female in the gynechophoric canal of male,</nobr></div> <div style="position:absolute;top:11684;left:135"><nobr><b>(b) </b>The anterior region of the body with an oval-shaped oral sucker having three notable regions</nobr></div> <div style="position:absolute;top:11703;left:135"><nobr>(rim, inner zone, and the mouth), a round-shaped <font color="#000000">ventral sucker distinguished into three regions</font></nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:14px;font-family:Times"> <div style="position:absolute;top:11722;left:135"><nobr>(rim, inner zone, and central zone), and hemispherical papillae on the tegument <font color="#0d0d0d">between the two</font></nobr></div> </span></font> <font size=3 color="#0d0d0d" face="Times"><span style="font-size:14px;font-family:Times;color:#0d0d0d"> <div style="position:absolute;top:11741;left:135"><nobr>suckers, as well as the beginning of the middle region of the body with gynechophoric canal and</nobr></div> <div style="position:absolute;top:11760;left:135"><nobr>tubercles, <b>(c) </b>The outer margin of the gynechophoric canal with tiny spines, <b>(d) </b>Inner surface of</nobr></div> <div style="position:absolute;top:11779;left:135"><nobr>the gynechophoric canal with folds and several spines, <b>(e) </b>Sponge-like tegument in the middle</nobr></div> <div style="position:absolute;top:11798;left:135"><nobr>part of the body of adult male covered with tubercles armed with apically directed spines and</nobr></div> <div style="position:absolute;top:11817;left:135"><nobr>appearance of macule papillae on the wrinkled tegument around them, and <b>(f) </b>Tegumental surface</nobr></div> <div style="position:absolute;top:11836;left:135"><nobr>of the body posterior region with spiny tubercles and macule papillae between them. (M) Male,</nobr></div> <div style="position:absolute;top:11854;left:135"><nobr>(F) Female, (GC) Gynechophoric canal, (OS) Oral sucker, (VS) Ventral sucker, (Mo) Mouth,</nobr></div> <div style="position:absolute;top:11874;left:135"><nobr>(Rm) Rim, (IZ) Inner zone, (CZ) Central zone, (HP) Hemispherical papillae, (Tu) Tubercles, (Sp)</nobr></div> <div style="position:absolute;top:11892;left:135"><nobr>Spines, (F) Folds, and (MP) Macule papillae </nobr></div> </span></font> <div style="position:absolute;top:12055;left:0"><hr><table border=0 width=100%><tr><td bgcolor=eeeeee align=right><font face=arial,sans-serif><a name=11><b>Page 11</b></a></font></td></tr></table></div><font size=3 color="#0d0d0d" face="Times"><span style="font-size:11px;font-family:Times;color:#0d0d0d"> <div style="position:absolute;top:12111;left:135"><nobr><b>Antischistosomal Effect of <font color="#000000">the Marine </font><i><font color="#000000">Hyrtios </font></i><font color="#000000">Sponge Extract </font>against <i>Schistosoma mansoni </i>Worms</b></nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:14px;font-family:Times"> <div style="position:absolute;top:12112;left:791"><nobr>809</nobr></div> </span></font> <font size=3 color="#0d0d0d" face="Times"><span style="font-size:16px;font-family:Times;color:#0d0d0d"> <div style="position:absolute;top:12868;left:135"><nobr><b>Fig. 3. </b>Scanning electron micrographs (SEM) of the female adult worms of <i>Schistosoma</i></nobr></div> <div style="position:absolute;top:12888;left:135"><nobr><i>mansoni </i>from the control group showing: <b>(a) </b>Cylindrical and elongated body of female</nobr></div> <div style="position:absolute;top:12909;left:135"><nobr>worm with oral and ventral suckers, <b>(b) </b>Magnification of triangular-shaped oral sucker</nobr></div> <div style="position:absolute;top:12930;left:135"><nobr>with three regions: thin rim, inner zone, and mouth and presence of numerous spines</nobr></div> <div style="position:absolute;top:12950;left:135"><nobr>oriented towards the mouth cavity and hemispherical papillae surrounding the oral</nobr></div> <div style="position:absolute;top:12971;left:135"><nobr>sucker, <b>(c) </b>Magnification of circular-shaped ventral sucker with three regions: rim, inner</nobr></div> <div style="position:absolute;top:12992;left:135"><nobr>zone, and central zone and presence of several spines with consistent shape and size, <b>(d)</b></nobr></div> <div style="position:absolute;top:13013;left:135"><nobr>An intact tegument of the dorsal surface with hemispherical and macule papillae, and <b>(e)</b></nobr></div> <div style="position:absolute;top:13033;left:135"><nobr>Tegumental surface of the posterior part of the body with numerous spines and</nobr></div> <div style="position:absolute;top:13054;left:135"><nobr>hemispherical papilla. (OS) Oral sucker, (VS) Ventral sucker, (Sp) Spines, (Mo) Mouth,</nobr></div> <div style="position:absolute;top:13075;left:135"><nobr>(Rm) Rim, (IZ) Inner zone, (HP) Hemispherical papillae, (CZ) Central zone, and (MP)</nobr></div> <div style="position:absolute;top:13095;left:135"><nobr>Macule papillae </nobr></div> </span></font> <div style="position:absolute;top:13243;left:0"><hr><table border=0 width=100%><tr><td bgcolor=eeeeee align=right><font face=arial,sans-serif><a name=12><b>Page 12</b></a></font></td></tr></table></div><font size=3 color="#0d0d0d" face="Times"><span style="font-size:12px;font-family:Times;color:#0d0d0d"> <div style="position:absolute;top:13300;left:387"><nobr><b>El-Abbassy <i><font color="#000000">et al.</font></i><font color="#000000">, 2024</font></b></nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:12px;font-family:Times"> <div style="position:absolute;top:13300;left:135"><nobr>810</nobr></div> </span></font> <font size=3 color="#0d0d0d" face="Times"><span style="font-size:16px;font-family:Times;color:#0d0d0d"> <div style="position:absolute;top:14095;left:135"><nobr><b>Fig. 4. </b>Scanning electron micrographs (SEM) of the male adult worms of <i>Schistosoma</i></nobr></div> <div style="position:absolute;top:14116;left:135"><nobr><i>mansoni </i>after 48h of <i>in vitro </i>treatment with the methanolic crude <i>Hyrtios </i>sponge extract</nobr></div> <div style="position:absolute;top:14136;left:135"><nobr>(HSE) at 100µg/ ml showing: <b>(a- b) </b>Intensive contraction of the swelling body, including</nobr></div> <div style="position:absolute;top:14157;left:135"><nobr>the gynechophoric canal and severe tegumental shrinkage with an obvious two folds</nobr></div> <div style="position:absolute;top:14178;left:135"><nobr>including an invagination on the anterior part of the dorsal side, <b>(c) </b>Self-coiling</nobr></div> <div style="position:absolute;top:14198;left:135"><nobr>phenomenon and deformation of gynechophoric canal, <b>(d) </b>Severe swelling of oral and</nobr></div> <div style="position:absolute;top:14219;left:135"><nobr>ventral suckers, <b>(e) </b>Completely naked swollen tubercles and <font color="#000000">presence of </font>blebs between</nobr></div> <div style="position:absolute;top:14240;left:135"><nobr>them, and <b>(f) </b>Collapsed tubercles and tegumental erosion. (White wide arrow)</nobr></div> <div style="position:absolute;top:14260;left:135"><nobr>Invagination, (F) Folds, (GC) Gynechophoric canal, (OS) Oral sucker, (VS) Ventral</nobr></div> <div style="position:absolute;top:14281;left:135"><nobr>sucker, (Tu) Tubercles, (B) Blebs, and (E) Erosion</nobr></div> </span></font> <div style="position:absolute;top:14431;left:0"><hr><table border=0 width=100%><tr><td bgcolor=eeeeee align=right><font face=arial,sans-serif><a name=13><b>Page 13</b></a></font></td></tr></table></div><font size=3 color="#0d0d0d" face="Times"><span style="font-size:11px;font-family:Times;color:#0d0d0d"> <div style="position:absolute;top:14487;left:135"><nobr><b>Antischistosomal Effect of <font color="#000000">the Marine </font><i><font color="#000000">Hyrtios </font></i><font color="#000000">Sponge Extract </font>against <i>Schistosoma mansoni </i>Worms</b></nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:14px;font-family:Times"> <div style="position:absolute;top:14488;left:791"><nobr>811</nobr></div> </span></font> <font size=3 color="#0d0d0d" face="Times"><span style="font-size:16px;font-family:Times;color:#0d0d0d"> <div style="position:absolute;top:15318;left:135"><nobr><b>Fig. 5. </b>Scanning electron micrographs (SEM) of the male adult worms of <i>Schistosoma</i></nobr></div> <div style="position:absolute;top:15338;left:135"><nobr><i>mansoni </i>after 48h of <i>in vitro </i>treatment with the methanolic crude <i>Hyrtios </i>sponge extract</nobr></div> <div style="position:absolute;top:15359;left:135"><nobr>(HSE) at 200 µg/ml showing: <b>(a) </b>Flattened body with loss of tubercles and spines, <b>(b)</b></nobr></div> <div style="position:absolute;top:15380;left:135"><nobr>Marked distortion of the suckers; oral and ventral suckers, <b>(c) </b>Male worm body with oral</nobr></div> <div style="position:absolute;top:15400;left:135"><nobr>and ventral suckers and deformation of gynechophoric canal, <b>(d) </b>Lateral side of the</nobr></div> <div style="position:absolute;top:15421;left:135"><nobr>gynechophoric canal with tegumental sloughing, and <b>(e- f) </b>Presence of collapsed and</nobr></div> <div style="position:absolute;top:15442;left:135"><nobr>ruptured tubercles arranged in irregular rows and have no spines. (FB) Flat body, (OS)</nobr></div> <div style="position:absolute;top:15462;left:135"><nobr>Oral sucker, (VS) Ventral sucker, (GC) Gynechophoric canal, (S) Sloughing, and (Tu)</nobr></div> <div style="position:absolute;top:15483;left:135"><nobr>Tubercles. </nobr></div> </span></font> <div style="position:absolute;top:15619;left:0"><hr><table border=0 width=100%><tr><td bgcolor=eeeeee align=right><font face=arial,sans-serif><a name=14><b>Page 14</b></a></font></td></tr></table></div><font size=3 color="#0d0d0d" face="Times"><span style="font-size:12px;font-family:Times;color:#0d0d0d"> <div style="position:absolute;top:15676;left:387"><nobr><b>El-Abbassy <i><font color="#000000">et al.</font></i><font color="#000000">, 2024</font></b></nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:12px;font-family:Times"> <div style="position:absolute;top:15676;left:135"><nobr>812</nobr></div> </span></font> <font size=3 color="#0d0d0d" face="Times"><span style="font-size:16px;font-family:Times;color:#0d0d0d"> <div style="position:absolute;top:16528;left:135"><nobr><b>Fig. 6. </b>Scanning electron micrographs (SEM) of the female adult worms of <i>Schistosoma</i></nobr></div> <div style="position:absolute;top:16549;left:135"><nobr><i>mansoni </i>after 48h of <i>in vitro </i>treatment with the methanolic crude <i>Hyrtios </i>sponge extract</nobr></div> <div style="position:absolute;top:16570;left:135"><nobr>(HSE) at 100 µg/ml showing: <b>(a- b) </b>Female body with oral sucker, <font color="#000000">shrinkage </font>of ventral</nobr></div> <div style="position:absolute;top:16590;left:135"><nobr>sucker, and tegumental pits with corrugation and invaginations, <b>(c- d) </b>Corrugation of the</nobr></div> <div style="position:absolute;top:16611;left:135"><nobr>tegument with tegumental folds and invaginations, <b>(e) </b>Oral sucker surrounding by</nobr></div> <div style="position:absolute;top:16632;left:135"><nobr>tegumental sloughing, and <b>(f) </b>Body with tegumental sloughing. (OS) Oral sucker, (VS)</nobr></div> <div style="position:absolute;top:16652;left:135"><nobr>Ventral sucker, (Asterisk) Corrugation of the tegument, (White wide arrow) Invagination,</nobr></div> <div style="position:absolute;top:16673;left:135"><nobr>(P) Pits, (F) Folds, and (S) Sloughing </nobr></div> </span></font> <div style="position:absolute;top:16807;left:0"><hr><table border=0 width=100%><tr><td bgcolor=eeeeee align=right><font face=arial,sans-serif><a name=15><b>Page 15</b></a></font></td></tr></table></div><font size=3 color="#0d0d0d" face="Times"><span style="font-size:11px;font-family:Times;color:#0d0d0d"> <div style="position:absolute;top:16863;left:135"><nobr><b>Antischistosomal Effect of <font color="#000000">the Marine </font><i><font color="#000000">Hyrtios </font></i><font color="#000000">Sponge Extract </font>against <i>Schistosoma mansoni </i>Worms</b></nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:14px;font-family:Times"> <div style="position:absolute;top:16864;left:791"><nobr>813</nobr></div> </span></font> <font size=3 color="#0d0d0d" face="Times"><span style="font-size:16px;font-family:Times;color:#0d0d0d"> <div style="position:absolute;top:17690;left:135"><nobr><b>Fig. 7. </b>Scanning electron micrographs (SEM) of the female adult worms of <i>Schistosoma</i></nobr></div> <div style="position:absolute;top:17710;left:135"><nobr><i>mansoni </i>after 48h of <i>in vitro </i>treatment with the methanolic crude <i>Hyrtios </i>sponge extract</nobr></div> <div style="position:absolute;top:17731;left:135"><nobr>(HSE) at 200µg/ ml showing: <b>(a- b) </b>Swelling of the whole body with wrinkles and</nobr></div> <div style="position:absolute;top:17752;left:135"><nobr>longitudinal invagination, <b>(c) </b>Swelling of the whole body along with abnormal oral</nobr></div> <div style="position:absolute;top:17772;left:135"><nobr>sucker, <font color="#000000">shrinkage </font>of ventral sucker, tegumental folds, and surface invaginations, <b>(d)</b></nobr></div> <div style="position:absolute;top:17793;left:135"><nobr>Blebs around the oral sucker, <b>(e) </b>Marked shrinkage in ventral sucker with pulling it inside</nobr></div> <div style="position:absolute;top:17814;left:135"><nobr>and tegumental erosion, and <b>(f) </b>Smooth tegument without spines and presence of blebs.</nobr></div> <div style="position:absolute;top:17835;left:135"><nobr>(White wide arrow) Invagination, (W) Wrinkles, (OS) Oral sucker, (VS) Ventral sucker,</nobr></div> <div style="position:absolute;top:17855;left:135"><nobr>(F) Folds, (B) Blebs, and (E) Erosion </nobr></div> </span></font> <div style="position:absolute;top:17995;left:0"><hr><table border=0 width=100%><tr><td bgcolor=eeeeee align=right><font face=arial,sans-serif><a name=16><b>Page 16</b></a></font></td></tr></table></div><font size=3 color="#0d0d0d" face="Times"><span style="font-size:12px;font-family:Times;color:#0d0d0d"> <div style="position:absolute;top:18052;left:387"><nobr><b>El-Abbassy <i><font color="#000000">et al.</font></i><font color="#000000">, 2024</font></b></nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:12px;font-family:Times"> <div style="position:absolute;top:18052;left:135"><nobr>814</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:18107;left:177"><nobr><b>DISCUSSION</b></nobr></div> </span></font> <font size=3 color="#0d0d0d" face="Times"><span style="font-size:16px;font-family:Times;color:#0d0d0d"> <div style="position:absolute;top:18148;left:171"><nobr>Schistosomiasis is one of the common diseases related to using water sources</nobr></div> <div style="position:absolute;top:18169;left:135"><nobr>contaminated by sewage and unsanitary practices. PZQ is the only drug that has been</nobr></div> <div style="position:absolute;top:18190;left:135"><nobr>used against schistosomiasis for decades, but regrettably, an emergence of PZQ-resistant</nobr></div> <div style="position:absolute;top:18210;left:135"><nobr>Schistosomes was recorded <b>(Melman <i>et al., </i>2009; Wang, 2012; Shaaban <i>et al.</i>, <font color="#000000">2019</font>).</b></nobr></div> <div style="position:absolute;top:18231;left:135"><nobr>Additionally, PZQ exhibits genotoxicity, mutagenic effects, and inadequate efficacy</nobr></div> <div style="position:absolute;top:18252;left:135"><nobr>against juvenile forms <b>(Vale <i>et al</i>., 2017)</b>. Therefore, efforts are focused on developing</nobr></div> <div style="position:absolute;top:18273;left:135"><nobr>different approaches to treat schistosomiasis alongside PZQ <b>(Lago <i>et al</i>., 2019)</b>. Natural</nobr></div> <div style="position:absolute;top:18293;left:135"><nobr>marine products have a great interest as possible sources of new drugs for schistosomiasis</nobr></div> <div style="position:absolute;top:18314;left:135"><nobr><b>(Mona <i>et al., </i>2012; Stein <i>et al., </i>2015; Abd-Allah <i>et al., </i>2022)</b>. Among marine animals,</nobr></div> <div style="position:absolute;top:18335;left:135"><nobr>sponges recorded a high rank in the priority of natural products research due to</nobr></div> <div style="position:absolute;top:18355;left:135"><nobr>identifying a variety of bioactive chemical components and secondary metabolites with</nobr></div> <div style="position:absolute;top:18376;left:135"><nobr>possible medicinal uses <b>(Perdicaris <i>et al</i>., 2013)</b>. <i>Hyrtios </i>marine sponge has different</nobr></div> <div style="position:absolute;top:18397;left:135"><nobr>chemicals with a broad spectrum of antiparasitic activities, such as antitrypanosomal</nobr></div> <div style="position:absolute;top:18417;left:135"><nobr><b>(Shady <i>et al.</i>, 2018) </b>and antiplasmodial activities <b>(Mahfur <i>et al</i>., 2022)</b>. However, this</nobr></div> <div style="position:absolute;top:18438;left:135"><nobr>study is considered the first one to assess the <i>in vitro </i>activity of the methanolic extract</nobr></div> <div style="position:absolute;top:18459;left:135"><nobr>prepared from <i>Hyrtios </i>sponge (HSE) of adult worms of <i>S</i>. <i>mansoni </i>by estimating the</nobr></div> <div style="position:absolute;top:18480;left:135"><nobr>mortality and observing the tegumental alterations.</nobr></div> <div style="position:absolute;top:18500;left:171"><nobr>The present study recorded different phytochemicals in HSE with a high level of</nobr></div> <div style="position:absolute;top:18521;left:135"><nobr>alkaloids content, followed by saponins, phenolics, and flavonoids, as reported in the</nobr></div> <div style="position:absolute;top:18542;left:135"><nobr>previous studies <b>(Salmoun <i>et al., </i>2002; Utkina, 2009; Youssef <i>et al., </i>2013; Wardany &</b></nobr></div> <div style="position:absolute;top:18562;left:135"><nobr><b>Nirmala, 2023)</b>. This finding is confirmed by the estimation of the antioxidant capacity</nobr></div> <div style="position:absolute;top:18583;left:135"><nobr>of this extract, which was 101.70mg AAE/ g of extract, as these phytochemicals are well-</nobr></div> <div style="position:absolute;top:18604;left:135"><nobr>known antioxidant secondary metabolites possessing free radical scavenging activity</nobr></div> <div style="position:absolute;top:18624;left:135"><nobr>through their redox potentials that enable them to function as reducing agents, through</nobr></div> <div style="position:absolute;top:18645;left:135"><nobr>donation of hydrogen and singlet oxygen quenching <b>(Francis <i>et al., </i>2002; Ozsoy <i>et al</i>.,</b></nobr></div> <div style="position:absolute;top:18666;left:135"><nobr><b>2009; Formagio <i>et al., </i>2014; Zou <i>et al., </i>2016)</b>.</nobr></div> <div style="position:absolute;top:18687;left:171"><nobr>The <i>in vitro </i>test is a helpful tool to investigate the antischistosomal effect of novel</nobr></div> <div style="position:absolute;top:18707;left:135"><nobr>drugs. It is considered an initial step to understand its mechanism of action against</nobr></div> <div style="position:absolute;top:18728;left:135"><nobr><i>Schistosoma </i><b>(Doenhoff <i>et al</i>., 2009)</b>. The current findings show that the mortality rates of</nobr></div> <div style="position:absolute;top:18749;left:135"><nobr>adult worms of <i>S. mansoni </i>increased with the duration of exposure to HSE and occurred</nobr></div> <div style="position:absolute;top:18769;left:135"><nobr>in a dose-dependent manner. The positive control group exposed to 0.2µg/ ml of PZQ</nobr></div> <div style="position:absolute;top:18790;left:135"><nobr>demonstrated a significant increase in worm mortality, scoring 100% death of adult</nobr></div> <div style="position:absolute;top:18811;left:135"><nobr>worms after 3h of incubation. The antischistosomal mechanism of PZQ is based on</nobr></div> <div style="position:absolute;top:18832;left:135"><nobr>embedding itself into the outer membrane of the worm tegument and reacting with it, thus</nobr></div> <div style="position:absolute;top:18852;left:135"><nobr>resulting in a lipid phase transition followed by a destabilizing effect on the membrane</nobr></div> <div style="position:absolute;top:18873;left:135"><nobr>and cutting it dramatically <b>(Abou El-Nour & Fadladdin, 2021)</b>.</nobr></div> <div style="position:absolute;top:18894;left:171"><nobr>At the same time, the mortality rates of adult worms of <i>S. mansoni </i>were more than</nobr></div> <div style="position:absolute;top:18914;left:135"><nobr>50% after 2 and 3h of exposure to HSE at 200µg/ ml, after 24h of exposure to HSE at 100</nobr></div> <div style="position:absolute;top:18935;left:135"><nobr>and 200µg/ ml, and after 36 and 48h of exposing to HSE at concentrations ranging from</nobr></div> <div style="position:absolute;top:18956;left:135"><nobr>60 to 200µg/ ml. Moreover, it achieved 100% mortality at 200µg/ ml after 48h. The</nobr></div> <div style="position:absolute;top:18976;left:135"><nobr>values of LC<font style="font-size:10px">50 </font>and LC<font style="font-size:10px">90 </font>of HSE against the <i>S. mansoni </i>adult worms were 115.40 and</nobr></div> <div style="position:absolute;top:18997;left:135"><nobr>241.19µg/ ml, respectively, after 24h of exposure time, and they were 59.36 and</nobr></div> <div style="position:absolute;top:19018;left:135"><nobr>144.11µg/ ml after 48h of exposure time, respectively. The antischistosomal efficiency of</nobr></div> <div style="position:absolute;top:19039;left:135"><nobr>HSE is considered better when compared with the findings observed in previous studies </nobr></div> </span></font> <div style="position:absolute;top:19183;left:0"><hr><table border=0 width=100%><tr><td bgcolor=eeeeee align=right><font face=arial,sans-serif><a name=17><b>Page 17</b></a></font></td></tr></table></div><font size=3 color="#0d0d0d" face="Times"><span style="font-size:11px;font-family:Times;color:#0d0d0d"> <div style="position:absolute;top:19239;left:135"><nobr><b>Antischistosomal Effect of <font color="#000000">the Marine </font><i><font color="#000000">Hyrtios </font></i><font color="#000000">Sponge Extract </font>against <i>Schistosoma mansoni </i>Worms</b></nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:14px;font-family:Times"> <div style="position:absolute;top:19240;left:791"><nobr>815</nobr></div> </span></font> <font size=3 color="#0d0d0d" face="Times"><span style="font-size:16px;font-family:Times;color:#0d0d0d"> <div style="position:absolute;top:19295;left:135"><nobr>on different types of extracts prepared from natural sources. <b>Reda <i>et al. </i>(2016) </b>recorded</nobr></div> <div style="position:absolute;top:19316;left:135"><nobr>that values of LC<font style="font-size:10px">50 </font>and LC<font style="font-size:10px">90 </font>of <i>Olea europaea </i>leaves extract were 121.4 and 436.17µg/</nobr></div> <div style="position:absolute;top:19336;left:135"><nobr>ml, respectively, after 24h of incubation of <i>S. mansoni </i>adult worms with this extract.</nobr></div> <div style="position:absolute;top:19357;left:135"><nobr><b>Tekwu <i>et al. </i>(2017) </b>reported that all adult worms exposed to 250µg/ ml of <i>Rauwolfia</i></nobr></div> <div style="position:absolute;top:19378;left:135"><nobr><i>vomitoria </i>root and stem bark died within 120h of incubation. <b>Nkondo <i>et al. </i>(2022) </b>found</nobr></div> <div style="position:absolute;top:19398;left:135"><nobr>that the value of LC<font style="font-size:10px">50 </font>of the hydroethanolic extract of <i>Pedilanthus tithymaloides </i>stem</nobr></div> <div style="position:absolute;top:19419;left:135"><nobr>bark was 731.17μg/ ml after 48h of <i>S. mansoni </i>adult worms exposure to this extract.</nobr></div> <div style="position:absolute;top:19440;left:171"><nobr>The development of new antischistosomal drugs requires sufficient knowledge of</nobr></div> <div style="position:absolute;top:19461;left:135"><nobr><i>Schistosoma </i>tegumental constituents since it functions as an intermediary between the</nobr></div> <div style="position:absolute;top:19481;left:135"><nobr>parasite body and the host's intravascular environment <b>(El-Shabasy <i>et al</i>., 2015; Kamel</b></nobr></div> <div style="position:absolute;top:19502;left:135"><nobr><b>& El-Shinnawy, 2015)</b>. The tegument also has a role in regulating the osmotic balance,</nobr></div> <div style="position:absolute;top:19523;left:135"><nobr>metabolite secretion, and nutrition intake <b>(Reda <i>et al</i>.<i>, </i>2016)</b>. Changes in the worm</nobr></div> <div style="position:absolute;top:19543;left:135"><nobr>tegument lead to impairment in the immunological efficiency of the worms, and thus, it</nobr></div> <div style="position:absolute;top:19564;left:135"><nobr>becomes easy for the host's immune system to attack <b>(Xiao <i>et al., </i>2000, 2002)</b>.</nobr></div> <div style="position:absolute;top:19585;left:135"><nobr>Furthermore, the tegumental alterations cause the worms to be unable to adhere to host</nobr></div> <div style="position:absolute;top:19605;left:135"><nobr>blood vessel walls, move by the bloodstream from mesenteric veins till they reach</nobr></div> <div style="position:absolute;top:19626;left:135"><nobr>intravenous hepatic capillaries, and become trapped in the liver, causing disintegration</nobr></div> <div style="position:absolute;top:19647;left:135"><nobr>and death of the worms <b>(Gnanasekar <i>et al., </i>2009; Riad <i>et al., </i>2009)</b>. Therefore, the</nobr></div> <div style="position:absolute;top:19668;left:135"><nobr>schistosomicidal activity of HSE in the present study was supported by the SEM study to</nobr></div> <div style="position:absolute;top:19688;left:135"><nobr>assess its effect on the <i>S. mansoni </i>tegument. </nobr></div> <div style="position:absolute;top:19709;left:171"><nobr>The present study showed that the structural characteristics of male and female <i>S</i>.</nobr></div> <div style="position:absolute;top:19730;left:135"><nobr><i>mansoni </i>in the control group remained unchanged and are consistent with those found in</nobr></div> <div style="position:absolute;top:19750;left:135"><nobr>previous studies <b>(Aboueldahab & Elhussieny, 2016; Ellakany <i>et al., </i>2019; Reda <i>et al.,</b></i></nobr></div> <div style="position:absolute;top:19771;left:135"><nobr><b>2019; El-Morsy <i>et al., </i>2021)</b>. Spines are widely dispersed on both oral and ventral</nobr></div> <div style="position:absolute;top:19792;left:135"><nobr>suckers, gynaecophoric canal, as well as along the tegumental surface of <i>S. mansoni </i>male</nobr></div> <div style="position:absolute;top:19812;left:135"><nobr>worms and some parts of the body of female worms.</nobr></div> <div style="position:absolute;top:19833;left:175"><nobr>Concerning the ultrastructural findings of the <i>S</i>. <i>mansoni </i>tegument in response to</nobr></div> <div style="position:absolute;top:19854;left:135"><nobr>HSE, the results showed that the male and female worms exhibited obvious modifications</nobr></div> <div style="position:absolute;top:19875;left:135"><nobr>in the structure of the suckers and tegument after 48h of incubation with a methanolic</nobr></div> <div style="position:absolute;top:19895;left:135"><nobr>extract of <i>Hyrtios </i>sponge in comparison to the negative control group. Among all tested</nobr></div> <div style="position:absolute;top:19916;left:135"><nobr>HSE concentrations, 100 and 200μg/ ml concentrations were selected for the SEM study</nobr></div> <div style="position:absolute;top:19937;left:135"><nobr>since they recorded the highest mortality rates of adult worms. The ultramorphological</nobr></div> <div style="position:absolute;top:19957;left:135"><nobr>alterations in mature worms were visible in these two concentrations. The morphological</nobr></div> <div style="position:absolute;top:19978;left:135"><nobr>changes in the male tegument included intensive contraction of the body with a self-</nobr></div> <div style="position:absolute;top:19999;left:135"><nobr>coiling phenomenon, swelling or flattening of the body, severe shrinkage and</nobr></div> <div style="position:absolute;top:20020;left:135"><nobr>invaginations in the worm body, deformation of the gynechophoric canal, marked</nobr></div> <div style="position:absolute;top:20040;left:135"><nobr>distortion of suckers, extensive tegumental erosion and sloughing, and clear appearance</nobr></div> <div style="position:absolute;top:20061;left:135"><nobr>of damaged smooth tubercles with blebs between them. At the same time, the treated</nobr></div> <div style="position:absolute;top:20082;left:135"><nobr>females of <i>S. mansoni </i>with HSE suffered from the swelling of the whole body, severe</nobr></div> <div style="position:absolute;top:20102;left:135"><nobr>shrinkage of the ventral sucker, tegumental pits, and other tegumental changes in the</nobr></div> <div style="position:absolute;top:20123;left:135"><nobr>form of folds with invaginations, corrugation, wrinkling, erosions, and sloughing.</nobr></div> <div style="position:absolute;top:20144;left:135"><nobr>Furthermore, the tegumental blebs with the absence of spines were recorded.</nobr></div> <div style="position:absolute;top:20164;left:171"><nobr>These alterations impede the worm's attachment to the blood vessel tissues and thus</nobr></div> <div style="position:absolute;top:20185;left:135"><nobr>affect feeding. The denaturation of the ventral and oral suckers and tubercle's spines</nobr></div> <div style="position:absolute;top:20206;left:135"><nobr>could result in an inability to adhere to blood vessel tissues. Thus, they would struggle to</nobr></div> <div style="position:absolute;top:20227;left:135"><nobr>acquire nutrients from the blood efficiently. Additionally, the worm's ability to select an </nobr></div> </span></font> <div style="position:absolute;top:20371;left:0"><hr><table border=0 width=100%><tr><td bgcolor=eeeeee align=right><font face=arial,sans-serif><a name=18><b>Page 18</b></a></font></td></tr></table></div><font size=3 color="#0d0d0d" face="Times"><span style="font-size:12px;font-family:Times;color:#0d0d0d"> <div style="position:absolute;top:20428;left:387"><nobr><b>El-Abbassy <i><font color="#000000">et al.</font></i><font color="#000000">, 2024</font></b></nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:12px;font-family:Times"> <div style="position:absolute;top:20428;left:135"><nobr>816</nobr></div> </span></font> <font size=3 color="#0d0d0d" face="Times"><span style="font-size:16px;font-family:Times;color:#0d0d0d"> <div style="position:absolute;top:20483;left:135"><nobr>appropriate attachment site during feeding is impacted by the elimination of the sensory</nobr></div> <div style="position:absolute;top:20504;left:135"><nobr>papillae on the oral sucker <b>(Reda <i>et al</i>., 2019)</b>. Additionally, tegumental damage may</nobr></div> <div style="position:absolute;top:20524;left:135"><nobr>affect the tegumental functions, causing damage to the defensive mechanism in the</nobr></div> <div style="position:absolute;top:20545;left:135"><nobr>worm, and therefore, it could be easily targeted by the host’s immune system <b>(Xiao <i>et</b></i></nobr></div> <div style="position:absolute;top:20566;left:135"><nobr><i><b>al., </i>2000; Kamel & El-Shinnawy, 2015; Shaaban <i>et al., </i>2019)</b>.</nobr></div> <div style="position:absolute;top:20586;left:171"><nobr>These findings are consistent with the comparable tegumental damages of <i>in vitro</i></nobr></div> <div style="position:absolute;top:20607;left:135"><nobr>and <i>in vivo </i>previous studies on adult worms of <i>S. mansoni </i>under exposure to natural</nobr></div> <div style="position:absolute;top:20628;left:135"><nobr>antischistosomal extracts. <b>Abdella <i>et al</i>. (2024) </b>demonstrated that by incubating adult <i>S.</i></nobr></div> <div style="position:absolute;top:20649;left:135"><nobr><i>mansoni </i>worms with ethanolic extracts of <i>Artemisia annua </i>at different concentrations for</nobr></div> <div style="position:absolute;top:20669;left:135"><nobr>24h, the worms showed contractions, atrophic changes, peeling of tubercles, i.e.,</nobr></div> <div style="position:absolute;top:20686;left:135"><nobr>tubercles became swollen with complete loss of spines, and sucker’s alteration or</nobr></div> <div style="position:absolute;top:20711;left:135"><nobr>destruction. Moreover, <b>Aboueldahab and Elhussieny (2016)</b>, <b>Hassan <i>et al. </i>(2016)</b>,</nobr></div> <div style="position:absolute;top:20731;left:135"><nobr><b>Shaaban <i>et al</i>. (2019)</b>, <b>Abou El-Nour and Fadladdin (2021) </b>and <b>El-Morsy <i>et al.</b></i></nobr></div> <div style="position:absolute;top:20752;left:135"><nobr><b>(2021) </b>coincided with these changes. </nobr></div> <div style="position:absolute;top:20773;left:171"><nobr>It is worth mentioning that the antischistosomal activity of HSE and its potency to</nobr></div> <div style="position:absolute;top:20793;left:135"><nobr>cause morphological alterations in the body of adult <i>S. mansoni </i>worms may be attributed</nobr></div> <div style="position:absolute;top:20814;left:135"><nobr>to the different phytochemical compounds in it, including alkaloids, saponins, phenolics,</nobr></div> <div style="position:absolute;top:20835;left:135"><nobr>and flavonoids. <i>In vitro </i>exposure to countless alkaloids displays antischistosomal activity</nobr></div> <div style="position:absolute;top:20856;left:135"><nobr>either by decreasing motor activity, separating all paired worms, or tegumental</nobr></div> <div style="position:absolute;top:20876;left:135"><nobr>sloughing, causing death to worms <b>(Neves <i>et al., </i>2015)</b>. Saponins, triterpene glycosides,</nobr></div> <div style="position:absolute;top:20897;left:135"><nobr>are potential natural compounds having hemolytic and anthelmintic activities based on</nobr></div> <div style="position:absolute;top:20918;left:135"><nobr>either adhesive defense or toxic mechanisms <b>(Melek <i>et al</i>., 2012; Shalaby <i>et al</i>., 2016;</b></nobr></div> <div style="position:absolute;top:20938;left:135"><nobr><b>Bahrami <i>et al., </i>2018)</b>. Flavonoid and phenolic compounds have been reported to exhibit</nobr></div> <div style="position:absolute;top:20959;left:135"><nobr>pronounced anthelmintic activity against nematode worms <b>(Garcia-Bustos <i>et al</i>., 2019)</b></nobr></div> <div style="position:absolute;top:20980;left:135"><nobr>and the <i>Opisthorchis felineus </i>liver flukes <b>(Mordvinov <i>et al</i>., 2021)</b>, respectively.</nobr></div> <div style="position:absolute;top:21000;left:135"><nobr>Moreover, flavonoids can cause a notable decrease in motor activity or pairing of the</nobr></div> <div style="position:absolute;top:21021;left:135"><nobr>adult <i>S. mansoni </i>worms <b>(Neves <i>et al., </i>2015)</b>.</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:21057;left:177"><nobr><b>CONCLUSION</b></nobr></div> </span></font> <font size=3 color="#0d0d0d" face="Times"><span style="font-size:16px;font-family:Times;color:#0d0d0d"> <div style="position:absolute;top:21098;left:171"><nobr>Given the obtained results, the current work concluded that the HSE has an effective</nobr></div> <div style="position:absolute;top:21119;left:135"><nobr><i>in vitro </i>antischistosomal impact against adult <i>S. mansoni </i>worms causing death and</nobr></div> <div style="position:absolute;top:21140;left:135"><nobr>tegumental alterations. Thus, it can be considered a promising adjuvant medication for</nobr></div> <div style="position:absolute;top:21160;left:135"><nobr>PZQ in <i>S. mansoni </i>infection. However, further <i>in vivo </i>studies are needed to examine its</nobr></div> <div style="position:absolute;top:21181;left:135"><nobr>effect on the liver tissues of the infected mice.</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:21223;left:177"><nobr><b>REFERENCES</b></nobr></div> <div style="position:absolute;top:21264;left:92"><nobr><b>Abd-Allah, M.; Abdelwahab, M.; Aly, N. S.; Amer, O. S. and Sarhan, M. </b>(2022). <i>In vitro</i></nobr></div> <div style="position:absolute;top:21285;left:135"><nobr>schistosomicidal activity of five common species of Red Sea cone snail muscle extracts.</nobr></div> <div style="position:absolute;top:21305;left:135"><nobr>Al-Azhar Bull. Sci., 33(2-C): 111-119.</nobr></div> </span></font> <font size=3 color="#0000ff" face="Times"><span style="font-size:16px;font-family:Times;color:#0000ff"> <div style="position:absolute;top:21305;left:512"><nobr>https://doi.<a href="https://doi.org/10.21608/absb.2022.140291.1187">org/10.21608/ABSB.2022.</a></nobr></div> <div style="position:absolute;top:21326;left:135"><nobr><a href="https://doi.org/10.21608/absb.2022.140291.1187">140291.1187</a></nobr></div> </span></font> <font size=3 color="#0d0d0d" face="Times"><span style="font-size:16px;font-family:Times;color:#0d0d0d"> <div style="position:absolute;top:21347;left:92"><nobr><b>Abdella, O. H.; Ahmed, D. H.; Abdel-Rahman, I. A.; El-Kady, A. M. <font color="#000000">and </font>Mohamed, Y.</b></nobr></div> <div style="position:absolute;top:21367;left:135"><nobr><b>M. </b>(2024). Tegumental alterations in adult <i>Schistosoma mansoni </i>treated with ethanolic</nobr></div> <div style="position:absolute;top:21388;left:135"><nobr>extracts of <i>Artemisia annua in vitro</i>. SVU Int. J. Med. 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F. <font color="#000000">and </font>Fadladdin, Y. </b>(2021). Antischistosomal activity of <i>Zingiber</i></nobr></div> <div style="position:absolute;top:21692;left:135"><nobr><i>officinale</i>, <i>Piper nigrum</i>, and <i>Coriandrum sativum </i>aqueous plant extracts on hamster</nobr></div> <div style="position:absolute;top:21712;left:135"><nobr>infected with</nobr></div> <div style="position:absolute;top:21712;left:292"><nobr><i>Schistosoma mansoni</i>. J. Parasitol. Res., 2021: 1-21.‏</nobr></div> </span></font> <font size=3 color="#0000ff" face="Times"><span style="font-size:16px;font-family:Times;color:#0000ff"> <div style="position:absolute;top:21733;left:139"><nobr><a href="https://doi.org/10.1155/2021/6628787">https://doi.org/10.1155/2021/6628787</a></nobr></div> </span></font> <font size=3 color="#0d0d0d" face="Times"><span style="font-size:16px;font-family:Times;color:#0d0d0d"> <div style="position:absolute;top:21754;left:92"><nobr><b>Aboueldahab, M. 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Drugs, 11(4): 1061-1070.‏</nobr></div> </span></font> <font size=3 color="#0000ff" face="Times"><span style="font-size:16px;font-family:Times;color:#0000ff"> <div style="position:absolute;top:28087;left:135"><nobr>https://doi.<a href="https://www.mdpi.com/1660-3397/11/4/1061">org/10.3390/md11041061</a> </nobr></div> </span></font> <font size=3 color="#0d0d0d" face="Times"><span style="font-size:16px;font-family:Times;color:#0d0d0d"> <div style="position:absolute;top:28108;left:92"><nobr><b>Youssef, D. T.; Yamaki, R. K.; Kelly, M. and Scheuer, P. J. </b>(2002). Salmahyrtisol a, a novel</nobr></div> <div style="position:absolute;top:28128;left:135"><nobr>cytotoxic sesterterpene from the red sea sponge <i>Hyrtios erecta</i>. J. Nat. Prod.<font color="#000000">, 65(1): 2-6.‏</font></nobr></div> </span></font> <font size=3 color="#0000ff" face="Times"><span style="font-size:16px;font-family:Times;color:#0000ff"> <div style="position:absolute;top:28149;left:135"><nobr><a href="https://doi.org/10.1021/np0101853">https://doi.org/10.1021/np0101853</a></nobr></div> </span></font> <font size=3 color="#0d0d0d" face="Times"><span style="font-size:16px;font-family:Times;color:#0d0d0d"> <div style="position:absolute;top:28170;left:92"><nobr><b>Zhishen, J.; Mengcheng, T. and Jianming, W. </b>(1999). The determination of flavonoid</nobr></div> <div style="position:absolute;top:28191;left:135"><nobr>contents in mulberry and their scavenging effects on superoxide radicals. Food Chem.,</nobr></div> <div style="position:absolute;top:28211;left:135"><nobr>64(4): 555-559.‏<a href="https://doi.org/10.1016/S0308-8146(98)00102-2"></a><font color="#0000ff"><a href="https://doi.org/10.1016/S0308-8146(98)00102-2">https://doi.org/10.1016/S0308-8146(98)00102-2</a></font></nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:28232;left:92"><nobr><b>Zou, Z.; Xi, W.; Hu, Y.; Nie, C. and Zhou, Z. </b>(2016). Antioxidant activity of citrus fruits.</nobr></div> <div style="position:absolute;top:28253;left:135"><nobr>Food Chem., 100(196): 885-896. <a href="https://doi.org/10.1016/j.foodchem.2015.09.072"></a><font color="#0000ff"><a href="https://doi.org/10.1016/j.foodchem.2015.09.072">https://doi.org/10.1016/j.foodchem.2015.09.072</a></font></nobr></div> </span></font> <div style="position:absolute;top:28687;left:0"><hr><table border=0 width=100%><tr><td bgcolor=eeeeee align=right><font face=arial,sans-serif><a name=25><b>Page 25</b></a></font></td></tr></table></div><font size=3 color="#0d0d0d" face="Times"><span style="font-size:11px;font-family:Times;color:#0d0d0d"> <div style="position:absolute;top:28743;left:135"><nobr><b>Antischistosomal Effect of <font color="#000000">the Marine </font><i><font color="#000000">Hyrtios </font></i><font color="#000000">Sponge Extract </font>against <i>Schistosoma mansoni </i>Worms</b></nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:14px;font-family:Times"> <div style="position:absolute;top:28744;left:791"><nobr>823</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:28820;left:426"><nobr><b>ARABIC SUMMARY</b></nobr></div> <div style="position:absolute;top:28892;left:550"><nobr><b>دﺎﺿﻣﻟا رﯾﺛﺄﺗﻟﻟ يرﺑﺗﺧﻣﻟا مﯾﯾﻘﺗﻟا</b></nobr></div> <div style="position:absolute;top:28892;left:488"><nobr><b>ﺎﯾﺳرﺎﮭﻟﺑﻟﻟ</b></nobr></div> <div style="position:absolute;top:28892;left:423"><nobr><b>صﻟﺧﺗﺳﻣﻟ</b></nobr></div> <div style="position:absolute;top:28892;left:383"><nobr><b>ﺞﻧﻔﺳإ</b></nobr></div> <div style="position:absolute;top:28896;left:297"><nobr><i><b>Hyrtios </i>sp.</b></nobr></div> <div style="position:absolute;top:28892;left:251"><nobr><b>ىرﺣﺑﻟا</b></nobr></div> <div style="position:absolute;top:28892;left:226"><nobr><b>دﺿ</b></nobr></div> <div style="position:absolute;top:28892;left:191"><nobr><b>نادﯾد</b></nobr></div> </span></font> <font size=3 color="#0d0d0d" face="Times"><span style="font-size:16px;font-family:Times;color:#0d0d0d"> <div style="position:absolute;top:28916;left:392"><nobr><i><b>Schistosoma mansoni</b></i></nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:28913;left:356"><nobr><b>ﺔﻐﻟﺎﺑﻟا</b></nobr></div> <div style="position:absolute;top:28958;left:123"><nobr><b>ىدﯾﻌﺻﻟا حﺎﺗﻔﻟا دﺑﻋ ىوﻟﺳ ,مﯾﻧﻏ ﯽﮐز لﻣأ ,ىرﺿﺧﻟا دوﻣﺣﻣ نﺎﮭﯾﺟ ,ىراوﮭﻟا ﯽﻟﻋ مﯾﻌﻧ ﺎﯾﻧد ,ﯽﺳﺎﺑﻌﻟا مﯾھارﺑا دﻣﺣأ رﯾﻣﺳ</b></nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:19px;font-family:Times"> <div style="position:absolute;top:28958;left:108"><nobr>*</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:14px;font-family:Times"> <div style="position:absolute;top:28994;left:512"><nobr>ُا٘ٞسىا‏ٌيع‏ٌسق</nobr></div> <div style="position:absolute;top:28994;left:502"><nobr>-‏</nobr></div> <div style="position:absolute;top:28994;left:448"><nobr>ً٘يعىا‏تٞيم</nobr></div> <div style="position:absolute;top:28994;left:439"><nobr>-‏</nobr></div> <div style="position:absolute;top:28994;left:391"><nobr>ٍد‏تعٍبخ</nobr></div> <div style="position:absolute;top:28994;left:365"><nobr>سْٖ٘</nobr></div> <div style="position:absolute;top:28994;left:356"><nobr>-‏</nobr></div> <div style="position:absolute;top:28994;left:323"><nobr>.شصٍ</nobr></div> </span></font> <font size=3 face="Times"><span style="font-size:16px;font-family:Times"> <div style="position:absolute;top:29031;left:748"><nobr>‏‏‏‏‏‏‏‏</nobr></div> <div style="position:absolute;top:29031;left:730"><nobr>ذعٝ</nobr></div> <div style="position:absolute;top:29031;left:719"><nobr>‏</nobr></div> <div style="position:absolute;top:29031;left:702"><nobr>ءاد</nobr></div> <div style="position:absolute;top:29031;left:691"><nobr>‏</nobr></div> <div style="position:absolute;top:29031;left:637"><nobr>بٞسسبٖيبىا</nobr></div> <div style="position:absolute;top:29031;left:627"><nobr>‏</nobr></div> <div style="position:absolute;top:29031;left:610"><nobr>ٍِ</nobr></div> <div style="position:absolute;top:29031;left:599"><nobr>‏</nobr></div> <div style="position:absolute;top:29031;left:546"><nobr>ضاشٍلأا</nobr></div> <div style="position:absolute;top:29031;left:535"><nobr>‏</nobr></div> <div style="position:absolute;top:29031;left:439"><nobr>ةششخَْىا‏ تٞئبب٘ىا</nobr></div> <div style="position:absolute;top:29031;left:412"><nobr>‏ٜف</nobr></div> <div style="position:absolute;top:29031;left:401"><nobr>‏</nobr></div> <div style="position:absolute;top:29031;left:370"><nobr>شصٍ</nobr></div> <div style="position:absolute;top:29031;left:366"><nobr>.</nobr></div> <div style="position:absolute;top:29031;left:325"><nobr>تدٞخّ</nobr></div> <div style="position:absolute;top:29031;left:315"><nobr>‏</nobr></div> <div style="position:absolute;top:29031;left:260"><nobr>ًاذخخسلاى</nobr></div> <div style="position:absolute;top:29031;left:250"><nobr>‏</nobr></div> <div style="position:absolute;top:29031;left:197"><nobr>ٛشصسىا</nobr></div> <div style="position:absolute;top:29031;left:186"><nobr>‏</nobr></div> <div style="position:absolute;top:29031;left:135"><nobr>سشنخَىاٗ</nobr></div> <div style="position:absolute;top:29031;left:130"><nobr>‏</nobr></div> <div style="position:absolute;top:29052;left:776"><nobr>يى</nobr></div> <div style="position:absolute;top:29052;left:710"><nobr>وخّا٘نٝصاشب</nobr></div> <div style="position:absolute;top:29052;left:703"><nobr>‏</nobr></div> <div style="position:absolute;top:29052;left:688"><nobr>ٜف</nobr></div> <div style="position:absolute;top:29052;left:681"><nobr>‏</nobr></div> <div style="position:absolute;top:29052;left:651"><nobr>جلاع</nobr></div> <div style="position:absolute;top:29052;left:645"><nobr>‏</nobr></div> <div style="position:absolute;top:29052;left:627"><nobr>ءاد</nobr></div> <div style="position:absolute;top:29052;left:621"><nobr>‏</nobr></div> <div style="position:absolute;top:29052;left:562"><nobr>،بٞسسبٖيبىا</nobr></div> <div style="position:absolute;top:29052;left:544"><nobr>‏ذق</nobr></div> <div style="position:absolute;top:29052;left:538"><nobr>‏</nobr></div> <div style="position:absolute;top:29052;left:505"><nobr>ربصٝ</nobr></div> <div style="position:absolute;top:29052;left:498"><nobr>‏</nobr></div> <div style="position:absolute;top:29052;left:457"><nobr>ٜيٞفطىا</nobr></div> <div style="position:absolute;top:29052;left:451"><nobr>‏</nobr></div> <div style="position:absolute;top:29052;left:416"><nobr>بًٍٗبقٍ</nobr></div> <div style="position:absolute;top:29052;left:409"><nobr>‏</nobr></div> <div style="position:absolute;top:29052;left:377"><nobr>ءاٗذيى</nobr></div> <div style="position:absolute;top:29052;left:366"><nobr>‏.</nobr></div> <div style="position:absolute;top:29052;left:338"><nobr>‏ًءبْبٗ</nobr></div> <div style="position:absolute;top:29052;left:331"><nobr>‏</nobr></div> <div style="position:absolute;top:29052;left:308"><nobr>ٚيع</nobr></div> <div style="position:absolute;top:29052;left:301"><nobr>‏</nobr></div> <div style="position:absolute;top:29052;left:275"><nobr>،لىر</nobr></div> <div style="position:absolute;top:29052;left:268"><nobr>‏</nobr></div> <div style="position:absolute;top:29052;left:232"><nobr>جَخٕا</nobr></div> <div style="position:absolute;top:29052;left:226"><nobr>‏</nobr></div> <div style="position:absolute;top:29052;left:182"><nobr>دبسبلأا</nobr></div> <div style="position:absolute;top:29052;left:171"><nobr>‏ب</nobr></div> <div style="position:absolute;top:29052;left:135"><nobr>شٝ٘طخ</nobr></div> <div style="position:absolute;top:29052;left:130"><nobr>‏</nobr></div> <div style="position:absolute;top:29073;left:755"><nobr>تٝٗدأ</nobr></div> <div style="position:absolute;top:29073;left:750"><nobr>‏</nobr></div> <div style="position:absolute;top:29073;left:719"><nobr>ةذٝذخ</nobr></div> <div style="position:absolute;top:29073;left:714"><nobr>‏</nobr></div> <div style="position:absolute;top:29073;left:676"><nobr>ةدبعٍ</nobr></div> <div style="position:absolute;top:29073;left:672"><nobr>‏</nobr></div> <div style="position:absolute;top:29073;left:618"><nobr>بٞسسبٖيبيى</nobr></div> <div style="position:absolute;top:29073;left:613"><nobr>‏</nobr></div> <div style="position:absolute;top:29073;left:597"><nobr>ٍِ</nobr></div> <div style="position:absolute;top:29073;left:592"><nobr>‏</nobr></div> <div style="position:absolute;top:29073;left:551"><nobr>سدبصٍ</nobr></div> <div style="position:absolute;top:29073;left:546"><nobr>‏</nobr></div> <div style="position:absolute;top:29073;left:520"><nobr>عٞبغ</nobr></div> <div style="position:absolute;top:29073;left:508"><nobr>تٞ</nobr></div> <div style="position:absolute;top:29073;left:499"><nobr>‏.</nobr></div> <div style="position:absolute;top:29073;left:462"><nobr>ٛ٘خسٝ</nobr></div> <div style="position:absolute;top:29073;left:457"><nobr>‏</nobr></div> <div style="position:absolute;top:29073;left:425"><nobr>حْفسإ</nobr></div> <div style="position:absolute;top:29073;left:420"><nobr>‏</nobr></div> <div style="position:absolute;top:29072;left:367"><nobr>ط٘ٞحشٕٞ</nobr></div> <div style="position:absolute;top:29073;left:363"><nobr>‏</nobr></div> <div style="position:absolute;top:29073;left:321"><nobr>ٛشسبىا</nobr></div> <div style="position:absolute;top:29073;left:316"><nobr>‏</nobr></div> <div style="position:absolute;top:29073;left:292"><nobr>ٚيع</nobr></div> <div style="position:absolute;top:29073;left:287"><nobr>‏</nobr></div> <div style="position:absolute;top:29073;left:243"><nobr>ثببمشٍ</nobr></div> <div style="position:absolute;top:29073;left:238"><nobr>‏</nobr></div> <div style="position:absolute;top:29073;left:207"><nobr>تطشّ</nobr></div> <div style="position:absolute;top:29073;left:202"><nobr>‏</nobr></div> <div style="position:absolute;top:29073;left:156"><nobr>بًٞخ٘ى٘ٞب</nobr></div> <div style="position:absolute;top:29073;left:151"><nobr>‏</nobr></div> <div style="position:absolute;top:29073;left:135"><nobr>ٍِ</nobr></div> <div style="position:absolute;top:29073;left:130"><nobr>‏</nobr></div> <div style="position:absolute;top:29093;left:757"><nobr>ثبئف</nobr></div> <div style="position:absolute;top:29093;left:752"><nobr>‏</nobr></div> <div style="position:absolute;top:29093;left:716"><nobr>تفيخخٍ</nobr></div> <div style="position:absolute;top:29093;left:711"><nobr>‏</nobr></div> <div style="position:absolute;top:29093;left:680"><nobr>ٔيٕؤح</nobr></div> <div style="position:absolute;top:29093;left:675"><nobr>‏</nobr></div> <div style="position:absolute;top:29093;left:617"><nobr>فبشنخسلا</nobr></div> <div style="position:absolute;top:29093;left:612"><nobr>‏</nobr></div> <div style="position:absolute;top:29093;left:583"><nobr>تٝٗدأ</nobr></div> <div style="position:absolute;top:29093;left:578"><nobr>‏</nobr></div> <div style="position:absolute;top:29093;left:547"><nobr>ةذٝذخ</nobr></div> <div style="position:absolute;top:29093;left:537"><nobr>‏.</nobr></div> <div style="position:absolute;top:29093;left:513"><nobr>ٍِٗ</nobr></div> <div style="position:absolute;top:29093;left:508"><nobr>‏</nobr></div> <div style="position:absolute;top:29093;left:491"><nobr>بْٕ</nobr></div> <div style="position:absolute;top:29093;left:486"><nobr>‏</nobr></div> <div style="position:absolute;top:29093;left:454"><nobr>جفذٕ</nobr></div> <div style="position:absolute;top:29093;left:450"><nobr>‏</nobr></div> <div style="position:absolute;top:29093;left:407"><nobr>تساسذىا</nobr></div> <div style="position:absolute;top:29093;left:402"><nobr>‏</nobr></div> <div style="position:absolute;top:29093;left:366"><nobr>تٞىبسىا</nobr></div> <div style="position:absolute;top:29093;left:358"><nobr>‏ى</nobr></div> <div style="position:absolute;top:29093;left:334"><nobr>ٌٞٞقخ</nobr></div> <div style="position:absolute;top:29093;left:329"><nobr>‏</nobr></div> <div style="position:absolute;top:29093;left:293"><nobr>غبشْىا</nobr></div> <div style="position:absolute;top:29093;left:288"><nobr>‏</nobr></div> <div style="position:absolute;top:29093;left:235"><nobr>ٛشبخخَىا</nobr></div> <div style="position:absolute;top:29093;left:230"><nobr>‏</nobr></div> <div style="position:absolute;top:29093;left:172"><nobr>صيخخسَى</nobr></div> <div style="position:absolute;top:29093;left:167"><nobr>‏</nobr></div> <div style="position:absolute;top:29093;left:135"><nobr>حْفسإ</nobr></div> <div style="position:absolute;top:29093;left:131"><nobr>‏</nobr></div> <div style="position:absolute;top:29113;left:731"><nobr>ط٘ٞحشٕٞ</nobr></div> <div style="position:absolute;top:29114;left:720"><nobr>(</nobr></div> <div style="position:absolute;top:29118;left:686"><nobr>HSE</nobr></div> <div style="position:absolute;top:29114;left:675"><nobr>‏)</nobr></div> <div style="position:absolute;top:29114;left:654"><nobr>ذظ</nobr></div> <div style="position:absolute;top:29114;left:649"><nobr>‏</nobr></div> <div style="position:absolute;top:29114;left:632"><nobr>ءاد</nobr></div> <div style="position:absolute;top:29114;left:627"><nobr>‏</nobr></div> <div style="position:absolute;top:29114;left:574"><nobr>بٞسسبٖيبىا</nobr></div> <div style="position:absolute;top:29114;left:569"><nobr>‏</nobr></div> <div style="position:absolute;top:29114;left:553"><nobr>ٍِ</nobr></div> <div style="position:absolute;top:29114;left:548"><nobr>‏</nobr></div> <div style="position:absolute;top:29114;left:518"><nobr>هلاخ</nobr></div> <div style="position:absolute;top:29114;left:514"><nobr>‏</nobr></div> <div style="position:absolute;top:29114;left:475"><nobr>فشنىا</nobr></div> <div style="position:absolute;top:29114;left:471"><nobr>‏</nobr></div> <div style="position:absolute;top:29114;left:452"><nobr>ِع</nobr></div> <div style="position:absolute;top:29114;left:447"><nobr>‏</nobr></div> <div style="position:absolute;top:29114;left:405"><nobr>ثبٞف٘ىا</nobr></div> <div style="position:absolute;top:29114;left:401"><nobr>‏</nobr></div> <div style="position:absolute;top:29114;left:381"><nobr>خىاٗ</nobr></div> <div style="position:absolute;top:29114;left:344"><nobr>ثاشٞغ</nobr></div> <div style="position:absolute;top:29114;left:339"><nobr>‏</nobr></div> <div style="position:absolute;top:29114;left:295"><nobr>ذٞيخ‏ٚف</nobr></div> <div style="position:absolute;top:29114;left:291"><nobr>‏</nobr></div> <div style="position:absolute;top:29114;left:253"><nobr>ُاذٝذىا</nobr></div> <div style="position:absolute;top:29114;left:249"><nobr>‏</nobr></div> <div style="position:absolute;top:29114;left:215"><nobr>تغىببىا</nobr></div> <div style="position:absolute;top:29114;left:199"><nobr>‏ىـي</nobr></div> <div style="position:absolute;top:29113;left:130"><nobr>‏بٍ٘س٘خسٞش</nobr></div> <div style="position:absolute;top:29134;left:736"><nobr>ّٚ٘سّبٍ</nobr></div> <div style="position:absolute;top:29135;left:726"><nobr>‏.</nobr></div> <div style="position:absolute;top:29135;left:698"><nobr>جّبم</nobr></div> <div style="position:absolute;top:29135;left:693"><nobr>‏</nobr></div> <div style="position:absolute;top:29135;left:650"><nobr>ثلاذعٍ</nobr></div> <div style="position:absolute;top:29135;left:645"><nobr>‏</nobr></div> <div style="position:absolute;top:29135;left:611"><nobr>ثبٞفٗ</nobr></div> <div style="position:absolute;top:29135;left:606"><nobr>‏</nobr></div> <div style="position:absolute;top:29135;left:576"><nobr>ُاذٝد</nobr></div> <div style="position:absolute;top:29135;left:571"><nobr>‏</nobr></div> <div style="position:absolute;top:29134;left:454"><nobr>ّٚ٘سّبٍ‏بٍ٘س٘خسٞش</nobr></div> <div style="position:absolute;top:29135;left:449"><nobr>‏</nobr></div> <div style="position:absolute;top:29135;left:425"><nobr>شثمأ</nobr></div> <div style="position:absolute;top:29135;left:420"><nobr>‏</nobr></div> <div style="position:absolute;top:29135;left:404"><nobr>ٍِ</nobr></div> <div style="position:absolute;top:29135;left:399"><nobr>‏</nobr></div> <div style="position:absolute;top:29135;left:381"><nobr>50</nobr></div> <div style="position:absolute;top:29135;left:361"><nobr>‏%</nobr></div> <div style="position:absolute;top:29135;left:343"><nobr>ذعب</nobr></div> <div style="position:absolute;top:29135;left:338"><nobr>‏</nobr></div> <div style="position:absolute;top:29135;left:297"><nobr>ِٞخعبس</nobr></div> <div style="position:absolute;top:29135;left:284"><nobr>‏ٗ</nobr></div> <div style="position:absolute;top:29135;left:270"><nobr>3‏</nobr></div> <div style="position:absolute;top:29135;left:230"><nobr>ثبعبس</nobr></div> <div style="position:absolute;top:29135;left:225"><nobr>‏</nobr></div> <div style="position:absolute;top:29135;left:205"><nobr>ذْع</nobr></div> <div style="position:absolute;top:29135;left:200"><nobr>‏</nobr></div> <div style="position:absolute;top:29135;left:167"><nobr>ضٞمشح</nobr></div> <div style="position:absolute;top:29135;left:162"><nobr>‏</nobr></div> <div style="position:absolute;top:29135;left:135"><nobr>200</nobr></div> <div style="position:absolute;top:29135;left:130"><nobr>‏</nobr></div> <div style="position:absolute;top:29156;left:749"><nobr>ٗشنٍٞ</nobr></div> <div style="position:absolute;top:29156;left:739"><nobr>خ</nobr></div> <div style="position:absolute;top:29156;left:720"><nobr>ًاش</nobr></div> <div style="position:absolute;top:29156;left:715"><nobr>/</nobr></div> <div style="position:absolute;top:29156;left:699"><nobr>وٍ</nobr></div> <div style="position:absolute;top:29156;left:693"><nobr>‏</nobr></div> <div style="position:absolute;top:29156;left:677"><nobr>ٍِ</nobr></div> <div style="position:absolute;top:29156;left:671"><nobr>‏</nobr></div> <div style="position:absolute;top:29159;left:637"><nobr>HSE</nobr></div> <div style="position:absolute;top:29156;left:618"><nobr>،‏ٗ</nobr></div> <div style="position:absolute;top:29156;left:613"><nobr>‏</nobr></div> <div style="position:absolute;top:29156;left:595"><nobr>ذعب</nobr></div> <div style="position:absolute;top:29156;left:590"><nobr>‏</nobr></div> <div style="position:absolute;top:29156;left:572"><nobr>24</nobr></div> <div style="position:absolute;top:29156;left:566"><nobr>‏</nobr></div> <div style="position:absolute;top:29156;left:536"><nobr>تعبس</nobr></div> <div style="position:absolute;top:29156;left:531"><nobr>‏</nobr></div> <div style="position:absolute;top:29156;left:511"><nobr>ذْع</nobr></div> <div style="position:absolute;top:29156;left:505"><nobr>‏</nobr></div> <div style="position:absolute;top:29156;left:478"><nobr>100</nobr></div> <div style="position:absolute;top:29156;left:465"><nobr>‏ٗ</nobr></div> <div style="position:absolute;top:29156;left:438"><nobr>200</nobr></div> <div style="position:absolute;top:29156;left:432"><nobr>‏</nobr></div> <div style="position:absolute;top:29156;left:398"><nobr>ٗشنٍٞ</nobr></div> <div style="position:absolute;top:29156;left:388"><nobr>خ</nobr></div> <div style="position:absolute;top:29156;left:369"><nobr>ًاش</nobr></div> <div style="position:absolute;top:29156;left:364"><nobr>/</nobr></div> <div style="position:absolute;top:29156;left:342"><nobr>،وٍ</nobr></div> <div style="position:absolute;top:29156;left:323"><nobr>‏ٗ‏</nobr></div> <div style="position:absolute;top:29156;left:306"><nobr>ذعب</nobr></div> <div style="position:absolute;top:29156;left:300"><nobr>‏</nobr></div> <div style="position:absolute;top:29156;left:282"><nobr>36</nobr></div> <div style="position:absolute;top:29156;left:269"><nobr>‏ٗ</nobr></div> <div style="position:absolute;top:29156;left:251"><nobr>44</nobr></div> <div style="position:absolute;top:29156;left:246"><nobr>‏</nobr></div> <div style="position:absolute;top:29156;left:216"><nobr>تعبس</nobr></div> <div style="position:absolute;top:29156;left:210"><nobr>‏</nobr></div> <div style="position:absolute;top:29156;left:190"><nobr>ذْع</nobr></div> <div style="position:absolute;top:29156;left:185"><nobr>‏</nobr></div> <div style="position:absolute;top:29156;left:135"><nobr>ثاضٞمشح</nobr></div> <div style="position:absolute;top:29156;left:130"><nobr>‏</nobr></div> <div style="position:absolute;top:29176;left:744"><nobr>ذٗاشخح</nobr></div> <div style="position:absolute;top:29176;left:738"><nobr>‏</nobr></div> <div style="position:absolute;top:29176;left:720"><nobr>ِٞب</nobr></div> <div style="position:absolute;top:29176;left:714"><nobr>‏</nobr></div> <div style="position:absolute;top:29176;left:696"><nobr>60</nobr></div> <div style="position:absolute;top:29176;left:690"><nobr>‏</nobr></div> <div style="position:absolute;top:29176;left:672"><nobr>ٚىإ</nobr></div> <div style="position:absolute;top:29176;left:666"><nobr>‏</nobr></div> <div style="position:absolute;top:29176;left:639"><nobr>200</nobr></div> <div style="position:absolute;top:29176;left:633"><nobr>‏</nobr></div> <div style="position:absolute;top:29176;left:570"><nobr>ًاشخٗشنٍٞ</nobr></div> <div style="position:absolute;top:29176;left:565"><nobr>/</nobr></div> <div style="position:absolute;top:29176;left:548"><nobr>وٍ</nobr></div> <div style="position:absolute;top:29176;left:538"><nobr>‏.</nobr></div> <div style="position:absolute;top:29176;left:505"><nobr>ةٗلاع</nobr></div> <div style="position:absolute;top:29176;left:499"><nobr>‏</nobr></div> <div style="position:absolute;top:29176;left:475"><nobr>ٚيع</nobr></div> <div style="position:absolute;top:29176;left:469"><nobr>‏</nobr></div> <div style="position:absolute;top:29176;left:443"><nobr>،لىر</nobr></div> <div style="position:absolute;top:29176;left:425"><nobr>‏عب</nobr></div> <div style="position:absolute;top:29176;left:413"><nobr>ذ‏</nobr></div> <div style="position:absolute;top:29176;left:395"><nobr>44</nobr></div> <div style="position:absolute;top:29176;left:389"><nobr>‏</nobr></div> <div style="position:absolute;top:29176;left:353"><nobr>،تعبس</nobr></div> <div style="position:absolute;top:29176;left:347"><nobr>‏</nobr></div> <div style="position:absolute;top:29176;left:315"><nobr>وصٗ</nobr></div> <div style="position:absolute;top:29176;left:309"><nobr>‏</nobr></div> <div style="position:absolute;top:29176;left:280"><nobr>هذعٍ</nobr></div> <div style="position:absolute;top:29176;left:274"><nobr>‏</nobr></div> <div style="position:absolute;top:29176;left:233"><nobr>ثبٞف٘ىا</nobr></div> <div style="position:absolute;top:29176;left:227"><nobr>‏</nobr></div> <div style="position:absolute;top:29176;left:209"><nobr>ٚىإ</nobr></div> <div style="position:absolute;top:29176;left:203"><nobr>‏</nobr></div> <div style="position:absolute;top:29176;left:176"><nobr>100</nobr></div> <div style="position:absolute;top:29176;left:155"><nobr>‏%</nobr></div> <div style="position:absolute;top:29176;left:135"><nobr>ذْع</nobr></div> <div style="position:absolute;top:29176;left:130"><nobr>‏</nobr></div> <div style="position:absolute;top:29197;left:749"><nobr>تعشخ</nobr></div> <div style="position:absolute;top:29197;left:740"><nobr>‏</nobr></div> <div style="position:absolute;top:29197;left:713"><nobr>200</nobr></div> <div style="position:absolute;top:29197;left:704"><nobr>‏</nobr></div> <div style="position:absolute;top:29197;left:669"><nobr>ٗشنٍٞ</nobr></div> <div style="position:absolute;top:29197;left:660"><nobr>خ</nobr></div> <div style="position:absolute;top:29197;left:641"><nobr>ًاش</nobr></div> <div style="position:absolute;top:29197;left:636"><nobr>/</nobr></div> <div style="position:absolute;top:29197;left:620"><nobr>وٍ</nobr></div> <div style="position:absolute;top:29197;left:607"><nobr>‏ .</nobr></div> <div style="position:absolute;top:29197;left:589"><nobr>ذعب</nobr></div> <div style="position:absolute;top:29197;left:580"><nobr>‏</nobr></div> <div style="position:absolute;top:29197;left:536"><nobr>طٝشعح</nobr></div> <div style="position:absolute;top:29197;left:527"><nobr>‏</nobr></div> <div style="position:absolute;top:29197;left:497"><nobr>ُاذٝد</nobr></div> <div style="position:absolute;top:29197;left:488"><nobr>‏</nobr></div> <div style="position:absolute;top:29196;left:368"><nobr>ّٚ٘سّبٍ‏ بٍ٘س٘خسٞش</nobr></div> <div style="position:absolute;top:29197;left:359"><nobr>‏</nobr></div> <div style="position:absolute;top:29197;left:341"><nobr>ٚىإ</nobr></div> <div style="position:absolute;top:29197;left:332"><nobr>‏</nobr></div> <div style="position:absolute;top:29201;left:298"><nobr>HSE</nobr></div> <div style="position:absolute;top:29197;left:289"><nobr>‏</nobr></div> <div style="position:absolute;top:29197;left:267"><nobr>ةذَى</nobr></div> <div style="position:absolute;top:29197;left:259"><nobr>‏</nobr></div> <div style="position:absolute;top:29197;left:241"><nobr>24</nobr></div> <div style="position:absolute;top:29197;left:232"><nobr>‏</nobr></div> <div style="position:absolute;top:29197;left:196"><nobr>،تعبس</nobr></div> <div style="position:absolute;top:29197;left:187"><nobr>‏</nobr></div> <div style="position:absolute;top:29197;left:159"><nobr>جّبم</nobr></div> <div style="position:absolute;top:29197;left:135"><nobr>‏ٌٞق</nobr></div> <div style="position:absolute;top:29197;left:130"><nobr>‏</nobr></div> <div style="position:absolute;top:29218;left:743"><nobr>ضٞمشخىا</nobr></div> <div style="position:absolute;top:29218;left:726"><nobr>ثا</nobr></div> <div style="position:absolute;top:29218;left:721"><nobr>‏</nobr></div> <div style="position:absolute;top:29218;left:684"><nobr>تخََٞىا</nobr></div> <div style="position:absolute;top:29218;left:673"><nobr>‏(</nobr></div> <div style="position:absolute;top:29221;left:638"><nobr>LC<font style="font-size:10px">50</font></nobr></div> <div style="position:absolute;top:29218;left:626"><nobr>‏ٗ</nobr></div> <div style="position:absolute;top:29221;left:580"><nobr>(LC<font style="font-size:10px">90</font></nobr></div> <div style="position:absolute;top:29218;left:521"><nobr>115.40</nobr></div> <div style="position:absolute;top:29218;left:503"><nobr>‏ٗ‏</nobr></div> <div style="position:absolute;top:29218;left:454"><nobr>241.12</nobr></div> <div style="position:absolute;top:29218;left:449"><nobr>‏</nobr></div> <div style="position:absolute;top:29218;left:414"><nobr>ٗشنٍٞ</nobr></div> <div style="position:absolute;top:29218;left:405"><nobr>خ</nobr></div> <div style="position:absolute;top:29218;left:386"><nobr>ًاش</nobr></div> <div style="position:absolute;top:29218;left:381"><nobr>/</nobr></div> <div style="position:absolute;top:29218;left:359"><nobr>،وٍ</nobr></div> <div style="position:absolute;top:29218;left:354"><nobr>‏</nobr></div> <div style="position:absolute;top:29218;left:330"><nobr>ٚيع</nobr></div> <div style="position:absolute;top:29218;left:326"><nobr>‏</nobr></div> <div style="position:absolute;top:29218;left:288"><nobr>ٜىا٘خىا</nobr></div> <div style="position:absolute;top:29218;left:278"><nobr>‏.</nobr></div> <div style="position:absolute;top:29218;left:242"><nobr>جّبمٗ</nobr></div> <div style="position:absolute;top:29218;left:238"><nobr>‏</nobr></div> <div style="position:absolute;top:29218;left:197"><nobr>52.36</nobr></div> <div style="position:absolute;top:29218;left:185"><nobr>‏ٗ</nobr></div> <div style="position:absolute;top:29218;left:135"><nobr>144.11</nobr></div> <div style="position:absolute;top:29218;left:130"><nobr>‏</nobr></div> <div style="position:absolute;top:29238;left:749"><nobr>ٗشنٍٞ</nobr></div> <div style="position:absolute;top:29238;left:739"><nobr>خ</nobr></div> <div style="position:absolute;top:29238;left:720"><nobr>ًاش</nobr></div> <div style="position:absolute;top:29238;left:715"><nobr>/</nobr></div> <div style="position:absolute;top:29238;left:699"><nobr>وٍ</nobr></div> <div style="position:absolute;top:29238;left:692"><nobr>‏</nobr></div> <div style="position:absolute;top:29238;left:675"><nobr>ذعب</nobr></div> <div style="position:absolute;top:29238;left:668"><nobr>‏</nobr></div> <div style="position:absolute;top:29238;left:650"><nobr>44</nobr></div> <div style="position:absolute;top:29238;left:644"><nobr>‏</nobr></div> <div style="position:absolute;top:29238;left:614"><nobr>تعبس</nobr></div> <div style="position:absolute;top:29238;left:607"><nobr>‏</nobr></div> <div style="position:absolute;top:29238;left:590"><nobr>ٍِ</nobr></div> <div style="position:absolute;top:29238;left:584"><nobr>‏</nobr></div> <div style="position:absolute;top:29238;left:558"><nobr>جقٗ</nobr></div> <div style="position:absolute;top:29238;left:552"><nobr>‏</nobr></div> <div style="position:absolute;top:29238;left:498"><nobr>،ضشعخىا</nobr></div> <div style="position:absolute;top:29238;left:492"><nobr>‏</nobr></div> <div style="position:absolute;top:29238;left:468"><nobr>ٚيع</nobr></div> <div style="position:absolute;top:29238;left:461"><nobr>‏</nobr></div> <div style="position:absolute;top:29238;left:423"><nobr>ٜىا٘خىا</nobr></div> <div style="position:absolute;top:29238;left:412"><nobr>‏.</nobr></div> <div style="position:absolute;top:29238;left:382"><nobr>فشم</nobr></div> <div style="position:absolute;top:29238;left:375"><nobr>‏</nobr></div> <div style="position:absolute;top:29238;left:333"><nobr>صسفىا</nobr></div> <div style="position:absolute;top:29238;left:327"><nobr>‏</nobr></div> <div style="position:absolute;top:29238;left:275"><nobr>ٛشٖدَىا</nobr></div> <div style="position:absolute;top:29238;left:269"><nobr>‏</nobr></div> <div style="position:absolute;top:29238;left:208"><nobr>ّٜٗشخنىلإا</nobr></div> <div style="position:absolute;top:29238;left:202"><nobr>‏</nobr></div> <div style="position:absolute;top:29238;left:183"><nobr>ِع</nobr></div> <div style="position:absolute;top:29238;left:176"><nobr>‏</nobr></div> <div style="position:absolute;top:29238;left:135"><nobr>ثاشٞغح</nobr></div> <div style="position:absolute;top:29238;left:130"><nobr>‏</nobr></div> <div style="position:absolute;top:29259;left:752"><nobr>تٞينش</nobr></div> <div style="position:absolute;top:29259;left:731"><nobr>‏ٜف</nobr></div> <div style="position:absolute;top:29259;left:726"><nobr>‏</nobr></div> <div style="position:absolute;top:29259;left:709"><nobr>لام</nobr></div> <div style="position:absolute;top:29259;left:703"><nobr>‏</nobr></div> <div style="position:absolute;top:29259;left:687"><nobr>ٍِ</nobr></div> <div style="position:absolute;top:29259;left:682"><nobr>‏</nobr></div> <div style="position:absolute;top:29259;left:644"><nobr>ُاذٝذىا</nobr></div> <div style="position:absolute;top:29259;left:639"><nobr>‏</nobr></div> <div style="position:absolute;top:29259;left:599"><nobr>تٝشمزىا</nobr></div> <div style="position:absolute;top:29259;left:594"><nobr>‏</nobr></div> <div style="position:absolute;top:29259;left:545"><nobr>تٝ٘ثّلأاٗ</nobr></div> <div style="position:absolute;top:29259;left:540"><nobr>‏</nobr></div> <div style="position:absolute;top:29259;left:522"><nobr>ذعب</nobr></div> <div style="position:absolute;top:29259;left:509"><nobr>‏ىا</nobr></div> <div style="position:absolute;top:29259;left:471"><nobr>تدىبعَ</nobr></div> <div style="position:absolute;top:29259;left:466"><nobr>‏</nobr></div> <div style="position:absolute;top:29259;left:413"><nobr>تٝشبخخَىا</nobr></div> <div style="position:absolute;top:29259;left:408"><nobr>‏</nobr></div> <div style="position:absolute;top:29259;left:374"><nobr>ُاذٝذى</nobr></div> <div style="position:absolute;top:29259;left:369"><nobr>‏</nobr></div> <div style="position:absolute;top:29258;left:252"><nobr>ّٚ٘سّبٍ‏بٍ٘س٘خسٞش</nobr></div> <div style="position:absolute;top:29259;left:247"><nobr>‏</nobr></div> <div style="position:absolute;top:29259;left:199"><nobr>ًاذخخسبب</nobr></div> <div style="position:absolute;top:29259;left:194"><nobr>‏</nobr></div> <div style="position:absolute;top:29263;left:160"><nobr>HSE</nobr></div> <div style="position:absolute;top:29259;left:155"><nobr>‏</nobr></div> <div style="position:absolute;top:29259;left:130"><nobr>‏ذْع</nobr></div> <div style="position:absolute;top:29280;left:734"><nobr>ثاضٞمشح</nobr></div> <div style="position:absolute;top:29280;left:722"><nobr>‏</nobr></div> <div style="position:absolute;top:29280;left:695"><nobr>100</nobr></div> <div style="position:absolute;top:29280;left:675"><nobr>‏ٗ</nobr></div> <div style="position:absolute;top:29280;left:648"><nobr>200</nobr></div> <div style="position:absolute;top:29280;left:636"><nobr>‏</nobr></div> <div style="position:absolute;top:29280;left:601"><nobr>ٗشنٍٞ</nobr></div> <div style="position:absolute;top:29280;left:592"><nobr>خ</nobr></div> <div style="position:absolute;top:29280;left:573"><nobr>ًاش</nobr></div> <div style="position:absolute;top:29280;left:568"><nobr>/</nobr></div> <div style="position:absolute;top:29280;left:552"><nobr>وٍ</nobr></div> <div style="position:absolute;top:29280;left:540"><nobr>‏</nobr></div> <div style="position:absolute;top:29280;left:518"><nobr>ةذَى</nobr></div> <div style="position:absolute;top:29280;left:506"><nobr>‏</nobr></div> <div style="position:absolute;top:29280;left:488"><nobr>44</nobr></div> <div style="position:absolute;top:29280;left:477"><nobr>‏</nobr></div> <div style="position:absolute;top:29280;left:441"><nobr>،تعبس</nobr></div> <div style="position:absolute;top:29280;left:429"><nobr>‏</nobr></div> <div style="position:absolute;top:29280;left:393"><nobr>‏ًتّسبقٍ</nobr></div> <div style="position:absolute;top:29280;left:381"><nobr>‏</nobr></div> <div style="position:absolute;top:29280;left:321"><nobr>تعَ٘دَىبب</nobr></div> <div style="position:absolute;top:29280;left:310"><nobr>‏</nobr></div> <div style="position:absolute;top:29280;left:261"><nobr>تطببعىا</nobr></div> <div style="position:absolute;top:29280;left:245"><nobr>‏ .</nobr></div> <div style="position:absolute;top:29280;left:201"><nobr>جَْعح</nobr></div> <div style="position:absolute;top:29280;left:189"><nobr>‏</nobr></div> <div style="position:absolute;top:29280;left:135"><nobr>ثبٕ٘شخىا</nobr></div> <div style="position:absolute;top:29280;left:130"><nobr>‏</nobr></div> <div style="position:absolute;top:29300;left:776"><nobr>ىا</nobr></div> <div style="position:absolute;top:29300;left:708"><nobr>تٞخ٘ى٘فسَ٘</nobr></div> <div style="position:absolute;top:29300;left:703"><nobr>‏</nobr></div> <div style="position:absolute;top:29300;left:683"><nobr>ٜف‏</nobr></div> <div style="position:absolute;top:29300;left:653"><nobr>س٘مر</nobr></div> <div style="position:absolute;top:29300;left:649"><nobr>‏</nobr></div> <div style="position:absolute;top:29300;left:612"><nobr>ُاذٝذىا</nobr></div> <div style="position:absolute;top:29300;left:607"><nobr>‏</nobr></div> <div style="position:absolute;top:29300;left:569"><nobr>بًٍبسخأ</nobr></div> <div style="position:absolute;top:29300;left:565"><nobr>‏</nobr></div> <div style="position:absolute;top:29300;left:528"><nobr>تخفخٍْ</nobr></div> <div style="position:absolute;top:29300;left:512"><nobr>‏ٗأ</nobr></div> <div style="position:absolute;top:29300;left:507"><nobr>‏</nobr></div> <div style="position:absolute;top:29300;left:458"><nobr>،تسطسٍ</nobr></div> <div style="position:absolute;top:29300;left:441"><nobr>‏ٗ‏</nobr></div> <div style="position:absolute;top:29300;left:334"><nobr>بٖسفّ‏ه٘ز‏تصيقخٍ</nobr></div> <div style="position:absolute;top:29300;left:324"><nobr>،‏</nobr></div> <div style="position:absolute;top:29300;left:130"><nobr>‏ثادبَغّاٗ‏ذٝذش‏شبَنّا‏د٘خٗ‏عٍ</nobr></div> <div style="position:absolute;top:29321;left:700"><nobr>ةدٗذىا‏ذٞيخ‏ٚف</nobr></div> <div style="position:absolute;top:29321;left:689"><nobr>،‏</nobr></div> <div style="position:absolute;top:29321;left:654"><nobr>ٓ٘شحٗ</nobr></div> <div style="position:absolute;top:29321;left:649"><nobr>‏</nobr></div> <div style="position:absolute;top:29321;left:630"><nobr>ةبْق</nobr></div> <div style="position:absolute;top:29321;left:625"><nobr>‏</nobr></div> <div style="position:absolute;top:29321;left:568"><nobr>ُبعخزلإا</nobr></div> <div style="position:absolute;top:29321;left:556"><nobr>،‏</nobr></div> <div style="position:absolute;top:29321;left:425"><nobr>ٓ٘شٍ‏ثبصٍَ‏د٘خٗ‏ٗ</nobr></div> <div style="position:absolute;top:29321;left:413"><nobr>،‏</nobr></div> <div style="position:absolute;top:29321;left:381"><nobr>ومآحٗ</nobr></div> <div style="position:absolute;top:29321;left:375"><nobr>‏</nobr></div> <div style="position:absolute;top:29321;left:286"><nobr>ذٞيدىا‏شلاسّا‏ٗ</nobr></div> <div style="position:absolute;top:29321;left:274"><nobr>،‏</nobr></div> <div style="position:absolute;top:29321;left:193"><nobr>ثبّسد‏د٘خٗٗ</nobr></div> <div style="position:absolute;top:29321;left:188"><nobr>‏</nobr></div> <div style="position:absolute;top:29321;left:156"><nobr>تَعبّ</nobr></div> <div style="position:absolute;top:29321;left:135"><nobr>‏عٍ</nobr></div> <div style="position:absolute;top:29321;left:130"><nobr>‏</nobr></div> <div style="position:absolute;top:29342;left:669"><nobr>بَٞف‏ثبخبفخّا‏سٖ٘ظ</nobr></div> <div style="position:absolute;top:29342;left:663"><nobr>‏</nobr></div> <div style="position:absolute;top:29342;left:638"><nobr>بْٖٞب</nobr></div> <div style="position:absolute;top:29342;left:627"><nobr>‏.</nobr></div> <div style="position:absolute;top:29342;left:597"><nobr>شٖظأ</nobr></div> <div style="position:absolute;top:29342;left:591"><nobr>‏</nobr></div> <div style="position:absolute;top:29342;left:475"><nobr>تٝ٘ثّلأا‏ُاذٝذىا‏ٌسخ</nobr></div> <div style="position:absolute;top:29342;left:457"><nobr>‏غح</nobr></div> <div style="position:absolute;top:29342;left:428"><nobr>ثاشٞ</nobr></div> <div style="position:absolute;top:29342;left:421"><nobr>‏</nobr></div> <div style="position:absolute;top:29342;left:384"><nobr>،تٞينش</nobr></div> <div style="position:absolute;top:29342;left:378"><nobr>‏</nobr></div> <div style="position:absolute;top:29342;left:362"><nobr>بَب</nobr></div> <div style="position:absolute;top:29342;left:341"><nobr>‏ٜف</nobr></div> <div style="position:absolute;top:29342;left:335"><nobr>‏</nobr></div> <div style="position:absolute;top:29342;left:314"><nobr>لىر</nobr></div> <div style="position:absolute;top:29342;left:308"><nobr>‏</nobr></div> <div style="position:absolute;top:29342;left:281"><nobr>ًس٘ح</nobr></div> <div style="position:absolute;top:29342;left:275"><nobr>‏</nobr></div> <div style="position:absolute;top:29342;left:242"><nobr>ٌسدىا</nobr></div> <div style="position:absolute;top:29342;left:236"><nobr>‏</nobr></div> <div style="position:absolute;top:29342;left:190"><nobr>،وٍبنىبب</nobr></div> <div style="position:absolute;top:29342;left:176"><nobr>‏ٗ</nobr></div> <div style="position:absolute;top:29342;left:130"><nobr>‏شبَنّا</nobr></div> <div style="position:absolute;top:29363;left:664"><nobr>ْٚطبىا‏صََيى‏ذٝذش</nobr></div> <div style="position:absolute;top:29363;left:651"><nobr>،‏</nobr></div> <div style="position:absolute;top:29363;left:636"><nobr>ٗ‏</nobr></div> <div style="position:absolute;top:29363;left:570"><nobr>ذٞيخ‏سٖ٘ظ</nobr></div> <div style="position:absolute;top:29363;left:563"><nobr>‏</nobr></div> <div style="position:absolute;top:29363;left:530"><nobr>س٘قٍْ</nobr></div> <div style="position:absolute;top:29363;left:508"><nobr>‏عٍ</nobr></div> <div style="position:absolute;top:29363;left:500"><nobr>‏</nobr></div> <div style="position:absolute;top:29363;left:467"><nobr>دَٝ٘ح</nobr></div> <div style="position:absolute;top:29363;left:450"><nobr>ثب</nobr></div> <div style="position:absolute;top:29363;left:437"><nobr>،‏</nobr></div> <div style="position:absolute;top:29363;left:392"><nobr>،ثبٞغٗ</nobr></div> <div style="position:absolute;top:29363;left:376"><nobr>‏ٗ</nobr></div> <div style="position:absolute;top:29363;left:327"><nobr>ثادبَغّا</nobr></div> <div style="position:absolute;top:29363;left:314"><nobr>،‏</nobr></div> <div style="position:absolute;top:29363;left:263"><nobr>،ذٞعبدحٗ</nobr></div> <div style="position:absolute;top:29363;left:255"><nobr>‏</nobr></div> <div style="position:absolute;top:29363;left:217"><nobr>،ومآحٗ</nobr></div> <div style="position:absolute;top:29363;left:194"><nobr>‏ٗ‏</nobr></div> <div style="position:absolute;top:29363;left:156"><nobr>شلاسّا</nobr></div> <div style="position:absolute;top:29363;left:143"><nobr>،‏</nobr></div> <div style="position:absolute;top:29363;left:130"><nobr>ٗ‏</nobr></div> <div style="position:absolute;top:29383;left:743"><nobr>ثبعبقف</nobr></div> <div style="position:absolute;top:29383;left:725"><nobr>،‏ٗ</nobr></div> <div style="position:absolute;top:29383;left:720"><nobr>‏</nobr></div> <div style="position:absolute;top:29383;left:662"><nobr>د٘خٗ‏ًذع</nobr></div> <div style="position:absolute;top:29383;left:657"><nobr>‏</nobr></div> <div style="position:absolute;top:29383;left:622"><nobr>كا٘شأ</nobr></div> <div style="position:absolute;top:29383;left:612"><nobr>‏.</nobr></div> <div style="position:absolute;top:29383;left:585"><nobr>ِنَٝ</nobr></div> <div style="position:absolute;top:29383;left:566"><nobr>‏ُأ</nobr></div> <div style="position:absolute;top:29383;left:562"><nobr>‏</nobr></div> <div style="position:absolute;top:29383;left:529"><nobr>ػبحشٝ</nobr></div> <div style="position:absolute;top:29383;left:524"><nobr>‏</nobr></div> <div style="position:absolute;top:29383;left:497"><nobr>ثٍ٘</nobr></div> <div style="position:absolute;top:29383;left:492"><nobr>‏</nobr></div> <div style="position:absolute;top:29383;left:462"><nobr>ُاذٝد</nobr></div> <div style="position:absolute;top:29383;left:457"><nobr>‏</nobr></div> <div style="position:absolute;top:29382;left:340"><nobr>ّٚ٘سّبٍ‏بٍ٘س٘خسٞش</nobr></div> <div style="position:absolute;top:29383;left:335"><nobr>‏</nobr></div> <div style="position:absolute;top:29383;left:304"><nobr>ٌخبْىا</nobr></div> <div style="position:absolute;top:29383;left:299"><nobr>‏</nobr></div> <div style="position:absolute;top:29383;left:280"><nobr>ِع</nobr></div> <div style="position:absolute;top:29383;left:275"><nobr>‏</nobr></div> <div style="position:absolute;top:29383;left:214"><nobr>ه‏بٖظشعح</nobr></div> <div style="position:absolute;top:29383;left:209"><nobr>‏</nobr></div> <div style="position:absolute;top:29387;left:175"><nobr>HSE</nobr></div> <div style="position:absolute;top:29383;left:170"><nobr>‏</nobr></div> <div style="position:absolute;top:29383;left:135"><nobr>ٓشٞثؤخب</nobr></div> <div style="position:absolute;top:29383;left:130"><nobr>‏</nobr></div> <div style="position:absolute;top:29404;left:760"><nobr>ٚيع</nobr></div> <div style="position:absolute;top:29404;left:752"><nobr>‏</nobr></div> <div style="position:absolute;top:29404;left:722"><nobr>رطس</nobr></div> <div style="position:absolute;top:29404;left:715"><nobr>‏</nobr></div> <div style="position:absolute;top:29404;left:698"><nobr>دىا</nobr></div> <div style="position:absolute;top:29404;left:614"><nobr>بٖى‏فيغَىا‏ذٞي</nobr></div> <div style="position:absolute;top:29404;left:591"><nobr>‏ٗ‏</nobr></div> <div style="position:absolute;top:29404;left:558"><nobr>‏ٙزىا</nobr></div> <div style="position:absolute;top:29404;left:540"><nobr>ذق‏</nobr></div> <div style="position:absolute;top:29404;left:511"><nobr>ُ٘نٝ</nobr></div> <div style="position:absolute;top:29404;left:504"><nobr>‏</nobr></div> <div style="position:absolute;top:29404;left:464"><nobr>بًطبحشٍ</nobr></div> <div style="position:absolute;top:29404;left:457"><nobr>‏</nobr></div> <div style="position:absolute;top:29404;left:421"><nobr>د٘خ٘ب</nobr></div> <div style="position:absolute;top:29404;left:413"><nobr>‏</nobr></div> <div style="position:absolute;top:29404;left:389"><nobr>داٍ٘</nobr></div> <div style="position:absolute;top:29404;left:381"><nobr>‏</nobr></div> <div style="position:absolute;top:29404;left:338"><nobr>تٞئبَٞٞم</nobr></div> <div style="position:absolute;top:29404;left:331"><nobr>‏</nobr></div> <div style="position:absolute;top:29404;left:302"><nobr>تٞحببّ</nobr></div> <div style="position:absolute;top:29404;left:295"><nobr>‏</nobr></div> <div style="position:absolute;top:29404;left:259"><nobr>تفيخخٍ</nobr></div> <div style="position:absolute;top:29404;left:251"><nobr>‏</nobr></div> <div style="position:absolute;top:29404;left:213"><nobr>ةدبعٍ</nobr></div> <div style="position:absolute;top:29404;left:206"><nobr>‏</nobr></div> <div style="position:absolute;top:29404;left:163"><nobr>ةذسملأى</nobr></div> <div style="position:absolute;top:29404;left:156"><nobr>‏</nobr></div> <div style="position:absolute;top:29404;left:135"><nobr>وثٍ</nobr></div> <div style="position:absolute;top:29404;left:130"><nobr>‏</nobr></div> <div style="position:absolute;top:29425;left:733"><nobr>ثاذٝ٘يقىا</nobr></div> <div style="position:absolute;top:29425;left:728"><nobr>‏</nobr></div> <div style="position:absolute;top:29425;left:663"><nobr>ِّٞ٘ببصىاٗ</nobr></div> <div style="position:absolute;top:29425;left:658"><nobr>‏</nobr></div> <div style="position:absolute;top:29425;left:599"><nobr>ثلاْ٘ٞفىاٗ</nobr></div> <div style="position:absolute;top:29425;left:594"><nobr>‏</nobr></div> <div style="position:absolute;top:29425;left:511"><nobr>ثاذّٝ٘٘فلافىاٗ</nobr></div> <div style="position:absolute;top:29425;left:507"><nobr>‏</nobr></div> <div style="position:absolute;top:29425;left:491"><nobr>ٜف</nobr></div> <div style="position:absolute;top:29425;left:486"><nobr>‏</nobr></div> <div style="position:absolute;top:29425;left:468"><nobr>ازٕ</nobr></div> <div style="position:absolute;top:29425;left:463"><nobr>‏</nobr></div> <div style="position:absolute;top:29425;left:402"><nobr>صيخخسَىا</nobr></div> <div style="position:absolute;top:29425;left:392"><nobr>‏.</nobr></div> <div style="position:absolute;top:29425;left:377"><nobr>ٜف</nobr></div> <div style="position:absolute;top:29425;left:372"><nobr>‏</nobr></div> <div style="position:absolute;top:29425;left:335"><nobr>،ًبخخىا</nobr></div> <div style="position:absolute;top:29425;left:330"><nobr>‏</nobr></div> <div style="position:absolute;top:29428;left:296"><nobr>HSE</nobr></div> <div style="position:absolute;top:29425;left:275"><nobr>‏ٔى‏</nobr></div> <div style="position:absolute;top:29425;left:250"><nobr>شٞثؤح</nobr></div> <div style="position:absolute;top:29425;left:245"><nobr>‏</nobr></div> <div style="position:absolute;top:29425;left:219"><nobr>هبعف</nobr></div> <div style="position:absolute;top:29425;left:215"><nobr>‏</nobr></div> <div style="position:absolute;top:29425;left:193"><nobr>ذظ</nobr></div> <div style="position:absolute;top:29425;left:188"><nobr>‏</nobr></div> <div style="position:absolute;top:29425;left:135"><nobr>بٞسسبٖيبىا</nobr></div> <div style="position:absolute;top:29425;left:131"><nobr>‏</nobr></div> <div style="position:absolute;top:29446;left:748"><nobr>ِنَٝٗ</nobr></div> <div style="position:absolute;top:29446;left:743"><nobr>‏</nobr></div> <div style="position:absolute;top:29446;left:703"><nobr>ٓسببخعا</nobr></div> <div style="position:absolute;top:29446;left:699"><nobr>‏</nobr></div> <div style="position:absolute;top:29446;left:674"><nobr>ءاٗد</nobr></div> <div style="position:absolute;top:29446;left:669"><nobr>‏</nobr></div> <div style="position:absolute;top:29446;left:633"><nobr>ذعبسٍ</nobr></div> <div style="position:absolute;top:29446;left:617"><nobr>‏ـىي</nobr></div> <div style="position:absolute;top:29446;left:531"><nobr>ٜف‏وخّا٘نٝصاشب</nobr></div> <div style="position:absolute;top:29446;left:526"><nobr>‏</nobr></div> <div style="position:absolute;top:29446;left:491"><nobr>ٙٗذع</nobr></div> <div style="position:absolute;top:29446;left:487"><nobr>‏</nobr></div> <div style="position:absolute;top:29445;left:411"><nobr>ٍ‏بٍ٘س٘خسٞش</nobr></div> <div style="position:absolute;top:29445;left:370"><nobr>ّٚ٘سّب</nobr></div> <div style="position:absolute;top:29446;left:366"><nobr>.</nobr></div> </span></font>  </body> </html>