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Antibilharzial agents from marine sponge extracts from Gulf of Aqaba, Red Sea, Egypt

Bilharziasis is a parasitic disease caused by platyhelminthes, affecting millions worldwide. Marine invertebrates generally and marine sponges especially are promising organisms for the synthesis of novel bioactive compounds. There is an urgent need to investigate and develop a new and potential antibilharzial agent instead of using synthetic drugs. The extracts of two marine sponges: Negombata magnifica (Nm) and Callyspongia siphonella (Cs) collected from Gulf of Aqaba have been investigated for their effect as anthelmintic agents. Extracts from both types of sponges were obtained by using CH2Cl2, C4H8O2 and CHCl3 solvent. Mice were divided into 6 groups; (G1-G3) include infected mice with cercaria and administrated orally for 2 days a dose of one-tenth of LC50 of each extract from Nm (7.85, 11.25 and 10, mg/kg body weight/mouse, respectively); (G4-G7) administrated each extract from Cs (12.32, 13.11 and 14.25mg/kg body weight/mouse, respectively). G7 includes infected mice treated with 200 mg/kg body weight praziquantel for 2 days; G8 is infected mice and not treated (control group). The effects of each extract on the worm recovery and total egg count were determined. Oral administration of extract to infected mice (G1, G2 and G3) at 9 weeks post treated (WPT), induced a highly significant reduction, in the mean numbers of male and female worms. The males being more affected than females at 9 WPT. Also, treated mice in (G1), (G2) and (G3) showed a significant reduction in the mean number of female worms compared to the infected untreated mice group (G8). The females being more affected than males at 8 WPT. Only (G1), (G2) and (G3), as well as (G7) produced significant decline in the tissues liver and intestine egg counts at 8 WPT. In conclusion the current data indicated that the investigated sponge extracts can be applied as potential agent to treat bilharziasis. Also, the results provide a basis for exploring extracts from marine sponge as sources for new bioactive agents.


INTRODUCTION
Bilharziasis is one of the most common diseases in the world, which caused by Platyhelminthes called Schistosoma (WHO, 2019) which is endemic in Africa and the middle east countries mainly Egypt, causing acute and chronic clinical pathogenicity to man (Saad et al.,2019;Bonnefond et al., 2019).
Its deaths was difficult to estimate due to hidden pathology as hepatic cancer and colorectal cancer (Osada et al.,2005;Neghina et al., 2009;WHO,2019). There are more than 240 million people infected and around 800 million are at risk to infected with this helminthes (Butrous, 2019;WHO, 2018WHO, , 2019. The strategy for controlling the Bilharziasis, only just one Ibraheem M. M. Gobaara drug is a praziquantel (Doenhoff et al., 2008;Riad et al., 2009). However, after many years of praziquantel usage, a decreased susceptibility to the drug and the emergence of drug-resistant strains was reported (Doenhoff et al., 2008;Riad et al., 2009;Campelo,et al.,2018). Besides, praziquantel has poor efficacy against juvenile forms, also genotoxicity and mutagenic effect (Vale et al, 2017). It is necessary to invest a novel biological pathways for development of new safe treatment (Sadref-ozalayi et al.,2018;Abu Almaaty et al., 2020) to circumvent challenges linked to drug resistant parasites and better than the risky praziquantel (Stelma et al., 1995;Keiser,2010;Zhang et al., 2016;Abou El Dahab et al.,2019).
The marine environment represents a rich resource of novel chemical defenses against various types of parasites and/or competitors in the habitat space (Gomes et al.,2016;Herath et al.,2019;Abu Almaaty et al.,2020). Marine animals in general and marine invertebrates in particular are promising organisms for synthesis of novel bioactive compounds (Sepcic et al., 1997;Van Soest et al.,2008). A lot of recent studies suggested that some bioactive compounds isolated from marine organisms are shown to exhibit antimicrobial, antifungal, antiinflammatory, anticancer, antiparasites and other pharmacological activities (Herath et al.,2019;Rady and Bashar 2020). Bioactive compounds obtained from marine fauna, 70% of which comes from sponges (Purushottama, et al., 2009;Mehbub et al., 2014). Although the marine ecosystem features a rich biodiversity, most of them are not yet been explored (Jiménez, 2018;Herath et al., 2019).Therefore, there's a desire to explore and develop a new anthelmintic candidates to bypass the widespread drug resistance problem that exists in populations of parasites around the world (Kaplan, andVidyashankar 2012 andHerath et al., 2019). The present work aims to investigate the effect of different extracts from two sponges; Negombata magnifica (Nm) and Callyspongia siphonella (Cs) from Gulf of Aqaba, Red Sea, Egypt as potential antibilharzial agents instead of using synthetic drugs to treat bilharzias.

Fecal samples:
These were collected from all mice and examined after 4 weeks post-infection, by light microscope for S. mansoni eggs. Mice were sacrificed at the 7 th , 8 th and the 9 th weeks post infection.

Worm burden:
Worms were recovered from tissues of control and treated mice by Wang et al. (2004) methodology. Immediately, after dissection of the animals, the worms were counted under a dissecting microscope and classified into males, females and copulated, each tissue of worms were added to glass watch in doses the same as used in vivo to show whether they was affected by endogenous factors of the mice which may decrease their effectiveness. Then the worms were compressed between two clean glass plates until the parenchyma was evenly strewn into a transparent layer (Kloetzel, 1967).

Egg load count:
Eggs were count from stool after cercarial infection to determine the presence of S. mansoni eggs. Liver and intestine were removed and located in a petri-dish after the mice scarified (Lago et al., 2019). Eggs were count from tissues under a microscope using 10X magnification. The egg count was multiplied after Smithers and Terry (1965).

Percentage of egg developmental stages (Oogram pattern):
These were examined in three samples/mouse (Tendler et al., 1986). Each piece was compressed between two clean glass slides and studied under a microscope (Mati and Melo, 2013). The mean of each stage/animal was obtained (Helmy et al, 2009).

Statistical analysis of data:
Results were expressed as means ± standard error of the means (SE). Differences between groups were analyzed by using one way analysis of variance (ANOVA).The mean number of worms and eggs recovered from the different groups were subjected to Student's t-test using Microsoft office 16 to determine their statistical significance in comparison with the control groups. The data were considered significant if P <0.05.

Fig. 1: Lethal dose (LD 50 ) values for infected mice after exposure time (one day) to Nm
and Cs sponge extractions.
In the current study, the results obtained showed that treatment of the infected mice by orally administrated extracts induced a highly significant reduction in the mean numbers of male worms (94.8%), (90.3%), (88.4%) and (95.3%) in (G 1 ), (G 2 ) and (G 3 ) as well as Praziquantel (G 7 ), respectively after 9 weeks post treatment (WPT). Also, the mean numbers of female worms showed a highly significant reduction, (91.6%) (90.2%), (87.5%) and (93.1%) in the same groups, respectively compared to the infected untreated mice group (G 8 ). Also, it was obvious that the reduction number of male is higher than that of female worms at 9 WPT. Moreover, sponge extractions of (G 1 ) , (G 3 ) and (G 7 ) caused a significant reduction in the mean numbers of coupled worms (75.7%), (70.4%) and (78.7%), respectively compared to the infected untreated mice group (G 8 ) (Table 1). Administration dose of (G 4 ), (G 5 ) and (G 6 ) to infected mice at 9 WPT, caused a significant reduction in the mean numbers of male worms (78.06%), (74.1%) and (75.5%), respectively and females (79.0%), (70.8%) and (74.5%), respectively neither no coupled worms burden compared to the infected untreated group (Table 1 and Fig. 2). Table 1. The mean number of worms recovered from tissues after treatment with groups and scarified 7, 8 and 9 weeks post treat.

Fig. 2. Reduction rate of worms recovered from tissues of infected mice and treated groups post scarified at 9 weeks
Oral administration of infected mice to Nm-CH 2 Cl 2 extract (G 1 ) and Praziquantel (G 7 ) at 8 WPT, showed a significant reduction efficiency (79.7%) and (81%), respectively in the mean number of male worms burden compared to the infected untreated mice group (G8).
No significant reduction in the mean numbers of separate male and female or couple worms induced in treated infected mice of (G 4 ), (G 5 ) and (G 6 ) at 8 WPT compared to the infected control group (G 8 ) (Table1 and Fig. 3). However, there was a reduction in number of male and female worms with females being more affected than males at 8 WPT.  The results obtained showed no significant reductions in mean number of worms burden infected mice 7 WPT in groups (G 1-G 6 ), as well as (G 7 ) compared with the Non-treated infected mice group (G 8 ) (Table1 and Fig. 4). However, females being more affected than males at 7 WPT. Treatment of infected mice by the investigated sponge extractions as well as by Praziquantel resulted in no significant reduction in the number of eggs in the fecal of mice in (G 1-G 6 ) and (G 7 ) compared with the (G 8 ) ( Table 2).
Also sponge extractions from G 1 to G 6 resulted in no significant reduction in the number of eggs in the tissues liver and intestine of the treated group at 7 WPT compared with the non-treated infected group (G 8 ). On the other hand, only (G 7 ) produced significant reduction (70 %) in egg count in the tissue liver and without significant in the intestine (66 %) at 7 WPT compared with the non-treated infected group (G 8 ) ( Table 2).

Oogram recorded:
The reduction rate of each stage changes in Oogram patterns in the liver and intestine were given Table (3) and Figures (5 & 6). In the liver tissues, the Nm-extractions treated group (G 1 ), (G 2 ), and (G 3 ) showed a significant in the mean number of dead eggs, immature and mature in the liver tissues at 9 WPT. The other groups (G4), (G5) and (G6) had a non-significant correlation. The treated G7 had recorded a highly significant in the mean number of dead eggs immature and mature compared with the non-treated infected group (G8). Oogram pattern in liver showed significant change in dead eggs higher than in intestine (Table 3 and Fig. 6).  .  Table 3. The mean number of developing egg, and worm stages recovered from tissues of treated mice in different groups after 9 weeks post treatment compared with untreated group. Antibilharzial agents from marine sponge extracts from Gulf of Aqaba, Red Sea, Egypt DISCUSSION Bilharziasis has a serious impacts on public health (deBrito et al., 2017;Lago et al., 2018) and there is one obtainable drug (Praziquantel, PZQ) used to treat it. PZQ has low efficacy against schistosomula and juveniles (Ammar et al., 2020) and immature stages (Doenhoff et al., 2008). However, it is efficient against all Schistosoma spp. with the recommended dose and its efficacy is low in heavily infective individuals (Raso et al., 2004;Utzinger et al., 2003). Therefore, efforts are dedicated to developing different approaches to treat bilharziasis alongside praziquantel (Silva et al., 2017;Lago et al., 2018). The hard part of new drug discovery is to seek out a substance that could expeditiously wreck the parasite whilst no longer harming the host (de-Moraes et al., 2012;Musili et al., 2015). The emergence of drug-resistant bilharzia strains has brought great attention toward natural bioactive compounds. In a recent publication, Barbosa deCastro et al., (2013) compiled various therapeutic from naturalist sources, which include saponins, flavonoids, alkaloids, phenolic compounds, and terpenoids. Other studies have been done to assess natural substances of plant origin as antibilharzial agents like Neem (Taher, et al., 2016), Curcuma longa (Rizk et al., 2000), garlic (Riad et al., 2009), andginger (Al-Sharkawi et al., 2007;Mostafa et al., 2011).
With regard to marine sources, some compounds have shown unique biological properties, in part due to their singular chemical identity, and are currently employed as new therapeutic agents, either unmodified or as prototypes for the designing and synthesis of new drugs (Eustáquio et al., 2011;Cherigo et al., 2015). The present study represents an initial trial to use the extraction of marine sponge as associate antibilharzial agent.
Researchers have demonstrated that marine organisms are a rich source of structurally bioactive compounds and novel and biologically active metabolites (Blunt et al., 2005;Ibrahim et al., 2017;El-Damhougy et al., 2017).
The effectiveness of Negombata magnifica (Nm) and Callyspongia siphonella (Cs) were tested in vivo in Schistosoma mansoni infected mice at various dose/day, on various strategies such as killing the worms, and also worm egg-laying inhibitor starting in the fourth week post infection and ended by ninth week post treated. The results showed a significant reduction in worm burden, liver and intestinal egg load, with a significant increase in the percentage of dead eggs in the oogram pattern. These results are in agreement with previous studies that showed a significant reduction of both the worm burden and egg count in when treated with chemical drugs as PZQtreated settings compared to the untreated group (Tansatit et al., 2012;Tekwu et al., 2017;Beshay et al., 2019). Both ethanolic and crude extracts of Negombata magnifica are extremely potential for anti bilharzial properties although ethanolic extract remains to require more information about its effects on host In the current study the sensitivity of (Nm) in male worms s. mansoni more sensitive than female as well as PZQ at 9 weeks post treatment this is agree with several previous studies have been reported of differences in drug sensitiveness between males and females of S. mansoni; male worms S. mansoni are often more sensitive than female worms in studies of praziquantel resistance (Pica-Mattoccia and Cioli 2004) and in studies that estimated bioactivity of ginger extract (Sanderson et al., 2002). But in contrast to our result, study demonstrated showed higher survival rates for males than for females, such as alkane thiosulfuric amino acids (de Oliveira et al., 2008). A previous study reported that the treatment with curcumin might, significantly, reduce the worm burden, but after three months of infection, compared to the results of untreated infected groups (El-Ansary et al., 2007), which may be due to used long duration of treatment Compared with the present study which used short duration treatment.
The present study showed that treatment of S. mansoni infected mice with sponge extracts induced significant reductions in the liver egg count, or the intestinal egg count. On the contrary to our results, a previous study reported that the treatment with curcumin might, significantly, reduce the egg count, compared to the results of untreated infected groups (El-Ansary et al.,2007).
The current result revealed that at four weeks post infection there are some worms still alive which agree with a previous investigation revealed that at the same time of infection and treated with PZQ, there were some male worms still alive (Melkus, 2020;Yang, 2009.).
In the current result, we reported the reduction of the number of eggs per gram of the S. mansoni tissue, beside, the abnormal development of stages of developing egg (oogram pattern) in the intestinal wall of the treated mice groups this is an agreement with Aly, (2017). the present study used treatment protocol of sponge with soluble of methylene chloride, ethyl acetate and chloroform, In another study, a treatment protocol with methanol extract and soluble glycoprotein fraction of Allium sativum (A. sativum) was applied to target the inhibition of ova production in the hosts exposed to infection by which the further damages of host tissues and organs have been avoided (Kamel, and El-Shinnawy, 2015). Another study used water as extract of Zingiber officinale, Piper nigrum, and Coriandrum sativum was chosen in order to water is a safe, nontoxic widespread dissolvent, and avoid the high toxicity of organic dissolvent (such as methanol, acetone, chloroform, and dichloromethane) to living cells (Kinuthia et al., 2015).
Finally, we concluded that sponge extracts is a remarkable non-toxic extraction with many medical properties, and efficacy as an anti-schistosomal drug is much as it might improve the pathogenic changes induced in S. mansoni infected mice, and induced regimens in the treatment of the S. mansoni infections.