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Antischistosomal activity from Brazilian marine algae

Abstract

Schistosomiasis may be caused by six different species of the genus Schistosoma. Current treatment is based only on two drugs: oxamniquine, which is only effective against the Schistosoma mansoni species, and praziquantel, which is ineffective against young parasites. Therefore, research on new drugs and their targets for the treatment of this disease is urgently needed. In the present work, the efficacies of several seaweeds extracts against S. mansoni were tested. Worm couples were incubated with different concentration of seaweed extracts for 120 h and monitored after the first 2 h and then every 24 h to evaluate death, mobility reduction and couple detachment. The extracts of 13 different seaweed species were tested in a first trial and the active extracts were further evaluated in lower concentrations. The extracts of Gracilaria ornata and species belonging to the genera Dictyota and Laurencia showed activity at relatively low concentrations. The active extracts were analyzed by LC–MS, and possible candidates are proposed.

Keywords
Antischistosomal; Seaweeds; Secondary metabolites; Schistosoma mansoni; Terpenes

Introduction

Schistosomiasis is the most serious form of parasitism by multicellular organisms and still remains on the list of the neglected diseases prioritized by the World Health Organization (WHO, 2015WHO, 2015. Neglected Tropical Disease, http://http://www.who.int/schistosomiasis/en/ (accessed March 2015).
http://http://www.who.int/schistosomiasi...
). The disease, also known as bilharzia or snail fever, is caused by an infection with blood flukes of the genus Schistosoma. It predominantly affects the poor population, representing one of the main public health problems in more than 70 developing countries. Among the Schistosoma species, Schistosoma mansoni is the most widely spread in Africa and Latin America. The infection occurs when the host's skin is penetrated by the cercaria, the infectious form of the parasite life cycle. Once inside the host they transform into schistosomula, mature and form couples in the venous system. The egg is responsible for parasite transmission and is also the main cause of the disease symptoms (Gryssels et al., 2006Gryssels, B., Polman, K., Clerinx, J., Kestens, L., 2006. Human schistosomiasis. Lancet 368, 1106-1118.).

The disease affects approximately 240 million people around the world, resulting in an annual mortality rate of 280 000 people. Disease treatment is based on two therapeutic drugs: praziquantel and oxamniquine (WHO, 2015WHO, 2015. Neglected Tropical Disease, http://http://www.who.int/schistosomiasis/en/ (accessed March 2015).
http://http://www.who.int/schistosomiasi...
). Praziquantel is ineffective against young parasites and larval-stage schistosomula, but is effective against one-month-old worms; while oxamniquine is only active against the S. mansoni species, but not against other members of the Schistosoma genus. Moreover, both drugs face the problem of resistance development in the parasites and side effects. Therefore, further studies on the targets and new drugs are desired.

Due to the ability to produce a wide range of chemicals with sophisticated structures, marine organisms are considered good candidates to provide new drugs with pharmaceutical activities, including chemicals to parasitic diseases (Torres et al., 2014Torres, F.A.E., Passalacqua, T.G., Velásquez, A.M.A., de Souza, R.A., Colepicolo, P., Graminha, M.A.S., 2014. New drugs with antiprotozoal activity from marine algae: a review. Rev. Bras. Farmacogn. 24, 265-276.). It is well established that marine and freshwater synthetize important metabolites with economic impact (Cardozo et al., 2006Cardozo, K.H.M., Carvalho, V.M., Pinto, E., Colepicolo, P., 2006. Fragmentation of mycosporine-like amino acids by hydrogen/deuterium exchange and electrospray ionisation tandem mass spectrometry. Rapid Commun. Mass Spectrom. 20, 253-258.; Gressler et al., 2011aGressler, V., Fujii, M.T., Martins, A.P., Colepicolo, P., Mancini-Filho, J., Pinto, E., 2011. Biochemical composition of two red seaweed species grown on the Brazilian coast. J. Sci. Food Agric. 91, 1687-1692.,bGressler, V., Stein, É.M., Dörr, F., Fujii, M.T., Colepicolo, P., Pinto, E., 2011. Sesquiterpenes from the essential oil of Laurencia dendroidea (Ceramiales, Rhodophyta): isolation, biological activities and distribution among seaweeds. Rev. Bras. Farmacogn. 21, 248-254.; Stein et al., 2011Stein, E.M., Colepicolo, P., Afonso, F.A.K., Fujii, M.T., 2011. Screening for antifungal activities of extracts of the Brazilian seaweed genus Laurencia (Ceramiales, Rhodophyta). Rev. Bras. Farmacogn. 21, 290-295.; Andreguetti et al., 2013Andreguetti, D., Stein, E.M., Pereira, C.M.P., Pinto, E., Colepicolo, P., 2013. Antioxidant properties and UV absorbance pattern of mycosporine-like amino acids analogs synthesized in an environmentally friendly manner. J. Biochem. Mol. Toxicol. 27, 305-312.; Machado et al., 2014Machado, L., de Carvalho, L., Young, M., Zambotti-Villela, L., Colepicolo, P., Andreguetti, D., Yokoya, N., 2014. Comparative chemical analysis and antifungal activity of Ochtodes secundiramea (Rhodophyta) extracts obtained using different biomass processing methods. J. Appl. Phycol. 26, 2029-2035.; Simas-Rodrigues et al., 2015Simas-Rodrigues, C., Villela, H.D.M., Martins, A.P., Marques, L.G., Colepicolo, P., Tonon, A.P., 2015. Microalgae for economic applications: advantages and perspectives for bioethanol. J. Exp. Bot., 4097-4108.), such as low-weight hydrocarbons, tannins, fatty acids, acetogenins, saponins, phenolic compounds, lignans, alkaloids and terpenoids (Crews et al., 1978Crews, P., Kho-Wiseman, E., Montana, P., 1978. Halogenated alicyclic monoterpenes from the red algae Plocamium. J. Org. Chem. 43, 116-120.; González et al., 1982González, A.G., Darias, V., Estévez, E., 1982. Chemotherapeutic activity of polyhalogenated terpenes from Spanish algae. Planta Med. 44, 44-46.; Laus, 2001Laus, G., 2001. Biological activities of natural halogen compounds. In: Atta-ur, R. (Ed.), Studies in Natural Products Chemistry. Elsevier, pp. 757–809.; Li et al., 2011Li, Y.-X., Wijesekara, I., Li, Y., Kim, S.-K., 2011. Phlorotannins as bioactive agents from brown algae. Process Biochem. 46, 2219-2224.). Specifically, the last five classes cited have been described for their anthelmintic properties.

In this paper, we show the efficacies of 13 macroalgae extracts against S. mansoni. The active extracts from the Gracilaria,Dictyota and Laurencia genera were analyzed by LC-MS, and possible candidates are proposed.

Materials and methods

Seaweed material

The species Chondria littoralis Harvey - SP 428.154, Dictyota dichotoma (Hudson) J.V.Lamouroux SP 469.031, Dictyota menstrualis (Hoyt) Schnetter, Hörnig & Weber-Peukert - SP 469.032, Plocamium brasiliense (Greville) M.Howe & W.R.Taylor - SP 428.163 and Spyridia hypnoides (Bory de Saint-Vincent) Papenfuss - SP 428.171 were collected at Ubu beach, Anchieta - ES while the species of Laurencia catarinensis Cordeiro-Marino & Fujii - SP 400.209, Laurencia dendroidea J.Agardh - SP 400.198 (designated as (a)) were collected at Castelhanos beach, Guarapari - ES in October 2011. The above seaweed identification was performed by Dr. Mutue T. Fujii from Instituto de Botânica and MSc. Erika M. Stein. The vouchers of each species were placed in Herbário Maria Eneida P. Kauffman Fidalgo at Instituto de Botânica, São Paulo (SP). Padina gymnospora (Kützing) Sonder - 18.849, Gracilaria ornata Areschoug - 37.629, L. dendroidea J.Agardh - 18.852 (designated as (b)), Ochtodes secundiramea (Montagne) M.A.Howe - 18.851, Dictyota mertensii (Martius) Kützing - SP 469.156 and Pterocladiella capillacea (S.G.Gmelin) Santelices & Hommersand - 18.853 were collected at Baleia beach, Vitória - ES in July 2013, and the identification was performed by Dr. Levi P. Machado and a voucher of each specie was deposited in the herbarium VIES at the Universidade Federal do Espírito Santo.

Extraction procedure

Extracts were prepared by using different procedures. C. littoralis,D. dichotoma,D. menstrualis,P. brasiliense and S. hypnoides were freeze-dried, powdered and extracted with supercritical CO2, with the addition of 4% ethyl alcohol as a modifier solvent. The extractions were performed for 3 h at 45 °C and a pressure of 280 bar with a CO2 flow of 12 ml/min. The species L. catarinensis and L. dendroidea (b) were also freeze-dried and powdered, and different extracts were obtained by the crescent order of solvent polarity: first hexane, followed by chloroform and then methanol, three times each for 24 h. The species P. gymnospora,G. ornata,L. dendroidea (a), O. secundiramea,D. mertensii and P. capillacea were freshly extracted with a 2:1 mixture of methylene chloride: methanol, at a proportion of 10 ml/g of seaweed with stirring for one week at a temperature of 20 °C.

Schistosomicidal screening

The life cycle of S. mansoni (Samboon, 1907Samboon, L.L., 1907. Descriptions of some new species of animal parasites. Proc. Zoo.Soc. Lon. 77 (1), 282–283.) (Trematoda: Schistosomatidae) (BH strain - Belo Horizonte, MG, Brazil) was maintained in Biomphalaria glabrata (Say, 1818) (Gastropoda: Planorbidae) snails and Mesocricetus auratus (Waterhouse, 1839) (Mammalia: Cricetidae) hamsters. Female hamsters were infected by subcutaneous injection of 300 cercariae, and six weeks later, S. mansoni adult worms were recovered by perfusion of the rodent's portal and mesenteric system. The worms were washed in RPMI 1640 medium (Invitrogen), pH 7.5, supplemented with sodium bicarbonate (2000 μg/ml), penicillin (100 UI/ml), streptomycin (100 μg/ml), amphotericin B (0.25 μg/ml) and 10% fetal bovine serum (Gibco BRL). Adult worm pairs (male and female) were transferred to each well of a 24-well culture plate containing 1 ml of the medium. The seaweeds extracts were dissolved in DMSO (dimethyl sulfoxide, at 1.5%), diluted in 1 ml of RPMI medium and added to the cultured worms to achieve a final concentration of 500 μg/ml. The parasites were kept for 120 h and monitored after the first 2 h and then every 24 h under a light microscope to evaluate motor activity and the mortality rate. The worms were considered to be dead when no movement was observed. RPMI 1640 with 1.5% DMSO was used in the negative control group, and 4.8 μM (1.5 μg/ml) praziquantel (PZQ) was used in the positive control group. The experiments were carried out in five replicates and repeated at least two times. The active extracts in the assay were re-tested at a lower concentration of 100 μg/ml.

UFLC-MS analysis

The analysis were carried out using an UFLC system (Shimadzu, USA) consisting of a solvent delivery pump (Model LC-20AD) and auto sampler (SIL-20Aht) with a 100-μl loop, degasser (DGU 20A3r) and column oven (CTO-20A) followed by a Bruker ESI-microTOF II mass spectrometer. System operation was performed using otofControl and HyStar V.3.2, while data collection and analysis were performed using Compass DataAnalysis V.4.1. Gradient elution was performed on a Phenomenex® Luna 3μ C18(2) 100A column (150 mm × 2.0 mm) at 30 °C. Mobile phase A consisted of a aqueous solution of 20 mM NH4Ac and 0.1% formic acid adjusted to pH 6.4, and solution B consisted of acetonitrile with 0.1% formic acid. Separations were effected by a gradient elution program as follows: solution B was maintained at 0% from 0 to 2 min, followed by a linear change from 0% to 100% from 2 to 75 min, B was keep isocratic at 100% from 75 to 80 min. Solution B was changed to 0% at 80.1 min and was held constant until 90 min. The mobile phase flow rate was 0.2 ml/min, and the injection volume was 10 μl. Mass detection was performed scanning the m/z between 50 and 1100 in positive mode with the following parameters: capillary 4500 V; end plate offset −500 V; nebulizer 2.0 Bar; dry heater 180 °C; and dry gas at a flow of 8 l/min.

Results

The active extracts from the Gracilaria,Dictyota and Laurencia genera displayed high activity against S. mansoni in comparison with the other extracts. Table 1 summarizes the extraction methods and in vitro effects of all of positive tested seaweed extracts, which have been classified into two main groups, red algae (Rhodophyta) and brown algae (Heterokontophyta). As previously mentioned, the extracts displaying activity at 500 μg/ml exposure, were considered to be active and subjected to further testing at a lower concentration (100 μg/ml). The killing time (24, 72 and 120 h) was recorded and Table 1 shows the 100% mortality rate. The supercritical extracts of the red algae P. brasiliense,C. littoralis and S. hypnoides were active only at the higher concentration (500 μg/ml) tested. The macroalgae P. capillacea,O. secundiramae and L. dendroidea extracted with dichloromethane/methanol (2:1), L. dendroidea extracted with methanol, and L. catarinensis extracted with chloroform did not present anthelmintic activity (data not shown).

Table 1
List of seaweeds utilized for screening, extraction methods and antischistosomal activity of the extracts at 500 and 100 μg/ml.

The red macroalgae P. brasiliense,S. hypnoides and C. littoralis submitted to supercritical extraction were active only at the higher concentrations (Table 1). Interestingly that L. dendroidea and L. catarinensis submitted to hexane extraction display distinct results against schistosoma worms. L. catarinensis hexanic extracts kill the worms in 24 h and L. dendroidea, killed the worms in females in 24 h and males in 72 h. In addition, chloroformic extract of L. dendroidea was also effective against male after 120 h incubation. G. ornata submitted to the dichlorometane/methanol extraction displays anthelmintic activity (100 μg/ml) only against male worms in after 120 h.

As observed in Table 1, the brown algae, the three Dictyota species showed activity at the two concentrations tested. The supercritical extract of D. menstrualis kills very effectively both male and female after 24 h exposure. Additionally, D. dichotoma submitted to supercritical extraction kills the schistosoma worms after 72 h. Surprisingly, D. mertensii extracted with dichloromethane/methanol kills male after 72 h and female after 120 h incubation. P. gymnospora extracted with dichloromethane/methanol did not show anthelmintic activity (data not shown).

In order to compare the chemical composition of the active extracts, samples were submitted to chromatographic analysis applying a UFLC-MS system. Fig. 1 shows extract chromatograms generated from the total ion chromatogram (TIC) mode, where the signal peaks correspond to the sum of each mass detected.

Fig. 1
Chromatograms obtained for the active seaweed extracts against S. mansoni. The X-axis represents the retention time for each peak.

The chromatograms were manually analyzed to identify similar compounds present in different samples. The chromatograms were stacked to standardize the retention times, and the mass spectra of similar peaks were analyzed individually. It was assumed that the same chemical structure was present in samples when a similar mass spectrum was detected at a similar retention time for both extracts. Table 2 summarizes common m/z values obtained in different extract with similar retention times.

Table 2
Common [M+H]+ ion at m / z values related to each seaweed species analyzed by UFLC–MS in different retention times.

Among the m/z values detected in more than one extract, we found an [M+H]+ ion at m/z 391.283 in five extracts, with a retention time of 76.1 min, indicating a similar secondary metabolite in the extracts of D. mentrualis,D. dichotoma,L. catarinensis and L. dendroidea (hexanic and chloroformic). As expected, similar m/z values were derived from species belonging to the same genus, Laurencia, even among different species or with different extraction methods. However, this pattern did not hold for the Dictyota genus, where only one metabolite ([M+H]+ ion at m/z 391.283) was common between the species D. dichotoma and D. mentrualis. Comparing the three genera - Dictyota,Laurencia and Gracilaria - one common metabolite [M+H]+ ion at m/z 494.564 was detected at 80.9 min. The dichlorometane/methanol extract of D. mertensii was the only sample that shared no similar metabolites with the other species. A comparison of the extracts from D. mentrualis and hexanic extracts from both L. catarinensis and L. dendroidea - the best three candidates from the antischistosomal screening -identified one common metabolite with a [M+H]+ ion at m/z 181.123 at 36.1 min.

Discussion

Over the past few decades, research on natural sources has identified chemical compounds with important pharmacological roles in medicinal fields. 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 design and synthesis of new drugs (Eustáquio et al., 2011Eustáquio, A.S., Nam, S.-J., Penn, K., Lechner, A., Wilson, M.C., Fenical, W., Jensen, P.R., Moore, B.S., 2011. The discovery of salinosporamide K from the marine bacterium Salinispora pacifica by genome mining gives insight into pathway evolution. ChemBioChem 12, 61-64.; Cherigo et al., 2015Cherigo, L., Lopez, D., Martinez-Luis, S., 2015. Marine natural products as breast cancer resistance protein inhibitors. Mar. Drugs 13, 2010-2029.). Seaweeds from the phyla of Rhodophyta and Heterokontophyta have led to the discovery of a wide variety of secondary metabolites belonging to different chemical classes, such as terpenoids (monoterpenes, sesquiterpenes, diterpenes, triterpenes, etc.) and their variants (cyclic, acyclic, halogenated) (Paul et al., 1988Paul, V.J., Hay, M.E., Duffy, J.E., Fenical, W., Gustafson, K., 1988. Chemical defense in the seaweed Ochtodes secundiramea (Montagne) Howe (Rhodophyta): effects of its monoterpenoid components upon diverse coral-reef herbivores. J. Exp. Mar. Biol. Ecol. 114, 249-260.; La Barre et al., 2010La Barre, S., Potin, P., Leblanc, C., Delage, L., 2010. The halogenated metabolism of brown algae (Phaeophyta) its biological importance and its environmental significance. Marine Drugs 8, 988-1010.), alkaloids (Güven et al., 2010Güven, K.C., Percot, A., Sezik, E., 2010. Alkaloids in marine algae. Mar. Drugs 8, 269-284.), steroids (Francavilla et al., 2013Francavilla, M., Franchi, M., Monteleone, M., Caroppo, C., 2013. The red seaweed Gracilaria gracilis as a multi products source. Mar. Drugs 11, 3754-3776.), saponins (Jeeva et al., 2012Jeeva, S., Antonisamy, J.M., Domettila, C., Anantham, B., Mahesh, M., 2012. Preliminary phytochemical studies on some selected seaweeds from Gulf of Mannar India. Asian Pac. J. Trop. Biomed. 2, S30-S33.), lignans, and phenolic compounds (Keyrouz et al., 2011Keyrouz, R., Abasq, M.L., Bourvellec, C.L., Blanc, N., Audibert, L., ArGall, E., Hauchard, D., 2011. Total phenolic contents, radical scavenging and cyclic voltammetry of seaweeds from Brittany. Food Chem. 126, 831-836.).

In recent years, considerable efforts have been dedicated to develop alternative approaches to treat schistosomiasis. In a recent publication, Barbosa de Castro et al. (2013)Barbosa de Castro, C.C., Dias, M.M., Pessoa de Rezende, T., Magalhães, L.G., Da Silva Filho, A.A., 2013. Chapter 8 - Natural Products with Activity Against Schistosoma Species. In: Mahendra, R., Kateryna, K. (Eds.), Fighting Multidrug Resistance with Herbal Extracts, Essential Oils and their Components. Academic Press, San Diego, pp. 109–134. compiled several therapeutic candidates from natural sources, including alkaloids, flavonoids, saponins, phenolic compounds and terpenoids. Therefore, the presence of those chemical classes in seaweeds justifies the importance of this study.

In antischistosomal assays, the most significant results were obtained when the parasites were exposed to extracts obtained from seaweed that belonged mainly to the three genera, Dictyota,Gracilaria and Laurencia. A commonality among these three genera is the terpenoid compounds (Peres et al., 2012Peres, J.C., Carvalho, L.R., Gonçalez, E., Berian, L.O.S., Felicio, J.D., 2012. Evaluation of antifungal activity of seaweed extracts. Ciên. Agrotec. 36, 294-299.), which may play an important role against S. mansoni. Nevertheless, there remains a possibility that the antischistosomal activity can be mediated by another molecule.

The active extracts were analyzed chromatographically in search of a common metabolite among all of the seaweeds. From the three most active extracts, defined for the total mortality reached in a short period of time (24 h) - D. menstrualis,L. dendroidea and L. catarinensis - a common [M+H]+ ion was found at m/z 181.123, which may coincide with the molar weight of a sesquiterpene or even a conjugated monoterpene (Al-Massarani, 2014Al-Massarani, A.M., 2014. Phytochemical and biological properties of sesquiterpene constituents from the marine red seaweed Laurencia: a review. Nat. Prod. Chem. Res. 2, 147, http://dx.doi.org/10.4172/2329-6836.1000147
http://dx.doi.org/10.4172/2329-6836.1000...
). Among all of the active extracts, the most common peak separated at 76.1 min and showed a [M+H]+ ion at m/z 391.283, which is compatible with the molar weight of a sesterterpene or a conjugated diterpene (Hirano et al., 2001Hirano, A., Sugiyama, E., Kondo, H., Suda, H., Ogawa, H., Kojiri, K., 2001. Sesterterpene derivatives exhibiting antifungal activities. Google Patents.).

Overall, the most active extracts were obtained by non-polar extraction solvents, such as hexane, chloroform, dichloromethane or supercritical CO2, and the pattern of the mass spectra (data not showed) obtained for the extracts revealed an abundance of low weight molecules (i.e., 95% of all m/z are under 500 Da for D. menstrualis) in all of the active extracts. This leads to our hypothesis that terpenes and/or their variants, such as mono-, di-, tri-, sesqui- or sesterterpenes, may be a promising source of new molecules with activity against S. mansoni and represent therapeutic alternatives to treat the disease.

Acknowledgements

The authors thank FAPESP (2011/18564-2 and 2011/10155-6), CAPES and CNPq for their financial support.

References

  • Al-Massarani, A.M., 2014. Phytochemical and biological properties of sesquiterpene constituents from the marine red seaweed Laurencia: a review. Nat. Prod. Chem. Res. 2, 147, http://dx.doi.org/10.4172/2329-6836.1000147
    » http://dx.doi.org/10.4172/2329-6836.1000147
  • Andreguetti, D., Stein, E.M., Pereira, C.M.P., Pinto, E., Colepicolo, P., 2013. Antioxidant properties and UV absorbance pattern of mycosporine-like amino acids analogs synthesized in an environmentally friendly manner. J. Biochem. Mol. Toxicol. 27, 305-312.
  • Barbosa de Castro, C.C., Dias, M.M., Pessoa de Rezende, T., Magalhães, L.G., Da Silva Filho, A.A., 2013. Chapter 8 - Natural Products with Activity Against Schistosoma Species. In: Mahendra, R., Kateryna, K. (Eds.), Fighting Multidrug Resistance with Herbal Extracts, Essential Oils and their Components. Academic Press, San Diego, pp. 109–134.
  • Cardozo, K.H.M., Carvalho, V.M., Pinto, E., Colepicolo, P., 2006. Fragmentation of mycosporine-like amino acids by hydrogen/deuterium exchange and electrospray ionisation tandem mass spectrometry. Rapid Commun. Mass Spectrom. 20, 253-258.
  • Cherigo, L., Lopez, D., Martinez-Luis, S., 2015. Marine natural products as breast cancer resistance protein inhibitors. Mar. Drugs 13, 2010-2029.
  • Crews, P., Kho-Wiseman, E., Montana, P., 1978. Halogenated alicyclic monoterpenes from the red algae Plocamium J. Org. Chem. 43, 116-120.
  • Eustáquio, A.S., Nam, S.-J., Penn, K., Lechner, A., Wilson, M.C., Fenical, W., Jensen, P.R., Moore, B.S., 2011. The discovery of salinosporamide K from the marine bacterium Salinispora pacifica by genome mining gives insight into pathway evolution. ChemBioChem 12, 61-64.
  • Francavilla, M., Franchi, M., Monteleone, M., Caroppo, C., 2013. The red seaweed Gracilaria gracilis as a multi products source. Mar. Drugs 11, 3754-3776.
  • González, A.G., Darias, V., Estévez, E., 1982. Chemotherapeutic activity of polyhalogenated terpenes from Spanish algae. Planta Med. 44, 44-46.
  • Gressler, V., Fujii, M.T., Martins, A.P., Colepicolo, P., Mancini-Filho, J., Pinto, E., 2011. Biochemical composition of two red seaweed species grown on the Brazilian coast. J. Sci. Food Agric. 91, 1687-1692.
  • Gressler, V., Stein, É.M., Dörr, F., Fujii, M.T., Colepicolo, P., Pinto, E., 2011. Sesquiterpenes from the essential oil of Laurencia dendroidea (Ceramiales, Rhodophyta): isolation, biological activities and distribution among seaweeds. Rev. Bras. Farmacogn. 21, 248-254.
  • Gryssels, B., Polman, K., Clerinx, J., Kestens, L., 2006. Human schistosomiasis. Lancet 368, 1106-1118.
  • Güven, K.C., Percot, A., Sezik, E., 2010. Alkaloids in marine algae. Mar. Drugs 8, 269-284.
  • Hirano, A., Sugiyama, E., Kondo, H., Suda, H., Ogawa, H., Kojiri, K., 2001. Sesterterpene derivatives exhibiting antifungal activities. Google Patents.
  • Jeeva, S., Antonisamy, J.M., Domettila, C., Anantham, B., Mahesh, M., 2012. Preliminary phytochemical studies on some selected seaweeds from Gulf of Mannar India. Asian Pac. J. Trop. Biomed. 2, S30-S33.
  • Keyrouz, R., Abasq, M.L., Bourvellec, C.L., Blanc, N., Audibert, L., ArGall, E., Hauchard, D., 2011. Total phenolic contents, radical scavenging and cyclic voltammetry of seaweeds from Brittany. Food Chem. 126, 831-836.
  • La Barre, S., Potin, P., Leblanc, C., Delage, L., 2010. The halogenated metabolism of brown algae (Phaeophyta) its biological importance and its environmental significance. Marine Drugs 8, 988-1010.
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  • Peres, J.C., Carvalho, L.R., Gonçalez, E., Berian, L.O.S., Felicio, J.D., 2012. Evaluation of antifungal activity of seaweed extracts. Ciên. Agrotec. 36, 294-299.
  • Samboon, L.L., 1907. Descriptions of some new species of animal parasites. Proc. Zoo.Soc. Lon. 77 (1), 282–283.
  • Simas-Rodrigues, C., Villela, H.D.M., Martins, A.P., Marques, L.G., Colepicolo, P., Tonon, A.P., 2015. Microalgae for economic applications: advantages and perspectives for bioethanol. J. Exp. Bot., 4097-4108.
  • Stein, E.M., Colepicolo, P., Afonso, F.A.K., Fujii, M.T., 2011. Screening for antifungal activities of extracts of the Brazilian seaweed genus Laurencia (Ceramiales, Rhodophyta). Rev. Bras. Farmacogn. 21, 290-295.
  • Torres, F.A.E., Passalacqua, T.G., Velásquez, A.M.A., de Souza, R.A., Colepicolo, P., Graminha, M.A.S., 2014. New drugs with antiprotozoal activity from marine algae: a review. Rev. Bras. Farmacogn. 24, 265-276.
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    » http://http://www.who.int/schistosomiasis/en/

Publication Dates

  • Publication in this collection
    Dec 2015

History

  • Received
    22 Apr 2015
  • Accepted
    7 Sept 2015
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