Molluscicidal Activity of Compounds Isolated from Euphorbia conspicua N. E. Br

O latex de Euphorbia conspicua foi fracionado nas frações triterpênica e irritantes I e II. Da fração triterpênica foram isolados 15 compostos já conhecidos e um novo triterpeno denominado 3β-(E)-cinamoileuforbol. A fração irritante II forneceu o 20-O-acetil-3-O-angeloil-ingenol. A atividade moluscicida dos compostos eufol, 3β-acetoxieufa-8,24-dieno, 3β-(E)-cinamoileuforbol e 20-O-acetil-3-O-angeloil-ingenol foi avaliada. O 20-O-acetil-3-O-angeloil-ingenol apresentou uma LC100 de 1 mg mL, a qual foi equivalente ao moluscicida padrão niclosamida. Os compostos eufol, 3β-acetoxieufa-8,24-dieno e 3β-(E)-cinamoileuforbol apresentaram uma fraca atividade moluscicida. O 3β-(E)-cinamoileuforbol foi submetido a testes de mutagenicidade (teste de Ames com TA 98, 100 e 102) na presença e ausência de ativação metabólica (mistura S9). Foram também realizados os ensaios de citotoxicidade (teste MTT) e genotoxicidade (teste dos micronúcleos, CBMN) com e sem mistura S9, em células V79 de Hamster chinês. O 3β-(E)-cinamoileuforbol revelou-se fracamente citotóxico e sem atividade mutagênica ou genotóxica.


Introduction
Euphorbia conspicua N. E. Br. 1 (Euphorbiaceae) is a succulent tree endemic to Angola and is traditionally used as a treatment for dermatitis and leprosy wounds. 2 The genus Euphorbia is the largest in the spurge family, with more than 1000 species divided into many subgenera and sections.][5] Some Euphorbia species have been studied for molluscicidal properties against schistosomiasis or bilharzia, leading to the discovery of milliamines, the most potent molluscicides identified so far. 6chistosomiasis is a major source of morbidity and mortality in developing countries.Three schistosome species infect humans: Schistosoma mansoni, S. haematobium and S. japonicum.Because freshwater snails are intermediate hosts for these parasites, 7 the use of molluscicides is desirable in the integrated control of schistosomiasis. 7urrently, only niclosamide is widely used in control programs, and it is highly active at all stages of the snail life cycle and on schistosome larvae.Natural molluscicidal compounds isolated from a large number of plants have received much attention in hope that they might provide cheap, biodegradable and effective control agents in rural areas where schistosomiasis is endemic. 8,9s part of our ongoing study of the plants of Angola, 2,10,11 we observed that Euphorbia conspicua latex exhibits strong molluscicidal activity 2 and may be a potential source of bioactive compounds.This work intended to isolate and describe the constituents of the active fractions and to evaluate their effects.As a result, sixteen compounds were characterized, including a new natural product named 3β-(E)cinnamoyleuphorbol (3).Compounds 1-3 and 16 were evaluated for their molluscicidal activity.The cytotoxicity, mutagenic and genotoxicity activities of compound 3 were evaluated using the MTT test, Ames test and cytokinesisblock micronucleus assay (CBMN), respectively.
Compound 3 was obtained as a white amorphous solid, and its molecular formula C 40 H 58 O 2 was established by HREIMS, showing a molecular ion peak m/z of 570.4417 [M + ] (calc.570.4436) and 12 degrees of unsaturation.The IR spectrum revealed absorption bands for an ester (1720 and 1153 cm -1 ), a terminal methylene group (890 cm -1 ), a fully substituted double bond (1640 cm -1 ) and an aromatic ring (1580, 850, 820 and 679 cm -1 ).The 1 H NMR spectrum ( suggested the existence of an isopropyl group in the molecule; moreover, the multiplicity of the methine as a septet implied that the isopropyl group was bonded directly to a quaternary carbon.The 13 C NMR spectrum (Table 1) showed signals of two sp 2 carbons of a tetrasubstituted double bond (d C 133.6 and 134.0) along with signals of a terminal methylene group (d C 156.9 and 106.0) and a cinnamate moiety (d C 166.9, 144.3 and 118.9).The EI mass spectrum revealed fragment ion peaks at m/z 555 ([M] + -Me), 407 ([M] + -Me -HO 2 CCH=CHC 6 H 5 ), 297 (loss of C 9 H 17 and HO 2 CCH=CHC 6 H 5 ), 255 (297 -C 3 H 6 ) and 241 (255 -CH 2 ), which corroborated the structure as a D 8-9 tetracyclic triterpene bearing a cinnamoyl group at the C-3 position and a side chain containing nine carbon atoms, including isopropyl and C-24 methylene groups.
Compounds 1-3 and 16 were evaluated for molluscicidal activity against Biomphalaria glabrata, a vector of S. mansoni (Table 2).The molluscicidal activity of compound 16, with an LC 100 of 1 mg mL -1 , was equivalent to that of niclosamide, the synthetic compound used for the control of mollusks (LC 100 1.5 mg mL -1 ). 24Compound 16 presented a dose dependent and continuous effect on adult snails after 24 h of exposure, while it was completely inactive against the egg masses.In contrast, the triterpenic compounds displayed weak activity.Compounds 1 and 3 caused 20% mortality, while compound 2 cause 40% of mortality at a concentration of 100 mg mL -1 .Due to a shortage of sample materials, the other triterpenic compounds were not tested.
The survival values in V79 cells are given in Table 3, highlighting that only a slight decrease of survival was observed for 250 µg per well.Ames assay outcomes (Table 4) revealed compound 3 as a non-mutagenic agent on the strains tested at doses up to 250 µg per plate.The results obtained for the induction of micronuclei in Chinese hamster cells (V79 cells) at concentrations up to 100 µg mL -1 revealed no significant increase compared to the negative control in the absence or presence of S9 mix (Table 5).as mean values ± standard deviations (SD) (n = 2); in each experiment 1000 binuleated cells were analyzed for the presence of micronuclei; % of binucleated cells (% BN) was used as index of cell proliferation; mytomicin C and cyclophosphamide as positive controls, dose 0 as negative control.

Conclusions
E. conspicua, a succulent tree endemic to Angola, was previously evaluated for its molluscicidal activity, 2 but the latex chemical composition was not determined.The present study revealed that E. conspicua latex is composed mainly of triterpenes with euphane and cycloartane skeletons along with other triterpenes and ingenane diterpenes and contains a new compound with a tirucallane skeleton, 3β-(E)-cinnamoyleuphorbol (3).Four of the isolated compounds, 1-3 and 16, were evaluated for their molluscicidal activity.Compound 16 was found to be the most active, with an LC 100 value of 1 mg mL -1 , which is similar to that for milliamine L, the most powerful molluscicide of plant origin characterized thus far. 25Although compound 16 and milliamine L are structurally differing from each other at the position C-3, where milliamine L bears a dianthraniloyl peptide group and compound 16 an angeloyl group, molluscidal activity remains.At this point compound 16 seems to be the major responsible for E. conspicua molluscicidal activity but further investigation is needed.Compound 16 was found to be totally inactive against the egg masses of B. glabrata.Compound 3 was evaluated for its cytotoxicity, mutagenic activity and genotoxicity using the MTT test, the Ames test and the cytokinesis-block micronucleus assay (CBMN), respectively.No mutagenic or genotoxic activity was detected and little or no cytotoxicity was found concluding that this particular compound as no potential risk regarding their future use as bioactive compound.
The irritant properties of the latex can explain the etnopharmacological use against leprosy wounds and dermatitis in general, but more studies are needed to validate this idea.
Biological and chemical evaluation of other components of E. conspicua latex will require large amounts of latex and will be the subject of a future study.

O p t i c a l r o t a t i o n s w e r e o b t a i n e d w i t h a
Bellingham+Stanley Ltd ADP 220 polarimeter.HREIMS measurements were conducted on a VG Autospec M and recorded at 70 eV.The mass spectrum of 3 was obtained from a GC-MS (Hewlett-Packard 5989 A) spectrometer.The IR spectra were recorded in a Unicam Mattson 5000 FTIR.NMR spectra of 3 were recorded in a Bruker Avance II 600 MHz ( 1 H NMR) and 150.9 MHz ( 13 C NMR) spectrometer in CDCl 3 .Spectra of compounds 1-2 and 4-16 were recorded in a Bruker AC 250P 250 MHz ( 1 H NMR) and 62.9 MHz ( 13 C NMR) spectrometer in CDCl 3 .Chemical shifts are given in d ppm and are referenced to residual CHCl 3 , 7.26 ppm for the 1 H and 77.0 ppm for 13 C. Two-dimensional experiments were performed with standard Bruker software.Column chromatography was conducted on silica gel 60 (70-230 mesh, Merck, Darmstadt, Germany).

Plant material
Plant material was collected at Cacuaco, Luanda and was identified by Professor Esperança da Costa from Agostinho Neto University (Biology Department).A voucher specimen (No 4498) has been deposited at the Luanda Herbarium, Luanda, Angola.
MTT assay is a cell viability assay based on conversion of MTT dye by mitochondrial enzymes of viable cells into a formazan which can be spectrophotometrically measured.In this assay the absorbance is proportional to the number of viable cells. 31he MTT assay was conducted on V79 Chinese hamster cells.Approximately 10 4 cells were grown at 37 °C in a 5% CO 2 atmosphere for 24 h in 96-well plates in 200 µL of Ham's F-10 medium supplemented with 10% newborn calf serum and 1% penicillin/streptomycin solution.Different doses of the compound (25, 50 and 250 µg per well) were added, and the cells were incubated for 3 h.The medium was removed, and cells were incubated for 3 h with MTT (0.5 mg mL -1 ).The cells were washed carefully with PBS, and then 200 µL of DMSO was added to each well.The absorbance of the converted dye was measured at 595 nm in a Zenith 3100 microplate reader.Cell viability was assessed by comparing the absorbance values of treated cells with that of the control.Absorbance values presented by V79 cell cultures without the addition of compound 3, i.e. control cultures, correspond to 100% of cell viability.Three independent experiments were performed.In each independent experiment four replicate cultures were used.

Ames assay
Mutagenicity testing was conducted by the plate incorporation assay described by Maron and Ames 32 using Salmonella typhimurium strains TA 98, TA 100 and TA 102 in the presence or absence of S9 mix. 32At least two independent experiments were performed for each assay.Quercetin and 4-nitroquinoline-1-oxide were used as positive controls and dose 0 as negative control.

Cytokinesis-block micronucleus assay (CBMN)
Approximately 5 × 10 5 V79 Chinese hamster cells were cultured for 24 h in 25 cm 2 culture flasks and then exposed to compound 3 at concentrations of 20 and 100 µg mL -1 .Mitomycin C (2.5 µg mL -1 ) or cyclophosphamide (2.0 µg mL -1 ) was used as a positive control with and without S9 mix and dose 0 as negative control, respectively.24 h after the genotoxic treatment, the cells were washed with fresh culture medium, and cytochalasin-B (Cyt-B) was added to a final concentration of 4.5 µg mL -1 .The cells were incubated for additional 16 h, harvested by trypsinization, rinsed and submitted to a mild hypotonic treatment as described elsewhere. 33The centrifuged cells were placed onto dry slides, and smears were made.After air-drying, the slides were fixed with cold methanol for 30 min.One day later, the slides were stained with Giemsa (4% (v/v) in 0.01 mol L -1 sodium phosphate buffer, pH 6.8) for 10 min.For each experimental point, 1000 binucleate V79 cells (BN) with well-preserved cytoplasm were scored.Micronuclei were identified under a light microscope using a magnification of 1250 × according to the criteria proposed by Caria et al. 34 We evaluated MN/BN (data not showed), which represents the average number of micronuclei per binucleated cell, and the frequency of micronucleated binucleated V79 cells (% MNBN), which represents the fraction of cytokinesis blocked (binucleated) cells with micronuclei, regardless of the number of micronuclei per BN cell. 35At least two independent experiments were performed for each assay.

Cell proliferation
The decrease in cell proliferation in the experiments described above was assessed by determining the frequency
13Spectra were recorded at 600 MHz for 1 H NMR and 150.9 MHz for13C NMR; 2D NMR experiments recorded in accordance; coupling constants (J Hz) are in parenthesis; b partially overlapped.Molluscicidal Activity of Compounds Isolated from Euphorbia conspicua N. E. Br.J. Braz. Cem. So.1882 Figure 1.

Table 2 .
Molluscicidal activity of terpenes from Euphorbia conspicua N. E. Br. latex on Biomphalaria glabrata Say

Table 3 .
Effect of compound 3 on cell viability of V79 Chinese hamster cells using the MTT assay

Table 4 .
Mutagenic activity of compound 3 in the Ames assay, revertants in three strains of Salmonella typhimurium (TA 98, TA100 and TA102) treated with different concentrations of compound 3 in the presence and absence of metabolic activation (S9)