Phytol, a Diterpene Alcohol from Chlorophyll, as a Drug against Neglected Tropical Disease Schistosomiasis Mansoni

<div><p>Background</p><p>Schistosomiasis is a major endemic disease that affects hundreds of millions worldwide. Since the treatment and control of this parasitic disease rely on a single drug, praziquantel, it is imperative that new effective drugs are developed. Here, we report that phytol, a diterpene alcohol from chlorophyll widely used as a food additive and in medicinal fields, possesses promising antischistosomal properties <i>in vitro</i> and in a mouse model of schistosomiasis mansoni.</p><p>Methods and findings</p><p><i>In vitro</i>, phytol reduced the motor activity of worms, caused their death and confocal laser scanning microscopy analysis showed extensive tegumental alterations in a concentration-dependent manner (50 to 100 µg/mL). Additionally, phytol at sublethal doses (25 µg/mL) reduced the number of <i>Schistosoma mansoni</i> eggs. <i>In vivo</i>, a single dose of phytol (40 mg/kg) administered orally to mice infected with adult <i>S. mansoni</i> resulted in total and female worm burden reductions of 51.2% and 70.3%, respectively. Moreover, phytol reduced the number of eggs in faeces (76.6%) and the frequency of immature eggs (oogram pattern) was significantly reduced. The oogram also showed increases in the proportion of dead eggs. Confocal microcopy studies revealed tegumental damage in adult <i>S. mansoni</i> recovered from mice, especially in female worms.</p><p>Conclusions</p><p>The significant reduction in parasite burden by this chlorophyll molecule validates phytol as a promising drug and offers the potential of a new direction for chemotherapy of human schistosomiasis. Phytol is a common food additive and nonmutagenic, with satisfactory safety. Thus, phytol has potential as a safe and cost-effective addition to antischistosomal therapy.</p></div

Quantitative analysis of morphological alterations on the tegument of <i>S. mansoni</i> after exposure to phytol.

<p>The quantification of the number of tubercles was performed using three-dimensional images obtained from laser scanning confocal microscopy. Indicated are numbers of intact tubercles and these numbers were measured in a 20,000 µm² of area in a dorsal region of adult male worm (see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002617#pntd-0002617-g002" target="_blank">Figure 2</a>), and calculated with the Zeiss LSM Image Browser software. Praziquantel (PZQ, 1 µg/mL) was used as positive control. A minimum of three tegument areas of each parasite were assessed. Values are means ± SD (bars) of ten male adult worms. **P<0.01 and ***P<0.001 compared with untreated groups.</p

Effect of phytol on <i>Schistosoma mansoni</i> oviposition.

<p>Adult worm couples were incubated in 24-well culture plates containing RPMI 1640 medium and phytol at the indicated concentrations. At the indicated time periods, the cumulative number of eggs per worm couple was scored using an inverted microscope. Values are the means ± SD (bars) of ten worm couples. *P<0.05, **P<0.01, and ***P<0.001 compared with untreated groups.</p

Microscopy investigation of <i>Schistosoma mansoni</i> female worm after <i>in vitro</i> incubation with phytol.

<p>In these experiments, pairs of adult worms were incubated in 24-well culture plates containing RPMI 1640 medium and treated with different concentrations of phytol. After 24 h of incubation or in the case of death, adult female worms were fixed in FAA solution and the fluorescent images were obtained using confocal laser scanning microscopy. <b>A:</b> Negative control (RPMI 1640 medium) after 120 h. Female schistosome showing an intra-uterine egg (arrow) and eggs laid by worms are seen (inset). <b>B:</b> Positive control (1 µg/mL praziquantel) after 24 h. <b>C:</b> Worm treated with 50 µg/mL phytol after 24 h; tegumental surface showing extensive sloughing (sh). Scale bars = 50 µm.</p

Microscopy investigation of <i>Schistosoma mansoni</i> male worm after <i>in vitro</i> incubation with phytol.

<p>In these experiments, pairs of adult worms were incubated in 24-well culture plates containing RPMI 1640 medium and treated with different concentrations of phytol. After 120 h of incubation or in the case of death, adult male worms were fixed in FAA solution and the fluorescent images were obtained using confocal laser scanning microscopy. <b>A:</b> Negative control (RPMI 1640 medium) after 120 h; dorsal tegumental surface showing tubercles (T). <b>B:</b> Positive control (1 µg/mL praziquantel) after 24 h. <b>C:</b> worm treated with 50 µg/mL of phytol after 96 h; tubercles (T) and the lateral tegument disintegrate (di). <b>D:</b> worm treated with 75 µg/mL phytol after 72 h; dorsal tegumental surface showing sloughing (sh). <b>E:</b> worm treated with 100 µg/mL phytol after 24 h; dorsal tegumental surface showing swollen (sw), sloughing (sh) and erosion (er). Scale bars = 50 µm.</p

Effect on worm burden of phytol 40/kg.

<p>These results represent the effect on worm burden of a single 40/kg oral dose of phytol administered to mice harboring adult <i>S. mansoni</i> infection, stratified by sex. Drug administration started at day 56 postinfection. The animals were killed at 2 weeks post-treatment. Points represent data from individual mice that were infected and treated with phytol, or infected and untreated (control). The horizontal bars represent median values. **P<0.01 and ***P<0.001 compared with untreated groups.</p

Chemical structure of phytol (3,7,11,15-tetramethylhexadec-2-en-1-ol).

<p>Chemical structure of phytol (3,7,11,15-tetramethylhexadec-2-en-1-ol).</p

<i>In vitro</i> effects of phytol against 56-day-old adult <i>Schistosoma mansoni</i>.

a<p>RPMI 1640.</p>*<p>Values correspond to the sum of the adult schistosomes obtained from three separate experiments performed in triplicate (n = 2) and quadruplicate (n = 1). Male parasite (M). Female parasite (F).</p

Effect on egg development stages (A), and stool egg load (B) of phytol 40 mg/kg.

<p>These results represent the effect on egg development stages (oogram pattern), and stool egg load of a single 40 mg/kg oral dose of phytol administered to mice harboring adult <i>S. mansoni</i> infection. Drug administration started at day 56 postinfection. The animals were killed at 2 weeks post-treatment. Points represent data from individual mice that were infected and treated with phytol, or infected and untreated (control). The horizontal bars represent median values. ***P<0.001 compared with untreated groups.</p

Microscopy observations of adult <i>Schistosoma mansoni</i> recovered from mice.

<p>Animals were treated with a single oral dose of phytol (40 mg/kg) at day 56 postinfection. The animals were killed at 48 h post-treatment and fluorescent images were obtained using confocal laser scanning microscopy. <b>A:</b> Male worms from infected and non-treated animal (control); dorsal tegumental surface showing tubercles (T). <b>B:</b> male worms from infected animals and treated with phytol; dorsal tegumental surface showing blebs (sb), <b>C:</b> female worms from infected and non-treated animal (control); female schistosome showing an intra-uterine egg (arrow). <b>D:</b> female worms from infected animals and treated with phytol; tegumental surface showing blebs (sb) and extensive sloughing (sh). Scale bars = 50 µm.</p