The World Health Organization (WHO) lists 17 Neglected Tropical Diseases (NTDs), most of which are caused by protozoa. These diseases cause serious health problems, affecting more than one billion people, most of who live in extreme poverty in tropical and subtropical regions. These diseases are widespread, with an incidence of over 10% in the world population, and yet, their treatments are highly toxic and sometimes ineffective1, presumably due to lack of attention from the scientific community.
Among these, leishmaniasis is responsible for high morbidity and mortality rates, with an average of 12 million infected people, and about 350 million living in at-risk areas in 98 countries on five continents1. During the chemotherapeutic treatment of leishmaniasis, pentavalent antimonials (Sb+5), pentamidines, and amphotericin B are used. However, these treatments have their limitations, including high cost, exclusive venous administration, cardiac alterations, hepatic and renal toxicity, and the emergence of strains resistant to treatment2.
Trypanosoma cruzi is a flagellate protozoan, responsible for causing Chagas disease. Fourteen million people are infected with T. cruzi worldwide, causing approximately 7000 deaths per year, with more than 25 million people living in at-risk areas1. Nifurtimox and benznidazole, the currently available treatment options, are relatively toxic, poorly effective in the chronic phase of the disease, and their prolonged administration encourages patient evasion leading to the emergence of resistant strains3.
In this group of diseases that pose public health challenges, malaria, caused by Plasmodium falciparum, stands out, especially in endemic areas such as Africa and Asia, where it is responsible for high rates of morbidity and mortality1. The development of resistance of these parasites to major therapeutic compounds, artemisinin and quinine derivatives, makes the search for new bioactive molecules important, especially those studied from a biodiversity perspective4.
Investing in the discovery of new molecules may have solutions in biodiversity. In this context, plants of the genus Piper L. (Piperaceae) are composed of components such as: alkaloids, flavonoids, arylpropanoids, terpenoids, phenylpropanoids, lignans, and cinnamic acid; which are characterized by antifungal, antioxidant, antiplasmodial, and trypanocidal bioactivity5,6,7. Among these compounds, cinnamic acid (CinAc) was selected as a model in this study’s search for synthetic analogs with trypanocidal and antimalarial action6,7. By using this leader molecule we intend to enable the development of more effective drugs with low toxicity, thus presenting a new alternative for treating such diseases.
Synthetic analogs are derivatives of CinAc, isolated from the plant P. tuberculatum Jacq. These compounds (Figure 1) were synthesized by Sigma (Sigma-Aldrich).
The analogs were diluted in dimethylsulfoxide-DMSO (Sigma-Aldrich) at concentrations at or below 0.6%. The drugs pentamidine, benznidazole, and artemisinin were used as negative controls (100% death) for Leishmanias spp., T. cruzi, and P. falciparum, respectively.
L. braziliensis (MHOM/BR/75/M2904) and L. infantum chagasi (MCAN/ES/92/BNC 83) promastigotes were maintained in vitro in RPMI 1640 medium (Sigma-Aldrich) supplemented with 25 mM HEPES (Sigma-Aldrich), 21 mM sodium bicarbonate (Sigma-Aldrich), 11 mM glucose (Sigma-Aldrich), 40 μg/mL of 10% gentamycin (v/v), and 10% previously inactivated fetal bovine serum (FBS) (Sigma-Aldrich). The parasites were kept in a BOD incubator at 26 ºC, and subcultured every five days.
Trypanosoma cruzi epimastigotes, “CL-B5” strain, were grown in liver infusion tryptose culture medium (LIT, Sigma-Aldrich) supplemented with 10% FBS; cultures were kept in a BOD incubator at 24 °C for weekly subculturing.
Chloroquine-resistant and mefloquine-sensitive P. falciparum (W2) strain derived from the Indochin III/CDC strain were cultured in vitro in human red blood cells with 2% hematocrit, diluted in RPMI 1640 culture medium supplemented with Albumax (Thermo Fisher Scientific). The cultures were maintained in desiccators at 37 °C in a gasogenic mixture containing 5% O2, 5% CO2, and 90% N2.
L. braziliensis and L. infantum chagasi promastigotes were plated at 1.5×106 parasites/mL in 96-well microplates, incubated at 26 °C for 72 h with varied concentrations (1260-9.8 μM) of the analogs. Subsequently, a 3 mM solution of alamarBlue (Sigma-Aldrich) was added and incubated at 26 °C for 4 h. Absorbance was monitored at 570 and 600 nm using a Synergy H1 (Biotek) multimodal spectrophotometer. Positive controls (parasites with no treatment) and negative controls in pentamidine, at varied concentrations (9-0.14 μM), and DMSO (0.6%) were used in order for the assay to be effective.
T. cruzi epimastigotes were plated at 2.5×105 parasites/mL on microplates and incubated at 24 °C for 72 h in serial concentrations of the analogs (1,260-9.8 μM). Subsequently, a solution of 200 μM chlorophenolRed-β-D-galactopyranoside (CPRG) was added followed by incubation at 37 °C for 4 h; absorbance was monitored using a Synergy H1 multimodal spectrophotometer (Biotek) at 570 nm8. Parasites incubated with culture medium were considered as the growth control; reference drug benznidazole (100-1.56 μM) was used as a negative control.
P. falciparum cultures (W2) were treated with 5% sorbitol (Sigma-Aldrich), for synchronization in young trophozoites; parasitemia (0.05%) was determined by optic microscopy using a Rapid Panoptic kit (Laborclin, BR). Analogs were added to microplates at varied concentrations (1,260-9.8 μM), and infected red cells (positive control) and varied concentrations (0.17-0.0026 μM) of Artemisinin (negative control) were used; the activity was evaluated for 48 h at 37 °C.
The antimalarial evaluation was performed using SYBR (SYBR Green I, Thermo Fiser). RBCs were washed with 1× PBS and centrifuged at 700 g for 10 min. Then, the supernatant was discarded and 100 μL of a SYBR Green I solution (0.001% v/v in lysis buffer) was added; the contents were transferred to microplates containing 100 μL of 1x PBS. The plates were incubated for 30 min at room temperature; fluorescence was obtained at an excitation of 485 nm and an emission of 535 nm9.
The cytotoxicity was assessed using NCTC 929 fibroblasts in an alamarBlue assay. Cells were plated in microplates with approximately 2.5×104 cells/well and incubated with varied concentrations of the analogs (1,260-9.8 μM) for 48 h in an incubator at 37 °C in a humid atmosphere containing 5% CO2. Subsequently, a 3 mM solution of alamarBlue was added followed by incubated at 37 °C for 4 h. Absorbance was monitored at 570 and 600 nm using a Synergy H1 (Biotek) multimodal spectrophotometer. The 50% cytotoxic concentration for cell growth (CC50) in the presence of the analogs and the control drugs was determined and compared with cells grown only in culture medium. The activity of the molecules tested was estimated by calculating the percentage of viable cells.
The 50% inhibitory concentration of parasite (IC50) and CC50 was determined by dose-response curves based on non-linear regression, applying the formula y = A1 + (A2-A1)/(1+10^((LOGx0-x)*p)), with determination of the level of significance at p < 0.05. The program Origin (OriginLab Corporation, Northampton, MA 01060, USA) was used. Standard deviation was calculated using MSExcel software. All experiments were performed in triplicates.
The selectivity index (SI) of samples was obtained by calculating the ratio between CC50 and IC50. Values > 10 were considered nontoxic, while substances with values below ten were considered toxic10.
Another way to assess the safety of a drug is to measure its hemolytic activity. Hemolytic assays were performed at varied concentrations (1,260-9.8 μM); 180 μL of erythrocytes (human) were added with 1% hematocrit in microplates with a U-bottom. The plates were incubated for 30 min at 37 °C with agitation after every 5 min. They were then centrifuged for 10 min. The supernatant was analyzed using a microplate spectrophotometer (Biochrom model: Expert plus) at 540 nm. Saponin (0.05%) was used as a positive control for hemolysis.
Chemical compounds isolated from plant extracts were studied because of their proven in vitro antiprotozoal activity, including the molecules derived from CinAc11. Among the synthetic analogs of CinAc, E5 presented the lowest IC50 value against L. infantum chagasi (212.75 μM); when compared to L. braziliensis (IC50 340.43 μM). When tested against T. cruzi, IC50 was 1071.5 μM. Another analog, I9, presented IC50 value of 248.56 μM against L. braziliensis; against L. infantum chagasi and T. cruzi, I9 showed IC50 values ranging between 582 and 723 μM. Analog G7 showed inhibitory concentration against L. infantum chagasi (IC50 642.9 μM) and against T. cruzi (IC50 667.11 μM). Analogs A1, B2, C3, D4, and F6 were not effective at a concentration of 1,260 μM (Table 1). The analogs, at concentration of 1,260 μM (the highest concentration tested), presented no antimalarial, hemolytic, or cytotoxic activity; thus, selectivity index calculations were based on this concentration.
|Compounds||IC50(µM) ± SD|
|L. braziliensis a||L. infantum b||T. cruzi c||P. falciparum d||SIe||SIf||SIg|
|A1||˃ 1,260||˃ 1,260||˃ 1,260||˃ 1,260||NC||NC||NC|
|B2||˃ 1,260||˃ 1,260||˃ 1,260||˃ 1,260||NC||NC||NC|
|C3||˃ 1,260||˃ 1,260||˃ 1,260||˃ 1,260||NC||NC||NC|
|D4||˃ 1,260||˃ 1,260||˃ 1,260||˃ 1,260||NC||NC||NC|
|E5||340.4 ± 0.93||212.7 ± 0.57||1071.6 ± 1.2||˃ 1,260||˃ 3.7||˃ 5.9||˃1.2|
|F6||˃ 1,260||˃ 1,260||˃ 1,260||˃ 1,260||NC||NC||NC|
|G7||˃ 1.260||642.9 ± 1||667.1 ± 2.9||˃ 1,260||NC||˃ 1.9||˃ 1.9|
|H8||1018.3 ± 0.8||772.8 ± 0.9||987.2 ± 2.3||˃ 1,260||˃ 1.2||˃ 1.6||˃ 1.3|
|I9||248.5 ± 0.8||582 ± 0.8||723 ± 3.3||˃ 1,260||˃ 5||˃ 2.2||˃1.7|
|J10||˃ 1.260||1110 ± 1.5||1133.9 ± 2.8||˃ 1,260||NC||˃ 1.1||˃ 1.1|
|PTM||0.76 ± 0.04||1.4 ± 0.26||NT||NT||NT||NT||NT|
|BZND||NT||NT||9.6 ± 0.9||NT||NT||NT||NT|
|ARTN||NT||NT||NT||0.02 ± 0.09||NT||NT||NT|
IC 50 : 50% inhibitory concentration/Inhibition of 50% of parasite; Mean ± SD: results correspond to the average of three trials for each sample and standard deviation. a L. braziliensis (MHOM/BR/75/M2904); b L. infantum chagasi (MCAN/ES/92/BNC 83); c T. cruzi (CL-B5 clone); d P. falciparum (Indochina III/CDC); SI:Selectivity Index, SI e : L. braziliensis; SI f : L. infantum chagasi; SI g : T. cruzi; Reference drugs: PTM– Pentamidine, BZND– Benznidazole, ARTN– Artemisinin, NT– Not tested, NC– Not calculated.
Analogs E5 (L. infantum chagasi) and I9 (L. braziliensis) presented the best IC50 of 212.7 and 248.5 μM, respectively. When we analyzed the leishmanicidal action among parasite species in relation to the treatment control pentamidine, greater efficacy was obtained against L. braziliensis (IC50 0.76 μM) than against L. infantum chagasi (IC50 1.4 μM).
Studies have described the fractionation of biomonitoring of Valeriana wallichii chloroform extract, which resulted in two active CinAc derivatives against L. major promastigotes with IC50 of 48.8 μM11. In this context, 3-(3,4,5-trimethoxyphenyl) propanoic acid obtained naturally from Piper tuberculatum Jacq, also presented activity against L. amazonensis promastigotes after 96 h hours of treatment6 with IC50 at the concentration of 145 μg/mL (608.63 µM). In another study, synthetic derivatives of CinAc (3,4,5-trimethoxicinnamic acid) were effective at the concentration of 2 mg/mL (8.39 mM) against promastigotes of L. amazonensis, causing death of 92% of the parasites when treated for 96 h12. The results of this study corroborate the results presented here, where the tests against Leishmania promastigotes were performed for 72 h and the maximum concentration was 1,260 μM.
The CinAc derivatives were inactive against the W2 (CQ-resistant) P. falciparum strain (IC50 > 1,260 μM). However, we expected high activity for this class since CinAc isolated from K. africana (β-hydroxycinnamic) showed IC50 ranging from 53.84 to 6.71 μM against other P. falciparum strains (W2, W2mef, CAM10, and SHF4)13. The main mechanism of antiplasmodial action of CinAc class is the decline in energy production (ATP) due to inhibition of lactate transporters, changes in mitochondrial respiration, and reduction of translocation of carbohydrates and amino acids in parasitized red blood cells. Previous studies have frequently suggested that structural modifications of CinAc may alter its effectiveness, thus, reducing its antiplasmodial activity14.
Derivatives of natural products of plant origin exhibiting trypanocidal activity have been considered valid alternatives for control of parasites15. In this context, studies performed with the species P. arboreum and P. tuberculatum, using the hexane extracts obtained from leaves against T. cruzi epimastigotes, resulted in an IC50of 13.3 μg/mL for P. arboreum and 17.2 μg/mL for P. tuberculatum5. Among the CinAc analogs tested, G7 showed the best trypanocidal activity, exhibiting IC50 of 667.1 μM and selectivity index of 1.9.
The results demonstrated that synthetic analogs of CinAc have antiprotozoal activity against L. braziliensis and L. infantum chagasi promastigotes, and T. cruzi epimastigotes. However, prior to performing the in vitro and in vivotests, it is suggested that the compounds undergo structural modifications in order to obtain a more satisfactory selectivity index. The analogs tested are promising in the development of new molecules with greater effectiveness and fewer side effects.