Activity of Piperaceae extracts and fractions in the control of Phytomonas serpens

Protozoa of the genus Phytomonas are harmful parasites to several agricultural crops of economic importance. Due to their recognized biological activity, crude extracts of Piper aduncum, P. crassinervium, P. hispidum, and P. amalago leaves, were tested using the microdilution plate technique to assess the antiparasitic potential against Phytomonas serpens. Results showed that the ethanolic crude extract of P. crassinervium and P. amalago presented the best inhibitory concentration for 50% of the cells (IC50), 16.5 μg mL -1 in chloroform phase, and 18 μg mL-1 in aqueous phase, respectively, after 48 h treatment. Cytotoxicity analyses were performed using the colorimetric method of sulforhodamine-B in LLCMK2 mammalian cells. The chloroform phase of P. crassinervium was subjected to the fractionation process, in which the ethyl acetate and dichloromethane fractions obtained better IC50 values. Scanning electron microscopy (SEM) images showed alterations in the cell membrane of the treated parasites. The data obtained indicate a potential antiparasitic effect of the Piper species analyzed against P. serpens, being considered promising candidates for formulations of bioproducts to control the parasite.


INTRODUCTION
Species of the Trypanosomatidae family are pathogenic protozoa that cause various diseases, affecting vertebrate animals, humans, and plants. In humans, these flagellates are etiological agents of Chagas disease (Trypanosoma cruzi) and Leishmaniasis (Leishmania spp.). In plants, Phytomonas is a genus of trypanosomatids, composed of several species recognized as significant pathogens, responsible for Cancini et al. producing diseases in tropical agricultural crops of great economic interest (MASLoV et al., 2018).
Studies showed that some species of Phytomonas spp. are capable of causing lethal plant diseases, while others cause minor damage, infecting different plant tissues such as dairy ducts, phloem, seeds, flowers, and fruits. Phytomonas are an example of dixenic trypanosomatids, which affect both the invertebrate host and plants as a promastigote (CAMARGO et al., 1999). Transmitted by phytophages, these hemipterous insects are popularly known as bedbugs, and they are responsible for spreading the infection to more than 100 plant species (KAUFER et al., 2017). Among them, important crops such as tomatoes, coffee, cassava, cocoa trees, and palm trees that produce oil and coconut (PoRCEL et al., 2014;FRoLoV et al., 2019).
For a long time there were doubts about correlating the genus Phytomonas, as being phytopathogen or only endophytic to plant hosts, because its differentiation is complex and it does not produce easily detectable effects on plant growth or yield (ABREU FILHo et al., 2001). Since initial research on the pathogenicity of Phytomonas begun, two main species producing plant diseases have been identified: Phytomonas staheli that causes sudden wilt in coconut trees (Cocos nucifera) and slow wilt in oil palm (Elaeis guineensis). In these cases, leaf wilt leads to root rot. Additionally, Phytomonas leptovasorum causes phloem necrosis in Arabica and Liberica coffee (JASKOWSKA et al., 2015). The species Phytomonas serpens is isolated only in tomato fruit. The fruit infected by the insects Phthia picta and Nezara viridula present yellowish spots that result in nutritional loss and; consequently, lead to commercial impracticability of the product (JANKEVICIUS et al., 1989). Phytomonas serpens is also attributed to the production of auxin, a class of phytohormones that ends up causing interference with the plant metabolism, thus affecting plant development (IENNE et al., 2014). Despite the damage caused to crops, P. serpens is not considered a phytopathogen parasite, since the promastigote form of the parasite remains restricted to the place of infection (oLIVEIRA et al., 2017).
Despite being globally present, such as in African, European, and Asian countries, species of Phytomonas are considered endemic in South America, with a significant number of species in Brazil, which makes them a food security risk for many economies around the world. (DA SILVA et al., 2013). the most significant losses are registered mainly in developing countries, such as Colombia, Brazil, Ecuador and Costa Rica, which concentrate their export economies on tropical crops affected by Phytomonas. Besides, there are no adequate chemical control and treatment against these microorganisms, which requires the felling of the sick plant or the removal of infected plant material (JASKOWSKA et al., 2015).
In this sense, many studies point to plants as a potential source of new phytochemical substances, whose extracts can be used as antimicrobial agents (PEREIRA et al., 2018). the genus Piper has around 2000 species, and it is common in tropical and subtropical regions of the Atlantic Forest (PRANDO et al., 2014). Traditionally, these plants are used by the population of these regions in the treatment of influenza, cough, and rheumatism once they have ethnomedicinal properties. they are popularly known as pepper plants or false jaborandi (LAGO and KATO, 2007).
Thus, the objective of this study was to evaluate the antimicrobial activity of the crude extract and fractions of Piper amalago, P. aduncum, P. hispidum, and P. crassinervium, against P. serpens, aiming the use of natural compounds with antiparasitic action.

Plant material
the plant specimens analyzed in this study were identified by Dr. Adriana Lenita Albiero from the State University of Maringá and deposited in its Herbarium. The leaves of (1) Piper amalago L.

Preparation of crude extract and fractionation
the fresh leaves were dried in air circulation greenhouse (QuimisR © , model Q-31, Diadema-SP, Brazil) at 40 °C. three days later, the material was crushed in a knife grinder (Tecnal MarconiR © , model TE048, Piracicaba, Brazil) and then powdered. Extracts of Piper species were obtained by the maceration method at room temperature with ethanol: water (9: 1, v/v), resulting in raw extracts. The extracts were filtered and evaporated under vacuum at 40 °C to obtain an aqueous extract. this freeze-dried aqueous extract was subjected to direct extraction with chloroform (without partition), generating a fraction soluble in chloroform, which was later removed generating the aqueous chloroform extract used in clinical tests (Figure 1). the extracts were kept in a freezer at approximately -20 °C. the active chloroform extract of P. crassinervium was processed by vacuum adsorption column chromatography on silica gel 60 (70 to 230 mesh) and eluted with hexane, hexane: dichloromethane (50: 50; v/v), dichloromethane, ethyl acetate, and methanol, yielding 5 fractions.

Antipromastigote activity
Crude extracts and fractions of Piper species assessed were dissolved in dimethylsulfoxide (DMSO) and Phosphate-bufferid saline (PBS), obtaining a stock solution of 10,000 µg mL -1 . Subsequently, dilutions were performed in Warren medium to obtain final concentrations of 1000, 500, 100, 50, and 10 µg mL -1 . In these concentrations, protozoa with 48 h culture (initial inoculum of 1x10 6 cells/mL) were added. the assays were performed in 24-well microplates, incubated at 28 ºC. After 48 h, growth was evaluated by counting the protozoa in the Neubauer chamber. Later, the percentage that inhibited 50% of protozoa growth was calculated (IC 50 ). Protozoa culture with no addition of extracts was used as a negative control.

Cytotoxicity assay
A suspension of 100 µL of LLCMK 2 cells (Macaca mulatta kidney epithelial cells) at a concentration of 2.5x10 5 cels/mL in DMEM (Dulbecco Modified Eagle Medium-GibcoR ® ) was seeded in 96well plates and incubated at 37 °C with a 5% CO 2 tension. After 24 h incubation, 100 μL of the various concentrations of crude extract solutions -P. aduncum, P. crassinervium, P. hispidum, and P. amalago -were added and incubated for additional 96 h. Cell growth was evaluated by the colorimetric method of sulforodamine B, according to SKEHAN et al. (1990). the reading was performed in an ELISA reader (Bio-Tek FL-600 Microplate Fluorescence Reader) at an optical density of 530 nm and then the CC 50 (cytotoxic concentration for 50% of cells) was calculated. the results were expressed as the percentage of growth inhibition with regards to the control.

Scanning electron microscopy (SEM)
Promastigotes (1×10 6 cells/mL) were treated with 18 μg mL -1 of P. amalago aqueous extract for 48 h at 28 ºC and then fixed in 2.5% glutaraldehyde in 0.1M sodium cacodylate buffer for 1 h. Afterwards, the parasites were adhered to L-lysine coated coverslips and dehydrated in increasing concentrations of ethanol. the samples were dried at a critical Co 2 point, gold coated, and observed with a Shimadzu SS-550 scanning electron microscope (Japan) (GARCIA et al., 2013).

Statistical analysis
All tests were carried out in triplicate, and the data were analyzed through Analysis of Variance (ANoVA). tukey's test was conducted, and a p-value of ≤0,05 was considered significant compared with the control group. the statistical analysis was performed with the program Graph-Pad Prism 4, USA.

RESULTS AND DISCUSSION
Among the tools that are most explored to biological control we have the use of plant extracts. Several studies have shown the wide biological activity of plant species that belong to the genus Piper.
Recently showed the antibacterial activity of crude extract and fractions of Piper peltatum and Piper marginatum against Alicyclobacillus acidoterrestris. the hexanedichloromethane fraction of P. peltatum was the one that provided the best effect.
In our research, we first evaluated antiproliferative activity of hydroalcoholic crude extracts -chloroform phase (Figure 2), and aqueous phase ( Figure 3) of P. hispidum, P. aduncum, P. amalago and P. crassinervium against promastigote forms of P. serpens, after 48 h incubation. The IC 50 values obtained are shown in table 1. The chloroform phase of P. crassinervium demonstrated the best antiproliferative effect against P. serpens when compared with the chloroform phase of other Piper species. Among the aqueous phase extracts, P. amalago presented the lowest IC 50 in P. serpens.
Similar results were presented by LoPES et al. (2008) in the assay with isolated compounds of P. crassinervium leaves that were evaluated against epimastigote forms of T. cruzi. The prenylated hydroquinone exhibited trypanocidal activity with IC 50 of 6.10 mg mL -1 . Likewise, CARRARA et al. (2012) demonstrated the activity against promastigote forms of L. amazonensis from P. amalago leaf extraction. the extract showed significant antiproliferative activity against promastigotes, with an IC 50 of 15 mg mL -1 .
An essential criterion in the research for active compounds with antiprotozoal activity is their toxicity on mammalian cells. For this purpose, the cytotoxicity of the extracts was evaluated on mammalian cells LLCMK 2 . After 96 h treatment, the cytotoxic concentration for 50% of the cells (CC 50 ) was determined using the colorimetric method of sulforhodamine-B. CC 50 values are exposed in Table  1. the CC 50 obtained in the chloroform phase for all evaluated Piper species showed moderate toxicity, whereas the aqueous extracts showed low toxicity, with CC 50 >850 mg mL -1 .
Extracts cytotoxicity of Piper species on LLCMK 2 mammalian cells was compared with the antiproliferative activity against promastigotes of P. serpens, using the selectivity index (SI), the ratio between CC 50 for LLCMK 2 cells and IC 50 for protozoa. According to table 1, the aqueous fraction of P. amalago extract presented the best SI among the Piper species analyzed, which means that it was 47.5 times more toxic to promastigote forms of P. serpens than LLCMK 2 cells.
Therefore, the best selectivity indexes were presented by P. amalago in aqueous phase and by P. crassinervium in chloroform phase. Most extracts were more toxic to the parasite than to mammalian cells.
In our results, the chloroform phase of P. crassinervium and the aqueous phase of P. amalago were selected for additional tests once they presented a more significant antiproliferative effect and a better selectivity index. The chloroform phase of P. crassinervium was submitted to the fractioning process. According to CARGNIN et al. (2013) andBAPELA et al. (2017) the compounds that present significant antiprotozoal activity are among those capable of being extracted by apolar solvents. Figure 4 shows the antiproliferative activity of the five fractions obtained from P. crassinervium (Hexane fraction, Hexan-Dichloromethane fraction (1:1), Dichloromethane fraction, Ethyl acetate fraction, and Methanol fraction). the most active fractions were dichloromethane and ethyl acetate, both with an IC 50 <10 mg mL -1 . the methanol fraction showed IC 50 of 13 mg mL -1 and the hexane, and hexane dichloromethane fractions presented IC 50 of 84 and 47 mg mL -1 , respectively. DMSo at the  Leishmania amazonensis. A progressive increase in the antileishmanial effect was observed in the course of the fractionation. the dichloromethane and ethyl acetate fractions exhibited the best action against protozoa with IC 50 values of 2,1 mg mL -1 for promastigotes and 24 mg mL -1 for amastigotes. these data corroborate the results of this study, since the same fractions showed higher antiprotozoal activity against P. serpens.
the action of the aqueous phase extracted from P. amalago was analyzed using scanning electron microscopy. the photomicrographs ( Figure  5) reveal the differences in the morphology of promastigote cells treated with the extract when compared with the control (untreated cells). the control cells presented typical morphology of P. serpens, with elongated cell body, smooth cell surface, and preserved terminal flagellum ( Figure  5A). on the contrary, when promastigotes were treated with their respective IC 50 values (Table 1), there were some changes in their structural integrity. the aqueous phase of P. amalago, induced changes in the cell membrane, detected by deformities on the cell surface ( Figure 5B), with a reduction in volume and rounding of the cell body ( Figure 5C). Besides, it caused shortening and/or loss of the flagellum ( Figure 5D). there are currently no control records for P. serpens, and significant research efforts have been made to achieve formulations that can be used in bioproducts for parasite management. According to SILVA et al. (2019), the essential oil of Varronia curassavica (Cordiaceae) has an antiprotozoal activity against P. serpens by causing changes in the permeability of the cytoplasmic membrane of promastigote cells, reaching up to 75% reduction in cellular proliferation of parasites exposed to the essential oil. MEDINA et al. (2015) investigated the toxicity of alkaloids produced by tomato plants, called tomatine and tomatidine, in inhibiting the growth of P. serpens. Results indicated that the tomatine is capable of disrupting the structure of the plasma cell membrane, causing the death of the parasite. However, tomatidine only interferes with growth due to the inhibition of sterol synthesis. Similar results have been described by EVANGELIStA et al. (2018).

CONCLUSION
Ethanolic crude extracts and Piperaceae fractions were effective against P. serpens, mainly because they showed higher selectivity to parasites than to LLCMK 2 mammalian cells. our results showed through the inhibitory concentrations obtained after P. crassinervium fractionation, an improvement in the antiprotozoal activity of dichloromethane and ethyl acetate fractions. Moreover, the damage detected by scanning electron microscopy confirmed the effect of treatment with the crude extract of P. amalago, which was able to generate evident morphological changes in promastigote cells of P. serpens.
Later studies are essential to determine the inhibiton in pathways caused by the extracts since the evaluation carried out in this study points out that extracts of the different Piper species can be considered promising sources for the bio-guided isolation of new active compounds, indicating biotechnological potential in the development of chemical controls.