Anti-trypanosomal screening of Salvadoran flora

Chagas disease is caused by the protozoan parasite Trypanosoma cruzi, and in Central America, it is considered one of the four most infectious diseases. This study aimed to screen the anti-trypanosomal activity of plant species from Salvadoran flora. Plants were selected through literature search for plants ethnobotanically used for antiparasitic and Chagas disease symptomatology, and reported in Museo de Historia Natural de El Salvador (MUHNES) database. T. cruzi was incubated for 72 h with 2 different concentrations of methanolic extracts of 38 species, among which four species, Piper jacquemontianum, Piper lacunosum, Trichilia havanensis, and Peperomia pseudopereskiifolia, showed the activity (≤ 52.0% viability) at 100 µg/mL. Separation of the methanolic extract of aerial parts from Piper jacquemontianum afforded a new flavanone (4) and four known compounds, 2,2-dimethyl-6-carboxymethoxychroman-4-one (1), 2,2-dimethyl-6-carboxychroman-4-one (2), cardamomin (3), and pinocembrin (5), among which cardamomin exhibited the highest anti-trypanosomal activity (IC50 = 66 µM). Detailed analyses of the spectral data revealed that the new compound 4, named as jaqueflavanone A, was a derivative of pinocembrin having a prenylated benzoate moiety at the 8-position of the A ring.


Introduction
Chagas disease is considered the fourth highest burden of infectious diseases in Central America, behind HIV/AIDS, acute respiratory infections, and acute diarrheal diseases [1]. Trypanosoma cruzi has been identified as the causal agent of Chagas disease. This protozoan parasite is mainly transmitted through contact with the feces of hematophagous triatomine insects and infects a broad range of mammalian species including humans. In Latin America, Chagas disease continues to be an important public and social health problem, and the World Health Organization [2] considers it as one of the neglected tropical diseases (NTDs).
El Salvador has a population of 6.5 million and approximately 39% of the population is at risk of contracting this disease [3]. The latest official data on Chagas disease, from the Health's Ministry of El Salvador, conclude that between 2014 and 2017, 771 chronic cases and 53 acute cases were reported and the prevalence of T. cruzi in blood banks is among the highest in the Americas, fluctuating between 1.5 and 3.9% in 2008-2016 [1].
At present, Chagas disease is being treated mainly with nifurtimox (NF) and benznidazole (BNZ). Nifurtimox and benznidazole achieve cure rates of 70% and 75%, respectively, in acute cases, and a 100% cure is obtained in congenital cases if the treatment is carried out during the first year of life. However, both drugs are administered for at least 30-60 days and produce side effects in 30% of the cases. Other drugs that have been administered are itraconazole and posaconazole. In acquired chronic cases, a 20% cure and 50% improvement of electrocardiographic changes are obtained with itraconazole [4].
The development of new, safer, and more effective trypanocidal drugs remains a current challenge, as drugs for Chagas disease are not considered a high priority by the R&D-based pharmaceutical industry [5]. Throughout the history of mankind, natural products have been decisive for the discovery of new drugs, since they have enormous structural diversity as compared to conventional synthetic molecules and the screening of these natural sources remains one of the most attractive routes for this purpose [6,7]. Various plant species have been tested in the search for natural products to combat Chagas disease caused by T. cruzi, exhibiting in many cases high trypanocidal activity and low toxicity [8][9][10][11].
The present work aimed to assess the in vitro activity of some Salvadoran plant extracts against the epimastigotes forms of T. cruzi. Thirty-eight species of plants belonging to 19 genera in 15 families were investigated. Trypanocidal activity was observed in the methanolic extracts obtained from Peperomia pseudopereskiifolia, Piper jacquemontianum, P. lacunosum (Piperaceae), and Trichilia havanensis (Meliaceae). Methanolic extract of aerial parts from P. jacquemontianum was fractionated, resulting in the isolation and structural elucidation of a new flavanone (4), together with four known compounds (1-3 and 5). Anti-trypanosomal activity of the isolated compounds was evaluated.

Results and discussion
Bibliographic research for plants ethnobotanically used for antiparasitic and Chagas disease symptomatology (fatigue, depression, constipation, and gastric pains) or heart complaints [12,13], and reported in Museo de Historia Natural de El Salvador (MUHNES) database, resulted in 350 species. From these species, 97 species belonging to promising botanical families and genera, from which characteristic compounds of proven trypanocidal potential have been reported, were selected as candidates for anti-trypanosomal screening. Among these species, 38 species were collected and used for the screening (Table 1).
To carry out the anti-trypanosomal screening, MeOH extracts of collected plants were prepared. The yields of the extract ranged between 5.8-18.1%, 2.7-11.5%, 10.4-48.8%, and 6.3% for aerial parts, leaves, stem bark, and roots, respectively. The activity of 38 plant species from El Salvador against T. cruzi is summarized in Fig. 1 (also see S3). Although there are no widely accepted criteria to consider a promising extract or compound [13], taking into account the criteria of Osorio et al. [14], an extract is considered moderately active if it reduces viablility of T. cruzi around 50% at 100 µg/mL. Among the 38 plant species, four species, Peperomia pseudopereskiifolia, Piper jacquemontianum, P. lacunosum (Piperaceae), and Trichilia havanensis (Meliaceae), showed moderate activity at 100 µg/mL.
The Piperaceae family includes a large number of plants used in tropical and subtropical regions. The Piper and Peperomia genera are the most representative of this botanical family and Piper species are commonly used in Latino-American traditional medicines for the treatment of protozoal diseases [15]. From the Piperaceae family, some species have been tested against T. cruzi and some bioactive compounds have been isolated such as flavonoids, terpenoids, lignans, chromanes, and alkaloids [13,15,16]. Concerning genus Peperomia, the main classes of compounds described are, phenylpropanoids, lignans, pyrones, aliphatic and aromatic amides, alkaloids, polyketides, benzoic acid derivatives, and chromenes, among which trypanocidal activity was reported for lignans and benzoic acid derivatives [17,18].
Genus Trichilia has been studied for different biological activities, including trypanocidal activity [19,20]. In a study of Meliaceae and Rutaceae family, the branches of T. ramalhoi were one of the most active extracts [21]. According to Pizzolatti et al. [19], the bark of T. catigua showed activity against trypomastigotes of T. cruzi. From T. havanensis some metabolites have been isolated, such as triterpenoids, tetranortriterpenoid, hydroxybutenolide derivative, and limonoids [22][23][24]. In a study carried out against Trypanosoma brucei, the bark extract of T. emetica showed activity attributed to the presence of limonoids [20].
In this study, we isolated the constituents of P. jacquemontianum. This plant is a shrub 1-4 m in high, and native to Central America and the Caribbean lowlands [25]. In various Latin American countries, P. jacquemontianum is used in folklore medicine to treat skin ailments, infections, anemia, and body aches [26,27]. In Panama, it is traditionally used as a remedy for fever, headache, and cold, nervousness, diabetes, stomachache [28]. Chemical study of this species has been mainly performed on essential oils. Linalool and E-nerolidol were reported as the major constituents of its essential oil [29,30], whereas a recent study revealed that the composition was variable and only E-nerolidol was detected as a common constituent among the eight cultivars from different places in Guatemala [31]. Although linalool has been reported to show anti-trypanosomal activity [32], nothing has been reported on anti-trypanosomal constituents of the MeOH extract of this species. Aerial parts of P. jaquemontianum (510 g) were extracted with MeOH at room temperature to give 83.5 g of the extract. As a preliminary experiment, the MeOH extract (1.0 g) was separated by a silica gel column chromatography with stepwise gradients of hexane and ethyl acetate and then MeOH to obtain nine fractions (D1-D9), among which D3-D6 inhibited the growth of epimastigotes of T. cruzi more than 50% at 100 mg/mL. The major constituents of the active fractions were isolated to obtain compounds 1 (58 mg) and 2 (8.6 mg) from D4 and D6, respectively.
The new compound 4 was obtained as white powder. Its 1 H NMR spectrum (Table 2) Table 2), suggested that the substituent on the A ring is a prenylated methyl benzoate derivative. The connectivity of these groups was concluded from the HMBC correlations as shown in Fig. 3. The HMBC spectrum also showed correlations from the methine proton [5.39 (1H, d, J = 8.2 Hz, H-8')] of the prenyl group to C-7, C-8 and C-9 carbons of the ring A of pinocembrin moiety (Fig. 3), confirming the substitution position of this group at the 8-position on the A ring. Thus, the structure of compound 4 was concluded as shown in Fig. 2. Flavanones and prenylated benzoic acid derivatives [30,35,[41][42][43] have been reported from various Piper species. However, a compound having both of these moieties has not been reported. As this is a new compound, it is named as jaqueflavanone A. In the HPLC purification of this compound, the presence of a closely related compound was observed. The 1 H NMR spectrum of a mixture of this compound and compound 4 suggested that it is a diastereomer of compound 4. However, this compound could not be obtained as a single compound. Compound 4 has two asymmetric carbons (C-2 and C-8'). As pinocembrin isolated from this extract was a racemic mixture, the stereochemistry of C-2 seems racemic. However, the stereochemistry of the other carbon could not be determined.

Conclusion
In the present study, in vitro anti-trypanosomal activities of thirty-eight species from Salvadoran flora are reported. The most active methanolic extracts were from Peperomia speudopereskiifolia (Piperaceae), Trichilia havanensis (Meliaceae), Piper lacunosum (Piperaceae), and Piper jacquemontianum (Piperaceae). These results confirmed the effectiveness of the ethnobotanically used plants.
The activity-guided fractionation of P. jacquemontianum resulted in the isolation of anti-trypanosomal compounds including cardamomin, whose activity against Trypanosoma has been reported. Constituents of the other active species are now under investigation. Considering these findings, we can conclude that Salvadoran flora is a potential source of anti-trypanosomal substances and that the most promising extracts are potential sources of compounds for the development of more effective drugs for the treatment of Chagas disease. Translation of some in vitro results into in vivo follow-up studies is recommended in the future.

Plant selection process
Bibliographic research was done to look for Salvadoran plants with ethnobotanical uses for antiparasitic and Chagas disease symptomatology (fatigue, depression, constipation, gastric pains) or heart complaints [12,13], resulting in 380 possible plant species. The selection was limited to the plant species reported in Museo de Historia Natural de El Salvador (MUHNES) database, resulting in 350 species. These species were investigated in scientific journals database to find promising botanical families and genera from which bioactive compounds have been reported, moreover, the plants are known to contain characteristic compounds of proven trypanocidal potential. Ninety-seven species resulted as a possible anti-trypanosomal species.

Plant materials
From the 97 possible species, only thirty-eight species were possible to collect for anti-trypanosomal screening. They were collected under the permission and resolution code MARN-DEV-GVS-040-2018 at protected natural areas of El Salvador in 2018 and identified by Jenny Elizabeth Menjívar Cruz, Curator of the Herbarium at the Museo de Historia Natural de El Salvador (Table 1). A voucher specimen has been deposited for each species in the Herbarium at the MUHNES.
To carried out the bioassay-guided isolation, the aerial parts of Piper jacquemontianum Kunth were collected at El Imposible National Park, Ahuachapán, El Salvador

Preparation of plant extracts
The collected plants were dried at 40 °C for 48-72 h in a circulating air oven (BIOBASE, China, model BOV-V225F) and milled to obtain a particle size ≤ 2 mm (Bel-Art products, USA, model micro-mill). Twenty grams of each sample were extracted with methanol (200 mL × 2) in a magnetic stirrer ultrasonic bath (VWR, USA, model 97,043-988, operating frequency at 35 kHz) for 90 min at 25 °C. Each extract was concentrated under reduced pressure at 40 °C (model RE801, Yamato Scientific Co., Ltd., Japan) to obtain the MeOH extracts.

Sample preparation for HPLC profile of crude extracts
Each extract (10.0 mg) was dissolved in 1 mL of methanol, using an ultrasonic bath (VWR, model 97,043-988, operating frequency at 35 kHz) at room temperature. A solution of 1 mg/mL was prepared for each extract solution using methanol. The sample solution was filtrated through a 0.45 µm membrane filter before being subjected to HPLC analysis. HPLC profiles of the extracts are shown in the supplemental materials ( Figure S7).

Anti-trypanosomal screening of crude extracts (MTT method)
Ninety-five microliters of epimastigotes (3 × 10 6 epimastigotes/mL, Tulahuen strain) suspended in GIT medium supplemented with hemin (12.4 µM) were added in each well of 96-well plate. These were incubated at 28 °C for 72 h, after the addition of 5 mL of extracts (100 and 10 µg/mL in DMSO). Benznidazole was used as a positive control. After the incubation time, 10 µL of MTT reagent (5 mg/mL in PBS) was added to each well and incubated for another 24 h. The medium was discarded and the precipitates of formazan were dissolved with 100 µL of DMSO. The measurement of absorbance was performed at 530 nm.

Anti-trypanosomal test for fractions and isolated compounds (luminescence method)
Epimastigotes of T. cruzi (1 × 10 5 parasites/well) in GIT medium (50 µL) and a sample dissolved in DMSO (0.5 mL) were added to each well of a 384-well white plate (Falcon, 353,988). The plate was incubated at 27 °C for 24 h. After the incubation, 20 µL of CellTiter-Glo reagent (Promega, G7570) were added to each well and the intensity of luminescence (500-670 nm) was measured by a luminometer (Infinite M200 Pro, Tecan). To compensate for the luminescence of the sample itself, each sample solution in GIT medium without epimastigotes was used as a blank. Tamoxifen was used as a positive control (IC 50 19.3 mM). To determine IC 50 values, six different concentrations of each sample were prepared.