Antiprotozoal Activity against Entamoeba histolytica of Plants Used in Northeast Mexican Traditional Medicine. Bioactive Compounds from Lippia graveolens and Ruta chalepensis

Amoebiasis caused by Entamoeba histolytica is associated with high morbidity and mortality is becoming a major public health problem worldwide, especially in developing countries. Because of the side-effects and the resistance that pathogenic protozoa build against the standard antiparasitic drugs, e.g., metronidazole, much recent attention has been paid to plants used in traditional medicine around the world in order to find new antiprotozoal agents. We collected 32 plants used in Northeast Mexican traditional medicine and the methanolic extracts of these species were screened for antiprotozoal activity against E. histolytica trophozoites using in vitro tests. Only 18 extracts showed a significant inhibiting activity and among them six plant extracts showed more than 80% growth inhibition against E. histolytica at a concentration of 150 µg/mL and the IC50 values of these extracts were determined. Lippia graveolens Kunth and Ruta chalepensis Pers. showed the more significant antiprotozoal activity (91.54% and 90.50% growth inhibition at a concentration of 150 µg/mL with IC50 values of 59.14 and 60.07 µg/mL, respectively). Bioassay-guided fractionation of the methanolic extracts from these two plants afforded carvacrol (1) and chalepensin (2), respectively, as bioactive compounds with antiprotozoal activity.


Introduction
An estimated billion people are infected with one or more neglected tropical diseases [1][2][3][4].Amoebiasis caused by Entamoeba histolytica, a protozoan of the family Endomoebidae [5,6], is associated with high morbidity and mortality and has become a major public health problem worldwide [7] and is therefore considered as the third parasitosis of medical importance after malaria and schistosomiasis [8].E. histolytica is still endemic in tropical and sub-tropical regions, causing a high incidence of infections in developing countries in Latin America, Asia and Africa [9], where poor sanitary conditions, population explosion and inadequate control of reservoirs intensify the development of these infections [10].The amoebiasis is prevalent throughout the developing nations with tropical ecosystems, at times reaching a prevalence of 50% of the general population and is estimated to cause more than 100,000 deaths per year [11,12].Symptomatic patients typically may develop abdominal pain and tenderness, diarrhea, and bloody stools, but the disease may spread to the liver and other organs resulting in death [13,14].
Currently metronidazole is the therapeutic drug of choice for the treatment of amoebiasis [15], but is experiencing drug resistance by E. histolytica [16,17], resulting in the need for increased doses to overcome the infection [18] and thus causing unpleasant side-effects, such as headache, nausea, dry mouth, and a metallic taste, as well as neurotoxicity [19,20].Owing to these undesired side effects and taking into account the development of resistant strains of E. histolytica against metronidazole, new, more effective and safer antiprotozoal agents are urgently required [20,21].Natural products have proved to be an important source of lead compounds in the development of new drugs and artemisinin, quinine and licochalcone A are all examples of plant-derived products with antiparasitic activity [22,23].Screening natural products provides the chance to discover new molecules of unique structure with high activity and selectivity [24].
Thus, with the purpose of searching for new antiprotozoal agents, 32 medicinal plants used in Northeast Mexican traditional medicine [25,26] were selected to evaluate the activity of their methanol crude extracts against E. histolytica trophozoites.The selection of the species was mainly based on a follow up of ethnobotanical uses for the treatment or relief of symptoms related with parasitic infections.

In Vitro Susceptibility Assays of Plant Extracts
In this work, we report the antiprotozoal activity of 32 crude methanolic extracts derived from plants used in Northeast Mexico for the treatment of gastrointestinal disorders.The yields after Soxhlet extraction of each plant are shown in Table 1.
Table 1.Soxhlet extraction of medicinal plants used in Northeast Mexico investigated for antiprotozoal activity.
In vitro susceptibility assays were performed for each crude extract.Table 2 summarizes the antiprotozoal activity on Entamoeba histolytica of the plant extracts and the control drug (metronidazole).Extracts from 18 out of the 32 samples tested showed significant growth inhibition of E. histolytica with percentage values ranging from 24.65 to 91.54 at a concentration of 150 µg/mL.The remaining 12 plants showed absolutely no activity.Lippia graveolens Kunth, Ruta chalepensis Pers, Capsicum annuum Linnaeus, Opuntia ficus-indica Linnaeus, Haematoxylon brasiletto Karsten and Schinus molle Linnaeus displayed more than 80% growth inhibition against E. histolytica at a concentration of 150 µg/mL, with IC50 values ranging from 32.45 to 98.75 µg/mL, far less effective than metronidazole (IC50 0.205 µg/mL), but these IC50 values are suitable as selection criterion for further investigation of these plant extracts as source of potential antiprotozoal agents [10].It is important to point out that the antiprotozoal activity of seven plants chosen for this work has been previously reported.However, we decided to evaluate these species again because the antiprotozoal activity was tested with different parasites or extracts.From Lippia graveolens the biological activity mainly of its essential oils against Giardia lamblia [27][28][29] and Leishmania infantum [30] has been reported.There have been previous reports describing the activity of methanolic extracts (macerated at room temperature) of Artemisia mexicana, Ocimum basilicum, Ruta chalepensis and Schinus molle against trophozoites of E. histolytica and G. lamblia [10], reporting IC50 values for these plants of 82.2, 41.7, 61.9 and 82.4 µg/mL, respectively, against E. histolytica.Although we observed a similar IC50 to the one reported by Calzada [10] for R. chalepensis, we did not notice any activity for A. mexicana and O. basilicum, but observed higher activity for S. molle, which could be explained by the fact that the plant material was provided by different regional suppliers.In addition, R. chalepensis also showed activity against L. infantum and L. major [31], and S. molle also showed activity against Plasmodium falciparum, Trypanosoma brucei, T. cruzi, and L. infantum [32].Melissa officinalis has demonstrated biological activity reported against cysts and trophozoites of Acanthamoeba castellanii [33].Some reports on Castela texana revealed that the ethanolic extract of aerial parts and the methanolic extract from roots have relevant amebicide activity [34][35][36].We also found good amebicide activity for this plant (73.82% growth inhibition at a concentration of 150 µg/mL) by using leaves for the preparation of a methanolic extract.
Neither chemical nor biological reports concerning antiprotozoal activity of Juglans mollis could be found in the literature, but from the related plant Juglans regia oleic, linoleic, α-linoleic and ellagic acid as well as the flavonoid juglamin have been isolated [70,71].
Bioassay-guided fractionation of the bioactive extracts from the plants included in this study will be carried out in order to isolate pure compounds related to their antiprotozoal activity.Lippia graveolens Kunth and Ruta chalepensis Pers showed the most significant antiprotozoal activity (91.50 and 90.50% growth inhibition at a concentration of 150 µg/mL with IC50 values of 59.14 and 60.07 µg/mL, respectively), therefore these plants were the first choice for subsequent work on the isolation of their active constituents.

Isolation and Structure Elucidation of Compounds with Antiprotozoal Activity
Bioassay-guided fractionation of the methanolic extract from L. graveolens rendered carvacrol (1) with 95%-98% inhibition against E. histolytica at a concentration of 150 µg/mL (IC50 44.3 µg/mL) and from the methanolic extract of R. chalepensis chalepensin (2) with 98.4% inhibition at a concentration of 150 µg/mL (IC50 45.95 µg/mL) was recovered.Identification of the isolated compounds was based on spectroscopic/spectrometric analyses (IR, 1 H-and 13 C-NMR; MS) and comparison with literature data.The corresponding chemical structures are shown in Figure 1.

Carvacrol from Lippia graveolens
Preliminary fractionation of the methanolic extract of Lippia graveolens by extraction with n-hexane led, after evaporation of solvent, to a residue with good activity against E. histolytica (90.9% growth inhibition at a concentration of 150 µg/mL).Chromatography of this hexane residue over a silica gel column led to the isolation of carvacrol (1) as a colorless oil.Spectroscopic data of 1 were in concordance with literature values [72][73][74].
The essential oil of Lippia graveolens contains many monoterpenes, sesquiterpenes and phenolic terpenes among which carvacrol and thymol are the most common components [75][76][77] and their average abundance establishes the chemotype that can be assigned to L. graveolens varieties [78][79][80].
Due to its acidic and hydrophobic nature, carvacrol tends to damage biological systems and for that reason is responsible for affecting a wide range of microorganisms, including bacteria, fungi, yeast and parasites [81][82][83][84][85].It also has been proposed as a therapeutic agent against some cancer cell lines due to its activity as an antiproliferative compound [86,87], DNA synthesis inhibitor [88] and by triggering apoptosis [89,90].
Furthermore, carvacrol was tested against many tropical parasites responsible for serious human diseases.Thus, the compound was evaluated for its trypanocidal activity against Trypanosoma cruzi and T. brucei rhodesiense, being very effective in both cases, with IC50 values under 30 µg/mL [101][102][103], but noteworthily an important inhibition effect was observed on the epimastigote form in T. cruzi isolates (IC50 3.0 µg/mL).Following this topic, carvacrol was evaluated against visceral parasites of the genus Leishmania and the results demonstrated an important effectiveness range over the promastigote form of Leishmania chagasi with IC50 values between 2.3 to 28 µg/mL [103,104].A weak activity was observed on Leishmania donovani with an IC50 value of 17.8 mg/mL, compared with the reference drug miltefosine (IC50 0.14 mg/mL).In addition, antimalarial activity against Plasmodium falciparum was tested, obtaining a very significant IC50 value of 7.9 mg/mL [85].
Prior to our investigation, amebicide evaluation of carvacrol had not been carried out on E. histolytica, but one clinical study was performed on 14 adult patients, whose stools tested positive for enteric parasites such as Entamoeba hartmanni, Endolimax nana and Blastocystis hominis.The patients were supplemented with an essential oil rich in carvacrol from Origanum vulgare and after 6 weeks of treatment total disappearance of E. hartmanni and E. nana was observed in all infected patient cases.B. hominis was not detected in five cases [105].Nevertheless this is the first report about the antiprotozoal activity of carvacrol against Entamoeba histolytica.

Chalepensin from Ruta chalepensis
The bioguided fractionation of the methanolic extract of Ruta chalepensis by partition between methanol and n-hexane followed by chromatography of the hexane residue (84.66% growth inhibition against E. histolytica at a concentration of 150 µg/mL) over a silica gel column afforded chalepensin (2) as colorless needles with a melting point of 75 °C.The spectroscopic data were identical with those reported for chalepensin [106][107][108][109][110][111][112][113], but we now report the complete, unambiguous assignment of the 13 C-NMR spectrum of chalepensin as the hydrogen and carbon connectivities in 2 were deduced from 1 H-1 H COSY, NOESY, HSQC and HMBC spectra.

Possible Antiprotozoal Mechanism of Action of Carvacrol and Chalepensin
Little knowledge exists about the antiprotozoal mechanism of action of carvacrol but the antimicrobial mechanisms of action of carvacrol have been thoroughly investigated [81][82][83][84][85].In the following, we describe some facts of these antimicrobial mechanisms which future research might reveal if these apply as well to protozoa.Carvacrol exhibits antimicrobial activity against the biological membranes of bacteria.It exerts its action by rapidly depleting the intracellular ATP pool by reducing ATP synthesis and increasing ATP hydrolysis.Reduction of transmembrane electric potential which is the driving force of ATP synthesis enhances proton permeability of the membrane.At 1 mM carvacrol lowers the internal pH of bacteria from 7.1 to 5.8 according to ion gradients of the cell membrane.Carvacrol (1 mM) decreases cell protein content from 12 mmol/mg to 0.99 mmol/mg by using potassium (K + ) of bacterial cells in a short time (5 min).Potassium (K + ) plays a role in the activation of cytoplasmic enzymes, in maintaining osmotic pressure and in the regulation of cytoplasmic pH.Leakage of K + out of the cell is a clear indication of membrane damage.Ultee et al. [82] hypothesized a scheme for the mechanism of action of carvacrol through the cytoplasmic membrane of bacteria.According to this hypothesis undissociated carvacrol diffuses through the cytoplasmic membrane and dissociates releasing its proton to the cytoplasm.It then returns undissociated through the membrane into the external environment carrying a potassium ion.Outside the cell carvacrol replaces potassium with a proton and reenters the cell the same way.
The mechanism of action of oregano oils has been shown to be related, especially, to the synthesis of structural components and to the disruption of a series of energy systems.The leakage of ions, ATP and amino acids from bacterial cells explains this phenomenon.Potassium and phosphate ion concentrations were affected at a rate much lower than their MIC values [85].
Carvacrol increases overall permeability of the cytoplasmic membrane by disrupting the outer membranes of Gram negative bacteria leading to the leakage of ATP from the cell.Carvacrol also inhibits ATPase [85].Similar alterations to those observed in bacteria [85] were also observed on Giardia lamblia exposed to essential oils from different sources, especially those where carvacrol had a dominant presence (over 70% of general composition) [28].The main ultrastructural alterations promoted by essential oils were deformations in typical trophozoite appearance, often roundly shaped, irregular dorsal and ventral surface, presence of membrane blebs, electrodense precipitates in the cytoplasm and nuclei and internalization of flagella and ventral disc.The data suggest that essential oils probably induced cell death by processes associated to the loss of osmoregulation caused by plasmatic membrane alterations [28].
To our knowledge there are no studies regarding the antiprotozoal mechanism of chalepensin but chalepin, a furocoumarin structural related to chalepensin, exerts a potent inhibitory activity against the recombinant enzyme TcGAPDH (glyceraldehyde-3-phosphate dehydrogenase) of Trypanosoma cruzi with a strong IC50 of 64 µM [129,130].Further studies are required to establish the antiprotozoal mechanism of carvacrol and chalepensin against E. histolytica.

General
Melting points were determined on an Electrothermal 9100 apparatus (Electrothermal Engineering Ltd., Southend on Sea, UK).IR spectra were recorded on a Frontier FT-IR spectrometer (PerkinElmer, Waltham, MA, USA) using an ATR accessory.NMR spectra were measured on a Avance DPX 400 Spectrometer (Bruker, Billerica, MA, USA) operating at 400.13 MHz for 1 H and 100.61MHz for 13 C. ESI HR mass spectra were measured on a 4.7 T FT-ICR Mass spectrometer (Bruker, Bremen, Germany).EI MS was recorded on a MAT 95 spectrometer (70 eV, Finnigan, San Jose, CA, USA).TLC was carried out on pre-coated silica gel glass plates 5 cm × 10 cm (Merck silica gel 60 F254, Darmstadt, Germany).Normal phase column chromatography was performed on silica gel (60-200 mesh) purchased from J. T. Baker (Phillipsburg, NJ, USA).

Plant Material
The plants were obtained from the field or purchased from Pacalli ® (pacalli.com.mx,Monterrey, Mexico).Reference vouchers of the plant material were deposited at the herbarium UNL of the Facultad de Ciencias Biológicas (Universidad Autónoma de Nuevo León).Plant species, botanical name, family, voucher specimens and plant parts used to obtain the extracts are summarized in Table 1.Vegetal material was dried and ground to powder.

Extraction and Isolation
Sixty grams of dried and powdered material from the respective plant was extracted with methanol (MeOH, 600 mL) by using a Soxhlet system for continuous extraction.After filtration, the solvent was evaporated under reduced pressure in a rotary evaporator [25].The different extracts were conserved in tightly sealed glass vials.The yields are shown in Table 1.

Test Microorganisms
Entamoeba histolytica strain HM-1:IMSS was obtained from the microorganism culture collection of the Centro de Investigación Biomédica del Noreste (CIBIN-IMSS) in Nuevo León, Mexico.The trophozoites were grown axenically and maintained in peptone, pancreas and liver extract plus bovine serum [131].The trophozoites were employed at log phase of growth (2 × 10 4 cells/mL) by all the performed bioassays [132,133].

In Vitro Assay for Entamoeba histolytica
The MeOH extract from each plant was dissolved in DMSO and adjusted to a concentration of 150 µg/mL in a suspension of E. histolytica trophozoites at logarithmic phase in PEHPS medium containing 10% of bovine serum.Vials were incubated for 72 h, then chilled in iced water for 20 min and the number of dead trophozoites per milliliter was counted by using a hemocytometer.Each extract assay was performed by triplicate [132,133].Each test included a positive control by using metronidazole and a negative control by using E. histolytica suspension in PEHPS medium with no extract added.The inhibition percentage was estimated as the number of dead cells compared with the untreated controls.
The same procedure was performed with fractions or pure isolated compounds.

In Vitro IC50 Determination
The MeOH extract from each plant was dissolved in DMSO and adjusted to 150, 75, 32.5 and 16.25 µg/mL with a suspension of E. histolytica trophozoites at logarithmic phase in PEHPS medium containing 10% of bovine serum.Vials were incubated for 72 h, then chilled in iced water for 20 min and the number of dead trophozoites per milliliter was determined by using a hemocytometer.Each extract assay was performed by triplicate.The 50% inhibitory (IC50) concentration of each extract was determined by using a Probit analysis with a 95% confidence level.The same procedure was performed with fractions or pure isolated compounds.

Conclusions
Entamoeba histolytica is the most common parasite to cause enteric protozoan infections.The drug of choice used to treat amoebic dysentery is metronidazole, which has been associated with unpleasant side effects [134][135][136], therefore alternative drugs are needed and medicinal plants may be an important alternative source of new antiamoebic compounds.The results of the antiprotozoal screening in this work support the popular uses of 18 of the studied species for the treatment of diarrhea and dysentery in Mexican traditional medicine.The extracts from both Lippia graveolens Kunth and Ruta chalepensis Pers showed the most significant antiprotozoal activity and were submitted to a bioguided fractionation.Structure elucidation of the isolated compounds was accomplished by spectroscopic and mass spectrometric data.The methanolic extract of L. graveolens rendered carvacrol (1) with 95%-98% inhibition against E. histolytica at a concentration of 150 µg/mL (IC50 44.3 µg/mL) and from the methanolic extract of R. chalepensis chalepensin (2) with 98.4% inhibition at a concentration of 150 µg/mL (IC50 45.95 µg/mL) was recovered.To our knowledge, this is the first report on the antiamoebic activity of carvacrol and chalepensin, both known compounds with other notable pharmacological activities.These compounds may also offer new opportunities for treating amoebiasis and other important and often neglected diseases [137] or be useful as lead compounds in the development of new antiprotozoal agents.Further work for isolation of other active constituents from these plants is under way.

Table 2 .
Antiprotozoal activity against Entamoeba histolytica of methanolic extracts a from selected plants.