Furanocoumarins from Ruta chalepensis with Amebicide Activity

Entamoeba histolytica (protozoan; family Endomoebidae) is the cause of amoebiasis, a disease related to high morbidity and mortality. Nowadays, this illness is considered a significant public health issue in developing countries. In addition, parasite resistance to conventional medicinal treatment has increased in recent years. Traditional medicine around the world represents a valuable source of alternative treatment for many parasite diseases. In a previous paper, we communicated about the antiprotozoal activity in vitro of the methanolic (MeOH) extract of Ruta chalepensis (Rutaceae) against E. histolytica. The plant is extensively employed in Mexican traditional medicine. The following workup of the MeOH extract of R. chalepensis afforded the furocoumarins rutamarin (1) and chalepin (2), which showed high antiprotozoal activity on Entamoeba histolytica trophozoites employing in vitro tests (IC50 values of 6.52 and 28.95 µg/mL, respectively). Therefore, we offer a full scientific report about the bioguided isolation and the amebicide activity of chalepin and rutamarin.


Introduction
Amoebiasis is the term for a parasitic infection triggered by the protozoan Entamoeba histolytica. This disease represents one of the most widespread parasite maladies in developing countries [1,2], making it a significant public health problem [3,4].
In Mexico, this infection is considered an endemic illness [5][6][7] and represents the most common parasitic disease found in the general population [8,9]. It is also more frequently found in the intestinal amoebiasis of Mexican children, especially newborn and school children [10].
Amoebiasis frequently produces an intestinal infection that can transform into an extraintestinal infection throughout the portal veins, affecting the liver and producing a more significant hepatic lesion, in addition to lesions in the lungs, skin, and brain [11,12]. Following malaria, amoebiasis represents the second leading factor of death caused by parasite infections [13]. Nearly 50 million people develop acute amoebiasis, and 40,000-100,000 deaths caused by this sickness happen per year worldwide [14,15].
It is important to highlight that the chemotherapy used against E. histolytica has shown significant advancement. Nevertheless, the treatment's abandonment produces chronic patients, illness propagators, and develops a multidrug resistance parasite [10,16].
The most utilized therapeutic drug for the medication of this infection is metronidazole, but, owing to the drug's undesired side effects [17,18] and considering the burgeon of resistant strains of E. histolytica against it, new antiprotozoal agents are required [19,20]. Plants are a good source of natural products that have been used in the treatment of protozoa illness [21][22][23][24], and Mexican traditional medicine can offer many plants that could be useful for developing treatment against E. histolytica [25]. In a previous paper, we discussed the antiamoebic activity in vitro of the MeOH extract of Ruta chalepensis [26]. Ruta chalepensis is a Mediterranean plant introduced in Mexico and used by different ethnic groups to treat gastrointestinal illnesses such as stomachache, diarrhea, dysentery, and nausea [27]. Additionally, there are reports of emmenagogue, anthelmintic, antirheumatic, antihypertensive, abortive, and anti-inflammatory activity for this plant [28,29]. R. chalepensis possess many known furanocoumarins, coumarins, alkaloids, quinoline alkaloids, and flavonoids distributed in leaves, stems, and roots [28,30,31].
This research aims to isolate and identify substances responsible for the amebicide activity of R. Chalepensis.

Bioguided Isolation of Furanocoumarins from Ruta chalepensis
The following diagram ( Figure 1) shows the number of chromatography columns implemented (silica gel or Sephadex), as well as the percentage of inhibition against E. histolytica of the most relevant fractions during the bioguided fractionation of the MeOH extract of R. chalepensis to obtain the furanocoumarins Rutamarin (1) and Chalepin (2) in high purity (TLC; see Supplementary Materials). Both compounds were characterized by different spectroscopic techniques. The unambiguous assignment of the 13 C-NMR spectrum of the isolated furanocoumarins  was obtained from 1 H-1 H COSY, NOESY, HSQC, and HMBC spectra (see Supplementary Material).
Both compounds are structurally related to chalepensin (3), a furanocoumarin also occurring in this plant [26]. See Figure 2.  50 for Rutamarin (1) and Chalepin (2) The in vitro assay for the isolated furanocoumarins against trophozoites of E. histolytica showed significant activity. Figures 3 and 4 show the 50% inhibitory concentration of each compound calculated using a probit analysis, considering a 95% confidence level.

Discussion
Ruta chalepensis is a medicinal plant used worldwide for its wide range of medicinal purposes [35]. However, the antiparasitic activity against Entamoeba histolytica of this plant has not been sufficiently disclosed in ethnobotanical reports until now. Antiparasitic activity of R. chalepensis has been previously evaluated on helminths of veterinary importance [36,37] and protozoa of clinical importance such as Leishmania infantum, L. major [38] and Giardia lamblia [39,40]. The antiparasitic activity over E. histolytica by the methanolic crude extract was previously reported [26], showing moderate activity with an IC 50 of 60.07 µg/mL. The antiparasitic activity of the furanocoumarin chalepensin (3), isolated from the hexane partition of R. chalepensis by Quintanilla-Licea et al. [26], was also moderate, showing an IC 50 of 45.95 µg/mL. The antiamoebic activity of rutamarin (1) and chalepin (2) isolated in this research work is slightly higher (IC 50 of 6.52 and 28.95 µg/mL, respectively) but far less effective than metronidazole (IC 50 0.205 µg/mL) [26]. We can therefore consider the three furanocoumarins as responsible for the antiparasitic activity of R. chalepensis. Furanocoumarins are part of the chemical components with the most significant presence in R. chalepensis [41]. Rutamarin (1) and chalepin (2) have been previously isolated from this plant [42]; our research group briefly described the amebicide activity of these furanocoumarins at a scientific congress [43,44]. We are now offering a full scientific report about the bioguided isolation and identification of these amebicide compounds. These results may support the use of R. chalepensis as an alternative treatment for amoebiasis in traditional Mexican medicine, as it is for other plants of equal importance [45,46]. The toxic activity of coumarins on eukaryotic cells has received an important analysis suggesting a partial mechanism of action for compounds of this nature where cell injury is caused by damage to DNA [47]. Chalepin (2) has shown specific toxicity on normal cells and cancer cell lines, for example HT29 human colon carcinoma cells with cytotoxic activity by 55.1 µM. Although rutamarin (1), in some analyses, showed selectivity with its cytotoxic activity at a concentration of 1.12 µM, it was also reported that it generates significant alterations in the cell growth of BCBL-1 cells at a concentration of 5.60 µM [48,49]. A certain toxicity on normal mammalian cells at the respective concentrations of the IC 50 of compounds 1 and 2 (18.29 and 92.08 µM, respectively) could be expected. The furanocoumarins isolated from R. chalepensis (1-3) have outstanding antiamoebic activity, but the presence of an acetyl group in rutmarin may increase the antiparasitic activity, producing a better complex with DNA and therefore causing more considerable cellular damage [47].

General Experimental Procedures
NMR spectra were acquired on an Avance DPX 400 Spectrometer (Bruker, Billerica, MA, USA) working at 400.13 MHz for 1 H and 100.61 MHz for 13 C. Melting points were measured on Electrothermal 9100 equipment (Electrothermal Engineering Ltd., Southendon-Sea, UK).
Thin-layer chromatography (TLC) was developed on pre-coated silica gel plates (Merck, Kenilworth, NJ, USA Silica Gel 60 F254). Visualization of the components of the crude extract or pure compounds was made using UV light. Open column chromatography was implemented with silica gel of a 60-200 mesh (J. T. Baker, Phillipsburg, NJ, USA) and Sephadex LH-20 (Sigma-Aldrich, St. Louis, MO, USA, LH20100-500G).

Plant Material
R. chalepensis, L. Rutaceae was collected near the city of Aramberri, Nuevo León State in northern Mexico, 24 • 19 13" N, 99 • 54 55" W. It was dried using a light chamber at 38 • C and was then powdered with a manual grain. Plant material was placed at the herbarium of the Facultad de Ciencias Biológicas in the Universidad Autónoma de Nuevo León, with a register number 025579.

Extraction of Plant Material from R. chalepensis
Six hundred grams of leaves and stems from R. chalepensis were dried and powdered, separated in packets of 60 g, and subjected to extraction with 600 mL of MeOH, each using Soxhlet equipment for 40 h. The MeOH was eliminated in a rotary evaporator. The crude extract was preserved at 4 • C until it was needed.

Microorganisms
The trophozoites of Entamoeba histolytica (strain HM-1:IMSS) were acquired from the Centro de Investigación Biomédica del Noreste (CIBIN) microorganism culture collection in Monterrey (Mexico). The parasites were grown axenically and kept up in peptone combined with pancreas extract, liver extract and bovine serum (PEHPS medium, so designated based on the Spanish initials of its major components). The microorganisms were utilized at the log phase of growth (2 × 10 4 cells/mL) by all of the tests carried out [51,52].
Next, 2 × 10 5 trophozoites of E. histolytica in 5 mL of the PEHPS medium (with the 10% bovine serum added) were inoculated in 13 mm × 100 mm screw cap test tubes and incubated at 36.5 • C for 120 h to establish the growth curve for E. histolytica. Every 24 h, the number of trophozoites was evaluated to determine the medium's growth parameters [53]. The procedure was performed in 3 separate assays per triplicate.

In Vitro Test for Entamoeba histolytica
Each sample was dissolved in the DMSO and standardized to 150 µg/mL by adding a mixture of E. histolytica trophozoites at a logarithmic phase in the PEHPS medium with the 10% bovine serum. Afterward, vials were incubated for 72 h and then introduced into iced water for 20 min. The number of dead trophozoites per milliliter was estimated by using a hemocytometer. Each assay was implemented in triplicate. The positive control was metronidazole. The negative control was an E. histolytica suspension in the PEHPS medium with no extract added. The percentage of inhibition was determined by evaluating the number of dead trophozoites in the samples and the negative controls [53].

In Vitro IC 50 Evaluation
Each sample was dissolved in the DMSO and standardized to 150, 75, 37.5, 18.75, and 9.375 µg/mL by adding a mixture of E. histolytica trophozoites at a logarithmic phase in the PEHPS medium with the 10% bovine serum. Afterward, vials were incubated for 72 h and then introduced into iced water for 20 min. The number of dead trophozoites per milliliter was estimated by using a hemocytometer. Each assay was implemented in triplicate. The positive control was Metronidazole. The negative control was an E. histolytica suspension in the PEHPS medium with no extract added. The percentage of inhibition was determined by evaluating the number of dead trophozoites in the samples and the negative controls. The 50% inhibitory concentration of each sample was determined using a probit analysis, taking into account a 95% confidence level [53].

Bioassay-guided Fractionation
The crude MeOH extract of R. chalepensis (124 g) with an amebicide activity of 90.50% at 150 µg/mL [26] was dissolved again in one L methanol and distributed into four portions of 250 mL, each to make a liquid-liquid partition with n-hexane (750 mL each portion). The hexane partition was evaporated under vacuum. Column chromatography on the silica gel of this hexane residuum led to the isolation of chalepensin (3), Figure 1

Conclusions
This study describes the bioguided isolation [54] of two amebicide constituents of R. chalepensis. The furanocoumarins rutamarin (1) and chalepin (2) represent the most active chemical components of R. chalepensis against the protozoa E. histolytica with IC 50 values of 6.52 and 28.95 µg/mL, respectively. To the best of our knowledge, this is the first report about the amebicide activity of chalepin and rutamarin. Even though the amebicide activity of both compounds are far less effective than metronidazole (IC 50 0.205 µg/mL), the IC 50 values obtained in our research for rutamarin and chalepin may also be used as the basis for incorporating Ruta chalepensis extracts into conventional and complementary medicine for the therapy of amoebiasis and other infectious diseases.  Figure S37. NMR Data of Rutamarin (1) and Chalepin (2), Figure S38. Antiprotozoal activity of Rutamarin (1) against trophozoite of E. histolytica. T = negative control consisting of culture medium and parasites, Figure S39. Antiprotozoal activity of Chalepin) (2) against trophozoite of E. histolytica. T = negative control consisting of culture medium and parasites, Figure S40. General scheme for the bioguided isolation of compounds with antiamoebic activity from Ruta chalepensis.  protozoa E. histolytica used in this research. We thank H. Laatsch of the University of Göttingen (Germany) for the spectroscopic measurements.