Metabolites from Roots of Colubrina greggii var. yucatanensis and Evaluation of their Antiprotozoan, Cytotoxic and Antiproliferative Activities

A purificação do extrato da raiz de Colubrina greggii var. yucatanensis levou ao isolamento e identificação do ácido 3-O-acetil ceanótico, um novo triterpeno natural, juntamente com os metabólitos já descritos: ácido ceanótico, ácido cenoténico, ácido betulínico, discarina B e crisofaneína. Os produtos naturais e os derivados semi-sintéticos éster de acetil dimetil ceanotato, dimetil ceanotato e peracetato de crisofaneína mostraram moderada a baixa atividade leishmanicida e tripanocida. Nenhum dos metabólitos mostrou ser citotóxicos ou ter atividade antiproliferativa. Os resultados também sugerem que o ácido betulínico contribui para a atividade antiplasmódica inicialmente detectada na raiz do extrato bruto de C. greggii var. yucatanensis.


Introduction
Leishmaniosis, trypanosomiosis and malaria are a group of protozoan diseases considered of significant importance due to their incidence and rate of mortality in developing countries. 1 The low effectiveness, limited availability and high toxicity of existing treatments for these illnesses emphasize the importance of continuing the search for new antiprotozoan pharmaceuticals. 2 Plants are considered an important source of biologically active natural products, 3 including a number of them with antiprotozoan activity. [4][5][6] During the screening of extracts from native Yucatecan medicinal plants as potential sources of bioactive metabolites, the root extract of Colubrina greggii S. Watson var. yucatanensis M. C. Johnst., a shrub used for the treatment of liver diseases, ulcerations, abscesses, asthma and tuberculosis, 7 showed trypanocidal, antimalarial, leishmanicidal and cytostatic activity. 8,9 Previous phytochemical studies of the genus Colubrina (Rhamnaceae), which include 31 species, 10 reported the presence of a wide structural diversity of metabolites, including ansa macrolides, saponins, aporphinic alkaloids, phenolic acids, flavones and triterpenoid acids. [11][12][13][14][15][16][17][18] To date, only chrysophanol, an anthraquinone with antimicrobial activity, has been reported as a metabolite from C. greggii. 19 In the present study we report the leishmanicidal, trypanocidal, antiplasmodial, cytotoxic and antiproliferative activity of the crude extract, semi-purified fractions and isolated metabolites from the root extract of C. greggii var. yucatanensis.
The FTIR spectrum of 1 showed absorption bands at 3073 (CH=C), 1731 (ester) and 1681 cm -1 (carboxyl). The pseudo molecular ion peak [M + Na] + at m/z 551.3524 in HRMS (high resolution mass spectrometry) indicated a molecular formula C 32 H 48 O 6 , implying nine degrees of unsaturation. Carbon multiplicity, deduced from HMQC (heteronuclear multiple quantum coherence) and DEPT (distortionless enhancement by polarization transfer) experiments, indicated the presence of seven methyl groups, nine methylene, seven methine, and nine quaternary carbons. The 1 H NMR spectrum of 1 (Table S1, Figure S1) displayed seven three-proton singlets at d 0. accounted for four degrees of unsaturation thus indicating that the five remaining unsaturation sites corresponded to a pentacyclic structure. The spectroscopic data of 1 proved to be very similar to those reported for ceanothic acid (2), a ceanothane triterpene also known as emmolic acid, originally isolated from Ceanothus americanus 20 and later identified from Colubrina granulosa. 13 However, the presence of a   suggested that 1 was the 3-O-acetyl derivative of ceanothic acid, which was confirmed when acetylation of 2 produced 1 as the only product. The presence of 1 in the original root extract of C. greggii, as detected by TCL ( Figure S2), ruled out its being an artifact of the isolation procedure. Discarine B (5), chrysophanein (6) and ceanothenic acid (3) were identified from comparison of their spectroscopic data (Tables S1, S2 and S3) with those reported in literature. [21][22][23] Betulinic acid (4) was identified by comparison with an authentic sample. 24 All of the isolated metabolites and the semisynthetic esters acetyl-dimethyl ceanothate (1a), dimethyl ceanothate (2a) and chrysophanein peracetate (6a) were evaluated for their in vitro antiprotozoan (leishmanicidal, trypanocidal, and antiplasmodial), cytotoxic and antiproliferative activities ( Table 1). The results showed a moderate leishmanicidal activity (IC 50 values of 20-28 µg mL -1 ) for natural ceanothanes 1 and 3 and semisynthetic derivatives 2a and 6a, whereas a low trypanocidal activity (IC 50 of 30-70 µg mL -1 ) was observed for the natural products 3-5 and the semisynthetic esters 1a and 6a. Betulinic acid (4) appeared to contribute to the antiplasmodial activity of the crude extract of C. greggii, with an IC 50 of 9.7 µg mL -1 . 25,26 Although the crude root extract, together with the low and medium polarity fractions showed cytotoxic activity against HEp-2 cells, none of the isolated metabolites displayed this type of activity (Table S4). Furthermore, none of the metabolites tested, with the exception of the peracetylated chrysophanein (6a), showed antiproliferative activity against KB cells (Table S5). It is interesting to point out that the cytotoxic activity of C. macrocarpa and C. texensis has been attributed to the presence of colubrinol and its acetate, 11,27 however these metabolites were not detected in the root extract of C. greggii.
To date, ceanothane triterpenes have only been reported to occur in species of the Rhamnaceae family, 20,23,28-36 and particularly in those belonging to the ziziphoids in the tribal classification reported by Richardson et al. 38 These results, together with our finding of ceanothanes in the root extract of C. greggii, support the possible use of this class of triterpenes as chemotaxonomic markers for a classification of Rhamnaceae based on a phylogenetic analysis.

General
Analytical TLC (thin layer chromatography) was carried out on aluminum-backed silica gel (60F 254 ) plates

Leishmanicidal assay
The growth inhibition of promastigotes was carried out following the procedure previously reported by Muñoz et al., 39 and Inchausti et al. 40 Briefly, a strain of L. amazonensis (IFLA/BR/75/PH8) was grown in Schneider culture medium with 10% fetal bovine serum (FBS), penicillin (100 IU mL -1 ) and streptomycin (100 mg mL -1 ) at 25 °C; parasites in the log phase of their growth cycle were then transferred to a microplate (96 wells; 1 × 10 5 parasites/well). Stock solutions of DMSO (blank), pentamidine (positive control), crude extract, semipurified fractions and pure metabolites were diluted in Schneider medium at ≤ 100 µg mL -1 , added to the plate, and incubated for 72 h. The percentages of inhibition were obtained by directed observation of each well with an inverted phase microscope. All the assays were carried out in triplicate.

Trypanocidal assay
Epimastigotes of Trypanosoma cruzi strain Tulahuen parasites were maintained in liver infusion tryptose (LIT) medium supplemented with 5% FBS, following the procedure modified by Chataing et al. 41 Briefly, parasites in the log phase of growth cycle were transferred to a microplate (96 wells; 1 × 10 6 parasites/well) together with stock solutions of benznidazole, DMSO (positive control and blank respectively), extract, semipurified fractions or pure metabolites prepared at different concentrations (≤ 100 µg mL -1 ). The microplates were incubated at 26 °C for 72 h.

Antiplasmodial assay
Plasmodium falciparum strain F32 was grown at 37 °C in RPMI medium with 10% of human serum and 4% of hematocrit (O, Rh+), under anaerobic conditions, according to a reported method. 41 Cultures with 1% parasitemic and 2% hematocrite (100 µL) were transferred to a 96 well plate. Stock solutions of chloroquine (positive control), DMSO (blank), extract, semipurified fractions or pure metabolites were diluted in RPMI medium to a concentration of < 10 µg mL -1 and added to each well. The plate was then incubated at 37 °C for 48 h.

Cytotoxicity assay
Human laryngeal carcinoma (HEp-2), human cervical adenocarcinoma (HeLa), human nasopharyngeal carcinoma (KB), and green monkey Vero kidney cells (VERO) were grown in DMEM (Gibco) media supplemented with 10% (v/v) FBS (Gibco), penicillin (100 IU mL -1 ), and streptomycin (100 mg mL -1 ). All the cell lines were maintained at 37 ºC in a 5% CO 2 atmosphere with 95% humidity. The cytotoxicity assay was performed according to a method described by Rahman et al. 43 Briefly, cell lines were transferred to a microplate (1.5 × 10 4 viable cells of each cell line) and incubated at 37 °C, with 95% humidity and 5% CO 2 in DMEM medium supplemented with 10% of FBS, penicillin (10000 IU), streptomycin (10 mg mL -1 ), and amphotericine B (5 mg mL -1 ). After 24 h, the medium was replaced by fresh medium with 0.05% DMSO (blank) or different concentrations of docetaxel (positive control, Sigma), crude extract, semipurified fractions or pure metabolites dissolved in DMSO (100, 50, 25, 12.5 and 6.25 µg mL -1 ), and the cells were incubated for 72 h under the conditions already described. The medium was removed and 200 µL of a 0.5% MTT (Sigma) solution in PBS (pH 7.2) were added to each well, and left to stand for 4 h at 37 °C. Then 100 µL of acidified isopropanol (0.4 mol L -1 HCl) were added to each well and the optical density (OD) measured at 540 nm using a Bioassay reader (Bio-Rad). The experiment was carried out in triplicate and each concentration was tested in duplicate.

Antiproliferative assay
The sulforhodamine B (SRB) assay was carried out according to the method reported by Rahman et al., 43 using DMEM medium with 10% FBS to induce cell proliferation. After 48 h of incubation, the medium was discarded and 100 µL of ice-cold 40% trichloroacetic acid (TCA, Aldrich) were added to fix the cells, incubating for 1 h at 4 °C. The cells were washed five times with water, left to dry, and then 50 µL of SRB stain (10 mg 1% acetic acid, Sigma) were added to each well and left to stand for 30 min. Finally, the cells were washed with 50 mL 1% acetic acid, and rinsed four times with water. The OD was measured at 540 nm using an ELISA reader (Bio-Rad model 450). The experiment was carried out in triplicate.